0.05). The average number of days between visits was 135 (± 18) days with the shortest interval being 100 days and the longest, 189 days. The interquartile range was 126 to 141 days. The mean toenail growth length across the average number of days between visits was 6.48 mm (135 days x 0.048 mm/day). The mean time period spanned by the three visits was 270 days (range, 226 to 350). Interquartile Hg-concentration ranges for the three visits were 0.33-0.67, 0.34-0.66 and 0.34-0.67 ng/mg, respectively. Respective 95th percentile confidence intervals were ± 0.12, 0.13 and 0.10. Arithmetic mean toenail-Hg values with standard deviations and coefficients of variation in parentheses for the 1st, 2nd and 3rd visits were 0.60 ±0.42 (0.70), 0.60 ±0.43 (0.72) and 0.56 ±0.35 (0.63) ng/mg with box plots depicting visit distributional data provided in Figure 1. No significant difference in toenail-Hg levels was observed among the three sampling visits, but the relatively small number of samples limited the power to detect a small difference. Pearson’s product–moment correlation coefficients of Hg concentration between sampling intervals were as follows: r = 0.92 between 1st and 2nd clinic visits, r = 0.87 between 2nd and 3rd visits and r = 0.75 between 1st and 3rd visits.\nToenail-Hg levels for each visit (n = 43). Medians are middle lines within box. Top and bottom of box represents upper and lower quartile values, respectively. Upper and lower whiskers represent sample maximum and minimum values, respectively. Distribution mean values do not differ significantly (p <0.05).\nThe absolute change in toenail-Hg levels on an individual basis between successive samples across the three visits is depicted in Figure 2. With the exception of three toenail-Hg levels (out of 129), all values for separate clinic visits were within 50% to 150% of the individual’s mean toenail-Hg level. The extent of intra-individual change among participants depicted as the ratio of highest to lowest toenail-Hg levels from the visits for each individual is provided as a histogram in Figure 3. Nearly all participants (39 of 43) had less than a two-fold change in toenail-Hg levels across the one-year time period encompassed by the three samples with half (22 of 43) not exceeding a 1.5-fold change. Range of change was 1.08 - 3.44.\nIntra-individual toenail-Hg variability across three successive clinic visits.\nRatio of intra-individual toenail-Hg levels (n = 43).\nChronologically matched sample evaluation was conducted by comparing toenail-Hg levels with hair-Hg values representing the same time period of exposure (n = 41). Chronological matching of samples was not possible for all individuals due to factors such as timing of sample collections and length of hair samples. A regression model of the relationship between chronologically matched toenail-Hg and hair-Hg samples reflecting the same period of exposure provided for a slope (Hg ng/mg) of 2.79 for hair relative to tonail (r=0.954) (Figure 4). For purposes of comparison with previously conducted studies, the regression line of hair-Hg on toenail-Hg using results from samples obtained at the same point in time (i.e. during a clinic visit) and thus not reflecting the same window of exposure (chronologically unmatched) was determined and yielded a slope of 2.39 (r = 0.871)(data not shown). A comparison of results on group mean biomarker-Hg levels identified from the literature are provided in Table 1. The table gives ratios of hair-Hg to toenail-Hg based on study mean values with the addition of slope values from regression models of hair-Hg on toenail-Hg provided from three of the studies [13,15, this study]. The ratio of hair- to toenail-Hg based on arithmetic mean values for this study was 3.08 and 2.77 based on chronologically matched and unmatched samples respectively. The unmatched sample ratio of 2.77 was similar to the ratio (2.56) obtained by Ohno and co-workers [15] within a similar population (non-occupationally exposed group of Japanese women) and also similar to ratios obtained from a control group (2.4) in a cohort study of dentists as well as from one of two cohorts (2.5) studied in Finland [13,26]. The slope from the regression model using chronologically unmatched sample results (2.39) was similar to the slope (2.44) obtained by Ohno and co-workers [15] and by Alfthan [13] in one of two cohorts (2.24). For comparative purposes, a ratio of 3.16 was derived and is presented in Table 1 using geometric mean values for hair-Hg and toenail-Hg from matched samples; 1.41 ng/mg and 0.45 ng/mg, respectively.\nRelationship between toenail- and hair- Hg levels (ng/mg) among participants (n= 41).\nHair-Hg to toenail-Hg ratios determined from regression models and mean values obtained from literature\na. Study data provided for blood-Hg and toenail-Hg values along with regression model. Hair-Hg to blood-Hg ratio of 250:1 was used to obtain hair-Hg: toenail-Hg and slope of regression model of blood-Hg on toenail-Hg [4].\nb. One individual had previous occupational exposure as a dental nurse.\nc. Ohno and co-workers [15] and Alfthan [13] provided regression models of hair-Hg on toenail-Hg with resulting slope factors. Slope in this study from linear regression model based on chronologically matched and unmatched samples are provided.\nd. For this study, geometric mean values for hair-Hg and toenail-Hg from matched samples were 1.410 ng/mg and 0.446 ng/mg, respectively."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The data presented in this study will allow for an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure. A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that has a MeHg body-burden level that is relatively stable over time. Intra-individual variation was less than two-fold, which may represent normal body burden fluctuation for individuals consuming elevated amounts of various fish species with different contaminant levels. The use of biomarker-based estimates reflecting body-burden levels that are chronologically distant from manifestation of deleterious outcome, or that are used as the sole indicator reflecting chronic exposure, need to be considered carefully when testing a hypothesis as individual body-burden levels may not remain constant across lengthy time periods such as a decade."},"other_sections_titles":{"kind":"list like","value":["Background","AMIBS","Hair and toenail sampling","Hair- and toenail-Hg analyses","Statistical analyses","Abbreviations","Competing interests","Authors’ contributions","Disclaimer"],"string":"[\n \"Background\",\n \"AMIBS\",\n \"Hair and toenail sampling\",\n \"Hair- and toenail-Hg analyses\",\n \"Statistical analyses\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Disclaimer\"\n]"},"other_sections_texts":{"kind":"list like","value":["As most human exposures to methylmercury (MeHg) result from fish consumption, federal and state consumption advisories have been released as a public health protective measure [1,2]. Most state and federal advice to fish consumers reflects consideration of the US EPA reference dose (RfD) for MeHg [3]. The RfD, as well as the general recognition of the hazardous nature of MeHg exposure is in turn based upon extensive neurological research that has been repeatedly summarized e.g., [4-6]. In addition to concerns over neurophysiological deficits that can occur in children exposed in utero to MeHg, concern has been raised about the possible role of MeHg in cardiovascular disease in adults. There have been several major epidemiological studies conducted that investigated the relationship between MeHg exposure and the incidence of myocardial infarction [7-11]. The findings from four of these five studies along with additional data relating MeHg exposure with, for example, heart rate variability and oxidative stress were critical to a panel assembled under contract to the US EPA that recommended the development of a dose–response function for myocardial infarction and MeHg exposure in a regulatory benefits analyses addressing Hg emissions [12]. Roman et al. [12] did not address findings from the subsequently published fifth study which did not observe an association between MeHg exposure and coronary heart disease or stroke [11].\nHair-Hg, blood-Hg and toenail-Hg levels have been used as biomarkers of exposure for MeHg with each biomarker having been significantly correlated with fish consumption which is the primary source of MeHg exposure [4,6,13-15]. Analyses of hair-Hg and blood-Hg levels in fish consumers and non-fish consumers have been conducted allowing for these compartments to be used to characterize exposure to MeHg within a population [4]. Hair- and blood- Hg concentration have been the biomarkers of MeHg exposure used most frequently in studies investigating the relationship between MeHg exposure and neurodevelopmental outcomes as well as in several of the major studies investigating cardiovascular effects [7,10,16-18]. However, several important epidemiological studies investigating the relationship between exposure and cardiovascular outcomes have relied on toenail-Hg levels as the biomarker for MeHg exposure [8,9,11]. Data on the use of toenail-Hg levels as a biomarker of exposure is less robust compared to data from hair and blood compartments. The foundation of the toenail compartment as a biomarker is primarily based on observed correlations with hair-Hg levels and fish consumption with several studies having assessed MeHg exposures using toenails and a few having compared measurements across multiple markers [13-15,19-26]. Several studies within this group have provided for data allowing hair-Hg to toenail-Hg ratios to be established based on mean values and have developed regression models examining the relationship between hair-Hg and toenail-Hg levels [13,15,21,22,26]. However, despite these available data comparing Hg concentrations between two compartments, data are not available comparing hair and toenail biomarker results from exposure that occurred over the same time period.\nAs part of the Arsenic Mercury Intake Biometric Study (AMIBS), we compared Hg levels from hair segments and toenail clippings reflecting the same time period of exposure and examined longitudinal data for toenail-Hg levels obtained at three time points within the population of Japanese women living in the Puget Sound area of Washington State (US). Previous work has examined fish intake and MeHg body burden in this population and results indicated that this Japanese population consumes substantially more fish than the national average and that MeHg exposure within this group is among the highest in the US [27,28]. The goals of this work were two-fold: to more accurately define the toxicokinetic variability of Hg levels observed between the hair and toenail compartment, and to examine variability in temporal toenail-Hg levels that were obtained over a period of approximately one year. This information will permit an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure and may be useful in future studies examining the relationship between neurological effects and MeHg exposure. It will also facilitate the comparison between the results of studies employing toenail- and hair-Hg levels as biomarkers of exposure.","As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.","To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.","Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).","A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).","AMIBS: Arsenic Mercury Intake Biometric Study; EPA: Environmental Protection Agency; GM: Geometric Mean; Hg: Mercury; MeHg: MethylMercury; RfD: Reference Dose; US: United States; WA: Washington State.","All authors declare they have no actual or potential competing financial or non-financial interests in this work.","TH conducted the hair-Hg and toenail-Hg analyses and provided the Supplemental Materials. AT was a significant contributor to almost all aspects of this project involving the Korean and Japanese populations. AMIBS would not have been as successful without her. AS aided in writing the manuscript. TMB contributed significantly to study design and data interpretation. EM aided in the writing of the manuscript and contributed to the acquisition of the data. All authors contributed substantially to the discussion of the data and their analyses, and provided editorial comments to the draft manuscript.","The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views or policies of the U.S. EPA, NIH/National Institute of Environmental Health, National Science Foundation, New Jersey Department of Environmental Protection, or Washington State Department of Health."],"string":"[\n \"As most human exposures to methylmercury (MeHg) result from fish consumption, federal and state consumption advisories have been released as a public health protective measure [1,2]. Most state and federal advice to fish consumers reflects consideration of the US EPA reference dose (RfD) for MeHg [3]. The RfD, as well as the general recognition of the hazardous nature of MeHg exposure is in turn based upon extensive neurological research that has been repeatedly summarized e.g., [4-6]. In addition to concerns over neurophysiological deficits that can occur in children exposed in utero to MeHg, concern has been raised about the possible role of MeHg in cardiovascular disease in adults. There have been several major epidemiological studies conducted that investigated the relationship between MeHg exposure and the incidence of myocardial infarction [7-11]. The findings from four of these five studies along with additional data relating MeHg exposure with, for example, heart rate variability and oxidative stress were critical to a panel assembled under contract to the US EPA that recommended the development of a dose–response function for myocardial infarction and MeHg exposure in a regulatory benefits analyses addressing Hg emissions [12]. Roman et al. [12] did not address findings from the subsequently published fifth study which did not observe an association between MeHg exposure and coronary heart disease or stroke [11].\\nHair-Hg, blood-Hg and toenail-Hg levels have been used as biomarkers of exposure for MeHg with each biomarker having been significantly correlated with fish consumption which is the primary source of MeHg exposure [4,6,13-15]. Analyses of hair-Hg and blood-Hg levels in fish consumers and non-fish consumers have been conducted allowing for these compartments to be used to characterize exposure to MeHg within a population [4]. Hair- and blood- Hg concentration have been the biomarkers of MeHg exposure used most frequently in studies investigating the relationship between MeHg exposure and neurodevelopmental outcomes as well as in several of the major studies investigating cardiovascular effects [7,10,16-18]. However, several important epidemiological studies investigating the relationship between exposure and cardiovascular outcomes have relied on toenail-Hg levels as the biomarker for MeHg exposure [8,9,11]. Data on the use of toenail-Hg levels as a biomarker of exposure is less robust compared to data from hair and blood compartments. The foundation of the toenail compartment as a biomarker is primarily based on observed correlations with hair-Hg levels and fish consumption with several studies having assessed MeHg exposures using toenails and a few having compared measurements across multiple markers [13-15,19-26]. Several studies within this group have provided for data allowing hair-Hg to toenail-Hg ratios to be established based on mean values and have developed regression models examining the relationship between hair-Hg and toenail-Hg levels [13,15,21,22,26]. However, despite these available data comparing Hg concentrations between two compartments, data are not available comparing hair and toenail biomarker results from exposure that occurred over the same time period.\\nAs part of the Arsenic Mercury Intake Biometric Study (AMIBS), we compared Hg levels from hair segments and toenail clippings reflecting the same time period of exposure and examined longitudinal data for toenail-Hg levels obtained at three time points within the population of Japanese women living in the Puget Sound area of Washington State (US). Previous work has examined fish intake and MeHg body burden in this population and results indicated that this Japanese population consumes substantially more fish than the national average and that MeHg exposure within this group is among the highest in the US [27,28]. The goals of this work were two-fold: to more accurately define the toxicokinetic variability of Hg levels observed between the hair and toenail compartment, and to examine variability in temporal toenail-Hg levels that were obtained over a period of approximately one year. This information will permit an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure and may be useful in future studies examining the relationship between neurological effects and MeHg exposure. It will also facilitate the comparison between the results of studies employing toenail- and hair-Hg levels as biomarkers of exposure.\",\n \"As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.\",\n \"To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.\",\n \"Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).\",\n \"A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).\",\n \"AMIBS: Arsenic Mercury Intake Biometric Study; EPA: Environmental Protection Agency; GM: Geometric Mean; Hg: Mercury; MeHg: MethylMercury; RfD: Reference Dose; US: United States; WA: Washington State.\",\n \"All authors declare they have no actual or potential competing financial or non-financial interests in this work.\",\n \"TH conducted the hair-Hg and toenail-Hg analyses and provided the Supplemental Materials. AT was a significant contributor to almost all aspects of this project involving the Korean and Japanese populations. AMIBS would not have been as successful without her. AS aided in writing the manuscript. TMB contributed significantly to study design and data interpretation. EM aided in the writing of the manuscript and contributed to the acquisition of the data. All authors contributed substantially to the discussion of the data and their analyses, and provided editorial comments to the draft manuscript.\",\n \"The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views or policies of the U.S. EPA, NIH/National Institute of Environmental Health, National Science Foundation, New Jersey Department of Environmental Protection, or Washington State Department of Health.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","AMIBS","Hair and toenail sampling","Hair- and toenail-Hg analyses","Statistical analyses","Results","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions","Disclaimer","Supplementary Material"],"string":"[\n \"Background\",\n \"Methods\",\n \"AMIBS\",\n \"Hair and toenail sampling\",\n \"Hair- and toenail-Hg analyses\",\n \"Statistical analyses\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Disclaimer\",\n \"Supplementary Material\"\n]"},"all_sections_texts":{"kind":"list like","value":["As most human exposures to methylmercury (MeHg) result from fish consumption, federal and state consumption advisories have been released as a public health protective measure [1,2]. Most state and federal advice to fish consumers reflects consideration of the US EPA reference dose (RfD) for MeHg [3]. The RfD, as well as the general recognition of the hazardous nature of MeHg exposure is in turn based upon extensive neurological research that has been repeatedly summarized e.g., [4-6]. In addition to concerns over neurophysiological deficits that can occur in children exposed in utero to MeHg, concern has been raised about the possible role of MeHg in cardiovascular disease in adults. There have been several major epidemiological studies conducted that investigated the relationship between MeHg exposure and the incidence of myocardial infarction [7-11]. The findings from four of these five studies along with additional data relating MeHg exposure with, for example, heart rate variability and oxidative stress were critical to a panel assembled under contract to the US EPA that recommended the development of a dose–response function for myocardial infarction and MeHg exposure in a regulatory benefits analyses addressing Hg emissions [12]. Roman et al. [12] did not address findings from the subsequently published fifth study which did not observe an association between MeHg exposure and coronary heart disease or stroke [11].\nHair-Hg, blood-Hg and toenail-Hg levels have been used as biomarkers of exposure for MeHg with each biomarker having been significantly correlated with fish consumption which is the primary source of MeHg exposure [4,6,13-15]. Analyses of hair-Hg and blood-Hg levels in fish consumers and non-fish consumers have been conducted allowing for these compartments to be used to characterize exposure to MeHg within a population [4]. Hair- and blood- Hg concentration have been the biomarkers of MeHg exposure used most frequently in studies investigating the relationship between MeHg exposure and neurodevelopmental outcomes as well as in several of the major studies investigating cardiovascular effects [7,10,16-18]. However, several important epidemiological studies investigating the relationship between exposure and cardiovascular outcomes have relied on toenail-Hg levels as the biomarker for MeHg exposure [8,9,11]. Data on the use of toenail-Hg levels as a biomarker of exposure is less robust compared to data from hair and blood compartments. The foundation of the toenail compartment as a biomarker is primarily based on observed correlations with hair-Hg levels and fish consumption with several studies having assessed MeHg exposures using toenails and a few having compared measurements across multiple markers [13-15,19-26]. Several studies within this group have provided for data allowing hair-Hg to toenail-Hg ratios to be established based on mean values and have developed regression models examining the relationship between hair-Hg and toenail-Hg levels [13,15,21,22,26]. However, despite these available data comparing Hg concentrations between two compartments, data are not available comparing hair and toenail biomarker results from exposure that occurred over the same time period.\nAs part of the Arsenic Mercury Intake Biometric Study (AMIBS), we compared Hg levels from hair segments and toenail clippings reflecting the same time period of exposure and examined longitudinal data for toenail-Hg levels obtained at three time points within the population of Japanese women living in the Puget Sound area of Washington State (US). Previous work has examined fish intake and MeHg body burden in this population and results indicated that this Japanese population consumes substantially more fish than the national average and that MeHg exposure within this group is among the highest in the US [27,28]. The goals of this work were two-fold: to more accurately define the toxicokinetic variability of Hg levels observed between the hair and toenail compartment, and to examine variability in temporal toenail-Hg levels that were obtained over a period of approximately one year. This information will permit an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure and may be useful in future studies examining the relationship between neurological effects and MeHg exposure. It will also facilitate the comparison between the results of studies employing toenail- and hair-Hg levels as biomarkers of exposure."," AMIBS As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.\nAs a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.\n Hair and toenail sampling To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.\nTo assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.\n Hair- and toenail-Hg analyses Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).\nSpecimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).\n Statistical analyses A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).\nA repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).","As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.","To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.","Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).","A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).","Toenail and hair samples were collected from 43 Japanese participants across three clinic visits. Mean toenail growth rate was 0.048 ±0.012 mm/day (22.4 ±6.5 days/mm) and mean hair growth rate was 0.42 ±0.07 mm/day (24.5 ±5.3 days/cm). No significant difference in toenail-Hg or in hair-Hg levels was observed between pregnant (n = 11) and non-pregnant participants (n = 32) (p > 0.05). The average number of days between visits was 135 (± 18) days with the shortest interval being 100 days and the longest, 189 days. The interquartile range was 126 to 141 days. The mean toenail growth length across the average number of days between visits was 6.48 mm (135 days x 0.048 mm/day). The mean time period spanned by the three visits was 270 days (range, 226 to 350). Interquartile Hg-concentration ranges for the three visits were 0.33-0.67, 0.34-0.66 and 0.34-0.67 ng/mg, respectively. Respective 95th percentile confidence intervals were ± 0.12, 0.13 and 0.10. Arithmetic mean toenail-Hg values with standard deviations and coefficients of variation in parentheses for the 1st, 2nd and 3rd visits were 0.60 ±0.42 (0.70), 0.60 ±0.43 (0.72) and 0.56 ±0.35 (0.63) ng/mg with box plots depicting visit distributional data provided in Figure 1. No significant difference in toenail-Hg levels was observed among the three sampling visits, but the relatively small number of samples limited the power to detect a small difference. Pearson’s product–moment correlation coefficients of Hg concentration between sampling intervals were as follows: r = 0.92 between 1st and 2nd clinic visits, r = 0.87 between 2nd and 3rd visits and r = 0.75 between 1st and 3rd visits.\nToenail-Hg levels for each visit (n = 43). Medians are middle lines within box. Top and bottom of box represents upper and lower quartile values, respectively. Upper and lower whiskers represent sample maximum and minimum values, respectively. Distribution mean values do not differ significantly (p <0.05).\nThe absolute change in toenail-Hg levels on an individual basis between successive samples across the three visits is depicted in Figure 2. With the exception of three toenail-Hg levels (out of 129), all values for separate clinic visits were within 50% to 150% of the individual’s mean toenail-Hg level. The extent of intra-individual change among participants depicted as the ratio of highest to lowest toenail-Hg levels from the visits for each individual is provided as a histogram in Figure 3. Nearly all participants (39 of 43) had less than a two-fold change in toenail-Hg levels across the one-year time period encompassed by the three samples with half (22 of 43) not exceeding a 1.5-fold change. Range of change was 1.08 - 3.44.\nIntra-individual toenail-Hg variability across three successive clinic visits.\nRatio of intra-individual toenail-Hg levels (n = 43).\nChronologically matched sample evaluation was conducted by comparing toenail-Hg levels with hair-Hg values representing the same time period of exposure (n = 41). Chronological matching of samples was not possible for all individuals due to factors such as timing of sample collections and length of hair samples. A regression model of the relationship between chronologically matched toenail-Hg and hair-Hg samples reflecting the same period of exposure provided for a slope (Hg ng/mg) of 2.79 for hair relative to tonail (r=0.954) (Figure 4). For purposes of comparison with previously conducted studies, the regression line of hair-Hg on toenail-Hg using results from samples obtained at the same point in time (i.e. during a clinic visit) and thus not reflecting the same window of exposure (chronologically unmatched) was determined and yielded a slope of 2.39 (r = 0.871)(data not shown). A comparison of results on group mean biomarker-Hg levels identified from the literature are provided in Table 1. The table gives ratios of hair-Hg to toenail-Hg based on study mean values with the addition of slope values from regression models of hair-Hg on toenail-Hg provided from three of the studies [13,15, this study]. The ratio of hair- to toenail-Hg based on arithmetic mean values for this study was 3.08 and 2.77 based on chronologically matched and unmatched samples respectively. The unmatched sample ratio of 2.77 was similar to the ratio (2.56) obtained by Ohno and co-workers [15] within a similar population (non-occupationally exposed group of Japanese women) and also similar to ratios obtained from a control group (2.4) in a cohort study of dentists as well as from one of two cohorts (2.5) studied in Finland [13,26]. The slope from the regression model using chronologically unmatched sample results (2.39) was similar to the slope (2.44) obtained by Ohno and co-workers [15] and by Alfthan [13] in one of two cohorts (2.24). For comparative purposes, a ratio of 3.16 was derived and is presented in Table 1 using geometric mean values for hair-Hg and toenail-Hg from matched samples; 1.41 ng/mg and 0.45 ng/mg, respectively.\nRelationship between toenail- and hair- Hg levels (ng/mg) among participants (n= 41).\nHair-Hg to toenail-Hg ratios determined from regression models and mean values obtained from literature\na. Study data provided for blood-Hg and toenail-Hg values along with regression model. Hair-Hg to blood-Hg ratio of 250:1 was used to obtain hair-Hg: toenail-Hg and slope of regression model of blood-Hg on toenail-Hg [4].\nb. One individual had previous occupational exposure as a dental nurse.\nc. Ohno and co-workers [15] and Alfthan [13] provided regression models of hair-Hg on toenail-Hg with resulting slope factors. Slope in this study from linear regression model based on chronologically matched and unmatched samples are provided.\nd. For this study, geometric mean values for hair-Hg and toenail-Hg from matched samples were 1.410 ng/mg and 0.446 ng/mg, respectively.","Toenail clippings from females were examined over a period of approximately one year to investigate variability in temporal intra-individual toenail-Hg levels, and chronologically matched hair and toenail samples were compared to more accurately define the toxicokinetic variability of MeHg levels observed between the two compartments.\nHair-Hg levels from this population of females have previously been investigated with results showing no significant change in MeHg body burden levels across the study period suggesting that if MeHg population-based body burden levels were changing they were doing so slowly and across a time period larger than the one investigated [35]. This study corroborates that conclusion and further, indicates that while there was temporal stability of the three toenail-Hg levels for the group as a whole, individuals had variable MeHg body-burden levels but the levels did not fluctuate by more than two-fold across the one-year period investigated. The fluctuation in individual body burden levels is likely due to changes in MeHg intake across sample intervals while intra-individual toxicokinetic variability may also be a contributing factor. The intra-individual change in Hg levels observed across the study period which did not exceed 1.5-fold for half of the participants and two-fold for nearly the entire study group can be considered normal behavioral variability that is non-directional fluctuation within longer term temporal stability. For individuals with stable body burden levels, a single data point value, such as toenail-Hg levels obtained at a given time, could be used to reflect long term MeHg body burden; with the individual’s mean value being within the interval defined by one-third to twice the obtained toenail-Hg level. It must be noted however, that for identifying body burden levels across a short time period, even within those having temporal stability, biomarker based measurements from a single time point used to estimate exposure can miss short term elevated MeHg exposure periods consisting of single exposures to those lasting weeks [36-38].\nFor toenails to be used as a biomarker for long term exposures such as years, there is a need to either verify temporal stability within the study population by comparing biomarkers reflecting body-burden levels against an empirically derived ratio as defined herein, or repeated biomarker determinations would be needed so as to understand long term body burden characteristics. If a population has stable MeHg body burden levels, or if long term averages can be identified, biomarkers such as toenail could be used to identify causal association even though the biomarker may be chronologically removed from disease manifestation. These determinants are relevant to the relationship between ongoing exposures and endpoints resulting from chronic exposure (i.e. cardiovascular disease) but would not be as applicable to circumstances associated with the population consisting of women of child bearing age. Long term variability will not be as relevant for gestational exposure as present day understanding suggests a finite window of susceptibility for women during pregnancy may exist that can result in deleterious effects; possibly even from a single exposure resulting in a large bolus dose at a critical time period during fetal development.\nA limitation of this work is that the intra-individual temporal variability observed in toenail-Hg levels, even though only two fold, could be a factor leading to imprecision in exposure measurement. Estimates of imprecision for biomarker parameters such as hair and blood that can impact dose estimations in dose–response models have already been provided and the toenail compartment as a reservoir reflecting MeHg body burden may include similar imprecision [39]. When investigating dose–response relationships, unbiased imprecision can lead to an underestimation of the relationship between dose and response. In the present work, where two toxicokinetic compartments are being compared, the imprecision will not lead to bias but will provide for a random error effect.\nChronologically matching hair with toenails provided for high correlation and yielded a slope of 2.79 based on a regression model (Table 1). Data that were obtained from samples collected at the same point in time (i.e. during a clinic visit) and thus not chronologically matched with respect to exposure period were also shown to correlate and were similar to results found in other studies comparing data from biomarkers collected at the same time but not representing the same period of exposure (Table 1). Nonetheless, the chronologically matched ratio should be used to estimate Hg levels in the hair compartment based on toenail levels. The chronologically matched ratio would allow for estimating the corresponding Hg levels in hair that have been widely used as a biomarker for investigating multiple toxicological effects, including cardiovascular outcomes and, would allow for comparison across studies as the hair-Hg data set within the literature is robust.\nFor any given population, if the hair- and toenail-Hg ratios from chronologically matched and unmatched samples are in reasonable agreement, we may conclude that their body burdens are relatively stable over time. From the existing population data sets (Table 1), this reasoning would suggest that the non-occupationally exposed Japanese women [15], one group of non-occupationally exposed Finns [13] and possibly the control group from within the dentists study [26] may have stable body burden levels. However, even if the body burden in a population is found to be, on average, stable over a given time period, it cannot necessarily be assumed that such stability can be extrapolated to much longer time periods. Thus, attempts at identifying stability in body burden within a study population should be made if biomarkers chronologically removed from disease manifestation are to be used to investigate a dose response relationship.","The data presented in this study will allow for an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure. A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that has a MeHg body-burden level that is relatively stable over time. Intra-individual variation was less than two-fold, which may represent normal body burden fluctuation for individuals consuming elevated amounts of various fish species with different contaminant levels. The use of biomarker-based estimates reflecting body-burden levels that are chronologically distant from manifestation of deleterious outcome, or that are used as the sole indicator reflecting chronic exposure, need to be considered carefully when testing a hypothesis as individual body-burden levels may not remain constant across lengthy time periods such as a decade.","AMIBS: Arsenic Mercury Intake Biometric Study; EPA: Environmental Protection Agency; GM: Geometric Mean; Hg: Mercury; MeHg: MethylMercury; RfD: Reference Dose; US: United States; WA: Washington State.","All authors declare they have no actual or potential competing financial or non-financial interests in this work.","TH conducted the hair-Hg and toenail-Hg analyses and provided the Supplemental Materials. AT was a significant contributor to almost all aspects of this project involving the Korean and Japanese populations. AMIBS would not have been as successful without her. AS aided in writing the manuscript. TMB contributed significantly to study design and data interpretation. EM aided in the writing of the manuscript and contributed to the acquisition of the data. All authors contributed substantially to the discussion of the data and their analyses, and provided editorial comments to the draft manuscript.","The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views or policies of the U.S. EPA, NIH/National Institute of Environmental Health, National Science Foundation, New Jersey Department of Environmental Protection, or Washington State Department of Health.","Supplemental Material. Provides additional text to description found in Methods section on hair-Hg representativeness for each hair sample.\nClick here for file"],"string":"[\n \"As most human exposures to methylmercury (MeHg) result from fish consumption, federal and state consumption advisories have been released as a public health protective measure [1,2]. Most state and federal advice to fish consumers reflects consideration of the US EPA reference dose (RfD) for MeHg [3]. The RfD, as well as the general recognition of the hazardous nature of MeHg exposure is in turn based upon extensive neurological research that has been repeatedly summarized e.g., [4-6]. In addition to concerns over neurophysiological deficits that can occur in children exposed in utero to MeHg, concern has been raised about the possible role of MeHg in cardiovascular disease in adults. There have been several major epidemiological studies conducted that investigated the relationship between MeHg exposure and the incidence of myocardial infarction [7-11]. The findings from four of these five studies along with additional data relating MeHg exposure with, for example, heart rate variability and oxidative stress were critical to a panel assembled under contract to the US EPA that recommended the development of a dose–response function for myocardial infarction and MeHg exposure in a regulatory benefits analyses addressing Hg emissions [12]. Roman et al. [12] did not address findings from the subsequently published fifth study which did not observe an association between MeHg exposure and coronary heart disease or stroke [11].\\nHair-Hg, blood-Hg and toenail-Hg levels have been used as biomarkers of exposure for MeHg with each biomarker having been significantly correlated with fish consumption which is the primary source of MeHg exposure [4,6,13-15]. Analyses of hair-Hg and blood-Hg levels in fish consumers and non-fish consumers have been conducted allowing for these compartments to be used to characterize exposure to MeHg within a population [4]. Hair- and blood- Hg concentration have been the biomarkers of MeHg exposure used most frequently in studies investigating the relationship between MeHg exposure and neurodevelopmental outcomes as well as in several of the major studies investigating cardiovascular effects [7,10,16-18]. However, several important epidemiological studies investigating the relationship between exposure and cardiovascular outcomes have relied on toenail-Hg levels as the biomarker for MeHg exposure [8,9,11]. Data on the use of toenail-Hg levels as a biomarker of exposure is less robust compared to data from hair and blood compartments. The foundation of the toenail compartment as a biomarker is primarily based on observed correlations with hair-Hg levels and fish consumption with several studies having assessed MeHg exposures using toenails and a few having compared measurements across multiple markers [13-15,19-26]. Several studies within this group have provided for data allowing hair-Hg to toenail-Hg ratios to be established based on mean values and have developed regression models examining the relationship between hair-Hg and toenail-Hg levels [13,15,21,22,26]. However, despite these available data comparing Hg concentrations between two compartments, data are not available comparing hair and toenail biomarker results from exposure that occurred over the same time period.\\nAs part of the Arsenic Mercury Intake Biometric Study (AMIBS), we compared Hg levels from hair segments and toenail clippings reflecting the same time period of exposure and examined longitudinal data for toenail-Hg levels obtained at three time points within the population of Japanese women living in the Puget Sound area of Washington State (US). Previous work has examined fish intake and MeHg body burden in this population and results indicated that this Japanese population consumes substantially more fish than the national average and that MeHg exposure within this group is among the highest in the US [27,28]. The goals of this work were two-fold: to more accurately define the toxicokinetic variability of Hg levels observed between the hair and toenail compartment, and to examine variability in temporal toenail-Hg levels that were obtained over a period of approximately one year. This information will permit an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure and may be useful in future studies examining the relationship between neurological effects and MeHg exposure. It will also facilitate the comparison between the results of studies employing toenail- and hair-Hg levels as biomarkers of exposure.\",\n \" AMIBS As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.\\nAs a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.\\n Hair and toenail sampling To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.\\nTo assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.\\n Hair- and toenail-Hg analyses Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).\\nSpecimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).\\n Statistical analyses A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).\\nA repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).\",\n \"As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.\",\n \"To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.\",\n \"Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).\",\n \"A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).\",\n \"Toenail and hair samples were collected from 43 Japanese participants across three clinic visits. Mean toenail growth rate was 0.048 ±0.012 mm/day (22.4 ±6.5 days/mm) and mean hair growth rate was 0.42 ±0.07 mm/day (24.5 ±5.3 days/cm). No significant difference in toenail-Hg or in hair-Hg levels was observed between pregnant (n = 11) and non-pregnant participants (n = 32) (p > 0.05). The average number of days between visits was 135 (± 18) days with the shortest interval being 100 days and the longest, 189 days. The interquartile range was 126 to 141 days. The mean toenail growth length across the average number of days between visits was 6.48 mm (135 days x 0.048 mm/day). The mean time period spanned by the three visits was 270 days (range, 226 to 350). Interquartile Hg-concentration ranges for the three visits were 0.33-0.67, 0.34-0.66 and 0.34-0.67 ng/mg, respectively. Respective 95th percentile confidence intervals were ± 0.12, 0.13 and 0.10. Arithmetic mean toenail-Hg values with standard deviations and coefficients of variation in parentheses for the 1st, 2nd and 3rd visits were 0.60 ±0.42 (0.70), 0.60 ±0.43 (0.72) and 0.56 ±0.35 (0.63) ng/mg with box plots depicting visit distributional data provided in Figure 1. No significant difference in toenail-Hg levels was observed among the three sampling visits, but the relatively small number of samples limited the power to detect a small difference. Pearson’s product–moment correlation coefficients of Hg concentration between sampling intervals were as follows: r = 0.92 between 1st and 2nd clinic visits, r = 0.87 between 2nd and 3rd visits and r = 0.75 between 1st and 3rd visits.\\nToenail-Hg levels for each visit (n = 43). Medians are middle lines within box. Top and bottom of box represents upper and lower quartile values, respectively. Upper and lower whiskers represent sample maximum and minimum values, respectively. Distribution mean values do not differ significantly (p <0.05).\\nThe absolute change in toenail-Hg levels on an individual basis between successive samples across the three visits is depicted in Figure 2. With the exception of three toenail-Hg levels (out of 129), all values for separate clinic visits were within 50% to 150% of the individual’s mean toenail-Hg level. The extent of intra-individual change among participants depicted as the ratio of highest to lowest toenail-Hg levels from the visits for each individual is provided as a histogram in Figure 3. Nearly all participants (39 of 43) had less than a two-fold change in toenail-Hg levels across the one-year time period encompassed by the three samples with half (22 of 43) not exceeding a 1.5-fold change. Range of change was 1.08 - 3.44.\\nIntra-individual toenail-Hg variability across three successive clinic visits.\\nRatio of intra-individual toenail-Hg levels (n = 43).\\nChronologically matched sample evaluation was conducted by comparing toenail-Hg levels with hair-Hg values representing the same time period of exposure (n = 41). Chronological matching of samples was not possible for all individuals due to factors such as timing of sample collections and length of hair samples. A regression model of the relationship between chronologically matched toenail-Hg and hair-Hg samples reflecting the same period of exposure provided for a slope (Hg ng/mg) of 2.79 for hair relative to tonail (r=0.954) (Figure 4). For purposes of comparison with previously conducted studies, the regression line of hair-Hg on toenail-Hg using results from samples obtained at the same point in time (i.e. during a clinic visit) and thus not reflecting the same window of exposure (chronologically unmatched) was determined and yielded a slope of 2.39 (r = 0.871)(data not shown). A comparison of results on group mean biomarker-Hg levels identified from the literature are provided in Table 1. The table gives ratios of hair-Hg to toenail-Hg based on study mean values with the addition of slope values from regression models of hair-Hg on toenail-Hg provided from three of the studies [13,15, this study]. The ratio of hair- to toenail-Hg based on arithmetic mean values for this study was 3.08 and 2.77 based on chronologically matched and unmatched samples respectively. The unmatched sample ratio of 2.77 was similar to the ratio (2.56) obtained by Ohno and co-workers [15] within a similar population (non-occupationally exposed group of Japanese women) and also similar to ratios obtained from a control group (2.4) in a cohort study of dentists as well as from one of two cohorts (2.5) studied in Finland [13,26]. The slope from the regression model using chronologically unmatched sample results (2.39) was similar to the slope (2.44) obtained by Ohno and co-workers [15] and by Alfthan [13] in one of two cohorts (2.24). For comparative purposes, a ratio of 3.16 was derived and is presented in Table 1 using geometric mean values for hair-Hg and toenail-Hg from matched samples; 1.41 ng/mg and 0.45 ng/mg, respectively.\\nRelationship between toenail- and hair- Hg levels (ng/mg) among participants (n= 41).\\nHair-Hg to toenail-Hg ratios determined from regression models and mean values obtained from literature\\na. Study data provided for blood-Hg and toenail-Hg values along with regression model. Hair-Hg to blood-Hg ratio of 250:1 was used to obtain hair-Hg: toenail-Hg and slope of regression model of blood-Hg on toenail-Hg [4].\\nb. One individual had previous occupational exposure as a dental nurse.\\nc. Ohno and co-workers [15] and Alfthan [13] provided regression models of hair-Hg on toenail-Hg with resulting slope factors. Slope in this study from linear regression model based on chronologically matched and unmatched samples are provided.\\nd. For this study, geometric mean values for hair-Hg and toenail-Hg from matched samples were 1.410 ng/mg and 0.446 ng/mg, respectively.\",\n \"Toenail clippings from females were examined over a period of approximately one year to investigate variability in temporal intra-individual toenail-Hg levels, and chronologically matched hair and toenail samples were compared to more accurately define the toxicokinetic variability of MeHg levels observed between the two compartments.\\nHair-Hg levels from this population of females have previously been investigated with results showing no significant change in MeHg body burden levels across the study period suggesting that if MeHg population-based body burden levels were changing they were doing so slowly and across a time period larger than the one investigated [35]. This study corroborates that conclusion and further, indicates that while there was temporal stability of the three toenail-Hg levels for the group as a whole, individuals had variable MeHg body-burden levels but the levels did not fluctuate by more than two-fold across the one-year period investigated. The fluctuation in individual body burden levels is likely due to changes in MeHg intake across sample intervals while intra-individual toxicokinetic variability may also be a contributing factor. The intra-individual change in Hg levels observed across the study period which did not exceed 1.5-fold for half of the participants and two-fold for nearly the entire study group can be considered normal behavioral variability that is non-directional fluctuation within longer term temporal stability. For individuals with stable body burden levels, a single data point value, such as toenail-Hg levels obtained at a given time, could be used to reflect long term MeHg body burden; with the individual’s mean value being within the interval defined by one-third to twice the obtained toenail-Hg level. It must be noted however, that for identifying body burden levels across a short time period, even within those having temporal stability, biomarker based measurements from a single time point used to estimate exposure can miss short term elevated MeHg exposure periods consisting of single exposures to those lasting weeks [36-38].\\nFor toenails to be used as a biomarker for long term exposures such as years, there is a need to either verify temporal stability within the study population by comparing biomarkers reflecting body-burden levels against an empirically derived ratio as defined herein, or repeated biomarker determinations would be needed so as to understand long term body burden characteristics. If a population has stable MeHg body burden levels, or if long term averages can be identified, biomarkers such as toenail could be used to identify causal association even though the biomarker may be chronologically removed from disease manifestation. These determinants are relevant to the relationship between ongoing exposures and endpoints resulting from chronic exposure (i.e. cardiovascular disease) but would not be as applicable to circumstances associated with the population consisting of women of child bearing age. Long term variability will not be as relevant for gestational exposure as present day understanding suggests a finite window of susceptibility for women during pregnancy may exist that can result in deleterious effects; possibly even from a single exposure resulting in a large bolus dose at a critical time period during fetal development.\\nA limitation of this work is that the intra-individual temporal variability observed in toenail-Hg levels, even though only two fold, could be a factor leading to imprecision in exposure measurement. Estimates of imprecision for biomarker parameters such as hair and blood that can impact dose estimations in dose–response models have already been provided and the toenail compartment as a reservoir reflecting MeHg body burden may include similar imprecision [39]. When investigating dose–response relationships, unbiased imprecision can lead to an underestimation of the relationship between dose and response. In the present work, where two toxicokinetic compartments are being compared, the imprecision will not lead to bias but will provide for a random error effect.\\nChronologically matching hair with toenails provided for high correlation and yielded a slope of 2.79 based on a regression model (Table 1). Data that were obtained from samples collected at the same point in time (i.e. during a clinic visit) and thus not chronologically matched with respect to exposure period were also shown to correlate and were similar to results found in other studies comparing data from biomarkers collected at the same time but not representing the same period of exposure (Table 1). Nonetheless, the chronologically matched ratio should be used to estimate Hg levels in the hair compartment based on toenail levels. The chronologically matched ratio would allow for estimating the corresponding Hg levels in hair that have been widely used as a biomarker for investigating multiple toxicological effects, including cardiovascular outcomes and, would allow for comparison across studies as the hair-Hg data set within the literature is robust.\\nFor any given population, if the hair- and toenail-Hg ratios from chronologically matched and unmatched samples are in reasonable agreement, we may conclude that their body burdens are relatively stable over time. From the existing population data sets (Table 1), this reasoning would suggest that the non-occupationally exposed Japanese women [15], one group of non-occupationally exposed Finns [13] and possibly the control group from within the dentists study [26] may have stable body burden levels. However, even if the body burden in a population is found to be, on average, stable over a given time period, it cannot necessarily be assumed that such stability can be extrapolated to much longer time periods. Thus, attempts at identifying stability in body burden within a study population should be made if biomarkers chronologically removed from disease manifestation are to be used to investigate a dose response relationship.\",\n \"The data presented in this study will allow for an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure. A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that has a MeHg body-burden level that is relatively stable over time. Intra-individual variation was less than two-fold, which may represent normal body burden fluctuation for individuals consuming elevated amounts of various fish species with different contaminant levels. The use of biomarker-based estimates reflecting body-burden levels that are chronologically distant from manifestation of deleterious outcome, or that are used as the sole indicator reflecting chronic exposure, need to be considered carefully when testing a hypothesis as individual body-burden levels may not remain constant across lengthy time periods such as a decade.\",\n \"AMIBS: Arsenic Mercury Intake Biometric Study; EPA: Environmental Protection Agency; GM: Geometric Mean; Hg: Mercury; MeHg: MethylMercury; RfD: Reference Dose; US: United States; WA: Washington State.\",\n \"All authors declare they have no actual or potential competing financial or non-financial interests in this work.\",\n \"TH conducted the hair-Hg and toenail-Hg analyses and provided the Supplemental Materials. AT was a significant contributor to almost all aspects of this project involving the Korean and Japanese populations. AMIBS would not have been as successful without her. AS aided in writing the manuscript. TMB contributed significantly to study design and data interpretation. EM aided in the writing of the manuscript and contributed to the acquisition of the data. All authors contributed substantially to the discussion of the data and their analyses, and provided editorial comments to the draft manuscript.\",\n \"The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views or policies of the U.S. EPA, NIH/National Institute of Environmental Health, National Science Foundation, New Jersey Department of Environmental Protection, or Washington State Department of Health.\",\n \"Supplemental Material. Provides additional text to description found in Methods section on hair-Hg representativeness for each hair sample.\\nClick here for file\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,"results","discussion","conclusions",null,null,null,null,"supplementary-material"],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null,\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["Mercury","Methylmercury","Cardiovascular","Hair","Toenail","Chronological","Biomarker"],"string":"[\n \"Mercury\",\n \"Methylmercury\",\n \"Cardiovascular\",\n \"Hair\",\n \"Toenail\",\n \"Chronological\",\n \"Biomarker\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nAs most human exposures to methylmercury (MeHg) result from fish consumption, federal and state consumption advisories have been released as a public health protective measure [1,2]. Most state and federal advice to fish consumers reflects consideration of the US EPA reference dose (RfD) for MeHg [3]. The RfD, as well as the general recognition of the hazardous nature of MeHg exposure is in turn based upon extensive neurological research that has been repeatedly summarized e.g., [4-6]. In addition to concerns over neurophysiological deficits that can occur in children exposed in utero to MeHg, concern has been raised about the possible role of MeHg in cardiovascular disease in adults. There have been several major epidemiological studies conducted that investigated the relationship between MeHg exposure and the incidence of myocardial infarction [7-11]. The findings from four of these five studies along with additional data relating MeHg exposure with, for example, heart rate variability and oxidative stress were critical to a panel assembled under contract to the US EPA that recommended the development of a dose–response function for myocardial infarction and MeHg exposure in a regulatory benefits analyses addressing Hg emissions [12]. Roman et al. [12] did not address findings from the subsequently published fifth study which did not observe an association between MeHg exposure and coronary heart disease or stroke [11].\nHair-Hg, blood-Hg and toenail-Hg levels have been used as biomarkers of exposure for MeHg with each biomarker having been significantly correlated with fish consumption which is the primary source of MeHg exposure [4,6,13-15]. Analyses of hair-Hg and blood-Hg levels in fish consumers and non-fish consumers have been conducted allowing for these compartments to be used to characterize exposure to MeHg within a population [4]. Hair- and blood- Hg concentration have been the biomarkers of MeHg exposure used most frequently in studies investigating the relationship between MeHg exposure and neurodevelopmental outcomes as well as in several of the major studies investigating cardiovascular effects [7,10,16-18]. However, several important epidemiological studies investigating the relationship between exposure and cardiovascular outcomes have relied on toenail-Hg levels as the biomarker for MeHg exposure [8,9,11]. Data on the use of toenail-Hg levels as a biomarker of exposure is less robust compared to data from hair and blood compartments. The foundation of the toenail compartment as a biomarker is primarily based on observed correlations with hair-Hg levels and fish consumption with several studies having assessed MeHg exposures using toenails and a few having compared measurements across multiple markers [13-15,19-26]. Several studies within this group have provided for data allowing hair-Hg to toenail-Hg ratios to be established based on mean values and have developed regression models examining the relationship between hair-Hg and toenail-Hg levels [13,15,21,22,26]. However, despite these available data comparing Hg concentrations between two compartments, data are not available comparing hair and toenail biomarker results from exposure that occurred over the same time period.\nAs part of the Arsenic Mercury Intake Biometric Study (AMIBS), we compared Hg levels from hair segments and toenail clippings reflecting the same time period of exposure and examined longitudinal data for toenail-Hg levels obtained at three time points within the population of Japanese women living in the Puget Sound area of Washington State (US). Previous work has examined fish intake and MeHg body burden in this population and results indicated that this Japanese population consumes substantially more fish than the national average and that MeHg exposure within this group is among the highest in the US [27,28]. The goals of this work were two-fold: to more accurately define the toxicokinetic variability of Hg levels observed between the hair and toenail compartment, and to examine variability in temporal toenail-Hg levels that were obtained over a period of approximately one year. This information will permit an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure and may be useful in future studies examining the relationship between neurological effects and MeHg exposure. It will also facilitate the comparison between the results of studies employing toenail- and hair-Hg levels as biomarkers of exposure.\n\nMethods:\n AMIBS As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.\nAs a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.\n Hair and toenail sampling To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.\nTo assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.\n Hair- and toenail-Hg analyses Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).\nSpecimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).\n Statistical analyses A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).\nA repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).\n\nAMIBS:\nAs a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.\n\nHair and toenail sampling:\nTo assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.\n\nHair- and toenail-Hg analyses:\nSpecimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).\n\nStatistical analyses:\nA repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).\n\nResults:\nToenail and hair samples were collected from 43 Japanese participants across three clinic visits. Mean toenail growth rate was 0.048 ±0.012 mm/day (22.4 ±6.5 days/mm) and mean hair growth rate was 0.42 ±0.07 mm/day (24.5 ±5.3 days/cm). No significant difference in toenail-Hg or in hair-Hg levels was observed between pregnant (n = 11) and non-pregnant participants (n = 32) (p > 0.05). The average number of days between visits was 135 (± 18) days with the shortest interval being 100 days and the longest, 189 days. The interquartile range was 126 to 141 days. The mean toenail growth length across the average number of days between visits was 6.48 mm (135 days x 0.048 mm/day). The mean time period spanned by the three visits was 270 days (range, 226 to 350). Interquartile Hg-concentration ranges for the three visits were 0.33-0.67, 0.34-0.66 and 0.34-0.67 ng/mg, respectively. Respective 95th percentile confidence intervals were ± 0.12, 0.13 and 0.10. Arithmetic mean toenail-Hg values with standard deviations and coefficients of variation in parentheses for the 1st, 2nd and 3rd visits were 0.60 ±0.42 (0.70), 0.60 ±0.43 (0.72) and 0.56 ±0.35 (0.63) ng/mg with box plots depicting visit distributional data provided in Figure 1. No significant difference in toenail-Hg levels was observed among the three sampling visits, but the relatively small number of samples limited the power to detect a small difference. Pearson’s product–moment correlation coefficients of Hg concentration between sampling intervals were as follows: r = 0.92 between 1st and 2nd clinic visits, r = 0.87 between 2nd and 3rd visits and r = 0.75 between 1st and 3rd visits.\nToenail-Hg levels for each visit (n = 43). Medians are middle lines within box. Top and bottom of box represents upper and lower quartile values, respectively. Upper and lower whiskers represent sample maximum and minimum values, respectively. Distribution mean values do not differ significantly (p <0.05).\nThe absolute change in toenail-Hg levels on an individual basis between successive samples across the three visits is depicted in Figure 2. With the exception of three toenail-Hg levels (out of 129), all values for separate clinic visits were within 50% to 150% of the individual’s mean toenail-Hg level. The extent of intra-individual change among participants depicted as the ratio of highest to lowest toenail-Hg levels from the visits for each individual is provided as a histogram in Figure 3. Nearly all participants (39 of 43) had less than a two-fold change in toenail-Hg levels across the one-year time period encompassed by the three samples with half (22 of 43) not exceeding a 1.5-fold change. Range of change was 1.08 - 3.44.\nIntra-individual toenail-Hg variability across three successive clinic visits.\nRatio of intra-individual toenail-Hg levels (n = 43).\nChronologically matched sample evaluation was conducted by comparing toenail-Hg levels with hair-Hg values representing the same time period of exposure (n = 41). Chronological matching of samples was not possible for all individuals due to factors such as timing of sample collections and length of hair samples. A regression model of the relationship between chronologically matched toenail-Hg and hair-Hg samples reflecting the same period of exposure provided for a slope (Hg ng/mg) of 2.79 for hair relative to tonail (r=0.954) (Figure 4). For purposes of comparison with previously conducted studies, the regression line of hair-Hg on toenail-Hg using results from samples obtained at the same point in time (i.e. during a clinic visit) and thus not reflecting the same window of exposure (chronologically unmatched) was determined and yielded a slope of 2.39 (r = 0.871)(data not shown). A comparison of results on group mean biomarker-Hg levels identified from the literature are provided in Table 1. The table gives ratios of hair-Hg to toenail-Hg based on study mean values with the addition of slope values from regression models of hair-Hg on toenail-Hg provided from three of the studies [13,15, this study]. The ratio of hair- to toenail-Hg based on arithmetic mean values for this study was 3.08 and 2.77 based on chronologically matched and unmatched samples respectively. The unmatched sample ratio of 2.77 was similar to the ratio (2.56) obtained by Ohno and co-workers [15] within a similar population (non-occupationally exposed group of Japanese women) and also similar to ratios obtained from a control group (2.4) in a cohort study of dentists as well as from one of two cohorts (2.5) studied in Finland [13,26]. The slope from the regression model using chronologically unmatched sample results (2.39) was similar to the slope (2.44) obtained by Ohno and co-workers [15] and by Alfthan [13] in one of two cohorts (2.24). For comparative purposes, a ratio of 3.16 was derived and is presented in Table 1 using geometric mean values for hair-Hg and toenail-Hg from matched samples; 1.41 ng/mg and 0.45 ng/mg, respectively.\nRelationship between toenail- and hair- Hg levels (ng/mg) among participants (n= 41).\nHair-Hg to toenail-Hg ratios determined from regression models and mean values obtained from literature\na. Study data provided for blood-Hg and toenail-Hg values along with regression model. Hair-Hg to blood-Hg ratio of 250:1 was used to obtain hair-Hg: toenail-Hg and slope of regression model of blood-Hg on toenail-Hg [4].\nb. One individual had previous occupational exposure as a dental nurse.\nc. Ohno and co-workers [15] and Alfthan [13] provided regression models of hair-Hg on toenail-Hg with resulting slope factors. Slope in this study from linear regression model based on chronologically matched and unmatched samples are provided.\nd. For this study, geometric mean values for hair-Hg and toenail-Hg from matched samples were 1.410 ng/mg and 0.446 ng/mg, respectively.\n\nDiscussion:\nToenail clippings from females were examined over a period of approximately one year to investigate variability in temporal intra-individual toenail-Hg levels, and chronologically matched hair and toenail samples were compared to more accurately define the toxicokinetic variability of MeHg levels observed between the two compartments.\nHair-Hg levels from this population of females have previously been investigated with results showing no significant change in MeHg body burden levels across the study period suggesting that if MeHg population-based body burden levels were changing they were doing so slowly and across a time period larger than the one investigated [35]. This study corroborates that conclusion and further, indicates that while there was temporal stability of the three toenail-Hg levels for the group as a whole, individuals had variable MeHg body-burden levels but the levels did not fluctuate by more than two-fold across the one-year period investigated. The fluctuation in individual body burden levels is likely due to changes in MeHg intake across sample intervals while intra-individual toxicokinetic variability may also be a contributing factor. The intra-individual change in Hg levels observed across the study period which did not exceed 1.5-fold for half of the participants and two-fold for nearly the entire study group can be considered normal behavioral variability that is non-directional fluctuation within longer term temporal stability. For individuals with stable body burden levels, a single data point value, such as toenail-Hg levels obtained at a given time, could be used to reflect long term MeHg body burden; with the individual’s mean value being within the interval defined by one-third to twice the obtained toenail-Hg level. It must be noted however, that for identifying body burden levels across a short time period, even within those having temporal stability, biomarker based measurements from a single time point used to estimate exposure can miss short term elevated MeHg exposure periods consisting of single exposures to those lasting weeks [36-38].\nFor toenails to be used as a biomarker for long term exposures such as years, there is a need to either verify temporal stability within the study population by comparing biomarkers reflecting body-burden levels against an empirically derived ratio as defined herein, or repeated biomarker determinations would be needed so as to understand long term body burden characteristics. If a population has stable MeHg body burden levels, or if long term averages can be identified, biomarkers such as toenail could be used to identify causal association even though the biomarker may be chronologically removed from disease manifestation. These determinants are relevant to the relationship between ongoing exposures and endpoints resulting from chronic exposure (i.e. cardiovascular disease) but would not be as applicable to circumstances associated with the population consisting of women of child bearing age. Long term variability will not be as relevant for gestational exposure as present day understanding suggests a finite window of susceptibility for women during pregnancy may exist that can result in deleterious effects; possibly even from a single exposure resulting in a large bolus dose at a critical time period during fetal development.\nA limitation of this work is that the intra-individual temporal variability observed in toenail-Hg levels, even though only two fold, could be a factor leading to imprecision in exposure measurement. Estimates of imprecision for biomarker parameters such as hair and blood that can impact dose estimations in dose–response models have already been provided and the toenail compartment as a reservoir reflecting MeHg body burden may include similar imprecision [39]. When investigating dose–response relationships, unbiased imprecision can lead to an underestimation of the relationship between dose and response. In the present work, where two toxicokinetic compartments are being compared, the imprecision will not lead to bias but will provide for a random error effect.\nChronologically matching hair with toenails provided for high correlation and yielded a slope of 2.79 based on a regression model (Table 1). Data that were obtained from samples collected at the same point in time (i.e. during a clinic visit) and thus not chronologically matched with respect to exposure period were also shown to correlate and were similar to results found in other studies comparing data from biomarkers collected at the same time but not representing the same period of exposure (Table 1). Nonetheless, the chronologically matched ratio should be used to estimate Hg levels in the hair compartment based on toenail levels. The chronologically matched ratio would allow for estimating the corresponding Hg levels in hair that have been widely used as a biomarker for investigating multiple toxicological effects, including cardiovascular outcomes and, would allow for comparison across studies as the hair-Hg data set within the literature is robust.\nFor any given population, if the hair- and toenail-Hg ratios from chronologically matched and unmatched samples are in reasonable agreement, we may conclude that their body burdens are relatively stable over time. From the existing population data sets (Table 1), this reasoning would suggest that the non-occupationally exposed Japanese women [15], one group of non-occupationally exposed Finns [13] and possibly the control group from within the dentists study [26] may have stable body burden levels. However, even if the body burden in a population is found to be, on average, stable over a given time period, it cannot necessarily be assumed that such stability can be extrapolated to much longer time periods. Thus, attempts at identifying stability in body burden within a study population should be made if biomarkers chronologically removed from disease manifestation are to be used to investigate a dose response relationship.\n\nConclusions:\nThe data presented in this study will allow for an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure. A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that has a MeHg body-burden level that is relatively stable over time. Intra-individual variation was less than two-fold, which may represent normal body burden fluctuation for individuals consuming elevated amounts of various fish species with different contaminant levels. The use of biomarker-based estimates reflecting body-burden levels that are chronologically distant from manifestation of deleterious outcome, or that are used as the sole indicator reflecting chronic exposure, need to be considered carefully when testing a hypothesis as individual body-burden levels may not remain constant across lengthy time periods such as a decade.\n\nAbbreviations:\nAMIBS: Arsenic Mercury Intake Biometric Study; EPA: Environmental Protection Agency; GM: Geometric Mean; Hg: Mercury; MeHg: MethylMercury; RfD: Reference Dose; US: United States; WA: Washington State.\n\nCompeting interests:\nAll authors declare they have no actual or potential competing financial or non-financial interests in this work.\n\nAuthors’ contributions:\nTH conducted the hair-Hg and toenail-Hg analyses and provided the Supplemental Materials. AT was a significant contributor to almost all aspects of this project involving the Korean and Japanese populations. AMIBS would not have been as successful without her. AS aided in writing the manuscript. TMB contributed significantly to study design and data interpretation. EM aided in the writing of the manuscript and contributed to the acquisition of the data. All authors contributed substantially to the discussion of the data and their analyses, and provided editorial comments to the draft manuscript.\n\nDisclaimer:\nThe contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views or policies of the U.S. EPA, NIH/National Institute of Environmental Health, National Science Foundation, New Jersey Department of Environmental Protection, or Washington State Department of Health.\n\nSupplementary Material:\nSupplemental Material. Provides additional text to description found in Methods section on hair-Hg representativeness for each hair sample.\nClick here for file\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nToenail-Hg levels are being used as a marker of methylmercury (MeHg) exposure in efforts to associate exposure with effects such as cardiovascular disease. There is a need to correlate this marker with more established biomarkers that presently underlie existing dose-response relationships in order to compare these relationships across studies.\n\nMethods:\nAs part of the Arsenic Mercury Intake Biometric Study, toenail clippings were collected at three time points over a period of one year amongst females from within the population of Japanese living near Puget Sound in Washington State (US). Variability in temporal intra-individual toenail-Hg levels was examined and chronologically matched hair and toenail samples were compared to more accurately define the toxicokinetic variability of Hg levels observed between the two compartments.\n\nResults:\nMean toenail-Hg values (n=43) for the 1st, 2nd and 3rd visits were 0.60, 0.60 and 0.56 ng/mg. Correlations were as follows: r=0.92 between 1st and 2nd clinic visits, r=0.75 between 1st and 3rd visits and r=0.87 between 2nd and 3rd visits. With few exceptions, toenail-Hg values from any visit were within 50-150% of the individual's mean toenail-Hg level. Nearly all participants had less than a two-fold change in toenail-Hg levels across the study period. A regression model of the relationship between toenail-Hg and hair-Hg (n = 41) levels representing the same time period of exposure, gave a slope (Hg ng/mg) of 2.79 for hair relative to toenail (r=0.954).\n\nConclusions:\nA chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that consumes fish regularly and in quantity. Intra-individual variation in toenail-Hg levels was less than two-fold and may represent dietary-based fluctuations in body burden for individuals consuming various fish species with different contaminant levels. The chronologically matched ratio will be useful for relating MeHg exposure and dose-response derived from toenail-Hg measurements to those derived from hair-Hg measurements in other studies, and may be useful in future investigations as an indicator of stable MeHg body burden within a population."},"background_conclusion_text":{"kind":"string","value":"Background:\nAs most human exposures to methylmercury (MeHg) result from fish consumption, federal and state consumption advisories have been released as a public health protective measure [1,2]. Most state and federal advice to fish consumers reflects consideration of the US EPA reference dose (RfD) for MeHg [3]. The RfD, as well as the general recognition of the hazardous nature of MeHg exposure is in turn based upon extensive neurological research that has been repeatedly summarized e.g., [4-6]. In addition to concerns over neurophysiological deficits that can occur in children exposed in utero to MeHg, concern has been raised about the possible role of MeHg in cardiovascular disease in adults. There have been several major epidemiological studies conducted that investigated the relationship between MeHg exposure and the incidence of myocardial infarction [7-11]. The findings from four of these five studies along with additional data relating MeHg exposure with, for example, heart rate variability and oxidative stress were critical to a panel assembled under contract to the US EPA that recommended the development of a dose–response function for myocardial infarction and MeHg exposure in a regulatory benefits analyses addressing Hg emissions [12]. Roman et al. [12] did not address findings from the subsequently published fifth study which did not observe an association between MeHg exposure and coronary heart disease or stroke [11].\nHair-Hg, blood-Hg and toenail-Hg levels have been used as biomarkers of exposure for MeHg with each biomarker having been significantly correlated with fish consumption which is the primary source of MeHg exposure [4,6,13-15]. Analyses of hair-Hg and blood-Hg levels in fish consumers and non-fish consumers have been conducted allowing for these compartments to be used to characterize exposure to MeHg within a population [4]. Hair- and blood- Hg concentration have been the biomarkers of MeHg exposure used most frequently in studies investigating the relationship between MeHg exposure and neurodevelopmental outcomes as well as in several of the major studies investigating cardiovascular effects [7,10,16-18]. However, several important epidemiological studies investigating the relationship between exposure and cardiovascular outcomes have relied on toenail-Hg levels as the biomarker for MeHg exposure [8,9,11]. Data on the use of toenail-Hg levels as a biomarker of exposure is less robust compared to data from hair and blood compartments. The foundation of the toenail compartment as a biomarker is primarily based on observed correlations with hair-Hg levels and fish consumption with several studies having assessed MeHg exposures using toenails and a few having compared measurements across multiple markers [13-15,19-26]. Several studies within this group have provided for data allowing hair-Hg to toenail-Hg ratios to be established based on mean values and have developed regression models examining the relationship between hair-Hg and toenail-Hg levels [13,15,21,22,26]. However, despite these available data comparing Hg concentrations between two compartments, data are not available comparing hair and toenail biomarker results from exposure that occurred over the same time period.\nAs part of the Arsenic Mercury Intake Biometric Study (AMIBS), we compared Hg levels from hair segments and toenail clippings reflecting the same time period of exposure and examined longitudinal data for toenail-Hg levels obtained at three time points within the population of Japanese women living in the Puget Sound area of Washington State (US). Previous work has examined fish intake and MeHg body burden in this population and results indicated that this Japanese population consumes substantially more fish than the national average and that MeHg exposure within this group is among the highest in the US [27,28]. The goals of this work were two-fold: to more accurately define the toxicokinetic variability of Hg levels observed between the hair and toenail compartment, and to examine variability in temporal toenail-Hg levels that were obtained over a period of approximately one year. This information will permit an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure and may be useful in future studies examining the relationship between neurological effects and MeHg exposure. It will also facilitate the comparison between the results of studies employing toenail- and hair-Hg levels as biomarkers of exposure.\n\nConclusions:\nThe data presented in this study will allow for an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure. A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that has a MeHg body-burden level that is relatively stable over time. Intra-individual variation was less than two-fold, which may represent normal body burden fluctuation for individuals consuming elevated amounts of various fish species with different contaminant levels. The use of biomarker-based estimates reflecting body-burden levels that are chronologically distant from manifestation of deleterious outcome, or that are used as the sole indicator reflecting chronic exposure, need to be considered carefully when testing a hypothesis as individual body-burden levels may not remain constant across lengthy time periods such as a decade."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nToenail-Hg levels are being used as a marker of methylmercury (MeHg) exposure in efforts to associate exposure with effects such as cardiovascular disease. There is a need to correlate this marker with more established biomarkers that presently underlie existing dose-response relationships in order to compare these relationships across studies.\n\nMethods:\nAs part of the Arsenic Mercury Intake Biometric Study, toenail clippings were collected at three time points over a period of one year amongst females from within the population of Japanese living near Puget Sound in Washington State (US). Variability in temporal intra-individual toenail-Hg levels was examined and chronologically matched hair and toenail samples were compared to more accurately define the toxicokinetic variability of Hg levels observed between the two compartments.\n\nResults:\nMean toenail-Hg values (n=43) for the 1st, 2nd and 3rd visits were 0.60, 0.60 and 0.56 ng/mg. Correlations were as follows: r=0.92 between 1st and 2nd clinic visits, r=0.75 between 1st and 3rd visits and r=0.87 between 2nd and 3rd visits. With few exceptions, toenail-Hg values from any visit were within 50-150% of the individual's mean toenail-Hg level. Nearly all participants had less than a two-fold change in toenail-Hg levels across the study period. A regression model of the relationship between toenail-Hg and hair-Hg (n = 41) levels representing the same time period of exposure, gave a slope (Hg ng/mg) of 2.79 for hair relative to toenail (r=0.954).\n\nConclusions:\nA chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that consumes fish regularly and in quantity. Intra-individual variation in toenail-Hg levels was less than two-fold and may represent dietary-based fluctuations in body burden for individuals consuming various fish species with different contaminant levels. The chronologically matched ratio will be useful for relating MeHg exposure and dose-response derived from toenail-Hg measurements to those derived from hair-Hg measurements in other studies, and may be useful in future investigations as an indicator of stable MeHg body burden within a population."},"whole_article_text_length":{"kind":"number","value":9675,"string":"9,675"},"whole_article_abstract_length":{"kind":"number","value":414,"string":"414"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["hair","toenail","hg","exposure","time","levels","growth","toenail hg","nail","sample"],"string":"[\n \"hair\",\n \"toenail\",\n \"hg\",\n \"exposure\",\n \"time\",\n \"levels\",\n \"growth\",\n \"toenail hg\",\n \"nail\",\n \"sample\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] mehg | exposure | mehg exposure | hg | hg levels | studies | levels | toenail | fish | hair [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hair | toenail | growth | nail | end | clipping | segment | distal | hair segment | time [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hg | toenail | toenail hg | values | visits | days | mean | hair | samples | hg levels [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] body | body burden | burden | exposure | mehg | body burden levels | burden levels | levels | mehg exposure | time [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] toenail | hair | hg | levels | exposure | hg levels | mehg | toenail hg | time | growth [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] toenail | hair | hg | levels | exposure | hg levels | mehg | toenail hg | time | growth [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Toenail-Hg | MeHg ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] the Arsenic Mercury Intake Biometric Study | three | one year | Japanese | Washington State | US ||| two [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] the 1st, 2nd | 3rd | 0.60 | 0.60 | 0.56 ng ||| between 1st and 2nd | between 1st and | 3rd | r=0.87 | between 2nd and 3rd ||| 50-150% ||| two-fold ||| 41 | 2.79 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Intra | less than two-fold ||| MeHg | MeHg [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Toenail-Hg | MeHg ||| ||| the Arsenic Mercury Intake Biometric Study | three | one year | Japanese | Washington State | US ||| two ||| the 1st, 2nd | 3rd | 0.60 | 0.60 | 0.56 ng ||| between 1st and 2nd | between 1st and | 3rd | r=0.87 | between 2nd and 3rd ||| 50-150% ||| two-fold ||| 41 | 2.79 ||| ||| Intra | less than two-fold ||| MeHg | MeHg [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Toenail-Hg | MeHg ||| ||| the Arsenic Mercury Intake Biometric Study | three | one year | Japanese | Washington State | US ||| two ||| the 1st, 2nd | 3rd | 0.60 | 0.60 | 0.56 ng ||| between 1st and 2nd | between 1st and | 3rd | r=0.87 | between 2nd and 3rd ||| 50-150% ||| two-fold ||| 41 | 2.79 ||| ||| Intra | less than two-fold ||| MeHg | MeHg [SUMMARY]"}}},{"rowIdx":257,"cells":{"title":{"kind":"string","value":"Comparison of double antigen sandwich and indirect enzyme-linked immunosorbent assay for the diagnosis of hepatitis C virus antibodies."},"pmid":{"kind":"string","value":"33245583"},"background_abstract":{"kind":"string","value":"The aim of this study is to compare double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA in the diagnosis of hepatitis C virus（HCV）infection."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A total of 176 samples from the Tumor Hospital Affiliated to Xin Jiang Medical University were utilized to comparison. All serum samples were tested using double-antigen sandwich ELISA and indirect ELISA. Cohen's kappa statistics were used to assess the agreement between the two assays, and multivariate analysis was used to evaluate risk factors for the discordance between the double-antigen ELISA and indirect ELISA."},"methods_abstract_label":{"kind":"string","value":"METHODS AND MATERIALS"},"results_abstract":{"kind":"string","value":"The positivities of indirect ELISA (Beijing Wantai), double-antigen sandwich ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 74.43%, 68.75%, and 73.30%, respectively. The agreement between the indirect ELISA (Beijing Wantai) and double-antigen sandwich ELISA (Beijing Wantai) was high (κ = 0.829;P < .001), and the agreement between the double-antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847;P < .001). Variables associated with discordant results between the double-antigen sandwich and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"In detection of HCV, high agreement was found between the double-antigen sandwich ELISA and indirect ELISA. Female, younger age, and suffer from malignant tumor were significant risk factors for the discordance. Based on double-antigen sandwich ELISA has distinct methodological advantages over indirect ELISA. It is recommended for the diagnosis of HCV infection."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Aged","Aged, 80 and over","Antigens, Viral","Enzyme-Linked Immunosorbent Assay","Female","Hepacivirus","Hepatitis C","Hepatitis C Antibodies","Humans","Immunologic Tests","Male","Middle Aged","Reproducibility of Results","Young Adult"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Antigens, Viral\",\n \"Enzyme-Linked Immunosorbent Assay\",\n \"Female\",\n \"Hepacivirus\",\n \"Hepatitis C\",\n \"Hepatitis C Antibodies\",\n \"Humans\",\n \"Immunologic Tests\",\n \"Male\",\n \"Middle Aged\",\n \"Reproducibility of Results\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"7676215"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"The virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.\n1\n, \n2\n The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.\n3\n, \n4\n Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.\n5\n For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.\n6\n WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.\n7\n People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.\n8\n Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.\n9\n, \n10\n\n\nDepending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.\n11\n In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":" Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO\n Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\n Discordance among the test results Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.\nVariables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin."},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"In detection of HCV infection, high agreement was found between the double‐antigen sandwich ELISA and indirect ELISA. Based on double‐antigen sandwich ELISA has distinct methodological advantages over indirect ELISA, considering double‐antigen sandwich ELISA has high clinical popularization value in the screening of HCV infection. Female, younger age, and malignancy were significant risk factors for the discordance. It is recommended that relevant laboratories should pay more attention to the existence of the above significant risk factors by using indirect ELISA and suggest to detect anti‐HCV by using double‐antigen sandwich ELISA if condition permits."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Subjects","Commercial assays","Statistical analysis","Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)","Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao)","Discordance among the test results"],"string":"[\n \"INTRODUCTION\",\n \"Subjects\",\n \"Commercial assays\",\n \"Statistical analysis\",\n \"Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\",\n \"Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao)\",\n \"Discordance among the test results\"\n]"},"other_sections_texts":{"kind":"list like","value":["The virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.\n1\n, \n2\n The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.\n3\n, \n4\n Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.\n5\n For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.\n6\n WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.\n7\n People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.\n8\n Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.\n9\n, \n10\n\n\nDepending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.\n11\n In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection.","A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.","Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.","Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.","Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO","Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).","Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin."],"string":"[\n \"The virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.\\n1\\n, \\n2\\n The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.\\n3\\n, \\n4\\n Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.\\n5\\n For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.\\n6\\n WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.\\n7\\n People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.\\n8\\n Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.\\n9\\n, \\n10\\n\\n\\nDepending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.\\n11\\n In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection.\",\n \"A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.\",\n \"Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.\",\n \"Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.\",\n \"Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\\nIndirect ELISA\\n(Wantai), number(%)\\nS/CO\\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\\nS/CO\",\n \"Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\\nHCV antibody detection by double‐antigen sandwich\\nIndirect ELISA\\n(Jinhao), number(%)\\nS/CO\\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\\nS/CO\\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\",\n \"Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIAL AND METHOD","Subjects","Commercial assays","Statistical analysis","RESULTS","Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)","Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao)","Discordance among the test results","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"MATERIAL AND METHOD\",\n \"Subjects\",\n \"Commercial assays\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\",\n \"Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao)\",\n \"Discordance among the test results\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["The virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.\n1\n, \n2\n The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.\n3\n, \n4\n Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.\n5\n For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.\n6\n WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.\n7\n People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.\n8\n Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.\n9\n, \n10\n\n\nDepending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.\n11\n In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection."," Subjects A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.\nA total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.\n Commercial assays Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.\nDouble‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.\n Statistical analysis Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.\nStatistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.","A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.","Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.","Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference."," Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO\n Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\n Discordance among the test results Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.\nVariables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.","Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO","Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).","Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.","There are no symptoms or only mild symptoms such as right upper abdominal discomfort and oil aversion in the incubation period after HCV infection, and it is likely to take 20‐40 years to developed into serious clinical complication such as liver fibrosis or even hepatocellular carcinoma.\n12\n, \n13\n The exact time of HCV infection is difficult to determine the symptoms are mild or absent, easily transform into chronic, and higher chronic rate was 55% to 85%. Additionally, the understanding degree of the disease at a lower level, autonomous screening rate is low and public lacks attention with this disease. It caused tremendous burden on both China and the world economy. Along with a deeper understanding of the HCV life cycle, direct antiviral drugs (DAAs) have become widely used since its introduction, which is highly effective, less adverse reactions and a short course of treatment available to fight HCV.\n14\n, \n15\n DAAs are working directly on the non‐structural protein (NS) 3/4A, 5A, and 5B to achieve virus clearance, so the deadlock of clinical treatment of viral hepatitis C was broken even promising to get complete cure; thus, WHO sets targets to eliminate hepatitis C as a public health threat by 2030.\n16\n, \n17\n However, related research suggests that DAAs are still at a high price, at the same time, reinfection cannot be avoided, and the cured patients still have the risk of hepatocellular carcinoma.\n18\n, \n19\n, \n20\n Vaccine is the most cost‐effective and effective measure to prevent and combat the spread of disease. However, there is not any effective vaccine to prevent HCV infection.\n21\n The successful development and clinical application of HCV vaccine are unpredictable.\n22\n In summary, the focal problems for hepatitis C are shifting from the therapeutic strategies to screening program launched in high‐risk groups.\nTo date, early screening and diagnosis of HCV infection are based on three different methods, which may be utilized alone or in combination. These are (a) detection of HCV core antigen (HCV‐Ag); (b) nucleic acid testing (NAT) to detect HCV‐RNA by PCR; and (c) detection of an IgG antibody by ELISA (anti‐HCV).\n23\n Related literature reports that there is a good correlation between the contents of HCV‐Ag and the quantity of HCV‐RNA as viral load exceeds 3000 IU/mL in the serum samples of HCV patients, so HCV core antigen can be used as early diagnostic indicators of HCV infection.\n24\n Meanwhile, detection of HCV‐Ag has the advantages of relatively low cost, handiness, and simplicity and not need to add special instruments. However, the structure of the HCV‐cAg is very special and the preparation of antibody is more difficult, and detection sensitivity of HCV‐cAg is lower than that of anti‐HCV by using ELISA. It can be used as a supplement to anti‐HCV detection but cannot replace the determination of antibody. The samples were tested HCV‐RNA positive are the reliable index of viral replication activity, and the HCV‐RNA methodology can be subdivided into qualitative and quantitative methods.\n25\n, \n26\n Qualitative analysis has played a key role in blood screening of blood banks, it as a means of indicating the presence or absence of viremia but not measure the viral load. In recent years, the real‐time PCR (RT‐PCR) is gradually replacing qualitative analysis as a common method used in detecting HCV‐RNA, as it features high sensitivity and specificity. However, it cannot be used as a routine method because of its complicated operation and higher experimental conditions needed. In particular, most hospitals, especially primary healthcare facilities, do not have the capacity to detect HCV‐RNA and provide accurate results.\n27\n\n\nAccording to guidelines for the prevention and treatment of hepatitis C (2019 version) revised by the Chinese Medical Association, along with WS 213—2018 Diagnosis for hepatitis C (National Health and Family Planning Commission of the People's Republic of China). Compared with (HCV‐Ag) and nucleic acid testing (NAT), the anti‐HCV immunoassays are the most frequently used laboratory tests for detection of HCV infection. And now it has the following advantages in the detection of HCV antibodies with the rapid development of genetic engineering technology: high sensitivity and specificity, better stability, automation, low cost, and convenience. Indirect ELISA had already gone through three reformation, the first generation anti‐HCV assay using recombinant c100‐3 epitope from the NS4 protein, which had limited sensitivity and specificity. Second generation assay was developed using epitopes from the core, NS3, and NS4 proteins, a multi‐antigen format that can reduce the time required and increase the sensitivity. More recently, an highly conserved NS5 antigen have adopted in third generation assays. If the sample contains anti‐HCV, incubate for a certain period of time, solid‐phase antigen will fully bind to the anti‐HCV in the serum, and then react with the added IgG‐HRP. Tetramethylbenzidine (TMB) color development or not to indicate present or absent of anti‐HCV in human serum or plasma.\n28\n However, indirect ELISA can always result in false positives due to interfering factors, including high gamma globulin levels, nephritic syndrome, pregnancy, sample hemolysis, the splashing, and pollution of the blood sample.\n29\n\n\nDouble‐antigen sandwich ELISA is a developing technology with great potential.\n30\n At the beginning of development, it faces two major obstacles, the HCV antigen labeled with HRP directly is easy to cause the space steric hindrance and ratio of enzyme‐labeled antigens is difficult to control. Subsequently, preparation of growth arm biotinylated HCV antigen as a sandwich antigen using genetic engineering, with horseradish peroxidase mildew affinity tag chain element as enzymes, so anti‐HCV could perform twice specific binding in the sample and amplification of the signal was achieved using biotin–streptavidin system. Related research shows that compared to traditional indirect ELISA, double‐antigen sandwich ELISA displays higher specificity and better sensitivity, and it can utilize to detect IgM.\n31\n Double‐antigen sandwich ELISA could make up for the methodological deficiencies inherent in indirect ELISA and has clinical utility in both screening and diagnosis of HCV infection.\nObjective of our study is to investigate the consistency of three commercially available anti‐HCV assays, and analysis of influencing factors to disagreement between the double‐antigen sandwich and indirect ELISA. So as to provide pertinent evidence for the selection of methods of detecting anti‐HCV in clinical laboratories. In our study, among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai), indirect ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 68.75%, 74.43%, and 73.30%, respectively. The agreement between the double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) was high (κ = 0.829; P < .001). The agreement between the double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847; P < .001). But there are still inconsistent results in the analysis of anti‐HCV by using double‐antigen sandwich and indirect ELISA. It was found that among the fifty‐five specimens that were tested negative using by double‐antigen sandwich ELISA (Beijing Wantai), eleven were positive using by indirect ELISA (Beijing Wantai) and ten were positive using by indirect ELISA (Beijing Jinhao), and the possible cause of this problem perhaps is that nonspecific immunoglobulin, rheumatoid factor, hydrophilic antibodies, and other interfering substances in the specimen, which can be nonspecifically combined with the enzyme‐labeled secondary antibody, lead to false‐positive results. Among one hundred and twenty‐one samples tested positive using by double‐antigen sandwich ELISA (Beijing Wantai), one was negative using by indirect ELISA (Beijing Wantai) and two were negative by using indirect ELISA (Beijing Jinhao). The probable reason is that the patients are in the window period of infection and the body has not yet produced IgG. Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR: 1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05). Analysis of possible reasons were as follows: (a) More women predisposed to autoimmune diseases and autoantibody positivity was higher than men; (b) positive antinuclear antibodies in malignancies than benign tumor; and (c) pregnant woman in people younger than 35 years old was high and the presence of some interfering substances may be affect the results significantly. These discrepancies emphasize the need for further research, and we will follow up in subsequent studies.","In detection of HCV infection, high agreement was found between the double‐antigen sandwich ELISA and indirect ELISA. Based on double‐antigen sandwich ELISA has distinct methodological advantages over indirect ELISA, considering double‐antigen sandwich ELISA has high clinical popularization value in the screening of HCV infection. Female, younger age, and malignancy were significant risk factors for the discordance. It is recommended that relevant laboratories should pay more attention to the existence of the above significant risk factors by using indirect ELISA and suggest to detect anti‐HCV by using double‐antigen sandwich ELISA if condition permits."],"string":"[\n \"The virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.\\n1\\n, \\n2\\n The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.\\n3\\n, \\n4\\n Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.\\n5\\n For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.\\n6\\n WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.\\n7\\n People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.\\n8\\n Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.\\n9\\n, \\n10\\n\\n\\nDepending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.\\n11\\n In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection.\",\n \" Subjects A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.\\nA total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.\\n Commercial assays Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.\\nDouble‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.\\n Statistical analysis Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.\\nStatistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.\",\n \"A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.\",\n \"Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.\",\n \"Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.\",\n \" Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\\nIndirect ELISA\\n(Wantai), number(%)\\nS/CO\\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\\nS/CO\\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\\nIndirect ELISA\\n(Wantai), number(%)\\nS/CO\\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\\nS/CO\\n Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\\nHCV antibody detection by double‐antigen sandwich\\nIndirect ELISA\\n(Jinhao), number(%)\\nS/CO\\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\\nS/CO\\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\\nHCV antibody detection by double‐antigen sandwich\\nIndirect ELISA\\n(Jinhao), number(%)\\nS/CO\\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\\nS/CO\\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\\n Discordance among the test results Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.\\nVariables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.\",\n \"Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\\nIndirect ELISA\\n(Wantai), number(%)\\nS/CO\\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\\nS/CO\",\n \"Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\\nHCV antibody detection by double‐antigen sandwich\\nIndirect ELISA\\n(Jinhao), number(%)\\nS/CO\\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\\nS/CO\\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\",\n \"Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.\",\n \"There are no symptoms or only mild symptoms such as right upper abdominal discomfort and oil aversion in the incubation period after HCV infection, and it is likely to take 20‐40 years to developed into serious clinical complication such as liver fibrosis or even hepatocellular carcinoma.\\n12\\n, \\n13\\n The exact time of HCV infection is difficult to determine the symptoms are mild or absent, easily transform into chronic, and higher chronic rate was 55% to 85%. Additionally, the understanding degree of the disease at a lower level, autonomous screening rate is low and public lacks attention with this disease. It caused tremendous burden on both China and the world economy. Along with a deeper understanding of the HCV life cycle, direct antiviral drugs (DAAs) have become widely used since its introduction, which is highly effective, less adverse reactions and a short course of treatment available to fight HCV.\\n14\\n, \\n15\\n DAAs are working directly on the non‐structural protein (NS) 3/4A, 5A, and 5B to achieve virus clearance, so the deadlock of clinical treatment of viral hepatitis C was broken even promising to get complete cure; thus, WHO sets targets to eliminate hepatitis C as a public health threat by 2030.\\n16\\n, \\n17\\n However, related research suggests that DAAs are still at a high price, at the same time, reinfection cannot be avoided, and the cured patients still have the risk of hepatocellular carcinoma.\\n18\\n, \\n19\\n, \\n20\\n Vaccine is the most cost‐effective and effective measure to prevent and combat the spread of disease. However, there is not any effective vaccine to prevent HCV infection.\\n21\\n The successful development and clinical application of HCV vaccine are unpredictable.\\n22\\n In summary, the focal problems for hepatitis C are shifting from the therapeutic strategies to screening program launched in high‐risk groups.\\nTo date, early screening and diagnosis of HCV infection are based on three different methods, which may be utilized alone or in combination. These are (a) detection of HCV core antigen (HCV‐Ag); (b) nucleic acid testing (NAT) to detect HCV‐RNA by PCR; and (c) detection of an IgG antibody by ELISA (anti‐HCV).\\n23\\n Related literature reports that there is a good correlation between the contents of HCV‐Ag and the quantity of HCV‐RNA as viral load exceeds 3000 IU/mL in the serum samples of HCV patients, so HCV core antigen can be used as early diagnostic indicators of HCV infection.\\n24\\n Meanwhile, detection of HCV‐Ag has the advantages of relatively low cost, handiness, and simplicity and not need to add special instruments. However, the structure of the HCV‐cAg is very special and the preparation of antibody is more difficult, and detection sensitivity of HCV‐cAg is lower than that of anti‐HCV by using ELISA. It can be used as a supplement to anti‐HCV detection but cannot replace the determination of antibody. The samples were tested HCV‐RNA positive are the reliable index of viral replication activity, and the HCV‐RNA methodology can be subdivided into qualitative and quantitative methods.\\n25\\n, \\n26\\n Qualitative analysis has played a key role in blood screening of blood banks, it as a means of indicating the presence or absence of viremia but not measure the viral load. In recent years, the real‐time PCR (RT‐PCR) is gradually replacing qualitative analysis as a common method used in detecting HCV‐RNA, as it features high sensitivity and specificity. However, it cannot be used as a routine method because of its complicated operation and higher experimental conditions needed. In particular, most hospitals, especially primary healthcare facilities, do not have the capacity to detect HCV‐RNA and provide accurate results.\\n27\\n\\n\\nAccording to guidelines for the prevention and treatment of hepatitis C (2019 version) revised by the Chinese Medical Association, along with WS 213—2018 Diagnosis for hepatitis C (National Health and Family Planning Commission of the People's Republic of China). Compared with (HCV‐Ag) and nucleic acid testing (NAT), the anti‐HCV immunoassays are the most frequently used laboratory tests for detection of HCV infection. And now it has the following advantages in the detection of HCV antibodies with the rapid development of genetic engineering technology: high sensitivity and specificity, better stability, automation, low cost, and convenience. Indirect ELISA had already gone through three reformation, the first generation anti‐HCV assay using recombinant c100‐3 epitope from the NS4 protein, which had limited sensitivity and specificity. Second generation assay was developed using epitopes from the core, NS3, and NS4 proteins, a multi‐antigen format that can reduce the time required and increase the sensitivity. More recently, an highly conserved NS5 antigen have adopted in third generation assays. If the sample contains anti‐HCV, incubate for a certain period of time, solid‐phase antigen will fully bind to the anti‐HCV in the serum, and then react with the added IgG‐HRP. Tetramethylbenzidine (TMB) color development or not to indicate present or absent of anti‐HCV in human serum or plasma.\\n28\\n However, indirect ELISA can always result in false positives due to interfering factors, including high gamma globulin levels, nephritic syndrome, pregnancy, sample hemolysis, the splashing, and pollution of the blood sample.\\n29\\n\\n\\nDouble‐antigen sandwich ELISA is a developing technology with great potential.\\n30\\n At the beginning of development, it faces two major obstacles, the HCV antigen labeled with HRP directly is easy to cause the space steric hindrance and ratio of enzyme‐labeled antigens is difficult to control. Subsequently, preparation of growth arm biotinylated HCV antigen as a sandwich antigen using genetic engineering, with horseradish peroxidase mildew affinity tag chain element as enzymes, so anti‐HCV could perform twice specific binding in the sample and amplification of the signal was achieved using biotin–streptavidin system. Related research shows that compared to traditional indirect ELISA, double‐antigen sandwich ELISA displays higher specificity and better sensitivity, and it can utilize to detect IgM.\\n31\\n Double‐antigen sandwich ELISA could make up for the methodological deficiencies inherent in indirect ELISA and has clinical utility in both screening and diagnosis of HCV infection.\\nObjective of our study is to investigate the consistency of three commercially available anti‐HCV assays, and analysis of influencing factors to disagreement between the double‐antigen sandwich and indirect ELISA. So as to provide pertinent evidence for the selection of methods of detecting anti‐HCV in clinical laboratories. In our study, among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai), indirect ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 68.75%, 74.43%, and 73.30%, respectively. The agreement between the double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) was high (κ = 0.829; P < .001). The agreement between the double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847; P < .001). But there are still inconsistent results in the analysis of anti‐HCV by using double‐antigen sandwich and indirect ELISA. It was found that among the fifty‐five specimens that were tested negative using by double‐antigen sandwich ELISA (Beijing Wantai), eleven were positive using by indirect ELISA (Beijing Wantai) and ten were positive using by indirect ELISA (Beijing Jinhao), and the possible cause of this problem perhaps is that nonspecific immunoglobulin, rheumatoid factor, hydrophilic antibodies, and other interfering substances in the specimen, which can be nonspecifically combined with the enzyme‐labeled secondary antibody, lead to false‐positive results. Among one hundred and twenty‐one samples tested positive using by double‐antigen sandwich ELISA (Beijing Wantai), one was negative using by indirect ELISA (Beijing Wantai) and two were negative by using indirect ELISA (Beijing Jinhao). The probable reason is that the patients are in the window period of infection and the body has not yet produced IgG. Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR: 1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05). Analysis of possible reasons were as follows: (a) More women predisposed to autoimmune diseases and autoantibody positivity was higher than men; (b) positive antinuclear antibodies in malignancies than benign tumor; and (c) pregnant woman in people younger than 35 years old was high and the presence of some interfering substances may be affect the results significantly. These discrepancies emphasize the need for further research, and we will follow up in subsequent studies.\",\n \"In detection of HCV infection, high agreement was found between the double‐antigen sandwich ELISA and indirect ELISA. Based on double‐antigen sandwich ELISA has distinct methodological advantages over indirect ELISA, considering double‐antigen sandwich ELISA has high clinical popularization value in the screening of HCV infection. Female, younger age, and malignancy were significant risk factors for the discordance. It is recommended that relevant laboratories should pay more attention to the existence of the above significant risk factors by using indirect ELISA and suggest to detect anti‐HCV by using double‐antigen sandwich ELISA if condition permits.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"materials-and-methods",null,null,null,"results",null,null,null,"discussion","conclusions"],"string":"[\n null,\n \"materials-and-methods\",\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["double‐antigen sandwich ELISA","hepatitis C antibodies","indirect ELISA"],"string":"[\n \"double‐antigen sandwich ELISA\",\n \"hepatitis C antibodies\",\n \"indirect ELISA\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nThe virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.\n1\n, \n2\n The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.\n3\n, \n4\n Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.\n5\n For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.\n6\n WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.\n7\n People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.\n8\n Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.\n9\n, \n10\n\n\nDepending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.\n11\n In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection.\n\nMATERIAL AND METHOD:\n Subjects A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.\nA total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.\n Commercial assays Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.\nDouble‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.\n Statistical analysis Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.\nStatistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.\n\nSubjects:\nA total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.\n\nCommercial assays:\nDouble‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.\n\nStatistical analysis:\nStatistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.\n\nRESULTS:\n Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO\n Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\n Discordance among the test results Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.\nVariables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.\n\nComparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai):\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO\n\nComparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao):\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\n\nDiscordance among the test results:\nVariables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.\n\nDISCUSSION:\nThere are no symptoms or only mild symptoms such as right upper abdominal discomfort and oil aversion in the incubation period after HCV infection, and it is likely to take 20‐40 years to developed into serious clinical complication such as liver fibrosis or even hepatocellular carcinoma.\n12\n, \n13\n The exact time of HCV infection is difficult to determine the symptoms are mild or absent, easily transform into chronic, and higher chronic rate was 55% to 85%. Additionally, the understanding degree of the disease at a lower level, autonomous screening rate is low and public lacks attention with this disease. It caused tremendous burden on both China and the world economy. Along with a deeper understanding of the HCV life cycle, direct antiviral drugs (DAAs) have become widely used since its introduction, which is highly effective, less adverse reactions and a short course of treatment available to fight HCV.\n14\n, \n15\n DAAs are working directly on the non‐structural protein (NS) 3/4A, 5A, and 5B to achieve virus clearance, so the deadlock of clinical treatment of viral hepatitis C was broken even promising to get complete cure; thus, WHO sets targets to eliminate hepatitis C as a public health threat by 2030.\n16\n, \n17\n However, related research suggests that DAAs are still at a high price, at the same time, reinfection cannot be avoided, and the cured patients still have the risk of hepatocellular carcinoma.\n18\n, \n19\n, \n20\n Vaccine is the most cost‐effective and effective measure to prevent and combat the spread of disease. However, there is not any effective vaccine to prevent HCV infection.\n21\n The successful development and clinical application of HCV vaccine are unpredictable.\n22\n In summary, the focal problems for hepatitis C are shifting from the therapeutic strategies to screening program launched in high‐risk groups.\nTo date, early screening and diagnosis of HCV infection are based on three different methods, which may be utilized alone or in combination. These are (a) detection of HCV core antigen (HCV‐Ag); (b) nucleic acid testing (NAT) to detect HCV‐RNA by PCR; and (c) detection of an IgG antibody by ELISA (anti‐HCV).\n23\n Related literature reports that there is a good correlation between the contents of HCV‐Ag and the quantity of HCV‐RNA as viral load exceeds 3000 IU/mL in the serum samples of HCV patients, so HCV core antigen can be used as early diagnostic indicators of HCV infection.\n24\n Meanwhile, detection of HCV‐Ag has the advantages of relatively low cost, handiness, and simplicity and not need to add special instruments. However, the structure of the HCV‐cAg is very special and the preparation of antibody is more difficult, and detection sensitivity of HCV‐cAg is lower than that of anti‐HCV by using ELISA. It can be used as a supplement to anti‐HCV detection but cannot replace the determination of antibody. The samples were tested HCV‐RNA positive are the reliable index of viral replication activity, and the HCV‐RNA methodology can be subdivided into qualitative and quantitative methods.\n25\n, \n26\n Qualitative analysis has played a key role in blood screening of blood banks, it as a means of indicating the presence or absence of viremia but not measure the viral load. In recent years, the real‐time PCR (RT‐PCR) is gradually replacing qualitative analysis as a common method used in detecting HCV‐RNA, as it features high sensitivity and specificity. However, it cannot be used as a routine method because of its complicated operation and higher experimental conditions needed. In particular, most hospitals, especially primary healthcare facilities, do not have the capacity to detect HCV‐RNA and provide accurate results.\n27\n\n\nAccording to guidelines for the prevention and treatment of hepatitis C (2019 version) revised by the Chinese Medical Association, along with WS 213—2018 Diagnosis for hepatitis C (National Health and Family Planning Commission of the People's Republic of China). Compared with (HCV‐Ag) and nucleic acid testing (NAT), the anti‐HCV immunoassays are the most frequently used laboratory tests for detection of HCV infection. And now it has the following advantages in the detection of HCV antibodies with the rapid development of genetic engineering technology: high sensitivity and specificity, better stability, automation, low cost, and convenience. Indirect ELISA had already gone through three reformation, the first generation anti‐HCV assay using recombinant c100‐3 epitope from the NS4 protein, which had limited sensitivity and specificity. Second generation assay was developed using epitopes from the core, NS3, and NS4 proteins, a multi‐antigen format that can reduce the time required and increase the sensitivity. More recently, an highly conserved NS5 antigen have adopted in third generation assays. If the sample contains anti‐HCV, incubate for a certain period of time, solid‐phase antigen will fully bind to the anti‐HCV in the serum, and then react with the added IgG‐HRP. Tetramethylbenzidine (TMB) color development or not to indicate present or absent of anti‐HCV in human serum or plasma.\n28\n However, indirect ELISA can always result in false positives due to interfering factors, including high gamma globulin levels, nephritic syndrome, pregnancy, sample hemolysis, the splashing, and pollution of the blood sample.\n29\n\n\nDouble‐antigen sandwich ELISA is a developing technology with great potential.\n30\n At the beginning of development, it faces two major obstacles, the HCV antigen labeled with HRP directly is easy to cause the space steric hindrance and ratio of enzyme‐labeled antigens is difficult to control. Subsequently, preparation of growth arm biotinylated HCV antigen as a sandwich antigen using genetic engineering, with horseradish peroxidase mildew affinity tag chain element as enzymes, so anti‐HCV could perform twice specific binding in the sample and amplification of the signal was achieved using biotin–streptavidin system. Related research shows that compared to traditional indirect ELISA, double‐antigen sandwich ELISA displays higher specificity and better sensitivity, and it can utilize to detect IgM.\n31\n Double‐antigen sandwich ELISA could make up for the methodological deficiencies inherent in indirect ELISA and has clinical utility in both screening and diagnosis of HCV infection.\nObjective of our study is to investigate the consistency of three commercially available anti‐HCV assays, and analysis of influencing factors to disagreement between the double‐antigen sandwich and indirect ELISA. So as to provide pertinent evidence for the selection of methods of detecting anti‐HCV in clinical laboratories. In our study, among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai), indirect ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 68.75%, 74.43%, and 73.30%, respectively. The agreement between the double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) was high (κ = 0.829; P < .001). The agreement between the double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847; P < .001). But there are still inconsistent results in the analysis of anti‐HCV by using double‐antigen sandwich and indirect ELISA. It was found that among the fifty‐five specimens that were tested negative using by double‐antigen sandwich ELISA (Beijing Wantai), eleven were positive using by indirect ELISA (Beijing Wantai) and ten were positive using by indirect ELISA (Beijing Jinhao), and the possible cause of this problem perhaps is that nonspecific immunoglobulin, rheumatoid factor, hydrophilic antibodies, and other interfering substances in the specimen, which can be nonspecifically combined with the enzyme‐labeled secondary antibody, lead to false‐positive results. Among one hundred and twenty‐one samples tested positive using by double‐antigen sandwich ELISA (Beijing Wantai), one was negative using by indirect ELISA (Beijing Wantai) and two were negative by using indirect ELISA (Beijing Jinhao). The probable reason is that the patients are in the window period of infection and the body has not yet produced IgG. Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR: 1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05). Analysis of possible reasons were as follows: (a) More women predisposed to autoimmune diseases and autoantibody positivity was higher than men; (b) positive antinuclear antibodies in malignancies than benign tumor; and (c) pregnant woman in people younger than 35 years old was high and the presence of some interfering substances may be affect the results significantly. These discrepancies emphasize the need for further research, and we will follow up in subsequent studies.\n\nCONCLUSION:\nIn detection of HCV infection, high agreement was found between the double‐antigen sandwich ELISA and indirect ELISA. Based on double‐antigen sandwich ELISA has distinct methodological advantages over indirect ELISA, considering double‐antigen sandwich ELISA has high clinical popularization value in the screening of HCV infection. Female, younger age, and malignancy were significant risk factors for the discordance. It is recommended that relevant laboratories should pay more attention to the existence of the above significant risk factors by using indirect ELISA and suggest to detect anti‐HCV by using double‐antigen sandwich ELISA if condition permits.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe aim of this study is to compare double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA in the diagnosis of hepatitis C virus（HCV）infection.\n\nMethods:\nA total of 176 samples from the Tumor Hospital Affiliated to Xin Jiang Medical University were utilized to comparison. All serum samples were tested using double-antigen sandwich ELISA and indirect ELISA. Cohen's kappa statistics were used to assess the agreement between the two assays, and multivariate analysis was used to evaluate risk factors for the discordance between the double-antigen ELISA and indirect ELISA.\n\nResults:\nThe positivities of indirect ELISA (Beijing Wantai), double-antigen sandwich ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 74.43%, 68.75%, and 73.30%, respectively. The agreement between the indirect ELISA (Beijing Wantai) and double-antigen sandwich ELISA (Beijing Wantai) was high (κ = 0.829;P < .001), and the agreement between the double-antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847;P < .001). Variables associated with discordant results between the double-antigen sandwich and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05).\n\nConclusions:\nIn detection of HCV, high agreement was found between the double-antigen sandwich ELISA and indirect ELISA. Female, younger age, and suffer from malignant tumor were significant risk factors for the discordance. Based on double-antigen sandwich ELISA has distinct methodological advantages over indirect ELISA. It is recommended for the diagnosis of HCV infection."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nThe virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.\n1\n, \n2\n The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.\n3\n, \n4\n Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.\n5\n For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.\n6\n WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.\n7\n People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.\n8\n Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.\n9\n, \n10\n\n\nDepending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.\n11\n In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection.\n\nCONCLUSION:\nIn detection of HCV infection, high agreement was found between the double‐antigen sandwich ELISA and indirect ELISA. Based on double‐antigen sandwich ELISA has distinct methodological advantages over indirect ELISA, considering double‐antigen sandwich ELISA has high clinical popularization value in the screening of HCV infection. Female, younger age, and malignancy were significant risk factors for the discordance. It is recommended that relevant laboratories should pay more attention to the existence of the above significant risk factors by using indirect ELISA and suggest to detect anti‐HCV by using double‐antigen sandwich ELISA if condition permits."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe aim of this study is to compare double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA in the diagnosis of hepatitis C virus（HCV）infection.\n\nMethods:\nA total of 176 samples from the Tumor Hospital Affiliated to Xin Jiang Medical University were utilized to comparison. All serum samples were tested using double-antigen sandwich ELISA and indirect ELISA. Cohen's kappa statistics were used to assess the agreement between the two assays, and multivariate analysis was used to evaluate risk factors for the discordance between the double-antigen ELISA and indirect ELISA.\n\nResults:\nThe positivities of indirect ELISA (Beijing Wantai), double-antigen sandwich ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 74.43%, 68.75%, and 73.30%, respectively. The agreement between the indirect ELISA (Beijing Wantai) and double-antigen sandwich ELISA (Beijing Wantai) was high (κ = 0.829;P < .001), and the agreement between the double-antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847;P < .001). Variables associated with discordant results between the double-antigen sandwich and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05).\n\nConclusions:\nIn detection of HCV, high agreement was found between the double-antigen sandwich ELISA and indirect ELISA. Female, younger age, and suffer from malignant tumor were significant risk factors for the discordance. Based on double-antigen sandwich ELISA has distinct methodological advantages over indirect ELISA. It is recommended for the diagnosis of HCV infection."},"whole_article_text_length":{"kind":"number","value":5573,"string":"5,573"},"whole_article_abstract_length":{"kind":"number","value":356,"string":"356"},"num_sections":{"kind":"number","value":11,"string":"11"},"most_frequent_words":{"kind":"list like","value":["elisa","hcv","beijing","antigen","indirect","indirect elisa","wantai","elisa beijing","sandwich","antigen sandwich"],"string":"[\n \"elisa\",\n \"hcv\",\n \"beijing\",\n \"antigen\",\n \"indirect\",\n \"indirect elisa\",\n \"wantai\",\n \"elisa beijing\",\n \"sandwich\",\n \"antigen sandwich\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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[SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] elisa | hcv | beijing | antigen | indirect | indirect elisa | wantai | elisa beijing | sandwich | antigen sandwich [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] elisa | hcv | beijing | antigen | indirect | indirect elisa | wantai | elisa beijing | sandwich | antigen sandwich [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] elisa | hcv | beijing | antigen | indirect | indirect elisa | wantai | elisa beijing | sandwich | antigen sandwich [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] elisa | hcv | beijing | antigen | indirect | indirect elisa | wantai | elisa beijing | sandwich | antigen sandwich [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hcv | virus | genotype | genotypes | diagnosis | daas | people | effective | hepatitis | screening [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] elisa | wantai | elisa beijing | beijing | beijing wantai indirect elisa | elisa beijing wantai indirect | wantai indirect | wantai indirect elisa | beijing wantai indirect | elisa beijing wantai [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] elisa | significant risk | significant risk factors | double antigen sandwich elisa | sandwich elisa | antigen sandwich elisa | risk factors | significant | double | sandwich [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] elisa | beijing | hcv | wantai | elisa beijing | antigen | indirect | beijing wantai | antigen sandwich | sandwich [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] elisa | beijing | hcv | wantai | elisa beijing | antigen | indirect | beijing wantai | antigen sandwich | sandwich [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ELISA | ELISA [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao | 74.43% | 68.75% | 73.30% ||| ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao ||| ELISA | OR:1.462 | 35 years old | OR:3.621 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ELISA | ELISA ||| ||| ELISA | ELISA ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ELISA | ELISA ||| 176 | the Tumor Hospital Affiliated | Xin Jiang Medical University ||| ELISA | ELISA ||| Cohen | the two assays | ELISA | ELISA ||| ||| ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao | 74.43% | 68.75% | 73.30% ||| ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao ||| ELISA | OR:1.462 | 35 years old | OR:3.621 ||| ELISA | ELISA ||| ||| ELISA | ELISA ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ELISA | ELISA ||| 176 | the Tumor Hospital Affiliated | Xin Jiang Medical University ||| ELISA | ELISA ||| Cohen | the two assays | ELISA | ELISA ||| ||| ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao | 74.43% | 68.75% | 73.30% ||| ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao ||| ELISA | OR:1.462 | 35 years old | OR:3.621 ||| ELISA | ELISA ||| ||| ELISA | ELISA ||| [SUMMARY]"}}},{"rowIdx":258,"cells":{"title":{"kind":"string","value":"Superior Effectiveness of Zidovudine Compared With Tenofovir When Combined With Nevirapine-based Antiretroviral Therapy in a Large Nigerian Cohort."},"pmid":{"kind":"string","value":"26561532"},"background_abstract":{"kind":"string","value":"Despite sparse efficacy data, tenofovir-emtricitabine or tenofovir-lamivudine plus nevirapine is used in many resource-constrained settings."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"This retrospective cohort study included patients initiating nevirapine-based antiretroviral therapy (ART) with either tenofovir-emtricitabine or lamivudine (tenofovir group) or zidovudine-lamivudine (zidovudine group). Clinical, virologic, and immunologic evaluations were performed at baseline and every 6 months. Virologic failure was defined as 2 consecutive human immunodeficiency virus (HIV)-RNA values >1000 copies/mL. Patients were included from ART initiation until time of failure, regimen switch, discontinuation, or last HIV-RNA measurement. Cox proportional hazards regression was used to model factors influencing time to failure. Bias due to dependent censoring was investigated via inverse probability weighted pooled logistic regression."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A total of 5547 patients were evaluated; 1484 (26.8%) were in the tenofovir group and 4063 (73.2%) were in the zidovudine group. In the adjusted model, tenofovir regimen (hazard ratio [HR], 1.47; 95% confidence interval [CI], 1.21-1.79) and higher baseline log10 HIV-RNA (HR, 1.15; 95% CI, 1.03-1.28) were associated with virologic failure. Higher baseline log10 CD4+ cell count (HR, 0.50; 95% CI, .40-.63) and increasing age (HR, 0.98; 95% CI, .97-.99) decreased the risk of virologic failure. Inverse probability weighting results were consistent with the primary analysis."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Compared with zidovudine-lamivudine, the use of tenofovir-lamivudine or emtricitabine in combination with nevirapine was a strong predictor of virologic failure in our cohort, which was not explained by other risk factors or criteria for regimen selection."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Anti-Retroviral Agents","Antiretroviral Therapy, Highly Active","CD4 Lymphocyte Count","Cohort Studies","Female","HIV Infections","Humans","Male","Nevirapine","Retrospective Studies","Tenofovir","Treatment Outcome","Viral Load","Young Adult","Zidovudine"],"string":"[\n \"Adult\",\n \"Anti-Retroviral Agents\",\n \"Antiretroviral Therapy, Highly Active\",\n \"CD4 Lymphocyte Count\",\n \"Cohort Studies\",\n \"Female\",\n \"HIV Infections\",\n \"Humans\",\n \"Male\",\n \"Nevirapine\",\n \"Retrospective Studies\",\n \"Tenofovir\",\n \"Treatment Outcome\",\n \"Viral Load\",\n \"Young Adult\",\n \"Zidovudine\"\n]"},"pmcid":{"kind":"string","value":"4725384"},"background_title":{"kind":"null"},"background_text":{"kind":"null"},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"In this retrospective cohort study, prospectively collected data from a large clinical cohort in Nigeria were used. Between June 2004 and February 2012, the Harvard T. H. Chan School of Public Health (Harvard) collaborated with the AIDS Prevention Initiative in Nigeria Ltd./Gte. (APIN) to support HIV prevention, care, and treatment to over 160 000 patients, funded by the President's Emergency Plan for AIDS Relief (PEPFAR). Upon program entry, patients underwent a clinical examination and baseline laboratory testing including hematology and chemistry, CD4+ cell count (Cyflow, Partec), HIV-RNA (Cobas Amplicor Monitor Assay, Roche Diagnostics), hepatitis B virus (HBV) surface antigen (Monolisa HBsAg Ultra3, BioRad), and hepatitis C virus (HCV) antibody (DIA.PRO Diagnostic, Bioprobes srl) assessments. Patients eligible for ART, based on guidelines at the time of program entry, initiated standard first-line ART with 1 NNRTI and 2 NRTIs [11, 12]. Prescription refills were obtained monthly; clinical visits and laboratory tests were performed every 6 months, or earlier if necessary. All patient data were maintained in previously described electronic databases [13].\n Ethical Considerations All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\nAll patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\n Patient Selection Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\nAntiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\n Study Definitions Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\nSex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\n Statistical Analyses Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nSubject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18]."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"During the study period, 6575 patients were identified for inclusion and 5547 (84%) had data available for all variables in the final model—4063 (73.2%) in the zidovudine group and 1484 (26.8%) in the tenofovir group (Figure 1). The most common reason for exclusion was missing HBV or HCV status (11.0%). Baseline participant characteristics are summarized in Table 1. Consistent with clinical considerations for selecting an NRTI, HBV coinfection was more common among those in the tenofovir vs zidovudine group (27.2% vs 14.9%; P < .001), as was a lower median baseline hemoglobin (100 vs 110 g/L, P < .001). Proportionally, more women, patients with HCV coinfection, and those with more advanced HIV disease received tenofovir. Median (interquartile range [IQR]) duration of time to event or censor was slightly longer for the zidovudine group compared with the tenofovir group (392 [244] vs 376 [170] days, P < .001).\nTable 1.Baseline Characteristics and Outcomes of Study ParticipantsTenofovir Group n = 1484Zidovudine Group n = 4063P ValueBaseline Characteristics Age (y)33 (11)32 (11).41 Female gender1255 (84.6)3085 (75.9)<.001 Hepatitis B virus coinfection404 (27.2)604 (14.9)<.001 Hepatitis C virus coinfection144 (9.7)294 (7.2).003 CD4 count (cells/mm3)122 (122)143 (115)<.001 Human immunodeficiency virus-RNA (log10 copies/mL)4.86 (1.1)4.70 (1.2)<.001 World Health Organization Stagen = 1335n = 3489<.001 1236 (17.7)923 (26.5) 2435 (32.6)1042 (29.9) 3494 (37.0)1045 (30.0) 4170 (12.7)479 (13.7) Tuberculosis coinfection294 (19.8)466 (11.5)<.001 Alanine transaminase (µkat/L)0.42 (0.37)0.40 (0.36).04 Hemoglobin (g/L)100 (24), n = 1462110 (20), n = 3948<.001 Creatinine (μmol/L)77 (31), n = 145877 (31), n = 3880.943Study Outcomes Virologic failure159 (10.7)298 (7.3)<.001 Antiretroviral therapy switch256 (17.3)622 (15.3).079 Discontinue308 (20.8)649 (16.0)<.001 No event761 (51.3)2494 (61.4)<.001Values shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.\nFigure 1.Flow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.\nBaseline Characteristics and Outcomes of Study Participants\nValues shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.\nFlow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.\nAmong patients in the tenofovir group, 1043 (70.2%) received emtricitabine as their second NRTI, 190 (12.8%) received lamivudine, and 252 (19.8%) switched between emtricitabine and lamivudine during the study period (median [IQR] time to switch was 172 [97] days). The number of virologic failure events did not differ significantly between patients receiving emtricitabine or lamivudine (P = .47); therefore, data were combined in the final analysis irrespective of emtricitabine or lamivudine use.\nOverall, the median ART adherence rate was 95.3%, which was similar between the zidovudine and tenofovir groups (95.2% vs 95.8%). Among those who experienced virologic failure, more patients had adherence <95% compared with ≥95% (56.4% vs 43.6%; P < .001), but there was no difference in median adherence between those patients with failure in the zidovudine or tenofovir groups (91.9% vs 93.6%; P = .36).\nPatients in the tenofovir group had a higher rate of virologic failure and discontinuation (Table 1). Figure 2 represents time to virologic failure between the groups. Of the 957 patients with a discontinuation event, 14 (1.5%) died, 849 (88.7%) were lost to follow-up, 92 (9.6%) transferred to another site, and 2 (0.2%) withdrew from the program. These distributions were similar between NRTI groups (P = .50).\nFigure 2.Kaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.\nKaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.\n Time to Virologic Failure After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\nAfter adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\n Evaluation of Other Outcome Differences or Dependent Censoring Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.\nAdditional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Ethical Considerations","Patient Selection","Study Definitions","Statistical Analyses","Time to Virologic Failure Regression Modeling","Evaluation of Dependent Censoring","Time to Virologic Failure","Evaluation of Other Outcome Differences or Dependent Censoring"],"string":"[\n \"Ethical Considerations\",\n \"Patient Selection\",\n \"Study Definitions\",\n \"Statistical Analyses\",\n \"Time to Virologic Failure Regression Modeling\",\n \"Evaluation of Dependent Censoring\",\n \"Time to Virologic Failure\",\n \"Evaluation of Other Outcome Differences or Dependent Censoring\"\n]"},"other_sections_texts":{"kind":"list like","value":["All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.","Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.","Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.","Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].","Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.","The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].","After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).","Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates."],"string":"[\n \"All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\",\n \"Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\",\n \"Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\",\n \"Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\",\n \"Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\",\n \"The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\",\n \"After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\\nAll risk factors were measured at baseline.\\nBold results indicate statistically significant results (P value <.05).\\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\",\n \"Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["METHODS","Ethical Considerations","Patient Selection","Study Definitions","Statistical Analyses","Time to Virologic Failure Regression Modeling","Evaluation of Dependent Censoring","RESULTS","Time to Virologic Failure","Evaluation of Other Outcome Differences or Dependent Censoring","DISCUSSION","Supplementary Data"],"string":"[\n \"METHODS\",\n \"Ethical Considerations\",\n \"Patient Selection\",\n \"Study Definitions\",\n \"Statistical Analyses\",\n \"Time to Virologic Failure Regression Modeling\",\n \"Evaluation of Dependent Censoring\",\n \"RESULTS\",\n \"Time to Virologic Failure\",\n \"Evaluation of Other Outcome Differences or Dependent Censoring\",\n \"DISCUSSION\",\n \"Supplementary Data\"\n]"},"all_sections_texts":{"kind":"list like","value":["In this retrospective cohort study, prospectively collected data from a large clinical cohort in Nigeria were used. Between June 2004 and February 2012, the Harvard T. H. Chan School of Public Health (Harvard) collaborated with the AIDS Prevention Initiative in Nigeria Ltd./Gte. (APIN) to support HIV prevention, care, and treatment to over 160 000 patients, funded by the President's Emergency Plan for AIDS Relief (PEPFAR). Upon program entry, patients underwent a clinical examination and baseline laboratory testing including hematology and chemistry, CD4+ cell count (Cyflow, Partec), HIV-RNA (Cobas Amplicor Monitor Assay, Roche Diagnostics), hepatitis B virus (HBV) surface antigen (Monolisa HBsAg Ultra3, BioRad), and hepatitis C virus (HCV) antibody (DIA.PRO Diagnostic, Bioprobes srl) assessments. Patients eligible for ART, based on guidelines at the time of program entry, initiated standard first-line ART with 1 NNRTI and 2 NRTIs [11, 12]. Prescription refills were obtained monthly; clinical visits and laboratory tests were performed every 6 months, or earlier if necessary. All patient data were maintained in previously described electronic databases [13].\n Ethical Considerations All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\nAll patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\n Patient Selection Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\nAntiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\n Study Definitions Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\nSex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\n Statistical Analyses Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nSubject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].","All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.","Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.","Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.","Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].","Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.","The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].","During the study period, 6575 patients were identified for inclusion and 5547 (84%) had data available for all variables in the final model—4063 (73.2%) in the zidovudine group and 1484 (26.8%) in the tenofovir group (Figure 1). The most common reason for exclusion was missing HBV or HCV status (11.0%). Baseline participant characteristics are summarized in Table 1. Consistent with clinical considerations for selecting an NRTI, HBV coinfection was more common among those in the tenofovir vs zidovudine group (27.2% vs 14.9%; P < .001), as was a lower median baseline hemoglobin (100 vs 110 g/L, P < .001). Proportionally, more women, patients with HCV coinfection, and those with more advanced HIV disease received tenofovir. Median (interquartile range [IQR]) duration of time to event or censor was slightly longer for the zidovudine group compared with the tenofovir group (392 [244] vs 376 [170] days, P < .001).\nTable 1.Baseline Characteristics and Outcomes of Study ParticipantsTenofovir Group n = 1484Zidovudine Group n = 4063P ValueBaseline Characteristics Age (y)33 (11)32 (11).41 Female gender1255 (84.6)3085 (75.9)<.001 Hepatitis B virus coinfection404 (27.2)604 (14.9)<.001 Hepatitis C virus coinfection144 (9.7)294 (7.2).003 CD4 count (cells/mm3)122 (122)143 (115)<.001 Human immunodeficiency virus-RNA (log10 copies/mL)4.86 (1.1)4.70 (1.2)<.001 World Health Organization Stagen = 1335n = 3489<.001 1236 (17.7)923 (26.5) 2435 (32.6)1042 (29.9) 3494 (37.0)1045 (30.0) 4170 (12.7)479 (13.7) Tuberculosis coinfection294 (19.8)466 (11.5)<.001 Alanine transaminase (µkat/L)0.42 (0.37)0.40 (0.36).04 Hemoglobin (g/L)100 (24), n = 1462110 (20), n = 3948<.001 Creatinine (μmol/L)77 (31), n = 145877 (31), n = 3880.943Study Outcomes Virologic failure159 (10.7)298 (7.3)<.001 Antiretroviral therapy switch256 (17.3)622 (15.3).079 Discontinue308 (20.8)649 (16.0)<.001 No event761 (51.3)2494 (61.4)<.001Values shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.\nFigure 1.Flow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.\nBaseline Characteristics and Outcomes of Study Participants\nValues shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.\nFlow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.\nAmong patients in the tenofovir group, 1043 (70.2%) received emtricitabine as their second NRTI, 190 (12.8%) received lamivudine, and 252 (19.8%) switched between emtricitabine and lamivudine during the study period (median [IQR] time to switch was 172 [97] days). The number of virologic failure events did not differ significantly between patients receiving emtricitabine or lamivudine (P = .47); therefore, data were combined in the final analysis irrespective of emtricitabine or lamivudine use.\nOverall, the median ART adherence rate was 95.3%, which was similar between the zidovudine and tenofovir groups (95.2% vs 95.8%). Among those who experienced virologic failure, more patients had adherence <95% compared with ≥95% (56.4% vs 43.6%; P < .001), but there was no difference in median adherence between those patients with failure in the zidovudine or tenofovir groups (91.9% vs 93.6%; P = .36).\nPatients in the tenofovir group had a higher rate of virologic failure and discontinuation (Table 1). Figure 2 represents time to virologic failure between the groups. Of the 957 patients with a discontinuation event, 14 (1.5%) died, 849 (88.7%) were lost to follow-up, 92 (9.6%) transferred to another site, and 2 (0.2%) withdrew from the program. These distributions were similar between NRTI groups (P = .50).\nFigure 2.Kaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.\nKaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.\n Time to Virologic Failure After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\nAfter adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\n Evaluation of Other Outcome Differences or Dependent Censoring Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.\nAdditional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.","After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).","Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.","The Harvard–APIN program initially identified a high rate of confirmed virologic failure among patients on nevirapine plus tenofovir and emtricitabine or lamivudine compared with other WHO-recommended or alternative first-line regimens [7]. To evaluate the specific impact of the NRTI combination with nevirapine, we focused on patients initiating nevirapine-based ART combined with either zidovudine or tenofovir using Cox proportional hazards, time varying analysis, and IPW modeling. These results indicate that among patients initiating nevirapine, the use of tenofovir carried a 47% greater risk of virologic failure compared with the use of zidovudine; this difference persisted after adjusting for other variables that may influence regimen selection and treatment outcomes (Table 2). Tenofovir benefits were not observed in other outcomes; specifically, the tenofovir group had higher risk for regimen switch and similar risk for discontinuation. Notably, we did not observe significantly higher virologic failure in the tenofovir group after 12 months of ART, suggesting that patients already virologically suppressed on nevirapine plus tenofovir may not be at increased risk for virologic failure. These data have significant clinical implications for adults living in low- and middle-income countries who require an alternative to efavirenz for first-line ART [1].\nOur results are consistent with those from other evaluations of outcomes among patients who received ART containing nevirapine and tenofovir, thus supporting the belief that NNRTI effectiveness differs by NRTI selection. The DAUFIN study, which evaluated the effectiveness and tolerability of nevirapine given either with zidovudine–lamivudine or tenofovir–lamivudine in antiretroviral-naive patients, was terminated early due to high rates of early virologic failure in the tenofovir arm [6]; 9 of 36 patients receiving tenofovir experienced virologic failure, 8 during the first 12 weeks of the study, compared with only 1 of 35 patients receiving zidovudine. Lapadula et al also reported early virologic failure in patients receiving tenofovir–emtricitabine plus nevirapine compared with ritonavir-boosted atazanavir [5]. In another pilot study that described virologic outcomes among 23 patients receiving once-daily nevirapine plus tenofovir–lamivudine, only 10 patients achieved HIV-RNA <75 copies/mL by week 24 [3]. Turning to large clinical cohorts that evaluated this question, among 10 256 antiretroviral-naive patients initiating either zidovudine or tenofovir in combination with nevirapine in Zambia, 90-day mortality was higher among patients who received tenofovir (aHR, 1.45; 95% CI, 1.03–2.06); however, this difference did not remain after sensitivity analyses, including time-varying analysis [9]. In adjusted models from a large cohort study from southern Africa, patients on nevirapine plus tenofovir–emtricitabine or tenofovir–lamivudine experienced significantly higher rates of mortality but lower rates of loss to follow-up when compared with patients taking efavirenz plus similar NRTIs [21]. Consistent with these data, a metaanalysis of 33 studies comparing virologic efficacy among the WHO-recommended tenofovir-containing first-line ART regimens highlighted poorer outcomes with the nevirapine plus tenofovir-containing regimens [22].\nThe reason for the poorer performance of nevirapine–tenofovir ART is unclear. One proposed mechanism is an intracellular drug–drug interaction observed in vitro between tenofovir and nevirapine, resulting in lower intracellular concentrations of both antiretrovirals [23]. Differences in lamivudine vs emtricitabine pharmacokinetics have also been proposed. An observational Dutch cohort study identified a higher risk of virologic failure when lamivudine vs emtricitabine was combined with tenofovir and either nevirapine (aHR 2.01; 95% CI, 1.36–2.98) or efavirenz (aHR, 2.35; 95%CI, 1.61–3.42) [24]. Notably, our data did not identify a difference in outcomes among patients in the tenofovir group who received lamivudine or emtricitabine. Our results are consistent with those from a metaanalysis of 12 randomized controlled trials that compared 4913 patients randomized to lamivudine or emtricitabine in addition to various ART combinations and found that the 2 agents are clinically interchangeable [25].\nOur findings are limited by the study's retrospective cohort design. At the time of data collection, WHO guidelines recommended the use of either nevirapine or efavirenz in combination with zidovudine and lamivudine or with tenofovir and emtricitabine or lamivudine for first-line ART [11]. Regimen selection may have been influenced by factors that we were unable to measure or control for in our analysis, indicated by differences in baseline characteristics (Table 1). To account for some potential biases from dependent censoring, we generated an IPW pooled logistic regression model, and the close correspondence between these models testifies to the robustness of the Cox estimates. Additionally, there may be limitations to measuring adherence based on prescription refill data, particularly as it relates to pill burden. Although all patients received nevirapine twice daily, there was a difference between groups in the total number of pills per day. The zidovudine group received nevirapine plus a twice-daily FDC tablet of zidovudine–lamivudine (4 pills daily) or, beginning early 2007, a twice-daily full regimen FDC tablet (2 pills daily). The tenofovir group received nevirapine plus a once-daily FDC tablet of tenofovir–emtricitabine or tenofovir–lamivudine (3 pills daily). For twice-daily regimens, higher pill burden has previously been associated with lower rates of both adherence and virologic suppression (Spearman correlation, −0.67 and −0.75, respectively; both P < .01) [26]. However, a difference in adherence was not identified between our study groups and did not influence the associated risk of virologic failure related to regimen selection.\nIn conclusion, ART that includes tenofovir and nevirapine should be initiated with caution. The WHO recommends nevirapine as the alternative for patients who cannot take efavirenz, and nevirapine remains widely used for first-line ART in low- and middle-income countries [1, 27]. These results offer important information to guide selection of the NRTI combination when designing an HIV treatment regimen, indicating a preference for zidovudine over tenofovir when combined with nevirapine. These findings support the need for systematic evaluations of ART combinations before their inclusion in ART guidelines and subsequent widespread implementation.","Supplementary materials are available at http://cid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author."],"string":"[\n \"In this retrospective cohort study, prospectively collected data from a large clinical cohort in Nigeria were used. Between June 2004 and February 2012, the Harvard T. H. Chan School of Public Health (Harvard) collaborated with the AIDS Prevention Initiative in Nigeria Ltd./Gte. (APIN) to support HIV prevention, care, and treatment to over 160 000 patients, funded by the President's Emergency Plan for AIDS Relief (PEPFAR). Upon program entry, patients underwent a clinical examination and baseline laboratory testing including hematology and chemistry, CD4+ cell count (Cyflow, Partec), HIV-RNA (Cobas Amplicor Monitor Assay, Roche Diagnostics), hepatitis B virus (HBV) surface antigen (Monolisa HBsAg Ultra3, BioRad), and hepatitis C virus (HCV) antibody (DIA.PRO Diagnostic, Bioprobes srl) assessments. Patients eligible for ART, based on guidelines at the time of program entry, initiated standard first-line ART with 1 NNRTI and 2 NRTIs [11, 12]. Prescription refills were obtained monthly; clinical visits and laboratory tests were performed every 6 months, or earlier if necessary. All patient data were maintained in previously described electronic databases [13].\\n Ethical Considerations All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\\nAll patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\\n Patient Selection Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\\nAntiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\\n Study Definitions Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\\nSex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\\n Statistical Analyses Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\\nSubject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\",\n \"All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\",\n \"Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\",\n \"Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\",\n \"Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\",\n \"Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\",\n \"The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\",\n \"During the study period, 6575 patients were identified for inclusion and 5547 (84%) had data available for all variables in the final model—4063 (73.2%) in the zidovudine group and 1484 (26.8%) in the tenofovir group (Figure 1). The most common reason for exclusion was missing HBV or HCV status (11.0%). Baseline participant characteristics are summarized in Table 1. Consistent with clinical considerations for selecting an NRTI, HBV coinfection was more common among those in the tenofovir vs zidovudine group (27.2% vs 14.9%; P < .001), as was a lower median baseline hemoglobin (100 vs 110 g/L, P < .001). Proportionally, more women, patients with HCV coinfection, and those with more advanced HIV disease received tenofovir. Median (interquartile range [IQR]) duration of time to event or censor was slightly longer for the zidovudine group compared with the tenofovir group (392 [244] vs 376 [170] days, P < .001).\\nTable 1.Baseline Characteristics and Outcomes of Study ParticipantsTenofovir Group n = 1484Zidovudine Group n = 4063P ValueBaseline Characteristics Age (y)33 (11)32 (11).41 Female gender1255 (84.6)3085 (75.9)<.001 Hepatitis B virus coinfection404 (27.2)604 (14.9)<.001 Hepatitis C virus coinfection144 (9.7)294 (7.2).003 CD4 count (cells/mm3)122 (122)143 (115)<.001 Human immunodeficiency virus-RNA (log10 copies/mL)4.86 (1.1)4.70 (1.2)<.001 World Health Organization Stagen = 1335n = 3489<.001 1236 (17.7)923 (26.5) 2435 (32.6)1042 (29.9) 3494 (37.0)1045 (30.0) 4170 (12.7)479 (13.7) Tuberculosis coinfection294 (19.8)466 (11.5)<.001 Alanine transaminase (µkat/L)0.42 (0.37)0.40 (0.36).04 Hemoglobin (g/L)100 (24), n = 1462110 (20), n = 3948<.001 Creatinine (μmol/L)77 (31), n = 145877 (31), n = 3880.943Study Outcomes Virologic failure159 (10.7)298 (7.3)<.001 Antiretroviral therapy switch256 (17.3)622 (15.3).079 Discontinue308 (20.8)649 (16.0)<.001 No event761 (51.3)2494 (61.4)<.001Values shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.\\nFigure 1.Flow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.\\nBaseline Characteristics and Outcomes of Study Participants\\nValues shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.\\nFlow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.\\nAmong patients in the tenofovir group, 1043 (70.2%) received emtricitabine as their second NRTI, 190 (12.8%) received lamivudine, and 252 (19.8%) switched between emtricitabine and lamivudine during the study period (median [IQR] time to switch was 172 [97] days). The number of virologic failure events did not differ significantly between patients receiving emtricitabine or lamivudine (P = .47); therefore, data were combined in the final analysis irrespective of emtricitabine or lamivudine use.\\nOverall, the median ART adherence rate was 95.3%, which was similar between the zidovudine and tenofovir groups (95.2% vs 95.8%). Among those who experienced virologic failure, more patients had adherence <95% compared with ≥95% (56.4% vs 43.6%; P < .001), but there was no difference in median adherence between those patients with failure in the zidovudine or tenofovir groups (91.9% vs 93.6%; P = .36).\\nPatients in the tenofovir group had a higher rate of virologic failure and discontinuation (Table 1). Figure 2 represents time to virologic failure between the groups. Of the 957 patients with a discontinuation event, 14 (1.5%) died, 849 (88.7%) were lost to follow-up, 92 (9.6%) transferred to another site, and 2 (0.2%) withdrew from the program. These distributions were similar between NRTI groups (P = .50).\\nFigure 2.Kaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.\\nKaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.\\n Time to Virologic Failure After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\\nAll risk factors were measured at baseline.\\nBold results indicate statistically significant results (P value <.05).\\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\\nAfter adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\\nAll risk factors were measured at baseline.\\nBold results indicate statistically significant results (P value <.05).\\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\\n Evaluation of Other Outcome Differences or Dependent Censoring Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.\\nAdditional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.\",\n \"After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\\nAll risk factors were measured at baseline.\\nBold results indicate statistically significant results (P value <.05).\\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\",\n \"Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.\",\n \"The Harvard–APIN program initially identified a high rate of confirmed virologic failure among patients on nevirapine plus tenofovir and emtricitabine or lamivudine compared with other WHO-recommended or alternative first-line regimens [7]. To evaluate the specific impact of the NRTI combination with nevirapine, we focused on patients initiating nevirapine-based ART combined with either zidovudine or tenofovir using Cox proportional hazards, time varying analysis, and IPW modeling. These results indicate that among patients initiating nevirapine, the use of tenofovir carried a 47% greater risk of virologic failure compared with the use of zidovudine; this difference persisted after adjusting for other variables that may influence regimen selection and treatment outcomes (Table 2). Tenofovir benefits were not observed in other outcomes; specifically, the tenofovir group had higher risk for regimen switch and similar risk for discontinuation. Notably, we did not observe significantly higher virologic failure in the tenofovir group after 12 months of ART, suggesting that patients already virologically suppressed on nevirapine plus tenofovir may not be at increased risk for virologic failure. These data have significant clinical implications for adults living in low- and middle-income countries who require an alternative to efavirenz for first-line ART [1].\\nOur results are consistent with those from other evaluations of outcomes among patients who received ART containing nevirapine and tenofovir, thus supporting the belief that NNRTI effectiveness differs by NRTI selection. The DAUFIN study, which evaluated the effectiveness and tolerability of nevirapine given either with zidovudine–lamivudine or tenofovir–lamivudine in antiretroviral-naive patients, was terminated early due to high rates of early virologic failure in the tenofovir arm [6]; 9 of 36 patients receiving tenofovir experienced virologic failure, 8 during the first 12 weeks of the study, compared with only 1 of 35 patients receiving zidovudine. Lapadula et al also reported early virologic failure in patients receiving tenofovir–emtricitabine plus nevirapine compared with ritonavir-boosted atazanavir [5]. In another pilot study that described virologic outcomes among 23 patients receiving once-daily nevirapine plus tenofovir–lamivudine, only 10 patients achieved HIV-RNA <75 copies/mL by week 24 [3]. Turning to large clinical cohorts that evaluated this question, among 10 256 antiretroviral-naive patients initiating either zidovudine or tenofovir in combination with nevirapine in Zambia, 90-day mortality was higher among patients who received tenofovir (aHR, 1.45; 95% CI, 1.03–2.06); however, this difference did not remain after sensitivity analyses, including time-varying analysis [9]. In adjusted models from a large cohort study from southern Africa, patients on nevirapine plus tenofovir–emtricitabine or tenofovir–lamivudine experienced significantly higher rates of mortality but lower rates of loss to follow-up when compared with patients taking efavirenz plus similar NRTIs [21]. Consistent with these data, a metaanalysis of 33 studies comparing virologic efficacy among the WHO-recommended tenofovir-containing first-line ART regimens highlighted poorer outcomes with the nevirapine plus tenofovir-containing regimens [22].\\nThe reason for the poorer performance of nevirapine–tenofovir ART is unclear. One proposed mechanism is an intracellular drug–drug interaction observed in vitro between tenofovir and nevirapine, resulting in lower intracellular concentrations of both antiretrovirals [23]. Differences in lamivudine vs emtricitabine pharmacokinetics have also been proposed. An observational Dutch cohort study identified a higher risk of virologic failure when lamivudine vs emtricitabine was combined with tenofovir and either nevirapine (aHR 2.01; 95% CI, 1.36–2.98) or efavirenz (aHR, 2.35; 95%CI, 1.61–3.42) [24]. Notably, our data did not identify a difference in outcomes among patients in the tenofovir group who received lamivudine or emtricitabine. Our results are consistent with those from a metaanalysis of 12 randomized controlled trials that compared 4913 patients randomized to lamivudine or emtricitabine in addition to various ART combinations and found that the 2 agents are clinically interchangeable [25].\\nOur findings are limited by the study's retrospective cohort design. At the time of data collection, WHO guidelines recommended the use of either nevirapine or efavirenz in combination with zidovudine and lamivudine or with tenofovir and emtricitabine or lamivudine for first-line ART [11]. Regimen selection may have been influenced by factors that we were unable to measure or control for in our analysis, indicated by differences in baseline characteristics (Table 1). To account for some potential biases from dependent censoring, we generated an IPW pooled logistic regression model, and the close correspondence between these models testifies to the robustness of the Cox estimates. Additionally, there may be limitations to measuring adherence based on prescription refill data, particularly as it relates to pill burden. Although all patients received nevirapine twice daily, there was a difference between groups in the total number of pills per day. The zidovudine group received nevirapine plus a twice-daily FDC tablet of zidovudine–lamivudine (4 pills daily) or, beginning early 2007, a twice-daily full regimen FDC tablet (2 pills daily). The tenofovir group received nevirapine plus a once-daily FDC tablet of tenofovir–emtricitabine or tenofovir–lamivudine (3 pills daily). For twice-daily regimens, higher pill burden has previously been associated with lower rates of both adherence and virologic suppression (Spearman correlation, −0.67 and −0.75, respectively; both P < .01) [26]. However, a difference in adherence was not identified between our study groups and did not influence the associated risk of virologic failure related to regimen selection.\\nIn conclusion, ART that includes tenofovir and nevirapine should be initiated with caution. The WHO recommends nevirapine as the alternative for patients who cannot take efavirenz, and nevirapine remains widely used for first-line ART in low- and middle-income countries [1, 27]. These results offer important information to guide selection of the NRTI combination when designing an HIV treatment regimen, indicating a preference for zidovudine over tenofovir when combined with nevirapine. These findings support the need for systematic evaluations of ART combinations before their inclusion in ART guidelines and subsequent widespread implementation.\",\n \"Supplementary materials are available at http://cid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["methods",null,null,null,null,null,null,"results",null,null,"discussion","supplementary-material"],"string":"[\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n \"discussion\",\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["zidovudine","tenofovir","nevirapine","virologic failure","antiretroviral therapy"],"string":"[\n \"zidovudine\",\n \"tenofovir\",\n \"nevirapine\",\n \"virologic failure\",\n \"antiretroviral therapy\"\n]"},"whole_article_text":{"kind":"string","value":"METHODS:\nIn this retrospective cohort study, prospectively collected data from a large clinical cohort in Nigeria were used. Between June 2004 and February 2012, the Harvard T. H. Chan School of Public Health (Harvard) collaborated with the AIDS Prevention Initiative in Nigeria Ltd./Gte. (APIN) to support HIV prevention, care, and treatment to over 160 000 patients, funded by the President's Emergency Plan for AIDS Relief (PEPFAR). Upon program entry, patients underwent a clinical examination and baseline laboratory testing including hematology and chemistry, CD4+ cell count (Cyflow, Partec), HIV-RNA (Cobas Amplicor Monitor Assay, Roche Diagnostics), hepatitis B virus (HBV) surface antigen (Monolisa HBsAg Ultra3, BioRad), and hepatitis C virus (HCV) antibody (DIA.PRO Diagnostic, Bioprobes srl) assessments. Patients eligible for ART, based on guidelines at the time of program entry, initiated standard first-line ART with 1 NNRTI and 2 NRTIs [11, 12]. Prescription refills were obtained monthly; clinical visits and laboratory tests were performed every 6 months, or earlier if necessary. All patient data were maintained in previously described electronic databases [13].\n Ethical Considerations All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\nAll patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\n Patient Selection Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\nAntiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\n Study Definitions Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\nSex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\n Statistical Analyses Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nSubject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\n\nEthical Considerations:\nAll patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\n\nPatient Selection:\nAntiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\n\nStudy Definitions:\nSex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\n\nStatistical Analyses:\nSubject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\n\nTime to Virologic Failure Regression Modeling:\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n\nEvaluation of Dependent Censoring:\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\n\nRESULTS:\nDuring the study period, 6575 patients were identified for inclusion and 5547 (84%) had data available for all variables in the final model—4063 (73.2%) in the zidovudine group and 1484 (26.8%) in the tenofovir group (Figure 1). The most common reason for exclusion was missing HBV or HCV status (11.0%). Baseline participant characteristics are summarized in Table 1. Consistent with clinical considerations for selecting an NRTI, HBV coinfection was more common among those in the tenofovir vs zidovudine group (27.2% vs 14.9%; P < .001), as was a lower median baseline hemoglobin (100 vs 110 g/L, P < .001). Proportionally, more women, patients with HCV coinfection, and those with more advanced HIV disease received tenofovir. Median (interquartile range [IQR]) duration of time to event or censor was slightly longer for the zidovudine group compared with the tenofovir group (392 [244] vs 376 [170] days, P < .001).\nTable 1.Baseline Characteristics and Outcomes of Study ParticipantsTenofovir Group n = 1484Zidovudine Group n = 4063P ValueBaseline Characteristics Age (y)33 (11)32 (11).41 Female gender1255 (84.6)3085 (75.9)<.001 Hepatitis B virus coinfection404 (27.2)604 (14.9)<.001 Hepatitis C virus coinfection144 (9.7)294 (7.2).003 CD4 count (cells/mm3)122 (122)143 (115)<.001 Human immunodeficiency virus-RNA (log10 copies/mL)4.86 (1.1)4.70 (1.2)<.001 World Health Organization Stagen = 1335n = 3489<.001 1236 (17.7)923 (26.5) 2435 (32.6)1042 (29.9) 3494 (37.0)1045 (30.0) 4170 (12.7)479 (13.7) Tuberculosis coinfection294 (19.8)466 (11.5)<.001 Alanine transaminase (µkat/L)0.42 (0.37)0.40 (0.36).04 Hemoglobin (g/L)100 (24), n = 1462110 (20), n = 3948<.001 Creatinine (μmol/L)77 (31), n = 145877 (31), n = 3880.943Study Outcomes Virologic failure159 (10.7)298 (7.3)<.001 Antiretroviral therapy switch256 (17.3)622 (15.3).079 Discontinue308 (20.8)649 (16.0)<.001 No event761 (51.3)2494 (61.4)<.001Values shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.\nFigure 1.Flow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.\nBaseline Characteristics and Outcomes of Study Participants\nValues shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.\nFlow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.\nAmong patients in the tenofovir group, 1043 (70.2%) received emtricitabine as their second NRTI, 190 (12.8%) received lamivudine, and 252 (19.8%) switched between emtricitabine and lamivudine during the study period (median [IQR] time to switch was 172 [97] days). The number of virologic failure events did not differ significantly between patients receiving emtricitabine or lamivudine (P = .47); therefore, data were combined in the final analysis irrespective of emtricitabine or lamivudine use.\nOverall, the median ART adherence rate was 95.3%, which was similar between the zidovudine and tenofovir groups (95.2% vs 95.8%). Among those who experienced virologic failure, more patients had adherence <95% compared with ≥95% (56.4% vs 43.6%; P < .001), but there was no difference in median adherence between those patients with failure in the zidovudine or tenofovir groups (91.9% vs 93.6%; P = .36).\nPatients in the tenofovir group had a higher rate of virologic failure and discontinuation (Table 1). Figure 2 represents time to virologic failure between the groups. Of the 957 patients with a discontinuation event, 14 (1.5%) died, 849 (88.7%) were lost to follow-up, 92 (9.6%) transferred to another site, and 2 (0.2%) withdrew from the program. These distributions were similar between NRTI groups (P = .50).\nFigure 2.Kaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.\nKaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.\n Time to Virologic Failure After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\nAfter adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\n Evaluation of Other Outcome Differences or Dependent Censoring Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.\nAdditional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.\n\nTime to Virologic Failure:\nAfter adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\n\nEvaluation of Other Outcome Differences or Dependent Censoring:\nAdditional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.\n\nDISCUSSION:\nThe Harvard–APIN program initially identified a high rate of confirmed virologic failure among patients on nevirapine plus tenofovir and emtricitabine or lamivudine compared with other WHO-recommended or alternative first-line regimens [7]. To evaluate the specific impact of the NRTI combination with nevirapine, we focused on patients initiating nevirapine-based ART combined with either zidovudine or tenofovir using Cox proportional hazards, time varying analysis, and IPW modeling. These results indicate that among patients initiating nevirapine, the use of tenofovir carried a 47% greater risk of virologic failure compared with the use of zidovudine; this difference persisted after adjusting for other variables that may influence regimen selection and treatment outcomes (Table 2). Tenofovir benefits were not observed in other outcomes; specifically, the tenofovir group had higher risk for regimen switch and similar risk for discontinuation. Notably, we did not observe significantly higher virologic failure in the tenofovir group after 12 months of ART, suggesting that patients already virologically suppressed on nevirapine plus tenofovir may not be at increased risk for virologic failure. These data have significant clinical implications for adults living in low- and middle-income countries who require an alternative to efavirenz for first-line ART [1].\nOur results are consistent with those from other evaluations of outcomes among patients who received ART containing nevirapine and tenofovir, thus supporting the belief that NNRTI effectiveness differs by NRTI selection. The DAUFIN study, which evaluated the effectiveness and tolerability of nevirapine given either with zidovudine–lamivudine or tenofovir–lamivudine in antiretroviral-naive patients, was terminated early due to high rates of early virologic failure in the tenofovir arm [6]; 9 of 36 patients receiving tenofovir experienced virologic failure, 8 during the first 12 weeks of the study, compared with only 1 of 35 patients receiving zidovudine. Lapadula et al also reported early virologic failure in patients receiving tenofovir–emtricitabine plus nevirapine compared with ritonavir-boosted atazanavir [5]. In another pilot study that described virologic outcomes among 23 patients receiving once-daily nevirapine plus tenofovir–lamivudine, only 10 patients achieved HIV-RNA <75 copies/mL by week 24 [3]. Turning to large clinical cohorts that evaluated this question, among 10 256 antiretroviral-naive patients initiating either zidovudine or tenofovir in combination with nevirapine in Zambia, 90-day mortality was higher among patients who received tenofovir (aHR, 1.45; 95% CI, 1.03–2.06); however, this difference did not remain after sensitivity analyses, including time-varying analysis [9]. In adjusted models from a large cohort study from southern Africa, patients on nevirapine plus tenofovir–emtricitabine or tenofovir–lamivudine experienced significantly higher rates of mortality but lower rates of loss to follow-up when compared with patients taking efavirenz plus similar NRTIs [21]. Consistent with these data, a metaanalysis of 33 studies comparing virologic efficacy among the WHO-recommended tenofovir-containing first-line ART regimens highlighted poorer outcomes with the nevirapine plus tenofovir-containing regimens [22].\nThe reason for the poorer performance of nevirapine–tenofovir ART is unclear. One proposed mechanism is an intracellular drug–drug interaction observed in vitro between tenofovir and nevirapine, resulting in lower intracellular concentrations of both antiretrovirals [23]. Differences in lamivudine vs emtricitabine pharmacokinetics have also been proposed. An observational Dutch cohort study identified a higher risk of virologic failure when lamivudine vs emtricitabine was combined with tenofovir and either nevirapine (aHR 2.01; 95% CI, 1.36–2.98) or efavirenz (aHR, 2.35; 95%CI, 1.61–3.42) [24]. Notably, our data did not identify a difference in outcomes among patients in the tenofovir group who received lamivudine or emtricitabine. Our results are consistent with those from a metaanalysis of 12 randomized controlled trials that compared 4913 patients randomized to lamivudine or emtricitabine in addition to various ART combinations and found that the 2 agents are clinically interchangeable [25].\nOur findings are limited by the study's retrospective cohort design. At the time of data collection, WHO guidelines recommended the use of either nevirapine or efavirenz in combination with zidovudine and lamivudine or with tenofovir and emtricitabine or lamivudine for first-line ART [11]. Regimen selection may have been influenced by factors that we were unable to measure or control for in our analysis, indicated by differences in baseline characteristics (Table 1). To account for some potential biases from dependent censoring, we generated an IPW pooled logistic regression model, and the close correspondence between these models testifies to the robustness of the Cox estimates. Additionally, there may be limitations to measuring adherence based on prescription refill data, particularly as it relates to pill burden. Although all patients received nevirapine twice daily, there was a difference between groups in the total number of pills per day. The zidovudine group received nevirapine plus a twice-daily FDC tablet of zidovudine–lamivudine (4 pills daily) or, beginning early 2007, a twice-daily full regimen FDC tablet (2 pills daily). The tenofovir group received nevirapine plus a once-daily FDC tablet of tenofovir–emtricitabine or tenofovir–lamivudine (3 pills daily). For twice-daily regimens, higher pill burden has previously been associated with lower rates of both adherence and virologic suppression (Spearman correlation, −0.67 and −0.75, respectively; both P < .01) [26]. However, a difference in adherence was not identified between our study groups and did not influence the associated risk of virologic failure related to regimen selection.\nIn conclusion, ART that includes tenofovir and nevirapine should be initiated with caution. The WHO recommends nevirapine as the alternative for patients who cannot take efavirenz, and nevirapine remains widely used for first-line ART in low- and middle-income countries [1, 27]. These results offer important information to guide selection of the NRTI combination when designing an HIV treatment regimen, indicating a preference for zidovudine over tenofovir when combined with nevirapine. These findings support the need for systematic evaluations of ART combinations before their inclusion in ART guidelines and subsequent widespread implementation.\n\nSupplementary Data:\nSupplementary materials are available at http://cid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nDespite sparse efficacy data, tenofovir-emtricitabine or tenofovir-lamivudine plus nevirapine is used in many resource-constrained settings.\n\nMethods:\nThis retrospective cohort study included patients initiating nevirapine-based antiretroviral therapy (ART) with either tenofovir-emtricitabine or lamivudine (tenofovir group) or zidovudine-lamivudine (zidovudine group). Clinical, virologic, and immunologic evaluations were performed at baseline and every 6 months. Virologic failure was defined as 2 consecutive human immunodeficiency virus (HIV)-RNA values >1000 copies/mL. Patients were included from ART initiation until time of failure, regimen switch, discontinuation, or last HIV-RNA measurement. Cox proportional hazards regression was used to model factors influencing time to failure. Bias due to dependent censoring was investigated via inverse probability weighted pooled logistic regression.\n\nResults:\nA total of 5547 patients were evaluated; 1484 (26.8%) were in the tenofovir group and 4063 (73.2%) were in the zidovudine group. In the adjusted model, tenofovir regimen (hazard ratio [HR], 1.47; 95% confidence interval [CI], 1.21-1.79) and higher baseline log10 HIV-RNA (HR, 1.15; 95% CI, 1.03-1.28) were associated with virologic failure. Higher baseline log10 CD4+ cell count (HR, 0.50; 95% CI, .40-.63) and increasing age (HR, 0.98; 95% CI, .97-.99) decreased the risk of virologic failure. Inverse probability weighting results were consistent with the primary analysis.\n\nConclusions:\nCompared with zidovudine-lamivudine, the use of tenofovir-lamivudine or emtricitabine in combination with nevirapine was a strong predictor of virologic failure in our cohort, which was not explained by other risk factors or criteria for regimen selection."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":9310,"string":"9,310"},"whole_article_abstract_length":{"kind":"number","value":336,"string":"336"},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["virologic","failure","model","virologic failure","art","cox","time","tenofovir","patients","adherence"],"string":"[\n \"virologic\",\n \"failure\",\n \"model\",\n \"virologic failure\",\n \"art\",\n \"cox\",\n \"time\",\n \"tenofovir\",\n \"patients\",\n \"adherence\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"null"},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"null"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] zidovudine | tenofovir | nevirapine | virologic failure | antiretroviral therapy [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] zidovudine | tenofovir | nevirapine | virologic failure | antiretroviral therapy [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] zidovudine | tenofovir | nevirapine | virologic failure | antiretroviral therapy [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"null"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Anti-Retroviral Agents | Antiretroviral Therapy, Highly Active | CD4 Lymphocyte Count | Cohort Studies | Female | HIV Infections | Humans | Male | Nevirapine | Retrospective Studies | Tenofovir | Treatment Outcome | Viral Load | Young Adult | Zidovudine [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Anti-Retroviral Agents | Antiretroviral Therapy, Highly Active | CD4 Lymphocyte Count | Cohort Studies | Female | HIV Infections | Humans | Male | Nevirapine | Retrospective Studies | Tenofovir | Treatment Outcome | Viral Load | Young Adult | Zidovudine [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Anti-Retroviral Agents | Antiretroviral Therapy, Highly Active | CD4 Lymphocyte Count | Cohort Studies | Female | HIV Infections | Humans | Male | Nevirapine | Retrospective Studies | Tenofovir | Treatment Outcome | Viral Load | Young Adult | Zidovudine [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"null"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"null"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] virologic | failure | model | virologic failure | art | cox | time | tenofovir | patients | adherence [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] virologic | failure | model | virologic failure | art | cox | time | tenofovir | patients | adherence [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] virologic | failure | model | virologic failure | art | cox | time | tenofovir | patients | adherence [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"null"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] art | variables | model | hiv | time | mg | evaluated | cox | covariates | hiv rna [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] failure | virologic | virologic failure | 95 | ci | risk | 001 | virus | group | ahr [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] virologic | art | model | failure | virologic failure | cox | tenofovir | time | variables | patients [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"null"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| every 6 months ||| Virologic | 2 | 1000 ||| ||| Bias [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 5547 | 1484 | 26.8% | 4063 | 73.2% ||| 1.47 | 95% | CI] | 1.21-1.79 | 1.15 | 95% | CI | 1.03-1.28 ||| 0.50 | 95% | CI | 0.98 | 95% | CI ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| every 6 months ||| Virologic | 2 | 1000 ||| ||| Bias ||| 5547 | 1484 | 26.8% | 4063 | 73.2% ||| 1.47 | 95% | CI] | 1.21-1.79 | 1.15 | 95% | CI | 1.03-1.28 ||| 0.50 | 95% | CI | 0.98 | 95% | CI ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":259,"cells":{"title":{"kind":"string","value":"Prognostic Value of Soluble ST2 During Hospitalization for ST-Segment Elevation Myocardial Infarction."},"pmid":{"kind":"string","value":"27139603"},"background_abstract":{"kind":"string","value":"Studying the role of soluble ST2 (sST2) during hospitalization for myocardial infarction (MI) can be helpful for predicting the course of the hospitalization and development of complications."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We included 88 patients with MI (median age, 58 yr). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. On days 1 and 12 after MI, serum sST2 and N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured by ELISA."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"On day 1, the concentrations of sST2 and NT-proBNP increased 2.4- and 4.5-fold, compared with the controls. Measurements on day 12 showed a significant decrease in the sST2 level (P=0.001), whereas the NT-proBNP level did not change. On day 1, the sST2 level in the unfavorable outcome group was 2-fold higher than that in the favorable outcome group and 3.7-fold higher than in the controls. On day 12, the marker level decreased in both groups. On day 1, the NT-proBNP level in the unfavorable outcome group was 6.8-fold higher than in the controls and 1.8-fold higher than in the favorable outcome group. On day 12, the level of NT-proBNP remained elevated in both groups. Determining the levels of both sST2 and NT-proBNP increases their diagnostic significance (odds ratio [OR], 1.92; 95% confidence interval [CI], 1.7-3.2; areas under curve [AUC] 0.89; P=0.004)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The level of sST2 is a more sensitive indicator during MI hospitalization than NT-proBNP."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Area Under Curve","Case-Control Studies","Electrocardiography","Enzyme-Linked Immunosorbent Assay","Female","Hospitalization","Humans","Logistic Models","Male","Middle Aged","Myocardial Infarction","Natriuretic Peptide, Brain","Odds Ratio","Peptide Fragments","Prognosis","Proportional Hazards Models","ROC Curve","Receptors, Somatostatin"],"string":"[\n \"Area Under Curve\",\n \"Case-Control Studies\",\n \"Electrocardiography\",\n \"Enzyme-Linked Immunosorbent Assay\",\n \"Female\",\n \"Hospitalization\",\n \"Humans\",\n \"Logistic Models\",\n \"Male\",\n \"Middle Aged\",\n \"Myocardial Infarction\",\n \"Natriuretic Peptide, Brain\",\n \"Odds Ratio\",\n \"Peptide Fragments\",\n \"Prognosis\",\n \"Proportional Hazards Models\",\n \"ROC Curve\",\n \"Receptors, Somatostatin\"\n]"},"pmcid":{"kind":"string","value":"4855050"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Myocardial infarction (MI) is accompanied by structural and geometric changes in the heart [1]. Early remodeling is characterized by the stretching and thinning of the myocardium and the dilation and spherification of the left ventricle. The acute stretching of the viable myocardium maintains the pumping function despite the decrease in its contracting function [1]. If more than 20% of the left ventricular mass is affected, the compensation is inadequate. A traditional indicator of the stretching of cardiomyocytes and the development of chronic heart failure is the level of the N-terminal pro-brain natriuretic peptide (NT-proBNP) [23]. However, the widespread application of this indicator is limited by its biological variation, as it varies according to sex, age, and body mass index. The levels of NT-proBNP may vary also in other pathologies, such as infections and kidney diseases [4].\nST2 is an early marker of myocardial remodeling, and this understudied growth-stimulating factor is expressed on macrovascular (aortic and coronary artery) and microvascular endothelial cells in the heart in humans [5] and on cardiomyocytes in rats and mice [3] when under biomechanical stress [6]; thus, it is a novel and promising marker. ST2 is a member of the family of interleukin (IL)-1 receptors. The main function of ST2, which potentiates IL-33, is to exert antihypertrophic and antifibrosing effects on cardiomyocytes that are under biomechanical stretching conditions [78]. However, an acute increase in the ST2 level has been observed when damage is accompanied by the inhibition of IL-33 and its favorable antihypertrophic effects. Studying the role of ST2 during hospitalization for MI can be helpful for predicting the course of the hospitalization and development of complications [391011]. The aim of this study was to determine the level of soluble ST2 (sST2) and its correlation with the level of NT-proBNP and with the clinical course of MI during hospitalization."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":" 1. Study population For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\nFor this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\n 2. Assays Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\nSerum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\n 3. Statistical analysis Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.\nStatistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"On day 1 of hospitalization for MI, the levels of sST2 and NT-proBNP increased by 2.4-fold and 4.5-fold, respectively, compared with the control group [44.75 (24.90; 93.56) ng/mL, P=0.002; 18.81 (15.12; 21.03) ng/mL] [36.84 (24.09; 89.26) fmol/mL, P=0.000; 8.23 (5.61; 11.12) fmol/mL)].\nBy day 12, the sST2 level significantly decreased by 2.5-fold [17.82 (15.30; 23.25 ng/mL, P=0.001], whereas the changes in the NT-proBNP level were not significant (Fig. 1).\nThe results of the correlation analyses indicated a moderate correlation between the levels of sST2 and NT-proBNP in both groups on days 1 (R=0.50, P=0.001) and 12 of MI hospitalization (R=0.55, P=0. 0002).\nAccording to experimental data, sST2 in animals is expressed only in cardiomyocytes during myocardial injury; nevertheless, we were interested in the correlation analysis between troponin T (the classic marker of cardiomyocyte damage) and sST2 concentrations during the MI hospitalization period. The results of the correlation analyses indicated a direct correlation between the levels of sST2 and troponin T in both groups at the time of admission (R=0.65, P=0.002).\nA comparative analysis of the levels of sST2 and NT-proBNP in both groups (favorable (n=58) and unfavorable (n=30) outcome) was performed, and the results are shown in Table 3. The level of sST2 in the unfavorable outcome group on day 1 was 2-fold higher than that in favorable outcome group. The sST2 levels increased by 1.9- and 3.7-fold in the favorable and unfavorable outcome groups, respectively, compared with those in the control group.\nOn day 12 after MI onset, the sST2 level was significantly lower (P=0.011) in subjects in both groups compared with the level in the control group.\nIn contrast to sST2, the level of NT-proBNP increased equally in both the favorable and unfavorable outcome groups up to day 12 of the study. The highest increase in the NT-proBNP level (6.8-fold) was observed in patients in the unfavorable outcome group on day 1.\nA level of sST2 higher than 35 ng/mL is considered an indicator of adverse outcome development in cardiovascular pathology [1013]. Thus, in the next stage of our study, we analyzed the differences in the clinical and anamnestic characteristics of the subjects by categorizing the sST2 level as lower (Group 1) or higher (Group 2) than the critical level of 35 ng/mL (Table 2). In Group 2 (sST2 concentration above 35 ng/mL), according to the clinical and anamnestic characteristics, complications [MI (44.6%) and diabetes mellitus (23%)] during hospitalization were more common. In Group 1 (sST2 level lower than 35 ng/mL), the percentage of adverse events and complications during hospitalization was significantly lower (P=0.021).\nConversely, analysis of individual patient data indicated that five patients (15.6%) with a poor prognosis had an sST2 concentration lower than 35 ng/mL, whereas 31 (55.4%) patients with a favorable prognosis had an sST2 level higher than 35 ng/mL.\nThe results of the evaluation of the NT-proBNP level as a function of sST2 are shown in Table 4. On day 1, in patients with sST2 concentrations above 35 ng/mL, the concentration of NT-proBNP increased by 6.7-fold compared with the controls. At the same time, in patients with a moderate increase in the concentration of sST2 (less than 35 ng/mL), the concentration of NT-proBNP also increased, although to a lesser degree (4.6-fold compared with the controls). At day 12, the level of NT-proBNP remained elevated in both groups and did not differ significantly between the two (Table 4).\nLogistic regression analysis showed that an increase in the concentration of sST2 increased the risk of complications during the hospitalization by 1.7-fold times (OR, 1.7; 95% CI, 1.6-2.8; areas under curve [AUC]=0.78; P=0.003), with sensitivity of 76.9% and a specificity of 69.4%. At the same time, the increase in the level of NT-proBNP was accompanied only by a 1.2-fold increase in adverse outcomes (OR, 1.2; 95% CI, 1.1-1.6; AUC=0.69; P=0.034), without affecting the high diagnostic sensitivity (69.6%) and specificity (65.3%).\nDetermining the level of sST2 in combination with NT-proBNP increases their diagnostic significance (OR, 1.92; 95% CI, 1.7-3.2). These indicators, when used together and measured at the early stages of MI, increased the quality of the model, with sensitivity of 81%, specificity of 72%, and AUC of 0.86."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["1. Study population","2. Assays","3. Statistical analysis"],"string":"[\n \"1. Study population\",\n \"2. Assays\",\n \"3. Statistical analysis\"\n]"},"other_sections_texts":{"kind":"list like","value":["For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.","Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.","Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant."],"string":"[\n \"For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \\\"Research Institute for Complex Issues of Cardiovascular Disease\\\" and was developed in accordance with the WMA Declaration of Helsinki \\\"Ethical principles for medical research involving human subjects\\\" (amended in 2000) and \\\"Rules for clinical practice in the Russian Federation\\\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\",\n \"Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\",\n \"Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null],"string":"[\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","1. Study population","2. Assays","3. Statistical analysis","RESULTS","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"1. Study population\",\n \"2. Assays\",\n \"3. Statistical analysis\",\n \"RESULTS\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Myocardial infarction (MI) is accompanied by structural and geometric changes in the heart [1]. Early remodeling is characterized by the stretching and thinning of the myocardium and the dilation and spherification of the left ventricle. The acute stretching of the viable myocardium maintains the pumping function despite the decrease in its contracting function [1]. If more than 20% of the left ventricular mass is affected, the compensation is inadequate. A traditional indicator of the stretching of cardiomyocytes and the development of chronic heart failure is the level of the N-terminal pro-brain natriuretic peptide (NT-proBNP) [23]. However, the widespread application of this indicator is limited by its biological variation, as it varies according to sex, age, and body mass index. The levels of NT-proBNP may vary also in other pathologies, such as infections and kidney diseases [4].\nST2 is an early marker of myocardial remodeling, and this understudied growth-stimulating factor is expressed on macrovascular (aortic and coronary artery) and microvascular endothelial cells in the heart in humans [5] and on cardiomyocytes in rats and mice [3] when under biomechanical stress [6]; thus, it is a novel and promising marker. ST2 is a member of the family of interleukin (IL)-1 receptors. The main function of ST2, which potentiates IL-33, is to exert antihypertrophic and antifibrosing effects on cardiomyocytes that are under biomechanical stretching conditions [78]. However, an acute increase in the ST2 level has been observed when damage is accompanied by the inhibition of IL-33 and its favorable antihypertrophic effects. Studying the role of ST2 during hospitalization for MI can be helpful for predicting the course of the hospitalization and development of complications [391011]. The aim of this study was to determine the level of soluble ST2 (sST2) and its correlation with the level of NT-proBNP and with the clinical course of MI during hospitalization."," 1. Study population For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\nFor this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\n 2. Assays Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\nSerum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\n 3. Statistical analysis Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.\nStatistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.","For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.","Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.","Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.","On day 1 of hospitalization for MI, the levels of sST2 and NT-proBNP increased by 2.4-fold and 4.5-fold, respectively, compared with the control group [44.75 (24.90; 93.56) ng/mL, P=0.002; 18.81 (15.12; 21.03) ng/mL] [36.84 (24.09; 89.26) fmol/mL, P=0.000; 8.23 (5.61; 11.12) fmol/mL)].\nBy day 12, the sST2 level significantly decreased by 2.5-fold [17.82 (15.30; 23.25 ng/mL, P=0.001], whereas the changes in the NT-proBNP level were not significant (Fig. 1).\nThe results of the correlation analyses indicated a moderate correlation between the levels of sST2 and NT-proBNP in both groups on days 1 (R=0.50, P=0.001) and 12 of MI hospitalization (R=0.55, P=0. 0002).\nAccording to experimental data, sST2 in animals is expressed only in cardiomyocytes during myocardial injury; nevertheless, we were interested in the correlation analysis between troponin T (the classic marker of cardiomyocyte damage) and sST2 concentrations during the MI hospitalization period. The results of the correlation analyses indicated a direct correlation between the levels of sST2 and troponin T in both groups at the time of admission (R=0.65, P=0.002).\nA comparative analysis of the levels of sST2 and NT-proBNP in both groups (favorable (n=58) and unfavorable (n=30) outcome) was performed, and the results are shown in Table 3. The level of sST2 in the unfavorable outcome group on day 1 was 2-fold higher than that in favorable outcome group. The sST2 levels increased by 1.9- and 3.7-fold in the favorable and unfavorable outcome groups, respectively, compared with those in the control group.\nOn day 12 after MI onset, the sST2 level was significantly lower (P=0.011) in subjects in both groups compared with the level in the control group.\nIn contrast to sST2, the level of NT-proBNP increased equally in both the favorable and unfavorable outcome groups up to day 12 of the study. The highest increase in the NT-proBNP level (6.8-fold) was observed in patients in the unfavorable outcome group on day 1.\nA level of sST2 higher than 35 ng/mL is considered an indicator of adverse outcome development in cardiovascular pathology [1013]. Thus, in the next stage of our study, we analyzed the differences in the clinical and anamnestic characteristics of the subjects by categorizing the sST2 level as lower (Group 1) or higher (Group 2) than the critical level of 35 ng/mL (Table 2). In Group 2 (sST2 concentration above 35 ng/mL), according to the clinical and anamnestic characteristics, complications [MI (44.6%) and diabetes mellitus (23%)] during hospitalization were more common. In Group 1 (sST2 level lower than 35 ng/mL), the percentage of adverse events and complications during hospitalization was significantly lower (P=0.021).\nConversely, analysis of individual patient data indicated that five patients (15.6%) with a poor prognosis had an sST2 concentration lower than 35 ng/mL, whereas 31 (55.4%) patients with a favorable prognosis had an sST2 level higher than 35 ng/mL.\nThe results of the evaluation of the NT-proBNP level as a function of sST2 are shown in Table 4. On day 1, in patients with sST2 concentrations above 35 ng/mL, the concentration of NT-proBNP increased by 6.7-fold compared with the controls. At the same time, in patients with a moderate increase in the concentration of sST2 (less than 35 ng/mL), the concentration of NT-proBNP also increased, although to a lesser degree (4.6-fold compared with the controls). At day 12, the level of NT-proBNP remained elevated in both groups and did not differ significantly between the two (Table 4).\nLogistic regression analysis showed that an increase in the concentration of sST2 increased the risk of complications during the hospitalization by 1.7-fold times (OR, 1.7; 95% CI, 1.6-2.8; areas under curve [AUC]=0.78; P=0.003), with sensitivity of 76.9% and a specificity of 69.4%. At the same time, the increase in the level of NT-proBNP was accompanied only by a 1.2-fold increase in adverse outcomes (OR, 1.2; 95% CI, 1.1-1.6; AUC=0.69; P=0.034), without affecting the high diagnostic sensitivity (69.6%) and specificity (65.3%).\nDetermining the level of sST2 in combination with NT-proBNP increases their diagnostic significance (OR, 1.92; 95% CI, 1.7-3.2). These indicators, when used together and measured at the early stages of MI, increased the quality of the model, with sensitivity of 81%, specificity of 72%, and AUC of 0.86.","MI is accompanied by the mechanical deformation of cardiomyocytes, which may undergo adaptive and maladaptive changes, leading to chronic heart failure [1]. In response to increased wall tension of the heart ventricles, the intracardiac pressure increases, resulting in increased cardiomyocyte volume and the synthesis of factors such as NT-proBNP and sST2, which function to increase myocardial performance [3].\nOur study showed that on day 1 after MI, the NT-proBNP concentration increased 4.5-fold (Fig. 1) and remained elevated up to day 12 of the study. It was shown in BNP transgenic mice that following artificially induced MI, the infarcted area increased by about 5-fold during the 48 hr following MI onset and remained at an increased level over the next three to four weeks [1415]. When the disease course was unfavorable, the NT-proBNP concentration increased even more, to 6.8-fold compared with the control values. In addition to the mechanical stretching of the ventricles, there may be other mechanisms that stimulate the production of NT-proBNP, such as ischemia in various locations, arrhythmias, myocardial hypertrophy, and endothelial dysfunction [16]. However, in this study, the NT-proBNP level had a low diagnostic sensitivity and specificity as an indicator of an unfavorable course following MI, with its increase indicating only a 1.2-fold increase in the risk for complications.\nThe stimulating growth factor sST2 had a higher sensitivity for the development of a poor prognosis. With its increase on day 1, there was a 1.7-fold increase in the risk of an unfavorable outcome following MI. Compared with the controls, the sST2 level increased by 1.9-fold in cases with a favorable course of MI and increased 3.7-fold in cases of unfavorable outcomes (Table 2). Elevated levels of sST2 have been associated with an increase in the synthesis of sST2 in cardiac myocytes and fibroblasts due to biomechanical stress [31718].\nST2 is a member of the superfamily of IL-1 receptors. It exists in two forms: a transmembrane receptor (ST2L) and soluble receptor-trap (sST2). The ST2 ligand is IL-33, which contributes to the process reduction of the fibrosis and hypertrophy of tissues under mechanical loads [19]. sST2 acts as a decoy receptor by binding free IL-33 and preventing its signaling through ST2L [2021]. A moderate increase in the concentration of sST2 probably exerts a protective effect, which manifests itself in patients with a favorable course of MI [39]. The transmembrane form protects the myocardium from overcharging, whereas the soluble form of ST2 prevents this protective mechanism, binds to IL-33, and blocks its cardioprotective effect [320]. Perhaps the increase in the concentration of sST2 in cases of unfavorable outcomes is associated with an increased content of the soluble form of the marker following release by damaged cardiomyocytes.\nBoth sST2 and troponin levels have been measured in several other studies. In the study by Mueller et al. [22] in 2015, in contrast to our study, no correlation between sST2 and troponin levels was found. This difference can be explained by the fact that we measured biomarkers in patients in the acute phase of MI, whereas the patients in the study by Mueller et al. [22] had heart failure.\nIn the present study, by day 12 following MI, sST2 concentrations decreased to the level of control values, thus making it difficult to distinguish between the favorable and unfavorable outcome groups. The dynamics of the sST2 changes are somewhat similar to those of C-reactive protein (CRP). CRP levels increased acutely during acute MI; however, this marker tended to decrease by day 12 after MI onset [23]. This similarity indicates their common inflammatory nature. These results are consistent with those reported in the study by Weinberg et al. [24], which was performed on an experimental MI model (in vivo) by using C57/BL6J mice. After ligating the coronary artery, the maximal transcriptional induction of sST2 in cardiac myocytes occurred within 2 hr, was maintained for 9 hr, and decreased after 15 hr.\nStudies have shown that the sST2 threshold in patients with chronic heart failure is 35 ng/mL. Above this value, the risk of death increases dramatically within one year of the event [91018]. Kohli et al. [10] reported similar results, showing that high levels of sST2 (>35 ng/mL) in patients suffering from acute coronary syndrome predicted a 3-fold higher risk of cardiovascular death and heart failure within 30 days and one year. It is of particular interest to study such patterns in a cohort of patients with MI. In our study, the adverse outcomes during the early period of MI were not associated with sST2 levels above 35 ng/mL, as there were complications in patients with levels below (15.5%) and above (55.4%) the threshold. These results were probably affected not only by the level of sST2 but also by the presence of other factors, specifically the increase in NT-proBNP. Indeed, the use of these two markers together significantly increases their sensitivity and specificity of predicting the risk of unfavorable MI outcomes [25].\nOur findings are consistent with the study by Sabatine et al. [18], in which unfavorable outcomes were observed with high levels of both markers (risk of death or developing heart failure was 6.5-fold higher) during the 30-day observation period. Using the levels of NT-proBNP and sST2 to construct the receiver operating characteristics curve, the identification of combined cardiovascular mortality or heart failure significantly improved to 0.78 (95% CI, 0.74-0.83; P=0.0025).\nIn conclusion, the concentration of sST2 is a more sensitive indicator of the course of hospitalization for MI than the traditional indicator of the NT-proBNP concentration. Increased concentrations of sST2 on day 1 after MI were followed by an unfavorable hospitalization course, including progressive angina, arrhythmias, MI, and recurrent symptomatic AHF (Killip class II-IV)."],"string":"[\n \"Myocardial infarction (MI) is accompanied by structural and geometric changes in the heart [1]. Early remodeling is characterized by the stretching and thinning of the myocardium and the dilation and spherification of the left ventricle. The acute stretching of the viable myocardium maintains the pumping function despite the decrease in its contracting function [1]. If more than 20% of the left ventricular mass is affected, the compensation is inadequate. A traditional indicator of the stretching of cardiomyocytes and the development of chronic heart failure is the level of the N-terminal pro-brain natriuretic peptide (NT-proBNP) [23]. However, the widespread application of this indicator is limited by its biological variation, as it varies according to sex, age, and body mass index. The levels of NT-proBNP may vary also in other pathologies, such as infections and kidney diseases [4].\\nST2 is an early marker of myocardial remodeling, and this understudied growth-stimulating factor is expressed on macrovascular (aortic and coronary artery) and microvascular endothelial cells in the heart in humans [5] and on cardiomyocytes in rats and mice [3] when under biomechanical stress [6]; thus, it is a novel and promising marker. ST2 is a member of the family of interleukin (IL)-1 receptors. The main function of ST2, which potentiates IL-33, is to exert antihypertrophic and antifibrosing effects on cardiomyocytes that are under biomechanical stretching conditions [78]. However, an acute increase in the ST2 level has been observed when damage is accompanied by the inhibition of IL-33 and its favorable antihypertrophic effects. Studying the role of ST2 during hospitalization for MI can be helpful for predicting the course of the hospitalization and development of complications [391011]. The aim of this study was to determine the level of soluble ST2 (sST2) and its correlation with the level of NT-proBNP and with the clinical course of MI during hospitalization.\",\n \" 1. Study population For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \\\"Research Institute for Complex Issues of Cardiovascular Disease\\\" and was developed in accordance with the WMA Declaration of Helsinki \\\"Ethical principles for medical research involving human subjects\\\" (amended in 2000) and \\\"Rules for clinical practice in the Russian Federation\\\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\\nFor this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \\\"Research Institute for Complex Issues of Cardiovascular Disease\\\" and was developed in accordance with the WMA Declaration of Helsinki \\\"Ethical principles for medical research involving human subjects\\\" (amended in 2000) and \\\"Rules for clinical practice in the Russian Federation\\\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\\n 2. Assays Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\\nSerum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\\n 3. Statistical analysis Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.\\nStatistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.\",\n \"For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \\\"Research Institute for Complex Issues of Cardiovascular Disease\\\" and was developed in accordance with the WMA Declaration of Helsinki \\\"Ethical principles for medical research involving human subjects\\\" (amended in 2000) and \\\"Rules for clinical practice in the Russian Federation\\\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\",\n \"Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\",\n \"Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.\",\n \"On day 1 of hospitalization for MI, the levels of sST2 and NT-proBNP increased by 2.4-fold and 4.5-fold, respectively, compared with the control group [44.75 (24.90; 93.56) ng/mL, P=0.002; 18.81 (15.12; 21.03) ng/mL] [36.84 (24.09; 89.26) fmol/mL, P=0.000; 8.23 (5.61; 11.12) fmol/mL)].\\nBy day 12, the sST2 level significantly decreased by 2.5-fold [17.82 (15.30; 23.25 ng/mL, P=0.001], whereas the changes in the NT-proBNP level were not significant (Fig. 1).\\nThe results of the correlation analyses indicated a moderate correlation between the levels of sST2 and NT-proBNP in both groups on days 1 (R=0.50, P=0.001) and 12 of MI hospitalization (R=0.55, P=0. 0002).\\nAccording to experimental data, sST2 in animals is expressed only in cardiomyocytes during myocardial injury; nevertheless, we were interested in the correlation analysis between troponin T (the classic marker of cardiomyocyte damage) and sST2 concentrations during the MI hospitalization period. The results of the correlation analyses indicated a direct correlation between the levels of sST2 and troponin T in both groups at the time of admission (R=0.65, P=0.002).\\nA comparative analysis of the levels of sST2 and NT-proBNP in both groups (favorable (n=58) and unfavorable (n=30) outcome) was performed, and the results are shown in Table 3. The level of sST2 in the unfavorable outcome group on day 1 was 2-fold higher than that in favorable outcome group. The sST2 levels increased by 1.9- and 3.7-fold in the favorable and unfavorable outcome groups, respectively, compared with those in the control group.\\nOn day 12 after MI onset, the sST2 level was significantly lower (P=0.011) in subjects in both groups compared with the level in the control group.\\nIn contrast to sST2, the level of NT-proBNP increased equally in both the favorable and unfavorable outcome groups up to day 12 of the study. The highest increase in the NT-proBNP level (6.8-fold) was observed in patients in the unfavorable outcome group on day 1.\\nA level of sST2 higher than 35 ng/mL is considered an indicator of adverse outcome development in cardiovascular pathology [1013]. Thus, in the next stage of our study, we analyzed the differences in the clinical and anamnestic characteristics of the subjects by categorizing the sST2 level as lower (Group 1) or higher (Group 2) than the critical level of 35 ng/mL (Table 2). In Group 2 (sST2 concentration above 35 ng/mL), according to the clinical and anamnestic characteristics, complications [MI (44.6%) and diabetes mellitus (23%)] during hospitalization were more common. In Group 1 (sST2 level lower than 35 ng/mL), the percentage of adverse events and complications during hospitalization was significantly lower (P=0.021).\\nConversely, analysis of individual patient data indicated that five patients (15.6%) with a poor prognosis had an sST2 concentration lower than 35 ng/mL, whereas 31 (55.4%) patients with a favorable prognosis had an sST2 level higher than 35 ng/mL.\\nThe results of the evaluation of the NT-proBNP level as a function of sST2 are shown in Table 4. On day 1, in patients with sST2 concentrations above 35 ng/mL, the concentration of NT-proBNP increased by 6.7-fold compared with the controls. At the same time, in patients with a moderate increase in the concentration of sST2 (less than 35 ng/mL), the concentration of NT-proBNP also increased, although to a lesser degree (4.6-fold compared with the controls). At day 12, the level of NT-proBNP remained elevated in both groups and did not differ significantly between the two (Table 4).\\nLogistic regression analysis showed that an increase in the concentration of sST2 increased the risk of complications during the hospitalization by 1.7-fold times (OR, 1.7; 95% CI, 1.6-2.8; areas under curve [AUC]=0.78; P=0.003), with sensitivity of 76.9% and a specificity of 69.4%. At the same time, the increase in the level of NT-proBNP was accompanied only by a 1.2-fold increase in adverse outcomes (OR, 1.2; 95% CI, 1.1-1.6; AUC=0.69; P=0.034), without affecting the high diagnostic sensitivity (69.6%) and specificity (65.3%).\\nDetermining the level of sST2 in combination with NT-proBNP increases their diagnostic significance (OR, 1.92; 95% CI, 1.7-3.2). These indicators, when used together and measured at the early stages of MI, increased the quality of the model, with sensitivity of 81%, specificity of 72%, and AUC of 0.86.\",\n \"MI is accompanied by the mechanical deformation of cardiomyocytes, which may undergo adaptive and maladaptive changes, leading to chronic heart failure [1]. In response to increased wall tension of the heart ventricles, the intracardiac pressure increases, resulting in increased cardiomyocyte volume and the synthesis of factors such as NT-proBNP and sST2, which function to increase myocardial performance [3].\\nOur study showed that on day 1 after MI, the NT-proBNP concentration increased 4.5-fold (Fig. 1) and remained elevated up to day 12 of the study. It was shown in BNP transgenic mice that following artificially induced MI, the infarcted area increased by about 5-fold during the 48 hr following MI onset and remained at an increased level over the next three to four weeks [1415]. When the disease course was unfavorable, the NT-proBNP concentration increased even more, to 6.8-fold compared with the control values. In addition to the mechanical stretching of the ventricles, there may be other mechanisms that stimulate the production of NT-proBNP, such as ischemia in various locations, arrhythmias, myocardial hypertrophy, and endothelial dysfunction [16]. However, in this study, the NT-proBNP level had a low diagnostic sensitivity and specificity as an indicator of an unfavorable course following MI, with its increase indicating only a 1.2-fold increase in the risk for complications.\\nThe stimulating growth factor sST2 had a higher sensitivity for the development of a poor prognosis. With its increase on day 1, there was a 1.7-fold increase in the risk of an unfavorable outcome following MI. Compared with the controls, the sST2 level increased by 1.9-fold in cases with a favorable course of MI and increased 3.7-fold in cases of unfavorable outcomes (Table 2). Elevated levels of sST2 have been associated with an increase in the synthesis of sST2 in cardiac myocytes and fibroblasts due to biomechanical stress [31718].\\nST2 is a member of the superfamily of IL-1 receptors. It exists in two forms: a transmembrane receptor (ST2L) and soluble receptor-trap (sST2). The ST2 ligand is IL-33, which contributes to the process reduction of the fibrosis and hypertrophy of tissues under mechanical loads [19]. sST2 acts as a decoy receptor by binding free IL-33 and preventing its signaling through ST2L [2021]. A moderate increase in the concentration of sST2 probably exerts a protective effect, which manifests itself in patients with a favorable course of MI [39]. The transmembrane form protects the myocardium from overcharging, whereas the soluble form of ST2 prevents this protective mechanism, binds to IL-33, and blocks its cardioprotective effect [320]. Perhaps the increase in the concentration of sST2 in cases of unfavorable outcomes is associated with an increased content of the soluble form of the marker following release by damaged cardiomyocytes.\\nBoth sST2 and troponin levels have been measured in several other studies. In the study by Mueller et al. [22] in 2015, in contrast to our study, no correlation between sST2 and troponin levels was found. This difference can be explained by the fact that we measured biomarkers in patients in the acute phase of MI, whereas the patients in the study by Mueller et al. [22] had heart failure.\\nIn the present study, by day 12 following MI, sST2 concentrations decreased to the level of control values, thus making it difficult to distinguish between the favorable and unfavorable outcome groups. The dynamics of the sST2 changes are somewhat similar to those of C-reactive protein (CRP). CRP levels increased acutely during acute MI; however, this marker tended to decrease by day 12 after MI onset [23]. This similarity indicates their common inflammatory nature. These results are consistent with those reported in the study by Weinberg et al. [24], which was performed on an experimental MI model (in vivo) by using C57/BL6J mice. After ligating the coronary artery, the maximal transcriptional induction of sST2 in cardiac myocytes occurred within 2 hr, was maintained for 9 hr, and decreased after 15 hr.\\nStudies have shown that the sST2 threshold in patients with chronic heart failure is 35 ng/mL. Above this value, the risk of death increases dramatically within one year of the event [91018]. Kohli et al. [10] reported similar results, showing that high levels of sST2 (>35 ng/mL) in patients suffering from acute coronary syndrome predicted a 3-fold higher risk of cardiovascular death and heart failure within 30 days and one year. It is of particular interest to study such patterns in a cohort of patients with MI. In our study, the adverse outcomes during the early period of MI were not associated with sST2 levels above 35 ng/mL, as there were complications in patients with levels below (15.5%) and above (55.4%) the threshold. These results were probably affected not only by the level of sST2 but also by the presence of other factors, specifically the increase in NT-proBNP. Indeed, the use of these two markers together significantly increases their sensitivity and specificity of predicting the risk of unfavorable MI outcomes [25].\\nOur findings are consistent with the study by Sabatine et al. [18], in which unfavorable outcomes were observed with high levels of both markers (risk of death or developing heart failure was 6.5-fold higher) during the 30-day observation period. Using the levels of NT-proBNP and sST2 to construct the receiver operating characteristics curve, the identification of combined cardiovascular mortality or heart failure significantly improved to 0.78 (95% CI, 0.74-0.83; P=0.0025).\\nIn conclusion, the concentration of sST2 is a more sensitive indicator of the course of hospitalization for MI than the traditional indicator of the NT-proBNP concentration. Increased concentrations of sST2 on day 1 after MI were followed by an unfavorable hospitalization course, including progressive angina, arrhythmias, MI, and recurrent symptomatic AHF (Killip class II-IV).\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,"results","discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Myocardial infarction","NT-proBNP","sST2"],"string":"[\n \"Myocardial infarction\",\n \"NT-proBNP\",\n \"sST2\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nMyocardial infarction (MI) is accompanied by structural and geometric changes in the heart [1]. Early remodeling is characterized by the stretching and thinning of the myocardium and the dilation and spherification of the left ventricle. The acute stretching of the viable myocardium maintains the pumping function despite the decrease in its contracting function [1]. If more than 20% of the left ventricular mass is affected, the compensation is inadequate. A traditional indicator of the stretching of cardiomyocytes and the development of chronic heart failure is the level of the N-terminal pro-brain natriuretic peptide (NT-proBNP) [23]. However, the widespread application of this indicator is limited by its biological variation, as it varies according to sex, age, and body mass index. The levels of NT-proBNP may vary also in other pathologies, such as infections and kidney diseases [4].\nST2 is an early marker of myocardial remodeling, and this understudied growth-stimulating factor is expressed on macrovascular (aortic and coronary artery) and microvascular endothelial cells in the heart in humans [5] and on cardiomyocytes in rats and mice [3] when under biomechanical stress [6]; thus, it is a novel and promising marker. ST2 is a member of the family of interleukin (IL)-1 receptors. The main function of ST2, which potentiates IL-33, is to exert antihypertrophic and antifibrosing effects on cardiomyocytes that are under biomechanical stretching conditions [78]. However, an acute increase in the ST2 level has been observed when damage is accompanied by the inhibition of IL-33 and its favorable antihypertrophic effects. Studying the role of ST2 during hospitalization for MI can be helpful for predicting the course of the hospitalization and development of complications [391011]. The aim of this study was to determine the level of soluble ST2 (sST2) and its correlation with the level of NT-proBNP and with the clinical course of MI during hospitalization.\n\nMETHODS:\n 1. Study population For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\nFor this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\n 2. Assays Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\nSerum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\n 3. Statistical analysis Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.\nStatistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.\n\n1. Study population:\nFor this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\n\n2. Assays:\nSerum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\n\n3. Statistical analysis:\nStatistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.\n\nRESULTS:\nOn day 1 of hospitalization for MI, the levels of sST2 and NT-proBNP increased by 2.4-fold and 4.5-fold, respectively, compared with the control group [44.75 (24.90; 93.56) ng/mL, P=0.002; 18.81 (15.12; 21.03) ng/mL] [36.84 (24.09; 89.26) fmol/mL, P=0.000; 8.23 (5.61; 11.12) fmol/mL)].\nBy day 12, the sST2 level significantly decreased by 2.5-fold [17.82 (15.30; 23.25 ng/mL, P=0.001], whereas the changes in the NT-proBNP level were not significant (Fig. 1).\nThe results of the correlation analyses indicated a moderate correlation between the levels of sST2 and NT-proBNP in both groups on days 1 (R=0.50, P=0.001) and 12 of MI hospitalization (R=0.55, P=0. 0002).\nAccording to experimental data, sST2 in animals is expressed only in cardiomyocytes during myocardial injury; nevertheless, we were interested in the correlation analysis between troponin T (the classic marker of cardiomyocyte damage) and sST2 concentrations during the MI hospitalization period. The results of the correlation analyses indicated a direct correlation between the levels of sST2 and troponin T in both groups at the time of admission (R=0.65, P=0.002).\nA comparative analysis of the levels of sST2 and NT-proBNP in both groups (favorable (n=58) and unfavorable (n=30) outcome) was performed, and the results are shown in Table 3. The level of sST2 in the unfavorable outcome group on day 1 was 2-fold higher than that in favorable outcome group. The sST2 levels increased by 1.9- and 3.7-fold in the favorable and unfavorable outcome groups, respectively, compared with those in the control group.\nOn day 12 after MI onset, the sST2 level was significantly lower (P=0.011) in subjects in both groups compared with the level in the control group.\nIn contrast to sST2, the level of NT-proBNP increased equally in both the favorable and unfavorable outcome groups up to day 12 of the study. The highest increase in the NT-proBNP level (6.8-fold) was observed in patients in the unfavorable outcome group on day 1.\nA level of sST2 higher than 35 ng/mL is considered an indicator of adverse outcome development in cardiovascular pathology [1013]. Thus, in the next stage of our study, we analyzed the differences in the clinical and anamnestic characteristics of the subjects by categorizing the sST2 level as lower (Group 1) or higher (Group 2) than the critical level of 35 ng/mL (Table 2). In Group 2 (sST2 concentration above 35 ng/mL), according to the clinical and anamnestic characteristics, complications [MI (44.6%) and diabetes mellitus (23%)] during hospitalization were more common. In Group 1 (sST2 level lower than 35 ng/mL), the percentage of adverse events and complications during hospitalization was significantly lower (P=0.021).\nConversely, analysis of individual patient data indicated that five patients (15.6%) with a poor prognosis had an sST2 concentration lower than 35 ng/mL, whereas 31 (55.4%) patients with a favorable prognosis had an sST2 level higher than 35 ng/mL.\nThe results of the evaluation of the NT-proBNP level as a function of sST2 are shown in Table 4. On day 1, in patients with sST2 concentrations above 35 ng/mL, the concentration of NT-proBNP increased by 6.7-fold compared with the controls. At the same time, in patients with a moderate increase in the concentration of sST2 (less than 35 ng/mL), the concentration of NT-proBNP also increased, although to a lesser degree (4.6-fold compared with the controls). At day 12, the level of NT-proBNP remained elevated in both groups and did not differ significantly between the two (Table 4).\nLogistic regression analysis showed that an increase in the concentration of sST2 increased the risk of complications during the hospitalization by 1.7-fold times (OR, 1.7; 95% CI, 1.6-2.8; areas under curve [AUC]=0.78; P=0.003), with sensitivity of 76.9% and a specificity of 69.4%. At the same time, the increase in the level of NT-proBNP was accompanied only by a 1.2-fold increase in adverse outcomes (OR, 1.2; 95% CI, 1.1-1.6; AUC=0.69; P=0.034), without affecting the high diagnostic sensitivity (69.6%) and specificity (65.3%).\nDetermining the level of sST2 in combination with NT-proBNP increases their diagnostic significance (OR, 1.92; 95% CI, 1.7-3.2). These indicators, when used together and measured at the early stages of MI, increased the quality of the model, with sensitivity of 81%, specificity of 72%, and AUC of 0.86.\n\nDISCUSSION:\nMI is accompanied by the mechanical deformation of cardiomyocytes, which may undergo adaptive and maladaptive changes, leading to chronic heart failure [1]. In response to increased wall tension of the heart ventricles, the intracardiac pressure increases, resulting in increased cardiomyocyte volume and the synthesis of factors such as NT-proBNP and sST2, which function to increase myocardial performance [3].\nOur study showed that on day 1 after MI, the NT-proBNP concentration increased 4.5-fold (Fig. 1) and remained elevated up to day 12 of the study. It was shown in BNP transgenic mice that following artificially induced MI, the infarcted area increased by about 5-fold during the 48 hr following MI onset and remained at an increased level over the next three to four weeks [1415]. When the disease course was unfavorable, the NT-proBNP concentration increased even more, to 6.8-fold compared with the control values. In addition to the mechanical stretching of the ventricles, there may be other mechanisms that stimulate the production of NT-proBNP, such as ischemia in various locations, arrhythmias, myocardial hypertrophy, and endothelial dysfunction [16]. However, in this study, the NT-proBNP level had a low diagnostic sensitivity and specificity as an indicator of an unfavorable course following MI, with its increase indicating only a 1.2-fold increase in the risk for complications.\nThe stimulating growth factor sST2 had a higher sensitivity for the development of a poor prognosis. With its increase on day 1, there was a 1.7-fold increase in the risk of an unfavorable outcome following MI. Compared with the controls, the sST2 level increased by 1.9-fold in cases with a favorable course of MI and increased 3.7-fold in cases of unfavorable outcomes (Table 2). Elevated levels of sST2 have been associated with an increase in the synthesis of sST2 in cardiac myocytes and fibroblasts due to biomechanical stress [31718].\nST2 is a member of the superfamily of IL-1 receptors. It exists in two forms: a transmembrane receptor (ST2L) and soluble receptor-trap (sST2). The ST2 ligand is IL-33, which contributes to the process reduction of the fibrosis and hypertrophy of tissues under mechanical loads [19]. sST2 acts as a decoy receptor by binding free IL-33 and preventing its signaling through ST2L [2021]. A moderate increase in the concentration of sST2 probably exerts a protective effect, which manifests itself in patients with a favorable course of MI [39]. The transmembrane form protects the myocardium from overcharging, whereas the soluble form of ST2 prevents this protective mechanism, binds to IL-33, and blocks its cardioprotective effect [320]. Perhaps the increase in the concentration of sST2 in cases of unfavorable outcomes is associated with an increased content of the soluble form of the marker following release by damaged cardiomyocytes.\nBoth sST2 and troponin levels have been measured in several other studies. In the study by Mueller et al. [22] in 2015, in contrast to our study, no correlation between sST2 and troponin levels was found. This difference can be explained by the fact that we measured biomarkers in patients in the acute phase of MI, whereas the patients in the study by Mueller et al. [22] had heart failure.\nIn the present study, by day 12 following MI, sST2 concentrations decreased to the level of control values, thus making it difficult to distinguish between the favorable and unfavorable outcome groups. The dynamics of the sST2 changes are somewhat similar to those of C-reactive protein (CRP). CRP levels increased acutely during acute MI; however, this marker tended to decrease by day 12 after MI onset [23]. This similarity indicates their common inflammatory nature. These results are consistent with those reported in the study by Weinberg et al. [24], which was performed on an experimental MI model (in vivo) by using C57/BL6J mice. After ligating the coronary artery, the maximal transcriptional induction of sST2 in cardiac myocytes occurred within 2 hr, was maintained for 9 hr, and decreased after 15 hr.\nStudies have shown that the sST2 threshold in patients with chronic heart failure is 35 ng/mL. Above this value, the risk of death increases dramatically within one year of the event [91018]. Kohli et al. [10] reported similar results, showing that high levels of sST2 (>35 ng/mL) in patients suffering from acute coronary syndrome predicted a 3-fold higher risk of cardiovascular death and heart failure within 30 days and one year. It is of particular interest to study such patterns in a cohort of patients with MI. In our study, the adverse outcomes during the early period of MI were not associated with sST2 levels above 35 ng/mL, as there were complications in patients with levels below (15.5%) and above (55.4%) the threshold. These results were probably affected not only by the level of sST2 but also by the presence of other factors, specifically the increase in NT-proBNP. Indeed, the use of these two markers together significantly increases their sensitivity and specificity of predicting the risk of unfavorable MI outcomes [25].\nOur findings are consistent with the study by Sabatine et al. [18], in which unfavorable outcomes were observed with high levels of both markers (risk of death or developing heart failure was 6.5-fold higher) during the 30-day observation period. Using the levels of NT-proBNP and sST2 to construct the receiver operating characteristics curve, the identification of combined cardiovascular mortality or heart failure significantly improved to 0.78 (95% CI, 0.74-0.83; P=0.0025).\nIn conclusion, the concentration of sST2 is a more sensitive indicator of the course of hospitalization for MI than the traditional indicator of the NT-proBNP concentration. Increased concentrations of sST2 on day 1 after MI were followed by an unfavorable hospitalization course, including progressive angina, arrhythmias, MI, and recurrent symptomatic AHF (Killip class II-IV).\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nStudying the role of soluble ST2 (sST2) during hospitalization for myocardial infarction (MI) can be helpful for predicting the course of the hospitalization and development of complications.\n\nMethods:\nWe included 88 patients with MI (median age, 58 yr). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. On days 1 and 12 after MI, serum sST2 and N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured by ELISA.\n\nResults:\nOn day 1, the concentrations of sST2 and NT-proBNP increased 2.4- and 4.5-fold, compared with the controls. Measurements on day 12 showed a significant decrease in the sST2 level (P=0.001), whereas the NT-proBNP level did not change. On day 1, the sST2 level in the unfavorable outcome group was 2-fold higher than that in the favorable outcome group and 3.7-fold higher than in the controls. On day 12, the marker level decreased in both groups. On day 1, the NT-proBNP level in the unfavorable outcome group was 6.8-fold higher than in the controls and 1.8-fold higher than in the favorable outcome group. On day 12, the level of NT-proBNP remained elevated in both groups. Determining the levels of both sST2 and NT-proBNP increases their diagnostic significance (odds ratio [OR], 1.92; 95% confidence interval [CI], 1.7-3.2; areas under curve [AUC] 0.89; P=0.004).\n\nConclusions:\nThe level of sST2 is a more sensitive indicator during MI hospitalization than NT-proBNP."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":5484,"string":"5,484"},"whole_article_abstract_length":{"kind":"number","value":329,"string":"329"},"num_sections":{"kind":"number","value":7,"string":"7"},"most_frequent_words":{"kind":"list like","value":["sst2","patients","mi","ml","ng","ng ml","study","level","nt","nt probnp"],"string":"[\n \"sst2\",\n \"patients\",\n \"mi\",\n \"ml\",\n \"ng\",\n \"ng ml\",\n \"study\",\n \"level\",\n \"nt\",\n \"nt probnp\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Myocardial infarction | NT-proBNP | sST2 [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Myocardial infarction | NT-proBNP | sST2 [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Myocardial infarction | NT-proBNP | sST2 [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Myocardial infarction | NT-proBNP | sST2 [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Area Under Curve | Case-Control Studies | Electrocardiography | Enzyme-Linked Immunosorbent Assay | Female | Hospitalization | Humans | Logistic Models | Male | Middle Aged | Myocardial Infarction | Natriuretic Peptide, Brain | Odds Ratio | Peptide Fragments | Prognosis | Proportional Hazards Models | ROC Curve | Receptors, Somatostatin [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Area Under Curve | Case-Control Studies | Electrocardiography | Enzyme-Linked Immunosorbent Assay | Female | Hospitalization | Humans | Logistic Models | Male | Middle Aged | Myocardial Infarction | Natriuretic Peptide, Brain | Odds Ratio | Peptide Fragments | Prognosis | Proportional Hazards Models | ROC Curve | Receptors, Somatostatin [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Area Under Curve | Case-Control Studies | Electrocardiography | Enzyme-Linked Immunosorbent Assay | Female | Hospitalization | Humans | Logistic Models | Male | Middle Aged | Myocardial Infarction | Natriuretic Peptide, Brain | Odds Ratio | Peptide Fragments | Prognosis | Proportional Hazards Models | ROC Curve | Receptors, Somatostatin [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Area Under Curve | Case-Control Studies | Electrocardiography | Enzyme-Linked Immunosorbent Assay | Female | Hospitalization | Humans | Logistic Models | Male | Middle Aged | Myocardial Infarction | Natriuretic Peptide, Brain | Odds Ratio | Peptide Fragments | Prognosis | Proportional Hazards Models | ROC Curve | Receptors, Somatostatin [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] sst2 | patients | mi | ml | ng | ng ml | study | level | nt | nt probnp [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] sst2 | patients | mi | ml | ng | ng ml | study | level | nt | nt probnp [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] sst2 | patients | mi | ml | ng | ng ml | study | level | nt | nt probnp [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] sst2 | patients | mi | ml | ng | ng ml | study | level | nt | nt probnp [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] st2 | stretching | level | cardiomyocytes | function | effects | mass | remodeling | antihypertrophic | il [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | ml | roche | ng | ng ml | diseases | russian | connection | diagnostics | included [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] sst2 | level | fold | ml | day | nt probnp | probnp | nt | group | ng ml [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | sst2 | ml | mi | ng ml | ng | level | nt probnp | nt | probnp [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 88 | 58 ||| two | n=58 ||| days 1 and 12 | ELISA [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] day 1 | 2.4- | 4.5-fold ||| day 12 ||| day 1 | 2-fold | 3.7-fold ||| day 12 ||| day 1 | 6.8-fold | 1.8-fold ||| day 12 | NT-proBNP ||| ||| 1.92 | 95% ||| CI | 1.7 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 88 | 58 ||| two | n=58 ||| days 1 and 12 | ELISA ||| ||| day 1 | 2.4- | 4.5-fold ||| day 12 ||| day 1 | 2-fold | 3.7-fold ||| day 12 ||| day 1 | 6.8-fold | 1.8-fold ||| day 12 | NT-proBNP ||| ||| 1.92 | 95% ||| CI | 1.7 ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":260,"cells":{"title":{"kind":"string","value":"Safety of combination antiretroviral prophylaxis in high-risk HIV-exposed newborns: a retrospective review of the Canadian experience."},"pmid":{"kind":"string","value":"26880241"},"background_abstract":{"kind":"string","value":"The optimal management of infants born to HIV-positive mothers who are untreated or have detectable viral load prior to delivery remains controversial. Despite the increasing use of combination antiretroviral therapy (cART) for post-exposure prophylaxis (PEP) of neonates at high risk of HIV infection, there is little safety and pharmacokinetic data to support this approach. The objective of this study was to evaluate the safety and tolerability of cART for PEP in HIV-exposed neonates."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"Retrospective study on 148 cART and 145 Zidovudine (ZDV) monotherapy-exposed infants identified from four Canadian centres where cART for PEP has routinely been prescribed in high-risk situations. Physician-reported adverse events and clinical outcomes were extracted by chart review. Haematological and growth parameters at birth, one and six months of age were compared between cART and ZDV-exposed infants using multivariate mixed effects modelling."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Non-specific signs and symptoms were reported in 10.2% of cART recipients versus none of the ZDV recipients. Treatment was discontinued prematurely in 9.5% of cART recipients versus 2.1% of ZDV recipients (p=0.01). In the multivariate model, cART recipients had lower mean haemoglobin (decrease of 2.07 g/L) over the 6-month period compared with ZDV recipients (p=0.04), but no effect was seen on absolute neutrophil count. cART recipients had lower weight and smaller head circumference at birth and one month of age compared with ZDV-exposed infants; these differences were no longer significant at six months of age."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"cART administered at treatment doses for PEP in neonates was generally well tolerated, though a higher incidence of non-specific signs and symptoms and early treatment discontinuation occurred among cART recipients."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Anti-HIV Agents","Canada","Drug Therapy, Combination","Female","HIV Infections","Humans","Infant, Newborn","Infectious Disease Transmission, Vertical","Male","Pregnancy","Retrospective Studies"],"string":"[\n \"Adult\",\n \"Anti-HIV Agents\",\n \"Canada\",\n \"Drug Therapy, Combination\",\n \"Female\",\n \"HIV Infections\",\n \"Humans\",\n \"Infant, Newborn\",\n \"Infectious Disease Transmission, Vertical\",\n \"Male\",\n \"Pregnancy\",\n \"Retrospective Studies\"\n]"},"pmcid":{"kind":"string","value":"4753845"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"While the overall risk of perinatal HIV transmission in the developed world has decreased to less than 1%, transmission still occurs, most often due to failure to diagnose HIV in pregnant women in a timely manner, or because of poor maternal drug adherence to, and/or refusal of, antiretroviral therapy [1]. Despite the relatively high risk of transmission in these situations, the optimal management of infants born to HIV-positive mothers, who are untreated or have detectable viral load prior to delivery despite receiving antenatal therapy, remains controversial. Current US Department of Health and Human Services (DHHS) guidelines recommend a six-week course of Zidovudine (ZDV), along with three doses of Nevirapine (NVP) in the first week of life for infants born to mothers who have not received any treatment [2]. However, in practice, many paediatric HIV specialists prescribe various combinations of three or more drugs to such “high-risk” neonates. In a national web-based survey of US care providers between December 2009 and January 2010, 62% reported the use of combination antiretroviral therapy (cART) during the previous year [3]. In a recent study from the United Kingdom, cART was prescribed to 71% of infants born to untreated mothers [4].\nDespite the increasing use of cART for prophylaxis in neonates at high risk of HIV infection, there is limited data on the safety of these different combination regimens, or on the appropriate dosing for the antiretroviral (ARV) medications used for this purpose. Transient haematological toxicity has been reported with the use of six-week post-natal ZDV [5], the recommended regimen in standard-risk situations, and a number of studies have suggested increased haematological toxicity associated with the addition of a second nucleoside analogue [6, 7]. Neither the safety nor the optimal dosing of additional drugs used for prophylaxis in neonates (non-nucleoside reverse transcriptase inhibitors or protease inhibitors (PIs)) has been well defined [8–13]. Furthermore, there is emerging concern about the long-term impact of increased ARV exposure on the growth of exposed infants [14, 15].\nIn four Canadian centres, triple cART at treatment doses has been routinely prescribed for the post-exposure prophylaxis (PEP) of neonates deemed at high risk of HIV infection, either because of detectable maternal viral load near delivery or in the absence of viral load results, clinical factors such as poor adherence or late diagnosis. In standard-risk situations, six weeks of ZDV monotherapy is prescribed. Given the limited data to support the use of cART, the primary objective of this study was to compare the safety and tolerability of cART versus ZDV monotherapy of PEP with respect to adverse events, haematological and growth parameters in exposed neonates."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"HIV-1 exposed children were eligible for this study if they were started on treatment doses of cART within 72 hours of birth because of incomplete maternal virologic suppression at delivery or, in the absence of maternal viral load results, a maternal history of incomplete adherence or non-adherence to cART, or late pregnancy initiation of cART. Neonatal cART was defined as any regimen of three or more antiretroviral agents, prescribed at treatment doses, for a minimum of six weeks. Regimens included ZDV (2 mg/kg every six hours prior to 2011, and 4 mg/kg twice daily in 2011–2013) for six weeks, Lamivudine (3TC) (2 mg/kg twice daily) for six weeks, with the addition of either NVP (150 mg/m2 daily for 14 days, followed by 150 mg/m2 twice daily for 14 days) or Nelfinavir (NFV) 40–50 mg/kg twice daily for six weeks) or LPV/Ritonavir (LPV/r) (300 mg/m2 twice daily) for six weeks. Combination regimens containing single or three doses of NVP were excluded.\nSubjects were identified for the period 1997–2013 from the clinical databases of four paediatric HIV-care institutions in Canada: The Hospital for Sick Children, Toronto; Children's Hospital of Eastern Ontario, Ottawa; Centre Hospitalier Universitaire (CHU) Sainte-Justine, Montreal; Stollery Children's Hospital, Edmonton. A control group consisting of all infants of HIV-positive mothers who were prescribed ZDV monotherapy during a three-year period (2010–2012) at the Hospital for Sick Children was selected (n=148). Approval for the study was obtained by the ethics review board of each participating institution; given the retrospective nature of the study, individual patient consent was not deemed necessary by the respective ethics review boards for the chart review.\n Statistical analysis Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).\nBaseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA)."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"One hundred and forty-eight infants received triple cART during the study period. NFV-based cART was the most common regimen (55%) followed by NVP-based cART (40%) and LPV/r-based cART (5%). The most common reason for prescribing cART was documented detectable maternal viral load (78.4%). Other overlapping factors included poor adherence (45%), late diagnosis (20%), no antenatal care (8%) and refusal of antenatal ARV therapy (8%). Fifteen of the cART-treated infants and five of the ZDV-treated infants had missing six-month data, either because they were lost to follow-up (n=8) or they did not attend the scheduled six-month visit (but were seen at 18 months of age or older) (n=12).\nBaseline maternal and infant characteristics according to treatment group are described in Table 1. Mothers of cART-treated infants were younger, had higher viral loads and lower CD4 counts at the time of delivery, and were more likely to deliver vaginally and prematurely than mothers of ZDV monotherapy-treated infants.\nBaseline demographic and clinical characteristics of mothers and infantsa\n\nReported means and standard deviation;\nChi Square test of proportions or Wilcoxon rank-sum.\nHematological and Growth Parameters by treatment group†\nReported means and standard deviation\nStudent T test, Wilcoxon rank sum, or Kruskal-Wallace test where appropriate.\nn* observations recorded for each parameter out of a total of 148 cART recipients at birth, and 145 ZDV recipients.\nThirteen of the cART recipients were perinatally infected with HIV (8.8%). None of the ZDV recipients were infected. Five of the 13 (38.5%) had HIV-1 detected by PCR within 48 hours of birth suggesting in utero infection according to diagnostic standards. For the remaining eight, the timing of infection could not be ascertained as initial testing took place after 48 hours of life. Of the 13 infected children, four achieved long-term sustained virologic suppression, the details of which have been described elsewhere [16].\nNon-specific signs and symptoms, including vomiting, diarrhoea, rash, jitteriness or irritability, potentially attributable to medication-related adverse effects, were reported in 10.2% of cART recipients and none of the ZDV recipients (p<0.001). ARV treatment was discontinued prematurely in 9.5% (n=14) of cART recipients (five NVP-treated and nine PI-treated infants) compared with 2.1% (n=3) ZDV recipients (p=0.01). Reasons given for premature treatment discontinuation among cART recipients included significant anaemia (n=3; haemoglobin at two, four and four weeks of 62, 76 and 92 g/L, respectively), neutropenia (n=1; neutrophil count 0.4 cells/mm3 at four weeks), non-specific symptoms including rash, vomiting, and diarrhoea (n=6), and parental decision (n=4). In the six cases of non-specific symptoms, treatment discontinuation was precautionary, as the treating physician indicated that the symptoms were most likely not medication related. In four cases of parental discontinuation, parents stopped the medication without evidence of toxicity or consultation with their healthcare provider. ZDV monotherapy was stopped early in three children, all because of significant anaemia (haemoglobin at four weeks of 76, 85 and 72 g/L).\nThe potential impact of cART on haematologic parameters was evaluated by comparing haemoglobin level and absolute neutrophil count for cART-treated and ZDV monotherapy-treated infants at one, two and six months of age (Table 2). In the unadjusted analysis, haemoglobin was lower in cART-treated infants compared with ZDV-treated infants at one month of age (mean 105±19 vs. 109±17 g/L, p=0.04), but not at two or six months of age (mean 104±12 vs. 105±9 g/L, p=0.31 and 118±11 vs. 121±8 g/L, p=0.15). In a subgroup analysis of specific treatment type, mean haemoglobin was lower among PI-treated infants compared with NVP-treated infants at one month of age (104±17 vs. 107±21, p=0.09), though only statistically significant at six months of age (mean 115±10 vs. 124±9 g/L, p<0.001) (Figure 1).\nHaematological parameters according to treatment group.\nGraphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.) \nTo study the overall effect of treatment type on haemoglobin over time, we constructed a multivariate model adjusting for chronological age and gestational age (Supplementary Table, not shown). Overall, the mean haemoglobin was 2.07 g/L lower among cART versus ZDV-treated infants (p=0.04) over the six-month period. In the subgroup analysis looking at specific treatment type, mean haemoglobin was 3.62 g/L lower in those receiving PI-based cART compared with those receiving ZDV monotherapy (p=0.002); no such effect was observed for NVP-based cART compared with ZDV monotherapy (p=0.82). There was no difference in absolute neutrophil count between NVP-based cART, PI-based cART and ZDV monotherapy-treated infants at one, two or six months of age in the unadjusted analysis; this finding was maintained in the multivariable model adjusting for visit number, ethnicity and gestational age.\nWeight, length and HC were compared between all cART-treated and ZDV-treated infants, and between PI-based cART, NVP-based cART and ZDV at birth, one and six months of age (Table 2 and Figure 2). In the unadjusted analysis, cART-treated infants were smaller compared with ZDV-treated infants at birth (weight 2.91±0.60 vs. 3.09±0.56 kg, p=0.01; length 49.0±3.6 vs. 50.2±3.8 cm, p=0.03, head circumference 33.2±2.4 vs.33.6±2.3 cm, p=0.16). By six months of age, there was no difference in weight (8.03±1.12 kg vs. 8.25±1.12 kg, p=0.14) or head circumference (43.7±1.8 cm vs. 44.0±1.7, p=0.13), although the cART-treated group remained shorter than ZDV-treated infants (67.1±2.9 cm vs. 68.1±3.2 cm, p=0.01). In the subgroup analysis looking at specific treatment type, both NVP-and PI-treated infants were smaller than ZDV-treated infants across all growth parameters at birth; only the difference in length remained significant at six months of age.\nGrowth parameters according to treatment group.\nGraphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.) \nTo study the overall effect of treatment type on growth parameters over time, we constructed a multivariate fixed effects model adjusting for birthweight, gestational age, chronological age and gender. Overall, there was a mean HC difference of −0.63 cm among cART compared with ZDV-treated infants over the six-month period (p<0.001) (Supplementary Table, not shown). A similar effect was seen with respect to weight and length, with a mean weight difference of −236 g and length difference of −0.92 cm over the six-month period (p<0.001 and p=0.002, respectively). In the subgroup multivariate analysis comparing PI-based cART versus ZDV and NVP-based cART versus ZDV, there was no discernable difference between the two cART treatment types and these growth parameters."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In summary, our study shows that cART at treatment doses administered as prophylaxis to “high-risk” HIV-exposed neonates is generally well tolerated and should be considered for newborn infants deemed at high risk of perinatally acquired HIV infection. However, close monitoring for anaemia, neutropenia and other side effects is important if such therapy is given. Until more robust pharmacokinetic data are available, the use of cART as prophylaxis in neonates may be limited to resource-rich settings where routine therapeutic drug monitoring is possible. However, further study is urgently needed in resource-limited settings where the vast majority of high-risk HIV-exposed infants are born. The potential benefits of cART, both to avert infection and, in case of transmission, preserve future therapeutic options and potentially limit the viral reservoir, may well outweigh the risks. Finally, if this strategy is used, counselling of parents and close follow-up is necessary to avoid premature treatment discontinuation and to ensure proper monitoring of cART-exposed infants."},"other_sections_titles":{"kind":"list like","value":["Statistical analysis"],"string":"[\n \"Statistical analysis\"\n]"},"other_sections_texts":{"kind":"list like","value":["Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA)."],"string":"[\n \"Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null],"string":"[\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Statistical analysis","Results","Discussion","Conclusions","Supplementary Material"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Statistical analysis\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Supplementary Material\"\n]"},"all_sections_texts":{"kind":"list like","value":["While the overall risk of perinatal HIV transmission in the developed world has decreased to less than 1%, transmission still occurs, most often due to failure to diagnose HIV in pregnant women in a timely manner, or because of poor maternal drug adherence to, and/or refusal of, antiretroviral therapy [1]. Despite the relatively high risk of transmission in these situations, the optimal management of infants born to HIV-positive mothers, who are untreated or have detectable viral load prior to delivery despite receiving antenatal therapy, remains controversial. Current US Department of Health and Human Services (DHHS) guidelines recommend a six-week course of Zidovudine (ZDV), along with three doses of Nevirapine (NVP) in the first week of life for infants born to mothers who have not received any treatment [2]. However, in practice, many paediatric HIV specialists prescribe various combinations of three or more drugs to such “high-risk” neonates. In a national web-based survey of US care providers between December 2009 and January 2010, 62% reported the use of combination antiretroviral therapy (cART) during the previous year [3]. In a recent study from the United Kingdom, cART was prescribed to 71% of infants born to untreated mothers [4].\nDespite the increasing use of cART for prophylaxis in neonates at high risk of HIV infection, there is limited data on the safety of these different combination regimens, or on the appropriate dosing for the antiretroviral (ARV) medications used for this purpose. Transient haematological toxicity has been reported with the use of six-week post-natal ZDV [5], the recommended regimen in standard-risk situations, and a number of studies have suggested increased haematological toxicity associated with the addition of a second nucleoside analogue [6, 7]. Neither the safety nor the optimal dosing of additional drugs used for prophylaxis in neonates (non-nucleoside reverse transcriptase inhibitors or protease inhibitors (PIs)) has been well defined [8–13]. Furthermore, there is emerging concern about the long-term impact of increased ARV exposure on the growth of exposed infants [14, 15].\nIn four Canadian centres, triple cART at treatment doses has been routinely prescribed for the post-exposure prophylaxis (PEP) of neonates deemed at high risk of HIV infection, either because of detectable maternal viral load near delivery or in the absence of viral load results, clinical factors such as poor adherence or late diagnosis. In standard-risk situations, six weeks of ZDV monotherapy is prescribed. Given the limited data to support the use of cART, the primary objective of this study was to compare the safety and tolerability of cART versus ZDV monotherapy of PEP with respect to adverse events, haematological and growth parameters in exposed neonates.","HIV-1 exposed children were eligible for this study if they were started on treatment doses of cART within 72 hours of birth because of incomplete maternal virologic suppression at delivery or, in the absence of maternal viral load results, a maternal history of incomplete adherence or non-adherence to cART, or late pregnancy initiation of cART. Neonatal cART was defined as any regimen of three or more antiretroviral agents, prescribed at treatment doses, for a minimum of six weeks. Regimens included ZDV (2 mg/kg every six hours prior to 2011, and 4 mg/kg twice daily in 2011–2013) for six weeks, Lamivudine (3TC) (2 mg/kg twice daily) for six weeks, with the addition of either NVP (150 mg/m2 daily for 14 days, followed by 150 mg/m2 twice daily for 14 days) or Nelfinavir (NFV) 40–50 mg/kg twice daily for six weeks) or LPV/Ritonavir (LPV/r) (300 mg/m2 twice daily) for six weeks. Combination regimens containing single or three doses of NVP were excluded.\nSubjects were identified for the period 1997–2013 from the clinical databases of four paediatric HIV-care institutions in Canada: The Hospital for Sick Children, Toronto; Children's Hospital of Eastern Ontario, Ottawa; Centre Hospitalier Universitaire (CHU) Sainte-Justine, Montreal; Stollery Children's Hospital, Edmonton. A control group consisting of all infants of HIV-positive mothers who were prescribed ZDV monotherapy during a three-year period (2010–2012) at the Hospital for Sick Children was selected (n=148). Approval for the study was obtained by the ethics review board of each participating institution; given the retrospective nature of the study, individual patient consent was not deemed necessary by the respective ethics review boards for the chart review.\n Statistical analysis Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).\nBaseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).","Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).","One hundred and forty-eight infants received triple cART during the study period. NFV-based cART was the most common regimen (55%) followed by NVP-based cART (40%) and LPV/r-based cART (5%). The most common reason for prescribing cART was documented detectable maternal viral load (78.4%). Other overlapping factors included poor adherence (45%), late diagnosis (20%), no antenatal care (8%) and refusal of antenatal ARV therapy (8%). Fifteen of the cART-treated infants and five of the ZDV-treated infants had missing six-month data, either because they were lost to follow-up (n=8) or they did not attend the scheduled six-month visit (but were seen at 18 months of age or older) (n=12).\nBaseline maternal and infant characteristics according to treatment group are described in Table 1. Mothers of cART-treated infants were younger, had higher viral loads and lower CD4 counts at the time of delivery, and were more likely to deliver vaginally and prematurely than mothers of ZDV monotherapy-treated infants.\nBaseline demographic and clinical characteristics of mothers and infantsa\n\nReported means and standard deviation;\nChi Square test of proportions or Wilcoxon rank-sum.\nHematological and Growth Parameters by treatment group†\nReported means and standard deviation\nStudent T test, Wilcoxon rank sum, or Kruskal-Wallace test where appropriate.\nn* observations recorded for each parameter out of a total of 148 cART recipients at birth, and 145 ZDV recipients.\nThirteen of the cART recipients were perinatally infected with HIV (8.8%). None of the ZDV recipients were infected. Five of the 13 (38.5%) had HIV-1 detected by PCR within 48 hours of birth suggesting in utero infection according to diagnostic standards. For the remaining eight, the timing of infection could not be ascertained as initial testing took place after 48 hours of life. Of the 13 infected children, four achieved long-term sustained virologic suppression, the details of which have been described elsewhere [16].\nNon-specific signs and symptoms, including vomiting, diarrhoea, rash, jitteriness or irritability, potentially attributable to medication-related adverse effects, were reported in 10.2% of cART recipients and none of the ZDV recipients (p<0.001). ARV treatment was discontinued prematurely in 9.5% (n=14) of cART recipients (five NVP-treated and nine PI-treated infants) compared with 2.1% (n=3) ZDV recipients (p=0.01). Reasons given for premature treatment discontinuation among cART recipients included significant anaemia (n=3; haemoglobin at two, four and four weeks of 62, 76 and 92 g/L, respectively), neutropenia (n=1; neutrophil count 0.4 cells/mm3 at four weeks), non-specific symptoms including rash, vomiting, and diarrhoea (n=6), and parental decision (n=4). In the six cases of non-specific symptoms, treatment discontinuation was precautionary, as the treating physician indicated that the symptoms were most likely not medication related. In four cases of parental discontinuation, parents stopped the medication without evidence of toxicity or consultation with their healthcare provider. ZDV monotherapy was stopped early in three children, all because of significant anaemia (haemoglobin at four weeks of 76, 85 and 72 g/L).\nThe potential impact of cART on haematologic parameters was evaluated by comparing haemoglobin level and absolute neutrophil count for cART-treated and ZDV monotherapy-treated infants at one, two and six months of age (Table 2). In the unadjusted analysis, haemoglobin was lower in cART-treated infants compared with ZDV-treated infants at one month of age (mean 105±19 vs. 109±17 g/L, p=0.04), but not at two or six months of age (mean 104±12 vs. 105±9 g/L, p=0.31 and 118±11 vs. 121±8 g/L, p=0.15). In a subgroup analysis of specific treatment type, mean haemoglobin was lower among PI-treated infants compared with NVP-treated infants at one month of age (104±17 vs. 107±21, p=0.09), though only statistically significant at six months of age (mean 115±10 vs. 124±9 g/L, p<0.001) (Figure 1).\nHaematological parameters according to treatment group.\nGraphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.) \nTo study the overall effect of treatment type on haemoglobin over time, we constructed a multivariate model adjusting for chronological age and gestational age (Supplementary Table, not shown). Overall, the mean haemoglobin was 2.07 g/L lower among cART versus ZDV-treated infants (p=0.04) over the six-month period. In the subgroup analysis looking at specific treatment type, mean haemoglobin was 3.62 g/L lower in those receiving PI-based cART compared with those receiving ZDV monotherapy (p=0.002); no such effect was observed for NVP-based cART compared with ZDV monotherapy (p=0.82). There was no difference in absolute neutrophil count between NVP-based cART, PI-based cART and ZDV monotherapy-treated infants at one, two or six months of age in the unadjusted analysis; this finding was maintained in the multivariable model adjusting for visit number, ethnicity and gestational age.\nWeight, length and HC were compared between all cART-treated and ZDV-treated infants, and between PI-based cART, NVP-based cART and ZDV at birth, one and six months of age (Table 2 and Figure 2). In the unadjusted analysis, cART-treated infants were smaller compared with ZDV-treated infants at birth (weight 2.91±0.60 vs. 3.09±0.56 kg, p=0.01; length 49.0±3.6 vs. 50.2±3.8 cm, p=0.03, head circumference 33.2±2.4 vs.33.6±2.3 cm, p=0.16). By six months of age, there was no difference in weight (8.03±1.12 kg vs. 8.25±1.12 kg, p=0.14) or head circumference (43.7±1.8 cm vs. 44.0±1.7, p=0.13), although the cART-treated group remained shorter than ZDV-treated infants (67.1±2.9 cm vs. 68.1±3.2 cm, p=0.01). In the subgroup analysis looking at specific treatment type, both NVP-and PI-treated infants were smaller than ZDV-treated infants across all growth parameters at birth; only the difference in length remained significant at six months of age.\nGrowth parameters according to treatment group.\nGraphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.) \nTo study the overall effect of treatment type on growth parameters over time, we constructed a multivariate fixed effects model adjusting for birthweight, gestational age, chronological age and gender. Overall, there was a mean HC difference of −0.63 cm among cART compared with ZDV-treated infants over the six-month period (p<0.001) (Supplementary Table, not shown). A similar effect was seen with respect to weight and length, with a mean weight difference of −236 g and length difference of −0.92 cm over the six-month period (p<0.001 and p=0.002, respectively). In the subgroup multivariate analysis comparing PI-based cART versus ZDV and NVP-based cART versus ZDV, there was no discernable difference between the two cART treatment types and these growth parameters.","In this retrospective study, cART initiated empirically for newborns deemed at high risk for perinatal HIV infection was generally well-tolerated, though adverse effects and consequent premature discontinuation of therapy did occur more often in the cART versus ZDV-treated group (9.5% vs. 2.1%). Among the 14 cART-treated children in whom medications were prematurely discontinued, four had their medications stopped by the parents without consultation with the healthcare provider and in the absence of any side effects, and six for precautionary reasons by the clinician even though the symptoms were not thought to be medication related, highlighting a lower threshold for treatment discontinuation by both parents and physicians among cART-treated infants.\nWith respect to haematological toxicity, the frequency and degree of anaemia observed in our cART-treated neonates was higher than that observed in the ZDV-monotherapy neonates. In the subgroup analysis of cART-treated infants, haemoglobin was lower at one month of age among PI versus NVP-treated infants, suggesting perhaps less haematological toxicity with NVP-based cART. The lower mean haemoglobin observed in cART-treated subjects compared with ZDV-monotherapy-treated subjects at one month of age was no longer evident at two and six months of age after stopping treatment, confirming the reversible nature of the anaemia. Reassuringly, compared with ZDV monotherapy, the use of cART was not associated with any discernible change in neutrophil count.\nWhile in the multivariate mixed effects model cART-treated infants had lower mean growth measures (HC, weight and length) than ZDV-monotherapy-treated infants over time, there were no absolute differences in growth measures for head circumference or weight at six months of age, indicating good catch-up growth among the cART-treated infants. The observed lower mean growth over time in cART recipients was therefore likely due to factors associated with the indications for the use of cART. Specifically, the higher overall incidence of prematurity among cART-treated infants was likely a result of poorly controlled maternal HIV, an established risk factor for low birthweight and preterm delivery [17]. The higher viral load and lower CD4 counts of mothers of cART recipients in our study supports this hypothesis. While we adjusted for preterm delivery and gestational age in the multivariate models for growth, we were unable to adjust for in utero drug exposure, social situation, maternal health, nutritional status and education, which may be important predictors of infant growth and development.\nBecause of the selective use of cART for infants at higher risk of perinatal transmission in our study, it is not appropriate to compare the risk of transmission among cART versus ZDV-treated infants as the latter group would have received better antenatal preventive management. The 8.8% overall transmission rate observed among cART recipients in our cohort was similar to that in a previous trial in which neonates of untreated mothers were randomized to different cART regimens, evidence of the high risk of transmission despite prophylaxis in these situations [6]. Key potential benefits of cART at treatment doses initiated as prophylaxis for neonates later found to be infected is that such treatment will preserve health by controlling viral replication more rapidly, reduce the risk of generating antiretroviral medication resistance, as seen with single or multiple dose NVP in non-cART strategies, and limit the size of HIV viral reservoirs, which could have significant implications for achieving HIV remission in the future [18–20]. Given the limited number of therapeutic drug regimens available for children in resource-limited settings, preventing drug resistance to first-line therapy given to prevent perinatal transmission is a particularly important consideration.\nOur study has a number of limitations, including its retrospective nature, relatively small sample size, and potential differences in the patient population at different study sites which may have influenced biological and growth outcomes. All control group infants were selected from a single centre, and the choice of PI-based versus NVP-based cART was also site specific. Given that these were the comparison groups of interest, we could not adjust for site and treatment type in the final analysis, thereby limiting our conclusions on specific cART treatment type.","In summary, our study shows that cART at treatment doses administered as prophylaxis to “high-risk” HIV-exposed neonates is generally well tolerated and should be considered for newborn infants deemed at high risk of perinatally acquired HIV infection. However, close monitoring for anaemia, neutropenia and other side effects is important if such therapy is given. Until more robust pharmacokinetic data are available, the use of cART as prophylaxis in neonates may be limited to resource-rich settings where routine therapeutic drug monitoring is possible. However, further study is urgently needed in resource-limited settings where the vast majority of high-risk HIV-exposed infants are born. The potential benefits of cART, both to avert infection and, in case of transmission, preserve future therapeutic options and potentially limit the viral reservoir, may well outweigh the risks. Finally, if this strategy is used, counselling of parents and close follow-up is necessary to avoid premature treatment discontinuation and to ensure proper monitoring of cART-exposed infants.","Click here for additional data file."],"string":"[\n \"While the overall risk of perinatal HIV transmission in the developed world has decreased to less than 1%, transmission still occurs, most often due to failure to diagnose HIV in pregnant women in a timely manner, or because of poor maternal drug adherence to, and/or refusal of, antiretroviral therapy [1]. Despite the relatively high risk of transmission in these situations, the optimal management of infants born to HIV-positive mothers, who are untreated or have detectable viral load prior to delivery despite receiving antenatal therapy, remains controversial. Current US Department of Health and Human Services (DHHS) guidelines recommend a six-week course of Zidovudine (ZDV), along with three doses of Nevirapine (NVP) in the first week of life for infants born to mothers who have not received any treatment [2]. However, in practice, many paediatric HIV specialists prescribe various combinations of three or more drugs to such “high-risk” neonates. In a national web-based survey of US care providers between December 2009 and January 2010, 62% reported the use of combination antiretroviral therapy (cART) during the previous year [3]. In a recent study from the United Kingdom, cART was prescribed to 71% of infants born to untreated mothers [4].\\nDespite the increasing use of cART for prophylaxis in neonates at high risk of HIV infection, there is limited data on the safety of these different combination regimens, or on the appropriate dosing for the antiretroviral (ARV) medications used for this purpose. Transient haematological toxicity has been reported with the use of six-week post-natal ZDV [5], the recommended regimen in standard-risk situations, and a number of studies have suggested increased haematological toxicity associated with the addition of a second nucleoside analogue [6, 7]. Neither the safety nor the optimal dosing of additional drugs used for prophylaxis in neonates (non-nucleoside reverse transcriptase inhibitors or protease inhibitors (PIs)) has been well defined [8–13]. Furthermore, there is emerging concern about the long-term impact of increased ARV exposure on the growth of exposed infants [14, 15].\\nIn four Canadian centres, triple cART at treatment doses has been routinely prescribed for the post-exposure prophylaxis (PEP) of neonates deemed at high risk of HIV infection, either because of detectable maternal viral load near delivery or in the absence of viral load results, clinical factors such as poor adherence or late diagnosis. In standard-risk situations, six weeks of ZDV monotherapy is prescribed. Given the limited data to support the use of cART, the primary objective of this study was to compare the safety and tolerability of cART versus ZDV monotherapy of PEP with respect to adverse events, haematological and growth parameters in exposed neonates.\",\n \"HIV-1 exposed children were eligible for this study if they were started on treatment doses of cART within 72 hours of birth because of incomplete maternal virologic suppression at delivery or, in the absence of maternal viral load results, a maternal history of incomplete adherence or non-adherence to cART, or late pregnancy initiation of cART. Neonatal cART was defined as any regimen of three or more antiretroviral agents, prescribed at treatment doses, for a minimum of six weeks. Regimens included ZDV (2 mg/kg every six hours prior to 2011, and 4 mg/kg twice daily in 2011–2013) for six weeks, Lamivudine (3TC) (2 mg/kg twice daily) for six weeks, with the addition of either NVP (150 mg/m2 daily for 14 days, followed by 150 mg/m2 twice daily for 14 days) or Nelfinavir (NFV) 40–50 mg/kg twice daily for six weeks) or LPV/Ritonavir (LPV/r) (300 mg/m2 twice daily) for six weeks. Combination regimens containing single or three doses of NVP were excluded.\\nSubjects were identified for the period 1997–2013 from the clinical databases of four paediatric HIV-care institutions in Canada: The Hospital for Sick Children, Toronto; Children's Hospital of Eastern Ontario, Ottawa; Centre Hospitalier Universitaire (CHU) Sainte-Justine, Montreal; Stollery Children's Hospital, Edmonton. A control group consisting of all infants of HIV-positive mothers who were prescribed ZDV monotherapy during a three-year period (2010–2012) at the Hospital for Sick Children was selected (n=148). Approval for the study was obtained by the ethics review board of each participating institution; given the retrospective nature of the study, individual patient consent was not deemed necessary by the respective ethics review boards for the chart review.\\n Statistical analysis Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).\\nBaseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).\",\n \"Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).\",\n \"One hundred and forty-eight infants received triple cART during the study period. NFV-based cART was the most common regimen (55%) followed by NVP-based cART (40%) and LPV/r-based cART (5%). The most common reason for prescribing cART was documented detectable maternal viral load (78.4%). Other overlapping factors included poor adherence (45%), late diagnosis (20%), no antenatal care (8%) and refusal of antenatal ARV therapy (8%). Fifteen of the cART-treated infants and five of the ZDV-treated infants had missing six-month data, either because they were lost to follow-up (n=8) or they did not attend the scheduled six-month visit (but were seen at 18 months of age or older) (n=12).\\nBaseline maternal and infant characteristics according to treatment group are described in Table 1. Mothers of cART-treated infants were younger, had higher viral loads and lower CD4 counts at the time of delivery, and were more likely to deliver vaginally and prematurely than mothers of ZDV monotherapy-treated infants.\\nBaseline demographic and clinical characteristics of mothers and infantsa\\n\\nReported means and standard deviation;\\nChi Square test of proportions or Wilcoxon rank-sum.\\nHematological and Growth Parameters by treatment group†\\nReported means and standard deviation\\nStudent T test, Wilcoxon rank sum, or Kruskal-Wallace test where appropriate.\\nn* observations recorded for each parameter out of a total of 148 cART recipients at birth, and 145 ZDV recipients.\\nThirteen of the cART recipients were perinatally infected with HIV (8.8%). None of the ZDV recipients were infected. Five of the 13 (38.5%) had HIV-1 detected by PCR within 48 hours of birth suggesting in utero infection according to diagnostic standards. For the remaining eight, the timing of infection could not be ascertained as initial testing took place after 48 hours of life. Of the 13 infected children, four achieved long-term sustained virologic suppression, the details of which have been described elsewhere [16].\\nNon-specific signs and symptoms, including vomiting, diarrhoea, rash, jitteriness or irritability, potentially attributable to medication-related adverse effects, were reported in 10.2% of cART recipients and none of the ZDV recipients (p<0.001). ARV treatment was discontinued prematurely in 9.5% (n=14) of cART recipients (five NVP-treated and nine PI-treated infants) compared with 2.1% (n=3) ZDV recipients (p=0.01). Reasons given for premature treatment discontinuation among cART recipients included significant anaemia (n=3; haemoglobin at two, four and four weeks of 62, 76 and 92 g/L, respectively), neutropenia (n=1; neutrophil count 0.4 cells/mm3 at four weeks), non-specific symptoms including rash, vomiting, and diarrhoea (n=6), and parental decision (n=4). In the six cases of non-specific symptoms, treatment discontinuation was precautionary, as the treating physician indicated that the symptoms were most likely not medication related. In four cases of parental discontinuation, parents stopped the medication without evidence of toxicity or consultation with their healthcare provider. ZDV monotherapy was stopped early in three children, all because of significant anaemia (haemoglobin at four weeks of 76, 85 and 72 g/L).\\nThe potential impact of cART on haematologic parameters was evaluated by comparing haemoglobin level and absolute neutrophil count for cART-treated and ZDV monotherapy-treated infants at one, two and six months of age (Table 2). In the unadjusted analysis, haemoglobin was lower in cART-treated infants compared with ZDV-treated infants at one month of age (mean 105±19 vs. 109±17 g/L, p=0.04), but not at two or six months of age (mean 104±12 vs. 105±9 g/L, p=0.31 and 118±11 vs. 121±8 g/L, p=0.15). In a subgroup analysis of specific treatment type, mean haemoglobin was lower among PI-treated infants compared with NVP-treated infants at one month of age (104±17 vs. 107±21, p=0.09), though only statistically significant at six months of age (mean 115±10 vs. 124±9 g/L, p<0.001) (Figure 1).\\nHaematological parameters according to treatment group.\\nGraphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.) \\nTo study the overall effect of treatment type on haemoglobin over time, we constructed a multivariate model adjusting for chronological age and gestational age (Supplementary Table, not shown). Overall, the mean haemoglobin was 2.07 g/L lower among cART versus ZDV-treated infants (p=0.04) over the six-month period. In the subgroup analysis looking at specific treatment type, mean haemoglobin was 3.62 g/L lower in those receiving PI-based cART compared with those receiving ZDV monotherapy (p=0.002); no such effect was observed for NVP-based cART compared with ZDV monotherapy (p=0.82). There was no difference in absolute neutrophil count between NVP-based cART, PI-based cART and ZDV monotherapy-treated infants at one, two or six months of age in the unadjusted analysis; this finding was maintained in the multivariable model adjusting for visit number, ethnicity and gestational age.\\nWeight, length and HC were compared between all cART-treated and ZDV-treated infants, and between PI-based cART, NVP-based cART and ZDV at birth, one and six months of age (Table 2 and Figure 2). In the unadjusted analysis, cART-treated infants were smaller compared with ZDV-treated infants at birth (weight 2.91±0.60 vs. 3.09±0.56 kg, p=0.01; length 49.0±3.6 vs. 50.2±3.8 cm, p=0.03, head circumference 33.2±2.4 vs.33.6±2.3 cm, p=0.16). By six months of age, there was no difference in weight (8.03±1.12 kg vs. 8.25±1.12 kg, p=0.14) or head circumference (43.7±1.8 cm vs. 44.0±1.7, p=0.13), although the cART-treated group remained shorter than ZDV-treated infants (67.1±2.9 cm vs. 68.1±3.2 cm, p=0.01). In the subgroup analysis looking at specific treatment type, both NVP-and PI-treated infants were smaller than ZDV-treated infants across all growth parameters at birth; only the difference in length remained significant at six months of age.\\nGrowth parameters according to treatment group.\\nGraphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.) \\nTo study the overall effect of treatment type on growth parameters over time, we constructed a multivariate fixed effects model adjusting for birthweight, gestational age, chronological age and gender. Overall, there was a mean HC difference of −0.63 cm among cART compared with ZDV-treated infants over the six-month period (p<0.001) (Supplementary Table, not shown). A similar effect was seen with respect to weight and length, with a mean weight difference of −236 g and length difference of −0.92 cm over the six-month period (p<0.001 and p=0.002, respectively). In the subgroup multivariate analysis comparing PI-based cART versus ZDV and NVP-based cART versus ZDV, there was no discernable difference between the two cART treatment types and these growth parameters.\",\n \"In this retrospective study, cART initiated empirically for newborns deemed at high risk for perinatal HIV infection was generally well-tolerated, though adverse effects and consequent premature discontinuation of therapy did occur more often in the cART versus ZDV-treated group (9.5% vs. 2.1%). Among the 14 cART-treated children in whom medications were prematurely discontinued, four had their medications stopped by the parents without consultation with the healthcare provider and in the absence of any side effects, and six for precautionary reasons by the clinician even though the symptoms were not thought to be medication related, highlighting a lower threshold for treatment discontinuation by both parents and physicians among cART-treated infants.\\nWith respect to haematological toxicity, the frequency and degree of anaemia observed in our cART-treated neonates was higher than that observed in the ZDV-monotherapy neonates. In the subgroup analysis of cART-treated infants, haemoglobin was lower at one month of age among PI versus NVP-treated infants, suggesting perhaps less haematological toxicity with NVP-based cART. The lower mean haemoglobin observed in cART-treated subjects compared with ZDV-monotherapy-treated subjects at one month of age was no longer evident at two and six months of age after stopping treatment, confirming the reversible nature of the anaemia. Reassuringly, compared with ZDV monotherapy, the use of cART was not associated with any discernible change in neutrophil count.\\nWhile in the multivariate mixed effects model cART-treated infants had lower mean growth measures (HC, weight and length) than ZDV-monotherapy-treated infants over time, there were no absolute differences in growth measures for head circumference or weight at six months of age, indicating good catch-up growth among the cART-treated infants. The observed lower mean growth over time in cART recipients was therefore likely due to factors associated with the indications for the use of cART. Specifically, the higher overall incidence of prematurity among cART-treated infants was likely a result of poorly controlled maternal HIV, an established risk factor for low birthweight and preterm delivery [17]. The higher viral load and lower CD4 counts of mothers of cART recipients in our study supports this hypothesis. While we adjusted for preterm delivery and gestational age in the multivariate models for growth, we were unable to adjust for in utero drug exposure, social situation, maternal health, nutritional status and education, which may be important predictors of infant growth and development.\\nBecause of the selective use of cART for infants at higher risk of perinatal transmission in our study, it is not appropriate to compare the risk of transmission among cART versus ZDV-treated infants as the latter group would have received better antenatal preventive management. The 8.8% overall transmission rate observed among cART recipients in our cohort was similar to that in a previous trial in which neonates of untreated mothers were randomized to different cART regimens, evidence of the high risk of transmission despite prophylaxis in these situations [6]. Key potential benefits of cART at treatment doses initiated as prophylaxis for neonates later found to be infected is that such treatment will preserve health by controlling viral replication more rapidly, reduce the risk of generating antiretroviral medication resistance, as seen with single or multiple dose NVP in non-cART strategies, and limit the size of HIV viral reservoirs, which could have significant implications for achieving HIV remission in the future [18–20]. Given the limited number of therapeutic drug regimens available for children in resource-limited settings, preventing drug resistance to first-line therapy given to prevent perinatal transmission is a particularly important consideration.\\nOur study has a number of limitations, including its retrospective nature, relatively small sample size, and potential differences in the patient population at different study sites which may have influenced biological and growth outcomes. All control group infants were selected from a single centre, and the choice of PI-based versus NVP-based cART was also site specific. Given that these were the comparison groups of interest, we could not adjust for site and treatment type in the final analysis, thereby limiting our conclusions on specific cART treatment type.\",\n \"In summary, our study shows that cART at treatment doses administered as prophylaxis to “high-risk” HIV-exposed neonates is generally well tolerated and should be considered for newborn infants deemed at high risk of perinatally acquired HIV infection. However, close monitoring for anaemia, neutropenia and other side effects is important if such therapy is given. Until more robust pharmacokinetic data are available, the use of cART as prophylaxis in neonates may be limited to resource-rich settings where routine therapeutic drug monitoring is possible. However, further study is urgently needed in resource-limited settings where the vast majority of high-risk HIV-exposed infants are born. The potential benefits of cART, both to avert infection and, in case of transmission, preserve future therapeutic options and potentially limit the viral reservoir, may well outweigh the risks. Finally, if this strategy is used, counselling of parents and close follow-up is necessary to avoid premature treatment discontinuation and to ensure proper monitoring of cART-exposed infants.\",\n \"Click here for additional data file.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,"results","discussion","conclusions","supplementary-material"],"string":"[\n \"intro\",\n \"methods\",\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["Prevention of mother-to-child transmission","HIV","neonatal prophylaxis","safety","combination antiretroviral therapy"],"string":"[\n \"Prevention of mother-to-child transmission\",\n \"HIV\",\n \"neonatal prophylaxis\",\n \"safety\",\n \"combination antiretroviral therapy\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nWhile the overall risk of perinatal HIV transmission in the developed world has decreased to less than 1%, transmission still occurs, most often due to failure to diagnose HIV in pregnant women in a timely manner, or because of poor maternal drug adherence to, and/or refusal of, antiretroviral therapy [1]. Despite the relatively high risk of transmission in these situations, the optimal management of infants born to HIV-positive mothers, who are untreated or have detectable viral load prior to delivery despite receiving antenatal therapy, remains controversial. Current US Department of Health and Human Services (DHHS) guidelines recommend a six-week course of Zidovudine (ZDV), along with three doses of Nevirapine (NVP) in the first week of life for infants born to mothers who have not received any treatment [2]. However, in practice, many paediatric HIV specialists prescribe various combinations of three or more drugs to such “high-risk” neonates. In a national web-based survey of US care providers between December 2009 and January 2010, 62% reported the use of combination antiretroviral therapy (cART) during the previous year [3]. In a recent study from the United Kingdom, cART was prescribed to 71% of infants born to untreated mothers [4].\nDespite the increasing use of cART for prophylaxis in neonates at high risk of HIV infection, there is limited data on the safety of these different combination regimens, or on the appropriate dosing for the antiretroviral (ARV) medications used for this purpose. Transient haematological toxicity has been reported with the use of six-week post-natal ZDV [5], the recommended regimen in standard-risk situations, and a number of studies have suggested increased haematological toxicity associated with the addition of a second nucleoside analogue [6, 7]. Neither the safety nor the optimal dosing of additional drugs used for prophylaxis in neonates (non-nucleoside reverse transcriptase inhibitors or protease inhibitors (PIs)) has been well defined [8–13]. Furthermore, there is emerging concern about the long-term impact of increased ARV exposure on the growth of exposed infants [14, 15].\nIn four Canadian centres, triple cART at treatment doses has been routinely prescribed for the post-exposure prophylaxis (PEP) of neonates deemed at high risk of HIV infection, either because of detectable maternal viral load near delivery or in the absence of viral load results, clinical factors such as poor adherence or late diagnosis. In standard-risk situations, six weeks of ZDV monotherapy is prescribed. Given the limited data to support the use of cART, the primary objective of this study was to compare the safety and tolerability of cART versus ZDV monotherapy of PEP with respect to adverse events, haematological and growth parameters in exposed neonates.\n\nMethods:\nHIV-1 exposed children were eligible for this study if they were started on treatment doses of cART within 72 hours of birth because of incomplete maternal virologic suppression at delivery or, in the absence of maternal viral load results, a maternal history of incomplete adherence or non-adherence to cART, or late pregnancy initiation of cART. Neonatal cART was defined as any regimen of three or more antiretroviral agents, prescribed at treatment doses, for a minimum of six weeks. Regimens included ZDV (2 mg/kg every six hours prior to 2011, and 4 mg/kg twice daily in 2011–2013) for six weeks, Lamivudine (3TC) (2 mg/kg twice daily) for six weeks, with the addition of either NVP (150 mg/m2 daily for 14 days, followed by 150 mg/m2 twice daily for 14 days) or Nelfinavir (NFV) 40–50 mg/kg twice daily for six weeks) or LPV/Ritonavir (LPV/r) (300 mg/m2 twice daily) for six weeks. Combination regimens containing single or three doses of NVP were excluded.\nSubjects were identified for the period 1997–2013 from the clinical databases of four paediatric HIV-care institutions in Canada: The Hospital for Sick Children, Toronto; Children's Hospital of Eastern Ontario, Ottawa; Centre Hospitalier Universitaire (CHU) Sainte-Justine, Montreal; Stollery Children's Hospital, Edmonton. A control group consisting of all infants of HIV-positive mothers who were prescribed ZDV monotherapy during a three-year period (2010–2012) at the Hospital for Sick Children was selected (n=148). Approval for the study was obtained by the ethics review board of each participating institution; given the retrospective nature of the study, individual patient consent was not deemed necessary by the respective ethics review boards for the chart review.\n Statistical analysis Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).\nBaseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).\n\nStatistical analysis:\nBaseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).\n\nResults:\nOne hundred and forty-eight infants received triple cART during the study period. NFV-based cART was the most common regimen (55%) followed by NVP-based cART (40%) and LPV/r-based cART (5%). The most common reason for prescribing cART was documented detectable maternal viral load (78.4%). Other overlapping factors included poor adherence (45%), late diagnosis (20%), no antenatal care (8%) and refusal of antenatal ARV therapy (8%). Fifteen of the cART-treated infants and five of the ZDV-treated infants had missing six-month data, either because they were lost to follow-up (n=8) or they did not attend the scheduled six-month visit (but were seen at 18 months of age or older) (n=12).\nBaseline maternal and infant characteristics according to treatment group are described in Table 1. Mothers of cART-treated infants were younger, had higher viral loads and lower CD4 counts at the time of delivery, and were more likely to deliver vaginally and prematurely than mothers of ZDV monotherapy-treated infants.\nBaseline demographic and clinical characteristics of mothers and infantsa\n\nReported means and standard deviation;\nChi Square test of proportions or Wilcoxon rank-sum.\nHematological and Growth Parameters by treatment group†\nReported means and standard deviation\nStudent T test, Wilcoxon rank sum, or Kruskal-Wallace test where appropriate.\nn* observations recorded for each parameter out of a total of 148 cART recipients at birth, and 145 ZDV recipients.\nThirteen of the cART recipients were perinatally infected with HIV (8.8%). None of the ZDV recipients were infected. Five of the 13 (38.5%) had HIV-1 detected by PCR within 48 hours of birth suggesting in utero infection according to diagnostic standards. For the remaining eight, the timing of infection could not be ascertained as initial testing took place after 48 hours of life. Of the 13 infected children, four achieved long-term sustained virologic suppression, the details of which have been described elsewhere [16].\nNon-specific signs and symptoms, including vomiting, diarrhoea, rash, jitteriness or irritability, potentially attributable to medication-related adverse effects, were reported in 10.2% of cART recipients and none of the ZDV recipients (p<0.001). ARV treatment was discontinued prematurely in 9.5% (n=14) of cART recipients (five NVP-treated and nine PI-treated infants) compared with 2.1% (n=3) ZDV recipients (p=0.01). Reasons given for premature treatment discontinuation among cART recipients included significant anaemia (n=3; haemoglobin at two, four and four weeks of 62, 76 and 92 g/L, respectively), neutropenia (n=1; neutrophil count 0.4 cells/mm3 at four weeks), non-specific symptoms including rash, vomiting, and diarrhoea (n=6), and parental decision (n=4). In the six cases of non-specific symptoms, treatment discontinuation was precautionary, as the treating physician indicated that the symptoms were most likely not medication related. In four cases of parental discontinuation, parents stopped the medication without evidence of toxicity or consultation with their healthcare provider. ZDV monotherapy was stopped early in three children, all because of significant anaemia (haemoglobin at four weeks of 76, 85 and 72 g/L).\nThe potential impact of cART on haematologic parameters was evaluated by comparing haemoglobin level and absolute neutrophil count for cART-treated and ZDV monotherapy-treated infants at one, two and six months of age (Table 2). In the unadjusted analysis, haemoglobin was lower in cART-treated infants compared with ZDV-treated infants at one month of age (mean 105±19 vs. 109±17 g/L, p=0.04), but not at two or six months of age (mean 104±12 vs. 105±9 g/L, p=0.31 and 118±11 vs. 121±8 g/L, p=0.15). In a subgroup analysis of specific treatment type, mean haemoglobin was lower among PI-treated infants compared with NVP-treated infants at one month of age (104±17 vs. 107±21, p=0.09), though only statistically significant at six months of age (mean 115±10 vs. 124±9 g/L, p<0.001) (Figure 1).\nHaematological parameters according to treatment group.\nGraphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.) \nTo study the overall effect of treatment type on haemoglobin over time, we constructed a multivariate model adjusting for chronological age and gestational age (Supplementary Table, not shown). Overall, the mean haemoglobin was 2.07 g/L lower among cART versus ZDV-treated infants (p=0.04) over the six-month period. In the subgroup analysis looking at specific treatment type, mean haemoglobin was 3.62 g/L lower in those receiving PI-based cART compared with those receiving ZDV monotherapy (p=0.002); no such effect was observed for NVP-based cART compared with ZDV monotherapy (p=0.82). There was no difference in absolute neutrophil count between NVP-based cART, PI-based cART and ZDV monotherapy-treated infants at one, two or six months of age in the unadjusted analysis; this finding was maintained in the multivariable model adjusting for visit number, ethnicity and gestational age.\nWeight, length and HC were compared between all cART-treated and ZDV-treated infants, and between PI-based cART, NVP-based cART and ZDV at birth, one and six months of age (Table 2 and Figure 2). In the unadjusted analysis, cART-treated infants were smaller compared with ZDV-treated infants at birth (weight 2.91±0.60 vs. 3.09±0.56 kg, p=0.01; length 49.0±3.6 vs. 50.2±3.8 cm, p=0.03, head circumference 33.2±2.4 vs.33.6±2.3 cm, p=0.16). By six months of age, there was no difference in weight (8.03±1.12 kg vs. 8.25±1.12 kg, p=0.14) or head circumference (43.7±1.8 cm vs. 44.0±1.7, p=0.13), although the cART-treated group remained shorter than ZDV-treated infants (67.1±2.9 cm vs. 68.1±3.2 cm, p=0.01). In the subgroup analysis looking at specific treatment type, both NVP-and PI-treated infants were smaller than ZDV-treated infants across all growth parameters at birth; only the difference in length remained significant at six months of age.\nGrowth parameters according to treatment group.\nGraphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.) \nTo study the overall effect of treatment type on growth parameters over time, we constructed a multivariate fixed effects model adjusting for birthweight, gestational age, chronological age and gender. Overall, there was a mean HC difference of −0.63 cm among cART compared with ZDV-treated infants over the six-month period (p<0.001) (Supplementary Table, not shown). A similar effect was seen with respect to weight and length, with a mean weight difference of −236 g and length difference of −0.92 cm over the six-month period (p<0.001 and p=0.002, respectively). In the subgroup multivariate analysis comparing PI-based cART versus ZDV and NVP-based cART versus ZDV, there was no discernable difference between the two cART treatment types and these growth parameters.\n\nDiscussion:\nIn this retrospective study, cART initiated empirically for newborns deemed at high risk for perinatal HIV infection was generally well-tolerated, though adverse effects and consequent premature discontinuation of therapy did occur more often in the cART versus ZDV-treated group (9.5% vs. 2.1%). Among the 14 cART-treated children in whom medications were prematurely discontinued, four had their medications stopped by the parents without consultation with the healthcare provider and in the absence of any side effects, and six for precautionary reasons by the clinician even though the symptoms were not thought to be medication related, highlighting a lower threshold for treatment discontinuation by both parents and physicians among cART-treated infants.\nWith respect to haematological toxicity, the frequency and degree of anaemia observed in our cART-treated neonates was higher than that observed in the ZDV-monotherapy neonates. In the subgroup analysis of cART-treated infants, haemoglobin was lower at one month of age among PI versus NVP-treated infants, suggesting perhaps less haematological toxicity with NVP-based cART. The lower mean haemoglobin observed in cART-treated subjects compared with ZDV-monotherapy-treated subjects at one month of age was no longer evident at two and six months of age after stopping treatment, confirming the reversible nature of the anaemia. Reassuringly, compared with ZDV monotherapy, the use of cART was not associated with any discernible change in neutrophil count.\nWhile in the multivariate mixed effects model cART-treated infants had lower mean growth measures (HC, weight and length) than ZDV-monotherapy-treated infants over time, there were no absolute differences in growth measures for head circumference or weight at six months of age, indicating good catch-up growth among the cART-treated infants. The observed lower mean growth over time in cART recipients was therefore likely due to factors associated with the indications for the use of cART. Specifically, the higher overall incidence of prematurity among cART-treated infants was likely a result of poorly controlled maternal HIV, an established risk factor for low birthweight and preterm delivery [17]. The higher viral load and lower CD4 counts of mothers of cART recipients in our study supports this hypothesis. While we adjusted for preterm delivery and gestational age in the multivariate models for growth, we were unable to adjust for in utero drug exposure, social situation, maternal health, nutritional status and education, which may be important predictors of infant growth and development.\nBecause of the selective use of cART for infants at higher risk of perinatal transmission in our study, it is not appropriate to compare the risk of transmission among cART versus ZDV-treated infants as the latter group would have received better antenatal preventive management. The 8.8% overall transmission rate observed among cART recipients in our cohort was similar to that in a previous trial in which neonates of untreated mothers were randomized to different cART regimens, evidence of the high risk of transmission despite prophylaxis in these situations [6]. Key potential benefits of cART at treatment doses initiated as prophylaxis for neonates later found to be infected is that such treatment will preserve health by controlling viral replication more rapidly, reduce the risk of generating antiretroviral medication resistance, as seen with single or multiple dose NVP in non-cART strategies, and limit the size of HIV viral reservoirs, which could have significant implications for achieving HIV remission in the future [18–20]. Given the limited number of therapeutic drug regimens available for children in resource-limited settings, preventing drug resistance to first-line therapy given to prevent perinatal transmission is a particularly important consideration.\nOur study has a number of limitations, including its retrospective nature, relatively small sample size, and potential differences in the patient population at different study sites which may have influenced biological and growth outcomes. All control group infants were selected from a single centre, and the choice of PI-based versus NVP-based cART was also site specific. Given that these were the comparison groups of interest, we could not adjust for site and treatment type in the final analysis, thereby limiting our conclusions on specific cART treatment type.\n\nConclusions:\nIn summary, our study shows that cART at treatment doses administered as prophylaxis to “high-risk” HIV-exposed neonates is generally well tolerated and should be considered for newborn infants deemed at high risk of perinatally acquired HIV infection. However, close monitoring for anaemia, neutropenia and other side effects is important if such therapy is given. Until more robust pharmacokinetic data are available, the use of cART as prophylaxis in neonates may be limited to resource-rich settings where routine therapeutic drug monitoring is possible. However, further study is urgently needed in resource-limited settings where the vast majority of high-risk HIV-exposed infants are born. The potential benefits of cART, both to avert infection and, in case of transmission, preserve future therapeutic options and potentially limit the viral reservoir, may well outweigh the risks. Finally, if this strategy is used, counselling of parents and close follow-up is necessary to avoid premature treatment discontinuation and to ensure proper monitoring of cART-exposed infants.\n\nSupplementary Material:\nClick here for additional data file.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe optimal management of infants born to HIV-positive mothers who are untreated or have detectable viral load prior to delivery remains controversial. Despite the increasing use of combination antiretroviral therapy (cART) for post-exposure prophylaxis (PEP) of neonates at high risk of HIV infection, there is little safety and pharmacokinetic data to support this approach. The objective of this study was to evaluate the safety and tolerability of cART for PEP in HIV-exposed neonates.\n\nMethods:\nRetrospective study on 148 cART and 145 Zidovudine (ZDV) monotherapy-exposed infants identified from four Canadian centres where cART for PEP has routinely been prescribed in high-risk situations. Physician-reported adverse events and clinical outcomes were extracted by chart review. Haematological and growth parameters at birth, one and six months of age were compared between cART and ZDV-exposed infants using multivariate mixed effects modelling.\n\nResults:\nNon-specific signs and symptoms were reported in 10.2% of cART recipients versus none of the ZDV recipients. Treatment was discontinued prematurely in 9.5% of cART recipients versus 2.1% of ZDV recipients (p=0.01). In the multivariate model, cART recipients had lower mean haemoglobin (decrease of 2.07 g/L) over the 6-month period compared with ZDV recipients (p=0.04), but no effect was seen on absolute neutrophil count. cART recipients had lower weight and smaller head circumference at birth and one month of age compared with ZDV-exposed infants; these differences were no longer significant at six months of age.\n\nConclusions:\ncART administered at treatment doses for PEP in neonates was generally well tolerated, though a higher incidence of non-specific signs and symptoms and early treatment discontinuation occurred among cART recipients."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nWhile the overall risk of perinatal HIV transmission in the developed world has decreased to less than 1%, transmission still occurs, most often due to failure to diagnose HIV in pregnant women in a timely manner, or because of poor maternal drug adherence to, and/or refusal of, antiretroviral therapy [1]. Despite the relatively high risk of transmission in these situations, the optimal management of infants born to HIV-positive mothers, who are untreated or have detectable viral load prior to delivery despite receiving antenatal therapy, remains controversial. Current US Department of Health and Human Services (DHHS) guidelines recommend a six-week course of Zidovudine (ZDV), along with three doses of Nevirapine (NVP) in the first week of life for infants born to mothers who have not received any treatment [2]. However, in practice, many paediatric HIV specialists prescribe various combinations of three or more drugs to such “high-risk” neonates. In a national web-based survey of US care providers between December 2009 and January 2010, 62% reported the use of combination antiretroviral therapy (cART) during the previous year [3]. In a recent study from the United Kingdom, cART was prescribed to 71% of infants born to untreated mothers [4].\nDespite the increasing use of cART for prophylaxis in neonates at high risk of HIV infection, there is limited data on the safety of these different combination regimens, or on the appropriate dosing for the antiretroviral (ARV) medications used for this purpose. Transient haematological toxicity has been reported with the use of six-week post-natal ZDV [5], the recommended regimen in standard-risk situations, and a number of studies have suggested increased haematological toxicity associated with the addition of a second nucleoside analogue [6, 7]. Neither the safety nor the optimal dosing of additional drugs used for prophylaxis in neonates (non-nucleoside reverse transcriptase inhibitors or protease inhibitors (PIs)) has been well defined [8–13]. Furthermore, there is emerging concern about the long-term impact of increased ARV exposure on the growth of exposed infants [14, 15].\nIn four Canadian centres, triple cART at treatment doses has been routinely prescribed for the post-exposure prophylaxis (PEP) of neonates deemed at high risk of HIV infection, either because of detectable maternal viral load near delivery or in the absence of viral load results, clinical factors such as poor adherence or late diagnosis. In standard-risk situations, six weeks of ZDV monotherapy is prescribed. Given the limited data to support the use of cART, the primary objective of this study was to compare the safety and tolerability of cART versus ZDV monotherapy of PEP with respect to adverse events, haematological and growth parameters in exposed neonates.\n\nConclusions:\nIn summary, our study shows that cART at treatment doses administered as prophylaxis to “high-risk” HIV-exposed neonates is generally well tolerated and should be considered for newborn infants deemed at high risk of perinatally acquired HIV infection. However, close monitoring for anaemia, neutropenia and other side effects is important if such therapy is given. Until more robust pharmacokinetic data are available, the use of cART as prophylaxis in neonates may be limited to resource-rich settings where routine therapeutic drug monitoring is possible. However, further study is urgently needed in resource-limited settings where the vast majority of high-risk HIV-exposed infants are born. The potential benefits of cART, both to avert infection and, in case of transmission, preserve future therapeutic options and potentially limit the viral reservoir, may well outweigh the risks. Finally, if this strategy is used, counselling of parents and close follow-up is necessary to avoid premature treatment discontinuation and to ensure proper monitoring of cART-exposed infants."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe optimal management of infants born to HIV-positive mothers who are untreated or have detectable viral load prior to delivery remains controversial. Despite the increasing use of combination antiretroviral therapy (cART) for post-exposure prophylaxis (PEP) of neonates at high risk of HIV infection, there is little safety and pharmacokinetic data to support this approach. The objective of this study was to evaluate the safety and tolerability of cART for PEP in HIV-exposed neonates.\n\nMethods:\nRetrospective study on 148 cART and 145 Zidovudine (ZDV) monotherapy-exposed infants identified from four Canadian centres where cART for PEP has routinely been prescribed in high-risk situations. Physician-reported adverse events and clinical outcomes were extracted by chart review. Haematological and growth parameters at birth, one and six months of age were compared between cART and ZDV-exposed infants using multivariate mixed effects modelling.\n\nResults:\nNon-specific signs and symptoms were reported in 10.2% of cART recipients versus none of the ZDV recipients. Treatment was discontinued prematurely in 9.5% of cART recipients versus 2.1% of ZDV recipients (p=0.01). In the multivariate model, cART recipients had lower mean haemoglobin (decrease of 2.07 g/L) over the 6-month period compared with ZDV recipients (p=0.04), but no effect was seen on absolute neutrophil count. cART recipients had lower weight and smaller head circumference at birth and one month of age compared with ZDV-exposed infants; these differences were no longer significant at six months of age.\n\nConclusions:\ncART administered at treatment doses for PEP in neonates was generally well tolerated, though a higher incidence of non-specific signs and symptoms and early treatment discontinuation occurred among cART recipients."},"whole_article_text_length":{"kind":"number","value":4271,"string":"4,271"},"whole_article_abstract_length":{"kind":"number","value":331,"string":"331"},"num_sections":{"kind":"number","value":7,"string":"7"},"most_frequent_words":{"kind":"list like","value":["cart","infants","zdv","age","treated","treatment","parameters","treated infants","growth","based"],"string":"[\n \"cart\",\n \"infants\",\n \"zdv\",\n \"age\",\n \"treated\",\n \"treatment\",\n \"parameters\",\n \"treated infants\",\n \"growth\",\n \"based\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] risk | neonates | week | safety | hiv | cart | high | high risk | use | born [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] parameters | age | mg | daily | cart | growth | twice | twice daily | growth parameters | zdv [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] treated | cart | treated infants | zdv | infants | age | vs | recipients | cm | difference [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] monitoring | risk | high risk | high | close | risk hiv exposed | high risk hiv exposed | cart | exposed | risk hiv [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cart | age | infants | parameters | zdv | treated | growth | treatment | risk | treated infants [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cart | age | infants | parameters | zdv | treated | growth | treatment | risk | treated infants [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 148 | 145 | ZDV | four | Canadian | PEP ||| ||| one and six months of age | ZDV [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 10.2% | ZDV ||| 9.5% | 2.1% | ZDV ||| 2.07 | 6-month | ZDV | p=0.04 ||| birth and one month of age | ZDV | six months of age [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| 148 | 145 | ZDV | four | Canadian | PEP ||| ||| one and six months of age | ZDV ||| 10.2% | ZDV ||| 9.5% | 2.1% | ZDV ||| 2.07 | 6-month | ZDV | p=0.04 ||| birth and one month of age | ZDV | six months of age ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| 148 | 145 | ZDV | four | Canadian | PEP ||| ||| one and six months of age | ZDV ||| 10.2% | ZDV ||| 9.5% | 2.1% | ZDV ||| 2.07 | 6-month | ZDV | p=0.04 ||| birth and one month of age | ZDV | six months of age ||| [SUMMARY]"}}},{"rowIdx":261,"cells":{"title":{"kind":"string","value":"Economic burden of stroke in a large county in Sweden."},"pmid":{"kind":"string","value":"23013284"},"background_abstract":{"kind":"string","value":"Stroke remains to be a major burden of disease, often causing death or physical impairment or disability. This paper estimates the economic burden of stroke in a large county of 1.5 million inhabitants in western Sweden."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"The economic burden of stroke was estimated from a societal perspective with an incidence approach. Data were collected from clinical registries and 3,074 patients were included. In the cost calculations, both direct and indirect costs were estimated and were based on costs for 12 months after a first-ever stroke."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The total excess costs in the first 12 months after the first-ever stroke for a population of 1.5 million was 629 million SEK (€69 million). Men consumed more acute care in hospitals, whereas women consumed more rehabilitation and long-term care provided by the municipalities. Younger patients brought a significantly higher burden on society compared with older patients due to the loss of productivity and the increased use of resources in health care."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The results of this cost-of-illness study were based on an improved calculation process in a number of fields and are consistent with previous studies. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-time care and informal care and productivity loss explains 10% of total cost for the stroke disease. The result of this study can be used for further development of the methods for economic analyses as well as for analysis of improvements and investments in health care."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Aged, 80 and over","Cost of Illness","Female","Health Care Costs","Health Services Research","Humans","Male","Middle Aged","Registries","Retrospective Studies","Stroke","Sweden"],"string":"[\n \"Aged\",\n \"Aged, 80 and over\",\n \"Cost of Illness\",\n \"Female\",\n \"Health Care Costs\",\n \"Health Services Research\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Registries\",\n \"Retrospective Studies\",\n \"Stroke\",\n \"Sweden\"\n]"},"pmcid":{"kind":"string","value":"3506527"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Stroke continues to be a major burden of disease\n[1,2] even when the risk of premature death has been reduced\n[3]. Several assessments of the direct and indirect costs have been published\n[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail\n[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population\n[11]. Thus, the financial burden of diseases such as stroke is important.\nThe Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure\n1). All units of care regardless of the provider are financed by local taxes.\nProviders in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.\nA health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.\nThe aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Epidemiology Stroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\nStroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\n Data source To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.\nTo identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.\n Direct costs in health care Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.\nData were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.\n Municipal cost As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\nAs this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\n Informal care volume Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\nEstimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\n Informal care costs The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).\nThe opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).\n Cost for loss of productivity Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\nEstimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\n Lifetime costs Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.\nEstimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Excess total costs This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nThis study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\n Excess cost per individual The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nThe average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\n Excess costs the first three years Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\nCosts after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\n Generalizability and comparability Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.\nCosts for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The results of this cost of illness study are consistent with previous studies, although we have the cost calculation process in a number of fields. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-term care, informal care and productivity loss explain 10% of total cost for the stroke disease. The results of this study can be used as further development as well as for improvements and investments in health care as well as for development of econometric methods."},"other_sections_titles":{"kind":"list like","value":["Background","Epidemiology","Data source","Direct costs in health care","Municipal cost","Informal care volume","Informal care costs","Cost for loss of productivity","Lifetime costs","Excess total costs","Excess cost per individual","Excess costs the first three years","Generalizability and comparability","Methodological considerations","Analysis of result","Comparison with other studies","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Epidemiology\",\n \"Data source\",\n \"Direct costs in health care\",\n \"Municipal cost\",\n \"Informal care volume\",\n \"Informal care costs\",\n \"Cost for loss of productivity\",\n \"Lifetime costs\",\n \"Excess total costs\",\n \"Excess cost per individual\",\n \"Excess costs the first three years\",\n \"Generalizability and comparability\",\n \"Methodological considerations\",\n \"Analysis of result\",\n \"Comparison with other studies\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Stroke continues to be a major burden of disease\n[1,2] even when the risk of premature death has been reduced\n[3]. Several assessments of the direct and indirect costs have been published\n[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail\n[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population\n[11]. Thus, the financial burden of diseases such as stroke is important.\nThe Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure\n1). All units of care regardless of the provider are financed by local taxes.\nProviders in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.\nA health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.\nThe aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke.","Stroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.","To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.","Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.","As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.","Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.","The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).","Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.","Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.","This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).","The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).","Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.","Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.","The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\n[11]. Thus, systematic data on informal care is an essential area for further research.","This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.","When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.","The study was performed upon request from the county council of Västra Götaland and the authors acknowledge financial support from the county council of Västra Götaland. However, none of the authors has received reimbursement, fees, funding, or salary from any other organisation. None of the authors hold any stock or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. None of the authors hold or are currently applying for any patents relating to the content of the manuscript or received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. None of the authors have any other financial or non-financial competing interests.","JP has written the manuscript and has made substantial contributions to analysis. JFN have made substantial contributions to analysis and revising the manuscript. IK have been involved in designing the study, drafting and revising the manuscript. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6963/12/341/prepub\n"],"string":"[\n \"Stroke continues to be a major burden of disease\\n[1,2] even when the risk of premature death has been reduced\\n[3]. Several assessments of the direct and indirect costs have been published\\n[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail\\n[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population\\n[11]. Thus, the financial burden of diseases such as stroke is important.\\nThe Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure\\n1). All units of care regardless of the provider are financed by local taxes.\\nProviders in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.\\nA health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.\\nThe aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke.\",\n \"Stroke is a clinical syndrome with several different pathologies\\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\\n[5]. The sex distribution in the whole group was equal (Table\\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\\n2).\\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\\nTotal number of patients n = 3,074.\\nData source: Local administrative register and The Stroke Register.\\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\\nTotal number of patients n = 3,074.\\nData source: The Stroke register.\\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\",\n \"To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\\nChain of health care for stroke survivors in Sweden.\",\n \"Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\\nPrices of year 2008 in SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\\nData source: see methods.\",\n \"As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\",\n \"Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\",\n \"The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\\n[20] of the production loss value (Table\\n3).\",\n \"Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\\n[19] (Table\\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\",\n \"Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.\",\n \"This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\",\n \"The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\\n5).\\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\",\n \"Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\",\n \"Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.\",\n \"The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\\n[11]. Thus, systematic data on informal care is an essential area for further research.\",\n \"This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.\",\n \"When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.\",\n \"The study was performed upon request from the county council of Västra Götaland and the authors acknowledge financial support from the county council of Västra Götaland. However, none of the authors has received reimbursement, fees, funding, or salary from any other organisation. None of the authors hold any stock or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. None of the authors hold or are currently applying for any patents relating to the content of the manuscript or received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. None of the authors have any other financial or non-financial competing interests.\",\n \"JP has written the manuscript and has made substantial contributions to analysis. JFN have made substantial contributions to analysis and revising the manuscript. IK have been involved in designing the study, drafting and revising the manuscript. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1472-6963/12/341/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Epidemiology","Data source","Direct costs in health care","Municipal cost","Informal care volume","Informal care costs","Cost for loss of productivity","Lifetime costs","Results","Excess total costs","Excess cost per individual","Excess costs the first three years","Generalizability and comparability","Discussion","Methodological considerations","Analysis of result","Comparison with other studies","Conclusions","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Epidemiology\",\n \"Data source\",\n \"Direct costs in health care\",\n \"Municipal cost\",\n \"Informal care volume\",\n \"Informal care costs\",\n \"Cost for loss of productivity\",\n \"Lifetime costs\",\n \"Results\",\n \"Excess total costs\",\n \"Excess cost per individual\",\n \"Excess costs the first three years\",\n \"Generalizability and comparability\",\n \"Discussion\",\n \"Methodological considerations\",\n \"Analysis of result\",\n \"Comparison with other studies\",\n \"Conclusions\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Stroke continues to be a major burden of disease\n[1,2] even when the risk of premature death has been reduced\n[3]. Several assessments of the direct and indirect costs have been published\n[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail\n[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population\n[11]. Thus, the financial burden of diseases such as stroke is important.\nThe Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure\n1). All units of care regardless of the provider are financed by local taxes.\nProviders in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.\nA health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.\nThe aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke."," Epidemiology Stroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\nStroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\n Data source To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.\nTo identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.\n Direct costs in health care Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.\nData were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.\n Municipal cost As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\nAs this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\n Informal care volume Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\nEstimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\n Informal care costs The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).\nThe opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).\n Cost for loss of productivity Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\nEstimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\n Lifetime costs Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.\nEstimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.","Stroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.","To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.","Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.","As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.","Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.","The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).","Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.","Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year."," Excess total costs This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nThis study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\n Excess cost per individual The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nThe average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\n Excess costs the first three years Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\nCosts after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\n Generalizability and comparability Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.\nCosts for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.","This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).","The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).","Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.","Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates."," Methodological considerations The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\n[11]. Thus, systematic data on informal care is an essential area for further research.\nThe estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\n[11]. Thus, systematic data on informal care is an essential area for further research.\n Analysis of result This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.\nThis study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.\n Comparison with other studies When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.\nWhen comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.","The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\n[11]. Thus, systematic data on informal care is an essential area for further research.","This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.","When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.","The results of this cost of illness study are consistent with previous studies, although we have the cost calculation process in a number of fields. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-term care, informal care and productivity loss explain 10% of total cost for the stroke disease. The results of this study can be used as further development as well as for improvements and investments in health care as well as for development of econometric methods.","The study was performed upon request from the county council of Västra Götaland and the authors acknowledge financial support from the county council of Västra Götaland. However, none of the authors has received reimbursement, fees, funding, or salary from any other organisation. None of the authors hold any stock or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. None of the authors hold or are currently applying for any patents relating to the content of the manuscript or received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. None of the authors have any other financial or non-financial competing interests.","JP has written the manuscript and has made substantial contributions to analysis. JFN have made substantial contributions to analysis and revising the manuscript. IK have been involved in designing the study, drafting and revising the manuscript. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6963/12/341/prepub\n"],"string":"[\n \"Stroke continues to be a major burden of disease\\n[1,2] even when the risk of premature death has been reduced\\n[3]. Several assessments of the direct and indirect costs have been published\\n[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail\\n[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population\\n[11]. Thus, the financial burden of diseases such as stroke is important.\\nThe Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure\\n1). All units of care regardless of the provider are financed by local taxes.\\nProviders in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.\\nA health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.\\nThe aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke.\",\n \" Epidemiology Stroke is a clinical syndrome with several different pathologies\\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\\n[5]. The sex distribution in the whole group was equal (Table\\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\\n2).\\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\\nTotal number of patients n = 3,074.\\nData source: Local administrative register and The Stroke Register.\\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\\nTotal number of patients n = 3,074.\\nData source: The Stroke register.\\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\\nStroke is a clinical syndrome with several different pathologies\\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\\n[5]. The sex distribution in the whole group was equal (Table\\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\\n2).\\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\\nTotal number of patients n = 3,074.\\nData source: Local administrative register and The Stroke Register.\\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\\nTotal number of patients n = 3,074.\\nData source: The Stroke register.\\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\\n Data source To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\\nChain of health care for stroke survivors in Sweden.\\nTo identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\\nChain of health care for stroke survivors in Sweden.\\n Direct costs in health care Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\\nPrices of year 2008 in SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\\nData source: see methods.\\nData were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\\nPrices of year 2008 in SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\\nData source: see methods.\\n Municipal cost As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\\nAs this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\\n Informal care volume Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\\nEstimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\\n Informal care costs The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\\n[20] of the production loss value (Table\\n3).\\nThe opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\\n[20] of the production loss value (Table\\n3).\\n Cost for loss of productivity Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\\n[19] (Table\\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\\nEstimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\\n[19] (Table\\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\\n Lifetime costs Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.\\nEstimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.\",\n \"Stroke is a clinical syndrome with several different pathologies\\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\\n[5]. The sex distribution in the whole group was equal (Table\\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\\n2).\\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\\nTotal number of patients n = 3,074.\\nData source: Local administrative register and The Stroke Register.\\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\\nTotal number of patients n = 3,074.\\nData source: The Stroke register.\\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\",\n \"To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\\nChain of health care for stroke survivors in Sweden.\",\n \"Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\\nPrices of year 2008 in SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\\nData source: see methods.\",\n \"As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\",\n \"Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\",\n \"The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\\n[20] of the production loss value (Table\\n3).\",\n \"Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\\n[19] (Table\\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\",\n \"Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.\",\n \" Excess total costs This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\\nThis study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\\n Excess cost per individual The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\\n5).\\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\\nThe average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\\n5).\\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\\n Excess costs the first three years Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\\nCosts after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\\n Generalizability and comparability Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.\\nCosts for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.\",\n \"This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\",\n \"The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\\n5).\\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\",\n \"Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\",\n \"Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.\",\n \" Methodological considerations The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\\n[11]. Thus, systematic data on informal care is an essential area for further research.\\nThe estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\\n[11]. Thus, systematic data on informal care is an essential area for further research.\\n Analysis of result This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.\\nThis study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.\\n Comparison with other studies When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.\\nWhen comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.\",\n \"The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\\n[11]. Thus, systematic data on informal care is an essential area for further research.\",\n \"This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.\",\n \"When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.\",\n \"The results of this cost of illness study are consistent with previous studies, although we have the cost calculation process in a number of fields. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-term care, informal care and productivity loss explain 10% of total cost for the stroke disease. The results of this study can be used as further development as well as for improvements and investments in health care as well as for development of econometric methods.\",\n \"The study was performed upon request from the county council of Västra Götaland and the authors acknowledge financial support from the county council of Västra Götaland. However, none of the authors has received reimbursement, fees, funding, or salary from any other organisation. None of the authors hold any stock or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. None of the authors hold or are currently applying for any patents relating to the content of the manuscript or received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. None of the authors have any other financial or non-financial competing interests.\",\n \"JP has written the manuscript and has made substantial contributions to analysis. JFN have made substantial contributions to analysis and revising the manuscript. IK have been involved in designing the study, drafting and revising the manuscript. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1472-6963/12/341/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,"results",null,null,null,null,"discussion",null,null,null,"conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n null,\n null,\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Burden","Cost of illness","Health economics","Incidence cost","Stroke","Sweden"],"string":"[\n \"Burden\",\n \"Cost of illness\",\n \"Health economics\",\n \"Incidence cost\",\n \"Stroke\",\n \"Sweden\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nStroke continues to be a major burden of disease\n[1,2] even when the risk of premature death has been reduced\n[3]. Several assessments of the direct and indirect costs have been published\n[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail\n[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population\n[11]. Thus, the financial burden of diseases such as stroke is important.\nThe Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure\n1). All units of care regardless of the provider are financed by local taxes.\nProviders in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.\nA health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.\nThe aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke.\n\nMethods:\n Epidemiology Stroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\nStroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\n Data source To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.\nTo identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.\n Direct costs in health care Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.\nData were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.\n Municipal cost As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\nAs this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\n Informal care volume Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\nEstimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\n Informal care costs The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).\nThe opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).\n Cost for loss of productivity Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\nEstimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\n Lifetime costs Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.\nEstimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.\n\nEpidemiology:\nStroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\n\nData source:\nTo identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.\n\nDirect costs in health care:\nData were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.\n\nMunicipal cost:\nAs this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\n\nInformal care volume:\nEstimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\n\nInformal care costs:\nThe opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).\n\nCost for loss of productivity:\nEstimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\n\nLifetime costs:\nEstimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.\n\nResults:\n Excess total costs This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nThis study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\n Excess cost per individual The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nThe average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\n Excess costs the first three years Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\nCosts after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\n Generalizability and comparability Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.\nCosts for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.\n\nExcess total costs:\nThis study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\n\nExcess cost per individual:\nThe average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\n\nExcess costs the first three years:\nCosts after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\n\nGeneralizability and comparability:\nCosts for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.\n\nDiscussion:\n Methodological considerations The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\n[11]. Thus, systematic data on informal care is an essential area for further research.\nThe estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\n[11]. Thus, systematic data on informal care is an essential area for further research.\n Analysis of result This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.\nThis study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.\n Comparison with other studies When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.\nWhen comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.\n\nMethodological considerations:\nThe estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\n[11]. Thus, systematic data on informal care is an essential area for further research.\n\nAnalysis of result:\nThis study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.\n\nComparison with other studies:\nWhen comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.\n\nConclusions:\nThe results of this cost of illness study are consistent with previous studies, although we have the cost calculation process in a number of fields. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-term care, informal care and productivity loss explain 10% of total cost for the stroke disease. The results of this study can be used as further development as well as for improvements and investments in health care as well as for development of econometric methods.\n\nCompeting interests:\nThe study was performed upon request from the county council of Västra Götaland and the authors acknowledge financial support from the county council of Västra Götaland. However, none of the authors has received reimbursement, fees, funding, or salary from any other organisation. None of the authors hold any stock or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. None of the authors hold or are currently applying for any patents relating to the content of the manuscript or received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. None of the authors have any other financial or non-financial competing interests.\n\nAuthors’ contributions:\nJP has written the manuscript and has made substantial contributions to analysis. JFN have made substantial contributions to analysis and revising the manuscript. IK have been involved in designing the study, drafting and revising the manuscript. All authors read and approved the final manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6963/12/341/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nStroke remains to be a major burden of disease, often causing death or physical impairment or disability. This paper estimates the economic burden of stroke in a large county of 1.5 million inhabitants in western Sweden.\n\nMethods:\nThe economic burden of stroke was estimated from a societal perspective with an incidence approach. Data were collected from clinical registries and 3,074 patients were included. In the cost calculations, both direct and indirect costs were estimated and were based on costs for 12 months after a first-ever stroke.\n\nResults:\nThe total excess costs in the first 12 months after the first-ever stroke for a population of 1.5 million was 629 million SEK (€69 million). Men consumed more acute care in hospitals, whereas women consumed more rehabilitation and long-term care provided by the municipalities. Younger patients brought a significantly higher burden on society compared with older patients due to the loss of productivity and the increased use of resources in health care.\n\nConclusions:\nThe results of this cost-of-illness study were based on an improved calculation process in a number of fields and are consistent with previous studies. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-time care and informal care and productivity loss explains 10% of total cost for the stroke disease. The result of this study can be used for further development of the methods for economic analyses as well as for analysis of improvements and investments in health care."},"background_conclusion_text":{"kind":"string","value":"Background:\nStroke continues to be a major burden of disease\n[1,2] even when the risk of premature death has been reduced\n[3]. Several assessments of the direct and indirect costs have been published\n[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail\n[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population\n[11]. Thus, the financial burden of diseases such as stroke is important.\nThe Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure\n1). All units of care regardless of the provider are financed by local taxes.\nProviders in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.\nA health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.\nThe aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke.\n\nConclusions:\nThe results of this cost of illness study are consistent with previous studies, although we have the cost calculation process in a number of fields. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-term care, informal care and productivity loss explain 10% of total cost for the stroke disease. The results of this study can be used as further development as well as for improvements and investments in health care as well as for development of econometric methods."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nStroke remains to be a major burden of disease, often causing death or physical impairment or disability. This paper estimates the economic burden of stroke in a large county of 1.5 million inhabitants in western Sweden.\n\nMethods:\nThe economic burden of stroke was estimated from a societal perspective with an incidence approach. Data were collected from clinical registries and 3,074 patients were included. In the cost calculations, both direct and indirect costs were estimated and were based on costs for 12 months after a first-ever stroke.\n\nResults:\nThe total excess costs in the first 12 months after the first-ever stroke for a population of 1.5 million was 629 million SEK (€69 million). Men consumed more acute care in hospitals, whereas women consumed more rehabilitation and long-term care provided by the municipalities. Younger patients brought a significantly higher burden on society compared with older patients due to the loss of productivity and the increased use of resources in health care.\n\nConclusions:\nThe results of this cost-of-illness study were based on an improved calculation process in a number of fields and are consistent with previous studies. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-time care and informal care and productivity loss explains 10% of total cost for the stroke disease. The result of this study can be used for further development of the methods for economic analyses as well as for analysis of improvements and investments in health care."},"whole_article_text_length":{"kind":"number","value":10986,"string":"10,986"},"whole_article_abstract_length":{"kind":"number","value":293,"string":"293"},"num_sections":{"kind":"number","value":23,"string":"23"},"most_frequent_words":{"kind":"list like","value":["care","costs","stroke","health","cost","health care","patients","data","study","year"],"string":"[\n \"care\",\n \"costs\",\n \"stroke\",\n \"health\",\n \"cost\",\n \"health care\",\n \"patients\",\n \"data\",\n \"study\",\n \"year\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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[SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Burden | Cost of illness | Health economics | Incidence cost | Stroke | Sweden [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Burden | Cost of illness | Health economics | Incidence cost | Stroke | Sweden [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Burden | Cost of illness | Health economics | Incidence cost | Stroke | Sweden [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cost of Illness | Female | Health Care Costs | Health Services Research | Humans | Male | Middle Aged | Registries | Retrospective Studies | Stroke | Sweden [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cost of Illness | Female | Health Care Costs | Health Services 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[SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] care | costs | stroke | health | cost | health care | patients | data | study | year [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] care | health | provide | health care | swedish | system | health care system | swedish health care | swedish health | care system [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | data | care | register | costs | based | patients | health | age | income [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] costs | care | total | total costs | stroke | year | excess | cost | sek | individual [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] care | development | results | cost | care development | process number fields | studies cost calculation | studies cost calculation process | process | process number [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] care | costs | stroke | health | cost | study | health care | year | data | patients [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] care | costs | stroke | health | cost | study | health care | year | data | patients [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 1.5 million | Sweden [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 3,074 ||| 12 months | first [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] the first 12 months | first | 1.5 million | 629 million | SEK | €69 million ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 50% | 40% | 10% ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 1.5 million | Sweden ||| ||| 3,074 ||| 12 months | first ||| ||| the first 12 months | first | 1.5 million | 629 million | SEK | €69 million ||| ||| ||| ||| 50% | 40% | 10% ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 1.5 million | Sweden ||| ||| 3,074 ||| 12 months | first ||| ||| the first 12 months | first | 1.5 million | 629 million | SEK | €69 million ||| ||| ||| ||| 50% | 40% | 10% ||| [SUMMARY]"}}},{"rowIdx":262,"cells":{"title":{"kind":"string","value":"Role of tight junction proteins in gastroesophageal reflux disease."},"pmid":{"kind":"string","value":"22994974"},"background_abstract":{"kind":"string","value":"Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function that is regulated by cell-cell contacts. The aim of the study was to investigate the expression pattern of selected components involved in the formation of tight junctions in relation to GERD."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Eighty-four patients with GERD-related symptoms with endoscopic signs (erosive: n = 47) or without them (non-erosive: n = 37) as well as 26 patients lacking GERD-specific symptoms as controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and adapted Ismeil-Beigi criteria, respectively. Mucosal biopsies from distal esophagus were taken for analysis by histopathology, immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (RT-PCR) of five genes encoding tight junction components [Occludin, Claudin-1, -2, Zona occludens (ZO-1, -2)]."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Histopathology confirmed GERD-specific alterations as dilated intercellular spaces in the esophageal mucosa of patients with GERD compared to controls (P < 0.05). Claudin-1 and -2 were 2- to 6-fold upregulation on transcript (P < 0.01) and in part on protein level (P < 0.015) in GERD, while subgroup analysis of revealed this upregulation for ERD only. In both erosive and non-erosive reflux disease, expression levels of Occludin and ZO-1,-2 were not significantly affected. Notably, the induced expression of both claudins did not correlate with histopathological parameters (basal cell hyperplasia, dilated intercellular spaces) in patients with GERD."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Taken together, the missing correlation between the expression of tight junction-related components and histomorphological GERD-specific alterations does not support a major role of the five proteins studied in the pathogenesis of GERD."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Esophagitis","Esophagoscopy","Female","Gastroesophageal Reflux","Gastroscopy","Gene Expression Regulation","Humans","Immunohistochemistry","Male","Middle Aged","Tight Junction Proteins","Young Adult"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Esophagitis\",\n \"Esophagoscopy\",\n \"Female\",\n \"Gastroesophageal Reflux\",\n \"Gastroscopy\",\n \"Gene Expression Regulation\",\n \"Humans\",\n \"Immunohistochemistry\",\n \"Male\",\n \"Middle Aged\",\n \"Tight Junction Proteins\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"3503771"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Gastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world\n[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)\n[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification\n[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD\n[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD\n[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium\n[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located\n[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD\n[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD\n[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function\n[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others\n[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma\n[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models\n[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4\n[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4\n[33].\nHere, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Study design and patients’ characteristics Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nBetween 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\n Endoscopy and histopathology The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].\nThe patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].\n Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.\nExtraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.\n Immunohistochemical analysis of tight junctional components Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\nImmunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\n Statistical analysis Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.\nData are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Patients and GERD-specific histomorphological changes The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\nThe three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\n Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease As exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\nAs exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\n Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).\nIn order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In summary, this study demonstrates a partial upregulation of tight junction-related components, in particular Claudin-1, in relation to GERD. Since identified molecular changes do not correlate with histomorphological alterations in general, a major role of Claudin-1 as of the other four tight junction-related proteins in the pathogenesis of GERD can not be concluded from our study."},"other_sections_titles":{"kind":"list like","value":["Background","Study design and patients’ characteristics","Inclusion criteria","Exclusion criteria","Endoscopy and histopathology","Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes","Immunohistochemical analysis of tight junctional components","Statistical analysis","Patients and GERD-specific histomorphological changes","Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease","Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa","Abbreviations","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Study design and patients’ characteristics\",\n \"Inclusion criteria\",\n \"Exclusion criteria\",\n \"Endoscopy and histopathology\",\n \"Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes\",\n \"Immunohistochemical analysis of tight junctional components\",\n \"Statistical analysis\",\n \"Patients and GERD-specific histomorphological changes\",\n \"Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease\",\n \"Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Gastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world\n[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)\n[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification\n[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD\n[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD\n[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium\n[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located\n[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD\n[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD\n[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function\n[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others\n[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma\n[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models\n[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4\n[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4\n[33].\nHere, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD.","Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.","Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.","Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.","The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].","Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.","Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.","Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.","The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.","As exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).","In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).","BE: Barrett’s esophagus; ERD: Erosive reflux disease; NERD: Nonerosive reflux disease; GEJ: Gastroesophageal junction; GERD: Gastroesophageal reflux disease; H2RA: Histamine-receptor antagonist; H. pylori: Helicobacter pylori; ICS: Intercellular spaces; INR: International normalized ratio; iNOS: Inducible nitro oxygen synthetase; JAM: Junctional adhesion molecule; NFκB: Nuclear factor kappa B; PAR-2: Protease activated receptor-2; PPI: Proton pump inhibitor; RDQ: Reflux disease questionnaire; ROS: Reactive oxygen species; RT-PCR: Reverse transcription polymerase chain reaction; ZO: Zona occludens.","The authors declare that they have no competing interest concerning the content of this article.","KM, PM and TW designed the study. KM, LCF enrolled the majority of patients. AK provided clinical data. TW coordinated and performed laboratory work. DK and AR provided histopathological and immunohistochemical data. TW and SK performed statistical analysis. The manuscript was drafted by TW, AK, DK, and reviewed for important intellectual content by KM and PM. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-230X/12/128/prepub\n"],"string":"[\n \"Gastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world\\n[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)\\n[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification\\n[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD\\n[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD\\n[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium\\n[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located\\n[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD\\n[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD\\n[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function\\n[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others\\n[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma\\n[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models\\n[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4\\n[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4\\n[33].\\nHere, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD.\",\n \"Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\\n1A and Table\\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\",\n \"Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\",\n \"Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\",\n \"The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\\n[34,36], molecules related to barrier functions\\n[37,38], desmosomal proteins\\n[39] and histopathological alterations\\n[34].\",\n \"Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\\nCharacteristics of primers, RT-PCR protocol and antibodies\\nmab: monoclonal antibody, fw: forward, rv: reverse.\",\n \"Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\",\n \"Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.\",\n \"The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\\n1). Histomorphological alterations are shown in Table\\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\\n3).\\nHistopathological parameters\\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\",\n \"As exemplarily demonstrated in figure\\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\\n4A and\\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\\n1).\\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\\n4A, and B.\\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\",\n \"In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\\n5B).\\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\\n5A, and 5B.\\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\\nPanel A: The histopathological scores (Table\\n3) were correlated with gene expression levels (transcript levels, Table\\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\\nIn addition to the correlation based on transcript levels (Figure\\n3, Table\\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\\n4B) and histopathological alterations (Table\\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).\",\n \"BE: Barrett’s esophagus; ERD: Erosive reflux disease; NERD: Nonerosive reflux disease; GEJ: Gastroesophageal junction; GERD: Gastroesophageal reflux disease; H2RA: Histamine-receptor antagonist; H. pylori: Helicobacter pylori; ICS: Intercellular spaces; INR: International normalized ratio; iNOS: Inducible nitro oxygen synthetase; JAM: Junctional adhesion molecule; NFκB: Nuclear factor kappa B; PAR-2: Protease activated receptor-2; PPI: Proton pump inhibitor; RDQ: Reflux disease questionnaire; ROS: Reactive oxygen species; RT-PCR: Reverse transcription polymerase chain reaction; ZO: Zona occludens.\",\n \"The authors declare that they have no competing interest concerning the content of this article.\",\n \"KM, PM and TW designed the study. KM, LCF enrolled the majority of patients. AK provided clinical data. TW coordinated and performed laboratory work. DK and AR provided histopathological and immunohistochemical data. TW and SK performed statistical analysis. The manuscript was drafted by TW, AK, DK, and reviewed for important intellectual content by KM and PM. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-230X/12/128/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study design and patients’ characteristics","Inclusion criteria","Exclusion criteria","Endoscopy and histopathology","Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes","Immunohistochemical analysis of tight junctional components","Statistical analysis","Results","Patients and GERD-specific histomorphological changes","Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease","Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study design and patients’ characteristics\",\n \"Inclusion criteria\",\n \"Exclusion criteria\",\n \"Endoscopy and histopathology\",\n \"Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes\",\n \"Immunohistochemical analysis of tight junctional components\",\n \"Statistical analysis\",\n \"Results\",\n \"Patients and GERD-specific histomorphological changes\",\n \"Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease\",\n \"Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Gastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world\n[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)\n[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification\n[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD\n[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD\n[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium\n[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located\n[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD\n[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD\n[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function\n[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others\n[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma\n[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models\n[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4\n[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4\n[33].\nHere, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD."," Study design and patients’ characteristics Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nBetween 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\n Endoscopy and histopathology The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].\nThe patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].\n Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.\nExtraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.\n Immunohistochemical analysis of tight junctional components Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\nImmunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\n Statistical analysis Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.\nData are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.","Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.","Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.","Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.","The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].","Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.","Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.","Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05."," Patients and GERD-specific histomorphological changes The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\nThe three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\n Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease As exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\nAs exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\n Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).\nIn order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).","The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.","As exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).","In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).","In this study, we demonstrated (I) distinct expression patterns of five genes encoding for proteins involved in the formation of tight junctions in esophageal mucosa. In particular Claudin-1 in ERD and to lesser extent Claudin-2 was expressed at higher levels in patients with GERD. In contrast, ZO-1, ZO-2, and Occludin were not affected by the presence of GERD. (II) In general, altered gene expression of Claudin-1/-2 did not correlate with the degree of histomorphological changes in the esophageal mucosa of patients with GERD.\nTight junctions are composed of transmembrane proteins such as Occludin, 24 Claudins, several junctional adhesion molecules (JAMs) with different isoforms, E-Cadherin as well as cytosolic binding partners\n[43,44]. The selection of the five genes studied was based on functional aspects. Occludin is critical for the formation of tight junctions in most tissues\n[45]. Claudin-1 is one of the numerous Claudins that seals intercellular space leading to higher barrier function\n[46], while Claudin-2 is the only pore-forming member of this family resulting in increased permeability\n[47]. Zonula occludens (ZO)-1 and-2 are cytosolic partners of tight junctions in most epithelial surfaces\n[48,49]. The selected genes present important components of the tight junctional complex, and were considered to allow assessment about alterations of tight junctions in relation to GERD. A comprehensive analysis concerning the general expression pattern of other junctional proteins was not performed.\nRecently, several studies demonstrated characteristic histopathological alterations in esophageal mucosa of patients with GERD and a proinflammatory response including the activation of related pathways such as NFκB, PAR-2, ROS and iNOS\n[50,51]. Several in vitro and animal studies have provided evidence that incubation of esophageal mucosa or squamous cell lines either with acidified media with/without bile acids or proinflammatory cytokines can provoke changes in transepithelial electric resistance and increased transepithelial permeability\n[52-55]. Notably, several studies demonstrated a cytokine-mediated change of tight junction-related molecules in various cell models. For instance, IL-6 markedly induces Claudin-2 expression via MEK and PI3K signaling leading to increased tight junction permeability\n[56]. In a rabbit model of GERD, elevated IL-6 expression correlated with induction of several tight junction-related proteins (Claudin-1, Occludin, JAM-1, ZO-1)\n[57] and altered the motogenic activity of smooth muscle cells\n[58].\nAll together, there is sufficient data showing that the exposure of mixed gastric or gastroduodenal refluxate causes altered esophageal epithelial barrier function, inflammation and cellular damage, although the timely order of these processes is a matter of debate\n[20]. As today, it is well accepted that impaired epithelial barrier function of the distal esophagus presents a major pathophysiological process in GERD.\nThis study shows an upregulation of tight junction-related proteins in relation to ERD and NERD in mucosal samples. In particular, Claudin-1 and Claudin-2, though mediating opposite functionally effects, were induced, while cytoplasmic adapters and Occludin were rather unchanged in relation to controls. The higher expression of Claudin-1 (both on transcript and protein level) was the only significant difference identified between patients with ERD and NERD. The fact that all other identified changes were similar between NERD and ERD supports the concept of similar pathophysiological mechanisms between both diseases. Unexpectedly, these changes did not correlate with histomorphological alterations, in particular with dilated ICS in esophageal mucosa. This finding is in contrast to the recently identified correlation between histopathological alterations, in particular basal cell hyperplasia, and elevated gene expression of desmosomal proteins\n[39]. In this study, few borderline correlations were found for basal cell hyperplasia and some genes only, but notably these findings were mostly restricted to reflux-negative controls, whereas patients with GERD did not reveal significant correlations between histopathological alterations and transcript levels of the five genes. Since correlation analyses were performed in an explorative way (without adjustment for multiple comparison), the few significant correlations (with borderline significance) do not support a general role of these findings for the pathophysiology of GERD. Taken into consideration this limitation and the fact that that the overall majority of our comparisons (17 out of 20) revealed no correlations, we conclude that our data do not give evidence for an association between the gene expression of the five genes studied and the histopathological changes in our study groups. It is well known that extent of basal cell hyperplasia reflects proliferative status of esophageal mucosa\n[9]. Since the identified correlations between gene expression levels and basal cell hyperplasia were mostly restricted to controls, it is unlikely that elevated Claudin-1 levels in ERD reflect tissue repair in context to mucosal damage caused by refluxate in these patients. Since we and others demonstrated more severe histomorphological alterations in ERD than NERD, the overall consistent changes of the 5 genes and their corresponding proteins in both diseases seem to be of limited relevance to the mucosal integrity and function. Furthermore, it is notable that some of the stainings revealed not the typical membrane-restricted expression pattern as demonstrated for these tight junction-related molecules im most gastrointestinal tissues\n[59,60]. However, cytoplasmic or diffuse membranous expression patterns have been identified for Claudin-2\n[60] and ZO-1\n[61] in human gastrointestinal tissue and for Claudin-1 in esophageal mucosa of rat\n[62]. Occludin staining pattern or expression in esophageal mucosa differs frequently also from those identified in gastric or intestinal mucosa\n[60,63]. Overall, the subcellular distribution of the 5 tight junction-related proteins seems to differ partially from those identified in columnar-lined epithelium. However, the study was not aimed to analyze the subcellular distribution pattern of the molecules in esophageal mucosa on the subcellular level. The presence of appropriate negative and positive control stainings in other tissues, and the good concordance between expression data on transcript and protein level in general provide further indirect evidence for the specificity of immunohistochemical stainings.\nBased on the descriptive study design, it remains open whether the altered gene expression levels of Claudin-1 and −2 contribute to GERD pathophysiology or merely are markers for the existing disease. Furthermore it is notable that the majority of patients received GERD medications (PPI, H2RA) in the past before entering study. Even a stop of at least 2 weeks was mandatory to enter the study, we can not exclude that the effects of long-term therapy in the past or the changes induced by the 2-week stop of medication (e.g. acid rebound)\n[64] could have affect the expression of the five genes studied. Another limitation is the assessment of protein expression by an immunohistochemical score that can be done semiquantitatively at best. Besides this methodological aspect, posttranscriptional regulatory mechanisms can lead to different findings between gene expression analysis performed on transcript and protein levels. But as mentioned above, overall we observed a good concordance between both levels even not all significant findings were confirmed by both methodologies. Since we studied five selected components of tight junction complexes in GERD only, general conclusions can not be made. Assessment of other tight junction related molecules (e.g. Claudins, JAMs, Tricellulin)\n[44,46,65] in regard to GERD needs to be performed.","In summary, this study demonstrates a partial upregulation of tight junction-related components, in particular Claudin-1, in relation to GERD. Since identified molecular changes do not correlate with histomorphological alterations in general, a major role of Claudin-1 as of the other four tight junction-related proteins in the pathogenesis of GERD can not be concluded from our study.","BE: Barrett’s esophagus; ERD: Erosive reflux disease; NERD: Nonerosive reflux disease; GEJ: Gastroesophageal junction; GERD: Gastroesophageal reflux disease; H2RA: Histamine-receptor antagonist; H. pylori: Helicobacter pylori; ICS: Intercellular spaces; INR: International normalized ratio; iNOS: Inducible nitro oxygen synthetase; JAM: Junctional adhesion molecule; NFκB: Nuclear factor kappa B; PAR-2: Protease activated receptor-2; PPI: Proton pump inhibitor; RDQ: Reflux disease questionnaire; ROS: Reactive oxygen species; RT-PCR: Reverse transcription polymerase chain reaction; ZO: Zona occludens.","The authors declare that they have no competing interest concerning the content of this article.","KM, PM and TW designed the study. KM, LCF enrolled the majority of patients. AK provided clinical data. TW coordinated and performed laboratory work. DK and AR provided histopathological and immunohistochemical data. TW and SK performed statistical analysis. The manuscript was drafted by TW, AK, DK, and reviewed for important intellectual content by KM and PM. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-230X/12/128/prepub\n"],"string":"[\n \"Gastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world\\n[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)\\n[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification\\n[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD\\n[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD\\n[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium\\n[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located\\n[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD\\n[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD\\n[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function\\n[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others\\n[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma\\n[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models\\n[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4\\n[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4\\n[33].\\nHere, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD.\",\n \" Study design and patients’ characteristics Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\\n1A and Table\\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\\nBetween 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\\n1A and Table\\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\\n Endoscopy and histopathology The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\\n[34,36], molecules related to barrier functions\\n[37,38], desmosomal proteins\\n[39] and histopathological alterations\\n[34].\\nThe patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\\n[34,36], molecules related to barrier functions\\n[37,38], desmosomal proteins\\n[39] and histopathological alterations\\n[34].\\n Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\\nCharacteristics of primers, RT-PCR protocol and antibodies\\nmab: monoclonal antibody, fw: forward, rv: reverse.\\nExtraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\\nCharacteristics of primers, RT-PCR protocol and antibodies\\nmab: monoclonal antibody, fw: forward, rv: reverse.\\n Immunohistochemical analysis of tight junctional components Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\\nImmunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\\n Statistical analysis Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.\\nData are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.\",\n \"Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\\n1A and Table\\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\",\n \"Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\",\n \"Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\",\n \"The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\\n[34,36], molecules related to barrier functions\\n[37,38], desmosomal proteins\\n[39] and histopathological alterations\\n[34].\",\n \"Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\\nCharacteristics of primers, RT-PCR protocol and antibodies\\nmab: monoclonal antibody, fw: forward, rv: reverse.\",\n \"Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\",\n \"Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.\",\n \" Patients and GERD-specific histomorphological changes The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\\n1). Histomorphological alterations are shown in Table\\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\\n3).\\nHistopathological parameters\\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\\nThe three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\\n1). Histomorphological alterations are shown in Table\\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\\n3).\\nHistopathological parameters\\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\\n Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease As exemplarily demonstrated in figure\\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\\n4A and\\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\\n1).\\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\\n4A, and B.\\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\\nAs exemplarily demonstrated in figure\\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\\n4A and\\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\\n1).\\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\\n4A, and B.\\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\\n Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\\n5B).\\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\\n5A, and 5B.\\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\\nPanel A: The histopathological scores (Table\\n3) were correlated with gene expression levels (transcript levels, Table\\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\\nIn addition to the correlation based on transcript levels (Figure\\n3, Table\\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\\n4B) and histopathological alterations (Table\\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).\\nIn order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\\n5B).\\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\\n5A, and 5B.\\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\\nPanel A: The histopathological scores (Table\\n3) were correlated with gene expression levels (transcript levels, Table\\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\\nIn addition to the correlation based on transcript levels (Figure\\n3, Table\\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\\n4B) and histopathological alterations (Table\\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).\",\n \"The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\\n1). Histomorphological alterations are shown in Table\\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\\n3).\\nHistopathological parameters\\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\",\n \"As exemplarily demonstrated in figure\\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\\n4A and\\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\\n1).\\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\\n4A, and B.\\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\",\n \"In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\\n5B).\\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\\n5A, and 5B.\\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\\nPanel A: The histopathological scores (Table\\n3) were correlated with gene expression levels (transcript levels, Table\\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\\nIn addition to the correlation based on transcript levels (Figure\\n3, Table\\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\\n4B) and histopathological alterations (Table\\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).\",\n \"In this study, we demonstrated (I) distinct expression patterns of five genes encoding for proteins involved in the formation of tight junctions in esophageal mucosa. In particular Claudin-1 in ERD and to lesser extent Claudin-2 was expressed at higher levels in patients with GERD. In contrast, ZO-1, ZO-2, and Occludin were not affected by the presence of GERD. (II) In general, altered gene expression of Claudin-1/-2 did not correlate with the degree of histomorphological changes in the esophageal mucosa of patients with GERD.\\nTight junctions are composed of transmembrane proteins such as Occludin, 24 Claudins, several junctional adhesion molecules (JAMs) with different isoforms, E-Cadherin as well as cytosolic binding partners\\n[43,44]. The selection of the five genes studied was based on functional aspects. Occludin is critical for the formation of tight junctions in most tissues\\n[45]. Claudin-1 is one of the numerous Claudins that seals intercellular space leading to higher barrier function\\n[46], while Claudin-2 is the only pore-forming member of this family resulting in increased permeability\\n[47]. Zonula occludens (ZO)-1 and-2 are cytosolic partners of tight junctions in most epithelial surfaces\\n[48,49]. The selected genes present important components of the tight junctional complex, and were considered to allow assessment about alterations of tight junctions in relation to GERD. A comprehensive analysis concerning the general expression pattern of other junctional proteins was not performed.\\nRecently, several studies demonstrated characteristic histopathological alterations in esophageal mucosa of patients with GERD and a proinflammatory response including the activation of related pathways such as NFκB, PAR-2, ROS and iNOS\\n[50,51]. Several in vitro and animal studies have provided evidence that incubation of esophageal mucosa or squamous cell lines either with acidified media with/without bile acids or proinflammatory cytokines can provoke changes in transepithelial electric resistance and increased transepithelial permeability\\n[52-55]. Notably, several studies demonstrated a cytokine-mediated change of tight junction-related molecules in various cell models. For instance, IL-6 markedly induces Claudin-2 expression via MEK and PI3K signaling leading to increased tight junction permeability\\n[56]. In a rabbit model of GERD, elevated IL-6 expression correlated with induction of several tight junction-related proteins (Claudin-1, Occludin, JAM-1, ZO-1)\\n[57] and altered the motogenic activity of smooth muscle cells\\n[58].\\nAll together, there is sufficient data showing that the exposure of mixed gastric or gastroduodenal refluxate causes altered esophageal epithelial barrier function, inflammation and cellular damage, although the timely order of these processes is a matter of debate\\n[20]. As today, it is well accepted that impaired epithelial barrier function of the distal esophagus presents a major pathophysiological process in GERD.\\nThis study shows an upregulation of tight junction-related proteins in relation to ERD and NERD in mucosal samples. In particular, Claudin-1 and Claudin-2, though mediating opposite functionally effects, were induced, while cytoplasmic adapters and Occludin were rather unchanged in relation to controls. The higher expression of Claudin-1 (both on transcript and protein level) was the only significant difference identified between patients with ERD and NERD. The fact that all other identified changes were similar between NERD and ERD supports the concept of similar pathophysiological mechanisms between both diseases. Unexpectedly, these changes did not correlate with histomorphological alterations, in particular with dilated ICS in esophageal mucosa. This finding is in contrast to the recently identified correlation between histopathological alterations, in particular basal cell hyperplasia, and elevated gene expression of desmosomal proteins\\n[39]. In this study, few borderline correlations were found for basal cell hyperplasia and some genes only, but notably these findings were mostly restricted to reflux-negative controls, whereas patients with GERD did not reveal significant correlations between histopathological alterations and transcript levels of the five genes. Since correlation analyses were performed in an explorative way (without adjustment for multiple comparison), the few significant correlations (with borderline significance) do not support a general role of these findings for the pathophysiology of GERD. Taken into consideration this limitation and the fact that that the overall majority of our comparisons (17 out of 20) revealed no correlations, we conclude that our data do not give evidence for an association between the gene expression of the five genes studied and the histopathological changes in our study groups. It is well known that extent of basal cell hyperplasia reflects proliferative status of esophageal mucosa\\n[9]. Since the identified correlations between gene expression levels and basal cell hyperplasia were mostly restricted to controls, it is unlikely that elevated Claudin-1 levels in ERD reflect tissue repair in context to mucosal damage caused by refluxate in these patients. Since we and others demonstrated more severe histomorphological alterations in ERD than NERD, the overall consistent changes of the 5 genes and their corresponding proteins in both diseases seem to be of limited relevance to the mucosal integrity and function. Furthermore, it is notable that some of the stainings revealed not the typical membrane-restricted expression pattern as demonstrated for these tight junction-related molecules im most gastrointestinal tissues\\n[59,60]. However, cytoplasmic or diffuse membranous expression patterns have been identified for Claudin-2\\n[60] and ZO-1\\n[61] in human gastrointestinal tissue and for Claudin-1 in esophageal mucosa of rat\\n[62]. Occludin staining pattern or expression in esophageal mucosa differs frequently also from those identified in gastric or intestinal mucosa\\n[60,63]. Overall, the subcellular distribution of the 5 tight junction-related proteins seems to differ partially from those identified in columnar-lined epithelium. However, the study was not aimed to analyze the subcellular distribution pattern of the molecules in esophageal mucosa on the subcellular level. The presence of appropriate negative and positive control stainings in other tissues, and the good concordance between expression data on transcript and protein level in general provide further indirect evidence for the specificity of immunohistochemical stainings.\\nBased on the descriptive study design, it remains open whether the altered gene expression levels of Claudin-1 and −2 contribute to GERD pathophysiology or merely are markers for the existing disease. Furthermore it is notable that the majority of patients received GERD medications (PPI, H2RA) in the past before entering study. Even a stop of at least 2 weeks was mandatory to enter the study, we can not exclude that the effects of long-term therapy in the past or the changes induced by the 2-week stop of medication (e.g. acid rebound)\\n[64] could have affect the expression of the five genes studied. Another limitation is the assessment of protein expression by an immunohistochemical score that can be done semiquantitatively at best. Besides this methodological aspect, posttranscriptional regulatory mechanisms can lead to different findings between gene expression analysis performed on transcript and protein levels. But as mentioned above, overall we observed a good concordance between both levels even not all significant findings were confirmed by both methodologies. Since we studied five selected components of tight junction complexes in GERD only, general conclusions can not be made. Assessment of other tight junction related molecules (e.g. Claudins, JAMs, Tricellulin)\\n[44,46,65] in regard to GERD needs to be performed.\",\n \"In summary, this study demonstrates a partial upregulation of tight junction-related components, in particular Claudin-1, in relation to GERD. Since identified molecular changes do not correlate with histomorphological alterations in general, a major role of Claudin-1 as of the other four tight junction-related proteins in the pathogenesis of GERD can not be concluded from our study.\",\n \"BE: Barrett’s esophagus; ERD: Erosive reflux disease; NERD: Nonerosive reflux disease; GEJ: Gastroesophageal junction; GERD: Gastroesophageal reflux disease; H2RA: Histamine-receptor antagonist; H. pylori: Helicobacter pylori; ICS: Intercellular spaces; INR: International normalized ratio; iNOS: Inducible nitro oxygen synthetase; JAM: Junctional adhesion molecule; NFκB: Nuclear factor kappa B; PAR-2: Protease activated receptor-2; PPI: Proton pump inhibitor; RDQ: Reflux disease questionnaire; ROS: Reactive oxygen species; RT-PCR: Reverse transcription polymerase chain reaction; ZO: Zona occludens.\",\n \"The authors declare that they have no competing interest concerning the content of this article.\",\n \"KM, PM and TW designed the study. KM, LCF enrolled the majority of patients. AK provided clinical data. TW coordinated and performed laboratory work. DK and AR provided histopathological and immunohistochemical data. TW and SK performed statistical analysis. The manuscript was drafted by TW, AK, DK, and reviewed for important intellectual content by KM and PM. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-230X/12/128/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,"results",null,null,null,"discussion","conclusions",null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Gastroesophageal reflux disease","Tight junction","Claudins","Esophagitis","Inflammation"],"string":"[\n \"Gastroesophageal reflux disease\",\n \"Tight junction\",\n \"Claudins\",\n \"Esophagitis\",\n \"Inflammation\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nGastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world\n[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)\n[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification\n[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD\n[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD\n[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium\n[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located\n[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD\n[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD\n[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function\n[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others\n[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma\n[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models\n[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4\n[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4\n[33].\nHere, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD.\n\nMethods:\n Study design and patients’ characteristics Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nBetween 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\n Endoscopy and histopathology The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].\nThe patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].\n Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.\nExtraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.\n Immunohistochemical analysis of tight junctional components Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\nImmunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\n Statistical analysis Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.\nData are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.\n\nStudy design and patients’ characteristics:\nBetween 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\n\nInclusion criteria:\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n\nExclusion criteria:\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\n\nEndoscopy and histopathology:\nThe patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].\n\nExtraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes:\nExtraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.\n\nImmunohistochemical analysis of tight junctional components:\nImmunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\n\nStatistical analysis:\nData are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.\n\nResults:\n Patients and GERD-specific histomorphological changes The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\nThe three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\n Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease As exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\nAs exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\n Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).\nIn order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).\n\nPatients and GERD-specific histomorphological changes:\nThe three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\n\nUpregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease:\nAs exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\n\nIncreased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa:\nIn order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).\n\nDiscussion:\nIn this study, we demonstrated (I) distinct expression patterns of five genes encoding for proteins involved in the formation of tight junctions in esophageal mucosa. In particular Claudin-1 in ERD and to lesser extent Claudin-2 was expressed at higher levels in patients with GERD. In contrast, ZO-1, ZO-2, and Occludin were not affected by the presence of GERD. (II) In general, altered gene expression of Claudin-1/-2 did not correlate with the degree of histomorphological changes in the esophageal mucosa of patients with GERD.\nTight junctions are composed of transmembrane proteins such as Occludin, 24 Claudins, several junctional adhesion molecules (JAMs) with different isoforms, E-Cadherin as well as cytosolic binding partners\n[43,44]. The selection of the five genes studied was based on functional aspects. Occludin is critical for the formation of tight junctions in most tissues\n[45]. Claudin-1 is one of the numerous Claudins that seals intercellular space leading to higher barrier function\n[46], while Claudin-2 is the only pore-forming member of this family resulting in increased permeability\n[47]. Zonula occludens (ZO)-1 and-2 are cytosolic partners of tight junctions in most epithelial surfaces\n[48,49]. The selected genes present important components of the tight junctional complex, and were considered to allow assessment about alterations of tight junctions in relation to GERD. A comprehensive analysis concerning the general expression pattern of other junctional proteins was not performed.\nRecently, several studies demonstrated characteristic histopathological alterations in esophageal mucosa of patients with GERD and a proinflammatory response including the activation of related pathways such as NFκB, PAR-2, ROS and iNOS\n[50,51]. Several in vitro and animal studies have provided evidence that incubation of esophageal mucosa or squamous cell lines either with acidified media with/without bile acids or proinflammatory cytokines can provoke changes in transepithelial electric resistance and increased transepithelial permeability\n[52-55]. Notably, several studies demonstrated a cytokine-mediated change of tight junction-related molecules in various cell models. For instance, IL-6 markedly induces Claudin-2 expression via MEK and PI3K signaling leading to increased tight junction permeability\n[56]. In a rabbit model of GERD, elevated IL-6 expression correlated with induction of several tight junction-related proteins (Claudin-1, Occludin, JAM-1, ZO-1)\n[57] and altered the motogenic activity of smooth muscle cells\n[58].\nAll together, there is sufficient data showing that the exposure of mixed gastric or gastroduodenal refluxate causes altered esophageal epithelial barrier function, inflammation and cellular damage, although the timely order of these processes is a matter of debate\n[20]. As today, it is well accepted that impaired epithelial barrier function of the distal esophagus presents a major pathophysiological process in GERD.\nThis study shows an upregulation of tight junction-related proteins in relation to ERD and NERD in mucosal samples. In particular, Claudin-1 and Claudin-2, though mediating opposite functionally effects, were induced, while cytoplasmic adapters and Occludin were rather unchanged in relation to controls. The higher expression of Claudin-1 (both on transcript and protein level) was the only significant difference identified between patients with ERD and NERD. The fact that all other identified changes were similar between NERD and ERD supports the concept of similar pathophysiological mechanisms between both diseases. Unexpectedly, these changes did not correlate with histomorphological alterations, in particular with dilated ICS in esophageal mucosa. This finding is in contrast to the recently identified correlation between histopathological alterations, in particular basal cell hyperplasia, and elevated gene expression of desmosomal proteins\n[39]. In this study, few borderline correlations were found for basal cell hyperplasia and some genes only, but notably these findings were mostly restricted to reflux-negative controls, whereas patients with GERD did not reveal significant correlations between histopathological alterations and transcript levels of the five genes. Since correlation analyses were performed in an explorative way (without adjustment for multiple comparison), the few significant correlations (with borderline significance) do not support a general role of these findings for the pathophysiology of GERD. Taken into consideration this limitation and the fact that that the overall majority of our comparisons (17 out of 20) revealed no correlations, we conclude that our data do not give evidence for an association between the gene expression of the five genes studied and the histopathological changes in our study groups. It is well known that extent of basal cell hyperplasia reflects proliferative status of esophageal mucosa\n[9]. Since the identified correlations between gene expression levels and basal cell hyperplasia were mostly restricted to controls, it is unlikely that elevated Claudin-1 levels in ERD reflect tissue repair in context to mucosal damage caused by refluxate in these patients. Since we and others demonstrated more severe histomorphological alterations in ERD than NERD, the overall consistent changes of the 5 genes and their corresponding proteins in both diseases seem to be of limited relevance to the mucosal integrity and function. Furthermore, it is notable that some of the stainings revealed not the typical membrane-restricted expression pattern as demonstrated for these tight junction-related molecules im most gastrointestinal tissues\n[59,60]. However, cytoplasmic or diffuse membranous expression patterns have been identified for Claudin-2\n[60] and ZO-1\n[61] in human gastrointestinal tissue and for Claudin-1 in esophageal mucosa of rat\n[62]. Occludin staining pattern or expression in esophageal mucosa differs frequently also from those identified in gastric or intestinal mucosa\n[60,63]. Overall, the subcellular distribution of the 5 tight junction-related proteins seems to differ partially from those identified in columnar-lined epithelium. However, the study was not aimed to analyze the subcellular distribution pattern of the molecules in esophageal mucosa on the subcellular level. The presence of appropriate negative and positive control stainings in other tissues, and the good concordance between expression data on transcript and protein level in general provide further indirect evidence for the specificity of immunohistochemical stainings.\nBased on the descriptive study design, it remains open whether the altered gene expression levels of Claudin-1 and −2 contribute to GERD pathophysiology or merely are markers for the existing disease. Furthermore it is notable that the majority of patients received GERD medications (PPI, H2RA) in the past before entering study. Even a stop of at least 2 weeks was mandatory to enter the study, we can not exclude that the effects of long-term therapy in the past or the changes induced by the 2-week stop of medication (e.g. acid rebound)\n[64] could have affect the expression of the five genes studied. Another limitation is the assessment of protein expression by an immunohistochemical score that can be done semiquantitatively at best. Besides this methodological aspect, posttranscriptional regulatory mechanisms can lead to different findings between gene expression analysis performed on transcript and protein levels. But as mentioned above, overall we observed a good concordance between both levels even not all significant findings were confirmed by both methodologies. Since we studied five selected components of tight junction complexes in GERD only, general conclusions can not be made. Assessment of other tight junction related molecules (e.g. Claudins, JAMs, Tricellulin)\n[44,46,65] in regard to GERD needs to be performed.\n\nConclusions:\nIn summary, this study demonstrates a partial upregulation of tight junction-related components, in particular Claudin-1, in relation to GERD. Since identified molecular changes do not correlate with histomorphological alterations in general, a major role of Claudin-1 as of the other four tight junction-related proteins in the pathogenesis of GERD can not be concluded from our study.\n\nAbbreviations:\nBE: Barrett’s esophagus; ERD: Erosive reflux disease; NERD: Nonerosive reflux disease; GEJ: Gastroesophageal junction; GERD: Gastroesophageal reflux disease; H2RA: Histamine-receptor antagonist; H. pylori: Helicobacter pylori; ICS: Intercellular spaces; INR: International normalized ratio; iNOS: Inducible nitro oxygen synthetase; JAM: Junctional adhesion molecule; NFκB: Nuclear factor kappa B; PAR-2: Protease activated receptor-2; PPI: Proton pump inhibitor; RDQ: Reflux disease questionnaire; ROS: Reactive oxygen species; RT-PCR: Reverse transcription polymerase chain reaction; ZO: Zona occludens.\n\nCompeting interests:\nThe authors declare that they have no competing interest concerning the content of this article.\n\nAuthors’ contributions:\nKM, PM and TW designed the study. KM, LCF enrolled the majority of patients. AK provided clinical data. TW coordinated and performed laboratory work. DK and AR provided histopathological and immunohistochemical data. TW and SK performed statistical analysis. The manuscript was drafted by TW, AK, DK, and reviewed for important intellectual content by KM and PM. All authors read and approved the final manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-230X/12/128/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nGastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function that is regulated by cell-cell contacts. The aim of the study was to investigate the expression pattern of selected components involved in the formation of tight junctions in relation to GERD.\n\nMethods:\nEighty-four patients with GERD-related symptoms with endoscopic signs (erosive: n = 47) or without them (non-erosive: n = 37) as well as 26 patients lacking GERD-specific symptoms as controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and adapted Ismeil-Beigi criteria, respectively. Mucosal biopsies from distal esophagus were taken for analysis by histopathology, immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (RT-PCR) of five genes encoding tight junction components [Occludin, Claudin-1, -2, Zona occludens (ZO-1, -2)].\n\nResults:\nHistopathology confirmed GERD-specific alterations as dilated intercellular spaces in the esophageal mucosa of patients with GERD compared to controls (P < 0.05). Claudin-1 and -2 were 2- to 6-fold upregulation on transcript (P < 0.01) and in part on protein level (P < 0.015) in GERD, while subgroup analysis of revealed this upregulation for ERD only. In both erosive and non-erosive reflux disease, expression levels of Occludin and ZO-1,-2 were not significantly affected. Notably, the induced expression of both claudins did not correlate with histopathological parameters (basal cell hyperplasia, dilated intercellular spaces) in patients with GERD.\n\nConclusions:\nTaken together, the missing correlation between the expression of tight junction-related components and histomorphological GERD-specific alterations does not support a major role of the five proteins studied in the pathogenesis of GERD."},"background_conclusion_text":{"kind":"string","value":"Background:\nGastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world\n[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)\n[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification\n[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD\n[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD\n[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium\n[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located\n[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD\n[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD\n[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function\n[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others\n[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma\n[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models\n[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4\n[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4\n[33].\nHere, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD.\n\nConclusions:\nIn summary, this study demonstrates a partial upregulation of tight junction-related components, in particular Claudin-1, in relation to GERD. Since identified molecular changes do not correlate with histomorphological alterations in general, a major role of Claudin-1 as of the other four tight junction-related proteins in the pathogenesis of GERD can not be concluded from our study."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nGastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function that is regulated by cell-cell contacts. The aim of the study was to investigate the expression pattern of selected components involved in the formation of tight junctions in relation to GERD.\n\nMethods:\nEighty-four patients with GERD-related symptoms with endoscopic signs (erosive: n = 47) or without them (non-erosive: n = 37) as well as 26 patients lacking GERD-specific symptoms as controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and adapted Ismeil-Beigi criteria, respectively. Mucosal biopsies from distal esophagus were taken for analysis by histopathology, immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (RT-PCR) of five genes encoding tight junction components [Occludin, Claudin-1, -2, Zona occludens (ZO-1, -2)].\n\nResults:\nHistopathology confirmed GERD-specific alterations as dilated intercellular spaces in the esophageal mucosa of patients with GERD compared to controls (P < 0.05). Claudin-1 and -2 were 2- to 6-fold upregulation on transcript (P < 0.01) and in part on protein level (P < 0.015) in GERD, while subgroup analysis of revealed this upregulation for ERD only. In both erosive and non-erosive reflux disease, expression levels of Occludin and ZO-1,-2 were not significantly affected. Notably, the induced expression of both claudins did not correlate with histopathological parameters (basal cell hyperplasia, dilated intercellular spaces) in patients with GERD.\n\nConclusions:\nTaken together, the missing correlation between the expression of tight junction-related components and histomorphological GERD-specific alterations does not support a major role of the five proteins studied in the pathogenesis of GERD."},"whole_article_text_length":{"kind":"number","value":11448,"string":"11,448"},"whole_article_abstract_length":{"kind":"number","value":346,"string":"346"},"num_sections":{"kind":"number","value":19,"string":"19"},"most_frequent_words":{"kind":"list like","value":["patients","claudin","expression","gerd","data","levels","table","gene","mucosa","controls"],"string":"[\n \"patients\",\n \"claudin\",\n \"expression\",\n \"gerd\",\n \"data\",\n \"levels\",\n \"table\",\n \"gene\",\n \"mucosa\",\n \"controls\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] gerd | esophageal | molecules | ics | esophageal mucosa | visible | structural | nerd | claudin | cell [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | symptoms | reflux | reflux symptoms | performed | score | 80 | gene | study | defined [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] claudin | expression | levels | table | transcript | correlations | panel | data | correlation | scores [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] tight | junction related | tight junction related | tight junction | claudin | junction | related | alterations general major | general major | alterations general [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | claudin | expression | gerd | symptoms | levels | reflux | data | table | study [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | claudin | expression | gerd | symptoms | levels | reflux | data | table | study [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] GERD ||| GERD [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Eighty-four | 47 | 37 | 26 | GERD ||| Los Angeles ||| Mucosal | RT-PCR | five ||| Zona [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] GERD | 0.05 ||| 6-fold | 0.01 | 0.015 | GERD | ERD ||| Occludin ||| GERD [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] five | GERD [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] GERD ||| GERD ||| Eighty-four | 47 | 37 | 26 | GERD ||| Los Angeles ||| Mucosal | RT-PCR | five | Occludin | Zona ||| ||| GERD | 0.05 ||| 6-fold | 0.01 | 0.015 | GERD | ERD ||| Occludin ||| GERD ||| five | GERD [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] GERD ||| GERD ||| Eighty-four | 47 | 37 | 26 | GERD ||| Los Angeles ||| Mucosal | RT-PCR | five | Occludin | Zona ||| ||| GERD | 0.05 ||| 6-fold | 0.01 | 0.015 | GERD | ERD ||| Occludin ||| GERD ||| five | GERD [SUMMARY]"}}},{"rowIdx":263,"cells":{"title":{"kind":"string","value":"Modification by hemochromatosis gene polymorphisms of the association between traffic-related air pollution and cognition in older men: a cohort study."},"pmid":{"kind":"string","value":"23413885"},"background_abstract":{"kind":"string","value":"Previous studies found effect modification of associations between traffic-related air pollution and cardiovascular outcomes by polymorphisms in the hemochromatosis gene (HFE). As traffic-related air pollution may impact cognition through effects on cardiovascular health or through mechanisms which may also influence cardiovascular outcomes, we hypothesized that HFE polymorphisms would also modify a previously observed association between traffic-related air pollution exposure and cognition in older men."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We considered data from 628 participants of the VA Normative Aging Study. We estimated long term exposure to black carbon (BC), a marker of traffic related air pollution, using a spatio-temporal land use regression model. We assessed cognition using the Mini-Mental State Examination (MMSE), a test of global function, and performance on a battery of other tests, covering a wide range of domains. We investigated whether variants of HFE C282Y and H63D modified the association between BC and having a low MMSE score using logistic models with generalized estimating equations and multiplicative interaction terms. Similarly, we assessed whether HFE variants modified the association between BC and performance on the cognitive battery using linear mixed models with multiplicative interaction terms."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Our results suggest modification of the BC-cognition association by HFE C282Y, although the test of interaction did not achieve statistical significance. In multivariable-adjusted models, participants who lacked a HFE C282Y variant (CC) exhibited an adverse association between BC and total cognition z-score (beta for a doubling in BC concentration: -0.061, 95% CI: -0.115, -0.007), while we did not observe an association in participants with at least one variant genotype (CY or YY) (beta for a doubling in BC concentration: 0.073, 95% CI: -0.081, 0.228; p-value for interaction: 0.11). The pattern of association was similar for analyses considering performance on the Mini-Mental State Examination. There was little evidence to support effect modification of the BC-cognition association by the HFE H63D genotype."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Our data suggest that older adults who lack an HFE C282Y variant may be more susceptible to an adverse effect of traffic-related air pollution exposure on cognition. This finding and the proposed biological mechanism require confirmation."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Air Pollutants","Cognition","Cohort Studies","Genotype","Hemochromatosis Protein","Histocompatibility Antigens Class I","Humans","Male","Membrane Proteins","Middle Aged","Multiplex Polymerase Chain Reaction","Polymorphism, Single Nucleotide","Soot","United States","Vehicle Emissions"],"string":"[\n \"Aged\",\n \"Air Pollutants\",\n \"Cognition\",\n \"Cohort Studies\",\n \"Genotype\",\n \"Hemochromatosis Protein\",\n \"Histocompatibility Antigens Class I\",\n \"Humans\",\n \"Male\",\n \"Membrane Proteins\",\n \"Middle Aged\",\n \"Multiplex Polymerase Chain Reaction\",\n \"Polymorphism, Single Nucleotide\",\n \"Soot\",\n \"United States\",\n \"Vehicle Emissions\"\n]"},"pmcid":{"kind":"string","value":"3599892"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"While we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large\n[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.\nTraffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men\n[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above\n[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women\n[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese\n[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children\n[6-11].\nThe hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease\n[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium\n[14-18].\nPrevious studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors\n[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Study sample The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.\nThe current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.\n Exposure assessment For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\nFor each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\n Cognitive testing The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\nThe content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\n HFE genotyping For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\nFor this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\n Statistical methods As the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).\nAs the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC)."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Six hundred twenty eight (92%) of the 680 men with data on black carbon exposure, cognition, and all covariates were successfully genotyped for both HFE SNPs. Table \n1 summarizes the sample characteristics for the current analysis in total and by HFE SNP genotypes. At baseline, our participants were, on average, 70 years old (SD: 7.1) and had 14.4 years of formal education (SD: 2.6). 540 (86%) of our participants lacked an HFE C282Y variant and 6 (1%) were homozygous for the C282Y variant. 469 (75%) lacked an HFE H63D variant and 18 (3%) were homozygous for the H63D variant. Both SNPs were in Hardy-Weinberg equilibrium (C282Y: χ2 = 2.05, p = 0.15; H63D: χ2 = 3.32, p = 0.07).\nBaseline characteristics of the cohort (n = 628)\nAbbreviations: HFE, hemochromatosis gene; SD, standard deviation; MET-hr/week, metabolic equivalent hours per week.\nIn the current study sample, the direction and magnitude of the association between black carbon and cognitive function in fully adjusted models was materially unchanged compared to previously reported analyses (Additional file\n1: Table S1). As expected, there was little evidence to support a main effect of either HFE variant on cognition. The presence of an HFE C282Y variant did not appear to be associated with total cognition (beta: -0.063, 95% CI: -0.142, 0.017) or MMSE performance (odds ratio (OR): 0.92, 95% CI: 0.64, 1.35). Similarly, presence of an HFE H63D variant was not associated with either total cognition (beta: 0.022, 95% CI: -0.041, 0.085) or MMSE performance (OR: 0.98, 95% CI: 0.75, 1.28).\nConsideration of regression models with interaction terms between HFE genotype and black carbon suggests that HFE C282Y, but not HFE H63D, modifies the association between black carbon concentrations and total cognitive function (Table \n2). Although the p-values for interaction did not achieve the threshold for statistical significance in any model, the adverse association between black carbon exposure and cognition appears to be exclusive to those lacking the HFE C282Y variant. While participants who lacked an HFE C282Y variant exhibited strong adverse associations between BC and total cognition, a magnitude of effect that is equivalent to approximately 2.1 years of age in our data, we did not observe an adverse association for participants with at least one variant genotype. Similarly, we found elevated odds of low MMSE scores for a doubling in BC concentrations for participants who lacked an HFE C282Y variant but little evidence to support an association in participants with at least one copy of the HFE C282Y variant (Table \n3).\nThe association between a doubling in BC concentration and total cognitive z-score by HFE polymorphisms\nAbbreviations: HFE, hemochromatosis gene; BC, black carbon.\naAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.\nThe association between a doubling in BC concentration and low MMSE scores by HFE polymorphisms\nAbbreviations: HFE, hemochromatosis gene; MMSE, Mini-Mental State Examination; BC, black carbon.\naAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.\nWe found little support for modification of the black carbon-cognition association by HFE H63D for either total cognition or MMSE performance. Although we observed statistically significant associations between black carbon and cognition within groups defined by HFE H63D genotype, the p-values for interaction did not achieve statistical significance in either model, and the pattern of association across those with and without the HFE H63D variant differed by outcome. In analyses of total cognition, we observed a statistically significant adverse association between black carbon concentrations among HFE H63D homozygous wild type participants, but not among those possessing at least one variant allele (Table \n2). However, we observed the opposite pattern in analyses considering MMSE scores (Table \n3), where we found a statistically significant adverse association in those with at least one H63D variant, but not in those who were homozygous wild type.\nIn sensitivity analyses, additional adjustment for hemoglobin, body mass index, smoking, or past lead exposure did not appreciably change effect estimates (data not shown)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In conclusion, our study suggests that persons who lack a HFE C282Y variant polymorphism may be more susceptible to adverse effects of exposure to traffic-related air pollution on cognition than those possessing a variant. The mechanism by which HFE modifies this association may involve metal transport and inflammation pathways. As with any candidate gene-environment interaction study, this analysis requires independent confirmation."},"other_sections_titles":{"kind":"list like","value":["Background","Study sample","Exposure assessment","Cognitive testing","HFE genotyping","Statistical methods","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Study sample\",\n \"Exposure assessment\",\n \"Cognitive testing\",\n \"HFE genotyping\",\n \"Statistical methods\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["While we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large\n[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.\nTraffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men\n[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above\n[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women\n[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese\n[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children\n[6-11].\nThe hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease\n[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium\n[14-18].\nPrevious studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors\n[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms.","The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.","For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.","The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.","For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.","As the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).","BC: Black carbon; BMI: Body mass index; HFE: Hemochromatosis gene; CI: Confidence interval; MET-hrs: Metabolic equivalent hours; MMSE: Mini-Mental State Examination; NAS: Normative Aging Study; OR: Odds ratio; QT interval: Heart-rate-corrected interval; VA: United States Department of Veterans Affairs.","The authors declare they have no competing interests.","MCP made substantial contributions to the study conception and design, analysis and interpretation of the data, and drafted the manuscript. MGW and JS made substantial contributions to the study conception and design, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. BAC, SEA, AS made substantial contributions to the acquisition of data and revised the manuscript critically for important intellectual content. ROW made substantial contributions to the analysis or interpretation of the data and revised the manuscript critically for important intellectual content. All authors have given final approval of the version to be published."],"string":"[\n \"While we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large\\n[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.\\nTraffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men\\n[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above\\n[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women\\n[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese\\n[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children\\n[6-11].\\nThe hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease\\n[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium\\n[14-18].\\nPrevious studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors\\n[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms.\",\n \"The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\\n[2] we refer readers to that paper for a more exhaustive description of our methods.\",\n \"For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\",\n \"The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\",\n \"For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\",\n \"As the current dataset excludes participants from the previously reported analyses\\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).\",\n \"BC: Black carbon; BMI: Body mass index; HFE: Hemochromatosis gene; CI: Confidence interval; MET-hrs: Metabolic equivalent hours; MMSE: Mini-Mental State Examination; NAS: Normative Aging Study; OR: Odds ratio; QT interval: Heart-rate-corrected interval; VA: United States Department of Veterans Affairs.\",\n \"The authors declare they have no competing interests.\",\n \"MCP made substantial contributions to the study conception and design, analysis and interpretation of the data, and drafted the manuscript. MGW and JS made substantial contributions to the study conception and design, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. BAC, SEA, AS made substantial contributions to the acquisition of data and revised the manuscript critically for important intellectual content. ROW made substantial contributions to the analysis or interpretation of the data and revised the manuscript critically for important intellectual content. All authors have given final approval of the version to be published.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study sample","Exposure assessment","Cognitive testing","HFE genotyping","Statistical methods","Results","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions","Supplementary Material"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study sample\",\n \"Exposure assessment\",\n \"Cognitive testing\",\n \"HFE genotyping\",\n \"Statistical methods\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Supplementary Material\"\n]"},"all_sections_texts":{"kind":"list like","value":["While we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large\n[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.\nTraffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men\n[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above\n[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women\n[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese\n[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children\n[6-11].\nThe hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease\n[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium\n[14-18].\nPrevious studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors\n[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms."," Study sample The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.\nThe current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.\n Exposure assessment For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\nFor each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\n Cognitive testing The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\nThe content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\n HFE genotyping For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\nFor this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\n Statistical methods As the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).\nAs the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).","The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.","For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.","The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.","For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.","As the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).","Six hundred twenty eight (92%) of the 680 men with data on black carbon exposure, cognition, and all covariates were successfully genotyped for both HFE SNPs. Table \n1 summarizes the sample characteristics for the current analysis in total and by HFE SNP genotypes. At baseline, our participants were, on average, 70 years old (SD: 7.1) and had 14.4 years of formal education (SD: 2.6). 540 (86%) of our participants lacked an HFE C282Y variant and 6 (1%) were homozygous for the C282Y variant. 469 (75%) lacked an HFE H63D variant and 18 (3%) were homozygous for the H63D variant. Both SNPs were in Hardy-Weinberg equilibrium (C282Y: χ2 = 2.05, p = 0.15; H63D: χ2 = 3.32, p = 0.07).\nBaseline characteristics of the cohort (n = 628)\nAbbreviations: HFE, hemochromatosis gene; SD, standard deviation; MET-hr/week, metabolic equivalent hours per week.\nIn the current study sample, the direction and magnitude of the association between black carbon and cognitive function in fully adjusted models was materially unchanged compared to previously reported analyses (Additional file\n1: Table S1). As expected, there was little evidence to support a main effect of either HFE variant on cognition. The presence of an HFE C282Y variant did not appear to be associated with total cognition (beta: -0.063, 95% CI: -0.142, 0.017) or MMSE performance (odds ratio (OR): 0.92, 95% CI: 0.64, 1.35). Similarly, presence of an HFE H63D variant was not associated with either total cognition (beta: 0.022, 95% CI: -0.041, 0.085) or MMSE performance (OR: 0.98, 95% CI: 0.75, 1.28).\nConsideration of regression models with interaction terms between HFE genotype and black carbon suggests that HFE C282Y, but not HFE H63D, modifies the association between black carbon concentrations and total cognitive function (Table \n2). Although the p-values for interaction did not achieve the threshold for statistical significance in any model, the adverse association between black carbon exposure and cognition appears to be exclusive to those lacking the HFE C282Y variant. While participants who lacked an HFE C282Y variant exhibited strong adverse associations between BC and total cognition, a magnitude of effect that is equivalent to approximately 2.1 years of age in our data, we did not observe an adverse association for participants with at least one variant genotype. Similarly, we found elevated odds of low MMSE scores for a doubling in BC concentrations for participants who lacked an HFE C282Y variant but little evidence to support an association in participants with at least one copy of the HFE C282Y variant (Table \n3).\nThe association between a doubling in BC concentration and total cognitive z-score by HFE polymorphisms\nAbbreviations: HFE, hemochromatosis gene; BC, black carbon.\naAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.\nThe association between a doubling in BC concentration and low MMSE scores by HFE polymorphisms\nAbbreviations: HFE, hemochromatosis gene; MMSE, Mini-Mental State Examination; BC, black carbon.\naAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.\nWe found little support for modification of the black carbon-cognition association by HFE H63D for either total cognition or MMSE performance. Although we observed statistically significant associations between black carbon and cognition within groups defined by HFE H63D genotype, the p-values for interaction did not achieve statistical significance in either model, and the pattern of association across those with and without the HFE H63D variant differed by outcome. In analyses of total cognition, we observed a statistically significant adverse association between black carbon concentrations among HFE H63D homozygous wild type participants, but not among those possessing at least one variant allele (Table \n2). However, we observed the opposite pattern in analyses considering MMSE scores (Table \n3), where we found a statistically significant adverse association in those with at least one H63D variant, but not in those who were homozygous wild type.\nIn sensitivity analyses, additional adjustment for hemoglobin, body mass index, smoking, or past lead exposure did not appreciably change effect estimates (data not shown).","Although the statistical evidence to support the claim that the HFE C282Y polymorphism modifies the association between traffic-related air pollution and cognitive function in our sample of older men is weak, the pattern of response across groups is intriguing and suggests the presence of effect modification by HFE C282Y. Participants possessing a variant HFE C282Y polymorphism appear to be protected from the adverse association between black carbon and cognition, while participants who lacked the C282Y variant exhibit strong adverse associations. We found little support for modification of the association between traffic-related air pollution and cognition by HFE H63D.\nThis is the first study to investigate effect modification by HFE of the air pollution-cognition relationship. These findings are in line with reported effect modification by HFE of the relationship between exposure to air pollution and cardiovascular outcomes or risk factors in the NAS cohort. Persons lacking HFE variant genotypes exhibit strong adverse associations between exposure to fine particulate matter (PM) and heart rate variability, while there is little evidence for an association among those possessing an HFE variant, and this pattern was driven primarily by the HFE C282Y SNP\n[20]. Similarly, higher exposures to black carbon or PM2.5 were associated with higher plasma homocysteine concentrations in participants who lacked the HFE C282Y variant, but no association was observed in participants possessing at least one HFE C282Y variant\n[19]. A similar pattern was observed for modification by HFE C282Y of the association between traffic-related air pollution and heart-rate-corrected (QT) interval, an electrocardiographic marker of ventricular repolarization that is associated with arrhythmia and cardiac death\n[21].\nThe mechanism by which exposure to traffic-related air pollution may contribute to cognitive impairment is unknown, but likely involves inhalation of particulates. In animal experiments, exposure to fine or ultrafine particulate matter appears to induce inflammation, lipid peroxidation, and neuronal degeneration in the central nervous system\n[25-28]. Metals, including iron, are often found adsorbed onto the surface of traffic-related particulates\n[29] and exposure to metals is associated with induction of oxidative stress and inflammation\n[30]. Oxidative stress and inflammation may contribute to the development of cognitive impairment directly or through inducement of cardiovascular problems.\nHFE modulates intracellular uptake of iron and other metals. Variants of HFE are associated with the disease hemochromatosis, which is characterized by iron overload due to excessive uptake of iron from the gastrointestinal tract\n[12]. Studies of HFE modulation of the health effects of air pollution, including the current study, generally find that variants in HFE protect against the adverse impact of air pollution exposure\n[19,20]. These findings may be attributable to HFE modulation of uptake of metals from the lung. Exposure to traffic-related air pollution occurs primarily through inhalation. In order for traffic-related air pollution to adversely impact human health, some component of traffic-related air pollution must enter the body, either through penetration of the respiratory epithelium, ingestion, or through translocation up the olfactory nerve. Variants of HFE C282Y and H63D are associated with increased iron uptake from the gut, and higher body stores of iron may down-regulate overall metal absorption, including absorption from the lung of metals adhered to inhaled air pollution. In animal models, rats fed a high-iron diet or exposed to iron oxide exhibited less absorption of manganese after intratracheal installation compared to those fed a regular diet\n[17,18]. This reduction in pulmonary absorption was paralleled by lower uptake by other organs, including the brain\n[18]. Therefore, HFE genotype, associated iron levels, and iron homeostatic mechanisms could influence the toxicity of exposure to traffic-related air pollution through modulation of metal uptake. Lower uptake of adsorbed metals would be expected to reduce the associated systemic and brain-based oxidative stress and inflammation and lessen any adverse effect of air pollution on cognition. This mechanistic hypothesis might explain why we did not see an adverse association between air pollution and cognition in carriers of the HFE C282Y variant.\nWe also note that the evidence to support effect modification of the association between air pollution and cognition was stronger when we considered the C282Y variant than when we considered the H63D variant, which is consistent with the relative degree of function of these SNPs. The HFE protein produced by the C282Y variant, but not the H63D variant protein, is often degraded before final processing and is not expressed on the cell surface\n[31] and does not associate with transferrin\n[32]. As such, the C282Y variant is associated with greater loss of function compared to the H63D variant, resulting in increased iron transfer in the gut. In this case, we may expect lower respiratory intake of adsorbed metals, and therefore lower levels of air pollution-related oxidative stress and inflammation, in those with the C282Y variant than in those with the H63D variant.\nWe acknowledge that our study has several limitations. Use of BC exposure estimates based on residential address may misclassify long-term exposure. However, given the age and residential stability of our cohort, misclassification of our exposure due to occupation, commuting, or relocation is expected to be minimal and largely unrelated to cognition or HFE status, suggesting bias towards the null. Non-differential misclassification of cognitive status is likely, but is mitigated by the use of all available cognitive data to consider the association between BC and total metrics of cognition. It is possible that residual confounding may bias our results. However, we were able to adjust for many known predictors of cognition, including several measures of socioeconomic status. Furthermore, we expect confounding by other classes of air pollution to be minimal, as the correlation between traffic-related air pollution and regional pollutants is relatively low due to differences in the spatial-temporal distribution of these types of pollutants. Selection bias is a concern, but because both poor cognition and exposure to traffic-related air pollution predict morbidity and mortality\n[33-36], we would expect selection bias to mask the expected adverse association or create a false protective association. While this may contribute to the absence of an adverse association among participants with HFE C282Y variants, it would not account for the strong adverse association observed within those who lacked HFE C282Y variants. While we are unable to concretely attribute our findings to a particular aspect of traffic-related exposure, modification of the BC-cognition association by HFE implicates metals adhered to particulate matter as a likely toxic agent. While population substructure may induce spurious associations in gene-environment interaction studies, this is unlikely to account for our findings given our homogeneous study sample of white men. Finally, our sample size and the number of participants with the variant HFE alleles are small, which makes it difficult to detect a subtle effect and increases the possibility that our findings are due to chance. This analysis requires replication in an independent sample.","In conclusion, our study suggests that persons who lack a HFE C282Y variant polymorphism may be more susceptible to adverse effects of exposure to traffic-related air pollution on cognition than those possessing a variant. The mechanism by which HFE modifies this association may involve metal transport and inflammation pathways. As with any candidate gene-environment interaction study, this analysis requires independent confirmation.","BC: Black carbon; BMI: Body mass index; HFE: Hemochromatosis gene; CI: Confidence interval; MET-hrs: Metabolic equivalent hours; MMSE: Mini-Mental State Examination; NAS: Normative Aging Study; OR: Odds ratio; QT interval: Heart-rate-corrected interval; VA: United States Department of Veterans Affairs.","The authors declare they have no competing interests.","MCP made substantial contributions to the study conception and design, analysis and interpretation of the data, and drafted the manuscript. MGW and JS made substantial contributions to the study conception and design, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. BAC, SEA, AS made substantial contributions to the acquisition of data and revised the manuscript critically for important intellectual content. ROW made substantial contributions to the analysis or interpretation of the data and revised the manuscript critically for important intellectual content. All authors have given final approval of the version to be published.","Comparison the association between a doubling in BC concentration and cognition in the HFE dataset to the previously reported association in the full dataset. Table showing the main effect of BC exposure in the full dataset used in the original analyses and the reduced dataset used in the current analyses.\nClick here for file"],"string":"[\n \"While we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large\\n[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.\\nTraffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men\\n[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above\\n[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women\\n[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese\\n[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children\\n[6-11].\\nThe hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease\\n[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium\\n[14-18].\\nPrevious studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors\\n[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms.\",\n \" Study sample The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\\n[2] we refer readers to that paper for a more exhaustive description of our methods.\\nThe current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\\n[2] we refer readers to that paper for a more exhaustive description of our methods.\\n Exposure assessment For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\\nFor each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\\n Cognitive testing The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\\nThe content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\\n HFE genotyping For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\\nFor this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\\n Statistical methods As the current dataset excludes participants from the previously reported analyses\\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).\\nAs the current dataset excludes participants from the previously reported analyses\\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).\",\n \"The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\\n[2] we refer readers to that paper for a more exhaustive description of our methods.\",\n \"For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\",\n \"The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\",\n \"For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\",\n \"As the current dataset excludes participants from the previously reported analyses\\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).\",\n \"Six hundred twenty eight (92%) of the 680 men with data on black carbon exposure, cognition, and all covariates were successfully genotyped for both HFE SNPs. Table \\n1 summarizes the sample characteristics for the current analysis in total and by HFE SNP genotypes. At baseline, our participants were, on average, 70 years old (SD: 7.1) and had 14.4 years of formal education (SD: 2.6). 540 (86%) of our participants lacked an HFE C282Y variant and 6 (1%) were homozygous for the C282Y variant. 469 (75%) lacked an HFE H63D variant and 18 (3%) were homozygous for the H63D variant. Both SNPs were in Hardy-Weinberg equilibrium (C282Y: χ2 = 2.05, p = 0.15; H63D: χ2 = 3.32, p = 0.07).\\nBaseline characteristics of the cohort (n = 628)\\nAbbreviations: HFE, hemochromatosis gene; SD, standard deviation; MET-hr/week, metabolic equivalent hours per week.\\nIn the current study sample, the direction and magnitude of the association between black carbon and cognitive function in fully adjusted models was materially unchanged compared to previously reported analyses (Additional file\\n1: Table S1). As expected, there was little evidence to support a main effect of either HFE variant on cognition. The presence of an HFE C282Y variant did not appear to be associated with total cognition (beta: -0.063, 95% CI: -0.142, 0.017) or MMSE performance (odds ratio (OR): 0.92, 95% CI: 0.64, 1.35). Similarly, presence of an HFE H63D variant was not associated with either total cognition (beta: 0.022, 95% CI: -0.041, 0.085) or MMSE performance (OR: 0.98, 95% CI: 0.75, 1.28).\\nConsideration of regression models with interaction terms between HFE genotype and black carbon suggests that HFE C282Y, but not HFE H63D, modifies the association between black carbon concentrations and total cognitive function (Table \\n2). Although the p-values for interaction did not achieve the threshold for statistical significance in any model, the adverse association between black carbon exposure and cognition appears to be exclusive to those lacking the HFE C282Y variant. While participants who lacked an HFE C282Y variant exhibited strong adverse associations between BC and total cognition, a magnitude of effect that is equivalent to approximately 2.1 years of age in our data, we did not observe an adverse association for participants with at least one variant genotype. Similarly, we found elevated odds of low MMSE scores for a doubling in BC concentrations for participants who lacked an HFE C282Y variant but little evidence to support an association in participants with at least one copy of the HFE C282Y variant (Table \\n3).\\nThe association between a doubling in BC concentration and total cognitive z-score by HFE polymorphisms\\nAbbreviations: HFE, hemochromatosis gene; BC, black carbon.\\naAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.\\nThe association between a doubling in BC concentration and low MMSE scores by HFE polymorphisms\\nAbbreviations: HFE, hemochromatosis gene; MMSE, Mini-Mental State Examination; BC, black carbon.\\naAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.\\nWe found little support for modification of the black carbon-cognition association by HFE H63D for either total cognition or MMSE performance. Although we observed statistically significant associations between black carbon and cognition within groups defined by HFE H63D genotype, the p-values for interaction did not achieve statistical significance in either model, and the pattern of association across those with and without the HFE H63D variant differed by outcome. In analyses of total cognition, we observed a statistically significant adverse association between black carbon concentrations among HFE H63D homozygous wild type participants, but not among those possessing at least one variant allele (Table \\n2). However, we observed the opposite pattern in analyses considering MMSE scores (Table \\n3), where we found a statistically significant adverse association in those with at least one H63D variant, but not in those who were homozygous wild type.\\nIn sensitivity analyses, additional adjustment for hemoglobin, body mass index, smoking, or past lead exposure did not appreciably change effect estimates (data not shown).\",\n \"Although the statistical evidence to support the claim that the HFE C282Y polymorphism modifies the association between traffic-related air pollution and cognitive function in our sample of older men is weak, the pattern of response across groups is intriguing and suggests the presence of effect modification by HFE C282Y. Participants possessing a variant HFE C282Y polymorphism appear to be protected from the adverse association between black carbon and cognition, while participants who lacked the C282Y variant exhibit strong adverse associations. We found little support for modification of the association between traffic-related air pollution and cognition by HFE H63D.\\nThis is the first study to investigate effect modification by HFE of the air pollution-cognition relationship. These findings are in line with reported effect modification by HFE of the relationship between exposure to air pollution and cardiovascular outcomes or risk factors in the NAS cohort. Persons lacking HFE variant genotypes exhibit strong adverse associations between exposure to fine particulate matter (PM) and heart rate variability, while there is little evidence for an association among those possessing an HFE variant, and this pattern was driven primarily by the HFE C282Y SNP\\n[20]. Similarly, higher exposures to black carbon or PM2.5 were associated with higher plasma homocysteine concentrations in participants who lacked the HFE C282Y variant, but no association was observed in participants possessing at least one HFE C282Y variant\\n[19]. A similar pattern was observed for modification by HFE C282Y of the association between traffic-related air pollution and heart-rate-corrected (QT) interval, an electrocardiographic marker of ventricular repolarization that is associated with arrhythmia and cardiac death\\n[21].\\nThe mechanism by which exposure to traffic-related air pollution may contribute to cognitive impairment is unknown, but likely involves inhalation of particulates. In animal experiments, exposure to fine or ultrafine particulate matter appears to induce inflammation, lipid peroxidation, and neuronal degeneration in the central nervous system\\n[25-28]. Metals, including iron, are often found adsorbed onto the surface of traffic-related particulates\\n[29] and exposure to metals is associated with induction of oxidative stress and inflammation\\n[30]. Oxidative stress and inflammation may contribute to the development of cognitive impairment directly or through inducement of cardiovascular problems.\\nHFE modulates intracellular uptake of iron and other metals. Variants of HFE are associated with the disease hemochromatosis, which is characterized by iron overload due to excessive uptake of iron from the gastrointestinal tract\\n[12]. Studies of HFE modulation of the health effects of air pollution, including the current study, generally find that variants in HFE protect against the adverse impact of air pollution exposure\\n[19,20]. These findings may be attributable to HFE modulation of uptake of metals from the lung. Exposure to traffic-related air pollution occurs primarily through inhalation. In order for traffic-related air pollution to adversely impact human health, some component of traffic-related air pollution must enter the body, either through penetration of the respiratory epithelium, ingestion, or through translocation up the olfactory nerve. Variants of HFE C282Y and H63D are associated with increased iron uptake from the gut, and higher body stores of iron may down-regulate overall metal absorption, including absorption from the lung of metals adhered to inhaled air pollution. In animal models, rats fed a high-iron diet or exposed to iron oxide exhibited less absorption of manganese after intratracheal installation compared to those fed a regular diet\\n[17,18]. This reduction in pulmonary absorption was paralleled by lower uptake by other organs, including the brain\\n[18]. Therefore, HFE genotype, associated iron levels, and iron homeostatic mechanisms could influence the toxicity of exposure to traffic-related air pollution through modulation of metal uptake. Lower uptake of adsorbed metals would be expected to reduce the associated systemic and brain-based oxidative stress and inflammation and lessen any adverse effect of air pollution on cognition. This mechanistic hypothesis might explain why we did not see an adverse association between air pollution and cognition in carriers of the HFE C282Y variant.\\nWe also note that the evidence to support effect modification of the association between air pollution and cognition was stronger when we considered the C282Y variant than when we considered the H63D variant, which is consistent with the relative degree of function of these SNPs. The HFE protein produced by the C282Y variant, but not the H63D variant protein, is often degraded before final processing and is not expressed on the cell surface\\n[31] and does not associate with transferrin\\n[32]. As such, the C282Y variant is associated with greater loss of function compared to the H63D variant, resulting in increased iron transfer in the gut. In this case, we may expect lower respiratory intake of adsorbed metals, and therefore lower levels of air pollution-related oxidative stress and inflammation, in those with the C282Y variant than in those with the H63D variant.\\nWe acknowledge that our study has several limitations. Use of BC exposure estimates based on residential address may misclassify long-term exposure. However, given the age and residential stability of our cohort, misclassification of our exposure due to occupation, commuting, or relocation is expected to be minimal and largely unrelated to cognition or HFE status, suggesting bias towards the null. Non-differential misclassification of cognitive status is likely, but is mitigated by the use of all available cognitive data to consider the association between BC and total metrics of cognition. It is possible that residual confounding may bias our results. However, we were able to adjust for many known predictors of cognition, including several measures of socioeconomic status. Furthermore, we expect confounding by other classes of air pollution to be minimal, as the correlation between traffic-related air pollution and regional pollutants is relatively low due to differences in the spatial-temporal distribution of these types of pollutants. Selection bias is a concern, but because both poor cognition and exposure to traffic-related air pollution predict morbidity and mortality\\n[33-36], we would expect selection bias to mask the expected adverse association or create a false protective association. While this may contribute to the absence of an adverse association among participants with HFE C282Y variants, it would not account for the strong adverse association observed within those who lacked HFE C282Y variants. While we are unable to concretely attribute our findings to a particular aspect of traffic-related exposure, modification of the BC-cognition association by HFE implicates metals adhered to particulate matter as a likely toxic agent. While population substructure may induce spurious associations in gene-environment interaction studies, this is unlikely to account for our findings given our homogeneous study sample of white men. Finally, our sample size and the number of participants with the variant HFE alleles are small, which makes it difficult to detect a subtle effect and increases the possibility that our findings are due to chance. This analysis requires replication in an independent sample.\",\n \"In conclusion, our study suggests that persons who lack a HFE C282Y variant polymorphism may be more susceptible to adverse effects of exposure to traffic-related air pollution on cognition than those possessing a variant. The mechanism by which HFE modifies this association may involve metal transport and inflammation pathways. As with any candidate gene-environment interaction study, this analysis requires independent confirmation.\",\n \"BC: Black carbon; BMI: Body mass index; HFE: Hemochromatosis gene; CI: Confidence interval; MET-hrs: Metabolic equivalent hours; MMSE: Mini-Mental State Examination; NAS: Normative Aging Study; OR: Odds ratio; QT interval: Heart-rate-corrected interval; VA: United States Department of Veterans Affairs.\",\n \"The authors declare they have no competing interests.\",\n \"MCP made substantial contributions to the study conception and design, analysis and interpretation of the data, and drafted the manuscript. MGW and JS made substantial contributions to the study conception and design, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. BAC, SEA, AS made substantial contributions to the acquisition of data and revised the manuscript critically for important intellectual content. ROW made substantial contributions to the analysis or interpretation of the data and revised the manuscript critically for important intellectual content. All authors have given final approval of the version to be published.\",\n \"Comparison the association between a doubling in BC concentration and cognition in the HFE dataset to the previously reported association in the full dataset. Table showing the main effect of BC exposure in the full dataset used in the original analyses and the reduced dataset used in the current analyses.\\nClick here for file\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,"results","discussion","conclusions",null,null,null,"supplementary-material"],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["Aging","Black carbon","Cognitive dysfunction","Epidemiology","Particulate matter","HFE","Hemochromatosis","Gene-environment interaction","Susceptible group"],"string":"[\n \"Aging\",\n \"Black carbon\",\n \"Cognitive dysfunction\",\n \"Epidemiology\",\n \"Particulate matter\",\n \"HFE\",\n \"Hemochromatosis\",\n \"Gene-environment interaction\",\n \"Susceptible group\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nWhile we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large\n[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.\nTraffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men\n[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above\n[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women\n[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese\n[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children\n[6-11].\nThe hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease\n[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium\n[14-18].\nPrevious studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors\n[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms.\n\nMethods:\n Study sample The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.\nThe current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.\n Exposure assessment For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\nFor each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\n Cognitive testing The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\nThe content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\n HFE genotyping For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\nFor this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\n Statistical methods As the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).\nAs the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).\n\nStudy sample:\nThe current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.\n\nExposure assessment:\nFor each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\n\nCognitive testing:\nThe content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\n\nHFE genotyping:\nFor this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\n\nStatistical methods:\nAs the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).\n\nResults:\nSix hundred twenty eight (92%) of the 680 men with data on black carbon exposure, cognition, and all covariates were successfully genotyped for both HFE SNPs. Table \n1 summarizes the sample characteristics for the current analysis in total and by HFE SNP genotypes. At baseline, our participants were, on average, 70 years old (SD: 7.1) and had 14.4 years of formal education (SD: 2.6). 540 (86%) of our participants lacked an HFE C282Y variant and 6 (1%) were homozygous for the C282Y variant. 469 (75%) lacked an HFE H63D variant and 18 (3%) were homozygous for the H63D variant. Both SNPs were in Hardy-Weinberg equilibrium (C282Y: χ2 = 2.05, p = 0.15; H63D: χ2 = 3.32, p = 0.07).\nBaseline characteristics of the cohort (n = 628)\nAbbreviations: HFE, hemochromatosis gene; SD, standard deviation; MET-hr/week, metabolic equivalent hours per week.\nIn the current study sample, the direction and magnitude of the association between black carbon and cognitive function in fully adjusted models was materially unchanged compared to previously reported analyses (Additional file\n1: Table S1). As expected, there was little evidence to support a main effect of either HFE variant on cognition. The presence of an HFE C282Y variant did not appear to be associated with total cognition (beta: -0.063, 95% CI: -0.142, 0.017) or MMSE performance (odds ratio (OR): 0.92, 95% CI: 0.64, 1.35). Similarly, presence of an HFE H63D variant was not associated with either total cognition (beta: 0.022, 95% CI: -0.041, 0.085) or MMSE performance (OR: 0.98, 95% CI: 0.75, 1.28).\nConsideration of regression models with interaction terms between HFE genotype and black carbon suggests that HFE C282Y, but not HFE H63D, modifies the association between black carbon concentrations and total cognitive function (Table \n2). Although the p-values for interaction did not achieve the threshold for statistical significance in any model, the adverse association between black carbon exposure and cognition appears to be exclusive to those lacking the HFE C282Y variant. While participants who lacked an HFE C282Y variant exhibited strong adverse associations between BC and total cognition, a magnitude of effect that is equivalent to approximately 2.1 years of age in our data, we did not observe an adverse association for participants with at least one variant genotype. Similarly, we found elevated odds of low MMSE scores for a doubling in BC concentrations for participants who lacked an HFE C282Y variant but little evidence to support an association in participants with at least one copy of the HFE C282Y variant (Table \n3).\nThe association between a doubling in BC concentration and total cognitive z-score by HFE polymorphisms\nAbbreviations: HFE, hemochromatosis gene; BC, black carbon.\naAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.\nThe association between a doubling in BC concentration and low MMSE scores by HFE polymorphisms\nAbbreviations: HFE, hemochromatosis gene; MMSE, Mini-Mental State Examination; BC, black carbon.\naAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.\nWe found little support for modification of the black carbon-cognition association by HFE H63D for either total cognition or MMSE performance. Although we observed statistically significant associations between black carbon and cognition within groups defined by HFE H63D genotype, the p-values for interaction did not achieve statistical significance in either model, and the pattern of association across those with and without the HFE H63D variant differed by outcome. In analyses of total cognition, we observed a statistically significant adverse association between black carbon concentrations among HFE H63D homozygous wild type participants, but not among those possessing at least one variant allele (Table \n2). However, we observed the opposite pattern in analyses considering MMSE scores (Table \n3), where we found a statistically significant adverse association in those with at least one H63D variant, but not in those who were homozygous wild type.\nIn sensitivity analyses, additional adjustment for hemoglobin, body mass index, smoking, or past lead exposure did not appreciably change effect estimates (data not shown).\n\nDiscussion:\nAlthough the statistical evidence to support the claim that the HFE C282Y polymorphism modifies the association between traffic-related air pollution and cognitive function in our sample of older men is weak, the pattern of response across groups is intriguing and suggests the presence of effect modification by HFE C282Y. Participants possessing a variant HFE C282Y polymorphism appear to be protected from the adverse association between black carbon and cognition, while participants who lacked the C282Y variant exhibit strong adverse associations. We found little support for modification of the association between traffic-related air pollution and cognition by HFE H63D.\nThis is the first study to investigate effect modification by HFE of the air pollution-cognition relationship. These findings are in line with reported effect modification by HFE of the relationship between exposure to air pollution and cardiovascular outcomes or risk factors in the NAS cohort. Persons lacking HFE variant genotypes exhibit strong adverse associations between exposure to fine particulate matter (PM) and heart rate variability, while there is little evidence for an association among those possessing an HFE variant, and this pattern was driven primarily by the HFE C282Y SNP\n[20]. Similarly, higher exposures to black carbon or PM2.5 were associated with higher plasma homocysteine concentrations in participants who lacked the HFE C282Y variant, but no association was observed in participants possessing at least one HFE C282Y variant\n[19]. A similar pattern was observed for modification by HFE C282Y of the association between traffic-related air pollution and heart-rate-corrected (QT) interval, an electrocardiographic marker of ventricular repolarization that is associated with arrhythmia and cardiac death\n[21].\nThe mechanism by which exposure to traffic-related air pollution may contribute to cognitive impairment is unknown, but likely involves inhalation of particulates. In animal experiments, exposure to fine or ultrafine particulate matter appears to induce inflammation, lipid peroxidation, and neuronal degeneration in the central nervous system\n[25-28]. Metals, including iron, are often found adsorbed onto the surface of traffic-related particulates\n[29] and exposure to metals is associated with induction of oxidative stress and inflammation\n[30]. Oxidative stress and inflammation may contribute to the development of cognitive impairment directly or through inducement of cardiovascular problems.\nHFE modulates intracellular uptake of iron and other metals. Variants of HFE are associated with the disease hemochromatosis, which is characterized by iron overload due to excessive uptake of iron from the gastrointestinal tract\n[12]. Studies of HFE modulation of the health effects of air pollution, including the current study, generally find that variants in HFE protect against the adverse impact of air pollution exposure\n[19,20]. These findings may be attributable to HFE modulation of uptake of metals from the lung. Exposure to traffic-related air pollution occurs primarily through inhalation. In order for traffic-related air pollution to adversely impact human health, some component of traffic-related air pollution must enter the body, either through penetration of the respiratory epithelium, ingestion, or through translocation up the olfactory nerve. Variants of HFE C282Y and H63D are associated with increased iron uptake from the gut, and higher body stores of iron may down-regulate overall metal absorption, including absorption from the lung of metals adhered to inhaled air pollution. In animal models, rats fed a high-iron diet or exposed to iron oxide exhibited less absorption of manganese after intratracheal installation compared to those fed a regular diet\n[17,18]. This reduction in pulmonary absorption was paralleled by lower uptake by other organs, including the brain\n[18]. Therefore, HFE genotype, associated iron levels, and iron homeostatic mechanisms could influence the toxicity of exposure to traffic-related air pollution through modulation of metal uptake. Lower uptake of adsorbed metals would be expected to reduce the associated systemic and brain-based oxidative stress and inflammation and lessen any adverse effect of air pollution on cognition. This mechanistic hypothesis might explain why we did not see an adverse association between air pollution and cognition in carriers of the HFE C282Y variant.\nWe also note that the evidence to support effect modification of the association between air pollution and cognition was stronger when we considered the C282Y variant than when we considered the H63D variant, which is consistent with the relative degree of function of these SNPs. The HFE protein produced by the C282Y variant, but not the H63D variant protein, is often degraded before final processing and is not expressed on the cell surface\n[31] and does not associate with transferrin\n[32]. As such, the C282Y variant is associated with greater loss of function compared to the H63D variant, resulting in increased iron transfer in the gut. In this case, we may expect lower respiratory intake of adsorbed metals, and therefore lower levels of air pollution-related oxidative stress and inflammation, in those with the C282Y variant than in those with the H63D variant.\nWe acknowledge that our study has several limitations. Use of BC exposure estimates based on residential address may misclassify long-term exposure. However, given the age and residential stability of our cohort, misclassification of our exposure due to occupation, commuting, or relocation is expected to be minimal and largely unrelated to cognition or HFE status, suggesting bias towards the null. Non-differential misclassification of cognitive status is likely, but is mitigated by the use of all available cognitive data to consider the association between BC and total metrics of cognition. It is possible that residual confounding may bias our results. However, we were able to adjust for many known predictors of cognition, including several measures of socioeconomic status. Furthermore, we expect confounding by other classes of air pollution to be minimal, as the correlation between traffic-related air pollution and regional pollutants is relatively low due to differences in the spatial-temporal distribution of these types of pollutants. Selection bias is a concern, but because both poor cognition and exposure to traffic-related air pollution predict morbidity and mortality\n[33-36], we would expect selection bias to mask the expected adverse association or create a false protective association. While this may contribute to the absence of an adverse association among participants with HFE C282Y variants, it would not account for the strong adverse association observed within those who lacked HFE C282Y variants. While we are unable to concretely attribute our findings to a particular aspect of traffic-related exposure, modification of the BC-cognition association by HFE implicates metals adhered to particulate matter as a likely toxic agent. While population substructure may induce spurious associations in gene-environment interaction studies, this is unlikely to account for our findings given our homogeneous study sample of white men. Finally, our sample size and the number of participants with the variant HFE alleles are small, which makes it difficult to detect a subtle effect and increases the possibility that our findings are due to chance. This analysis requires replication in an independent sample.\n\nConclusions:\nIn conclusion, our study suggests that persons who lack a HFE C282Y variant polymorphism may be more susceptible to adverse effects of exposure to traffic-related air pollution on cognition than those possessing a variant. The mechanism by which HFE modifies this association may involve metal transport and inflammation pathways. As with any candidate gene-environment interaction study, this analysis requires independent confirmation.\n\nAbbreviations:\nBC: Black carbon; BMI: Body mass index; HFE: Hemochromatosis gene; CI: Confidence interval; MET-hrs: Metabolic equivalent hours; MMSE: Mini-Mental State Examination; NAS: Normative Aging Study; OR: Odds ratio; QT interval: Heart-rate-corrected interval; VA: United States Department of Veterans Affairs.\n\nCompeting interests:\nThe authors declare they have no competing interests.\n\nAuthors’ contributions:\nMCP made substantial contributions to the study conception and design, analysis and interpretation of the data, and drafted the manuscript. MGW and JS made substantial contributions to the study conception and design, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. BAC, SEA, AS made substantial contributions to the acquisition of data and revised the manuscript critically for important intellectual content. ROW made substantial contributions to the analysis or interpretation of the data and revised the manuscript critically for important intellectual content. All authors have given final approval of the version to be published.\n\nSupplementary Material:\nComparison the association between a doubling in BC concentration and cognition in the HFE dataset to the previously reported association in the full dataset. Table showing the main effect of BC exposure in the full dataset used in the original analyses and the reduced dataset used in the current analyses.\nClick here for file\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nPrevious studies found effect modification of associations between traffic-related air pollution and cardiovascular outcomes by polymorphisms in the hemochromatosis gene (HFE). As traffic-related air pollution may impact cognition through effects on cardiovascular health or through mechanisms which may also influence cardiovascular outcomes, we hypothesized that HFE polymorphisms would also modify a previously observed association between traffic-related air pollution exposure and cognition in older men.\n\nMethods:\nWe considered data from 628 participants of the VA Normative Aging Study. We estimated long term exposure to black carbon (BC), a marker of traffic related air pollution, using a spatio-temporal land use regression model. We assessed cognition using the Mini-Mental State Examination (MMSE), a test of global function, and performance on a battery of other tests, covering a wide range of domains. We investigated whether variants of HFE C282Y and H63D modified the association between BC and having a low MMSE score using logistic models with generalized estimating equations and multiplicative interaction terms. Similarly, we assessed whether HFE variants modified the association between BC and performance on the cognitive battery using linear mixed models with multiplicative interaction terms.\n\nResults:\nOur results suggest modification of the BC-cognition association by HFE C282Y, although the test of interaction did not achieve statistical significance. In multivariable-adjusted models, participants who lacked a HFE C282Y variant (CC) exhibited an adverse association between BC and total cognition z-score (beta for a doubling in BC concentration: -0.061, 95% CI: -0.115, -0.007), while we did not observe an association in participants with at least one variant genotype (CY or YY) (beta for a doubling in BC concentration: 0.073, 95% CI: -0.081, 0.228; p-value for interaction: 0.11). The pattern of association was similar for analyses considering performance on the Mini-Mental State Examination. There was little evidence to support effect modification of the BC-cognition association by the HFE H63D genotype.\n\nConclusions:\nOur data suggest that older adults who lack an HFE C282Y variant may be more susceptible to an adverse effect of traffic-related air pollution exposure on cognition. This finding and the proposed biological mechanism require confirmation."},"background_conclusion_text":{"kind":"string","value":"Background:\nWhile we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large\n[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.\nTraffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men\n[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above\n[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women\n[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese\n[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children\n[6-11].\nThe hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease\n[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium\n[14-18].\nPrevious studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors\n[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms.\n\nConclusions:\nIn conclusion, our study suggests that persons who lack a HFE C282Y variant polymorphism may be more susceptible to adverse effects of exposure to traffic-related air pollution on cognition than those possessing a variant. The mechanism by which HFE modifies this association may involve metal transport and inflammation pathways. As with any candidate gene-environment interaction study, this analysis requires independent confirmation."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nPrevious studies found effect modification of associations between traffic-related air pollution and cardiovascular outcomes by polymorphisms in the hemochromatosis gene (HFE). As traffic-related air pollution may impact cognition through effects on cardiovascular health or through mechanisms which may also influence cardiovascular outcomes, we hypothesized that HFE polymorphisms would also modify a previously observed association between traffic-related air pollution exposure and cognition in older men.\n\nMethods:\nWe considered data from 628 participants of the VA Normative Aging Study. We estimated long term exposure to black carbon (BC), a marker of traffic related air pollution, using a spatio-temporal land use regression model. We assessed cognition using the Mini-Mental State Examination (MMSE), a test of global function, and performance on a battery of other tests, covering a wide range of domains. We investigated whether variants of HFE C282Y and H63D modified the association between BC and having a low MMSE score using logistic models with generalized estimating equations and multiplicative interaction terms. Similarly, we assessed whether HFE variants modified the association between BC and performance on the cognitive battery using linear mixed models with multiplicative interaction terms.\n\nResults:\nOur results suggest modification of the BC-cognition association by HFE C282Y, although the test of interaction did not achieve statistical significance. In multivariable-adjusted models, participants who lacked a HFE C282Y variant (CC) exhibited an adverse association between BC and total cognition z-score (beta for a doubling in BC concentration: -0.061, 95% CI: -0.115, -0.007), while we did not observe an association in participants with at least one variant genotype (CY or YY) (beta for a doubling in BC concentration: 0.073, 95% CI: -0.081, 0.228; p-value for interaction: 0.11). The pattern of association was similar for analyses considering performance on the Mini-Mental State Examination. There was little evidence to support effect modification of the BC-cognition association by the HFE H63D genotype.\n\nConclusions:\nOur data suggest that older adults who lack an HFE C282Y variant may be more susceptible to an adverse effect of traffic-related air pollution exposure on cognition. This finding and the proposed biological mechanism require confirmation."},"whole_article_text_length":{"kind":"number","value":7485,"string":"7,485"},"whole_article_abstract_length":{"kind":"number","value":427,"string":"427"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["hfe","cognitive","black","black carbon","carbon","cognition","exposure","association","participants","variant"],"string":"[\n \"hfe\",\n \"cognitive\",\n \"black\",\n \"black carbon\",\n \"carbon\",\n \"cognition\",\n \"exposure\",\n \"association\",\n \"participants\",\n \"variant\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] air | pollution | air pollution | exposure | groups | associated | polymorphisms | hfe polymorphisms | disease | susceptible [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cognitive | assessment | carbon | black carbon | black | participant | tests | hfe | mmse | lead [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hfe | variant | association | h63d | c282y variant | table | total | black carbon | carbon | cognition [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] variant | hfe modifies | pollution cognition possessing variant | pollution cognition possessing | mechanism hfe modifies association | mechanism hfe modifies | mechanism hfe | hfe modifies association involve | hfe modifies association | effects exposure traffic [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hfe | cognitive | black carbon | black | carbon | association | cognition | variant | exposure | air [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hfe | cognitive | black carbon | black | carbon | association | cognition | variant | exposure | air [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 628 | the VA Normative Aging Study ||| BC ||| the Mini-Mental State Examination ||| H63D | BC ||| BC | linear [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] BC ||| CC | BC | BC | 95% | CI | at least one | YY | BC | 0.073 | 95% | CI | 0.228 | 0.11 ||| the Mini-Mental State Examination ||| BC [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| 628 | the VA Normative Aging Study ||| BC ||| the Mini-Mental State Examination ||| H63D | BC ||| BC | linear ||| ||| BC ||| CC | BC | BC | 95% | CI | at least one | YY | BC | 0.073 | 95% | CI | 0.228 | 0.11 ||| the Mini-Mental State Examination ||| BC ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| 628 | the VA Normative Aging Study ||| BC ||| the Mini-Mental State Examination ||| H63D | BC ||| BC | linear ||| ||| BC ||| CC | BC | BC | 95% | CI | at least one | YY | BC | 0.073 | 95% | CI | 0.228 | 0.11 ||| the Mini-Mental State Examination ||| BC ||| ||| [SUMMARY]"}}},{"rowIdx":264,"cells":{"title":{"kind":"string","value":"Atrioventricular block in coronary artery bypass surgery: perioperative predictors and impact on mortality."},"pmid":{"kind":"string","value":"26107447"},"background_abstract":{"kind":"string","value":"Disturbances of the cardiac conduction system are frequent in the postoperative period of coronary artery bypass surgery. They are mostly reversible and associated with some injury of the conduction tissue, caused by the ischemic heart disease itself or by perioperative factors."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"Analysis of a retrospective cohort of patients submitted to coronary artery bypass surgery from the database of the Postoperative Heart Surgery Unit of the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande do Sul, using the logistic regression method."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In the period from January 1996 to December 2012, 3532 coronary artery bypass surgery were carried out. Two hundred and eighty-eight (8.15% of the total sample) patients had atrioventricular block during the postoperative period of coronary artery bypass surgery, requiring temporary pacing. Eight of those who had atrioventricular block progressed to implantation of a permanent pacemaker (0.23% of the total sample). Multivariate analysis revealed a significant association of atrioventricular block with age above 60 years (OR=2.34; CI 95% 1.75-3.12; P<0.0001), female gender (OR=1.37; CI 95% 1.06-1.77; P=0.015), chronic kidney disease (OR=2.05; CI 95% 1.49-2.81; P<0.0001), atrial fibrillation (OR=2.06; CI 95% 1.16-3.66; P=0.014), functional class III and IV of the New York Heart Association (OR=1.43; CI 95% 1.03-1.98; P=0.031), perioperative acute myocardial infarction (OR=1.70; CI 95% 1.26-2.29; P<0.0001) and with the use of the intra-aortic balloon in the postoperative period of coronary artery bypass surgery (OR=1.92; CI 95% 1.21-3.05; P=0.006). The presence of atrioventricular block resulted in a significant increase in mortality (17.9% vs. 7.3% in those who did not develop atrioventricular block) (OR=2.09; CI 95% 1.46-2.99; P<0.0001) and a longer hospital stay (12.75 days x 10.53 days for those who didn't develop atrioventricular block) (OR=1.01; CI 95% 1.00-1.02; P=0.01)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"In most cases, atrioventricular block in the postoperative period of coronary artery bypass surgery is transient and associated with several perioperative factors: age above 60 years, female sex, chronic kidney disease, atrial fibrillation, New York Heart Association functional class III or IV, perioperative acute myocardial infarction and use of an intra-aortic balloon. Its occurrence prolongs hospitalization and, above all, doubles the risk of mortality."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Age Factors","Atrioventricular Block","Cardiopulmonary Bypass","Coronary Artery Bypass","Epidemiologic Methods","Female","Hospital Mortality","Humans","Length of Stay","Male","Middle Aged","Pacemaker, Artificial","Perioperative Period","Postoperative Complications","Risk Factors","Sex Factors","Time Factors","Treatment Outcome"],"string":"[\n \"Age Factors\",\n \"Atrioventricular Block\",\n \"Cardiopulmonary Bypass\",\n \"Coronary Artery Bypass\",\n \"Epidemiologic Methods\",\n \"Female\",\n \"Hospital Mortality\",\n \"Humans\",\n \"Length of Stay\",\n \"Male\",\n \"Middle Aged\",\n \"Pacemaker, Artificial\",\n \"Perioperative Period\",\n \"Postoperative Complications\",\n \"Risk Factors\",\n \"Sex Factors\",\n \"Time Factors\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"4462961"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Disturbances of the cardiac conduction system are relatively frequent in the\npostoperative period (PO) of coronary artery bypass grafting (CABG), with an\nincidence ranging from 18 to 55% of cases[1-6].\nAtrioventricular block (AVB) is one of these conduction disturbances and its\nincidence ranges from 0.5 to 16%[3,5-9]. Most patients have transitory and\nreversible conduction disorders that require temporary pacing (TP). 0.4 to 1.1% of\npatients, however, when faced with the irreversibility of the condition, will have\nto undergo a permanent pacemaker (PPM) implant during their hospital\nstay[10].\nThis study, which is unprecedented in the national literature, tries to identify the\nrelationship between pre-, intra and postoperative (perioperative) factors\nassociated with the emergence of AVB, the need for TP and, if the case, the\nimplantation of a PPM in the PO of CABG."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":" Population and sample Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).\nBetween January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).\n Study Design Historical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.\nHistorical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.\n Inclusion Criteria Patients with age equal to or greater than 18 years who were submitted to\nisolated CABG.\nPatients with age equal to or greater than 18 years who were submitted to\nisolated CABG.\n Exclusion Criteria Patients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.\nPatients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.\n Study Variables Age - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.\nAge - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.\n Outcome Development of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.\nDevelopment of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.\n Procedures The CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.\nThe CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.\n Statistical Analysis The data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.\nThe data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.\n Ethical Considerations The design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.\nThe design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"Of the 3532 patients undergoing CABG in the period under analysis, 288 (8.15%)\npresented the clinical and electrocardiographic signs of AVB during the\npostoperative period, with an indication for temporary pacing (TP).\nTable 1 shows the demographic profile of the\npatients studied. The univariate analysis of the preoperative data revealed a\ngreater need for TP in the PO of CABG in patients above 60 years of age (OR=2.48; CI\n95% 1.90-3.24; P<0.0001), of the female gender (OR=1.03; CI 95%\n1.00-1.05; P=0.012), with CKD (OR=2.03; CI 95% 1.55-2.65;\nP<0.0001), the presence of AF (OR=2.38; CI 95% 1.49-3.72;\nP<0.0001) and in patients with NYHA functional class III or\nIV (OR=1.60; CI 95% 1.21-2.12; P=0.001).\nPreoperative characteristics of the groups and univariate analysis.\nAF=atrial fibrillation; AMI=acute myocardial infarction;\nBB=beta-blockers; CI=confidence interval; CKD=chronic kidney disease;\nDM=diabetes mellitus; EF=left ventricular ejection fraction;\nFC=functional class; NYHA=New York Heart Association; NTP=no use of\ntemporary pacing; OR=odds ratio; P=statistical significance;\nTP=temporary pacing\nTable 2 presents the trans and postoperative\ndata, together with their univariate analysis. Here we can observe the association\nof AVB with the need of TP in patients who presented calcification of the aorta,\nperioperative AMI and the need for the use of an IAB. Of statistically significant\nrelevance, the univariate analysis also revealed the association of TP caused by AVB\nwith increased mortality (17.9% vs. 7.3%) and with a longer hospital stay (mean\nhospitalization time of 12.75 days compared to 10.53 days for those who did not\nrequire TP).\nTrans and postoperative data of groups and univariate analysis.\nCalcification Ao=calcification of the aorta; CPBT=cardiopulmonary bypass\ntime; IAB=intra-aortic balloon; Peri AMI=perioperative acute myocardial\ninfarction; Tclamping=aortic clamping time; Others: see Table 1\nThese data were submitted to multivariate analysis (Table 3), which revealed a higher risk of AVB in the PO of CABG in\npatients with: age > 60 years, female sex, CKD, AF, NYHA functional class III or\nIV, perioperative AMI and with the use of an IAB. Patients with EF≤40 %, DM,\nthe use of beta-blockers, statins and other antiarrhythmic drugs, prior AMI and CPB\nand aortic clamping times didn't prove to be independent risk variables for the\ndevelopment of AVB in the PO of CABG.\nMultivariate analysis of the risk factors and outcomes of AVB in the PO of\nCABG.\nAMI=acute myocardial infarction; AVB=atrioventricular block; CABG=coronary\nartery bypass grafting; CKD=chronic kidney disease; FC=functional class;\nIAB=intra-aortic balloon; PO=postoperative\nIn the multivariate analysis, the presence of AVB resulted in a longer hospital stay\n(12.75 days vs. 10.53 days for those who didn't develop AVB) (OR=1.01; CI 95%\n1.00-1.02; P=0.01) and in a significant increase in the risk of\nmortality (17.9% vs. 7.3% for patients without AVB) (OR=2.09; CI 95% 1.46-2.99;\nP<0.0001).\nIn the subgroup of 288 patients who had AVB and who had undergone TP, 08 (2.78 %)\nrequired a PPM implant, corresponding to 0.23% of the total cohort analyzed. The\naverage time elapsed since the surgery until the PPM implant was 12.25 days."},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"This work sheds light on the risk factors associated with the development of AVB in\nthe PO of CABG and the consequent need for TP and a definitive pacemaker. Based on\nthis we could establish that female patients, 60 years of age or more, with the\ndiagnosis of AF and CKD, in stages III and IV of the functional class, who had\nperioperative AMI and required the use of an IAB, have a higher risk of developing\nAVB in the PO of CABG. AVB determines a more prolonged hospitalization and, what is\nmore important, doubles the risk of mortality."},"other_sections_titles":{"kind":"list like","value":["Population and sample","Study Design","Inclusion Criteria","Exclusion Criteria","Study Variables","Outcome","Procedures","Statistical Analysis","Ethical Considerations","Limitations of the Study"],"string":"[\n \"Population and sample\",\n \"Study Design\",\n \"Inclusion Criteria\",\n \"Exclusion Criteria\",\n \"Study Variables\",\n \"Outcome\",\n \"Procedures\",\n \"Statistical Analysis\",\n \"Ethical Considerations\",\n \"Limitations of the Study\"\n]"},"other_sections_texts":{"kind":"list like","value":["Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).","Historical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.","Patients with age equal to or greater than 18 years who were submitted to\nisolated CABG.","Patients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.","Age - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.","Development of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.","The CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.","The data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.","The design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.","The limitations of this study are those inherent to a retrospective database\nanalysis, but they reflect the significant years of experience of an academic\ninstitution. Within these limitations we can cite the relative difficulty of\naccessing the full data, which causes a potential risk of not measuring some\nrandom variables. The fact that the results come from the sample of a single\ncenter can also represent some degree of bias in the treatment. Another\nlimitation of this study is the absence of more precise information regarding\nthe height of the atrioventricular conduction disorder and the existence or not\nof any escape rhythm.\nRegarding the PPM implants performed in our study, they followed the\nrecommendations of Brazilian, American and European guidelines almost strictly.\nIn this small group of patients a more thorough analysis was compromised, but\nthis could be the target of a more detailed study to be developed in the\nfuture."],"string":"[\n \"Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\\nUniversity of Rio Grande do Sul (PUCRS).\",\n \"Historical cohort observation study. Data were collected prospectively and\\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\\nSão Lucas Hospital of PUCRS.\",\n \"Patients with age equal to or greater than 18 years who were submitted to\\nisolated CABG.\",\n \"Patients who had been undergo valvular surgery, for left ventricular\\naneurysmectomy or correction of the interventricular communication associated\\nwith CABG.\",\n \"Age - the mean age was calculated and also divided into groups for analysis: less\\nthan 60 years and greater than or equal to 60 years, according to the reference\\nin the literature[11,12]; gender (male and\\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\\nor radiocardiography, with the values being subdivided for analysis into\\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\\nserum creatinine level > 1.5 mg/dl, according to the reference in the\\nliterature[11,12]; Diabetes Mellitus\\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\\nfunctional (NYHA) class; presence of calcification of the aorta; time of\\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\\nhospital stay and hospital death.\",\n \"Development of AVB in the PO of CABG and the need for TP and implantation of a\\nPPM.\",\n \"The CABGs were performed under general anesthesia. In all cases, a hyperkalemic\\ncardiac arrest was induced using a cold cardioplegic blood solution in the\\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\\ntransferred to the ICU of the POHS with mechanical ventilation.\",\n \"The data was plotted in a digital Microsoft Access® spreadsheet\\nand analyzed using version 17.0 of the statistical software SPSS. The\\ndescriptive analysis was performed through frequency and mean ± standard\\ndeviation analysis, according to the case. For the univariate analysis the\\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\\nordinal variables and the Student's T-test for quantitative data. The\\nmultivariate analysis was performed using logistic regression (backward\\nconditional method). The difference was considered as statistically\\nsignificant for the value of P<0.05.\",\n \"The design of this study was submitted to the Research Ethics Committee of the\\nFaculty of Medicine of the PUCRS, under registration number 060/3478.\",\n \"The limitations of this study are those inherent to a retrospective database\\nanalysis, but they reflect the significant years of experience of an academic\\ninstitution. Within these limitations we can cite the relative difficulty of\\naccessing the full data, which causes a potential risk of not measuring some\\nrandom variables. The fact that the results come from the sample of a single\\ncenter can also represent some degree of bias in the treatment. Another\\nlimitation of this study is the absence of more precise information regarding\\nthe height of the atrioventricular conduction disorder and the existence or not\\nof any escape rhythm.\\nRegarding the PPM implants performed in our study, they followed the\\nrecommendations of Brazilian, American and European guidelines almost strictly.\\nIn this small group of patients a more thorough analysis was compromised, but\\nthis could be the target of a more detailed study to be developed in the\\nfuture.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","Population and sample","Study Design","Inclusion Criteria","Exclusion Criteria","Study Variables","Outcome","Procedures","Statistical Analysis","Ethical Considerations","RESULTS","DISCUSSION","Limitations of the Study","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"Population and sample\",\n \"Study Design\",\n \"Inclusion Criteria\",\n \"Exclusion Criteria\",\n \"Study Variables\",\n \"Outcome\",\n \"Procedures\",\n \"Statistical Analysis\",\n \"Ethical Considerations\",\n \"RESULTS\",\n \"DISCUSSION\",\n \"Limitations of the Study\",\n \"CONCLUSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Disturbances of the cardiac conduction system are relatively frequent in the\npostoperative period (PO) of coronary artery bypass grafting (CABG), with an\nincidence ranging from 18 to 55% of cases[1-6].\nAtrioventricular block (AVB) is one of these conduction disturbances and its\nincidence ranges from 0.5 to 16%[3,5-9]. Most patients have transitory and\nreversible conduction disorders that require temporary pacing (TP). 0.4 to 1.1% of\npatients, however, when faced with the irreversibility of the condition, will have\nto undergo a permanent pacemaker (PPM) implant during their hospital\nstay[10].\nThis study, which is unprecedented in the national literature, tries to identify the\nrelationship between pre-, intra and postoperative (perioperative) factors\nassociated with the emergence of AVB, the need for TP and, if the case, the\nimplantation of a PPM in the PO of CABG."," Population and sample Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).\nBetween January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).\n Study Design Historical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.\nHistorical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.\n Inclusion Criteria Patients with age equal to or greater than 18 years who were submitted to\nisolated CABG.\nPatients with age equal to or greater than 18 years who were submitted to\nisolated CABG.\n Exclusion Criteria Patients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.\nPatients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.\n Study Variables Age - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.\nAge - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.\n Outcome Development of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.\nDevelopment of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.\n Procedures The CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.\nThe CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.\n Statistical Analysis The data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.\nThe data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.\n Ethical Considerations The design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.\nThe design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.","Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).","Historical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.","Patients with age equal to or greater than 18 years who were submitted to\nisolated CABG.","Patients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.","Age - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.","Development of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.","The CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.","The data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.","The design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.","Of the 3532 patients undergoing CABG in the period under analysis, 288 (8.15%)\npresented the clinical and electrocardiographic signs of AVB during the\npostoperative period, with an indication for temporary pacing (TP).\nTable 1 shows the demographic profile of the\npatients studied. The univariate analysis of the preoperative data revealed a\ngreater need for TP in the PO of CABG in patients above 60 years of age (OR=2.48; CI\n95% 1.90-3.24; P<0.0001), of the female gender (OR=1.03; CI 95%\n1.00-1.05; P=0.012), with CKD (OR=2.03; CI 95% 1.55-2.65;\nP<0.0001), the presence of AF (OR=2.38; CI 95% 1.49-3.72;\nP<0.0001) and in patients with NYHA functional class III or\nIV (OR=1.60; CI 95% 1.21-2.12; P=0.001).\nPreoperative characteristics of the groups and univariate analysis.\nAF=atrial fibrillation; AMI=acute myocardial infarction;\nBB=beta-blockers; CI=confidence interval; CKD=chronic kidney disease;\nDM=diabetes mellitus; EF=left ventricular ejection fraction;\nFC=functional class; NYHA=New York Heart Association; NTP=no use of\ntemporary pacing; OR=odds ratio; P=statistical significance;\nTP=temporary pacing\nTable 2 presents the trans and postoperative\ndata, together with their univariate analysis. Here we can observe the association\nof AVB with the need of TP in patients who presented calcification of the aorta,\nperioperative AMI and the need for the use of an IAB. Of statistically significant\nrelevance, the univariate analysis also revealed the association of TP caused by AVB\nwith increased mortality (17.9% vs. 7.3%) and with a longer hospital stay (mean\nhospitalization time of 12.75 days compared to 10.53 days for those who did not\nrequire TP).\nTrans and postoperative data of groups and univariate analysis.\nCalcification Ao=calcification of the aorta; CPBT=cardiopulmonary bypass\ntime; IAB=intra-aortic balloon; Peri AMI=perioperative acute myocardial\ninfarction; Tclamping=aortic clamping time; Others: see Table 1\nThese data were submitted to multivariate analysis (Table 3), which revealed a higher risk of AVB in the PO of CABG in\npatients with: age > 60 years, female sex, CKD, AF, NYHA functional class III or\nIV, perioperative AMI and with the use of an IAB. Patients with EF≤40 %, DM,\nthe use of beta-blockers, statins and other antiarrhythmic drugs, prior AMI and CPB\nand aortic clamping times didn't prove to be independent risk variables for the\ndevelopment of AVB in the PO of CABG.\nMultivariate analysis of the risk factors and outcomes of AVB in the PO of\nCABG.\nAMI=acute myocardial infarction; AVB=atrioventricular block; CABG=coronary\nartery bypass grafting; CKD=chronic kidney disease; FC=functional class;\nIAB=intra-aortic balloon; PO=postoperative\nIn the multivariate analysis, the presence of AVB resulted in a longer hospital stay\n(12.75 days vs. 10.53 days for those who didn't develop AVB) (OR=1.01; CI 95%\n1.00-1.02; P=0.01) and in a significant increase in the risk of\nmortality (17.9% vs. 7.3% for patients without AVB) (OR=2.09; CI 95% 1.46-2.99;\nP<0.0001).\nIn the subgroup of 288 patients who had AVB and who had undergone TP, 08 (2.78 %)\nrequired a PPM implant, corresponding to 0.23% of the total cohort analyzed. The\naverage time elapsed since the surgery until the PPM implant was 12.25 days.","CABG is a proven therapeutic strategy for the treatment of Coronary Artery Disease\n(CAD). Although it is well tolerated by most patients, perioperative complications\ncan occur, among which we find disturbances in the cardiac conduction system in\nvarying degrees, including AVB.\nPrevious studies have reported an incidence of conduction disturbances (CD) after\nCABG that varies from 18 to 55% of cases[1-6], with the\nright bundle branch block being the most common[4]. Atrioventricular block (AVB) is one of\nthese conduction disturbances and its incidence ranges from 0.5 to\n16%[3,5-9]. Our incidence of AVB is in line with this data, since\n8.15% of our patients developed AVB in the PO of CABG.\nThe etiology of AVB seems to be multifactorial. The patient's age (>60 years),\nhypertension, number of revascularized vessels, aortic clamping time, total time of\nCPB, use of digitalis and beta-blockers, type of cardioplegia and previously\nexisting left bundle branch block may be related to its appearance[1-4,8,9,13,14].\nMyocardial ischemia seems to be the factor that is most implicated in the emergence\nof AVB, since there is a correlation with coronary artery disease (CAD) and\npreoperative AMI[4].\nStudies[3,10] have demonstrated that\nperioperative AMI also increases the incidence of AVB in the PO of CABG. Caspi et\nal.[7]\nreported a higher occurrence of AVB in patients with AMI in the PO of CABG (12% vs.\n2 %, P<0.05).\nHowever, AMI before CABG was not a significant factor for the appearance of AVB in\nour study, which is consistent with the world literature[3,7,8,15,16].\nThis shows there is no difference in the incidence of AVB between patients who had\npreoperative AMI and those who didn't, regardless of its electrocardiographic\nlocation.\nCaspi et al.[7] have\nshown that the combination of left main disease and proximal obstruction of a\ndominant right coronary artery was more frequent in patients who exhibited AVB (32%)\nthan in those who without it (12%, P<0.05). The explanation for\nthis effect is the fact that the cardioplegic solution is not properly distributed\nto the coronary beds because of their high degree of obstruction, which compromises\nmyocardial protection and, in some cases, because of the impossibility of bypassing\nthe right coronary artery.\nThe impairment of myocardial irrigation gets worse with age, just as the frequency of\ndegenerative diseases of the conduction system, increasing the probability of\nAVB[9,11,15,17-19]. In this scenario, our patients above 60\nyears of age presented a significant risk (OR=2.34; CI 95% 1.75-3.12;\nP<0.0001) for the development of AVB in the PO of CABG,\ncorroborating the findings of other studies[7,8,13,14].\nThe electrical cardiac conduction tissue differs from cardiac myocytes by being less\ntolerant to the effects of ischemia, hyperkalemia and hypothermia (whether these are\nsystemic or, mostly, induced by a cardioplegic solution that is cold and rich in\npotassium). This may cause a transient block of the conduction\nsystem[11].\nThe advent of cold cardioplegia as a method of myocardial protection has increased\nthe incidence of CD from 20 to 58%[13]. The more significant incidence of conduction\ndisturbances occurred in patients who received cold cardioplegia, as opposed to warm\n(19.6% vs. 1.7 %, respectively)[13], a finding that has also been described by Sirlak\net al.[20].\nSpecifically with respect to AVB, the incidence was of 3.8% in the hypothermia group\nand zero in the normothermal group[13]. All patients in our study underwent surgery with\nthe myocardial protection performed by infusion of a cold cardioplegic blood\nsolution at the root of the aorta every 20 minutes, which contributed to the genesis\nof AVB cases.\nAs such, the perfusion injury determined by the myocardial ischemia and the\nhypothermic injury caused by the cardioplegic solution are the mechanisms that are\nmost involved in the genesis of AVB, acting on the proximal portions of the bundle\nof His, which are more sensitive to this type of aggression than the more distal\nconduction tissue, determining the emergence of bundle branch blocks and increasing\nthe risk of AVB[4].\nIn this scenario, the extent of the CAD, the duration of CPB and the aortic clamping\ntime could compromise myocardial protection during surgery, increasing the risk of\nan ischemic injury and of metabolic damage to the conduction\ntissue[11].\nHowever, our CPB time of ≥ 90 min and aortic clamping time of ≥ 40 min\nshowed no influence on the development of AVB, which is supported by the\nliterature[5-7,16,19]. Baerman\net al.[1], however,\ndemonstrated that patients with lower CPB (101±32min x 121±34min;\nP<0,01) and aortic clamping (44±19min x\n53±17min; P<0.05) times didn't show evidence of AVB in\nthe PO of CABG.\nOur study has shown that the female gender is a risk factor for the occurrence of AVB\n(OR=1.37, CI 95% 1.06-1.77; P=0.015), which contrasts with the\nresults of Gordon et al.[19] who observed a higher need for PPM implants in men\n(P=0.041). Other studies[3,8,15,20], however, didn't point to any of the genders as\nrisk factor. Cadore et al.[12] had already pointed to the female gender as a risk\npredictor for mortality in CABG, which can be an expression of the greater severity\nof the ischemic impairment in this gender and explain their greater tendency for\ndeveloping the block, as seen in our study.\nThe presence of CKD was also verified to be a risk factor for the development of AVB\n(OR = 2.05; CI 95% 1.49-2.81; P<0.0001). A previous\nstudy[19]\nindicated the presence of CKD as more significant among those patients who required\nPPM implantation in the PO of CABG. Like the female gender variable, CKD was also\nfound to be a predictor of mortality in patients underwent CABG according to the\nscore by Cadore et al.[12], expressing its potential for increasing the risk of\ncomplications in the PO of CABG.\nAnother risk predictor for the occurrence of AVB was the more advanced functional\nclass of the NYHA (III and IV) (OR = 1.43; CI 95% 1.03-1.98;\nP=0.031). Studies[18,19] have\ncorroborated this finding, indicating that patients who underwent heart surgery and\nneeding a PPM implant were in the more severe functional class of the NYHA (III and\nIV) when compared to patients who did not require such an implant (57% vs. 35%,\nrespectively, P<0.0001)[18]. Bateman et al.[6] showed that of those\npatients who passed away within the first 30 days of the PO of CABG and who had\ndeveloped some degree of blockage, 90% were into class IV of the NYHA in the\npreoperative period.\nThe patients in this study who had EF≤40% did not present a significant risk\nfor the appearance of AVB in the PO of CABG (OR=1.11; CI 95% 0.85-1.45;\nP=0.44), a finding supported by Gordon et al.[19] who didn't observe any\nsignificant impact of EF on the need for PPM implantation in the PO of isolated\nCABG. Caspi et al.[7], however, found a greater susceptibility to the\ndevelopment of atrioventricular block in patients submitted to CABG with a lower\nEF.\nAlthough Merin et al.[18] mention that the use of antiarrhythmic agents is more\nfrequent in the group of patients that develops blockage after heart surgery (CABG,\nvalve or combined), our data does not reflect this influence. Regarding the use of\nbeta-blockers, we also didn't find any association with the development of AVB,\nwhich has already been described by other authors[2,3,16].\nThe need for the use of an IAB in the PO of CABG occurred in 141 patients (4%) of the\ntotal sample of 3532 patients, of which 20.6% developed AVB, leading to the need for\nTP (OR=1.92; CI 95% 1.21-3.05; P=0.006). The need for the use of\nthe IAB has been associated with a greater probability of developing blocking and\nhas been indicated as a predictor of its occurrence and of the need for a PPM\nimplant[6,17,19]. Probably because its use is an expression of a\nmore significant ischemic cardiopathy, i.e., of patients with more severe\ncompromising. This finding is important because the patients did not have AVB in the\npreoperative period, presumably reflecting a greater perioperative myocardial\ninjury[6].\nPerioperative AMI was a risk factor for the emergence of AVB (OR=1.70; CI 95%\n1.26-2.29; P<0.0001), and this was corroborated by the study of\nCaspi et al.[7] who\nidentified the occurrence of low cardiac output (34% vs. 3%) and perioperative AMI\n(12% x 2%) as risk factors for AVB in the PO of CABG. Perioperative AMI also\nincreases the need for a PPM implant[10], reflecting acute ischemic damage of the conduction\ntissue.\nThe need for TP showed a significant association with mortality (OR=2.09; CI 95%\n1.46-2.99; P<0.0001), which was 17.7% for patients with AVB and\n7.2% for those who didn't develop it. Zeldis et al.[3] had already reported a\nmortality of 19.2% in the group of patients who developed block of the left\nconduction system (left bundle branch block or left anterior hemiblock or both),\ncompared with a 7% mortality rate in the group of patients without such block.\nSpecifically with respect to AVB, Caspi et al.[7] observed a significantly higher mortality\nin the group of patients who developed AVB (7% vs. 0.6%). On the other hand,\npatients who develop right bundle branch block or fascicular block have a more\nfavorable prognosis, because these are more transient disorders and because they do\nnot increase mortality[6,21].\nThe patients who developed AVB had a significantly longer hospital stay (mean\nhospitalization time of 12.75 days compared to 10.53 days for those who did not need\nTP for AVB (OR=1.01 CI 95% 1.00-1.02; P=0.01). Gordon et\nal.[19] have\nshown that the need for a PPM implant significantly increased hospital stay\n(23.3±18.7 days vs. 9.6±9.0 days for patients without need of implant,\nP=0.0001) and ICU stay (5.6±10.5 days vs. 2.2±3.3\ndays, P=0.0258). Other studies[11,18]\nalso corroborate this finding of a longer hospital stay in the presence of AVB and\nthe need of TP.\nIn our study, the need for a PPM implant occurred in 08 of the 3532 patients studied\n(0.23%), which is lower than the rate found in the literature, which points to the\nneed for PPM implants in 0.49% of AVB cases[8]. Gordon et al.[19] implanted PPMs in 50 of\ntheir 6859 patients submitted to CABG (0.73%). When other types of post-CABG\nconduction blocks are considered, the incidence of implants rises and ranges from\n0.4 to 1.1%[10]. The\ncalculated risk for need of a PPM implant in the PO of non-complicated CABG is\n0.9%[19].\nNascimento et al.[22]\ncouldn't identify any prognosis criterion for the reversibility of AVB in the PO of\nheart surgery. The ideal moment for the implantation of a PPM in the PO of CABG\nhasn't yet been properly established. According to the Brazilian Guidelines for\nImplantable Electronic Heart Devices[23], patients with asymptomatic AVB with wide QRS after\ncardiac surgery that persists after 15 days, are indicated for a PPM implantation\n(Class I, level of evidence C). In the cases of asymptomatic AVB persisting after 15\ndays, resulting from cardiac surgery, with narrow QRS or nodal escape rhythm and\ngood chronotropic response, and in those cases without the prospect of reversal\n(< 15 days) PPM implantation is also indicated (Class IIa, level C).\nAccording to the criteria of the American College of Cardiology and\nthe American Heart Association, a PPM implant is indicated in\n3rd and advanced 2nd degree AVB in the postoperative\nperiod of heart surgery, in addition to cases without expectation of resolution. The\ndecision regarding the time of the implant should be taken by the\nphysician[24].\nThe European Society of Cardiology recommends a waiting period of 5 to 7 days for the\nresolution of transient bradyarrhythmias after cardiac surgery, before the decision\nfor the implant is made[25].\nAccording to Pires et al.[13] and Merin et al.[18], the decision to perform the implant\nshould be taken between the 4th and 5th day of the PO, because\nif the AVB or dysfunction of the sinus node are still present up to this moment,\nthen they tend to be permanent. This would facilitate the early mobilization of\npatients and shorten their hospitalization time.\nOf the 288 patients in our study who had AVB, 08 received a PPM implant after an\naverage of 12.25 days into the PO, which is in line with the Brazilian (Class IIa,\nlevel of evidence C), American and European (Class I, level C) guidelines. Emlein et\nal.[8]\ndescribed a series of 8 patients who underwent a PPM implant after developing AVB\nwith an average of 10.5±6.5 days into the PO.\n Limitations of the Study The limitations of this study are those inherent to a retrospective database\nanalysis, but they reflect the significant years of experience of an academic\ninstitution. Within these limitations we can cite the relative difficulty of\naccessing the full data, which causes a potential risk of not measuring some\nrandom variables. The fact that the results come from the sample of a single\ncenter can also represent some degree of bias in the treatment. Another\nlimitation of this study is the absence of more precise information regarding\nthe height of the atrioventricular conduction disorder and the existence or not\nof any escape rhythm.\nRegarding the PPM implants performed in our study, they followed the\nrecommendations of Brazilian, American and European guidelines almost strictly.\nIn this small group of patients a more thorough analysis was compromised, but\nthis could be the target of a more detailed study to be developed in the\nfuture.\nThe limitations of this study are those inherent to a retrospective database\nanalysis, but they reflect the significant years of experience of an academic\ninstitution. Within these limitations we can cite the relative difficulty of\naccessing the full data, which causes a potential risk of not measuring some\nrandom variables. The fact that the results come from the sample of a single\ncenter can also represent some degree of bias in the treatment. Another\nlimitation of this study is the absence of more precise information regarding\nthe height of the atrioventricular conduction disorder and the existence or not\nof any escape rhythm.\nRegarding the PPM implants performed in our study, they followed the\nrecommendations of Brazilian, American and European guidelines almost strictly.\nIn this small group of patients a more thorough analysis was compromised, but\nthis could be the target of a more detailed study to be developed in the\nfuture.","The limitations of this study are those inherent to a retrospective database\nanalysis, but they reflect the significant years of experience of an academic\ninstitution. Within these limitations we can cite the relative difficulty of\naccessing the full data, which causes a potential risk of not measuring some\nrandom variables. The fact that the results come from the sample of a single\ncenter can also represent some degree of bias in the treatment. Another\nlimitation of this study is the absence of more precise information regarding\nthe height of the atrioventricular conduction disorder and the existence or not\nof any escape rhythm.\nRegarding the PPM implants performed in our study, they followed the\nrecommendations of Brazilian, American and European guidelines almost strictly.\nIn this small group of patients a more thorough analysis was compromised, but\nthis could be the target of a more detailed study to be developed in the\nfuture.","This work sheds light on the risk factors associated with the development of AVB in\nthe PO of CABG and the consequent need for TP and a definitive pacemaker. Based on\nthis we could establish that female patients, 60 years of age or more, with the\ndiagnosis of AF and CKD, in stages III and IV of the functional class, who had\nperioperative AMI and required the use of an IAB, have a higher risk of developing\nAVB in the PO of CABG. AVB determines a more prolonged hospitalization and, what is\nmore important, doubles the risk of mortality."],"string":"[\n \"Disturbances of the cardiac conduction system are relatively frequent in the\\npostoperative period (PO) of coronary artery bypass grafting (CABG), with an\\nincidence ranging from 18 to 55% of cases[1-6].\\nAtrioventricular block (AVB) is one of these conduction disturbances and its\\nincidence ranges from 0.5 to 16%[3,5-9]. Most patients have transitory and\\nreversible conduction disorders that require temporary pacing (TP). 0.4 to 1.1% of\\npatients, however, when faced with the irreversibility of the condition, will have\\nto undergo a permanent pacemaker (PPM) implant during their hospital\\nstay[10].\\nThis study, which is unprecedented in the national literature, tries to identify the\\nrelationship between pre-, intra and postoperative (perioperative) factors\\nassociated with the emergence of AVB, the need for TP and, if the case, the\\nimplantation of a PPM in the PO of CABG.\",\n \" Population and sample Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\\nUniversity of Rio Grande do Sul (PUCRS).\\nBetween January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\\nUniversity of Rio Grande do Sul (PUCRS).\\n Study Design Historical cohort observation study. Data were collected prospectively and\\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\\nSão Lucas Hospital of PUCRS.\\nHistorical cohort observation study. Data were collected prospectively and\\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\\nSão Lucas Hospital of PUCRS.\\n Inclusion Criteria Patients with age equal to or greater than 18 years who were submitted to\\nisolated CABG.\\nPatients with age equal to or greater than 18 years who were submitted to\\nisolated CABG.\\n Exclusion Criteria Patients who had been undergo valvular surgery, for left ventricular\\naneurysmectomy or correction of the interventricular communication associated\\nwith CABG.\\nPatients who had been undergo valvular surgery, for left ventricular\\naneurysmectomy or correction of the interventricular communication associated\\nwith CABG.\\n Study Variables Age - the mean age was calculated and also divided into groups for analysis: less\\nthan 60 years and greater than or equal to 60 years, according to the reference\\nin the literature[11,12]; gender (male and\\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\\nor radiocardiography, with the values being subdivided for analysis into\\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\\nserum creatinine level > 1.5 mg/dl, according to the reference in the\\nliterature[11,12]; Diabetes Mellitus\\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\\nfunctional (NYHA) class; presence of calcification of the aorta; time of\\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\\nhospital stay and hospital death.\\nAge - the mean age was calculated and also divided into groups for analysis: less\\nthan 60 years and greater than or equal to 60 years, according to the reference\\nin the literature[11,12]; gender (male and\\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\\nor radiocardiography, with the values being subdivided for analysis into\\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\\nserum creatinine level > 1.5 mg/dl, according to the reference in the\\nliterature[11,12]; Diabetes Mellitus\\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\\nfunctional (NYHA) class; presence of calcification of the aorta; time of\\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\\nhospital stay and hospital death.\\n Outcome Development of AVB in the PO of CABG and the need for TP and implantation of a\\nPPM.\\nDevelopment of AVB in the PO of CABG and the need for TP and implantation of a\\nPPM.\\n Procedures The CABGs were performed under general anesthesia. In all cases, a hyperkalemic\\ncardiac arrest was induced using a cold cardioplegic blood solution in the\\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\\ntransferred to the ICU of the POHS with mechanical ventilation.\\nThe CABGs were performed under general anesthesia. In all cases, a hyperkalemic\\ncardiac arrest was induced using a cold cardioplegic blood solution in the\\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\\ntransferred to the ICU of the POHS with mechanical ventilation.\\n Statistical Analysis The data was plotted in a digital Microsoft Access® spreadsheet\\nand analyzed using version 17.0 of the statistical software SPSS. The\\ndescriptive analysis was performed through frequency and mean ± standard\\ndeviation analysis, according to the case. For the univariate analysis the\\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\\nordinal variables and the Student's T-test for quantitative data. The\\nmultivariate analysis was performed using logistic regression (backward\\nconditional method). The difference was considered as statistically\\nsignificant for the value of P<0.05.\\nThe data was plotted in a digital Microsoft Access® spreadsheet\\nand analyzed using version 17.0 of the statistical software SPSS. The\\ndescriptive analysis was performed through frequency and mean ± standard\\ndeviation analysis, according to the case. For the univariate analysis the\\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\\nordinal variables and the Student's T-test for quantitative data. The\\nmultivariate analysis was performed using logistic regression (backward\\nconditional method). The difference was considered as statistically\\nsignificant for the value of P<0.05.\\n Ethical Considerations The design of this study was submitted to the Research Ethics Committee of the\\nFaculty of Medicine of the PUCRS, under registration number 060/3478.\\nThe design of this study was submitted to the Research Ethics Committee of the\\nFaculty of Medicine of the PUCRS, under registration number 060/3478.\",\n \"Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\\nUniversity of Rio Grande do Sul (PUCRS).\",\n \"Historical cohort observation study. Data were collected prospectively and\\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\\nSão Lucas Hospital of PUCRS.\",\n \"Patients with age equal to or greater than 18 years who were submitted to\\nisolated CABG.\",\n \"Patients who had been undergo valvular surgery, for left ventricular\\naneurysmectomy or correction of the interventricular communication associated\\nwith CABG.\",\n \"Age - the mean age was calculated and also divided into groups for analysis: less\\nthan 60 years and greater than or equal to 60 years, according to the reference\\nin the literature[11,12]; gender (male and\\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\\nor radiocardiography, with the values being subdivided for analysis into\\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\\nserum creatinine level > 1.5 mg/dl, according to the reference in the\\nliterature[11,12]; Diabetes Mellitus\\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\\nfunctional (NYHA) class; presence of calcification of the aorta; time of\\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\\nhospital stay and hospital death.\",\n \"Development of AVB in the PO of CABG and the need for TP and implantation of a\\nPPM.\",\n \"The CABGs were performed under general anesthesia. In all cases, a hyperkalemic\\ncardiac arrest was induced using a cold cardioplegic blood solution in the\\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\\ntransferred to the ICU of the POHS with mechanical ventilation.\",\n \"The data was plotted in a digital Microsoft Access® spreadsheet\\nand analyzed using version 17.0 of the statistical software SPSS. The\\ndescriptive analysis was performed through frequency and mean ± standard\\ndeviation analysis, according to the case. For the univariate analysis the\\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\\nordinal variables and the Student's T-test for quantitative data. The\\nmultivariate analysis was performed using logistic regression (backward\\nconditional method). The difference was considered as statistically\\nsignificant for the value of P<0.05.\",\n \"The design of this study was submitted to the Research Ethics Committee of the\\nFaculty of Medicine of the PUCRS, under registration number 060/3478.\",\n \"Of the 3532 patients undergoing CABG in the period under analysis, 288 (8.15%)\\npresented the clinical and electrocardiographic signs of AVB during the\\npostoperative period, with an indication for temporary pacing (TP).\\nTable 1 shows the demographic profile of the\\npatients studied. The univariate analysis of the preoperative data revealed a\\ngreater need for TP in the PO of CABG in patients above 60 years of age (OR=2.48; CI\\n95% 1.90-3.24; P<0.0001), of the female gender (OR=1.03; CI 95%\\n1.00-1.05; P=0.012), with CKD (OR=2.03; CI 95% 1.55-2.65;\\nP<0.0001), the presence of AF (OR=2.38; CI 95% 1.49-3.72;\\nP<0.0001) and in patients with NYHA functional class III or\\nIV (OR=1.60; CI 95% 1.21-2.12; P=0.001).\\nPreoperative characteristics of the groups and univariate analysis.\\nAF=atrial fibrillation; AMI=acute myocardial infarction;\\nBB=beta-blockers; CI=confidence interval; CKD=chronic kidney disease;\\nDM=diabetes mellitus; EF=left ventricular ejection fraction;\\nFC=functional class; NYHA=New York Heart Association; NTP=no use of\\ntemporary pacing; OR=odds ratio; P=statistical significance;\\nTP=temporary pacing\\nTable 2 presents the trans and postoperative\\ndata, together with their univariate analysis. Here we can observe the association\\nof AVB with the need of TP in patients who presented calcification of the aorta,\\nperioperative AMI and the need for the use of an IAB. Of statistically significant\\nrelevance, the univariate analysis also revealed the association of TP caused by AVB\\nwith increased mortality (17.9% vs. 7.3%) and with a longer hospital stay (mean\\nhospitalization time of 12.75 days compared to 10.53 days for those who did not\\nrequire TP).\\nTrans and postoperative data of groups and univariate analysis.\\nCalcification Ao=calcification of the aorta; CPBT=cardiopulmonary bypass\\ntime; IAB=intra-aortic balloon; Peri AMI=perioperative acute myocardial\\ninfarction; Tclamping=aortic clamping time; Others: see Table 1\\nThese data were submitted to multivariate analysis (Table 3), which revealed a higher risk of AVB in the PO of CABG in\\npatients with: age > 60 years, female sex, CKD, AF, NYHA functional class III or\\nIV, perioperative AMI and with the use of an IAB. Patients with EF≤40 %, DM,\\nthe use of beta-blockers, statins and other antiarrhythmic drugs, prior AMI and CPB\\nand aortic clamping times didn't prove to be independent risk variables for the\\ndevelopment of AVB in the PO of CABG.\\nMultivariate analysis of the risk factors and outcomes of AVB in the PO of\\nCABG.\\nAMI=acute myocardial infarction; AVB=atrioventricular block; CABG=coronary\\nartery bypass grafting; CKD=chronic kidney disease; FC=functional class;\\nIAB=intra-aortic balloon; PO=postoperative\\nIn the multivariate analysis, the presence of AVB resulted in a longer hospital stay\\n(12.75 days vs. 10.53 days for those who didn't develop AVB) (OR=1.01; CI 95%\\n1.00-1.02; P=0.01) and in a significant increase in the risk of\\nmortality (17.9% vs. 7.3% for patients without AVB) (OR=2.09; CI 95% 1.46-2.99;\\nP<0.0001).\\nIn the subgroup of 288 patients who had AVB and who had undergone TP, 08 (2.78 %)\\nrequired a PPM implant, corresponding to 0.23% of the total cohort analyzed. The\\naverage time elapsed since the surgery until the PPM implant was 12.25 days.\",\n \"CABG is a proven therapeutic strategy for the treatment of Coronary Artery Disease\\n(CAD). Although it is well tolerated by most patients, perioperative complications\\ncan occur, among which we find disturbances in the cardiac conduction system in\\nvarying degrees, including AVB.\\nPrevious studies have reported an incidence of conduction disturbances (CD) after\\nCABG that varies from 18 to 55% of cases[1-6], with the\\nright bundle branch block being the most common[4]. Atrioventricular block (AVB) is one of\\nthese conduction disturbances and its incidence ranges from 0.5 to\\n16%[3,5-9]. Our incidence of AVB is in line with this data, since\\n8.15% of our patients developed AVB in the PO of CABG.\\nThe etiology of AVB seems to be multifactorial. The patient's age (>60 years),\\nhypertension, number of revascularized vessels, aortic clamping time, total time of\\nCPB, use of digitalis and beta-blockers, type of cardioplegia and previously\\nexisting left bundle branch block may be related to its appearance[1-4,8,9,13,14].\\nMyocardial ischemia seems to be the factor that is most implicated in the emergence\\nof AVB, since there is a correlation with coronary artery disease (CAD) and\\npreoperative AMI[4].\\nStudies[3,10] have demonstrated that\\nperioperative AMI also increases the incidence of AVB in the PO of CABG. Caspi et\\nal.[7]\\nreported a higher occurrence of AVB in patients with AMI in the PO of CABG (12% vs.\\n2 %, P<0.05).\\nHowever, AMI before CABG was not a significant factor for the appearance of AVB in\\nour study, which is consistent with the world literature[3,7,8,15,16].\\nThis shows there is no difference in the incidence of AVB between patients who had\\npreoperative AMI and those who didn't, regardless of its electrocardiographic\\nlocation.\\nCaspi et al.[7] have\\nshown that the combination of left main disease and proximal obstruction of a\\ndominant right coronary artery was more frequent in patients who exhibited AVB (32%)\\nthan in those who without it (12%, P<0.05). The explanation for\\nthis effect is the fact that the cardioplegic solution is not properly distributed\\nto the coronary beds because of their high degree of obstruction, which compromises\\nmyocardial protection and, in some cases, because of the impossibility of bypassing\\nthe right coronary artery.\\nThe impairment of myocardial irrigation gets worse with age, just as the frequency of\\ndegenerative diseases of the conduction system, increasing the probability of\\nAVB[9,11,15,17-19]. In this scenario, our patients above 60\\nyears of age presented a significant risk (OR=2.34; CI 95% 1.75-3.12;\\nP<0.0001) for the development of AVB in the PO of CABG,\\ncorroborating the findings of other studies[7,8,13,14].\\nThe electrical cardiac conduction tissue differs from cardiac myocytes by being less\\ntolerant to the effects of ischemia, hyperkalemia and hypothermia (whether these are\\nsystemic or, mostly, induced by a cardioplegic solution that is cold and rich in\\npotassium). This may cause a transient block of the conduction\\nsystem[11].\\nThe advent of cold cardioplegia as a method of myocardial protection has increased\\nthe incidence of CD from 20 to 58%[13]. The more significant incidence of conduction\\ndisturbances occurred in patients who received cold cardioplegia, as opposed to warm\\n(19.6% vs. 1.7 %, respectively)[13], a finding that has also been described by Sirlak\\net al.[20].\\nSpecifically with respect to AVB, the incidence was of 3.8% in the hypothermia group\\nand zero in the normothermal group[13]. All patients in our study underwent surgery with\\nthe myocardial protection performed by infusion of a cold cardioplegic blood\\nsolution at the root of the aorta every 20 minutes, which contributed to the genesis\\nof AVB cases.\\nAs such, the perfusion injury determined by the myocardial ischemia and the\\nhypothermic injury caused by the cardioplegic solution are the mechanisms that are\\nmost involved in the genesis of AVB, acting on the proximal portions of the bundle\\nof His, which are more sensitive to this type of aggression than the more distal\\nconduction tissue, determining the emergence of bundle branch blocks and increasing\\nthe risk of AVB[4].\\nIn this scenario, the extent of the CAD, the duration of CPB and the aortic clamping\\ntime could compromise myocardial protection during surgery, increasing the risk of\\nan ischemic injury and of metabolic damage to the conduction\\ntissue[11].\\nHowever, our CPB time of ≥ 90 min and aortic clamping time of ≥ 40 min\\nshowed no influence on the development of AVB, which is supported by the\\nliterature[5-7,16,19]. Baerman\\net al.[1], however,\\ndemonstrated that patients with lower CPB (101±32min x 121±34min;\\nP<0,01) and aortic clamping (44±19min x\\n53±17min; P<0.05) times didn't show evidence of AVB in\\nthe PO of CABG.\\nOur study has shown that the female gender is a risk factor for the occurrence of AVB\\n(OR=1.37, CI 95% 1.06-1.77; P=0.015), which contrasts with the\\nresults of Gordon et al.[19] who observed a higher need for PPM implants in men\\n(P=0.041). Other studies[3,8,15,20], however, didn't point to any of the genders as\\nrisk factor. Cadore et al.[12] had already pointed to the female gender as a risk\\npredictor for mortality in CABG, which can be an expression of the greater severity\\nof the ischemic impairment in this gender and explain their greater tendency for\\ndeveloping the block, as seen in our study.\\nThe presence of CKD was also verified to be a risk factor for the development of AVB\\n(OR = 2.05; CI 95% 1.49-2.81; P<0.0001). A previous\\nstudy[19]\\nindicated the presence of CKD as more significant among those patients who required\\nPPM implantation in the PO of CABG. Like the female gender variable, CKD was also\\nfound to be a predictor of mortality in patients underwent CABG according to the\\nscore by Cadore et al.[12], expressing its potential for increasing the risk of\\ncomplications in the PO of CABG.\\nAnother risk predictor for the occurrence of AVB was the more advanced functional\\nclass of the NYHA (III and IV) (OR = 1.43; CI 95% 1.03-1.98;\\nP=0.031). Studies[18,19] have\\ncorroborated this finding, indicating that patients who underwent heart surgery and\\nneeding a PPM implant were in the more severe functional class of the NYHA (III and\\nIV) when compared to patients who did not require such an implant (57% vs. 35%,\\nrespectively, P<0.0001)[18]. Bateman et al.[6] showed that of those\\npatients who passed away within the first 30 days of the PO of CABG and who had\\ndeveloped some degree of blockage, 90% were into class IV of the NYHA in the\\npreoperative period.\\nThe patients in this study who had EF≤40% did not present a significant risk\\nfor the appearance of AVB in the PO of CABG (OR=1.11; CI 95% 0.85-1.45;\\nP=0.44), a finding supported by Gordon et al.[19] who didn't observe any\\nsignificant impact of EF on the need for PPM implantation in the PO of isolated\\nCABG. Caspi et al.[7], however, found a greater susceptibility to the\\ndevelopment of atrioventricular block in patients submitted to CABG with a lower\\nEF.\\nAlthough Merin et al.[18] mention that the use of antiarrhythmic agents is more\\nfrequent in the group of patients that develops blockage after heart surgery (CABG,\\nvalve or combined), our data does not reflect this influence. Regarding the use of\\nbeta-blockers, we also didn't find any association with the development of AVB,\\nwhich has already been described by other authors[2,3,16].\\nThe need for the use of an IAB in the PO of CABG occurred in 141 patients (4%) of the\\ntotal sample of 3532 patients, of which 20.6% developed AVB, leading to the need for\\nTP (OR=1.92; CI 95% 1.21-3.05; P=0.006). The need for the use of\\nthe IAB has been associated with a greater probability of developing blocking and\\nhas been indicated as a predictor of its occurrence and of the need for a PPM\\nimplant[6,17,19]. Probably because its use is an expression of a\\nmore significant ischemic cardiopathy, i.e., of patients with more severe\\ncompromising. This finding is important because the patients did not have AVB in the\\npreoperative period, presumably reflecting a greater perioperative myocardial\\ninjury[6].\\nPerioperative AMI was a risk factor for the emergence of AVB (OR=1.70; CI 95%\\n1.26-2.29; P<0.0001), and this was corroborated by the study of\\nCaspi et al.[7] who\\nidentified the occurrence of low cardiac output (34% vs. 3%) and perioperative AMI\\n(12% x 2%) as risk factors for AVB in the PO of CABG. Perioperative AMI also\\nincreases the need for a PPM implant[10], reflecting acute ischemic damage of the conduction\\ntissue.\\nThe need for TP showed a significant association with mortality (OR=2.09; CI 95%\\n1.46-2.99; P<0.0001), which was 17.7% for patients with AVB and\\n7.2% for those who didn't develop it. Zeldis et al.[3] had already reported a\\nmortality of 19.2% in the group of patients who developed block of the left\\nconduction system (left bundle branch block or left anterior hemiblock or both),\\ncompared with a 7% mortality rate in the group of patients without such block.\\nSpecifically with respect to AVB, Caspi et al.[7] observed a significantly higher mortality\\nin the group of patients who developed AVB (7% vs. 0.6%). On the other hand,\\npatients who develop right bundle branch block or fascicular block have a more\\nfavorable prognosis, because these are more transient disorders and because they do\\nnot increase mortality[6,21].\\nThe patients who developed AVB had a significantly longer hospital stay (mean\\nhospitalization time of 12.75 days compared to 10.53 days for those who did not need\\nTP for AVB (OR=1.01 CI 95% 1.00-1.02; P=0.01). Gordon et\\nal.[19] have\\nshown that the need for a PPM implant significantly increased hospital stay\\n(23.3±18.7 days vs. 9.6±9.0 days for patients without need of implant,\\nP=0.0001) and ICU stay (5.6±10.5 days vs. 2.2±3.3\\ndays, P=0.0258). Other studies[11,18]\\nalso corroborate this finding of a longer hospital stay in the presence of AVB and\\nthe need of TP.\\nIn our study, the need for a PPM implant occurred in 08 of the 3532 patients studied\\n(0.23%), which is lower than the rate found in the literature, which points to the\\nneed for PPM implants in 0.49% of AVB cases[8]. Gordon et al.[19] implanted PPMs in 50 of\\ntheir 6859 patients submitted to CABG (0.73%). When other types of post-CABG\\nconduction blocks are considered, the incidence of implants rises and ranges from\\n0.4 to 1.1%[10]. The\\ncalculated risk for need of a PPM implant in the PO of non-complicated CABG is\\n0.9%[19].\\nNascimento et al.[22]\\ncouldn't identify any prognosis criterion for the reversibility of AVB in the PO of\\nheart surgery. The ideal moment for the implantation of a PPM in the PO of CABG\\nhasn't yet been properly established. According to the Brazilian Guidelines for\\nImplantable Electronic Heart Devices[23], patients with asymptomatic AVB with wide QRS after\\ncardiac surgery that persists after 15 days, are indicated for a PPM implantation\\n(Class I, level of evidence C). In the cases of asymptomatic AVB persisting after 15\\ndays, resulting from cardiac surgery, with narrow QRS or nodal escape rhythm and\\ngood chronotropic response, and in those cases without the prospect of reversal\\n(< 15 days) PPM implantation is also indicated (Class IIa, level C).\\nAccording to the criteria of the American College of Cardiology and\\nthe American Heart Association, a PPM implant is indicated in\\n3rd and advanced 2nd degree AVB in the postoperative\\nperiod of heart surgery, in addition to cases without expectation of resolution. The\\ndecision regarding the time of the implant should be taken by the\\nphysician[24].\\nThe European Society of Cardiology recommends a waiting period of 5 to 7 days for the\\nresolution of transient bradyarrhythmias after cardiac surgery, before the decision\\nfor the implant is made[25].\\nAccording to Pires et al.[13] and Merin et al.[18], the decision to perform the implant\\nshould be taken between the 4th and 5th day of the PO, because\\nif the AVB or dysfunction of the sinus node are still present up to this moment,\\nthen they tend to be permanent. This would facilitate the early mobilization of\\npatients and shorten their hospitalization time.\\nOf the 288 patients in our study who had AVB, 08 received a PPM implant after an\\naverage of 12.25 days into the PO, which is in line with the Brazilian (Class IIa,\\nlevel of evidence C), American and European (Class I, level C) guidelines. Emlein et\\nal.[8]\\ndescribed a series of 8 patients who underwent a PPM implant after developing AVB\\nwith an average of 10.5±6.5 days into the PO.\\n Limitations of the Study The limitations of this study are those inherent to a retrospective database\\nanalysis, but they reflect the significant years of experience of an academic\\ninstitution. Within these limitations we can cite the relative difficulty of\\naccessing the full data, which causes a potential risk of not measuring some\\nrandom variables. The fact that the results come from the sample of a single\\ncenter can also represent some degree of bias in the treatment. Another\\nlimitation of this study is the absence of more precise information regarding\\nthe height of the atrioventricular conduction disorder and the existence or not\\nof any escape rhythm.\\nRegarding the PPM implants performed in our study, they followed the\\nrecommendations of Brazilian, American and European guidelines almost strictly.\\nIn this small group of patients a more thorough analysis was compromised, but\\nthis could be the target of a more detailed study to be developed in the\\nfuture.\\nThe limitations of this study are those inherent to a retrospective database\\nanalysis, but they reflect the significant years of experience of an academic\\ninstitution. Within these limitations we can cite the relative difficulty of\\naccessing the full data, which causes a potential risk of not measuring some\\nrandom variables. The fact that the results come from the sample of a single\\ncenter can also represent some degree of bias in the treatment. Another\\nlimitation of this study is the absence of more precise information regarding\\nthe height of the atrioventricular conduction disorder and the existence or not\\nof any escape rhythm.\\nRegarding the PPM implants performed in our study, they followed the\\nrecommendations of Brazilian, American and European guidelines almost strictly.\\nIn this small group of patients a more thorough analysis was compromised, but\\nthis could be the target of a more detailed study to be developed in the\\nfuture.\",\n \"The limitations of this study are those inherent to a retrospective database\\nanalysis, but they reflect the significant years of experience of an academic\\ninstitution. Within these limitations we can cite the relative difficulty of\\naccessing the full data, which causes a potential risk of not measuring some\\nrandom variables. The fact that the results come from the sample of a single\\ncenter can also represent some degree of bias in the treatment. Another\\nlimitation of this study is the absence of more precise information regarding\\nthe height of the atrioventricular conduction disorder and the existence or not\\nof any escape rhythm.\\nRegarding the PPM implants performed in our study, they followed the\\nrecommendations of Brazilian, American and European guidelines almost strictly.\\nIn this small group of patients a more thorough analysis was compromised, but\\nthis could be the target of a more detailed study to be developed in the\\nfuture.\",\n \"This work sheds light on the risk factors associated with the development of AVB in\\nthe PO of CABG and the consequent need for TP and a definitive pacemaker. Based on\\nthis we could establish that female patients, 60 years of age or more, with the\\ndiagnosis of AF and CKD, in stages III and IV of the functional class, who had\\nperioperative AMI and required the use of an IAB, have a higher risk of developing\\nAVB in the PO of CABG. AVB determines a more prolonged hospitalization and, what is\\nmore important, doubles the risk of mortality.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,null,null,null,null,"results","discussion",null,"conclusions"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n null,\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["Atrioventricular block","Artificial Pacemaker","Coronary Artery Bypass","Postoperative Complications"],"string":"[\n \"Atrioventricular block\",\n \"Artificial Pacemaker\",\n \"Coronary Artery Bypass\",\n \"Postoperative Complications\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nDisturbances of the cardiac conduction system are relatively frequent in the\npostoperative period (PO) of coronary artery bypass grafting (CABG), with an\nincidence ranging from 18 to 55% of cases[1-6].\nAtrioventricular block (AVB) is one of these conduction disturbances and its\nincidence ranges from 0.5 to 16%[3,5-9]. Most patients have transitory and\nreversible conduction disorders that require temporary pacing (TP). 0.4 to 1.1% of\npatients, however, when faced with the irreversibility of the condition, will have\nto undergo a permanent pacemaker (PPM) implant during their hospital\nstay[10].\nThis study, which is unprecedented in the national literature, tries to identify the\nrelationship between pre-, intra and postoperative (perioperative) factors\nassociated with the emergence of AVB, the need for TP and, if the case, the\nimplantation of a PPM in the PO of CABG.\n\nMETHODS:\n Population and sample Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).\nBetween January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).\n Study Design Historical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.\nHistorical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.\n Inclusion Criteria Patients with age equal to or greater than 18 years who were submitted to\nisolated CABG.\nPatients with age equal to or greater than 18 years who were submitted to\nisolated CABG.\n Exclusion Criteria Patients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.\nPatients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.\n Study Variables Age - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.\nAge - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.\n Outcome Development of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.\nDevelopment of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.\n Procedures The CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.\nThe CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.\n Statistical Analysis The data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.\nThe data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.\n Ethical Considerations The design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.\nThe design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.\n\nPopulation and sample:\nBetween January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).\n\nStudy Design:\nHistorical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.\n\nInclusion Criteria:\nPatients with age equal to or greater than 18 years who were submitted to\nisolated CABG.\n\nExclusion Criteria:\nPatients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.\n\nStudy Variables:\nAge - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.\n\nOutcome:\nDevelopment of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.\n\nProcedures:\nThe CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.\n\nStatistical Analysis:\nThe data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.\n\nEthical Considerations:\nThe design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.\n\nRESULTS:\nOf the 3532 patients undergoing CABG in the period under analysis, 288 (8.15%)\npresented the clinical and electrocardiographic signs of AVB during the\npostoperative period, with an indication for temporary pacing (TP).\nTable 1 shows the demographic profile of the\npatients studied. The univariate analysis of the preoperative data revealed a\ngreater need for TP in the PO of CABG in patients above 60 years of age (OR=2.48; CI\n95% 1.90-3.24; P<0.0001), of the female gender (OR=1.03; CI 95%\n1.00-1.05; P=0.012), with CKD (OR=2.03; CI 95% 1.55-2.65;\nP<0.0001), the presence of AF (OR=2.38; CI 95% 1.49-3.72;\nP<0.0001) and in patients with NYHA functional class III or\nIV (OR=1.60; CI 95% 1.21-2.12; P=0.001).\nPreoperative characteristics of the groups and univariate analysis.\nAF=atrial fibrillation; AMI=acute myocardial infarction;\nBB=beta-blockers; CI=confidence interval; CKD=chronic kidney disease;\nDM=diabetes mellitus; EF=left ventricular ejection fraction;\nFC=functional class; NYHA=New York Heart Association; NTP=no use of\ntemporary pacing; OR=odds ratio; P=statistical significance;\nTP=temporary pacing\nTable 2 presents the trans and postoperative\ndata, together with their univariate analysis. Here we can observe the association\nof AVB with the need of TP in patients who presented calcification of the aorta,\nperioperative AMI and the need for the use of an IAB. Of statistically significant\nrelevance, the univariate analysis also revealed the association of TP caused by AVB\nwith increased mortality (17.9% vs. 7.3%) and with a longer hospital stay (mean\nhospitalization time of 12.75 days compared to 10.53 days for those who did not\nrequire TP).\nTrans and postoperative data of groups and univariate analysis.\nCalcification Ao=calcification of the aorta; CPBT=cardiopulmonary bypass\ntime; IAB=intra-aortic balloon; Peri AMI=perioperative acute myocardial\ninfarction; Tclamping=aortic clamping time; Others: see Table 1\nThese data were submitted to multivariate analysis (Table 3), which revealed a higher risk of AVB in the PO of CABG in\npatients with: age > 60 years, female sex, CKD, AF, NYHA functional class III or\nIV, perioperative AMI and with the use of an IAB. Patients with EF≤40 %, DM,\nthe use of beta-blockers, statins and other antiarrhythmic drugs, prior AMI and CPB\nand aortic clamping times didn't prove to be independent risk variables for the\ndevelopment of AVB in the PO of CABG.\nMultivariate analysis of the risk factors and outcomes of AVB in the PO of\nCABG.\nAMI=acute myocardial infarction; AVB=atrioventricular block; CABG=coronary\nartery bypass grafting; CKD=chronic kidney disease; FC=functional class;\nIAB=intra-aortic balloon; PO=postoperative\nIn the multivariate analysis, the presence of AVB resulted in a longer hospital stay\n(12.75 days vs. 10.53 days for those who didn't develop AVB) (OR=1.01; CI 95%\n1.00-1.02; P=0.01) and in a significant increase in the risk of\nmortality (17.9% vs. 7.3% for patients without AVB) (OR=2.09; CI 95% 1.46-2.99;\nP<0.0001).\nIn the subgroup of 288 patients who had AVB and who had undergone TP, 08 (2.78 %)\nrequired a PPM implant, corresponding to 0.23% of the total cohort analyzed. The\naverage time elapsed since the surgery until the PPM implant was 12.25 days.\n\nDISCUSSION:\nCABG is a proven therapeutic strategy for the treatment of Coronary Artery Disease\n(CAD). Although it is well tolerated by most patients, perioperative complications\ncan occur, among which we find disturbances in the cardiac conduction system in\nvarying degrees, including AVB.\nPrevious studies have reported an incidence of conduction disturbances (CD) after\nCABG that varies from 18 to 55% of cases[1-6], with the\nright bundle branch block being the most common[4]. Atrioventricular block (AVB) is one of\nthese conduction disturbances and its incidence ranges from 0.5 to\n16%[3,5-9]. Our incidence of AVB is in line with this data, since\n8.15% of our patients developed AVB in the PO of CABG.\nThe etiology of AVB seems to be multifactorial. The patient's age (>60 years),\nhypertension, number of revascularized vessels, aortic clamping time, total time of\nCPB, use of digitalis and beta-blockers, type of cardioplegia and previously\nexisting left bundle branch block may be related to its appearance[1-4,8,9,13,14].\nMyocardial ischemia seems to be the factor that is most implicated in the emergence\nof AVB, since there is a correlation with coronary artery disease (CAD) and\npreoperative AMI[4].\nStudies[3,10] have demonstrated that\nperioperative AMI also increases the incidence of AVB in the PO of CABG. Caspi et\nal.[7]\nreported a higher occurrence of AVB in patients with AMI in the PO of CABG (12% vs.\n2 %, P<0.05).\nHowever, AMI before CABG was not a significant factor for the appearance of AVB in\nour study, which is consistent with the world literature[3,7,8,15,16].\nThis shows there is no difference in the incidence of AVB between patients who had\npreoperative AMI and those who didn't, regardless of its electrocardiographic\nlocation.\nCaspi et al.[7] have\nshown that the combination of left main disease and proximal obstruction of a\ndominant right coronary artery was more frequent in patients who exhibited AVB (32%)\nthan in those who without it (12%, P<0.05). The explanation for\nthis effect is the fact that the cardioplegic solution is not properly distributed\nto the coronary beds because of their high degree of obstruction, which compromises\nmyocardial protection and, in some cases, because of the impossibility of bypassing\nthe right coronary artery.\nThe impairment of myocardial irrigation gets worse with age, just as the frequency of\ndegenerative diseases of the conduction system, increasing the probability of\nAVB[9,11,15,17-19]. In this scenario, our patients above 60\nyears of age presented a significant risk (OR=2.34; CI 95% 1.75-3.12;\nP<0.0001) for the development of AVB in the PO of CABG,\ncorroborating the findings of other studies[7,8,13,14].\nThe electrical cardiac conduction tissue differs from cardiac myocytes by being less\ntolerant to the effects of ischemia, hyperkalemia and hypothermia (whether these are\nsystemic or, mostly, induced by a cardioplegic solution that is cold and rich in\npotassium). This may cause a transient block of the conduction\nsystem[11].\nThe advent of cold cardioplegia as a method of myocardial protection has increased\nthe incidence of CD from 20 to 58%[13]. The more significant incidence of conduction\ndisturbances occurred in patients who received cold cardioplegia, as opposed to warm\n(19.6% vs. 1.7 %, respectively)[13], a finding that has also been described by Sirlak\net al.[20].\nSpecifically with respect to AVB, the incidence was of 3.8% in the hypothermia group\nand zero in the normothermal group[13]. All patients in our study underwent surgery with\nthe myocardial protection performed by infusion of a cold cardioplegic blood\nsolution at the root of the aorta every 20 minutes, which contributed to the genesis\nof AVB cases.\nAs such, the perfusion injury determined by the myocardial ischemia and the\nhypothermic injury caused by the cardioplegic solution are the mechanisms that are\nmost involved in the genesis of AVB, acting on the proximal portions of the bundle\nof His, which are more sensitive to this type of aggression than the more distal\nconduction tissue, determining the emergence of bundle branch blocks and increasing\nthe risk of AVB[4].\nIn this scenario, the extent of the CAD, the duration of CPB and the aortic clamping\ntime could compromise myocardial protection during surgery, increasing the risk of\nan ischemic injury and of metabolic damage to the conduction\ntissue[11].\nHowever, our CPB time of ≥ 90 min and aortic clamping time of ≥ 40 min\nshowed no influence on the development of AVB, which is supported by the\nliterature[5-7,16,19]. Baerman\net al.[1], however,\ndemonstrated that patients with lower CPB (101±32min x 121±34min;\nP<0,01) and aortic clamping (44±19min x\n53±17min; P<0.05) times didn't show evidence of AVB in\nthe PO of CABG.\nOur study has shown that the female gender is a risk factor for the occurrence of AVB\n(OR=1.37, CI 95% 1.06-1.77; P=0.015), which contrasts with the\nresults of Gordon et al.[19] who observed a higher need for PPM implants in men\n(P=0.041). Other studies[3,8,15,20], however, didn't point to any of the genders as\nrisk factor. Cadore et al.[12] had already pointed to the female gender as a risk\npredictor for mortality in CABG, which can be an expression of the greater severity\nof the ischemic impairment in this gender and explain their greater tendency for\ndeveloping the block, as seen in our study.\nThe presence of CKD was also verified to be a risk factor for the development of AVB\n(OR = 2.05; CI 95% 1.49-2.81; P<0.0001). A previous\nstudy[19]\nindicated the presence of CKD as more significant among those patients who required\nPPM implantation in the PO of CABG. Like the female gender variable, CKD was also\nfound to be a predictor of mortality in patients underwent CABG according to the\nscore by Cadore et al.[12], expressing its potential for increasing the risk of\ncomplications in the PO of CABG.\nAnother risk predictor for the occurrence of AVB was the more advanced functional\nclass of the NYHA (III and IV) (OR = 1.43; CI 95% 1.03-1.98;\nP=0.031). Studies[18,19] have\ncorroborated this finding, indicating that patients who underwent heart surgery and\nneeding a PPM implant were in the more severe functional class of the NYHA (III and\nIV) when compared to patients who did not require such an implant (57% vs. 35%,\nrespectively, P<0.0001)[18]. Bateman et al.[6] showed that of those\npatients who passed away within the first 30 days of the PO of CABG and who had\ndeveloped some degree of blockage, 90% were into class IV of the NYHA in the\npreoperative period.\nThe patients in this study who had EF≤40% did not present a significant risk\nfor the appearance of AVB in the PO of CABG (OR=1.11; CI 95% 0.85-1.45;\nP=0.44), a finding supported by Gordon et al.[19] who didn't observe any\nsignificant impact of EF on the need for PPM implantation in the PO of isolated\nCABG. Caspi et al.[7], however, found a greater susceptibility to the\ndevelopment of atrioventricular block in patients submitted to CABG with a lower\nEF.\nAlthough Merin et al.[18] mention that the use of antiarrhythmic agents is more\nfrequent in the group of patients that develops blockage after heart surgery (CABG,\nvalve or combined), our data does not reflect this influence. Regarding the use of\nbeta-blockers, we also didn't find any association with the development of AVB,\nwhich has already been described by other authors[2,3,16].\nThe need for the use of an IAB in the PO of CABG occurred in 141 patients (4%) of the\ntotal sample of 3532 patients, of which 20.6% developed AVB, leading to the need for\nTP (OR=1.92; CI 95% 1.21-3.05; P=0.006). The need for the use of\nthe IAB has been associated with a greater probability of developing blocking and\nhas been indicated as a predictor of its occurrence and of the need for a PPM\nimplant[6,17,19]. Probably because its use is an expression of a\nmore significant ischemic cardiopathy, i.e., of patients with more severe\ncompromising. This finding is important because the patients did not have AVB in the\npreoperative period, presumably reflecting a greater perioperative myocardial\ninjury[6].\nPerioperative AMI was a risk factor for the emergence of AVB (OR=1.70; CI 95%\n1.26-2.29; P<0.0001), and this was corroborated by the study of\nCaspi et al.[7] who\nidentified the occurrence of low cardiac output (34% vs. 3%) and perioperative AMI\n(12% x 2%) as risk factors for AVB in the PO of CABG. Perioperative AMI also\nincreases the need for a PPM implant[10], reflecting acute ischemic damage of the conduction\ntissue.\nThe need for TP showed a significant association with mortality (OR=2.09; CI 95%\n1.46-2.99; P<0.0001), which was 17.7% for patients with AVB and\n7.2% for those who didn't develop it. Zeldis et al.[3] had already reported a\nmortality of 19.2% in the group of patients who developed block of the left\nconduction system (left bundle branch block or left anterior hemiblock or both),\ncompared with a 7% mortality rate in the group of patients without such block.\nSpecifically with respect to AVB, Caspi et al.[7] observed a significantly higher mortality\nin the group of patients who developed AVB (7% vs. 0.6%). On the other hand,\npatients who develop right bundle branch block or fascicular block have a more\nfavorable prognosis, because these are more transient disorders and because they do\nnot increase mortality[6,21].\nThe patients who developed AVB had a significantly longer hospital stay (mean\nhospitalization time of 12.75 days compared to 10.53 days for those who did not need\nTP for AVB (OR=1.01 CI 95% 1.00-1.02; P=0.01). Gordon et\nal.[19] have\nshown that the need for a PPM implant significantly increased hospital stay\n(23.3±18.7 days vs. 9.6±9.0 days for patients without need of implant,\nP=0.0001) and ICU stay (5.6±10.5 days vs. 2.2±3.3\ndays, P=0.0258). Other studies[11,18]\nalso corroborate this finding of a longer hospital stay in the presence of AVB and\nthe need of TP.\nIn our study, the need for a PPM implant occurred in 08 of the 3532 patients studied\n(0.23%), which is lower than the rate found in the literature, which points to the\nneed for PPM implants in 0.49% of AVB cases[8]. Gordon et al.[19] implanted PPMs in 50 of\ntheir 6859 patients submitted to CABG (0.73%). When other types of post-CABG\nconduction blocks are considered, the incidence of implants rises and ranges from\n0.4 to 1.1%[10]. The\ncalculated risk for need of a PPM implant in the PO of non-complicated CABG is\n0.9%[19].\nNascimento et al.[22]\ncouldn't identify any prognosis criterion for the reversibility of AVB in the PO of\nheart surgery. The ideal moment for the implantation of a PPM in the PO of CABG\nhasn't yet been properly established. According to the Brazilian Guidelines for\nImplantable Electronic Heart Devices[23], patients with asymptomatic AVB with wide QRS after\ncardiac surgery that persists after 15 days, are indicated for a PPM implantation\n(Class I, level of evidence C). In the cases of asymptomatic AVB persisting after 15\ndays, resulting from cardiac surgery, with narrow QRS or nodal escape rhythm and\ngood chronotropic response, and in those cases without the prospect of reversal\n(< 15 days) PPM implantation is also indicated (Class IIa, level C).\nAccording to the criteria of the American College of Cardiology and\nthe American Heart Association, a PPM implant is indicated in\n3rd and advanced 2nd degree AVB in the postoperative\nperiod of heart surgery, in addition to cases without expectation of resolution. The\ndecision regarding the time of the implant should be taken by the\nphysician[24].\nThe European Society of Cardiology recommends a waiting period of 5 to 7 days for the\nresolution of transient bradyarrhythmias after cardiac surgery, before the decision\nfor the implant is made[25].\nAccording to Pires et al.[13] and Merin et al.[18], the decision to perform the implant\nshould be taken between the 4th and 5th day of the PO, because\nif the AVB or dysfunction of the sinus node are still present up to this moment,\nthen they tend to be permanent. This would facilitate the early mobilization of\npatients and shorten their hospitalization time.\nOf the 288 patients in our study who had AVB, 08 received a PPM implant after an\naverage of 12.25 days into the PO, which is in line with the Brazilian (Class IIa,\nlevel of evidence C), American and European (Class I, level C) guidelines. Emlein et\nal.[8]\ndescribed a series of 8 patients who underwent a PPM implant after developing AVB\nwith an average of 10.5±6.5 days into the PO.\n Limitations of the Study The limitations of this study are those inherent to a retrospective database\nanalysis, but they reflect the significant years of experience of an academic\ninstitution. Within these limitations we can cite the relative difficulty of\naccessing the full data, which causes a potential risk of not measuring some\nrandom variables. The fact that the results come from the sample of a single\ncenter can also represent some degree of bias in the treatment. Another\nlimitation of this study is the absence of more precise information regarding\nthe height of the atrioventricular conduction disorder and the existence or not\nof any escape rhythm.\nRegarding the PPM implants performed in our study, they followed the\nrecommendations of Brazilian, American and European guidelines almost strictly.\nIn this small group of patients a more thorough analysis was compromised, but\nthis could be the target of a more detailed study to be developed in the\nfuture.\nThe limitations of this study are those inherent to a retrospective database\nanalysis, but they reflect the significant years of experience of an academic\ninstitution. Within these limitations we can cite the relative difficulty of\naccessing the full data, which causes a potential risk of not measuring some\nrandom variables. The fact that the results come from the sample of a single\ncenter can also represent some degree of bias in the treatment. Another\nlimitation of this study is the absence of more precise information regarding\nthe height of the atrioventricular conduction disorder and the existence or not\nof any escape rhythm.\nRegarding the PPM implants performed in our study, they followed the\nrecommendations of Brazilian, American and European guidelines almost strictly.\nIn this small group of patients a more thorough analysis was compromised, but\nthis could be the target of a more detailed study to be developed in the\nfuture.\n\nLimitations of the Study:\nThe limitations of this study are those inherent to a retrospective database\nanalysis, but they reflect the significant years of experience of an academic\ninstitution. Within these limitations we can cite the relative difficulty of\naccessing the full data, which causes a potential risk of not measuring some\nrandom variables. The fact that the results come from the sample of a single\ncenter can also represent some degree of bias in the treatment. Another\nlimitation of this study is the absence of more precise information regarding\nthe height of the atrioventricular conduction disorder and the existence or not\nof any escape rhythm.\nRegarding the PPM implants performed in our study, they followed the\nrecommendations of Brazilian, American and European guidelines almost strictly.\nIn this small group of patients a more thorough analysis was compromised, but\nthis could be the target of a more detailed study to be developed in the\nfuture.\n\nCONCLUSION:\nThis work sheds light on the risk factors associated with the development of AVB in\nthe PO of CABG and the consequent need for TP and a definitive pacemaker. Based on\nthis we could establish that female patients, 60 years of age or more, with the\ndiagnosis of AF and CKD, in stages III and IV of the functional class, who had\nperioperative AMI and required the use of an IAB, have a higher risk of developing\nAVB in the PO of CABG. AVB determines a more prolonged hospitalization and, what is\nmore important, doubles the risk of mortality.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nDisturbances of the cardiac conduction system are frequent in the postoperative period of coronary artery bypass surgery. They are mostly reversible and associated with some injury of the conduction tissue, caused by the ischemic heart disease itself or by perioperative factors.\n\nMethods:\nAnalysis of a retrospective cohort of patients submitted to coronary artery bypass surgery from the database of the Postoperative Heart Surgery Unit of the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande do Sul, using the logistic regression method.\n\nResults:\nIn the period from January 1996 to December 2012, 3532 coronary artery bypass surgery were carried out. Two hundred and eighty-eight (8.15% of the total sample) patients had atrioventricular block during the postoperative period of coronary artery bypass surgery, requiring temporary pacing. Eight of those who had atrioventricular block progressed to implantation of a permanent pacemaker (0.23% of the total sample). Multivariate analysis revealed a significant association of atrioventricular block with age above 60 years (OR=2.34; CI 95% 1.75-3.12; P<0.0001), female gender (OR=1.37; CI 95% 1.06-1.77; P=0.015), chronic kidney disease (OR=2.05; CI 95% 1.49-2.81; P<0.0001), atrial fibrillation (OR=2.06; CI 95% 1.16-3.66; P=0.014), functional class III and IV of the New York Heart Association (OR=1.43; CI 95% 1.03-1.98; P=0.031), perioperative acute myocardial infarction (OR=1.70; CI 95% 1.26-2.29; P<0.0001) and with the use of the intra-aortic balloon in the postoperative period of coronary artery bypass surgery (OR=1.92; CI 95% 1.21-3.05; P=0.006). The presence of atrioventricular block resulted in a significant increase in mortality (17.9% vs. 7.3% in those who did not develop atrioventricular block) (OR=2.09; CI 95% 1.46-2.99; P<0.0001) and a longer hospital stay (12.75 days x 10.53 days for those who didn't develop atrioventricular block) (OR=1.01; CI 95% 1.00-1.02; P=0.01).\n\nConclusions:\nIn most cases, atrioventricular block in the postoperative period of coronary artery bypass surgery is transient and associated with several perioperative factors: age above 60 years, female sex, chronic kidney disease, atrial fibrillation, New York Heart Association functional class III or IV, perioperative acute myocardial infarction and use of an intra-aortic balloon. Its occurrence prolongs hospitalization and, above all, doubles the risk of mortality."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nDisturbances of the cardiac conduction system are relatively frequent in the\npostoperative period (PO) of coronary artery bypass grafting (CABG), with an\nincidence ranging from 18 to 55% of cases[1-6].\nAtrioventricular block (AVB) is one of these conduction disturbances and its\nincidence ranges from 0.5 to 16%[3,5-9]. Most patients have transitory and\nreversible conduction disorders that require temporary pacing (TP). 0.4 to 1.1% of\npatients, however, when faced with the irreversibility of the condition, will have\nto undergo a permanent pacemaker (PPM) implant during their hospital\nstay[10].\nThis study, which is unprecedented in the national literature, tries to identify the\nrelationship between pre-, intra and postoperative (perioperative) factors\nassociated with the emergence of AVB, the need for TP and, if the case, the\nimplantation of a PPM in the PO of CABG.\n\nCONCLUSION:\nThis work sheds light on the risk factors associated with the development of AVB in\nthe PO of CABG and the consequent need for TP and a definitive pacemaker. Based on\nthis we could establish that female patients, 60 years of age or more, with the\ndiagnosis of AF and CKD, in stages III and IV of the functional class, who had\nperioperative AMI and required the use of an IAB, have a higher risk of developing\nAVB in the PO of CABG. AVB determines a more prolonged hospitalization and, what is\nmore important, doubles the risk of mortality."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nDisturbances of the cardiac conduction system are frequent in the postoperative period of coronary artery bypass surgery. They are mostly reversible and associated with some injury of the conduction tissue, caused by the ischemic heart disease itself or by perioperative factors.\n\nMethods:\nAnalysis of a retrospective cohort of patients submitted to coronary artery bypass surgery from the database of the Postoperative Heart Surgery Unit of the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande do Sul, using the logistic regression method.\n\nResults:\nIn the period from January 1996 to December 2012, 3532 coronary artery bypass surgery were carried out. Two hundred and eighty-eight (8.15% of the total sample) patients had atrioventricular block during the postoperative period of coronary artery bypass surgery, requiring temporary pacing. Eight of those who had atrioventricular block progressed to implantation of a permanent pacemaker (0.23% of the total sample). Multivariate analysis revealed a significant association of atrioventricular block with age above 60 years (OR=2.34; CI 95% 1.75-3.12; P<0.0001), female gender (OR=1.37; CI 95% 1.06-1.77; P=0.015), chronic kidney disease (OR=2.05; CI 95% 1.49-2.81; P<0.0001), atrial fibrillation (OR=2.06; CI 95% 1.16-3.66; P=0.014), functional class III and IV of the New York Heart Association (OR=1.43; CI 95% 1.03-1.98; P=0.031), perioperative acute myocardial infarction (OR=1.70; CI 95% 1.26-2.29; P<0.0001) and with the use of the intra-aortic balloon in the postoperative period of coronary artery bypass surgery (OR=1.92; CI 95% 1.21-3.05; P=0.006). The presence of atrioventricular block resulted in a significant increase in mortality (17.9% vs. 7.3% in those who did not develop atrioventricular block) (OR=2.09; CI 95% 1.46-2.99; P<0.0001) and a longer hospital stay (12.75 days x 10.53 days for those who didn't develop atrioventricular block) (OR=1.01; CI 95% 1.00-1.02; P=0.01).\n\nConclusions:\nIn most cases, atrioventricular block in the postoperative period of coronary artery bypass surgery is transient and associated with several perioperative factors: age above 60 years, female sex, chronic kidney disease, atrial fibrillation, New York Heart Association functional class III or IV, perioperative acute myocardial infarction and use of an intra-aortic balloon. Its occurrence prolongs hospitalization and, above all, doubles the risk of mortality."},"whole_article_text_length":{"kind":"number","value":5959,"string":"5,959"},"whole_article_abstract_length":{"kind":"number","value":471,"string":"471"},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["avb","patients","cabg","analysis","study","po","need","ppm","po cabg","use"],"string":"[\n \"avb\",\n \"patients\",\n \"cabg\",\n \"analysis\",\n \"study\",\n \"po\",\n \"need\",\n \"ppm\",\n \"po cabg\",\n \"use\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] conduction | incidence | disturbances | postoperative | ppm | tp | avb | po | period po coronary | period po [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] analysis | performed | previous use | use | cabg | previous | hospital | according | pucrs | study [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ci | avb | 95 | ci 95 | analysis | days | patients | tp | table | univariate analysis [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] risk | avb | avb po | avb po cabg | po cabg | po | use iab higher risk | avb determines prolonged hospitalization | ami required | ami required use [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] avb | cabg | patients | analysis | study | po | po cabg | performed | ppm | tp [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] avb | cabg | patients | analysis | study | po | po cabg | performed | ppm | tp [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] the Postoperative Heart Surgery Unit | the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] the period | January 1996 to December 2012 | 3532 ||| Two hundred and eighty-eight | 8.15% ||| Eight | 0.23% ||| age above 60 years | OR=2.34 | CI | 95% | 1.75 | CI | 95% | 1.06 | OR=2.05 | CI | 95% | 1.49 | OR=2.06 | CI | 95% | 1.16-3.66 | IV | the New York Heart Association | CI | 95% | 1.03 | OR=1.70 | CI | 95% | 1.26-2.29 | OR=1.92 | CI | 95% | 1.21 ||| 17.9% | 7.3% | CI | 95% | 1.46 | 12.75 days | 10.53 days | OR=1.01 | CI | 95% | 1.00-1.02 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] age above 60 years | New York Heart Association | IV ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| the Postoperative Heart Surgery Unit | the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande ||| ||| the period | January 1996 to December 2012 | 3532 ||| Two hundred and eighty-eight | 8.15% ||| Eight | 0.23% ||| age above 60 years | OR=2.34 | CI | 95% | 1.75 | CI | 95% | 1.06 | OR=2.05 | CI | 95% | 1.49 | OR=2.06 | CI | 95% | 1.16-3.66 | IV | the New York Heart Association | CI | 95% | 1.03 | OR=1.70 | CI | 95% | 1.26-2.29 | OR=1.92 | CI | 95% | 1.21 ||| 17.9% | 7.3% | CI | 95% | 1.46 | 12.75 days | 10.53 days | OR=1.01 | CI | 95% | 1.00-1.02 ||| age above 60 years | New York Heart Association | IV ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| the Postoperative Heart Surgery Unit | the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande ||| ||| the period | January 1996 to December 2012 | 3532 ||| Two hundred and eighty-eight | 8.15% ||| Eight | 0.23% ||| age above 60 years | OR=2.34 | CI | 95% | 1.75 | CI | 95% | 1.06 | OR=2.05 | CI | 95% | 1.49 | OR=2.06 | CI | 95% | 1.16-3.66 | IV | the New York Heart Association | CI | 95% | 1.03 | OR=1.70 | CI | 95% | 1.26-2.29 | OR=1.92 | CI | 95% | 1.21 ||| 17.9% | 7.3% | CI | 95% | 1.46 | 12.75 days | 10.53 days | OR=1.01 | CI | 95% | 1.00-1.02 ||| age above 60 years | New York Heart Association | IV ||| [SUMMARY]"}}},{"rowIdx":265,"cells":{"title":{"kind":"string","value":"Performance of two rapid diagnostic tests for malaria diagnosis at the China-Myanmar border area."},"pmid":{"kind":"string","value":"23433230"},"background_abstract":{"kind":"string","value":"Rapid diagnostic tests (RDTs) have become an essential tool in the contemporary malaria control and management programmes in the world. This study aims to evaluate the performance of two commonly used RDTs for malaria diagnosis in the China-Myanmar border area."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A total 606 febrile patients in the China-Myanmar border were recruited to this study and were diagnosed for malaria infections by microscopy, two RDTs tests (Pf/Pan device, and Pv/Pf device) and nested PCR."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Malaria parasites were found in 143 patients by microscopy, of which 51, 73, and 19 were Plasmodium falciparum, Plasmodium vivax and P. falciparum/P. vivax mixed infections, respectively. Compared to microscopy, the sensitivity of the Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4%. For a subset of 350 patients, the sensitivity of the Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively. The specificity of the Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively. Nested PCR detected malaria parasites in 174 of 606 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. Compared to nested PCR, all other methods had sensitivity below 80%, suggesting that a significant number of cases were missed."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Compared to PCR, both microscopy and RDTs had lower sensitivities. RDTs had similar performance to microscopy for P. falciparum diagnosis, but performed worse for P. vivax diagnosis. Other RDT products should be selected with higher sensitivity (and good specificity) for both P. falciparum and P. vivax diagnosis."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Aged","Aged, 80 and over","Child","Child, Preschool","China","Clinical Laboratory Techniques","Diagnostic Tests, Routine","Female","Humans","Infant","Malaria","Male","Middle Aged","Myanmar","Parasitology","Plasmodium falciparum","Plasmodium ovale","Plasmodium vivax","Sensitivity and Specificity","Young Adult"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Child\",\n \"Child, Preschool\",\n \"China\",\n \"Clinical Laboratory Techniques\",\n \"Diagnostic Tests, Routine\",\n \"Female\",\n \"Humans\",\n \"Infant\",\n \"Malaria\",\n \"Male\",\n \"Middle Aged\",\n \"Myanmar\",\n \"Parasitology\",\n \"Plasmodium falciparum\",\n \"Plasmodium ovale\",\n \"Plasmodium vivax\",\n \"Sensitivity and Specificity\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"3599043"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Malaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually\n[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms\n[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease\n[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.\nMicroscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately\n[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific\n[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine\n[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings\n[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign\n[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.\nRDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results\n[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported\n[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria\n[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Study site and patients The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\nThe study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\n Microscopic examinations Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\nThick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\n RDTs for malaria The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\nThe RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\n Diagnosis by PCR Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\nFresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\n Statistical analysis The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.\nThe RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Blood samples were collected from a total of 606 patients. All samples were evaluated by the Pf/Pan test device, while a subset of 350 were also evaluated by the Pv/Pf test device. Of the 606 samples, malaria parasites were found in 143 by microscopy; 51, 73, and 19 were P. falciparum, P. vivax and P. falciparum/P. vivax mixed infections, respectively (Table \n3). No other malaria parasite species were detected by microscopy. The parasite density ranges of P. falciparum were 40–105,920 parasites/μL. For P. vivax, the majority of cases had parasite density ranging from 80 to 17,800 parasites/μL. One P. vivax case had a deviant parasite density of >200,000 parasites/μL based on leucocyte number probably due to a low leucocyte count in this patient. Because P. falciparum single infections and mixed species infections containing P. falciparum cannot be differentiated by the Pf/Pan device, the microscopy results were grouped into “P. falciparum and mixed infections with P. falciparum” and “Non-falciparum”. Based on this interpretation, the Pf/Pan device detected 33 single P. falciparum infections (only the PfHRP2 line visible), 56 non-falciparum cases (only the pan-pLDH line visible) and 70 P. falciparum infections/potentially mixed infections (both lines visible) (Table \n3). Compared with the results by microscopy, the Pf/Pan device detected 62 and 51 samples as true positives for P. falciparum and non-falciparum, respectively. From this result, the sensitivity of Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4% (Table \n4).\nDetection results of malaria infections by the Malaria Pf/Pan test in comparison with microscopy (N=606)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\n\nPerformance of two RDTs for detection of \n\nP. falciparum \n\nand \n\nP. vivax \n\nwith microscopy as the gold standard\n\nData are presented as percentage (95% confidence interval; CI).\nFor the subset of 350 samples, microscopy detected 37 samples containing P. falciparum, 50 samples containing P. vivax and 11 were diagnosed as mixed species infections (Table \n5). Since the Pv/Pf device is able to differentiate P. falciparum and P. vivax infections, the microscopy results were grouped into “infections containing P. falciparum” and “infections containing P. vivax”. The Pv/Pf device detected 40 single P. falciparum infections, 42 P. vivax infections, and 8 mixed species infections (Table \n5). Compared to microscopy, the Pv/Pf device detected 44 and 45 samples as true positives for P. falciparum and P. vivax, respectively, whereas the Pf/Pan device detected 42 and 36 samples as true positives for P. falciparum and P. vivax, respectively. The sensitivity of Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively (Table \n4). The specificity of Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively (Table \n4).\nDetection results of malaria infections by Malaria Pv/Pf test and Malaria Pf/Pan test in comparison with the microscopic method (N=350)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nIt has been observed that some RDTs do not perform well when the parasite density is below 500 parasites/μL. For P. falciparum, both RDTs had significantly higher sensitivity for cases with a parasite density above 500 parasites/μL (>92%) than those with a density below 500 parasites/μL (<75.0%) (Table \n6). However, for P. vivax malaria, regardless of the parasite density, both RDTs displayed sensitivity of below 79%. Both RDTs had similar detection limits; they were >240 and >320 parasites/μL for P. falciparum and P. vivax, respectively.\n\nComparative sensitivity of the two RDTs for detection of \n\nP. falciparum \n\nand \n\nP. vivax \n\nin comparison with the microscopic method, categorized according to parasite density\n\nSensitivity is presented as percentage (95% confidence interval; CI). TP true positive.\nTo further evaluate the performance of these two RDTs, all 606 samples were examined by nested PCR. Nested PCR detected malaria parasites in 174 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. No P. knowlesi infections were detected by PCR (Table \n7). For the subset of 350 samples, 113 samples were identified by nested PCR as infection with malaria parasites. Of which, 40, 52, and 21 were P. falciparum, P. vivax, and P. falciparum/P. vivax mixed infections, respectively (Table \n8). Among all detection methods, PCR was the most sensitive one. If PCR was used as the gold standard, the sensitivity of microscopy for P. falciparum and P. vivax was 71.0% and 73.3% in 606 detected samples, and 75.4% and 74.0% in the 350 subset samples, respectively (Table \n9). The sensitivity of Pf/Pan test for P. falciparum and P. vivax was 81.7% and 64.6% in 606 detected samples, and 77.1% and 63.5% in the 350 subset samples, respectively (Table \n9). For the subset of 350 samples analyzed by Pv/Pf devices, the sensitivity was 72.1% for P. falciparum and 58.9% for P. vivax, respectively (Table \n9). Microscopy and the two RDTs had similar levels of specificity ranging from 94.7% to 96.3% (Table \n9). It is noteworthy that although the Pf/Pan device is designed to identify all human malaria parasite species, the two P. ovale infections identified by PCR were missed by this device and by microscopy, possibly due to the low parasitaemia in the P. ovale infections (containing 120 parasites/μL and 320 parasites/μL, respectively). Re-examination of the P. ovale slides by microscopy did detect P. ovale-parasitized erythrocytes (Figure \n1).\nBlood smears showing parasitized erythrocytes by P. ovale in two malaria patients. The two patients with febrile illness attended a malaria clinic at the China-Myanmar border and were diagnosed for malaria. Both cases were only detected by PCR but missed by microscopy and RDTs.\nDetection results of malaria infections by microscopy and Malaria Pf/Pan test in comparison with the Nested PCR (N=606)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nDetection results of malaria infections by microscopy, Malaria Pf/Pan test and Malaria Pv/Pf test in comparison with the Nested PCR (N=350)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nPerformance of different diagnosis methods with the nested PCR method as the gold standard\nData are presented as percentage (95% confidence interval; CI).\nAccording to the microscopy results corrected by PCR, four and eight cases were false negative in 606 samples examined by the Pf/Pan test device for P. falciparum and P. vivax, respectively (Table \n3). When mixed infections were excluded, three out of four P. falciparum cases had lower parasite density (<480 parasites/μL). Only one P. falciparum case with higher parasite density (8,800 parasites/μL) was not detected by the Pf/Pan test device, but was positive by the Pv/Pf test device. Five out of eight P. vivax cases were lower parasite density (<560 parasites/μL). The remaining three P. vivax cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by the Pf/Pan test device. In the subset of 350 samples examined by the Pv/Pf test device (mixed infection not included, Table \n5), two P. falciparum cases with lower parasite density (<480 parasites/μL) were false negative. Nine P. vivax cases were the false negative, of which three had lower parasite density (<320 parasites/μL), and three cases (parasite density of 1,320/μL, 2080/μL, 7,280/μL) were positive by the Pf/Pan test device. The other three cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by both RDTs.\nFalse positive and wrong species identification were observed with both RDTs. In all 606 samples (identified by microscopy and corrected by PCR, mixed infection not included) examined by the Pf/Pan test device, one P. vivax case, one P. ovale case, and eight negative cases showed the PfHRP2 line. Ten P. vivax cases showed not only the pan-pLDH line, but the PfHRP2 line. Seven negative cases showed both PfHRP2 and pan-pLDH lines (Table \n7). In the subset of 350 samples analysed by the Pv/Pf test, two negative cases showed the PfHRP2 line. One P. falciparum case showed the Pv-pLDH line only. Two P. vivax cases showed not only the Pv-pLDH line, but the PfHRP2-line. Six P. falciparum cases (parasite density ranging from 240 to 34,400 parasites/μL) showed not only the PfHRP2 line, but also the Pv-pLDH line (Table \n8). It has been reported that P. falciparum infections with high parasite densities may generate a false positive Pv-pLDH line\n[22-25]. For the Pv/Pf test devices, six out of 40 P. falciparum (corrected by PCR) with the Pv-pLDH line visible showed that cross reactions between different species occurred although parasite densities of some P. falciparum infections were not high. From these comparisons, the specificities of these devices need further improvement for areas with both P. falciparum and P. vivax malaria.\nSince P. falciparum and P. vivax infections are treated with different drugs, it would be important to compare the different methods for detecting mixed species infections. PCR, microscopy and Pv/Pf test device detected 21, 11, and eight mixed-species infections in the 350 subset samples (Table \n8), corresponding to 18.6%, 11.2% and 8.9% of positive cases detected by the respective methods."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The aim of this study was to evaluate the performance of two RDTs for malaria diagnosis at the China-Myanmar border area. PCR method was much more sensitive than microscopy and RDTs. Compared to microscopy, both RDTs demonstrated similar sensitivities for detecting P. falciparum infections but reduced sensitivity for detecting P. vivax infections. Neither of them had satisfactory results in detecting mixed species infections. Therefore, methods with improved sensitivity for diagnosing P. vivax malaria are needed. False negative results for the diagnosis of P. falciparum malaria call for further investigations on potential deletion of pfhrp2 gene in this malaria-endemic region."},"other_sections_titles":{"kind":"list like","value":["Background","Study site and patients","Microscopic examinations","RDTs for malaria","Diagnosis by PCR","Statistical analysis","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Study site and patients\",\n \"Microscopic examinations\",\n \"RDTs for malaria\",\n \"Diagnosis by PCR\",\n \"Statistical analysis\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["Malaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually\n[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms\n[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease\n[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.\nMicroscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately\n[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific\n[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine\n[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings\n[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign\n[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.\nRDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results\n[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported\n[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria\n[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria.","The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.","Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.","The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.","Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections","The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.","The authors declare that they have no competing interests.","YJ, LN, WX, LP and ZZ carried out the experimental work and data analysis.WL, LS and ZG participated in data analysis. YJ performed manuscript writing. YC, QF and LC conceived the study and participated in the design of the study. All authors read and approved the final manuscript."],"string":"[\n \"Malaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually\\n[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms\\n[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease\\n[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.\\nMicroscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately\\n[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific\\n[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine\\n[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings\\n[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign\\n[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.\\nRDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results\\n[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported\\n[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria\\n[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria.\",\n \"The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\",\n \"Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\",\n \"The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\",\n \"Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\\n[20,21]. Plasmodium genus-specific primers were shown in Table \\n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \\n1.\\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\",\n \"The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \\n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\\nInterpretation of the results for the Pf/Pan RDT\\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.\",\n \"The authors declare that they have no competing interests.\",\n \"YJ, LN, WX, LP and ZZ carried out the experimental work and data analysis.WL, LS and ZG participated in data analysis. YJ performed manuscript writing. YC, QF and LC conceived the study and participated in the design of the study. All authors read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study site and patients","Microscopic examinations","RDTs for malaria","Diagnosis by PCR","Statistical analysis","Results","Discussion","Conclusions","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study site and patients\",\n \"Microscopic examinations\",\n \"RDTs for malaria\",\n \"Diagnosis by PCR\",\n \"Statistical analysis\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Malaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually\n[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms\n[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease\n[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.\nMicroscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately\n[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific\n[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine\n[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings\n[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign\n[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.\nRDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results\n[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported\n[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria\n[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria."," Study site and patients The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\nThe study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\n Microscopic examinations Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\nThick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\n RDTs for malaria The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\nThe RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\n Diagnosis by PCR Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\nFresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\n Statistical analysis The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.\nThe RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.","The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.","Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.","The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.","Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections","The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.","Blood samples were collected from a total of 606 patients. All samples were evaluated by the Pf/Pan test device, while a subset of 350 were also evaluated by the Pv/Pf test device. Of the 606 samples, malaria parasites were found in 143 by microscopy; 51, 73, and 19 were P. falciparum, P. vivax and P. falciparum/P. vivax mixed infections, respectively (Table \n3). No other malaria parasite species were detected by microscopy. The parasite density ranges of P. falciparum were 40–105,920 parasites/μL. For P. vivax, the majority of cases had parasite density ranging from 80 to 17,800 parasites/μL. One P. vivax case had a deviant parasite density of >200,000 parasites/μL based on leucocyte number probably due to a low leucocyte count in this patient. Because P. falciparum single infections and mixed species infections containing P. falciparum cannot be differentiated by the Pf/Pan device, the microscopy results were grouped into “P. falciparum and mixed infections with P. falciparum” and “Non-falciparum”. Based on this interpretation, the Pf/Pan device detected 33 single P. falciparum infections (only the PfHRP2 line visible), 56 non-falciparum cases (only the pan-pLDH line visible) and 70 P. falciparum infections/potentially mixed infections (both lines visible) (Table \n3). Compared with the results by microscopy, the Pf/Pan device detected 62 and 51 samples as true positives for P. falciparum and non-falciparum, respectively. From this result, the sensitivity of Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4% (Table \n4).\nDetection results of malaria infections by the Malaria Pf/Pan test in comparison with microscopy (N=606)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\n\nPerformance of two RDTs for detection of \n\nP. falciparum \n\nand \n\nP. vivax \n\nwith microscopy as the gold standard\n\nData are presented as percentage (95% confidence interval; CI).\nFor the subset of 350 samples, microscopy detected 37 samples containing P. falciparum, 50 samples containing P. vivax and 11 were diagnosed as mixed species infections (Table \n5). Since the Pv/Pf device is able to differentiate P. falciparum and P. vivax infections, the microscopy results were grouped into “infections containing P. falciparum” and “infections containing P. vivax”. The Pv/Pf device detected 40 single P. falciparum infections, 42 P. vivax infections, and 8 mixed species infections (Table \n5). Compared to microscopy, the Pv/Pf device detected 44 and 45 samples as true positives for P. falciparum and P. vivax, respectively, whereas the Pf/Pan device detected 42 and 36 samples as true positives for P. falciparum and P. vivax, respectively. The sensitivity of Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively (Table \n4). The specificity of Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively (Table \n4).\nDetection results of malaria infections by Malaria Pv/Pf test and Malaria Pf/Pan test in comparison with the microscopic method (N=350)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nIt has been observed that some RDTs do not perform well when the parasite density is below 500 parasites/μL. For P. falciparum, both RDTs had significantly higher sensitivity for cases with a parasite density above 500 parasites/μL (>92%) than those with a density below 500 parasites/μL (<75.0%) (Table \n6). However, for P. vivax malaria, regardless of the parasite density, both RDTs displayed sensitivity of below 79%. Both RDTs had similar detection limits; they were >240 and >320 parasites/μL for P. falciparum and P. vivax, respectively.\n\nComparative sensitivity of the two RDTs for detection of \n\nP. falciparum \n\nand \n\nP. vivax \n\nin comparison with the microscopic method, categorized according to parasite density\n\nSensitivity is presented as percentage (95% confidence interval; CI). TP true positive.\nTo further evaluate the performance of these two RDTs, all 606 samples were examined by nested PCR. Nested PCR detected malaria parasites in 174 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. No P. knowlesi infections were detected by PCR (Table \n7). For the subset of 350 samples, 113 samples were identified by nested PCR as infection with malaria parasites. Of which, 40, 52, and 21 were P. falciparum, P. vivax, and P. falciparum/P. vivax mixed infections, respectively (Table \n8). Among all detection methods, PCR was the most sensitive one. If PCR was used as the gold standard, the sensitivity of microscopy for P. falciparum and P. vivax was 71.0% and 73.3% in 606 detected samples, and 75.4% and 74.0% in the 350 subset samples, respectively (Table \n9). The sensitivity of Pf/Pan test for P. falciparum and P. vivax was 81.7% and 64.6% in 606 detected samples, and 77.1% and 63.5% in the 350 subset samples, respectively (Table \n9). For the subset of 350 samples analyzed by Pv/Pf devices, the sensitivity was 72.1% for P. falciparum and 58.9% for P. vivax, respectively (Table \n9). Microscopy and the two RDTs had similar levels of specificity ranging from 94.7% to 96.3% (Table \n9). It is noteworthy that although the Pf/Pan device is designed to identify all human malaria parasite species, the two P. ovale infections identified by PCR were missed by this device and by microscopy, possibly due to the low parasitaemia in the P. ovale infections (containing 120 parasites/μL and 320 parasites/μL, respectively). Re-examination of the P. ovale slides by microscopy did detect P. ovale-parasitized erythrocytes (Figure \n1).\nBlood smears showing parasitized erythrocytes by P. ovale in two malaria patients. The two patients with febrile illness attended a malaria clinic at the China-Myanmar border and were diagnosed for malaria. Both cases were only detected by PCR but missed by microscopy and RDTs.\nDetection results of malaria infections by microscopy and Malaria Pf/Pan test in comparison with the Nested PCR (N=606)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nDetection results of malaria infections by microscopy, Malaria Pf/Pan test and Malaria Pv/Pf test in comparison with the Nested PCR (N=350)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nPerformance of different diagnosis methods with the nested PCR method as the gold standard\nData are presented as percentage (95% confidence interval; CI).\nAccording to the microscopy results corrected by PCR, four and eight cases were false negative in 606 samples examined by the Pf/Pan test device for P. falciparum and P. vivax, respectively (Table \n3). When mixed infections were excluded, three out of four P. falciparum cases had lower parasite density (<480 parasites/μL). Only one P. falciparum case with higher parasite density (8,800 parasites/μL) was not detected by the Pf/Pan test device, but was positive by the Pv/Pf test device. Five out of eight P. vivax cases were lower parasite density (<560 parasites/μL). The remaining three P. vivax cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by the Pf/Pan test device. In the subset of 350 samples examined by the Pv/Pf test device (mixed infection not included, Table \n5), two P. falciparum cases with lower parasite density (<480 parasites/μL) were false negative. Nine P. vivax cases were the false negative, of which three had lower parasite density (<320 parasites/μL), and three cases (parasite density of 1,320/μL, 2080/μL, 7,280/μL) were positive by the Pf/Pan test device. The other three cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by both RDTs.\nFalse positive and wrong species identification were observed with both RDTs. In all 606 samples (identified by microscopy and corrected by PCR, mixed infection not included) examined by the Pf/Pan test device, one P. vivax case, one P. ovale case, and eight negative cases showed the PfHRP2 line. Ten P. vivax cases showed not only the pan-pLDH line, but the PfHRP2 line. Seven negative cases showed both PfHRP2 and pan-pLDH lines (Table \n7). In the subset of 350 samples analysed by the Pv/Pf test, two negative cases showed the PfHRP2 line. One P. falciparum case showed the Pv-pLDH line only. Two P. vivax cases showed not only the Pv-pLDH line, but the PfHRP2-line. Six P. falciparum cases (parasite density ranging from 240 to 34,400 parasites/μL) showed not only the PfHRP2 line, but also the Pv-pLDH line (Table \n8). It has been reported that P. falciparum infections with high parasite densities may generate a false positive Pv-pLDH line\n[22-25]. For the Pv/Pf test devices, six out of 40 P. falciparum (corrected by PCR) with the Pv-pLDH line visible showed that cross reactions between different species occurred although parasite densities of some P. falciparum infections were not high. From these comparisons, the specificities of these devices need further improvement for areas with both P. falciparum and P. vivax malaria.\nSince P. falciparum and P. vivax infections are treated with different drugs, it would be important to compare the different methods for detecting mixed species infections. PCR, microscopy and Pv/Pf test device detected 21, 11, and eight mixed-species infections in the 350 subset samples (Table \n8), corresponding to 18.6%, 11.2% and 8.9% of positive cases detected by the respective methods.","RDTs are playing an essential role in the current malaria control/elimination campaign worldwide. In this study, the performance of two RDT devices commonly used at the China-Myanmar border area was evaluated. Compared to diagnosis by microscopy, both the Pf/Pan and Pv/Pf devices showed higher sensitivity (>87%) for detecting P. falciparum infections, whereas the sensitivity for detecting P. vivax infections was much lower (<74%). Both RDTs had similar levels of detection limits, and their performance was poor for infections with parasite densities below 500 parasites/μL for P. falciparum infections. Besides, since both devices rely on the PfHRP2 antigen for the detection of P. falciparum, pfhrp2 gene deletions will lead to false-negative results\n[26]. A recent report showing up to 40% of P. falciparum parasites with pfhrp2 deletions in South America\n[27] indicate that such RDTs would not perform well in these regions. It certainly warrants more detailed investigations in other malaria endemic regions.\nWhen PCR was used as the gold standard, the detection sensitivity of both RDTs was reduced especially for the detection of P. vivax infections (<64%), although the specificity of the all detection methods remained above 92%. In addition, the sensitivity of conventional microscopy was also low, ranging from 71.0% to 77.4% for detecting both parasite species. This result is worrisome; since nearly 30% of the malaria cases was not correctly diagnosed by microscopy or RDTs and subsequently treated, these patients may constitute an important reservoir for transmission. Whereas low parasite density might be the most important limiting factor for microscopic diagnosis\n[6,28], the varying skills and experience of microscopists may also be responsible for the lower sensitivity of diagnosis\n[29]. In the past, the Pf/Pan test (Wondfo) has been evaluated for diagnosis of P. falciparum only and showed highly satisfactory result\n[30]. Recently, a CareStart™ kit (Pf/Pan) was evaluated in the nearby Yunnan province of China, which showed comparable results to microscopy for diagnosing P. falciparum and P. vivax infections\n[31]. Therefore, further evaluations of RDTs are needed to select better diagnostic tests with improved accuracy in this region.\nOverall, both RDTs have good sensitivity for detecting P. falciparum infections, but the sensitivity for detecting P. vivax was much lower. This could be due to lower parasite density for P. vivax since this parasite selectively invades reticulocytes. However, comparison of the two RDTs showed that the sensitivities for P. vivax detection were not different between the high and low parasite densities (Table \n6). This observation is difficult to explain and could be due to the lower number of infections analyzed in the <500 parasites/μL group. Furthermore, the arbitrary selection of 500 parasites/μL as the border line between high and low parasite densities may not be appropriate for P. vivax. In addition, the sensitivity to P. vivax may be affected by other factors. Therefore, better RDTs with significantly improved sensitivity for P. vivax are needed.\nOne important criterion for the selection of RDTs in the GMS is the ability to detect other human malaria species in addition to P. falciparum. The advantage of the Pf/Pan test is that it is designed to detect four human parasite species, which co-exist in the GMS. However, this device failed to detect the two P. ovale infections, suggesting that further improvement in sensitivity for detecting other malaria species is needed. It is also worth to note that the PCR method failed to identify any P. knowlesi infections, which is in stark contrast to the eaerlier report of some 30% of malaria cases in this region as mixed infections with P. knowlesi[16]. Therefore, the significantly lower prevalence of other malaria parasite species in this region and the generally low sensitivity of the Pf/Pan test for diagnosis of P. ovale and P. malariae suggest that such an advantage is hardly of any importance in this region. In comparison, the Pv/Pf test is specific for P. falciparum and P. vivax, which make up the majority of malaria infections in the GMS, although it would certainly miss detecting P. malariae and P. ovale infections. Because P. falciparum and P. vivax require different antimalarial treatments, correct diagnosis of mixed infections should be an important criterion for the selection of an RDT. Even though the Pv/Pf device is able to detect P. falciparum and P. vivax mixed infection, the detection rate was low and the eight mix infections detected by this device were all misdiagnosed when compared to the PCR result. This result may be due to reduced density of one parasite species in the presence of another due to competition in the same host. Consequently, with the current anti-malarial drug policy of this region, misdiagnosis of mixed species infections as single species infections would lead to improper drug treatments.","The aim of this study was to evaluate the performance of two RDTs for malaria diagnosis at the China-Myanmar border area. PCR method was much more sensitive than microscopy and RDTs. Compared to microscopy, both RDTs demonstrated similar sensitivities for detecting P. falciparum infections but reduced sensitivity for detecting P. vivax infections. Neither of them had satisfactory results in detecting mixed species infections. Therefore, methods with improved sensitivity for diagnosing P. vivax malaria are needed. False negative results for the diagnosis of P. falciparum malaria call for further investigations on potential deletion of pfhrp2 gene in this malaria-endemic region.","The authors declare that they have no competing interests.","YJ, LN, WX, LP and ZZ carried out the experimental work and data analysis.WL, LS and ZG participated in data analysis. YJ performed manuscript writing. YC, QF and LC conceived the study and participated in the design of the study. All authors read and approved the final manuscript."],"string":"[\n \"Malaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually\\n[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms\\n[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease\\n[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.\\nMicroscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately\\n[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific\\n[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine\\n[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings\\n[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign\\n[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.\\nRDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results\\n[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported\\n[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria\\n[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria.\",\n \" Study site and patients The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\\nThe study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\\n Microscopic examinations Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\\nThick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\\n RDTs for malaria The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\\nThe RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\\n Diagnosis by PCR Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\\n[20,21]. Plasmodium genus-specific primers were shown in Table \\n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \\n1.\\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\\nFresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\\n[20,21]. Plasmodium genus-specific primers were shown in Table \\n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \\n1.\\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\\n Statistical analysis The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \\n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\\nInterpretation of the results for the Pf/Pan RDT\\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.\\nThe RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \\n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\\nInterpretation of the results for the Pf/Pan RDT\\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.\",\n \"The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\",\n \"Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\",\n \"The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\",\n \"Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\\n[20,21]. Plasmodium genus-specific primers were shown in Table \\n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \\n1.\\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\",\n \"The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \\n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\\nInterpretation of the results for the Pf/Pan RDT\\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.\",\n \"Blood samples were collected from a total of 606 patients. All samples were evaluated by the Pf/Pan test device, while a subset of 350 were also evaluated by the Pv/Pf test device. Of the 606 samples, malaria parasites were found in 143 by microscopy; 51, 73, and 19 were P. falciparum, P. vivax and P. falciparum/P. vivax mixed infections, respectively (Table \\n3). No other malaria parasite species were detected by microscopy. The parasite density ranges of P. falciparum were 40–105,920 parasites/μL. For P. vivax, the majority of cases had parasite density ranging from 80 to 17,800 parasites/μL. One P. vivax case had a deviant parasite density of >200,000 parasites/μL based on leucocyte number probably due to a low leucocyte count in this patient. Because P. falciparum single infections and mixed species infections containing P. falciparum cannot be differentiated by the Pf/Pan device, the microscopy results were grouped into “P. falciparum and mixed infections with P. falciparum” and “Non-falciparum”. Based on this interpretation, the Pf/Pan device detected 33 single P. falciparum infections (only the PfHRP2 line visible), 56 non-falciparum cases (only the pan-pLDH line visible) and 70 P. falciparum infections/potentially mixed infections (both lines visible) (Table \\n3). Compared with the results by microscopy, the Pf/Pan device detected 62 and 51 samples as true positives for P. falciparum and non-falciparum, respectively. From this result, the sensitivity of Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4% (Table \\n4).\\nDetection results of malaria infections by the Malaria Pf/Pan test in comparison with microscopy (N=606)\\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\\n\\nPerformance of two RDTs for detection of \\n\\nP. falciparum \\n\\nand \\n\\nP. vivax \\n\\nwith microscopy as the gold standard\\n\\nData are presented as percentage (95% confidence interval; CI).\\nFor the subset of 350 samples, microscopy detected 37 samples containing P. falciparum, 50 samples containing P. vivax and 11 were diagnosed as mixed species infections (Table \\n5). Since the Pv/Pf device is able to differentiate P. falciparum and P. vivax infections, the microscopy results were grouped into “infections containing P. falciparum” and “infections containing P. vivax”. The Pv/Pf device detected 40 single P. falciparum infections, 42 P. vivax infections, and 8 mixed species infections (Table \\n5). Compared to microscopy, the Pv/Pf device detected 44 and 45 samples as true positives for P. falciparum and P. vivax, respectively, whereas the Pf/Pan device detected 42 and 36 samples as true positives for P. falciparum and P. vivax, respectively. The sensitivity of Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively (Table \\n4). The specificity of Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively (Table \\n4).\\nDetection results of malaria infections by Malaria Pv/Pf test and Malaria Pf/Pan test in comparison with the microscopic method (N=350)\\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\\nIt has been observed that some RDTs do not perform well when the parasite density is below 500 parasites/μL. For P. falciparum, both RDTs had significantly higher sensitivity for cases with a parasite density above 500 parasites/μL (>92%) than those with a density below 500 parasites/μL (<75.0%) (Table \\n6). However, for P. vivax malaria, regardless of the parasite density, both RDTs displayed sensitivity of below 79%. Both RDTs had similar detection limits; they were >240 and >320 parasites/μL for P. falciparum and P. vivax, respectively.\\n\\nComparative sensitivity of the two RDTs for detection of \\n\\nP. falciparum \\n\\nand \\n\\nP. vivax \\n\\nin comparison with the microscopic method, categorized according to parasite density\\n\\nSensitivity is presented as percentage (95% confidence interval; CI). TP true positive.\\nTo further evaluate the performance of these two RDTs, all 606 samples were examined by nested PCR. Nested PCR detected malaria parasites in 174 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. No P. knowlesi infections were detected by PCR (Table \\n7). For the subset of 350 samples, 113 samples were identified by nested PCR as infection with malaria parasites. Of which, 40, 52, and 21 were P. falciparum, P. vivax, and P. falciparum/P. vivax mixed infections, respectively (Table \\n8). Among all detection methods, PCR was the most sensitive one. If PCR was used as the gold standard, the sensitivity of microscopy for P. falciparum and P. vivax was 71.0% and 73.3% in 606 detected samples, and 75.4% and 74.0% in the 350 subset samples, respectively (Table \\n9). The sensitivity of Pf/Pan test for P. falciparum and P. vivax was 81.7% and 64.6% in 606 detected samples, and 77.1% and 63.5% in the 350 subset samples, respectively (Table \\n9). For the subset of 350 samples analyzed by Pv/Pf devices, the sensitivity was 72.1% for P. falciparum and 58.9% for P. vivax, respectively (Table \\n9). Microscopy and the two RDTs had similar levels of specificity ranging from 94.7% to 96.3% (Table \\n9). It is noteworthy that although the Pf/Pan device is designed to identify all human malaria parasite species, the two P. ovale infections identified by PCR were missed by this device and by microscopy, possibly due to the low parasitaemia in the P. ovale infections (containing 120 parasites/μL and 320 parasites/μL, respectively). Re-examination of the P. ovale slides by microscopy did detect P. ovale-parasitized erythrocytes (Figure \\n1).\\nBlood smears showing parasitized erythrocytes by P. ovale in two malaria patients. The two patients with febrile illness attended a malaria clinic at the China-Myanmar border and were diagnosed for malaria. Both cases were only detected by PCR but missed by microscopy and RDTs.\\nDetection results of malaria infections by microscopy and Malaria Pf/Pan test in comparison with the Nested PCR (N=606)\\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\\nDetection results of malaria infections by microscopy, Malaria Pf/Pan test and Malaria Pv/Pf test in comparison with the Nested PCR (N=350)\\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\\nPerformance of different diagnosis methods with the nested PCR method as the gold standard\\nData are presented as percentage (95% confidence interval; CI).\\nAccording to the microscopy results corrected by PCR, four and eight cases were false negative in 606 samples examined by the Pf/Pan test device for P. falciparum and P. vivax, respectively (Table \\n3). When mixed infections were excluded, three out of four P. falciparum cases had lower parasite density (<480 parasites/μL). Only one P. falciparum case with higher parasite density (8,800 parasites/μL) was not detected by the Pf/Pan test device, but was positive by the Pv/Pf test device. Five out of eight P. vivax cases were lower parasite density (<560 parasites/μL). The remaining three P. vivax cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by the Pf/Pan test device. In the subset of 350 samples examined by the Pv/Pf test device (mixed infection not included, Table \\n5), two P. falciparum cases with lower parasite density (<480 parasites/μL) were false negative. Nine P. vivax cases were the false negative, of which three had lower parasite density (<320 parasites/μL), and three cases (parasite density of 1,320/μL, 2080/μL, 7,280/μL) were positive by the Pf/Pan test device. The other three cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by both RDTs.\\nFalse positive and wrong species identification were observed with both RDTs. In all 606 samples (identified by microscopy and corrected by PCR, mixed infection not included) examined by the Pf/Pan test device, one P. vivax case, one P. ovale case, and eight negative cases showed the PfHRP2 line. Ten P. vivax cases showed not only the pan-pLDH line, but the PfHRP2 line. Seven negative cases showed both PfHRP2 and pan-pLDH lines (Table \\n7). In the subset of 350 samples analysed by the Pv/Pf test, two negative cases showed the PfHRP2 line. One P. falciparum case showed the Pv-pLDH line only. Two P. vivax cases showed not only the Pv-pLDH line, but the PfHRP2-line. Six P. falciparum cases (parasite density ranging from 240 to 34,400 parasites/μL) showed not only the PfHRP2 line, but also the Pv-pLDH line (Table \\n8). It has been reported that P. falciparum infections with high parasite densities may generate a false positive Pv-pLDH line\\n[22-25]. For the Pv/Pf test devices, six out of 40 P. falciparum (corrected by PCR) with the Pv-pLDH line visible showed that cross reactions between different species occurred although parasite densities of some P. falciparum infections were not high. From these comparisons, the specificities of these devices need further improvement for areas with both P. falciparum and P. vivax malaria.\\nSince P. falciparum and P. vivax infections are treated with different drugs, it would be important to compare the different methods for detecting mixed species infections. PCR, microscopy and Pv/Pf test device detected 21, 11, and eight mixed-species infections in the 350 subset samples (Table \\n8), corresponding to 18.6%, 11.2% and 8.9% of positive cases detected by the respective methods.\",\n \"RDTs are playing an essential role in the current malaria control/elimination campaign worldwide. In this study, the performance of two RDT devices commonly used at the China-Myanmar border area was evaluated. Compared to diagnosis by microscopy, both the Pf/Pan and Pv/Pf devices showed higher sensitivity (>87%) for detecting P. falciparum infections, whereas the sensitivity for detecting P. vivax infections was much lower (<74%). Both RDTs had similar levels of detection limits, and their performance was poor for infections with parasite densities below 500 parasites/μL for P. falciparum infections. Besides, since both devices rely on the PfHRP2 antigen for the detection of P. falciparum, pfhrp2 gene deletions will lead to false-negative results\\n[26]. A recent report showing up to 40% of P. falciparum parasites with pfhrp2 deletions in South America\\n[27] indicate that such RDTs would not perform well in these regions. It certainly warrants more detailed investigations in other malaria endemic regions.\\nWhen PCR was used as the gold standard, the detection sensitivity of both RDTs was reduced especially for the detection of P. vivax infections (<64%), although the specificity of the all detection methods remained above 92%. In addition, the sensitivity of conventional microscopy was also low, ranging from 71.0% to 77.4% for detecting both parasite species. This result is worrisome; since nearly 30% of the malaria cases was not correctly diagnosed by microscopy or RDTs and subsequently treated, these patients may constitute an important reservoir for transmission. Whereas low parasite density might be the most important limiting factor for microscopic diagnosis\\n[6,28], the varying skills and experience of microscopists may also be responsible for the lower sensitivity of diagnosis\\n[29]. In the past, the Pf/Pan test (Wondfo) has been evaluated for diagnosis of P. falciparum only and showed highly satisfactory result\\n[30]. Recently, a CareStart™ kit (Pf/Pan) was evaluated in the nearby Yunnan province of China, which showed comparable results to microscopy for diagnosing P. falciparum and P. vivax infections\\n[31]. Therefore, further evaluations of RDTs are needed to select better diagnostic tests with improved accuracy in this region.\\nOverall, both RDTs have good sensitivity for detecting P. falciparum infections, but the sensitivity for detecting P. vivax was much lower. This could be due to lower parasite density for P. vivax since this parasite selectively invades reticulocytes. However, comparison of the two RDTs showed that the sensitivities for P. vivax detection were not different between the high and low parasite densities (Table \\n6). This observation is difficult to explain and could be due to the lower number of infections analyzed in the <500 parasites/μL group. Furthermore, the arbitrary selection of 500 parasites/μL as the border line between high and low parasite densities may not be appropriate for P. vivax. In addition, the sensitivity to P. vivax may be affected by other factors. Therefore, better RDTs with significantly improved sensitivity for P. vivax are needed.\\nOne important criterion for the selection of RDTs in the GMS is the ability to detect other human malaria species in addition to P. falciparum. The advantage of the Pf/Pan test is that it is designed to detect four human parasite species, which co-exist in the GMS. However, this device failed to detect the two P. ovale infections, suggesting that further improvement in sensitivity for detecting other malaria species is needed. It is also worth to note that the PCR method failed to identify any P. knowlesi infections, which is in stark contrast to the eaerlier report of some 30% of malaria cases in this region as mixed infections with P. knowlesi[16]. Therefore, the significantly lower prevalence of other malaria parasite species in this region and the generally low sensitivity of the Pf/Pan test for diagnosis of P. ovale and P. malariae suggest that such an advantage is hardly of any importance in this region. In comparison, the Pv/Pf test is specific for P. falciparum and P. vivax, which make up the majority of malaria infections in the GMS, although it would certainly miss detecting P. malariae and P. ovale infections. Because P. falciparum and P. vivax require different antimalarial treatments, correct diagnosis of mixed infections should be an important criterion for the selection of an RDT. Even though the Pv/Pf device is able to detect P. falciparum and P. vivax mixed infection, the detection rate was low and the eight mix infections detected by this device were all misdiagnosed when compared to the PCR result. This result may be due to reduced density of one parasite species in the presence of another due to competition in the same host. Consequently, with the current anti-malarial drug policy of this region, misdiagnosis of mixed species infections as single species infections would lead to improper drug treatments.\",\n \"The aim of this study was to evaluate the performance of two RDTs for malaria diagnosis at the China-Myanmar border area. PCR method was much more sensitive than microscopy and RDTs. Compared to microscopy, both RDTs demonstrated similar sensitivities for detecting P. falciparum infections but reduced sensitivity for detecting P. vivax infections. Neither of them had satisfactory results in detecting mixed species infections. Therefore, methods with improved sensitivity for diagnosing P. vivax malaria are needed. False negative results for the diagnosis of P. falciparum malaria call for further investigations on potential deletion of pfhrp2 gene in this malaria-endemic region.\",\n \"The authors declare that they have no competing interests.\",\n \"YJ, LN, WX, LP and ZZ carried out the experimental work and data analysis.WL, LS and ZG participated in data analysis. YJ performed manuscript writing. YC, QF and LC conceived the study and participated in the design of the study. All authors read and approved the final manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,"results","discussion","conclusions",null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Rapid diagnostic tests (RDTs)","Malaria diagnosis","Microscopy","PCR","Sensitivity","Specificity"],"string":"[\n \"Rapid diagnostic tests (RDTs)\",\n \"Malaria diagnosis\",\n \"Microscopy\",\n \"PCR\",\n \"Sensitivity\",\n \"Specificity\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nMalaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually\n[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms\n[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease\n[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.\nMicroscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately\n[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific\n[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine\n[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings\n[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign\n[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.\nRDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results\n[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported\n[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria\n[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria.\n\nMethods:\n Study site and patients The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\nThe study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\n Microscopic examinations Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\nThick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\n RDTs for malaria The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\nThe RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\n Diagnosis by PCR Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\nFresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\n Statistical analysis The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.\nThe RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.\n\nStudy site and patients:\nThe study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\n\nMicroscopic examinations:\nThick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\n\nRDTs for malaria:\nThe RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\n\nDiagnosis by PCR:\nFresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\n\nStatistical analysis:\nThe RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.\n\nResults:\nBlood samples were collected from a total of 606 patients. All samples were evaluated by the Pf/Pan test device, while a subset of 350 were also evaluated by the Pv/Pf test device. Of the 606 samples, malaria parasites were found in 143 by microscopy; 51, 73, and 19 were P. falciparum, P. vivax and P. falciparum/P. vivax mixed infections, respectively (Table \n3). No other malaria parasite species were detected by microscopy. The parasite density ranges of P. falciparum were 40–105,920 parasites/μL. For P. vivax, the majority of cases had parasite density ranging from 80 to 17,800 parasites/μL. One P. vivax case had a deviant parasite density of >200,000 parasites/μL based on leucocyte number probably due to a low leucocyte count in this patient. Because P. falciparum single infections and mixed species infections containing P. falciparum cannot be differentiated by the Pf/Pan device, the microscopy results were grouped into “P. falciparum and mixed infections with P. falciparum” and “Non-falciparum”. Based on this interpretation, the Pf/Pan device detected 33 single P. falciparum infections (only the PfHRP2 line visible), 56 non-falciparum cases (only the pan-pLDH line visible) and 70 P. falciparum infections/potentially mixed infections (both lines visible) (Table \n3). Compared with the results by microscopy, the Pf/Pan device detected 62 and 51 samples as true positives for P. falciparum and non-falciparum, respectively. From this result, the sensitivity of Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4% (Table \n4).\nDetection results of malaria infections by the Malaria Pf/Pan test in comparison with microscopy (N=606)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\n\nPerformance of two RDTs for detection of \n\nP. falciparum \n\nand \n\nP. vivax \n\nwith microscopy as the gold standard\n\nData are presented as percentage (95% confidence interval; CI).\nFor the subset of 350 samples, microscopy detected 37 samples containing P. falciparum, 50 samples containing P. vivax and 11 were diagnosed as mixed species infections (Table \n5). Since the Pv/Pf device is able to differentiate P. falciparum and P. vivax infections, the microscopy results were grouped into “infections containing P. falciparum” and “infections containing P. vivax”. The Pv/Pf device detected 40 single P. falciparum infections, 42 P. vivax infections, and 8 mixed species infections (Table \n5). Compared to microscopy, the Pv/Pf device detected 44 and 45 samples as true positives for P. falciparum and P. vivax, respectively, whereas the Pf/Pan device detected 42 and 36 samples as true positives for P. falciparum and P. vivax, respectively. The sensitivity of Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively (Table \n4). The specificity of Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively (Table \n4).\nDetection results of malaria infections by Malaria Pv/Pf test and Malaria Pf/Pan test in comparison with the microscopic method (N=350)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nIt has been observed that some RDTs do not perform well when the parasite density is below 500 parasites/μL. For P. falciparum, both RDTs had significantly higher sensitivity for cases with a parasite density above 500 parasites/μL (>92%) than those with a density below 500 parasites/μL (<75.0%) (Table \n6). However, for P. vivax malaria, regardless of the parasite density, both RDTs displayed sensitivity of below 79%. Both RDTs had similar detection limits; they were >240 and >320 parasites/μL for P. falciparum and P. vivax, respectively.\n\nComparative sensitivity of the two RDTs for detection of \n\nP. falciparum \n\nand \n\nP. vivax \n\nin comparison with the microscopic method, categorized according to parasite density\n\nSensitivity is presented as percentage (95% confidence interval; CI). TP true positive.\nTo further evaluate the performance of these two RDTs, all 606 samples were examined by nested PCR. Nested PCR detected malaria parasites in 174 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. No P. knowlesi infections were detected by PCR (Table \n7). For the subset of 350 samples, 113 samples were identified by nested PCR as infection with malaria parasites. Of which, 40, 52, and 21 were P. falciparum, P. vivax, and P. falciparum/P. vivax mixed infections, respectively (Table \n8). Among all detection methods, PCR was the most sensitive one. If PCR was used as the gold standard, the sensitivity of microscopy for P. falciparum and P. vivax was 71.0% and 73.3% in 606 detected samples, and 75.4% and 74.0% in the 350 subset samples, respectively (Table \n9). The sensitivity of Pf/Pan test for P. falciparum and P. vivax was 81.7% and 64.6% in 606 detected samples, and 77.1% and 63.5% in the 350 subset samples, respectively (Table \n9). For the subset of 350 samples analyzed by Pv/Pf devices, the sensitivity was 72.1% for P. falciparum and 58.9% for P. vivax, respectively (Table \n9). Microscopy and the two RDTs had similar levels of specificity ranging from 94.7% to 96.3% (Table \n9). It is noteworthy that although the Pf/Pan device is designed to identify all human malaria parasite species, the two P. ovale infections identified by PCR were missed by this device and by microscopy, possibly due to the low parasitaemia in the P. ovale infections (containing 120 parasites/μL and 320 parasites/μL, respectively). Re-examination of the P. ovale slides by microscopy did detect P. ovale-parasitized erythrocytes (Figure \n1).\nBlood smears showing parasitized erythrocytes by P. ovale in two malaria patients. The two patients with febrile illness attended a malaria clinic at the China-Myanmar border and were diagnosed for malaria. Both cases were only detected by PCR but missed by microscopy and RDTs.\nDetection results of malaria infections by microscopy and Malaria Pf/Pan test in comparison with the Nested PCR (N=606)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nDetection results of malaria infections by microscopy, Malaria Pf/Pan test and Malaria Pv/Pf test in comparison with the Nested PCR (N=350)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nPerformance of different diagnosis methods with the nested PCR method as the gold standard\nData are presented as percentage (95% confidence interval; CI).\nAccording to the microscopy results corrected by PCR, four and eight cases were false negative in 606 samples examined by the Pf/Pan test device for P. falciparum and P. vivax, respectively (Table \n3). When mixed infections were excluded, three out of four P. falciparum cases had lower parasite density (<480 parasites/μL). Only one P. falciparum case with higher parasite density (8,800 parasites/μL) was not detected by the Pf/Pan test device, but was positive by the Pv/Pf test device. Five out of eight P. vivax cases were lower parasite density (<560 parasites/μL). The remaining three P. vivax cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by the Pf/Pan test device. In the subset of 350 samples examined by the Pv/Pf test device (mixed infection not included, Table \n5), two P. falciparum cases with lower parasite density (<480 parasites/μL) were false negative. Nine P. vivax cases were the false negative, of which three had lower parasite density (<320 parasites/μL), and three cases (parasite density of 1,320/μL, 2080/μL, 7,280/μL) were positive by the Pf/Pan test device. The other three cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by both RDTs.\nFalse positive and wrong species identification were observed with both RDTs. In all 606 samples (identified by microscopy and corrected by PCR, mixed infection not included) examined by the Pf/Pan test device, one P. vivax case, one P. ovale case, and eight negative cases showed the PfHRP2 line. Ten P. vivax cases showed not only the pan-pLDH line, but the PfHRP2 line. Seven negative cases showed both PfHRP2 and pan-pLDH lines (Table \n7). In the subset of 350 samples analysed by the Pv/Pf test, two negative cases showed the PfHRP2 line. One P. falciparum case showed the Pv-pLDH line only. Two P. vivax cases showed not only the Pv-pLDH line, but the PfHRP2-line. Six P. falciparum cases (parasite density ranging from 240 to 34,400 parasites/μL) showed not only the PfHRP2 line, but also the Pv-pLDH line (Table \n8). It has been reported that P. falciparum infections with high parasite densities may generate a false positive Pv-pLDH line\n[22-25]. For the Pv/Pf test devices, six out of 40 P. falciparum (corrected by PCR) with the Pv-pLDH line visible showed that cross reactions between different species occurred although parasite densities of some P. falciparum infections were not high. From these comparisons, the specificities of these devices need further improvement for areas with both P. falciparum and P. vivax malaria.\nSince P. falciparum and P. vivax infections are treated with different drugs, it would be important to compare the different methods for detecting mixed species infections. PCR, microscopy and Pv/Pf test device detected 21, 11, and eight mixed-species infections in the 350 subset samples (Table \n8), corresponding to 18.6%, 11.2% and 8.9% of positive cases detected by the respective methods.\n\nDiscussion:\nRDTs are playing an essential role in the current malaria control/elimination campaign worldwide. In this study, the performance of two RDT devices commonly used at the China-Myanmar border area was evaluated. Compared to diagnosis by microscopy, both the Pf/Pan and Pv/Pf devices showed higher sensitivity (>87%) for detecting P. falciparum infections, whereas the sensitivity for detecting P. vivax infections was much lower (<74%). Both RDTs had similar levels of detection limits, and their performance was poor for infections with parasite densities below 500 parasites/μL for P. falciparum infections. Besides, since both devices rely on the PfHRP2 antigen for the detection of P. falciparum, pfhrp2 gene deletions will lead to false-negative results\n[26]. A recent report showing up to 40% of P. falciparum parasites with pfhrp2 deletions in South America\n[27] indicate that such RDTs would not perform well in these regions. It certainly warrants more detailed investigations in other malaria endemic regions.\nWhen PCR was used as the gold standard, the detection sensitivity of both RDTs was reduced especially for the detection of P. vivax infections (<64%), although the specificity of the all detection methods remained above 92%. In addition, the sensitivity of conventional microscopy was also low, ranging from 71.0% to 77.4% for detecting both parasite species. This result is worrisome; since nearly 30% of the malaria cases was not correctly diagnosed by microscopy or RDTs and subsequently treated, these patients may constitute an important reservoir for transmission. Whereas low parasite density might be the most important limiting factor for microscopic diagnosis\n[6,28], the varying skills and experience of microscopists may also be responsible for the lower sensitivity of diagnosis\n[29]. In the past, the Pf/Pan test (Wondfo) has been evaluated for diagnosis of P. falciparum only and showed highly satisfactory result\n[30]. Recently, a CareStart™ kit (Pf/Pan) was evaluated in the nearby Yunnan province of China, which showed comparable results to microscopy for diagnosing P. falciparum and P. vivax infections\n[31]. Therefore, further evaluations of RDTs are needed to select better diagnostic tests with improved accuracy in this region.\nOverall, both RDTs have good sensitivity for detecting P. falciparum infections, but the sensitivity for detecting P. vivax was much lower. This could be due to lower parasite density for P. vivax since this parasite selectively invades reticulocytes. However, comparison of the two RDTs showed that the sensitivities for P. vivax detection were not different between the high and low parasite densities (Table \n6). This observation is difficult to explain and could be due to the lower number of infections analyzed in the <500 parasites/μL group. Furthermore, the arbitrary selection of 500 parasites/μL as the border line between high and low parasite densities may not be appropriate for P. vivax. In addition, the sensitivity to P. vivax may be affected by other factors. Therefore, better RDTs with significantly improved sensitivity for P. vivax are needed.\nOne important criterion for the selection of RDTs in the GMS is the ability to detect other human malaria species in addition to P. falciparum. The advantage of the Pf/Pan test is that it is designed to detect four human parasite species, which co-exist in the GMS. However, this device failed to detect the two P. ovale infections, suggesting that further improvement in sensitivity for detecting other malaria species is needed. It is also worth to note that the PCR method failed to identify any P. knowlesi infections, which is in stark contrast to the eaerlier report of some 30% of malaria cases in this region as mixed infections with P. knowlesi[16]. Therefore, the significantly lower prevalence of other malaria parasite species in this region and the generally low sensitivity of the Pf/Pan test for diagnosis of P. ovale and P. malariae suggest that such an advantage is hardly of any importance in this region. In comparison, the Pv/Pf test is specific for P. falciparum and P. vivax, which make up the majority of malaria infections in the GMS, although it would certainly miss detecting P. malariae and P. ovale infections. Because P. falciparum and P. vivax require different antimalarial treatments, correct diagnosis of mixed infections should be an important criterion for the selection of an RDT. Even though the Pv/Pf device is able to detect P. falciparum and P. vivax mixed infection, the detection rate was low and the eight mix infections detected by this device were all misdiagnosed when compared to the PCR result. This result may be due to reduced density of one parasite species in the presence of another due to competition in the same host. Consequently, with the current anti-malarial drug policy of this region, misdiagnosis of mixed species infections as single species infections would lead to improper drug treatments.\n\nConclusions:\nThe aim of this study was to evaluate the performance of two RDTs for malaria diagnosis at the China-Myanmar border area. PCR method was much more sensitive than microscopy and RDTs. Compared to microscopy, both RDTs demonstrated similar sensitivities for detecting P. falciparum infections but reduced sensitivity for detecting P. vivax infections. Neither of them had satisfactory results in detecting mixed species infections. Therefore, methods with improved sensitivity for diagnosing P. vivax malaria are needed. False negative results for the diagnosis of P. falciparum malaria call for further investigations on potential deletion of pfhrp2 gene in this malaria-endemic region.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nYJ, LN, WX, LP and ZZ carried out the experimental work and data analysis.WL, LS and ZG participated in data analysis. YJ performed manuscript writing. YC, QF and LC conceived the study and participated in the design of the study. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nRapid diagnostic tests (RDTs) have become an essential tool in the contemporary malaria control and management programmes in the world. This study aims to evaluate the performance of two commonly used RDTs for malaria diagnosis in the China-Myanmar border area.\n\nMethods:\nA total 606 febrile patients in the China-Myanmar border were recruited to this study and were diagnosed for malaria infections by microscopy, two RDTs tests (Pf/Pan device, and Pv/Pf device) and nested PCR.\n\nResults:\nMalaria parasites were found in 143 patients by microscopy, of which 51, 73, and 19 were Plasmodium falciparum, Plasmodium vivax and P. falciparum/P. vivax mixed infections, respectively. Compared to microscopy, the sensitivity of the Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4%. For a subset of 350 patients, the sensitivity of the Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively. The specificity of the Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively. Nested PCR detected malaria parasites in 174 of 606 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. Compared to nested PCR, all other methods had sensitivity below 80%, suggesting that a significant number of cases were missed.\n\nConclusions:\nCompared to PCR, both microscopy and RDTs had lower sensitivities. RDTs had similar performance to microscopy for P. falciparum diagnosis, but performed worse for P. vivax diagnosis. Other RDT products should be selected with higher sensitivity (and good specificity) for both P. falciparum and P. vivax diagnosis."},"background_conclusion_text":{"kind":"string","value":"Background:\nMalaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually\n[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms\n[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease\n[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.\nMicroscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately\n[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific\n[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine\n[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings\n[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign\n[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.\nRDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results\n[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported\n[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria\n[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria.\n\nConclusions:\nThe aim of this study was to evaluate the performance of two RDTs for malaria diagnosis at the China-Myanmar border area. PCR method was much more sensitive than microscopy and RDTs. Compared to microscopy, both RDTs demonstrated similar sensitivities for detecting P. falciparum infections but reduced sensitivity for detecting P. vivax infections. Neither of them had satisfactory results in detecting mixed species infections. Therefore, methods with improved sensitivity for diagnosing P. vivax malaria are needed. False negative results for the diagnosis of P. falciparum malaria call for further investigations on potential deletion of pfhrp2 gene in this malaria-endemic region."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nRapid diagnostic tests (RDTs) have become an essential tool in the contemporary malaria control and management programmes in the world. This study aims to evaluate the performance of two commonly used RDTs for malaria diagnosis in the China-Myanmar border area.\n\nMethods:\nA total 606 febrile patients in the China-Myanmar border were recruited to this study and were diagnosed for malaria infections by microscopy, two RDTs tests (Pf/Pan device, and Pv/Pf device) and nested PCR.\n\nResults:\nMalaria parasites were found in 143 patients by microscopy, of which 51, 73, and 19 were Plasmodium falciparum, Plasmodium vivax and P. falciparum/P. vivax mixed infections, respectively. Compared to microscopy, the sensitivity of the Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4%. For a subset of 350 patients, the sensitivity of the Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively. The specificity of the Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively. Nested PCR detected malaria parasites in 174 of 606 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. Compared to nested PCR, all other methods had sensitivity below 80%, suggesting that a significant number of cases were missed.\n\nConclusions:\nCompared to PCR, both microscopy and RDTs had lower sensitivities. RDTs had similar performance to microscopy for P. falciparum diagnosis, but performed worse for P. vivax diagnosis. Other RDT products should be selected with higher sensitivity (and good specificity) for both P. falciparum and P. vivax diagnosis."},"whole_article_text_length":{"kind":"number","value":7424,"string":"7,424"},"whole_article_abstract_length":{"kind":"number","value":360,"string":"360"},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["falciparum","malaria","pf","vivax","infections","test","pcr","pan","device","rdts"],"string":"[\n \"falciparum\",\n \"malaria\",\n \"pf\",\n \"vivax\",\n \"infections\",\n \"test\",\n \"pcr\",\n \"pan\",\n \"device\",\n \"rdts\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Child | Child, Preschool | China | Clinical Laboratory Techniques | Diagnostic Tests, Routine | Female | Humans | Infant | Malaria | Male | Middle Aged | Myanmar | Parasitology | Plasmodium falciparum | Plasmodium ovale | Plasmodium vivax | Sensitivity and Specificity | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Child | Child, Preschool | China | Clinical Laboratory Techniques | Diagnostic Tests, Routine | Female | Humans | Infant | Malaria | Male | Middle Aged | Myanmar | Parasitology | Plasmodium falciparum | Plasmodium ovale | Plasmodium vivax | Sensitivity and Specificity | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Child | Child, Preschool | China | Clinical Laboratory Techniques | Diagnostic Tests, Routine | Female | Humans | Infant | Malaria | Male | Middle Aged | Myanmar | Parasitology | Plasmodium falciparum | Plasmodium ovale | Plasmodium vivax | Sensitivity and Specificity | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Aged 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[SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] malaria | vivax | plasmodium | diagnosis | areas | rdts | falciparum | specific | endemic | different [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | blood | falciparum | pcr | pf | malaria | positive | test device | min | pan [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] falciparum | pf | infections | device | vivax | samples | pan | detected | cases | μl [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] malaria | detecting | infections | microscopy rdts | rdts | sensitivity | diagnosis | vivax | microscopy | rdts demonstrated [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] falciparum | malaria | pf | test | vivax | infections | rdts | blood | parasite | pcr [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] falciparum | malaria | pf | test | vivax | infections | rdts | blood | parasite | pcr [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| two | China | Myanmar [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 606 | China | Myanmar | two | Pv/Pf | PCR [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria | 143 | 51 | 73 | 19 | Plasmodium | Plasmodium | P. ||| the Pf/Pan | 88.6% | 69.9% | 90.4% ||| 350 | the Pf/Pan | Pv/Pf | 87.5% | 91.7% | 72.0% | 73.8% ||| Pv/Pf | 94.3% | 96.5% ||| 174 | 606 | 67 | 79 | two | 26 ||| PCR | 80% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] PCR ||| P. ||| RDT | P. [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| two | China | Myanmar ||| 606 | China | Myanmar | two | Pv/Pf | PCR ||| ||| Malaria | 143 | 51 | 73 | 19 | Plasmodium | Plasmodium | P. ||| the Pf/Pan | 88.6% | 69.9% | 90.4% ||| 350 | the Pf/Pan | Pv/Pf | 87.5% | 91.7% | 72.0% | 73.8% ||| Pv/Pf | 94.3% | 96.5% ||| 174 | 606 | 67 | 79 | two | 26 ||| PCR | 80% ||| PCR ||| P. ||| RDT | P. [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| two | China | Myanmar ||| 606 | China | Myanmar | two | Pv/Pf | PCR ||| ||| Malaria | 143 | 51 | 73 | 19 | Plasmodium | Plasmodium | P. ||| the Pf/Pan | 88.6% | 69.9% | 90.4% ||| 350 | the Pf/Pan | Pv/Pf | 87.5% | 91.7% | 72.0% | 73.8% ||| Pv/Pf | 94.3% | 96.5% ||| 174 | 606 | 67 | 79 | two | 26 ||| PCR | 80% ||| PCR ||| P. ||| RDT | P. [SUMMARY]"}}},{"rowIdx":266,"cells":{"title":{"kind":"string","value":"Time from Self-Detection of Symptoms to Seeking Definitive Care among Cervical Cancer Patients."},"pmid":{"kind":"string","value":"33247688"},"background_abstract":{"kind":"string","value":"India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one-third of global cervical cancer deaths during the year 2018. Cervical cancer is the leading cause of cancer mortality in India. The present study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking care and different barriers for the possible time lag in seeking care."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A cross-sectional study was undertaken from April 2017 to September 2017 in a regional cancer centre in the south of India. The centre has both a population and a hospital-based cancer registry. Cervical cancer cases (N= 210) with histological confirmation were interviewed at the hospital using a pre-tested semi-structured questionnaire."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The median time interval between the self-detection of cervical cancer symptoms and first contact with the general physician was 80 [IQR 45-150] days. The overall median time interval between the self-detection of symptoms to the initiation of primary treatment was 123[IQR 83-205] days. The major perceived reason for not seeking medical care was a lack of awareness in identifying cervical cancer symptoms in 183(92.9%) women."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The median time of 80 days was observed from the self-detection of cervical cancer symptoms to the first contact with a general physician. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care.
."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Cross-Sectional Studies","Female","Follow-Up Studies","Health Knowledge, Attitudes, Practice","Humans","India","Middle Aged","Patient Acceptance of Health Care","Prognosis","Retrospective Studies","Self-Assessment","Surveys and Questionnaires","Time Factors","Time-to-Treatment","Uterine Cervical Neoplasms"],"string":"[\n \"Cross-Sectional Studies\",\n \"Female\",\n \"Follow-Up Studies\",\n \"Health Knowledge, Attitudes, Practice\",\n \"Humans\",\n \"India\",\n \"Middle Aged\",\n \"Patient Acceptance of Health Care\",\n \"Prognosis\",\n \"Retrospective Studies\",\n \"Self-Assessment\",\n \"Surveys and Questionnaires\",\n \"Time Factors\",\n \"Time-to-Treatment\",\n \"Uterine Cervical Neoplasms\"\n]"},"pmcid":{"kind":"string","value":"8033105"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Globally, an estimated 570,000 new cases and 311,000 deaths has been reported due to cervical cancer in 2018 and it is the fourth most common cancer in women. The Incidence and mortality rank second behind breast cancer in lower human development index (HDI) settings. Eight out of 10 women diagnosed and nine of 10 women who die from cervical cancer live in a low- or middle-income countries (Bray et al., 2018) “ Cervical cancer continues to be a major public health problem affecting middle-aged women, particularly in less-resourced countries” (Arbyn et al., 2020). India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one third of global cervical cancer deaths during the year 2018 (“Globocan 2018,” 2018). Cervical cancer is the leading cause of cancer mortality in India which accounts for 17% of all cancer deaths among women aged 30 to 69 years.(Bobdey et al., 2016, “The Challenge Ahead,” 2014) One third of cervical cancer cases was reported among women aged 50 -59 years ( 27.4%) (Bobdey et al., 2016). The higher mortality and morbidity due to cancer is because most patients seek/get treatment at advanced stage and therefore have a higher risk of dying from the disease (Bray et al., 2018).\nCervical cancer is a preventable disease primarily because of its long lead time (women develop precancerous lesions years before it becomes cancerous). Cervical cancer can be detected in the pre-cancerous phase by screening and thereby the development of cancer prevented. Cervical cancer can also be treated successfully if diagnosed at an early stage. Routine screening by visual inspection of cervix through ‘See-and-treat’ approach among the female population has been suggested for early diagnosis and control of cervical cancer (Sankaranarayanan, 2012). Periodic Pap or HPV samples are difficult to organize in low- or middle-income countries like India and therefore large-scale routine screening would be difficult and not affordable (“HTA_CaCx Screening in India.pdf,” n.d.) Millions of cancer patients could be saved from premature death and disability, if they had timely access to early detection and treatment (“WHO,” n.d.). Studies have shown that it is possible to reduce the need of aggressive treatment of cancer is detected at an early stage. A long interval between start of disease symptoms to having a definite diagnosis is highly critical and associated with low survival among cervical cancer patients.(Chen et al., 2019) \nIn India, the present existing public health care delivery system is a three-level system. The primary level is primary health centres (PHCs). In India, PHCs focuses on eight elements of primary health care outlined in the Alma-Ata declaration and provides mainly outpatient services. In the secondary level the district hospitals provide outpatient, inpatient and emergency services. At the tertiary level, medical college hospitals, apex institutions and regional cancer centres provide specialized services. Under the programme of “National programme for prevention and control of cancer, diabetes, cardiovascular diseases and stroke (NPCDCS)”, cancer screening is done at the primary level by visual inspection of the cervix with acetic acid (VIA) and those who are suspected for cancer are referred to secondary or tertiary care level for further management. In addition, a health programme for non-communicable diseases is carried out with a cancer care component implemented at all the health care levels (Gulia et al., 2016) \nThis study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking for care and different barriers for the possible time lag in seeking care."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The distribution of socio-demographic and clinical data of the 210 cervical cancer study subjects are presented along with data pertaining to the cases not enrolled for analysis (Table 1). Among 210 enrolled patients, the majority 106 (50.5%) of women were between the age 46 to 59 years where in not enrolled patients 89 (40.4%) were of same age group. It was noted 167 (63.0%) and 179 (81.4%) were not literate among enrolled and respectively. Among enrolled patients, 150 (71.4%) were from rural areas and 158 (71.4%) were financially dependent for livelihood on the family. Only 19 women (9.0%) had any health insurance policy. The majority of patients 197 (93.8%) were not aware of symptoms of cervical cancer and only 7 (3.3%) patients had a family history of cancer.\nOf the symptoms which led for seeking medical care, the majority of women, 172 (81.9%), indicated lower abdominal pain, and 137 (65.2%) of the respondents experienced abnormal vaginal discharge/bleeding. Among all patients, 106 (50.5%) were diagnosed at an early stage of the disease (stages I, IIA and IIB).\nThe median time interval between self-detection of cervical cancer symptoms and first contact with general physician (T1) was 80 [IQR 45-150] days, while 74 patients (35.2%) had a time lag of 90 days or more. The median time taken for reaching diagnosis after the first contact (T2) was 25 [IQR 15 - 45] days. The overall median time between self-detection of symptoms to initiation of primary treatment (T5) was 123[IQR 83-205] days. The majority of patients, 145 (66.2%), had initiation of primary treatment only after 90 days from self-detection of cervical cancer symptoms (Table 2). \nFactors associated with delay in seeking medical care of 90 days and more (patient delay) were analyzed in relation to socio-demographic characteristics and clinical symptoms (Table 3). In multivariate analysis after adjusting to all the variables in the study the odds of patient delay in the age group ≥ 60 years was 3.2 times higher compared to patients in the age group below 45 years (95% CI: 1.2 – 8.4). The other factors which were associated with patient delay were being not literate (OR 2.3, 95% CI: 1.0 – 5.4), from rural area (OR = 2.3, 95% CI: 1.1 – 4.9), being financially dependent for livelihood (OR = 4.5, 95% CI: 1.9 – 10.6) and not having any abnormal vaginal discharge / bleeding (OR = 2.4, 95% CI: 1.3 – 4.6). Family history of cancer, presence of any health insurance, knowledge regarding cancer cure by early seeking for care, knowledge of symptoms of cervical cancer and having lower abdominal pain was not statistically related to delay in seeking medical care. \nInformation was sought regarding perceived reasons from 197 (93.8%) patients who did not seek care within one week from self-detection of cancer symptoms. The major reasons (table 4) were lack of awareness in identifying cervical cancer symptoms in 183 (92.9%) women and 120 (60.9%) patients assuming that the symptom would resolve by itself. The other common reasons were absence of pain, changes in the body attributed to common illness and vague symptoms. \nThe most common reason for delay in seeking for definite diagnosis at tertiary care hospitals was due to non-affordability 82 (41.6%). The other common reasons were visiting multiple medical practitioners before presenting at tertiary care 60 (30.1%) and not suspected for cancer by physician at first contact. \nThe delay in seeking for treatment after diagnosis at tertiary care was due to long treatment procedure 57 (27.1%), fear of treatment 50 (23.8%), financial dependence on the family 48 (22.9%), fear of disfigurement of the body 26 (12.4%) and stigma attached with the disease (Table 4).\nDistribution of Socio-Demographic, Clinical and Other Variables Among the Subject Enrolled and Not Enrolled to the Study\nDistribution of Time Interval Between Self-Detection of Cancer Symptoms to Various Stages (T1 To T5) for Seeking Treatment\nFactors Associated with Delay in Seeking Medical Care: Univariate and Multivariate Logistic Regression Results\nDistribution of Perceived Reasons for Delay in Seeking Medical Care at Various Stages (T1 – T4)\n*, Responses were elicited from those who had not sought specific care for more than one week from the onset of symptoms; #, Responses was elicited from those who had not sought treatment on specified time by doctor \nRecruitment of Participants for the Study"},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":[],"string":"[]"},"other_sections_texts":{"kind":"list like","value":[],"string":"[]"},"other_sections_sec_types":{"kind":"list like","value":[],"string":"[]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and Methods","Results","Discussion"],"string":"[\n \"Introduction\",\n \"Materials and Methods\",\n \"Results\",\n \"Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Globally, an estimated 570,000 new cases and 311,000 deaths has been reported due to cervical cancer in 2018 and it is the fourth most common cancer in women. The Incidence and mortality rank second behind breast cancer in lower human development index (HDI) settings. Eight out of 10 women diagnosed and nine of 10 women who die from cervical cancer live in a low- or middle-income countries (Bray et al., 2018) “ Cervical cancer continues to be a major public health problem affecting middle-aged women, particularly in less-resourced countries” (Arbyn et al., 2020). India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one third of global cervical cancer deaths during the year 2018 (“Globocan 2018,” 2018). Cervical cancer is the leading cause of cancer mortality in India which accounts for 17% of all cancer deaths among women aged 30 to 69 years.(Bobdey et al., 2016, “The Challenge Ahead,” 2014) One third of cervical cancer cases was reported among women aged 50 -59 years ( 27.4%) (Bobdey et al., 2016). The higher mortality and morbidity due to cancer is because most patients seek/get treatment at advanced stage and therefore have a higher risk of dying from the disease (Bray et al., 2018).\nCervical cancer is a preventable disease primarily because of its long lead time (women develop precancerous lesions years before it becomes cancerous). Cervical cancer can be detected in the pre-cancerous phase by screening and thereby the development of cancer prevented. Cervical cancer can also be treated successfully if diagnosed at an early stage. Routine screening by visual inspection of cervix through ‘See-and-treat’ approach among the female population has been suggested for early diagnosis and control of cervical cancer (Sankaranarayanan, 2012). Periodic Pap or HPV samples are difficult to organize in low- or middle-income countries like India and therefore large-scale routine screening would be difficult and not affordable (“HTA_CaCx Screening in India.pdf,” n.d.) Millions of cancer patients could be saved from premature death and disability, if they had timely access to early detection and treatment (“WHO,” n.d.). Studies have shown that it is possible to reduce the need of aggressive treatment of cancer is detected at an early stage. A long interval between start of disease symptoms to having a definite diagnosis is highly critical and associated with low survival among cervical cancer patients.(Chen et al., 2019) \nIn India, the present existing public health care delivery system is a three-level system. The primary level is primary health centres (PHCs). In India, PHCs focuses on eight elements of primary health care outlined in the Alma-Ata declaration and provides mainly outpatient services. In the secondary level the district hospitals provide outpatient, inpatient and emergency services. At the tertiary level, medical college hospitals, apex institutions and regional cancer centres provide specialized services. Under the programme of “National programme for prevention and control of cancer, diabetes, cardiovascular diseases and stroke (NPCDCS)”, cancer screening is done at the primary level by visual inspection of the cervix with acetic acid (VIA) and those who are suspected for cancer are referred to secondary or tertiary care level for further management. In addition, a health programme for non-communicable diseases is carried out with a cancer care component implemented at all the health care levels (Gulia et al., 2016) \nThis study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking for care and different barriers for the possible time lag in seeking care.","The data was collected from a regional cancer centre from south India, Bengaluru, which has a population-based cancer registry (PBCR) and a hospital-based cancer registry (HBCR). This tertiary care centre has state of art facilities for diagnosis, treatment and research. Cervical cancer cases with histological confirmation were recruited from the hospital-based cancer registry of this Institution during the time period of April 2017 to September 2017 using cross sectional study design. We identified 430 cervical cancer patients during the period from April 2017 to September 2017. Of these, 52 were ineligible due to referral to terminal/palliative care. Of 378 eligible women, 96 were not included for the analysis as patients moved to different hospital or not being available for interview, and 38 were not willing to participate in the study. In the 244 patients who gave their consent, 34 patient’s had incomplete information in the records. Finally, 210 patients were included for the present study (Figure 1) \nThe cervical cancers were mostly squamous cell carcinomas (SCC) and few adenocarcinomas (AC). They were divided in two stage based on FIGO classification (Bhatla et al., 2019). Stage I and II were categorized as early stage; III and IV were considered as late stage of cervical cancer. \nPatient delay in seeking for medical care refers to a long interval between detecting of first/initial symptoms and seeking for appointment with a general physician. The patients were further divided into two groups, those who sought medical care within 90 days and those who sought care 90 days and above (patient delay) after self-detection of the first symptoms of cervical cancer. \nSample size was calculated based on the study conducted by Rudd et al., (2017) where 39.3% patients had delayed diagnosis from the onset of self-detection of cervical cancer symptoms. In this study the sample size was estimated for patient related delay, from symptoms to first contact with a general practitioner, as it was our major concern. To get an absolute precision of 6.6% with 95% confidence level, the study required a minimum sample of 210 subjects. Based on hospital record collection, it was noted that six months’ time period was sufficient to achieve the required sample size. \nExpert opinion from oncologists, biostatisticians and epidemiologists were obtained using the Delphi technique (Skulmoski et al., 2007). After obtaining the written informed consent, patients were interviewed in person using the questionnaire and their medical records was used to collect the baseline information as well as key components of the time interval between self-detection of cervical cancer symptom to seeking medical care. More details of the collection of data and the different components of time intervals are described elsewhere.(Somanna et al., 2020)\nIndependent variables included socio-demographic characteristics, such as age at diagnosis, availability of health insurance, place of residence (rural/urban), history of cancer literacy, and financially dependent for livelihood and clinical variables, such as personal history of cancer, and family history of cancer.\nThe time interval between various stages was measured in days as reported by the patients and by review of hospital case sheets as indicated in earlier study.(Somanna et al., 2018) \na) Time interval between self-detection of symptoms and the first presentation at general practitioner (T1). \nb) Time interval from referral by the general practitioner to attending tertiary care centre (T2). \nc) Time interval from first consultation at tertiary care for diagnosis to definite diagnosis date (T3). \nd) Time interval from definite diagnosis to initiation of primary treatment (T4). \ne) Total time elapsed from self-detection of symptoms to initiation of primary treatment (T5).\n\nStatistical methods\n\nDescriptive statistics of time intervals were summarized with median and inter-quartile range as data were not normally distributed (Kolmogrov-Smirnov and Shapiro test). Univariate odds ratios (OR) with 95% confidence intervals (CI) were estimated between the factors and patient delay. The patient delay was categorised into < 90 days and ≥ 90 days. Considering ≥ 90 days as patient delay, the association of various factors were estimated considering the low risk group as the reference. Variables showing a univariate association with patient delay (at P < 0.20) were included in multivariate unconditional logistic stepwise forward regression model to adjust for confounders. The analysis was carried out using SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago. \nEthical approval was sought from the ethical review committee of the study Institution. ","The distribution of socio-demographic and clinical data of the 210 cervical cancer study subjects are presented along with data pertaining to the cases not enrolled for analysis (Table 1). Among 210 enrolled patients, the majority 106 (50.5%) of women were between the age 46 to 59 years where in not enrolled patients 89 (40.4%) were of same age group. It was noted 167 (63.0%) and 179 (81.4%) were not literate among enrolled and respectively. Among enrolled patients, 150 (71.4%) were from rural areas and 158 (71.4%) were financially dependent for livelihood on the family. Only 19 women (9.0%) had any health insurance policy. The majority of patients 197 (93.8%) were not aware of symptoms of cervical cancer and only 7 (3.3%) patients had a family history of cancer.\nOf the symptoms which led for seeking medical care, the majority of women, 172 (81.9%), indicated lower abdominal pain, and 137 (65.2%) of the respondents experienced abnormal vaginal discharge/bleeding. Among all patients, 106 (50.5%) were diagnosed at an early stage of the disease (stages I, IIA and IIB).\nThe median time interval between self-detection of cervical cancer symptoms and first contact with general physician (T1) was 80 [IQR 45-150] days, while 74 patients (35.2%) had a time lag of 90 days or more. The median time taken for reaching diagnosis after the first contact (T2) was 25 [IQR 15 - 45] days. The overall median time between self-detection of symptoms to initiation of primary treatment (T5) was 123[IQR 83-205] days. The majority of patients, 145 (66.2%), had initiation of primary treatment only after 90 days from self-detection of cervical cancer symptoms (Table 2). \nFactors associated with delay in seeking medical care of 90 days and more (patient delay) were analyzed in relation to socio-demographic characteristics and clinical symptoms (Table 3). In multivariate analysis after adjusting to all the variables in the study the odds of patient delay in the age group ≥ 60 years was 3.2 times higher compared to patients in the age group below 45 years (95% CI: 1.2 – 8.4). The other factors which were associated with patient delay were being not literate (OR 2.3, 95% CI: 1.0 – 5.4), from rural area (OR = 2.3, 95% CI: 1.1 – 4.9), being financially dependent for livelihood (OR = 4.5, 95% CI: 1.9 – 10.6) and not having any abnormal vaginal discharge / bleeding (OR = 2.4, 95% CI: 1.3 – 4.6). Family history of cancer, presence of any health insurance, knowledge regarding cancer cure by early seeking for care, knowledge of symptoms of cervical cancer and having lower abdominal pain was not statistically related to delay in seeking medical care. \nInformation was sought regarding perceived reasons from 197 (93.8%) patients who did not seek care within one week from self-detection of cancer symptoms. The major reasons (table 4) were lack of awareness in identifying cervical cancer symptoms in 183 (92.9%) women and 120 (60.9%) patients assuming that the symptom would resolve by itself. The other common reasons were absence of pain, changes in the body attributed to common illness and vague symptoms. \nThe most common reason for delay in seeking for definite diagnosis at tertiary care hospitals was due to non-affordability 82 (41.6%). The other common reasons were visiting multiple medical practitioners before presenting at tertiary care 60 (30.1%) and not suspected for cancer by physician at first contact. \nThe delay in seeking for treatment after diagnosis at tertiary care was due to long treatment procedure 57 (27.1%), fear of treatment 50 (23.8%), financial dependence on the family 48 (22.9%), fear of disfigurement of the body 26 (12.4%) and stigma attached with the disease (Table 4).\nDistribution of Socio-Demographic, Clinical and Other Variables Among the Subject Enrolled and Not Enrolled to the Study\nDistribution of Time Interval Between Self-Detection of Cancer Symptoms to Various Stages (T1 To T5) for Seeking Treatment\nFactors Associated with Delay in Seeking Medical Care: Univariate and Multivariate Logistic Regression Results\nDistribution of Perceived Reasons for Delay in Seeking Medical Care at Various Stages (T1 – T4)\n*, Responses were elicited from those who had not sought specific care for more than one week from the onset of symptoms; #, Responses was elicited from those who had not sought treatment on specified time by doctor \nRecruitment of Participants for the Study","The study centre was a regional cancer care centre having both population and hospital-based cancer registry and where a majority of cervix cancer patients from the entire state seek cancer care. The median age in enrolled and not enrolled patients was 50 and 56 years, respectively. Among all the patients, the proportion of not literate patients was higher (81%) in not enrolled patients compared to enrolled patients (63%). The study by Deshmukh and Rathod, (2017) observed that the average age of the patients was 57 years and 81% were not literate. Our result concur with the study by Panda et al., 2019 where the average age of the study participants was 52.1 ± 10.9 years. \nThe overall median time between the self-detection of cervical cancer symptoms and initiation of primary treatment was 123[IQR 83-205] days and the median time taken for first contact with general physician from self-detection of symptoms was 80 [IQR 45-150] days. Overall, 66% women delayed (≥ 90 days) for initiation of primary treatment from the onset of symptoms which is potentially concerning, because longer duration between diagnosis and treatment is related with higher risk of death. Among patients who started their treatment after an interval of >180 days, the risk of death was reported to be 36% higher compared to those who started treatment within 6 months from diagnosis (Chen et al., 2019) \nThe present study assessed the time interval between self-detection of symptoms to treatment of cervical cancer and patient delay. The median time interval between self-detection of symptoms and the first presentation at general practitioner (T1) was 80 days with 35.4% of the patients reporting 90 days or more. The present study observation was consistent with a study conducted in India on patients attending tertiary care centre, showing that 39% of patients had a diagnostic delay (Panda et al., 2019) The median time between symptoms and presenting at health care provider was 69 days from two tertiary care centres in Nepal (Gyenwali et al., 2014). It was noted from the study by Lim et al., (2014) that only 28% of patients had a delay of more than 3 months in all national health service (NHS) hospitals diagnosing cervical cancer in England. There was a mean delay of 23.1 weeks between onset of symptoms from the study in tertiary care centre of Sothern Malawi (Rudd et al., 2017). Results from our study were in line with studies from low- or middle-income countries but differed from studies in developed countries. This difference may be partially due to regular screening programs in many developed countries and differing rates of literacy (IARC, 2005). \nOverall median time from self-detection of symptoms to initiation of primary treatment (T5) was 123 days in the current study. Another study by Henriques, (2016) in India showed on an average six months duration from first symptom to cervical cancer treatment. Such a long time may be explained by the fact that lack of awareness of patients pertaining to cancer symptoms was high (93%) in present study. In another study, women who delayed, had mistaken the symptoms of cervix cancer as symptoms of sexually transmitted diseases (STD) and were scared to report (Deshmukh and Rathod, 2017). This shows that the lack of awareness of cervix cancer symptoms is a major factor and warrants improvement in public awareness. Our study results concur closely with other studies from similar settings and in low- or middle-income countries (Benemariya et al., 2018; Gyenwali et al., 2014).\nThe patients who were aged 60 years and above, residing in rural areas, not literate, financially dependent for livelihood and did not had abnormal vaginal discharge/ bleeding were independently associated with patient delay. Similar results were observed in a study from Nepal where not literate was found to be independently associated with late diagnosis and women having abnormal vaginal bleeding as symptom was protective factor for late diagnosis (Gyenwali et al., 2013). Previous studies from low- or middle-income countries also concur with our result (Dunyo et al., 2018)(Ouasmani et al., 2016)(Henriques, 2016).\nThe present study has been conducted in a population-based cancer registry (PBCR) where the data are included in International Agency for Research on Cancer (IARC) and data collection procedure involves standard quality control measures. The study included only patients who had been diagnosed with standard clinical and histopathological criteria. The information was obtained using pre-tested validated questionnaire by the investigator. Additional information pertaining to clinical evaluation and histological diagnosis were obtained from case sheets. All efforts were made to reduce the recall bias by local events calendar and standardization of terms employed in the questionnaire. \n\nLimitations\n\nPossible selection bias could have occurred as the subjects who were not enrolled for the study due to various reasons were older women and not literate compared to subjects in the study. Socio-demographic profile of population who availed the services at this centre may be different from private tertiary cancer care centre which would biases the result. The sample in study represent only those who availed the treatment at the centre and does not represent the entire population of cervical cancer patients. Thus, it may have some restrictions for external generalization. Possibility of recall bias exist as past history of cancer symptoms were assessed retrospectively. \nIn conclusion, the median time of 80 days was observed from self-detection of cervical cancer symptoms to first contact with general physician. Further, a median time of another 45 days was observed for obtaining primary treatment. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care. Interventions like raising awareness of cervical cancer symptoms and improving health seeking behavior of women should be focused."],"string":"[\n \"Globally, an estimated 570,000 new cases and 311,000 deaths has been reported due to cervical cancer in 2018 and it is the fourth most common cancer in women. The Incidence and mortality rank second behind breast cancer in lower human development index (HDI) settings. Eight out of 10 women diagnosed and nine of 10 women who die from cervical cancer live in a low- or middle-income countries (Bray et al., 2018) “ Cervical cancer continues to be a major public health problem affecting middle-aged women, particularly in less-resourced countries” (Arbyn et al., 2020). India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one third of global cervical cancer deaths during the year 2018 (“Globocan 2018,” 2018). Cervical cancer is the leading cause of cancer mortality in India which accounts for 17% of all cancer deaths among women aged 30 to 69 years.(Bobdey et al., 2016, “The Challenge Ahead,” 2014) One third of cervical cancer cases was reported among women aged 50 -59 years ( 27.4%) (Bobdey et al., 2016). The higher mortality and morbidity due to cancer is because most patients seek/get treatment at advanced stage and therefore have a higher risk of dying from the disease (Bray et al., 2018).\\nCervical cancer is a preventable disease primarily because of its long lead time (women develop precancerous lesions years before it becomes cancerous). Cervical cancer can be detected in the pre-cancerous phase by screening and thereby the development of cancer prevented. Cervical cancer can also be treated successfully if diagnosed at an early stage. Routine screening by visual inspection of cervix through ‘See-and-treat’ approach among the female population has been suggested for early diagnosis and control of cervical cancer (Sankaranarayanan, 2012). Periodic Pap or HPV samples are difficult to organize in low- or middle-income countries like India and therefore large-scale routine screening would be difficult and not affordable (“HTA_CaCx Screening in India.pdf,” n.d.) Millions of cancer patients could be saved from premature death and disability, if they had timely access to early detection and treatment (“WHO,” n.d.). Studies have shown that it is possible to reduce the need of aggressive treatment of cancer is detected at an early stage. A long interval between start of disease symptoms to having a definite diagnosis is highly critical and associated with low survival among cervical cancer patients.(Chen et al., 2019) \\nIn India, the present existing public health care delivery system is a three-level system. The primary level is primary health centres (PHCs). In India, PHCs focuses on eight elements of primary health care outlined in the Alma-Ata declaration and provides mainly outpatient services. In the secondary level the district hospitals provide outpatient, inpatient and emergency services. At the tertiary level, medical college hospitals, apex institutions and regional cancer centres provide specialized services. Under the programme of “National programme for prevention and control of cancer, diabetes, cardiovascular diseases and stroke (NPCDCS)”, cancer screening is done at the primary level by visual inspection of the cervix with acetic acid (VIA) and those who are suspected for cancer are referred to secondary or tertiary care level for further management. In addition, a health programme for non-communicable diseases is carried out with a cancer care component implemented at all the health care levels (Gulia et al., 2016) \\nThis study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking for care and different barriers for the possible time lag in seeking care.\",\n \"The data was collected from a regional cancer centre from south India, Bengaluru, which has a population-based cancer registry (PBCR) and a hospital-based cancer registry (HBCR). This tertiary care centre has state of art facilities for diagnosis, treatment and research. Cervical cancer cases with histological confirmation were recruited from the hospital-based cancer registry of this Institution during the time period of April 2017 to September 2017 using cross sectional study design. We identified 430 cervical cancer patients during the period from April 2017 to September 2017. Of these, 52 were ineligible due to referral to terminal/palliative care. Of 378 eligible women, 96 were not included for the analysis as patients moved to different hospital or not being available for interview, and 38 were not willing to participate in the study. In the 244 patients who gave their consent, 34 patient’s had incomplete information in the records. Finally, 210 patients were included for the present study (Figure 1) \\nThe cervical cancers were mostly squamous cell carcinomas (SCC) and few adenocarcinomas (AC). They were divided in two stage based on FIGO classification (Bhatla et al., 2019). Stage I and II were categorized as early stage; III and IV were considered as late stage of cervical cancer. \\nPatient delay in seeking for medical care refers to a long interval between detecting of first/initial symptoms and seeking for appointment with a general physician. The patients were further divided into two groups, those who sought medical care within 90 days and those who sought care 90 days and above (patient delay) after self-detection of the first symptoms of cervical cancer. \\nSample size was calculated based on the study conducted by Rudd et al., (2017) where 39.3% patients had delayed diagnosis from the onset of self-detection of cervical cancer symptoms. In this study the sample size was estimated for patient related delay, from symptoms to first contact with a general practitioner, as it was our major concern. To get an absolute precision of 6.6% with 95% confidence level, the study required a minimum sample of 210 subjects. Based on hospital record collection, it was noted that six months’ time period was sufficient to achieve the required sample size. \\nExpert opinion from oncologists, biostatisticians and epidemiologists were obtained using the Delphi technique (Skulmoski et al., 2007). After obtaining the written informed consent, patients were interviewed in person using the questionnaire and their medical records was used to collect the baseline information as well as key components of the time interval between self-detection of cervical cancer symptom to seeking medical care. More details of the collection of data and the different components of time intervals are described elsewhere.(Somanna et al., 2020)\\nIndependent variables included socio-demographic characteristics, such as age at diagnosis, availability of health insurance, place of residence (rural/urban), history of cancer literacy, and financially dependent for livelihood and clinical variables, such as personal history of cancer, and family history of cancer.\\nThe time interval between various stages was measured in days as reported by the patients and by review of hospital case sheets as indicated in earlier study.(Somanna et al., 2018) \\na) Time interval between self-detection of symptoms and the first presentation at general practitioner (T1). \\nb) Time interval from referral by the general practitioner to attending tertiary care centre (T2). \\nc) Time interval from first consultation at tertiary care for diagnosis to definite diagnosis date (T3). \\nd) Time interval from definite diagnosis to initiation of primary treatment (T4). \\ne) Total time elapsed from self-detection of symptoms to initiation of primary treatment (T5).\\n\\nStatistical methods\\n\\nDescriptive statistics of time intervals were summarized with median and inter-quartile range as data were not normally distributed (Kolmogrov-Smirnov and Shapiro test). Univariate odds ratios (OR) with 95% confidence intervals (CI) were estimated between the factors and patient delay. The patient delay was categorised into < 90 days and ≥ 90 days. Considering ≥ 90 days as patient delay, the association of various factors were estimated considering the low risk group as the reference. Variables showing a univariate association with patient delay (at P < 0.20) were included in multivariate unconditional logistic stepwise forward regression model to adjust for confounders. The analysis was carried out using SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago. \\nEthical approval was sought from the ethical review committee of the study Institution. \",\n \"The distribution of socio-demographic and clinical data of the 210 cervical cancer study subjects are presented along with data pertaining to the cases not enrolled for analysis (Table 1). Among 210 enrolled patients, the majority 106 (50.5%) of women were between the age 46 to 59 years where in not enrolled patients 89 (40.4%) were of same age group. It was noted 167 (63.0%) and 179 (81.4%) were not literate among enrolled and respectively. Among enrolled patients, 150 (71.4%) were from rural areas and 158 (71.4%) were financially dependent for livelihood on the family. Only 19 women (9.0%) had any health insurance policy. The majority of patients 197 (93.8%) were not aware of symptoms of cervical cancer and only 7 (3.3%) patients had a family history of cancer.\\nOf the symptoms which led for seeking medical care, the majority of women, 172 (81.9%), indicated lower abdominal pain, and 137 (65.2%) of the respondents experienced abnormal vaginal discharge/bleeding. Among all patients, 106 (50.5%) were diagnosed at an early stage of the disease (stages I, IIA and IIB).\\nThe median time interval between self-detection of cervical cancer symptoms and first contact with general physician (T1) was 80 [IQR 45-150] days, while 74 patients (35.2%) had a time lag of 90 days or more. The median time taken for reaching diagnosis after the first contact (T2) was 25 [IQR 15 - 45] days. The overall median time between self-detection of symptoms to initiation of primary treatment (T5) was 123[IQR 83-205] days. The majority of patients, 145 (66.2%), had initiation of primary treatment only after 90 days from self-detection of cervical cancer symptoms (Table 2). \\nFactors associated with delay in seeking medical care of 90 days and more (patient delay) were analyzed in relation to socio-demographic characteristics and clinical symptoms (Table 3). In multivariate analysis after adjusting to all the variables in the study the odds of patient delay in the age group ≥ 60 years was 3.2 times higher compared to patients in the age group below 45 years (95% CI: 1.2 – 8.4). The other factors which were associated with patient delay were being not literate (OR 2.3, 95% CI: 1.0 – 5.4), from rural area (OR = 2.3, 95% CI: 1.1 – 4.9), being financially dependent for livelihood (OR = 4.5, 95% CI: 1.9 – 10.6) and not having any abnormal vaginal discharge / bleeding (OR = 2.4, 95% CI: 1.3 – 4.6). Family history of cancer, presence of any health insurance, knowledge regarding cancer cure by early seeking for care, knowledge of symptoms of cervical cancer and having lower abdominal pain was not statistically related to delay in seeking medical care. \\nInformation was sought regarding perceived reasons from 197 (93.8%) patients who did not seek care within one week from self-detection of cancer symptoms. The major reasons (table 4) were lack of awareness in identifying cervical cancer symptoms in 183 (92.9%) women and 120 (60.9%) patients assuming that the symptom would resolve by itself. The other common reasons were absence of pain, changes in the body attributed to common illness and vague symptoms. \\nThe most common reason for delay in seeking for definite diagnosis at tertiary care hospitals was due to non-affordability 82 (41.6%). The other common reasons were visiting multiple medical practitioners before presenting at tertiary care 60 (30.1%) and not suspected for cancer by physician at first contact. \\nThe delay in seeking for treatment after diagnosis at tertiary care was due to long treatment procedure 57 (27.1%), fear of treatment 50 (23.8%), financial dependence on the family 48 (22.9%), fear of disfigurement of the body 26 (12.4%) and stigma attached with the disease (Table 4).\\nDistribution of Socio-Demographic, Clinical and Other Variables Among the Subject Enrolled and Not Enrolled to the Study\\nDistribution of Time Interval Between Self-Detection of Cancer Symptoms to Various Stages (T1 To T5) for Seeking Treatment\\nFactors Associated with Delay in Seeking Medical Care: Univariate and Multivariate Logistic Regression Results\\nDistribution of Perceived Reasons for Delay in Seeking Medical Care at Various Stages (T1 – T4)\\n*, Responses were elicited from those who had not sought specific care for more than one week from the onset of symptoms; #, Responses was elicited from those who had not sought treatment on specified time by doctor \\nRecruitment of Participants for the Study\",\n \"The study centre was a regional cancer care centre having both population and hospital-based cancer registry and where a majority of cervix cancer patients from the entire state seek cancer care. The median age in enrolled and not enrolled patients was 50 and 56 years, respectively. Among all the patients, the proportion of not literate patients was higher (81%) in not enrolled patients compared to enrolled patients (63%). The study by Deshmukh and Rathod, (2017) observed that the average age of the patients was 57 years and 81% were not literate. Our result concur with the study by Panda et al., 2019 where the average age of the study participants was 52.1 ± 10.9 years. \\nThe overall median time between the self-detection of cervical cancer symptoms and initiation of primary treatment was 123[IQR 83-205] days and the median time taken for first contact with general physician from self-detection of symptoms was 80 [IQR 45-150] days. Overall, 66% women delayed (≥ 90 days) for initiation of primary treatment from the onset of symptoms which is potentially concerning, because longer duration between diagnosis and treatment is related with higher risk of death. Among patients who started their treatment after an interval of >180 days, the risk of death was reported to be 36% higher compared to those who started treatment within 6 months from diagnosis (Chen et al., 2019) \\nThe present study assessed the time interval between self-detection of symptoms to treatment of cervical cancer and patient delay. The median time interval between self-detection of symptoms and the first presentation at general practitioner (T1) was 80 days with 35.4% of the patients reporting 90 days or more. The present study observation was consistent with a study conducted in India on patients attending tertiary care centre, showing that 39% of patients had a diagnostic delay (Panda et al., 2019) The median time between symptoms and presenting at health care provider was 69 days from two tertiary care centres in Nepal (Gyenwali et al., 2014). It was noted from the study by Lim et al., (2014) that only 28% of patients had a delay of more than 3 months in all national health service (NHS) hospitals diagnosing cervical cancer in England. There was a mean delay of 23.1 weeks between onset of symptoms from the study in tertiary care centre of Sothern Malawi (Rudd et al., 2017). Results from our study were in line with studies from low- or middle-income countries but differed from studies in developed countries. This difference may be partially due to regular screening programs in many developed countries and differing rates of literacy (IARC, 2005). \\nOverall median time from self-detection of symptoms to initiation of primary treatment (T5) was 123 days in the current study. Another study by Henriques, (2016) in India showed on an average six months duration from first symptom to cervical cancer treatment. Such a long time may be explained by the fact that lack of awareness of patients pertaining to cancer symptoms was high (93%) in present study. In another study, women who delayed, had mistaken the symptoms of cervix cancer as symptoms of sexually transmitted diseases (STD) and were scared to report (Deshmukh and Rathod, 2017). This shows that the lack of awareness of cervix cancer symptoms is a major factor and warrants improvement in public awareness. Our study results concur closely with other studies from similar settings and in low- or middle-income countries (Benemariya et al., 2018; Gyenwali et al., 2014).\\nThe patients who were aged 60 years and above, residing in rural areas, not literate, financially dependent for livelihood and did not had abnormal vaginal discharge/ bleeding were independently associated with patient delay. Similar results were observed in a study from Nepal where not literate was found to be independently associated with late diagnosis and women having abnormal vaginal bleeding as symptom was protective factor for late diagnosis (Gyenwali et al., 2013). Previous studies from low- or middle-income countries also concur with our result (Dunyo et al., 2018)(Ouasmani et al., 2016)(Henriques, 2016).\\nThe present study has been conducted in a population-based cancer registry (PBCR) where the data are included in International Agency for Research on Cancer (IARC) and data collection procedure involves standard quality control measures. The study included only patients who had been diagnosed with standard clinical and histopathological criteria. The information was obtained using pre-tested validated questionnaire by the investigator. Additional information pertaining to clinical evaluation and histological diagnosis were obtained from case sheets. All efforts were made to reduce the recall bias by local events calendar and standardization of terms employed in the questionnaire. \\n\\nLimitations\\n\\nPossible selection bias could have occurred as the subjects who were not enrolled for the study due to various reasons were older women and not literate compared to subjects in the study. Socio-demographic profile of population who availed the services at this centre may be different from private tertiary cancer care centre which would biases the result. The sample in study represent only those who availed the treatment at the centre and does not represent the entire population of cervical cancer patients. Thus, it may have some restrictions for external generalization. Possibility of recall bias exist as past history of cancer symptoms were assessed retrospectively. \\nIn conclusion, the median time of 80 days was observed from self-detection of cervical cancer symptoms to first contact with general physician. Further, a median time of another 45 days was observed for obtaining primary treatment. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care. Interventions like raising awareness of cervical cancer symptoms and improving health seeking behavior of women should be focused.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","materials|methods","results","discussion"],"string":"[\n \"intro\",\n \"materials|methods\",\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Cervical cancer","time interval","cancer symptoms","seeking cancer care"],"string":"[\n \"Cervical cancer\",\n \"time interval\",\n \"cancer symptoms\",\n \"seeking cancer care\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nGlobally, an estimated 570,000 new cases and 311,000 deaths has been reported due to cervical cancer in 2018 and it is the fourth most common cancer in women. The Incidence and mortality rank second behind breast cancer in lower human development index (HDI) settings. Eight out of 10 women diagnosed and nine of 10 women who die from cervical cancer live in a low- or middle-income countries (Bray et al., 2018) “ Cervical cancer continues to be a major public health problem affecting middle-aged women, particularly in less-resourced countries” (Arbyn et al., 2020). India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one third of global cervical cancer deaths during the year 2018 (“Globocan 2018,” 2018). Cervical cancer is the leading cause of cancer mortality in India which accounts for 17% of all cancer deaths among women aged 30 to 69 years.(Bobdey et al., 2016, “The Challenge Ahead,” 2014) One third of cervical cancer cases was reported among women aged 50 -59 years ( 27.4%) (Bobdey et al., 2016). The higher mortality and morbidity due to cancer is because most patients seek/get treatment at advanced stage and therefore have a higher risk of dying from the disease (Bray et al., 2018).\nCervical cancer is a preventable disease primarily because of its long lead time (women develop precancerous lesions years before it becomes cancerous). Cervical cancer can be detected in the pre-cancerous phase by screening and thereby the development of cancer prevented. Cervical cancer can also be treated successfully if diagnosed at an early stage. Routine screening by visual inspection of cervix through ‘See-and-treat’ approach among the female population has been suggested for early diagnosis and control of cervical cancer (Sankaranarayanan, 2012). Periodic Pap or HPV samples are difficult to organize in low- or middle-income countries like India and therefore large-scale routine screening would be difficult and not affordable (“HTA_CaCx Screening in India.pdf,” n.d.) Millions of cancer patients could be saved from premature death and disability, if they had timely access to early detection and treatment (“WHO,” n.d.). Studies have shown that it is possible to reduce the need of aggressive treatment of cancer is detected at an early stage. A long interval between start of disease symptoms to having a definite diagnosis is highly critical and associated with low survival among cervical cancer patients.(Chen et al., 2019) \nIn India, the present existing public health care delivery system is a three-level system. The primary level is primary health centres (PHCs). In India, PHCs focuses on eight elements of primary health care outlined in the Alma-Ata declaration and provides mainly outpatient services. In the secondary level the district hospitals provide outpatient, inpatient and emergency services. At the tertiary level, medical college hospitals, apex institutions and regional cancer centres provide specialized services. Under the programme of “National programme for prevention and control of cancer, diabetes, cardiovascular diseases and stroke (NPCDCS)”, cancer screening is done at the primary level by visual inspection of the cervix with acetic acid (VIA) and those who are suspected for cancer are referred to secondary or tertiary care level for further management. In addition, a health programme for non-communicable diseases is carried out with a cancer care component implemented at all the health care levels (Gulia et al., 2016) \nThis study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking for care and different barriers for the possible time lag in seeking care.\n\nMaterials and Methods:\nThe data was collected from a regional cancer centre from south India, Bengaluru, which has a population-based cancer registry (PBCR) and a hospital-based cancer registry (HBCR). This tertiary care centre has state of art facilities for diagnosis, treatment and research. Cervical cancer cases with histological confirmation were recruited from the hospital-based cancer registry of this Institution during the time period of April 2017 to September 2017 using cross sectional study design. We identified 430 cervical cancer patients during the period from April 2017 to September 2017. Of these, 52 were ineligible due to referral to terminal/palliative care. Of 378 eligible women, 96 were not included for the analysis as patients moved to different hospital or not being available for interview, and 38 were not willing to participate in the study. In the 244 patients who gave their consent, 34 patient’s had incomplete information in the records. Finally, 210 patients were included for the present study (Figure 1) \nThe cervical cancers were mostly squamous cell carcinomas (SCC) and few adenocarcinomas (AC). They were divided in two stage based on FIGO classification (Bhatla et al., 2019). Stage I and II were categorized as early stage; III and IV were considered as late stage of cervical cancer. \nPatient delay in seeking for medical care refers to a long interval between detecting of first/initial symptoms and seeking for appointment with a general physician. The patients were further divided into two groups, those who sought medical care within 90 days and those who sought care 90 days and above (patient delay) after self-detection of the first symptoms of cervical cancer. \nSample size was calculated based on the study conducted by Rudd et al., (2017) where 39.3% patients had delayed diagnosis from the onset of self-detection of cervical cancer symptoms. In this study the sample size was estimated for patient related delay, from symptoms to first contact with a general practitioner, as it was our major concern. To get an absolute precision of 6.6% with 95% confidence level, the study required a minimum sample of 210 subjects. Based on hospital record collection, it was noted that six months’ time period was sufficient to achieve the required sample size. \nExpert opinion from oncologists, biostatisticians and epidemiologists were obtained using the Delphi technique (Skulmoski et al., 2007). After obtaining the written informed consent, patients were interviewed in person using the questionnaire and their medical records was used to collect the baseline information as well as key components of the time interval between self-detection of cervical cancer symptom to seeking medical care. More details of the collection of data and the different components of time intervals are described elsewhere.(Somanna et al., 2020)\nIndependent variables included socio-demographic characteristics, such as age at diagnosis, availability of health insurance, place of residence (rural/urban), history of cancer literacy, and financially dependent for livelihood and clinical variables, such as personal history of cancer, and family history of cancer.\nThe time interval between various stages was measured in days as reported by the patients and by review of hospital case sheets as indicated in earlier study.(Somanna et al., 2018) \na) Time interval between self-detection of symptoms and the first presentation at general practitioner (T1). \nb) Time interval from referral by the general practitioner to attending tertiary care centre (T2). \nc) Time interval from first consultation at tertiary care for diagnosis to definite diagnosis date (T3). \nd) Time interval from definite diagnosis to initiation of primary treatment (T4). \ne) Total time elapsed from self-detection of symptoms to initiation of primary treatment (T5).\n\nStatistical methods\n\nDescriptive statistics of time intervals were summarized with median and inter-quartile range as data were not normally distributed (Kolmogrov-Smirnov and Shapiro test). Univariate odds ratios (OR) with 95% confidence intervals (CI) were estimated between the factors and patient delay. The patient delay was categorised into < 90 days and ≥ 90 days. Considering ≥ 90 days as patient delay, the association of various factors were estimated considering the low risk group as the reference. Variables showing a univariate association with patient delay (at P < 0.20) were included in multivariate unconditional logistic stepwise forward regression model to adjust for confounders. The analysis was carried out using SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago. \nEthical approval was sought from the ethical review committee of the study Institution. \n\nResults:\nThe distribution of socio-demographic and clinical data of the 210 cervical cancer study subjects are presented along with data pertaining to the cases not enrolled for analysis (Table 1). Among 210 enrolled patients, the majority 106 (50.5%) of women were between the age 46 to 59 years where in not enrolled patients 89 (40.4%) were of same age group. It was noted 167 (63.0%) and 179 (81.4%) were not literate among enrolled and respectively. Among enrolled patients, 150 (71.4%) were from rural areas and 158 (71.4%) were financially dependent for livelihood on the family. Only 19 women (9.0%) had any health insurance policy. The majority of patients 197 (93.8%) were not aware of symptoms of cervical cancer and only 7 (3.3%) patients had a family history of cancer.\nOf the symptoms which led for seeking medical care, the majority of women, 172 (81.9%), indicated lower abdominal pain, and 137 (65.2%) of the respondents experienced abnormal vaginal discharge/bleeding. Among all patients, 106 (50.5%) were diagnosed at an early stage of the disease (stages I, IIA and IIB).\nThe median time interval between self-detection of cervical cancer symptoms and first contact with general physician (T1) was 80 [IQR 45-150] days, while 74 patients (35.2%) had a time lag of 90 days or more. The median time taken for reaching diagnosis after the first contact (T2) was 25 [IQR 15 - 45] days. The overall median time between self-detection of symptoms to initiation of primary treatment (T5) was 123[IQR 83-205] days. The majority of patients, 145 (66.2%), had initiation of primary treatment only after 90 days from self-detection of cervical cancer symptoms (Table 2). \nFactors associated with delay in seeking medical care of 90 days and more (patient delay) were analyzed in relation to socio-demographic characteristics and clinical symptoms (Table 3). In multivariate analysis after adjusting to all the variables in the study the odds of patient delay in the age group ≥ 60 years was 3.2 times higher compared to patients in the age group below 45 years (95% CI: 1.2 – 8.4). The other factors which were associated with patient delay were being not literate (OR 2.3, 95% CI: 1.0 – 5.4), from rural area (OR = 2.3, 95% CI: 1.1 – 4.9), being financially dependent for livelihood (OR = 4.5, 95% CI: 1.9 – 10.6) and not having any abnormal vaginal discharge / bleeding (OR = 2.4, 95% CI: 1.3 – 4.6). Family history of cancer, presence of any health insurance, knowledge regarding cancer cure by early seeking for care, knowledge of symptoms of cervical cancer and having lower abdominal pain was not statistically related to delay in seeking medical care. \nInformation was sought regarding perceived reasons from 197 (93.8%) patients who did not seek care within one week from self-detection of cancer symptoms. The major reasons (table 4) were lack of awareness in identifying cervical cancer symptoms in 183 (92.9%) women and 120 (60.9%) patients assuming that the symptom would resolve by itself. The other common reasons were absence of pain, changes in the body attributed to common illness and vague symptoms. \nThe most common reason for delay in seeking for definite diagnosis at tertiary care hospitals was due to non-affordability 82 (41.6%). The other common reasons were visiting multiple medical practitioners before presenting at tertiary care 60 (30.1%) and not suspected for cancer by physician at first contact. \nThe delay in seeking for treatment after diagnosis at tertiary care was due to long treatment procedure 57 (27.1%), fear of treatment 50 (23.8%), financial dependence on the family 48 (22.9%), fear of disfigurement of the body 26 (12.4%) and stigma attached with the disease (Table 4).\nDistribution of Socio-Demographic, Clinical and Other Variables Among the Subject Enrolled and Not Enrolled to the Study\nDistribution of Time Interval Between Self-Detection of Cancer Symptoms to Various Stages (T1 To T5) for Seeking Treatment\nFactors Associated with Delay in Seeking Medical Care: Univariate and Multivariate Logistic Regression Results\nDistribution of Perceived Reasons for Delay in Seeking Medical Care at Various Stages (T1 – T4)\n*, Responses were elicited from those who had not sought specific care for more than one week from the onset of symptoms; #, Responses was elicited from those who had not sought treatment on specified time by doctor \nRecruitment of Participants for the Study\n\nDiscussion:\nThe study centre was a regional cancer care centre having both population and hospital-based cancer registry and where a majority of cervix cancer patients from the entire state seek cancer care. The median age in enrolled and not enrolled patients was 50 and 56 years, respectively. Among all the patients, the proportion of not literate patients was higher (81%) in not enrolled patients compared to enrolled patients (63%). The study by Deshmukh and Rathod, (2017) observed that the average age of the patients was 57 years and 81% were not literate. Our result concur with the study by Panda et al., 2019 where the average age of the study participants was 52.1 ± 10.9 years. \nThe overall median time between the self-detection of cervical cancer symptoms and initiation of primary treatment was 123[IQR 83-205] days and the median time taken for first contact with general physician from self-detection of symptoms was 80 [IQR 45-150] days. Overall, 66% women delayed (≥ 90 days) for initiation of primary treatment from the onset of symptoms which is potentially concerning, because longer duration between diagnosis and treatment is related with higher risk of death. Among patients who started their treatment after an interval of >180 days, the risk of death was reported to be 36% higher compared to those who started treatment within 6 months from diagnosis (Chen et al., 2019) \nThe present study assessed the time interval between self-detection of symptoms to treatment of cervical cancer and patient delay. The median time interval between self-detection of symptoms and the first presentation at general practitioner (T1) was 80 days with 35.4% of the patients reporting 90 days or more. The present study observation was consistent with a study conducted in India on patients attending tertiary care centre, showing that 39% of patients had a diagnostic delay (Panda et al., 2019) The median time between symptoms and presenting at health care provider was 69 days from two tertiary care centres in Nepal (Gyenwali et al., 2014). It was noted from the study by Lim et al., (2014) that only 28% of patients had a delay of more than 3 months in all national health service (NHS) hospitals diagnosing cervical cancer in England. There was a mean delay of 23.1 weeks between onset of symptoms from the study in tertiary care centre of Sothern Malawi (Rudd et al., 2017). Results from our study were in line with studies from low- or middle-income countries but differed from studies in developed countries. This difference may be partially due to regular screening programs in many developed countries and differing rates of literacy (IARC, 2005). \nOverall median time from self-detection of symptoms to initiation of primary treatment (T5) was 123 days in the current study. Another study by Henriques, (2016) in India showed on an average six months duration from first symptom to cervical cancer treatment. Such a long time may be explained by the fact that lack of awareness of patients pertaining to cancer symptoms was high (93%) in present study. In another study, women who delayed, had mistaken the symptoms of cervix cancer as symptoms of sexually transmitted diseases (STD) and were scared to report (Deshmukh and Rathod, 2017). This shows that the lack of awareness of cervix cancer symptoms is a major factor and warrants improvement in public awareness. Our study results concur closely with other studies from similar settings and in low- or middle-income countries (Benemariya et al., 2018; Gyenwali et al., 2014).\nThe patients who were aged 60 years and above, residing in rural areas, not literate, financially dependent for livelihood and did not had abnormal vaginal discharge/ bleeding were independently associated with patient delay. Similar results were observed in a study from Nepal where not literate was found to be independently associated with late diagnosis and women having abnormal vaginal bleeding as symptom was protective factor for late diagnosis (Gyenwali et al., 2013). Previous studies from low- or middle-income countries also concur with our result (Dunyo et al., 2018)(Ouasmani et al., 2016)(Henriques, 2016).\nThe present study has been conducted in a population-based cancer registry (PBCR) where the data are included in International Agency for Research on Cancer (IARC) and data collection procedure involves standard quality control measures. The study included only patients who had been diagnosed with standard clinical and histopathological criteria. The information was obtained using pre-tested validated questionnaire by the investigator. Additional information pertaining to clinical evaluation and histological diagnosis were obtained from case sheets. All efforts were made to reduce the recall bias by local events calendar and standardization of terms employed in the questionnaire. \n\nLimitations\n\nPossible selection bias could have occurred as the subjects who were not enrolled for the study due to various reasons were older women and not literate compared to subjects in the study. Socio-demographic profile of population who availed the services at this centre may be different from private tertiary cancer care centre which would biases the result. The sample in study represent only those who availed the treatment at the centre and does not represent the entire population of cervical cancer patients. Thus, it may have some restrictions for external generalization. Possibility of recall bias exist as past history of cancer symptoms were assessed retrospectively. \nIn conclusion, the median time of 80 days was observed from self-detection of cervical cancer symptoms to first contact with general physician. Further, a median time of another 45 days was observed for obtaining primary treatment. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care. Interventions like raising awareness of cervical cancer symptoms and improving health seeking behavior of women should be focused.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIndia had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one-third of global cervical cancer deaths during the year 2018. Cervical cancer is the leading cause of cancer mortality in India. The present study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking care and different barriers for the possible time lag in seeking care.\n\nMethods:\nA cross-sectional study was undertaken from April 2017 to September 2017 in a regional cancer centre in the south of India. The centre has both a population and a hospital-based cancer registry. Cervical cancer cases (N= 210) with histological confirmation were interviewed at the hospital using a pre-tested semi-structured questionnaire.\n\nResults:\nThe median time interval between the self-detection of cervical cancer symptoms and first contact with the general physician was 80 [IQR 45-150] days. The overall median time interval between the self-detection of symptoms to the initiation of primary treatment was 123[IQR 83-205] days. The major perceived reason for not seeking medical care was a lack of awareness in identifying cervical cancer symptoms in 183(92.9%) women.\n\nConclusions:\nThe median time of 80 days was observed from the self-detection of cervical cancer symptoms to the first contact with a general physician. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care.
."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":3637,"string":"3,637"},"whole_article_abstract_length":{"kind":"number","value":278,"string":"278"},"num_sections":{"kind":"number","value":4,"string":"4"},"most_frequent_words":{"kind":"list like","value":["cancer","patients","symptoms","study","care","cervical","cervical cancer","time","treatment","days"],"string":"[\n \"cancer\",\n \"patients\",\n \"symptoms\",\n \"study\",\n \"care\",\n \"cervical\",\n \"cervical cancer\",\n \"time\",\n \"treatment\",\n \"days\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Cervical cancer | time interval | cancer symptoms | seeking cancer care [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Cervical cancer | time interval | cancer symptoms | seeking cancer care [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Cervical cancer | time interval | cancer symptoms | seeking cancer care [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Cross-Sectional Studies | Female | Follow-Up Studies | Health Knowledge, Attitudes, Practice | Humans | India | Middle Aged | Patient Acceptance of Health Care | Prognosis | Retrospective Studies | Self-Assessment | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Uterine Cervical Neoplasms [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Cross-Sectional Studies | Female | Follow-Up Studies | Health Knowledge, Attitudes, Practice | Humans | India | Middle Aged | Patient Acceptance of Health Care | Prognosis | Retrospective Studies | Self-Assessment | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Uterine Cervical Neoplasms [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Cross-Sectional Studies | Female | Follow-Up Studies | Health Knowledge, Attitudes, Practice | Humans | India | Middle Aged | Patient Acceptance of Health Care | Prognosis | Retrospective Studies | Self-Assessment | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Uterine Cervical Neoplasms [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | patients | symptoms | study | care | cervical | cervical cancer | time | treatment | days [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | patients | symptoms | study | care | cervical | cervical cancer | time | treatment | days [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | patients | symptoms | study | care | cervical | cervical cancer | time | treatment | days [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | cervical cancer | cervical | level | 000 | deaths | screening | 2018 | india | care [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | symptoms | delay | patients | care | enrolled | 95 ci | table | delay seeking | seeking [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | patients | symptoms | care | cervical | cervical cancer | study | time | days | delay [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] India | 97,000 | 60,000 | nearly one-third | the year 2018 ||| India ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] first | 80 ||| ||| 123[IQR | 83 ||| 183(92.9% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] India | 97,000 | 60,000 | nearly one-third | the year 2018 ||| India ||| ||| April 2017 to September 2017 | India ||| ||| 210 ||| ||| first | 80 ||| ||| 123[IQR | 83 ||| 183(92.9% ||| 80 days | first ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":267,"cells":{"title":{"kind":"string","value":"Alteration of Thyroid Hormone among Patients with Ischemic Stroke visiting a Tertiary Care Hospital: A Descriptive Cross-sectional Study."},"pmid":{"kind":"string","value":"34508483"},"background_abstract":{"kind":"string","value":"Stroke is broadly classified as cerebral infarction, intracerebral hemorrhage and subarachnoid hemorrhage. Neuroendocrine profile is altered in acute ischemic stroke and there is a link between hypothyroidism and atherosclerosis which in turn may lead to stroke. The objective of this study was to find out the prevalence of alteration of thyroid hormones in patients with ischemic stroke in a tertiary care center."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"This descriptive cross-sectional study was conducted from June to December 2019 in a tertiary care center. Ethical approval was taken from Institutional review board of National Academy of Medical Sciences (reference number: IM 175). Patients with a diagnosis of stroke, without evidence of cardioembolic source, history of liver disease, renal failure and thyroid disease and who do not use thyroidal supplementation within 180 days prior the event were included. Convenience sampling was done. Data was entered in Microsoft Excel and analyzed using Statistical Package for the Social Sciences version 22. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The prevalence of altered thyroid levels among 73 patients was 13 (17.8%) (90% Confidence Interval= 10.44-25.16). Among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Among severity of hypothyroid cases, subclinical hypothyroidism grade IA was seen in 51 (70%), subclinical hypothyroidism grade IB was seen in 22 (30%)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The prevalence of altered thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Brain Ischemia","Cross-Sectional Studies","Humans","Ischemic Stroke","Stroke","Tertiary Care Centers","Thyroid Hormones"],"string":"[\n \"Brain Ischemia\",\n \"Cross-Sectional Studies\",\n \"Humans\",\n \"Ischemic Stroke\",\n \"Stroke\",\n \"Tertiary Care Centers\",\n \"Thyroid Hormones\"\n]"},"pmcid":{"kind":"string","value":"9107842"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Stroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4\nThyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4\nThe aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"This descriptive cross-sectional study was conducted from June to December 2019 in National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu. Ethical approval from Institutional review board of NAMS (reference number. IM 175). Convenience sampling was done and the sample size of cases to be enrolled was calculated by using the formula,\nn = Z2 × p × q / e2\n = (1.645)2 × (0.5) × (1-0.5) / (0.1)2\n = 67.65\nWhere,\nn = minimum required sample size,\nZ = 1.645 at 90% Confidence Interval (CI),\np = prevalence taken as 50% for maximum sample size,\nq = 1-p\ne = margin of error, 10%\nHence, the required sample size was 67.65. Adding a 10% nonresponse rate, we enrolled 73 patients in our study. Patients with clinically diagnosed stroke, presenting within 48 hours of onset of symptoms with CT or MRI findings were included in the study. Patients with prior use of thyroid supplements recently within last 180 days and with a history of liver disease, renal failure and known thyroid disease and who don't give consent were excluded from the study. Informed written consent was taken either in Nepali or in English language, in whichever they felt comfortable. Confidentiality was maintained to the utmost. For those who are not able to give consent because of their clinical state, informed consent was taken from their close relative.\nProforma was translated in Nepali verbally if needed depending upon the cases. For diagnosing the type of stroke, every patient underwent neuroimaging with non-contrast CT of the head. Cerebral infarction was diagnosed if neurological deficits are accompanied by a hypo dense lesion >15 mm in diameter in an appropriate area on a cranial CT scan. Lacunar infarction were diagnosed if the patient presented with a pure motor stroke, a pure sensory stroke, ataxic hemiparesis or a sensorimotor stroke in the absence of a visual field defect and evidence of higher cerebral dysfunction and a hypo dense lesion of < 15 mm in diameter or normal CT scan. In the laboratory, fully automatic analyzer VITROSECi/ECiQ Immunodiagnostic Systems, VITROSTSH was measured using a chemiluminescent immunometric assay with a detection range from 0.01 to 100 m IU/L. TSH values between 0.46 and 4.68 m IU/L were considered the normal reference range (euthyroid). Specimens with TSH values above or below the normal reference range were tested for free thyroxine (FT4). FT4 was measured using a competitive chemiluminescent assay with a detection range of 0.1-12.0 ng/dL. FT4 concentrations within 0.70-2.19 ng/dL was within the normal reference range. All samples included in the study met quality control criteria. Samples with TSH values > 4.69 m IU/L and normal FT4 levels were classified as indicating SCH.\nThe collected data was stored and analyzed with the Statistical Package for the Social Sciences (SPSS) version 22.0 for windows. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was 13 (17.8%) and among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Minimum age of patient in the study was 26 years and maximum age was 115 years. The mean age of presentation of cerebral infarction in this study was 63.30±19.217.\nAmong the total 73 cases, 49 were male and 24 were female. Among 49 males, 37 were euthyroid, 11 were hypothyroid and one male was hyperthyroid. Among 24 females, 23 were euthyroid and one female was hyperthyroid. On further categorization of altered thyroid status, 10 (13.7%) were subclinical hypothyroidism, 1 (1.4%) cases were of overt hypothyroidism and 2 (2.7%) cases had subclinical hyperthyroidism. With regards to the severity of subclinical hypothyroidism, out of the total 10 cases of subclinical hypothyroidism, 70% had grade IA, while the remaining 30% had grade IB severity of the status (Figure 1). In the study 49 (67.12%) of ischemic stroke were male and 24 (32.88%) were female.\nAmong euthyroid cases mean total cholesterol was 139.55 mg/dl which was higher than the mean cholesterol level in overt hypothyroid cases with 110 mg/dl. But mean total cholesterol level was lower among overt hypothyroid cases compared to euthyroid and overt hypothyroid cases. Mean TC was highest among subclinical hyperthyroid cases. Mean LDLc was highest among euthyroid cases with mean value of 73.94 mg/dl and lowest among overt hypothyroid cases with mean value of 41.00 mg/dl. TG was highest among overt hypothyroid cases with mean value of 199 mg/dl. HDLc was highest among euthyroid cases with mean value of 43.42 mg/dl. Subclinical hypothyroid cases had mean HDL value of 39.90 mg/dl (Table 1)."},"conclusions_title":{"kind":"string","value":"CONCLUSIONS"},"conclusions_text":{"kind":"string","value":"The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke."},"other_sections_titles":{"kind":"list like","value":[],"string":"[]"},"other_sections_texts":{"kind":"list like","value":[],"string":"[]"},"other_sections_sec_types":{"kind":"list like","value":[],"string":"[]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","RESULTS","DISCUSSION","CONCLUSIONS"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"RESULTS\",\n \"DISCUSSION\",\n \"CONCLUSIONS\"\n]"},"all_sections_texts":{"kind":"list like","value":["Stroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4\nThyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4\nThe aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.","This descriptive cross-sectional study was conducted from June to December 2019 in National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu. Ethical approval from Institutional review board of NAMS (reference number. IM 175). Convenience sampling was done and the sample size of cases to be enrolled was calculated by using the formula,\nn = Z2 × p × q / e2\n = (1.645)2 × (0.5) × (1-0.5) / (0.1)2\n = 67.65\nWhere,\nn = minimum required sample size,\nZ = 1.645 at 90% Confidence Interval (CI),\np = prevalence taken as 50% for maximum sample size,\nq = 1-p\ne = margin of error, 10%\nHence, the required sample size was 67.65. Adding a 10% nonresponse rate, we enrolled 73 patients in our study. Patients with clinically diagnosed stroke, presenting within 48 hours of onset of symptoms with CT or MRI findings were included in the study. Patients with prior use of thyroid supplements recently within last 180 days and with a history of liver disease, renal failure and known thyroid disease and who don't give consent were excluded from the study. Informed written consent was taken either in Nepali or in English language, in whichever they felt comfortable. Confidentiality was maintained to the utmost. For those who are not able to give consent because of their clinical state, informed consent was taken from their close relative.\nProforma was translated in Nepali verbally if needed depending upon the cases. For diagnosing the type of stroke, every patient underwent neuroimaging with non-contrast CT of the head. Cerebral infarction was diagnosed if neurological deficits are accompanied by a hypo dense lesion >15 mm in diameter in an appropriate area on a cranial CT scan. Lacunar infarction were diagnosed if the patient presented with a pure motor stroke, a pure sensory stroke, ataxic hemiparesis or a sensorimotor stroke in the absence of a visual field defect and evidence of higher cerebral dysfunction and a hypo dense lesion of < 15 mm in diameter or normal CT scan. In the laboratory, fully automatic analyzer VITROSECi/ECiQ Immunodiagnostic Systems, VITROSTSH was measured using a chemiluminescent immunometric assay with a detection range from 0.01 to 100 m IU/L. TSH values between 0.46 and 4.68 m IU/L were considered the normal reference range (euthyroid). Specimens with TSH values above or below the normal reference range were tested for free thyroxine (FT4). FT4 was measured using a competitive chemiluminescent assay with a detection range of 0.1-12.0 ng/dL. FT4 concentrations within 0.70-2.19 ng/dL was within the normal reference range. All samples included in the study met quality control criteria. Samples with TSH values > 4.69 m IU/L and normal FT4 levels were classified as indicating SCH.\nThe collected data was stored and analyzed with the Statistical Package for the Social Sciences (SPSS) version 22.0 for windows. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.","The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was 13 (17.8%) and among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Minimum age of patient in the study was 26 years and maximum age was 115 years. The mean age of presentation of cerebral infarction in this study was 63.30±19.217.\nAmong the total 73 cases, 49 were male and 24 were female. Among 49 males, 37 were euthyroid, 11 were hypothyroid and one male was hyperthyroid. Among 24 females, 23 were euthyroid and one female was hyperthyroid. On further categorization of altered thyroid status, 10 (13.7%) were subclinical hypothyroidism, 1 (1.4%) cases were of overt hypothyroidism and 2 (2.7%) cases had subclinical hyperthyroidism. With regards to the severity of subclinical hypothyroidism, out of the total 10 cases of subclinical hypothyroidism, 70% had grade IA, while the remaining 30% had grade IB severity of the status (Figure 1). In the study 49 (67.12%) of ischemic stroke were male and 24 (32.88%) were female.\nAmong euthyroid cases mean total cholesterol was 139.55 mg/dl which was higher than the mean cholesterol level in overt hypothyroid cases with 110 mg/dl. But mean total cholesterol level was lower among overt hypothyroid cases compared to euthyroid and overt hypothyroid cases. Mean TC was highest among subclinical hyperthyroid cases. Mean LDLc was highest among euthyroid cases with mean value of 73.94 mg/dl and lowest among overt hypothyroid cases with mean value of 41.00 mg/dl. TG was highest among overt hypothyroid cases with mean value of 199 mg/dl. HDLc was highest among euthyroid cases with mean value of 43.42 mg/dl. Subclinical hypothyroid cases had mean HDL value of 39.90 mg/dl (Table 1).","Stroke is a form of acute stress with detrimental effect on various neurophysiological pathways.7 Hypothyroidism is a possible risk factor for stroke.8 A number of comorbidities have been associated with increased mortality in acute stroke patients. It is not known whether hypothyroidism (either clinical or subclinical) affects outcome in patients with acute cerebrovascular disease. A neuroprotective role of hypothyroidism has been shown in acute stroke patients.8 Low T3 appeared to be associated with stroke severity and short-term outcome.9 In addition, strokes of undetermined etiology accounted for one-third to one-quarter of ischemic strokes among young people and among cases of stroke of undetermined etiology hyperthyroidism could be the underlying cause.10,11 So, TFT screening is useful in patients with acute ischemic stroke (IS) because thyroid disorders are known risk factors for cerebrovascular diseases.6 In our study we observed 17.8% of the cases had thyroid dysfunction. Our finding is similar to the findings of the retrospective study done by Xu XY , et al. in 893 patients with acute ischemic stroke in which 19% of the cases had abnormal thyroid function tests with at least one of the thyroid related hormones below or above the normal ranges.12 Unknown overt or subclinical hyperthyroidism is associated with cardio-embolic stroke.13 Thyroid dysfunction in our study was lower than the study done by Dimopolou, et al. in 33 critically ill patients under mechanical ventilation due to acute stroke; study concluded 36% of the cases had thyroid dysfunction.14 In our study we found that 1.4% of the cases had overt hypothyroidism, this is comparable to the study done by O'keefe L.M , et al. in 129 patients with acute ischemic stroke. 2.32 % of the cases in the study had hypothyroidism.1513.7 % of our study population had subclinical hypothyroidism.\nOur result is similar to the study done by Pande, et al. to establish a relation between the variation in thyroid profile and ischemic CVAs in 75 patients within 48 hours of the event, which concluded that 12% of the cases had subclinical hypothyroidism.7 Prospective cohort study done by Chakler L, et al. on 47,573 adults (3451 subclinical hypothyroidism) from 17 cohorts concluded an increased risk of stroke on subjects younger than 65 years and those with higher TSH concentrations. Subclinical hypothyroidism (SCH) is postulated to increase stroke risk via atherogenic changes associated with abnormal thyroid function.16 The study concluded that hyperthyroidism is associated with an increased risk for ischemic stroke among young adults.11\nIn our study HDLc was low (<40 mg/dl) in all the case with thyroid dysfunction (18% cases) this is much lower compared to the study done by Pokharel , et al., among 281 stroke patients, in which 45% had their HDLc levels below 40 mg/dl.17 TG was elevated (>150 mg/dl) in cases with overt hypothyroidism (1.4%) this is consistent with the study done by Abrams JJ , et al. which concluded TG metabolism is not grossly deranged in hypothyroidism.18 In our study we found that stroke occurred more commonly among males (67.12%) compared to females (32.88%). Women have lower stroke incidence than men, it might be due to positive effects of estrogen on the cerebral circulation. A lifetime exposure to ovarian estrogens may protect against ischemic stroke.19 Seventy-two percent of the cases in our study were smokers. Wang, et al. reported that 48% of patients with stroke smoked.20 Tsai, et al. reported 38% of the cases with ischemic stroke were smokers.21\nOur results might not be generalized to all stroke patients as we recruited study participants from a single hospital only. As many of our patients presented to us after 48 hours of the occurrence of stroke events, we could not involve them in our study.","The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke."],"string":"[\n \"Stroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4\\nThyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4\\nThe aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.\",\n \"This descriptive cross-sectional study was conducted from June to December 2019 in National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu. Ethical approval from Institutional review board of NAMS (reference number. IM 175). Convenience sampling was done and the sample size of cases to be enrolled was calculated by using the formula,\\nn = Z2 × p × q / e2\\n = (1.645)2 × (0.5) × (1-0.5) / (0.1)2\\n = 67.65\\nWhere,\\nn = minimum required sample size,\\nZ = 1.645 at 90% Confidence Interval (CI),\\np = prevalence taken as 50% for maximum sample size,\\nq = 1-p\\ne = margin of error, 10%\\nHence, the required sample size was 67.65. Adding a 10% nonresponse rate, we enrolled 73 patients in our study. Patients with clinically diagnosed stroke, presenting within 48 hours of onset of symptoms with CT or MRI findings were included in the study. Patients with prior use of thyroid supplements recently within last 180 days and with a history of liver disease, renal failure and known thyroid disease and who don't give consent were excluded from the study. Informed written consent was taken either in Nepali or in English language, in whichever they felt comfortable. Confidentiality was maintained to the utmost. For those who are not able to give consent because of their clinical state, informed consent was taken from their close relative.\\nProforma was translated in Nepali verbally if needed depending upon the cases. For diagnosing the type of stroke, every patient underwent neuroimaging with non-contrast CT of the head. Cerebral infarction was diagnosed if neurological deficits are accompanied by a hypo dense lesion >15 mm in diameter in an appropriate area on a cranial CT scan. Lacunar infarction were diagnosed if the patient presented with a pure motor stroke, a pure sensory stroke, ataxic hemiparesis or a sensorimotor stroke in the absence of a visual field defect and evidence of higher cerebral dysfunction and a hypo dense lesion of < 15 mm in diameter or normal CT scan. In the laboratory, fully automatic analyzer VITROSECi/ECiQ Immunodiagnostic Systems, VITROSTSH was measured using a chemiluminescent immunometric assay with a detection range from 0.01 to 100 m IU/L. TSH values between 0.46 and 4.68 m IU/L were considered the normal reference range (euthyroid). Specimens with TSH values above or below the normal reference range were tested for free thyroxine (FT4). FT4 was measured using a competitive chemiluminescent assay with a detection range of 0.1-12.0 ng/dL. FT4 concentrations within 0.70-2.19 ng/dL was within the normal reference range. All samples included in the study met quality control criteria. Samples with TSH values > 4.69 m IU/L and normal FT4 levels were classified as indicating SCH.\\nThe collected data was stored and analyzed with the Statistical Package for the Social Sciences (SPSS) version 22.0 for windows. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.\",\n \"The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was 13 (17.8%) and among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Minimum age of patient in the study was 26 years and maximum age was 115 years. The mean age of presentation of cerebral infarction in this study was 63.30±19.217.\\nAmong the total 73 cases, 49 were male and 24 were female. Among 49 males, 37 were euthyroid, 11 were hypothyroid and one male was hyperthyroid. Among 24 females, 23 were euthyroid and one female was hyperthyroid. On further categorization of altered thyroid status, 10 (13.7%) were subclinical hypothyroidism, 1 (1.4%) cases were of overt hypothyroidism and 2 (2.7%) cases had subclinical hyperthyroidism. With regards to the severity of subclinical hypothyroidism, out of the total 10 cases of subclinical hypothyroidism, 70% had grade IA, while the remaining 30% had grade IB severity of the status (Figure 1). In the study 49 (67.12%) of ischemic stroke were male and 24 (32.88%) were female.\\nAmong euthyroid cases mean total cholesterol was 139.55 mg/dl which was higher than the mean cholesterol level in overt hypothyroid cases with 110 mg/dl. But mean total cholesterol level was lower among overt hypothyroid cases compared to euthyroid and overt hypothyroid cases. Mean TC was highest among subclinical hyperthyroid cases. Mean LDLc was highest among euthyroid cases with mean value of 73.94 mg/dl and lowest among overt hypothyroid cases with mean value of 41.00 mg/dl. TG was highest among overt hypothyroid cases with mean value of 199 mg/dl. HDLc was highest among euthyroid cases with mean value of 43.42 mg/dl. Subclinical hypothyroid cases had mean HDL value of 39.90 mg/dl (Table 1).\",\n \"Stroke is a form of acute stress with detrimental effect on various neurophysiological pathways.7 Hypothyroidism is a possible risk factor for stroke.8 A number of comorbidities have been associated with increased mortality in acute stroke patients. It is not known whether hypothyroidism (either clinical or subclinical) affects outcome in patients with acute cerebrovascular disease. A neuroprotective role of hypothyroidism has been shown in acute stroke patients.8 Low T3 appeared to be associated with stroke severity and short-term outcome.9 In addition, strokes of undetermined etiology accounted for one-third to one-quarter of ischemic strokes among young people and among cases of stroke of undetermined etiology hyperthyroidism could be the underlying cause.10,11 So, TFT screening is useful in patients with acute ischemic stroke (IS) because thyroid disorders are known risk factors for cerebrovascular diseases.6 In our study we observed 17.8% of the cases had thyroid dysfunction. Our finding is similar to the findings of the retrospective study done by Xu XY , et al. in 893 patients with acute ischemic stroke in which 19% of the cases had abnormal thyroid function tests with at least one of the thyroid related hormones below or above the normal ranges.12 Unknown overt or subclinical hyperthyroidism is associated with cardio-embolic stroke.13 Thyroid dysfunction in our study was lower than the study done by Dimopolou, et al. in 33 critically ill patients under mechanical ventilation due to acute stroke; study concluded 36% of the cases had thyroid dysfunction.14 In our study we found that 1.4% of the cases had overt hypothyroidism, this is comparable to the study done by O'keefe L.M , et al. in 129 patients with acute ischemic stroke. 2.32 % of the cases in the study had hypothyroidism.1513.7 % of our study population had subclinical hypothyroidism.\\nOur result is similar to the study done by Pande, et al. to establish a relation between the variation in thyroid profile and ischemic CVAs in 75 patients within 48 hours of the event, which concluded that 12% of the cases had subclinical hypothyroidism.7 Prospective cohort study done by Chakler L, et al. on 47,573 adults (3451 subclinical hypothyroidism) from 17 cohorts concluded an increased risk of stroke on subjects younger than 65 years and those with higher TSH concentrations. Subclinical hypothyroidism (SCH) is postulated to increase stroke risk via atherogenic changes associated with abnormal thyroid function.16 The study concluded that hyperthyroidism is associated with an increased risk for ischemic stroke among young adults.11\\nIn our study HDLc was low (<40 mg/dl) in all the case with thyroid dysfunction (18% cases) this is much lower compared to the study done by Pokharel , et al., among 281 stroke patients, in which 45% had their HDLc levels below 40 mg/dl.17 TG was elevated (>150 mg/dl) in cases with overt hypothyroidism (1.4%) this is consistent with the study done by Abrams JJ , et al. which concluded TG metabolism is not grossly deranged in hypothyroidism.18 In our study we found that stroke occurred more commonly among males (67.12%) compared to females (32.88%). Women have lower stroke incidence than men, it might be due to positive effects of estrogen on the cerebral circulation. A lifetime exposure to ovarian estrogens may protect against ischemic stroke.19 Seventy-two percent of the cases in our study were smokers. Wang, et al. reported that 48% of patients with stroke smoked.20 Tsai, et al. reported 38% of the cases with ischemic stroke were smokers.21\\nOur results might not be generalized to all stroke patients as we recruited study participants from a single hospital only. As many of our patients presented to us after 48 hours of the occurrence of stroke events, we could not involve them in our study.\",\n \"The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods","results","discussion","conclusions"],"string":"[\n \"intro\",\n \"methods\",\n \"results\",\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["\nhyperthyroidism\n","\nhypothyroidism\n","\nischemic stroke\n"],"string":"[\n \"\\nhyperthyroidism\\n\",\n \"\\nhypothyroidism\\n\",\n \"\\nischemic stroke\\n\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nStroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4\nThyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4\nThe aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.\n\nMETHODS:\nThis descriptive cross-sectional study was conducted from June to December 2019 in National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu. Ethical approval from Institutional review board of NAMS (reference number. IM 175). Convenience sampling was done and the sample size of cases to be enrolled was calculated by using the formula,\nn = Z2 × p × q / e2\n = (1.645)2 × (0.5) × (1-0.5) / (0.1)2\n = 67.65\nWhere,\nn = minimum required sample size,\nZ = 1.645 at 90% Confidence Interval (CI),\np = prevalence taken as 50% for maximum sample size,\nq = 1-p\ne = margin of error, 10%\nHence, the required sample size was 67.65. Adding a 10% nonresponse rate, we enrolled 73 patients in our study. Patients with clinically diagnosed stroke, presenting within 48 hours of onset of symptoms with CT or MRI findings were included in the study. Patients with prior use of thyroid supplements recently within last 180 days and with a history of liver disease, renal failure and known thyroid disease and who don't give consent were excluded from the study. Informed written consent was taken either in Nepali or in English language, in whichever they felt comfortable. Confidentiality was maintained to the utmost. For those who are not able to give consent because of their clinical state, informed consent was taken from their close relative.\nProforma was translated in Nepali verbally if needed depending upon the cases. For diagnosing the type of stroke, every patient underwent neuroimaging with non-contrast CT of the head. Cerebral infarction was diagnosed if neurological deficits are accompanied by a hypo dense lesion >15 mm in diameter in an appropriate area on a cranial CT scan. Lacunar infarction were diagnosed if the patient presented with a pure motor stroke, a pure sensory stroke, ataxic hemiparesis or a sensorimotor stroke in the absence of a visual field defect and evidence of higher cerebral dysfunction and a hypo dense lesion of < 15 mm in diameter or normal CT scan. In the laboratory, fully automatic analyzer VITROSECi/ECiQ Immunodiagnostic Systems, VITROSTSH was measured using a chemiluminescent immunometric assay with a detection range from 0.01 to 100 m IU/L. TSH values between 0.46 and 4.68 m IU/L were considered the normal reference range (euthyroid). Specimens with TSH values above or below the normal reference range were tested for free thyroxine (FT4). FT4 was measured using a competitive chemiluminescent assay with a detection range of 0.1-12.0 ng/dL. FT4 concentrations within 0.70-2.19 ng/dL was within the normal reference range. All samples included in the study met quality control criteria. Samples with TSH values > 4.69 m IU/L and normal FT4 levels were classified as indicating SCH.\nThe collected data was stored and analyzed with the Statistical Package for the Social Sciences (SPSS) version 22.0 for windows. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.\n\nRESULTS:\nThe prevalence of altered in thyroid levels among patients undergoing ischemic stroke was 13 (17.8%) and among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Minimum age of patient in the study was 26 years and maximum age was 115 years. The mean age of presentation of cerebral infarction in this study was 63.30±19.217.\nAmong the total 73 cases, 49 were male and 24 were female. Among 49 males, 37 were euthyroid, 11 were hypothyroid and one male was hyperthyroid. Among 24 females, 23 were euthyroid and one female was hyperthyroid. On further categorization of altered thyroid status, 10 (13.7%) were subclinical hypothyroidism, 1 (1.4%) cases were of overt hypothyroidism and 2 (2.7%) cases had subclinical hyperthyroidism. With regards to the severity of subclinical hypothyroidism, out of the total 10 cases of subclinical hypothyroidism, 70% had grade IA, while the remaining 30% had grade IB severity of the status (Figure 1). In the study 49 (67.12%) of ischemic stroke were male and 24 (32.88%) were female.\nAmong euthyroid cases mean total cholesterol was 139.55 mg/dl which was higher than the mean cholesterol level in overt hypothyroid cases with 110 mg/dl. But mean total cholesterol level was lower among overt hypothyroid cases compared to euthyroid and overt hypothyroid cases. Mean TC was highest among subclinical hyperthyroid cases. Mean LDLc was highest among euthyroid cases with mean value of 73.94 mg/dl and lowest among overt hypothyroid cases with mean value of 41.00 mg/dl. TG was highest among overt hypothyroid cases with mean value of 199 mg/dl. HDLc was highest among euthyroid cases with mean value of 43.42 mg/dl. Subclinical hypothyroid cases had mean HDL value of 39.90 mg/dl (Table 1).\n\nDISCUSSION:\nStroke is a form of acute stress with detrimental effect on various neurophysiological pathways.7 Hypothyroidism is a possible risk factor for stroke.8 A number of comorbidities have been associated with increased mortality in acute stroke patients. It is not known whether hypothyroidism (either clinical or subclinical) affects outcome in patients with acute cerebrovascular disease. A neuroprotective role of hypothyroidism has been shown in acute stroke patients.8 Low T3 appeared to be associated with stroke severity and short-term outcome.9 In addition, strokes of undetermined etiology accounted for one-third to one-quarter of ischemic strokes among young people and among cases of stroke of undetermined etiology hyperthyroidism could be the underlying cause.10,11 So, TFT screening is useful in patients with acute ischemic stroke (IS) because thyroid disorders are known risk factors for cerebrovascular diseases.6 In our study we observed 17.8% of the cases had thyroid dysfunction. Our finding is similar to the findings of the retrospective study done by Xu XY , et al. in 893 patients with acute ischemic stroke in which 19% of the cases had abnormal thyroid function tests with at least one of the thyroid related hormones below or above the normal ranges.12 Unknown overt or subclinical hyperthyroidism is associated with cardio-embolic stroke.13 Thyroid dysfunction in our study was lower than the study done by Dimopolou, et al. in 33 critically ill patients under mechanical ventilation due to acute stroke; study concluded 36% of the cases had thyroid dysfunction.14 In our study we found that 1.4% of the cases had overt hypothyroidism, this is comparable to the study done by O'keefe L.M , et al. in 129 patients with acute ischemic stroke. 2.32 % of the cases in the study had hypothyroidism.1513.7 % of our study population had subclinical hypothyroidism.\nOur result is similar to the study done by Pande, et al. to establish a relation between the variation in thyroid profile and ischemic CVAs in 75 patients within 48 hours of the event, which concluded that 12% of the cases had subclinical hypothyroidism.7 Prospective cohort study done by Chakler L, et al. on 47,573 adults (3451 subclinical hypothyroidism) from 17 cohorts concluded an increased risk of stroke on subjects younger than 65 years and those with higher TSH concentrations. Subclinical hypothyroidism (SCH) is postulated to increase stroke risk via atherogenic changes associated with abnormal thyroid function.16 The study concluded that hyperthyroidism is associated with an increased risk for ischemic stroke among young adults.11\nIn our study HDLc was low (<40 mg/dl) in all the case with thyroid dysfunction (18% cases) this is much lower compared to the study done by Pokharel , et al., among 281 stroke patients, in which 45% had their HDLc levels below 40 mg/dl.17 TG was elevated (>150 mg/dl) in cases with overt hypothyroidism (1.4%) this is consistent with the study done by Abrams JJ , et al. which concluded TG metabolism is not grossly deranged in hypothyroidism.18 In our study we found that stroke occurred more commonly among males (67.12%) compared to females (32.88%). Women have lower stroke incidence than men, it might be due to positive effects of estrogen on the cerebral circulation. A lifetime exposure to ovarian estrogens may protect against ischemic stroke.19 Seventy-two percent of the cases in our study were smokers. Wang, et al. reported that 48% of patients with stroke smoked.20 Tsai, et al. reported 38% of the cases with ischemic stroke were smokers.21\nOur results might not be generalized to all stroke patients as we recruited study participants from a single hospital only. As many of our patients presented to us after 48 hours of the occurrence of stroke events, we could not involve them in our study.\n\nCONCLUSIONS:\nThe prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nStroke is broadly classified as cerebral infarction, intracerebral hemorrhage and subarachnoid hemorrhage. Neuroendocrine profile is altered in acute ischemic stroke and there is a link between hypothyroidism and atherosclerosis which in turn may lead to stroke. The objective of this study was to find out the prevalence of alteration of thyroid hormones in patients with ischemic stroke in a tertiary care center.\n\nMethods:\nThis descriptive cross-sectional study was conducted from June to December 2019 in a tertiary care center. Ethical approval was taken from Institutional review board of National Academy of Medical Sciences (reference number: IM 175). Patients with a diagnosis of stroke, without evidence of cardioembolic source, history of liver disease, renal failure and thyroid disease and who do not use thyroidal supplementation within 180 days prior the event were included. Convenience sampling was done. Data was entered in Microsoft Excel and analyzed using Statistical Package for the Social Sciences version 22. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.\n\nResults:\nThe prevalence of altered thyroid levels among 73 patients was 13 (17.8%) (90% Confidence Interval= 10.44-25.16). Among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Among severity of hypothyroid cases, subclinical hypothyroidism grade IA was seen in 51 (70%), subclinical hypothyroidism grade IB was seen in 22 (30%).\n\nConclusions:\nThe prevalence of altered thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nStroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4\nThyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4\nThe aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.\n\nCONCLUSIONS:\nThe prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nStroke is broadly classified as cerebral infarction, intracerebral hemorrhage and subarachnoid hemorrhage. Neuroendocrine profile is altered in acute ischemic stroke and there is a link between hypothyroidism and atherosclerosis which in turn may lead to stroke. The objective of this study was to find out the prevalence of alteration of thyroid hormones in patients with ischemic stroke in a tertiary care center.\n\nMethods:\nThis descriptive cross-sectional study was conducted from June to December 2019 in a tertiary care center. Ethical approval was taken from Institutional review board of National Academy of Medical Sciences (reference number: IM 175). Patients with a diagnosis of stroke, without evidence of cardioembolic source, history of liver disease, renal failure and thyroid disease and who do not use thyroidal supplementation within 180 days prior the event were included. Convenience sampling was done. Data was entered in Microsoft Excel and analyzed using Statistical Package for the Social Sciences version 22. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.\n\nResults:\nThe prevalence of altered thyroid levels among 73 patients was 13 (17.8%) (90% Confidence Interval= 10.44-25.16). Among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Among severity of hypothyroid cases, subclinical hypothyroidism grade IA was seen in 51 (70%), subclinical hypothyroidism grade IB was seen in 22 (30%).\n\nConclusions:\nThe prevalence of altered thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies."},"whole_article_text_length":{"kind":"number","value":1919,"string":"1,919"},"whole_article_abstract_length":{"kind":"number","value":298,"string":"298"},"num_sections":{"kind":"number","value":5,"string":"5"},"most_frequent_words":{"kind":"list like","value":["stroke","study","cases","patients","thyroid","hypothyroidism","ischemic","dl","subclinical","mean"],"string":"[\n \"stroke\",\n \"study\",\n \"cases\",\n \"patients\",\n \"thyroid\",\n \"hypothyroidism\",\n \"ischemic\",\n \"dl\",\n \"subclinical\",\n \"mean\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | death | year | 000 | stroke year | hemorrhage | ischemic | cause | cvas | associated [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] range | normal | sample | ct | consent | size | ft4 | reference | sample size | iu [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] mean | cases | cases mean | hypothyroid | hypothyroid cases | mg dl | mg | overt hypothyroid cases | overt hypothyroid | value [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] modifiable | stroke | risk | non | risk factors | factors | prevalence | factors great | impact reduction morbidity | non communicable diseases world [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | cases | study | mean | hypothyroidism | patients | ischemic | subclinical | thyroid | risk [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | cases | study | mean | hypothyroidism | patients | ischemic | subclinical | thyroid | risk [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| tertiary [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] June to December 2019 | tertiary ||| Institutional review board | National Academy of Medical Sciences | IM | 175 ||| 180 days ||| ||| Microsoft Excel | Statistical Package | Social Sciences | 22 ||| Point | 90% [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 73 | 13 | 17.8% | 90% | 10.44-25.16 ||| 11 | 15.1% | 2 | 2.7% ||| IA | 51 | 70% | IB | 22 | 30% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| tertiary ||| June to December 2019 | tertiary ||| Institutional review board | National Academy of Medical Sciences | IM | 175 ||| 180 days ||| ||| Microsoft Excel | Statistical Package | Social Sciences | 22 ||| Point | 90% ||| ||| 73 | 13 | 17.8% | 90% | 10.44-25.16 ||| 11 | 15.1% | 2 | 2.7% ||| IA | 51 | 70% | IB | 22 | 30% ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| tertiary ||| June to December 2019 | tertiary ||| Institutional review board | National Academy of Medical Sciences | IM | 175 ||| 180 days ||| ||| Microsoft Excel | Statistical Package | Social Sciences | 22 ||| Point | 90% ||| ||| 73 | 13 | 17.8% | 90% | 10.44-25.16 ||| 11 | 15.1% | 2 | 2.7% ||| IA | 51 | 70% | IB | 22 | 30% ||| [SUMMARY]"}}},{"rowIdx":268,"cells":{"title":{"kind":"string","value":"Post-Transplant Lymphoproliferative Diseases in Pediatric Kidney Allograft Recipients with Epstein-Barr Virus Viremia."},"pmid":{"kind":"string","value":"31373185"},"background_abstract":{"kind":"string","value":"Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, especially in children with Epstein-Barr virus (EBV) viremia (EV). We performed a retrospective study to evaluate risk factors for PTLD in children with EV."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Among 199 pediatric kidney transplantation (KT) recipients at our center from January 2001 to October 2015, records of those with EBV viral loads of > 1,000 copies/mL and/or PTLD were reviewed."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Diagnosis of PTLD was made in seven patients (PTLD group), and 39 patients had EV only (EV only group). The median time from KT to EV and PTLD diagnosis was 6.7 (range 0.4-47.8) months and 8.2 (range, 2.8-98.9) months, respectively. There were no significant differences between the groups in terms of sex, age at transplantation, donor type, EBV viral load, or EV-free duration after KT. Higher tacrolimus level before EV (hazard ratio, 44.5; P = 0.003) was an independent risk factor for PTLD in multivariate Cox regression analysis. Six patients with a high EBV load (median 171,639 copies/mL) were treated with preemptive rituximab (RTX) therapy, resulting in transient reduction of EBV load. None of these patients developed PTLD (median follow-up 51.5 months); however, two had neutropenia and two developed infection requiring hospital admission."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"In pediatric KT recipients, higher tacrolimus levels were associated with a higher incidence of PTLD. Conversely, those who received preemptive RTX for EV did not develop PTLD."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Antineoplastic Agents, Immunological","Child, Preschool","Female","Herpesvirus 4, Human","Humans","Infant","Kidney Transplantation","Lymphoproliferative Disorders","Male","Neutropenia","Proportional Hazards Models","Retrospective Studies","Risk Factors","Rituximab","Transplantation, Homologous","Viral Load","Viremia"],"string":"[\n \"Antineoplastic Agents, Immunological\",\n \"Child, Preschool\",\n \"Female\",\n \"Herpesvirus 4, Human\",\n \"Humans\",\n \"Infant\",\n \"Kidney Transplantation\",\n \"Lymphoproliferative Disorders\",\n \"Male\",\n \"Neutropenia\",\n \"Proportional Hazards Models\",\n \"Retrospective Studies\",\n \"Risk Factors\",\n \"Rituximab\",\n \"Transplantation, Homologous\",\n \"Viral Load\",\n \"Viremia\"\n]"},"pmcid":{"kind":"string","value":"6676002"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, with an incidence ranging from 1% to 16% depending on the allograft organ.1 This incidence is 0.2%–2.5% in liver and kidney transplantation cases, which is relatively lower than that in other organ transplant cases.23 Incidence rates of PTLD are higher in pediatric kidney transplant recipients than in adult kidney transplant recipients, ranging from 1.2% to 10%.456 More than 90% of pediatric PTLDs are due to Epstein-Barr virus (EBV)-positive B-cell proliferation. Previously reported risk factors for EBV-associated PTLD include recipient EBV seronegativity, degree of immunosuppression, acute rejection episode, use of OKT3 or tacrolimus, recipient age and race, allograft type, host genetic variations, and especially Epstein-Barr virus viremia (EV).789101112 While adult allograft recipients usually have acquired-immunity to EBV at the time of transplantation, pediatric allograft recipients often experience primary EBV infection after transplantation. Although EBV-infected transformed B cells are highly immunogenic and rapidly eliminated by EBV-specific T cells in healthy hosts, if immunosuppressed pediatric patients have primary EBV infection, then an inadequate immune response may result in massive infection of B cells. Primary EBV infection increases the chance of developing PTLD 6–76 fold.13141516\nEBV infection and/or reactivation, which can be detected as increasing copy numbers of EBV DNA in the peripheral blood, usually precede PTLD. Therefore, it is recommended that EBV titer be regularly monitored after solid-organ transplantation in patients at a higher risk for PTLD. The Kidney Disease Improving Global Outcomes clinical practice guideline for the care of kidney transplant recipients recommended the following monitoring regimen for EBV viral load monitoring in EBV-seronegative patients who received an allograft from a seropositive donor: every 1 week in the first 3 months after transplantation, at least monthly for 3–6 months, and then every 3 months for the rest of the first year.17 Once EBV infection and/or reactivation is noted, transplantation physicians reduce immunosuppression to prevent EBV-associated PTLD. However, EBV infection often persists and progresses despite reduction in immunosuppressive drug regimen, and there is no anti-viral agent with proven efficacy against EBV.8 In contrast, during stem cell transplantation, pre-emptive treatment with rituximab is often used to eradicate B lymphocytes, the reservoir of EBV, thereby to prevent PTLD.1819 A similar approach has been attempted in solid organ transplantation in patients at high-risk for EBV-associated PTLD.20\nIn this study, the risk factors for PTLD were assessed in pediatric kidney allograft recipients with EV, including the effect of pre-emptive rituximab treatment to prevent PTLD."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":" Patients and data collection We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\nWe performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\n Immunosuppression In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\nIn our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\n EV monitoring Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\nPrincipally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\n Pre-emptive rituximab therapy Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\nSome of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\n Statistical analysis To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\nTo determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\n Ethics statement The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\nThe study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"During the study period, 199 children underwent kidney transplantation in our center. Of these, 46 (23.1%) had viremia defined as an EBV load greater than 1,000 copies/mL in whole blood for 2 consecutive tests during a median follow-up period of 5.3 years (Fig. 1). Viremia of all patients (EBV > 1,000 copies/mL) was first detected at a median of 6.7 months (range, 0.4–47.8 months) after kidney transplantation. The diagnosis of PTLD was made in seven patients (PTLD group) at a median of 8.2 months (2.8–98.9 months) after transplantation. The other 39 patients had EV only (EV only group).\nEBV = Epstein-Barr virus, PTLD = post-transplant lymphoproliferative disease, RTX = rituximab.\n Clinical course of PTLD Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\nPatients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\n Risk factors for PTLD Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\nTable 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\n Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\nSix patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Patients and data collection","Immunosuppression","EV monitoring","Pre-emptive rituximab therapy","Statistical analysis","Ethics statement","Clinical course of PTLD","Risk factors for PTLD","Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant"],"string":"[\n \"Patients and data collection\",\n \"Immunosuppression\",\n \"EV monitoring\",\n \"Pre-emptive rituximab therapy\",\n \"Statistical analysis\",\n \"Ethics statement\",\n \"Clinical course of PTLD\",\n \"Risk factors for PTLD\",\n \"Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant\"\n]"},"other_sections_texts":{"kind":"list like","value":["We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.","In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.","Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.","Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.","To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).","The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.","Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.","Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.","Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab."],"string":"[\n \"We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\",\n \"In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\",\n \"Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\",\n \"Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\",\n \"To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\",\n \"The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\",\n \"Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\",\n \"Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\\nValues are expressed as numbers (%) and median (range).\\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\",\n \"Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\\naGenetic disorder caused by WT1 mutation.\\nEBV = Epstein-Barr virus, RTX = rituximab.\\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","Patients and data collection","Immunosuppression","EV monitoring","Pre-emptive rituximab therapy","Statistical analysis","Ethics statement","RESULTS","Clinical course of PTLD","Risk factors for PTLD","Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"Patients and data collection\",\n \"Immunosuppression\",\n \"EV monitoring\",\n \"Pre-emptive rituximab therapy\",\n \"Statistical analysis\",\n \"Ethics statement\",\n \"RESULTS\",\n \"Clinical course of PTLD\",\n \"Risk factors for PTLD\",\n \"Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, with an incidence ranging from 1% to 16% depending on the allograft organ.1 This incidence is 0.2%–2.5% in liver and kidney transplantation cases, which is relatively lower than that in other organ transplant cases.23 Incidence rates of PTLD are higher in pediatric kidney transplant recipients than in adult kidney transplant recipients, ranging from 1.2% to 10%.456 More than 90% of pediatric PTLDs are due to Epstein-Barr virus (EBV)-positive B-cell proliferation. Previously reported risk factors for EBV-associated PTLD include recipient EBV seronegativity, degree of immunosuppression, acute rejection episode, use of OKT3 or tacrolimus, recipient age and race, allograft type, host genetic variations, and especially Epstein-Barr virus viremia (EV).789101112 While adult allograft recipients usually have acquired-immunity to EBV at the time of transplantation, pediatric allograft recipients often experience primary EBV infection after transplantation. Although EBV-infected transformed B cells are highly immunogenic and rapidly eliminated by EBV-specific T cells in healthy hosts, if immunosuppressed pediatric patients have primary EBV infection, then an inadequate immune response may result in massive infection of B cells. Primary EBV infection increases the chance of developing PTLD 6–76 fold.13141516\nEBV infection and/or reactivation, which can be detected as increasing copy numbers of EBV DNA in the peripheral blood, usually precede PTLD. Therefore, it is recommended that EBV titer be regularly monitored after solid-organ transplantation in patients at a higher risk for PTLD. The Kidney Disease Improving Global Outcomes clinical practice guideline for the care of kidney transplant recipients recommended the following monitoring regimen for EBV viral load monitoring in EBV-seronegative patients who received an allograft from a seropositive donor: every 1 week in the first 3 months after transplantation, at least monthly for 3–6 months, and then every 3 months for the rest of the first year.17 Once EBV infection and/or reactivation is noted, transplantation physicians reduce immunosuppression to prevent EBV-associated PTLD. However, EBV infection often persists and progresses despite reduction in immunosuppressive drug regimen, and there is no anti-viral agent with proven efficacy against EBV.8 In contrast, during stem cell transplantation, pre-emptive treatment with rituximab is often used to eradicate B lymphocytes, the reservoir of EBV, thereby to prevent PTLD.1819 A similar approach has been attempted in solid organ transplantation in patients at high-risk for EBV-associated PTLD.20\nIn this study, the risk factors for PTLD were assessed in pediatric kidney allograft recipients with EV, including the effect of pre-emptive rituximab treatment to prevent PTLD."," Patients and data collection We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\nWe performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\n Immunosuppression In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\nIn our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\n EV monitoring Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\nPrincipally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\n Pre-emptive rituximab therapy Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\nSome of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\n Statistical analysis To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\nTo determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\n Ethics statement The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\nThe study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.","We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.","In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.","Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.","Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.","To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).","The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.","During the study period, 199 children underwent kidney transplantation in our center. Of these, 46 (23.1%) had viremia defined as an EBV load greater than 1,000 copies/mL in whole blood for 2 consecutive tests during a median follow-up period of 5.3 years (Fig. 1). Viremia of all patients (EBV > 1,000 copies/mL) was first detected at a median of 6.7 months (range, 0.4–47.8 months) after kidney transplantation. The diagnosis of PTLD was made in seven patients (PTLD group) at a median of 8.2 months (2.8–98.9 months) after transplantation. The other 39 patients had EV only (EV only group).\nEBV = Epstein-Barr virus, PTLD = post-transplant lymphoproliferative disease, RTX = rituximab.\n Clinical course of PTLD Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\nPatients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\n Risk factors for PTLD Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\nTable 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\n Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\nSix patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.","Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.","Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.","Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.","During the last 15 years, among the almost 200 pediatric kidney transplantation recipients at our center, seven developed EBV-associated PTLD (3.5%). In our study, the risk factor for PTLD in pediatric kidney transplant recipients with EV was a high tacrolimus level before EV. Twenty (43.4%) out of 46 recipients who had viremia were EBV seronegative, which is similar to the rates reported in previous studies in North America and Europe (19%–57%).212223 Pre-transplant recipient EBV seronegativity is a well-known risk factor for PTLD. In adult transplant studies, the rate of developing PTLD is 5–12 fold higher in EBV-seronegative patients than in EBV-seropositive patients.2324 McDonald et al.6 reported that EBV-seronegative pediatric subjects have a 4.7-fold higher relative HR than EBV-positive subjects. In our study, recipient EBV seronegativity did not increase the risk of PTLD in multivariate Cox regression analysis. While transplant from an EBV-seropositive donor to a seronegative recipient has been associated with the development of PTLD,25 there was no statistically significant difference in EBV serostatus (donor/recipient) in the present study. This finding was attributed mainly to the fact that there were no EBV seropositive recipients in PTLD group and we did not have serostatus information for the majority of donors (67.3%).\nThe majority of kidney allograft recipients in this study were given tacrolimus, and mycophenolate was used in more than 80% of patients instead of azathioprine.26 By the late 2000s, monoclonal interleukin-2 receptor antibodies were used as induction therapy in up to 80% of patients. Basiliximab, a monoclonal antibody which targets activated T lymphocytes, was not related to PTLD risk in our study, as previously reported.427 Several studies have suggested that higher tacrolimus levels are associated with higher risk for PTLD, and others have reported that the net state of immunosuppression, rather than any individual agent, increases the risk for PTLD.282930 In our study, all patients received tacrolimus for maintenance immunosuppression, and we found that a higher pre-EV tacrolimus level in the PTLD group compared with the EV only group was a risk factor for PTLD.\nRegular monitoring of EBV viral load and early recognition of recipients at high risk of PTLD have been identified as clinical priorities in recent years.31 Previous studies have shown that elevated levels of EBV DNA and persistent high EBV loads are risk factors for PTLD,122032 but no clear cut-off point of EBV viral load for the prediction of PTLD development has been determined. We did not find a significant relationship between EV and PTLD in this study; however, six patients with a high EBV titer were treated with preemptive RTX and they did not develop PTLD. Because they were included in analysis, this could have confounded the causality of high EBV titer and PTLD development.\nTreatment strategies for organ transplant recipients with EV include reduction of immunosuppression with/without antiviral agents, immunoglobulin, or RTX.33 These treatments are still undergoing clinical studies. Preemptive administration of RTX is widely used and has been demonstrated to reduce the incidence of PTLD in stem cell transplant recipients with a high EBV viral load.183435 Pre-emptive RTX therapy has been reported in 14.5% of global transplant programs, and in more than 60% of pediatric transplant patients worldwide.33 However, only one study reported the use of RTX in five pediatric renal allograft recipients,20 and there have been no prospective studies on the efficacy of pre-emptive RTX therapy in solid organ transplantation. Rituximab is a murine/human chimeric monoclonal anti-CD20 antibody and is able to deplete circulating B cells rapidly, including those infected with EBV. Although RTX was effective for reducing EBV viral load in our patients, this reduction was not permanent in line with previous reports.20 In addition, significant adverse effects, such as infection and neutropenia, accompanied RTX administration. In patients with stem cell transplant (SCT), preemptive RTX was not associated with an increase in infectious complications.1819 This is important because, while SCT recipients are able to discontinue their immunosuppressive agents within 6–9 months after SCT, recipients of solid organ transplantation have to be on immunosuppressive agents for as long as their allografts are functioning. Therefore, the long-term use of immunosuppressive agents may explain why infection and neutropenia are common clinical findings after RTX therapy in this patient population.\nAlthough the small sample size of the current study precludes us from drawing any definite conclusions, our observations suggest that preemptive RTX treatment may effectively reduce high EBV viral load in pediatric recipients of solid organ transplants. Because RTX therapy eradicates B-lymphocytes including transformed lymphocytes, the risk of PTLD might be reduced at least during the period of B cell depletion. However, one should take into account that this treatment significantly increases the risk of neutropenia and infection. More research into the influence of preemptive RTX therapy on PTLD development is needed.\nThe occurrence of PTLD in kidney transplant recipients follows a bimodal distribution, with one peak in the first year and the second in the later post-transplantation period. Early PTLD, occurring within the first year of transplantation, is associated with EBV infection and tends to occur more commonly in children than adults.123637 In this study, while six patients in the PTLD group were diagnosed with PTLD within the first year of renal transplantation, one patient with WT1 mutation developed PTLD later than 8.2 years after renal transplantation. We suspect that in this patient, the WT1 mutation of a tumor suppressor gene might have increased the risk of PTLD, and especially that of late-onset. The occurrence of PTLD in patients with WT1 mutation has been reported previously, within the first year in two cases and later than the first year in another.383940 Based on the limited availability of clinical data, the association between WT1 mutation and development of PTLD requires further investigation in future clinical studies and is beyond the scope of the current study.\nThere are several limitations of this study. First, this is a retrospective observational study of a single center and the number of the patients observed was therefore small. The limitations of this small sample size might have affected the outcome of multivariate analysis. In addition, data of donor EBV serology were not uniformly available in our study. Data on donor serology status was only available for 33% of participants. Therefore, our study did not show any association between PTLD and EBV-donor/recipient serostatus. Finally, the number of patients who were treated with RTX was not large enough to draw any definitive conclusions.\nIn summary, this study demonstrates that a higher tacrolimus level before EV is correlated with the development of PTLD. Preemptive RTX appears to be effective for reducing EBV viral load in pediatric kidney transplant recipients. However, the reduction of EBV viral load was not persistent, and adverse effects of RTX, namely infection and neutropenia, were clinically significant."],"string":"[\n \"Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, with an incidence ranging from 1% to 16% depending on the allograft organ.1 This incidence is 0.2%–2.5% in liver and kidney transplantation cases, which is relatively lower than that in other organ transplant cases.23 Incidence rates of PTLD are higher in pediatric kidney transplant recipients than in adult kidney transplant recipients, ranging from 1.2% to 10%.456 More than 90% of pediatric PTLDs are due to Epstein-Barr virus (EBV)-positive B-cell proliferation. Previously reported risk factors for EBV-associated PTLD include recipient EBV seronegativity, degree of immunosuppression, acute rejection episode, use of OKT3 or tacrolimus, recipient age and race, allograft type, host genetic variations, and especially Epstein-Barr virus viremia (EV).789101112 While adult allograft recipients usually have acquired-immunity to EBV at the time of transplantation, pediatric allograft recipients often experience primary EBV infection after transplantation. Although EBV-infected transformed B cells are highly immunogenic and rapidly eliminated by EBV-specific T cells in healthy hosts, if immunosuppressed pediatric patients have primary EBV infection, then an inadequate immune response may result in massive infection of B cells. Primary EBV infection increases the chance of developing PTLD 6–76 fold.13141516\\nEBV infection and/or reactivation, which can be detected as increasing copy numbers of EBV DNA in the peripheral blood, usually precede PTLD. Therefore, it is recommended that EBV titer be regularly monitored after solid-organ transplantation in patients at a higher risk for PTLD. The Kidney Disease Improving Global Outcomes clinical practice guideline for the care of kidney transplant recipients recommended the following monitoring regimen for EBV viral load monitoring in EBV-seronegative patients who received an allograft from a seropositive donor: every 1 week in the first 3 months after transplantation, at least monthly for 3–6 months, and then every 3 months for the rest of the first year.17 Once EBV infection and/or reactivation is noted, transplantation physicians reduce immunosuppression to prevent EBV-associated PTLD. However, EBV infection often persists and progresses despite reduction in immunosuppressive drug regimen, and there is no anti-viral agent with proven efficacy against EBV.8 In contrast, during stem cell transplantation, pre-emptive treatment with rituximab is often used to eradicate B lymphocytes, the reservoir of EBV, thereby to prevent PTLD.1819 A similar approach has been attempted in solid organ transplantation in patients at high-risk for EBV-associated PTLD.20\\nIn this study, the risk factors for PTLD were assessed in pediatric kidney allograft recipients with EV, including the effect of pre-emptive rituximab treatment to prevent PTLD.\",\n \" Patients and data collection We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\\nWe performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\\n Immunosuppression In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\\nIn our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\\n EV monitoring Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\\nPrincipally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\\n Pre-emptive rituximab therapy Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\\nSome of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\\n Statistical analysis To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\\nTo determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\\n Ethics statement The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\\nThe study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\",\n \"We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\",\n \"In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\",\n \"Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\",\n \"Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\",\n \"To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\",\n \"The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\",\n \"During the study period, 199 children underwent kidney transplantation in our center. Of these, 46 (23.1%) had viremia defined as an EBV load greater than 1,000 copies/mL in whole blood for 2 consecutive tests during a median follow-up period of 5.3 years (Fig. 1). Viremia of all patients (EBV > 1,000 copies/mL) was first detected at a median of 6.7 months (range, 0.4–47.8 months) after kidney transplantation. The diagnosis of PTLD was made in seven patients (PTLD group) at a median of 8.2 months (2.8–98.9 months) after transplantation. The other 39 patients had EV only (EV only group).\\nEBV = Epstein-Barr virus, PTLD = post-transplant lymphoproliferative disease, RTX = rituximab.\\n Clinical course of PTLD Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\\nPatients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\\n Risk factors for PTLD Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\\nValues are expressed as numbers (%) and median (range).\\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\\nTable 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\\nValues are expressed as numbers (%) and median (range).\\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\\n Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\\naGenetic disorder caused by WT1 mutation.\\nEBV = Epstein-Barr virus, RTX = rituximab.\\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\\nSix patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\\naGenetic disorder caused by WT1 mutation.\\nEBV = Epstein-Barr virus, RTX = rituximab.\\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\",\n \"Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\",\n \"Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\\nValues are expressed as numbers (%) and median (range).\\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\",\n \"Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\\naGenetic disorder caused by WT1 mutation.\\nEBV = Epstein-Barr virus, RTX = rituximab.\\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\",\n \"During the last 15 years, among the almost 200 pediatric kidney transplantation recipients at our center, seven developed EBV-associated PTLD (3.5%). In our study, the risk factor for PTLD in pediatric kidney transplant recipients with EV was a high tacrolimus level before EV. Twenty (43.4%) out of 46 recipients who had viremia were EBV seronegative, which is similar to the rates reported in previous studies in North America and Europe (19%–57%).212223 Pre-transplant recipient EBV seronegativity is a well-known risk factor for PTLD. In adult transplant studies, the rate of developing PTLD is 5–12 fold higher in EBV-seronegative patients than in EBV-seropositive patients.2324 McDonald et al.6 reported that EBV-seronegative pediatric subjects have a 4.7-fold higher relative HR than EBV-positive subjects. In our study, recipient EBV seronegativity did not increase the risk of PTLD in multivariate Cox regression analysis. While transplant from an EBV-seropositive donor to a seronegative recipient has been associated with the development of PTLD,25 there was no statistically significant difference in EBV serostatus (donor/recipient) in the present study. This finding was attributed mainly to the fact that there were no EBV seropositive recipients in PTLD group and we did not have serostatus information for the majority of donors (67.3%).\\nThe majority of kidney allograft recipients in this study were given tacrolimus, and mycophenolate was used in more than 80% of patients instead of azathioprine.26 By the late 2000s, monoclonal interleukin-2 receptor antibodies were used as induction therapy in up to 80% of patients. Basiliximab, a monoclonal antibody which targets activated T lymphocytes, was not related to PTLD risk in our study, as previously reported.427 Several studies have suggested that higher tacrolimus levels are associated with higher risk for PTLD, and others have reported that the net state of immunosuppression, rather than any individual agent, increases the risk for PTLD.282930 In our study, all patients received tacrolimus for maintenance immunosuppression, and we found that a higher pre-EV tacrolimus level in the PTLD group compared with the EV only group was a risk factor for PTLD.\\nRegular monitoring of EBV viral load and early recognition of recipients at high risk of PTLD have been identified as clinical priorities in recent years.31 Previous studies have shown that elevated levels of EBV DNA and persistent high EBV loads are risk factors for PTLD,122032 but no clear cut-off point of EBV viral load for the prediction of PTLD development has been determined. We did not find a significant relationship between EV and PTLD in this study; however, six patients with a high EBV titer were treated with preemptive RTX and they did not develop PTLD. Because they were included in analysis, this could have confounded the causality of high EBV titer and PTLD development.\\nTreatment strategies for organ transplant recipients with EV include reduction of immunosuppression with/without antiviral agents, immunoglobulin, or RTX.33 These treatments are still undergoing clinical studies. Preemptive administration of RTX is widely used and has been demonstrated to reduce the incidence of PTLD in stem cell transplant recipients with a high EBV viral load.183435 Pre-emptive RTX therapy has been reported in 14.5% of global transplant programs, and in more than 60% of pediatric transplant patients worldwide.33 However, only one study reported the use of RTX in five pediatric renal allograft recipients,20 and there have been no prospective studies on the efficacy of pre-emptive RTX therapy in solid organ transplantation. Rituximab is a murine/human chimeric monoclonal anti-CD20 antibody and is able to deplete circulating B cells rapidly, including those infected with EBV. Although RTX was effective for reducing EBV viral load in our patients, this reduction was not permanent in line with previous reports.20 In addition, significant adverse effects, such as infection and neutropenia, accompanied RTX administration. In patients with stem cell transplant (SCT), preemptive RTX was not associated with an increase in infectious complications.1819 This is important because, while SCT recipients are able to discontinue their immunosuppressive agents within 6–9 months after SCT, recipients of solid organ transplantation have to be on immunosuppressive agents for as long as their allografts are functioning. Therefore, the long-term use of immunosuppressive agents may explain why infection and neutropenia are common clinical findings after RTX therapy in this patient population.\\nAlthough the small sample size of the current study precludes us from drawing any definite conclusions, our observations suggest that preemptive RTX treatment may effectively reduce high EBV viral load in pediatric recipients of solid organ transplants. Because RTX therapy eradicates B-lymphocytes including transformed lymphocytes, the risk of PTLD might be reduced at least during the period of B cell depletion. However, one should take into account that this treatment significantly increases the risk of neutropenia and infection. More research into the influence of preemptive RTX therapy on PTLD development is needed.\\nThe occurrence of PTLD in kidney transplant recipients follows a bimodal distribution, with one peak in the first year and the second in the later post-transplantation period. Early PTLD, occurring within the first year of transplantation, is associated with EBV infection and tends to occur more commonly in children than adults.123637 In this study, while six patients in the PTLD group were diagnosed with PTLD within the first year of renal transplantation, one patient with WT1 mutation developed PTLD later than 8.2 years after renal transplantation. We suspect that in this patient, the WT1 mutation of a tumor suppressor gene might have increased the risk of PTLD, and especially that of late-onset. The occurrence of PTLD in patients with WT1 mutation has been reported previously, within the first year in two cases and later than the first year in another.383940 Based on the limited availability of clinical data, the association between WT1 mutation and development of PTLD requires further investigation in future clinical studies and is beyond the scope of the current study.\\nThere are several limitations of this study. First, this is a retrospective observational study of a single center and the number of the patients observed was therefore small. The limitations of this small sample size might have affected the outcome of multivariate analysis. In addition, data of donor EBV serology were not uniformly available in our study. Data on donor serology status was only available for 33% of participants. Therefore, our study did not show any association between PTLD and EBV-donor/recipient serostatus. Finally, the number of patients who were treated with RTX was not large enough to draw any definitive conclusions.\\nIn summary, this study demonstrates that a higher tacrolimus level before EV is correlated with the development of PTLD. Preemptive RTX appears to be effective for reducing EBV viral load in pediatric kidney transplant recipients. However, the reduction of EBV viral load was not persistent, and adverse effects of RTX, namely infection and neutropenia, were clinically significant.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,null,"results",null,null,null,"discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Post-Transplant Lymphoproliferative Disease","Kidney Transplantation","Epstein-Barr Virus","Rituximab"],"string":"[\n \"Post-Transplant Lymphoproliferative Disease\",\n \"Kidney Transplantation\",\n \"Epstein-Barr Virus\",\n \"Rituximab\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nPost-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, with an incidence ranging from 1% to 16% depending on the allograft organ.1 This incidence is 0.2%–2.5% in liver and kidney transplantation cases, which is relatively lower than that in other organ transplant cases.23 Incidence rates of PTLD are higher in pediatric kidney transplant recipients than in adult kidney transplant recipients, ranging from 1.2% to 10%.456 More than 90% of pediatric PTLDs are due to Epstein-Barr virus (EBV)-positive B-cell proliferation. Previously reported risk factors for EBV-associated PTLD include recipient EBV seronegativity, degree of immunosuppression, acute rejection episode, use of OKT3 or tacrolimus, recipient age and race, allograft type, host genetic variations, and especially Epstein-Barr virus viremia (EV).789101112 While adult allograft recipients usually have acquired-immunity to EBV at the time of transplantation, pediatric allograft recipients often experience primary EBV infection after transplantation. Although EBV-infected transformed B cells are highly immunogenic and rapidly eliminated by EBV-specific T cells in healthy hosts, if immunosuppressed pediatric patients have primary EBV infection, then an inadequate immune response may result in massive infection of B cells. Primary EBV infection increases the chance of developing PTLD 6–76 fold.13141516\nEBV infection and/or reactivation, which can be detected as increasing copy numbers of EBV DNA in the peripheral blood, usually precede PTLD. Therefore, it is recommended that EBV titer be regularly monitored after solid-organ transplantation in patients at a higher risk for PTLD. The Kidney Disease Improving Global Outcomes clinical practice guideline for the care of kidney transplant recipients recommended the following monitoring regimen for EBV viral load monitoring in EBV-seronegative patients who received an allograft from a seropositive donor: every 1 week in the first 3 months after transplantation, at least monthly for 3–6 months, and then every 3 months for the rest of the first year.17 Once EBV infection and/or reactivation is noted, transplantation physicians reduce immunosuppression to prevent EBV-associated PTLD. However, EBV infection often persists and progresses despite reduction in immunosuppressive drug regimen, and there is no anti-viral agent with proven efficacy against EBV.8 In contrast, during stem cell transplantation, pre-emptive treatment with rituximab is often used to eradicate B lymphocytes, the reservoir of EBV, thereby to prevent PTLD.1819 A similar approach has been attempted in solid organ transplantation in patients at high-risk for EBV-associated PTLD.20\nIn this study, the risk factors for PTLD were assessed in pediatric kidney allograft recipients with EV, including the effect of pre-emptive rituximab treatment to prevent PTLD.\n\nMETHODS:\n Patients and data collection We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\nWe performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\n Immunosuppression In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\nIn our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\n EV monitoring Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\nPrincipally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\n Pre-emptive rituximab therapy Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\nSome of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\n Statistical analysis To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\nTo determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\n Ethics statement The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\nThe study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\n\nPatients and data collection:\nWe performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\n\nImmunosuppression:\nIn our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\n\nEV monitoring:\nPrincipally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\n\nPre-emptive rituximab therapy:\nSome of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\n\nStatistical analysis:\nTo determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\n\nEthics statement:\nThe study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\n\nRESULTS:\nDuring the study period, 199 children underwent kidney transplantation in our center. Of these, 46 (23.1%) had viremia defined as an EBV load greater than 1,000 copies/mL in whole blood for 2 consecutive tests during a median follow-up period of 5.3 years (Fig. 1). Viremia of all patients (EBV > 1,000 copies/mL) was first detected at a median of 6.7 months (range, 0.4–47.8 months) after kidney transplantation. The diagnosis of PTLD was made in seven patients (PTLD group) at a median of 8.2 months (2.8–98.9 months) after transplantation. The other 39 patients had EV only (EV only group).\nEBV = Epstein-Barr virus, PTLD = post-transplant lymphoproliferative disease, RTX = rituximab.\n Clinical course of PTLD Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\nPatients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\n Risk factors for PTLD Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\nTable 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\n Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\nSix patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\n\nClinical course of PTLD:\nPatients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\n\nRisk factors for PTLD:\nTable 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\n\nEfficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant:\nSix patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\n\nDISCUSSION:\nDuring the last 15 years, among the almost 200 pediatric kidney transplantation recipients at our center, seven developed EBV-associated PTLD (3.5%). In our study, the risk factor for PTLD in pediatric kidney transplant recipients with EV was a high tacrolimus level before EV. Twenty (43.4%) out of 46 recipients who had viremia were EBV seronegative, which is similar to the rates reported in previous studies in North America and Europe (19%–57%).212223 Pre-transplant recipient EBV seronegativity is a well-known risk factor for PTLD. In adult transplant studies, the rate of developing PTLD is 5–12 fold higher in EBV-seronegative patients than in EBV-seropositive patients.2324 McDonald et al.6 reported that EBV-seronegative pediatric subjects have a 4.7-fold higher relative HR than EBV-positive subjects. In our study, recipient EBV seronegativity did not increase the risk of PTLD in multivariate Cox regression analysis. While transplant from an EBV-seropositive donor to a seronegative recipient has been associated with the development of PTLD,25 there was no statistically significant difference in EBV serostatus (donor/recipient) in the present study. This finding was attributed mainly to the fact that there were no EBV seropositive recipients in PTLD group and we did not have serostatus information for the majority of donors (67.3%).\nThe majority of kidney allograft recipients in this study were given tacrolimus, and mycophenolate was used in more than 80% of patients instead of azathioprine.26 By the late 2000s, monoclonal interleukin-2 receptor antibodies were used as induction therapy in up to 80% of patients. Basiliximab, a monoclonal antibody which targets activated T lymphocytes, was not related to PTLD risk in our study, as previously reported.427 Several studies have suggested that higher tacrolimus levels are associated with higher risk for PTLD, and others have reported that the net state of immunosuppression, rather than any individual agent, increases the risk for PTLD.282930 In our study, all patients received tacrolimus for maintenance immunosuppression, and we found that a higher pre-EV tacrolimus level in the PTLD group compared with the EV only group was a risk factor for PTLD.\nRegular monitoring of EBV viral load and early recognition of recipients at high risk of PTLD have been identified as clinical priorities in recent years.31 Previous studies have shown that elevated levels of EBV DNA and persistent high EBV loads are risk factors for PTLD,122032 but no clear cut-off point of EBV viral load for the prediction of PTLD development has been determined. We did not find a significant relationship between EV and PTLD in this study; however, six patients with a high EBV titer were treated with preemptive RTX and they did not develop PTLD. Because they were included in analysis, this could have confounded the causality of high EBV titer and PTLD development.\nTreatment strategies for organ transplant recipients with EV include reduction of immunosuppression with/without antiviral agents, immunoglobulin, or RTX.33 These treatments are still undergoing clinical studies. Preemptive administration of RTX is widely used and has been demonstrated to reduce the incidence of PTLD in stem cell transplant recipients with a high EBV viral load.183435 Pre-emptive RTX therapy has been reported in 14.5% of global transplant programs, and in more than 60% of pediatric transplant patients worldwide.33 However, only one study reported the use of RTX in five pediatric renal allograft recipients,20 and there have been no prospective studies on the efficacy of pre-emptive RTX therapy in solid organ transplantation. Rituximab is a murine/human chimeric monoclonal anti-CD20 antibody and is able to deplete circulating B cells rapidly, including those infected with EBV. Although RTX was effective for reducing EBV viral load in our patients, this reduction was not permanent in line with previous reports.20 In addition, significant adverse effects, such as infection and neutropenia, accompanied RTX administration. In patients with stem cell transplant (SCT), preemptive RTX was not associated with an increase in infectious complications.1819 This is important because, while SCT recipients are able to discontinue their immunosuppressive agents within 6–9 months after SCT, recipients of solid organ transplantation have to be on immunosuppressive agents for as long as their allografts are functioning. Therefore, the long-term use of immunosuppressive agents may explain why infection and neutropenia are common clinical findings after RTX therapy in this patient population.\nAlthough the small sample size of the current study precludes us from drawing any definite conclusions, our observations suggest that preemptive RTX treatment may effectively reduce high EBV viral load in pediatric recipients of solid organ transplants. Because RTX therapy eradicates B-lymphocytes including transformed lymphocytes, the risk of PTLD might be reduced at least during the period of B cell depletion. However, one should take into account that this treatment significantly increases the risk of neutropenia and infection. More research into the influence of preemptive RTX therapy on PTLD development is needed.\nThe occurrence of PTLD in kidney transplant recipients follows a bimodal distribution, with one peak in the first year and the second in the later post-transplantation period. Early PTLD, occurring within the first year of transplantation, is associated with EBV infection and tends to occur more commonly in children than adults.123637 In this study, while six patients in the PTLD group were diagnosed with PTLD within the first year of renal transplantation, one patient with WT1 mutation developed PTLD later than 8.2 years after renal transplantation. We suspect that in this patient, the WT1 mutation of a tumor suppressor gene might have increased the risk of PTLD, and especially that of late-onset. The occurrence of PTLD in patients with WT1 mutation has been reported previously, within the first year in two cases and later than the first year in another.383940 Based on the limited availability of clinical data, the association between WT1 mutation and development of PTLD requires further investigation in future clinical studies and is beyond the scope of the current study.\nThere are several limitations of this study. First, this is a retrospective observational study of a single center and the number of the patients observed was therefore small. The limitations of this small sample size might have affected the outcome of multivariate analysis. In addition, data of donor EBV serology were not uniformly available in our study. Data on donor serology status was only available for 33% of participants. Therefore, our study did not show any association between PTLD and EBV-donor/recipient serostatus. Finally, the number of patients who were treated with RTX was not large enough to draw any definitive conclusions.\nIn summary, this study demonstrates that a higher tacrolimus level before EV is correlated with the development of PTLD. Preemptive RTX appears to be effective for reducing EBV viral load in pediatric kidney transplant recipients. However, the reduction of EBV viral load was not persistent, and adverse effects of RTX, namely infection and neutropenia, were clinically significant.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nPost-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, especially in children with Epstein-Barr virus (EBV) viremia (EV). We performed a retrospective study to evaluate risk factors for PTLD in children with EV.\n\nMethods:\nAmong 199 pediatric kidney transplantation (KT) recipients at our center from January 2001 to October 2015, records of those with EBV viral loads of > 1,000 copies/mL and/or PTLD were reviewed.\n\nResults:\nDiagnosis of PTLD was made in seven patients (PTLD group), and 39 patients had EV only (EV only group). The median time from KT to EV and PTLD diagnosis was 6.7 (range 0.4-47.8) months and 8.2 (range, 2.8-98.9) months, respectively. There were no significant differences between the groups in terms of sex, age at transplantation, donor type, EBV viral load, or EV-free duration after KT. Higher tacrolimus level before EV (hazard ratio, 44.5; P = 0.003) was an independent risk factor for PTLD in multivariate Cox regression analysis. Six patients with a high EBV load (median 171,639 copies/mL) were treated with preemptive rituximab (RTX) therapy, resulting in transient reduction of EBV load. None of these patients developed PTLD (median follow-up 51.5 months); however, two had neutropenia and two developed infection requiring hospital admission.\n\nConclusions:\nIn pediatric KT recipients, higher tacrolimus levels were associated with a higher incidence of PTLD. Conversely, those who received preemptive RTX for EV did not develop PTLD."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":8529,"string":"8,529"},"whole_article_abstract_length":{"kind":"number","value":310,"string":"310"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["ebv","ptld","patients","rtx","ev","transplantation","months","kidney","therapy","ml"],"string":"[\n \"ebv\",\n \"ptld\",\n \"patients\",\n \"rtx\",\n \"ev\",\n \"transplantation\",\n \"months\",\n \"kidney\",\n \"therapy\",\n \"ml\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Post-Transplant Lymphoproliferative Disease | Kidney Transplantation | Epstein-Barr Virus | Rituximab [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Post-Transplant Lymphoproliferative Disease | Kidney Transplantation | Epstein-Barr Virus | Rituximab [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Post-Transplant Lymphoproliferative Disease | Kidney Transplantation | Epstein-Barr Virus | Rituximab [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Post-Transplant Lymphoproliferative Disease | Kidney Transplantation | Epstein-Barr Virus | Rituximab [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Antineoplastic Agents, Immunological | Child, Preschool | Female | Herpesvirus 4, Human | Humans | Infant | Kidney Transplantation | Lymphoproliferative Disorders | Male | Neutropenia | Proportional Hazards Models | Retrospective Studies | Risk Factors | Rituximab | Transplantation, Homologous | Viral Load | Viremia [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Antineoplastic Agents, Immunological | Child, Preschool | Female | Herpesvirus 4, Human | Humans | Infant | Kidney Transplantation | Lymphoproliferative Disorders | Male | Neutropenia | Proportional Hazards Models | Retrospective Studies | Risk Factors | Rituximab | Transplantation, Homologous | Viral Load | Viremia [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Antineoplastic Agents, Immunological | Child, Preschool | Female | Herpesvirus 4, Human | Humans | Infant | Kidney Transplantation | Lymphoproliferative Disorders | Male | Neutropenia | Proportional Hazards Models | Retrospective Studies | Risk Factors | Rituximab | Transplantation, Homologous | Viral Load | Viremia [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Antineoplastic Agents, Immunological | Child, Preschool | Female | Herpesvirus 4, Human | Humans | Infant | Kidney Transplantation | Lymphoproliferative Disorders | Male | Neutropenia | Proportional Hazards Models | Retrospective Studies | Risk Factors | Rituximab | Transplantation, Homologous | Viral Load | Viremia [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | ptld | patients | rtx | ev | transplantation | months | kidney | therapy | ml [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | ptld | patients | rtx | ev | transplantation | months | kidney | therapy | ml [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | ptld | patients | rtx | ev | transplantation | months | kidney | therapy | ml [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | ptld | patients | rtx | ev | transplantation | months | kidney | therapy | ml [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | ptld | ebv infection | transplantation | infection | pediatric | allograft | recipients | primary ebv infection | primary ebv [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | ebv | transplantation | patients | therapy | months | ev | variables | tacrolimus | ml [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] rtx | ebv | patients | ptld | ev | months | group | median | ml | lymph [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | ptld | patients | rtx | months | ev | transplantation | ml | kidney | therapy [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Epstein-Barr | EBV | EV ||| EV [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 199 | KT | January 2001 to October 2015 | EBV | 1,000 [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] seven | 39 | EV | EV ||| KT | EV | 6.7 | 0.4-47.8 | months | 8.2 | 2.8 | months ||| EV | KT ||| EV | 44.5 | P = | 0.003 ||| Six | EBV | 171,639 | EBV ||| 51.5 months | two | two [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Epstein-Barr | EBV | EV ||| EV ||| 199 | KT | January 2001 to October 2015 | EBV | 1,000 ||| seven | 39 | EV | EV ||| KT | EV | 6.7 | 0.4-47.8 | months | 8.2 | 2.8 | months ||| EV | KT ||| EV | 44.5 | P = | 0.003 ||| Six | EBV | 171,639 | EBV ||| 51.5 months | two | two ||| KT ||| RTX | EV [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":269,"cells":{"title":{"kind":"string","value":"Influence of ischemia before vein grafting on early hyperplasia of the graft and the dynamic changes of the intima after grafting."},"pmid":{"kind":"string","value":"23006637"},"background_abstract":{"kind":"string","value":"To investigate both the influence of ischemia before grafting on early hyperplasia of the vein grafts, and the dynamic changes of the intima after grafting in a rabbit model of vein graft disease."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We performed paired vein graft experiments under different ischemic conditions (15 vs. 60 min; 15 vs. 90 min) in the neck of the rabbits and compared the differences between the grafts. Clopidogrel, an anti-platelet agent, was administered before and after surgery. Twenty-eight days after the grafting procedure, the veins were evaluated microscopically. The dynamic changes of the intima after grafting were evaluated by scanning electron microscopy over time."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The vein grafts subjected to 60- or 90-min ischemia exhibited no differences compared to those subjected to 15-min ischemia in terms of the mean thickness of the intimal, medial, and adventitial layers of the graft. Similarly, there was no difference in the Ki-67 labeling index (proliferation marker) between the vein grafts. Vein grafts with 15-min ischemia lost endothelial cells (ECs) but healed by 3 days post graft, whereas vein grafts with 90-min ischemia suffered serious EC loss, which was restored with new ECs during days 2 to 14 post graft."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Ninety-minute ischemia before vein grafting can cause serious EC loss, but does not increase early intimal hyperplasia when clopidogrel is administered. Protecting the vein from ischemia and reperfusion injury preserves ECs."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Blood Vessel Prosthesis","Blood Vessel Prosthesis Implantation","Cell Proliferation","Clopidogrel","Endothelial Cells","Histocytochemistry","Hyperplasia","Ischemia","Ki-67 Antigen","Microscopy, Electron, Scanning","Platelet Aggregation Inhibitors","Rabbits","Ticlopidine","Tunica Intima"],"string":"[\n \"Animals\",\n \"Blood Vessel Prosthesis\",\n \"Blood Vessel Prosthesis Implantation\",\n \"Cell Proliferation\",\n \"Clopidogrel\",\n \"Endothelial Cells\",\n \"Histocytochemistry\",\n \"Hyperplasia\",\n \"Ischemia\",\n \"Ki-67 Antigen\",\n \"Microscopy, Electron, Scanning\",\n \"Platelet Aggregation Inhibitors\",\n \"Rabbits\",\n \"Ticlopidine\",\n \"Tunica Intima\"\n]"},"pmcid":{"kind":"string","value":"3495780"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Animals and grouping New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\nNew Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\n Vein graft surgery Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nAnesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\n Morphometric analysis The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\nThe vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\n Immunocytochemistry and cell proliferation analysis Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\nCell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\n Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\nSamples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\n Statistical analysis All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\nAll data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Graft patency All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\nAll grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\n Morphometric analysis Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\nUsing the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\n Immunocytochemistry and cell proliferation analysis Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\nMany Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\n Scanning electron microscopy observation of intima SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\nSEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs."},"other_sections_titles":{"kind":"list like","value":["Background","Animals and grouping","Vein graft surgery","Morphometric analysis","Immunocytochemistry and cell proliferation analysis","Scanning electron microscopy","Statistical analysis","Graft patency","Morphometric analysis","Immunocytochemistry and cell proliferation analysis","Scanning electron microscopy observation of intima","Competing interest","Authors' contributions"],"string":"[\n \"Background\",\n \"Animals and grouping\",\n \"Vein graft surgery\",\n \"Morphometric analysis\",\n \"Immunocytochemistry and cell proliferation analysis\",\n \"Scanning electron microscopy\",\n \"Statistical analysis\",\n \"Graft patency\",\n \"Morphometric analysis\",\n \"Immunocytochemistry and cell proliferation analysis\",\n \"Scanning electron microscopy observation of intima\",\n \"Competing interest\",\n \"Authors' contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.","New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.","Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.","The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.","Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.","Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).","All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.","All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.","Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).","Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.","SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.","The authors declare that they have no competing interests.","RJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript."],"string":"[\n \"The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\\n[1-3].\\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.\",\n \"New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\",\n \"Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\",\n \"The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\",\n \"Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\",\n \"Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\",\n \"All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\",\n \"All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\",\n \"Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\\n1, Figure\\n2).\\nComparison of vein grafts under different ischemic conditions\\nValues are mean ± standard deviation.\\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\",\n \"Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\\n3), as in the mean thickness results.\\nThe Ki-67 labeling index of the vein grafts.\",\n \"SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\\n4), likely due to the infusion pressure.\\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\\n5).\\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\",\n \"The authors declare that they have no competing interests.\",\n \"RJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Animals and grouping","Vein graft surgery","Morphometric analysis","Immunocytochemistry and cell proliferation analysis","Scanning electron microscopy","Statistical analysis","Results","Graft patency","Morphometric analysis","Immunocytochemistry and cell proliferation analysis","Scanning electron microscopy observation of intima","Discussion","Conclusions","Competing interest","Authors' contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Animals and grouping\",\n \"Vein graft surgery\",\n \"Morphometric analysis\",\n \"Immunocytochemistry and cell proliferation analysis\",\n \"Scanning electron microscopy\",\n \"Statistical analysis\",\n \"Results\",\n \"Graft patency\",\n \"Morphometric analysis\",\n \"Immunocytochemistry and cell proliferation analysis\",\n \"Scanning electron microscopy observation of intima\",\n \"Discussion\",\n \"Conclusions\",\n \"Competing interest\",\n \"Authors' contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used."," Animals and grouping New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\nNew Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\n Vein graft surgery Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nAnesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\n Morphometric analysis The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\nThe vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\n Immunocytochemistry and cell proliferation analysis Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\nCell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\n Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\nSamples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\n Statistical analysis All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\nAll data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.","New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.","Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.","The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.","Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.","Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).","All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant."," Graft patency All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\nAll grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\n Morphometric analysis Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\nUsing the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\n Immunocytochemistry and cell proliferation analysis Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\nMany Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\n Scanning electron microscopy observation of intima SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\nSEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.","All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.","Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).","Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.","SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.","Ischemic injury before grafting and reperfusion injury after grafting may injure ECs, and subsequently activate platelets and trigger the coagulation cascade\n[10]. Various solutions and heparinized autologous blood have been used previously for vein preservation to reduce ischemia and reperfusion (IR) injury. For instance, Cavallari et al. used autologous whole blood (AWB), 0.9% normal saline solution (NS), and University of Wisconsin solution (UWs) individually to treat autogenous vein grafts for 45 min at 4°C before grafting, and found the veins stored in UWs had a similar intimal thickness with that of control samples, whereas the veins stored in AWB or NS had a thicker intima layer after grafting\n[11]. However, Solberg et al. found that veins stored in cell culture media at 4°C show a marked increase in EC dysjunction and an 18% loss of EC number, compared to only a 4% loss at 20°C, indicating that hypothermy may injure ECs\n[12]. In typical situations, the veins are placed in saline containing heparin sodium at 20°C after being removed. Therefore, we designed this study to investigate the influence of pre-grafting ischemia on early hyperplasia after grafting under these conditions, and found the ischemic tolerance of the vein. After the step, we planed to investigate how long the veins can be preserved in the solutions (such as UWs) or how long the AWB should be renewed. The cuff technique was used in this study for the vein graft model because it can limit the ischemic time caused by the procedure to 15 min, avoiding IR injury. In one side, the veins were grafted immediately after removal to offer controls, whereas, in the other side, the veins were stored before being used. We also included clopidogrel treatment to reduce the risk of thrombus or mural thrombus and to make the vein graft model more similar to the clinical situation.\nProspective controlled trials have demonstrated a graft patency benefit when aspirin was started 1, 7, or 24 h postoperatively, a benefit which was lost when started after 48 h\n[9]. Aspirin has been shown to significantly reduce postoperative mortality, myocardial infarction, stroke, renal failure, and bowel infarction when administered within 48 h of CABG\n[8]. However, in patients with atherosclerotic vascular disease, clopidogrel is more effective than aspirin in reducing the combined risk of stroke, myocardial infarction, and vascular death\n[13].\nBesides having anti-thrombotic properties, inhibitors of platelet aggregation attenuate platelet function, prevent platelet activation and release of peptide growth factors and nonpeptide substances, and thereby inhibit intimal hyperplasia\n[14]. Atherosclerosis is described as an inflammatory disease\n[15]. Clopidogrel inhibits the expression of platelet activation markers and the interaction of platelets and leukocytes\n[16], and is powerful in decreasing the levels of inflammatory factors\n[17].\nIn our present study, SEM revealed that 90-min ischemia caused serious injury of the intima, including the exposed subendothelial collagen fibers that may aggregate and activate platelets and the frequently adherent leukocytes, which may trigger vascular inflammation. Clopidogrel was used and avoided thrombus or mural thrombus successfully. Moreover, results show no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia in term of thickness and the hyperplasia intensity of the intima, media, or adventitia at 4 weeks after the grafting procedure. It seems ischemia up to 90 min does not increase early intimal hyperplasia of the vein graft when clopidogrel was used effectively. Approximately 90 min is a long enough time period for surgeons to complete 3 or 4 bypasses; therefore, when using clopidogrel, it is seemingly unnecessary to consider the influence of ischemia before grafting on early hyperplasia of the vein grafts.\nThe recovery of the intima was also observed post operation. EC coverage has been used in some studies\n[18,19], which is easily gained and facilitates statistical analysis. However, based on our study, it is apparently unreliable because fibrin and micro thrombi covered the surface of the intima when there was IR injury according to SEM analysis. Therefore, we only reported the characteristics of the intima in this study.\nAccording to our observations, the intimae of vein grafts with 15-min ischemia only lost a portion of their ECs and reintegrated at 3 days post operation. However, the intimae of vein grafts with 90-min ischemia lost all their ECs and redintegrated at 14 days post operation. Furthermore, the anterior had less risk of thrombosis. Reducing ischemia and reperfusion injury is valuable. It has been suggested previously that UWs preserves the vein before grafting because of its excellent effect on protecting the endothelium and its functions\n[20].\nHowever, vein grafts with 15-min ischemia, which had almost no IR injury, likely face a widely torn intima caused by systolic pressure 1 h after grafting. Furthermore, the vein grafts with 90-min ischemia likely face more serious loss of ECs until new ECs are present, which come predominantly from extrinsic cells such as bone marrow-derived cells\n[21], and form an intima layer with integrity. Thus, it seems antithrombotic therapy should be administered as early as possible post operation.\nGavaghan et al. found that administering aspirin within 1 h of CABG did not increase chest-tube blood loss, the red cell transfusion requirements, or the re-exploration rate\n[22]. However, Goldman et al. reported that aspirin use on the day before CABG has a similar effect on early graft patency within 6 h after surgery, and increased bleeding complications\n[23]. Considering that the number of platelets and the levels of blood coagulation factors vary and change with time after CABG, we recommend starting anti-platelet therapy when the postoperative chest tube drainage is ≤50 ml/h for 2 consecutive hours, as presented by both Halkos et al. and Chan et al.\n[24,25].","In summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs.","The authors declare that they have no competing interests.","RJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript."],"string":"[\n \"The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\\n[1-3].\\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.\",\n \" Animals and grouping New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\\nNew Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\\n Vein graft surgery Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\nAnesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\n Morphometric analysis The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\\nThe vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\\n Immunocytochemistry and cell proliferation analysis Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\\nCell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\\n Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\\nSamples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\\n Statistical analysis All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\\nAll data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\",\n \"New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\",\n \"Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\",\n \"The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\",\n \"Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\",\n \"Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\",\n \"All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\",\n \" Graft patency All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\\nAll grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\\n Morphometric analysis Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\\n1, Figure\\n2).\\nComparison of vein grafts under different ischemic conditions\\nValues are mean ± standard deviation.\\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\\nUsing the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\\n1, Figure\\n2).\\nComparison of vein grafts under different ischemic conditions\\nValues are mean ± standard deviation.\\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\\n Immunocytochemistry and cell proliferation analysis Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\\n3), as in the mean thickness results.\\nThe Ki-67 labeling index of the vein grafts.\\nMany Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\\n3), as in the mean thickness results.\\nThe Ki-67 labeling index of the vein grafts.\\n Scanning electron microscopy observation of intima SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\\n4), likely due to the infusion pressure.\\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\\n5).\\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\\nSEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\\n4), likely due to the infusion pressure.\\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\\n5).\\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\",\n \"All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\",\n \"Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\\n1, Figure\\n2).\\nComparison of vein grafts under different ischemic conditions\\nValues are mean ± standard deviation.\\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\",\n \"Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\\n3), as in the mean thickness results.\\nThe Ki-67 labeling index of the vein grafts.\",\n \"SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\\n4), likely due to the infusion pressure.\\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\\n5).\\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\",\n \"Ischemic injury before grafting and reperfusion injury after grafting may injure ECs, and subsequently activate platelets and trigger the coagulation cascade\\n[10]. Various solutions and heparinized autologous blood have been used previously for vein preservation to reduce ischemia and reperfusion (IR) injury. For instance, Cavallari et al. used autologous whole blood (AWB), 0.9% normal saline solution (NS), and University of Wisconsin solution (UWs) individually to treat autogenous vein grafts for 45 min at 4°C before grafting, and found the veins stored in UWs had a similar intimal thickness with that of control samples, whereas the veins stored in AWB or NS had a thicker intima layer after grafting\\n[11]. However, Solberg et al. found that veins stored in cell culture media at 4°C show a marked increase in EC dysjunction and an 18% loss of EC number, compared to only a 4% loss at 20°C, indicating that hypothermy may injure ECs\\n[12]. In typical situations, the veins are placed in saline containing heparin sodium at 20°C after being removed. Therefore, we designed this study to investigate the influence of pre-grafting ischemia on early hyperplasia after grafting under these conditions, and found the ischemic tolerance of the vein. After the step, we planed to investigate how long the veins can be preserved in the solutions (such as UWs) or how long the AWB should be renewed. The cuff technique was used in this study for the vein graft model because it can limit the ischemic time caused by the procedure to 15 min, avoiding IR injury. In one side, the veins were grafted immediately after removal to offer controls, whereas, in the other side, the veins were stored before being used. We also included clopidogrel treatment to reduce the risk of thrombus or mural thrombus and to make the vein graft model more similar to the clinical situation.\\nProspective controlled trials have demonstrated a graft patency benefit when aspirin was started 1, 7, or 24 h postoperatively, a benefit which was lost when started after 48 h\\n[9]. Aspirin has been shown to significantly reduce postoperative mortality, myocardial infarction, stroke, renal failure, and bowel infarction when administered within 48 h of CABG\\n[8]. However, in patients with atherosclerotic vascular disease, clopidogrel is more effective than aspirin in reducing the combined risk of stroke, myocardial infarction, and vascular death\\n[13].\\nBesides having anti-thrombotic properties, inhibitors of platelet aggregation attenuate platelet function, prevent platelet activation and release of peptide growth factors and nonpeptide substances, and thereby inhibit intimal hyperplasia\\n[14]. Atherosclerosis is described as an inflammatory disease\\n[15]. Clopidogrel inhibits the expression of platelet activation markers and the interaction of platelets and leukocytes\\n[16], and is powerful in decreasing the levels of inflammatory factors\\n[17].\\nIn our present study, SEM revealed that 90-min ischemia caused serious injury of the intima, including the exposed subendothelial collagen fibers that may aggregate and activate platelets and the frequently adherent leukocytes, which may trigger vascular inflammation. Clopidogrel was used and avoided thrombus or mural thrombus successfully. Moreover, results show no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia in term of thickness and the hyperplasia intensity of the intima, media, or adventitia at 4 weeks after the grafting procedure. It seems ischemia up to 90 min does not increase early intimal hyperplasia of the vein graft when clopidogrel was used effectively. Approximately 90 min is a long enough time period for surgeons to complete 3 or 4 bypasses; therefore, when using clopidogrel, it is seemingly unnecessary to consider the influence of ischemia before grafting on early hyperplasia of the vein grafts.\\nThe recovery of the intima was also observed post operation. EC coverage has been used in some studies\\n[18,19], which is easily gained and facilitates statistical analysis. However, based on our study, it is apparently unreliable because fibrin and micro thrombi covered the surface of the intima when there was IR injury according to SEM analysis. Therefore, we only reported the characteristics of the intima in this study.\\nAccording to our observations, the intimae of vein grafts with 15-min ischemia only lost a portion of their ECs and reintegrated at 3 days post operation. However, the intimae of vein grafts with 90-min ischemia lost all their ECs and redintegrated at 14 days post operation. Furthermore, the anterior had less risk of thrombosis. Reducing ischemia and reperfusion injury is valuable. It has been suggested previously that UWs preserves the vein before grafting because of its excellent effect on protecting the endothelium and its functions\\n[20].\\nHowever, vein grafts with 15-min ischemia, which had almost no IR injury, likely face a widely torn intima caused by systolic pressure 1 h after grafting. Furthermore, the vein grafts with 90-min ischemia likely face more serious loss of ECs until new ECs are present, which come predominantly from extrinsic cells such as bone marrow-derived cells\\n[21], and form an intima layer with integrity. Thus, it seems antithrombotic therapy should be administered as early as possible post operation.\\nGavaghan et al. found that administering aspirin within 1 h of CABG did not increase chest-tube blood loss, the red cell transfusion requirements, or the re-exploration rate\\n[22]. However, Goldman et al. reported that aspirin use on the day before CABG has a similar effect on early graft patency within 6 h after surgery, and increased bleeding complications\\n[23]. Considering that the number of platelets and the levels of blood coagulation factors vary and change with time after CABG, we recommend starting anti-platelet therapy when the postoperative chest tube drainage is ≤50 ml/h for 2 consecutive hours, as presented by both Halkos et al. and Chan et al.\\n[24,25].\",\n \"In summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs.\",\n \"The authors declare that they have no competing interests.\",\n \"RJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,"results",null,null,null,null,"discussion","conclusions",null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Vein graft","Ischemia","Intimal hyperplasia","Endothelial cell"],"string":"[\n \"Vein graft\",\n \"Ischemia\",\n \"Intimal hyperplasia\",\n \"Endothelial cell\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nThe autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.\n\nMethods:\n Animals and grouping New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\nNew Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\n Vein graft surgery Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nAnesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\n Morphometric analysis The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\nThe vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\n Immunocytochemistry and cell proliferation analysis Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\nCell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\n Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\nSamples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\n Statistical analysis All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\nAll data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\n\nAnimals and grouping:\nNew Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\n\nVein graft surgery:\nAnesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\n\nMorphometric analysis:\nThe vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\n\nImmunocytochemistry and cell proliferation analysis:\nCell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\n\nScanning electron microscopy:\nSamples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\n\nStatistical analysis:\nAll data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\n\nResults:\n Graft patency All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\nAll grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\n Morphometric analysis Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\nUsing the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\n Immunocytochemistry and cell proliferation analysis Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\nMany Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\n Scanning electron microscopy observation of intima SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\nSEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\n\nGraft patency:\nAll grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\n\nMorphometric analysis:\nUsing the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\n\nImmunocytochemistry and cell proliferation analysis:\nMany Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\n\nScanning electron microscopy observation of intima:\nSEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\n\nDiscussion:\nIschemic injury before grafting and reperfusion injury after grafting may injure ECs, and subsequently activate platelets and trigger the coagulation cascade\n[10]. Various solutions and heparinized autologous blood have been used previously for vein preservation to reduce ischemia and reperfusion (IR) injury. For instance, Cavallari et al. used autologous whole blood (AWB), 0.9% normal saline solution (NS), and University of Wisconsin solution (UWs) individually to treat autogenous vein grafts for 45 min at 4°C before grafting, and found the veins stored in UWs had a similar intimal thickness with that of control samples, whereas the veins stored in AWB or NS had a thicker intima layer after grafting\n[11]. However, Solberg et al. found that veins stored in cell culture media at 4°C show a marked increase in EC dysjunction and an 18% loss of EC number, compared to only a 4% loss at 20°C, indicating that hypothermy may injure ECs\n[12]. In typical situations, the veins are placed in saline containing heparin sodium at 20°C after being removed. Therefore, we designed this study to investigate the influence of pre-grafting ischemia on early hyperplasia after grafting under these conditions, and found the ischemic tolerance of the vein. After the step, we planed to investigate how long the veins can be preserved in the solutions (such as UWs) or how long the AWB should be renewed. The cuff technique was used in this study for the vein graft model because it can limit the ischemic time caused by the procedure to 15 min, avoiding IR injury. In one side, the veins were grafted immediately after removal to offer controls, whereas, in the other side, the veins were stored before being used. We also included clopidogrel treatment to reduce the risk of thrombus or mural thrombus and to make the vein graft model more similar to the clinical situation.\nProspective controlled trials have demonstrated a graft patency benefit when aspirin was started 1, 7, or 24 h postoperatively, a benefit which was lost when started after 48 h\n[9]. Aspirin has been shown to significantly reduce postoperative mortality, myocardial infarction, stroke, renal failure, and bowel infarction when administered within 48 h of CABG\n[8]. However, in patients with atherosclerotic vascular disease, clopidogrel is more effective than aspirin in reducing the combined risk of stroke, myocardial infarction, and vascular death\n[13].\nBesides having anti-thrombotic properties, inhibitors of platelet aggregation attenuate platelet function, prevent platelet activation and release of peptide growth factors and nonpeptide substances, and thereby inhibit intimal hyperplasia\n[14]. Atherosclerosis is described as an inflammatory disease\n[15]. Clopidogrel inhibits the expression of platelet activation markers and the interaction of platelets and leukocytes\n[16], and is powerful in decreasing the levels of inflammatory factors\n[17].\nIn our present study, SEM revealed that 90-min ischemia caused serious injury of the intima, including the exposed subendothelial collagen fibers that may aggregate and activate platelets and the frequently adherent leukocytes, which may trigger vascular inflammation. Clopidogrel was used and avoided thrombus or mural thrombus successfully. Moreover, results show no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia in term of thickness and the hyperplasia intensity of the intima, media, or adventitia at 4 weeks after the grafting procedure. It seems ischemia up to 90 min does not increase early intimal hyperplasia of the vein graft when clopidogrel was used effectively. Approximately 90 min is a long enough time period for surgeons to complete 3 or 4 bypasses; therefore, when using clopidogrel, it is seemingly unnecessary to consider the influence of ischemia before grafting on early hyperplasia of the vein grafts.\nThe recovery of the intima was also observed post operation. EC coverage has been used in some studies\n[18,19], which is easily gained and facilitates statistical analysis. However, based on our study, it is apparently unreliable because fibrin and micro thrombi covered the surface of the intima when there was IR injury according to SEM analysis. Therefore, we only reported the characteristics of the intima in this study.\nAccording to our observations, the intimae of vein grafts with 15-min ischemia only lost a portion of their ECs and reintegrated at 3 days post operation. However, the intimae of vein grafts with 90-min ischemia lost all their ECs and redintegrated at 14 days post operation. Furthermore, the anterior had less risk of thrombosis. Reducing ischemia and reperfusion injury is valuable. It has been suggested previously that UWs preserves the vein before grafting because of its excellent effect on protecting the endothelium and its functions\n[20].\nHowever, vein grafts with 15-min ischemia, which had almost no IR injury, likely face a widely torn intima caused by systolic pressure 1 h after grafting. Furthermore, the vein grafts with 90-min ischemia likely face more serious loss of ECs until new ECs are present, which come predominantly from extrinsic cells such as bone marrow-derived cells\n[21], and form an intima layer with integrity. Thus, it seems antithrombotic therapy should be administered as early as possible post operation.\nGavaghan et al. found that administering aspirin within 1 h of CABG did not increase chest-tube blood loss, the red cell transfusion requirements, or the re-exploration rate\n[22]. However, Goldman et al. reported that aspirin use on the day before CABG has a similar effect on early graft patency within 6 h after surgery, and increased bleeding complications\n[23]. Considering that the number of platelets and the levels of blood coagulation factors vary and change with time after CABG, we recommend starting anti-platelet therapy when the postoperative chest tube drainage is ≤50 ml/h for 2 consecutive hours, as presented by both Halkos et al. and Chan et al.\n[24,25].\n\nConclusions:\nIn summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs.\n\nCompeting interest:\nThe authors declare that they have no competing interests.\n\nAuthors' contributions:\nRJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nTo investigate both the influence of ischemia before grafting on early hyperplasia of the vein grafts, and the dynamic changes of the intima after grafting in a rabbit model of vein graft disease.\n\nMethods:\nWe performed paired vein graft experiments under different ischemic conditions (15 vs. 60 min; 15 vs. 90 min) in the neck of the rabbits and compared the differences between the grafts. Clopidogrel, an anti-platelet agent, was administered before and after surgery. Twenty-eight days after the grafting procedure, the veins were evaluated microscopically. The dynamic changes of the intima after grafting were evaluated by scanning electron microscopy over time.\n\nResults:\nThe vein grafts subjected to 60- or 90-min ischemia exhibited no differences compared to those subjected to 15-min ischemia in terms of the mean thickness of the intimal, medial, and adventitial layers of the graft. Similarly, there was no difference in the Ki-67 labeling index (proliferation marker) between the vein grafts. Vein grafts with 15-min ischemia lost endothelial cells (ECs) but healed by 3 days post graft, whereas vein grafts with 90-min ischemia suffered serious EC loss, which was restored with new ECs during days 2 to 14 post graft.\n\nConclusions:\nNinety-minute ischemia before vein grafting can cause serious EC loss, but does not increase early intimal hyperplasia when clopidogrel is administered. Protecting the vein from ischemia and reperfusion injury preserves ECs."},"background_conclusion_text":{"kind":"string","value":"Background:\nThe autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.\n\nConclusions:\nIn summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nTo investigate both the influence of ischemia before grafting on early hyperplasia of the vein grafts, and the dynamic changes of the intima after grafting in a rabbit model of vein graft disease.\n\nMethods:\nWe performed paired vein graft experiments under different ischemic conditions (15 vs. 60 min; 15 vs. 90 min) in the neck of the rabbits and compared the differences between the grafts. Clopidogrel, an anti-platelet agent, was administered before and after surgery. Twenty-eight days after the grafting procedure, the veins were evaluated microscopically. The dynamic changes of the intima after grafting were evaluated by scanning electron microscopy over time.\n\nResults:\nThe vein grafts subjected to 60- or 90-min ischemia exhibited no differences compared to those subjected to 15-min ischemia in terms of the mean thickness of the intimal, medial, and adventitial layers of the graft. Similarly, there was no difference in the Ki-67 labeling index (proliferation marker) between the vein grafts. Vein grafts with 15-min ischemia lost endothelial cells (ECs) but healed by 3 days post graft, whereas vein grafts with 90-min ischemia suffered serious EC loss, which was restored with new ECs during days 2 to 14 post graft.\n\nConclusions:\nNinety-minute ischemia before vein grafting can cause serious EC loss, but does not increase early intimal hyperplasia when clopidogrel is administered. Protecting the vein from ischemia and reperfusion injury preserves ECs."},"whole_article_text_length":{"kind":"number","value":7886,"string":"7,886"},"whole_article_abstract_length":{"kind":"number","value":278,"string":"278"},"num_sections":{"kind":"number","value":17,"string":"17"},"most_frequent_words":{"kind":"list like","value":["vein","min","ischemia","min ischemia","grafts","15","intima","vein grafts","90","90 min"],"string":"[\n \"vein\",\n \"min\",\n \"ischemia\",\n \"min ischemia\",\n \"grafts\",\n \"15\",\n \"intima\",\n \"vein grafts\",\n \"90\",\n \"90 min\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] thrombosis | vein | grafting | acute thrombosis | acute | hyperplasia | drugs | stress | use | risk [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] min | rabbits | vein | saline | removed | shanghai | jugular | internal | internal jugular | rinsed [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min ischemia | min | ischemia | grafts | vein grafts | intima | 15 min ischemia | 15 | ecs [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] injury | study | study indicates clopidogrel administered | study indicates clopidogrel | cause ec loss avoiding | grafts long term study | grafts long term | grafts long | furthermore ir | loss avoiding [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | grafts | ischemia | min ischemia | vein grafts | 15 | 90 | 90 min | intima [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | grafts | ischemia | min ischemia | vein grafts | 15 | 90 | 90 min | intima [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 15 | 60 | 15 | 90 ||| Clopidogrel ||| Twenty-eight days ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 60- | 90 | 15 ||| ||| 15 | 3 days | 90 | EC | days 2 | 14 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Ninety-minute | EC ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 15 | 60 | 15 | 90 ||| Clopidogrel ||| Twenty-eight days ||| ||| ||| 60- | 90 | 15 ||| ||| 15 | 3 days | 90 | EC | days 2 | 14 ||| Ninety-minute | EC ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 15 | 60 | 15 | 90 ||| Clopidogrel ||| Twenty-eight days ||| ||| ||| 60- | 90 | 15 ||| ||| 15 | 3 days | 90 | EC | days 2 | 14 ||| Ninety-minute | EC ||| [SUMMARY]"}}},{"rowIdx":270,"cells":{"title":{"kind":"string","value":"Cognitive awareness of carbohydrate intake does not alter exercise-induced lymphocyte apoptosis."},"pmid":{"kind":"string","value":"21484033"},"background_abstract":{"kind":"string","value":"Carbohydrate supplementation during aerobic exercise generally protects against the immunosuppressive effects of exercise. It is not currently known whether carbohydrate consumption or simply the knowledge of carbohydrate consumption also has that effect."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"Endurance trained male and female (N = 10) athletes were randomly assigned to one of two groups based on either a correct or incorrect cognitive awareness of carbohydrate intake. In the incorrect group, the subjects were informed that they were receiving the carbohydrate beverage but actually received the placebo beverage. Participants completed a 60-min ride on a cycle ergometer at 80% VO₂peak under carbohydrate and placebo supplemented conditions. Venous blood samples were collected at rest and immediately after exercise and were used to determine the plasma glucose concentration, lymphocyte count, and extent of lymphocyte apoptosis. Cognitive awareness, either correct or incorrect, did not have an effect on any of the measured variables."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Carbohydrate supplementation during exercise did not have an effect on lymphocyte count or apoptotic index. Independent of drink type, exercise resulted in significant lymphocytosis and lymphocyte apoptosis (apoptotic index at rest = 6.3 ± 3% and apoptotic index following exercise = 11.6 ± 3%, P < 0.01)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Neither carbohydrate nor placebo supplementation altered the typical lymphocyte apoptotic response following exercise. While carbohydrate supplementation generally has an immune-boosting effect during exercise, it appears that this influence does not extend to the mechanisms that govern exercise-induced lymphocyte cell death."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Analysis of Variance","Apoptosis","Awareness","Beverages","Blood Glucose","Cognition","Dietary Carbohydrates","Female","Humans","Lymphocytes","Male","Physical Endurance","Statistics, Nonparametric","Stress, Physiological"],"string":"[\n \"Adult\",\n \"Analysis of Variance\",\n \"Apoptosis\",\n \"Awareness\",\n \"Beverages\",\n \"Blood Glucose\",\n \"Cognition\",\n \"Dietary Carbohydrates\",\n \"Female\",\n \"Humans\",\n \"Lymphocytes\",\n \"Male\",\n \"Physical Endurance\",\n \"Statistics, Nonparametric\",\n \"Stress, Physiological\"\n]"},"pmcid":{"kind":"string","value":"3059873"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Acute exercise has a marked effect on the immune system response. Increased neutrophil, monocyte, and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and short‐term swim exercise.3 Two hours of cycling at 55% VO2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30‐min at 60% VO2max increased lymphocyte proliferation and plasma interleukin‐6 concentration (IL‐6).2 Significant increases in tumor necrosis factor‐α (TNF‐α) have been reported after a 30‐min high‐intensity run at 85% VO2max4 and following 15‐min of moderate‐intensity swimming.5 Therefore, it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors.\nWhile acute exercise significantly increases immune parameters, carbohydrate (CHO) supplementation has been shown to have an immunomodulating effect on many of these responses. CHO supplementation prior to 2.5 h of cycling at 65% VO2max resulted in reduced cell numbers of neutrophils, monocytes, and lymphocytes compared to placebo,6 as well as a significant decrease in plasma IL‐6 concentration.7 Similar reductions in immune cell counts and IL‐6 have been reported in trained individuals who received CHO supplementation during a 4‐h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8 \nRegarding the lymphocyte response to acute exercise, an increase in cell concentration is observed with the performance of high intensity exercise, followed by a reduction in concentration that is significantly lower than resting levels.9,10 A portion of this post‐exercise decrease is likely due to programmed cell death, or apoptosis. Previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 To our knowledge, only one previous study has assessed the effect that CHO supplementation has on lymphocyte apoptosis. Green et al. reported that CHO supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in CD4 and CD8 cells in culture taken from endurance‐trained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 This finding is consistent with previously cited research that CHO supplementation may reduce the immunoendocrine changes associated with exercise.6-8 \nA wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress (see review by Black).15 Neuroendocrine responses to stress are mediated by hypothalamic corticotropin‐releasing factor16 which acts on the hypothalamic‐pituitary‐adrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 In addition, there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 Therefore, it is possible that the immune system can be modified not only by nutritional supplementation as discussed above, but also independently by neural and cognitive factors. The influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume, suggesting potential deletion through a cell death mechanism (apoptosis).\nWhile carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo, it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise. It was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise. This would hold true whether participants had a correct or incorrect knowledge of supplementation type. Therefore, the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exercise‐induced lymphocyte apoptotic response, independent of actual carbohydrate intake."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":" Glucose There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\nThere was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\n Lymphocyte Concentration No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\nNo interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\n 3.3 Lymphocyte Apoptosis Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\nCognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Participants","2.2 Protocol","2.3 Statistical Analysis","Glucose","Lymphocyte Concentration","3.3 Lymphocyte Apoptosis","Acknowledgements"],"string":"[\n \"Participants\",\n \"2.2 Protocol\",\n \"2.3 Statistical Analysis\",\n \"Glucose\",\n \"Lymphocyte Concentration\",\n \"3.3 Lymphocyte Apoptosis\",\n \"Acknowledgements\"\n]"},"other_sections_texts":{"kind":"list like","value":["Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.","Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.","Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.","There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.","No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).","Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).","The authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation."],"string":"[\n \"Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\",\n \"Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\",\n \"Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\",\n \"There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\",\n \"No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\",\n \"Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\",\n \"The authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS AND MATERIALS","Participants","2.2 Protocol","2.3 Statistical Analysis","RESULTS","Glucose","Lymphocyte Concentration","3.3 Lymphocyte Apoptosis","DISCUSSION","Acknowledgements"],"string":"[\n \"INTRODUCTION\",\n \"METHODS AND MATERIALS\",\n \"Participants\",\n \"2.2 Protocol\",\n \"2.3 Statistical Analysis\",\n \"RESULTS\",\n \"Glucose\",\n \"Lymphocyte Concentration\",\n \"3.3 Lymphocyte Apoptosis\",\n \"DISCUSSION\",\n \"Acknowledgements\"\n]"},"all_sections_texts":{"kind":"list like","value":["Acute exercise has a marked effect on the immune system response. Increased neutrophil, monocyte, and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and short‐term swim exercise.3 Two hours of cycling at 55% VO2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30‐min at 60% VO2max increased lymphocyte proliferation and plasma interleukin‐6 concentration (IL‐6).2 Significant increases in tumor necrosis factor‐α (TNF‐α) have been reported after a 30‐min high‐intensity run at 85% VO2max4 and following 15‐min of moderate‐intensity swimming.5 Therefore, it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors.\nWhile acute exercise significantly increases immune parameters, carbohydrate (CHO) supplementation has been shown to have an immunomodulating effect on many of these responses. CHO supplementation prior to 2.5 h of cycling at 65% VO2max resulted in reduced cell numbers of neutrophils, monocytes, and lymphocytes compared to placebo,6 as well as a significant decrease in plasma IL‐6 concentration.7 Similar reductions in immune cell counts and IL‐6 have been reported in trained individuals who received CHO supplementation during a 4‐h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8 \nRegarding the lymphocyte response to acute exercise, an increase in cell concentration is observed with the performance of high intensity exercise, followed by a reduction in concentration that is significantly lower than resting levels.9,10 A portion of this post‐exercise decrease is likely due to programmed cell death, or apoptosis. Previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 To our knowledge, only one previous study has assessed the effect that CHO supplementation has on lymphocyte apoptosis. Green et al. reported that CHO supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in CD4 and CD8 cells in culture taken from endurance‐trained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 This finding is consistent with previously cited research that CHO supplementation may reduce the immunoendocrine changes associated with exercise.6-8 \nA wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress (see review by Black).15 Neuroendocrine responses to stress are mediated by hypothalamic corticotropin‐releasing factor16 which acts on the hypothalamic‐pituitary‐adrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 In addition, there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 Therefore, it is possible that the immune system can be modified not only by nutritional supplementation as discussed above, but also independently by neural and cognitive factors. The influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume, suggesting potential deletion through a cell death mechanism (apoptosis).\nWhile carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo, it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise. It was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise. This would hold true whether participants had a correct or incorrect knowledge of supplementation type. Therefore, the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exercise‐induced lymphocyte apoptotic response, independent of actual carbohydrate intake."," Participants Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\nEndurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\n 2.2 Protocol Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\nPeak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\n 2.3 Statistical Analysis Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\nBased on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.","Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.","Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.","Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test."," Glucose There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\nThere was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\n Lymphocyte Concentration No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\nNo interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\n 3.3 Lymphocyte Apoptosis Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\nCognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).","There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.","No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).","Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).","The main finding of this investigation was that cognitive awareness of drink type had no physiological effect on selected immune parameters following high‐intensity aerobic exercise. Individuals who had correct cognitive awareness of consumed supplementation (CHO or PLA) had statistically similar measures for plasma glucose, lymphocyte concentration, and lymphocyte apoptosis compared to individuals who had an incorrect cognitive awareness of the ingested supplement. In addition, we observed no effect of CHO supplementation on either lymphocyte concentration or apoptotic index following 60‐min of cycle exercise at 80% VO2peak.\nCarbohydrate supplementation significantly increased plasma glucose following exercise regardless of correct or incorrect cognitive awareness of drink type. The rise in glucose response has been confirmed by a number of other investigations.14,22-24 We are aware of only one other deception study in which subjects have received an incorrect cognitive awareness of carbohydrate or placebo supplementation prior to exercise.25 As we have shown, cognitive awareness of drink type (whether correct or incorrect) did not have an effect on the blood glucose response. The lack of a difference likely means that stimulating factors facilitating the release of glucose into the bloodstream (i.e. glucagon, epinephrine, etc.) are not affected directly by cognitive awareness during a bout high‐intensity aerobic exercise.\nAn exercise‐induced lymphocytosis was evident following both CHO and PLA supplemented bouts. An increase in lymphocyte concentration following exercise has been reported in numerous investigations.1-3,5-6 Thirty minutes of walking at 60% VO2max increased lymphocytosis by 28% in human subjects2 and 120‐min of cycling at 65% VO2max resulted in a 20% increase.6 Using an animal model, Prestes et al. observed increases in lymphocyte counts of 98% and 165% in male Wistar rats who performed unloaded swimming for 5 and 15‐min, and increases of 252% and 191% in animals who swam for 5 and 15‐min with a 5% load.5 The magnitude of lymphocytosis in the present study was 93% in endurance trained participants who completed 60‐min of cycle ergometry exercise at 80% VO2peak. An explanation for the lymphocytosis observed with exercise of increasing intensity is the greater expression of adhesion/activation molecules which can serve to facilitate movement of lymphocytes from the marginal pools into the circulation.26,27 \nWhile others have reported a significant decrease in lymphocyte count with CHO supplementation compared to PLA,6 our results did not show a difference. As part of an investigation to assess the effect of carbohydrate supplementation following caffeine ingestion, the participants of Walker et al. consumed either caffeine or placebo prior to exercise, and either carbohydrate or placebo during a 2 h cycle ride at 65% VO2max.6 Using only the trials in which placebo was consumed prior to exercise (and excluding the effect of caffeine), it was reported that supplementation with CHO during exercise reduced lymphocyte count by 21% compared with the trial in which placebo was administered during exercise.6 The common explanation for immunomodulation associated with CHO supplementation are effects through hypothalamic‐pituitary‐adrenal (HPA) activation and a muted stress hormone response.28 In the present study, CHO was only ingested prior to exercise and did not result in suppressed lymphocyte cell count compared to PLA. There are two possibilities to explain the differences between the study by Walker et al.6 and our findings. First, our participants completed only a 1h cycle ride compared to 2 h. It is possible that the extended exercise duration differentially affected the immune response through increased HPA activation compared to our subjects who completed only half of the duration. Second, the combination of supplemented glucose during the 2 h cycle exercise by the subjects of Walker et al. could have resulted in a greater prolonged effect toward reducing immune parameters compared to our participants who received a single bolus of carbohydrate prior to exercise, but did not receive supplementation at any other point during their exercise trial. Thus it is possible that blood glucose concentration plays a modulating role on cells of the immune system during exercise. In addition, it is likely that CHO has a different effect depending on the immune cell subset. While an overall decrease in leukocyte numbers have been reported in various CHO supplementation investigations,8,24-25 ingestion appears to affect neutrophils to a greater extent than lymphocytes. Nieman et al. and Sharhag et al. both found carbohydrate supplementation to reduce neutrophil count, but to have no effect on numbers of lymphocytes.8,24 \nNeither correct, nor incorrect, cognitive awareness of drink type altered immunity in terms of lymphocyte apoptosis following exercise. To our knowledge, only one other study has attempted to evaluate the effect of carbohydrate supplementation on exercise‐induced lymphocyte cell death.14 Green et al. used carboxyfluorescein succinimidylester fluorescence to estimate lymphocytes undergoing mitosis or cell death using iterative processes until all cells could be accounted for.14 They reported the estimated cell death rates of T‐cell subsets to be significantly reduced in CHO supplemented trials compared to PLA bouts in six well‐trained male cyclists. In the present study, carbohydrate supplementation did not have an effect on lymphocyte apoptosis. Recently, exercise‐induced lymphocyte apoptosis has been measured using either biochemical markers,4,29-30 or a morphological technique.11-13 In the current investigation, we employed the aforementioned morphological method which we have proposed is potentially more sensitive to cells displaying a wider range of apoptotic characteristics compared to a single apoptotic biomarker. We acknowledge that this method does have drawbacks (i.e. highly subjective, low number of cells that are evaluated per sample, etc.); however the methodology was the gold standard to which the current biomarker techniques were validated against.31 Using a direct evaluation of cells displaying morphological characteristics of apoptosis, we found that carbohydrate supplementation during exercise did not alter exercise‐induced cell death compared to the placebo supplemented trial. While carbohydrate supplementation may alter certain immune parameters, it appears that ingestion has no effect on the mechanisms governing cell death induction during exercise.\nWhile we were unable to ascertain differences in select immune parameters due to cognitive awareness of carbohydrate in the present investigation, our study design could be modified to make detection of such differences more likely. The utilization of a single group cross‐over intervention in which subjects randomly complete the conditions of exercise alone, exercise with carbohydrate supplementation, and exercise in conjunction with placebo, would represent an acceptable experimental design in this regard. It should also be noted that the findings of the present study can only be applied to young and middle‐aged individuals who are highly aerobically trained, and should not be extended to other populations. In this regard, other investigations are warranted.\nIn conclusion, our findings indicate that cognitive awareness (either correct or incorrect, of carbohydrate or placebo) has no effect on blood glucose concentration, lymphocyte concentration, or exercise‐induced lymphocyte apoptosis. While glucose intake itself may reduce the magnitude of various immune responses, knowledge of supplementation and indeed the carbohydrate supplement itself has no effect on altering immunity in terms of lymphocyte cell death following exercise.","The authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation."],"string":"[\n \"Acute exercise has a marked effect on the immune system response. Increased neutrophil, monocyte, and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and short‐term swim exercise.3 Two hours of cycling at 55% VO2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30‐min at 60% VO2max increased lymphocyte proliferation and plasma interleukin‐6 concentration (IL‐6).2 Significant increases in tumor necrosis factor‐α (TNF‐α) have been reported after a 30‐min high‐intensity run at 85% VO2max4 and following 15‐min of moderate‐intensity swimming.5 Therefore, it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors.\\nWhile acute exercise significantly increases immune parameters, carbohydrate (CHO) supplementation has been shown to have an immunomodulating effect on many of these responses. CHO supplementation prior to 2.5 h of cycling at 65% VO2max resulted in reduced cell numbers of neutrophils, monocytes, and lymphocytes compared to placebo,6 as well as a significant decrease in plasma IL‐6 concentration.7 Similar reductions in immune cell counts and IL‐6 have been reported in trained individuals who received CHO supplementation during a 4‐h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8 \\nRegarding the lymphocyte response to acute exercise, an increase in cell concentration is observed with the performance of high intensity exercise, followed by a reduction in concentration that is significantly lower than resting levels.9,10 A portion of this post‐exercise decrease is likely due to programmed cell death, or apoptosis. Previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 To our knowledge, only one previous study has assessed the effect that CHO supplementation has on lymphocyte apoptosis. Green et al. reported that CHO supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in CD4 and CD8 cells in culture taken from endurance‐trained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 This finding is consistent with previously cited research that CHO supplementation may reduce the immunoendocrine changes associated with exercise.6-8 \\nA wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress (see review by Black).15 Neuroendocrine responses to stress are mediated by hypothalamic corticotropin‐releasing factor16 which acts on the hypothalamic‐pituitary‐adrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 In addition, there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 Therefore, it is possible that the immune system can be modified not only by nutritional supplementation as discussed above, but also independently by neural and cognitive factors. The influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume, suggesting potential deletion through a cell death mechanism (apoptosis).\\nWhile carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo, it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise. It was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise. This would hold true whether participants had a correct or incorrect knowledge of supplementation type. Therefore, the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exercise‐induced lymphocyte apoptotic response, independent of actual carbohydrate intake.\",\n \" Participants Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\\nEndurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\\n 2.2 Protocol Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\\nPeak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\\n 2.3 Statistical Analysis Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\\nBased on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\",\n \"Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\",\n \"Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\",\n \"Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\",\n \" Glucose There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\\nThere was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\\n Lymphocyte Concentration No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\\nNo interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\\n 3.3 Lymphocyte Apoptosis Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\\nCognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\",\n \"There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\",\n \"No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\",\n \"Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\",\n \"The main finding of this investigation was that cognitive awareness of drink type had no physiological effect on selected immune parameters following high‐intensity aerobic exercise. Individuals who had correct cognitive awareness of consumed supplementation (CHO or PLA) had statistically similar measures for plasma glucose, lymphocyte concentration, and lymphocyte apoptosis compared to individuals who had an incorrect cognitive awareness of the ingested supplement. In addition, we observed no effect of CHO supplementation on either lymphocyte concentration or apoptotic index following 60‐min of cycle exercise at 80% VO2peak.\\nCarbohydrate supplementation significantly increased plasma glucose following exercise regardless of correct or incorrect cognitive awareness of drink type. The rise in glucose response has been confirmed by a number of other investigations.14,22-24 We are aware of only one other deception study in which subjects have received an incorrect cognitive awareness of carbohydrate or placebo supplementation prior to exercise.25 As we have shown, cognitive awareness of drink type (whether correct or incorrect) did not have an effect on the blood glucose response. The lack of a difference likely means that stimulating factors facilitating the release of glucose into the bloodstream (i.e. glucagon, epinephrine, etc.) are not affected directly by cognitive awareness during a bout high‐intensity aerobic exercise.\\nAn exercise‐induced lymphocytosis was evident following both CHO and PLA supplemented bouts. An increase in lymphocyte concentration following exercise has been reported in numerous investigations.1-3,5-6 Thirty minutes of walking at 60% VO2max increased lymphocytosis by 28% in human subjects2 and 120‐min of cycling at 65% VO2max resulted in a 20% increase.6 Using an animal model, Prestes et al. observed increases in lymphocyte counts of 98% and 165% in male Wistar rats who performed unloaded swimming for 5 and 15‐min, and increases of 252% and 191% in animals who swam for 5 and 15‐min with a 5% load.5 The magnitude of lymphocytosis in the present study was 93% in endurance trained participants who completed 60‐min of cycle ergometry exercise at 80% VO2peak. An explanation for the lymphocytosis observed with exercise of increasing intensity is the greater expression of adhesion/activation molecules which can serve to facilitate movement of lymphocytes from the marginal pools into the circulation.26,27 \\nWhile others have reported a significant decrease in lymphocyte count with CHO supplementation compared to PLA,6 our results did not show a difference. As part of an investigation to assess the effect of carbohydrate supplementation following caffeine ingestion, the participants of Walker et al. consumed either caffeine or placebo prior to exercise, and either carbohydrate or placebo during a 2 h cycle ride at 65% VO2max.6 Using only the trials in which placebo was consumed prior to exercise (and excluding the effect of caffeine), it was reported that supplementation with CHO during exercise reduced lymphocyte count by 21% compared with the trial in which placebo was administered during exercise.6 The common explanation for immunomodulation associated with CHO supplementation are effects through hypothalamic‐pituitary‐adrenal (HPA) activation and a muted stress hormone response.28 In the present study, CHO was only ingested prior to exercise and did not result in suppressed lymphocyte cell count compared to PLA. There are two possibilities to explain the differences between the study by Walker et al.6 and our findings. First, our participants completed only a 1h cycle ride compared to 2 h. It is possible that the extended exercise duration differentially affected the immune response through increased HPA activation compared to our subjects who completed only half of the duration. Second, the combination of supplemented glucose during the 2 h cycle exercise by the subjects of Walker et al. could have resulted in a greater prolonged effect toward reducing immune parameters compared to our participants who received a single bolus of carbohydrate prior to exercise, but did not receive supplementation at any other point during their exercise trial. Thus it is possible that blood glucose concentration plays a modulating role on cells of the immune system during exercise. In addition, it is likely that CHO has a different effect depending on the immune cell subset. While an overall decrease in leukocyte numbers have been reported in various CHO supplementation investigations,8,24-25 ingestion appears to affect neutrophils to a greater extent than lymphocytes. Nieman et al. and Sharhag et al. both found carbohydrate supplementation to reduce neutrophil count, but to have no effect on numbers of lymphocytes.8,24 \\nNeither correct, nor incorrect, cognitive awareness of drink type altered immunity in terms of lymphocyte apoptosis following exercise. To our knowledge, only one other study has attempted to evaluate the effect of carbohydrate supplementation on exercise‐induced lymphocyte cell death.14 Green et al. used carboxyfluorescein succinimidylester fluorescence to estimate lymphocytes undergoing mitosis or cell death using iterative processes until all cells could be accounted for.14 They reported the estimated cell death rates of T‐cell subsets to be significantly reduced in CHO supplemented trials compared to PLA bouts in six well‐trained male cyclists. In the present study, carbohydrate supplementation did not have an effect on lymphocyte apoptosis. Recently, exercise‐induced lymphocyte apoptosis has been measured using either biochemical markers,4,29-30 or a morphological technique.11-13 In the current investigation, we employed the aforementioned morphological method which we have proposed is potentially more sensitive to cells displaying a wider range of apoptotic characteristics compared to a single apoptotic biomarker. We acknowledge that this method does have drawbacks (i.e. highly subjective, low number of cells that are evaluated per sample, etc.); however the methodology was the gold standard to which the current biomarker techniques were validated against.31 Using a direct evaluation of cells displaying morphological characteristics of apoptosis, we found that carbohydrate supplementation during exercise did not alter exercise‐induced cell death compared to the placebo supplemented trial. While carbohydrate supplementation may alter certain immune parameters, it appears that ingestion has no effect on the mechanisms governing cell death induction during exercise.\\nWhile we were unable to ascertain differences in select immune parameters due to cognitive awareness of carbohydrate in the present investigation, our study design could be modified to make detection of such differences more likely. The utilization of a single group cross‐over intervention in which subjects randomly complete the conditions of exercise alone, exercise with carbohydrate supplementation, and exercise in conjunction with placebo, would represent an acceptable experimental design in this regard. It should also be noted that the findings of the present study can only be applied to young and middle‐aged individuals who are highly aerobically trained, and should not be extended to other populations. In this regard, other investigations are warranted.\\nIn conclusion, our findings indicate that cognitive awareness (either correct or incorrect, of carbohydrate or placebo) has no effect on blood glucose concentration, lymphocyte concentration, or exercise‐induced lymphocyte apoptosis. While glucose intake itself may reduce the magnitude of various immune responses, knowledge of supplementation and indeed the carbohydrate supplement itself has no effect on altering immunity in terms of lymphocyte cell death following exercise.\",\n \"The authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","materials|methods",null,null,null,"results",null,null,null,"discussion",null],"string":"[\n \"intro\",\n \"materials|methods\",\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n null\n]"},"keywords":{"kind":"list like","value":["Programmed cell death","High‐intensity aerobic exercise","Supplementation","Deception investigation","Immune system"],"string":"[\n \"Programmed cell death\",\n \"High‐intensity aerobic exercise\",\n \"Supplementation\",\n \"Deception investigation\",\n \"Immune system\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nAcute exercise has a marked effect on the immune system response. Increased neutrophil, monocyte, and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and short‐term swim exercise.3 Two hours of cycling at 55% VO2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30‐min at 60% VO2max increased lymphocyte proliferation and plasma interleukin‐6 concentration (IL‐6).2 Significant increases in tumor necrosis factor‐α (TNF‐α) have been reported after a 30‐min high‐intensity run at 85% VO2max4 and following 15‐min of moderate‐intensity swimming.5 Therefore, it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors.\nWhile acute exercise significantly increases immune parameters, carbohydrate (CHO) supplementation has been shown to have an immunomodulating effect on many of these responses. CHO supplementation prior to 2.5 h of cycling at 65% VO2max resulted in reduced cell numbers of neutrophils, monocytes, and lymphocytes compared to placebo,6 as well as a significant decrease in plasma IL‐6 concentration.7 Similar reductions in immune cell counts and IL‐6 have been reported in trained individuals who received CHO supplementation during a 4‐h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8 \nRegarding the lymphocyte response to acute exercise, an increase in cell concentration is observed with the performance of high intensity exercise, followed by a reduction in concentration that is significantly lower than resting levels.9,10 A portion of this post‐exercise decrease is likely due to programmed cell death, or apoptosis. Previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 To our knowledge, only one previous study has assessed the effect that CHO supplementation has on lymphocyte apoptosis. Green et al. reported that CHO supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in CD4 and CD8 cells in culture taken from endurance‐trained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 This finding is consistent with previously cited research that CHO supplementation may reduce the immunoendocrine changes associated with exercise.6-8 \nA wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress (see review by Black).15 Neuroendocrine responses to stress are mediated by hypothalamic corticotropin‐releasing factor16 which acts on the hypothalamic‐pituitary‐adrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 In addition, there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 Therefore, it is possible that the immune system can be modified not only by nutritional supplementation as discussed above, but also independently by neural and cognitive factors. The influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume, suggesting potential deletion through a cell death mechanism (apoptosis).\nWhile carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo, it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise. It was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise. This would hold true whether participants had a correct or incorrect knowledge of supplementation type. Therefore, the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exercise‐induced lymphocyte apoptotic response, independent of actual carbohydrate intake.\n\nMETHODS AND MATERIALS:\n Participants Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\nEndurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\n 2.2 Protocol Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\nPeak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\n 2.3 Statistical Analysis Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\nBased on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\n\nParticipants:\nEndurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\n\n2.2 Protocol:\nPeak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\n\n2.3 Statistical Analysis:\nBased on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\n\nRESULTS:\n Glucose There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\nThere was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\n Lymphocyte Concentration No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\nNo interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\n 3.3 Lymphocyte Apoptosis Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\nCognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\n\nGlucose:\nThere was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\n\nLymphocyte Concentration:\nNo interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\n\n3.3 Lymphocyte Apoptosis:\nCognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\n\nDISCUSSION:\nThe main finding of this investigation was that cognitive awareness of drink type had no physiological effect on selected immune parameters following high‐intensity aerobic exercise. Individuals who had correct cognitive awareness of consumed supplementation (CHO or PLA) had statistically similar measures for plasma glucose, lymphocyte concentration, and lymphocyte apoptosis compared to individuals who had an incorrect cognitive awareness of the ingested supplement. In addition, we observed no effect of CHO supplementation on either lymphocyte concentration or apoptotic index following 60‐min of cycle exercise at 80% VO2peak.\nCarbohydrate supplementation significantly increased plasma glucose following exercise regardless of correct or incorrect cognitive awareness of drink type. The rise in glucose response has been confirmed by a number of other investigations.14,22-24 We are aware of only one other deception study in which subjects have received an incorrect cognitive awareness of carbohydrate or placebo supplementation prior to exercise.25 As we have shown, cognitive awareness of drink type (whether correct or incorrect) did not have an effect on the blood glucose response. The lack of a difference likely means that stimulating factors facilitating the release of glucose into the bloodstream (i.e. glucagon, epinephrine, etc.) are not affected directly by cognitive awareness during a bout high‐intensity aerobic exercise.\nAn exercise‐induced lymphocytosis was evident following both CHO and PLA supplemented bouts. An increase in lymphocyte concentration following exercise has been reported in numerous investigations.1-3,5-6 Thirty minutes of walking at 60% VO2max increased lymphocytosis by 28% in human subjects2 and 120‐min of cycling at 65% VO2max resulted in a 20% increase.6 Using an animal model, Prestes et al. observed increases in lymphocyte counts of 98% and 165% in male Wistar rats who performed unloaded swimming for 5 and 15‐min, and increases of 252% and 191% in animals who swam for 5 and 15‐min with a 5% load.5 The magnitude of lymphocytosis in the present study was 93% in endurance trained participants who completed 60‐min of cycle ergometry exercise at 80% VO2peak. An explanation for the lymphocytosis observed with exercise of increasing intensity is the greater expression of adhesion/activation molecules which can serve to facilitate movement of lymphocytes from the marginal pools into the circulation.26,27 \nWhile others have reported a significant decrease in lymphocyte count with CHO supplementation compared to PLA,6 our results did not show a difference. As part of an investigation to assess the effect of carbohydrate supplementation following caffeine ingestion, the participants of Walker et al. consumed either caffeine or placebo prior to exercise, and either carbohydrate or placebo during a 2 h cycle ride at 65% VO2max.6 Using only the trials in which placebo was consumed prior to exercise (and excluding the effect of caffeine), it was reported that supplementation with CHO during exercise reduced lymphocyte count by 21% compared with the trial in which placebo was administered during exercise.6 The common explanation for immunomodulation associated with CHO supplementation are effects through hypothalamic‐pituitary‐adrenal (HPA) activation and a muted stress hormone response.28 In the present study, CHO was only ingested prior to exercise and did not result in suppressed lymphocyte cell count compared to PLA. There are two possibilities to explain the differences between the study by Walker et al.6 and our findings. First, our participants completed only a 1h cycle ride compared to 2 h. It is possible that the extended exercise duration differentially affected the immune response through increased HPA activation compared to our subjects who completed only half of the duration. Second, the combination of supplemented glucose during the 2 h cycle exercise by the subjects of Walker et al. could have resulted in a greater prolonged effect toward reducing immune parameters compared to our participants who received a single bolus of carbohydrate prior to exercise, but did not receive supplementation at any other point during their exercise trial. Thus it is possible that blood glucose concentration plays a modulating role on cells of the immune system during exercise. In addition, it is likely that CHO has a different effect depending on the immune cell subset. While an overall decrease in leukocyte numbers have been reported in various CHO supplementation investigations,8,24-25 ingestion appears to affect neutrophils to a greater extent than lymphocytes. Nieman et al. and Sharhag et al. both found carbohydrate supplementation to reduce neutrophil count, but to have no effect on numbers of lymphocytes.8,24 \nNeither correct, nor incorrect, cognitive awareness of drink type altered immunity in terms of lymphocyte apoptosis following exercise. To our knowledge, only one other study has attempted to evaluate the effect of carbohydrate supplementation on exercise‐induced lymphocyte cell death.14 Green et al. used carboxyfluorescein succinimidylester fluorescence to estimate lymphocytes undergoing mitosis or cell death using iterative processes until all cells could be accounted for.14 They reported the estimated cell death rates of T‐cell subsets to be significantly reduced in CHO supplemented trials compared to PLA bouts in six well‐trained male cyclists. In the present study, carbohydrate supplementation did not have an effect on lymphocyte apoptosis. Recently, exercise‐induced lymphocyte apoptosis has been measured using either biochemical markers,4,29-30 or a morphological technique.11-13 In the current investigation, we employed the aforementioned morphological method which we have proposed is potentially more sensitive to cells displaying a wider range of apoptotic characteristics compared to a single apoptotic biomarker. We acknowledge that this method does have drawbacks (i.e. highly subjective, low number of cells that are evaluated per sample, etc.); however the methodology was the gold standard to which the current biomarker techniques were validated against.31 Using a direct evaluation of cells displaying morphological characteristics of apoptosis, we found that carbohydrate supplementation during exercise did not alter exercise‐induced cell death compared to the placebo supplemented trial. While carbohydrate supplementation may alter certain immune parameters, it appears that ingestion has no effect on the mechanisms governing cell death induction during exercise.\nWhile we were unable to ascertain differences in select immune parameters due to cognitive awareness of carbohydrate in the present investigation, our study design could be modified to make detection of such differences more likely. The utilization of a single group cross‐over intervention in which subjects randomly complete the conditions of exercise alone, exercise with carbohydrate supplementation, and exercise in conjunction with placebo, would represent an acceptable experimental design in this regard. It should also be noted that the findings of the present study can only be applied to young and middle‐aged individuals who are highly aerobically trained, and should not be extended to other populations. In this regard, other investigations are warranted.\nIn conclusion, our findings indicate that cognitive awareness (either correct or incorrect, of carbohydrate or placebo) has no effect on blood glucose concentration, lymphocyte concentration, or exercise‐induced lymphocyte apoptosis. While glucose intake itself may reduce the magnitude of various immune responses, knowledge of supplementation and indeed the carbohydrate supplement itself has no effect on altering immunity in terms of lymphocyte cell death following exercise.\n\nAcknowledgements:\nThe authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nCarbohydrate supplementation during aerobic exercise generally protects against the immunosuppressive effects of exercise. It is not currently known whether carbohydrate consumption or simply the knowledge of carbohydrate consumption also has that effect.\n\nMethods:\nEndurance trained male and female (N = 10) athletes were randomly assigned to one of two groups based on either a correct or incorrect cognitive awareness of carbohydrate intake. In the incorrect group, the subjects were informed that they were receiving the carbohydrate beverage but actually received the placebo beverage. Participants completed a 60-min ride on a cycle ergometer at 80% VO₂peak under carbohydrate and placebo supplemented conditions. Venous blood samples were collected at rest and immediately after exercise and were used to determine the plasma glucose concentration, lymphocyte count, and extent of lymphocyte apoptosis. Cognitive awareness, either correct or incorrect, did not have an effect on any of the measured variables.\n\nResults:\nCarbohydrate supplementation during exercise did not have an effect on lymphocyte count or apoptotic index. Independent of drink type, exercise resulted in significant lymphocytosis and lymphocyte apoptosis (apoptotic index at rest = 6.3 ± 3% and apoptotic index following exercise = 11.6 ± 3%, P < 0.01).\n\nConclusions:\nNeither carbohydrate nor placebo supplementation altered the typical lymphocyte apoptotic response following exercise. While carbohydrate supplementation generally has an immune-boosting effect during exercise, it appears that this influence does not extend to the mechanisms that govern exercise-induced lymphocyte cell death."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":5943,"string":"5,943"},"whole_article_abstract_length":{"kind":"number","value":278,"string":"278"},"num_sections":{"kind":"number","value":11,"string":"11"},"most_frequent_words":{"kind":"list like","value":["exercise","lymphocyte","cho","min","supplementation","effect","apoptotic","apoptosis","cell","cognitive"],"string":"[\n \"exercise\",\n \"lymphocyte\",\n \"cho\",\n \"min\",\n \"supplementation\",\n \"effect\",\n \"apoptotic\",\n \"apoptosis\",\n \"cell\",\n \"cognitive\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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[SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 6.3 ± | 3% | 11.6 | 3% | 0.01 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| 10 | one | two ||| ||| 60 | 80% ||| ||| ||| ||| 6.3 ± | 3% | 11.6 | 3% | 0.01 ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":271,"cells":{"title":{"kind":"string","value":"Plasma Metabolite Profiles of Red Meat, Poultry, and Fish Consumption, and Their Associations with Colorectal Cancer Risk."},"pmid":{"kind":"string","value":"35267954"},"background_abstract":{"kind":"string","value":"Red and processed meat consumption has been consistently associated with increased risk of colorectal cancer (CRC), but the association for fish intake is unclear. Evidence using objective dietary assessment approaches to evaluate these associations is sparse."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We measured plasma metabolites among 5269 participants from the Nurses' Health Study (NHS), NHSII, and Health Professionals Follow-Up study (HPFS). We calculated partial Spearman correlations between each metabolite and self-reported intake of seven red meat, poultry, and fish groups. Metabolite profile scores correlated to self-reported dietary intakes were developed using elastic net regression. Associations between self-reported intakes, metabolite profile scores, and subsequent CRC risk were further evaluated using conditional logistic regression among 559 matched (1:1) case-control pairs in NHS/HPFS and replicated among 266 pairs in Women's Health Study."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Plasma metabolites, especially highly unsaturated lipids, were differentially associated with red meat and fish groups. Metabolite profile scores for each food group were significantly correlated with the corresponding self-reported dietary intake. A higher dietary intake of processed red meat was associated with a higher risk of CRC (pooled OR per 1 SD, 1.15; 95% CI: 1.03, 1.29). In contrast, higher metabolite profile scores for all fish groups, not dietary intakes, were consistently associated with a lower CRC risk: the pooled OR per 1 SD was 0.86 (95% CI: 0.78, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were found for other food groups."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Red meat and fish intake exhibited systematically different plasma metabolite profiles. Plasma metabolite profile of fish intake was inversely associated with CRC risk."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Colorectal Neoplasms","Female","Follow-Up Studies","Logistic Models","Poultry","Red Meat"],"string":"[\n \"Animals\",\n \"Colorectal Neoplasms\",\n \"Female\",\n \"Follow-Up Studies\",\n \"Logistic Models\",\n \"Poultry\",\n \"Red Meat\"\n]"},"pmcid":{"kind":"string","value":"8912563"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Colorectal cancer (CRC) remains the second most commonly occurring cancer in women and the third in men worldwide [1]. CRC incidence is largely affected by screening and modifiable dietary and lifestyle factors [2]. Among these factors, meat and fish consumption has been the subject of many investigations. The consumption of red meat, especially processed red meat, is consistently associated with an increased risk of CRC [3]. However, there is still uncertainty regarding the association between fish intake and CRC risk. The World Cancer Research Fund and American Institute for Cancer Research (WCRF/AICR) concluded that there was only “limited evidence” for the beneficial effect of fish intake in CRC prevention [4]. Recent studies of fish intake and CRC risk also generated inconsistent results [5,6,7,8].\nPrevious epidemiological studies examining the association of meat and fish intake with CRC risk mainly used self-reported dietary data. Rapid developments in high-throughput metabolomics are leading to a new era in nutritional epidemiological research. By measuring the small-molecule metabolites in biological samples, metabolomics may provide an objective picture of food intake and its related biological consequences [9]. Feeding trials and observational studies have demonstrated that plasma and urinary metabolites differed between meat and fish consumption [10,11,12,13,14]. However, it is unknown whether the metabolite profiles related to meat and fish intake are associated with CRC risk and whether these profiles could be used as a complementary approach to evaluate the association between meat and fish intake and CRC risk.\nTherefore, we examined the associations of red meat, poultry, and fish consumption with plasma metabolite profiles among participants from the Nurses’ Health Study (NHS), NHSII, and Health Professionals Follow-up Study (HPFS). We also developed metabolite profile scores that were correlated to the intakes of these meat and fish groups and evaluated the prospective associations of metabolite profile scores with CRC risk. The results for metabolite profile scores were further replicated in an external validation cohort (Women’s Health Study, WHS)."},"methods_title":{"kind":"string","value":"2. Methods"},"methods_text":{"kind":"string","value":" Study Population Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\nOur primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required."},"results_title":{"kind":"string","value":"6. Results"},"results_text":{"kind":"string","value":"In NHS/NHSII/HPFS, participants were predominately white and middle-aged (mean age 53 years), with an average BMI of 25.4 kg/m2 (Table 1). The percentage energy intake from protein was 18%, and animal protein was 13%. Participants with a higher total red meat intake were slightly more likely to have a lower total fish intake (Pearson r = −0.07). In the analysis of CRC, participants had similar demographic characteristics but were a little older (mean age 61 years) and had a lower proportion of females. Compared to control participants, CRC cases were less likely to use multivitamins and aspirin, receive endoscopy screening, and be physically active, but were more likely to smoke and have a family history of CRC. They also had a somewhat higher intake of total red meat (mainly processed red meat) and a lower fish intake. Similarly, CRC cases in WHS were less likely to be physically active and more likely to smoke and have a family history of CRC. However, they had a lower intake of total red meat (mainly unprocessed red meat) than control participants.\nOf the 287 known metabolites, 85 were significantly correlated with total red meat intake, 36 with unprocessed red meat, 51 with processed red meat, 28 with poultry, 60 with total fish, 60 with dark meat fish, and 27 with canned tuna fish after Bonferroni correction (Figure 2). Similar metabolites correlated with total red meat intake, unprocessed red meat, and processed red meat. All three red meat groups were positively associated with creatine, hydroxyproline, coenzyme Q10, myristoleic acid, acylcarnitines, and plasmalogens with a number of double bonds ≤6 but were negatively associated with highly unsaturated lipid species including triglycerides (TAGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylcholines (LPCs). Poultry intake was positively correlated with creatine, ectoine, a few PCs, TAGs, and PE plasmalogens after Bonferroni correction. In contrast to red meat intake, total fish intake, dark meat fish, and canned tuna fish were all positively correlated with highly unsaturated lipid species. When we restricted the analysis to fasting participants and participants selected as controls in the original sub-studies, the correlation results were similar (Supplementary Figures S2 and S3).\nOf the 2561 unknown metabolites, 702 were significantly correlated with total red meat intake after Bonferroni correction, 222 with unprocessed red meat, 462 with processed red meat, 65 with poultry, 284 with total fish intake, 414 with dark meat fish, and 119 with canned tuna fish (Supplementary Table S2). Among these, the absolute partial Spearman rho exceeded 0.3 for 11 unknown metabolites: one with total red meat intake, 10 with total fish intake, and 5 with dark meat fish intake. Pearson correlation analysis between these 11 unknown metabolites and those known metabolites (130 in total) that were significantly associated with total red meat, total fish, or dark meat fish consumption identified several unknown metabolites being strongly correlated with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC (all Pearson r > 0.8) (Supplementary Figure S4).\nA total of 53 known metabolites were selected in the elastic net regression for total red meat intake, 55 for unprocessed red meat, 36 for processed red meat, 7 for poultry, 18 for total fish, 27 for dark meat fish, and 11 for canned tuna fish (Table 2). Each metabolite profile score based on the selected known metabolites was significantly correlated with the corresponding self-reported dietary intake in both NHS/NHSII/HPFS and WHS (r = 0.33–0.46 for total red meat; r = 0.36–0.42 for unprocessed red meat; r = 0.19–0.33 for processed red meat; r = 0.12–0.21 for poultry; r = 0.31–0.40 for total fish; r = 0.32–0.42 for dark meat fish; and r = 0.14–0.22 for canned tuna fish) (Table 2). Adding unknown metabolites into the metabolite profile scores did not materially improve their correlations with the dietary intake (Supplementary Table S3). As a sensitivity analysis, we adopted the random forest algorithm to impute the missingness and re-derived the metabolomic profile score. The newly derived scores were highly correlated to the original scores, with a Pearson r = 0.98 for the total red meat score, 0.98 for the unprocessed red meat, 0.96 for the processed red meat score, 0.92 for the poultry score, 0.99 for the total fish score, 0.99 for the dark meat fish score, and 0.97 for the canned tuna fish score.\nAfter adjusting for potential confounders, dietary intake of processed red meat was significantly associated with CRC risk. The pooled OR for 1 SD higher intake was 1.15 (95% CI: 1.03, 1.29) (Table 3). We did not observe consistent associations for dietary intake of three fish groups across NHS/HPFS and WHS. An inverse association for total fish and canned tuna fish was only found in NHS/HPFS and for dark meat fish in WHS. In contrast, metabolite profile scores for these three fish groups were inversely associated with CRC risk in both NHS/HPFS and WHS (Table 3). The pooled OR per 1 SD higher was 0.86 (95% CI: 0.77, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were observed for other meat intakes or metabolite profile scores. The results remained similar after adjusting for menopausal status or ethnicity in NHS/HPFS (data not shown). In the analysis by tumor site, we observed that the above associations were stronger for rectal cancer than those for colon cancer (Supplementary Table S4).\nAmong metabolites selected in metabolite profile scores of all three fish groups, five metabolites (C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) were positively correlated to fish intake but inversely associated with CRC risk; two metabolites (C20:4 LPE and C22:5 LPC) were inversely correlated to fish intake but positively associated with CRC risk (Supplementary Figure S5)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Study Population","3. Dietary Assessment","4. Metabolomics Measurement","5. Nondietary Covariates","Statistical Analyses"],"string":"[\n \"Study Population\",\n \"3. Dietary Assessment\",\n \"4. Metabolomics Measurement\",\n \"5. Nondietary Covariates\",\n \"Statistical Analyses\"\n]"},"other_sections_texts":{"kind":"list like","value":["Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.","Validated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.","Profiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.","In NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.","We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1."],"string":"[\n \"Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\",\n \"Validated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.\",\n \"Profiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.\",\n \"In NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\",\n \"We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Methods","Study Population","3. Dietary Assessment","4. Metabolomics Measurement","5. Nondietary Covariates","Statistical Analyses","6. Results","7. Discussion"],"string":"[\n \"1. Introduction\",\n \"2. Methods\",\n \"Study Population\",\n \"3. Dietary Assessment\",\n \"4. Metabolomics Measurement\",\n \"5. Nondietary Covariates\",\n \"Statistical Analyses\",\n \"6. Results\",\n \"7. Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Colorectal cancer (CRC) remains the second most commonly occurring cancer in women and the third in men worldwide [1]. CRC incidence is largely affected by screening and modifiable dietary and lifestyle factors [2]. Among these factors, meat and fish consumption has been the subject of many investigations. The consumption of red meat, especially processed red meat, is consistently associated with an increased risk of CRC [3]. However, there is still uncertainty regarding the association between fish intake and CRC risk. The World Cancer Research Fund and American Institute for Cancer Research (WCRF/AICR) concluded that there was only “limited evidence” for the beneficial effect of fish intake in CRC prevention [4]. Recent studies of fish intake and CRC risk also generated inconsistent results [5,6,7,8].\nPrevious epidemiological studies examining the association of meat and fish intake with CRC risk mainly used self-reported dietary data. Rapid developments in high-throughput metabolomics are leading to a new era in nutritional epidemiological research. By measuring the small-molecule metabolites in biological samples, metabolomics may provide an objective picture of food intake and its related biological consequences [9]. Feeding trials and observational studies have demonstrated that plasma and urinary metabolites differed between meat and fish consumption [10,11,12,13,14]. However, it is unknown whether the metabolite profiles related to meat and fish intake are associated with CRC risk and whether these profiles could be used as a complementary approach to evaluate the association between meat and fish intake and CRC risk.\nTherefore, we examined the associations of red meat, poultry, and fish consumption with plasma metabolite profiles among participants from the Nurses’ Health Study (NHS), NHSII, and Health Professionals Follow-up Study (HPFS). We also developed metabolite profile scores that were correlated to the intakes of these meat and fish groups and evaluated the prospective associations of metabolite profile scores with CRC risk. The results for metabolite profile scores were further replicated in an external validation cohort (Women’s Health Study, WHS)."," Study Population Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\nOur primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.","Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.","Validated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.","Profiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.","In NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.","We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.","In NHS/NHSII/HPFS, participants were predominately white and middle-aged (mean age 53 years), with an average BMI of 25.4 kg/m2 (Table 1). The percentage energy intake from protein was 18%, and animal protein was 13%. Participants with a higher total red meat intake were slightly more likely to have a lower total fish intake (Pearson r = −0.07). In the analysis of CRC, participants had similar demographic characteristics but were a little older (mean age 61 years) and had a lower proportion of females. Compared to control participants, CRC cases were less likely to use multivitamins and aspirin, receive endoscopy screening, and be physically active, but were more likely to smoke and have a family history of CRC. They also had a somewhat higher intake of total red meat (mainly processed red meat) and a lower fish intake. Similarly, CRC cases in WHS were less likely to be physically active and more likely to smoke and have a family history of CRC. However, they had a lower intake of total red meat (mainly unprocessed red meat) than control participants.\nOf the 287 known metabolites, 85 were significantly correlated with total red meat intake, 36 with unprocessed red meat, 51 with processed red meat, 28 with poultry, 60 with total fish, 60 with dark meat fish, and 27 with canned tuna fish after Bonferroni correction (Figure 2). Similar metabolites correlated with total red meat intake, unprocessed red meat, and processed red meat. All three red meat groups were positively associated with creatine, hydroxyproline, coenzyme Q10, myristoleic acid, acylcarnitines, and plasmalogens with a number of double bonds ≤6 but were negatively associated with highly unsaturated lipid species including triglycerides (TAGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylcholines (LPCs). Poultry intake was positively correlated with creatine, ectoine, a few PCs, TAGs, and PE plasmalogens after Bonferroni correction. In contrast to red meat intake, total fish intake, dark meat fish, and canned tuna fish were all positively correlated with highly unsaturated lipid species. When we restricted the analysis to fasting participants and participants selected as controls in the original sub-studies, the correlation results were similar (Supplementary Figures S2 and S3).\nOf the 2561 unknown metabolites, 702 were significantly correlated with total red meat intake after Bonferroni correction, 222 with unprocessed red meat, 462 with processed red meat, 65 with poultry, 284 with total fish intake, 414 with dark meat fish, and 119 with canned tuna fish (Supplementary Table S2). Among these, the absolute partial Spearman rho exceeded 0.3 for 11 unknown metabolites: one with total red meat intake, 10 with total fish intake, and 5 with dark meat fish intake. Pearson correlation analysis between these 11 unknown metabolites and those known metabolites (130 in total) that were significantly associated with total red meat, total fish, or dark meat fish consumption identified several unknown metabolites being strongly correlated with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC (all Pearson r > 0.8) (Supplementary Figure S4).\nA total of 53 known metabolites were selected in the elastic net regression for total red meat intake, 55 for unprocessed red meat, 36 for processed red meat, 7 for poultry, 18 for total fish, 27 for dark meat fish, and 11 for canned tuna fish (Table 2). Each metabolite profile score based on the selected known metabolites was significantly correlated with the corresponding self-reported dietary intake in both NHS/NHSII/HPFS and WHS (r = 0.33–0.46 for total red meat; r = 0.36–0.42 for unprocessed red meat; r = 0.19–0.33 for processed red meat; r = 0.12–0.21 for poultry; r = 0.31–0.40 for total fish; r = 0.32–0.42 for dark meat fish; and r = 0.14–0.22 for canned tuna fish) (Table 2). Adding unknown metabolites into the metabolite profile scores did not materially improve their correlations with the dietary intake (Supplementary Table S3). As a sensitivity analysis, we adopted the random forest algorithm to impute the missingness and re-derived the metabolomic profile score. The newly derived scores were highly correlated to the original scores, with a Pearson r = 0.98 for the total red meat score, 0.98 for the unprocessed red meat, 0.96 for the processed red meat score, 0.92 for the poultry score, 0.99 for the total fish score, 0.99 for the dark meat fish score, and 0.97 for the canned tuna fish score.\nAfter adjusting for potential confounders, dietary intake of processed red meat was significantly associated with CRC risk. The pooled OR for 1 SD higher intake was 1.15 (95% CI: 1.03, 1.29) (Table 3). We did not observe consistent associations for dietary intake of three fish groups across NHS/HPFS and WHS. An inverse association for total fish and canned tuna fish was only found in NHS/HPFS and for dark meat fish in WHS. In contrast, metabolite profile scores for these three fish groups were inversely associated with CRC risk in both NHS/HPFS and WHS (Table 3). The pooled OR per 1 SD higher was 0.86 (95% CI: 0.77, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were observed for other meat intakes or metabolite profile scores. The results remained similar after adjusting for menopausal status or ethnicity in NHS/HPFS (data not shown). In the analysis by tumor site, we observed that the above associations were stronger for rectal cancer than those for colon cancer (Supplementary Table S4).\nAmong metabolites selected in metabolite profile scores of all three fish groups, five metabolites (C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) were positively correlated to fish intake but inversely associated with CRC risk; two metabolites (C20:4 LPE and C22:5 LPC) were inversely correlated to fish intake but positively associated with CRC risk (Supplementary Figure S5).","Leveraging metabolomics data from four studies, we found systematic differences in plasma metabolite profiles according to various types of meat and fish consumption. We also developed metabolite profile scores that were correlated to the consumption of red meat, poultry, and fish. Higher metabolite profile scores for all three fish groups (total fish, dark meat fish, and canned tuna fish), rather than their self-reported dietary intake, were consistently associated with a lower CRC risk in both NHS/HPFS and WHS. These findings suggest the promising use of metabolomics in complementing traditional dietary assessments to evaluate the association between dietary exposure and health outcomes.\nConsistent with previous studies, we found that consumption of all three red meat groups was positively correlated with several PE and PC plasmalogens, all of which have a low degree of unsaturation and contain acid moieties derived from animal tissues [14,29]. In contrast, total fish intake was positively correlated with highly unsaturated lipids, including TAGs, PCs, PEs, LPCs, LPEs, CEs, and plasmalogens. Fish intake, especially marine fish, is the principal dietary contributor of highly n-3 polyunsaturated fatty acids [30], and thus, are dietary sources for these lipids and their downstream metabolites.\nApart from lipid metabolites, we also observed several other metabolites correlated with meat and fish consumption. Creatine, an amino acid metabolite abundant in skeletal muscle [31], was positively correlated with the intake of red meat, poultry, and fish. Hydroxyproline, an amino acid that forms part of collagen [32], was positively correlated with red meat and poultry intake. Additionally, we found a positive correlation between poultry intake and ectoine (an α-amino acid), in line with one previous study [14]. Ectoine can be synthesized and released by several species in the genus Halomonas [33], the dominant microbial genus in the chicken embryo [34], which might lead to the presence of ectoine in chicken.\nMost previous metabolomic studies mainly focused on known metabolites, which are only a small percent of the data obtained in a typical metabolomics measurement (~10%) [35]. In the present study, we analyzed the unknown metabolites and identified a set of unknown metabolites that were significantly correlated with meat or fish consumption. Several unknown metabolites even exhibited a higher correlation with meat and fish intake (partial Spearman rho > 0.3) compared to known metabolites. By further examining the correlation between these unknown metabolites and the known metabolites, strong correlations were found with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC, indicating their potential biological links. However, whether these unknown metabolites are metabolites of the known metabolites or unrelated molecules affected by the same food is not clear. A critical next step is to structurally identify the promising and influential unknown metabolites. It should also be noted that despite the many statistically significant correlations between meat and fish groups and the unknown metabolites, these unknown metabolites added minimally to the correlations between meat and fish intakes and metabolite profile score based only on the known metabolites. The possibility remains that other metabolomics platforms may add further independent prediction of intake.\nThe human metabolome reflects a holistic biological status, which can be influenced by various exogenous and endogenous factors, such as dietary intakes, gut microbiota, health status, and genotype [36,37]. The metabolites selected in the metabolite profile score for meat or fish consumption could represent, in part, the components of food, such as the eicosapentaenoic acid/docosahexaenoic acid-containing lipids for the fish intake and hydroxyproline for the intake of red meat and poultry. Other metabolites, for example, C20:4 LPE and C22:5 LPC, could be related to the metabolic processes after fish intake, as fish is one of the main sources of arachidonic acid (C20:4) [38] and docosapentaenoic acid (C22:5) [39], but it was inversely correlated with these two lipids. Thus, metabolite profile score can be a comprehensive way to reflect both genuine compounds from foods as well as markers of complex metabolomic responses to dietary exposures.\nNot surprisingly, we found a significant positive association between processed red meat intake and CRC risk. However, the metabolite profile score for processed red meat consumption was not significantly associated with CRC risk. One possible explanation could be that the increased CRC risk associated with high processed red meat intake was mainly due to the carcinogenic compounds [40], such as polycyclic aromatic hydrocarbons, heterocyclic amines, and N-nitroso compounds, which were not measured in our metabolomics platforms. On the contrary, we did not observe a consistent inverse association between dietary fish intake and CRC risk, but inverse associations were found for the metabolite profile scores of all three fish groups in both NHS/HPFS and WHS. Results from previous studies of fish intake and CRC risk were generally inconsistent, some indicating an inverse association [7,8], but others not [5,6]. Unlike self-reported dietary intake data widely used in previous studies, the metabolomics data objectively captures the metabolites related to dietary exposures in the human body, which might be more directly associated with health outcomes. The seven metabolites (C20:4 LPE, C22:5 LPC, C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) selected in metabolite profile scores of all three fish groups could represent the beneficial metabolomic response to fish intake, and this response led to a lower risk of CRC.\nThe main strengths of our study are the large sample size, comprehensive metabolite profiles including both known and unknown metabolites, prospectively collected dietary data before CRC diagnosis, and the use of the elastic net model, which performs well in high-dimensional data where there are high correlations between predictors [41]. Moreover, the robustness of our metabolite profile scores was validated in an independent study. Nevertheless, our results also have several limitations. First, we did not have data on the type of red meat. Red meat subtypes (beef, lamb, or pork) may differ in their associations with CRC risk [42]. Second, only one metabolomics measurement was performed for each participant. However, our previous pilot study indicated that most metabolites were highly stable over 1–2 years within individuals [26]. Third, the metabolites measured in our study focused on lipids, amino acids, and amino acid derivatives and did not include all established biomarkers of food intake. Finally, participants in our study were mainly white women; replication of our findings in men and other racial groups is needed.\nIn conclusion, we identified a panel of metabolites differentially correlated with the intake of red meat, poultry, and fish. We also developed metabolite profile scores associated with the consumption of meat and fish and observed consistent inverse associations between metabolite profile scores of fish intake and CRC risk. Our results suggest the potential utility of metabolomics in complementing traditional dietary assessments to investigate the diet-disease association and disentangle the metabolomic responses linking higher fish intake with reduced CRC risk."],"string":"[\n \"Colorectal cancer (CRC) remains the second most commonly occurring cancer in women and the third in men worldwide [1]. CRC incidence is largely affected by screening and modifiable dietary and lifestyle factors [2]. Among these factors, meat and fish consumption has been the subject of many investigations. The consumption of red meat, especially processed red meat, is consistently associated with an increased risk of CRC [3]. However, there is still uncertainty regarding the association between fish intake and CRC risk. The World Cancer Research Fund and American Institute for Cancer Research (WCRF/AICR) concluded that there was only “limited evidence” for the beneficial effect of fish intake in CRC prevention [4]. Recent studies of fish intake and CRC risk also generated inconsistent results [5,6,7,8].\\nPrevious epidemiological studies examining the association of meat and fish intake with CRC risk mainly used self-reported dietary data. Rapid developments in high-throughput metabolomics are leading to a new era in nutritional epidemiological research. By measuring the small-molecule metabolites in biological samples, metabolomics may provide an objective picture of food intake and its related biological consequences [9]. Feeding trials and observational studies have demonstrated that plasma and urinary metabolites differed between meat and fish consumption [10,11,12,13,14]. However, it is unknown whether the metabolite profiles related to meat and fish intake are associated with CRC risk and whether these profiles could be used as a complementary approach to evaluate the association between meat and fish intake and CRC risk.\\nTherefore, we examined the associations of red meat, poultry, and fish consumption with plasma metabolite profiles among participants from the Nurses’ Health Study (NHS), NHSII, and Health Professionals Follow-up Study (HPFS). We also developed metabolite profile scores that were correlated to the intakes of these meat and fish groups and evaluated the prospective associations of metabolite profile scores with CRC risk. The results for metabolite profile scores were further replicated in an external validation cohort (Women’s Health Study, WHS).\",\n \" Study Population Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\\nOur primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\",\n \"Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\",\n \"Validated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.\",\n \"Profiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.\",\n \"In NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\",\n \"We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\",\n \"In NHS/NHSII/HPFS, participants were predominately white and middle-aged (mean age 53 years), with an average BMI of 25.4 kg/m2 (Table 1). The percentage energy intake from protein was 18%, and animal protein was 13%. Participants with a higher total red meat intake were slightly more likely to have a lower total fish intake (Pearson r = −0.07). In the analysis of CRC, participants had similar demographic characteristics but were a little older (mean age 61 years) and had a lower proportion of females. Compared to control participants, CRC cases were less likely to use multivitamins and aspirin, receive endoscopy screening, and be physically active, but were more likely to smoke and have a family history of CRC. They also had a somewhat higher intake of total red meat (mainly processed red meat) and a lower fish intake. Similarly, CRC cases in WHS were less likely to be physically active and more likely to smoke and have a family history of CRC. However, they had a lower intake of total red meat (mainly unprocessed red meat) than control participants.\\nOf the 287 known metabolites, 85 were significantly correlated with total red meat intake, 36 with unprocessed red meat, 51 with processed red meat, 28 with poultry, 60 with total fish, 60 with dark meat fish, and 27 with canned tuna fish after Bonferroni correction (Figure 2). Similar metabolites correlated with total red meat intake, unprocessed red meat, and processed red meat. All three red meat groups were positively associated with creatine, hydroxyproline, coenzyme Q10, myristoleic acid, acylcarnitines, and plasmalogens with a number of double bonds ≤6 but were negatively associated with highly unsaturated lipid species including triglycerides (TAGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylcholines (LPCs). Poultry intake was positively correlated with creatine, ectoine, a few PCs, TAGs, and PE plasmalogens after Bonferroni correction. In contrast to red meat intake, total fish intake, dark meat fish, and canned tuna fish were all positively correlated with highly unsaturated lipid species. When we restricted the analysis to fasting participants and participants selected as controls in the original sub-studies, the correlation results were similar (Supplementary Figures S2 and S3).\\nOf the 2561 unknown metabolites, 702 were significantly correlated with total red meat intake after Bonferroni correction, 222 with unprocessed red meat, 462 with processed red meat, 65 with poultry, 284 with total fish intake, 414 with dark meat fish, and 119 with canned tuna fish (Supplementary Table S2). Among these, the absolute partial Spearman rho exceeded 0.3 for 11 unknown metabolites: one with total red meat intake, 10 with total fish intake, and 5 with dark meat fish intake. Pearson correlation analysis between these 11 unknown metabolites and those known metabolites (130 in total) that were significantly associated with total red meat, total fish, or dark meat fish consumption identified several unknown metabolites being strongly correlated with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC (all Pearson r > 0.8) (Supplementary Figure S4).\\nA total of 53 known metabolites were selected in the elastic net regression for total red meat intake, 55 for unprocessed red meat, 36 for processed red meat, 7 for poultry, 18 for total fish, 27 for dark meat fish, and 11 for canned tuna fish (Table 2). Each metabolite profile score based on the selected known metabolites was significantly correlated with the corresponding self-reported dietary intake in both NHS/NHSII/HPFS and WHS (r = 0.33–0.46 for total red meat; r = 0.36–0.42 for unprocessed red meat; r = 0.19–0.33 for processed red meat; r = 0.12–0.21 for poultry; r = 0.31–0.40 for total fish; r = 0.32–0.42 for dark meat fish; and r = 0.14–0.22 for canned tuna fish) (Table 2). Adding unknown metabolites into the metabolite profile scores did not materially improve their correlations with the dietary intake (Supplementary Table S3). As a sensitivity analysis, we adopted the random forest algorithm to impute the missingness and re-derived the metabolomic profile score. The newly derived scores were highly correlated to the original scores, with a Pearson r = 0.98 for the total red meat score, 0.98 for the unprocessed red meat, 0.96 for the processed red meat score, 0.92 for the poultry score, 0.99 for the total fish score, 0.99 for the dark meat fish score, and 0.97 for the canned tuna fish score.\\nAfter adjusting for potential confounders, dietary intake of processed red meat was significantly associated with CRC risk. The pooled OR for 1 SD higher intake was 1.15 (95% CI: 1.03, 1.29) (Table 3). We did not observe consistent associations for dietary intake of three fish groups across NHS/HPFS and WHS. An inverse association for total fish and canned tuna fish was only found in NHS/HPFS and for dark meat fish in WHS. In contrast, metabolite profile scores for these three fish groups were inversely associated with CRC risk in both NHS/HPFS and WHS (Table 3). The pooled OR per 1 SD higher was 0.86 (95% CI: 0.77, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were observed for other meat intakes or metabolite profile scores. The results remained similar after adjusting for menopausal status or ethnicity in NHS/HPFS (data not shown). In the analysis by tumor site, we observed that the above associations were stronger for rectal cancer than those for colon cancer (Supplementary Table S4).\\nAmong metabolites selected in metabolite profile scores of all three fish groups, five metabolites (C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) were positively correlated to fish intake but inversely associated with CRC risk; two metabolites (C20:4 LPE and C22:5 LPC) were inversely correlated to fish intake but positively associated with CRC risk (Supplementary Figure S5).\",\n \"Leveraging metabolomics data from four studies, we found systematic differences in plasma metabolite profiles according to various types of meat and fish consumption. We also developed metabolite profile scores that were correlated to the consumption of red meat, poultry, and fish. Higher metabolite profile scores for all three fish groups (total fish, dark meat fish, and canned tuna fish), rather than their self-reported dietary intake, were consistently associated with a lower CRC risk in both NHS/HPFS and WHS. These findings suggest the promising use of metabolomics in complementing traditional dietary assessments to evaluate the association between dietary exposure and health outcomes.\\nConsistent with previous studies, we found that consumption of all three red meat groups was positively correlated with several PE and PC plasmalogens, all of which have a low degree of unsaturation and contain acid moieties derived from animal tissues [14,29]. In contrast, total fish intake was positively correlated with highly unsaturated lipids, including TAGs, PCs, PEs, LPCs, LPEs, CEs, and plasmalogens. Fish intake, especially marine fish, is the principal dietary contributor of highly n-3 polyunsaturated fatty acids [30], and thus, are dietary sources for these lipids and their downstream metabolites.\\nApart from lipid metabolites, we also observed several other metabolites correlated with meat and fish consumption. Creatine, an amino acid metabolite abundant in skeletal muscle [31], was positively correlated with the intake of red meat, poultry, and fish. Hydroxyproline, an amino acid that forms part of collagen [32], was positively correlated with red meat and poultry intake. Additionally, we found a positive correlation between poultry intake and ectoine (an α-amino acid), in line with one previous study [14]. Ectoine can be synthesized and released by several species in the genus Halomonas [33], the dominant microbial genus in the chicken embryo [34], which might lead to the presence of ectoine in chicken.\\nMost previous metabolomic studies mainly focused on known metabolites, which are only a small percent of the data obtained in a typical metabolomics measurement (~10%) [35]. In the present study, we analyzed the unknown metabolites and identified a set of unknown metabolites that were significantly correlated with meat or fish consumption. Several unknown metabolites even exhibited a higher correlation with meat and fish intake (partial Spearman rho > 0.3) compared to known metabolites. By further examining the correlation between these unknown metabolites and the known metabolites, strong correlations were found with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC, indicating their potential biological links. However, whether these unknown metabolites are metabolites of the known metabolites or unrelated molecules affected by the same food is not clear. A critical next step is to structurally identify the promising and influential unknown metabolites. It should also be noted that despite the many statistically significant correlations between meat and fish groups and the unknown metabolites, these unknown metabolites added minimally to the correlations between meat and fish intakes and metabolite profile score based only on the known metabolites. The possibility remains that other metabolomics platforms may add further independent prediction of intake.\\nThe human metabolome reflects a holistic biological status, which can be influenced by various exogenous and endogenous factors, such as dietary intakes, gut microbiota, health status, and genotype [36,37]. The metabolites selected in the metabolite profile score for meat or fish consumption could represent, in part, the components of food, such as the eicosapentaenoic acid/docosahexaenoic acid-containing lipids for the fish intake and hydroxyproline for the intake of red meat and poultry. Other metabolites, for example, C20:4 LPE and C22:5 LPC, could be related to the metabolic processes after fish intake, as fish is one of the main sources of arachidonic acid (C20:4) [38] and docosapentaenoic acid (C22:5) [39], but it was inversely correlated with these two lipids. Thus, metabolite profile score can be a comprehensive way to reflect both genuine compounds from foods as well as markers of complex metabolomic responses to dietary exposures.\\nNot surprisingly, we found a significant positive association between processed red meat intake and CRC risk. However, the metabolite profile score for processed red meat consumption was not significantly associated with CRC risk. One possible explanation could be that the increased CRC risk associated with high processed red meat intake was mainly due to the carcinogenic compounds [40], such as polycyclic aromatic hydrocarbons, heterocyclic amines, and N-nitroso compounds, which were not measured in our metabolomics platforms. On the contrary, we did not observe a consistent inverse association between dietary fish intake and CRC risk, but inverse associations were found for the metabolite profile scores of all three fish groups in both NHS/HPFS and WHS. Results from previous studies of fish intake and CRC risk were generally inconsistent, some indicating an inverse association [7,8], but others not [5,6]. Unlike self-reported dietary intake data widely used in previous studies, the metabolomics data objectively captures the metabolites related to dietary exposures in the human body, which might be more directly associated with health outcomes. The seven metabolites (C20:4 LPE, C22:5 LPC, C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) selected in metabolite profile scores of all three fish groups could represent the beneficial metabolomic response to fish intake, and this response led to a lower risk of CRC.\\nThe main strengths of our study are the large sample size, comprehensive metabolite profiles including both known and unknown metabolites, prospectively collected dietary data before CRC diagnosis, and the use of the elastic net model, which performs well in high-dimensional data where there are high correlations between predictors [41]. Moreover, the robustness of our metabolite profile scores was validated in an independent study. Nevertheless, our results also have several limitations. First, we did not have data on the type of red meat. Red meat subtypes (beef, lamb, or pork) may differ in their associations with CRC risk [42]. Second, only one metabolomics measurement was performed for each participant. However, our previous pilot study indicated that most metabolites were highly stable over 1–2 years within individuals [26]. Third, the metabolites measured in our study focused on lipids, amino acids, and amino acid derivatives and did not include all established biomarkers of food intake. Finally, participants in our study were mainly white women; replication of our findings in men and other racial groups is needed.\\nIn conclusion, we identified a panel of metabolites differentially correlated with the intake of red meat, poultry, and fish. We also developed metabolite profile scores associated with the consumption of meat and fish and observed consistent inverse associations between metabolite profile scores of fish intake and CRC risk. Our results suggest the potential utility of metabolomics in complementing traditional dietary assessments to investigate the diet-disease association and disentangle the metabolomic responses linking higher fish intake with reduced CRC risk.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,"results","discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["red meat","fish","plasma metabolomics","colorectal cancer"],"string":"[\n \"red meat\",\n \"fish\",\n \"plasma metabolomics\",\n \"colorectal cancer\"\n]"},"whole_article_text":{"kind":"string","value":"1. Introduction:\nColorectal cancer (CRC) remains the second most commonly occurring cancer in women and the third in men worldwide [1]. CRC incidence is largely affected by screening and modifiable dietary and lifestyle factors [2]. Among these factors, meat and fish consumption has been the subject of many investigations. The consumption of red meat, especially processed red meat, is consistently associated with an increased risk of CRC [3]. However, there is still uncertainty regarding the association between fish intake and CRC risk. The World Cancer Research Fund and American Institute for Cancer Research (WCRF/AICR) concluded that there was only “limited evidence” for the beneficial effect of fish intake in CRC prevention [4]. Recent studies of fish intake and CRC risk also generated inconsistent results [5,6,7,8].\nPrevious epidemiological studies examining the association of meat and fish intake with CRC risk mainly used self-reported dietary data. Rapid developments in high-throughput metabolomics are leading to a new era in nutritional epidemiological research. By measuring the small-molecule metabolites in biological samples, metabolomics may provide an objective picture of food intake and its related biological consequences [9]. Feeding trials and observational studies have demonstrated that plasma and urinary metabolites differed between meat and fish consumption [10,11,12,13,14]. However, it is unknown whether the metabolite profiles related to meat and fish intake are associated with CRC risk and whether these profiles could be used as a complementary approach to evaluate the association between meat and fish intake and CRC risk.\nTherefore, we examined the associations of red meat, poultry, and fish consumption with plasma metabolite profiles among participants from the Nurses’ Health Study (NHS), NHSII, and Health Professionals Follow-up Study (HPFS). We also developed metabolite profile scores that were correlated to the intakes of these meat and fish groups and evaluated the prospective associations of metabolite profile scores with CRC risk. The results for metabolite profile scores were further replicated in an external validation cohort (Women’s Health Study, WHS).\n\n2. Methods:\n Study Population Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\nOur primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\n\nStudy Population:\nOur primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\n\n3. Dietary Assessment:\nValidated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.\n\n4. Metabolomics Measurement:\nProfiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.\n\n5. Nondietary Covariates:\nIn NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\n\nStatistical Analyses:\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\n\n6. Results:\nIn NHS/NHSII/HPFS, participants were predominately white and middle-aged (mean age 53 years), with an average BMI of 25.4 kg/m2 (Table 1). The percentage energy intake from protein was 18%, and animal protein was 13%. Participants with a higher total red meat intake were slightly more likely to have a lower total fish intake (Pearson r = −0.07). In the analysis of CRC, participants had similar demographic characteristics but were a little older (mean age 61 years) and had a lower proportion of females. Compared to control participants, CRC cases were less likely to use multivitamins and aspirin, receive endoscopy screening, and be physically active, but were more likely to smoke and have a family history of CRC. They also had a somewhat higher intake of total red meat (mainly processed red meat) and a lower fish intake. Similarly, CRC cases in WHS were less likely to be physically active and more likely to smoke and have a family history of CRC. However, they had a lower intake of total red meat (mainly unprocessed red meat) than control participants.\nOf the 287 known metabolites, 85 were significantly correlated with total red meat intake, 36 with unprocessed red meat, 51 with processed red meat, 28 with poultry, 60 with total fish, 60 with dark meat fish, and 27 with canned tuna fish after Bonferroni correction (Figure 2). Similar metabolites correlated with total red meat intake, unprocessed red meat, and processed red meat. All three red meat groups were positively associated with creatine, hydroxyproline, coenzyme Q10, myristoleic acid, acylcarnitines, and plasmalogens with a number of double bonds ≤6 but were negatively associated with highly unsaturated lipid species including triglycerides (TAGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylcholines (LPCs). Poultry intake was positively correlated with creatine, ectoine, a few PCs, TAGs, and PE plasmalogens after Bonferroni correction. In contrast to red meat intake, total fish intake, dark meat fish, and canned tuna fish were all positively correlated with highly unsaturated lipid species. When we restricted the analysis to fasting participants and participants selected as controls in the original sub-studies, the correlation results were similar (Supplementary Figures S2 and S3).\nOf the 2561 unknown metabolites, 702 were significantly correlated with total red meat intake after Bonferroni correction, 222 with unprocessed red meat, 462 with processed red meat, 65 with poultry, 284 with total fish intake, 414 with dark meat fish, and 119 with canned tuna fish (Supplementary Table S2). Among these, the absolute partial Spearman rho exceeded 0.3 for 11 unknown metabolites: one with total red meat intake, 10 with total fish intake, and 5 with dark meat fish intake. Pearson correlation analysis between these 11 unknown metabolites and those known metabolites (130 in total) that were significantly associated with total red meat, total fish, or dark meat fish consumption identified several unknown metabolites being strongly correlated with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC (all Pearson r > 0.8) (Supplementary Figure S4).\nA total of 53 known metabolites were selected in the elastic net regression for total red meat intake, 55 for unprocessed red meat, 36 for processed red meat, 7 for poultry, 18 for total fish, 27 for dark meat fish, and 11 for canned tuna fish (Table 2). Each metabolite profile score based on the selected known metabolites was significantly correlated with the corresponding self-reported dietary intake in both NHS/NHSII/HPFS and WHS (r = 0.33–0.46 for total red meat; r = 0.36–0.42 for unprocessed red meat; r = 0.19–0.33 for processed red meat; r = 0.12–0.21 for poultry; r = 0.31–0.40 for total fish; r = 0.32–0.42 for dark meat fish; and r = 0.14–0.22 for canned tuna fish) (Table 2). Adding unknown metabolites into the metabolite profile scores did not materially improve their correlations with the dietary intake (Supplementary Table S3). As a sensitivity analysis, we adopted the random forest algorithm to impute the missingness and re-derived the metabolomic profile score. The newly derived scores were highly correlated to the original scores, with a Pearson r = 0.98 for the total red meat score, 0.98 for the unprocessed red meat, 0.96 for the processed red meat score, 0.92 for the poultry score, 0.99 for the total fish score, 0.99 for the dark meat fish score, and 0.97 for the canned tuna fish score.\nAfter adjusting for potential confounders, dietary intake of processed red meat was significantly associated with CRC risk. The pooled OR for 1 SD higher intake was 1.15 (95% CI: 1.03, 1.29) (Table 3). We did not observe consistent associations for dietary intake of three fish groups across NHS/HPFS and WHS. An inverse association for total fish and canned tuna fish was only found in NHS/HPFS and for dark meat fish in WHS. In contrast, metabolite profile scores for these three fish groups were inversely associated with CRC risk in both NHS/HPFS and WHS (Table 3). The pooled OR per 1 SD higher was 0.86 (95% CI: 0.77, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were observed for other meat intakes or metabolite profile scores. The results remained similar after adjusting for menopausal status or ethnicity in NHS/HPFS (data not shown). In the analysis by tumor site, we observed that the above associations were stronger for rectal cancer than those for colon cancer (Supplementary Table S4).\nAmong metabolites selected in metabolite profile scores of all three fish groups, five metabolites (C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) were positively correlated to fish intake but inversely associated with CRC risk; two metabolites (C20:4 LPE and C22:5 LPC) were inversely correlated to fish intake but positively associated with CRC risk (Supplementary Figure S5).\n\n7. Discussion:\nLeveraging metabolomics data from four studies, we found systematic differences in plasma metabolite profiles according to various types of meat and fish consumption. We also developed metabolite profile scores that were correlated to the consumption of red meat, poultry, and fish. Higher metabolite profile scores for all three fish groups (total fish, dark meat fish, and canned tuna fish), rather than their self-reported dietary intake, were consistently associated with a lower CRC risk in both NHS/HPFS and WHS. These findings suggest the promising use of metabolomics in complementing traditional dietary assessments to evaluate the association between dietary exposure and health outcomes.\nConsistent with previous studies, we found that consumption of all three red meat groups was positively correlated with several PE and PC plasmalogens, all of which have a low degree of unsaturation and contain acid moieties derived from animal tissues [14,29]. In contrast, total fish intake was positively correlated with highly unsaturated lipids, including TAGs, PCs, PEs, LPCs, LPEs, CEs, and plasmalogens. Fish intake, especially marine fish, is the principal dietary contributor of highly n-3 polyunsaturated fatty acids [30], and thus, are dietary sources for these lipids and their downstream metabolites.\nApart from lipid metabolites, we also observed several other metabolites correlated with meat and fish consumption. Creatine, an amino acid metabolite abundant in skeletal muscle [31], was positively correlated with the intake of red meat, poultry, and fish. Hydroxyproline, an amino acid that forms part of collagen [32], was positively correlated with red meat and poultry intake. Additionally, we found a positive correlation between poultry intake and ectoine (an α-amino acid), in line with one previous study [14]. Ectoine can be synthesized and released by several species in the genus Halomonas [33], the dominant microbial genus in the chicken embryo [34], which might lead to the presence of ectoine in chicken.\nMost previous metabolomic studies mainly focused on known metabolites, which are only a small percent of the data obtained in a typical metabolomics measurement (~10%) [35]. In the present study, we analyzed the unknown metabolites and identified a set of unknown metabolites that were significantly correlated with meat or fish consumption. Several unknown metabolites even exhibited a higher correlation with meat and fish intake (partial Spearman rho > 0.3) compared to known metabolites. By further examining the correlation between these unknown metabolites and the known metabolites, strong correlations were found with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC, indicating their potential biological links. However, whether these unknown metabolites are metabolites of the known metabolites or unrelated molecules affected by the same food is not clear. A critical next step is to structurally identify the promising and influential unknown metabolites. It should also be noted that despite the many statistically significant correlations between meat and fish groups and the unknown metabolites, these unknown metabolites added minimally to the correlations between meat and fish intakes and metabolite profile score based only on the known metabolites. The possibility remains that other metabolomics platforms may add further independent prediction of intake.\nThe human metabolome reflects a holistic biological status, which can be influenced by various exogenous and endogenous factors, such as dietary intakes, gut microbiota, health status, and genotype [36,37]. The metabolites selected in the metabolite profile score for meat or fish consumption could represent, in part, the components of food, such as the eicosapentaenoic acid/docosahexaenoic acid-containing lipids for the fish intake and hydroxyproline for the intake of red meat and poultry. Other metabolites, for example, C20:4 LPE and C22:5 LPC, could be related to the metabolic processes after fish intake, as fish is one of the main sources of arachidonic acid (C20:4) [38] and docosapentaenoic acid (C22:5) [39], but it was inversely correlated with these two lipids. Thus, metabolite profile score can be a comprehensive way to reflect both genuine compounds from foods as well as markers of complex metabolomic responses to dietary exposures.\nNot surprisingly, we found a significant positive association between processed red meat intake and CRC risk. However, the metabolite profile score for processed red meat consumption was not significantly associated with CRC risk. One possible explanation could be that the increased CRC risk associated with high processed red meat intake was mainly due to the carcinogenic compounds [40], such as polycyclic aromatic hydrocarbons, heterocyclic amines, and N-nitroso compounds, which were not measured in our metabolomics platforms. On the contrary, we did not observe a consistent inverse association between dietary fish intake and CRC risk, but inverse associations were found for the metabolite profile scores of all three fish groups in both NHS/HPFS and WHS. Results from previous studies of fish intake and CRC risk were generally inconsistent, some indicating an inverse association [7,8], but others not [5,6]. Unlike self-reported dietary intake data widely used in previous studies, the metabolomics data objectively captures the metabolites related to dietary exposures in the human body, which might be more directly associated with health outcomes. The seven metabolites (C20:4 LPE, C22:5 LPC, C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) selected in metabolite profile scores of all three fish groups could represent the beneficial metabolomic response to fish intake, and this response led to a lower risk of CRC.\nThe main strengths of our study are the large sample size, comprehensive metabolite profiles including both known and unknown metabolites, prospectively collected dietary data before CRC diagnosis, and the use of the elastic net model, which performs well in high-dimensional data where there are high correlations between predictors [41]. Moreover, the robustness of our metabolite profile scores was validated in an independent study. Nevertheless, our results also have several limitations. First, we did not have data on the type of red meat. Red meat subtypes (beef, lamb, or pork) may differ in their associations with CRC risk [42]. Second, only one metabolomics measurement was performed for each participant. However, our previous pilot study indicated that most metabolites were highly stable over 1–2 years within individuals [26]. Third, the metabolites measured in our study focused on lipids, amino acids, and amino acid derivatives and did not include all established biomarkers of food intake. Finally, participants in our study were mainly white women; replication of our findings in men and other racial groups is needed.\nIn conclusion, we identified a panel of metabolites differentially correlated with the intake of red meat, poultry, and fish. We also developed metabolite profile scores associated with the consumption of meat and fish and observed consistent inverse associations between metabolite profile scores of fish intake and CRC risk. Our results suggest the potential utility of metabolomics in complementing traditional dietary assessments to investigate the diet-disease association and disentangle the metabolomic responses linking higher fish intake with reduced CRC risk.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nRed and processed meat consumption has been consistently associated with increased risk of colorectal cancer (CRC), but the association for fish intake is unclear. Evidence using objective dietary assessment approaches to evaluate these associations is sparse.\n\nMethods:\nWe measured plasma metabolites among 5269 participants from the Nurses' Health Study (NHS), NHSII, and Health Professionals Follow-Up study (HPFS). We calculated partial Spearman correlations between each metabolite and self-reported intake of seven red meat, poultry, and fish groups. Metabolite profile scores correlated to self-reported dietary intakes were developed using elastic net regression. Associations between self-reported intakes, metabolite profile scores, and subsequent CRC risk were further evaluated using conditional logistic regression among 559 matched (1:1) case-control pairs in NHS/HPFS and replicated among 266 pairs in Women's Health Study.\n\nResults:\nPlasma metabolites, especially highly unsaturated lipids, were differentially associated with red meat and fish groups. Metabolite profile scores for each food group were significantly correlated with the corresponding self-reported dietary intake. A higher dietary intake of processed red meat was associated with a higher risk of CRC (pooled OR per 1 SD, 1.15; 95% CI: 1.03, 1.29). In contrast, higher metabolite profile scores for all fish groups, not dietary intakes, were consistently associated with a lower CRC risk: the pooled OR per 1 SD was 0.86 (95% CI: 0.78, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were found for other food groups.\n\nConclusions:\nRed meat and fish intake exhibited systematically different plasma metabolite profiles. Plasma metabolite profile of fish intake was inversely associated with CRC risk."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":6815,"string":"6,815"},"whole_article_abstract_length":{"kind":"number","value":357,"string":"357"},"num_sections":{"kind":"number","value":9,"string":"9"},"most_frequent_words":{"kind":"list like","value":["fish","meat","metabolites","intake","red meat","red","metabolite","crc","dietary","profile"],"string":"[\n \"fish\",\n \"meat\",\n \"metabolites\",\n \"intake\",\n \"red meat\",\n \"red\",\n \"metabolite\",\n \"crc\",\n \"dietary\",\n \"profile\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | crc | meat | fish intake | intake crc | fish intake crc | risk | intake | crc risk | intake crc risk [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] blood | aged | cancer | blood collection | collection | samples | blood samples | years | included | matched [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] meat | fish | red meat | red | intake | total | total red meat | total red | total fish | dark [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | meat | metabolites | intake | metabolite | red | red meat | crc | metabolite profile | meat fish [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] CRC ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 5269 | NHSII | Health Professionals Follow-Up ||| Spearman | seven ||| ||| CRC | 559 | 1:1 | NHS | 266 | Women's Health Study [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Plasma metabolites ||| ||| CRC | 1 SD | 1.15 | 95% | CI | 1.03 | 1.29 ||| CRC | 0.86 | 95% | CI | 0.78 | 0.96 | 0.86 | 95% | CI | 0.77 | 0.96 | 0.87 | 95% | CI | 0.78 | 0.97 ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] CRC ||| ||| 5269 | NHSII | Health Professionals Follow-Up ||| Spearman | seven ||| ||| CRC | 559 | 1:1 | NHS | 266 | Women's Health Study ||| ||| Plasma metabolites ||| ||| CRC | 1 SD | 1.15 | 95% | CI | 1.03 | 1.29 ||| CRC | 0.86 | 95% | CI | 0.78 | 0.96 | 0.86 | 95% | CI | 0.77 | 0.96 | 0.87 | 95% | CI | 0.78 | 0.97 ||| ||| ||| Plasma | CRC [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":272,"cells":{"title":{"kind":"string","value":"Sericin cream reduces pruritus in hemodialysis patients: a randomized, double-blind, placebo-controlled experimental study."},"pmid":{"kind":"string","value":"23006933"},"background_abstract":{"kind":"string","value":"Uremic pruritus (UP) is a significant complication in ESRD patients and substantially impairs their quality of life. UP is considered to be a skin manifestation of chronic inflammation. Because sericin can suppress the release of pro-inflammatory cytokines, the purpose of this study was to investigate the short-term safety and efficacy of sericin cream for treating UP in hemodialysis patients."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"This study used a double-blind design to investigate the effects of random topical administration of sericin cream and cream base (placebo) on either the right or left extremities of hemodialysis patients for 6 weeks. Skin hydration, irritation and pigmentation were evaluated every 2 weeks using Skin Diagnostic SD27. The visual analog scale for itching was also evaluated every 2 weeks, and the Kidney Disease Quality of Life Short Form was performed on the day of each patient's enrollment and after 6 weeks of treatment."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Fifty dialysis patients were enrolled, 47 of which completed the study. The hydration of the skin of the patients' extremities increased significantly after administration of sericin cream; significant differences were found between sericin treatment and control after 6 weeks of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Moreover, a significant difference was also found in skin irritation between the two treatments (p = 0.013 for arms and p = 0.027 for legs, respectively). At the end of the study, the skin pigmentation level was significantly reduced on both the arms (p = 0.032) and legs (p = 0.021) of the sericin-treated side compared with the side treated with cream base. The mean itching score decreased significantly from moderate to severe at the time of enrollment to mild pruritus after 6 weeks of treatment (p = 0.002). A better quality of life was found in all domains tested although statistically significant differences before and after treatment was found only in the patients' pain scores, the effect of kidney disease on daily life, sleep quality and symptoms or problems related to kidney disease."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"We conclude that sericin cream has a high potential for reducing UP in hemodialysis patients.The trial registration number of this study is ISRCTN16019033; its public title is \"sericin cream reduces pruritus in hemodialysis patients\"."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Administration, Topical","Double-Blind Method","Female","Humans","Male","Middle Aged","Placebo Effect","Pruritus","Quality of Life","Renal Dialysis","Sericins","Skin Cream","Treatment Outcome"],"string":"[\n \"Administration, Topical\",\n \"Double-Blind Method\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Placebo Effect\",\n \"Pruritus\",\n \"Quality of Life\",\n \"Renal Dialysis\",\n \"Sericins\",\n \"Skin Cream\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"3472272"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Preparation of silk sericin cream Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\nBecause commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\n Molecular weight determination of sericin To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\nTo determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\n Amino acid analysis of sericin The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\nThe amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\n Study design Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\nSkin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\n Study population Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Study treatment In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\nIn 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\n Measurement and outcome The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\nThe level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\n Patients’ quality of life The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\nThe patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\n Safety monitoring The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\nThe occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\n Statistical analysis The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\nThe results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Molecular weight and amino acid composition of sericin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\n Study population Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.\nFifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Our study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients."},"other_sections_titles":{"kind":"list like","value":["Background","Molecular weight and amino acid composition of sericin","Study population","Preparation of silk sericin cream","Molecular weight determination of sericin","Amino acid analysis of sericin","Study design","Study population","Inclusion criteria","Exclusion criteria","Study treatment","Measurement and outcome","Patients’ quality of life","Safety monitoring","Statistical analysis","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Molecular weight and amino acid composition of sericin\",\n \"Study population\",\n \"Preparation of silk sericin cream\",\n \"Molecular weight determination of sericin\",\n \"Amino acid analysis of sericin\",\n \"Study design\",\n \"Study population\",\n \"Inclusion criteria\",\n \"Exclusion criteria\",\n \"Study treatment\",\n \"Measurement and outcome\",\n \"Patients’ quality of life\",\n \"Safety monitoring\",\n \"Statistical analysis\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.","Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.","Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.","Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.","To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.","The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.","Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention."," Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.","All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.","Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.","In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.","The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].","The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].","The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.","The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.","The authors declare that they have no competing interests.","PA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\n"],"string":"[\n \"Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.\",\n \"Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\\nAmino acid composition of sericin\\nThese mean values were obtained by triplicate analysis.\",\n \"Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \\nBaseline characteristics of subjects (N = 47)\\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\\nSS = silk sericin.\\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \\nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\\n* Indicates significant differences compared to baseline, p < 0.05.\",\n \"Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\",\n \"To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\",\n \"The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\",\n \"Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\",\n \" Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\",\n \"All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\",\n \"Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\",\n \"In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\",\n \"The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\\n\\n\\n(1)\\n\\n\\n%\\nchanges in each parameter\\n=\\n\\n\\n\\n\\n\\nP\\nt\\n\\n−\\n\\nP\\n0\\n\\n\\n\\n/\\n\\nP\\n0\\n\\n\\n\\n×\\n100\\n,\\n\\n\\n\\n\\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\",\n \"The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\",\n \"The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\",\n \"The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\",\n \"The authors declare that they have no competing interests.\",\n \"PA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Results","Molecular weight and amino acid composition of sericin","Study population","Discussion","Conclusions","Methods","Preparation of silk sericin cream","Molecular weight determination of sericin","Amino acid analysis of sericin","Study design","Study population","Inclusion criteria","Exclusion criteria","Study treatment","Measurement and outcome","Patients’ quality of life","Safety monitoring","Statistical analysis","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Results\",\n \"Molecular weight and amino acid composition of sericin\",\n \"Study population\",\n \"Discussion\",\n \"Conclusions\",\n \"Methods\",\n \"Preparation of silk sericin cream\",\n \"Molecular weight determination of sericin\",\n \"Amino acid analysis of sericin\",\n \"Study design\",\n \"Study population\",\n \"Inclusion criteria\",\n \"Exclusion criteria\",\n \"Study treatment\",\n \"Measurement and outcome\",\n \"Patients’ quality of life\",\n \"Safety monitoring\",\n \"Statistical analysis\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated."," Molecular weight and amino acid composition of sericin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\n Study population Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.\nFifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.","Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.","Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.","This study has shown that the use of sericin cream can reduce UP in hemodialysis patients according to their VAS scores. Sericin cream can also significantly increase skin hydration and reduce skin irritation and skin pigmentation in patients. The molecular weight analysis of the sericin used in this study gave results that agree with those reported by Sprague, who indicated that sericin is a mixture of at least 15 different polypeptide chains ranging in size from 20–220 kDa [19]. Similar to the earlier report by Kato et al., serine was the most abundant amino acid found in sericin [20].\nAlthough antihistamines, anti-allergic agents and topical corticosteroids are commonly used to treat UP in ESRD patients, their effectiveness is sometimes limited [2,8,21,22]. Alternative and effective treatments for intractable pruritus that generate low levels of adverse reactions, particularly in sensitive patients such as the ESRD group, must therefore be developed. This clinical study is the first to suggest that a sericin cream is beneficial for treating pruritus in hemodialysis patients. The benefits of this treatment apparently result from increases in skin hydration and suppression of pro-inflammatory cytokines, as shown in our earlier study [17,23], thereby resulting in less skin irritation without any allergic reactions.\nPruritus associated with chronic kidney disease has been shown to be associated with elevated levels of C-reactive protein and other inflammatory cytokines. This evidence suggests that there is an inflammatory component in this form of pruritus [24]. Some authors consider UP to be a skin manifestation of chronic inflammation [25]. Recently, the “persistent microinflammation” theory, in which it is suggested that inflammation may be related to the genesis of UP, was proposed [26]. The skin of ESRD patients with UP contains an increased number of mast cells, and these cells can release various substances such as histamine, interleukins (IL) and tumor necrosis factor (TNF) [27-30].\nPruritic skin can appear normal except for dryness; because the severity of pruritus is closely correlated with skin dryness [31], xerosis treatment normally begins with a topical agent such as a gentle moisturizing cream. Because sericin is well known for its moisturizing effect and its ability to reduce the generation of pro-inflammatory cytokines by fibroblasts, this study assessed the usefulness of sericin cream in treating UP. Evaluating the degree of pruritus was difficult because it is a subjective symptom; therefore, functional measurements of skin conditions known to be associated with pruritus, such as skin hydration and skin irritation, were considered to be objective indices. We found that both sericin cream and the cream base used to prepare the sericin cream increased the moisture content of the stratum corneum; however, the level of hydration was significantly higher in the skin treated with sericin cream. The side of the body treated with sericin cream had significantly lower levels of post-treatment dryness compared to pretreatment dryness and lower total dermatological severity scores in parameters such as skin pigmentation.\nThe pathogenesis of ESRD itself is associated with inflammation and oxidative stress, as reflected in studies of plasma biochemistry, including the levels of the cytokines IL and TNF-α [32-34], classic markers of inflammatory processes that are strongly affected by pathological conditions [35]. These cytokines are upregulated in the plasma of ESRD patients [32,36]. The skin inflammation associated with the release of IL and TNF-α is further complicated by the inflammatory lesions that occur secondary to scratching [37]. Our results indicate that improvement in skin hydration and suppression of the release of pro-inflammatory cytokines in skin might be the mechanism by which sericin improves UP. Skin irritation, which can be caused by either skin inflammation or by scratching, was substantially reduced in the sericin group compared with the cream base group after 6 weeks of treatment. This result confirmed that sericin reduces skin irritation in patients with UP and suggested that it might be due to suppression of the inflammatory cytokines in skin. Similar results have been obtained in animal studies [23].\nSecondary skin lesions that can result from the itch-scratch cycle include excoriations, hyperpigmentation, lichenification, prurigo nodules and scars [38]. Chronic renal failure is usually accompanied by a variety of cutaneous manifestations; dermal manifestations, including hyperpigmentation, have also been reported, and these can exact a considerable toll on the quality of life [39-41]. Although skin pigmentation is not related to specific clinical symptoms, it has an important effect on patient satisfaction. Sericin has been reported to have activity against tyrosinase [15], an enzyme related to melanin production; as this study shows, treatment with sericin results in significant reduction in the skin color of patients.\nThe pruritus/mortality relationship may be strongly attributed to sleep disturbances, as previously mentioned in the DOPPS [8,9,42]. Many previous studies have found a strong association between inflammation, as measured by C-reactive protein or inflammatory cytokines, and sleep disturbances in dialysis patients [43-45]. The significant effects of pruritus on sleep, mood and social functioning require further investigation with the goal of improving the available treatments for this serious ESRD complication. We found that reduction in itching intensity from moderate to severe pruritus at the time of enrollment to mild pruritus after 6 weeks of treatment was associated with a better quality of life in all of the measured domains, including the mental, physical and kidney disease components. The KDQOL-SF score in the ESRD patients indicated improvements in several domains regarding the patients’ quality of life, particularly pain and sleep, which are relevant to itching. A better quality of sleep may reflect some degree of relief from itching achieved through the treatments.\nThis study has certain limitations. First, the study design was an in-subject controlled study; it cannot be determined whether the improvements in the itching evaluation and the quality of life were from the sample (sericin cream) or the placebo (cream base). Moreover, due to the in-subject controlled design, the biochemical parameters could not be evaluated at the end of the study. Second, the study included a small number of patients, and these were followed for a relatively short time. A long-term study with a larger sample size is necessary. In addition to studies of hemodialysis patients, similar studies of other ESRD patients, such as peritoneal dialysis and kidney transplant patients, are also necessary to confirm the findings presented here.","Our study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients."," Preparation of silk sericin cream Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\nBecause commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\n Molecular weight determination of sericin To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\nTo determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\n Amino acid analysis of sericin The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\nThe amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\n Study design Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\nSkin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\n Study population Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Study treatment In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\nIn 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\n Measurement and outcome The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\nThe level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\n Patients’ quality of life The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\nThe patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\n Safety monitoring The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\nThe occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\n Statistical analysis The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\nThe results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.","Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.","To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.","The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.","Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention."," Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.","All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.","Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.","In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.","The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].","The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].","The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.","The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.","The authors declare that they have no competing interests.","PA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\n"],"string":"[\n \"Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.\",\n \" Molecular weight and amino acid composition of sericin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\\nAmino acid composition of sericin\\nThese mean values were obtained by triplicate analysis.\\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\\nAmino acid composition of sericin\\nThese mean values were obtained by triplicate analysis.\\n Study population Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \\nBaseline characteristics of subjects (N = 47)\\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\\nSS = silk sericin.\\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \\nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\\n* Indicates significant differences compared to baseline, p < 0.05.\\nFifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \\nBaseline characteristics of subjects (N = 47)\\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\\nSS = silk sericin.\\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \\nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\\n* Indicates significant differences compared to baseline, p < 0.05.\",\n \"Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\\nAmino acid composition of sericin\\nThese mean values were obtained by triplicate analysis.\",\n \"Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \\nBaseline characteristics of subjects (N = 47)\\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\\nSS = silk sericin.\\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \\nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\\n* Indicates significant differences compared to baseline, p < 0.05.\",\n \"This study has shown that the use of sericin cream can reduce UP in hemodialysis patients according to their VAS scores. Sericin cream can also significantly increase skin hydration and reduce skin irritation and skin pigmentation in patients. The molecular weight analysis of the sericin used in this study gave results that agree with those reported by Sprague, who indicated that sericin is a mixture of at least 15 different polypeptide chains ranging in size from 20–220 kDa [19]. Similar to the earlier report by Kato et al., serine was the most abundant amino acid found in sericin [20].\\nAlthough antihistamines, anti-allergic agents and topical corticosteroids are commonly used to treat UP in ESRD patients, their effectiveness is sometimes limited [2,8,21,22]. Alternative and effective treatments for intractable pruritus that generate low levels of adverse reactions, particularly in sensitive patients such as the ESRD group, must therefore be developed. This clinical study is the first to suggest that a sericin cream is beneficial for treating pruritus in hemodialysis patients. The benefits of this treatment apparently result from increases in skin hydration and suppression of pro-inflammatory cytokines, as shown in our earlier study [17,23], thereby resulting in less skin irritation without any allergic reactions.\\nPruritus associated with chronic kidney disease has been shown to be associated with elevated levels of C-reactive protein and other inflammatory cytokines. This evidence suggests that there is an inflammatory component in this form of pruritus [24]. Some authors consider UP to be a skin manifestation of chronic inflammation [25]. Recently, the “persistent microinflammation” theory, in which it is suggested that inflammation may be related to the genesis of UP, was proposed [26]. The skin of ESRD patients with UP contains an increased number of mast cells, and these cells can release various substances such as histamine, interleukins (IL) and tumor necrosis factor (TNF) [27-30].\\nPruritic skin can appear normal except for dryness; because the severity of pruritus is closely correlated with skin dryness [31], xerosis treatment normally begins with a topical agent such as a gentle moisturizing cream. Because sericin is well known for its moisturizing effect and its ability to reduce the generation of pro-inflammatory cytokines by fibroblasts, this study assessed the usefulness of sericin cream in treating UP. Evaluating the degree of pruritus was difficult because it is a subjective symptom; therefore, functional measurements of skin conditions known to be associated with pruritus, such as skin hydration and skin irritation, were considered to be objective indices. We found that both sericin cream and the cream base used to prepare the sericin cream increased the moisture content of the stratum corneum; however, the level of hydration was significantly higher in the skin treated with sericin cream. The side of the body treated with sericin cream had significantly lower levels of post-treatment dryness compared to pretreatment dryness and lower total dermatological severity scores in parameters such as skin pigmentation.\\nThe pathogenesis of ESRD itself is associated with inflammation and oxidative stress, as reflected in studies of plasma biochemistry, including the levels of the cytokines IL and TNF-α [32-34], classic markers of inflammatory processes that are strongly affected by pathological conditions [35]. These cytokines are upregulated in the plasma of ESRD patients [32,36]. The skin inflammation associated with the release of IL and TNF-α is further complicated by the inflammatory lesions that occur secondary to scratching [37]. Our results indicate that improvement in skin hydration and suppression of the release of pro-inflammatory cytokines in skin might be the mechanism by which sericin improves UP. Skin irritation, which can be caused by either skin inflammation or by scratching, was substantially reduced in the sericin group compared with the cream base group after 6 weeks of treatment. This result confirmed that sericin reduces skin irritation in patients with UP and suggested that it might be due to suppression of the inflammatory cytokines in skin. Similar results have been obtained in animal studies [23].\\nSecondary skin lesions that can result from the itch-scratch cycle include excoriations, hyperpigmentation, lichenification, prurigo nodules and scars [38]. Chronic renal failure is usually accompanied by a variety of cutaneous manifestations; dermal manifestations, including hyperpigmentation, have also been reported, and these can exact a considerable toll on the quality of life [39-41]. Although skin pigmentation is not related to specific clinical symptoms, it has an important effect on patient satisfaction. Sericin has been reported to have activity against tyrosinase [15], an enzyme related to melanin production; as this study shows, treatment with sericin results in significant reduction in the skin color of patients.\\nThe pruritus/mortality relationship may be strongly attributed to sleep disturbances, as previously mentioned in the DOPPS [8,9,42]. Many previous studies have found a strong association between inflammation, as measured by C-reactive protein or inflammatory cytokines, and sleep disturbances in dialysis patients [43-45]. The significant effects of pruritus on sleep, mood and social functioning require further investigation with the goal of improving the available treatments for this serious ESRD complication. We found that reduction in itching intensity from moderate to severe pruritus at the time of enrollment to mild pruritus after 6 weeks of treatment was associated with a better quality of life in all of the measured domains, including the mental, physical and kidney disease components. The KDQOL-SF score in the ESRD patients indicated improvements in several domains regarding the patients’ quality of life, particularly pain and sleep, which are relevant to itching. A better quality of sleep may reflect some degree of relief from itching achieved through the treatments.\\nThis study has certain limitations. First, the study design was an in-subject controlled study; it cannot be determined whether the improvements in the itching evaluation and the quality of life were from the sample (sericin cream) or the placebo (cream base). Moreover, due to the in-subject controlled design, the biochemical parameters could not be evaluated at the end of the study. Second, the study included a small number of patients, and these were followed for a relatively short time. A long-term study with a larger sample size is necessary. In addition to studies of hemodialysis patients, similar studies of other ESRD patients, such as peritoneal dialysis and kidney transplant patients, are also necessary to confirm the findings presented here.\",\n \"Our study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients.\",\n \" Preparation of silk sericin cream Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\\nBecause commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\\n Molecular weight determination of sericin To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\\nTo determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\\n Amino acid analysis of sericin The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\\nThe amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\\n Study design Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\\nSkin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\\n Study population Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\n Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\n Study treatment In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\\nIn 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\\n Measurement and outcome The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\\n\\n\\n(1)\\n\\n\\n%\\nchanges in each parameter\\n=\\n\\n\\n\\n\\n\\nP\\nt\\n\\n−\\n\\nP\\n0\\n\\n\\n\\n/\\n\\nP\\n0\\n\\n\\n\\n×\\n100\\n,\\n\\n\\n\\n\\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\\nThe level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\\n\\n\\n(1)\\n\\n\\n%\\nchanges in each parameter\\n=\\n\\n\\n\\n\\n\\nP\\nt\\n\\n−\\n\\nP\\n0\\n\\n\\n\\n/\\n\\nP\\n0\\n\\n\\n\\n×\\n100\\n,\\n\\n\\n\\n\\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\\n Patients’ quality of life The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\\nThe patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\\n Safety monitoring The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\\nThe occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\\n Statistical analysis The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\\nThe results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\",\n \"Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\",\n \"To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\",\n \"The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\",\n \"Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\",\n \" Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\",\n \"All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\",\n \"Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\",\n \"In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\",\n \"The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\\n\\n\\n(1)\\n\\n\\n%\\nchanges in each parameter\\n=\\n\\n\\n\\n\\n\\nP\\nt\\n\\n−\\n\\nP\\n0\\n\\n\\n\\n/\\n\\nP\\n0\\n\\n\\n\\n×\\n100\\n,\\n\\n\\n\\n\\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\",\n \"The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\",\n \"The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\",\n \"The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\",\n \"The authors declare that they have no competing interests.\",\n \"PA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"results",null,null,"discussion","conclusions","methods",null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"results\",\n null,\n null,\n \"discussion\",\n \"conclusions\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Pruritus","Hemodialysis","Sericin","Hydration","Inflammation","Pigmentation"],"string":"[\n \"Pruritus\",\n \"Hemodialysis\",\n \"Sericin\",\n \"Hydration\",\n \"Inflammation\",\n \"Pigmentation\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nItching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.\n\nResults:\n Molecular weight and amino acid composition of sericin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\n Study population Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.\nFifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.\n\nMolecular weight and amino acid composition of sericin:\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\n\nStudy population:\nFifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.\n\nDiscussion:\nThis study has shown that the use of sericin cream can reduce UP in hemodialysis patients according to their VAS scores. Sericin cream can also significantly increase skin hydration and reduce skin irritation and skin pigmentation in patients. The molecular weight analysis of the sericin used in this study gave results that agree with those reported by Sprague, who indicated that sericin is a mixture of at least 15 different polypeptide chains ranging in size from 20–220 kDa [19]. Similar to the earlier report by Kato et al., serine was the most abundant amino acid found in sericin [20].\nAlthough antihistamines, anti-allergic agents and topical corticosteroids are commonly used to treat UP in ESRD patients, their effectiveness is sometimes limited [2,8,21,22]. Alternative and effective treatments for intractable pruritus that generate low levels of adverse reactions, particularly in sensitive patients such as the ESRD group, must therefore be developed. This clinical study is the first to suggest that a sericin cream is beneficial for treating pruritus in hemodialysis patients. The benefits of this treatment apparently result from increases in skin hydration and suppression of pro-inflammatory cytokines, as shown in our earlier study [17,23], thereby resulting in less skin irritation without any allergic reactions.\nPruritus associated with chronic kidney disease has been shown to be associated with elevated levels of C-reactive protein and other inflammatory cytokines. This evidence suggests that there is an inflammatory component in this form of pruritus [24]. Some authors consider UP to be a skin manifestation of chronic inflammation [25]. Recently, the “persistent microinflammation” theory, in which it is suggested that inflammation may be related to the genesis of UP, was proposed [26]. The skin of ESRD patients with UP contains an increased number of mast cells, and these cells can release various substances such as histamine, interleukins (IL) and tumor necrosis factor (TNF) [27-30].\nPruritic skin can appear normal except for dryness; because the severity of pruritus is closely correlated with skin dryness [31], xerosis treatment normally begins with a topical agent such as a gentle moisturizing cream. Because sericin is well known for its moisturizing effect and its ability to reduce the generation of pro-inflammatory cytokines by fibroblasts, this study assessed the usefulness of sericin cream in treating UP. Evaluating the degree of pruritus was difficult because it is a subjective symptom; therefore, functional measurements of skin conditions known to be associated with pruritus, such as skin hydration and skin irritation, were considered to be objective indices. We found that both sericin cream and the cream base used to prepare the sericin cream increased the moisture content of the stratum corneum; however, the level of hydration was significantly higher in the skin treated with sericin cream. The side of the body treated with sericin cream had significantly lower levels of post-treatment dryness compared to pretreatment dryness and lower total dermatological severity scores in parameters such as skin pigmentation.\nThe pathogenesis of ESRD itself is associated with inflammation and oxidative stress, as reflected in studies of plasma biochemistry, including the levels of the cytokines IL and TNF-α [32-34], classic markers of inflammatory processes that are strongly affected by pathological conditions [35]. These cytokines are upregulated in the plasma of ESRD patients [32,36]. The skin inflammation associated with the release of IL and TNF-α is further complicated by the inflammatory lesions that occur secondary to scratching [37]. Our results indicate that improvement in skin hydration and suppression of the release of pro-inflammatory cytokines in skin might be the mechanism by which sericin improves UP. Skin irritation, which can be caused by either skin inflammation or by scratching, was substantially reduced in the sericin group compared with the cream base group after 6 weeks of treatment. This result confirmed that sericin reduces skin irritation in patients with UP and suggested that it might be due to suppression of the inflammatory cytokines in skin. Similar results have been obtained in animal studies [23].\nSecondary skin lesions that can result from the itch-scratch cycle include excoriations, hyperpigmentation, lichenification, prurigo nodules and scars [38]. Chronic renal failure is usually accompanied by a variety of cutaneous manifestations; dermal manifestations, including hyperpigmentation, have also been reported, and these can exact a considerable toll on the quality of life [39-41]. Although skin pigmentation is not related to specific clinical symptoms, it has an important effect on patient satisfaction. Sericin has been reported to have activity against tyrosinase [15], an enzyme related to melanin production; as this study shows, treatment with sericin results in significant reduction in the skin color of patients.\nThe pruritus/mortality relationship may be strongly attributed to sleep disturbances, as previously mentioned in the DOPPS [8,9,42]. Many previous studies have found a strong association between inflammation, as measured by C-reactive protein or inflammatory cytokines, and sleep disturbances in dialysis patients [43-45]. The significant effects of pruritus on sleep, mood and social functioning require further investigation with the goal of improving the available treatments for this serious ESRD complication. We found that reduction in itching intensity from moderate to severe pruritus at the time of enrollment to mild pruritus after 6 weeks of treatment was associated with a better quality of life in all of the measured domains, including the mental, physical and kidney disease components. The KDQOL-SF score in the ESRD patients indicated improvements in several domains regarding the patients’ quality of life, particularly pain and sleep, which are relevant to itching. A better quality of sleep may reflect some degree of relief from itching achieved through the treatments.\nThis study has certain limitations. First, the study design was an in-subject controlled study; it cannot be determined whether the improvements in the itching evaluation and the quality of life were from the sample (sericin cream) or the placebo (cream base). Moreover, due to the in-subject controlled design, the biochemical parameters could not be evaluated at the end of the study. Second, the study included a small number of patients, and these were followed for a relatively short time. A long-term study with a larger sample size is necessary. In addition to studies of hemodialysis patients, similar studies of other ESRD patients, such as peritoneal dialysis and kidney transplant patients, are also necessary to confirm the findings presented here.\n\nConclusions:\nOur study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients.\n\nMethods:\n Preparation of silk sericin cream Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\nBecause commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\n Molecular weight determination of sericin To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\nTo determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\n Amino acid analysis of sericin The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\nThe amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\n Study design Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\nSkin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\n Study population Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Study treatment In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\nIn 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\n Measurement and outcome The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\nThe level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\n Patients’ quality of life The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\nThe patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\n Safety monitoring The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\nThe occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\n Statistical analysis The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\nThe results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\n\nPreparation of silk sericin cream:\nBecause commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\n\nMolecular weight determination of sericin:\nTo determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\n\nAmino acid analysis of sericin:\nThe amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\n\nStudy design:\nSkin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\n\nStudy population:\n Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n\nInclusion criteria:\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n\nExclusion criteria:\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n\nStudy treatment:\nIn 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\n\nMeasurement and outcome:\nThe level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\n\nPatients’ quality of life:\nThe patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\n\nSafety monitoring:\nThe occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\n\nStatistical analysis:\nThe results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nPA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nUremic pruritus (UP) is a significant complication in ESRD patients and substantially impairs their quality of life. UP is considered to be a skin manifestation of chronic inflammation. Because sericin can suppress the release of pro-inflammatory cytokines, the purpose of this study was to investigate the short-term safety and efficacy of sericin cream for treating UP in hemodialysis patients.\n\nMethods:\nThis study used a double-blind design to investigate the effects of random topical administration of sericin cream and cream base (placebo) on either the right or left extremities of hemodialysis patients for 6 weeks. Skin hydration, irritation and pigmentation were evaluated every 2 weeks using Skin Diagnostic SD27. The visual analog scale for itching was also evaluated every 2 weeks, and the Kidney Disease Quality of Life Short Form was performed on the day of each patient's enrollment and after 6 weeks of treatment.\n\nResults:\nFifty dialysis patients were enrolled, 47 of which completed the study. The hydration of the skin of the patients' extremities increased significantly after administration of sericin cream; significant differences were found between sericin treatment and control after 6 weeks of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Moreover, a significant difference was also found in skin irritation between the two treatments (p = 0.013 for arms and p = 0.027 for legs, respectively). At the end of the study, the skin pigmentation level was significantly reduced on both the arms (p = 0.032) and legs (p = 0.021) of the sericin-treated side compared with the side treated with cream base. The mean itching score decreased significantly from moderate to severe at the time of enrollment to mild pruritus after 6 weeks of treatment (p = 0.002). A better quality of life was found in all domains tested although statistically significant differences before and after treatment was found only in the patients' pain scores, the effect of kidney disease on daily life, sleep quality and symptoms or problems related to kidney disease.\n\nConclusions:\nWe conclude that sericin cream has a high potential for reducing UP in hemodialysis patients.The trial registration number of this study is ISRCTN16019033; its public title is \"sericin cream reduces pruritus in hemodialysis patients\"."},"background_conclusion_text":{"kind":"string","value":"Background:\nItching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.\n\nConclusions:\nOur study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nUremic pruritus (UP) is a significant complication in ESRD patients and substantially impairs their quality of life. UP is considered to be a skin manifestation of chronic inflammation. Because sericin can suppress the release of pro-inflammatory cytokines, the purpose of this study was to investigate the short-term safety and efficacy of sericin cream for treating UP in hemodialysis patients.\n\nMethods:\nThis study used a double-blind design to investigate the effects of random topical administration of sericin cream and cream base (placebo) on either the right or left extremities of hemodialysis patients for 6 weeks. Skin hydration, irritation and pigmentation were evaluated every 2 weeks using Skin Diagnostic SD27. The visual analog scale for itching was also evaluated every 2 weeks, and the Kidney Disease Quality of Life Short Form was performed on the day of each patient's enrollment and after 6 weeks of treatment.\n\nResults:\nFifty dialysis patients were enrolled, 47 of which completed the study. The hydration of the skin of the patients' extremities increased significantly after administration of sericin cream; significant differences were found between sericin treatment and control after 6 weeks of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Moreover, a significant difference was also found in skin irritation between the two treatments (p = 0.013 for arms and p = 0.027 for legs, respectively). At the end of the study, the skin pigmentation level was significantly reduced on both the arms (p = 0.032) and legs (p = 0.021) of the sericin-treated side compared with the side treated with cream base. The mean itching score decreased significantly from moderate to severe at the time of enrollment to mild pruritus after 6 weeks of treatment (p = 0.002). A better quality of life was found in all domains tested although statistically significant differences before and after treatment was found only in the patients' pain scores, the effect of kidney disease on daily life, sleep quality and symptoms or problems related to kidney disease.\n\nConclusions:\nWe conclude that sericin cream has a high potential for reducing UP in hemodialysis patients.The trial registration number of this study is ISRCTN16019033; its public title is \"sericin cream reduces pruritus in hemodialysis patients\"."},"whole_article_text_length":{"kind":"number","value":16613,"string":"16,613"},"whole_article_abstract_length":{"kind":"number","value":447,"string":"447"},"num_sections":{"kind":"number","value":22,"string":"22"},"most_frequent_words":{"kind":"list like","value":["skin","cream","patients","treatment","sericin","study","weeks","sericin cream","baseline","base"],"string":"[\n \"skin\",\n \"cream\",\n \"patients\",\n \"treatment\",\n \"sericin\",\n \"study\",\n \"weeks\",\n \"sericin cream\",\n \"baseline\",\n \"base\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Topical | Double-Blind Method | Female | Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome 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Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Topical | Double-Blind Method | Female | Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test 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[SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | cream | patients | treatment | sericin | study | weeks | sericin cream | baseline | base [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | cream | patients | treatment | sericin | study | weeks | sericin cream | baseline | base [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] inflammatory | sericin | uremic | pruritus | hypothesis | opioid | immune | associated | pro | pro inflammatory [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | skin | study | cream | sericin | treatment | excluded | weeks | sericin cream | itching [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cream | skin | baseline | significant | legs | arms | treatment | hydration | weeks | sericin [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] use | cream | baseline use cream | baseline use cream base | compared baseline use | compared baseline use cream | treatment compared baseline use | baseline use | significantly | sericin [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cream | patients | skin | sericin | study | treatment | sericin cream | weeks | pruritus | baseline [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cream | patients | skin | sericin | study | treatment | sericin cream | weeks | pruritus | baseline [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ESRD ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 6 weeks ||| every 2 weeks | Skin Diagnostic ||| every 2 weeks | the Kidney Disease Quality of Life Short Form | the day | 6 weeks [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Fifty | 47 ||| 6 weeks | 0.041 | 0.022 ||| two | 0.013 | 0.027 ||| 0.032 | 0.021 ||| 6 weeks | 0.002 ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ESRD ||| ||| ||| 6 weeks ||| every 2 weeks | Skin Diagnostic ||| every 2 weeks | the Kidney Disease Quality of Life Short Form | the day | 6 weeks ||| ||| Fifty | 47 ||| 6 weeks | 0.041 | 0.022 ||| two | 0.013 | 0.027 ||| 0.032 | 0.021 ||| 6 weeks | 0.002 ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ESRD ||| ||| ||| 6 weeks ||| every 2 weeks | Skin Diagnostic ||| every 2 weeks | the Kidney Disease Quality of Life Short Form | the day | 6 weeks ||| ||| Fifty | 47 ||| 6 weeks | 0.041 | 0.022 ||| two | 0.013 | 0.027 ||| 0.032 | 0.021 ||| 6 weeks | 0.002 ||| ||| ||| [SUMMARY]"}}},{"rowIdx":273,"cells":{"title":{"kind":"string","value":"Particulate matter air pollution and respiratory symptoms in individuals having either asthma or chronic obstructive pulmonary disease: a European multicentre panel study."},"pmid":{"kind":"string","value":"23039312"},"background_abstract":{"kind":"string","value":"Particulate matter air pollution has been associated with adverse health effects. The fraction of ambient particles that are mainly responsible for the observed health effects is still a matter of controversy. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.The RUPIOH study, an EU-funded multicentre study, was designed to examine the distribution of various ambient particle metrics in four European cities (Amsterdam, Athens, Birmingham, Helsinki) and assess their health effects in participants with asthma or COPD, based on a detailed exposure assessment. In this paper the association of central site measurements with respiratory symptoms and restriction of activities is examined."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"At each centre a panel of participants with either asthma or COPD recorded respiratory symptoms and restriction of activities in a diary for six months. Exposure assessment included simultaneous measurements of coarse, fine and ultrafine particles at a central site. Data on gaseous pollutants were also collected. The associations of the 24-hour average concentrations of air pollution indices with the health outcomes were assessed in a hierarchical modelling approach. A city specific analysis controlling for potential confounders was followed by a meta-analysis to provide overall effect estimates."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A 10 μg/m3 increase in previous day coarse particles concentrations was positively associated with most symptoms (an increase of 0.6 to 0.7% in average) and limitation in walking (OR= 1.076, 95% CI: 1.026-1.128). Same day, previous day and previous two days ozone concentrations were positively associated with cough (OR= 1.061, 95% CI: 1.013-1.111; OR= 1.049, 95% CI: 1.016-1.083 and OR= 1.059, 95% CI: 1.027-1.091, respectively). No consistent associations were observed between fine particle concentrations, nitrogen dioxide and respiratory health effects. As for particle number concentrations negative association (mostly non-significant at the nominal level) was observed with most symptoms whilst the positive association with limitation of activities did not reach the nominal level of significance."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The observed associations with coarse particles are in agreement with the findings of toxicological studies. Together they suggest it is prudent to regulate also coarse particles in addition to fine particles."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Aged, 80 and over","Air Pollutants","Air Pollution","Asthma","Cities","Europe","Female","Humans","Male","Middle Aged","Nitrogen Dioxide","Odds Ratio","Ozone","Particulate Matter","Pulmonary Disease, Chronic Obstructive","Respiration Disorders","Walking"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Air Pollutants\",\n \"Air Pollution\",\n \"Asthma\",\n \"Cities\",\n \"Europe\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Nitrogen Dioxide\",\n \"Odds Ratio\",\n \"Ozone\",\n \"Particulate Matter\",\n \"Pulmonary Disease, Chronic Obstructive\",\n \"Respiration Disorders\",\n \"Walking\"\n]"},"pmcid":{"kind":"string","value":"3509003"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20]."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Study design In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\nIn the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\n Study population Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\nInclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\n Symptom diary The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\nThe diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\n Air pollution exposure Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\nExposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\n Confounder data Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\nTime trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\n Quality assurance/quality control Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\nAir pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\n Statistical analysis Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].\nData analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27]."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Panel characteristics A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\nA brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\n Symptoms In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\nIn total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\n Air pollution concentrations Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\nHelsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\n Air pollution effects on symptoms-limitation in activities due to breathing problems Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Incidence analyses Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.\nPatterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Our study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles."},"other_sections_titles":{"kind":"list like","value":["Background","Study design","Study population","Symptom diary","Air pollution exposure","Confounder data","Quality assurance/quality control","Statistical analysis","Panel characteristics","Symptoms","Air pollution concentrations","Air pollution effects on symptoms-limitation in activities due to breathing problems","Prevalence analyses","Incidence analyses","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Study design\",\n \"Study population\",\n \"Symptom diary\",\n \"Air pollution exposure\",\n \"Confounder data\",\n \"Quality assurance/quality control\",\n \"Statistical analysis\",\n \"Panel characteristics\",\n \"Symptoms\",\n \"Air pollution concentrations\",\n \"Air pollution effects on symptoms-limitation in activities due to breathing problems\",\n \"Prevalence analyses\",\n \"Incidence analyses\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20].","In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.","Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.","The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.","Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.","Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).","Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.","Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].","A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.","In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.","Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities"," Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n","We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n","Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.","AIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.","The authors declare that they have no competing interests.","All authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions."],"string":"[\n \"Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\\n[9,12-14].\\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\\n[16-20].\",\n \"In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\",\n \"Inclusion criteria and recruitment procedures have been described in detail before\\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\",\n \"The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\",\n \"Exposure assessment has been described in previous publications\\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\",\n \"Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\",\n \"Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\",\n \"Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\\n[27].\",\n \"A brief description of the study population is presented in Table\\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\\nCharacteristics of four European panels of asthmatic/COPD patients\\na Total participants in panel.\\nb Asthma + COPD or chronic non-specific lung disease.\\nc Given as mean and [range].\\nd Chronic non-specific lung disease.\\ne Environmental tobacco smoke.\\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\",\n \"In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\\n2).\\nPerson days with symptoms in the diary (n = number of expected person days)\\na due to breathing problems.\",\n \"Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\\n3).\\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\",\n \" Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\nWe observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\",\n \"We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\",\n \"Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\\n7).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith incidence of symptoms in the four panels (random effects pooled estimates)\\n\\nBold are significant pooled effects.\",\n \"AIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.\",\n \"The authors declare that they have no competing interests.\",\n \"All authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study design","Study population","Symptom diary","Air pollution exposure","Confounder data","Quality assurance/quality control","Statistical analysis","Results","Panel characteristics","Symptoms","Air pollution concentrations","Air pollution effects on symptoms-limitation in activities due to breathing problems","Prevalence analyses","Incidence analyses","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study design\",\n \"Study population\",\n \"Symptom diary\",\n \"Air pollution exposure\",\n \"Confounder data\",\n \"Quality assurance/quality control\",\n \"Statistical analysis\",\n \"Results\",\n \"Panel characteristics\",\n \"Symptoms\",\n \"Air pollution concentrations\",\n \"Air pollution effects on symptoms-limitation in activities due to breathing problems\",\n \"Prevalence analyses\",\n \"Incidence analyses\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20]."," Study design In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\nIn the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\n Study population Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\nInclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\n Symptom diary The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\nThe diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\n Air pollution exposure Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\nExposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\n Confounder data Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\nTime trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\n Quality assurance/quality control Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\nAir pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\n Statistical analysis Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].\nData analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].","In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.","Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.","The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.","Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.","Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).","Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.","Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27]."," Panel characteristics A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\nA brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\n Symptoms In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\nIn total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\n Air pollution concentrations Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\nHelsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\n Air pollution effects on symptoms-limitation in activities due to breathing problems Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Incidence analyses Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.\nPatterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.","A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.","In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.","Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities"," Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n","We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n","Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.","In this multicentre study we found consistent positive associations between coarse particles central sites concentrations and prevalence of respiratory symptoms, as recorded in a 6-month diary, in four panels of participants with predominantly mild to moderate asthma or COPD in four European cities participating in the RUPIOH study. We also found a significant association of ozone with cough and woken with breathing problems, but not with other symptoms. Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst positive associations with woken with breathing problems and cough as well as with limitation of vigorous and moderate activities due to breathing problems, did not reach the nominal level of significance. Interestingly, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant at the nominal level. An analysis of the asthmatic subgroup showed generally lower odds ratios for PM10-2.5.\nOne particularity and strength of the RUPIOH study is the in depth assessment of particulate air pollution by measuring PM10, PM2.5 (then deriving coarse particles), filters absorbance as well as the number of ultrafine particles. Previous work from RUPIOH that included air pollution monitoring for one week inside and directly outside participants’ homes reported no association with lung function\n[19]. As the authors stated a potential explanation could be the high prevalence of medication use, the short period of measurements (one week) that limited the ability to assess lagged effects over several days or absence of an effect. The high prevalence of medication use may also have covered some associations in the present study.\nA limitation of the study is the inclusion of both COPD and asthma patients. COPD and asthma are two diseases with different underlying pathophysiological mechanisms and day to day variability in their symptoms\n[22,23]. Mixing of the two diseases does not create bias in the analysis in the full population as we adjusted for differences in health status between individuals. The generalizability of the size of the effect estimates is more affected by the population. Though asthma and COPD are different diseases, we are not aware of studies that have demonstrated differences in the magnitude of response to air pollution. In our recently accepted paper in the same panel we did not find any difference in the effect of PM10-2.5 on total nitrate and nitrite concentrations in exhaled breath condensate (EBC NOx), a marker of oxidative stress between asthma and COPD patients\n[28]. In that study we could evaluate disease status as the outcome was a continuous variable. Unfortunately, an analysis restricted to COPD patients was not possible due to the small number of COPD patients participating in Helsinki and Birmingham. Hence, we also could not test whether the smaller PM10-2.5 effect in the asthmatic subgroup differed significantly from the COPD subgroup.\nOur coarse particle findings are however consistent with the observation that in the RUPIOH study only the PM10-2.5 concentration at central sites was significantly associated with increased EBC NOx collected during the same week as the spirometry\n[28]. EBC NOx has been suggested as a reliable marker of oxidative stress\n[29-31]. The link between PM10-2.5 with oxidative stress and airway inflammation may explain the increase in respiratory symptoms we found.\nIn this study, we also report significant positive associations of ozone with cough throughout most of the examined lags both in the analysis of total participants and the subgroup of the asthmatics that are consistent with previous epidemiological and toxicological studies\n[32]. In addition, positive associations, but not significant in the nominal level, were observed with most of symptoms when total participants were included in the analysis. However, when we restricted the analysis to the subgroup of asthmatics, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2. Negative associations were also observed with woken with breathing problems, wheezing and with limitation in activities due to breathing problems, although non significant. Factors like high medication use, intrinsic differences in responsiveness to ozone among individuals, adaptation to ozone issues or other spurious effects may have been responsible for these findings\n[32].\nIn the last two decades a substantial body of literature has focused on the harmful health effects of PM10 and PM2.5[15,32]. As a result guideline values have been recommended by the U.S. Environmental Protection Agency and World Health Organization for both indicators of PM pollution to protect public health\n[2,3]. However, from recent studies there is increasing evidence that the health effects of coarse particles should not be underestimated. In a systematic review of epidemiological studies that have analyzed fine and coarse PM jointly, Brunekreef and Forsberg examined the epidemiological evidence for effects of coarse particles on health\n[15]. They concluded that the effects of PM10-2.5 were stronger than or as strong as PM2.5 on short-term respiratory morbidity. Furthermore, in a national multicity study, Zanobetti and Schwartz found a strong association of both fine and coarse particles with daily deaths in 112 U.S. cities\n[33]. A 10 μg/m3 increase in PM10-2.5 was significantly associated with total mortality, stroke, cardiovascular, and respiratory mortality, the latter of which showing the largest effect (a 1.2% increase). Mechanistically, these effects may be due either to biogenic factors or to metals carried by PM10-2.5 by activation of inflammatory and oxidative stress pathways\n[34-36]. The findings of our study support previous epidemiological and toxicological evidence that health effects due to the coarse fraction may be substantial\n[37].\nThe large number of calculations we have done could have given some statistically significant associations by chance. However, multiple testing is an unlikely explanation of the findings in the current study. In the full study population we found 14 significant associations of which 10 were positive; in the asthmatics subgroup we found 12 significant associations of which 8 were positive. The consistency of associations (e.g. for ozone and cough) further argues against chance as the main explanation for our findings. Additionally, in the full study population the significant associations for PM10-2.5 were supported by elevated though not nominally significant ORs for other lags and symptoms. Finally, ORs were mainly homogeneous across centers. Moreover, the modest correlations between PM10-2.5 and PM2.5 did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5.\nThe majority of studies that investigated health effects of particulate pollutants have expressed results on a mass basis. It has been suggested that when taking into consideration particle number or surface area, the pulmonary dose of toxic material related to PM2.5 may be much larger than the dose related to PM10-2.5 that for this reason alone, comparison on a mass basis may be less informative\n[15]. In our study we separately investigated the mass and the number effect. Neither central site PM2.5 nor PNC were consistently associated with symptoms. The association we observed with PM10-2.5, if not by chance, may also imply that a central measurement site is more appropriate for measurements of mass concentrations than for PNC. The analysis of RUPIOH data by Puustinen et al. showed generally high correlations between 24 hour average central site and residential outdoor concentrations for PM2.5 and soot with a lesser median correlation for PM10 and a lower correlation for PNC and PM10-2.5[17]. For PM10-2.5 correlations between central site and home outdoor measurements were 0.66, 0.74, 0.89 and 0.64 in Helsinki, Athens, Amsterdam and Birmingham respectively. A central site thus provides a reasonably good estimate of more local exposures even for coarse particles.\nThe relatively high divergence of PM10-2.5 concentrations between proximate sites in the UK has recently been confirmed by Liu and Harrison\n[38]. Consequently, for both PNC and PM10-2.5, there is a higher probability of exposure misclassification than for PM2.5 or soot. The finding of significant associations with respiratory health outcomes for PM10-2.5 but not for PNC is therefore quite striking but consistent with the recent findings of a time series study in London which found significant associations between PNC and cardiovascular health outcomes whilst PM mass metrics were associated with respiratory outcomes\n[39]. A plausible explanation could be the existence of different biological and pathophysiological mechanisms through which PM10-2.5 and PNC exert their adverse effects or different target organs. The results of recent toxicological studies support the theory that PM10-2.5 exert their effects at the site of deposition in the airways whereas PNC, after crossing the alveolar epithelial barrier, enter into the systemic circulation and affect cardiovascular function\n[40,41]. This theory could explain the positive associations we found between PM10-2.5 and respiratory symptoms.\nIn summary, our study contributes to the literature on the health effects of PM in respiratory patients. Moreover, the results of our study are in agreement with the findings of recent epidemiological and toxicological studies and provide enough evidence to conclude that it is prudent to keep PM10-2.5 regulated in addition to fine particles.","Our study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles.","AIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.","The authors declare that they have no competing interests.","All authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions."],"string":"[\n \"Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\\n[9,12-14].\\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\\n[16-20].\",\n \" Study design In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\\nIn the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\\n Study population Inclusion criteria and recruitment procedures have been described in detail before\\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\\nInclusion criteria and recruitment procedures have been described in detail before\\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\\n Symptom diary The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\\nThe diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\\n Air pollution exposure Exposure assessment has been described in previous publications\\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\\nExposure assessment has been described in previous publications\\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\\n Confounder data Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\\nTime trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\\n Quality assurance/quality control Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\\nAir pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\\n Statistical analysis Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\\n[27].\\nData analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\\n[27].\",\n \"In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\",\n \"Inclusion criteria and recruitment procedures have been described in detail before\\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\",\n \"The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\",\n \"Exposure assessment has been described in previous publications\\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\",\n \"Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\",\n \"Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\",\n \"Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\\n[27].\",\n \" Panel characteristics A brief description of the study population is presented in Table\\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\\nCharacteristics of four European panels of asthmatic/COPD patients\\na Total participants in panel.\\nb Asthma + COPD or chronic non-specific lung disease.\\nc Given as mean and [range].\\nd Chronic non-specific lung disease.\\ne Environmental tobacco smoke.\\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\\nA brief description of the study population is presented in Table\\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\\nCharacteristics of four European panels of asthmatic/COPD patients\\na Total participants in panel.\\nb Asthma + COPD or chronic non-specific lung disease.\\nc Given as mean and [range].\\nd Chronic non-specific lung disease.\\ne Environmental tobacco smoke.\\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\\n Symptoms In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\\n2).\\nPerson days with symptoms in the diary (n = number of expected person days)\\na due to breathing problems.\\nIn total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\\n2).\\nPerson days with symptoms in the diary (n = number of expected person days)\\na due to breathing problems.\\n Air pollution concentrations Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\\n3).\\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\\nHelsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\\n3).\\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\\n Air pollution effects on symptoms-limitation in activities due to breathing problems Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\nWe observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\n Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\nWe observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\n Incidence analyses Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\\n7).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith incidence of symptoms in the four panels (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nPatterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\\n7).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith incidence of symptoms in the four panels (random effects pooled estimates)\\n\\nBold are significant pooled effects.\",\n \"A brief description of the study population is presented in Table\\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\\nCharacteristics of four European panels of asthmatic/COPD patients\\na Total participants in panel.\\nb Asthma + COPD or chronic non-specific lung disease.\\nc Given as mean and [range].\\nd Chronic non-specific lung disease.\\ne Environmental tobacco smoke.\\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\",\n \"In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\\n2).\\nPerson days with symptoms in the diary (n = number of expected person days)\\na due to breathing problems.\",\n \"Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\\n3).\\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\",\n \" Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\nWe observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\",\n \"We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\",\n \"Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\\n7).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith incidence of symptoms in the four panels (random effects pooled estimates)\\n\\nBold are significant pooled effects.\",\n \"In this multicentre study we found consistent positive associations between coarse particles central sites concentrations and prevalence of respiratory symptoms, as recorded in a 6-month diary, in four panels of participants with predominantly mild to moderate asthma or COPD in four European cities participating in the RUPIOH study. We also found a significant association of ozone with cough and woken with breathing problems, but not with other symptoms. Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst positive associations with woken with breathing problems and cough as well as with limitation of vigorous and moderate activities due to breathing problems, did not reach the nominal level of significance. Interestingly, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant at the nominal level. An analysis of the asthmatic subgroup showed generally lower odds ratios for PM10-2.5.\\nOne particularity and strength of the RUPIOH study is the in depth assessment of particulate air pollution by measuring PM10, PM2.5 (then deriving coarse particles), filters absorbance as well as the number of ultrafine particles. Previous work from RUPIOH that included air pollution monitoring for one week inside and directly outside participants’ homes reported no association with lung function\\n[19]. As the authors stated a potential explanation could be the high prevalence of medication use, the short period of measurements (one week) that limited the ability to assess lagged effects over several days or absence of an effect. The high prevalence of medication use may also have covered some associations in the present study.\\nA limitation of the study is the inclusion of both COPD and asthma patients. COPD and asthma are two diseases with different underlying pathophysiological mechanisms and day to day variability in their symptoms\\n[22,23]. Mixing of the two diseases does not create bias in the analysis in the full population as we adjusted for differences in health status between individuals. The generalizability of the size of the effect estimates is more affected by the population. Though asthma and COPD are different diseases, we are not aware of studies that have demonstrated differences in the magnitude of response to air pollution. In our recently accepted paper in the same panel we did not find any difference in the effect of PM10-2.5 on total nitrate and nitrite concentrations in exhaled breath condensate (EBC NOx), a marker of oxidative stress between asthma and COPD patients\\n[28]. In that study we could evaluate disease status as the outcome was a continuous variable. Unfortunately, an analysis restricted to COPD patients was not possible due to the small number of COPD patients participating in Helsinki and Birmingham. Hence, we also could not test whether the smaller PM10-2.5 effect in the asthmatic subgroup differed significantly from the COPD subgroup.\\nOur coarse particle findings are however consistent with the observation that in the RUPIOH study only the PM10-2.5 concentration at central sites was significantly associated with increased EBC NOx collected during the same week as the spirometry\\n[28]. EBC NOx has been suggested as a reliable marker of oxidative stress\\n[29-31]. The link between PM10-2.5 with oxidative stress and airway inflammation may explain the increase in respiratory symptoms we found.\\nIn this study, we also report significant positive associations of ozone with cough throughout most of the examined lags both in the analysis of total participants and the subgroup of the asthmatics that are consistent with previous epidemiological and toxicological studies\\n[32]. In addition, positive associations, but not significant in the nominal level, were observed with most of symptoms when total participants were included in the analysis. However, when we restricted the analysis to the subgroup of asthmatics, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2. Negative associations were also observed with woken with breathing problems, wheezing and with limitation in activities due to breathing problems, although non significant. Factors like high medication use, intrinsic differences in responsiveness to ozone among individuals, adaptation to ozone issues or other spurious effects may have been responsible for these findings\\n[32].\\nIn the last two decades a substantial body of literature has focused on the harmful health effects of PM10 and PM2.5[15,32]. As a result guideline values have been recommended by the U.S. Environmental Protection Agency and World Health Organization for both indicators of PM pollution to protect public health\\n[2,3]. However, from recent studies there is increasing evidence that the health effects of coarse particles should not be underestimated. In a systematic review of epidemiological studies that have analyzed fine and coarse PM jointly, Brunekreef and Forsberg examined the epidemiological evidence for effects of coarse particles on health\\n[15]. They concluded that the effects of PM10-2.5 were stronger than or as strong as PM2.5 on short-term respiratory morbidity. Furthermore, in a national multicity study, Zanobetti and Schwartz found a strong association of both fine and coarse particles with daily deaths in 112 U.S. cities\\n[33]. A 10 μg/m3 increase in PM10-2.5 was significantly associated with total mortality, stroke, cardiovascular, and respiratory mortality, the latter of which showing the largest effect (a 1.2% increase). Mechanistically, these effects may be due either to biogenic factors or to metals carried by PM10-2.5 by activation of inflammatory and oxidative stress pathways\\n[34-36]. The findings of our study support previous epidemiological and toxicological evidence that health effects due to the coarse fraction may be substantial\\n[37].\\nThe large number of calculations we have done could have given some statistically significant associations by chance. However, multiple testing is an unlikely explanation of the findings in the current study. In the full study population we found 14 significant associations of which 10 were positive; in the asthmatics subgroup we found 12 significant associations of which 8 were positive. The consistency of associations (e.g. for ozone and cough) further argues against chance as the main explanation for our findings. Additionally, in the full study population the significant associations for PM10-2.5 were supported by elevated though not nominally significant ORs for other lags and symptoms. Finally, ORs were mainly homogeneous across centers. Moreover, the modest correlations between PM10-2.5 and PM2.5 did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5.\\nThe majority of studies that investigated health effects of particulate pollutants have expressed results on a mass basis. It has been suggested that when taking into consideration particle number or surface area, the pulmonary dose of toxic material related to PM2.5 may be much larger than the dose related to PM10-2.5 that for this reason alone, comparison on a mass basis may be less informative\\n[15]. In our study we separately investigated the mass and the number effect. Neither central site PM2.5 nor PNC were consistently associated with symptoms. The association we observed with PM10-2.5, if not by chance, may also imply that a central measurement site is more appropriate for measurements of mass concentrations than for PNC. The analysis of RUPIOH data by Puustinen et al. showed generally high correlations between 24 hour average central site and residential outdoor concentrations for PM2.5 and soot with a lesser median correlation for PM10 and a lower correlation for PNC and PM10-2.5[17]. For PM10-2.5 correlations between central site and home outdoor measurements were 0.66, 0.74, 0.89 and 0.64 in Helsinki, Athens, Amsterdam and Birmingham respectively. A central site thus provides a reasonably good estimate of more local exposures even for coarse particles.\\nThe relatively high divergence of PM10-2.5 concentrations between proximate sites in the UK has recently been confirmed by Liu and Harrison\\n[38]. Consequently, for both PNC and PM10-2.5, there is a higher probability of exposure misclassification than for PM2.5 or soot. The finding of significant associations with respiratory health outcomes for PM10-2.5 but not for PNC is therefore quite striking but consistent with the recent findings of a time series study in London which found significant associations between PNC and cardiovascular health outcomes whilst PM mass metrics were associated with respiratory outcomes\\n[39]. A plausible explanation could be the existence of different biological and pathophysiological mechanisms through which PM10-2.5 and PNC exert their adverse effects or different target organs. The results of recent toxicological studies support the theory that PM10-2.5 exert their effects at the site of deposition in the airways whereas PNC, after crossing the alveolar epithelial barrier, enter into the systemic circulation and affect cardiovascular function\\n[40,41]. This theory could explain the positive associations we found between PM10-2.5 and respiratory symptoms.\\nIn summary, our study contributes to the literature on the health effects of PM in respiratory patients. Moreover, the results of our study are in agreement with the findings of recent epidemiological and toxicological studies and provide enough evidence to conclude that it is prudent to keep PM10-2.5 regulated in addition to fine particles.\",\n \"Our study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles.\",\n \"AIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.\",\n \"The authors declare that they have no competing interests.\",\n \"All authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,"results",null,null,null,null,null,null,"discussion","conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Air pollution","Asthma","Chronic obstructive pulmonary disease","Coarse particles","Particle number concentration","Respiratory health"],"string":"[\n \"Air pollution\",\n \"Asthma\",\n \"Chronic obstructive pulmonary disease\",\n \"Coarse particles\",\n \"Particle number concentration\",\n \"Respiratory health\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nOver the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20].\n\nMethods:\n Study design In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\nIn the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\n Study population Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\nInclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\n Symptom diary The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\nThe diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\n Air pollution exposure Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\nExposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\n Confounder data Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\nTime trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\n Quality assurance/quality control Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\nAir pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\n Statistical analysis Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].\nData analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].\n\nStudy design:\nIn the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\n\nStudy population:\nInclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\n\nSymptom diary:\nThe diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\n\nAir pollution exposure:\nExposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\n\nConfounder data:\nTime trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\n\nQuality assurance/quality control:\nAir pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\n\nStatistical analysis:\nData analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].\n\nResults:\n Panel characteristics A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\nA brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\n Symptoms In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\nIn total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\n Air pollution concentrations Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\nHelsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\n Air pollution effects on symptoms-limitation in activities due to breathing problems Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Incidence analyses Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.\nPatterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nPanel characteristics:\nA brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\n\nSymptoms:\nIn total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\n\nAir pollution concentrations:\nHelsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\n\nAir pollution effects on symptoms-limitation in activities due to breathing problems:\n Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n\nPrevalence analyses:\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n\nIncidence analyses:\nPatterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nDiscussion:\nIn this multicentre study we found consistent positive associations between coarse particles central sites concentrations and prevalence of respiratory symptoms, as recorded in a 6-month diary, in four panels of participants with predominantly mild to moderate asthma or COPD in four European cities participating in the RUPIOH study. We also found a significant association of ozone with cough and woken with breathing problems, but not with other symptoms. Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst positive associations with woken with breathing problems and cough as well as with limitation of vigorous and moderate activities due to breathing problems, did not reach the nominal level of significance. Interestingly, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant at the nominal level. An analysis of the asthmatic subgroup showed generally lower odds ratios for PM10-2.5.\nOne particularity and strength of the RUPIOH study is the in depth assessment of particulate air pollution by measuring PM10, PM2.5 (then deriving coarse particles), filters absorbance as well as the number of ultrafine particles. Previous work from RUPIOH that included air pollution monitoring for one week inside and directly outside participants’ homes reported no association with lung function\n[19]. As the authors stated a potential explanation could be the high prevalence of medication use, the short period of measurements (one week) that limited the ability to assess lagged effects over several days or absence of an effect. The high prevalence of medication use may also have covered some associations in the present study.\nA limitation of the study is the inclusion of both COPD and asthma patients. COPD and asthma are two diseases with different underlying pathophysiological mechanisms and day to day variability in their symptoms\n[22,23]. Mixing of the two diseases does not create bias in the analysis in the full population as we adjusted for differences in health status between individuals. The generalizability of the size of the effect estimates is more affected by the population. Though asthma and COPD are different diseases, we are not aware of studies that have demonstrated differences in the magnitude of response to air pollution. In our recently accepted paper in the same panel we did not find any difference in the effect of PM10-2.5 on total nitrate and nitrite concentrations in exhaled breath condensate (EBC NOx), a marker of oxidative stress between asthma and COPD patients\n[28]. In that study we could evaluate disease status as the outcome was a continuous variable. Unfortunately, an analysis restricted to COPD patients was not possible due to the small number of COPD patients participating in Helsinki and Birmingham. Hence, we also could not test whether the smaller PM10-2.5 effect in the asthmatic subgroup differed significantly from the COPD subgroup.\nOur coarse particle findings are however consistent with the observation that in the RUPIOH study only the PM10-2.5 concentration at central sites was significantly associated with increased EBC NOx collected during the same week as the spirometry\n[28]. EBC NOx has been suggested as a reliable marker of oxidative stress\n[29-31]. The link between PM10-2.5 with oxidative stress and airway inflammation may explain the increase in respiratory symptoms we found.\nIn this study, we also report significant positive associations of ozone with cough throughout most of the examined lags both in the analysis of total participants and the subgroup of the asthmatics that are consistent with previous epidemiological and toxicological studies\n[32]. In addition, positive associations, but not significant in the nominal level, were observed with most of symptoms when total participants were included in the analysis. However, when we restricted the analysis to the subgroup of asthmatics, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2. Negative associations were also observed with woken with breathing problems, wheezing and with limitation in activities due to breathing problems, although non significant. Factors like high medication use, intrinsic differences in responsiveness to ozone among individuals, adaptation to ozone issues or other spurious effects may have been responsible for these findings\n[32].\nIn the last two decades a substantial body of literature has focused on the harmful health effects of PM10 and PM2.5[15,32]. As a result guideline values have been recommended by the U.S. Environmental Protection Agency and World Health Organization for both indicators of PM pollution to protect public health\n[2,3]. However, from recent studies there is increasing evidence that the health effects of coarse particles should not be underestimated. In a systematic review of epidemiological studies that have analyzed fine and coarse PM jointly, Brunekreef and Forsberg examined the epidemiological evidence for effects of coarse particles on health\n[15]. They concluded that the effects of PM10-2.5 were stronger than or as strong as PM2.5 on short-term respiratory morbidity. Furthermore, in a national multicity study, Zanobetti and Schwartz found a strong association of both fine and coarse particles with daily deaths in 112 U.S. cities\n[33]. A 10 μg/m3 increase in PM10-2.5 was significantly associated with total mortality, stroke, cardiovascular, and respiratory mortality, the latter of which showing the largest effect (a 1.2% increase). Mechanistically, these effects may be due either to biogenic factors or to metals carried by PM10-2.5 by activation of inflammatory and oxidative stress pathways\n[34-36]. The findings of our study support previous epidemiological and toxicological evidence that health effects due to the coarse fraction may be substantial\n[37].\nThe large number of calculations we have done could have given some statistically significant associations by chance. However, multiple testing is an unlikely explanation of the findings in the current study. In the full study population we found 14 significant associations of which 10 were positive; in the asthmatics subgroup we found 12 significant associations of which 8 were positive. The consistency of associations (e.g. for ozone and cough) further argues against chance as the main explanation for our findings. Additionally, in the full study population the significant associations for PM10-2.5 were supported by elevated though not nominally significant ORs for other lags and symptoms. Finally, ORs were mainly homogeneous across centers. Moreover, the modest correlations between PM10-2.5 and PM2.5 did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5.\nThe majority of studies that investigated health effects of particulate pollutants have expressed results on a mass basis. It has been suggested that when taking into consideration particle number or surface area, the pulmonary dose of toxic material related to PM2.5 may be much larger than the dose related to PM10-2.5 that for this reason alone, comparison on a mass basis may be less informative\n[15]. In our study we separately investigated the mass and the number effect. Neither central site PM2.5 nor PNC were consistently associated with symptoms. The association we observed with PM10-2.5, if not by chance, may also imply that a central measurement site is more appropriate for measurements of mass concentrations than for PNC. The analysis of RUPIOH data by Puustinen et al. showed generally high correlations between 24 hour average central site and residential outdoor concentrations for PM2.5 and soot with a lesser median correlation for PM10 and a lower correlation for PNC and PM10-2.5[17]. For PM10-2.5 correlations between central site and home outdoor measurements were 0.66, 0.74, 0.89 and 0.64 in Helsinki, Athens, Amsterdam and Birmingham respectively. A central site thus provides a reasonably good estimate of more local exposures even for coarse particles.\nThe relatively high divergence of PM10-2.5 concentrations between proximate sites in the UK has recently been confirmed by Liu and Harrison\n[38]. Consequently, for both PNC and PM10-2.5, there is a higher probability of exposure misclassification than for PM2.5 or soot. The finding of significant associations with respiratory health outcomes for PM10-2.5 but not for PNC is therefore quite striking but consistent with the recent findings of a time series study in London which found significant associations between PNC and cardiovascular health outcomes whilst PM mass metrics were associated with respiratory outcomes\n[39]. A plausible explanation could be the existence of different biological and pathophysiological mechanisms through which PM10-2.5 and PNC exert their adverse effects or different target organs. The results of recent toxicological studies support the theory that PM10-2.5 exert their effects at the site of deposition in the airways whereas PNC, after crossing the alveolar epithelial barrier, enter into the systemic circulation and affect cardiovascular function\n[40,41]. This theory could explain the positive associations we found between PM10-2.5 and respiratory symptoms.\nIn summary, our study contributes to the literature on the health effects of PM in respiratory patients. Moreover, the results of our study are in agreement with the findings of recent epidemiological and toxicological studies and provide enough evidence to conclude that it is prudent to keep PM10-2.5 regulated in addition to fine particles.\n\nConclusions:\nOur study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles.\n\nAbbreviations:\nAIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nAll authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nParticulate matter air pollution has been associated with adverse health effects. The fraction of ambient particles that are mainly responsible for the observed health effects is still a matter of controversy. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.The RUPIOH study, an EU-funded multicentre study, was designed to examine the distribution of various ambient particle metrics in four European cities (Amsterdam, Athens, Birmingham, Helsinki) and assess their health effects in participants with asthma or COPD, based on a detailed exposure assessment. In this paper the association of central site measurements with respiratory symptoms and restriction of activities is examined.\n\nMethods:\nAt each centre a panel of participants with either asthma or COPD recorded respiratory symptoms and restriction of activities in a diary for six months. Exposure assessment included simultaneous measurements of coarse, fine and ultrafine particles at a central site. Data on gaseous pollutants were also collected. The associations of the 24-hour average concentrations of air pollution indices with the health outcomes were assessed in a hierarchical modelling approach. A city specific analysis controlling for potential confounders was followed by a meta-analysis to provide overall effect estimates.\n\nResults:\nA 10 μg/m3 increase in previous day coarse particles concentrations was positively associated with most symptoms (an increase of 0.6 to 0.7% in average) and limitation in walking (OR= 1.076, 95% CI: 1.026-1.128). Same day, previous day and previous two days ozone concentrations were positively associated with cough (OR= 1.061, 95% CI: 1.013-1.111; OR= 1.049, 95% CI: 1.016-1.083 and OR= 1.059, 95% CI: 1.027-1.091, respectively). No consistent associations were observed between fine particle concentrations, nitrogen dioxide and respiratory health effects. As for particle number concentrations negative association (mostly non-significant at the nominal level) was observed with most symptoms whilst the positive association with limitation of activities did not reach the nominal level of significance.\n\nConclusions:\nThe observed associations with coarse particles are in agreement with the findings of toxicological studies. Together they suggest it is prudent to regulate also coarse particles in addition to fine particles."},"background_conclusion_text":{"kind":"string","value":"Background:\nOver the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20].\n\nConclusions:\nOur study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nParticulate matter air pollution has been associated with adverse health effects. The fraction of ambient particles that are mainly responsible for the observed health effects is still a matter of controversy. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.The RUPIOH study, an EU-funded multicentre study, was designed to examine the distribution of various ambient particle metrics in four European cities (Amsterdam, Athens, Birmingham, Helsinki) and assess their health effects in participants with asthma or COPD, based on a detailed exposure assessment. In this paper the association of central site measurements with respiratory symptoms and restriction of activities is examined.\n\nMethods:\nAt each centre a panel of participants with either asthma or COPD recorded respiratory symptoms and restriction of activities in a diary for six months. Exposure assessment included simultaneous measurements of coarse, fine and ultrafine particles at a central site. Data on gaseous pollutants were also collected. The associations of the 24-hour average concentrations of air pollution indices with the health outcomes were assessed in a hierarchical modelling approach. A city specific analysis controlling for potential confounders was followed by a meta-analysis to provide overall effect estimates.\n\nResults:\nA 10 μg/m3 increase in previous day coarse particles concentrations was positively associated with most symptoms (an increase of 0.6 to 0.7% in average) and limitation in walking (OR= 1.076, 95% CI: 1.026-1.128). Same day, previous day and previous two days ozone concentrations were positively associated with cough (OR= 1.061, 95% CI: 1.013-1.111; OR= 1.049, 95% CI: 1.016-1.083 and OR= 1.059, 95% CI: 1.027-1.091, respectively). No consistent associations were observed between fine particle concentrations, nitrogen dioxide and respiratory health effects. As for particle number concentrations negative association (mostly non-significant at the nominal level) was observed with most symptoms whilst the positive association with limitation of activities did not reach the nominal level of significance.\n\nConclusions:\nThe observed associations with coarse particles are in agreement with the findings of toxicological studies. Together they suggest it is prudent to regulate also coarse particles in addition to fine particles."},"whole_article_text_length":{"kind":"number","value":17122,"string":"17,122"},"whole_article_abstract_length":{"kind":"number","value":432,"string":"432"},"num_sections":{"kind":"number","value":21,"string":"21"},"most_frequent_words":{"kind":"list like","value":["effects","pm10","associations","significant","symptoms","lag","participants","problems","breathing","breathing problems"],"string":"[\n \"effects\",\n \"pm10\",\n \"associations\",\n \"significant\",\n \"symptoms\",\n \"lag\",\n \"participants\",\n \"problems\",\n \"breathing\",\n \"breathing problems\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Air pollution | Asthma | Chronic obstructive pulmonary disease | Coarse particles | Particle number concentration | Respiratory health [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Air pollution | Asthma | Chronic obstructive pulmonary disease | Coarse particles | Particle number concentration | Respiratory health [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 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| Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ultrafine | health | particles | μm | ultrafine particles | intensive | effects | based | fine | outdoor [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] analysis | study | day | criteria | symptoms | participants | city | patients | models | effects [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] significant | lag | activities | pooled | associations | effects | problems | breathing problems | breathing | limitation [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] studies | pm | clarify possible different | toxicological studies health effects | limited existing evidence recent | limited existing evidence | limited existing | effects coarse fraction ambient | coarse particles addition | coarse particles addition fine [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] pm10 | effects | significant | associations | lag | symptoms | participants | study | problems | pm2 [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] pm10 | effects | significant | associations | lag | symptoms | participants | study | problems | pm2 [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| EU | four | European | Amsterdam | Athens | Birmingham | Helsinki | COPD ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] six months ||| ||| ||| 24-hour ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 10 | previous day | 0.6 to 0.7% | 1.076 | 95% | CI | 1.026 ||| previous day | two days | 1.061 | 95% | CI | 1.013 | 1.049 | 95% | CI | 1.016-1.083 | 1.059 | 95% | CI | 1.027 ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| EU | four | European | Amsterdam | Athens | Birmingham | Helsinki | COPD ||| ||| six months ||| ||| ||| 24-hour ||| ||| ||| 10 | previous day | 0.6 to 0.7% | 1.076 | 95% | CI | 1.026 ||| previous day | two days | 1.061 | 95% | CI | 1.013 | 1.049 | 95% | CI | 1.016-1.083 | 1.059 | 95% | CI | 1.027 ||| ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| EU | four | European | Amsterdam | Athens | Birmingham | Helsinki | COPD ||| ||| six months ||| ||| ||| 24-hour ||| ||| ||| 10 | previous day | 0.6 to 0.7% | 1.076 | 95% | CI | 1.026 ||| previous day | two days | 1.061 | 95% | CI | 1.013 | 1.049 | 95% | CI | 1.016-1.083 | 1.059 | 95% | CI | 1.027 ||| ||| ||| ||| [SUMMARY]"}}},{"rowIdx":274,"cells":{"title":{"kind":"string","value":"DRAIN AMYLASE ON THE FIRST POSTOPERATIVE DAY OF WHIPPLE SURGERY: WHAT VALUE IS THE BEST PREDICTOR FOR EARLY DRAIN REMOVAL?"},"pmid":{"kind":"string","value":"29513806"},"background_abstract":{"kind":"string","value":"The value of drain amylase on the first postoperative day after pancreatic resections has been described as an efficient predictor of pancreatic fistula. In spite of this, the cut-off point below which the drains can be removed early remains controversial."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Were included patients undergoing Whipple surgery in the period of 2007 to 2016. Group 1 enrolled the ones who did not develop fistula and those who developed biochemical fistula for less than seven days postoperatively and group 2 included patients who developed persistent biochemical fistula between seven and 21 days and those with grade B and C fistula."},"methods_abstract_label":{"kind":"string","value":"METHOD"},"results_abstract":{"kind":"string","value":"Sixty-one patients were included, 41 comprised group 1 and 20 group 2. The incidence of abdominal collections, need for reoperation and time of hospitalization were for group 1 and 2, respectively: 17.1%, 17.1% and 9.5 days, and 65%, 40% and 21.1 days. The median of the amylase from the drain at 1st postoperative day was in group 1 and 2, respectively: 175 U/l and 3172.5 U/l (p=0.001). Using a cut-off of 180 to predict the group to which the patient would belong there was obtained sensitivity, specificity, positive predictive value and negative predictive value of 100%, 48.8%, 50% and 100% respectively."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"It was validated the cut-off value of 180 U/l as appropriate to early drain removal."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Amylases","Drainage","Female","Humans","Male","Middle Aged","Pancreatic Fistula","Pancreaticoduodenectomy","Postoperative Care","Postoperative Complications","Predictive Value of Tests","Prospective Studies"],"string":"[\n \"Amylases\",\n \"Drainage\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Pancreatic Fistula\",\n \"Pancreaticoduodenectomy\",\n \"Postoperative Care\",\n \"Postoperative Complications\",\n \"Predictive Value of Tests\",\n \"Prospective Studies\"\n]"},"pmcid":{"kind":"string","value":"5863991"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Historically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them\n16\n. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis\n13\n\n,\n\n11\n. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results\n8\n\n,\n\n18\n\n,\n\n19\n, which has been a limiting factor in the practice of not using drains in this type of procedure. \nDescription of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L\n6\n and 90 U/l\n14\n have been suggested. \nThe objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients."},"methods_title":{"kind":"string","value":"METHOD"},"methods_text":{"kind":"string","value":"The project was submitted and approved by the Human Research Ethics Committee, under nº 59716616.9.0000.5292 in the Plataforma Brasil.\nSixty-three patients were analyzed from a prospective database, who had undergone Kausch-Whipple surgery performed by the 1st author of this study (ECA), for the treatment of pancreatic or peripancreatic diseases. The time period was between June 2007 and September 2016, in the following hospitals located in the city of Natal: Hospital Universitário Onofre Lopes, Liga Norte Riograndense Contra o Câncer, Casa de Saúde São Lucas and Natal Hospital Center.\nThe technical steps for the Whipple technique as performed by the author are detailed in previous studies\n1\n\n,\n\n2\n. Three techniques for pancreatojejunal anastomosis were utilized. For pancreas with main pancreatic duct ≥5 mm, two-layer pancreatojejunal terminolateral duct to mucosa anastomosis was performed. For pancreas with normal or slightly dilated (diameter <5 mm) main pancreatic duct, terminoterminal pancreatojejunal anastomosis (“telescopic”) or terminolateral pancreatojejunal anastomosis (“invagination”) were carried out, the latter performed systematically after the 33rd patient from the casebook. At the end of the surgery, two drains, preferably laminar silicone drains, were placed in the cavity and exteriorized at each flank. \nIn the majority of patients, subcutaneous octreotide was utilized as prophylaxis for the prevention of PF, at 0.3 mg/day, fractioned in 8 h intervals, during seven days. Debits were recorded along with amylase dosage of the drained liquid on the 1st, 3rd, 5th, 7th and sometimes, on the 9th postoperative day. The value of AD1PO vas defined from the highest amylase value of both drains, measured from a sample of liquid obtained on the first day after surgery. \nPF diagnosis utilized the 2016 revised criterion of GIEDFP\n3\n. PF was defined when on the 3rd postoperative (PO) day the value of amylase in the drained liquid was three times higher than the normal upper limit for serum amylase. Patients that did not develop fistula or those with transient biochemical fistula (<7 days PO) were characterized as group 1, and patients with persistent biochemical fistula (between 7 and 21 days PO) and fistula grade B and C were characterized as group 2. In most cases, on the 9th day after surgery, control ultrasound or tomography tests were carried out. Removal of drains occurred in those cases with low amylase values for the drained liquid (under three times the upper limit for normal serum amylase) and when image tests did not show abdominal collections. Intra-hospital mortality was defined as death occurring within 90 days of surgery.\nThe Receiver Operating Characteristic (ROC) curve was utilized to identify an adequate cut-off point for AD1PO and verify its predictive characteristic in patients of both groups. The ROC value was generated after sensitivity, specificity and accuracy calculations. Additionally, the cut-off point 578 U/l was also selected because of its closeness to the value utilized by Fong e cols \n9\n, for comparison purposes. \n Statistical analysis After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \nAfter determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. "},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"From the initial sample of 63 patients, two were excluded. One had AD1PO collected incorrectly on the 2nd day after surgery, and the other passed within the 1st week PO. Therefore analysis covered 61 patients. Group 1 was constituted of 41 patients, of which 36 did not develop fistula and five transient biochemical fistula (grade A). Group 2 was constituted of 20 patients, of which six presented persistent biochemical fistula (grade A), eight degree B fistula, and six grade C fistula. The index of clinical fistula (grades B + C) in the overall sample was 22.9%. \nThe demographic and clinical characteristics of the patients, as well as respective tests, are shown in Table 1. There was no statistical difference between groups regarding age, gender and type of illness. However, regarding the size of main pancreatic duct and type of anastomosis, a statistically significant difference was detected. All patients with dilated main pancreatic ducts as well as those submitted to duct to mucosa anastomosis belonged to group 1.\n\nTABLE 1Associations between demographic and clinical characteristics and patient groups Variables Group 1 Group 2 pAge 58.0±13.06I\n 57.1±12.1I\n 0.706II\n\n n % n %\nGender\n\n\n\n\nMale 19 63.3 11 36.7 0.525III\nFemale 22 71.0 9 29.0Disease\n\n\n\n\nPancreatic adenocarcinoma 17 65.4 9 34.6 0.264III\nPapillary adenocarcinoma 12 60.0 8 40.0Duodenal adenocarcinoma 1 33.3 2 66.7Colangiocarcinoma 4 100.0 0 0.0Frantz 5 100.0 0 0.0Others 2 66.7 1 33.3Size of duct\n\n\n\n\n< 5 31 60.8 20 39.2 0.023IV\n> 5 10 100.0 0 0.0Anastomosis*\n\n\n\n\nT-L (Invagination) 14 50.0 14 50.0 0.011III\nT-T 17 73.9 6 26.1T-L (DM) 10 100.0 0 0.0I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\n\nI=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\nRegarding postoperative evolution, group 2 was associated with a higher level of general complications, postoperative collections, longer permanence of abdominal drain and hospitalization time. There was also an association between high resurgery index and patients of group 2. Nevertheless, despite all these worse result factors related to group 2, there was no statistically significant difference when comparing mortality across groups (Table 2). \n\nTABLE 2Associations between postoperative clinical findings and patient groups, along with results for drain and hospitalization times, per group Variable Group 1 Group 2 p n % n %\nCollection\n\n\n\n\nYes733.31466.7 <0.001I\nNo3485.0615.0Postoperative complications\n\n\n\n\nYes2050.020.050.0 <0.001II\nNo21100.000.0Resurgery\n\n\n\n\nYes746.7853.3 0.051I\nNo3473.91226.1Time with drain (days) 9.5±2.60III29.1±13.58III <0.001IV\nHospitalization time (days) 16.7±17.9III34.7±16.7III <0.001IV\nMortality49.815.0 1.000II\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\n\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\nMortality of the series was 8.19%: four patients belonged to group 1 and one patient to group 2. The mortality causes in group 1 were: kidney insufficiency due to coagulopathy; dehiscence of gastrojejunal anastomosis and sepsis, pneumonia due to broncoaspiration and necrosis of the pancreaticojejunostomy loop associated with sepsis. The only patient of group 2 passed due to necrotic pancreatitis associated with pancreatic fistula and sepsis. \nRegarding the AD1PO value, the median was 175 U/l (48.5-954) in group 1 and 2172.5 U/l (833.5-6421.0) in group 2. This difference was statistically significant (p=0.001).\nThe area under the ROC curve presented an exactness index of 83.0% (p=0.001). When considering a 180 U/l cut-off point for AD1PO, the following results were obtained: sensitivity 100%, specificity 48.8%, positive predictor value 50%, and negative predictor value 100%. There was an association between the arbitrated AD1PO value from the ROC curve (>180 U/l) with groups 1 and 2 (p<0.001). With a cut-off point of 578 U/l, the results were: sensitivity 80%, specificity 39%, positive predictor value 50% and negative predictor value 86.2%. There was also an association between the arbitrated AD1PO value from the ROC curve (>578 U/l) with groups 1 and 2 (p<0.003). Patients presenting AD1PO values higher than 180 U/l and 578 U/l were 2.0 and 6.25 times more likely to belong to group 2, and therefore require postoperative drainage. Table 3 shows the comparison between the two AD1PO values. \n\nTABLE 3Precision and exactness measurements, per AD1PO cut-off point Parameters Cut-off point (U/l) 180 578Sensitivity 100% 80%Specificity 48.8% 39%VPP 50% 50%VPN 100% 86.2%\n"},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"Was validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients. "},"other_sections_titles":{"kind":"list like","value":["Statistical analysis"],"string":"[\n \"Statistical analysis\"\n]"},"other_sections_texts":{"kind":"list like","value":["After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. "],"string":"[\n \"After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null],"string":"[\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHOD","Statistical analysis","RESULTS","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"METHOD\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Historically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them\n16\n. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis\n13\n\n,\n\n11\n. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results\n8\n\n,\n\n18\n\n,\n\n19\n, which has been a limiting factor in the practice of not using drains in this type of procedure. \nDescription of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L\n6\n and 90 U/l\n14\n have been suggested. \nThe objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.","The project was submitted and approved by the Human Research Ethics Committee, under nº 59716616.9.0000.5292 in the Plataforma Brasil.\nSixty-three patients were analyzed from a prospective database, who had undergone Kausch-Whipple surgery performed by the 1st author of this study (ECA), for the treatment of pancreatic or peripancreatic diseases. The time period was between June 2007 and September 2016, in the following hospitals located in the city of Natal: Hospital Universitário Onofre Lopes, Liga Norte Riograndense Contra o Câncer, Casa de Saúde São Lucas and Natal Hospital Center.\nThe technical steps for the Whipple technique as performed by the author are detailed in previous studies\n1\n\n,\n\n2\n. Three techniques for pancreatojejunal anastomosis were utilized. For pancreas with main pancreatic duct ≥5 mm, two-layer pancreatojejunal terminolateral duct to mucosa anastomosis was performed. For pancreas with normal or slightly dilated (diameter <5 mm) main pancreatic duct, terminoterminal pancreatojejunal anastomosis (“telescopic”) or terminolateral pancreatojejunal anastomosis (“invagination”) were carried out, the latter performed systematically after the 33rd patient from the casebook. At the end of the surgery, two drains, preferably laminar silicone drains, were placed in the cavity and exteriorized at each flank. \nIn the majority of patients, subcutaneous octreotide was utilized as prophylaxis for the prevention of PF, at 0.3 mg/day, fractioned in 8 h intervals, during seven days. Debits were recorded along with amylase dosage of the drained liquid on the 1st, 3rd, 5th, 7th and sometimes, on the 9th postoperative day. The value of AD1PO vas defined from the highest amylase value of both drains, measured from a sample of liquid obtained on the first day after surgery. \nPF diagnosis utilized the 2016 revised criterion of GIEDFP\n3\n. PF was defined when on the 3rd postoperative (PO) day the value of amylase in the drained liquid was three times higher than the normal upper limit for serum amylase. Patients that did not develop fistula or those with transient biochemical fistula (<7 days PO) were characterized as group 1, and patients with persistent biochemical fistula (between 7 and 21 days PO) and fistula grade B and C were characterized as group 2. In most cases, on the 9th day after surgery, control ultrasound or tomography tests were carried out. Removal of drains occurred in those cases with low amylase values for the drained liquid (under three times the upper limit for normal serum amylase) and when image tests did not show abdominal collections. Intra-hospital mortality was defined as death occurring within 90 days of surgery.\nThe Receiver Operating Characteristic (ROC) curve was utilized to identify an adequate cut-off point for AD1PO and verify its predictive characteristic in patients of both groups. The ROC value was generated after sensitivity, specificity and accuracy calculations. Additionally, the cut-off point 578 U/l was also selected because of its closeness to the value utilized by Fong e cols \n9\n, for comparison purposes. \n Statistical analysis After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \nAfter determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. ","After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. ","From the initial sample of 63 patients, two were excluded. One had AD1PO collected incorrectly on the 2nd day after surgery, and the other passed within the 1st week PO. Therefore analysis covered 61 patients. Group 1 was constituted of 41 patients, of which 36 did not develop fistula and five transient biochemical fistula (grade A). Group 2 was constituted of 20 patients, of which six presented persistent biochemical fistula (grade A), eight degree B fistula, and six grade C fistula. The index of clinical fistula (grades B + C) in the overall sample was 22.9%. \nThe demographic and clinical characteristics of the patients, as well as respective tests, are shown in Table 1. There was no statistical difference between groups regarding age, gender and type of illness. However, regarding the size of main pancreatic duct and type of anastomosis, a statistically significant difference was detected. All patients with dilated main pancreatic ducts as well as those submitted to duct to mucosa anastomosis belonged to group 1.\n\nTABLE 1Associations between demographic and clinical characteristics and patient groups Variables Group 1 Group 2 pAge 58.0±13.06I\n 57.1±12.1I\n 0.706II\n\n n % n %\nGender\n\n\n\n\nMale 19 63.3 11 36.7 0.525III\nFemale 22 71.0 9 29.0Disease\n\n\n\n\nPancreatic adenocarcinoma 17 65.4 9 34.6 0.264III\nPapillary adenocarcinoma 12 60.0 8 40.0Duodenal adenocarcinoma 1 33.3 2 66.7Colangiocarcinoma 4 100.0 0 0.0Frantz 5 100.0 0 0.0Others 2 66.7 1 33.3Size of duct\n\n\n\n\n< 5 31 60.8 20 39.2 0.023IV\n> 5 10 100.0 0 0.0Anastomosis*\n\n\n\n\nT-L (Invagination) 14 50.0 14 50.0 0.011III\nT-T 17 73.9 6 26.1T-L (DM) 10 100.0 0 0.0I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\n\nI=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\nRegarding postoperative evolution, group 2 was associated with a higher level of general complications, postoperative collections, longer permanence of abdominal drain and hospitalization time. There was also an association between high resurgery index and patients of group 2. Nevertheless, despite all these worse result factors related to group 2, there was no statistically significant difference when comparing mortality across groups (Table 2). \n\nTABLE 2Associations between postoperative clinical findings and patient groups, along with results for drain and hospitalization times, per group Variable Group 1 Group 2 p n % n %\nCollection\n\n\n\n\nYes733.31466.7 <0.001I\nNo3485.0615.0Postoperative complications\n\n\n\n\nYes2050.020.050.0 <0.001II\nNo21100.000.0Resurgery\n\n\n\n\nYes746.7853.3 0.051I\nNo3473.91226.1Time with drain (days) 9.5±2.60III29.1±13.58III <0.001IV\nHospitalization time (days) 16.7±17.9III34.7±16.7III <0.001IV\nMortality49.815.0 1.000II\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\n\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\nMortality of the series was 8.19%: four patients belonged to group 1 and one patient to group 2. The mortality causes in group 1 were: kidney insufficiency due to coagulopathy; dehiscence of gastrojejunal anastomosis and sepsis, pneumonia due to broncoaspiration and necrosis of the pancreaticojejunostomy loop associated with sepsis. The only patient of group 2 passed due to necrotic pancreatitis associated with pancreatic fistula and sepsis. \nRegarding the AD1PO value, the median was 175 U/l (48.5-954) in group 1 and 2172.5 U/l (833.5-6421.0) in group 2. This difference was statistically significant (p=0.001).\nThe area under the ROC curve presented an exactness index of 83.0% (p=0.001). When considering a 180 U/l cut-off point for AD1PO, the following results were obtained: sensitivity 100%, specificity 48.8%, positive predictor value 50%, and negative predictor value 100%. There was an association between the arbitrated AD1PO value from the ROC curve (>180 U/l) with groups 1 and 2 (p<0.001). With a cut-off point of 578 U/l, the results were: sensitivity 80%, specificity 39%, positive predictor value 50% and negative predictor value 86.2%. There was also an association between the arbitrated AD1PO value from the ROC curve (>578 U/l) with groups 1 and 2 (p<0.003). Patients presenting AD1PO values higher than 180 U/l and 578 U/l were 2.0 and 6.25 times more likely to belong to group 2, and therefore require postoperative drainage. Table 3 shows the comparison between the two AD1PO values. \n\nTABLE 3Precision and exactness measurements, per AD1PO cut-off point Parameters Cut-off point (U/l) 180 578Sensitivity 100% 80%Specificity 48.8% 39%VPP 50% 50%VPN 100% 86.2%\n","AD1PO as a predictor of post-pancreatectomy PF was described by Yamaguchi e cols in 2003\n20\n. The authors analyzed the amylase values measured in abdominal drains of 26 patients submitted to pancreatectomy, and observed that values were already high on the first day PO in those who developed clinical PF after. At least three meta-analysis\n10\n\n,\n\n15\n\n,\n\n21\n have validated the correlation between high AD1PO values and the development of PF. However, there is still controversy on the adequate cut-off point for AD1PO to predict PF, and on its applicability to the removal of prophylactic drains inserted after pancreatic resections. Verona’s group was the first to establish a strategy for the early removal of abdominal drains based on AD1PO values\n6\n. The authors carried out a prospective study on 114 patients submitted to pancreatic resection and randomized those with AD1PO <5000 U/l to remove the drain on the 3rd or 5th day PO. Early removal of drains was associated with a lower rate of pancreatic fistula (1.8% vs. 26%), lower rate of abdominal complications (12.2% vs. 52.6%), lower rate of pulmonary complications (26.3% vs. 52.6%), shorter hospitalization times (8.7 (±4) vs. 10.8 (±6.9) and lower hospital readmission rates (0% vs. 8.8%). Although the study suggested that early removal of abdominal drains was safe with AD1PO under 5000 U/l, other authors reported hypothetical PF indexes between 25-48% if drains were removed according to this suggestion\n7\n\n,\n\n12\n.\nFor those patients submitted to duodenopancreatectomy and that received prophylactic drains, the most important question is to know which patients will not develop PF - and not the other way around. On one hand, knowing which patients will not develop PF is useful because it allows for early drain removal, which leads to shorter hospitalization times. On the other hand, predicting which patients will develop PF after surgery, when the abdominal cavity is being drained, is not as relevant because PF can be easily diagnosed by measuring amylase in the drained liquid. In most cases, treatment is simple maintenance of drains, associated with fasting and nutritional support. For this reason the study herein presented analyzed two low AD1PO cut-off points (180 and 578) that were associated with higher specificity and consequently, with a high negative predictive factor, in detriment to a high cut-off point associated with higher sensitivity to PF diagnosis. Another reason for the selection is related to the impossibility of obtaining daily interventionist radiology services in the public hospital where the majority of the patients studied underwent surgery. These services include percutaneous drainage of abdominal collections/abscesses, which is indicated in urgent cases where there are abdominal complications as a consequence of undrained anastomotic leaks. \nThe methodology of this study utilized the PF definition based on the revised classification by ISGPS\n5\n, which no longer considers grade A of the previous classification to be a true PF\n4\n. However, as this study aimed at identifying a group of patients in which early removal of drains could be beneficial, it was important to include not only the concept of PF, but also grades B and C and those patients with biochemical fistula that persisted with amylase-rich debit between 7-21 days PO. In these patients, early removal of drains would increase susceptibility to abdominal collections or abscesses. With the 180 U/l cut-off point, an excellent correlation was obtained between AD1PO and absence of fistula. Therefore it was possible to identify 1/3 of patients that would have been benefitted by early drain removal. Besides, with the same cut-off, none of the patients that developed clinically relevant fistula or even persistent biochemical fistula would have had their drains removed early. \nData obtained herein were capable of validating previous publications that utilized low cut-off points\n7\n\n,\n\n12\n\n,\n\n14\n\n,\n\n17\n. Differently from these studies, however, the first series was entirely prospective herein, including only patients submitted consecutively to duodenopancreatectomy by a single team of surgeons, where PF definition followed ISGPS criteria. The cut-off point 180 U/l obtained herein was lower than the one found by Fong et al\n9\n at the Massachusetts General Hospital. They divided the study in two cohorts: the first involved 126 patients, and the 612 U/l cut-off point was defined as the one presenting best accuracy, sensitivity and specificity; the second had the objective of validating a 600 U/l cut-off point, involving 369 patients submitted to duodenopancreatectomy within January 2009 and December 2012. Almost two-thirds of patients (62.1%) presented AD1PO lower than 600 U/l and only two PF cases (0.9%) were diagnosed. This was different than the other patients with AD1PO of at least 600 U/l, with PF incidence of 31.4%. When considering herein a cut-off point close to Fong et al., 578 U/l, 13.8% of patients with PF diagnosis would have been affected by early removal of drains (negative predictor value: 86.2%). In the reality considered herein, there is no non-invasive treatment for intracavitary collections/abscesses available all the time at the public hospital, and therefore increasing the cut-off point would be a high price to pay. \nThis study presents some limitations. The first concerns the low number of included patients, which did not hinder statistical analysis. The second is related to the non-verification of the real benefits of drains in patients who developed PF, as all patients received drains. The high index of abdominal collections (65%) and necessity of re-surgery (40%) in group 2 suggested that, for some patients, drains were not totally efficient; nevertheless, the objective of the study was to find a group of patients in which drains could be removed early and not confirm the non-efficiency of drains in the group that developed PF.","Was validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients. "],"string":"[\n \"Historically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them\\n16\\n. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis\\n13\\n\\n,\\n\\n11\\n. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results\\n8\\n\\n,\\n\\n18\\n\\n,\\n\\n19\\n, which has been a limiting factor in the practice of not using drains in this type of procedure. \\nDescription of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L\\n6\\n and 90 U/l\\n14\\n have been suggested. \\nThe objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.\",\n \"The project was submitted and approved by the Human Research Ethics Committee, under nº 59716616.9.0000.5292 in the Plataforma Brasil.\\nSixty-three patients were analyzed from a prospective database, who had undergone Kausch-Whipple surgery performed by the 1st author of this study (ECA), for the treatment of pancreatic or peripancreatic diseases. The time period was between June 2007 and September 2016, in the following hospitals located in the city of Natal: Hospital Universitário Onofre Lopes, Liga Norte Riograndense Contra o Câncer, Casa de Saúde São Lucas and Natal Hospital Center.\\nThe technical steps for the Whipple technique as performed by the author are detailed in previous studies\\n1\\n\\n,\\n\\n2\\n. Three techniques for pancreatojejunal anastomosis were utilized. For pancreas with main pancreatic duct ≥5 mm, two-layer pancreatojejunal terminolateral duct to mucosa anastomosis was performed. For pancreas with normal or slightly dilated (diameter <5 mm) main pancreatic duct, terminoterminal pancreatojejunal anastomosis (“telescopic”) or terminolateral pancreatojejunal anastomosis (“invagination”) were carried out, the latter performed systematically after the 33rd patient from the casebook. At the end of the surgery, two drains, preferably laminar silicone drains, were placed in the cavity and exteriorized at each flank. \\nIn the majority of patients, subcutaneous octreotide was utilized as prophylaxis for the prevention of PF, at 0.3 mg/day, fractioned in 8 h intervals, during seven days. Debits were recorded along with amylase dosage of the drained liquid on the 1st, 3rd, 5th, 7th and sometimes, on the 9th postoperative day. The value of AD1PO vas defined from the highest amylase value of both drains, measured from a sample of liquid obtained on the first day after surgery. \\nPF diagnosis utilized the 2016 revised criterion of GIEDFP\\n3\\n. PF was defined when on the 3rd postoperative (PO) day the value of amylase in the drained liquid was three times higher than the normal upper limit for serum amylase. Patients that did not develop fistula or those with transient biochemical fistula (<7 days PO) were characterized as group 1, and patients with persistent biochemical fistula (between 7 and 21 days PO) and fistula grade B and C were characterized as group 2. In most cases, on the 9th day after surgery, control ultrasound or tomography tests were carried out. Removal of drains occurred in those cases with low amylase values for the drained liquid (under three times the upper limit for normal serum amylase) and when image tests did not show abdominal collections. Intra-hospital mortality was defined as death occurring within 90 days of surgery.\\nThe Receiver Operating Characteristic (ROC) curve was utilized to identify an adequate cut-off point for AD1PO and verify its predictive characteristic in patients of both groups. The ROC value was generated after sensitivity, specificity and accuracy calculations. Additionally, the cut-off point 578 U/l was also selected because of its closeness to the value utilized by Fong e cols \\n9\\n, for comparison purposes. \\n Statistical analysis After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \\nAfter determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \",\n \"After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \",\n \"From the initial sample of 63 patients, two were excluded. One had AD1PO collected incorrectly on the 2nd day after surgery, and the other passed within the 1st week PO. Therefore analysis covered 61 patients. Group 1 was constituted of 41 patients, of which 36 did not develop fistula and five transient biochemical fistula (grade A). Group 2 was constituted of 20 patients, of which six presented persistent biochemical fistula (grade A), eight degree B fistula, and six grade C fistula. The index of clinical fistula (grades B + C) in the overall sample was 22.9%. \\nThe demographic and clinical characteristics of the patients, as well as respective tests, are shown in Table 1. There was no statistical difference between groups regarding age, gender and type of illness. However, regarding the size of main pancreatic duct and type of anastomosis, a statistically significant difference was detected. All patients with dilated main pancreatic ducts as well as those submitted to duct to mucosa anastomosis belonged to group 1.\\n\\nTABLE 1Associations between demographic and clinical characteristics and patient groups Variables Group 1 Group 2 pAge 58.0±13.06I\\n 57.1±12.1I\\n 0.706II\\n\\n n % n %\\nGender\\n\\n\\n\\n\\nMale 19 63.3 11 36.7 0.525III\\nFemale 22 71.0 9 29.0Disease\\n\\n\\n\\n\\nPancreatic adenocarcinoma 17 65.4 9 34.6 0.264III\\nPapillary adenocarcinoma 12 60.0 8 40.0Duodenal adenocarcinoma 1 33.3 2 66.7Colangiocarcinoma 4 100.0 0 0.0Frantz 5 100.0 0 0.0Others 2 66.7 1 33.3Size of duct\\n\\n\\n\\n\\n< 5 31 60.8 20 39.2 0.023IV\\n> 5 10 100.0 0 0.0Anastomosis*\\n\\n\\n\\n\\nT-L (Invagination) 14 50.0 14 50.0 0.011III\\nT-T 17 73.9 6 26.1T-L (DM) 10 100.0 0 0.0I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\\n\\nI=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\\nRegarding postoperative evolution, group 2 was associated with a higher level of general complications, postoperative collections, longer permanence of abdominal drain and hospitalization time. There was also an association between high resurgery index and patients of group 2. Nevertheless, despite all these worse result factors related to group 2, there was no statistically significant difference when comparing mortality across groups (Table 2). \\n\\nTABLE 2Associations between postoperative clinical findings and patient groups, along with results for drain and hospitalization times, per group Variable Group 1 Group 2 p n % n %\\nCollection\\n\\n\\n\\n\\nYes733.31466.7 <0.001I\\nNo3485.0615.0Postoperative complications\\n\\n\\n\\n\\nYes2050.020.050.0 <0.001II\\nNo21100.000.0Resurgery\\n\\n\\n\\n\\nYes746.7853.3 0.051I\\nNo3473.91226.1Time with drain (days) 9.5±2.60III29.1±13.58III <0.001IV\\nHospitalization time (days) 16.7±17.9III34.7±16.7III <0.001IV\\nMortality49.815.0 1.000II\\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\\n\\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\\nMortality of the series was 8.19%: four patients belonged to group 1 and one patient to group 2. The mortality causes in group 1 were: kidney insufficiency due to coagulopathy; dehiscence of gastrojejunal anastomosis and sepsis, pneumonia due to broncoaspiration and necrosis of the pancreaticojejunostomy loop associated with sepsis. The only patient of group 2 passed due to necrotic pancreatitis associated with pancreatic fistula and sepsis. \\nRegarding the AD1PO value, the median was 175 U/l (48.5-954) in group 1 and 2172.5 U/l (833.5-6421.0) in group 2. This difference was statistically significant (p=0.001).\\nThe area under the ROC curve presented an exactness index of 83.0% (p=0.001). When considering a 180 U/l cut-off point for AD1PO, the following results were obtained: sensitivity 100%, specificity 48.8%, positive predictor value 50%, and negative predictor value 100%. There was an association between the arbitrated AD1PO value from the ROC curve (>180 U/l) with groups 1 and 2 (p<0.001). With a cut-off point of 578 U/l, the results were: sensitivity 80%, specificity 39%, positive predictor value 50% and negative predictor value 86.2%. There was also an association between the arbitrated AD1PO value from the ROC curve (>578 U/l) with groups 1 and 2 (p<0.003). Patients presenting AD1PO values higher than 180 U/l and 578 U/l were 2.0 and 6.25 times more likely to belong to group 2, and therefore require postoperative drainage. Table 3 shows the comparison between the two AD1PO values. \\n\\nTABLE 3Precision and exactness measurements, per AD1PO cut-off point Parameters Cut-off point (U/l) 180 578Sensitivity 100% 80%Specificity 48.8% 39%VPP 50% 50%VPN 100% 86.2%\\n\",\n \"AD1PO as a predictor of post-pancreatectomy PF was described by Yamaguchi e cols in 2003\\n20\\n. The authors analyzed the amylase values measured in abdominal drains of 26 patients submitted to pancreatectomy, and observed that values were already high on the first day PO in those who developed clinical PF after. At least three meta-analysis\\n10\\n\\n,\\n\\n15\\n\\n,\\n\\n21\\n have validated the correlation between high AD1PO values and the development of PF. However, there is still controversy on the adequate cut-off point for AD1PO to predict PF, and on its applicability to the removal of prophylactic drains inserted after pancreatic resections. Verona’s group was the first to establish a strategy for the early removal of abdominal drains based on AD1PO values\\n6\\n. The authors carried out a prospective study on 114 patients submitted to pancreatic resection and randomized those with AD1PO <5000 U/l to remove the drain on the 3rd or 5th day PO. Early removal of drains was associated with a lower rate of pancreatic fistula (1.8% vs. 26%), lower rate of abdominal complications (12.2% vs. 52.6%), lower rate of pulmonary complications (26.3% vs. 52.6%), shorter hospitalization times (8.7 (±4) vs. 10.8 (±6.9) and lower hospital readmission rates (0% vs. 8.8%). Although the study suggested that early removal of abdominal drains was safe with AD1PO under 5000 U/l, other authors reported hypothetical PF indexes between 25-48% if drains were removed according to this suggestion\\n7\\n\\n,\\n\\n12\\n.\\nFor those patients submitted to duodenopancreatectomy and that received prophylactic drains, the most important question is to know which patients will not develop PF - and not the other way around. On one hand, knowing which patients will not develop PF is useful because it allows for early drain removal, which leads to shorter hospitalization times. On the other hand, predicting which patients will develop PF after surgery, when the abdominal cavity is being drained, is not as relevant because PF can be easily diagnosed by measuring amylase in the drained liquid. In most cases, treatment is simple maintenance of drains, associated with fasting and nutritional support. For this reason the study herein presented analyzed two low AD1PO cut-off points (180 and 578) that were associated with higher specificity and consequently, with a high negative predictive factor, in detriment to a high cut-off point associated with higher sensitivity to PF diagnosis. Another reason for the selection is related to the impossibility of obtaining daily interventionist radiology services in the public hospital where the majority of the patients studied underwent surgery. These services include percutaneous drainage of abdominal collections/abscesses, which is indicated in urgent cases where there are abdominal complications as a consequence of undrained anastomotic leaks. \\nThe methodology of this study utilized the PF definition based on the revised classification by ISGPS\\n5\\n, which no longer considers grade A of the previous classification to be a true PF\\n4\\n. However, as this study aimed at identifying a group of patients in which early removal of drains could be beneficial, it was important to include not only the concept of PF, but also grades B and C and those patients with biochemical fistula that persisted with amylase-rich debit between 7-21 days PO. In these patients, early removal of drains would increase susceptibility to abdominal collections or abscesses. With the 180 U/l cut-off point, an excellent correlation was obtained between AD1PO and absence of fistula. Therefore it was possible to identify 1/3 of patients that would have been benefitted by early drain removal. Besides, with the same cut-off, none of the patients that developed clinically relevant fistula or even persistent biochemical fistula would have had their drains removed early. \\nData obtained herein were capable of validating previous publications that utilized low cut-off points\\n7\\n\\n,\\n\\n12\\n\\n,\\n\\n14\\n\\n,\\n\\n17\\n. Differently from these studies, however, the first series was entirely prospective herein, including only patients submitted consecutively to duodenopancreatectomy by a single team of surgeons, where PF definition followed ISGPS criteria. The cut-off point 180 U/l obtained herein was lower than the one found by Fong et al\\n9\\n at the Massachusetts General Hospital. They divided the study in two cohorts: the first involved 126 patients, and the 612 U/l cut-off point was defined as the one presenting best accuracy, sensitivity and specificity; the second had the objective of validating a 600 U/l cut-off point, involving 369 patients submitted to duodenopancreatectomy within January 2009 and December 2012. Almost two-thirds of patients (62.1%) presented AD1PO lower than 600 U/l and only two PF cases (0.9%) were diagnosed. This was different than the other patients with AD1PO of at least 600 U/l, with PF incidence of 31.4%. When considering herein a cut-off point close to Fong et al., 578 U/l, 13.8% of patients with PF diagnosis would have been affected by early removal of drains (negative predictor value: 86.2%). In the reality considered herein, there is no non-invasive treatment for intracavitary collections/abscesses available all the time at the public hospital, and therefore increasing the cut-off point would be a high price to pay. \\nThis study presents some limitations. The first concerns the low number of included patients, which did not hinder statistical analysis. The second is related to the non-verification of the real benefits of drains in patients who developed PF, as all patients received drains. The high index of abdominal collections (65%) and necessity of re-surgery (40%) in group 2 suggested that, for some patients, drains were not totally efficient; nevertheless, the objective of the study was to find a group of patients in which drains could be removed early and not confirm the non-efficiency of drains in the group that developed PF.\",\n \"Was validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients. \"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,"results","discussion","conclusions"],"string":"[\n \"intro\",\n \"methods\",\n null,\n \"results\",\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["Amylase","Postoperative complications","Pancreatic fistula","Pancreatoduodenectomy","Amilase","Complicações pós-operatórias","Fístula pancreática","Pancreatoduodenectomia"],"string":"[\n \"Amylase\",\n \"Postoperative complications\",\n \"Pancreatic fistula\",\n \"Pancreatoduodenectomy\",\n \"Amilase\",\n \"Complicações pós-operatórias\",\n \"Fístula pancreática\",\n \"Pancreatoduodenectomia\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nHistorically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them\n16\n. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis\n13\n\n,\n\n11\n. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results\n8\n\n,\n\n18\n\n,\n\n19\n, which has been a limiting factor in the practice of not using drains in this type of procedure. \nDescription of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L\n6\n and 90 U/l\n14\n have been suggested. \nThe objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.\n\nMETHOD:\nThe project was submitted and approved by the Human Research Ethics Committee, under nº 59716616.9.0000.5292 in the Plataforma Brasil.\nSixty-three patients were analyzed from a prospective database, who had undergone Kausch-Whipple surgery performed by the 1st author of this study (ECA), for the treatment of pancreatic or peripancreatic diseases. The time period was between June 2007 and September 2016, in the following hospitals located in the city of Natal: Hospital Universitário Onofre Lopes, Liga Norte Riograndense Contra o Câncer, Casa de Saúde São Lucas and Natal Hospital Center.\nThe technical steps for the Whipple technique as performed by the author are detailed in previous studies\n1\n\n,\n\n2\n. Three techniques for pancreatojejunal anastomosis were utilized. For pancreas with main pancreatic duct ≥5 mm, two-layer pancreatojejunal terminolateral duct to mucosa anastomosis was performed. For pancreas with normal or slightly dilated (diameter <5 mm) main pancreatic duct, terminoterminal pancreatojejunal anastomosis (“telescopic”) or terminolateral pancreatojejunal anastomosis (“invagination”) were carried out, the latter performed systematically after the 33rd patient from the casebook. At the end of the surgery, two drains, preferably laminar silicone drains, were placed in the cavity and exteriorized at each flank. \nIn the majority of patients, subcutaneous octreotide was utilized as prophylaxis for the prevention of PF, at 0.3 mg/day, fractioned in 8 h intervals, during seven days. Debits were recorded along with amylase dosage of the drained liquid on the 1st, 3rd, 5th, 7th and sometimes, on the 9th postoperative day. The value of AD1PO vas defined from the highest amylase value of both drains, measured from a sample of liquid obtained on the first day after surgery. \nPF diagnosis utilized the 2016 revised criterion of GIEDFP\n3\n. PF was defined when on the 3rd postoperative (PO) day the value of amylase in the drained liquid was three times higher than the normal upper limit for serum amylase. Patients that did not develop fistula or those with transient biochemical fistula (<7 days PO) were characterized as group 1, and patients with persistent biochemical fistula (between 7 and 21 days PO) and fistula grade B and C were characterized as group 2. In most cases, on the 9th day after surgery, control ultrasound or tomography tests were carried out. Removal of drains occurred in those cases with low amylase values for the drained liquid (under three times the upper limit for normal serum amylase) and when image tests did not show abdominal collections. Intra-hospital mortality was defined as death occurring within 90 days of surgery.\nThe Receiver Operating Characteristic (ROC) curve was utilized to identify an adequate cut-off point for AD1PO and verify its predictive characteristic in patients of both groups. The ROC value was generated after sensitivity, specificity and accuracy calculations. Additionally, the cut-off point 578 U/l was also selected because of its closeness to the value utilized by Fong e cols \n9\n, for comparison purposes. \n Statistical analysis After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \nAfter determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \n\nStatistical analysis:\nAfter determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \n\nRESULTS:\nFrom the initial sample of 63 patients, two were excluded. One had AD1PO collected incorrectly on the 2nd day after surgery, and the other passed within the 1st week PO. Therefore analysis covered 61 patients. Group 1 was constituted of 41 patients, of which 36 did not develop fistula and five transient biochemical fistula (grade A). Group 2 was constituted of 20 patients, of which six presented persistent biochemical fistula (grade A), eight degree B fistula, and six grade C fistula. The index of clinical fistula (grades B + C) in the overall sample was 22.9%. \nThe demographic and clinical characteristics of the patients, as well as respective tests, are shown in Table 1. There was no statistical difference between groups regarding age, gender and type of illness. However, regarding the size of main pancreatic duct and type of anastomosis, a statistically significant difference was detected. All patients with dilated main pancreatic ducts as well as those submitted to duct to mucosa anastomosis belonged to group 1.\n\nTABLE 1Associations between demographic and clinical characteristics and patient groups Variables Group 1 Group 2 pAge 58.0±13.06I\n 57.1±12.1I\n 0.706II\n\n n % n %\nGender\n\n\n\n\nMale 19 63.3 11 36.7 0.525III\nFemale 22 71.0 9 29.0Disease\n\n\n\n\nPancreatic adenocarcinoma 17 65.4 9 34.6 0.264III\nPapillary adenocarcinoma 12 60.0 8 40.0Duodenal adenocarcinoma 1 33.3 2 66.7Colangiocarcinoma 4 100.0 0 0.0Frantz 5 100.0 0 0.0Others 2 66.7 1 33.3Size of duct\n\n\n\n\n< 5 31 60.8 20 39.2 0.023IV\n> 5 10 100.0 0 0.0Anastomosis*\n\n\n\n\nT-L (Invagination) 14 50.0 14 50.0 0.011III\nT-T 17 73.9 6 26.1T-L (DM) 10 100.0 0 0.0I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\n\nI=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\nRegarding postoperative evolution, group 2 was associated with a higher level of general complications, postoperative collections, longer permanence of abdominal drain and hospitalization time. There was also an association between high resurgery index and patients of group 2. Nevertheless, despite all these worse result factors related to group 2, there was no statistically significant difference when comparing mortality across groups (Table 2). \n\nTABLE 2Associations between postoperative clinical findings and patient groups, along with results for drain and hospitalization times, per group Variable Group 1 Group 2 p n % n %\nCollection\n\n\n\n\nYes733.31466.7 <0.001I\nNo3485.0615.0Postoperative complications\n\n\n\n\nYes2050.020.050.0 <0.001II\nNo21100.000.0Resurgery\n\n\n\n\nYes746.7853.3 0.051I\nNo3473.91226.1Time with drain (days) 9.5±2.60III29.1±13.58III <0.001IV\nHospitalization time (days) 16.7±17.9III34.7±16.7III <0.001IV\nMortality49.815.0 1.000II\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\n\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\nMortality of the series was 8.19%: four patients belonged to group 1 and one patient to group 2. The mortality causes in group 1 were: kidney insufficiency due to coagulopathy; dehiscence of gastrojejunal anastomosis and sepsis, pneumonia due to broncoaspiration and necrosis of the pancreaticojejunostomy loop associated with sepsis. The only patient of group 2 passed due to necrotic pancreatitis associated with pancreatic fistula and sepsis. \nRegarding the AD1PO value, the median was 175 U/l (48.5-954) in group 1 and 2172.5 U/l (833.5-6421.0) in group 2. This difference was statistically significant (p=0.001).\nThe area under the ROC curve presented an exactness index of 83.0% (p=0.001). When considering a 180 U/l cut-off point for AD1PO, the following results were obtained: sensitivity 100%, specificity 48.8%, positive predictor value 50%, and negative predictor value 100%. There was an association between the arbitrated AD1PO value from the ROC curve (>180 U/l) with groups 1 and 2 (p<0.001). With a cut-off point of 578 U/l, the results were: sensitivity 80%, specificity 39%, positive predictor value 50% and negative predictor value 86.2%. There was also an association between the arbitrated AD1PO value from the ROC curve (>578 U/l) with groups 1 and 2 (p<0.003). Patients presenting AD1PO values higher than 180 U/l and 578 U/l were 2.0 and 6.25 times more likely to belong to group 2, and therefore require postoperative drainage. Table 3 shows the comparison between the two AD1PO values. \n\nTABLE 3Precision and exactness measurements, per AD1PO cut-off point Parameters Cut-off point (U/l) 180 578Sensitivity 100% 80%Specificity 48.8% 39%VPP 50% 50%VPN 100% 86.2%\n\n\nDISCUSSION:\nAD1PO as a predictor of post-pancreatectomy PF was described by Yamaguchi e cols in 2003\n20\n. The authors analyzed the amylase values measured in abdominal drains of 26 patients submitted to pancreatectomy, and observed that values were already high on the first day PO in those who developed clinical PF after. At least three meta-analysis\n10\n\n,\n\n15\n\n,\n\n21\n have validated the correlation between high AD1PO values and the development of PF. However, there is still controversy on the adequate cut-off point for AD1PO to predict PF, and on its applicability to the removal of prophylactic drains inserted after pancreatic resections. Verona’s group was the first to establish a strategy for the early removal of abdominal drains based on AD1PO values\n6\n. The authors carried out a prospective study on 114 patients submitted to pancreatic resection and randomized those with AD1PO <5000 U/l to remove the drain on the 3rd or 5th day PO. Early removal of drains was associated with a lower rate of pancreatic fistula (1.8% vs. 26%), lower rate of abdominal complications (12.2% vs. 52.6%), lower rate of pulmonary complications (26.3% vs. 52.6%), shorter hospitalization times (8.7 (±4) vs. 10.8 (±6.9) and lower hospital readmission rates (0% vs. 8.8%). Although the study suggested that early removal of abdominal drains was safe with AD1PO under 5000 U/l, other authors reported hypothetical PF indexes between 25-48% if drains were removed according to this suggestion\n7\n\n,\n\n12\n.\nFor those patients submitted to duodenopancreatectomy and that received prophylactic drains, the most important question is to know which patients will not develop PF - and not the other way around. On one hand, knowing which patients will not develop PF is useful because it allows for early drain removal, which leads to shorter hospitalization times. On the other hand, predicting which patients will develop PF after surgery, when the abdominal cavity is being drained, is not as relevant because PF can be easily diagnosed by measuring amylase in the drained liquid. In most cases, treatment is simple maintenance of drains, associated with fasting and nutritional support. For this reason the study herein presented analyzed two low AD1PO cut-off points (180 and 578) that were associated with higher specificity and consequently, with a high negative predictive factor, in detriment to a high cut-off point associated with higher sensitivity to PF diagnosis. Another reason for the selection is related to the impossibility of obtaining daily interventionist radiology services in the public hospital where the majority of the patients studied underwent surgery. These services include percutaneous drainage of abdominal collections/abscesses, which is indicated in urgent cases where there are abdominal complications as a consequence of undrained anastomotic leaks. \nThe methodology of this study utilized the PF definition based on the revised classification by ISGPS\n5\n, which no longer considers grade A of the previous classification to be a true PF\n4\n. However, as this study aimed at identifying a group of patients in which early removal of drains could be beneficial, it was important to include not only the concept of PF, but also grades B and C and those patients with biochemical fistula that persisted with amylase-rich debit between 7-21 days PO. In these patients, early removal of drains would increase susceptibility to abdominal collections or abscesses. With the 180 U/l cut-off point, an excellent correlation was obtained between AD1PO and absence of fistula. Therefore it was possible to identify 1/3 of patients that would have been benefitted by early drain removal. Besides, with the same cut-off, none of the patients that developed clinically relevant fistula or even persistent biochemical fistula would have had their drains removed early. \nData obtained herein were capable of validating previous publications that utilized low cut-off points\n7\n\n,\n\n12\n\n,\n\n14\n\n,\n\n17\n. Differently from these studies, however, the first series was entirely prospective herein, including only patients submitted consecutively to duodenopancreatectomy by a single team of surgeons, where PF definition followed ISGPS criteria. The cut-off point 180 U/l obtained herein was lower than the one found by Fong et al\n9\n at the Massachusetts General Hospital. They divided the study in two cohorts: the first involved 126 patients, and the 612 U/l cut-off point was defined as the one presenting best accuracy, sensitivity and specificity; the second had the objective of validating a 600 U/l cut-off point, involving 369 patients submitted to duodenopancreatectomy within January 2009 and December 2012. Almost two-thirds of patients (62.1%) presented AD1PO lower than 600 U/l and only two PF cases (0.9%) were diagnosed. This was different than the other patients with AD1PO of at least 600 U/l, with PF incidence of 31.4%. When considering herein a cut-off point close to Fong et al., 578 U/l, 13.8% of patients with PF diagnosis would have been affected by early removal of drains (negative predictor value: 86.2%). In the reality considered herein, there is no non-invasive treatment for intracavitary collections/abscesses available all the time at the public hospital, and therefore increasing the cut-off point would be a high price to pay. \nThis study presents some limitations. The first concerns the low number of included patients, which did not hinder statistical analysis. The second is related to the non-verification of the real benefits of drains in patients who developed PF, as all patients received drains. The high index of abdominal collections (65%) and necessity of re-surgery (40%) in group 2 suggested that, for some patients, drains were not totally efficient; nevertheless, the objective of the study was to find a group of patients in which drains could be removed early and not confirm the non-efficiency of drains in the group that developed PF.\n\nCONCLUSION:\nWas validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients. \n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe value of drain amylase on the first postoperative day after pancreatic resections has been described as an efficient predictor of pancreatic fistula. In spite of this, the cut-off point below which the drains can be removed early remains controversial.\n\nMethods:\nWere included patients undergoing Whipple surgery in the period of 2007 to 2016. Group 1 enrolled the ones who did not develop fistula and those who developed biochemical fistula for less than seven days postoperatively and group 2 included patients who developed persistent biochemical fistula between seven and 21 days and those with grade B and C fistula.\n\nResults:\nSixty-one patients were included, 41 comprised group 1 and 20 group 2. The incidence of abdominal collections, need for reoperation and time of hospitalization were for group 1 and 2, respectively: 17.1%, 17.1% and 9.5 days, and 65%, 40% and 21.1 days. The median of the amylase from the drain at 1st postoperative day was in group 1 and 2, respectively: 175 U/l and 3172.5 U/l (p=0.001). Using a cut-off of 180 to predict the group to which the patient would belong there was obtained sensitivity, specificity, positive predictive value and negative predictive value of 100%, 48.8%, 50% and 100% respectively.\n\nConclusions:\nIt was validated the cut-off value of 180 U/l as appropriate to early drain removal."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nHistorically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them\n16\n. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis\n13\n\n,\n\n11\n. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results\n8\n\n,\n\n18\n\n,\n\n19\n, which has been a limiting factor in the practice of not using drains in this type of procedure. \nDescription of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L\n6\n and 90 U/l\n14\n have been suggested. \nThe objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.\n\nCONCLUSION:\nWas validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients. "},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe value of drain amylase on the first postoperative day after pancreatic resections has been described as an efficient predictor of pancreatic fistula. In spite of this, the cut-off point below which the drains can be removed early remains controversial.\n\nMethods:\nWere included patients undergoing Whipple surgery in the period of 2007 to 2016. Group 1 enrolled the ones who did not develop fistula and those who developed biochemical fistula for less than seven days postoperatively and group 2 included patients who developed persistent biochemical fistula between seven and 21 days and those with grade B and C fistula.\n\nResults:\nSixty-one patients were included, 41 comprised group 1 and 20 group 2. The incidence of abdominal collections, need for reoperation and time of hospitalization were for group 1 and 2, respectively: 17.1%, 17.1% and 9.5 days, and 65%, 40% and 21.1 days. The median of the amylase from the drain at 1st postoperative day was in group 1 and 2, respectively: 175 U/l and 3172.5 U/l (p=0.001). Using a cut-off of 180 to predict the group to which the patient would belong there was obtained sensitivity, specificity, positive predictive value and negative predictive value of 100%, 48.8%, 50% and 100% respectively.\n\nConclusions:\nIt was validated the cut-off value of 180 U/l as appropriate to early drain removal."},"whole_article_text_length":{"kind":"number","value":3414,"string":"3,414"},"whole_article_abstract_length":{"kind":"number","value":276,"string":"276"},"num_sections":{"kind":"number","value":6,"string":"6"},"most_frequent_words":{"kind":"list like","value":["patients","drains","ad1po","pf","group","cut","cut point","point","test","fistula"],"string":"[\n \"patients\",\n \"drains\",\n \"ad1po\",\n \"pf\",\n \"group\",\n \"cut\",\n \"cut point\",\n \"point\",\n \"test\",\n \"fistula\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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[SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] drains | pancreatic | studies | systematic | manner | use | pf | correlation | objective | resections [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] amylase | tests | performed | pancreatojejunal | utilized | value | surgery | groups | liquid | applied [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] group | test | 100 | table | 50 | fistula | value | patients | groups | duct [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ideal cut | ideal | ideal cut point | cut | point | cut point | patients | pf benefits faster | service | compensate risk [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | pf | cut | test | cut point | point | ad1po | group | groups [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | pf | cut | test | cut point | point | ad1po | group | groups [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] the first postoperative day ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Whipple | the period of 2007 to 2016 ||| Group 1 | less than seven days | 2 | between seven and 21 days [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Sixty-one | 41 | 1 | 20 | 2 ||| 1 | 2 | 17.1% | 17.1% | 9.5 days | 65% | 40% | 21.1 days ||| 1st postoperative day | 1 | 2 | 175 | 3172.5 | U/l (p=0.001 ||| 180 | 100% | 48.8% | 50% and 100% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] 180 [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] the first postoperative day ||| ||| Whipple | the period of 2007 to 2016 ||| Group 1 | less than seven days | 2 | between seven and 21 days ||| ||| Sixty-one | 41 | 1 | 20 | 2 ||| 1 | 2 | 17.1% | 17.1% | 9.5 days | 65% | 40% | 21.1 days ||| 1st postoperative day | 1 | 2 | 175 | 3172.5 | U/l (p=0.001 ||| 180 | 100% | 48.8% | 50% and 100% ||| 180 [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] the first postoperative day ||| ||| Whipple | the period of 2007 to 2016 ||| Group 1 | less than seven days | 2 | between seven and 21 days ||| ||| Sixty-one | 41 | 1 | 20 | 2 ||| 1 | 2 | 17.1% | 17.1% | 9.5 days | 65% | 40% | 21.1 days ||| 1st postoperative day | 1 | 2 | 175 | 3172.5 | U/l (p=0.001 ||| 180 | 100% | 48.8% | 50% and 100% ||| 180 [SUMMARY]"}}},{"rowIdx":275,"cells":{"title":{"kind":"string","value":"Does a continuous local anaesthetic pain treatment after immediate tissue expander reconstruction in breast carcinoma patients more efficiently reduce acute postoperative pain--a prospective randomised study."},"pmid":{"kind":"string","value":"24433317"},"background_abstract":{"kind":"string","value":"Immediate breast reconstruction with an expander is a reasonable option for properly selected patients. After reconstruction, patients have severe postoperative pain, which responds poorly to opioids. Our aim was to evaluate if continuous wound infusion of a local anaesthetic into the surgical wound reduces postoperative pain, consumption of opioids and incidence of chronic pain compared to standard intravenous piritramide after primary breast reconstruction in breast carcinoma patients."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Altogether, 60 patients were enrolled in our study; one half in the group with wound infusion of a local anaesthetic, and the other half in the standard (piritramide) group. Parameters measured included: pain intensity (visual analogue scale), drug requirements, alertness, hospitalisation, side-effects and late complications. A p-value of < 0.05 was considered statistically significant."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In the recovery room, the test group reported less acute pain at rest (P = 0.03) and at activity (P = 0.01), and on the day of the surgical procedure they reported less pain at activity (P = 0.003). Consumption of piritramide and metoclopramide was lower in this group (P < 0.0001), but their alertness after the surgical procedure was higher compared to the standard group (P < 0.001). After three months, the test group reported less chronic pain (P = 0.01)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"After primary tissue expander breast reconstruction, wound infusion of a local anaesthetic significantly reduces acute pain and enables reduced opioid consumption, resulting in less postoperative sedation and reduced need for antiemetic drugs. Wound infusion of a local anaesthetic reduces chronic pain."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Acute Disease","Aged","Anesthetics, Local","Breast Neoplasms","Carcinoma, Ductal, Breast","Carcinoma, Intraductal, Noninfiltrating","Carcinoma, Lobular","Case-Control Studies","Catheters, Indwelling","Female","Follow-Up Studies","Humans","Lymphatic Metastasis","Mammaplasty","Mastectomy","Middle Aged","Neoplasm Grading","Neoplasm Staging","Pain, Postoperative","Postoperative Complications","Prognosis","Prospective Studies","Tissue Expansion Devices"],"string":"[\n \"Acute Disease\",\n \"Aged\",\n \"Anesthetics, Local\",\n \"Breast Neoplasms\",\n \"Carcinoma, Ductal, Breast\",\n \"Carcinoma, Intraductal, Noninfiltrating\",\n \"Carcinoma, Lobular\",\n \"Case-Control Studies\",\n \"Catheters, Indwelling\",\n \"Female\",\n \"Follow-Up Studies\",\n \"Humans\",\n \"Lymphatic Metastasis\",\n \"Mammaplasty\",\n \"Mastectomy\",\n \"Middle Aged\",\n \"Neoplasm Grading\",\n \"Neoplasm Staging\",\n \"Pain, Postoperative\",\n \"Postoperative Complications\",\n \"Prognosis\",\n \"Prospective Studies\",\n \"Tissue Expansion Devices\"\n]"},"pmcid":{"kind":"string","value":"3899444"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Breast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Altogether, 60 breast carcinoma patients were enrolled in the study protocol between January 2011 and May 2012: 72 patients who were eligible for inclusion were offered the opportunity to participate in our study during the recruitment period; 12 of them declined participation, and the remaining 60 were included in the study. Patients listed for mastectomy and primary reconstruction with a tissue expander because of the presence of breast carcinoma or for prophylactic mastectomy, were randomized during the routine preoperative anaesthetic assessment. Anaesthesia for the procedure was the same for all study subjects. We used the same protocol as already reported for patients with axillary dissection\n[14]. The first study arm was treated with continuous local analgesia, and the other (control) arm received standard intravenous analgesia. Allergies to local anaesthetics and chronic pre-operative opioid consumption were the criteria to exclude patients from our study.\nAfter informed consent was obtained the randomization was made by research nurses from the Clinical Research Unit of the Institute of Oncology Ljubljana. The research nurse performed randomization using random numbers generated by a computer and then informed the principal investigator about the treatment allocation of the patient.\nSurgical procedures were performed by ten experienced oncology surgeons and ten plastic surgeons. Sentinel lymph node biopsy was performed in 55 patients. In one patient with positive imprint cytology of the sentinel lymph node, axillary lymphadenectomy was performed immediately.\nThe study was reviewed by the The National Medical Ethics Committee of the Republic of Slovenia and performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. Our study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and conducted with the understanding and consent of all subjects involved. Study was entirely financed by the Institute of Oncology as a part of public service.\n Test group of patients Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\nBefore wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\n Control group of patients The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\nThe control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\n Both groups of patients Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\nPatient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\n Pain measurement Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\nData about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\n Statistical analysis In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\nIn order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The mean age of patients was 47.8 years (range 25 to 64 y), height was 166 cm (range 153 to 178 cm), weight was 61.7 kg (range 45 to 85 kg), and the body mass index (BMI) was 22.4 (range 17 to 31.2). There were no significant differences between the study groups in BMI, American Society of Anesthesiology (ASA) score, comorbidities (Table \n1), type and length of surgical procedure, extent of lymph node dissection, adjuvant therapy, complications, or hospital stay (Table \n2).\nPatient characteristics\nASA, American Society of Anesthesiology; HER-2, human epidermal growth factor receptor-2; n, number of patients.\nTreatment of patients, adjuvant therapy, hospital stay and complications\nValues are numbers of patients unless stated otherwise.\n Acute pain Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\nData on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\n Opioid consumption Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\nConsumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\n Nausea Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\nPatients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\n Complications No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\nNo local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\n Hospital stay The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\nThe average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\n Unilateral and bilateral tissue expander In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\nIn order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\n Late complications (three months after primary reconstruction) Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\nThree months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Our current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia."},"other_sections_titles":{"kind":"list like","value":["Background","Test group of patients","Control group of patients","Both groups of patients","Pain measurement","Statistical analysis","Acute pain","Opioid consumption","Nausea","Complications","Hospital stay","Unilateral and bilateral tissue expander","Late complications (three months after primary reconstruction)","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Test group of patients\",\n \"Control group of patients\",\n \"Both groups of patients\",\n \"Pain measurement\",\n \"Statistical analysis\",\n \"Acute pain\",\n \"Opioid consumption\",\n \"Nausea\",\n \"Complications\",\n \"Hospital stay\",\n \"Unilateral and bilateral tissue expander\",\n \"Late complications (three months after primary reconstruction)\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["Breast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.","Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.","The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.","Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.","Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.","In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.","Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.","Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.","Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).","No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.","The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.","In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).","Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.","BMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.","The authors declare that they have no competing interests.","BS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript."],"string":"[\n \"Breast cancer is the most common cancer in women both in the developed and the developing countries\\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\\n[4].\\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\\n[6].\\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.\",\n \"Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\",\n \"The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\",\n \"Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\",\n \"Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\\n[19] in a composite way, as we did in our previous study\\n[14].\\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\",\n \"In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\",\n \"Data on postoperative pain are presented in Table \\n3.\\nPain in the local anaesthetic group and the standard group of patients\\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\",\n \"Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \\n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\\nOAA/S - observer's assessment of alertness/sedation.\",\n \"Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\",\n \"No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\",\n \"The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\",\n \"In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\",\n \"Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\",\n \"BMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.\",\n \"The authors declare that they have no competing interests.\",\n \"BS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Test group of patients","Control group of patients","Both groups of patients","Pain measurement","Statistical analysis","Results","Acute pain","Opioid consumption","Nausea","Complications","Hospital stay","Unilateral and bilateral tissue expander","Late complications (three months after primary reconstruction)","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Test group of patients\",\n \"Control group of patients\",\n \"Both groups of patients\",\n \"Pain measurement\",\n \"Statistical analysis\",\n \"Results\",\n \"Acute pain\",\n \"Opioid consumption\",\n \"Nausea\",\n \"Complications\",\n \"Hospital stay\",\n \"Unilateral and bilateral tissue expander\",\n \"Late complications (three months after primary reconstruction)\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Breast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.","Altogether, 60 breast carcinoma patients were enrolled in the study protocol between January 2011 and May 2012: 72 patients who were eligible for inclusion were offered the opportunity to participate in our study during the recruitment period; 12 of them declined participation, and the remaining 60 were included in the study. Patients listed for mastectomy and primary reconstruction with a tissue expander because of the presence of breast carcinoma or for prophylactic mastectomy, were randomized during the routine preoperative anaesthetic assessment. Anaesthesia for the procedure was the same for all study subjects. We used the same protocol as already reported for patients with axillary dissection\n[14]. The first study arm was treated with continuous local analgesia, and the other (control) arm received standard intravenous analgesia. Allergies to local anaesthetics and chronic pre-operative opioid consumption were the criteria to exclude patients from our study.\nAfter informed consent was obtained the randomization was made by research nurses from the Clinical Research Unit of the Institute of Oncology Ljubljana. The research nurse performed randomization using random numbers generated by a computer and then informed the principal investigator about the treatment allocation of the patient.\nSurgical procedures were performed by ten experienced oncology surgeons and ten plastic surgeons. Sentinel lymph node biopsy was performed in 55 patients. In one patient with positive imprint cytology of the sentinel lymph node, axillary lymphadenectomy was performed immediately.\nThe study was reviewed by the The National Medical Ethics Committee of the Republic of Slovenia and performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. Our study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and conducted with the understanding and consent of all subjects involved. Study was entirely financed by the Institute of Oncology as a part of public service.\n Test group of patients Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\nBefore wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\n Control group of patients The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\nThe control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\n Both groups of patients Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\nPatient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\n Pain measurement Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\nData about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\n Statistical analysis In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\nIn order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.","Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.","The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.","Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.","Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.","In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.","The mean age of patients was 47.8 years (range 25 to 64 y), height was 166 cm (range 153 to 178 cm), weight was 61.7 kg (range 45 to 85 kg), and the body mass index (BMI) was 22.4 (range 17 to 31.2). There were no significant differences between the study groups in BMI, American Society of Anesthesiology (ASA) score, comorbidities (Table \n1), type and length of surgical procedure, extent of lymph node dissection, adjuvant therapy, complications, or hospital stay (Table \n2).\nPatient characteristics\nASA, American Society of Anesthesiology; HER-2, human epidermal growth factor receptor-2; n, number of patients.\nTreatment of patients, adjuvant therapy, hospital stay and complications\nValues are numbers of patients unless stated otherwise.\n Acute pain Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\nData on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\n Opioid consumption Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\nConsumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\n Nausea Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\nPatients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\n Complications No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\nNo local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\n Hospital stay The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\nThe average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\n Unilateral and bilateral tissue expander In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\nIn order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\n Late complications (three months after primary reconstruction) Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\nThree months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.","Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.","Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.","Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).","No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.","The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.","In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).","Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.","Ideally the study should have been placebo double-blinded to eliminate all biases. However, the cost of the elastomeric pump and wound catheter was 175 euro, so we were not able to perform a double-blind study. However, the cost of an elastomeric pump and wound catheter was not the only reason that precluded us from performing such a study. We had also ethical reasons for not carrying out a placebo-controlled study. Namely, a reconstruction with a tissue expander is a very painful procedure for the majority of patients, so we felt that it is not ethical to have a control group treated by placebo only. Instead, we offered our control group of patients the best available treatment, that is, intravenous analgesia. Based on the good results of our preliminary data on the use of continuous local anaesthetics for treatment of pain, our patients were willing to participate in our study because they were offered state-of-the-art pain treatment versus potentially even more effective pain treatment.\nSub-pectoral breast augmentation and breast reconstruction with an expander or an implant are associated with considerable pain in the early postoperative period\n[6,9,10,20-24]. Legeby et al.\n[6] found that pain in the first 3 h after immediate breast reconstruction with a tissue expander was more severe than pain after mastectomy or axillary dissection. The mean VAS value for pain in the first 3 h after breast reconstruction was 4.9 and 5.0, if axillary dissection was also performed, whereas the values were 2.1 for mastectomy and 3.0 for mastectomy with axillary dissection\n[6]. Similarly, after tissue-expander implantation, our standard analgesia group of patients also suffered pain in the recovery room, which was by about 3.0 VAS units larger than in patients who underwent breast surgery with axillary dissection without reconstruction, as reported by Strazisar et al.\n[14]. The mean VAS score after immediate reconstruction was 4.9 in the standard analgesia group, whereas it was only 3.4 in the local anaesthetic group. In our previous study, the mean VAS score in recovery after breast cancer surgery with axillary dissection was 2.0 in the standard control group and 0.5 in the local anaesthetic group\n[14]. In both our studies, the local anaesthetic reduced pain by 1.5 VAS units more than standard treatment alone.\nThe most positive result of our study is the observation that 77% of patients treated with local anaesthetics did not experience severe pain. To our knowledge, there are no reports in the literature about similarly effective early postoperative analgesia after the implantation of a breast-tissue expander. Legeby et al. reported that even 48% of patients treated with local anaesthetics experienced unacceptably severe pain (VAS 4.7 to 10.0) in the early postoperative period\n[6]. Legeby et al. used an epidural catheter with only a few holes at its tip and instilled local anaesthetic every 3 h. They probably did not establish an equal distribution of the local anaesthetic along the entire length of the muscle and the wound. On the other hand, we used quality wound-catheters that were perforated 15 cm at the distal end, which enabled a better distribution of the local anaesthetic. Furthermore, we used an elastomeric pump, which enabled a continuous infusion of the local anaesthetic. That is probably the reason why only seven patients (23.1%) in the test group reported pain above a VAS score of 5.0, whereas in the control group, such pain levels were reported by thirteen patients (42.9%).\nLegeby et al. found that the use of opioids was about three times higher after the implantation of a tissue expander compared to other breast surgical procedures\n[6]. After the tissue-expander implantation with or without axillary dissection, the consumption of opioids was 12.8 mg and 11.8 mg, whereas after mastectomy or mastectomy with axillary dissection, the mean consumption of opioids was 3.2 mg and 5.5 mg, respectively\n[6]. Our patients with standard analgesia after the tissue-expander implantation needed 5.6 times more opioids than patients who underwent axillary dissection alone\n[14]. Local anaesthetics can reduce consumption of opioids after mastectomy and axillary dissection\n[11,13,14] and also after breast augmentation with implants\n[7]. Our study shows that the use of continuous local anaesthesia can effectively reduce opioid consumption. Our patients treated with a local anaesthetic needed about three times less opioids than the standard analgesia group. Subsequently, patients from the local anaesthesia group experienced nausea less often, thus they had a lower consumption of metoclopramide than the standard analgesia group. Furthermore, the use of local anaesthetics resulted in less sedated and more alert patients in the recovery room and 6 h after the operation. The complication rate did not differ between the two groups of patients. This is in agreement with the reports of other authors who used local anaesthetics\n[11-13,18,21,25-30]. Some authors report that local anaesthetics appear to reduce the incidence of inflammation\n[31-33].\nThere was no difference in pain on the first postoperative day between the study and control group of patients. It means that local anaesthesia was no longer as effective as it was on the day of surgery. A possible explanation is that muscle overstretching caused by a tissue expander is no longer as painful as it was. Other explanations are that in some patients the position of the wound catheter might have changed during movement or that there was a different perfusion region in the erect, prone or supine position, or that continuous infusion was flawed because of the wound drainage. Unfortunately, in our study we did not test a rescue bolus-injection of levobupivacaine in the case of severe pain. However, we have good experience with application of a rescue bolus of levobupivacaine in patients with neuropathic pain after axillary lymphadenectomy. Many times we have observed that injection of a bolus of 15 mL of 0.25% levobupivacaine into a drain and clamping of wound drainage inhibited neuropathic pain for a couple of hours.\nMemory of severe postoperative pain is the most important risk factor for the development of chronic pain\n[34]. Patients who were treated with patient-controlled analgesia had three times less chronic pain than the traditionally nurse-administered patients\n[6]. The results of our study support this observation. Only 17% of patients treated with a local anaesthetic experienced chronic pain. This is significantly less than in the standard analgesia group of patients, of whom 50% suffered chronic pain. The proportion of patients treated with axillary lymphadenectomy, chemotherapy, hormone therapy and/or irradiation was similar in both study arms. Hence, we think that inadequately treated acute pain had an impact on the frequency of chronic pain in the standard analgesia group.","Our current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia.","BMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.","The authors declare that they have no competing interests.","BS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript."],"string":"[\n \"Breast cancer is the most common cancer in women both in the developed and the developing countries\\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\\n[4].\\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\\n[6].\\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.\",\n \"Altogether, 60 breast carcinoma patients were enrolled in the study protocol between January 2011 and May 2012: 72 patients who were eligible for inclusion were offered the opportunity to participate in our study during the recruitment period; 12 of them declined participation, and the remaining 60 were included in the study. Patients listed for mastectomy and primary reconstruction with a tissue expander because of the presence of breast carcinoma or for prophylactic mastectomy, were randomized during the routine preoperative anaesthetic assessment. Anaesthesia for the procedure was the same for all study subjects. We used the same protocol as already reported for patients with axillary dissection\\n[14]. The first study arm was treated with continuous local analgesia, and the other (control) arm received standard intravenous analgesia. Allergies to local anaesthetics and chronic pre-operative opioid consumption were the criteria to exclude patients from our study.\\nAfter informed consent was obtained the randomization was made by research nurses from the Clinical Research Unit of the Institute of Oncology Ljubljana. The research nurse performed randomization using random numbers generated by a computer and then informed the principal investigator about the treatment allocation of the patient.\\nSurgical procedures were performed by ten experienced oncology surgeons and ten plastic surgeons. Sentinel lymph node biopsy was performed in 55 patients. In one patient with positive imprint cytology of the sentinel lymph node, axillary lymphadenectomy was performed immediately.\\nThe study was reviewed by the The National Medical Ethics Committee of the Republic of Slovenia and performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. Our study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and conducted with the understanding and consent of all subjects involved. Study was entirely financed by the Institute of Oncology as a part of public service.\\n Test group of patients Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\\nBefore wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\\n Control group of patients The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\\nThe control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\\n Both groups of patients Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\\nPatient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\\n Pain measurement Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\\n[19] in a composite way, as we did in our previous study\\n[14].\\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\\nData about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\\n[19] in a composite way, as we did in our previous study\\n[14].\\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\\n Statistical analysis In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\\nIn order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\",\n \"Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\",\n \"The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\",\n \"Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\",\n \"Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\\n[19] in a composite way, as we did in our previous study\\n[14].\\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\",\n \"In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\",\n \"The mean age of patients was 47.8 years (range 25 to 64 y), height was 166 cm (range 153 to 178 cm), weight was 61.7 kg (range 45 to 85 kg), and the body mass index (BMI) was 22.4 (range 17 to 31.2). There were no significant differences between the study groups in BMI, American Society of Anesthesiology (ASA) score, comorbidities (Table \\n1), type and length of surgical procedure, extent of lymph node dissection, adjuvant therapy, complications, or hospital stay (Table \\n2).\\nPatient characteristics\\nASA, American Society of Anesthesiology; HER-2, human epidermal growth factor receptor-2; n, number of patients.\\nTreatment of patients, adjuvant therapy, hospital stay and complications\\nValues are numbers of patients unless stated otherwise.\\n Acute pain Data on postoperative pain are presented in Table \\n3.\\nPain in the local anaesthetic group and the standard group of patients\\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\\nData on postoperative pain are presented in Table \\n3.\\nPain in the local anaesthetic group and the standard group of patients\\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\\n Opioid consumption Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \\n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\\nOAA/S - observer's assessment of alertness/sedation.\\nConsumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \\n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\\nOAA/S - observer's assessment of alertness/sedation.\\n Nausea Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\\nPatients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\\n Complications No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\\nNo local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\\n Hospital stay The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\\nThe average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\\n Unilateral and bilateral tissue expander In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\\nIn order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\\n Late complications (three months after primary reconstruction) Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\\nThree months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\",\n \"Data on postoperative pain are presented in Table \\n3.\\nPain in the local anaesthetic group and the standard group of patients\\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\",\n \"Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \\n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\\nOAA/S - observer's assessment of alertness/sedation.\",\n \"Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\",\n \"No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\",\n \"The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\",\n \"In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\",\n \"Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\",\n \"Ideally the study should have been placebo double-blinded to eliminate all biases. However, the cost of the elastomeric pump and wound catheter was 175 euro, so we were not able to perform a double-blind study. However, the cost of an elastomeric pump and wound catheter was not the only reason that precluded us from performing such a study. We had also ethical reasons for not carrying out a placebo-controlled study. Namely, a reconstruction with a tissue expander is a very painful procedure for the majority of patients, so we felt that it is not ethical to have a control group treated by placebo only. Instead, we offered our control group of patients the best available treatment, that is, intravenous analgesia. Based on the good results of our preliminary data on the use of continuous local anaesthetics for treatment of pain, our patients were willing to participate in our study because they were offered state-of-the-art pain treatment versus potentially even more effective pain treatment.\\nSub-pectoral breast augmentation and breast reconstruction with an expander or an implant are associated with considerable pain in the early postoperative period\\n[6,9,10,20-24]. Legeby et al.\\n[6] found that pain in the first 3 h after immediate breast reconstruction with a tissue expander was more severe than pain after mastectomy or axillary dissection. The mean VAS value for pain in the first 3 h after breast reconstruction was 4.9 and 5.0, if axillary dissection was also performed, whereas the values were 2.1 for mastectomy and 3.0 for mastectomy with axillary dissection\\n[6]. Similarly, after tissue-expander implantation, our standard analgesia group of patients also suffered pain in the recovery room, which was by about 3.0 VAS units larger than in patients who underwent breast surgery with axillary dissection without reconstruction, as reported by Strazisar et al.\\n[14]. The mean VAS score after immediate reconstruction was 4.9 in the standard analgesia group, whereas it was only 3.4 in the local anaesthetic group. In our previous study, the mean VAS score in recovery after breast cancer surgery with axillary dissection was 2.0 in the standard control group and 0.5 in the local anaesthetic group\\n[14]. In both our studies, the local anaesthetic reduced pain by 1.5 VAS units more than standard treatment alone.\\nThe most positive result of our study is the observation that 77% of patients treated with local anaesthetics did not experience severe pain. To our knowledge, there are no reports in the literature about similarly effective early postoperative analgesia after the implantation of a breast-tissue expander. Legeby et al. reported that even 48% of patients treated with local anaesthetics experienced unacceptably severe pain (VAS 4.7 to 10.0) in the early postoperative period\\n[6]. Legeby et al. used an epidural catheter with only a few holes at its tip and instilled local anaesthetic every 3 h. They probably did not establish an equal distribution of the local anaesthetic along the entire length of the muscle and the wound. On the other hand, we used quality wound-catheters that were perforated 15 cm at the distal end, which enabled a better distribution of the local anaesthetic. Furthermore, we used an elastomeric pump, which enabled a continuous infusion of the local anaesthetic. That is probably the reason why only seven patients (23.1%) in the test group reported pain above a VAS score of 5.0, whereas in the control group, such pain levels were reported by thirteen patients (42.9%).\\nLegeby et al. found that the use of opioids was about three times higher after the implantation of a tissue expander compared to other breast surgical procedures\\n[6]. After the tissue-expander implantation with or without axillary dissection, the consumption of opioids was 12.8 mg and 11.8 mg, whereas after mastectomy or mastectomy with axillary dissection, the mean consumption of opioids was 3.2 mg and 5.5 mg, respectively\\n[6]. Our patients with standard analgesia after the tissue-expander implantation needed 5.6 times more opioids than patients who underwent axillary dissection alone\\n[14]. Local anaesthetics can reduce consumption of opioids after mastectomy and axillary dissection\\n[11,13,14] and also after breast augmentation with implants\\n[7]. Our study shows that the use of continuous local anaesthesia can effectively reduce opioid consumption. Our patients treated with a local anaesthetic needed about three times less opioids than the standard analgesia group. Subsequently, patients from the local anaesthesia group experienced nausea less often, thus they had a lower consumption of metoclopramide than the standard analgesia group. Furthermore, the use of local anaesthetics resulted in less sedated and more alert patients in the recovery room and 6 h after the operation. The complication rate did not differ between the two groups of patients. This is in agreement with the reports of other authors who used local anaesthetics\\n[11-13,18,21,25-30]. Some authors report that local anaesthetics appear to reduce the incidence of inflammation\\n[31-33].\\nThere was no difference in pain on the first postoperative day between the study and control group of patients. It means that local anaesthesia was no longer as effective as it was on the day of surgery. A possible explanation is that muscle overstretching caused by a tissue expander is no longer as painful as it was. Other explanations are that in some patients the position of the wound catheter might have changed during movement or that there was a different perfusion region in the erect, prone or supine position, or that continuous infusion was flawed because of the wound drainage. Unfortunately, in our study we did not test a rescue bolus-injection of levobupivacaine in the case of severe pain. However, we have good experience with application of a rescue bolus of levobupivacaine in patients with neuropathic pain after axillary lymphadenectomy. Many times we have observed that injection of a bolus of 15 mL of 0.25% levobupivacaine into a drain and clamping of wound drainage inhibited neuropathic pain for a couple of hours.\\nMemory of severe postoperative pain is the most important risk factor for the development of chronic pain\\n[34]. Patients who were treated with patient-controlled analgesia had three times less chronic pain than the traditionally nurse-administered patients\\n[6]. The results of our study support this observation. Only 17% of patients treated with a local anaesthetic experienced chronic pain. This is significantly less than in the standard analgesia group of patients, of whom 50% suffered chronic pain. The proportion of patients treated with axillary lymphadenectomy, chemotherapy, hormone therapy and/or irradiation was similar in both study arms. Hence, we think that inadequately treated acute pain had an impact on the frequency of chronic pain in the standard analgesia group.\",\n \"Our current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia.\",\n \"BMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.\",\n \"The authors declare that they have no competing interests.\",\n \"BS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,"results",null,null,null,null,null,null,null,"discussion","conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Breast carcinoma","Primary reconstruction with tissue expander","Pain treatment","Wound infusion of local anaesthetic","Elastomeric pump"],"string":"[\n \"Breast carcinoma\",\n \"Primary reconstruction with tissue expander\",\n \"Pain treatment\",\n \"Wound infusion of local anaesthetic\",\n \"Elastomeric pump\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nBreast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.\n\nMethods:\nAltogether, 60 breast carcinoma patients were enrolled in the study protocol between January 2011 and May 2012: 72 patients who were eligible for inclusion were offered the opportunity to participate in our study during the recruitment period; 12 of them declined participation, and the remaining 60 were included in the study. Patients listed for mastectomy and primary reconstruction with a tissue expander because of the presence of breast carcinoma or for prophylactic mastectomy, were randomized during the routine preoperative anaesthetic assessment. Anaesthesia for the procedure was the same for all study subjects. We used the same protocol as already reported for patients with axillary dissection\n[14]. The first study arm was treated with continuous local analgesia, and the other (control) arm received standard intravenous analgesia. Allergies to local anaesthetics and chronic pre-operative opioid consumption were the criteria to exclude patients from our study.\nAfter informed consent was obtained the randomization was made by research nurses from the Clinical Research Unit of the Institute of Oncology Ljubljana. The research nurse performed randomization using random numbers generated by a computer and then informed the principal investigator about the treatment allocation of the patient.\nSurgical procedures were performed by ten experienced oncology surgeons and ten plastic surgeons. Sentinel lymph node biopsy was performed in 55 patients. In one patient with positive imprint cytology of the sentinel lymph node, axillary lymphadenectomy was performed immediately.\nThe study was reviewed by the The National Medical Ethics Committee of the Republic of Slovenia and performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. Our study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and conducted with the understanding and consent of all subjects involved. Study was entirely financed by the Institute of Oncology as a part of public service.\n Test group of patients Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\nBefore wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\n Control group of patients The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\nThe control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\n Both groups of patients Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\nPatient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\n Pain measurement Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\nData about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\n Statistical analysis In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\nIn order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\n\nTest group of patients:\nBefore wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\n\nControl group of patients:\nThe control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\n\nBoth groups of patients:\nPatient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\n\nPain measurement:\nData about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\n\nStatistical analysis:\nIn order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\n\nResults:\nThe mean age of patients was 47.8 years (range 25 to 64 y), height was 166 cm (range 153 to 178 cm), weight was 61.7 kg (range 45 to 85 kg), and the body mass index (BMI) was 22.4 (range 17 to 31.2). There were no significant differences between the study groups in BMI, American Society of Anesthesiology (ASA) score, comorbidities (Table \n1), type and length of surgical procedure, extent of lymph node dissection, adjuvant therapy, complications, or hospital stay (Table \n2).\nPatient characteristics\nASA, American Society of Anesthesiology; HER-2, human epidermal growth factor receptor-2; n, number of patients.\nTreatment of patients, adjuvant therapy, hospital stay and complications\nValues are numbers of patients unless stated otherwise.\n Acute pain Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\nData on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\n Opioid consumption Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\nConsumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\n Nausea Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\nPatients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\n Complications No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\nNo local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\n Hospital stay The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\nThe average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\n Unilateral and bilateral tissue expander In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\nIn order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\n Late complications (three months after primary reconstruction) Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\nThree months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\n\nAcute pain:\nData on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\n\nOpioid consumption:\nConsumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\n\nNausea:\nPatients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\n\nComplications:\nNo local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\n\nHospital stay:\nThe average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\n\nUnilateral and bilateral tissue expander:\nIn order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\n\nLate complications (three months after primary reconstruction):\nThree months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\n\nDiscussion:\nIdeally the study should have been placebo double-blinded to eliminate all biases. However, the cost of the elastomeric pump and wound catheter was 175 euro, so we were not able to perform a double-blind study. However, the cost of an elastomeric pump and wound catheter was not the only reason that precluded us from performing such a study. We had also ethical reasons for not carrying out a placebo-controlled study. Namely, a reconstruction with a tissue expander is a very painful procedure for the majority of patients, so we felt that it is not ethical to have a control group treated by placebo only. Instead, we offered our control group of patients the best available treatment, that is, intravenous analgesia. Based on the good results of our preliminary data on the use of continuous local anaesthetics for treatment of pain, our patients were willing to participate in our study because they were offered state-of-the-art pain treatment versus potentially even more effective pain treatment.\nSub-pectoral breast augmentation and breast reconstruction with an expander or an implant are associated with considerable pain in the early postoperative period\n[6,9,10,20-24]. Legeby et al.\n[6] found that pain in the first 3 h after immediate breast reconstruction with a tissue expander was more severe than pain after mastectomy or axillary dissection. The mean VAS value for pain in the first 3 h after breast reconstruction was 4.9 and 5.0, if axillary dissection was also performed, whereas the values were 2.1 for mastectomy and 3.0 for mastectomy with axillary dissection\n[6]. Similarly, after tissue-expander implantation, our standard analgesia group of patients also suffered pain in the recovery room, which was by about 3.0 VAS units larger than in patients who underwent breast surgery with axillary dissection without reconstruction, as reported by Strazisar et al.\n[14]. The mean VAS score after immediate reconstruction was 4.9 in the standard analgesia group, whereas it was only 3.4 in the local anaesthetic group. In our previous study, the mean VAS score in recovery after breast cancer surgery with axillary dissection was 2.0 in the standard control group and 0.5 in the local anaesthetic group\n[14]. In both our studies, the local anaesthetic reduced pain by 1.5 VAS units more than standard treatment alone.\nThe most positive result of our study is the observation that 77% of patients treated with local anaesthetics did not experience severe pain. To our knowledge, there are no reports in the literature about similarly effective early postoperative analgesia after the implantation of a breast-tissue expander. Legeby et al. reported that even 48% of patients treated with local anaesthetics experienced unacceptably severe pain (VAS 4.7 to 10.0) in the early postoperative period\n[6]. Legeby et al. used an epidural catheter with only a few holes at its tip and instilled local anaesthetic every 3 h. They probably did not establish an equal distribution of the local anaesthetic along the entire length of the muscle and the wound. On the other hand, we used quality wound-catheters that were perforated 15 cm at the distal end, which enabled a better distribution of the local anaesthetic. Furthermore, we used an elastomeric pump, which enabled a continuous infusion of the local anaesthetic. That is probably the reason why only seven patients (23.1%) in the test group reported pain above a VAS score of 5.0, whereas in the control group, such pain levels were reported by thirteen patients (42.9%).\nLegeby et al. found that the use of opioids was about three times higher after the implantation of a tissue expander compared to other breast surgical procedures\n[6]. After the tissue-expander implantation with or without axillary dissection, the consumption of opioids was 12.8 mg and 11.8 mg, whereas after mastectomy or mastectomy with axillary dissection, the mean consumption of opioids was 3.2 mg and 5.5 mg, respectively\n[6]. Our patients with standard analgesia after the tissue-expander implantation needed 5.6 times more opioids than patients who underwent axillary dissection alone\n[14]. Local anaesthetics can reduce consumption of opioids after mastectomy and axillary dissection\n[11,13,14] and also after breast augmentation with implants\n[7]. Our study shows that the use of continuous local anaesthesia can effectively reduce opioid consumption. Our patients treated with a local anaesthetic needed about three times less opioids than the standard analgesia group. Subsequently, patients from the local anaesthesia group experienced nausea less often, thus they had a lower consumption of metoclopramide than the standard analgesia group. Furthermore, the use of local anaesthetics resulted in less sedated and more alert patients in the recovery room and 6 h after the operation. The complication rate did not differ between the two groups of patients. This is in agreement with the reports of other authors who used local anaesthetics\n[11-13,18,21,25-30]. Some authors report that local anaesthetics appear to reduce the incidence of inflammation\n[31-33].\nThere was no difference in pain on the first postoperative day between the study and control group of patients. It means that local anaesthesia was no longer as effective as it was on the day of surgery. A possible explanation is that muscle overstretching caused by a tissue expander is no longer as painful as it was. Other explanations are that in some patients the position of the wound catheter might have changed during movement or that there was a different perfusion region in the erect, prone or supine position, or that continuous infusion was flawed because of the wound drainage. Unfortunately, in our study we did not test a rescue bolus-injection of levobupivacaine in the case of severe pain. However, we have good experience with application of a rescue bolus of levobupivacaine in patients with neuropathic pain after axillary lymphadenectomy. Many times we have observed that injection of a bolus of 15 mL of 0.25% levobupivacaine into a drain and clamping of wound drainage inhibited neuropathic pain for a couple of hours.\nMemory of severe postoperative pain is the most important risk factor for the development of chronic pain\n[34]. Patients who were treated with patient-controlled analgesia had three times less chronic pain than the traditionally nurse-administered patients\n[6]. The results of our study support this observation. Only 17% of patients treated with a local anaesthetic experienced chronic pain. This is significantly less than in the standard analgesia group of patients, of whom 50% suffered chronic pain. The proportion of patients treated with axillary lymphadenectomy, chemotherapy, hormone therapy and/or irradiation was similar in both study arms. Hence, we think that inadequately treated acute pain had an impact on the frequency of chronic pain in the standard analgesia group.\n\nConclusions:\nOur current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia.\n\nAbbreviations:\nBMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nBS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nImmediate breast reconstruction with an expander is a reasonable option for properly selected patients. After reconstruction, patients have severe postoperative pain, which responds poorly to opioids. Our aim was to evaluate if continuous wound infusion of a local anaesthetic into the surgical wound reduces postoperative pain, consumption of opioids and incidence of chronic pain compared to standard intravenous piritramide after primary breast reconstruction in breast carcinoma patients.\n\nMethods:\nAltogether, 60 patients were enrolled in our study; one half in the group with wound infusion of a local anaesthetic, and the other half in the standard (piritramide) group. Parameters measured included: pain intensity (visual analogue scale), drug requirements, alertness, hospitalisation, side-effects and late complications. A p-value of < 0.05 was considered statistically significant.\n\nResults:\nIn the recovery room, the test group reported less acute pain at rest (P = 0.03) and at activity (P = 0.01), and on the day of the surgical procedure they reported less pain at activity (P = 0.003). Consumption of piritramide and metoclopramide was lower in this group (P < 0.0001), but their alertness after the surgical procedure was higher compared to the standard group (P < 0.001). After three months, the test group reported less chronic pain (P = 0.01).\n\nConclusions:\nAfter primary tissue expander breast reconstruction, wound infusion of a local anaesthetic significantly reduces acute pain and enables reduced opioid consumption, resulting in less postoperative sedation and reduced need for antiemetic drugs. Wound infusion of a local anaesthetic reduces chronic pain."},"background_conclusion_text":{"kind":"string","value":"Background:\nBreast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.\n\nConclusions:\nOur current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nImmediate breast reconstruction with an expander is a reasonable option for properly selected patients. After reconstruction, patients have severe postoperative pain, which responds poorly to opioids. Our aim was to evaluate if continuous wound infusion of a local anaesthetic into the surgical wound reduces postoperative pain, consumption of opioids and incidence of chronic pain compared to standard intravenous piritramide after primary breast reconstruction in breast carcinoma patients.\n\nMethods:\nAltogether, 60 patients were enrolled in our study; one half in the group with wound infusion of a local anaesthetic, and the other half in the standard (piritramide) group. Parameters measured included: pain intensity (visual analogue scale), drug requirements, alertness, hospitalisation, side-effects and late complications. A p-value of < 0.05 was considered statistically significant.\n\nResults:\nIn the recovery room, the test group reported less acute pain at rest (P = 0.03) and at activity (P = 0.01), and on the day of the surgical procedure they reported less pain at activity (P = 0.003). Consumption of piritramide and metoclopramide was lower in this group (P < 0.0001), but their alertness after the surgical procedure was higher compared to the standard group (P < 0.001). After three months, the test group reported less chronic pain (P = 0.01).\n\nConclusions:\nAfter primary tissue expander breast reconstruction, wound infusion of a local anaesthetic significantly reduces acute pain and enables reduced opioid consumption, resulting in less postoperative sedation and reduced need for antiemetic drugs. Wound infusion of a local anaesthetic reduces chronic pain."},"whole_article_text_length":{"kind":"number","value":7759,"string":"7,759"},"whole_article_abstract_length":{"kind":"number","value":307,"string":"307"},"num_sections":{"kind":"number","value":20,"string":"20"},"most_frequent_words":{"kind":"list like","value":["patients","group","pain","local","control","test","surgical","reconstruction","wound","standard"],"string":"[\n \"patients\",\n \"group\",\n \"pain\",\n \"local\",\n \"control\",\n \"test\",\n \"surgical\",\n \"reconstruction\",\n \"wound\",\n \"standard\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] breast | local | use | mastectomy | pain | anaesthetics | local anaesthetics | breast reconstruction | postoperative pain | reconstruction [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | pain | catheter | wound | mg | subjects | ml | intravenous | fenestrated | day [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] group | patients | unilateral | bilateral | test | control group | test group | control | reconstruction | standard group [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients treated | surgical | pain | treated | lower | local anaesthetic | anaesthetic | surgical procedure | procedure | local [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] group | patients | pain | test | control | surgical | local | surgical procedure | standard | procedure [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] group | patients | pain | test | control | surgical | local | surgical procedure | standard | procedure [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 60 | one half | half ||| ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 0.03 | 0.01 | the day | 0.003 ||| ||| three months | 0.01 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| 60 | one half | half ||| ||| ||| ||| 0.03 | 0.01 | the day | 0.003 ||| ||| three months | 0.01 ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| 60 | one half | half ||| ||| ||| ||| 0.03 | 0.01 | the day | 0.003 ||| ||| three months | 0.01 ||| ||| [SUMMARY]"}}},{"rowIdx":276,"cells":{"title":{"kind":"string","value":"Lung transplantation as therapeutic option in acute respiratory distress syndrome for coronavirus disease 2019-related pulmonary fibrosis."},"pmid":{"kind":"string","value":"32251003"},"background_abstract":{"kind":"string","value":"Critical patients with the coronavirus disease 2019 (COVID-19), even those whose nucleic acid test results had turned negative and those receiving maximal medical support, have been noted to progress to irreversible fatal respiratory failure. Lung transplantation (LT) as the sole therapy for end-stage pulmonary fibrosis related to acute respiratory distress syndrome has been considered as the ultimate rescue therapy for these patients."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"From February 10 to March 10, 2020, three male patients were urgently assessed and listed for transplantation. After conducting a full ethical review and after obtaining assent from the family of the patients, we performed three LT procedures for COVID-19 patients with illness durations of more than one month and extremely high sequential organ failure assessment scores."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Two of the three recipients survived post-LT and started participating in a rehabilitation program. Pearls of the LT team collaboration and perioperative logistics were summarized and continually improved. The pathological results of the explanted lungs were concordant with the critical clinical manifestation, and provided insight towards better understanding of the disease. Government health affair systems, virology detection tools, and modern communication technology all play key roles towards the survival of the patients and their rehabilitation."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"LT can be performed in end-stage patients with respiratory failure due to COVID-19-related pulmonary fibrosis. If confirmed positive-turned-negative virology status without organ dysfunction that could contraindicate LT, LT provided the final option for these patients to avoid certain death, with proper protection of transplant surgeons and medical staffs. By ensuring instant seamless care for both patients and medical teams, the goal of reducing the mortality rate and salvaging the lives of patients with COVID-19 can be attained."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Betacoronavirus","COVID-19","Coronavirus Infections","Extracorporeal Membrane Oxygenation","Humans","Lung Transplantation","Male","Middle Aged","Pandemics","Pneumonia, Viral","Pulmonary Fibrosis","Respiratory Distress Syndrome","SARS-CoV-2"],"string":"[\n \"Aged\",\n \"Betacoronavirus\",\n \"COVID-19\",\n \"Coronavirus Infections\",\n \"Extracorporeal Membrane Oxygenation\",\n \"Humans\",\n \"Lung Transplantation\",\n \"Male\",\n \"Middle Aged\",\n \"Pandemics\",\n \"Pneumonia, Viral\",\n \"Pulmonary Fibrosis\",\n \"Respiratory Distress Syndrome\",\n \"SARS-CoV-2\"\n]"},"pmcid":{"kind":"string","value":"7339336"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"The outbreak of the coronavirus disease 2019 (COVID-19) caused more than 200,000 reported cases and more than 8000 people have lost their lives,[1] and thus, was declared a global pandemic by the World Health Organization.[2] As reported by Zhong et al,[3] 5.0% of 1099 patients were admitted to the intensive care unit (ICU) and 2.3% received invasive mechanical ventilation (MV). Publications have reported the general characteristics of disease progression with analysis of outcomes, including those of the recovered and mortality cases.[4] Some patients have prolonged ICU stays due to severe complications, such as acute respiratory distress syndrome (ARDS) and related pulmonary fibrosis, even after results of repeated virological tests are confirmed to be negative. Even with maximal support with MV and extra-corporeal membrane oxygenation (ECMO), such patients showed irreversible deterioration of pulmonary function. We performed lung transplantation (LT) for end-stage patients and hereby present the early results of LT in these recipients. Issues related to the urgent candidate listing of post-COVID-19 critical patients and peri-LT management are further discussed."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Ethical approval The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\nThe lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\n Patient information and data collection This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\nThis is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\n Construction of surgical facilities with high protection level In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\nIn view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" General characteristics before LT All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\nAll the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\n Pearls of peri-operative management All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\nAll the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\n Post-LT survival status and explant pathology Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.\nBoth patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Ethical approval","Patient information and data collection","Construction of surgical facilities with high protection level","General characteristics before LT","Pearls of peri-operative management","Post-LT survival status and explant pathology","Key points on determination of LT candidacy","Best practices for the protection of the medical team involved","Factors important for post-LT survival","Perspective","Funding"],"string":"[\n \"Ethical approval\",\n \"Patient information and data collection\",\n \"Construction of surgical facilities with high protection level\",\n \"General characteristics before LT\",\n \"Pearls of peri-operative management\",\n \"Post-LT survival status and explant pathology\",\n \"Key points on determination of LT candidacy\",\n \"Best practices for the protection of the medical team involved\",\n \"Factors important for post-LT survival\",\n \"Perspective\",\n \"Funding\"\n]"},"other_sections_texts":{"kind":"list like","value":["The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]","This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.","In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.","All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.","All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.","Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.","The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.","For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.","During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.","With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.","This study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City."],"string":"[\n \"The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\",\n \"This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\",\n \"In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\",\n \"All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\\nGeneral characteristics of the three urgently listed LT recipients.\",\n \"All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\",\n \"Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\\nRepresentative comparison of explant pathologies.\",\n \"The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\",\n \"For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\\nConsiderations and comparisons of surgical preparations.\\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\",\n \"During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\",\n \"With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\",\n \"This study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,"subjects",null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"subjects\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Ethical approval","Patient information and data collection","Construction of surgical facilities with high protection level","Results","General characteristics before LT","Pearls of peri-operative management","Post-LT survival status and explant pathology","Discussion","Best time to perform transplantation in critical patients","Key points on determination of LT candidacy","Best practices for the protection of the medical team involved","Factors important for post-LT survival","Perspective","Funding","Conflicts of interest"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Ethical approval\",\n \"Patient information and data collection\",\n \"Construction of surgical facilities with high protection level\",\n \"Results\",\n \"General characteristics before LT\",\n \"Pearls of peri-operative management\",\n \"Post-LT survival status and explant pathology\",\n \"Discussion\",\n \"Best time to perform transplantation in critical patients\",\n \"Key points on determination of LT candidacy\",\n \"Best practices for the protection of the medical team involved\",\n \"Factors important for post-LT survival\",\n \"Perspective\",\n \"Funding\",\n \"Conflicts of interest\"\n]"},"all_sections_texts":{"kind":"list like","value":["The outbreak of the coronavirus disease 2019 (COVID-19) caused more than 200,000 reported cases and more than 8000 people have lost their lives,[1] and thus, was declared a global pandemic by the World Health Organization.[2] As reported by Zhong et al,[3] 5.0% of 1099 patients were admitted to the intensive care unit (ICU) and 2.3% received invasive mechanical ventilation (MV). Publications have reported the general characteristics of disease progression with analysis of outcomes, including those of the recovered and mortality cases.[4] Some patients have prolonged ICU stays due to severe complications, such as acute respiratory distress syndrome (ARDS) and related pulmonary fibrosis, even after results of repeated virological tests are confirmed to be negative. Even with maximal support with MV and extra-corporeal membrane oxygenation (ECMO), such patients showed irreversible deterioration of pulmonary function. We performed lung transplantation (LT) for end-stage patients and hereby present the early results of LT in these recipients. Issues related to the urgent candidate listing of post-COVID-19 critical patients and peri-LT management are further discussed."," Ethical approval The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\nThe lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\n Patient information and data collection This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\nThis is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\n Construction of surgical facilities with high protection level In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\nIn view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.","The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]","This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.","In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems."," General characteristics before LT All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\nAll the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\n Pearls of peri-operative management All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\nAll the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\n Post-LT survival status and explant pathology Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.\nBoth patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.","All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.","All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.","Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies."," Best time to perform transplantation in critical patients This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\nThis is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\n Key points on determination of LT candidacy The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\nThe following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\n Best practices for the protection of the medical team involved For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\nFor the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\n Factors important for post-LT survival During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\nDuring the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\n Perspective With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\nWith careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.","This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.","The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.","For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.","During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.","With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.","This study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City.","None."],"string":"[\n \"The outbreak of the coronavirus disease 2019 (COVID-19) caused more than 200,000 reported cases and more than 8000 people have lost their lives,[1] and thus, was declared a global pandemic by the World Health Organization.[2] As reported by Zhong et al,[3] 5.0% of 1099 patients were admitted to the intensive care unit (ICU) and 2.3% received invasive mechanical ventilation (MV). Publications have reported the general characteristics of disease progression with analysis of outcomes, including those of the recovered and mortality cases.[4] Some patients have prolonged ICU stays due to severe complications, such as acute respiratory distress syndrome (ARDS) and related pulmonary fibrosis, even after results of repeated virological tests are confirmed to be negative. Even with maximal support with MV and extra-corporeal membrane oxygenation (ECMO), such patients showed irreversible deterioration of pulmonary function. We performed lung transplantation (LT) for end-stage patients and hereby present the early results of LT in these recipients. Issues related to the urgent candidate listing of post-COVID-19 critical patients and peri-LT management are further discussed.\",\n \" Ethical approval The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\\nThe lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\\n Patient information and data collection This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\\nThis is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\\n Construction of surgical facilities with high protection level In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\\nIn view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\",\n \"The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\",\n \"This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\",\n \"In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\",\n \" General characteristics before LT All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\\nGeneral characteristics of the three urgently listed LT recipients.\\nAll the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\\nGeneral characteristics of the three urgently listed LT recipients.\\n Pearls of peri-operative management All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\\nAll the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\\n Post-LT survival status and explant pathology Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\\nRepresentative comparison of explant pathologies.\\nBoth patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\\nRepresentative comparison of explant pathologies.\",\n \"All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\\nGeneral characteristics of the three urgently listed LT recipients.\",\n \"All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\",\n \"Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\\nRepresentative comparison of explant pathologies.\",\n \" Best time to perform transplantation in critical patients This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\\nThis is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\\n Key points on determination of LT candidacy The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\\nThe following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\\n Best practices for the protection of the medical team involved For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\\nConsiderations and comparisons of surgical preparations.\\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\\nFor the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\\nConsiderations and comparisons of surgical preparations.\\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\\n Factors important for post-LT survival During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\\nDuring the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\\n Perspective With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\\nWith careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\",\n \"This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\",\n \"The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\",\n \"For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\\nConsiderations and comparisons of surgical preparations.\\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\",\n \"During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\",\n \"With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\",\n \"This study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City.\",\n \"None.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,"subjects",null,"results",null,null,null,"discussion","subjects",null,null,null,null,null,"COI-statement"],"string":"[\n \"intro\",\n \"methods\",\n null,\n \"subjects\",\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n \"subjects\",\n null,\n null,\n null,\n null,\n null,\n \"COI-statement\"\n]"},"keywords":{"kind":"list like","value":["Coronavirus disease 2019","Lung transplantation","Acute respiratory distress syndrome","Pulmonary fibrosis","Sequential Organ Failure Assessment score"],"string":"[\n \"Coronavirus disease 2019\",\n \"Lung transplantation\",\n \"Acute respiratory distress syndrome\",\n \"Pulmonary fibrosis\",\n \"Sequential Organ Failure Assessment score\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nThe outbreak of the coronavirus disease 2019 (COVID-19) caused more than 200,000 reported cases and more than 8000 people have lost their lives,[1] and thus, was declared a global pandemic by the World Health Organization.[2] As reported by Zhong et al,[3] 5.0% of 1099 patients were admitted to the intensive care unit (ICU) and 2.3% received invasive mechanical ventilation (MV). Publications have reported the general characteristics of disease progression with analysis of outcomes, including those of the recovered and mortality cases.[4] Some patients have prolonged ICU stays due to severe complications, such as acute respiratory distress syndrome (ARDS) and related pulmonary fibrosis, even after results of repeated virological tests are confirmed to be negative. Even with maximal support with MV and extra-corporeal membrane oxygenation (ECMO), such patients showed irreversible deterioration of pulmonary function. We performed lung transplantation (LT) for end-stage patients and hereby present the early results of LT in these recipients. Issues related to the urgent candidate listing of post-COVID-19 critical patients and peri-LT management are further discussed.\n\nMethods:\n Ethical approval The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\nThe lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\n Patient information and data collection This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\nThis is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\n Construction of surgical facilities with high protection level In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\nIn view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\n\nEthical approval:\nThe lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\n\nPatient information and data collection:\nThis is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\n\nConstruction of surgical facilities with high protection level:\nIn view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\n\nResults:\n General characteristics before LT All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\nAll the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\n Pearls of peri-operative management All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\nAll the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\n Post-LT survival status and explant pathology Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.\nBoth patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.\n\nGeneral characteristics before LT:\nAll the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\n\nPearls of peri-operative management:\nAll the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\n\nPost-LT survival status and explant pathology:\nBoth patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.\n\nDiscussion:\n Best time to perform transplantation in critical patients This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\nThis is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\n Key points on determination of LT candidacy The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\nThe following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\n Best practices for the protection of the medical team involved For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\nFor the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\n Factors important for post-LT survival During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\nDuring the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\n Perspective With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\nWith careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\n\nBest time to perform transplantation in critical patients:\nThis is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\n\nKey points on determination of LT candidacy:\nThe following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\n\nBest practices for the protection of the medical team involved:\nFor the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\n\nFactors important for post-LT survival:\nDuring the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\n\nPerspective:\nWith careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\n\nFunding:\nThis study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City.\n\nConflicts of interest:\nNone.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nCritical patients with the coronavirus disease 2019 (COVID-19), even those whose nucleic acid test results had turned negative and those receiving maximal medical support, have been noted to progress to irreversible fatal respiratory failure. Lung transplantation (LT) as the sole therapy for end-stage pulmonary fibrosis related to acute respiratory distress syndrome has been considered as the ultimate rescue therapy for these patients.\n\nMethods:\nFrom February 10 to March 10, 2020, three male patients were urgently assessed and listed for transplantation. After conducting a full ethical review and after obtaining assent from the family of the patients, we performed three LT procedures for COVID-19 patients with illness durations of more than one month and extremely high sequential organ failure assessment scores.\n\nResults:\nTwo of the three recipients survived post-LT and started participating in a rehabilitation program. Pearls of the LT team collaboration and perioperative logistics were summarized and continually improved. The pathological results of the explanted lungs were concordant with the critical clinical manifestation, and provided insight towards better understanding of the disease. Government health affair systems, virology detection tools, and modern communication technology all play key roles towards the survival of the patients and their rehabilitation.\n\nConclusions:\nLT can be performed in end-stage patients with respiratory failure due to COVID-19-related pulmonary fibrosis. If confirmed positive-turned-negative virology status without organ dysfunction that could contraindicate LT, LT provided the final option for these patients to avoid certain death, with proper protection of transplant surgeons and medical staffs. By ensuring instant seamless care for both patients and medical teams, the goal of reducing the mortality rate and salvaging the lives of patients with COVID-19 can be attained."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":10633,"string":"10,633"},"whole_article_abstract_length":{"kind":"number","value":324,"string":"324"},"num_sections":{"kind":"number","value":17,"string":"17"},"most_frequent_words":{"kind":"list like","value":["lt","patients","patient","post","lung","covid","covid 19","19","negative","team"],"string":"[\n \"lt\",\n \"patients\",\n \"patient\",\n \"post\",\n \"lung\",\n \"covid\",\n \"covid 19\",\n \"19\",\n \"negative\",\n \"team\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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of the ageing process. Complete programme evaluations are a necessary prerequisite to continuous quality improvements. Being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly in order to support its continuous improvement is, therefore, crucial. Thus, the aim of this study was to develop a self-assessment tool for PA programmes for the elderly."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A 3-round Delphi process was conducted via the Internet with 43 national experts in PA for the elderly, management and delivery of PA programmes for the elderly, sports management, quality management and gerontology, asking experts to identify the propositions that they considered relevant for inclusion in the self-assessment tool. Experts reviewed a list of proposed statements, based on the criteria and sub-criteria from the European Foundation for Quality Management Excellence Model (EFQM) and PA guidelines for older adults and rated each proposition from 1 to 8 (disagree to agree) and modified and/or added propositions. Propositions receiving either bottom or top scores of greater than 70% were considered to have achieved consensus to drop or retain, respectively."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93. In round 2, a total of 104 propositions were presented, of which experts modified 39 and achieved consensus on 53. In the last round, of 51 proposed statements, the experts achieved consensus on 19. After 3 rounds of rating, experts had not achieved consensus on 32 propositions. The resulting tool consisted of 165 statements that assess nine management areas involved in the development of PA programmes for the elderly."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Based on experts' opinions, a self-assessment tool was found in order to access quality of PA programmes for the elderly. Information obtained with evaluations would be useful to organizations seeking to improve their services, customer satisfaction and, consequently, adherence to PA programmes, targeting the ageing population."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Aging","Consensus","Delivery of Health Care","Delphi Technique","Europe","Exercise","Geriatrics","Health Promotion","Humans","Internet","Practice Guidelines as Topic","Program Evaluation","Quality Control","Sports"],"string":"[\n \"Aged\",\n \"Aging\",\n \"Consensus\",\n \"Delivery of Health Care\",\n \"Delphi Technique\",\n \"Europe\",\n \"Exercise\",\n \"Geriatrics\",\n \"Health Promotion\",\n \"Humans\",\n \"Internet\",\n \"Practice Guidelines as Topic\",\n \"Program Evaluation\",\n \"Quality Control\",\n \"Sports\"\n]"},"pmcid":{"kind":"string","value":"3278362"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"A modified Delphi process was conducted using the Internet, from October 2009 to September 2010. The Delphi technique was developed in the 1950s by scientists at the Rand Corporation as a method of making informed decisions based on expert opinion [23]. Since then, it has been used to clarify a variety of problems in different sectors [24-29]. Despite having undergone some modifications, it remains a viable approach for gathering expert opinions through a structured iterative process that builds consensus [30]. This process involves multiple interactions with participants who usually complete two or more rounds in a reasonable amount of time [31] - even when participants are in geographically-distinct locations, since rounds can be conducted by mail or email [32,33]. The results of previous iterations can be modified by participants in later iterations, as they are able to review comments and feedbacks provided by other experts in earlier rounds [31]. Furthermore, the Delphi technique offers a number of specific advantages and is particularly helpful because it avoids the barriers commonly observed in other group discussions, such as interpersonal influence, time pressure and group demands [31,34,35]. This is due to the fact that respondents are not aware of the identities of other respondents and are, therefore, freed of personal and social constraints [30]. They are also able to complete the Delphi rounds in ways that suit them best because they participate in the rounds asynchronously [36]. The Delphi technique is also advantageous because a variety of statistical analysis techniques can be used to interpret the data its generates [37].\nThe Delphi process was conducted in three rounds [38,39] (Figure 1). Following each step listed in the previous figure, our main question was: Which quality practices must be included in a quality self-assessment tool for PA programmes for the elderly?\nSteps of the modified Delphi process used in the present study.\nUsing criteria and sub-criteria from the EFQM Excellence Model [13] and PA guidelines for older adults [3,40] as a starting point, we reviewed the literature to identify best practice principles and generate a list of statements. Our review was undertaken using PubMed (1980-2010), B-On (1980-2010), and Google™. We searched a variety of combinations of key words related to PA programmes for the elderly, quality management and the EFQM Excellence Model, such as: 'evaluation', 'guidelines', 'recommendations', 'exercise', 'physical activity', 'programmes', 'elderly', 'old', 'review', 'framework', 'EFQM', 'assess' and 'quality'.\nAfter identifying a list of statements, an online questionnaire was developed and tested with 5 PA programme coordinators for comments on readability and functionality. Some adjustments were made to make the affirmations included in the questionnaire clearer and more relevant to this case. We established that statements that received greater than 70% of experts' votes had achieved consensus [41-43] in both the bottom scores (i.e., reached consensus to drop) and top scores (i.e., reached consensus to include/retain). Statements that were dropped were not included in subsequent rounds of ratings. The remaining items were included in the next rounds, until a consensus was achieved to either drop or retain. At the end of three rounds, the statements on which experts had not reached consensus were also not included in the output list.\nThe fourth phase of the process involved nominating experts to participate in the Delphi rounds. National experts in research on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology were identified. Our decisions were based on expertise or/and breadth of scientific work [44]. The DeGóis Curricula Platform1 assisted us in this process. A list of 63 potential participants was generated, along with key contacts for each. This group included 34 PhD scientists and academics (11 in PA for the elderly, 4 in sports management, 18 in quality management and 1 in gerontology), 3 non-PhD academics (1 in PA for the elderly and 2 in sports management) and 26 senior technicians (22 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). Previous information containing details about the EFQM Excellence Model, the Delphi process and the purpose of our study was provided. Of those invited to participate, 5 did not respond and 3 declined, due to lack of time (all PhD scientists and academics in quality management). Thus, 55 experts (30 females and 25 males) responded to our initial invitation and agreed to participate. Those who accepted our invitation were informed that they were required to respond to three online rounds of ratings.\nThe rounds were performed using Survey Monkey, a web-based survey and data collection system. In every round, participants were asked to rate their level of agreement with each proposition, from 1 to 8 ('strongly disagree' to 'strongly agree'), suggest modifications to proposed definitions and/or add propositions that would be useful in a quality self-assessment tool for PA programmes for the elderly. The 8-point Likert scale was selected to bring out more variability in responses [45]. After each round, the frequency and mean of the panel's ratings and the percentage of scores ≥ 7 were calculated. Based on this data, a new questionnaire was designed and placed online for the next round. We asked participants to review all the information sent and re-rate each statement.\nAfter round 3, we gathered all our data and developed a list of statements that did and did not reach consensus."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"1 It is an instrument for gathering, supplying and analyze the intellectual and scientific production of the Portuguese researchers."},"other_sections_titles":{"kind":"list like","value":["Background","Results","Discussion","Strengths and Limitations","Conclusion"],"string":"[\n \"Background\",\n \"Results\",\n \"Discussion\",\n \"Strengths and Limitations\",\n \"Conclusion\"\n]"},"other_sections_texts":{"kind":"list like","value":["Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.","Eight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\nResults of the three rounds by criterion\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\" to \"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).","The main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\" and \"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\nThroughout the Delphi process, it was suggested that the proposition \"The programme involves a multidisciplinary team of professionals\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \"external partnerships\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\" and \"Regular and formal communication procedures are established with partners\".\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \"Market research is used to determine the needs and expectations of future customers\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \"The programme has measures of perception and/or performance indicators regarding employees' performance\" and \"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.","To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.","Our Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results)."],"string":"[\n \"Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.\",\n \"Eight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\\nResults of the three rounds by criterion\\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \\\"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\\\" to \\\"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\\\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\",\n \"The main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \\\"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\\\" and \\\"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\\\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\\nThroughout the Delphi process, it was suggested that the proposition \\\"The programme involves a multidisciplinary team of professionals\\\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \\\"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\\\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \\\"external partnerships\\\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \\\"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\\\" and \\\"Regular and formal communication procedures are established with partners\\\".\\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \\\"Market research is used to determine the needs and expectations of future customers\\\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \\\"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\\\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \\\"The programme has measures of perception and/or performance indicators regarding employees' performance\\\" and \\\"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\\\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\",\n \"To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\",\n \"Our Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Results","Discussion","Strengths and Limitations","Conclusion","Supplementary Material"],"string":"[\n \"Background\",\n \"Methods\",\n \"Results\",\n \"Discussion\",\n \"Strengths and Limitations\",\n \"Conclusion\",\n \"Supplementary Material\"\n]"},"all_sections_texts":{"kind":"list like","value":["Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.","A modified Delphi process was conducted using the Internet, from October 2009 to September 2010. The Delphi technique was developed in the 1950s by scientists at the Rand Corporation as a method of making informed decisions based on expert opinion [23]. Since then, it has been used to clarify a variety of problems in different sectors [24-29]. Despite having undergone some modifications, it remains a viable approach for gathering expert opinions through a structured iterative process that builds consensus [30]. This process involves multiple interactions with participants who usually complete two or more rounds in a reasonable amount of time [31] - even when participants are in geographically-distinct locations, since rounds can be conducted by mail or email [32,33]. The results of previous iterations can be modified by participants in later iterations, as they are able to review comments and feedbacks provided by other experts in earlier rounds [31]. Furthermore, the Delphi technique offers a number of specific advantages and is particularly helpful because it avoids the barriers commonly observed in other group discussions, such as interpersonal influence, time pressure and group demands [31,34,35]. This is due to the fact that respondents are not aware of the identities of other respondents and are, therefore, freed of personal and social constraints [30]. They are also able to complete the Delphi rounds in ways that suit them best because they participate in the rounds asynchronously [36]. The Delphi technique is also advantageous because a variety of statistical analysis techniques can be used to interpret the data its generates [37].\nThe Delphi process was conducted in three rounds [38,39] (Figure 1). Following each step listed in the previous figure, our main question was: Which quality practices must be included in a quality self-assessment tool for PA programmes for the elderly?\nSteps of the modified Delphi process used in the present study.\nUsing criteria and sub-criteria from the EFQM Excellence Model [13] and PA guidelines for older adults [3,40] as a starting point, we reviewed the literature to identify best practice principles and generate a list of statements. Our review was undertaken using PubMed (1980-2010), B-On (1980-2010), and Google™. We searched a variety of combinations of key words related to PA programmes for the elderly, quality management and the EFQM Excellence Model, such as: 'evaluation', 'guidelines', 'recommendations', 'exercise', 'physical activity', 'programmes', 'elderly', 'old', 'review', 'framework', 'EFQM', 'assess' and 'quality'.\nAfter identifying a list of statements, an online questionnaire was developed and tested with 5 PA programme coordinators for comments on readability and functionality. Some adjustments were made to make the affirmations included in the questionnaire clearer and more relevant to this case. We established that statements that received greater than 70% of experts' votes had achieved consensus [41-43] in both the bottom scores (i.e., reached consensus to drop) and top scores (i.e., reached consensus to include/retain). Statements that were dropped were not included in subsequent rounds of ratings. The remaining items were included in the next rounds, until a consensus was achieved to either drop or retain. At the end of three rounds, the statements on which experts had not reached consensus were also not included in the output list.\nThe fourth phase of the process involved nominating experts to participate in the Delphi rounds. National experts in research on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology were identified. Our decisions were based on expertise or/and breadth of scientific work [44]. The DeGóis Curricula Platform1 assisted us in this process. A list of 63 potential participants was generated, along with key contacts for each. This group included 34 PhD scientists and academics (11 in PA for the elderly, 4 in sports management, 18 in quality management and 1 in gerontology), 3 non-PhD academics (1 in PA for the elderly and 2 in sports management) and 26 senior technicians (22 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). Previous information containing details about the EFQM Excellence Model, the Delphi process and the purpose of our study was provided. Of those invited to participate, 5 did not respond and 3 declined, due to lack of time (all PhD scientists and academics in quality management). Thus, 55 experts (30 females and 25 males) responded to our initial invitation and agreed to participate. Those who accepted our invitation were informed that they were required to respond to three online rounds of ratings.\nThe rounds were performed using Survey Monkey, a web-based survey and data collection system. In every round, participants were asked to rate their level of agreement with each proposition, from 1 to 8 ('strongly disagree' to 'strongly agree'), suggest modifications to proposed definitions and/or add propositions that would be useful in a quality self-assessment tool for PA programmes for the elderly. The 8-point Likert scale was selected to bring out more variability in responses [45]. After each round, the frequency and mean of the panel's ratings and the percentage of scores ≥ 7 were calculated. Based on this data, a new questionnaire was designed and placed online for the next round. We asked participants to review all the information sent and re-rate each statement.\nAfter round 3, we gathered all our data and developed a list of statements that did and did not reach consensus.","Eight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\nResults of the three rounds by criterion\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\" to \"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).","The main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\" and \"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\nThroughout the Delphi process, it was suggested that the proposition \"The programme involves a multidisciplinary team of professionals\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \"external partnerships\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\" and \"Regular and formal communication procedures are established with partners\".\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \"Market research is used to determine the needs and expectations of future customers\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \"The programme has measures of perception and/or performance indicators regarding employees' performance\" and \"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.","To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.","Our Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).","Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors). the file presents the resulting tool - named Q-STEPS - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly.\nClick here for file"],"string":"[\n \"Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.\",\n \"A modified Delphi process was conducted using the Internet, from October 2009 to September 2010. The Delphi technique was developed in the 1950s by scientists at the Rand Corporation as a method of making informed decisions based on expert opinion [23]. Since then, it has been used to clarify a variety of problems in different sectors [24-29]. Despite having undergone some modifications, it remains a viable approach for gathering expert opinions through a structured iterative process that builds consensus [30]. This process involves multiple interactions with participants who usually complete two or more rounds in a reasonable amount of time [31] - even when participants are in geographically-distinct locations, since rounds can be conducted by mail or email [32,33]. The results of previous iterations can be modified by participants in later iterations, as they are able to review comments and feedbacks provided by other experts in earlier rounds [31]. Furthermore, the Delphi technique offers a number of specific advantages and is particularly helpful because it avoids the barriers commonly observed in other group discussions, such as interpersonal influence, time pressure and group demands [31,34,35]. This is due to the fact that respondents are not aware of the identities of other respondents and are, therefore, freed of personal and social constraints [30]. They are also able to complete the Delphi rounds in ways that suit them best because they participate in the rounds asynchronously [36]. The Delphi technique is also advantageous because a variety of statistical analysis techniques can be used to interpret the data its generates [37].\\nThe Delphi process was conducted in three rounds [38,39] (Figure 1). Following each step listed in the previous figure, our main question was: Which quality practices must be included in a quality self-assessment tool for PA programmes for the elderly?\\nSteps of the modified Delphi process used in the present study.\\nUsing criteria and sub-criteria from the EFQM Excellence Model [13] and PA guidelines for older adults [3,40] as a starting point, we reviewed the literature to identify best practice principles and generate a list of statements. Our review was undertaken using PubMed (1980-2010), B-On (1980-2010), and Google™. We searched a variety of combinations of key words related to PA programmes for the elderly, quality management and the EFQM Excellence Model, such as: 'evaluation', 'guidelines', 'recommendations', 'exercise', 'physical activity', 'programmes', 'elderly', 'old', 'review', 'framework', 'EFQM', 'assess' and 'quality'.\\nAfter identifying a list of statements, an online questionnaire was developed and tested with 5 PA programme coordinators for comments on readability and functionality. Some adjustments were made to make the affirmations included in the questionnaire clearer and more relevant to this case. We established that statements that received greater than 70% of experts' votes had achieved consensus [41-43] in both the bottom scores (i.e., reached consensus to drop) and top scores (i.e., reached consensus to include/retain). Statements that were dropped were not included in subsequent rounds of ratings. The remaining items were included in the next rounds, until a consensus was achieved to either drop or retain. At the end of three rounds, the statements on which experts had not reached consensus were also not included in the output list.\\nThe fourth phase of the process involved nominating experts to participate in the Delphi rounds. National experts in research on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology were identified. Our decisions were based on expertise or/and breadth of scientific work [44]. The DeGóis Curricula Platform1 assisted us in this process. A list of 63 potential participants was generated, along with key contacts for each. This group included 34 PhD scientists and academics (11 in PA for the elderly, 4 in sports management, 18 in quality management and 1 in gerontology), 3 non-PhD academics (1 in PA for the elderly and 2 in sports management) and 26 senior technicians (22 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). Previous information containing details about the EFQM Excellence Model, the Delphi process and the purpose of our study was provided. Of those invited to participate, 5 did not respond and 3 declined, due to lack of time (all PhD scientists and academics in quality management). Thus, 55 experts (30 females and 25 males) responded to our initial invitation and agreed to participate. Those who accepted our invitation were informed that they were required to respond to three online rounds of ratings.\\nThe rounds were performed using Survey Monkey, a web-based survey and data collection system. In every round, participants were asked to rate their level of agreement with each proposition, from 1 to 8 ('strongly disagree' to 'strongly agree'), suggest modifications to proposed definitions and/or add propositions that would be useful in a quality self-assessment tool for PA programmes for the elderly. The 8-point Likert scale was selected to bring out more variability in responses [45]. After each round, the frequency and mean of the panel's ratings and the percentage of scores ≥ 7 were calculated. Based on this data, a new questionnaire was designed and placed online for the next round. We asked participants to review all the information sent and re-rate each statement.\\nAfter round 3, we gathered all our data and developed a list of statements that did and did not reach consensus.\",\n \"Eight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\\nResults of the three rounds by criterion\\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \\\"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\\\" to \\\"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\\\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\",\n \"The main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \\\"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\\\" and \\\"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\\\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\\nThroughout the Delphi process, it was suggested that the proposition \\\"The programme involves a multidisciplinary team of professionals\\\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \\\"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\\\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \\\"external partnerships\\\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \\\"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\\\" and \\\"Regular and formal communication procedures are established with partners\\\".\\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \\\"Market research is used to determine the needs and expectations of future customers\\\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \\\"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\\\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \\\"The programme has measures of perception and/or performance indicators regarding employees' performance\\\" and \\\"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\\\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\",\n \"To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\",\n \"Our Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\",\n \"Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors). the file presents the resulting tool - named Q-STEPS - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly.\\nClick here for file\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,"supplementary-material"],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["physical activity","programmes","elderly","tool","evaluation","quality","adherence"],"string":"[\n \"physical activity\",\n \"programmes\",\n \"elderly\",\n \"tool\",\n \"evaluation\",\n \"quality\",\n \"adherence\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nPhysical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.\n\nMethods:\nA modified Delphi process was conducted using the Internet, from October 2009 to September 2010. The Delphi technique was developed in the 1950s by scientists at the Rand Corporation as a method of making informed decisions based on expert opinion [23]. Since then, it has been used to clarify a variety of problems in different sectors [24-29]. Despite having undergone some modifications, it remains a viable approach for gathering expert opinions through a structured iterative process that builds consensus [30]. This process involves multiple interactions with participants who usually complete two or more rounds in a reasonable amount of time [31] - even when participants are in geographically-distinct locations, since rounds can be conducted by mail or email [32,33]. The results of previous iterations can be modified by participants in later iterations, as they are able to review comments and feedbacks provided by other experts in earlier rounds [31]. Furthermore, the Delphi technique offers a number of specific advantages and is particularly helpful because it avoids the barriers commonly observed in other group discussions, such as interpersonal influence, time pressure and group demands [31,34,35]. This is due to the fact that respondents are not aware of the identities of other respondents and are, therefore, freed of personal and social constraints [30]. They are also able to complete the Delphi rounds in ways that suit them best because they participate in the rounds asynchronously [36]. The Delphi technique is also advantageous because a variety of statistical analysis techniques can be used to interpret the data its generates [37].\nThe Delphi process was conducted in three rounds [38,39] (Figure 1). Following each step listed in the previous figure, our main question was: Which quality practices must be included in a quality self-assessment tool for PA programmes for the elderly?\nSteps of the modified Delphi process used in the present study.\nUsing criteria and sub-criteria from the EFQM Excellence Model [13] and PA guidelines for older adults [3,40] as a starting point, we reviewed the literature to identify best practice principles and generate a list of statements. Our review was undertaken using PubMed (1980-2010), B-On (1980-2010), and Google™. We searched a variety of combinations of key words related to PA programmes for the elderly, quality management and the EFQM Excellence Model, such as: 'evaluation', 'guidelines', 'recommendations', 'exercise', 'physical activity', 'programmes', 'elderly', 'old', 'review', 'framework', 'EFQM', 'assess' and 'quality'.\nAfter identifying a list of statements, an online questionnaire was developed and tested with 5 PA programme coordinators for comments on readability and functionality. Some adjustments were made to make the affirmations included in the questionnaire clearer and more relevant to this case. We established that statements that received greater than 70% of experts' votes had achieved consensus [41-43] in both the bottom scores (i.e., reached consensus to drop) and top scores (i.e., reached consensus to include/retain). Statements that were dropped were not included in subsequent rounds of ratings. The remaining items were included in the next rounds, until a consensus was achieved to either drop or retain. At the end of three rounds, the statements on which experts had not reached consensus were also not included in the output list.\nThe fourth phase of the process involved nominating experts to participate in the Delphi rounds. National experts in research on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology were identified. Our decisions were based on expertise or/and breadth of scientific work [44]. The DeGóis Curricula Platform1 assisted us in this process. A list of 63 potential participants was generated, along with key contacts for each. This group included 34 PhD scientists and academics (11 in PA for the elderly, 4 in sports management, 18 in quality management and 1 in gerontology), 3 non-PhD academics (1 in PA for the elderly and 2 in sports management) and 26 senior technicians (22 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). Previous information containing details about the EFQM Excellence Model, the Delphi process and the purpose of our study was provided. Of those invited to participate, 5 did not respond and 3 declined, due to lack of time (all PhD scientists and academics in quality management). Thus, 55 experts (30 females and 25 males) responded to our initial invitation and agreed to participate. Those who accepted our invitation were informed that they were required to respond to three online rounds of ratings.\nThe rounds were performed using Survey Monkey, a web-based survey and data collection system. In every round, participants were asked to rate their level of agreement with each proposition, from 1 to 8 ('strongly disagree' to 'strongly agree'), suggest modifications to proposed definitions and/or add propositions that would be useful in a quality self-assessment tool for PA programmes for the elderly. The 8-point Likert scale was selected to bring out more variability in responses [45]. After each round, the frequency and mean of the panel's ratings and the percentage of scores ≥ 7 were calculated. Based on this data, a new questionnaire was designed and placed online for the next round. We asked participants to review all the information sent and re-rate each statement.\nAfter round 3, we gathered all our data and developed a list of statements that did and did not reach consensus.\n\nResults:\nEight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\nResults of the three rounds by criterion\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\" to \"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\n\nDiscussion:\nThe main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\" and \"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\nThroughout the Delphi process, it was suggested that the proposition \"The programme involves a multidisciplinary team of professionals\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \"external partnerships\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\" and \"Regular and formal communication procedures are established with partners\".\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \"Market research is used to determine the needs and expectations of future customers\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \"The programme has measures of perception and/or performance indicators regarding employees' performance\" and \"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\n\nStrengths and Limitations:\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\n\nConclusion:\nOur Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\n\nSupplementary Material:\nQ-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors). the file presents the resulting tool - named Q-STEPS - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly.\nClick here for file\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThere has been a growing concern in designing physical activity (PA) programmes for elderly people, since evidence suggests that such health promotion interventions may reduce the deleterious effects of the ageing process. Complete programme evaluations are a necessary prerequisite to continuous quality improvements. Being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly in order to support its continuous improvement is, therefore, crucial. Thus, the aim of this study was to develop a self-assessment tool for PA programmes for the elderly.\n\nMethods:\nA 3-round Delphi process was conducted via the Internet with 43 national experts in PA for the elderly, management and delivery of PA programmes for the elderly, sports management, quality management and gerontology, asking experts to identify the propositions that they considered relevant for inclusion in the self-assessment tool. Experts reviewed a list of proposed statements, based on the criteria and sub-criteria from the European Foundation for Quality Management Excellence Model (EFQM) and PA guidelines for older adults and rated each proposition from 1 to 8 (disagree to agree) and modified and/or added propositions. Propositions receiving either bottom or top scores of greater than 70% were considered to have achieved consensus to drop or retain, respectively.\n\nResults:\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93. In round 2, a total of 104 propositions were presented, of which experts modified 39 and achieved consensus on 53. In the last round, of 51 proposed statements, the experts achieved consensus on 19. After 3 rounds of rating, experts had not achieved consensus on 32 propositions. The resulting tool consisted of 165 statements that assess nine management areas involved in the development of PA programmes for the elderly.\n\nConclusions:\nBased on experts' opinions, a self-assessment tool was found in order to access quality of PA programmes for the elderly. Information obtained with evaluations would be useful to organizations seeking to improve their services, customer satisfaction and, consequently, adherence to PA programmes, targeting the ageing population."},"background_conclusion_text":{"kind":"string","value":"Background:\nPhysical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.\n\nConclusion:\n1 It is an instrument for gathering, supplying and analyze the intellectual and scientific production of the Portuguese researchers."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThere has been a growing concern in designing physical activity (PA) programmes for elderly people, since evidence suggests that such health promotion interventions may reduce the deleterious effects of the ageing process. Complete programme evaluations are a necessary prerequisite to continuous quality improvements. Being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly in order to support its continuous improvement is, therefore, crucial. Thus, the aim of this study was to develop a self-assessment tool for PA programmes for the elderly.\n\nMethods:\nA 3-round Delphi process was conducted via the Internet with 43 national experts in PA for the elderly, management and delivery of PA programmes for the elderly, sports management, quality management and gerontology, asking experts to identify the propositions that they considered relevant for inclusion in the self-assessment tool. Experts reviewed a list of proposed statements, based on the criteria and sub-criteria from the European Foundation for Quality Management Excellence Model (EFQM) and PA guidelines for older adults and rated each proposition from 1 to 8 (disagree to agree) and modified and/or added propositions. Propositions receiving either bottom or top scores of greater than 70% were considered to have achieved consensus to drop or retain, respectively.\n\nResults:\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93. In round 2, a total of 104 propositions were presented, of which experts modified 39 and achieved consensus on 53. In the last round, of 51 proposed statements, the experts achieved consensus on 19. After 3 rounds of rating, experts had not achieved consensus on 32 propositions. The resulting tool consisted of 165 statements that assess nine management areas involved in the development of PA programmes for the elderly.\n\nConclusions:\nBased on experts' opinions, a self-assessment tool was found in order to access quality of PA programmes for the elderly. Information obtained with evaluations would be useful to organizations seeking to improve their services, customer satisfaction and, consequently, adherence to PA programmes, targeting the ageing population."},"whole_article_text_length":{"kind":"number","value":5170,"string":"5,170"},"whole_article_abstract_length":{"kind":"number","value":421,"string":"421"},"num_sections":{"kind":"number","value":7,"string":"7"},"most_frequent_words":{"kind":"list like","value":["programmes","pa","quality","consensus","elderly","pa programmes","results","experts","management","statements"],"string":"[\n \"programmes\",\n \"pa\",\n \"quality\",\n \"consensus\",\n \"elderly\",\n \"pa programmes\",\n \"results\",\n \"experts\",\n \"management\",\n \"statements\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] health | programmes | quality | important | pa | elderly | pa programmes | improve | satisfaction | efqm [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] rounds | delphi | management | list | included | participants | consensus | pa | process | quality [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] results | criteria assess | people | quality | programmes | assess | criteria | essential assessments quality pa | tool assesses areas | essential assessments quality [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | results | elderly | consensus | pa programmes | experts | statements | tool [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | results | elderly | consensus | pa programmes | experts | statements | tool [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 3 | Delphi | 43 | PA ||| the European Foundation for Quality Management Excellence Model | PA | 1 to 8 ||| greater than 70% [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| ||| 3 | Delphi | 43 | PA ||| the European Foundation for Quality Management Excellence Model | PA | 1 to 8 ||| greater than 70% ||| ||| 1 | 196 | 41 | 1 | 93 ||| 2 | 104 | 39 | 53 ||| 51 | 19 ||| 3 | 32 ||| 165 | nine ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| ||| 3 | Delphi | 43 | PA ||| the European Foundation for Quality Management Excellence Model | PA | 1 to 8 ||| greater than 70% ||| ||| 1 | 196 | 41 | 1 | 93 ||| 2 | 104 | 39 | 53 ||| 51 | 19 ||| 3 | 32 ||| 165 | nine ||| ||| [SUMMARY]"}}},{"rowIdx":278,"cells":{"title":{"kind":"string","value":"miR-223 increases gallbladder cancer cell sensitivity to docetaxel by downregulating STMN1."},"pmid":{"kind":"string","value":"27577078"},"background_abstract":{"kind":"string","value":"MicroRNAs (miRs) are involved in cancer carcinogenesis, and certain regulatory miRs could provide promising therapeutic methods for refractory malignancies, such as gallbladder cancer (GBC). miR-223 was found to play a pivotal role in enhancing chemotherapeutic effects, therefore evoking interest in the role of miR-223 in GBC."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We examined miR-223 expression in GBC tissue and GBC cell lines using qRT-PCR. The effects of modulated miR-223 expression in GBC cells were assayed using Cell Counting Kit-8 (CCK8), flow cytometry, and wound-healing and invasion assays. Susceptibility to docetaxel was evaluated in miR-223/STMN1-modulated GBC cells and xenograft tumor models. The protein expression of relevant genes was examined by Western blotting."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"miR-223 was decreased in GBC tissues and cell lines, and ectopic miR- 223 expression exhibited multiple anti-tumorigenic effects in GBC cells, including decreased proliferation, migration and invasion in vitro. However, treatment with a miR-223 inhibitor increased cell viability. We determined that STMN1 was negatively correlated with and regulated by miR-223 in GBC. miR-223 increased GBC sensitivity to docetaxel in vitro and in vivo, and the induced sensitivity to docetaxel was suppressed by the restoration of STMN1 expression."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"These findings indicated that miR-223 might serve as an onco-suppressor that enhances susceptibility to docetaxel by downregulating STMN1 in GBC, highlighting its promising therapeutic value."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Antineoplastic Agents","Cell Line, Tumor","Cell Proliferation","Cell Survival","Docetaxel","Down-Regulation","Flow Cytometry","Gallbladder","Gallbladder Neoplasms","Gene Expression Regulation, Neoplastic","Genes, Tumor Suppressor","Humans","Immunohistochemistry","Mice","Mice, Nude","MicroRNAs","Real-Time Polymerase Chain Reaction","Stathmin","Taxoids","Xenograft Model Antitumor Assays"],"string":"[\n \"Animals\",\n \"Antineoplastic Agents\",\n \"Cell Line, Tumor\",\n \"Cell Proliferation\",\n \"Cell Survival\",\n \"Docetaxel\",\n \"Down-Regulation\",\n \"Flow Cytometry\",\n \"Gallbladder\",\n \"Gallbladder Neoplasms\",\n \"Gene Expression Regulation, Neoplastic\",\n \"Genes, Tumor Suppressor\",\n \"Humans\",\n \"Immunohistochemistry\",\n \"Mice\",\n \"Mice, Nude\",\n \"MicroRNAs\",\n \"Real-Time Polymerase Chain Reaction\",\n \"Stathmin\",\n \"Taxoids\",\n \"Xenograft Model Antitumor Assays\"\n]"},"pmcid":{"kind":"string","value":"5308733"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications."},"methods_title":{"kind":"string","value":"MATERIALS AND METHODS"},"methods_text":{"kind":"string","value":" Reagents The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\nThe CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\n GBC tissue samples and cell lines Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\nFresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\n Tissue immunohistochemistry for STMN1 and in situ hybridization for miRNA Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\nImmunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\n Western blotting After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\nAfter treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\n RNA isolation and qRT-PCR Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\nTotal RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\n Cell biology experimental procedures In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\nIn these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\n DAPI staining cell apoptosis test Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\nTreated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\n Animal experiments To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\nTo investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\n Statistical analyses Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\nStatistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001)."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"DISCUSSION"},"conclusions_text":{"kind":"string","value":"Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001)."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","RESULTS","Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation","miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression","Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation","miR-223 overexpression inhibits GBC cell migration and invasion","GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics","The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"RESULTS\",\n \"Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation\",\n \"miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression\",\n \"Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation\",\n \"miR-223 overexpression inhibits GBC cell migration and invasion\",\n \"GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics\",\n \"The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid\",\n \"DISCUSSION\"\n]"},"other_sections_texts":{"kind":"list like","value":["Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications."," Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.","To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.","To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.","To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.","Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).","GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).","The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.","GBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach."],"string":"[\n \"Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.\",\n \" Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\",\n \"To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\",\n \"To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\",\n \"To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\",\n \"Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\",\n \"GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\",\n \"The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\",\n \"GBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","RESULTS","Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation","miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression","Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation","miR-223 overexpression inhibits GBC cell migration and invasion","GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics","The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid","DISCUSSION","MATERIALS AND METHODS","SUPPLEMENTARY MATERIALS FIGURES"],"string":"[\n \"INTRODUCTION\",\n \"RESULTS\",\n \"Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation\",\n \"miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression\",\n \"Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation\",\n \"miR-223 overexpression inhibits GBC cell migration and invasion\",\n \"GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics\",\n \"The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid\",\n \"DISCUSSION\",\n \"MATERIALS AND METHODS\",\n \"SUPPLEMENTARY MATERIALS FIGURES\"\n]"},"all_sections_texts":{"kind":"list like","value":["Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications."," Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.","To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.","To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.","To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.","Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).","GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).","The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.","GBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach."," Reagents The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\nThe CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\n GBC tissue samples and cell lines Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\nFresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\n Tissue immunohistochemistry for STMN1 and in situ hybridization for miRNA Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\nImmunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\n Western blotting After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\nAfter treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\n RNA isolation and qRT-PCR Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\nTotal RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\n Cell biology experimental procedures In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\nIn these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\n DAPI staining cell apoptosis test Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\nTreated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\n Animal experiments To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\nTo investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\n Statistical analyses Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\nStatistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).",""],"string":"[\n \"Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.\",\n \" Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\",\n \"To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\",\n \"To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\",\n \"To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\",\n \"Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\",\n \"GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\",\n \"The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\",\n \"GBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach.\",\n \" Reagents The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\\nThe CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\\n GBC tissue samples and cell lines Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\\nFresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\\n Tissue immunohistochemistry for STMN1 and in situ hybridization for miRNA Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\\nImmunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\\n Western blotting After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\\nAfter treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\\n RNA isolation and qRT-PCR Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\\nSTMN1 - CT\\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\\nTotal RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\\nSTMN1 - CT\\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\\n Cell biology experimental procedures In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\\nIn these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\\n DAPI staining cell apoptosis test Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\\nTreated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\\n Animal experiments To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\\nTo investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\\n Statistical analyses Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\\nStatistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\",\n \"\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,"methods","supplementary-material"],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"methods\",\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["miR-223","gallbladder cancer","malignancy","STMN1"],"string":"[\n \"miR-223\",\n \"gallbladder cancer\",\n \"malignancy\",\n \"STMN1\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nGallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.\n\nRESULTS:\n Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\n\nAberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation:\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\n\nmiR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression:\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\n\nEctopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation:\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\n\nmiR-223 overexpression inhibits GBC cell migration and invasion:\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\n\nGBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics:\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\n\nThe chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid:\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\n\nDISCUSSION:\nGBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach.\n\nMATERIALS AND METHODS:\n Reagents The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\nThe CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\n GBC tissue samples and cell lines Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\nFresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\n Tissue immunohistochemistry for STMN1 and in situ hybridization for miRNA Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\nImmunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\n Western blotting After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\nAfter treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\n RNA isolation and qRT-PCR Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\nTotal RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\n Cell biology experimental procedures In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\nIn these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\n DAPI staining cell apoptosis test Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\nTreated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\n Animal experiments To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\nTo investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\n Statistical analyses Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\nStatistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\n\nSUPPLEMENTARY MATERIALS FIGURES:\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nMicroRNAs (miRs) are involved in cancer carcinogenesis, and certain regulatory miRs could provide promising therapeutic methods for refractory malignancies, such as gallbladder cancer (GBC). miR-223 was found to play a pivotal role in enhancing chemotherapeutic effects, therefore evoking interest in the role of miR-223 in GBC.\n\nMethods:\nWe examined miR-223 expression in GBC tissue and GBC cell lines using qRT-PCR. The effects of modulated miR-223 expression in GBC cells were assayed using Cell Counting Kit-8 (CCK8), flow cytometry, and wound-healing and invasion assays. Susceptibility to docetaxel was evaluated in miR-223/STMN1-modulated GBC cells and xenograft tumor models. The protein expression of relevant genes was examined by Western blotting.\n\nResults:\nmiR-223 was decreased in GBC tissues and cell lines, and ectopic miR- 223 expression exhibited multiple anti-tumorigenic effects in GBC cells, including decreased proliferation, migration and invasion in vitro. However, treatment with a miR-223 inhibitor increased cell viability. We determined that STMN1 was negatively correlated with and regulated by miR-223 in GBC. miR-223 increased GBC sensitivity to docetaxel in vitro and in vivo, and the induced sensitivity to docetaxel was suppressed by the restoration of STMN1 expression.\n\nConclusions:\nThese findings indicated that miR-223 might serve as an onco-suppressor that enhances susceptibility to docetaxel by downregulating STMN1 in GBC, highlighting its promising therapeutic value."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nGallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.\n\nDISCUSSION:\nStatistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001)."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nMicroRNAs (miRs) are involved in cancer carcinogenesis, and certain regulatory miRs could provide promising therapeutic methods for refractory malignancies, such as gallbladder cancer (GBC). miR-223 was found to play a pivotal role in enhancing chemotherapeutic effects, therefore evoking interest in the role of miR-223 in GBC.\n\nMethods:\nWe examined miR-223 expression in GBC tissue and GBC cell lines using qRT-PCR. The effects of modulated miR-223 expression in GBC cells were assayed using Cell Counting Kit-8 (CCK8), flow cytometry, and wound-healing and invasion assays. Susceptibility to docetaxel was evaluated in miR-223/STMN1-modulated GBC cells and xenograft tumor models. The protein expression of relevant genes was examined by Western blotting.\n\nResults:\nmiR-223 was decreased in GBC tissues and cell lines, and ectopic miR- 223 expression exhibited multiple anti-tumorigenic effects in GBC cells, including decreased proliferation, migration and invasion in vitro. However, treatment with a miR-223 inhibitor increased cell viability. We determined that STMN1 was negatively correlated with and regulated by miR-223 in GBC. miR-223 increased GBC sensitivity to docetaxel in vitro and in vivo, and the induced sensitivity to docetaxel was suppressed by the restoration of STMN1 expression.\n\nConclusions:\nThese findings indicated that miR-223 might serve as an onco-suppressor that enhances susceptibility to docetaxel by downregulating STMN1 in GBC, highlighting its promising therapeutic value."},"whole_article_text_length":{"kind":"number","value":12789,"string":"12,789"},"whole_article_abstract_length":{"kind":"number","value":263,"string":"263"},"num_sections":{"kind":"number","value":11,"string":"11"},"most_frequent_words":{"kind":"list like","value":["mir","mir 223","223","gbc","cells","cell","stmn1","expression","sd","noz"],"string":"[\n \"mir\",\n \"mir 223\",\n \"223\",\n \"gbc\",\n \"cells\",\n \"cell\",\n \"stmn1\",\n \"expression\",\n \"sd\",\n \"noz\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | mir 223 | 223 | gbc | cells | cell | stmn1 | expression | sd | noz [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | mir 223 | 223 | gbc | cells | cell | stmn1 | expression | sd | noz 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[SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | mir 223 | 223 | gbc | stmn1 | carcinogenesis | expression | cell | microtubule | role [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | 223 | mir 223 | gbc | cells | stmn1 | expression | sd | cell | gbc sd [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | 223 | mir 223 | gbc | cells | stmn1 | expression | sd | cell | gbc sd [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| STMN1 ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] STMN1 | GBC [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| ||| STMN1 ||| ||| 223 ||| ||| STMN1 ||| STMN1 ||| STMN1 | GBC [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| ||| STMN1 ||| ||| 223 ||| ||| STMN1 ||| STMN1 ||| STMN1 | GBC [SUMMARY]"}}},{"rowIdx":279,"cells":{"title":{"kind":"string","value":"Harm avoidance is associated with progression of parkinsonism in community-dwelling older adults: a prospective cohort study."},"pmid":{"kind":"string","value":"24754876"},"background_abstract":{"kind":"string","value":"We tested the hypothesis that harm avoidance, a trait associated with behavioral inhibition, is associated with the rate of change in parkinsonism in older adults."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"At baseline harm avoidance was assessed with a standard self-report instrument in 969 older people without dementia participating in the Rush Memory and Aging Project, a longitudinal community-based cohort study. Parkinsonism was assessed annually with a modified version of the motor section of the Unified Parkinson's Disease Rating Scale (mUPDRS)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Average follow-up was 5 years. A linear mixed-effects model controlling for age, sex and education showed that for an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10), the overall severity of parkinsonism increased by about 0.05 unit/ year (Estimate, 0.054, S.E., 0.007, p <0.001) and that the level of harm avoidance was associated with the progression of parkinsonism (Estimate, 0.004, S.E., 0.001, p <0.001). Thus, for an average participant, every 6 point (~1 SD) increase in harm avoidance score at baseline, the rate of progression of parkinsonism increased about 50% compared to an individual with an average harm avoidance score. This amount of change in parkinsonism over the course of the study was associated with about a 5% increased risk of death. The association between harm avoidance and progression of parkinsonism persisted when controlling for cognitive function, depressive symptoms, loneliness, neuroticism, late-life cognitive, social and physical activities and chronic health conditions."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"A higher level of the harm avoidance trait is associated with a more rapid progression of parkinsonism in older adults."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Aged, 80 and over","Cohort Studies","Disease Progression","Female","Follow-Up Studies","Harm Reduction","Humans","Longitudinal Studies","Male","Parkinsonian Disorders","Prospective Studies","Residence Characteristics"],"string":"[\n \"Aged\",\n \"Aged, 80 and over\",\n \"Cohort Studies\",\n \"Disease Progression\",\n \"Female\",\n \"Follow-Up Studies\",\n \"Harm Reduction\",\n \"Humans\",\n \"Longitudinal Studies\",\n \"Male\",\n \"Parkinsonian Disorders\",\n \"Prospective Studies\",\n \"Residence Characteristics\"\n]"},"pmcid":{"kind":"string","value":"4022545"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Participants Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\nParticipants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\n Clinical diagnoses Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\nClinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\n Assessment of harm avoidance Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\nStudy participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\n Assessment of parkinsonism Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\nTrained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\n Assessment of other covariates Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\nAge in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\n Statistical analyses The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\nThe global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23]."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Descriptive properties of harm avoidance measure The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\nThe clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\n Harm avoidance and change in parkinsonism Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\nBaseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\n Potential confounders of harm avoidance and change in parkinsonism Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\nPrior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\n Clinical significance of the loss of motor function associated with personality To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\nTo determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging."},"other_sections_titles":{"kind":"list like","value":["Background","Participants","Clinical diagnoses","Assessment of harm avoidance","Assessment of parkinsonism","Assessment of other covariates","Statistical analyses","Descriptive properties of harm avoidance measure","Harm avoidance and change in parkinsonism","Potential confounders of harm avoidance and change in parkinsonism","Clinical significance of the loss of motor function associated with personality","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Participants\",\n \"Clinical diagnoses\",\n \"Assessment of harm avoidance\",\n \"Assessment of parkinsonism\",\n \"Assessment of other covariates\",\n \"Statistical analyses\",\n \"Descriptive properties of harm avoidance measure\",\n \"Harm avoidance and change in parkinsonism\",\n \"Potential confounders of harm avoidance and change in parkinsonism\",\n \"Clinical significance of the loss of motor function associated with personality\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.","Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.","Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].","Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].","Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].","Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].","The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].","The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).","Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].","Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).","To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).","The authors declare that they have no competing interests.","ASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\n"],"string":"[\n \"Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.\",\n \"Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\",\n \"Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\",\n \"Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\",\n \"Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\",\n \"Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\",\n \"The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\",\n \"The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\\nClinical characteristics of the participants included in these analyses at this study’s baseline\\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\",\n \"Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\",\n \"Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\",\n \"To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\",\n \"The authors declare that they have no competing interests.\",\n \"ASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Participants","Clinical diagnoses","Assessment of harm avoidance","Assessment of parkinsonism","Assessment of other covariates","Statistical analyses","Results","Descriptive properties of harm avoidance measure","Harm avoidance and change in parkinsonism","Potential confounders of harm avoidance and change in parkinsonism","Clinical significance of the loss of motor function associated with personality","Discussion","Conclusions","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Participants\",\n \"Clinical diagnoses\",\n \"Assessment of harm avoidance\",\n \"Assessment of parkinsonism\",\n \"Assessment of other covariates\",\n \"Statistical analyses\",\n \"Results\",\n \"Descriptive properties of harm avoidance measure\",\n \"Harm avoidance and change in parkinsonism\",\n \"Potential confounders of harm avoidance and change in parkinsonism\",\n \"Clinical significance of the loss of motor function associated with personality\",\n \"Discussion\",\n \"Conclusions\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles."," Participants Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\nParticipants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\n Clinical diagnoses Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\nClinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\n Assessment of harm avoidance Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\nStudy participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\n Assessment of parkinsonism Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\nTrained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\n Assessment of other covariates Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\nAge in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\n Statistical analyses The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\nThe global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].","Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.","Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].","Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].","Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].","Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].","The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23]."," Descriptive properties of harm avoidance measure The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\nThe clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\n Harm avoidance and change in parkinsonism Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\nBaseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\n Potential confounders of harm avoidance and change in parkinsonism Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\nPrior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\n Clinical significance of the loss of motor function associated with personality To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\nTo determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).","The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).","Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].","Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).","To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).","Harm avoidance is a broad anxiety-related trait. People with a high level of the trait tend to be pessimistic, apprehensive, shy, and easily fatigued, behaviorally inhibited and to avoid new and potentially aversive situations [3]. In a cohort of more than 900 older adults, those with a high level of the trait showed a more rapid rate of progression of parkinsonism as compared to older adults with a low level of the trait. This association persisted after controlling for other psychosocial factors including depressive symptoms, loneliness and neuroticism, late-life activities including physical, social and cognitive activities, global cognitive function and chronic health conditions. Together these data suggest that the level of the trait of harm avoidance, may identify older individuals at higher risk for more rapidly progressive parkinsonism and provides evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.\nPrior studies have reported that higher levels of harm avoidance are associated with adverse health consequences in older adults including: incident disability, [6] and late-life cognitive impairments including incident mild cognitive impairment (MCI) and Alzheimer’s disease (AD) as well as cognitive decline [7,24,25]. This study extends these findings by showing that harm avoidance is also associated with both the level and rate of progression of parkinsonism in older adults without overt neurologic diseases including dementia, stroke and Parkinson’s disease (PD). Analyses of the sub-scale components of harm avoidance showed that all of its constituent factors are associated with the rate of progressive parkinsonism. The association of harm avoidance and parkinsonism, was robust and remained significant after accounting for related personality traits and psychosocial factors, global cognition, a wide range of late-life activities and chronic health conditions. The results of the current study have important translational implications as they suggest that personality traits need to be considered for our understanding of individual differences in the progression of parkinsonism in older adults. Furthermore, understanding the biologic basis of the association has the potential to lead to new therapeutic targets to reduce the burden of late-life motor impairment.\nThe basis of the association between harm avoidance and parkinsonism is likely to be complex. While, harm avoidance is related to other personality traits and psychosocial factors, [26] the association of harm avoidance and parkinsonism was unchanged when we adjusted for several of these factors in the current study (Table 3) [27]. Personality traits may affect for lifestyle choices i.e. physical and other late-life activities and could thus link harm avoidance with parkinsonism. In contrast to other personality traits, [17,28] statistical adjustment for late-life physical, cognitive and social activities in the current study did not affect the association of harm avoidance with progressive parkinsonism. Both harm avoidance and motor function in older people are preferentially associated with structural changes in specific brain regions including cortical and cerebellar structures which might account for their association in this study [29-33]. Thus, higher levels of harm avoidance may be lead to stress-related changes in neurotransmitters (e.g., cortisol or dopamine) causing brain atrophy, damaging motor-related brain regions or decreasing the brain’s capacity (motor reserve) to tolerate ongoing neurodegeneration and the accumulation of other neuropathologies [34-37].\nOver the years there have been many reports suggesting that PD may be associated with a distinctive personality which may manifest many years before clinical evidence of parkinsonism, but empirical studies have failed to support this suggestion [38,39]. Nonethelss, building on imaging data linking harm avoidance with brain dopamine receptors, recent studies have reported higher levels of harm avoidance occur in individuals with early clinical manifestations of PD such as REM sleep disorder which can manifest years before a clinical diagnosis of PD [40-43]. Studies in the current cohort have suggested that PD pathology is found in up to 40% of older adults without a clinical diagnoses of PD, and these lesions are associated with the severity of parkinsonism proximate to death [14]. Further work is needed to determine if the accumulation of subclinical PD pathology might contribute to the association between harm avoidance and the rate of change in the severity of parkinsonism. Alternatively, lower harm avoidance is associated with resilience, optimism, composure, and energy which may facilitate adaptation to accumulating neurodegeneration and the accumulation of neuropathology in older adults [10,44]. Further work to understand the neurobiologic basis for the current findings has the potential to identify new targets and pathways, for interventions to decrease the growing burden of progressive parkinsonism in older adults.\nOur study has some limitations. While some studies have suggested that personality traits such as harm avoidance are stable even in old age, others suggest that personality changes may occur with age but there are few studies which have focused on individuals older than 80 years [45-48]. Further, it is possible that a third unmeasured variable is related to both harm avoidance and progressive parkinsonism. A precondition for participation in the current study was consent to annual exam and organ donation at death, so given the selected nature of the cohort, our findings will need replication in other cohorts. This study was large since on an individual level the effect sizes for the association of harm avoidance and progressive parkinsonism are small. Similar effect sizes have been reported in prior reports of other psychosocial factors with motor decline [17]. Nonetheless, from a public policy perspective even the modest effect sizes observed in the current study are likely to be important.\nHowever, several factors increase confidence in our findings. Perhaps most importantly, the study enjoys high follow-up participation reducing bias due to attrition. In addition, personality traits were assessed among people without dementia and parkinsonism was evaluated as part of a uniform clinical evaluation which incorporated other widely accepted personality, affect and cognitive measures. In addition, a relatively large number of older people were studied, so that there was adequate statistical power to identify the association of interest while controlling for several potentially confounding variables. Results were similar with total score and subscores of the trait of harm avoidance.","The growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.","The authors declare that they have no competing interests.","ASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\n"],"string":"[\n \"Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.\",\n \" Participants Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\\nParticipants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\\n Clinical diagnoses Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\\nClinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\\n Assessment of harm avoidance Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\\nStudy participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\\n Assessment of parkinsonism Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\\nTrained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\\n Assessment of other covariates Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\\nAge in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\\n Statistical analyses The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\\nThe global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\",\n \"Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\",\n \"Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\",\n \"Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\",\n \"Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\",\n \"Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\",\n \"The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\",\n \" Descriptive properties of harm avoidance measure The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\\nClinical characteristics of the participants included in these analyses at this study’s baseline\\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\\nThe clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\\nClinical characteristics of the participants included in these analyses at this study’s baseline\\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\\n Harm avoidance and change in parkinsonism Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\\nBaseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\\n Potential confounders of harm avoidance and change in parkinsonism Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\\nPrior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\\n Clinical significance of the loss of motor function associated with personality To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\\nTo determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\",\n \"The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\\nClinical characteristics of the participants included in these analyses at this study’s baseline\\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\",\n \"Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\",\n \"Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\",\n \"To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\",\n \"Harm avoidance is a broad anxiety-related trait. People with a high level of the trait tend to be pessimistic, apprehensive, shy, and easily fatigued, behaviorally inhibited and to avoid new and potentially aversive situations [3]. In a cohort of more than 900 older adults, those with a high level of the trait showed a more rapid rate of progression of parkinsonism as compared to older adults with a low level of the trait. This association persisted after controlling for other psychosocial factors including depressive symptoms, loneliness and neuroticism, late-life activities including physical, social and cognitive activities, global cognitive function and chronic health conditions. Together these data suggest that the level of the trait of harm avoidance, may identify older individuals at higher risk for more rapidly progressive parkinsonism and provides evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.\\nPrior studies have reported that higher levels of harm avoidance are associated with adverse health consequences in older adults including: incident disability, [6] and late-life cognitive impairments including incident mild cognitive impairment (MCI) and Alzheimer’s disease (AD) as well as cognitive decline [7,24,25]. This study extends these findings by showing that harm avoidance is also associated with both the level and rate of progression of parkinsonism in older adults without overt neurologic diseases including dementia, stroke and Parkinson’s disease (PD). Analyses of the sub-scale components of harm avoidance showed that all of its constituent factors are associated with the rate of progressive parkinsonism. The association of harm avoidance and parkinsonism, was robust and remained significant after accounting for related personality traits and psychosocial factors, global cognition, a wide range of late-life activities and chronic health conditions. The results of the current study have important translational implications as they suggest that personality traits need to be considered for our understanding of individual differences in the progression of parkinsonism in older adults. Furthermore, understanding the biologic basis of the association has the potential to lead to new therapeutic targets to reduce the burden of late-life motor impairment.\\nThe basis of the association between harm avoidance and parkinsonism is likely to be complex. While, harm avoidance is related to other personality traits and psychosocial factors, [26] the association of harm avoidance and parkinsonism was unchanged when we adjusted for several of these factors in the current study (Table 3) [27]. Personality traits may affect for lifestyle choices i.e. physical and other late-life activities and could thus link harm avoidance with parkinsonism. In contrast to other personality traits, [17,28] statistical adjustment for late-life physical, cognitive and social activities in the current study did not affect the association of harm avoidance with progressive parkinsonism. Both harm avoidance and motor function in older people are preferentially associated with structural changes in specific brain regions including cortical and cerebellar structures which might account for their association in this study [29-33]. Thus, higher levels of harm avoidance may be lead to stress-related changes in neurotransmitters (e.g., cortisol or dopamine) causing brain atrophy, damaging motor-related brain regions or decreasing the brain’s capacity (motor reserve) to tolerate ongoing neurodegeneration and the accumulation of other neuropathologies [34-37].\\nOver the years there have been many reports suggesting that PD may be associated with a distinctive personality which may manifest many years before clinical evidence of parkinsonism, but empirical studies have failed to support this suggestion [38,39]. Nonethelss, building on imaging data linking harm avoidance with brain dopamine receptors, recent studies have reported higher levels of harm avoidance occur in individuals with early clinical manifestations of PD such as REM sleep disorder which can manifest years before a clinical diagnosis of PD [40-43]. Studies in the current cohort have suggested that PD pathology is found in up to 40% of older adults without a clinical diagnoses of PD, and these lesions are associated with the severity of parkinsonism proximate to death [14]. Further work is needed to determine if the accumulation of subclinical PD pathology might contribute to the association between harm avoidance and the rate of change in the severity of parkinsonism. Alternatively, lower harm avoidance is associated with resilience, optimism, composure, and energy which may facilitate adaptation to accumulating neurodegeneration and the accumulation of neuropathology in older adults [10,44]. Further work to understand the neurobiologic basis for the current findings has the potential to identify new targets and pathways, for interventions to decrease the growing burden of progressive parkinsonism in older adults.\\nOur study has some limitations. While some studies have suggested that personality traits such as harm avoidance are stable even in old age, others suggest that personality changes may occur with age but there are few studies which have focused on individuals older than 80 years [45-48]. Further, it is possible that a third unmeasured variable is related to both harm avoidance and progressive parkinsonism. A precondition for participation in the current study was consent to annual exam and organ donation at death, so given the selected nature of the cohort, our findings will need replication in other cohorts. This study was large since on an individual level the effect sizes for the association of harm avoidance and progressive parkinsonism are small. Similar effect sizes have been reported in prior reports of other psychosocial factors with motor decline [17]. Nonetheless, from a public policy perspective even the modest effect sizes observed in the current study are likely to be important.\\nHowever, several factors increase confidence in our findings. Perhaps most importantly, the study enjoys high follow-up participation reducing bias due to attrition. In addition, personality traits were assessed among people without dementia and parkinsonism was evaluated as part of a uniform clinical evaluation which incorporated other widely accepted personality, affect and cognitive measures. In addition, a relatively large number of older people were studied, so that there was adequate statistical power to identify the association of interest while controlling for several potentially confounding variables. Results were similar with total score and subscores of the trait of harm avoidance.\",\n \"The growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.\",\n \"The authors declare that they have no competing interests.\",\n \"ASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,"results",null,null,null,null,"discussion","conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Late-life motor impairment","Aging","Parkinsonism","Harm avoidance"],"string":"[\n \"Late-life motor impairment\",\n \"Aging\",\n \"Parkinsonism\",\n \"Harm avoidance\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nLate-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.\n\nMethods:\n Participants Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\nParticipants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\n Clinical diagnoses Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\nClinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\n Assessment of harm avoidance Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\nStudy participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\n Assessment of parkinsonism Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\nTrained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\n Assessment of other covariates Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\nAge in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\n Statistical analyses The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\nThe global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\n\nParticipants:\nParticipants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\n\nClinical diagnoses:\nClinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\n\nAssessment of harm avoidance:\nStudy participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\n\nAssessment of parkinsonism:\nTrained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\n\nAssessment of other covariates:\nAge in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\n\nStatistical analyses:\nThe global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\n\nResults:\n Descriptive properties of harm avoidance measure The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\nThe clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\n Harm avoidance and change in parkinsonism Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\nBaseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\n Potential confounders of harm avoidance and change in parkinsonism Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\nPrior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\n Clinical significance of the loss of motor function associated with personality To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\nTo determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\n\nDescriptive properties of harm avoidance measure:\nThe clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\n\nHarm avoidance and change in parkinsonism:\nBaseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\n\nPotential confounders of harm avoidance and change in parkinsonism:\nPrior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\n\nClinical significance of the loss of motor function associated with personality:\nTo determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\n\nDiscussion:\nHarm avoidance is a broad anxiety-related trait. People with a high level of the trait tend to be pessimistic, apprehensive, shy, and easily fatigued, behaviorally inhibited and to avoid new and potentially aversive situations [3]. In a cohort of more than 900 older adults, those with a high level of the trait showed a more rapid rate of progression of parkinsonism as compared to older adults with a low level of the trait. This association persisted after controlling for other psychosocial factors including depressive symptoms, loneliness and neuroticism, late-life activities including physical, social and cognitive activities, global cognitive function and chronic health conditions. Together these data suggest that the level of the trait of harm avoidance, may identify older individuals at higher risk for more rapidly progressive parkinsonism and provides evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.\nPrior studies have reported that higher levels of harm avoidance are associated with adverse health consequences in older adults including: incident disability, [6] and late-life cognitive impairments including incident mild cognitive impairment (MCI) and Alzheimer’s disease (AD) as well as cognitive decline [7,24,25]. This study extends these findings by showing that harm avoidance is also associated with both the level and rate of progression of parkinsonism in older adults without overt neurologic diseases including dementia, stroke and Parkinson’s disease (PD). Analyses of the sub-scale components of harm avoidance showed that all of its constituent factors are associated with the rate of progressive parkinsonism. The association of harm avoidance and parkinsonism, was robust and remained significant after accounting for related personality traits and psychosocial factors, global cognition, a wide range of late-life activities and chronic health conditions. The results of the current study have important translational implications as they suggest that personality traits need to be considered for our understanding of individual differences in the progression of parkinsonism in older adults. Furthermore, understanding the biologic basis of the association has the potential to lead to new therapeutic targets to reduce the burden of late-life motor impairment.\nThe basis of the association between harm avoidance and parkinsonism is likely to be complex. While, harm avoidance is related to other personality traits and psychosocial factors, [26] the association of harm avoidance and parkinsonism was unchanged when we adjusted for several of these factors in the current study (Table 3) [27]. Personality traits may affect for lifestyle choices i.e. physical and other late-life activities and could thus link harm avoidance with parkinsonism. In contrast to other personality traits, [17,28] statistical adjustment for late-life physical, cognitive and social activities in the current study did not affect the association of harm avoidance with progressive parkinsonism. Both harm avoidance and motor function in older people are preferentially associated with structural changes in specific brain regions including cortical and cerebellar structures which might account for their association in this study [29-33]. Thus, higher levels of harm avoidance may be lead to stress-related changes in neurotransmitters (e.g., cortisol or dopamine) causing brain atrophy, damaging motor-related brain regions or decreasing the brain’s capacity (motor reserve) to tolerate ongoing neurodegeneration and the accumulation of other neuropathologies [34-37].\nOver the years there have been many reports suggesting that PD may be associated with a distinctive personality which may manifest many years before clinical evidence of parkinsonism, but empirical studies have failed to support this suggestion [38,39]. Nonethelss, building on imaging data linking harm avoidance with brain dopamine receptors, recent studies have reported higher levels of harm avoidance occur in individuals with early clinical manifestations of PD such as REM sleep disorder which can manifest years before a clinical diagnosis of PD [40-43]. Studies in the current cohort have suggested that PD pathology is found in up to 40% of older adults without a clinical diagnoses of PD, and these lesions are associated with the severity of parkinsonism proximate to death [14]. Further work is needed to determine if the accumulation of subclinical PD pathology might contribute to the association between harm avoidance and the rate of change in the severity of parkinsonism. Alternatively, lower harm avoidance is associated with resilience, optimism, composure, and energy which may facilitate adaptation to accumulating neurodegeneration and the accumulation of neuropathology in older adults [10,44]. Further work to understand the neurobiologic basis for the current findings has the potential to identify new targets and pathways, for interventions to decrease the growing burden of progressive parkinsonism in older adults.\nOur study has some limitations. While some studies have suggested that personality traits such as harm avoidance are stable even in old age, others suggest that personality changes may occur with age but there are few studies which have focused on individuals older than 80 years [45-48]. Further, it is possible that a third unmeasured variable is related to both harm avoidance and progressive parkinsonism. A precondition for participation in the current study was consent to annual exam and organ donation at death, so given the selected nature of the cohort, our findings will need replication in other cohorts. This study was large since on an individual level the effect sizes for the association of harm avoidance and progressive parkinsonism are small. Similar effect sizes have been reported in prior reports of other psychosocial factors with motor decline [17]. Nonetheless, from a public policy perspective even the modest effect sizes observed in the current study are likely to be important.\nHowever, several factors increase confidence in our findings. Perhaps most importantly, the study enjoys high follow-up participation reducing bias due to attrition. In addition, personality traits were assessed among people without dementia and parkinsonism was evaluated as part of a uniform clinical evaluation which incorporated other widely accepted personality, affect and cognitive measures. In addition, a relatively large number of older people were studied, so that there was adequate statistical power to identify the association of interest while controlling for several potentially confounding variables. Results were similar with total score and subscores of the trait of harm avoidance.\n\nConclusions:\nThe growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWe tested the hypothesis that harm avoidance, a trait associated with behavioral inhibition, is associated with the rate of change in parkinsonism in older adults.\n\nMethods:\nAt baseline harm avoidance was assessed with a standard self-report instrument in 969 older people without dementia participating in the Rush Memory and Aging Project, a longitudinal community-based cohort study. Parkinsonism was assessed annually with a modified version of the motor section of the Unified Parkinson's Disease Rating Scale (mUPDRS).\n\nResults:\nAverage follow-up was 5 years. A linear mixed-effects model controlling for age, sex and education showed that for an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10), the overall severity of parkinsonism increased by about 0.05 unit/ year (Estimate, 0.054, S.E., 0.007, p <0.001) and that the level of harm avoidance was associated with the progression of parkinsonism (Estimate, 0.004, S.E., 0.001, p <0.001). Thus, for an average participant, every 6 point (~1 SD) increase in harm avoidance score at baseline, the rate of progression of parkinsonism increased about 50% compared to an individual with an average harm avoidance score. This amount of change in parkinsonism over the course of the study was associated with about a 5% increased risk of death. The association between harm avoidance and progression of parkinsonism persisted when controlling for cognitive function, depressive symptoms, loneliness, neuroticism, late-life cognitive, social and physical activities and chronic health conditions.\n\nConclusions:\nA higher level of the harm avoidance trait is associated with a more rapid progression of parkinsonism in older adults."},"background_conclusion_text":{"kind":"string","value":"Background:\nLate-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.\n\nConclusions:\nThe growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nWe tested the hypothesis that harm avoidance, a trait associated with behavioral inhibition, is associated with the rate of change in parkinsonism in older adults.\n\nMethods:\nAt baseline harm avoidance was assessed with a standard self-report instrument in 969 older people without dementia participating in the Rush Memory and Aging Project, a longitudinal community-based cohort study. Parkinsonism was assessed annually with a modified version of the motor section of the Unified Parkinson's Disease Rating Scale (mUPDRS).\n\nResults:\nAverage follow-up was 5 years. A linear mixed-effects model controlling for age, sex and education showed that for an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10), the overall severity of parkinsonism increased by about 0.05 unit/ year (Estimate, 0.054, S.E., 0.007, p <0.001) and that the level of harm avoidance was associated with the progression of parkinsonism (Estimate, 0.004, S.E., 0.001, p <0.001). Thus, for an average participant, every 6 point (~1 SD) increase in harm avoidance score at baseline, the rate of progression of parkinsonism increased about 50% compared to an individual with an average harm avoidance score. This amount of change in parkinsonism over the course of the study was associated with about a 5% increased risk of death. The association between harm avoidance and progression of parkinsonism persisted when controlling for cognitive function, depressive symptoms, loneliness, neuroticism, late-life cognitive, social and physical activities and chronic health conditions.\n\nConclusions:\nA higher level of the harm avoidance trait is associated with a more rapid progression of parkinsonism in older adults."},"whole_article_text_length":{"kind":"number","value":13063,"string":"13,063"},"whole_article_abstract_length":{"kind":"number","value":330,"string":"330"},"num_sections":{"kind":"number","value":18,"string":"18"},"most_frequent_words":{"kind":"list like","value":["parkinsonism","harm avoidance","harm","avoidance","baseline","change","rate","score","change parkinsonism","rate change"],"string":"[\n \"parkinsonism\",\n \"harm avoidance\",\n \"harm\",\n \"avoidance\",\n \"baseline\",\n \"change\",\n \"rate\",\n \"score\",\n \"change parkinsonism\",\n \"rate change\"\n]"},"keybert_topics":{"kind":"list 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[SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] harm | avoidance | harm avoidance | parkinsonism | adults | older | trait | related | associated | level [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] avoidance | harm avoidance | harm | parkinsonism | participants | items | baseline | score | models | change [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | avoidance | harm avoidance | harm | change | change parkinsonism | 001 | rate change | rate change parkinsonism | rate [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] adults | older | older adults | level trait | motor | traits | growing | personality traits | level | level trait harm [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | score | change | rate | association | change parkinsonism [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | score | change | rate | association | change parkinsonism [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 969 | the Rush Memory and Aging Project ||| annually | the Unified Parkinson's [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 5 years ||| 80 years old | 14 years | 10 | about 0.05 unit/ year | Estimate | 0.054 | S.E. | 0.007 | p <0.001 | Estimate | 0.004 | S.E. | 0.001 | p <0.001 ||| 6 | about 50% ||| 5% ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 969 | the Rush Memory and Aging Project ||| annually | the Unified Parkinson's ||| 5 years ||| 80 years old | 14 years | 10 | about 0.05 unit/ year | Estimate | 0.054 | S.E. | 0.007 | p <0.001 | Estimate | 0.004 | S.E. | 0.001 | p <0.001 ||| 6 | about 50% ||| 5% ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 969 | the Rush Memory and Aging Project ||| annually | the Unified Parkinson's ||| 5 years ||| 80 years old | 14 years | 10 | about 0.05 unit/ year | Estimate | 0.054 | S.E. | 0.007 | p <0.001 | Estimate | 0.004 | S.E. | 0.001 | p <0.001 ||| 6 | about 50% ||| 5% ||| ||| [SUMMARY]"}}},{"rowIdx":280,"cells":{"title":{"kind":"string","value":"Secular Trends in Dietary Intake over a 20-Year Period in People with Type 2 Diabetes in Japan: A Comparative Study of Two Nationwide Registries; Japan Diabetes Complications Study (JDCS) and Japan Diabetes Clinical Data Management Study (JDDM)."},"pmid":{"kind":"string","value":"34684444"},"background_abstract":{"kind":"string","value":"In order to provide effective dietary guidance, it is necessary to consider dietary intake, which can change over time. This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We compared the results of two dietary surveys that used the food frequency questionnaire format. The first was conducted in 1996 by the Japan Diabetes Complications Study (JDCS) (n = 1509; males 53.3%), and the second in 2014-2018 by the Japan Diabetes Clinical Data Management Study (JDDM) (n = 1145; males 65.6%). Both are nationwide representative registries of outpatients with type 2 diabetes in Japan."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Over a 20-year period, both men and women with type 2 diabetes had a significant increase in body mass index (BMI). Nonetheless, there was only a small change in energy intake. Conversely, there was a significant increase in fat intake and thus in the fat-to-energy ratio. With regard to food groups, there was a significant increase in meat intake and a decrease in the intake of fish, soybeans/soy products, vegetables, and fruits, with a particularly significant decrease in vegetables."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Even in Japan, an industrialized country with a stable socioeconomic environment, there were many significant changes in the dietary intake of patients with type 2 diabetes over the 20-year period."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Biomarkers","Body Mass Index","Diabetes Mellitus, Type 2","Diet","Disease Susceptibility","Energy Intake","Feeding Behavior","Female","Humans","Japan","Male","Middle Aged","Public Health Surveillance","Registries"],"string":"[\n \"Aged\",\n \"Biomarkers\",\n \"Body Mass Index\",\n \"Diabetes Mellitus, Type 2\",\n \"Diet\",\n \"Disease Susceptibility\",\n \"Energy Intake\",\n \"Feeding Behavior\",\n \"Female\",\n \"Humans\",\n \"Japan\",\n \"Male\",\n \"Middle Aged\",\n \"Public Health Surveillance\",\n \"Registries\"\n]"},"pmcid":{"kind":"string","value":"8538089"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Diet plays a central role in diabetes self-management education and support [1]. To provide effective dietary guidance and/or to establish contemporary dietary guidelines, we must understand and take into account the changes in dietary intake and pattern over time. Dietary habits, including dietary content, are strongly influenced by changes in socioeconomic conditions and the environment [2]. The National Health and Nutrition Survey in Japan revealed decreases in intakes of 151 kcal (−7.4%) of energy, 24.9 g (−8.9%) of carbohydrates, 12.7 g (−15.5%) of protein, and 3.8 g (−27.7%) of salt (as sodium chloride) over a period of 20 years (1996–2016).\nPeople with diabetes often receive nutritional guidance in the early stages of the disease, so their knowledge, as well as dietary content and habits, may differ from those who do not have diabetes. Thus, dietary changes in people with type 2 diabetes may differ from the general population. However, very few studies have investigated this change. In a study reporting on the dietary changes made by people with type 2 diabetes in the United States over the past 25 years, Casagrande et al. [3] reported 206 kcal (+12.2%) and 339 mg (+11.2%) increases in energy and sodium, respectively, as well as a 0.9 g (−8.7%) decrease in dietary fiber. In addition, according to the Dietary Survey of Type 2 Diabetes in Spain [4], although the sample size was small at 200, protein intake decreased by 6.9% in men and 1.4% in women, and fat intake increased by 3.2% in men and 5.5% in women in 2000 compared to 1993. However, as these studies focused on nutrient changes, it was unclear what changes in foods contributed to these nutrient changes. Dietary intake by food groups in those with type 2 diabetes has been previously reported [5], but to the best of our knowledge, there have been no studies on changes in dietary intake over time.\nThe diabetic population in the Asian region is rapidly increasing, with those in East Asia accounting for approximately a quarter of the world diabetes population [6]. Therefore, from the perspective of global epidemiology, it is important to elucidate changes in the nutrient intake of Asian people with diabetes over time. Previously, we reported [7] on the nutritional status of Japanese patients with type 2 diabetes from a survey conducted in 1996. However, no study thus far has examined changes in nutrient intake in the Asian diabetic population over time on a nationwide scale.\nThe purpose of this study was to reveal the detailed changes in dietary intake in Japanese patients with type 2 diabetes over a 20-year period beginning in 1996. In the present study, we conducted a nutritional survey, almost identical to our previous survey, which focused on a newly established nationwide registry of Japanese patients with type 2 diabetes. We then compared the results with those of our previous study. The results of this study reveal the importance of understanding which areas need to be focused on for efficient and effective dietary guidance, and thus for the creation of contemporary dietary guidelines. In addition, the results may provide fundamental insights into the differences in dietary advice received by patients of Asian and Western origins."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":"Table 1 shows the clinical features of participants according to registry and sex. Although statistically significant differences were found between JDCS and JDDM patients in most variables, the differences in age and diabetes duration were too small to be considered clinically different. Variables that were considered to be clinically different between registries included BMI, HbA1c, blood pressure, and smoking rate among men. The BMI was significantly higher (+3.1 kg/m2 in men and +2.7 kg/m2 in women) in JDDM participants compared to JDCS participants. Comparing the mean BMI between JDCS and JDDM participants, the proportion of obese patients defined by BMI ≥ 25 kg/m2 according to Japanese criterion was significantly greater in JDDM participants, being approximately 20% to 50% higher in men and approximately 30% to 50% higher in women. HbA1c was significantly lower by approximately 1% and systolic blood pressure by approximately 5 mmHg for both men and women among JDDM participants, and the difference in the smoking rate for men was approximately 15% lower in JDDM participants than in JDCS participants.\nWhen participants were divided into two groups based on the median age of 60 years, the characteristics were similar to those identified in the comparison of all groups, regardless of age. However, there was a dramatic increase in the BMI of JDDM participants younger than 60 years compared to JDCS participants in the same age group (Supplementary Table S1).\nTable 2 shows a comparison of daily nutrient intake between patients in both studies. In Model 2, which was adjusted for age, HbA1c, BMI, insulin use, and oral medication use, there was no significant difference in energy intake in men between the two studies, but in women, there was a slight, but significant, increase of approximately 5% in the JDDM compared to the JDCS. Regarding proportions of macronutrients, the fat-to-energy ratio increased significantly, by about 3%, and the carbohydrate-to-energy ratio and protein-to-energy ratio decreased significantly for both men and women from the JDCS to the JDDM. Regarding changes in fat intake, the proportion of energy derived from saturated fatty acids increased significantly from 7.6% to 9.0% in men and from 8.3% to 9.4% in women in the interval between the surveys, whereas the proportion of energy derived from polyunsaturated fatty acids decreased significantly. Other variables that showed nutritionally meaningful differences between the two studies included significant decreases in dietary fiber, by 1.7 g for men and 1.6 g for women, and in salt, by 1.9 g for men and 2.7 g for women. The results were similar to the comparison of all groups, with fat-to-energy ratio and saturated fatty acid-to-energy ratio significantly increased in JDDM patients regardless of age, and polyunsaturated fatty acid-to-energy ratio, dietary fiber, and salt significantly decreased in JDDM patients (Supplementary Table S2).\nTable 3 shows a comparison of daily food intakes categorized by food groups. In terms of grains intake, from the JDCS to the JDDM, there was a slight, but significant, decrease of 7.4% in men, but no significant difference was found in women. Meat intake by JDDM patients was significantly increased, by about 60%, for both men and women compared to JDCS patients, and intake of fish and soybean/soy products was significantly decreased in JDDM patients. In particular, changes in meat and fish intake were remarkable, with an increase of about 30 g of meat in the JDDM participants and a decrease of about 30 g of fish in the JDDM participants, for both men and women. In summary, the JDCS patients consumed more fish and soybean/soy products than meat, while the JDDM patients consumed more meat than fish and soybean/soy products; thus, the composition of the major protein food sources was reversed over the 20-year period. In addition, dairy intake decreased by about 20%, and intake of sweets and snacks was about three times higher in that interval. On the other hand, the intake of vegetables and fruits among JDDM patients was dramatically reduced by about 30% in both men and women compared with JDCS patients. Both intakes of green-yellow vegetables and other vegetables significantly decreased from the time of the JDCS survey to the JDDM survey. In particular, intake of green-yellow vegetables was markedly reduced between the two surveys by 42.0% for men and 35.0% for women. Regardless of age, meat intake increased in JDDM patients, while fish and soybean/soy products showed a decrease. However, there was no trend toward a reversal of the composition of the major protein food sources in patients aged 60 years and older. In both the under 60 and over 60 age groups, there was an increase in sweets and snacks and a decrease in fruits and total vegetable intake (Supplementary Table S3)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["2. Materials and Methods"],"string":"[\n \"2. Materials and Methods\"\n]"},"other_sections_texts":{"kind":"list like","value":["This study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study."],"string":"[\n \"This study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null],"string":"[\n null\n]"},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Materials and Methods","3. Results","4. Discussion"],"string":"[\n \"1. Introduction\",\n \"2. Materials and Methods\",\n \"3. Results\",\n \"4. Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Diet plays a central role in diabetes self-management education and support [1]. To provide effective dietary guidance and/or to establish contemporary dietary guidelines, we must understand and take into account the changes in dietary intake and pattern over time. Dietary habits, including dietary content, are strongly influenced by changes in socioeconomic conditions and the environment [2]. The National Health and Nutrition Survey in Japan revealed decreases in intakes of 151 kcal (−7.4%) of energy, 24.9 g (−8.9%) of carbohydrates, 12.7 g (−15.5%) of protein, and 3.8 g (−27.7%) of salt (as sodium chloride) over a period of 20 years (1996–2016).\nPeople with diabetes often receive nutritional guidance in the early stages of the disease, so their knowledge, as well as dietary content and habits, may differ from those who do not have diabetes. Thus, dietary changes in people with type 2 diabetes may differ from the general population. However, very few studies have investigated this change. In a study reporting on the dietary changes made by people with type 2 diabetes in the United States over the past 25 years, Casagrande et al. [3] reported 206 kcal (+12.2%) and 339 mg (+11.2%) increases in energy and sodium, respectively, as well as a 0.9 g (−8.7%) decrease in dietary fiber. In addition, according to the Dietary Survey of Type 2 Diabetes in Spain [4], although the sample size was small at 200, protein intake decreased by 6.9% in men and 1.4% in women, and fat intake increased by 3.2% in men and 5.5% in women in 2000 compared to 1993. However, as these studies focused on nutrient changes, it was unclear what changes in foods contributed to these nutrient changes. Dietary intake by food groups in those with type 2 diabetes has been previously reported [5], but to the best of our knowledge, there have been no studies on changes in dietary intake over time.\nThe diabetic population in the Asian region is rapidly increasing, with those in East Asia accounting for approximately a quarter of the world diabetes population [6]. Therefore, from the perspective of global epidemiology, it is important to elucidate changes in the nutrient intake of Asian people with diabetes over time. Previously, we reported [7] on the nutritional status of Japanese patients with type 2 diabetes from a survey conducted in 1996. However, no study thus far has examined changes in nutrient intake in the Asian diabetic population over time on a nationwide scale.\nThe purpose of this study was to reveal the detailed changes in dietary intake in Japanese patients with type 2 diabetes over a 20-year period beginning in 1996. In the present study, we conducted a nutritional survey, almost identical to our previous survey, which focused on a newly established nationwide registry of Japanese patients with type 2 diabetes. We then compared the results with those of our previous study. The results of this study reveal the importance of understanding which areas need to be focused on for efficient and effective dietary guidance, and thus for the creation of contemporary dietary guidelines. In addition, the results may provide fundamental insights into the differences in dietary advice received by patients of Asian and Western origins.","This study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study.","Table 1 shows the clinical features of participants according to registry and sex. Although statistically significant differences were found between JDCS and JDDM patients in most variables, the differences in age and diabetes duration were too small to be considered clinically different. Variables that were considered to be clinically different between registries included BMI, HbA1c, blood pressure, and smoking rate among men. The BMI was significantly higher (+3.1 kg/m2 in men and +2.7 kg/m2 in women) in JDDM participants compared to JDCS participants. Comparing the mean BMI between JDCS and JDDM participants, the proportion of obese patients defined by BMI ≥ 25 kg/m2 according to Japanese criterion was significantly greater in JDDM participants, being approximately 20% to 50% higher in men and approximately 30% to 50% higher in women. HbA1c was significantly lower by approximately 1% and systolic blood pressure by approximately 5 mmHg for both men and women among JDDM participants, and the difference in the smoking rate for men was approximately 15% lower in JDDM participants than in JDCS participants.\nWhen participants were divided into two groups based on the median age of 60 years, the characteristics were similar to those identified in the comparison of all groups, regardless of age. However, there was a dramatic increase in the BMI of JDDM participants younger than 60 years compared to JDCS participants in the same age group (Supplementary Table S1).\nTable 2 shows a comparison of daily nutrient intake between patients in both studies. In Model 2, which was adjusted for age, HbA1c, BMI, insulin use, and oral medication use, there was no significant difference in energy intake in men between the two studies, but in women, there was a slight, but significant, increase of approximately 5% in the JDDM compared to the JDCS. Regarding proportions of macronutrients, the fat-to-energy ratio increased significantly, by about 3%, and the carbohydrate-to-energy ratio and protein-to-energy ratio decreased significantly for both men and women from the JDCS to the JDDM. Regarding changes in fat intake, the proportion of energy derived from saturated fatty acids increased significantly from 7.6% to 9.0% in men and from 8.3% to 9.4% in women in the interval between the surveys, whereas the proportion of energy derived from polyunsaturated fatty acids decreased significantly. Other variables that showed nutritionally meaningful differences between the two studies included significant decreases in dietary fiber, by 1.7 g for men and 1.6 g for women, and in salt, by 1.9 g for men and 2.7 g for women. The results were similar to the comparison of all groups, with fat-to-energy ratio and saturated fatty acid-to-energy ratio significantly increased in JDDM patients regardless of age, and polyunsaturated fatty acid-to-energy ratio, dietary fiber, and salt significantly decreased in JDDM patients (Supplementary Table S2).\nTable 3 shows a comparison of daily food intakes categorized by food groups. In terms of grains intake, from the JDCS to the JDDM, there was a slight, but significant, decrease of 7.4% in men, but no significant difference was found in women. Meat intake by JDDM patients was significantly increased, by about 60%, for both men and women compared to JDCS patients, and intake of fish and soybean/soy products was significantly decreased in JDDM patients. In particular, changes in meat and fish intake were remarkable, with an increase of about 30 g of meat in the JDDM participants and a decrease of about 30 g of fish in the JDDM participants, for both men and women. In summary, the JDCS patients consumed more fish and soybean/soy products than meat, while the JDDM patients consumed more meat than fish and soybean/soy products; thus, the composition of the major protein food sources was reversed over the 20-year period. In addition, dairy intake decreased by about 20%, and intake of sweets and snacks was about three times higher in that interval. On the other hand, the intake of vegetables and fruits among JDDM patients was dramatically reduced by about 30% in both men and women compared with JDCS patients. Both intakes of green-yellow vegetables and other vegetables significantly decreased from the time of the JDCS survey to the JDDM survey. In particular, intake of green-yellow vegetables was markedly reduced between the two surveys by 42.0% for men and 35.0% for women. Regardless of age, meat intake increased in JDDM patients, while fish and soybean/soy products showed a decrease. However, there was no trend toward a reversal of the composition of the major protein food sources in patients aged 60 years and older. In both the under 60 and over 60 age groups, there was an increase in sweets and snacks and a decrease in fruits and total vegetable intake (Supplementary Table S3).","This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period using two surveys with essentially the same format. Although the backgrounds of the patients, including age and duration of diabetes, were quite similar between the two studies, BMI and obesity rates were markedly increased in the later study. Regarding dietary content, fat intake increased, including saturated fatty acids, along with significant increases in the consumption of meat and sweets and snacks, and a significant decrease in vegetable and fish intake. To date, there have been no studies on the changes in dietary intake by persons with type 2 diabetes in Asia, except for a report on adherence to dietary recommendations in Korea [12]. To our knowledge, the changes in nutrient and food intake have not been reported. Therefore, this is the first study to clarify the secular changes in the dietary status of patients with type 2 diabetes in Asia.\nSelf-management education and support for diet and physical activity are essential in the care of overweight or obese people with type 2 diabetes. A previous study observing the secular trends in the United States from 1988 to 2012 [3] showed a trend of increases in BMI and energy intake in patients with type 2 diabetes, which was also the case even in adults without diabetes. On the other hand, in our study of East Asians, the BMI markedly increased from 22.7 kg/m2 to 25.8 kg/m2 in men and 23.2 kg/m2 to 25.9 kg/m2 in women, although no such remarkable changes were found in the Japanese general population according to the Japan National Health and Nutrition Survey performed during a similar period (1996 and 2016) (from 23.3 kg/ m2 to 24.0 kg/m2 in men and 23.2 kg m2 to 22.6 kg/m2 in women) [13]. This suggests that secular changes in BMI and obesity were different between people with and without type 2 diabetes and between the East and the West. According to the National Health and Nutrition Surveys, from 1996 to 2016, the energy intake by the general population aged 40–69 years mildly declined from 2284 kcal to 2145 kcal (−139 kcal) for men and from 1880 kcal to 1722 kcal (−158 kcal) for women, although the dietary survey method was different from our method [14].\nWe previously reported, using JDCS baseline data, that Western populations with diabetes are mostly obese and are also more obese than the general population, whereas Japanese people with diabetes not only have a mean BMI within the normal range, but also it is almost the same as in the general population [15]. The results of the current study, including the JDDM results 20 years after the initial JDCS survey, strongly support the fact that obesity among those with type 2 diabetes has recently increased in Japan, as is the case in Western countries [16]. On the other hand, it is not clear from this study alone why the increase in BMI in Japanese patients with type 2 diabetes was significantly greater than in the general population, even though there was no significant increase in energy intake between the two studies. Our previous study [17] suggested that there are racial differences in the relationship between energy intake and obesity, but several other hypotheses are also possible.\nThe first possible explanation for the significant increase in BMI without a significant increase in energy intake in Japanese patients with type 2 diabetes may be the effect of decreased energy expenditure due to decreased physical activity. A comparison of the activity levels between people with diabetes and the general population found no significant difference in physical activity levels and leisure-time physical activity patterns, and reported that neither groups achieved the recommended physical activity goals [18,19]. The decrease in the number of steps by the general Japanese population from 1996 to 2016 was remarkable for both men and women [13], and it was pointed out that the decrease in physical activity was due to the development of transportation [20]. In this study, direct comparisons were difficult because of the differences in the studies’ survey methods regarding the amount of physical activity; however, 44.4% of patients in the JDDM reported low levels of overall physical activity in their daily lives, which was calculated from activity time and activity intensity. Future research on secular trends in the amount of physical activity in patients with type 2 diabetes is expected.\nThe second theory as to why BMI increased without an increase in energy intake is that it could be due to the effect of smoking rates and medications. The BMI of smokers was reportedly lower than that of non-smokers [21,22], and the smoking rate of men in the JDDM with a high BMI was significantly lower than that in the JDCS. In addition, the increase in the rate of insulin use over time that was observed between these studies may be a factor in weight gain. The fact that there was a significant decrease in the rate of use of oral hypoglycemic agents may have contributed to weight gain or weight loss, depending on the type of oral hypoglycemic agent [23]. Further studies, including a detailed follow-up of the status of the medications, are necessary.\nAs a third conjecture, the ratio of macronutrients may have affected these findings. Regarding the energy-producing nutrients, both men and women in the JDDM had higher fat-to-energy ratios than those in the JDCS. In the JDDM compared to the JDCS, men had a lower carbohydrate-to-energy ratio and protein-to-energy ratio and women had a lower protein-to-energy ratio. Women in the JDDM had an increase in energy intake, which could be attributed to an increased fat intake as their carbohydrate and protein intakes did not increase. In the Japanese National Health and Nutrition Survey, which was of a similar duration (1996 and 2016), the fat-to-energy ratio increased (men: +2.6%, women: +2.9%), but carbohydrate and protein intakes tended to decrease (carbohydrate: −22.6 g/day for men, −33.4 g/day for women, protein: −15.9 g/day for men, −13.0 g/day for women), and fat intake was almost unchanged, suggesting that the fat-to-energy ratio was relatively increased. Increased fat-to-energy ratios due to an increased fat intake in patients with type 2 diabetes may have contributed to the increased BMI. However, previous studies have not provided a unified consensus on the association between the fat-to-energy ratio and obesity, which needs to be clarified in the future [24].\nIt is well known that, regardless of body weight, a high-fat diet, especially an excessive intake of saturated fatty acids and a high saturated fatty acid-to-energy ratio, is significantly associated with a higher risk of cardiovascular events in patients with type 2 diabetes [25]. According to the Japanese practice guidelines for diabetes [26], a reduction in saturated fatty acids is recommended when the lipid-to-energy ratio exceeds 25%. In addition, Dietary Reference Intakes for Japanese [27] set a target saturated fatty acid-to-energy ratio of ≤7%. Among our patients with a fat-to-energy ratio of ≥25%, only 9.5% of JDCS patients and 4.4% of JDDM patients had a saturated fatty acid-to-energy ratio of ≤7%, suggesting the necessity for enhanced guidance to reduce the saturated fatty acid-to-energy ratio. The saturated fatty acid-to-energy ratio for Americans with type 2 diabetes was approximately 11%, which was still higher than the level (<10%) recommended by the U.S. Dietary Guidelines, but it did not increase between 1988 and 2012 [28].\nIn contrast, in our Japanese patients, the saturated fatty acid-to-energy ratio increased significantly over the 20 years to approximately 9% in both men and women, and the percentage of patients with a saturated fatty acid-to-energy ratio of more than 10% increased significantly from 8.2% in men and 13.3% in women in the JDCS to 28.4% in men and 36.6% in women in the JDDM. This suggests that the fat intake pattern of Japanese people with type 2 diabetes is approaching that of Western patients. Therefore, correcting excessive lipid intake while taking into account the content and quality of lipids is considered to be a crucial issue.\nIn terms of secular changes in food group intake patterns, an increase in meat intake and a decrease in the consumption of fish and dairy products were observed as sources of protein intake. JDCS patients had higher intakes of fish and soybeans/soy products than meat, whereas JDDM patients had higher intakes of meat than fish and soybeans/soy products. The National Health and Nutrition Survey [29] also showed the same secular changes in food group intake patterns as this study.\nIt is indicated that patients with type 2 diabetes in Japan had a major change in the composition of their protein sources, as did the general Japanese population, which was a change to a meat-centered diet. In general, among people in Western countries, the circumstances in which meat intake is higher than fish intake continues. It is inferred that the diet of Japanese patients with type 2 diabetes has been transformed into a diet centered on meat due to the influx of various lifestyle patterns and dietary cultures from Western countries, as is the case with the general Japanese population. It has been reported that meat was associated with cardiovascular complications and mortality in people with type 2 diabetes in a study in the US [30] and in our previous study [31]. Thus, it is considered necessary to strengthen the guidance on protein sources to prevent complications in the future.\nMeat is the food with the highest contribution to fat intake by Japanese people. Therefore, it is thought that an increased meat intake may also affect fat-to-energy ratios and saturated fatty acid increases in JDDM patients. Aside from meat, saturated fatty acids are found in the Japanese diet in milk, butter, and coconut oil, which are commonly used as ingredients in Western sweets rather than in traditional Japanese ones (which are mostly made with sugar, rice, and beans [32]). Although the type of sweets could not be identified in this survey, as their intake more than doubled from the JDCS to the JDDM, it is inferred that in conjunction with meat, the increase in the intake of sweets contributed to the increase in saturated fatty acid consumption.\nContrary to meat, the intake of fruits and vegetables decreased significantly between the two studies. As vegetables and fruits are good sources of dietary fiber, fiber intake decreased significantly as vegetable and fruit intakes decreased. A higher intake of dietary fiber is known to improve glycemic control, and we previously reported that it was significantly associated with a reduced risk of stroke [33] and retinopathy [34] in JDCS patients. However, despite its importance, the intake of dietary fiber was lower than the recommended goal of 20 g/day or higher in 1996, and even declined in the following 20 years. A decreased intake of fruits and vegetables is apparent in the Japanese general population [29], and is considered to be an important issue in dietary guidance.\nAs to the background of the decrease in the consumption of vegetables and fruits, survey reports have suggested that differences in lifestyle, such as economic conditions [35] and living alone, exist [36] and that diabetic patients who work may choose a less diversified diet [37]. It was also reported that the consumption of vegetables and fruits tends to be higher with a lower frequency of eating meals away from home [38]. As to the proportions of food expenditure in Japan from 1995 to 2015, the consumption of fresh foods has decreased, and eating out and consuming processed foods have increased from 65.4% to 72.6% [39]. This suggests that the increase in eating out and buying processed foods may influence the decrease in fruit and vegetable intake, not only in the general population, but also in those with type 2 diabetes. This Westernization of dietary habits has been widely adopted, and advice on the dietary treatment of diabetes should consider the current dietary situation.\nThis study has several limitations. First, both the JDCS and the JDDM were comprised of patients from different cohorts, although they were both multicenter studies. However, both studies were conducted at outpatient clinics throughout Japan specializing in diabetes, and the clinical settings were similar except for the time period studied. An additional limitation is that the number of participants that were included in both surveys was different from the potential candidates. Of the 2033 JDCS patients, 1588 responded to a baseline dietary survey, and 524 patients who had missing values in the dietary survey were excluded. In the JDDM patients, 1145 of 2337 patients met the eligibility criteria for the JDCS and responded to the baseline dietary survey. However, there were no notable differences in baseline characteristics between the responders and non-responders [8], and consequently, no selection bias was found. Finally, the participants’ dietary intake was obtained from a self-administered questionnaire, which could be an underestimation. As for the long-term comparison of 20 years, changes had been made in the food composition tables and in the FFQg version used in both studies. However, those changes corresponded to changes in dietary habits and food ingredients due to secular trends, and there was little effect on energy intake and intake of macronutrients due to the changes in the food composition tables.\nThe results of this study clarify the changes in the dietary intake of Japanese patients with type 2 diabetes over 20 years, beginning in 1996. Despite a lack of significant change in the total energy intake during the period, both men and women with type 2 diabetes had a large increase in BMI. A rise in the fat-to-energy ratio due to an increase in fat intake was also observed, particularly in men. In terms of intake by food group, both men and women had clearly unfavorable changes in terms of complication prevention. These included an increase in the intake of meat and a decrease in the intake of fish, soybean/soy products, vegetables, and fruits. The results of our study strongly suggest that, in order to prevent complications in Japanese patients with type 2 diabetes in the future, the provision of nutritional guidance, in line with these chronological changes in dietary intake, is necessary."],"string":"[\n \"Diet plays a central role in diabetes self-management education and support [1]. To provide effective dietary guidance and/or to establish contemporary dietary guidelines, we must understand and take into account the changes in dietary intake and pattern over time. Dietary habits, including dietary content, are strongly influenced by changes in socioeconomic conditions and the environment [2]. The National Health and Nutrition Survey in Japan revealed decreases in intakes of 151 kcal (−7.4%) of energy, 24.9 g (−8.9%) of carbohydrates, 12.7 g (−15.5%) of protein, and 3.8 g (−27.7%) of salt (as sodium chloride) over a period of 20 years (1996–2016).\\nPeople with diabetes often receive nutritional guidance in the early stages of the disease, so their knowledge, as well as dietary content and habits, may differ from those who do not have diabetes. Thus, dietary changes in people with type 2 diabetes may differ from the general population. However, very few studies have investigated this change. In a study reporting on the dietary changes made by people with type 2 diabetes in the United States over the past 25 years, Casagrande et al. [3] reported 206 kcal (+12.2%) and 339 mg (+11.2%) increases in energy and sodium, respectively, as well as a 0.9 g (−8.7%) decrease in dietary fiber. In addition, according to the Dietary Survey of Type 2 Diabetes in Spain [4], although the sample size was small at 200, protein intake decreased by 6.9% in men and 1.4% in women, and fat intake increased by 3.2% in men and 5.5% in women in 2000 compared to 1993. However, as these studies focused on nutrient changes, it was unclear what changes in foods contributed to these nutrient changes. Dietary intake by food groups in those with type 2 diabetes has been previously reported [5], but to the best of our knowledge, there have been no studies on changes in dietary intake over time.\\nThe diabetic population in the Asian region is rapidly increasing, with those in East Asia accounting for approximately a quarter of the world diabetes population [6]. Therefore, from the perspective of global epidemiology, it is important to elucidate changes in the nutrient intake of Asian people with diabetes over time. Previously, we reported [7] on the nutritional status of Japanese patients with type 2 diabetes from a survey conducted in 1996. However, no study thus far has examined changes in nutrient intake in the Asian diabetic population over time on a nationwide scale.\\nThe purpose of this study was to reveal the detailed changes in dietary intake in Japanese patients with type 2 diabetes over a 20-year period beginning in 1996. In the present study, we conducted a nutritional survey, almost identical to our previous survey, which focused on a newly established nationwide registry of Japanese patients with type 2 diabetes. We then compared the results with those of our previous study. The results of this study reveal the importance of understanding which areas need to be focused on for efficient and effective dietary guidance, and thus for the creation of contemporary dietary guidelines. In addition, the results may provide fundamental insights into the differences in dietary advice received by patients of Asian and Western origins.\",\n \"This study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study.\",\n \"Table 1 shows the clinical features of participants according to registry and sex. Although statistically significant differences were found between JDCS and JDDM patients in most variables, the differences in age and diabetes duration were too small to be considered clinically different. Variables that were considered to be clinically different between registries included BMI, HbA1c, blood pressure, and smoking rate among men. The BMI was significantly higher (+3.1 kg/m2 in men and +2.7 kg/m2 in women) in JDDM participants compared to JDCS participants. Comparing the mean BMI between JDCS and JDDM participants, the proportion of obese patients defined by BMI ≥ 25 kg/m2 according to Japanese criterion was significantly greater in JDDM participants, being approximately 20% to 50% higher in men and approximately 30% to 50% higher in women. HbA1c was significantly lower by approximately 1% and systolic blood pressure by approximately 5 mmHg for both men and women among JDDM participants, and the difference in the smoking rate for men was approximately 15% lower in JDDM participants than in JDCS participants.\\nWhen participants were divided into two groups based on the median age of 60 years, the characteristics were similar to those identified in the comparison of all groups, regardless of age. However, there was a dramatic increase in the BMI of JDDM participants younger than 60 years compared to JDCS participants in the same age group (Supplementary Table S1).\\nTable 2 shows a comparison of daily nutrient intake between patients in both studies. In Model 2, which was adjusted for age, HbA1c, BMI, insulin use, and oral medication use, there was no significant difference in energy intake in men between the two studies, but in women, there was a slight, but significant, increase of approximately 5% in the JDDM compared to the JDCS. Regarding proportions of macronutrients, the fat-to-energy ratio increased significantly, by about 3%, and the carbohydrate-to-energy ratio and protein-to-energy ratio decreased significantly for both men and women from the JDCS to the JDDM. Regarding changes in fat intake, the proportion of energy derived from saturated fatty acids increased significantly from 7.6% to 9.0% in men and from 8.3% to 9.4% in women in the interval between the surveys, whereas the proportion of energy derived from polyunsaturated fatty acids decreased significantly. Other variables that showed nutritionally meaningful differences between the two studies included significant decreases in dietary fiber, by 1.7 g for men and 1.6 g for women, and in salt, by 1.9 g for men and 2.7 g for women. The results were similar to the comparison of all groups, with fat-to-energy ratio and saturated fatty acid-to-energy ratio significantly increased in JDDM patients regardless of age, and polyunsaturated fatty acid-to-energy ratio, dietary fiber, and salt significantly decreased in JDDM patients (Supplementary Table S2).\\nTable 3 shows a comparison of daily food intakes categorized by food groups. In terms of grains intake, from the JDCS to the JDDM, there was a slight, but significant, decrease of 7.4% in men, but no significant difference was found in women. Meat intake by JDDM patients was significantly increased, by about 60%, for both men and women compared to JDCS patients, and intake of fish and soybean/soy products was significantly decreased in JDDM patients. In particular, changes in meat and fish intake were remarkable, with an increase of about 30 g of meat in the JDDM participants and a decrease of about 30 g of fish in the JDDM participants, for both men and women. In summary, the JDCS patients consumed more fish and soybean/soy products than meat, while the JDDM patients consumed more meat than fish and soybean/soy products; thus, the composition of the major protein food sources was reversed over the 20-year period. In addition, dairy intake decreased by about 20%, and intake of sweets and snacks was about three times higher in that interval. On the other hand, the intake of vegetables and fruits among JDDM patients was dramatically reduced by about 30% in both men and women compared with JDCS patients. Both intakes of green-yellow vegetables and other vegetables significantly decreased from the time of the JDCS survey to the JDDM survey. In particular, intake of green-yellow vegetables was markedly reduced between the two surveys by 42.0% for men and 35.0% for women. Regardless of age, meat intake increased in JDDM patients, while fish and soybean/soy products showed a decrease. However, there was no trend toward a reversal of the composition of the major protein food sources in patients aged 60 years and older. In both the under 60 and over 60 age groups, there was an increase in sweets and snacks and a decrease in fruits and total vegetable intake (Supplementary Table S3).\",\n \"This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period using two surveys with essentially the same format. Although the backgrounds of the patients, including age and duration of diabetes, were quite similar between the two studies, BMI and obesity rates were markedly increased in the later study. Regarding dietary content, fat intake increased, including saturated fatty acids, along with significant increases in the consumption of meat and sweets and snacks, and a significant decrease in vegetable and fish intake. To date, there have been no studies on the changes in dietary intake by persons with type 2 diabetes in Asia, except for a report on adherence to dietary recommendations in Korea [12]. To our knowledge, the changes in nutrient and food intake have not been reported. Therefore, this is the first study to clarify the secular changes in the dietary status of patients with type 2 diabetes in Asia.\\nSelf-management education and support for diet and physical activity are essential in the care of overweight or obese people with type 2 diabetes. A previous study observing the secular trends in the United States from 1988 to 2012 [3] showed a trend of increases in BMI and energy intake in patients with type 2 diabetes, which was also the case even in adults without diabetes. On the other hand, in our study of East Asians, the BMI markedly increased from 22.7 kg/m2 to 25.8 kg/m2 in men and 23.2 kg/m2 to 25.9 kg/m2 in women, although no such remarkable changes were found in the Japanese general population according to the Japan National Health and Nutrition Survey performed during a similar period (1996 and 2016) (from 23.3 kg/ m2 to 24.0 kg/m2 in men and 23.2 kg m2 to 22.6 kg/m2 in women) [13]. This suggests that secular changes in BMI and obesity were different between people with and without type 2 diabetes and between the East and the West. According to the National Health and Nutrition Surveys, from 1996 to 2016, the energy intake by the general population aged 40–69 years mildly declined from 2284 kcal to 2145 kcal (−139 kcal) for men and from 1880 kcal to 1722 kcal (−158 kcal) for women, although the dietary survey method was different from our method [14].\\nWe previously reported, using JDCS baseline data, that Western populations with diabetes are mostly obese and are also more obese than the general population, whereas Japanese people with diabetes not only have a mean BMI within the normal range, but also it is almost the same as in the general population [15]. The results of the current study, including the JDDM results 20 years after the initial JDCS survey, strongly support the fact that obesity among those with type 2 diabetes has recently increased in Japan, as is the case in Western countries [16]. On the other hand, it is not clear from this study alone why the increase in BMI in Japanese patients with type 2 diabetes was significantly greater than in the general population, even though there was no significant increase in energy intake between the two studies. Our previous study [17] suggested that there are racial differences in the relationship between energy intake and obesity, but several other hypotheses are also possible.\\nThe first possible explanation for the significant increase in BMI without a significant increase in energy intake in Japanese patients with type 2 diabetes may be the effect of decreased energy expenditure due to decreased physical activity. A comparison of the activity levels between people with diabetes and the general population found no significant difference in physical activity levels and leisure-time physical activity patterns, and reported that neither groups achieved the recommended physical activity goals [18,19]. The decrease in the number of steps by the general Japanese population from 1996 to 2016 was remarkable for both men and women [13], and it was pointed out that the decrease in physical activity was due to the development of transportation [20]. In this study, direct comparisons were difficult because of the differences in the studies’ survey methods regarding the amount of physical activity; however, 44.4% of patients in the JDDM reported low levels of overall physical activity in their daily lives, which was calculated from activity time and activity intensity. Future research on secular trends in the amount of physical activity in patients with type 2 diabetes is expected.\\nThe second theory as to why BMI increased without an increase in energy intake is that it could be due to the effect of smoking rates and medications. The BMI of smokers was reportedly lower than that of non-smokers [21,22], and the smoking rate of men in the JDDM with a high BMI was significantly lower than that in the JDCS. In addition, the increase in the rate of insulin use over time that was observed between these studies may be a factor in weight gain. The fact that there was a significant decrease in the rate of use of oral hypoglycemic agents may have contributed to weight gain or weight loss, depending on the type of oral hypoglycemic agent [23]. Further studies, including a detailed follow-up of the status of the medications, are necessary.\\nAs a third conjecture, the ratio of macronutrients may have affected these findings. Regarding the energy-producing nutrients, both men and women in the JDDM had higher fat-to-energy ratios than those in the JDCS. In the JDDM compared to the JDCS, men had a lower carbohydrate-to-energy ratio and protein-to-energy ratio and women had a lower protein-to-energy ratio. Women in the JDDM had an increase in energy intake, which could be attributed to an increased fat intake as their carbohydrate and protein intakes did not increase. In the Japanese National Health and Nutrition Survey, which was of a similar duration (1996 and 2016), the fat-to-energy ratio increased (men: +2.6%, women: +2.9%), but carbohydrate and protein intakes tended to decrease (carbohydrate: −22.6 g/day for men, −33.4 g/day for women, protein: −15.9 g/day for men, −13.0 g/day for women), and fat intake was almost unchanged, suggesting that the fat-to-energy ratio was relatively increased. Increased fat-to-energy ratios due to an increased fat intake in patients with type 2 diabetes may have contributed to the increased BMI. However, previous studies have not provided a unified consensus on the association between the fat-to-energy ratio and obesity, which needs to be clarified in the future [24].\\nIt is well known that, regardless of body weight, a high-fat diet, especially an excessive intake of saturated fatty acids and a high saturated fatty acid-to-energy ratio, is significantly associated with a higher risk of cardiovascular events in patients with type 2 diabetes [25]. According to the Japanese practice guidelines for diabetes [26], a reduction in saturated fatty acids is recommended when the lipid-to-energy ratio exceeds 25%. In addition, Dietary Reference Intakes for Japanese [27] set a target saturated fatty acid-to-energy ratio of ≤7%. Among our patients with a fat-to-energy ratio of ≥25%, only 9.5% of JDCS patients and 4.4% of JDDM patients had a saturated fatty acid-to-energy ratio of ≤7%, suggesting the necessity for enhanced guidance to reduce the saturated fatty acid-to-energy ratio. The saturated fatty acid-to-energy ratio for Americans with type 2 diabetes was approximately 11%, which was still higher than the level (<10%) recommended by the U.S. Dietary Guidelines, but it did not increase between 1988 and 2012 [28].\\nIn contrast, in our Japanese patients, the saturated fatty acid-to-energy ratio increased significantly over the 20 years to approximately 9% in both men and women, and the percentage of patients with a saturated fatty acid-to-energy ratio of more than 10% increased significantly from 8.2% in men and 13.3% in women in the JDCS to 28.4% in men and 36.6% in women in the JDDM. This suggests that the fat intake pattern of Japanese people with type 2 diabetes is approaching that of Western patients. Therefore, correcting excessive lipid intake while taking into account the content and quality of lipids is considered to be a crucial issue.\\nIn terms of secular changes in food group intake patterns, an increase in meat intake and a decrease in the consumption of fish and dairy products were observed as sources of protein intake. JDCS patients had higher intakes of fish and soybeans/soy products than meat, whereas JDDM patients had higher intakes of meat than fish and soybeans/soy products. The National Health and Nutrition Survey [29] also showed the same secular changes in food group intake patterns as this study.\\nIt is indicated that patients with type 2 diabetes in Japan had a major change in the composition of their protein sources, as did the general Japanese population, which was a change to a meat-centered diet. In general, among people in Western countries, the circumstances in which meat intake is higher than fish intake continues. It is inferred that the diet of Japanese patients with type 2 diabetes has been transformed into a diet centered on meat due to the influx of various lifestyle patterns and dietary cultures from Western countries, as is the case with the general Japanese population. It has been reported that meat was associated with cardiovascular complications and mortality in people with type 2 diabetes in a study in the US [30] and in our previous study [31]. Thus, it is considered necessary to strengthen the guidance on protein sources to prevent complications in the future.\\nMeat is the food with the highest contribution to fat intake by Japanese people. Therefore, it is thought that an increased meat intake may also affect fat-to-energy ratios and saturated fatty acid increases in JDDM patients. Aside from meat, saturated fatty acids are found in the Japanese diet in milk, butter, and coconut oil, which are commonly used as ingredients in Western sweets rather than in traditional Japanese ones (which are mostly made with sugar, rice, and beans [32]). Although the type of sweets could not be identified in this survey, as their intake more than doubled from the JDCS to the JDDM, it is inferred that in conjunction with meat, the increase in the intake of sweets contributed to the increase in saturated fatty acid consumption.\\nContrary to meat, the intake of fruits and vegetables decreased significantly between the two studies. As vegetables and fruits are good sources of dietary fiber, fiber intake decreased significantly as vegetable and fruit intakes decreased. A higher intake of dietary fiber is known to improve glycemic control, and we previously reported that it was significantly associated with a reduced risk of stroke [33] and retinopathy [34] in JDCS patients. However, despite its importance, the intake of dietary fiber was lower than the recommended goal of 20 g/day or higher in 1996, and even declined in the following 20 years. A decreased intake of fruits and vegetables is apparent in the Japanese general population [29], and is considered to be an important issue in dietary guidance.\\nAs to the background of the decrease in the consumption of vegetables and fruits, survey reports have suggested that differences in lifestyle, such as economic conditions [35] and living alone, exist [36] and that diabetic patients who work may choose a less diversified diet [37]. It was also reported that the consumption of vegetables and fruits tends to be higher with a lower frequency of eating meals away from home [38]. As to the proportions of food expenditure in Japan from 1995 to 2015, the consumption of fresh foods has decreased, and eating out and consuming processed foods have increased from 65.4% to 72.6% [39]. This suggests that the increase in eating out and buying processed foods may influence the decrease in fruit and vegetable intake, not only in the general population, but also in those with type 2 diabetes. This Westernization of dietary habits has been widely adopted, and advice on the dietary treatment of diabetes should consider the current dietary situation.\\nThis study has several limitations. First, both the JDCS and the JDDM were comprised of patients from different cohorts, although they were both multicenter studies. However, both studies were conducted at outpatient clinics throughout Japan specializing in diabetes, and the clinical settings were similar except for the time period studied. An additional limitation is that the number of participants that were included in both surveys was different from the potential candidates. Of the 2033 JDCS patients, 1588 responded to a baseline dietary survey, and 524 patients who had missing values in the dietary survey were excluded. In the JDDM patients, 1145 of 2337 patients met the eligibility criteria for the JDCS and responded to the baseline dietary survey. However, there were no notable differences in baseline characteristics between the responders and non-responders [8], and consequently, no selection bias was found. Finally, the participants’ dietary intake was obtained from a self-administered questionnaire, which could be an underestimation. As for the long-term comparison of 20 years, changes had been made in the food composition tables and in the FFQg version used in both studies. However, those changes corresponded to changes in dietary habits and food ingredients due to secular trends, and there was little effect on energy intake and intake of macronutrients due to the changes in the food composition tables.\\nThe results of this study clarify the changes in the dietary intake of Japanese patients with type 2 diabetes over 20 years, beginning in 1996. Despite a lack of significant change in the total energy intake during the period, both men and women with type 2 diabetes had a large increase in BMI. A rise in the fat-to-energy ratio due to an increase in fat intake was also observed, particularly in men. In terms of intake by food group, both men and women had clearly unfavorable changes in terms of complication prevention. These included an increase in the intake of meat and a decrease in the intake of fish, soybean/soy products, vegetables, and fruits. The results of our study strongly suggest that, in order to prevent complications in Japanese patients with type 2 diabetes in the future, the provision of nutritional guidance, in line with these chronological changes in dietary intake, is necessary.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,"results","discussion"],"string":"[\n \"intro\",\n null,\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["food intake","type 2 diabetes","obesity","diabetes mellitus","Asia"],"string":"[\n \"food intake\",\n \"type 2 diabetes\",\n \"obesity\",\n \"diabetes mellitus\",\n \"Asia\"\n]"},"whole_article_text":{"kind":"string","value":"1. Introduction:\nDiet plays a central role in diabetes self-management education and support [1]. To provide effective dietary guidance and/or to establish contemporary dietary guidelines, we must understand and take into account the changes in dietary intake and pattern over time. Dietary habits, including dietary content, are strongly influenced by changes in socioeconomic conditions and the environment [2]. The National Health and Nutrition Survey in Japan revealed decreases in intakes of 151 kcal (−7.4%) of energy, 24.9 g (−8.9%) of carbohydrates, 12.7 g (−15.5%) of protein, and 3.8 g (−27.7%) of salt (as sodium chloride) over a period of 20 years (1996–2016).\nPeople with diabetes often receive nutritional guidance in the early stages of the disease, so their knowledge, as well as dietary content and habits, may differ from those who do not have diabetes. Thus, dietary changes in people with type 2 diabetes may differ from the general population. However, very few studies have investigated this change. In a study reporting on the dietary changes made by people with type 2 diabetes in the United States over the past 25 years, Casagrande et al. [3] reported 206 kcal (+12.2%) and 339 mg (+11.2%) increases in energy and sodium, respectively, as well as a 0.9 g (−8.7%) decrease in dietary fiber. In addition, according to the Dietary Survey of Type 2 Diabetes in Spain [4], although the sample size was small at 200, protein intake decreased by 6.9% in men and 1.4% in women, and fat intake increased by 3.2% in men and 5.5% in women in 2000 compared to 1993. However, as these studies focused on nutrient changes, it was unclear what changes in foods contributed to these nutrient changes. Dietary intake by food groups in those with type 2 diabetes has been previously reported [5], but to the best of our knowledge, there have been no studies on changes in dietary intake over time.\nThe diabetic population in the Asian region is rapidly increasing, with those in East Asia accounting for approximately a quarter of the world diabetes population [6]. Therefore, from the perspective of global epidemiology, it is important to elucidate changes in the nutrient intake of Asian people with diabetes over time. Previously, we reported [7] on the nutritional status of Japanese patients with type 2 diabetes from a survey conducted in 1996. However, no study thus far has examined changes in nutrient intake in the Asian diabetic population over time on a nationwide scale.\nThe purpose of this study was to reveal the detailed changes in dietary intake in Japanese patients with type 2 diabetes over a 20-year period beginning in 1996. In the present study, we conducted a nutritional survey, almost identical to our previous survey, which focused on a newly established nationwide registry of Japanese patients with type 2 diabetes. We then compared the results with those of our previous study. The results of this study reveal the importance of understanding which areas need to be focused on for efficient and effective dietary guidance, and thus for the creation of contemporary dietary guidelines. In addition, the results may provide fundamental insights into the differences in dietary advice received by patients of Asian and Western origins.\n\n2. Materials and Methods:\nThis study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study.\n\n3. Results:\nTable 1 shows the clinical features of participants according to registry and sex. Although statistically significant differences were found between JDCS and JDDM patients in most variables, the differences in age and diabetes duration were too small to be considered clinically different. Variables that were considered to be clinically different between registries included BMI, HbA1c, blood pressure, and smoking rate among men. The BMI was significantly higher (+3.1 kg/m2 in men and +2.7 kg/m2 in women) in JDDM participants compared to JDCS participants. Comparing the mean BMI between JDCS and JDDM participants, the proportion of obese patients defined by BMI ≥ 25 kg/m2 according to Japanese criterion was significantly greater in JDDM participants, being approximately 20% to 50% higher in men and approximately 30% to 50% higher in women. HbA1c was significantly lower by approximately 1% and systolic blood pressure by approximately 5 mmHg for both men and women among JDDM participants, and the difference in the smoking rate for men was approximately 15% lower in JDDM participants than in JDCS participants.\nWhen participants were divided into two groups based on the median age of 60 years, the characteristics were similar to those identified in the comparison of all groups, regardless of age. However, there was a dramatic increase in the BMI of JDDM participants younger than 60 years compared to JDCS participants in the same age group (Supplementary Table S1).\nTable 2 shows a comparison of daily nutrient intake between patients in both studies. In Model 2, which was adjusted for age, HbA1c, BMI, insulin use, and oral medication use, there was no significant difference in energy intake in men between the two studies, but in women, there was a slight, but significant, increase of approximately 5% in the JDDM compared to the JDCS. Regarding proportions of macronutrients, the fat-to-energy ratio increased significantly, by about 3%, and the carbohydrate-to-energy ratio and protein-to-energy ratio decreased significantly for both men and women from the JDCS to the JDDM. Regarding changes in fat intake, the proportion of energy derived from saturated fatty acids increased significantly from 7.6% to 9.0% in men and from 8.3% to 9.4% in women in the interval between the surveys, whereas the proportion of energy derived from polyunsaturated fatty acids decreased significantly. Other variables that showed nutritionally meaningful differences between the two studies included significant decreases in dietary fiber, by 1.7 g for men and 1.6 g for women, and in salt, by 1.9 g for men and 2.7 g for women. The results were similar to the comparison of all groups, with fat-to-energy ratio and saturated fatty acid-to-energy ratio significantly increased in JDDM patients regardless of age, and polyunsaturated fatty acid-to-energy ratio, dietary fiber, and salt significantly decreased in JDDM patients (Supplementary Table S2).\nTable 3 shows a comparison of daily food intakes categorized by food groups. In terms of grains intake, from the JDCS to the JDDM, there was a slight, but significant, decrease of 7.4% in men, but no significant difference was found in women. Meat intake by JDDM patients was significantly increased, by about 60%, for both men and women compared to JDCS patients, and intake of fish and soybean/soy products was significantly decreased in JDDM patients. In particular, changes in meat and fish intake were remarkable, with an increase of about 30 g of meat in the JDDM participants and a decrease of about 30 g of fish in the JDDM participants, for both men and women. In summary, the JDCS patients consumed more fish and soybean/soy products than meat, while the JDDM patients consumed more meat than fish and soybean/soy products; thus, the composition of the major protein food sources was reversed over the 20-year period. In addition, dairy intake decreased by about 20%, and intake of sweets and snacks was about three times higher in that interval. On the other hand, the intake of vegetables and fruits among JDDM patients was dramatically reduced by about 30% in both men and women compared with JDCS patients. Both intakes of green-yellow vegetables and other vegetables significantly decreased from the time of the JDCS survey to the JDDM survey. In particular, intake of green-yellow vegetables was markedly reduced between the two surveys by 42.0% for men and 35.0% for women. Regardless of age, meat intake increased in JDDM patients, while fish and soybean/soy products showed a decrease. However, there was no trend toward a reversal of the composition of the major protein food sources in patients aged 60 years and older. In both the under 60 and over 60 age groups, there was an increase in sweets and snacks and a decrease in fruits and total vegetable intake (Supplementary Table S3).\n\n4. Discussion:\nThis study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period using two surveys with essentially the same format. Although the backgrounds of the patients, including age and duration of diabetes, were quite similar between the two studies, BMI and obesity rates were markedly increased in the later study. Regarding dietary content, fat intake increased, including saturated fatty acids, along with significant increases in the consumption of meat and sweets and snacks, and a significant decrease in vegetable and fish intake. To date, there have been no studies on the changes in dietary intake by persons with type 2 diabetes in Asia, except for a report on adherence to dietary recommendations in Korea [12]. To our knowledge, the changes in nutrient and food intake have not been reported. Therefore, this is the first study to clarify the secular changes in the dietary status of patients with type 2 diabetes in Asia.\nSelf-management education and support for diet and physical activity are essential in the care of overweight or obese people with type 2 diabetes. A previous study observing the secular trends in the United States from 1988 to 2012 [3] showed a trend of increases in BMI and energy intake in patients with type 2 diabetes, which was also the case even in adults without diabetes. On the other hand, in our study of East Asians, the BMI markedly increased from 22.7 kg/m2 to 25.8 kg/m2 in men and 23.2 kg/m2 to 25.9 kg/m2 in women, although no such remarkable changes were found in the Japanese general population according to the Japan National Health and Nutrition Survey performed during a similar period (1996 and 2016) (from 23.3 kg/ m2 to 24.0 kg/m2 in men and 23.2 kg m2 to 22.6 kg/m2 in women) [13]. This suggests that secular changes in BMI and obesity were different between people with and without type 2 diabetes and between the East and the West. According to the National Health and Nutrition Surveys, from 1996 to 2016, the energy intake by the general population aged 40–69 years mildly declined from 2284 kcal to 2145 kcal (−139 kcal) for men and from 1880 kcal to 1722 kcal (−158 kcal) for women, although the dietary survey method was different from our method [14].\nWe previously reported, using JDCS baseline data, that Western populations with diabetes are mostly obese and are also more obese than the general population, whereas Japanese people with diabetes not only have a mean BMI within the normal range, but also it is almost the same as in the general population [15]. The results of the current study, including the JDDM results 20 years after the initial JDCS survey, strongly support the fact that obesity among those with type 2 diabetes has recently increased in Japan, as is the case in Western countries [16]. On the other hand, it is not clear from this study alone why the increase in BMI in Japanese patients with type 2 diabetes was significantly greater than in the general population, even though there was no significant increase in energy intake between the two studies. Our previous study [17] suggested that there are racial differences in the relationship between energy intake and obesity, but several other hypotheses are also possible.\nThe first possible explanation for the significant increase in BMI without a significant increase in energy intake in Japanese patients with type 2 diabetes may be the effect of decreased energy expenditure due to decreased physical activity. A comparison of the activity levels between people with diabetes and the general population found no significant difference in physical activity levels and leisure-time physical activity patterns, and reported that neither groups achieved the recommended physical activity goals [18,19]. The decrease in the number of steps by the general Japanese population from 1996 to 2016 was remarkable for both men and women [13], and it was pointed out that the decrease in physical activity was due to the development of transportation [20]. In this study, direct comparisons were difficult because of the differences in the studies’ survey methods regarding the amount of physical activity; however, 44.4% of patients in the JDDM reported low levels of overall physical activity in their daily lives, which was calculated from activity time and activity intensity. Future research on secular trends in the amount of physical activity in patients with type 2 diabetes is expected.\nThe second theory as to why BMI increased without an increase in energy intake is that it could be due to the effect of smoking rates and medications. The BMI of smokers was reportedly lower than that of non-smokers [21,22], and the smoking rate of men in the JDDM with a high BMI was significantly lower than that in the JDCS. In addition, the increase in the rate of insulin use over time that was observed between these studies may be a factor in weight gain. The fact that there was a significant decrease in the rate of use of oral hypoglycemic agents may have contributed to weight gain or weight loss, depending on the type of oral hypoglycemic agent [23]. Further studies, including a detailed follow-up of the status of the medications, are necessary.\nAs a third conjecture, the ratio of macronutrients may have affected these findings. Regarding the energy-producing nutrients, both men and women in the JDDM had higher fat-to-energy ratios than those in the JDCS. In the JDDM compared to the JDCS, men had a lower carbohydrate-to-energy ratio and protein-to-energy ratio and women had a lower protein-to-energy ratio. Women in the JDDM had an increase in energy intake, which could be attributed to an increased fat intake as their carbohydrate and protein intakes did not increase. In the Japanese National Health and Nutrition Survey, which was of a similar duration (1996 and 2016), the fat-to-energy ratio increased (men: +2.6%, women: +2.9%), but carbohydrate and protein intakes tended to decrease (carbohydrate: −22.6 g/day for men, −33.4 g/day for women, protein: −15.9 g/day for men, −13.0 g/day for women), and fat intake was almost unchanged, suggesting that the fat-to-energy ratio was relatively increased. Increased fat-to-energy ratios due to an increased fat intake in patients with type 2 diabetes may have contributed to the increased BMI. However, previous studies have not provided a unified consensus on the association between the fat-to-energy ratio and obesity, which needs to be clarified in the future [24].\nIt is well known that, regardless of body weight, a high-fat diet, especially an excessive intake of saturated fatty acids and a high saturated fatty acid-to-energy ratio, is significantly associated with a higher risk of cardiovascular events in patients with type 2 diabetes [25]. According to the Japanese practice guidelines for diabetes [26], a reduction in saturated fatty acids is recommended when the lipid-to-energy ratio exceeds 25%. In addition, Dietary Reference Intakes for Japanese [27] set a target saturated fatty acid-to-energy ratio of ≤7%. Among our patients with a fat-to-energy ratio of ≥25%, only 9.5% of JDCS patients and 4.4% of JDDM patients had a saturated fatty acid-to-energy ratio of ≤7%, suggesting the necessity for enhanced guidance to reduce the saturated fatty acid-to-energy ratio. The saturated fatty acid-to-energy ratio for Americans with type 2 diabetes was approximately 11%, which was still higher than the level (<10%) recommended by the U.S. Dietary Guidelines, but it did not increase between 1988 and 2012 [28].\nIn contrast, in our Japanese patients, the saturated fatty acid-to-energy ratio increased significantly over the 20 years to approximately 9% in both men and women, and the percentage of patients with a saturated fatty acid-to-energy ratio of more than 10% increased significantly from 8.2% in men and 13.3% in women in the JDCS to 28.4% in men and 36.6% in women in the JDDM. This suggests that the fat intake pattern of Japanese people with type 2 diabetes is approaching that of Western patients. Therefore, correcting excessive lipid intake while taking into account the content and quality of lipids is considered to be a crucial issue.\nIn terms of secular changes in food group intake patterns, an increase in meat intake and a decrease in the consumption of fish and dairy products were observed as sources of protein intake. JDCS patients had higher intakes of fish and soybeans/soy products than meat, whereas JDDM patients had higher intakes of meat than fish and soybeans/soy products. The National Health and Nutrition Survey [29] also showed the same secular changes in food group intake patterns as this study.\nIt is indicated that patients with type 2 diabetes in Japan had a major change in the composition of their protein sources, as did the general Japanese population, which was a change to a meat-centered diet. In general, among people in Western countries, the circumstances in which meat intake is higher than fish intake continues. It is inferred that the diet of Japanese patients with type 2 diabetes has been transformed into a diet centered on meat due to the influx of various lifestyle patterns and dietary cultures from Western countries, as is the case with the general Japanese population. It has been reported that meat was associated with cardiovascular complications and mortality in people with type 2 diabetes in a study in the US [30] and in our previous study [31]. Thus, it is considered necessary to strengthen the guidance on protein sources to prevent complications in the future.\nMeat is the food with the highest contribution to fat intake by Japanese people. Therefore, it is thought that an increased meat intake may also affect fat-to-energy ratios and saturated fatty acid increases in JDDM patients. Aside from meat, saturated fatty acids are found in the Japanese diet in milk, butter, and coconut oil, which are commonly used as ingredients in Western sweets rather than in traditional Japanese ones (which are mostly made with sugar, rice, and beans [32]). Although the type of sweets could not be identified in this survey, as their intake more than doubled from the JDCS to the JDDM, it is inferred that in conjunction with meat, the increase in the intake of sweets contributed to the increase in saturated fatty acid consumption.\nContrary to meat, the intake of fruits and vegetables decreased significantly between the two studies. As vegetables and fruits are good sources of dietary fiber, fiber intake decreased significantly as vegetable and fruit intakes decreased. A higher intake of dietary fiber is known to improve glycemic control, and we previously reported that it was significantly associated with a reduced risk of stroke [33] and retinopathy [34] in JDCS patients. However, despite its importance, the intake of dietary fiber was lower than the recommended goal of 20 g/day or higher in 1996, and even declined in the following 20 years. A decreased intake of fruits and vegetables is apparent in the Japanese general population [29], and is considered to be an important issue in dietary guidance.\nAs to the background of the decrease in the consumption of vegetables and fruits, survey reports have suggested that differences in lifestyle, such as economic conditions [35] and living alone, exist [36] and that diabetic patients who work may choose a less diversified diet [37]. It was also reported that the consumption of vegetables and fruits tends to be higher with a lower frequency of eating meals away from home [38]. As to the proportions of food expenditure in Japan from 1995 to 2015, the consumption of fresh foods has decreased, and eating out and consuming processed foods have increased from 65.4% to 72.6% [39]. This suggests that the increase in eating out and buying processed foods may influence the decrease in fruit and vegetable intake, not only in the general population, but also in those with type 2 diabetes. This Westernization of dietary habits has been widely adopted, and advice on the dietary treatment of diabetes should consider the current dietary situation.\nThis study has several limitations. First, both the JDCS and the JDDM were comprised of patients from different cohorts, although they were both multicenter studies. However, both studies were conducted at outpatient clinics throughout Japan specializing in diabetes, and the clinical settings were similar except for the time period studied. An additional limitation is that the number of participants that were included in both surveys was different from the potential candidates. Of the 2033 JDCS patients, 1588 responded to a baseline dietary survey, and 524 patients who had missing values in the dietary survey were excluded. In the JDDM patients, 1145 of 2337 patients met the eligibility criteria for the JDCS and responded to the baseline dietary survey. However, there were no notable differences in baseline characteristics between the responders and non-responders [8], and consequently, no selection bias was found. Finally, the participants’ dietary intake was obtained from a self-administered questionnaire, which could be an underestimation. As for the long-term comparison of 20 years, changes had been made in the food composition tables and in the FFQg version used in both studies. However, those changes corresponded to changes in dietary habits and food ingredients due to secular trends, and there was little effect on energy intake and intake of macronutrients due to the changes in the food composition tables.\nThe results of this study clarify the changes in the dietary intake of Japanese patients with type 2 diabetes over 20 years, beginning in 1996. Despite a lack of significant change in the total energy intake during the period, both men and women with type 2 diabetes had a large increase in BMI. A rise in the fat-to-energy ratio due to an increase in fat intake was also observed, particularly in men. In terms of intake by food group, both men and women had clearly unfavorable changes in terms of complication prevention. These included an increase in the intake of meat and a decrease in the intake of fish, soybean/soy products, vegetables, and fruits. The results of our study strongly suggest that, in order to prevent complications in Japanese patients with type 2 diabetes in the future, the provision of nutritional guidance, in line with these chronological changes in dietary intake, is necessary.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIn order to provide effective dietary guidance, it is necessary to consider dietary intake, which can change over time. This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period.\n\nMethods:\nWe compared the results of two dietary surveys that used the food frequency questionnaire format. The first was conducted in 1996 by the Japan Diabetes Complications Study (JDCS) (n = 1509; males 53.3%), and the second in 2014-2018 by the Japan Diabetes Clinical Data Management Study (JDDM) (n = 1145; males 65.6%). Both are nationwide representative registries of outpatients with type 2 diabetes in Japan.\n\nResults:\nOver a 20-year period, both men and women with type 2 diabetes had a significant increase in body mass index (BMI). Nonetheless, there was only a small change in energy intake. Conversely, there was a significant increase in fat intake and thus in the fat-to-energy ratio. With regard to food groups, there was a significant increase in meat intake and a decrease in the intake of fish, soybeans/soy products, vegetables, and fruits, with a particularly significant decrease in vegetables.\n\nConclusions:\nEven in Japan, an industrialized country with a stable socioeconomic environment, there were many significant changes in the dietary intake of patients with type 2 diabetes over the 20-year period."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":5052,"string":"5,052"},"whole_article_abstract_length":{"kind":"number","value":277,"string":"277"},"num_sections":{"kind":"number","value":4,"string":"4"},"most_frequent_words":{"kind":"list like","value":["intake","patients","diabetes","dietary","jddm","energy","jdcs","men","type","type diabetes"],"string":"[\n \"intake\",\n \"patients\",\n \"diabetes\",\n \"dietary\",\n \"jddm\",\n \"energy\",\n \"jdcs\",\n \"men\",\n \"type\",\n \"type diabetes\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] food intake | type 2 diabetes | obesity | diabetes mellitus | Asia [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] food intake | type 2 diabetes | obesity | diabetes mellitus | Asia [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] food intake | type 2 diabetes | obesity | diabetes mellitus | Asia [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Biomarkers | Body Mass Index | Diabetes Mellitus, Type 2 | Diet | Disease Susceptibility | Energy Intake | Feeding Behavior | Female | Humans | Japan | Male | Middle Aged | Public Health Surveillance | Registries [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Biomarkers | Body Mass Index | Diabetes Mellitus, Type 2 | Diet | Disease Susceptibility | Energy Intake | Feeding Behavior | Female | Humans | Japan | Male | Middle Aged | Public Health Surveillance | Registries [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Biomarkers | Body Mass Index | Diabetes Mellitus, Type 2 | Diet | Disease Susceptibility | Energy Intake | Feeding Behavior | Female | Humans | Japan | Male | Middle Aged | Public Health Surveillance | Registries [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] intake | patients | diabetes | dietary | jddm | energy | jdcs | men | type | type diabetes [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] intake | patients | diabetes | dietary | jddm | energy | jdcs | men | type | type diabetes [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] intake | patients | diabetes | dietary | jddm | energy | jdcs | men | type | type diabetes [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] dietary | changes | diabetes | type diabetes | type | intake | asian | study | changes dietary | people [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] jddm | men | significantly | women | participants | jddm participants | jdcs | patients | intake | jddm patients [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] intake | jddm | dietary | diabetes | jdcs | patients | energy | type | men | changes [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Japanese | 2 | 20-year [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 20-year | 2 | BMI ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Japanese | 2 | 20-year ||| two ||| first | 1996 | the Japan Diabetes Complications Study | JDCS | 1509 | 53.3% | second | 2014-2018 | the Japan Diabetes Clinical Data Management Study | 1145 | 65.6% ||| 2 | Japan ||| 20-year | 2 | BMI ||| ||| ||| ||| Japan | 2 | 20-year [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":281,"cells":{"title":{"kind":"string","value":"Are pre-existing markers of chronic kidney disease associated with short-term mortality following acute community-acquired pneumonia and sepsis? A cohort study among older people with diabetes using electronic health records."},"pmid":{"kind":"string","value":"25605811"},"background_abstract":{"kind":"string","value":"We aimed to examine whether pre-existing impaired estimated glomerular filtration rate (eGFR) and proteinuria were associated with mortality following community-acquired pneumonia or sepsis among people aged ≥ 65 years with diabetes mellitus, without end-stage renal disease."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Patients were followed up from onset of first community-acquired pneumonia or sepsis episode in a cohort study using large, linked electronic health databases. Follow-up was for up to 90 days, unlimited by hospital discharge. We used generalized linear models with log link, normal distribution and robust standard errors to calculate risk ratios (RRs) for all-cause 28- and 90-day mortality according to two markers of chronic kidney disease: eGFR and proteinuria."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"All-cause mortality among the 4743 patients with pneumonia was 29.6% after 28 days and 37.4% after 90 days. Among the 1058 patients with sepsis, all-cause 28- and 90-day mortality were 35.6 and 44.2%, respectively. eGFR <30 mL/min/1.73 m(2) was a risk marker of higher 28-day mortality for pneumonia (RR 1.27: 95% CI 1.12-1.43) and sepsis (RR 1.32: 95% CI 1.07-1.64), adjusted for age, sex, socio-economic status, smoking status and co-morbidities. Neither moderately impaired eGFR nor proteinuria were associated with short-term mortality following either infection."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"People with pre-existing low eGFR but not on dialysis are at higher risk of death following pneumonia and sepsis. This association was not explained by existing co-morbidities. These patients need to be carefully monitored to prevent modifiable causes of death."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Age Factors","Aged","Aged, 80 and over","Biomarkers","Cohort Studies","Community-Acquired Infections","Comorbidity","Diabetes Mellitus","Electronic Health Records","Female","Humans","Kidney Failure, Chronic","Male","Pneumonia","Proteinuria","Renal Dialysis","Risk Factors","Sepsis","Survival Rate"],"string":"[\n \"Age Factors\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Biomarkers\",\n \"Cohort Studies\",\n \"Community-Acquired Infections\",\n \"Comorbidity\",\n \"Diabetes Mellitus\",\n \"Electronic Health Records\",\n \"Female\",\n \"Humans\",\n \"Kidney Failure, Chronic\",\n \"Male\",\n \"Pneumonia\",\n \"Proteinuria\",\n \"Renal Dialysis\",\n \"Risk Factors\",\n \"Sepsis\",\n \"Survival Rate\"\n]"},"pmcid":{"kind":"string","value":"4438741"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Chronic kidney disease (CKD) affects an estimated 1.8 million people in England, 98% of whom do not require renal replacement therapy [1]. CKD is defined by reduced estimated glomerular filtration rate (eGFR) or evidence of kidney damage such as proteinuria and is commonest among older people [2, 3]. Most patients with CKD are managed in primary care [4].\nInfection is an important cause of mortality among older people [5, 6]. Both reduced eGFR and proteinuria are associated with an increased rate of infection-related mortality, which could be partly explained by increased incidence of infection [7–9]. It is less clear whether CKD is also associated with poorer prognosis following infection. When clinicians assess patients with community-acquired infection, developing complications such as acute kidney injury (AKI) may not yet be apparent, but they will know which patients have pre-existing CKD. If pre-existing CKD is a risk marker for short-term mortality, this would be useful for risk stratification and clinical management of patients with infections, especially in primary care where clinicians may not have access to immediate laboratory tests. While the implications of acute changes in eGFR during infection are a focus of current research, few studies have investigated the role of pre-existing CKD [10]. Low baseline eGFR has been found to be associated with mortality following sepsis and community-acquired pneumonia, but rarely examined according to clinically meaningful categories of eGFR [11–15]. To the best of our knowledge, proteinuria has not been examined as a potential risk marker for mortality following infection [12, 15, 16].\nAmong older people, CKD frequently co-exists with other co-morbidities [3]. An association of CKD with poor prognosis of infections could thus be due to confounding from these co-morbidities. For example, CKD is strongly associated with cardiovascular disease, which may be complicated by infection, resulting in post-infection mortality driven by the underlying cardiovascular disease [17]. Such deaths would largely follow hospitalization for cardiovascular events. CKD is associated with healthcare-associated pneumonia, which carries a worse prognosis than community-acquired pneumonia [18, 19]. Focusing on community-acquired infections should exclude infections arising as short-term sequelae of cardiovascular events and improve understanding of the relationship between pre-existing CKD markers and infection prognosis.\nOlder people with diabetes mellitus form a large and growing population in primary care who suffer a high incidence of community-acquired pneumonia and sepsis [20]. Forty per cent of adults with diabetes have CKD, of whom three-quarters have proteinuria, and CKD among these patients is associated with a greater all-cause excess mortality than among patients without diabetes [21]. If proteinuria is a risk marker for mortality among older patients with diabetes who develop community-acquired pneumonia or sepsis, this could inform clinical management of a large primary care patient population with appreciable mortality following infection [20].\nThis study aimed to examine whether baseline eGFR and proteinuria were independent risk markers for short-term mortality following community-acquired pneumonia or sepsis among older people with diabetes mellitus, using large, linked electronic health record databases."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"We identified 4957 individuals with community-acquired pneumonia and 1114 individuals with community-acquired sepsis. Data were missing (for smoking status and/or HbA1C results) for 212/4957 individuals with pneumonia (4.3%) and 56/1114 individuals with sepsis (5.0%). These patients were excluded. Among patients with pneumonia, patients with missing data were older (median age 83 years, IQR: 78–88) compared with those included (median age 80 years, IQR: 74–85), with a higher 28-day mortality (excluded 88/214, 41.1%: included 1406/4743, 29.6%) but a similar distribution of baseline eGFR. A similar pattern was seen for sepsis.\nEstimated GFR was based on the higher of two results for 4029 patients with pneumonia (84.9%) and 919 patients with sepsis (86.9%); for the remaining patients, only a single valid serum creatinine result was available. CKD prevalence was high: almost half of the patients had eGFR <60 mL/min/1.73 m2 and a third had proteinuria. Patients with eGFR <60 mL/min/1.73 m2 were older, with a higher prevalence of ischaemic heart disease and congestive heart failure than patients with eGFR ≥60 mL/min/1.73 m2 (Table 1).Table 1.Baseline characteristics of the study populationPneumoniaSepsiseGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2eGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2Age (years) Median (IQR)82 (77–87)78 (72–83)81 (75–86)76 (71–82)n (col %)n (col %)n (col %)n (col %)Gender Female1231 (53.0)1106 (42.0)294 (55.7)268 (45.7)Onset prior to 1 April 2004584 (25.1)527 (20.0)124 (23.5)120 (20.5)Socio-economic status (IMD quintile)a 1 (least deprived)383 (16.5)438 (16.6)109 (20.6)106 (18.1) 2554 (23.8)595 (22.6)116 (22.0)127 (21.7) 3494 (21.3)582 (22.1)120 (22.7)137 (23.4) 4502 (21.6)574 (21.8)110 (20.8)108 (18.4) 5 (most deprived)391 (16.8)444 (16.9)73 (13.8)108 (18.4)Smoking status Current321 (13.8)549 (20.9)70 (13.3)103 (17.6) Ex-smoker1185 (51.0)1351 (51.3)235 (44.5)284 (48.5) Non-smoker776 (33.4)698 (26.5)207 (39.2)193 (32.9) Missing42 (1.8)35 (1.3)16 (3.0)6 (1.0)Comorbidities Chronic lung disease503 (21.6)686 (26.1)59 (11.2)95 (16.2) Hypertension1623 (69.8)1662 (63.1)372 (70.5)381 (65.0) Congestive heart failure734 (31.6)430 (16.3)147 (27.8)95 (16.2) Ischaemic heart disease966 (41.6)887 (33.7)223 (42.2)207 (35.3) Cerebrovascular disease692 (29.8)638 (24.2)152 (28.8)145 (24.7) Other dementia174 (7.5)190 (7.2)42 (8.0)23 (3.9) Cancer382 (16.4)474 (18.0)99 (18.8)120 (20.5) Connective tissue disorders228 (9.8)215 (8.2)36 (6.8)52 (8.9)HbA1C Good <7%1176 (50.6)1401 (53.2)251 (47.5)308 (52.6) Borderline 7–10%966 (41.6)1033 (39.2)213 (40.3)230 (39.3) Poor >10%108 (4.7)131 (5.0)41 (7.8)34 (5.8) None recorded74 (3.2)68 (2.6)23 (4.4)14 (2.4)Prior antidiabetes medication Insulin122 (5.3)126 (4.8)34 (6.4)23 (3.9) Oral medications1139 (49.0)1426 (54.2)247 (46.8)312 (53.2) Both462 (19.9)384 (14.6)130 (24.6)114 (19.5) None601 (25.9)697 (26.5)117 (22.2)137 (23.4) Total23242633528586eGFR, estimated glomerular filtration rate.aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nBaseline characteristics of the study population\neGFR, estimated glomerular filtration rate.\naIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nPatients with pneumonia experienced 29.6% 28-day all-cause mortality (1406 deaths). Patients with sepsis experienced 35.6% 28-day all-cause mortality (377 deaths) (Table 2). Survival curves showed high mortality at infection onset, declining over ∼30 days to a more stable rate for the next 60 days following both pneumonia and sepsis (Figure 1). RRs for 28-day mortality were higher among people with eGFR <30 mL/min/1.73 m2 compared with people with eGFR ≥60 mL/min/1.73 m2 for pneumonia (RR = 1.27; 95% CI 1.10–1.47) and sepsis (RR = 1.42; 1.10–1.84), adjusted for age, sex, socio-economic status and onset prior to April 2004. Adjustment for smoking status, co-morbidities and characteristics of diabetes had minimal effect on these RRs for pneumonia (fully adjusted RR = 1.27; 1.12–1.43) or sepsis (fully adjusted RR = 1.32; 1.07–1.64). There was no evidence of associations between intermediate levels of eGFR and 28-day mortality for either infection, nor for an association between proteinuria and 28-day mortality. The pattern of associations of eGFR and proteinuria with 90-day mortality was similar to those for 28-day mortality (Table 2). Results were unchanged by additional adjustment for peripheral vascular disease. There was no good evidence of effect modification between eGFR and proteinuria, and in particular no evidence of any association of proteinuria with 28-day mortality within any category of eGFR status (data not shown).Table 2.Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)aNumber (column %)28-Day mortality (row %)90-Day mortality (row %)RRs for 28-day mortality (95% CI)RRs for 90-day mortality (95% CI)Adjusted for demographicsbadjusted for non-CVD co-morbiditiescFully adjusteddAdjusted for demographicsbAdjusted for non-CVD co-morbiditiescFully adjusteddPneumonia Proteinuria (adjusted for eGFR) Yes1611 (34.0)499 (31.0)625 (38.8)1.07 (0.97–1.17)1.07 (0.98–1.18)1.07 (0.98–1.18)1.05 (0.97–1.13)1.06 (0.98–1.14)1.05 (0.98–1.14) No3132 (66.0)907 (29.0)1150 (36.7)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<1523 (0.5)12 (52.2)12 (52.2)1.27 (1.10–1.47)1.31 (1.13–1.52)1.30 (1.12–1.52)1.26 (1.12–1.42)1.29 (1.14–1.45)1.27 (1.12–1.43)15–29263 (5.6)110 (41.8)139 (52.9) 30–44764 (16.1)265 (34.7)336 (44.0)0.98 (0.86–1.10)0.99 (0.88–1.13)0.99 (0.88–1.12)1.02 (0.93–1.13)1.04 (0.94–1.15)1.03 (0.94–1.14) 45–601162 (24.5)332 (28.6)418 (36.0)0.91 (0.82–1.02)0.94 (0.84–1.04)0.93 (0.84–1.04)0.91 (0.83–1.00)0.93 (0.85–1.02)0.92 (0.84–1.01) ≥602531 (53.4)687 (27.1)870 (34.4)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total47431406 (29.6)1775 (37.4)Sepsis Proteinuria (adjusted for eGFR) Yes358 (33.8)128 (35.8)159 (44.4)0.98 (0.82–1.17)1.01 (0.85–1.21)1.01 (0.85–1.21)0.98 (0.86–1.13)0.96 (0.84–1.10)0.96 (0.84–1.10) No700 (66.2)249 (35.6)309 (44.1)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<158 (0.8)2 (25.0)5 (62.5)1.42 (1.10–1.84)1.41 (1.08–1.84)1.37 (1.05–1.79)1.39 (1.14–1.70)1.32 (1.07–1.63)1.32 (1.07–1.64)15–2962 (5.9)32 (51.6)39 (62.9) 30–44190 (18.0)88 (46.3)103 (54.2)1.25 (1.01–1.55)1.24 (0.99–1.55)1.24 (0.99–1.54)1.14 (0.96–1.36)1.12 (0.94–1.33)1.11 (0.94–1.32) 45–60232 (21.9)76 (32.8)95 (41.0)0.95 (0.75–1.19)0.91 (0.72–1.15)0.91 (0.72–1.14)0.92 (0.77–1.11)0.89 (0.74–1.07)0.89 (0.75–1.07) ≥60566 (53.5)179 (31.6)226 (39.9)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total1058377 (35.6)468 (44.2)aExcluding patients with missing smoking or HbA1C data.bAge, gender, socio-economic status, onset prior to 1 April 2004.cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.FIGURE 1:Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nShort-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)a\naExcluding patients with missing smoking or HbA1C data.\nbAge, gender, socio-economic status, onset prior to 1 April 2004.\ncAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.\ndAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.\nSurvival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nThe underlying causes of death following pneumonia and sepsis were similar for patients with eGFR above and below 60 mL/min/1.73 m2. Causes of 28-day mortality following sepsis were predominantly sources of infection (Table 3). Following pneumonia onset, pneumonia was recorded as an underlying or contributory cause of death for 83.9% (1191/1419) of deaths within 28 days and 76.3% (1366/1790) of deaths within 90 days. Among patients with eGFR <60 mL/min/1.73 m2, renal disease was recorded as an underlying or contributory cause for 10.6% (77/724) of those who died within 28 days of pneumonia onset and 16.6% (152/913) of those who died within 90 days. Recording of CKD as a cause of death increased with lower eGFR (Table 4).Table 3.Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)aeGFReGFR <60 mL/min/1.73 m2eGFR ≥60 mL/min/1.73 m228-Day mortality following pneumonia, n = 1419J18 Pneumonia, organism unspecified, n = 256, 35.4%J44 Other chronic obstructive pulmonary disease, n = 63, 8.7%I25 Chronic ischaemic heart disease, n = 47, 6.5%I50 Heart failure, n = 43, 5.9%I64 Stroke, not specified as haemorrhage or infarction, n = 37, 5.1%J18 Pneumonia, organism unspecified, n = 216, 31.1%J44 Other chronic obstructive pulmonary disease, n = 63, 9.1%I64 Stroke, not specified as haemorrhage or infarction, n = 35, 5.0%C34 Malignant neoplasm of bronchus and lung, n = 34, 4.9%I25 Chronic ischaemic heart disease, n = 29, 4.2%Total = 724Total = 69529–90 Day mortality following pneumonia, n = 371J18 Pneumonia, organism unspecified, n = 38, 20.1%I25 Chronic ischaemic heart disease, n = 24, 12.7%J44 Other chronic obstructive pulmonary disease, n = 13, 6.9%C34 Malignant neoplasm of bronchus and lung, n = 12, 6.4%I21 Acute myocardial infarction, n = 12, 6.4%J18 Pneumonia, organism unspecified, n = 20, 11.0%C34 Malignant neoplasm of bronchus and lung, n = 20, 11.0%J44 Other chronic obstructive pulmonary disease, n = 19, 10.4%I25 Chronic ischaemic heart disease, n = 9, 5.0%I64 Stroke, not specified as haemorrhage or infarction, n = 9, 5.0%Total = 189Total = 18228-Day mortality following sepsis, n = 387N39 Other disorders of urinary systemb, n = 33, 15.9%J18 Pneumonia, organism unspecified, n = 30, 14.4%E14 Unspecified diabetes mellitus, n = 12, 5.8%A41 Other sepsis, n = 11, 5.3%L03 Cellulitis, n = 11, 5.3%N39 Other disorders of urinary systemb, n = 20, 11.2%A41 Other sepsis, n = 18, 10.1%J18 Pneumonia, organism unspecified, n = 18, 10.1%E14 Unspecified diabetes mellitus, n = 10, 5.6%J44 Other chronic obstructive pulmonary disease, n = 6, 3.4%=K55 Vascular disorders of intestine, n = 6, 3.4%=L03 Cellulitis, n = 6, 3.4%Total = 208Total = 179aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.Table 4.Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFReGFR (mL/min/1.73 m2)Deaths within 28 days of pneumonia onsetDeaths within 90 days of pneumonia onsetTotalN18 CKD, n (%)Renal disease,b\nn (%)TotalN18 CKD, n (%)Renal disease,b\nn (%)<1586 (75.0)7 (87.5)86 (75.0)7 (87.5)15–2910919 (17.4)41 (37.6)13926 (18.7)53 (38.1)30–4427514 (5.1)2 (15.3)34720 (5.8)56 (16.1)35–593328 (2.4)27 (8.1)41910 (2.4)36 (8.6)Total72447 (6.5)77 (10.6)91362 (6.8)152 (16.6)aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\nTop five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)a\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.\nRecording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFR\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Data sources","Study population","Definition of infections","Study outcomes","Definition of CKD","Other variables","Data analysis","Ethics"],"string":"[\n \"Data sources\",\n \"Study population\",\n \"Definition of infections\",\n \"Study outcomes\",\n \"Definition of CKD\",\n \"Other variables\",\n \"Data analysis\",\n \"Ethics\"\n]"},"other_sections_texts":{"kind":"list like","value":["The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].","The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.","Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.","The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.","CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.","Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.","Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.","The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116)."],"string":"[\n \"The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\",\n \"The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\",\n \"Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\",\n \"The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\",\n \"CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\",\n \"Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\",\n \"Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\\nStata version 13.1 was used for data analysis. All code lists are available on request.\",\n \"The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Data sources","Study population","Definition of infections","Study outcomes","Definition of CKD","Other variables","Data analysis","Ethics","RESULTS","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Data sources\",\n \"Study population\",\n \"Definition of infections\",\n \"Study outcomes\",\n \"Definition of CKD\",\n \"Other variables\",\n \"Data analysis\",\n \"Ethics\",\n \"RESULTS\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Chronic kidney disease (CKD) affects an estimated 1.8 million people in England, 98% of whom do not require renal replacement therapy [1]. CKD is defined by reduced estimated glomerular filtration rate (eGFR) or evidence of kidney damage such as proteinuria and is commonest among older people [2, 3]. Most patients with CKD are managed in primary care [4].\nInfection is an important cause of mortality among older people [5, 6]. Both reduced eGFR and proteinuria are associated with an increased rate of infection-related mortality, which could be partly explained by increased incidence of infection [7–9]. It is less clear whether CKD is also associated with poorer prognosis following infection. When clinicians assess patients with community-acquired infection, developing complications such as acute kidney injury (AKI) may not yet be apparent, but they will know which patients have pre-existing CKD. If pre-existing CKD is a risk marker for short-term mortality, this would be useful for risk stratification and clinical management of patients with infections, especially in primary care where clinicians may not have access to immediate laboratory tests. While the implications of acute changes in eGFR during infection are a focus of current research, few studies have investigated the role of pre-existing CKD [10]. Low baseline eGFR has been found to be associated with mortality following sepsis and community-acquired pneumonia, but rarely examined according to clinically meaningful categories of eGFR [11–15]. To the best of our knowledge, proteinuria has not been examined as a potential risk marker for mortality following infection [12, 15, 16].\nAmong older people, CKD frequently co-exists with other co-morbidities [3]. An association of CKD with poor prognosis of infections could thus be due to confounding from these co-morbidities. For example, CKD is strongly associated with cardiovascular disease, which may be complicated by infection, resulting in post-infection mortality driven by the underlying cardiovascular disease [17]. Such deaths would largely follow hospitalization for cardiovascular events. CKD is associated with healthcare-associated pneumonia, which carries a worse prognosis than community-acquired pneumonia [18, 19]. Focusing on community-acquired infections should exclude infections arising as short-term sequelae of cardiovascular events and improve understanding of the relationship between pre-existing CKD markers and infection prognosis.\nOlder people with diabetes mellitus form a large and growing population in primary care who suffer a high incidence of community-acquired pneumonia and sepsis [20]. Forty per cent of adults with diabetes have CKD, of whom three-quarters have proteinuria, and CKD among these patients is associated with a greater all-cause excess mortality than among patients without diabetes [21]. If proteinuria is a risk marker for mortality among older patients with diabetes who develop community-acquired pneumonia or sepsis, this could inform clinical management of a large primary care patient population with appreciable mortality following infection [20].\nThis study aimed to examine whether baseline eGFR and proteinuria were independent risk markers for short-term mortality following community-acquired pneumonia or sepsis among older people with diabetes mellitus, using large, linked electronic health record databases."," Data sources The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\nThe Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\n Study population The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\nThe study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\n Definition of infections Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\nInfection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\n Study outcomes The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\nThe outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\n Definition of CKD CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\nCKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\n Other variables Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\nAge was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\n Data analysis Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\nPneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\n Ethics The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\nThe study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).","The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].","The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.","Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.","The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.","CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.","Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.","Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.","The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).","We identified 4957 individuals with community-acquired pneumonia and 1114 individuals with community-acquired sepsis. Data were missing (for smoking status and/or HbA1C results) for 212/4957 individuals with pneumonia (4.3%) and 56/1114 individuals with sepsis (5.0%). These patients were excluded. Among patients with pneumonia, patients with missing data were older (median age 83 years, IQR: 78–88) compared with those included (median age 80 years, IQR: 74–85), with a higher 28-day mortality (excluded 88/214, 41.1%: included 1406/4743, 29.6%) but a similar distribution of baseline eGFR. A similar pattern was seen for sepsis.\nEstimated GFR was based on the higher of two results for 4029 patients with pneumonia (84.9%) and 919 patients with sepsis (86.9%); for the remaining patients, only a single valid serum creatinine result was available. CKD prevalence was high: almost half of the patients had eGFR <60 mL/min/1.73 m2 and a third had proteinuria. Patients with eGFR <60 mL/min/1.73 m2 were older, with a higher prevalence of ischaemic heart disease and congestive heart failure than patients with eGFR ≥60 mL/min/1.73 m2 (Table 1).Table 1.Baseline characteristics of the study populationPneumoniaSepsiseGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2eGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2Age (years) Median (IQR)82 (77–87)78 (72–83)81 (75–86)76 (71–82)n (col %)n (col %)n (col %)n (col %)Gender Female1231 (53.0)1106 (42.0)294 (55.7)268 (45.7)Onset prior to 1 April 2004584 (25.1)527 (20.0)124 (23.5)120 (20.5)Socio-economic status (IMD quintile)a 1 (least deprived)383 (16.5)438 (16.6)109 (20.6)106 (18.1) 2554 (23.8)595 (22.6)116 (22.0)127 (21.7) 3494 (21.3)582 (22.1)120 (22.7)137 (23.4) 4502 (21.6)574 (21.8)110 (20.8)108 (18.4) 5 (most deprived)391 (16.8)444 (16.9)73 (13.8)108 (18.4)Smoking status Current321 (13.8)549 (20.9)70 (13.3)103 (17.6) Ex-smoker1185 (51.0)1351 (51.3)235 (44.5)284 (48.5) Non-smoker776 (33.4)698 (26.5)207 (39.2)193 (32.9) Missing42 (1.8)35 (1.3)16 (3.0)6 (1.0)Comorbidities Chronic lung disease503 (21.6)686 (26.1)59 (11.2)95 (16.2) Hypertension1623 (69.8)1662 (63.1)372 (70.5)381 (65.0) Congestive heart failure734 (31.6)430 (16.3)147 (27.8)95 (16.2) Ischaemic heart disease966 (41.6)887 (33.7)223 (42.2)207 (35.3) Cerebrovascular disease692 (29.8)638 (24.2)152 (28.8)145 (24.7) Other dementia174 (7.5)190 (7.2)42 (8.0)23 (3.9) Cancer382 (16.4)474 (18.0)99 (18.8)120 (20.5) Connective tissue disorders228 (9.8)215 (8.2)36 (6.8)52 (8.9)HbA1C Good <7%1176 (50.6)1401 (53.2)251 (47.5)308 (52.6) Borderline 7–10%966 (41.6)1033 (39.2)213 (40.3)230 (39.3) Poor >10%108 (4.7)131 (5.0)41 (7.8)34 (5.8) None recorded74 (3.2)68 (2.6)23 (4.4)14 (2.4)Prior antidiabetes medication Insulin122 (5.3)126 (4.8)34 (6.4)23 (3.9) Oral medications1139 (49.0)1426 (54.2)247 (46.8)312 (53.2) Both462 (19.9)384 (14.6)130 (24.6)114 (19.5) None601 (25.9)697 (26.5)117 (22.2)137 (23.4) Total23242633528586eGFR, estimated glomerular filtration rate.aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nBaseline characteristics of the study population\neGFR, estimated glomerular filtration rate.\naIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nPatients with pneumonia experienced 29.6% 28-day all-cause mortality (1406 deaths). Patients with sepsis experienced 35.6% 28-day all-cause mortality (377 deaths) (Table 2). Survival curves showed high mortality at infection onset, declining over ∼30 days to a more stable rate for the next 60 days following both pneumonia and sepsis (Figure 1). RRs for 28-day mortality were higher among people with eGFR <30 mL/min/1.73 m2 compared with people with eGFR ≥60 mL/min/1.73 m2 for pneumonia (RR = 1.27; 95% CI 1.10–1.47) and sepsis (RR = 1.42; 1.10–1.84), adjusted for age, sex, socio-economic status and onset prior to April 2004. Adjustment for smoking status, co-morbidities and characteristics of diabetes had minimal effect on these RRs for pneumonia (fully adjusted RR = 1.27; 1.12–1.43) or sepsis (fully adjusted RR = 1.32; 1.07–1.64). There was no evidence of associations between intermediate levels of eGFR and 28-day mortality for either infection, nor for an association between proteinuria and 28-day mortality. The pattern of associations of eGFR and proteinuria with 90-day mortality was similar to those for 28-day mortality (Table 2). Results were unchanged by additional adjustment for peripheral vascular disease. There was no good evidence of effect modification between eGFR and proteinuria, and in particular no evidence of any association of proteinuria with 28-day mortality within any category of eGFR status (data not shown).Table 2.Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)aNumber (column %)28-Day mortality (row %)90-Day mortality (row %)RRs for 28-day mortality (95% CI)RRs for 90-day mortality (95% CI)Adjusted for demographicsbadjusted for non-CVD co-morbiditiescFully adjusteddAdjusted for demographicsbAdjusted for non-CVD co-morbiditiescFully adjusteddPneumonia Proteinuria (adjusted for eGFR) Yes1611 (34.0)499 (31.0)625 (38.8)1.07 (0.97–1.17)1.07 (0.98–1.18)1.07 (0.98–1.18)1.05 (0.97–1.13)1.06 (0.98–1.14)1.05 (0.98–1.14) No3132 (66.0)907 (29.0)1150 (36.7)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<1523 (0.5)12 (52.2)12 (52.2)1.27 (1.10–1.47)1.31 (1.13–1.52)1.30 (1.12–1.52)1.26 (1.12–1.42)1.29 (1.14–1.45)1.27 (1.12–1.43)15–29263 (5.6)110 (41.8)139 (52.9) 30–44764 (16.1)265 (34.7)336 (44.0)0.98 (0.86–1.10)0.99 (0.88–1.13)0.99 (0.88–1.12)1.02 (0.93–1.13)1.04 (0.94–1.15)1.03 (0.94–1.14) 45–601162 (24.5)332 (28.6)418 (36.0)0.91 (0.82–1.02)0.94 (0.84–1.04)0.93 (0.84–1.04)0.91 (0.83–1.00)0.93 (0.85–1.02)0.92 (0.84–1.01) ≥602531 (53.4)687 (27.1)870 (34.4)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total47431406 (29.6)1775 (37.4)Sepsis Proteinuria (adjusted for eGFR) Yes358 (33.8)128 (35.8)159 (44.4)0.98 (0.82–1.17)1.01 (0.85–1.21)1.01 (0.85–1.21)0.98 (0.86–1.13)0.96 (0.84–1.10)0.96 (0.84–1.10) No700 (66.2)249 (35.6)309 (44.1)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<158 (0.8)2 (25.0)5 (62.5)1.42 (1.10–1.84)1.41 (1.08–1.84)1.37 (1.05–1.79)1.39 (1.14–1.70)1.32 (1.07–1.63)1.32 (1.07–1.64)15–2962 (5.9)32 (51.6)39 (62.9) 30–44190 (18.0)88 (46.3)103 (54.2)1.25 (1.01–1.55)1.24 (0.99–1.55)1.24 (0.99–1.54)1.14 (0.96–1.36)1.12 (0.94–1.33)1.11 (0.94–1.32) 45–60232 (21.9)76 (32.8)95 (41.0)0.95 (0.75–1.19)0.91 (0.72–1.15)0.91 (0.72–1.14)0.92 (0.77–1.11)0.89 (0.74–1.07)0.89 (0.75–1.07) ≥60566 (53.5)179 (31.6)226 (39.9)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total1058377 (35.6)468 (44.2)aExcluding patients with missing smoking or HbA1C data.bAge, gender, socio-economic status, onset prior to 1 April 2004.cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.FIGURE 1:Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nShort-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)a\naExcluding patients with missing smoking or HbA1C data.\nbAge, gender, socio-economic status, onset prior to 1 April 2004.\ncAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.\ndAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.\nSurvival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nThe underlying causes of death following pneumonia and sepsis were similar for patients with eGFR above and below 60 mL/min/1.73 m2. Causes of 28-day mortality following sepsis were predominantly sources of infection (Table 3). Following pneumonia onset, pneumonia was recorded as an underlying or contributory cause of death for 83.9% (1191/1419) of deaths within 28 days and 76.3% (1366/1790) of deaths within 90 days. Among patients with eGFR <60 mL/min/1.73 m2, renal disease was recorded as an underlying or contributory cause for 10.6% (77/724) of those who died within 28 days of pneumonia onset and 16.6% (152/913) of those who died within 90 days. Recording of CKD as a cause of death increased with lower eGFR (Table 4).Table 3.Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)aeGFReGFR <60 mL/min/1.73 m2eGFR ≥60 mL/min/1.73 m228-Day mortality following pneumonia, n = 1419J18 Pneumonia, organism unspecified, n = 256, 35.4%J44 Other chronic obstructive pulmonary disease, n = 63, 8.7%I25 Chronic ischaemic heart disease, n = 47, 6.5%I50 Heart failure, n = 43, 5.9%I64 Stroke, not specified as haemorrhage or infarction, n = 37, 5.1%J18 Pneumonia, organism unspecified, n = 216, 31.1%J44 Other chronic obstructive pulmonary disease, n = 63, 9.1%I64 Stroke, not specified as haemorrhage or infarction, n = 35, 5.0%C34 Malignant neoplasm of bronchus and lung, n = 34, 4.9%I25 Chronic ischaemic heart disease, n = 29, 4.2%Total = 724Total = 69529–90 Day mortality following pneumonia, n = 371J18 Pneumonia, organism unspecified, n = 38, 20.1%I25 Chronic ischaemic heart disease, n = 24, 12.7%J44 Other chronic obstructive pulmonary disease, n = 13, 6.9%C34 Malignant neoplasm of bronchus and lung, n = 12, 6.4%I21 Acute myocardial infarction, n = 12, 6.4%J18 Pneumonia, organism unspecified, n = 20, 11.0%C34 Malignant neoplasm of bronchus and lung, n = 20, 11.0%J44 Other chronic obstructive pulmonary disease, n = 19, 10.4%I25 Chronic ischaemic heart disease, n = 9, 5.0%I64 Stroke, not specified as haemorrhage or infarction, n = 9, 5.0%Total = 189Total = 18228-Day mortality following sepsis, n = 387N39 Other disorders of urinary systemb, n = 33, 15.9%J18 Pneumonia, organism unspecified, n = 30, 14.4%E14 Unspecified diabetes mellitus, n = 12, 5.8%A41 Other sepsis, n = 11, 5.3%L03 Cellulitis, n = 11, 5.3%N39 Other disorders of urinary systemb, n = 20, 11.2%A41 Other sepsis, n = 18, 10.1%J18 Pneumonia, organism unspecified, n = 18, 10.1%E14 Unspecified diabetes mellitus, n = 10, 5.6%J44 Other chronic obstructive pulmonary disease, n = 6, 3.4%=K55 Vascular disorders of intestine, n = 6, 3.4%=L03 Cellulitis, n = 6, 3.4%Total = 208Total = 179aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.Table 4.Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFReGFR (mL/min/1.73 m2)Deaths within 28 days of pneumonia onsetDeaths within 90 days of pneumonia onsetTotalN18 CKD, n (%)Renal disease,b\nn (%)TotalN18 CKD, n (%)Renal disease,b\nn (%)<1586 (75.0)7 (87.5)86 (75.0)7 (87.5)15–2910919 (17.4)41 (37.6)13926 (18.7)53 (38.1)30–4427514 (5.1)2 (15.3)34720 (5.8)56 (16.1)35–593328 (2.4)27 (8.1)41910 (2.4)36 (8.6)Total72447 (6.5)77 (10.6)91362 (6.8)152 (16.6)aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\nTop five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)a\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.\nRecording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFR\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.","Among this population of older people with diabetes mellitus, eGFR <30 mL/min/1.73 m2 was a risk marker of higher 28- and 90-day mortality following community-acquired pneumonia and sepsis, compared with patients with eGFR ≥60 mL/min/1.73 m2. The relationship between eGFR and mortality did not change with adjustment for co-morbidities. Neither moderately impaired eGFR nor proteinuria was associated with higher short-term mortality following either infection.\nThe strengths of this study follow from the analysis of a focused question using large, linked datasets for a highly monitored primary care population with a cohort study design. Our study identifies that the association between eGFR and post-infection mortality persists when patients with end-stage renal disease (ESRD) are excluded (and is not explained by renal replacement therapy), when considering fixed-term rather than in-hospital mortality (thus is not due to differences in hospital stay) and when exclusively community-acquired infections are considered (so does not result from increased risk of healthcare-associated infections). The linked datasets allowed us to identify infections both among patients presenting directly to hospital and those managed in the community, maximized ascertainment of mortality and enabled description of the causes of death. The highly monitored population allowed good ascertainment of CKD status. The cohort study design has less potential for selection bias than an equivalent case–control study.\nA limitation is our assumption that the absence of a record implies a negative status for proteinuria and co-morbidities. Under-ascertainment of co-morbidities could result in residual confounding, with unpredictable effects, but the high prevalence of co-morbidities observed suggests that ascertainment was not markedly incomplete. We observed a high prevalence of proteinuria, and this is a highly monitored population (with financial incentives for standardized recording of proteinuria since 2004), but under-ascertainment of proteinuria could result in underestimation of any association between proteinuria and mortality [24]. Residual confounding from undiagnosed cardiovascular disease should have been minimized by adjustment for cardiovascular disease risk factors including smoking, hypertension and characteristics of diabetes.\nOur findings for eGFR provide further detail to build on previous findings that baseline eGFR <60 mL/min/1.73 m2 or renal disease are risk factors for short-term mortality following (hospital- or community-acquired) sepsis and for in-hospital mortality following community-acquired pneumonia (including patients receiving dialysis) [11–15]. A more comparable Canadian study examined the associations between eGFR and 30-day mortality following community-acquired pneumonia among the general population aged ≥65 years, excluding patients with ESRD [16]. Fully adjusted hazard ratios for 30-day mortality were 1.22 (95% CI 1.01–1.49) for eGFR 45–59, 2.03 (1.64–2.50) for eGFR 30–44 and 4.94 (3.94–6.19) for eGFR < 30, compared with eGFR 60–104 mL/min/1.73 m2. These are somewhat greater than the associations we observed. The difference may be explained by the different study populations. Both studies required a baseline serum creatinine result for inclusion. Our study population of older people with diabetes were routinely monitored for CKD (with financial incentivization in primary care) [32]. Creatinine testing of the Canadian study population may have been encouraged by co-morbidities or health-behaviours associated with CKD (such as smoking), which increase post-infection mortality, resulting in over-estimation of the association of eGFR and post-infection mortality. Our study population is less vulnerable to differential ascertainment of CKD. Alternatively, the association between eGFR and post-infection mortality may be smaller among patients with diabetes.\nTo the best of our knowledge, our examination of any association between proteinuria and mortality following community-acquired pneumonia and sepsis is novel. A history of proteinuria, although a marker for mortality in general, does not appear to be a risk marker for short-term mortality following community-acquired infection. This is unlikely to be due to chance, as the study was large, with findings consistent across both infections. We designed our study to produce conservative estimates and may have under-estimated the association between proteinuria and post-infection mortality due to under-ascertainment of proteinuria. Alternatively, any potential relationship between proteinuria and mortality may have been mitigated by clinical care of patients with infection who had pre-existing proteinuria, for example, through swift recognition of AKI.\nThe survival curves demonstrate a steep initial mortality following infection onset, and a high proportion of deaths had the underlying cause assigned to infection. Since 2001 in England, co-morbidities are assigned as the underlying cause of death when pneumonia has occurred in the context of, for example, malignancy or respiratory disease [25]. This suggests that the associations we observed are driven by an association between eGFR and infection prognosis, not merely high underlying baseline mortality among patients with impaired eGFR. This is supported by previous research which found 7.7-fold elevated mortality in the 30 days following community-acquired pneumonia [19]. Estimates for associations between eGFR and mortality were not substantially altered by adjustment for co-morbidities, suggesting that any causal relationship between eGFR and mortality is not mediated through co-morbidities.\nOur findings apply to the large population of older patients with community-acquired pneumonia or sepsis with diabetes mellitus who do not have ESRD. Inclusion criteria are unlikely to have limited generalizability appreciably. Practices which consent to data linkage could be more research oriented, providing good primary care management of risk factors for infection-related mortality (such as smoking cessation), but this is unlikely to affect the relationship between CKD and short-term mortality post-infection. Lack of pre-existing creatinine test results is likely to reflect limited time potentially eligible for the study rather than CKD status among this highly monitored population. Missing data on smoking status and HbA1C may be a marker of low patient engagement: caution should be used in generalizing our results to patients who are not actively managed in primary care.\nWe found that CKD is a useful clinical risk marker for post-infectious mortality. Whether this relationship is causal is less clear; but the association does not appear to be explained by age, co-morbidities or hospital attendance. Potential mechanisms include immune system dysfunction, but also more preventable complications such as AKI. Combinations of risk factors may be important: for example, patients with post-operative AKI have higher mortality if they also have pre-existing CKD [30].\nOur results have implications for patient management and future research. Patients with baseline eGFR <30 mL/min/1.73 m2 and community-acquired infection need careful monitoring, particularly in the 28 days following infection. Future research should investigate preventable mechanisms by which low baseline eGFR could be related to post-infection mortality, for example, fluid management, AKI and drug dosage in patients with low renal clearance."],"string":"[\n \"Chronic kidney disease (CKD) affects an estimated 1.8 million people in England, 98% of whom do not require renal replacement therapy [1]. CKD is defined by reduced estimated glomerular filtration rate (eGFR) or evidence of kidney damage such as proteinuria and is commonest among older people [2, 3]. Most patients with CKD are managed in primary care [4].\\nInfection is an important cause of mortality among older people [5, 6]. Both reduced eGFR and proteinuria are associated with an increased rate of infection-related mortality, which could be partly explained by increased incidence of infection [7–9]. It is less clear whether CKD is also associated with poorer prognosis following infection. When clinicians assess patients with community-acquired infection, developing complications such as acute kidney injury (AKI) may not yet be apparent, but they will know which patients have pre-existing CKD. If pre-existing CKD is a risk marker for short-term mortality, this would be useful for risk stratification and clinical management of patients with infections, especially in primary care where clinicians may not have access to immediate laboratory tests. While the implications of acute changes in eGFR during infection are a focus of current research, few studies have investigated the role of pre-existing CKD [10]. Low baseline eGFR has been found to be associated with mortality following sepsis and community-acquired pneumonia, but rarely examined according to clinically meaningful categories of eGFR [11–15]. To the best of our knowledge, proteinuria has not been examined as a potential risk marker for mortality following infection [12, 15, 16].\\nAmong older people, CKD frequently co-exists with other co-morbidities [3]. An association of CKD with poor prognosis of infections could thus be due to confounding from these co-morbidities. For example, CKD is strongly associated with cardiovascular disease, which may be complicated by infection, resulting in post-infection mortality driven by the underlying cardiovascular disease [17]. Such deaths would largely follow hospitalization for cardiovascular events. CKD is associated with healthcare-associated pneumonia, which carries a worse prognosis than community-acquired pneumonia [18, 19]. Focusing on community-acquired infections should exclude infections arising as short-term sequelae of cardiovascular events and improve understanding of the relationship between pre-existing CKD markers and infection prognosis.\\nOlder people with diabetes mellitus form a large and growing population in primary care who suffer a high incidence of community-acquired pneumonia and sepsis [20]. Forty per cent of adults with diabetes have CKD, of whom three-quarters have proteinuria, and CKD among these patients is associated with a greater all-cause excess mortality than among patients without diabetes [21]. If proteinuria is a risk marker for mortality among older patients with diabetes who develop community-acquired pneumonia or sepsis, this could inform clinical management of a large primary care patient population with appreciable mortality following infection [20].\\nThis study aimed to examine whether baseline eGFR and proteinuria were independent risk markers for short-term mortality following community-acquired pneumonia or sepsis among older people with diabetes mellitus, using large, linked electronic health record databases.\",\n \" Data sources The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\\nThe Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\\n Study population The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\\nThe study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\\n Definition of infections Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\\nInfection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\\n Study outcomes The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\\nThe outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\\n Definition of CKD CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\\nCKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\\n Other variables Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\\nAge was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\\n Data analysis Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\\nStata version 13.1 was used for data analysis. All code lists are available on request.\\nPneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\\nStata version 13.1 was used for data analysis. All code lists are available on request.\\n Ethics The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\\nThe study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\",\n \"The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\",\n \"The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\",\n \"Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\",\n \"The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\",\n \"CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\",\n \"Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\",\n \"Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\\nStata version 13.1 was used for data analysis. All code lists are available on request.\",\n \"The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\",\n \"We identified 4957 individuals with community-acquired pneumonia and 1114 individuals with community-acquired sepsis. Data were missing (for smoking status and/or HbA1C results) for 212/4957 individuals with pneumonia (4.3%) and 56/1114 individuals with sepsis (5.0%). These patients were excluded. Among patients with pneumonia, patients with missing data were older (median age 83 years, IQR: 78–88) compared with those included (median age 80 years, IQR: 74–85), with a higher 28-day mortality (excluded 88/214, 41.1%: included 1406/4743, 29.6%) but a similar distribution of baseline eGFR. A similar pattern was seen for sepsis.\\nEstimated GFR was based on the higher of two results for 4029 patients with pneumonia (84.9%) and 919 patients with sepsis (86.9%); for the remaining patients, only a single valid serum creatinine result was available. CKD prevalence was high: almost half of the patients had eGFR <60 mL/min/1.73 m2 and a third had proteinuria. Patients with eGFR <60 mL/min/1.73 m2 were older, with a higher prevalence of ischaemic heart disease and congestive heart failure than patients with eGFR ≥60 mL/min/1.73 m2 (Table 1).Table 1.Baseline characteristics of the study populationPneumoniaSepsiseGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2eGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2Age (years) Median (IQR)82 (77–87)78 (72–83)81 (75–86)76 (71–82)n (col %)n (col %)n (col %)n (col %)Gender Female1231 (53.0)1106 (42.0)294 (55.7)268 (45.7)Onset prior to 1 April 2004584 (25.1)527 (20.0)124 (23.5)120 (20.5)Socio-economic status (IMD quintile)a 1 (least deprived)383 (16.5)438 (16.6)109 (20.6)106 (18.1) 2554 (23.8)595 (22.6)116 (22.0)127 (21.7) 3494 (21.3)582 (22.1)120 (22.7)137 (23.4) 4502 (21.6)574 (21.8)110 (20.8)108 (18.4) 5 (most deprived)391 (16.8)444 (16.9)73 (13.8)108 (18.4)Smoking status Current321 (13.8)549 (20.9)70 (13.3)103 (17.6) Ex-smoker1185 (51.0)1351 (51.3)235 (44.5)284 (48.5) Non-smoker776 (33.4)698 (26.5)207 (39.2)193 (32.9) Missing42 (1.8)35 (1.3)16 (3.0)6 (1.0)Comorbidities Chronic lung disease503 (21.6)686 (26.1)59 (11.2)95 (16.2) Hypertension1623 (69.8)1662 (63.1)372 (70.5)381 (65.0) Congestive heart failure734 (31.6)430 (16.3)147 (27.8)95 (16.2) Ischaemic heart disease966 (41.6)887 (33.7)223 (42.2)207 (35.3) Cerebrovascular disease692 (29.8)638 (24.2)152 (28.8)145 (24.7) Other dementia174 (7.5)190 (7.2)42 (8.0)23 (3.9) Cancer382 (16.4)474 (18.0)99 (18.8)120 (20.5) Connective tissue disorders228 (9.8)215 (8.2)36 (6.8)52 (8.9)HbA1C Good <7%1176 (50.6)1401 (53.2)251 (47.5)308 (52.6) Borderline 7–10%966 (41.6)1033 (39.2)213 (40.3)230 (39.3) Poor >10%108 (4.7)131 (5.0)41 (7.8)34 (5.8) None recorded74 (3.2)68 (2.6)23 (4.4)14 (2.4)Prior antidiabetes medication Insulin122 (5.3)126 (4.8)34 (6.4)23 (3.9) Oral medications1139 (49.0)1426 (54.2)247 (46.8)312 (53.2) Both462 (19.9)384 (14.6)130 (24.6)114 (19.5) None601 (25.9)697 (26.5)117 (22.2)137 (23.4) Total23242633528586eGFR, estimated glomerular filtration rate.aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\\nBaseline characteristics of the study population\\neGFR, estimated glomerular filtration rate.\\naIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\\nPatients with pneumonia experienced 29.6% 28-day all-cause mortality (1406 deaths). Patients with sepsis experienced 35.6% 28-day all-cause mortality (377 deaths) (Table 2). Survival curves showed high mortality at infection onset, declining over ∼30 days to a more stable rate for the next 60 days following both pneumonia and sepsis (Figure 1). RRs for 28-day mortality were higher among people with eGFR <30 mL/min/1.73 m2 compared with people with eGFR ≥60 mL/min/1.73 m2 for pneumonia (RR = 1.27; 95% CI 1.10–1.47) and sepsis (RR = 1.42; 1.10–1.84), adjusted for age, sex, socio-economic status and onset prior to April 2004. Adjustment for smoking status, co-morbidities and characteristics of diabetes had minimal effect on these RRs for pneumonia (fully adjusted RR = 1.27; 1.12–1.43) or sepsis (fully adjusted RR = 1.32; 1.07–1.64). There was no evidence of associations between intermediate levels of eGFR and 28-day mortality for either infection, nor for an association between proteinuria and 28-day mortality. The pattern of associations of eGFR and proteinuria with 90-day mortality was similar to those for 28-day mortality (Table 2). Results were unchanged by additional adjustment for peripheral vascular disease. There was no good evidence of effect modification between eGFR and proteinuria, and in particular no evidence of any association of proteinuria with 28-day mortality within any category of eGFR status (data not shown).Table 2.Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)aNumber (column %)28-Day mortality (row %)90-Day mortality (row %)RRs for 28-day mortality (95% CI)RRs for 90-day mortality (95% CI)Adjusted for demographicsbadjusted for non-CVD co-morbiditiescFully adjusteddAdjusted for demographicsbAdjusted for non-CVD co-morbiditiescFully adjusteddPneumonia Proteinuria (adjusted for eGFR) Yes1611 (34.0)499 (31.0)625 (38.8)1.07 (0.97–1.17)1.07 (0.98–1.18)1.07 (0.98–1.18)1.05 (0.97–1.13)1.06 (0.98–1.14)1.05 (0.98–1.14) No3132 (66.0)907 (29.0)1150 (36.7)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<1523 (0.5)12 (52.2)12 (52.2)1.27 (1.10–1.47)1.31 (1.13–1.52)1.30 (1.12–1.52)1.26 (1.12–1.42)1.29 (1.14–1.45)1.27 (1.12–1.43)15–29263 (5.6)110 (41.8)139 (52.9) 30–44764 (16.1)265 (34.7)336 (44.0)0.98 (0.86–1.10)0.99 (0.88–1.13)0.99 (0.88–1.12)1.02 (0.93–1.13)1.04 (0.94–1.15)1.03 (0.94–1.14) 45–601162 (24.5)332 (28.6)418 (36.0)0.91 (0.82–1.02)0.94 (0.84–1.04)0.93 (0.84–1.04)0.91 (0.83–1.00)0.93 (0.85–1.02)0.92 (0.84–1.01) ≥602531 (53.4)687 (27.1)870 (34.4)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total47431406 (29.6)1775 (37.4)Sepsis Proteinuria (adjusted for eGFR) Yes358 (33.8)128 (35.8)159 (44.4)0.98 (0.82–1.17)1.01 (0.85–1.21)1.01 (0.85–1.21)0.98 (0.86–1.13)0.96 (0.84–1.10)0.96 (0.84–1.10) No700 (66.2)249 (35.6)309 (44.1)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<158 (0.8)2 (25.0)5 (62.5)1.42 (1.10–1.84)1.41 (1.08–1.84)1.37 (1.05–1.79)1.39 (1.14–1.70)1.32 (1.07–1.63)1.32 (1.07–1.64)15–2962 (5.9)32 (51.6)39 (62.9) 30–44190 (18.0)88 (46.3)103 (54.2)1.25 (1.01–1.55)1.24 (0.99–1.55)1.24 (0.99–1.54)1.14 (0.96–1.36)1.12 (0.94–1.33)1.11 (0.94–1.32) 45–60232 (21.9)76 (32.8)95 (41.0)0.95 (0.75–1.19)0.91 (0.72–1.15)0.91 (0.72–1.14)0.92 (0.77–1.11)0.89 (0.74–1.07)0.89 (0.75–1.07) ≥60566 (53.5)179 (31.6)226 (39.9)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total1058377 (35.6)468 (44.2)aExcluding patients with missing smoking or HbA1C data.bAge, gender, socio-economic status, onset prior to 1 April 2004.cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.FIGURE 1:Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\\nShort-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)a\\naExcluding patients with missing smoking or HbA1C data.\\nbAge, gender, socio-economic status, onset prior to 1 April 2004.\\ncAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.\\ndAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.\\nSurvival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\\nThe underlying causes of death following pneumonia and sepsis were similar for patients with eGFR above and below 60 mL/min/1.73 m2. Causes of 28-day mortality following sepsis were predominantly sources of infection (Table 3). Following pneumonia onset, pneumonia was recorded as an underlying or contributory cause of death for 83.9% (1191/1419) of deaths within 28 days and 76.3% (1366/1790) of deaths within 90 days. Among patients with eGFR <60 mL/min/1.73 m2, renal disease was recorded as an underlying or contributory cause for 10.6% (77/724) of those who died within 28 days of pneumonia onset and 16.6% (152/913) of those who died within 90 days. Recording of CKD as a cause of death increased with lower eGFR (Table 4).Table 3.Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)aeGFReGFR <60 mL/min/1.73 m2eGFR ≥60 mL/min/1.73 m228-Day mortality following pneumonia, n = 1419J18 Pneumonia, organism unspecified, n = 256, 35.4%J44 Other chronic obstructive pulmonary disease, n = 63, 8.7%I25 Chronic ischaemic heart disease, n = 47, 6.5%I50 Heart failure, n = 43, 5.9%I64 Stroke, not specified as haemorrhage or infarction, n = 37, 5.1%J18 Pneumonia, organism unspecified, n = 216, 31.1%J44 Other chronic obstructive pulmonary disease, n = 63, 9.1%I64 Stroke, not specified as haemorrhage or infarction, n = 35, 5.0%C34 Malignant neoplasm of bronchus and lung, n = 34, 4.9%I25 Chronic ischaemic heart disease, n = 29, 4.2%Total = 724Total = 69529–90 Day mortality following pneumonia, n = 371J18 Pneumonia, organism unspecified, n = 38, 20.1%I25 Chronic ischaemic heart disease, n = 24, 12.7%J44 Other chronic obstructive pulmonary disease, n = 13, 6.9%C34 Malignant neoplasm of bronchus and lung, n = 12, 6.4%I21 Acute myocardial infarction, n = 12, 6.4%J18 Pneumonia, organism unspecified, n = 20, 11.0%C34 Malignant neoplasm of bronchus and lung, n = 20, 11.0%J44 Other chronic obstructive pulmonary disease, n = 19, 10.4%I25 Chronic ischaemic heart disease, n = 9, 5.0%I64 Stroke, not specified as haemorrhage or infarction, n = 9, 5.0%Total = 189Total = 18228-Day mortality following sepsis, n = 387N39 Other disorders of urinary systemb, n = 33, 15.9%J18 Pneumonia, organism unspecified, n = 30, 14.4%E14 Unspecified diabetes mellitus, n = 12, 5.8%A41 Other sepsis, n = 11, 5.3%L03 Cellulitis, n = 11, 5.3%N39 Other disorders of urinary systemb, n = 20, 11.2%A41 Other sepsis, n = 18, 10.1%J18 Pneumonia, organism unspecified, n = 18, 10.1%E14 Unspecified diabetes mellitus, n = 10, 5.6%J44 Other chronic obstructive pulmonary disease, n = 6, 3.4%=K55 Vascular disorders of intestine, n = 6, 3.4%=L03 Cellulitis, n = 6, 3.4%Total = 208Total = 179aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.Table 4.Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFReGFR (mL/min/1.73 m2)Deaths within 28 days of pneumonia onsetDeaths within 90 days of pneumonia onsetTotalN18 CKD, n (%)Renal disease,b\\nn (%)TotalN18 CKD, n (%)Renal disease,b\\nn (%)<1586 (75.0)7 (87.5)86 (75.0)7 (87.5)15–2910919 (17.4)41 (37.6)13926 (18.7)53 (38.1)30–4427514 (5.1)2 (15.3)34720 (5.8)56 (16.1)35–593328 (2.4)27 (8.1)41910 (2.4)36 (8.6)Total72447 (6.5)77 (10.6)91362 (6.8)152 (16.6)aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\\nTop five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)a\\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\\nbAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.\\nRecording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFR\\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\\nbAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\",\n \"Among this population of older people with diabetes mellitus, eGFR <30 mL/min/1.73 m2 was a risk marker of higher 28- and 90-day mortality following community-acquired pneumonia and sepsis, compared with patients with eGFR ≥60 mL/min/1.73 m2. The relationship between eGFR and mortality did not change with adjustment for co-morbidities. Neither moderately impaired eGFR nor proteinuria was associated with higher short-term mortality following either infection.\\nThe strengths of this study follow from the analysis of a focused question using large, linked datasets for a highly monitored primary care population with a cohort study design. Our study identifies that the association between eGFR and post-infection mortality persists when patients with end-stage renal disease (ESRD) are excluded (and is not explained by renal replacement therapy), when considering fixed-term rather than in-hospital mortality (thus is not due to differences in hospital stay) and when exclusively community-acquired infections are considered (so does not result from increased risk of healthcare-associated infections). The linked datasets allowed us to identify infections both among patients presenting directly to hospital and those managed in the community, maximized ascertainment of mortality and enabled description of the causes of death. The highly monitored population allowed good ascertainment of CKD status. The cohort study design has less potential for selection bias than an equivalent case–control study.\\nA limitation is our assumption that the absence of a record implies a negative status for proteinuria and co-morbidities. Under-ascertainment of co-morbidities could result in residual confounding, with unpredictable effects, but the high prevalence of co-morbidities observed suggests that ascertainment was not markedly incomplete. We observed a high prevalence of proteinuria, and this is a highly monitored population (with financial incentives for standardized recording of proteinuria since 2004), but under-ascertainment of proteinuria could result in underestimation of any association between proteinuria and mortality [24]. Residual confounding from undiagnosed cardiovascular disease should have been minimized by adjustment for cardiovascular disease risk factors including smoking, hypertension and characteristics of diabetes.\\nOur findings for eGFR provide further detail to build on previous findings that baseline eGFR <60 mL/min/1.73 m2 or renal disease are risk factors for short-term mortality following (hospital- or community-acquired) sepsis and for in-hospital mortality following community-acquired pneumonia (including patients receiving dialysis) [11–15]. A more comparable Canadian study examined the associations between eGFR and 30-day mortality following community-acquired pneumonia among the general population aged ≥65 years, excluding patients with ESRD [16]. Fully adjusted hazard ratios for 30-day mortality were 1.22 (95% CI 1.01–1.49) for eGFR 45–59, 2.03 (1.64–2.50) for eGFR 30–44 and 4.94 (3.94–6.19) for eGFR < 30, compared with eGFR 60–104 mL/min/1.73 m2. These are somewhat greater than the associations we observed. The difference may be explained by the different study populations. Both studies required a baseline serum creatinine result for inclusion. Our study population of older people with diabetes were routinely monitored for CKD (with financial incentivization in primary care) [32]. Creatinine testing of the Canadian study population may have been encouraged by co-morbidities or health-behaviours associated with CKD (such as smoking), which increase post-infection mortality, resulting in over-estimation of the association of eGFR and post-infection mortality. Our study population is less vulnerable to differential ascertainment of CKD. Alternatively, the association between eGFR and post-infection mortality may be smaller among patients with diabetes.\\nTo the best of our knowledge, our examination of any association between proteinuria and mortality following community-acquired pneumonia and sepsis is novel. A history of proteinuria, although a marker for mortality in general, does not appear to be a risk marker for short-term mortality following community-acquired infection. This is unlikely to be due to chance, as the study was large, with findings consistent across both infections. We designed our study to produce conservative estimates and may have under-estimated the association between proteinuria and post-infection mortality due to under-ascertainment of proteinuria. Alternatively, any potential relationship between proteinuria and mortality may have been mitigated by clinical care of patients with infection who had pre-existing proteinuria, for example, through swift recognition of AKI.\\nThe survival curves demonstrate a steep initial mortality following infection onset, and a high proportion of deaths had the underlying cause assigned to infection. Since 2001 in England, co-morbidities are assigned as the underlying cause of death when pneumonia has occurred in the context of, for example, malignancy or respiratory disease [25]. This suggests that the associations we observed are driven by an association between eGFR and infection prognosis, not merely high underlying baseline mortality among patients with impaired eGFR. This is supported by previous research which found 7.7-fold elevated mortality in the 30 days following community-acquired pneumonia [19]. Estimates for associations between eGFR and mortality were not substantially altered by adjustment for co-morbidities, suggesting that any causal relationship between eGFR and mortality is not mediated through co-morbidities.\\nOur findings apply to the large population of older patients with community-acquired pneumonia or sepsis with diabetes mellitus who do not have ESRD. Inclusion criteria are unlikely to have limited generalizability appreciably. Practices which consent to data linkage could be more research oriented, providing good primary care management of risk factors for infection-related mortality (such as smoking cessation), but this is unlikely to affect the relationship between CKD and short-term mortality post-infection. Lack of pre-existing creatinine test results is likely to reflect limited time potentially eligible for the study rather than CKD status among this highly monitored population. Missing data on smoking status and HbA1C may be a marker of low patient engagement: caution should be used in generalizing our results to patients who are not actively managed in primary care.\\nWe found that CKD is a useful clinical risk marker for post-infectious mortality. Whether this relationship is causal is less clear; but the association does not appear to be explained by age, co-morbidities or hospital attendance. Potential mechanisms include immune system dysfunction, but also more preventable complications such as AKI. Combinations of risk factors may be important: for example, patients with post-operative AKI have higher mortality if they also have pre-existing CKD [30].\\nOur results have implications for patient management and future research. Patients with baseline eGFR <30 mL/min/1.73 m2 and community-acquired infection need careful monitoring, particularly in the 28 days following infection. Future research should investigate preventable mechanisms by which low baseline eGFR could be related to post-infection mortality, for example, fluid management, AKI and drug dosage in patients with low renal clearance.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","materials|methods",null,null,null,null,null,null,null,null,"results","discussion"],"string":"[\n \"intro\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["chronic kidney disease","community-acquired infections","electronic health records","infection/mortality","proteinuria"],"string":"[\n \"chronic kidney disease\",\n \"community-acquired infections\",\n \"electronic health records\",\n \"infection/mortality\",\n \"proteinuria\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nChronic kidney disease (CKD) affects an estimated 1.8 million people in England, 98% of whom do not require renal replacement therapy [1]. CKD is defined by reduced estimated glomerular filtration rate (eGFR) or evidence of kidney damage such as proteinuria and is commonest among older people [2, 3]. Most patients with CKD are managed in primary care [4].\nInfection is an important cause of mortality among older people [5, 6]. Both reduced eGFR and proteinuria are associated with an increased rate of infection-related mortality, which could be partly explained by increased incidence of infection [7–9]. It is less clear whether CKD is also associated with poorer prognosis following infection. When clinicians assess patients with community-acquired infection, developing complications such as acute kidney injury (AKI) may not yet be apparent, but they will know which patients have pre-existing CKD. If pre-existing CKD is a risk marker for short-term mortality, this would be useful for risk stratification and clinical management of patients with infections, especially in primary care where clinicians may not have access to immediate laboratory tests. While the implications of acute changes in eGFR during infection are a focus of current research, few studies have investigated the role of pre-existing CKD [10]. Low baseline eGFR has been found to be associated with mortality following sepsis and community-acquired pneumonia, but rarely examined according to clinically meaningful categories of eGFR [11–15]. To the best of our knowledge, proteinuria has not been examined as a potential risk marker for mortality following infection [12, 15, 16].\nAmong older people, CKD frequently co-exists with other co-morbidities [3]. An association of CKD with poor prognosis of infections could thus be due to confounding from these co-morbidities. For example, CKD is strongly associated with cardiovascular disease, which may be complicated by infection, resulting in post-infection mortality driven by the underlying cardiovascular disease [17]. Such deaths would largely follow hospitalization for cardiovascular events. CKD is associated with healthcare-associated pneumonia, which carries a worse prognosis than community-acquired pneumonia [18, 19]. Focusing on community-acquired infections should exclude infections arising as short-term sequelae of cardiovascular events and improve understanding of the relationship between pre-existing CKD markers and infection prognosis.\nOlder people with diabetes mellitus form a large and growing population in primary care who suffer a high incidence of community-acquired pneumonia and sepsis [20]. Forty per cent of adults with diabetes have CKD, of whom three-quarters have proteinuria, and CKD among these patients is associated with a greater all-cause excess mortality than among patients without diabetes [21]. If proteinuria is a risk marker for mortality among older patients with diabetes who develop community-acquired pneumonia or sepsis, this could inform clinical management of a large primary care patient population with appreciable mortality following infection [20].\nThis study aimed to examine whether baseline eGFR and proteinuria were independent risk markers for short-term mortality following community-acquired pneumonia or sepsis among older people with diabetes mellitus, using large, linked electronic health record databases.\n\nMATERIALS AND METHODS:\n Data sources The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\nThe Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\n Study population The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\nThe study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\n Definition of infections Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\nInfection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\n Study outcomes The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\nThe outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\n Definition of CKD CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\nCKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\n Other variables Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\nAge was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\n Data analysis Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\nPneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\n Ethics The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\nThe study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\n\nData sources:\nThe Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\n\nStudy population:\nThe study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\n\nDefinition of infections:\nInfection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\n\nStudy outcomes:\nThe outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\n\nDefinition of CKD:\nCKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\n\nOther variables:\nAge was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\n\nData analysis:\nPneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\n\nEthics:\nThe study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\n\nRESULTS:\nWe identified 4957 individuals with community-acquired pneumonia and 1114 individuals with community-acquired sepsis. Data were missing (for smoking status and/or HbA1C results) for 212/4957 individuals with pneumonia (4.3%) and 56/1114 individuals with sepsis (5.0%). These patients were excluded. Among patients with pneumonia, patients with missing data were older (median age 83 years, IQR: 78–88) compared with those included (median age 80 years, IQR: 74–85), with a higher 28-day mortality (excluded 88/214, 41.1%: included 1406/4743, 29.6%) but a similar distribution of baseline eGFR. A similar pattern was seen for sepsis.\nEstimated GFR was based on the higher of two results for 4029 patients with pneumonia (84.9%) and 919 patients with sepsis (86.9%); for the remaining patients, only a single valid serum creatinine result was available. CKD prevalence was high: almost half of the patients had eGFR <60 mL/min/1.73 m2 and a third had proteinuria. Patients with eGFR <60 mL/min/1.73 m2 were older, with a higher prevalence of ischaemic heart disease and congestive heart failure than patients with eGFR ≥60 mL/min/1.73 m2 (Table 1).Table 1.Baseline characteristics of the study populationPneumoniaSepsiseGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2eGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2Age (years) Median (IQR)82 (77–87)78 (72–83)81 (75–86)76 (71–82)n (col %)n (col %)n (col %)n (col %)Gender Female1231 (53.0)1106 (42.0)294 (55.7)268 (45.7)Onset prior to 1 April 2004584 (25.1)527 (20.0)124 (23.5)120 (20.5)Socio-economic status (IMD quintile)a 1 (least deprived)383 (16.5)438 (16.6)109 (20.6)106 (18.1) 2554 (23.8)595 (22.6)116 (22.0)127 (21.7) 3494 (21.3)582 (22.1)120 (22.7)137 (23.4) 4502 (21.6)574 (21.8)110 (20.8)108 (18.4) 5 (most deprived)391 (16.8)444 (16.9)73 (13.8)108 (18.4)Smoking status Current321 (13.8)549 (20.9)70 (13.3)103 (17.6) Ex-smoker1185 (51.0)1351 (51.3)235 (44.5)284 (48.5) Non-smoker776 (33.4)698 (26.5)207 (39.2)193 (32.9) Missing42 (1.8)35 (1.3)16 (3.0)6 (1.0)Comorbidities Chronic lung disease503 (21.6)686 (26.1)59 (11.2)95 (16.2) Hypertension1623 (69.8)1662 (63.1)372 (70.5)381 (65.0) Congestive heart failure734 (31.6)430 (16.3)147 (27.8)95 (16.2) Ischaemic heart disease966 (41.6)887 (33.7)223 (42.2)207 (35.3) Cerebrovascular disease692 (29.8)638 (24.2)152 (28.8)145 (24.7) Other dementia174 (7.5)190 (7.2)42 (8.0)23 (3.9) Cancer382 (16.4)474 (18.0)99 (18.8)120 (20.5) Connective tissue disorders228 (9.8)215 (8.2)36 (6.8)52 (8.9)HbA1C Good <7%1176 (50.6)1401 (53.2)251 (47.5)308 (52.6) Borderline 7–10%966 (41.6)1033 (39.2)213 (40.3)230 (39.3) Poor >10%108 (4.7)131 (5.0)41 (7.8)34 (5.8) None recorded74 (3.2)68 (2.6)23 (4.4)14 (2.4)Prior antidiabetes medication Insulin122 (5.3)126 (4.8)34 (6.4)23 (3.9) Oral medications1139 (49.0)1426 (54.2)247 (46.8)312 (53.2) Both462 (19.9)384 (14.6)130 (24.6)114 (19.5) None601 (25.9)697 (26.5)117 (22.2)137 (23.4) Total23242633528586eGFR, estimated glomerular filtration rate.aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nBaseline characteristics of the study population\neGFR, estimated glomerular filtration rate.\naIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nPatients with pneumonia experienced 29.6% 28-day all-cause mortality (1406 deaths). Patients with sepsis experienced 35.6% 28-day all-cause mortality (377 deaths) (Table 2). Survival curves showed high mortality at infection onset, declining over ∼30 days to a more stable rate for the next 60 days following both pneumonia and sepsis (Figure 1). RRs for 28-day mortality were higher among people with eGFR <30 mL/min/1.73 m2 compared with people with eGFR ≥60 mL/min/1.73 m2 for pneumonia (RR = 1.27; 95% CI 1.10–1.47) and sepsis (RR = 1.42; 1.10–1.84), adjusted for age, sex, socio-economic status and onset prior to April 2004. Adjustment for smoking status, co-morbidities and characteristics of diabetes had minimal effect on these RRs for pneumonia (fully adjusted RR = 1.27; 1.12–1.43) or sepsis (fully adjusted RR = 1.32; 1.07–1.64). There was no evidence of associations between intermediate levels of eGFR and 28-day mortality for either infection, nor for an association between proteinuria and 28-day mortality. The pattern of associations of eGFR and proteinuria with 90-day mortality was similar to those for 28-day mortality (Table 2). Results were unchanged by additional adjustment for peripheral vascular disease. There was no good evidence of effect modification between eGFR and proteinuria, and in particular no evidence of any association of proteinuria with 28-day mortality within any category of eGFR status (data not shown).Table 2.Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)aNumber (column %)28-Day mortality (row %)90-Day mortality (row %)RRs for 28-day mortality (95% CI)RRs for 90-day mortality (95% CI)Adjusted for demographicsbadjusted for non-CVD co-morbiditiescFully adjusteddAdjusted for demographicsbAdjusted for non-CVD co-morbiditiescFully adjusteddPneumonia Proteinuria (adjusted for eGFR) Yes1611 (34.0)499 (31.0)625 (38.8)1.07 (0.97–1.17)1.07 (0.98–1.18)1.07 (0.98–1.18)1.05 (0.97–1.13)1.06 (0.98–1.14)1.05 (0.98–1.14) No3132 (66.0)907 (29.0)1150 (36.7)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<1523 (0.5)12 (52.2)12 (52.2)1.27 (1.10–1.47)1.31 (1.13–1.52)1.30 (1.12–1.52)1.26 (1.12–1.42)1.29 (1.14–1.45)1.27 (1.12–1.43)15–29263 (5.6)110 (41.8)139 (52.9) 30–44764 (16.1)265 (34.7)336 (44.0)0.98 (0.86–1.10)0.99 (0.88–1.13)0.99 (0.88–1.12)1.02 (0.93–1.13)1.04 (0.94–1.15)1.03 (0.94–1.14) 45–601162 (24.5)332 (28.6)418 (36.0)0.91 (0.82–1.02)0.94 (0.84–1.04)0.93 (0.84–1.04)0.91 (0.83–1.00)0.93 (0.85–1.02)0.92 (0.84–1.01) ≥602531 (53.4)687 (27.1)870 (34.4)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total47431406 (29.6)1775 (37.4)Sepsis Proteinuria (adjusted for eGFR) Yes358 (33.8)128 (35.8)159 (44.4)0.98 (0.82–1.17)1.01 (0.85–1.21)1.01 (0.85–1.21)0.98 (0.86–1.13)0.96 (0.84–1.10)0.96 (0.84–1.10) No700 (66.2)249 (35.6)309 (44.1)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<158 (0.8)2 (25.0)5 (62.5)1.42 (1.10–1.84)1.41 (1.08–1.84)1.37 (1.05–1.79)1.39 (1.14–1.70)1.32 (1.07–1.63)1.32 (1.07–1.64)15–2962 (5.9)32 (51.6)39 (62.9) 30–44190 (18.0)88 (46.3)103 (54.2)1.25 (1.01–1.55)1.24 (0.99–1.55)1.24 (0.99–1.54)1.14 (0.96–1.36)1.12 (0.94–1.33)1.11 (0.94–1.32) 45–60232 (21.9)76 (32.8)95 (41.0)0.95 (0.75–1.19)0.91 (0.72–1.15)0.91 (0.72–1.14)0.92 (0.77–1.11)0.89 (0.74–1.07)0.89 (0.75–1.07) ≥60566 (53.5)179 (31.6)226 (39.9)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total1058377 (35.6)468 (44.2)aExcluding patients with missing smoking or HbA1C data.bAge, gender, socio-economic status, onset prior to 1 April 2004.cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.FIGURE 1:Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nShort-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)a\naExcluding patients with missing smoking or HbA1C data.\nbAge, gender, socio-economic status, onset prior to 1 April 2004.\ncAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.\ndAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.\nSurvival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nThe underlying causes of death following pneumonia and sepsis were similar for patients with eGFR above and below 60 mL/min/1.73 m2. Causes of 28-day mortality following sepsis were predominantly sources of infection (Table 3). Following pneumonia onset, pneumonia was recorded as an underlying or contributory cause of death for 83.9% (1191/1419) of deaths within 28 days and 76.3% (1366/1790) of deaths within 90 days. Among patients with eGFR <60 mL/min/1.73 m2, renal disease was recorded as an underlying or contributory cause for 10.6% (77/724) of those who died within 28 days of pneumonia onset and 16.6% (152/913) of those who died within 90 days. Recording of CKD as a cause of death increased with lower eGFR (Table 4).Table 3.Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)aeGFReGFR <60 mL/min/1.73 m2eGFR ≥60 mL/min/1.73 m228-Day mortality following pneumonia, n = 1419J18 Pneumonia, organism unspecified, n = 256, 35.4%J44 Other chronic obstructive pulmonary disease, n = 63, 8.7%I25 Chronic ischaemic heart disease, n = 47, 6.5%I50 Heart failure, n = 43, 5.9%I64 Stroke, not specified as haemorrhage or infarction, n = 37, 5.1%J18 Pneumonia, organism unspecified, n = 216, 31.1%J44 Other chronic obstructive pulmonary disease, n = 63, 9.1%I64 Stroke, not specified as haemorrhage or infarction, n = 35, 5.0%C34 Malignant neoplasm of bronchus and lung, n = 34, 4.9%I25 Chronic ischaemic heart disease, n = 29, 4.2%Total = 724Total = 69529–90 Day mortality following pneumonia, n = 371J18 Pneumonia, organism unspecified, n = 38, 20.1%I25 Chronic ischaemic heart disease, n = 24, 12.7%J44 Other chronic obstructive pulmonary disease, n = 13, 6.9%C34 Malignant neoplasm of bronchus and lung, n = 12, 6.4%I21 Acute myocardial infarction, n = 12, 6.4%J18 Pneumonia, organism unspecified, n = 20, 11.0%C34 Malignant neoplasm of bronchus and lung, n = 20, 11.0%J44 Other chronic obstructive pulmonary disease, n = 19, 10.4%I25 Chronic ischaemic heart disease, n = 9, 5.0%I64 Stroke, not specified as haemorrhage or infarction, n = 9, 5.0%Total = 189Total = 18228-Day mortality following sepsis, n = 387N39 Other disorders of urinary systemb, n = 33, 15.9%J18 Pneumonia, organism unspecified, n = 30, 14.4%E14 Unspecified diabetes mellitus, n = 12, 5.8%A41 Other sepsis, n = 11, 5.3%L03 Cellulitis, n = 11, 5.3%N39 Other disorders of urinary systemb, n = 20, 11.2%A41 Other sepsis, n = 18, 10.1%J18 Pneumonia, organism unspecified, n = 18, 10.1%E14 Unspecified diabetes mellitus, n = 10, 5.6%J44 Other chronic obstructive pulmonary disease, n = 6, 3.4%=K55 Vascular disorders of intestine, n = 6, 3.4%=L03 Cellulitis, n = 6, 3.4%Total = 208Total = 179aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.Table 4.Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFReGFR (mL/min/1.73 m2)Deaths within 28 days of pneumonia onsetDeaths within 90 days of pneumonia onsetTotalN18 CKD, n (%)Renal disease,b\nn (%)TotalN18 CKD, n (%)Renal disease,b\nn (%)<1586 (75.0)7 (87.5)86 (75.0)7 (87.5)15–2910919 (17.4)41 (37.6)13926 (18.7)53 (38.1)30–4427514 (5.1)2 (15.3)34720 (5.8)56 (16.1)35–593328 (2.4)27 (8.1)41910 (2.4)36 (8.6)Total72447 (6.5)77 (10.6)91362 (6.8)152 (16.6)aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\nTop five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)a\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.\nRecording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFR\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\n\nDISCUSSION:\nAmong this population of older people with diabetes mellitus, eGFR <30 mL/min/1.73 m2 was a risk marker of higher 28- and 90-day mortality following community-acquired pneumonia and sepsis, compared with patients with eGFR ≥60 mL/min/1.73 m2. The relationship between eGFR and mortality did not change with adjustment for co-morbidities. Neither moderately impaired eGFR nor proteinuria was associated with higher short-term mortality following either infection.\nThe strengths of this study follow from the analysis of a focused question using large, linked datasets for a highly monitored primary care population with a cohort study design. Our study identifies that the association between eGFR and post-infection mortality persists when patients with end-stage renal disease (ESRD) are excluded (and is not explained by renal replacement therapy), when considering fixed-term rather than in-hospital mortality (thus is not due to differences in hospital stay) and when exclusively community-acquired infections are considered (so does not result from increased risk of healthcare-associated infections). The linked datasets allowed us to identify infections both among patients presenting directly to hospital and those managed in the community, maximized ascertainment of mortality and enabled description of the causes of death. The highly monitored population allowed good ascertainment of CKD status. The cohort study design has less potential for selection bias than an equivalent case–control study.\nA limitation is our assumption that the absence of a record implies a negative status for proteinuria and co-morbidities. Under-ascertainment of co-morbidities could result in residual confounding, with unpredictable effects, but the high prevalence of co-morbidities observed suggests that ascertainment was not markedly incomplete. We observed a high prevalence of proteinuria, and this is a highly monitored population (with financial incentives for standardized recording of proteinuria since 2004), but under-ascertainment of proteinuria could result in underestimation of any association between proteinuria and mortality [24]. Residual confounding from undiagnosed cardiovascular disease should have been minimized by adjustment for cardiovascular disease risk factors including smoking, hypertension and characteristics of diabetes.\nOur findings for eGFR provide further detail to build on previous findings that baseline eGFR <60 mL/min/1.73 m2 or renal disease are risk factors for short-term mortality following (hospital- or community-acquired) sepsis and for in-hospital mortality following community-acquired pneumonia (including patients receiving dialysis) [11–15]. A more comparable Canadian study examined the associations between eGFR and 30-day mortality following community-acquired pneumonia among the general population aged ≥65 years, excluding patients with ESRD [16]. Fully adjusted hazard ratios for 30-day mortality were 1.22 (95% CI 1.01–1.49) for eGFR 45–59, 2.03 (1.64–2.50) for eGFR 30–44 and 4.94 (3.94–6.19) for eGFR < 30, compared with eGFR 60–104 mL/min/1.73 m2. These are somewhat greater than the associations we observed. The difference may be explained by the different study populations. Both studies required a baseline serum creatinine result for inclusion. Our study population of older people with diabetes were routinely monitored for CKD (with financial incentivization in primary care) [32]. Creatinine testing of the Canadian study population may have been encouraged by co-morbidities or health-behaviours associated with CKD (such as smoking), which increase post-infection mortality, resulting in over-estimation of the association of eGFR and post-infection mortality. Our study population is less vulnerable to differential ascertainment of CKD. Alternatively, the association between eGFR and post-infection mortality may be smaller among patients with diabetes.\nTo the best of our knowledge, our examination of any association between proteinuria and mortality following community-acquired pneumonia and sepsis is novel. A history of proteinuria, although a marker for mortality in general, does not appear to be a risk marker for short-term mortality following community-acquired infection. This is unlikely to be due to chance, as the study was large, with findings consistent across both infections. We designed our study to produce conservative estimates and may have under-estimated the association between proteinuria and post-infection mortality due to under-ascertainment of proteinuria. Alternatively, any potential relationship between proteinuria and mortality may have been mitigated by clinical care of patients with infection who had pre-existing proteinuria, for example, through swift recognition of AKI.\nThe survival curves demonstrate a steep initial mortality following infection onset, and a high proportion of deaths had the underlying cause assigned to infection. Since 2001 in England, co-morbidities are assigned as the underlying cause of death when pneumonia has occurred in the context of, for example, malignancy or respiratory disease [25]. This suggests that the associations we observed are driven by an association between eGFR and infection prognosis, not merely high underlying baseline mortality among patients with impaired eGFR. This is supported by previous research which found 7.7-fold elevated mortality in the 30 days following community-acquired pneumonia [19]. Estimates for associations between eGFR and mortality were not substantially altered by adjustment for co-morbidities, suggesting that any causal relationship between eGFR and mortality is not mediated through co-morbidities.\nOur findings apply to the large population of older patients with community-acquired pneumonia or sepsis with diabetes mellitus who do not have ESRD. Inclusion criteria are unlikely to have limited generalizability appreciably. Practices which consent to data linkage could be more research oriented, providing good primary care management of risk factors for infection-related mortality (such as smoking cessation), but this is unlikely to affect the relationship between CKD and short-term mortality post-infection. Lack of pre-existing creatinine test results is likely to reflect limited time potentially eligible for the study rather than CKD status among this highly monitored population. Missing data on smoking status and HbA1C may be a marker of low patient engagement: caution should be used in generalizing our results to patients who are not actively managed in primary care.\nWe found that CKD is a useful clinical risk marker for post-infectious mortality. Whether this relationship is causal is less clear; but the association does not appear to be explained by age, co-morbidities or hospital attendance. Potential mechanisms include immune system dysfunction, but also more preventable complications such as AKI. Combinations of risk factors may be important: for example, patients with post-operative AKI have higher mortality if they also have pre-existing CKD [30].\nOur results have implications for patient management and future research. Patients with baseline eGFR <30 mL/min/1.73 m2 and community-acquired infection need careful monitoring, particularly in the 28 days following infection. Future research should investigate preventable mechanisms by which low baseline eGFR could be related to post-infection mortality, for example, fluid management, AKI and drug dosage in patients with low renal clearance.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWe aimed to examine whether pre-existing impaired estimated glomerular filtration rate (eGFR) and proteinuria were associated with mortality following community-acquired pneumonia or sepsis among people aged ≥ 65 years with diabetes mellitus, without end-stage renal disease.\n\nMethods:\nPatients were followed up from onset of first community-acquired pneumonia or sepsis episode in a cohort study using large, linked electronic health databases. Follow-up was for up to 90 days, unlimited by hospital discharge. We used generalized linear models with log link, normal distribution and robust standard errors to calculate risk ratios (RRs) for all-cause 28- and 90-day mortality according to two markers of chronic kidney disease: eGFR and proteinuria.\n\nResults:\nAll-cause mortality among the 4743 patients with pneumonia was 29.6% after 28 days and 37.4% after 90 days. Among the 1058 patients with sepsis, all-cause 28- and 90-day mortality were 35.6 and 44.2%, respectively. eGFR <30 mL/min/1.73 m(2) was a risk marker of higher 28-day mortality for pneumonia (RR 1.27: 95% CI 1.12-1.43) and sepsis (RR 1.32: 95% CI 1.07-1.64), adjusted for age, sex, socio-economic status, smoking status and co-morbidities. Neither moderately impaired eGFR nor proteinuria were associated with short-term mortality following either infection.\n\nConclusions:\nPeople with pre-existing low eGFR but not on dialysis are at higher risk of death following pneumonia and sepsis. This association was not explained by existing co-morbidities. These patients need to be carefully monitored to prevent modifiable causes of death."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":8630,"string":"8,630"},"whole_article_abstract_length":{"kind":"number","value":322,"string":"322"},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["infection","mortality","egfr","patients","ckd","pneumonia","disease","status","28","onset"],"string":"[\n \"infection\",\n \"mortality\",\n \"egfr\",\n \"patients\",\n \"ckd\",\n \"pneumonia\",\n \"disease\",\n \"status\",\n \"28\",\n \"onset\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] chronic kidney disease | community-acquired infections | electronic health records | infection/mortality | proteinuria [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] chronic kidney disease | community-acquired infections | electronic health records | infection/mortality | proteinuria [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] chronic kidney disease | community-acquired infections | electronic health records | infection/mortality | proteinuria [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Aged | Aged, 80 and over | Biomarkers | Cohort Studies | Community-Acquired Infections | Comorbidity | Diabetes Mellitus | Electronic Health Records | Female | Humans | Kidney Failure, Chronic | Male | Pneumonia | Proteinuria | Renal Dialysis | Risk Factors | Sepsis | Survival Rate [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Aged | Aged, 80 and over | Biomarkers | Cohort Studies | Community-Acquired Infections | Comorbidity | Diabetes Mellitus | Electronic Health Records | Female | Humans | Kidney Failure, Chronic | Male | Pneumonia | Proteinuria | Renal Dialysis | Risk Factors | Sepsis | Survival Rate [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Aged | Aged, 80 and over | Biomarkers | Cohort Studies | Community-Acquired Infections | Comorbidity | Diabetes Mellitus | Electronic Health Records | Female | Humans | Kidney Failure, Chronic | Male | Pneumonia | Proteinuria | Renal Dialysis | Risk Factors | Sepsis | Survival Rate [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | mortality | egfr | patients | ckd | pneumonia | disease | status | 28 | onset [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | mortality | egfr | patients | ckd | pneumonia | disease | status | 28 | onset [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | mortality | egfr | patients | ckd | pneumonia | disease | status | 28 | onset [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ckd | associated | mortality | infection | older | older people | community | community acquired | acquired | prognosis [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] reference reference | reference | pneumonia | reference reference reference | day mortality | mortality | disease | reference reference reference reference | 10 | 73 [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | mortality | egfr | ckd | patients | days | data | 28 | acquired | status [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ≥ | 65 years [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 4743 | 29.6% | 28 days | 37.4% | 90 days ||| 1058 | 28- | 90-day | 35.6 | 44.2% ||| m(2 | 28-day | 1.27 | 95% | CI | 1.12-1.43 | 1.32 | 95% | CI | 1.07-1.64 ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ≥ | 65 years ||| first ||| up to 90 days ||| linear | 28- | 90-day | two | proteinuria ||| 4743 | 29.6% | 28 days | 37.4% | 90 days ||| 1058 | 28- | 90-day | 35.6 | 44.2% ||| m(2 | 28-day | 1.27 | 95% | CI | 1.12-1.43 | 1.32 | 95% | CI | 1.07-1.64 ||| ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":282,"cells":{"title":{"kind":"string","value":"Endometrial intraepithelial carcinoma in association with polyp: review of eight cases."},"pmid":{"kind":"string","value":"23414240"},"background_abstract":{"kind":"string","value":"The uterine endometrial polyp (EMP) has a potential risk of developing malignant tumors especially in postmenopausal women. These malignancies include endometrial intraepithelial carcinoma (EIC)."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Eight patients with EIC in the EMP, who were postmenopausal with ages ranging from 49 to 76 years (av. 62), were cytologically reviewed in comparison with histological findings."},"methods_abstract_label":{"kind":"string","value":"PATIENTS AND METHODS"},"results_abstract":{"kind":"string","value":"The endometrial cytological findings were summarized as follows: mucous and watery diathesis as a background lacking or with little necrotic inflammatory changes; micropapillary cluster formation; abrupt transition between carcinoma cells and normal cells; nuclear enlargement; high N/C ratio; and single or a few prominent nucleoli. Histologically, one case had EIC alone in the EMP; three cases had EIC with stromal invasion confined to the EMP; and four cases had EIC in the atrophic endometrium in addition to EIC in the EMP. Seven patients have taken a disease-free course after surgical resection, but one patient died 44 months following the initial diagnosis because of the massive tumor extending over her peritoneal cavity."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Endometrial cytology may be helpful for the detection of early endometrial adenocarcinomas with serous features including EIC. Some early stage endometrial adenocarcinomas represented by EIC exceptionally take an aggressive clinical course irrespective of a lack of extrauterine lesions."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adenocarcinoma","Aged","Carcinoma in Situ","Disease Progression","Early Detection of Cancer","Endometrial Neoplasms","Female","Humans","Middle Aged","Polyps","Predictive Value of Tests","Time Factors","Treatment Outcome"],"string":"[\n \"Adenocarcinoma\",\n \"Aged\",\n \"Carcinoma in Situ\",\n \"Disease Progression\",\n \"Early Detection of Cancer\",\n \"Endometrial Neoplasms\",\n \"Female\",\n \"Humans\",\n \"Middle Aged\",\n \"Polyps\",\n \"Predictive Value of Tests\",\n \"Time Factors\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"3599099"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The EMPs including EIC with / without stromal invasion measured less than 1 cm in the great diameter in all of the patients. The cytological features are summarized in Table 1 and representatively depicted in Figures 1, 2, and 3, including histological findings of three patients. The tumor diathesis was characterized as being predominantly mucous for 3 patients, watery for 3 patients, and bloody for 2 patients, but with no prominent necrotic inflammatory changes. The tumor cell clusters predominantly took a micropapillary pattern and occasionally a sheet-like pattern. Overlapping of the tumor cell nuclei was noted in a variable degree: often for 2 patients, and occasionally for 5 patients. There were varying amounts of normal epithelial cells, which served as a hallmark in distinction from the malignancy because of a significant difference in the nuclear size. Using Isis ver. 5 (MetaSystems GmbH, Altlussheim, Germany), the nuclear size (greatest diameter) of tumor cells were measured as well as normal atrophic cells in the cytological preparations. The numbers of counted cells for the former and the latter ranged from 50 to 70 in each case. On the total average of 8 cases, the tumor cell size was 12.5 μm and the relative ratio to the corresponding normal atrophic cells was 2.3 (Table 1). The chromatin was considerably dense, coarse or granular. The number of prominent nucleoli was as follows: 1 for 4 patients; 1 or 2 for 4 patients. Calcified deposits of psammoma body were not apparently observed in any patient.\nCytological features of endometrial intraepithelial carcinomas\na: average of largest diameter of tumor cells (relative value compared to normal nucleus).\nCase 2 possessing EIC in both endometrial polyp and endometrium (A. Pap × 20, B. Pap × 60, C. loupe view, D. HE × 20, E. HE × 60, F. HE × 60). A. Papillary-structured carcinoma cell cluster is accompanied by a watery background (Pap. ×20). B. The N/C ratio is fairly high and the chromatin is coarsely granular (Pap. ×60). C. Loupe findings show an endometrial polyp arising in the uterine fundus, measuring 1.5 cm in longitudinal length. The endometrium is markedly thin and atrophic. The area marked by an arrow and that marked by an arrowhead are depicted respectively in E and F. D. In the polyp, the glands composed of carcinoma cells are found to be aggregated in the fibrotic stroma. E. Carcinoma cells are replacing the glands in the polyp and stratified into micropapillary protrusions, lacking apparent stromal invasion (HE × 20). F. Carcinoma nests similar to those in the endometrial polyp (D) are also present in the endometrial gland (HE × 60).\nCase 5 possessing EIC in both endometrial polyp and endometrium, but with stromal invasion in the polyp (A. Pap × 20, B. Pap × 60, C. HE × 20, D. HE × 60). A. A carcinoma cell cluster is accompanied by a bloody background. B. Carcinoma cells having enlarged nuclei appear to be transitional with normal atrophic cells having small-sized nuclei (arrows). C. The polyp has many atypical glands with intraluminal papillary protrusion of the carcinoma cells. D. The border between the carcinoma cells and normal cells is clearly discernible (arrows). There are small carcinoma nests showing stromal invasion (arrowheads).\nCase 6 possessing EIC confined to endometrial polyp (A. Pap × 20, B. Pap × 60, C. HE × 60, D. HE × 60). A. Large-sized carcinoma cell clusters are accompanied by a non-necroinflammatory background. B. Carcinoma cells mainly appear in a flat and sheet-like cluster. The nucleus has an enlarged eosinophilic nucleolus. C. The atypical glands are normal-sized, accompanied by cystic dilatation of atrophic normal glands. D. Carcinoma cells are replacing pre-existing glands and are arranged in a single layer (compare with normal gland marked by an arrow).\nThe clinicopathological profiles are summarized in Table 2. All the patients had an EMP in which atypical tumor cells were arranged predominantly in a tubular structure with micropapillary protrusions, replacing the pre-existing endometrial glands. Micropapillary protrusions and small tufts were accompanied with or without thin fibrovascular cores. These tumor cells showed conspicuous nuclear pleomorphism and hyperchromatism, frequent mitotic figures, high N/C ratio, and also one or two prominent nucleoli. The tumor front was abruptly made into normal glandular epithelium and surface-lining epithelium in the EMP. Minimal stromal invasion of the tumor cells limited to the EMP was recognized in 3 patients. In addition, similar tumor cells also were also found to replace some atrophic endometrial glands in 4 patients, lacking stromal invasion. In the background, cystic dilatation of atrophic normal glands was evident especially in the EMP.\nClinicopathological profiles of endometrial intraepithelial carcinomas\na:+, strongly positive cells more than 80%.\nb: index of positive cells(%), c: +, positive cells more than 80%.\nd: +, positive cells more than 80%.\ne: since initial diagnosis.\nf: history of breast cancer with Tamoxifen administration.\ng: autopsy performed.\nh: with stromal invasion, i: disease free, j: death on disease.\nImmunohistochemical expressions of p53 (DO7, 1:50, Dako, Glostrup, Denmark), Ki-67 (MIB-1, 1:100, Dako, Glostrup, Denmark), ER (SP-1, 1:1, Ventana, AZ, USA), and PgR (1E2, 1:1, Ventana, AZ, USA) were examined (Figure 4). Six patients showed marked p53 expression with a positive ratio of more than 80%. The labeling index of Ki-67 ranged from 25 to 70% (av. approximately 40). ER was mildly expressed in one patient and PgR expression also was mildly observed in 2 patients.\nImmunohistochemical staining profiles represented by Case 5 (A. p53 × 40, B. MIB-1 × 40, C. ER × 20, D. PgR × 20). A. Almost all of the carcinoma cells are strongly positive. B. More than half the carcinoma cells are labeled. C. The expression is observed in the cystically dilated normal glands and stromal cells but not in the carcinoma cells. D. The expression behavior is basically similar to the ER expression.\nFIGO stage was determined at IA for all the patients. Neither adjuvant chemotherapy nor irradiation was performed. Seven patients have been taking an uneventful clinical course (follow-up period ranging from 12 to 66 months, av. 41), but one patient died of the recurrent disease massively occupying the abdominal cavity 44 months after the initial diagnosis."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Introduction","Patients","Patients’ consents","Ethical approval","Competing interests","Authors’ contributions"],"string":"[\n \"Introduction\",\n \"Patients\",\n \"Patients’ consents\",\n \"Ethical approval\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.","The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.","Written informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.","This review was performed based on compliance with the Helsinki Declaration.","All of the authors declare that they have no competing interests.","MY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript."],"string":"[\n \"The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\",\n \"The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\",\n \"Written informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.\",\n \"This review was performed based on compliance with the Helsinki Declaration.\",\n \"All of the authors declare that they have no competing interests.\",\n \"MY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Patients","Results","Discussion","Patients’ consents","Ethical approval","Competing interests","Authors’ contributions"],"string":"[\n \"Introduction\",\n \"Patients\",\n \"Results\",\n \"Discussion\",\n \"Patients’ consents\",\n \"Ethical approval\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.","The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.","The EMPs including EIC with / without stromal invasion measured less than 1 cm in the great diameter in all of the patients. The cytological features are summarized in Table 1 and representatively depicted in Figures 1, 2, and 3, including histological findings of three patients. The tumor diathesis was characterized as being predominantly mucous for 3 patients, watery for 3 patients, and bloody for 2 patients, but with no prominent necrotic inflammatory changes. The tumor cell clusters predominantly took a micropapillary pattern and occasionally a sheet-like pattern. Overlapping of the tumor cell nuclei was noted in a variable degree: often for 2 patients, and occasionally for 5 patients. There were varying amounts of normal epithelial cells, which served as a hallmark in distinction from the malignancy because of a significant difference in the nuclear size. Using Isis ver. 5 (MetaSystems GmbH, Altlussheim, Germany), the nuclear size (greatest diameter) of tumor cells were measured as well as normal atrophic cells in the cytological preparations. The numbers of counted cells for the former and the latter ranged from 50 to 70 in each case. On the total average of 8 cases, the tumor cell size was 12.5 μm and the relative ratio to the corresponding normal atrophic cells was 2.3 (Table 1). The chromatin was considerably dense, coarse or granular. The number of prominent nucleoli was as follows: 1 for 4 patients; 1 or 2 for 4 patients. Calcified deposits of psammoma body were not apparently observed in any patient.\nCytological features of endometrial intraepithelial carcinomas\na: average of largest diameter of tumor cells (relative value compared to normal nucleus).\nCase 2 possessing EIC in both endometrial polyp and endometrium (A. Pap × 20, B. Pap × 60, C. loupe view, D. HE × 20, E. HE × 60, F. HE × 60). A. Papillary-structured carcinoma cell cluster is accompanied by a watery background (Pap. ×20). B. The N/C ratio is fairly high and the chromatin is coarsely granular (Pap. ×60). C. Loupe findings show an endometrial polyp arising in the uterine fundus, measuring 1.5 cm in longitudinal length. The endometrium is markedly thin and atrophic. The area marked by an arrow and that marked by an arrowhead are depicted respectively in E and F. D. In the polyp, the glands composed of carcinoma cells are found to be aggregated in the fibrotic stroma. E. Carcinoma cells are replacing the glands in the polyp and stratified into micropapillary protrusions, lacking apparent stromal invasion (HE × 20). F. Carcinoma nests similar to those in the endometrial polyp (D) are also present in the endometrial gland (HE × 60).\nCase 5 possessing EIC in both endometrial polyp and endometrium, but with stromal invasion in the polyp (A. Pap × 20, B. Pap × 60, C. HE × 20, D. HE × 60). A. A carcinoma cell cluster is accompanied by a bloody background. B. Carcinoma cells having enlarged nuclei appear to be transitional with normal atrophic cells having small-sized nuclei (arrows). C. The polyp has many atypical glands with intraluminal papillary protrusion of the carcinoma cells. D. The border between the carcinoma cells and normal cells is clearly discernible (arrows). There are small carcinoma nests showing stromal invasion (arrowheads).\nCase 6 possessing EIC confined to endometrial polyp (A. Pap × 20, B. Pap × 60, C. HE × 60, D. HE × 60). A. Large-sized carcinoma cell clusters are accompanied by a non-necroinflammatory background. B. Carcinoma cells mainly appear in a flat and sheet-like cluster. The nucleus has an enlarged eosinophilic nucleolus. C. The atypical glands are normal-sized, accompanied by cystic dilatation of atrophic normal glands. D. Carcinoma cells are replacing pre-existing glands and are arranged in a single layer (compare with normal gland marked by an arrow).\nThe clinicopathological profiles are summarized in Table 2. All the patients had an EMP in which atypical tumor cells were arranged predominantly in a tubular structure with micropapillary protrusions, replacing the pre-existing endometrial glands. Micropapillary protrusions and small tufts were accompanied with or without thin fibrovascular cores. These tumor cells showed conspicuous nuclear pleomorphism and hyperchromatism, frequent mitotic figures, high N/C ratio, and also one or two prominent nucleoli. The tumor front was abruptly made into normal glandular epithelium and surface-lining epithelium in the EMP. Minimal stromal invasion of the tumor cells limited to the EMP was recognized in 3 patients. In addition, similar tumor cells also were also found to replace some atrophic endometrial glands in 4 patients, lacking stromal invasion. In the background, cystic dilatation of atrophic normal glands was evident especially in the EMP.\nClinicopathological profiles of endometrial intraepithelial carcinomas\na:+, strongly positive cells more than 80%.\nb: index of positive cells(%), c: +, positive cells more than 80%.\nd: +, positive cells more than 80%.\ne: since initial diagnosis.\nf: history of breast cancer with Tamoxifen administration.\ng: autopsy performed.\nh: with stromal invasion, i: disease free, j: death on disease.\nImmunohistochemical expressions of p53 (DO7, 1:50, Dako, Glostrup, Denmark), Ki-67 (MIB-1, 1:100, Dako, Glostrup, Denmark), ER (SP-1, 1:1, Ventana, AZ, USA), and PgR (1E2, 1:1, Ventana, AZ, USA) were examined (Figure 4). Six patients showed marked p53 expression with a positive ratio of more than 80%. The labeling index of Ki-67 ranged from 25 to 70% (av. approximately 40). ER was mildly expressed in one patient and PgR expression also was mildly observed in 2 patients.\nImmunohistochemical staining profiles represented by Case 5 (A. p53 × 40, B. MIB-1 × 40, C. ER × 20, D. PgR × 20). A. Almost all of the carcinoma cells are strongly positive. B. More than half the carcinoma cells are labeled. C. The expression is observed in the cystically dilated normal glands and stromal cells but not in the carcinoma cells. D. The expression behavior is basically similar to the ER expression.\nFIGO stage was determined at IA for all the patients. Neither adjuvant chemotherapy nor irradiation was performed. Seven patients have been taking an uneventful clinical course (follow-up period ranging from 12 to 66 months, av. 41), but one patient died of the recurrent disease massively occupying the abdominal cavity 44 months after the initial diagnosis.","Endometrial polyp is a common benign disease of the uterus. The overall incidence where cancer arises in the endometrial polyp has been reported as only a few percent, while postmenopausal women with EMP are at an increased risk of malignancy, compared to premenopausal women [10,13-15]. As risk factors for development of the malignancy in EMP, hypertension, obesity, and unopposed estrogen therapy have been indicated in addition to postmenopausal status [15]. There are unique or specific pathological and clinical features for EIC as a putative precursor of uterine body serous adenocarcinoma, as follows [1,4,5,7,16-20]: close association with an EMP; multicentric occurrence in the inactive or resting thin endometrium, lacking alteration in the architecture in the endometrium lacking an intervening phase of endometrial hyperplasia; extrauterine invasive extent, irrespective of an absence of stromal invasion of the uterus; and controversial outcome, whether taking a favorable or unfavorable clinical course. Development of EIC associated with an EMP may also be explained in part by Tamoxifen administration following breast cancer treatment [6,17]. However, little has been documented about the endometrial cytological features of minimal serous adenocarcinoma of the uterine body including EIC, mainly due to the fact that the disease is not frequently encountered in the routine practice. To the best of our knowledge, there is a case report cytologically referring to serous adenocarcinoma of the uterine body associated with an EMP [21], but here the patient already showed peritoneal dissemination at the initial presentation. In another report, serous adenocarcinomas of the uterine body were clinico-cytologically reviewed but they were all at advanced stages [22]. The latter report mentions that cytologically diagnostic findings of uterine body serous adenocarcinoma would be based on the following features: papillary cluster as well as tubular cluster, arborescent structure, frequent exfoliation, psammoma body, eccentric enlarged nuclei, coarsely granular chromatin, numerous nucleoli, and diameter of greater than G1 endometrioid adenocarcinoma [22]. In our review, 5 patients were purely non-invasive as they were composed of only EIC existing in an EMP, with few or no necroinflammatory changes. The background was found to be characteristically watery or mucous (Figure 1) in a way of simulating the immigration of extrauterine carcinoma cells via the fallopian tubes [12]. Many clusters were arranged in micropapillary architecture without fine vascular stroma, and others were occasionally tubular. An abrupt transition between the EIC cells and atrophic endometrial cells was noted by front formation (Figure 2). Conspicuous nucleoli were found in 3 patients (Figure 3). Even though it seems difficult to reach a definite diagnosis, these findings may serve as a diagnostic indicator for the uterine body serous adenocarcinoma, whether being at an early stage or an advanced stage.\nDifferential diagnoses for atypical epithelial sheets in the normal-appearing endometrial background include variable interpretations as follows: EIC, endometrial glandular dysplasia, endometrial intraepithelial neoplasia, hyperplastic polyp, and metastatic carcinoma [9,23]. In addition to these, isolated atypical glands with morphological and immunohistochemical features of a typical endometrial hyperplasia or type I endometrial adenocarcinoma may be encountered in grossly normal postmenopausal endometrium of asymptomatic patients [24]. Especially in the postmenopausal state, thin and smooth endometrium with atrophy is composed of hypocellular and flattened glands lined by cells with reduced or absent mitosis and sparse fibrous stroma [25]. It is supposed that the abrupt transition of the atrophic endometrial cells to atypical cells may indicate EIC in postmenopausal women.\nOverexpression of p53 is closely linked to the rapid growth of uterine body serous adenocarcinoma [6]. In EIC, too, frequent p53 mutation is reported to be associated with LOH of 17p [26]. Thus, immunohistochemistry for p53 is also adjunctively available in the diagnosis of uterine body serous adenocarcinoma, irrespective of whether it is at an early stage or advanced stage [27], in the discrimination between uterine body serous adenocarcinoma and benign morphologic mimics [28]. Negativity of ER and PgR staining is more reasonable with the diagnosis of serous adenocarcinoma including EIC.\nIn summary, EMP arising in postmenopausal women is at a high risk of giving birth to the tumorigenetic background for endometrial carcinomas such as endometrioid adenocarcinoma and serous adenocarcinoma including EIC. Endometrial cytology may become a helpful diagnostic approach for serous adenocarcinoma, whether it is at early or advanced stage, from the viewpoint of the distinctive cytological features. Above all, it should be noted that some EIC behaves in a more aggressive fashion regardless of whether or not it lacks apparent stroma invasion and extrauterine extent.","Written informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.","This review was performed based on compliance with the Helsinki Declaration.","All of the authors declare that they have no competing interests.","MY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript."],"string":"[\n \"The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\",\n \"The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\",\n \"The EMPs including EIC with / without stromal invasion measured less than 1 cm in the great diameter in all of the patients. The cytological features are summarized in Table 1 and representatively depicted in Figures 1, 2, and 3, including histological findings of three patients. The tumor diathesis was characterized as being predominantly mucous for 3 patients, watery for 3 patients, and bloody for 2 patients, but with no prominent necrotic inflammatory changes. The tumor cell clusters predominantly took a micropapillary pattern and occasionally a sheet-like pattern. Overlapping of the tumor cell nuclei was noted in a variable degree: often for 2 patients, and occasionally for 5 patients. There were varying amounts of normal epithelial cells, which served as a hallmark in distinction from the malignancy because of a significant difference in the nuclear size. Using Isis ver. 5 (MetaSystems GmbH, Altlussheim, Germany), the nuclear size (greatest diameter) of tumor cells were measured as well as normal atrophic cells in the cytological preparations. The numbers of counted cells for the former and the latter ranged from 50 to 70 in each case. On the total average of 8 cases, the tumor cell size was 12.5 μm and the relative ratio to the corresponding normal atrophic cells was 2.3 (Table 1). The chromatin was considerably dense, coarse or granular. The number of prominent nucleoli was as follows: 1 for 4 patients; 1 or 2 for 4 patients. Calcified deposits of psammoma body were not apparently observed in any patient.\\nCytological features of endometrial intraepithelial carcinomas\\na: average of largest diameter of tumor cells (relative value compared to normal nucleus).\\nCase 2 possessing EIC in both endometrial polyp and endometrium (A. Pap × 20, B. Pap × 60, C. loupe view, D. HE × 20, E. HE × 60, F. HE × 60). A. Papillary-structured carcinoma cell cluster is accompanied by a watery background (Pap. ×20). B. The N/C ratio is fairly high and the chromatin is coarsely granular (Pap. ×60). C. Loupe findings show an endometrial polyp arising in the uterine fundus, measuring 1.5 cm in longitudinal length. The endometrium is markedly thin and atrophic. The area marked by an arrow and that marked by an arrowhead are depicted respectively in E and F. D. In the polyp, the glands composed of carcinoma cells are found to be aggregated in the fibrotic stroma. E. Carcinoma cells are replacing the glands in the polyp and stratified into micropapillary protrusions, lacking apparent stromal invasion (HE × 20). F. Carcinoma nests similar to those in the endometrial polyp (D) are also present in the endometrial gland (HE × 60).\\nCase 5 possessing EIC in both endometrial polyp and endometrium, but with stromal invasion in the polyp (A. Pap × 20, B. Pap × 60, C. HE × 20, D. HE × 60). A. A carcinoma cell cluster is accompanied by a bloody background. B. Carcinoma cells having enlarged nuclei appear to be transitional with normal atrophic cells having small-sized nuclei (arrows). C. The polyp has many atypical glands with intraluminal papillary protrusion of the carcinoma cells. D. The border between the carcinoma cells and normal cells is clearly discernible (arrows). There are small carcinoma nests showing stromal invasion (arrowheads).\\nCase 6 possessing EIC confined to endometrial polyp (A. Pap × 20, B. Pap × 60, C. HE × 60, D. HE × 60). A. Large-sized carcinoma cell clusters are accompanied by a non-necroinflammatory background. B. Carcinoma cells mainly appear in a flat and sheet-like cluster. The nucleus has an enlarged eosinophilic nucleolus. C. The atypical glands are normal-sized, accompanied by cystic dilatation of atrophic normal glands. D. Carcinoma cells are replacing pre-existing glands and are arranged in a single layer (compare with normal gland marked by an arrow).\\nThe clinicopathological profiles are summarized in Table 2. All the patients had an EMP in which atypical tumor cells were arranged predominantly in a tubular structure with micropapillary protrusions, replacing the pre-existing endometrial glands. Micropapillary protrusions and small tufts were accompanied with or without thin fibrovascular cores. These tumor cells showed conspicuous nuclear pleomorphism and hyperchromatism, frequent mitotic figures, high N/C ratio, and also one or two prominent nucleoli. The tumor front was abruptly made into normal glandular epithelium and surface-lining epithelium in the EMP. Minimal stromal invasion of the tumor cells limited to the EMP was recognized in 3 patients. In addition, similar tumor cells also were also found to replace some atrophic endometrial glands in 4 patients, lacking stromal invasion. In the background, cystic dilatation of atrophic normal glands was evident especially in the EMP.\\nClinicopathological profiles of endometrial intraepithelial carcinomas\\na:+, strongly positive cells more than 80%.\\nb: index of positive cells(%), c: +, positive cells more than 80%.\\nd: +, positive cells more than 80%.\\ne: since initial diagnosis.\\nf: history of breast cancer with Tamoxifen administration.\\ng: autopsy performed.\\nh: with stromal invasion, i: disease free, j: death on disease.\\nImmunohistochemical expressions of p53 (DO7, 1:50, Dako, Glostrup, Denmark), Ki-67 (MIB-1, 1:100, Dako, Glostrup, Denmark), ER (SP-1, 1:1, Ventana, AZ, USA), and PgR (1E2, 1:1, Ventana, AZ, USA) were examined (Figure 4). Six patients showed marked p53 expression with a positive ratio of more than 80%. The labeling index of Ki-67 ranged from 25 to 70% (av. approximately 40). ER was mildly expressed in one patient and PgR expression also was mildly observed in 2 patients.\\nImmunohistochemical staining profiles represented by Case 5 (A. p53 × 40, B. MIB-1 × 40, C. ER × 20, D. PgR × 20). A. Almost all of the carcinoma cells are strongly positive. B. More than half the carcinoma cells are labeled. C. The expression is observed in the cystically dilated normal glands and stromal cells but not in the carcinoma cells. D. The expression behavior is basically similar to the ER expression.\\nFIGO stage was determined at IA for all the patients. Neither adjuvant chemotherapy nor irradiation was performed. Seven patients have been taking an uneventful clinical course (follow-up period ranging from 12 to 66 months, av. 41), but one patient died of the recurrent disease massively occupying the abdominal cavity 44 months after the initial diagnosis.\",\n \"Endometrial polyp is a common benign disease of the uterus. The overall incidence where cancer arises in the endometrial polyp has been reported as only a few percent, while postmenopausal women with EMP are at an increased risk of malignancy, compared to premenopausal women [10,13-15]. As risk factors for development of the malignancy in EMP, hypertension, obesity, and unopposed estrogen therapy have been indicated in addition to postmenopausal status [15]. There are unique or specific pathological and clinical features for EIC as a putative precursor of uterine body serous adenocarcinoma, as follows [1,4,5,7,16-20]: close association with an EMP; multicentric occurrence in the inactive or resting thin endometrium, lacking alteration in the architecture in the endometrium lacking an intervening phase of endometrial hyperplasia; extrauterine invasive extent, irrespective of an absence of stromal invasion of the uterus; and controversial outcome, whether taking a favorable or unfavorable clinical course. Development of EIC associated with an EMP may also be explained in part by Tamoxifen administration following breast cancer treatment [6,17]. However, little has been documented about the endometrial cytological features of minimal serous adenocarcinoma of the uterine body including EIC, mainly due to the fact that the disease is not frequently encountered in the routine practice. To the best of our knowledge, there is a case report cytologically referring to serous adenocarcinoma of the uterine body associated with an EMP [21], but here the patient already showed peritoneal dissemination at the initial presentation. In another report, serous adenocarcinomas of the uterine body were clinico-cytologically reviewed but they were all at advanced stages [22]. The latter report mentions that cytologically diagnostic findings of uterine body serous adenocarcinoma would be based on the following features: papillary cluster as well as tubular cluster, arborescent structure, frequent exfoliation, psammoma body, eccentric enlarged nuclei, coarsely granular chromatin, numerous nucleoli, and diameter of greater than G1 endometrioid adenocarcinoma [22]. In our review, 5 patients were purely non-invasive as they were composed of only EIC existing in an EMP, with few or no necroinflammatory changes. The background was found to be characteristically watery or mucous (Figure 1) in a way of simulating the immigration of extrauterine carcinoma cells via the fallopian tubes [12]. Many clusters were arranged in micropapillary architecture without fine vascular stroma, and others were occasionally tubular. An abrupt transition between the EIC cells and atrophic endometrial cells was noted by front formation (Figure 2). Conspicuous nucleoli were found in 3 patients (Figure 3). Even though it seems difficult to reach a definite diagnosis, these findings may serve as a diagnostic indicator for the uterine body serous adenocarcinoma, whether being at an early stage or an advanced stage.\\nDifferential diagnoses for atypical epithelial sheets in the normal-appearing endometrial background include variable interpretations as follows: EIC, endometrial glandular dysplasia, endometrial intraepithelial neoplasia, hyperplastic polyp, and metastatic carcinoma [9,23]. In addition to these, isolated atypical glands with morphological and immunohistochemical features of a typical endometrial hyperplasia or type I endometrial adenocarcinoma may be encountered in grossly normal postmenopausal endometrium of asymptomatic patients [24]. Especially in the postmenopausal state, thin and smooth endometrium with atrophy is composed of hypocellular and flattened glands lined by cells with reduced or absent mitosis and sparse fibrous stroma [25]. It is supposed that the abrupt transition of the atrophic endometrial cells to atypical cells may indicate EIC in postmenopausal women.\\nOverexpression of p53 is closely linked to the rapid growth of uterine body serous adenocarcinoma [6]. In EIC, too, frequent p53 mutation is reported to be associated with LOH of 17p [26]. Thus, immunohistochemistry for p53 is also adjunctively available in the diagnosis of uterine body serous adenocarcinoma, irrespective of whether it is at an early stage or advanced stage [27], in the discrimination between uterine body serous adenocarcinoma and benign morphologic mimics [28]. Negativity of ER and PgR staining is more reasonable with the diagnosis of serous adenocarcinoma including EIC.\\nIn summary, EMP arising in postmenopausal women is at a high risk of giving birth to the tumorigenetic background for endometrial carcinomas such as endometrioid adenocarcinoma and serous adenocarcinoma including EIC. Endometrial cytology may become a helpful diagnostic approach for serous adenocarcinoma, whether it is at early or advanced stage, from the viewpoint of the distinctive cytological features. Above all, it should be noted that some EIC behaves in a more aggressive fashion regardless of whether or not it lacks apparent stroma invasion and extrauterine extent.\",\n \"Written informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.\",\n \"This review was performed based on compliance with the Helsinki Declaration.\",\n \"All of the authors declare that they have no competing interests.\",\n \"MY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,"results","discussion",null,null,null,null],"string":"[\n null,\n null,\n \"results\",\n \"discussion\",\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Endometrial intraepithelial carcinoma (EIC)","Endometrial polyp","Cytology"],"string":"[\n \"Endometrial intraepithelial carcinoma (EIC)\",\n \"Endometrial polyp\",\n \"Cytology\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nThe uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\n\nPatients:\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\n\nResults:\nThe EMPs including EIC with / without stromal invasion measured less than 1 cm in the great diameter in all of the patients. The cytological features are summarized in Table 1 and representatively depicted in Figures 1, 2, and 3, including histological findings of three patients. The tumor diathesis was characterized as being predominantly mucous for 3 patients, watery for 3 patients, and bloody for 2 patients, but with no prominent necrotic inflammatory changes. The tumor cell clusters predominantly took a micropapillary pattern and occasionally a sheet-like pattern. Overlapping of the tumor cell nuclei was noted in a variable degree: often for 2 patients, and occasionally for 5 patients. There were varying amounts of normal epithelial cells, which served as a hallmark in distinction from the malignancy because of a significant difference in the nuclear size. Using Isis ver. 5 (MetaSystems GmbH, Altlussheim, Germany), the nuclear size (greatest diameter) of tumor cells were measured as well as normal atrophic cells in the cytological preparations. The numbers of counted cells for the former and the latter ranged from 50 to 70 in each case. On the total average of 8 cases, the tumor cell size was 12.5 μm and the relative ratio to the corresponding normal atrophic cells was 2.3 (Table 1). The chromatin was considerably dense, coarse or granular. The number of prominent nucleoli was as follows: 1 for 4 patients; 1 or 2 for 4 patients. Calcified deposits of psammoma body were not apparently observed in any patient.\nCytological features of endometrial intraepithelial carcinomas\na: average of largest diameter of tumor cells (relative value compared to normal nucleus).\nCase 2 possessing EIC in both endometrial polyp and endometrium (A. Pap × 20, B. Pap × 60, C. loupe view, D. HE × 20, E. HE × 60, F. HE × 60). A. Papillary-structured carcinoma cell cluster is accompanied by a watery background (Pap. ×20). B. The N/C ratio is fairly high and the chromatin is coarsely granular (Pap. ×60). C. Loupe findings show an endometrial polyp arising in the uterine fundus, measuring 1.5 cm in longitudinal length. The endometrium is markedly thin and atrophic. The area marked by an arrow and that marked by an arrowhead are depicted respectively in E and F. D. In the polyp, the glands composed of carcinoma cells are found to be aggregated in the fibrotic stroma. E. Carcinoma cells are replacing the glands in the polyp and stratified into micropapillary protrusions, lacking apparent stromal invasion (HE × 20). F. Carcinoma nests similar to those in the endometrial polyp (D) are also present in the endometrial gland (HE × 60).\nCase 5 possessing EIC in both endometrial polyp and endometrium, but with stromal invasion in the polyp (A. Pap × 20, B. Pap × 60, C. HE × 20, D. HE × 60). A. A carcinoma cell cluster is accompanied by a bloody background. B. Carcinoma cells having enlarged nuclei appear to be transitional with normal atrophic cells having small-sized nuclei (arrows). C. The polyp has many atypical glands with intraluminal papillary protrusion of the carcinoma cells. D. The border between the carcinoma cells and normal cells is clearly discernible (arrows). There are small carcinoma nests showing stromal invasion (arrowheads).\nCase 6 possessing EIC confined to endometrial polyp (A. Pap × 20, B. Pap × 60, C. HE × 60, D. HE × 60). A. Large-sized carcinoma cell clusters are accompanied by a non-necroinflammatory background. B. Carcinoma cells mainly appear in a flat and sheet-like cluster. The nucleus has an enlarged eosinophilic nucleolus. C. The atypical glands are normal-sized, accompanied by cystic dilatation of atrophic normal glands. D. Carcinoma cells are replacing pre-existing glands and are arranged in a single layer (compare with normal gland marked by an arrow).\nThe clinicopathological profiles are summarized in Table 2. All the patients had an EMP in which atypical tumor cells were arranged predominantly in a tubular structure with micropapillary protrusions, replacing the pre-existing endometrial glands. Micropapillary protrusions and small tufts were accompanied with or without thin fibrovascular cores. These tumor cells showed conspicuous nuclear pleomorphism and hyperchromatism, frequent mitotic figures, high N/C ratio, and also one or two prominent nucleoli. The tumor front was abruptly made into normal glandular epithelium and surface-lining epithelium in the EMP. Minimal stromal invasion of the tumor cells limited to the EMP was recognized in 3 patients. In addition, similar tumor cells also were also found to replace some atrophic endometrial glands in 4 patients, lacking stromal invasion. In the background, cystic dilatation of atrophic normal glands was evident especially in the EMP.\nClinicopathological profiles of endometrial intraepithelial carcinomas\na:+, strongly positive cells more than 80%.\nb: index of positive cells(%), c: +, positive cells more than 80%.\nd: +, positive cells more than 80%.\ne: since initial diagnosis.\nf: history of breast cancer with Tamoxifen administration.\ng: autopsy performed.\nh: with stromal invasion, i: disease free, j: death on disease.\nImmunohistochemical expressions of p53 (DO7, 1:50, Dako, Glostrup, Denmark), Ki-67 (MIB-1, 1:100, Dako, Glostrup, Denmark), ER (SP-1, 1:1, Ventana, AZ, USA), and PgR (1E2, 1:1, Ventana, AZ, USA) were examined (Figure 4). Six patients showed marked p53 expression with a positive ratio of more than 80%. The labeling index of Ki-67 ranged from 25 to 70% (av. approximately 40). ER was mildly expressed in one patient and PgR expression also was mildly observed in 2 patients.\nImmunohistochemical staining profiles represented by Case 5 (A. p53 × 40, B. MIB-1 × 40, C. ER × 20, D. PgR × 20). A. Almost all of the carcinoma cells are strongly positive. B. More than half the carcinoma cells are labeled. C. The expression is observed in the cystically dilated normal glands and stromal cells but not in the carcinoma cells. D. The expression behavior is basically similar to the ER expression.\nFIGO stage was determined at IA for all the patients. Neither adjuvant chemotherapy nor irradiation was performed. Seven patients have been taking an uneventful clinical course (follow-up period ranging from 12 to 66 months, av. 41), but one patient died of the recurrent disease massively occupying the abdominal cavity 44 months after the initial diagnosis.\n\nDiscussion:\nEndometrial polyp is a common benign disease of the uterus. The overall incidence where cancer arises in the endometrial polyp has been reported as only a few percent, while postmenopausal women with EMP are at an increased risk of malignancy, compared to premenopausal women [10,13-15]. As risk factors for development of the malignancy in EMP, hypertension, obesity, and unopposed estrogen therapy have been indicated in addition to postmenopausal status [15]. There are unique or specific pathological and clinical features for EIC as a putative precursor of uterine body serous adenocarcinoma, as follows [1,4,5,7,16-20]: close association with an EMP; multicentric occurrence in the inactive or resting thin endometrium, lacking alteration in the architecture in the endometrium lacking an intervening phase of endometrial hyperplasia; extrauterine invasive extent, irrespective of an absence of stromal invasion of the uterus; and controversial outcome, whether taking a favorable or unfavorable clinical course. Development of EIC associated with an EMP may also be explained in part by Tamoxifen administration following breast cancer treatment [6,17]. However, little has been documented about the endometrial cytological features of minimal serous adenocarcinoma of the uterine body including EIC, mainly due to the fact that the disease is not frequently encountered in the routine practice. To the best of our knowledge, there is a case report cytologically referring to serous adenocarcinoma of the uterine body associated with an EMP [21], but here the patient already showed peritoneal dissemination at the initial presentation. In another report, serous adenocarcinomas of the uterine body were clinico-cytologically reviewed but they were all at advanced stages [22]. The latter report mentions that cytologically diagnostic findings of uterine body serous adenocarcinoma would be based on the following features: papillary cluster as well as tubular cluster, arborescent structure, frequent exfoliation, psammoma body, eccentric enlarged nuclei, coarsely granular chromatin, numerous nucleoli, and diameter of greater than G1 endometrioid adenocarcinoma [22]. In our review, 5 patients were purely non-invasive as they were composed of only EIC existing in an EMP, with few or no necroinflammatory changes. The background was found to be characteristically watery or mucous (Figure 1) in a way of simulating the immigration of extrauterine carcinoma cells via the fallopian tubes [12]. Many clusters were arranged in micropapillary architecture without fine vascular stroma, and others were occasionally tubular. An abrupt transition between the EIC cells and atrophic endometrial cells was noted by front formation (Figure 2). Conspicuous nucleoli were found in 3 patients (Figure 3). Even though it seems difficult to reach a definite diagnosis, these findings may serve as a diagnostic indicator for the uterine body serous adenocarcinoma, whether being at an early stage or an advanced stage.\nDifferential diagnoses for atypical epithelial sheets in the normal-appearing endometrial background include variable interpretations as follows: EIC, endometrial glandular dysplasia, endometrial intraepithelial neoplasia, hyperplastic polyp, and metastatic carcinoma [9,23]. In addition to these, isolated atypical glands with morphological and immunohistochemical features of a typical endometrial hyperplasia or type I endometrial adenocarcinoma may be encountered in grossly normal postmenopausal endometrium of asymptomatic patients [24]. Especially in the postmenopausal state, thin and smooth endometrium with atrophy is composed of hypocellular and flattened glands lined by cells with reduced or absent mitosis and sparse fibrous stroma [25]. It is supposed that the abrupt transition of the atrophic endometrial cells to atypical cells may indicate EIC in postmenopausal women.\nOverexpression of p53 is closely linked to the rapid growth of uterine body serous adenocarcinoma [6]. In EIC, too, frequent p53 mutation is reported to be associated with LOH of 17p [26]. Thus, immunohistochemistry for p53 is also adjunctively available in the diagnosis of uterine body serous adenocarcinoma, irrespective of whether it is at an early stage or advanced stage [27], in the discrimination between uterine body serous adenocarcinoma and benign morphologic mimics [28]. Negativity of ER and PgR staining is more reasonable with the diagnosis of serous adenocarcinoma including EIC.\nIn summary, EMP arising in postmenopausal women is at a high risk of giving birth to the tumorigenetic background for endometrial carcinomas such as endometrioid adenocarcinoma and serous adenocarcinoma including EIC. Endometrial cytology may become a helpful diagnostic approach for serous adenocarcinoma, whether it is at early or advanced stage, from the viewpoint of the distinctive cytological features. Above all, it should be noted that some EIC behaves in a more aggressive fashion regardless of whether or not it lacks apparent stroma invasion and extrauterine extent.\n\nPatients’ consents:\nWritten informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.\n\nEthical approval:\nThis review was performed based on compliance with the Helsinki Declaration.\n\nCompeting interests:\nAll of the authors declare that they have no competing interests.\n\nAuthors’ contributions:\nMY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe uterine endometrial polyp (EMP) has a potential risk of developing malignant tumors especially in postmenopausal women. These malignancies include endometrial intraepithelial carcinoma (EIC).\n\nMethods:\nEight patients with EIC in the EMP, who were postmenopausal with ages ranging from 49 to 76 years (av. 62), were cytologically reviewed in comparison with histological findings.\n\nResults:\nThe endometrial cytological findings were summarized as follows: mucous and watery diathesis as a background lacking or with little necrotic inflammatory changes; micropapillary cluster formation; abrupt transition between carcinoma cells and normal cells; nuclear enlargement; high N/C ratio; and single or a few prominent nucleoli. Histologically, one case had EIC alone in the EMP; three cases had EIC with stromal invasion confined to the EMP; and four cases had EIC in the atrophic endometrium in addition to EIC in the EMP. Seven patients have taken a disease-free course after surgical resection, but one patient died 44 months following the initial diagnosis because of the massive tumor extending over her peritoneal cavity.\n\nConclusions:\nEndometrial cytology may be helpful for the detection of early endometrial adenocarcinomas with serous features including EIC. Some early stage endometrial adenocarcinomas represented by EIC exceptionally take an aggressive clinical course irrespective of a lack of extrauterine lesions."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":3319,"string":"3,319"},"whole_article_abstract_length":{"kind":"number","value":247,"string":"247"},"num_sections":{"kind":"number","value":8,"string":"8"},"most_frequent_words":{"kind":"list like","value":["patients","endometrial","cells","adenocarcinoma","eic","carcinoma","serous","serous adenocarcinoma","body","normal"],"string":"[\n \"patients\",\n \"endometrial\",\n \"cells\",\n \"adenocarcinoma\",\n \"eic\",\n \"carcinoma\",\n \"serous\",\n \"serous adenocarcinoma\",\n \"body\",\n \"normal\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Endometrial intraepithelial carcinoma (EIC) | Endometrial polyp | Cytology [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Endometrial intraepithelial carcinoma (EIC) | Endometrial polyp | Cytology [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Endometrial intraepithelial carcinoma (EIC) | Endometrial polyp | Cytology [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Aged | Carcinoma in Situ | Disease Progression | Early Detection of Cancer | Endometrial Neoplasms | Female | Humans | Middle Aged | Polyps | Predictive Value of Tests | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Aged | Carcinoma in Situ | Disease Progression | Early Detection of Cancer | Endometrial Neoplasms | Female | Humans | Middle Aged | Polyps | Predictive Value of Tests | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Aged | Carcinoma in Situ | Disease Progression | Early Detection of Cancer | Endometrial Neoplasms | Female | Humans | Middle Aged | Polyps | Predictive Value of Tests | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | endometrial | cells | adenocarcinoma | eic | carcinoma | serous | serous adenocarcinoma | body | normal [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | endometrial | cells | adenocarcinoma | eic | carcinoma | serous | serous adenocarcinoma | body | normal [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | endometrial | cells | adenocarcinoma | eic | carcinoma | serous | serous adenocarcinoma | body | normal [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] adenocarcinoma | patients | endometrial | eic | serous | type | specimen | patients endometrial | hysterectomy | years [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cells | normal | 60 | carcinoma | tumor | patients | carcinoma cells | glands | pap | 20 [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | adenocarcinoma | endometrial | cells | eic | authors | authors declare competing | competing interests | competing | declare [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] EMP ||| EIC [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| one | EIC | EMP | three | EIC | EMP | four | EIC | EIC | EMP ||| Seven | 44 months [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] EMP ||| EIC ||| Eight | EIC | EMP | 49 to 76 years | 62 ||| ||| ||| one | EIC | EMP | three | EIC | EMP | four | EIC | EIC | EMP ||| Seven | 44 months ||| EIC ||| EIC [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":283,"cells":{"title":{"kind":"string","value":"Changes in specialized blood vessels in lymph nodes and their role in cancer metastasis."},"pmid":{"kind":"string","value":"23035663"},"background_abstract":{"kind":"string","value":"High endothelial venules (HEV) have been recognized to play a role in metastasis by its changes seen in the cancer microenvironment of lymph nodes (LN) and solid cancers. Squamous cell carcinoma (SCC) of the tongue is a prevalent tumor of the head and neck region with high propensity for LN metastasis. The extent of LN metastasis is the most reliable adverse prognostic factor. Primary tumors can induce vasculature reorganization within sentinel LN before the arrival of tumor cells and HEV represents these remodelled vessels. This study aims to evaluate the cancer induced vascular changes in regional lymph nodes (LN) of patients by studying the morphological and functional alterations of HEV and its correlation with clinical outcome and pathological features."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"This study was based on 65 patients with SCC tongue who underwent primary surgical treatment including neck dissection. The patients were categorized into 2 groups based on the presence of malignancy in their cervical lymph nodes. A review of the patients' pathological and clinical data was performed from a prospective database. Immunohistochemical staining of the tissue blocks for HEV and high-power-field image analysis were performed and analyzed with correlation to the patients' clinical and pathological features."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The total number of HEV was found to be significantly associated to disease-free interval. There was a similar association comparing the HEV parameters to overall survival. The density of abnormal HEV was significantly higher in patients with established metastases in their lymph nodes and HEV was shown to be a better prognosis factor than conventional tumor staging. The HEV morphological metamorphosis demonstrates a spectrum that correlates well with disease progression and clinical outcome."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The results suggest that the HEV displays a spectrum of morphological changes in the presence of cancer and LN metastasis, and that HEV is possibly involved in the process of cancer metastasis. We revealed the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environments in regional lymph nodes of tongue cancer patients in relation to clinical outcomes. The significant observation of modified dilated HEV containing red blood cells in lymph nodal basin of a cancer suggests the shifting of its function from one primarily of immune response to that of a blood carrying vessel. It also demonstrated potential prognostic value. More studies are needed to elucidate its potential role in cancer immunotherapy and as a potential novel therapeutic approach to preventing metastasis by manipulating the remodelling processes of HEV."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Case-Control Studies","Disease-Free Survival","Endothelium, Vascular","Humans","Kaplan-Meier Estimate","Lymph Nodes","Lymphatic Metastasis","Neoplasms","Venules"],"string":"[\n \"Case-Control Studies\",\n \"Disease-Free Survival\",\n \"Endothelium, Vascular\",\n \"Humans\",\n \"Kaplan-Meier Estimate\",\n \"Lymph Nodes\",\n \"Lymphatic Metastasis\",\n \"Neoplasms\",\n \"Venules\"\n]"},"pmcid":{"kind":"string","value":"3551724"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Approval was obtained from the Institutional Review Boards of both institutions involved in the study (2007/464/B). The study population consisted of 175 consecutive patients with SCC of the head and neck who had received primary surgical treatment at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Center, Singapore, from January 2001 through to December 2005. The patients’ pathological and clinical data including follow-up information was reviewed from a prospective database. The inclusion criteria included all surgically treated patients with histologically proven SCC tongue who underwent a neck dissection, with a minimum of 2-year follow up period. Patients with a second primary cancer and patients who did not have a neck dissection as part of their primary treament were excluded. There were a total 65 patients that met the study inclusion criteria. There were 35 patients in the group with primary SCC tongue without pathologically proven lymph node metastases in their neck dissection specimen who were designated as “cases”, and 30 patients in the group with primary SCC tongue with pathologically proven lymph node metastases in the neck dissection specimens, who were designated as “controls”. All patients included had radical excision of the primary tongue lesion and a neck dissection according to departmental protocol. In both groups, all patients had radical enbloc excision of the primary tongue lesion with or without resection of adjacent structures with either a unilateral or a bilateral, supraomohyoid neck dissection or a modified radical neck dissection. Tumours were classified according to the AJCC TNM Staging Classification, based on a pre-operative clinical evaluation and this determined the type of neck dissection performed by the primary surgeon\n[19]. All cases were discussed at a multi-discplinary head and neck tumor board. Intra-operative frozen sections were performed for margins of the tongue lesion and further resection of the tongue was performed in event that the surgical margins were involved. Upon discharge from the hospital, they were followed up at regular intervals of monthly, 3-monthly, 6-monthly and yearly at a progessive rate. Inpatient and outpatient clincal data, pathological, radiological and operative records were retrieved from a central medical records office and from the Department of Pathology, Singapore General Hospital. Mortality data was obtained and confirmed from the hospital central medical records office as well as the Singapore Registry of Births & Deaths.\n Immunohistochemistry A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\nA histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\n Computer assisted image analysis The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\nThe digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\n Definitions of the HEV’s parameters and ratios This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\nThis is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\n Statistical analysis Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\nCorrelating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The association of HEV and clinical outcome was studied by initially analyzing the OS and DFI of the 2 groups prior to individually analyzing the various HEV parameters (A, B, C, B/A, C/B, C/A) against OS and DFI. The details of the preliminary results comparing HEV parameters and patient outcomes have been previously published in an earlier paper\n[20]. In the secondary analysis, we further analyzed the tumor pathological characteristics (i.e. primary tumor volume, the stage of the disease and grade of the tumor) against clinical outcome (OS and DFI), to determine if these conventional tumor factors were prognostic in nature. We further investigated the role of HEV and its prognostic value by comparing and analyzing the HEV parameters with respect to the tumor volume, grade and stage. These analyses were repeated without controlling for the group factor (i.e. taking the sample population as a single cohort). Finally, we investigated the HEV parameters in each group to determine if there was a difference in the HEV values in the presence or absence of metastasis in the LN.\nWe analyzed the DFI and OS of the 65 patients with respect to their groups (35 cases versus 30 controls) to estimate the relative risk of DFI and OS associated with the group.\n Overall survival analysis The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\nThe relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\n Disease free interval analysis Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\nSimilarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\n Secondary analysis Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups\nTumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups"},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"This study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets."},"other_sections_titles":{"kind":"list like","value":["Background","High endothelial venules and its role in cancer metastasis","Objectives of study","Immunohistochemistry","Computer assisted image analysis","Definitions of the HEV’s parameters and ratios","Statistical analysis","Overall survival analysis","Disease free interval analysis","Secondary analysis","Limitations","Abbreviations","Competing interests","Authors’ contribution"],"string":"[\n \"Background\",\n \"High endothelial venules and its role in cancer metastasis\",\n \"Objectives of study\",\n \"Immunohistochemistry\",\n \"Computer assisted image analysis\",\n \"Definitions of the HEV’s parameters and ratios\",\n \"Statistical analysis\",\n \"Overall survival analysis\",\n \"Disease free interval analysis\",\n \"Secondary analysis\",\n \"Limitations\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contribution\"\n]"},"other_sections_texts":{"kind":"list like","value":["Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.","There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].","We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.","A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].","The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.","This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A","Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.","The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results","Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).","Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups","Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.","HEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.","The authors declare that they have no competing interests, financial or non-financial.","LSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript."],"string":"[\n \"Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\\n[6,7].\\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\",\n \"There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\",\n \"We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\",\n \"A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\\n[5,20].\",\n \"The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\",\n \"This is represented by the following alphabets A, B and C (Figure\\n1).\\n• number of all HEV : A\\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\\n• Ratio of dilated HEV to the total number of HEV : B/A\\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\",\n \"Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\",\n \"The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\\n1).\\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\\nSummary of results\",\n \"Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\\n3).\\nDisease free interval curves for the two groups (Cases vs. Controls).\\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\\n1).\",\n \"Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\\n3).\\nSummary of secondary analysis results\\nComparing high endothelial venules parameters in the 2 patient groups\",\n \"Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\",\n \"HEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.\",\n \"The authors declare that they have no competing interests, financial or non-financial.\",\n \"LSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","High endothelial venules and its role in cancer metastasis","Objectives of study","Methods","Immunohistochemistry","Computer assisted image analysis","Definitions of the HEV’s parameters and ratios","Statistical analysis","Results","Overall survival analysis","Disease free interval analysis","Secondary analysis","Discussion","Limitations","Conclusion","Abbreviations","Competing interests","Authors’ contribution"],"string":"[\n \"Background\",\n \"High endothelial venules and its role in cancer metastasis\",\n \"Objectives of study\",\n \"Methods\",\n \"Immunohistochemistry\",\n \"Computer assisted image analysis\",\n \"Definitions of the HEV’s parameters and ratios\",\n \"Statistical analysis\",\n \"Results\",\n \"Overall survival analysis\",\n \"Disease free interval analysis\",\n \"Secondary analysis\",\n \"Discussion\",\n \"Limitations\",\n \"Conclusion\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contribution\"\n]"},"all_sections_texts":{"kind":"list like","value":["Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.","There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].","We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.","Approval was obtained from the Institutional Review Boards of both institutions involved in the study (2007/464/B). The study population consisted of 175 consecutive patients with SCC of the head and neck who had received primary surgical treatment at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Center, Singapore, from January 2001 through to December 2005. The patients’ pathological and clinical data including follow-up information was reviewed from a prospective database. The inclusion criteria included all surgically treated patients with histologically proven SCC tongue who underwent a neck dissection, with a minimum of 2-year follow up period. Patients with a second primary cancer and patients who did not have a neck dissection as part of their primary treament were excluded. There were a total 65 patients that met the study inclusion criteria. There were 35 patients in the group with primary SCC tongue without pathologically proven lymph node metastases in their neck dissection specimen who were designated as “cases”, and 30 patients in the group with primary SCC tongue with pathologically proven lymph node metastases in the neck dissection specimens, who were designated as “controls”. All patients included had radical excision of the primary tongue lesion and a neck dissection according to departmental protocol. In both groups, all patients had radical enbloc excision of the primary tongue lesion with or without resection of adjacent structures with either a unilateral or a bilateral, supraomohyoid neck dissection or a modified radical neck dissection. Tumours were classified according to the AJCC TNM Staging Classification, based on a pre-operative clinical evaluation and this determined the type of neck dissection performed by the primary surgeon\n[19]. All cases were discussed at a multi-discplinary head and neck tumor board. Intra-operative frozen sections were performed for margins of the tongue lesion and further resection of the tongue was performed in event that the surgical margins were involved. Upon discharge from the hospital, they were followed up at regular intervals of monthly, 3-monthly, 6-monthly and yearly at a progessive rate. Inpatient and outpatient clincal data, pathological, radiological and operative records were retrieved from a central medical records office and from the Department of Pathology, Singapore General Hospital. Mortality data was obtained and confirmed from the hospital central medical records office as well as the Singapore Registry of Births & Deaths.\n Immunohistochemistry A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\nA histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\n Computer assisted image analysis The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\nThe digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\n Definitions of the HEV’s parameters and ratios This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\nThis is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\n Statistical analysis Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\nCorrelating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.","A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].","The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.","This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A","Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.","The association of HEV and clinical outcome was studied by initially analyzing the OS and DFI of the 2 groups prior to individually analyzing the various HEV parameters (A, B, C, B/A, C/B, C/A) against OS and DFI. The details of the preliminary results comparing HEV parameters and patient outcomes have been previously published in an earlier paper\n[20]. In the secondary analysis, we further analyzed the tumor pathological characteristics (i.e. primary tumor volume, the stage of the disease and grade of the tumor) against clinical outcome (OS and DFI), to determine if these conventional tumor factors were prognostic in nature. We further investigated the role of HEV and its prognostic value by comparing and analyzing the HEV parameters with respect to the tumor volume, grade and stage. These analyses were repeated without controlling for the group factor (i.e. taking the sample population as a single cohort). Finally, we investigated the HEV parameters in each group to determine if there was a difference in the HEV values in the presence or absence of metastasis in the LN.\nWe analyzed the DFI and OS of the 65 patients with respect to their groups (35 cases versus 30 controls) to estimate the relative risk of DFI and OS associated with the group.\n Overall survival analysis The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\nThe relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\n Disease free interval analysis Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\nSimilarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\n Secondary analysis Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups\nTumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups","The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results","Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).","Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups","Squamous cell carcinoma of the tongue has one of the fastest rising rate today affecting the young with a high propensity for LN metastases.\n[22] The presence of metastatic cells in regional LN, along with extracapsular spread are the most important prognostic factors in patients with squamous cell carcinoma (SCC) of the tongue\n[22-25]. The prognosis is worse compared to other equivalent carcinomas of the head and neck region.\nTumor cell metastasis to regional LN marks one of the initial steps in the cascade of tumor metastasis after tumor cell progression in the primary tumor. The lymphatic metastatic cascade is a series of complex interrelated steps and processes\n[26]. As the tumor grows and enlarges, cytokines are secreted to promote lymphangiogenesis\n[27]. As malignant cells invade the extracellular matrix, they enter the lumen of the lymphatics and they move in clusters or as single cells to the first echelon of regional LN, otherwise known as the SLN\n[28,29]. The structural properties, such as the lack of basement membrane in terminal lymphatics, the presence of the intercellular clefts, the traumatic environment in the blood including shear forces of blood turbulence and tumor antigenicity detection by host immune cells, contribute and account for the observation that in many solid cancers, lymphatic metastasis precedes metastasis via the vascular system\n[26,30]. The relationship between angiogenesis and lymphangiogenesis has been extensively studied in cancer research\n[14,26,31,32]. This is pertinent in the biology of LN metastasis where the two systems literally lie side by side. There is extensive evidence showing members of the Vascular endothelial growth factor (VEGF) family, namely VEGF-C, VEGF-D and VEGF-A\n[33], are not only important regulators of lymph vessel growth in vivo, but may also be involved in the process of promoting lymphatic metastasis, such as VEGF-C\n[27,34-36]. This is significant because there is evidence that tumor can activate both LN lymphangiogenesis and angiogenesis before metastasis, and this would translate into logical efforts in the field of anti-cancer research specifically targeting pathways of tumor lymphangiogenesis and angiogenesis. There have been encouraging results from therapeutic targeting of the various VEGF member pathways to inhibit lymph node metastasis, reduce lymphatic and vascular density in LN as well as to reduce overall tumor burden in the lymph nodes and distant metastases\n[34,37,38].\nTargeted therapy such as anti-angiogenesis drugs has shown positive results in clinical studies. Bevacizumab (AvastinTM), a monoclonal antibody to VEGF-A, was approved by United States Food and Drug Administration (FDA) in 2004 and has proven clinical benefits in treatment of metastatic colorectal cancer when the drug was added to standard chemotherapy and also has positive results in metastatic kidney cancer and advanced lung cancer. There are many ongoing trials including Phase III clinical trials in advanced or metastatic renal cell carcinoma, pancreatic cancer, and ovarian cancer for AvastinTM[39]. Other molecular pathways that harbor potential targets which have varying degrees of anti-angiogenesis and anti-lymphangiogenesis properties, are ligands such as hepatocyte growth factor (HGF), receptors like Neuropilin 2, PDGFRα/β and others such as Angiostatin, interferon α/β and platelet-factor 4\n[40]. Two other antiangiogenic drugs, Sorafenib (Nexavar™, Bayer) and Sunitinib (Sutent™, Pfizer), have also been approved by the FDA for various aspects of cancer treatment, for example, Sorafenib and Sunitinib have been beneficial in the treatment of metastatic renal-cell cancer and advanced hepatocellular carcinoma\n[41,42]. They target multiple receptor tyrosine kinases, including VEGF receptors and platelet-derived growth factor (PDGF) receptors\n[43].\nIn our series, we detected a statistical significance when we analyzed the OS but not in DFI in the patients with or without LN metastasis (cases vs. controls) (Figures\n2 and\n3). This may be attributed to our limited sample size. It has been proposed and is currently being validated, that SLN biopsy may play the next stage in the evolution in the neck management and treatment of tongue SCC\n[44]. Exploration and understanding of this concept coupled with advances and optimistic results in anti-angiogenesis therapy involving VEGF anti-metabolites translates the next logical exploration to be invested in the study of the pathogenesis of lymph node metastasis. It was shown that the lumen of the lymphatic sinuses and blood vessels were dilated in SLN before metastasis\n[5]. We have demonstrated that lymph nodes are transformed by the primary tongue tumor to become a functional blood vessel–enriched organ before and independent of metastasis, with the changes in the morphology of the HEV to become main blood flow carrier in the lymph node (Figure\n4). This process of vascularization in the LN (HEV morphological alterations) appears similar and consistent in human tissues and previous animal models\n[5,20]. This study presents functional and structural data that the primary tumor can possibly manipulate the microenvironment and biology of lymph nodes to potentially facilitate the proliferation of subsequent tumor deposits leading to established metastases. The morphological alteration of HEV in the presence of a cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shift and phenotypic change of HEV from its known function as a lymphocyte recruitment modulator to one as a blood carrier.\nDilated HEVs with red blood cells in its lumen (high power field).\nIn previous studies, the degree of lymphatic dilation in the SLN significantly correlated with the primary tumor weight\n[5,10]. This observation suggests that the lymphatic fluid from the primary tumor induced the persistent alteration of the lymph channel in SLN. These results are consistent with findings that demonstrate, in contrast to angiogenesis, in which blood flow proceeds only after the vessel develops, lymphangiogenesis can be induced by interstitial fluid channeling\n[45]. In this aspect, this is analogous to immunology studies showing the alteration of lymph channels in SLN can also occur during an inflammatory reaction, which facilitates the migration of inflammatory cells\n[46,47] The difference in cancer metastasis biology and inflammatory process starts here. In endotoxin induced experiments and immunology studies, the dilated lymphatic sinuses and vessels in the associated reactive LN were full of lymphocytes. In contrast in tumor-reactive lymphadenopathy, the lymphatics contained minimum or no cellular material, suggesting different roles of the SLN lymphatic channels in different pathologic processes. It has been well characterized that the HEV of lymph nodes play an important role in recruiting lymphocytes for the generation of immune responses\n[9,11]. By expressing homing receptors on their surface, which blood lymphocytes can recognize as they pass in circulation, HEV provide a unique location where naive lymphocytes can enter the lymph node\n[47,48].\nIn our previous study\n[5], we found that the role of HEV was transformed from a lymphocyte recruiter to become the main blood flow carrier in the SLN prior to metastasis. We have shown in this study that in the regional cervical LN, not only could the morphology of individual HEV change dramatically to increase blood flow, but the proliferation rate of HEV endothelial cells was also increased before metastasis. This transformation of HEV as a lymphocytic carrier in LN to a blood-flow carrier can be seen in the large quantity of red blood cells visible in the HEV even in pre-metastatic regional LN. More significantly, this phenomenon is also more pronounced in the regional LN of the patients with established LN metastasis. This is consistent with the findings of Chung and colleagues demonstrating the increased HEV density in SLN of oral SCC before the arrival of tumor cells, the HEV density in metastatic SLN was also higher than that of non-metastatic SLN\n[6]. We have shown this in our supplementary data that patients with lymph node metastases have more dilated HEV with RBC in their LN as compared to patients without LN metastasis. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV(C/B) in the control (pN+) group is statistically significantly higher than that in case group (p-value = 0.0318).\nIn addition, with the benefit of patients’ pathological and follow-up information, we have demonstrated that even with just an increase of 1 HEV in a high-power-field (HPF), regardless of the group, the risk is 1.024 times worse in terms of overall survival. This is consistent with clinical knowledge and natural history of the disease. It is well established that patients without LN metastases in their regional LN (cases in our cohort) have a significantly better prognosis, and; this fact is also reflected in our analysis\n[22,49-51] (Figure\n2). Cases had a 0.428 times the risk of Controls with regards to overall survival if they had the same number of HEV. It is crucial to understand that this risk is exponential in nature. To illustrate the significance of this result, an example will be used: Patient A with tongue SCC has proven LN metastases in the cervical LN. On further immunohistochemical staining, it is found that he has 0 HEV in a random high-power-field. Comparing this to patient B who has the same grade, stage of tumor, degree of LN metastases as patient A but has 100 HEV in a HPF, patient B will have a [ (1.024)100 = 10.715 ] 10 times worse prognosis in OS as compared to patient A. We will then consider patient C who has tongue SCC with no LN metastasis. If the HEV amount in a HPF is the same as either patient A or B, patient C’s risk as compared to either patient A or B will be 2.34 times (1/0.428= 2.34) better in terms of overall survival (Table\n4). Although this did not reach statistical significance (p=0.471), we believe this to be a factor of a small sample size.\nDifferences in OS and DFI relative risk in the 2 groups\nAssuming that our hypothesis is true, HEV transformation from a normal immunological mediator to a tumor metastasis mediator is reflected by morphological changes from a normal appearing HEV, to a dilated HEV, and then to a dilated HEV containing RBC. We believe that this metamorphosis occurs on a spectrum, and this process begins with the HEV increasing in absolute numbers, then each becoming more dilated before progressing into a functional vessel carrying blood (Figure\n5). Hence we analyzed our data looking at the 3 different stages of HEV transformation and the ratio comparing each parameter in a systematic way to demonstrate this spectrum (Figure\n1).\nMetamorphosis of High endothelial venules in a tumor microenvironment. This process begins with the high endothelial venules increasing in absolute numbers, subsequently each of them becoming more dilated and lastly every one of them will become a function vessel carrying blood.\nWe recall that that in the last example we illustrated that a patient’s OS risk is 1.024 times worse than an equivalent patient if there is 1 more HEV in one HPF of his regional LN, the risk is increased to 1.071 if the HEV is a dilated HEV. The risk is even higher at 1.116 times if the HEV is a dilated HEV containing RBC (Table\n1)\n[20]. This trend is also seen when all patients are considered in a single cohort. A patient’s OS risk is worsened by 1.004 times if there is an increase of 1 dilated HEV in a HPF. If that dilated HEV contains RBC, the OS risk is increased to 1.008 times.\nIn the treatment of tongue carcinomas, DFI is important as it signifies the failure of loco-regional control for which there is little effective therapy. Further surgery e.g. neck dissection to remove loco-regional recurrences is plagued with high morbidity and mortality. This, coupled with poor chemotherapy and radiotherapy response rates in recurrences, often equates to rapid deterioration of the patient’s condition. Disease free interval was also analyzed in the same manner to the three different HEV morphological phenotypes.\nWe found statistical significance in the relationship between the total number of HEV (A) and DFI when we controlled for the group (i.e. taking in the presence of LN metastasis as a factor). (p=0.022). This significance is preserved when we analyzed all the patients as a cohort (p=0.023)\n[20]. This means that the quantity of HEV is inversely related to DFI. If you consider the LN status and take it into statistical consideration, if there is one more HEV in a HPF, the DFI is 1.051 times worse than zero HEV. It is again important to note that the relationship of the quantity of HEV with respect to DFI is exponential in nature. For example, a patient (patient B) with 100 more HEV (A) in one HPF has a {(1.051)100 =144.634} 144 times shorter DFI as compared to an equivalent patient with only one HEV per HPF (patient A) (Table\n4). Excluding the group effect and considering all the patients as a cohort, a patient with 1 more HEV per HPF will have a DFI that is 1.051 times worse than a patient with zero HEV in a HPF (p=0.023).\nWe looked at the different ratios of abnormal HEV and compared them to several clinic-pathologic parameters namely overall survival, disease–free interval, tumor volume, stage and grade of the tumor. The details are specified in the secondary results (Table\n2).\nThere is a general trend observed. The more advanced the disease, the higher the ratio/percentage of abnormality of HEV. This can be seen in when we analyzed OS and the 2 ratios. The OS relative risk worsens by 1.078 if the ratio B/A (ratio of dilated HEV to the total number of HEV) increased by 1, while the OS relative risk worsens by 3.624 times if the ratio of dilated HEVs with RBC to the total number of HEV increases by 1. Most importantly, if we consider the most abnormal form of HEV (dilated HEV with RBC within its lumen, C) and look at it as a ratio to the total no. of HEV, a patient’s OS relative risk worsens by 17.884 times if this ratio (C/A) increases by a factor of 1. This observation approaches marginal significance. (p = 0.171) (Figure\n6).\nOverall survival relative risk with respect to the different high endothelial venules ratios.\nIn this study, we have shown the relationship of HEV and their transformation in a cancerous environment. We also note that in previous studies the HEV morphology did not alter at all in endotoxin-induced lymphadenopathy, implying a selective reaction of HEV in the cancerous condition\n[5]. Distinct, differentiated gene expression has also been reported when the endothelial cells respond to the changing of their microenvironment\n[52]. In a previous study, it was shown that the cellular morphology of the tall endothelial cells forming HEV changed dramatically to become flat endothelial cells in the presence of cancer. As a consequence, the HEV was remodelled from a thick-walled, endothelial vessel with a small lumen to a thin walled, large-lumen vessel shifting its function from recruitment of lymphocytes to becoming a blood vessel (Figure\n4)\n[10]. These facts indicate that the blood vessel endothelium has tremendous potential to adapt its environment. The confined lymph and blood channel alterations within the SLN, but not in the next station lymph node, imply that an inducer from the primary tumor is functioning locally with the existence of an active primary cancer. VEGF-A has been found to be an inducer of lymphangiogenesis in SLN\n[33]. Other studies have shown that serum level of VEGF-A was elevated in patients with late stage NPC\n[21] VEGF-A is a secreted protein factor that can travel in blood to other lymph nodes, these findings suggest that there may be other inducers involved. It is also of interest to explore the role of HEV after the establishment of a metastatic tumor nest. The enlarged, remodelled HEV could integrate into the metastatic tumor vasculature with further differentiation, characterized by the gradual loss of their specific marker MECA-79 from the tumor margin to the central part of the metastatic tumor nest.\n[5,10] It has been explained that, compared with primary tumors, the more rapid growth of metastatic lesions in the cervical lymph nodes of NPC patients was due to clonal selection of the cancer cells during metastases, with highly proliferative clones disseminated to the cervical nodes. However, based on our findings, the metastatic tumor vasculature in lymph nodes consists of many large blood vessels derived from normal HEVs, suggesting that the efficiency of nutrition and oxygen supplies could be better for the metastatic tumor cells in the involved lymph node. The enrichment of the blood supply in the lymph node before and after metastasis may favor the growth of newly arriving metastatic cancer cells. Consequently, as evident clinically in the long term follow-up of cancer patients, the involved regional lymph nodes may become manifest first, whereas the primary tumor remain clinically occult for years\n[53]. Moreover, the high density of functioning blood vessels in lymph nodes may subsequently facilitate the metastasis of cancer cell to distant organs. Conversely, consistent with its role in lymphocyte trafficking, if the HEV density is increased in the tumor themselves as opposed to LN, it has favorable prognostic value, Martinet et al. reported that in 146 breast cancers, the density of tumor HEV was an independent prognostic risk factor for DFI and OS\n[8]. This further suggests the tumor HEV function as major gateway for lymphocyte infiltration into tumors and represents potential targets for cancer immunotherapy. It will also be important to elucidate if the highly vascularized premetastatic SLN is associated with an increased metastatic potential. Control of lymphatic fluid movement may also be a target to consider in preventing metastases.\nAssuming our hypothesis is true with regards to the role of HEV in cancer metastasis, a potential therapeutic approach is to interrupt or block this remodelling process and evaluate if there is an effect on metastasis. This will confirm the HEV’s central role in the pathogenesis of metastasis.\nFurther studies need to designed and performed to elucidate the pathways of HEV transformation. A control gene for the growth and differentiation of HEV remains to be identified\n[9]. With regards to this, a recently identified nuclear factor (NF), NF-HEV has potential because it is preferentially expressed by HEV\n[54]. Dendritic cells, well known for their antigen presentation roles has recently been shown to modulate the phenotype of HEV and control entry of naive lymphocytes to LN, their role in a cancer microenvironment is also an area of potential research\n[13]. There are recent experiments in mouse models investigating conditional gene targeting of HEV as well\n[55]. Identification and characterization the molecular pathways and the control genes would have considerable clinical applications. For example, it would enable the design of experiments and studies to induce or retard the formation of HEV in various tissues, including tumors and their LN, improving vaccination strategies against pathogens and cancers. Therefore, there is a wealth of translational applications with regards to targeting HEV as a ligand for cancer investigational therapeutics. It paves the journey in the discovery of many unknown genes and molecules with potential clinical importance. Lastly, more work can be performed to validate our findings in other solid cancers.\n Limitations Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\nOur study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.","Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.","This study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets.","HEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.","The authors declare that they have no competing interests, financial or non-financial.","LSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript."],"string":"[\n \"Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\\n[6,7].\\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\",\n \"There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\",\n \"We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\",\n \"Approval was obtained from the Institutional Review Boards of both institutions involved in the study (2007/464/B). The study population consisted of 175 consecutive patients with SCC of the head and neck who had received primary surgical treatment at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Center, Singapore, from January 2001 through to December 2005. The patients’ pathological and clinical data including follow-up information was reviewed from a prospective database. The inclusion criteria included all surgically treated patients with histologically proven SCC tongue who underwent a neck dissection, with a minimum of 2-year follow up period. Patients with a second primary cancer and patients who did not have a neck dissection as part of their primary treament were excluded. There were a total 65 patients that met the study inclusion criteria. There were 35 patients in the group with primary SCC tongue without pathologically proven lymph node metastases in their neck dissection specimen who were designated as “cases”, and 30 patients in the group with primary SCC tongue with pathologically proven lymph node metastases in the neck dissection specimens, who were designated as “controls”. All patients included had radical excision of the primary tongue lesion and a neck dissection according to departmental protocol. In both groups, all patients had radical enbloc excision of the primary tongue lesion with or without resection of adjacent structures with either a unilateral or a bilateral, supraomohyoid neck dissection or a modified radical neck dissection. Tumours were classified according to the AJCC TNM Staging Classification, based on a pre-operative clinical evaluation and this determined the type of neck dissection performed by the primary surgeon\\n[19]. All cases were discussed at a multi-discplinary head and neck tumor board. Intra-operative frozen sections were performed for margins of the tongue lesion and further resection of the tongue was performed in event that the surgical margins were involved. Upon discharge from the hospital, they were followed up at regular intervals of monthly, 3-monthly, 6-monthly and yearly at a progessive rate. Inpatient and outpatient clincal data, pathological, radiological and operative records were retrieved from a central medical records office and from the Department of Pathology, Singapore General Hospital. Mortality data was obtained and confirmed from the hospital central medical records office as well as the Singapore Registry of Births & Deaths.\\n Immunohistochemistry A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\\n[5,20].\\nA histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\\n[5,20].\\n Computer assisted image analysis The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\\nThe digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\\n Definitions of the HEV’s parameters and ratios This is represented by the following alphabets A, B and C (Figure\\n1).\\n• number of all HEV : A\\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\\n• Ratio of dilated HEV to the total number of HEV : B/A\\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\\nThis is represented by the following alphabets A, B and C (Figure\\n1).\\n• number of all HEV : A\\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\\n• Ratio of dilated HEV to the total number of HEV : B/A\\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\\n Statistical analysis Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\\nCorrelating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\",\n \"A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\\n[5,20].\",\n \"The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\",\n \"This is represented by the following alphabets A, B and C (Figure\\n1).\\n• number of all HEV : A\\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\\n• Ratio of dilated HEV to the total number of HEV : B/A\\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\",\n \"Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\",\n \"The association of HEV and clinical outcome was studied by initially analyzing the OS and DFI of the 2 groups prior to individually analyzing the various HEV parameters (A, B, C, B/A, C/B, C/A) against OS and DFI. The details of the preliminary results comparing HEV parameters and patient outcomes have been previously published in an earlier paper\\n[20]. In the secondary analysis, we further analyzed the tumor pathological characteristics (i.e. primary tumor volume, the stage of the disease and grade of the tumor) against clinical outcome (OS and DFI), to determine if these conventional tumor factors were prognostic in nature. We further investigated the role of HEV and its prognostic value by comparing and analyzing the HEV parameters with respect to the tumor volume, grade and stage. These analyses were repeated without controlling for the group factor (i.e. taking the sample population as a single cohort). Finally, we investigated the HEV parameters in each group to determine if there was a difference in the HEV values in the presence or absence of metastasis in the LN.\\nWe analyzed the DFI and OS of the 65 patients with respect to their groups (35 cases versus 30 controls) to estimate the relative risk of DFI and OS associated with the group.\\n Overall survival analysis The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\\n1).\\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\\nSummary of results\\nThe relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\\n1).\\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\\nSummary of results\\n Disease free interval analysis Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\\n3).\\nDisease free interval curves for the two groups (Cases vs. Controls).\\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\\n1).\\nSimilarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\\n3).\\nDisease free interval curves for the two groups (Cases vs. Controls).\\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\\n1).\\n Secondary analysis Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\\n3).\\nSummary of secondary analysis results\\nComparing high endothelial venules parameters in the 2 patient groups\\nTumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\\n3).\\nSummary of secondary analysis results\\nComparing high endothelial venules parameters in the 2 patient groups\",\n \"The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\\n1).\\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\\nSummary of results\",\n \"Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\\n3).\\nDisease free interval curves for the two groups (Cases vs. Controls).\\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\\n1).\",\n \"Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\\n3).\\nSummary of secondary analysis results\\nComparing high endothelial venules parameters in the 2 patient groups\",\n \"Squamous cell carcinoma of the tongue has one of the fastest rising rate today affecting the young with a high propensity for LN metastases.\\n[22] The presence of metastatic cells in regional LN, along with extracapsular spread are the most important prognostic factors in patients with squamous cell carcinoma (SCC) of the tongue\\n[22-25]. The prognosis is worse compared to other equivalent carcinomas of the head and neck region.\\nTumor cell metastasis to regional LN marks one of the initial steps in the cascade of tumor metastasis after tumor cell progression in the primary tumor. The lymphatic metastatic cascade is a series of complex interrelated steps and processes\\n[26]. As the tumor grows and enlarges, cytokines are secreted to promote lymphangiogenesis\\n[27]. As malignant cells invade the extracellular matrix, they enter the lumen of the lymphatics and they move in clusters or as single cells to the first echelon of regional LN, otherwise known as the SLN\\n[28,29]. The structural properties, such as the lack of basement membrane in terminal lymphatics, the presence of the intercellular clefts, the traumatic environment in the blood including shear forces of blood turbulence and tumor antigenicity detection by host immune cells, contribute and account for the observation that in many solid cancers, lymphatic metastasis precedes metastasis via the vascular system\\n[26,30]. The relationship between angiogenesis and lymphangiogenesis has been extensively studied in cancer research\\n[14,26,31,32]. This is pertinent in the biology of LN metastasis where the two systems literally lie side by side. There is extensive evidence showing members of the Vascular endothelial growth factor (VEGF) family, namely VEGF-C, VEGF-D and VEGF-A\\n[33], are not only important regulators of lymph vessel growth in vivo, but may also be involved in the process of promoting lymphatic metastasis, such as VEGF-C\\n[27,34-36]. This is significant because there is evidence that tumor can activate both LN lymphangiogenesis and angiogenesis before metastasis, and this would translate into logical efforts in the field of anti-cancer research specifically targeting pathways of tumor lymphangiogenesis and angiogenesis. There have been encouraging results from therapeutic targeting of the various VEGF member pathways to inhibit lymph node metastasis, reduce lymphatic and vascular density in LN as well as to reduce overall tumor burden in the lymph nodes and distant metastases\\n[34,37,38].\\nTargeted therapy such as anti-angiogenesis drugs has shown positive results in clinical studies. Bevacizumab (AvastinTM), a monoclonal antibody to VEGF-A, was approved by United States Food and Drug Administration (FDA) in 2004 and has proven clinical benefits in treatment of metastatic colorectal cancer when the drug was added to standard chemotherapy and also has positive results in metastatic kidney cancer and advanced lung cancer. There are many ongoing trials including Phase III clinical trials in advanced or metastatic renal cell carcinoma, pancreatic cancer, and ovarian cancer for AvastinTM[39]. Other molecular pathways that harbor potential targets which have varying degrees of anti-angiogenesis and anti-lymphangiogenesis properties, are ligands such as hepatocyte growth factor (HGF), receptors like Neuropilin 2, PDGFRα/β and others such as Angiostatin, interferon α/β and platelet-factor 4\\n[40]. Two other antiangiogenic drugs, Sorafenib (Nexavar™, Bayer) and Sunitinib (Sutent™, Pfizer), have also been approved by the FDA for various aspects of cancer treatment, for example, Sorafenib and Sunitinib have been beneficial in the treatment of metastatic renal-cell cancer and advanced hepatocellular carcinoma\\n[41,42]. They target multiple receptor tyrosine kinases, including VEGF receptors and platelet-derived growth factor (PDGF) receptors\\n[43].\\nIn our series, we detected a statistical significance when we analyzed the OS but not in DFI in the patients with or without LN metastasis (cases vs. controls) (Figures\\n2 and\\n3). This may be attributed to our limited sample size. It has been proposed and is currently being validated, that SLN biopsy may play the next stage in the evolution in the neck management and treatment of tongue SCC\\n[44]. Exploration and understanding of this concept coupled with advances and optimistic results in anti-angiogenesis therapy involving VEGF anti-metabolites translates the next logical exploration to be invested in the study of the pathogenesis of lymph node metastasis. It was shown that the lumen of the lymphatic sinuses and blood vessels were dilated in SLN before metastasis\\n[5]. We have demonstrated that lymph nodes are transformed by the primary tongue tumor to become a functional blood vessel–enriched organ before and independent of metastasis, with the changes in the morphology of the HEV to become main blood flow carrier in the lymph node (Figure\\n4). This process of vascularization in the LN (HEV morphological alterations) appears similar and consistent in human tissues and previous animal models\\n[5,20]. This study presents functional and structural data that the primary tumor can possibly manipulate the microenvironment and biology of lymph nodes to potentially facilitate the proliferation of subsequent tumor deposits leading to established metastases. The morphological alteration of HEV in the presence of a cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shift and phenotypic change of HEV from its known function as a lymphocyte recruitment modulator to one as a blood carrier.\\nDilated HEVs with red blood cells in its lumen (high power field).\\nIn previous studies, the degree of lymphatic dilation in the SLN significantly correlated with the primary tumor weight\\n[5,10]. This observation suggests that the lymphatic fluid from the primary tumor induced the persistent alteration of the lymph channel in SLN. These results are consistent with findings that demonstrate, in contrast to angiogenesis, in which blood flow proceeds only after the vessel develops, lymphangiogenesis can be induced by interstitial fluid channeling\\n[45]. In this aspect, this is analogous to immunology studies showing the alteration of lymph channels in SLN can also occur during an inflammatory reaction, which facilitates the migration of inflammatory cells\\n[46,47] The difference in cancer metastasis biology and inflammatory process starts here. In endotoxin induced experiments and immunology studies, the dilated lymphatic sinuses and vessels in the associated reactive LN were full of lymphocytes. In contrast in tumor-reactive lymphadenopathy, the lymphatics contained minimum or no cellular material, suggesting different roles of the SLN lymphatic channels in different pathologic processes. It has been well characterized that the HEV of lymph nodes play an important role in recruiting lymphocytes for the generation of immune responses\\n[9,11]. By expressing homing receptors on their surface, which blood lymphocytes can recognize as they pass in circulation, HEV provide a unique location where naive lymphocytes can enter the lymph node\\n[47,48].\\nIn our previous study\\n[5], we found that the role of HEV was transformed from a lymphocyte recruiter to become the main blood flow carrier in the SLN prior to metastasis. We have shown in this study that in the regional cervical LN, not only could the morphology of individual HEV change dramatically to increase blood flow, but the proliferation rate of HEV endothelial cells was also increased before metastasis. This transformation of HEV as a lymphocytic carrier in LN to a blood-flow carrier can be seen in the large quantity of red blood cells visible in the HEV even in pre-metastatic regional LN. More significantly, this phenomenon is also more pronounced in the regional LN of the patients with established LN metastasis. This is consistent with the findings of Chung and colleagues demonstrating the increased HEV density in SLN of oral SCC before the arrival of tumor cells, the HEV density in metastatic SLN was also higher than that of non-metastatic SLN\\n[6]. We have shown this in our supplementary data that patients with lymph node metastases have more dilated HEV with RBC in their LN as compared to patients without LN metastasis. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV(C/B) in the control (pN+) group is statistically significantly higher than that in case group (p-value = 0.0318).\\nIn addition, with the benefit of patients’ pathological and follow-up information, we have demonstrated that even with just an increase of 1 HEV in a high-power-field (HPF), regardless of the group, the risk is 1.024 times worse in terms of overall survival. This is consistent with clinical knowledge and natural history of the disease. It is well established that patients without LN metastases in their regional LN (cases in our cohort) have a significantly better prognosis, and; this fact is also reflected in our analysis\\n[22,49-51] (Figure\\n2). Cases had a 0.428 times the risk of Controls with regards to overall survival if they had the same number of HEV. It is crucial to understand that this risk is exponential in nature. To illustrate the significance of this result, an example will be used: Patient A with tongue SCC has proven LN metastases in the cervical LN. On further immunohistochemical staining, it is found that he has 0 HEV in a random high-power-field. Comparing this to patient B who has the same grade, stage of tumor, degree of LN metastases as patient A but has 100 HEV in a HPF, patient B will have a [ (1.024)100 = 10.715 ] 10 times worse prognosis in OS as compared to patient A. We will then consider patient C who has tongue SCC with no LN metastasis. If the HEV amount in a HPF is the same as either patient A or B, patient C’s risk as compared to either patient A or B will be 2.34 times (1/0.428= 2.34) better in terms of overall survival (Table\\n4). Although this did not reach statistical significance (p=0.471), we believe this to be a factor of a small sample size.\\nDifferences in OS and DFI relative risk in the 2 groups\\nAssuming that our hypothesis is true, HEV transformation from a normal immunological mediator to a tumor metastasis mediator is reflected by morphological changes from a normal appearing HEV, to a dilated HEV, and then to a dilated HEV containing RBC. We believe that this metamorphosis occurs on a spectrum, and this process begins with the HEV increasing in absolute numbers, then each becoming more dilated before progressing into a functional vessel carrying blood (Figure\\n5). Hence we analyzed our data looking at the 3 different stages of HEV transformation and the ratio comparing each parameter in a systematic way to demonstrate this spectrum (Figure\\n1).\\nMetamorphosis of High endothelial venules in a tumor microenvironment. This process begins with the high endothelial venules increasing in absolute numbers, subsequently each of them becoming more dilated and lastly every one of them will become a function vessel carrying blood.\\nWe recall that that in the last example we illustrated that a patient’s OS risk is 1.024 times worse than an equivalent patient if there is 1 more HEV in one HPF of his regional LN, the risk is increased to 1.071 if the HEV is a dilated HEV. The risk is even higher at 1.116 times if the HEV is a dilated HEV containing RBC (Table\\n1)\\n[20]. This trend is also seen when all patients are considered in a single cohort. A patient’s OS risk is worsened by 1.004 times if there is an increase of 1 dilated HEV in a HPF. If that dilated HEV contains RBC, the OS risk is increased to 1.008 times.\\nIn the treatment of tongue carcinomas, DFI is important as it signifies the failure of loco-regional control for which there is little effective therapy. Further surgery e.g. neck dissection to remove loco-regional recurrences is plagued with high morbidity and mortality. This, coupled with poor chemotherapy and radiotherapy response rates in recurrences, often equates to rapid deterioration of the patient’s condition. Disease free interval was also analyzed in the same manner to the three different HEV morphological phenotypes.\\nWe found statistical significance in the relationship between the total number of HEV (A) and DFI when we controlled for the group (i.e. taking in the presence of LN metastasis as a factor). (p=0.022). This significance is preserved when we analyzed all the patients as a cohort (p=0.023)\\n[20]. This means that the quantity of HEV is inversely related to DFI. If you consider the LN status and take it into statistical consideration, if there is one more HEV in a HPF, the DFI is 1.051 times worse than zero HEV. It is again important to note that the relationship of the quantity of HEV with respect to DFI is exponential in nature. For example, a patient (patient B) with 100 more HEV (A) in one HPF has a {(1.051)100 =144.634} 144 times shorter DFI as compared to an equivalent patient with only one HEV per HPF (patient A) (Table\\n4). Excluding the group effect and considering all the patients as a cohort, a patient with 1 more HEV per HPF will have a DFI that is 1.051 times worse than a patient with zero HEV in a HPF (p=0.023).\\nWe looked at the different ratios of abnormal HEV and compared them to several clinic-pathologic parameters namely overall survival, disease–free interval, tumor volume, stage and grade of the tumor. The details are specified in the secondary results (Table\\n2).\\nThere is a general trend observed. The more advanced the disease, the higher the ratio/percentage of abnormality of HEV. This can be seen in when we analyzed OS and the 2 ratios. The OS relative risk worsens by 1.078 if the ratio B/A (ratio of dilated HEV to the total number of HEV) increased by 1, while the OS relative risk worsens by 3.624 times if the ratio of dilated HEVs with RBC to the total number of HEV increases by 1. Most importantly, if we consider the most abnormal form of HEV (dilated HEV with RBC within its lumen, C) and look at it as a ratio to the total no. of HEV, a patient’s OS relative risk worsens by 17.884 times if this ratio (C/A) increases by a factor of 1. This observation approaches marginal significance. (p = 0.171) (Figure\\n6).\\nOverall survival relative risk with respect to the different high endothelial venules ratios.\\nIn this study, we have shown the relationship of HEV and their transformation in a cancerous environment. We also note that in previous studies the HEV morphology did not alter at all in endotoxin-induced lymphadenopathy, implying a selective reaction of HEV in the cancerous condition\\n[5]. Distinct, differentiated gene expression has also been reported when the endothelial cells respond to the changing of their microenvironment\\n[52]. In a previous study, it was shown that the cellular morphology of the tall endothelial cells forming HEV changed dramatically to become flat endothelial cells in the presence of cancer. As a consequence, the HEV was remodelled from a thick-walled, endothelial vessel with a small lumen to a thin walled, large-lumen vessel shifting its function from recruitment of lymphocytes to becoming a blood vessel (Figure\\n4)\\n[10]. These facts indicate that the blood vessel endothelium has tremendous potential to adapt its environment. The confined lymph and blood channel alterations within the SLN, but not in the next station lymph node, imply that an inducer from the primary tumor is functioning locally with the existence of an active primary cancer. VEGF-A has been found to be an inducer of lymphangiogenesis in SLN\\n[33]. Other studies have shown that serum level of VEGF-A was elevated in patients with late stage NPC\\n[21] VEGF-A is a secreted protein factor that can travel in blood to other lymph nodes, these findings suggest that there may be other inducers involved. It is also of interest to explore the role of HEV after the establishment of a metastatic tumor nest. The enlarged, remodelled HEV could integrate into the metastatic tumor vasculature with further differentiation, characterized by the gradual loss of their specific marker MECA-79 from the tumor margin to the central part of the metastatic tumor nest.\\n[5,10] It has been explained that, compared with primary tumors, the more rapid growth of metastatic lesions in the cervical lymph nodes of NPC patients was due to clonal selection of the cancer cells during metastases, with highly proliferative clones disseminated to the cervical nodes. However, based on our findings, the metastatic tumor vasculature in lymph nodes consists of many large blood vessels derived from normal HEVs, suggesting that the efficiency of nutrition and oxygen supplies could be better for the metastatic tumor cells in the involved lymph node. The enrichment of the blood supply in the lymph node before and after metastasis may favor the growth of newly arriving metastatic cancer cells. Consequently, as evident clinically in the long term follow-up of cancer patients, the involved regional lymph nodes may become manifest first, whereas the primary tumor remain clinically occult for years\\n[53]. Moreover, the high density of functioning blood vessels in lymph nodes may subsequently facilitate the metastasis of cancer cell to distant organs. Conversely, consistent with its role in lymphocyte trafficking, if the HEV density is increased in the tumor themselves as opposed to LN, it has favorable prognostic value, Martinet et al. reported that in 146 breast cancers, the density of tumor HEV was an independent prognostic risk factor for DFI and OS\\n[8]. This further suggests the tumor HEV function as major gateway for lymphocyte infiltration into tumors and represents potential targets for cancer immunotherapy. It will also be important to elucidate if the highly vascularized premetastatic SLN is associated with an increased metastatic potential. Control of lymphatic fluid movement may also be a target to consider in preventing metastases.\\nAssuming our hypothesis is true with regards to the role of HEV in cancer metastasis, a potential therapeutic approach is to interrupt or block this remodelling process and evaluate if there is an effect on metastasis. This will confirm the HEV’s central role in the pathogenesis of metastasis.\\nFurther studies need to designed and performed to elucidate the pathways of HEV transformation. A control gene for the growth and differentiation of HEV remains to be identified\\n[9]. With regards to this, a recently identified nuclear factor (NF), NF-HEV has potential because it is preferentially expressed by HEV\\n[54]. Dendritic cells, well known for their antigen presentation roles has recently been shown to modulate the phenotype of HEV and control entry of naive lymphocytes to LN, their role in a cancer microenvironment is also an area of potential research\\n[13]. There are recent experiments in mouse models investigating conditional gene targeting of HEV as well\\n[55]. Identification and characterization the molecular pathways and the control genes would have considerable clinical applications. For example, it would enable the design of experiments and studies to induce or retard the formation of HEV in various tissues, including tumors and their LN, improving vaccination strategies against pathogens and cancers. Therefore, there is a wealth of translational applications with regards to targeting HEV as a ligand for cancer investigational therapeutics. It paves the journey in the discovery of many unknown genes and molecules with potential clinical importance. Lastly, more work can be performed to validate our findings in other solid cancers.\\n Limitations Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\\nOur study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\",\n \"Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\",\n \"This study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets.\",\n \"HEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.\",\n \"The authors declare that they have no competing interests, financial or non-financial.\",\n \"LSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,"methods",null,null,null,null,"results",null,null,null,"discussion",null,"conclusions",null,null,null],"string":"[\n null,\n null,\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n null,\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["High endothelial venules","Cancer metastasis","Angiogenesis","Lymph nodes"],"string":"[\n \"High endothelial venules\",\n \"Cancer metastasis\",\n \"Angiogenesis\",\n \"Lymph nodes\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nCancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\n\nHigh endothelial venules and its role in cancer metastasis:\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n\nObjectives of study:\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\n\nMethods:\nApproval was obtained from the Institutional Review Boards of both institutions involved in the study (2007/464/B). The study population consisted of 175 consecutive patients with SCC of the head and neck who had received primary surgical treatment at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Center, Singapore, from January 2001 through to December 2005. The patients’ pathological and clinical data including follow-up information was reviewed from a prospective database. The inclusion criteria included all surgically treated patients with histologically proven SCC tongue who underwent a neck dissection, with a minimum of 2-year follow up period. Patients with a second primary cancer and patients who did not have a neck dissection as part of their primary treament were excluded. There were a total 65 patients that met the study inclusion criteria. There were 35 patients in the group with primary SCC tongue without pathologically proven lymph node metastases in their neck dissection specimen who were designated as “cases”, and 30 patients in the group with primary SCC tongue with pathologically proven lymph node metastases in the neck dissection specimens, who were designated as “controls”. All patients included had radical excision of the primary tongue lesion and a neck dissection according to departmental protocol. In both groups, all patients had radical enbloc excision of the primary tongue lesion with or without resection of adjacent structures with either a unilateral or a bilateral, supraomohyoid neck dissection or a modified radical neck dissection. Tumours were classified according to the AJCC TNM Staging Classification, based on a pre-operative clinical evaluation and this determined the type of neck dissection performed by the primary surgeon\n[19]. All cases were discussed at a multi-discplinary head and neck tumor board. Intra-operative frozen sections were performed for margins of the tongue lesion and further resection of the tongue was performed in event that the surgical margins were involved. Upon discharge from the hospital, they were followed up at regular intervals of monthly, 3-monthly, 6-monthly and yearly at a progessive rate. Inpatient and outpatient clincal data, pathological, radiological and operative records were retrieved from a central medical records office and from the Department of Pathology, Singapore General Hospital. Mortality data was obtained and confirmed from the hospital central medical records office as well as the Singapore Registry of Births & Deaths.\n Immunohistochemistry A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\nA histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\n Computer assisted image analysis The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\nThe digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\n Definitions of the HEV’s parameters and ratios This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\nThis is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\n Statistical analysis Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\nCorrelating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\n\nImmunohistochemistry:\nA histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\n\nComputer assisted image analysis:\nThe digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\n\nDefinitions of the HEV’s parameters and ratios:\nThis is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\n\nStatistical analysis:\nCorrelating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\n\nResults:\nThe association of HEV and clinical outcome was studied by initially analyzing the OS and DFI of the 2 groups prior to individually analyzing the various HEV parameters (A, B, C, B/A, C/B, C/A) against OS and DFI. The details of the preliminary results comparing HEV parameters and patient outcomes have been previously published in an earlier paper\n[20]. In the secondary analysis, we further analyzed the tumor pathological characteristics (i.e. primary tumor volume, the stage of the disease and grade of the tumor) against clinical outcome (OS and DFI), to determine if these conventional tumor factors were prognostic in nature. We further investigated the role of HEV and its prognostic value by comparing and analyzing the HEV parameters with respect to the tumor volume, grade and stage. These analyses were repeated without controlling for the group factor (i.e. taking the sample population as a single cohort). Finally, we investigated the HEV parameters in each group to determine if there was a difference in the HEV values in the presence or absence of metastasis in the LN.\nWe analyzed the DFI and OS of the 65 patients with respect to their groups (35 cases versus 30 controls) to estimate the relative risk of DFI and OS associated with the group.\n Overall survival analysis The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\nThe relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\n Disease free interval analysis Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\nSimilarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\n Secondary analysis Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups\nTumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups\n\nOverall survival analysis:\nThe relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\n\nDisease free interval analysis:\nSimilarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\n\nSecondary analysis:\nTumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups\n\nDiscussion:\nSquamous cell carcinoma of the tongue has one of the fastest rising rate today affecting the young with a high propensity for LN metastases.\n[22] The presence of metastatic cells in regional LN, along with extracapsular spread are the most important prognostic factors in patients with squamous cell carcinoma (SCC) of the tongue\n[22-25]. The prognosis is worse compared to other equivalent carcinomas of the head and neck region.\nTumor cell metastasis to regional LN marks one of the initial steps in the cascade of tumor metastasis after tumor cell progression in the primary tumor. The lymphatic metastatic cascade is a series of complex interrelated steps and processes\n[26]. As the tumor grows and enlarges, cytokines are secreted to promote lymphangiogenesis\n[27]. As malignant cells invade the extracellular matrix, they enter the lumen of the lymphatics and they move in clusters or as single cells to the first echelon of regional LN, otherwise known as the SLN\n[28,29]. The structural properties, such as the lack of basement membrane in terminal lymphatics, the presence of the intercellular clefts, the traumatic environment in the blood including shear forces of blood turbulence and tumor antigenicity detection by host immune cells, contribute and account for the observation that in many solid cancers, lymphatic metastasis precedes metastasis via the vascular system\n[26,30]. The relationship between angiogenesis and lymphangiogenesis has been extensively studied in cancer research\n[14,26,31,32]. This is pertinent in the biology of LN metastasis where the two systems literally lie side by side. There is extensive evidence showing members of the Vascular endothelial growth factor (VEGF) family, namely VEGF-C, VEGF-D and VEGF-A\n[33], are not only important regulators of lymph vessel growth in vivo, but may also be involved in the process of promoting lymphatic metastasis, such as VEGF-C\n[27,34-36]. This is significant because there is evidence that tumor can activate both LN lymphangiogenesis and angiogenesis before metastasis, and this would translate into logical efforts in the field of anti-cancer research specifically targeting pathways of tumor lymphangiogenesis and angiogenesis. There have been encouraging results from therapeutic targeting of the various VEGF member pathways to inhibit lymph node metastasis, reduce lymphatic and vascular density in LN as well as to reduce overall tumor burden in the lymph nodes and distant metastases\n[34,37,38].\nTargeted therapy such as anti-angiogenesis drugs has shown positive results in clinical studies. Bevacizumab (AvastinTM), a monoclonal antibody to VEGF-A, was approved by United States Food and Drug Administration (FDA) in 2004 and has proven clinical benefits in treatment of metastatic colorectal cancer when the drug was added to standard chemotherapy and also has positive results in metastatic kidney cancer and advanced lung cancer. There are many ongoing trials including Phase III clinical trials in advanced or metastatic renal cell carcinoma, pancreatic cancer, and ovarian cancer for AvastinTM[39]. Other molecular pathways that harbor potential targets which have varying degrees of anti-angiogenesis and anti-lymphangiogenesis properties, are ligands such as hepatocyte growth factor (HGF), receptors like Neuropilin 2, PDGFRα/β and others such as Angiostatin, interferon α/β and platelet-factor 4\n[40]. Two other antiangiogenic drugs, Sorafenib (Nexavar™, Bayer) and Sunitinib (Sutent™, Pfizer), have also been approved by the FDA for various aspects of cancer treatment, for example, Sorafenib and Sunitinib have been beneficial in the treatment of metastatic renal-cell cancer and advanced hepatocellular carcinoma\n[41,42]. They target multiple receptor tyrosine kinases, including VEGF receptors and platelet-derived growth factor (PDGF) receptors\n[43].\nIn our series, we detected a statistical significance when we analyzed the OS but not in DFI in the patients with or without LN metastasis (cases vs. controls) (Figures\n2 and\n3). This may be attributed to our limited sample size. It has been proposed and is currently being validated, that SLN biopsy may play the next stage in the evolution in the neck management and treatment of tongue SCC\n[44]. Exploration and understanding of this concept coupled with advances and optimistic results in anti-angiogenesis therapy involving VEGF anti-metabolites translates the next logical exploration to be invested in the study of the pathogenesis of lymph node metastasis. It was shown that the lumen of the lymphatic sinuses and blood vessels were dilated in SLN before metastasis\n[5]. We have demonstrated that lymph nodes are transformed by the primary tongue tumor to become a functional blood vessel–enriched organ before and independent of metastasis, with the changes in the morphology of the HEV to become main blood flow carrier in the lymph node (Figure\n4). This process of vascularization in the LN (HEV morphological alterations) appears similar and consistent in human tissues and previous animal models\n[5,20]. This study presents functional and structural data that the primary tumor can possibly manipulate the microenvironment and biology of lymph nodes to potentially facilitate the proliferation of subsequent tumor deposits leading to established metastases. The morphological alteration of HEV in the presence of a cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shift and phenotypic change of HEV from its known function as a lymphocyte recruitment modulator to one as a blood carrier.\nDilated HEVs with red blood cells in its lumen (high power field).\nIn previous studies, the degree of lymphatic dilation in the SLN significantly correlated with the primary tumor weight\n[5,10]. This observation suggests that the lymphatic fluid from the primary tumor induced the persistent alteration of the lymph channel in SLN. These results are consistent with findings that demonstrate, in contrast to angiogenesis, in which blood flow proceeds only after the vessel develops, lymphangiogenesis can be induced by interstitial fluid channeling\n[45]. In this aspect, this is analogous to immunology studies showing the alteration of lymph channels in SLN can also occur during an inflammatory reaction, which facilitates the migration of inflammatory cells\n[46,47] The difference in cancer metastasis biology and inflammatory process starts here. In endotoxin induced experiments and immunology studies, the dilated lymphatic sinuses and vessels in the associated reactive LN were full of lymphocytes. In contrast in tumor-reactive lymphadenopathy, the lymphatics contained minimum or no cellular material, suggesting different roles of the SLN lymphatic channels in different pathologic processes. It has been well characterized that the HEV of lymph nodes play an important role in recruiting lymphocytes for the generation of immune responses\n[9,11]. By expressing homing receptors on their surface, which blood lymphocytes can recognize as they pass in circulation, HEV provide a unique location where naive lymphocytes can enter the lymph node\n[47,48].\nIn our previous study\n[5], we found that the role of HEV was transformed from a lymphocyte recruiter to become the main blood flow carrier in the SLN prior to metastasis. We have shown in this study that in the regional cervical LN, not only could the morphology of individual HEV change dramatically to increase blood flow, but the proliferation rate of HEV endothelial cells was also increased before metastasis. This transformation of HEV as a lymphocytic carrier in LN to a blood-flow carrier can be seen in the large quantity of red blood cells visible in the HEV even in pre-metastatic regional LN. More significantly, this phenomenon is also more pronounced in the regional LN of the patients with established LN metastasis. This is consistent with the findings of Chung and colleagues demonstrating the increased HEV density in SLN of oral SCC before the arrival of tumor cells, the HEV density in metastatic SLN was also higher than that of non-metastatic SLN\n[6]. We have shown this in our supplementary data that patients with lymph node metastases have more dilated HEV with RBC in their LN as compared to patients without LN metastasis. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV(C/B) in the control (pN+) group is statistically significantly higher than that in case group (p-value = 0.0318).\nIn addition, with the benefit of patients’ pathological and follow-up information, we have demonstrated that even with just an increase of 1 HEV in a high-power-field (HPF), regardless of the group, the risk is 1.024 times worse in terms of overall survival. This is consistent with clinical knowledge and natural history of the disease. It is well established that patients without LN metastases in their regional LN (cases in our cohort) have a significantly better prognosis, and; this fact is also reflected in our analysis\n[22,49-51] (Figure\n2). Cases had a 0.428 times the risk of Controls with regards to overall survival if they had the same number of HEV. It is crucial to understand that this risk is exponential in nature. To illustrate the significance of this result, an example will be used: Patient A with tongue SCC has proven LN metastases in the cervical LN. On further immunohistochemical staining, it is found that he has 0 HEV in a random high-power-field. Comparing this to patient B who has the same grade, stage of tumor, degree of LN metastases as patient A but has 100 HEV in a HPF, patient B will have a [ (1.024)100 = 10.715 ] 10 times worse prognosis in OS as compared to patient A. We will then consider patient C who has tongue SCC with no LN metastasis. If the HEV amount in a HPF is the same as either patient A or B, patient C’s risk as compared to either patient A or B will be 2.34 times (1/0.428= 2.34) better in terms of overall survival (Table\n4). Although this did not reach statistical significance (p=0.471), we believe this to be a factor of a small sample size.\nDifferences in OS and DFI relative risk in the 2 groups\nAssuming that our hypothesis is true, HEV transformation from a normal immunological mediator to a tumor metastasis mediator is reflected by morphological changes from a normal appearing HEV, to a dilated HEV, and then to a dilated HEV containing RBC. We believe that this metamorphosis occurs on a spectrum, and this process begins with the HEV increasing in absolute numbers, then each becoming more dilated before progressing into a functional vessel carrying blood (Figure\n5). Hence we analyzed our data looking at the 3 different stages of HEV transformation and the ratio comparing each parameter in a systematic way to demonstrate this spectrum (Figure\n1).\nMetamorphosis of High endothelial venules in a tumor microenvironment. This process begins with the high endothelial venules increasing in absolute numbers, subsequently each of them becoming more dilated and lastly every one of them will become a function vessel carrying blood.\nWe recall that that in the last example we illustrated that a patient’s OS risk is 1.024 times worse than an equivalent patient if there is 1 more HEV in one HPF of his regional LN, the risk is increased to 1.071 if the HEV is a dilated HEV. The risk is even higher at 1.116 times if the HEV is a dilated HEV containing RBC (Table\n1)\n[20]. This trend is also seen when all patients are considered in a single cohort. A patient’s OS risk is worsened by 1.004 times if there is an increase of 1 dilated HEV in a HPF. If that dilated HEV contains RBC, the OS risk is increased to 1.008 times.\nIn the treatment of tongue carcinomas, DFI is important as it signifies the failure of loco-regional control for which there is little effective therapy. Further surgery e.g. neck dissection to remove loco-regional recurrences is plagued with high morbidity and mortality. This, coupled with poor chemotherapy and radiotherapy response rates in recurrences, often equates to rapid deterioration of the patient’s condition. Disease free interval was also analyzed in the same manner to the three different HEV morphological phenotypes.\nWe found statistical significance in the relationship between the total number of HEV (A) and DFI when we controlled for the group (i.e. taking in the presence of LN metastasis as a factor). (p=0.022). This significance is preserved when we analyzed all the patients as a cohort (p=0.023)\n[20]. This means that the quantity of HEV is inversely related to DFI. If you consider the LN status and take it into statistical consideration, if there is one more HEV in a HPF, the DFI is 1.051 times worse than zero HEV. It is again important to note that the relationship of the quantity of HEV with respect to DFI is exponential in nature. For example, a patient (patient B) with 100 more HEV (A) in one HPF has a {(1.051)100 =144.634} 144 times shorter DFI as compared to an equivalent patient with only one HEV per HPF (patient A) (Table\n4). Excluding the group effect and considering all the patients as a cohort, a patient with 1 more HEV per HPF will have a DFI that is 1.051 times worse than a patient with zero HEV in a HPF (p=0.023).\nWe looked at the different ratios of abnormal HEV and compared them to several clinic-pathologic parameters namely overall survival, disease–free interval, tumor volume, stage and grade of the tumor. The details are specified in the secondary results (Table\n2).\nThere is a general trend observed. The more advanced the disease, the higher the ratio/percentage of abnormality of HEV. This can be seen in when we analyzed OS and the 2 ratios. The OS relative risk worsens by 1.078 if the ratio B/A (ratio of dilated HEV to the total number of HEV) increased by 1, while the OS relative risk worsens by 3.624 times if the ratio of dilated HEVs with RBC to the total number of HEV increases by 1. Most importantly, if we consider the most abnormal form of HEV (dilated HEV with RBC within its lumen, C) and look at it as a ratio to the total no. of HEV, a patient’s OS relative risk worsens by 17.884 times if this ratio (C/A) increases by a factor of 1. This observation approaches marginal significance. (p = 0.171) (Figure\n6).\nOverall survival relative risk with respect to the different high endothelial venules ratios.\nIn this study, we have shown the relationship of HEV and their transformation in a cancerous environment. We also note that in previous studies the HEV morphology did not alter at all in endotoxin-induced lymphadenopathy, implying a selective reaction of HEV in the cancerous condition\n[5]. Distinct, differentiated gene expression has also been reported when the endothelial cells respond to the changing of their microenvironment\n[52]. In a previous study, it was shown that the cellular morphology of the tall endothelial cells forming HEV changed dramatically to become flat endothelial cells in the presence of cancer. As a consequence, the HEV was remodelled from a thick-walled, endothelial vessel with a small lumen to a thin walled, large-lumen vessel shifting its function from recruitment of lymphocytes to becoming a blood vessel (Figure\n4)\n[10]. These facts indicate that the blood vessel endothelium has tremendous potential to adapt its environment. The confined lymph and blood channel alterations within the SLN, but not in the next station lymph node, imply that an inducer from the primary tumor is functioning locally with the existence of an active primary cancer. VEGF-A has been found to be an inducer of lymphangiogenesis in SLN\n[33]. Other studies have shown that serum level of VEGF-A was elevated in patients with late stage NPC\n[21] VEGF-A is a secreted protein factor that can travel in blood to other lymph nodes, these findings suggest that there may be other inducers involved. It is also of interest to explore the role of HEV after the establishment of a metastatic tumor nest. The enlarged, remodelled HEV could integrate into the metastatic tumor vasculature with further differentiation, characterized by the gradual loss of their specific marker MECA-79 from the tumor margin to the central part of the metastatic tumor nest.\n[5,10] It has been explained that, compared with primary tumors, the more rapid growth of metastatic lesions in the cervical lymph nodes of NPC patients was due to clonal selection of the cancer cells during metastases, with highly proliferative clones disseminated to the cervical nodes. However, based on our findings, the metastatic tumor vasculature in lymph nodes consists of many large blood vessels derived from normal HEVs, suggesting that the efficiency of nutrition and oxygen supplies could be better for the metastatic tumor cells in the involved lymph node. The enrichment of the blood supply in the lymph node before and after metastasis may favor the growth of newly arriving metastatic cancer cells. Consequently, as evident clinically in the long term follow-up of cancer patients, the involved regional lymph nodes may become manifest first, whereas the primary tumor remain clinically occult for years\n[53]. Moreover, the high density of functioning blood vessels in lymph nodes may subsequently facilitate the metastasis of cancer cell to distant organs. Conversely, consistent with its role in lymphocyte trafficking, if the HEV density is increased in the tumor themselves as opposed to LN, it has favorable prognostic value, Martinet et al. reported that in 146 breast cancers, the density of tumor HEV was an independent prognostic risk factor for DFI and OS\n[8]. This further suggests the tumor HEV function as major gateway for lymphocyte infiltration into tumors and represents potential targets for cancer immunotherapy. It will also be important to elucidate if the highly vascularized premetastatic SLN is associated with an increased metastatic potential. Control of lymphatic fluid movement may also be a target to consider in preventing metastases.\nAssuming our hypothesis is true with regards to the role of HEV in cancer metastasis, a potential therapeutic approach is to interrupt or block this remodelling process and evaluate if there is an effect on metastasis. This will confirm the HEV’s central role in the pathogenesis of metastasis.\nFurther studies need to designed and performed to elucidate the pathways of HEV transformation. A control gene for the growth and differentiation of HEV remains to be identified\n[9]. With regards to this, a recently identified nuclear factor (NF), NF-HEV has potential because it is preferentially expressed by HEV\n[54]. Dendritic cells, well known for their antigen presentation roles has recently been shown to modulate the phenotype of HEV and control entry of naive lymphocytes to LN, their role in a cancer microenvironment is also an area of potential research\n[13]. There are recent experiments in mouse models investigating conditional gene targeting of HEV as well\n[55]. Identification and characterization the molecular pathways and the control genes would have considerable clinical applications. For example, it would enable the design of experiments and studies to induce or retard the formation of HEV in various tissues, including tumors and their LN, improving vaccination strategies against pathogens and cancers. Therefore, there is a wealth of translational applications with regards to targeting HEV as a ligand for cancer investigational therapeutics. It paves the journey in the discovery of many unknown genes and molecules with potential clinical importance. Lastly, more work can be performed to validate our findings in other solid cancers.\n Limitations Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\nOur study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\n\nLimitations:\nOur study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\n\nConclusion:\nThis study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets.\n\nAbbreviations:\nHEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.\n\nCompeting interests:\nThe authors declare that they have no competing interests, financial or non-financial.\n\nAuthors’ contribution:\nLSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nHigh endothelial venules (HEV) have been recognized to play a role in metastasis by its changes seen in the cancer microenvironment of lymph nodes (LN) and solid cancers. Squamous cell carcinoma (SCC) of the tongue is a prevalent tumor of the head and neck region with high propensity for LN metastasis. The extent of LN metastasis is the most reliable adverse prognostic factor. Primary tumors can induce vasculature reorganization within sentinel LN before the arrival of tumor cells and HEV represents these remodelled vessels. This study aims to evaluate the cancer induced vascular changes in regional lymph nodes (LN) of patients by studying the morphological and functional alterations of HEV and its correlation with clinical outcome and pathological features.\n\nMethods:\nThis study was based on 65 patients with SCC tongue who underwent primary surgical treatment including neck dissection. The patients were categorized into 2 groups based on the presence of malignancy in their cervical lymph nodes. A review of the patients' pathological and clinical data was performed from a prospective database. Immunohistochemical staining of the tissue blocks for HEV and high-power-field image analysis were performed and analyzed with correlation to the patients' clinical and pathological features.\n\nResults:\nThe total number of HEV was found to be significantly associated to disease-free interval. There was a similar association comparing the HEV parameters to overall survival. The density of abnormal HEV was significantly higher in patients with established metastases in their lymph nodes and HEV was shown to be a better prognosis factor than conventional tumor staging. The HEV morphological metamorphosis demonstrates a spectrum that correlates well with disease progression and clinical outcome.\n\nConclusions:\nThe results suggest that the HEV displays a spectrum of morphological changes in the presence of cancer and LN metastasis, and that HEV is possibly involved in the process of cancer metastasis. We revealed the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environments in regional lymph nodes of tongue cancer patients in relation to clinical outcomes. The significant observation of modified dilated HEV containing red blood cells in lymph nodal basin of a cancer suggests the shifting of its function from one primarily of immune response to that of a blood carrying vessel. It also demonstrated potential prognostic value. More studies are needed to elucidate its potential role in cancer immunotherapy and as a potential novel therapeutic approach to preventing metastasis by manipulating the remodelling processes of HEV."},"background_conclusion_text":{"kind":"string","value":"Background:\nCancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\n\nConclusion:\nThis study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nHigh endothelial venules (HEV) have been recognized to play a role in metastasis by its changes seen in the cancer microenvironment of lymph nodes (LN) and solid cancers. Squamous cell carcinoma (SCC) of the tongue is a prevalent tumor of the head and neck region with high propensity for LN metastasis. The extent of LN metastasis is the most reliable adverse prognostic factor. Primary tumors can induce vasculature reorganization within sentinel LN before the arrival of tumor cells and HEV represents these remodelled vessels. This study aims to evaluate the cancer induced vascular changes in regional lymph nodes (LN) of patients by studying the morphological and functional alterations of HEV and its correlation with clinical outcome and pathological features.\n\nMethods:\nThis study was based on 65 patients with SCC tongue who underwent primary surgical treatment including neck dissection. The patients were categorized into 2 groups based on the presence of malignancy in their cervical lymph nodes. A review of the patients' pathological and clinical data was performed from a prospective database. Immunohistochemical staining of the tissue blocks for HEV and high-power-field image analysis were performed and analyzed with correlation to the patients' clinical and pathological features.\n\nResults:\nThe total number of HEV was found to be significantly associated to disease-free interval. There was a similar association comparing the HEV parameters to overall survival. The density of abnormal HEV was significantly higher in patients with established metastases in their lymph nodes and HEV was shown to be a better prognosis factor than conventional tumor staging. The HEV morphological metamorphosis demonstrates a spectrum that correlates well with disease progression and clinical outcome.\n\nConclusions:\nThe results suggest that the HEV displays a spectrum of morphological changes in the presence of cancer and LN metastasis, and that HEV is possibly involved in the process of cancer metastasis. We revealed the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environments in regional lymph nodes of tongue cancer patients in relation to clinical outcomes. The significant observation of modified dilated HEV containing red blood cells in lymph nodal basin of a cancer suggests the shifting of its function from one primarily of immune response to that of a blood carrying vessel. It also demonstrated potential prognostic value. More studies are needed to elucidate its potential role in cancer immunotherapy and as a potential novel therapeutic approach to preventing metastasis by manipulating the remodelling processes of HEV."},"whole_article_text_length":{"kind":"number","value":14568,"string":"14,568"},"whole_article_abstract_length":{"kind":"number","value":451,"string":"451"},"num_sections":{"kind":"number","value":18,"string":"18"},"most_frequent_words":{"kind":"list like","value":["hev","tumor","ln","cancer","patients","blood","study","lymph","cells","dilated"],"string":"[\n \"hev\",\n \"tumor\",\n \"ln\",\n \"cancer\",\n \"patients\",\n \"blood\",\n \"study\",\n \"lymph\",\n \"cells\",\n \"dilated\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | cancer | lymphatic | cells | tumor | blood | ln | route | role | vessels [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | neck | test | number | neck dissection | parameters | dissection | dilated hev | total number | number hev [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | hev | dfi | hev parameters | parameters | risk | grade | cases | stage | groups [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] shortcut | lymph | circulation | sln | flow | metastatic | hev | role elusive junction | role elusive junction providing | pre metastatic metastatic environment [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | parameters | cancer | dilated | lymph | dilated hev | patients | ln | cases [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | parameters | cancer | dilated | lymph | dilated hev | patients | ln | cases [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] LN ||| LN ||| LN ||| ||| LN | HEV [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 65 ||| 2 ||| ||| HEV [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] HEV ||| HEV ||| HEV | HEV ||| HEV [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] HEV | LN | HEV ||| HEV ||| ||| ||| HEV [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] LN ||| LN ||| LN ||| ||| LN | HEV ||| 65 ||| 2 ||| ||| HEV ||| ||| HEV ||| HEV ||| HEV | HEV ||| HEV ||| HEV | LN | HEV ||| HEV ||| ||| ||| HEV [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] LN ||| LN ||| LN ||| ||| LN | HEV ||| 65 ||| 2 ||| ||| HEV ||| ||| HEV ||| HEV ||| HEV | HEV ||| HEV ||| HEV | LN | HEV ||| HEV ||| ||| ||| HEV [SUMMARY]"}}},{"rowIdx":284,"cells":{"title":{"kind":"string","value":"Preoperative therapy restores ventilatory parameters and reduces length of stay in patients undergoing myocardial revascularization."},"pmid":{"kind":"string","value":"25140472"},"background_abstract":{"kind":"string","value":"The frequency of surgical procedures has increased steadily in recent decades, including the myocardial revascularization."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"We conducted a prospective study with patients undergoing myocardial revascularization, the Hospital das Clínicas da Universidade Estadual Paulista (UNESP)/Botucatu - SP. We evaluated 70 patients of both genders, aged between 40 and 75 years, subdivided into two groups: group I - 35 patients of both genders, who received a written protocol guidance, breathing exercises and respiratory muscle training in the preoperative period and group II - 35 patients of both genders, who received only orientation of the ward on the day of surgery. This study was approved by the Ethics Committee of UNESP / Botucatu - SP."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Maximal inspiratory pressure in third postoperative day and fifth postoperative day and significant difference between groups, being better for the intervention group. Expiratory pressure was significant in fifth postoperative day in the intervention group compared to controls. The difference of length of hospital stay in the postoperative was found between the groups with shorter hospital stay in the group receiving preoperative therapy."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Physical therapy plays an important role in the preoperative period, so that individuals in the intervention group more readily restored the parameters evaluated before surgery, in addition, there was a decrease in the time of the postoperative hospital stay. Thus, it is thought the cost-effectiveness of a program of preoperative physiotherapy."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Breathing Exercises","Female","Humans","Inspiratory Capacity","Length of Stay","Male","Middle Aged","Muscle Strength","Muscle Stretching Exercises","Myocardial Revascularization","Preoperative Care","Preoperative Period","Prospective Studies","Reference Values","Reproducibility of Results","Respiratory Muscles","Respiratory Rate","Statistics, Nonparametric","Tidal Volume","Time Factors","Treatment Outcome"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Breathing Exercises\",\n \"Female\",\n \"Humans\",\n \"Inspiratory Capacity\",\n \"Length of Stay\",\n \"Male\",\n \"Middle Aged\",\n \"Muscle Strength\",\n \"Muscle Stretching Exercises\",\n \"Myocardial Revascularization\",\n \"Preoperative Care\",\n \"Preoperative Period\",\n \"Prospective Studies\",\n \"Reference Values\",\n \"Reproducibility of Results\",\n \"Respiratory Muscles\",\n \"Respiratory Rate\",\n \"Statistics, Nonparametric\",\n \"Tidal Volume\",\n \"Time Factors\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"4389447"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"The cardiac surgery was always clothed with great interest, curiosity, and in some\nmoments, mysticism, and fact understandable when we analyzed the noble importance of\nthis component for the proper functioning of the human body[1].\nIn recent decades the frequency of surgical procedures has progressively increased,\namong them the myocardial revascularization (MR). Cardiovascular diseases are a serious\npublic health problem in Brazil[2].\nIn individuals over 70 years old it is estimated an incidence of 70% of coronary artery\ndisease. As the age is a determinant of coronary atherosclerosis, a growing number of\nelderly people will be subjected to MR and/or other therapeutic methods[3,4].\nThe main risk factors that contribute to cardiac diseases are: smoking, high levels of\nlow-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density\nlipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,\nfamily history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors\nrelated to atherosclerosis and its clinical manifestations[4,5].\nThe coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment\nof ischemic heart disease that is manifested clinically by angina and acute myocardial\ninfarction. The CABG surgery creates a new route for the blood flow and, is used for\nmaking a diversion of blood to the portions of the coronary artery distal to the\nobstructive atherosclerotic involvement[1,5].\nWith the advancements in care of physical therapy in the preoperative period, surgical\ntechniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,\nanesthesia and intensive care in the post-operative period, there was a decrease in\nmorbidity and mortality of MR which caused the surgical indication in groups of patients\nincreasingly complex[6,7].\nObserving the fact that they are common pulmonary complications of CABG with CPB was\njustified to carry out this research, there is great importance in properly carrying out\nevaluations, guidelines and breathing exercises in the period before surgery, and\naddition, observe the influence and duration of physical therapy assistance in the\npreoperative period and also its behavior in the postoperative period.\n Objective To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\nTo demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"We performed a prospective clinical study, simple blind, in the period from July 2009 to\nSeptember 2011 in the ward of cardiac and thoracic surgery at Hospital das Clínicas da\nUniversidade Estadual Paulista (UNESP), Botucatu-SP. We evaluated 70 patients of both\ngender, age ranging from 40 to 75 years, undergoing CABG with use of CPB, randomized by\nmeans of a computerized software (STATTREK) and delivered in a sealed envelope to the\nphysical therapist responsible for primary health care, subdividing into two groups:\n- Group I (GI) - Thirty-five patients, undergoing physiotherapeutic intervention,\nperformed under supervision, once a day, during the period that preceded the surgery.\nThe protocol consisted of breathing exercises (breathing in time, deep breathing\nfollowed by prolonged expiration, sustained maximal inspiration with apnea of 6 seconds,\nand diaphragmatic breathing associated with the mobilization of the upper limbs) and\nbreathing exercises with threshold - IMT® (Threshold - IMT®\nInspiratory Muscle Trainer, HealthscanProducts Inc.) at an intensity of 40% of the\ninitial MIP with three sets of ten repetitions, respecting two-minute intervals between\neach series.\n- Group II (GII) - thirty-five patients who did not undergo the physical therapy\nprotocol during the preoperative period, receiving only guidelines ward routine.\nWe included patients of both sexes, aged 40-75 years with coronary artery disease\nadmitted for cardiac surgery with cardiopulmonary bypass (CPB) MR, who agreed to\nparticipate in the study. We excluded patients who require the use of intra-aortic\nballoon, carrying pulmonary pathology, musculoskeletal impairment and severe\nneurological surgery performed without CPB, emergency surgery, homodynamic instability,\nand any event that threatens the integrity of patient or the fidelity of the measures\nanalyzed.\n Delimitation All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\nAll patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\n Procedures/treatment Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\nPatients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\n Statistical Analysis The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.\nThe sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"Seventy patients were divided into two groups, Group I composed of 35 patients (12\nfemale and 23 male) and Group II, with 35 patients (six female and 29 male), as\nillustrated in Figure 1.\nCharacterization of groups by gender\nNo significant difference was noticed between groups for the variables age (GI 58.9±9.53\nyears and GII 61.4±8.43 years; P=0.26) and body mass index (GI\n26.8±3.96 kg/m2 and G II 26±3.86 kg/m2) (Table 1).\nPatient characteristics regarding age and BMI.\nBMI: body mass index; Wilcoxon test. * P<0.05\nThe groups were homogenous concerning presence of active smoking, ex-smokers and\nnonsmokers, as illustrated in Table 2.\nNumber and proportion of patients in each group according to the presence of\nactive smoking, ex-smokers and nonsmokers.\nTwo proportions followed by the same lowercase letter do not differ in groups\n(rows). Two proportions followed by the same capital letter do not differ in\nthe types of smoking (P>0.05). Goldman test to compare proportions between\nand within populations multinomina.\nBetween the GI and GII, no significant difference regarding the proportion of\nex-smokers, smokers and nonsmokers, in other words, the groups were homogeneous as\ndemonstrated below (Table 2).\n Maximum Inspiratory Pressure (MIP) The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\nThe mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\n Maximum Expiratory Pressure (MEP) The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\nThe mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\n Minute Volume (MV) The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\nThe analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\n Tidal Volume Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\nFigure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\n Respiratory rate (RR) The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative\nThe median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative"},"conclusions_title":{"kind":"string","value":"CONCLUSIONS"},"conclusions_text":{"kind":"string","value":"The physical therapy has an important role in the preoperative period, restoring greater\nreadiness ventilatory parameters of patients undergoing coronary artery bypass grafting\nwith cardiopulmonary bypass, resulting in a decrease in length of hospital stay after\nsurgery."},"other_sections_titles":{"kind":"list like","value":["Objective","Delimitation","Procedures/treatment","Statistical Analysis","Maximum Inspiratory Pressure (MIP)","Maximum Expiratory Pressure (MEP)","Minute Volume (MV)","Tidal Volume","Respiratory rate (RR)","Limitation of the study"],"string":"[\n \"Objective\",\n \"Delimitation\",\n \"Procedures/treatment\",\n \"Statistical Analysis\",\n \"Maximum Inspiratory Pressure (MIP)\",\n \"Maximum Expiratory Pressure (MEP)\",\n \"Minute Volume (MV)\",\n \"Tidal Volume\",\n \"Respiratory rate (RR)\",\n \"Limitation of the study\"\n]"},"other_sections_texts":{"kind":"list like","value":["To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.","All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].","Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.","The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.","The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.","The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.","The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.","Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.","The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative","The major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately."],"string":"[\n \"To demonstrate the importance of the role of physical therapy in the preoperative\\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\\nsurgery.\",\n \"All patients were evaluated by the same evaluator (preoperative period - PRE), third\\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\\nphysiotherapeutical treatment, according to their needs in the postoperative period\\nby physical therapy team in the hospital and only Group I received physiotherapy in\\nthe preoperative period.\\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\\nanesthesia time and the time that the patient remained with the endotracheal tube in\\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\\nICU stay (when the patient arrived in the intensive care unit until discharged to the\\nward bed) and length of stay in the OP (obtained from the time that the patient\\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\\nwere obtained by means of the daily developments of medical and nursing staff.\\nThe assessment of respiratory muscle strength was obtained by measurement of\\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\\nforceps according to the technique described by Nephew et al. (2008). The MIP\\nmeasurement was performed from residual volume, and MEP was obtained from total lung\\ncapacity. Three measurements were taken technically satisfactory, and if there were\\nmore than 20% difference between the measurements, a fourth and even fifth\\nmeasurement would be performed, being always considered the highest value obtained.\\nThe MIP was performed for evaluation of inspiratory muscle strength in three\\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\\nthe buccinator muscles during MIP maneuver[9].\\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\\nminute (cpm) and obtained by direct observation of respiratory cycles during the\\nevaluation of the minute volume[7].\",\n \"Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\\nof breathing exercises (breathing in time, deep breath and then exhaling long,\\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\\nassociated with mobilization of the upper limb, with three series out of ten for each\\nbreathing exercise) and breathing exercises with the device Threshold -\\nIMT®. We conducted three sets of ten repetitions with an interval of\\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\\nThe GII patients received only guidelines ward routine before surgery, but\\npostoperatively, both groups performed physical therapy as needed by staff\\nphysiotherapy service.\",\n \"The sample should be made with at least 26 patients who were randomly divided into\\ntwo study groups (with physical therapy preoperatively and control patients who did\\nnot receive preoperative physiotherapy). We considered the level of significance of\\n5% and a test power in the order of 80% for a minimum difference in the order of\\ntwo.\\nThe comparison between the groups for age and body mass index (BMI) was performed by\\nWilcoxon test for independent samples. The smoking variable was analyzed using\\nGoldman to compare proportions within and among multinomial populations. The\\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\\nhospital stay were obtained in minutes and analyzed by Student's t test or the\\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\\ntests for comparison of the moments in each group and Mann Whitney test to compare\\ngroups at each time, adopting a significance level of 5% probability of rejecting\\nnull hypothesis.\\nThis study was approved by the Research Ethics Committee of Universidade Estadual\\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\\nprotocol 3223) and all patients signed an informed consent form.\",\n \"The mean values the for maximum inspiratory pressures evaluated in the preoperative\\nperiod and 3PO showed no significant difference between groups\\n(P=0.276 and 0.065; respectively), however, we observed greater\\ninspiratory muscle strength for GI in relation to GII in 5PO\\n(P=0.001) (Figure 2).\\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\\noperation of each group analyzed (with and without physical therapy\\npreoperatively). Friedman test for comparison of moments in each group and Mann\\nWhitney test to compare the groups at each time, *P<0.05.\",\n \"The mean value for the maximum expiratory pressure obtained in the preoperative\\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\\ncmH2O) for GII, not presenting significant difference between groups\\n(P=0.704). However, the values referring MEP measured in the 5PO\\nshowed significant difference between groups (P=0.001), as\\nillustrated in Figure 3.\\nMedian for maximum expiratory pressure (MEP) variable at different times of the\\noperation in the different groups. Friedman test for comparison of moments in\\neach group and Mann Whitney test to compare the groups at each time,\\n*P<0.05.\",\n \"The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\\nP=0.001). However, these values have not showed significative\\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\\nrespectively).\\nMedian minute volume (VM ) variable at different times of the operation of each\\ngroup analyzed. Friedman test for comparison of the moments in each group and\\nthe Mann Whitney test for comparison between groups at any time,\\n*P<0.05.\",\n \"Figure 5 illustrates the analysis of variable\\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\\nvalues were not significantly different for the 3PO and 5 PO\\n(P=0.059 and P=0.549; respectively).\\nMedian tidal volume variable at different times for the group treated with\\npreoperative physiotherapy and without physical therapy preoperatively.\\nFriedman test for comparison of moments in each group and Mann Whitney test to\\ncompare the groups at each time, *P<0.05.\",\n \"The median refers to the respiratory rate obtained preoperatively for GI and GII\\nshowed no significant difference (16 cpm and 18 cpm, respectively,\\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\\nshowed no significant difference between groups (P=0.090 and\\nP=0.886, respectively), as shown in Figure 6.\\nMedian variable respiratory rate at different times of the operation of each\\ngroup analyzed (with and without physical therapy preoperatively). Friedman\\ntest for comparison of moments in each group and Mann Whitney test to compare\\nthe groups at each time, *P<0.05.\\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\\nhospital stay after surgery.\\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\\nstay did not significantly differ between groups, however, significant difference was\\nobserved for the length of stay variable in the postoperative period\\n(P=0.001), as shown in Table\\n3.\\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\\n5PO.\\nValues are shown as mean ± standard deviation or median (minimum and maximum\\nvalues).\\nMann and Whitney\\nStudent's t test.\\nSignificant difference between groups (P <0.05). ICU: intensive care\\nunit; PO: postoperative\",\n \"The major limitation was the short period of time that patients received preoperative\\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\\nrelevance even in a short period, so the study may add knowledge of response to\\ntherapy performed during intraoperative CABG surgery.\\nNot performing spirometry can be mentioned as a limitation on the analysis of\\nventilation parameters, however, due to the conditions offered by our service, we\\ncould not perform this evaluation.\\nOn the other hand, the results showed that even with limited time monitoring in the\\npreoperative period and respiratory variables obtained only by respirometry and\\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\\npatients undergoing surgery CABG.\\nWe understand that the results may open up new avenues of future research related to\\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\\nfurther studies should be conducted using tools such as spirometry, to assess and\\nquantify the volume and lung capacity more accurately.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","Objective","METHODS","Delimitation","Procedures/treatment","Statistical Analysis","RESULTS","Maximum Inspiratory Pressure (MIP)","Maximum Expiratory Pressure (MEP)","Minute Volume (MV)","Tidal Volume","Respiratory rate (RR)","DISCUSSION","Limitation of the study","CONCLUSIONS"],"string":"[\n \"INTRODUCTION\",\n \"Objective\",\n \"METHODS\",\n \"Delimitation\",\n \"Procedures/treatment\",\n \"Statistical Analysis\",\n \"RESULTS\",\n \"Maximum Inspiratory Pressure (MIP)\",\n \"Maximum Expiratory Pressure (MEP)\",\n \"Minute Volume (MV)\",\n \"Tidal Volume\",\n \"Respiratory rate (RR)\",\n \"DISCUSSION\",\n \"Limitation of the study\",\n \"CONCLUSIONS\"\n]"},"all_sections_texts":{"kind":"list like","value":["The cardiac surgery was always clothed with great interest, curiosity, and in some\nmoments, mysticism, and fact understandable when we analyzed the noble importance of\nthis component for the proper functioning of the human body[1].\nIn recent decades the frequency of surgical procedures has progressively increased,\namong them the myocardial revascularization (MR). Cardiovascular diseases are a serious\npublic health problem in Brazil[2].\nIn individuals over 70 years old it is estimated an incidence of 70% of coronary artery\ndisease. As the age is a determinant of coronary atherosclerosis, a growing number of\nelderly people will be subjected to MR and/or other therapeutic methods[3,4].\nThe main risk factors that contribute to cardiac diseases are: smoking, high levels of\nlow-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density\nlipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,\nfamily history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors\nrelated to atherosclerosis and its clinical manifestations[4,5].\nThe coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment\nof ischemic heart disease that is manifested clinically by angina and acute myocardial\ninfarction. The CABG surgery creates a new route for the blood flow and, is used for\nmaking a diversion of blood to the portions of the coronary artery distal to the\nobstructive atherosclerotic involvement[1,5].\nWith the advancements in care of physical therapy in the preoperative period, surgical\ntechniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,\nanesthesia and intensive care in the post-operative period, there was a decrease in\nmorbidity and mortality of MR which caused the surgical indication in groups of patients\nincreasingly complex[6,7].\nObserving the fact that they are common pulmonary complications of CABG with CPB was\njustified to carry out this research, there is great importance in properly carrying out\nevaluations, guidelines and breathing exercises in the period before surgery, and\naddition, observe the influence and duration of physical therapy assistance in the\npreoperative period and also its behavior in the postoperative period.\n Objective To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\nTo demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.","To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.","We performed a prospective clinical study, simple blind, in the period from July 2009 to\nSeptember 2011 in the ward of cardiac and thoracic surgery at Hospital das Clínicas da\nUniversidade Estadual Paulista (UNESP), Botucatu-SP. We evaluated 70 patients of both\ngender, age ranging from 40 to 75 years, undergoing CABG with use of CPB, randomized by\nmeans of a computerized software (STATTREK) and delivered in a sealed envelope to the\nphysical therapist responsible for primary health care, subdividing into two groups:\n- Group I (GI) - Thirty-five patients, undergoing physiotherapeutic intervention,\nperformed under supervision, once a day, during the period that preceded the surgery.\nThe protocol consisted of breathing exercises (breathing in time, deep breathing\nfollowed by prolonged expiration, sustained maximal inspiration with apnea of 6 seconds,\nand diaphragmatic breathing associated with the mobilization of the upper limbs) and\nbreathing exercises with threshold - IMT® (Threshold - IMT®\nInspiratory Muscle Trainer, HealthscanProducts Inc.) at an intensity of 40% of the\ninitial MIP with three sets of ten repetitions, respecting two-minute intervals between\neach series.\n- Group II (GII) - thirty-five patients who did not undergo the physical therapy\nprotocol during the preoperative period, receiving only guidelines ward routine.\nWe included patients of both sexes, aged 40-75 years with coronary artery disease\nadmitted for cardiac surgery with cardiopulmonary bypass (CPB) MR, who agreed to\nparticipate in the study. We excluded patients who require the use of intra-aortic\nballoon, carrying pulmonary pathology, musculoskeletal impairment and severe\nneurological surgery performed without CPB, emergency surgery, homodynamic instability,\nand any event that threatens the integrity of patient or the fidelity of the measures\nanalyzed.\n Delimitation All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\nAll patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\n Procedures/treatment Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\nPatients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\n Statistical Analysis The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.\nThe sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.","All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].","Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.","The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.","Seventy patients were divided into two groups, Group I composed of 35 patients (12\nfemale and 23 male) and Group II, with 35 patients (six female and 29 male), as\nillustrated in Figure 1.\nCharacterization of groups by gender\nNo significant difference was noticed between groups for the variables age (GI 58.9±9.53\nyears and GII 61.4±8.43 years; P=0.26) and body mass index (GI\n26.8±3.96 kg/m2 and G II 26±3.86 kg/m2) (Table 1).\nPatient characteristics regarding age and BMI.\nBMI: body mass index; Wilcoxon test. * P<0.05\nThe groups were homogenous concerning presence of active smoking, ex-smokers and\nnonsmokers, as illustrated in Table 2.\nNumber and proportion of patients in each group according to the presence of\nactive smoking, ex-smokers and nonsmokers.\nTwo proportions followed by the same lowercase letter do not differ in groups\n(rows). Two proportions followed by the same capital letter do not differ in\nthe types of smoking (P>0.05). Goldman test to compare proportions between\nand within populations multinomina.\nBetween the GI and GII, no significant difference regarding the proportion of\nex-smokers, smokers and nonsmokers, in other words, the groups were homogeneous as\ndemonstrated below (Table 2).\n Maximum Inspiratory Pressure (MIP) The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\nThe mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\n Maximum Expiratory Pressure (MEP) The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\nThe mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\n Minute Volume (MV) The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\nThe analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\n Tidal Volume Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\nFigure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\n Respiratory rate (RR) The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative\nThe median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative","The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.","The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.","The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.","Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.","The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative","Studies by Feltrim et al.[10] show\nthat performing preoperative physiotherapy is more effective in reducing respiratory\ncomplications in patients with moderate or higher risk than those whose risk was\nlow.\nIn the present study, we observed no significant difference between the subjects of\nGroup I and Group II as the characteristics (age, BMI, smoking and number of co\nmorbidities), which suggests that the study subjects in both groups were\nhomogeneous.\nIn the study by Leguisamo et al.[9],\nwith 86 patients undergoing CABG, divided into two groups: the intervention group (44\npatients) who were assessed and received physiotherapeutic guidance at least 15 days\nbefore surgery. The control group received routine care on the day of\nhospitalization.\nThe average MIP between groups showed neither significant difference in the preoperative\nperiod nor in the 1PO and 6PO.\nIn the present study it was observed that there was no significant difference between\ngroups to measure MIP in the preoperative period, but now in times of 3PO and 5PO\nsignificant difference between groups was found. Similarly, no significant difference in\nthe amount of MEP preoperatively in the 3PO and between groups. However in 5PO, there\nwas significant difference between the groups with the best values of the group that\nperformed physical therapy before surgery.\nBarros et al.[11] evaluated the MIP\nand MEP in the preoperative period, the first day after surgery and in 38 patients\nundergoing CABG with CPB who were divided into two groups: 23 patients in group I\n(respiratory muscle training) and 15 in group II (control). Group I received\nconventional physiotherapy over respiratory muscle training and Group II the\nconventional physiotherapy. The authors found that the values obtained for MIP and MEP\nin hospital discharge were higher in group I.\nThere was no decrease in maximal respiratory pressures, transdiaphragmatic pressure and\ndiaphragm pressure, indicating weakness of respiratory muscle strength[12,13].\nAgreeing with the authors cited above, we observed in the present study, after cardiac\nsurgery, MIP values in GI and GII which decreased in 3PO, however the value of MIP in\n5PO increase was observed for GI values compared to those in PRE and 3PO since the GII\nvalues remained stable in 3PO. The authors noted the drop in MEP in 3PO in both groups,\nhowever, in 5PO MEP has increased in GI. Regarding PRE and 3PO in GII no return value\nwas observed in PRE.\nArcêncio et al.[12] conducted a study\nwith 30 patients, aged 50 years-old and candidates for CABG and / or heart valve,\ndividing into two groups. Intervention group comprised 15 patients who underwent at\nleast a two weeks inspiratory muscle training using incentive spirometry (\"Threshold -\nIMT®) with 40% charge of the MIP and control group with 15 patients who\nreceived only general guidelines. The authors compared the spirometric values before and\nafter training and showed no significant difference in the values of MIP and\nMEP[14].\nElias et al.[15] studied 42 patients\naged 18 to 75 years-old with coronary artery disease, in the pre-and postoperative\nperiod, divided into two groups. Intervention group received TMR with Threshold -\nIMT® and control group received only guidelines. The TMR both pre and\npost-operative period consisted of three sets of 10 inspiratory exercises once a day for\nthree consecutive days. It was observed that after cardiac surgery there was a\nsignificant reduction in MIP and MEP in both groups.\nMorsch et al.[13] conducted a study to\nevaluate 108 patients undergoing elective CABG surgery from April 2006 to February 2007\n(preoperatively and postoperatively).\nThe average values of MIP in the preoperative period were significantly decreased\ncompared to 6 days after the surgery (P<0.001). The authors also\nobserved a significant decrease in the values of MEP obtained on the 6th\npostoperative day compared to the preoperative period (P<0.001).\nPatients with respiratory muscle weakness have higher risk of postoperative pulmonary\ncomplications. The respiratory muscle training in the pre-operative may prevent\npulmonary complications in patients undergoing thoracic surgery[16]. There may be a reduction of all lung\nvolumes resulting from factors such as diaphragmatic dysfunction, pain, absence of deep\nbreaths, pulmonary changes and ribcage. The functional residual capacity (FRC) decreases\nbecause of the reduction of both the residual volume (RV) and expiratory reserve volume.\nThe ventilation is affected by VC reduction by approximately 20%, and the increase in\nFR(22-24). The sum of these factors cause changes in the mechanics of\nrespiration, which leads to a shallow breathing pattern of decreased lung\nvolume[13].\nPatients undergoing CABG develop mostly PO pulmonary dysfunction with a significant\nreduction in lung volumes, impairments in respiratory function, decreased lung\ncompliance and increased work of breathing. The reduction in lung volumes and capacities\ncontributes to changes in gas exchange, resulting in hypoxemia[17].\nIn this study, the VM showed significant difference regarding the groups I and II in the\npreoperative period but not significant between groups in 3PO. Likewise, the mean value\nof the VM for the GI 5PO, there was no significant difference in the GII. The mean value\nof VC in PRE in GI was significant, as the mean value of the VC in GII patients, however\nin GI and GII we observed in 5PO and 3PO that there was no significant difference. It\nwas observed in GI at different times that the increase of VM will be very likely due to\nincreased FR, since the current VC remained without many changes between different\ntimes.\nBarros et al.[11] demonstrated a\nsignificant decrease in lung volumes in patients undergoing cardiac surgery in the\npostoperative period.\nCarvalho et al.[18] observed a\ndecrease in the values of minute volume and tidal volume measured on the 2nd\npostoperative day, however, these values showed gradual improvement returning to the\nvalues obtained at baseline (preoperative period) at the time of hospital discharge.\nIn a study, Guizilini et al.[19]\nevaluated 30 patients with a mean age of 56 years and divided into two groups, group A\n(n=15) without CPB and group B (n=15) with CPB. All patients underwent pulmonary\nfunction. In both groups, there was a decrease in FVC until the fifth postoperative day\n(P<0.05).\nRegarding the respiratory rate in the present study, we can verify that there was an\nincrease in both groups, with a higher peak on the 3rd postoperative day and\ndecreased on the 6th postoperative day, but the respiratory rate did not\nreturn to the preoperative values, Carvalho et al.[18] already mentioned above that they concluded in their studies\nthat the mean respiratory frequency had a significant increase between pre and\n2nd PO, 3rd PO, decreasing on the 4th postoperative\nday and increased again on the 5th postoperative day and decreased in\nhospital discharge, but patients did not return to the preoperative values.\nAccording to the results obtained in this study, we can affirm that there was no\nsignificant difference between the groups regarding the time variables: anesthesia,\nsurgery, perfusion, anoxia, mechanical ventilation, intubation. This suggests that the\ngroups were quite homogeneous. As for the time of postoperative hospital stay, there was\na decrease of approximately 25 hours, comparing patients who underwent physical therapy\nbefore surgery with patients who did not.\nIn contrast to the findings of this study, Patman et al.[20] performed a randomized study of 236 patients to\nevaluate the effects of physical therapy interventions performed early versus only\nstarted after extubation in patients undergoing cardiac surgery and no significant\ndifferences were found in the time of intubation, ICU stay and length of hospital stay\nbetween the groups.\nHowever, Celli et al.[21] conducted a\nstudy with 172 patients and showed a decrease in length of hospital stay in the group\nthat had received guidelines breathing exercises (9.6±3.2 days) compared to the control\ngroup (13±5 days).\nCorroborating these findings, as well as those obtained in the present study, Leguisamo\net al.[9] performed a prospective\nstudy to evaluate the effectiveness of a physiotherapy program start in the preoperative\nperiod in reducing the length of hospital stay and showed a significant reduction in\nlength of hospital stay for the intervention group compared to the control group\n(P<0.05).\nThese data suggest that physical therapy initiated before surgery can improve conditions\nof patients, reestablishing early respiratory pressures, reducing the length of stay and\ndecreased hospital costs for the period of hospitalization.\n Limitation of the study The major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.\nThe major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.","The major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.","The physical therapy has an important role in the preoperative period, restoring greater\nreadiness ventilatory parameters of patients undergoing coronary artery bypass grafting\nwith cardiopulmonary bypass, resulting in a decrease in length of hospital stay after\nsurgery."],"string":"[\n \"The cardiac surgery was always clothed with great interest, curiosity, and in some\\nmoments, mysticism, and fact understandable when we analyzed the noble importance of\\nthis component for the proper functioning of the human body[1].\\nIn recent decades the frequency of surgical procedures has progressively increased,\\namong them the myocardial revascularization (MR). Cardiovascular diseases are a serious\\npublic health problem in Brazil[2].\\nIn individuals over 70 years old it is estimated an incidence of 70% of coronary artery\\ndisease. As the age is a determinant of coronary atherosclerosis, a growing number of\\nelderly people will be subjected to MR and/or other therapeutic methods[3,4].\\nThe main risk factors that contribute to cardiac diseases are: smoking, high levels of\\nlow-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density\\nlipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,\\nfamily history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors\\nrelated to atherosclerosis and its clinical manifestations[4,5].\\nThe coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment\\nof ischemic heart disease that is manifested clinically by angina and acute myocardial\\ninfarction. The CABG surgery creates a new route for the blood flow and, is used for\\nmaking a diversion of blood to the portions of the coronary artery distal to the\\nobstructive atherosclerotic involvement[1,5].\\nWith the advancements in care of physical therapy in the preoperative period, surgical\\ntechniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,\\nanesthesia and intensive care in the post-operative period, there was a decrease in\\nmorbidity and mortality of MR which caused the surgical indication in groups of patients\\nincreasingly complex[6,7].\\nObserving the fact that they are common pulmonary complications of CABG with CPB was\\njustified to carry out this research, there is great importance in properly carrying out\\nevaluations, guidelines and breathing exercises in the period before surgery, and\\naddition, observe the influence and duration of physical therapy assistance in the\\npreoperative period and also its behavior in the postoperative period.\\n Objective To demonstrate the importance of the role of physical therapy in the preoperative\\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\\nsurgery.\\nTo demonstrate the importance of the role of physical therapy in the preoperative\\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\\nsurgery.\",\n \"To demonstrate the importance of the role of physical therapy in the preoperative\\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\\nsurgery.\",\n \"We performed a prospective clinical study, simple blind, in the period from July 2009 to\\nSeptember 2011 in the ward of cardiac and thoracic surgery at Hospital das Clínicas da\\nUniversidade Estadual Paulista (UNESP), Botucatu-SP. We evaluated 70 patients of both\\ngender, age ranging from 40 to 75 years, undergoing CABG with use of CPB, randomized by\\nmeans of a computerized software (STATTREK) and delivered in a sealed envelope to the\\nphysical therapist responsible for primary health care, subdividing into two groups:\\n- Group I (GI) - Thirty-five patients, undergoing physiotherapeutic intervention,\\nperformed under supervision, once a day, during the period that preceded the surgery.\\nThe protocol consisted of breathing exercises (breathing in time, deep breathing\\nfollowed by prolonged expiration, sustained maximal inspiration with apnea of 6 seconds,\\nand diaphragmatic breathing associated with the mobilization of the upper limbs) and\\nbreathing exercises with threshold - IMT® (Threshold - IMT®\\nInspiratory Muscle Trainer, HealthscanProducts Inc.) at an intensity of 40% of the\\ninitial MIP with three sets of ten repetitions, respecting two-minute intervals between\\neach series.\\n- Group II (GII) - thirty-five patients who did not undergo the physical therapy\\nprotocol during the preoperative period, receiving only guidelines ward routine.\\nWe included patients of both sexes, aged 40-75 years with coronary artery disease\\nadmitted for cardiac surgery with cardiopulmonary bypass (CPB) MR, who agreed to\\nparticipate in the study. We excluded patients who require the use of intra-aortic\\nballoon, carrying pulmonary pathology, musculoskeletal impairment and severe\\nneurological surgery performed without CPB, emergency surgery, homodynamic instability,\\nand any event that threatens the integrity of patient or the fidelity of the measures\\nanalyzed.\\n Delimitation All patients were evaluated by the same evaluator (preoperative period - PRE), third\\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\\nphysiotherapeutical treatment, according to their needs in the postoperative period\\nby physical therapy team in the hospital and only Group I received physiotherapy in\\nthe preoperative period.\\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\\nanesthesia time and the time that the patient remained with the endotracheal tube in\\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\\nICU stay (when the patient arrived in the intensive care unit until discharged to the\\nward bed) and length of stay in the OP (obtained from the time that the patient\\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\\nwere obtained by means of the daily developments of medical and nursing staff.\\nThe assessment of respiratory muscle strength was obtained by measurement of\\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\\nforceps according to the technique described by Nephew et al. (2008). The MIP\\nmeasurement was performed from residual volume, and MEP was obtained from total lung\\ncapacity. Three measurements were taken technically satisfactory, and if there were\\nmore than 20% difference between the measurements, a fourth and even fifth\\nmeasurement would be performed, being always considered the highest value obtained.\\nThe MIP was performed for evaluation of inspiratory muscle strength in three\\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\\nthe buccinator muscles during MIP maneuver[9].\\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\\nminute (cpm) and obtained by direct observation of respiratory cycles during the\\nevaluation of the minute volume[7].\\nAll patients were evaluated by the same evaluator (preoperative period - PRE), third\\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\\nphysiotherapeutical treatment, according to their needs in the postoperative period\\nby physical therapy team in the hospital and only Group I received physiotherapy in\\nthe preoperative period.\\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\\nanesthesia time and the time that the patient remained with the endotracheal tube in\\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\\nICU stay (when the patient arrived in the intensive care unit until discharged to the\\nward bed) and length of stay in the OP (obtained from the time that the patient\\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\\nwere obtained by means of the daily developments of medical and nursing staff.\\nThe assessment of respiratory muscle strength was obtained by measurement of\\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\\nforceps according to the technique described by Nephew et al. (2008). The MIP\\nmeasurement was performed from residual volume, and MEP was obtained from total lung\\ncapacity. Three measurements were taken technically satisfactory, and if there were\\nmore than 20% difference between the measurements, a fourth and even fifth\\nmeasurement would be performed, being always considered the highest value obtained.\\nThe MIP was performed for evaluation of inspiratory muscle strength in three\\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\\nthe buccinator muscles during MIP maneuver[9].\\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\\nminute (cpm) and obtained by direct observation of respiratory cycles during the\\nevaluation of the minute volume[7].\\n Procedures/treatment Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\\nof breathing exercises (breathing in time, deep breath and then exhaling long,\\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\\nassociated with mobilization of the upper limb, with three series out of ten for each\\nbreathing exercise) and breathing exercises with the device Threshold -\\nIMT®. We conducted three sets of ten repetitions with an interval of\\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\\nThe GII patients received only guidelines ward routine before surgery, but\\npostoperatively, both groups performed physical therapy as needed by staff\\nphysiotherapy service.\\nPatients allocated to the GI protocol underwent preoperative physiotherapy consisting\\nof breathing exercises (breathing in time, deep breath and then exhaling long,\\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\\nassociated with mobilization of the upper limb, with three series out of ten for each\\nbreathing exercise) and breathing exercises with the device Threshold -\\nIMT®. We conducted three sets of ten repetitions with an interval of\\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\\nThe GII patients received only guidelines ward routine before surgery, but\\npostoperatively, both groups performed physical therapy as needed by staff\\nphysiotherapy service.\\n Statistical Analysis The sample should be made with at least 26 patients who were randomly divided into\\ntwo study groups (with physical therapy preoperatively and control patients who did\\nnot receive preoperative physiotherapy). We considered the level of significance of\\n5% and a test power in the order of 80% for a minimum difference in the order of\\ntwo.\\nThe comparison between the groups for age and body mass index (BMI) was performed by\\nWilcoxon test for independent samples. The smoking variable was analyzed using\\nGoldman to compare proportions within and among multinomial populations. The\\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\\nhospital stay were obtained in minutes and analyzed by Student's t test or the\\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\\ntests for comparison of the moments in each group and Mann Whitney test to compare\\ngroups at each time, adopting a significance level of 5% probability of rejecting\\nnull hypothesis.\\nThis study was approved by the Research Ethics Committee of Universidade Estadual\\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\\nprotocol 3223) and all patients signed an informed consent form.\\nThe sample should be made with at least 26 patients who were randomly divided into\\ntwo study groups (with physical therapy preoperatively and control patients who did\\nnot receive preoperative physiotherapy). We considered the level of significance of\\n5% and a test power in the order of 80% for a minimum difference in the order of\\ntwo.\\nThe comparison between the groups for age and body mass index (BMI) was performed by\\nWilcoxon test for independent samples. The smoking variable was analyzed using\\nGoldman to compare proportions within and among multinomial populations. The\\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\\nhospital stay were obtained in minutes and analyzed by Student's t test or the\\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\\ntests for comparison of the moments in each group and Mann Whitney test to compare\\ngroups at each time, adopting a significance level of 5% probability of rejecting\\nnull hypothesis.\\nThis study was approved by the Research Ethics Committee of Universidade Estadual\\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\\nprotocol 3223) and all patients signed an informed consent form.\",\n \"All patients were evaluated by the same evaluator (preoperative period - PRE), third\\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\\nphysiotherapeutical treatment, according to their needs in the postoperative period\\nby physical therapy team in the hospital and only Group I received physiotherapy in\\nthe preoperative period.\\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\\nanesthesia time and the time that the patient remained with the endotracheal tube in\\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\\nICU stay (when the patient arrived in the intensive care unit until discharged to the\\nward bed) and length of stay in the OP (obtained from the time that the patient\\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\\nwere obtained by means of the daily developments of medical and nursing staff.\\nThe assessment of respiratory muscle strength was obtained by measurement of\\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\\nforceps according to the technique described by Nephew et al. (2008). The MIP\\nmeasurement was performed from residual volume, and MEP was obtained from total lung\\ncapacity. Three measurements were taken technically satisfactory, and if there were\\nmore than 20% difference between the measurements, a fourth and even fifth\\nmeasurement would be performed, being always considered the highest value obtained.\\nThe MIP was performed for evaluation of inspiratory muscle strength in three\\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\\nthe buccinator muscles during MIP maneuver[9].\\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\\nminute (cpm) and obtained by direct observation of respiratory cycles during the\\nevaluation of the minute volume[7].\",\n \"Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\\nof breathing exercises (breathing in time, deep breath and then exhaling long,\\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\\nassociated with mobilization of the upper limb, with three series out of ten for each\\nbreathing exercise) and breathing exercises with the device Threshold -\\nIMT®. We conducted three sets of ten repetitions with an interval of\\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\\nThe GII patients received only guidelines ward routine before surgery, but\\npostoperatively, both groups performed physical therapy as needed by staff\\nphysiotherapy service.\",\n \"The sample should be made with at least 26 patients who were randomly divided into\\ntwo study groups (with physical therapy preoperatively and control patients who did\\nnot receive preoperative physiotherapy). We considered the level of significance of\\n5% and a test power in the order of 80% for a minimum difference in the order of\\ntwo.\\nThe comparison between the groups for age and body mass index (BMI) was performed by\\nWilcoxon test for independent samples. The smoking variable was analyzed using\\nGoldman to compare proportions within and among multinomial populations. The\\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\\nhospital stay were obtained in minutes and analyzed by Student's t test or the\\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\\ntests for comparison of the moments in each group and Mann Whitney test to compare\\ngroups at each time, adopting a significance level of 5% probability of rejecting\\nnull hypothesis.\\nThis study was approved by the Research Ethics Committee of Universidade Estadual\\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\\nprotocol 3223) and all patients signed an informed consent form.\",\n \"Seventy patients were divided into two groups, Group I composed of 35 patients (12\\nfemale and 23 male) and Group II, with 35 patients (six female and 29 male), as\\nillustrated in Figure 1.\\nCharacterization of groups by gender\\nNo significant difference was noticed between groups for the variables age (GI 58.9±9.53\\nyears and GII 61.4±8.43 years; P=0.26) and body mass index (GI\\n26.8±3.96 kg/m2 and G II 26±3.86 kg/m2) (Table 1).\\nPatient characteristics regarding age and BMI.\\nBMI: body mass index; Wilcoxon test. * P<0.05\\nThe groups were homogenous concerning presence of active smoking, ex-smokers and\\nnonsmokers, as illustrated in Table 2.\\nNumber and proportion of patients in each group according to the presence of\\nactive smoking, ex-smokers and nonsmokers.\\nTwo proportions followed by the same lowercase letter do not differ in groups\\n(rows). Two proportions followed by the same capital letter do not differ in\\nthe types of smoking (P>0.05). Goldman test to compare proportions between\\nand within populations multinomina.\\nBetween the GI and GII, no significant difference regarding the proportion of\\nex-smokers, smokers and nonsmokers, in other words, the groups were homogeneous as\\ndemonstrated below (Table 2).\\n Maximum Inspiratory Pressure (MIP) The mean values the for maximum inspiratory pressures evaluated in the preoperative\\nperiod and 3PO showed no significant difference between groups\\n(P=0.276 and 0.065; respectively), however, we observed greater\\ninspiratory muscle strength for GI in relation to GII in 5PO\\n(P=0.001) (Figure 2).\\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\\noperation of each group analyzed (with and without physical therapy\\npreoperatively). Friedman test for comparison of moments in each group and Mann\\nWhitney test to compare the groups at each time, *P<0.05.\\nThe mean values the for maximum inspiratory pressures evaluated in the preoperative\\nperiod and 3PO showed no significant difference between groups\\n(P=0.276 and 0.065; respectively), however, we observed greater\\ninspiratory muscle strength for GI in relation to GII in 5PO\\n(P=0.001) (Figure 2).\\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\\noperation of each group analyzed (with and without physical therapy\\npreoperatively). Friedman test for comparison of moments in each group and Mann\\nWhitney test to compare the groups at each time, *P<0.05.\\n Maximum Expiratory Pressure (MEP) The mean value for the maximum expiratory pressure obtained in the preoperative\\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\\ncmH2O) for GII, not presenting significant difference between groups\\n(P=0.704). However, the values referring MEP measured in the 5PO\\nshowed significant difference between groups (P=0.001), as\\nillustrated in Figure 3.\\nMedian for maximum expiratory pressure (MEP) variable at different times of the\\noperation in the different groups. Friedman test for comparison of moments in\\neach group and Mann Whitney test to compare the groups at each time,\\n*P<0.05.\\nThe mean value for the maximum expiratory pressure obtained in the preoperative\\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\\ncmH2O) for GII, not presenting significant difference between groups\\n(P=0.704). However, the values referring MEP measured in the 5PO\\nshowed significant difference between groups (P=0.001), as\\nillustrated in Figure 3.\\nMedian for maximum expiratory pressure (MEP) variable at different times of the\\noperation in the different groups. Friedman test for comparison of moments in\\neach group and Mann Whitney test to compare the groups at each time,\\n*P<0.05.\\n Minute Volume (MV) The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\\nP=0.001). However, these values have not showed significative\\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\\nrespectively).\\nMedian minute volume (VM ) variable at different times of the operation of each\\ngroup analyzed. Friedman test for comparison of the moments in each group and\\nthe Mann Whitney test for comparison between groups at any time,\\n*P<0.05.\\nThe analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\\nP=0.001). However, these values have not showed significative\\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\\nrespectively).\\nMedian minute volume (VM ) variable at different times of the operation of each\\ngroup analyzed. Friedman test for comparison of the moments in each group and\\nthe Mann Whitney test for comparison between groups at any time,\\n*P<0.05.\\n Tidal Volume Figure 5 illustrates the analysis of variable\\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\\nvalues were not significantly different for the 3PO and 5 PO\\n(P=0.059 and P=0.549; respectively).\\nMedian tidal volume variable at different times for the group treated with\\npreoperative physiotherapy and without physical therapy preoperatively.\\nFriedman test for comparison of moments in each group and Mann Whitney test to\\ncompare the groups at each time, *P<0.05.\\nFigure 5 illustrates the analysis of variable\\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\\nvalues were not significantly different for the 3PO and 5 PO\\n(P=0.059 and P=0.549; respectively).\\nMedian tidal volume variable at different times for the group treated with\\npreoperative physiotherapy and without physical therapy preoperatively.\\nFriedman test for comparison of moments in each group and Mann Whitney test to\\ncompare the groups at each time, *P<0.05.\\n Respiratory rate (RR) The median refers to the respiratory rate obtained preoperatively for GI and GII\\nshowed no significant difference (16 cpm and 18 cpm, respectively,\\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\\nshowed no significant difference between groups (P=0.090 and\\nP=0.886, respectively), as shown in Figure 6.\\nMedian variable respiratory rate at different times of the operation of each\\ngroup analyzed (with and without physical therapy preoperatively). Friedman\\ntest for comparison of moments in each group and Mann Whitney test to compare\\nthe groups at each time, *P<0.05.\\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\\nhospital stay after surgery.\\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\\nstay did not significantly differ between groups, however, significant difference was\\nobserved for the length of stay variable in the postoperative period\\n(P=0.001), as shown in Table\\n3.\\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\\n5PO.\\nValues are shown as mean ± standard deviation or median (minimum and maximum\\nvalues).\\nMann and Whitney\\nStudent's t test.\\nSignificant difference between groups (P <0.05). ICU: intensive care\\nunit; PO: postoperative\\nThe median refers to the respiratory rate obtained preoperatively for GI and GII\\nshowed no significant difference (16 cpm and 18 cpm, respectively,\\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\\nshowed no significant difference between groups (P=0.090 and\\nP=0.886, respectively), as shown in Figure 6.\\nMedian variable respiratory rate at different times of the operation of each\\ngroup analyzed (with and without physical therapy preoperatively). Friedman\\ntest for comparison of moments in each group and Mann Whitney test to compare\\nthe groups at each time, *P<0.05.\\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\\nhospital stay after surgery.\\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\\nstay did not significantly differ between groups, however, significant difference was\\nobserved for the length of stay variable in the postoperative period\\n(P=0.001), as shown in Table\\n3.\\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\\n5PO.\\nValues are shown as mean ± standard deviation or median (minimum and maximum\\nvalues).\\nMann and Whitney\\nStudent's t test.\\nSignificant difference between groups (P <0.05). ICU: intensive care\\nunit; PO: postoperative\",\n \"The mean values the for maximum inspiratory pressures evaluated in the preoperative\\nperiod and 3PO showed no significant difference between groups\\n(P=0.276 and 0.065; respectively), however, we observed greater\\ninspiratory muscle strength for GI in relation to GII in 5PO\\n(P=0.001) (Figure 2).\\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\\noperation of each group analyzed (with and without physical therapy\\npreoperatively). Friedman test for comparison of moments in each group and Mann\\nWhitney test to compare the groups at each time, *P<0.05.\",\n \"The mean value for the maximum expiratory pressure obtained in the preoperative\\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\\ncmH2O) for GII, not presenting significant difference between groups\\n(P=0.704). However, the values referring MEP measured in the 5PO\\nshowed significant difference between groups (P=0.001), as\\nillustrated in Figure 3.\\nMedian for maximum expiratory pressure (MEP) variable at different times of the\\noperation in the different groups. Friedman test for comparison of moments in\\neach group and Mann Whitney test to compare the groups at each time,\\n*P<0.05.\",\n \"The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\\nP=0.001). However, these values have not showed significative\\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\\nrespectively).\\nMedian minute volume (VM ) variable at different times of the operation of each\\ngroup analyzed. Friedman test for comparison of the moments in each group and\\nthe Mann Whitney test for comparison between groups at any time,\\n*P<0.05.\",\n \"Figure 5 illustrates the analysis of variable\\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\\nvalues were not significantly different for the 3PO and 5 PO\\n(P=0.059 and P=0.549; respectively).\\nMedian tidal volume variable at different times for the group treated with\\npreoperative physiotherapy and without physical therapy preoperatively.\\nFriedman test for comparison of moments in each group and Mann Whitney test to\\ncompare the groups at each time, *P<0.05.\",\n \"The median refers to the respiratory rate obtained preoperatively for GI and GII\\nshowed no significant difference (16 cpm and 18 cpm, respectively,\\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\\nshowed no significant difference between groups (P=0.090 and\\nP=0.886, respectively), as shown in Figure 6.\\nMedian variable respiratory rate at different times of the operation of each\\ngroup analyzed (with and without physical therapy preoperatively). Friedman\\ntest for comparison of moments in each group and Mann Whitney test to compare\\nthe groups at each time, *P<0.05.\\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\\nhospital stay after surgery.\\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\\nstay did not significantly differ between groups, however, significant difference was\\nobserved for the length of stay variable in the postoperative period\\n(P=0.001), as shown in Table\\n3.\\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\\n5PO.\\nValues are shown as mean ± standard deviation or median (minimum and maximum\\nvalues).\\nMann and Whitney\\nStudent's t test.\\nSignificant difference between groups (P <0.05). ICU: intensive care\\nunit; PO: postoperative\",\n \"Studies by Feltrim et al.[10] show\\nthat performing preoperative physiotherapy is more effective in reducing respiratory\\ncomplications in patients with moderate or higher risk than those whose risk was\\nlow.\\nIn the present study, we observed no significant difference between the subjects of\\nGroup I and Group II as the characteristics (age, BMI, smoking and number of co\\nmorbidities), which suggests that the study subjects in both groups were\\nhomogeneous.\\nIn the study by Leguisamo et al.[9],\\nwith 86 patients undergoing CABG, divided into two groups: the intervention group (44\\npatients) who were assessed and received physiotherapeutic guidance at least 15 days\\nbefore surgery. The control group received routine care on the day of\\nhospitalization.\\nThe average MIP between groups showed neither significant difference in the preoperative\\nperiod nor in the 1PO and 6PO.\\nIn the present study it was observed that there was no significant difference between\\ngroups to measure MIP in the preoperative period, but now in times of 3PO and 5PO\\nsignificant difference between groups was found. Similarly, no significant difference in\\nthe amount of MEP preoperatively in the 3PO and between groups. However in 5PO, there\\nwas significant difference between the groups with the best values of the group that\\nperformed physical therapy before surgery.\\nBarros et al.[11] evaluated the MIP\\nand MEP in the preoperative period, the first day after surgery and in 38 patients\\nundergoing CABG with CPB who were divided into two groups: 23 patients in group I\\n(respiratory muscle training) and 15 in group II (control). Group I received\\nconventional physiotherapy over respiratory muscle training and Group II the\\nconventional physiotherapy. The authors found that the values obtained for MIP and MEP\\nin hospital discharge were higher in group I.\\nThere was no decrease in maximal respiratory pressures, transdiaphragmatic pressure and\\ndiaphragm pressure, indicating weakness of respiratory muscle strength[12,13].\\nAgreeing with the authors cited above, we observed in the present study, after cardiac\\nsurgery, MIP values in GI and GII which decreased in 3PO, however the value of MIP in\\n5PO increase was observed for GI values compared to those in PRE and 3PO since the GII\\nvalues remained stable in 3PO. The authors noted the drop in MEP in 3PO in both groups,\\nhowever, in 5PO MEP has increased in GI. Regarding PRE and 3PO in GII no return value\\nwas observed in PRE.\\nArcêncio et al.[12] conducted a study\\nwith 30 patients, aged 50 years-old and candidates for CABG and / or heart valve,\\ndividing into two groups. Intervention group comprised 15 patients who underwent at\\nleast a two weeks inspiratory muscle training using incentive spirometry (\\\"Threshold -\\nIMT®) with 40% charge of the MIP and control group with 15 patients who\\nreceived only general guidelines. The authors compared the spirometric values before and\\nafter training and showed no significant difference in the values of MIP and\\nMEP[14].\\nElias et al.[15] studied 42 patients\\naged 18 to 75 years-old with coronary artery disease, in the pre-and postoperative\\nperiod, divided into two groups. Intervention group received TMR with Threshold -\\nIMT® and control group received only guidelines. The TMR both pre and\\npost-operative period consisted of three sets of 10 inspiratory exercises once a day for\\nthree consecutive days. It was observed that after cardiac surgery there was a\\nsignificant reduction in MIP and MEP in both groups.\\nMorsch et al.[13] conducted a study to\\nevaluate 108 patients undergoing elective CABG surgery from April 2006 to February 2007\\n(preoperatively and postoperatively).\\nThe average values of MIP in the preoperative period were significantly decreased\\ncompared to 6 days after the surgery (P<0.001). The authors also\\nobserved a significant decrease in the values of MEP obtained on the 6th\\npostoperative day compared to the preoperative period (P<0.001).\\nPatients with respiratory muscle weakness have higher risk of postoperative pulmonary\\ncomplications. The respiratory muscle training in the pre-operative may prevent\\npulmonary complications in patients undergoing thoracic surgery[16]. There may be a reduction of all lung\\nvolumes resulting from factors such as diaphragmatic dysfunction, pain, absence of deep\\nbreaths, pulmonary changes and ribcage. The functional residual capacity (FRC) decreases\\nbecause of the reduction of both the residual volume (RV) and expiratory reserve volume.\\nThe ventilation is affected by VC reduction by approximately 20%, and the increase in\\nFR(22-24). The sum of these factors cause changes in the mechanics of\\nrespiration, which leads to a shallow breathing pattern of decreased lung\\nvolume[13].\\nPatients undergoing CABG develop mostly PO pulmonary dysfunction with a significant\\nreduction in lung volumes, impairments in respiratory function, decreased lung\\ncompliance and increased work of breathing. The reduction in lung volumes and capacities\\ncontributes to changes in gas exchange, resulting in hypoxemia[17].\\nIn this study, the VM showed significant difference regarding the groups I and II in the\\npreoperative period but not significant between groups in 3PO. Likewise, the mean value\\nof the VM for the GI 5PO, there was no significant difference in the GII. The mean value\\nof VC in PRE in GI was significant, as the mean value of the VC in GII patients, however\\nin GI and GII we observed in 5PO and 3PO that there was no significant difference. It\\nwas observed in GI at different times that the increase of VM will be very likely due to\\nincreased FR, since the current VC remained without many changes between different\\ntimes.\\nBarros et al.[11] demonstrated a\\nsignificant decrease in lung volumes in patients undergoing cardiac surgery in the\\npostoperative period.\\nCarvalho et al.[18] observed a\\ndecrease in the values of minute volume and tidal volume measured on the 2nd\\npostoperative day, however, these values showed gradual improvement returning to the\\nvalues obtained at baseline (preoperative period) at the time of hospital discharge.\\nIn a study, Guizilini et al.[19]\\nevaluated 30 patients with a mean age of 56 years and divided into two groups, group A\\n(n=15) without CPB and group B (n=15) with CPB. All patients underwent pulmonary\\nfunction. In both groups, there was a decrease in FVC until the fifth postoperative day\\n(P<0.05).\\nRegarding the respiratory rate in the present study, we can verify that there was an\\nincrease in both groups, with a higher peak on the 3rd postoperative day and\\ndecreased on the 6th postoperative day, but the respiratory rate did not\\nreturn to the preoperative values, Carvalho et al.[18] already mentioned above that they concluded in their studies\\nthat the mean respiratory frequency had a significant increase between pre and\\n2nd PO, 3rd PO, decreasing on the 4th postoperative\\nday and increased again on the 5th postoperative day and decreased in\\nhospital discharge, but patients did not return to the preoperative values.\\nAccording to the results obtained in this study, we can affirm that there was no\\nsignificant difference between the groups regarding the time variables: anesthesia,\\nsurgery, perfusion, anoxia, mechanical ventilation, intubation. This suggests that the\\ngroups were quite homogeneous. As for the time of postoperative hospital stay, there was\\na decrease of approximately 25 hours, comparing patients who underwent physical therapy\\nbefore surgery with patients who did not.\\nIn contrast to the findings of this study, Patman et al.[20] performed a randomized study of 236 patients to\\nevaluate the effects of physical therapy interventions performed early versus only\\nstarted after extubation in patients undergoing cardiac surgery and no significant\\ndifferences were found in the time of intubation, ICU stay and length of hospital stay\\nbetween the groups.\\nHowever, Celli et al.[21] conducted a\\nstudy with 172 patients and showed a decrease in length of hospital stay in the group\\nthat had received guidelines breathing exercises (9.6±3.2 days) compared to the control\\ngroup (13±5 days).\\nCorroborating these findings, as well as those obtained in the present study, Leguisamo\\net al.[9] performed a prospective\\nstudy to evaluate the effectiveness of a physiotherapy program start in the preoperative\\nperiod in reducing the length of hospital stay and showed a significant reduction in\\nlength of hospital stay for the intervention group compared to the control group\\n(P<0.05).\\nThese data suggest that physical therapy initiated before surgery can improve conditions\\nof patients, reestablishing early respiratory pressures, reducing the length of stay and\\ndecreased hospital costs for the period of hospitalization.\\n Limitation of the study The major limitation was the short period of time that patients received preoperative\\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\\nrelevance even in a short period, so the study may add knowledge of response to\\ntherapy performed during intraoperative CABG surgery.\\nNot performing spirometry can be mentioned as a limitation on the analysis of\\nventilation parameters, however, due to the conditions offered by our service, we\\ncould not perform this evaluation.\\nOn the other hand, the results showed that even with limited time monitoring in the\\npreoperative period and respiratory variables obtained only by respirometry and\\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\\npatients undergoing surgery CABG.\\nWe understand that the results may open up new avenues of future research related to\\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\\nfurther studies should be conducted using tools such as spirometry, to assess and\\nquantify the volume and lung capacity more accurately.\\nThe major limitation was the short period of time that patients received preoperative\\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\\nrelevance even in a short period, so the study may add knowledge of response to\\ntherapy performed during intraoperative CABG surgery.\\nNot performing spirometry can be mentioned as a limitation on the analysis of\\nventilation parameters, however, due to the conditions offered by our service, we\\ncould not perform this evaluation.\\nOn the other hand, the results showed that even with limited time monitoring in the\\npreoperative period and respiratory variables obtained only by respirometry and\\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\\npatients undergoing surgery CABG.\\nWe understand that the results may open up new avenues of future research related to\\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\\nfurther studies should be conducted using tools such as spirometry, to assess and\\nquantify the volume and lung capacity more accurately.\",\n \"The major limitation was the short period of time that patients received preoperative\\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\\nrelevance even in a short period, so the study may add knowledge of response to\\ntherapy performed during intraoperative CABG surgery.\\nNot performing spirometry can be mentioned as a limitation on the analysis of\\nventilation parameters, however, due to the conditions offered by our service, we\\ncould not perform this evaluation.\\nOn the other hand, the results showed that even with limited time monitoring in the\\npreoperative period and respiratory variables obtained only by respirometry and\\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\\npatients undergoing surgery CABG.\\nWe understand that the results may open up new avenues of future research related to\\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\\nfurther studies should be conducted using tools such as spirometry, to assess and\\nquantify the volume and lung capacity more accurately.\",\n \"The physical therapy has an important role in the preoperative period, restoring greater\\nreadiness ventilatory parameters of patients undergoing coronary artery bypass grafting\\nwith cardiopulmonary bypass, resulting in a decrease in length of hospital stay after\\nsurgery.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,"methods",null,null,null,"results",null,null,null,null,null,"discussion",null,"conclusions"],"string":"[\n \"intro\",\n null,\n \"methods\",\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["Thoracic Surgery","Breathing Exercises","Physical Therapy (Specialty)"],"string":"[\n \"Thoracic Surgery\",\n \"Breathing Exercises\",\n \"Physical Therapy (Specialty)\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nThe cardiac surgery was always clothed with great interest, curiosity, and in some\nmoments, mysticism, and fact understandable when we analyzed the noble importance of\nthis component for the proper functioning of the human body[1].\nIn recent decades the frequency of surgical procedures has progressively increased,\namong them the myocardial revascularization (MR). Cardiovascular diseases are a serious\npublic health problem in Brazil[2].\nIn individuals over 70 years old it is estimated an incidence of 70% of coronary artery\ndisease. As the age is a determinant of coronary atherosclerosis, a growing number of\nelderly people will be subjected to MR and/or other therapeutic methods[3,4].\nThe main risk factors that contribute to cardiac diseases are: smoking, high levels of\nlow-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density\nlipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,\nfamily history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors\nrelated to atherosclerosis and its clinical manifestations[4,5].\nThe coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment\nof ischemic heart disease that is manifested clinically by angina and acute myocardial\ninfarction. The CABG surgery creates a new route for the blood flow and, is used for\nmaking a diversion of blood to the portions of the coronary artery distal to the\nobstructive atherosclerotic involvement[1,5].\nWith the advancements in care of physical therapy in the preoperative period, surgical\ntechniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,\nanesthesia and intensive care in the post-operative period, there was a decrease in\nmorbidity and mortality of MR which caused the surgical indication in groups of patients\nincreasingly complex[6,7].\nObserving the fact that they are common pulmonary complications of CABG with CPB was\njustified to carry out this research, there is great importance in properly carrying out\nevaluations, guidelines and breathing exercises in the period before surgery, and\naddition, observe the influence and duration of physical therapy assistance in the\npreoperative period and also its behavior in the postoperative period.\n Objective To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\nTo demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\n\nObjective:\nTo demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\n\nMETHODS:\nWe performed a prospective clinical study, simple blind, in the period from July 2009 to\nSeptember 2011 in the ward of cardiac and thoracic surgery at Hospital das Clínicas da\nUniversidade Estadual Paulista (UNESP), Botucatu-SP. We evaluated 70 patients of both\ngender, age ranging from 40 to 75 years, undergoing CABG with use of CPB, randomized by\nmeans of a computerized software (STATTREK) and delivered in a sealed envelope to the\nphysical therapist responsible for primary health care, subdividing into two groups:\n- Group I (GI) - Thirty-five patients, undergoing physiotherapeutic intervention,\nperformed under supervision, once a day, during the period that preceded the surgery.\nThe protocol consisted of breathing exercises (breathing in time, deep breathing\nfollowed by prolonged expiration, sustained maximal inspiration with apnea of 6 seconds,\nand diaphragmatic breathing associated with the mobilization of the upper limbs) and\nbreathing exercises with threshold - IMT® (Threshold - IMT®\nInspiratory Muscle Trainer, HealthscanProducts Inc.) at an intensity of 40% of the\ninitial MIP with three sets of ten repetitions, respecting two-minute intervals between\neach series.\n- Group II (GII) - thirty-five patients who did not undergo the physical therapy\nprotocol during the preoperative period, receiving only guidelines ward routine.\nWe included patients of both sexes, aged 40-75 years with coronary artery disease\nadmitted for cardiac surgery with cardiopulmonary bypass (CPB) MR, who agreed to\nparticipate in the study. We excluded patients who require the use of intra-aortic\nballoon, carrying pulmonary pathology, musculoskeletal impairment and severe\nneurological surgery performed without CPB, emergency surgery, homodynamic instability,\nand any event that threatens the integrity of patient or the fidelity of the measures\nanalyzed.\n Delimitation All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\nAll patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\n Procedures/treatment Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\nPatients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\n Statistical Analysis The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.\nThe sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.\n\nDelimitation:\nAll patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\n\nProcedures/treatment:\nPatients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\n\nStatistical Analysis:\nThe sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.\n\nRESULTS:\nSeventy patients were divided into two groups, Group I composed of 35 patients (12\nfemale and 23 male) and Group II, with 35 patients (six female and 29 male), as\nillustrated in Figure 1.\nCharacterization of groups by gender\nNo significant difference was noticed between groups for the variables age (GI 58.9±9.53\nyears and GII 61.4±8.43 years; P=0.26) and body mass index (GI\n26.8±3.96 kg/m2 and G II 26±3.86 kg/m2) (Table 1).\nPatient characteristics regarding age and BMI.\nBMI: body mass index; Wilcoxon test. * P<0.05\nThe groups were homogenous concerning presence of active smoking, ex-smokers and\nnonsmokers, as illustrated in Table 2.\nNumber and proportion of patients in each group according to the presence of\nactive smoking, ex-smokers and nonsmokers.\nTwo proportions followed by the same lowercase letter do not differ in groups\n(rows). Two proportions followed by the same capital letter do not differ in\nthe types of smoking (P>0.05). Goldman test to compare proportions between\nand within populations multinomina.\nBetween the GI and GII, no significant difference regarding the proportion of\nex-smokers, smokers and nonsmokers, in other words, the groups were homogeneous as\ndemonstrated below (Table 2).\n Maximum Inspiratory Pressure (MIP) The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\nThe mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\n Maximum Expiratory Pressure (MEP) The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\nThe mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\n Minute Volume (MV) The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\nThe analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\n Tidal Volume Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\nFigure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\n Respiratory rate (RR) The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative\nThe median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative\n\nMaximum Inspiratory Pressure (MIP):\nThe mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\n\nMaximum Expiratory Pressure (MEP):\nThe mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\n\nMinute Volume (MV):\nThe analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\n\nTidal Volume:\nFigure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\n\nRespiratory rate (RR):\nThe median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative\n\nDISCUSSION:\nStudies by Feltrim et al.[10] show\nthat performing preoperative physiotherapy is more effective in reducing respiratory\ncomplications in patients with moderate or higher risk than those whose risk was\nlow.\nIn the present study, we observed no significant difference between the subjects of\nGroup I and Group II as the characteristics (age, BMI, smoking and number of co\nmorbidities), which suggests that the study subjects in both groups were\nhomogeneous.\nIn the study by Leguisamo et al.[9],\nwith 86 patients undergoing CABG, divided into two groups: the intervention group (44\npatients) who were assessed and received physiotherapeutic guidance at least 15 days\nbefore surgery. The control group received routine care on the day of\nhospitalization.\nThe average MIP between groups showed neither significant difference in the preoperative\nperiod nor in the 1PO and 6PO.\nIn the present study it was observed that there was no significant difference between\ngroups to measure MIP in the preoperative period, but now in times of 3PO and 5PO\nsignificant difference between groups was found. Similarly, no significant difference in\nthe amount of MEP preoperatively in the 3PO and between groups. However in 5PO, there\nwas significant difference between the groups with the best values of the group that\nperformed physical therapy before surgery.\nBarros et al.[11] evaluated the MIP\nand MEP in the preoperative period, the first day after surgery and in 38 patients\nundergoing CABG with CPB who were divided into two groups: 23 patients in group I\n(respiratory muscle training) and 15 in group II (control). Group I received\nconventional physiotherapy over respiratory muscle training and Group II the\nconventional physiotherapy. The authors found that the values obtained for MIP and MEP\nin hospital discharge were higher in group I.\nThere was no decrease in maximal respiratory pressures, transdiaphragmatic pressure and\ndiaphragm pressure, indicating weakness of respiratory muscle strength[12,13].\nAgreeing with the authors cited above, we observed in the present study, after cardiac\nsurgery, MIP values in GI and GII which decreased in 3PO, however the value of MIP in\n5PO increase was observed for GI values compared to those in PRE and 3PO since the GII\nvalues remained stable in 3PO. The authors noted the drop in MEP in 3PO in both groups,\nhowever, in 5PO MEP has increased in GI. Regarding PRE and 3PO in GII no return value\nwas observed in PRE.\nArcêncio et al.[12] conducted a study\nwith 30 patients, aged 50 years-old and candidates for CABG and / or heart valve,\ndividing into two groups. Intervention group comprised 15 patients who underwent at\nleast a two weeks inspiratory muscle training using incentive spirometry (\"Threshold -\nIMT®) with 40% charge of the MIP and control group with 15 patients who\nreceived only general guidelines. The authors compared the spirometric values before and\nafter training and showed no significant difference in the values of MIP and\nMEP[14].\nElias et al.[15] studied 42 patients\naged 18 to 75 years-old with coronary artery disease, in the pre-and postoperative\nperiod, divided into two groups. Intervention group received TMR with Threshold -\nIMT® and control group received only guidelines. The TMR both pre and\npost-operative period consisted of three sets of 10 inspiratory exercises once a day for\nthree consecutive days. It was observed that after cardiac surgery there was a\nsignificant reduction in MIP and MEP in both groups.\nMorsch et al.[13] conducted a study to\nevaluate 108 patients undergoing elective CABG surgery from April 2006 to February 2007\n(preoperatively and postoperatively).\nThe average values of MIP in the preoperative period were significantly decreased\ncompared to 6 days after the surgery (P<0.001). The authors also\nobserved a significant decrease in the values of MEP obtained on the 6th\npostoperative day compared to the preoperative period (P<0.001).\nPatients with respiratory muscle weakness have higher risk of postoperative pulmonary\ncomplications. The respiratory muscle training in the pre-operative may prevent\npulmonary complications in patients undergoing thoracic surgery[16]. There may be a reduction of all lung\nvolumes resulting from factors such as diaphragmatic dysfunction, pain, absence of deep\nbreaths, pulmonary changes and ribcage. The functional residual capacity (FRC) decreases\nbecause of the reduction of both the residual volume (RV) and expiratory reserve volume.\nThe ventilation is affected by VC reduction by approximately 20%, and the increase in\nFR(22-24). The sum of these factors cause changes in the mechanics of\nrespiration, which leads to a shallow breathing pattern of decreased lung\nvolume[13].\nPatients undergoing CABG develop mostly PO pulmonary dysfunction with a significant\nreduction in lung volumes, impairments in respiratory function, decreased lung\ncompliance and increased work of breathing. The reduction in lung volumes and capacities\ncontributes to changes in gas exchange, resulting in hypoxemia[17].\nIn this study, the VM showed significant difference regarding the groups I and II in the\npreoperative period but not significant between groups in 3PO. Likewise, the mean value\nof the VM for the GI 5PO, there was no significant difference in the GII. The mean value\nof VC in PRE in GI was significant, as the mean value of the VC in GII patients, however\nin GI and GII we observed in 5PO and 3PO that there was no significant difference. It\nwas observed in GI at different times that the increase of VM will be very likely due to\nincreased FR, since the current VC remained without many changes between different\ntimes.\nBarros et al.[11] demonstrated a\nsignificant decrease in lung volumes in patients undergoing cardiac surgery in the\npostoperative period.\nCarvalho et al.[18] observed a\ndecrease in the values of minute volume and tidal volume measured on the 2nd\npostoperative day, however, these values showed gradual improvement returning to the\nvalues obtained at baseline (preoperative period) at the time of hospital discharge.\nIn a study, Guizilini et al.[19]\nevaluated 30 patients with a mean age of 56 years and divided into two groups, group A\n(n=15) without CPB and group B (n=15) with CPB. All patients underwent pulmonary\nfunction. In both groups, there was a decrease in FVC until the fifth postoperative day\n(P<0.05).\nRegarding the respiratory rate in the present study, we can verify that there was an\nincrease in both groups, with a higher peak on the 3rd postoperative day and\ndecreased on the 6th postoperative day, but the respiratory rate did not\nreturn to the preoperative values, Carvalho et al.[18] already mentioned above that they concluded in their studies\nthat the mean respiratory frequency had a significant increase between pre and\n2nd PO, 3rd PO, decreasing on the 4th postoperative\nday and increased again on the 5th postoperative day and decreased in\nhospital discharge, but patients did not return to the preoperative values.\nAccording to the results obtained in this study, we can affirm that there was no\nsignificant difference between the groups regarding the time variables: anesthesia,\nsurgery, perfusion, anoxia, mechanical ventilation, intubation. This suggests that the\ngroups were quite homogeneous. As for the time of postoperative hospital stay, there was\na decrease of approximately 25 hours, comparing patients who underwent physical therapy\nbefore surgery with patients who did not.\nIn contrast to the findings of this study, Patman et al.[20] performed a randomized study of 236 patients to\nevaluate the effects of physical therapy interventions performed early versus only\nstarted after extubation in patients undergoing cardiac surgery and no significant\ndifferences were found in the time of intubation, ICU stay and length of hospital stay\nbetween the groups.\nHowever, Celli et al.[21] conducted a\nstudy with 172 patients and showed a decrease in length of hospital stay in the group\nthat had received guidelines breathing exercises (9.6±3.2 days) compared to the control\ngroup (13±5 days).\nCorroborating these findings, as well as those obtained in the present study, Leguisamo\net al.[9] performed a prospective\nstudy to evaluate the effectiveness of a physiotherapy program start in the preoperative\nperiod in reducing the length of hospital stay and showed a significant reduction in\nlength of hospital stay for the intervention group compared to the control group\n(P<0.05).\nThese data suggest that physical therapy initiated before surgery can improve conditions\nof patients, reestablishing early respiratory pressures, reducing the length of stay and\ndecreased hospital costs for the period of hospitalization.\n Limitation of the study The major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.\nThe major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.\n\nLimitation of the study:\nThe major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.\n\nCONCLUSIONS:\nThe physical therapy has an important role in the preoperative period, restoring greater\nreadiness ventilatory parameters of patients undergoing coronary artery bypass grafting\nwith cardiopulmonary bypass, resulting in a decrease in length of hospital stay after\nsurgery.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe frequency of surgical procedures has increased steadily in recent decades, including the myocardial revascularization.\n\nMethods:\nWe conducted a prospective study with patients undergoing myocardial revascularization, the Hospital das Clínicas da Universidade Estadual Paulista (UNESP)/Botucatu - SP. We evaluated 70 patients of both genders, aged between 40 and 75 years, subdivided into two groups: group I - 35 patients of both genders, who received a written protocol guidance, breathing exercises and respiratory muscle training in the preoperative period and group II - 35 patients of both genders, who received only orientation of the ward on the day of surgery. This study was approved by the Ethics Committee of UNESP / Botucatu - SP.\n\nResults:\nMaximal inspiratory pressure in third postoperative day and fifth postoperative day and significant difference between groups, being better for the intervention group. Expiratory pressure was significant in fifth postoperative day in the intervention group compared to controls. The difference of length of hospital stay in the postoperative was found between the groups with shorter hospital stay in the group receiving preoperative therapy.\n\nConclusions:\nPhysical therapy plays an important role in the preoperative period, so that individuals in the intervention group more readily restored the parameters evaluated before surgery, in addition, there was a decrease in the time of the postoperative hospital stay. Thus, it is thought the cost-effectiveness of a program of preoperative physiotherapy."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nThe cardiac surgery was always clothed with great interest, curiosity, and in some\nmoments, mysticism, and fact understandable when we analyzed the noble importance of\nthis component for the proper functioning of the human body[1].\nIn recent decades the frequency of surgical procedures has progressively increased,\namong them the myocardial revascularization (MR). Cardiovascular diseases are a serious\npublic health problem in Brazil[2].\nIn individuals over 70 years old it is estimated an incidence of 70% of coronary artery\ndisease. As the age is a determinant of coronary atherosclerosis, a growing number of\nelderly people will be subjected to MR and/or other therapeutic methods[3,4].\nThe main risk factors that contribute to cardiac diseases are: smoking, high levels of\nlow-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density\nlipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,\nfamily history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors\nrelated to atherosclerosis and its clinical manifestations[4,5].\nThe coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment\nof ischemic heart disease that is manifested clinically by angina and acute myocardial\ninfarction. The CABG surgery creates a new route for the blood flow and, is used for\nmaking a diversion of blood to the portions of the coronary artery distal to the\nobstructive atherosclerotic involvement[1,5].\nWith the advancements in care of physical therapy in the preoperative period, surgical\ntechniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,\nanesthesia and intensive care in the post-operative period, there was a decrease in\nmorbidity and mortality of MR which caused the surgical indication in groups of patients\nincreasingly complex[6,7].\nObserving the fact that they are common pulmonary complications of CABG with CPB was\njustified to carry out this research, there is great importance in properly carrying out\nevaluations, guidelines and breathing exercises in the period before surgery, and\naddition, observe the influence and duration of physical therapy assistance in the\npreoperative period and also its behavior in the postoperative period.\n Objective To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\nTo demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\n\nCONCLUSIONS:\nThe physical therapy has an important role in the preoperative period, restoring greater\nreadiness ventilatory parameters of patients undergoing coronary artery bypass grafting\nwith cardiopulmonary bypass, resulting in a decrease in length of hospital stay after\nsurgery."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe frequency of surgical procedures has increased steadily in recent decades, including the myocardial revascularization.\n\nMethods:\nWe conducted a prospective study with patients undergoing myocardial revascularization, the Hospital das Clínicas da Universidade Estadual Paulista (UNESP)/Botucatu - SP. We evaluated 70 patients of both genders, aged between 40 and 75 years, subdivided into two groups: group I - 35 patients of both genders, who received a written protocol guidance, breathing exercises and respiratory muscle training in the preoperative period and group II - 35 patients of both genders, who received only orientation of the ward on the day of surgery. This study was approved by the Ethics Committee of UNESP / Botucatu - SP.\n\nResults:\nMaximal inspiratory pressure in third postoperative day and fifth postoperative day and significant difference between groups, being better for the intervention group. Expiratory pressure was significant in fifth postoperative day in the intervention group compared to controls. The difference of length of hospital stay in the postoperative was found between the groups with shorter hospital stay in the group receiving preoperative therapy.\n\nConclusions:\nPhysical therapy plays an important role in the preoperative period, so that individuals in the intervention group more readily restored the parameters evaluated before surgery, in addition, there was a decrease in the time of the postoperative hospital stay. Thus, it is thought the cost-effectiveness of a program of preoperative physiotherapy."},"whole_article_text_length":{"kind":"number","value":8719,"string":"8,719"},"whole_article_abstract_length":{"kind":"number","value":265,"string":"265"},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["groups","surgery","time","patients","period","preoperative","group","obtained","test","significant"],"string":"[\n \"groups\",\n \"surgery\",\n \"time\",\n \"patients\",\n \"period\",\n \"preoperative\",\n \"group\",\n \"obtained\",\n \"test\",\n \"significant\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cholesterol | importance | surgery | surgical | cardiac | coronary | period | duration | mr | myocardial [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] breathing | patients | performed | patient | obtained | mip | time | minute | pre | threshold [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | test | significant | significant difference | difference | group | values | maximum | median | variable [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] bypass | coronary artery bypass grafting | grafting cardiopulmonary bypass resulting | undergoing coronary | role preoperative period restoring | grafting | grafting cardiopulmonary | grafting cardiopulmonary bypass | ventilatory parameters patients undergoing | undergoing coronary artery bypass [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | test | surgery | period | group | patients | significant | time | preoperative | difference [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | test | surgery | period | group | patients | significant | time | preoperative | difference [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] recent decades [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Estadual Paulista | UNESP)/Botucatu - SP ||| 70 | between 40 and 75 years | two | II - 35 | the day ||| the Ethics Committee of UNESP / Botucatu - SP [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] third postoperative day | fifth ||| fifth ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] recent decades ||| Estadual Paulista | UNESP)/Botucatu - SP ||| 70 | between 40 and 75 years | two | II - 35 | the day ||| the Ethics Committee of UNESP / Botucatu - SP ||| ||| third postoperative day | fifth ||| fifth ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] recent decades ||| Estadual Paulista | UNESP)/Botucatu - SP ||| 70 | between 40 and 75 years | two | II - 35 | the day ||| the Ethics Committee of UNESP / Botucatu - SP ||| ||| third postoperative day | fifth ||| fifth ||| ||| ||| [SUMMARY]"}}},{"rowIdx":285,"cells":{"title":{"kind":"string","value":"DNA Methylation status of Wnt antagonist SFRP5 can predict the response to the EGFR-tyrosine kinase inhibitor therapy in non-small cell lung cancer."},"pmid":{"kind":"string","value":"23009178"},"background_abstract":{"kind":"string","value":"It is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Tumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"We found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Patients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adaptor Proteins, Signal Transducing","Aged","Biomarkers, Pharmacological","Carcinoma, Non-Small-Cell Lung","DNA Methylation","Disease-Free Survival","ErbB Receptors","Eye Proteins","Female","Humans","Male","Membrane Proteins","Middle Aged","Mutation","Neoplasm Staging","Protein Kinase Inhibitors","Treatment Outcome","Wnt Signaling Pathway"],"string":"[\n \"Adaptor Proteins, Signal Transducing\",\n \"Aged\",\n \"Biomarkers, Pharmacological\",\n \"Carcinoma, Non-Small-Cell Lung\",\n \"DNA Methylation\",\n \"Disease-Free Survival\",\n \"ErbB Receptors\",\n \"Eye Proteins\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Membrane Proteins\",\n \"Middle Aged\",\n \"Mutation\",\n \"Neoplasm Staging\",\n \"Protein Kinase Inhibitors\",\n \"Treatment Outcome\",\n \"Wnt Signaling Pathway\"\n]"},"pmcid":{"kind":"string","value":"3524045"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Lung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Patients 155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n DNA extraction and methylation-specific PCR Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\nGenomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\n Mutation detection The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\nThe denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\n Statistical analysis All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\nAll data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Characteristics of study patients Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\nTable\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\n Epigenotype of Wnt antagonists in NSCLC Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\nGenomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\n Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\nThe RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\n Epigenotype of Wnt antagonists and progression-free survival (PFS) We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\nWe next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\n Epigenotype of Wnt antagonists and overall survival rate (OS) To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\nTo test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\n Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\nIn order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development\n[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC."},"other_sections_titles":{"kind":"list like","value":["Background","Patients","DNA extraction and methylation-specific PCR","Mutation detection","Statistical analysis","Characteristics of study patients","Epigenotype of Wnt antagonists in NSCLC","Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy","Epigenotype of Wnt antagonists and progression-free survival (PFS)","Epigenotype of Wnt antagonists and overall survival rate (OS)","Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy","Abbreviations","Competing interests","Authors’ contributions","Authors’ informations"],"string":"[\n \"Background\",\n \"Patients\",\n \"DNA extraction and methylation-specific PCR\",\n \"Mutation detection\",\n \"Statistical analysis\",\n \"Characteristics of study patients\",\n \"Epigenotype of Wnt antagonists in NSCLC\",\n \"Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy\",\n \"Epigenotype of Wnt antagonists and progression-free survival (PFS)\",\n \"Epigenotype of Wnt antagonists and overall survival rate (OS)\",\n \"Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Authors’ informations\"\n]"},"other_sections_texts":{"kind":"list like","value":["Lung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.","155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.","Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.","The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].","All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.","Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.","Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.","The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).","We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).","To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n","In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.","EGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.","The authors declare that they have no competing interests.","JZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.","Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22)."],"string":"[\n \"Lung cancer is the leading cause of cancer death worldwide\\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\\n[7-11].\\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\\n[10,16,17]. In addition, previous studies reported that both T790M mutation\\n[18] and c-MET amplification\\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.\",\n \"155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\",\n \"Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\",\n \"The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\\n[28].\",\n \"All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\",\n \"Table\\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\\nMethylation and mutation profile of NSCLC\\n*The frequency of this group is significantly higher than their counterparts.\",\n \"Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\\n2).\\nP value among methylated genes and EGFR mutation\\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \\n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\",\n \"The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\",\n \"We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \\n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \\n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\\n\\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), different genotype of \\n\\nEGFR \\n\\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\\n\\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\\nSimilar to the previous discovery\\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \\n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \\n2D).\",\n \"To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \\n3B), while the epigenotypes of SFRP5 (Figure \\n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \\n3C) were not associated with OS in our patients.\\n\\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), or different genotype of \\n\\nEGFR \\n\\n(C).\\n\",\n \"In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\",\n \"EGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.\",\n \"The authors declare that they have no competing interests.\",\n \"JZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.\",\n \"Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Patients","DNA extraction and methylation-specific PCR","Mutation detection","Statistical analysis","Results","Characteristics of study patients","Epigenotype of Wnt antagonists in NSCLC","Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy","Epigenotype of Wnt antagonists and progression-free survival (PFS)","Epigenotype of Wnt antagonists and overall survival rate (OS)","Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions","Authors’ informations","Supplementary Material"],"string":"[\n \"Background\",\n \"Methods\",\n \"Patients\",\n \"DNA extraction and methylation-specific PCR\",\n \"Mutation detection\",\n \"Statistical analysis\",\n \"Results\",\n \"Characteristics of study patients\",\n \"Epigenotype of Wnt antagonists in NSCLC\",\n \"Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy\",\n \"Epigenotype of Wnt antagonists and progression-free survival (PFS)\",\n \"Epigenotype of Wnt antagonists and overall survival rate (OS)\",\n \"Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Authors’ informations\",\n \"Supplementary Material\"\n]"},"all_sections_texts":{"kind":"list like","value":["Lung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists."," Patients 155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n DNA extraction and methylation-specific PCR Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\nGenomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\n Mutation detection The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\nThe denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\n Statistical analysis All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\nAll data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.","155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.","Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.","The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].","All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant."," Characteristics of study patients Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\nTable\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\n Epigenotype of Wnt antagonists in NSCLC Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\nGenomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\n Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\nThe RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\n Epigenotype of Wnt antagonists and progression-free survival (PFS) We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\nWe next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\n Epigenotype of Wnt antagonists and overall survival rate (OS) To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\nTo test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\n Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\nIn order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.","Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.","Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.","The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).","We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).","To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n","In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.","Recent studies have demonstrated that cancer is as much an epigenetic disease as it is a genetic disease (Iacobuzio-Donahue). Therefore, in addition to genetic alterations, changes in epigenetic features such as CpG DNA methylation status of specific gene loci also mark the progress of cancers. Our current study showed that methylation of Wnt antagonist SFRP5 gene before treatment, independent of the genotype of EGFR gene, correlated with decreased progression free survival rate in NSCLC patients in response to the EGFR-TKI therapy. To our knowledge, this is the first report indicating that DNA methylation at specific gene loci in patient may predict drug response to the EGFT-TKI therapy.\nBoth genetic and epigenetic risk factors for NSCLC have been studied extensively. Suzuki et al\n[23] has reported that methylation of the Wnt antagonist DKK3 correlated with low survival rate in NSCLC patients, despite of the different therapies patients received. However, in our study, we did not find significant difference in the EGFR-TKI responses between patient groups with or without methylated DKK3 (Additional file\n1: Figure S2 and S3). In contrast, our results suggested epigenotype of SFRP5 provide better prognostic estimation for the EGFR-TKI response, comparing to other Wnt antagonists.\nSFRP5 is a member of the SFRP protein family containing a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins. It acts as soluble antagonist of Wnt signaling and is highly expressed in the retinal pigment epithelium, and moderately expressed in the pancreas (\"Entrez Gene: SFRP5 secreted frizzled-related protein 5\"). Previous studies has identified association of SFRP5 promoter hypermethylation with Acute myeloid leukemia\n[29], ovarian cancer\n[30], gastric cancer\n[31], oral squamous cell carcinoma\n[32], pancreatic cancer\n[33] and breast cancer\n[34].\nWe found that hypermethylation of SFRP5 predicted worse outcomes of the EGFR-TKI therapy. Therefore, SFRP5 DNA methylation status may serve as a prognostic molecular marker for appropriately predicting whether NSCLC patients would benefit from the EGFR-TKI therapy. Especially, it is interesting that in the subgroup with adenocarcinoma and EGFR mutation, patients with sFRP5 methylation have a significantly shorter PFS than those without sFRP5 methylation, While in nonsmokers without EGFR mutation, patients without sFRP1 methylation have a longer PFS compared with patients with its methylation(9.7 ms vs 2.0 ms, p = 0.05). Based on these results, we can make a hypothesis that activation of Wnt signaling by antagonist methylation could confer tumors the characters of stem cell, which consequently causes tumors resistant to EGFR TKIs therapy by generating acquired resistance, such as MET amplification or changes of PTEN tumor suppressor activity and so on. Further study is needed to validate this hypothesis.","In conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development\n[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC.","EGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.","The authors declare that they have no competing interests.","JZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.","Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22).","Figure S1. Methylated and unmethyalted bands of Wnt antagonist genes and wild/mutant EGFR. S1: The example graphs of methylated and unmethyalted bands of Wnt antagonist genes (A) and EGFR wild (B) and mutation types (C, D) by methylation specific PCR and DHPLC respectively. Figure S2 PFS with different epigenotypes of Wnt antagonist genes. Figure2S A-F.Kaplan-Meier curves of comparing the progression free survival of patients with different epigenotypes of SFRP1(A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F). Figure S3 OS with different epigenotypes of Wnt antagonist genes. Figure3S A-F. Kaplan-Meier curves of comparing the overall survival of patients with different epigenotypes of SFRP1 (A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F).\nClick here for file"],"string":"[\n \"Lung cancer is the leading cause of cancer death worldwide\\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\\n[7-11].\\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\\n[10,16,17]. In addition, previous studies reported that both T790M mutation\\n[18] and c-MET amplification\\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.\",\n \" Patients 155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\\n155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\\n DNA extraction and methylation-specific PCR Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\\nGenomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\\n Mutation detection The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\\n[28].\\nThe denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\\n[28].\\n Statistical analysis All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\\nAll data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\",\n \"155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\",\n \"Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\",\n \"The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\\n[28].\",\n \"All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\",\n \" Characteristics of study patients Table\\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\\nMethylation and mutation profile of NSCLC\\n*The frequency of this group is significantly higher than their counterparts.\\nTable\\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\\nMethylation and mutation profile of NSCLC\\n*The frequency of this group is significantly higher than their counterparts.\\n Epigenotype of Wnt antagonists in NSCLC Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\\n2).\\nP value among methylated genes and EGFR mutation\\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \\n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\\nGenomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\\n2).\\nP value among methylated genes and EGFR mutation\\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \\n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\\n Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\\nThe RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\\n Epigenotype of Wnt antagonists and progression-free survival (PFS) We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \\n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \\n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\\n\\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), different genotype of \\n\\nEGFR \\n\\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\\n\\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\\nSimilar to the previous discovery\\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \\n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \\n2D).\\nWe next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \\n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \\n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\\n\\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), different genotype of \\n\\nEGFR \\n\\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\\n\\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\\nSimilar to the previous discovery\\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \\n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \\n2D).\\n Epigenotype of Wnt antagonists and overall survival rate (OS) To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \\n3B), while the epigenotypes of SFRP5 (Figure \\n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \\n3C) were not associated with OS in our patients.\\n\\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), or different genotype of \\n\\nEGFR \\n\\n(C).\\n\\nTo test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \\n3B), while the epigenotypes of SFRP5 (Figure \\n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \\n3C) were not associated with OS in our patients.\\n\\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), or different genotype of \\n\\nEGFR \\n\\n(C).\\n\\n Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\\nIn order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\",\n \"Table\\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\\nMethylation and mutation profile of NSCLC\\n*The frequency of this group is significantly higher than their counterparts.\",\n \"Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\\n2).\\nP value among methylated genes and EGFR mutation\\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \\n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\",\n \"The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\",\n \"We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \\n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \\n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\\n\\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), different genotype of \\n\\nEGFR \\n\\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\\n\\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\\nSimilar to the previous discovery\\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \\n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \\n2D).\",\n \"To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \\n3B), while the epigenotypes of SFRP5 (Figure \\n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \\n3C) were not associated with OS in our patients.\\n\\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), or different genotype of \\n\\nEGFR \\n\\n(C).\\n\",\n \"In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\",\n \"Recent studies have demonstrated that cancer is as much an epigenetic disease as it is a genetic disease (Iacobuzio-Donahue). Therefore, in addition to genetic alterations, changes in epigenetic features such as CpG DNA methylation status of specific gene loci also mark the progress of cancers. Our current study showed that methylation of Wnt antagonist SFRP5 gene before treatment, independent of the genotype of EGFR gene, correlated with decreased progression free survival rate in NSCLC patients in response to the EGFR-TKI therapy. To our knowledge, this is the first report indicating that DNA methylation at specific gene loci in patient may predict drug response to the EGFT-TKI therapy.\\nBoth genetic and epigenetic risk factors for NSCLC have been studied extensively. Suzuki et al\\n[23] has reported that methylation of the Wnt antagonist DKK3 correlated with low survival rate in NSCLC patients, despite of the different therapies patients received. However, in our study, we did not find significant difference in the EGFR-TKI responses between patient groups with or without methylated DKK3 (Additional file\\n1: Figure S2 and S3). In contrast, our results suggested epigenotype of SFRP5 provide better prognostic estimation for the EGFR-TKI response, comparing to other Wnt antagonists.\\nSFRP5 is a member of the SFRP protein family containing a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins. It acts as soluble antagonist of Wnt signaling and is highly expressed in the retinal pigment epithelium, and moderately expressed in the pancreas (\\\"Entrez Gene: SFRP5 secreted frizzled-related protein 5\\\"). Previous studies has identified association of SFRP5 promoter hypermethylation with Acute myeloid leukemia\\n[29], ovarian cancer\\n[30], gastric cancer\\n[31], oral squamous cell carcinoma\\n[32], pancreatic cancer\\n[33] and breast cancer\\n[34].\\nWe found that hypermethylation of SFRP5 predicted worse outcomes of the EGFR-TKI therapy. Therefore, SFRP5 DNA methylation status may serve as a prognostic molecular marker for appropriately predicting whether NSCLC patients would benefit from the EGFR-TKI therapy. Especially, it is interesting that in the subgroup with adenocarcinoma and EGFR mutation, patients with sFRP5 methylation have a significantly shorter PFS than those without sFRP5 methylation, While in nonsmokers without EGFR mutation, patients without sFRP1 methylation have a longer PFS compared with patients with its methylation(9.7 ms vs 2.0 ms, p = 0.05). Based on these results, we can make a hypothesis that activation of Wnt signaling by antagonist methylation could confer tumors the characters of stem cell, which consequently causes tumors resistant to EGFR TKIs therapy by generating acquired resistance, such as MET amplification or changes of PTEN tumor suppressor activity and so on. Further study is needed to validate this hypothesis.\",\n \"In conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development\\n[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC.\",\n \"EGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.\",\n \"The authors declare that they have no competing interests.\",\n \"JZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.\",\n \"Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22).\",\n \"Figure S1. Methylated and unmethyalted bands of Wnt antagonist genes and wild/mutant EGFR. S1: The example graphs of methylated and unmethyalted bands of Wnt antagonist genes (A) and EGFR wild (B) and mutation types (C, D) by methylation specific PCR and DHPLC respectively. Figure S2 PFS with different epigenotypes of Wnt antagonist genes. Figure2S A-F.Kaplan-Meier curves of comparing the progression free survival of patients with different epigenotypes of SFRP1(A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F). Figure S3 OS with different epigenotypes of Wnt antagonist genes. Figure3S A-F. Kaplan-Meier curves of comparing the overall survival of patients with different epigenotypes of SFRP1 (A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F).\\nClick here for file\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,"results",null,null,null,null,null,null,"discussion","conclusions",null,null,null,null,"supplementary-material"],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null,\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["DNA methylation","EGFR-TKI","Wnt antagonists","Non-small cell lung cancer"],"string":"[\n \"DNA methylation\",\n \"EGFR-TKI\",\n \"Wnt antagonists\",\n \"Non-small cell lung cancer\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nLung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.\n\nMethods:\n Patients 155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n DNA extraction and methylation-specific PCR Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\nGenomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\n Mutation detection The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\nThe denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\n Statistical analysis All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\nAll data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\n\nPatients:\n155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n\nDNA extraction and methylation-specific PCR:\nGenomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\n\nMutation detection:\nThe denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\n\nStatistical analysis:\nAll data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\n\nResults:\n Characteristics of study patients Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\nTable\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\n Epigenotype of Wnt antagonists in NSCLC Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\nGenomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\n Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\nThe RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\n Epigenotype of Wnt antagonists and progression-free survival (PFS) We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\nWe next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\n Epigenotype of Wnt antagonists and overall survival rate (OS) To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\nTo test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\n Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\nIn order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\n\nCharacteristics of study patients:\nTable\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\n\nEpigenotype of Wnt antagonists in NSCLC:\nGenomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\n\nEpigenotype of Wnt antagonist genes and clinical responses to TKI therapy:\nThe RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\n\nEpigenotype of Wnt antagonists and progression-free survival (PFS):\nWe next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\n\nEpigenotype of Wnt antagonists and overall survival rate (OS):\nTo test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\n\nCorrelation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy:\nIn order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\n\nDiscussion:\nRecent studies have demonstrated that cancer is as much an epigenetic disease as it is a genetic disease (Iacobuzio-Donahue). Therefore, in addition to genetic alterations, changes in epigenetic features such as CpG DNA methylation status of specific gene loci also mark the progress of cancers. Our current study showed that methylation of Wnt antagonist SFRP5 gene before treatment, independent of the genotype of EGFR gene, correlated with decreased progression free survival rate in NSCLC patients in response to the EGFR-TKI therapy. To our knowledge, this is the first report indicating that DNA methylation at specific gene loci in patient may predict drug response to the EGFT-TKI therapy.\nBoth genetic and epigenetic risk factors for NSCLC have been studied extensively. Suzuki et al\n[23] has reported that methylation of the Wnt antagonist DKK3 correlated with low survival rate in NSCLC patients, despite of the different therapies patients received. However, in our study, we did not find significant difference in the EGFR-TKI responses between patient groups with or without methylated DKK3 (Additional file\n1: Figure S2 and S3). In contrast, our results suggested epigenotype of SFRP5 provide better prognostic estimation for the EGFR-TKI response, comparing to other Wnt antagonists.\nSFRP5 is a member of the SFRP protein family containing a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins. It acts as soluble antagonist of Wnt signaling and is highly expressed in the retinal pigment epithelium, and moderately expressed in the pancreas (\"Entrez Gene: SFRP5 secreted frizzled-related protein 5\"). Previous studies has identified association of SFRP5 promoter hypermethylation with Acute myeloid leukemia\n[29], ovarian cancer\n[30], gastric cancer\n[31], oral squamous cell carcinoma\n[32], pancreatic cancer\n[33] and breast cancer\n[34].\nWe found that hypermethylation of SFRP5 predicted worse outcomes of the EGFR-TKI therapy. Therefore, SFRP5 DNA methylation status may serve as a prognostic molecular marker for appropriately predicting whether NSCLC patients would benefit from the EGFR-TKI therapy. Especially, it is interesting that in the subgroup with adenocarcinoma and EGFR mutation, patients with sFRP5 methylation have a significantly shorter PFS than those without sFRP5 methylation, While in nonsmokers without EGFR mutation, patients without sFRP1 methylation have a longer PFS compared with patients with its methylation(9.7 ms vs 2.0 ms, p = 0.05). Based on these results, we can make a hypothesis that activation of Wnt signaling by antagonist methylation could confer tumors the characters of stem cell, which consequently causes tumors resistant to EGFR TKIs therapy by generating acquired resistance, such as MET amplification or changes of PTEN tumor suppressor activity and so on. Further study is needed to validate this hypothesis.\n\nConclusions:\nIn conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development\n[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC.\n\nAbbreviations:\nEGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nJZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.\n\nAuthors’ informations:\nSupported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22).\n\nSupplementary Material:\nFigure S1. Methylated and unmethyalted bands of Wnt antagonist genes and wild/mutant EGFR. S1: The example graphs of methylated and unmethyalted bands of Wnt antagonist genes (A) and EGFR wild (B) and mutation types (C, D) by methylation specific PCR and DHPLC respectively. Figure S2 PFS with different epigenotypes of Wnt antagonist genes. Figure2S A-F.Kaplan-Meier curves of comparing the progression free survival of patients with different epigenotypes of SFRP1(A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F). Figure S3 OS with different epigenotypes of Wnt antagonist genes. Figure3S A-F. Kaplan-Meier curves of comparing the overall survival of patients with different epigenotypes of SFRP1 (A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F).\nClick here for file\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIt is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy.\n\nMethods:\nTumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC).\n\nResults:\nWe found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype.\n\nConclusions:\nPatients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy."},"background_conclusion_text":{"kind":"string","value":"Background:\nLung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.\n\nConclusions:\nIn conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development\n[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nIt is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy.\n\nMethods:\nTumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC).\n\nResults:\nWe found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype.\n\nConclusions:\nPatients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy."},"whole_article_text_length":{"kind":"number","value":10366,"string":"10,366"},"whole_article_abstract_length":{"kind":"number","value":266,"string":"266"},"num_sections":{"kind":"number","value":20,"string":"20"},"most_frequent_words":{"kind":"list like","value":["egfr","patients","methylation","wnt","sfrp5","tki","therapy","pfs","95","95 ci"],"string":"[\n \"egfr\",\n \"patients\",\n \"methylation\",\n \"wnt\",\n \"sfrp5\",\n \"tki\",\n \"therapy\",\n \"pfs\",\n \"95\",\n \"95 ci\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | lung | lung cancer | cancer | therapy | wnt | tki | signaling | egfr tki | tki therapy [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] unmethylatedf | dna | defined | disease | pcr | pairs | methylatedf | primer pairs | primer | specific [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ci | 95 ci | 95 | patients | egfr | sfrp5 | pfs | months | wnt | significantly [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] drugs | detection | dna methylation | dna | egfr tki | protein rna | sfrp5 independent | pfs long | conclusion study revealed sfrp5 | conclusion study revealed [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | wnt | methylation | tki | sfrp5 | 95 | 95 ci | ci | therapy [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | wnt | methylation | tki | sfrp5 | 95 | 95 ci | ci | therapy [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] EGFR | EGFR ||| EGFR [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 155 | EGFR | Wnt | SFRP1 | SFRP5 | DKK3 | APC | PCR | MSP ||| EGFR | Denaturing High Performance Liquid Chromatography (DHPLC [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Wnt ||| EGFR | 0.005 | 0.011 ||| Wnt | EGFR ||| SFRP5 | SFRP5 | EGFR | 0.002 | EGFR [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] SFRP5 | EGFR [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] EGFR | EGFR ||| EGFR ||| 155 | EGFR | Wnt | SFRP1 | SFRP5 | DKK3 | APC | PCR | MSP ||| EGFR | Denaturing High Performance Liquid Chromatography (DHPLC ||| ||| Wnt ||| EGFR | 0.005 | 0.011 ||| Wnt | EGFR ||| SFRP5 | SFRP5 | EGFR | 0.002 | EGFR ||| SFRP5 | EGFR [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] EGFR | EGFR ||| EGFR ||| 155 | EGFR | Wnt | SFRP1 | SFRP5 | DKK3 | APC | PCR | MSP ||| EGFR | Denaturing High Performance Liquid Chromatography (DHPLC ||| ||| Wnt ||| EGFR | 0.005 | 0.011 ||| Wnt | EGFR ||| SFRP5 | SFRP5 | EGFR | 0.002 | EGFR ||| SFRP5 | EGFR [SUMMARY]"}}},{"rowIdx":286,"cells":{"title":{"kind":"string","value":"Factors Associated with Increased Alpha-Tocopherol Content in Milk in Response to Maternal Supplementation with 800 IU of Vitamin E."},"pmid":{"kind":"string","value":"31013594"},"background_abstract":{"kind":"string","value":"Vitamin E supplementation might represent an efficient strategy to increase the vitamin E content in milk. The present study aimed to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol content of milk and the factors associated with the increase in vitamin E."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Randomized clinical trial with 79 lactating women from Brazil, who were assigned to the control group, or to the supplemented group (800 IU of RRR-alpha-tocopherol). Milk and serum were collected between 30 and 90 days after delivery (collection 1), and on the next day (collection 2). Alpha-tocopherol was analyzed using high-performance liquid chromatography."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In the supplemented group, the alpha-tocopherol content in serum and milk increased after supplementation (p < 0.001). In the multivariate analysis, only alpha-tocopherol in milk (collection 1) was associated with the level of this vitamin in milk after supplementation (β = 0.927, p < 0.001), and binary logistic regression showed that the dietary intake was the only determinant for the greater effect of supplementation in milk."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The pre-existing vitamin level in milk and diet are determinants for the efficacy of supplementation in milk, suggesting that in populations with vitamin E deficiency, high-dose supplementation can be used to restore its level in milk."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Breast Feeding","Dietary Supplements","Female","Humans","Logistic Models","Maternal Nutritional Physiological Phenomena","Milk, Human","Vitamin E","Young Adult","alpha-Tocopherol"],"string":"[\n \"Adult\",\n \"Breast Feeding\",\n \"Dietary Supplements\",\n \"Female\",\n \"Humans\",\n \"Logistic Models\",\n \"Maternal Nutritional Physiological Phenomena\",\n \"Milk, Human\",\n \"Vitamin E\",\n \"Young Adult\",\n \"alpha-Tocopherol\"\n]"},"pmcid":{"kind":"string","value":"6520676"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Breast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].\nStudies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].\nOne strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].\nSingle-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.\nInterestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.\nThus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":" 3.1. General Characteristics of the Population The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\nThe socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\n 3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\nAt collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\n 3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\nIn the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively)."},"conclusions_title":{"kind":"string","value":"5. Conclusions"},"conclusions_text":{"kind":"string","value":"Vitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E."},"other_sections_titles":{"kind":"list like","value":["2. Materials and Methods","2.2. Data Collection","2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples","2.4. Dietary Intake of Vitamin E","2.5. Statistical Analysis","3.1. General Characteristics of the Population","3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk","3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation"],"string":"[\n \"2. Materials and Methods\",\n \"2.2. Data Collection\",\n \"2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples\",\n \"2.4. Dietary Intake of Vitamin E\",\n \"2.5. Statistical Analysis\",\n \"3.1. General Characteristics of the Population\",\n \"3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk\",\n \"3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation\"\n]"},"other_sections_texts":{"kind":"list like","value":[" 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.","A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.","The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].","Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.","Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.","The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).","At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.","In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively)."],"string":"[\n \" 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\",\n \"A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\",\n \"The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\",\n \"Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\",\n \"Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\",\n \"The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\",\n \"At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\",\n \"In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Materials and Methods","2.1. Participants and Intervention","2.2. Data Collection","2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples","2.4. Dietary Intake of Vitamin E","2.5. Statistical Analysis","3. Results","3.1. General Characteristics of the Population","3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk","3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation","4. Discussion","5. Conclusions"],"string":"[\n \"1. Introduction\",\n \"2. Materials and Methods\",\n \"2.1. Participants and Intervention\",\n \"2.2. Data Collection\",\n \"2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples\",\n \"2.4. Dietary Intake of Vitamin E\",\n \"2.5. Statistical Analysis\",\n \"3. Results\",\n \"3.1. General Characteristics of the Population\",\n \"3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk\",\n \"3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation\",\n \"4. Discussion\",\n \"5. Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Breast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].\nStudies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].\nOne strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].\nSingle-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.\nInterestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.\nThus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period."," 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.","The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).","A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.","The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].","Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.","Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05."," 3.1. General Characteristics of the Population The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\nThe socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\n 3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\nAt collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\n 3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\nIn the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).","The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).","At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.","In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).","Mature milk is the most stable stage of lactation, in which the content of alpha-tocopherol is not influenced by pregnancy-related factors, as occurs in the colostrum [2,7]. It should be emphasized that the mature milk presents a higher concentration of lipids and in contrast, there is a lower secretion of alpha-tocopherol, suggesting that there are distinct mechanisms involved in the transfer of this vitamin into breast milk [2,35,36].\nIn the present study, the lactating women had adequate vitamin E status, in accordance with other studies considering the same stage of lactation [8,21]. However, a low dietary intake of vitamin E was noted (Table 1), which could trigger the mobilization of alpha-tocopherol from maternal reserves, such as the adipose tissue, into breast milk [15,19]. This low consumption of vitamin E was also reported in other populations in Brazil, Greece and Poland [21,25,36], which suggests a frequent inadequacy in vitamin E consumption during lactation.\nEven in situations of inadequate consumption, the vitamin E concentration in milk was not influenced by diet and circulating maternal levels [8,20,21,37,38,39,40]. However, this present study was the first to identify a positive association between alpha-tocopherol levels in milk and ingested vitamin E (diet + supplementation) and with serum alpha-tocopherol (Figure 2d,c). This suggested that in high-consumption situations, the ingested and circulating maternal levels are the main factors responsible for the vitamin level in milk. It is likely that in situations of low vitamin E consumption (as found in the studies cited), milk vitamin E might originate from other sources, such as the body’s reserve [4], explaining the absence of a relationship between those variables.\nTo prevent of VED in infants, it is necessary for breast milk to contain adequate levels of vitamin E, so that children can obtain the benefits of the micronutrient, through the creation of vitamin reserves in the body and its antioxidant action [4,19]. Some studies that evaluated this vitamin in mature milk observed values below the nutritional requirements of infants [20,23,36,37], which suggested that maternal vitamin E supplementation could be an important strategy to increase milk vitamin contents [17,20,21].\nIn this study, supplementation with 800 IU of alpha-tocopherol caused a 183% increase in serum alpha-tocopherol, and a 124% increase in breast milk alpha-tocopherol (Table 2). Other clinical trials using a lower dose (400 IU alpha-tocopherol) found an increase of 60% to 80% in the vitamin content in milk after supplementation [18,20,21,22], showing a reduced effect compared with that shown in the present study. These findings suggested that the response to supplementation might be influenced by both the dosage used and the determinant factors. However, trials have not evaluated the factors associated with the different responses to large vitamin E doses [17,20], being important to understand how this vitamin is transferred into the mammary gland.\nWhen analyzing the factors associated with a better response in milk after supplementation, it is important to highlight that only the content of this vitamin in the basal milk (before supplementation) and the dietary intake were demonstrated to increase the levels of this micronutrient in milk (Table 3), which suggested that the higher the consumption of vitamin E and its levels in milk, the greater the transfer of alpha-tocopherol from the supplement to the mammary gland.\nAssessment of the profile of lactating women in the supplemented group, showed that the participants with the highest supplementation effect (quartiles 2–4) had a higher intake of calories, alpha-tocopherol and total fat (Table 4). These findings suggested that the amount of fat available in the diet may improve the bioavailability of the vitamin in the body, such as its absorption and distribution to tissues, and in this case, to the mammary gland, as reported in [41,42,43].\nSuch evidence also demonstrated that in mature milk, the Michaelis–Menten kinetic theory could not be applied, as it proposes that the transfer of vitamin to milk occurs through active transport, characterized by a saturation of the lipoprotein receptors in the breast tissue in situations of large contents of vitamin E, which would prevent the continuous transfer of the vitamin into the breast milk after supplementation [38,44]. Notably, in a study of dairy cows, Weiss and Wyatt (2003) [45] suggested that the ability of lipoproteins to carry alpha-tocopherol could determine the uptake of this vitamin by the breast tissue, and that the limiting factor for this mechanism would be the maximum content of vitamin E in the lipoproteins.\nTo further investigate this relationship between lipoproteins and vitamin E transport, we determined the circulating lipoproteins and the serum cholesterol and triglycerides profiles; however, no relation between them and the response to supplementation was found (Table 2). In fact, the mechanism of transport of vitamin E to the mammary gland is poorly understood [2,46,47]. Circulating lipoproteins are responsible for this transfer, with LDL being the main carrier [44,48]; however, transport may occur in the presence or absence of its receptors in the mammary gland [2,44]. Other receptors are found in breast tissue, such as Scavenger Receptor B-1 (SR-B1), which has binding sites for both LDL and HDL, and CD36, which has high affinity binding sites for HDL, LDL, and very low-density lipoprotein (VLDL) [2,48]. It has also been suggested the participation of lipoprotein lipase (LPL), which may show increased activity during lactation, contributes to the greater circulation of alpha-tocopherol and its uptake [49].\nThese findings provide important information to understand the mechanisms by which vitamin E is transferred into the mammary gland, demonstrating that, in situations of supplementation with 800 IU of vitamin E and its effect in mature milk, the better the vitamin E status (considering the milk and dietary intake), the more effective uptake into the mammary gland will be, regardless of receptor saturation. Further investigation into how this transport occurs is required, by means of in vitro and in vivo studies and using labeled isotopes of alpha-tocopherol, for example, to investigate its biotransformation. Notably, in populations with dietary inadequacy and low contents of vitamin E in milk, supplementation with higher doses of the vitamin, such as 800 IU alpha-tocopherol, might be required to obtain a more effective intervention. The analysis of a single dose during the day allowed us to investigate possible factors that could interfere with the response to supplementation; however, it is necessary to analyze how long the effect of this supplementation could be sustained, its contribution to maternal and infant nutritional status, and the use of smaller daily doses.\nTherefore, supplementation associated with an adequate intake of vitamin E is an effective strategy to increase vitamin E levels in breast milk and prevent cases of vitamin E deficiency in infants, especially premature infants [2,4,11].","Vitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E."],"string":"[\n \"Breast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].\\nStudies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].\\nOne strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].\\nSingle-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.\\nInterestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.\\nThus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period.\",\n \" 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\",\n \"The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\",\n \"A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\",\n \"The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\",\n \"Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\",\n \"Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\",\n \" 3.1. General Characteristics of the Population The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\\nThe socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\\n 3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\\nAt collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\\n 3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\\nIn the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\",\n \"The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\",\n \"At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\",\n \"In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\",\n \"Mature milk is the most stable stage of lactation, in which the content of alpha-tocopherol is not influenced by pregnancy-related factors, as occurs in the colostrum [2,7]. It should be emphasized that the mature milk presents a higher concentration of lipids and in contrast, there is a lower secretion of alpha-tocopherol, suggesting that there are distinct mechanisms involved in the transfer of this vitamin into breast milk [2,35,36].\\nIn the present study, the lactating women had adequate vitamin E status, in accordance with other studies considering the same stage of lactation [8,21]. However, a low dietary intake of vitamin E was noted (Table 1), which could trigger the mobilization of alpha-tocopherol from maternal reserves, such as the adipose tissue, into breast milk [15,19]. This low consumption of vitamin E was also reported in other populations in Brazil, Greece and Poland [21,25,36], which suggests a frequent inadequacy in vitamin E consumption during lactation.\\nEven in situations of inadequate consumption, the vitamin E concentration in milk was not influenced by diet and circulating maternal levels [8,20,21,37,38,39,40]. However, this present study was the first to identify a positive association between alpha-tocopherol levels in milk and ingested vitamin E (diet + supplementation) and with serum alpha-tocopherol (Figure 2d,c). This suggested that in high-consumption situations, the ingested and circulating maternal levels are the main factors responsible for the vitamin level in milk. It is likely that in situations of low vitamin E consumption (as found in the studies cited), milk vitamin E might originate from other sources, such as the body’s reserve [4], explaining the absence of a relationship between those variables.\\nTo prevent of VED in infants, it is necessary for breast milk to contain adequate levels of vitamin E, so that children can obtain the benefits of the micronutrient, through the creation of vitamin reserves in the body and its antioxidant action [4,19]. Some studies that evaluated this vitamin in mature milk observed values below the nutritional requirements of infants [20,23,36,37], which suggested that maternal vitamin E supplementation could be an important strategy to increase milk vitamin contents [17,20,21].\\nIn this study, supplementation with 800 IU of alpha-tocopherol caused a 183% increase in serum alpha-tocopherol, and a 124% increase in breast milk alpha-tocopherol (Table 2). Other clinical trials using a lower dose (400 IU alpha-tocopherol) found an increase of 60% to 80% in the vitamin content in milk after supplementation [18,20,21,22], showing a reduced effect compared with that shown in the present study. These findings suggested that the response to supplementation might be influenced by both the dosage used and the determinant factors. However, trials have not evaluated the factors associated with the different responses to large vitamin E doses [17,20], being important to understand how this vitamin is transferred into the mammary gland.\\nWhen analyzing the factors associated with a better response in milk after supplementation, it is important to highlight that only the content of this vitamin in the basal milk (before supplementation) and the dietary intake were demonstrated to increase the levels of this micronutrient in milk (Table 3), which suggested that the higher the consumption of vitamin E and its levels in milk, the greater the transfer of alpha-tocopherol from the supplement to the mammary gland.\\nAssessment of the profile of lactating women in the supplemented group, showed that the participants with the highest supplementation effect (quartiles 2–4) had a higher intake of calories, alpha-tocopherol and total fat (Table 4). These findings suggested that the amount of fat available in the diet may improve the bioavailability of the vitamin in the body, such as its absorption and distribution to tissues, and in this case, to the mammary gland, as reported in [41,42,43].\\nSuch evidence also demonstrated that in mature milk, the Michaelis–Menten kinetic theory could not be applied, as it proposes that the transfer of vitamin to milk occurs through active transport, characterized by a saturation of the lipoprotein receptors in the breast tissue in situations of large contents of vitamin E, which would prevent the continuous transfer of the vitamin into the breast milk after supplementation [38,44]. Notably, in a study of dairy cows, Weiss and Wyatt (2003) [45] suggested that the ability of lipoproteins to carry alpha-tocopherol could determine the uptake of this vitamin by the breast tissue, and that the limiting factor for this mechanism would be the maximum content of vitamin E in the lipoproteins.\\nTo further investigate this relationship between lipoproteins and vitamin E transport, we determined the circulating lipoproteins and the serum cholesterol and triglycerides profiles; however, no relation between them and the response to supplementation was found (Table 2). In fact, the mechanism of transport of vitamin E to the mammary gland is poorly understood [2,46,47]. Circulating lipoproteins are responsible for this transfer, with LDL being the main carrier [44,48]; however, transport may occur in the presence or absence of its receptors in the mammary gland [2,44]. Other receptors are found in breast tissue, such as Scavenger Receptor B-1 (SR-B1), which has binding sites for both LDL and HDL, and CD36, which has high affinity binding sites for HDL, LDL, and very low-density lipoprotein (VLDL) [2,48]. It has also been suggested the participation of lipoprotein lipase (LPL), which may show increased activity during lactation, contributes to the greater circulation of alpha-tocopherol and its uptake [49].\\nThese findings provide important information to understand the mechanisms by which vitamin E is transferred into the mammary gland, demonstrating that, in situations of supplementation with 800 IU of vitamin E and its effect in mature milk, the better the vitamin E status (considering the milk and dietary intake), the more effective uptake into the mammary gland will be, regardless of receptor saturation. Further investigation into how this transport occurs is required, by means of in vitro and in vivo studies and using labeled isotopes of alpha-tocopherol, for example, to investigate its biotransformation. Notably, in populations with dietary inadequacy and low contents of vitamin E in milk, supplementation with higher doses of the vitamin, such as 800 IU alpha-tocopherol, might be required to obtain a more effective intervention. The analysis of a single dose during the day allowed us to investigate possible factors that could interfere with the response to supplementation; however, it is necessary to analyze how long the effect of this supplementation could be sustained, its contribution to maternal and infant nutritional status, and the use of smaller daily doses.\\nTherefore, supplementation associated with an adequate intake of vitamin E is an effective strategy to increase vitamin E levels in breast milk and prevent cases of vitamin E deficiency in infants, especially premature infants [2,4,11].\",\n \"Vitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,"subjects",null,null,null,null,"results",null,null,null,"discussion","conclusions"],"string":"[\n \"intro\",\n null,\n \"subjects\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["clinical trial","lactation","infants","breastfeeding","lactating women"],"string":"[\n \"clinical trial\",\n \"lactation\",\n \"infants\",\n \"breastfeeding\",\n \"lactating women\"\n]"},"whole_article_text":{"kind":"string","value":"1. Introduction:\nBreast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].\nStudies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].\nOne strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].\nSingle-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.\nInterestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.\nThus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period.\n\n2. Materials and Methods:\n 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\n\n2.1. Participants and Intervention:\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\n\n2.2. Data Collection:\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\n\n2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples:\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\n\n2.4. Dietary Intake of Vitamin E:\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\n\n2.5. Statistical Analysis:\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\n\n3. Results:\n 3.1. General Characteristics of the Population The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\nThe socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\n 3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\nAt collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\n 3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\nIn the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\n\n3.1. General Characteristics of the Population:\nThe socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\n\n3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk:\nAt collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\n\n3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation:\nIn the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\n\n4. Discussion:\nMature milk is the most stable stage of lactation, in which the content of alpha-tocopherol is not influenced by pregnancy-related factors, as occurs in the colostrum [2,7]. It should be emphasized that the mature milk presents a higher concentration of lipids and in contrast, there is a lower secretion of alpha-tocopherol, suggesting that there are distinct mechanisms involved in the transfer of this vitamin into breast milk [2,35,36].\nIn the present study, the lactating women had adequate vitamin E status, in accordance with other studies considering the same stage of lactation [8,21]. However, a low dietary intake of vitamin E was noted (Table 1), which could trigger the mobilization of alpha-tocopherol from maternal reserves, such as the adipose tissue, into breast milk [15,19]. This low consumption of vitamin E was also reported in other populations in Brazil, Greece and Poland [21,25,36], which suggests a frequent inadequacy in vitamin E consumption during lactation.\nEven in situations of inadequate consumption, the vitamin E concentration in milk was not influenced by diet and circulating maternal levels [8,20,21,37,38,39,40]. However, this present study was the first to identify a positive association between alpha-tocopherol levels in milk and ingested vitamin E (diet + supplementation) and with serum alpha-tocopherol (Figure 2d,c). This suggested that in high-consumption situations, the ingested and circulating maternal levels are the main factors responsible for the vitamin level in milk. It is likely that in situations of low vitamin E consumption (as found in the studies cited), milk vitamin E might originate from other sources, such as the body’s reserve [4], explaining the absence of a relationship between those variables.\nTo prevent of VED in infants, it is necessary for breast milk to contain adequate levels of vitamin E, so that children can obtain the benefits of the micronutrient, through the creation of vitamin reserves in the body and its antioxidant action [4,19]. Some studies that evaluated this vitamin in mature milk observed values below the nutritional requirements of infants [20,23,36,37], which suggested that maternal vitamin E supplementation could be an important strategy to increase milk vitamin contents [17,20,21].\nIn this study, supplementation with 800 IU of alpha-tocopherol caused a 183% increase in serum alpha-tocopherol, and a 124% increase in breast milk alpha-tocopherol (Table 2). Other clinical trials using a lower dose (400 IU alpha-tocopherol) found an increase of 60% to 80% in the vitamin content in milk after supplementation [18,20,21,22], showing a reduced effect compared with that shown in the present study. These findings suggested that the response to supplementation might be influenced by both the dosage used and the determinant factors. However, trials have not evaluated the factors associated with the different responses to large vitamin E doses [17,20], being important to understand how this vitamin is transferred into the mammary gland.\nWhen analyzing the factors associated with a better response in milk after supplementation, it is important to highlight that only the content of this vitamin in the basal milk (before supplementation) and the dietary intake were demonstrated to increase the levels of this micronutrient in milk (Table 3), which suggested that the higher the consumption of vitamin E and its levels in milk, the greater the transfer of alpha-tocopherol from the supplement to the mammary gland.\nAssessment of the profile of lactating women in the supplemented group, showed that the participants with the highest supplementation effect (quartiles 2–4) had a higher intake of calories, alpha-tocopherol and total fat (Table 4). These findings suggested that the amount of fat available in the diet may improve the bioavailability of the vitamin in the body, such as its absorption and distribution to tissues, and in this case, to the mammary gland, as reported in [41,42,43].\nSuch evidence also demonstrated that in mature milk, the Michaelis–Menten kinetic theory could not be applied, as it proposes that the transfer of vitamin to milk occurs through active transport, characterized by a saturation of the lipoprotein receptors in the breast tissue in situations of large contents of vitamin E, which would prevent the continuous transfer of the vitamin into the breast milk after supplementation [38,44]. Notably, in a study of dairy cows, Weiss and Wyatt (2003) [45] suggested that the ability of lipoproteins to carry alpha-tocopherol could determine the uptake of this vitamin by the breast tissue, and that the limiting factor for this mechanism would be the maximum content of vitamin E in the lipoproteins.\nTo further investigate this relationship between lipoproteins and vitamin E transport, we determined the circulating lipoproteins and the serum cholesterol and triglycerides profiles; however, no relation between them and the response to supplementation was found (Table 2). In fact, the mechanism of transport of vitamin E to the mammary gland is poorly understood [2,46,47]. Circulating lipoproteins are responsible for this transfer, with LDL being the main carrier [44,48]; however, transport may occur in the presence or absence of its receptors in the mammary gland [2,44]. Other receptors are found in breast tissue, such as Scavenger Receptor B-1 (SR-B1), which has binding sites for both LDL and HDL, and CD36, which has high affinity binding sites for HDL, LDL, and very low-density lipoprotein (VLDL) [2,48]. It has also been suggested the participation of lipoprotein lipase (LPL), which may show increased activity during lactation, contributes to the greater circulation of alpha-tocopherol and its uptake [49].\nThese findings provide important information to understand the mechanisms by which vitamin E is transferred into the mammary gland, demonstrating that, in situations of supplementation with 800 IU of vitamin E and its effect in mature milk, the better the vitamin E status (considering the milk and dietary intake), the more effective uptake into the mammary gland will be, regardless of receptor saturation. Further investigation into how this transport occurs is required, by means of in vitro and in vivo studies and using labeled isotopes of alpha-tocopherol, for example, to investigate its biotransformation. Notably, in populations with dietary inadequacy and low contents of vitamin E in milk, supplementation with higher doses of the vitamin, such as 800 IU alpha-tocopherol, might be required to obtain a more effective intervention. The analysis of a single dose during the day allowed us to investigate possible factors that could interfere with the response to supplementation; however, it is necessary to analyze how long the effect of this supplementation could be sustained, its contribution to maternal and infant nutritional status, and the use of smaller daily doses.\nTherefore, supplementation associated with an adequate intake of vitamin E is an effective strategy to increase vitamin E levels in breast milk and prevent cases of vitamin E deficiency in infants, especially premature infants [2,4,11].\n\n5. Conclusions:\nVitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nVitamin E supplementation might represent an efficient strategy to increase the vitamin E content in milk. The present study aimed to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol content of milk and the factors associated with the increase in vitamin E.\n\nMethods:\nRandomized clinical trial with 79 lactating women from Brazil, who were assigned to the control group, or to the supplemented group (800 IU of RRR-alpha-tocopherol). Milk and serum were collected between 30 and 90 days after delivery (collection 1), and on the next day (collection 2). Alpha-tocopherol was analyzed using high-performance liquid chromatography.\n\nResults:\nIn the supplemented group, the alpha-tocopherol content in serum and milk increased after supplementation (p < 0.001). In the multivariate analysis, only alpha-tocopherol in milk (collection 1) was associated with the level of this vitamin in milk after supplementation (β = 0.927, p < 0.001), and binary logistic regression showed that the dietary intake was the only determinant for the greater effect of supplementation in milk.\n\nConclusions:\nThe pre-existing vitamin level in milk and diet are determinants for the efficacy of supplementation in milk, suggesting that in populations with vitamin E deficiency, high-dose supplementation can be used to restore its level in milk."},"background_conclusion_text":{"kind":"string","value":"1. Introduction:\nBreast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].\nStudies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].\nOne strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].\nSingle-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.\nInterestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.\nThus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period.\n\n5. Conclusions:\nVitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nVitamin E supplementation might represent an efficient strategy to increase the vitamin E content in milk. The present study aimed to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol content of milk and the factors associated with the increase in vitamin E.\n\nMethods:\nRandomized clinical trial with 79 lactating women from Brazil, who were assigned to the control group, or to the supplemented group (800 IU of RRR-alpha-tocopherol). Milk and serum were collected between 30 and 90 days after delivery (collection 1), and on the next day (collection 2). Alpha-tocopherol was analyzed using high-performance liquid chromatography.\n\nResults:\nIn the supplemented group, the alpha-tocopherol content in serum and milk increased after supplementation (p < 0.001). In the multivariate analysis, only alpha-tocopherol in milk (collection 1) was associated with the level of this vitamin in milk after supplementation (β = 0.927, p < 0.001), and binary logistic regression showed that the dietary intake was the only determinant for the greater effect of supplementation in milk.\n\nConclusions:\nThe pre-existing vitamin level in milk and diet are determinants for the efficacy of supplementation in milk, suggesting that in populations with vitamin E deficiency, high-dose supplementation can be used to restore its level in milk."},"whole_article_text_length":{"kind":"number","value":9199,"string":"9,199"},"whole_article_abstract_length":{"kind":"number","value":266,"string":"266"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["vitamin","milk","alpha","tocopherol","alpha tocopherol","supplementation","collection","group","serum","intake"],"string":"[\n \"vitamin\",\n \"milk\",\n \"alpha\",\n 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Alpha ||| ||| ||| 1 | 0.927 ||| [SUMMARY]"}}},{"rowIdx":287,"cells":{"title":{"kind":"string","value":"Mobile Phone Apps to Promote Weight Loss and Increase Physical Activity: A Systematic Review and Meta-Analysis."},"pmid":{"kind":"string","value":"26554314"},"background_abstract":{"kind":"string","value":"To our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We conducted a systematic review and meta-analysis of relevant studies identified by a search of PubMed, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), and Scopus from their inception through to August 2015. Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted data. We included all controlled studies that assessed a mobile phone app intervention with weight-related health measures (ie, body weight, body mass index, or waist circumference) or physical activity outcomes. Net change estimates comparing the intervention group with the control group were pooled across studies using random-effects models."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"We included 12 articles in this systematic review and meta-analysis. Compared with the control group, use of a mobile phone app was associated with significant changes in body weight (kg) and body mass index (kg/m(2)) of -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) and -0.43 kg/m(2) (95% CI -0.74 to -0.13; I2 = 50%), respectively. Moreover, a nonsignificant difference in physical activity was observed between the two groups (standardized mean difference 0.40, 95% CI -0.07 to 0.87; I2 = 93%). These findings were remarkably robust in the sensitivity analysis. No publication bias was shown."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Evidence from this study shows that mobile phone app-based interventions may be useful tools for weight loss."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Cell Phone","Humans","Mobile Applications","Motor Activity","Obesity","Weight Loss"],"string":"[\n \"Cell Phone\",\n \"Humans\",\n \"Mobile Applications\",\n \"Motor Activity\",\n \"Obesity\",\n \"Weight Loss\"\n]"},"pmcid":{"kind":"string","value":"4704965"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Overweight and obesity are a global public health issue and an important feature in discussions about strategies for primary and secondary health care. Developing since the 1960s and now gathering pace rapidly, the issue is contributing, together with population aging, to major increases in the prevalence of high blood pressure and cholesterol levels, type 2 diabetes, and cancers [1]. Mortality rates are increasing with increasing degrees of overweight, as measured by body mass index (BMI) [2].\nIn 2008, 35% of adults older than 20 years were overweight (BMI ≥ 25 kg/m2) and the worldwide prevalence of obesity (BMI ≥30 kg/m2) had nearly doubled since 1980, from 5% of men and 8% of women to 10% and 14%, respectively [2]. An estimated 205 million men and 297 million women were obese—a total of more than half a billion adults worldwide [3]. For these reasons, identifying effective interventions is an important component in public health efforts to curb obesity, but the most effective strategies for weight loss remain unclear.\nWith the extensive market penetration of mobile phones, the International Telecommunications Union (ITU) reports that as of 2015, there are more than 7 billion mobile cellular subscriptions worldwide, corresponding to a 97% penetration rate—defined by ITU as mobile cellular telephone subscribers per 100 inhabitants [4]. Advanced-feature mobile phones (those with computer operating systems) have broadened the functions of mobile phones considerably. Mobile phone apps meet a variety of user needs, and are designed and adapted for each type of mobile device; therefore, they are applicable in nearly all social and economic sectors and environments. At present, these apps, apart from their recreational function, are becoming instruments of patient education and support and are also helpful to health care professionals [5]. Nonetheless, the market for health care apps is very fragmented because many of them are very specific or directed at minority diseases or specialties. The world market for medical apps for mobile phones and tablets multiplied seven times over in 2011 alone, reaching a total of US $718 million according to a market analysis by the American firm research2guidance [6]. A recent analysis of app store catalogs identified more than 97,000 mHealth apps, most of them dealing with general health and physical fitness; in general, they facilitate the monitoring of various parameters by individual users and provide general information and support related to those topics [5]. Previous research has suggested that mobile apps may be beneficial in asthma control [7] and diabetes management [8,9].\nTo our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity. The objective of this study was to perform a systematic review and meta-analysis of published studies to evaluate the efficacy of interventions that included mobile phone apps compared with other interventions to reduce weight and increase physical activity in populations of children and adults."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Search Strategy We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\nWe conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\n Study Selection Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\nTwo members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\n Data Extraction and Quality Assessment Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\nTwo investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\n Statistical Methods For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\nFor each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration)."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Study Selection The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\nThe search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\n Meta-Analysis of Mobile App Intervention and Body Weight Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Body Mass Index Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Physical Activity Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Risk of Bias in Included Studies Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\nRandomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Search Strategy","Study Selection","Data Extraction and Quality Assessment","Statistical Methods","Study Selection","Meta-Analysis of Mobile App Intervention and Body Weight","Meta-Analysis of Mobile App Intervention and Body Mass Index","Meta-Analysis of Mobile App Intervention and Physical Activity","Risk of Bias in Included Studies"],"string":"[\n \"Search Strategy\",\n \"Study Selection\",\n \"Data Extraction and Quality Assessment\",\n \"Statistical Methods\",\n \"Study Selection\",\n \"Meta-Analysis of Mobile App Intervention and Body Weight\",\n \"Meta-Analysis of Mobile App Intervention and Body Mass Index\",\n \"Meta-Analysis of Mobile App Intervention and Physical Activity\",\n \"Risk of Bias in Included Studies\"\n]"},"other_sections_texts":{"kind":"list like","value":["We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.","Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.","Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.","For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).","The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.","Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study."],"string":"[\n \"We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\",\n \"Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\\nFlowchart for the selection of the articles in this meta-analysis.\",\n \"Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\",\n \"For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\\nSD2\\ndiff= SD2\\npre+ SD2\\npost- 2×ρ×SDpre×SDpost\\n\\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\",\n \"The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\\nCharacteristics of included clinical trials.\\n\\naSS: sample size.\\n\\nbCCS: case-control study.\\n\\ncN/A: not applicable.\\n\\ndBMI: body mass index.\\n\\neRCT: randomized controlled trial.\\n\\nfMCCT: matched case-control trial.\\n\\ngMPA: moderate physical activity.\\n\\nhVPA: vigorous physical activity.\\nCharacteristics of intervention types and description of apps.\\n\\naBMI: body mass index.\\n\\nbMPA: moderate physical activity.\\n\\ncVPA: vigorous physical activity.\\n\\ndSMS: short message service.\",\n \"Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Search Strategy","Study Selection","Data Extraction and Quality Assessment","Statistical Methods","Results","Study Selection","Meta-Analysis of Mobile App Intervention and Body Weight","Meta-Analysis of Mobile App Intervention and Body Mass Index","Meta-Analysis of Mobile App Intervention and Physical Activity","Risk of Bias in Included Studies","Discussion"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Search Strategy\",\n \"Study Selection\",\n \"Data Extraction and Quality Assessment\",\n \"Statistical Methods\",\n \"Results\",\n \"Study Selection\",\n \"Meta-Analysis of Mobile App Intervention and Body Weight\",\n \"Meta-Analysis of Mobile App Intervention and Body Mass Index\",\n \"Meta-Analysis of Mobile App Intervention and Physical Activity\",\n \"Risk of Bias in Included Studies\",\n \"Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Overweight and obesity are a global public health issue and an important feature in discussions about strategies for primary and secondary health care. Developing since the 1960s and now gathering pace rapidly, the issue is contributing, together with population aging, to major increases in the prevalence of high blood pressure and cholesterol levels, type 2 diabetes, and cancers [1]. Mortality rates are increasing with increasing degrees of overweight, as measured by body mass index (BMI) [2].\nIn 2008, 35% of adults older than 20 years were overweight (BMI ≥ 25 kg/m2) and the worldwide prevalence of obesity (BMI ≥30 kg/m2) had nearly doubled since 1980, from 5% of men and 8% of women to 10% and 14%, respectively [2]. An estimated 205 million men and 297 million women were obese—a total of more than half a billion adults worldwide [3]. For these reasons, identifying effective interventions is an important component in public health efforts to curb obesity, but the most effective strategies for weight loss remain unclear.\nWith the extensive market penetration of mobile phones, the International Telecommunications Union (ITU) reports that as of 2015, there are more than 7 billion mobile cellular subscriptions worldwide, corresponding to a 97% penetration rate—defined by ITU as mobile cellular telephone subscribers per 100 inhabitants [4]. Advanced-feature mobile phones (those with computer operating systems) have broadened the functions of mobile phones considerably. Mobile phone apps meet a variety of user needs, and are designed and adapted for each type of mobile device; therefore, they are applicable in nearly all social and economic sectors and environments. At present, these apps, apart from their recreational function, are becoming instruments of patient education and support and are also helpful to health care professionals [5]. Nonetheless, the market for health care apps is very fragmented because many of them are very specific or directed at minority diseases or specialties. The world market for medical apps for mobile phones and tablets multiplied seven times over in 2011 alone, reaching a total of US $718 million according to a market analysis by the American firm research2guidance [6]. A recent analysis of app store catalogs identified more than 97,000 mHealth apps, most of them dealing with general health and physical fitness; in general, they facilitate the monitoring of various parameters by individual users and provide general information and support related to those topics [5]. Previous research has suggested that mobile apps may be beneficial in asthma control [7] and diabetes management [8,9].\nTo our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity. The objective of this study was to perform a systematic review and meta-analysis of published studies to evaluate the efficacy of interventions that included mobile phone apps compared with other interventions to reduce weight and increase physical activity in populations of children and adults."," Search Strategy We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\nWe conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\n Study Selection Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\nTwo members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\n Data Extraction and Quality Assessment Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\nTwo investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\n Statistical Methods For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\nFor each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).","We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.","Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.","Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.","For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration)."," Study Selection The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\nThe search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\n Meta-Analysis of Mobile App Intervention and Body Weight Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Body Mass Index Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Physical Activity Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Risk of Bias in Included Studies Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\nRandomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.","The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.","Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.","The current meta-analysis suggested that mobile phone app interventions compared with various control interventions significantly reduced body weight by 1.04 kg, reduced BMI by 0.43 kg/m2, and nonsignificantly increased physical activity by an SMD of 0.40. Our findings were robust across sensitivity analyses. Although the mean reductions in body weight and BMI were modest, it would not be expected for a single change in weight-loss interventions, such as mobile phone apps, to cause clinically meaningful weight loss compared with other control interventions [25]. Many of the control group treatments were other interventions. This could dilute the analysis, as it is possible that in some of the studies the treatment group showed a significant change, while the control group also showed a similar significant result. In our sensitivity analyses, the results were not modified when we excluded one study that did not describe if the control group had received any intervention [13].\nSome of the most recognizable research in mobile interventions has focused on text-messaging interventions, or SMS. A previous meta-analysis [26] found that mobile phone interventions were associated with significant changes in body weight and BMI compared with the control group (-1.44 kg and -0.24 units, respectively). This meta-analysis included only mobile interventions based on contacts by SMS and multimedia message services (MMS). A previous systematic review found strong evidence from the included RCT that weight loss occurs in the short term because of mobile technology interventions [27]. Another systematic review that included seven articles demonstrated a beneficial impact of text messaging or a mobile app for reducing physical inactivity and/or overweight/obesity [28]. Finally, a recent systematic review found that most apps that are focused on weight loss have inconsistent outcomes [29].\nTo our knowledge, this study is the first meta-analysis to summarize the evidence to date regarding effects of mobile phone app interventions compared with various control interventions. We excluded from our meta-analysis interventions based only on text messaging and focused solely on mobile phone apps because text-message interventions do not utilize the full potential of mobile phone technologies. Well-designed apps expand the potential for technology-based health interventions to impact populations in ways that previously were not possible and cannot be achieved without the capabilities of mobile phone software. Therefore, the need to regulate this growing market is becoming a concern, with increased advertising claims about effectiveness and researchers emphasizing the need for studies that will contribute scientific evidence about the true impact of these types of apps. The portability of mobile phones enables users to have access 24 hours a day, making possible the long-term management and reinforcement of health behaviors through a variety of communications and apps. Fitness and weight-loss mobile phone apps allow for the tracking of diet, weight, and physical activity; making grocery and restaurant decisions; cooking healthy meals [28]; or gamification of the intervention. Moreover, participants do not need to carry an extra piece of purchased technology, such as a pedometer, to track physical activity.\nSeveral limitations have been noted. Only a small number of available studies assess the effectiveness of mobile phone apps in weight-loss programs, and they included small sample sizes and short follow-up periods. The use of apps for improving physical activity and reducing anthropometric measures is relatively new. More randomized controlled trials with larger sample sizes and longer follow-up periods are needed to determine the effectiveness of mobile apps in improving health outcomes.\nA recent study aimed to evaluate diet/nutrition and anthropometric apps based on incorporation of features consistent with theories of behavior change; all apps were found to be very low in theoretical content or use of theory to guide behavior change [28]. The studies included in this meta-analysis also varied in the content and theoretical basis of the intervention. Further investigation into the effective features of the mobile phone apps and the interventions’ consistencies with theories of behavior change was not possible; this should be considered an area for future research.\nThe risk of bias was high in most of the studies and future research should improve on several issues, such as the use of blinding or improving attrition rate. All studies, except for one [19], failed to conceal the blinding of participants and personnel, and only one study [24] blinded the research staff collecting the data on outcomes as to the allocation of participants. Given the nature of the intervention, blinding of participants and personnel is difficult. However, it is important to recognize the possible influence of patient and personnel expectations. Therefore, adoption of blinding techniques, such as the use of sham procedures, blinding participants to the study hypothesis, or using a blinded centralized assessment of primary outcomes, will improve the quality of the evidence [30].\nA large attrition rate was noted in some of the studies [17,20] included in this meta-analysis. High attrition rates are common in weight-loss interventions, and the reasons for this are likely complex and varied [31]. Attrition has an obvious impact on the validity of results obtained and can introduce bias; for example, those more motivated to reduce their weight or increase physical activity may remain in the trial. Moreover, several studies found that most participants rarely used the app after the first month of the study [20,23]. As with other weight-loss interventions, the most effective app may be one that can engage people for the longest period. It is known that adherence to self-monitoring of food intake is associated with twice as much weight loss as infrequent monitoring [32]. Without the participant’s active engagement, the app is not likely to be used and as a result will not be effective [28]. The most highly ranked engagement strategies identified are (in order of preference) ease of use, design aesthetic, feedback, function, ability to change design to suit own preference, tailored information, and unique mobile phone features [33]. Weight-loss apps may need to be substantially more engaging or less time-consuming to produce weight reduction in the average individual. It would be useful to design strategies to increase “app appeal” before implementing this type of intervention. Gamification of the app, financial incentives, or delivering the app in a setting of group competition could be important adjuncts to increase motivation to use the app and lose weight [20,34].\nTo our knowledge, this is the first meta-analysis to summarize the effectiveness of mobile apps designed to improve physical activity and reduce anthropometric measures. Our meta-analysis highlighted the need to perform larger, high-quality, randomized controlled clinical trials with longer follow-up. The number of available mobile phone apps is growing steadily, and mobile phones are constantly undergoing updates so the features have changed over time. Incorporating features consistent with theories of behavior change into health-related apps would be useful to improve weight-loss outcomes [35]. We searched several databases in order to avoid publication bias, which is a concern in meta-analyses that only include published studies. Using funnel plots in our meta-analysis made it possible to exclude publication bias with some confidence.\nAs the world’s understanding of health and how to empower individuals to take better care of their health changes, health professionals must treat this change as progress. However, we must ensure that the patient is using a mobile app with appropriate quality guarantees. This meta-analysis aimed to provide a rigorous, systematic, and quantitative review of the studies that have analyzed the effectiveness of mobile apps and attempted to measure their influence on lifestyle changes. Bombarded by information overload in all arenas, health professionals and managers have a need for the insights provided by a tool like the meta-analysis; this could help them make decisions and decide what direction we should be moving in our efforts to promote weight loss, increase physical activity, and confront the public health crisis presented by overweight and obesity.\nIn summary, the results from this meta-analysis demonstrated that interventions based on mobile phone apps are associated with more weight loss than other types of interventions. Furthermore, a nonsignificant increase in physical activity was detected. Evidence form this meta-analysis shows that mobile phone app-based intervention may be useful tools for weight loss."],"string":"[\n \"Overweight and obesity are a global public health issue and an important feature in discussions about strategies for primary and secondary health care. Developing since the 1960s and now gathering pace rapidly, the issue is contributing, together with population aging, to major increases in the prevalence of high blood pressure and cholesterol levels, type 2 diabetes, and cancers [1]. Mortality rates are increasing with increasing degrees of overweight, as measured by body mass index (BMI) [2].\\nIn 2008, 35% of adults older than 20 years were overweight (BMI ≥ 25 kg/m2) and the worldwide prevalence of obesity (BMI ≥30 kg/m2) had nearly doubled since 1980, from 5% of men and 8% of women to 10% and 14%, respectively [2]. An estimated 205 million men and 297 million women were obese—a total of more than half a billion adults worldwide [3]. For these reasons, identifying effective interventions is an important component in public health efforts to curb obesity, but the most effective strategies for weight loss remain unclear.\\nWith the extensive market penetration of mobile phones, the International Telecommunications Union (ITU) reports that as of 2015, there are more than 7 billion mobile cellular subscriptions worldwide, corresponding to a 97% penetration rate—defined by ITU as mobile cellular telephone subscribers per 100 inhabitants [4]. Advanced-feature mobile phones (those with computer operating systems) have broadened the functions of mobile phones considerably. Mobile phone apps meet a variety of user needs, and are designed and adapted for each type of mobile device; therefore, they are applicable in nearly all social and economic sectors and environments. At present, these apps, apart from their recreational function, are becoming instruments of patient education and support and are also helpful to health care professionals [5]. Nonetheless, the market for health care apps is very fragmented because many of them are very specific or directed at minority diseases or specialties. The world market for medical apps for mobile phones and tablets multiplied seven times over in 2011 alone, reaching a total of US $718 million according to a market analysis by the American firm research2guidance [6]. A recent analysis of app store catalogs identified more than 97,000 mHealth apps, most of them dealing with general health and physical fitness; in general, they facilitate the monitoring of various parameters by individual users and provide general information and support related to those topics [5]. Previous research has suggested that mobile apps may be beneficial in asthma control [7] and diabetes management [8,9].\\nTo our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity. The objective of this study was to perform a systematic review and meta-analysis of published studies to evaluate the efficacy of interventions that included mobile phone apps compared with other interventions to reduce weight and increase physical activity in populations of children and adults.\",\n \" Search Strategy We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\\nWe conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\\n Study Selection Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\\nFlowchart for the selection of the articles in this meta-analysis.\\nTwo members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\\nFlowchart for the selection of the articles in this meta-analysis.\\n Data Extraction and Quality Assessment Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\\nTwo investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\\n Statistical Methods For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\\nSD2\\ndiff= SD2\\npre+ SD2\\npost- 2×ρ×SDpre×SDpost\\n\\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\\nFor each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\\nSD2\\ndiff= SD2\\npre+ SD2\\npost- 2×ρ×SDpre×SDpost\\n\\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\",\n \"We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\",\n \"Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\\nFlowchart for the selection of the articles in this meta-analysis.\",\n \"Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\",\n \"For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\\nSD2\\ndiff= SD2\\npre+ SD2\\npost- 2×ρ×SDpre×SDpost\\n\\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\",\n \" Study Selection The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\\nCharacteristics of included clinical trials.\\n\\naSS: sample size.\\n\\nbCCS: case-control study.\\n\\ncN/A: not applicable.\\n\\ndBMI: body mass index.\\n\\neRCT: randomized controlled trial.\\n\\nfMCCT: matched case-control trial.\\n\\ngMPA: moderate physical activity.\\n\\nhVPA: vigorous physical activity.\\nCharacteristics of intervention types and description of apps.\\n\\naBMI: body mass index.\\n\\nbMPA: moderate physical activity.\\n\\ncVPA: vigorous physical activity.\\n\\ndSMS: short message service.\\nThe search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\\nCharacteristics of included clinical trials.\\n\\naSS: sample size.\\n\\nbCCS: case-control study.\\n\\ncN/A: not applicable.\\n\\ndBMI: body mass index.\\n\\neRCT: randomized controlled trial.\\n\\nfMCCT: matched case-control trial.\\n\\ngMPA: moderate physical activity.\\n\\nhVPA: vigorous physical activity.\\nCharacteristics of intervention types and description of apps.\\n\\naBMI: body mass index.\\n\\nbMPA: moderate physical activity.\\n\\ncVPA: vigorous physical activity.\\n\\ndSMS: short message service.\\n Meta-Analysis of Mobile App Intervention and Body Weight Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\nData from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\n Meta-Analysis of Mobile App Intervention and Body Mass Index Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\nData from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\n Meta-Analysis of Mobile App Intervention and Physical Activity Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\nData from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\n Risk of Bias in Included Studies Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\\nRandomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\",\n \"The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\\nCharacteristics of included clinical trials.\\n\\naSS: sample size.\\n\\nbCCS: case-control study.\\n\\ncN/A: not applicable.\\n\\ndBMI: body mass index.\\n\\neRCT: randomized controlled trial.\\n\\nfMCCT: matched case-control trial.\\n\\ngMPA: moderate physical activity.\\n\\nhVPA: vigorous physical activity.\\nCharacteristics of intervention types and description of apps.\\n\\naBMI: body mass index.\\n\\nbMPA: moderate physical activity.\\n\\ncVPA: vigorous physical activity.\\n\\ndSMS: short message service.\",\n \"Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\",\n \"The current meta-analysis suggested that mobile phone app interventions compared with various control interventions significantly reduced body weight by 1.04 kg, reduced BMI by 0.43 kg/m2, and nonsignificantly increased physical activity by an SMD of 0.40. Our findings were robust across sensitivity analyses. Although the mean reductions in body weight and BMI were modest, it would not be expected for a single change in weight-loss interventions, such as mobile phone apps, to cause clinically meaningful weight loss compared with other control interventions [25]. Many of the control group treatments were other interventions. This could dilute the analysis, as it is possible that in some of the studies the treatment group showed a significant change, while the control group also showed a similar significant result. In our sensitivity analyses, the results were not modified when we excluded one study that did not describe if the control group had received any intervention [13].\\nSome of the most recognizable research in mobile interventions has focused on text-messaging interventions, or SMS. A previous meta-analysis [26] found that mobile phone interventions were associated with significant changes in body weight and BMI compared with the control group (-1.44 kg and -0.24 units, respectively). This meta-analysis included only mobile interventions based on contacts by SMS and multimedia message services (MMS). A previous systematic review found strong evidence from the included RCT that weight loss occurs in the short term because of mobile technology interventions [27]. Another systematic review that included seven articles demonstrated a beneficial impact of text messaging or a mobile app for reducing physical inactivity and/or overweight/obesity [28]. Finally, a recent systematic review found that most apps that are focused on weight loss have inconsistent outcomes [29].\\nTo our knowledge, this study is the first meta-analysis to summarize the evidence to date regarding effects of mobile phone app interventions compared with various control interventions. We excluded from our meta-analysis interventions based only on text messaging and focused solely on mobile phone apps because text-message interventions do not utilize the full potential of mobile phone technologies. Well-designed apps expand the potential for technology-based health interventions to impact populations in ways that previously were not possible and cannot be achieved without the capabilities of mobile phone software. Therefore, the need to regulate this growing market is becoming a concern, with increased advertising claims about effectiveness and researchers emphasizing the need for studies that will contribute scientific evidence about the true impact of these types of apps. The portability of mobile phones enables users to have access 24 hours a day, making possible the long-term management and reinforcement of health behaviors through a variety of communications and apps. Fitness and weight-loss mobile phone apps allow for the tracking of diet, weight, and physical activity; making grocery and restaurant decisions; cooking healthy meals [28]; or gamification of the intervention. Moreover, participants do not need to carry an extra piece of purchased technology, such as a pedometer, to track physical activity.\\nSeveral limitations have been noted. Only a small number of available studies assess the effectiveness of mobile phone apps in weight-loss programs, and they included small sample sizes and short follow-up periods. The use of apps for improving physical activity and reducing anthropometric measures is relatively new. More randomized controlled trials with larger sample sizes and longer follow-up periods are needed to determine the effectiveness of mobile apps in improving health outcomes.\\nA recent study aimed to evaluate diet/nutrition and anthropometric apps based on incorporation of features consistent with theories of behavior change; all apps were found to be very low in theoretical content or use of theory to guide behavior change [28]. The studies included in this meta-analysis also varied in the content and theoretical basis of the intervention. Further investigation into the effective features of the mobile phone apps and the interventions’ consistencies with theories of behavior change was not possible; this should be considered an area for future research.\\nThe risk of bias was high in most of the studies and future research should improve on several issues, such as the use of blinding or improving attrition rate. All studies, except for one [19], failed to conceal the blinding of participants and personnel, and only one study [24] blinded the research staff collecting the data on outcomes as to the allocation of participants. Given the nature of the intervention, blinding of participants and personnel is difficult. However, it is important to recognize the possible influence of patient and personnel expectations. Therefore, adoption of blinding techniques, such as the use of sham procedures, blinding participants to the study hypothesis, or using a blinded centralized assessment of primary outcomes, will improve the quality of the evidence [30].\\nA large attrition rate was noted in some of the studies [17,20] included in this meta-analysis. High attrition rates are common in weight-loss interventions, and the reasons for this are likely complex and varied [31]. Attrition has an obvious impact on the validity of results obtained and can introduce bias; for example, those more motivated to reduce their weight or increase physical activity may remain in the trial. Moreover, several studies found that most participants rarely used the app after the first month of the study [20,23]. As with other weight-loss interventions, the most effective app may be one that can engage people for the longest period. It is known that adherence to self-monitoring of food intake is associated with twice as much weight loss as infrequent monitoring [32]. Without the participant’s active engagement, the app is not likely to be used and as a result will not be effective [28]. The most highly ranked engagement strategies identified are (in order of preference) ease of use, design aesthetic, feedback, function, ability to change design to suit own preference, tailored information, and unique mobile phone features [33]. Weight-loss apps may need to be substantially more engaging or less time-consuming to produce weight reduction in the average individual. It would be useful to design strategies to increase “app appeal” before implementing this type of intervention. Gamification of the app, financial incentives, or delivering the app in a setting of group competition could be important adjuncts to increase motivation to use the app and lose weight [20,34].\\nTo our knowledge, this is the first meta-analysis to summarize the effectiveness of mobile apps designed to improve physical activity and reduce anthropometric measures. Our meta-analysis highlighted the need to perform larger, high-quality, randomized controlled clinical trials with longer follow-up. The number of available mobile phone apps is growing steadily, and mobile phones are constantly undergoing updates so the features have changed over time. Incorporating features consistent with theories of behavior change into health-related apps would be useful to improve weight-loss outcomes [35]. We searched several databases in order to avoid publication bias, which is a concern in meta-analyses that only include published studies. Using funnel plots in our meta-analysis made it possible to exclude publication bias with some confidence.\\nAs the world’s understanding of health and how to empower individuals to take better care of their health changes, health professionals must treat this change as progress. However, we must ensure that the patient is using a mobile app with appropriate quality guarantees. This meta-analysis aimed to provide a rigorous, systematic, and quantitative review of the studies that have analyzed the effectiveness of mobile apps and attempted to measure their influence on lifestyle changes. Bombarded by information overload in all arenas, health professionals and managers have a need for the insights provided by a tool like the meta-analysis; this could help them make decisions and decide what direction we should be moving in our efforts to promote weight loss, increase physical activity, and confront the public health crisis presented by overweight and obesity.\\nIn summary, the results from this meta-analysis demonstrated that interventions based on mobile phone apps are associated with more weight loss than other types of interventions. Furthermore, a nonsignificant increase in physical activity was detected. Evidence form this meta-analysis shows that mobile phone app-based intervention may be useful tools for weight loss.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["introduction","methods",null,null,null,null,"results",null,null,null,null,null,"discussion"],"string":"[\n \"introduction\",\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["mHealth","mobile phone","apps","obesity","physical activity","intervention"],"string":"[\n \"mHealth\",\n \"mobile phone\",\n \"apps\",\n \"obesity\",\n \"physical activity\",\n \"intervention\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nOverweight and obesity are a global public health issue and an important feature in discussions about strategies for primary and secondary health care. Developing since the 1960s and now gathering pace rapidly, the issue is contributing, together with population aging, to major increases in the prevalence of high blood pressure and cholesterol levels, type 2 diabetes, and cancers [1]. Mortality rates are increasing with increasing degrees of overweight, as measured by body mass index (BMI) [2].\nIn 2008, 35% of adults older than 20 years were overweight (BMI ≥ 25 kg/m2) and the worldwide prevalence of obesity (BMI ≥30 kg/m2) had nearly doubled since 1980, from 5% of men and 8% of women to 10% and 14%, respectively [2]. An estimated 205 million men and 297 million women were obese—a total of more than half a billion adults worldwide [3]. For these reasons, identifying effective interventions is an important component in public health efforts to curb obesity, but the most effective strategies for weight loss remain unclear.\nWith the extensive market penetration of mobile phones, the International Telecommunications Union (ITU) reports that as of 2015, there are more than 7 billion mobile cellular subscriptions worldwide, corresponding to a 97% penetration rate—defined by ITU as mobile cellular telephone subscribers per 100 inhabitants [4]. Advanced-feature mobile phones (those with computer operating systems) have broadened the functions of mobile phones considerably. Mobile phone apps meet a variety of user needs, and are designed and adapted for each type of mobile device; therefore, they are applicable in nearly all social and economic sectors and environments. At present, these apps, apart from their recreational function, are becoming instruments of patient education and support and are also helpful to health care professionals [5]. Nonetheless, the market for health care apps is very fragmented because many of them are very specific or directed at minority diseases or specialties. The world market for medical apps for mobile phones and tablets multiplied seven times over in 2011 alone, reaching a total of US $718 million according to a market analysis by the American firm research2guidance [6]. A recent analysis of app store catalogs identified more than 97,000 mHealth apps, most of them dealing with general health and physical fitness; in general, they facilitate the monitoring of various parameters by individual users and provide general information and support related to those topics [5]. Previous research has suggested that mobile apps may be beneficial in asthma control [7] and diabetes management [8,9].\nTo our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity. The objective of this study was to perform a systematic review and meta-analysis of published studies to evaluate the efficacy of interventions that included mobile phone apps compared with other interventions to reduce weight and increase physical activity in populations of children and adults.\n\nMethods:\n Search Strategy We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\nWe conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\n Study Selection Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\nTwo members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\n Data Extraction and Quality Assessment Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\nTwo investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\n Statistical Methods For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\nFor each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\n\nSearch Strategy:\nWe conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\n\nStudy Selection:\nTwo members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\n\nData Extraction and Quality Assessment:\nTwo investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\n\nStatistical Methods:\nFor each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\n\nResults:\n Study Selection The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\nThe search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\n Meta-Analysis of Mobile App Intervention and Body Weight Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Body Mass Index Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Physical Activity Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Risk of Bias in Included Studies Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\nRandomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\n\nStudy Selection:\nThe search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\n\nMeta-Analysis of Mobile App Intervention and Body Weight:\nData from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n\nMeta-Analysis of Mobile App Intervention and Body Mass Index:\nData from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n\nMeta-Analysis of Mobile App Intervention and Physical Activity:\nData from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n\nRisk of Bias in Included Studies:\nRandomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\n\nDiscussion:\nThe current meta-analysis suggested that mobile phone app interventions compared with various control interventions significantly reduced body weight by 1.04 kg, reduced BMI by 0.43 kg/m2, and nonsignificantly increased physical activity by an SMD of 0.40. Our findings were robust across sensitivity analyses. Although the mean reductions in body weight and BMI were modest, it would not be expected for a single change in weight-loss interventions, such as mobile phone apps, to cause clinically meaningful weight loss compared with other control interventions [25]. Many of the control group treatments were other interventions. This could dilute the analysis, as it is possible that in some of the studies the treatment group showed a significant change, while the control group also showed a similar significant result. In our sensitivity analyses, the results were not modified when we excluded one study that did not describe if the control group had received any intervention [13].\nSome of the most recognizable research in mobile interventions has focused on text-messaging interventions, or SMS. A previous meta-analysis [26] found that mobile phone interventions were associated with significant changes in body weight and BMI compared with the control group (-1.44 kg and -0.24 units, respectively). This meta-analysis included only mobile interventions based on contacts by SMS and multimedia message services (MMS). A previous systematic review found strong evidence from the included RCT that weight loss occurs in the short term because of mobile technology interventions [27]. Another systematic review that included seven articles demonstrated a beneficial impact of text messaging or a mobile app for reducing physical inactivity and/or overweight/obesity [28]. Finally, a recent systematic review found that most apps that are focused on weight loss have inconsistent outcomes [29].\nTo our knowledge, this study is the first meta-analysis to summarize the evidence to date regarding effects of mobile phone app interventions compared with various control interventions. We excluded from our meta-analysis interventions based only on text messaging and focused solely on mobile phone apps because text-message interventions do not utilize the full potential of mobile phone technologies. Well-designed apps expand the potential for technology-based health interventions to impact populations in ways that previously were not possible and cannot be achieved without the capabilities of mobile phone software. Therefore, the need to regulate this growing market is becoming a concern, with increased advertising claims about effectiveness and researchers emphasizing the need for studies that will contribute scientific evidence about the true impact of these types of apps. The portability of mobile phones enables users to have access 24 hours a day, making possible the long-term management and reinforcement of health behaviors through a variety of communications and apps. Fitness and weight-loss mobile phone apps allow for the tracking of diet, weight, and physical activity; making grocery and restaurant decisions; cooking healthy meals [28]; or gamification of the intervention. Moreover, participants do not need to carry an extra piece of purchased technology, such as a pedometer, to track physical activity.\nSeveral limitations have been noted. Only a small number of available studies assess the effectiveness of mobile phone apps in weight-loss programs, and they included small sample sizes and short follow-up periods. The use of apps for improving physical activity and reducing anthropometric measures is relatively new. More randomized controlled trials with larger sample sizes and longer follow-up periods are needed to determine the effectiveness of mobile apps in improving health outcomes.\nA recent study aimed to evaluate diet/nutrition and anthropometric apps based on incorporation of features consistent with theories of behavior change; all apps were found to be very low in theoretical content or use of theory to guide behavior change [28]. The studies included in this meta-analysis also varied in the content and theoretical basis of the intervention. Further investigation into the effective features of the mobile phone apps and the interventions’ consistencies with theories of behavior change was not possible; this should be considered an area for future research.\nThe risk of bias was high in most of the studies and future research should improve on several issues, such as the use of blinding or improving attrition rate. All studies, except for one [19], failed to conceal the blinding of participants and personnel, and only one study [24] blinded the research staff collecting the data on outcomes as to the allocation of participants. Given the nature of the intervention, blinding of participants and personnel is difficult. However, it is important to recognize the possible influence of patient and personnel expectations. Therefore, adoption of blinding techniques, such as the use of sham procedures, blinding participants to the study hypothesis, or using a blinded centralized assessment of primary outcomes, will improve the quality of the evidence [30].\nA large attrition rate was noted in some of the studies [17,20] included in this meta-analysis. High attrition rates are common in weight-loss interventions, and the reasons for this are likely complex and varied [31]. Attrition has an obvious impact on the validity of results obtained and can introduce bias; for example, those more motivated to reduce their weight or increase physical activity may remain in the trial. Moreover, several studies found that most participants rarely used the app after the first month of the study [20,23]. As with other weight-loss interventions, the most effective app may be one that can engage people for the longest period. It is known that adherence to self-monitoring of food intake is associated with twice as much weight loss as infrequent monitoring [32]. Without the participant’s active engagement, the app is not likely to be used and as a result will not be effective [28]. The most highly ranked engagement strategies identified are (in order of preference) ease of use, design aesthetic, feedback, function, ability to change design to suit own preference, tailored information, and unique mobile phone features [33]. Weight-loss apps may need to be substantially more engaging or less time-consuming to produce weight reduction in the average individual. It would be useful to design strategies to increase “app appeal” before implementing this type of intervention. Gamification of the app, financial incentives, or delivering the app in a setting of group competition could be important adjuncts to increase motivation to use the app and lose weight [20,34].\nTo our knowledge, this is the first meta-analysis to summarize the effectiveness of mobile apps designed to improve physical activity and reduce anthropometric measures. Our meta-analysis highlighted the need to perform larger, high-quality, randomized controlled clinical trials with longer follow-up. The number of available mobile phone apps is growing steadily, and mobile phones are constantly undergoing updates so the features have changed over time. Incorporating features consistent with theories of behavior change into health-related apps would be useful to improve weight-loss outcomes [35]. We searched several databases in order to avoid publication bias, which is a concern in meta-analyses that only include published studies. Using funnel plots in our meta-analysis made it possible to exclude publication bias with some confidence.\nAs the world’s understanding of health and how to empower individuals to take better care of their health changes, health professionals must treat this change as progress. However, we must ensure that the patient is using a mobile app with appropriate quality guarantees. This meta-analysis aimed to provide a rigorous, systematic, and quantitative review of the studies that have analyzed the effectiveness of mobile apps and attempted to measure their influence on lifestyle changes. Bombarded by information overload in all arenas, health professionals and managers have a need for the insights provided by a tool like the meta-analysis; this could help them make decisions and decide what direction we should be moving in our efforts to promote weight loss, increase physical activity, and confront the public health crisis presented by overweight and obesity.\nIn summary, the results from this meta-analysis demonstrated that interventions based on mobile phone apps are associated with more weight loss than other types of interventions. Furthermore, a nonsignificant increase in physical activity was detected. Evidence form this meta-analysis shows that mobile phone app-based intervention may be useful tools for weight loss.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nTo our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity.\n\nMethods:\nWe conducted a systematic review and meta-analysis of relevant studies identified by a search of PubMed, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), and Scopus from their inception through to August 2015. Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted data. We included all controlled studies that assessed a mobile phone app intervention with weight-related health measures (ie, body weight, body mass index, or waist circumference) or physical activity outcomes. Net change estimates comparing the intervention group with the control group were pooled across studies using random-effects models.\n\nResults:\nWe included 12 articles in this systematic review and meta-analysis. Compared with the control group, use of a mobile phone app was associated with significant changes in body weight (kg) and body mass index (kg/m(2)) of -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) and -0.43 kg/m(2) (95% CI -0.74 to -0.13; I2 = 50%), respectively. Moreover, a nonsignificant difference in physical activity was observed between the two groups (standardized mean difference 0.40, 95% CI -0.07 to 0.87; I2 = 93%). These findings were remarkably robust in the sensitivity analysis. No publication bias was shown.\n\nConclusions:\nEvidence from this study shows that mobile phone app-based interventions may be useful tools for weight loss."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":8752,"string":"8,752"},"whole_article_abstract_length":{"kind":"number","value":320,"string":"320"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["study","mobile","intervention","studies","weight","physical","analysis","activity","physical activity","control"],"string":"[\n \"study\",\n \"mobile\",\n \"intervention\",\n \"studies\",\n \"weight\",\n \"physical\",\n \"analysis\",\n \"activity\",\n \"physical activity\",\n \"control\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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| Humans | Mobile Applications | Motor Activity | Obesity | Weight Loss [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] study | mobile | intervention | studies | weight | physical | analysis | activity | physical activity | control 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[SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] intervention | sd | study | baseline | end | end intervention | weight | body weight | search | baseline end intervention [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] mobile | pooled | kg | change | intervention | study | trials | 95 | app | 13 [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mobile | study | intervention | physical | weight | studies | physical activity | activity | change | app [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] PubMed | Allied Health Literature (CINAHL | August 2015 ||| Two ||| ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 12 ||| kg/m(2 | -1.04 kg | 95% | CI | I2 | 41% | 95% | CI | I2 | 50% ||| two | 0.40 | 95% | CI | 0.87 | I2 | 93% ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| PubMed | Allied Health Literature (CINAHL | August 2015 ||| Two ||| ||| ||| ||| 12 ||| kg/m(2 | -1.04 kg | 95% | CI | I2 | 41% | 95% | CI | I2 | 50% ||| two | 0.40 | 95% | CI | 0.87 | I2 | 93% ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":288,"cells":{"title":{"kind":"string","value":"Association of simultaneously measured four-limb blood pressures with cardiovascular function: a cross-sectional study."},"pmid":{"kind":"string","value":"28155695"},"background_abstract":{"kind":"string","value":"Simultaneous measurement of four-limb blood pressures can improve the accuracy of cardiovascular disease diagnosis. This study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the non-invasive diagnostic method of cardiovascular disease in primary care."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled. Four-limb blood pressure measurements were simultaneously performed using a blood pressure and pulse monitor device in the supine position. Cardiac functional parameters were also measured by using a cardiac hemodynamic detector in the same position. Data were statistically analyzed with SPSS15.0."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The mean age of the 229 subjects was 60.56 ± 8.68 years. Cardiovascular functional parameters decreased with age and body mass index (BMI), only the total peripheral resistance (TPR) was in contrast. Age, BMI, left ankle diastolic pressure (LADP), high arm mean arterial pressure (HARMAP), left arm diastolic pressure (LARDP) and right ankle diastolic pressure (RADP) were significantly correlated with cardiovascular functional parameters. Cardiovascular functional parameters have significant differences with inter-arm difference in systolic blood pressure (SBP) between ≥10 and <10 mmHg, inter-ankle difference in SBP between ≥15 and ≥20 mmHg, inter-ankle difference in SBP between ≥15 and <10 mmHg and right ankle brachial index (RABI) between ≤0.9 and ≥1.0. After excluding 99 hypertensive patients, a part of cardiovascular functional parameters has still significant differences with inter-arm difference in SBP between ≥10 and ≥15 mmHg and RABI between ≤0.9 and ≥1.0."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Age, BMI, LADP, HARMAP, LARDP and RADP were the determinants of cardiovascular functional parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in SBP ≥10 mmHg, inter-ankle difference in SBP ≥15 mmHg and RABI ≤0.9, while these differences still existed after excluding 99 hypertensive patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the non-invasive diagnostic method of cardiovascular disease in primary care."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Ankle Brachial Index","Arterial Pressure","Blood Pressure","Blood Pressure Determination","Cardiovascular Diseases","Cardiovascular System","Computer Simulation","Cross-Sectional Studies","Female","Hemodynamics","Humans","Hypertension","Linear Models","Male","Middle Aged","Models, Statistical","Primary Health Care","Risk Assessment"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Ankle Brachial Index\",\n \"Arterial Pressure\",\n \"Blood Pressure\",\n \"Blood Pressure Determination\",\n \"Cardiovascular Diseases\",\n \"Cardiovascular System\",\n \"Computer Simulation\",\n \"Cross-Sectional Studies\",\n \"Female\",\n \"Hemodynamics\",\n \"Humans\",\n \"Hypertension\",\n \"Linear Models\",\n \"Male\",\n \"Middle Aged\",\n \"Models, Statistical\",\n \"Primary Health Care\",\n \"Risk Assessment\"\n]"},"pmcid":{"kind":"string","value":"5259996"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Baseline characteristics of subjects The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\nThe mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\n Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\nPearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\n Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\nMultiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\n Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\nIn additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\nSince cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"LADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care."},"other_sections_titles":{"kind":"list like","value":["Background","Subjects","Four-limb blood pressure measurements","Cardiovascular function measurements","Statistical analysis","Baseline characteristics of subjects","Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters","Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters","Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters","Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup"],"string":"[\n \"Background\",\n \"Subjects\",\n \"Four-limb blood pressure measurements\",\n \"Cardiovascular function measurements\",\n \"Statistical analysis\",\n \"Baseline characteristics of subjects\",\n \"Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters\",\n \"Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters\",\n \"Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters\",\n \"Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup\"\n]"},"other_sections_texts":{"kind":"list like","value":["Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.","This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.","Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.","Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.","Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.","The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants","Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.","Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters","In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences","Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0."],"string":"[\n \"Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.\",\n \"This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\",\n \"Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\",\n \"Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\n\\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\",\n \"Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\",\n \"The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\\n\\nBaseline characteristics of the study participants\",\n \"Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\",\n \"Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\\n\\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\",\n \"In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\n\\nThe distribution of four-limb blood pressure difference in the subjects\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\",\n \"Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Subjects","Four-limb blood pressure measurements","Cardiovascular function measurements","Statistical analysis","Results","Baseline characteristics of subjects","Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters","Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters","Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters","Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup","Discussion","Conclusion"],"string":"[\n \"Background\",\n \"Methods\",\n \"Subjects\",\n \"Four-limb blood pressure measurements\",\n \"Cardiovascular function measurements\",\n \"Statistical analysis\",\n \"Results\",\n \"Baseline characteristics of subjects\",\n \"Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters\",\n \"Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters\",\n \"Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters\",\n \"Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup\",\n \"Discussion\",\n \"Conclusion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care."," Subjects This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\nThis study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\n Four-limb blood pressure measurements Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\nFour-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\n Cardiovascular function measurements Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\nCardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\n Statistical analysis Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\nData were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.","This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.","Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.","Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.","Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05."," Baseline characteristics of subjects The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\nThe mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\n Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\nPearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\n Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\nMultiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\n Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\nIn additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\nSince cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.","The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants","Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.","Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters","In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences","Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.","In this cross-sectional study, by using a simultaneous measurement technique, the association between four-limb blood pressure and cardiovascular function and related risk factors was evaluated. Cardiovascular functional parameters illustrated the cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity. Thus, this study has more systematicness and pertinence than previous studies. This study suggested that cardiovascular functional parameters (CO, SV, CI, SVI, EF, IC, HI, EDV, LVEDP, SWI, AC and TPR) have all a significant difference with HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP, while there was a negative correlation between them except TPR. Meanwhile, the determinants of cardiac functional parameter were age, BMI, LADP, HARMAP LARDP, HARPPI ADBPD, RARSP, RADP and LARSP, while the TPR was positively correlated with age, BMI, LADP and HARMAP.\nPrevious studies demonstrated that population aging has become the uncontrolled risk factor of cardiovascular disease [16–18]. It is a physiology problem that the effect between blood pressure and cardiovascular function is mutual. The cardiovascular function decreased gradually with age, especially arteries can produce physiological degeneration. The thickening of vessels wall, the decrease or even rupture and calcification of elastic fibers, the increase of peripheral resistance, the decrease of blood vessel compliance, all these factors will result in the systolic blood pressure and diastolic blood pressure increasing with age. In addition, it is a fluid mechanics problem that different arteries away from heart have different resistance and pressure. According to the Poiseuille’s law, brachial artery resistance is relatively small and the blood pressure is low because heart is close to the upper limb, while the phenomenon of the ankle artery is in contrast. Hence blood pressures in four limbs are different. In addition, the distance from the heart affects pressure wave propagation and reflection because of the geometry tapper and elasticity tapper, and augments the pressure of peripheral vascular compared with that of upper limb.\nPrevious studies have reported that inter-arm or inter-ankle blood pressure difference predicted cardiovascular mortality [1, 2, 8–13, 15]. These results suggested that the simultaneous measurement of four-limb blood pressure is needed to improve diagnostic accuracy between cardiovascular disease and blood pressure difference [2–5]. The present study demonstrated the significant correlation between cardiovascular functional parameters and four-limb blood pressure, while cardiovascular functional parameters had no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI. However, the mean plots of cardiovascular functional parameters present obvious change trend in four-limb systolic blood pressure difference and diastolic blood pressure difference ≥10 or ≥15 mmHg. These results were different form previous studies. Verberk et al. [19] reported that the prevalence of inter-arm difference in systolic blood pressure of 10 mmHg or more was roughly doubled when diagnosis measurements method used a sequential approach, or used manual approach rather than automated measurements approach. Thus, simultaneous measurement of four-limb blood pressure and calculation of four-limb blood pressure difference may be helpful and necessary in evaluating the heart function and predicting patients with cardiovascular disease. Further study, the subgroup analysis showed that the predictive value of four limbs blood pressure for cardiovascular function. Subgroup analysis found that a part of cardiovascular functional parameter has still significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and with RABI between ≤0.9 and ≥1.0. However, the exact reason is unclear now, hypertension is the influence factors of cardiovascular function parameters, and four-limb blood pressure differences are also applicable to diagnose hypertensive patients. As mentioned above, four limbs blood pressures were not only influence factors of cardiovascular function parameters, but a predictor for cardiovascular disease, evaluating cardiovascular function parameters was important in clinical practice and epidemiological studies. In addition, the better way for cardiovascular function parameters was simultaneous blood pressures for four limbs.\nA clinical device, the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China), has been developed to automatically and simultaneously measure blood pressure in four limbs, and the measurement can be easily obtained. The similar device was reported by previous studies, for example, VP-1000 ABI—form device (Colin Co. Ltd., Komaki, Japan) and VP-1000 device (Omron, Kyoto, Japan) were used to automatically and simultaneously measure four limbs’ blood pressures. The validation in measurement technology of device (Boundless Horizon Company, Shandong, China) might be prone for measurement errors, especially in multinomial detection of cardiac function parameters rather than single detection. Although it could not give very precise measurements for the clinical value of cardiac function parameters, it can provide objective evaluation of health in the early stage of cardiovascular diseases. Thus, these devices can be applied for the epidemiological evaluation between four limbs’ blood pressures and cardiac function parameters.\nIn this study, by using a simultaneous and noninvasive measurement technique, we measured four limbs blood pressures. But this technique was not a popular one to measure blood pressure in daily clinical practice. Hence, although four limbs blood pressure with simultaneous measurement could improve the predictive value for cardiovascular disease, our results might be changed if daily clinical measurement was used. In addition, the subjects were mainly from retired people, whose health care consciousness is better than the serving officer. The factors of smoking, drinking, salting and movement have no significant difference in this study subjects. Hence, the clinical utility of this study may be limited in community people.","LADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care."],"string":"[\n \"Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.\",\n \" Subjects This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\\nThis study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\\n Four-limb blood pressure measurements Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\\nFour-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\\n Cardiovascular function measurements Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\n\\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\\nCardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\n\\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\\n Statistical analysis Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\\nData were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\",\n \"This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\",\n \"Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\",\n \"Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\n\\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\",\n \"Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\",\n \" Baseline characteristics of subjects The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\\n\\nBaseline characteristics of the study participants\\nThe mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\\n\\nBaseline characteristics of the study participants\\n Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\\nPearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\\n Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\\n\\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\\nMultiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\\n\\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\\n Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\n\\nThe distribution of four-limb blood pressure difference in the subjects\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\nIn additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\n\\nThe distribution of four-limb blood pressure difference in the subjects\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\n Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\\nSince cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\",\n \"The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\\n\\nBaseline characteristics of the study participants\",\n \"Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\",\n \"Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\\n\\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\",\n \"In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\n\\nThe distribution of four-limb blood pressure difference in the subjects\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\",\n \"Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\",\n \"In this cross-sectional study, by using a simultaneous measurement technique, the association between four-limb blood pressure and cardiovascular function and related risk factors was evaluated. Cardiovascular functional parameters illustrated the cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity. Thus, this study has more systematicness and pertinence than previous studies. This study suggested that cardiovascular functional parameters (CO, SV, CI, SVI, EF, IC, HI, EDV, LVEDP, SWI, AC and TPR) have all a significant difference with HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP, while there was a negative correlation between them except TPR. Meanwhile, the determinants of cardiac functional parameter were age, BMI, LADP, HARMAP LARDP, HARPPI ADBPD, RARSP, RADP and LARSP, while the TPR was positively correlated with age, BMI, LADP and HARMAP.\\nPrevious studies demonstrated that population aging has become the uncontrolled risk factor of cardiovascular disease [16–18]. It is a physiology problem that the effect between blood pressure and cardiovascular function is mutual. The cardiovascular function decreased gradually with age, especially arteries can produce physiological degeneration. The thickening of vessels wall, the decrease or even rupture and calcification of elastic fibers, the increase of peripheral resistance, the decrease of blood vessel compliance, all these factors will result in the systolic blood pressure and diastolic blood pressure increasing with age. In addition, it is a fluid mechanics problem that different arteries away from heart have different resistance and pressure. According to the Poiseuille’s law, brachial artery resistance is relatively small and the blood pressure is low because heart is close to the upper limb, while the phenomenon of the ankle artery is in contrast. Hence blood pressures in four limbs are different. In addition, the distance from the heart affects pressure wave propagation and reflection because of the geometry tapper and elasticity tapper, and augments the pressure of peripheral vascular compared with that of upper limb.\\nPrevious studies have reported that inter-arm or inter-ankle blood pressure difference predicted cardiovascular mortality [1, 2, 8–13, 15]. These results suggested that the simultaneous measurement of four-limb blood pressure is needed to improve diagnostic accuracy between cardiovascular disease and blood pressure difference [2–5]. The present study demonstrated the significant correlation between cardiovascular functional parameters and four-limb blood pressure, while cardiovascular functional parameters had no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI. However, the mean plots of cardiovascular functional parameters present obvious change trend in four-limb systolic blood pressure difference and diastolic blood pressure difference ≥10 or ≥15 mmHg. These results were different form previous studies. Verberk et al. [19] reported that the prevalence of inter-arm difference in systolic blood pressure of 10 mmHg or more was roughly doubled when diagnosis measurements method used a sequential approach, or used manual approach rather than automated measurements approach. Thus, simultaneous measurement of four-limb blood pressure and calculation of four-limb blood pressure difference may be helpful and necessary in evaluating the heart function and predicting patients with cardiovascular disease. Further study, the subgroup analysis showed that the predictive value of four limbs blood pressure for cardiovascular function. Subgroup analysis found that a part of cardiovascular functional parameter has still significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and with RABI between ≤0.9 and ≥1.0. However, the exact reason is unclear now, hypertension is the influence factors of cardiovascular function parameters, and four-limb blood pressure differences are also applicable to diagnose hypertensive patients. As mentioned above, four limbs blood pressures were not only influence factors of cardiovascular function parameters, but a predictor for cardiovascular disease, evaluating cardiovascular function parameters was important in clinical practice and epidemiological studies. In addition, the better way for cardiovascular function parameters was simultaneous blood pressures for four limbs.\\nA clinical device, the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China), has been developed to automatically and simultaneously measure blood pressure in four limbs, and the measurement can be easily obtained. The similar device was reported by previous studies, for example, VP-1000 ABI—form device (Colin Co. Ltd., Komaki, Japan) and VP-1000 device (Omron, Kyoto, Japan) were used to automatically and simultaneously measure four limbs’ blood pressures. The validation in measurement technology of device (Boundless Horizon Company, Shandong, China) might be prone for measurement errors, especially in multinomial detection of cardiac function parameters rather than single detection. Although it could not give very precise measurements for the clinical value of cardiac function parameters, it can provide objective evaluation of health in the early stage of cardiovascular diseases. Thus, these devices can be applied for the epidemiological evaluation between four limbs’ blood pressures and cardiac function parameters.\\nIn this study, by using a simultaneous and noninvasive measurement technique, we measured four limbs blood pressures. But this technique was not a popular one to measure blood pressure in daily clinical practice. Hence, although four limbs blood pressure with simultaneous measurement could improve the predictive value for cardiovascular disease, our results might be changed if daily clinical measurement was used. In addition, the subjects were mainly from retired people, whose health care consciousness is better than the serving officer. The factors of smoking, drinking, salting and movement have no significant difference in this study subjects. Hence, the clinical utility of this study may be limited in community people.\",\n \"LADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"materials|methods",null,null,null,null,"results",null,null,null,null,null,"discussion","conclusion"],"string":"[\n null,\n \"materials|methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusion\"\n]"},"keywords":{"kind":"list like","value":["Cardiovascular function","Four limbs","Blood pressure difference","Simultaneous measurement"],"string":"[\n \"Cardiovascular function\",\n \"Four limbs\",\n \"Blood pressure difference\",\n \"Simultaneous measurement\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nAccurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.\n\nMethods:\n Subjects This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\nThis study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\n Four-limb blood pressure measurements Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\nFour-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\n Cardiovascular function measurements Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\nCardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\n Statistical analysis Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\nData were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\n\nSubjects:\nThis study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\n\nFour-limb blood pressure measurements:\nFour-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\n\nCardiovascular function measurements:\nCardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\n\nStatistical analysis:\nData were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\n\nResults:\n Baseline characteristics of subjects The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\nThe mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\n Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\nPearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\n Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\nMultiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\n Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\nIn additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\nSince cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\n\nBaseline characteristics of subjects:\nThe mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\n\nPearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters:\nPearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\n\nMultiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters:\nMultiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\n\nVariance analysis between four-limb blood pressure differences and cardiovascular functional parameters:\nIn additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nAnalysis between four-limb blood pressures and cardiovascular functional parameters in subgroup:\nSince cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\n\nDiscussion:\nIn this cross-sectional study, by using a simultaneous measurement technique, the association between four-limb blood pressure and cardiovascular function and related risk factors was evaluated. Cardiovascular functional parameters illustrated the cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity. Thus, this study has more systematicness and pertinence than previous studies. This study suggested that cardiovascular functional parameters (CO, SV, CI, SVI, EF, IC, HI, EDV, LVEDP, SWI, AC and TPR) have all a significant difference with HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP, while there was a negative correlation between them except TPR. Meanwhile, the determinants of cardiac functional parameter were age, BMI, LADP, HARMAP LARDP, HARPPI ADBPD, RARSP, RADP and LARSP, while the TPR was positively correlated with age, BMI, LADP and HARMAP.\nPrevious studies demonstrated that population aging has become the uncontrolled risk factor of cardiovascular disease [16–18]. It is a physiology problem that the effect between blood pressure and cardiovascular function is mutual. The cardiovascular function decreased gradually with age, especially arteries can produce physiological degeneration. The thickening of vessels wall, the decrease or even rupture and calcification of elastic fibers, the increase of peripheral resistance, the decrease of blood vessel compliance, all these factors will result in the systolic blood pressure and diastolic blood pressure increasing with age. In addition, it is a fluid mechanics problem that different arteries away from heart have different resistance and pressure. According to the Poiseuille’s law, brachial artery resistance is relatively small and the blood pressure is low because heart is close to the upper limb, while the phenomenon of the ankle artery is in contrast. Hence blood pressures in four limbs are different. In addition, the distance from the heart affects pressure wave propagation and reflection because of the geometry tapper and elasticity tapper, and augments the pressure of peripheral vascular compared with that of upper limb.\nPrevious studies have reported that inter-arm or inter-ankle blood pressure difference predicted cardiovascular mortality [1, 2, 8–13, 15]. These results suggested that the simultaneous measurement of four-limb blood pressure is needed to improve diagnostic accuracy between cardiovascular disease and blood pressure difference [2–5]. The present study demonstrated the significant correlation between cardiovascular functional parameters and four-limb blood pressure, while cardiovascular functional parameters had no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI. However, the mean plots of cardiovascular functional parameters present obvious change trend in four-limb systolic blood pressure difference and diastolic blood pressure difference ≥10 or ≥15 mmHg. These results were different form previous studies. Verberk et al. [19] reported that the prevalence of inter-arm difference in systolic blood pressure of 10 mmHg or more was roughly doubled when diagnosis measurements method used a sequential approach, or used manual approach rather than automated measurements approach. Thus, simultaneous measurement of four-limb blood pressure and calculation of four-limb blood pressure difference may be helpful and necessary in evaluating the heart function and predicting patients with cardiovascular disease. Further study, the subgroup analysis showed that the predictive value of four limbs blood pressure for cardiovascular function. Subgroup analysis found that a part of cardiovascular functional parameter has still significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and with RABI between ≤0.9 and ≥1.0. However, the exact reason is unclear now, hypertension is the influence factors of cardiovascular function parameters, and four-limb blood pressure differences are also applicable to diagnose hypertensive patients. As mentioned above, four limbs blood pressures were not only influence factors of cardiovascular function parameters, but a predictor for cardiovascular disease, evaluating cardiovascular function parameters was important in clinical practice and epidemiological studies. In addition, the better way for cardiovascular function parameters was simultaneous blood pressures for four limbs.\nA clinical device, the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China), has been developed to automatically and simultaneously measure blood pressure in four limbs, and the measurement can be easily obtained. The similar device was reported by previous studies, for example, VP-1000 ABI—form device (Colin Co. Ltd., Komaki, Japan) and VP-1000 device (Omron, Kyoto, Japan) were used to automatically and simultaneously measure four limbs’ blood pressures. The validation in measurement technology of device (Boundless Horizon Company, Shandong, China) might be prone for measurement errors, especially in multinomial detection of cardiac function parameters rather than single detection. Although it could not give very precise measurements for the clinical value of cardiac function parameters, it can provide objective evaluation of health in the early stage of cardiovascular diseases. Thus, these devices can be applied for the epidemiological evaluation between four limbs’ blood pressures and cardiac function parameters.\nIn this study, by using a simultaneous and noninvasive measurement technique, we measured four limbs blood pressures. But this technique was not a popular one to measure blood pressure in daily clinical practice. Hence, although four limbs blood pressure with simultaneous measurement could improve the predictive value for cardiovascular disease, our results might be changed if daily clinical measurement was used. In addition, the subjects were mainly from retired people, whose health care consciousness is better than the serving officer. The factors of smoking, drinking, salting and movement have no significant difference in this study subjects. Hence, the clinical utility of this study may be limited in community people.\n\nConclusion:\nLADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nSimultaneous measurement of four-limb blood pressures can improve the accuracy of cardiovascular disease diagnosis. This study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the non-invasive diagnostic method of cardiovascular disease in primary care.\n\nMethods:\n229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled. Four-limb blood pressure measurements were simultaneously performed using a blood pressure and pulse monitor device in the supine position. Cardiac functional parameters were also measured by using a cardiac hemodynamic detector in the same position. Data were statistically analyzed with SPSS15.0.\n\nResults:\nThe mean age of the 229 subjects was 60.56 ± 8.68 years. Cardiovascular functional parameters decreased with age and body mass index (BMI), only the total peripheral resistance (TPR) was in contrast. Age, BMI, left ankle diastolic pressure (LADP), high arm mean arterial pressure (HARMAP), left arm diastolic pressure (LARDP) and right ankle diastolic pressure (RADP) were significantly correlated with cardiovascular functional parameters. Cardiovascular functional parameters have significant differences with inter-arm difference in systolic blood pressure (SBP) between ≥10 and <10 mmHg, inter-ankle difference in SBP between ≥15 and ≥20 mmHg, inter-ankle difference in SBP between ≥15 and <10 mmHg and right ankle brachial index (RABI) between ≤0.9 and ≥1.0. After excluding 99 hypertensive patients, a part of cardiovascular functional parameters has still significant differences with inter-arm difference in SBP between ≥10 and ≥15 mmHg and RABI between ≤0.9 and ≥1.0.\n\nConclusions:\nAge, BMI, LADP, HARMAP, LARDP and RADP were the determinants of cardiovascular functional parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in SBP ≥10 mmHg, inter-ankle difference in SBP ≥15 mmHg and RABI ≤0.9, while these differences still existed after excluding 99 hypertensive patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the non-invasive diagnostic method of cardiovascular disease in primary care."},"background_conclusion_text":{"kind":"string","value":"Background:\nAccurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.\n\nConclusion:\nLADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nSimultaneous measurement of four-limb blood pressures can improve the accuracy of cardiovascular disease diagnosis. This study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the non-invasive diagnostic method of cardiovascular disease in primary care.\n\nMethods:\n229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled. Four-limb blood pressure measurements were simultaneously performed using a blood pressure and pulse monitor device in the supine position. Cardiac functional parameters were also measured by using a cardiac hemodynamic detector in the same position. Data were statistically analyzed with SPSS15.0.\n\nResults:\nThe mean age of the 229 subjects was 60.56 ± 8.68 years. Cardiovascular functional parameters decreased with age and body mass index (BMI), only the total peripheral resistance (TPR) was in contrast. Age, BMI, left ankle diastolic pressure (LADP), high arm mean arterial pressure (HARMAP), left arm diastolic pressure (LARDP) and right ankle diastolic pressure (RADP) were significantly correlated with cardiovascular functional parameters. Cardiovascular functional parameters have significant differences with inter-arm difference in systolic blood pressure (SBP) between ≥10 and <10 mmHg, inter-ankle difference in SBP between ≥15 and ≥20 mmHg, inter-ankle difference in SBP between ≥15 and <10 mmHg and right ankle brachial index (RABI) between ≤0.9 and ≥1.0. After excluding 99 hypertensive patients, a part of cardiovascular functional parameters has still significant differences with inter-arm difference in SBP between ≥10 and ≥15 mmHg and RABI between ≤0.9 and ≥1.0.\n\nConclusions:\nAge, BMI, LADP, HARMAP, LARDP and RADP were the determinants of cardiovascular functional parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in SBP ≥10 mmHg, inter-ankle difference in SBP ≥15 mmHg and RABI ≤0.9, while these differences still existed after excluding 99 hypertensive patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the non-invasive diagnostic method of cardiovascular disease in primary care."},"whole_article_text_length":{"kind":"number","value":13813,"string":"13,813"},"whole_article_abstract_length":{"kind":"number","value":427,"string":"427"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["pressure","blood","blood pressure","limb","limb blood","differences","limb blood pressure","parameters","mean","cardiovascular"],"string":"[\n \"pressure\",\n \"blood\",\n \"blood pressure\",\n \"limb\",\n \"limb blood\",\n \"differences\",\n \"limb blood pressure\",\n \"parameters\",\n \"mean\",\n \"cardiovascular\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Cardiovascular function | Four limbs | Blood pressure difference | Simultaneous measurement [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Cardiovascular function | Four limbs | Blood pressure difference | Simultaneous measurement [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Cardiovascular function | Four 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[SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Ankle Brachial Index | Arterial Pressure | Blood Pressure | Blood Pressure Determination | Cardiovascular Diseases | Cardiovascular System | Computer Simulation | Cross-Sectional Studies | Female | Hemodynamics | Humans | Hypertension | Linear Models | Male | Middle Aged | Models, Statistical | Primary Health Care | Risk Assessment [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Ankle Brachial Index | Arterial Pressure | Blood Pressure | Blood Pressure Determination | Cardiovascular Diseases | Cardiovascular System | Computer Simulation | Cross-Sectional Studies | Female | Hemodynamics | Humans | Hypertension | Linear Models | Male | Middle Aged | Models, Statistical | Primary Health Care | Risk Assessment [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | 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| mean | cardiovascular [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] pressure | blood | blood pressure | limb | limb blood | differences | limb blood pressure | parameters | mean | cardiovascular [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] pressure | blood | blood pressure | limb | limb blood | differences | limb blood pressure | parameters | mean | cardiovascular [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] pressure | blood | blood pressure | limb | limb blood | differences | limb blood pressure | parameters | mean | cardiovascular [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] blood | blood pressure | pressure | disease | mortality | cardiovascular | cardiovascular disease | cardiovascular mortality | systolic blood pressure difference | pressure difference [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] distribution | mean distribution | 000 | pressure | limb blood pressure differences | pressure differences mean distribution | pressure differences mean | differences mean distribution | blood pressure differences mean | differences mean [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cardiovascular | difference systolic blood | functional parameter | cardiovascular functional parameter | parameter | blood | mmhg | difference systolic | systolic blood | age body mass index [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] pressure | blood | blood pressure | parameters | cardiovascular | cardiac | differences | 000 | limb blood pressure | limb blood [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] pressure | blood | blood pressure | parameters | cardiovascular | cardiac | differences | 000 | limb blood pressure | limb blood [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] four ||| four [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 229 | 60.56 | 8.68 years ||| BMI | TPR ||| BMI | RADP ||| SBP | 10 | SBP | SBP | 10 | RABI | ≥1.0 ||| 99 | SBP | RABI | ≥1.0 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] BMI | HARMAP | LARDP | RADP ||| SBP | SBP | RABI | 99 ||| four [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] four ||| four ||| 229 | 62 | 63.50 | 11.13 years | 167 | 59.47 | 7.33 years ||| Four ||| Cardiac ||| ||| ||| 229 | 60.56 | 8.68 years ||| BMI | TPR ||| BMI | RADP ||| SBP | 10 | SBP | SBP | 10 | RABI | ≥1.0 ||| 99 | SBP | RABI | ≥1.0 ||| BMI | HARMAP | LARDP | RADP ||| SBP | SBP | RABI | 99 ||| four [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] four ||| four ||| 229 | 62 | 63.50 | 11.13 years | 167 | 59.47 | 7.33 years ||| Four ||| Cardiac ||| ||| ||| 229 | 60.56 | 8.68 years ||| BMI | TPR ||| BMI | RADP ||| SBP | 10 | SBP | SBP | 10 | RABI | ≥1.0 ||| 99 | SBP | RABI | ≥1.0 ||| BMI | HARMAP | LARDP | RADP ||| SBP | SBP | RABI | 99 ||| four [SUMMARY]"}}},{"rowIdx":289,"cells":{"title":{"kind":"string","value":"Incidence of symptoms and accidents during baths and showers among the Japanese general public."},"pmid":{"kind":"string","value":"21478641"},"background_abstract":{"kind":"string","value":"Bathing is a deeply ingrained custom among Japanese; however, data on the incidence rate of symptoms and accidents during bathing have not yet been reported for the Japanese general public."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We conducted a population-based cross-sectional study of 617 Japanese adults who attended a specialized health checkup. Participants completed a self-administered questionnaire to assess weekly frequencies of bathtub bathing and showering and the frequency of symptoms/accidents (falling, loss of consciousness, and other) during these activities in the past year. We calculated the incidence rates of accidents per 10 000 baths/showers and 95% confidence intervals (CIs) and compared the clinical characteristics of participants who had symptoms/accidents with those who did not."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22-0.84) and 0.24 (95% CI: 0.04-1.37). Although these rates are low, there were 740 000 bathtub bathing-related accidents in Japan, due to the fact that bathing is an almost-daily habit. There was no significant difference in clinical characteristics between groups."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"We collected basic information on the incidence of bathing-related accidents in Japan. Falls and loss of consciousness during bathing or showering can potentially lead to a serious accident, so the general public should be educated about the possibility of such accidents during bathing."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Accidental Falls","Accidents, Home","Adult","Aged","Baths","Confidence Intervals","Cross-Sectional Studies","Female","Health Surveys","Humans","Incidence","Japan","Male","Middle Aged","Public Health","Self-Assessment","Surveys and Questionnaires"],"string":"[\n \"Accidental Falls\",\n \"Accidents, Home\",\n \"Adult\",\n \"Aged\",\n \"Baths\",\n \"Confidence Intervals\",\n \"Cross-Sectional Studies\",\n \"Female\",\n \"Health Surveys\",\n \"Humans\",\n \"Incidence\",\n \"Japan\",\n \"Male\",\n \"Middle Aged\",\n \"Public Health\",\n \"Self-Assessment\",\n \"Surveys and Questionnaires\"\n]"},"pmcid":{"kind":"string","value":"3899424"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"This cross-sectional study enrolled adults aged 40 to 74 years who underwent a specialized health checkup provided in October 2008 in the district of Kawane in Shimada city, Shizuoka. The investigation utilized self-administered questionnaires, measurement of height and weight, blood pressure examination, and blood testing for triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, L-aspartate aminotransferase, L-alanine aminotransferase, and hemoglobin A1c, based on a program standardized by the Japanese Ministry of Health, Labour and Welfare.9 From a total of 1319 eligible adults, 617 who actually completed the health checkup and gave consent participated in the study. Participants completed a self-administered questionnaire to assess the weekly frequencies of bathtub bathing and showering during summer (July through September) and winter (December through February). In addition, we investigated the frequency of symptoms/accidents (falling, loss of consciousness, pallor, nausea/vomiting, and other) during bathtub bathing and showering in the past year: the list of symptoms/accidents consisted of those frequently identified in previous research.7\nFirst, we calculated the weekly frequencies of bathtub bathing and showering by averaging the frequencies between the 2 seasons, after which we assessed the incidence of each type of accident during bathtub bathing and showering in the past year for all participants and in 2 groups divided by the median age of the participants (ie, 66 years). Next, based on the weekly frequencies of bathtub bathing and showering, we estimated the overall frequencies of bathtub bathing and showering during the past year for all participants. We then divided the total number of accidents by the annual frequencies of bathtub bathing and showering for all participants and calculated the incidence rates and 95% confidence intervals (CIs) of accidents per 10 000 baths/showers. We calculated 95% CIs using a binomial distribution according to Wilson’s method.10 Then, to estimate the annual frequencies of bathtub bathing and showering among Japanese aged 40 to 74 years, we used the 2008 Vital Statistics of Japan to determine how many Japanese were included in this age group.11 By multiplying these estimates by the incidence rates for accidents (which were obtained earlier), we estimated the total annual numbers of accidents that were associated with bathtub bathing and showering in Japanese aged 40 to 74 years.\nAfter dividing the participants into 2 groups according to their accident status, ie, cases with and without bath-related accidents, we used the t-test to compare mean values for age, body mass index, blood pressure, and blood chemistry findings and Fisher’s exact test to compare differences in sex, medication use, and past medical history.\nThe study protocol was approved by the institutional review board (IRB) of Hamamatsu University School of Medicine (No. 20-55). The nature of the study was explained to the participants using written statements approved by the IRB. Voluntary informed consent was obtained from all participants before beginning the study. Completion of the questionnaire was deemed as indicating consent to participate in the study. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (SPSS Japan Inc., version 17.0, Tokyo, Japan)."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"All 617 individuals (266 men, 351 women) who had completed the health checkup and were recruited for the study agreed to participate. The frequencies of bathtub bathing and showering were calculated based on summer and winter frequencies (mean ± SD) of 5.82 ± 2.09 and 1.29 ± 1.81 times per week, respectively (Table 1). The overall annual numbers of bathtub baths and showers were estimated based on the weekly frequencies and were approximately 190 000 and 41 000, respectively. Of the 617 individuals, 588 completed a self-administered questionnaire about accidents. Of these, 5 reported a total of 8 bathtub bathing-related accidents over the past year, including 5 cases of facial pallor, 2 cases of loss of consciousness, and 1 case of falling. In contrast, there was only 1 shower-related accident (loss of consciousness). The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22–0.84) and 0.24 (95% CI: 0.04–1.37), respectively. According to estimates based on the Japanese population of adults aged 40 to 74 years, people in this age group bathe a total of 1.7 × 1010 times per year and have a total of 740 000 (95% CI: 380 000 to 1 500 000) accidents. They shower 3.8 × 109 times per year and have a total of 93 000 (95% CI: 16 000 to 530 000) accidents.\nSD: standard deviation; CI: confidence interval.\nA comparison of the characteristics of participants who did and did not have bath-related accidents are shown in Table 2. There was no significant difference in the characteristics of the 5 participants who had accidents and those who did not.\nBMI: Body mass index; SBP: Systolic blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; AST: L-aspartate aminotransferase; ALT: L-alanine aminotransferase."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION"],"string":"[\n \"INTRODUCTION\"\n]"},"other_sections_texts":{"kind":"list like","value":["Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public."],"string":"[\n \"Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null],"string":"[\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","RESULTS","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"RESULTS\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public.","This cross-sectional study enrolled adults aged 40 to 74 years who underwent a specialized health checkup provided in October 2008 in the district of Kawane in Shimada city, Shizuoka. The investigation utilized self-administered questionnaires, measurement of height and weight, blood pressure examination, and blood testing for triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, L-aspartate aminotransferase, L-alanine aminotransferase, and hemoglobin A1c, based on a program standardized by the Japanese Ministry of Health, Labour and Welfare.9 From a total of 1319 eligible adults, 617 who actually completed the health checkup and gave consent participated in the study. Participants completed a self-administered questionnaire to assess the weekly frequencies of bathtub bathing and showering during summer (July through September) and winter (December through February). In addition, we investigated the frequency of symptoms/accidents (falling, loss of consciousness, pallor, nausea/vomiting, and other) during bathtub bathing and showering in the past year: the list of symptoms/accidents consisted of those frequently identified in previous research.7\nFirst, we calculated the weekly frequencies of bathtub bathing and showering by averaging the frequencies between the 2 seasons, after which we assessed the incidence of each type of accident during bathtub bathing and showering in the past year for all participants and in 2 groups divided by the median age of the participants (ie, 66 years). Next, based on the weekly frequencies of bathtub bathing and showering, we estimated the overall frequencies of bathtub bathing and showering during the past year for all participants. We then divided the total number of accidents by the annual frequencies of bathtub bathing and showering for all participants and calculated the incidence rates and 95% confidence intervals (CIs) of accidents per 10 000 baths/showers. We calculated 95% CIs using a binomial distribution according to Wilson’s method.10 Then, to estimate the annual frequencies of bathtub bathing and showering among Japanese aged 40 to 74 years, we used the 2008 Vital Statistics of Japan to determine how many Japanese were included in this age group.11 By multiplying these estimates by the incidence rates for accidents (which were obtained earlier), we estimated the total annual numbers of accidents that were associated with bathtub bathing and showering in Japanese aged 40 to 74 years.\nAfter dividing the participants into 2 groups according to their accident status, ie, cases with and without bath-related accidents, we used the t-test to compare mean values for age, body mass index, blood pressure, and blood chemistry findings and Fisher’s exact test to compare differences in sex, medication use, and past medical history.\nThe study protocol was approved by the institutional review board (IRB) of Hamamatsu University School of Medicine (No. 20-55). The nature of the study was explained to the participants using written statements approved by the IRB. Voluntary informed consent was obtained from all participants before beginning the study. Completion of the questionnaire was deemed as indicating consent to participate in the study. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (SPSS Japan Inc., version 17.0, Tokyo, Japan).","All 617 individuals (266 men, 351 women) who had completed the health checkup and were recruited for the study agreed to participate. The frequencies of bathtub bathing and showering were calculated based on summer and winter frequencies (mean ± SD) of 5.82 ± 2.09 and 1.29 ± 1.81 times per week, respectively (Table 1). The overall annual numbers of bathtub baths and showers were estimated based on the weekly frequencies and were approximately 190 000 and 41 000, respectively. Of the 617 individuals, 588 completed a self-administered questionnaire about accidents. Of these, 5 reported a total of 8 bathtub bathing-related accidents over the past year, including 5 cases of facial pallor, 2 cases of loss of consciousness, and 1 case of falling. In contrast, there was only 1 shower-related accident (loss of consciousness). The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22–0.84) and 0.24 (95% CI: 0.04–1.37), respectively. According to estimates based on the Japanese population of adults aged 40 to 74 years, people in this age group bathe a total of 1.7 × 1010 times per year and have a total of 740 000 (95% CI: 380 000 to 1 500 000) accidents. They shower 3.8 × 109 times per year and have a total of 93 000 (95% CI: 16 000 to 530 000) accidents.\nSD: standard deviation; CI: confidence interval.\nA comparison of the characteristics of participants who did and did not have bath-related accidents are shown in Table 2. There was no significant difference in the characteristics of the 5 participants who had accidents and those who did not.\nBMI: Body mass index; SBP: Systolic blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; AST: L-aspartate aminotransferase; ALT: L-alanine aminotransferase.","The present study is the first to ascertain the incidence rate of bathing-related accidents among the Japanese general public. The incidence rate in the present study was somewhat higher than that noted in an investigation of public welfare facilities for the aged (0.067 and 0.204 per 10 000 in-facility baths and home-visit baths, respectively).8 The somewhat lower incidence rate of accidents in bathing services provided by welfare facilities may be attributable to the following facts. First, in welfare services, a physical checkup is routinely performed before bathing, which may prevent accidents. Second, bathing in the presence of a caregiver may allow for early detection of potential accidents and serious symptoms. Third, the incidence rate was likely to have been underreported in the previous study,8 as it was based only on the objective findings of caregivers.\nIn the present study, the incidence rate of accidents during showering was lower than that during bathtub bathing. It has been suggested that, as compared with bathtub bathing, the lower water pressure used during showering imposes a lower burden on the body.12 However, as habitual bathing is reported to be associated with good self-rated health and sleep quality,13 it would not be wise to suggest that bathtub bathing is, in general, more hazardous to general health than showering.\nWhile the incidence rate of accidents in the present study is small, the total number of bathtub bathing-related accidents in Japan was as high as 740 000, because bathing is an almost-daily habit. Although no accidents resulted in death in this study, falls and loss of consciousness could potentially lead to a serious accident.14 One participant who reported falling in the bath lost consciousness twice. Therefore, the general public must be educated about accidents such as falls during bathing.\nThis study has several limitations. First, the use of self-report questionnaires may have caused underreporting due to obscured memory. Second, the sample size was relatively small, and the study was conducted within 1 geographic region. It is very difficult to use a small sample in a specific area to estimate the incidence for the general population. To determine the characteristics of the accident group, we analyzed the relationship between disease history and the results of the health checkup using additional data from the health checkup. However, there were no characteristic findings. Third, participants had all sought a health checkup, suggesting possible selection bias. A previous study reported high incidence rates of drowning among the very old.3 It is possible that, in the present study, recall bias explains why incidence rates were lower among the very old than among middle-aged adults. Although there were some limitations in the present report, it is the first to collect information on the incidence of bathing-related accidents among the Japanese general public."],"string":"[\n \"Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public.\",\n \"This cross-sectional study enrolled adults aged 40 to 74 years who underwent a specialized health checkup provided in October 2008 in the district of Kawane in Shimada city, Shizuoka. The investigation utilized self-administered questionnaires, measurement of height and weight, blood pressure examination, and blood testing for triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, L-aspartate aminotransferase, L-alanine aminotransferase, and hemoglobin A1c, based on a program standardized by the Japanese Ministry of Health, Labour and Welfare.9 From a total of 1319 eligible adults, 617 who actually completed the health checkup and gave consent participated in the study. Participants completed a self-administered questionnaire to assess the weekly frequencies of bathtub bathing and showering during summer (July through September) and winter (December through February). In addition, we investigated the frequency of symptoms/accidents (falling, loss of consciousness, pallor, nausea/vomiting, and other) during bathtub bathing and showering in the past year: the list of symptoms/accidents consisted of those frequently identified in previous research.7\\nFirst, we calculated the weekly frequencies of bathtub bathing and showering by averaging the frequencies between the 2 seasons, after which we assessed the incidence of each type of accident during bathtub bathing and showering in the past year for all participants and in 2 groups divided by the median age of the participants (ie, 66 years). Next, based on the weekly frequencies of bathtub bathing and showering, we estimated the overall frequencies of bathtub bathing and showering during the past year for all participants. We then divided the total number of accidents by the annual frequencies of bathtub bathing and showering for all participants and calculated the incidence rates and 95% confidence intervals (CIs) of accidents per 10 000 baths/showers. We calculated 95% CIs using a binomial distribution according to Wilson’s method.10 Then, to estimate the annual frequencies of bathtub bathing and showering among Japanese aged 40 to 74 years, we used the 2008 Vital Statistics of Japan to determine how many Japanese were included in this age group.11 By multiplying these estimates by the incidence rates for accidents (which were obtained earlier), we estimated the total annual numbers of accidents that were associated with bathtub bathing and showering in Japanese aged 40 to 74 years.\\nAfter dividing the participants into 2 groups according to their accident status, ie, cases with and without bath-related accidents, we used the t-test to compare mean values for age, body mass index, blood pressure, and blood chemistry findings and Fisher’s exact test to compare differences in sex, medication use, and past medical history.\\nThe study protocol was approved by the institutional review board (IRB) of Hamamatsu University School of Medicine (No. 20-55). The nature of the study was explained to the participants using written statements approved by the IRB. Voluntary informed consent was obtained from all participants before beginning the study. Completion of the questionnaire was deemed as indicating consent to participate in the study. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (SPSS Japan Inc., version 17.0, Tokyo, Japan).\",\n \"All 617 individuals (266 men, 351 women) who had completed the health checkup and were recruited for the study agreed to participate. The frequencies of bathtub bathing and showering were calculated based on summer and winter frequencies (mean ± SD) of 5.82 ± 2.09 and 1.29 ± 1.81 times per week, respectively (Table 1). The overall annual numbers of bathtub baths and showers were estimated based on the weekly frequencies and were approximately 190 000 and 41 000, respectively. Of the 617 individuals, 588 completed a self-administered questionnaire about accidents. Of these, 5 reported a total of 8 bathtub bathing-related accidents over the past year, including 5 cases of facial pallor, 2 cases of loss of consciousness, and 1 case of falling. In contrast, there was only 1 shower-related accident (loss of consciousness). The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22–0.84) and 0.24 (95% CI: 0.04–1.37), respectively. According to estimates based on the Japanese population of adults aged 40 to 74 years, people in this age group bathe a total of 1.7 × 1010 times per year and have a total of 740 000 (95% CI: 380 000 to 1 500 000) accidents. They shower 3.8 × 109 times per year and have a total of 93 000 (95% CI: 16 000 to 530 000) accidents.\\nSD: standard deviation; CI: confidence interval.\\nA comparison of the characteristics of participants who did and did not have bath-related accidents are shown in Table 2. There was no significant difference in the characteristics of the 5 participants who had accidents and those who did not.\\nBMI: Body mass index; SBP: Systolic blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; AST: L-aspartate aminotransferase; ALT: L-alanine aminotransferase.\",\n \"The present study is the first to ascertain the incidence rate of bathing-related accidents among the Japanese general public. The incidence rate in the present study was somewhat higher than that noted in an investigation of public welfare facilities for the aged (0.067 and 0.204 per 10 000 in-facility baths and home-visit baths, respectively).8 The somewhat lower incidence rate of accidents in bathing services provided by welfare facilities may be attributable to the following facts. First, in welfare services, a physical checkup is routinely performed before bathing, which may prevent accidents. Second, bathing in the presence of a caregiver may allow for early detection of potential accidents and serious symptoms. Third, the incidence rate was likely to have been underreported in the previous study,8 as it was based only on the objective findings of caregivers.\\nIn the present study, the incidence rate of accidents during showering was lower than that during bathtub bathing. It has been suggested that, as compared with bathtub bathing, the lower water pressure used during showering imposes a lower burden on the body.12 However, as habitual bathing is reported to be associated with good self-rated health and sleep quality,13 it would not be wise to suggest that bathtub bathing is, in general, more hazardous to general health than showering.\\nWhile the incidence rate of accidents in the present study is small, the total number of bathtub bathing-related accidents in Japan was as high as 740 000, because bathing is an almost-daily habit. Although no accidents resulted in death in this study, falls and loss of consciousness could potentially lead to a serious accident.14 One participant who reported falling in the bath lost consciousness twice. Therefore, the general public must be educated about accidents such as falls during bathing.\\nThis study has several limitations. First, the use of self-report questionnaires may have caused underreporting due to obscured memory. Second, the sample size was relatively small, and the study was conducted within 1 geographic region. It is very difficult to use a small sample in a specific area to estimate the incidence for the general population. To determine the characteristics of the accident group, we analyzed the relationship between disease history and the results of the health checkup using additional data from the health checkup. However, there were no characteristic findings. Third, participants had all sought a health checkup, suggesting possible selection bias. A previous study reported high incidence rates of drowning among the very old.3 It is possible that, in the present study, recall bias explains why incidence rates were lower among the very old than among middle-aged adults. Although there were some limitations in the present report, it is the first to collect information on the incidence of bathing-related accidents among the Japanese general public.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods","results","discussion"],"string":"[\n null,\n \"methods\",\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["baths","shower","incidence","accidents","population-based study"],"string":"[\n \"baths\",\n \"shower\",\n \"incidence\",\n \"accidents\",\n \"population-based study\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nBathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public.\n\nMETHODS:\nThis cross-sectional study enrolled adults aged 40 to 74 years who underwent a specialized health checkup provided in October 2008 in the district of Kawane in Shimada city, Shizuoka. The investigation utilized self-administered questionnaires, measurement of height and weight, blood pressure examination, and blood testing for triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, L-aspartate aminotransferase, L-alanine aminotransferase, and hemoglobin A1c, based on a program standardized by the Japanese Ministry of Health, Labour and Welfare.9 From a total of 1319 eligible adults, 617 who actually completed the health checkup and gave consent participated in the study. Participants completed a self-administered questionnaire to assess the weekly frequencies of bathtub bathing and showering during summer (July through September) and winter (December through February). In addition, we investigated the frequency of symptoms/accidents (falling, loss of consciousness, pallor, nausea/vomiting, and other) during bathtub bathing and showering in the past year: the list of symptoms/accidents consisted of those frequently identified in previous research.7\nFirst, we calculated the weekly frequencies of bathtub bathing and showering by averaging the frequencies between the 2 seasons, after which we assessed the incidence of each type of accident during bathtub bathing and showering in the past year for all participants and in 2 groups divided by the median age of the participants (ie, 66 years). Next, based on the weekly frequencies of bathtub bathing and showering, we estimated the overall frequencies of bathtub bathing and showering during the past year for all participants. We then divided the total number of accidents by the annual frequencies of bathtub bathing and showering for all participants and calculated the incidence rates and 95% confidence intervals (CIs) of accidents per 10 000 baths/showers. We calculated 95% CIs using a binomial distribution according to Wilson’s method.10 Then, to estimate the annual frequencies of bathtub bathing and showering among Japanese aged 40 to 74 years, we used the 2008 Vital Statistics of Japan to determine how many Japanese were included in this age group.11 By multiplying these estimates by the incidence rates for accidents (which were obtained earlier), we estimated the total annual numbers of accidents that were associated with bathtub bathing and showering in Japanese aged 40 to 74 years.\nAfter dividing the participants into 2 groups according to their accident status, ie, cases with and without bath-related accidents, we used the t-test to compare mean values for age, body mass index, blood pressure, and blood chemistry findings and Fisher’s exact test to compare differences in sex, medication use, and past medical history.\nThe study protocol was approved by the institutional review board (IRB) of Hamamatsu University School of Medicine (No. 20-55). The nature of the study was explained to the participants using written statements approved by the IRB. Voluntary informed consent was obtained from all participants before beginning the study. Completion of the questionnaire was deemed as indicating consent to participate in the study. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (SPSS Japan Inc., version 17.0, Tokyo, Japan).\n\nRESULTS:\nAll 617 individuals (266 men, 351 women) who had completed the health checkup and were recruited for the study agreed to participate. The frequencies of bathtub bathing and showering were calculated based on summer and winter frequencies (mean ± SD) of 5.82 ± 2.09 and 1.29 ± 1.81 times per week, respectively (Table 1). The overall annual numbers of bathtub baths and showers were estimated based on the weekly frequencies and were approximately 190 000 and 41 000, respectively. Of the 617 individuals, 588 completed a self-administered questionnaire about accidents. Of these, 5 reported a total of 8 bathtub bathing-related accidents over the past year, including 5 cases of facial pallor, 2 cases of loss of consciousness, and 1 case of falling. In contrast, there was only 1 shower-related accident (loss of consciousness). The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22–0.84) and 0.24 (95% CI: 0.04–1.37), respectively. According to estimates based on the Japanese population of adults aged 40 to 74 years, people in this age group bathe a total of 1.7 × 1010 times per year and have a total of 740 000 (95% CI: 380 000 to 1 500 000) accidents. They shower 3.8 × 109 times per year and have a total of 93 000 (95% CI: 16 000 to 530 000) accidents.\nSD: standard deviation; CI: confidence interval.\nA comparison of the characteristics of participants who did and did not have bath-related accidents are shown in Table 2. There was no significant difference in the characteristics of the 5 participants who had accidents and those who did not.\nBMI: Body mass index; SBP: Systolic blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; AST: L-aspartate aminotransferase; ALT: L-alanine aminotransferase.\n\nDISCUSSION:\nThe present study is the first to ascertain the incidence rate of bathing-related accidents among the Japanese general public. The incidence rate in the present study was somewhat higher than that noted in an investigation of public welfare facilities for the aged (0.067 and 0.204 per 10 000 in-facility baths and home-visit baths, respectively).8 The somewhat lower incidence rate of accidents in bathing services provided by welfare facilities may be attributable to the following facts. First, in welfare services, a physical checkup is routinely performed before bathing, which may prevent accidents. Second, bathing in the presence of a caregiver may allow for early detection of potential accidents and serious symptoms. Third, the incidence rate was likely to have been underreported in the previous study,8 as it was based only on the objective findings of caregivers.\nIn the present study, the incidence rate of accidents during showering was lower than that during bathtub bathing. It has been suggested that, as compared with bathtub bathing, the lower water pressure used during showering imposes a lower burden on the body.12 However, as habitual bathing is reported to be associated with good self-rated health and sleep quality,13 it would not be wise to suggest that bathtub bathing is, in general, more hazardous to general health than showering.\nWhile the incidence rate of accidents in the present study is small, the total number of bathtub bathing-related accidents in Japan was as high as 740 000, because bathing is an almost-daily habit. Although no accidents resulted in death in this study, falls and loss of consciousness could potentially lead to a serious accident.14 One participant who reported falling in the bath lost consciousness twice. Therefore, the general public must be educated about accidents such as falls during bathing.\nThis study has several limitations. First, the use of self-report questionnaires may have caused underreporting due to obscured memory. Second, the sample size was relatively small, and the study was conducted within 1 geographic region. It is very difficult to use a small sample in a specific area to estimate the incidence for the general population. To determine the characteristics of the accident group, we analyzed the relationship between disease history and the results of the health checkup using additional data from the health checkup. However, there were no characteristic findings. Third, participants had all sought a health checkup, suggesting possible selection bias. A previous study reported high incidence rates of drowning among the very old.3 It is possible that, in the present study, recall bias explains why incidence rates were lower among the very old than among middle-aged adults. Although there were some limitations in the present report, it is the first to collect information on the incidence of bathing-related accidents among the Japanese general public.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nBathing is a deeply ingrained custom among Japanese; however, data on the incidence rate of symptoms and accidents during bathing have not yet been reported for the Japanese general public.\n\nMethods:\nWe conducted a population-based cross-sectional study of 617 Japanese adults who attended a specialized health checkup. Participants completed a self-administered questionnaire to assess weekly frequencies of bathtub bathing and showering and the frequency of symptoms/accidents (falling, loss of consciousness, and other) during these activities in the past year. We calculated the incidence rates of accidents per 10 000 baths/showers and 95% confidence intervals (CIs) and compared the clinical characteristics of participants who had symptoms/accidents with those who did not.\n\nResults:\nThe incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22-0.84) and 0.24 (95% CI: 0.04-1.37). Although these rates are low, there were 740 000 bathtub bathing-related accidents in Japan, due to the fact that bathing is an almost-daily habit. There was no significant difference in clinical characteristics between groups.\n\nConclusions:\nWe collected basic information on the incidence of bathing-related accidents in Japan. Falls and loss of consciousness during bathing or showering can potentially lead to a serious accident, so the general public should be educated about the possibility of such accidents during bathing."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":1754,"string":"1,754"},"whole_article_abstract_length":{"kind":"number","value":274,"string":"274"},"num_sections":{"kind":"number","value":4,"string":"4"},"most_frequent_words":{"kind":"list like","value":["bathing","accidents","study","bathtub","incidence","bathtub bathing","showering","000","japanese","participants"],"string":"[\n \"bathing\",\n \"accidents\",\n \"study\",\n \"bathtub\",\n \"incidence\",\n \"bathtub bathing\",\n \"showering\",\n \"000\",\n \"japanese\",\n \"participants\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] baths | shower | incidence | accidents | population-based study [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] baths | shower | incidence | accidents | population-based study [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] baths | shower | incidence | accidents | population-based study [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] baths | shower | incidence | accidents | population-based study [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | japanese | bathing related | incidence rate | rate | public | general public | general | related [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing showering | bathtub bathing showering | showering | bathtub bathing | frequencies | participants | frequencies bathtub bathing | frequencies bathtub bathing showering | frequencies bathtub | bathing [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 000 | ci | 95 ci | accidents | 95 | times | total | respectively | frequencies | bathtub [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | 000 | general | rate | incidence rate [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Japanese | Japanese [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 617 | Japanese ||| weekly | the past year ||| 10 000 | 95% [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 10 | 95% | 0.22-0.84 | 0.24 | 95% | CI | 0.04 ||| 740 | Japan ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Japanese | Japanese ||| 617 | Japanese ||| weekly | the past year ||| 10 000 | 95% ||| ||| 10 | 95% | 0.22-0.84 | 0.24 | 95% | CI | 0.04 ||| 740 | Japan ||| ||| Japan ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":290,"cells":{"title":{"kind":"string","value":"Effect of probiotics on length of hospitalization in mild acute pancreatitis: A randomized, double-blind, placebo-controlled trial."},"pmid":{"kind":"string","value":"33510561"},"background_abstract":{"kind":"string","value":"Acute pancreatitis is the leading cause of hospitalization for acute gastrointestinal disease worldwide. The effects of probiotics in mild acute pancreatitis have not been studied. We hypothesized that the administration of probiotics may accelerate the recovery of intestinal function and shorten the length of hospital stay (LOS) in patients with mild pancreatitis."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We conducted a double-blind randomized clinical trial to evaluate the effects of probiotics administered to patients with mild acute pancreatitis at a tertiary medical center. The patients were given probiotics capsules (a mixed preparation of Bacillus subtilis and Enterococcus faecium) or placebo. The primary study endpoint was the LOS. The secondary endpoints included time to abdominal pain relief, recurrent abdominal pain, and time to successful oral feeding."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A total of 128 patients were included, with 64 patients in each arm. The severity of illness and the etiological distribution of disease were similar in the two groups. There was a significant reduction in the LOS in the probiotics treatment group vs the placebo group (5.36 ± 0.15 vs 6.02 ± 0.17 d, P < 0.05). The probiotics group was associated with a shorter time to abdominal pain relief and time to successful oral feeding (P < 0.01 for both) than the placebo group. No statistical difference was found in recurrent abdominal pain between the two groups."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The study results showed that the administration of probiotics capsules is associated with a shorter duration of hospitalization in patients with mild acute pancreatitis."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Acute Disease","Double-Blind Method","Hospitalization","Humans","Pancreatitis","Probiotics","Treatment Outcome"],"string":"[\n \"Acute Disease\",\n \"Double-Blind Method\",\n \"Hospitalization\",\n \"Humans\",\n \"Pancreatitis\",\n \"Probiotics\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"7807297"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis."},"methods_title":{"kind":"string","value":"MATERIALS AND METHODS"},"methods_text":{"kind":"string","value":" Study design This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\nThis 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\n Inclusion and exclusion criteria The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\nThe inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\n Data collection We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \nWe extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \n Outcomes We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \nWe used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \n Sample size estimation According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\nAccording to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\n Statistical analysis Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\nContinuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States)."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"Probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Study design","Inclusion and exclusion criteria","Data collection","Outcomes","Sample size estimation","Statistical analysis","RESULTS","Participant characteristics ","Primary endpoint","Secondary end points","Safety and harm","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"Study design\",\n \"Inclusion and exclusion criteria\",\n \"Data collection\",\n \"Outcomes\",\n \"Sample size estimation\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Participant characteristics \",\n \"Primary endpoint\",\n \"Secondary end points\",\n \"Safety and harm\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]"},"other_sections_texts":{"kind":"list like","value":["Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.","This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.","The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.","We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. ","We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. ","According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.","Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States)."," Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups","We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.","The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.","The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups","The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups","The present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.","In conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases."],"string":"[\n \"Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.\",\n \"This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\",\n \"The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\",\n \"We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \",\n \"We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \",\n \"According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\",\n \"Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\",\n \" Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\",\n \"We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\",\n \"The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\",\n \"The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\",\n \"The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\",\n \"The present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \\nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.\",\n \"In conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Study design","Inclusion and exclusion criteria","Data collection","Outcomes","Sample size estimation","Statistical analysis","RESULTS","Participant characteristics ","Primary endpoint","Secondary end points","Safety and harm","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Study design\",\n \"Inclusion and exclusion criteria\",\n \"Data collection\",\n \"Outcomes\",\n \"Sample size estimation\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Participant characteristics \",\n \"Primary endpoint\",\n \"Secondary end points\",\n \"Safety and harm\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis."," Study design This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\nThis 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\n Inclusion and exclusion criteria The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\nThe inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\n Data collection We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \nWe extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \n Outcomes We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \nWe used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \n Sample size estimation According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\nAccording to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\n Statistical analysis Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\nContinuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).","This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.","The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.","We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. ","We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. ","According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.","Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States)."," Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups","We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.","The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.","The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups","The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups","The present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.","In conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases."],"string":"[\n \"Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.\",\n \" Study design This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\\nThis 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\\n Inclusion and exclusion criteria The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\\nThe inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\\n Data collection We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \\nWe extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \\n Outcomes We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \\nWe used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \\n Sample size estimation According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\\nAccording to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\\n Statistical analysis Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\\nContinuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\",\n \"This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\",\n \"The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\",\n \"We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \",\n \"We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \",\n \"According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\",\n \"Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\",\n \" Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\",\n \"We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\",\n \"The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\",\n \"The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\",\n \"The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\",\n \"The present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \\nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.\",\n \"In conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Acute pancreatitis","Probiotics","Length of hospitalization","Randomized study","Placebo","Mild"],"string":"[\n \"Acute pancreatitis\",\n \"Probiotics\",\n \"Length of hospitalization\",\n \"Randomized study\",\n \"Placebo\",\n \"Mild\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nAcute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.\n\nMATERIALS AND METHODS:\n Study design This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\nThis 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\n Inclusion and exclusion criteria The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\nThe inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\n Data collection We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \nWe extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \n Outcomes We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \nWe used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \n Sample size estimation According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\nAccording to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\n Statistical analysis Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\nContinuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\n\nStudy design:\nThis 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\n\nInclusion and exclusion criteria:\nThe inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\n\nData collection:\nWe extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \n\nOutcomes:\nWe used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \n\nSample size estimation:\nAccording to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\n\nStatistical analysis:\nContinuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\n\nRESULTS:\n Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\n\nParticipant characteristics :\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\n\nPrimary endpoint:\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\n\nSecondary end points:\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\n\nSafety and harm:\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\n\nDISCUSSION:\nThe present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.\n\nCONCLUSION:\nIn conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nAcute pancreatitis is the leading cause of hospitalization for acute gastrointestinal disease worldwide. The effects of probiotics in mild acute pancreatitis have not been studied. We hypothesized that the administration of probiotics may accelerate the recovery of intestinal function and shorten the length of hospital stay (LOS) in patients with mild pancreatitis.\n\nMethods:\nWe conducted a double-blind randomized clinical trial to evaluate the effects of probiotics administered to patients with mild acute pancreatitis at a tertiary medical center. The patients were given probiotics capsules (a mixed preparation of Bacillus subtilis and Enterococcus faecium) or placebo. The primary study endpoint was the LOS. The secondary endpoints included time to abdominal pain relief, recurrent abdominal pain, and time to successful oral feeding.\n\nResults:\nA total of 128 patients were included, with 64 patients in each arm. The severity of illness and the etiological distribution of disease were similar in the two groups. There was a significant reduction in the LOS in the probiotics treatment group vs the placebo group (5.36 ± 0.15 vs 6.02 ± 0.17 d, P < 0.05). The probiotics group was associated with a shorter time to abdominal pain relief and time to successful oral feeding (P < 0.01 for both) than the placebo group. No statistical difference was found in recurrent abdominal pain between the two groups.\n\nConclusions:\nThe study results showed that the administration of probiotics capsules is associated with a shorter duration of hospitalization in patients with mild acute pancreatitis."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nAcute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.\n\nCONCLUSION:\nProbiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nAcute pancreatitis is the leading cause of hospitalization for acute gastrointestinal disease worldwide. The effects of probiotics in mild acute pancreatitis have not been studied. We hypothesized that the administration of probiotics may accelerate the recovery of intestinal function and shorten the length of hospital stay (LOS) in patients with mild pancreatitis.\n\nMethods:\nWe conducted a double-blind randomized clinical trial to evaluate the effects of probiotics administered to patients with mild acute pancreatitis at a tertiary medical center. The patients were given probiotics capsules (a mixed preparation of Bacillus subtilis and Enterococcus faecium) or placebo. The primary study endpoint was the LOS. The secondary endpoints included time to abdominal pain relief, recurrent abdominal pain, and time to successful oral feeding.\n\nResults:\nA total of 128 patients were included, with 64 patients in each arm. The severity of illness and the etiological distribution of disease were similar in the two groups. There was a significant reduction in the LOS in the probiotics treatment group vs the placebo group (5.36 ± 0.15 vs 6.02 ± 0.17 d, P < 0.05). The probiotics group was associated with a shorter time to abdominal pain relief and time to successful oral feeding (P < 0.01 for both) than the placebo group. No statistical difference was found in recurrent abdominal pain between the two groups.\n\nConclusions:\nThe study results showed that the administration of probiotics capsules is associated with a shorter duration of hospitalization in patients with mild acute pancreatitis."},"whole_article_text_length":{"kind":"number","value":6334,"string":"6,334"},"whole_article_abstract_length":{"kind":"number","value":283,"string":"283"},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["probiotics","patients","abdominal","pancreatitis","study","group","pain","abdominal pain","placebo","acute"],"string":"[\n \"probiotics\",\n \"patients\",\n \"abdominal\",\n \"pancreatitis\",\n \"study\",\n \"group\",\n \"pain\",\n \"abdominal pain\",\n \"placebo\",\n \"acute\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Double-Blind Method | Hospitalization | Humans | Pancreatitis | Probiotics | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Double-Blind Method | Hospitalization | Humans | Pancreatitis | Probiotics | Treatment Outcome 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[SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] acute | pancreatitis | acute pancreatitis | disease | probiotics | microbiota | intestinal | patients | mild | analysis [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] abdominal | abdominal pain | pain | patients | data | capsules | time | study | normally distributed | test [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] sample | probiotics | randomized | acute | acute pancreatitis | pancreatitis double blinded randomized | follow time short | time short study future | time short study | time short [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | abdominal | group | patients | pancreatitis | placebo | study | acute | pain | abdominal pain [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | abdominal | group | patients | pancreatitis | placebo | study | acute | pain | abdominal pain [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] tertiary ||| Enterococcus ||| ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| tertiary ||| Enterococcus ||| ||| ||| ||| 128 | 64 ||| two ||| 5.36 ± | 0.15 | 6.02 | 0.17 ||| ||| two ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| tertiary ||| Enterococcus ||| ||| ||| ||| 128 | 64 ||| two ||| 5.36 ± | 0.15 | 6.02 | 0.17 ||| ||| two ||| [SUMMARY]"}}},{"rowIdx":291,"cells":{"title":{"kind":"string","value":"Extract of Corallodiscus flabellata attenuates renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS signaling pathway."},"pmid":{"kind":"string","value":"35227255"},"background_abstract":{"kind":"string","value":"Fibrosis is one of the most common pathological features of the aging process of the kidney, and fibrosis in aging kidneys also aggravates the process of chronic kidney disease (CKD). Corallodiscus flabellata B. L. Burtt (C. flabellata, CF) is a commonly used botanical drug in Chinese folklore. However, few studies have reported its pharmacological effects. This study aimed to explore the effect of CF ethanol extract on renal fibrosis in SAMP8 mice and identify potentially active compounds."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Senescence-accelerated mouse-prone 8 (SAMP8) were used as animal models, and different doses of CF were given by gavage for one month. To observe the degree of renal aging in mice using β-galactosidase staining. Masson staining and the expression levels of Col-I, α-SMA, and FN were used to evaluate the renal fibrosis in mice. The protein expression levels of Nrf2 pathway and Wnt/β-catenin/RAS pathway in the kidney were measured. And β-galactosidase (β-gal) induced NRK-52E cells as an in vitro model to screen the active components of CF."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The CF ethanol extract significantly inhibited the activity of renal β-galactosidase and the expression levels of Col-I, α-SMA, and FN in SAMP8 mice, and improved Masson staining in SAMP8 mice. CF remarkably reduced urinary protein, creatinine, urea nitrogen and serum levels of TNF-α and IL-1β in SAMP8 mice, and significantly increased the levels of SOD and GSH-Px. Moreover, CF activated the Nrf2 pathway and blocked the Wnt/β-catenin/RAS pathway in the kidneys of mice. Besides, 3,4-dihydroxyphenylethanol (SDC-0-14, 16) and (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (SDC-1-8) were isolated from CF, which reduced the senescence of NRK-52E cells, and maybe the active ingredients of CF playing the anti-aging role."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Our experiments illuminated that CF ethanol extract may ameliorate renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS pathway. And SDC-0-14,16 and SDC-1-8 may be the material basis for CF to exert anti-renal senescence-related effects."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Fibrosis","Mice","Plant Extracts","Renal Insufficiency, Chronic","Wnt Signaling Pathway","beta Catenin"],"string":"[\n \"Animals\",\n \"Fibrosis\",\n \"Mice\",\n \"Plant Extracts\",\n \"Renal Insufficiency, Chronic\",\n \"Wnt Signaling Pathway\",\n \"beta Catenin\"\n]"},"pmcid":{"kind":"string","value":"8887028"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"The global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.\nCF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" CF improved the aging and fibrosis of the kidneys in SAMP8 mice To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nTo investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\n CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nBesides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\n CF activated the Nrf2 pathway in the kidney of SAMP8 mice The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nThe expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\n CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nIn order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\n CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nD-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\n CF improved PCA analysis of kidney tissue in SAMP8 mice Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPrincipal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF."},"other_sections_titles":{"kind":"list like","value":["Collection and extraction of the plant material","Animals and administration","Sample collection","Cell culture and in vitro study","Senescence-associated β-galactosidase staining","Histological analysis","Biochemical measurements","Immunohistochemical analysis","Western blot analysis","UPLC-Q-TOF-MS analysis for kidney samples","Statistical analysis","CF improved the aging and fibrosis of the kidneys in SAMP8 mice","CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice","CF activated the Nrf2 pathway in the kidney of SAMP8 mice","CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice","CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal","CF improved PCA analysis of kidney tissue in SAMP8 mice",""],"string":"[\n \"Collection and extraction of the plant material\",\n \"Animals and administration\",\n \"Sample collection\",\n \"Cell culture and in vitro study\",\n \"Senescence-associated β-galactosidase staining\",\n \"Histological analysis\",\n \"Biochemical measurements\",\n \"Immunohistochemical analysis\",\n \"Western blot analysis\",\n \"UPLC-Q-TOF-MS analysis for kidney samples\",\n \"Statistical analysis\",\n \"CF improved the aging and fibrosis of the kidneys in SAMP8 mice\",\n \"CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice\",\n \"CF activated the Nrf2 pathway in the kidney of SAMP8 mice\",\n \"CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice\",\n \"CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal\",\n \"CF improved PCA analysis of kidney tissue in SAMP8 mice\",\n \"\"\n]"},"other_sections_texts":{"kind":"list like","value":["“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).","Six-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.","At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.","NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.","Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.","The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.","The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.","The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.","Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.","The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.","The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.","To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)","Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)","The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)","In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)","D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group","Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;","\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3."],"string":"[\n \"“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\",\n \"Six-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\",\n \"At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\",\n \"NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\",\n \"Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\",\n \"The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\",\n \"The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\",\n \"The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\",\n \"Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\",\n \"The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\",\n \"The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\",\n \"To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\\n\\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\",\n \"Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\\n\\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\",\n \"The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\\n\\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\",\n \"In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\\n\\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\",\n \"D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\\n\\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\",\n \"Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\\n\\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\",\n \"\\nAdditional file 1.\\nAdditional file 1.\\n\\nAdditional file 2.\\nAdditional file 2.\\n\\nAdditional file 3.\\nAdditional file 3.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and methods","Collection and extraction of the plant material","Animals and administration","Sample collection","Cell culture and in vitro study","Senescence-associated β-galactosidase staining","Histological analysis","Biochemical measurements","Immunohistochemical analysis","Western blot analysis","UPLC-Q-TOF-MS analysis for kidney samples","Statistical analysis","Results","CF improved the aging and fibrosis of the kidneys in SAMP8 mice","CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice","CF activated the Nrf2 pathway in the kidney of SAMP8 mice","CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice","CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal","CF improved PCA analysis of kidney tissue in SAMP8 mice","Discussion","Conclusions","Supplementary Information",""],"string":"[\n \"Introduction\",\n \"Materials and methods\",\n \"Collection and extraction of the plant material\",\n \"Animals and administration\",\n \"Sample collection\",\n \"Cell culture and in vitro study\",\n \"Senescence-associated β-galactosidase staining\",\n \"Histological analysis\",\n \"Biochemical measurements\",\n \"Immunohistochemical analysis\",\n \"Western blot analysis\",\n \"UPLC-Q-TOF-MS analysis for kidney samples\",\n \"Statistical analysis\",\n \"Results\",\n \"CF improved the aging and fibrosis of the kidneys in SAMP8 mice\",\n \"CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice\",\n \"CF activated the Nrf2 pathway in the kidney of SAMP8 mice\",\n \"CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice\",\n \"CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal\",\n \"CF improved PCA analysis of kidney tissue in SAMP8 mice\",\n \"Discussion\",\n \"Conclusions\",\n \"Supplementary Information\",\n \"\"\n]"},"all_sections_texts":{"kind":"list like","value":["The global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.\nCF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books."," Collection and extraction of the plant material “Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\n“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).","“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).","Six-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.","At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.","NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.","Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.","The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.","The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.","The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.","Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.","The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.","The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant."," CF improved the aging and fibrosis of the kidneys in SAMP8 mice To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nTo investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\n CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nBesides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\n CF activated the Nrf2 pathway in the kidney of SAMP8 mice The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nThe expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\n CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nIn order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\n CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nD-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\n CF improved PCA analysis of kidney tissue in SAMP8 mice Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPrincipal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;","To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)","Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)","The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)","In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)","D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group","Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;","Aging plays an important role in the progression of CKD [19]. As CKD fibrosis progresses, senescent cells express and secrete pro-fibrotic factors (TGF-β, CTGF, and so forth) and pro-inflammatory factors (IL-1β, IL-6, TNF-α, and so forth), which are senescence-associated secretory phenotype factors, thereby accelerating renal fibrosis [20, 21]. At present, traditional Chinese medicine has achieved good results in treating renal fibrosis with fewer side effects [22]. This study explored the interventional effects of CF extract on renal fibrosis in SAMP8 mice.\nThe mouse strain selected for this study was SAMP8, which was developed based on the lifespan, senescence, and pathological phenotypic grading scores of AKR/J mice and was an accelerated aging model solely of genetic origin [23, 24]. Studies have shown that the changes in the renal pathology of SAMP8 mice (9 months) include tubulointerstitial fibrosis and focal segmental glomerulosclerosis [25]. And it was found that renal fibrosis in SAMP8 mice was age-related [6]. Therefore, we tested the activity of β-galactosidase and the level of renal fibrosis in SAMP8 mice, and found that CF-L and CF-M improved kidney fibrosis and β-galactosidase activity in SAMP8 mice (Fig. 1). And we also tested the urine output of the mice. Consistent with previously published studies indicating that CF obtained by two different processes had diuretic effects [13], the extract significantly increased the urine volume of SAMP8 mice. Also, the present study showed that supplementation with CF improved the renal function, the activity of antioxidant enzymes, and levels of inflammatory factors in SAMP8 mice (Fig. 2).\nThe Keap1–Nrf2 system gained the attention of many scholars in recent years due to its antioxidant and anti-inflammatory properties. Its pharmacological potential in treating kidney diseases had been extensively studied in nonclinical and clinical studies [26]. Nrf2 is a master transcriptional regulator for genes related to redox status and antioxidant effects [27]. Studies have shown that phosphorylation is required for Nrf2 activation and target gene induction [28]. Activating Nrf2 improved CKD progression by preventing oxidative stress and maintaining cellular redox homeostasis [29]. As a key transcription factor, Nrf2 plays a crucial role in defense against oxidative stress by regulating its downstream antioxidants and detoxification enzymes [30]. Kim et al. reported that resveratrol, as a potent Nrf2 activator, ameliorated aging-related progressive renal injury [31]. In the present study, CF improved the attenuation of Nrf2 in the kidney of SAMP8 mice without affecting the expression level of Keap1, suggesting that CF crude extract might improve oxidative damage in the kidneys by activating the Nrf2 pathway (Fig. 3).\nRenal fibrosis is characterized by excessive extracellular matrix (ECM) deposition leading to the formation of scars in the renal parenchyma [32]. Transforming growth factor-β (TGF-β) is thought to be a key cytokine in fibroblast overactivation [33, 34]. Of course, targeting only the TGF-β signaling pathway is insufficient to reduce renal fibrosis. Some studies indicated that CTGF, Wnt/β-catenin, renin-angiotensin system, oxidative stress, and so forth, were implicated in renal fibrosis [35–37]. Wnt/β-catenin is an evolutionarily conserved signaling pathway involved in the regulation of tissue homeostasis, organ development, and injury repair [38]. Cisternas et al. discussed the pro-fibrotic effect of Wnt signaling in both skeletal muscle and kidney [39]. Mounting evidence established that the Wnt/β-catenin signaling pathway plays a crucial role in regulating the development and progression of renal fibrotic lesions following injury [40–42]. The Wnt/β-catenin signal is relatively silent in the kidneys of healthy adults and is activated once the kidneys are subjected to various kinds of damages [43]. In mammals, the Wnt family has at least 19 family members critical for kidney development. And at least 15 of these family members are differentially upregulated in the aging kidney, including Wnt4 [6]. The results of the present study showed that the expression level of Wnt4 protein in the kidneys of SAMP8 mice was significantly higher than that in SAMR1 mice, which was verified by both Western blot and immunohistochemical analyses (Fig. 4a, d and f). Coincidentally, the expression level of β-catenin protein was also upregulated. Also, both Wnt4 and β-catenin were expressed in renal tubular epithelial cells, as revealed by the immunohistochemical analysis (Fig. 4a and f). Wnt/β-catenin elicited renal fibrosis by inducing multiple fibrogenic genes such as RAS components, matrix metalloproteinase-7 (MMP-7), plasminogen activator inhibitor 1 (PAI-1), and Snail1 [37]. Zhou et al. described Wnt/β-catenin as the major upstream regulator, which controls the expression of all tested RAS components in the kidneys [44]. Zhou et al. used a 5/6 nephrectomy (5/6 NX) rat model to show that the expression levels of major components of RAS in the brain and kidneys, such as angiotensinogen, angiotensin-converting enzyme, and angiotensin II AT1-receptor, was significantly upregulated. The upregulated expression level was inhibited by a central blocker of Wnt, which was an adeno-associated virus vector overexpressing the DKK1 gene [45]. Similarly, the present study found that AGTR1 was still expressed in the renal tubular epithelium, and the expression levels of renin and AGTR1 in the kidney tissues of SAMP8 mice were significantly greater than those in SAMR1 mice (Fig. 4a and d g). These results indicated that the Wnt/β-catenin/RAS signaling pathway was activated in the kidneys of SAMP8 mice, and the CF effectively inhibited the activation of this pathway.\nCTGF exerts multiple biological functions, including promoting mitosis of chemotactic cells, inducing adhesion, and promoting cell proliferation and ECM synthesis [35, 46]. CCN2 (CTGF) modulated Wnt signaling by binding to low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to further mediate fibrosis [39, 47]. Other studies used gene silencing to discover and confirm that Nrf2 regulated the Wnt pathway by regulating the expression level of CTGF, affecting renal interstitial disease. Also, Nrf2 regulated the CTGF transcription level mainly via CTGF transcriptional regulator c-Fos [48]. The immunohistochemical analysis revealed that CTGF and p-c-Fos were expressed mainly in renal tubular epithelial cells, and p-c-Fos was also distributed in glomeruli. The quantitative analysis showed that the expression levels of both proteins was inhibited by CF (Figs. 3b and e and 4a and c). Finally, the PCA analysis was used to further verify that CF improved kidney damage in SAMP8 mice (Fig. 6). The present study provided evidence that the CF not only activated Nrf2 signaling but also relied on Nrf2 to balance oxidative stress and inflammation, inhibited Wnt/β-catenin/RAS signaling, and improved kidney aging and renal fibrogenesis. However, we also found inconsistent improvement in the effect of CF on Masson and SA-β-gal staining in this experiment. It is speculated that this may be due to the inconsistent progression of renal aging and renal fibrosis in SAMP8 mice. The degree of renal aging in the mice in this experiment may be more serious than fibrosis. Therefore, the ameliorating effect of CF-H on renal aging in SAMP8 mice was not as obvious as that of renal fibrosis.\nAs a reducing monosaccharide, D-galactose is widely used in various age-related diseases in vivo and in vitro. D-gal was found to cause senescence and injury in NRK-52E cells [49], induce senescence of human kidney proximal tubular epithelial cells (HKC-8 cells), and increase the expression levels of two renal fibrosis marker proteins FN and α-SMA [6]. In this study, D-gal was used to induce NRK-52E cells in vitro. Using MTT assay and β-galactosidase staining to detect the compounds isolated from CF, it was found that SDC-0-14,16 and SDC-1-8 enhanced the cell viability induced by D-gal and inhibited D-gal induced cell senescence (Fig. 5). These suggested that SDC-0-14,16 and SDC-1-8 may be the material basis for CF to delay kidney aging. SDC-1-8 is one of the phenylethanoid glycosides. phenylethanoid glycosides were found to extend the life span of Caenorhabditis Elegans [50], with anti-aging, [51] and neuroprotective effects [52–54]. These provide a direction for our follow-up study on the pharmacological effects of CF.","In conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF."," \nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.\n\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.","\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3."],"string":"[\n \"The global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.\\nCF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books.\",\n \" Collection and extraction of the plant material “Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\\n“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\",\n \"“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\",\n \"Six-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\",\n \"At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\",\n \"NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\",\n \"Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\",\n \"The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\",\n \"The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\",\n \"The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\",\n \"Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\",\n \"The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\",\n \"The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\",\n \" CF improved the aging and fibrosis of the kidneys in SAMP8 mice To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\\n\\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\nTo investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\\n\\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\n CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\\n\\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\nBesides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\\n\\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\n CF activated the Nrf2 pathway in the kidney of SAMP8 mice The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\\n\\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\nThe expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\\n\\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\n CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\\n\\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\nIn order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\\n\\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\n CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\\n\\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\nD-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\\n\\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\n CF improved PCA analysis of kidney tissue in SAMP8 mice Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\\n\\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\\nPrincipal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\\n\\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\",\n \"To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\\n\\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\",\n \"Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\\n\\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\",\n \"The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\\n\\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\",\n \"In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\\n\\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\",\n \"D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\\n\\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\",\n \"Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\\n\\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\",\n \"Aging plays an important role in the progression of CKD [19]. As CKD fibrosis progresses, senescent cells express and secrete pro-fibrotic factors (TGF-β, CTGF, and so forth) and pro-inflammatory factors (IL-1β, IL-6, TNF-α, and so forth), which are senescence-associated secretory phenotype factors, thereby accelerating renal fibrosis [20, 21]. At present, traditional Chinese medicine has achieved good results in treating renal fibrosis with fewer side effects [22]. This study explored the interventional effects of CF extract on renal fibrosis in SAMP8 mice.\\nThe mouse strain selected for this study was SAMP8, which was developed based on the lifespan, senescence, and pathological phenotypic grading scores of AKR/J mice and was an accelerated aging model solely of genetic origin [23, 24]. Studies have shown that the changes in the renal pathology of SAMP8 mice (9 months) include tubulointerstitial fibrosis and focal segmental glomerulosclerosis [25]. And it was found that renal fibrosis in SAMP8 mice was age-related [6]. Therefore, we tested the activity of β-galactosidase and the level of renal fibrosis in SAMP8 mice, and found that CF-L and CF-M improved kidney fibrosis and β-galactosidase activity in SAMP8 mice (Fig. 1). And we also tested the urine output of the mice. Consistent with previously published studies indicating that CF obtained by two different processes had diuretic effects [13], the extract significantly increased the urine volume of SAMP8 mice. Also, the present study showed that supplementation with CF improved the renal function, the activity of antioxidant enzymes, and levels of inflammatory factors in SAMP8 mice (Fig. 2).\\nThe Keap1–Nrf2 system gained the attention of many scholars in recent years due to its antioxidant and anti-inflammatory properties. Its pharmacological potential in treating kidney diseases had been extensively studied in nonclinical and clinical studies [26]. Nrf2 is a master transcriptional regulator for genes related to redox status and antioxidant effects [27]. Studies have shown that phosphorylation is required for Nrf2 activation and target gene induction [28]. Activating Nrf2 improved CKD progression by preventing oxidative stress and maintaining cellular redox homeostasis [29]. As a key transcription factor, Nrf2 plays a crucial role in defense against oxidative stress by regulating its downstream antioxidants and detoxification enzymes [30]. Kim et al. reported that resveratrol, as a potent Nrf2 activator, ameliorated aging-related progressive renal injury [31]. In the present study, CF improved the attenuation of Nrf2 in the kidney of SAMP8 mice without affecting the expression level of Keap1, suggesting that CF crude extract might improve oxidative damage in the kidneys by activating the Nrf2 pathway (Fig. 3).\\nRenal fibrosis is characterized by excessive extracellular matrix (ECM) deposition leading to the formation of scars in the renal parenchyma [32]. Transforming growth factor-β (TGF-β) is thought to be a key cytokine in fibroblast overactivation [33, 34]. Of course, targeting only the TGF-β signaling pathway is insufficient to reduce renal fibrosis. Some studies indicated that CTGF, Wnt/β-catenin, renin-angiotensin system, oxidative stress, and so forth, were implicated in renal fibrosis [35–37]. Wnt/β-catenin is an evolutionarily conserved signaling pathway involved in the regulation of tissue homeostasis, organ development, and injury repair [38]. Cisternas et al. discussed the pro-fibrotic effect of Wnt signaling in both skeletal muscle and kidney [39]. Mounting evidence established that the Wnt/β-catenin signaling pathway plays a crucial role in regulating the development and progression of renal fibrotic lesions following injury [40–42]. The Wnt/β-catenin signal is relatively silent in the kidneys of healthy adults and is activated once the kidneys are subjected to various kinds of damages [43]. In mammals, the Wnt family has at least 19 family members critical for kidney development. And at least 15 of these family members are differentially upregulated in the aging kidney, including Wnt4 [6]. The results of the present study showed that the expression level of Wnt4 protein in the kidneys of SAMP8 mice was significantly higher than that in SAMR1 mice, which was verified by both Western blot and immunohistochemical analyses (Fig. 4a, d and f). Coincidentally, the expression level of β-catenin protein was also upregulated. Also, both Wnt4 and β-catenin were expressed in renal tubular epithelial cells, as revealed by the immunohistochemical analysis (Fig. 4a and f). Wnt/β-catenin elicited renal fibrosis by inducing multiple fibrogenic genes such as RAS components, matrix metalloproteinase-7 (MMP-7), plasminogen activator inhibitor 1 (PAI-1), and Snail1 [37]. Zhou et al. described Wnt/β-catenin as the major upstream regulator, which controls the expression of all tested RAS components in the kidneys [44]. Zhou et al. used a 5/6 nephrectomy (5/6 NX) rat model to show that the expression levels of major components of RAS in the brain and kidneys, such as angiotensinogen, angiotensin-converting enzyme, and angiotensin II AT1-receptor, was significantly upregulated. The upregulated expression level was inhibited by a central blocker of Wnt, which was an adeno-associated virus vector overexpressing the DKK1 gene [45]. Similarly, the present study found that AGTR1 was still expressed in the renal tubular epithelium, and the expression levels of renin and AGTR1 in the kidney tissues of SAMP8 mice were significantly greater than those in SAMR1 mice (Fig. 4a and d g). These results indicated that the Wnt/β-catenin/RAS signaling pathway was activated in the kidneys of SAMP8 mice, and the CF effectively inhibited the activation of this pathway.\\nCTGF exerts multiple biological functions, including promoting mitosis of chemotactic cells, inducing adhesion, and promoting cell proliferation and ECM synthesis [35, 46]. CCN2 (CTGF) modulated Wnt signaling by binding to low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to further mediate fibrosis [39, 47]. Other studies used gene silencing to discover and confirm that Nrf2 regulated the Wnt pathway by regulating the expression level of CTGF, affecting renal interstitial disease. Also, Nrf2 regulated the CTGF transcription level mainly via CTGF transcriptional regulator c-Fos [48]. The immunohistochemical analysis revealed that CTGF and p-c-Fos were expressed mainly in renal tubular epithelial cells, and p-c-Fos was also distributed in glomeruli. The quantitative analysis showed that the expression levels of both proteins was inhibited by CF (Figs. 3b and e and 4a and c). Finally, the PCA analysis was used to further verify that CF improved kidney damage in SAMP8 mice (Fig. 6). The present study provided evidence that the CF not only activated Nrf2 signaling but also relied on Nrf2 to balance oxidative stress and inflammation, inhibited Wnt/β-catenin/RAS signaling, and improved kidney aging and renal fibrogenesis. However, we also found inconsistent improvement in the effect of CF on Masson and SA-β-gal staining in this experiment. It is speculated that this may be due to the inconsistent progression of renal aging and renal fibrosis in SAMP8 mice. The degree of renal aging in the mice in this experiment may be more serious than fibrosis. Therefore, the ameliorating effect of CF-H on renal aging in SAMP8 mice was not as obvious as that of renal fibrosis.\\nAs a reducing monosaccharide, D-galactose is widely used in various age-related diseases in vivo and in vitro. D-gal was found to cause senescence and injury in NRK-52E cells [49], induce senescence of human kidney proximal tubular epithelial cells (HKC-8 cells), and increase the expression levels of two renal fibrosis marker proteins FN and α-SMA [6]. In this study, D-gal was used to induce NRK-52E cells in vitro. Using MTT assay and β-galactosidase staining to detect the compounds isolated from CF, it was found that SDC-0-14,16 and SDC-1-8 enhanced the cell viability induced by D-gal and inhibited D-gal induced cell senescence (Fig. 5). These suggested that SDC-0-14,16 and SDC-1-8 may be the material basis for CF to delay kidney aging. SDC-1-8 is one of the phenylethanoid glycosides. phenylethanoid glycosides were found to extend the life span of Caenorhabditis Elegans [50], with anti-aging, [51] and neuroprotective effects [52–54]. These provide a direction for our follow-up study on the pharmacological effects of CF.\",\n \"In conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF.\",\n \" \\nAdditional file 1.\\nAdditional file 1.\\n\\nAdditional file 2.\\nAdditional file 2.\\n\\nAdditional file 3.\\nAdditional file 3.\\n\\nAdditional file 1.\\nAdditional file 1.\\n\\nAdditional file 2.\\nAdditional file 2.\\n\\nAdditional file 3.\\nAdditional file 3.\",\n \"\\nAdditional file 1.\\nAdditional file 1.\\n\\nAdditional file 2.\\nAdditional file 2.\\n\\nAdditional file 3.\\nAdditional file 3.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["introduction","materials|methods",null,null,null,null,null,null,null,null,null,null,null,"results",null,null,null,null,null,null,"discussion","conclusion","supplementary-material",null],"string":"[\n \"introduction\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusion\",\n \"supplementary-material\",\n null\n]"},"keywords":{"kind":"list like","value":["Aging","\nCorallodiscus flabellata\n","Kidney","Renal fibrosis","SAMP8","Senescence","Wnt/β-catenin/RAS"],"string":"[\n \"Aging\",\n \"\\nCorallodiscus flabellata\\n\",\n \"Kidney\",\n \"Renal fibrosis\",\n \"SAMP8\",\n \"Senescence\",\n \"Wnt/β-catenin/RAS\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nThe global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.\nCF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books.\n\nMaterials and methods:\n Collection and extraction of the plant material “Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\n“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\n\nCollection and extraction of the plant material:\n“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\n\nAnimals and administration:\nSix-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\n\nSample collection:\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\n\nCell culture and in vitro study:\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\n\nSenescence-associated β-galactosidase staining:\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\n\nHistological analysis:\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\n\nBiochemical measurements:\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\n\nImmunohistochemical analysis:\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\n\nWestern blot analysis:\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\n\nUPLC-Q-TOF-MS analysis for kidney samples:\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\n\nStatistical analysis:\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\n\nResults:\n CF improved the aging and fibrosis of the kidneys in SAMP8 mice To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nTo investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\n CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nBesides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\n CF activated the Nrf2 pathway in the kidney of SAMP8 mice The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nThe expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\n CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nIn order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\n CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nD-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\n CF improved PCA analysis of kidney tissue in SAMP8 mice Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPrincipal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\n\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice:\nTo investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\n\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice:\nBesides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\n\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice:\nThe expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\n\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice:\nIn order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\n\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal:\nD-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\n\nCF improved PCA analysis of kidney tissue in SAMP8 mice:\nPrincipal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\n\nDiscussion:\nAging plays an important role in the progression of CKD [19]. As CKD fibrosis progresses, senescent cells express and secrete pro-fibrotic factors (TGF-β, CTGF, and so forth) and pro-inflammatory factors (IL-1β, IL-6, TNF-α, and so forth), which are senescence-associated secretory phenotype factors, thereby accelerating renal fibrosis [20, 21]. At present, traditional Chinese medicine has achieved good results in treating renal fibrosis with fewer side effects [22]. This study explored the interventional effects of CF extract on renal fibrosis in SAMP8 mice.\nThe mouse strain selected for this study was SAMP8, which was developed based on the lifespan, senescence, and pathological phenotypic grading scores of AKR/J mice and was an accelerated aging model solely of genetic origin [23, 24]. Studies have shown that the changes in the renal pathology of SAMP8 mice (9 months) include tubulointerstitial fibrosis and focal segmental glomerulosclerosis [25]. And it was found that renal fibrosis in SAMP8 mice was age-related [6]. Therefore, we tested the activity of β-galactosidase and the level of renal fibrosis in SAMP8 mice, and found that CF-L and CF-M improved kidney fibrosis and β-galactosidase activity in SAMP8 mice (Fig. 1). And we also tested the urine output of the mice. Consistent with previously published studies indicating that CF obtained by two different processes had diuretic effects [13], the extract significantly increased the urine volume of SAMP8 mice. Also, the present study showed that supplementation with CF improved the renal function, the activity of antioxidant enzymes, and levels of inflammatory factors in SAMP8 mice (Fig. 2).\nThe Keap1–Nrf2 system gained the attention of many scholars in recent years due to its antioxidant and anti-inflammatory properties. Its pharmacological potential in treating kidney diseases had been extensively studied in nonclinical and clinical studies [26]. Nrf2 is a master transcriptional regulator for genes related to redox status and antioxidant effects [27]. Studies have shown that phosphorylation is required for Nrf2 activation and target gene induction [28]. Activating Nrf2 improved CKD progression by preventing oxidative stress and maintaining cellular redox homeostasis [29]. As a key transcription factor, Nrf2 plays a crucial role in defense against oxidative stress by regulating its downstream antioxidants and detoxification enzymes [30]. Kim et al. reported that resveratrol, as a potent Nrf2 activator, ameliorated aging-related progressive renal injury [31]. In the present study, CF improved the attenuation of Nrf2 in the kidney of SAMP8 mice without affecting the expression level of Keap1, suggesting that CF crude extract might improve oxidative damage in the kidneys by activating the Nrf2 pathway (Fig. 3).\nRenal fibrosis is characterized by excessive extracellular matrix (ECM) deposition leading to the formation of scars in the renal parenchyma [32]. Transforming growth factor-β (TGF-β) is thought to be a key cytokine in fibroblast overactivation [33, 34]. Of course, targeting only the TGF-β signaling pathway is insufficient to reduce renal fibrosis. Some studies indicated that CTGF, Wnt/β-catenin, renin-angiotensin system, oxidative stress, and so forth, were implicated in renal fibrosis [35–37]. Wnt/β-catenin is an evolutionarily conserved signaling pathway involved in the regulation of tissue homeostasis, organ development, and injury repair [38]. Cisternas et al. discussed the pro-fibrotic effect of Wnt signaling in both skeletal muscle and kidney [39]. Mounting evidence established that the Wnt/β-catenin signaling pathway plays a crucial role in regulating the development and progression of renal fibrotic lesions following injury [40–42]. The Wnt/β-catenin signal is relatively silent in the kidneys of healthy adults and is activated once the kidneys are subjected to various kinds of damages [43]. In mammals, the Wnt family has at least 19 family members critical for kidney development. And at least 15 of these family members are differentially upregulated in the aging kidney, including Wnt4 [6]. The results of the present study showed that the expression level of Wnt4 protein in the kidneys of SAMP8 mice was significantly higher than that in SAMR1 mice, which was verified by both Western blot and immunohistochemical analyses (Fig. 4a, d and f). Coincidentally, the expression level of β-catenin protein was also upregulated. Also, both Wnt4 and β-catenin were expressed in renal tubular epithelial cells, as revealed by the immunohistochemical analysis (Fig. 4a and f). Wnt/β-catenin elicited renal fibrosis by inducing multiple fibrogenic genes such as RAS components, matrix metalloproteinase-7 (MMP-7), plasminogen activator inhibitor 1 (PAI-1), and Snail1 [37]. Zhou et al. described Wnt/β-catenin as the major upstream regulator, which controls the expression of all tested RAS components in the kidneys [44]. Zhou et al. used a 5/6 nephrectomy (5/6 NX) rat model to show that the expression levels of major components of RAS in the brain and kidneys, such as angiotensinogen, angiotensin-converting enzyme, and angiotensin II AT1-receptor, was significantly upregulated. The upregulated expression level was inhibited by a central blocker of Wnt, which was an adeno-associated virus vector overexpressing the DKK1 gene [45]. Similarly, the present study found that AGTR1 was still expressed in the renal tubular epithelium, and the expression levels of renin and AGTR1 in the kidney tissues of SAMP8 mice were significantly greater than those in SAMR1 mice (Fig. 4a and d g). These results indicated that the Wnt/β-catenin/RAS signaling pathway was activated in the kidneys of SAMP8 mice, and the CF effectively inhibited the activation of this pathway.\nCTGF exerts multiple biological functions, including promoting mitosis of chemotactic cells, inducing adhesion, and promoting cell proliferation and ECM synthesis [35, 46]. CCN2 (CTGF) modulated Wnt signaling by binding to low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to further mediate fibrosis [39, 47]. Other studies used gene silencing to discover and confirm that Nrf2 regulated the Wnt pathway by regulating the expression level of CTGF, affecting renal interstitial disease. Also, Nrf2 regulated the CTGF transcription level mainly via CTGF transcriptional regulator c-Fos [48]. The immunohistochemical analysis revealed that CTGF and p-c-Fos were expressed mainly in renal tubular epithelial cells, and p-c-Fos was also distributed in glomeruli. The quantitative analysis showed that the expression levels of both proteins was inhibited by CF (Figs. 3b and e and 4a and c). Finally, the PCA analysis was used to further verify that CF improved kidney damage in SAMP8 mice (Fig. 6). The present study provided evidence that the CF not only activated Nrf2 signaling but also relied on Nrf2 to balance oxidative stress and inflammation, inhibited Wnt/β-catenin/RAS signaling, and improved kidney aging and renal fibrogenesis. However, we also found inconsistent improvement in the effect of CF on Masson and SA-β-gal staining in this experiment. It is speculated that this may be due to the inconsistent progression of renal aging and renal fibrosis in SAMP8 mice. The degree of renal aging in the mice in this experiment may be more serious than fibrosis. Therefore, the ameliorating effect of CF-H on renal aging in SAMP8 mice was not as obvious as that of renal fibrosis.\nAs a reducing monosaccharide, D-galactose is widely used in various age-related diseases in vivo and in vitro. D-gal was found to cause senescence and injury in NRK-52E cells [49], induce senescence of human kidney proximal tubular epithelial cells (HKC-8 cells), and increase the expression levels of two renal fibrosis marker proteins FN and α-SMA [6]. In this study, D-gal was used to induce NRK-52E cells in vitro. Using MTT assay and β-galactosidase staining to detect the compounds isolated from CF, it was found that SDC-0-14,16 and SDC-1-8 enhanced the cell viability induced by D-gal and inhibited D-gal induced cell senescence (Fig. 5). These suggested that SDC-0-14,16 and SDC-1-8 may be the material basis for CF to delay kidney aging. SDC-1-8 is one of the phenylethanoid glycosides. phenylethanoid glycosides were found to extend the life span of Caenorhabditis Elegans [50], with anti-aging, [51] and neuroprotective effects [52–54]. These provide a direction for our follow-up study on the pharmacological effects of CF.\n\nConclusions:\nIn conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF.\n\nSupplementary Information:\n \nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.\n\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.\n\n:\n\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nFibrosis is one of the most common pathological features of the aging process of the kidney, and fibrosis in aging kidneys also aggravates the process of chronic kidney disease (CKD). Corallodiscus flabellata B. L. Burtt (C. flabellata, CF) is a commonly used botanical drug in Chinese folklore. However, few studies have reported its pharmacological effects. This study aimed to explore the effect of CF ethanol extract on renal fibrosis in SAMP8 mice and identify potentially active compounds.\n\nMethods:\nSenescence-accelerated mouse-prone 8 (SAMP8) were used as animal models, and different doses of CF were given by gavage for one month. To observe the degree of renal aging in mice using β-galactosidase staining. Masson staining and the expression levels of Col-I, α-SMA, and FN were used to evaluate the renal fibrosis in mice. The protein expression levels of Nrf2 pathway and Wnt/β-catenin/RAS pathway in the kidney were measured. And β-galactosidase (β-gal) induced NRK-52E cells as an in vitro model to screen the active components of CF.\n\nResults:\nThe CF ethanol extract significantly inhibited the activity of renal β-galactosidase and the expression levels of Col-I, α-SMA, and FN in SAMP8 mice, and improved Masson staining in SAMP8 mice. CF remarkably reduced urinary protein, creatinine, urea nitrogen and serum levels of TNF-α and IL-1β in SAMP8 mice, and significantly increased the levels of SOD and GSH-Px. Moreover, CF activated the Nrf2 pathway and blocked the Wnt/β-catenin/RAS pathway in the kidneys of mice. Besides, 3,4-dihydroxyphenylethanol (SDC-0-14, 16) and (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (SDC-1-8) were isolated from CF, which reduced the senescence of NRK-52E cells, and maybe the active ingredients of CF playing the anti-aging role.\n\nConclusions:\nOur experiments illuminated that CF ethanol extract may ameliorate renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS pathway. And SDC-0-14,16 and SDC-1-8 may be the material basis for CF to exert anti-renal senescence-related effects."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nThe global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.\nCF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books.\n\nConclusions:\nIn conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nFibrosis is one of the most common pathological features of the aging process of the kidney, and fibrosis in aging kidneys also aggravates the process of chronic kidney disease (CKD). Corallodiscus flabellata B. L. Burtt (C. flabellata, CF) is a commonly used botanical drug in Chinese folklore. However, few studies have reported its pharmacological effects. This study aimed to explore the effect of CF ethanol extract on renal fibrosis in SAMP8 mice and identify potentially active compounds.\n\nMethods:\nSenescence-accelerated mouse-prone 8 (SAMP8) were used as animal models, and different doses of CF were given by gavage for one month. To observe the degree of renal aging in mice using β-galactosidase staining. Masson staining and the expression levels of Col-I, α-SMA, and FN were used to evaluate the renal fibrosis in mice. The protein expression levels of Nrf2 pathway and Wnt/β-catenin/RAS pathway in the kidney were measured. And β-galactosidase (β-gal) induced NRK-52E cells as an in vitro model to screen the active components of CF.\n\nResults:\nThe CF ethanol extract significantly inhibited the activity of renal β-galactosidase and the expression levels of Col-I, α-SMA, and FN in SAMP8 mice, and improved Masson staining in SAMP8 mice. CF remarkably reduced urinary protein, creatinine, urea nitrogen and serum levels of TNF-α and IL-1β in SAMP8 mice, and significantly increased the levels of SOD and GSH-Px. Moreover, CF activated the Nrf2 pathway and blocked the Wnt/β-catenin/RAS pathway in the kidneys of mice. Besides, 3,4-dihydroxyphenylethanol (SDC-0-14, 16) and (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (SDC-1-8) were isolated from CF, which reduced the senescence of NRK-52E cells, and maybe the active ingredients of CF playing the anti-aging role.\n\nConclusions:\nOur experiments illuminated that CF ethanol extract may ameliorate renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS pathway. And SDC-0-14,16 and SDC-1-8 may be the material basis for CF to exert anti-renal senescence-related effects."},"whole_article_text_length":{"kind":"number","value":17396,"string":"17,396"},"whole_article_abstract_length":{"kind":"number","value":442,"string":"442"},"num_sections":{"kind":"number","value":24,"string":"24"},"most_frequent_words":{"kind":"list like","value":["cf","mice","extract","kidney","samp8","ethanol","samp8 mice","treated","dose","ethanol extract"],"string":"[\n \"cf\",\n \"mice\",\n \"extract\",\n \"kidney\",\n \"samp8\",\n \"ethanol\",\n \"samp8 mice\",\n \"treated\",\n \"dose\",\n \"ethanol extract\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | \nCorallodiscus flabellata\n | Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | \nCorallodiscus flabellata\n | Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | \nCorallodiscus flabellata\n | Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | \nCorallodiscus flabellata\n | Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | \nCorallodiscus flabellata\n | Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] fibrosis | renal fibrosis | catenin | renal | ras | disease | lef | cf | wnt catenin | wnt [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | cf treated | dose cf ethanol | dose cf ethanol extract | cf ethanol extract | dose cf | ethanol extract | cf ethanol | dose [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ingredients | active | active ingredients | cf | pharmacological support treating | research active | experiments findings provided pharmacological | experiments findings provided | experiments findings | ingredients found [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | ethanol | extract | china | additional | additional file | file | kidney | samp8 [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | ethanol | extract | china | additional | additional file | file | kidney | samp8 [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| B. L. Burtt | CF | Chinese ||| ||| CF | SAMP8 [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] CF | SAMP8 | Masson | SAMP8 ||| TNF | IL-1β | SAMP8 | SOD | GSH-Px ||| Nrf2 | Wnt ||| 3,4 | SDC-0-14 | 16 | 3,4 | SDC-1-8) | CF | NRK-52E | CF [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] CF | SAMP8 | Wnt ||| SDC-0-14,16 | SDC-1-8 | CF [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| B. L. Burtt | CF | Chinese ||| ||| CF | SAMP8 ||| CF | one month ||| ||| Masson ||| Nrf2 | Wnt ||| NRK-52E | CF ||| ||| CF | SAMP8 | Masson | SAMP8 ||| TNF | IL-1β | SAMP8 | SOD | GSH-Px ||| Nrf2 | Wnt ||| 3,4 | SDC-0-14 | 16 | 3,4 | SDC-1-8) | CF | NRK-52E | CF ||| CF | SAMP8 | Wnt ||| SDC-0-14,16 | SDC-1-8 | CF [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| B. L. Burtt | CF | Chinese ||| ||| CF | SAMP8 ||| CF | one month ||| ||| Masson ||| Nrf2 | Wnt ||| NRK-52E | CF ||| ||| CF | SAMP8 | Masson | SAMP8 ||| TNF | IL-1β | SAMP8 | SOD | GSH-Px ||| Nrf2 | Wnt ||| 3,4 | SDC-0-14 | 16 | 3,4 | SDC-1-8) | CF | NRK-52E | CF ||| CF | SAMP8 | Wnt ||| SDC-0-14,16 | SDC-1-8 | CF [SUMMARY]"}}},{"rowIdx":292,"cells":{"title":{"kind":"string","value":"The plant coumarins auraptene and lacinartin as potential multifunctional therapeutic agents for treating periodontal disease."},"pmid":{"kind":"string","value":"22742512"},"background_abstract":{"kind":"string","value":"Periodontal diseases are bacterial infections leading to chronic inflammation disorders that are frequently observed in adults. In the present study, we evaluated the effect of auraptene and lacinartin, two natural oxyprenylated coumarins, on the growth, adherence properties, and collagenase activity of Porphyromonas gingivalis. We also investigated the capacity of these compounds to reduce cytokine and matrix metalloproteinase (MMP) secretion by lipopolysaccharide (LPS)-stimulated macrophages and to inhibit MMP-9 activity."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Microplate dilution assays were performed to determine the effect of auraptene and lacinartin on P. gingivalis growth as well as biofilm formation stained with crystal violet. Adhesion of FITC-labeled P. gingivalis to oral epithelial cells was monitored by fluorometry. The effects of auraptene and lacinartin on LPS-induced cytokine and MMP secretion by macrophages were determined by immunological assays. Fluorogenic assays were used to evaluate the capacity of the two coumarins to inhibit the activity of P. gingivalis collagenase and MMP-9."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Only lacinartin completely inhibited P. gingivalis growth in a complex culture medium. However, under iron-limiting conditions, auraptene and lacinartin both inhibited the growth of P. gingivalis. Lacinartin also inhibited biofilm formation by P. gingivalis and promoted biofilm desorption. Both compounds prevented the adherence of P. gingivalis to oral epithelial cells, dose-dependently reduced the secretion of cytokines (IL-8 and TNF-α) and MMP-8 and MMP-9 by LPS-stimulated macrophages, and inhibited MMP-9 activity. Lacinartin also inhibited P. gingivalis collagenase activity."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"By acting on multiple targets, including pathogenic bacteria, tissue-destructive enzymes, and the host inflammatory response, auraptene and lacinartin may be promising natural compounds for preventing and treating periodontal diseases."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Bacterial Adhesion","Biofilms","Cell Line","Coumarins","Epithelial Cells","Humans","Macrophages","Periodontal Diseases","Plant Extracts","Porphyromonas gingivalis"],"string":"[\n \"Bacterial Adhesion\",\n \"Biofilms\",\n \"Cell Line\",\n \"Coumarins\",\n \"Epithelial Cells\",\n \"Humans\",\n \"Macrophages\",\n \"Periodontal Diseases\",\n \"Plant Extracts\",\n \"Porphyromonas gingivalis\"\n]"},"pmcid":{"kind":"string","value":"3489859"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Lacinartin (50 and 100 μg/ml) almost completely inhibited the growth of P. gingivalis in THB-HK (Figure.\n2B), while the highest concentration of auraptene tested (100 μg/ml) only reduced growth by 42% (Figure.\n2A). Bacterial growth was slightly lower in MBB-H (OD660 = 0.59), which represents an iron poor condition for P. gingivalis, than in THB-HK (OD660 = 0.78) (Figure.\n2). The lowest concentration of auraptene tested (12.5 μg/ml) inhibited growth by 58% in MBB-H, while such a concentration had no significant inhibitory effect in THB-HK (Figure.\n2A). Almost complete inhibition was observed at higher concentrations (50 and 100 μg/ml). As for auraptene, lacinartin seemed to be more effective for inhibiting growth of P. gingivalis in iron-limiting conditions.\n Effect of auraptene (A) and lacinartin (B) on the growth of P. gingivalis in a complex medium (THB-HK) and under iron-limiting conditions in MBB-H. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed using the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene had no obvious inhibitory effect on P. gingivalis biofilm formation or desorption (Figure.\n3). On the contrary, high concentrations (50 and 100 μg/ml) of auraptene appear to increase biofilm formation (Figure.\n3A). Lacinartin at 50 and 100 μg/ml inhibited biofilm formation by P. gingivalis by approximately 75% (Figure.\n3A). In addition, lacinartin (12.5-100 μg/ml) caused approximately one-third of the biofilm to desorb (Figure.\n3B).\n Effect of auraptene and lacinartin on P. gingivalis biofilm formation (A) and on desorption of a pre-formed P. gingivalis biofilm (B). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene and lacinartin both dose-dependently inhibited bacterial adhesion to oral epithelial cells (Figure.\n4). At the lowest concentration tested (12.5 μg/ml), auraptene and lacinartin reduced the adherence of P. gingivalis to epithelial cells by 33% and 43%, respectively, while 100 μg/ml of auraptene and lacinartin reduced adherence by 37% and 71%, respectively (Figure.\n4).\n Effect of auraptene and lacinartin on the adherence of P. gingivalis to human oral epithelial cells. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. A value of 100% was assigned to the control (no auraptene; no lacinartin). Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nThe highest non-cytotoxic concentrations of auraptene and lacinartin that can be used to evaluate their effect on the inflammatory response of a human macrophage model stimulated with LPS were 25 and 50 μg/ml, respectively (data not shown). Following a 2-h pretreatment of the model with auraptene (6.25, 12.5, and 25 μg/ml) or lacinartin (12.5, 25, and 50 μg/ml), macrophages were stimulated with LPS to induce an inflammatory response (cytokine and MMP secretion). Auraptene and lacinartin both had a significant inhibitory effect on IL-8 and TNF-α secretion. At the lowest concentration tested (6.25 μg/ml), auraptene reduced IL-8 and TNF-α secretion by 22% and 37%, respectively, compared to untreated cells, while at the highest concentration tested (25 μg/ml), it inhibited IL-8 and TNF-α secretion by 92% and 85%, respectively (Figure.\n5). As shown in Fig.\n5, the highest concentration of lacinartin tested (50 μg/ml) reduced IL-8 and TNF-α secretion by 95% and 99%, respectively, while 12.5 and 25 μg/ml of lacinartin increased the secretion of both cytokines, likely due to a synergistic effect of LPS and lacinartin, since lacinartin alone had no effect (data not shown). Auraptene and lacinartin both reduced MMP-8 and MMP-9 secretion, sometimes below basal levels (Figure.\n6). Auraptene reduced MMP-8 secretion by 63% (6.25 μg/ml), 69% (12.5 μg/ml), and 73% (25 μg/ml), while lacinartin reduced MMP-8 secretion by 79% (12.5 μg/ml), 83% (25 μg/ml), and 89% (50 μg/ml) (Figure.\n6A). At their highest concentrations tested, auraptene (25 μg/ml) reduced MMP-9 secretion by 76% (25 μg/ml) (Figure.\n6B), while lacinartin (50 μg/ml) reduced MMP-9 secretion by 82% (Figure.\n6B).\n Effect of auraptene and lacinartin on the secretion of IL-8 (A) and TNF-α (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control, †: p ≤ 0.05 vs. control without auraptene or lacinartin).\n Effect of auraptene and lacinartin on the secretion of MMP-8 (A) and MMP-9 (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control).\nAfter demonstrating that auraptene and lacinartin can decrease MMP secretion in a macrophage model, we evaluated their effect on proteinase activity. Both auraptene and lacinartin (12.5 μg/ml) reduced MMP-9 activity by 74% (Table\n1). Auraptene had no inhibitory effect on P. gingivalis collagenase activity while 12.5 μg/ml and 100 μg/ml of lacinartin reduced collagenase activity by 20% and 64%, respectively (Table\n1).\n\nEffect of auraptene and lacinartin on the activity of MMP-9 and \n\nP. gingivalis\n\n collagenase\n\n1Inhibitor of MMP-9: GM6001 (0.025 μM); inhibitor of P. gingivalis collagenase: leupeptin (1 μM)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity."},"other_sections_titles":{"kind":"list like","value":["Introduction","Compounds","Effect on Porphyromonas gingivalis growth","Effect on P. gingivalis biofilm formation/desorption","Effect on P. gingivalis adherence to oral epithelial cells","Anti-inflammatory properties in a macrophage model","Inhibition of MMP-9 and P. gingivalis collagenase activity","Statistical analysis","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Introduction\",\n \"Compounds\",\n \"Effect on Porphyromonas gingivalis growth\",\n \"Effect on P. gingivalis biofilm formation/desorption\",\n \"Effect on P. gingivalis adherence to oral epithelial cells\",\n \"Anti-inflammatory properties in a macrophage model\",\n \"Inhibition of MMP-9 and P. gingivalis collagenase activity\",\n \"Statistical analysis\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.","Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.","P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.","P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.","P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.","U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.","Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.","Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.","The authors declare that they have no competing interests.","All authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\n"],"string":"[\n \"Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\\n[6,7].\\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\\n[18,19] while little is known about lacinartin. \\n Chemical structures of auraptene (A) and lacinartin (B).\\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.\",\n \"Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\",\n \"P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\",\n \"P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\",\n \"P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\",\n \"U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\",\n \"Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\",\n \"Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\",\n \"The authors declare that they have no competing interests.\",\n \"All authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and methods","Compounds","Effect on Porphyromonas gingivalis growth","Effect on P. gingivalis biofilm formation/desorption","Effect on P. gingivalis adherence to oral epithelial cells","Anti-inflammatory properties in a macrophage model","Inhibition of MMP-9 and P. gingivalis collagenase activity","Statistical analysis","Results","Discussion","Conclusions","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Introduction\",\n \"Materials and methods\",\n \"Compounds\",\n \"Effect on Porphyromonas gingivalis growth\",\n \"Effect on P. gingivalis biofilm formation/desorption\",\n \"Effect on P. gingivalis adherence to oral epithelial cells\",\n \"Anti-inflammatory properties in a macrophage model\",\n \"Inhibition of MMP-9 and P. gingivalis collagenase activity\",\n \"Statistical analysis\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase."," Compounds Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\nAuraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\n Effect on Porphyromonas gingivalis growth P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\nP. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\n Effect on P. gingivalis biofilm formation/desorption P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\nP. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\n Effect on P. gingivalis adherence to oral epithelial cells P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\nP. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\n Anti-inflammatory properties in a macrophage model U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\nU937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\n Inhibition of MMP-9 and P. gingivalis collagenase activity Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\nHuman recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\n Statistical analysis Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\nResults are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.","Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.","P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.","P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.","P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.","U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.","Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.","Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.","Lacinartin (50 and 100 μg/ml) almost completely inhibited the growth of P. gingivalis in THB-HK (Figure.\n2B), while the highest concentration of auraptene tested (100 μg/ml) only reduced growth by 42% (Figure.\n2A). Bacterial growth was slightly lower in MBB-H (OD660 = 0.59), which represents an iron poor condition for P. gingivalis, than in THB-HK (OD660 = 0.78) (Figure.\n2). The lowest concentration of auraptene tested (12.5 μg/ml) inhibited growth by 58% in MBB-H, while such a concentration had no significant inhibitory effect in THB-HK (Figure.\n2A). Almost complete inhibition was observed at higher concentrations (50 and 100 μg/ml). As for auraptene, lacinartin seemed to be more effective for inhibiting growth of P. gingivalis in iron-limiting conditions.\n Effect of auraptene (A) and lacinartin (B) on the growth of P. gingivalis in a complex medium (THB-HK) and under iron-limiting conditions in MBB-H. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed using the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene had no obvious inhibitory effect on P. gingivalis biofilm formation or desorption (Figure.\n3). On the contrary, high concentrations (50 and 100 μg/ml) of auraptene appear to increase biofilm formation (Figure.\n3A). Lacinartin at 50 and 100 μg/ml inhibited biofilm formation by P. gingivalis by approximately 75% (Figure.\n3A). In addition, lacinartin (12.5-100 μg/ml) caused approximately one-third of the biofilm to desorb (Figure.\n3B).\n Effect of auraptene and lacinartin on P. gingivalis biofilm formation (A) and on desorption of a pre-formed P. gingivalis biofilm (B). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene and lacinartin both dose-dependently inhibited bacterial adhesion to oral epithelial cells (Figure.\n4). At the lowest concentration tested (12.5 μg/ml), auraptene and lacinartin reduced the adherence of P. gingivalis to epithelial cells by 33% and 43%, respectively, while 100 μg/ml of auraptene and lacinartin reduced adherence by 37% and 71%, respectively (Figure.\n4).\n Effect of auraptene and lacinartin on the adherence of P. gingivalis to human oral epithelial cells. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. A value of 100% was assigned to the control (no auraptene; no lacinartin). Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nThe highest non-cytotoxic concentrations of auraptene and lacinartin that can be used to evaluate their effect on the inflammatory response of a human macrophage model stimulated with LPS were 25 and 50 μg/ml, respectively (data not shown). Following a 2-h pretreatment of the model with auraptene (6.25, 12.5, and 25 μg/ml) or lacinartin (12.5, 25, and 50 μg/ml), macrophages were stimulated with LPS to induce an inflammatory response (cytokine and MMP secretion). Auraptene and lacinartin both had a significant inhibitory effect on IL-8 and TNF-α secretion. At the lowest concentration tested (6.25 μg/ml), auraptene reduced IL-8 and TNF-α secretion by 22% and 37%, respectively, compared to untreated cells, while at the highest concentration tested (25 μg/ml), it inhibited IL-8 and TNF-α secretion by 92% and 85%, respectively (Figure.\n5). As shown in Fig.\n5, the highest concentration of lacinartin tested (50 μg/ml) reduced IL-8 and TNF-α secretion by 95% and 99%, respectively, while 12.5 and 25 μg/ml of lacinartin increased the secretion of both cytokines, likely due to a synergistic effect of LPS and lacinartin, since lacinartin alone had no effect (data not shown). Auraptene and lacinartin both reduced MMP-8 and MMP-9 secretion, sometimes below basal levels (Figure.\n6). Auraptene reduced MMP-8 secretion by 63% (6.25 μg/ml), 69% (12.5 μg/ml), and 73% (25 μg/ml), while lacinartin reduced MMP-8 secretion by 79% (12.5 μg/ml), 83% (25 μg/ml), and 89% (50 μg/ml) (Figure.\n6A). At their highest concentrations tested, auraptene (25 μg/ml) reduced MMP-9 secretion by 76% (25 μg/ml) (Figure.\n6B), while lacinartin (50 μg/ml) reduced MMP-9 secretion by 82% (Figure.\n6B).\n Effect of auraptene and lacinartin on the secretion of IL-8 (A) and TNF-α (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control, †: p ≤ 0.05 vs. control without auraptene or lacinartin).\n Effect of auraptene and lacinartin on the secretion of MMP-8 (A) and MMP-9 (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control).\nAfter demonstrating that auraptene and lacinartin can decrease MMP secretion in a macrophage model, we evaluated their effect on proteinase activity. Both auraptene and lacinartin (12.5 μg/ml) reduced MMP-9 activity by 74% (Table\n1). Auraptene had no inhibitory effect on P. gingivalis collagenase activity while 12.5 μg/ml and 100 μg/ml of lacinartin reduced collagenase activity by 20% and 64%, respectively (Table\n1).\n\nEffect of auraptene and lacinartin on the activity of MMP-9 and \n\nP. gingivalis\n\n collagenase\n\n1Inhibitor of MMP-9: GM6001 (0.025 μM); inhibitor of P. gingivalis collagenase: leupeptin (1 μM).","Periodontal diseases are polymicrobial infections and are the most common chronic inflammatory disorders in adults\n[22]. Periodontitis is induced by a specific group of Gram-negative anaerobic bacteria and is the major cause of tooth loss in adults\n[23]. Over the past two decades, natural compounds with antibacterial and anti-inflammatory properties have received considerable attention as new therapeutic agents for the treatment of periodontal infections. In this study, we investigated the potential of auraptene and lacinartin for preventing and treating periodontal diseases.\nWe first showed that lacinartin and to a lesser extent auraptene reduced P. gingivalis growth. This is the first report indicating that lacinartin possesses anti-bacterial properties. Previous studies have shown that auraptene has antibacterial properties against Helicobacter pylori[24,25]. The exact mechanism by which lacinartin and auraptene inhibit bacterial growth is unknown. However, other natural coumarins (novobiocin and clorobiocin) inhibit deoxyribonuclease gyrase activity, which results in bacteria death\n[14,26]. In addition, we showed that auraptene and lacinartin inhibit growth more effectively under iron-limiting conditions, requiring much lower concentrations to significantly reduce the growth of P. gingivalis. Our results are in agreement with those of Mladnka et al.\n[27], who showed that coumarins possess iron-chelating properties. Additional studies are required to investigate interactions between iron and auraptene and lacinartin.\nWe also showed that lacinartin, but not auraptene, inhibits biofilm formation by P. gingivalis. Lacinartin also caused the desorption of a pre-formed P. gingivalis biofilm. To the best of our knowledge, this is the first report regarding the inhibitory effects of lacinartin on bacterial biofilms. Auraptene and lacinartin prevented the adherence of P. gingivalis to oral epithelial cells to a significant degree. Epithelial cells act as a physical barrier, and bacterial adherence to these host cells may be a critical step for the initiation of periodontal diseases\n[28]. Given its ability to reduce growth of P. gingivalis and its adherence to epithelial cells, lacinartin may be a promising therapeutic candidate through its action on different targets.\nPolyphenols reduce inflammatory mediator secretion and, as such, inflammation-mediated damage\n[29]. We showed that auraptene markedly reduces IL-8 and TNF-α secretion by LPS-stimulated macrophages. Our results are in agreement with those of Genovese et al., who reported that auraptene inhibits the release of TNF-α by RAW 264.7 macrophages\n[30]. To our knowledge, no one has investigated the anti-inflammatory properties of lacinartin. We showed that 12.5 μg/ml of lacinartin induced IL-8 and TNF-α secretion, likely due to a synergistic interaction between LPS and lacinartin. On the other hand, 50 μg/ml of lacinartin significantly inhibited IL-8 and TNF-α secretion. We also showed that auraptene and lacinartin reduced MMP-8 and MMP-9 secretion. These results are in agreement with those of a study by Epifano et al., who reported that auraptene inhibits MMP-7 secretion by HT-29 epithelial cells\n[18]. Since MMP release and cytokine secretion are associated with tooth-supporting tissue destruction, our results suggested that both compounds may contribute to reducing host cell damage, including bone resorption\n[5,31]. The mechanisms by which auraptene and lacinartin reduce inflammatory mediator secretion are unknown, but previous studies have shown that coumarins can block the activation of nuclear factor-κB and inhibit kinase pathways (Akt/PKB)\n[26]. Considering that gingival fibroblasts may also play a significant role in periodontal tissue destruction through cytokine-inducible MMP secretion, future studies should investigate the effects of auraptene and lacinartin on this cell type.\nWe further showed that auraptene and lacinartin reduce MMP-9 activity while only lacinartin inhibits P. gingivalis collagenase activity. Auraptene has previously been shown to inhibit MMP-7 activity\n[32]. These observations suggest that these coumarins may contribute to reducing tissue destruction.","In conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity.","The authors declare that they have no competing interests.","All authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\n"],"string":"[\n \"Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\\n[6,7].\\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\\n[18,19] while little is known about lacinartin. \\n Chemical structures of auraptene (A) and lacinartin (B).\\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.\",\n \" Compounds Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\\nAuraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\\n Effect on Porphyromonas gingivalis growth P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\\nP. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\\n Effect on P. gingivalis biofilm formation/desorption P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\\nP. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\\n Effect on P. gingivalis adherence to oral epithelial cells P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\\nP. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\\n Anti-inflammatory properties in a macrophage model U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\\nU937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\\n Inhibition of MMP-9 and P. gingivalis collagenase activity Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\\nHuman recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\\n Statistical analysis Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\\nResults are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\",\n \"Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\",\n \"P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\",\n \"P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\",\n \"P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\",\n \"U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\",\n \"Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\",\n \"Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\",\n \"Lacinartin (50 and 100 μg/ml) almost completely inhibited the growth of P. gingivalis in THB-HK (Figure.\\n2B), while the highest concentration of auraptene tested (100 μg/ml) only reduced growth by 42% (Figure.\\n2A). Bacterial growth was slightly lower in MBB-H (OD660 = 0.59), which represents an iron poor condition for P. gingivalis, than in THB-HK (OD660 = 0.78) (Figure.\\n2). The lowest concentration of auraptene tested (12.5 μg/ml) inhibited growth by 58% in MBB-H, while such a concentration had no significant inhibitory effect in THB-HK (Figure.\\n2A). Almost complete inhibition was observed at higher concentrations (50 and 100 μg/ml). As for auraptene, lacinartin seemed to be more effective for inhibiting growth of P. gingivalis in iron-limiting conditions.\\n Effect of auraptene (A) and lacinartin (B) on the growth of P. gingivalis in a complex medium (THB-HK) and under iron-limiting conditions in MBB-H. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed using the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\\nAuraptene had no obvious inhibitory effect on P. gingivalis biofilm formation or desorption (Figure.\\n3). On the contrary, high concentrations (50 and 100 μg/ml) of auraptene appear to increase biofilm formation (Figure.\\n3A). Lacinartin at 50 and 100 μg/ml inhibited biofilm formation by P. gingivalis by approximately 75% (Figure.\\n3A). In addition, lacinartin (12.5-100 μg/ml) caused approximately one-third of the biofilm to desorb (Figure.\\n3B).\\n Effect of auraptene and lacinartin on P. gingivalis biofilm formation (A) and on desorption of a pre-formed P. gingivalis biofilm (B). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\\nAuraptene and lacinartin both dose-dependently inhibited bacterial adhesion to oral epithelial cells (Figure.\\n4). At the lowest concentration tested (12.5 μg/ml), auraptene and lacinartin reduced the adherence of P. gingivalis to epithelial cells by 33% and 43%, respectively, while 100 μg/ml of auraptene and lacinartin reduced adherence by 37% and 71%, respectively (Figure.\\n4).\\n Effect of auraptene and lacinartin on the adherence of P. gingivalis to human oral epithelial cells. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. A value of 100% was assigned to the control (no auraptene; no lacinartin). Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\\nThe highest non-cytotoxic concentrations of auraptene and lacinartin that can be used to evaluate their effect on the inflammatory response of a human macrophage model stimulated with LPS were 25 and 50 μg/ml, respectively (data not shown). Following a 2-h pretreatment of the model with auraptene (6.25, 12.5, and 25 μg/ml) or lacinartin (12.5, 25, and 50 μg/ml), macrophages were stimulated with LPS to induce an inflammatory response (cytokine and MMP secretion). Auraptene and lacinartin both had a significant inhibitory effect on IL-8 and TNF-α secretion. At the lowest concentration tested (6.25 μg/ml), auraptene reduced IL-8 and TNF-α secretion by 22% and 37%, respectively, compared to untreated cells, while at the highest concentration tested (25 μg/ml), it inhibited IL-8 and TNF-α secretion by 92% and 85%, respectively (Figure.\\n5). As shown in Fig.\\n5, the highest concentration of lacinartin tested (50 μg/ml) reduced IL-8 and TNF-α secretion by 95% and 99%, respectively, while 12.5 and 25 μg/ml of lacinartin increased the secretion of both cytokines, likely due to a synergistic effect of LPS and lacinartin, since lacinartin alone had no effect (data not shown). Auraptene and lacinartin both reduced MMP-8 and MMP-9 secretion, sometimes below basal levels (Figure.\\n6). Auraptene reduced MMP-8 secretion by 63% (6.25 μg/ml), 69% (12.5 μg/ml), and 73% (25 μg/ml), while lacinartin reduced MMP-8 secretion by 79% (12.5 μg/ml), 83% (25 μg/ml), and 89% (50 μg/ml) (Figure.\\n6A). At their highest concentrations tested, auraptene (25 μg/ml) reduced MMP-9 secretion by 76% (25 μg/ml) (Figure.\\n6B), while lacinartin (50 μg/ml) reduced MMP-9 secretion by 82% (Figure.\\n6B).\\n Effect of auraptene and lacinartin on the secretion of IL-8 (A) and TNF-α (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control, †: p ≤ 0.05 vs. control without auraptene or lacinartin).\\n Effect of auraptene and lacinartin on the secretion of MMP-8 (A) and MMP-9 (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control).\\nAfter demonstrating that auraptene and lacinartin can decrease MMP secretion in a macrophage model, we evaluated their effect on proteinase activity. Both auraptene and lacinartin (12.5 μg/ml) reduced MMP-9 activity by 74% (Table\\n1). Auraptene had no inhibitory effect on P. gingivalis collagenase activity while 12.5 μg/ml and 100 μg/ml of lacinartin reduced collagenase activity by 20% and 64%, respectively (Table\\n1).\\n\\nEffect of auraptene and lacinartin on the activity of MMP-9 and \\n\\nP. gingivalis\\n\\n collagenase\\n\\n1Inhibitor of MMP-9: GM6001 (0.025 μM); inhibitor of P. gingivalis collagenase: leupeptin (1 μM).\",\n \"Periodontal diseases are polymicrobial infections and are the most common chronic inflammatory disorders in adults\\n[22]. Periodontitis is induced by a specific group of Gram-negative anaerobic bacteria and is the major cause of tooth loss in adults\\n[23]. Over the past two decades, natural compounds with antibacterial and anti-inflammatory properties have received considerable attention as new therapeutic agents for the treatment of periodontal infections. In this study, we investigated the potential of auraptene and lacinartin for preventing and treating periodontal diseases.\\nWe first showed that lacinartin and to a lesser extent auraptene reduced P. gingivalis growth. This is the first report indicating that lacinartin possesses anti-bacterial properties. Previous studies have shown that auraptene has antibacterial properties against Helicobacter pylori[24,25]. The exact mechanism by which lacinartin and auraptene inhibit bacterial growth is unknown. However, other natural coumarins (novobiocin and clorobiocin) inhibit deoxyribonuclease gyrase activity, which results in bacteria death\\n[14,26]. In addition, we showed that auraptene and lacinartin inhibit growth more effectively under iron-limiting conditions, requiring much lower concentrations to significantly reduce the growth of P. gingivalis. Our results are in agreement with those of Mladnka et al.\\n[27], who showed that coumarins possess iron-chelating properties. Additional studies are required to investigate interactions between iron and auraptene and lacinartin.\\nWe also showed that lacinartin, but not auraptene, inhibits biofilm formation by P. gingivalis. Lacinartin also caused the desorption of a pre-formed P. gingivalis biofilm. To the best of our knowledge, this is the first report regarding the inhibitory effects of lacinartin on bacterial biofilms. Auraptene and lacinartin prevented the adherence of P. gingivalis to oral epithelial cells to a significant degree. Epithelial cells act as a physical barrier, and bacterial adherence to these host cells may be a critical step for the initiation of periodontal diseases\\n[28]. Given its ability to reduce growth of P. gingivalis and its adherence to epithelial cells, lacinartin may be a promising therapeutic candidate through its action on different targets.\\nPolyphenols reduce inflammatory mediator secretion and, as such, inflammation-mediated damage\\n[29]. We showed that auraptene markedly reduces IL-8 and TNF-α secretion by LPS-stimulated macrophages. Our results are in agreement with those of Genovese et al., who reported that auraptene inhibits the release of TNF-α by RAW 264.7 macrophages\\n[30]. To our knowledge, no one has investigated the anti-inflammatory properties of lacinartin. We showed that 12.5 μg/ml of lacinartin induced IL-8 and TNF-α secretion, likely due to a synergistic interaction between LPS and lacinartin. On the other hand, 50 μg/ml of lacinartin significantly inhibited IL-8 and TNF-α secretion. We also showed that auraptene and lacinartin reduced MMP-8 and MMP-9 secretion. These results are in agreement with those of a study by Epifano et al., who reported that auraptene inhibits MMP-7 secretion by HT-29 epithelial cells\\n[18]. Since MMP release and cytokine secretion are associated with tooth-supporting tissue destruction, our results suggested that both compounds may contribute to reducing host cell damage, including bone resorption\\n[5,31]. The mechanisms by which auraptene and lacinartin reduce inflammatory mediator secretion are unknown, but previous studies have shown that coumarins can block the activation of nuclear factor-κB and inhibit kinase pathways (Akt/PKB)\\n[26]. Considering that gingival fibroblasts may also play a significant role in periodontal tissue destruction through cytokine-inducible MMP secretion, future studies should investigate the effects of auraptene and lacinartin on this cell type.\\nWe further showed that auraptene and lacinartin reduce MMP-9 activity while only lacinartin inhibits P. gingivalis collagenase activity. Auraptene has previously been shown to inhibit MMP-7 activity\\n[32]. These observations suggest that these coumarins may contribute to reducing tissue destruction.\",\n \"In conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity.\",\n \"The authors declare that they have no competing interests.\",\n \"All authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"materials|methods",null,null,null,null,null,null,null,"results","discussion","conclusions",null,null,null],"string":"[\n null,\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Coumarins","Auraptene","Lacinartin","Antibacterial","Anti-adherence","Anti-inflammatory"],"string":"[\n \"Coumarins\",\n \"Auraptene\",\n \"Lacinartin\",\n \"Antibacterial\",\n \"Anti-adherence\",\n \"Anti-inflammatory\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nPeriodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.\n\nMaterials and methods:\n Compounds Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\nAuraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\n Effect on Porphyromonas gingivalis growth P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\nP. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\n Effect on P. gingivalis biofilm formation/desorption P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\nP. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\n Effect on P. gingivalis adherence to oral epithelial cells P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\nP. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\n Anti-inflammatory properties in a macrophage model U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\nU937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\n Inhibition of MMP-9 and P. gingivalis collagenase activity Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\nHuman recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\n Statistical analysis Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\nResults are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\n\nCompounds:\nAuraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\n\nEffect on Porphyromonas gingivalis growth:\nP. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\n\nEffect on P. gingivalis biofilm formation/desorption:\nP. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\n\nEffect on P. gingivalis adherence to oral epithelial cells:\nP. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\n\nAnti-inflammatory properties in a macrophage model:\nU937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\n\nInhibition of MMP-9 and P. gingivalis collagenase activity:\nHuman recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\n\nStatistical analysis:\nResults are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\n\nResults:\nLacinartin (50 and 100 μg/ml) almost completely inhibited the growth of P. gingivalis in THB-HK (Figure.\n2B), while the highest concentration of auraptene tested (100 μg/ml) only reduced growth by 42% (Figure.\n2A). Bacterial growth was slightly lower in MBB-H (OD660 = 0.59), which represents an iron poor condition for P. gingivalis, than in THB-HK (OD660 = 0.78) (Figure.\n2). The lowest concentration of auraptene tested (12.5 μg/ml) inhibited growth by 58% in MBB-H, while such a concentration had no significant inhibitory effect in THB-HK (Figure.\n2A). Almost complete inhibition was observed at higher concentrations (50 and 100 μg/ml). As for auraptene, lacinartin seemed to be more effective for inhibiting growth of P. gingivalis in iron-limiting conditions.\n Effect of auraptene (A) and lacinartin (B) on the growth of P. gingivalis in a complex medium (THB-HK) and under iron-limiting conditions in MBB-H. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed using the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene had no obvious inhibitory effect on P. gingivalis biofilm formation or desorption (Figure.\n3). On the contrary, high concentrations (50 and 100 μg/ml) of auraptene appear to increase biofilm formation (Figure.\n3A). Lacinartin at 50 and 100 μg/ml inhibited biofilm formation by P. gingivalis by approximately 75% (Figure.\n3A). In addition, lacinartin (12.5-100 μg/ml) caused approximately one-third of the biofilm to desorb (Figure.\n3B).\n Effect of auraptene and lacinartin on P. gingivalis biofilm formation (A) and on desorption of a pre-formed P. gingivalis biofilm (B). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene and lacinartin both dose-dependently inhibited bacterial adhesion to oral epithelial cells (Figure.\n4). At the lowest concentration tested (12.5 μg/ml), auraptene and lacinartin reduced the adherence of P. gingivalis to epithelial cells by 33% and 43%, respectively, while 100 μg/ml of auraptene and lacinartin reduced adherence by 37% and 71%, respectively (Figure.\n4).\n Effect of auraptene and lacinartin on the adherence of P. gingivalis to human oral epithelial cells. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. A value of 100% was assigned to the control (no auraptene; no lacinartin). Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nThe highest non-cytotoxic concentrations of auraptene and lacinartin that can be used to evaluate their effect on the inflammatory response of a human macrophage model stimulated with LPS were 25 and 50 μg/ml, respectively (data not shown). Following a 2-h pretreatment of the model with auraptene (6.25, 12.5, and 25 μg/ml) or lacinartin (12.5, 25, and 50 μg/ml), macrophages were stimulated with LPS to induce an inflammatory response (cytokine and MMP secretion). Auraptene and lacinartin both had a significant inhibitory effect on IL-8 and TNF-α secretion. At the lowest concentration tested (6.25 μg/ml), auraptene reduced IL-8 and TNF-α secretion by 22% and 37%, respectively, compared to untreated cells, while at the highest concentration tested (25 μg/ml), it inhibited IL-8 and TNF-α secretion by 92% and 85%, respectively (Figure.\n5). As shown in Fig.\n5, the highest concentration of lacinartin tested (50 μg/ml) reduced IL-8 and TNF-α secretion by 95% and 99%, respectively, while 12.5 and 25 μg/ml of lacinartin increased the secretion of both cytokines, likely due to a synergistic effect of LPS and lacinartin, since lacinartin alone had no effect (data not shown). Auraptene and lacinartin both reduced MMP-8 and MMP-9 secretion, sometimes below basal levels (Figure.\n6). Auraptene reduced MMP-8 secretion by 63% (6.25 μg/ml), 69% (12.5 μg/ml), and 73% (25 μg/ml), while lacinartin reduced MMP-8 secretion by 79% (12.5 μg/ml), 83% (25 μg/ml), and 89% (50 μg/ml) (Figure.\n6A). At their highest concentrations tested, auraptene (25 μg/ml) reduced MMP-9 secretion by 76% (25 μg/ml) (Figure.\n6B), while lacinartin (50 μg/ml) reduced MMP-9 secretion by 82% (Figure.\n6B).\n Effect of auraptene and lacinartin on the secretion of IL-8 (A) and TNF-α (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control, †: p ≤ 0.05 vs. control without auraptene or lacinartin).\n Effect of auraptene and lacinartin on the secretion of MMP-8 (A) and MMP-9 (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control).\nAfter demonstrating that auraptene and lacinartin can decrease MMP secretion in a macrophage model, we evaluated their effect on proteinase activity. Both auraptene and lacinartin (12.5 μg/ml) reduced MMP-9 activity by 74% (Table\n1). Auraptene had no inhibitory effect on P. gingivalis collagenase activity while 12.5 μg/ml and 100 μg/ml of lacinartin reduced collagenase activity by 20% and 64%, respectively (Table\n1).\n\nEffect of auraptene and lacinartin on the activity of MMP-9 and \n\nP. gingivalis\n\n collagenase\n\n1Inhibitor of MMP-9: GM6001 (0.025 μM); inhibitor of P. gingivalis collagenase: leupeptin (1 μM).\n\nDiscussion:\nPeriodontal diseases are polymicrobial infections and are the most common chronic inflammatory disorders in adults\n[22]. Periodontitis is induced by a specific group of Gram-negative anaerobic bacteria and is the major cause of tooth loss in adults\n[23]. Over the past two decades, natural compounds with antibacterial and anti-inflammatory properties have received considerable attention as new therapeutic agents for the treatment of periodontal infections. In this study, we investigated the potential of auraptene and lacinartin for preventing and treating periodontal diseases.\nWe first showed that lacinartin and to a lesser extent auraptene reduced P. gingivalis growth. This is the first report indicating that lacinartin possesses anti-bacterial properties. Previous studies have shown that auraptene has antibacterial properties against Helicobacter pylori[24,25]. The exact mechanism by which lacinartin and auraptene inhibit bacterial growth is unknown. However, other natural coumarins (novobiocin and clorobiocin) inhibit deoxyribonuclease gyrase activity, which results in bacteria death\n[14,26]. In addition, we showed that auraptene and lacinartin inhibit growth more effectively under iron-limiting conditions, requiring much lower concentrations to significantly reduce the growth of P. gingivalis. Our results are in agreement with those of Mladnka et al.\n[27], who showed that coumarins possess iron-chelating properties. Additional studies are required to investigate interactions between iron and auraptene and lacinartin.\nWe also showed that lacinartin, but not auraptene, inhibits biofilm formation by P. gingivalis. Lacinartin also caused the desorption of a pre-formed P. gingivalis biofilm. To the best of our knowledge, this is the first report regarding the inhibitory effects of lacinartin on bacterial biofilms. Auraptene and lacinartin prevented the adherence of P. gingivalis to oral epithelial cells to a significant degree. Epithelial cells act as a physical barrier, and bacterial adherence to these host cells may be a critical step for the initiation of periodontal diseases\n[28]. Given its ability to reduce growth of P. gingivalis and its adherence to epithelial cells, lacinartin may be a promising therapeutic candidate through its action on different targets.\nPolyphenols reduce inflammatory mediator secretion and, as such, inflammation-mediated damage\n[29]. We showed that auraptene markedly reduces IL-8 and TNF-α secretion by LPS-stimulated macrophages. Our results are in agreement with those of Genovese et al., who reported that auraptene inhibits the release of TNF-α by RAW 264.7 macrophages\n[30]. To our knowledge, no one has investigated the anti-inflammatory properties of lacinartin. We showed that 12.5 μg/ml of lacinartin induced IL-8 and TNF-α secretion, likely due to a synergistic interaction between LPS and lacinartin. On the other hand, 50 μg/ml of lacinartin significantly inhibited IL-8 and TNF-α secretion. We also showed that auraptene and lacinartin reduced MMP-8 and MMP-9 secretion. These results are in agreement with those of a study by Epifano et al., who reported that auraptene inhibits MMP-7 secretion by HT-29 epithelial cells\n[18]. Since MMP release and cytokine secretion are associated with tooth-supporting tissue destruction, our results suggested that both compounds may contribute to reducing host cell damage, including bone resorption\n[5,31]. The mechanisms by which auraptene and lacinartin reduce inflammatory mediator secretion are unknown, but previous studies have shown that coumarins can block the activation of nuclear factor-κB and inhibit kinase pathways (Akt/PKB)\n[26]. Considering that gingival fibroblasts may also play a significant role in periodontal tissue destruction through cytokine-inducible MMP secretion, future studies should investigate the effects of auraptene and lacinartin on this cell type.\nWe further showed that auraptene and lacinartin reduce MMP-9 activity while only lacinartin inhibits P. gingivalis collagenase activity. Auraptene has previously been shown to inhibit MMP-7 activity\n[32]. These observations suggest that these coumarins may contribute to reducing tissue destruction.\n\nConclusions:\nIn conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nAll authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nPeriodontal diseases are bacterial infections leading to chronic inflammation disorders that are frequently observed in adults. In the present study, we evaluated the effect of auraptene and lacinartin, two natural oxyprenylated coumarins, on the growth, adherence properties, and collagenase activity of Porphyromonas gingivalis. We also investigated the capacity of these compounds to reduce cytokine and matrix metalloproteinase (MMP) secretion by lipopolysaccharide (LPS)-stimulated macrophages and to inhibit MMP-9 activity.\n\nMethods:\nMicroplate dilution assays were performed to determine the effect of auraptene and lacinartin on P. gingivalis growth as well as biofilm formation stained with crystal violet. Adhesion of FITC-labeled P. gingivalis to oral epithelial cells was monitored by fluorometry. The effects of auraptene and lacinartin on LPS-induced cytokine and MMP secretion by macrophages were determined by immunological assays. Fluorogenic assays were used to evaluate the capacity of the two coumarins to inhibit the activity of P. gingivalis collagenase and MMP-9.\n\nResults:\nOnly lacinartin completely inhibited P. gingivalis growth in a complex culture medium. However, under iron-limiting conditions, auraptene and lacinartin both inhibited the growth of P. gingivalis. Lacinartin also inhibited biofilm formation by P. gingivalis and promoted biofilm desorption. Both compounds prevented the adherence of P. gingivalis to oral epithelial cells, dose-dependently reduced the secretion of cytokines (IL-8 and TNF-α) and MMP-8 and MMP-9 by LPS-stimulated macrophages, and inhibited MMP-9 activity. Lacinartin also inhibited P. gingivalis collagenase activity.\n\nConclusions:\nBy acting on multiple targets, including pathogenic bacteria, tissue-destructive enzymes, and the host inflammatory response, auraptene and lacinartin may be promising natural compounds for preventing and treating periodontal diseases."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nPeriodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.\n\nConclusions:\nIn conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nPeriodontal diseases are bacterial infections leading to chronic inflammation disorders that are frequently observed in adults. In the present study, we evaluated the effect of auraptene and lacinartin, two natural oxyprenylated coumarins, on the growth, adherence properties, and collagenase activity of Porphyromonas gingivalis. We also investigated the capacity of these compounds to reduce cytokine and matrix metalloproteinase (MMP) secretion by lipopolysaccharide (LPS)-stimulated macrophages and to inhibit MMP-9 activity.\n\nMethods:\nMicroplate dilution assays were performed to determine the effect of auraptene and lacinartin on P. gingivalis growth as well as biofilm formation stained with crystal violet. Adhesion of FITC-labeled P. gingivalis to oral epithelial cells was monitored by fluorometry. The effects of auraptene and lacinartin on LPS-induced cytokine and MMP secretion by macrophages were determined by immunological assays. Fluorogenic assays were used to evaluate the capacity of the two coumarins to inhibit the activity of P. gingivalis collagenase and MMP-9.\n\nResults:\nOnly lacinartin completely inhibited P. gingivalis growth in a complex culture medium. However, under iron-limiting conditions, auraptene and lacinartin both inhibited the growth of P. gingivalis. Lacinartin also inhibited biofilm formation by P. gingivalis and promoted biofilm desorption. Both compounds prevented the adherence of P. gingivalis to oral epithelial cells, dose-dependently reduced the secretion of cytokines (IL-8 and TNF-α) and MMP-8 and MMP-9 by LPS-stimulated macrophages, and inhibited MMP-9 activity. Lacinartin also inhibited P. gingivalis collagenase activity.\n\nConclusions:\nBy acting on multiple targets, including pathogenic bacteria, tissue-destructive enzymes, and the host inflammatory response, auraptene and lacinartin may be promising natural compounds for preventing and treating periodontal diseases."},"whole_article_text_length":{"kind":"number","value":8512,"string":"8,512"},"whole_article_abstract_length":{"kind":"number","value":314,"string":"314"},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["lacinartin","auraptene","ml","auraptene lacinartin","gingivalis","μg","μg ml","100","cells","mmp"],"string":"[\n \"lacinartin\",\n \"auraptene\",\n \"ml\",\n \"auraptene lacinartin\",\n \"gingivalis\",\n \"μg\",\n \"μg ml\",\n \"100\",\n \"cells\",\n \"mmp\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] diseases | compounds | inflammatory | periodontal | known | immune | properties | anti | natural | polyphenols [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] μg | μg ml | ml | figure | lacinartin | secretion | auraptene | reduced | effect | 25 μg ml [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] anti | inflammatory | mechanisms | periodontal | periodontal diseases | diseases | anti inflammatory | protease | properties useful prevention treatment | properties useful prevention [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | 100 | μg | μg ml | authors | wells [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | 100 | μg | μg ml | authors | wells [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| lacinartin | two | Porphyromonas ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin ||| lacinartin ||| Lacinartin ||| TNF | MMP-8 | MMP-9 | LPS ||| Lacinartin [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| lacinartin | two | Porphyromonas ||| ||| ||| ||| lacinartin | LPS ||| two | MMP-9 ||| ||| lacinartin ||| Lacinartin ||| TNF | MMP-8 | MMP-9 | LPS ||| Lacinartin ||| lacinartin [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| lacinartin | two | Porphyromonas ||| ||| ||| ||| lacinartin | LPS ||| two | MMP-9 ||| ||| lacinartin ||| Lacinartin ||| TNF | MMP-8 | MMP-9 | LPS ||| Lacinartin ||| lacinartin [SUMMARY]"}}},{"rowIdx":293,"cells":{"title":{"kind":"string","value":"A pilot prospective study to evaluate whether the bladder morphology in cystography and/or urodynamic may help predict the response to botulinum toxin a injection in neurogenic bladder refractory to anticholinergics."},"pmid":{"kind":"string","value":"25123234"},"background_abstract":{"kind":"string","value":"We have observed different clinical responses to botulinum toxin A (BTX-A) in patients who had similar urodynamic parameters before the procedure. Furthermore, some bladders evaluated by cystography and cystoscopy during the procedure had different characteristics that could influence the outcome of the treatment. The aim of this study was to assess whether cystography and urodynamic parameters could help predict which patients with neurogenic detrusor overactivity (NDO) refractory to anticholinergics respond better to treatment with injection of BTX-A."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"In total, 34 patients with spinal cord injury were prospectively evaluated. All patients emptied their bladder by clean intermittent catheterization (CIC) and had incontinence and NDO, despite using 40 mg or more of intravesical oxybutynin and undergoing detrusor injection of BTX-A (300 IU). Pretreatment evaluation included urodynamic, and cystography. Follow-up consisted of urodynamic and ambulatory visits four months after treatment. The cystography parameters used were bladder shape, capacity and presence of diverticula. Urodynamic parameters used for assessment were maximum cystometric capacity (MCC), maximum detrusor pressure (MDP), compliance and reflex volume (RV)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"After injection of BTX-A, 70% of the patients had success, with 4 months or more of continence. Before the treatment, there were significant differences in most urodynamic parameters between those who responded successfully compared to those who did not. Patients who responded successfully had greater MCC (p = 0.019), higher RV (p = 0.041), and greater compliance (p = 0.043). There was no significant difference in the MDP (0.691). The cystography parameters were not significantly different between these groups bladder shape (p = 0.271), capacity (p > 0.720) and presence of diverticula (p > 0.999). Statistical analyses were performed using SPSS (version 20.0) and included Student's t-test for two paired samples and Fisher's exact test, with a significance threshold of 0.05."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"This study suggests that the cystography parameters evaluated cannot be used to help predict the response to injection of BTX-A in the treatment of refractory NDO. However, the urodynamic parameters were significantly different in patients who responded to the treatment, with the exception of the MDP."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Botulinum Toxins, Type A","Female","Humans","Male","Mandelic Acids","Middle Aged","Muscarinic Antagonists","Neuromuscular Agents","Pilot Projects","Prospective Studies","Radiography","Urinary Bladder","Urinary Bladder, Neurogenic","Urinary Bladder, Overactive","Urodynamics","Young Adult"],"string":"[\n \"Adult\",\n \"Botulinum Toxins, Type A\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Mandelic Acids\",\n \"Middle Aged\",\n \"Muscarinic Antagonists\",\n \"Neuromuscular Agents\",\n \"Pilot Projects\",\n \"Prospective Studies\",\n \"Radiography\",\n \"Urinary Bladder\",\n \"Urinary Bladder, Neurogenic\",\n \"Urinary Bladder, Overactive\",\n \"Urodynamics\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"4139716"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Thirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7 [8] for two dependent groups (matched pairs). Parameters used in the calculations were as follows: effect size, 0.50; significance probability, 0.05; and power test, 0.80. Of the 34 patients, 23 were male and 11 were female. All methods and definitions were based on the standardization of terminology of lower urinary tract function, as described by Abrams et al. [9]. The study was approved by the ethics committee of Sarah Hospital in Brasilia (CAAE 24188413.3.0000.0022). Informed consent was obtained from all patients participating in the study.The inclusion criteria for the injection of BTX-A were those patients who emptied their bladder by CIC and had urinary incontinence due to hyperactivity refractory to intravesical oxybutynin doses equal or greater than 40 mg. An evaluation was conducted before the procedure and included a clinical history, physical examination, ultrasonography of the kidneys and urinary tract, cystography and urodynamic tests (Multichannel Urodynamics - Medtronic Duet systems, version 8.20, Minneapolis). The following urodynamic parameters were measured: reflex volume (RV), maximum detrusor pressure (MDP), bladder compliance, and maximum cystometric capacity (MCC). For better assessment of compliance, only the study bladders with a capacity ≥ 150 ml were used and compliance was measured five minutes after the end of bladder filling to allow for stabilization of the detrusor pressure. During cystography, the bladder filling was stopped when the maximum capacity had been reached, urinary leakage started, or there was supra pubic discomfort. Outcome was assessed in relation to the bladder shape and capacity and the presence of diverticula. The shape of the bladder was characterized as either “rounded” or “pear-shaped” and/or “pine” (see Figure 1). The cystometric capacity was measured in milliliters. Diverticula were characterized as absent or present, with one group consisting of patients with a diverticula number <10 and another group with a diverticula number ≥ 10 (Figure 1).\nThe shape of the bladder (“rounded” shape, “pear-shaped” or “pine”).\nAll procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A (Westport Allergan Pharmaceuticals Ireland - Ireland) was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. [4]. Patients were instructed to continue using the anticholinergic medication and to gradually reduce the dose after the procedure, if it was successful and suspend its use if possible. The treatment was considered successful if the patient remained continent for four months or more, with complete absence of urinary leak, regardless of the use of the anticholinergic medication. Clinical and urodynamic control assessments were performed 4 months after treatment. Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test with a significance threshold of 0.05. Before each t-test, the hypothesis of equality of variances was checked using Levene’s test. Differences between the groups were compared with the Mann–Whitney U test for two independent samples."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Of the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366) (Table 1). There were significant differences in most of the pretreatment urodynamic parameters in patients with a successful response compared with those with an unsuccessful response. The MCC (p = 0.019), RV (0.041) and compliance (p = 0.043) were higher in patients with a successful response. The MDP was not significantly different between groups (p = 0.691) (Table 2).\nUrodynamic assessment before and after botulinum toxin injection\n*Paired, two-sided Student’s t-test.\nUrodynamic parameters before botulinum toxin injection\n*Mann–Whitney U Test for two independent samples.\nRegarding cystography, before the procedure, 24 patients (70%) had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) (Table 3).\nResults and cystography parameters\n*Mann–Whitney U Test for two independent samples.\nAfter 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 of these patients were able to decrease the dose of the anticholinergic drugs after the procedure (Table 5).\nAnticholinergic use after botulinum toxin injection (Total)\nAnticholinergic use after botulinum toxin injection (success)"},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"This study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work."},"other_sections_titles":{"kind":"list like","value":["Background","Abbreviations","Competing interests","Author’s contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Author’s contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.","NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.","The authors declare that they have no competing interests.","RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\n"],"string":"[\n \"Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.\",\n \"NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.\",\n \"The authors declare that they have no competing interests.\",\n \"RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Results","Discussion","Conclusion","Abbreviations","Competing interests","Author’s contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Results\",\n \"Discussion\",\n \"Conclusion\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Author’s contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.","Thirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7 [8] for two dependent groups (matched pairs). Parameters used in the calculations were as follows: effect size, 0.50; significance probability, 0.05; and power test, 0.80. Of the 34 patients, 23 were male and 11 were female. All methods and definitions were based on the standardization of terminology of lower urinary tract function, as described by Abrams et al. [9]. The study was approved by the ethics committee of Sarah Hospital in Brasilia (CAAE 24188413.3.0000.0022). Informed consent was obtained from all patients participating in the study.The inclusion criteria for the injection of BTX-A were those patients who emptied their bladder by CIC and had urinary incontinence due to hyperactivity refractory to intravesical oxybutynin doses equal or greater than 40 mg. An evaluation was conducted before the procedure and included a clinical history, physical examination, ultrasonography of the kidneys and urinary tract, cystography and urodynamic tests (Multichannel Urodynamics - Medtronic Duet systems, version 8.20, Minneapolis). The following urodynamic parameters were measured: reflex volume (RV), maximum detrusor pressure (MDP), bladder compliance, and maximum cystometric capacity (MCC). For better assessment of compliance, only the study bladders with a capacity ≥ 150 ml were used and compliance was measured five minutes after the end of bladder filling to allow for stabilization of the detrusor pressure. During cystography, the bladder filling was stopped when the maximum capacity had been reached, urinary leakage started, or there was supra pubic discomfort. Outcome was assessed in relation to the bladder shape and capacity and the presence of diverticula. The shape of the bladder was characterized as either “rounded” or “pear-shaped” and/or “pine” (see Figure 1). The cystometric capacity was measured in milliliters. Diverticula were characterized as absent or present, with one group consisting of patients with a diverticula number <10 and another group with a diverticula number ≥ 10 (Figure 1).\nThe shape of the bladder (“rounded” shape, “pear-shaped” or “pine”).\nAll procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A (Westport Allergan Pharmaceuticals Ireland - Ireland) was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. [4]. Patients were instructed to continue using the anticholinergic medication and to gradually reduce the dose after the procedure, if it was successful and suspend its use if possible. The treatment was considered successful if the patient remained continent for four months or more, with complete absence of urinary leak, regardless of the use of the anticholinergic medication. Clinical and urodynamic control assessments were performed 4 months after treatment. Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test with a significance threshold of 0.05. Before each t-test, the hypothesis of equality of variances was checked using Levene’s test. Differences between the groups were compared with the Mann–Whitney U test for two independent samples.","Of the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366) (Table 1). There were significant differences in most of the pretreatment urodynamic parameters in patients with a successful response compared with those with an unsuccessful response. The MCC (p = 0.019), RV (0.041) and compliance (p = 0.043) were higher in patients with a successful response. The MDP was not significantly different between groups (p = 0.691) (Table 2).\nUrodynamic assessment before and after botulinum toxin injection\n*Paired, two-sided Student’s t-test.\nUrodynamic parameters before botulinum toxin injection\n*Mann–Whitney U Test for two independent samples.\nRegarding cystography, before the procedure, 24 patients (70%) had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) (Table 3).\nResults and cystography parameters\n*Mann–Whitney U Test for two independent samples.\nAfter 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 of these patients were able to decrease the dose of the anticholinergic drugs after the procedure (Table 5).\nAnticholinergic use after botulinum toxin injection (Total)\nAnticholinergic use after botulinum toxin injection (success)","BTX-A injections into the detrusor muscle provide a clinically significant improvement in patients with NDO refractory to anticholinergics and are very well tolerated (5;6). In this study, continence was observed over a period of more than 4 months in 70% of the patients undergoing treatment with BTX-A. In studies with similar populations of patients, the percentage of patients with continence after injection of the toxin ranged from 42 to 87% [10]. Karsenty et al. reported that anticholinergic agents may be discontinued in 28% to 58% of patients after treatment with BTX-A and the dose can be substantially reduced in the remaining patients [10]. In this study, after 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) discontinued the use and nine (26.5%) did not change the dose of medication (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 patients were able to decrease the dose of anticholinergic drugs after the procedure (Table 5). We note that some of these patients did not reduce the dose because they were afraid that the urinary losses would return after being continent. According to previous studies, some factors that may be related to the efficacy of BTX-A injections into the detrusor muscle include, for example, whether the doses of anticholinergics used before the procedure were considered high (refractory bladder) [7], what the optimal dose of BTX-A is [11,12], the formulations used [13,14] and the injection technique [11]. These factors may explain some of the differences observed in the results across different studies. The results obtained in this study were similar to earlier studies. Among patients presenting with NDO that is refractory to anticholinergic agents, there was a percentage with unsuccessful results.\nAlthough it has been reported that alterations can occur in the detrusor muscle of the NDO [15,16], it was postulated that the morphology of the bladder (shape and presence of diverticula) could be a result of these alterations, and could, in turn, influence the response to BTX-A injections. These changes in bladder shape are most likely the result of smooth muscle hypertrophy and changes in the connective tissue matrix that do not respond to conservative treatment. However, the present study did not demonstrate that the cystography parameters could predict which cases would be more likely to have a better response to BTX-A injections.\nRegarding urodynamic parameters, we observed in this study that the bladders that had better compliance, greater capacity and increased reflex volume before treatment showed a better response to treatment. Furthermore, there was no significant change in compliance after surgery (p = 0.366), suggesting that compliance is related to alterations in the bladder wall, is unresponsive to drug therapy, and is, therefore, directly related to the treatment response. This is in contrast to a study conducted by Klaphajone J [17], in which a small number of patients did not show this relationship.","This study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work.","NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.","The authors declare that they have no competing interests.","RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\n"],"string":"[\n \"Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.\",\n \"Thirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7 [8] for two dependent groups (matched pairs). Parameters used in the calculations were as follows: effect size, 0.50; significance probability, 0.05; and power test, 0.80. Of the 34 patients, 23 were male and 11 were female. All methods and definitions were based on the standardization of terminology of lower urinary tract function, as described by Abrams et al. [9]. The study was approved by the ethics committee of Sarah Hospital in Brasilia (CAAE 24188413.3.0000.0022). Informed consent was obtained from all patients participating in the study.The inclusion criteria for the injection of BTX-A were those patients who emptied their bladder by CIC and had urinary incontinence due to hyperactivity refractory to intravesical oxybutynin doses equal or greater than 40 mg. An evaluation was conducted before the procedure and included a clinical history, physical examination, ultrasonography of the kidneys and urinary tract, cystography and urodynamic tests (Multichannel Urodynamics - Medtronic Duet systems, version 8.20, Minneapolis). The following urodynamic parameters were measured: reflex volume (RV), maximum detrusor pressure (MDP), bladder compliance, and maximum cystometric capacity (MCC). For better assessment of compliance, only the study bladders with a capacity ≥ 150 ml were used and compliance was measured five minutes after the end of bladder filling to allow for stabilization of the detrusor pressure. During cystography, the bladder filling was stopped when the maximum capacity had been reached, urinary leakage started, or there was supra pubic discomfort. Outcome was assessed in relation to the bladder shape and capacity and the presence of diverticula. The shape of the bladder was characterized as either “rounded” or “pear-shaped” and/or “pine” (see Figure 1). The cystometric capacity was measured in milliliters. Diverticula were characterized as absent or present, with one group consisting of patients with a diverticula number <10 and another group with a diverticula number ≥ 10 (Figure 1).\\nThe shape of the bladder (“rounded” shape, “pear-shaped” or “pine”).\\nAll procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A (Westport Allergan Pharmaceuticals Ireland - Ireland) was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. [4]. Patients were instructed to continue using the anticholinergic medication and to gradually reduce the dose after the procedure, if it was successful and suspend its use if possible. The treatment was considered successful if the patient remained continent for four months or more, with complete absence of urinary leak, regardless of the use of the anticholinergic medication. Clinical and urodynamic control assessments were performed 4 months after treatment. Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test with a significance threshold of 0.05. Before each t-test, the hypothesis of equality of variances was checked using Levene’s test. Differences between the groups were compared with the Mann–Whitney U test for two independent samples.\",\n \"Of the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366) (Table 1). There were significant differences in most of the pretreatment urodynamic parameters in patients with a successful response compared with those with an unsuccessful response. The MCC (p = 0.019), RV (0.041) and compliance (p = 0.043) were higher in patients with a successful response. The MDP was not significantly different between groups (p = 0.691) (Table 2).\\nUrodynamic assessment before and after botulinum toxin injection\\n*Paired, two-sided Student’s t-test.\\nUrodynamic parameters before botulinum toxin injection\\n*Mann–Whitney U Test for two independent samples.\\nRegarding cystography, before the procedure, 24 patients (70%) had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) (Table 3).\\nResults and cystography parameters\\n*Mann–Whitney U Test for two independent samples.\\nAfter 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 of these patients were able to decrease the dose of the anticholinergic drugs after the procedure (Table 5).\\nAnticholinergic use after botulinum toxin injection (Total)\\nAnticholinergic use after botulinum toxin injection (success)\",\n \"BTX-A injections into the detrusor muscle provide a clinically significant improvement in patients with NDO refractory to anticholinergics and are very well tolerated (5;6). In this study, continence was observed over a period of more than 4 months in 70% of the patients undergoing treatment with BTX-A. In studies with similar populations of patients, the percentage of patients with continence after injection of the toxin ranged from 42 to 87% [10]. Karsenty et al. reported that anticholinergic agents may be discontinued in 28% to 58% of patients after treatment with BTX-A and the dose can be substantially reduced in the remaining patients [10]. In this study, after 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) discontinued the use and nine (26.5%) did not change the dose of medication (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 patients were able to decrease the dose of anticholinergic drugs after the procedure (Table 5). We note that some of these patients did not reduce the dose because they were afraid that the urinary losses would return after being continent. According to previous studies, some factors that may be related to the efficacy of BTX-A injections into the detrusor muscle include, for example, whether the doses of anticholinergics used before the procedure were considered high (refractory bladder) [7], what the optimal dose of BTX-A is [11,12], the formulations used [13,14] and the injection technique [11]. These factors may explain some of the differences observed in the results across different studies. The results obtained in this study were similar to earlier studies. Among patients presenting with NDO that is refractory to anticholinergic agents, there was a percentage with unsuccessful results.\\nAlthough it has been reported that alterations can occur in the detrusor muscle of the NDO [15,16], it was postulated that the morphology of the bladder (shape and presence of diverticula) could be a result of these alterations, and could, in turn, influence the response to BTX-A injections. These changes in bladder shape are most likely the result of smooth muscle hypertrophy and changes in the connective tissue matrix that do not respond to conservative treatment. However, the present study did not demonstrate that the cystography parameters could predict which cases would be more likely to have a better response to BTX-A injections.\\nRegarding urodynamic parameters, we observed in this study that the bladders that had better compliance, greater capacity and increased reflex volume before treatment showed a better response to treatment. Furthermore, there was no significant change in compliance after surgery (p = 0.366), suggesting that compliance is related to alterations in the bladder wall, is unresponsive to drug therapy, and is, therefore, directly related to the treatment response. This is in contrast to a study conducted by Klaphajone J [17], in which a small number of patients did not show this relationship.\",\n \"This study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work.\",\n \"NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.\",\n \"The authors declare that they have no competing interests.\",\n \"RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods","results","discussion","conclusions",null,null,null,null],"string":"[\n null,\n \"methods\",\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Neurogenic detrusor overactivity","Botulinum toxin A","Cystography","Urodynamic","Neurogenic bladder"],"string":"[\n \"Neurogenic detrusor overactivity\",\n \"Botulinum toxin A\",\n \"Cystography\",\n \"Urodynamic\",\n \"Neurogenic bladder\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nBotulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.\n\nMethods:\nThirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7 [8] for two dependent groups (matched pairs). Parameters used in the calculations were as follows: effect size, 0.50; significance probability, 0.05; and power test, 0.80. Of the 34 patients, 23 were male and 11 were female. All methods and definitions were based on the standardization of terminology of lower urinary tract function, as described by Abrams et al. [9]. The study was approved by the ethics committee of Sarah Hospital in Brasilia (CAAE 24188413.3.0000.0022). Informed consent was obtained from all patients participating in the study.The inclusion criteria for the injection of BTX-A were those patients who emptied their bladder by CIC and had urinary incontinence due to hyperactivity refractory to intravesical oxybutynin doses equal or greater than 40 mg. An evaluation was conducted before the procedure and included a clinical history, physical examination, ultrasonography of the kidneys and urinary tract, cystography and urodynamic tests (Multichannel Urodynamics - Medtronic Duet systems, version 8.20, Minneapolis). The following urodynamic parameters were measured: reflex volume (RV), maximum detrusor pressure (MDP), bladder compliance, and maximum cystometric capacity (MCC). For better assessment of compliance, only the study bladders with a capacity ≥ 150 ml were used and compliance was measured five minutes after the end of bladder filling to allow for stabilization of the detrusor pressure. During cystography, the bladder filling was stopped when the maximum capacity had been reached, urinary leakage started, or there was supra pubic discomfort. Outcome was assessed in relation to the bladder shape and capacity and the presence of diverticula. The shape of the bladder was characterized as either “rounded” or “pear-shaped” and/or “pine” (see Figure 1). The cystometric capacity was measured in milliliters. Diverticula were characterized as absent or present, with one group consisting of patients with a diverticula number <10 and another group with a diverticula number ≥ 10 (Figure 1).\nThe shape of the bladder (“rounded” shape, “pear-shaped” or “pine”).\nAll procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A (Westport Allergan Pharmaceuticals Ireland - Ireland) was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. [4]. Patients were instructed to continue using the anticholinergic medication and to gradually reduce the dose after the procedure, if it was successful and suspend its use if possible. The treatment was considered successful if the patient remained continent for four months or more, with complete absence of urinary leak, regardless of the use of the anticholinergic medication. Clinical and urodynamic control assessments were performed 4 months after treatment. Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test with a significance threshold of 0.05. Before each t-test, the hypothesis of equality of variances was checked using Levene’s test. Differences between the groups were compared with the Mann–Whitney U test for two independent samples.\n\nResults:\nOf the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366) (Table 1). There were significant differences in most of the pretreatment urodynamic parameters in patients with a successful response compared with those with an unsuccessful response. The MCC (p = 0.019), RV (0.041) and compliance (p = 0.043) were higher in patients with a successful response. The MDP was not significantly different between groups (p = 0.691) (Table 2).\nUrodynamic assessment before and after botulinum toxin injection\n*Paired, two-sided Student’s t-test.\nUrodynamic parameters before botulinum toxin injection\n*Mann–Whitney U Test for two independent samples.\nRegarding cystography, before the procedure, 24 patients (70%) had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) (Table 3).\nResults and cystography parameters\n*Mann–Whitney U Test for two independent samples.\nAfter 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 of these patients were able to decrease the dose of the anticholinergic drugs after the procedure (Table 5).\nAnticholinergic use after botulinum toxin injection (Total)\nAnticholinergic use after botulinum toxin injection (success)\n\nDiscussion:\nBTX-A injections into the detrusor muscle provide a clinically significant improvement in patients with NDO refractory to anticholinergics and are very well tolerated (5;6). In this study, continence was observed over a period of more than 4 months in 70% of the patients undergoing treatment with BTX-A. In studies with similar populations of patients, the percentage of patients with continence after injection of the toxin ranged from 42 to 87% [10]. Karsenty et al. reported that anticholinergic agents may be discontinued in 28% to 58% of patients after treatment with BTX-A and the dose can be substantially reduced in the remaining patients [10]. In this study, after 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) discontinued the use and nine (26.5%) did not change the dose of medication (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 patients were able to decrease the dose of anticholinergic drugs after the procedure (Table 5). We note that some of these patients did not reduce the dose because they were afraid that the urinary losses would return after being continent. According to previous studies, some factors that may be related to the efficacy of BTX-A injections into the detrusor muscle include, for example, whether the doses of anticholinergics used before the procedure were considered high (refractory bladder) [7], what the optimal dose of BTX-A is [11,12], the formulations used [13,14] and the injection technique [11]. These factors may explain some of the differences observed in the results across different studies. The results obtained in this study were similar to earlier studies. Among patients presenting with NDO that is refractory to anticholinergic agents, there was a percentage with unsuccessful results.\nAlthough it has been reported that alterations can occur in the detrusor muscle of the NDO [15,16], it was postulated that the morphology of the bladder (shape and presence of diverticula) could be a result of these alterations, and could, in turn, influence the response to BTX-A injections. These changes in bladder shape are most likely the result of smooth muscle hypertrophy and changes in the connective tissue matrix that do not respond to conservative treatment. However, the present study did not demonstrate that the cystography parameters could predict which cases would be more likely to have a better response to BTX-A injections.\nRegarding urodynamic parameters, we observed in this study that the bladders that had better compliance, greater capacity and increased reflex volume before treatment showed a better response to treatment. Furthermore, there was no significant change in compliance after surgery (p = 0.366), suggesting that compliance is related to alterations in the bladder wall, is unresponsive to drug therapy, and is, therefore, directly related to the treatment response. This is in contrast to a study conducted by Klaphajone J [17], in which a small number of patients did not show this relationship.\n\nConclusion:\nThis study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work.\n\nAbbreviations:\nNDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthor’s contributions:\nRAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWe have observed different clinical responses to botulinum toxin A (BTX-A) in patients who had similar urodynamic parameters before the procedure. Furthermore, some bladders evaluated by cystography and cystoscopy during the procedure had different characteristics that could influence the outcome of the treatment. The aim of this study was to assess whether cystography and urodynamic parameters could help predict which patients with neurogenic detrusor overactivity (NDO) refractory to anticholinergics respond better to treatment with injection of BTX-A.\n\nMethods:\nIn total, 34 patients with spinal cord injury were prospectively evaluated. All patients emptied their bladder by clean intermittent catheterization (CIC) and had incontinence and NDO, despite using 40 mg or more of intravesical oxybutynin and undergoing detrusor injection of BTX-A (300 IU). Pretreatment evaluation included urodynamic, and cystography. Follow-up consisted of urodynamic and ambulatory visits four months after treatment. The cystography parameters used were bladder shape, capacity and presence of diverticula. Urodynamic parameters used for assessment were maximum cystometric capacity (MCC), maximum detrusor pressure (MDP), compliance and reflex volume (RV).\n\nResults:\nAfter injection of BTX-A, 70% of the patients had success, with 4 months or more of continence. Before the treatment, there were significant differences in most urodynamic parameters between those who responded successfully compared to those who did not. Patients who responded successfully had greater MCC (p = 0.019), higher RV (p = 0.041), and greater compliance (p = 0.043). There was no significant difference in the MDP (0.691). The cystography parameters were not significantly different between these groups bladder shape (p = 0.271), capacity (p > 0.720) and presence of diverticula (p > 0.999). Statistical analyses were performed using SPSS (version 20.0) and included Student's t-test for two paired samples and Fisher's exact test, with a significance threshold of 0.05.\n\nConclusions:\nThis study suggests that the cystography parameters evaluated cannot be used to help predict the response to injection of BTX-A in the treatment of refractory NDO. However, the urodynamic parameters were significantly different in patients who responded to the treatment, with the exception of the MDP."},"background_conclusion_text":{"kind":"string","value":"Background:\nBotulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.\n\nConclusion:\nThis study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nWe have observed different clinical responses to botulinum toxin A (BTX-A) in patients who had similar urodynamic parameters before the procedure. Furthermore, some bladders evaluated by cystography and cystoscopy during the procedure had different characteristics that could influence the outcome of the treatment. The aim of this study was to assess whether cystography and urodynamic parameters could help predict which patients with neurogenic detrusor overactivity (NDO) refractory to anticholinergics respond better to treatment with injection of BTX-A.\n\nMethods:\nIn total, 34 patients with spinal cord injury were prospectively evaluated. All patients emptied their bladder by clean intermittent catheterization (CIC) and had incontinence and NDO, despite using 40 mg or more of intravesical oxybutynin and undergoing detrusor injection of BTX-A (300 IU). Pretreatment evaluation included urodynamic, and cystography. Follow-up consisted of urodynamic and ambulatory visits four months after treatment. The cystography parameters used were bladder shape, capacity and presence of diverticula. Urodynamic parameters used for assessment were maximum cystometric capacity (MCC), maximum detrusor pressure (MDP), compliance and reflex volume (RV).\n\nResults:\nAfter injection of BTX-A, 70% of the patients had success, with 4 months or more of continence. Before the treatment, there were significant differences in most urodynamic parameters between those who responded successfully compared to those who did not. Patients who responded successfully had greater MCC (p = 0.019), higher RV (p = 0.041), and greater compliance (p = 0.043). There was no significant difference in the MDP (0.691). The cystography parameters were not significantly different between these groups bladder shape (p = 0.271), capacity (p > 0.720) and presence of diverticula (p > 0.999). Statistical analyses were performed using SPSS (version 20.0) and included Student's t-test for two paired samples and Fisher's exact test, with a significance threshold of 0.05.\n\nConclusions:\nThis study suggests that the cystography parameters evaluated cannot be used to help predict the response to injection of BTX-A in the treatment of refractory NDO. However, the urodynamic parameters were significantly different in patients who responded to the treatment, with the exception of the MDP."},"whole_article_text_length":{"kind":"number","value":2504,"string":"2,504"},"whole_article_abstract_length":{"kind":"number","value":430,"string":"430"},"num_sections":{"kind":"number","value":9,"string":"9"},"most_frequent_words":{"kind":"list like","value":["patients","btx","treatment","bladder","study","parameters","detrusor","urodynamic","procedure","capacity"],"string":"[\n \"patients\",\n \"btx\",\n \"treatment\",\n \"bladder\",\n \"study\",\n \"parameters\",\n \"detrusor\",\n \"urodynamic\",\n \"procedure\",\n \"capacity\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] btx | treatment | invasive | muscle | evaluated | detrusor | clinical | release | acetylcholine | serotypes [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | patients | bladder | performed | urinary | measured | ml | version | capacity | diverticula [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | table | toxin injection | years | botulinum toxin injection | response | months | test | 001 | mean [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] compliance showed | showed | greater | compliance | results | treatment | study | parameters | analysis appropriate clarify results | bladders higher cystometric capacity [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | study | authors | detrusor | bladder | authors declare competing interests | competing interests | declare competing interests [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | study | authors | detrusor | bladder | authors declare competing interests | competing interests | declare competing interests [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| NDO | BTX-A. [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 34 ||| CIC | NDO | 40 | BTX-A ||| ||| four months ||| ||| MDP | RV [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] BTX-A | 70% | 4 months ||| ||| MCC | 0.019 | RV | 0.041 | 0.043 ||| MDP | 0.691 ||| 0.271 | 0.720 | 0.999 ||| SPSS | 20.0 | Student | two | Fisher | 0.05 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] BTX-A | NDO ||| MDP [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| NDO | BTX-A. Methods | 34 ||| CIC | NDO | 40 | BTX-A ||| ||| four months ||| ||| MDP | RV ||| ||| BTX-A | 70% | 4 months ||| ||| MCC | 0.019 | RV | 0.041 | 0.043 ||| MDP | 0.691 ||| 0.271 | 0.720 | 0.999 ||| SPSS | 20.0 | Student | two | Fisher | 0.05 ||| BTX-A | NDO ||| MDP [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| NDO | BTX-A. Methods | 34 ||| CIC | NDO | 40 | BTX-A ||| ||| four months ||| ||| MDP | RV ||| ||| BTX-A | 70% | 4 months ||| ||| MCC | 0.019 | RV | 0.041 | 0.043 ||| MDP | 0.691 ||| 0.271 | 0.720 | 0.999 ||| SPSS | 20.0 | Student | two | Fisher | 0.05 ||| BTX-A | NDO ||| MDP [SUMMARY]"}}},{"rowIdx":294,"cells":{"title":{"kind":"string","value":"Efficacy and safety of risedronate 150-mg once a month in the treatment of postmenopausal osteoporosis: 2-year data."},"pmid":{"kind":"string","value":"22752050"},"background_abstract":{"kind":"string","value":"Risedronate is effective in the treatment of postmenopausal osteoporosis in oral daily, weekly, or on two consecutive days per month doses. This 2-year randomized, double-blind, multicenter study assesses the efficacy and safety of a single risedronate 150-mg once-a-month oral dose compared with the 5-mg daily regimen."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"Women with postmenopausal osteoporosis were randomly assigned to receive risedronate 5-mg daily (n = 642) or 150-mg once a month (n = 650) for 2 years. Bone mineral density (BMD), bone turnover markers, new vertebral fractures, and adverse events were evaluated. The primary efficacy endpoint was the mean percent change from baseline in lumbar spine BMD after 1 year."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Four hundred ninety-eight subjects in the daily group (77.6 %) and 513 subjects in the once-a-month group (78.9 %) completed the study. After 24 months, the mean percent change in lumbar spine BMD was 3.9 % (95 % confidence interval [CI], 3.43 to 4.42 %) and 4.2 % (95 % CI, 3.68 to 4.65 %) in the daily and once-a-month groups, respectively. The once-a-month regimen was determined to be non-inferior to the daily regimen. The mean percent changes in BMD at the hip were similar in both dose groups, as were changes in biochemical markers of bone turnover. The incidence of adverse events, adverse events leading to withdrawal, and upper gastrointestinal tract adverse events were similar in the two treatment groups."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"After 2 years, treatment with risedronate 150-mg once a month provided similar efficacy and tolerability to daily dosing and provides an alternative for patients who prefer once-a-month oral dosing."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Administration, Oral","Aged","Biomarkers","Bone Density","Bone Density Conservation Agents","Double-Blind Method","Drug Administration Schedule","Etidronic Acid","Female","Femur","Humans","Lumbar Vertebrae","Middle Aged","Osteoporosis, Postmenopausal","Risedronic Acid","Treatment Outcome"],"string":"[\n \"Administration, Oral\",\n \"Aged\",\n \"Biomarkers\",\n \"Bone Density\",\n \"Bone Density Conservation Agents\",\n \"Double-Blind Method\",\n \"Drug Administration Schedule\",\n \"Etidronic Acid\",\n \"Female\",\n \"Femur\",\n \"Humans\",\n \"Lumbar Vertebrae\",\n \"Middle Aged\",\n \"Osteoporosis, Postmenopausal\",\n \"Risedronic Acid\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"3536944"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Risedronate is a pyridinyl bisphosphonate that has been shown in prospective studies to reduce the risk of vertebral, nonvertebral, and hip fractures [1–3]. Like other bisphosphonates, risedronate remains active on the surface of bone for long periods after dosing, providing the opportunity to develop a range of dosing schedules.\nThe original risedronate dosing regimen for postmenopausal osteoporosis was an oral dose of 5-mg daily [1–3]. It was later demonstrated that risedronate 35-mg once a week and 75-mg each day for two consecutive days a month provided similar efficacy and safety to the daily regimen [4, 5]. The efficacy and tolerability of risedronate once-a-month dosing (150-mg) was compared with risedronate daily dosing (5-mg) in women with osteoporosis with changes in lumbar spine bone mineral density (BMD) as the primary endpoint. After 1 year of treatment, published previously, the efficacy of risedronate 150-mg once-a-month regimen was non-inferior to the 5-mg daily regimen [6]. The once-a-month regimen also had a similar tolerability profile as the daily regimen after 1 year of treatment. This study continued for an additional year of treatment, and the results of the complete study over 2 years are presented here."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Subjects From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\nFrom the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\n Efficacy assessments The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\nThe within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n Safety assessments Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\nOverall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Study design","Patients","Treatments","Efficacy assessments","Safety assessments","Statistical analysis","Subjects","Efficacy assessments","Safety assessments"],"string":"[\n \"Study design\",\n \"Patients\",\n \"Treatments\",\n \"Efficacy assessments\",\n \"Safety assessments\",\n \"Statistical analysis\",\n \"Subjects\",\n \"Efficacy assessments\",\n \"Safety assessments\"\n]"},"other_sections_texts":{"kind":"list like","value":["This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.","Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].","Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.","Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.","Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).","The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.","From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density","The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)","Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups."],"string":"[\n \"This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\",\n \"Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\",\n \"Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\",\n \"Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\",\n \"Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\",\n \"The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\",\n \"From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\\n\\nDisposition of subjects. BMD bone mineral density\",\n \"The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\n\\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\n\\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\",\n \"Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\\nn (%)\\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\\n23 (3.6)25 (3.8)\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\nAE adverse event\\n\\nSummary of adverse events\\n\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\n\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\n\\nAE adverse event\\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and methods","Study design","Patients","Treatments","Efficacy assessments","Safety assessments","Statistical analysis","Results","Subjects","Efficacy assessments","Safety assessments","Discussion"],"string":"[\n \"Introduction\",\n \"Materials and methods\",\n \"Study design\",\n \"Patients\",\n \"Treatments\",\n \"Efficacy assessments\",\n \"Safety assessments\",\n \"Statistical analysis\",\n \"Results\",\n \"Subjects\",\n \"Efficacy assessments\",\n \"Safety assessments\",\n \"Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Risedronate is a pyridinyl bisphosphonate that has been shown in prospective studies to reduce the risk of vertebral, nonvertebral, and hip fractures [1–3]. Like other bisphosphonates, risedronate remains active on the surface of bone for long periods after dosing, providing the opportunity to develop a range of dosing schedules.\nThe original risedronate dosing regimen for postmenopausal osteoporosis was an oral dose of 5-mg daily [1–3]. It was later demonstrated that risedronate 35-mg once a week and 75-mg each day for two consecutive days a month provided similar efficacy and safety to the daily regimen [4, 5]. The efficacy and tolerability of risedronate once-a-month dosing (150-mg) was compared with risedronate daily dosing (5-mg) in women with osteoporosis with changes in lumbar spine bone mineral density (BMD) as the primary endpoint. After 1 year of treatment, published previously, the efficacy of risedronate 150-mg once-a-month regimen was non-inferior to the 5-mg daily regimen [6]. The once-a-month regimen also had a similar tolerability profile as the daily regimen after 1 year of treatment. This study continued for an additional year of treatment, and the results of the complete study over 2 years are presented here."," Study design This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\nThis randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\n Patients Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\nEligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\n Treatments Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\nSubjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\n Efficacy assessments Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\nDual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\n Safety assessments Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\nPhysical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\n Statistical analysis The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\nThe primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.","This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.","Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].","Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.","Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.","Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).","The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group."," Subjects From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\nFrom the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\n Efficacy assessments The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\nThe within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n Safety assessments Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\nOverall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.","From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density","The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)","Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.","Risedronate is a widely used osteoporosis treatment with proven vertebral and nonvertebral antifracture efficacy and a minimum wait of 30 min after dosing before eating or drinking anything other than water. A 5-mg daily regimen was developed originally, but less frequent dose regimens have now been developed. This study was a preplanned 2-year study comparing a dose of risedronate 150-mg once a month to the 5-mg daily dose. These 2-year data show that the 150-mg once-a-month dose continues to produce clinical effects that are similar to those seen with the 5-mg daily dose. Specifically, the mean percent change in lumbar spine BMD at 24 months in the monthly group was non-inferior to the mean percent change in lumbar spine BMD in the daily group. Changes in secondary efficacy parameters, including BMD at the hip, bone turnover markers at endpoint, and morphometric vertebral fractures, were also similar in both groups. Statistically significant differences between treatment groups were observed for all three bone turnover markers at month 3, but persisted at months 6 and 12 for CTX only. No statistically significant differences between groups were observed for any marker at month 24 or endpoint. That the CTX response did not differ between treatment groups at month 24 might be explained by the small number of subjects at month 24 that would limit statistical power to observe difference. It is not likely that these small differences between groups in bone turnover markers are clinically meaningful.\nThe risedronate 150-mg once-a-month dose was well tolerated over 2 years, with a safety profile similar to that seen with the 5-mg daily regimen. The low incidences of subjects with vertebral and nonvertebral clinical fractures were similar between groups and consistent with rates previously observed with the 5-mg daily dose [1–3].\nChange in BMD is an appropriate endpoint when evaluating a new dosing schedule of a bisphosphonate for which a fracture benefit has already been established. Similar non-inferiority trials have been conducted previously to evaluate new dosing regimens of oral bisphosphonates [4, 8, 9], and this approach has been accepted by both the US Food and Drug Administration and the European Medicines Agency [10] for approval of new regimens of established agents. The magnitude of BMD change associated with the vertebral and nonvertebral antifracture efficacy of risedronate has been established in multiple large studies that had fracture as the primary endpoint [1–3]. This study has demonstrated that the 150-mg once-a-month dose reduces bone turnover and increases BMD to a degree comparable to that observed with the 5-mg daily dose in these fracture studies.\nThe results of this study after 2 years are consistent with the findings at month 12 [6], demonstrating the persistent similarity between risedronate 150-mg once-a-month and the 5-mg daily dosing regimens. Additionally, these results are consistent with the favorable tolerability and efficacy profiles observed in large placebo-controlled clinical trials of the risedronate 5-mg daily regimen [1–3]. The findings are also consistent with previous studies of less frequent dosing with risedronate. Such studies showed that the treatment effects of risedronate 35-mg weekly and 75-mg on two consecutive days each month were similar to the effects of daily dosing [4, 5].\nRisedronate 150-mg once a month, taken for 2 years, is similar in efficacy and tolerability to the 5-mg daily dosing regimen that had been proven to reduce the incidence of vertebral and nonvertebral fractures. The addition of this dosing regimen to the therapeutic armamentarium will provide women with postmenopausal osteoporosis a full range of risedronate oral dosing options, from daily to weekly to monthly."],"string":"[\n \"Risedronate is a pyridinyl bisphosphonate that has been shown in prospective studies to reduce the risk of vertebral, nonvertebral, and hip fractures [1–3]. Like other bisphosphonates, risedronate remains active on the surface of bone for long periods after dosing, providing the opportunity to develop a range of dosing schedules.\\nThe original risedronate dosing regimen for postmenopausal osteoporosis was an oral dose of 5-mg daily [1–3]. It was later demonstrated that risedronate 35-mg once a week and 75-mg each day for two consecutive days a month provided similar efficacy and safety to the daily regimen [4, 5]. The efficacy and tolerability of risedronate once-a-month dosing (150-mg) was compared with risedronate daily dosing (5-mg) in women with osteoporosis with changes in lumbar spine bone mineral density (BMD) as the primary endpoint. After 1 year of treatment, published previously, the efficacy of risedronate 150-mg once-a-month regimen was non-inferior to the 5-mg daily regimen [6]. The once-a-month regimen also had a similar tolerability profile as the daily regimen after 1 year of treatment. This study continued for an additional year of treatment, and the results of the complete study over 2 years are presented here.\",\n \" Study design This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\\nThis randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\\n Patients Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\\nEligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\\n Treatments Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\\nSubjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\\n Efficacy assessments Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\\nDual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\\n Safety assessments Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\\nPhysical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\\n Statistical analysis The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\\nThe primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\",\n \"This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\",\n \"Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\",\n \"Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\",\n \"Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\",\n \"Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\",\n \"The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\",\n \" Subjects From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\\n\\nDisposition of subjects. BMD bone mineral density\\nFrom the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\\n\\nDisposition of subjects. BMD bone mineral density\\n Efficacy assessments The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\n\\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\n\\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\nThe within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\n\\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\n\\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\n Safety assessments Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\\nn (%)\\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\\n23 (3.6)25 (3.8)\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\nAE adverse event\\n\\nSummary of adverse events\\n\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\n\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\n\\nAE adverse event\\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\\nOverall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\\nn (%)\\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\\n23 (3.6)25 (3.8)\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\nAE adverse event\\n\\nSummary of adverse events\\n\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\n\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\n\\nAE adverse event\\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\",\n \"From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\\n\\nDisposition of subjects. BMD bone mineral density\",\n \"The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\n\\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\n\\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\",\n \"Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\\nn (%)\\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\\n23 (3.6)25 (3.8)\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\nAE adverse event\\n\\nSummary of adverse events\\n\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\n\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\n\\nAE adverse event\\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\",\n \"Risedronate is a widely used osteoporosis treatment with proven vertebral and nonvertebral antifracture efficacy and a minimum wait of 30 min after dosing before eating or drinking anything other than water. A 5-mg daily regimen was developed originally, but less frequent dose regimens have now been developed. This study was a preplanned 2-year study comparing a dose of risedronate 150-mg once a month to the 5-mg daily dose. These 2-year data show that the 150-mg once-a-month dose continues to produce clinical effects that are similar to those seen with the 5-mg daily dose. Specifically, the mean percent change in lumbar spine BMD at 24 months in the monthly group was non-inferior to the mean percent change in lumbar spine BMD in the daily group. Changes in secondary efficacy parameters, including BMD at the hip, bone turnover markers at endpoint, and morphometric vertebral fractures, were also similar in both groups. Statistically significant differences between treatment groups were observed for all three bone turnover markers at month 3, but persisted at months 6 and 12 for CTX only. No statistically significant differences between groups were observed for any marker at month 24 or endpoint. That the CTX response did not differ between treatment groups at month 24 might be explained by the small number of subjects at month 24 that would limit statistical power to observe difference. It is not likely that these small differences between groups in bone turnover markers are clinically meaningful.\\nThe risedronate 150-mg once-a-month dose was well tolerated over 2 years, with a safety profile similar to that seen with the 5-mg daily regimen. The low incidences of subjects with vertebral and nonvertebral clinical fractures were similar between groups and consistent with rates previously observed with the 5-mg daily dose [1–3].\\nChange in BMD is an appropriate endpoint when evaluating a new dosing schedule of a bisphosphonate for which a fracture benefit has already been established. Similar non-inferiority trials have been conducted previously to evaluate new dosing regimens of oral bisphosphonates [4, 8, 9], and this approach has been accepted by both the US Food and Drug Administration and the European Medicines Agency [10] for approval of new regimens of established agents. The magnitude of BMD change associated with the vertebral and nonvertebral antifracture efficacy of risedronate has been established in multiple large studies that had fracture as the primary endpoint [1–3]. This study has demonstrated that the 150-mg once-a-month dose reduces bone turnover and increases BMD to a degree comparable to that observed with the 5-mg daily dose in these fracture studies.\\nThe results of this study after 2 years are consistent with the findings at month 12 [6], demonstrating the persistent similarity between risedronate 150-mg once-a-month and the 5-mg daily dosing regimens. Additionally, these results are consistent with the favorable tolerability and efficacy profiles observed in large placebo-controlled clinical trials of the risedronate 5-mg daily regimen [1–3]. The findings are also consistent with previous studies of less frequent dosing with risedronate. Such studies showed that the treatment effects of risedronate 35-mg weekly and 75-mg on two consecutive days each month were similar to the effects of daily dosing [4, 5].\\nRisedronate 150-mg once a month, taken for 2 years, is similar in efficacy and tolerability to the 5-mg daily dosing regimen that had been proven to reduce the incidence of vertebral and nonvertebral fractures. The addition of this dosing regimen to the therapeutic armamentarium will provide women with postmenopausal osteoporosis a full range of risedronate oral dosing options, from daily to weekly to monthly.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["introduction","materials|methods",null,null,null,null,null,null,"results",null,null,null,"discussion"],"string":"[\n \"introduction\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Bone mineral density","Fracture risk","Monthly","Osteoporosis","Risedronate"],"string":"[\n \"Bone mineral density\",\n \"Fracture risk\",\n \"Monthly\",\n \"Osteoporosis\",\n \"Risedronate\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nRisedronate is a pyridinyl bisphosphonate that has been shown in prospective studies to reduce the risk of vertebral, nonvertebral, and hip fractures [1–3]. Like other bisphosphonates, risedronate remains active on the surface of bone for long periods after dosing, providing the opportunity to develop a range of dosing schedules.\nThe original risedronate dosing regimen for postmenopausal osteoporosis was an oral dose of 5-mg daily [1–3]. It was later demonstrated that risedronate 35-mg once a week and 75-mg each day for two consecutive days a month provided similar efficacy and safety to the daily regimen [4, 5]. The efficacy and tolerability of risedronate once-a-month dosing (150-mg) was compared with risedronate daily dosing (5-mg) in women with osteoporosis with changes in lumbar spine bone mineral density (BMD) as the primary endpoint. After 1 year of treatment, published previously, the efficacy of risedronate 150-mg once-a-month regimen was non-inferior to the 5-mg daily regimen [6]. The once-a-month regimen also had a similar tolerability profile as the daily regimen after 1 year of treatment. This study continued for an additional year of treatment, and the results of the complete study over 2 years are presented here.\n\nMaterials and methods:\n Study design This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\nThis randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\n Patients Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\nEligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\n Treatments Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\nSubjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\n Efficacy assessments Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\nDual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\n Safety assessments Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\nPhysical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\n Statistical analysis The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\nThe primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\n\nStudy design:\nThis randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\n\nPatients:\nEligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\n\nTreatments:\nSubjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\n\nEfficacy assessments:\nDual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\n\nSafety assessments:\nPhysical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\n\nStatistical analysis:\nThe primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\n\nResults:\n Subjects From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\nFrom the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\n Efficacy assessments The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\nThe within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n Safety assessments Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\nOverall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\n\nSubjects:\nFrom the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\n\nEfficacy assessments:\nThe within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nSafety assessments:\nOverall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\n\nDiscussion:\nRisedronate is a widely used osteoporosis treatment with proven vertebral and nonvertebral antifracture efficacy and a minimum wait of 30 min after dosing before eating or drinking anything other than water. A 5-mg daily regimen was developed originally, but less frequent dose regimens have now been developed. This study was a preplanned 2-year study comparing a dose of risedronate 150-mg once a month to the 5-mg daily dose. These 2-year data show that the 150-mg once-a-month dose continues to produce clinical effects that are similar to those seen with the 5-mg daily dose. Specifically, the mean percent change in lumbar spine BMD at 24 months in the monthly group was non-inferior to the mean percent change in lumbar spine BMD in the daily group. Changes in secondary efficacy parameters, including BMD at the hip, bone turnover markers at endpoint, and morphometric vertebral fractures, were also similar in both groups. Statistically significant differences between treatment groups were observed for all three bone turnover markers at month 3, but persisted at months 6 and 12 for CTX only. No statistically significant differences between groups were observed for any marker at month 24 or endpoint. That the CTX response did not differ between treatment groups at month 24 might be explained by the small number of subjects at month 24 that would limit statistical power to observe difference. It is not likely that these small differences between groups in bone turnover markers are clinically meaningful.\nThe risedronate 150-mg once-a-month dose was well tolerated over 2 years, with a safety profile similar to that seen with the 5-mg daily regimen. The low incidences of subjects with vertebral and nonvertebral clinical fractures were similar between groups and consistent with rates previously observed with the 5-mg daily dose [1–3].\nChange in BMD is an appropriate endpoint when evaluating a new dosing schedule of a bisphosphonate for which a fracture benefit has already been established. Similar non-inferiority trials have been conducted previously to evaluate new dosing regimens of oral bisphosphonates [4, 8, 9], and this approach has been accepted by both the US Food and Drug Administration and the European Medicines Agency [10] for approval of new regimens of established agents. The magnitude of BMD change associated with the vertebral and nonvertebral antifracture efficacy of risedronate has been established in multiple large studies that had fracture as the primary endpoint [1–3]. This study has demonstrated that the 150-mg once-a-month dose reduces bone turnover and increases BMD to a degree comparable to that observed with the 5-mg daily dose in these fracture studies.\nThe results of this study after 2 years are consistent with the findings at month 12 [6], demonstrating the persistent similarity between risedronate 150-mg once-a-month and the 5-mg daily dosing regimens. Additionally, these results are consistent with the favorable tolerability and efficacy profiles observed in large placebo-controlled clinical trials of the risedronate 5-mg daily regimen [1–3]. The findings are also consistent with previous studies of less frequent dosing with risedronate. Such studies showed that the treatment effects of risedronate 35-mg weekly and 75-mg on two consecutive days each month were similar to the effects of daily dosing [4, 5].\nRisedronate 150-mg once a month, taken for 2 years, is similar in efficacy and tolerability to the 5-mg daily dosing regimen that had been proven to reduce the incidence of vertebral and nonvertebral fractures. The addition of this dosing regimen to the therapeutic armamentarium will provide women with postmenopausal osteoporosis a full range of risedronate oral dosing options, from daily to weekly to monthly.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nRisedronate is effective in the treatment of postmenopausal osteoporosis in oral daily, weekly, or on two consecutive days per month doses. This 2-year randomized, double-blind, multicenter study assesses the efficacy and safety of a single risedronate 150-mg once-a-month oral dose compared with the 5-mg daily regimen.\n\nMethods:\nWomen with postmenopausal osteoporosis were randomly assigned to receive risedronate 5-mg daily (n = 642) or 150-mg once a month (n = 650) for 2 years. Bone mineral density (BMD), bone turnover markers, new vertebral fractures, and adverse events were evaluated. The primary efficacy endpoint was the mean percent change from baseline in lumbar spine BMD after 1 year.\n\nResults:\nFour hundred ninety-eight subjects in the daily group (77.6 %) and 513 subjects in the once-a-month group (78.9 %) completed the study. After 24 months, the mean percent change in lumbar spine BMD was 3.9 % (95 % confidence interval [CI], 3.43 to 4.42 %) and 4.2 % (95 % CI, 3.68 to 4.65 %) in the daily and once-a-month groups, respectively. The once-a-month regimen was determined to be non-inferior to the daily regimen. The mean percent changes in BMD at the hip were similar in both dose groups, as were changes in biochemical markers of bone turnover. The incidence of adverse events, adverse events leading to withdrawal, and upper gastrointestinal tract adverse events were similar in the two treatment groups.\n\nConclusions:\nAfter 2 years, treatment with risedronate 150-mg once a month provided similar efficacy and tolerability to daily dosing and provides an alternative for patients who prefer once-a-month oral dosing."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":10390,"string":"10,390"},"whole_article_abstract_length":{"kind":"number","value":365,"string":"365"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["month","mg","treatment","24","groups","daily","subjects","group","baseline","mg daily"],"string":"[\n \"month\",\n \"mg\",\n \"treatment\",\n \"24\",\n \"groups\",\n \"daily\",\n \"subjects\",\n \"group\",\n \"baseline\",\n \"mg daily\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Bone mineral density | Fracture risk | Monthly | Osteoporosis | Risedronate [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Bone mineral density | Fracture risk | Monthly | Osteoporosis | Risedronate [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Bone mineral density | Fracture risk | Monthly | Osteoporosis | Risedronate [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Oral | Aged | Biomarkers | Bone Density | Bone Density Conservation Agents | Double-Blind Method | Drug Administration Schedule | Etidronic Acid | Female | Femur | Humans | Lumbar Vertebrae | Middle Aged | Osteoporosis, Postmenopausal | Risedronic Acid | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Oral | Aged | Biomarkers | Bone Density | Bone Density Conservation Agents | Double-Blind Method | Drug Administration Schedule | Etidronic Acid | Female | Femur | Humans | Lumbar Vertebrae | Middle Aged | Osteoporosis, Postmenopausal | Risedronic Acid | Treatment Outcome [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Oral | Aged | Biomarkers | Bone Density | Bone Density Conservation Agents | Double-Blind Method | Drug Administration Schedule | Etidronic Acid | Female | Femur | Humans | Lumbar Vertebrae | Middle Aged | Osteoporosis, Postmenopausal | Risedronic Acid | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] month | mg | treatment | 24 | groups | daily | subjects | group | baseline | mg daily [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] month | mg | treatment | 24 | groups | daily | subjects | group | baseline | mg daily [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] month | mg | treatment | 24 | groups | daily | subjects | group | baseline | mg daily [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] regimen | risedronate | dosing | mg | year treatment | daily | efficacy | daily regimen | tolerability | year [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] mg | groups | month | group | treatment | adverse | treatment groups | significant | events | line [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mg | month | treatment | 24 | daily | subjects | groups | group | baseline | adverse [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] daily | two consecutive days per month ||| 2-year | 150 | 5 | daily [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Four hundred ninety-eight | daily | 77.6 % | 513 | 78.9 % ||| 24 months | the mean percent | BMD | 3.9 % | 95 % | CI | 3.43 | 4.42 % | 4.2 % | 95 % | CI | 3.68 | 4.65 % | daily ||| daily ||| The mean percent | BMD ||| two [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] daily | two consecutive days per month ||| 2-year | 150 | 5 | daily ||| 5 | daily | 642 | 150 | 650 | 2 years ||| BMD ||| the mean percent | BMD | 1 year ||| Four hundred ninety-eight | daily | 77.6 % | 513 | 78.9 % ||| 24 months | the mean percent | BMD | 3.9 % | 95 % | CI | 3.43 | 4.42 % | 4.2 % | 95 % | CI | 3.68 | 4.65 % | daily ||| daily ||| The mean percent | BMD ||| two ||| 2 years | 150 | daily [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":295,"cells":{"title":{"kind":"string","value":"Survival Trend of Tuberculosis Patients and Risk Factors Associated with Mortality and Developing Drug-Resistant Tuberculosis in Hospital Pulau Pinang, Malaysia: A Retrospective Study."},"pmid":{"kind":"string","value":"36412638"},"background_abstract":{"kind":"string","value":"Multidrug resistance TB (MDR-TB) has emerged as a public health issue worldwide, and the mortality rate is worrying. Therefore, this study was conducted to investigate the factors related to MDR-TB occurrence and the survival experience of TB patients."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A retrospective cohort study was conducted at Hospital Pulau Pinang in Malaysia. Medical records of active TB patients from 2014-2018 were reviewed. Cox regression was used to identify the factors associated with MDR-TB development and mortality among TB patients."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The patients had a mean age of 48.84 ± 16.713 years, and a majority of the Chinese race (46.4%). Out of 351 TB patients, 325 (92.6%) were drug-susceptible TB, and 26 (7.4%) were diagnosed with MDR-TB. Among drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, and 73 (22.5%) passed away. In multivariable Cox regression, drug addiction, levels of white blood cells, urea, platelets, and albumin were significantly associated with death. Relapsed TB, alcohol consumption, and being single were significant risk factors for MDR-TB development."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Patients achieved a success rate of 75.4%, which is encouraging but still far below the WHO target (at least an 85% success rate) and has room for further improvement."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Humans","Adult","Middle Aged","Aged","Retrospective Studies","Antitubercular Agents","Malaysia","Tuberculosis, Multidrug-Resistant","Risk Factors","Hospitals"],"string":"[\n \"Humans\",\n \"Adult\",\n \"Middle Aged\",\n \"Aged\",\n \"Retrospective Studies\",\n \"Antitubercular Agents\",\n \"Malaysia\",\n \"Tuberculosis, Multidrug-Resistant\",\n \"Risk Factors\",\n \"Hospitals\"\n]"},"pmcid":{"kind":"string","value":"9774739"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Tuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":" 3.1. Sociodemographic and Clinical Characteristics At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\nAt the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\n 3.2. Drug Resistant Patterns of TB Patients The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\nThe study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\n 3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\nOf 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\n 3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\nAmong the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10)."},"conclusions_title":{"kind":"string","value":"5. Conclusions"},"conclusions_text":{"kind":"string","value":"Eventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single."},"other_sections_titles":{"kind":"list like","value":["2. Materials and Methods","2.1. Study Design and Population","2.2. Diagnosis and Treatment","2.3. Data Collection","2.4. Statistical Analysis","3.1. Sociodemographic and Clinical Characteristics","3.2. Drug Resistant Patterns of TB Patients","3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB"],"string":"[\n \"2. Materials and Methods\",\n \"2.1. Study Design and Population\",\n \"2.2. Diagnosis and Treatment\",\n \"2.3. Data Collection\",\n \"2.4. Statistical Analysis\",\n \"3.1. Sociodemographic and Clinical Characteristics\",\n \"3.2. Drug Resistant Patterns of TB Patients\",\n \"3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB\"\n]"},"other_sections_texts":{"kind":"list like","value":[" 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. ","A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. ","Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. ","Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. ","To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. ","At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).","The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).","Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10)."],"string":"[\n \" 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \\nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \\n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \\nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \\n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \\nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \\n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \\nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \",\n \"A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \",\n \"Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \",\n \"Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \",\n \"To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \",\n \"At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\",\n \"The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\",\n \"Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,"subjects",null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n \"subjects\",\n null\n]"},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Materials and Methods","2.1. Study Design and Population","2.2. Diagnosis and Treatment","2.3. Data Collection","2.4. Statistical Analysis","3. Results","3.1. Sociodemographic and Clinical Characteristics","3.2. Drug Resistant Patterns of TB Patients","3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients","3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB","4. Discussion","5. Conclusions"],"string":"[\n \"1. Introduction\",\n \"2. Materials and Methods\",\n \"2.1. Study Design and Population\",\n \"2.2. Diagnosis and Treatment\",\n \"2.3. Data Collection\",\n \"2.4. Statistical Analysis\",\n \"3. Results\",\n \"3.1. Sociodemographic and Clinical Characteristics\",\n \"3.2. Drug Resistant Patterns of TB Patients\",\n \"3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients\",\n \"3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB\",\n \"4. Discussion\",\n \"5. Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Tuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients."," 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. ","A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. ","Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. ","Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. ","To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. "," 3.1. Sociodemographic and Clinical Characteristics At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\nAt the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\n 3.2. Drug Resistant Patterns of TB Patients The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\nThe study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\n 3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\nOf 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\n 3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\nAmong the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).","At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).","The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).","Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).","Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).","Our research looked into the factors that contribute to mortality among TB patients, as well as the factors associated with MDR-TB development. Since then, tuberculosis treatment has been problematic and difficult to apply properly because of long duration and compliance issues due to anti-tubercular drugs’ side effects. Some of the side effects are self-limiting, but others necessitate treatment discontinuation. Although rare, thrombocytopenia is one of the most common and serious life-threatening side effects. Thrombocytopenia is caused by the formation of antibodies that bind to platelets and initiate the complement cascade, causing these cells to lyse. These antibodies also suppress the production of platelets [8]. This study demonstrated that thrombocytopenia significantly increased the risk of mortality among TB patients. [9] reported a low platelet count as a significant risk factor for mortality.\nMortality among patients suffering from any infection has been linked to WBC count [10]. As a result, elevated WBC counts in TB patients may be related to the severity of lung involvement and provide an estimated risk of mortality [11]. During TB infection, the body produces leukocytes and macrophages as part of the immune mechanism to defend the body against invading Mycobacterium tuberculosis, which raises the total number of WBC [12]. The current study findings documented that every 103 cells/µL increment in WBC above the normal range increased the risk of death by 1.102. In line with our results, a study conducted in Malaysia pointed out that the risk of death increased by 1.12 times for every 103 cells per microliter unit increase in total WBC [13]. Other studies have indicated that WBC levels are a significant risk factor correlated with TB mortality [11,14]. Pneumonia ranked the second cause of death among the study population. The WBC count elevation has been reported as a predictor of the severity of pneumonia, one of the leading causes of death in TB patients [11].\nThe present findings indicated that the level of urea was a strong significant predictor of death. Increased levels of urea were found to be significantly associated with mortality [11]. We can relate elevated urea levels to kidney dysfunction, where renal clearance is reduced due to renal impairment/failure or chronic kidney disease (CKD). Urea levels may also increase in other conditions, such as upper GI bleeding [15]. As the prevalence of CKD in our cohort was 5.69%, where 20 patients developed CKD while receiving anti-tuberculosis treatment, further research is needed to determine the relationship between CKD and TB. Strategies for the prevention, detection, and treatment of TB-CKD cannot be refined without advanced understanding.\nHypoalbuminemia showed a statistically significant relationship with death; patients who had hypoalbuminemia were more likely to pass away than patients who did not. Matching our results, lower albumin levels showed an association with non-survivors (p< 0.001) compared to survivors. Low levels of albumin were found to be significantly correlated with the hazard of death [9,16]. Furthermore, hypoalbuminemia was considered a predictor of mortality in community-acquired pneumonia (CAP), which is not surprising considering that albumin is involved in inflammation and indicates malnutrition [17]. The initial value of serum albumin might also be a result of malnutrition or an underlying disease that can worsen the nutritional status of the patient [18]. As a result of malnutrition, deceased patients’ immune systems were suppressed and were therefore sicker upon initial presentation.\nAs for drug addiction characteristics, the findings of this study revealed that patients who had a history of addiction or were current addicts had a 1.895 times higher risk of death than those who had never abused drugs. In line with our findings, deceased patients were more likely to abuse heroin IV than living patients, and addiction was correlated with all causes of death in tuberculosis patients [19,20]. Supporting our results, a history of addiction was a statistically significant risk factor for death within the first year after diagnosis in Hong Kong, Russia, and Iran [21,22,23]. Poor adherence of drug users, default treatment, and poor access to health services can create challenges for the treatment of the drug addict population [24]. As drug users have a higher likelihood of default treatment than non-drug users, the increased hazard of death can be attributed to the increased prevalence of alcohol misuse, chronic viral hepatitis, and anti-tubercular drug-induced hepatotoxicity that injectable drug users exhibit [25,26].\nDrug resistance TB has emerged as a serious health issue undermining global efforts to control TB and reduce the spread of the disease, in addition to making TB treatment more complicated. Thus, we aimed to study the risk factors associated with the development of MDR-TB in TB patients. In the current study, relapsed TB cases were more likely to develop MDR-TB than newly registered cases that had never had exposure to anti-TB treatment. In agreement with our findings, a history of treatment with anti-tubercular drugs has been mentioned in the previous literature as a risk factor for MDR-TB development in common with other risk factors [27,28,29,30]. Relapsed TB cases were found to be more likely to achieve unsuccessful treatment outcomes [31,32]. As the history of being treated with anti-TB drugs raises the hazard of resistance to many drugs, more attention must be paid to identifying the underlying causes of treatment failure and default treatment, which may decrease the incidence of retreatment among tuberculosis patients [28]. The link between prior anti-TB exposure and the emergence of drug resistance could be due to the fact that prior anti-TB exposure suppresses only the growth of susceptible bacilli. On the other hand, it may allow for the multiplication of pre-existing drug-resistant mutants [33].\nOne of the sociodemographic characteristics related to tuberculosis is marital status. Our findings reported that single patients were at 6.817 times higher risk of developing MDR-TB than married patients, which was in agreement with other studies that revealed that single individuals were at higher risk of developing MDR-TB than married individuals [34,35]. Even though there is no biological relationship between the marital status of the patients and TB, when compared to married people, single people are more likely to be infected with tuberculosis or MDR-TB strains. Being at high risk can still be due to a lack of social support or participation in high-risk behaviors, such as alcohol and drug addiction [36].\nAside from the previously mentioned risk factors, alcohol consumption has been significantly associated with developing MDR-TB. Patients who had a history of drinking or were current drinkers had a 7.591 times higher risk of MDR-TB than those who had never drank. Drinkers had a higher risk of MDR-TB, according to the results of a study conducted in China and Uganda [37,38]. Alcohol use was a contributing factor in TB treatment interruption, which is the main factor in MDR-TB development [39]. Many explanations could stand behind the impact of alcohol consumption on MDR-TB. Poorer treatment outcomes, including default treatment, mortality, and treatment failure, have been observed in TB patients who have consumed alcohol and MDR-TB, while treatment failure has been identified as a significant risk factor for drug resistance [38]. In addition, the consequences of alcohol ingestion can result in liver damage, weakened immunity, and nutritional deficiency, contributing to sensitive and resistant TB infection [40,41,42].\nThe findings of the current study highlighted patients’ groups that are at high risk of achieving unsuccessful TB treatment outcomes. As MDR-TB has proven to be a major influencer on patients’ health and safety, more attention must be paid to the factors leading to relapse and overcoming them in order to minimize the risk of MDR-TB occurrence in TB patients. A must improve public awareness about the worsening effect of alcohol on TB treatment and the importance of psychological support from the patient’s family to adhere to TB treatment. Moreover, the government should implement more restricted rules to minimize the availability of drugs in the population hands and to increase the awareness of people regarding the hazard of drugs. Additionally, malnutrition status was a risk factor for death among TB patients, so monthly counseling and material support in the form of food is suggested.\nThe study has many limitations that must be recognized. This study targeted only patients registered with HPP, which may not cover the true proportion of the Malaysian population. Being a unicentral study and a convenient sample technique limits the generalization of the results to the entire Malaysian population. An unequal proportionality among several variables, including age, ethnicity, and gender, might influence the final results in comparison with randomized control trials (selection bias). Due to the retrospective nature of the present study, it was impossible to record certain sociodemographic characteristics of the patients who passed away and default treatment (information bias), which might enhance the findings of the study. It was difficult to set a control group that needed further clinical trials to support the study results. The aforementioned limitations might affect the extent to which the current findings are accurate and consistent and can be reproduced under the same methodology.","Eventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single."],"string":"[\n \"Tuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients.\",\n \" 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \\nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \\n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \\nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \\n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \\nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \\n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \\nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \",\n \"A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \",\n \"Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \",\n \"Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \",\n \"To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \",\n \" 3.1. Sociodemographic and Clinical Characteristics At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\\nAt the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\\n 3.2. Drug Resistant Patterns of TB Patients The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\\nThe study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\\n 3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\\nOf 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\\n 3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\\nAmong the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\",\n \"At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\",\n \"The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\",\n \"Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\",\n \"Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\",\n \"Our research looked into the factors that contribute to mortality among TB patients, as well as the factors associated with MDR-TB development. Since then, tuberculosis treatment has been problematic and difficult to apply properly because of long duration and compliance issues due to anti-tubercular drugs’ side effects. Some of the side effects are self-limiting, but others necessitate treatment discontinuation. Although rare, thrombocytopenia is one of the most common and serious life-threatening side effects. Thrombocytopenia is caused by the formation of antibodies that bind to platelets and initiate the complement cascade, causing these cells to lyse. These antibodies also suppress the production of platelets [8]. This study demonstrated that thrombocytopenia significantly increased the risk of mortality among TB patients. [9] reported a low platelet count as a significant risk factor for mortality.\\nMortality among patients suffering from any infection has been linked to WBC count [10]. As a result, elevated WBC counts in TB patients may be related to the severity of lung involvement and provide an estimated risk of mortality [11]. During TB infection, the body produces leukocytes and macrophages as part of the immune mechanism to defend the body against invading Mycobacterium tuberculosis, which raises the total number of WBC [12]. The current study findings documented that every 103 cells/µL increment in WBC above the normal range increased the risk of death by 1.102. In line with our results, a study conducted in Malaysia pointed out that the risk of death increased by 1.12 times for every 103 cells per microliter unit increase in total WBC [13]. Other studies have indicated that WBC levels are a significant risk factor correlated with TB mortality [11,14]. Pneumonia ranked the second cause of death among the study population. The WBC count elevation has been reported as a predictor of the severity of pneumonia, one of the leading causes of death in TB patients [11].\\nThe present findings indicated that the level of urea was a strong significant predictor of death. Increased levels of urea were found to be significantly associated with mortality [11]. We can relate elevated urea levels to kidney dysfunction, where renal clearance is reduced due to renal impairment/failure or chronic kidney disease (CKD). Urea levels may also increase in other conditions, such as upper GI bleeding [15]. As the prevalence of CKD in our cohort was 5.69%, where 20 patients developed CKD while receiving anti-tuberculosis treatment, further research is needed to determine the relationship between CKD and TB. Strategies for the prevention, detection, and treatment of TB-CKD cannot be refined without advanced understanding.\\nHypoalbuminemia showed a statistically significant relationship with death; patients who had hypoalbuminemia were more likely to pass away than patients who did not. Matching our results, lower albumin levels showed an association with non-survivors (p< 0.001) compared to survivors. Low levels of albumin were found to be significantly correlated with the hazard of death [9,16]. Furthermore, hypoalbuminemia was considered a predictor of mortality in community-acquired pneumonia (CAP), which is not surprising considering that albumin is involved in inflammation and indicates malnutrition [17]. The initial value of serum albumin might also be a result of malnutrition or an underlying disease that can worsen the nutritional status of the patient [18]. As a result of malnutrition, deceased patients’ immune systems were suppressed and were therefore sicker upon initial presentation.\\nAs for drug addiction characteristics, the findings of this study revealed that patients who had a history of addiction or were current addicts had a 1.895 times higher risk of death than those who had never abused drugs. In line with our findings, deceased patients were more likely to abuse heroin IV than living patients, and addiction was correlated with all causes of death in tuberculosis patients [19,20]. Supporting our results, a history of addiction was a statistically significant risk factor for death within the first year after diagnosis in Hong Kong, Russia, and Iran [21,22,23]. Poor adherence of drug users, default treatment, and poor access to health services can create challenges for the treatment of the drug addict population [24]. As drug users have a higher likelihood of default treatment than non-drug users, the increased hazard of death can be attributed to the increased prevalence of alcohol misuse, chronic viral hepatitis, and anti-tubercular drug-induced hepatotoxicity that injectable drug users exhibit [25,26].\\nDrug resistance TB has emerged as a serious health issue undermining global efforts to control TB and reduce the spread of the disease, in addition to making TB treatment more complicated. Thus, we aimed to study the risk factors associated with the development of MDR-TB in TB patients. In the current study, relapsed TB cases were more likely to develop MDR-TB than newly registered cases that had never had exposure to anti-TB treatment. In agreement with our findings, a history of treatment with anti-tubercular drugs has been mentioned in the previous literature as a risk factor for MDR-TB development in common with other risk factors [27,28,29,30]. Relapsed TB cases were found to be more likely to achieve unsuccessful treatment outcomes [31,32]. As the history of being treated with anti-TB drugs raises the hazard of resistance to many drugs, more attention must be paid to identifying the underlying causes of treatment failure and default treatment, which may decrease the incidence of retreatment among tuberculosis patients [28]. The link between prior anti-TB exposure and the emergence of drug resistance could be due to the fact that prior anti-TB exposure suppresses only the growth of susceptible bacilli. On the other hand, it may allow for the multiplication of pre-existing drug-resistant mutants [33].\\nOne of the sociodemographic characteristics related to tuberculosis is marital status. Our findings reported that single patients were at 6.817 times higher risk of developing MDR-TB than married patients, which was in agreement with other studies that revealed that single individuals were at higher risk of developing MDR-TB than married individuals [34,35]. Even though there is no biological relationship between the marital status of the patients and TB, when compared to married people, single people are more likely to be infected with tuberculosis or MDR-TB strains. Being at high risk can still be due to a lack of social support or participation in high-risk behaviors, such as alcohol and drug addiction [36].\\nAside from the previously mentioned risk factors, alcohol consumption has been significantly associated with developing MDR-TB. Patients who had a history of drinking or were current drinkers had a 7.591 times higher risk of MDR-TB than those who had never drank. Drinkers had a higher risk of MDR-TB, according to the results of a study conducted in China and Uganda [37,38]. Alcohol use was a contributing factor in TB treatment interruption, which is the main factor in MDR-TB development [39]. Many explanations could stand behind the impact of alcohol consumption on MDR-TB. Poorer treatment outcomes, including default treatment, mortality, and treatment failure, have been observed in TB patients who have consumed alcohol and MDR-TB, while treatment failure has been identified as a significant risk factor for drug resistance [38]. In addition, the consequences of alcohol ingestion can result in liver damage, weakened immunity, and nutritional deficiency, contributing to sensitive and resistant TB infection [40,41,42].\\nThe findings of the current study highlighted patients’ groups that are at high risk of achieving unsuccessful TB treatment outcomes. As MDR-TB has proven to be a major influencer on patients’ health and safety, more attention must be paid to the factors leading to relapse and overcoming them in order to minimize the risk of MDR-TB occurrence in TB patients. A must improve public awareness about the worsening effect of alcohol on TB treatment and the importance of psychological support from the patient’s family to adhere to TB treatment. Moreover, the government should implement more restricted rules to minimize the availability of drugs in the population hands and to increase the awareness of people regarding the hazard of drugs. Additionally, malnutrition status was a risk factor for death among TB patients, so monthly counseling and material support in the form of food is suggested.\\nThe study has many limitations that must be recognized. This study targeted only patients registered with HPP, which may not cover the true proportion of the Malaysian population. Being a unicentral study and a convenient sample technique limits the generalization of the results to the entire Malaysian population. An unequal proportionality among several variables, including age, ethnicity, and gender, might influence the final results in comparison with randomized control trials (selection bias). Due to the retrospective nature of the present study, it was impossible to record certain sociodemographic characteristics of the patients who passed away and default treatment (information bias), which might enhance the findings of the study. It was difficult to set a control group that needed further clinical trials to support the study results. The aforementioned limitations might affect the extent to which the current findings are accurate and consistent and can be reproduced under the same methodology.\",\n \"Eventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,null,null,null,null,"results",null,"subjects","subjects",null,"discussion","conclusions"],"string":"[\n \"intro\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n \"subjects\",\n \"subjects\",\n null,\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["multidrug-resistant","mortality","death","treatment outcomes","survival","unsuccessful treatment"],"string":"[\n \"multidrug-resistant\",\n \"mortality\",\n \"death\",\n \"treatment outcomes\",\n \"survival\",\n \"unsuccessful treatment\"\n]"},"whole_article_text":{"kind":"string","value":"1. Introduction:\nTuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients.\n\n2. Materials and Methods:\n 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \n\n2.1. Study Design and Population:\nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \n\n2.2. Diagnosis and Treatment:\nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \n\n2.3. Data Collection:\nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \n\n2.4. Statistical Analysis:\nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \n\n3. Results:\n 3.1. Sociodemographic and Clinical Characteristics At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\nAt the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\n 3.2. Drug Resistant Patterns of TB Patients The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\nThe study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\n 3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\nOf 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\n 3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\nAmong the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\n\n3.1. Sociodemographic and Clinical Characteristics:\nAt the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\n\n3.2. Drug Resistant Patterns of TB Patients:\nThe study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\n\n3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients:\nOf 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\n\n3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB:\nAmong the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\n\n4. Discussion:\nOur research looked into the factors that contribute to mortality among TB patients, as well as the factors associated with MDR-TB development. Since then, tuberculosis treatment has been problematic and difficult to apply properly because of long duration and compliance issues due to anti-tubercular drugs’ side effects. Some of the side effects are self-limiting, but others necessitate treatment discontinuation. Although rare, thrombocytopenia is one of the most common and serious life-threatening side effects. Thrombocytopenia is caused by the formation of antibodies that bind to platelets and initiate the complement cascade, causing these cells to lyse. These antibodies also suppress the production of platelets [8]. This study demonstrated that thrombocytopenia significantly increased the risk of mortality among TB patients. [9] reported a low platelet count as a significant risk factor for mortality.\nMortality among patients suffering from any infection has been linked to WBC count [10]. As a result, elevated WBC counts in TB patients may be related to the severity of lung involvement and provide an estimated risk of mortality [11]. During TB infection, the body produces leukocytes and macrophages as part of the immune mechanism to defend the body against invading Mycobacterium tuberculosis, which raises the total number of WBC [12]. The current study findings documented that every 103 cells/µL increment in WBC above the normal range increased the risk of death by 1.102. In line with our results, a study conducted in Malaysia pointed out that the risk of death increased by 1.12 times for every 103 cells per microliter unit increase in total WBC [13]. Other studies have indicated that WBC levels are a significant risk factor correlated with TB mortality [11,14]. Pneumonia ranked the second cause of death among the study population. The WBC count elevation has been reported as a predictor of the severity of pneumonia, one of the leading causes of death in TB patients [11].\nThe present findings indicated that the level of urea was a strong significant predictor of death. Increased levels of urea were found to be significantly associated with mortality [11]. We can relate elevated urea levels to kidney dysfunction, where renal clearance is reduced due to renal impairment/failure or chronic kidney disease (CKD). Urea levels may also increase in other conditions, such as upper GI bleeding [15]. As the prevalence of CKD in our cohort was 5.69%, where 20 patients developed CKD while receiving anti-tuberculosis treatment, further research is needed to determine the relationship between CKD and TB. Strategies for the prevention, detection, and treatment of TB-CKD cannot be refined without advanced understanding.\nHypoalbuminemia showed a statistically significant relationship with death; patients who had hypoalbuminemia were more likely to pass away than patients who did not. Matching our results, lower albumin levels showed an association with non-survivors (p< 0.001) compared to survivors. Low levels of albumin were found to be significantly correlated with the hazard of death [9,16]. Furthermore, hypoalbuminemia was considered a predictor of mortality in community-acquired pneumonia (CAP), which is not surprising considering that albumin is involved in inflammation and indicates malnutrition [17]. The initial value of serum albumin might also be a result of malnutrition or an underlying disease that can worsen the nutritional status of the patient [18]. As a result of malnutrition, deceased patients’ immune systems were suppressed and were therefore sicker upon initial presentation.\nAs for drug addiction characteristics, the findings of this study revealed that patients who had a history of addiction or were current addicts had a 1.895 times higher risk of death than those who had never abused drugs. In line with our findings, deceased patients were more likely to abuse heroin IV than living patients, and addiction was correlated with all causes of death in tuberculosis patients [19,20]. Supporting our results, a history of addiction was a statistically significant risk factor for death within the first year after diagnosis in Hong Kong, Russia, and Iran [21,22,23]. Poor adherence of drug users, default treatment, and poor access to health services can create challenges for the treatment of the drug addict population [24]. As drug users have a higher likelihood of default treatment than non-drug users, the increased hazard of death can be attributed to the increased prevalence of alcohol misuse, chronic viral hepatitis, and anti-tubercular drug-induced hepatotoxicity that injectable drug users exhibit [25,26].\nDrug resistance TB has emerged as a serious health issue undermining global efforts to control TB and reduce the spread of the disease, in addition to making TB treatment more complicated. Thus, we aimed to study the risk factors associated with the development of MDR-TB in TB patients. In the current study, relapsed TB cases were more likely to develop MDR-TB than newly registered cases that had never had exposure to anti-TB treatment. In agreement with our findings, a history of treatment with anti-tubercular drugs has been mentioned in the previous literature as a risk factor for MDR-TB development in common with other risk factors [27,28,29,30]. Relapsed TB cases were found to be more likely to achieve unsuccessful treatment outcomes [31,32]. As the history of being treated with anti-TB drugs raises the hazard of resistance to many drugs, more attention must be paid to identifying the underlying causes of treatment failure and default treatment, which may decrease the incidence of retreatment among tuberculosis patients [28]. The link between prior anti-TB exposure and the emergence of drug resistance could be due to the fact that prior anti-TB exposure suppresses only the growth of susceptible bacilli. On the other hand, it may allow for the multiplication of pre-existing drug-resistant mutants [33].\nOne of the sociodemographic characteristics related to tuberculosis is marital status. Our findings reported that single patients were at 6.817 times higher risk of developing MDR-TB than married patients, which was in agreement with other studies that revealed that single individuals were at higher risk of developing MDR-TB than married individuals [34,35]. Even though there is no biological relationship between the marital status of the patients and TB, when compared to married people, single people are more likely to be infected with tuberculosis or MDR-TB strains. Being at high risk can still be due to a lack of social support or participation in high-risk behaviors, such as alcohol and drug addiction [36].\nAside from the previously mentioned risk factors, alcohol consumption has been significantly associated with developing MDR-TB. Patients who had a history of drinking or were current drinkers had a 7.591 times higher risk of MDR-TB than those who had never drank. Drinkers had a higher risk of MDR-TB, according to the results of a study conducted in China and Uganda [37,38]. Alcohol use was a contributing factor in TB treatment interruption, which is the main factor in MDR-TB development [39]. Many explanations could stand behind the impact of alcohol consumption on MDR-TB. Poorer treatment outcomes, including default treatment, mortality, and treatment failure, have been observed in TB patients who have consumed alcohol and MDR-TB, while treatment failure has been identified as a significant risk factor for drug resistance [38]. In addition, the consequences of alcohol ingestion can result in liver damage, weakened immunity, and nutritional deficiency, contributing to sensitive and resistant TB infection [40,41,42].\nThe findings of the current study highlighted patients’ groups that are at high risk of achieving unsuccessful TB treatment outcomes. As MDR-TB has proven to be a major influencer on patients’ health and safety, more attention must be paid to the factors leading to relapse and overcoming them in order to minimize the risk of MDR-TB occurrence in TB patients. A must improve public awareness about the worsening effect of alcohol on TB treatment and the importance of psychological support from the patient’s family to adhere to TB treatment. Moreover, the government should implement more restricted rules to minimize the availability of drugs in the population hands and to increase the awareness of people regarding the hazard of drugs. Additionally, malnutrition status was a risk factor for death among TB patients, so monthly counseling and material support in the form of food is suggested.\nThe study has many limitations that must be recognized. This study targeted only patients registered with HPP, which may not cover the true proportion of the Malaysian population. Being a unicentral study and a convenient sample technique limits the generalization of the results to the entire Malaysian population. An unequal proportionality among several variables, including age, ethnicity, and gender, might influence the final results in comparison with randomized control trials (selection bias). Due to the retrospective nature of the present study, it was impossible to record certain sociodemographic characteristics of the patients who passed away and default treatment (information bias), which might enhance the findings of the study. It was difficult to set a control group that needed further clinical trials to support the study results. The aforementioned limitations might affect the extent to which the current findings are accurate and consistent and can be reproduced under the same methodology.\n\n5. Conclusions:\nEventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nMultidrug resistance TB (MDR-TB) has emerged as a public health issue worldwide, and the mortality rate is worrying. Therefore, this study was conducted to investigate the factors related to MDR-TB occurrence and the survival experience of TB patients.\n\nMethods:\nA retrospective cohort study was conducted at Hospital Pulau Pinang in Malaysia. Medical records of active TB patients from 2014-2018 were reviewed. Cox regression was used to identify the factors associated with MDR-TB development and mortality among TB patients.\n\nResults:\nThe patients had a mean age of 48.84 ± 16.713 years, and a majority of the Chinese race (46.4%). Out of 351 TB patients, 325 (92.6%) were drug-susceptible TB, and 26 (7.4%) were diagnosed with MDR-TB. Among drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, and 73 (22.5%) passed away. In multivariable Cox regression, drug addiction, levels of white blood cells, urea, platelets, and albumin were significantly associated with death. Relapsed TB, alcohol consumption, and being single were significant risk factors for MDR-TB development.\n\nConclusions:\nPatients achieved a success rate of 75.4%, which is encouraging but still far below the WHO target (at least an 85% success rate) and has room for further improvement."},"background_conclusion_text":{"kind":"string","value":"1. Introduction:\nTuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients.\n\n5. Conclusions:\nEventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nMultidrug resistance TB (MDR-TB) has emerged as a public health issue worldwide, and the mortality rate is worrying. Therefore, this study was conducted to investigate the factors related to MDR-TB occurrence and the survival experience of TB patients.\n\nMethods:\nA retrospective cohort study was conducted at Hospital Pulau Pinang in Malaysia. Medical records of active TB patients from 2014-2018 were reviewed. Cox regression was used to identify the factors associated with MDR-TB development and mortality among TB patients.\n\nResults:\nThe patients had a mean age of 48.84 ± 16.713 years, and a majority of the Chinese race (46.4%). Out of 351 TB patients, 325 (92.6%) were drug-susceptible TB, and 26 (7.4%) were diagnosed with MDR-TB. Among drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, and 73 (22.5%) passed away. In multivariable Cox regression, drug addiction, levels of white blood cells, urea, platelets, and albumin were significantly associated with death. Relapsed TB, alcohol consumption, and being single were significant risk factors for MDR-TB development.\n\nConclusions:\nPatients achieved a success rate of 75.4%, which is encouraging but still far below the WHO target (at least an 85% success rate) and has room for further improvement."},"whole_article_text_length":{"kind":"number","value":9383,"string":"9,383"},"whole_article_abstract_length":{"kind":"number","value":268,"string":"268"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["tb","patients","treatment","hr","ci","95 ci","95","mdr tb","mdr","drug"],"string":"[\n \"tb\",\n \"patients\",\n \"treatment\",\n \"hr\",\n \"ci\",\n \"95 ci\",\n \"95\",\n \"mdr tb\",\n \"mdr\",\n \"drug\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | mdr tb | mdr | 2019 | disease | people | 000 | million | mortality | 2020 [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 95 | 95 ci | ci | hr | table | tb | associated | risk | patients | drug [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | levels | addiction | eventually patients history addiction | patients drug addiction | related relapsed cases alcohol | tuberculosis developed | tuberculosis developed 26 | tuberculosis developed 26 351 | patients drug addiction white [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | 95 | ci | 95 ci | hr | treatment | mdr tb | mdr | drug [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | 95 | ci | 95 ci | hr | treatment | mdr tb | mdr | drug [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] TB ||| MDR | TB [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 16.713 years | Chinese | 46.4% ||| 351 | 325 | 92.6% | TB | 26 | 7.4% | MDR ||| 245 | 75.4% | 73 | 22.5% ||| ||| TB | MDR [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] 75.4% | WHO | at least an | 85% [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] TB ||| MDR | TB ||| Hospital Pulau Pinang | Malaysia ||| TB | 2014-2018 ||| MDR | TB ||| ||| 16.713 years | Chinese | 46.4% ||| 351 | 325 | 92.6% | TB | 26 | 7.4% | MDR ||| 245 | 75.4% | 73 | 22.5% ||| ||| TB | MDR ||| 75.4% | WHO | at least an | 85% [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] TB ||| MDR | TB ||| Hospital Pulau Pinang | Malaysia ||| TB | 2014-2018 ||| MDR | TB ||| ||| 16.713 years | Chinese | 46.4% ||| 351 | 325 | 92.6% | TB | 26 | 7.4% | MDR ||| 245 | 75.4% | 73 | 22.5% ||| ||| TB | MDR ||| 75.4% | WHO | at least an | 85% [SUMMARY]"}}},{"rowIdx":296,"cells":{"title":{"kind":"string","value":"Is the Association between Age and Fertility Problems Modified by Diet Quality? Findings from a National Study of Reproductive Age Women in Australia."},"pmid":{"kind":"string","value":"36297039"},"background_abstract":{"kind":"string","value":"Increasing age is a strong risk factor for infertility, and there is accumulating evidence of the importance of a healthier diet for fertility. Whether a healthier diet modifies the association between increasing age and infertility has not been investigated. This study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Data were from Surveys 3 and 5 of the 1973-1978 birth cohort of the Australian Longitudinal Study on Women's Health. Cross-sectional analysis with multivariable generalized linear models were used to examine the association between age and fertility status, adjusted for various confounders. Multiplicative and additive effect modification by diet quality was assessed, with additive effect modification evaluated with the relative risk for interaction (RERI)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In total, 3387 women were included from Survey 3 (age range 24-31 years) and 5614 women from Survey 5 (age range 30-38 years); 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems in the respective surveys. In Survey 3, compared to younger women with a good-quality diet, older women with a poor-quality diet had a 43% increased risk for fertility problems, with risk increasing after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62). In Survey 5 in younger women (<33.9 years), there was no association between diet quality and risk for infertility problems. The RERI (across different adjusted models) was between -0.08 (-0.70, 0.55) to -0.39 (-1.40, 0.62) in survey 3 and 0.07 (-0.17, 0.31) to 0.08 (-0.17, 0.32) in Survey 5."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"There is little evidence to suggest effect modification on the effect of age and fertility problems with diet quality."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Female","Humans","Aged","Young Adult","Adult","Infertility, Female","Longitudinal Studies","Cross-Sectional Studies","Australia","Fertility","Diet","Risk Factors"],"string":"[\n \"Female\",\n \"Humans\",\n \"Aged\",\n \"Young Adult\",\n \"Adult\",\n \"Infertility, Female\",\n \"Longitudinal Studies\",\n \"Cross-Sectional Studies\",\n \"Australia\",\n \"Fertility\",\n \"Diet\",\n \"Risk Factors\"\n]"},"pmcid":{"kind":"string","value":"9606952"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Infertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].\nIncreasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].\nAlthough increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].\nThere is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":" 3.1. Participants A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\nA total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\n 3.2. Effect Modification of Diet on Age and Fertility Problems Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\nTable 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\n 3.3. Dietary Change and Fertility Status Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\nTable 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5)."},"conclusions_title":{"kind":"string","value":"5. Conclusions"},"conclusions_text":{"kind":"string","value":"In conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age."},"other_sections_titles":{"kind":"list like","value":["2. Materials and Methods","2.1. Study Design","2.2. Study Population","2.3. Exposure Variables","2.4. Outcome Variable","2.5. Confounding Variables","2.6. Statistical Analysis","3.2. Effect Modification of Diet on Age and Fertility Problems","3.3. Dietary Change and Fertility Status"],"string":"[\n \"2. Materials and Methods\",\n \"2.1. Study Design\",\n \"2.2. Study Population\",\n \"2.3. Exposure Variables\",\n \"2.4. Outcome Variable\",\n \"2.5. Confounding Variables\",\n \"2.6. Statistical Analysis\",\n \"3.2. Effect Modification of Diet on Age and Fertility Problems\",\n \"3.3. Dietary Change and Fertility Status\"\n]"},"other_sections_texts":{"kind":"list like","value":[" 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).","Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.","This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).","The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.","Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.","A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].","Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).","Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.","Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5)."],"string":"[\n \" 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\",\n \"Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\",\n \"This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\",\n \"The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\",\n \"Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\",\n \"A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\",\n \"Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\",\n \"Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\",\n \"Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Materials and Methods","2.1. Study Design","2.2. Study Population","2.3. Exposure Variables","2.4. Outcome Variable","2.5. Confounding Variables","2.6. Statistical Analysis","3. Results","3.1. Participants","3.2. Effect Modification of Diet on Age and Fertility Problems","3.3. Dietary Change and Fertility Status","4. Discussion","5. Conclusions"],"string":"[\n \"1. Introduction\",\n \"2. Materials and Methods\",\n \"2.1. Study Design\",\n \"2.2. Study Population\",\n \"2.3. Exposure Variables\",\n \"2.4. Outcome Variable\",\n \"2.5. Confounding Variables\",\n \"2.6. Statistical Analysis\",\n \"3. Results\",\n \"3.1. Participants\",\n \"3.2. Effect Modification of Diet on Age and Fertility Problems\",\n \"3.3. Dietary Change and Fertility Status\",\n \"4. Discussion\",\n \"5. Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Infertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].\nIncreasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].\nAlthough increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].\nThere is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality."," 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).","Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.","This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).","The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.","Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.","A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].","Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA)."," 3.1. Participants A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\nA total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\n 3.2. Effect Modification of Diet on Age and Fertility Problems Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\nTable 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\n 3.3. Dietary Change and Fertility Status Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\nTable 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).","A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.","Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.","Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).","Among a large cohort of Australian women, we examined whether diet modified the relationship between age and infertility, and whether a change in diet impacts fertility. Our results do not support our hypothesis; that is, in both Survey 3 and in Survey 5, there was little evidence that diet quality modified the association between age and fertility problems. Although increasing age (from Survey 3 to Survey 5) was associated with an approximate 5% higher rate of fertility problems, a change in diet quality over this time was not associated with fertility problems at Survey 5.\nAmong Survey 3 participants who were aged between 24–31 years, independent associations were found, such that older woman (28.2 to 30.8 years) who had a poor-quality diet had higher risk for infertility problems compared to the younger women (24.4 to <28.2 years) with a good-quality diet. At Survey 5 when women were aged 30–38 years, there was no difference in risk for fertility problems by diet quality or age. This was an unexpected finding. At Survey 5, there was an approximate 10% higher percentage of women with a good-quality diet; thus, with increasing age, the importance of a healthier diet may be realised, irrespective of fertility status. Yet, the older women at Survey 5 did appear to drink slightly more alcohol and perform less metabolic minutes of exercise, which are both associated with higher likelihood for infertility [6,36]. Interestingly, the mean BMI among women with and without fertility problems was consistent over the 5-year period from Survey 3 to Survey 5, with the women with infertility problems maintaining the approximate two-unit higher BMI than the women without infertility problems. The combination of some infertility risk factors increasing over time, but others remaining consistent, may partly explain the lack of association in this slightly older age group of women. Nevertheless, our results might suggest a small window of opportunity in women aged 28–31 years to optimise their diet to support fertility, whereas after this age, the current data do not support this. Although many studies have reported on the independent effects of increasing age with infertility, and increasing evidence shows the importance of a healthy diet for reproductive health, qualitative data reveals that women do not have a clear understanding of the age at which fertility begins to decline [37]. There are also no specific recommendations for an appropriate diet to optimise fertility. The knowledge gaps and misconceptions surrounding reproductive health and nutrition, highlights a critical opportunity that is needed to support patient awareness, counselling to support dietary behaviours, and that involves pregnancy planning.\nOur findings provide little evidence of effect modification by diet at either survey. Whilst at Survey 3 there was a suggestion of a negative effect measure modification, such that the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet, the wide 95% CIs are consistent with positive effect modification by diet. At Survey 5, the RERI was positive, suggesting that the effect of age interacting with diet quality was higher than the sum of the independent effects of age and diet, but the wide confidence intervals are also consistent with positive effect modification. Thus, we cannot conclude that better quality diet could attenuate the decline in fertility with age over the observed periods. These findings suggest that the effect of a poor-quality diet and older age on fertility may not be as pronounced as expected, but also that a poor diet quality is not appreciably leading to reduced fertility in older women.\nWhilst our results are unexpected, they build the foundation for further studies to assess the interactions between age and diet quality. Although the additive effect modification RERI is the more relevant public health measure, and we would theorise that the targeted group for intervention would be older women with a poor-quality diet, in this instance, we cannot confidently draw this conclusion. Importantly, our results do not mean that a good-quality diet is adverse in any way but implies that a good-quality diet does not attenuate the relationship between age and infertility, and that the joint effect of the two exposures is less than their main effects combined. The indication of a positive RERI at Survey 5 supports the promotion of improved diet quality in older women.\nOverall diet quality of women was low in both surveys, which is consistent with previous studies reporting that a poor-quality diet is common amongst women of reproductive age [23,38,39,40,41]. Despite the slight increase in proportion of women having a good-quality diet from Survey 3 to Survey 5, there was no evidence that a change in diet, whether that was an improvement or worsening of diet quality over time, was associated with fertility problems. However, we acknowledge that this change in diet quality is very small and may be equivalent to only replacing white bread with brown (whole meal or multigrain) as the usual bread choice. In line with our findings, a review of studies assessing pregnancy intentions and diet or physical activity behaviours in the preconception and antenatal periods found that women intending to become pregnant do not report different preconception dietary or physical activity behaviours compared with women without pregnancy intentions [42]. This reflects the common barriers observed in women of reproductive age to achieve successful health behaviour change [43]. In our study, the lack of association between a change in diet and fertility may also relate to reverse causation. That is, following a diagnosis of fertility problems, women may alter their behaviours towards healthier choices, or their improvement in dietary intake may not be sufficient to alter the effect on fertility problems.\nTo the best of our knowledge, this is the first study to assess whether better diet quality can reduce the age-related decline in fertility. A strength of this study is the use of data from a nationally representative cohort of Australian women at two different time points, increasing the generalisability of our findings to all Australian women. We used robust statistical measures, including calculation of the RERI, whereby effect measure modification of diet on the additive scale provides a clear comparison of the effect of diet on the association between one of the major risk factors of fertility (i.e., age) and fertility problems among women. Some limitations are also worth mentioning. A validated, semi-quantitative FFQ was used to capture women’s dietary consumption, which was designed for use in the Australian community. Although the ARFS is an acceptable tool for assessing diet quality, for most foods the scoring was based on ‘at least once per week’. Additional response options might be helpful to quantify diet quality more accurately. While the analysis included a large cohort of women, it may not be sufficiently powered to identify interactions; thus, further work in this area, with larger populations and covering wider age ranges, is warranted. Lastly, the current study included self-reported fertility problems and dietary intakes that could be subject to recall bias.","In conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age."],"string":"[\n \"Infertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].\\nIncreasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].\\nAlthough increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].\\nThere is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality.\",\n \" 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\",\n \"Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\",\n \"This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\",\n \"The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\",\n \"Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\",\n \"A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\",\n \"Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\",\n \" 3.1. Participants A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\\nA total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\\n 3.2. Effect Modification of Diet on Age and Fertility Problems Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\\nTable 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\\n 3.3. Dietary Change and Fertility Status Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\\nTable 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\",\n \"A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\",\n \"Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\",\n \"Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\",\n \"Among a large cohort of Australian women, we examined whether diet modified the relationship between age and infertility, and whether a change in diet impacts fertility. Our results do not support our hypothesis; that is, in both Survey 3 and in Survey 5, there was little evidence that diet quality modified the association between age and fertility problems. Although increasing age (from Survey 3 to Survey 5) was associated with an approximate 5% higher rate of fertility problems, a change in diet quality over this time was not associated with fertility problems at Survey 5.\\nAmong Survey 3 participants who were aged between 24–31 years, independent associations were found, such that older woman (28.2 to 30.8 years) who had a poor-quality diet had higher risk for infertility problems compared to the younger women (24.4 to <28.2 years) with a good-quality diet. At Survey 5 when women were aged 30–38 years, there was no difference in risk for fertility problems by diet quality or age. This was an unexpected finding. At Survey 5, there was an approximate 10% higher percentage of women with a good-quality diet; thus, with increasing age, the importance of a healthier diet may be realised, irrespective of fertility status. Yet, the older women at Survey 5 did appear to drink slightly more alcohol and perform less metabolic minutes of exercise, which are both associated with higher likelihood for infertility [6,36]. Interestingly, the mean BMI among women with and without fertility problems was consistent over the 5-year period from Survey 3 to Survey 5, with the women with infertility problems maintaining the approximate two-unit higher BMI than the women without infertility problems. The combination of some infertility risk factors increasing over time, but others remaining consistent, may partly explain the lack of association in this slightly older age group of women. Nevertheless, our results might suggest a small window of opportunity in women aged 28–31 years to optimise their diet to support fertility, whereas after this age, the current data do not support this. Although many studies have reported on the independent effects of increasing age with infertility, and increasing evidence shows the importance of a healthy diet for reproductive health, qualitative data reveals that women do not have a clear understanding of the age at which fertility begins to decline [37]. There are also no specific recommendations for an appropriate diet to optimise fertility. The knowledge gaps and misconceptions surrounding reproductive health and nutrition, highlights a critical opportunity that is needed to support patient awareness, counselling to support dietary behaviours, and that involves pregnancy planning.\\nOur findings provide little evidence of effect modification by diet at either survey. Whilst at Survey 3 there was a suggestion of a negative effect measure modification, such that the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet, the wide 95% CIs are consistent with positive effect modification by diet. At Survey 5, the RERI was positive, suggesting that the effect of age interacting with diet quality was higher than the sum of the independent effects of age and diet, but the wide confidence intervals are also consistent with positive effect modification. Thus, we cannot conclude that better quality diet could attenuate the decline in fertility with age over the observed periods. These findings suggest that the effect of a poor-quality diet and older age on fertility may not be as pronounced as expected, but also that a poor diet quality is not appreciably leading to reduced fertility in older women.\\nWhilst our results are unexpected, they build the foundation for further studies to assess the interactions between age and diet quality. Although the additive effect modification RERI is the more relevant public health measure, and we would theorise that the targeted group for intervention would be older women with a poor-quality diet, in this instance, we cannot confidently draw this conclusion. Importantly, our results do not mean that a good-quality diet is adverse in any way but implies that a good-quality diet does not attenuate the relationship between age and infertility, and that the joint effect of the two exposures is less than their main effects combined. The indication of a positive RERI at Survey 5 supports the promotion of improved diet quality in older women.\\nOverall diet quality of women was low in both surveys, which is consistent with previous studies reporting that a poor-quality diet is common amongst women of reproductive age [23,38,39,40,41]. Despite the slight increase in proportion of women having a good-quality diet from Survey 3 to Survey 5, there was no evidence that a change in diet, whether that was an improvement or worsening of diet quality over time, was associated with fertility problems. However, we acknowledge that this change in diet quality is very small and may be equivalent to only replacing white bread with brown (whole meal or multigrain) as the usual bread choice. In line with our findings, a review of studies assessing pregnancy intentions and diet or physical activity behaviours in the preconception and antenatal periods found that women intending to become pregnant do not report different preconception dietary or physical activity behaviours compared with women without pregnancy intentions [42]. This reflects the common barriers observed in women of reproductive age to achieve successful health behaviour change [43]. In our study, the lack of association between a change in diet and fertility may also relate to reverse causation. That is, following a diagnosis of fertility problems, women may alter their behaviours towards healthier choices, or their improvement in dietary intake may not be sufficient to alter the effect on fertility problems.\\nTo the best of our knowledge, this is the first study to assess whether better diet quality can reduce the age-related decline in fertility. A strength of this study is the use of data from a nationally representative cohort of Australian women at two different time points, increasing the generalisability of our findings to all Australian women. We used robust statistical measures, including calculation of the RERI, whereby effect measure modification of diet on the additive scale provides a clear comparison of the effect of diet on the association between one of the major risk factors of fertility (i.e., age) and fertility problems among women. Some limitations are also worth mentioning. A validated, semi-quantitative FFQ was used to capture women’s dietary consumption, which was designed for use in the Australian community. Although the ARFS is an acceptable tool for assessing diet quality, for most foods the scoring was based on ‘at least once per week’. Additional response options might be helpful to quantify diet quality more accurately. While the analysis included a large cohort of women, it may not be sufficiently powered to identify interactions; thus, further work in this area, with larger populations and covering wider age ranges, is warranted. Lastly, the current study included self-reported fertility problems and dietary intakes that could be subject to recall bias.\",\n \"In conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,null,null,null,null,null,null,"results","subjects",null,null,"discussion","conclusions"],"string":"[\n \"intro\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"subjects\",\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["age","diet quality","reproductive age","Australia","survey","infertility","women"],"string":"[\n \"age\",\n \"diet quality\",\n \"reproductive age\",\n \"Australia\",\n \"survey\",\n \"infertility\",\n \"women\"\n]"},"whole_article_text":{"kind":"string","value":"1. Introduction:\nInfertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].\nIncreasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].\nAlthough increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].\nThere is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality.\n\n2. Materials and Methods:\n 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\n\n2.1. Study Design:\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\n\n2.2. Study Population:\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\n\n2.3. Exposure Variables:\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\n\n2.4. Outcome Variable:\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\n\n2.5. Confounding Variables:\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\n\n2.6. Statistical Analysis:\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\n\n3. Results:\n 3.1. Participants A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\nA total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\n 3.2. Effect Modification of Diet on Age and Fertility Problems Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\nTable 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\n 3.3. Dietary Change and Fertility Status Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\nTable 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\n\n3.1. Participants:\nA total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\n\n3.2. Effect Modification of Diet on Age and Fertility Problems:\nTable 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\n\n3.3. Dietary Change and Fertility Status:\nTable 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\n\n4. Discussion:\nAmong a large cohort of Australian women, we examined whether diet modified the relationship between age and infertility, and whether a change in diet impacts fertility. Our results do not support our hypothesis; that is, in both Survey 3 and in Survey 5, there was little evidence that diet quality modified the association between age and fertility problems. Although increasing age (from Survey 3 to Survey 5) was associated with an approximate 5% higher rate of fertility problems, a change in diet quality over this time was not associated with fertility problems at Survey 5.\nAmong Survey 3 participants who were aged between 24–31 years, independent associations were found, such that older woman (28.2 to 30.8 years) who had a poor-quality diet had higher risk for infertility problems compared to the younger women (24.4 to <28.2 years) with a good-quality diet. At Survey 5 when women were aged 30–38 years, there was no difference in risk for fertility problems by diet quality or age. This was an unexpected finding. At Survey 5, there was an approximate 10% higher percentage of women with a good-quality diet; thus, with increasing age, the importance of a healthier diet may be realised, irrespective of fertility status. Yet, the older women at Survey 5 did appear to drink slightly more alcohol and perform less metabolic minutes of exercise, which are both associated with higher likelihood for infertility [6,36]. Interestingly, the mean BMI among women with and without fertility problems was consistent over the 5-year period from Survey 3 to Survey 5, with the women with infertility problems maintaining the approximate two-unit higher BMI than the women without infertility problems. The combination of some infertility risk factors increasing over time, but others remaining consistent, may partly explain the lack of association in this slightly older age group of women. Nevertheless, our results might suggest a small window of opportunity in women aged 28–31 years to optimise their diet to support fertility, whereas after this age, the current data do not support this. Although many studies have reported on the independent effects of increasing age with infertility, and increasing evidence shows the importance of a healthy diet for reproductive health, qualitative data reveals that women do not have a clear understanding of the age at which fertility begins to decline [37]. There are also no specific recommendations for an appropriate diet to optimise fertility. The knowledge gaps and misconceptions surrounding reproductive health and nutrition, highlights a critical opportunity that is needed to support patient awareness, counselling to support dietary behaviours, and that involves pregnancy planning.\nOur findings provide little evidence of effect modification by diet at either survey. Whilst at Survey 3 there was a suggestion of a negative effect measure modification, such that the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet, the wide 95% CIs are consistent with positive effect modification by diet. At Survey 5, the RERI was positive, suggesting that the effect of age interacting with diet quality was higher than the sum of the independent effects of age and diet, but the wide confidence intervals are also consistent with positive effect modification. Thus, we cannot conclude that better quality diet could attenuate the decline in fertility with age over the observed periods. These findings suggest that the effect of a poor-quality diet and older age on fertility may not be as pronounced as expected, but also that a poor diet quality is not appreciably leading to reduced fertility in older women.\nWhilst our results are unexpected, they build the foundation for further studies to assess the interactions between age and diet quality. Although the additive effect modification RERI is the more relevant public health measure, and we would theorise that the targeted group for intervention would be older women with a poor-quality diet, in this instance, we cannot confidently draw this conclusion. Importantly, our results do not mean that a good-quality diet is adverse in any way but implies that a good-quality diet does not attenuate the relationship between age and infertility, and that the joint effect of the two exposures is less than their main effects combined. The indication of a positive RERI at Survey 5 supports the promotion of improved diet quality in older women.\nOverall diet quality of women was low in both surveys, which is consistent with previous studies reporting that a poor-quality diet is common amongst women of reproductive age [23,38,39,40,41]. Despite the slight increase in proportion of women having a good-quality diet from Survey 3 to Survey 5, there was no evidence that a change in diet, whether that was an improvement or worsening of diet quality over time, was associated with fertility problems. However, we acknowledge that this change in diet quality is very small and may be equivalent to only replacing white bread with brown (whole meal or multigrain) as the usual bread choice. In line with our findings, a review of studies assessing pregnancy intentions and diet or physical activity behaviours in the preconception and antenatal periods found that women intending to become pregnant do not report different preconception dietary or physical activity behaviours compared with women without pregnancy intentions [42]. This reflects the common barriers observed in women of reproductive age to achieve successful health behaviour change [43]. In our study, the lack of association between a change in diet and fertility may also relate to reverse causation. That is, following a diagnosis of fertility problems, women may alter their behaviours towards healthier choices, or their improvement in dietary intake may not be sufficient to alter the effect on fertility problems.\nTo the best of our knowledge, this is the first study to assess whether better diet quality can reduce the age-related decline in fertility. A strength of this study is the use of data from a nationally representative cohort of Australian women at two different time points, increasing the generalisability of our findings to all Australian women. We used robust statistical measures, including calculation of the RERI, whereby effect measure modification of diet on the additive scale provides a clear comparison of the effect of diet on the association between one of the major risk factors of fertility (i.e., age) and fertility problems among women. Some limitations are also worth mentioning. A validated, semi-quantitative FFQ was used to capture women’s dietary consumption, which was designed for use in the Australian community. Although the ARFS is an acceptable tool for assessing diet quality, for most foods the scoring was based on ‘at least once per week’. Additional response options might be helpful to quantify diet quality more accurately. While the analysis included a large cohort of women, it may not be sufficiently powered to identify interactions; thus, further work in this area, with larger populations and covering wider age ranges, is warranted. Lastly, the current study included self-reported fertility problems and dietary intakes that could be subject to recall bias.\n\n5. Conclusions:\nIn conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIncreasing age is a strong risk factor for infertility, and there is accumulating evidence of the importance of a healthier diet for fertility. Whether a healthier diet modifies the association between increasing age and infertility has not been investigated. This study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems.\n\nMethods:\nData were from Surveys 3 and 5 of the 1973-1978 birth cohort of the Australian Longitudinal Study on Women's Health. Cross-sectional analysis with multivariable generalized linear models were used to examine the association between age and fertility status, adjusted for various confounders. Multiplicative and additive effect modification by diet quality was assessed, with additive effect modification evaluated with the relative risk for interaction (RERI).\n\nResults:\nIn total, 3387 women were included from Survey 3 (age range 24-31 years) and 5614 women from Survey 5 (age range 30-38 years); 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems in the respective surveys. In Survey 3, compared to younger women with a good-quality diet, older women with a poor-quality diet had a 43% increased risk for fertility problems, with risk increasing after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62). In Survey 5 in younger women (<33.9 years), there was no association between diet quality and risk for infertility problems. The RERI (across different adjusted models) was between -0.08 (-0.70, 0.55) to -0.39 (-1.40, 0.62) in survey 3 and 0.07 (-0.17, 0.31) to 0.08 (-0.17, 0.32) in Survey 5.\n\nConclusions:\nThere is little evidence to suggest effect modification on the effect of age and fertility problems with diet quality."},"background_conclusion_text":{"kind":"string","value":"1. Introduction:\nInfertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].\nIncreasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].\nAlthough increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].\nThere is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality.\n\n5. Conclusions:\nIn conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nIncreasing age is a strong risk factor for infertility, and there is accumulating evidence of the importance of a healthier diet for fertility. Whether a healthier diet modifies the association between increasing age and infertility has not been investigated. This study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems.\n\nMethods:\nData were from Surveys 3 and 5 of the 1973-1978 birth cohort of the Australian Longitudinal Study on Women's Health. Cross-sectional analysis with multivariable generalized linear models were used to examine the association between age and fertility status, adjusted for various confounders. Multiplicative and additive effect modification by diet quality was assessed, with additive effect modification evaluated with the relative risk for interaction (RERI).\n\nResults:\nIn total, 3387 women were included from Survey 3 (age range 24-31 years) and 5614 women from Survey 5 (age range 30-38 years); 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems in the respective surveys. In Survey 3, compared to younger women with a good-quality diet, older women with a poor-quality diet had a 43% increased risk for fertility problems, with risk increasing after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62). In Survey 5 in younger women (<33.9 years), there was no association between diet quality and risk for infertility problems. The RERI (across different adjusted models) was between -0.08 (-0.70, 0.55) to -0.39 (-1.40, 0.62) in survey 3 and 0.07 (-0.17, 0.31) to 0.08 (-0.17, 0.32) in Survey 5.\n\nConclusions:\nThere is little evidence to suggest effect modification on the effect of age and fertility problems with diet quality."},"whole_article_text_length":{"kind":"number","value":9634,"string":"9,634"},"whole_article_abstract_length":{"kind":"number","value":386,"string":"386"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["diet","survey","women","fertility","quality","age","effect","problems","years","fertility problems"],"string":"[\n \"diet\",\n \"survey\",\n \"women\",\n \"fertility\",\n \"quality\",\n \"age\",\n \"effect\",\n \"problems\",\n \"years\",\n \"fertility problems\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] age | infertility | women | fertility | factor | diet | pregnancy | study | years | associated [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | women | fertility | fertility problems | survey | quality | effect | quality diet | problems | table [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet | findings | helpful | diet quality older | quality older | age range | older | older age | quality [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 3387 | 3 | 24-31 years | 5614 | Survey 5 | 30-38 years | 588 | 17.4% | 1321 | 23.4% ||| Survey 3 | 43% | BMI | 1.59 | 95% | CI | 1.07 | 2.37 | PCOS (RR: 1.74 | 95% | CI | 1.15 | 2.62 ||| Survey 5 ||| -0.70 | 0.55 | 0.62 | 3 | 0.31 | 0.08 | 0.32 | Survey 5 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| Surveys 3 and 5 | 1973-1978 | the Australian Longitudinal Study on Women's Health ||| linear ||| ||| ||| 3387 | 3 | 24-31 years | 5614 | Survey 5 | 30-38 years | 588 | 17.4% | 1321 | 23.4% ||| Survey 3 | 43% | BMI | 1.59 | 95% | CI | 1.07 | 2.37 | PCOS (RR: 1.74 | 95% | CI | 1.15 | 2.62 ||| Survey 5 ||| -0.70 | 0.55 | 0.62 | 3 | 0.31 | 0.08 | 0.32 | Survey 5 [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| Surveys 3 and 5 | 1973-1978 | the Australian Longitudinal Study on Women's Health ||| linear ||| ||| ||| 3387 | 3 | 24-31 years | 5614 | Survey 5 | 30-38 years | 588 | 17.4% | 1321 | 23.4% ||| Survey 3 | 43% | BMI | 1.59 | 95% | CI | 1.07 | 2.37 | PCOS (RR: 1.74 | 95% | CI | 1.15 | 2.62 ||| Survey 5 ||| -0.70 | 0.55 | 0.62 | 3 | 0.31 | 0.08 | 0.32 | Survey 5 [SUMMARY]"}}},{"rowIdx":297,"cells":{"title":{"kind":"string","value":"The I405V and Taq1B polymorphisms of the CETP gene differentially affect sub-clinical carotid atherosclerosis."},"pmid":{"kind":"string","value":"23039379"},"background_abstract":{"kind":"string","value":"Cholesteryl ester transfer protein (CETP) plays a major role in lipid metabolism, but studies on the association of CETP polymorphisms with risks of cardiovascular disease are inconsistent. This study investigated whether the CETP gene I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian population sample."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"The polymorphisms were analyzed using polymerase chain reaction in 207 adult volunteers. Serum lipid profiles, oxLDL Ab titers, C-reactive protein and tumor necrosis factor-α concentrations and CETP and phospholipid transfer protein (PLTP) activities were determined, and common carotid artery intima-media thickness (cIMT) was measured using ultrasonography."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"No differences in cIMT were observed between the presence or absence of the minor B2 and V alleles in either polymorphism. However, inverse correlations between mean cIMT and CETP activity in the presence of these polymorphisms were observed, and positive correlations of these polymorphisms with PLTP activity and oxLDL Ab titers were identified. Moreover, logistic multivariate analysis revealed that the presence of the B2 allele was associated with a 5.1-fold (CI 95%, OR: 1.26 - 21.06) increased risk for cIMT, which was equal and above the 66th percentile and positively interacted with age. However, no associations with the V allele or CETP and PLTP activities were observed."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"None of the studied parameters, including CETP activity, explained the different relationships between these polymorphisms and cIMT, suggesting that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Autoantibodies","Brazil","C-Reactive Protein","Carotid Artery Diseases","Carotid Intima-Media Thickness","Cholesterol Ester Transfer Proteins","Female","Gene Frequency","Humans","Lipids","Lipoproteins, LDL","Male","Middle Aged","Phospholipid Transfer Proteins","Polymorphism, Genetic","Tumor Necrosis Factor-alpha","Young Adult"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Autoantibodies\",\n \"Brazil\",\n \"C-Reactive Protein\",\n \"Carotid Artery Diseases\",\n \"Carotid Intima-Media Thickness\",\n \"Cholesterol Ester Transfer Proteins\",\n \"Female\",\n \"Gene Frequency\",\n \"Humans\",\n \"Lipids\",\n \"Lipoproteins, LDL\",\n \"Male\",\n \"Middle Aged\",\n \"Phospholipid Transfer Proteins\",\n \"Polymorphism, Genetic\",\n \"Tumor Necrosis Factor-alpha\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"3503625"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Study subjects A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nA total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\n Polymorphism detection Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\nGenomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\n Measurement of cIMT A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\nA single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\n Statistical analysis Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].\nMann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15]."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The frequencies of I405V and Taq1B genotypes in our population sample, represented by the presence (VV, IV) and absence (II) of the V allele and the presence (B2B2, B1B2) and absence (B1B1) of the B2 allele, are presented in Table \n1. The frequencies of the II, IV and VV genotypes were 28%, 53% and 19%, respectively, and the frequencies of the B1B1, B1B2 and B2B2 were 33%, 41% and 26%, respectively.\nBiochemical parameters in the presence and absence of minor alleles\nData are presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values: Mann–Whitney U test for comparisons between the presence and absence of the minor alleles (V and B2). Significant differences: p≤0.05 in bold. CETP (%) = activity of cholesterol ester transfer protein; HDL-C = high-density lipoprotein cholesterol; PLTP (%) = activity of phospholipid transfer protein; oxLDL Ab = antibodies against oxidized LDL titers; (OD)= optical density.\nAllele frequencies were consistent with Hardy–Weinberg equilibrium for the I405V polymorphism (p=0.36) but not the Taq1B (p=0.02), and the MAFs (minor allele frequency) were <45% and <46%, respectively. The linkage disequilibrium between the two polymorphisms was very weak, as evidenced by the D`=0.17.\nClinical and anthropometric characteristics and biochemical data of the investigated population are presented in Tables \n2 and\n1.\nClinical and anthropometric characteristics in the presence and absence of minor alleles\nData presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values = Mann–Whitney U test for comparisons between the presence and absence of the alleles (V and B2); gender comparisons, Chi-Square test. Significant differences: p≤0.05 in bold. BMI= Body Mass Index; WC= waist circumference; SBP= systolic blood pressure; DBP= diastolic blood pressure; cIMT= carotid intima-media thickness.\nTable \n3 presents the significant correlations between mean cIMT, oxLDL Ab titers and CETP and PLTP activities.\nSignificant correlations between cIMT, oxLDL Ab, CETP (%) and PLTP (%) in the presence of minor alleles\nr= Spearman´s coefficient; Significant differences: p≤0.05 in bold. cIMT= carotid intima-media thickness; oxLDL Ab= antibodies against oxidized LDL titers; (OD) = optical density; CETP(%) = activity of cholesteryl ester transfer protein; PLTP (%) = activity of phospholipid transfer protein.\nThe univariate analyses (Table \n4) indicated that the female gender, age, HDL-C, BMI and SBP were significantly associated (p value ≤0.05) with higher cIMT.\n\nUnivariate Logistic Regression analyses for higher cIMT (above 66 percentile) (\n\nn=\n\n118)\n\ncIMT below and above the 66th percentile, <0.795 (n=76) and >0.795 (n=42), respectively. (ref).=reference level. (cont).= continue variable. OR= Odds ratio for higher cIMT, above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for the Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism, V = minor allele of I405V polymorphism, HDL-C = high-density lipoprotein cholesterol, BMI: body mass index, SBP: systolic blood pressure.\nThe multivariate analysis (Table \n5) demonstrated that age and the presence of the B2 allele were positively related to higher cIMT. Individuals with increased risk exhibited the B2 allele (5.1-fold increase) and higher age (21.2% increase/each year of age).\n\nMultivariate Logistic Regression* analyses for higher cIMT (above 66\n\nth\n\npercentile) (\n\nn=\n\n114)\n\n*Stepwise criteria for selection of variables. cIMT below and above 66th percentile, <0.795 (n=73) and >0.795 (n=41), respectively. (ref).= reference level; (cont).=continue variable. OR= Odds ratio for higher cIMT above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism.\nA Chi-Square analysis indicated an interaction between the presence of the B2 allele and age. The combination between age, categorized in ≥ and <50 years (median value), and the presence or absence B2 allele formed 4 groups of individuals who were crossed with cIMTs above and below 0.795 mm (data not shown)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved."},"other_sections_titles":{"kind":"list like","value":["Background","Study subjects","Biochemical analysis","Polymorphism detection","Measurement of cIMT","Statistical analysis","Abbreviations","Misc","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Study subjects\",\n \"Biochemical analysis\",\n \"Polymorphism detection\",\n \"Measurement of cIMT\",\n \"Statistical analysis\",\n \"Abbreviations\",\n \"Misc\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.","A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].","The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].","Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.","A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.","Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].","CETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.","Eliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.","The authors state that they have no conflicts of interest.","ESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript."],"string":"[\n \"Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\\n[3].\\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\\n[5], and their association with subclinical CVD are less well characterized.\\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.\",\n \"A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\",\n \"The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\",\n \"Genomic DNA was extracted\\n[11] and amplified using polymerase chain reaction to identify the Taq1B\\n[12] and I405V\\n[13] polymorphisms.\",\n \"A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\",\n \"Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\\n[15].\",\n \"CETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.\",\n \"Eliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.\",\n \"The authors state that they have no conflicts of interest.\",\n \"ESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study subjects","Biochemical analysis","Polymorphism detection","Measurement of cIMT","Statistical analysis","Results","Discussion","Conclusions","Abbreviations","Misc","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study subjects\",\n \"Biochemical analysis\",\n \"Polymorphism detection\",\n \"Measurement of cIMT\",\n \"Statistical analysis\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Misc\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population."," Study subjects A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nA total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\n Polymorphism detection Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\nGenomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\n Measurement of cIMT A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\nA single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\n Statistical analysis Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].\nMann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].","A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].","The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].","Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.","A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.","Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].","The frequencies of I405V and Taq1B genotypes in our population sample, represented by the presence (VV, IV) and absence (II) of the V allele and the presence (B2B2, B1B2) and absence (B1B1) of the B2 allele, are presented in Table \n1. The frequencies of the II, IV and VV genotypes were 28%, 53% and 19%, respectively, and the frequencies of the B1B1, B1B2 and B2B2 were 33%, 41% and 26%, respectively.\nBiochemical parameters in the presence and absence of minor alleles\nData are presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values: Mann–Whitney U test for comparisons between the presence and absence of the minor alleles (V and B2). Significant differences: p≤0.05 in bold. CETP (%) = activity of cholesterol ester transfer protein; HDL-C = high-density lipoprotein cholesterol; PLTP (%) = activity of phospholipid transfer protein; oxLDL Ab = antibodies against oxidized LDL titers; (OD)= optical density.\nAllele frequencies were consistent with Hardy–Weinberg equilibrium for the I405V polymorphism (p=0.36) but not the Taq1B (p=0.02), and the MAFs (minor allele frequency) were <45% and <46%, respectively. The linkage disequilibrium between the two polymorphisms was very weak, as evidenced by the D`=0.17.\nClinical and anthropometric characteristics and biochemical data of the investigated population are presented in Tables \n2 and\n1.\nClinical and anthropometric characteristics in the presence and absence of minor alleles\nData presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values = Mann–Whitney U test for comparisons between the presence and absence of the alleles (V and B2); gender comparisons, Chi-Square test. Significant differences: p≤0.05 in bold. BMI= Body Mass Index; WC= waist circumference; SBP= systolic blood pressure; DBP= diastolic blood pressure; cIMT= carotid intima-media thickness.\nTable \n3 presents the significant correlations between mean cIMT, oxLDL Ab titers and CETP and PLTP activities.\nSignificant correlations between cIMT, oxLDL Ab, CETP (%) and PLTP (%) in the presence of minor alleles\nr= Spearman´s coefficient; Significant differences: p≤0.05 in bold. cIMT= carotid intima-media thickness; oxLDL Ab= antibodies against oxidized LDL titers; (OD) = optical density; CETP(%) = activity of cholesteryl ester transfer protein; PLTP (%) = activity of phospholipid transfer protein.\nThe univariate analyses (Table \n4) indicated that the female gender, age, HDL-C, BMI and SBP were significantly associated (p value ≤0.05) with higher cIMT.\n\nUnivariate Logistic Regression analyses for higher cIMT (above 66 percentile) (\n\nn=\n\n118)\n\ncIMT below and above the 66th percentile, <0.795 (n=76) and >0.795 (n=42), respectively. (ref).=reference level. (cont).= continue variable. OR= Odds ratio for higher cIMT, above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for the Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism, V = minor allele of I405V polymorphism, HDL-C = high-density lipoprotein cholesterol, BMI: body mass index, SBP: systolic blood pressure.\nThe multivariate analysis (Table \n5) demonstrated that age and the presence of the B2 allele were positively related to higher cIMT. Individuals with increased risk exhibited the B2 allele (5.1-fold increase) and higher age (21.2% increase/each year of age).\n\nMultivariate Logistic Regression* analyses for higher cIMT (above 66\n\nth\n\npercentile) (\n\nn=\n\n114)\n\n*Stepwise criteria for selection of variables. cIMT below and above 66th percentile, <0.795 (n=73) and >0.795 (n=41), respectively. (ref).= reference level; (cont).=continue variable. OR= Odds ratio for higher cIMT above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism.\nA Chi-Square analysis indicated an interaction between the presence of the B2 allele and age. The combination between age, categorized in ≥ and <50 years (median value), and the presence or absence B2 allele formed 4 groups of individuals who were crossed with cIMTs above and below 0.795 mm (data not shown).","This study investigated whether the I405V and Taq1B polymorphisms modify subclinical atherosclerosis in an asymptomatic population sample.\nPrevious studies present controversial data on the role of Taq1B and I405V in atherosclerosis and CVD. The B2 allele is associated with lower internal carotid artery IMT in men\n[16], but other studies do not identify associations between the Taq1B polymorphism and cIMT\n[17,18]. The present study demonstrated that cIMT did not differ between either allele of the polymorphisms. However, inverse correlations between cIMT and CETP activity were demonstrated in both polymorphisms (Table \n3), which suggest a potential atheroprotective role for CETP in these individuals. A potential anti-inflammatory role for CETP was demonstrated previously\n[19] wherein the induction of acute inflammation produced lower mortality due to sepsis and lower cytokine circulation in mice that express human CETP. Reductions in CETP concentrations in human patients with sepsis are more pronounced in non-survivors\n[20].\nWe identified positive correlations between cIMT and PLTP and oxLDL Ab in the presence of the V and B2 alleles. Previous studies have suggested that PLTP increases the risk of cardiovascular disease in humans\n[21], and the balance of evidence suggests a pro-atherogenic role for oxLDL Ab titers\n[22] in the development of atherosclerosis. These positive correlations and the lack of correlations of these variables in both common B1B1 and II allele homozygotes (CETP p≤0.318 and 0.091, oxLDL Ab p≤ 0.709 and 0.386 and PLTP p≤0.823 and 0.470, respectively) (data not shown) suggest a possible pro-atherogenic role of these markers associated with the polymorphisms. Our sample size was relatively small and different between groups. Therefore, further studies are underway in our lab to further confirm these correlations.\nThe presence of the V allele was associated with lower CETP (24%) and PLTP (29%) activities and higher HDL2-C (13%) concentrations. Patients with coronary disease typically exhibit fewer large HDL particles (HDL2), and the concentration of these large particles is a better predictor of future cardiovascular events than HDL-C concentration\n[23].\nLipoprotein (a) plasma levels were also significantly increased in the presence of the B2 allele. High levels of lipoprotein (a) are significantly associated with the presence, severity and extent of carotid atherosclerosis\n[24], but this increase was not observed as a possible pro-atherogenic factor.\nNo significant differences in CETP activity and HDL-C levels were observed in Taq1B polymorphism, despite a 15.4% reduction in CETP activity in presence of the B2 allele. These results are consistent with a previous study in another Brazilian population, in which no association between Taq1B genotypes and HDL-C levels was observed\n[25].\nA univariate analysis (Table \n4) followed by a multivariate logistic stepwise analysis (Table \n5) demonstrated that the presence of the B2 allele was independently associated with a mean 5.1-fold increased risk for higher cIMT and age interacted with the allele effect. The presence of the B2 allele in the ≥ 50 years age group exhibited the highest risk of higher cIMT in the Chi-Square analysis (data not shown).\nThe effects of the Taq1B variant on circulating plasma lipids may be related to other functional mutations within the CETP gene locus that are in LD\n[26], which may explain the association in our study. However, other factors than the CETP activity may exert different atherosclerotic repercussions in Taq1B and I405V polymorphisms. A multivariate analysis was performed to verify the independence of the associations between the qualitative binary variable cIMT above or below 66th percentile (0.795mm), CETP and PLTP plasma activities and the potential confounding factors, including gender, age, BMI, SBP and HDL-C. Only age exhibited a significant statistical association (OR: 1.247, CI 95%OR: 1.129 – 1.377), which was a strong determinant of cIMT (data not shown). The absence of interactions of other tested parameters with higher cIMT in this study suggests that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.\nOur results focused on several biomarkers that were assessed for the first time in the Brazilian population, and the results were partially based on the correlations between a relatively small numbers of individuals. These results merit further exploration due to the high frequency of the investigated polymorphisms in the present Brazilian sample. Identifying the impact of these polymorphisms is an ongoing aim of our research group.","The I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved.","CETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.","Eliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.","The authors state that they have no conflicts of interest.","ESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript."],"string":"[\n \"Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\\n[3].\\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\\n[5], and their association with subclinical CVD are less well characterized.\\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.\",\n \" Study subjects A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\nA total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\n Polymorphism detection Genomic DNA was extracted\\n[11] and amplified using polymerase chain reaction to identify the Taq1B\\n[12] and I405V\\n[13] polymorphisms.\\nGenomic DNA was extracted\\n[11] and amplified using polymerase chain reaction to identify the Taq1B\\n[12] and I405V\\n[13] polymorphisms.\\n Measurement of cIMT A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\\nA single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\\n Statistical analysis Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\\n[15].\\nMann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\\n[15].\",\n \"A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\",\n \"The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\",\n \"Genomic DNA was extracted\\n[11] and amplified using polymerase chain reaction to identify the Taq1B\\n[12] and I405V\\n[13] polymorphisms.\",\n \"A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\",\n \"Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\\n[15].\",\n \"The frequencies of I405V and Taq1B genotypes in our population sample, represented by the presence (VV, IV) and absence (II) of the V allele and the presence (B2B2, B1B2) and absence (B1B1) of the B2 allele, are presented in Table \\n1. The frequencies of the II, IV and VV genotypes were 28%, 53% and 19%, respectively, and the frequencies of the B1B1, B1B2 and B2B2 were 33%, 41% and 26%, respectively.\\nBiochemical parameters in the presence and absence of minor alleles\\nData are presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values: Mann–Whitney U test for comparisons between the presence and absence of the minor alleles (V and B2). Significant differences: p≤0.05 in bold. CETP (%) = activity of cholesterol ester transfer protein; HDL-C = high-density lipoprotein cholesterol; PLTP (%) = activity of phospholipid transfer protein; oxLDL Ab = antibodies against oxidized LDL titers; (OD)= optical density.\\nAllele frequencies were consistent with Hardy–Weinberg equilibrium for the I405V polymorphism (p=0.36) but not the Taq1B (p=0.02), and the MAFs (minor allele frequency) were <45% and <46%, respectively. The linkage disequilibrium between the two polymorphisms was very weak, as evidenced by the D`=0.17.\\nClinical and anthropometric characteristics and biochemical data of the investigated population are presented in Tables \\n2 and\\n1.\\nClinical and anthropometric characteristics in the presence and absence of minor alleles\\nData presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values = Mann–Whitney U test for comparisons between the presence and absence of the alleles (V and B2); gender comparisons, Chi-Square test. Significant differences: p≤0.05 in bold. BMI= Body Mass Index; WC= waist circumference; SBP= systolic blood pressure; DBP= diastolic blood pressure; cIMT= carotid intima-media thickness.\\nTable \\n3 presents the significant correlations between mean cIMT, oxLDL Ab titers and CETP and PLTP activities.\\nSignificant correlations between cIMT, oxLDL Ab, CETP (%) and PLTP (%) in the presence of minor alleles\\nr= Spearman´s coefficient; Significant differences: p≤0.05 in bold. cIMT= carotid intima-media thickness; oxLDL Ab= antibodies against oxidized LDL titers; (OD) = optical density; CETP(%) = activity of cholesteryl ester transfer protein; PLTP (%) = activity of phospholipid transfer protein.\\nThe univariate analyses (Table \\n4) indicated that the female gender, age, HDL-C, BMI and SBP were significantly associated (p value ≤0.05) with higher cIMT.\\n\\nUnivariate Logistic Regression analyses for higher cIMT (above 66 percentile) (\\n\\nn=\\n\\n118)\\n\\ncIMT below and above the 66th percentile, <0.795 (n=76) and >0.795 (n=42), respectively. (ref).=reference level. (cont).= continue variable. OR= Odds ratio for higher cIMT, above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for the Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism, V = minor allele of I405V polymorphism, HDL-C = high-density lipoprotein cholesterol, BMI: body mass index, SBP: systolic blood pressure.\\nThe multivariate analysis (Table \\n5) demonstrated that age and the presence of the B2 allele were positively related to higher cIMT. Individuals with increased risk exhibited the B2 allele (5.1-fold increase) and higher age (21.2% increase/each year of age).\\n\\nMultivariate Logistic Regression* analyses for higher cIMT (above 66\\n\\nth\\n\\npercentile) (\\n\\nn=\\n\\n114)\\n\\n*Stepwise criteria for selection of variables. cIMT below and above 66th percentile, <0.795 (n=73) and >0.795 (n=41), respectively. (ref).= reference level; (cont).=continue variable. OR= Odds ratio for higher cIMT above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism.\\nA Chi-Square analysis indicated an interaction between the presence of the B2 allele and age. The combination between age, categorized in ≥ and <50 years (median value), and the presence or absence B2 allele formed 4 groups of individuals who were crossed with cIMTs above and below 0.795 mm (data not shown).\",\n \"This study investigated whether the I405V and Taq1B polymorphisms modify subclinical atherosclerosis in an asymptomatic population sample.\\nPrevious studies present controversial data on the role of Taq1B and I405V in atherosclerosis and CVD. The B2 allele is associated with lower internal carotid artery IMT in men\\n[16], but other studies do not identify associations between the Taq1B polymorphism and cIMT\\n[17,18]. The present study demonstrated that cIMT did not differ between either allele of the polymorphisms. However, inverse correlations between cIMT and CETP activity were demonstrated in both polymorphisms (Table \\n3), which suggest a potential atheroprotective role for CETP in these individuals. A potential anti-inflammatory role for CETP was demonstrated previously\\n[19] wherein the induction of acute inflammation produced lower mortality due to sepsis and lower cytokine circulation in mice that express human CETP. Reductions in CETP concentrations in human patients with sepsis are more pronounced in non-survivors\\n[20].\\nWe identified positive correlations between cIMT and PLTP and oxLDL Ab in the presence of the V and B2 alleles. Previous studies have suggested that PLTP increases the risk of cardiovascular disease in humans\\n[21], and the balance of evidence suggests a pro-atherogenic role for oxLDL Ab titers\\n[22] in the development of atherosclerosis. These positive correlations and the lack of correlations of these variables in both common B1B1 and II allele homozygotes (CETP p≤0.318 and 0.091, oxLDL Ab p≤ 0.709 and 0.386 and PLTP p≤0.823 and 0.470, respectively) (data not shown) suggest a possible pro-atherogenic role of these markers associated with the polymorphisms. Our sample size was relatively small and different between groups. Therefore, further studies are underway in our lab to further confirm these correlations.\\nThe presence of the V allele was associated with lower CETP (24%) and PLTP (29%) activities and higher HDL2-C (13%) concentrations. Patients with coronary disease typically exhibit fewer large HDL particles (HDL2), and the concentration of these large particles is a better predictor of future cardiovascular events than HDL-C concentration\\n[23].\\nLipoprotein (a) plasma levels were also significantly increased in the presence of the B2 allele. High levels of lipoprotein (a) are significantly associated with the presence, severity and extent of carotid atherosclerosis\\n[24], but this increase was not observed as a possible pro-atherogenic factor.\\nNo significant differences in CETP activity and HDL-C levels were observed in Taq1B polymorphism, despite a 15.4% reduction in CETP activity in presence of the B2 allele. These results are consistent with a previous study in another Brazilian population, in which no association between Taq1B genotypes and HDL-C levels was observed\\n[25].\\nA univariate analysis (Table \\n4) followed by a multivariate logistic stepwise analysis (Table \\n5) demonstrated that the presence of the B2 allele was independently associated with a mean 5.1-fold increased risk for higher cIMT and age interacted with the allele effect. The presence of the B2 allele in the ≥ 50 years age group exhibited the highest risk of higher cIMT in the Chi-Square analysis (data not shown).\\nThe effects of the Taq1B variant on circulating plasma lipids may be related to other functional mutations within the CETP gene locus that are in LD\\n[26], which may explain the association in our study. However, other factors than the CETP activity may exert different atherosclerotic repercussions in Taq1B and I405V polymorphisms. A multivariate analysis was performed to verify the independence of the associations between the qualitative binary variable cIMT above or below 66th percentile (0.795mm), CETP and PLTP plasma activities and the potential confounding factors, including gender, age, BMI, SBP and HDL-C. Only age exhibited a significant statistical association (OR: 1.247, CI 95%OR: 1.129 – 1.377), which was a strong determinant of cIMT (data not shown). The absence of interactions of other tested parameters with higher cIMT in this study suggests that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.\\nOur results focused on several biomarkers that were assessed for the first time in the Brazilian population, and the results were partially based on the correlations between a relatively small numbers of individuals. These results merit further exploration due to the high frequency of the investigated polymorphisms in the present Brazilian sample. Identifying the impact of these polymorphisms is an ongoing aim of our research group.\",\n \"The I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved.\",\n \"CETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.\",\n \"Eliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.\",\n \"The authors state that they have no conflicts of interest.\",\n \"ESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,"results","discussion","conclusions",null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Carotid atherosclerosis","Carotid intima-media thickness","Cholesteryl ester transfer protein","Genetic polymorphism"],"string":"[\n \"Carotid atherosclerosis\",\n \"Carotid intima-media thickness\",\n \"Cholesteryl ester transfer protein\",\n \"Genetic polymorphism\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nAtherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.\n\nMethods:\n Study subjects A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nA total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\n Polymorphism detection Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\nGenomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\n Measurement of cIMT A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\nA single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\n Statistical analysis Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].\nMann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].\n\nStudy subjects:\nA total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\n\nBiochemical analysis:\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\n\nPolymorphism detection:\nGenomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\n\nMeasurement of cIMT:\nA single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\n\nStatistical analysis:\nMann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].\n\nResults:\nThe frequencies of I405V and Taq1B genotypes in our population sample, represented by the presence (VV, IV) and absence (II) of the V allele and the presence (B2B2, B1B2) and absence (B1B1) of the B2 allele, are presented in Table \n1. The frequencies of the II, IV and VV genotypes were 28%, 53% and 19%, respectively, and the frequencies of the B1B1, B1B2 and B2B2 were 33%, 41% and 26%, respectively.\nBiochemical parameters in the presence and absence of minor alleles\nData are presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values: Mann–Whitney U test for comparisons between the presence and absence of the minor alleles (V and B2). Significant differences: p≤0.05 in bold. CETP (%) = activity of cholesterol ester transfer protein; HDL-C = high-density lipoprotein cholesterol; PLTP (%) = activity of phospholipid transfer protein; oxLDL Ab = antibodies against oxidized LDL titers; (OD)= optical density.\nAllele frequencies were consistent with Hardy–Weinberg equilibrium for the I405V polymorphism (p=0.36) but not the Taq1B (p=0.02), and the MAFs (minor allele frequency) were <45% and <46%, respectively. The linkage disequilibrium between the two polymorphisms was very weak, as evidenced by the D`=0.17.\nClinical and anthropometric characteristics and biochemical data of the investigated population are presented in Tables \n2 and\n1.\nClinical and anthropometric characteristics in the presence and absence of minor alleles\nData presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values = Mann–Whitney U test for comparisons between the presence and absence of the alleles (V and B2); gender comparisons, Chi-Square test. Significant differences: p≤0.05 in bold. BMI= Body Mass Index; WC= waist circumference; SBP= systolic blood pressure; DBP= diastolic blood pressure; cIMT= carotid intima-media thickness.\nTable \n3 presents the significant correlations between mean cIMT, oxLDL Ab titers and CETP and PLTP activities.\nSignificant correlations between cIMT, oxLDL Ab, CETP (%) and PLTP (%) in the presence of minor alleles\nr= Spearman´s coefficient; Significant differences: p≤0.05 in bold. cIMT= carotid intima-media thickness; oxLDL Ab= antibodies against oxidized LDL titers; (OD) = optical density; CETP(%) = activity of cholesteryl ester transfer protein; PLTP (%) = activity of phospholipid transfer protein.\nThe univariate analyses (Table \n4) indicated that the female gender, age, HDL-C, BMI and SBP were significantly associated (p value ≤0.05) with higher cIMT.\n\nUnivariate Logistic Regression analyses for higher cIMT (above 66 percentile) (\n\nn=\n\n118)\n\ncIMT below and above the 66th percentile, <0.795 (n=76) and >0.795 (n=42), respectively. (ref).=reference level. (cont).= continue variable. OR= Odds ratio for higher cIMT, above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for the Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism, V = minor allele of I405V polymorphism, HDL-C = high-density lipoprotein cholesterol, BMI: body mass index, SBP: systolic blood pressure.\nThe multivariate analysis (Table \n5) demonstrated that age and the presence of the B2 allele were positively related to higher cIMT. Individuals with increased risk exhibited the B2 allele (5.1-fold increase) and higher age (21.2% increase/each year of age).\n\nMultivariate Logistic Regression* analyses for higher cIMT (above 66\n\nth\n\npercentile) (\n\nn=\n\n114)\n\n*Stepwise criteria for selection of variables. cIMT below and above 66th percentile, <0.795 (n=73) and >0.795 (n=41), respectively. (ref).= reference level; (cont).=continue variable. OR= Odds ratio for higher cIMT above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism.\nA Chi-Square analysis indicated an interaction between the presence of the B2 allele and age. The combination between age, categorized in ≥ and <50 years (median value), and the presence or absence B2 allele formed 4 groups of individuals who were crossed with cIMTs above and below 0.795 mm (data not shown).\n\nDiscussion:\nThis study investigated whether the I405V and Taq1B polymorphisms modify subclinical atherosclerosis in an asymptomatic population sample.\nPrevious studies present controversial data on the role of Taq1B and I405V in atherosclerosis and CVD. The B2 allele is associated with lower internal carotid artery IMT in men\n[16], but other studies do not identify associations between the Taq1B polymorphism and cIMT\n[17,18]. The present study demonstrated that cIMT did not differ between either allele of the polymorphisms. However, inverse correlations between cIMT and CETP activity were demonstrated in both polymorphisms (Table \n3), which suggest a potential atheroprotective role for CETP in these individuals. A potential anti-inflammatory role for CETP was demonstrated previously\n[19] wherein the induction of acute inflammation produced lower mortality due to sepsis and lower cytokine circulation in mice that express human CETP. Reductions in CETP concentrations in human patients with sepsis are more pronounced in non-survivors\n[20].\nWe identified positive correlations between cIMT and PLTP and oxLDL Ab in the presence of the V and B2 alleles. Previous studies have suggested that PLTP increases the risk of cardiovascular disease in humans\n[21], and the balance of evidence suggests a pro-atherogenic role for oxLDL Ab titers\n[22] in the development of atherosclerosis. These positive correlations and the lack of correlations of these variables in both common B1B1 and II allele homozygotes (CETP p≤0.318 and 0.091, oxLDL Ab p≤ 0.709 and 0.386 and PLTP p≤0.823 and 0.470, respectively) (data not shown) suggest a possible pro-atherogenic role of these markers associated with the polymorphisms. Our sample size was relatively small and different between groups. Therefore, further studies are underway in our lab to further confirm these correlations.\nThe presence of the V allele was associated with lower CETP (24%) and PLTP (29%) activities and higher HDL2-C (13%) concentrations. Patients with coronary disease typically exhibit fewer large HDL particles (HDL2), and the concentration of these large particles is a better predictor of future cardiovascular events than HDL-C concentration\n[23].\nLipoprotein (a) plasma levels were also significantly increased in the presence of the B2 allele. High levels of lipoprotein (a) are significantly associated with the presence, severity and extent of carotid atherosclerosis\n[24], but this increase was not observed as a possible pro-atherogenic factor.\nNo significant differences in CETP activity and HDL-C levels were observed in Taq1B polymorphism, despite a 15.4% reduction in CETP activity in presence of the B2 allele. These results are consistent with a previous study in another Brazilian population, in which no association between Taq1B genotypes and HDL-C levels was observed\n[25].\nA univariate analysis (Table \n4) followed by a multivariate logistic stepwise analysis (Table \n5) demonstrated that the presence of the B2 allele was independently associated with a mean 5.1-fold increased risk for higher cIMT and age interacted with the allele effect. The presence of the B2 allele in the ≥ 50 years age group exhibited the highest risk of higher cIMT in the Chi-Square analysis (data not shown).\nThe effects of the Taq1B variant on circulating plasma lipids may be related to other functional mutations within the CETP gene locus that are in LD\n[26], which may explain the association in our study. However, other factors than the CETP activity may exert different atherosclerotic repercussions in Taq1B and I405V polymorphisms. A multivariate analysis was performed to verify the independence of the associations between the qualitative binary variable cIMT above or below 66th percentile (0.795mm), CETP and PLTP plasma activities and the potential confounding factors, including gender, age, BMI, SBP and HDL-C. Only age exhibited a significant statistical association (OR: 1.247, CI 95%OR: 1.129 – 1.377), which was a strong determinant of cIMT (data not shown). The absence of interactions of other tested parameters with higher cIMT in this study suggests that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.\nOur results focused on several biomarkers that were assessed for the first time in the Brazilian population, and the results were partially based on the correlations between a relatively small numbers of individuals. These results merit further exploration due to the high frequency of the investigated polymorphisms in the present Brazilian sample. Identifying the impact of these polymorphisms is an ongoing aim of our research group.\n\nConclusions:\nThe I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved.\n\nAbbreviations:\nCETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.\n\nMisc:\nEliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.\n\nCompeting interests:\nThe authors state that they have no conflicts of interest.\n\nAuthors’ contributions:\nESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nCholesteryl ester transfer protein (CETP) plays a major role in lipid metabolism, but studies on the association of CETP polymorphisms with risks of cardiovascular disease are inconsistent. This study investigated whether the CETP gene I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian population sample.\n\nMethods:\nThe polymorphisms were analyzed using polymerase chain reaction in 207 adult volunteers. Serum lipid profiles, oxLDL Ab titers, C-reactive protein and tumor necrosis factor-α concentrations and CETP and phospholipid transfer protein (PLTP) activities were determined, and common carotid artery intima-media thickness (cIMT) was measured using ultrasonography.\n\nResults:\nNo differences in cIMT were observed between the presence or absence of the minor B2 and V alleles in either polymorphism. However, inverse correlations between mean cIMT and CETP activity in the presence of these polymorphisms were observed, and positive correlations of these polymorphisms with PLTP activity and oxLDL Ab titers were identified. Moreover, logistic multivariate analysis revealed that the presence of the B2 allele was associated with a 5.1-fold (CI 95%, OR: 1.26 - 21.06) increased risk for cIMT, which was equal and above the 66th percentile and positively interacted with age. However, no associations with the V allele or CETP and PLTP activities were observed.\n\nConclusions:\nNone of the studied parameters, including CETP activity, explained the different relationships between these polymorphisms and cIMT, suggesting that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease."},"background_conclusion_text":{"kind":"string","value":"Background:\nAtherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.\n\nConclusions:\nThe I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nCholesteryl ester transfer protein (CETP) plays a major role in lipid metabolism, but studies on the association of CETP polymorphisms with risks of cardiovascular disease are inconsistent. This study investigated whether the CETP gene I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian population sample.\n\nMethods:\nThe polymorphisms were analyzed using polymerase chain reaction in 207 adult volunteers. Serum lipid profiles, oxLDL Ab titers, C-reactive protein and tumor necrosis factor-α concentrations and CETP and phospholipid transfer protein (PLTP) activities were determined, and common carotid artery intima-media thickness (cIMT) was measured using ultrasonography.\n\nResults:\nNo differences in cIMT were observed between the presence or absence of the minor B2 and V alleles in either polymorphism. However, inverse correlations between mean cIMT and CETP activity in the presence of these polymorphisms were observed, and positive correlations of these polymorphisms with PLTP activity and oxLDL Ab titers were identified. Moreover, logistic multivariate analysis revealed that the presence of the B2 allele was associated with a 5.1-fold (CI 95%, OR: 1.26 - 21.06) increased risk for cIMT, which was equal and above the 66th percentile and positively interacted with age. However, no associations with the V allele or CETP and PLTP activities were observed.\n\nConclusions:\nNone of the studied parameters, including CETP activity, explained the different relationships between these polymorphisms and cIMT, suggesting that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease."},"whole_article_text_length":{"kind":"number","value":5263,"string":"5,263"},"whole_article_abstract_length":{"kind":"number","value":290,"string":"290"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["cimt","allele","hdl","cetp","performed","tg","protein","cholesterol","b2","presence"],"string":"[\n \"cimt\",\n \"allele\",\n \"hdl\",\n \"cetp\",\n \"performed\",\n \"tg\",\n \"protein\",\n \"cholesterol\",\n \"b2\",\n \"presence\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] polymorphisms | cholesterol | association | atherosclerosis | subclinical | disease | cvd | i405v | taq1b | cetp [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] tg | performed | usa | mg dl | dl | plus | diagnostics | pap | obtained | nas [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | presence | significant | absence | 05 | minor | 795 | b2 | significant differences 05 bold [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] allele | factors | cimt | studied | associated | b2 allele | polymorphisms | b2 | explained different | explained different relationships [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | polymorphisms | performed | cholesterol | protein | tg | cetp | b2 | taq1b [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | polymorphisms | performed | cholesterol | protein | tg | cetp | b2 | taq1b [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| CETP | Brazilian [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 207 ||| Ab | CETP | PLTP [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| CETP | PLTP ||| ||| 5.1-fold | CI | 95% | 1.26 - 21.06 | 66th ||| CETP | PLTP [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] CETP [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Cholesteryl ||| CETP | Brazilian ||| 207 ||| Ab | CETP | PLTP ||| ||| CETP | PLTP ||| ||| 5.1-fold | CI | 95% | 1.26 - 21.06 | 66th ||| CETP | PLTP ||| CETP [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Cholesteryl ||| CETP | Brazilian ||| 207 ||| Ab | CETP | PLTP ||| ||| CETP | PLTP ||| ||| 5.1-fold | CI | 95% | 1.26 - 21.06 | 66th ||| CETP | PLTP ||| CETP [SUMMARY]"}}},{"rowIdx":298,"cells":{"title":{"kind":"string","value":"Subjective Evaluation of the Results of Injectable Hyaluronic Acid Fillers for the Face."},"pmid":{"kind":"string","value":"32021131"},"background_abstract":{"kind":"string","value":"Skin ageing is a physiological process, progressive and irreversible. Hyaluronic acid injection treatments are used to correct the signs of skin ageing."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Hyaluronic acid was implanted in the area of the cheek and the forehead aesthetic units in 57 women, aged 35-55 years. Apart from the clinical observation, self-assessment of the therapeutic results was conducted. The \"My skin\" questionnaire was used for subjective evaluation of the treatment results."},"methods_abstract_label":{"kind":"string","value":"MATERIAL AND METHODS"},"results_abstract":{"kind":"string","value":"Mean wrinkle score in the pre-menopausal group changed after the treatment, from 3.2±0.6 to 1.1±0.3 and from 3.2±0.6 to 0.8±0.6 for the forehead and the cheek esthetic units, respectively. In the post-menopausal group, the score decreased from 3.8±0.4 to 1.7± 0.7 and from 3.2±0.617 to 0.8± 0.6 for the forehead and the cheek esthetic units, respectively. The changes were age-dependent. Improved appearance of the facial skin - higher satisfaction with skin tone and scent - was reported after hyaluronic acid injections. Higher subjective perception of improvement corresponded to older age, irrespectively of the menopausal status. Correlations between age and the effect of the treatment on maintaining proper skin hydration as well as between improved appearance of the forehead area and feelings of autonomy and well-being were found."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Hyaluronic acid injections significantly improved the subjective perception and overall assessment of the scent and appearance of the facial skin."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Cosmetic Techniques","Face","Female","Humans","Hyaluronic Acid","Injections","Middle Aged","Patient Satisfaction","Rejuvenation","Skin Aging","Treatment Outcome","Viscoelastic Substances"],"string":"[\n \"Adult\",\n \"Cosmetic Techniques\",\n \"Face\",\n \"Female\",\n \"Humans\",\n \"Hyaluronic Acid\",\n \"Injections\",\n \"Middle Aged\",\n \"Patient Satisfaction\",\n \"Rejuvenation\",\n \"Skin Aging\",\n \"Treatment Outcome\",\n \"Viscoelastic Substances\"\n]"},"pmcid":{"kind":"string","value":"6968800"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"The skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.\nSkin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being."},"other_sections_titles":{"kind":"list like","value":["Objectives","Materials and Methods","Results","Discussion","Conclusions"],"string":"[\n \"Objectives\",\n \"Materials and Methods\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\"\n]"},"other_sections_texts":{"kind":"list like","value":["The aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.","A total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).","Mean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\n\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.","The condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47","HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being."],"string":"[\n \"The aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.\",\n \"A total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).\",\n \"Mean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\\n\\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.\",\n \"The condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47\",\n \"HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Objectives","Materials and Methods","Results","Discussion","Conclusions"],"string":"[\n \"Introduction\",\n \"Objectives\",\n \"Materials and Methods\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["The skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.\nSkin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.","The aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.","A total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).","Mean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\n\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.","The condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47","HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being."],"string":"[\n \"The skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.\\nSkin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.\",\n \"The aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.\",\n \"A total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).\",\n \"Mean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\\n\\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.\",\n \"The condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47\",\n \"HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,null,null,null,null],"string":"[\n \"intro\",\n null,\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["hyaluronic acid fillers","skin","women","subjective evaluation"],"string":"[\n \"hyaluronic acid fillers\",\n \"skin\",\n \"women\",\n \"subjective evaluation\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nThe skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.\nSkin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.\n\nObjectives:\nThe aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.\n\nMaterials and Methods:\nA total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).\n\nResults:\nMean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\n\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.\n\nDiscussion:\nThe condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47\n\nConclusions:\nHA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nSkin ageing is a physiological process, progressive and irreversible. Hyaluronic acid injection treatments are used to correct the signs of skin ageing.\n\nMethods:\nHyaluronic acid was implanted in the area of the cheek and the forehead aesthetic units in 57 women, aged 35-55 years. Apart from the clinical observation, self-assessment of the therapeutic results was conducted. The \"My skin\" questionnaire was used for subjective evaluation of the treatment results.\n\nResults:\nMean wrinkle score in the pre-menopausal group changed after the treatment, from 3.2±0.6 to 1.1±0.3 and from 3.2±0.6 to 0.8±0.6 for the forehead and the cheek esthetic units, respectively. In the post-menopausal group, the score decreased from 3.8±0.4 to 1.7± 0.7 and from 3.2±0.617 to 0.8± 0.6 for the forehead and the cheek esthetic units, respectively. The changes were age-dependent. Improved appearance of the facial skin - higher satisfaction with skin tone and scent - was reported after hyaluronic acid injections. Higher subjective perception of improvement corresponded to older age, irrespectively of the menopausal status. Correlations between age and the effect of the treatment on maintaining proper skin hydration as well as between improved appearance of the forehead area and feelings of autonomy and well-being were found.\n\nConclusions:\nHyaluronic acid injections significantly improved the subjective perception and overall assessment of the scent and appearance of the facial skin."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nThe skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.\nSkin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.\n\nConclusions:\nHA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nSkin ageing is a physiological process, progressive and irreversible. Hyaluronic acid injection treatments are used to correct the signs of skin ageing.\n\nMethods:\nHyaluronic acid was implanted in the area of the cheek and the forehead aesthetic units in 57 women, aged 35-55 years. Apart from the clinical observation, self-assessment of the therapeutic results was conducted. The \"My skin\" questionnaire was used for subjective evaluation of the treatment results.\n\nResults:\nMean wrinkle score in the pre-menopausal group changed after the treatment, from 3.2±0.6 to 1.1±0.3 and from 3.2±0.6 to 0.8±0.6 for the forehead and the cheek esthetic units, respectively. In the post-menopausal group, the score decreased from 3.8±0.4 to 1.7± 0.7 and from 3.2±0.617 to 0.8± 0.6 for the forehead and the cheek esthetic units, respectively. The changes were age-dependent. Improved appearance of the facial skin - higher satisfaction with skin tone and scent - was reported after hyaluronic acid injections. Higher subjective perception of improvement corresponded to older age, irrespectively of the menopausal status. Correlations between age and the effect of the treatment on maintaining proper skin hydration as well as between improved appearance of the forehead area and feelings of autonomy and well-being were found.\n\nConclusions:\nHyaluronic acid injections significantly improved the subjective perception and overall assessment of the scent and appearance of the facial skin."},"whole_article_text_length":{"kind":"number","value":3976,"string":"3,976"},"whole_article_abstract_length":{"kind":"number","value":264,"string":"264"},"num_sections":{"kind":"number","value":6,"string":"6"},"most_frequent_words":{"kind":"list like","value":["skin","appearance","treatment","age","menopausal","changes","ha","facial","skin appearance","significant"],"string":"[\n \"skin\",\n \"appearance\",\n \"treatment\",\n \"age\",\n \"menopausal\",\n \"changes\",\n \"ha\",\n \"facial\",\n \"skin appearance\",\n \"significant\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | changes | effectiveness | indicative | biophysical | body | number | associated | physiological | treatments [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ha injections connected | injections connected | evaluation | ha | positive | self | injections | evaluation skin | ha injections | connected [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | treatment | appearance | ha | age | menopausal | evaluation | changes | facial | haf [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | treatment | appearance | ha | age | menopausal | evaluation | changes | facial | haf [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| 57 | 35-55 years ||| ||| ||| 3.2±0.6 | 1.1±0.3 | 3.2±0.6 ||| 3.8±0.4 | 0.7 | 0.8± 0.6 ||| ||| ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| 57 | 35-55 years ||| ||| ||| 3.2±0.6 | 1.1±0.3 | 3.2±0.6 ||| 3.8±0.4 | 0.7 | 0.8± 0.6 ||| ||| ||| ||| ||| [SUMMARY]"}}},{"rowIdx":299,"cells":{"title":{"kind":"string","value":"Function and regulation annotation of up-regulated long non-coding RNA LINC01234 in gastric cancer."},"pmid":{"kind":"string","value":"32011780"},"background_abstract":{"kind":"string","value":"Accumulated evidences indicate that long non-coding RNAs (lncRNAs) participate in many biological mechanisms. Moreover, it acts as an essential regulator in various human diseases such as gastric cancer (GC). Nevertheless, the comprehensive regulatory roles and clinical significance of most lncRNAs in GC are not fully understood."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"In this research, our aim was to investigate the underlying mechanism of lncRNA LINC01234 in GC. Firstly, the usage of qRT-PCR helped to establish expression pattern of LINC01234 in GC tissues. Following this, appropriate statistical tests were applied to analyze the relation between expression level and clinicopathological factors. Ultimately, potential functions and regulatory network of LINC01234 were concluded via GSEA and a series of bioinformatics tools or databases, respectively."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Consequently, at the end of research we found LINC01234 is up-regulated in GC tissues in comparison with adjacent normal tissues. Furthermore, its expression level is correlated with differentiation of patients with GC. It is also important to highlight bioinformatics analysis revealed that LINC01234 is involved in cancer-associated pathways such as cell cycle and mismatch repair. Also, regulatory network of LINC01234 presented a probability in the involvement of tumorigenesis through regulating cancer-associated genes."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Overall, our results suggested that LINC01234 may play a crucial role in GC."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adenocarcinoma","Aged","Biomarkers, Tumor","Female","Gene Expression Regulation, Neoplastic","Gene Regulatory Networks","Humans","Male","MicroRNAs","Middle Aged","RNA, Long Noncoding","Stomach Neoplasms","Up-Regulation"],"string":"[\n \"Adenocarcinoma\",\n \"Aged\",\n \"Biomarkers, Tumor\",\n \"Female\",\n \"Gene Expression Regulation, Neoplastic\",\n \"Gene Regulatory Networks\",\n \"Humans\",\n \"Male\",\n \"MicroRNAs\",\n \"Middle Aged\",\n \"RNA, Long Noncoding\",\n \"Stomach Neoplasms\",\n \"Up-Regulation\"\n]"},"pmcid":{"kind":"string","value":"7246363"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":" Experimental verification of LINC01234 up‐regulation in GC tissues Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\nFirstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\n Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\nIn the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\n Potential functions of LINC01234\n To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\nTo explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\n Regulatory network of LINC01234\n LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nLncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Differently expression analysis of lncRNAs in STAD from TCGA","Collection of GC samples and patients' clinical information","Total RNA extraction and qRT‐PCR","Gene set enrichment analysis of LINC01234\n","Construction of LINC01234 regulatory network","TF‐LINC01234 regulation","miRNA‐LINC01234 interactions","RBP‐LINC01234 interactions","Statistical analysis","Experimental verification of LINC01234 up‐regulation in GC tissues","Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients","Potential functions of LINC01234\n","Regulatory network of LINC01234\n","TF‐LINC01234 regulation","miRNA‐LINC01234 regulation","RBP‐LINC01234 regulation"],"string":"[\n \"INTRODUCTION\",\n \"Differently expression analysis of lncRNAs in STAD from TCGA\",\n \"Collection of GC samples and patients' clinical information\",\n \"Total RNA extraction and qRT‐PCR\",\n \"Gene set enrichment analysis of LINC01234\\n\",\n \"Construction of LINC01234 regulatory network\",\n \"TF‐LINC01234 regulation\",\n \"miRNA‐LINC01234 interactions\",\n \"RBP‐LINC01234 interactions\",\n \"Statistical analysis\",\n \"Experimental verification of LINC01234 up‐regulation in GC tissues\",\n \"Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients\",\n \"Potential functions of LINC01234\\n\",\n \"Regulatory network of LINC01234\\n\",\n \"TF‐LINC01234 regulation\",\n \"miRNA‐LINC01234 regulation\",\n \"RBP‐LINC01234 regulation\"\n]"},"other_sections_texts":{"kind":"list like","value":["Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.","Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.","Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.","The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.","By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant."," TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.","We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.","The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n","Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.","IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.","Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues","In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR","To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)","LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).","Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).","A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n","A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4)."],"string":"[\n \"Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.\",\n \"Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\",\n \"Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\",\n \"The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\",\n \"By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\",\n \" TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\",\n \"We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\",\n \"The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\",\n \"Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\",\n \"IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\",\n \"Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\",\n \"In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\\nAbbreviation: SE, standard error.\\nThe sample size is so small that the result may be unbelievable even though P < .05.\\n\\nP < .05.\\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\",\n \"To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\",\n \"LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\",\n \"Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\",\n \"A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\",\n \"A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Differently expression analysis of lncRNAs in STAD from TCGA","Collection of GC samples and patients' clinical information","Total RNA extraction and qRT‐PCR","Gene set enrichment analysis of LINC01234\n","Construction of LINC01234 regulatory network","TF‐LINC01234 regulation","miRNA‐LINC01234 interactions","RBP‐LINC01234 interactions","Statistical analysis","RESULTS","Experimental verification of LINC01234 up‐regulation in GC tissues","Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients","Potential functions of LINC01234\n","Regulatory network of LINC01234\n","TF‐LINC01234 regulation","miRNA‐LINC01234 regulation","RBP‐LINC01234 regulation","DISCUSSION","Supporting information"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Differently expression analysis of lncRNAs in STAD from TCGA\",\n \"Collection of GC samples and patients' clinical information\",\n \"Total RNA extraction and qRT‐PCR\",\n \"Gene set enrichment analysis of LINC01234\\n\",\n \"Construction of LINC01234 regulatory network\",\n \"TF‐LINC01234 regulation\",\n \"miRNA‐LINC01234 interactions\",\n \"RBP‐LINC01234 interactions\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Experimental verification of LINC01234 up‐regulation in GC tissues\",\n \"Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients\",\n \"Potential functions of LINC01234\\n\",\n \"Regulatory network of LINC01234\\n\",\n \"TF‐LINC01234 regulation\",\n \"miRNA‐LINC01234 regulation\",\n \"RBP‐LINC01234 regulation\",\n \"DISCUSSION\",\n \"Supporting information\"\n]"},"all_sections_texts":{"kind":"list like","value":["Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC."," Differently expression analysis of lncRNAs in STAD from TCGA Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\nFragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\n Collection of GC samples and patients' clinical information Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\nPaired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\n Total RNA extraction and qRT‐PCR The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\nThe methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\n Gene set enrichment analysis of LINC01234\n By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\nBy using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\n Construction of LINC01234 regulatory network TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n Statistical analysis IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\nIBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.","Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.","Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.","The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.","By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant."," TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.","We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.","The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n","Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.","IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances."," Experimental verification of LINC01234 up‐regulation in GC tissues Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\nFirstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\n Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\nIn the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\n Potential functions of LINC01234\n To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\nTo explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\n Regulatory network of LINC01234\n LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nLncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).","Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues","In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR","To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)","LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).","Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).","A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n","A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).","LncRNA is a type of non‐coding RNAs without the ability of encoding proteins; despite this, it has a regulating gene expression at chromatin modification, transcriptional, or post‐transcriptional levels.35 In addition, the polymorphisms in lncRNAs could be a risk of disease or cancer.36 In fact, an increasing number of lncRNAs have been identified to be related to numerous kinds of diseases,37 including GC. For example, through the expression analysis of metabolic pathway‐related lncRNAs and protein‐coding genes, a dozen of lncRNAs were functionally annotated and discovered to be important in GC.38\nDGCR9, another lncRNA up‐regulated in GC, was shown to promote the tumorigenesis of GC.\n39 Some of lncRNAs were even indeed considered as biomarkers for diagnosis or prognosis of GC. For instance, a metabolism‐related lncRNA, RP11‐555H23.1, was found to be a potential diagnostic biomarker in GC.40 Also, the expression level of H19 in plasma could be served as a biomarker for patients with GC.41 Due to the regulatory role of lncRNAs, exploring new lncRNA biomarkers can help to explain the initiation and progression mechanism of GC. Nevertheless, still there are many unknown lncRNAs in GC nowadays.\nThe previous study found LINC01234 is highly expressed in esophageal squamous cell carcinoma (ESCC). Also, it is one of the three lncRNAs that can be a signature to predict the survival time of ESCC patients accurately.42, 43, 44 Besides, it was likewise discovered to be up‐regulated in the GC in prior study.26 However, the functions and regulatory role of LINC01234 need to be studied.\nIn this research, we identified that LINC01234 was also highly expressed in GC tissues compared with adjacent non‐cancerous tissues. Later, we explored the associations between the expression level of LINC01234 and clinical features through which we found LINC01234 was correlated with differentiation of GC. LncRNAs usually interact with other kinds of molecules to involve in multiple biological processes. For example, the binding of lncRNA OLC8 and IL‐11 will impair the degradation of IL‐11 mRNAs to accelerate GC development.45 Besides, combination of lncRNA and its target may increase the diagnostic value of lncRNA. Just as it was found by previous report that the combined use of RP11‐19P22.6‐001 and its target NOS2 may be useful to diagnose patients with GC.46 Thus, we further explored the potential functions and regulatory network of LINC01234. We identified 218 relationships of LINC01234 in total. Among them, 17 associations including two pairs of TF regulation and 17 pairs of RBP interaction were found to be co‐expressed. One of 2 TFs, ELK1, is an important regulator and known to activate many lncRNAs including TRPM2‐AS, MIR100HG, and HOXA10‐AS in cancer until now,47, 48, 49 indicating ELK1 may induce expression of LINC01234 to promote tumor progression in GC too.\nIn conclusion, the results of this present study indicated that LINC01234 expression is linked with the diagnostics of patients with GC and similarly may be involved in differentiation in GC through cell cycle or other cancer‐related pathways."," \nClick here for additional data file.\n \nClick here for additional data file.\n \nClick here for additional data file.\n \nClick here for additional data file."],"string":"[\n \"Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.\",\n \" Differently expression analysis of lncRNAs in STAD from TCGA Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\\nFragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\\n Collection of GC samples and patients' clinical information Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\\nPaired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\\n Total RNA extraction and qRT‐PCR The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\\nThe methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\\n Gene set enrichment analysis of LINC01234\\n By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\\nBy using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\\n Construction of LINC01234 regulatory network TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\n TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\n Statistical analysis IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\\nIBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\",\n \"Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\",\n \"Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\",\n \"The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\",\n \"By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\",\n \" TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\",\n \"We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\",\n \"The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\",\n \"Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\",\n \"IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\",\n \" Experimental verification of LINC01234 up‐regulation in GC tissues Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\\nFirstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\\n Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\\nAbbreviation: SE, standard error.\\nThe sample size is so small that the result may be unbelievable even though P < .05.\\n\\nP < .05.\\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\\nIn the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\\nAbbreviation: SE, standard error.\\nThe sample size is so small that the result may be unbelievable even though P < .05.\\n\\nP < .05.\\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\\n Potential functions of LINC01234\\n To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\\nTo explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\\n Regulatory network of LINC01234\\n LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\\nLncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\",\n \"Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\",\n \"In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\\nAbbreviation: SE, standard error.\\nThe sample size is so small that the result may be unbelievable even though P < .05.\\n\\nP < .05.\\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\",\n \"To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\",\n \"LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\",\n \"Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\",\n \"A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\",\n \"A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\",\n \"LncRNA is a type of non‐coding RNAs without the ability of encoding proteins; despite this, it has a regulating gene expression at chromatin modification, transcriptional, or post‐transcriptional levels.35 In addition, the polymorphisms in lncRNAs could be a risk of disease or cancer.36 In fact, an increasing number of lncRNAs have been identified to be related to numerous kinds of diseases,37 including GC. For example, through the expression analysis of metabolic pathway‐related lncRNAs and protein‐coding genes, a dozen of lncRNAs were functionally annotated and discovered to be important in GC.38\\nDGCR9, another lncRNA up‐regulated in GC, was shown to promote the tumorigenesis of GC.\\n39 Some of lncRNAs were even indeed considered as biomarkers for diagnosis or prognosis of GC. For instance, a metabolism‐related lncRNA, RP11‐555H23.1, was found to be a potential diagnostic biomarker in GC.40 Also, the expression level of H19 in plasma could be served as a biomarker for patients with GC.41 Due to the regulatory role of lncRNAs, exploring new lncRNA biomarkers can help to explain the initiation and progression mechanism of GC. Nevertheless, still there are many unknown lncRNAs in GC nowadays.\\nThe previous study found LINC01234 is highly expressed in esophageal squamous cell carcinoma (ESCC). Also, it is one of the three lncRNAs that can be a signature to predict the survival time of ESCC patients accurately.42, 43, 44 Besides, it was likewise discovered to be up‐regulated in the GC in prior study.26 However, the functions and regulatory role of LINC01234 need to be studied.\\nIn this research, we identified that LINC01234 was also highly expressed in GC tissues compared with adjacent non‐cancerous tissues. Later, we explored the associations between the expression level of LINC01234 and clinical features through which we found LINC01234 was correlated with differentiation of GC. LncRNAs usually interact with other kinds of molecules to involve in multiple biological processes. For example, the binding of lncRNA OLC8 and IL‐11 will impair the degradation of IL‐11 mRNAs to accelerate GC development.45 Besides, combination of lncRNA and its target may increase the diagnostic value of lncRNA. Just as it was found by previous report that the combined use of RP11‐19P22.6‐001 and its target NOS2 may be useful to diagnose patients with GC.46 Thus, we further explored the potential functions and regulatory network of LINC01234. We identified 218 relationships of LINC01234 in total. Among them, 17 associations including two pairs of TF regulation and 17 pairs of RBP interaction were found to be co‐expressed. One of 2 TFs, ELK1, is an important regulator and known to activate many lncRNAs including TRPM2‐AS, MIR100HG, and HOXA10‐AS in cancer until now,47, 48, 49 indicating ELK1 may induce expression of LINC01234 to promote tumor progression in GC too.\\nIn conclusion, the results of this present study indicated that LINC01234 expression is linked with the diagnostics of patients with GC and similarly may be involved in differentiation in GC through cell cycle or other cancer‐related pathways.\",\n \" \\nClick here for additional data file.\\n \\nClick here for additional data file.\\n \\nClick here for additional data file.\\n \\nClick here for additional data file.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"materials-and-methods",null,null,null,null,null,null,null,null,null,"results",null,null,null,null,null,null,null,"discussion","supplementary-material"],"string":"[\n null,\n \"materials-and-methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["gastric cancer","\nLINC01234\n","long non‐coding RNA","regulatory network"],"string":"[\n \"gastric cancer\",\n \"\\nLINC01234\\n\",\n \"long non‐coding RNA\",\n \"regulatory network\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nGastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.\n\nMATERIALS AND METHODS:\n Differently expression analysis of lncRNAs in STAD from TCGA Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\nFragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\n Collection of GC samples and patients' clinical information Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\nPaired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\n Total RNA extraction and qRT‐PCR The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\nThe methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\n Gene set enrichment analysis of LINC01234\n By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\nBy using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\n Construction of LINC01234 regulatory network TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n Statistical analysis IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\nIBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\n\nDifferently expression analysis of lncRNAs in STAD from TCGA:\nFragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\n\nCollection of GC samples and patients' clinical information:\nPaired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\n\nTotal RNA extraction and qRT‐PCR:\nThe methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\n\nGene set enrichment analysis of LINC01234\n:\nBy using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\n\nConstruction of LINC01234 regulatory network:\n TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n\nTF‐LINC01234 regulation:\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n\nmiRNA‐LINC01234 interactions:\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n\nRBP‐LINC01234 interactions:\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n\nStatistical analysis:\nIBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\n\nRESULTS:\n Experimental verification of LINC01234 up‐regulation in GC tissues Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\nFirstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\n Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\nIn the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\n Potential functions of LINC01234\n To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\nTo explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\n Regulatory network of LINC01234\n LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nLncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\n\nExperimental verification of LINC01234 up‐regulation in GC tissues:\nFirstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\n\nAssociation analysis between expression level of LINC01234 and clinicopathological factors in GC patients:\nIn the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\n\nPotential functions of LINC01234\n:\nTo explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\n\nRegulatory network of LINC01234\n:\nLncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\n\nTF‐LINC01234 regulation:\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n\nmiRNA‐LINC01234 regulation:\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n\nRBP‐LINC01234 regulation:\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\n\nDISCUSSION:\nLncRNA is a type of non‐coding RNAs without the ability of encoding proteins; despite this, it has a regulating gene expression at chromatin modification, transcriptional, or post‐transcriptional levels.35 In addition, the polymorphisms in lncRNAs could be a risk of disease or cancer.36 In fact, an increasing number of lncRNAs have been identified to be related to numerous kinds of diseases,37 including GC. For example, through the expression analysis of metabolic pathway‐related lncRNAs and protein‐coding genes, a dozen of lncRNAs were functionally annotated and discovered to be important in GC.38\nDGCR9, another lncRNA up‐regulated in GC, was shown to promote the tumorigenesis of GC.\n39 Some of lncRNAs were even indeed considered as biomarkers for diagnosis or prognosis of GC. For instance, a metabolism‐related lncRNA, RP11‐555H23.1, was found to be a potential diagnostic biomarker in GC.40 Also, the expression level of H19 in plasma could be served as a biomarker for patients with GC.41 Due to the regulatory role of lncRNAs, exploring new lncRNA biomarkers can help to explain the initiation and progression mechanism of GC. Nevertheless, still there are many unknown lncRNAs in GC nowadays.\nThe previous study found LINC01234 is highly expressed in esophageal squamous cell carcinoma (ESCC). Also, it is one of the three lncRNAs that can be a signature to predict the survival time of ESCC patients accurately.42, 43, 44 Besides, it was likewise discovered to be up‐regulated in the GC in prior study.26 However, the functions and regulatory role of LINC01234 need to be studied.\nIn this research, we identified that LINC01234 was also highly expressed in GC tissues compared with adjacent non‐cancerous tissues. Later, we explored the associations between the expression level of LINC01234 and clinical features through which we found LINC01234 was correlated with differentiation of GC. LncRNAs usually interact with other kinds of molecules to involve in multiple biological processes. For example, the binding of lncRNA OLC8 and IL‐11 will impair the degradation of IL‐11 mRNAs to accelerate GC development.45 Besides, combination of lncRNA and its target may increase the diagnostic value of lncRNA. Just as it was found by previous report that the combined use of RP11‐19P22.6‐001 and its target NOS2 may be useful to diagnose patients with GC.46 Thus, we further explored the potential functions and regulatory network of LINC01234. We identified 218 relationships of LINC01234 in total. Among them, 17 associations including two pairs of TF regulation and 17 pairs of RBP interaction were found to be co‐expressed. One of 2 TFs, ELK1, is an important regulator and known to activate many lncRNAs including TRPM2‐AS, MIR100HG, and HOXA10‐AS in cancer until now,47, 48, 49 indicating ELK1 may induce expression of LINC01234 to promote tumor progression in GC too.\nIn conclusion, the results of this present study indicated that LINC01234 expression is linked with the diagnostics of patients with GC and similarly may be involved in differentiation in GC through cell cycle or other cancer‐related pathways.\n\nSupporting information:\n \nClick here for additional data file.\n \nClick here for additional data file.\n \nClick here for additional data file.\n \nClick here for additional data file.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nAccumulated evidences indicate that long non-coding RNAs (lncRNAs) participate in many biological mechanisms. Moreover, it acts as an essential regulator in various human diseases such as gastric cancer (GC). Nevertheless, the comprehensive regulatory roles and clinical significance of most lncRNAs in GC are not fully understood.\n\nMethods:\nIn this research, our aim was to investigate the underlying mechanism of lncRNA LINC01234 in GC. Firstly, the usage of qRT-PCR helped to establish expression pattern of LINC01234 in GC tissues. Following this, appropriate statistical tests were applied to analyze the relation between expression level and clinicopathological factors. Ultimately, potential functions and regulatory network of LINC01234 were concluded via GSEA and a series of bioinformatics tools or databases, respectively.\n\nResults:\nConsequently, at the end of research we found LINC01234 is up-regulated in GC tissues in comparison with adjacent normal tissues. Furthermore, its expression level is correlated with differentiation of patients with GC. It is also important to highlight bioinformatics analysis revealed that LINC01234 is involved in cancer-associated pathways such as cell cycle and mismatch repair. Also, regulatory network of LINC01234 presented a probability in the involvement of tumorigenesis through regulating cancer-associated genes.\n\nConclusions:\nOverall, our results suggested that LINC01234 may play a crucial role in GC."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":9795,"string":"9,795"},"whole_article_abstract_length":{"kind":"number","value":252,"string":"252"},"num_sections":{"kind":"number","value":21,"string":"21"},"most_frequent_words":{"kind":"list like","value":["gc","linc01234","expression","tissues","regulation","cell","stad","lncrnas","patients","cancer"],"string":"[\n \"gc\",\n \"linc01234\",\n \"expression\",\n \"tissues\",\n \"regulation\",\n \"cell\",\n \"stad\",\n \"lncrnas\",\n \"patients\",\n \"cancer\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] gastric cancer | \nLINC01234\n | long non‐coding RNA | regulatory network [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] gastric cancer | \nLINC01234\n | long non‐coding RNA | regulatory network [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] gastric cancer | \nLINC01234\n | long non‐coding RNA | regulatory network [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Aged | Biomarkers, Tumor | Female | Gene Expression Regulation, Neoplastic | Gene Regulatory Networks | Humans | Male | MicroRNAs | Middle Aged | RNA, Long Noncoding | Stomach Neoplasms | Up-Regulation [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Aged | Biomarkers, Tumor | Female | Gene Expression Regulation, Neoplastic | Gene Regulatory Networks | Humans | Male | MicroRNAs | Middle Aged | RNA, Long Noncoding | Stomach Neoplasms | Up-Regulation [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Aged | Biomarkers, Tumor | Female | Gene Expression Regulation, Neoplastic | Gene Regulatory Networks | Humans | Male | MicroRNAs | Middle Aged | RNA, Long Noncoding | Stomach Neoplasms | Up-Regulation [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] gc | linc01234 | expression | tissues | regulation | cell | stad | lncrnas | patients | cancer [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] gc | linc01234 | expression | tissues | regulation | cell | stad | lncrnas | patients | cancer [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] gc | linc01234 | expression | tissues | regulation | cell | stad | lncrnas | patients | cancer [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] gc | lncrnas | transcriptome | gene | rna | kinds | patients | cancerous tissues | tissues | non [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] gc | linc01234 | expression | cell | tissues | figure | regulation | related | proliferation | mir [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] gc | linc01234 | expression | tissues | cell | lncrnas | regulation | set | 05 | stad [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| GC ||| GC [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] GC ||| GC ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| GC ||| GC ||| GC ||| Firstly | GC ||| ||| ||| ||| GC ||| GC ||| ||| ||| GC [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}}],"truncated":false,"partial":false},"paginationData":{"pageIndex":2,"numItemsPerPage":100,"numTotalItems":8129,"offset":200,"length":100}},"jwt":"eyJhbGciOiJFZERTQSJ9.eyJyZWFkIjp0cnVlLCJwZXJtaXNzaW9ucyI6eyJyZXBvLmNvbnRlbnQucmVhZCI6dHJ1ZX0sImlhdCI6MTc1NDAyNTYxMCwic3ViIjoiL2RhdGFzZXRzL2FjbWMvYmVhbWl0LWFubm90YXRlZC1mdWxsLXRleHRzLWRhdGFzZXQiLCJleHAiOjE3NTQwMjkyMTAsImlzcyI6Imh0dHBzOi8vaHVnZ2luZ2ZhY2UuY28ifQ.yKk_OF86ndoke-zh5__bQ3wJjB7osLEclUEUEBQtFFLHhTy-i9LBnDUheFRBiVTMv-iKUfmm2MXuRSi7Vt9xBQ","displayUrls":true},"discussionsStats":{"closed":6,"open":11,"total":17},"fullWidth":true,"hasGatedAccess":true,"hasFullAccess":true,"isEmbedded":false,"savedQueries":{"community":[],"user":[]}}">
."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Cross-Sectional Studies","Female","Follow-Up Studies","Health Knowledge, Attitudes, Practice","Humans","India","Middle Aged","Patient Acceptance of Health Care","Prognosis","Retrospective Studies","Self-Assessment","Surveys and Questionnaires","Time Factors","Time-to-Treatment","Uterine Cervical Neoplasms"],"string":"[\n \"Cross-Sectional Studies\",\n \"Female\",\n \"Follow-Up Studies\",\n \"Health Knowledge, Attitudes, Practice\",\n \"Humans\",\n \"India\",\n \"Middle Aged\",\n \"Patient Acceptance of Health Care\",\n \"Prognosis\",\n \"Retrospective Studies\",\n \"Self-Assessment\",\n \"Surveys and Questionnaires\",\n \"Time Factors\",\n \"Time-to-Treatment\",\n \"Uterine Cervical Neoplasms\"\n]"},"pmcid":{"kind":"string","value":"8033105"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Globally, an estimated 570,000 new cases and 311,000 deaths has been reported due to cervical cancer in 2018 and it is the fourth most common cancer in women. The Incidence and mortality rank second behind breast cancer in lower human development index (HDI) settings. Eight out of 10 women diagnosed and nine of 10 women who die from cervical cancer live in a low- or middle-income countries (Bray et al., 2018) “ Cervical cancer continues to be a major public health problem affecting middle-aged women, particularly in less-resourced countries” (Arbyn et al., 2020). India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one third of global cervical cancer deaths during the year 2018 (“Globocan 2018,” 2018). Cervical cancer is the leading cause of cancer mortality in India which accounts for 17% of all cancer deaths among women aged 30 to 69 years.(Bobdey et al., 2016, “The Challenge Ahead,” 2014) One third of cervical cancer cases was reported among women aged 50 -59 years ( 27.4%) (Bobdey et al., 2016). The higher mortality and morbidity due to cancer is because most patients seek/get treatment at advanced stage and therefore have a higher risk of dying from the disease (Bray et al., 2018).\nCervical cancer is a preventable disease primarily because of its long lead time (women develop precancerous lesions years before it becomes cancerous). Cervical cancer can be detected in the pre-cancerous phase by screening and thereby the development of cancer prevented. Cervical cancer can also be treated successfully if diagnosed at an early stage. Routine screening by visual inspection of cervix through ‘See-and-treat’ approach among the female population has been suggested for early diagnosis and control of cervical cancer (Sankaranarayanan, 2012). Periodic Pap or HPV samples are difficult to organize in low- or middle-income countries like India and therefore large-scale routine screening would be difficult and not affordable (“HTA_CaCx Screening in India.pdf,” n.d.) Millions of cancer patients could be saved from premature death and disability, if they had timely access to early detection and treatment (“WHO,” n.d.). Studies have shown that it is possible to reduce the need of aggressive treatment of cancer is detected at an early stage. A long interval between start of disease symptoms to having a definite diagnosis is highly critical and associated with low survival among cervical cancer patients.(Chen et al., 2019) \nIn India, the present existing public health care delivery system is a three-level system. The primary level is primary health centres (PHCs). In India, PHCs focuses on eight elements of primary health care outlined in the Alma-Ata declaration and provides mainly outpatient services. In the secondary level the district hospitals provide outpatient, inpatient and emergency services. At the tertiary level, medical college hospitals, apex institutions and regional cancer centres provide specialized services. Under the programme of “National programme for prevention and control of cancer, diabetes, cardiovascular diseases and stroke (NPCDCS)”, cancer screening is done at the primary level by visual inspection of the cervix with acetic acid (VIA) and those who are suspected for cancer are referred to secondary or tertiary care level for further management. In addition, a health programme for non-communicable diseases is carried out with a cancer care component implemented at all the health care levels (Gulia et al., 2016) \nThis study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking for care and different barriers for the possible time lag in seeking care."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The distribution of socio-demographic and clinical data of the 210 cervical cancer study subjects are presented along with data pertaining to the cases not enrolled for analysis (Table 1). Among 210 enrolled patients, the majority 106 (50.5%) of women were between the age 46 to 59 years where in not enrolled patients 89 (40.4%) were of same age group. It was noted 167 (63.0%) and 179 (81.4%) were not literate among enrolled and respectively. Among enrolled patients, 150 (71.4%) were from rural areas and 158 (71.4%) were financially dependent for livelihood on the family. Only 19 women (9.0%) had any health insurance policy. The majority of patients 197 (93.8%) were not aware of symptoms of cervical cancer and only 7 (3.3%) patients had a family history of cancer.\nOf the symptoms which led for seeking medical care, the majority of women, 172 (81.9%), indicated lower abdominal pain, and 137 (65.2%) of the respondents experienced abnormal vaginal discharge/bleeding. Among all patients, 106 (50.5%) were diagnosed at an early stage of the disease (stages I, IIA and IIB).\nThe median time interval between self-detection of cervical cancer symptoms and first contact with general physician (T1) was 80 [IQR 45-150] days, while 74 patients (35.2%) had a time lag of 90 days or more. The median time taken for reaching diagnosis after the first contact (T2) was 25 [IQR 15 - 45] days. The overall median time between self-detection of symptoms to initiation of primary treatment (T5) was 123[IQR 83-205] days. The majority of patients, 145 (66.2%), had initiation of primary treatment only after 90 days from self-detection of cervical cancer symptoms (Table 2). \nFactors associated with delay in seeking medical care of 90 days and more (patient delay) were analyzed in relation to socio-demographic characteristics and clinical symptoms (Table 3). In multivariate analysis after adjusting to all the variables in the study the odds of patient delay in the age group ≥ 60 years was 3.2 times higher compared to patients in the age group below 45 years (95% CI: 1.2 – 8.4). The other factors which were associated with patient delay were being not literate (OR 2.3, 95% CI: 1.0 – 5.4), from rural area (OR = 2.3, 95% CI: 1.1 – 4.9), being financially dependent for livelihood (OR = 4.5, 95% CI: 1.9 – 10.6) and not having any abnormal vaginal discharge / bleeding (OR = 2.4, 95% CI: 1.3 – 4.6). Family history of cancer, presence of any health insurance, knowledge regarding cancer cure by early seeking for care, knowledge of symptoms of cervical cancer and having lower abdominal pain was not statistically related to delay in seeking medical care. \nInformation was sought regarding perceived reasons from 197 (93.8%) patients who did not seek care within one week from self-detection of cancer symptoms. The major reasons (table 4) were lack of awareness in identifying cervical cancer symptoms in 183 (92.9%) women and 120 (60.9%) patients assuming that the symptom would resolve by itself. The other common reasons were absence of pain, changes in the body attributed to common illness and vague symptoms. \nThe most common reason for delay in seeking for definite diagnosis at tertiary care hospitals was due to non-affordability 82 (41.6%). The other common reasons were visiting multiple medical practitioners before presenting at tertiary care 60 (30.1%) and not suspected for cancer by physician at first contact. \nThe delay in seeking for treatment after diagnosis at tertiary care was due to long treatment procedure 57 (27.1%), fear of treatment 50 (23.8%), financial dependence on the family 48 (22.9%), fear of disfigurement of the body 26 (12.4%) and stigma attached with the disease (Table 4).\nDistribution of Socio-Demographic, Clinical and Other Variables Among the Subject Enrolled and Not Enrolled to the Study\nDistribution of Time Interval Between Self-Detection of Cancer Symptoms to Various Stages (T1 To T5) for Seeking Treatment\nFactors Associated with Delay in Seeking Medical Care: Univariate and Multivariate Logistic Regression Results\nDistribution of Perceived Reasons for Delay in Seeking Medical Care at Various Stages (T1 – T4)\n*, Responses were elicited from those who had not sought specific care for more than one week from the onset of symptoms; #, Responses was elicited from those who had not sought treatment on specified time by doctor \nRecruitment of Participants for the Study"},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":[],"string":"[]"},"other_sections_texts":{"kind":"list like","value":[],"string":"[]"},"other_sections_sec_types":{"kind":"list like","value":[],"string":"[]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and Methods","Results","Discussion"],"string":"[\n \"Introduction\",\n \"Materials and Methods\",\n \"Results\",\n \"Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Globally, an estimated 570,000 new cases and 311,000 deaths has been reported due to cervical cancer in 2018 and it is the fourth most common cancer in women. The Incidence and mortality rank second behind breast cancer in lower human development index (HDI) settings. Eight out of 10 women diagnosed and nine of 10 women who die from cervical cancer live in a low- or middle-income countries (Bray et al., 2018) “ Cervical cancer continues to be a major public health problem affecting middle-aged women, particularly in less-resourced countries” (Arbyn et al., 2020). India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one third of global cervical cancer deaths during the year 2018 (“Globocan 2018,” 2018). Cervical cancer is the leading cause of cancer mortality in India which accounts for 17% of all cancer deaths among women aged 30 to 69 years.(Bobdey et al., 2016, “The Challenge Ahead,” 2014) One third of cervical cancer cases was reported among women aged 50 -59 years ( 27.4%) (Bobdey et al., 2016). The higher mortality and morbidity due to cancer is because most patients seek/get treatment at advanced stage and therefore have a higher risk of dying from the disease (Bray et al., 2018).\nCervical cancer is a preventable disease primarily because of its long lead time (women develop precancerous lesions years before it becomes cancerous). Cervical cancer can be detected in the pre-cancerous phase by screening and thereby the development of cancer prevented. Cervical cancer can also be treated successfully if diagnosed at an early stage. Routine screening by visual inspection of cervix through ‘See-and-treat’ approach among the female population has been suggested for early diagnosis and control of cervical cancer (Sankaranarayanan, 2012). Periodic Pap or HPV samples are difficult to organize in low- or middle-income countries like India and therefore large-scale routine screening would be difficult and not affordable (“HTA_CaCx Screening in India.pdf,” n.d.) Millions of cancer patients could be saved from premature death and disability, if they had timely access to early detection and treatment (“WHO,” n.d.). Studies have shown that it is possible to reduce the need of aggressive treatment of cancer is detected at an early stage. A long interval between start of disease symptoms to having a definite diagnosis is highly critical and associated with low survival among cervical cancer patients.(Chen et al., 2019) \nIn India, the present existing public health care delivery system is a three-level system. The primary level is primary health centres (PHCs). In India, PHCs focuses on eight elements of primary health care outlined in the Alma-Ata declaration and provides mainly outpatient services. In the secondary level the district hospitals provide outpatient, inpatient and emergency services. At the tertiary level, medical college hospitals, apex institutions and regional cancer centres provide specialized services. Under the programme of “National programme for prevention and control of cancer, diabetes, cardiovascular diseases and stroke (NPCDCS)”, cancer screening is done at the primary level by visual inspection of the cervix with acetic acid (VIA) and those who are suspected for cancer are referred to secondary or tertiary care level for further management. In addition, a health programme for non-communicable diseases is carried out with a cancer care component implemented at all the health care levels (Gulia et al., 2016) \nThis study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking for care and different barriers for the possible time lag in seeking care.","The data was collected from a regional cancer centre from south India, Bengaluru, which has a population-based cancer registry (PBCR) and a hospital-based cancer registry (HBCR). This tertiary care centre has state of art facilities for diagnosis, treatment and research. Cervical cancer cases with histological confirmation were recruited from the hospital-based cancer registry of this Institution during the time period of April 2017 to September 2017 using cross sectional study design. We identified 430 cervical cancer patients during the period from April 2017 to September 2017. Of these, 52 were ineligible due to referral to terminal/palliative care. Of 378 eligible women, 96 were not included for the analysis as patients moved to different hospital or not being available for interview, and 38 were not willing to participate in the study. In the 244 patients who gave their consent, 34 patient’s had incomplete information in the records. Finally, 210 patients were included for the present study (Figure 1) \nThe cervical cancers were mostly squamous cell carcinomas (SCC) and few adenocarcinomas (AC). They were divided in two stage based on FIGO classification (Bhatla et al., 2019). Stage I and II were categorized as early stage; III and IV were considered as late stage of cervical cancer. \nPatient delay in seeking for medical care refers to a long interval between detecting of first/initial symptoms and seeking for appointment with a general physician. The patients were further divided into two groups, those who sought medical care within 90 days and those who sought care 90 days and above (patient delay) after self-detection of the first symptoms of cervical cancer. \nSample size was calculated based on the study conducted by Rudd et al., (2017) where 39.3% patients had delayed diagnosis from the onset of self-detection of cervical cancer symptoms. In this study the sample size was estimated for patient related delay, from symptoms to first contact with a general practitioner, as it was our major concern. To get an absolute precision of 6.6% with 95% confidence level, the study required a minimum sample of 210 subjects. Based on hospital record collection, it was noted that six months’ time period was sufficient to achieve the required sample size. \nExpert opinion from oncologists, biostatisticians and epidemiologists were obtained using the Delphi technique (Skulmoski et al., 2007). After obtaining the written informed consent, patients were interviewed in person using the questionnaire and their medical records was used to collect the baseline information as well as key components of the time interval between self-detection of cervical cancer symptom to seeking medical care. More details of the collection of data and the different components of time intervals are described elsewhere.(Somanna et al., 2020)\nIndependent variables included socio-demographic characteristics, such as age at diagnosis, availability of health insurance, place of residence (rural/urban), history of cancer literacy, and financially dependent for livelihood and clinical variables, such as personal history of cancer, and family history of cancer.\nThe time interval between various stages was measured in days as reported by the patients and by review of hospital case sheets as indicated in earlier study.(Somanna et al., 2018) \na) Time interval between self-detection of symptoms and the first presentation at general practitioner (T1). \nb) Time interval from referral by the general practitioner to attending tertiary care centre (T2). \nc) Time interval from first consultation at tertiary care for diagnosis to definite diagnosis date (T3). \nd) Time interval from definite diagnosis to initiation of primary treatment (T4). \ne) Total time elapsed from self-detection of symptoms to initiation of primary treatment (T5).\n\nStatistical methods\n\nDescriptive statistics of time intervals were summarized with median and inter-quartile range as data were not normally distributed (Kolmogrov-Smirnov and Shapiro test). Univariate odds ratios (OR) with 95% confidence intervals (CI) were estimated between the factors and patient delay. The patient delay was categorised into < 90 days and ≥ 90 days. Considering ≥ 90 days as patient delay, the association of various factors were estimated considering the low risk group as the reference. Variables showing a univariate association with patient delay (at P < 0.20) were included in multivariate unconditional logistic stepwise forward regression model to adjust for confounders. The analysis was carried out using SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago. \nEthical approval was sought from the ethical review committee of the study Institution. ","The distribution of socio-demographic and clinical data of the 210 cervical cancer study subjects are presented along with data pertaining to the cases not enrolled for analysis (Table 1). Among 210 enrolled patients, the majority 106 (50.5%) of women were between the age 46 to 59 years where in not enrolled patients 89 (40.4%) were of same age group. It was noted 167 (63.0%) and 179 (81.4%) were not literate among enrolled and respectively. Among enrolled patients, 150 (71.4%) were from rural areas and 158 (71.4%) were financially dependent for livelihood on the family. Only 19 women (9.0%) had any health insurance policy. The majority of patients 197 (93.8%) were not aware of symptoms of cervical cancer and only 7 (3.3%) patients had a family history of cancer.\nOf the symptoms which led for seeking medical care, the majority of women, 172 (81.9%), indicated lower abdominal pain, and 137 (65.2%) of the respondents experienced abnormal vaginal discharge/bleeding. Among all patients, 106 (50.5%) were diagnosed at an early stage of the disease (stages I, IIA and IIB).\nThe median time interval between self-detection of cervical cancer symptoms and first contact with general physician (T1) was 80 [IQR 45-150] days, while 74 patients (35.2%) had a time lag of 90 days or more. The median time taken for reaching diagnosis after the first contact (T2) was 25 [IQR 15 - 45] days. The overall median time between self-detection of symptoms to initiation of primary treatment (T5) was 123[IQR 83-205] days. The majority of patients, 145 (66.2%), had initiation of primary treatment only after 90 days from self-detection of cervical cancer symptoms (Table 2). \nFactors associated with delay in seeking medical care of 90 days and more (patient delay) were analyzed in relation to socio-demographic characteristics and clinical symptoms (Table 3). In multivariate analysis after adjusting to all the variables in the study the odds of patient delay in the age group ≥ 60 years was 3.2 times higher compared to patients in the age group below 45 years (95% CI: 1.2 – 8.4). The other factors which were associated with patient delay were being not literate (OR 2.3, 95% CI: 1.0 – 5.4), from rural area (OR = 2.3, 95% CI: 1.1 – 4.9), being financially dependent for livelihood (OR = 4.5, 95% CI: 1.9 – 10.6) and not having any abnormal vaginal discharge / bleeding (OR = 2.4, 95% CI: 1.3 – 4.6). Family history of cancer, presence of any health insurance, knowledge regarding cancer cure by early seeking for care, knowledge of symptoms of cervical cancer and having lower abdominal pain was not statistically related to delay in seeking medical care. \nInformation was sought regarding perceived reasons from 197 (93.8%) patients who did not seek care within one week from self-detection of cancer symptoms. The major reasons (table 4) were lack of awareness in identifying cervical cancer symptoms in 183 (92.9%) women and 120 (60.9%) patients assuming that the symptom would resolve by itself. The other common reasons were absence of pain, changes in the body attributed to common illness and vague symptoms. \nThe most common reason for delay in seeking for definite diagnosis at tertiary care hospitals was due to non-affordability 82 (41.6%). The other common reasons were visiting multiple medical practitioners before presenting at tertiary care 60 (30.1%) and not suspected for cancer by physician at first contact. \nThe delay in seeking for treatment after diagnosis at tertiary care was due to long treatment procedure 57 (27.1%), fear of treatment 50 (23.8%), financial dependence on the family 48 (22.9%), fear of disfigurement of the body 26 (12.4%) and stigma attached with the disease (Table 4).\nDistribution of Socio-Demographic, Clinical and Other Variables Among the Subject Enrolled and Not Enrolled to the Study\nDistribution of Time Interval Between Self-Detection of Cancer Symptoms to Various Stages (T1 To T5) for Seeking Treatment\nFactors Associated with Delay in Seeking Medical Care: Univariate and Multivariate Logistic Regression Results\nDistribution of Perceived Reasons for Delay in Seeking Medical Care at Various Stages (T1 – T4)\n*, Responses were elicited from those who had not sought specific care for more than one week from the onset of symptoms; #, Responses was elicited from those who had not sought treatment on specified time by doctor \nRecruitment of Participants for the Study","The study centre was a regional cancer care centre having both population and hospital-based cancer registry and where a majority of cervix cancer patients from the entire state seek cancer care. The median age in enrolled and not enrolled patients was 50 and 56 years, respectively. Among all the patients, the proportion of not literate patients was higher (81%) in not enrolled patients compared to enrolled patients (63%). The study by Deshmukh and Rathod, (2017) observed that the average age of the patients was 57 years and 81% were not literate. Our result concur with the study by Panda et al., 2019 where the average age of the study participants was 52.1 ± 10.9 years. \nThe overall median time between the self-detection of cervical cancer symptoms and initiation of primary treatment was 123[IQR 83-205] days and the median time taken for first contact with general physician from self-detection of symptoms was 80 [IQR 45-150] days. Overall, 66% women delayed (≥ 90 days) for initiation of primary treatment from the onset of symptoms which is potentially concerning, because longer duration between diagnosis and treatment is related with higher risk of death. Among patients who started their treatment after an interval of >180 days, the risk of death was reported to be 36% higher compared to those who started treatment within 6 months from diagnosis (Chen et al., 2019) \nThe present study assessed the time interval between self-detection of symptoms to treatment of cervical cancer and patient delay. The median time interval between self-detection of symptoms and the first presentation at general practitioner (T1) was 80 days with 35.4% of the patients reporting 90 days or more. The present study observation was consistent with a study conducted in India on patients attending tertiary care centre, showing that 39% of patients had a diagnostic delay (Panda et al., 2019) The median time between symptoms and presenting at health care provider was 69 days from two tertiary care centres in Nepal (Gyenwali et al., 2014). It was noted from the study by Lim et al., (2014) that only 28% of patients had a delay of more than 3 months in all national health service (NHS) hospitals diagnosing cervical cancer in England. There was a mean delay of 23.1 weeks between onset of symptoms from the study in tertiary care centre of Sothern Malawi (Rudd et al., 2017). Results from our study were in line with studies from low- or middle-income countries but differed from studies in developed countries. This difference may be partially due to regular screening programs in many developed countries and differing rates of literacy (IARC, 2005). \nOverall median time from self-detection of symptoms to initiation of primary treatment (T5) was 123 days in the current study. Another study by Henriques, (2016) in India showed on an average six months duration from first symptom to cervical cancer treatment. Such a long time may be explained by the fact that lack of awareness of patients pertaining to cancer symptoms was high (93%) in present study. In another study, women who delayed, had mistaken the symptoms of cervix cancer as symptoms of sexually transmitted diseases (STD) and were scared to report (Deshmukh and Rathod, 2017). This shows that the lack of awareness of cervix cancer symptoms is a major factor and warrants improvement in public awareness. Our study results concur closely with other studies from similar settings and in low- or middle-income countries (Benemariya et al., 2018; Gyenwali et al., 2014).\nThe patients who were aged 60 years and above, residing in rural areas, not literate, financially dependent for livelihood and did not had abnormal vaginal discharge/ bleeding were independently associated with patient delay. Similar results were observed in a study from Nepal where not literate was found to be independently associated with late diagnosis and women having abnormal vaginal bleeding as symptom was protective factor for late diagnosis (Gyenwali et al., 2013). Previous studies from low- or middle-income countries also concur with our result (Dunyo et al., 2018)(Ouasmani et al., 2016)(Henriques, 2016).\nThe present study has been conducted in a population-based cancer registry (PBCR) where the data are included in International Agency for Research on Cancer (IARC) and data collection procedure involves standard quality control measures. The study included only patients who had been diagnosed with standard clinical and histopathological criteria. The information was obtained using pre-tested validated questionnaire by the investigator. Additional information pertaining to clinical evaluation and histological diagnosis were obtained from case sheets. All efforts were made to reduce the recall bias by local events calendar and standardization of terms employed in the questionnaire. \n\nLimitations\n\nPossible selection bias could have occurred as the subjects who were not enrolled for the study due to various reasons were older women and not literate compared to subjects in the study. Socio-demographic profile of population who availed the services at this centre may be different from private tertiary cancer care centre which would biases the result. The sample in study represent only those who availed the treatment at the centre and does not represent the entire population of cervical cancer patients. Thus, it may have some restrictions for external generalization. Possibility of recall bias exist as past history of cancer symptoms were assessed retrospectively. \nIn conclusion, the median time of 80 days was observed from self-detection of cervical cancer symptoms to first contact with general physician. Further, a median time of another 45 days was observed for obtaining primary treatment. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care. Interventions like raising awareness of cervical cancer symptoms and improving health seeking behavior of women should be focused."],"string":"[\n \"Globally, an estimated 570,000 new cases and 311,000 deaths has been reported due to cervical cancer in 2018 and it is the fourth most common cancer in women. The Incidence and mortality rank second behind breast cancer in lower human development index (HDI) settings. Eight out of 10 women diagnosed and nine of 10 women who die from cervical cancer live in a low- or middle-income countries (Bray et al., 2018) “ Cervical cancer continues to be a major public health problem affecting middle-aged women, particularly in less-resourced countries” (Arbyn et al., 2020). India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one third of global cervical cancer deaths during the year 2018 (“Globocan 2018,” 2018). Cervical cancer is the leading cause of cancer mortality in India which accounts for 17% of all cancer deaths among women aged 30 to 69 years.(Bobdey et al., 2016, “The Challenge Ahead,” 2014) One third of cervical cancer cases was reported among women aged 50 -59 years ( 27.4%) (Bobdey et al., 2016). The higher mortality and morbidity due to cancer is because most patients seek/get treatment at advanced stage and therefore have a higher risk of dying from the disease (Bray et al., 2018).\\nCervical cancer is a preventable disease primarily because of its long lead time (women develop precancerous lesions years before it becomes cancerous). Cervical cancer can be detected in the pre-cancerous phase by screening and thereby the development of cancer prevented. Cervical cancer can also be treated successfully if diagnosed at an early stage. Routine screening by visual inspection of cervix through ‘See-and-treat’ approach among the female population has been suggested for early diagnosis and control of cervical cancer (Sankaranarayanan, 2012). Periodic Pap or HPV samples are difficult to organize in low- or middle-income countries like India and therefore large-scale routine screening would be difficult and not affordable (“HTA_CaCx Screening in India.pdf,” n.d.) Millions of cancer patients could be saved from premature death and disability, if they had timely access to early detection and treatment (“WHO,” n.d.). Studies have shown that it is possible to reduce the need of aggressive treatment of cancer is detected at an early stage. A long interval between start of disease symptoms to having a definite diagnosis is highly critical and associated with low survival among cervical cancer patients.(Chen et al., 2019) \\nIn India, the present existing public health care delivery system is a three-level system. The primary level is primary health centres (PHCs). In India, PHCs focuses on eight elements of primary health care outlined in the Alma-Ata declaration and provides mainly outpatient services. In the secondary level the district hospitals provide outpatient, inpatient and emergency services. At the tertiary level, medical college hospitals, apex institutions and regional cancer centres provide specialized services. Under the programme of “National programme for prevention and control of cancer, diabetes, cardiovascular diseases and stroke (NPCDCS)”, cancer screening is done at the primary level by visual inspection of the cervix with acetic acid (VIA) and those who are suspected for cancer are referred to secondary or tertiary care level for further management. In addition, a health programme for non-communicable diseases is carried out with a cancer care component implemented at all the health care levels (Gulia et al., 2016) \\nThis study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking for care and different barriers for the possible time lag in seeking care.\",\n \"The data was collected from a regional cancer centre from south India, Bengaluru, which has a population-based cancer registry (PBCR) and a hospital-based cancer registry (HBCR). This tertiary care centre has state of art facilities for diagnosis, treatment and research. Cervical cancer cases with histological confirmation were recruited from the hospital-based cancer registry of this Institution during the time period of April 2017 to September 2017 using cross sectional study design. We identified 430 cervical cancer patients during the period from April 2017 to September 2017. Of these, 52 were ineligible due to referral to terminal/palliative care. Of 378 eligible women, 96 were not included for the analysis as patients moved to different hospital or not being available for interview, and 38 were not willing to participate in the study. In the 244 patients who gave their consent, 34 patient’s had incomplete information in the records. Finally, 210 patients were included for the present study (Figure 1) \\nThe cervical cancers were mostly squamous cell carcinomas (SCC) and few adenocarcinomas (AC). They were divided in two stage based on FIGO classification (Bhatla et al., 2019). Stage I and II were categorized as early stage; III and IV were considered as late stage of cervical cancer. \\nPatient delay in seeking for medical care refers to a long interval between detecting of first/initial symptoms and seeking for appointment with a general physician. The patients were further divided into two groups, those who sought medical care within 90 days and those who sought care 90 days and above (patient delay) after self-detection of the first symptoms of cervical cancer. \\nSample size was calculated based on the study conducted by Rudd et al., (2017) where 39.3% patients had delayed diagnosis from the onset of self-detection of cervical cancer symptoms. In this study the sample size was estimated for patient related delay, from symptoms to first contact with a general practitioner, as it was our major concern. To get an absolute precision of 6.6% with 95% confidence level, the study required a minimum sample of 210 subjects. Based on hospital record collection, it was noted that six months’ time period was sufficient to achieve the required sample size. \\nExpert opinion from oncologists, biostatisticians and epidemiologists were obtained using the Delphi technique (Skulmoski et al., 2007). After obtaining the written informed consent, patients were interviewed in person using the questionnaire and their medical records was used to collect the baseline information as well as key components of the time interval between self-detection of cervical cancer symptom to seeking medical care. More details of the collection of data and the different components of time intervals are described elsewhere.(Somanna et al., 2020)\\nIndependent variables included socio-demographic characteristics, such as age at diagnosis, availability of health insurance, place of residence (rural/urban), history of cancer literacy, and financially dependent for livelihood and clinical variables, such as personal history of cancer, and family history of cancer.\\nThe time interval between various stages was measured in days as reported by the patients and by review of hospital case sheets as indicated in earlier study.(Somanna et al., 2018) \\na) Time interval between self-detection of symptoms and the first presentation at general practitioner (T1). \\nb) Time interval from referral by the general practitioner to attending tertiary care centre (T2). \\nc) Time interval from first consultation at tertiary care for diagnosis to definite diagnosis date (T3). \\nd) Time interval from definite diagnosis to initiation of primary treatment (T4). \\ne) Total time elapsed from self-detection of symptoms to initiation of primary treatment (T5).\\n\\nStatistical methods\\n\\nDescriptive statistics of time intervals were summarized with median and inter-quartile range as data were not normally distributed (Kolmogrov-Smirnov and Shapiro test). Univariate odds ratios (OR) with 95% confidence intervals (CI) were estimated between the factors and patient delay. The patient delay was categorised into < 90 days and ≥ 90 days. Considering ≥ 90 days as patient delay, the association of various factors were estimated considering the low risk group as the reference. Variables showing a univariate association with patient delay (at P < 0.20) were included in multivariate unconditional logistic stepwise forward regression model to adjust for confounders. The analysis was carried out using SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago. \\nEthical approval was sought from the ethical review committee of the study Institution. \",\n \"The distribution of socio-demographic and clinical data of the 210 cervical cancer study subjects are presented along with data pertaining to the cases not enrolled for analysis (Table 1). Among 210 enrolled patients, the majority 106 (50.5%) of women were between the age 46 to 59 years where in not enrolled patients 89 (40.4%) were of same age group. It was noted 167 (63.0%) and 179 (81.4%) were not literate among enrolled and respectively. Among enrolled patients, 150 (71.4%) were from rural areas and 158 (71.4%) were financially dependent for livelihood on the family. Only 19 women (9.0%) had any health insurance policy. The majority of patients 197 (93.8%) were not aware of symptoms of cervical cancer and only 7 (3.3%) patients had a family history of cancer.\\nOf the symptoms which led for seeking medical care, the majority of women, 172 (81.9%), indicated lower abdominal pain, and 137 (65.2%) of the respondents experienced abnormal vaginal discharge/bleeding. Among all patients, 106 (50.5%) were diagnosed at an early stage of the disease (stages I, IIA and IIB).\\nThe median time interval between self-detection of cervical cancer symptoms and first contact with general physician (T1) was 80 [IQR 45-150] days, while 74 patients (35.2%) had a time lag of 90 days or more. The median time taken for reaching diagnosis after the first contact (T2) was 25 [IQR 15 - 45] days. The overall median time between self-detection of symptoms to initiation of primary treatment (T5) was 123[IQR 83-205] days. The majority of patients, 145 (66.2%), had initiation of primary treatment only after 90 days from self-detection of cervical cancer symptoms (Table 2). \\nFactors associated with delay in seeking medical care of 90 days and more (patient delay) were analyzed in relation to socio-demographic characteristics and clinical symptoms (Table 3). In multivariate analysis after adjusting to all the variables in the study the odds of patient delay in the age group ≥ 60 years was 3.2 times higher compared to patients in the age group below 45 years (95% CI: 1.2 – 8.4). The other factors which were associated with patient delay were being not literate (OR 2.3, 95% CI: 1.0 – 5.4), from rural area (OR = 2.3, 95% CI: 1.1 – 4.9), being financially dependent for livelihood (OR = 4.5, 95% CI: 1.9 – 10.6) and not having any abnormal vaginal discharge / bleeding (OR = 2.4, 95% CI: 1.3 – 4.6). Family history of cancer, presence of any health insurance, knowledge regarding cancer cure by early seeking for care, knowledge of symptoms of cervical cancer and having lower abdominal pain was not statistically related to delay in seeking medical care. \\nInformation was sought regarding perceived reasons from 197 (93.8%) patients who did not seek care within one week from self-detection of cancer symptoms. The major reasons (table 4) were lack of awareness in identifying cervical cancer symptoms in 183 (92.9%) women and 120 (60.9%) patients assuming that the symptom would resolve by itself. The other common reasons were absence of pain, changes in the body attributed to common illness and vague symptoms. \\nThe most common reason for delay in seeking for definite diagnosis at tertiary care hospitals was due to non-affordability 82 (41.6%). The other common reasons were visiting multiple medical practitioners before presenting at tertiary care 60 (30.1%) and not suspected for cancer by physician at first contact. \\nThe delay in seeking for treatment after diagnosis at tertiary care was due to long treatment procedure 57 (27.1%), fear of treatment 50 (23.8%), financial dependence on the family 48 (22.9%), fear of disfigurement of the body 26 (12.4%) and stigma attached with the disease (Table 4).\\nDistribution of Socio-Demographic, Clinical and Other Variables Among the Subject Enrolled and Not Enrolled to the Study\\nDistribution of Time Interval Between Self-Detection of Cancer Symptoms to Various Stages (T1 To T5) for Seeking Treatment\\nFactors Associated with Delay in Seeking Medical Care: Univariate and Multivariate Logistic Regression Results\\nDistribution of Perceived Reasons for Delay in Seeking Medical Care at Various Stages (T1 – T4)\\n*, Responses were elicited from those who had not sought specific care for more than one week from the onset of symptoms; #, Responses was elicited from those who had not sought treatment on specified time by doctor \\nRecruitment of Participants for the Study\",\n \"The study centre was a regional cancer care centre having both population and hospital-based cancer registry and where a majority of cervix cancer patients from the entire state seek cancer care. The median age in enrolled and not enrolled patients was 50 and 56 years, respectively. Among all the patients, the proportion of not literate patients was higher (81%) in not enrolled patients compared to enrolled patients (63%). The study by Deshmukh and Rathod, (2017) observed that the average age of the patients was 57 years and 81% were not literate. Our result concur with the study by Panda et al., 2019 where the average age of the study participants was 52.1 ± 10.9 years. \\nThe overall median time between the self-detection of cervical cancer symptoms and initiation of primary treatment was 123[IQR 83-205] days and the median time taken for first contact with general physician from self-detection of symptoms was 80 [IQR 45-150] days. Overall, 66% women delayed (≥ 90 days) for initiation of primary treatment from the onset of symptoms which is potentially concerning, because longer duration between diagnosis and treatment is related with higher risk of death. Among patients who started their treatment after an interval of >180 days, the risk of death was reported to be 36% higher compared to those who started treatment within 6 months from diagnosis (Chen et al., 2019) \\nThe present study assessed the time interval between self-detection of symptoms to treatment of cervical cancer and patient delay. The median time interval between self-detection of symptoms and the first presentation at general practitioner (T1) was 80 days with 35.4% of the patients reporting 90 days or more. The present study observation was consistent with a study conducted in India on patients attending tertiary care centre, showing that 39% of patients had a diagnostic delay (Panda et al., 2019) The median time between symptoms and presenting at health care provider was 69 days from two tertiary care centres in Nepal (Gyenwali et al., 2014). It was noted from the study by Lim et al., (2014) that only 28% of patients had a delay of more than 3 months in all national health service (NHS) hospitals diagnosing cervical cancer in England. There was a mean delay of 23.1 weeks between onset of symptoms from the study in tertiary care centre of Sothern Malawi (Rudd et al., 2017). Results from our study were in line with studies from low- or middle-income countries but differed from studies in developed countries. This difference may be partially due to regular screening programs in many developed countries and differing rates of literacy (IARC, 2005). \\nOverall median time from self-detection of symptoms to initiation of primary treatment (T5) was 123 days in the current study. Another study by Henriques, (2016) in India showed on an average six months duration from first symptom to cervical cancer treatment. Such a long time may be explained by the fact that lack of awareness of patients pertaining to cancer symptoms was high (93%) in present study. In another study, women who delayed, had mistaken the symptoms of cervix cancer as symptoms of sexually transmitted diseases (STD) and were scared to report (Deshmukh and Rathod, 2017). This shows that the lack of awareness of cervix cancer symptoms is a major factor and warrants improvement in public awareness. Our study results concur closely with other studies from similar settings and in low- or middle-income countries (Benemariya et al., 2018; Gyenwali et al., 2014).\\nThe patients who were aged 60 years and above, residing in rural areas, not literate, financially dependent for livelihood and did not had abnormal vaginal discharge/ bleeding were independently associated with patient delay. Similar results were observed in a study from Nepal where not literate was found to be independently associated with late diagnosis and women having abnormal vaginal bleeding as symptom was protective factor for late diagnosis (Gyenwali et al., 2013). Previous studies from low- or middle-income countries also concur with our result (Dunyo et al., 2018)(Ouasmani et al., 2016)(Henriques, 2016).\\nThe present study has been conducted in a population-based cancer registry (PBCR) where the data are included in International Agency for Research on Cancer (IARC) and data collection procedure involves standard quality control measures. The study included only patients who had been diagnosed with standard clinical and histopathological criteria. The information was obtained using pre-tested validated questionnaire by the investigator. Additional information pertaining to clinical evaluation and histological diagnosis were obtained from case sheets. All efforts were made to reduce the recall bias by local events calendar and standardization of terms employed in the questionnaire. \\n\\nLimitations\\n\\nPossible selection bias could have occurred as the subjects who were not enrolled for the study due to various reasons were older women and not literate compared to subjects in the study. Socio-demographic profile of population who availed the services at this centre may be different from private tertiary cancer care centre which would biases the result. The sample in study represent only those who availed the treatment at the centre and does not represent the entire population of cervical cancer patients. Thus, it may have some restrictions for external generalization. Possibility of recall bias exist as past history of cancer symptoms were assessed retrospectively. \\nIn conclusion, the median time of 80 days was observed from self-detection of cervical cancer symptoms to first contact with general physician. Further, a median time of another 45 days was observed for obtaining primary treatment. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care. Interventions like raising awareness of cervical cancer symptoms and improving health seeking behavior of women should be focused.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","materials|methods","results","discussion"],"string":"[\n \"intro\",\n \"materials|methods\",\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Cervical cancer","time interval","cancer symptoms","seeking cancer care"],"string":"[\n \"Cervical cancer\",\n \"time interval\",\n \"cancer symptoms\",\n \"seeking cancer care\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nGlobally, an estimated 570,000 new cases and 311,000 deaths has been reported due to cervical cancer in 2018 and it is the fourth most common cancer in women. The Incidence and mortality rank second behind breast cancer in lower human development index (HDI) settings. Eight out of 10 women diagnosed and nine of 10 women who die from cervical cancer live in a low- or middle-income countries (Bray et al., 2018) “ Cervical cancer continues to be a major public health problem affecting middle-aged women, particularly in less-resourced countries” (Arbyn et al., 2020). India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one third of global cervical cancer deaths during the year 2018 (“Globocan 2018,” 2018). Cervical cancer is the leading cause of cancer mortality in India which accounts for 17% of all cancer deaths among women aged 30 to 69 years.(Bobdey et al., 2016, “The Challenge Ahead,” 2014) One third of cervical cancer cases was reported among women aged 50 -59 years ( 27.4%) (Bobdey et al., 2016). The higher mortality and morbidity due to cancer is because most patients seek/get treatment at advanced stage and therefore have a higher risk of dying from the disease (Bray et al., 2018).\nCervical cancer is a preventable disease primarily because of its long lead time (women develop precancerous lesions years before it becomes cancerous). Cervical cancer can be detected in the pre-cancerous phase by screening and thereby the development of cancer prevented. Cervical cancer can also be treated successfully if diagnosed at an early stage. Routine screening by visual inspection of cervix through ‘See-and-treat’ approach among the female population has been suggested for early diagnosis and control of cervical cancer (Sankaranarayanan, 2012). Periodic Pap or HPV samples are difficult to organize in low- or middle-income countries like India and therefore large-scale routine screening would be difficult and not affordable (“HTA_CaCx Screening in India.pdf,” n.d.) Millions of cancer patients could be saved from premature death and disability, if they had timely access to early detection and treatment (“WHO,” n.d.). Studies have shown that it is possible to reduce the need of aggressive treatment of cancer is detected at an early stage. A long interval between start of disease symptoms to having a definite diagnosis is highly critical and associated with low survival among cervical cancer patients.(Chen et al., 2019) \nIn India, the present existing public health care delivery system is a three-level system. The primary level is primary health centres (PHCs). In India, PHCs focuses on eight elements of primary health care outlined in the Alma-Ata declaration and provides mainly outpatient services. In the secondary level the district hospitals provide outpatient, inpatient and emergency services. At the tertiary level, medical college hospitals, apex institutions and regional cancer centres provide specialized services. Under the programme of “National programme for prevention and control of cancer, diabetes, cardiovascular diseases and stroke (NPCDCS)”, cancer screening is done at the primary level by visual inspection of the cervix with acetic acid (VIA) and those who are suspected for cancer are referred to secondary or tertiary care level for further management. In addition, a health programme for non-communicable diseases is carried out with a cancer care component implemented at all the health care levels (Gulia et al., 2016) \nThis study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking for care and different barriers for the possible time lag in seeking care.\n\nMaterials and Methods:\nThe data was collected from a regional cancer centre from south India, Bengaluru, which has a population-based cancer registry (PBCR) and a hospital-based cancer registry (HBCR). This tertiary care centre has state of art facilities for diagnosis, treatment and research. Cervical cancer cases with histological confirmation were recruited from the hospital-based cancer registry of this Institution during the time period of April 2017 to September 2017 using cross sectional study design. We identified 430 cervical cancer patients during the period from April 2017 to September 2017. Of these, 52 were ineligible due to referral to terminal/palliative care. Of 378 eligible women, 96 were not included for the analysis as patients moved to different hospital or not being available for interview, and 38 were not willing to participate in the study. In the 244 patients who gave their consent, 34 patient’s had incomplete information in the records. Finally, 210 patients were included for the present study (Figure 1) \nThe cervical cancers were mostly squamous cell carcinomas (SCC) and few adenocarcinomas (AC). They were divided in two stage based on FIGO classification (Bhatla et al., 2019). Stage I and II were categorized as early stage; III and IV were considered as late stage of cervical cancer. \nPatient delay in seeking for medical care refers to a long interval between detecting of first/initial symptoms and seeking for appointment with a general physician. The patients were further divided into two groups, those who sought medical care within 90 days and those who sought care 90 days and above (patient delay) after self-detection of the first symptoms of cervical cancer. \nSample size was calculated based on the study conducted by Rudd et al., (2017) where 39.3% patients had delayed diagnosis from the onset of self-detection of cervical cancer symptoms. In this study the sample size was estimated for patient related delay, from symptoms to first contact with a general practitioner, as it was our major concern. To get an absolute precision of 6.6% with 95% confidence level, the study required a minimum sample of 210 subjects. Based on hospital record collection, it was noted that six months’ time period was sufficient to achieve the required sample size. \nExpert opinion from oncologists, biostatisticians and epidemiologists were obtained using the Delphi technique (Skulmoski et al., 2007). After obtaining the written informed consent, patients were interviewed in person using the questionnaire and their medical records was used to collect the baseline information as well as key components of the time interval between self-detection of cervical cancer symptom to seeking medical care. More details of the collection of data and the different components of time intervals are described elsewhere.(Somanna et al., 2020)\nIndependent variables included socio-demographic characteristics, such as age at diagnosis, availability of health insurance, place of residence (rural/urban), history of cancer literacy, and financially dependent for livelihood and clinical variables, such as personal history of cancer, and family history of cancer.\nThe time interval between various stages was measured in days as reported by the patients and by review of hospital case sheets as indicated in earlier study.(Somanna et al., 2018) \na) Time interval between self-detection of symptoms and the first presentation at general practitioner (T1). \nb) Time interval from referral by the general practitioner to attending tertiary care centre (T2). \nc) Time interval from first consultation at tertiary care for diagnosis to definite diagnosis date (T3). \nd) Time interval from definite diagnosis to initiation of primary treatment (T4). \ne) Total time elapsed from self-detection of symptoms to initiation of primary treatment (T5).\n\nStatistical methods\n\nDescriptive statistics of time intervals were summarized with median and inter-quartile range as data were not normally distributed (Kolmogrov-Smirnov and Shapiro test). Univariate odds ratios (OR) with 95% confidence intervals (CI) were estimated between the factors and patient delay. The patient delay was categorised into < 90 days and ≥ 90 days. Considering ≥ 90 days as patient delay, the association of various factors were estimated considering the low risk group as the reference. Variables showing a univariate association with patient delay (at P < 0.20) were included in multivariate unconditional logistic stepwise forward regression model to adjust for confounders. The analysis was carried out using SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago. \nEthical approval was sought from the ethical review committee of the study Institution. \n\nResults:\nThe distribution of socio-demographic and clinical data of the 210 cervical cancer study subjects are presented along with data pertaining to the cases not enrolled for analysis (Table 1). Among 210 enrolled patients, the majority 106 (50.5%) of women were between the age 46 to 59 years where in not enrolled patients 89 (40.4%) were of same age group. It was noted 167 (63.0%) and 179 (81.4%) were not literate among enrolled and respectively. Among enrolled patients, 150 (71.4%) were from rural areas and 158 (71.4%) were financially dependent for livelihood on the family. Only 19 women (9.0%) had any health insurance policy. The majority of patients 197 (93.8%) were not aware of symptoms of cervical cancer and only 7 (3.3%) patients had a family history of cancer.\nOf the symptoms which led for seeking medical care, the majority of women, 172 (81.9%), indicated lower abdominal pain, and 137 (65.2%) of the respondents experienced abnormal vaginal discharge/bleeding. Among all patients, 106 (50.5%) were diagnosed at an early stage of the disease (stages I, IIA and IIB).\nThe median time interval between self-detection of cervical cancer symptoms and first contact with general physician (T1) was 80 [IQR 45-150] days, while 74 patients (35.2%) had a time lag of 90 days or more. The median time taken for reaching diagnosis after the first contact (T2) was 25 [IQR 15 - 45] days. The overall median time between self-detection of symptoms to initiation of primary treatment (T5) was 123[IQR 83-205] days. The majority of patients, 145 (66.2%), had initiation of primary treatment only after 90 days from self-detection of cervical cancer symptoms (Table 2). \nFactors associated with delay in seeking medical care of 90 days and more (patient delay) were analyzed in relation to socio-demographic characteristics and clinical symptoms (Table 3). In multivariate analysis after adjusting to all the variables in the study the odds of patient delay in the age group ≥ 60 years was 3.2 times higher compared to patients in the age group below 45 years (95% CI: 1.2 – 8.4). The other factors which were associated with patient delay were being not literate (OR 2.3, 95% CI: 1.0 – 5.4), from rural area (OR = 2.3, 95% CI: 1.1 – 4.9), being financially dependent for livelihood (OR = 4.5, 95% CI: 1.9 – 10.6) and not having any abnormal vaginal discharge / bleeding (OR = 2.4, 95% CI: 1.3 – 4.6). Family history of cancer, presence of any health insurance, knowledge regarding cancer cure by early seeking for care, knowledge of symptoms of cervical cancer and having lower abdominal pain was not statistically related to delay in seeking medical care. \nInformation was sought regarding perceived reasons from 197 (93.8%) patients who did not seek care within one week from self-detection of cancer symptoms. The major reasons (table 4) were lack of awareness in identifying cervical cancer symptoms in 183 (92.9%) women and 120 (60.9%) patients assuming that the symptom would resolve by itself. The other common reasons were absence of pain, changes in the body attributed to common illness and vague symptoms. \nThe most common reason for delay in seeking for definite diagnosis at tertiary care hospitals was due to non-affordability 82 (41.6%). The other common reasons were visiting multiple medical practitioners before presenting at tertiary care 60 (30.1%) and not suspected for cancer by physician at first contact. \nThe delay in seeking for treatment after diagnosis at tertiary care was due to long treatment procedure 57 (27.1%), fear of treatment 50 (23.8%), financial dependence on the family 48 (22.9%), fear of disfigurement of the body 26 (12.4%) and stigma attached with the disease (Table 4).\nDistribution of Socio-Demographic, Clinical and Other Variables Among the Subject Enrolled and Not Enrolled to the Study\nDistribution of Time Interval Between Self-Detection of Cancer Symptoms to Various Stages (T1 To T5) for Seeking Treatment\nFactors Associated with Delay in Seeking Medical Care: Univariate and Multivariate Logistic Regression Results\nDistribution of Perceived Reasons for Delay in Seeking Medical Care at Various Stages (T1 – T4)\n*, Responses were elicited from those who had not sought specific care for more than one week from the onset of symptoms; #, Responses was elicited from those who had not sought treatment on specified time by doctor \nRecruitment of Participants for the Study\n\nDiscussion:\nThe study centre was a regional cancer care centre having both population and hospital-based cancer registry and where a majority of cervix cancer patients from the entire state seek cancer care. The median age in enrolled and not enrolled patients was 50 and 56 years, respectively. Among all the patients, the proportion of not literate patients was higher (81%) in not enrolled patients compared to enrolled patients (63%). The study by Deshmukh and Rathod, (2017) observed that the average age of the patients was 57 years and 81% were not literate. Our result concur with the study by Panda et al., 2019 where the average age of the study participants was 52.1 ± 10.9 years. \nThe overall median time between the self-detection of cervical cancer symptoms and initiation of primary treatment was 123[IQR 83-205] days and the median time taken for first contact with general physician from self-detection of symptoms was 80 [IQR 45-150] days. Overall, 66% women delayed (≥ 90 days) for initiation of primary treatment from the onset of symptoms which is potentially concerning, because longer duration between diagnosis and treatment is related with higher risk of death. Among patients who started their treatment after an interval of >180 days, the risk of death was reported to be 36% higher compared to those who started treatment within 6 months from diagnosis (Chen et al., 2019) \nThe present study assessed the time interval between self-detection of symptoms to treatment of cervical cancer and patient delay. The median time interval between self-detection of symptoms and the first presentation at general practitioner (T1) was 80 days with 35.4% of the patients reporting 90 days or more. The present study observation was consistent with a study conducted in India on patients attending tertiary care centre, showing that 39% of patients had a diagnostic delay (Panda et al., 2019) The median time between symptoms and presenting at health care provider was 69 days from two tertiary care centres in Nepal (Gyenwali et al., 2014). It was noted from the study by Lim et al., (2014) that only 28% of patients had a delay of more than 3 months in all national health service (NHS) hospitals diagnosing cervical cancer in England. There was a mean delay of 23.1 weeks between onset of symptoms from the study in tertiary care centre of Sothern Malawi (Rudd et al., 2017). Results from our study were in line with studies from low- or middle-income countries but differed from studies in developed countries. This difference may be partially due to regular screening programs in many developed countries and differing rates of literacy (IARC, 2005). \nOverall median time from self-detection of symptoms to initiation of primary treatment (T5) was 123 days in the current study. Another study by Henriques, (2016) in India showed on an average six months duration from first symptom to cervical cancer treatment. Such a long time may be explained by the fact that lack of awareness of patients pertaining to cancer symptoms was high (93%) in present study. In another study, women who delayed, had mistaken the symptoms of cervix cancer as symptoms of sexually transmitted diseases (STD) and were scared to report (Deshmukh and Rathod, 2017). This shows that the lack of awareness of cervix cancer symptoms is a major factor and warrants improvement in public awareness. Our study results concur closely with other studies from similar settings and in low- or middle-income countries (Benemariya et al., 2018; Gyenwali et al., 2014).\nThe patients who were aged 60 years and above, residing in rural areas, not literate, financially dependent for livelihood and did not had abnormal vaginal discharge/ bleeding were independently associated with patient delay. Similar results were observed in a study from Nepal where not literate was found to be independently associated with late diagnosis and women having abnormal vaginal bleeding as symptom was protective factor for late diagnosis (Gyenwali et al., 2013). Previous studies from low- or middle-income countries also concur with our result (Dunyo et al., 2018)(Ouasmani et al., 2016)(Henriques, 2016).\nThe present study has been conducted in a population-based cancer registry (PBCR) where the data are included in International Agency for Research on Cancer (IARC) and data collection procedure involves standard quality control measures. The study included only patients who had been diagnosed with standard clinical and histopathological criteria. The information was obtained using pre-tested validated questionnaire by the investigator. Additional information pertaining to clinical evaluation and histological diagnosis were obtained from case sheets. All efforts were made to reduce the recall bias by local events calendar and standardization of terms employed in the questionnaire. \n\nLimitations\n\nPossible selection bias could have occurred as the subjects who were not enrolled for the study due to various reasons were older women and not literate compared to subjects in the study. Socio-demographic profile of population who availed the services at this centre may be different from private tertiary cancer care centre which would biases the result. The sample in study represent only those who availed the treatment at the centre and does not represent the entire population of cervical cancer patients. Thus, it may have some restrictions for external generalization. Possibility of recall bias exist as past history of cancer symptoms were assessed retrospectively. \nIn conclusion, the median time of 80 days was observed from self-detection of cervical cancer symptoms to first contact with general physician. Further, a median time of another 45 days was observed for obtaining primary treatment. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care. Interventions like raising awareness of cervical cancer symptoms and improving health seeking behavior of women should be focused.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIndia had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one-third of global cervical cancer deaths during the year 2018. Cervical cancer is the leading cause of cancer mortality in India. The present study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking care and different barriers for the possible time lag in seeking care.\n\nMethods:\nA cross-sectional study was undertaken from April 2017 to September 2017 in a regional cancer centre in the south of India. The centre has both a population and a hospital-based cancer registry. Cervical cancer cases (N= 210) with histological confirmation were interviewed at the hospital using a pre-tested semi-structured questionnaire.\n\nResults:\nThe median time interval between the self-detection of cervical cancer symptoms and first contact with the general physician was 80 [IQR 45-150] days. The overall median time interval between the self-detection of symptoms to the initiation of primary treatment was 123[IQR 83-205] days. The major perceived reason for not seeking medical care was a lack of awareness in identifying cervical cancer symptoms in 183(92.9%) women.\n\nConclusions:\nThe median time of 80 days was observed from the self-detection of cervical cancer symptoms to the first contact with a general physician. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care.
."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":3637,"string":"3,637"},"whole_article_abstract_length":{"kind":"number","value":278,"string":"278"},"num_sections":{"kind":"number","value":4,"string":"4"},"most_frequent_words":{"kind":"list like","value":["cancer","patients","symptoms","study","care","cervical","cervical cancer","time","treatment","days"],"string":"[\n \"cancer\",\n \"patients\",\n \"symptoms\",\n \"study\",\n \"care\",\n \"cervical\",\n \"cervical cancer\",\n \"time\",\n \"treatment\",\n \"days\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Cervical cancer | time interval | cancer symptoms | seeking cancer care [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Cervical cancer | time interval | cancer symptoms | seeking cancer care [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Cervical cancer | time interval | cancer symptoms | seeking cancer care [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Cross-Sectional Studies | Female | Follow-Up Studies | Health Knowledge, Attitudes, Practice | Humans | India | Middle Aged | Patient Acceptance of Health Care | Prognosis | Retrospective Studies | Self-Assessment | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Uterine Cervical Neoplasms [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Cross-Sectional Studies | Female | Follow-Up Studies | Health Knowledge, Attitudes, Practice | Humans | India | Middle Aged | Patient Acceptance of Health Care | Prognosis | Retrospective Studies | Self-Assessment | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Uterine Cervical Neoplasms [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Cross-Sectional Studies | Female | Follow-Up Studies | Health Knowledge, Attitudes, Practice | Humans | India | Middle Aged | Patient Acceptance of Health Care | Prognosis | Retrospective Studies | Self-Assessment | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Uterine Cervical Neoplasms [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | patients | symptoms | study | care | cervical | cervical cancer | time | treatment | days [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | patients | symptoms | study | care | cervical | cervical cancer | time | treatment | days [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | patients | symptoms | study | care | cervical | cervical cancer | time | treatment | days [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | cervical cancer | cervical | level | 000 | deaths | screening | 2018 | india | care [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | symptoms | delay | patients | care | enrolled | 95 ci | table | delay seeking | seeking [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | patients | symptoms | care | cervical | cervical cancer | study | time | days | delay [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] India | 97,000 | 60,000 | nearly one-third | the year 2018 ||| India ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] first | 80 ||| ||| 123[IQR | 83 ||| 183(92.9% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] India | 97,000 | 60,000 | nearly one-third | the year 2018 ||| India ||| ||| April 2017 to September 2017 | India ||| ||| 210 ||| ||| first | 80 ||| ||| 123[IQR | 83 ||| 183(92.9% ||| 80 days | first ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":267,"cells":{"title":{"kind":"string","value":"Alteration of Thyroid Hormone among Patients with Ischemic Stroke visiting a Tertiary Care Hospital: A Descriptive Cross-sectional Study."},"pmid":{"kind":"string","value":"34508483"},"background_abstract":{"kind":"string","value":"Stroke is broadly classified as cerebral infarction, intracerebral hemorrhage and subarachnoid hemorrhage. Neuroendocrine profile is altered in acute ischemic stroke and there is a link between hypothyroidism and atherosclerosis which in turn may lead to stroke. The objective of this study was to find out the prevalence of alteration of thyroid hormones in patients with ischemic stroke in a tertiary care center."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"This descriptive cross-sectional study was conducted from June to December 2019 in a tertiary care center. Ethical approval was taken from Institutional review board of National Academy of Medical Sciences (reference number: IM 175). Patients with a diagnosis of stroke, without evidence of cardioembolic source, history of liver disease, renal failure and thyroid disease and who do not use thyroidal supplementation within 180 days prior the event were included. Convenience sampling was done. Data was entered in Microsoft Excel and analyzed using Statistical Package for the Social Sciences version 22. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The prevalence of altered thyroid levels among 73 patients was 13 (17.8%) (90% Confidence Interval= 10.44-25.16). Among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Among severity of hypothyroid cases, subclinical hypothyroidism grade IA was seen in 51 (70%), subclinical hypothyroidism grade IB was seen in 22 (30%)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The prevalence of altered thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Brain Ischemia","Cross-Sectional Studies","Humans","Ischemic Stroke","Stroke","Tertiary Care Centers","Thyroid Hormones"],"string":"[\n \"Brain Ischemia\",\n \"Cross-Sectional Studies\",\n \"Humans\",\n \"Ischemic Stroke\",\n \"Stroke\",\n \"Tertiary Care Centers\",\n \"Thyroid Hormones\"\n]"},"pmcid":{"kind":"string","value":"9107842"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Stroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4\nThyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4\nThe aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"This descriptive cross-sectional study was conducted from June to December 2019 in National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu. Ethical approval from Institutional review board of NAMS (reference number. IM 175). Convenience sampling was done and the sample size of cases to be enrolled was calculated by using the formula,\nn = Z2 × p × q / e2\n = (1.645)2 × (0.5) × (1-0.5) / (0.1)2\n = 67.65\nWhere,\nn = minimum required sample size,\nZ = 1.645 at 90% Confidence Interval (CI),\np = prevalence taken as 50% for maximum sample size,\nq = 1-p\ne = margin of error, 10%\nHence, the required sample size was 67.65. Adding a 10% nonresponse rate, we enrolled 73 patients in our study. Patients with clinically diagnosed stroke, presenting within 48 hours of onset of symptoms with CT or MRI findings were included in the study. Patients with prior use of thyroid supplements recently within last 180 days and with a history of liver disease, renal failure and known thyroid disease and who don't give consent were excluded from the study. Informed written consent was taken either in Nepali or in English language, in whichever they felt comfortable. Confidentiality was maintained to the utmost. For those who are not able to give consent because of their clinical state, informed consent was taken from their close relative.\nProforma was translated in Nepali verbally if needed depending upon the cases. For diagnosing the type of stroke, every patient underwent neuroimaging with non-contrast CT of the head. Cerebral infarction was diagnosed if neurological deficits are accompanied by a hypo dense lesion >15 mm in diameter in an appropriate area on a cranial CT scan. Lacunar infarction were diagnosed if the patient presented with a pure motor stroke, a pure sensory stroke, ataxic hemiparesis or a sensorimotor stroke in the absence of a visual field defect and evidence of higher cerebral dysfunction and a hypo dense lesion of < 15 mm in diameter or normal CT scan. In the laboratory, fully automatic analyzer VITROSECi/ECiQ Immunodiagnostic Systems, VITROSTSH was measured using a chemiluminescent immunometric assay with a detection range from 0.01 to 100 m IU/L. TSH values between 0.46 and 4.68 m IU/L were considered the normal reference range (euthyroid). Specimens with TSH values above or below the normal reference range were tested for free thyroxine (FT4). FT4 was measured using a competitive chemiluminescent assay with a detection range of 0.1-12.0 ng/dL. FT4 concentrations within 0.70-2.19 ng/dL was within the normal reference range. All samples included in the study met quality control criteria. Samples with TSH values > 4.69 m IU/L and normal FT4 levels were classified as indicating SCH.\nThe collected data was stored and analyzed with the Statistical Package for the Social Sciences (SPSS) version 22.0 for windows. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was 13 (17.8%) and among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Minimum age of patient in the study was 26 years and maximum age was 115 years. The mean age of presentation of cerebral infarction in this study was 63.30±19.217.\nAmong the total 73 cases, 49 were male and 24 were female. Among 49 males, 37 were euthyroid, 11 were hypothyroid and one male was hyperthyroid. Among 24 females, 23 were euthyroid and one female was hyperthyroid. On further categorization of altered thyroid status, 10 (13.7%) were subclinical hypothyroidism, 1 (1.4%) cases were of overt hypothyroidism and 2 (2.7%) cases had subclinical hyperthyroidism. With regards to the severity of subclinical hypothyroidism, out of the total 10 cases of subclinical hypothyroidism, 70% had grade IA, while the remaining 30% had grade IB severity of the status (Figure 1). In the study 49 (67.12%) of ischemic stroke were male and 24 (32.88%) were female.\nAmong euthyroid cases mean total cholesterol was 139.55 mg/dl which was higher than the mean cholesterol level in overt hypothyroid cases with 110 mg/dl. But mean total cholesterol level was lower among overt hypothyroid cases compared to euthyroid and overt hypothyroid cases. Mean TC was highest among subclinical hyperthyroid cases. Mean LDLc was highest among euthyroid cases with mean value of 73.94 mg/dl and lowest among overt hypothyroid cases with mean value of 41.00 mg/dl. TG was highest among overt hypothyroid cases with mean value of 199 mg/dl. HDLc was highest among euthyroid cases with mean value of 43.42 mg/dl. Subclinical hypothyroid cases had mean HDL value of 39.90 mg/dl (Table 1)."},"conclusions_title":{"kind":"string","value":"CONCLUSIONS"},"conclusions_text":{"kind":"string","value":"The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke."},"other_sections_titles":{"kind":"list like","value":[],"string":"[]"},"other_sections_texts":{"kind":"list like","value":[],"string":"[]"},"other_sections_sec_types":{"kind":"list like","value":[],"string":"[]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","RESULTS","DISCUSSION","CONCLUSIONS"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"RESULTS\",\n \"DISCUSSION\",\n \"CONCLUSIONS\"\n]"},"all_sections_texts":{"kind":"list like","value":["Stroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4\nThyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4\nThe aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.","This descriptive cross-sectional study was conducted from June to December 2019 in National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu. Ethical approval from Institutional review board of NAMS (reference number. IM 175). Convenience sampling was done and the sample size of cases to be enrolled was calculated by using the formula,\nn = Z2 × p × q / e2\n = (1.645)2 × (0.5) × (1-0.5) / (0.1)2\n = 67.65\nWhere,\nn = minimum required sample size,\nZ = 1.645 at 90% Confidence Interval (CI),\np = prevalence taken as 50% for maximum sample size,\nq = 1-p\ne = margin of error, 10%\nHence, the required sample size was 67.65. Adding a 10% nonresponse rate, we enrolled 73 patients in our study. Patients with clinically diagnosed stroke, presenting within 48 hours of onset of symptoms with CT or MRI findings were included in the study. Patients with prior use of thyroid supplements recently within last 180 days and with a history of liver disease, renal failure and known thyroid disease and who don't give consent were excluded from the study. Informed written consent was taken either in Nepali or in English language, in whichever they felt comfortable. Confidentiality was maintained to the utmost. For those who are not able to give consent because of their clinical state, informed consent was taken from their close relative.\nProforma was translated in Nepali verbally if needed depending upon the cases. For diagnosing the type of stroke, every patient underwent neuroimaging with non-contrast CT of the head. Cerebral infarction was diagnosed if neurological deficits are accompanied by a hypo dense lesion >15 mm in diameter in an appropriate area on a cranial CT scan. Lacunar infarction were diagnosed if the patient presented with a pure motor stroke, a pure sensory stroke, ataxic hemiparesis or a sensorimotor stroke in the absence of a visual field defect and evidence of higher cerebral dysfunction and a hypo dense lesion of < 15 mm in diameter or normal CT scan. In the laboratory, fully automatic analyzer VITROSECi/ECiQ Immunodiagnostic Systems, VITROSTSH was measured using a chemiluminescent immunometric assay with a detection range from 0.01 to 100 m IU/L. TSH values between 0.46 and 4.68 m IU/L were considered the normal reference range (euthyroid). Specimens with TSH values above or below the normal reference range were tested for free thyroxine (FT4). FT4 was measured using a competitive chemiluminescent assay with a detection range of 0.1-12.0 ng/dL. FT4 concentrations within 0.70-2.19 ng/dL was within the normal reference range. All samples included in the study met quality control criteria. Samples with TSH values > 4.69 m IU/L and normal FT4 levels were classified as indicating SCH.\nThe collected data was stored and analyzed with the Statistical Package for the Social Sciences (SPSS) version 22.0 for windows. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.","The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was 13 (17.8%) and among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Minimum age of patient in the study was 26 years and maximum age was 115 years. The mean age of presentation of cerebral infarction in this study was 63.30±19.217.\nAmong the total 73 cases, 49 were male and 24 were female. Among 49 males, 37 were euthyroid, 11 were hypothyroid and one male was hyperthyroid. Among 24 females, 23 were euthyroid and one female was hyperthyroid. On further categorization of altered thyroid status, 10 (13.7%) were subclinical hypothyroidism, 1 (1.4%) cases were of overt hypothyroidism and 2 (2.7%) cases had subclinical hyperthyroidism. With regards to the severity of subclinical hypothyroidism, out of the total 10 cases of subclinical hypothyroidism, 70% had grade IA, while the remaining 30% had grade IB severity of the status (Figure 1). In the study 49 (67.12%) of ischemic stroke were male and 24 (32.88%) were female.\nAmong euthyroid cases mean total cholesterol was 139.55 mg/dl which was higher than the mean cholesterol level in overt hypothyroid cases with 110 mg/dl. But mean total cholesterol level was lower among overt hypothyroid cases compared to euthyroid and overt hypothyroid cases. Mean TC was highest among subclinical hyperthyroid cases. Mean LDLc was highest among euthyroid cases with mean value of 73.94 mg/dl and lowest among overt hypothyroid cases with mean value of 41.00 mg/dl. TG was highest among overt hypothyroid cases with mean value of 199 mg/dl. HDLc was highest among euthyroid cases with mean value of 43.42 mg/dl. Subclinical hypothyroid cases had mean HDL value of 39.90 mg/dl (Table 1).","Stroke is a form of acute stress with detrimental effect on various neurophysiological pathways.7 Hypothyroidism is a possible risk factor for stroke.8 A number of comorbidities have been associated with increased mortality in acute stroke patients. It is not known whether hypothyroidism (either clinical or subclinical) affects outcome in patients with acute cerebrovascular disease. A neuroprotective role of hypothyroidism has been shown in acute stroke patients.8 Low T3 appeared to be associated with stroke severity and short-term outcome.9 In addition, strokes of undetermined etiology accounted for one-third to one-quarter of ischemic strokes among young people and among cases of stroke of undetermined etiology hyperthyroidism could be the underlying cause.10,11 So, TFT screening is useful in patients with acute ischemic stroke (IS) because thyroid disorders are known risk factors for cerebrovascular diseases.6 In our study we observed 17.8% of the cases had thyroid dysfunction. Our finding is similar to the findings of the retrospective study done by Xu XY , et al. in 893 patients with acute ischemic stroke in which 19% of the cases had abnormal thyroid function tests with at least one of the thyroid related hormones below or above the normal ranges.12 Unknown overt or subclinical hyperthyroidism is associated with cardio-embolic stroke.13 Thyroid dysfunction in our study was lower than the study done by Dimopolou, et al. in 33 critically ill patients under mechanical ventilation due to acute stroke; study concluded 36% of the cases had thyroid dysfunction.14 In our study we found that 1.4% of the cases had overt hypothyroidism, this is comparable to the study done by O'keefe L.M , et al. in 129 patients with acute ischemic stroke. 2.32 % of the cases in the study had hypothyroidism.1513.7 % of our study population had subclinical hypothyroidism.\nOur result is similar to the study done by Pande, et al. to establish a relation between the variation in thyroid profile and ischemic CVAs in 75 patients within 48 hours of the event, which concluded that 12% of the cases had subclinical hypothyroidism.7 Prospective cohort study done by Chakler L, et al. on 47,573 adults (3451 subclinical hypothyroidism) from 17 cohorts concluded an increased risk of stroke on subjects younger than 65 years and those with higher TSH concentrations. Subclinical hypothyroidism (SCH) is postulated to increase stroke risk via atherogenic changes associated with abnormal thyroid function.16 The study concluded that hyperthyroidism is associated with an increased risk for ischemic stroke among young adults.11\nIn our study HDLc was low (<40 mg/dl) in all the case with thyroid dysfunction (18% cases) this is much lower compared to the study done by Pokharel , et al., among 281 stroke patients, in which 45% had their HDLc levels below 40 mg/dl.17 TG was elevated (>150 mg/dl) in cases with overt hypothyroidism (1.4%) this is consistent with the study done by Abrams JJ , et al. which concluded TG metabolism is not grossly deranged in hypothyroidism.18 In our study we found that stroke occurred more commonly among males (67.12%) compared to females (32.88%). Women have lower stroke incidence than men, it might be due to positive effects of estrogen on the cerebral circulation. A lifetime exposure to ovarian estrogens may protect against ischemic stroke.19 Seventy-two percent of the cases in our study were smokers. Wang, et al. reported that 48% of patients with stroke smoked.20 Tsai, et al. reported 38% of the cases with ischemic stroke were smokers.21\nOur results might not be generalized to all stroke patients as we recruited study participants from a single hospital only. As many of our patients presented to us after 48 hours of the occurrence of stroke events, we could not involve them in our study.","The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke."],"string":"[\n \"Stroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4\\nThyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4\\nThe aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.\",\n \"This descriptive cross-sectional study was conducted from June to December 2019 in National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu. Ethical approval from Institutional review board of NAMS (reference number. IM 175). Convenience sampling was done and the sample size of cases to be enrolled was calculated by using the formula,\\nn = Z2 × p × q / e2\\n = (1.645)2 × (0.5) × (1-0.5) / (0.1)2\\n = 67.65\\nWhere,\\nn = minimum required sample size,\\nZ = 1.645 at 90% Confidence Interval (CI),\\np = prevalence taken as 50% for maximum sample size,\\nq = 1-p\\ne = margin of error, 10%\\nHence, the required sample size was 67.65. Adding a 10% nonresponse rate, we enrolled 73 patients in our study. Patients with clinically diagnosed stroke, presenting within 48 hours of onset of symptoms with CT or MRI findings were included in the study. Patients with prior use of thyroid supplements recently within last 180 days and with a history of liver disease, renal failure and known thyroid disease and who don't give consent were excluded from the study. Informed written consent was taken either in Nepali or in English language, in whichever they felt comfortable. Confidentiality was maintained to the utmost. For those who are not able to give consent because of their clinical state, informed consent was taken from their close relative.\\nProforma was translated in Nepali verbally if needed depending upon the cases. For diagnosing the type of stroke, every patient underwent neuroimaging with non-contrast CT of the head. Cerebral infarction was diagnosed if neurological deficits are accompanied by a hypo dense lesion >15 mm in diameter in an appropriate area on a cranial CT scan. Lacunar infarction were diagnosed if the patient presented with a pure motor stroke, a pure sensory stroke, ataxic hemiparesis or a sensorimotor stroke in the absence of a visual field defect and evidence of higher cerebral dysfunction and a hypo dense lesion of < 15 mm in diameter or normal CT scan. In the laboratory, fully automatic analyzer VITROSECi/ECiQ Immunodiagnostic Systems, VITROSTSH was measured using a chemiluminescent immunometric assay with a detection range from 0.01 to 100 m IU/L. TSH values between 0.46 and 4.68 m IU/L were considered the normal reference range (euthyroid). Specimens with TSH values above or below the normal reference range were tested for free thyroxine (FT4). FT4 was measured using a competitive chemiluminescent assay with a detection range of 0.1-12.0 ng/dL. FT4 concentrations within 0.70-2.19 ng/dL was within the normal reference range. All samples included in the study met quality control criteria. Samples with TSH values > 4.69 m IU/L and normal FT4 levels were classified as indicating SCH.\\nThe collected data was stored and analyzed with the Statistical Package for the Social Sciences (SPSS) version 22.0 for windows. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.\",\n \"The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was 13 (17.8%) and among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Minimum age of patient in the study was 26 years and maximum age was 115 years. The mean age of presentation of cerebral infarction in this study was 63.30±19.217.\\nAmong the total 73 cases, 49 were male and 24 were female. Among 49 males, 37 were euthyroid, 11 were hypothyroid and one male was hyperthyroid. Among 24 females, 23 were euthyroid and one female was hyperthyroid. On further categorization of altered thyroid status, 10 (13.7%) were subclinical hypothyroidism, 1 (1.4%) cases were of overt hypothyroidism and 2 (2.7%) cases had subclinical hyperthyroidism. With regards to the severity of subclinical hypothyroidism, out of the total 10 cases of subclinical hypothyroidism, 70% had grade IA, while the remaining 30% had grade IB severity of the status (Figure 1). In the study 49 (67.12%) of ischemic stroke were male and 24 (32.88%) were female.\\nAmong euthyroid cases mean total cholesterol was 139.55 mg/dl which was higher than the mean cholesterol level in overt hypothyroid cases with 110 mg/dl. But mean total cholesterol level was lower among overt hypothyroid cases compared to euthyroid and overt hypothyroid cases. Mean TC was highest among subclinical hyperthyroid cases. Mean LDLc was highest among euthyroid cases with mean value of 73.94 mg/dl and lowest among overt hypothyroid cases with mean value of 41.00 mg/dl. TG was highest among overt hypothyroid cases with mean value of 199 mg/dl. HDLc was highest among euthyroid cases with mean value of 43.42 mg/dl. Subclinical hypothyroid cases had mean HDL value of 39.90 mg/dl (Table 1).\",\n \"Stroke is a form of acute stress with detrimental effect on various neurophysiological pathways.7 Hypothyroidism is a possible risk factor for stroke.8 A number of comorbidities have been associated with increased mortality in acute stroke patients. It is not known whether hypothyroidism (either clinical or subclinical) affects outcome in patients with acute cerebrovascular disease. A neuroprotective role of hypothyroidism has been shown in acute stroke patients.8 Low T3 appeared to be associated with stroke severity and short-term outcome.9 In addition, strokes of undetermined etiology accounted for one-third to one-quarter of ischemic strokes among young people and among cases of stroke of undetermined etiology hyperthyroidism could be the underlying cause.10,11 So, TFT screening is useful in patients with acute ischemic stroke (IS) because thyroid disorders are known risk factors for cerebrovascular diseases.6 In our study we observed 17.8% of the cases had thyroid dysfunction. Our finding is similar to the findings of the retrospective study done by Xu XY , et al. in 893 patients with acute ischemic stroke in which 19% of the cases had abnormal thyroid function tests with at least one of the thyroid related hormones below or above the normal ranges.12 Unknown overt or subclinical hyperthyroidism is associated with cardio-embolic stroke.13 Thyroid dysfunction in our study was lower than the study done by Dimopolou, et al. in 33 critically ill patients under mechanical ventilation due to acute stroke; study concluded 36% of the cases had thyroid dysfunction.14 In our study we found that 1.4% of the cases had overt hypothyroidism, this is comparable to the study done by O'keefe L.M , et al. in 129 patients with acute ischemic stroke. 2.32 % of the cases in the study had hypothyroidism.1513.7 % of our study population had subclinical hypothyroidism.\\nOur result is similar to the study done by Pande, et al. to establish a relation between the variation in thyroid profile and ischemic CVAs in 75 patients within 48 hours of the event, which concluded that 12% of the cases had subclinical hypothyroidism.7 Prospective cohort study done by Chakler L, et al. on 47,573 adults (3451 subclinical hypothyroidism) from 17 cohorts concluded an increased risk of stroke on subjects younger than 65 years and those with higher TSH concentrations. Subclinical hypothyroidism (SCH) is postulated to increase stroke risk via atherogenic changes associated with abnormal thyroid function.16 The study concluded that hyperthyroidism is associated with an increased risk for ischemic stroke among young adults.11\\nIn our study HDLc was low (<40 mg/dl) in all the case with thyroid dysfunction (18% cases) this is much lower compared to the study done by Pokharel , et al., among 281 stroke patients, in which 45% had their HDLc levels below 40 mg/dl.17 TG was elevated (>150 mg/dl) in cases with overt hypothyroidism (1.4%) this is consistent with the study done by Abrams JJ , et al. which concluded TG metabolism is not grossly deranged in hypothyroidism.18 In our study we found that stroke occurred more commonly among males (67.12%) compared to females (32.88%). Women have lower stroke incidence than men, it might be due to positive effects of estrogen on the cerebral circulation. A lifetime exposure to ovarian estrogens may protect against ischemic stroke.19 Seventy-two percent of the cases in our study were smokers. Wang, et al. reported that 48% of patients with stroke smoked.20 Tsai, et al. reported 38% of the cases with ischemic stroke were smokers.21\\nOur results might not be generalized to all stroke patients as we recruited study participants from a single hospital only. As many of our patients presented to us after 48 hours of the occurrence of stroke events, we could not involve them in our study.\",\n \"The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods","results","discussion","conclusions"],"string":"[\n \"intro\",\n \"methods\",\n \"results\",\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["\nhyperthyroidism\n","\nhypothyroidism\n","\nischemic stroke\n"],"string":"[\n \"\\nhyperthyroidism\\n\",\n \"\\nhypothyroidism\\n\",\n \"\\nischemic stroke\\n\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nStroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4\nThyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4\nThe aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.\n\nMETHODS:\nThis descriptive cross-sectional study was conducted from June to December 2019 in National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu. Ethical approval from Institutional review board of NAMS (reference number. IM 175). Convenience sampling was done and the sample size of cases to be enrolled was calculated by using the formula,\nn = Z2 × p × q / e2\n = (1.645)2 × (0.5) × (1-0.5) / (0.1)2\n = 67.65\nWhere,\nn = minimum required sample size,\nZ = 1.645 at 90% Confidence Interval (CI),\np = prevalence taken as 50% for maximum sample size,\nq = 1-p\ne = margin of error, 10%\nHence, the required sample size was 67.65. Adding a 10% nonresponse rate, we enrolled 73 patients in our study. Patients with clinically diagnosed stroke, presenting within 48 hours of onset of symptoms with CT or MRI findings were included in the study. Patients with prior use of thyroid supplements recently within last 180 days and with a history of liver disease, renal failure and known thyroid disease and who don't give consent were excluded from the study. Informed written consent was taken either in Nepali or in English language, in whichever they felt comfortable. Confidentiality was maintained to the utmost. For those who are not able to give consent because of their clinical state, informed consent was taken from their close relative.\nProforma was translated in Nepali verbally if needed depending upon the cases. For diagnosing the type of stroke, every patient underwent neuroimaging with non-contrast CT of the head. Cerebral infarction was diagnosed if neurological deficits are accompanied by a hypo dense lesion >15 mm in diameter in an appropriate area on a cranial CT scan. Lacunar infarction were diagnosed if the patient presented with a pure motor stroke, a pure sensory stroke, ataxic hemiparesis or a sensorimotor stroke in the absence of a visual field defect and evidence of higher cerebral dysfunction and a hypo dense lesion of < 15 mm in diameter or normal CT scan. In the laboratory, fully automatic analyzer VITROSECi/ECiQ Immunodiagnostic Systems, VITROSTSH was measured using a chemiluminescent immunometric assay with a detection range from 0.01 to 100 m IU/L. TSH values between 0.46 and 4.68 m IU/L were considered the normal reference range (euthyroid). Specimens with TSH values above or below the normal reference range were tested for free thyroxine (FT4). FT4 was measured using a competitive chemiluminescent assay with a detection range of 0.1-12.0 ng/dL. FT4 concentrations within 0.70-2.19 ng/dL was within the normal reference range. All samples included in the study met quality control criteria. Samples with TSH values > 4.69 m IU/L and normal FT4 levels were classified as indicating SCH.\nThe collected data was stored and analyzed with the Statistical Package for the Social Sciences (SPSS) version 22.0 for windows. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.\n\nRESULTS:\nThe prevalence of altered in thyroid levels among patients undergoing ischemic stroke was 13 (17.8%) and among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Minimum age of patient in the study was 26 years and maximum age was 115 years. The mean age of presentation of cerebral infarction in this study was 63.30±19.217.\nAmong the total 73 cases, 49 were male and 24 were female. Among 49 males, 37 were euthyroid, 11 were hypothyroid and one male was hyperthyroid. Among 24 females, 23 were euthyroid and one female was hyperthyroid. On further categorization of altered thyroid status, 10 (13.7%) were subclinical hypothyroidism, 1 (1.4%) cases were of overt hypothyroidism and 2 (2.7%) cases had subclinical hyperthyroidism. With regards to the severity of subclinical hypothyroidism, out of the total 10 cases of subclinical hypothyroidism, 70% had grade IA, while the remaining 30% had grade IB severity of the status (Figure 1). In the study 49 (67.12%) of ischemic stroke were male and 24 (32.88%) were female.\nAmong euthyroid cases mean total cholesterol was 139.55 mg/dl which was higher than the mean cholesterol level in overt hypothyroid cases with 110 mg/dl. But mean total cholesterol level was lower among overt hypothyroid cases compared to euthyroid and overt hypothyroid cases. Mean TC was highest among subclinical hyperthyroid cases. Mean LDLc was highest among euthyroid cases with mean value of 73.94 mg/dl and lowest among overt hypothyroid cases with mean value of 41.00 mg/dl. TG was highest among overt hypothyroid cases with mean value of 199 mg/dl. HDLc was highest among euthyroid cases with mean value of 43.42 mg/dl. Subclinical hypothyroid cases had mean HDL value of 39.90 mg/dl (Table 1).\n\nDISCUSSION:\nStroke is a form of acute stress with detrimental effect on various neurophysiological pathways.7 Hypothyroidism is a possible risk factor for stroke.8 A number of comorbidities have been associated with increased mortality in acute stroke patients. It is not known whether hypothyroidism (either clinical or subclinical) affects outcome in patients with acute cerebrovascular disease. A neuroprotective role of hypothyroidism has been shown in acute stroke patients.8 Low T3 appeared to be associated with stroke severity and short-term outcome.9 In addition, strokes of undetermined etiology accounted for one-third to one-quarter of ischemic strokes among young people and among cases of stroke of undetermined etiology hyperthyroidism could be the underlying cause.10,11 So, TFT screening is useful in patients with acute ischemic stroke (IS) because thyroid disorders are known risk factors for cerebrovascular diseases.6 In our study we observed 17.8% of the cases had thyroid dysfunction. Our finding is similar to the findings of the retrospective study done by Xu XY , et al. in 893 patients with acute ischemic stroke in which 19% of the cases had abnormal thyroid function tests with at least one of the thyroid related hormones below or above the normal ranges.12 Unknown overt or subclinical hyperthyroidism is associated with cardio-embolic stroke.13 Thyroid dysfunction in our study was lower than the study done by Dimopolou, et al. in 33 critically ill patients under mechanical ventilation due to acute stroke; study concluded 36% of the cases had thyroid dysfunction.14 In our study we found that 1.4% of the cases had overt hypothyroidism, this is comparable to the study done by O'keefe L.M , et al. in 129 patients with acute ischemic stroke. 2.32 % of the cases in the study had hypothyroidism.1513.7 % of our study population had subclinical hypothyroidism.\nOur result is similar to the study done by Pande, et al. to establish a relation between the variation in thyroid profile and ischemic CVAs in 75 patients within 48 hours of the event, which concluded that 12% of the cases had subclinical hypothyroidism.7 Prospective cohort study done by Chakler L, et al. on 47,573 adults (3451 subclinical hypothyroidism) from 17 cohorts concluded an increased risk of stroke on subjects younger than 65 years and those with higher TSH concentrations. Subclinical hypothyroidism (SCH) is postulated to increase stroke risk via atherogenic changes associated with abnormal thyroid function.16 The study concluded that hyperthyroidism is associated with an increased risk for ischemic stroke among young adults.11\nIn our study HDLc was low (<40 mg/dl) in all the case with thyroid dysfunction (18% cases) this is much lower compared to the study done by Pokharel , et al., among 281 stroke patients, in which 45% had their HDLc levels below 40 mg/dl.17 TG was elevated (>150 mg/dl) in cases with overt hypothyroidism (1.4%) this is consistent with the study done by Abrams JJ , et al. which concluded TG metabolism is not grossly deranged in hypothyroidism.18 In our study we found that stroke occurred more commonly among males (67.12%) compared to females (32.88%). Women have lower stroke incidence than men, it might be due to positive effects of estrogen on the cerebral circulation. A lifetime exposure to ovarian estrogens may protect against ischemic stroke.19 Seventy-two percent of the cases in our study were smokers. Wang, et al. reported that 48% of patients with stroke smoked.20 Tsai, et al. reported 38% of the cases with ischemic stroke were smokers.21\nOur results might not be generalized to all stroke patients as we recruited study participants from a single hospital only. As many of our patients presented to us after 48 hours of the occurrence of stroke events, we could not involve them in our study.\n\nCONCLUSIONS:\nThe prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nStroke is broadly classified as cerebral infarction, intracerebral hemorrhage and subarachnoid hemorrhage. Neuroendocrine profile is altered in acute ischemic stroke and there is a link between hypothyroidism and atherosclerosis which in turn may lead to stroke. The objective of this study was to find out the prevalence of alteration of thyroid hormones in patients with ischemic stroke in a tertiary care center.\n\nMethods:\nThis descriptive cross-sectional study was conducted from June to December 2019 in a tertiary care center. Ethical approval was taken from Institutional review board of National Academy of Medical Sciences (reference number: IM 175). Patients with a diagnosis of stroke, without evidence of cardioembolic source, history of liver disease, renal failure and thyroid disease and who do not use thyroidal supplementation within 180 days prior the event were included. Convenience sampling was done. Data was entered in Microsoft Excel and analyzed using Statistical Package for the Social Sciences version 22. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.\n\nResults:\nThe prevalence of altered thyroid levels among 73 patients was 13 (17.8%) (90% Confidence Interval= 10.44-25.16). Among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Among severity of hypothyroid cases, subclinical hypothyroidism grade IA was seen in 51 (70%), subclinical hypothyroidism grade IB was seen in 22 (30%).\n\nConclusions:\nThe prevalence of altered thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nStroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4\nThyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4\nThe aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.\n\nCONCLUSIONS:\nThe prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nStroke is broadly classified as cerebral infarction, intracerebral hemorrhage and subarachnoid hemorrhage. Neuroendocrine profile is altered in acute ischemic stroke and there is a link between hypothyroidism and atherosclerosis which in turn may lead to stroke. The objective of this study was to find out the prevalence of alteration of thyroid hormones in patients with ischemic stroke in a tertiary care center.\n\nMethods:\nThis descriptive cross-sectional study was conducted from June to December 2019 in a tertiary care center. Ethical approval was taken from Institutional review board of National Academy of Medical Sciences (reference number: IM 175). Patients with a diagnosis of stroke, without evidence of cardioembolic source, history of liver disease, renal failure and thyroid disease and who do not use thyroidal supplementation within 180 days prior the event were included. Convenience sampling was done. Data was entered in Microsoft Excel and analyzed using Statistical Package for the Social Sciences version 22. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.\n\nResults:\nThe prevalence of altered thyroid levels among 73 patients was 13 (17.8%) (90% Confidence Interval= 10.44-25.16). Among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Among severity of hypothyroid cases, subclinical hypothyroidism grade IA was seen in 51 (70%), subclinical hypothyroidism grade IB was seen in 22 (30%).\n\nConclusions:\nThe prevalence of altered thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies."},"whole_article_text_length":{"kind":"number","value":1919,"string":"1,919"},"whole_article_abstract_length":{"kind":"number","value":298,"string":"298"},"num_sections":{"kind":"number","value":5,"string":"5"},"most_frequent_words":{"kind":"list like","value":["stroke","study","cases","patients","thyroid","hypothyroidism","ischemic","dl","subclinical","mean"],"string":"[\n \"stroke\",\n \"study\",\n \"cases\",\n \"patients\",\n \"thyroid\",\n \"hypothyroidism\",\n \"ischemic\",\n \"dl\",\n \"subclinical\",\n \"mean\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] \nhyperthyroidism\n | \nhypothyroidism\n | \nischemic stroke\n [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | death | year | 000 | stroke year | hemorrhage | ischemic | cause | cvas | associated [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] range | normal | sample | ct | consent | size | ft4 | reference | sample size | iu [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] mean | cases | cases mean | hypothyroid | hypothyroid cases | mg dl | mg | overt hypothyroid cases | overt hypothyroid | value [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] modifiable | stroke | risk | non | risk factors | factors | prevalence | factors great | impact reduction morbidity | non communicable diseases world [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | cases | study | mean | hypothyroidism | patients | ischemic | subclinical | thyroid | risk [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | cases | study | mean | hypothyroidism | patients | ischemic | subclinical | thyroid | risk [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| tertiary [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] June to December 2019 | tertiary ||| Institutional review board | National Academy of Medical Sciences | IM | 175 ||| 180 days ||| ||| Microsoft Excel | Statistical Package | Social Sciences | 22 ||| Point | 90% [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 73 | 13 | 17.8% | 90% | 10.44-25.16 ||| 11 | 15.1% | 2 | 2.7% ||| IA | 51 | 70% | IB | 22 | 30% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| tertiary ||| June to December 2019 | tertiary ||| Institutional review board | National Academy of Medical Sciences | IM | 175 ||| 180 days ||| ||| Microsoft Excel | Statistical Package | Social Sciences | 22 ||| Point | 90% ||| ||| 73 | 13 | 17.8% | 90% | 10.44-25.16 ||| 11 | 15.1% | 2 | 2.7% ||| IA | 51 | 70% | IB | 22 | 30% ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| tertiary ||| June to December 2019 | tertiary ||| Institutional review board | National Academy of Medical Sciences | IM | 175 ||| 180 days ||| ||| Microsoft Excel | Statistical Package | Social Sciences | 22 ||| Point | 90% ||| ||| 73 | 13 | 17.8% | 90% | 10.44-25.16 ||| 11 | 15.1% | 2 | 2.7% ||| IA | 51 | 70% | IB | 22 | 30% ||| [SUMMARY]"}}},{"rowIdx":268,"cells":{"title":{"kind":"string","value":"Post-Transplant Lymphoproliferative Diseases in Pediatric Kidney Allograft Recipients with Epstein-Barr Virus Viremia."},"pmid":{"kind":"string","value":"31373185"},"background_abstract":{"kind":"string","value":"Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, especially in children with Epstein-Barr virus (EBV) viremia (EV). We performed a retrospective study to evaluate risk factors for PTLD in children with EV."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Among 199 pediatric kidney transplantation (KT) recipients at our center from January 2001 to October 2015, records of those with EBV viral loads of > 1,000 copies/mL and/or PTLD were reviewed."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Diagnosis of PTLD was made in seven patients (PTLD group), and 39 patients had EV only (EV only group). The median time from KT to EV and PTLD diagnosis was 6.7 (range 0.4-47.8) months and 8.2 (range, 2.8-98.9) months, respectively. There were no significant differences between the groups in terms of sex, age at transplantation, donor type, EBV viral load, or EV-free duration after KT. Higher tacrolimus level before EV (hazard ratio, 44.5; P = 0.003) was an independent risk factor for PTLD in multivariate Cox regression analysis. Six patients with a high EBV load (median 171,639 copies/mL) were treated with preemptive rituximab (RTX) therapy, resulting in transient reduction of EBV load. None of these patients developed PTLD (median follow-up 51.5 months); however, two had neutropenia and two developed infection requiring hospital admission."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"In pediatric KT recipients, higher tacrolimus levels were associated with a higher incidence of PTLD. Conversely, those who received preemptive RTX for EV did not develop PTLD."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Antineoplastic Agents, Immunological","Child, Preschool","Female","Herpesvirus 4, Human","Humans","Infant","Kidney Transplantation","Lymphoproliferative Disorders","Male","Neutropenia","Proportional Hazards Models","Retrospective Studies","Risk Factors","Rituximab","Transplantation, Homologous","Viral Load","Viremia"],"string":"[\n \"Antineoplastic Agents, Immunological\",\n \"Child, Preschool\",\n \"Female\",\n \"Herpesvirus 4, Human\",\n \"Humans\",\n \"Infant\",\n \"Kidney Transplantation\",\n \"Lymphoproliferative Disorders\",\n \"Male\",\n \"Neutropenia\",\n \"Proportional Hazards Models\",\n \"Retrospective Studies\",\n \"Risk Factors\",\n \"Rituximab\",\n \"Transplantation, Homologous\",\n \"Viral Load\",\n \"Viremia\"\n]"},"pmcid":{"kind":"string","value":"6676002"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, with an incidence ranging from 1% to 16% depending on the allograft organ.1 This incidence is 0.2%–2.5% in liver and kidney transplantation cases, which is relatively lower than that in other organ transplant cases.23 Incidence rates of PTLD are higher in pediatric kidney transplant recipients than in adult kidney transplant recipients, ranging from 1.2% to 10%.456 More than 90% of pediatric PTLDs are due to Epstein-Barr virus (EBV)-positive B-cell proliferation. Previously reported risk factors for EBV-associated PTLD include recipient EBV seronegativity, degree of immunosuppression, acute rejection episode, use of OKT3 or tacrolimus, recipient age and race, allograft type, host genetic variations, and especially Epstein-Barr virus viremia (EV).789101112 While adult allograft recipients usually have acquired-immunity to EBV at the time of transplantation, pediatric allograft recipients often experience primary EBV infection after transplantation. Although EBV-infected transformed B cells are highly immunogenic and rapidly eliminated by EBV-specific T cells in healthy hosts, if immunosuppressed pediatric patients have primary EBV infection, then an inadequate immune response may result in massive infection of B cells. Primary EBV infection increases the chance of developing PTLD 6–76 fold.13141516\nEBV infection and/or reactivation, which can be detected as increasing copy numbers of EBV DNA in the peripheral blood, usually precede PTLD. Therefore, it is recommended that EBV titer be regularly monitored after solid-organ transplantation in patients at a higher risk for PTLD. The Kidney Disease Improving Global Outcomes clinical practice guideline for the care of kidney transplant recipients recommended the following monitoring regimen for EBV viral load monitoring in EBV-seronegative patients who received an allograft from a seropositive donor: every 1 week in the first 3 months after transplantation, at least monthly for 3–6 months, and then every 3 months for the rest of the first year.17 Once EBV infection and/or reactivation is noted, transplantation physicians reduce immunosuppression to prevent EBV-associated PTLD. However, EBV infection often persists and progresses despite reduction in immunosuppressive drug regimen, and there is no anti-viral agent with proven efficacy against EBV.8 In contrast, during stem cell transplantation, pre-emptive treatment with rituximab is often used to eradicate B lymphocytes, the reservoir of EBV, thereby to prevent PTLD.1819 A similar approach has been attempted in solid organ transplantation in patients at high-risk for EBV-associated PTLD.20\nIn this study, the risk factors for PTLD were assessed in pediatric kidney allograft recipients with EV, including the effect of pre-emptive rituximab treatment to prevent PTLD."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":" Patients and data collection We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\nWe performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\n Immunosuppression In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\nIn our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\n EV monitoring Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\nPrincipally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\n Pre-emptive rituximab therapy Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\nSome of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\n Statistical analysis To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\nTo determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\n Ethics statement The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\nThe study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"During the study period, 199 children underwent kidney transplantation in our center. Of these, 46 (23.1%) had viremia defined as an EBV load greater than 1,000 copies/mL in whole blood for 2 consecutive tests during a median follow-up period of 5.3 years (Fig. 1). Viremia of all patients (EBV > 1,000 copies/mL) was first detected at a median of 6.7 months (range, 0.4–47.8 months) after kidney transplantation. The diagnosis of PTLD was made in seven patients (PTLD group) at a median of 8.2 months (2.8–98.9 months) after transplantation. The other 39 patients had EV only (EV only group).\nEBV = Epstein-Barr virus, PTLD = post-transplant lymphoproliferative disease, RTX = rituximab.\n Clinical course of PTLD Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\nPatients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\n Risk factors for PTLD Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\nTable 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\n Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\nSix patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Patients and data collection","Immunosuppression","EV monitoring","Pre-emptive rituximab therapy","Statistical analysis","Ethics statement","Clinical course of PTLD","Risk factors for PTLD","Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant"],"string":"[\n \"Patients and data collection\",\n \"Immunosuppression\",\n \"EV monitoring\",\n \"Pre-emptive rituximab therapy\",\n \"Statistical analysis\",\n \"Ethics statement\",\n \"Clinical course of PTLD\",\n \"Risk factors for PTLD\",\n \"Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant\"\n]"},"other_sections_texts":{"kind":"list like","value":["We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.","In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.","Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.","Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.","To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).","The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.","Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.","Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.","Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab."],"string":"[\n \"We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\",\n \"In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\",\n \"Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\",\n \"Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\",\n \"To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\",\n \"The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\",\n \"Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\",\n \"Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\\nValues are expressed as numbers (%) and median (range).\\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\",\n \"Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\\naGenetic disorder caused by WT1 mutation.\\nEBV = Epstein-Barr virus, RTX = rituximab.\\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","Patients and data collection","Immunosuppression","EV monitoring","Pre-emptive rituximab therapy","Statistical analysis","Ethics statement","RESULTS","Clinical course of PTLD","Risk factors for PTLD","Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"Patients and data collection\",\n \"Immunosuppression\",\n \"EV monitoring\",\n \"Pre-emptive rituximab therapy\",\n \"Statistical analysis\",\n \"Ethics statement\",\n \"RESULTS\",\n \"Clinical course of PTLD\",\n \"Risk factors for PTLD\",\n \"Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, with an incidence ranging from 1% to 16% depending on the allograft organ.1 This incidence is 0.2%–2.5% in liver and kidney transplantation cases, which is relatively lower than that in other organ transplant cases.23 Incidence rates of PTLD are higher in pediatric kidney transplant recipients than in adult kidney transplant recipients, ranging from 1.2% to 10%.456 More than 90% of pediatric PTLDs are due to Epstein-Barr virus (EBV)-positive B-cell proliferation. Previously reported risk factors for EBV-associated PTLD include recipient EBV seronegativity, degree of immunosuppression, acute rejection episode, use of OKT3 or tacrolimus, recipient age and race, allograft type, host genetic variations, and especially Epstein-Barr virus viremia (EV).789101112 While adult allograft recipients usually have acquired-immunity to EBV at the time of transplantation, pediatric allograft recipients often experience primary EBV infection after transplantation. Although EBV-infected transformed B cells are highly immunogenic and rapidly eliminated by EBV-specific T cells in healthy hosts, if immunosuppressed pediatric patients have primary EBV infection, then an inadequate immune response may result in massive infection of B cells. Primary EBV infection increases the chance of developing PTLD 6–76 fold.13141516\nEBV infection and/or reactivation, which can be detected as increasing copy numbers of EBV DNA in the peripheral blood, usually precede PTLD. Therefore, it is recommended that EBV titer be regularly monitored after solid-organ transplantation in patients at a higher risk for PTLD. The Kidney Disease Improving Global Outcomes clinical practice guideline for the care of kidney transplant recipients recommended the following monitoring regimen for EBV viral load monitoring in EBV-seronegative patients who received an allograft from a seropositive donor: every 1 week in the first 3 months after transplantation, at least monthly for 3–6 months, and then every 3 months for the rest of the first year.17 Once EBV infection and/or reactivation is noted, transplantation physicians reduce immunosuppression to prevent EBV-associated PTLD. However, EBV infection often persists and progresses despite reduction in immunosuppressive drug regimen, and there is no anti-viral agent with proven efficacy against EBV.8 In contrast, during stem cell transplantation, pre-emptive treatment with rituximab is often used to eradicate B lymphocytes, the reservoir of EBV, thereby to prevent PTLD.1819 A similar approach has been attempted in solid organ transplantation in patients at high-risk for EBV-associated PTLD.20\nIn this study, the risk factors for PTLD were assessed in pediatric kidney allograft recipients with EV, including the effect of pre-emptive rituximab treatment to prevent PTLD."," Patients and data collection We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\nWe performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\n Immunosuppression In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\nIn our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\n EV monitoring Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\nPrincipally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\n Pre-emptive rituximab therapy Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\nSome of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\n Statistical analysis To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\nTo determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\n Ethics statement The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\nThe study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.","We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.","In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.","Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.","Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.","To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).","The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.","During the study period, 199 children underwent kidney transplantation in our center. Of these, 46 (23.1%) had viremia defined as an EBV load greater than 1,000 copies/mL in whole blood for 2 consecutive tests during a median follow-up period of 5.3 years (Fig. 1). Viremia of all patients (EBV > 1,000 copies/mL) was first detected at a median of 6.7 months (range, 0.4–47.8 months) after kidney transplantation. The diagnosis of PTLD was made in seven patients (PTLD group) at a median of 8.2 months (2.8–98.9 months) after transplantation. The other 39 patients had EV only (EV only group).\nEBV = Epstein-Barr virus, PTLD = post-transplant lymphoproliferative disease, RTX = rituximab.\n Clinical course of PTLD Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\nPatients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\n Risk factors for PTLD Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\nTable 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\n Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\nSix patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.","Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.","Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.","Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.","During the last 15 years, among the almost 200 pediatric kidney transplantation recipients at our center, seven developed EBV-associated PTLD (3.5%). In our study, the risk factor for PTLD in pediatric kidney transplant recipients with EV was a high tacrolimus level before EV. Twenty (43.4%) out of 46 recipients who had viremia were EBV seronegative, which is similar to the rates reported in previous studies in North America and Europe (19%–57%).212223 Pre-transplant recipient EBV seronegativity is a well-known risk factor for PTLD. In adult transplant studies, the rate of developing PTLD is 5–12 fold higher in EBV-seronegative patients than in EBV-seropositive patients.2324 McDonald et al.6 reported that EBV-seronegative pediatric subjects have a 4.7-fold higher relative HR than EBV-positive subjects. In our study, recipient EBV seronegativity did not increase the risk of PTLD in multivariate Cox regression analysis. While transplant from an EBV-seropositive donor to a seronegative recipient has been associated with the development of PTLD,25 there was no statistically significant difference in EBV serostatus (donor/recipient) in the present study. This finding was attributed mainly to the fact that there were no EBV seropositive recipients in PTLD group and we did not have serostatus information for the majority of donors (67.3%).\nThe majority of kidney allograft recipients in this study were given tacrolimus, and mycophenolate was used in more than 80% of patients instead of azathioprine.26 By the late 2000s, monoclonal interleukin-2 receptor antibodies were used as induction therapy in up to 80% of patients. Basiliximab, a monoclonal antibody which targets activated T lymphocytes, was not related to PTLD risk in our study, as previously reported.427 Several studies have suggested that higher tacrolimus levels are associated with higher risk for PTLD, and others have reported that the net state of immunosuppression, rather than any individual agent, increases the risk for PTLD.282930 In our study, all patients received tacrolimus for maintenance immunosuppression, and we found that a higher pre-EV tacrolimus level in the PTLD group compared with the EV only group was a risk factor for PTLD.\nRegular monitoring of EBV viral load and early recognition of recipients at high risk of PTLD have been identified as clinical priorities in recent years.31 Previous studies have shown that elevated levels of EBV DNA and persistent high EBV loads are risk factors for PTLD,122032 but no clear cut-off point of EBV viral load for the prediction of PTLD development has been determined. We did not find a significant relationship between EV and PTLD in this study; however, six patients with a high EBV titer were treated with preemptive RTX and they did not develop PTLD. Because they were included in analysis, this could have confounded the causality of high EBV titer and PTLD development.\nTreatment strategies for organ transplant recipients with EV include reduction of immunosuppression with/without antiviral agents, immunoglobulin, or RTX.33 These treatments are still undergoing clinical studies. Preemptive administration of RTX is widely used and has been demonstrated to reduce the incidence of PTLD in stem cell transplant recipients with a high EBV viral load.183435 Pre-emptive RTX therapy has been reported in 14.5% of global transplant programs, and in more than 60% of pediatric transplant patients worldwide.33 However, only one study reported the use of RTX in five pediatric renal allograft recipients,20 and there have been no prospective studies on the efficacy of pre-emptive RTX therapy in solid organ transplantation. Rituximab is a murine/human chimeric monoclonal anti-CD20 antibody and is able to deplete circulating B cells rapidly, including those infected with EBV. Although RTX was effective for reducing EBV viral load in our patients, this reduction was not permanent in line with previous reports.20 In addition, significant adverse effects, such as infection and neutropenia, accompanied RTX administration. In patients with stem cell transplant (SCT), preemptive RTX was not associated with an increase in infectious complications.1819 This is important because, while SCT recipients are able to discontinue their immunosuppressive agents within 6–9 months after SCT, recipients of solid organ transplantation have to be on immunosuppressive agents for as long as their allografts are functioning. Therefore, the long-term use of immunosuppressive agents may explain why infection and neutropenia are common clinical findings after RTX therapy in this patient population.\nAlthough the small sample size of the current study precludes us from drawing any definite conclusions, our observations suggest that preemptive RTX treatment may effectively reduce high EBV viral load in pediatric recipients of solid organ transplants. Because RTX therapy eradicates B-lymphocytes including transformed lymphocytes, the risk of PTLD might be reduced at least during the period of B cell depletion. However, one should take into account that this treatment significantly increases the risk of neutropenia and infection. More research into the influence of preemptive RTX therapy on PTLD development is needed.\nThe occurrence of PTLD in kidney transplant recipients follows a bimodal distribution, with one peak in the first year and the second in the later post-transplantation period. Early PTLD, occurring within the first year of transplantation, is associated with EBV infection and tends to occur more commonly in children than adults.123637 In this study, while six patients in the PTLD group were diagnosed with PTLD within the first year of renal transplantation, one patient with WT1 mutation developed PTLD later than 8.2 years after renal transplantation. We suspect that in this patient, the WT1 mutation of a tumor suppressor gene might have increased the risk of PTLD, and especially that of late-onset. The occurrence of PTLD in patients with WT1 mutation has been reported previously, within the first year in two cases and later than the first year in another.383940 Based on the limited availability of clinical data, the association between WT1 mutation and development of PTLD requires further investigation in future clinical studies and is beyond the scope of the current study.\nThere are several limitations of this study. First, this is a retrospective observational study of a single center and the number of the patients observed was therefore small. The limitations of this small sample size might have affected the outcome of multivariate analysis. In addition, data of donor EBV serology were not uniformly available in our study. Data on donor serology status was only available for 33% of participants. Therefore, our study did not show any association between PTLD and EBV-donor/recipient serostatus. Finally, the number of patients who were treated with RTX was not large enough to draw any definitive conclusions.\nIn summary, this study demonstrates that a higher tacrolimus level before EV is correlated with the development of PTLD. Preemptive RTX appears to be effective for reducing EBV viral load in pediatric kidney transplant recipients. However, the reduction of EBV viral load was not persistent, and adverse effects of RTX, namely infection and neutropenia, were clinically significant."],"string":"[\n \"Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, with an incidence ranging from 1% to 16% depending on the allograft organ.1 This incidence is 0.2%–2.5% in liver and kidney transplantation cases, which is relatively lower than that in other organ transplant cases.23 Incidence rates of PTLD are higher in pediatric kidney transplant recipients than in adult kidney transplant recipients, ranging from 1.2% to 10%.456 More than 90% of pediatric PTLDs are due to Epstein-Barr virus (EBV)-positive B-cell proliferation. Previously reported risk factors for EBV-associated PTLD include recipient EBV seronegativity, degree of immunosuppression, acute rejection episode, use of OKT3 or tacrolimus, recipient age and race, allograft type, host genetic variations, and especially Epstein-Barr virus viremia (EV).789101112 While adult allograft recipients usually have acquired-immunity to EBV at the time of transplantation, pediatric allograft recipients often experience primary EBV infection after transplantation. Although EBV-infected transformed B cells are highly immunogenic and rapidly eliminated by EBV-specific T cells in healthy hosts, if immunosuppressed pediatric patients have primary EBV infection, then an inadequate immune response may result in massive infection of B cells. Primary EBV infection increases the chance of developing PTLD 6–76 fold.13141516\\nEBV infection and/or reactivation, which can be detected as increasing copy numbers of EBV DNA in the peripheral blood, usually precede PTLD. Therefore, it is recommended that EBV titer be regularly monitored after solid-organ transplantation in patients at a higher risk for PTLD. The Kidney Disease Improving Global Outcomes clinical practice guideline for the care of kidney transplant recipients recommended the following monitoring regimen for EBV viral load monitoring in EBV-seronegative patients who received an allograft from a seropositive donor: every 1 week in the first 3 months after transplantation, at least monthly for 3–6 months, and then every 3 months for the rest of the first year.17 Once EBV infection and/or reactivation is noted, transplantation physicians reduce immunosuppression to prevent EBV-associated PTLD. However, EBV infection often persists and progresses despite reduction in immunosuppressive drug regimen, and there is no anti-viral agent with proven efficacy against EBV.8 In contrast, during stem cell transplantation, pre-emptive treatment with rituximab is often used to eradicate B lymphocytes, the reservoir of EBV, thereby to prevent PTLD.1819 A similar approach has been attempted in solid organ transplantation in patients at high-risk for EBV-associated PTLD.20\\nIn this study, the risk factors for PTLD were assessed in pediatric kidney allograft recipients with EV, including the effect of pre-emptive rituximab treatment to prevent PTLD.\",\n \" Patients and data collection We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\\nWe performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\\n Immunosuppression In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\\nIn our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\\n EV monitoring Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\\nPrincipally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\\n Pre-emptive rituximab therapy Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\\nSome of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\\n Statistical analysis To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\\nTo determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\\n Ethics statement The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\\nThe study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\",\n \"We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\",\n \"In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\",\n \"Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\",\n \"Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\",\n \"To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\",\n \"The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\",\n \"During the study period, 199 children underwent kidney transplantation in our center. Of these, 46 (23.1%) had viremia defined as an EBV load greater than 1,000 copies/mL in whole blood for 2 consecutive tests during a median follow-up period of 5.3 years (Fig. 1). Viremia of all patients (EBV > 1,000 copies/mL) was first detected at a median of 6.7 months (range, 0.4–47.8 months) after kidney transplantation. The diagnosis of PTLD was made in seven patients (PTLD group) at a median of 8.2 months (2.8–98.9 months) after transplantation. The other 39 patients had EV only (EV only group).\\nEBV = Epstein-Barr virus, PTLD = post-transplant lymphoproliferative disease, RTX = rituximab.\\n Clinical course of PTLD Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\\nPatients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\\n Risk factors for PTLD Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\\nValues are expressed as numbers (%) and median (range).\\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\\nTable 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\\nValues are expressed as numbers (%) and median (range).\\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\\n Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\\naGenetic disorder caused by WT1 mutation.\\nEBV = Epstein-Barr virus, RTX = rituximab.\\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\\nSix patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\\naGenetic disorder caused by WT1 mutation.\\nEBV = Epstein-Barr virus, RTX = rituximab.\\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\",\n \"Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\",\n \"Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\\nValues are expressed as numbers (%) and median (range).\\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\",\n \"Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\\naGenetic disorder caused by WT1 mutation.\\nEBV = Epstein-Barr virus, RTX = rituximab.\\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\",\n \"During the last 15 years, among the almost 200 pediatric kidney transplantation recipients at our center, seven developed EBV-associated PTLD (3.5%). In our study, the risk factor for PTLD in pediatric kidney transplant recipients with EV was a high tacrolimus level before EV. Twenty (43.4%) out of 46 recipients who had viremia were EBV seronegative, which is similar to the rates reported in previous studies in North America and Europe (19%–57%).212223 Pre-transplant recipient EBV seronegativity is a well-known risk factor for PTLD. In adult transplant studies, the rate of developing PTLD is 5–12 fold higher in EBV-seronegative patients than in EBV-seropositive patients.2324 McDonald et al.6 reported that EBV-seronegative pediatric subjects have a 4.7-fold higher relative HR than EBV-positive subjects. In our study, recipient EBV seronegativity did not increase the risk of PTLD in multivariate Cox regression analysis. While transplant from an EBV-seropositive donor to a seronegative recipient has been associated with the development of PTLD,25 there was no statistically significant difference in EBV serostatus (donor/recipient) in the present study. This finding was attributed mainly to the fact that there were no EBV seropositive recipients in PTLD group and we did not have serostatus information for the majority of donors (67.3%).\\nThe majority of kidney allograft recipients in this study were given tacrolimus, and mycophenolate was used in more than 80% of patients instead of azathioprine.26 By the late 2000s, monoclonal interleukin-2 receptor antibodies were used as induction therapy in up to 80% of patients. Basiliximab, a monoclonal antibody which targets activated T lymphocytes, was not related to PTLD risk in our study, as previously reported.427 Several studies have suggested that higher tacrolimus levels are associated with higher risk for PTLD, and others have reported that the net state of immunosuppression, rather than any individual agent, increases the risk for PTLD.282930 In our study, all patients received tacrolimus for maintenance immunosuppression, and we found that a higher pre-EV tacrolimus level in the PTLD group compared with the EV only group was a risk factor for PTLD.\\nRegular monitoring of EBV viral load and early recognition of recipients at high risk of PTLD have been identified as clinical priorities in recent years.31 Previous studies have shown that elevated levels of EBV DNA and persistent high EBV loads are risk factors for PTLD,122032 but no clear cut-off point of EBV viral load for the prediction of PTLD development has been determined. We did not find a significant relationship between EV and PTLD in this study; however, six patients with a high EBV titer were treated with preemptive RTX and they did not develop PTLD. Because they were included in analysis, this could have confounded the causality of high EBV titer and PTLD development.\\nTreatment strategies for organ transplant recipients with EV include reduction of immunosuppression with/without antiviral agents, immunoglobulin, or RTX.33 These treatments are still undergoing clinical studies. Preemptive administration of RTX is widely used and has been demonstrated to reduce the incidence of PTLD in stem cell transplant recipients with a high EBV viral load.183435 Pre-emptive RTX therapy has been reported in 14.5% of global transplant programs, and in more than 60% of pediatric transplant patients worldwide.33 However, only one study reported the use of RTX in five pediatric renal allograft recipients,20 and there have been no prospective studies on the efficacy of pre-emptive RTX therapy in solid organ transplantation. Rituximab is a murine/human chimeric monoclonal anti-CD20 antibody and is able to deplete circulating B cells rapidly, including those infected with EBV. Although RTX was effective for reducing EBV viral load in our patients, this reduction was not permanent in line with previous reports.20 In addition, significant adverse effects, such as infection and neutropenia, accompanied RTX administration. In patients with stem cell transplant (SCT), preemptive RTX was not associated with an increase in infectious complications.1819 This is important because, while SCT recipients are able to discontinue their immunosuppressive agents within 6–9 months after SCT, recipients of solid organ transplantation have to be on immunosuppressive agents for as long as their allografts are functioning. Therefore, the long-term use of immunosuppressive agents may explain why infection and neutropenia are common clinical findings after RTX therapy in this patient population.\\nAlthough the small sample size of the current study precludes us from drawing any definite conclusions, our observations suggest that preemptive RTX treatment may effectively reduce high EBV viral load in pediatric recipients of solid organ transplants. Because RTX therapy eradicates B-lymphocytes including transformed lymphocytes, the risk of PTLD might be reduced at least during the period of B cell depletion. However, one should take into account that this treatment significantly increases the risk of neutropenia and infection. More research into the influence of preemptive RTX therapy on PTLD development is needed.\\nThe occurrence of PTLD in kidney transplant recipients follows a bimodal distribution, with one peak in the first year and the second in the later post-transplantation period. Early PTLD, occurring within the first year of transplantation, is associated with EBV infection and tends to occur more commonly in children than adults.123637 In this study, while six patients in the PTLD group were diagnosed with PTLD within the first year of renal transplantation, one patient with WT1 mutation developed PTLD later than 8.2 years after renal transplantation. We suspect that in this patient, the WT1 mutation of a tumor suppressor gene might have increased the risk of PTLD, and especially that of late-onset. The occurrence of PTLD in patients with WT1 mutation has been reported previously, within the first year in two cases and later than the first year in another.383940 Based on the limited availability of clinical data, the association between WT1 mutation and development of PTLD requires further investigation in future clinical studies and is beyond the scope of the current study.\\nThere are several limitations of this study. First, this is a retrospective observational study of a single center and the number of the patients observed was therefore small. The limitations of this small sample size might have affected the outcome of multivariate analysis. In addition, data of donor EBV serology were not uniformly available in our study. Data on donor serology status was only available for 33% of participants. Therefore, our study did not show any association between PTLD and EBV-donor/recipient serostatus. Finally, the number of patients who were treated with RTX was not large enough to draw any definitive conclusions.\\nIn summary, this study demonstrates that a higher tacrolimus level before EV is correlated with the development of PTLD. Preemptive RTX appears to be effective for reducing EBV viral load in pediatric kidney transplant recipients. However, the reduction of EBV viral load was not persistent, and adverse effects of RTX, namely infection and neutropenia, were clinically significant.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,null,"results",null,null,null,"discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Post-Transplant Lymphoproliferative Disease","Kidney Transplantation","Epstein-Barr Virus","Rituximab"],"string":"[\n \"Post-Transplant Lymphoproliferative Disease\",\n \"Kidney Transplantation\",\n \"Epstein-Barr Virus\",\n \"Rituximab\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nPost-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, with an incidence ranging from 1% to 16% depending on the allograft organ.1 This incidence is 0.2%–2.5% in liver and kidney transplantation cases, which is relatively lower than that in other organ transplant cases.23 Incidence rates of PTLD are higher in pediatric kidney transplant recipients than in adult kidney transplant recipients, ranging from 1.2% to 10%.456 More than 90% of pediatric PTLDs are due to Epstein-Barr virus (EBV)-positive B-cell proliferation. Previously reported risk factors for EBV-associated PTLD include recipient EBV seronegativity, degree of immunosuppression, acute rejection episode, use of OKT3 or tacrolimus, recipient age and race, allograft type, host genetic variations, and especially Epstein-Barr virus viremia (EV).789101112 While adult allograft recipients usually have acquired-immunity to EBV at the time of transplantation, pediatric allograft recipients often experience primary EBV infection after transplantation. Although EBV-infected transformed B cells are highly immunogenic and rapidly eliminated by EBV-specific T cells in healthy hosts, if immunosuppressed pediatric patients have primary EBV infection, then an inadequate immune response may result in massive infection of B cells. Primary EBV infection increases the chance of developing PTLD 6–76 fold.13141516\nEBV infection and/or reactivation, which can be detected as increasing copy numbers of EBV DNA in the peripheral blood, usually precede PTLD. Therefore, it is recommended that EBV titer be regularly monitored after solid-organ transplantation in patients at a higher risk for PTLD. The Kidney Disease Improving Global Outcomes clinical practice guideline for the care of kidney transplant recipients recommended the following monitoring regimen for EBV viral load monitoring in EBV-seronegative patients who received an allograft from a seropositive donor: every 1 week in the first 3 months after transplantation, at least monthly for 3–6 months, and then every 3 months for the rest of the first year.17 Once EBV infection and/or reactivation is noted, transplantation physicians reduce immunosuppression to prevent EBV-associated PTLD. However, EBV infection often persists and progresses despite reduction in immunosuppressive drug regimen, and there is no anti-viral agent with proven efficacy against EBV.8 In contrast, during stem cell transplantation, pre-emptive treatment with rituximab is often used to eradicate B lymphocytes, the reservoir of EBV, thereby to prevent PTLD.1819 A similar approach has been attempted in solid organ transplantation in patients at high-risk for EBV-associated PTLD.20\nIn this study, the risk factors for PTLD were assessed in pediatric kidney allograft recipients with EV, including the effect of pre-emptive rituximab treatment to prevent PTLD.\n\nMETHODS:\n Patients and data collection We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\nWe performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\n Immunosuppression In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\nIn our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\n EV monitoring Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\nPrincipally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\n Pre-emptive rituximab therapy Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\nSome of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\n Statistical analysis To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\nTo determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\n Ethics statement The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\nThe study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\n\nPatients and data collection:\nWe performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\n\nImmunosuppression:\nIn our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\n\nEV monitoring:\nPrincipally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\n\nPre-emptive rituximab therapy:\nSome of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\n\nStatistical analysis:\nTo determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\n\nEthics statement:\nThe study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\n\nRESULTS:\nDuring the study period, 199 children underwent kidney transplantation in our center. Of these, 46 (23.1%) had viremia defined as an EBV load greater than 1,000 copies/mL in whole blood for 2 consecutive tests during a median follow-up period of 5.3 years (Fig. 1). Viremia of all patients (EBV > 1,000 copies/mL) was first detected at a median of 6.7 months (range, 0.4–47.8 months) after kidney transplantation. The diagnosis of PTLD was made in seven patients (PTLD group) at a median of 8.2 months (2.8–98.9 months) after transplantation. The other 39 patients had EV only (EV only group).\nEBV = Epstein-Barr virus, PTLD = post-transplant lymphoproliferative disease, RTX = rituximab.\n Clinical course of PTLD Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\nPatients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\n Risk factors for PTLD Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\nTable 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\n Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\nSix patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\n\nClinical course of PTLD:\nPatients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\n\nRisk factors for PTLD:\nTable 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\n\nEfficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant:\nSix patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\n\nDISCUSSION:\nDuring the last 15 years, among the almost 200 pediatric kidney transplantation recipients at our center, seven developed EBV-associated PTLD (3.5%). In our study, the risk factor for PTLD in pediatric kidney transplant recipients with EV was a high tacrolimus level before EV. Twenty (43.4%) out of 46 recipients who had viremia were EBV seronegative, which is similar to the rates reported in previous studies in North America and Europe (19%–57%).212223 Pre-transplant recipient EBV seronegativity is a well-known risk factor for PTLD. In adult transplant studies, the rate of developing PTLD is 5–12 fold higher in EBV-seronegative patients than in EBV-seropositive patients.2324 McDonald et al.6 reported that EBV-seronegative pediatric subjects have a 4.7-fold higher relative HR than EBV-positive subjects. In our study, recipient EBV seronegativity did not increase the risk of PTLD in multivariate Cox regression analysis. While transplant from an EBV-seropositive donor to a seronegative recipient has been associated with the development of PTLD,25 there was no statistically significant difference in EBV serostatus (donor/recipient) in the present study. This finding was attributed mainly to the fact that there were no EBV seropositive recipients in PTLD group and we did not have serostatus information for the majority of donors (67.3%).\nThe majority of kidney allograft recipients in this study were given tacrolimus, and mycophenolate was used in more than 80% of patients instead of azathioprine.26 By the late 2000s, monoclonal interleukin-2 receptor antibodies were used as induction therapy in up to 80% of patients. Basiliximab, a monoclonal antibody which targets activated T lymphocytes, was not related to PTLD risk in our study, as previously reported.427 Several studies have suggested that higher tacrolimus levels are associated with higher risk for PTLD, and others have reported that the net state of immunosuppression, rather than any individual agent, increases the risk for PTLD.282930 In our study, all patients received tacrolimus for maintenance immunosuppression, and we found that a higher pre-EV tacrolimus level in the PTLD group compared with the EV only group was a risk factor for PTLD.\nRegular monitoring of EBV viral load and early recognition of recipients at high risk of PTLD have been identified as clinical priorities in recent years.31 Previous studies have shown that elevated levels of EBV DNA and persistent high EBV loads are risk factors for PTLD,122032 but no clear cut-off point of EBV viral load for the prediction of PTLD development has been determined. We did not find a significant relationship between EV and PTLD in this study; however, six patients with a high EBV titer were treated with preemptive RTX and they did not develop PTLD. Because they were included in analysis, this could have confounded the causality of high EBV titer and PTLD development.\nTreatment strategies for organ transplant recipients with EV include reduction of immunosuppression with/without antiviral agents, immunoglobulin, or RTX.33 These treatments are still undergoing clinical studies. Preemptive administration of RTX is widely used and has been demonstrated to reduce the incidence of PTLD in stem cell transplant recipients with a high EBV viral load.183435 Pre-emptive RTX therapy has been reported in 14.5% of global transplant programs, and in more than 60% of pediatric transplant patients worldwide.33 However, only one study reported the use of RTX in five pediatric renal allograft recipients,20 and there have been no prospective studies on the efficacy of pre-emptive RTX therapy in solid organ transplantation. Rituximab is a murine/human chimeric monoclonal anti-CD20 antibody and is able to deplete circulating B cells rapidly, including those infected with EBV. Although RTX was effective for reducing EBV viral load in our patients, this reduction was not permanent in line with previous reports.20 In addition, significant adverse effects, such as infection and neutropenia, accompanied RTX administration. In patients with stem cell transplant (SCT), preemptive RTX was not associated with an increase in infectious complications.1819 This is important because, while SCT recipients are able to discontinue their immunosuppressive agents within 6–9 months after SCT, recipients of solid organ transplantation have to be on immunosuppressive agents for as long as their allografts are functioning. Therefore, the long-term use of immunosuppressive agents may explain why infection and neutropenia are common clinical findings after RTX therapy in this patient population.\nAlthough the small sample size of the current study precludes us from drawing any definite conclusions, our observations suggest that preemptive RTX treatment may effectively reduce high EBV viral load in pediatric recipients of solid organ transplants. Because RTX therapy eradicates B-lymphocytes including transformed lymphocytes, the risk of PTLD might be reduced at least during the period of B cell depletion. However, one should take into account that this treatment significantly increases the risk of neutropenia and infection. More research into the influence of preemptive RTX therapy on PTLD development is needed.\nThe occurrence of PTLD in kidney transplant recipients follows a bimodal distribution, with one peak in the first year and the second in the later post-transplantation period. Early PTLD, occurring within the first year of transplantation, is associated with EBV infection and tends to occur more commonly in children than adults.123637 In this study, while six patients in the PTLD group were diagnosed with PTLD within the first year of renal transplantation, one patient with WT1 mutation developed PTLD later than 8.2 years after renal transplantation. We suspect that in this patient, the WT1 mutation of a tumor suppressor gene might have increased the risk of PTLD, and especially that of late-onset. The occurrence of PTLD in patients with WT1 mutation has been reported previously, within the first year in two cases and later than the first year in another.383940 Based on the limited availability of clinical data, the association between WT1 mutation and development of PTLD requires further investigation in future clinical studies and is beyond the scope of the current study.\nThere are several limitations of this study. First, this is a retrospective observational study of a single center and the number of the patients observed was therefore small. The limitations of this small sample size might have affected the outcome of multivariate analysis. In addition, data of donor EBV serology were not uniformly available in our study. Data on donor serology status was only available for 33% of participants. Therefore, our study did not show any association between PTLD and EBV-donor/recipient serostatus. Finally, the number of patients who were treated with RTX was not large enough to draw any definitive conclusions.\nIn summary, this study demonstrates that a higher tacrolimus level before EV is correlated with the development of PTLD. Preemptive RTX appears to be effective for reducing EBV viral load in pediatric kidney transplant recipients. However, the reduction of EBV viral load was not persistent, and adverse effects of RTX, namely infection and neutropenia, were clinically significant.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nPost-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, especially in children with Epstein-Barr virus (EBV) viremia (EV). We performed a retrospective study to evaluate risk factors for PTLD in children with EV.\n\nMethods:\nAmong 199 pediatric kidney transplantation (KT) recipients at our center from January 2001 to October 2015, records of those with EBV viral loads of > 1,000 copies/mL and/or PTLD were reviewed.\n\nResults:\nDiagnosis of PTLD was made in seven patients (PTLD group), and 39 patients had EV only (EV only group). The median time from KT to EV and PTLD diagnosis was 6.7 (range 0.4-47.8) months and 8.2 (range, 2.8-98.9) months, respectively. There were no significant differences between the groups in terms of sex, age at transplantation, donor type, EBV viral load, or EV-free duration after KT. Higher tacrolimus level before EV (hazard ratio, 44.5; P = 0.003) was an independent risk factor for PTLD in multivariate Cox regression analysis. Six patients with a high EBV load (median 171,639 copies/mL) were treated with preemptive rituximab (RTX) therapy, resulting in transient reduction of EBV load. None of these patients developed PTLD (median follow-up 51.5 months); however, two had neutropenia and two developed infection requiring hospital admission.\n\nConclusions:\nIn pediatric KT recipients, higher tacrolimus levels were associated with a higher incidence of PTLD. Conversely, those who received preemptive RTX for EV did not develop PTLD."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":8529,"string":"8,529"},"whole_article_abstract_length":{"kind":"number","value":310,"string":"310"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["ebv","ptld","patients","rtx","ev","transplantation","months","kidney","therapy","ml"],"string":"[\n \"ebv\",\n \"ptld\",\n \"patients\",\n \"rtx\",\n \"ev\",\n \"transplantation\",\n \"months\",\n \"kidney\",\n \"therapy\",\n \"ml\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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171,639 | EBV ||| 51.5 months | two | two [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Epstein-Barr | EBV | EV ||| EV ||| 199 | KT | January 2001 to October 2015 | EBV | 1,000 ||| seven | 39 | EV | EV ||| KT | EV | 6.7 | 0.4-47.8 | months | 8.2 | 2.8 | months ||| EV | KT ||| EV | 44.5 | P = | 0.003 ||| Six | EBV | 171,639 | EBV ||| 51.5 months | two | two ||| KT ||| RTX | EV [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":269,"cells":{"title":{"kind":"string","value":"Influence of ischemia before vein grafting on early hyperplasia of the graft and the dynamic changes of the intima after grafting."},"pmid":{"kind":"string","value":"23006637"},"background_abstract":{"kind":"string","value":"To investigate both the influence of ischemia before grafting on early hyperplasia of the vein grafts, and the dynamic changes of the intima after grafting in a rabbit model of vein graft disease."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We performed paired vein graft experiments under different ischemic conditions (15 vs. 60 min; 15 vs. 90 min) in the neck of the rabbits and compared the differences between the grafts. Clopidogrel, an anti-platelet agent, was administered before and after surgery. Twenty-eight days after the grafting procedure, the veins were evaluated microscopically. The dynamic changes of the intima after grafting were evaluated by scanning electron microscopy over time."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The vein grafts subjected to 60- or 90-min ischemia exhibited no differences compared to those subjected to 15-min ischemia in terms of the mean thickness of the intimal, medial, and adventitial layers of the graft. Similarly, there was no difference in the Ki-67 labeling index (proliferation marker) between the vein grafts. Vein grafts with 15-min ischemia lost endothelial cells (ECs) but healed by 3 days post graft, whereas vein grafts with 90-min ischemia suffered serious EC loss, which was restored with new ECs during days 2 to 14 post graft."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Ninety-minute ischemia before vein grafting can cause serious EC loss, but does not increase early intimal hyperplasia when clopidogrel is administered. Protecting the vein from ischemia and reperfusion injury preserves ECs."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Blood Vessel Prosthesis","Blood Vessel Prosthesis Implantation","Cell Proliferation","Clopidogrel","Endothelial Cells","Histocytochemistry","Hyperplasia","Ischemia","Ki-67 Antigen","Microscopy, Electron, Scanning","Platelet Aggregation Inhibitors","Rabbits","Ticlopidine","Tunica Intima"],"string":"[\n \"Animals\",\n \"Blood Vessel Prosthesis\",\n \"Blood Vessel Prosthesis Implantation\",\n \"Cell Proliferation\",\n \"Clopidogrel\",\n \"Endothelial Cells\",\n \"Histocytochemistry\",\n \"Hyperplasia\",\n \"Ischemia\",\n \"Ki-67 Antigen\",\n \"Microscopy, Electron, Scanning\",\n \"Platelet Aggregation Inhibitors\",\n \"Rabbits\",\n \"Ticlopidine\",\n \"Tunica Intima\"\n]"},"pmcid":{"kind":"string","value":"3495780"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Animals and grouping New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\nNew Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\n Vein graft surgery Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nAnesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\n Morphometric analysis The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\nThe vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\n Immunocytochemistry and cell proliferation analysis Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\nCell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\n Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\nSamples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\n Statistical analysis All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\nAll data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Graft patency All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\nAll grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\n Morphometric analysis Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\nUsing the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\n Immunocytochemistry and cell proliferation analysis Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\nMany Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\n Scanning electron microscopy observation of intima SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\nSEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs."},"other_sections_titles":{"kind":"list like","value":["Background","Animals and grouping","Vein graft surgery","Morphometric analysis","Immunocytochemistry and cell proliferation analysis","Scanning electron microscopy","Statistical analysis","Graft patency","Morphometric analysis","Immunocytochemistry and cell proliferation analysis","Scanning electron microscopy observation of intima","Competing interest","Authors' contributions"],"string":"[\n \"Background\",\n \"Animals and grouping\",\n \"Vein graft surgery\",\n \"Morphometric analysis\",\n \"Immunocytochemistry and cell proliferation analysis\",\n \"Scanning electron microscopy\",\n \"Statistical analysis\",\n \"Graft patency\",\n \"Morphometric analysis\",\n \"Immunocytochemistry and cell proliferation analysis\",\n \"Scanning electron microscopy observation of intima\",\n \"Competing interest\",\n \"Authors' contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.","New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.","Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.","The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.","Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.","Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).","All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.","All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.","Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).","Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.","SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.","The authors declare that they have no competing interests.","RJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript."],"string":"[\n \"The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\\n[1-3].\\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.\",\n \"New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\",\n \"Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\",\n \"The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\",\n \"Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\",\n \"Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\",\n \"All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\",\n \"All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\",\n \"Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\\n1, Figure\\n2).\\nComparison of vein grafts under different ischemic conditions\\nValues are mean ± standard deviation.\\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\",\n \"Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\\n3), as in the mean thickness results.\\nThe Ki-67 labeling index of the vein grafts.\",\n \"SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\\n4), likely due to the infusion pressure.\\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\\n5).\\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\",\n \"The authors declare that they have no competing interests.\",\n \"RJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Animals and grouping","Vein graft surgery","Morphometric analysis","Immunocytochemistry and cell proliferation analysis","Scanning electron microscopy","Statistical analysis","Results","Graft patency","Morphometric analysis","Immunocytochemistry and cell proliferation analysis","Scanning electron microscopy observation of intima","Discussion","Conclusions","Competing interest","Authors' contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Animals and grouping\",\n \"Vein graft surgery\",\n \"Morphometric analysis\",\n \"Immunocytochemistry and cell proliferation analysis\",\n \"Scanning electron microscopy\",\n \"Statistical analysis\",\n \"Results\",\n \"Graft patency\",\n \"Morphometric analysis\",\n \"Immunocytochemistry and cell proliferation analysis\",\n \"Scanning electron microscopy observation of intima\",\n \"Discussion\",\n \"Conclusions\",\n \"Competing interest\",\n \"Authors' contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used."," Animals and grouping New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\nNew Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\n Vein graft surgery Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nAnesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\n Morphometric analysis The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\nThe vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\n Immunocytochemistry and cell proliferation analysis Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\nCell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\n Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\nSamples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\n Statistical analysis All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\nAll data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.","New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.","Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.","The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.","Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.","Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).","All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant."," Graft patency All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\nAll grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\n Morphometric analysis Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\nUsing the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\n Immunocytochemistry and cell proliferation analysis Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\nMany Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\n Scanning electron microscopy observation of intima SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\nSEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.","All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.","Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).","Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.","SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.","Ischemic injury before grafting and reperfusion injury after grafting may injure ECs, and subsequently activate platelets and trigger the coagulation cascade\n[10]. Various solutions and heparinized autologous blood have been used previously for vein preservation to reduce ischemia and reperfusion (IR) injury. For instance, Cavallari et al. used autologous whole blood (AWB), 0.9% normal saline solution (NS), and University of Wisconsin solution (UWs) individually to treat autogenous vein grafts for 45 min at 4°C before grafting, and found the veins stored in UWs had a similar intimal thickness with that of control samples, whereas the veins stored in AWB or NS had a thicker intima layer after grafting\n[11]. However, Solberg et al. found that veins stored in cell culture media at 4°C show a marked increase in EC dysjunction and an 18% loss of EC number, compared to only a 4% loss at 20°C, indicating that hypothermy may injure ECs\n[12]. In typical situations, the veins are placed in saline containing heparin sodium at 20°C after being removed. Therefore, we designed this study to investigate the influence of pre-grafting ischemia on early hyperplasia after grafting under these conditions, and found the ischemic tolerance of the vein. After the step, we planed to investigate how long the veins can be preserved in the solutions (such as UWs) or how long the AWB should be renewed. The cuff technique was used in this study for the vein graft model because it can limit the ischemic time caused by the procedure to 15 min, avoiding IR injury. In one side, the veins were grafted immediately after removal to offer controls, whereas, in the other side, the veins were stored before being used. We also included clopidogrel treatment to reduce the risk of thrombus or mural thrombus and to make the vein graft model more similar to the clinical situation.\nProspective controlled trials have demonstrated a graft patency benefit when aspirin was started 1, 7, or 24 h postoperatively, a benefit which was lost when started after 48 h\n[9]. Aspirin has been shown to significantly reduce postoperative mortality, myocardial infarction, stroke, renal failure, and bowel infarction when administered within 48 h of CABG\n[8]. However, in patients with atherosclerotic vascular disease, clopidogrel is more effective than aspirin in reducing the combined risk of stroke, myocardial infarction, and vascular death\n[13].\nBesides having anti-thrombotic properties, inhibitors of platelet aggregation attenuate platelet function, prevent platelet activation and release of peptide growth factors and nonpeptide substances, and thereby inhibit intimal hyperplasia\n[14]. Atherosclerosis is described as an inflammatory disease\n[15]. Clopidogrel inhibits the expression of platelet activation markers and the interaction of platelets and leukocytes\n[16], and is powerful in decreasing the levels of inflammatory factors\n[17].\nIn our present study, SEM revealed that 90-min ischemia caused serious injury of the intima, including the exposed subendothelial collagen fibers that may aggregate and activate platelets and the frequently adherent leukocytes, which may trigger vascular inflammation. Clopidogrel was used and avoided thrombus or mural thrombus successfully. Moreover, results show no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia in term of thickness and the hyperplasia intensity of the intima, media, or adventitia at 4 weeks after the grafting procedure. It seems ischemia up to 90 min does not increase early intimal hyperplasia of the vein graft when clopidogrel was used effectively. Approximately 90 min is a long enough time period for surgeons to complete 3 or 4 bypasses; therefore, when using clopidogrel, it is seemingly unnecessary to consider the influence of ischemia before grafting on early hyperplasia of the vein grafts.\nThe recovery of the intima was also observed post operation. EC coverage has been used in some studies\n[18,19], which is easily gained and facilitates statistical analysis. However, based on our study, it is apparently unreliable because fibrin and micro thrombi covered the surface of the intima when there was IR injury according to SEM analysis. Therefore, we only reported the characteristics of the intima in this study.\nAccording to our observations, the intimae of vein grafts with 15-min ischemia only lost a portion of their ECs and reintegrated at 3 days post operation. However, the intimae of vein grafts with 90-min ischemia lost all their ECs and redintegrated at 14 days post operation. Furthermore, the anterior had less risk of thrombosis. Reducing ischemia and reperfusion injury is valuable. It has been suggested previously that UWs preserves the vein before grafting because of its excellent effect on protecting the endothelium and its functions\n[20].\nHowever, vein grafts with 15-min ischemia, which had almost no IR injury, likely face a widely torn intima caused by systolic pressure 1 h after grafting. Furthermore, the vein grafts with 90-min ischemia likely face more serious loss of ECs until new ECs are present, which come predominantly from extrinsic cells such as bone marrow-derived cells\n[21], and form an intima layer with integrity. Thus, it seems antithrombotic therapy should be administered as early as possible post operation.\nGavaghan et al. found that administering aspirin within 1 h of CABG did not increase chest-tube blood loss, the red cell transfusion requirements, or the re-exploration rate\n[22]. However, Goldman et al. reported that aspirin use on the day before CABG has a similar effect on early graft patency within 6 h after surgery, and increased bleeding complications\n[23]. Considering that the number of platelets and the levels of blood coagulation factors vary and change with time after CABG, we recommend starting anti-platelet therapy when the postoperative chest tube drainage is ≤50 ml/h for 2 consecutive hours, as presented by both Halkos et al. and Chan et al.\n[24,25].","In summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs.","The authors declare that they have no competing interests.","RJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript."],"string":"[\n \"The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\\n[1-3].\\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.\",\n \" Animals and grouping New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\\nNew Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\\n Vein graft surgery Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\nAnesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\n Morphometric analysis The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\\nThe vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\\n Immunocytochemistry and cell proliferation analysis Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\\nCell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\\n Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\\nSamples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\\n Statistical analysis All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\\nAll data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\",\n \"New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\",\n \"Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\",\n \"The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\",\n \"Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\",\n \"Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\",\n \"All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\",\n \" Graft patency All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\\nAll grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\\n Morphometric analysis Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\\n1, Figure\\n2).\\nComparison of vein grafts under different ischemic conditions\\nValues are mean ± standard deviation.\\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\\nUsing the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\\n1, Figure\\n2).\\nComparison of vein grafts under different ischemic conditions\\nValues are mean ± standard deviation.\\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\\n Immunocytochemistry and cell proliferation analysis Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\\n3), as in the mean thickness results.\\nThe Ki-67 labeling index of the vein grafts.\\nMany Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\\n3), as in the mean thickness results.\\nThe Ki-67 labeling index of the vein grafts.\\n Scanning electron microscopy observation of intima SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\\n4), likely due to the infusion pressure.\\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\\n5).\\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\\nSEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\\n4), likely due to the infusion pressure.\\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\\n5).\\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\",\n \"All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\",\n \"Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\\n1, Figure\\n2).\\nComparison of vein grafts under different ischemic conditions\\nValues are mean ± standard deviation.\\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\",\n \"Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\\n3), as in the mean thickness results.\\nThe Ki-67 labeling index of the vein grafts.\",\n \"SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\\n4), likely due to the infusion pressure.\\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\\n5).\\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\",\n \"Ischemic injury before grafting and reperfusion injury after grafting may injure ECs, and subsequently activate platelets and trigger the coagulation cascade\\n[10]. Various solutions and heparinized autologous blood have been used previously for vein preservation to reduce ischemia and reperfusion (IR) injury. For instance, Cavallari et al. used autologous whole blood (AWB), 0.9% normal saline solution (NS), and University of Wisconsin solution (UWs) individually to treat autogenous vein grafts for 45 min at 4°C before grafting, and found the veins stored in UWs had a similar intimal thickness with that of control samples, whereas the veins stored in AWB or NS had a thicker intima layer after grafting\\n[11]. However, Solberg et al. found that veins stored in cell culture media at 4°C show a marked increase in EC dysjunction and an 18% loss of EC number, compared to only a 4% loss at 20°C, indicating that hypothermy may injure ECs\\n[12]. In typical situations, the veins are placed in saline containing heparin sodium at 20°C after being removed. Therefore, we designed this study to investigate the influence of pre-grafting ischemia on early hyperplasia after grafting under these conditions, and found the ischemic tolerance of the vein. After the step, we planed to investigate how long the veins can be preserved in the solutions (such as UWs) or how long the AWB should be renewed. The cuff technique was used in this study for the vein graft model because it can limit the ischemic time caused by the procedure to 15 min, avoiding IR injury. In one side, the veins were grafted immediately after removal to offer controls, whereas, in the other side, the veins were stored before being used. We also included clopidogrel treatment to reduce the risk of thrombus or mural thrombus and to make the vein graft model more similar to the clinical situation.\\nProspective controlled trials have demonstrated a graft patency benefit when aspirin was started 1, 7, or 24 h postoperatively, a benefit which was lost when started after 48 h\\n[9]. Aspirin has been shown to significantly reduce postoperative mortality, myocardial infarction, stroke, renal failure, and bowel infarction when administered within 48 h of CABG\\n[8]. However, in patients with atherosclerotic vascular disease, clopidogrel is more effective than aspirin in reducing the combined risk of stroke, myocardial infarction, and vascular death\\n[13].\\nBesides having anti-thrombotic properties, inhibitors of platelet aggregation attenuate platelet function, prevent platelet activation and release of peptide growth factors and nonpeptide substances, and thereby inhibit intimal hyperplasia\\n[14]. Atherosclerosis is described as an inflammatory disease\\n[15]. Clopidogrel inhibits the expression of platelet activation markers and the interaction of platelets and leukocytes\\n[16], and is powerful in decreasing the levels of inflammatory factors\\n[17].\\nIn our present study, SEM revealed that 90-min ischemia caused serious injury of the intima, including the exposed subendothelial collagen fibers that may aggregate and activate platelets and the frequently adherent leukocytes, which may trigger vascular inflammation. Clopidogrel was used and avoided thrombus or mural thrombus successfully. Moreover, results show no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia in term of thickness and the hyperplasia intensity of the intima, media, or adventitia at 4 weeks after the grafting procedure. It seems ischemia up to 90 min does not increase early intimal hyperplasia of the vein graft when clopidogrel was used effectively. Approximately 90 min is a long enough time period for surgeons to complete 3 or 4 bypasses; therefore, when using clopidogrel, it is seemingly unnecessary to consider the influence of ischemia before grafting on early hyperplasia of the vein grafts.\\nThe recovery of the intima was also observed post operation. EC coverage has been used in some studies\\n[18,19], which is easily gained and facilitates statistical analysis. However, based on our study, it is apparently unreliable because fibrin and micro thrombi covered the surface of the intima when there was IR injury according to SEM analysis. Therefore, we only reported the characteristics of the intima in this study.\\nAccording to our observations, the intimae of vein grafts with 15-min ischemia only lost a portion of their ECs and reintegrated at 3 days post operation. However, the intimae of vein grafts with 90-min ischemia lost all their ECs and redintegrated at 14 days post operation. Furthermore, the anterior had less risk of thrombosis. Reducing ischemia and reperfusion injury is valuable. It has been suggested previously that UWs preserves the vein before grafting because of its excellent effect on protecting the endothelium and its functions\\n[20].\\nHowever, vein grafts with 15-min ischemia, which had almost no IR injury, likely face a widely torn intima caused by systolic pressure 1 h after grafting. Furthermore, the vein grafts with 90-min ischemia likely face more serious loss of ECs until new ECs are present, which come predominantly from extrinsic cells such as bone marrow-derived cells\\n[21], and form an intima layer with integrity. Thus, it seems antithrombotic therapy should be administered as early as possible post operation.\\nGavaghan et al. found that administering aspirin within 1 h of CABG did not increase chest-tube blood loss, the red cell transfusion requirements, or the re-exploration rate\\n[22]. However, Goldman et al. reported that aspirin use on the day before CABG has a similar effect on early graft patency within 6 h after surgery, and increased bleeding complications\\n[23]. Considering that the number of platelets and the levels of blood coagulation factors vary and change with time after CABG, we recommend starting anti-platelet therapy when the postoperative chest tube drainage is ≤50 ml/h for 2 consecutive hours, as presented by both Halkos et al. and Chan et al.\\n[24,25].\",\n \"In summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs.\",\n \"The authors declare that they have no competing interests.\",\n \"RJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,"results",null,null,null,null,"discussion","conclusions",null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Vein graft","Ischemia","Intimal hyperplasia","Endothelial cell"],"string":"[\n \"Vein graft\",\n \"Ischemia\",\n \"Intimal hyperplasia\",\n \"Endothelial cell\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nThe autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.\n\nMethods:\n Animals and grouping New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\nNew Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\n Vein graft surgery Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nAnesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\n Morphometric analysis The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\nThe vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\n Immunocytochemistry and cell proliferation analysis Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\nCell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\n Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\nSamples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\n Statistical analysis All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\nAll data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\n\nAnimals and grouping:\nNew Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\n\nVein graft surgery:\nAnesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\n\nMorphometric analysis:\nThe vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\n\nImmunocytochemistry and cell proliferation analysis:\nCell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\n\nScanning electron microscopy:\nSamples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\n\nStatistical analysis:\nAll data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\n\nResults:\n Graft patency All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\nAll grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\n Morphometric analysis Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\nUsing the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\n Immunocytochemistry and cell proliferation analysis Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\nMany Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\n Scanning electron microscopy observation of intima SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\nSEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\n\nGraft patency:\nAll grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\n\nMorphometric analysis:\nUsing the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\n\nImmunocytochemistry and cell proliferation analysis:\nMany Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\n\nScanning electron microscopy observation of intima:\nSEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\n\nDiscussion:\nIschemic injury before grafting and reperfusion injury after grafting may injure ECs, and subsequently activate platelets and trigger the coagulation cascade\n[10]. Various solutions and heparinized autologous blood have been used previously for vein preservation to reduce ischemia and reperfusion (IR) injury. For instance, Cavallari et al. used autologous whole blood (AWB), 0.9% normal saline solution (NS), and University of Wisconsin solution (UWs) individually to treat autogenous vein grafts for 45 min at 4°C before grafting, and found the veins stored in UWs had a similar intimal thickness with that of control samples, whereas the veins stored in AWB or NS had a thicker intima layer after grafting\n[11]. However, Solberg et al. found that veins stored in cell culture media at 4°C show a marked increase in EC dysjunction and an 18% loss of EC number, compared to only a 4% loss at 20°C, indicating that hypothermy may injure ECs\n[12]. In typical situations, the veins are placed in saline containing heparin sodium at 20°C after being removed. Therefore, we designed this study to investigate the influence of pre-grafting ischemia on early hyperplasia after grafting under these conditions, and found the ischemic tolerance of the vein. After the step, we planed to investigate how long the veins can be preserved in the solutions (such as UWs) or how long the AWB should be renewed. The cuff technique was used in this study for the vein graft model because it can limit the ischemic time caused by the procedure to 15 min, avoiding IR injury. In one side, the veins were grafted immediately after removal to offer controls, whereas, in the other side, the veins were stored before being used. We also included clopidogrel treatment to reduce the risk of thrombus or mural thrombus and to make the vein graft model more similar to the clinical situation.\nProspective controlled trials have demonstrated a graft patency benefit when aspirin was started 1, 7, or 24 h postoperatively, a benefit which was lost when started after 48 h\n[9]. Aspirin has been shown to significantly reduce postoperative mortality, myocardial infarction, stroke, renal failure, and bowel infarction when administered within 48 h of CABG\n[8]. However, in patients with atherosclerotic vascular disease, clopidogrel is more effective than aspirin in reducing the combined risk of stroke, myocardial infarction, and vascular death\n[13].\nBesides having anti-thrombotic properties, inhibitors of platelet aggregation attenuate platelet function, prevent platelet activation and release of peptide growth factors and nonpeptide substances, and thereby inhibit intimal hyperplasia\n[14]. Atherosclerosis is described as an inflammatory disease\n[15]. Clopidogrel inhibits the expression of platelet activation markers and the interaction of platelets and leukocytes\n[16], and is powerful in decreasing the levels of inflammatory factors\n[17].\nIn our present study, SEM revealed that 90-min ischemia caused serious injury of the intima, including the exposed subendothelial collagen fibers that may aggregate and activate platelets and the frequently adherent leukocytes, which may trigger vascular inflammation. Clopidogrel was used and avoided thrombus or mural thrombus successfully. Moreover, results show no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia in term of thickness and the hyperplasia intensity of the intima, media, or adventitia at 4 weeks after the grafting procedure. It seems ischemia up to 90 min does not increase early intimal hyperplasia of the vein graft when clopidogrel was used effectively. Approximately 90 min is a long enough time period for surgeons to complete 3 or 4 bypasses; therefore, when using clopidogrel, it is seemingly unnecessary to consider the influence of ischemia before grafting on early hyperplasia of the vein grafts.\nThe recovery of the intima was also observed post operation. EC coverage has been used in some studies\n[18,19], which is easily gained and facilitates statistical analysis. However, based on our study, it is apparently unreliable because fibrin and micro thrombi covered the surface of the intima when there was IR injury according to SEM analysis. Therefore, we only reported the characteristics of the intima in this study.\nAccording to our observations, the intimae of vein grafts with 15-min ischemia only lost a portion of their ECs and reintegrated at 3 days post operation. However, the intimae of vein grafts with 90-min ischemia lost all their ECs and redintegrated at 14 days post operation. Furthermore, the anterior had less risk of thrombosis. Reducing ischemia and reperfusion injury is valuable. It has been suggested previously that UWs preserves the vein before grafting because of its excellent effect on protecting the endothelium and its functions\n[20].\nHowever, vein grafts with 15-min ischemia, which had almost no IR injury, likely face a widely torn intima caused by systolic pressure 1 h after grafting. Furthermore, the vein grafts with 90-min ischemia likely face more serious loss of ECs until new ECs are present, which come predominantly from extrinsic cells such as bone marrow-derived cells\n[21], and form an intima layer with integrity. Thus, it seems antithrombotic therapy should be administered as early as possible post operation.\nGavaghan et al. found that administering aspirin within 1 h of CABG did not increase chest-tube blood loss, the red cell transfusion requirements, or the re-exploration rate\n[22]. However, Goldman et al. reported that aspirin use on the day before CABG has a similar effect on early graft patency within 6 h after surgery, and increased bleeding complications\n[23]. Considering that the number of platelets and the levels of blood coagulation factors vary and change with time after CABG, we recommend starting anti-platelet therapy when the postoperative chest tube drainage is ≤50 ml/h for 2 consecutive hours, as presented by both Halkos et al. and Chan et al.\n[24,25].\n\nConclusions:\nIn summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs.\n\nCompeting interest:\nThe authors declare that they have no competing interests.\n\nAuthors' contributions:\nRJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nTo investigate both the influence of ischemia before grafting on early hyperplasia of the vein grafts, and the dynamic changes of the intima after grafting in a rabbit model of vein graft disease.\n\nMethods:\nWe performed paired vein graft experiments under different ischemic conditions (15 vs. 60 min; 15 vs. 90 min) in the neck of the rabbits and compared the differences between the grafts. Clopidogrel, an anti-platelet agent, was administered before and after surgery. Twenty-eight days after the grafting procedure, the veins were evaluated microscopically. The dynamic changes of the intima after grafting were evaluated by scanning electron microscopy over time.\n\nResults:\nThe vein grafts subjected to 60- or 90-min ischemia exhibited no differences compared to those subjected to 15-min ischemia in terms of the mean thickness of the intimal, medial, and adventitial layers of the graft. Similarly, there was no difference in the Ki-67 labeling index (proliferation marker) between the vein grafts. Vein grafts with 15-min ischemia lost endothelial cells (ECs) but healed by 3 days post graft, whereas vein grafts with 90-min ischemia suffered serious EC loss, which was restored with new ECs during days 2 to 14 post graft.\n\nConclusions:\nNinety-minute ischemia before vein grafting can cause serious EC loss, but does not increase early intimal hyperplasia when clopidogrel is administered. Protecting the vein from ischemia and reperfusion injury preserves ECs."},"background_conclusion_text":{"kind":"string","value":"Background:\nThe autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.\n\nConclusions:\nIn summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nTo investigate both the influence of ischemia before grafting on early hyperplasia of the vein grafts, and the dynamic changes of the intima after grafting in a rabbit model of vein graft disease.\n\nMethods:\nWe performed paired vein graft experiments under different ischemic conditions (15 vs. 60 min; 15 vs. 90 min) in the neck of the rabbits and compared the differences between the grafts. Clopidogrel, an anti-platelet agent, was administered before and after surgery. Twenty-eight days after the grafting procedure, the veins were evaluated microscopically. The dynamic changes of the intima after grafting were evaluated by scanning electron microscopy over time.\n\nResults:\nThe vein grafts subjected to 60- or 90-min ischemia exhibited no differences compared to those subjected to 15-min ischemia in terms of the mean thickness of the intimal, medial, and adventitial layers of the graft. Similarly, there was no difference in the Ki-67 labeling index (proliferation marker) between the vein grafts. Vein grafts with 15-min ischemia lost endothelial cells (ECs) but healed by 3 days post graft, whereas vein grafts with 90-min ischemia suffered serious EC loss, which was restored with new ECs during days 2 to 14 post graft.\n\nConclusions:\nNinety-minute ischemia before vein grafting can cause serious EC loss, but does not increase early intimal hyperplasia when clopidogrel is administered. Protecting the vein from ischemia and reperfusion injury preserves ECs."},"whole_article_text_length":{"kind":"number","value":7886,"string":"7,886"},"whole_article_abstract_length":{"kind":"number","value":278,"string":"278"},"num_sections":{"kind":"number","value":17,"string":"17"},"most_frequent_words":{"kind":"list like","value":["vein","min","ischemia","min ischemia","grafts","15","intima","vein grafts","90","90 min"],"string":"[\n \"vein\",\n \"min\",\n \"ischemia\",\n \"min ischemia\",\n \"grafts\",\n \"15\",\n \"intima\",\n \"vein grafts\",\n \"90\",\n \"90 min\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] thrombosis | vein | grafting | acute thrombosis | acute | hyperplasia | drugs | stress | use | risk [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] min | rabbits | vein | saline | removed | shanghai | jugular | internal | internal jugular | rinsed [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min ischemia | min | ischemia | grafts | vein grafts | intima | 15 min ischemia | 15 | ecs [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] injury | study | study indicates clopidogrel administered | study indicates clopidogrel | cause ec loss avoiding | grafts long term study | grafts long term | grafts long | furthermore ir | loss avoiding [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | grafts | ischemia | min ischemia | vein grafts | 15 | 90 | 90 min | intima [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] vein | min | grafts | ischemia | min ischemia | vein grafts | 15 | 90 | 90 min | intima [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 15 | 60 | 15 | 90 ||| Clopidogrel ||| Twenty-eight days ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 60- | 90 | 15 ||| ||| 15 | 3 days | 90 | EC | days 2 | 14 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Ninety-minute | EC ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 15 | 60 | 15 | 90 ||| Clopidogrel ||| Twenty-eight days ||| ||| ||| 60- | 90 | 15 ||| ||| 15 | 3 days | 90 | EC | days 2 | 14 ||| Ninety-minute | EC ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 15 | 60 | 15 | 90 ||| Clopidogrel ||| Twenty-eight days ||| ||| ||| 60- | 90 | 15 ||| ||| 15 | 3 days | 90 | EC | days 2 | 14 ||| Ninety-minute | EC ||| [SUMMARY]"}}},{"rowIdx":270,"cells":{"title":{"kind":"string","value":"Cognitive awareness of carbohydrate intake does not alter exercise-induced lymphocyte apoptosis."},"pmid":{"kind":"string","value":"21484033"},"background_abstract":{"kind":"string","value":"Carbohydrate supplementation during aerobic exercise generally protects against the immunosuppressive effects of exercise. It is not currently known whether carbohydrate consumption or simply the knowledge of carbohydrate consumption also has that effect."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"Endurance trained male and female (N = 10) athletes were randomly assigned to one of two groups based on either a correct or incorrect cognitive awareness of carbohydrate intake. In the incorrect group, the subjects were informed that they were receiving the carbohydrate beverage but actually received the placebo beverage. Participants completed a 60-min ride on a cycle ergometer at 80% VO₂peak under carbohydrate and placebo supplemented conditions. Venous blood samples were collected at rest and immediately after exercise and were used to determine the plasma glucose concentration, lymphocyte count, and extent of lymphocyte apoptosis. Cognitive awareness, either correct or incorrect, did not have an effect on any of the measured variables."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Carbohydrate supplementation during exercise did not have an effect on lymphocyte count or apoptotic index. Independent of drink type, exercise resulted in significant lymphocytosis and lymphocyte apoptosis (apoptotic index at rest = 6.3 ± 3% and apoptotic index following exercise = 11.6 ± 3%, P < 0.01)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Neither carbohydrate nor placebo supplementation altered the typical lymphocyte apoptotic response following exercise. While carbohydrate supplementation generally has an immune-boosting effect during exercise, it appears that this influence does not extend to the mechanisms that govern exercise-induced lymphocyte cell death."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Analysis of Variance","Apoptosis","Awareness","Beverages","Blood Glucose","Cognition","Dietary Carbohydrates","Female","Humans","Lymphocytes","Male","Physical Endurance","Statistics, Nonparametric","Stress, Physiological"],"string":"[\n \"Adult\",\n \"Analysis of Variance\",\n \"Apoptosis\",\n \"Awareness\",\n \"Beverages\",\n \"Blood Glucose\",\n \"Cognition\",\n \"Dietary Carbohydrates\",\n \"Female\",\n \"Humans\",\n \"Lymphocytes\",\n \"Male\",\n \"Physical Endurance\",\n \"Statistics, Nonparametric\",\n \"Stress, Physiological\"\n]"},"pmcid":{"kind":"string","value":"3059873"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Acute exercise has a marked effect on the immune system response. Increased neutrophil, monocyte, and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and short‐term swim exercise.3 Two hours of cycling at 55% VO2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30‐min at 60% VO2max increased lymphocyte proliferation and plasma interleukin‐6 concentration (IL‐6).2 Significant increases in tumor necrosis factor‐α (TNF‐α) have been reported after a 30‐min high‐intensity run at 85% VO2max4 and following 15‐min of moderate‐intensity swimming.5 Therefore, it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors.\nWhile acute exercise significantly increases immune parameters, carbohydrate (CHO) supplementation has been shown to have an immunomodulating effect on many of these responses. CHO supplementation prior to 2.5 h of cycling at 65% VO2max resulted in reduced cell numbers of neutrophils, monocytes, and lymphocytes compared to placebo,6 as well as a significant decrease in plasma IL‐6 concentration.7 Similar reductions in immune cell counts and IL‐6 have been reported in trained individuals who received CHO supplementation during a 4‐h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8 \nRegarding the lymphocyte response to acute exercise, an increase in cell concentration is observed with the performance of high intensity exercise, followed by a reduction in concentration that is significantly lower than resting levels.9,10 A portion of this post‐exercise decrease is likely due to programmed cell death, or apoptosis. Previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 To our knowledge, only one previous study has assessed the effect that CHO supplementation has on lymphocyte apoptosis. Green et al. reported that CHO supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in CD4 and CD8 cells in culture taken from endurance‐trained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 This finding is consistent with previously cited research that CHO supplementation may reduce the immunoendocrine changes associated with exercise.6-8 \nA wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress (see review by Black).15 Neuroendocrine responses to stress are mediated by hypothalamic corticotropin‐releasing factor16 which acts on the hypothalamic‐pituitary‐adrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 In addition, there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 Therefore, it is possible that the immune system can be modified not only by nutritional supplementation as discussed above, but also independently by neural and cognitive factors. The influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume, suggesting potential deletion through a cell death mechanism (apoptosis).\nWhile carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo, it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise. It was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise. This would hold true whether participants had a correct or incorrect knowledge of supplementation type. Therefore, the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exercise‐induced lymphocyte apoptotic response, independent of actual carbohydrate intake."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":" Glucose There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\nThere was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\n Lymphocyte Concentration No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\nNo interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\n 3.3 Lymphocyte Apoptosis Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\nCognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Participants","2.2 Protocol","2.3 Statistical Analysis","Glucose","Lymphocyte Concentration","3.3 Lymphocyte Apoptosis","Acknowledgements"],"string":"[\n \"Participants\",\n \"2.2 Protocol\",\n \"2.3 Statistical Analysis\",\n \"Glucose\",\n \"Lymphocyte Concentration\",\n \"3.3 Lymphocyte Apoptosis\",\n \"Acknowledgements\"\n]"},"other_sections_texts":{"kind":"list like","value":["Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.","Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.","Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.","There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.","No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).","Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).","The authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation."],"string":"[\n \"Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\",\n \"Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\",\n \"Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\",\n \"There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\",\n \"No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\",\n \"Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\",\n \"The authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS AND MATERIALS","Participants","2.2 Protocol","2.3 Statistical Analysis","RESULTS","Glucose","Lymphocyte Concentration","3.3 Lymphocyte Apoptosis","DISCUSSION","Acknowledgements"],"string":"[\n \"INTRODUCTION\",\n \"METHODS AND MATERIALS\",\n \"Participants\",\n \"2.2 Protocol\",\n \"2.3 Statistical Analysis\",\n \"RESULTS\",\n \"Glucose\",\n \"Lymphocyte Concentration\",\n \"3.3 Lymphocyte Apoptosis\",\n \"DISCUSSION\",\n \"Acknowledgements\"\n]"},"all_sections_texts":{"kind":"list like","value":["Acute exercise has a marked effect on the immune system response. Increased neutrophil, monocyte, and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and short‐term swim exercise.3 Two hours of cycling at 55% VO2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30‐min at 60% VO2max increased lymphocyte proliferation and plasma interleukin‐6 concentration (IL‐6).2 Significant increases in tumor necrosis factor‐α (TNF‐α) have been reported after a 30‐min high‐intensity run at 85% VO2max4 and following 15‐min of moderate‐intensity swimming.5 Therefore, it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors.\nWhile acute exercise significantly increases immune parameters, carbohydrate (CHO) supplementation has been shown to have an immunomodulating effect on many of these responses. CHO supplementation prior to 2.5 h of cycling at 65% VO2max resulted in reduced cell numbers of neutrophils, monocytes, and lymphocytes compared to placebo,6 as well as a significant decrease in plasma IL‐6 concentration.7 Similar reductions in immune cell counts and IL‐6 have been reported in trained individuals who received CHO supplementation during a 4‐h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8 \nRegarding the lymphocyte response to acute exercise, an increase in cell concentration is observed with the performance of high intensity exercise, followed by a reduction in concentration that is significantly lower than resting levels.9,10 A portion of this post‐exercise decrease is likely due to programmed cell death, or apoptosis. Previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 To our knowledge, only one previous study has assessed the effect that CHO supplementation has on lymphocyte apoptosis. Green et al. reported that CHO supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in CD4 and CD8 cells in culture taken from endurance‐trained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 This finding is consistent with previously cited research that CHO supplementation may reduce the immunoendocrine changes associated with exercise.6-8 \nA wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress (see review by Black).15 Neuroendocrine responses to stress are mediated by hypothalamic corticotropin‐releasing factor16 which acts on the hypothalamic‐pituitary‐adrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 In addition, there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 Therefore, it is possible that the immune system can be modified not only by nutritional supplementation as discussed above, but also independently by neural and cognitive factors. The influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume, suggesting potential deletion through a cell death mechanism (apoptosis).\nWhile carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo, it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise. It was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise. This would hold true whether participants had a correct or incorrect knowledge of supplementation type. Therefore, the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exercise‐induced lymphocyte apoptotic response, independent of actual carbohydrate intake."," Participants Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\nEndurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\n 2.2 Protocol Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\nPeak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\n 2.3 Statistical Analysis Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\nBased on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.","Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.","Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.","Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test."," Glucose There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\nThere was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\n Lymphocyte Concentration No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\nNo interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\n 3.3 Lymphocyte Apoptosis Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\nCognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).","There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.","No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).","Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).","The main finding of this investigation was that cognitive awareness of drink type had no physiological effect on selected immune parameters following high‐intensity aerobic exercise. Individuals who had correct cognitive awareness of consumed supplementation (CHO or PLA) had statistically similar measures for plasma glucose, lymphocyte concentration, and lymphocyte apoptosis compared to individuals who had an incorrect cognitive awareness of the ingested supplement. In addition, we observed no effect of CHO supplementation on either lymphocyte concentration or apoptotic index following 60‐min of cycle exercise at 80% VO2peak.\nCarbohydrate supplementation significantly increased plasma glucose following exercise regardless of correct or incorrect cognitive awareness of drink type. The rise in glucose response has been confirmed by a number of other investigations.14,22-24 We are aware of only one other deception study in which subjects have received an incorrect cognitive awareness of carbohydrate or placebo supplementation prior to exercise.25 As we have shown, cognitive awareness of drink type (whether correct or incorrect) did not have an effect on the blood glucose response. The lack of a difference likely means that stimulating factors facilitating the release of glucose into the bloodstream (i.e. glucagon, epinephrine, etc.) are not affected directly by cognitive awareness during a bout high‐intensity aerobic exercise.\nAn exercise‐induced lymphocytosis was evident following both CHO and PLA supplemented bouts. An increase in lymphocyte concentration following exercise has been reported in numerous investigations.1-3,5-6 Thirty minutes of walking at 60% VO2max increased lymphocytosis by 28% in human subjects2 and 120‐min of cycling at 65% VO2max resulted in a 20% increase.6 Using an animal model, Prestes et al. observed increases in lymphocyte counts of 98% and 165% in male Wistar rats who performed unloaded swimming for 5 and 15‐min, and increases of 252% and 191% in animals who swam for 5 and 15‐min with a 5% load.5 The magnitude of lymphocytosis in the present study was 93% in endurance trained participants who completed 60‐min of cycle ergometry exercise at 80% VO2peak. An explanation for the lymphocytosis observed with exercise of increasing intensity is the greater expression of adhesion/activation molecules which can serve to facilitate movement of lymphocytes from the marginal pools into the circulation.26,27 \nWhile others have reported a significant decrease in lymphocyte count with CHO supplementation compared to PLA,6 our results did not show a difference. As part of an investigation to assess the effect of carbohydrate supplementation following caffeine ingestion, the participants of Walker et al. consumed either caffeine or placebo prior to exercise, and either carbohydrate or placebo during a 2 h cycle ride at 65% VO2max.6 Using only the trials in which placebo was consumed prior to exercise (and excluding the effect of caffeine), it was reported that supplementation with CHO during exercise reduced lymphocyte count by 21% compared with the trial in which placebo was administered during exercise.6 The common explanation for immunomodulation associated with CHO supplementation are effects through hypothalamic‐pituitary‐adrenal (HPA) activation and a muted stress hormone response.28 In the present study, CHO was only ingested prior to exercise and did not result in suppressed lymphocyte cell count compared to PLA. There are two possibilities to explain the differences between the study by Walker et al.6 and our findings. First, our participants completed only a 1h cycle ride compared to 2 h. It is possible that the extended exercise duration differentially affected the immune response through increased HPA activation compared to our subjects who completed only half of the duration. Second, the combination of supplemented glucose during the 2 h cycle exercise by the subjects of Walker et al. could have resulted in a greater prolonged effect toward reducing immune parameters compared to our participants who received a single bolus of carbohydrate prior to exercise, but did not receive supplementation at any other point during their exercise trial. Thus it is possible that blood glucose concentration plays a modulating role on cells of the immune system during exercise. In addition, it is likely that CHO has a different effect depending on the immune cell subset. While an overall decrease in leukocyte numbers have been reported in various CHO supplementation investigations,8,24-25 ingestion appears to affect neutrophils to a greater extent than lymphocytes. Nieman et al. and Sharhag et al. both found carbohydrate supplementation to reduce neutrophil count, but to have no effect on numbers of lymphocytes.8,24 \nNeither correct, nor incorrect, cognitive awareness of drink type altered immunity in terms of lymphocyte apoptosis following exercise. To our knowledge, only one other study has attempted to evaluate the effect of carbohydrate supplementation on exercise‐induced lymphocyte cell death.14 Green et al. used carboxyfluorescein succinimidylester fluorescence to estimate lymphocytes undergoing mitosis or cell death using iterative processes until all cells could be accounted for.14 They reported the estimated cell death rates of T‐cell subsets to be significantly reduced in CHO supplemented trials compared to PLA bouts in six well‐trained male cyclists. In the present study, carbohydrate supplementation did not have an effect on lymphocyte apoptosis. Recently, exercise‐induced lymphocyte apoptosis has been measured using either biochemical markers,4,29-30 or a morphological technique.11-13 In the current investigation, we employed the aforementioned morphological method which we have proposed is potentially more sensitive to cells displaying a wider range of apoptotic characteristics compared to a single apoptotic biomarker. We acknowledge that this method does have drawbacks (i.e. highly subjective, low number of cells that are evaluated per sample, etc.); however the methodology was the gold standard to which the current biomarker techniques were validated against.31 Using a direct evaluation of cells displaying morphological characteristics of apoptosis, we found that carbohydrate supplementation during exercise did not alter exercise‐induced cell death compared to the placebo supplemented trial. While carbohydrate supplementation may alter certain immune parameters, it appears that ingestion has no effect on the mechanisms governing cell death induction during exercise.\nWhile we were unable to ascertain differences in select immune parameters due to cognitive awareness of carbohydrate in the present investigation, our study design could be modified to make detection of such differences more likely. The utilization of a single group cross‐over intervention in which subjects randomly complete the conditions of exercise alone, exercise with carbohydrate supplementation, and exercise in conjunction with placebo, would represent an acceptable experimental design in this regard. It should also be noted that the findings of the present study can only be applied to young and middle‐aged individuals who are highly aerobically trained, and should not be extended to other populations. In this regard, other investigations are warranted.\nIn conclusion, our findings indicate that cognitive awareness (either correct or incorrect, of carbohydrate or placebo) has no effect on blood glucose concentration, lymphocyte concentration, or exercise‐induced lymphocyte apoptosis. While glucose intake itself may reduce the magnitude of various immune responses, knowledge of supplementation and indeed the carbohydrate supplement itself has no effect on altering immunity in terms of lymphocyte cell death following exercise.","The authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation."],"string":"[\n \"Acute exercise has a marked effect on the immune system response. Increased neutrophil, monocyte, and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and short‐term swim exercise.3 Two hours of cycling at 55% VO2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30‐min at 60% VO2max increased lymphocyte proliferation and plasma interleukin‐6 concentration (IL‐6).2 Significant increases in tumor necrosis factor‐α (TNF‐α) have been reported after a 30‐min high‐intensity run at 85% VO2max4 and following 15‐min of moderate‐intensity swimming.5 Therefore, it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors.\\nWhile acute exercise significantly increases immune parameters, carbohydrate (CHO) supplementation has been shown to have an immunomodulating effect on many of these responses. CHO supplementation prior to 2.5 h of cycling at 65% VO2max resulted in reduced cell numbers of neutrophils, monocytes, and lymphocytes compared to placebo,6 as well as a significant decrease in plasma IL‐6 concentration.7 Similar reductions in immune cell counts and IL‐6 have been reported in trained individuals who received CHO supplementation during a 4‐h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8 \\nRegarding the lymphocyte response to acute exercise, an increase in cell concentration is observed with the performance of high intensity exercise, followed by a reduction in concentration that is significantly lower than resting levels.9,10 A portion of this post‐exercise decrease is likely due to programmed cell death, or apoptosis. Previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 To our knowledge, only one previous study has assessed the effect that CHO supplementation has on lymphocyte apoptosis. Green et al. reported that CHO supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in CD4 and CD8 cells in culture taken from endurance‐trained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 This finding is consistent with previously cited research that CHO supplementation may reduce the immunoendocrine changes associated with exercise.6-8 \\nA wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress (see review by Black).15 Neuroendocrine responses to stress are mediated by hypothalamic corticotropin‐releasing factor16 which acts on the hypothalamic‐pituitary‐adrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 In addition, there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 Therefore, it is possible that the immune system can be modified not only by nutritional supplementation as discussed above, but also independently by neural and cognitive factors. The influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume, suggesting potential deletion through a cell death mechanism (apoptosis).\\nWhile carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo, it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise. It was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise. This would hold true whether participants had a correct or incorrect knowledge of supplementation type. Therefore, the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exercise‐induced lymphocyte apoptotic response, independent of actual carbohydrate intake.\",\n \" Participants Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\\nEndurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\\n 2.2 Protocol Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\\nPeak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\\n 2.3 Statistical Analysis Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\\nBased on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\",\n \"Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\",\n \"Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\",\n \"Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\",\n \" Glucose There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\\nThere was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\\n Lymphocyte Concentration No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\\nNo interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\\n 3.3 Lymphocyte Apoptosis Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\\nCognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\",\n \"There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\",\n \"No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\",\n \"Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\",\n \"The main finding of this investigation was that cognitive awareness of drink type had no physiological effect on selected immune parameters following high‐intensity aerobic exercise. Individuals who had correct cognitive awareness of consumed supplementation (CHO or PLA) had statistically similar measures for plasma glucose, lymphocyte concentration, and lymphocyte apoptosis compared to individuals who had an incorrect cognitive awareness of the ingested supplement. In addition, we observed no effect of CHO supplementation on either lymphocyte concentration or apoptotic index following 60‐min of cycle exercise at 80% VO2peak.\\nCarbohydrate supplementation significantly increased plasma glucose following exercise regardless of correct or incorrect cognitive awareness of drink type. The rise in glucose response has been confirmed by a number of other investigations.14,22-24 We are aware of only one other deception study in which subjects have received an incorrect cognitive awareness of carbohydrate or placebo supplementation prior to exercise.25 As we have shown, cognitive awareness of drink type (whether correct or incorrect) did not have an effect on the blood glucose response. The lack of a difference likely means that stimulating factors facilitating the release of glucose into the bloodstream (i.e. glucagon, epinephrine, etc.) are not affected directly by cognitive awareness during a bout high‐intensity aerobic exercise.\\nAn exercise‐induced lymphocytosis was evident following both CHO and PLA supplemented bouts. An increase in lymphocyte concentration following exercise has been reported in numerous investigations.1-3,5-6 Thirty minutes of walking at 60% VO2max increased lymphocytosis by 28% in human subjects2 and 120‐min of cycling at 65% VO2max resulted in a 20% increase.6 Using an animal model, Prestes et al. observed increases in lymphocyte counts of 98% and 165% in male Wistar rats who performed unloaded swimming for 5 and 15‐min, and increases of 252% and 191% in animals who swam for 5 and 15‐min with a 5% load.5 The magnitude of lymphocytosis in the present study was 93% in endurance trained participants who completed 60‐min of cycle ergometry exercise at 80% VO2peak. An explanation for the lymphocytosis observed with exercise of increasing intensity is the greater expression of adhesion/activation molecules which can serve to facilitate movement of lymphocytes from the marginal pools into the circulation.26,27 \\nWhile others have reported a significant decrease in lymphocyte count with CHO supplementation compared to PLA,6 our results did not show a difference. As part of an investigation to assess the effect of carbohydrate supplementation following caffeine ingestion, the participants of Walker et al. consumed either caffeine or placebo prior to exercise, and either carbohydrate or placebo during a 2 h cycle ride at 65% VO2max.6 Using only the trials in which placebo was consumed prior to exercise (and excluding the effect of caffeine), it was reported that supplementation with CHO during exercise reduced lymphocyte count by 21% compared with the trial in which placebo was administered during exercise.6 The common explanation for immunomodulation associated with CHO supplementation are effects through hypothalamic‐pituitary‐adrenal (HPA) activation and a muted stress hormone response.28 In the present study, CHO was only ingested prior to exercise and did not result in suppressed lymphocyte cell count compared to PLA. There are two possibilities to explain the differences between the study by Walker et al.6 and our findings. First, our participants completed only a 1h cycle ride compared to 2 h. It is possible that the extended exercise duration differentially affected the immune response through increased HPA activation compared to our subjects who completed only half of the duration. Second, the combination of supplemented glucose during the 2 h cycle exercise by the subjects of Walker et al. could have resulted in a greater prolonged effect toward reducing immune parameters compared to our participants who received a single bolus of carbohydrate prior to exercise, but did not receive supplementation at any other point during their exercise trial. Thus it is possible that blood glucose concentration plays a modulating role on cells of the immune system during exercise. In addition, it is likely that CHO has a different effect depending on the immune cell subset. While an overall decrease in leukocyte numbers have been reported in various CHO supplementation investigations,8,24-25 ingestion appears to affect neutrophils to a greater extent than lymphocytes. Nieman et al. and Sharhag et al. both found carbohydrate supplementation to reduce neutrophil count, but to have no effect on numbers of lymphocytes.8,24 \\nNeither correct, nor incorrect, cognitive awareness of drink type altered immunity in terms of lymphocyte apoptosis following exercise. To our knowledge, only one other study has attempted to evaluate the effect of carbohydrate supplementation on exercise‐induced lymphocyte cell death.14 Green et al. used carboxyfluorescein succinimidylester fluorescence to estimate lymphocytes undergoing mitosis or cell death using iterative processes until all cells could be accounted for.14 They reported the estimated cell death rates of T‐cell subsets to be significantly reduced in CHO supplemented trials compared to PLA bouts in six well‐trained male cyclists. In the present study, carbohydrate supplementation did not have an effect on lymphocyte apoptosis. Recently, exercise‐induced lymphocyte apoptosis has been measured using either biochemical markers,4,29-30 or a morphological technique.11-13 In the current investigation, we employed the aforementioned morphological method which we have proposed is potentially more sensitive to cells displaying a wider range of apoptotic characteristics compared to a single apoptotic biomarker. We acknowledge that this method does have drawbacks (i.e. highly subjective, low number of cells that are evaluated per sample, etc.); however the methodology was the gold standard to which the current biomarker techniques were validated against.31 Using a direct evaluation of cells displaying morphological characteristics of apoptosis, we found that carbohydrate supplementation during exercise did not alter exercise‐induced cell death compared to the placebo supplemented trial. While carbohydrate supplementation may alter certain immune parameters, it appears that ingestion has no effect on the mechanisms governing cell death induction during exercise.\\nWhile we were unable to ascertain differences in select immune parameters due to cognitive awareness of carbohydrate in the present investigation, our study design could be modified to make detection of such differences more likely. The utilization of a single group cross‐over intervention in which subjects randomly complete the conditions of exercise alone, exercise with carbohydrate supplementation, and exercise in conjunction with placebo, would represent an acceptable experimental design in this regard. It should also be noted that the findings of the present study can only be applied to young and middle‐aged individuals who are highly aerobically trained, and should not be extended to other populations. In this regard, other investigations are warranted.\\nIn conclusion, our findings indicate that cognitive awareness (either correct or incorrect, of carbohydrate or placebo) has no effect on blood glucose concentration, lymphocyte concentration, or exercise‐induced lymphocyte apoptosis. While glucose intake itself may reduce the magnitude of various immune responses, knowledge of supplementation and indeed the carbohydrate supplement itself has no effect on altering immunity in terms of lymphocyte cell death following exercise.\",\n \"The authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","materials|methods",null,null,null,"results",null,null,null,"discussion",null],"string":"[\n \"intro\",\n \"materials|methods\",\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n null\n]"},"keywords":{"kind":"list like","value":["Programmed cell death","High‐intensity aerobic exercise","Supplementation","Deception investigation","Immune system"],"string":"[\n \"Programmed cell death\",\n \"High‐intensity aerobic exercise\",\n \"Supplementation\",\n \"Deception investigation\",\n \"Immune system\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nAcute exercise has a marked effect on the immune system response. Increased neutrophil, monocyte, and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and short‐term swim exercise.3 Two hours of cycling at 55% VO2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30‐min at 60% VO2max increased lymphocyte proliferation and plasma interleukin‐6 concentration (IL‐6).2 Significant increases in tumor necrosis factor‐α (TNF‐α) have been reported after a 30‐min high‐intensity run at 85% VO2max4 and following 15‐min of moderate‐intensity swimming.5 Therefore, it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors.\nWhile acute exercise significantly increases immune parameters, carbohydrate (CHO) supplementation has been shown to have an immunomodulating effect on many of these responses. CHO supplementation prior to 2.5 h of cycling at 65% VO2max resulted in reduced cell numbers of neutrophils, monocytes, and lymphocytes compared to placebo,6 as well as a significant decrease in plasma IL‐6 concentration.7 Similar reductions in immune cell counts and IL‐6 have been reported in trained individuals who received CHO supplementation during a 4‐h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8 \nRegarding the lymphocyte response to acute exercise, an increase in cell concentration is observed with the performance of high intensity exercise, followed by a reduction in concentration that is significantly lower than resting levels.9,10 A portion of this post‐exercise decrease is likely due to programmed cell death, or apoptosis. Previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 To our knowledge, only one previous study has assessed the effect that CHO supplementation has on lymphocyte apoptosis. Green et al. reported that CHO supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in CD4 and CD8 cells in culture taken from endurance‐trained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 This finding is consistent with previously cited research that CHO supplementation may reduce the immunoendocrine changes associated with exercise.6-8 \nA wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress (see review by Black).15 Neuroendocrine responses to stress are mediated by hypothalamic corticotropin‐releasing factor16 which acts on the hypothalamic‐pituitary‐adrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 In addition, there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 Therefore, it is possible that the immune system can be modified not only by nutritional supplementation as discussed above, but also independently by neural and cognitive factors. The influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume, suggesting potential deletion through a cell death mechanism (apoptosis).\nWhile carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo, it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise. It was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise. This would hold true whether participants had a correct or incorrect knowledge of supplementation type. Therefore, the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exercise‐induced lymphocyte apoptotic response, independent of actual carbohydrate intake.\n\nMETHODS AND MATERIALS:\n Participants Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\nEndurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\n 2.2 Protocol Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\nPeak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\n 2.3 Statistical Analysis Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\nBased on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\n\nParticipants:\nEndurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\n\n2.2 Protocol:\nPeak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\n\n2.3 Statistical Analysis:\nBased on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\n\nRESULTS:\n Glucose There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\nThere was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\n Lymphocyte Concentration No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\nNo interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\n 3.3 Lymphocyte Apoptosis Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\nCognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\n\nGlucose:\nThere was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\n\nLymphocyte Concentration:\nNo interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\n\n3.3 Lymphocyte Apoptosis:\nCognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\n\nDISCUSSION:\nThe main finding of this investigation was that cognitive awareness of drink type had no physiological effect on selected immune parameters following high‐intensity aerobic exercise. Individuals who had correct cognitive awareness of consumed supplementation (CHO or PLA) had statistically similar measures for plasma glucose, lymphocyte concentration, and lymphocyte apoptosis compared to individuals who had an incorrect cognitive awareness of the ingested supplement. In addition, we observed no effect of CHO supplementation on either lymphocyte concentration or apoptotic index following 60‐min of cycle exercise at 80% VO2peak.\nCarbohydrate supplementation significantly increased plasma glucose following exercise regardless of correct or incorrect cognitive awareness of drink type. The rise in glucose response has been confirmed by a number of other investigations.14,22-24 We are aware of only one other deception study in which subjects have received an incorrect cognitive awareness of carbohydrate or placebo supplementation prior to exercise.25 As we have shown, cognitive awareness of drink type (whether correct or incorrect) did not have an effect on the blood glucose response. The lack of a difference likely means that stimulating factors facilitating the release of glucose into the bloodstream (i.e. glucagon, epinephrine, etc.) are not affected directly by cognitive awareness during a bout high‐intensity aerobic exercise.\nAn exercise‐induced lymphocytosis was evident following both CHO and PLA supplemented bouts. An increase in lymphocyte concentration following exercise has been reported in numerous investigations.1-3,5-6 Thirty minutes of walking at 60% VO2max increased lymphocytosis by 28% in human subjects2 and 120‐min of cycling at 65% VO2max resulted in a 20% increase.6 Using an animal model, Prestes et al. observed increases in lymphocyte counts of 98% and 165% in male Wistar rats who performed unloaded swimming for 5 and 15‐min, and increases of 252% and 191% in animals who swam for 5 and 15‐min with a 5% load.5 The magnitude of lymphocytosis in the present study was 93% in endurance trained participants who completed 60‐min of cycle ergometry exercise at 80% VO2peak. An explanation for the lymphocytosis observed with exercise of increasing intensity is the greater expression of adhesion/activation molecules which can serve to facilitate movement of lymphocytes from the marginal pools into the circulation.26,27 \nWhile others have reported a significant decrease in lymphocyte count with CHO supplementation compared to PLA,6 our results did not show a difference. As part of an investigation to assess the effect of carbohydrate supplementation following caffeine ingestion, the participants of Walker et al. consumed either caffeine or placebo prior to exercise, and either carbohydrate or placebo during a 2 h cycle ride at 65% VO2max.6 Using only the trials in which placebo was consumed prior to exercise (and excluding the effect of caffeine), it was reported that supplementation with CHO during exercise reduced lymphocyte count by 21% compared with the trial in which placebo was administered during exercise.6 The common explanation for immunomodulation associated with CHO supplementation are effects through hypothalamic‐pituitary‐adrenal (HPA) activation and a muted stress hormone response.28 In the present study, CHO was only ingested prior to exercise and did not result in suppressed lymphocyte cell count compared to PLA. There are two possibilities to explain the differences between the study by Walker et al.6 and our findings. First, our participants completed only a 1h cycle ride compared to 2 h. It is possible that the extended exercise duration differentially affected the immune response through increased HPA activation compared to our subjects who completed only half of the duration. Second, the combination of supplemented glucose during the 2 h cycle exercise by the subjects of Walker et al. could have resulted in a greater prolonged effect toward reducing immune parameters compared to our participants who received a single bolus of carbohydrate prior to exercise, but did not receive supplementation at any other point during their exercise trial. Thus it is possible that blood glucose concentration plays a modulating role on cells of the immune system during exercise. In addition, it is likely that CHO has a different effect depending on the immune cell subset. While an overall decrease in leukocyte numbers have been reported in various CHO supplementation investigations,8,24-25 ingestion appears to affect neutrophils to a greater extent than lymphocytes. Nieman et al. and Sharhag et al. both found carbohydrate supplementation to reduce neutrophil count, but to have no effect on numbers of lymphocytes.8,24 \nNeither correct, nor incorrect, cognitive awareness of drink type altered immunity in terms of lymphocyte apoptosis following exercise. To our knowledge, only one other study has attempted to evaluate the effect of carbohydrate supplementation on exercise‐induced lymphocyte cell death.14 Green et al. used carboxyfluorescein succinimidylester fluorescence to estimate lymphocytes undergoing mitosis or cell death using iterative processes until all cells could be accounted for.14 They reported the estimated cell death rates of T‐cell subsets to be significantly reduced in CHO supplemented trials compared to PLA bouts in six well‐trained male cyclists. In the present study, carbohydrate supplementation did not have an effect on lymphocyte apoptosis. Recently, exercise‐induced lymphocyte apoptosis has been measured using either biochemical markers,4,29-30 or a morphological technique.11-13 In the current investigation, we employed the aforementioned morphological method which we have proposed is potentially more sensitive to cells displaying a wider range of apoptotic characteristics compared to a single apoptotic biomarker. We acknowledge that this method does have drawbacks (i.e. highly subjective, low number of cells that are evaluated per sample, etc.); however the methodology was the gold standard to which the current biomarker techniques were validated against.31 Using a direct evaluation of cells displaying morphological characteristics of apoptosis, we found that carbohydrate supplementation during exercise did not alter exercise‐induced cell death compared to the placebo supplemented trial. While carbohydrate supplementation may alter certain immune parameters, it appears that ingestion has no effect on the mechanisms governing cell death induction during exercise.\nWhile we were unable to ascertain differences in select immune parameters due to cognitive awareness of carbohydrate in the present investigation, our study design could be modified to make detection of such differences more likely. The utilization of a single group cross‐over intervention in which subjects randomly complete the conditions of exercise alone, exercise with carbohydrate supplementation, and exercise in conjunction with placebo, would represent an acceptable experimental design in this regard. It should also be noted that the findings of the present study can only be applied to young and middle‐aged individuals who are highly aerobically trained, and should not be extended to other populations. In this regard, other investigations are warranted.\nIn conclusion, our findings indicate that cognitive awareness (either correct or incorrect, of carbohydrate or placebo) has no effect on blood glucose concentration, lymphocyte concentration, or exercise‐induced lymphocyte apoptosis. While glucose intake itself may reduce the magnitude of various immune responses, knowledge of supplementation and indeed the carbohydrate supplement itself has no effect on altering immunity in terms of lymphocyte cell death following exercise.\n\nAcknowledgements:\nThe authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nCarbohydrate supplementation during aerobic exercise generally protects against the immunosuppressive effects of exercise. It is not currently known whether carbohydrate consumption or simply the knowledge of carbohydrate consumption also has that effect.\n\nMethods:\nEndurance trained male and female (N = 10) athletes were randomly assigned to one of two groups based on either a correct or incorrect cognitive awareness of carbohydrate intake. In the incorrect group, the subjects were informed that they were receiving the carbohydrate beverage but actually received the placebo beverage. Participants completed a 60-min ride on a cycle ergometer at 80% VO₂peak under carbohydrate and placebo supplemented conditions. Venous blood samples were collected at rest and immediately after exercise and were used to determine the plasma glucose concentration, lymphocyte count, and extent of lymphocyte apoptosis. Cognitive awareness, either correct or incorrect, did not have an effect on any of the measured variables.\n\nResults:\nCarbohydrate supplementation during exercise did not have an effect on lymphocyte count or apoptotic index. Independent of drink type, exercise resulted in significant lymphocytosis and lymphocyte apoptosis (apoptotic index at rest = 6.3 ± 3% and apoptotic index following exercise = 11.6 ± 3%, P < 0.01).\n\nConclusions:\nNeither carbohydrate nor placebo supplementation altered the typical lymphocyte apoptotic response following exercise. While carbohydrate supplementation generally has an immune-boosting effect during exercise, it appears that this influence does not extend to the mechanisms that govern exercise-induced lymphocyte cell death."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":5943,"string":"5,943"},"whole_article_abstract_length":{"kind":"number","value":278,"string":"278"},"num_sections":{"kind":"number","value":11,"string":"11"},"most_frequent_words":{"kind":"list like","value":["exercise","lymphocyte","cho","min","supplementation","effect","apoptotic","apoptosis","cell","cognitive"],"string":"[\n \"exercise\",\n \"lymphocyte\",\n \"cho\",\n \"min\",\n \"supplementation\",\n \"effect\",\n \"apoptotic\",\n \"apoptosis\",\n \"cell\",\n \"cognitive\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Programmed cell death | High‐intensity aerobic exercise | Supplementation | Deception investigation | Immune system [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Programmed cell death | High‐intensity aerobic exercise | Supplementation | Deception investigation | Immune system [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Programmed cell death | High‐intensity aerobic exercise | Supplementation | Deception investigation | Immune system [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Analysis of Variance | Apoptosis | Awareness | Beverages | Blood Glucose | Cognition | Dietary Carbohydrates | Female | Humans | Lymphocytes | Male | Physical Endurance | Statistics, Nonparametric | Stress, Physiological [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Analysis of Variance | Apoptosis | Awareness | Beverages | Blood Glucose | Cognition | Dietary Carbohydrates | Female | Humans | Lymphocytes | Male | Physical Endurance | Statistics, Nonparametric | Stress, Physiological [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Analysis of Variance | Apoptosis | Awareness | Beverages | Blood Glucose | Cognition | Dietary Carbohydrates | Female | Humans | Lymphocytes | Male | Physical Endurance | Statistics, Nonparametric | Stress, Physiological [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test 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exercise | immune | supplementation | system | carbohydrate | cho supplementation | acute | cell | lymphocyte | effect [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] lymphocyte | glucose | exercise | fig | significant main | significant main effect | type affect | main effect | significant | concentration [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] exercise | lymphocyte | glucose | effect | cho | fig | supplementation | test | significant | min [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 6.3 ± | 3% | 11.6 | 3% | 0.01 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| 10 | one | two ||| ||| 60 | 80% ||| ||| ||| ||| 6.3 ± | 3% | 11.6 | 3% | 0.01 ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":271,"cells":{"title":{"kind":"string","value":"Plasma Metabolite Profiles of Red Meat, Poultry, and Fish Consumption, and Their Associations with Colorectal Cancer Risk."},"pmid":{"kind":"string","value":"35267954"},"background_abstract":{"kind":"string","value":"Red and processed meat consumption has been consistently associated with increased risk of colorectal cancer (CRC), but the association for fish intake is unclear. Evidence using objective dietary assessment approaches to evaluate these associations is sparse."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We measured plasma metabolites among 5269 participants from the Nurses' Health Study (NHS), NHSII, and Health Professionals Follow-Up study (HPFS). We calculated partial Spearman correlations between each metabolite and self-reported intake of seven red meat, poultry, and fish groups. Metabolite profile scores correlated to self-reported dietary intakes were developed using elastic net regression. Associations between self-reported intakes, metabolite profile scores, and subsequent CRC risk were further evaluated using conditional logistic regression among 559 matched (1:1) case-control pairs in NHS/HPFS and replicated among 266 pairs in Women's Health Study."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Plasma metabolites, especially highly unsaturated lipids, were differentially associated with red meat and fish groups. Metabolite profile scores for each food group were significantly correlated with the corresponding self-reported dietary intake. A higher dietary intake of processed red meat was associated with a higher risk of CRC (pooled OR per 1 SD, 1.15; 95% CI: 1.03, 1.29). In contrast, higher metabolite profile scores for all fish groups, not dietary intakes, were consistently associated with a lower CRC risk: the pooled OR per 1 SD was 0.86 (95% CI: 0.78, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were found for other food groups."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Red meat and fish intake exhibited systematically different plasma metabolite profiles. Plasma metabolite profile of fish intake was inversely associated with CRC risk."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Colorectal Neoplasms","Female","Follow-Up Studies","Logistic Models","Poultry","Red Meat"],"string":"[\n \"Animals\",\n \"Colorectal Neoplasms\",\n \"Female\",\n \"Follow-Up Studies\",\n \"Logistic Models\",\n \"Poultry\",\n \"Red Meat\"\n]"},"pmcid":{"kind":"string","value":"8912563"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Colorectal cancer (CRC) remains the second most commonly occurring cancer in women and the third in men worldwide [1]. CRC incidence is largely affected by screening and modifiable dietary and lifestyle factors [2]. Among these factors, meat and fish consumption has been the subject of many investigations. The consumption of red meat, especially processed red meat, is consistently associated with an increased risk of CRC [3]. However, there is still uncertainty regarding the association between fish intake and CRC risk. The World Cancer Research Fund and American Institute for Cancer Research (WCRF/AICR) concluded that there was only “limited evidence” for the beneficial effect of fish intake in CRC prevention [4]. Recent studies of fish intake and CRC risk also generated inconsistent results [5,6,7,8].\nPrevious epidemiological studies examining the association of meat and fish intake with CRC risk mainly used self-reported dietary data. Rapid developments in high-throughput metabolomics are leading to a new era in nutritional epidemiological research. By measuring the small-molecule metabolites in biological samples, metabolomics may provide an objective picture of food intake and its related biological consequences [9]. Feeding trials and observational studies have demonstrated that plasma and urinary metabolites differed between meat and fish consumption [10,11,12,13,14]. However, it is unknown whether the metabolite profiles related to meat and fish intake are associated with CRC risk and whether these profiles could be used as a complementary approach to evaluate the association between meat and fish intake and CRC risk.\nTherefore, we examined the associations of red meat, poultry, and fish consumption with plasma metabolite profiles among participants from the Nurses’ Health Study (NHS), NHSII, and Health Professionals Follow-up Study (HPFS). We also developed metabolite profile scores that were correlated to the intakes of these meat and fish groups and evaluated the prospective associations of metabolite profile scores with CRC risk. The results for metabolite profile scores were further replicated in an external validation cohort (Women’s Health Study, WHS)."},"methods_title":{"kind":"string","value":"2. Methods"},"methods_text":{"kind":"string","value":" Study Population Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\nOur primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required."},"results_title":{"kind":"string","value":"6. Results"},"results_text":{"kind":"string","value":"In NHS/NHSII/HPFS, participants were predominately white and middle-aged (mean age 53 years), with an average BMI of 25.4 kg/m2 (Table 1). The percentage energy intake from protein was 18%, and animal protein was 13%. Participants with a higher total red meat intake were slightly more likely to have a lower total fish intake (Pearson r = −0.07). In the analysis of CRC, participants had similar demographic characteristics but were a little older (mean age 61 years) and had a lower proportion of females. Compared to control participants, CRC cases were less likely to use multivitamins and aspirin, receive endoscopy screening, and be physically active, but were more likely to smoke and have a family history of CRC. They also had a somewhat higher intake of total red meat (mainly processed red meat) and a lower fish intake. Similarly, CRC cases in WHS were less likely to be physically active and more likely to smoke and have a family history of CRC. However, they had a lower intake of total red meat (mainly unprocessed red meat) than control participants.\nOf the 287 known metabolites, 85 were significantly correlated with total red meat intake, 36 with unprocessed red meat, 51 with processed red meat, 28 with poultry, 60 with total fish, 60 with dark meat fish, and 27 with canned tuna fish after Bonferroni correction (Figure 2). Similar metabolites correlated with total red meat intake, unprocessed red meat, and processed red meat. All three red meat groups were positively associated with creatine, hydroxyproline, coenzyme Q10, myristoleic acid, acylcarnitines, and plasmalogens with a number of double bonds ≤6 but were negatively associated with highly unsaturated lipid species including triglycerides (TAGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylcholines (LPCs). Poultry intake was positively correlated with creatine, ectoine, a few PCs, TAGs, and PE plasmalogens after Bonferroni correction. In contrast to red meat intake, total fish intake, dark meat fish, and canned tuna fish were all positively correlated with highly unsaturated lipid species. When we restricted the analysis to fasting participants and participants selected as controls in the original sub-studies, the correlation results were similar (Supplementary Figures S2 and S3).\nOf the 2561 unknown metabolites, 702 were significantly correlated with total red meat intake after Bonferroni correction, 222 with unprocessed red meat, 462 with processed red meat, 65 with poultry, 284 with total fish intake, 414 with dark meat fish, and 119 with canned tuna fish (Supplementary Table S2). Among these, the absolute partial Spearman rho exceeded 0.3 for 11 unknown metabolites: one with total red meat intake, 10 with total fish intake, and 5 with dark meat fish intake. Pearson correlation analysis between these 11 unknown metabolites and those known metabolites (130 in total) that were significantly associated with total red meat, total fish, or dark meat fish consumption identified several unknown metabolites being strongly correlated with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC (all Pearson r > 0.8) (Supplementary Figure S4).\nA total of 53 known metabolites were selected in the elastic net regression for total red meat intake, 55 for unprocessed red meat, 36 for processed red meat, 7 for poultry, 18 for total fish, 27 for dark meat fish, and 11 for canned tuna fish (Table 2). Each metabolite profile score based on the selected known metabolites was significantly correlated with the corresponding self-reported dietary intake in both NHS/NHSII/HPFS and WHS (r = 0.33–0.46 for total red meat; r = 0.36–0.42 for unprocessed red meat; r = 0.19–0.33 for processed red meat; r = 0.12–0.21 for poultry; r = 0.31–0.40 for total fish; r = 0.32–0.42 for dark meat fish; and r = 0.14–0.22 for canned tuna fish) (Table 2). Adding unknown metabolites into the metabolite profile scores did not materially improve their correlations with the dietary intake (Supplementary Table S3). As a sensitivity analysis, we adopted the random forest algorithm to impute the missingness and re-derived the metabolomic profile score. The newly derived scores were highly correlated to the original scores, with a Pearson r = 0.98 for the total red meat score, 0.98 for the unprocessed red meat, 0.96 for the processed red meat score, 0.92 for the poultry score, 0.99 for the total fish score, 0.99 for the dark meat fish score, and 0.97 for the canned tuna fish score.\nAfter adjusting for potential confounders, dietary intake of processed red meat was significantly associated with CRC risk. The pooled OR for 1 SD higher intake was 1.15 (95% CI: 1.03, 1.29) (Table 3). We did not observe consistent associations for dietary intake of three fish groups across NHS/HPFS and WHS. An inverse association for total fish and canned tuna fish was only found in NHS/HPFS and for dark meat fish in WHS. In contrast, metabolite profile scores for these three fish groups were inversely associated with CRC risk in both NHS/HPFS and WHS (Table 3). The pooled OR per 1 SD higher was 0.86 (95% CI: 0.77, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were observed for other meat intakes or metabolite profile scores. The results remained similar after adjusting for menopausal status or ethnicity in NHS/HPFS (data not shown). In the analysis by tumor site, we observed that the above associations were stronger for rectal cancer than those for colon cancer (Supplementary Table S4).\nAmong metabolites selected in metabolite profile scores of all three fish groups, five metabolites (C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) were positively correlated to fish intake but inversely associated with CRC risk; two metabolites (C20:4 LPE and C22:5 LPC) were inversely correlated to fish intake but positively associated with CRC risk (Supplementary Figure S5)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Study Population","3. Dietary Assessment","4. Metabolomics Measurement","5. Nondietary Covariates","Statistical Analyses"],"string":"[\n \"Study Population\",\n \"3. Dietary Assessment\",\n \"4. Metabolomics Measurement\",\n \"5. Nondietary Covariates\",\n \"Statistical Analyses\"\n]"},"other_sections_texts":{"kind":"list like","value":["Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.","Validated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.","Profiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.","In NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.","We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1."],"string":"[\n \"Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\",\n \"Validated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.\",\n \"Profiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.\",\n \"In NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\",\n \"We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Methods","Study Population","3. Dietary Assessment","4. Metabolomics Measurement","5. Nondietary Covariates","Statistical Analyses","6. Results","7. Discussion"],"string":"[\n \"1. Introduction\",\n \"2. Methods\",\n \"Study Population\",\n \"3. Dietary Assessment\",\n \"4. Metabolomics Measurement\",\n \"5. Nondietary Covariates\",\n \"Statistical Analyses\",\n \"6. Results\",\n \"7. Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Colorectal cancer (CRC) remains the second most commonly occurring cancer in women and the third in men worldwide [1]. CRC incidence is largely affected by screening and modifiable dietary and lifestyle factors [2]. Among these factors, meat and fish consumption has been the subject of many investigations. The consumption of red meat, especially processed red meat, is consistently associated with an increased risk of CRC [3]. However, there is still uncertainty regarding the association between fish intake and CRC risk. The World Cancer Research Fund and American Institute for Cancer Research (WCRF/AICR) concluded that there was only “limited evidence” for the beneficial effect of fish intake in CRC prevention [4]. Recent studies of fish intake and CRC risk also generated inconsistent results [5,6,7,8].\nPrevious epidemiological studies examining the association of meat and fish intake with CRC risk mainly used self-reported dietary data. Rapid developments in high-throughput metabolomics are leading to a new era in nutritional epidemiological research. By measuring the small-molecule metabolites in biological samples, metabolomics may provide an objective picture of food intake and its related biological consequences [9]. Feeding trials and observational studies have demonstrated that plasma and urinary metabolites differed between meat and fish consumption [10,11,12,13,14]. However, it is unknown whether the metabolite profiles related to meat and fish intake are associated with CRC risk and whether these profiles could be used as a complementary approach to evaluate the association between meat and fish intake and CRC risk.\nTherefore, we examined the associations of red meat, poultry, and fish consumption with plasma metabolite profiles among participants from the Nurses’ Health Study (NHS), NHSII, and Health Professionals Follow-up Study (HPFS). We also developed metabolite profile scores that were correlated to the intakes of these meat and fish groups and evaluated the prospective associations of metabolite profile scores with CRC risk. The results for metabolite profile scores were further replicated in an external validation cohort (Women’s Health Study, WHS)."," Study Population Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\nOur primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.","Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.","Validated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.","Profiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.","In NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.","We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.","In NHS/NHSII/HPFS, participants were predominately white and middle-aged (mean age 53 years), with an average BMI of 25.4 kg/m2 (Table 1). The percentage energy intake from protein was 18%, and animal protein was 13%. Participants with a higher total red meat intake were slightly more likely to have a lower total fish intake (Pearson r = −0.07). In the analysis of CRC, participants had similar demographic characteristics but were a little older (mean age 61 years) and had a lower proportion of females. Compared to control participants, CRC cases were less likely to use multivitamins and aspirin, receive endoscopy screening, and be physically active, but were more likely to smoke and have a family history of CRC. They also had a somewhat higher intake of total red meat (mainly processed red meat) and a lower fish intake. Similarly, CRC cases in WHS were less likely to be physically active and more likely to smoke and have a family history of CRC. However, they had a lower intake of total red meat (mainly unprocessed red meat) than control participants.\nOf the 287 known metabolites, 85 were significantly correlated with total red meat intake, 36 with unprocessed red meat, 51 with processed red meat, 28 with poultry, 60 with total fish, 60 with dark meat fish, and 27 with canned tuna fish after Bonferroni correction (Figure 2). Similar metabolites correlated with total red meat intake, unprocessed red meat, and processed red meat. All three red meat groups were positively associated with creatine, hydroxyproline, coenzyme Q10, myristoleic acid, acylcarnitines, and plasmalogens with a number of double bonds ≤6 but were negatively associated with highly unsaturated lipid species including triglycerides (TAGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylcholines (LPCs). Poultry intake was positively correlated with creatine, ectoine, a few PCs, TAGs, and PE plasmalogens after Bonferroni correction. In contrast to red meat intake, total fish intake, dark meat fish, and canned tuna fish were all positively correlated with highly unsaturated lipid species. When we restricted the analysis to fasting participants and participants selected as controls in the original sub-studies, the correlation results were similar (Supplementary Figures S2 and S3).\nOf the 2561 unknown metabolites, 702 were significantly correlated with total red meat intake after Bonferroni correction, 222 with unprocessed red meat, 462 with processed red meat, 65 with poultry, 284 with total fish intake, 414 with dark meat fish, and 119 with canned tuna fish (Supplementary Table S2). Among these, the absolute partial Spearman rho exceeded 0.3 for 11 unknown metabolites: one with total red meat intake, 10 with total fish intake, and 5 with dark meat fish intake. Pearson correlation analysis between these 11 unknown metabolites and those known metabolites (130 in total) that were significantly associated with total red meat, total fish, or dark meat fish consumption identified several unknown metabolites being strongly correlated with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC (all Pearson r > 0.8) (Supplementary Figure S4).\nA total of 53 known metabolites were selected in the elastic net regression for total red meat intake, 55 for unprocessed red meat, 36 for processed red meat, 7 for poultry, 18 for total fish, 27 for dark meat fish, and 11 for canned tuna fish (Table 2). Each metabolite profile score based on the selected known metabolites was significantly correlated with the corresponding self-reported dietary intake in both NHS/NHSII/HPFS and WHS (r = 0.33–0.46 for total red meat; r = 0.36–0.42 for unprocessed red meat; r = 0.19–0.33 for processed red meat; r = 0.12–0.21 for poultry; r = 0.31–0.40 for total fish; r = 0.32–0.42 for dark meat fish; and r = 0.14–0.22 for canned tuna fish) (Table 2). Adding unknown metabolites into the metabolite profile scores did not materially improve their correlations with the dietary intake (Supplementary Table S3). As a sensitivity analysis, we adopted the random forest algorithm to impute the missingness and re-derived the metabolomic profile score. The newly derived scores were highly correlated to the original scores, with a Pearson r = 0.98 for the total red meat score, 0.98 for the unprocessed red meat, 0.96 for the processed red meat score, 0.92 for the poultry score, 0.99 for the total fish score, 0.99 for the dark meat fish score, and 0.97 for the canned tuna fish score.\nAfter adjusting for potential confounders, dietary intake of processed red meat was significantly associated with CRC risk. The pooled OR for 1 SD higher intake was 1.15 (95% CI: 1.03, 1.29) (Table 3). We did not observe consistent associations for dietary intake of three fish groups across NHS/HPFS and WHS. An inverse association for total fish and canned tuna fish was only found in NHS/HPFS and for dark meat fish in WHS. In contrast, metabolite profile scores for these three fish groups were inversely associated with CRC risk in both NHS/HPFS and WHS (Table 3). The pooled OR per 1 SD higher was 0.86 (95% CI: 0.77, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were observed for other meat intakes or metabolite profile scores. The results remained similar after adjusting for menopausal status or ethnicity in NHS/HPFS (data not shown). In the analysis by tumor site, we observed that the above associations were stronger for rectal cancer than those for colon cancer (Supplementary Table S4).\nAmong metabolites selected in metabolite profile scores of all three fish groups, five metabolites (C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) were positively correlated to fish intake but inversely associated with CRC risk; two metabolites (C20:4 LPE and C22:5 LPC) were inversely correlated to fish intake but positively associated with CRC risk (Supplementary Figure S5).","Leveraging metabolomics data from four studies, we found systematic differences in plasma metabolite profiles according to various types of meat and fish consumption. We also developed metabolite profile scores that were correlated to the consumption of red meat, poultry, and fish. Higher metabolite profile scores for all three fish groups (total fish, dark meat fish, and canned tuna fish), rather than their self-reported dietary intake, were consistently associated with a lower CRC risk in both NHS/HPFS and WHS. These findings suggest the promising use of metabolomics in complementing traditional dietary assessments to evaluate the association between dietary exposure and health outcomes.\nConsistent with previous studies, we found that consumption of all three red meat groups was positively correlated with several PE and PC plasmalogens, all of which have a low degree of unsaturation and contain acid moieties derived from animal tissues [14,29]. In contrast, total fish intake was positively correlated with highly unsaturated lipids, including TAGs, PCs, PEs, LPCs, LPEs, CEs, and plasmalogens. Fish intake, especially marine fish, is the principal dietary contributor of highly n-3 polyunsaturated fatty acids [30], and thus, are dietary sources for these lipids and their downstream metabolites.\nApart from lipid metabolites, we also observed several other metabolites correlated with meat and fish consumption. Creatine, an amino acid metabolite abundant in skeletal muscle [31], was positively correlated with the intake of red meat, poultry, and fish. Hydroxyproline, an amino acid that forms part of collagen [32], was positively correlated with red meat and poultry intake. Additionally, we found a positive correlation between poultry intake and ectoine (an α-amino acid), in line with one previous study [14]. Ectoine can be synthesized and released by several species in the genus Halomonas [33], the dominant microbial genus in the chicken embryo [34], which might lead to the presence of ectoine in chicken.\nMost previous metabolomic studies mainly focused on known metabolites, which are only a small percent of the data obtained in a typical metabolomics measurement (~10%) [35]. In the present study, we analyzed the unknown metabolites and identified a set of unknown metabolites that were significantly correlated with meat or fish consumption. Several unknown metabolites even exhibited a higher correlation with meat and fish intake (partial Spearman rho > 0.3) compared to known metabolites. By further examining the correlation between these unknown metabolites and the known metabolites, strong correlations were found with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC, indicating their potential biological links. However, whether these unknown metabolites are metabolites of the known metabolites or unrelated molecules affected by the same food is not clear. A critical next step is to structurally identify the promising and influential unknown metabolites. It should also be noted that despite the many statistically significant correlations between meat and fish groups and the unknown metabolites, these unknown metabolites added minimally to the correlations between meat and fish intakes and metabolite profile score based only on the known metabolites. The possibility remains that other metabolomics platforms may add further independent prediction of intake.\nThe human metabolome reflects a holistic biological status, which can be influenced by various exogenous and endogenous factors, such as dietary intakes, gut microbiota, health status, and genotype [36,37]. The metabolites selected in the metabolite profile score for meat or fish consumption could represent, in part, the components of food, such as the eicosapentaenoic acid/docosahexaenoic acid-containing lipids for the fish intake and hydroxyproline for the intake of red meat and poultry. Other metabolites, for example, C20:4 LPE and C22:5 LPC, could be related to the metabolic processes after fish intake, as fish is one of the main sources of arachidonic acid (C20:4) [38] and docosapentaenoic acid (C22:5) [39], but it was inversely correlated with these two lipids. Thus, metabolite profile score can be a comprehensive way to reflect both genuine compounds from foods as well as markers of complex metabolomic responses to dietary exposures.\nNot surprisingly, we found a significant positive association between processed red meat intake and CRC risk. However, the metabolite profile score for processed red meat consumption was not significantly associated with CRC risk. One possible explanation could be that the increased CRC risk associated with high processed red meat intake was mainly due to the carcinogenic compounds [40], such as polycyclic aromatic hydrocarbons, heterocyclic amines, and N-nitroso compounds, which were not measured in our metabolomics platforms. On the contrary, we did not observe a consistent inverse association between dietary fish intake and CRC risk, but inverse associations were found for the metabolite profile scores of all three fish groups in both NHS/HPFS and WHS. Results from previous studies of fish intake and CRC risk were generally inconsistent, some indicating an inverse association [7,8], but others not [5,6]. Unlike self-reported dietary intake data widely used in previous studies, the metabolomics data objectively captures the metabolites related to dietary exposures in the human body, which might be more directly associated with health outcomes. The seven metabolites (C20:4 LPE, C22:5 LPC, C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) selected in metabolite profile scores of all three fish groups could represent the beneficial metabolomic response to fish intake, and this response led to a lower risk of CRC.\nThe main strengths of our study are the large sample size, comprehensive metabolite profiles including both known and unknown metabolites, prospectively collected dietary data before CRC diagnosis, and the use of the elastic net model, which performs well in high-dimensional data where there are high correlations between predictors [41]. Moreover, the robustness of our metabolite profile scores was validated in an independent study. Nevertheless, our results also have several limitations. First, we did not have data on the type of red meat. Red meat subtypes (beef, lamb, or pork) may differ in their associations with CRC risk [42]. Second, only one metabolomics measurement was performed for each participant. However, our previous pilot study indicated that most metabolites were highly stable over 1–2 years within individuals [26]. Third, the metabolites measured in our study focused on lipids, amino acids, and amino acid derivatives and did not include all established biomarkers of food intake. Finally, participants in our study were mainly white women; replication of our findings in men and other racial groups is needed.\nIn conclusion, we identified a panel of metabolites differentially correlated with the intake of red meat, poultry, and fish. We also developed metabolite profile scores associated with the consumption of meat and fish and observed consistent inverse associations between metabolite profile scores of fish intake and CRC risk. Our results suggest the potential utility of metabolomics in complementing traditional dietary assessments to investigate the diet-disease association and disentangle the metabolomic responses linking higher fish intake with reduced CRC risk."],"string":"[\n \"Colorectal cancer (CRC) remains the second most commonly occurring cancer in women and the third in men worldwide [1]. CRC incidence is largely affected by screening and modifiable dietary and lifestyle factors [2]. Among these factors, meat and fish consumption has been the subject of many investigations. The consumption of red meat, especially processed red meat, is consistently associated with an increased risk of CRC [3]. However, there is still uncertainty regarding the association between fish intake and CRC risk. The World Cancer Research Fund and American Institute for Cancer Research (WCRF/AICR) concluded that there was only “limited evidence” for the beneficial effect of fish intake in CRC prevention [4]. Recent studies of fish intake and CRC risk also generated inconsistent results [5,6,7,8].\\nPrevious epidemiological studies examining the association of meat and fish intake with CRC risk mainly used self-reported dietary data. Rapid developments in high-throughput metabolomics are leading to a new era in nutritional epidemiological research. By measuring the small-molecule metabolites in biological samples, metabolomics may provide an objective picture of food intake and its related biological consequences [9]. Feeding trials and observational studies have demonstrated that plasma and urinary metabolites differed between meat and fish consumption [10,11,12,13,14]. However, it is unknown whether the metabolite profiles related to meat and fish intake are associated with CRC risk and whether these profiles could be used as a complementary approach to evaluate the association between meat and fish intake and CRC risk.\\nTherefore, we examined the associations of red meat, poultry, and fish consumption with plasma metabolite profiles among participants from the Nurses’ Health Study (NHS), NHSII, and Health Professionals Follow-up Study (HPFS). We also developed metabolite profile scores that were correlated to the intakes of these meat and fish groups and evaluated the prospective associations of metabolite profile scores with CRC risk. The results for metabolite profile scores were further replicated in an external validation cohort (Women’s Health Study, WHS).\",\n \" Study Population Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\\nOur primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\",\n \"Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\",\n \"Validated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.\",\n \"Profiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.\",\n \"In NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\",\n \"We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\",\n \"In NHS/NHSII/HPFS, participants were predominately white and middle-aged (mean age 53 years), with an average BMI of 25.4 kg/m2 (Table 1). The percentage energy intake from protein was 18%, and animal protein was 13%. Participants with a higher total red meat intake were slightly more likely to have a lower total fish intake (Pearson r = −0.07). In the analysis of CRC, participants had similar demographic characteristics but were a little older (mean age 61 years) and had a lower proportion of females. Compared to control participants, CRC cases were less likely to use multivitamins and aspirin, receive endoscopy screening, and be physically active, but were more likely to smoke and have a family history of CRC. They also had a somewhat higher intake of total red meat (mainly processed red meat) and a lower fish intake. Similarly, CRC cases in WHS were less likely to be physically active and more likely to smoke and have a family history of CRC. However, they had a lower intake of total red meat (mainly unprocessed red meat) than control participants.\\nOf the 287 known metabolites, 85 were significantly correlated with total red meat intake, 36 with unprocessed red meat, 51 with processed red meat, 28 with poultry, 60 with total fish, 60 with dark meat fish, and 27 with canned tuna fish after Bonferroni correction (Figure 2). Similar metabolites correlated with total red meat intake, unprocessed red meat, and processed red meat. All three red meat groups were positively associated with creatine, hydroxyproline, coenzyme Q10, myristoleic acid, acylcarnitines, and plasmalogens with a number of double bonds ≤6 but were negatively associated with highly unsaturated lipid species including triglycerides (TAGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylcholines (LPCs). Poultry intake was positively correlated with creatine, ectoine, a few PCs, TAGs, and PE plasmalogens after Bonferroni correction. In contrast to red meat intake, total fish intake, dark meat fish, and canned tuna fish were all positively correlated with highly unsaturated lipid species. When we restricted the analysis to fasting participants and participants selected as controls in the original sub-studies, the correlation results were similar (Supplementary Figures S2 and S3).\\nOf the 2561 unknown metabolites, 702 were significantly correlated with total red meat intake after Bonferroni correction, 222 with unprocessed red meat, 462 with processed red meat, 65 with poultry, 284 with total fish intake, 414 with dark meat fish, and 119 with canned tuna fish (Supplementary Table S2). Among these, the absolute partial Spearman rho exceeded 0.3 for 11 unknown metabolites: one with total red meat intake, 10 with total fish intake, and 5 with dark meat fish intake. Pearson correlation analysis between these 11 unknown metabolites and those known metabolites (130 in total) that were significantly associated with total red meat, total fish, or dark meat fish consumption identified several unknown metabolites being strongly correlated with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC (all Pearson r > 0.8) (Supplementary Figure S4).\\nA total of 53 known metabolites were selected in the elastic net regression for total red meat intake, 55 for unprocessed red meat, 36 for processed red meat, 7 for poultry, 18 for total fish, 27 for dark meat fish, and 11 for canned tuna fish (Table 2). Each metabolite profile score based on the selected known metabolites was significantly correlated with the corresponding self-reported dietary intake in both NHS/NHSII/HPFS and WHS (r = 0.33–0.46 for total red meat; r = 0.36–0.42 for unprocessed red meat; r = 0.19–0.33 for processed red meat; r = 0.12–0.21 for poultry; r = 0.31–0.40 for total fish; r = 0.32–0.42 for dark meat fish; and r = 0.14–0.22 for canned tuna fish) (Table 2). Adding unknown metabolites into the metabolite profile scores did not materially improve their correlations with the dietary intake (Supplementary Table S3). As a sensitivity analysis, we adopted the random forest algorithm to impute the missingness and re-derived the metabolomic profile score. The newly derived scores were highly correlated to the original scores, with a Pearson r = 0.98 for the total red meat score, 0.98 for the unprocessed red meat, 0.96 for the processed red meat score, 0.92 for the poultry score, 0.99 for the total fish score, 0.99 for the dark meat fish score, and 0.97 for the canned tuna fish score.\\nAfter adjusting for potential confounders, dietary intake of processed red meat was significantly associated with CRC risk. The pooled OR for 1 SD higher intake was 1.15 (95% CI: 1.03, 1.29) (Table 3). We did not observe consistent associations for dietary intake of three fish groups across NHS/HPFS and WHS. An inverse association for total fish and canned tuna fish was only found in NHS/HPFS and for dark meat fish in WHS. In contrast, metabolite profile scores for these three fish groups were inversely associated with CRC risk in both NHS/HPFS and WHS (Table 3). The pooled OR per 1 SD higher was 0.86 (95% CI: 0.77, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were observed for other meat intakes or metabolite profile scores. The results remained similar after adjusting for menopausal status or ethnicity in NHS/HPFS (data not shown). In the analysis by tumor site, we observed that the above associations were stronger for rectal cancer than those for colon cancer (Supplementary Table S4).\\nAmong metabolites selected in metabolite profile scores of all three fish groups, five metabolites (C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) were positively correlated to fish intake but inversely associated with CRC risk; two metabolites (C20:4 LPE and C22:5 LPC) were inversely correlated to fish intake but positively associated with CRC risk (Supplementary Figure S5).\",\n \"Leveraging metabolomics data from four studies, we found systematic differences in plasma metabolite profiles according to various types of meat and fish consumption. We also developed metabolite profile scores that were correlated to the consumption of red meat, poultry, and fish. Higher metabolite profile scores for all three fish groups (total fish, dark meat fish, and canned tuna fish), rather than their self-reported dietary intake, were consistently associated with a lower CRC risk in both NHS/HPFS and WHS. These findings suggest the promising use of metabolomics in complementing traditional dietary assessments to evaluate the association between dietary exposure and health outcomes.\\nConsistent with previous studies, we found that consumption of all three red meat groups was positively correlated with several PE and PC plasmalogens, all of which have a low degree of unsaturation and contain acid moieties derived from animal tissues [14,29]. In contrast, total fish intake was positively correlated with highly unsaturated lipids, including TAGs, PCs, PEs, LPCs, LPEs, CEs, and plasmalogens. Fish intake, especially marine fish, is the principal dietary contributor of highly n-3 polyunsaturated fatty acids [30], and thus, are dietary sources for these lipids and their downstream metabolites.\\nApart from lipid metabolites, we also observed several other metabolites correlated with meat and fish consumption. Creatine, an amino acid metabolite abundant in skeletal muscle [31], was positively correlated with the intake of red meat, poultry, and fish. Hydroxyproline, an amino acid that forms part of collagen [32], was positively correlated with red meat and poultry intake. Additionally, we found a positive correlation between poultry intake and ectoine (an α-amino acid), in line with one previous study [14]. Ectoine can be synthesized and released by several species in the genus Halomonas [33], the dominant microbial genus in the chicken embryo [34], which might lead to the presence of ectoine in chicken.\\nMost previous metabolomic studies mainly focused on known metabolites, which are only a small percent of the data obtained in a typical metabolomics measurement (~10%) [35]. In the present study, we analyzed the unknown metabolites and identified a set of unknown metabolites that were significantly correlated with meat or fish consumption. Several unknown metabolites even exhibited a higher correlation with meat and fish intake (partial Spearman rho > 0.3) compared to known metabolites. By further examining the correlation between these unknown metabolites and the known metabolites, strong correlations were found with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC, indicating their potential biological links. However, whether these unknown metabolites are metabolites of the known metabolites or unrelated molecules affected by the same food is not clear. A critical next step is to structurally identify the promising and influential unknown metabolites. It should also be noted that despite the many statistically significant correlations between meat and fish groups and the unknown metabolites, these unknown metabolites added minimally to the correlations between meat and fish intakes and metabolite profile score based only on the known metabolites. The possibility remains that other metabolomics platforms may add further independent prediction of intake.\\nThe human metabolome reflects a holistic biological status, which can be influenced by various exogenous and endogenous factors, such as dietary intakes, gut microbiota, health status, and genotype [36,37]. The metabolites selected in the metabolite profile score for meat or fish consumption could represent, in part, the components of food, such as the eicosapentaenoic acid/docosahexaenoic acid-containing lipids for the fish intake and hydroxyproline for the intake of red meat and poultry. Other metabolites, for example, C20:4 LPE and C22:5 LPC, could be related to the metabolic processes after fish intake, as fish is one of the main sources of arachidonic acid (C20:4) [38] and docosapentaenoic acid (C22:5) [39], but it was inversely correlated with these two lipids. Thus, metabolite profile score can be a comprehensive way to reflect both genuine compounds from foods as well as markers of complex metabolomic responses to dietary exposures.\\nNot surprisingly, we found a significant positive association between processed red meat intake and CRC risk. However, the metabolite profile score for processed red meat consumption was not significantly associated with CRC risk. One possible explanation could be that the increased CRC risk associated with high processed red meat intake was mainly due to the carcinogenic compounds [40], such as polycyclic aromatic hydrocarbons, heterocyclic amines, and N-nitroso compounds, which were not measured in our metabolomics platforms. On the contrary, we did not observe a consistent inverse association between dietary fish intake and CRC risk, but inverse associations were found for the metabolite profile scores of all three fish groups in both NHS/HPFS and WHS. Results from previous studies of fish intake and CRC risk were generally inconsistent, some indicating an inverse association [7,8], but others not [5,6]. Unlike self-reported dietary intake data widely used in previous studies, the metabolomics data objectively captures the metabolites related to dietary exposures in the human body, which might be more directly associated with health outcomes. The seven metabolites (C20:4 LPE, C22:5 LPC, C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) selected in metabolite profile scores of all three fish groups could represent the beneficial metabolomic response to fish intake, and this response led to a lower risk of CRC.\\nThe main strengths of our study are the large sample size, comprehensive metabolite profiles including both known and unknown metabolites, prospectively collected dietary data before CRC diagnosis, and the use of the elastic net model, which performs well in high-dimensional data where there are high correlations between predictors [41]. Moreover, the robustness of our metabolite profile scores was validated in an independent study. Nevertheless, our results also have several limitations. First, we did not have data on the type of red meat. Red meat subtypes (beef, lamb, or pork) may differ in their associations with CRC risk [42]. Second, only one metabolomics measurement was performed for each participant. However, our previous pilot study indicated that most metabolites were highly stable over 1–2 years within individuals [26]. Third, the metabolites measured in our study focused on lipids, amino acids, and amino acid derivatives and did not include all established biomarkers of food intake. Finally, participants in our study were mainly white women; replication of our findings in men and other racial groups is needed.\\nIn conclusion, we identified a panel of metabolites differentially correlated with the intake of red meat, poultry, and fish. We also developed metabolite profile scores associated with the consumption of meat and fish and observed consistent inverse associations between metabolite profile scores of fish intake and CRC risk. Our results suggest the potential utility of metabolomics in complementing traditional dietary assessments to investigate the diet-disease association and disentangle the metabolomic responses linking higher fish intake with reduced CRC risk.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,"results","discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["red meat","fish","plasma metabolomics","colorectal cancer"],"string":"[\n \"red meat\",\n \"fish\",\n \"plasma metabolomics\",\n \"colorectal cancer\"\n]"},"whole_article_text":{"kind":"string","value":"1. Introduction:\nColorectal cancer (CRC) remains the second most commonly occurring cancer in women and the third in men worldwide [1]. CRC incidence is largely affected by screening and modifiable dietary and lifestyle factors [2]. Among these factors, meat and fish consumption has been the subject of many investigations. The consumption of red meat, especially processed red meat, is consistently associated with an increased risk of CRC [3]. However, there is still uncertainty regarding the association between fish intake and CRC risk. The World Cancer Research Fund and American Institute for Cancer Research (WCRF/AICR) concluded that there was only “limited evidence” for the beneficial effect of fish intake in CRC prevention [4]. Recent studies of fish intake and CRC risk also generated inconsistent results [5,6,7,8].\nPrevious epidemiological studies examining the association of meat and fish intake with CRC risk mainly used self-reported dietary data. Rapid developments in high-throughput metabolomics are leading to a new era in nutritional epidemiological research. By measuring the small-molecule metabolites in biological samples, metabolomics may provide an objective picture of food intake and its related biological consequences [9]. Feeding trials and observational studies have demonstrated that plasma and urinary metabolites differed between meat and fish consumption [10,11,12,13,14]. However, it is unknown whether the metabolite profiles related to meat and fish intake are associated with CRC risk and whether these profiles could be used as a complementary approach to evaluate the association between meat and fish intake and CRC risk.\nTherefore, we examined the associations of red meat, poultry, and fish consumption with plasma metabolite profiles among participants from the Nurses’ Health Study (NHS), NHSII, and Health Professionals Follow-up Study (HPFS). We also developed metabolite profile scores that were correlated to the intakes of these meat and fish groups and evaluated the prospective associations of metabolite profile scores with CRC risk. The results for metabolite profile scores were further replicated in an external validation cohort (Women’s Health Study, WHS).\n\n2. Methods:\n Study Population Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\nOur primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\n\nStudy Population:\nOur primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\n\n3. Dietary Assessment:\nValidated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.\n\n4. Metabolomics Measurement:\nProfiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.\n\n5. Nondietary Covariates:\nIn NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\n\nStatistical Analyses:\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\n\n6. Results:\nIn NHS/NHSII/HPFS, participants were predominately white and middle-aged (mean age 53 years), with an average BMI of 25.4 kg/m2 (Table 1). The percentage energy intake from protein was 18%, and animal protein was 13%. Participants with a higher total red meat intake were slightly more likely to have a lower total fish intake (Pearson r = −0.07). In the analysis of CRC, participants had similar demographic characteristics but were a little older (mean age 61 years) and had a lower proportion of females. Compared to control participants, CRC cases were less likely to use multivitamins and aspirin, receive endoscopy screening, and be physically active, but were more likely to smoke and have a family history of CRC. They also had a somewhat higher intake of total red meat (mainly processed red meat) and a lower fish intake. Similarly, CRC cases in WHS were less likely to be physically active and more likely to smoke and have a family history of CRC. However, they had a lower intake of total red meat (mainly unprocessed red meat) than control participants.\nOf the 287 known metabolites, 85 were significantly correlated with total red meat intake, 36 with unprocessed red meat, 51 with processed red meat, 28 with poultry, 60 with total fish, 60 with dark meat fish, and 27 with canned tuna fish after Bonferroni correction (Figure 2). Similar metabolites correlated with total red meat intake, unprocessed red meat, and processed red meat. All three red meat groups were positively associated with creatine, hydroxyproline, coenzyme Q10, myristoleic acid, acylcarnitines, and plasmalogens with a number of double bonds ≤6 but were negatively associated with highly unsaturated lipid species including triglycerides (TAGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylcholines (LPCs). Poultry intake was positively correlated with creatine, ectoine, a few PCs, TAGs, and PE plasmalogens after Bonferroni correction. In contrast to red meat intake, total fish intake, dark meat fish, and canned tuna fish were all positively correlated with highly unsaturated lipid species. When we restricted the analysis to fasting participants and participants selected as controls in the original sub-studies, the correlation results were similar (Supplementary Figures S2 and S3).\nOf the 2561 unknown metabolites, 702 were significantly correlated with total red meat intake after Bonferroni correction, 222 with unprocessed red meat, 462 with processed red meat, 65 with poultry, 284 with total fish intake, 414 with dark meat fish, and 119 with canned tuna fish (Supplementary Table S2). Among these, the absolute partial Spearman rho exceeded 0.3 for 11 unknown metabolites: one with total red meat intake, 10 with total fish intake, and 5 with dark meat fish intake. Pearson correlation analysis between these 11 unknown metabolites and those known metabolites (130 in total) that were significantly associated with total red meat, total fish, or dark meat fish consumption identified several unknown metabolites being strongly correlated with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC (all Pearson r > 0.8) (Supplementary Figure S4).\nA total of 53 known metabolites were selected in the elastic net regression for total red meat intake, 55 for unprocessed red meat, 36 for processed red meat, 7 for poultry, 18 for total fish, 27 for dark meat fish, and 11 for canned tuna fish (Table 2). Each metabolite profile score based on the selected known metabolites was significantly correlated with the corresponding self-reported dietary intake in both NHS/NHSII/HPFS and WHS (r = 0.33–0.46 for total red meat; r = 0.36–0.42 for unprocessed red meat; r = 0.19–0.33 for processed red meat; r = 0.12–0.21 for poultry; r = 0.31–0.40 for total fish; r = 0.32–0.42 for dark meat fish; and r = 0.14–0.22 for canned tuna fish) (Table 2). Adding unknown metabolites into the metabolite profile scores did not materially improve their correlations with the dietary intake (Supplementary Table S3). As a sensitivity analysis, we adopted the random forest algorithm to impute the missingness and re-derived the metabolomic profile score. The newly derived scores were highly correlated to the original scores, with a Pearson r = 0.98 for the total red meat score, 0.98 for the unprocessed red meat, 0.96 for the processed red meat score, 0.92 for the poultry score, 0.99 for the total fish score, 0.99 for the dark meat fish score, and 0.97 for the canned tuna fish score.\nAfter adjusting for potential confounders, dietary intake of processed red meat was significantly associated with CRC risk. The pooled OR for 1 SD higher intake was 1.15 (95% CI: 1.03, 1.29) (Table 3). We did not observe consistent associations for dietary intake of three fish groups across NHS/HPFS and WHS. An inverse association for total fish and canned tuna fish was only found in NHS/HPFS and for dark meat fish in WHS. In contrast, metabolite profile scores for these three fish groups were inversely associated with CRC risk in both NHS/HPFS and WHS (Table 3). The pooled OR per 1 SD higher was 0.86 (95% CI: 0.77, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were observed for other meat intakes or metabolite profile scores. The results remained similar after adjusting for menopausal status or ethnicity in NHS/HPFS (data not shown). In the analysis by tumor site, we observed that the above associations were stronger for rectal cancer than those for colon cancer (Supplementary Table S4).\nAmong metabolites selected in metabolite profile scores of all three fish groups, five metabolites (C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) were positively correlated to fish intake but inversely associated with CRC risk; two metabolites (C20:4 LPE and C22:5 LPC) were inversely correlated to fish intake but positively associated with CRC risk (Supplementary Figure S5).\n\n7. Discussion:\nLeveraging metabolomics data from four studies, we found systematic differences in plasma metabolite profiles according to various types of meat and fish consumption. We also developed metabolite profile scores that were correlated to the consumption of red meat, poultry, and fish. Higher metabolite profile scores for all three fish groups (total fish, dark meat fish, and canned tuna fish), rather than their self-reported dietary intake, were consistently associated with a lower CRC risk in both NHS/HPFS and WHS. These findings suggest the promising use of metabolomics in complementing traditional dietary assessments to evaluate the association between dietary exposure and health outcomes.\nConsistent with previous studies, we found that consumption of all three red meat groups was positively correlated with several PE and PC plasmalogens, all of which have a low degree of unsaturation and contain acid moieties derived from animal tissues [14,29]. In contrast, total fish intake was positively correlated with highly unsaturated lipids, including TAGs, PCs, PEs, LPCs, LPEs, CEs, and plasmalogens. Fish intake, especially marine fish, is the principal dietary contributor of highly n-3 polyunsaturated fatty acids [30], and thus, are dietary sources for these lipids and their downstream metabolites.\nApart from lipid metabolites, we also observed several other metabolites correlated with meat and fish consumption. Creatine, an amino acid metabolite abundant in skeletal muscle [31], was positively correlated with the intake of red meat, poultry, and fish. Hydroxyproline, an amino acid that forms part of collagen [32], was positively correlated with red meat and poultry intake. Additionally, we found a positive correlation between poultry intake and ectoine (an α-amino acid), in line with one previous study [14]. Ectoine can be synthesized and released by several species in the genus Halomonas [33], the dominant microbial genus in the chicken embryo [34], which might lead to the presence of ectoine in chicken.\nMost previous metabolomic studies mainly focused on known metabolites, which are only a small percent of the data obtained in a typical metabolomics measurement (~10%) [35]. In the present study, we analyzed the unknown metabolites and identified a set of unknown metabolites that were significantly correlated with meat or fish consumption. Several unknown metabolites even exhibited a higher correlation with meat and fish intake (partial Spearman rho > 0.3) compared to known metabolites. By further examining the correlation between these unknown metabolites and the known metabolites, strong correlations were found with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC, indicating their potential biological links. However, whether these unknown metabolites are metabolites of the known metabolites or unrelated molecules affected by the same food is not clear. A critical next step is to structurally identify the promising and influential unknown metabolites. It should also be noted that despite the many statistically significant correlations between meat and fish groups and the unknown metabolites, these unknown metabolites added minimally to the correlations between meat and fish intakes and metabolite profile score based only on the known metabolites. The possibility remains that other metabolomics platforms may add further independent prediction of intake.\nThe human metabolome reflects a holistic biological status, which can be influenced by various exogenous and endogenous factors, such as dietary intakes, gut microbiota, health status, and genotype [36,37]. The metabolites selected in the metabolite profile score for meat or fish consumption could represent, in part, the components of food, such as the eicosapentaenoic acid/docosahexaenoic acid-containing lipids for the fish intake and hydroxyproline for the intake of red meat and poultry. Other metabolites, for example, C20:4 LPE and C22:5 LPC, could be related to the metabolic processes after fish intake, as fish is one of the main sources of arachidonic acid (C20:4) [38] and docosapentaenoic acid (C22:5) [39], but it was inversely correlated with these two lipids. Thus, metabolite profile score can be a comprehensive way to reflect both genuine compounds from foods as well as markers of complex metabolomic responses to dietary exposures.\nNot surprisingly, we found a significant positive association between processed red meat intake and CRC risk. However, the metabolite profile score for processed red meat consumption was not significantly associated with CRC risk. One possible explanation could be that the increased CRC risk associated with high processed red meat intake was mainly due to the carcinogenic compounds [40], such as polycyclic aromatic hydrocarbons, heterocyclic amines, and N-nitroso compounds, which were not measured in our metabolomics platforms. On the contrary, we did not observe a consistent inverse association between dietary fish intake and CRC risk, but inverse associations were found for the metabolite profile scores of all three fish groups in both NHS/HPFS and WHS. Results from previous studies of fish intake and CRC risk were generally inconsistent, some indicating an inverse association [7,8], but others not [5,6]. Unlike self-reported dietary intake data widely used in previous studies, the metabolomics data objectively captures the metabolites related to dietary exposures in the human body, which might be more directly associated with health outcomes. The seven metabolites (C20:4 LPE, C22:5 LPC, C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) selected in metabolite profile scores of all three fish groups could represent the beneficial metabolomic response to fish intake, and this response led to a lower risk of CRC.\nThe main strengths of our study are the large sample size, comprehensive metabolite profiles including both known and unknown metabolites, prospectively collected dietary data before CRC diagnosis, and the use of the elastic net model, which performs well in high-dimensional data where there are high correlations between predictors [41]. Moreover, the robustness of our metabolite profile scores was validated in an independent study. Nevertheless, our results also have several limitations. First, we did not have data on the type of red meat. Red meat subtypes (beef, lamb, or pork) may differ in their associations with CRC risk [42]. Second, only one metabolomics measurement was performed for each participant. However, our previous pilot study indicated that most metabolites were highly stable over 1–2 years within individuals [26]. Third, the metabolites measured in our study focused on lipids, amino acids, and amino acid derivatives and did not include all established biomarkers of food intake. Finally, participants in our study were mainly white women; replication of our findings in men and other racial groups is needed.\nIn conclusion, we identified a panel of metabolites differentially correlated with the intake of red meat, poultry, and fish. We also developed metabolite profile scores associated with the consumption of meat and fish and observed consistent inverse associations between metabolite profile scores of fish intake and CRC risk. Our results suggest the potential utility of metabolomics in complementing traditional dietary assessments to investigate the diet-disease association and disentangle the metabolomic responses linking higher fish intake with reduced CRC risk.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nRed and processed meat consumption has been consistently associated with increased risk of colorectal cancer (CRC), but the association for fish intake is unclear. Evidence using objective dietary assessment approaches to evaluate these associations is sparse.\n\nMethods:\nWe measured plasma metabolites among 5269 participants from the Nurses' Health Study (NHS), NHSII, and Health Professionals Follow-Up study (HPFS). We calculated partial Spearman correlations between each metabolite and self-reported intake of seven red meat, poultry, and fish groups. Metabolite profile scores correlated to self-reported dietary intakes were developed using elastic net regression. Associations between self-reported intakes, metabolite profile scores, and subsequent CRC risk were further evaluated using conditional logistic regression among 559 matched (1:1) case-control pairs in NHS/HPFS and replicated among 266 pairs in Women's Health Study.\n\nResults:\nPlasma metabolites, especially highly unsaturated lipids, were differentially associated with red meat and fish groups. Metabolite profile scores for each food group were significantly correlated with the corresponding self-reported dietary intake. A higher dietary intake of processed red meat was associated with a higher risk of CRC (pooled OR per 1 SD, 1.15; 95% CI: 1.03, 1.29). In contrast, higher metabolite profile scores for all fish groups, not dietary intakes, were consistently associated with a lower CRC risk: the pooled OR per 1 SD was 0.86 (95% CI: 0.78, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were found for other food groups.\n\nConclusions:\nRed meat and fish intake exhibited systematically different plasma metabolite profiles. Plasma metabolite profile of fish intake was inversely associated with CRC risk."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":6815,"string":"6,815"},"whole_article_abstract_length":{"kind":"number","value":357,"string":"357"},"num_sections":{"kind":"number","value":9,"string":"9"},"most_frequent_words":{"kind":"list like","value":["fish","meat","metabolites","intake","red meat","red","metabolite","crc","dietary","profile"],"string":"[\n \"fish\",\n \"meat\",\n \"metabolites\",\n \"intake\",\n \"red meat\",\n \"red\",\n \"metabolite\",\n \"crc\",\n \"dietary\",\n \"profile\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | crc | meat | fish intake | intake crc | fish intake crc | risk | intake | crc risk | intake crc risk [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] blood | aged | cancer | blood collection | collection | samples | blood samples | years | included | matched [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] meat | fish | red meat | red | intake | total | total red meat | total red | total fish | dark [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] fish | meat | metabolites | intake | metabolite | red | red meat | crc | metabolite profile | meat fish [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] CRC ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 5269 | NHSII | Health Professionals Follow-Up ||| Spearman | seven ||| ||| CRC | 559 | 1:1 | NHS | 266 | Women's Health Study [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Plasma metabolites ||| ||| CRC | 1 SD | 1.15 | 95% | CI | 1.03 | 1.29 ||| CRC | 0.86 | 95% | CI | 0.78 | 0.96 | 0.86 | 95% | CI | 0.77 | 0.96 | 0.87 | 95% | CI | 0.78 | 0.97 ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] CRC ||| ||| 5269 | NHSII | Health Professionals Follow-Up ||| Spearman | seven ||| ||| CRC | 559 | 1:1 | NHS | 266 | Women's Health Study ||| ||| Plasma metabolites ||| ||| CRC | 1 SD | 1.15 | 95% | CI | 1.03 | 1.29 ||| CRC | 0.86 | 95% | CI | 0.78 | 0.96 | 0.86 | 95% | CI | 0.77 | 0.96 | 0.87 | 95% | CI | 0.78 | 0.97 ||| ||| ||| Plasma | CRC [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":272,"cells":{"title":{"kind":"string","value":"Sericin cream reduces pruritus in hemodialysis patients: a randomized, double-blind, placebo-controlled experimental study."},"pmid":{"kind":"string","value":"23006933"},"background_abstract":{"kind":"string","value":"Uremic pruritus (UP) is a significant complication in ESRD patients and substantially impairs their quality of life. UP is considered to be a skin manifestation of chronic inflammation. Because sericin can suppress the release of pro-inflammatory cytokines, the purpose of this study was to investigate the short-term safety and efficacy of sericin cream for treating UP in hemodialysis patients."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"This study used a double-blind design to investigate the effects of random topical administration of sericin cream and cream base (placebo) on either the right or left extremities of hemodialysis patients for 6 weeks. Skin hydration, irritation and pigmentation were evaluated every 2 weeks using Skin Diagnostic SD27. The visual analog scale for itching was also evaluated every 2 weeks, and the Kidney Disease Quality of Life Short Form was performed on the day of each patient's enrollment and after 6 weeks of treatment."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Fifty dialysis patients were enrolled, 47 of which completed the study. The hydration of the skin of the patients' extremities increased significantly after administration of sericin cream; significant differences were found between sericin treatment and control after 6 weeks of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Moreover, a significant difference was also found in skin irritation between the two treatments (p = 0.013 for arms and p = 0.027 for legs, respectively). At the end of the study, the skin pigmentation level was significantly reduced on both the arms (p = 0.032) and legs (p = 0.021) of the sericin-treated side compared with the side treated with cream base. The mean itching score decreased significantly from moderate to severe at the time of enrollment to mild pruritus after 6 weeks of treatment (p = 0.002). A better quality of life was found in all domains tested although statistically significant differences before and after treatment was found only in the patients' pain scores, the effect of kidney disease on daily life, sleep quality and symptoms or problems related to kidney disease."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"We conclude that sericin cream has a high potential for reducing UP in hemodialysis patients.The trial registration number of this study is ISRCTN16019033; its public title is \"sericin cream reduces pruritus in hemodialysis patients\"."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Administration, Topical","Double-Blind Method","Female","Humans","Male","Middle Aged","Placebo Effect","Pruritus","Quality of Life","Renal Dialysis","Sericins","Skin Cream","Treatment Outcome"],"string":"[\n \"Administration, Topical\",\n \"Double-Blind Method\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Placebo Effect\",\n \"Pruritus\",\n \"Quality of Life\",\n \"Renal Dialysis\",\n \"Sericins\",\n \"Skin Cream\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"3472272"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Preparation of silk sericin cream Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\nBecause commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\n Molecular weight determination of sericin To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\nTo determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\n Amino acid analysis of sericin The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\nThe amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\n Study design Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\nSkin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\n Study population Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Study treatment In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\nIn 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\n Measurement and outcome The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\nThe level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\n Patients’ quality of life The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\nThe patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\n Safety monitoring The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\nThe occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\n Statistical analysis The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\nThe results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Molecular weight and amino acid composition of sericin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\n Study population Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.\nFifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Our study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients."},"other_sections_titles":{"kind":"list like","value":["Background","Molecular weight and amino acid composition of sericin","Study population","Preparation of silk sericin cream","Molecular weight determination of sericin","Amino acid analysis of sericin","Study design","Study population","Inclusion criteria","Exclusion criteria","Study treatment","Measurement and outcome","Patients’ quality of life","Safety monitoring","Statistical analysis","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Molecular weight and amino acid composition of sericin\",\n \"Study population\",\n \"Preparation of silk sericin cream\",\n \"Molecular weight determination of sericin\",\n \"Amino acid analysis of sericin\",\n \"Study design\",\n \"Study population\",\n \"Inclusion criteria\",\n \"Exclusion criteria\",\n \"Study treatment\",\n \"Measurement and outcome\",\n \"Patients’ quality of life\",\n \"Safety monitoring\",\n \"Statistical analysis\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.","Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.","Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.","Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.","To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.","The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.","Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention."," Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.","All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.","Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.","In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.","The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].","The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].","The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.","The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.","The authors declare that they have no competing interests.","PA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\n"],"string":"[\n \"Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.\",\n \"Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\\nAmino acid composition of sericin\\nThese mean values were obtained by triplicate analysis.\",\n \"Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \\nBaseline characteristics of subjects (N = 47)\\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\\nSS = silk sericin.\\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \\nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\\n* Indicates significant differences compared to baseline, p < 0.05.\",\n \"Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\",\n \"To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\",\n \"The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\",\n \"Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\",\n \" Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\",\n \"All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\",\n \"Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\",\n \"In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\",\n \"The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\\n\\n\\n(1)\\n\\n\\n%\\nchanges in each parameter\\n=\\n\\n\\n\\n\\n\\nP\\nt\\n\\n−\\n\\nP\\n0\\n\\n\\n\\n/\\n\\nP\\n0\\n\\n\\n\\n×\\n100\\n,\\n\\n\\n\\n\\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\",\n \"The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\",\n \"The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\",\n \"The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\",\n \"The authors declare that they have no competing interests.\",\n \"PA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Results","Molecular weight and amino acid composition of sericin","Study population","Discussion","Conclusions","Methods","Preparation of silk sericin cream","Molecular weight determination of sericin","Amino acid analysis of sericin","Study design","Study population","Inclusion criteria","Exclusion criteria","Study treatment","Measurement and outcome","Patients’ quality of life","Safety monitoring","Statistical analysis","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Results\",\n \"Molecular weight and amino acid composition of sericin\",\n \"Study population\",\n \"Discussion\",\n \"Conclusions\",\n \"Methods\",\n \"Preparation of silk sericin cream\",\n \"Molecular weight determination of sericin\",\n \"Amino acid analysis of sericin\",\n \"Study design\",\n \"Study population\",\n \"Inclusion criteria\",\n \"Exclusion criteria\",\n \"Study treatment\",\n \"Measurement and outcome\",\n \"Patients’ quality of life\",\n \"Safety monitoring\",\n \"Statistical analysis\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated."," Molecular weight and amino acid composition of sericin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\n Study population Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.\nFifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.","Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.","Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.","This study has shown that the use of sericin cream can reduce UP in hemodialysis patients according to their VAS scores. Sericin cream can also significantly increase skin hydration and reduce skin irritation and skin pigmentation in patients. The molecular weight analysis of the sericin used in this study gave results that agree with those reported by Sprague, who indicated that sericin is a mixture of at least 15 different polypeptide chains ranging in size from 20–220 kDa [19]. Similar to the earlier report by Kato et al., serine was the most abundant amino acid found in sericin [20].\nAlthough antihistamines, anti-allergic agents and topical corticosteroids are commonly used to treat UP in ESRD patients, their effectiveness is sometimes limited [2,8,21,22]. Alternative and effective treatments for intractable pruritus that generate low levels of adverse reactions, particularly in sensitive patients such as the ESRD group, must therefore be developed. This clinical study is the first to suggest that a sericin cream is beneficial for treating pruritus in hemodialysis patients. The benefits of this treatment apparently result from increases in skin hydration and suppression of pro-inflammatory cytokines, as shown in our earlier study [17,23], thereby resulting in less skin irritation without any allergic reactions.\nPruritus associated with chronic kidney disease has been shown to be associated with elevated levels of C-reactive protein and other inflammatory cytokines. This evidence suggests that there is an inflammatory component in this form of pruritus [24]. Some authors consider UP to be a skin manifestation of chronic inflammation [25]. Recently, the “persistent microinflammation” theory, in which it is suggested that inflammation may be related to the genesis of UP, was proposed [26]. The skin of ESRD patients with UP contains an increased number of mast cells, and these cells can release various substances such as histamine, interleukins (IL) and tumor necrosis factor (TNF) [27-30].\nPruritic skin can appear normal except for dryness; because the severity of pruritus is closely correlated with skin dryness [31], xerosis treatment normally begins with a topical agent such as a gentle moisturizing cream. Because sericin is well known for its moisturizing effect and its ability to reduce the generation of pro-inflammatory cytokines by fibroblasts, this study assessed the usefulness of sericin cream in treating UP. Evaluating the degree of pruritus was difficult because it is a subjective symptom; therefore, functional measurements of skin conditions known to be associated with pruritus, such as skin hydration and skin irritation, were considered to be objective indices. We found that both sericin cream and the cream base used to prepare the sericin cream increased the moisture content of the stratum corneum; however, the level of hydration was significantly higher in the skin treated with sericin cream. The side of the body treated with sericin cream had significantly lower levels of post-treatment dryness compared to pretreatment dryness and lower total dermatological severity scores in parameters such as skin pigmentation.\nThe pathogenesis of ESRD itself is associated with inflammation and oxidative stress, as reflected in studies of plasma biochemistry, including the levels of the cytokines IL and TNF-α [32-34], classic markers of inflammatory processes that are strongly affected by pathological conditions [35]. These cytokines are upregulated in the plasma of ESRD patients [32,36]. The skin inflammation associated with the release of IL and TNF-α is further complicated by the inflammatory lesions that occur secondary to scratching [37]. Our results indicate that improvement in skin hydration and suppression of the release of pro-inflammatory cytokines in skin might be the mechanism by which sericin improves UP. Skin irritation, which can be caused by either skin inflammation or by scratching, was substantially reduced in the sericin group compared with the cream base group after 6 weeks of treatment. This result confirmed that sericin reduces skin irritation in patients with UP and suggested that it might be due to suppression of the inflammatory cytokines in skin. Similar results have been obtained in animal studies [23].\nSecondary skin lesions that can result from the itch-scratch cycle include excoriations, hyperpigmentation, lichenification, prurigo nodules and scars [38]. Chronic renal failure is usually accompanied by a variety of cutaneous manifestations; dermal manifestations, including hyperpigmentation, have also been reported, and these can exact a considerable toll on the quality of life [39-41]. Although skin pigmentation is not related to specific clinical symptoms, it has an important effect on patient satisfaction. Sericin has been reported to have activity against tyrosinase [15], an enzyme related to melanin production; as this study shows, treatment with sericin results in significant reduction in the skin color of patients.\nThe pruritus/mortality relationship may be strongly attributed to sleep disturbances, as previously mentioned in the DOPPS [8,9,42]. Many previous studies have found a strong association between inflammation, as measured by C-reactive protein or inflammatory cytokines, and sleep disturbances in dialysis patients [43-45]. The significant effects of pruritus on sleep, mood and social functioning require further investigation with the goal of improving the available treatments for this serious ESRD complication. We found that reduction in itching intensity from moderate to severe pruritus at the time of enrollment to mild pruritus after 6 weeks of treatment was associated with a better quality of life in all of the measured domains, including the mental, physical and kidney disease components. The KDQOL-SF score in the ESRD patients indicated improvements in several domains regarding the patients’ quality of life, particularly pain and sleep, which are relevant to itching. A better quality of sleep may reflect some degree of relief from itching achieved through the treatments.\nThis study has certain limitations. First, the study design was an in-subject controlled study; it cannot be determined whether the improvements in the itching evaluation and the quality of life were from the sample (sericin cream) or the placebo (cream base). Moreover, due to the in-subject controlled design, the biochemical parameters could not be evaluated at the end of the study. Second, the study included a small number of patients, and these were followed for a relatively short time. A long-term study with a larger sample size is necessary. In addition to studies of hemodialysis patients, similar studies of other ESRD patients, such as peritoneal dialysis and kidney transplant patients, are also necessary to confirm the findings presented here.","Our study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients."," Preparation of silk sericin cream Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\nBecause commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\n Molecular weight determination of sericin To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\nTo determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\n Amino acid analysis of sericin The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\nThe amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\n Study design Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\nSkin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\n Study population Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Study treatment In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\nIn 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\n Measurement and outcome The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\nThe level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\n Patients’ quality of life The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\nThe patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\n Safety monitoring The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\nThe occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\n Statistical analysis The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\nThe results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.","Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.","To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.","The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.","Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention."," Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.","All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.","Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.","In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.","The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].","The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].","The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.","The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.","The authors declare that they have no competing interests.","PA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\n"],"string":"[\n \"Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.\",\n \" Molecular weight and amino acid composition of sericin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\\nAmino acid composition of sericin\\nThese mean values were obtained by triplicate analysis.\\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\\nAmino acid composition of sericin\\nThese mean values were obtained by triplicate analysis.\\n Study population Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \\nBaseline characteristics of subjects (N = 47)\\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\\nSS = silk sericin.\\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \\nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\\n* Indicates significant differences compared to baseline, p < 0.05.\\nFifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \\nBaseline characteristics of subjects (N = 47)\\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\\nSS = silk sericin.\\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \\nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\\n* Indicates significant differences compared to baseline, p < 0.05.\",\n \"Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\\nAmino acid composition of sericin\\nThese mean values were obtained by triplicate analysis.\",\n \"Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \\nBaseline characteristics of subjects (N = 47)\\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\\nSS = silk sericin.\\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \\nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\\n* Indicates significant differences compared to baseline, p < 0.05.\",\n \"This study has shown that the use of sericin cream can reduce UP in hemodialysis patients according to their VAS scores. Sericin cream can also significantly increase skin hydration and reduce skin irritation and skin pigmentation in patients. The molecular weight analysis of the sericin used in this study gave results that agree with those reported by Sprague, who indicated that sericin is a mixture of at least 15 different polypeptide chains ranging in size from 20–220 kDa [19]. Similar to the earlier report by Kato et al., serine was the most abundant amino acid found in sericin [20].\\nAlthough antihistamines, anti-allergic agents and topical corticosteroids are commonly used to treat UP in ESRD patients, their effectiveness is sometimes limited [2,8,21,22]. Alternative and effective treatments for intractable pruritus that generate low levels of adverse reactions, particularly in sensitive patients such as the ESRD group, must therefore be developed. This clinical study is the first to suggest that a sericin cream is beneficial for treating pruritus in hemodialysis patients. The benefits of this treatment apparently result from increases in skin hydration and suppression of pro-inflammatory cytokines, as shown in our earlier study [17,23], thereby resulting in less skin irritation without any allergic reactions.\\nPruritus associated with chronic kidney disease has been shown to be associated with elevated levels of C-reactive protein and other inflammatory cytokines. This evidence suggests that there is an inflammatory component in this form of pruritus [24]. Some authors consider UP to be a skin manifestation of chronic inflammation [25]. Recently, the “persistent microinflammation” theory, in which it is suggested that inflammation may be related to the genesis of UP, was proposed [26]. The skin of ESRD patients with UP contains an increased number of mast cells, and these cells can release various substances such as histamine, interleukins (IL) and tumor necrosis factor (TNF) [27-30].\\nPruritic skin can appear normal except for dryness; because the severity of pruritus is closely correlated with skin dryness [31], xerosis treatment normally begins with a topical agent such as a gentle moisturizing cream. Because sericin is well known for its moisturizing effect and its ability to reduce the generation of pro-inflammatory cytokines by fibroblasts, this study assessed the usefulness of sericin cream in treating UP. Evaluating the degree of pruritus was difficult because it is a subjective symptom; therefore, functional measurements of skin conditions known to be associated with pruritus, such as skin hydration and skin irritation, were considered to be objective indices. We found that both sericin cream and the cream base used to prepare the sericin cream increased the moisture content of the stratum corneum; however, the level of hydration was significantly higher in the skin treated with sericin cream. The side of the body treated with sericin cream had significantly lower levels of post-treatment dryness compared to pretreatment dryness and lower total dermatological severity scores in parameters such as skin pigmentation.\\nThe pathogenesis of ESRD itself is associated with inflammation and oxidative stress, as reflected in studies of plasma biochemistry, including the levels of the cytokines IL and TNF-α [32-34], classic markers of inflammatory processes that are strongly affected by pathological conditions [35]. These cytokines are upregulated in the plasma of ESRD patients [32,36]. The skin inflammation associated with the release of IL and TNF-α is further complicated by the inflammatory lesions that occur secondary to scratching [37]. Our results indicate that improvement in skin hydration and suppression of the release of pro-inflammatory cytokines in skin might be the mechanism by which sericin improves UP. Skin irritation, which can be caused by either skin inflammation or by scratching, was substantially reduced in the sericin group compared with the cream base group after 6 weeks of treatment. This result confirmed that sericin reduces skin irritation in patients with UP and suggested that it might be due to suppression of the inflammatory cytokines in skin. Similar results have been obtained in animal studies [23].\\nSecondary skin lesions that can result from the itch-scratch cycle include excoriations, hyperpigmentation, lichenification, prurigo nodules and scars [38]. Chronic renal failure is usually accompanied by a variety of cutaneous manifestations; dermal manifestations, including hyperpigmentation, have also been reported, and these can exact a considerable toll on the quality of life [39-41]. Although skin pigmentation is not related to specific clinical symptoms, it has an important effect on patient satisfaction. Sericin has been reported to have activity against tyrosinase [15], an enzyme related to melanin production; as this study shows, treatment with sericin results in significant reduction in the skin color of patients.\\nThe pruritus/mortality relationship may be strongly attributed to sleep disturbances, as previously mentioned in the DOPPS [8,9,42]. Many previous studies have found a strong association between inflammation, as measured by C-reactive protein or inflammatory cytokines, and sleep disturbances in dialysis patients [43-45]. The significant effects of pruritus on sleep, mood and social functioning require further investigation with the goal of improving the available treatments for this serious ESRD complication. We found that reduction in itching intensity from moderate to severe pruritus at the time of enrollment to mild pruritus after 6 weeks of treatment was associated with a better quality of life in all of the measured domains, including the mental, physical and kidney disease components. The KDQOL-SF score in the ESRD patients indicated improvements in several domains regarding the patients’ quality of life, particularly pain and sleep, which are relevant to itching. A better quality of sleep may reflect some degree of relief from itching achieved through the treatments.\\nThis study has certain limitations. First, the study design was an in-subject controlled study; it cannot be determined whether the improvements in the itching evaluation and the quality of life were from the sample (sericin cream) or the placebo (cream base). Moreover, due to the in-subject controlled design, the biochemical parameters could not be evaluated at the end of the study. Second, the study included a small number of patients, and these were followed for a relatively short time. A long-term study with a larger sample size is necessary. In addition to studies of hemodialysis patients, similar studies of other ESRD patients, such as peritoneal dialysis and kidney transplant patients, are also necessary to confirm the findings presented here.\",\n \"Our study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients.\",\n \" Preparation of silk sericin cream Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\\nBecause commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\\n Molecular weight determination of sericin To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\\nTo determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\\n Amino acid analysis of sericin The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\\nThe amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\\n Study design Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\\nSkin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\\n Study population Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\n Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\n Study treatment In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\\nIn 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\\n Measurement and outcome The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\\n\\n\\n(1)\\n\\n\\n%\\nchanges in each parameter\\n=\\n\\n\\n\\n\\n\\nP\\nt\\n\\n−\\n\\nP\\n0\\n\\n\\n\\n/\\n\\nP\\n0\\n\\n\\n\\n×\\n100\\n,\\n\\n\\n\\n\\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\\nThe level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\\n\\n\\n(1)\\n\\n\\n%\\nchanges in each parameter\\n=\\n\\n\\n\\n\\n\\nP\\nt\\n\\n−\\n\\nP\\n0\\n\\n\\n\\n/\\n\\nP\\n0\\n\\n\\n\\n×\\n100\\n,\\n\\n\\n\\n\\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\\n Patients’ quality of life The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\\nThe patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\\n Safety monitoring The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\\nThe occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\\n Statistical analysis The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\\nThe results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\",\n \"Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\",\n \"To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\",\n \"The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\",\n \"Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\",\n \" Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\",\n \"All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\",\n \"Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\",\n \"In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\",\n \"The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\\n\\n\\n(1)\\n\\n\\n%\\nchanges in each parameter\\n=\\n\\n\\n\\n\\n\\nP\\nt\\n\\n−\\n\\nP\\n0\\n\\n\\n\\n/\\n\\nP\\n0\\n\\n\\n\\n×\\n100\\n,\\n\\n\\n\\n\\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\",\n \"The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\",\n \"The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\",\n \"The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\",\n \"The authors declare that they have no competing interests.\",\n \"PA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"results",null,null,"discussion","conclusions","methods",null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"results\",\n null,\n null,\n \"discussion\",\n \"conclusions\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Pruritus","Hemodialysis","Sericin","Hydration","Inflammation","Pigmentation"],"string":"[\n \"Pruritus\",\n \"Hemodialysis\",\n \"Sericin\",\n \"Hydration\",\n \"Inflammation\",\n \"Pigmentation\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nItching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.\n\nResults:\n Molecular weight and amino acid composition of sericin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\n Study population Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.\nFifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.\n\nMolecular weight and amino acid composition of sericin:\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\n\nStudy population:\nFifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.\n\nDiscussion:\nThis study has shown that the use of sericin cream can reduce UP in hemodialysis patients according to their VAS scores. Sericin cream can also significantly increase skin hydration and reduce skin irritation and skin pigmentation in patients. The molecular weight analysis of the sericin used in this study gave results that agree with those reported by Sprague, who indicated that sericin is a mixture of at least 15 different polypeptide chains ranging in size from 20–220 kDa [19]. Similar to the earlier report by Kato et al., serine was the most abundant amino acid found in sericin [20].\nAlthough antihistamines, anti-allergic agents and topical corticosteroids are commonly used to treat UP in ESRD patients, their effectiveness is sometimes limited [2,8,21,22]. Alternative and effective treatments for intractable pruritus that generate low levels of adverse reactions, particularly in sensitive patients such as the ESRD group, must therefore be developed. This clinical study is the first to suggest that a sericin cream is beneficial for treating pruritus in hemodialysis patients. The benefits of this treatment apparently result from increases in skin hydration and suppression of pro-inflammatory cytokines, as shown in our earlier study [17,23], thereby resulting in less skin irritation without any allergic reactions.\nPruritus associated with chronic kidney disease has been shown to be associated with elevated levels of C-reactive protein and other inflammatory cytokines. This evidence suggests that there is an inflammatory component in this form of pruritus [24]. Some authors consider UP to be a skin manifestation of chronic inflammation [25]. Recently, the “persistent microinflammation” theory, in which it is suggested that inflammation may be related to the genesis of UP, was proposed [26]. The skin of ESRD patients with UP contains an increased number of mast cells, and these cells can release various substances such as histamine, interleukins (IL) and tumor necrosis factor (TNF) [27-30].\nPruritic skin can appear normal except for dryness; because the severity of pruritus is closely correlated with skin dryness [31], xerosis treatment normally begins with a topical agent such as a gentle moisturizing cream. Because sericin is well known for its moisturizing effect and its ability to reduce the generation of pro-inflammatory cytokines by fibroblasts, this study assessed the usefulness of sericin cream in treating UP. Evaluating the degree of pruritus was difficult because it is a subjective symptom; therefore, functional measurements of skin conditions known to be associated with pruritus, such as skin hydration and skin irritation, were considered to be objective indices. We found that both sericin cream and the cream base used to prepare the sericin cream increased the moisture content of the stratum corneum; however, the level of hydration was significantly higher in the skin treated with sericin cream. The side of the body treated with sericin cream had significantly lower levels of post-treatment dryness compared to pretreatment dryness and lower total dermatological severity scores in parameters such as skin pigmentation.\nThe pathogenesis of ESRD itself is associated with inflammation and oxidative stress, as reflected in studies of plasma biochemistry, including the levels of the cytokines IL and TNF-α [32-34], classic markers of inflammatory processes that are strongly affected by pathological conditions [35]. These cytokines are upregulated in the plasma of ESRD patients [32,36]. The skin inflammation associated with the release of IL and TNF-α is further complicated by the inflammatory lesions that occur secondary to scratching [37]. Our results indicate that improvement in skin hydration and suppression of the release of pro-inflammatory cytokines in skin might be the mechanism by which sericin improves UP. Skin irritation, which can be caused by either skin inflammation or by scratching, was substantially reduced in the sericin group compared with the cream base group after 6 weeks of treatment. This result confirmed that sericin reduces skin irritation in patients with UP and suggested that it might be due to suppression of the inflammatory cytokines in skin. Similar results have been obtained in animal studies [23].\nSecondary skin lesions that can result from the itch-scratch cycle include excoriations, hyperpigmentation, lichenification, prurigo nodules and scars [38]. Chronic renal failure is usually accompanied by a variety of cutaneous manifestations; dermal manifestations, including hyperpigmentation, have also been reported, and these can exact a considerable toll on the quality of life [39-41]. Although skin pigmentation is not related to specific clinical symptoms, it has an important effect on patient satisfaction. Sericin has been reported to have activity against tyrosinase [15], an enzyme related to melanin production; as this study shows, treatment with sericin results in significant reduction in the skin color of patients.\nThe pruritus/mortality relationship may be strongly attributed to sleep disturbances, as previously mentioned in the DOPPS [8,9,42]. Many previous studies have found a strong association between inflammation, as measured by C-reactive protein or inflammatory cytokines, and sleep disturbances in dialysis patients [43-45]. The significant effects of pruritus on sleep, mood and social functioning require further investigation with the goal of improving the available treatments for this serious ESRD complication. We found that reduction in itching intensity from moderate to severe pruritus at the time of enrollment to mild pruritus after 6 weeks of treatment was associated with a better quality of life in all of the measured domains, including the mental, physical and kidney disease components. The KDQOL-SF score in the ESRD patients indicated improvements in several domains regarding the patients’ quality of life, particularly pain and sleep, which are relevant to itching. A better quality of sleep may reflect some degree of relief from itching achieved through the treatments.\nThis study has certain limitations. First, the study design was an in-subject controlled study; it cannot be determined whether the improvements in the itching evaluation and the quality of life were from the sample (sericin cream) or the placebo (cream base). Moreover, due to the in-subject controlled design, the biochemical parameters could not be evaluated at the end of the study. Second, the study included a small number of patients, and these were followed for a relatively short time. A long-term study with a larger sample size is necessary. In addition to studies of hemodialysis patients, similar studies of other ESRD patients, such as peritoneal dialysis and kidney transplant patients, are also necessary to confirm the findings presented here.\n\nConclusions:\nOur study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients.\n\nMethods:\n Preparation of silk sericin cream Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\nBecause commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\n Molecular weight determination of sericin To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\nTo determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\n Amino acid analysis of sericin The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\nThe amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\n Study design Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\nSkin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\n Study population Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Study treatment In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\nIn 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\n Measurement and outcome The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\nThe level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\n Patients’ quality of life The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\nThe patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\n Safety monitoring The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\nThe occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\n Statistical analysis The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\nThe results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\n\nPreparation of silk sericin cream:\nBecause commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\n\nMolecular weight determination of sericin:\nTo determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\n\nAmino acid analysis of sericin:\nThe amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\n\nStudy design:\nSkin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\n\nStudy population:\n Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n\nInclusion criteria:\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n\nExclusion criteria:\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n\nStudy treatment:\nIn 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\n\nMeasurement and outcome:\nThe level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\n\nPatients’ quality of life:\nThe patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\n\nSafety monitoring:\nThe occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\n\nStatistical analysis:\nThe results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nPA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nUremic pruritus (UP) is a significant complication in ESRD patients and substantially impairs their quality of life. UP is considered to be a skin manifestation of chronic inflammation. Because sericin can suppress the release of pro-inflammatory cytokines, the purpose of this study was to investigate the short-term safety and efficacy of sericin cream for treating UP in hemodialysis patients.\n\nMethods:\nThis study used a double-blind design to investigate the effects of random topical administration of sericin cream and cream base (placebo) on either the right or left extremities of hemodialysis patients for 6 weeks. Skin hydration, irritation and pigmentation were evaluated every 2 weeks using Skin Diagnostic SD27. The visual analog scale for itching was also evaluated every 2 weeks, and the Kidney Disease Quality of Life Short Form was performed on the day of each patient's enrollment and after 6 weeks of treatment.\n\nResults:\nFifty dialysis patients were enrolled, 47 of which completed the study. The hydration of the skin of the patients' extremities increased significantly after administration of sericin cream; significant differences were found between sericin treatment and control after 6 weeks of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Moreover, a significant difference was also found in skin irritation between the two treatments (p = 0.013 for arms and p = 0.027 for legs, respectively). At the end of the study, the skin pigmentation level was significantly reduced on both the arms (p = 0.032) and legs (p = 0.021) of the sericin-treated side compared with the side treated with cream base. The mean itching score decreased significantly from moderate to severe at the time of enrollment to mild pruritus after 6 weeks of treatment (p = 0.002). A better quality of life was found in all domains tested although statistically significant differences before and after treatment was found only in the patients' pain scores, the effect of kidney disease on daily life, sleep quality and symptoms or problems related to kidney disease.\n\nConclusions:\nWe conclude that sericin cream has a high potential for reducing UP in hemodialysis patients.The trial registration number of this study is ISRCTN16019033; its public title is \"sericin cream reduces pruritus in hemodialysis patients\"."},"background_conclusion_text":{"kind":"string","value":"Background:\nItching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.\n\nConclusions:\nOur study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nUremic pruritus (UP) is a significant complication in ESRD patients and substantially impairs their quality of life. UP is considered to be a skin manifestation of chronic inflammation. Because sericin can suppress the release of pro-inflammatory cytokines, the purpose of this study was to investigate the short-term safety and efficacy of sericin cream for treating UP in hemodialysis patients.\n\nMethods:\nThis study used a double-blind design to investigate the effects of random topical administration of sericin cream and cream base (placebo) on either the right or left extremities of hemodialysis patients for 6 weeks. Skin hydration, irritation and pigmentation were evaluated every 2 weeks using Skin Diagnostic SD27. The visual analog scale for itching was also evaluated every 2 weeks, and the Kidney Disease Quality of Life Short Form was performed on the day of each patient's enrollment and after 6 weeks of treatment.\n\nResults:\nFifty dialysis patients were enrolled, 47 of which completed the study. The hydration of the skin of the patients' extremities increased significantly after administration of sericin cream; significant differences were found between sericin treatment and control after 6 weeks of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Moreover, a significant difference was also found in skin irritation between the two treatments (p = 0.013 for arms and p = 0.027 for legs, respectively). At the end of the study, the skin pigmentation level was significantly reduced on both the arms (p = 0.032) and legs (p = 0.021) of the sericin-treated side compared with the side treated with cream base. The mean itching score decreased significantly from moderate to severe at the time of enrollment to mild pruritus after 6 weeks of treatment (p = 0.002). A better quality of life was found in all domains tested although statistically significant differences before and after treatment was found only in the patients' pain scores, the effect of kidney disease on daily life, sleep quality and symptoms or problems related to kidney disease.\n\nConclusions:\nWe conclude that sericin cream has a high potential for reducing UP in hemodialysis patients.The trial registration number of this study is ISRCTN16019033; its public title is \"sericin cream reduces pruritus in hemodialysis patients\"."},"whole_article_text_length":{"kind":"number","value":16613,"string":"16,613"},"whole_article_abstract_length":{"kind":"number","value":447,"string":"447"},"num_sections":{"kind":"number","value":22,"string":"22"},"most_frequent_words":{"kind":"list like","value":["skin","cream","patients","treatment","sericin","study","weeks","sericin cream","baseline","base"],"string":"[\n \"skin\",\n \"cream\",\n \"patients\",\n \"treatment\",\n \"sericin\",\n \"study\",\n \"weeks\",\n \"sericin cream\",\n \"baseline\",\n \"base\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Topical | Double-Blind Method | Female | Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome 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Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Topical | Double-Blind Method | Female | Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test 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[SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | cream | patients | treatment | sericin | study | weeks | sericin cream | baseline | base [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | cream | patients | treatment | sericin | study | weeks | sericin cream | baseline | base [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] inflammatory | sericin | uremic | pruritus | hypothesis | opioid | immune | associated | pro | pro inflammatory [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | skin | study | cream | sericin | treatment | excluded | weeks | sericin cream | itching [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cream | skin | baseline | significant | legs | arms | treatment | hydration | weeks | sericin [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] use | cream | baseline use cream | baseline use cream base | compared baseline use | compared baseline use cream | treatment compared baseline use | baseline use | significantly | sericin [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cream | patients | skin | sericin | study | treatment | sericin cream | weeks | pruritus | baseline [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cream | patients | skin | sericin | study | treatment | sericin cream | weeks | pruritus | baseline [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ESRD ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 6 weeks ||| every 2 weeks | Skin Diagnostic ||| every 2 weeks | the Kidney Disease Quality of Life Short Form | the day | 6 weeks [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Fifty | 47 ||| 6 weeks | 0.041 | 0.022 ||| two | 0.013 | 0.027 ||| 0.032 | 0.021 ||| 6 weeks | 0.002 ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ESRD ||| ||| ||| 6 weeks ||| every 2 weeks | Skin Diagnostic ||| every 2 weeks | the Kidney Disease Quality of Life Short Form | the day | 6 weeks ||| ||| Fifty | 47 ||| 6 weeks | 0.041 | 0.022 ||| two | 0.013 | 0.027 ||| 0.032 | 0.021 ||| 6 weeks | 0.002 ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ESRD ||| ||| ||| 6 weeks ||| every 2 weeks | Skin Diagnostic ||| every 2 weeks | the Kidney Disease Quality of Life Short Form | the day | 6 weeks ||| ||| Fifty | 47 ||| 6 weeks | 0.041 | 0.022 ||| two | 0.013 | 0.027 ||| 0.032 | 0.021 ||| 6 weeks | 0.002 ||| ||| ||| [SUMMARY]"}}},{"rowIdx":273,"cells":{"title":{"kind":"string","value":"Particulate matter air pollution and respiratory symptoms in individuals having either asthma or chronic obstructive pulmonary disease: a European multicentre panel study."},"pmid":{"kind":"string","value":"23039312"},"background_abstract":{"kind":"string","value":"Particulate matter air pollution has been associated with adverse health effects. The fraction of ambient particles that are mainly responsible for the observed health effects is still a matter of controversy. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.The RUPIOH study, an EU-funded multicentre study, was designed to examine the distribution of various ambient particle metrics in four European cities (Amsterdam, Athens, Birmingham, Helsinki) and assess their health effects in participants with asthma or COPD, based on a detailed exposure assessment. In this paper the association of central site measurements with respiratory symptoms and restriction of activities is examined."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"At each centre a panel of participants with either asthma or COPD recorded respiratory symptoms and restriction of activities in a diary for six months. Exposure assessment included simultaneous measurements of coarse, fine and ultrafine particles at a central site. Data on gaseous pollutants were also collected. The associations of the 24-hour average concentrations of air pollution indices with the health outcomes were assessed in a hierarchical modelling approach. A city specific analysis controlling for potential confounders was followed by a meta-analysis to provide overall effect estimates."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A 10 μg/m3 increase in previous day coarse particles concentrations was positively associated with most symptoms (an increase of 0.6 to 0.7% in average) and limitation in walking (OR= 1.076, 95% CI: 1.026-1.128). Same day, previous day and previous two days ozone concentrations were positively associated with cough (OR= 1.061, 95% CI: 1.013-1.111; OR= 1.049, 95% CI: 1.016-1.083 and OR= 1.059, 95% CI: 1.027-1.091, respectively). No consistent associations were observed between fine particle concentrations, nitrogen dioxide and respiratory health effects. As for particle number concentrations negative association (mostly non-significant at the nominal level) was observed with most symptoms whilst the positive association with limitation of activities did not reach the nominal level of significance."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The observed associations with coarse particles are in agreement with the findings of toxicological studies. Together they suggest it is prudent to regulate also coarse particles in addition to fine particles."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Aged, 80 and over","Air Pollutants","Air Pollution","Asthma","Cities","Europe","Female","Humans","Male","Middle Aged","Nitrogen Dioxide","Odds Ratio","Ozone","Particulate Matter","Pulmonary Disease, Chronic Obstructive","Respiration Disorders","Walking"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Air Pollutants\",\n \"Air Pollution\",\n \"Asthma\",\n \"Cities\",\n \"Europe\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Nitrogen Dioxide\",\n \"Odds Ratio\",\n \"Ozone\",\n \"Particulate Matter\",\n \"Pulmonary Disease, Chronic Obstructive\",\n \"Respiration Disorders\",\n \"Walking\"\n]"},"pmcid":{"kind":"string","value":"3509003"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20]."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Study design In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\nIn the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\n Study population Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\nInclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\n Symptom diary The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\nThe diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\n Air pollution exposure Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\nExposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\n Confounder data Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\nTime trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\n Quality assurance/quality control Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\nAir pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\n Statistical analysis Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].\nData analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27]."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Panel characteristics A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\nA brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\n Symptoms In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\nIn total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\n Air pollution concentrations Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\nHelsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\n Air pollution effects on symptoms-limitation in activities due to breathing problems Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Incidence analyses Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.\nPatterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Our study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles."},"other_sections_titles":{"kind":"list like","value":["Background","Study design","Study population","Symptom diary","Air pollution exposure","Confounder data","Quality assurance/quality control","Statistical analysis","Panel characteristics","Symptoms","Air pollution concentrations","Air pollution effects on symptoms-limitation in activities due to breathing problems","Prevalence analyses","Incidence analyses","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Study design\",\n \"Study population\",\n \"Symptom diary\",\n \"Air pollution exposure\",\n \"Confounder data\",\n \"Quality assurance/quality control\",\n \"Statistical analysis\",\n \"Panel characteristics\",\n \"Symptoms\",\n \"Air pollution concentrations\",\n \"Air pollution effects on symptoms-limitation in activities due to breathing problems\",\n \"Prevalence analyses\",\n \"Incidence analyses\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20].","In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.","Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.","The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.","Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.","Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).","Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.","Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].","A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.","In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.","Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities"," Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n","We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n","Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.","AIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.","The authors declare that they have no competing interests.","All authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions."],"string":"[\n \"Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\\n[9,12-14].\\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\\n[16-20].\",\n \"In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\",\n \"Inclusion criteria and recruitment procedures have been described in detail before\\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\",\n \"The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\",\n \"Exposure assessment has been described in previous publications\\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\",\n \"Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\",\n \"Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\",\n \"Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\\n[27].\",\n \"A brief description of the study population is presented in Table\\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\\nCharacteristics of four European panels of asthmatic/COPD patients\\na Total participants in panel.\\nb Asthma + COPD or chronic non-specific lung disease.\\nc Given as mean and [range].\\nd Chronic non-specific lung disease.\\ne Environmental tobacco smoke.\\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\",\n \"In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\\n2).\\nPerson days with symptoms in the diary (n = number of expected person days)\\na due to breathing problems.\",\n \"Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\\n3).\\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\",\n \" Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\nWe observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\",\n \"We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\",\n \"Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\\n7).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith incidence of symptoms in the four panels (random effects pooled estimates)\\n\\nBold are significant pooled effects.\",\n \"AIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.\",\n \"The authors declare that they have no competing interests.\",\n \"All authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study design","Study population","Symptom diary","Air pollution exposure","Confounder data","Quality assurance/quality control","Statistical analysis","Results","Panel characteristics","Symptoms","Air pollution concentrations","Air pollution effects on symptoms-limitation in activities due to breathing problems","Prevalence analyses","Incidence analyses","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study design\",\n \"Study population\",\n \"Symptom diary\",\n \"Air pollution exposure\",\n \"Confounder data\",\n \"Quality assurance/quality control\",\n \"Statistical analysis\",\n \"Results\",\n \"Panel characteristics\",\n \"Symptoms\",\n \"Air pollution concentrations\",\n \"Air pollution effects on symptoms-limitation in activities due to breathing problems\",\n \"Prevalence analyses\",\n \"Incidence analyses\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20]."," Study design In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\nIn the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\n Study population Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\nInclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\n Symptom diary The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\nThe diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\n Air pollution exposure Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\nExposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\n Confounder data Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\nTime trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\n Quality assurance/quality control Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\nAir pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\n Statistical analysis Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].\nData analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].","In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.","Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.","The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.","Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.","Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).","Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.","Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27]."," Panel characteristics A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\nA brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\n Symptoms In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\nIn total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\n Air pollution concentrations Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\nHelsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\n Air pollution effects on symptoms-limitation in activities due to breathing problems Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Incidence analyses Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.\nPatterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.","A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.","In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.","Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities"," Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n","We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n","Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.","In this multicentre study we found consistent positive associations between coarse particles central sites concentrations and prevalence of respiratory symptoms, as recorded in a 6-month diary, in four panels of participants with predominantly mild to moderate asthma or COPD in four European cities participating in the RUPIOH study. We also found a significant association of ozone with cough and woken with breathing problems, but not with other symptoms. Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst positive associations with woken with breathing problems and cough as well as with limitation of vigorous and moderate activities due to breathing problems, did not reach the nominal level of significance. Interestingly, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant at the nominal level. An analysis of the asthmatic subgroup showed generally lower odds ratios for PM10-2.5.\nOne particularity and strength of the RUPIOH study is the in depth assessment of particulate air pollution by measuring PM10, PM2.5 (then deriving coarse particles), filters absorbance as well as the number of ultrafine particles. Previous work from RUPIOH that included air pollution monitoring for one week inside and directly outside participants’ homes reported no association with lung function\n[19]. As the authors stated a potential explanation could be the high prevalence of medication use, the short period of measurements (one week) that limited the ability to assess lagged effects over several days or absence of an effect. The high prevalence of medication use may also have covered some associations in the present study.\nA limitation of the study is the inclusion of both COPD and asthma patients. COPD and asthma are two diseases with different underlying pathophysiological mechanisms and day to day variability in their symptoms\n[22,23]. Mixing of the two diseases does not create bias in the analysis in the full population as we adjusted for differences in health status between individuals. The generalizability of the size of the effect estimates is more affected by the population. Though asthma and COPD are different diseases, we are not aware of studies that have demonstrated differences in the magnitude of response to air pollution. In our recently accepted paper in the same panel we did not find any difference in the effect of PM10-2.5 on total nitrate and nitrite concentrations in exhaled breath condensate (EBC NOx), a marker of oxidative stress between asthma and COPD patients\n[28]. In that study we could evaluate disease status as the outcome was a continuous variable. Unfortunately, an analysis restricted to COPD patients was not possible due to the small number of COPD patients participating in Helsinki and Birmingham. Hence, we also could not test whether the smaller PM10-2.5 effect in the asthmatic subgroup differed significantly from the COPD subgroup.\nOur coarse particle findings are however consistent with the observation that in the RUPIOH study only the PM10-2.5 concentration at central sites was significantly associated with increased EBC NOx collected during the same week as the spirometry\n[28]. EBC NOx has been suggested as a reliable marker of oxidative stress\n[29-31]. The link between PM10-2.5 with oxidative stress and airway inflammation may explain the increase in respiratory symptoms we found.\nIn this study, we also report significant positive associations of ozone with cough throughout most of the examined lags both in the analysis of total participants and the subgroup of the asthmatics that are consistent with previous epidemiological and toxicological studies\n[32]. In addition, positive associations, but not significant in the nominal level, were observed with most of symptoms when total participants were included in the analysis. However, when we restricted the analysis to the subgroup of asthmatics, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2. Negative associations were also observed with woken with breathing problems, wheezing and with limitation in activities due to breathing problems, although non significant. Factors like high medication use, intrinsic differences in responsiveness to ozone among individuals, adaptation to ozone issues or other spurious effects may have been responsible for these findings\n[32].\nIn the last two decades a substantial body of literature has focused on the harmful health effects of PM10 and PM2.5[15,32]. As a result guideline values have been recommended by the U.S. Environmental Protection Agency and World Health Organization for both indicators of PM pollution to protect public health\n[2,3]. However, from recent studies there is increasing evidence that the health effects of coarse particles should not be underestimated. In a systematic review of epidemiological studies that have analyzed fine and coarse PM jointly, Brunekreef and Forsberg examined the epidemiological evidence for effects of coarse particles on health\n[15]. They concluded that the effects of PM10-2.5 were stronger than or as strong as PM2.5 on short-term respiratory morbidity. Furthermore, in a national multicity study, Zanobetti and Schwartz found a strong association of both fine and coarse particles with daily deaths in 112 U.S. cities\n[33]. A 10 μg/m3 increase in PM10-2.5 was significantly associated with total mortality, stroke, cardiovascular, and respiratory mortality, the latter of which showing the largest effect (a 1.2% increase). Mechanistically, these effects may be due either to biogenic factors or to metals carried by PM10-2.5 by activation of inflammatory and oxidative stress pathways\n[34-36]. The findings of our study support previous epidemiological and toxicological evidence that health effects due to the coarse fraction may be substantial\n[37].\nThe large number of calculations we have done could have given some statistically significant associations by chance. However, multiple testing is an unlikely explanation of the findings in the current study. In the full study population we found 14 significant associations of which 10 were positive; in the asthmatics subgroup we found 12 significant associations of which 8 were positive. The consistency of associations (e.g. for ozone and cough) further argues against chance as the main explanation for our findings. Additionally, in the full study population the significant associations for PM10-2.5 were supported by elevated though not nominally significant ORs for other lags and symptoms. Finally, ORs were mainly homogeneous across centers. Moreover, the modest correlations between PM10-2.5 and PM2.5 did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5.\nThe majority of studies that investigated health effects of particulate pollutants have expressed results on a mass basis. It has been suggested that when taking into consideration particle number or surface area, the pulmonary dose of toxic material related to PM2.5 may be much larger than the dose related to PM10-2.5 that for this reason alone, comparison on a mass basis may be less informative\n[15]. In our study we separately investigated the mass and the number effect. Neither central site PM2.5 nor PNC were consistently associated with symptoms. The association we observed with PM10-2.5, if not by chance, may also imply that a central measurement site is more appropriate for measurements of mass concentrations than for PNC. The analysis of RUPIOH data by Puustinen et al. showed generally high correlations between 24 hour average central site and residential outdoor concentrations for PM2.5 and soot with a lesser median correlation for PM10 and a lower correlation for PNC and PM10-2.5[17]. For PM10-2.5 correlations between central site and home outdoor measurements were 0.66, 0.74, 0.89 and 0.64 in Helsinki, Athens, Amsterdam and Birmingham respectively. A central site thus provides a reasonably good estimate of more local exposures even for coarse particles.\nThe relatively high divergence of PM10-2.5 concentrations between proximate sites in the UK has recently been confirmed by Liu and Harrison\n[38]. Consequently, for both PNC and PM10-2.5, there is a higher probability of exposure misclassification than for PM2.5 or soot. The finding of significant associations with respiratory health outcomes for PM10-2.5 but not for PNC is therefore quite striking but consistent with the recent findings of a time series study in London which found significant associations between PNC and cardiovascular health outcomes whilst PM mass metrics were associated with respiratory outcomes\n[39]. A plausible explanation could be the existence of different biological and pathophysiological mechanisms through which PM10-2.5 and PNC exert their adverse effects or different target organs. The results of recent toxicological studies support the theory that PM10-2.5 exert their effects at the site of deposition in the airways whereas PNC, after crossing the alveolar epithelial barrier, enter into the systemic circulation and affect cardiovascular function\n[40,41]. This theory could explain the positive associations we found between PM10-2.5 and respiratory symptoms.\nIn summary, our study contributes to the literature on the health effects of PM in respiratory patients. Moreover, the results of our study are in agreement with the findings of recent epidemiological and toxicological studies and provide enough evidence to conclude that it is prudent to keep PM10-2.5 regulated in addition to fine particles.","Our study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles.","AIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.","The authors declare that they have no competing interests.","All authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions."],"string":"[\n \"Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\\n[9,12-14].\\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\\n[16-20].\",\n \" Study design In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\\nIn the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\\n Study population Inclusion criteria and recruitment procedures have been described in detail before\\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\\nInclusion criteria and recruitment procedures have been described in detail before\\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\\n Symptom diary The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\\nThe diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\\n Air pollution exposure Exposure assessment has been described in previous publications\\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\\nExposure assessment has been described in previous publications\\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\\n Confounder data Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\\nTime trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\\n Quality assurance/quality control Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\\nAir pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\\n Statistical analysis Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\\n[27].\\nData analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\\n[27].\",\n \"In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\",\n \"Inclusion criteria and recruitment procedures have been described in detail before\\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\",\n \"The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\",\n \"Exposure assessment has been described in previous publications\\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\",\n \"Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\",\n \"Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\",\n \"Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\\n[27].\",\n \" Panel characteristics A brief description of the study population is presented in Table\\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\\nCharacteristics of four European panels of asthmatic/COPD patients\\na Total participants in panel.\\nb Asthma + COPD or chronic non-specific lung disease.\\nc Given as mean and [range].\\nd Chronic non-specific lung disease.\\ne Environmental tobacco smoke.\\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\\nA brief description of the study population is presented in Table\\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\\nCharacteristics of four European panels of asthmatic/COPD patients\\na Total participants in panel.\\nb Asthma + COPD or chronic non-specific lung disease.\\nc Given as mean and [range].\\nd Chronic non-specific lung disease.\\ne Environmental tobacco smoke.\\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\\n Symptoms In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\\n2).\\nPerson days with symptoms in the diary (n = number of expected person days)\\na due to breathing problems.\\nIn total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\\n2).\\nPerson days with symptoms in the diary (n = number of expected person days)\\na due to breathing problems.\\n Air pollution concentrations Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\\n3).\\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\\nHelsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\\n3).\\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\\n Air pollution effects on symptoms-limitation in activities due to breathing problems Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\nWe observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\n Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\nWe observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\n Incidence analyses Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\\n7).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith incidence of symptoms in the four panels (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nPatterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\\n7).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith incidence of symptoms in the four panels (random effects pooled estimates)\\n\\nBold are significant pooled effects.\",\n \"A brief description of the study population is presented in Table\\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\\nCharacteristics of four European panels of asthmatic/COPD patients\\na Total participants in panel.\\nb Asthma + COPD or chronic non-specific lung disease.\\nc Given as mean and [range].\\nd Chronic non-specific lung disease.\\ne Environmental tobacco smoke.\\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\",\n \"In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\\n2).\\nPerson days with symptoms in the diary (n = number of expected person days)\\na due to breathing problems.\",\n \"Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\\n3).\\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\",\n \" Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\\nWe observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\",\n \"We observed very small differences in fixed and random effects combined estimates. In Tables\\n4 and\\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\\n6).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\n\\nAssociations of PM\\n\\n10-2.5 \\n\\nand PM\\n\\n2.5 \\n\\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\\n\\nBold are significant pooled effects.\\nThe above-mentioned positive associations with PM10-2.5 (Tables\\n4 and\\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\\n4 and\\n5).\\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\\n4 and\\n5).\\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\\n\\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\\n\\n3 \\n\\nin previous day (lag1) concentrations of each pollutant (10,000/cm\\n\\n3 \\n\\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\\n\",\n \"Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\\n7).\\n\\nAssociations of particulate matter indices, NO\\n\\n2 \\n\\nand O\\n\\n3 \\n\\nwith incidence of symptoms in the four panels (random effects pooled estimates)\\n\\nBold are significant pooled effects.\",\n \"In this multicentre study we found consistent positive associations between coarse particles central sites concentrations and prevalence of respiratory symptoms, as recorded in a 6-month diary, in four panels of participants with predominantly mild to moderate asthma or COPD in four European cities participating in the RUPIOH study. We also found a significant association of ozone with cough and woken with breathing problems, but not with other symptoms. Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst positive associations with woken with breathing problems and cough as well as with limitation of vigorous and moderate activities due to breathing problems, did not reach the nominal level of significance. Interestingly, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant at the nominal level. An analysis of the asthmatic subgroup showed generally lower odds ratios for PM10-2.5.\\nOne particularity and strength of the RUPIOH study is the in depth assessment of particulate air pollution by measuring PM10, PM2.5 (then deriving coarse particles), filters absorbance as well as the number of ultrafine particles. Previous work from RUPIOH that included air pollution monitoring for one week inside and directly outside participants’ homes reported no association with lung function\\n[19]. As the authors stated a potential explanation could be the high prevalence of medication use, the short period of measurements (one week) that limited the ability to assess lagged effects over several days or absence of an effect. The high prevalence of medication use may also have covered some associations in the present study.\\nA limitation of the study is the inclusion of both COPD and asthma patients. COPD and asthma are two diseases with different underlying pathophysiological mechanisms and day to day variability in their symptoms\\n[22,23]. Mixing of the two diseases does not create bias in the analysis in the full population as we adjusted for differences in health status between individuals. The generalizability of the size of the effect estimates is more affected by the population. Though asthma and COPD are different diseases, we are not aware of studies that have demonstrated differences in the magnitude of response to air pollution. In our recently accepted paper in the same panel we did not find any difference in the effect of PM10-2.5 on total nitrate and nitrite concentrations in exhaled breath condensate (EBC NOx), a marker of oxidative stress between asthma and COPD patients\\n[28]. In that study we could evaluate disease status as the outcome was a continuous variable. Unfortunately, an analysis restricted to COPD patients was not possible due to the small number of COPD patients participating in Helsinki and Birmingham. Hence, we also could not test whether the smaller PM10-2.5 effect in the asthmatic subgroup differed significantly from the COPD subgroup.\\nOur coarse particle findings are however consistent with the observation that in the RUPIOH study only the PM10-2.5 concentration at central sites was significantly associated with increased EBC NOx collected during the same week as the spirometry\\n[28]. EBC NOx has been suggested as a reliable marker of oxidative stress\\n[29-31]. The link between PM10-2.5 with oxidative stress and airway inflammation may explain the increase in respiratory symptoms we found.\\nIn this study, we also report significant positive associations of ozone with cough throughout most of the examined lags both in the analysis of total participants and the subgroup of the asthmatics that are consistent with previous epidemiological and toxicological studies\\n[32]. In addition, positive associations, but not significant in the nominal level, were observed with most of symptoms when total participants were included in the analysis. However, when we restricted the analysis to the subgroup of asthmatics, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2. Negative associations were also observed with woken with breathing problems, wheezing and with limitation in activities due to breathing problems, although non significant. Factors like high medication use, intrinsic differences in responsiveness to ozone among individuals, adaptation to ozone issues or other spurious effects may have been responsible for these findings\\n[32].\\nIn the last two decades a substantial body of literature has focused on the harmful health effects of PM10 and PM2.5[15,32]. As a result guideline values have been recommended by the U.S. Environmental Protection Agency and World Health Organization for both indicators of PM pollution to protect public health\\n[2,3]. However, from recent studies there is increasing evidence that the health effects of coarse particles should not be underestimated. In a systematic review of epidemiological studies that have analyzed fine and coarse PM jointly, Brunekreef and Forsberg examined the epidemiological evidence for effects of coarse particles on health\\n[15]. They concluded that the effects of PM10-2.5 were stronger than or as strong as PM2.5 on short-term respiratory morbidity. Furthermore, in a national multicity study, Zanobetti and Schwartz found a strong association of both fine and coarse particles with daily deaths in 112 U.S. cities\\n[33]. A 10 μg/m3 increase in PM10-2.5 was significantly associated with total mortality, stroke, cardiovascular, and respiratory mortality, the latter of which showing the largest effect (a 1.2% increase). Mechanistically, these effects may be due either to biogenic factors or to metals carried by PM10-2.5 by activation of inflammatory and oxidative stress pathways\\n[34-36]. The findings of our study support previous epidemiological and toxicological evidence that health effects due to the coarse fraction may be substantial\\n[37].\\nThe large number of calculations we have done could have given some statistically significant associations by chance. However, multiple testing is an unlikely explanation of the findings in the current study. In the full study population we found 14 significant associations of which 10 were positive; in the asthmatics subgroup we found 12 significant associations of which 8 were positive. The consistency of associations (e.g. for ozone and cough) further argues against chance as the main explanation for our findings. Additionally, in the full study population the significant associations for PM10-2.5 were supported by elevated though not nominally significant ORs for other lags and symptoms. Finally, ORs were mainly homogeneous across centers. Moreover, the modest correlations between PM10-2.5 and PM2.5 did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5.\\nThe majority of studies that investigated health effects of particulate pollutants have expressed results on a mass basis. It has been suggested that when taking into consideration particle number or surface area, the pulmonary dose of toxic material related to PM2.5 may be much larger than the dose related to PM10-2.5 that for this reason alone, comparison on a mass basis may be less informative\\n[15]. In our study we separately investigated the mass and the number effect. Neither central site PM2.5 nor PNC were consistently associated with symptoms. The association we observed with PM10-2.5, if not by chance, may also imply that a central measurement site is more appropriate for measurements of mass concentrations than for PNC. The analysis of RUPIOH data by Puustinen et al. showed generally high correlations between 24 hour average central site and residential outdoor concentrations for PM2.5 and soot with a lesser median correlation for PM10 and a lower correlation for PNC and PM10-2.5[17]. For PM10-2.5 correlations between central site and home outdoor measurements were 0.66, 0.74, 0.89 and 0.64 in Helsinki, Athens, Amsterdam and Birmingham respectively. A central site thus provides a reasonably good estimate of more local exposures even for coarse particles.\\nThe relatively high divergence of PM10-2.5 concentrations between proximate sites in the UK has recently been confirmed by Liu and Harrison\\n[38]. Consequently, for both PNC and PM10-2.5, there is a higher probability of exposure misclassification than for PM2.5 or soot. The finding of significant associations with respiratory health outcomes for PM10-2.5 but not for PNC is therefore quite striking but consistent with the recent findings of a time series study in London which found significant associations between PNC and cardiovascular health outcomes whilst PM mass metrics were associated with respiratory outcomes\\n[39]. A plausible explanation could be the existence of different biological and pathophysiological mechanisms through which PM10-2.5 and PNC exert their adverse effects or different target organs. The results of recent toxicological studies support the theory that PM10-2.5 exert their effects at the site of deposition in the airways whereas PNC, after crossing the alveolar epithelial barrier, enter into the systemic circulation and affect cardiovascular function\\n[40,41]. This theory could explain the positive associations we found between PM10-2.5 and respiratory symptoms.\\nIn summary, our study contributes to the literature on the health effects of PM in respiratory patients. Moreover, the results of our study are in agreement with the findings of recent epidemiological and toxicological studies and provide enough evidence to conclude that it is prudent to keep PM10-2.5 regulated in addition to fine particles.\",\n \"Our study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles.\",\n \"AIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.\",\n \"The authors declare that they have no competing interests.\",\n \"All authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,"results",null,null,null,null,null,null,"discussion","conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Air pollution","Asthma","Chronic obstructive pulmonary disease","Coarse particles","Particle number concentration","Respiratory health"],"string":"[\n \"Air pollution\",\n \"Asthma\",\n \"Chronic obstructive pulmonary disease\",\n \"Coarse particles\",\n \"Particle number concentration\",\n \"Respiratory health\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nOver the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20].\n\nMethods:\n Study design In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\nIn the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\n Study population Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\nInclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\n Symptom diary The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\nThe diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\n Air pollution exposure Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\nExposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\n Confounder data Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\nTime trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\n Quality assurance/quality control Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\nAir pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\n Statistical analysis Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].\nData analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].\n\nStudy design:\nIn the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\n\nStudy population:\nInclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\n\nSymptom diary:\nThe diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\n\nAir pollution exposure:\nExposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\n\nConfounder data:\nTime trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\n\nQuality assurance/quality control:\nAir pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\n\nStatistical analysis:\nData analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].\n\nResults:\n Panel characteristics A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\nA brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\n Symptoms In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\nIn total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\n Air pollution concentrations Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\nHelsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\n Air pollution effects on symptoms-limitation in activities due to breathing problems Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Incidence analyses Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.\nPatterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nPanel characteristics:\nA brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\n\nSymptoms:\nIn total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\n\nAir pollution concentrations:\nHelsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\n\nAir pollution effects on symptoms-limitation in activities due to breathing problems:\n Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n\nPrevalence analyses:\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n\nIncidence analyses:\nPatterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nDiscussion:\nIn this multicentre study we found consistent positive associations between coarse particles central sites concentrations and prevalence of respiratory symptoms, as recorded in a 6-month diary, in four panels of participants with predominantly mild to moderate asthma or COPD in four European cities participating in the RUPIOH study. We also found a significant association of ozone with cough and woken with breathing problems, but not with other symptoms. Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst positive associations with woken with breathing problems and cough as well as with limitation of vigorous and moderate activities due to breathing problems, did not reach the nominal level of significance. Interestingly, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant at the nominal level. An analysis of the asthmatic subgroup showed generally lower odds ratios for PM10-2.5.\nOne particularity and strength of the RUPIOH study is the in depth assessment of particulate air pollution by measuring PM10, PM2.5 (then deriving coarse particles), filters absorbance as well as the number of ultrafine particles. Previous work from RUPIOH that included air pollution monitoring for one week inside and directly outside participants’ homes reported no association with lung function\n[19]. As the authors stated a potential explanation could be the high prevalence of medication use, the short period of measurements (one week) that limited the ability to assess lagged effects over several days or absence of an effect. The high prevalence of medication use may also have covered some associations in the present study.\nA limitation of the study is the inclusion of both COPD and asthma patients. COPD and asthma are two diseases with different underlying pathophysiological mechanisms and day to day variability in their symptoms\n[22,23]. Mixing of the two diseases does not create bias in the analysis in the full population as we adjusted for differences in health status between individuals. The generalizability of the size of the effect estimates is more affected by the population. Though asthma and COPD are different diseases, we are not aware of studies that have demonstrated differences in the magnitude of response to air pollution. In our recently accepted paper in the same panel we did not find any difference in the effect of PM10-2.5 on total nitrate and nitrite concentrations in exhaled breath condensate (EBC NOx), a marker of oxidative stress between asthma and COPD patients\n[28]. In that study we could evaluate disease status as the outcome was a continuous variable. Unfortunately, an analysis restricted to COPD patients was not possible due to the small number of COPD patients participating in Helsinki and Birmingham. Hence, we also could not test whether the smaller PM10-2.5 effect in the asthmatic subgroup differed significantly from the COPD subgroup.\nOur coarse particle findings are however consistent with the observation that in the RUPIOH study only the PM10-2.5 concentration at central sites was significantly associated with increased EBC NOx collected during the same week as the spirometry\n[28]. EBC NOx has been suggested as a reliable marker of oxidative stress\n[29-31]. The link between PM10-2.5 with oxidative stress and airway inflammation may explain the increase in respiratory symptoms we found.\nIn this study, we also report significant positive associations of ozone with cough throughout most of the examined lags both in the analysis of total participants and the subgroup of the asthmatics that are consistent with previous epidemiological and toxicological studies\n[32]. In addition, positive associations, but not significant in the nominal level, were observed with most of symptoms when total participants were included in the analysis. However, when we restricted the analysis to the subgroup of asthmatics, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2. Negative associations were also observed with woken with breathing problems, wheezing and with limitation in activities due to breathing problems, although non significant. Factors like high medication use, intrinsic differences in responsiveness to ozone among individuals, adaptation to ozone issues or other spurious effects may have been responsible for these findings\n[32].\nIn the last two decades a substantial body of literature has focused on the harmful health effects of PM10 and PM2.5[15,32]. As a result guideline values have been recommended by the U.S. Environmental Protection Agency and World Health Organization for both indicators of PM pollution to protect public health\n[2,3]. However, from recent studies there is increasing evidence that the health effects of coarse particles should not be underestimated. In a systematic review of epidemiological studies that have analyzed fine and coarse PM jointly, Brunekreef and Forsberg examined the epidemiological evidence for effects of coarse particles on health\n[15]. They concluded that the effects of PM10-2.5 were stronger than or as strong as PM2.5 on short-term respiratory morbidity. Furthermore, in a national multicity study, Zanobetti and Schwartz found a strong association of both fine and coarse particles with daily deaths in 112 U.S. cities\n[33]. A 10 μg/m3 increase in PM10-2.5 was significantly associated with total mortality, stroke, cardiovascular, and respiratory mortality, the latter of which showing the largest effect (a 1.2% increase). Mechanistically, these effects may be due either to biogenic factors or to metals carried by PM10-2.5 by activation of inflammatory and oxidative stress pathways\n[34-36]. The findings of our study support previous epidemiological and toxicological evidence that health effects due to the coarse fraction may be substantial\n[37].\nThe large number of calculations we have done could have given some statistically significant associations by chance. However, multiple testing is an unlikely explanation of the findings in the current study. In the full study population we found 14 significant associations of which 10 were positive; in the asthmatics subgroup we found 12 significant associations of which 8 were positive. The consistency of associations (e.g. for ozone and cough) further argues against chance as the main explanation for our findings. Additionally, in the full study population the significant associations for PM10-2.5 were supported by elevated though not nominally significant ORs for other lags and symptoms. Finally, ORs were mainly homogeneous across centers. Moreover, the modest correlations between PM10-2.5 and PM2.5 did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5.\nThe majority of studies that investigated health effects of particulate pollutants have expressed results on a mass basis. It has been suggested that when taking into consideration particle number or surface area, the pulmonary dose of toxic material related to PM2.5 may be much larger than the dose related to PM10-2.5 that for this reason alone, comparison on a mass basis may be less informative\n[15]. In our study we separately investigated the mass and the number effect. Neither central site PM2.5 nor PNC were consistently associated with symptoms. The association we observed with PM10-2.5, if not by chance, may also imply that a central measurement site is more appropriate for measurements of mass concentrations than for PNC. The analysis of RUPIOH data by Puustinen et al. showed generally high correlations between 24 hour average central site and residential outdoor concentrations for PM2.5 and soot with a lesser median correlation for PM10 and a lower correlation for PNC and PM10-2.5[17]. For PM10-2.5 correlations between central site and home outdoor measurements were 0.66, 0.74, 0.89 and 0.64 in Helsinki, Athens, Amsterdam and Birmingham respectively. A central site thus provides a reasonably good estimate of more local exposures even for coarse particles.\nThe relatively high divergence of PM10-2.5 concentrations between proximate sites in the UK has recently been confirmed by Liu and Harrison\n[38]. Consequently, for both PNC and PM10-2.5, there is a higher probability of exposure misclassification than for PM2.5 or soot. The finding of significant associations with respiratory health outcomes for PM10-2.5 but not for PNC is therefore quite striking but consistent with the recent findings of a time series study in London which found significant associations between PNC and cardiovascular health outcomes whilst PM mass metrics were associated with respiratory outcomes\n[39]. A plausible explanation could be the existence of different biological and pathophysiological mechanisms through which PM10-2.5 and PNC exert their adverse effects or different target organs. The results of recent toxicological studies support the theory that PM10-2.5 exert their effects at the site of deposition in the airways whereas PNC, after crossing the alveolar epithelial barrier, enter into the systemic circulation and affect cardiovascular function\n[40,41]. This theory could explain the positive associations we found between PM10-2.5 and respiratory symptoms.\nIn summary, our study contributes to the literature on the health effects of PM in respiratory patients. Moreover, the results of our study are in agreement with the findings of recent epidemiological and toxicological studies and provide enough evidence to conclude that it is prudent to keep PM10-2.5 regulated in addition to fine particles.\n\nConclusions:\nOur study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles.\n\nAbbreviations:\nAIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nAll authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nParticulate matter air pollution has been associated with adverse health effects. The fraction of ambient particles that are mainly responsible for the observed health effects is still a matter of controversy. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.The RUPIOH study, an EU-funded multicentre study, was designed to examine the distribution of various ambient particle metrics in four European cities (Amsterdam, Athens, Birmingham, Helsinki) and assess their health effects in participants with asthma or COPD, based on a detailed exposure assessment. In this paper the association of central site measurements with respiratory symptoms and restriction of activities is examined.\n\nMethods:\nAt each centre a panel of participants with either asthma or COPD recorded respiratory symptoms and restriction of activities in a diary for six months. Exposure assessment included simultaneous measurements of coarse, fine and ultrafine particles at a central site. Data on gaseous pollutants were also collected. The associations of the 24-hour average concentrations of air pollution indices with the health outcomes were assessed in a hierarchical modelling approach. A city specific analysis controlling for potential confounders was followed by a meta-analysis to provide overall effect estimates.\n\nResults:\nA 10 μg/m3 increase in previous day coarse particles concentrations was positively associated with most symptoms (an increase of 0.6 to 0.7% in average) and limitation in walking (OR= 1.076, 95% CI: 1.026-1.128). Same day, previous day and previous two days ozone concentrations were positively associated with cough (OR= 1.061, 95% CI: 1.013-1.111; OR= 1.049, 95% CI: 1.016-1.083 and OR= 1.059, 95% CI: 1.027-1.091, respectively). No consistent associations were observed between fine particle concentrations, nitrogen dioxide and respiratory health effects. As for particle number concentrations negative association (mostly non-significant at the nominal level) was observed with most symptoms whilst the positive association with limitation of activities did not reach the nominal level of significance.\n\nConclusions:\nThe observed associations with coarse particles are in agreement with the findings of toxicological studies. Together they suggest it is prudent to regulate also coarse particles in addition to fine particles."},"background_conclusion_text":{"kind":"string","value":"Background:\nOver the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20].\n\nConclusions:\nOur study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nParticulate matter air pollution has been associated with adverse health effects. The fraction of ambient particles that are mainly responsible for the observed health effects is still a matter of controversy. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.The RUPIOH study, an EU-funded multicentre study, was designed to examine the distribution of various ambient particle metrics in four European cities (Amsterdam, Athens, Birmingham, Helsinki) and assess their health effects in participants with asthma or COPD, based on a detailed exposure assessment. In this paper the association of central site measurements with respiratory symptoms and restriction of activities is examined.\n\nMethods:\nAt each centre a panel of participants with either asthma or COPD recorded respiratory symptoms and restriction of activities in a diary for six months. Exposure assessment included simultaneous measurements of coarse, fine and ultrafine particles at a central site. Data on gaseous pollutants were also collected. The associations of the 24-hour average concentrations of air pollution indices with the health outcomes were assessed in a hierarchical modelling approach. A city specific analysis controlling for potential confounders was followed by a meta-analysis to provide overall effect estimates.\n\nResults:\nA 10 μg/m3 increase in previous day coarse particles concentrations was positively associated with most symptoms (an increase of 0.6 to 0.7% in average) and limitation in walking (OR= 1.076, 95% CI: 1.026-1.128). Same day, previous day and previous two days ozone concentrations were positively associated with cough (OR= 1.061, 95% CI: 1.013-1.111; OR= 1.049, 95% CI: 1.016-1.083 and OR= 1.059, 95% CI: 1.027-1.091, respectively). No consistent associations were observed between fine particle concentrations, nitrogen dioxide and respiratory health effects. As for particle number concentrations negative association (mostly non-significant at the nominal level) was observed with most symptoms whilst the positive association with limitation of activities did not reach the nominal level of significance.\n\nConclusions:\nThe observed associations with coarse particles are in agreement with the findings of toxicological studies. Together they suggest it is prudent to regulate also coarse particles in addition to fine particles."},"whole_article_text_length":{"kind":"number","value":17122,"string":"17,122"},"whole_article_abstract_length":{"kind":"number","value":432,"string":"432"},"num_sections":{"kind":"number","value":21,"string":"21"},"most_frequent_words":{"kind":"list like","value":["effects","pm10","associations","significant","symptoms","lag","participants","problems","breathing","breathing problems"],"string":"[\n \"effects\",\n \"pm10\",\n \"associations\",\n \"significant\",\n \"symptoms\",\n \"lag\",\n \"participants\",\n \"problems\",\n \"breathing\",\n \"breathing problems\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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| Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking 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fraction ambient | coarse particles addition | coarse particles addition fine [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] pm10 | effects | significant | associations | lag | symptoms | participants | study | problems | pm2 [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] pm10 | effects | significant | associations | lag | symptoms | participants | study | problems | pm2 [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| EU | four | European | Amsterdam | Athens | Birmingham | Helsinki | COPD ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] six months ||| ||| ||| 24-hour ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 10 | previous day | 0.6 to 0.7% | 1.076 | 95% | CI | 1.026 ||| previous day | two days | 1.061 | 95% | CI | 1.013 | 1.049 | 95% | CI | 1.016-1.083 | 1.059 | 95% | CI | 1.027 ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| EU | four | European | Amsterdam | Athens | Birmingham | Helsinki | COPD ||| ||| six months ||| ||| ||| 24-hour ||| ||| ||| 10 | previous day | 0.6 to 0.7% | 1.076 | 95% | CI | 1.026 ||| previous day | two days | 1.061 | 95% | CI | 1.013 | 1.049 | 95% | CI | 1.016-1.083 | 1.059 | 95% | CI | 1.027 ||| ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| EU | four | European | Amsterdam | Athens | Birmingham | Helsinki | COPD ||| ||| six months ||| ||| ||| 24-hour ||| ||| ||| 10 | previous day | 0.6 to 0.7% | 1.076 | 95% | CI | 1.026 ||| previous day | two days | 1.061 | 95% | CI | 1.013 | 1.049 | 95% | CI | 1.016-1.083 | 1.059 | 95% | CI | 1.027 ||| ||| ||| ||| [SUMMARY]"}}},{"rowIdx":274,"cells":{"title":{"kind":"string","value":"DRAIN AMYLASE ON THE FIRST POSTOPERATIVE DAY OF WHIPPLE SURGERY: WHAT VALUE IS THE BEST PREDICTOR FOR EARLY DRAIN REMOVAL?"},"pmid":{"kind":"string","value":"29513806"},"background_abstract":{"kind":"string","value":"The value of drain amylase on the first postoperative day after pancreatic resections has been described as an efficient predictor of pancreatic fistula. In spite of this, the cut-off point below which the drains can be removed early remains controversial."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Were included patients undergoing Whipple surgery in the period of 2007 to 2016. Group 1 enrolled the ones who did not develop fistula and those who developed biochemical fistula for less than seven days postoperatively and group 2 included patients who developed persistent biochemical fistula between seven and 21 days and those with grade B and C fistula."},"methods_abstract_label":{"kind":"string","value":"METHOD"},"results_abstract":{"kind":"string","value":"Sixty-one patients were included, 41 comprised group 1 and 20 group 2. The incidence of abdominal collections, need for reoperation and time of hospitalization were for group 1 and 2, respectively: 17.1%, 17.1% and 9.5 days, and 65%, 40% and 21.1 days. The median of the amylase from the drain at 1st postoperative day was in group 1 and 2, respectively: 175 U/l and 3172.5 U/l (p=0.001). Using a cut-off of 180 to predict the group to which the patient would belong there was obtained sensitivity, specificity, positive predictive value and negative predictive value of 100%, 48.8%, 50% and 100% respectively."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"It was validated the cut-off value of 180 U/l as appropriate to early drain removal."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Amylases","Drainage","Female","Humans","Male","Middle Aged","Pancreatic Fistula","Pancreaticoduodenectomy","Postoperative Care","Postoperative Complications","Predictive Value of Tests","Prospective Studies"],"string":"[\n \"Amylases\",\n \"Drainage\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Pancreatic Fistula\",\n \"Pancreaticoduodenectomy\",\n \"Postoperative Care\",\n \"Postoperative Complications\",\n \"Predictive Value of Tests\",\n \"Prospective Studies\"\n]"},"pmcid":{"kind":"string","value":"5863991"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Historically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them\n16\n. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis\n13\n\n,\n\n11\n. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results\n8\n\n,\n\n18\n\n,\n\n19\n, which has been a limiting factor in the practice of not using drains in this type of procedure. \nDescription of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L\n6\n and 90 U/l\n14\n have been suggested. \nThe objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients."},"methods_title":{"kind":"string","value":"METHOD"},"methods_text":{"kind":"string","value":"The project was submitted and approved by the Human Research Ethics Committee, under nº 59716616.9.0000.5292 in the Plataforma Brasil.\nSixty-three patients were analyzed from a prospective database, who had undergone Kausch-Whipple surgery performed by the 1st author of this study (ECA), for the treatment of pancreatic or peripancreatic diseases. The time period was between June 2007 and September 2016, in the following hospitals located in the city of Natal: Hospital Universitário Onofre Lopes, Liga Norte Riograndense Contra o Câncer, Casa de Saúde São Lucas and Natal Hospital Center.\nThe technical steps for the Whipple technique as performed by the author are detailed in previous studies\n1\n\n,\n\n2\n. Three techniques for pancreatojejunal anastomosis were utilized. For pancreas with main pancreatic duct ≥5 mm, two-layer pancreatojejunal terminolateral duct to mucosa anastomosis was performed. For pancreas with normal or slightly dilated (diameter <5 mm) main pancreatic duct, terminoterminal pancreatojejunal anastomosis (“telescopic”) or terminolateral pancreatojejunal anastomosis (“invagination”) were carried out, the latter performed systematically after the 33rd patient from the casebook. At the end of the surgery, two drains, preferably laminar silicone drains, were placed in the cavity and exteriorized at each flank. \nIn the majority of patients, subcutaneous octreotide was utilized as prophylaxis for the prevention of PF, at 0.3 mg/day, fractioned in 8 h intervals, during seven days. Debits were recorded along with amylase dosage of the drained liquid on the 1st, 3rd, 5th, 7th and sometimes, on the 9th postoperative day. The value of AD1PO vas defined from the highest amylase value of both drains, measured from a sample of liquid obtained on the first day after surgery. \nPF diagnosis utilized the 2016 revised criterion of GIEDFP\n3\n. PF was defined when on the 3rd postoperative (PO) day the value of amylase in the drained liquid was three times higher than the normal upper limit for serum amylase. Patients that did not develop fistula or those with transient biochemical fistula (<7 days PO) were characterized as group 1, and patients with persistent biochemical fistula (between 7 and 21 days PO) and fistula grade B and C were characterized as group 2. In most cases, on the 9th day after surgery, control ultrasound or tomography tests were carried out. Removal of drains occurred in those cases with low amylase values for the drained liquid (under three times the upper limit for normal serum amylase) and when image tests did not show abdominal collections. Intra-hospital mortality was defined as death occurring within 90 days of surgery.\nThe Receiver Operating Characteristic (ROC) curve was utilized to identify an adequate cut-off point for AD1PO and verify its predictive characteristic in patients of both groups. The ROC value was generated after sensitivity, specificity and accuracy calculations. Additionally, the cut-off point 578 U/l was also selected because of its closeness to the value utilized by Fong e cols \n9\n, for comparison purposes. \n Statistical analysis After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \nAfter determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. "},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"From the initial sample of 63 patients, two were excluded. One had AD1PO collected incorrectly on the 2nd day after surgery, and the other passed within the 1st week PO. Therefore analysis covered 61 patients. Group 1 was constituted of 41 patients, of which 36 did not develop fistula and five transient biochemical fistula (grade A). Group 2 was constituted of 20 patients, of which six presented persistent biochemical fistula (grade A), eight degree B fistula, and six grade C fistula. The index of clinical fistula (grades B + C) in the overall sample was 22.9%. \nThe demographic and clinical characteristics of the patients, as well as respective tests, are shown in Table 1. There was no statistical difference between groups regarding age, gender and type of illness. However, regarding the size of main pancreatic duct and type of anastomosis, a statistically significant difference was detected. All patients with dilated main pancreatic ducts as well as those submitted to duct to mucosa anastomosis belonged to group 1.\n\nTABLE 1Associations between demographic and clinical characteristics and patient groups Variables Group 1 Group 2 pAge 58.0±13.06I\n 57.1±12.1I\n 0.706II\n\n n % n %\nGender\n\n\n\n\nMale 19 63.3 11 36.7 0.525III\nFemale 22 71.0 9 29.0Disease\n\n\n\n\nPancreatic adenocarcinoma 17 65.4 9 34.6 0.264III\nPapillary adenocarcinoma 12 60.0 8 40.0Duodenal adenocarcinoma 1 33.3 2 66.7Colangiocarcinoma 4 100.0 0 0.0Frantz 5 100.0 0 0.0Others 2 66.7 1 33.3Size of duct\n\n\n\n\n< 5 31 60.8 20 39.2 0.023IV\n> 5 10 100.0 0 0.0Anastomosis*\n\n\n\n\nT-L (Invagination) 14 50.0 14 50.0 0.011III\nT-T 17 73.9 6 26.1T-L (DM) 10 100.0 0 0.0I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\n\nI=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\nRegarding postoperative evolution, group 2 was associated with a higher level of general complications, postoperative collections, longer permanence of abdominal drain and hospitalization time. There was also an association between high resurgery index and patients of group 2. Nevertheless, despite all these worse result factors related to group 2, there was no statistically significant difference when comparing mortality across groups (Table 2). \n\nTABLE 2Associations between postoperative clinical findings and patient groups, along with results for drain and hospitalization times, per group Variable Group 1 Group 2 p n % n %\nCollection\n\n\n\n\nYes733.31466.7 <0.001I\nNo3485.0615.0Postoperative complications\n\n\n\n\nYes2050.020.050.0 <0.001II\nNo21100.000.0Resurgery\n\n\n\n\nYes746.7853.3 0.051I\nNo3473.91226.1Time with drain (days) 9.5±2.60III29.1±13.58III <0.001IV\nHospitalization time (days) 16.7±17.9III34.7±16.7III <0.001IV\nMortality49.815.0 1.000II\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\n\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\nMortality of the series was 8.19%: four patients belonged to group 1 and one patient to group 2. The mortality causes in group 1 were: kidney insufficiency due to coagulopathy; dehiscence of gastrojejunal anastomosis and sepsis, pneumonia due to broncoaspiration and necrosis of the pancreaticojejunostomy loop associated with sepsis. The only patient of group 2 passed due to necrotic pancreatitis associated with pancreatic fistula and sepsis. \nRegarding the AD1PO value, the median was 175 U/l (48.5-954) in group 1 and 2172.5 U/l (833.5-6421.0) in group 2. This difference was statistically significant (p=0.001).\nThe area under the ROC curve presented an exactness index of 83.0% (p=0.001). When considering a 180 U/l cut-off point for AD1PO, the following results were obtained: sensitivity 100%, specificity 48.8%, positive predictor value 50%, and negative predictor value 100%. There was an association between the arbitrated AD1PO value from the ROC curve (>180 U/l) with groups 1 and 2 (p<0.001). With a cut-off point of 578 U/l, the results were: sensitivity 80%, specificity 39%, positive predictor value 50% and negative predictor value 86.2%. There was also an association between the arbitrated AD1PO value from the ROC curve (>578 U/l) with groups 1 and 2 (p<0.003). Patients presenting AD1PO values higher than 180 U/l and 578 U/l were 2.0 and 6.25 times more likely to belong to group 2, and therefore require postoperative drainage. Table 3 shows the comparison between the two AD1PO values. \n\nTABLE 3Precision and exactness measurements, per AD1PO cut-off point Parameters Cut-off point (U/l) 180 578Sensitivity 100% 80%Specificity 48.8% 39%VPP 50% 50%VPN 100% 86.2%\n"},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"Was validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients. "},"other_sections_titles":{"kind":"list like","value":["Statistical analysis"],"string":"[\n \"Statistical analysis\"\n]"},"other_sections_texts":{"kind":"list like","value":["After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. "],"string":"[\n \"After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null],"string":"[\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHOD","Statistical analysis","RESULTS","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"METHOD\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Historically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them\n16\n. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis\n13\n\n,\n\n11\n. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results\n8\n\n,\n\n18\n\n,\n\n19\n, which has been a limiting factor in the practice of not using drains in this type of procedure. \nDescription of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L\n6\n and 90 U/l\n14\n have been suggested. \nThe objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.","The project was submitted and approved by the Human Research Ethics Committee, under nº 59716616.9.0000.5292 in the Plataforma Brasil.\nSixty-three patients were analyzed from a prospective database, who had undergone Kausch-Whipple surgery performed by the 1st author of this study (ECA), for the treatment of pancreatic or peripancreatic diseases. The time period was between June 2007 and September 2016, in the following hospitals located in the city of Natal: Hospital Universitário Onofre Lopes, Liga Norte Riograndense Contra o Câncer, Casa de Saúde São Lucas and Natal Hospital Center.\nThe technical steps for the Whipple technique as performed by the author are detailed in previous studies\n1\n\n,\n\n2\n. Three techniques for pancreatojejunal anastomosis were utilized. For pancreas with main pancreatic duct ≥5 mm, two-layer pancreatojejunal terminolateral duct to mucosa anastomosis was performed. For pancreas with normal or slightly dilated (diameter <5 mm) main pancreatic duct, terminoterminal pancreatojejunal anastomosis (“telescopic”) or terminolateral pancreatojejunal anastomosis (“invagination”) were carried out, the latter performed systematically after the 33rd patient from the casebook. At the end of the surgery, two drains, preferably laminar silicone drains, were placed in the cavity and exteriorized at each flank. \nIn the majority of patients, subcutaneous octreotide was utilized as prophylaxis for the prevention of PF, at 0.3 mg/day, fractioned in 8 h intervals, during seven days. Debits were recorded along with amylase dosage of the drained liquid on the 1st, 3rd, 5th, 7th and sometimes, on the 9th postoperative day. The value of AD1PO vas defined from the highest amylase value of both drains, measured from a sample of liquid obtained on the first day after surgery. \nPF diagnosis utilized the 2016 revised criterion of GIEDFP\n3\n. PF was defined when on the 3rd postoperative (PO) day the value of amylase in the drained liquid was three times higher than the normal upper limit for serum amylase. Patients that did not develop fistula or those with transient biochemical fistula (<7 days PO) were characterized as group 1, and patients with persistent biochemical fistula (between 7 and 21 days PO) and fistula grade B and C were characterized as group 2. In most cases, on the 9th day after surgery, control ultrasound or tomography tests were carried out. Removal of drains occurred in those cases with low amylase values for the drained liquid (under three times the upper limit for normal serum amylase) and when image tests did not show abdominal collections. Intra-hospital mortality was defined as death occurring within 90 days of surgery.\nThe Receiver Operating Characteristic (ROC) curve was utilized to identify an adequate cut-off point for AD1PO and verify its predictive characteristic in patients of both groups. The ROC value was generated after sensitivity, specificity and accuracy calculations. Additionally, the cut-off point 578 U/l was also selected because of its closeness to the value utilized by Fong e cols \n9\n, for comparison purposes. \n Statistical analysis After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \nAfter determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. ","After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. ","From the initial sample of 63 patients, two were excluded. One had AD1PO collected incorrectly on the 2nd day after surgery, and the other passed within the 1st week PO. Therefore analysis covered 61 patients. Group 1 was constituted of 41 patients, of which 36 did not develop fistula and five transient biochemical fistula (grade A). Group 2 was constituted of 20 patients, of which six presented persistent biochemical fistula (grade A), eight degree B fistula, and six grade C fistula. The index of clinical fistula (grades B + C) in the overall sample was 22.9%. \nThe demographic and clinical characteristics of the patients, as well as respective tests, are shown in Table 1. There was no statistical difference between groups regarding age, gender and type of illness. However, regarding the size of main pancreatic duct and type of anastomosis, a statistically significant difference was detected. All patients with dilated main pancreatic ducts as well as those submitted to duct to mucosa anastomosis belonged to group 1.\n\nTABLE 1Associations between demographic and clinical characteristics and patient groups Variables Group 1 Group 2 pAge 58.0±13.06I\n 57.1±12.1I\n 0.706II\n\n n % n %\nGender\n\n\n\n\nMale 19 63.3 11 36.7 0.525III\nFemale 22 71.0 9 29.0Disease\n\n\n\n\nPancreatic adenocarcinoma 17 65.4 9 34.6 0.264III\nPapillary adenocarcinoma 12 60.0 8 40.0Duodenal adenocarcinoma 1 33.3 2 66.7Colangiocarcinoma 4 100.0 0 0.0Frantz 5 100.0 0 0.0Others 2 66.7 1 33.3Size of duct\n\n\n\n\n< 5 31 60.8 20 39.2 0.023IV\n> 5 10 100.0 0 0.0Anastomosis*\n\n\n\n\nT-L (Invagination) 14 50.0 14 50.0 0.011III\nT-T 17 73.9 6 26.1T-L (DM) 10 100.0 0 0.0I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\n\nI=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\nRegarding postoperative evolution, group 2 was associated with a higher level of general complications, postoperative collections, longer permanence of abdominal drain and hospitalization time. There was also an association between high resurgery index and patients of group 2. Nevertheless, despite all these worse result factors related to group 2, there was no statistically significant difference when comparing mortality across groups (Table 2). \n\nTABLE 2Associations between postoperative clinical findings and patient groups, along with results for drain and hospitalization times, per group Variable Group 1 Group 2 p n % n %\nCollection\n\n\n\n\nYes733.31466.7 <0.001I\nNo3485.0615.0Postoperative complications\n\n\n\n\nYes2050.020.050.0 <0.001II\nNo21100.000.0Resurgery\n\n\n\n\nYes746.7853.3 0.051I\nNo3473.91226.1Time with drain (days) 9.5±2.60III29.1±13.58III <0.001IV\nHospitalization time (days) 16.7±17.9III34.7±16.7III <0.001IV\nMortality49.815.0 1.000II\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\n\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\nMortality of the series was 8.19%: four patients belonged to group 1 and one patient to group 2. The mortality causes in group 1 were: kidney insufficiency due to coagulopathy; dehiscence of gastrojejunal anastomosis and sepsis, pneumonia due to broncoaspiration and necrosis of the pancreaticojejunostomy loop associated with sepsis. The only patient of group 2 passed due to necrotic pancreatitis associated with pancreatic fistula and sepsis. \nRegarding the AD1PO value, the median was 175 U/l (48.5-954) in group 1 and 2172.5 U/l (833.5-6421.0) in group 2. This difference was statistically significant (p=0.001).\nThe area under the ROC curve presented an exactness index of 83.0% (p=0.001). When considering a 180 U/l cut-off point for AD1PO, the following results were obtained: sensitivity 100%, specificity 48.8%, positive predictor value 50%, and negative predictor value 100%. There was an association between the arbitrated AD1PO value from the ROC curve (>180 U/l) with groups 1 and 2 (p<0.001). With a cut-off point of 578 U/l, the results were: sensitivity 80%, specificity 39%, positive predictor value 50% and negative predictor value 86.2%. There was also an association between the arbitrated AD1PO value from the ROC curve (>578 U/l) with groups 1 and 2 (p<0.003). Patients presenting AD1PO values higher than 180 U/l and 578 U/l were 2.0 and 6.25 times more likely to belong to group 2, and therefore require postoperative drainage. Table 3 shows the comparison between the two AD1PO values. \n\nTABLE 3Precision and exactness measurements, per AD1PO cut-off point Parameters Cut-off point (U/l) 180 578Sensitivity 100% 80%Specificity 48.8% 39%VPP 50% 50%VPN 100% 86.2%\n","AD1PO as a predictor of post-pancreatectomy PF was described by Yamaguchi e cols in 2003\n20\n. The authors analyzed the amylase values measured in abdominal drains of 26 patients submitted to pancreatectomy, and observed that values were already high on the first day PO in those who developed clinical PF after. At least three meta-analysis\n10\n\n,\n\n15\n\n,\n\n21\n have validated the correlation between high AD1PO values and the development of PF. However, there is still controversy on the adequate cut-off point for AD1PO to predict PF, and on its applicability to the removal of prophylactic drains inserted after pancreatic resections. Verona’s group was the first to establish a strategy for the early removal of abdominal drains based on AD1PO values\n6\n. The authors carried out a prospective study on 114 patients submitted to pancreatic resection and randomized those with AD1PO <5000 U/l to remove the drain on the 3rd or 5th day PO. Early removal of drains was associated with a lower rate of pancreatic fistula (1.8% vs. 26%), lower rate of abdominal complications (12.2% vs. 52.6%), lower rate of pulmonary complications (26.3% vs. 52.6%), shorter hospitalization times (8.7 (±4) vs. 10.8 (±6.9) and lower hospital readmission rates (0% vs. 8.8%). Although the study suggested that early removal of abdominal drains was safe with AD1PO under 5000 U/l, other authors reported hypothetical PF indexes between 25-48% if drains were removed according to this suggestion\n7\n\n,\n\n12\n.\nFor those patients submitted to duodenopancreatectomy and that received prophylactic drains, the most important question is to know which patients will not develop PF - and not the other way around. On one hand, knowing which patients will not develop PF is useful because it allows for early drain removal, which leads to shorter hospitalization times. On the other hand, predicting which patients will develop PF after surgery, when the abdominal cavity is being drained, is not as relevant because PF can be easily diagnosed by measuring amylase in the drained liquid. In most cases, treatment is simple maintenance of drains, associated with fasting and nutritional support. For this reason the study herein presented analyzed two low AD1PO cut-off points (180 and 578) that were associated with higher specificity and consequently, with a high negative predictive factor, in detriment to a high cut-off point associated with higher sensitivity to PF diagnosis. Another reason for the selection is related to the impossibility of obtaining daily interventionist radiology services in the public hospital where the majority of the patients studied underwent surgery. These services include percutaneous drainage of abdominal collections/abscesses, which is indicated in urgent cases where there are abdominal complications as a consequence of undrained anastomotic leaks. \nThe methodology of this study utilized the PF definition based on the revised classification by ISGPS\n5\n, which no longer considers grade A of the previous classification to be a true PF\n4\n. However, as this study aimed at identifying a group of patients in which early removal of drains could be beneficial, it was important to include not only the concept of PF, but also grades B and C and those patients with biochemical fistula that persisted with amylase-rich debit between 7-21 days PO. In these patients, early removal of drains would increase susceptibility to abdominal collections or abscesses. With the 180 U/l cut-off point, an excellent correlation was obtained between AD1PO and absence of fistula. Therefore it was possible to identify 1/3 of patients that would have been benefitted by early drain removal. Besides, with the same cut-off, none of the patients that developed clinically relevant fistula or even persistent biochemical fistula would have had their drains removed early. \nData obtained herein were capable of validating previous publications that utilized low cut-off points\n7\n\n,\n\n12\n\n,\n\n14\n\n,\n\n17\n. Differently from these studies, however, the first series was entirely prospective herein, including only patients submitted consecutively to duodenopancreatectomy by a single team of surgeons, where PF definition followed ISGPS criteria. The cut-off point 180 U/l obtained herein was lower than the one found by Fong et al\n9\n at the Massachusetts General Hospital. They divided the study in two cohorts: the first involved 126 patients, and the 612 U/l cut-off point was defined as the one presenting best accuracy, sensitivity and specificity; the second had the objective of validating a 600 U/l cut-off point, involving 369 patients submitted to duodenopancreatectomy within January 2009 and December 2012. Almost two-thirds of patients (62.1%) presented AD1PO lower than 600 U/l and only two PF cases (0.9%) were diagnosed. This was different than the other patients with AD1PO of at least 600 U/l, with PF incidence of 31.4%. When considering herein a cut-off point close to Fong et al., 578 U/l, 13.8% of patients with PF diagnosis would have been affected by early removal of drains (negative predictor value: 86.2%). In the reality considered herein, there is no non-invasive treatment for intracavitary collections/abscesses available all the time at the public hospital, and therefore increasing the cut-off point would be a high price to pay. \nThis study presents some limitations. The first concerns the low number of included patients, which did not hinder statistical analysis. The second is related to the non-verification of the real benefits of drains in patients who developed PF, as all patients received drains. The high index of abdominal collections (65%) and necessity of re-surgery (40%) in group 2 suggested that, for some patients, drains were not totally efficient; nevertheless, the objective of the study was to find a group of patients in which drains could be removed early and not confirm the non-efficiency of drains in the group that developed PF.","Was validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients. "],"string":"[\n \"Historically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them\\n16\\n. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis\\n13\\n\\n,\\n\\n11\\n. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results\\n8\\n\\n,\\n\\n18\\n\\n,\\n\\n19\\n, which has been a limiting factor in the practice of not using drains in this type of procedure. \\nDescription of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L\\n6\\n and 90 U/l\\n14\\n have been suggested. \\nThe objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.\",\n \"The project was submitted and approved by the Human Research Ethics Committee, under nº 59716616.9.0000.5292 in the Plataforma Brasil.\\nSixty-three patients were analyzed from a prospective database, who had undergone Kausch-Whipple surgery performed by the 1st author of this study (ECA), for the treatment of pancreatic or peripancreatic diseases. The time period was between June 2007 and September 2016, in the following hospitals located in the city of Natal: Hospital Universitário Onofre Lopes, Liga Norte Riograndense Contra o Câncer, Casa de Saúde São Lucas and Natal Hospital Center.\\nThe technical steps for the Whipple technique as performed by the author are detailed in previous studies\\n1\\n\\n,\\n\\n2\\n. Three techniques for pancreatojejunal anastomosis were utilized. For pancreas with main pancreatic duct ≥5 mm, two-layer pancreatojejunal terminolateral duct to mucosa anastomosis was performed. For pancreas with normal or slightly dilated (diameter <5 mm) main pancreatic duct, terminoterminal pancreatojejunal anastomosis (“telescopic”) or terminolateral pancreatojejunal anastomosis (“invagination”) were carried out, the latter performed systematically after the 33rd patient from the casebook. At the end of the surgery, two drains, preferably laminar silicone drains, were placed in the cavity and exteriorized at each flank. \\nIn the majority of patients, subcutaneous octreotide was utilized as prophylaxis for the prevention of PF, at 0.3 mg/day, fractioned in 8 h intervals, during seven days. Debits were recorded along with amylase dosage of the drained liquid on the 1st, 3rd, 5th, 7th and sometimes, on the 9th postoperative day. The value of AD1PO vas defined from the highest amylase value of both drains, measured from a sample of liquid obtained on the first day after surgery. \\nPF diagnosis utilized the 2016 revised criterion of GIEDFP\\n3\\n. PF was defined when on the 3rd postoperative (PO) day the value of amylase in the drained liquid was three times higher than the normal upper limit for serum amylase. Patients that did not develop fistula or those with transient biochemical fistula (<7 days PO) were characterized as group 1, and patients with persistent biochemical fistula (between 7 and 21 days PO) and fistula grade B and C were characterized as group 2. In most cases, on the 9th day after surgery, control ultrasound or tomography tests were carried out. Removal of drains occurred in those cases with low amylase values for the drained liquid (under three times the upper limit for normal serum amylase) and when image tests did not show abdominal collections. Intra-hospital mortality was defined as death occurring within 90 days of surgery.\\nThe Receiver Operating Characteristic (ROC) curve was utilized to identify an adequate cut-off point for AD1PO and verify its predictive characteristic in patients of both groups. The ROC value was generated after sensitivity, specificity and accuracy calculations. Additionally, the cut-off point 578 U/l was also selected because of its closeness to the value utilized by Fong e cols \\n9\\n, for comparison purposes. \\n Statistical analysis After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \\nAfter determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \",\n \"After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \",\n \"From the initial sample of 63 patients, two were excluded. One had AD1PO collected incorrectly on the 2nd day after surgery, and the other passed within the 1st week PO. Therefore analysis covered 61 patients. Group 1 was constituted of 41 patients, of which 36 did not develop fistula and five transient biochemical fistula (grade A). Group 2 was constituted of 20 patients, of which six presented persistent biochemical fistula (grade A), eight degree B fistula, and six grade C fistula. The index of clinical fistula (grades B + C) in the overall sample was 22.9%. \\nThe demographic and clinical characteristics of the patients, as well as respective tests, are shown in Table 1. There was no statistical difference between groups regarding age, gender and type of illness. However, regarding the size of main pancreatic duct and type of anastomosis, a statistically significant difference was detected. All patients with dilated main pancreatic ducts as well as those submitted to duct to mucosa anastomosis belonged to group 1.\\n\\nTABLE 1Associations between demographic and clinical characteristics and patient groups Variables Group 1 Group 2 pAge 58.0±13.06I\\n 57.1±12.1I\\n 0.706II\\n\\n n % n %\\nGender\\n\\n\\n\\n\\nMale 19 63.3 11 36.7 0.525III\\nFemale 22 71.0 9 29.0Disease\\n\\n\\n\\n\\nPancreatic adenocarcinoma 17 65.4 9 34.6 0.264III\\nPapillary adenocarcinoma 12 60.0 8 40.0Duodenal adenocarcinoma 1 33.3 2 66.7Colangiocarcinoma 4 100.0 0 0.0Frantz 5 100.0 0 0.0Others 2 66.7 1 33.3Size of duct\\n\\n\\n\\n\\n< 5 31 60.8 20 39.2 0.023IV\\n> 5 10 100.0 0 0.0Anastomosis*\\n\\n\\n\\n\\nT-L (Invagination) 14 50.0 14 50.0 0.011III\\nT-T 17 73.9 6 26.1T-L (DM) 10 100.0 0 0.0I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\\n\\nI=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\\nRegarding postoperative evolution, group 2 was associated with a higher level of general complications, postoperative collections, longer permanence of abdominal drain and hospitalization time. There was also an association between high resurgery index and patients of group 2. Nevertheless, despite all these worse result factors related to group 2, there was no statistically significant difference when comparing mortality across groups (Table 2). \\n\\nTABLE 2Associations between postoperative clinical findings and patient groups, along with results for drain and hospitalization times, per group Variable Group 1 Group 2 p n % n %\\nCollection\\n\\n\\n\\n\\nYes733.31466.7 <0.001I\\nNo3485.0615.0Postoperative complications\\n\\n\\n\\n\\nYes2050.020.050.0 <0.001II\\nNo21100.000.0Resurgery\\n\\n\\n\\n\\nYes746.7853.3 0.051I\\nNo3473.91226.1Time with drain (days) 9.5±2.60III29.1±13.58III <0.001IV\\nHospitalization time (days) 16.7±17.9III34.7±16.7III <0.001IV\\nMortality49.815.0 1.000II\\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\\n\\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\\nMortality of the series was 8.19%: four patients belonged to group 1 and one patient to group 2. The mortality causes in group 1 were: kidney insufficiency due to coagulopathy; dehiscence of gastrojejunal anastomosis and sepsis, pneumonia due to broncoaspiration and necrosis of the pancreaticojejunostomy loop associated with sepsis. The only patient of group 2 passed due to necrotic pancreatitis associated with pancreatic fistula and sepsis. \\nRegarding the AD1PO value, the median was 175 U/l (48.5-954) in group 1 and 2172.5 U/l (833.5-6421.0) in group 2. This difference was statistically significant (p=0.001).\\nThe area under the ROC curve presented an exactness index of 83.0% (p=0.001). When considering a 180 U/l cut-off point for AD1PO, the following results were obtained: sensitivity 100%, specificity 48.8%, positive predictor value 50%, and negative predictor value 100%. There was an association between the arbitrated AD1PO value from the ROC curve (>180 U/l) with groups 1 and 2 (p<0.001). With a cut-off point of 578 U/l, the results were: sensitivity 80%, specificity 39%, positive predictor value 50% and negative predictor value 86.2%. There was also an association between the arbitrated AD1PO value from the ROC curve (>578 U/l) with groups 1 and 2 (p<0.003). Patients presenting AD1PO values higher than 180 U/l and 578 U/l were 2.0 and 6.25 times more likely to belong to group 2, and therefore require postoperative drainage. Table 3 shows the comparison between the two AD1PO values. \\n\\nTABLE 3Precision and exactness measurements, per AD1PO cut-off point Parameters Cut-off point (U/l) 180 578Sensitivity 100% 80%Specificity 48.8% 39%VPP 50% 50%VPN 100% 86.2%\\n\",\n \"AD1PO as a predictor of post-pancreatectomy PF was described by Yamaguchi e cols in 2003\\n20\\n. The authors analyzed the amylase values measured in abdominal drains of 26 patients submitted to pancreatectomy, and observed that values were already high on the first day PO in those who developed clinical PF after. At least three meta-analysis\\n10\\n\\n,\\n\\n15\\n\\n,\\n\\n21\\n have validated the correlation between high AD1PO values and the development of PF. However, there is still controversy on the adequate cut-off point for AD1PO to predict PF, and on its applicability to the removal of prophylactic drains inserted after pancreatic resections. Verona’s group was the first to establish a strategy for the early removal of abdominal drains based on AD1PO values\\n6\\n. The authors carried out a prospective study on 114 patients submitted to pancreatic resection and randomized those with AD1PO <5000 U/l to remove the drain on the 3rd or 5th day PO. Early removal of drains was associated with a lower rate of pancreatic fistula (1.8% vs. 26%), lower rate of abdominal complications (12.2% vs. 52.6%), lower rate of pulmonary complications (26.3% vs. 52.6%), shorter hospitalization times (8.7 (±4) vs. 10.8 (±6.9) and lower hospital readmission rates (0% vs. 8.8%). Although the study suggested that early removal of abdominal drains was safe with AD1PO under 5000 U/l, other authors reported hypothetical PF indexes between 25-48% if drains were removed according to this suggestion\\n7\\n\\n,\\n\\n12\\n.\\nFor those patients submitted to duodenopancreatectomy and that received prophylactic drains, the most important question is to know which patients will not develop PF - and not the other way around. On one hand, knowing which patients will not develop PF is useful because it allows for early drain removal, which leads to shorter hospitalization times. On the other hand, predicting which patients will develop PF after surgery, when the abdominal cavity is being drained, is not as relevant because PF can be easily diagnosed by measuring amylase in the drained liquid. In most cases, treatment is simple maintenance of drains, associated with fasting and nutritional support. For this reason the study herein presented analyzed two low AD1PO cut-off points (180 and 578) that were associated with higher specificity and consequently, with a high negative predictive factor, in detriment to a high cut-off point associated with higher sensitivity to PF diagnosis. Another reason for the selection is related to the impossibility of obtaining daily interventionist radiology services in the public hospital where the majority of the patients studied underwent surgery. These services include percutaneous drainage of abdominal collections/abscesses, which is indicated in urgent cases where there are abdominal complications as a consequence of undrained anastomotic leaks. \\nThe methodology of this study utilized the PF definition based on the revised classification by ISGPS\\n5\\n, which no longer considers grade A of the previous classification to be a true PF\\n4\\n. However, as this study aimed at identifying a group of patients in which early removal of drains could be beneficial, it was important to include not only the concept of PF, but also grades B and C and those patients with biochemical fistula that persisted with amylase-rich debit between 7-21 days PO. In these patients, early removal of drains would increase susceptibility to abdominal collections or abscesses. With the 180 U/l cut-off point, an excellent correlation was obtained between AD1PO and absence of fistula. Therefore it was possible to identify 1/3 of patients that would have been benefitted by early drain removal. Besides, with the same cut-off, none of the patients that developed clinically relevant fistula or even persistent biochemical fistula would have had their drains removed early. \\nData obtained herein were capable of validating previous publications that utilized low cut-off points\\n7\\n\\n,\\n\\n12\\n\\n,\\n\\n14\\n\\n,\\n\\n17\\n. Differently from these studies, however, the first series was entirely prospective herein, including only patients submitted consecutively to duodenopancreatectomy by a single team of surgeons, where PF definition followed ISGPS criteria. The cut-off point 180 U/l obtained herein was lower than the one found by Fong et al\\n9\\n at the Massachusetts General Hospital. They divided the study in two cohorts: the first involved 126 patients, and the 612 U/l cut-off point was defined as the one presenting best accuracy, sensitivity and specificity; the second had the objective of validating a 600 U/l cut-off point, involving 369 patients submitted to duodenopancreatectomy within January 2009 and December 2012. Almost two-thirds of patients (62.1%) presented AD1PO lower than 600 U/l and only two PF cases (0.9%) were diagnosed. This was different than the other patients with AD1PO of at least 600 U/l, with PF incidence of 31.4%. When considering herein a cut-off point close to Fong et al., 578 U/l, 13.8% of patients with PF diagnosis would have been affected by early removal of drains (negative predictor value: 86.2%). In the reality considered herein, there is no non-invasive treatment for intracavitary collections/abscesses available all the time at the public hospital, and therefore increasing the cut-off point would be a high price to pay. \\nThis study presents some limitations. The first concerns the low number of included patients, which did not hinder statistical analysis. The second is related to the non-verification of the real benefits of drains in patients who developed PF, as all patients received drains. The high index of abdominal collections (65%) and necessity of re-surgery (40%) in group 2 suggested that, for some patients, drains were not totally efficient; nevertheless, the objective of the study was to find a group of patients in which drains could be removed early and not confirm the non-efficiency of drains in the group that developed PF.\",\n \"Was validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients. \"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,"results","discussion","conclusions"],"string":"[\n \"intro\",\n \"methods\",\n null,\n \"results\",\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["Amylase","Postoperative complications","Pancreatic fistula","Pancreatoduodenectomy","Amilase","Complicações pós-operatórias","Fístula pancreática","Pancreatoduodenectomia"],"string":"[\n \"Amylase\",\n \"Postoperative complications\",\n \"Pancreatic fistula\",\n \"Pancreatoduodenectomy\",\n \"Amilase\",\n \"Complicações pós-operatórias\",\n \"Fístula pancreática\",\n \"Pancreatoduodenectomia\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nHistorically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them\n16\n. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis\n13\n\n,\n\n11\n. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results\n8\n\n,\n\n18\n\n,\n\n19\n, which has been a limiting factor in the practice of not using drains in this type of procedure. \nDescription of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L\n6\n and 90 U/l\n14\n have been suggested. \nThe objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.\n\nMETHOD:\nThe project was submitted and approved by the Human Research Ethics Committee, under nº 59716616.9.0000.5292 in the Plataforma Brasil.\nSixty-three patients were analyzed from a prospective database, who had undergone Kausch-Whipple surgery performed by the 1st author of this study (ECA), for the treatment of pancreatic or peripancreatic diseases. The time period was between June 2007 and September 2016, in the following hospitals located in the city of Natal: Hospital Universitário Onofre Lopes, Liga Norte Riograndense Contra o Câncer, Casa de Saúde São Lucas and Natal Hospital Center.\nThe technical steps for the Whipple technique as performed by the author are detailed in previous studies\n1\n\n,\n\n2\n. Three techniques for pancreatojejunal anastomosis were utilized. For pancreas with main pancreatic duct ≥5 mm, two-layer pancreatojejunal terminolateral duct to mucosa anastomosis was performed. For pancreas with normal or slightly dilated (diameter <5 mm) main pancreatic duct, terminoterminal pancreatojejunal anastomosis (“telescopic”) or terminolateral pancreatojejunal anastomosis (“invagination”) were carried out, the latter performed systematically after the 33rd patient from the casebook. At the end of the surgery, two drains, preferably laminar silicone drains, were placed in the cavity and exteriorized at each flank. \nIn the majority of patients, subcutaneous octreotide was utilized as prophylaxis for the prevention of PF, at 0.3 mg/day, fractioned in 8 h intervals, during seven days. Debits were recorded along with amylase dosage of the drained liquid on the 1st, 3rd, 5th, 7th and sometimes, on the 9th postoperative day. The value of AD1PO vas defined from the highest amylase value of both drains, measured from a sample of liquid obtained on the first day after surgery. \nPF diagnosis utilized the 2016 revised criterion of GIEDFP\n3\n. PF was defined when on the 3rd postoperative (PO) day the value of amylase in the drained liquid was three times higher than the normal upper limit for serum amylase. Patients that did not develop fistula or those with transient biochemical fistula (<7 days PO) were characterized as group 1, and patients with persistent biochemical fistula (between 7 and 21 days PO) and fistula grade B and C were characterized as group 2. In most cases, on the 9th day after surgery, control ultrasound or tomography tests were carried out. Removal of drains occurred in those cases with low amylase values for the drained liquid (under three times the upper limit for normal serum amylase) and when image tests did not show abdominal collections. Intra-hospital mortality was defined as death occurring within 90 days of surgery.\nThe Receiver Operating Characteristic (ROC) curve was utilized to identify an adequate cut-off point for AD1PO and verify its predictive characteristic in patients of both groups. The ROC value was generated after sensitivity, specificity and accuracy calculations. Additionally, the cut-off point 578 U/l was also selected because of its closeness to the value utilized by Fong e cols \n9\n, for comparison purposes. \n Statistical analysis After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \nAfter determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \n\nStatistical analysis:\nAfter determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \n\nRESULTS:\nFrom the initial sample of 63 patients, two were excluded. One had AD1PO collected incorrectly on the 2nd day after surgery, and the other passed within the 1st week PO. Therefore analysis covered 61 patients. Group 1 was constituted of 41 patients, of which 36 did not develop fistula and five transient biochemical fistula (grade A). Group 2 was constituted of 20 patients, of which six presented persistent biochemical fistula (grade A), eight degree B fistula, and six grade C fistula. The index of clinical fistula (grades B + C) in the overall sample was 22.9%. \nThe demographic and clinical characteristics of the patients, as well as respective tests, are shown in Table 1. There was no statistical difference between groups regarding age, gender and type of illness. However, regarding the size of main pancreatic duct and type of anastomosis, a statistically significant difference was detected. All patients with dilated main pancreatic ducts as well as those submitted to duct to mucosa anastomosis belonged to group 1.\n\nTABLE 1Associations between demographic and clinical characteristics and patient groups Variables Group 1 Group 2 pAge 58.0±13.06I\n 57.1±12.1I\n 0.706II\n\n n % n %\nGender\n\n\n\n\nMale 19 63.3 11 36.7 0.525III\nFemale 22 71.0 9 29.0Disease\n\n\n\n\nPancreatic adenocarcinoma 17 65.4 9 34.6 0.264III\nPapillary adenocarcinoma 12 60.0 8 40.0Duodenal adenocarcinoma 1 33.3 2 66.7Colangiocarcinoma 4 100.0 0 0.0Frantz 5 100.0 0 0.0Others 2 66.7 1 33.3Size of duct\n\n\n\n\n< 5 31 60.8 20 39.2 0.023IV\n> 5 10 100.0 0 0.0Anastomosis*\n\n\n\n\nT-L (Invagination) 14 50.0 14 50.0 0.011III\nT-T 17 73.9 6 26.1T-L (DM) 10 100.0 0 0.0I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\n\nI=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\nRegarding postoperative evolution, group 2 was associated with a higher level of general complications, postoperative collections, longer permanence of abdominal drain and hospitalization time. There was also an association between high resurgery index and patients of group 2. Nevertheless, despite all these worse result factors related to group 2, there was no statistically significant difference when comparing mortality across groups (Table 2). \n\nTABLE 2Associations between postoperative clinical findings and patient groups, along with results for drain and hospitalization times, per group Variable Group 1 Group 2 p n % n %\nCollection\n\n\n\n\nYes733.31466.7 <0.001I\nNo3485.0615.0Postoperative complications\n\n\n\n\nYes2050.020.050.0 <0.001II\nNo21100.000.0Resurgery\n\n\n\n\nYes746.7853.3 0.051I\nNo3473.91226.1Time with drain (days) 9.5±2.60III29.1±13.58III <0.001IV\nHospitalization time (days) 16.7±17.9III34.7±16.7III <0.001IV\nMortality49.815.0 1.000II\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\n\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\nMortality of the series was 8.19%: four patients belonged to group 1 and one patient to group 2. The mortality causes in group 1 were: kidney insufficiency due to coagulopathy; dehiscence of gastrojejunal anastomosis and sepsis, pneumonia due to broncoaspiration and necrosis of the pancreaticojejunostomy loop associated with sepsis. The only patient of group 2 passed due to necrotic pancreatitis associated with pancreatic fistula and sepsis. \nRegarding the AD1PO value, the median was 175 U/l (48.5-954) in group 1 and 2172.5 U/l (833.5-6421.0) in group 2. This difference was statistically significant (p=0.001).\nThe area under the ROC curve presented an exactness index of 83.0% (p=0.001). When considering a 180 U/l cut-off point for AD1PO, the following results were obtained: sensitivity 100%, specificity 48.8%, positive predictor value 50%, and negative predictor value 100%. There was an association between the arbitrated AD1PO value from the ROC curve (>180 U/l) with groups 1 and 2 (p<0.001). With a cut-off point of 578 U/l, the results were: sensitivity 80%, specificity 39%, positive predictor value 50% and negative predictor value 86.2%. There was also an association between the arbitrated AD1PO value from the ROC curve (>578 U/l) with groups 1 and 2 (p<0.003). Patients presenting AD1PO values higher than 180 U/l and 578 U/l were 2.0 and 6.25 times more likely to belong to group 2, and therefore require postoperative drainage. Table 3 shows the comparison between the two AD1PO values. \n\nTABLE 3Precision and exactness measurements, per AD1PO cut-off point Parameters Cut-off point (U/l) 180 578Sensitivity 100% 80%Specificity 48.8% 39%VPP 50% 50%VPN 100% 86.2%\n\n\nDISCUSSION:\nAD1PO as a predictor of post-pancreatectomy PF was described by Yamaguchi e cols in 2003\n20\n. The authors analyzed the amylase values measured in abdominal drains of 26 patients submitted to pancreatectomy, and observed that values were already high on the first day PO in those who developed clinical PF after. At least three meta-analysis\n10\n\n,\n\n15\n\n,\n\n21\n have validated the correlation between high AD1PO values and the development of PF. However, there is still controversy on the adequate cut-off point for AD1PO to predict PF, and on its applicability to the removal of prophylactic drains inserted after pancreatic resections. Verona’s group was the first to establish a strategy for the early removal of abdominal drains based on AD1PO values\n6\n. The authors carried out a prospective study on 114 patients submitted to pancreatic resection and randomized those with AD1PO <5000 U/l to remove the drain on the 3rd or 5th day PO. Early removal of drains was associated with a lower rate of pancreatic fistula (1.8% vs. 26%), lower rate of abdominal complications (12.2% vs. 52.6%), lower rate of pulmonary complications (26.3% vs. 52.6%), shorter hospitalization times (8.7 (±4) vs. 10.8 (±6.9) and lower hospital readmission rates (0% vs. 8.8%). Although the study suggested that early removal of abdominal drains was safe with AD1PO under 5000 U/l, other authors reported hypothetical PF indexes between 25-48% if drains were removed according to this suggestion\n7\n\n,\n\n12\n.\nFor those patients submitted to duodenopancreatectomy and that received prophylactic drains, the most important question is to know which patients will not develop PF - and not the other way around. On one hand, knowing which patients will not develop PF is useful because it allows for early drain removal, which leads to shorter hospitalization times. On the other hand, predicting which patients will develop PF after surgery, when the abdominal cavity is being drained, is not as relevant because PF can be easily diagnosed by measuring amylase in the drained liquid. In most cases, treatment is simple maintenance of drains, associated with fasting and nutritional support. For this reason the study herein presented analyzed two low AD1PO cut-off points (180 and 578) that were associated with higher specificity and consequently, with a high negative predictive factor, in detriment to a high cut-off point associated with higher sensitivity to PF diagnosis. Another reason for the selection is related to the impossibility of obtaining daily interventionist radiology services in the public hospital where the majority of the patients studied underwent surgery. These services include percutaneous drainage of abdominal collections/abscesses, which is indicated in urgent cases where there are abdominal complications as a consequence of undrained anastomotic leaks. \nThe methodology of this study utilized the PF definition based on the revised classification by ISGPS\n5\n, which no longer considers grade A of the previous classification to be a true PF\n4\n. However, as this study aimed at identifying a group of patients in which early removal of drains could be beneficial, it was important to include not only the concept of PF, but also grades B and C and those patients with biochemical fistula that persisted with amylase-rich debit between 7-21 days PO. In these patients, early removal of drains would increase susceptibility to abdominal collections or abscesses. With the 180 U/l cut-off point, an excellent correlation was obtained between AD1PO and absence of fistula. Therefore it was possible to identify 1/3 of patients that would have been benefitted by early drain removal. Besides, with the same cut-off, none of the patients that developed clinically relevant fistula or even persistent biochemical fistula would have had their drains removed early. \nData obtained herein were capable of validating previous publications that utilized low cut-off points\n7\n\n,\n\n12\n\n,\n\n14\n\n,\n\n17\n. Differently from these studies, however, the first series was entirely prospective herein, including only patients submitted consecutively to duodenopancreatectomy by a single team of surgeons, where PF definition followed ISGPS criteria. The cut-off point 180 U/l obtained herein was lower than the one found by Fong et al\n9\n at the Massachusetts General Hospital. They divided the study in two cohorts: the first involved 126 patients, and the 612 U/l cut-off point was defined as the one presenting best accuracy, sensitivity and specificity; the second had the objective of validating a 600 U/l cut-off point, involving 369 patients submitted to duodenopancreatectomy within January 2009 and December 2012. Almost two-thirds of patients (62.1%) presented AD1PO lower than 600 U/l and only two PF cases (0.9%) were diagnosed. This was different than the other patients with AD1PO of at least 600 U/l, with PF incidence of 31.4%. When considering herein a cut-off point close to Fong et al., 578 U/l, 13.8% of patients with PF diagnosis would have been affected by early removal of drains (negative predictor value: 86.2%). In the reality considered herein, there is no non-invasive treatment for intracavitary collections/abscesses available all the time at the public hospital, and therefore increasing the cut-off point would be a high price to pay. \nThis study presents some limitations. The first concerns the low number of included patients, which did not hinder statistical analysis. The second is related to the non-verification of the real benefits of drains in patients who developed PF, as all patients received drains. The high index of abdominal collections (65%) and necessity of re-surgery (40%) in group 2 suggested that, for some patients, drains were not totally efficient; nevertheless, the objective of the study was to find a group of patients in which drains could be removed early and not confirm the non-efficiency of drains in the group that developed PF.\n\nCONCLUSION:\nWas validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients. \n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe value of drain amylase on the first postoperative day after pancreatic resections has been described as an efficient predictor of pancreatic fistula. In spite of this, the cut-off point below which the drains can be removed early remains controversial.\n\nMethods:\nWere included patients undergoing Whipple surgery in the period of 2007 to 2016. Group 1 enrolled the ones who did not develop fistula and those who developed biochemical fistula for less than seven days postoperatively and group 2 included patients who developed persistent biochemical fistula between seven and 21 days and those with grade B and C fistula.\n\nResults:\nSixty-one patients were included, 41 comprised group 1 and 20 group 2. The incidence of abdominal collections, need for reoperation and time of hospitalization were for group 1 and 2, respectively: 17.1%, 17.1% and 9.5 days, and 65%, 40% and 21.1 days. The median of the amylase from the drain at 1st postoperative day was in group 1 and 2, respectively: 175 U/l and 3172.5 U/l (p=0.001). Using a cut-off of 180 to predict the group to which the patient would belong there was obtained sensitivity, specificity, positive predictive value and negative predictive value of 100%, 48.8%, 50% and 100% respectively.\n\nConclusions:\nIt was validated the cut-off value of 180 U/l as appropriate to early drain removal."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nHistorically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them\n16\n. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis\n13\n\n,\n\n11\n. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results\n8\n\n,\n\n18\n\n,\n\n19\n, which has been a limiting factor in the practice of not using drains in this type of procedure. \nDescription of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L\n6\n and 90 U/l\n14\n have been suggested. \nThe objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.\n\nCONCLUSION:\nWas validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients. "},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe value of drain amylase on the first postoperative day after pancreatic resections has been described as an efficient predictor of pancreatic fistula. In spite of this, the cut-off point below which the drains can be removed early remains controversial.\n\nMethods:\nWere included patients undergoing Whipple surgery in the period of 2007 to 2016. Group 1 enrolled the ones who did not develop fistula and those who developed biochemical fistula for less than seven days postoperatively and group 2 included patients who developed persistent biochemical fistula between seven and 21 days and those with grade B and C fistula.\n\nResults:\nSixty-one patients were included, 41 comprised group 1 and 20 group 2. The incidence of abdominal collections, need for reoperation and time of hospitalization were for group 1 and 2, respectively: 17.1%, 17.1% and 9.5 days, and 65%, 40% and 21.1 days. The median of the amylase from the drain at 1st postoperative day was in group 1 and 2, respectively: 175 U/l and 3172.5 U/l (p=0.001). Using a cut-off of 180 to predict the group to which the patient would belong there was obtained sensitivity, specificity, positive predictive value and negative predictive value of 100%, 48.8%, 50% and 100% respectively.\n\nConclusions:\nIt was validated the cut-off value of 180 U/l as appropriate to early drain removal."},"whole_article_text_length":{"kind":"number","value":3414,"string":"3,414"},"whole_article_abstract_length":{"kind":"number","value":276,"string":"276"},"num_sections":{"kind":"number","value":6,"string":"6"},"most_frequent_words":{"kind":"list like","value":["patients","drains","ad1po","pf","group","cut","cut point","point","test","fistula"],"string":"[\n \"patients\",\n \"drains\",\n \"ad1po\",\n \"pf\",\n \"group\",\n \"cut\",\n \"cut point\",\n \"point\",\n \"test\",\n \"fistula\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] drains | pancreatic | studies | systematic | manner | use | pf | correlation | objective | resections [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] amylase | tests | performed | pancreatojejunal | utilized | value | surgery | groups | liquid | applied [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] group | test | 100 | table | 50 | fistula | value | patients | groups | duct [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ideal cut | ideal | ideal cut point | cut | point | cut point | patients | pf benefits faster | service | compensate risk [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | pf | cut | test | cut point | point | ad1po | group | groups [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | drains | pf | cut | test | cut point | point | ad1po | group | groups [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] the first postoperative day ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Whipple | the period of 2007 to 2016 ||| Group 1 | less than seven days | 2 | between seven and 21 days [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Sixty-one | 41 | 1 | 20 | 2 ||| 1 | 2 | 17.1% | 17.1% | 9.5 days | 65% | 40% | 21.1 days ||| 1st postoperative day | 1 | 2 | 175 | 3172.5 | U/l (p=0.001 ||| 180 | 100% | 48.8% | 50% and 100% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] 180 [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] the first postoperative day ||| ||| Whipple | the period of 2007 to 2016 ||| Group 1 | less than seven days | 2 | between seven and 21 days ||| ||| Sixty-one | 41 | 1 | 20 | 2 ||| 1 | 2 | 17.1% | 17.1% | 9.5 days | 65% | 40% | 21.1 days ||| 1st postoperative day | 1 | 2 | 175 | 3172.5 | U/l (p=0.001 ||| 180 | 100% | 48.8% | 50% and 100% ||| 180 [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] the first postoperative day ||| ||| Whipple | the period of 2007 to 2016 ||| Group 1 | less than seven days | 2 | between seven and 21 days ||| ||| Sixty-one | 41 | 1 | 20 | 2 ||| 1 | 2 | 17.1% | 17.1% | 9.5 days | 65% | 40% | 21.1 days ||| 1st postoperative day | 1 | 2 | 175 | 3172.5 | U/l (p=0.001 ||| 180 | 100% | 48.8% | 50% and 100% ||| 180 [SUMMARY]"}}},{"rowIdx":275,"cells":{"title":{"kind":"string","value":"Does a continuous local anaesthetic pain treatment after immediate tissue expander reconstruction in breast carcinoma patients more efficiently reduce acute postoperative pain--a prospective randomised study."},"pmid":{"kind":"string","value":"24433317"},"background_abstract":{"kind":"string","value":"Immediate breast reconstruction with an expander is a reasonable option for properly selected patients. After reconstruction, patients have severe postoperative pain, which responds poorly to opioids. Our aim was to evaluate if continuous wound infusion of a local anaesthetic into the surgical wound reduces postoperative pain, consumption of opioids and incidence of chronic pain compared to standard intravenous piritramide after primary breast reconstruction in breast carcinoma patients."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Altogether, 60 patients were enrolled in our study; one half in the group with wound infusion of a local anaesthetic, and the other half in the standard (piritramide) group. Parameters measured included: pain intensity (visual analogue scale), drug requirements, alertness, hospitalisation, side-effects and late complications. A p-value of < 0.05 was considered statistically significant."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In the recovery room, the test group reported less acute pain at rest (P = 0.03) and at activity (P = 0.01), and on the day of the surgical procedure they reported less pain at activity (P = 0.003). Consumption of piritramide and metoclopramide was lower in this group (P < 0.0001), but their alertness after the surgical procedure was higher compared to the standard group (P < 0.001). After three months, the test group reported less chronic pain (P = 0.01)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"After primary tissue expander breast reconstruction, wound infusion of a local anaesthetic significantly reduces acute pain and enables reduced opioid consumption, resulting in less postoperative sedation and reduced need for antiemetic drugs. Wound infusion of a local anaesthetic reduces chronic pain."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Acute Disease","Aged","Anesthetics, Local","Breast Neoplasms","Carcinoma, Ductal, Breast","Carcinoma, Intraductal, Noninfiltrating","Carcinoma, Lobular","Case-Control Studies","Catheters, Indwelling","Female","Follow-Up Studies","Humans","Lymphatic Metastasis","Mammaplasty","Mastectomy","Middle Aged","Neoplasm Grading","Neoplasm Staging","Pain, Postoperative","Postoperative Complications","Prognosis","Prospective Studies","Tissue Expansion Devices"],"string":"[\n \"Acute Disease\",\n \"Aged\",\n \"Anesthetics, Local\",\n \"Breast Neoplasms\",\n \"Carcinoma, Ductal, Breast\",\n \"Carcinoma, Intraductal, Noninfiltrating\",\n \"Carcinoma, Lobular\",\n \"Case-Control Studies\",\n \"Catheters, Indwelling\",\n \"Female\",\n \"Follow-Up Studies\",\n \"Humans\",\n \"Lymphatic Metastasis\",\n \"Mammaplasty\",\n \"Mastectomy\",\n \"Middle Aged\",\n \"Neoplasm Grading\",\n \"Neoplasm Staging\",\n \"Pain, Postoperative\",\n \"Postoperative Complications\",\n \"Prognosis\",\n \"Prospective Studies\",\n \"Tissue Expansion Devices\"\n]"},"pmcid":{"kind":"string","value":"3899444"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Breast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Altogether, 60 breast carcinoma patients were enrolled in the study protocol between January 2011 and May 2012: 72 patients who were eligible for inclusion were offered the opportunity to participate in our study during the recruitment period; 12 of them declined participation, and the remaining 60 were included in the study. Patients listed for mastectomy and primary reconstruction with a tissue expander because of the presence of breast carcinoma or for prophylactic mastectomy, were randomized during the routine preoperative anaesthetic assessment. Anaesthesia for the procedure was the same for all study subjects. We used the same protocol as already reported for patients with axillary dissection\n[14]. The first study arm was treated with continuous local analgesia, and the other (control) arm received standard intravenous analgesia. Allergies to local anaesthetics and chronic pre-operative opioid consumption were the criteria to exclude patients from our study.\nAfter informed consent was obtained the randomization was made by research nurses from the Clinical Research Unit of the Institute of Oncology Ljubljana. The research nurse performed randomization using random numbers generated by a computer and then informed the principal investigator about the treatment allocation of the patient.\nSurgical procedures were performed by ten experienced oncology surgeons and ten plastic surgeons. Sentinel lymph node biopsy was performed in 55 patients. In one patient with positive imprint cytology of the sentinel lymph node, axillary lymphadenectomy was performed immediately.\nThe study was reviewed by the The National Medical Ethics Committee of the Republic of Slovenia and performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. Our study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and conducted with the understanding and consent of all subjects involved. Study was entirely financed by the Institute of Oncology as a part of public service.\n Test group of patients Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\nBefore wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\n Control group of patients The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\nThe control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\n Both groups of patients Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\nPatient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\n Pain measurement Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\nData about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\n Statistical analysis In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\nIn order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The mean age of patients was 47.8 years (range 25 to 64 y), height was 166 cm (range 153 to 178 cm), weight was 61.7 kg (range 45 to 85 kg), and the body mass index (BMI) was 22.4 (range 17 to 31.2). There were no significant differences between the study groups in BMI, American Society of Anesthesiology (ASA) score, comorbidities (Table \n1), type and length of surgical procedure, extent of lymph node dissection, adjuvant therapy, complications, or hospital stay (Table \n2).\nPatient characteristics\nASA, American Society of Anesthesiology; HER-2, human epidermal growth factor receptor-2; n, number of patients.\nTreatment of patients, adjuvant therapy, hospital stay and complications\nValues are numbers of patients unless stated otherwise.\n Acute pain Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\nData on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\n Opioid consumption Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\nConsumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\n Nausea Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\nPatients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\n Complications No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\nNo local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\n Hospital stay The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\nThe average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\n Unilateral and bilateral tissue expander In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\nIn order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\n Late complications (three months after primary reconstruction) Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\nThree months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Our current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia."},"other_sections_titles":{"kind":"list like","value":["Background","Test group of patients","Control group of patients","Both groups of patients","Pain measurement","Statistical analysis","Acute pain","Opioid consumption","Nausea","Complications","Hospital stay","Unilateral and bilateral tissue expander","Late complications (three months after primary reconstruction)","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Test group of patients\",\n \"Control group of patients\",\n \"Both groups of patients\",\n \"Pain measurement\",\n \"Statistical analysis\",\n \"Acute pain\",\n \"Opioid consumption\",\n \"Nausea\",\n \"Complications\",\n \"Hospital stay\",\n \"Unilateral and bilateral tissue expander\",\n \"Late complications (three months after primary reconstruction)\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["Breast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.","Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.","The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.","Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.","Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.","In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.","Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.","Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.","Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).","No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.","The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.","In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).","Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.","BMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.","The authors declare that they have no competing interests.","BS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript."],"string":"[\n \"Breast cancer is the most common cancer in women both in the developed and the developing countries\\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\\n[4].\\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\\n[6].\\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.\",\n \"Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\",\n \"The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\",\n \"Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\",\n \"Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\\n[19] in a composite way, as we did in our previous study\\n[14].\\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\",\n \"In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\",\n \"Data on postoperative pain are presented in Table \\n3.\\nPain in the local anaesthetic group and the standard group of patients\\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\",\n \"Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \\n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\\nOAA/S - observer's assessment of alertness/sedation.\",\n \"Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\",\n \"No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\",\n \"The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\",\n \"In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\",\n \"Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\",\n \"BMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.\",\n \"The authors declare that they have no competing interests.\",\n \"BS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Test group of patients","Control group of patients","Both groups of patients","Pain measurement","Statistical analysis","Results","Acute pain","Opioid consumption","Nausea","Complications","Hospital stay","Unilateral and bilateral tissue expander","Late complications (three months after primary reconstruction)","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Test group of patients\",\n \"Control group of patients\",\n \"Both groups of patients\",\n \"Pain measurement\",\n \"Statistical analysis\",\n \"Results\",\n \"Acute pain\",\n \"Opioid consumption\",\n \"Nausea\",\n \"Complications\",\n \"Hospital stay\",\n \"Unilateral and bilateral tissue expander\",\n \"Late complications (three months after primary reconstruction)\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Breast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.","Altogether, 60 breast carcinoma patients were enrolled in the study protocol between January 2011 and May 2012: 72 patients who were eligible for inclusion were offered the opportunity to participate in our study during the recruitment period; 12 of them declined participation, and the remaining 60 were included in the study. Patients listed for mastectomy and primary reconstruction with a tissue expander because of the presence of breast carcinoma or for prophylactic mastectomy, were randomized during the routine preoperative anaesthetic assessment. Anaesthesia for the procedure was the same for all study subjects. We used the same protocol as already reported for patients with axillary dissection\n[14]. The first study arm was treated with continuous local analgesia, and the other (control) arm received standard intravenous analgesia. Allergies to local anaesthetics and chronic pre-operative opioid consumption were the criteria to exclude patients from our study.\nAfter informed consent was obtained the randomization was made by research nurses from the Clinical Research Unit of the Institute of Oncology Ljubljana. The research nurse performed randomization using random numbers generated by a computer and then informed the principal investigator about the treatment allocation of the patient.\nSurgical procedures were performed by ten experienced oncology surgeons and ten plastic surgeons. Sentinel lymph node biopsy was performed in 55 patients. In one patient with positive imprint cytology of the sentinel lymph node, axillary lymphadenectomy was performed immediately.\nThe study was reviewed by the The National Medical Ethics Committee of the Republic of Slovenia and performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. Our study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and conducted with the understanding and consent of all subjects involved. Study was entirely financed by the Institute of Oncology as a part of public service.\n Test group of patients Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\nBefore wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\n Control group of patients The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\nThe control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\n Both groups of patients Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\nPatient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\n Pain measurement Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\nData about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\n Statistical analysis In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\nIn order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.","Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.","The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.","Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.","Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.","In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.","The mean age of patients was 47.8 years (range 25 to 64 y), height was 166 cm (range 153 to 178 cm), weight was 61.7 kg (range 45 to 85 kg), and the body mass index (BMI) was 22.4 (range 17 to 31.2). There were no significant differences between the study groups in BMI, American Society of Anesthesiology (ASA) score, comorbidities (Table \n1), type and length of surgical procedure, extent of lymph node dissection, adjuvant therapy, complications, or hospital stay (Table \n2).\nPatient characteristics\nASA, American Society of Anesthesiology; HER-2, human epidermal growth factor receptor-2; n, number of patients.\nTreatment of patients, adjuvant therapy, hospital stay and complications\nValues are numbers of patients unless stated otherwise.\n Acute pain Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\nData on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\n Opioid consumption Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\nConsumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\n Nausea Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\nPatients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\n Complications No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\nNo local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\n Hospital stay The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\nThe average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\n Unilateral and bilateral tissue expander In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\nIn order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\n Late complications (three months after primary reconstruction) Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\nThree months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.","Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.","Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.","Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).","No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.","The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.","In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).","Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.","Ideally the study should have been placebo double-blinded to eliminate all biases. However, the cost of the elastomeric pump and wound catheter was 175 euro, so we were not able to perform a double-blind study. However, the cost of an elastomeric pump and wound catheter was not the only reason that precluded us from performing such a study. We had also ethical reasons for not carrying out a placebo-controlled study. Namely, a reconstruction with a tissue expander is a very painful procedure for the majority of patients, so we felt that it is not ethical to have a control group treated by placebo only. Instead, we offered our control group of patients the best available treatment, that is, intravenous analgesia. Based on the good results of our preliminary data on the use of continuous local anaesthetics for treatment of pain, our patients were willing to participate in our study because they were offered state-of-the-art pain treatment versus potentially even more effective pain treatment.\nSub-pectoral breast augmentation and breast reconstruction with an expander or an implant are associated with considerable pain in the early postoperative period\n[6,9,10,20-24]. Legeby et al.\n[6] found that pain in the first 3 h after immediate breast reconstruction with a tissue expander was more severe than pain after mastectomy or axillary dissection. The mean VAS value for pain in the first 3 h after breast reconstruction was 4.9 and 5.0, if axillary dissection was also performed, whereas the values were 2.1 for mastectomy and 3.0 for mastectomy with axillary dissection\n[6]. Similarly, after tissue-expander implantation, our standard analgesia group of patients also suffered pain in the recovery room, which was by about 3.0 VAS units larger than in patients who underwent breast surgery with axillary dissection without reconstruction, as reported by Strazisar et al.\n[14]. The mean VAS score after immediate reconstruction was 4.9 in the standard analgesia group, whereas it was only 3.4 in the local anaesthetic group. In our previous study, the mean VAS score in recovery after breast cancer surgery with axillary dissection was 2.0 in the standard control group and 0.5 in the local anaesthetic group\n[14]. In both our studies, the local anaesthetic reduced pain by 1.5 VAS units more than standard treatment alone.\nThe most positive result of our study is the observation that 77% of patients treated with local anaesthetics did not experience severe pain. To our knowledge, there are no reports in the literature about similarly effective early postoperative analgesia after the implantation of a breast-tissue expander. Legeby et al. reported that even 48% of patients treated with local anaesthetics experienced unacceptably severe pain (VAS 4.7 to 10.0) in the early postoperative period\n[6]. Legeby et al. used an epidural catheter with only a few holes at its tip and instilled local anaesthetic every 3 h. They probably did not establish an equal distribution of the local anaesthetic along the entire length of the muscle and the wound. On the other hand, we used quality wound-catheters that were perforated 15 cm at the distal end, which enabled a better distribution of the local anaesthetic. Furthermore, we used an elastomeric pump, which enabled a continuous infusion of the local anaesthetic. That is probably the reason why only seven patients (23.1%) in the test group reported pain above a VAS score of 5.0, whereas in the control group, such pain levels were reported by thirteen patients (42.9%).\nLegeby et al. found that the use of opioids was about three times higher after the implantation of a tissue expander compared to other breast surgical procedures\n[6]. After the tissue-expander implantation with or without axillary dissection, the consumption of opioids was 12.8 mg and 11.8 mg, whereas after mastectomy or mastectomy with axillary dissection, the mean consumption of opioids was 3.2 mg and 5.5 mg, respectively\n[6]. Our patients with standard analgesia after the tissue-expander implantation needed 5.6 times more opioids than patients who underwent axillary dissection alone\n[14]. Local anaesthetics can reduce consumption of opioids after mastectomy and axillary dissection\n[11,13,14] and also after breast augmentation with implants\n[7]. Our study shows that the use of continuous local anaesthesia can effectively reduce opioid consumption. Our patients treated with a local anaesthetic needed about three times less opioids than the standard analgesia group. Subsequently, patients from the local anaesthesia group experienced nausea less often, thus they had a lower consumption of metoclopramide than the standard analgesia group. Furthermore, the use of local anaesthetics resulted in less sedated and more alert patients in the recovery room and 6 h after the operation. The complication rate did not differ between the two groups of patients. This is in agreement with the reports of other authors who used local anaesthetics\n[11-13,18,21,25-30]. Some authors report that local anaesthetics appear to reduce the incidence of inflammation\n[31-33].\nThere was no difference in pain on the first postoperative day between the study and control group of patients. It means that local anaesthesia was no longer as effective as it was on the day of surgery. A possible explanation is that muscle overstretching caused by a tissue expander is no longer as painful as it was. Other explanations are that in some patients the position of the wound catheter might have changed during movement or that there was a different perfusion region in the erect, prone or supine position, or that continuous infusion was flawed because of the wound drainage. Unfortunately, in our study we did not test a rescue bolus-injection of levobupivacaine in the case of severe pain. However, we have good experience with application of a rescue bolus of levobupivacaine in patients with neuropathic pain after axillary lymphadenectomy. Many times we have observed that injection of a bolus of 15 mL of 0.25% levobupivacaine into a drain and clamping of wound drainage inhibited neuropathic pain for a couple of hours.\nMemory of severe postoperative pain is the most important risk factor for the development of chronic pain\n[34]. Patients who were treated with patient-controlled analgesia had three times less chronic pain than the traditionally nurse-administered patients\n[6]. The results of our study support this observation. Only 17% of patients treated with a local anaesthetic experienced chronic pain. This is significantly less than in the standard analgesia group of patients, of whom 50% suffered chronic pain. The proportion of patients treated with axillary lymphadenectomy, chemotherapy, hormone therapy and/or irradiation was similar in both study arms. Hence, we think that inadequately treated acute pain had an impact on the frequency of chronic pain in the standard analgesia group.","Our current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia.","BMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.","The authors declare that they have no competing interests.","BS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript."],"string":"[\n \"Breast cancer is the most common cancer in women both in the developed and the developing countries\\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\\n[4].\\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\\n[6].\\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.\",\n \"Altogether, 60 breast carcinoma patients were enrolled in the study protocol between January 2011 and May 2012: 72 patients who were eligible for inclusion were offered the opportunity to participate in our study during the recruitment period; 12 of them declined participation, and the remaining 60 were included in the study. Patients listed for mastectomy and primary reconstruction with a tissue expander because of the presence of breast carcinoma or for prophylactic mastectomy, were randomized during the routine preoperative anaesthetic assessment. Anaesthesia for the procedure was the same for all study subjects. We used the same protocol as already reported for patients with axillary dissection\\n[14]. The first study arm was treated with continuous local analgesia, and the other (control) arm received standard intravenous analgesia. Allergies to local anaesthetics and chronic pre-operative opioid consumption were the criteria to exclude patients from our study.\\nAfter informed consent was obtained the randomization was made by research nurses from the Clinical Research Unit of the Institute of Oncology Ljubljana. The research nurse performed randomization using random numbers generated by a computer and then informed the principal investigator about the treatment allocation of the patient.\\nSurgical procedures were performed by ten experienced oncology surgeons and ten plastic surgeons. Sentinel lymph node biopsy was performed in 55 patients. In one patient with positive imprint cytology of the sentinel lymph node, axillary lymphadenectomy was performed immediately.\\nThe study was reviewed by the The National Medical Ethics Committee of the Republic of Slovenia and performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. Our study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and conducted with the understanding and consent of all subjects involved. Study was entirely financed by the Institute of Oncology as a part of public service.\\n Test group of patients Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\\nBefore wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\\n Control group of patients The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\\nThe control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\\n Both groups of patients Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\\nPatient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\\n Pain measurement Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\\n[19] in a composite way, as we did in our previous study\\n[14].\\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\\nData about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\\n[19] in a composite way, as we did in our previous study\\n[14].\\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\\n Statistical analysis In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\\nIn order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\",\n \"Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\",\n \"The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\",\n \"Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\",\n \"Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\\n[19] in a composite way, as we did in our previous study\\n[14].\\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\",\n \"In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\",\n \"The mean age of patients was 47.8 years (range 25 to 64 y), height was 166 cm (range 153 to 178 cm), weight was 61.7 kg (range 45 to 85 kg), and the body mass index (BMI) was 22.4 (range 17 to 31.2). There were no significant differences between the study groups in BMI, American Society of Anesthesiology (ASA) score, comorbidities (Table \\n1), type and length of surgical procedure, extent of lymph node dissection, adjuvant therapy, complications, or hospital stay (Table \\n2).\\nPatient characteristics\\nASA, American Society of Anesthesiology; HER-2, human epidermal growth factor receptor-2; n, number of patients.\\nTreatment of patients, adjuvant therapy, hospital stay and complications\\nValues are numbers of patients unless stated otherwise.\\n Acute pain Data on postoperative pain are presented in Table \\n3.\\nPain in the local anaesthetic group and the standard group of patients\\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\\nData on postoperative pain are presented in Table \\n3.\\nPain in the local anaesthetic group and the standard group of patients\\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\\n Opioid consumption Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \\n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\\nOAA/S - observer's assessment of alertness/sedation.\\nConsumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \\n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\\nOAA/S - observer's assessment of alertness/sedation.\\n Nausea Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\\nPatients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\\n Complications No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\\nNo local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\\n Hospital stay The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\\nThe average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\\n Unilateral and bilateral tissue expander In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\\nIn order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\\n Late complications (three months after primary reconstruction) Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\\nThree months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\",\n \"Data on postoperative pain are presented in Table \\n3.\\nPain in the local anaesthetic group and the standard group of patients\\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\",\n \"Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \\n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\\nOAA/S - observer's assessment of alertness/sedation.\",\n \"Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\",\n \"No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\",\n \"The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\",\n \"In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\",\n \"Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\",\n \"Ideally the study should have been placebo double-blinded to eliminate all biases. However, the cost of the elastomeric pump and wound catheter was 175 euro, so we were not able to perform a double-blind study. However, the cost of an elastomeric pump and wound catheter was not the only reason that precluded us from performing such a study. We had also ethical reasons for not carrying out a placebo-controlled study. Namely, a reconstruction with a tissue expander is a very painful procedure for the majority of patients, so we felt that it is not ethical to have a control group treated by placebo only. Instead, we offered our control group of patients the best available treatment, that is, intravenous analgesia. Based on the good results of our preliminary data on the use of continuous local anaesthetics for treatment of pain, our patients were willing to participate in our study because they were offered state-of-the-art pain treatment versus potentially even more effective pain treatment.\\nSub-pectoral breast augmentation and breast reconstruction with an expander or an implant are associated with considerable pain in the early postoperative period\\n[6,9,10,20-24]. Legeby et al.\\n[6] found that pain in the first 3 h after immediate breast reconstruction with a tissue expander was more severe than pain after mastectomy or axillary dissection. The mean VAS value for pain in the first 3 h after breast reconstruction was 4.9 and 5.0, if axillary dissection was also performed, whereas the values were 2.1 for mastectomy and 3.0 for mastectomy with axillary dissection\\n[6]. Similarly, after tissue-expander implantation, our standard analgesia group of patients also suffered pain in the recovery room, which was by about 3.0 VAS units larger than in patients who underwent breast surgery with axillary dissection without reconstruction, as reported by Strazisar et al.\\n[14]. The mean VAS score after immediate reconstruction was 4.9 in the standard analgesia group, whereas it was only 3.4 in the local anaesthetic group. In our previous study, the mean VAS score in recovery after breast cancer surgery with axillary dissection was 2.0 in the standard control group and 0.5 in the local anaesthetic group\\n[14]. In both our studies, the local anaesthetic reduced pain by 1.5 VAS units more than standard treatment alone.\\nThe most positive result of our study is the observation that 77% of patients treated with local anaesthetics did not experience severe pain. To our knowledge, there are no reports in the literature about similarly effective early postoperative analgesia after the implantation of a breast-tissue expander. Legeby et al. reported that even 48% of patients treated with local anaesthetics experienced unacceptably severe pain (VAS 4.7 to 10.0) in the early postoperative period\\n[6]. Legeby et al. used an epidural catheter with only a few holes at its tip and instilled local anaesthetic every 3 h. They probably did not establish an equal distribution of the local anaesthetic along the entire length of the muscle and the wound. On the other hand, we used quality wound-catheters that were perforated 15 cm at the distal end, which enabled a better distribution of the local anaesthetic. Furthermore, we used an elastomeric pump, which enabled a continuous infusion of the local anaesthetic. That is probably the reason why only seven patients (23.1%) in the test group reported pain above a VAS score of 5.0, whereas in the control group, such pain levels were reported by thirteen patients (42.9%).\\nLegeby et al. found that the use of opioids was about three times higher after the implantation of a tissue expander compared to other breast surgical procedures\\n[6]. After the tissue-expander implantation with or without axillary dissection, the consumption of opioids was 12.8 mg and 11.8 mg, whereas after mastectomy or mastectomy with axillary dissection, the mean consumption of opioids was 3.2 mg and 5.5 mg, respectively\\n[6]. Our patients with standard analgesia after the tissue-expander implantation needed 5.6 times more opioids than patients who underwent axillary dissection alone\\n[14]. Local anaesthetics can reduce consumption of opioids after mastectomy and axillary dissection\\n[11,13,14] and also after breast augmentation with implants\\n[7]. Our study shows that the use of continuous local anaesthesia can effectively reduce opioid consumption. Our patients treated with a local anaesthetic needed about three times less opioids than the standard analgesia group. Subsequently, patients from the local anaesthesia group experienced nausea less often, thus they had a lower consumption of metoclopramide than the standard analgesia group. Furthermore, the use of local anaesthetics resulted in less sedated and more alert patients in the recovery room and 6 h after the operation. The complication rate did not differ between the two groups of patients. This is in agreement with the reports of other authors who used local anaesthetics\\n[11-13,18,21,25-30]. Some authors report that local anaesthetics appear to reduce the incidence of inflammation\\n[31-33].\\nThere was no difference in pain on the first postoperative day between the study and control group of patients. It means that local anaesthesia was no longer as effective as it was on the day of surgery. A possible explanation is that muscle overstretching caused by a tissue expander is no longer as painful as it was. Other explanations are that in some patients the position of the wound catheter might have changed during movement or that there was a different perfusion region in the erect, prone or supine position, or that continuous infusion was flawed because of the wound drainage. Unfortunately, in our study we did not test a rescue bolus-injection of levobupivacaine in the case of severe pain. However, we have good experience with application of a rescue bolus of levobupivacaine in patients with neuropathic pain after axillary lymphadenectomy. Many times we have observed that injection of a bolus of 15 mL of 0.25% levobupivacaine into a drain and clamping of wound drainage inhibited neuropathic pain for a couple of hours.\\nMemory of severe postoperative pain is the most important risk factor for the development of chronic pain\\n[34]. Patients who were treated with patient-controlled analgesia had three times less chronic pain than the traditionally nurse-administered patients\\n[6]. The results of our study support this observation. Only 17% of patients treated with a local anaesthetic experienced chronic pain. This is significantly less than in the standard analgesia group of patients, of whom 50% suffered chronic pain. The proportion of patients treated with axillary lymphadenectomy, chemotherapy, hormone therapy and/or irradiation was similar in both study arms. Hence, we think that inadequately treated acute pain had an impact on the frequency of chronic pain in the standard analgesia group.\",\n \"Our current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia.\",\n \"BMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.\",\n \"The authors declare that they have no competing interests.\",\n \"BS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,"results",null,null,null,null,null,null,null,"discussion","conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Breast carcinoma","Primary reconstruction with tissue expander","Pain treatment","Wound infusion of local anaesthetic","Elastomeric pump"],"string":"[\n \"Breast carcinoma\",\n \"Primary reconstruction with tissue expander\",\n \"Pain treatment\",\n \"Wound infusion of local anaesthetic\",\n \"Elastomeric pump\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nBreast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.\n\nMethods:\nAltogether, 60 breast carcinoma patients were enrolled in the study protocol between January 2011 and May 2012: 72 patients who were eligible for inclusion were offered the opportunity to participate in our study during the recruitment period; 12 of them declined participation, and the remaining 60 were included in the study. Patients listed for mastectomy and primary reconstruction with a tissue expander because of the presence of breast carcinoma or for prophylactic mastectomy, were randomized during the routine preoperative anaesthetic assessment. Anaesthesia for the procedure was the same for all study subjects. We used the same protocol as already reported for patients with axillary dissection\n[14]. The first study arm was treated with continuous local analgesia, and the other (control) arm received standard intravenous analgesia. Allergies to local anaesthetics and chronic pre-operative opioid consumption were the criteria to exclude patients from our study.\nAfter informed consent was obtained the randomization was made by research nurses from the Clinical Research Unit of the Institute of Oncology Ljubljana. The research nurse performed randomization using random numbers generated by a computer and then informed the principal investigator about the treatment allocation of the patient.\nSurgical procedures were performed by ten experienced oncology surgeons and ten plastic surgeons. Sentinel lymph node biopsy was performed in 55 patients. In one patient with positive imprint cytology of the sentinel lymph node, axillary lymphadenectomy was performed immediately.\nThe study was reviewed by the The National Medical Ethics Committee of the Republic of Slovenia and performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. Our study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and conducted with the understanding and consent of all subjects involved. Study was entirely financed by the Institute of Oncology as a part of public service.\n Test group of patients Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\nBefore wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\n Control group of patients The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\nThe control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\n Both groups of patients Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\nPatient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\n Pain measurement Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\nData about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\n Statistical analysis In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\nIn order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\n\nTest group of patients:\nBefore wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\n\nControl group of patients:\nThe control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\n\nBoth groups of patients:\nPatient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\n\nPain measurement:\nData about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\n\nStatistical analysis:\nIn order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\n\nResults:\nThe mean age of patients was 47.8 years (range 25 to 64 y), height was 166 cm (range 153 to 178 cm), weight was 61.7 kg (range 45 to 85 kg), and the body mass index (BMI) was 22.4 (range 17 to 31.2). There were no significant differences between the study groups in BMI, American Society of Anesthesiology (ASA) score, comorbidities (Table \n1), type and length of surgical procedure, extent of lymph node dissection, adjuvant therapy, complications, or hospital stay (Table \n2).\nPatient characteristics\nASA, American Society of Anesthesiology; HER-2, human epidermal growth factor receptor-2; n, number of patients.\nTreatment of patients, adjuvant therapy, hospital stay and complications\nValues are numbers of patients unless stated otherwise.\n Acute pain Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\nData on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\n Opioid consumption Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\nConsumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\n Nausea Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\nPatients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\n Complications No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\nNo local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\n Hospital stay The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\nThe average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\n Unilateral and bilateral tissue expander In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\nIn order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\n Late complications (three months after primary reconstruction) Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\nThree months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\n\nAcute pain:\nData on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\n\nOpioid consumption:\nConsumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\n\nNausea:\nPatients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\n\nComplications:\nNo local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\n\nHospital stay:\nThe average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\n\nUnilateral and bilateral tissue expander:\nIn order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\n\nLate complications (three months after primary reconstruction):\nThree months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\n\nDiscussion:\nIdeally the study should have been placebo double-blinded to eliminate all biases. However, the cost of the elastomeric pump and wound catheter was 175 euro, so we were not able to perform a double-blind study. However, the cost of an elastomeric pump and wound catheter was not the only reason that precluded us from performing such a study. We had also ethical reasons for not carrying out a placebo-controlled study. Namely, a reconstruction with a tissue expander is a very painful procedure for the majority of patients, so we felt that it is not ethical to have a control group treated by placebo only. Instead, we offered our control group of patients the best available treatment, that is, intravenous analgesia. Based on the good results of our preliminary data on the use of continuous local anaesthetics for treatment of pain, our patients were willing to participate in our study because they were offered state-of-the-art pain treatment versus potentially even more effective pain treatment.\nSub-pectoral breast augmentation and breast reconstruction with an expander or an implant are associated with considerable pain in the early postoperative period\n[6,9,10,20-24]. Legeby et al.\n[6] found that pain in the first 3 h after immediate breast reconstruction with a tissue expander was more severe than pain after mastectomy or axillary dissection. The mean VAS value for pain in the first 3 h after breast reconstruction was 4.9 and 5.0, if axillary dissection was also performed, whereas the values were 2.1 for mastectomy and 3.0 for mastectomy with axillary dissection\n[6]. Similarly, after tissue-expander implantation, our standard analgesia group of patients also suffered pain in the recovery room, which was by about 3.0 VAS units larger than in patients who underwent breast surgery with axillary dissection without reconstruction, as reported by Strazisar et al.\n[14]. The mean VAS score after immediate reconstruction was 4.9 in the standard analgesia group, whereas it was only 3.4 in the local anaesthetic group. In our previous study, the mean VAS score in recovery after breast cancer surgery with axillary dissection was 2.0 in the standard control group and 0.5 in the local anaesthetic group\n[14]. In both our studies, the local anaesthetic reduced pain by 1.5 VAS units more than standard treatment alone.\nThe most positive result of our study is the observation that 77% of patients treated with local anaesthetics did not experience severe pain. To our knowledge, there are no reports in the literature about similarly effective early postoperative analgesia after the implantation of a breast-tissue expander. Legeby et al. reported that even 48% of patients treated with local anaesthetics experienced unacceptably severe pain (VAS 4.7 to 10.0) in the early postoperative period\n[6]. Legeby et al. used an epidural catheter with only a few holes at its tip and instilled local anaesthetic every 3 h. They probably did not establish an equal distribution of the local anaesthetic along the entire length of the muscle and the wound. On the other hand, we used quality wound-catheters that were perforated 15 cm at the distal end, which enabled a better distribution of the local anaesthetic. Furthermore, we used an elastomeric pump, which enabled a continuous infusion of the local anaesthetic. That is probably the reason why only seven patients (23.1%) in the test group reported pain above a VAS score of 5.0, whereas in the control group, such pain levels were reported by thirteen patients (42.9%).\nLegeby et al. found that the use of opioids was about three times higher after the implantation of a tissue expander compared to other breast surgical procedures\n[6]. After the tissue-expander implantation with or without axillary dissection, the consumption of opioids was 12.8 mg and 11.8 mg, whereas after mastectomy or mastectomy with axillary dissection, the mean consumption of opioids was 3.2 mg and 5.5 mg, respectively\n[6]. Our patients with standard analgesia after the tissue-expander implantation needed 5.6 times more opioids than patients who underwent axillary dissection alone\n[14]. Local anaesthetics can reduce consumption of opioids after mastectomy and axillary dissection\n[11,13,14] and also after breast augmentation with implants\n[7]. Our study shows that the use of continuous local anaesthesia can effectively reduce opioid consumption. Our patients treated with a local anaesthetic needed about three times less opioids than the standard analgesia group. Subsequently, patients from the local anaesthesia group experienced nausea less often, thus they had a lower consumption of metoclopramide than the standard analgesia group. Furthermore, the use of local anaesthetics resulted in less sedated and more alert patients in the recovery room and 6 h after the operation. The complication rate did not differ between the two groups of patients. This is in agreement with the reports of other authors who used local anaesthetics\n[11-13,18,21,25-30]. Some authors report that local anaesthetics appear to reduce the incidence of inflammation\n[31-33].\nThere was no difference in pain on the first postoperative day between the study and control group of patients. It means that local anaesthesia was no longer as effective as it was on the day of surgery. A possible explanation is that muscle overstretching caused by a tissue expander is no longer as painful as it was. Other explanations are that in some patients the position of the wound catheter might have changed during movement or that there was a different perfusion region in the erect, prone or supine position, or that continuous infusion was flawed because of the wound drainage. Unfortunately, in our study we did not test a rescue bolus-injection of levobupivacaine in the case of severe pain. However, we have good experience with application of a rescue bolus of levobupivacaine in patients with neuropathic pain after axillary lymphadenectomy. Many times we have observed that injection of a bolus of 15 mL of 0.25% levobupivacaine into a drain and clamping of wound drainage inhibited neuropathic pain for a couple of hours.\nMemory of severe postoperative pain is the most important risk factor for the development of chronic pain\n[34]. Patients who were treated with patient-controlled analgesia had three times less chronic pain than the traditionally nurse-administered patients\n[6]. The results of our study support this observation. Only 17% of patients treated with a local anaesthetic experienced chronic pain. This is significantly less than in the standard analgesia group of patients, of whom 50% suffered chronic pain. The proportion of patients treated with axillary lymphadenectomy, chemotherapy, hormone therapy and/or irradiation was similar in both study arms. Hence, we think that inadequately treated acute pain had an impact on the frequency of chronic pain in the standard analgesia group.\n\nConclusions:\nOur current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia.\n\nAbbreviations:\nBMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nBS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nImmediate breast reconstruction with an expander is a reasonable option for properly selected patients. After reconstruction, patients have severe postoperative pain, which responds poorly to opioids. Our aim was to evaluate if continuous wound infusion of a local anaesthetic into the surgical wound reduces postoperative pain, consumption of opioids and incidence of chronic pain compared to standard intravenous piritramide after primary breast reconstruction in breast carcinoma patients.\n\nMethods:\nAltogether, 60 patients were enrolled in our study; one half in the group with wound infusion of a local anaesthetic, and the other half in the standard (piritramide) group. Parameters measured included: pain intensity (visual analogue scale), drug requirements, alertness, hospitalisation, side-effects and late complications. A p-value of < 0.05 was considered statistically significant.\n\nResults:\nIn the recovery room, the test group reported less acute pain at rest (P = 0.03) and at activity (P = 0.01), and on the day of the surgical procedure they reported less pain at activity (P = 0.003). Consumption of piritramide and metoclopramide was lower in this group (P < 0.0001), but their alertness after the surgical procedure was higher compared to the standard group (P < 0.001). After three months, the test group reported less chronic pain (P = 0.01).\n\nConclusions:\nAfter primary tissue expander breast reconstruction, wound infusion of a local anaesthetic significantly reduces acute pain and enables reduced opioid consumption, resulting in less postoperative sedation and reduced need for antiemetic drugs. Wound infusion of a local anaesthetic reduces chronic pain."},"background_conclusion_text":{"kind":"string","value":"Background:\nBreast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.\n\nConclusions:\nOur current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nImmediate breast reconstruction with an expander is a reasonable option for properly selected patients. After reconstruction, patients have severe postoperative pain, which responds poorly to opioids. Our aim was to evaluate if continuous wound infusion of a local anaesthetic into the surgical wound reduces postoperative pain, consumption of opioids and incidence of chronic pain compared to standard intravenous piritramide after primary breast reconstruction in breast carcinoma patients.\n\nMethods:\nAltogether, 60 patients were enrolled in our study; one half in the group with wound infusion of a local anaesthetic, and the other half in the standard (piritramide) group. Parameters measured included: pain intensity (visual analogue scale), drug requirements, alertness, hospitalisation, side-effects and late complications. A p-value of < 0.05 was considered statistically significant.\n\nResults:\nIn the recovery room, the test group reported less acute pain at rest (P = 0.03) and at activity (P = 0.01), and on the day of the surgical procedure they reported less pain at activity (P = 0.003). Consumption of piritramide and metoclopramide was lower in this group (P < 0.0001), but their alertness after the surgical procedure was higher compared to the standard group (P < 0.001). After three months, the test group reported less chronic pain (P = 0.01).\n\nConclusions:\nAfter primary tissue expander breast reconstruction, wound infusion of a local anaesthetic significantly reduces acute pain and enables reduced opioid consumption, resulting in less postoperative sedation and reduced need for antiemetic drugs. Wound infusion of a local anaesthetic reduces chronic pain."},"whole_article_text_length":{"kind":"number","value":7759,"string":"7,759"},"whole_article_abstract_length":{"kind":"number","value":307,"string":"307"},"num_sections":{"kind":"number","value":20,"string":"20"},"most_frequent_words":{"kind":"list like","value":["patients","group","pain","local","control","test","surgical","reconstruction","wound","standard"],"string":"[\n \"patients\",\n \"group\",\n \"pain\",\n \"local\",\n \"control\",\n \"test\",\n \"surgical\",\n \"reconstruction\",\n \"wound\",\n \"standard\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] breast | local | use | mastectomy | pain | anaesthetics | local anaesthetics | breast reconstruction | postoperative pain | reconstruction [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | pain | catheter | wound | mg | subjects | ml | intravenous | fenestrated | day [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] group | patients | unilateral | bilateral | test | control group | test group | control | reconstruction | standard group [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients treated | surgical | pain | treated | lower | local anaesthetic | anaesthetic | surgical procedure | procedure | local [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] group | patients | pain | test | control | surgical | local | surgical procedure | standard | procedure [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] group | patients | pain | test | control | surgical | local | surgical procedure | standard | procedure [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 60 | one half | half ||| ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 0.03 | 0.01 | the day | 0.003 ||| ||| three months | 0.01 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| 60 | one half | half ||| ||| ||| ||| 0.03 | 0.01 | the day | 0.003 ||| ||| three months | 0.01 ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| 60 | one half | half ||| ||| ||| ||| 0.03 | 0.01 | the day | 0.003 ||| ||| three months | 0.01 ||| ||| [SUMMARY]"}}},{"rowIdx":276,"cells":{"title":{"kind":"string","value":"Lung transplantation as therapeutic option in acute respiratory distress syndrome for coronavirus disease 2019-related pulmonary fibrosis."},"pmid":{"kind":"string","value":"32251003"},"background_abstract":{"kind":"string","value":"Critical patients with the coronavirus disease 2019 (COVID-19), even those whose nucleic acid test results had turned negative and those receiving maximal medical support, have been noted to progress to irreversible fatal respiratory failure. Lung transplantation (LT) as the sole therapy for end-stage pulmonary fibrosis related to acute respiratory distress syndrome has been considered as the ultimate rescue therapy for these patients."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"From February 10 to March 10, 2020, three male patients were urgently assessed and listed for transplantation. After conducting a full ethical review and after obtaining assent from the family of the patients, we performed three LT procedures for COVID-19 patients with illness durations of more than one month and extremely high sequential organ failure assessment scores."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Two of the three recipients survived post-LT and started participating in a rehabilitation program. Pearls of the LT team collaboration and perioperative logistics were summarized and continually improved. The pathological results of the explanted lungs were concordant with the critical clinical manifestation, and provided insight towards better understanding of the disease. Government health affair systems, virology detection tools, and modern communication technology all play key roles towards the survival of the patients and their rehabilitation."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"LT can be performed in end-stage patients with respiratory failure due to COVID-19-related pulmonary fibrosis. If confirmed positive-turned-negative virology status without organ dysfunction that could contraindicate LT, LT provided the final option for these patients to avoid certain death, with proper protection of transplant surgeons and medical staffs. By ensuring instant seamless care for both patients and medical teams, the goal of reducing the mortality rate and salvaging the lives of patients with COVID-19 can be attained."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Betacoronavirus","COVID-19","Coronavirus Infections","Extracorporeal Membrane Oxygenation","Humans","Lung Transplantation","Male","Middle Aged","Pandemics","Pneumonia, Viral","Pulmonary Fibrosis","Respiratory Distress Syndrome","SARS-CoV-2"],"string":"[\n \"Aged\",\n \"Betacoronavirus\",\n \"COVID-19\",\n \"Coronavirus Infections\",\n \"Extracorporeal Membrane Oxygenation\",\n \"Humans\",\n \"Lung Transplantation\",\n \"Male\",\n \"Middle Aged\",\n \"Pandemics\",\n \"Pneumonia, Viral\",\n \"Pulmonary Fibrosis\",\n \"Respiratory Distress Syndrome\",\n \"SARS-CoV-2\"\n]"},"pmcid":{"kind":"string","value":"7339336"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"The outbreak of the coronavirus disease 2019 (COVID-19) caused more than 200,000 reported cases and more than 8000 people have lost their lives,[1] and thus, was declared a global pandemic by the World Health Organization.[2] As reported by Zhong et al,[3] 5.0% of 1099 patients were admitted to the intensive care unit (ICU) and 2.3% received invasive mechanical ventilation (MV). Publications have reported the general characteristics of disease progression with analysis of outcomes, including those of the recovered and mortality cases.[4] Some patients have prolonged ICU stays due to severe complications, such as acute respiratory distress syndrome (ARDS) and related pulmonary fibrosis, even after results of repeated virological tests are confirmed to be negative. Even with maximal support with MV and extra-corporeal membrane oxygenation (ECMO), such patients showed irreversible deterioration of pulmonary function. We performed lung transplantation (LT) for end-stage patients and hereby present the early results of LT in these recipients. Issues related to the urgent candidate listing of post-COVID-19 critical patients and peri-LT management are further discussed."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Ethical approval The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\nThe lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\n Patient information and data collection This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\nThis is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\n Construction of surgical facilities with high protection level In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\nIn view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" General characteristics before LT All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\nAll the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\n Pearls of peri-operative management All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\nAll the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\n Post-LT survival status and explant pathology Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.\nBoth patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Ethical approval","Patient information and data collection","Construction of surgical facilities with high protection level","General characteristics before LT","Pearls of peri-operative management","Post-LT survival status and explant pathology","Key points on determination of LT candidacy","Best practices for the protection of the medical team involved","Factors important for post-LT survival","Perspective","Funding"],"string":"[\n \"Ethical approval\",\n \"Patient information and data collection\",\n \"Construction of surgical facilities with high protection level\",\n \"General characteristics before LT\",\n \"Pearls of peri-operative management\",\n \"Post-LT survival status and explant pathology\",\n \"Key points on determination of LT candidacy\",\n \"Best practices for the protection of the medical team involved\",\n \"Factors important for post-LT survival\",\n \"Perspective\",\n \"Funding\"\n]"},"other_sections_texts":{"kind":"list like","value":["The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]","This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.","In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.","All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.","All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.","Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.","The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.","For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.","During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.","With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.","This study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City."],"string":"[\n \"The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\",\n \"This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\",\n \"In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\",\n \"All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\\nGeneral characteristics of the three urgently listed LT recipients.\",\n \"All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\",\n \"Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\\nRepresentative comparison of explant pathologies.\",\n \"The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\",\n \"For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\\nConsiderations and comparisons of surgical preparations.\\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\",\n \"During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\",\n \"With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\",\n \"This study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,"subjects",null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"subjects\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Ethical approval","Patient information and data collection","Construction of surgical facilities with high protection level","Results","General characteristics before LT","Pearls of peri-operative management","Post-LT survival status and explant pathology","Discussion","Best time to perform transplantation in critical patients","Key points on determination of LT candidacy","Best practices for the protection of the medical team involved","Factors important for post-LT survival","Perspective","Funding","Conflicts of interest"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Ethical approval\",\n \"Patient information and data collection\",\n \"Construction of surgical facilities with high protection level\",\n \"Results\",\n \"General characteristics before LT\",\n \"Pearls of peri-operative management\",\n \"Post-LT survival status and explant pathology\",\n \"Discussion\",\n \"Best time to perform transplantation in critical patients\",\n \"Key points on determination of LT candidacy\",\n \"Best practices for the protection of the medical team involved\",\n \"Factors important for post-LT survival\",\n \"Perspective\",\n \"Funding\",\n \"Conflicts of interest\"\n]"},"all_sections_texts":{"kind":"list like","value":["The outbreak of the coronavirus disease 2019 (COVID-19) caused more than 200,000 reported cases and more than 8000 people have lost their lives,[1] and thus, was declared a global pandemic by the World Health Organization.[2] As reported by Zhong et al,[3] 5.0% of 1099 patients were admitted to the intensive care unit (ICU) and 2.3% received invasive mechanical ventilation (MV). Publications have reported the general characteristics of disease progression with analysis of outcomes, including those of the recovered and mortality cases.[4] Some patients have prolonged ICU stays due to severe complications, such as acute respiratory distress syndrome (ARDS) and related pulmonary fibrosis, even after results of repeated virological tests are confirmed to be negative. Even with maximal support with MV and extra-corporeal membrane oxygenation (ECMO), such patients showed irreversible deterioration of pulmonary function. We performed lung transplantation (LT) for end-stage patients and hereby present the early results of LT in these recipients. Issues related to the urgent candidate listing of post-COVID-19 critical patients and peri-LT management are further discussed."," Ethical approval The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\nThe lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\n Patient information and data collection This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\nThis is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\n Construction of surgical facilities with high protection level In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\nIn view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.","The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]","This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.","In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems."," General characteristics before LT All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\nAll the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\n Pearls of peri-operative management All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\nAll the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\n Post-LT survival status and explant pathology Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.\nBoth patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.","All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.","All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.","Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies."," Best time to perform transplantation in critical patients This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\nThis is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\n Key points on determination of LT candidacy The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\nThe following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\n Best practices for the protection of the medical team involved For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\nFor the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\n Factors important for post-LT survival During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\nDuring the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\n Perspective With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\nWith careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.","This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.","The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.","For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.","During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.","With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.","This study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City.","None."],"string":"[\n \"The outbreak of the coronavirus disease 2019 (COVID-19) caused more than 200,000 reported cases and more than 8000 people have lost their lives,[1] and thus, was declared a global pandemic by the World Health Organization.[2] As reported by Zhong et al,[3] 5.0% of 1099 patients were admitted to the intensive care unit (ICU) and 2.3% received invasive mechanical ventilation (MV). Publications have reported the general characteristics of disease progression with analysis of outcomes, including those of the recovered and mortality cases.[4] Some patients have prolonged ICU stays due to severe complications, such as acute respiratory distress syndrome (ARDS) and related pulmonary fibrosis, even after results of repeated virological tests are confirmed to be negative. Even with maximal support with MV and extra-corporeal membrane oxygenation (ECMO), such patients showed irreversible deterioration of pulmonary function. We performed lung transplantation (LT) for end-stage patients and hereby present the early results of LT in these recipients. Issues related to the urgent candidate listing of post-COVID-19 critical patients and peri-LT management are further discussed.\",\n \" Ethical approval The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\\nThe lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\\n Patient information and data collection This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\\nThis is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\\n Construction of surgical facilities with high protection level In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\\nIn view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\",\n \"The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\",\n \"This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\",\n \"In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\",\n \" General characteristics before LT All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\\nGeneral characteristics of the three urgently listed LT recipients.\\nAll the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\\nGeneral characteristics of the three urgently listed LT recipients.\\n Pearls of peri-operative management All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\\nAll the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\\n Post-LT survival status and explant pathology Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\\nRepresentative comparison of explant pathologies.\\nBoth patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\\nRepresentative comparison of explant pathologies.\",\n \"All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\\nGeneral characteristics of the three urgently listed LT recipients.\",\n \"All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\",\n \"Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\\nRepresentative comparison of explant pathologies.\",\n \" Best time to perform transplantation in critical patients This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\\nThis is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\\n Key points on determination of LT candidacy The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\\nThe following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\\n Best practices for the protection of the medical team involved For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\\nConsiderations and comparisons of surgical preparations.\\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\\nFor the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\\nConsiderations and comparisons of surgical preparations.\\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\\n Factors important for post-LT survival During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\\nDuring the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\\n Perspective With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\\nWith careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\",\n \"This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\",\n \"The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\",\n \"For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\\nConsiderations and comparisons of surgical preparations.\\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\",\n \"During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\",\n \"With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\",\n \"This study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City.\",\n \"None.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,"subjects",null,"results",null,null,null,"discussion","subjects",null,null,null,null,null,"COI-statement"],"string":"[\n \"intro\",\n \"methods\",\n null,\n \"subjects\",\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n \"subjects\",\n null,\n null,\n null,\n null,\n null,\n \"COI-statement\"\n]"},"keywords":{"kind":"list like","value":["Coronavirus disease 2019","Lung transplantation","Acute respiratory distress syndrome","Pulmonary fibrosis","Sequential Organ Failure Assessment score"],"string":"[\n \"Coronavirus disease 2019\",\n \"Lung transplantation\",\n \"Acute respiratory distress syndrome\",\n \"Pulmonary fibrosis\",\n \"Sequential Organ Failure Assessment score\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nThe outbreak of the coronavirus disease 2019 (COVID-19) caused more than 200,000 reported cases and more than 8000 people have lost their lives,[1] and thus, was declared a global pandemic by the World Health Organization.[2] As reported by Zhong et al,[3] 5.0% of 1099 patients were admitted to the intensive care unit (ICU) and 2.3% received invasive mechanical ventilation (MV). Publications have reported the general characteristics of disease progression with analysis of outcomes, including those of the recovered and mortality cases.[4] Some patients have prolonged ICU stays due to severe complications, such as acute respiratory distress syndrome (ARDS) and related pulmonary fibrosis, even after results of repeated virological tests are confirmed to be negative. Even with maximal support with MV and extra-corporeal membrane oxygenation (ECMO), such patients showed irreversible deterioration of pulmonary function. We performed lung transplantation (LT) for end-stage patients and hereby present the early results of LT in these recipients. Issues related to the urgent candidate listing of post-COVID-19 critical patients and peri-LT management are further discussed.\n\nMethods:\n Ethical approval The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\nThe lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\n Patient information and data collection This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\nThis is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\n Construction of surgical facilities with high protection level In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\nIn view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\n\nEthical approval:\nThe lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\n\nPatient information and data collection:\nThis is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\n\nConstruction of surgical facilities with high protection level:\nIn view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\n\nResults:\n General characteristics before LT All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\nAll the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\n Pearls of peri-operative management All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\nAll the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\n Post-LT survival status and explant pathology Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.\nBoth patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.\n\nGeneral characteristics before LT:\nAll the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\n\nPearls of peri-operative management:\nAll the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\n\nPost-LT survival status and explant pathology:\nBoth patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.\n\nDiscussion:\n Best time to perform transplantation in critical patients This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\nThis is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\n Key points on determination of LT candidacy The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\nThe following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\n Best practices for the protection of the medical team involved For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\nFor the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\n Factors important for post-LT survival During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\nDuring the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\n Perspective With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\nWith careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\n\nBest time to perform transplantation in critical patients:\nThis is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\n\nKey points on determination of LT candidacy:\nThe following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\n\nBest practices for the protection of the medical team involved:\nFor the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\n\nFactors important for post-LT survival:\nDuring the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\n\nPerspective:\nWith careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\n\nFunding:\nThis study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City.\n\nConflicts of interest:\nNone.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nCritical patients with the coronavirus disease 2019 (COVID-19), even those whose nucleic acid test results had turned negative and those receiving maximal medical support, have been noted to progress to irreversible fatal respiratory failure. Lung transplantation (LT) as the sole therapy for end-stage pulmonary fibrosis related to acute respiratory distress syndrome has been considered as the ultimate rescue therapy for these patients.\n\nMethods:\nFrom February 10 to March 10, 2020, three male patients were urgently assessed and listed for transplantation. After conducting a full ethical review and after obtaining assent from the family of the patients, we performed three LT procedures for COVID-19 patients with illness durations of more than one month and extremely high sequential organ failure assessment scores.\n\nResults:\nTwo of the three recipients survived post-LT and started participating in a rehabilitation program. Pearls of the LT team collaboration and perioperative logistics were summarized and continually improved. The pathological results of the explanted lungs were concordant with the critical clinical manifestation, and provided insight towards better understanding of the disease. Government health affair systems, virology detection tools, and modern communication technology all play key roles towards the survival of the patients and their rehabilitation.\n\nConclusions:\nLT can be performed in end-stage patients with respiratory failure due to COVID-19-related pulmonary fibrosis. If confirmed positive-turned-negative virology status without organ dysfunction that could contraindicate LT, LT provided the final option for these patients to avoid certain death, with proper protection of transplant surgeons and medical staffs. By ensuring instant seamless care for both patients and medical teams, the goal of reducing the mortality rate and salvaging the lives of patients with COVID-19 can be attained."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":10633,"string":"10,633"},"whole_article_abstract_length":{"kind":"number","value":324,"string":"324"},"num_sections":{"kind":"number","value":17,"string":"17"},"most_frequent_words":{"kind":"list like","value":["lt","patients","patient","post","lung","covid","covid 19","19","negative","team"],"string":"[\n \"lt\",\n \"patients\",\n \"patient\",\n \"post\",\n \"lung\",\n \"covid\",\n \"covid 19\",\n \"19\",\n \"negative\",\n \"team\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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[SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patient | pod | lt | patients | left | heart | ecmo | post | day | operative [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] lt | patients | patient | post | day | 19 | covid | covid 19 | lung | ecmo [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] 2019 | COVID-19 ||| Lung [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] February 10 to March 10, 2020 | three ||| three | COVID-19 | more than one month [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Two | three ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] 2019 | COVID-19 ||| Lung ||| February 10 to March 10, 2020 | three ||| three | COVID-19 | more than one month ||| Two | three ||| ||| ||| ||| COVID-19 ||| ||| COVID-19 [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":277,"cells":{"title":{"kind":"string","value":"A proposed adaptation of the European Foundation for Quality Management Excellence Model to physical activity programmes for the elderly - development of a quality self-assessment tool using a modified Delphi process."},"pmid":{"kind":"string","value":"21958203"},"background_abstract":{"kind":"string","value":"There has been a growing concern in designing physical activity (PA) programmes for elderly people, since evidence suggests that such health promotion interventions may reduce the deleterious effects of the ageing process. Complete programme evaluations are a necessary prerequisite to continuous quality improvements. Being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly in order to support its continuous improvement is, therefore, crucial. Thus, the aim of this study was to develop a self-assessment tool for PA programmes for the elderly."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A 3-round Delphi process was conducted via the Internet with 43 national experts in PA for the elderly, management and delivery of PA programmes for the elderly, sports management, quality management and gerontology, asking experts to identify the propositions that they considered relevant for inclusion in the self-assessment tool. Experts reviewed a list of proposed statements, based on the criteria and sub-criteria from the European Foundation for Quality Management Excellence Model (EFQM) and PA guidelines for older adults and rated each proposition from 1 to 8 (disagree to agree) and modified and/or added propositions. Propositions receiving either bottom or top scores of greater than 70% were considered to have achieved consensus to drop or retain, respectively."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93. In round 2, a total of 104 propositions were presented, of which experts modified 39 and achieved consensus on 53. In the last round, of 51 proposed statements, the experts achieved consensus on 19. After 3 rounds of rating, experts had not achieved consensus on 32 propositions. The resulting tool consisted of 165 statements that assess nine management areas involved in the development of PA programmes for the elderly."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Based on experts' opinions, a self-assessment tool was found in order to access quality of PA programmes for the elderly. Information obtained with evaluations would be useful to organizations seeking to improve their services, customer satisfaction and, consequently, adherence to PA programmes, targeting the ageing population."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Aging","Consensus","Delivery of Health Care","Delphi Technique","Europe","Exercise","Geriatrics","Health Promotion","Humans","Internet","Practice Guidelines as Topic","Program Evaluation","Quality Control","Sports"],"string":"[\n \"Aged\",\n \"Aging\",\n \"Consensus\",\n \"Delivery of Health Care\",\n \"Delphi Technique\",\n \"Europe\",\n \"Exercise\",\n \"Geriatrics\",\n \"Health Promotion\",\n \"Humans\",\n \"Internet\",\n \"Practice Guidelines as Topic\",\n \"Program Evaluation\",\n \"Quality Control\",\n \"Sports\"\n]"},"pmcid":{"kind":"string","value":"3278362"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"A modified Delphi process was conducted using the Internet, from October 2009 to September 2010. The Delphi technique was developed in the 1950s by scientists at the Rand Corporation as a method of making informed decisions based on expert opinion [23]. Since then, it has been used to clarify a variety of problems in different sectors [24-29]. Despite having undergone some modifications, it remains a viable approach for gathering expert opinions through a structured iterative process that builds consensus [30]. This process involves multiple interactions with participants who usually complete two or more rounds in a reasonable amount of time [31] - even when participants are in geographically-distinct locations, since rounds can be conducted by mail or email [32,33]. The results of previous iterations can be modified by participants in later iterations, as they are able to review comments and feedbacks provided by other experts in earlier rounds [31]. Furthermore, the Delphi technique offers a number of specific advantages and is particularly helpful because it avoids the barriers commonly observed in other group discussions, such as interpersonal influence, time pressure and group demands [31,34,35]. This is due to the fact that respondents are not aware of the identities of other respondents and are, therefore, freed of personal and social constraints [30]. They are also able to complete the Delphi rounds in ways that suit them best because they participate in the rounds asynchronously [36]. The Delphi technique is also advantageous because a variety of statistical analysis techniques can be used to interpret the data its generates [37].\nThe Delphi process was conducted in three rounds [38,39] (Figure 1). Following each step listed in the previous figure, our main question was: Which quality practices must be included in a quality self-assessment tool for PA programmes for the elderly?\nSteps of the modified Delphi process used in the present study.\nUsing criteria and sub-criteria from the EFQM Excellence Model [13] and PA guidelines for older adults [3,40] as a starting point, we reviewed the literature to identify best practice principles and generate a list of statements. Our review was undertaken using PubMed (1980-2010), B-On (1980-2010), and Google™. We searched a variety of combinations of key words related to PA programmes for the elderly, quality management and the EFQM Excellence Model, such as: 'evaluation', 'guidelines', 'recommendations', 'exercise', 'physical activity', 'programmes', 'elderly', 'old', 'review', 'framework', 'EFQM', 'assess' and 'quality'.\nAfter identifying a list of statements, an online questionnaire was developed and tested with 5 PA programme coordinators for comments on readability and functionality. Some adjustments were made to make the affirmations included in the questionnaire clearer and more relevant to this case. We established that statements that received greater than 70% of experts' votes had achieved consensus [41-43] in both the bottom scores (i.e., reached consensus to drop) and top scores (i.e., reached consensus to include/retain). Statements that were dropped were not included in subsequent rounds of ratings. The remaining items were included in the next rounds, until a consensus was achieved to either drop or retain. At the end of three rounds, the statements on which experts had not reached consensus were also not included in the output list.\nThe fourth phase of the process involved nominating experts to participate in the Delphi rounds. National experts in research on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology were identified. Our decisions were based on expertise or/and breadth of scientific work [44]. The DeGóis Curricula Platform1 assisted us in this process. A list of 63 potential participants was generated, along with key contacts for each. This group included 34 PhD scientists and academics (11 in PA for the elderly, 4 in sports management, 18 in quality management and 1 in gerontology), 3 non-PhD academics (1 in PA for the elderly and 2 in sports management) and 26 senior technicians (22 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). Previous information containing details about the EFQM Excellence Model, the Delphi process and the purpose of our study was provided. Of those invited to participate, 5 did not respond and 3 declined, due to lack of time (all PhD scientists and academics in quality management). Thus, 55 experts (30 females and 25 males) responded to our initial invitation and agreed to participate. Those who accepted our invitation were informed that they were required to respond to three online rounds of ratings.\nThe rounds were performed using Survey Monkey, a web-based survey and data collection system. In every round, participants were asked to rate their level of agreement with each proposition, from 1 to 8 ('strongly disagree' to 'strongly agree'), suggest modifications to proposed definitions and/or add propositions that would be useful in a quality self-assessment tool for PA programmes for the elderly. The 8-point Likert scale was selected to bring out more variability in responses [45]. After each round, the frequency and mean of the panel's ratings and the percentage of scores ≥ 7 were calculated. Based on this data, a new questionnaire was designed and placed online for the next round. We asked participants to review all the information sent and re-rate each statement.\nAfter round 3, we gathered all our data and developed a list of statements that did and did not reach consensus."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"1 It is an instrument for gathering, supplying and analyze the intellectual and scientific production of the Portuguese researchers."},"other_sections_titles":{"kind":"list like","value":["Background","Results","Discussion","Strengths and Limitations","Conclusion"],"string":"[\n \"Background\",\n \"Results\",\n \"Discussion\",\n \"Strengths and Limitations\",\n \"Conclusion\"\n]"},"other_sections_texts":{"kind":"list like","value":["Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.","Eight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\nResults of the three rounds by criterion\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\" to \"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).","The main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\" and \"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\nThroughout the Delphi process, it was suggested that the proposition \"The programme involves a multidisciplinary team of professionals\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \"external partnerships\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\" and \"Regular and formal communication procedures are established with partners\".\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \"Market research is used to determine the needs and expectations of future customers\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \"The programme has measures of perception and/or performance indicators regarding employees' performance\" and \"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.","To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.","Our Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results)."],"string":"[\n \"Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.\",\n \"Eight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\\nResults of the three rounds by criterion\\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \\\"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\\\" to \\\"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\\\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\",\n \"The main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \\\"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\\\" and \\\"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\\\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\\nThroughout the Delphi process, it was suggested that the proposition \\\"The programme involves a multidisciplinary team of professionals\\\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \\\"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\\\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \\\"external partnerships\\\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \\\"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\\\" and \\\"Regular and formal communication procedures are established with partners\\\".\\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \\\"Market research is used to determine the needs and expectations of future customers\\\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \\\"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\\\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \\\"The programme has measures of perception and/or performance indicators regarding employees' performance\\\" and \\\"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\\\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\",\n \"To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\",\n \"Our Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Results","Discussion","Strengths and Limitations","Conclusion","Supplementary Material"],"string":"[\n \"Background\",\n \"Methods\",\n \"Results\",\n \"Discussion\",\n \"Strengths and Limitations\",\n \"Conclusion\",\n \"Supplementary Material\"\n]"},"all_sections_texts":{"kind":"list like","value":["Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.","A modified Delphi process was conducted using the Internet, from October 2009 to September 2010. The Delphi technique was developed in the 1950s by scientists at the Rand Corporation as a method of making informed decisions based on expert opinion [23]. Since then, it has been used to clarify a variety of problems in different sectors [24-29]. Despite having undergone some modifications, it remains a viable approach for gathering expert opinions through a structured iterative process that builds consensus [30]. This process involves multiple interactions with participants who usually complete two or more rounds in a reasonable amount of time [31] - even when participants are in geographically-distinct locations, since rounds can be conducted by mail or email [32,33]. The results of previous iterations can be modified by participants in later iterations, as they are able to review comments and feedbacks provided by other experts in earlier rounds [31]. Furthermore, the Delphi technique offers a number of specific advantages and is particularly helpful because it avoids the barriers commonly observed in other group discussions, such as interpersonal influence, time pressure and group demands [31,34,35]. This is due to the fact that respondents are not aware of the identities of other respondents and are, therefore, freed of personal and social constraints [30]. They are also able to complete the Delphi rounds in ways that suit them best because they participate in the rounds asynchronously [36]. The Delphi technique is also advantageous because a variety of statistical analysis techniques can be used to interpret the data its generates [37].\nThe Delphi process was conducted in three rounds [38,39] (Figure 1). Following each step listed in the previous figure, our main question was: Which quality practices must be included in a quality self-assessment tool for PA programmes for the elderly?\nSteps of the modified Delphi process used in the present study.\nUsing criteria and sub-criteria from the EFQM Excellence Model [13] and PA guidelines for older adults [3,40] as a starting point, we reviewed the literature to identify best practice principles and generate a list of statements. Our review was undertaken using PubMed (1980-2010), B-On (1980-2010), and Google™. We searched a variety of combinations of key words related to PA programmes for the elderly, quality management and the EFQM Excellence Model, such as: 'evaluation', 'guidelines', 'recommendations', 'exercise', 'physical activity', 'programmes', 'elderly', 'old', 'review', 'framework', 'EFQM', 'assess' and 'quality'.\nAfter identifying a list of statements, an online questionnaire was developed and tested with 5 PA programme coordinators for comments on readability and functionality. Some adjustments were made to make the affirmations included in the questionnaire clearer and more relevant to this case. We established that statements that received greater than 70% of experts' votes had achieved consensus [41-43] in both the bottom scores (i.e., reached consensus to drop) and top scores (i.e., reached consensus to include/retain). Statements that were dropped were not included in subsequent rounds of ratings. The remaining items were included in the next rounds, until a consensus was achieved to either drop or retain. At the end of three rounds, the statements on which experts had not reached consensus were also not included in the output list.\nThe fourth phase of the process involved nominating experts to participate in the Delphi rounds. National experts in research on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology were identified. Our decisions were based on expertise or/and breadth of scientific work [44]. The DeGóis Curricula Platform1 assisted us in this process. A list of 63 potential participants was generated, along with key contacts for each. This group included 34 PhD scientists and academics (11 in PA for the elderly, 4 in sports management, 18 in quality management and 1 in gerontology), 3 non-PhD academics (1 in PA for the elderly and 2 in sports management) and 26 senior technicians (22 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). Previous information containing details about the EFQM Excellence Model, the Delphi process and the purpose of our study was provided. Of those invited to participate, 5 did not respond and 3 declined, due to lack of time (all PhD scientists and academics in quality management). Thus, 55 experts (30 females and 25 males) responded to our initial invitation and agreed to participate. Those who accepted our invitation were informed that they were required to respond to three online rounds of ratings.\nThe rounds were performed using Survey Monkey, a web-based survey and data collection system. In every round, participants were asked to rate their level of agreement with each proposition, from 1 to 8 ('strongly disagree' to 'strongly agree'), suggest modifications to proposed definitions and/or add propositions that would be useful in a quality self-assessment tool for PA programmes for the elderly. The 8-point Likert scale was selected to bring out more variability in responses [45]. After each round, the frequency and mean of the panel's ratings and the percentage of scores ≥ 7 were calculated. Based on this data, a new questionnaire was designed and placed online for the next round. We asked participants to review all the information sent and re-rate each statement.\nAfter round 3, we gathered all our data and developed a list of statements that did and did not reach consensus.","Eight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\nResults of the three rounds by criterion\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\" to \"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).","The main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\" and \"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\nThroughout the Delphi process, it was suggested that the proposition \"The programme involves a multidisciplinary team of professionals\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \"external partnerships\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\" and \"Regular and formal communication procedures are established with partners\".\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \"Market research is used to determine the needs and expectations of future customers\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \"The programme has measures of perception and/or performance indicators regarding employees' performance\" and \"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.","To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.","Our Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).","Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors). the file presents the resulting tool - named Q-STEPS - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly.\nClick here for file"],"string":"[\n \"Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.\",\n \"A modified Delphi process was conducted using the Internet, from October 2009 to September 2010. The Delphi technique was developed in the 1950s by scientists at the Rand Corporation as a method of making informed decisions based on expert opinion [23]. Since then, it has been used to clarify a variety of problems in different sectors [24-29]. Despite having undergone some modifications, it remains a viable approach for gathering expert opinions through a structured iterative process that builds consensus [30]. This process involves multiple interactions with participants who usually complete two or more rounds in a reasonable amount of time [31] - even when participants are in geographically-distinct locations, since rounds can be conducted by mail or email [32,33]. The results of previous iterations can be modified by participants in later iterations, as they are able to review comments and feedbacks provided by other experts in earlier rounds [31]. Furthermore, the Delphi technique offers a number of specific advantages and is particularly helpful because it avoids the barriers commonly observed in other group discussions, such as interpersonal influence, time pressure and group demands [31,34,35]. This is due to the fact that respondents are not aware of the identities of other respondents and are, therefore, freed of personal and social constraints [30]. They are also able to complete the Delphi rounds in ways that suit them best because they participate in the rounds asynchronously [36]. The Delphi technique is also advantageous because a variety of statistical analysis techniques can be used to interpret the data its generates [37].\\nThe Delphi process was conducted in three rounds [38,39] (Figure 1). Following each step listed in the previous figure, our main question was: Which quality practices must be included in a quality self-assessment tool for PA programmes for the elderly?\\nSteps of the modified Delphi process used in the present study.\\nUsing criteria and sub-criteria from the EFQM Excellence Model [13] and PA guidelines for older adults [3,40] as a starting point, we reviewed the literature to identify best practice principles and generate a list of statements. Our review was undertaken using PubMed (1980-2010), B-On (1980-2010), and Google™. We searched a variety of combinations of key words related to PA programmes for the elderly, quality management and the EFQM Excellence Model, such as: 'evaluation', 'guidelines', 'recommendations', 'exercise', 'physical activity', 'programmes', 'elderly', 'old', 'review', 'framework', 'EFQM', 'assess' and 'quality'.\\nAfter identifying a list of statements, an online questionnaire was developed and tested with 5 PA programme coordinators for comments on readability and functionality. Some adjustments were made to make the affirmations included in the questionnaire clearer and more relevant to this case. We established that statements that received greater than 70% of experts' votes had achieved consensus [41-43] in both the bottom scores (i.e., reached consensus to drop) and top scores (i.e., reached consensus to include/retain). Statements that were dropped were not included in subsequent rounds of ratings. The remaining items were included in the next rounds, until a consensus was achieved to either drop or retain. At the end of three rounds, the statements on which experts had not reached consensus were also not included in the output list.\\nThe fourth phase of the process involved nominating experts to participate in the Delphi rounds. National experts in research on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology were identified. Our decisions were based on expertise or/and breadth of scientific work [44]. The DeGóis Curricula Platform1 assisted us in this process. A list of 63 potential participants was generated, along with key contacts for each. This group included 34 PhD scientists and academics (11 in PA for the elderly, 4 in sports management, 18 in quality management and 1 in gerontology), 3 non-PhD academics (1 in PA for the elderly and 2 in sports management) and 26 senior technicians (22 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). Previous information containing details about the EFQM Excellence Model, the Delphi process and the purpose of our study was provided. Of those invited to participate, 5 did not respond and 3 declined, due to lack of time (all PhD scientists and academics in quality management). Thus, 55 experts (30 females and 25 males) responded to our initial invitation and agreed to participate. Those who accepted our invitation were informed that they were required to respond to three online rounds of ratings.\\nThe rounds were performed using Survey Monkey, a web-based survey and data collection system. In every round, participants were asked to rate their level of agreement with each proposition, from 1 to 8 ('strongly disagree' to 'strongly agree'), suggest modifications to proposed definitions and/or add propositions that would be useful in a quality self-assessment tool for PA programmes for the elderly. The 8-point Likert scale was selected to bring out more variability in responses [45]. After each round, the frequency and mean of the panel's ratings and the percentage of scores ≥ 7 were calculated. Based on this data, a new questionnaire was designed and placed online for the next round. We asked participants to review all the information sent and re-rate each statement.\\nAfter round 3, we gathered all our data and developed a list of statements that did and did not reach consensus.\",\n \"Eight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\\nResults of the three rounds by criterion\\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \\\"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\\\" to \\\"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\\\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\",\n \"The main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \\\"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\\\" and \\\"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\\\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\\nThroughout the Delphi process, it was suggested that the proposition \\\"The programme involves a multidisciplinary team of professionals\\\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \\\"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\\\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \\\"external partnerships\\\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \\\"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\\\" and \\\"Regular and formal communication procedures are established with partners\\\".\\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \\\"Market research is used to determine the needs and expectations of future customers\\\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \\\"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\\\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \\\"The programme has measures of perception and/or performance indicators regarding employees' performance\\\" and \\\"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\\\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\",\n \"To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\",\n \"Our Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\",\n \"Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors). the file presents the resulting tool - named Q-STEPS - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly.\\nClick here for file\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,"supplementary-material"],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["physical activity","programmes","elderly","tool","evaluation","quality","adherence"],"string":"[\n \"physical activity\",\n \"programmes\",\n \"elderly\",\n \"tool\",\n \"evaluation\",\n \"quality\",\n \"adherence\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nPhysical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.\n\nMethods:\nA modified Delphi process was conducted using the Internet, from October 2009 to September 2010. The Delphi technique was developed in the 1950s by scientists at the Rand Corporation as a method of making informed decisions based on expert opinion [23]. Since then, it has been used to clarify a variety of problems in different sectors [24-29]. Despite having undergone some modifications, it remains a viable approach for gathering expert opinions through a structured iterative process that builds consensus [30]. This process involves multiple interactions with participants who usually complete two or more rounds in a reasonable amount of time [31] - even when participants are in geographically-distinct locations, since rounds can be conducted by mail or email [32,33]. The results of previous iterations can be modified by participants in later iterations, as they are able to review comments and feedbacks provided by other experts in earlier rounds [31]. Furthermore, the Delphi technique offers a number of specific advantages and is particularly helpful because it avoids the barriers commonly observed in other group discussions, such as interpersonal influence, time pressure and group demands [31,34,35]. This is due to the fact that respondents are not aware of the identities of other respondents and are, therefore, freed of personal and social constraints [30]. They are also able to complete the Delphi rounds in ways that suit them best because they participate in the rounds asynchronously [36]. The Delphi technique is also advantageous because a variety of statistical analysis techniques can be used to interpret the data its generates [37].\nThe Delphi process was conducted in three rounds [38,39] (Figure 1). Following each step listed in the previous figure, our main question was: Which quality practices must be included in a quality self-assessment tool for PA programmes for the elderly?\nSteps of the modified Delphi process used in the present study.\nUsing criteria and sub-criteria from the EFQM Excellence Model [13] and PA guidelines for older adults [3,40] as a starting point, we reviewed the literature to identify best practice principles and generate a list of statements. Our review was undertaken using PubMed (1980-2010), B-On (1980-2010), and Google™. We searched a variety of combinations of key words related to PA programmes for the elderly, quality management and the EFQM Excellence Model, such as: 'evaluation', 'guidelines', 'recommendations', 'exercise', 'physical activity', 'programmes', 'elderly', 'old', 'review', 'framework', 'EFQM', 'assess' and 'quality'.\nAfter identifying a list of statements, an online questionnaire was developed and tested with 5 PA programme coordinators for comments on readability and functionality. Some adjustments were made to make the affirmations included in the questionnaire clearer and more relevant to this case. We established that statements that received greater than 70% of experts' votes had achieved consensus [41-43] in both the bottom scores (i.e., reached consensus to drop) and top scores (i.e., reached consensus to include/retain). Statements that were dropped were not included in subsequent rounds of ratings. The remaining items were included in the next rounds, until a consensus was achieved to either drop or retain. At the end of three rounds, the statements on which experts had not reached consensus were also not included in the output list.\nThe fourth phase of the process involved nominating experts to participate in the Delphi rounds. National experts in research on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology were identified. Our decisions were based on expertise or/and breadth of scientific work [44]. The DeGóis Curricula Platform1 assisted us in this process. A list of 63 potential participants was generated, along with key contacts for each. This group included 34 PhD scientists and academics (11 in PA for the elderly, 4 in sports management, 18 in quality management and 1 in gerontology), 3 non-PhD academics (1 in PA for the elderly and 2 in sports management) and 26 senior technicians (22 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). Previous information containing details about the EFQM Excellence Model, the Delphi process and the purpose of our study was provided. Of those invited to participate, 5 did not respond and 3 declined, due to lack of time (all PhD scientists and academics in quality management). Thus, 55 experts (30 females and 25 males) responded to our initial invitation and agreed to participate. Those who accepted our invitation were informed that they were required to respond to three online rounds of ratings.\nThe rounds were performed using Survey Monkey, a web-based survey and data collection system. In every round, participants were asked to rate their level of agreement with each proposition, from 1 to 8 ('strongly disagree' to 'strongly agree'), suggest modifications to proposed definitions and/or add propositions that would be useful in a quality self-assessment tool for PA programmes for the elderly. The 8-point Likert scale was selected to bring out more variability in responses [45]. After each round, the frequency and mean of the panel's ratings and the percentage of scores ≥ 7 were calculated. Based on this data, a new questionnaire was designed and placed online for the next round. We asked participants to review all the information sent and re-rate each statement.\nAfter round 3, we gathered all our data and developed a list of statements that did and did not reach consensus.\n\nResults:\nEight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\nResults of the three rounds by criterion\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\" to \"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\n\nDiscussion:\nThe main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\" and \"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\nThroughout the Delphi process, it was suggested that the proposition \"The programme involves a multidisciplinary team of professionals\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \"external partnerships\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\" and \"Regular and formal communication procedures are established with partners\".\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \"Market research is used to determine the needs and expectations of future customers\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \"The programme has measures of perception and/or performance indicators regarding employees' performance\" and \"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\n\nStrengths and Limitations:\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\n\nConclusion:\nOur Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).\n\nSupplementary Material:\nQ-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors). the file presents the resulting tool - named Q-STEPS - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly.\nClick here for file\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThere has been a growing concern in designing physical activity (PA) programmes for elderly people, since evidence suggests that such health promotion interventions may reduce the deleterious effects of the ageing process. Complete programme evaluations are a necessary prerequisite to continuous quality improvements. Being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly in order to support its continuous improvement is, therefore, crucial. Thus, the aim of this study was to develop a self-assessment tool for PA programmes for the elderly.\n\nMethods:\nA 3-round Delphi process was conducted via the Internet with 43 national experts in PA for the elderly, management and delivery of PA programmes for the elderly, sports management, quality management and gerontology, asking experts to identify the propositions that they considered relevant for inclusion in the self-assessment tool. Experts reviewed a list of proposed statements, based on the criteria and sub-criteria from the European Foundation for Quality Management Excellence Model (EFQM) and PA guidelines for older adults and rated each proposition from 1 to 8 (disagree to agree) and modified and/or added propositions. Propositions receiving either bottom or top scores of greater than 70% were considered to have achieved consensus to drop or retain, respectively.\n\nResults:\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93. In round 2, a total of 104 propositions were presented, of which experts modified 39 and achieved consensus on 53. In the last round, of 51 proposed statements, the experts achieved consensus on 19. After 3 rounds of rating, experts had not achieved consensus on 32 propositions. The resulting tool consisted of 165 statements that assess nine management areas involved in the development of PA programmes for the elderly.\n\nConclusions:\nBased on experts' opinions, a self-assessment tool was found in order to access quality of PA programmes for the elderly. Information obtained with evaluations would be useful to organizations seeking to improve their services, customer satisfaction and, consequently, adherence to PA programmes, targeting the ageing population."},"background_conclusion_text":{"kind":"string","value":"Background:\nPhysical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.\n\nConclusion:\n1 It is an instrument for gathering, supplying and analyze the intellectual and scientific production of the Portuguese researchers."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThere has been a growing concern in designing physical activity (PA) programmes for elderly people, since evidence suggests that such health promotion interventions may reduce the deleterious effects of the ageing process. Complete programme evaluations are a necessary prerequisite to continuous quality improvements. Being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly in order to support its continuous improvement is, therefore, crucial. Thus, the aim of this study was to develop a self-assessment tool for PA programmes for the elderly.\n\nMethods:\nA 3-round Delphi process was conducted via the Internet with 43 national experts in PA for the elderly, management and delivery of PA programmes for the elderly, sports management, quality management and gerontology, asking experts to identify the propositions that they considered relevant for inclusion in the self-assessment tool. Experts reviewed a list of proposed statements, based on the criteria and sub-criteria from the European Foundation for Quality Management Excellence Model (EFQM) and PA guidelines for older adults and rated each proposition from 1 to 8 (disagree to agree) and modified and/or added propositions. Propositions receiving either bottom or top scores of greater than 70% were considered to have achieved consensus to drop or retain, respectively.\n\nResults:\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93. In round 2, a total of 104 propositions were presented, of which experts modified 39 and achieved consensus on 53. In the last round, of 51 proposed statements, the experts achieved consensus on 19. After 3 rounds of rating, experts had not achieved consensus on 32 propositions. The resulting tool consisted of 165 statements that assess nine management areas involved in the development of PA programmes for the elderly.\n\nConclusions:\nBased on experts' opinions, a self-assessment tool was found in order to access quality of PA programmes for the elderly. Information obtained with evaluations would be useful to organizations seeking to improve their services, customer satisfaction and, consequently, adherence to PA programmes, targeting the ageing population."},"whole_article_text_length":{"kind":"number","value":5170,"string":"5,170"},"whole_article_abstract_length":{"kind":"number","value":421,"string":"421"},"num_sections":{"kind":"number","value":7,"string":"7"},"most_frequent_words":{"kind":"list like","value":["programmes","pa","quality","consensus","elderly","pa programmes","results","experts","management","statements"],"string":"[\n \"programmes\",\n \"pa\",\n \"quality\",\n \"consensus\",\n \"elderly\",\n \"pa programmes\",\n \"results\",\n \"experts\",\n \"management\",\n \"statements\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] health | programmes | quality | important | pa | elderly | pa programmes | improve | satisfaction | efqm [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] rounds | delphi | management | list | included | participants | consensus | pa | process | quality [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] results | criteria assess | people | quality | programmes | assess | criteria | essential assessments quality pa | tool assesses areas | essential assessments quality [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | results | elderly | consensus | pa programmes | experts | statements | tool [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] programmes | pa | quality | results | elderly | consensus | pa programmes | experts | statements | tool [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 3 | Delphi | 43 | PA ||| the European Foundation for Quality Management Excellence Model | PA | 1 to 8 ||| greater than 70% [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| ||| 3 | Delphi | 43 | PA ||| the European Foundation for Quality Management Excellence Model | PA | 1 to 8 ||| greater than 70% ||| ||| 1 | 196 | 41 | 1 | 93 ||| 2 | 104 | 39 | 53 ||| 51 | 19 ||| 3 | 32 ||| 165 | nine ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| ||| 3 | Delphi | 43 | PA ||| the European Foundation for Quality Management Excellence Model | PA | 1 to 8 ||| greater than 70% ||| ||| 1 | 196 | 41 | 1 | 93 ||| 2 | 104 | 39 | 53 ||| 51 | 19 ||| 3 | 32 ||| 165 | nine ||| ||| [SUMMARY]"}}},{"rowIdx":278,"cells":{"title":{"kind":"string","value":"miR-223 increases gallbladder cancer cell sensitivity to docetaxel by downregulating STMN1."},"pmid":{"kind":"string","value":"27577078"},"background_abstract":{"kind":"string","value":"MicroRNAs (miRs) are involved in cancer carcinogenesis, and certain regulatory miRs could provide promising therapeutic methods for refractory malignancies, such as gallbladder cancer (GBC). miR-223 was found to play a pivotal role in enhancing chemotherapeutic effects, therefore evoking interest in the role of miR-223 in GBC."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We examined miR-223 expression in GBC tissue and GBC cell lines using qRT-PCR. The effects of modulated miR-223 expression in GBC cells were assayed using Cell Counting Kit-8 (CCK8), flow cytometry, and wound-healing and invasion assays. Susceptibility to docetaxel was evaluated in miR-223/STMN1-modulated GBC cells and xenograft tumor models. The protein expression of relevant genes was examined by Western blotting."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"miR-223 was decreased in GBC tissues and cell lines, and ectopic miR- 223 expression exhibited multiple anti-tumorigenic effects in GBC cells, including decreased proliferation, migration and invasion in vitro. However, treatment with a miR-223 inhibitor increased cell viability. We determined that STMN1 was negatively correlated with and regulated by miR-223 in GBC. miR-223 increased GBC sensitivity to docetaxel in vitro and in vivo, and the induced sensitivity to docetaxel was suppressed by the restoration of STMN1 expression."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"These findings indicated that miR-223 might serve as an onco-suppressor that enhances susceptibility to docetaxel by downregulating STMN1 in GBC, highlighting its promising therapeutic value."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Antineoplastic Agents","Cell Line, Tumor","Cell Proliferation","Cell Survival","Docetaxel","Down-Regulation","Flow Cytometry","Gallbladder","Gallbladder Neoplasms","Gene Expression Regulation, Neoplastic","Genes, Tumor Suppressor","Humans","Immunohistochemistry","Mice","Mice, Nude","MicroRNAs","Real-Time Polymerase Chain Reaction","Stathmin","Taxoids","Xenograft Model Antitumor Assays"],"string":"[\n \"Animals\",\n \"Antineoplastic Agents\",\n \"Cell Line, Tumor\",\n \"Cell Proliferation\",\n \"Cell Survival\",\n \"Docetaxel\",\n \"Down-Regulation\",\n \"Flow Cytometry\",\n \"Gallbladder\",\n \"Gallbladder Neoplasms\",\n \"Gene Expression Regulation, Neoplastic\",\n \"Genes, Tumor Suppressor\",\n \"Humans\",\n \"Immunohistochemistry\",\n \"Mice\",\n \"Mice, Nude\",\n \"MicroRNAs\",\n \"Real-Time Polymerase Chain Reaction\",\n \"Stathmin\",\n \"Taxoids\",\n \"Xenograft Model Antitumor Assays\"\n]"},"pmcid":{"kind":"string","value":"5308733"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications."},"methods_title":{"kind":"string","value":"MATERIALS AND METHODS"},"methods_text":{"kind":"string","value":" Reagents The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\nThe CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\n GBC tissue samples and cell lines Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\nFresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\n Tissue immunohistochemistry for STMN1 and in situ hybridization for miRNA Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\nImmunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\n Western blotting After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\nAfter treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\n RNA isolation and qRT-PCR Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\nTotal RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\n Cell biology experimental procedures In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\nIn these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\n DAPI staining cell apoptosis test Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\nTreated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\n Animal experiments To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\nTo investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\n Statistical analyses Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\nStatistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001)."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"DISCUSSION"},"conclusions_text":{"kind":"string","value":"Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001)."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","RESULTS","Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation","miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression","Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation","miR-223 overexpression inhibits GBC cell migration and invasion","GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics","The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"RESULTS\",\n \"Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation\",\n \"miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression\",\n \"Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation\",\n \"miR-223 overexpression inhibits GBC cell migration and invasion\",\n \"GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics\",\n \"The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid\",\n \"DISCUSSION\"\n]"},"other_sections_texts":{"kind":"list like","value":["Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications."," Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.","To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.","To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.","To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.","Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).","GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).","The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.","GBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach."],"string":"[\n \"Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.\",\n \" Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\",\n \"To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\",\n \"To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\",\n \"To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\",\n \"Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\",\n \"GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\",\n \"The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\",\n \"GBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","RESULTS","Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation","miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression","Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation","miR-223 overexpression inhibits GBC cell migration and invasion","GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics","The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid","DISCUSSION","MATERIALS AND METHODS","SUPPLEMENTARY MATERIALS FIGURES"],"string":"[\n \"INTRODUCTION\",\n \"RESULTS\",\n \"Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation\",\n \"miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression\",\n \"Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation\",\n \"miR-223 overexpression inhibits GBC cell migration and invasion\",\n \"GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics\",\n \"The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid\",\n \"DISCUSSION\",\n \"MATERIALS AND METHODS\",\n \"SUPPLEMENTARY MATERIALS FIGURES\"\n]"},"all_sections_texts":{"kind":"list like","value":["Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications."," Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.","To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.","To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.","To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.","Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).","GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).","The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.","GBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach."," Reagents The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\nThe CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\n GBC tissue samples and cell lines Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\nFresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\n Tissue immunohistochemistry for STMN1 and in situ hybridization for miRNA Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\nImmunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\n Western blotting After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\nAfter treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\n RNA isolation and qRT-PCR Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\nTotal RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\n Cell biology experimental procedures In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\nIn these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\n DAPI staining cell apoptosis test Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\nTreated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\n Animal experiments To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\nTo investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\n Statistical analyses Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\nStatistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).",""],"string":"[\n \"Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.\",\n \" Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\",\n \"To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\",\n \"To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\",\n \"To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\",\n \"Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\",\n \"GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\",\n \"The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\",\n \"GBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach.\",\n \" Reagents The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\\nThe CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\\n GBC tissue samples and cell lines Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\\nFresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\\n Tissue immunohistochemistry for STMN1 and in situ hybridization for miRNA Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\\nImmunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\\n Western blotting After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\\nAfter treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\\n RNA isolation and qRT-PCR Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\\nSTMN1 - CT\\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\\nTotal RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\\nSTMN1 - CT\\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\\n Cell biology experimental procedures In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\\nIn these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\\n DAPI staining cell apoptosis test Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\\nTreated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\\n Animal experiments To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\\nTo investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\\n Statistical analyses Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\\nStatistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\",\n \"\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,"methods","supplementary-material"],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"methods\",\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["miR-223","gallbladder cancer","malignancy","STMN1"],"string":"[\n \"miR-223\",\n \"gallbladder cancer\",\n \"malignancy\",\n \"STMN1\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nGallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.\n\nRESULTS:\n Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\n\nAberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation:\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\n\nmiR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression:\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\n\nEctopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation:\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\n\nmiR-223 overexpression inhibits GBC cell migration and invasion:\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\n\nGBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics:\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\n\nThe chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid:\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\n\nDISCUSSION:\nGBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach.\n\nMATERIALS AND METHODS:\n Reagents The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\nThe CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\n GBC tissue samples and cell lines Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\nFresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\n Tissue immunohistochemistry for STMN1 and in situ hybridization for miRNA Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\nImmunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\n Western blotting After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\nAfter treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\n RNA isolation and qRT-PCR Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\nTotal RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\n Cell biology experimental procedures In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\nIn these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\n DAPI staining cell apoptosis test Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\nTreated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\n Animal experiments To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\nTo investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\n Statistical analyses Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\nStatistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\n\nSUPPLEMENTARY MATERIALS FIGURES:\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nMicroRNAs (miRs) are involved in cancer carcinogenesis, and certain regulatory miRs could provide promising therapeutic methods for refractory malignancies, such as gallbladder cancer (GBC). miR-223 was found to play a pivotal role in enhancing chemotherapeutic effects, therefore evoking interest in the role of miR-223 in GBC.\n\nMethods:\nWe examined miR-223 expression in GBC tissue and GBC cell lines using qRT-PCR. The effects of modulated miR-223 expression in GBC cells were assayed using Cell Counting Kit-8 (CCK8), flow cytometry, and wound-healing and invasion assays. Susceptibility to docetaxel was evaluated in miR-223/STMN1-modulated GBC cells and xenograft tumor models. The protein expression of relevant genes was examined by Western blotting.\n\nResults:\nmiR-223 was decreased in GBC tissues and cell lines, and ectopic miR- 223 expression exhibited multiple anti-tumorigenic effects in GBC cells, including decreased proliferation, migration and invasion in vitro. However, treatment with a miR-223 inhibitor increased cell viability. We determined that STMN1 was negatively correlated with and regulated by miR-223 in GBC. miR-223 increased GBC sensitivity to docetaxel in vitro and in vivo, and the induced sensitivity to docetaxel was suppressed by the restoration of STMN1 expression.\n\nConclusions:\nThese findings indicated that miR-223 might serve as an onco-suppressor that enhances susceptibility to docetaxel by downregulating STMN1 in GBC, highlighting its promising therapeutic value."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nGallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.\n\nDISCUSSION:\nStatistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001)."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nMicroRNAs (miRs) are involved in cancer carcinogenesis, and certain regulatory miRs could provide promising therapeutic methods for refractory malignancies, such as gallbladder cancer (GBC). miR-223 was found to play a pivotal role in enhancing chemotherapeutic effects, therefore evoking interest in the role of miR-223 in GBC.\n\nMethods:\nWe examined miR-223 expression in GBC tissue and GBC cell lines using qRT-PCR. The effects of modulated miR-223 expression in GBC cells were assayed using Cell Counting Kit-8 (CCK8), flow cytometry, and wound-healing and invasion assays. Susceptibility to docetaxel was evaluated in miR-223/STMN1-modulated GBC cells and xenograft tumor models. The protein expression of relevant genes was examined by Western blotting.\n\nResults:\nmiR-223 was decreased in GBC tissues and cell lines, and ectopic miR- 223 expression exhibited multiple anti-tumorigenic effects in GBC cells, including decreased proliferation, migration and invasion in vitro. However, treatment with a miR-223 inhibitor increased cell viability. We determined that STMN1 was negatively correlated with and regulated by miR-223 in GBC. miR-223 increased GBC sensitivity to docetaxel in vitro and in vivo, and the induced sensitivity to docetaxel was suppressed by the restoration of STMN1 expression.\n\nConclusions:\nThese findings indicated that miR-223 might serve as an onco-suppressor that enhances susceptibility to docetaxel by downregulating STMN1 in GBC, highlighting its promising therapeutic value."},"whole_article_text_length":{"kind":"number","value":12789,"string":"12,789"},"whole_article_abstract_length":{"kind":"number","value":263,"string":"263"},"num_sections":{"kind":"number","value":11,"string":"11"},"most_frequent_words":{"kind":"list like","value":["mir","mir 223","223","gbc","cells","cell","stmn1","expression","sd","noz"],"string":"[\n \"mir\",\n \"mir 223\",\n \"223\",\n \"gbc\",\n \"cells\",\n \"cell\",\n \"stmn1\",\n \"expression\",\n \"sd\",\n \"noz\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | mir 223 | 223 | gbc | cells | cell | stmn1 | expression | sd | noz [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | mir 223 | 223 | gbc | cells | cell | stmn1 | expression | sd | noz [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | mir 223 | 223 | gbc | cells | cell | stmn1 | expression | sd | noz [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | mir 223 | 223 | gbc | cells | cell | stmn1 | expression | sd | noz [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | mir 223 | 223 | gbc | cells | cell | stmn1 | expression | sd | noz [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | 223 | mir 223 | mirs | gbc | studies | leukemia | cancer | carcinoma | advanced [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] min | purchased | sections | antibody | anti | mir | tissue | obtained | assay | 10 [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | mir 223 | 223 | gbc | stmn1 | carcinogenesis | expression | cell | microtubule | role [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | 223 | mir 223 | gbc | cells | stmn1 | expression | sd | cell | gbc sd [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | 223 | mir 223 | gbc | cells | stmn1 | expression | sd | cell | gbc sd [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| STMN1 ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] STMN1 | GBC [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| ||| STMN1 ||| ||| 223 ||| ||| STMN1 ||| STMN1 ||| STMN1 | GBC [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| ||| STMN1 ||| ||| 223 ||| ||| STMN1 ||| STMN1 ||| STMN1 | GBC [SUMMARY]"}}},{"rowIdx":279,"cells":{"title":{"kind":"string","value":"Harm avoidance is associated with progression of parkinsonism in community-dwelling older adults: a prospective cohort study."},"pmid":{"kind":"string","value":"24754876"},"background_abstract":{"kind":"string","value":"We tested the hypothesis that harm avoidance, a trait associated with behavioral inhibition, is associated with the rate of change in parkinsonism in older adults."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"At baseline harm avoidance was assessed with a standard self-report instrument in 969 older people without dementia participating in the Rush Memory and Aging Project, a longitudinal community-based cohort study. Parkinsonism was assessed annually with a modified version of the motor section of the Unified Parkinson's Disease Rating Scale (mUPDRS)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Average follow-up was 5 years. A linear mixed-effects model controlling for age, sex and education showed that for an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10), the overall severity of parkinsonism increased by about 0.05 unit/ year (Estimate, 0.054, S.E., 0.007, p <0.001) and that the level of harm avoidance was associated with the progression of parkinsonism (Estimate, 0.004, S.E., 0.001, p <0.001). Thus, for an average participant, every 6 point (~1 SD) increase in harm avoidance score at baseline, the rate of progression of parkinsonism increased about 50% compared to an individual with an average harm avoidance score. This amount of change in parkinsonism over the course of the study was associated with about a 5% increased risk of death. The association between harm avoidance and progression of parkinsonism persisted when controlling for cognitive function, depressive symptoms, loneliness, neuroticism, late-life cognitive, social and physical activities and chronic health conditions."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"A higher level of the harm avoidance trait is associated with a more rapid progression of parkinsonism in older adults."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Aged, 80 and over","Cohort Studies","Disease Progression","Female","Follow-Up Studies","Harm Reduction","Humans","Longitudinal Studies","Male","Parkinsonian Disorders","Prospective Studies","Residence Characteristics"],"string":"[\n \"Aged\",\n \"Aged, 80 and over\",\n \"Cohort Studies\",\n \"Disease Progression\",\n \"Female\",\n \"Follow-Up Studies\",\n \"Harm Reduction\",\n \"Humans\",\n \"Longitudinal Studies\",\n \"Male\",\n \"Parkinsonian Disorders\",\n \"Prospective Studies\",\n \"Residence Characteristics\"\n]"},"pmcid":{"kind":"string","value":"4022545"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Participants Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\nParticipants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\n Clinical diagnoses Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\nClinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\n Assessment of harm avoidance Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\nStudy participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\n Assessment of parkinsonism Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\nTrained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\n Assessment of other covariates Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\nAge in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\n Statistical analyses The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\nThe global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23]."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Descriptive properties of harm avoidance measure The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\nThe clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\n Harm avoidance and change in parkinsonism Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\nBaseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\n Potential confounders of harm avoidance and change in parkinsonism Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\nPrior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\n Clinical significance of the loss of motor function associated with personality To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\nTo determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging."},"other_sections_titles":{"kind":"list like","value":["Background","Participants","Clinical diagnoses","Assessment of harm avoidance","Assessment of parkinsonism","Assessment of other covariates","Statistical analyses","Descriptive properties of harm avoidance measure","Harm avoidance and change in parkinsonism","Potential confounders of harm avoidance and change in parkinsonism","Clinical significance of the loss of motor function associated with personality","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Participants\",\n \"Clinical diagnoses\",\n \"Assessment of harm avoidance\",\n \"Assessment of parkinsonism\",\n \"Assessment of other covariates\",\n \"Statistical analyses\",\n \"Descriptive properties of harm avoidance measure\",\n \"Harm avoidance and change in parkinsonism\",\n \"Potential confounders of harm avoidance and change in parkinsonism\",\n \"Clinical significance of the loss of motor function associated with personality\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.","Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.","Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].","Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].","Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].","Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].","The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].","The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).","Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].","Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).","To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).","The authors declare that they have no competing interests.","ASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\n"],"string":"[\n \"Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.\",\n \"Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\",\n \"Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\",\n \"Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\",\n \"Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\",\n \"Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\",\n \"The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\",\n \"The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\\nClinical characteristics of the participants included in these analyses at this study’s baseline\\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\",\n \"Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\",\n \"Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\",\n \"To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\",\n \"The authors declare that they have no competing interests.\",\n \"ASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Participants","Clinical diagnoses","Assessment of harm avoidance","Assessment of parkinsonism","Assessment of other covariates","Statistical analyses","Results","Descriptive properties of harm avoidance measure","Harm avoidance and change in parkinsonism","Potential confounders of harm avoidance and change in parkinsonism","Clinical significance of the loss of motor function associated with personality","Discussion","Conclusions","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Participants\",\n \"Clinical diagnoses\",\n \"Assessment of harm avoidance\",\n \"Assessment of parkinsonism\",\n \"Assessment of other covariates\",\n \"Statistical analyses\",\n \"Results\",\n \"Descriptive properties of harm avoidance measure\",\n \"Harm avoidance and change in parkinsonism\",\n \"Potential confounders of harm avoidance and change in parkinsonism\",\n \"Clinical significance of the loss of motor function associated with personality\",\n \"Discussion\",\n \"Conclusions\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles."," Participants Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\nParticipants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\n Clinical diagnoses Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\nClinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\n Assessment of harm avoidance Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\nStudy participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\n Assessment of parkinsonism Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\nTrained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\n Assessment of other covariates Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\nAge in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\n Statistical analyses The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\nThe global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].","Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.","Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].","Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].","Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].","Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].","The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23]."," Descriptive properties of harm avoidance measure The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\nThe clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\n Harm avoidance and change in parkinsonism Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\nBaseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\n Potential confounders of harm avoidance and change in parkinsonism Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\nPrior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\n Clinical significance of the loss of motor function associated with personality To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\nTo determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).","The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).","Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].","Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).","To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).","Harm avoidance is a broad anxiety-related trait. People with a high level of the trait tend to be pessimistic, apprehensive, shy, and easily fatigued, behaviorally inhibited and to avoid new and potentially aversive situations [3]. In a cohort of more than 900 older adults, those with a high level of the trait showed a more rapid rate of progression of parkinsonism as compared to older adults with a low level of the trait. This association persisted after controlling for other psychosocial factors including depressive symptoms, loneliness and neuroticism, late-life activities including physical, social and cognitive activities, global cognitive function and chronic health conditions. Together these data suggest that the level of the trait of harm avoidance, may identify older individuals at higher risk for more rapidly progressive parkinsonism and provides evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.\nPrior studies have reported that higher levels of harm avoidance are associated with adverse health consequences in older adults including: incident disability, [6] and late-life cognitive impairments including incident mild cognitive impairment (MCI) and Alzheimer’s disease (AD) as well as cognitive decline [7,24,25]. This study extends these findings by showing that harm avoidance is also associated with both the level and rate of progression of parkinsonism in older adults without overt neurologic diseases including dementia, stroke and Parkinson’s disease (PD). Analyses of the sub-scale components of harm avoidance showed that all of its constituent factors are associated with the rate of progressive parkinsonism. The association of harm avoidance and parkinsonism, was robust and remained significant after accounting for related personality traits and psychosocial factors, global cognition, a wide range of late-life activities and chronic health conditions. The results of the current study have important translational implications as they suggest that personality traits need to be considered for our understanding of individual differences in the progression of parkinsonism in older adults. Furthermore, understanding the biologic basis of the association has the potential to lead to new therapeutic targets to reduce the burden of late-life motor impairment.\nThe basis of the association between harm avoidance and parkinsonism is likely to be complex. While, harm avoidance is related to other personality traits and psychosocial factors, [26] the association of harm avoidance and parkinsonism was unchanged when we adjusted for several of these factors in the current study (Table 3) [27]. Personality traits may affect for lifestyle choices i.e. physical and other late-life activities and could thus link harm avoidance with parkinsonism. In contrast to other personality traits, [17,28] statistical adjustment for late-life physical, cognitive and social activities in the current study did not affect the association of harm avoidance with progressive parkinsonism. Both harm avoidance and motor function in older people are preferentially associated with structural changes in specific brain regions including cortical and cerebellar structures which might account for their association in this study [29-33]. Thus, higher levels of harm avoidance may be lead to stress-related changes in neurotransmitters (e.g., cortisol or dopamine) causing brain atrophy, damaging motor-related brain regions or decreasing the brain’s capacity (motor reserve) to tolerate ongoing neurodegeneration and the accumulation of other neuropathologies [34-37].\nOver the years there have been many reports suggesting that PD may be associated with a distinctive personality which may manifest many years before clinical evidence of parkinsonism, but empirical studies have failed to support this suggestion [38,39]. Nonethelss, building on imaging data linking harm avoidance with brain dopamine receptors, recent studies have reported higher levels of harm avoidance occur in individuals with early clinical manifestations of PD such as REM sleep disorder which can manifest years before a clinical diagnosis of PD [40-43]. Studies in the current cohort have suggested that PD pathology is found in up to 40% of older adults without a clinical diagnoses of PD, and these lesions are associated with the severity of parkinsonism proximate to death [14]. Further work is needed to determine if the accumulation of subclinical PD pathology might contribute to the association between harm avoidance and the rate of change in the severity of parkinsonism. Alternatively, lower harm avoidance is associated with resilience, optimism, composure, and energy which may facilitate adaptation to accumulating neurodegeneration and the accumulation of neuropathology in older adults [10,44]. Further work to understand the neurobiologic basis for the current findings has the potential to identify new targets and pathways, for interventions to decrease the growing burden of progressive parkinsonism in older adults.\nOur study has some limitations. While some studies have suggested that personality traits such as harm avoidance are stable even in old age, others suggest that personality changes may occur with age but there are few studies which have focused on individuals older than 80 years [45-48]. Further, it is possible that a third unmeasured variable is related to both harm avoidance and progressive parkinsonism. A precondition for participation in the current study was consent to annual exam and organ donation at death, so given the selected nature of the cohort, our findings will need replication in other cohorts. This study was large since on an individual level the effect sizes for the association of harm avoidance and progressive parkinsonism are small. Similar effect sizes have been reported in prior reports of other psychosocial factors with motor decline [17]. Nonetheless, from a public policy perspective even the modest effect sizes observed in the current study are likely to be important.\nHowever, several factors increase confidence in our findings. Perhaps most importantly, the study enjoys high follow-up participation reducing bias due to attrition. In addition, personality traits were assessed among people without dementia and parkinsonism was evaluated as part of a uniform clinical evaluation which incorporated other widely accepted personality, affect and cognitive measures. In addition, a relatively large number of older people were studied, so that there was adequate statistical power to identify the association of interest while controlling for several potentially confounding variables. Results were similar with total score and subscores of the trait of harm avoidance.","The growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.","The authors declare that they have no competing interests.","ASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\n"],"string":"[\n \"Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.\",\n \" Participants Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\\nParticipants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\\n Clinical diagnoses Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\\nClinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\\n Assessment of harm avoidance Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\\nStudy participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\\n Assessment of parkinsonism Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\\nTrained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\\n Assessment of other covariates Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\\nAge in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\\n Statistical analyses The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\\nThe global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\",\n \"Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\",\n \"Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\",\n \"Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\",\n \"Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\",\n \"Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\",\n \"The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\",\n \" Descriptive properties of harm avoidance measure The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\\nClinical characteristics of the participants included in these analyses at this study’s baseline\\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\\nThe clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\\nClinical characteristics of the participants included in these analyses at this study’s baseline\\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\\n Harm avoidance and change in parkinsonism Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\\nBaseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\\n Potential confounders of harm avoidance and change in parkinsonism Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\\nPrior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\\n Clinical significance of the loss of motor function associated with personality To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\\nTo determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\",\n \"The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\\nClinical characteristics of the participants included in these analyses at this study’s baseline\\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\",\n \"Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\",\n \"Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\",\n \"To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\",\n \"Harm avoidance is a broad anxiety-related trait. People with a high level of the trait tend to be pessimistic, apprehensive, shy, and easily fatigued, behaviorally inhibited and to avoid new and potentially aversive situations [3]. In a cohort of more than 900 older adults, those with a high level of the trait showed a more rapid rate of progression of parkinsonism as compared to older adults with a low level of the trait. This association persisted after controlling for other psychosocial factors including depressive symptoms, loneliness and neuroticism, late-life activities including physical, social and cognitive activities, global cognitive function and chronic health conditions. Together these data suggest that the level of the trait of harm avoidance, may identify older individuals at higher risk for more rapidly progressive parkinsonism and provides evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.\\nPrior studies have reported that higher levels of harm avoidance are associated with adverse health consequences in older adults including: incident disability, [6] and late-life cognitive impairments including incident mild cognitive impairment (MCI) and Alzheimer’s disease (AD) as well as cognitive decline [7,24,25]. This study extends these findings by showing that harm avoidance is also associated with both the level and rate of progression of parkinsonism in older adults without overt neurologic diseases including dementia, stroke and Parkinson’s disease (PD). Analyses of the sub-scale components of harm avoidance showed that all of its constituent factors are associated with the rate of progressive parkinsonism. The association of harm avoidance and parkinsonism, was robust and remained significant after accounting for related personality traits and psychosocial factors, global cognition, a wide range of late-life activities and chronic health conditions. The results of the current study have important translational implications as they suggest that personality traits need to be considered for our understanding of individual differences in the progression of parkinsonism in older adults. Furthermore, understanding the biologic basis of the association has the potential to lead to new therapeutic targets to reduce the burden of late-life motor impairment.\\nThe basis of the association between harm avoidance and parkinsonism is likely to be complex. While, harm avoidance is related to other personality traits and psychosocial factors, [26] the association of harm avoidance and parkinsonism was unchanged when we adjusted for several of these factors in the current study (Table 3) [27]. Personality traits may affect for lifestyle choices i.e. physical and other late-life activities and could thus link harm avoidance with parkinsonism. In contrast to other personality traits, [17,28] statistical adjustment for late-life physical, cognitive and social activities in the current study did not affect the association of harm avoidance with progressive parkinsonism. Both harm avoidance and motor function in older people are preferentially associated with structural changes in specific brain regions including cortical and cerebellar structures which might account for their association in this study [29-33]. Thus, higher levels of harm avoidance may be lead to stress-related changes in neurotransmitters (e.g., cortisol or dopamine) causing brain atrophy, damaging motor-related brain regions or decreasing the brain’s capacity (motor reserve) to tolerate ongoing neurodegeneration and the accumulation of other neuropathologies [34-37].\\nOver the years there have been many reports suggesting that PD may be associated with a distinctive personality which may manifest many years before clinical evidence of parkinsonism, but empirical studies have failed to support this suggestion [38,39]. Nonethelss, building on imaging data linking harm avoidance with brain dopamine receptors, recent studies have reported higher levels of harm avoidance occur in individuals with early clinical manifestations of PD such as REM sleep disorder which can manifest years before a clinical diagnosis of PD [40-43]. Studies in the current cohort have suggested that PD pathology is found in up to 40% of older adults without a clinical diagnoses of PD, and these lesions are associated with the severity of parkinsonism proximate to death [14]. Further work is needed to determine if the accumulation of subclinical PD pathology might contribute to the association between harm avoidance and the rate of change in the severity of parkinsonism. Alternatively, lower harm avoidance is associated with resilience, optimism, composure, and energy which may facilitate adaptation to accumulating neurodegeneration and the accumulation of neuropathology in older adults [10,44]. Further work to understand the neurobiologic basis for the current findings has the potential to identify new targets and pathways, for interventions to decrease the growing burden of progressive parkinsonism in older adults.\\nOur study has some limitations. While some studies have suggested that personality traits such as harm avoidance are stable even in old age, others suggest that personality changes may occur with age but there are few studies which have focused on individuals older than 80 years [45-48]. Further, it is possible that a third unmeasured variable is related to both harm avoidance and progressive parkinsonism. A precondition for participation in the current study was consent to annual exam and organ donation at death, so given the selected nature of the cohort, our findings will need replication in other cohorts. This study was large since on an individual level the effect sizes for the association of harm avoidance and progressive parkinsonism are small. Similar effect sizes have been reported in prior reports of other psychosocial factors with motor decline [17]. Nonetheless, from a public policy perspective even the modest effect sizes observed in the current study are likely to be important.\\nHowever, several factors increase confidence in our findings. Perhaps most importantly, the study enjoys high follow-up participation reducing bias due to attrition. In addition, personality traits were assessed among people without dementia and parkinsonism was evaluated as part of a uniform clinical evaluation which incorporated other widely accepted personality, affect and cognitive measures. In addition, a relatively large number of older people were studied, so that there was adequate statistical power to identify the association of interest while controlling for several potentially confounding variables. Results were similar with total score and subscores of the trait of harm avoidance.\",\n \"The growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.\",\n \"The authors declare that they have no competing interests.\",\n \"ASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,"results",null,null,null,null,"discussion","conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Late-life motor impairment","Aging","Parkinsonism","Harm avoidance"],"string":"[\n \"Late-life motor impairment\",\n \"Aging\",\n \"Parkinsonism\",\n \"Harm avoidance\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nLate-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.\n\nMethods:\n Participants Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\nParticipants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\n Clinical diagnoses Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\nClinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\n Assessment of harm avoidance Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\nStudy participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\n Assessment of parkinsonism Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\nTrained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\n Assessment of other covariates Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\nAge in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\n Statistical analyses The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\nThe global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\n\nParticipants:\nParticipants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\n\nClinical diagnoses:\nClinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\n\nAssessment of harm avoidance:\nStudy participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\n\nAssessment of parkinsonism:\nTrained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\n\nAssessment of other covariates:\nAge in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\n\nStatistical analyses:\nThe global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\n\nResults:\n Descriptive properties of harm avoidance measure The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\nThe clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\n Harm avoidance and change in parkinsonism Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\nBaseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\n Potential confounders of harm avoidance and change in parkinsonism Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\nPrior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\n Clinical significance of the loss of motor function associated with personality To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\nTo determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\n\nDescriptive properties of harm avoidance measure:\nThe clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\n\nHarm avoidance and change in parkinsonism:\nBaseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\n\nPotential confounders of harm avoidance and change in parkinsonism:\nPrior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\n\nClinical significance of the loss of motor function associated with personality:\nTo determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\n\nDiscussion:\nHarm avoidance is a broad anxiety-related trait. People with a high level of the trait tend to be pessimistic, apprehensive, shy, and easily fatigued, behaviorally inhibited and to avoid new and potentially aversive situations [3]. In a cohort of more than 900 older adults, those with a high level of the trait showed a more rapid rate of progression of parkinsonism as compared to older adults with a low level of the trait. This association persisted after controlling for other psychosocial factors including depressive symptoms, loneliness and neuroticism, late-life activities including physical, social and cognitive activities, global cognitive function and chronic health conditions. Together these data suggest that the level of the trait of harm avoidance, may identify older individuals at higher risk for more rapidly progressive parkinsonism and provides evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.\nPrior studies have reported that higher levels of harm avoidance are associated with adverse health consequences in older adults including: incident disability, [6] and late-life cognitive impairments including incident mild cognitive impairment (MCI) and Alzheimer’s disease (AD) as well as cognitive decline [7,24,25]. This study extends these findings by showing that harm avoidance is also associated with both the level and rate of progression of parkinsonism in older adults without overt neurologic diseases including dementia, stroke and Parkinson’s disease (PD). Analyses of the sub-scale components of harm avoidance showed that all of its constituent factors are associated with the rate of progressive parkinsonism. The association of harm avoidance and parkinsonism, was robust and remained significant after accounting for related personality traits and psychosocial factors, global cognition, a wide range of late-life activities and chronic health conditions. The results of the current study have important translational implications as they suggest that personality traits need to be considered for our understanding of individual differences in the progression of parkinsonism in older adults. Furthermore, understanding the biologic basis of the association has the potential to lead to new therapeutic targets to reduce the burden of late-life motor impairment.\nThe basis of the association between harm avoidance and parkinsonism is likely to be complex. While, harm avoidance is related to other personality traits and psychosocial factors, [26] the association of harm avoidance and parkinsonism was unchanged when we adjusted for several of these factors in the current study (Table 3) [27]. Personality traits may affect for lifestyle choices i.e. physical and other late-life activities and could thus link harm avoidance with parkinsonism. In contrast to other personality traits, [17,28] statistical adjustment for late-life physical, cognitive and social activities in the current study did not affect the association of harm avoidance with progressive parkinsonism. Both harm avoidance and motor function in older people are preferentially associated with structural changes in specific brain regions including cortical and cerebellar structures which might account for their association in this study [29-33]. Thus, higher levels of harm avoidance may be lead to stress-related changes in neurotransmitters (e.g., cortisol or dopamine) causing brain atrophy, damaging motor-related brain regions or decreasing the brain’s capacity (motor reserve) to tolerate ongoing neurodegeneration and the accumulation of other neuropathologies [34-37].\nOver the years there have been many reports suggesting that PD may be associated with a distinctive personality which may manifest many years before clinical evidence of parkinsonism, but empirical studies have failed to support this suggestion [38,39]. Nonethelss, building on imaging data linking harm avoidance with brain dopamine receptors, recent studies have reported higher levels of harm avoidance occur in individuals with early clinical manifestations of PD such as REM sleep disorder which can manifest years before a clinical diagnosis of PD [40-43]. Studies in the current cohort have suggested that PD pathology is found in up to 40% of older adults without a clinical diagnoses of PD, and these lesions are associated with the severity of parkinsonism proximate to death [14]. Further work is needed to determine if the accumulation of subclinical PD pathology might contribute to the association between harm avoidance and the rate of change in the severity of parkinsonism. Alternatively, lower harm avoidance is associated with resilience, optimism, composure, and energy which may facilitate adaptation to accumulating neurodegeneration and the accumulation of neuropathology in older adults [10,44]. Further work to understand the neurobiologic basis for the current findings has the potential to identify new targets and pathways, for interventions to decrease the growing burden of progressive parkinsonism in older adults.\nOur study has some limitations. While some studies have suggested that personality traits such as harm avoidance are stable even in old age, others suggest that personality changes may occur with age but there are few studies which have focused on individuals older than 80 years [45-48]. Further, it is possible that a third unmeasured variable is related to both harm avoidance and progressive parkinsonism. A precondition for participation in the current study was consent to annual exam and organ donation at death, so given the selected nature of the cohort, our findings will need replication in other cohorts. This study was large since on an individual level the effect sizes for the association of harm avoidance and progressive parkinsonism are small. Similar effect sizes have been reported in prior reports of other psychosocial factors with motor decline [17]. Nonetheless, from a public policy perspective even the modest effect sizes observed in the current study are likely to be important.\nHowever, several factors increase confidence in our findings. Perhaps most importantly, the study enjoys high follow-up participation reducing bias due to attrition. In addition, personality traits were assessed among people without dementia and parkinsonism was evaluated as part of a uniform clinical evaluation which incorporated other widely accepted personality, affect and cognitive measures. In addition, a relatively large number of older people were studied, so that there was adequate statistical power to identify the association of interest while controlling for several potentially confounding variables. Results were similar with total score and subscores of the trait of harm avoidance.\n\nConclusions:\nThe growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWe tested the hypothesis that harm avoidance, a trait associated with behavioral inhibition, is associated with the rate of change in parkinsonism in older adults.\n\nMethods:\nAt baseline harm avoidance was assessed with a standard self-report instrument in 969 older people without dementia participating in the Rush Memory and Aging Project, a longitudinal community-based cohort study. Parkinsonism was assessed annually with a modified version of the motor section of the Unified Parkinson's Disease Rating Scale (mUPDRS).\n\nResults:\nAverage follow-up was 5 years. A linear mixed-effects model controlling for age, sex and education showed that for an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10), the overall severity of parkinsonism increased by about 0.05 unit/ year (Estimate, 0.054, S.E., 0.007, p <0.001) and that the level of harm avoidance was associated with the progression of parkinsonism (Estimate, 0.004, S.E., 0.001, p <0.001). Thus, for an average participant, every 6 point (~1 SD) increase in harm avoidance score at baseline, the rate of progression of parkinsonism increased about 50% compared to an individual with an average harm avoidance score. This amount of change in parkinsonism over the course of the study was associated with about a 5% increased risk of death. The association between harm avoidance and progression of parkinsonism persisted when controlling for cognitive function, depressive symptoms, loneliness, neuroticism, late-life cognitive, social and physical activities and chronic health conditions.\n\nConclusions:\nA higher level of the harm avoidance trait is associated with a more rapid progression of parkinsonism in older adults."},"background_conclusion_text":{"kind":"string","value":"Background:\nLate-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.\n\nConclusions:\nThe growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nWe tested the hypothesis that harm avoidance, a trait associated with behavioral inhibition, is associated with the rate of change in parkinsonism in older adults.\n\nMethods:\nAt baseline harm avoidance was assessed with a standard self-report instrument in 969 older people without dementia participating in the Rush Memory and Aging Project, a longitudinal community-based cohort study. Parkinsonism was assessed annually with a modified version of the motor section of the Unified Parkinson's Disease Rating Scale (mUPDRS).\n\nResults:\nAverage follow-up was 5 years. A linear mixed-effects model controlling for age, sex and education showed that for an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10), the overall severity of parkinsonism increased by about 0.05 unit/ year (Estimate, 0.054, S.E., 0.007, p <0.001) and that the level of harm avoidance was associated with the progression of parkinsonism (Estimate, 0.004, S.E., 0.001, p <0.001). Thus, for an average participant, every 6 point (~1 SD) increase in harm avoidance score at baseline, the rate of progression of parkinsonism increased about 50% compared to an individual with an average harm avoidance score. This amount of change in parkinsonism over the course of the study was associated with about a 5% increased risk of death. The association between harm avoidance and progression of parkinsonism persisted when controlling for cognitive function, depressive symptoms, loneliness, neuroticism, late-life cognitive, social and physical activities and chronic health conditions.\n\nConclusions:\nA higher level of the harm avoidance trait is associated with a more rapid progression of parkinsonism in older adults."},"whole_article_text_length":{"kind":"number","value":13063,"string":"13,063"},"whole_article_abstract_length":{"kind":"number","value":330,"string":"330"},"num_sections":{"kind":"number","value":18,"string":"18"},"most_frequent_words":{"kind":"list like","value":["parkinsonism","harm avoidance","harm","avoidance","baseline","change","rate","score","change parkinsonism","rate change"],"string":"[\n \"parkinsonism\",\n \"harm avoidance\",\n \"harm\",\n \"avoidance\",\n \"baseline\",\n \"change\",\n \"rate\",\n \"score\",\n \"change parkinsonism\",\n \"rate change\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] harm | avoidance | harm avoidance | parkinsonism | adults | older | trait | related | associated | level [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] avoidance | harm avoidance | harm | parkinsonism | participants | items | baseline | score | models | change [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | avoidance | harm avoidance | harm | change | change parkinsonism | 001 | rate change | rate change parkinsonism | rate [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] adults | older | older adults | level trait | motor | traits | growing | personality traits | level | level trait harm [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | score | change | rate | association | change parkinsonism [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | score | change | rate | association | change parkinsonism [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 969 | the Rush Memory and Aging Project ||| annually | the Unified Parkinson's [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 5 years ||| 80 years old | 14 years | 10 | about 0.05 unit/ year | Estimate | 0.054 | S.E. | 0.007 | p <0.001 | Estimate | 0.004 | S.E. | 0.001 | p <0.001 ||| 6 | about 50% ||| 5% ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 969 | the Rush Memory and Aging Project ||| annually | the Unified Parkinson's ||| 5 years ||| 80 years old | 14 years | 10 | about 0.05 unit/ year | Estimate | 0.054 | S.E. | 0.007 | p <0.001 | Estimate | 0.004 | S.E. | 0.001 | p <0.001 ||| 6 | about 50% ||| 5% ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 969 | the Rush Memory and Aging Project ||| annually | the Unified Parkinson's ||| 5 years ||| 80 years old | 14 years | 10 | about 0.05 unit/ year | Estimate | 0.054 | S.E. | 0.007 | p <0.001 | Estimate | 0.004 | S.E. | 0.001 | p <0.001 ||| 6 | about 50% ||| 5% ||| ||| [SUMMARY]"}}},{"rowIdx":280,"cells":{"title":{"kind":"string","value":"Secular Trends in Dietary Intake over a 20-Year Period in People with Type 2 Diabetes in Japan: A Comparative Study of Two Nationwide Registries; Japan Diabetes Complications Study (JDCS) and Japan Diabetes Clinical Data Management Study (JDDM)."},"pmid":{"kind":"string","value":"34684444"},"background_abstract":{"kind":"string","value":"In order to provide effective dietary guidance, it is necessary to consider dietary intake, which can change over time. This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We compared the results of two dietary surveys that used the food frequency questionnaire format. The first was conducted in 1996 by the Japan Diabetes Complications Study (JDCS) (n = 1509; males 53.3%), and the second in 2014-2018 by the Japan Diabetes Clinical Data Management Study (JDDM) (n = 1145; males 65.6%). Both are nationwide representative registries of outpatients with type 2 diabetes in Japan."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Over a 20-year period, both men and women with type 2 diabetes had a significant increase in body mass index (BMI). Nonetheless, there was only a small change in energy intake. Conversely, there was a significant increase in fat intake and thus in the fat-to-energy ratio. With regard to food groups, there was a significant increase in meat intake and a decrease in the intake of fish, soybeans/soy products, vegetables, and fruits, with a particularly significant decrease in vegetables."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Even in Japan, an industrialized country with a stable socioeconomic environment, there were many significant changes in the dietary intake of patients with type 2 diabetes over the 20-year period."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Biomarkers","Body Mass Index","Diabetes Mellitus, Type 2","Diet","Disease Susceptibility","Energy Intake","Feeding Behavior","Female","Humans","Japan","Male","Middle Aged","Public Health Surveillance","Registries"],"string":"[\n \"Aged\",\n \"Biomarkers\",\n \"Body Mass Index\",\n \"Diabetes Mellitus, Type 2\",\n \"Diet\",\n \"Disease Susceptibility\",\n \"Energy Intake\",\n \"Feeding Behavior\",\n \"Female\",\n \"Humans\",\n \"Japan\",\n \"Male\",\n \"Middle Aged\",\n \"Public Health Surveillance\",\n \"Registries\"\n]"},"pmcid":{"kind":"string","value":"8538089"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Diet plays a central role in diabetes self-management education and support [1]. To provide effective dietary guidance and/or to establish contemporary dietary guidelines, we must understand and take into account the changes in dietary intake and pattern over time. Dietary habits, including dietary content, are strongly influenced by changes in socioeconomic conditions and the environment [2]. The National Health and Nutrition Survey in Japan revealed decreases in intakes of 151 kcal (−7.4%) of energy, 24.9 g (−8.9%) of carbohydrates, 12.7 g (−15.5%) of protein, and 3.8 g (−27.7%) of salt (as sodium chloride) over a period of 20 years (1996–2016).\nPeople with diabetes often receive nutritional guidance in the early stages of the disease, so their knowledge, as well as dietary content and habits, may differ from those who do not have diabetes. Thus, dietary changes in people with type 2 diabetes may differ from the general population. However, very few studies have investigated this change. In a study reporting on the dietary changes made by people with type 2 diabetes in the United States over the past 25 years, Casagrande et al. [3] reported 206 kcal (+12.2%) and 339 mg (+11.2%) increases in energy and sodium, respectively, as well as a 0.9 g (−8.7%) decrease in dietary fiber. In addition, according to the Dietary Survey of Type 2 Diabetes in Spain [4], although the sample size was small at 200, protein intake decreased by 6.9% in men and 1.4% in women, and fat intake increased by 3.2% in men and 5.5% in women in 2000 compared to 1993. However, as these studies focused on nutrient changes, it was unclear what changes in foods contributed to these nutrient changes. Dietary intake by food groups in those with type 2 diabetes has been previously reported [5], but to the best of our knowledge, there have been no studies on changes in dietary intake over time.\nThe diabetic population in the Asian region is rapidly increasing, with those in East Asia accounting for approximately a quarter of the world diabetes population [6]. Therefore, from the perspective of global epidemiology, it is important to elucidate changes in the nutrient intake of Asian people with diabetes over time. Previously, we reported [7] on the nutritional status of Japanese patients with type 2 diabetes from a survey conducted in 1996. However, no study thus far has examined changes in nutrient intake in the Asian diabetic population over time on a nationwide scale.\nThe purpose of this study was to reveal the detailed changes in dietary intake in Japanese patients with type 2 diabetes over a 20-year period beginning in 1996. In the present study, we conducted a nutritional survey, almost identical to our previous survey, which focused on a newly established nationwide registry of Japanese patients with type 2 diabetes. We then compared the results with those of our previous study. The results of this study reveal the importance of understanding which areas need to be focused on for efficient and effective dietary guidance, and thus for the creation of contemporary dietary guidelines. In addition, the results may provide fundamental insights into the differences in dietary advice received by patients of Asian and Western origins."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":"Table 1 shows the clinical features of participants according to registry and sex. Although statistically significant differences were found between JDCS and JDDM patients in most variables, the differences in age and diabetes duration were too small to be considered clinically different. Variables that were considered to be clinically different between registries included BMI, HbA1c, blood pressure, and smoking rate among men. The BMI was significantly higher (+3.1 kg/m2 in men and +2.7 kg/m2 in women) in JDDM participants compared to JDCS participants. Comparing the mean BMI between JDCS and JDDM participants, the proportion of obese patients defined by BMI ≥ 25 kg/m2 according to Japanese criterion was significantly greater in JDDM participants, being approximately 20% to 50% higher in men and approximately 30% to 50% higher in women. HbA1c was significantly lower by approximately 1% and systolic blood pressure by approximately 5 mmHg for both men and women among JDDM participants, and the difference in the smoking rate for men was approximately 15% lower in JDDM participants than in JDCS participants.\nWhen participants were divided into two groups based on the median age of 60 years, the characteristics were similar to those identified in the comparison of all groups, regardless of age. However, there was a dramatic increase in the BMI of JDDM participants younger than 60 years compared to JDCS participants in the same age group (Supplementary Table S1).\nTable 2 shows a comparison of daily nutrient intake between patients in both studies. In Model 2, which was adjusted for age, HbA1c, BMI, insulin use, and oral medication use, there was no significant difference in energy intake in men between the two studies, but in women, there was a slight, but significant, increase of approximately 5% in the JDDM compared to the JDCS. Regarding proportions of macronutrients, the fat-to-energy ratio increased significantly, by about 3%, and the carbohydrate-to-energy ratio and protein-to-energy ratio decreased significantly for both men and women from the JDCS to the JDDM. Regarding changes in fat intake, the proportion of energy derived from saturated fatty acids increased significantly from 7.6% to 9.0% in men and from 8.3% to 9.4% in women in the interval between the surveys, whereas the proportion of energy derived from polyunsaturated fatty acids decreased significantly. Other variables that showed nutritionally meaningful differences between the two studies included significant decreases in dietary fiber, by 1.7 g for men and 1.6 g for women, and in salt, by 1.9 g for men and 2.7 g for women. The results were similar to the comparison of all groups, with fat-to-energy ratio and saturated fatty acid-to-energy ratio significantly increased in JDDM patients regardless of age, and polyunsaturated fatty acid-to-energy ratio, dietary fiber, and salt significantly decreased in JDDM patients (Supplementary Table S2).\nTable 3 shows a comparison of daily food intakes categorized by food groups. In terms of grains intake, from the JDCS to the JDDM, there was a slight, but significant, decrease of 7.4% in men, but no significant difference was found in women. Meat intake by JDDM patients was significantly increased, by about 60%, for both men and women compared to JDCS patients, and intake of fish and soybean/soy products was significantly decreased in JDDM patients. In particular, changes in meat and fish intake were remarkable, with an increase of about 30 g of meat in the JDDM participants and a decrease of about 30 g of fish in the JDDM participants, for both men and women. In summary, the JDCS patients consumed more fish and soybean/soy products than meat, while the JDDM patients consumed more meat than fish and soybean/soy products; thus, the composition of the major protein food sources was reversed over the 20-year period. In addition, dairy intake decreased by about 20%, and intake of sweets and snacks was about three times higher in that interval. On the other hand, the intake of vegetables and fruits among JDDM patients was dramatically reduced by about 30% in both men and women compared with JDCS patients. Both intakes of green-yellow vegetables and other vegetables significantly decreased from the time of the JDCS survey to the JDDM survey. In particular, intake of green-yellow vegetables was markedly reduced between the two surveys by 42.0% for men and 35.0% for women. Regardless of age, meat intake increased in JDDM patients, while fish and soybean/soy products showed a decrease. However, there was no trend toward a reversal of the composition of the major protein food sources in patients aged 60 years and older. In both the under 60 and over 60 age groups, there was an increase in sweets and snacks and a decrease in fruits and total vegetable intake (Supplementary Table S3)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["2. Materials and Methods"],"string":"[\n \"2. Materials and Methods\"\n]"},"other_sections_texts":{"kind":"list like","value":["This study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study."],"string":"[\n \"This study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null],"string":"[\n null\n]"},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Materials and Methods","3. Results","4. Discussion"],"string":"[\n \"1. Introduction\",\n \"2. Materials and Methods\",\n \"3. Results\",\n \"4. Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Diet plays a central role in diabetes self-management education and support [1]. To provide effective dietary guidance and/or to establish contemporary dietary guidelines, we must understand and take into account the changes in dietary intake and pattern over time. Dietary habits, including dietary content, are strongly influenced by changes in socioeconomic conditions and the environment [2]. The National Health and Nutrition Survey in Japan revealed decreases in intakes of 151 kcal (−7.4%) of energy, 24.9 g (−8.9%) of carbohydrates, 12.7 g (−15.5%) of protein, and 3.8 g (−27.7%) of salt (as sodium chloride) over a period of 20 years (1996–2016).\nPeople with diabetes often receive nutritional guidance in the early stages of the disease, so their knowledge, as well as dietary content and habits, may differ from those who do not have diabetes. Thus, dietary changes in people with type 2 diabetes may differ from the general population. However, very few studies have investigated this change. In a study reporting on the dietary changes made by people with type 2 diabetes in the United States over the past 25 years, Casagrande et al. [3] reported 206 kcal (+12.2%) and 339 mg (+11.2%) increases in energy and sodium, respectively, as well as a 0.9 g (−8.7%) decrease in dietary fiber. In addition, according to the Dietary Survey of Type 2 Diabetes in Spain [4], although the sample size was small at 200, protein intake decreased by 6.9% in men and 1.4% in women, and fat intake increased by 3.2% in men and 5.5% in women in 2000 compared to 1993. However, as these studies focused on nutrient changes, it was unclear what changes in foods contributed to these nutrient changes. Dietary intake by food groups in those with type 2 diabetes has been previously reported [5], but to the best of our knowledge, there have been no studies on changes in dietary intake over time.\nThe diabetic population in the Asian region is rapidly increasing, with those in East Asia accounting for approximately a quarter of the world diabetes population [6]. Therefore, from the perspective of global epidemiology, it is important to elucidate changes in the nutrient intake of Asian people with diabetes over time. Previously, we reported [7] on the nutritional status of Japanese patients with type 2 diabetes from a survey conducted in 1996. However, no study thus far has examined changes in nutrient intake in the Asian diabetic population over time on a nationwide scale.\nThe purpose of this study was to reveal the detailed changes in dietary intake in Japanese patients with type 2 diabetes over a 20-year period beginning in 1996. In the present study, we conducted a nutritional survey, almost identical to our previous survey, which focused on a newly established nationwide registry of Japanese patients with type 2 diabetes. We then compared the results with those of our previous study. The results of this study reveal the importance of understanding which areas need to be focused on for efficient and effective dietary guidance, and thus for the creation of contemporary dietary guidelines. In addition, the results may provide fundamental insights into the differences in dietary advice received by patients of Asian and Western origins.","This study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study.","Table 1 shows the clinical features of participants according to registry and sex. Although statistically significant differences were found between JDCS and JDDM patients in most variables, the differences in age and diabetes duration were too small to be considered clinically different. Variables that were considered to be clinically different between registries included BMI, HbA1c, blood pressure, and smoking rate among men. The BMI was significantly higher (+3.1 kg/m2 in men and +2.7 kg/m2 in women) in JDDM participants compared to JDCS participants. Comparing the mean BMI between JDCS and JDDM participants, the proportion of obese patients defined by BMI ≥ 25 kg/m2 according to Japanese criterion was significantly greater in JDDM participants, being approximately 20% to 50% higher in men and approximately 30% to 50% higher in women. HbA1c was significantly lower by approximately 1% and systolic blood pressure by approximately 5 mmHg for both men and women among JDDM participants, and the difference in the smoking rate for men was approximately 15% lower in JDDM participants than in JDCS participants.\nWhen participants were divided into two groups based on the median age of 60 years, the characteristics were similar to those identified in the comparison of all groups, regardless of age. However, there was a dramatic increase in the BMI of JDDM participants younger than 60 years compared to JDCS participants in the same age group (Supplementary Table S1).\nTable 2 shows a comparison of daily nutrient intake between patients in both studies. In Model 2, which was adjusted for age, HbA1c, BMI, insulin use, and oral medication use, there was no significant difference in energy intake in men between the two studies, but in women, there was a slight, but significant, increase of approximately 5% in the JDDM compared to the JDCS. Regarding proportions of macronutrients, the fat-to-energy ratio increased significantly, by about 3%, and the carbohydrate-to-energy ratio and protein-to-energy ratio decreased significantly for both men and women from the JDCS to the JDDM. Regarding changes in fat intake, the proportion of energy derived from saturated fatty acids increased significantly from 7.6% to 9.0% in men and from 8.3% to 9.4% in women in the interval between the surveys, whereas the proportion of energy derived from polyunsaturated fatty acids decreased significantly. Other variables that showed nutritionally meaningful differences between the two studies included significant decreases in dietary fiber, by 1.7 g for men and 1.6 g for women, and in salt, by 1.9 g for men and 2.7 g for women. The results were similar to the comparison of all groups, with fat-to-energy ratio and saturated fatty acid-to-energy ratio significantly increased in JDDM patients regardless of age, and polyunsaturated fatty acid-to-energy ratio, dietary fiber, and salt significantly decreased in JDDM patients (Supplementary Table S2).\nTable 3 shows a comparison of daily food intakes categorized by food groups. In terms of grains intake, from the JDCS to the JDDM, there was a slight, but significant, decrease of 7.4% in men, but no significant difference was found in women. Meat intake by JDDM patients was significantly increased, by about 60%, for both men and women compared to JDCS patients, and intake of fish and soybean/soy products was significantly decreased in JDDM patients. In particular, changes in meat and fish intake were remarkable, with an increase of about 30 g of meat in the JDDM participants and a decrease of about 30 g of fish in the JDDM participants, for both men and women. In summary, the JDCS patients consumed more fish and soybean/soy products than meat, while the JDDM patients consumed more meat than fish and soybean/soy products; thus, the composition of the major protein food sources was reversed over the 20-year period. In addition, dairy intake decreased by about 20%, and intake of sweets and snacks was about three times higher in that interval. On the other hand, the intake of vegetables and fruits among JDDM patients was dramatically reduced by about 30% in both men and women compared with JDCS patients. Both intakes of green-yellow vegetables and other vegetables significantly decreased from the time of the JDCS survey to the JDDM survey. In particular, intake of green-yellow vegetables was markedly reduced between the two surveys by 42.0% for men and 35.0% for women. Regardless of age, meat intake increased in JDDM patients, while fish and soybean/soy products showed a decrease. However, there was no trend toward a reversal of the composition of the major protein food sources in patients aged 60 years and older. In both the under 60 and over 60 age groups, there was an increase in sweets and snacks and a decrease in fruits and total vegetable intake (Supplementary Table S3).","This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period using two surveys with essentially the same format. Although the backgrounds of the patients, including age and duration of diabetes, were quite similar between the two studies, BMI and obesity rates were markedly increased in the later study. Regarding dietary content, fat intake increased, including saturated fatty acids, along with significant increases in the consumption of meat and sweets and snacks, and a significant decrease in vegetable and fish intake. To date, there have been no studies on the changes in dietary intake by persons with type 2 diabetes in Asia, except for a report on adherence to dietary recommendations in Korea [12]. To our knowledge, the changes in nutrient and food intake have not been reported. Therefore, this is the first study to clarify the secular changes in the dietary status of patients with type 2 diabetes in Asia.\nSelf-management education and support for diet and physical activity are essential in the care of overweight or obese people with type 2 diabetes. A previous study observing the secular trends in the United States from 1988 to 2012 [3] showed a trend of increases in BMI and energy intake in patients with type 2 diabetes, which was also the case even in adults without diabetes. On the other hand, in our study of East Asians, the BMI markedly increased from 22.7 kg/m2 to 25.8 kg/m2 in men and 23.2 kg/m2 to 25.9 kg/m2 in women, although no such remarkable changes were found in the Japanese general population according to the Japan National Health and Nutrition Survey performed during a similar period (1996 and 2016) (from 23.3 kg/ m2 to 24.0 kg/m2 in men and 23.2 kg m2 to 22.6 kg/m2 in women) [13]. This suggests that secular changes in BMI and obesity were different between people with and without type 2 diabetes and between the East and the West. According to the National Health and Nutrition Surveys, from 1996 to 2016, the energy intake by the general population aged 40–69 years mildly declined from 2284 kcal to 2145 kcal (−139 kcal) for men and from 1880 kcal to 1722 kcal (−158 kcal) for women, although the dietary survey method was different from our method [14].\nWe previously reported, using JDCS baseline data, that Western populations with diabetes are mostly obese and are also more obese than the general population, whereas Japanese people with diabetes not only have a mean BMI within the normal range, but also it is almost the same as in the general population [15]. The results of the current study, including the JDDM results 20 years after the initial JDCS survey, strongly support the fact that obesity among those with type 2 diabetes has recently increased in Japan, as is the case in Western countries [16]. On the other hand, it is not clear from this study alone why the increase in BMI in Japanese patients with type 2 diabetes was significantly greater than in the general population, even though there was no significant increase in energy intake between the two studies. Our previous study [17] suggested that there are racial differences in the relationship between energy intake and obesity, but several other hypotheses are also possible.\nThe first possible explanation for the significant increase in BMI without a significant increase in energy intake in Japanese patients with type 2 diabetes may be the effect of decreased energy expenditure due to decreased physical activity. A comparison of the activity levels between people with diabetes and the general population found no significant difference in physical activity levels and leisure-time physical activity patterns, and reported that neither groups achieved the recommended physical activity goals [18,19]. The decrease in the number of steps by the general Japanese population from 1996 to 2016 was remarkable for both men and women [13], and it was pointed out that the decrease in physical activity was due to the development of transportation [20]. In this study, direct comparisons were difficult because of the differences in the studies’ survey methods regarding the amount of physical activity; however, 44.4% of patients in the JDDM reported low levels of overall physical activity in their daily lives, which was calculated from activity time and activity intensity. Future research on secular trends in the amount of physical activity in patients with type 2 diabetes is expected.\nThe second theory as to why BMI increased without an increase in energy intake is that it could be due to the effect of smoking rates and medications. The BMI of smokers was reportedly lower than that of non-smokers [21,22], and the smoking rate of men in the JDDM with a high BMI was significantly lower than that in the JDCS. In addition, the increase in the rate of insulin use over time that was observed between these studies may be a factor in weight gain. The fact that there was a significant decrease in the rate of use of oral hypoglycemic agents may have contributed to weight gain or weight loss, depending on the type of oral hypoglycemic agent [23]. Further studies, including a detailed follow-up of the status of the medications, are necessary.\nAs a third conjecture, the ratio of macronutrients may have affected these findings. Regarding the energy-producing nutrients, both men and women in the JDDM had higher fat-to-energy ratios than those in the JDCS. In the JDDM compared to the JDCS, men had a lower carbohydrate-to-energy ratio and protein-to-energy ratio and women had a lower protein-to-energy ratio. Women in the JDDM had an increase in energy intake, which could be attributed to an increased fat intake as their carbohydrate and protein intakes did not increase. In the Japanese National Health and Nutrition Survey, which was of a similar duration (1996 and 2016), the fat-to-energy ratio increased (men: +2.6%, women: +2.9%), but carbohydrate and protein intakes tended to decrease (carbohydrate: −22.6 g/day for men, −33.4 g/day for women, protein: −15.9 g/day for men, −13.0 g/day for women), and fat intake was almost unchanged, suggesting that the fat-to-energy ratio was relatively increased. Increased fat-to-energy ratios due to an increased fat intake in patients with type 2 diabetes may have contributed to the increased BMI. However, previous studies have not provided a unified consensus on the association between the fat-to-energy ratio and obesity, which needs to be clarified in the future [24].\nIt is well known that, regardless of body weight, a high-fat diet, especially an excessive intake of saturated fatty acids and a high saturated fatty acid-to-energy ratio, is significantly associated with a higher risk of cardiovascular events in patients with type 2 diabetes [25]. According to the Japanese practice guidelines for diabetes [26], a reduction in saturated fatty acids is recommended when the lipid-to-energy ratio exceeds 25%. In addition, Dietary Reference Intakes for Japanese [27] set a target saturated fatty acid-to-energy ratio of ≤7%. Among our patients with a fat-to-energy ratio of ≥25%, only 9.5% of JDCS patients and 4.4% of JDDM patients had a saturated fatty acid-to-energy ratio of ≤7%, suggesting the necessity for enhanced guidance to reduce the saturated fatty acid-to-energy ratio. The saturated fatty acid-to-energy ratio for Americans with type 2 diabetes was approximately 11%, which was still higher than the level (<10%) recommended by the U.S. Dietary Guidelines, but it did not increase between 1988 and 2012 [28].\nIn contrast, in our Japanese patients, the saturated fatty acid-to-energy ratio increased significantly over the 20 years to approximately 9% in both men and women, and the percentage of patients with a saturated fatty acid-to-energy ratio of more than 10% increased significantly from 8.2% in men and 13.3% in women in the JDCS to 28.4% in men and 36.6% in women in the JDDM. This suggests that the fat intake pattern of Japanese people with type 2 diabetes is approaching that of Western patients. Therefore, correcting excessive lipid intake while taking into account the content and quality of lipids is considered to be a crucial issue.\nIn terms of secular changes in food group intake patterns, an increase in meat intake and a decrease in the consumption of fish and dairy products were observed as sources of protein intake. JDCS patients had higher intakes of fish and soybeans/soy products than meat, whereas JDDM patients had higher intakes of meat than fish and soybeans/soy products. The National Health and Nutrition Survey [29] also showed the same secular changes in food group intake patterns as this study.\nIt is indicated that patients with type 2 diabetes in Japan had a major change in the composition of their protein sources, as did the general Japanese population, which was a change to a meat-centered diet. In general, among people in Western countries, the circumstances in which meat intake is higher than fish intake continues. It is inferred that the diet of Japanese patients with type 2 diabetes has been transformed into a diet centered on meat due to the influx of various lifestyle patterns and dietary cultures from Western countries, as is the case with the general Japanese population. It has been reported that meat was associated with cardiovascular complications and mortality in people with type 2 diabetes in a study in the US [30] and in our previous study [31]. Thus, it is considered necessary to strengthen the guidance on protein sources to prevent complications in the future.\nMeat is the food with the highest contribution to fat intake by Japanese people. Therefore, it is thought that an increased meat intake may also affect fat-to-energy ratios and saturated fatty acid increases in JDDM patients. Aside from meat, saturated fatty acids are found in the Japanese diet in milk, butter, and coconut oil, which are commonly used as ingredients in Western sweets rather than in traditional Japanese ones (which are mostly made with sugar, rice, and beans [32]). Although the type of sweets could not be identified in this survey, as their intake more than doubled from the JDCS to the JDDM, it is inferred that in conjunction with meat, the increase in the intake of sweets contributed to the increase in saturated fatty acid consumption.\nContrary to meat, the intake of fruits and vegetables decreased significantly between the two studies. As vegetables and fruits are good sources of dietary fiber, fiber intake decreased significantly as vegetable and fruit intakes decreased. A higher intake of dietary fiber is known to improve glycemic control, and we previously reported that it was significantly associated with a reduced risk of stroke [33] and retinopathy [34] in JDCS patients. However, despite its importance, the intake of dietary fiber was lower than the recommended goal of 20 g/day or higher in 1996, and even declined in the following 20 years. A decreased intake of fruits and vegetables is apparent in the Japanese general population [29], and is considered to be an important issue in dietary guidance.\nAs to the background of the decrease in the consumption of vegetables and fruits, survey reports have suggested that differences in lifestyle, such as economic conditions [35] and living alone, exist [36] and that diabetic patients who work may choose a less diversified diet [37]. It was also reported that the consumption of vegetables and fruits tends to be higher with a lower frequency of eating meals away from home [38]. As to the proportions of food expenditure in Japan from 1995 to 2015, the consumption of fresh foods has decreased, and eating out and consuming processed foods have increased from 65.4% to 72.6% [39]. This suggests that the increase in eating out and buying processed foods may influence the decrease in fruit and vegetable intake, not only in the general population, but also in those with type 2 diabetes. This Westernization of dietary habits has been widely adopted, and advice on the dietary treatment of diabetes should consider the current dietary situation.\nThis study has several limitations. First, both the JDCS and the JDDM were comprised of patients from different cohorts, although they were both multicenter studies. However, both studies were conducted at outpatient clinics throughout Japan specializing in diabetes, and the clinical settings were similar except for the time period studied. An additional limitation is that the number of participants that were included in both surveys was different from the potential candidates. Of the 2033 JDCS patients, 1588 responded to a baseline dietary survey, and 524 patients who had missing values in the dietary survey were excluded. In the JDDM patients, 1145 of 2337 patients met the eligibility criteria for the JDCS and responded to the baseline dietary survey. However, there were no notable differences in baseline characteristics between the responders and non-responders [8], and consequently, no selection bias was found. Finally, the participants’ dietary intake was obtained from a self-administered questionnaire, which could be an underestimation. As for the long-term comparison of 20 years, changes had been made in the food composition tables and in the FFQg version used in both studies. However, those changes corresponded to changes in dietary habits and food ingredients due to secular trends, and there was little effect on energy intake and intake of macronutrients due to the changes in the food composition tables.\nThe results of this study clarify the changes in the dietary intake of Japanese patients with type 2 diabetes over 20 years, beginning in 1996. Despite a lack of significant change in the total energy intake during the period, both men and women with type 2 diabetes had a large increase in BMI. A rise in the fat-to-energy ratio due to an increase in fat intake was also observed, particularly in men. In terms of intake by food group, both men and women had clearly unfavorable changes in terms of complication prevention. These included an increase in the intake of meat and a decrease in the intake of fish, soybean/soy products, vegetables, and fruits. The results of our study strongly suggest that, in order to prevent complications in Japanese patients with type 2 diabetes in the future, the provision of nutritional guidance, in line with these chronological changes in dietary intake, is necessary."],"string":"[\n \"Diet plays a central role in diabetes self-management education and support [1]. To provide effective dietary guidance and/or to establish contemporary dietary guidelines, we must understand and take into account the changes in dietary intake and pattern over time. Dietary habits, including dietary content, are strongly influenced by changes in socioeconomic conditions and the environment [2]. The National Health and Nutrition Survey in Japan revealed decreases in intakes of 151 kcal (−7.4%) of energy, 24.9 g (−8.9%) of carbohydrates, 12.7 g (−15.5%) of protein, and 3.8 g (−27.7%) of salt (as sodium chloride) over a period of 20 years (1996–2016).\\nPeople with diabetes often receive nutritional guidance in the early stages of the disease, so their knowledge, as well as dietary content and habits, may differ from those who do not have diabetes. Thus, dietary changes in people with type 2 diabetes may differ from the general population. However, very few studies have investigated this change. In a study reporting on the dietary changes made by people with type 2 diabetes in the United States over the past 25 years, Casagrande et al. [3] reported 206 kcal (+12.2%) and 339 mg (+11.2%) increases in energy and sodium, respectively, as well as a 0.9 g (−8.7%) decrease in dietary fiber. In addition, according to the Dietary Survey of Type 2 Diabetes in Spain [4], although the sample size was small at 200, protein intake decreased by 6.9% in men and 1.4% in women, and fat intake increased by 3.2% in men and 5.5% in women in 2000 compared to 1993. However, as these studies focused on nutrient changes, it was unclear what changes in foods contributed to these nutrient changes. Dietary intake by food groups in those with type 2 diabetes has been previously reported [5], but to the best of our knowledge, there have been no studies on changes in dietary intake over time.\\nThe diabetic population in the Asian region is rapidly increasing, with those in East Asia accounting for approximately a quarter of the world diabetes population [6]. Therefore, from the perspective of global epidemiology, it is important to elucidate changes in the nutrient intake of Asian people with diabetes over time. Previously, we reported [7] on the nutritional status of Japanese patients with type 2 diabetes from a survey conducted in 1996. However, no study thus far has examined changes in nutrient intake in the Asian diabetic population over time on a nationwide scale.\\nThe purpose of this study was to reveal the detailed changes in dietary intake in Japanese patients with type 2 diabetes over a 20-year period beginning in 1996. In the present study, we conducted a nutritional survey, almost identical to our previous survey, which focused on a newly established nationwide registry of Japanese patients with type 2 diabetes. We then compared the results with those of our previous study. The results of this study reveal the importance of understanding which areas need to be focused on for efficient and effective dietary guidance, and thus for the creation of contemporary dietary guidelines. In addition, the results may provide fundamental insights into the differences in dietary advice received by patients of Asian and Western origins.\",\n \"This study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study.\",\n \"Table 1 shows the clinical features of participants according to registry and sex. Although statistically significant differences were found between JDCS and JDDM patients in most variables, the differences in age and diabetes duration were too small to be considered clinically different. Variables that were considered to be clinically different between registries included BMI, HbA1c, blood pressure, and smoking rate among men. The BMI was significantly higher (+3.1 kg/m2 in men and +2.7 kg/m2 in women) in JDDM participants compared to JDCS participants. Comparing the mean BMI between JDCS and JDDM participants, the proportion of obese patients defined by BMI ≥ 25 kg/m2 according to Japanese criterion was significantly greater in JDDM participants, being approximately 20% to 50% higher in men and approximately 30% to 50% higher in women. HbA1c was significantly lower by approximately 1% and systolic blood pressure by approximately 5 mmHg for both men and women among JDDM participants, and the difference in the smoking rate for men was approximately 15% lower in JDDM participants than in JDCS participants.\\nWhen participants were divided into two groups based on the median age of 60 years, the characteristics were similar to those identified in the comparison of all groups, regardless of age. However, there was a dramatic increase in the BMI of JDDM participants younger than 60 years compared to JDCS participants in the same age group (Supplementary Table S1).\\nTable 2 shows a comparison of daily nutrient intake between patients in both studies. In Model 2, which was adjusted for age, HbA1c, BMI, insulin use, and oral medication use, there was no significant difference in energy intake in men between the two studies, but in women, there was a slight, but significant, increase of approximately 5% in the JDDM compared to the JDCS. Regarding proportions of macronutrients, the fat-to-energy ratio increased significantly, by about 3%, and the carbohydrate-to-energy ratio and protein-to-energy ratio decreased significantly for both men and women from the JDCS to the JDDM. Regarding changes in fat intake, the proportion of energy derived from saturated fatty acids increased significantly from 7.6% to 9.0% in men and from 8.3% to 9.4% in women in the interval between the surveys, whereas the proportion of energy derived from polyunsaturated fatty acids decreased significantly. Other variables that showed nutritionally meaningful differences between the two studies included significant decreases in dietary fiber, by 1.7 g for men and 1.6 g for women, and in salt, by 1.9 g for men and 2.7 g for women. The results were similar to the comparison of all groups, with fat-to-energy ratio and saturated fatty acid-to-energy ratio significantly increased in JDDM patients regardless of age, and polyunsaturated fatty acid-to-energy ratio, dietary fiber, and salt significantly decreased in JDDM patients (Supplementary Table S2).\\nTable 3 shows a comparison of daily food intakes categorized by food groups. In terms of grains intake, from the JDCS to the JDDM, there was a slight, but significant, decrease of 7.4% in men, but no significant difference was found in women. Meat intake by JDDM patients was significantly increased, by about 60%, for both men and women compared to JDCS patients, and intake of fish and soybean/soy products was significantly decreased in JDDM patients. In particular, changes in meat and fish intake were remarkable, with an increase of about 30 g of meat in the JDDM participants and a decrease of about 30 g of fish in the JDDM participants, for both men and women. In summary, the JDCS patients consumed more fish and soybean/soy products than meat, while the JDDM patients consumed more meat than fish and soybean/soy products; thus, the composition of the major protein food sources was reversed over the 20-year period. In addition, dairy intake decreased by about 20%, and intake of sweets and snacks was about three times higher in that interval. On the other hand, the intake of vegetables and fruits among JDDM patients was dramatically reduced by about 30% in both men and women compared with JDCS patients. Both intakes of green-yellow vegetables and other vegetables significantly decreased from the time of the JDCS survey to the JDDM survey. In particular, intake of green-yellow vegetables was markedly reduced between the two surveys by 42.0% for men and 35.0% for women. Regardless of age, meat intake increased in JDDM patients, while fish and soybean/soy products showed a decrease. However, there was no trend toward a reversal of the composition of the major protein food sources in patients aged 60 years and older. In both the under 60 and over 60 age groups, there was an increase in sweets and snacks and a decrease in fruits and total vegetable intake (Supplementary Table S3).\",\n \"This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period using two surveys with essentially the same format. Although the backgrounds of the patients, including age and duration of diabetes, were quite similar between the two studies, BMI and obesity rates were markedly increased in the later study. Regarding dietary content, fat intake increased, including saturated fatty acids, along with significant increases in the consumption of meat and sweets and snacks, and a significant decrease in vegetable and fish intake. To date, there have been no studies on the changes in dietary intake by persons with type 2 diabetes in Asia, except for a report on adherence to dietary recommendations in Korea [12]. To our knowledge, the changes in nutrient and food intake have not been reported. Therefore, this is the first study to clarify the secular changes in the dietary status of patients with type 2 diabetes in Asia.\\nSelf-management education and support for diet and physical activity are essential in the care of overweight or obese people with type 2 diabetes. A previous study observing the secular trends in the United States from 1988 to 2012 [3] showed a trend of increases in BMI and energy intake in patients with type 2 diabetes, which was also the case even in adults without diabetes. On the other hand, in our study of East Asians, the BMI markedly increased from 22.7 kg/m2 to 25.8 kg/m2 in men and 23.2 kg/m2 to 25.9 kg/m2 in women, although no such remarkable changes were found in the Japanese general population according to the Japan National Health and Nutrition Survey performed during a similar period (1996 and 2016) (from 23.3 kg/ m2 to 24.0 kg/m2 in men and 23.2 kg m2 to 22.6 kg/m2 in women) [13]. This suggests that secular changes in BMI and obesity were different between people with and without type 2 diabetes and between the East and the West. According to the National Health and Nutrition Surveys, from 1996 to 2016, the energy intake by the general population aged 40–69 years mildly declined from 2284 kcal to 2145 kcal (−139 kcal) for men and from 1880 kcal to 1722 kcal (−158 kcal) for women, although the dietary survey method was different from our method [14].\\nWe previously reported, using JDCS baseline data, that Western populations with diabetes are mostly obese and are also more obese than the general population, whereas Japanese people with diabetes not only have a mean BMI within the normal range, but also it is almost the same as in the general population [15]. The results of the current study, including the JDDM results 20 years after the initial JDCS survey, strongly support the fact that obesity among those with type 2 diabetes has recently increased in Japan, as is the case in Western countries [16]. On the other hand, it is not clear from this study alone why the increase in BMI in Japanese patients with type 2 diabetes was significantly greater than in the general population, even though there was no significant increase in energy intake between the two studies. Our previous study [17] suggested that there are racial differences in the relationship between energy intake and obesity, but several other hypotheses are also possible.\\nThe first possible explanation for the significant increase in BMI without a significant increase in energy intake in Japanese patients with type 2 diabetes may be the effect of decreased energy expenditure due to decreased physical activity. A comparison of the activity levels between people with diabetes and the general population found no significant difference in physical activity levels and leisure-time physical activity patterns, and reported that neither groups achieved the recommended physical activity goals [18,19]. The decrease in the number of steps by the general Japanese population from 1996 to 2016 was remarkable for both men and women [13], and it was pointed out that the decrease in physical activity was due to the development of transportation [20]. In this study, direct comparisons were difficult because of the differences in the studies’ survey methods regarding the amount of physical activity; however, 44.4% of patients in the JDDM reported low levels of overall physical activity in their daily lives, which was calculated from activity time and activity intensity. Future research on secular trends in the amount of physical activity in patients with type 2 diabetes is expected.\\nThe second theory as to why BMI increased without an increase in energy intake is that it could be due to the effect of smoking rates and medications. The BMI of smokers was reportedly lower than that of non-smokers [21,22], and the smoking rate of men in the JDDM with a high BMI was significantly lower than that in the JDCS. In addition, the increase in the rate of insulin use over time that was observed between these studies may be a factor in weight gain. The fact that there was a significant decrease in the rate of use of oral hypoglycemic agents may have contributed to weight gain or weight loss, depending on the type of oral hypoglycemic agent [23]. Further studies, including a detailed follow-up of the status of the medications, are necessary.\\nAs a third conjecture, the ratio of macronutrients may have affected these findings. Regarding the energy-producing nutrients, both men and women in the JDDM had higher fat-to-energy ratios than those in the JDCS. In the JDDM compared to the JDCS, men had a lower carbohydrate-to-energy ratio and protein-to-energy ratio and women had a lower protein-to-energy ratio. Women in the JDDM had an increase in energy intake, which could be attributed to an increased fat intake as their carbohydrate and protein intakes did not increase. In the Japanese National Health and Nutrition Survey, which was of a similar duration (1996 and 2016), the fat-to-energy ratio increased (men: +2.6%, women: +2.9%), but carbohydrate and protein intakes tended to decrease (carbohydrate: −22.6 g/day for men, −33.4 g/day for women, protein: −15.9 g/day for men, −13.0 g/day for women), and fat intake was almost unchanged, suggesting that the fat-to-energy ratio was relatively increased. Increased fat-to-energy ratios due to an increased fat intake in patients with type 2 diabetes may have contributed to the increased BMI. However, previous studies have not provided a unified consensus on the association between the fat-to-energy ratio and obesity, which needs to be clarified in the future [24].\\nIt is well known that, regardless of body weight, a high-fat diet, especially an excessive intake of saturated fatty acids and a high saturated fatty acid-to-energy ratio, is significantly associated with a higher risk of cardiovascular events in patients with type 2 diabetes [25]. According to the Japanese practice guidelines for diabetes [26], a reduction in saturated fatty acids is recommended when the lipid-to-energy ratio exceeds 25%. In addition, Dietary Reference Intakes for Japanese [27] set a target saturated fatty acid-to-energy ratio of ≤7%. Among our patients with a fat-to-energy ratio of ≥25%, only 9.5% of JDCS patients and 4.4% of JDDM patients had a saturated fatty acid-to-energy ratio of ≤7%, suggesting the necessity for enhanced guidance to reduce the saturated fatty acid-to-energy ratio. The saturated fatty acid-to-energy ratio for Americans with type 2 diabetes was approximately 11%, which was still higher than the level (<10%) recommended by the U.S. Dietary Guidelines, but it did not increase between 1988 and 2012 [28].\\nIn contrast, in our Japanese patients, the saturated fatty acid-to-energy ratio increased significantly over the 20 years to approximately 9% in both men and women, and the percentage of patients with a saturated fatty acid-to-energy ratio of more than 10% increased significantly from 8.2% in men and 13.3% in women in the JDCS to 28.4% in men and 36.6% in women in the JDDM. This suggests that the fat intake pattern of Japanese people with type 2 diabetes is approaching that of Western patients. Therefore, correcting excessive lipid intake while taking into account the content and quality of lipids is considered to be a crucial issue.\\nIn terms of secular changes in food group intake patterns, an increase in meat intake and a decrease in the consumption of fish and dairy products were observed as sources of protein intake. JDCS patients had higher intakes of fish and soybeans/soy products than meat, whereas JDDM patients had higher intakes of meat than fish and soybeans/soy products. The National Health and Nutrition Survey [29] also showed the same secular changes in food group intake patterns as this study.\\nIt is indicated that patients with type 2 diabetes in Japan had a major change in the composition of their protein sources, as did the general Japanese population, which was a change to a meat-centered diet. In general, among people in Western countries, the circumstances in which meat intake is higher than fish intake continues. It is inferred that the diet of Japanese patients with type 2 diabetes has been transformed into a diet centered on meat due to the influx of various lifestyle patterns and dietary cultures from Western countries, as is the case with the general Japanese population. It has been reported that meat was associated with cardiovascular complications and mortality in people with type 2 diabetes in a study in the US [30] and in our previous study [31]. Thus, it is considered necessary to strengthen the guidance on protein sources to prevent complications in the future.\\nMeat is the food with the highest contribution to fat intake by Japanese people. Therefore, it is thought that an increased meat intake may also affect fat-to-energy ratios and saturated fatty acid increases in JDDM patients. Aside from meat, saturated fatty acids are found in the Japanese diet in milk, butter, and coconut oil, which are commonly used as ingredients in Western sweets rather than in traditional Japanese ones (which are mostly made with sugar, rice, and beans [32]). Although the type of sweets could not be identified in this survey, as their intake more than doubled from the JDCS to the JDDM, it is inferred that in conjunction with meat, the increase in the intake of sweets contributed to the increase in saturated fatty acid consumption.\\nContrary to meat, the intake of fruits and vegetables decreased significantly between the two studies. As vegetables and fruits are good sources of dietary fiber, fiber intake decreased significantly as vegetable and fruit intakes decreased. A higher intake of dietary fiber is known to improve glycemic control, and we previously reported that it was significantly associated with a reduced risk of stroke [33] and retinopathy [34] in JDCS patients. However, despite its importance, the intake of dietary fiber was lower than the recommended goal of 20 g/day or higher in 1996, and even declined in the following 20 years. A decreased intake of fruits and vegetables is apparent in the Japanese general population [29], and is considered to be an important issue in dietary guidance.\\nAs to the background of the decrease in the consumption of vegetables and fruits, survey reports have suggested that differences in lifestyle, such as economic conditions [35] and living alone, exist [36] and that diabetic patients who work may choose a less diversified diet [37]. It was also reported that the consumption of vegetables and fruits tends to be higher with a lower frequency of eating meals away from home [38]. As to the proportions of food expenditure in Japan from 1995 to 2015, the consumption of fresh foods has decreased, and eating out and consuming processed foods have increased from 65.4% to 72.6% [39]. This suggests that the increase in eating out and buying processed foods may influence the decrease in fruit and vegetable intake, not only in the general population, but also in those with type 2 diabetes. This Westernization of dietary habits has been widely adopted, and advice on the dietary treatment of diabetes should consider the current dietary situation.\\nThis study has several limitations. First, both the JDCS and the JDDM were comprised of patients from different cohorts, although they were both multicenter studies. However, both studies were conducted at outpatient clinics throughout Japan specializing in diabetes, and the clinical settings were similar except for the time period studied. An additional limitation is that the number of participants that were included in both surveys was different from the potential candidates. Of the 2033 JDCS patients, 1588 responded to a baseline dietary survey, and 524 patients who had missing values in the dietary survey were excluded. In the JDDM patients, 1145 of 2337 patients met the eligibility criteria for the JDCS and responded to the baseline dietary survey. However, there were no notable differences in baseline characteristics between the responders and non-responders [8], and consequently, no selection bias was found. Finally, the participants’ dietary intake was obtained from a self-administered questionnaire, which could be an underestimation. As for the long-term comparison of 20 years, changes had been made in the food composition tables and in the FFQg version used in both studies. However, those changes corresponded to changes in dietary habits and food ingredients due to secular trends, and there was little effect on energy intake and intake of macronutrients due to the changes in the food composition tables.\\nThe results of this study clarify the changes in the dietary intake of Japanese patients with type 2 diabetes over 20 years, beginning in 1996. Despite a lack of significant change in the total energy intake during the period, both men and women with type 2 diabetes had a large increase in BMI. A rise in the fat-to-energy ratio due to an increase in fat intake was also observed, particularly in men. In terms of intake by food group, both men and women had clearly unfavorable changes in terms of complication prevention. These included an increase in the intake of meat and a decrease in the intake of fish, soybean/soy products, vegetables, and fruits. The results of our study strongly suggest that, in order to prevent complications in Japanese patients with type 2 diabetes in the future, the provision of nutritional guidance, in line with these chronological changes in dietary intake, is necessary.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,"results","discussion"],"string":"[\n \"intro\",\n null,\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["food intake","type 2 diabetes","obesity","diabetes mellitus","Asia"],"string":"[\n \"food intake\",\n \"type 2 diabetes\",\n \"obesity\",\n \"diabetes mellitus\",\n \"Asia\"\n]"},"whole_article_text":{"kind":"string","value":"1. Introduction:\nDiet plays a central role in diabetes self-management education and support [1]. To provide effective dietary guidance and/or to establish contemporary dietary guidelines, we must understand and take into account the changes in dietary intake and pattern over time. Dietary habits, including dietary content, are strongly influenced by changes in socioeconomic conditions and the environment [2]. The National Health and Nutrition Survey in Japan revealed decreases in intakes of 151 kcal (−7.4%) of energy, 24.9 g (−8.9%) of carbohydrates, 12.7 g (−15.5%) of protein, and 3.8 g (−27.7%) of salt (as sodium chloride) over a period of 20 years (1996–2016).\nPeople with diabetes often receive nutritional guidance in the early stages of the disease, so their knowledge, as well as dietary content and habits, may differ from those who do not have diabetes. Thus, dietary changes in people with type 2 diabetes may differ from the general population. However, very few studies have investigated this change. In a study reporting on the dietary changes made by people with type 2 diabetes in the United States over the past 25 years, Casagrande et al. [3] reported 206 kcal (+12.2%) and 339 mg (+11.2%) increases in energy and sodium, respectively, as well as a 0.9 g (−8.7%) decrease in dietary fiber. In addition, according to the Dietary Survey of Type 2 Diabetes in Spain [4], although the sample size was small at 200, protein intake decreased by 6.9% in men and 1.4% in women, and fat intake increased by 3.2% in men and 5.5% in women in 2000 compared to 1993. However, as these studies focused on nutrient changes, it was unclear what changes in foods contributed to these nutrient changes. Dietary intake by food groups in those with type 2 diabetes has been previously reported [5], but to the best of our knowledge, there have been no studies on changes in dietary intake over time.\nThe diabetic population in the Asian region is rapidly increasing, with those in East Asia accounting for approximately a quarter of the world diabetes population [6]. Therefore, from the perspective of global epidemiology, it is important to elucidate changes in the nutrient intake of Asian people with diabetes over time. Previously, we reported [7] on the nutritional status of Japanese patients with type 2 diabetes from a survey conducted in 1996. However, no study thus far has examined changes in nutrient intake in the Asian diabetic population over time on a nationwide scale.\nThe purpose of this study was to reveal the detailed changes in dietary intake in Japanese patients with type 2 diabetes over a 20-year period beginning in 1996. In the present study, we conducted a nutritional survey, almost identical to our previous survey, which focused on a newly established nationwide registry of Japanese patients with type 2 diabetes. We then compared the results with those of our previous study. The results of this study reveal the importance of understanding which areas need to be focused on for efficient and effective dietary guidance, and thus for the creation of contemporary dietary guidelines. In addition, the results may provide fundamental insights into the differences in dietary advice received by patients of Asian and Western origins.\n\n2. Materials and Methods:\nThis study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study.\n\n3. Results:\nTable 1 shows the clinical features of participants according to registry and sex. Although statistically significant differences were found between JDCS and JDDM patients in most variables, the differences in age and diabetes duration were too small to be considered clinically different. Variables that were considered to be clinically different between registries included BMI, HbA1c, blood pressure, and smoking rate among men. The BMI was significantly higher (+3.1 kg/m2 in men and +2.7 kg/m2 in women) in JDDM participants compared to JDCS participants. Comparing the mean BMI between JDCS and JDDM participants, the proportion of obese patients defined by BMI ≥ 25 kg/m2 according to Japanese criterion was significantly greater in JDDM participants, being approximately 20% to 50% higher in men and approximately 30% to 50% higher in women. HbA1c was significantly lower by approximately 1% and systolic blood pressure by approximately 5 mmHg for both men and women among JDDM participants, and the difference in the smoking rate for men was approximately 15% lower in JDDM participants than in JDCS participants.\nWhen participants were divided into two groups based on the median age of 60 years, the characteristics were similar to those identified in the comparison of all groups, regardless of age. However, there was a dramatic increase in the BMI of JDDM participants younger than 60 years compared to JDCS participants in the same age group (Supplementary Table S1).\nTable 2 shows a comparison of daily nutrient intake between patients in both studies. In Model 2, which was adjusted for age, HbA1c, BMI, insulin use, and oral medication use, there was no significant difference in energy intake in men between the two studies, but in women, there was a slight, but significant, increase of approximately 5% in the JDDM compared to the JDCS. Regarding proportions of macronutrients, the fat-to-energy ratio increased significantly, by about 3%, and the carbohydrate-to-energy ratio and protein-to-energy ratio decreased significantly for both men and women from the JDCS to the JDDM. Regarding changes in fat intake, the proportion of energy derived from saturated fatty acids increased significantly from 7.6% to 9.0% in men and from 8.3% to 9.4% in women in the interval between the surveys, whereas the proportion of energy derived from polyunsaturated fatty acids decreased significantly. Other variables that showed nutritionally meaningful differences between the two studies included significant decreases in dietary fiber, by 1.7 g for men and 1.6 g for women, and in salt, by 1.9 g for men and 2.7 g for women. The results were similar to the comparison of all groups, with fat-to-energy ratio and saturated fatty acid-to-energy ratio significantly increased in JDDM patients regardless of age, and polyunsaturated fatty acid-to-energy ratio, dietary fiber, and salt significantly decreased in JDDM patients (Supplementary Table S2).\nTable 3 shows a comparison of daily food intakes categorized by food groups. In terms of grains intake, from the JDCS to the JDDM, there was a slight, but significant, decrease of 7.4% in men, but no significant difference was found in women. Meat intake by JDDM patients was significantly increased, by about 60%, for both men and women compared to JDCS patients, and intake of fish and soybean/soy products was significantly decreased in JDDM patients. In particular, changes in meat and fish intake were remarkable, with an increase of about 30 g of meat in the JDDM participants and a decrease of about 30 g of fish in the JDDM participants, for both men and women. In summary, the JDCS patients consumed more fish and soybean/soy products than meat, while the JDDM patients consumed more meat than fish and soybean/soy products; thus, the composition of the major protein food sources was reversed over the 20-year period. In addition, dairy intake decreased by about 20%, and intake of sweets and snacks was about three times higher in that interval. On the other hand, the intake of vegetables and fruits among JDDM patients was dramatically reduced by about 30% in both men and women compared with JDCS patients. Both intakes of green-yellow vegetables and other vegetables significantly decreased from the time of the JDCS survey to the JDDM survey. In particular, intake of green-yellow vegetables was markedly reduced between the two surveys by 42.0% for men and 35.0% for women. Regardless of age, meat intake increased in JDDM patients, while fish and soybean/soy products showed a decrease. However, there was no trend toward a reversal of the composition of the major protein food sources in patients aged 60 years and older. In both the under 60 and over 60 age groups, there was an increase in sweets and snacks and a decrease in fruits and total vegetable intake (Supplementary Table S3).\n\n4. Discussion:\nThis study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period using two surveys with essentially the same format. Although the backgrounds of the patients, including age and duration of diabetes, were quite similar between the two studies, BMI and obesity rates were markedly increased in the later study. Regarding dietary content, fat intake increased, including saturated fatty acids, along with significant increases in the consumption of meat and sweets and snacks, and a significant decrease in vegetable and fish intake. To date, there have been no studies on the changes in dietary intake by persons with type 2 diabetes in Asia, except for a report on adherence to dietary recommendations in Korea [12]. To our knowledge, the changes in nutrient and food intake have not been reported. Therefore, this is the first study to clarify the secular changes in the dietary status of patients with type 2 diabetes in Asia.\nSelf-management education and support for diet and physical activity are essential in the care of overweight or obese people with type 2 diabetes. A previous study observing the secular trends in the United States from 1988 to 2012 [3] showed a trend of increases in BMI and energy intake in patients with type 2 diabetes, which was also the case even in adults without diabetes. On the other hand, in our study of East Asians, the BMI markedly increased from 22.7 kg/m2 to 25.8 kg/m2 in men and 23.2 kg/m2 to 25.9 kg/m2 in women, although no such remarkable changes were found in the Japanese general population according to the Japan National Health and Nutrition Survey performed during a similar period (1996 and 2016) (from 23.3 kg/ m2 to 24.0 kg/m2 in men and 23.2 kg m2 to 22.6 kg/m2 in women) [13]. This suggests that secular changes in BMI and obesity were different between people with and without type 2 diabetes and between the East and the West. According to the National Health and Nutrition Surveys, from 1996 to 2016, the energy intake by the general population aged 40–69 years mildly declined from 2284 kcal to 2145 kcal (−139 kcal) for men and from 1880 kcal to 1722 kcal (−158 kcal) for women, although the dietary survey method was different from our method [14].\nWe previously reported, using JDCS baseline data, that Western populations with diabetes are mostly obese and are also more obese than the general population, whereas Japanese people with diabetes not only have a mean BMI within the normal range, but also it is almost the same as in the general population [15]. The results of the current study, including the JDDM results 20 years after the initial JDCS survey, strongly support the fact that obesity among those with type 2 diabetes has recently increased in Japan, as is the case in Western countries [16]. On the other hand, it is not clear from this study alone why the increase in BMI in Japanese patients with type 2 diabetes was significantly greater than in the general population, even though there was no significant increase in energy intake between the two studies. Our previous study [17] suggested that there are racial differences in the relationship between energy intake and obesity, but several other hypotheses are also possible.\nThe first possible explanation for the significant increase in BMI without a significant increase in energy intake in Japanese patients with type 2 diabetes may be the effect of decreased energy expenditure due to decreased physical activity. A comparison of the activity levels between people with diabetes and the general population found no significant difference in physical activity levels and leisure-time physical activity patterns, and reported that neither groups achieved the recommended physical activity goals [18,19]. The decrease in the number of steps by the general Japanese population from 1996 to 2016 was remarkable for both men and women [13], and it was pointed out that the decrease in physical activity was due to the development of transportation [20]. In this study, direct comparisons were difficult because of the differences in the studies’ survey methods regarding the amount of physical activity; however, 44.4% of patients in the JDDM reported low levels of overall physical activity in their daily lives, which was calculated from activity time and activity intensity. Future research on secular trends in the amount of physical activity in patients with type 2 diabetes is expected.\nThe second theory as to why BMI increased without an increase in energy intake is that it could be due to the effect of smoking rates and medications. The BMI of smokers was reportedly lower than that of non-smokers [21,22], and the smoking rate of men in the JDDM with a high BMI was significantly lower than that in the JDCS. In addition, the increase in the rate of insulin use over time that was observed between these studies may be a factor in weight gain. The fact that there was a significant decrease in the rate of use of oral hypoglycemic agents may have contributed to weight gain or weight loss, depending on the type of oral hypoglycemic agent [23]. Further studies, including a detailed follow-up of the status of the medications, are necessary.\nAs a third conjecture, the ratio of macronutrients may have affected these findings. Regarding the energy-producing nutrients, both men and women in the JDDM had higher fat-to-energy ratios than those in the JDCS. In the JDDM compared to the JDCS, men had a lower carbohydrate-to-energy ratio and protein-to-energy ratio and women had a lower protein-to-energy ratio. Women in the JDDM had an increase in energy intake, which could be attributed to an increased fat intake as their carbohydrate and protein intakes did not increase. In the Japanese National Health and Nutrition Survey, which was of a similar duration (1996 and 2016), the fat-to-energy ratio increased (men: +2.6%, women: +2.9%), but carbohydrate and protein intakes tended to decrease (carbohydrate: −22.6 g/day for men, −33.4 g/day for women, protein: −15.9 g/day for men, −13.0 g/day for women), and fat intake was almost unchanged, suggesting that the fat-to-energy ratio was relatively increased. Increased fat-to-energy ratios due to an increased fat intake in patients with type 2 diabetes may have contributed to the increased BMI. However, previous studies have not provided a unified consensus on the association between the fat-to-energy ratio and obesity, which needs to be clarified in the future [24].\nIt is well known that, regardless of body weight, a high-fat diet, especially an excessive intake of saturated fatty acids and a high saturated fatty acid-to-energy ratio, is significantly associated with a higher risk of cardiovascular events in patients with type 2 diabetes [25]. According to the Japanese practice guidelines for diabetes [26], a reduction in saturated fatty acids is recommended when the lipid-to-energy ratio exceeds 25%. In addition, Dietary Reference Intakes for Japanese [27] set a target saturated fatty acid-to-energy ratio of ≤7%. Among our patients with a fat-to-energy ratio of ≥25%, only 9.5% of JDCS patients and 4.4% of JDDM patients had a saturated fatty acid-to-energy ratio of ≤7%, suggesting the necessity for enhanced guidance to reduce the saturated fatty acid-to-energy ratio. The saturated fatty acid-to-energy ratio for Americans with type 2 diabetes was approximately 11%, which was still higher than the level (<10%) recommended by the U.S. Dietary Guidelines, but it did not increase between 1988 and 2012 [28].\nIn contrast, in our Japanese patients, the saturated fatty acid-to-energy ratio increased significantly over the 20 years to approximately 9% in both men and women, and the percentage of patients with a saturated fatty acid-to-energy ratio of more than 10% increased significantly from 8.2% in men and 13.3% in women in the JDCS to 28.4% in men and 36.6% in women in the JDDM. This suggests that the fat intake pattern of Japanese people with type 2 diabetes is approaching that of Western patients. Therefore, correcting excessive lipid intake while taking into account the content and quality of lipids is considered to be a crucial issue.\nIn terms of secular changes in food group intake patterns, an increase in meat intake and a decrease in the consumption of fish and dairy products were observed as sources of protein intake. JDCS patients had higher intakes of fish and soybeans/soy products than meat, whereas JDDM patients had higher intakes of meat than fish and soybeans/soy products. The National Health and Nutrition Survey [29] also showed the same secular changes in food group intake patterns as this study.\nIt is indicated that patients with type 2 diabetes in Japan had a major change in the composition of their protein sources, as did the general Japanese population, which was a change to a meat-centered diet. In general, among people in Western countries, the circumstances in which meat intake is higher than fish intake continues. It is inferred that the diet of Japanese patients with type 2 diabetes has been transformed into a diet centered on meat due to the influx of various lifestyle patterns and dietary cultures from Western countries, as is the case with the general Japanese population. It has been reported that meat was associated with cardiovascular complications and mortality in people with type 2 diabetes in a study in the US [30] and in our previous study [31]. Thus, it is considered necessary to strengthen the guidance on protein sources to prevent complications in the future.\nMeat is the food with the highest contribution to fat intake by Japanese people. Therefore, it is thought that an increased meat intake may also affect fat-to-energy ratios and saturated fatty acid increases in JDDM patients. Aside from meat, saturated fatty acids are found in the Japanese diet in milk, butter, and coconut oil, which are commonly used as ingredients in Western sweets rather than in traditional Japanese ones (which are mostly made with sugar, rice, and beans [32]). Although the type of sweets could not be identified in this survey, as their intake more than doubled from the JDCS to the JDDM, it is inferred that in conjunction with meat, the increase in the intake of sweets contributed to the increase in saturated fatty acid consumption.\nContrary to meat, the intake of fruits and vegetables decreased significantly between the two studies. As vegetables and fruits are good sources of dietary fiber, fiber intake decreased significantly as vegetable and fruit intakes decreased. A higher intake of dietary fiber is known to improve glycemic control, and we previously reported that it was significantly associated with a reduced risk of stroke [33] and retinopathy [34] in JDCS patients. However, despite its importance, the intake of dietary fiber was lower than the recommended goal of 20 g/day or higher in 1996, and even declined in the following 20 years. A decreased intake of fruits and vegetables is apparent in the Japanese general population [29], and is considered to be an important issue in dietary guidance.\nAs to the background of the decrease in the consumption of vegetables and fruits, survey reports have suggested that differences in lifestyle, such as economic conditions [35] and living alone, exist [36] and that diabetic patients who work may choose a less diversified diet [37]. It was also reported that the consumption of vegetables and fruits tends to be higher with a lower frequency of eating meals away from home [38]. As to the proportions of food expenditure in Japan from 1995 to 2015, the consumption of fresh foods has decreased, and eating out and consuming processed foods have increased from 65.4% to 72.6% [39]. This suggests that the increase in eating out and buying processed foods may influence the decrease in fruit and vegetable intake, not only in the general population, but also in those with type 2 diabetes. This Westernization of dietary habits has been widely adopted, and advice on the dietary treatment of diabetes should consider the current dietary situation.\nThis study has several limitations. First, both the JDCS and the JDDM were comprised of patients from different cohorts, although they were both multicenter studies. However, both studies were conducted at outpatient clinics throughout Japan specializing in diabetes, and the clinical settings were similar except for the time period studied. An additional limitation is that the number of participants that were included in both surveys was different from the potential candidates. Of the 2033 JDCS patients, 1588 responded to a baseline dietary survey, and 524 patients who had missing values in the dietary survey were excluded. In the JDDM patients, 1145 of 2337 patients met the eligibility criteria for the JDCS and responded to the baseline dietary survey. However, there were no notable differences in baseline characteristics between the responders and non-responders [8], and consequently, no selection bias was found. Finally, the participants’ dietary intake was obtained from a self-administered questionnaire, which could be an underestimation. As for the long-term comparison of 20 years, changes had been made in the food composition tables and in the FFQg version used in both studies. However, those changes corresponded to changes in dietary habits and food ingredients due to secular trends, and there was little effect on energy intake and intake of macronutrients due to the changes in the food composition tables.\nThe results of this study clarify the changes in the dietary intake of Japanese patients with type 2 diabetes over 20 years, beginning in 1996. Despite a lack of significant change in the total energy intake during the period, both men and women with type 2 diabetes had a large increase in BMI. A rise in the fat-to-energy ratio due to an increase in fat intake was also observed, particularly in men. In terms of intake by food group, both men and women had clearly unfavorable changes in terms of complication prevention. These included an increase in the intake of meat and a decrease in the intake of fish, soybean/soy products, vegetables, and fruits. The results of our study strongly suggest that, in order to prevent complications in Japanese patients with type 2 diabetes in the future, the provision of nutritional guidance, in line with these chronological changes in dietary intake, is necessary.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIn order to provide effective dietary guidance, it is necessary to consider dietary intake, which can change over time. This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period.\n\nMethods:\nWe compared the results of two dietary surveys that used the food frequency questionnaire format. The first was conducted in 1996 by the Japan Diabetes Complications Study (JDCS) (n = 1509; males 53.3%), and the second in 2014-2018 by the Japan Diabetes Clinical Data Management Study (JDDM) (n = 1145; males 65.6%). Both are nationwide representative registries of outpatients with type 2 diabetes in Japan.\n\nResults:\nOver a 20-year period, both men and women with type 2 diabetes had a significant increase in body mass index (BMI). Nonetheless, there was only a small change in energy intake. Conversely, there was a significant increase in fat intake and thus in the fat-to-energy ratio. With regard to food groups, there was a significant increase in meat intake and a decrease in the intake of fish, soybeans/soy products, vegetables, and fruits, with a particularly significant decrease in vegetables.\n\nConclusions:\nEven in Japan, an industrialized country with a stable socioeconomic environment, there were many significant changes in the dietary intake of patients with type 2 diabetes over the 20-year period."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":5052,"string":"5,052"},"whole_article_abstract_length":{"kind":"number","value":277,"string":"277"},"num_sections":{"kind":"number","value":4,"string":"4"},"most_frequent_words":{"kind":"list like","value":["intake","patients","diabetes","dietary","jddm","energy","jdcs","men","type","type diabetes"],"string":"[\n \"intake\",\n \"patients\",\n \"diabetes\",\n \"dietary\",\n \"jddm\",\n \"energy\",\n \"jdcs\",\n \"men\",\n \"type\",\n \"type diabetes\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] food intake | type 2 diabetes | obesity | diabetes mellitus | Asia [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] food intake | type 2 diabetes | obesity | diabetes mellitus | Asia [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] food intake | type 2 diabetes | obesity | diabetes mellitus | Asia [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Biomarkers | Body Mass Index | Diabetes Mellitus, Type 2 | Diet | Disease Susceptibility | Energy Intake | Feeding Behavior | Female | Humans | Japan | Male | Middle Aged | Public Health Surveillance | Registries [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Biomarkers | Body Mass Index | Diabetes Mellitus, Type 2 | Diet | Disease Susceptibility | Energy Intake | Feeding Behavior | Female | Humans | Japan | Male | Middle Aged | Public Health Surveillance | Registries [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Biomarkers | Body Mass Index | Diabetes Mellitus, Type 2 | Diet | Disease Susceptibility | Energy Intake | Feeding Behavior | Female | Humans | Japan | Male | Middle Aged | Public Health Surveillance | Registries [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] intake | patients | diabetes | dietary | jddm | energy | jdcs | men | type | type diabetes [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] intake | patients | diabetes | dietary | jddm | energy | jdcs | men | type | type diabetes [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] intake | patients | diabetes | dietary | jddm | energy | jdcs | men | type | type diabetes [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] dietary | changes | diabetes | type diabetes | type | intake | asian | study | changes dietary | people [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] jddm | men | significantly | women | participants | jddm participants | jdcs | patients | intake | jddm patients [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] intake | jddm | dietary | diabetes | jdcs | patients | energy | type | men | changes [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Japanese | 2 | 20-year [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 20-year | 2 | BMI ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Japanese | 2 | 20-year ||| two ||| first | 1996 | the Japan Diabetes Complications Study | JDCS | 1509 | 53.3% | second | 2014-2018 | the Japan Diabetes Clinical Data Management Study | 1145 | 65.6% ||| 2 | Japan ||| 20-year | 2 | BMI ||| ||| ||| ||| Japan | 2 | 20-year [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":281,"cells":{"title":{"kind":"string","value":"Are pre-existing markers of chronic kidney disease associated with short-term mortality following acute community-acquired pneumonia and sepsis? A cohort study among older people with diabetes using electronic health records."},"pmid":{"kind":"string","value":"25605811"},"background_abstract":{"kind":"string","value":"We aimed to examine whether pre-existing impaired estimated glomerular filtration rate (eGFR) and proteinuria were associated with mortality following community-acquired pneumonia or sepsis among people aged ≥ 65 years with diabetes mellitus, without end-stage renal disease."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Patients were followed up from onset of first community-acquired pneumonia or sepsis episode in a cohort study using large, linked electronic health databases. Follow-up was for up to 90 days, unlimited by hospital discharge. We used generalized linear models with log link, normal distribution and robust standard errors to calculate risk ratios (RRs) for all-cause 28- and 90-day mortality according to two markers of chronic kidney disease: eGFR and proteinuria."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"All-cause mortality among the 4743 patients with pneumonia was 29.6% after 28 days and 37.4% after 90 days. Among the 1058 patients with sepsis, all-cause 28- and 90-day mortality were 35.6 and 44.2%, respectively. eGFR <30 mL/min/1.73 m(2) was a risk marker of higher 28-day mortality for pneumonia (RR 1.27: 95% CI 1.12-1.43) and sepsis (RR 1.32: 95% CI 1.07-1.64), adjusted for age, sex, socio-economic status, smoking status and co-morbidities. Neither moderately impaired eGFR nor proteinuria were associated with short-term mortality following either infection."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"People with pre-existing low eGFR but not on dialysis are at higher risk of death following pneumonia and sepsis. This association was not explained by existing co-morbidities. These patients need to be carefully monitored to prevent modifiable causes of death."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Age Factors","Aged","Aged, 80 and over","Biomarkers","Cohort Studies","Community-Acquired Infections","Comorbidity","Diabetes Mellitus","Electronic Health Records","Female","Humans","Kidney Failure, Chronic","Male","Pneumonia","Proteinuria","Renal Dialysis","Risk Factors","Sepsis","Survival Rate"],"string":"[\n \"Age Factors\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Biomarkers\",\n \"Cohort Studies\",\n \"Community-Acquired Infections\",\n \"Comorbidity\",\n \"Diabetes Mellitus\",\n \"Electronic Health Records\",\n \"Female\",\n \"Humans\",\n \"Kidney Failure, Chronic\",\n \"Male\",\n \"Pneumonia\",\n \"Proteinuria\",\n \"Renal Dialysis\",\n \"Risk Factors\",\n \"Sepsis\",\n \"Survival Rate\"\n]"},"pmcid":{"kind":"string","value":"4438741"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Chronic kidney disease (CKD) affects an estimated 1.8 million people in England, 98% of whom do not require renal replacement therapy [1]. CKD is defined by reduced estimated glomerular filtration rate (eGFR) or evidence of kidney damage such as proteinuria and is commonest among older people [2, 3]. Most patients with CKD are managed in primary care [4].\nInfection is an important cause of mortality among older people [5, 6]. Both reduced eGFR and proteinuria are associated with an increased rate of infection-related mortality, which could be partly explained by increased incidence of infection [7–9]. It is less clear whether CKD is also associated with poorer prognosis following infection. When clinicians assess patients with community-acquired infection, developing complications such as acute kidney injury (AKI) may not yet be apparent, but they will know which patients have pre-existing CKD. If pre-existing CKD is a risk marker for short-term mortality, this would be useful for risk stratification and clinical management of patients with infections, especially in primary care where clinicians may not have access to immediate laboratory tests. While the implications of acute changes in eGFR during infection are a focus of current research, few studies have investigated the role of pre-existing CKD [10]. Low baseline eGFR has been found to be associated with mortality following sepsis and community-acquired pneumonia, but rarely examined according to clinically meaningful categories of eGFR [11–15]. To the best of our knowledge, proteinuria has not been examined as a potential risk marker for mortality following infection [12, 15, 16].\nAmong older people, CKD frequently co-exists with other co-morbidities [3]. An association of CKD with poor prognosis of infections could thus be due to confounding from these co-morbidities. For example, CKD is strongly associated with cardiovascular disease, which may be complicated by infection, resulting in post-infection mortality driven by the underlying cardiovascular disease [17]. Such deaths would largely follow hospitalization for cardiovascular events. CKD is associated with healthcare-associated pneumonia, which carries a worse prognosis than community-acquired pneumonia [18, 19]. Focusing on community-acquired infections should exclude infections arising as short-term sequelae of cardiovascular events and improve understanding of the relationship between pre-existing CKD markers and infection prognosis.\nOlder people with diabetes mellitus form a large and growing population in primary care who suffer a high incidence of community-acquired pneumonia and sepsis [20]. Forty per cent of adults with diabetes have CKD, of whom three-quarters have proteinuria, and CKD among these patients is associated with a greater all-cause excess mortality than among patients without diabetes [21]. If proteinuria is a risk marker for mortality among older patients with diabetes who develop community-acquired pneumonia or sepsis, this could inform clinical management of a large primary care patient population with appreciable mortality following infection [20].\nThis study aimed to examine whether baseline eGFR and proteinuria were independent risk markers for short-term mortality following community-acquired pneumonia or sepsis among older people with diabetes mellitus, using large, linked electronic health record databases."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"We identified 4957 individuals with community-acquired pneumonia and 1114 individuals with community-acquired sepsis. Data were missing (for smoking status and/or HbA1C results) for 212/4957 individuals with pneumonia (4.3%) and 56/1114 individuals with sepsis (5.0%). These patients were excluded. Among patients with pneumonia, patients with missing data were older (median age 83 years, IQR: 78–88) compared with those included (median age 80 years, IQR: 74–85), with a higher 28-day mortality (excluded 88/214, 41.1%: included 1406/4743, 29.6%) but a similar distribution of baseline eGFR. A similar pattern was seen for sepsis.\nEstimated GFR was based on the higher of two results for 4029 patients with pneumonia (84.9%) and 919 patients with sepsis (86.9%); for the remaining patients, only a single valid serum creatinine result was available. CKD prevalence was high: almost half of the patients had eGFR <60 mL/min/1.73 m2 and a third had proteinuria. Patients with eGFR <60 mL/min/1.73 m2 were older, with a higher prevalence of ischaemic heart disease and congestive heart failure than patients with eGFR ≥60 mL/min/1.73 m2 (Table 1).Table 1.Baseline characteristics of the study populationPneumoniaSepsiseGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2eGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2Age (years) Median (IQR)82 (77–87)78 (72–83)81 (75–86)76 (71–82)n (col %)n (col %)n (col %)n (col %)Gender Female1231 (53.0)1106 (42.0)294 (55.7)268 (45.7)Onset prior to 1 April 2004584 (25.1)527 (20.0)124 (23.5)120 (20.5)Socio-economic status (IMD quintile)a 1 (least deprived)383 (16.5)438 (16.6)109 (20.6)106 (18.1) 2554 (23.8)595 (22.6)116 (22.0)127 (21.7) 3494 (21.3)582 (22.1)120 (22.7)137 (23.4) 4502 (21.6)574 (21.8)110 (20.8)108 (18.4) 5 (most deprived)391 (16.8)444 (16.9)73 (13.8)108 (18.4)Smoking status Current321 (13.8)549 (20.9)70 (13.3)103 (17.6) Ex-smoker1185 (51.0)1351 (51.3)235 (44.5)284 (48.5) Non-smoker776 (33.4)698 (26.5)207 (39.2)193 (32.9) Missing42 (1.8)35 (1.3)16 (3.0)6 (1.0)Comorbidities Chronic lung disease503 (21.6)686 (26.1)59 (11.2)95 (16.2) Hypertension1623 (69.8)1662 (63.1)372 (70.5)381 (65.0) Congestive heart failure734 (31.6)430 (16.3)147 (27.8)95 (16.2) Ischaemic heart disease966 (41.6)887 (33.7)223 (42.2)207 (35.3) Cerebrovascular disease692 (29.8)638 (24.2)152 (28.8)145 (24.7) Other dementia174 (7.5)190 (7.2)42 (8.0)23 (3.9) Cancer382 (16.4)474 (18.0)99 (18.8)120 (20.5) Connective tissue disorders228 (9.8)215 (8.2)36 (6.8)52 (8.9)HbA1C Good <7%1176 (50.6)1401 (53.2)251 (47.5)308 (52.6) Borderline 7–10%966 (41.6)1033 (39.2)213 (40.3)230 (39.3) Poor >10%108 (4.7)131 (5.0)41 (7.8)34 (5.8) None recorded74 (3.2)68 (2.6)23 (4.4)14 (2.4)Prior antidiabetes medication Insulin122 (5.3)126 (4.8)34 (6.4)23 (3.9) Oral medications1139 (49.0)1426 (54.2)247 (46.8)312 (53.2) Both462 (19.9)384 (14.6)130 (24.6)114 (19.5) None601 (25.9)697 (26.5)117 (22.2)137 (23.4) Total23242633528586eGFR, estimated glomerular filtration rate.aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nBaseline characteristics of the study population\neGFR, estimated glomerular filtration rate.\naIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nPatients with pneumonia experienced 29.6% 28-day all-cause mortality (1406 deaths). Patients with sepsis experienced 35.6% 28-day all-cause mortality (377 deaths) (Table 2). Survival curves showed high mortality at infection onset, declining over ∼30 days to a more stable rate for the next 60 days following both pneumonia and sepsis (Figure 1). RRs for 28-day mortality were higher among people with eGFR <30 mL/min/1.73 m2 compared with people with eGFR ≥60 mL/min/1.73 m2 for pneumonia (RR = 1.27; 95% CI 1.10–1.47) and sepsis (RR = 1.42; 1.10–1.84), adjusted for age, sex, socio-economic status and onset prior to April 2004. Adjustment for smoking status, co-morbidities and characteristics of diabetes had minimal effect on these RRs for pneumonia (fully adjusted RR = 1.27; 1.12–1.43) or sepsis (fully adjusted RR = 1.32; 1.07–1.64). There was no evidence of associations between intermediate levels of eGFR and 28-day mortality for either infection, nor for an association between proteinuria and 28-day mortality. The pattern of associations of eGFR and proteinuria with 90-day mortality was similar to those for 28-day mortality (Table 2). Results were unchanged by additional adjustment for peripheral vascular disease. There was no good evidence of effect modification between eGFR and proteinuria, and in particular no evidence of any association of proteinuria with 28-day mortality within any category of eGFR status (data not shown).Table 2.Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)aNumber (column %)28-Day mortality (row %)90-Day mortality (row %)RRs for 28-day mortality (95% CI)RRs for 90-day mortality (95% CI)Adjusted for demographicsbadjusted for non-CVD co-morbiditiescFully adjusteddAdjusted for demographicsbAdjusted for non-CVD co-morbiditiescFully adjusteddPneumonia Proteinuria (adjusted for eGFR) Yes1611 (34.0)499 (31.0)625 (38.8)1.07 (0.97–1.17)1.07 (0.98–1.18)1.07 (0.98–1.18)1.05 (0.97–1.13)1.06 (0.98–1.14)1.05 (0.98–1.14) No3132 (66.0)907 (29.0)1150 (36.7)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<1523 (0.5)12 (52.2)12 (52.2)1.27 (1.10–1.47)1.31 (1.13–1.52)1.30 (1.12–1.52)1.26 (1.12–1.42)1.29 (1.14–1.45)1.27 (1.12–1.43)15–29263 (5.6)110 (41.8)139 (52.9) 30–44764 (16.1)265 (34.7)336 (44.0)0.98 (0.86–1.10)0.99 (0.88–1.13)0.99 (0.88–1.12)1.02 (0.93–1.13)1.04 (0.94–1.15)1.03 (0.94–1.14) 45–601162 (24.5)332 (28.6)418 (36.0)0.91 (0.82–1.02)0.94 (0.84–1.04)0.93 (0.84–1.04)0.91 (0.83–1.00)0.93 (0.85–1.02)0.92 (0.84–1.01) ≥602531 (53.4)687 (27.1)870 (34.4)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total47431406 (29.6)1775 (37.4)Sepsis Proteinuria (adjusted for eGFR) Yes358 (33.8)128 (35.8)159 (44.4)0.98 (0.82–1.17)1.01 (0.85–1.21)1.01 (0.85–1.21)0.98 (0.86–1.13)0.96 (0.84–1.10)0.96 (0.84–1.10) No700 (66.2)249 (35.6)309 (44.1)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<158 (0.8)2 (25.0)5 (62.5)1.42 (1.10–1.84)1.41 (1.08–1.84)1.37 (1.05–1.79)1.39 (1.14–1.70)1.32 (1.07–1.63)1.32 (1.07–1.64)15–2962 (5.9)32 (51.6)39 (62.9) 30–44190 (18.0)88 (46.3)103 (54.2)1.25 (1.01–1.55)1.24 (0.99–1.55)1.24 (0.99–1.54)1.14 (0.96–1.36)1.12 (0.94–1.33)1.11 (0.94–1.32) 45–60232 (21.9)76 (32.8)95 (41.0)0.95 (0.75–1.19)0.91 (0.72–1.15)0.91 (0.72–1.14)0.92 (0.77–1.11)0.89 (0.74–1.07)0.89 (0.75–1.07) ≥60566 (53.5)179 (31.6)226 (39.9)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total1058377 (35.6)468 (44.2)aExcluding patients with missing smoking or HbA1C data.bAge, gender, socio-economic status, onset prior to 1 April 2004.cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.FIGURE 1:Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nShort-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)a\naExcluding patients with missing smoking or HbA1C data.\nbAge, gender, socio-economic status, onset prior to 1 April 2004.\ncAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.\ndAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.\nSurvival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nThe underlying causes of death following pneumonia and sepsis were similar for patients with eGFR above and below 60 mL/min/1.73 m2. Causes of 28-day mortality following sepsis were predominantly sources of infection (Table 3). Following pneumonia onset, pneumonia was recorded as an underlying or contributory cause of death for 83.9% (1191/1419) of deaths within 28 days and 76.3% (1366/1790) of deaths within 90 days. Among patients with eGFR <60 mL/min/1.73 m2, renal disease was recorded as an underlying or contributory cause for 10.6% (77/724) of those who died within 28 days of pneumonia onset and 16.6% (152/913) of those who died within 90 days. Recording of CKD as a cause of death increased with lower eGFR (Table 4).Table 3.Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)aeGFReGFR <60 mL/min/1.73 m2eGFR ≥60 mL/min/1.73 m228-Day mortality following pneumonia, n = 1419J18 Pneumonia, organism unspecified, n = 256, 35.4%J44 Other chronic obstructive pulmonary disease, n = 63, 8.7%I25 Chronic ischaemic heart disease, n = 47, 6.5%I50 Heart failure, n = 43, 5.9%I64 Stroke, not specified as haemorrhage or infarction, n = 37, 5.1%J18 Pneumonia, organism unspecified, n = 216, 31.1%J44 Other chronic obstructive pulmonary disease, n = 63, 9.1%I64 Stroke, not specified as haemorrhage or infarction, n = 35, 5.0%C34 Malignant neoplasm of bronchus and lung, n = 34, 4.9%I25 Chronic ischaemic heart disease, n = 29, 4.2%Total = 724Total = 69529–90 Day mortality following pneumonia, n = 371J18 Pneumonia, organism unspecified, n = 38, 20.1%I25 Chronic ischaemic heart disease, n = 24, 12.7%J44 Other chronic obstructive pulmonary disease, n = 13, 6.9%C34 Malignant neoplasm of bronchus and lung, n = 12, 6.4%I21 Acute myocardial infarction, n = 12, 6.4%J18 Pneumonia, organism unspecified, n = 20, 11.0%C34 Malignant neoplasm of bronchus and lung, n = 20, 11.0%J44 Other chronic obstructive pulmonary disease, n = 19, 10.4%I25 Chronic ischaemic heart disease, n = 9, 5.0%I64 Stroke, not specified as haemorrhage or infarction, n = 9, 5.0%Total = 189Total = 18228-Day mortality following sepsis, n = 387N39 Other disorders of urinary systemb, n = 33, 15.9%J18 Pneumonia, organism unspecified, n = 30, 14.4%E14 Unspecified diabetes mellitus, n = 12, 5.8%A41 Other sepsis, n = 11, 5.3%L03 Cellulitis, n = 11, 5.3%N39 Other disorders of urinary systemb, n = 20, 11.2%A41 Other sepsis, n = 18, 10.1%J18 Pneumonia, organism unspecified, n = 18, 10.1%E14 Unspecified diabetes mellitus, n = 10, 5.6%J44 Other chronic obstructive pulmonary disease, n = 6, 3.4%=K55 Vascular disorders of intestine, n = 6, 3.4%=L03 Cellulitis, n = 6, 3.4%Total = 208Total = 179aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.Table 4.Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFReGFR (mL/min/1.73 m2)Deaths within 28 days of pneumonia onsetDeaths within 90 days of pneumonia onsetTotalN18 CKD, n (%)Renal disease,b\nn (%)TotalN18 CKD, n (%)Renal disease,b\nn (%)<1586 (75.0)7 (87.5)86 (75.0)7 (87.5)15–2910919 (17.4)41 (37.6)13926 (18.7)53 (38.1)30–4427514 (5.1)2 (15.3)34720 (5.8)56 (16.1)35–593328 (2.4)27 (8.1)41910 (2.4)36 (8.6)Total72447 (6.5)77 (10.6)91362 (6.8)152 (16.6)aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\nTop five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)a\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.\nRecording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFR\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Data sources","Study population","Definition of infections","Study outcomes","Definition of CKD","Other variables","Data analysis","Ethics"],"string":"[\n \"Data sources\",\n \"Study population\",\n \"Definition of infections\",\n \"Study outcomes\",\n \"Definition of CKD\",\n \"Other variables\",\n \"Data analysis\",\n \"Ethics\"\n]"},"other_sections_texts":{"kind":"list like","value":["The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].","The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.","Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.","The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.","CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.","Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.","Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.","The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116)."],"string":"[\n \"The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\",\n \"The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\",\n \"Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\",\n \"The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\",\n \"CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\",\n \"Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\",\n \"Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\\nStata version 13.1 was used for data analysis. All code lists are available on request.\",\n \"The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Data sources","Study population","Definition of infections","Study outcomes","Definition of CKD","Other variables","Data analysis","Ethics","RESULTS","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Data sources\",\n \"Study population\",\n \"Definition of infections\",\n \"Study outcomes\",\n \"Definition of CKD\",\n \"Other variables\",\n \"Data analysis\",\n \"Ethics\",\n \"RESULTS\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Chronic kidney disease (CKD) affects an estimated 1.8 million people in England, 98% of whom do not require renal replacement therapy [1]. CKD is defined by reduced estimated glomerular filtration rate (eGFR) or evidence of kidney damage such as proteinuria and is commonest among older people [2, 3]. Most patients with CKD are managed in primary care [4].\nInfection is an important cause of mortality among older people [5, 6]. Both reduced eGFR and proteinuria are associated with an increased rate of infection-related mortality, which could be partly explained by increased incidence of infection [7–9]. It is less clear whether CKD is also associated with poorer prognosis following infection. When clinicians assess patients with community-acquired infection, developing complications such as acute kidney injury (AKI) may not yet be apparent, but they will know which patients have pre-existing CKD. If pre-existing CKD is a risk marker for short-term mortality, this would be useful for risk stratification and clinical management of patients with infections, especially in primary care where clinicians may not have access to immediate laboratory tests. While the implications of acute changes in eGFR during infection are a focus of current research, few studies have investigated the role of pre-existing CKD [10]. Low baseline eGFR has been found to be associated with mortality following sepsis and community-acquired pneumonia, but rarely examined according to clinically meaningful categories of eGFR [11–15]. To the best of our knowledge, proteinuria has not been examined as a potential risk marker for mortality following infection [12, 15, 16].\nAmong older people, CKD frequently co-exists with other co-morbidities [3]. An association of CKD with poor prognosis of infections could thus be due to confounding from these co-morbidities. For example, CKD is strongly associated with cardiovascular disease, which may be complicated by infection, resulting in post-infection mortality driven by the underlying cardiovascular disease [17]. Such deaths would largely follow hospitalization for cardiovascular events. CKD is associated with healthcare-associated pneumonia, which carries a worse prognosis than community-acquired pneumonia [18, 19]. Focusing on community-acquired infections should exclude infections arising as short-term sequelae of cardiovascular events and improve understanding of the relationship between pre-existing CKD markers and infection prognosis.\nOlder people with diabetes mellitus form a large and growing population in primary care who suffer a high incidence of community-acquired pneumonia and sepsis [20]. Forty per cent of adults with diabetes have CKD, of whom three-quarters have proteinuria, and CKD among these patients is associated with a greater all-cause excess mortality than among patients without diabetes [21]. If proteinuria is a risk marker for mortality among older patients with diabetes who develop community-acquired pneumonia or sepsis, this could inform clinical management of a large primary care patient population with appreciable mortality following infection [20].\nThis study aimed to examine whether baseline eGFR and proteinuria were independent risk markers for short-term mortality following community-acquired pneumonia or sepsis among older people with diabetes mellitus, using large, linked electronic health record databases."," Data sources The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\nThe Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\n Study population The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\nThe study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\n Definition of infections Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\nInfection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\n Study outcomes The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\nThe outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\n Definition of CKD CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\nCKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\n Other variables Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\nAge was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\n Data analysis Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\nPneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\n Ethics The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\nThe study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).","The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].","The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.","Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.","The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.","CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.","Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.","Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.","The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).","We identified 4957 individuals with community-acquired pneumonia and 1114 individuals with community-acquired sepsis. Data were missing (for smoking status and/or HbA1C results) for 212/4957 individuals with pneumonia (4.3%) and 56/1114 individuals with sepsis (5.0%). These patients were excluded. Among patients with pneumonia, patients with missing data were older (median age 83 years, IQR: 78–88) compared with those included (median age 80 years, IQR: 74–85), with a higher 28-day mortality (excluded 88/214, 41.1%: included 1406/4743, 29.6%) but a similar distribution of baseline eGFR. A similar pattern was seen for sepsis.\nEstimated GFR was based on the higher of two results for 4029 patients with pneumonia (84.9%) and 919 patients with sepsis (86.9%); for the remaining patients, only a single valid serum creatinine result was available. CKD prevalence was high: almost half of the patients had eGFR <60 mL/min/1.73 m2 and a third had proteinuria. Patients with eGFR <60 mL/min/1.73 m2 were older, with a higher prevalence of ischaemic heart disease and congestive heart failure than patients with eGFR ≥60 mL/min/1.73 m2 (Table 1).Table 1.Baseline characteristics of the study populationPneumoniaSepsiseGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2eGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2Age (years) Median (IQR)82 (77–87)78 (72–83)81 (75–86)76 (71–82)n (col %)n (col %)n (col %)n (col %)Gender Female1231 (53.0)1106 (42.0)294 (55.7)268 (45.7)Onset prior to 1 April 2004584 (25.1)527 (20.0)124 (23.5)120 (20.5)Socio-economic status (IMD quintile)a 1 (least deprived)383 (16.5)438 (16.6)109 (20.6)106 (18.1) 2554 (23.8)595 (22.6)116 (22.0)127 (21.7) 3494 (21.3)582 (22.1)120 (22.7)137 (23.4) 4502 (21.6)574 (21.8)110 (20.8)108 (18.4) 5 (most deprived)391 (16.8)444 (16.9)73 (13.8)108 (18.4)Smoking status Current321 (13.8)549 (20.9)70 (13.3)103 (17.6) Ex-smoker1185 (51.0)1351 (51.3)235 (44.5)284 (48.5) Non-smoker776 (33.4)698 (26.5)207 (39.2)193 (32.9) Missing42 (1.8)35 (1.3)16 (3.0)6 (1.0)Comorbidities Chronic lung disease503 (21.6)686 (26.1)59 (11.2)95 (16.2) Hypertension1623 (69.8)1662 (63.1)372 (70.5)381 (65.0) Congestive heart failure734 (31.6)430 (16.3)147 (27.8)95 (16.2) Ischaemic heart disease966 (41.6)887 (33.7)223 (42.2)207 (35.3) Cerebrovascular disease692 (29.8)638 (24.2)152 (28.8)145 (24.7) Other dementia174 (7.5)190 (7.2)42 (8.0)23 (3.9) Cancer382 (16.4)474 (18.0)99 (18.8)120 (20.5) Connective tissue disorders228 (9.8)215 (8.2)36 (6.8)52 (8.9)HbA1C Good <7%1176 (50.6)1401 (53.2)251 (47.5)308 (52.6) Borderline 7–10%966 (41.6)1033 (39.2)213 (40.3)230 (39.3) Poor >10%108 (4.7)131 (5.0)41 (7.8)34 (5.8) None recorded74 (3.2)68 (2.6)23 (4.4)14 (2.4)Prior antidiabetes medication Insulin122 (5.3)126 (4.8)34 (6.4)23 (3.9) Oral medications1139 (49.0)1426 (54.2)247 (46.8)312 (53.2) Both462 (19.9)384 (14.6)130 (24.6)114 (19.5) None601 (25.9)697 (26.5)117 (22.2)137 (23.4) Total23242633528586eGFR, estimated glomerular filtration rate.aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nBaseline characteristics of the study population\neGFR, estimated glomerular filtration rate.\naIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nPatients with pneumonia experienced 29.6% 28-day all-cause mortality (1406 deaths). Patients with sepsis experienced 35.6% 28-day all-cause mortality (377 deaths) (Table 2). Survival curves showed high mortality at infection onset, declining over ∼30 days to a more stable rate for the next 60 days following both pneumonia and sepsis (Figure 1). RRs for 28-day mortality were higher among people with eGFR <30 mL/min/1.73 m2 compared with people with eGFR ≥60 mL/min/1.73 m2 for pneumonia (RR = 1.27; 95% CI 1.10–1.47) and sepsis (RR = 1.42; 1.10–1.84), adjusted for age, sex, socio-economic status and onset prior to April 2004. Adjustment for smoking status, co-morbidities and characteristics of diabetes had minimal effect on these RRs for pneumonia (fully adjusted RR = 1.27; 1.12–1.43) or sepsis (fully adjusted RR = 1.32; 1.07–1.64). There was no evidence of associations between intermediate levels of eGFR and 28-day mortality for either infection, nor for an association between proteinuria and 28-day mortality. The pattern of associations of eGFR and proteinuria with 90-day mortality was similar to those for 28-day mortality (Table 2). Results were unchanged by additional adjustment for peripheral vascular disease. There was no good evidence of effect modification between eGFR and proteinuria, and in particular no evidence of any association of proteinuria with 28-day mortality within any category of eGFR status (data not shown).Table 2.Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)aNumber (column %)28-Day mortality (row %)90-Day mortality (row %)RRs for 28-day mortality (95% CI)RRs for 90-day mortality (95% CI)Adjusted for demographicsbadjusted for non-CVD co-morbiditiescFully adjusteddAdjusted for demographicsbAdjusted for non-CVD co-morbiditiescFully adjusteddPneumonia Proteinuria (adjusted for eGFR) Yes1611 (34.0)499 (31.0)625 (38.8)1.07 (0.97–1.17)1.07 (0.98–1.18)1.07 (0.98–1.18)1.05 (0.97–1.13)1.06 (0.98–1.14)1.05 (0.98–1.14) No3132 (66.0)907 (29.0)1150 (36.7)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<1523 (0.5)12 (52.2)12 (52.2)1.27 (1.10–1.47)1.31 (1.13–1.52)1.30 (1.12–1.52)1.26 (1.12–1.42)1.29 (1.14–1.45)1.27 (1.12–1.43)15–29263 (5.6)110 (41.8)139 (52.9) 30–44764 (16.1)265 (34.7)336 (44.0)0.98 (0.86–1.10)0.99 (0.88–1.13)0.99 (0.88–1.12)1.02 (0.93–1.13)1.04 (0.94–1.15)1.03 (0.94–1.14) 45–601162 (24.5)332 (28.6)418 (36.0)0.91 (0.82–1.02)0.94 (0.84–1.04)0.93 (0.84–1.04)0.91 (0.83–1.00)0.93 (0.85–1.02)0.92 (0.84–1.01) ≥602531 (53.4)687 (27.1)870 (34.4)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total47431406 (29.6)1775 (37.4)Sepsis Proteinuria (adjusted for eGFR) Yes358 (33.8)128 (35.8)159 (44.4)0.98 (0.82–1.17)1.01 (0.85–1.21)1.01 (0.85–1.21)0.98 (0.86–1.13)0.96 (0.84–1.10)0.96 (0.84–1.10) No700 (66.2)249 (35.6)309 (44.1)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<158 (0.8)2 (25.0)5 (62.5)1.42 (1.10–1.84)1.41 (1.08–1.84)1.37 (1.05–1.79)1.39 (1.14–1.70)1.32 (1.07–1.63)1.32 (1.07–1.64)15–2962 (5.9)32 (51.6)39 (62.9) 30–44190 (18.0)88 (46.3)103 (54.2)1.25 (1.01–1.55)1.24 (0.99–1.55)1.24 (0.99–1.54)1.14 (0.96–1.36)1.12 (0.94–1.33)1.11 (0.94–1.32) 45–60232 (21.9)76 (32.8)95 (41.0)0.95 (0.75–1.19)0.91 (0.72–1.15)0.91 (0.72–1.14)0.92 (0.77–1.11)0.89 (0.74–1.07)0.89 (0.75–1.07) ≥60566 (53.5)179 (31.6)226 (39.9)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total1058377 (35.6)468 (44.2)aExcluding patients with missing smoking or HbA1C data.bAge, gender, socio-economic status, onset prior to 1 April 2004.cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.FIGURE 1:Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nShort-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)a\naExcluding patients with missing smoking or HbA1C data.\nbAge, gender, socio-economic status, onset prior to 1 April 2004.\ncAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.\ndAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.\nSurvival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nThe underlying causes of death following pneumonia and sepsis were similar for patients with eGFR above and below 60 mL/min/1.73 m2. Causes of 28-day mortality following sepsis were predominantly sources of infection (Table 3). Following pneumonia onset, pneumonia was recorded as an underlying or contributory cause of death for 83.9% (1191/1419) of deaths within 28 days and 76.3% (1366/1790) of deaths within 90 days. Among patients with eGFR <60 mL/min/1.73 m2, renal disease was recorded as an underlying or contributory cause for 10.6% (77/724) of those who died within 28 days of pneumonia onset and 16.6% (152/913) of those who died within 90 days. Recording of CKD as a cause of death increased with lower eGFR (Table 4).Table 3.Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)aeGFReGFR <60 mL/min/1.73 m2eGFR ≥60 mL/min/1.73 m228-Day mortality following pneumonia, n = 1419J18 Pneumonia, organism unspecified, n = 256, 35.4%J44 Other chronic obstructive pulmonary disease, n = 63, 8.7%I25 Chronic ischaemic heart disease, n = 47, 6.5%I50 Heart failure, n = 43, 5.9%I64 Stroke, not specified as haemorrhage or infarction, n = 37, 5.1%J18 Pneumonia, organism unspecified, n = 216, 31.1%J44 Other chronic obstructive pulmonary disease, n = 63, 9.1%I64 Stroke, not specified as haemorrhage or infarction, n = 35, 5.0%C34 Malignant neoplasm of bronchus and lung, n = 34, 4.9%I25 Chronic ischaemic heart disease, n = 29, 4.2%Total = 724Total = 69529–90 Day mortality following pneumonia, n = 371J18 Pneumonia, organism unspecified, n = 38, 20.1%I25 Chronic ischaemic heart disease, n = 24, 12.7%J44 Other chronic obstructive pulmonary disease, n = 13, 6.9%C34 Malignant neoplasm of bronchus and lung, n = 12, 6.4%I21 Acute myocardial infarction, n = 12, 6.4%J18 Pneumonia, organism unspecified, n = 20, 11.0%C34 Malignant neoplasm of bronchus and lung, n = 20, 11.0%J44 Other chronic obstructive pulmonary disease, n = 19, 10.4%I25 Chronic ischaemic heart disease, n = 9, 5.0%I64 Stroke, not specified as haemorrhage or infarction, n = 9, 5.0%Total = 189Total = 18228-Day mortality following sepsis, n = 387N39 Other disorders of urinary systemb, n = 33, 15.9%J18 Pneumonia, organism unspecified, n = 30, 14.4%E14 Unspecified diabetes mellitus, n = 12, 5.8%A41 Other sepsis, n = 11, 5.3%L03 Cellulitis, n = 11, 5.3%N39 Other disorders of urinary systemb, n = 20, 11.2%A41 Other sepsis, n = 18, 10.1%J18 Pneumonia, organism unspecified, n = 18, 10.1%E14 Unspecified diabetes mellitus, n = 10, 5.6%J44 Other chronic obstructive pulmonary disease, n = 6, 3.4%=K55 Vascular disorders of intestine, n = 6, 3.4%=L03 Cellulitis, n = 6, 3.4%Total = 208Total = 179aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.Table 4.Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFReGFR (mL/min/1.73 m2)Deaths within 28 days of pneumonia onsetDeaths within 90 days of pneumonia onsetTotalN18 CKD, n (%)Renal disease,b\nn (%)TotalN18 CKD, n (%)Renal disease,b\nn (%)<1586 (75.0)7 (87.5)86 (75.0)7 (87.5)15–2910919 (17.4)41 (37.6)13926 (18.7)53 (38.1)30–4427514 (5.1)2 (15.3)34720 (5.8)56 (16.1)35–593328 (2.4)27 (8.1)41910 (2.4)36 (8.6)Total72447 (6.5)77 (10.6)91362 (6.8)152 (16.6)aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\nTop five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)a\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.\nRecording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFR\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.","Among this population of older people with diabetes mellitus, eGFR <30 mL/min/1.73 m2 was a risk marker of higher 28- and 90-day mortality following community-acquired pneumonia and sepsis, compared with patients with eGFR ≥60 mL/min/1.73 m2. The relationship between eGFR and mortality did not change with adjustment for co-morbidities. Neither moderately impaired eGFR nor proteinuria was associated with higher short-term mortality following either infection.\nThe strengths of this study follow from the analysis of a focused question using large, linked datasets for a highly monitored primary care population with a cohort study design. Our study identifies that the association between eGFR and post-infection mortality persists when patients with end-stage renal disease (ESRD) are excluded (and is not explained by renal replacement therapy), when considering fixed-term rather than in-hospital mortality (thus is not due to differences in hospital stay) and when exclusively community-acquired infections are considered (so does not result from increased risk of healthcare-associated infections). The linked datasets allowed us to identify infections both among patients presenting directly to hospital and those managed in the community, maximized ascertainment of mortality and enabled description of the causes of death. The highly monitored population allowed good ascertainment of CKD status. The cohort study design has less potential for selection bias than an equivalent case–control study.\nA limitation is our assumption that the absence of a record implies a negative status for proteinuria and co-morbidities. Under-ascertainment of co-morbidities could result in residual confounding, with unpredictable effects, but the high prevalence of co-morbidities observed suggests that ascertainment was not markedly incomplete. We observed a high prevalence of proteinuria, and this is a highly monitored population (with financial incentives for standardized recording of proteinuria since 2004), but under-ascertainment of proteinuria could result in underestimation of any association between proteinuria and mortality [24]. Residual confounding from undiagnosed cardiovascular disease should have been minimized by adjustment for cardiovascular disease risk factors including smoking, hypertension and characteristics of diabetes.\nOur findings for eGFR provide further detail to build on previous findings that baseline eGFR <60 mL/min/1.73 m2 or renal disease are risk factors for short-term mortality following (hospital- or community-acquired) sepsis and for in-hospital mortality following community-acquired pneumonia (including patients receiving dialysis) [11–15]. A more comparable Canadian study examined the associations between eGFR and 30-day mortality following community-acquired pneumonia among the general population aged ≥65 years, excluding patients with ESRD [16]. Fully adjusted hazard ratios for 30-day mortality were 1.22 (95% CI 1.01–1.49) for eGFR 45–59, 2.03 (1.64–2.50) for eGFR 30–44 and 4.94 (3.94–6.19) for eGFR < 30, compared with eGFR 60–104 mL/min/1.73 m2. These are somewhat greater than the associations we observed. The difference may be explained by the different study populations. Both studies required a baseline serum creatinine result for inclusion. Our study population of older people with diabetes were routinely monitored for CKD (with financial incentivization in primary care) [32]. Creatinine testing of the Canadian study population may have been encouraged by co-morbidities or health-behaviours associated with CKD (such as smoking), which increase post-infection mortality, resulting in over-estimation of the association of eGFR and post-infection mortality. Our study population is less vulnerable to differential ascertainment of CKD. Alternatively, the association between eGFR and post-infection mortality may be smaller among patients with diabetes.\nTo the best of our knowledge, our examination of any association between proteinuria and mortality following community-acquired pneumonia and sepsis is novel. A history of proteinuria, although a marker for mortality in general, does not appear to be a risk marker for short-term mortality following community-acquired infection. This is unlikely to be due to chance, as the study was large, with findings consistent across both infections. We designed our study to produce conservative estimates and may have under-estimated the association between proteinuria and post-infection mortality due to under-ascertainment of proteinuria. Alternatively, any potential relationship between proteinuria and mortality may have been mitigated by clinical care of patients with infection who had pre-existing proteinuria, for example, through swift recognition of AKI.\nThe survival curves demonstrate a steep initial mortality following infection onset, and a high proportion of deaths had the underlying cause assigned to infection. Since 2001 in England, co-morbidities are assigned as the underlying cause of death when pneumonia has occurred in the context of, for example, malignancy or respiratory disease [25]. This suggests that the associations we observed are driven by an association between eGFR and infection prognosis, not merely high underlying baseline mortality among patients with impaired eGFR. This is supported by previous research which found 7.7-fold elevated mortality in the 30 days following community-acquired pneumonia [19]. Estimates for associations between eGFR and mortality were not substantially altered by adjustment for co-morbidities, suggesting that any causal relationship between eGFR and mortality is not mediated through co-morbidities.\nOur findings apply to the large population of older patients with community-acquired pneumonia or sepsis with diabetes mellitus who do not have ESRD. Inclusion criteria are unlikely to have limited generalizability appreciably. Practices which consent to data linkage could be more research oriented, providing good primary care management of risk factors for infection-related mortality (such as smoking cessation), but this is unlikely to affect the relationship between CKD and short-term mortality post-infection. Lack of pre-existing creatinine test results is likely to reflect limited time potentially eligible for the study rather than CKD status among this highly monitored population. Missing data on smoking status and HbA1C may be a marker of low patient engagement: caution should be used in generalizing our results to patients who are not actively managed in primary care.\nWe found that CKD is a useful clinical risk marker for post-infectious mortality. Whether this relationship is causal is less clear; but the association does not appear to be explained by age, co-morbidities or hospital attendance. Potential mechanisms include immune system dysfunction, but also more preventable complications such as AKI. Combinations of risk factors may be important: for example, patients with post-operative AKI have higher mortality if they also have pre-existing CKD [30].\nOur results have implications for patient management and future research. Patients with baseline eGFR <30 mL/min/1.73 m2 and community-acquired infection need careful monitoring, particularly in the 28 days following infection. Future research should investigate preventable mechanisms by which low baseline eGFR could be related to post-infection mortality, for example, fluid management, AKI and drug dosage in patients with low renal clearance."],"string":"[\n \"Chronic kidney disease (CKD) affects an estimated 1.8 million people in England, 98% of whom do not require renal replacement therapy [1]. CKD is defined by reduced estimated glomerular filtration rate (eGFR) or evidence of kidney damage such as proteinuria and is commonest among older people [2, 3]. Most patients with CKD are managed in primary care [4].\\nInfection is an important cause of mortality among older people [5, 6]. Both reduced eGFR and proteinuria are associated with an increased rate of infection-related mortality, which could be partly explained by increased incidence of infection [7–9]. It is less clear whether CKD is also associated with poorer prognosis following infection. When clinicians assess patients with community-acquired infection, developing complications such as acute kidney injury (AKI) may not yet be apparent, but they will know which patients have pre-existing CKD. If pre-existing CKD is a risk marker for short-term mortality, this would be useful for risk stratification and clinical management of patients with infections, especially in primary care where clinicians may not have access to immediate laboratory tests. While the implications of acute changes in eGFR during infection are a focus of current research, few studies have investigated the role of pre-existing CKD [10]. Low baseline eGFR has been found to be associated with mortality following sepsis and community-acquired pneumonia, but rarely examined according to clinically meaningful categories of eGFR [11–15]. To the best of our knowledge, proteinuria has not been examined as a potential risk marker for mortality following infection [12, 15, 16].\\nAmong older people, CKD frequently co-exists with other co-morbidities [3]. An association of CKD with poor prognosis of infections could thus be due to confounding from these co-morbidities. For example, CKD is strongly associated with cardiovascular disease, which may be complicated by infection, resulting in post-infection mortality driven by the underlying cardiovascular disease [17]. Such deaths would largely follow hospitalization for cardiovascular events. CKD is associated with healthcare-associated pneumonia, which carries a worse prognosis than community-acquired pneumonia [18, 19]. Focusing on community-acquired infections should exclude infections arising as short-term sequelae of cardiovascular events and improve understanding of the relationship between pre-existing CKD markers and infection prognosis.\\nOlder people with diabetes mellitus form a large and growing population in primary care who suffer a high incidence of community-acquired pneumonia and sepsis [20]. Forty per cent of adults with diabetes have CKD, of whom three-quarters have proteinuria, and CKD among these patients is associated with a greater all-cause excess mortality than among patients without diabetes [21]. If proteinuria is a risk marker for mortality among older patients with diabetes who develop community-acquired pneumonia or sepsis, this could inform clinical management of a large primary care patient population with appreciable mortality following infection [20].\\nThis study aimed to examine whether baseline eGFR and proteinuria were independent risk markers for short-term mortality following community-acquired pneumonia or sepsis among older people with diabetes mellitus, using large, linked electronic health record databases.\",\n \" Data sources The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\\nThe Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\\n Study population The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\\nThe study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\\n Definition of infections Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\\nInfection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\\n Study outcomes The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\\nThe outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\\n Definition of CKD CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\\nCKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\\n Other variables Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\\nAge was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\\n Data analysis Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\\nStata version 13.1 was used for data analysis. All code lists are available on request.\\nPneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\\nStata version 13.1 was used for data analysis. All code lists are available on request.\\n Ethics The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\\nThe study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\",\n \"The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\",\n \"The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\",\n \"Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\",\n \"The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\",\n \"CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\",\n \"Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\",\n \"Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\\nStata version 13.1 was used for data analysis. All code lists are available on request.\",\n \"The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\",\n \"We identified 4957 individuals with community-acquired pneumonia and 1114 individuals with community-acquired sepsis. Data were missing (for smoking status and/or HbA1C results) for 212/4957 individuals with pneumonia (4.3%) and 56/1114 individuals with sepsis (5.0%). These patients were excluded. Among patients with pneumonia, patients with missing data were older (median age 83 years, IQR: 78–88) compared with those included (median age 80 years, IQR: 74–85), with a higher 28-day mortality (excluded 88/214, 41.1%: included 1406/4743, 29.6%) but a similar distribution of baseline eGFR. A similar pattern was seen for sepsis.\\nEstimated GFR was based on the higher of two results for 4029 patients with pneumonia (84.9%) and 919 patients with sepsis (86.9%); for the remaining patients, only a single valid serum creatinine result was available. CKD prevalence was high: almost half of the patients had eGFR <60 mL/min/1.73 m2 and a third had proteinuria. Patients with eGFR <60 mL/min/1.73 m2 were older, with a higher prevalence of ischaemic heart disease and congestive heart failure than patients with eGFR ≥60 mL/min/1.73 m2 (Table 1).Table 1.Baseline characteristics of the study populationPneumoniaSepsiseGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2eGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2Age (years) Median (IQR)82 (77–87)78 (72–83)81 (75–86)76 (71–82)n (col %)n (col %)n (col %)n (col %)Gender Female1231 (53.0)1106 (42.0)294 (55.7)268 (45.7)Onset prior to 1 April 2004584 (25.1)527 (20.0)124 (23.5)120 (20.5)Socio-economic status (IMD quintile)a 1 (least deprived)383 (16.5)438 (16.6)109 (20.6)106 (18.1) 2554 (23.8)595 (22.6)116 (22.0)127 (21.7) 3494 (21.3)582 (22.1)120 (22.7)137 (23.4) 4502 (21.6)574 (21.8)110 (20.8)108 (18.4) 5 (most deprived)391 (16.8)444 (16.9)73 (13.8)108 (18.4)Smoking status Current321 (13.8)549 (20.9)70 (13.3)103 (17.6) Ex-smoker1185 (51.0)1351 (51.3)235 (44.5)284 (48.5) Non-smoker776 (33.4)698 (26.5)207 (39.2)193 (32.9) Missing42 (1.8)35 (1.3)16 (3.0)6 (1.0)Comorbidities Chronic lung disease503 (21.6)686 (26.1)59 (11.2)95 (16.2) Hypertension1623 (69.8)1662 (63.1)372 (70.5)381 (65.0) Congestive heart failure734 (31.6)430 (16.3)147 (27.8)95 (16.2) Ischaemic heart disease966 (41.6)887 (33.7)223 (42.2)207 (35.3) Cerebrovascular disease692 (29.8)638 (24.2)152 (28.8)145 (24.7) Other dementia174 (7.5)190 (7.2)42 (8.0)23 (3.9) Cancer382 (16.4)474 (18.0)99 (18.8)120 (20.5) Connective tissue disorders228 (9.8)215 (8.2)36 (6.8)52 (8.9)HbA1C Good <7%1176 (50.6)1401 (53.2)251 (47.5)308 (52.6) Borderline 7–10%966 (41.6)1033 (39.2)213 (40.3)230 (39.3) Poor >10%108 (4.7)131 (5.0)41 (7.8)34 (5.8) None recorded74 (3.2)68 (2.6)23 (4.4)14 (2.4)Prior antidiabetes medication Insulin122 (5.3)126 (4.8)34 (6.4)23 (3.9) Oral medications1139 (49.0)1426 (54.2)247 (46.8)312 (53.2) Both462 (19.9)384 (14.6)130 (24.6)114 (19.5) None601 (25.9)697 (26.5)117 (22.2)137 (23.4) Total23242633528586eGFR, estimated glomerular filtration rate.aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\\nBaseline characteristics of the study population\\neGFR, estimated glomerular filtration rate.\\naIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\\nPatients with pneumonia experienced 29.6% 28-day all-cause mortality (1406 deaths). Patients with sepsis experienced 35.6% 28-day all-cause mortality (377 deaths) (Table 2). Survival curves showed high mortality at infection onset, declining over ∼30 days to a more stable rate for the next 60 days following both pneumonia and sepsis (Figure 1). RRs for 28-day mortality were higher among people with eGFR <30 mL/min/1.73 m2 compared with people with eGFR ≥60 mL/min/1.73 m2 for pneumonia (RR = 1.27; 95% CI 1.10–1.47) and sepsis (RR = 1.42; 1.10–1.84), adjusted for age, sex, socio-economic status and onset prior to April 2004. Adjustment for smoking status, co-morbidities and characteristics of diabetes had minimal effect on these RRs for pneumonia (fully adjusted RR = 1.27; 1.12–1.43) or sepsis (fully adjusted RR = 1.32; 1.07–1.64). There was no evidence of associations between intermediate levels of eGFR and 28-day mortality for either infection, nor for an association between proteinuria and 28-day mortality. The pattern of associations of eGFR and proteinuria with 90-day mortality was similar to those for 28-day mortality (Table 2). Results were unchanged by additional adjustment for peripheral vascular disease. There was no good evidence of effect modification between eGFR and proteinuria, and in particular no evidence of any association of proteinuria with 28-day mortality within any category of eGFR status (data not shown).Table 2.Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)aNumber (column %)28-Day mortality (row %)90-Day mortality (row %)RRs for 28-day mortality (95% CI)RRs for 90-day mortality (95% CI)Adjusted for demographicsbadjusted for non-CVD co-morbiditiescFully adjusteddAdjusted for demographicsbAdjusted for non-CVD co-morbiditiescFully adjusteddPneumonia Proteinuria (adjusted for eGFR) Yes1611 (34.0)499 (31.0)625 (38.8)1.07 (0.97–1.17)1.07 (0.98–1.18)1.07 (0.98–1.18)1.05 (0.97–1.13)1.06 (0.98–1.14)1.05 (0.98–1.14) No3132 (66.0)907 (29.0)1150 (36.7)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<1523 (0.5)12 (52.2)12 (52.2)1.27 (1.10–1.47)1.31 (1.13–1.52)1.30 (1.12–1.52)1.26 (1.12–1.42)1.29 (1.14–1.45)1.27 (1.12–1.43)15–29263 (5.6)110 (41.8)139 (52.9) 30–44764 (16.1)265 (34.7)336 (44.0)0.98 (0.86–1.10)0.99 (0.88–1.13)0.99 (0.88–1.12)1.02 (0.93–1.13)1.04 (0.94–1.15)1.03 (0.94–1.14) 45–601162 (24.5)332 (28.6)418 (36.0)0.91 (0.82–1.02)0.94 (0.84–1.04)0.93 (0.84–1.04)0.91 (0.83–1.00)0.93 (0.85–1.02)0.92 (0.84–1.01) ≥602531 (53.4)687 (27.1)870 (34.4)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total47431406 (29.6)1775 (37.4)Sepsis Proteinuria (adjusted for eGFR) Yes358 (33.8)128 (35.8)159 (44.4)0.98 (0.82–1.17)1.01 (0.85–1.21)1.01 (0.85–1.21)0.98 (0.86–1.13)0.96 (0.84–1.10)0.96 (0.84–1.10) No700 (66.2)249 (35.6)309 (44.1)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<158 (0.8)2 (25.0)5 (62.5)1.42 (1.10–1.84)1.41 (1.08–1.84)1.37 (1.05–1.79)1.39 (1.14–1.70)1.32 (1.07–1.63)1.32 (1.07–1.64)15–2962 (5.9)32 (51.6)39 (62.9) 30–44190 (18.0)88 (46.3)103 (54.2)1.25 (1.01–1.55)1.24 (0.99–1.55)1.24 (0.99–1.54)1.14 (0.96–1.36)1.12 (0.94–1.33)1.11 (0.94–1.32) 45–60232 (21.9)76 (32.8)95 (41.0)0.95 (0.75–1.19)0.91 (0.72–1.15)0.91 (0.72–1.14)0.92 (0.77–1.11)0.89 (0.74–1.07)0.89 (0.75–1.07) ≥60566 (53.5)179 (31.6)226 (39.9)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total1058377 (35.6)468 (44.2)aExcluding patients with missing smoking or HbA1C data.bAge, gender, socio-economic status, onset prior to 1 April 2004.cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.FIGURE 1:Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\\nShort-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)a\\naExcluding patients with missing smoking or HbA1C data.\\nbAge, gender, socio-economic status, onset prior to 1 April 2004.\\ncAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.\\ndAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.\\nSurvival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\\nThe underlying causes of death following pneumonia and sepsis were similar for patients with eGFR above and below 60 mL/min/1.73 m2. Causes of 28-day mortality following sepsis were predominantly sources of infection (Table 3). Following pneumonia onset, pneumonia was recorded as an underlying or contributory cause of death for 83.9% (1191/1419) of deaths within 28 days and 76.3% (1366/1790) of deaths within 90 days. Among patients with eGFR <60 mL/min/1.73 m2, renal disease was recorded as an underlying or contributory cause for 10.6% (77/724) of those who died within 28 days of pneumonia onset and 16.6% (152/913) of those who died within 90 days. Recording of CKD as a cause of death increased with lower eGFR (Table 4).Table 3.Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)aeGFReGFR <60 mL/min/1.73 m2eGFR ≥60 mL/min/1.73 m228-Day mortality following pneumonia, n = 1419J18 Pneumonia, organism unspecified, n = 256, 35.4%J44 Other chronic obstructive pulmonary disease, n = 63, 8.7%I25 Chronic ischaemic heart disease, n = 47, 6.5%I50 Heart failure, n = 43, 5.9%I64 Stroke, not specified as haemorrhage or infarction, n = 37, 5.1%J18 Pneumonia, organism unspecified, n = 216, 31.1%J44 Other chronic obstructive pulmonary disease, n = 63, 9.1%I64 Stroke, not specified as haemorrhage or infarction, n = 35, 5.0%C34 Malignant neoplasm of bronchus and lung, n = 34, 4.9%I25 Chronic ischaemic heart disease, n = 29, 4.2%Total = 724Total = 69529–90 Day mortality following pneumonia, n = 371J18 Pneumonia, organism unspecified, n = 38, 20.1%I25 Chronic ischaemic heart disease, n = 24, 12.7%J44 Other chronic obstructive pulmonary disease, n = 13, 6.9%C34 Malignant neoplasm of bronchus and lung, n = 12, 6.4%I21 Acute myocardial infarction, n = 12, 6.4%J18 Pneumonia, organism unspecified, n = 20, 11.0%C34 Malignant neoplasm of bronchus and lung, n = 20, 11.0%J44 Other chronic obstructive pulmonary disease, n = 19, 10.4%I25 Chronic ischaemic heart disease, n = 9, 5.0%I64 Stroke, not specified as haemorrhage or infarction, n = 9, 5.0%Total = 189Total = 18228-Day mortality following sepsis, n = 387N39 Other disorders of urinary systemb, n = 33, 15.9%J18 Pneumonia, organism unspecified, n = 30, 14.4%E14 Unspecified diabetes mellitus, n = 12, 5.8%A41 Other sepsis, n = 11, 5.3%L03 Cellulitis, n = 11, 5.3%N39 Other disorders of urinary systemb, n = 20, 11.2%A41 Other sepsis, n = 18, 10.1%J18 Pneumonia, organism unspecified, n = 18, 10.1%E14 Unspecified diabetes mellitus, n = 10, 5.6%J44 Other chronic obstructive pulmonary disease, n = 6, 3.4%=K55 Vascular disorders of intestine, n = 6, 3.4%=L03 Cellulitis, n = 6, 3.4%Total = 208Total = 179aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.Table 4.Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFReGFR (mL/min/1.73 m2)Deaths within 28 days of pneumonia onsetDeaths within 90 days of pneumonia onsetTotalN18 CKD, n (%)Renal disease,b\\nn (%)TotalN18 CKD, n (%)Renal disease,b\\nn (%)<1586 (75.0)7 (87.5)86 (75.0)7 (87.5)15–2910919 (17.4)41 (37.6)13926 (18.7)53 (38.1)30–4427514 (5.1)2 (15.3)34720 (5.8)56 (16.1)35–593328 (2.4)27 (8.1)41910 (2.4)36 (8.6)Total72447 (6.5)77 (10.6)91362 (6.8)152 (16.6)aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\\nTop five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)a\\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\\nbAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.\\nRecording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFR\\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\\nbAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\",\n \"Among this population of older people with diabetes mellitus, eGFR <30 mL/min/1.73 m2 was a risk marker of higher 28- and 90-day mortality following community-acquired pneumonia and sepsis, compared with patients with eGFR ≥60 mL/min/1.73 m2. The relationship between eGFR and mortality did not change with adjustment for co-morbidities. Neither moderately impaired eGFR nor proteinuria was associated with higher short-term mortality following either infection.\\nThe strengths of this study follow from the analysis of a focused question using large, linked datasets for a highly monitored primary care population with a cohort study design. Our study identifies that the association between eGFR and post-infection mortality persists when patients with end-stage renal disease (ESRD) are excluded (and is not explained by renal replacement therapy), when considering fixed-term rather than in-hospital mortality (thus is not due to differences in hospital stay) and when exclusively community-acquired infections are considered (so does not result from increased risk of healthcare-associated infections). The linked datasets allowed us to identify infections both among patients presenting directly to hospital and those managed in the community, maximized ascertainment of mortality and enabled description of the causes of death. The highly monitored population allowed good ascertainment of CKD status. The cohort study design has less potential for selection bias than an equivalent case–control study.\\nA limitation is our assumption that the absence of a record implies a negative status for proteinuria and co-morbidities. Under-ascertainment of co-morbidities could result in residual confounding, with unpredictable effects, but the high prevalence of co-morbidities observed suggests that ascertainment was not markedly incomplete. We observed a high prevalence of proteinuria, and this is a highly monitored population (with financial incentives for standardized recording of proteinuria since 2004), but under-ascertainment of proteinuria could result in underestimation of any association between proteinuria and mortality [24]. Residual confounding from undiagnosed cardiovascular disease should have been minimized by adjustment for cardiovascular disease risk factors including smoking, hypertension and characteristics of diabetes.\\nOur findings for eGFR provide further detail to build on previous findings that baseline eGFR <60 mL/min/1.73 m2 or renal disease are risk factors for short-term mortality following (hospital- or community-acquired) sepsis and for in-hospital mortality following community-acquired pneumonia (including patients receiving dialysis) [11–15]. A more comparable Canadian study examined the associations between eGFR and 30-day mortality following community-acquired pneumonia among the general population aged ≥65 years, excluding patients with ESRD [16]. Fully adjusted hazard ratios for 30-day mortality were 1.22 (95% CI 1.01–1.49) for eGFR 45–59, 2.03 (1.64–2.50) for eGFR 30–44 and 4.94 (3.94–6.19) for eGFR < 30, compared with eGFR 60–104 mL/min/1.73 m2. These are somewhat greater than the associations we observed. The difference may be explained by the different study populations. Both studies required a baseline serum creatinine result for inclusion. Our study population of older people with diabetes were routinely monitored for CKD (with financial incentivization in primary care) [32]. Creatinine testing of the Canadian study population may have been encouraged by co-morbidities or health-behaviours associated with CKD (such as smoking), which increase post-infection mortality, resulting in over-estimation of the association of eGFR and post-infection mortality. Our study population is less vulnerable to differential ascertainment of CKD. Alternatively, the association between eGFR and post-infection mortality may be smaller among patients with diabetes.\\nTo the best of our knowledge, our examination of any association between proteinuria and mortality following community-acquired pneumonia and sepsis is novel. A history of proteinuria, although a marker for mortality in general, does not appear to be a risk marker for short-term mortality following community-acquired infection. This is unlikely to be due to chance, as the study was large, with findings consistent across both infections. We designed our study to produce conservative estimates and may have under-estimated the association between proteinuria and post-infection mortality due to under-ascertainment of proteinuria. Alternatively, any potential relationship between proteinuria and mortality may have been mitigated by clinical care of patients with infection who had pre-existing proteinuria, for example, through swift recognition of AKI.\\nThe survival curves demonstrate a steep initial mortality following infection onset, and a high proportion of deaths had the underlying cause assigned to infection. Since 2001 in England, co-morbidities are assigned as the underlying cause of death when pneumonia has occurred in the context of, for example, malignancy or respiratory disease [25]. This suggests that the associations we observed are driven by an association between eGFR and infection prognosis, not merely high underlying baseline mortality among patients with impaired eGFR. This is supported by previous research which found 7.7-fold elevated mortality in the 30 days following community-acquired pneumonia [19]. Estimates for associations between eGFR and mortality were not substantially altered by adjustment for co-morbidities, suggesting that any causal relationship between eGFR and mortality is not mediated through co-morbidities.\\nOur findings apply to the large population of older patients with community-acquired pneumonia or sepsis with diabetes mellitus who do not have ESRD. Inclusion criteria are unlikely to have limited generalizability appreciably. Practices which consent to data linkage could be more research oriented, providing good primary care management of risk factors for infection-related mortality (such as smoking cessation), but this is unlikely to affect the relationship between CKD and short-term mortality post-infection. Lack of pre-existing creatinine test results is likely to reflect limited time potentially eligible for the study rather than CKD status among this highly monitored population. Missing data on smoking status and HbA1C may be a marker of low patient engagement: caution should be used in generalizing our results to patients who are not actively managed in primary care.\\nWe found that CKD is a useful clinical risk marker for post-infectious mortality. Whether this relationship is causal is less clear; but the association does not appear to be explained by age, co-morbidities or hospital attendance. Potential mechanisms include immune system dysfunction, but also more preventable complications such as AKI. Combinations of risk factors may be important: for example, patients with post-operative AKI have higher mortality if they also have pre-existing CKD [30].\\nOur results have implications for patient management and future research. Patients with baseline eGFR <30 mL/min/1.73 m2 and community-acquired infection need careful monitoring, particularly in the 28 days following infection. Future research should investigate preventable mechanisms by which low baseline eGFR could be related to post-infection mortality, for example, fluid management, AKI and drug dosage in patients with low renal clearance.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","materials|methods",null,null,null,null,null,null,null,null,"results","discussion"],"string":"[\n \"intro\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["chronic kidney disease","community-acquired infections","electronic health records","infection/mortality","proteinuria"],"string":"[\n \"chronic kidney disease\",\n \"community-acquired infections\",\n \"electronic health records\",\n \"infection/mortality\",\n \"proteinuria\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nChronic kidney disease (CKD) affects an estimated 1.8 million people in England, 98% of whom do not require renal replacement therapy [1]. CKD is defined by reduced estimated glomerular filtration rate (eGFR) or evidence of kidney damage such as proteinuria and is commonest among older people [2, 3]. Most patients with CKD are managed in primary care [4].\nInfection is an important cause of mortality among older people [5, 6]. Both reduced eGFR and proteinuria are associated with an increased rate of infection-related mortality, which could be partly explained by increased incidence of infection [7–9]. It is less clear whether CKD is also associated with poorer prognosis following infection. When clinicians assess patients with community-acquired infection, developing complications such as acute kidney injury (AKI) may not yet be apparent, but they will know which patients have pre-existing CKD. If pre-existing CKD is a risk marker for short-term mortality, this would be useful for risk stratification and clinical management of patients with infections, especially in primary care where clinicians may not have access to immediate laboratory tests. While the implications of acute changes in eGFR during infection are a focus of current research, few studies have investigated the role of pre-existing CKD [10]. Low baseline eGFR has been found to be associated with mortality following sepsis and community-acquired pneumonia, but rarely examined according to clinically meaningful categories of eGFR [11–15]. To the best of our knowledge, proteinuria has not been examined as a potential risk marker for mortality following infection [12, 15, 16].\nAmong older people, CKD frequently co-exists with other co-morbidities [3]. An association of CKD with poor prognosis of infections could thus be due to confounding from these co-morbidities. For example, CKD is strongly associated with cardiovascular disease, which may be complicated by infection, resulting in post-infection mortality driven by the underlying cardiovascular disease [17]. Such deaths would largely follow hospitalization for cardiovascular events. CKD is associated with healthcare-associated pneumonia, which carries a worse prognosis than community-acquired pneumonia [18, 19]. Focusing on community-acquired infections should exclude infections arising as short-term sequelae of cardiovascular events and improve understanding of the relationship between pre-existing CKD markers and infection prognosis.\nOlder people with diabetes mellitus form a large and growing population in primary care who suffer a high incidence of community-acquired pneumonia and sepsis [20]. Forty per cent of adults with diabetes have CKD, of whom three-quarters have proteinuria, and CKD among these patients is associated with a greater all-cause excess mortality than among patients without diabetes [21]. If proteinuria is a risk marker for mortality among older patients with diabetes who develop community-acquired pneumonia or sepsis, this could inform clinical management of a large primary care patient population with appreciable mortality following infection [20].\nThis study aimed to examine whether baseline eGFR and proteinuria were independent risk markers for short-term mortality following community-acquired pneumonia or sepsis among older people with diabetes mellitus, using large, linked electronic health record databases.\n\nMATERIALS AND METHODS:\n Data sources The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\nThe Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\n Study population The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\nThe study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\n Definition of infections Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\nInfection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\n Study outcomes The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\nThe outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\n Definition of CKD CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\nCKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\n Other variables Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\nAge was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\n Data analysis Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\nPneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\n Ethics The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\nThe study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\n\nData sources:\nThe Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\n\nStudy population:\nThe study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\n\nDefinition of infections:\nInfection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\n\nStudy outcomes:\nThe outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\n\nDefinition of CKD:\nCKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\n\nOther variables:\nAge was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\n\nData analysis:\nPneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\n\nEthics:\nThe study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\n\nRESULTS:\nWe identified 4957 individuals with community-acquired pneumonia and 1114 individuals with community-acquired sepsis. Data were missing (for smoking status and/or HbA1C results) for 212/4957 individuals with pneumonia (4.3%) and 56/1114 individuals with sepsis (5.0%). These patients were excluded. Among patients with pneumonia, patients with missing data were older (median age 83 years, IQR: 78–88) compared with those included (median age 80 years, IQR: 74–85), with a higher 28-day mortality (excluded 88/214, 41.1%: included 1406/4743, 29.6%) but a similar distribution of baseline eGFR. A similar pattern was seen for sepsis.\nEstimated GFR was based on the higher of two results for 4029 patients with pneumonia (84.9%) and 919 patients with sepsis (86.9%); for the remaining patients, only a single valid serum creatinine result was available. CKD prevalence was high: almost half of the patients had eGFR <60 mL/min/1.73 m2 and a third had proteinuria. Patients with eGFR <60 mL/min/1.73 m2 were older, with a higher prevalence of ischaemic heart disease and congestive heart failure than patients with eGFR ≥60 mL/min/1.73 m2 (Table 1).Table 1.Baseline characteristics of the study populationPneumoniaSepsiseGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2eGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2Age (years) Median (IQR)82 (77–87)78 (72–83)81 (75–86)76 (71–82)n (col %)n (col %)n (col %)n (col %)Gender Female1231 (53.0)1106 (42.0)294 (55.7)268 (45.7)Onset prior to 1 April 2004584 (25.1)527 (20.0)124 (23.5)120 (20.5)Socio-economic status (IMD quintile)a 1 (least deprived)383 (16.5)438 (16.6)109 (20.6)106 (18.1) 2554 (23.8)595 (22.6)116 (22.0)127 (21.7) 3494 (21.3)582 (22.1)120 (22.7)137 (23.4) 4502 (21.6)574 (21.8)110 (20.8)108 (18.4) 5 (most deprived)391 (16.8)444 (16.9)73 (13.8)108 (18.4)Smoking status Current321 (13.8)549 (20.9)70 (13.3)103 (17.6) Ex-smoker1185 (51.0)1351 (51.3)235 (44.5)284 (48.5) Non-smoker776 (33.4)698 (26.5)207 (39.2)193 (32.9) Missing42 (1.8)35 (1.3)16 (3.0)6 (1.0)Comorbidities Chronic lung disease503 (21.6)686 (26.1)59 (11.2)95 (16.2) Hypertension1623 (69.8)1662 (63.1)372 (70.5)381 (65.0) Congestive heart failure734 (31.6)430 (16.3)147 (27.8)95 (16.2) Ischaemic heart disease966 (41.6)887 (33.7)223 (42.2)207 (35.3) Cerebrovascular disease692 (29.8)638 (24.2)152 (28.8)145 (24.7) Other dementia174 (7.5)190 (7.2)42 (8.0)23 (3.9) Cancer382 (16.4)474 (18.0)99 (18.8)120 (20.5) Connective tissue disorders228 (9.8)215 (8.2)36 (6.8)52 (8.9)HbA1C Good <7%1176 (50.6)1401 (53.2)251 (47.5)308 (52.6) Borderline 7–10%966 (41.6)1033 (39.2)213 (40.3)230 (39.3) Poor >10%108 (4.7)131 (5.0)41 (7.8)34 (5.8) None recorded74 (3.2)68 (2.6)23 (4.4)14 (2.4)Prior antidiabetes medication Insulin122 (5.3)126 (4.8)34 (6.4)23 (3.9) Oral medications1139 (49.0)1426 (54.2)247 (46.8)312 (53.2) Both462 (19.9)384 (14.6)130 (24.6)114 (19.5) None601 (25.9)697 (26.5)117 (22.2)137 (23.4) Total23242633528586eGFR, estimated glomerular filtration rate.aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nBaseline characteristics of the study population\neGFR, estimated glomerular filtration rate.\naIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nPatients with pneumonia experienced 29.6% 28-day all-cause mortality (1406 deaths). Patients with sepsis experienced 35.6% 28-day all-cause mortality (377 deaths) (Table 2). Survival curves showed high mortality at infection onset, declining over ∼30 days to a more stable rate for the next 60 days following both pneumonia and sepsis (Figure 1). RRs for 28-day mortality were higher among people with eGFR <30 mL/min/1.73 m2 compared with people with eGFR ≥60 mL/min/1.73 m2 for pneumonia (RR = 1.27; 95% CI 1.10–1.47) and sepsis (RR = 1.42; 1.10–1.84), adjusted for age, sex, socio-economic status and onset prior to April 2004. Adjustment for smoking status, co-morbidities and characteristics of diabetes had minimal effect on these RRs for pneumonia (fully adjusted RR = 1.27; 1.12–1.43) or sepsis (fully adjusted RR = 1.32; 1.07–1.64). There was no evidence of associations between intermediate levels of eGFR and 28-day mortality for either infection, nor for an association between proteinuria and 28-day mortality. The pattern of associations of eGFR and proteinuria with 90-day mortality was similar to those for 28-day mortality (Table 2). Results were unchanged by additional adjustment for peripheral vascular disease. There was no good evidence of effect modification between eGFR and proteinuria, and in particular no evidence of any association of proteinuria with 28-day mortality within any category of eGFR status (data not shown).Table 2.Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)aNumber (column %)28-Day mortality (row %)90-Day mortality (row %)RRs for 28-day mortality (95% CI)RRs for 90-day mortality (95% CI)Adjusted for demographicsbadjusted for non-CVD co-morbiditiescFully adjusteddAdjusted for demographicsbAdjusted for non-CVD co-morbiditiescFully adjusteddPneumonia Proteinuria (adjusted for eGFR) Yes1611 (34.0)499 (31.0)625 (38.8)1.07 (0.97–1.17)1.07 (0.98–1.18)1.07 (0.98–1.18)1.05 (0.97–1.13)1.06 (0.98–1.14)1.05 (0.98–1.14) No3132 (66.0)907 (29.0)1150 (36.7)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<1523 (0.5)12 (52.2)12 (52.2)1.27 (1.10–1.47)1.31 (1.13–1.52)1.30 (1.12–1.52)1.26 (1.12–1.42)1.29 (1.14–1.45)1.27 (1.12–1.43)15–29263 (5.6)110 (41.8)139 (52.9) 30–44764 (16.1)265 (34.7)336 (44.0)0.98 (0.86–1.10)0.99 (0.88–1.13)0.99 (0.88–1.12)1.02 (0.93–1.13)1.04 (0.94–1.15)1.03 (0.94–1.14) 45–601162 (24.5)332 (28.6)418 (36.0)0.91 (0.82–1.02)0.94 (0.84–1.04)0.93 (0.84–1.04)0.91 (0.83–1.00)0.93 (0.85–1.02)0.92 (0.84–1.01) ≥602531 (53.4)687 (27.1)870 (34.4)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total47431406 (29.6)1775 (37.4)Sepsis Proteinuria (adjusted for eGFR) Yes358 (33.8)128 (35.8)159 (44.4)0.98 (0.82–1.17)1.01 (0.85–1.21)1.01 (0.85–1.21)0.98 (0.86–1.13)0.96 (0.84–1.10)0.96 (0.84–1.10) No700 (66.2)249 (35.6)309 (44.1)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<158 (0.8)2 (25.0)5 (62.5)1.42 (1.10–1.84)1.41 (1.08–1.84)1.37 (1.05–1.79)1.39 (1.14–1.70)1.32 (1.07–1.63)1.32 (1.07–1.64)15–2962 (5.9)32 (51.6)39 (62.9) 30–44190 (18.0)88 (46.3)103 (54.2)1.25 (1.01–1.55)1.24 (0.99–1.55)1.24 (0.99–1.54)1.14 (0.96–1.36)1.12 (0.94–1.33)1.11 (0.94–1.32) 45–60232 (21.9)76 (32.8)95 (41.0)0.95 (0.75–1.19)0.91 (0.72–1.15)0.91 (0.72–1.14)0.92 (0.77–1.11)0.89 (0.74–1.07)0.89 (0.75–1.07) ≥60566 (53.5)179 (31.6)226 (39.9)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total1058377 (35.6)468 (44.2)aExcluding patients with missing smoking or HbA1C data.bAge, gender, socio-economic status, onset prior to 1 April 2004.cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.FIGURE 1:Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nShort-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)a\naExcluding patients with missing smoking or HbA1C data.\nbAge, gender, socio-economic status, onset prior to 1 April 2004.\ncAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.\ndAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.\nSurvival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nThe underlying causes of death following pneumonia and sepsis were similar for patients with eGFR above and below 60 mL/min/1.73 m2. Causes of 28-day mortality following sepsis were predominantly sources of infection (Table 3). Following pneumonia onset, pneumonia was recorded as an underlying or contributory cause of death for 83.9% (1191/1419) of deaths within 28 days and 76.3% (1366/1790) of deaths within 90 days. Among patients with eGFR <60 mL/min/1.73 m2, renal disease was recorded as an underlying or contributory cause for 10.6% (77/724) of those who died within 28 days of pneumonia onset and 16.6% (152/913) of those who died within 90 days. Recording of CKD as a cause of death increased with lower eGFR (Table 4).Table 3.Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)aeGFReGFR <60 mL/min/1.73 m2eGFR ≥60 mL/min/1.73 m228-Day mortality following pneumonia, n = 1419J18 Pneumonia, organism unspecified, n = 256, 35.4%J44 Other chronic obstructive pulmonary disease, n = 63, 8.7%I25 Chronic ischaemic heart disease, n = 47, 6.5%I50 Heart failure, n = 43, 5.9%I64 Stroke, not specified as haemorrhage or infarction, n = 37, 5.1%J18 Pneumonia, organism unspecified, n = 216, 31.1%J44 Other chronic obstructive pulmonary disease, n = 63, 9.1%I64 Stroke, not specified as haemorrhage or infarction, n = 35, 5.0%C34 Malignant neoplasm of bronchus and lung, n = 34, 4.9%I25 Chronic ischaemic heart disease, n = 29, 4.2%Total = 724Total = 69529–90 Day mortality following pneumonia, n = 371J18 Pneumonia, organism unspecified, n = 38, 20.1%I25 Chronic ischaemic heart disease, n = 24, 12.7%J44 Other chronic obstructive pulmonary disease, n = 13, 6.9%C34 Malignant neoplasm of bronchus and lung, n = 12, 6.4%I21 Acute myocardial infarction, n = 12, 6.4%J18 Pneumonia, organism unspecified, n = 20, 11.0%C34 Malignant neoplasm of bronchus and lung, n = 20, 11.0%J44 Other chronic obstructive pulmonary disease, n = 19, 10.4%I25 Chronic ischaemic heart disease, n = 9, 5.0%I64 Stroke, not specified as haemorrhage or infarction, n = 9, 5.0%Total = 189Total = 18228-Day mortality following sepsis, n = 387N39 Other disorders of urinary systemb, n = 33, 15.9%J18 Pneumonia, organism unspecified, n = 30, 14.4%E14 Unspecified diabetes mellitus, n = 12, 5.8%A41 Other sepsis, n = 11, 5.3%L03 Cellulitis, n = 11, 5.3%N39 Other disorders of urinary systemb, n = 20, 11.2%A41 Other sepsis, n = 18, 10.1%J18 Pneumonia, organism unspecified, n = 18, 10.1%E14 Unspecified diabetes mellitus, n = 10, 5.6%J44 Other chronic obstructive pulmonary disease, n = 6, 3.4%=K55 Vascular disorders of intestine, n = 6, 3.4%=L03 Cellulitis, n = 6, 3.4%Total = 208Total = 179aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.Table 4.Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFReGFR (mL/min/1.73 m2)Deaths within 28 days of pneumonia onsetDeaths within 90 days of pneumonia onsetTotalN18 CKD, n (%)Renal disease,b\nn (%)TotalN18 CKD, n (%)Renal disease,b\nn (%)<1586 (75.0)7 (87.5)86 (75.0)7 (87.5)15–2910919 (17.4)41 (37.6)13926 (18.7)53 (38.1)30–4427514 (5.1)2 (15.3)34720 (5.8)56 (16.1)35–593328 (2.4)27 (8.1)41910 (2.4)36 (8.6)Total72447 (6.5)77 (10.6)91362 (6.8)152 (16.6)aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\nTop five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)a\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.\nRecording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFR\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\n\nDISCUSSION:\nAmong this population of older people with diabetes mellitus, eGFR <30 mL/min/1.73 m2 was a risk marker of higher 28- and 90-day mortality following community-acquired pneumonia and sepsis, compared with patients with eGFR ≥60 mL/min/1.73 m2. The relationship between eGFR and mortality did not change with adjustment for co-morbidities. Neither moderately impaired eGFR nor proteinuria was associated with higher short-term mortality following either infection.\nThe strengths of this study follow from the analysis of a focused question using large, linked datasets for a highly monitored primary care population with a cohort study design. Our study identifies that the association between eGFR and post-infection mortality persists when patients with end-stage renal disease (ESRD) are excluded (and is not explained by renal replacement therapy), when considering fixed-term rather than in-hospital mortality (thus is not due to differences in hospital stay) and when exclusively community-acquired infections are considered (so does not result from increased risk of healthcare-associated infections). The linked datasets allowed us to identify infections both among patients presenting directly to hospital and those managed in the community, maximized ascertainment of mortality and enabled description of the causes of death. The highly monitored population allowed good ascertainment of CKD status. The cohort study design has less potential for selection bias than an equivalent case–control study.\nA limitation is our assumption that the absence of a record implies a negative status for proteinuria and co-morbidities. Under-ascertainment of co-morbidities could result in residual confounding, with unpredictable effects, but the high prevalence of co-morbidities observed suggests that ascertainment was not markedly incomplete. We observed a high prevalence of proteinuria, and this is a highly monitored population (with financial incentives for standardized recording of proteinuria since 2004), but under-ascertainment of proteinuria could result in underestimation of any association between proteinuria and mortality [24]. Residual confounding from undiagnosed cardiovascular disease should have been minimized by adjustment for cardiovascular disease risk factors including smoking, hypertension and characteristics of diabetes.\nOur findings for eGFR provide further detail to build on previous findings that baseline eGFR <60 mL/min/1.73 m2 or renal disease are risk factors for short-term mortality following (hospital- or community-acquired) sepsis and for in-hospital mortality following community-acquired pneumonia (including patients receiving dialysis) [11–15]. A more comparable Canadian study examined the associations between eGFR and 30-day mortality following community-acquired pneumonia among the general population aged ≥65 years, excluding patients with ESRD [16]. Fully adjusted hazard ratios for 30-day mortality were 1.22 (95% CI 1.01–1.49) for eGFR 45–59, 2.03 (1.64–2.50) for eGFR 30–44 and 4.94 (3.94–6.19) for eGFR < 30, compared with eGFR 60–104 mL/min/1.73 m2. These are somewhat greater than the associations we observed. The difference may be explained by the different study populations. Both studies required a baseline serum creatinine result for inclusion. Our study population of older people with diabetes were routinely monitored for CKD (with financial incentivization in primary care) [32]. Creatinine testing of the Canadian study population may have been encouraged by co-morbidities or health-behaviours associated with CKD (such as smoking), which increase post-infection mortality, resulting in over-estimation of the association of eGFR and post-infection mortality. Our study population is less vulnerable to differential ascertainment of CKD. Alternatively, the association between eGFR and post-infection mortality may be smaller among patients with diabetes.\nTo the best of our knowledge, our examination of any association between proteinuria and mortality following community-acquired pneumonia and sepsis is novel. A history of proteinuria, although a marker for mortality in general, does not appear to be a risk marker for short-term mortality following community-acquired infection. This is unlikely to be due to chance, as the study was large, with findings consistent across both infections. We designed our study to produce conservative estimates and may have under-estimated the association between proteinuria and post-infection mortality due to under-ascertainment of proteinuria. Alternatively, any potential relationship between proteinuria and mortality may have been mitigated by clinical care of patients with infection who had pre-existing proteinuria, for example, through swift recognition of AKI.\nThe survival curves demonstrate a steep initial mortality following infection onset, and a high proportion of deaths had the underlying cause assigned to infection. Since 2001 in England, co-morbidities are assigned as the underlying cause of death when pneumonia has occurred in the context of, for example, malignancy or respiratory disease [25]. This suggests that the associations we observed are driven by an association between eGFR and infection prognosis, not merely high underlying baseline mortality among patients with impaired eGFR. This is supported by previous research which found 7.7-fold elevated mortality in the 30 days following community-acquired pneumonia [19]. Estimates for associations between eGFR and mortality were not substantially altered by adjustment for co-morbidities, suggesting that any causal relationship between eGFR and mortality is not mediated through co-morbidities.\nOur findings apply to the large population of older patients with community-acquired pneumonia or sepsis with diabetes mellitus who do not have ESRD. Inclusion criteria are unlikely to have limited generalizability appreciably. Practices which consent to data linkage could be more research oriented, providing good primary care management of risk factors for infection-related mortality (such as smoking cessation), but this is unlikely to affect the relationship between CKD and short-term mortality post-infection. Lack of pre-existing creatinine test results is likely to reflect limited time potentially eligible for the study rather than CKD status among this highly monitored population. Missing data on smoking status and HbA1C may be a marker of low patient engagement: caution should be used in generalizing our results to patients who are not actively managed in primary care.\nWe found that CKD is a useful clinical risk marker for post-infectious mortality. Whether this relationship is causal is less clear; but the association does not appear to be explained by age, co-morbidities or hospital attendance. Potential mechanisms include immune system dysfunction, but also more preventable complications such as AKI. Combinations of risk factors may be important: for example, patients with post-operative AKI have higher mortality if they also have pre-existing CKD [30].\nOur results have implications for patient management and future research. Patients with baseline eGFR <30 mL/min/1.73 m2 and community-acquired infection need careful monitoring, particularly in the 28 days following infection. Future research should investigate preventable mechanisms by which low baseline eGFR could be related to post-infection mortality, for example, fluid management, AKI and drug dosage in patients with low renal clearance.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWe aimed to examine whether pre-existing impaired estimated glomerular filtration rate (eGFR) and proteinuria were associated with mortality following community-acquired pneumonia or sepsis among people aged ≥ 65 years with diabetes mellitus, without end-stage renal disease.\n\nMethods:\nPatients were followed up from onset of first community-acquired pneumonia or sepsis episode in a cohort study using large, linked electronic health databases. Follow-up was for up to 90 days, unlimited by hospital discharge. We used generalized linear models with log link, normal distribution and robust standard errors to calculate risk ratios (RRs) for all-cause 28- and 90-day mortality according to two markers of chronic kidney disease: eGFR and proteinuria.\n\nResults:\nAll-cause mortality among the 4743 patients with pneumonia was 29.6% after 28 days and 37.4% after 90 days. Among the 1058 patients with sepsis, all-cause 28- and 90-day mortality were 35.6 and 44.2%, respectively. eGFR <30 mL/min/1.73 m(2) was a risk marker of higher 28-day mortality for pneumonia (RR 1.27: 95% CI 1.12-1.43) and sepsis (RR 1.32: 95% CI 1.07-1.64), adjusted for age, sex, socio-economic status, smoking status and co-morbidities. Neither moderately impaired eGFR nor proteinuria were associated with short-term mortality following either infection.\n\nConclusions:\nPeople with pre-existing low eGFR but not on dialysis are at higher risk of death following pneumonia and sepsis. This association was not explained by existing co-morbidities. These patients need to be carefully monitored to prevent modifiable causes of death."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":8630,"string":"8,630"},"whole_article_abstract_length":{"kind":"number","value":322,"string":"322"},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["infection","mortality","egfr","patients","ckd","pneumonia","disease","status","28","onset"],"string":"[\n \"infection\",\n \"mortality\",\n \"egfr\",\n \"patients\",\n \"ckd\",\n \"pneumonia\",\n \"disease\",\n \"status\",\n \"28\",\n \"onset\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] chronic kidney disease | community-acquired infections | electronic health records | infection/mortality | proteinuria [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] chronic kidney disease | community-acquired infections | electronic health records | infection/mortality | proteinuria [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] chronic kidney disease | community-acquired infections | electronic health records | infection/mortality | proteinuria [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Aged | Aged, 80 and over | Biomarkers | Cohort Studies | Community-Acquired Infections | Comorbidity | Diabetes Mellitus | Electronic Health Records | Female | Humans | Kidney Failure, Chronic | Male | Pneumonia | Proteinuria | Renal Dialysis | Risk Factors | Sepsis | Survival Rate [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Aged | Aged, 80 and over | Biomarkers | Cohort Studies | Community-Acquired Infections | Comorbidity | Diabetes Mellitus | Electronic Health Records | Female | Humans | Kidney Failure, Chronic | Male | Pneumonia | Proteinuria | Renal Dialysis | Risk Factors | Sepsis | Survival Rate [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Age Factors | Aged | Aged, 80 and over | Biomarkers | Cohort Studies | Community-Acquired Infections | Comorbidity | Diabetes Mellitus | Electronic Health Records | Female | Humans | Kidney Failure, Chronic | Male | Pneumonia | Proteinuria | Renal Dialysis | Risk Factors | Sepsis | Survival Rate [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | mortality | egfr | patients | ckd | pneumonia | disease | status | 28 | onset [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | mortality | egfr | patients | ckd | pneumonia | disease | status | 28 | onset [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | mortality | egfr | patients | ckd | pneumonia | disease | status | 28 | onset [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ckd | associated | mortality | infection | older | older people | community | community acquired | acquired | prognosis [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] reference reference | reference | pneumonia | reference reference reference | day mortality | mortality | disease | reference reference reference reference | 10 | 73 [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | mortality | egfr | ckd | patients | days | data | 28 | acquired | status [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ≥ | 65 years [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 4743 | 29.6% | 28 days | 37.4% | 90 days ||| 1058 | 28- | 90-day | 35.6 | 44.2% ||| m(2 | 28-day | 1.27 | 95% | CI | 1.12-1.43 | 1.32 | 95% | CI | 1.07-1.64 ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ≥ | 65 years ||| first ||| up to 90 days ||| linear | 28- | 90-day | two | proteinuria ||| 4743 | 29.6% | 28 days | 37.4% | 90 days ||| 1058 | 28- | 90-day | 35.6 | 44.2% ||| m(2 | 28-day | 1.27 | 95% | CI | 1.12-1.43 | 1.32 | 95% | CI | 1.07-1.64 ||| ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":282,"cells":{"title":{"kind":"string","value":"Endometrial intraepithelial carcinoma in association with polyp: review of eight cases."},"pmid":{"kind":"string","value":"23414240"},"background_abstract":{"kind":"string","value":"The uterine endometrial polyp (EMP) has a potential risk of developing malignant tumors especially in postmenopausal women. These malignancies include endometrial intraepithelial carcinoma (EIC)."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Eight patients with EIC in the EMP, who were postmenopausal with ages ranging from 49 to 76 years (av. 62), were cytologically reviewed in comparison with histological findings."},"methods_abstract_label":{"kind":"string","value":"PATIENTS AND METHODS"},"results_abstract":{"kind":"string","value":"The endometrial cytological findings were summarized as follows: mucous and watery diathesis as a background lacking or with little necrotic inflammatory changes; micropapillary cluster formation; abrupt transition between carcinoma cells and normal cells; nuclear enlargement; high N/C ratio; and single or a few prominent nucleoli. Histologically, one case had EIC alone in the EMP; three cases had EIC with stromal invasion confined to the EMP; and four cases had EIC in the atrophic endometrium in addition to EIC in the EMP. Seven patients have taken a disease-free course after surgical resection, but one patient died 44 months following the initial diagnosis because of the massive tumor extending over her peritoneal cavity."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Endometrial cytology may be helpful for the detection of early endometrial adenocarcinomas with serous features including EIC. Some early stage endometrial adenocarcinomas represented by EIC exceptionally take an aggressive clinical course irrespective of a lack of extrauterine lesions."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adenocarcinoma","Aged","Carcinoma in Situ","Disease Progression","Early Detection of Cancer","Endometrial Neoplasms","Female","Humans","Middle Aged","Polyps","Predictive Value of Tests","Time Factors","Treatment Outcome"],"string":"[\n \"Adenocarcinoma\",\n \"Aged\",\n \"Carcinoma in Situ\",\n \"Disease Progression\",\n \"Early Detection of Cancer\",\n \"Endometrial Neoplasms\",\n \"Female\",\n \"Humans\",\n \"Middle Aged\",\n \"Polyps\",\n \"Predictive Value of Tests\",\n \"Time Factors\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"3599099"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The EMPs including EIC with / without stromal invasion measured less than 1 cm in the great diameter in all of the patients. The cytological features are summarized in Table 1 and representatively depicted in Figures 1, 2, and 3, including histological findings of three patients. The tumor diathesis was characterized as being predominantly mucous for 3 patients, watery for 3 patients, and bloody for 2 patients, but with no prominent necrotic inflammatory changes. The tumor cell clusters predominantly took a micropapillary pattern and occasionally a sheet-like pattern. Overlapping of the tumor cell nuclei was noted in a variable degree: often for 2 patients, and occasionally for 5 patients. There were varying amounts of normal epithelial cells, which served as a hallmark in distinction from the malignancy because of a significant difference in the nuclear size. Using Isis ver. 5 (MetaSystems GmbH, Altlussheim, Germany), the nuclear size (greatest diameter) of tumor cells were measured as well as normal atrophic cells in the cytological preparations. The numbers of counted cells for the former and the latter ranged from 50 to 70 in each case. On the total average of 8 cases, the tumor cell size was 12.5 μm and the relative ratio to the corresponding normal atrophic cells was 2.3 (Table 1). The chromatin was considerably dense, coarse or granular. The number of prominent nucleoli was as follows: 1 for 4 patients; 1 or 2 for 4 patients. Calcified deposits of psammoma body were not apparently observed in any patient.\nCytological features of endometrial intraepithelial carcinomas\na: average of largest diameter of tumor cells (relative value compared to normal nucleus).\nCase 2 possessing EIC in both endometrial polyp and endometrium (A. Pap × 20, B. Pap × 60, C. loupe view, D. HE × 20, E. HE × 60, F. HE × 60). A. Papillary-structured carcinoma cell cluster is accompanied by a watery background (Pap. ×20). B. The N/C ratio is fairly high and the chromatin is coarsely granular (Pap. ×60). C. Loupe findings show an endometrial polyp arising in the uterine fundus, measuring 1.5 cm in longitudinal length. The endometrium is markedly thin and atrophic. The area marked by an arrow and that marked by an arrowhead are depicted respectively in E and F. D. In the polyp, the glands composed of carcinoma cells are found to be aggregated in the fibrotic stroma. E. Carcinoma cells are replacing the glands in the polyp and stratified into micropapillary protrusions, lacking apparent stromal invasion (HE × 20). F. Carcinoma nests similar to those in the endometrial polyp (D) are also present in the endometrial gland (HE × 60).\nCase 5 possessing EIC in both endometrial polyp and endometrium, but with stromal invasion in the polyp (A. Pap × 20, B. Pap × 60, C. HE × 20, D. HE × 60). A. A carcinoma cell cluster is accompanied by a bloody background. B. Carcinoma cells having enlarged nuclei appear to be transitional with normal atrophic cells having small-sized nuclei (arrows). C. The polyp has many atypical glands with intraluminal papillary protrusion of the carcinoma cells. D. The border between the carcinoma cells and normal cells is clearly discernible (arrows). There are small carcinoma nests showing stromal invasion (arrowheads).\nCase 6 possessing EIC confined to endometrial polyp (A. Pap × 20, B. Pap × 60, C. HE × 60, D. HE × 60). A. Large-sized carcinoma cell clusters are accompanied by a non-necroinflammatory background. B. Carcinoma cells mainly appear in a flat and sheet-like cluster. The nucleus has an enlarged eosinophilic nucleolus. C. The atypical glands are normal-sized, accompanied by cystic dilatation of atrophic normal glands. D. Carcinoma cells are replacing pre-existing glands and are arranged in a single layer (compare with normal gland marked by an arrow).\nThe clinicopathological profiles are summarized in Table 2. All the patients had an EMP in which atypical tumor cells were arranged predominantly in a tubular structure with micropapillary protrusions, replacing the pre-existing endometrial glands. Micropapillary protrusions and small tufts were accompanied with or without thin fibrovascular cores. These tumor cells showed conspicuous nuclear pleomorphism and hyperchromatism, frequent mitotic figures, high N/C ratio, and also one or two prominent nucleoli. The tumor front was abruptly made into normal glandular epithelium and surface-lining epithelium in the EMP. Minimal stromal invasion of the tumor cells limited to the EMP was recognized in 3 patients. In addition, similar tumor cells also were also found to replace some atrophic endometrial glands in 4 patients, lacking stromal invasion. In the background, cystic dilatation of atrophic normal glands was evident especially in the EMP.\nClinicopathological profiles of endometrial intraepithelial carcinomas\na:+, strongly positive cells more than 80%.\nb: index of positive cells(%), c: +, positive cells more than 80%.\nd: +, positive cells more than 80%.\ne: since initial diagnosis.\nf: history of breast cancer with Tamoxifen administration.\ng: autopsy performed.\nh: with stromal invasion, i: disease free, j: death on disease.\nImmunohistochemical expressions of p53 (DO7, 1:50, Dako, Glostrup, Denmark), Ki-67 (MIB-1, 1:100, Dako, Glostrup, Denmark), ER (SP-1, 1:1, Ventana, AZ, USA), and PgR (1E2, 1:1, Ventana, AZ, USA) were examined (Figure 4). Six patients showed marked p53 expression with a positive ratio of more than 80%. The labeling index of Ki-67 ranged from 25 to 70% (av. approximately 40). ER was mildly expressed in one patient and PgR expression also was mildly observed in 2 patients.\nImmunohistochemical staining profiles represented by Case 5 (A. p53 × 40, B. MIB-1 × 40, C. ER × 20, D. PgR × 20). A. Almost all of the carcinoma cells are strongly positive. B. More than half the carcinoma cells are labeled. C. The expression is observed in the cystically dilated normal glands and stromal cells but not in the carcinoma cells. D. The expression behavior is basically similar to the ER expression.\nFIGO stage was determined at IA for all the patients. Neither adjuvant chemotherapy nor irradiation was performed. Seven patients have been taking an uneventful clinical course (follow-up period ranging from 12 to 66 months, av. 41), but one patient died of the recurrent disease massively occupying the abdominal cavity 44 months after the initial diagnosis."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Introduction","Patients","Patients’ consents","Ethical approval","Competing interests","Authors’ contributions"],"string":"[\n \"Introduction\",\n \"Patients\",\n \"Patients’ consents\",\n \"Ethical approval\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.","The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.","Written informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.","This review was performed based on compliance with the Helsinki Declaration.","All of the authors declare that they have no competing interests.","MY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript."],"string":"[\n \"The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\",\n \"The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\",\n \"Written informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.\",\n \"This review was performed based on compliance with the Helsinki Declaration.\",\n \"All of the authors declare that they have no competing interests.\",\n \"MY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Patients","Results","Discussion","Patients’ consents","Ethical approval","Competing interests","Authors’ contributions"],"string":"[\n \"Introduction\",\n \"Patients\",\n \"Results\",\n \"Discussion\",\n \"Patients’ consents\",\n \"Ethical approval\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.","The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.","The EMPs including EIC with / without stromal invasion measured less than 1 cm in the great diameter in all of the patients. The cytological features are summarized in Table 1 and representatively depicted in Figures 1, 2, and 3, including histological findings of three patients. The tumor diathesis was characterized as being predominantly mucous for 3 patients, watery for 3 patients, and bloody for 2 patients, but with no prominent necrotic inflammatory changes. The tumor cell clusters predominantly took a micropapillary pattern and occasionally a sheet-like pattern. Overlapping of the tumor cell nuclei was noted in a variable degree: often for 2 patients, and occasionally for 5 patients. There were varying amounts of normal epithelial cells, which served as a hallmark in distinction from the malignancy because of a significant difference in the nuclear size. Using Isis ver. 5 (MetaSystems GmbH, Altlussheim, Germany), the nuclear size (greatest diameter) of tumor cells were measured as well as normal atrophic cells in the cytological preparations. The numbers of counted cells for the former and the latter ranged from 50 to 70 in each case. On the total average of 8 cases, the tumor cell size was 12.5 μm and the relative ratio to the corresponding normal atrophic cells was 2.3 (Table 1). The chromatin was considerably dense, coarse or granular. The number of prominent nucleoli was as follows: 1 for 4 patients; 1 or 2 for 4 patients. Calcified deposits of psammoma body were not apparently observed in any patient.\nCytological features of endometrial intraepithelial carcinomas\na: average of largest diameter of tumor cells (relative value compared to normal nucleus).\nCase 2 possessing EIC in both endometrial polyp and endometrium (A. Pap × 20, B. Pap × 60, C. loupe view, D. HE × 20, E. HE × 60, F. HE × 60). A. Papillary-structured carcinoma cell cluster is accompanied by a watery background (Pap. ×20). B. The N/C ratio is fairly high and the chromatin is coarsely granular (Pap. ×60). C. Loupe findings show an endometrial polyp arising in the uterine fundus, measuring 1.5 cm in longitudinal length. The endometrium is markedly thin and atrophic. The area marked by an arrow and that marked by an arrowhead are depicted respectively in E and F. D. In the polyp, the glands composed of carcinoma cells are found to be aggregated in the fibrotic stroma. E. Carcinoma cells are replacing the glands in the polyp and stratified into micropapillary protrusions, lacking apparent stromal invasion (HE × 20). F. Carcinoma nests similar to those in the endometrial polyp (D) are also present in the endometrial gland (HE × 60).\nCase 5 possessing EIC in both endometrial polyp and endometrium, but with stromal invasion in the polyp (A. Pap × 20, B. Pap × 60, C. HE × 20, D. HE × 60). A. A carcinoma cell cluster is accompanied by a bloody background. B. Carcinoma cells having enlarged nuclei appear to be transitional with normal atrophic cells having small-sized nuclei (arrows). C. The polyp has many atypical glands with intraluminal papillary protrusion of the carcinoma cells. D. The border between the carcinoma cells and normal cells is clearly discernible (arrows). There are small carcinoma nests showing stromal invasion (arrowheads).\nCase 6 possessing EIC confined to endometrial polyp (A. Pap × 20, B. Pap × 60, C. HE × 60, D. HE × 60). A. Large-sized carcinoma cell clusters are accompanied by a non-necroinflammatory background. B. Carcinoma cells mainly appear in a flat and sheet-like cluster. The nucleus has an enlarged eosinophilic nucleolus. C. The atypical glands are normal-sized, accompanied by cystic dilatation of atrophic normal glands. D. Carcinoma cells are replacing pre-existing glands and are arranged in a single layer (compare with normal gland marked by an arrow).\nThe clinicopathological profiles are summarized in Table 2. All the patients had an EMP in which atypical tumor cells were arranged predominantly in a tubular structure with micropapillary protrusions, replacing the pre-existing endometrial glands. Micropapillary protrusions and small tufts were accompanied with or without thin fibrovascular cores. These tumor cells showed conspicuous nuclear pleomorphism and hyperchromatism, frequent mitotic figures, high N/C ratio, and also one or two prominent nucleoli. The tumor front was abruptly made into normal glandular epithelium and surface-lining epithelium in the EMP. Minimal stromal invasion of the tumor cells limited to the EMP was recognized in 3 patients. In addition, similar tumor cells also were also found to replace some atrophic endometrial glands in 4 patients, lacking stromal invasion. In the background, cystic dilatation of atrophic normal glands was evident especially in the EMP.\nClinicopathological profiles of endometrial intraepithelial carcinomas\na:+, strongly positive cells more than 80%.\nb: index of positive cells(%), c: +, positive cells more than 80%.\nd: +, positive cells more than 80%.\ne: since initial diagnosis.\nf: history of breast cancer with Tamoxifen administration.\ng: autopsy performed.\nh: with stromal invasion, i: disease free, j: death on disease.\nImmunohistochemical expressions of p53 (DO7, 1:50, Dako, Glostrup, Denmark), Ki-67 (MIB-1, 1:100, Dako, Glostrup, Denmark), ER (SP-1, 1:1, Ventana, AZ, USA), and PgR (1E2, 1:1, Ventana, AZ, USA) were examined (Figure 4). Six patients showed marked p53 expression with a positive ratio of more than 80%. The labeling index of Ki-67 ranged from 25 to 70% (av. approximately 40). ER was mildly expressed in one patient and PgR expression also was mildly observed in 2 patients.\nImmunohistochemical staining profiles represented by Case 5 (A. p53 × 40, B. MIB-1 × 40, C. ER × 20, D. PgR × 20). A. Almost all of the carcinoma cells are strongly positive. B. More than half the carcinoma cells are labeled. C. The expression is observed in the cystically dilated normal glands and stromal cells but not in the carcinoma cells. D. The expression behavior is basically similar to the ER expression.\nFIGO stage was determined at IA for all the patients. Neither adjuvant chemotherapy nor irradiation was performed. Seven patients have been taking an uneventful clinical course (follow-up period ranging from 12 to 66 months, av. 41), but one patient died of the recurrent disease massively occupying the abdominal cavity 44 months after the initial diagnosis.","Endometrial polyp is a common benign disease of the uterus. The overall incidence where cancer arises in the endometrial polyp has been reported as only a few percent, while postmenopausal women with EMP are at an increased risk of malignancy, compared to premenopausal women [10,13-15]. As risk factors for development of the malignancy in EMP, hypertension, obesity, and unopposed estrogen therapy have been indicated in addition to postmenopausal status [15]. There are unique or specific pathological and clinical features for EIC as a putative precursor of uterine body serous adenocarcinoma, as follows [1,4,5,7,16-20]: close association with an EMP; multicentric occurrence in the inactive or resting thin endometrium, lacking alteration in the architecture in the endometrium lacking an intervening phase of endometrial hyperplasia; extrauterine invasive extent, irrespective of an absence of stromal invasion of the uterus; and controversial outcome, whether taking a favorable or unfavorable clinical course. Development of EIC associated with an EMP may also be explained in part by Tamoxifen administration following breast cancer treatment [6,17]. However, little has been documented about the endometrial cytological features of minimal serous adenocarcinoma of the uterine body including EIC, mainly due to the fact that the disease is not frequently encountered in the routine practice. To the best of our knowledge, there is a case report cytologically referring to serous adenocarcinoma of the uterine body associated with an EMP [21], but here the patient already showed peritoneal dissemination at the initial presentation. In another report, serous adenocarcinomas of the uterine body were clinico-cytologically reviewed but they were all at advanced stages [22]. The latter report mentions that cytologically diagnostic findings of uterine body serous adenocarcinoma would be based on the following features: papillary cluster as well as tubular cluster, arborescent structure, frequent exfoliation, psammoma body, eccentric enlarged nuclei, coarsely granular chromatin, numerous nucleoli, and diameter of greater than G1 endometrioid adenocarcinoma [22]. In our review, 5 patients were purely non-invasive as they were composed of only EIC existing in an EMP, with few or no necroinflammatory changes. The background was found to be characteristically watery or mucous (Figure 1) in a way of simulating the immigration of extrauterine carcinoma cells via the fallopian tubes [12]. Many clusters were arranged in micropapillary architecture without fine vascular stroma, and others were occasionally tubular. An abrupt transition between the EIC cells and atrophic endometrial cells was noted by front formation (Figure 2). Conspicuous nucleoli were found in 3 patients (Figure 3). Even though it seems difficult to reach a definite diagnosis, these findings may serve as a diagnostic indicator for the uterine body serous adenocarcinoma, whether being at an early stage or an advanced stage.\nDifferential diagnoses for atypical epithelial sheets in the normal-appearing endometrial background include variable interpretations as follows: EIC, endometrial glandular dysplasia, endometrial intraepithelial neoplasia, hyperplastic polyp, and metastatic carcinoma [9,23]. In addition to these, isolated atypical glands with morphological and immunohistochemical features of a typical endometrial hyperplasia or type I endometrial adenocarcinoma may be encountered in grossly normal postmenopausal endometrium of asymptomatic patients [24]. Especially in the postmenopausal state, thin and smooth endometrium with atrophy is composed of hypocellular and flattened glands lined by cells with reduced or absent mitosis and sparse fibrous stroma [25]. It is supposed that the abrupt transition of the atrophic endometrial cells to atypical cells may indicate EIC in postmenopausal women.\nOverexpression of p53 is closely linked to the rapid growth of uterine body serous adenocarcinoma [6]. In EIC, too, frequent p53 mutation is reported to be associated with LOH of 17p [26]. Thus, immunohistochemistry for p53 is also adjunctively available in the diagnosis of uterine body serous adenocarcinoma, irrespective of whether it is at an early stage or advanced stage [27], in the discrimination between uterine body serous adenocarcinoma and benign morphologic mimics [28]. Negativity of ER and PgR staining is more reasonable with the diagnosis of serous adenocarcinoma including EIC.\nIn summary, EMP arising in postmenopausal women is at a high risk of giving birth to the tumorigenetic background for endometrial carcinomas such as endometrioid adenocarcinoma and serous adenocarcinoma including EIC. Endometrial cytology may become a helpful diagnostic approach for serous adenocarcinoma, whether it is at early or advanced stage, from the viewpoint of the distinctive cytological features. Above all, it should be noted that some EIC behaves in a more aggressive fashion regardless of whether or not it lacks apparent stroma invasion and extrauterine extent.","Written informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.","This review was performed based on compliance with the Helsinki Declaration.","All of the authors declare that they have no competing interests.","MY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript."],"string":"[\n \"The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\",\n \"The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\",\n \"The EMPs including EIC with / without stromal invasion measured less than 1 cm in the great diameter in all of the patients. The cytological features are summarized in Table 1 and representatively depicted in Figures 1, 2, and 3, including histological findings of three patients. The tumor diathesis was characterized as being predominantly mucous for 3 patients, watery for 3 patients, and bloody for 2 patients, but with no prominent necrotic inflammatory changes. The tumor cell clusters predominantly took a micropapillary pattern and occasionally a sheet-like pattern. Overlapping of the tumor cell nuclei was noted in a variable degree: often for 2 patients, and occasionally for 5 patients. There were varying amounts of normal epithelial cells, which served as a hallmark in distinction from the malignancy because of a significant difference in the nuclear size. Using Isis ver. 5 (MetaSystems GmbH, Altlussheim, Germany), the nuclear size (greatest diameter) of tumor cells were measured as well as normal atrophic cells in the cytological preparations. The numbers of counted cells for the former and the latter ranged from 50 to 70 in each case. On the total average of 8 cases, the tumor cell size was 12.5 μm and the relative ratio to the corresponding normal atrophic cells was 2.3 (Table 1). The chromatin was considerably dense, coarse or granular. The number of prominent nucleoli was as follows: 1 for 4 patients; 1 or 2 for 4 patients. Calcified deposits of psammoma body were not apparently observed in any patient.\\nCytological features of endometrial intraepithelial carcinomas\\na: average of largest diameter of tumor cells (relative value compared to normal nucleus).\\nCase 2 possessing EIC in both endometrial polyp and endometrium (A. Pap × 20, B. Pap × 60, C. loupe view, D. HE × 20, E. HE × 60, F. HE × 60). A. Papillary-structured carcinoma cell cluster is accompanied by a watery background (Pap. ×20). B. The N/C ratio is fairly high and the chromatin is coarsely granular (Pap. ×60). C. Loupe findings show an endometrial polyp arising in the uterine fundus, measuring 1.5 cm in longitudinal length. The endometrium is markedly thin and atrophic. The area marked by an arrow and that marked by an arrowhead are depicted respectively in E and F. D. In the polyp, the glands composed of carcinoma cells are found to be aggregated in the fibrotic stroma. E. Carcinoma cells are replacing the glands in the polyp and stratified into micropapillary protrusions, lacking apparent stromal invasion (HE × 20). F. Carcinoma nests similar to those in the endometrial polyp (D) are also present in the endometrial gland (HE × 60).\\nCase 5 possessing EIC in both endometrial polyp and endometrium, but with stromal invasion in the polyp (A. Pap × 20, B. Pap × 60, C. HE × 20, D. HE × 60). A. A carcinoma cell cluster is accompanied by a bloody background. B. Carcinoma cells having enlarged nuclei appear to be transitional with normal atrophic cells having small-sized nuclei (arrows). C. The polyp has many atypical glands with intraluminal papillary protrusion of the carcinoma cells. D. The border between the carcinoma cells and normal cells is clearly discernible (arrows). There are small carcinoma nests showing stromal invasion (arrowheads).\\nCase 6 possessing EIC confined to endometrial polyp (A. Pap × 20, B. Pap × 60, C. HE × 60, D. HE × 60). A. Large-sized carcinoma cell clusters are accompanied by a non-necroinflammatory background. B. Carcinoma cells mainly appear in a flat and sheet-like cluster. The nucleus has an enlarged eosinophilic nucleolus. C. The atypical glands are normal-sized, accompanied by cystic dilatation of atrophic normal glands. D. Carcinoma cells are replacing pre-existing glands and are arranged in a single layer (compare with normal gland marked by an arrow).\\nThe clinicopathological profiles are summarized in Table 2. All the patients had an EMP in which atypical tumor cells were arranged predominantly in a tubular structure with micropapillary protrusions, replacing the pre-existing endometrial glands. Micropapillary protrusions and small tufts were accompanied with or without thin fibrovascular cores. These tumor cells showed conspicuous nuclear pleomorphism and hyperchromatism, frequent mitotic figures, high N/C ratio, and also one or two prominent nucleoli. The tumor front was abruptly made into normal glandular epithelium and surface-lining epithelium in the EMP. Minimal stromal invasion of the tumor cells limited to the EMP was recognized in 3 patients. In addition, similar tumor cells also were also found to replace some atrophic endometrial glands in 4 patients, lacking stromal invasion. In the background, cystic dilatation of atrophic normal glands was evident especially in the EMP.\\nClinicopathological profiles of endometrial intraepithelial carcinomas\\na:+, strongly positive cells more than 80%.\\nb: index of positive cells(%), c: +, positive cells more than 80%.\\nd: +, positive cells more than 80%.\\ne: since initial diagnosis.\\nf: history of breast cancer with Tamoxifen administration.\\ng: autopsy performed.\\nh: with stromal invasion, i: disease free, j: death on disease.\\nImmunohistochemical expressions of p53 (DO7, 1:50, Dako, Glostrup, Denmark), Ki-67 (MIB-1, 1:100, Dako, Glostrup, Denmark), ER (SP-1, 1:1, Ventana, AZ, USA), and PgR (1E2, 1:1, Ventana, AZ, USA) were examined (Figure 4). Six patients showed marked p53 expression with a positive ratio of more than 80%. The labeling index of Ki-67 ranged from 25 to 70% (av. approximately 40). ER was mildly expressed in one patient and PgR expression also was mildly observed in 2 patients.\\nImmunohistochemical staining profiles represented by Case 5 (A. p53 × 40, B. MIB-1 × 40, C. ER × 20, D. PgR × 20). A. Almost all of the carcinoma cells are strongly positive. B. More than half the carcinoma cells are labeled. C. The expression is observed in the cystically dilated normal glands and stromal cells but not in the carcinoma cells. D. The expression behavior is basically similar to the ER expression.\\nFIGO stage was determined at IA for all the patients. Neither adjuvant chemotherapy nor irradiation was performed. Seven patients have been taking an uneventful clinical course (follow-up period ranging from 12 to 66 months, av. 41), but one patient died of the recurrent disease massively occupying the abdominal cavity 44 months after the initial diagnosis.\",\n \"Endometrial polyp is a common benign disease of the uterus. The overall incidence where cancer arises in the endometrial polyp has been reported as only a few percent, while postmenopausal women with EMP are at an increased risk of malignancy, compared to premenopausal women [10,13-15]. As risk factors for development of the malignancy in EMP, hypertension, obesity, and unopposed estrogen therapy have been indicated in addition to postmenopausal status [15]. There are unique or specific pathological and clinical features for EIC as a putative precursor of uterine body serous adenocarcinoma, as follows [1,4,5,7,16-20]: close association with an EMP; multicentric occurrence in the inactive or resting thin endometrium, lacking alteration in the architecture in the endometrium lacking an intervening phase of endometrial hyperplasia; extrauterine invasive extent, irrespective of an absence of stromal invasion of the uterus; and controversial outcome, whether taking a favorable or unfavorable clinical course. Development of EIC associated with an EMP may also be explained in part by Tamoxifen administration following breast cancer treatment [6,17]. However, little has been documented about the endometrial cytological features of minimal serous adenocarcinoma of the uterine body including EIC, mainly due to the fact that the disease is not frequently encountered in the routine practice. To the best of our knowledge, there is a case report cytologically referring to serous adenocarcinoma of the uterine body associated with an EMP [21], but here the patient already showed peritoneal dissemination at the initial presentation. In another report, serous adenocarcinomas of the uterine body were clinico-cytologically reviewed but they were all at advanced stages [22]. The latter report mentions that cytologically diagnostic findings of uterine body serous adenocarcinoma would be based on the following features: papillary cluster as well as tubular cluster, arborescent structure, frequent exfoliation, psammoma body, eccentric enlarged nuclei, coarsely granular chromatin, numerous nucleoli, and diameter of greater than G1 endometrioid adenocarcinoma [22]. In our review, 5 patients were purely non-invasive as they were composed of only EIC existing in an EMP, with few or no necroinflammatory changes. The background was found to be characteristically watery or mucous (Figure 1) in a way of simulating the immigration of extrauterine carcinoma cells via the fallopian tubes [12]. Many clusters were arranged in micropapillary architecture without fine vascular stroma, and others were occasionally tubular. An abrupt transition between the EIC cells and atrophic endometrial cells was noted by front formation (Figure 2). Conspicuous nucleoli were found in 3 patients (Figure 3). Even though it seems difficult to reach a definite diagnosis, these findings may serve as a diagnostic indicator for the uterine body serous adenocarcinoma, whether being at an early stage or an advanced stage.\\nDifferential diagnoses for atypical epithelial sheets in the normal-appearing endometrial background include variable interpretations as follows: EIC, endometrial glandular dysplasia, endometrial intraepithelial neoplasia, hyperplastic polyp, and metastatic carcinoma [9,23]. In addition to these, isolated atypical glands with morphological and immunohistochemical features of a typical endometrial hyperplasia or type I endometrial adenocarcinoma may be encountered in grossly normal postmenopausal endometrium of asymptomatic patients [24]. Especially in the postmenopausal state, thin and smooth endometrium with atrophy is composed of hypocellular and flattened glands lined by cells with reduced or absent mitosis and sparse fibrous stroma [25]. It is supposed that the abrupt transition of the atrophic endometrial cells to atypical cells may indicate EIC in postmenopausal women.\\nOverexpression of p53 is closely linked to the rapid growth of uterine body serous adenocarcinoma [6]. In EIC, too, frequent p53 mutation is reported to be associated with LOH of 17p [26]. Thus, immunohistochemistry for p53 is also adjunctively available in the diagnosis of uterine body serous adenocarcinoma, irrespective of whether it is at an early stage or advanced stage [27], in the discrimination between uterine body serous adenocarcinoma and benign morphologic mimics [28]. Negativity of ER and PgR staining is more reasonable with the diagnosis of serous adenocarcinoma including EIC.\\nIn summary, EMP arising in postmenopausal women is at a high risk of giving birth to the tumorigenetic background for endometrial carcinomas such as endometrioid adenocarcinoma and serous adenocarcinoma including EIC. Endometrial cytology may become a helpful diagnostic approach for serous adenocarcinoma, whether it is at early or advanced stage, from the viewpoint of the distinctive cytological features. Above all, it should be noted that some EIC behaves in a more aggressive fashion regardless of whether or not it lacks apparent stroma invasion and extrauterine extent.\",\n \"Written informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.\",\n \"This review was performed based on compliance with the Helsinki Declaration.\",\n \"All of the authors declare that they have no competing interests.\",\n \"MY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,"results","discussion",null,null,null,null],"string":"[\n null,\n null,\n \"results\",\n \"discussion\",\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Endometrial intraepithelial carcinoma (EIC)","Endometrial polyp","Cytology"],"string":"[\n \"Endometrial intraepithelial carcinoma (EIC)\",\n \"Endometrial polyp\",\n \"Cytology\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nThe uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\n\nPatients:\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\n\nResults:\nThe EMPs including EIC with / without stromal invasion measured less than 1 cm in the great diameter in all of the patients. The cytological features are summarized in Table 1 and representatively depicted in Figures 1, 2, and 3, including histological findings of three patients. The tumor diathesis was characterized as being predominantly mucous for 3 patients, watery for 3 patients, and bloody for 2 patients, but with no prominent necrotic inflammatory changes. The tumor cell clusters predominantly took a micropapillary pattern and occasionally a sheet-like pattern. Overlapping of the tumor cell nuclei was noted in a variable degree: often for 2 patients, and occasionally for 5 patients. There were varying amounts of normal epithelial cells, which served as a hallmark in distinction from the malignancy because of a significant difference in the nuclear size. Using Isis ver. 5 (MetaSystems GmbH, Altlussheim, Germany), the nuclear size (greatest diameter) of tumor cells were measured as well as normal atrophic cells in the cytological preparations. The numbers of counted cells for the former and the latter ranged from 50 to 70 in each case. On the total average of 8 cases, the tumor cell size was 12.5 μm and the relative ratio to the corresponding normal atrophic cells was 2.3 (Table 1). The chromatin was considerably dense, coarse or granular. The number of prominent nucleoli was as follows: 1 for 4 patients; 1 or 2 for 4 patients. Calcified deposits of psammoma body were not apparently observed in any patient.\nCytological features of endometrial intraepithelial carcinomas\na: average of largest diameter of tumor cells (relative value compared to normal nucleus).\nCase 2 possessing EIC in both endometrial polyp and endometrium (A. Pap × 20, B. Pap × 60, C. loupe view, D. HE × 20, E. HE × 60, F. HE × 60). A. Papillary-structured carcinoma cell cluster is accompanied by a watery background (Pap. ×20). B. The N/C ratio is fairly high and the chromatin is coarsely granular (Pap. ×60). C. Loupe findings show an endometrial polyp arising in the uterine fundus, measuring 1.5 cm in longitudinal length. The endometrium is markedly thin and atrophic. The area marked by an arrow and that marked by an arrowhead are depicted respectively in E and F. D. In the polyp, the glands composed of carcinoma cells are found to be aggregated in the fibrotic stroma. E. Carcinoma cells are replacing the glands in the polyp and stratified into micropapillary protrusions, lacking apparent stromal invasion (HE × 20). F. Carcinoma nests similar to those in the endometrial polyp (D) are also present in the endometrial gland (HE × 60).\nCase 5 possessing EIC in both endometrial polyp and endometrium, but with stromal invasion in the polyp (A. Pap × 20, B. Pap × 60, C. HE × 20, D. HE × 60). A. A carcinoma cell cluster is accompanied by a bloody background. B. Carcinoma cells having enlarged nuclei appear to be transitional with normal atrophic cells having small-sized nuclei (arrows). C. The polyp has many atypical glands with intraluminal papillary protrusion of the carcinoma cells. D. The border between the carcinoma cells and normal cells is clearly discernible (arrows). There are small carcinoma nests showing stromal invasion (arrowheads).\nCase 6 possessing EIC confined to endometrial polyp (A. Pap × 20, B. Pap × 60, C. HE × 60, D. HE × 60). A. Large-sized carcinoma cell clusters are accompanied by a non-necroinflammatory background. B. Carcinoma cells mainly appear in a flat and sheet-like cluster. The nucleus has an enlarged eosinophilic nucleolus. C. The atypical glands are normal-sized, accompanied by cystic dilatation of atrophic normal glands. D. Carcinoma cells are replacing pre-existing glands and are arranged in a single layer (compare with normal gland marked by an arrow).\nThe clinicopathological profiles are summarized in Table 2. All the patients had an EMP in which atypical tumor cells were arranged predominantly in a tubular structure with micropapillary protrusions, replacing the pre-existing endometrial glands. Micropapillary protrusions and small tufts were accompanied with or without thin fibrovascular cores. These tumor cells showed conspicuous nuclear pleomorphism and hyperchromatism, frequent mitotic figures, high N/C ratio, and also one or two prominent nucleoli. The tumor front was abruptly made into normal glandular epithelium and surface-lining epithelium in the EMP. Minimal stromal invasion of the tumor cells limited to the EMP was recognized in 3 patients. In addition, similar tumor cells also were also found to replace some atrophic endometrial glands in 4 patients, lacking stromal invasion. In the background, cystic dilatation of atrophic normal glands was evident especially in the EMP.\nClinicopathological profiles of endometrial intraepithelial carcinomas\na:+, strongly positive cells more than 80%.\nb: index of positive cells(%), c: +, positive cells more than 80%.\nd: +, positive cells more than 80%.\ne: since initial diagnosis.\nf: history of breast cancer with Tamoxifen administration.\ng: autopsy performed.\nh: with stromal invasion, i: disease free, j: death on disease.\nImmunohistochemical expressions of p53 (DO7, 1:50, Dako, Glostrup, Denmark), Ki-67 (MIB-1, 1:100, Dako, Glostrup, Denmark), ER (SP-1, 1:1, Ventana, AZ, USA), and PgR (1E2, 1:1, Ventana, AZ, USA) were examined (Figure 4). Six patients showed marked p53 expression with a positive ratio of more than 80%. The labeling index of Ki-67 ranged from 25 to 70% (av. approximately 40). ER was mildly expressed in one patient and PgR expression also was mildly observed in 2 patients.\nImmunohistochemical staining profiles represented by Case 5 (A. p53 × 40, B. MIB-1 × 40, C. ER × 20, D. PgR × 20). A. Almost all of the carcinoma cells are strongly positive. B. More than half the carcinoma cells are labeled. C. The expression is observed in the cystically dilated normal glands and stromal cells but not in the carcinoma cells. D. The expression behavior is basically similar to the ER expression.\nFIGO stage was determined at IA for all the patients. Neither adjuvant chemotherapy nor irradiation was performed. Seven patients have been taking an uneventful clinical course (follow-up period ranging from 12 to 66 months, av. 41), but one patient died of the recurrent disease massively occupying the abdominal cavity 44 months after the initial diagnosis.\n\nDiscussion:\nEndometrial polyp is a common benign disease of the uterus. The overall incidence where cancer arises in the endometrial polyp has been reported as only a few percent, while postmenopausal women with EMP are at an increased risk of malignancy, compared to premenopausal women [10,13-15]. As risk factors for development of the malignancy in EMP, hypertension, obesity, and unopposed estrogen therapy have been indicated in addition to postmenopausal status [15]. There are unique or specific pathological and clinical features for EIC as a putative precursor of uterine body serous adenocarcinoma, as follows [1,4,5,7,16-20]: close association with an EMP; multicentric occurrence in the inactive or resting thin endometrium, lacking alteration in the architecture in the endometrium lacking an intervening phase of endometrial hyperplasia; extrauterine invasive extent, irrespective of an absence of stromal invasion of the uterus; and controversial outcome, whether taking a favorable or unfavorable clinical course. Development of EIC associated with an EMP may also be explained in part by Tamoxifen administration following breast cancer treatment [6,17]. However, little has been documented about the endometrial cytological features of minimal serous adenocarcinoma of the uterine body including EIC, mainly due to the fact that the disease is not frequently encountered in the routine practice. To the best of our knowledge, there is a case report cytologically referring to serous adenocarcinoma of the uterine body associated with an EMP [21], but here the patient already showed peritoneal dissemination at the initial presentation. In another report, serous adenocarcinomas of the uterine body were clinico-cytologically reviewed but they were all at advanced stages [22]. The latter report mentions that cytologically diagnostic findings of uterine body serous adenocarcinoma would be based on the following features: papillary cluster as well as tubular cluster, arborescent structure, frequent exfoliation, psammoma body, eccentric enlarged nuclei, coarsely granular chromatin, numerous nucleoli, and diameter of greater than G1 endometrioid adenocarcinoma [22]. In our review, 5 patients were purely non-invasive as they were composed of only EIC existing in an EMP, with few or no necroinflammatory changes. The background was found to be characteristically watery or mucous (Figure 1) in a way of simulating the immigration of extrauterine carcinoma cells via the fallopian tubes [12]. Many clusters were arranged in micropapillary architecture without fine vascular stroma, and others were occasionally tubular. An abrupt transition between the EIC cells and atrophic endometrial cells was noted by front formation (Figure 2). Conspicuous nucleoli were found in 3 patients (Figure 3). Even though it seems difficult to reach a definite diagnosis, these findings may serve as a diagnostic indicator for the uterine body serous adenocarcinoma, whether being at an early stage or an advanced stage.\nDifferential diagnoses for atypical epithelial sheets in the normal-appearing endometrial background include variable interpretations as follows: EIC, endometrial glandular dysplasia, endometrial intraepithelial neoplasia, hyperplastic polyp, and metastatic carcinoma [9,23]. In addition to these, isolated atypical glands with morphological and immunohistochemical features of a typical endometrial hyperplasia or type I endometrial adenocarcinoma may be encountered in grossly normal postmenopausal endometrium of asymptomatic patients [24]. Especially in the postmenopausal state, thin and smooth endometrium with atrophy is composed of hypocellular and flattened glands lined by cells with reduced or absent mitosis and sparse fibrous stroma [25]. It is supposed that the abrupt transition of the atrophic endometrial cells to atypical cells may indicate EIC in postmenopausal women.\nOverexpression of p53 is closely linked to the rapid growth of uterine body serous adenocarcinoma [6]. In EIC, too, frequent p53 mutation is reported to be associated with LOH of 17p [26]. Thus, immunohistochemistry for p53 is also adjunctively available in the diagnosis of uterine body serous adenocarcinoma, irrespective of whether it is at an early stage or advanced stage [27], in the discrimination between uterine body serous adenocarcinoma and benign morphologic mimics [28]. Negativity of ER and PgR staining is more reasonable with the diagnosis of serous adenocarcinoma including EIC.\nIn summary, EMP arising in postmenopausal women is at a high risk of giving birth to the tumorigenetic background for endometrial carcinomas such as endometrioid adenocarcinoma and serous adenocarcinoma including EIC. Endometrial cytology may become a helpful diagnostic approach for serous adenocarcinoma, whether it is at early or advanced stage, from the viewpoint of the distinctive cytological features. Above all, it should be noted that some EIC behaves in a more aggressive fashion regardless of whether or not it lacks apparent stroma invasion and extrauterine extent.\n\nPatients’ consents:\nWritten informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.\n\nEthical approval:\nThis review was performed based on compliance with the Helsinki Declaration.\n\nCompeting interests:\nAll of the authors declare that they have no competing interests.\n\nAuthors’ contributions:\nMY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe uterine endometrial polyp (EMP) has a potential risk of developing malignant tumors especially in postmenopausal women. These malignancies include endometrial intraepithelial carcinoma (EIC).\n\nMethods:\nEight patients with EIC in the EMP, who were postmenopausal with ages ranging from 49 to 76 years (av. 62), were cytologically reviewed in comparison with histological findings.\n\nResults:\nThe endometrial cytological findings were summarized as follows: mucous and watery diathesis as a background lacking or with little necrotic inflammatory changes; micropapillary cluster formation; abrupt transition between carcinoma cells and normal cells; nuclear enlargement; high N/C ratio; and single or a few prominent nucleoli. Histologically, one case had EIC alone in the EMP; three cases had EIC with stromal invasion confined to the EMP; and four cases had EIC in the atrophic endometrium in addition to EIC in the EMP. Seven patients have taken a disease-free course after surgical resection, but one patient died 44 months following the initial diagnosis because of the massive tumor extending over her peritoneal cavity.\n\nConclusions:\nEndometrial cytology may be helpful for the detection of early endometrial adenocarcinomas with serous features including EIC. Some early stage endometrial adenocarcinomas represented by EIC exceptionally take an aggressive clinical course irrespective of a lack of extrauterine lesions."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":3319,"string":"3,319"},"whole_article_abstract_length":{"kind":"number","value":247,"string":"247"},"num_sections":{"kind":"number","value":8,"string":"8"},"most_frequent_words":{"kind":"list like","value":["patients","endometrial","cells","adenocarcinoma","eic","carcinoma","serous","serous adenocarcinoma","body","normal"],"string":"[\n \"patients\",\n \"endometrial\",\n \"cells\",\n \"adenocarcinoma\",\n \"eic\",\n \"carcinoma\",\n \"serous\",\n \"serous adenocarcinoma\",\n \"body\",\n \"normal\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Endometrial intraepithelial carcinoma (EIC) | Endometrial polyp | Cytology [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Endometrial intraepithelial carcinoma (EIC) | Endometrial polyp | Cytology [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Endometrial intraepithelial carcinoma (EIC) | Endometrial polyp | Cytology [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Aged | Carcinoma in Situ | Disease Progression | Early Detection of Cancer | Endometrial Neoplasms | Female | Humans | Middle Aged | Polyps | Predictive Value of Tests | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Aged | Carcinoma in Situ | Disease Progression | Early Detection of Cancer | Endometrial Neoplasms | Female | Humans | Middle Aged | Polyps | Predictive Value of Tests | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Aged | Carcinoma in Situ | Disease Progression | Early Detection of Cancer | Endometrial Neoplasms | Female | Humans | Middle Aged | Polyps | Predictive Value of Tests | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | endometrial | cells | adenocarcinoma | eic | carcinoma | serous | serous adenocarcinoma | body | normal [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | endometrial | cells | adenocarcinoma | eic | carcinoma | serous | serous adenocarcinoma | body | normal [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | endometrial | cells | adenocarcinoma | eic | carcinoma | serous | serous adenocarcinoma | body | normal [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] adenocarcinoma | patients | endometrial | eic | serous | type | specimen | patients endometrial | hysterectomy | years [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cells | normal | 60 | carcinoma | tumor | patients | carcinoma cells | glands | pap | 20 [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | adenocarcinoma | endometrial | cells | eic | authors | authors declare competing | competing interests | competing | declare [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] EMP ||| EIC [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| one | EIC | EMP | three | EIC | EMP | four | EIC | EIC | EMP ||| Seven | 44 months [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] EMP ||| EIC ||| Eight | EIC | EMP | 49 to 76 years | 62 ||| ||| ||| one | EIC | EMP | three | EIC | EMP | four | EIC | EIC | EMP ||| Seven | 44 months ||| EIC ||| EIC [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":283,"cells":{"title":{"kind":"string","value":"Changes in specialized blood vessels in lymph nodes and their role in cancer metastasis."},"pmid":{"kind":"string","value":"23035663"},"background_abstract":{"kind":"string","value":"High endothelial venules (HEV) have been recognized to play a role in metastasis by its changes seen in the cancer microenvironment of lymph nodes (LN) and solid cancers. Squamous cell carcinoma (SCC) of the tongue is a prevalent tumor of the head and neck region with high propensity for LN metastasis. The extent of LN metastasis is the most reliable adverse prognostic factor. Primary tumors can induce vasculature reorganization within sentinel LN before the arrival of tumor cells and HEV represents these remodelled vessels. This study aims to evaluate the cancer induced vascular changes in regional lymph nodes (LN) of patients by studying the morphological and functional alterations of HEV and its correlation with clinical outcome and pathological features."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"This study was based on 65 patients with SCC tongue who underwent primary surgical treatment including neck dissection. The patients were categorized into 2 groups based on the presence of malignancy in their cervical lymph nodes. A review of the patients' pathological and clinical data was performed from a prospective database. Immunohistochemical staining of the tissue blocks for HEV and high-power-field image analysis were performed and analyzed with correlation to the patients' clinical and pathological features."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The total number of HEV was found to be significantly associated to disease-free interval. There was a similar association comparing the HEV parameters to overall survival. The density of abnormal HEV was significantly higher in patients with established metastases in their lymph nodes and HEV was shown to be a better prognosis factor than conventional tumor staging. The HEV morphological metamorphosis demonstrates a spectrum that correlates well with disease progression and clinical outcome."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The results suggest that the HEV displays a spectrum of morphological changes in the presence of cancer and LN metastasis, and that HEV is possibly involved in the process of cancer metastasis. We revealed the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environments in regional lymph nodes of tongue cancer patients in relation to clinical outcomes. The significant observation of modified dilated HEV containing red blood cells in lymph nodal basin of a cancer suggests the shifting of its function from one primarily of immune response to that of a blood carrying vessel. It also demonstrated potential prognostic value. More studies are needed to elucidate its potential role in cancer immunotherapy and as a potential novel therapeutic approach to preventing metastasis by manipulating the remodelling processes of HEV."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Case-Control Studies","Disease-Free Survival","Endothelium, Vascular","Humans","Kaplan-Meier Estimate","Lymph Nodes","Lymphatic Metastasis","Neoplasms","Venules"],"string":"[\n \"Case-Control Studies\",\n \"Disease-Free Survival\",\n \"Endothelium, Vascular\",\n \"Humans\",\n \"Kaplan-Meier Estimate\",\n \"Lymph Nodes\",\n \"Lymphatic Metastasis\",\n \"Neoplasms\",\n \"Venules\"\n]"},"pmcid":{"kind":"string","value":"3551724"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Approval was obtained from the Institutional Review Boards of both institutions involved in the study (2007/464/B). The study population consisted of 175 consecutive patients with SCC of the head and neck who had received primary surgical treatment at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Center, Singapore, from January 2001 through to December 2005. The patients’ pathological and clinical data including follow-up information was reviewed from a prospective database. The inclusion criteria included all surgically treated patients with histologically proven SCC tongue who underwent a neck dissection, with a minimum of 2-year follow up period. Patients with a second primary cancer and patients who did not have a neck dissection as part of their primary treament were excluded. There were a total 65 patients that met the study inclusion criteria. There were 35 patients in the group with primary SCC tongue without pathologically proven lymph node metastases in their neck dissection specimen who were designated as “cases”, and 30 patients in the group with primary SCC tongue with pathologically proven lymph node metastases in the neck dissection specimens, who were designated as “controls”. All patients included had radical excision of the primary tongue lesion and a neck dissection according to departmental protocol. In both groups, all patients had radical enbloc excision of the primary tongue lesion with or without resection of adjacent structures with either a unilateral or a bilateral, supraomohyoid neck dissection or a modified radical neck dissection. Tumours were classified according to the AJCC TNM Staging Classification, based on a pre-operative clinical evaluation and this determined the type of neck dissection performed by the primary surgeon\n[19]. All cases were discussed at a multi-discplinary head and neck tumor board. Intra-operative frozen sections were performed for margins of the tongue lesion and further resection of the tongue was performed in event that the surgical margins were involved. Upon discharge from the hospital, they were followed up at regular intervals of monthly, 3-monthly, 6-monthly and yearly at a progessive rate. Inpatient and outpatient clincal data, pathological, radiological and operative records were retrieved from a central medical records office and from the Department of Pathology, Singapore General Hospital. Mortality data was obtained and confirmed from the hospital central medical records office as well as the Singapore Registry of Births & Deaths.\n Immunohistochemistry A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\nA histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\n Computer assisted image analysis The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\nThe digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\n Definitions of the HEV’s parameters and ratios This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\nThis is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\n Statistical analysis Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\nCorrelating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The association of HEV and clinical outcome was studied by initially analyzing the OS and DFI of the 2 groups prior to individually analyzing the various HEV parameters (A, B, C, B/A, C/B, C/A) against OS and DFI. The details of the preliminary results comparing HEV parameters and patient outcomes have been previously published in an earlier paper\n[20]. In the secondary analysis, we further analyzed the tumor pathological characteristics (i.e. primary tumor volume, the stage of the disease and grade of the tumor) against clinical outcome (OS and DFI), to determine if these conventional tumor factors were prognostic in nature. We further investigated the role of HEV and its prognostic value by comparing and analyzing the HEV parameters with respect to the tumor volume, grade and stage. These analyses were repeated without controlling for the group factor (i.e. taking the sample population as a single cohort). Finally, we investigated the HEV parameters in each group to determine if there was a difference in the HEV values in the presence or absence of metastasis in the LN.\nWe analyzed the DFI and OS of the 65 patients with respect to their groups (35 cases versus 30 controls) to estimate the relative risk of DFI and OS associated with the group.\n Overall survival analysis The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\nThe relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\n Disease free interval analysis Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\nSimilarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\n Secondary analysis Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups\nTumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups"},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"This study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets."},"other_sections_titles":{"kind":"list like","value":["Background","High endothelial venules and its role in cancer metastasis","Objectives of study","Immunohistochemistry","Computer assisted image analysis","Definitions of the HEV’s parameters and ratios","Statistical analysis","Overall survival analysis","Disease free interval analysis","Secondary analysis","Limitations","Abbreviations","Competing interests","Authors’ contribution"],"string":"[\n \"Background\",\n \"High endothelial venules and its role in cancer metastasis\",\n \"Objectives of study\",\n \"Immunohistochemistry\",\n \"Computer assisted image analysis\",\n \"Definitions of the HEV’s parameters and ratios\",\n \"Statistical analysis\",\n \"Overall survival analysis\",\n \"Disease free interval analysis\",\n \"Secondary analysis\",\n \"Limitations\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contribution\"\n]"},"other_sections_texts":{"kind":"list like","value":["Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.","There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].","We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.","A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].","The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.","This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A","Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.","The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results","Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).","Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups","Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.","HEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.","The authors declare that they have no competing interests, financial or non-financial.","LSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript."],"string":"[\n \"Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\\n[6,7].\\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\",\n \"There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\",\n \"We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\",\n \"A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\\n[5,20].\",\n \"The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\",\n \"This is represented by the following alphabets A, B and C (Figure\\n1).\\n• number of all HEV : A\\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\\n• Ratio of dilated HEV to the total number of HEV : B/A\\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\",\n \"Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\",\n \"The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\\n1).\\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\\nSummary of results\",\n \"Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\\n3).\\nDisease free interval curves for the two groups (Cases vs. Controls).\\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\\n1).\",\n \"Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\\n3).\\nSummary of secondary analysis results\\nComparing high endothelial venules parameters in the 2 patient groups\",\n \"Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\",\n \"HEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.\",\n \"The authors declare that they have no competing interests, financial or non-financial.\",\n \"LSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","High endothelial venules and its role in cancer metastasis","Objectives of study","Methods","Immunohistochemistry","Computer assisted image analysis","Definitions of the HEV’s parameters and ratios","Statistical analysis","Results","Overall survival analysis","Disease free interval analysis","Secondary analysis","Discussion","Limitations","Conclusion","Abbreviations","Competing interests","Authors’ contribution"],"string":"[\n \"Background\",\n \"High endothelial venules and its role in cancer metastasis\",\n \"Objectives of study\",\n \"Methods\",\n \"Immunohistochemistry\",\n \"Computer assisted image analysis\",\n \"Definitions of the HEV’s parameters and ratios\",\n \"Statistical analysis\",\n \"Results\",\n \"Overall survival analysis\",\n \"Disease free interval analysis\",\n \"Secondary analysis\",\n \"Discussion\",\n \"Limitations\",\n \"Conclusion\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contribution\"\n]"},"all_sections_texts":{"kind":"list like","value":["Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.","There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].","We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.","Approval was obtained from the Institutional Review Boards of both institutions involved in the study (2007/464/B). The study population consisted of 175 consecutive patients with SCC of the head and neck who had received primary surgical treatment at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Center, Singapore, from January 2001 through to December 2005. The patients’ pathological and clinical data including follow-up information was reviewed from a prospective database. The inclusion criteria included all surgically treated patients with histologically proven SCC tongue who underwent a neck dissection, with a minimum of 2-year follow up period. Patients with a second primary cancer and patients who did not have a neck dissection as part of their primary treament were excluded. There were a total 65 patients that met the study inclusion criteria. There were 35 patients in the group with primary SCC tongue without pathologically proven lymph node metastases in their neck dissection specimen who were designated as “cases”, and 30 patients in the group with primary SCC tongue with pathologically proven lymph node metastases in the neck dissection specimens, who were designated as “controls”. All patients included had radical excision of the primary tongue lesion and a neck dissection according to departmental protocol. In both groups, all patients had radical enbloc excision of the primary tongue lesion with or without resection of adjacent structures with either a unilateral or a bilateral, supraomohyoid neck dissection or a modified radical neck dissection. Tumours were classified according to the AJCC TNM Staging Classification, based on a pre-operative clinical evaluation and this determined the type of neck dissection performed by the primary surgeon\n[19]. All cases were discussed at a multi-discplinary head and neck tumor board. Intra-operative frozen sections were performed for margins of the tongue lesion and further resection of the tongue was performed in event that the surgical margins were involved. Upon discharge from the hospital, they were followed up at regular intervals of monthly, 3-monthly, 6-monthly and yearly at a progessive rate. Inpatient and outpatient clincal data, pathological, radiological and operative records were retrieved from a central medical records office and from the Department of Pathology, Singapore General Hospital. Mortality data was obtained and confirmed from the hospital central medical records office as well as the Singapore Registry of Births & Deaths.\n Immunohistochemistry A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\nA histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\n Computer assisted image analysis The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\nThe digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\n Definitions of the HEV’s parameters and ratios This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\nThis is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\n Statistical analysis Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\nCorrelating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.","A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].","The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.","This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A","Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.","The association of HEV and clinical outcome was studied by initially analyzing the OS and DFI of the 2 groups prior to individually analyzing the various HEV parameters (A, B, C, B/A, C/B, C/A) against OS and DFI. The details of the preliminary results comparing HEV parameters and patient outcomes have been previously published in an earlier paper\n[20]. In the secondary analysis, we further analyzed the tumor pathological characteristics (i.e. primary tumor volume, the stage of the disease and grade of the tumor) against clinical outcome (OS and DFI), to determine if these conventional tumor factors were prognostic in nature. We further investigated the role of HEV and its prognostic value by comparing and analyzing the HEV parameters with respect to the tumor volume, grade and stage. These analyses were repeated without controlling for the group factor (i.e. taking the sample population as a single cohort). Finally, we investigated the HEV parameters in each group to determine if there was a difference in the HEV values in the presence or absence of metastasis in the LN.\nWe analyzed the DFI and OS of the 65 patients with respect to their groups (35 cases versus 30 controls) to estimate the relative risk of DFI and OS associated with the group.\n Overall survival analysis The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\nThe relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\n Disease free interval analysis Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\nSimilarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\n Secondary analysis Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups\nTumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups","The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results","Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).","Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups","Squamous cell carcinoma of the tongue has one of the fastest rising rate today affecting the young with a high propensity for LN metastases.\n[22] The presence of metastatic cells in regional LN, along with extracapsular spread are the most important prognostic factors in patients with squamous cell carcinoma (SCC) of the tongue\n[22-25]. The prognosis is worse compared to other equivalent carcinomas of the head and neck region.\nTumor cell metastasis to regional LN marks one of the initial steps in the cascade of tumor metastasis after tumor cell progression in the primary tumor. The lymphatic metastatic cascade is a series of complex interrelated steps and processes\n[26]. As the tumor grows and enlarges, cytokines are secreted to promote lymphangiogenesis\n[27]. As malignant cells invade the extracellular matrix, they enter the lumen of the lymphatics and they move in clusters or as single cells to the first echelon of regional LN, otherwise known as the SLN\n[28,29]. The structural properties, such as the lack of basement membrane in terminal lymphatics, the presence of the intercellular clefts, the traumatic environment in the blood including shear forces of blood turbulence and tumor antigenicity detection by host immune cells, contribute and account for the observation that in many solid cancers, lymphatic metastasis precedes metastasis via the vascular system\n[26,30]. The relationship between angiogenesis and lymphangiogenesis has been extensively studied in cancer research\n[14,26,31,32]. This is pertinent in the biology of LN metastasis where the two systems literally lie side by side. There is extensive evidence showing members of the Vascular endothelial growth factor (VEGF) family, namely VEGF-C, VEGF-D and VEGF-A\n[33], are not only important regulators of lymph vessel growth in vivo, but may also be involved in the process of promoting lymphatic metastasis, such as VEGF-C\n[27,34-36]. This is significant because there is evidence that tumor can activate both LN lymphangiogenesis and angiogenesis before metastasis, and this would translate into logical efforts in the field of anti-cancer research specifically targeting pathways of tumor lymphangiogenesis and angiogenesis. There have been encouraging results from therapeutic targeting of the various VEGF member pathways to inhibit lymph node metastasis, reduce lymphatic and vascular density in LN as well as to reduce overall tumor burden in the lymph nodes and distant metastases\n[34,37,38].\nTargeted therapy such as anti-angiogenesis drugs has shown positive results in clinical studies. Bevacizumab (AvastinTM), a monoclonal antibody to VEGF-A, was approved by United States Food and Drug Administration (FDA) in 2004 and has proven clinical benefits in treatment of metastatic colorectal cancer when the drug was added to standard chemotherapy and also has positive results in metastatic kidney cancer and advanced lung cancer. There are many ongoing trials including Phase III clinical trials in advanced or metastatic renal cell carcinoma, pancreatic cancer, and ovarian cancer for AvastinTM[39]. Other molecular pathways that harbor potential targets which have varying degrees of anti-angiogenesis and anti-lymphangiogenesis properties, are ligands such as hepatocyte growth factor (HGF), receptors like Neuropilin 2, PDGFRα/β and others such as Angiostatin, interferon α/β and platelet-factor 4\n[40]. Two other antiangiogenic drugs, Sorafenib (Nexavar™, Bayer) and Sunitinib (Sutent™, Pfizer), have also been approved by the FDA for various aspects of cancer treatment, for example, Sorafenib and Sunitinib have been beneficial in the treatment of metastatic renal-cell cancer and advanced hepatocellular carcinoma\n[41,42]. They target multiple receptor tyrosine kinases, including VEGF receptors and platelet-derived growth factor (PDGF) receptors\n[43].\nIn our series, we detected a statistical significance when we analyzed the OS but not in DFI in the patients with or without LN metastasis (cases vs. controls) (Figures\n2 and\n3). This may be attributed to our limited sample size. It has been proposed and is currently being validated, that SLN biopsy may play the next stage in the evolution in the neck management and treatment of tongue SCC\n[44]. Exploration and understanding of this concept coupled with advances and optimistic results in anti-angiogenesis therapy involving VEGF anti-metabolites translates the next logical exploration to be invested in the study of the pathogenesis of lymph node metastasis. It was shown that the lumen of the lymphatic sinuses and blood vessels were dilated in SLN before metastasis\n[5]. We have demonstrated that lymph nodes are transformed by the primary tongue tumor to become a functional blood vessel–enriched organ before and independent of metastasis, with the changes in the morphology of the HEV to become main blood flow carrier in the lymph node (Figure\n4). This process of vascularization in the LN (HEV morphological alterations) appears similar and consistent in human tissues and previous animal models\n[5,20]. This study presents functional and structural data that the primary tumor can possibly manipulate the microenvironment and biology of lymph nodes to potentially facilitate the proliferation of subsequent tumor deposits leading to established metastases. The morphological alteration of HEV in the presence of a cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shift and phenotypic change of HEV from its known function as a lymphocyte recruitment modulator to one as a blood carrier.\nDilated HEVs with red blood cells in its lumen (high power field).\nIn previous studies, the degree of lymphatic dilation in the SLN significantly correlated with the primary tumor weight\n[5,10]. This observation suggests that the lymphatic fluid from the primary tumor induced the persistent alteration of the lymph channel in SLN. These results are consistent with findings that demonstrate, in contrast to angiogenesis, in which blood flow proceeds only after the vessel develops, lymphangiogenesis can be induced by interstitial fluid channeling\n[45]. In this aspect, this is analogous to immunology studies showing the alteration of lymph channels in SLN can also occur during an inflammatory reaction, which facilitates the migration of inflammatory cells\n[46,47] The difference in cancer metastasis biology and inflammatory process starts here. In endotoxin induced experiments and immunology studies, the dilated lymphatic sinuses and vessels in the associated reactive LN were full of lymphocytes. In contrast in tumor-reactive lymphadenopathy, the lymphatics contained minimum or no cellular material, suggesting different roles of the SLN lymphatic channels in different pathologic processes. It has been well characterized that the HEV of lymph nodes play an important role in recruiting lymphocytes for the generation of immune responses\n[9,11]. By expressing homing receptors on their surface, which blood lymphocytes can recognize as they pass in circulation, HEV provide a unique location where naive lymphocytes can enter the lymph node\n[47,48].\nIn our previous study\n[5], we found that the role of HEV was transformed from a lymphocyte recruiter to become the main blood flow carrier in the SLN prior to metastasis. We have shown in this study that in the regional cervical LN, not only could the morphology of individual HEV change dramatically to increase blood flow, but the proliferation rate of HEV endothelial cells was also increased before metastasis. This transformation of HEV as a lymphocytic carrier in LN to a blood-flow carrier can be seen in the large quantity of red blood cells visible in the HEV even in pre-metastatic regional LN. More significantly, this phenomenon is also more pronounced in the regional LN of the patients with established LN metastasis. This is consistent with the findings of Chung and colleagues demonstrating the increased HEV density in SLN of oral SCC before the arrival of tumor cells, the HEV density in metastatic SLN was also higher than that of non-metastatic SLN\n[6]. We have shown this in our supplementary data that patients with lymph node metastases have more dilated HEV with RBC in their LN as compared to patients without LN metastasis. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV(C/B) in the control (pN+) group is statistically significantly higher than that in case group (p-value = 0.0318).\nIn addition, with the benefit of patients’ pathological and follow-up information, we have demonstrated that even with just an increase of 1 HEV in a high-power-field (HPF), regardless of the group, the risk is 1.024 times worse in terms of overall survival. This is consistent with clinical knowledge and natural history of the disease. It is well established that patients without LN metastases in their regional LN (cases in our cohort) have a significantly better prognosis, and; this fact is also reflected in our analysis\n[22,49-51] (Figure\n2). Cases had a 0.428 times the risk of Controls with regards to overall survival if they had the same number of HEV. It is crucial to understand that this risk is exponential in nature. To illustrate the significance of this result, an example will be used: Patient A with tongue SCC has proven LN metastases in the cervical LN. On further immunohistochemical staining, it is found that he has 0 HEV in a random high-power-field. Comparing this to patient B who has the same grade, stage of tumor, degree of LN metastases as patient A but has 100 HEV in a HPF, patient B will have a [ (1.024)100 = 10.715 ] 10 times worse prognosis in OS as compared to patient A. We will then consider patient C who has tongue SCC with no LN metastasis. If the HEV amount in a HPF is the same as either patient A or B, patient C’s risk as compared to either patient A or B will be 2.34 times (1/0.428= 2.34) better in terms of overall survival (Table\n4). Although this did not reach statistical significance (p=0.471), we believe this to be a factor of a small sample size.\nDifferences in OS and DFI relative risk in the 2 groups\nAssuming that our hypothesis is true, HEV transformation from a normal immunological mediator to a tumor metastasis mediator is reflected by morphological changes from a normal appearing HEV, to a dilated HEV, and then to a dilated HEV containing RBC. We believe that this metamorphosis occurs on a spectrum, and this process begins with the HEV increasing in absolute numbers, then each becoming more dilated before progressing into a functional vessel carrying blood (Figure\n5). Hence we analyzed our data looking at the 3 different stages of HEV transformation and the ratio comparing each parameter in a systematic way to demonstrate this spectrum (Figure\n1).\nMetamorphosis of High endothelial venules in a tumor microenvironment. This process begins with the high endothelial venules increasing in absolute numbers, subsequently each of them becoming more dilated and lastly every one of them will become a function vessel carrying blood.\nWe recall that that in the last example we illustrated that a patient’s OS risk is 1.024 times worse than an equivalent patient if there is 1 more HEV in one HPF of his regional LN, the risk is increased to 1.071 if the HEV is a dilated HEV. The risk is even higher at 1.116 times if the HEV is a dilated HEV containing RBC (Table\n1)\n[20]. This trend is also seen when all patients are considered in a single cohort. A patient’s OS risk is worsened by 1.004 times if there is an increase of 1 dilated HEV in a HPF. If that dilated HEV contains RBC, the OS risk is increased to 1.008 times.\nIn the treatment of tongue carcinomas, DFI is important as it signifies the failure of loco-regional control for which there is little effective therapy. Further surgery e.g. neck dissection to remove loco-regional recurrences is plagued with high morbidity and mortality. This, coupled with poor chemotherapy and radiotherapy response rates in recurrences, often equates to rapid deterioration of the patient’s condition. Disease free interval was also analyzed in the same manner to the three different HEV morphological phenotypes.\nWe found statistical significance in the relationship between the total number of HEV (A) and DFI when we controlled for the group (i.e. taking in the presence of LN metastasis as a factor). (p=0.022). This significance is preserved when we analyzed all the patients as a cohort (p=0.023)\n[20]. This means that the quantity of HEV is inversely related to DFI. If you consider the LN status and take it into statistical consideration, if there is one more HEV in a HPF, the DFI is 1.051 times worse than zero HEV. It is again important to note that the relationship of the quantity of HEV with respect to DFI is exponential in nature. For example, a patient (patient B) with 100 more HEV (A) in one HPF has a {(1.051)100 =144.634} 144 times shorter DFI as compared to an equivalent patient with only one HEV per HPF (patient A) (Table\n4). Excluding the group effect and considering all the patients as a cohort, a patient with 1 more HEV per HPF will have a DFI that is 1.051 times worse than a patient with zero HEV in a HPF (p=0.023).\nWe looked at the different ratios of abnormal HEV and compared them to several clinic-pathologic parameters namely overall survival, disease–free interval, tumor volume, stage and grade of the tumor. The details are specified in the secondary results (Table\n2).\nThere is a general trend observed. The more advanced the disease, the higher the ratio/percentage of abnormality of HEV. This can be seen in when we analyzed OS and the 2 ratios. The OS relative risk worsens by 1.078 if the ratio B/A (ratio of dilated HEV to the total number of HEV) increased by 1, while the OS relative risk worsens by 3.624 times if the ratio of dilated HEVs with RBC to the total number of HEV increases by 1. Most importantly, if we consider the most abnormal form of HEV (dilated HEV with RBC within its lumen, C) and look at it as a ratio to the total no. of HEV, a patient’s OS relative risk worsens by 17.884 times if this ratio (C/A) increases by a factor of 1. This observation approaches marginal significance. (p = 0.171) (Figure\n6).\nOverall survival relative risk with respect to the different high endothelial venules ratios.\nIn this study, we have shown the relationship of HEV and their transformation in a cancerous environment. We also note that in previous studies the HEV morphology did not alter at all in endotoxin-induced lymphadenopathy, implying a selective reaction of HEV in the cancerous condition\n[5]. Distinct, differentiated gene expression has also been reported when the endothelial cells respond to the changing of their microenvironment\n[52]. In a previous study, it was shown that the cellular morphology of the tall endothelial cells forming HEV changed dramatically to become flat endothelial cells in the presence of cancer. As a consequence, the HEV was remodelled from a thick-walled, endothelial vessel with a small lumen to a thin walled, large-lumen vessel shifting its function from recruitment of lymphocytes to becoming a blood vessel (Figure\n4)\n[10]. These facts indicate that the blood vessel endothelium has tremendous potential to adapt its environment. The confined lymph and blood channel alterations within the SLN, but not in the next station lymph node, imply that an inducer from the primary tumor is functioning locally with the existence of an active primary cancer. VEGF-A has been found to be an inducer of lymphangiogenesis in SLN\n[33]. Other studies have shown that serum level of VEGF-A was elevated in patients with late stage NPC\n[21] VEGF-A is a secreted protein factor that can travel in blood to other lymph nodes, these findings suggest that there may be other inducers involved. It is also of interest to explore the role of HEV after the establishment of a metastatic tumor nest. The enlarged, remodelled HEV could integrate into the metastatic tumor vasculature with further differentiation, characterized by the gradual loss of their specific marker MECA-79 from the tumor margin to the central part of the metastatic tumor nest.\n[5,10] It has been explained that, compared with primary tumors, the more rapid growth of metastatic lesions in the cervical lymph nodes of NPC patients was due to clonal selection of the cancer cells during metastases, with highly proliferative clones disseminated to the cervical nodes. However, based on our findings, the metastatic tumor vasculature in lymph nodes consists of many large blood vessels derived from normal HEVs, suggesting that the efficiency of nutrition and oxygen supplies could be better for the metastatic tumor cells in the involved lymph node. The enrichment of the blood supply in the lymph node before and after metastasis may favor the growth of newly arriving metastatic cancer cells. Consequently, as evident clinically in the long term follow-up of cancer patients, the involved regional lymph nodes may become manifest first, whereas the primary tumor remain clinically occult for years\n[53]. Moreover, the high density of functioning blood vessels in lymph nodes may subsequently facilitate the metastasis of cancer cell to distant organs. Conversely, consistent with its role in lymphocyte trafficking, if the HEV density is increased in the tumor themselves as opposed to LN, it has favorable prognostic value, Martinet et al. reported that in 146 breast cancers, the density of tumor HEV was an independent prognostic risk factor for DFI and OS\n[8]. This further suggests the tumor HEV function as major gateway for lymphocyte infiltration into tumors and represents potential targets for cancer immunotherapy. It will also be important to elucidate if the highly vascularized premetastatic SLN is associated with an increased metastatic potential. Control of lymphatic fluid movement may also be a target to consider in preventing metastases.\nAssuming our hypothesis is true with regards to the role of HEV in cancer metastasis, a potential therapeutic approach is to interrupt or block this remodelling process and evaluate if there is an effect on metastasis. This will confirm the HEV’s central role in the pathogenesis of metastasis.\nFurther studies need to designed and performed to elucidate the pathways of HEV transformation. A control gene for the growth and differentiation of HEV remains to be identified\n[9]. With regards to this, a recently identified nuclear factor (NF), NF-HEV has potential because it is preferentially expressed by HEV\n[54]. Dendritic cells, well known for their antigen presentation roles has recently been shown to modulate the phenotype of HEV and control entry of naive lymphocytes to LN, their role in a cancer microenvironment is also an area of potential research\n[13]. There are recent experiments in mouse models investigating conditional gene targeting of HEV as well\n[55]. Identification and characterization the molecular pathways and the control genes would have considerable clinical applications. For example, it would enable the design of experiments and studies to induce or retard the formation of HEV in various tissues, including tumors and their LN, improving vaccination strategies against pathogens and cancers. Therefore, there is a wealth of translational applications with regards to targeting HEV as a ligand for cancer investigational therapeutics. It paves the journey in the discovery of many unknown genes and molecules with potential clinical importance. Lastly, more work can be performed to validate our findings in other solid cancers.\n Limitations Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\nOur study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.","Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.","This study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets.","HEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.","The authors declare that they have no competing interests, financial or non-financial.","LSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript."],"string":"[\n \"Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\\n[6,7].\\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\",\n \"There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\\n[6].\\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\\n[5,10].\",\n \"We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\",\n \"Approval was obtained from the Institutional Review Boards of both institutions involved in the study (2007/464/B). The study population consisted of 175 consecutive patients with SCC of the head and neck who had received primary surgical treatment at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Center, Singapore, from January 2001 through to December 2005. The patients’ pathological and clinical data including follow-up information was reviewed from a prospective database. The inclusion criteria included all surgically treated patients with histologically proven SCC tongue who underwent a neck dissection, with a minimum of 2-year follow up period. Patients with a second primary cancer and patients who did not have a neck dissection as part of their primary treament were excluded. There were a total 65 patients that met the study inclusion criteria. There were 35 patients in the group with primary SCC tongue without pathologically proven lymph node metastases in their neck dissection specimen who were designated as “cases”, and 30 patients in the group with primary SCC tongue with pathologically proven lymph node metastases in the neck dissection specimens, who were designated as “controls”. All patients included had radical excision of the primary tongue lesion and a neck dissection according to departmental protocol. In both groups, all patients had radical enbloc excision of the primary tongue lesion with or without resection of adjacent structures with either a unilateral or a bilateral, supraomohyoid neck dissection or a modified radical neck dissection. Tumours were classified according to the AJCC TNM Staging Classification, based on a pre-operative clinical evaluation and this determined the type of neck dissection performed by the primary surgeon\\n[19]. All cases were discussed at a multi-discplinary head and neck tumor board. Intra-operative frozen sections were performed for margins of the tongue lesion and further resection of the tongue was performed in event that the surgical margins were involved. Upon discharge from the hospital, they were followed up at regular intervals of monthly, 3-monthly, 6-monthly and yearly at a progessive rate. Inpatient and outpatient clincal data, pathological, radiological and operative records were retrieved from a central medical records office and from the Department of Pathology, Singapore General Hospital. Mortality data was obtained and confirmed from the hospital central medical records office as well as the Singapore Registry of Births & Deaths.\\n Immunohistochemistry A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\\n[5,20].\\nA histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\\n[5,20].\\n Computer assisted image analysis The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\\nThe digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\\n Definitions of the HEV’s parameters and ratios This is represented by the following alphabets A, B and C (Figure\\n1).\\n• number of all HEV : A\\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\\n• Ratio of dilated HEV to the total number of HEV : B/A\\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\\nThis is represented by the following alphabets A, B and C (Figure\\n1).\\n• number of all HEV : A\\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\\n• Ratio of dilated HEV to the total number of HEV : B/A\\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\\n Statistical analysis Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\\nCorrelating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\",\n \"A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\\n[5,20].\",\n \"The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\",\n \"This is represented by the following alphabets A, B and C (Figure\\n1).\\n• number of all HEV : A\\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\\n• Ratio of dilated HEV to the total number of HEV : B/A\\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\",\n \"Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\",\n \"The association of HEV and clinical outcome was studied by initially analyzing the OS and DFI of the 2 groups prior to individually analyzing the various HEV parameters (A, B, C, B/A, C/B, C/A) against OS and DFI. The details of the preliminary results comparing HEV parameters and patient outcomes have been previously published in an earlier paper\\n[20]. In the secondary analysis, we further analyzed the tumor pathological characteristics (i.e. primary tumor volume, the stage of the disease and grade of the tumor) against clinical outcome (OS and DFI), to determine if these conventional tumor factors were prognostic in nature. We further investigated the role of HEV and its prognostic value by comparing and analyzing the HEV parameters with respect to the tumor volume, grade and stage. These analyses were repeated without controlling for the group factor (i.e. taking the sample population as a single cohort). Finally, we investigated the HEV parameters in each group to determine if there was a difference in the HEV values in the presence or absence of metastasis in the LN.\\nWe analyzed the DFI and OS of the 65 patients with respect to their groups (35 cases versus 30 controls) to estimate the relative risk of DFI and OS associated with the group.\\n Overall survival analysis The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\\n1).\\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\\nSummary of results\\nThe relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\\n1).\\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\\nSummary of results\\n Disease free interval analysis Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\\n3).\\nDisease free interval curves for the two groups (Cases vs. Controls).\\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\\n1).\\nSimilarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\\n3).\\nDisease free interval curves for the two groups (Cases vs. Controls).\\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\\n1).\\n Secondary analysis Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\\n3).\\nSummary of secondary analysis results\\nComparing high endothelial venules parameters in the 2 patient groups\\nTumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\\n3).\\nSummary of secondary analysis results\\nComparing high endothelial venules parameters in the 2 patient groups\",\n \"The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\\n1).\\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\\nSummary of results\",\n \"Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\\n3).\\nDisease free interval curves for the two groups (Cases vs. Controls).\\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\\n1).\",\n \"Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\\n3).\\nSummary of secondary analysis results\\nComparing high endothelial venules parameters in the 2 patient groups\",\n \"Squamous cell carcinoma of the tongue has one of the fastest rising rate today affecting the young with a high propensity for LN metastases.\\n[22] The presence of metastatic cells in regional LN, along with extracapsular spread are the most important prognostic factors in patients with squamous cell carcinoma (SCC) of the tongue\\n[22-25]. The prognosis is worse compared to other equivalent carcinomas of the head and neck region.\\nTumor cell metastasis to regional LN marks one of the initial steps in the cascade of tumor metastasis after tumor cell progression in the primary tumor. The lymphatic metastatic cascade is a series of complex interrelated steps and processes\\n[26]. As the tumor grows and enlarges, cytokines are secreted to promote lymphangiogenesis\\n[27]. As malignant cells invade the extracellular matrix, they enter the lumen of the lymphatics and they move in clusters or as single cells to the first echelon of regional LN, otherwise known as the SLN\\n[28,29]. The structural properties, such as the lack of basement membrane in terminal lymphatics, the presence of the intercellular clefts, the traumatic environment in the blood including shear forces of blood turbulence and tumor antigenicity detection by host immune cells, contribute and account for the observation that in many solid cancers, lymphatic metastasis precedes metastasis via the vascular system\\n[26,30]. The relationship between angiogenesis and lymphangiogenesis has been extensively studied in cancer research\\n[14,26,31,32]. This is pertinent in the biology of LN metastasis where the two systems literally lie side by side. There is extensive evidence showing members of the Vascular endothelial growth factor (VEGF) family, namely VEGF-C, VEGF-D and VEGF-A\\n[33], are not only important regulators of lymph vessel growth in vivo, but may also be involved in the process of promoting lymphatic metastasis, such as VEGF-C\\n[27,34-36]. This is significant because there is evidence that tumor can activate both LN lymphangiogenesis and angiogenesis before metastasis, and this would translate into logical efforts in the field of anti-cancer research specifically targeting pathways of tumor lymphangiogenesis and angiogenesis. There have been encouraging results from therapeutic targeting of the various VEGF member pathways to inhibit lymph node metastasis, reduce lymphatic and vascular density in LN as well as to reduce overall tumor burden in the lymph nodes and distant metastases\\n[34,37,38].\\nTargeted therapy such as anti-angiogenesis drugs has shown positive results in clinical studies. Bevacizumab (AvastinTM), a monoclonal antibody to VEGF-A, was approved by United States Food and Drug Administration (FDA) in 2004 and has proven clinical benefits in treatment of metastatic colorectal cancer when the drug was added to standard chemotherapy and also has positive results in metastatic kidney cancer and advanced lung cancer. There are many ongoing trials including Phase III clinical trials in advanced or metastatic renal cell carcinoma, pancreatic cancer, and ovarian cancer for AvastinTM[39]. Other molecular pathways that harbor potential targets which have varying degrees of anti-angiogenesis and anti-lymphangiogenesis properties, are ligands such as hepatocyte growth factor (HGF), receptors like Neuropilin 2, PDGFRα/β and others such as Angiostatin, interferon α/β and platelet-factor 4\\n[40]. Two other antiangiogenic drugs, Sorafenib (Nexavar™, Bayer) and Sunitinib (Sutent™, Pfizer), have also been approved by the FDA for various aspects of cancer treatment, for example, Sorafenib and Sunitinib have been beneficial in the treatment of metastatic renal-cell cancer and advanced hepatocellular carcinoma\\n[41,42]. They target multiple receptor tyrosine kinases, including VEGF receptors and platelet-derived growth factor (PDGF) receptors\\n[43].\\nIn our series, we detected a statistical significance when we analyzed the OS but not in DFI in the patients with or without LN metastasis (cases vs. controls) (Figures\\n2 and\\n3). This may be attributed to our limited sample size. It has been proposed and is currently being validated, that SLN biopsy may play the next stage in the evolution in the neck management and treatment of tongue SCC\\n[44]. Exploration and understanding of this concept coupled with advances and optimistic results in anti-angiogenesis therapy involving VEGF anti-metabolites translates the next logical exploration to be invested in the study of the pathogenesis of lymph node metastasis. It was shown that the lumen of the lymphatic sinuses and blood vessels were dilated in SLN before metastasis\\n[5]. We have demonstrated that lymph nodes are transformed by the primary tongue tumor to become a functional blood vessel–enriched organ before and independent of metastasis, with the changes in the morphology of the HEV to become main blood flow carrier in the lymph node (Figure\\n4). This process of vascularization in the LN (HEV morphological alterations) appears similar and consistent in human tissues and previous animal models\\n[5,20]. This study presents functional and structural data that the primary tumor can possibly manipulate the microenvironment and biology of lymph nodes to potentially facilitate the proliferation of subsequent tumor deposits leading to established metastases. The morphological alteration of HEV in the presence of a cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shift and phenotypic change of HEV from its known function as a lymphocyte recruitment modulator to one as a blood carrier.\\nDilated HEVs with red blood cells in its lumen (high power field).\\nIn previous studies, the degree of lymphatic dilation in the SLN significantly correlated with the primary tumor weight\\n[5,10]. This observation suggests that the lymphatic fluid from the primary tumor induced the persistent alteration of the lymph channel in SLN. These results are consistent with findings that demonstrate, in contrast to angiogenesis, in which blood flow proceeds only after the vessel develops, lymphangiogenesis can be induced by interstitial fluid channeling\\n[45]. In this aspect, this is analogous to immunology studies showing the alteration of lymph channels in SLN can also occur during an inflammatory reaction, which facilitates the migration of inflammatory cells\\n[46,47] The difference in cancer metastasis biology and inflammatory process starts here. In endotoxin induced experiments and immunology studies, the dilated lymphatic sinuses and vessels in the associated reactive LN were full of lymphocytes. In contrast in tumor-reactive lymphadenopathy, the lymphatics contained minimum or no cellular material, suggesting different roles of the SLN lymphatic channels in different pathologic processes. It has been well characterized that the HEV of lymph nodes play an important role in recruiting lymphocytes for the generation of immune responses\\n[9,11]. By expressing homing receptors on their surface, which blood lymphocytes can recognize as they pass in circulation, HEV provide a unique location where naive lymphocytes can enter the lymph node\\n[47,48].\\nIn our previous study\\n[5], we found that the role of HEV was transformed from a lymphocyte recruiter to become the main blood flow carrier in the SLN prior to metastasis. We have shown in this study that in the regional cervical LN, not only could the morphology of individual HEV change dramatically to increase blood flow, but the proliferation rate of HEV endothelial cells was also increased before metastasis. This transformation of HEV as a lymphocytic carrier in LN to a blood-flow carrier can be seen in the large quantity of red blood cells visible in the HEV even in pre-metastatic regional LN. More significantly, this phenomenon is also more pronounced in the regional LN of the patients with established LN metastasis. This is consistent with the findings of Chung and colleagues demonstrating the increased HEV density in SLN of oral SCC before the arrival of tumor cells, the HEV density in metastatic SLN was also higher than that of non-metastatic SLN\\n[6]. We have shown this in our supplementary data that patients with lymph node metastases have more dilated HEV with RBC in their LN as compared to patients without LN metastasis. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV(C/B) in the control (pN+) group is statistically significantly higher than that in case group (p-value = 0.0318).\\nIn addition, with the benefit of patients’ pathological and follow-up information, we have demonstrated that even with just an increase of 1 HEV in a high-power-field (HPF), regardless of the group, the risk is 1.024 times worse in terms of overall survival. This is consistent with clinical knowledge and natural history of the disease. It is well established that patients without LN metastases in their regional LN (cases in our cohort) have a significantly better prognosis, and; this fact is also reflected in our analysis\\n[22,49-51] (Figure\\n2). Cases had a 0.428 times the risk of Controls with regards to overall survival if they had the same number of HEV. It is crucial to understand that this risk is exponential in nature. To illustrate the significance of this result, an example will be used: Patient A with tongue SCC has proven LN metastases in the cervical LN. On further immunohistochemical staining, it is found that he has 0 HEV in a random high-power-field. Comparing this to patient B who has the same grade, stage of tumor, degree of LN metastases as patient A but has 100 HEV in a HPF, patient B will have a [ (1.024)100 = 10.715 ] 10 times worse prognosis in OS as compared to patient A. We will then consider patient C who has tongue SCC with no LN metastasis. If the HEV amount in a HPF is the same as either patient A or B, patient C’s risk as compared to either patient A or B will be 2.34 times (1/0.428= 2.34) better in terms of overall survival (Table\\n4). Although this did not reach statistical significance (p=0.471), we believe this to be a factor of a small sample size.\\nDifferences in OS and DFI relative risk in the 2 groups\\nAssuming that our hypothesis is true, HEV transformation from a normal immunological mediator to a tumor metastasis mediator is reflected by morphological changes from a normal appearing HEV, to a dilated HEV, and then to a dilated HEV containing RBC. We believe that this metamorphosis occurs on a spectrum, and this process begins with the HEV increasing in absolute numbers, then each becoming more dilated before progressing into a functional vessel carrying blood (Figure\\n5). Hence we analyzed our data looking at the 3 different stages of HEV transformation and the ratio comparing each parameter in a systematic way to demonstrate this spectrum (Figure\\n1).\\nMetamorphosis of High endothelial venules in a tumor microenvironment. This process begins with the high endothelial venules increasing in absolute numbers, subsequently each of them becoming more dilated and lastly every one of them will become a function vessel carrying blood.\\nWe recall that that in the last example we illustrated that a patient’s OS risk is 1.024 times worse than an equivalent patient if there is 1 more HEV in one HPF of his regional LN, the risk is increased to 1.071 if the HEV is a dilated HEV. The risk is even higher at 1.116 times if the HEV is a dilated HEV containing RBC (Table\\n1)\\n[20]. This trend is also seen when all patients are considered in a single cohort. A patient’s OS risk is worsened by 1.004 times if there is an increase of 1 dilated HEV in a HPF. If that dilated HEV contains RBC, the OS risk is increased to 1.008 times.\\nIn the treatment of tongue carcinomas, DFI is important as it signifies the failure of loco-regional control for which there is little effective therapy. Further surgery e.g. neck dissection to remove loco-regional recurrences is plagued with high morbidity and mortality. This, coupled with poor chemotherapy and radiotherapy response rates in recurrences, often equates to rapid deterioration of the patient’s condition. Disease free interval was also analyzed in the same manner to the three different HEV morphological phenotypes.\\nWe found statistical significance in the relationship between the total number of HEV (A) and DFI when we controlled for the group (i.e. taking in the presence of LN metastasis as a factor). (p=0.022). This significance is preserved when we analyzed all the patients as a cohort (p=0.023)\\n[20]. This means that the quantity of HEV is inversely related to DFI. If you consider the LN status and take it into statistical consideration, if there is one more HEV in a HPF, the DFI is 1.051 times worse than zero HEV. It is again important to note that the relationship of the quantity of HEV with respect to DFI is exponential in nature. For example, a patient (patient B) with 100 more HEV (A) in one HPF has a {(1.051)100 =144.634} 144 times shorter DFI as compared to an equivalent patient with only one HEV per HPF (patient A) (Table\\n4). Excluding the group effect and considering all the patients as a cohort, a patient with 1 more HEV per HPF will have a DFI that is 1.051 times worse than a patient with zero HEV in a HPF (p=0.023).\\nWe looked at the different ratios of abnormal HEV and compared them to several clinic-pathologic parameters namely overall survival, disease–free interval, tumor volume, stage and grade of the tumor. The details are specified in the secondary results (Table\\n2).\\nThere is a general trend observed. The more advanced the disease, the higher the ratio/percentage of abnormality of HEV. This can be seen in when we analyzed OS and the 2 ratios. The OS relative risk worsens by 1.078 if the ratio B/A (ratio of dilated HEV to the total number of HEV) increased by 1, while the OS relative risk worsens by 3.624 times if the ratio of dilated HEVs with RBC to the total number of HEV increases by 1. Most importantly, if we consider the most abnormal form of HEV (dilated HEV with RBC within its lumen, C) and look at it as a ratio to the total no. of HEV, a patient’s OS relative risk worsens by 17.884 times if this ratio (C/A) increases by a factor of 1. This observation approaches marginal significance. (p = 0.171) (Figure\\n6).\\nOverall survival relative risk with respect to the different high endothelial venules ratios.\\nIn this study, we have shown the relationship of HEV and their transformation in a cancerous environment. We also note that in previous studies the HEV morphology did not alter at all in endotoxin-induced lymphadenopathy, implying a selective reaction of HEV in the cancerous condition\\n[5]. Distinct, differentiated gene expression has also been reported when the endothelial cells respond to the changing of their microenvironment\\n[52]. In a previous study, it was shown that the cellular morphology of the tall endothelial cells forming HEV changed dramatically to become flat endothelial cells in the presence of cancer. As a consequence, the HEV was remodelled from a thick-walled, endothelial vessel with a small lumen to a thin walled, large-lumen vessel shifting its function from recruitment of lymphocytes to becoming a blood vessel (Figure\\n4)\\n[10]. These facts indicate that the blood vessel endothelium has tremendous potential to adapt its environment. The confined lymph and blood channel alterations within the SLN, but not in the next station lymph node, imply that an inducer from the primary tumor is functioning locally with the existence of an active primary cancer. VEGF-A has been found to be an inducer of lymphangiogenesis in SLN\\n[33]. Other studies have shown that serum level of VEGF-A was elevated in patients with late stage NPC\\n[21] VEGF-A is a secreted protein factor that can travel in blood to other lymph nodes, these findings suggest that there may be other inducers involved. It is also of interest to explore the role of HEV after the establishment of a metastatic tumor nest. The enlarged, remodelled HEV could integrate into the metastatic tumor vasculature with further differentiation, characterized by the gradual loss of their specific marker MECA-79 from the tumor margin to the central part of the metastatic tumor nest.\\n[5,10] It has been explained that, compared with primary tumors, the more rapid growth of metastatic lesions in the cervical lymph nodes of NPC patients was due to clonal selection of the cancer cells during metastases, with highly proliferative clones disseminated to the cervical nodes. However, based on our findings, the metastatic tumor vasculature in lymph nodes consists of many large blood vessels derived from normal HEVs, suggesting that the efficiency of nutrition and oxygen supplies could be better for the metastatic tumor cells in the involved lymph node. The enrichment of the blood supply in the lymph node before and after metastasis may favor the growth of newly arriving metastatic cancer cells. Consequently, as evident clinically in the long term follow-up of cancer patients, the involved regional lymph nodes may become manifest first, whereas the primary tumor remain clinically occult for years\\n[53]. Moreover, the high density of functioning blood vessels in lymph nodes may subsequently facilitate the metastasis of cancer cell to distant organs. Conversely, consistent with its role in lymphocyte trafficking, if the HEV density is increased in the tumor themselves as opposed to LN, it has favorable prognostic value, Martinet et al. reported that in 146 breast cancers, the density of tumor HEV was an independent prognostic risk factor for DFI and OS\\n[8]. This further suggests the tumor HEV function as major gateway for lymphocyte infiltration into tumors and represents potential targets for cancer immunotherapy. It will also be important to elucidate if the highly vascularized premetastatic SLN is associated with an increased metastatic potential. Control of lymphatic fluid movement may also be a target to consider in preventing metastases.\\nAssuming our hypothesis is true with regards to the role of HEV in cancer metastasis, a potential therapeutic approach is to interrupt or block this remodelling process and evaluate if there is an effect on metastasis. This will confirm the HEV’s central role in the pathogenesis of metastasis.\\nFurther studies need to designed and performed to elucidate the pathways of HEV transformation. A control gene for the growth and differentiation of HEV remains to be identified\\n[9]. With regards to this, a recently identified nuclear factor (NF), NF-HEV has potential because it is preferentially expressed by HEV\\n[54]. Dendritic cells, well known for their antigen presentation roles has recently been shown to modulate the phenotype of HEV and control entry of naive lymphocytes to LN, their role in a cancer microenvironment is also an area of potential research\\n[13]. There are recent experiments in mouse models investigating conditional gene targeting of HEV as well\\n[55]. Identification and characterization the molecular pathways and the control genes would have considerable clinical applications. For example, it would enable the design of experiments and studies to induce or retard the formation of HEV in various tissues, including tumors and their LN, improving vaccination strategies against pathogens and cancers. Therefore, there is a wealth of translational applications with regards to targeting HEV as a ligand for cancer investigational therapeutics. It paves the journey in the discovery of many unknown genes and molecules with potential clinical importance. Lastly, more work can be performed to validate our findings in other solid cancers.\\n Limitations Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\\nOur study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\",\n \"Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\",\n \"This study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets.\",\n \"HEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.\",\n \"The authors declare that they have no competing interests, financial or non-financial.\",\n \"LSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,"methods",null,null,null,null,"results",null,null,null,"discussion",null,"conclusions",null,null,null],"string":"[\n null,\n null,\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n null,\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["High endothelial venules","Cancer metastasis","Angiogenesis","Lymph nodes"],"string":"[\n \"High endothelial venules\",\n \"Cancer metastasis\",\n \"Angiogenesis\",\n \"Lymph nodes\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nCancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\n\nHigh endothelial venules and its role in cancer metastasis:\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n\nObjectives of study:\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\n\nMethods:\nApproval was obtained from the Institutional Review Boards of both institutions involved in the study (2007/464/B). The study population consisted of 175 consecutive patients with SCC of the head and neck who had received primary surgical treatment at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Center, Singapore, from January 2001 through to December 2005. The patients’ pathological and clinical data including follow-up information was reviewed from a prospective database. The inclusion criteria included all surgically treated patients with histologically proven SCC tongue who underwent a neck dissection, with a minimum of 2-year follow up period. Patients with a second primary cancer and patients who did not have a neck dissection as part of their primary treament were excluded. There were a total 65 patients that met the study inclusion criteria. There were 35 patients in the group with primary SCC tongue without pathologically proven lymph node metastases in their neck dissection specimen who were designated as “cases”, and 30 patients in the group with primary SCC tongue with pathologically proven lymph node metastases in the neck dissection specimens, who were designated as “controls”. All patients included had radical excision of the primary tongue lesion and a neck dissection according to departmental protocol. In both groups, all patients had radical enbloc excision of the primary tongue lesion with or without resection of adjacent structures with either a unilateral or a bilateral, supraomohyoid neck dissection or a modified radical neck dissection. Tumours were classified according to the AJCC TNM Staging Classification, based on a pre-operative clinical evaluation and this determined the type of neck dissection performed by the primary surgeon\n[19]. All cases were discussed at a multi-discplinary head and neck tumor board. Intra-operative frozen sections were performed for margins of the tongue lesion and further resection of the tongue was performed in event that the surgical margins were involved. Upon discharge from the hospital, they were followed up at regular intervals of monthly, 3-monthly, 6-monthly and yearly at a progessive rate. Inpatient and outpatient clincal data, pathological, radiological and operative records were retrieved from a central medical records office and from the Department of Pathology, Singapore General Hospital. Mortality data was obtained and confirmed from the hospital central medical records office as well as the Singapore Registry of Births & Deaths.\n Immunohistochemistry A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\nA histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\n Computer assisted image analysis The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\nThe digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\n Definitions of the HEV’s parameters and ratios This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\nThis is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\n Statistical analysis Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\nCorrelating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\n\nImmunohistochemistry:\nA histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\n\nComputer assisted image analysis:\nThe digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\n\nDefinitions of the HEV’s parameters and ratios:\nThis is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\n\nStatistical analysis:\nCorrelating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\n\nResults:\nThe association of HEV and clinical outcome was studied by initially analyzing the OS and DFI of the 2 groups prior to individually analyzing the various HEV parameters (A, B, C, B/A, C/B, C/A) against OS and DFI. The details of the preliminary results comparing HEV parameters and patient outcomes have been previously published in an earlier paper\n[20]. In the secondary analysis, we further analyzed the tumor pathological characteristics (i.e. primary tumor volume, the stage of the disease and grade of the tumor) against clinical outcome (OS and DFI), to determine if these conventional tumor factors were prognostic in nature. We further investigated the role of HEV and its prognostic value by comparing and analyzing the HEV parameters with respect to the tumor volume, grade and stage. These analyses were repeated without controlling for the group factor (i.e. taking the sample population as a single cohort). Finally, we investigated the HEV parameters in each group to determine if there was a difference in the HEV values in the presence or absence of metastasis in the LN.\nWe analyzed the DFI and OS of the 65 patients with respect to their groups (35 cases versus 30 controls) to estimate the relative risk of DFI and OS associated with the group.\n Overall survival analysis The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\nThe relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\n Disease free interval analysis Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\nSimilarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\n Secondary analysis Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups\nTumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups\n\nOverall survival analysis:\nThe relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\n\nDisease free interval analysis:\nSimilarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\n\nSecondary analysis:\nTumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups\n\nDiscussion:\nSquamous cell carcinoma of the tongue has one of the fastest rising rate today affecting the young with a high propensity for LN metastases.\n[22] The presence of metastatic cells in regional LN, along with extracapsular spread are the most important prognostic factors in patients with squamous cell carcinoma (SCC) of the tongue\n[22-25]. The prognosis is worse compared to other equivalent carcinomas of the head and neck region.\nTumor cell metastasis to regional LN marks one of the initial steps in the cascade of tumor metastasis after tumor cell progression in the primary tumor. The lymphatic metastatic cascade is a series of complex interrelated steps and processes\n[26]. As the tumor grows and enlarges, cytokines are secreted to promote lymphangiogenesis\n[27]. As malignant cells invade the extracellular matrix, they enter the lumen of the lymphatics and they move in clusters or as single cells to the first echelon of regional LN, otherwise known as the SLN\n[28,29]. The structural properties, such as the lack of basement membrane in terminal lymphatics, the presence of the intercellular clefts, the traumatic environment in the blood including shear forces of blood turbulence and tumor antigenicity detection by host immune cells, contribute and account for the observation that in many solid cancers, lymphatic metastasis precedes metastasis via the vascular system\n[26,30]. The relationship between angiogenesis and lymphangiogenesis has been extensively studied in cancer research\n[14,26,31,32]. This is pertinent in the biology of LN metastasis where the two systems literally lie side by side. There is extensive evidence showing members of the Vascular endothelial growth factor (VEGF) family, namely VEGF-C, VEGF-D and VEGF-A\n[33], are not only important regulators of lymph vessel growth in vivo, but may also be involved in the process of promoting lymphatic metastasis, such as VEGF-C\n[27,34-36]. This is significant because there is evidence that tumor can activate both LN lymphangiogenesis and angiogenesis before metastasis, and this would translate into logical efforts in the field of anti-cancer research specifically targeting pathways of tumor lymphangiogenesis and angiogenesis. There have been encouraging results from therapeutic targeting of the various VEGF member pathways to inhibit lymph node metastasis, reduce lymphatic and vascular density in LN as well as to reduce overall tumor burden in the lymph nodes and distant metastases\n[34,37,38].\nTargeted therapy such as anti-angiogenesis drugs has shown positive results in clinical studies. Bevacizumab (AvastinTM), a monoclonal antibody to VEGF-A, was approved by United States Food and Drug Administration (FDA) in 2004 and has proven clinical benefits in treatment of metastatic colorectal cancer when the drug was added to standard chemotherapy and also has positive results in metastatic kidney cancer and advanced lung cancer. There are many ongoing trials including Phase III clinical trials in advanced or metastatic renal cell carcinoma, pancreatic cancer, and ovarian cancer for AvastinTM[39]. Other molecular pathways that harbor potential targets which have varying degrees of anti-angiogenesis and anti-lymphangiogenesis properties, are ligands such as hepatocyte growth factor (HGF), receptors like Neuropilin 2, PDGFRα/β and others such as Angiostatin, interferon α/β and platelet-factor 4\n[40]. Two other antiangiogenic drugs, Sorafenib (Nexavar™, Bayer) and Sunitinib (Sutent™, Pfizer), have also been approved by the FDA for various aspects of cancer treatment, for example, Sorafenib and Sunitinib have been beneficial in the treatment of metastatic renal-cell cancer and advanced hepatocellular carcinoma\n[41,42]. They target multiple receptor tyrosine kinases, including VEGF receptors and platelet-derived growth factor (PDGF) receptors\n[43].\nIn our series, we detected a statistical significance when we analyzed the OS but not in DFI in the patients with or without LN metastasis (cases vs. controls) (Figures\n2 and\n3). This may be attributed to our limited sample size. It has been proposed and is currently being validated, that SLN biopsy may play the next stage in the evolution in the neck management and treatment of tongue SCC\n[44]. Exploration and understanding of this concept coupled with advances and optimistic results in anti-angiogenesis therapy involving VEGF anti-metabolites translates the next logical exploration to be invested in the study of the pathogenesis of lymph node metastasis. It was shown that the lumen of the lymphatic sinuses and blood vessels were dilated in SLN before metastasis\n[5]. We have demonstrated that lymph nodes are transformed by the primary tongue tumor to become a functional blood vessel–enriched organ before and independent of metastasis, with the changes in the morphology of the HEV to become main blood flow carrier in the lymph node (Figure\n4). This process of vascularization in the LN (HEV morphological alterations) appears similar and consistent in human tissues and previous animal models\n[5,20]. This study presents functional and structural data that the primary tumor can possibly manipulate the microenvironment and biology of lymph nodes to potentially facilitate the proliferation of subsequent tumor deposits leading to established metastases. The morphological alteration of HEV in the presence of a cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shift and phenotypic change of HEV from its known function as a lymphocyte recruitment modulator to one as a blood carrier.\nDilated HEVs with red blood cells in its lumen (high power field).\nIn previous studies, the degree of lymphatic dilation in the SLN significantly correlated with the primary tumor weight\n[5,10]. This observation suggests that the lymphatic fluid from the primary tumor induced the persistent alteration of the lymph channel in SLN. These results are consistent with findings that demonstrate, in contrast to angiogenesis, in which blood flow proceeds only after the vessel develops, lymphangiogenesis can be induced by interstitial fluid channeling\n[45]. In this aspect, this is analogous to immunology studies showing the alteration of lymph channels in SLN can also occur during an inflammatory reaction, which facilitates the migration of inflammatory cells\n[46,47] The difference in cancer metastasis biology and inflammatory process starts here. In endotoxin induced experiments and immunology studies, the dilated lymphatic sinuses and vessels in the associated reactive LN were full of lymphocytes. In contrast in tumor-reactive lymphadenopathy, the lymphatics contained minimum or no cellular material, suggesting different roles of the SLN lymphatic channels in different pathologic processes. It has been well characterized that the HEV of lymph nodes play an important role in recruiting lymphocytes for the generation of immune responses\n[9,11]. By expressing homing receptors on their surface, which blood lymphocytes can recognize as they pass in circulation, HEV provide a unique location where naive lymphocytes can enter the lymph node\n[47,48].\nIn our previous study\n[5], we found that the role of HEV was transformed from a lymphocyte recruiter to become the main blood flow carrier in the SLN prior to metastasis. We have shown in this study that in the regional cervical LN, not only could the morphology of individual HEV change dramatically to increase blood flow, but the proliferation rate of HEV endothelial cells was also increased before metastasis. This transformation of HEV as a lymphocytic carrier in LN to a blood-flow carrier can be seen in the large quantity of red blood cells visible in the HEV even in pre-metastatic regional LN. More significantly, this phenomenon is also more pronounced in the regional LN of the patients with established LN metastasis. This is consistent with the findings of Chung and colleagues demonstrating the increased HEV density in SLN of oral SCC before the arrival of tumor cells, the HEV density in metastatic SLN was also higher than that of non-metastatic SLN\n[6]. We have shown this in our supplementary data that patients with lymph node metastases have more dilated HEV with RBC in their LN as compared to patients without LN metastasis. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV(C/B) in the control (pN+) group is statistically significantly higher than that in case group (p-value = 0.0318).\nIn addition, with the benefit of patients’ pathological and follow-up information, we have demonstrated that even with just an increase of 1 HEV in a high-power-field (HPF), regardless of the group, the risk is 1.024 times worse in terms of overall survival. This is consistent with clinical knowledge and natural history of the disease. It is well established that patients without LN metastases in their regional LN (cases in our cohort) have a significantly better prognosis, and; this fact is also reflected in our analysis\n[22,49-51] (Figure\n2). Cases had a 0.428 times the risk of Controls with regards to overall survival if they had the same number of HEV. It is crucial to understand that this risk is exponential in nature. To illustrate the significance of this result, an example will be used: Patient A with tongue SCC has proven LN metastases in the cervical LN. On further immunohistochemical staining, it is found that he has 0 HEV in a random high-power-field. Comparing this to patient B who has the same grade, stage of tumor, degree of LN metastases as patient A but has 100 HEV in a HPF, patient B will have a [ (1.024)100 = 10.715 ] 10 times worse prognosis in OS as compared to patient A. We will then consider patient C who has tongue SCC with no LN metastasis. If the HEV amount in a HPF is the same as either patient A or B, patient C’s risk as compared to either patient A or B will be 2.34 times (1/0.428= 2.34) better in terms of overall survival (Table\n4). Although this did not reach statistical significance (p=0.471), we believe this to be a factor of a small sample size.\nDifferences in OS and DFI relative risk in the 2 groups\nAssuming that our hypothesis is true, HEV transformation from a normal immunological mediator to a tumor metastasis mediator is reflected by morphological changes from a normal appearing HEV, to a dilated HEV, and then to a dilated HEV containing RBC. We believe that this metamorphosis occurs on a spectrum, and this process begins with the HEV increasing in absolute numbers, then each becoming more dilated before progressing into a functional vessel carrying blood (Figure\n5). Hence we analyzed our data looking at the 3 different stages of HEV transformation and the ratio comparing each parameter in a systematic way to demonstrate this spectrum (Figure\n1).\nMetamorphosis of High endothelial venules in a tumor microenvironment. This process begins with the high endothelial venules increasing in absolute numbers, subsequently each of them becoming more dilated and lastly every one of them will become a function vessel carrying blood.\nWe recall that that in the last example we illustrated that a patient’s OS risk is 1.024 times worse than an equivalent patient if there is 1 more HEV in one HPF of his regional LN, the risk is increased to 1.071 if the HEV is a dilated HEV. The risk is even higher at 1.116 times if the HEV is a dilated HEV containing RBC (Table\n1)\n[20]. This trend is also seen when all patients are considered in a single cohort. A patient’s OS risk is worsened by 1.004 times if there is an increase of 1 dilated HEV in a HPF. If that dilated HEV contains RBC, the OS risk is increased to 1.008 times.\nIn the treatment of tongue carcinomas, DFI is important as it signifies the failure of loco-regional control for which there is little effective therapy. Further surgery e.g. neck dissection to remove loco-regional recurrences is plagued with high morbidity and mortality. This, coupled with poor chemotherapy and radiotherapy response rates in recurrences, often equates to rapid deterioration of the patient’s condition. Disease free interval was also analyzed in the same manner to the three different HEV morphological phenotypes.\nWe found statistical significance in the relationship between the total number of HEV (A) and DFI when we controlled for the group (i.e. taking in the presence of LN metastasis as a factor). (p=0.022). This significance is preserved when we analyzed all the patients as a cohort (p=0.023)\n[20]. This means that the quantity of HEV is inversely related to DFI. If you consider the LN status and take it into statistical consideration, if there is one more HEV in a HPF, the DFI is 1.051 times worse than zero HEV. It is again important to note that the relationship of the quantity of HEV with respect to DFI is exponential in nature. For example, a patient (patient B) with 100 more HEV (A) in one HPF has a {(1.051)100 =144.634} 144 times shorter DFI as compared to an equivalent patient with only one HEV per HPF (patient A) (Table\n4). Excluding the group effect and considering all the patients as a cohort, a patient with 1 more HEV per HPF will have a DFI that is 1.051 times worse than a patient with zero HEV in a HPF (p=0.023).\nWe looked at the different ratios of abnormal HEV and compared them to several clinic-pathologic parameters namely overall survival, disease–free interval, tumor volume, stage and grade of the tumor. The details are specified in the secondary results (Table\n2).\nThere is a general trend observed. The more advanced the disease, the higher the ratio/percentage of abnormality of HEV. This can be seen in when we analyzed OS and the 2 ratios. The OS relative risk worsens by 1.078 if the ratio B/A (ratio of dilated HEV to the total number of HEV) increased by 1, while the OS relative risk worsens by 3.624 times if the ratio of dilated HEVs with RBC to the total number of HEV increases by 1. Most importantly, if we consider the most abnormal form of HEV (dilated HEV with RBC within its lumen, C) and look at it as a ratio to the total no. of HEV, a patient’s OS relative risk worsens by 17.884 times if this ratio (C/A) increases by a factor of 1. This observation approaches marginal significance. (p = 0.171) (Figure\n6).\nOverall survival relative risk with respect to the different high endothelial venules ratios.\nIn this study, we have shown the relationship of HEV and their transformation in a cancerous environment. We also note that in previous studies the HEV morphology did not alter at all in endotoxin-induced lymphadenopathy, implying a selective reaction of HEV in the cancerous condition\n[5]. Distinct, differentiated gene expression has also been reported when the endothelial cells respond to the changing of their microenvironment\n[52]. In a previous study, it was shown that the cellular morphology of the tall endothelial cells forming HEV changed dramatically to become flat endothelial cells in the presence of cancer. As a consequence, the HEV was remodelled from a thick-walled, endothelial vessel with a small lumen to a thin walled, large-lumen vessel shifting its function from recruitment of lymphocytes to becoming a blood vessel (Figure\n4)\n[10]. These facts indicate that the blood vessel endothelium has tremendous potential to adapt its environment. The confined lymph and blood channel alterations within the SLN, but not in the next station lymph node, imply that an inducer from the primary tumor is functioning locally with the existence of an active primary cancer. VEGF-A has been found to be an inducer of lymphangiogenesis in SLN\n[33]. Other studies have shown that serum level of VEGF-A was elevated in patients with late stage NPC\n[21] VEGF-A is a secreted protein factor that can travel in blood to other lymph nodes, these findings suggest that there may be other inducers involved. It is also of interest to explore the role of HEV after the establishment of a metastatic tumor nest. The enlarged, remodelled HEV could integrate into the metastatic tumor vasculature with further differentiation, characterized by the gradual loss of their specific marker MECA-79 from the tumor margin to the central part of the metastatic tumor nest.\n[5,10] It has been explained that, compared with primary tumors, the more rapid growth of metastatic lesions in the cervical lymph nodes of NPC patients was due to clonal selection of the cancer cells during metastases, with highly proliferative clones disseminated to the cervical nodes. However, based on our findings, the metastatic tumor vasculature in lymph nodes consists of many large blood vessels derived from normal HEVs, suggesting that the efficiency of nutrition and oxygen supplies could be better for the metastatic tumor cells in the involved lymph node. The enrichment of the blood supply in the lymph node before and after metastasis may favor the growth of newly arriving metastatic cancer cells. Consequently, as evident clinically in the long term follow-up of cancer patients, the involved regional lymph nodes may become manifest first, whereas the primary tumor remain clinically occult for years\n[53]. Moreover, the high density of functioning blood vessels in lymph nodes may subsequently facilitate the metastasis of cancer cell to distant organs. Conversely, consistent with its role in lymphocyte trafficking, if the HEV density is increased in the tumor themselves as opposed to LN, it has favorable prognostic value, Martinet et al. reported that in 146 breast cancers, the density of tumor HEV was an independent prognostic risk factor for DFI and OS\n[8]. This further suggests the tumor HEV function as major gateway for lymphocyte infiltration into tumors and represents potential targets for cancer immunotherapy. It will also be important to elucidate if the highly vascularized premetastatic SLN is associated with an increased metastatic potential. Control of lymphatic fluid movement may also be a target to consider in preventing metastases.\nAssuming our hypothesis is true with regards to the role of HEV in cancer metastasis, a potential therapeutic approach is to interrupt or block this remodelling process and evaluate if there is an effect on metastasis. This will confirm the HEV’s central role in the pathogenesis of metastasis.\nFurther studies need to designed and performed to elucidate the pathways of HEV transformation. A control gene for the growth and differentiation of HEV remains to be identified\n[9]. With regards to this, a recently identified nuclear factor (NF), NF-HEV has potential because it is preferentially expressed by HEV\n[54]. Dendritic cells, well known for their antigen presentation roles has recently been shown to modulate the phenotype of HEV and control entry of naive lymphocytes to LN, their role in a cancer microenvironment is also an area of potential research\n[13]. There are recent experiments in mouse models investigating conditional gene targeting of HEV as well\n[55]. Identification and characterization the molecular pathways and the control genes would have considerable clinical applications. For example, it would enable the design of experiments and studies to induce or retard the formation of HEV in various tissues, including tumors and their LN, improving vaccination strategies against pathogens and cancers. Therefore, there is a wealth of translational applications with regards to targeting HEV as a ligand for cancer investigational therapeutics. It paves the journey in the discovery of many unknown genes and molecules with potential clinical importance. Lastly, more work can be performed to validate our findings in other solid cancers.\n Limitations Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\nOur study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\n\nLimitations:\nOur study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\n\nConclusion:\nThis study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets.\n\nAbbreviations:\nHEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.\n\nCompeting interests:\nThe authors declare that they have no competing interests, financial or non-financial.\n\nAuthors’ contribution:\nLSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nHigh endothelial venules (HEV) have been recognized to play a role in metastasis by its changes seen in the cancer microenvironment of lymph nodes (LN) and solid cancers. Squamous cell carcinoma (SCC) of the tongue is a prevalent tumor of the head and neck region with high propensity for LN metastasis. The extent of LN metastasis is the most reliable adverse prognostic factor. Primary tumors can induce vasculature reorganization within sentinel LN before the arrival of tumor cells and HEV represents these remodelled vessels. This study aims to evaluate the cancer induced vascular changes in regional lymph nodes (LN) of patients by studying the morphological and functional alterations of HEV and its correlation with clinical outcome and pathological features.\n\nMethods:\nThis study was based on 65 patients with SCC tongue who underwent primary surgical treatment including neck dissection. The patients were categorized into 2 groups based on the presence of malignancy in their cervical lymph nodes. A review of the patients' pathological and clinical data was performed from a prospective database. Immunohistochemical staining of the tissue blocks for HEV and high-power-field image analysis were performed and analyzed with correlation to the patients' clinical and pathological features.\n\nResults:\nThe total number of HEV was found to be significantly associated to disease-free interval. There was a similar association comparing the HEV parameters to overall survival. The density of abnormal HEV was significantly higher in patients with established metastases in their lymph nodes and HEV was shown to be a better prognosis factor than conventional tumor staging. The HEV morphological metamorphosis demonstrates a spectrum that correlates well with disease progression and clinical outcome.\n\nConclusions:\nThe results suggest that the HEV displays a spectrum of morphological changes in the presence of cancer and LN metastasis, and that HEV is possibly involved in the process of cancer metastasis. We revealed the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environments in regional lymph nodes of tongue cancer patients in relation to clinical outcomes. The significant observation of modified dilated HEV containing red blood cells in lymph nodal basin of a cancer suggests the shifting of its function from one primarily of immune response to that of a blood carrying vessel. It also demonstrated potential prognostic value. More studies are needed to elucidate its potential role in cancer immunotherapy and as a potential novel therapeutic approach to preventing metastasis by manipulating the remodelling processes of HEV."},"background_conclusion_text":{"kind":"string","value":"Background:\nCancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\n\nConclusion:\nThis study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nHigh endothelial venules (HEV) have been recognized to play a role in metastasis by its changes seen in the cancer microenvironment of lymph nodes (LN) and solid cancers. Squamous cell carcinoma (SCC) of the tongue is a prevalent tumor of the head and neck region with high propensity for LN metastasis. The extent of LN metastasis is the most reliable adverse prognostic factor. Primary tumors can induce vasculature reorganization within sentinel LN before the arrival of tumor cells and HEV represents these remodelled vessels. This study aims to evaluate the cancer induced vascular changes in regional lymph nodes (LN) of patients by studying the morphological and functional alterations of HEV and its correlation with clinical outcome and pathological features.\n\nMethods:\nThis study was based on 65 patients with SCC tongue who underwent primary surgical treatment including neck dissection. The patients were categorized into 2 groups based on the presence of malignancy in their cervical lymph nodes. A review of the patients' pathological and clinical data was performed from a prospective database. Immunohistochemical staining of the tissue blocks for HEV and high-power-field image analysis were performed and analyzed with correlation to the patients' clinical and pathological features.\n\nResults:\nThe total number of HEV was found to be significantly associated to disease-free interval. There was a similar association comparing the HEV parameters to overall survival. The density of abnormal HEV was significantly higher in patients with established metastases in their lymph nodes and HEV was shown to be a better prognosis factor than conventional tumor staging. The HEV morphological metamorphosis demonstrates a spectrum that correlates well with disease progression and clinical outcome.\n\nConclusions:\nThe results suggest that the HEV displays a spectrum of morphological changes in the presence of cancer and LN metastasis, and that HEV is possibly involved in the process of cancer metastasis. We revealed the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environments in regional lymph nodes of tongue cancer patients in relation to clinical outcomes. The significant observation of modified dilated HEV containing red blood cells in lymph nodal basin of a cancer suggests the shifting of its function from one primarily of immune response to that of a blood carrying vessel. It also demonstrated potential prognostic value. More studies are needed to elucidate its potential role in cancer immunotherapy and as a potential novel therapeutic approach to preventing metastasis by manipulating the remodelling processes of HEV."},"whole_article_text_length":{"kind":"number","value":14568,"string":"14,568"},"whole_article_abstract_length":{"kind":"number","value":451,"string":"451"},"num_sections":{"kind":"number","value":18,"string":"18"},"most_frequent_words":{"kind":"list like","value":["hev","tumor","ln","cancer","patients","blood","study","lymph","cells","dilated"],"string":"[\n \"hev\",\n \"tumor\",\n \"ln\",\n \"cancer\",\n \"patients\",\n \"blood\",\n \"study\",\n \"lymph\",\n \"cells\",\n \"dilated\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | cancer | lymphatic | cells | tumor | blood | ln | route | role | vessels [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | neck | test | number | neck dissection | parameters | dissection | dilated hev | total number | number hev [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | hev | dfi | hev parameters | parameters | risk | grade | cases | stage | groups [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] shortcut | lymph | circulation | sln | flow | metastatic | hev | role elusive junction | role elusive junction providing | pre metastatic metastatic environment [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | parameters | cancer | dilated | lymph | dilated hev | patients | ln | cases [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hev | tumor | parameters | cancer | dilated | lymph | dilated hev | patients | ln | cases [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] LN ||| LN ||| LN ||| ||| LN | HEV [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 65 ||| 2 ||| ||| HEV [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] HEV ||| HEV ||| HEV | HEV ||| HEV [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] HEV | LN | HEV ||| HEV ||| ||| ||| HEV [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] LN ||| LN ||| LN ||| ||| LN | HEV ||| 65 ||| 2 ||| ||| HEV ||| ||| HEV ||| HEV ||| HEV | HEV ||| HEV ||| HEV | LN | HEV ||| HEV ||| ||| ||| HEV [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] LN ||| LN ||| LN ||| ||| LN | HEV ||| 65 ||| 2 ||| ||| HEV ||| ||| HEV ||| HEV ||| HEV | HEV ||| HEV ||| HEV | LN | HEV ||| HEV ||| ||| ||| HEV [SUMMARY]"}}},{"rowIdx":284,"cells":{"title":{"kind":"string","value":"Preoperative therapy restores ventilatory parameters and reduces length of stay in patients undergoing myocardial revascularization."},"pmid":{"kind":"string","value":"25140472"},"background_abstract":{"kind":"string","value":"The frequency of surgical procedures has increased steadily in recent decades, including the myocardial revascularization."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"We conducted a prospective study with patients undergoing myocardial revascularization, the Hospital das Clínicas da Universidade Estadual Paulista (UNESP)/Botucatu - SP. We evaluated 70 patients of both genders, aged between 40 and 75 years, subdivided into two groups: group I - 35 patients of both genders, who received a written protocol guidance, breathing exercises and respiratory muscle training in the preoperative period and group II - 35 patients of both genders, who received only orientation of the ward on the day of surgery. This study was approved by the Ethics Committee of UNESP / Botucatu - SP."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Maximal inspiratory pressure in third postoperative day and fifth postoperative day and significant difference between groups, being better for the intervention group. Expiratory pressure was significant in fifth postoperative day in the intervention group compared to controls. The difference of length of hospital stay in the postoperative was found between the groups with shorter hospital stay in the group receiving preoperative therapy."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Physical therapy plays an important role in the preoperative period, so that individuals in the intervention group more readily restored the parameters evaluated before surgery, in addition, there was a decrease in the time of the postoperative hospital stay. Thus, it is thought the cost-effectiveness of a program of preoperative physiotherapy."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Breathing Exercises","Female","Humans","Inspiratory Capacity","Length of Stay","Male","Middle Aged","Muscle Strength","Muscle Stretching Exercises","Myocardial Revascularization","Preoperative Care","Preoperative Period","Prospective Studies","Reference Values","Reproducibility of Results","Respiratory Muscles","Respiratory Rate","Statistics, Nonparametric","Tidal Volume","Time Factors","Treatment Outcome"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Breathing Exercises\",\n \"Female\",\n \"Humans\",\n \"Inspiratory Capacity\",\n \"Length of Stay\",\n \"Male\",\n \"Middle Aged\",\n \"Muscle Strength\",\n \"Muscle Stretching Exercises\",\n \"Myocardial Revascularization\",\n \"Preoperative Care\",\n \"Preoperative Period\",\n \"Prospective Studies\",\n \"Reference Values\",\n \"Reproducibility of Results\",\n \"Respiratory Muscles\",\n \"Respiratory Rate\",\n \"Statistics, Nonparametric\",\n \"Tidal Volume\",\n \"Time Factors\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"4389447"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"The cardiac surgery was always clothed with great interest, curiosity, and in some\nmoments, mysticism, and fact understandable when we analyzed the noble importance of\nthis component for the proper functioning of the human body[1].\nIn recent decades the frequency of surgical procedures has progressively increased,\namong them the myocardial revascularization (MR). Cardiovascular diseases are a serious\npublic health problem in Brazil[2].\nIn individuals over 70 years old it is estimated an incidence of 70% of coronary artery\ndisease. As the age is a determinant of coronary atherosclerosis, a growing number of\nelderly people will be subjected to MR and/or other therapeutic methods[3,4].\nThe main risk factors that contribute to cardiac diseases are: smoking, high levels of\nlow-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density\nlipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,\nfamily history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors\nrelated to atherosclerosis and its clinical manifestations[4,5].\nThe coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment\nof ischemic heart disease that is manifested clinically by angina and acute myocardial\ninfarction. The CABG surgery creates a new route for the blood flow and, is used for\nmaking a diversion of blood to the portions of the coronary artery distal to the\nobstructive atherosclerotic involvement[1,5].\nWith the advancements in care of physical therapy in the preoperative period, surgical\ntechniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,\nanesthesia and intensive care in the post-operative period, there was a decrease in\nmorbidity and mortality of MR which caused the surgical indication in groups of patients\nincreasingly complex[6,7].\nObserving the fact that they are common pulmonary complications of CABG with CPB was\njustified to carry out this research, there is great importance in properly carrying out\nevaluations, guidelines and breathing exercises in the period before surgery, and\naddition, observe the influence and duration of physical therapy assistance in the\npreoperative period and also its behavior in the postoperative period.\n Objective To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\nTo demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"We performed a prospective clinical study, simple blind, in the period from July 2009 to\nSeptember 2011 in the ward of cardiac and thoracic surgery at Hospital das Clínicas da\nUniversidade Estadual Paulista (UNESP), Botucatu-SP. We evaluated 70 patients of both\ngender, age ranging from 40 to 75 years, undergoing CABG with use of CPB, randomized by\nmeans of a computerized software (STATTREK) and delivered in a sealed envelope to the\nphysical therapist responsible for primary health care, subdividing into two groups:\n- Group I (GI) - Thirty-five patients, undergoing physiotherapeutic intervention,\nperformed under supervision, once a day, during the period that preceded the surgery.\nThe protocol consisted of breathing exercises (breathing in time, deep breathing\nfollowed by prolonged expiration, sustained maximal inspiration with apnea of 6 seconds,\nand diaphragmatic breathing associated with the mobilization of the upper limbs) and\nbreathing exercises with threshold - IMT® (Threshold - IMT®\nInspiratory Muscle Trainer, HealthscanProducts Inc.) at an intensity of 40% of the\ninitial MIP with three sets of ten repetitions, respecting two-minute intervals between\neach series.\n- Group II (GII) - thirty-five patients who did not undergo the physical therapy\nprotocol during the preoperative period, receiving only guidelines ward routine.\nWe included patients of both sexes, aged 40-75 years with coronary artery disease\nadmitted for cardiac surgery with cardiopulmonary bypass (CPB) MR, who agreed to\nparticipate in the study. We excluded patients who require the use of intra-aortic\nballoon, carrying pulmonary pathology, musculoskeletal impairment and severe\nneurological surgery performed without CPB, emergency surgery, homodynamic instability,\nand any event that threatens the integrity of patient or the fidelity of the measures\nanalyzed.\n Delimitation All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\nAll patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\n Procedures/treatment Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\nPatients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\n Statistical Analysis The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.\nThe sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"Seventy patients were divided into two groups, Group I composed of 35 patients (12\nfemale and 23 male) and Group II, with 35 patients (six female and 29 male), as\nillustrated in Figure 1.\nCharacterization of groups by gender\nNo significant difference was noticed between groups for the variables age (GI 58.9±9.53\nyears and GII 61.4±8.43 years; P=0.26) and body mass index (GI\n26.8±3.96 kg/m2 and G II 26±3.86 kg/m2) (Table 1).\nPatient characteristics regarding age and BMI.\nBMI: body mass index; Wilcoxon test. * P<0.05\nThe groups were homogenous concerning presence of active smoking, ex-smokers and\nnonsmokers, as illustrated in Table 2.\nNumber and proportion of patients in each group according to the presence of\nactive smoking, ex-smokers and nonsmokers.\nTwo proportions followed by the same lowercase letter do not differ in groups\n(rows). Two proportions followed by the same capital letter do not differ in\nthe types of smoking (P>0.05). Goldman test to compare proportions between\nand within populations multinomina.\nBetween the GI and GII, no significant difference regarding the proportion of\nex-smokers, smokers and nonsmokers, in other words, the groups were homogeneous as\ndemonstrated below (Table 2).\n Maximum Inspiratory Pressure (MIP) The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\nThe mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\n Maximum Expiratory Pressure (MEP) The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\nThe mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\n Minute Volume (MV) The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\nThe analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\n Tidal Volume Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\nFigure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\n Respiratory rate (RR) The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative\nThe median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative"},"conclusions_title":{"kind":"string","value":"CONCLUSIONS"},"conclusions_text":{"kind":"string","value":"The physical therapy has an important role in the preoperative period, restoring greater\nreadiness ventilatory parameters of patients undergoing coronary artery bypass grafting\nwith cardiopulmonary bypass, resulting in a decrease in length of hospital stay after\nsurgery."},"other_sections_titles":{"kind":"list like","value":["Objective","Delimitation","Procedures/treatment","Statistical Analysis","Maximum Inspiratory Pressure (MIP)","Maximum Expiratory Pressure (MEP)","Minute Volume (MV)","Tidal Volume","Respiratory rate (RR)","Limitation of the study"],"string":"[\n \"Objective\",\n \"Delimitation\",\n \"Procedures/treatment\",\n \"Statistical Analysis\",\n \"Maximum Inspiratory Pressure (MIP)\",\n \"Maximum Expiratory Pressure (MEP)\",\n \"Minute Volume (MV)\",\n \"Tidal Volume\",\n \"Respiratory rate (RR)\",\n \"Limitation of the study\"\n]"},"other_sections_texts":{"kind":"list like","value":["To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.","All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].","Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.","The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.","The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.","The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.","The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.","Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.","The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative","The major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately."],"string":"[\n \"To demonstrate the importance of the role of physical therapy in the preoperative\\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\\nsurgery.\",\n \"All patients were evaluated by the same evaluator (preoperative period - PRE), third\\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\\nphysiotherapeutical treatment, according to their needs in the postoperative period\\nby physical therapy team in the hospital and only Group I received physiotherapy in\\nthe preoperative period.\\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\\nanesthesia time and the time that the patient remained with the endotracheal tube in\\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\\nICU stay (when the patient arrived in the intensive care unit until discharged to the\\nward bed) and length of stay in the OP (obtained from the time that the patient\\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\\nwere obtained by means of the daily developments of medical and nursing staff.\\nThe assessment of respiratory muscle strength was obtained by measurement of\\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\\nforceps according to the technique described by Nephew et al. (2008). The MIP\\nmeasurement was performed from residual volume, and MEP was obtained from total lung\\ncapacity. Three measurements were taken technically satisfactory, and if there were\\nmore than 20% difference between the measurements, a fourth and even fifth\\nmeasurement would be performed, being always considered the highest value obtained.\\nThe MIP was performed for evaluation of inspiratory muscle strength in three\\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\\nthe buccinator muscles during MIP maneuver[9].\\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\\nminute (cpm) and obtained by direct observation of respiratory cycles during the\\nevaluation of the minute volume[7].\",\n \"Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\\nof breathing exercises (breathing in time, deep breath and then exhaling long,\\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\\nassociated with mobilization of the upper limb, with three series out of ten for each\\nbreathing exercise) and breathing exercises with the device Threshold -\\nIMT®. We conducted three sets of ten repetitions with an interval of\\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\\nThe GII patients received only guidelines ward routine before surgery, but\\npostoperatively, both groups performed physical therapy as needed by staff\\nphysiotherapy service.\",\n \"The sample should be made with at least 26 patients who were randomly divided into\\ntwo study groups (with physical therapy preoperatively and control patients who did\\nnot receive preoperative physiotherapy). We considered the level of significance of\\n5% and a test power in the order of 80% for a minimum difference in the order of\\ntwo.\\nThe comparison between the groups for age and body mass index (BMI) was performed by\\nWilcoxon test for independent samples. The smoking variable was analyzed using\\nGoldman to compare proportions within and among multinomial populations. The\\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\\nhospital stay were obtained in minutes and analyzed by Student's t test or the\\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\\ntests for comparison of the moments in each group and Mann Whitney test to compare\\ngroups at each time, adopting a significance level of 5% probability of rejecting\\nnull hypothesis.\\nThis study was approved by the Research Ethics Committee of Universidade Estadual\\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\\nprotocol 3223) and all patients signed an informed consent form.\",\n \"The mean values the for maximum inspiratory pressures evaluated in the preoperative\\nperiod and 3PO showed no significant difference between groups\\n(P=0.276 and 0.065; respectively), however, we observed greater\\ninspiratory muscle strength for GI in relation to GII in 5PO\\n(P=0.001) (Figure 2).\\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\\noperation of each group analyzed (with and without physical therapy\\npreoperatively). Friedman test for comparison of moments in each group and Mann\\nWhitney test to compare the groups at each time, *P<0.05.\",\n \"The mean value for the maximum expiratory pressure obtained in the preoperative\\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\\ncmH2O) for GII, not presenting significant difference between groups\\n(P=0.704). However, the values referring MEP measured in the 5PO\\nshowed significant difference between groups (P=0.001), as\\nillustrated in Figure 3.\\nMedian for maximum expiratory pressure (MEP) variable at different times of the\\noperation in the different groups. Friedman test for comparison of moments in\\neach group and Mann Whitney test to compare the groups at each time,\\n*P<0.05.\",\n \"The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\\nP=0.001). However, these values have not showed significative\\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\\nrespectively).\\nMedian minute volume (VM ) variable at different times of the operation of each\\ngroup analyzed. Friedman test for comparison of the moments in each group and\\nthe Mann Whitney test for comparison between groups at any time,\\n*P<0.05.\",\n \"Figure 5 illustrates the analysis of variable\\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\\nvalues were not significantly different for the 3PO and 5 PO\\n(P=0.059 and P=0.549; respectively).\\nMedian tidal volume variable at different times for the group treated with\\npreoperative physiotherapy and without physical therapy preoperatively.\\nFriedman test for comparison of moments in each group and Mann Whitney test to\\ncompare the groups at each time, *P<0.05.\",\n \"The median refers to the respiratory rate obtained preoperatively for GI and GII\\nshowed no significant difference (16 cpm and 18 cpm, respectively,\\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\\nshowed no significant difference between groups (P=0.090 and\\nP=0.886, respectively), as shown in Figure 6.\\nMedian variable respiratory rate at different times of the operation of each\\ngroup analyzed (with and without physical therapy preoperatively). Friedman\\ntest for comparison of moments in each group and Mann Whitney test to compare\\nthe groups at each time, *P<0.05.\\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\\nhospital stay after surgery.\\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\\nstay did not significantly differ between groups, however, significant difference was\\nobserved for the length of stay variable in the postoperative period\\n(P=0.001), as shown in Table\\n3.\\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\\n5PO.\\nValues are shown as mean ± standard deviation or median (minimum and maximum\\nvalues).\\nMann and Whitney\\nStudent's t test.\\nSignificant difference between groups (P <0.05). ICU: intensive care\\nunit; PO: postoperative\",\n \"The major limitation was the short period of time that patients received preoperative\\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\\nrelevance even in a short period, so the study may add knowledge of response to\\ntherapy performed during intraoperative CABG surgery.\\nNot performing spirometry can be mentioned as a limitation on the analysis of\\nventilation parameters, however, due to the conditions offered by our service, we\\ncould not perform this evaluation.\\nOn the other hand, the results showed that even with limited time monitoring in the\\npreoperative period and respiratory variables obtained only by respirometry and\\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\\npatients undergoing surgery CABG.\\nWe understand that the results may open up new avenues of future research related to\\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\\nfurther studies should be conducted using tools such as spirometry, to assess and\\nquantify the volume and lung capacity more accurately.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","Objective","METHODS","Delimitation","Procedures/treatment","Statistical Analysis","RESULTS","Maximum Inspiratory Pressure (MIP)","Maximum Expiratory Pressure (MEP)","Minute Volume (MV)","Tidal Volume","Respiratory rate (RR)","DISCUSSION","Limitation of the study","CONCLUSIONS"],"string":"[\n \"INTRODUCTION\",\n \"Objective\",\n \"METHODS\",\n \"Delimitation\",\n \"Procedures/treatment\",\n \"Statistical Analysis\",\n \"RESULTS\",\n \"Maximum Inspiratory Pressure (MIP)\",\n \"Maximum Expiratory Pressure (MEP)\",\n \"Minute Volume (MV)\",\n \"Tidal Volume\",\n \"Respiratory rate (RR)\",\n \"DISCUSSION\",\n \"Limitation of the study\",\n \"CONCLUSIONS\"\n]"},"all_sections_texts":{"kind":"list like","value":["The cardiac surgery was always clothed with great interest, curiosity, and in some\nmoments, mysticism, and fact understandable when we analyzed the noble importance of\nthis component for the proper functioning of the human body[1].\nIn recent decades the frequency of surgical procedures has progressively increased,\namong them the myocardial revascularization (MR). Cardiovascular diseases are a serious\npublic health problem in Brazil[2].\nIn individuals over 70 years old it is estimated an incidence of 70% of coronary artery\ndisease. As the age is a determinant of coronary atherosclerosis, a growing number of\nelderly people will be subjected to MR and/or other therapeutic methods[3,4].\nThe main risk factors that contribute to cardiac diseases are: smoking, high levels of\nlow-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density\nlipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,\nfamily history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors\nrelated to atherosclerosis and its clinical manifestations[4,5].\nThe coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment\nof ischemic heart disease that is manifested clinically by angina and acute myocardial\ninfarction. The CABG surgery creates a new route for the blood flow and, is used for\nmaking a diversion of blood to the portions of the coronary artery distal to the\nobstructive atherosclerotic involvement[1,5].\nWith the advancements in care of physical therapy in the preoperative period, surgical\ntechniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,\nanesthesia and intensive care in the post-operative period, there was a decrease in\nmorbidity and mortality of MR which caused the surgical indication in groups of patients\nincreasingly complex[6,7].\nObserving the fact that they are common pulmonary complications of CABG with CPB was\njustified to carry out this research, there is great importance in properly carrying out\nevaluations, guidelines and breathing exercises in the period before surgery, and\naddition, observe the influence and duration of physical therapy assistance in the\npreoperative period and also its behavior in the postoperative period.\n Objective To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\nTo demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.","To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.","We performed a prospective clinical study, simple blind, in the period from July 2009 to\nSeptember 2011 in the ward of cardiac and thoracic surgery at Hospital das Clínicas da\nUniversidade Estadual Paulista (UNESP), Botucatu-SP. We evaluated 70 patients of both\ngender, age ranging from 40 to 75 years, undergoing CABG with use of CPB, randomized by\nmeans of a computerized software (STATTREK) and delivered in a sealed envelope to the\nphysical therapist responsible for primary health care, subdividing into two groups:\n- Group I (GI) - Thirty-five patients, undergoing physiotherapeutic intervention,\nperformed under supervision, once a day, during the period that preceded the surgery.\nThe protocol consisted of breathing exercises (breathing in time, deep breathing\nfollowed by prolonged expiration, sustained maximal inspiration with apnea of 6 seconds,\nand diaphragmatic breathing associated with the mobilization of the upper limbs) and\nbreathing exercises with threshold - IMT® (Threshold - IMT®\nInspiratory Muscle Trainer, HealthscanProducts Inc.) at an intensity of 40% of the\ninitial MIP with three sets of ten repetitions, respecting two-minute intervals between\neach series.\n- Group II (GII) - thirty-five patients who did not undergo the physical therapy\nprotocol during the preoperative period, receiving only guidelines ward routine.\nWe included patients of both sexes, aged 40-75 years with coronary artery disease\nadmitted for cardiac surgery with cardiopulmonary bypass (CPB) MR, who agreed to\nparticipate in the study. We excluded patients who require the use of intra-aortic\nballoon, carrying pulmonary pathology, musculoskeletal impairment and severe\nneurological surgery performed without CPB, emergency surgery, homodynamic instability,\nand any event that threatens the integrity of patient or the fidelity of the measures\nanalyzed.\n Delimitation All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\nAll patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\n Procedures/treatment Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\nPatients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\n Statistical Analysis The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.\nThe sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.","All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].","Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.","The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.","Seventy patients were divided into two groups, Group I composed of 35 patients (12\nfemale and 23 male) and Group II, with 35 patients (six female and 29 male), as\nillustrated in Figure 1.\nCharacterization of groups by gender\nNo significant difference was noticed between groups for the variables age (GI 58.9±9.53\nyears and GII 61.4±8.43 years; P=0.26) and body mass index (GI\n26.8±3.96 kg/m2 and G II 26±3.86 kg/m2) (Table 1).\nPatient characteristics regarding age and BMI.\nBMI: body mass index; Wilcoxon test. * P<0.05\nThe groups were homogenous concerning presence of active smoking, ex-smokers and\nnonsmokers, as illustrated in Table 2.\nNumber and proportion of patients in each group according to the presence of\nactive smoking, ex-smokers and nonsmokers.\nTwo proportions followed by the same lowercase letter do not differ in groups\n(rows). Two proportions followed by the same capital letter do not differ in\nthe types of smoking (P>0.05). Goldman test to compare proportions between\nand within populations multinomina.\nBetween the GI and GII, no significant difference regarding the proportion of\nex-smokers, smokers and nonsmokers, in other words, the groups were homogeneous as\ndemonstrated below (Table 2).\n Maximum Inspiratory Pressure (MIP) The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\nThe mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\n Maximum Expiratory Pressure (MEP) The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\nThe mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\n Minute Volume (MV) The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\nThe analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\n Tidal Volume Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\nFigure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\n Respiratory rate (RR) The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative\nThe median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative","The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.","The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.","The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.","Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.","The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative","Studies by Feltrim et al.[10] show\nthat performing preoperative physiotherapy is more effective in reducing respiratory\ncomplications in patients with moderate or higher risk than those whose risk was\nlow.\nIn the present study, we observed no significant difference between the subjects of\nGroup I and Group II as the characteristics (age, BMI, smoking and number of co\nmorbidities), which suggests that the study subjects in both groups were\nhomogeneous.\nIn the study by Leguisamo et al.[9],\nwith 86 patients undergoing CABG, divided into two groups: the intervention group (44\npatients) who were assessed and received physiotherapeutic guidance at least 15 days\nbefore surgery. The control group received routine care on the day of\nhospitalization.\nThe average MIP between groups showed neither significant difference in the preoperative\nperiod nor in the 1PO and 6PO.\nIn the present study it was observed that there was no significant difference between\ngroups to measure MIP in the preoperative period, but now in times of 3PO and 5PO\nsignificant difference between groups was found. Similarly, no significant difference in\nthe amount of MEP preoperatively in the 3PO and between groups. However in 5PO, there\nwas significant difference between the groups with the best values of the group that\nperformed physical therapy before surgery.\nBarros et al.[11] evaluated the MIP\nand MEP in the preoperative period, the first day after surgery and in 38 patients\nundergoing CABG with CPB who were divided into two groups: 23 patients in group I\n(respiratory muscle training) and 15 in group II (control). Group I received\nconventional physiotherapy over respiratory muscle training and Group II the\nconventional physiotherapy. The authors found that the values obtained for MIP and MEP\nin hospital discharge were higher in group I.\nThere was no decrease in maximal respiratory pressures, transdiaphragmatic pressure and\ndiaphragm pressure, indicating weakness of respiratory muscle strength[12,13].\nAgreeing with the authors cited above, we observed in the present study, after cardiac\nsurgery, MIP values in GI and GII which decreased in 3PO, however the value of MIP in\n5PO increase was observed for GI values compared to those in PRE and 3PO since the GII\nvalues remained stable in 3PO. The authors noted the drop in MEP in 3PO in both groups,\nhowever, in 5PO MEP has increased in GI. Regarding PRE and 3PO in GII no return value\nwas observed in PRE.\nArcêncio et al.[12] conducted a study\nwith 30 patients, aged 50 years-old and candidates for CABG and / or heart valve,\ndividing into two groups. Intervention group comprised 15 patients who underwent at\nleast a two weeks inspiratory muscle training using incentive spirometry (\"Threshold -\nIMT®) with 40% charge of the MIP and control group with 15 patients who\nreceived only general guidelines. The authors compared the spirometric values before and\nafter training and showed no significant difference in the values of MIP and\nMEP[14].\nElias et al.[15] studied 42 patients\naged 18 to 75 years-old with coronary artery disease, in the pre-and postoperative\nperiod, divided into two groups. Intervention group received TMR with Threshold -\nIMT® and control group received only guidelines. The TMR both pre and\npost-operative period consisted of three sets of 10 inspiratory exercises once a day for\nthree consecutive days. It was observed that after cardiac surgery there was a\nsignificant reduction in MIP and MEP in both groups.\nMorsch et al.[13] conducted a study to\nevaluate 108 patients undergoing elective CABG surgery from April 2006 to February 2007\n(preoperatively and postoperatively).\nThe average values of MIP in the preoperative period were significantly decreased\ncompared to 6 days after the surgery (P<0.001). The authors also\nobserved a significant decrease in the values of MEP obtained on the 6th\npostoperative day compared to the preoperative period (P<0.001).\nPatients with respiratory muscle weakness have higher risk of postoperative pulmonary\ncomplications. The respiratory muscle training in the pre-operative may prevent\npulmonary complications in patients undergoing thoracic surgery[16]. There may be a reduction of all lung\nvolumes resulting from factors such as diaphragmatic dysfunction, pain, absence of deep\nbreaths, pulmonary changes and ribcage. The functional residual capacity (FRC) decreases\nbecause of the reduction of both the residual volume (RV) and expiratory reserve volume.\nThe ventilation is affected by VC reduction by approximately 20%, and the increase in\nFR(22-24). The sum of these factors cause changes in the mechanics of\nrespiration, which leads to a shallow breathing pattern of decreased lung\nvolume[13].\nPatients undergoing CABG develop mostly PO pulmonary dysfunction with a significant\nreduction in lung volumes, impairments in respiratory function, decreased lung\ncompliance and increased work of breathing. The reduction in lung volumes and capacities\ncontributes to changes in gas exchange, resulting in hypoxemia[17].\nIn this study, the VM showed significant difference regarding the groups I and II in the\npreoperative period but not significant between groups in 3PO. Likewise, the mean value\nof the VM for the GI 5PO, there was no significant difference in the GII. The mean value\nof VC in PRE in GI was significant, as the mean value of the VC in GII patients, however\nin GI and GII we observed in 5PO and 3PO that there was no significant difference. It\nwas observed in GI at different times that the increase of VM will be very likely due to\nincreased FR, since the current VC remained without many changes between different\ntimes.\nBarros et al.[11] demonstrated a\nsignificant decrease in lung volumes in patients undergoing cardiac surgery in the\npostoperative period.\nCarvalho et al.[18] observed a\ndecrease in the values of minute volume and tidal volume measured on the 2nd\npostoperative day, however, these values showed gradual improvement returning to the\nvalues obtained at baseline (preoperative period) at the time of hospital discharge.\nIn a study, Guizilini et al.[19]\nevaluated 30 patients with a mean age of 56 years and divided into two groups, group A\n(n=15) without CPB and group B (n=15) with CPB. All patients underwent pulmonary\nfunction. In both groups, there was a decrease in FVC until the fifth postoperative day\n(P<0.05).\nRegarding the respiratory rate in the present study, we can verify that there was an\nincrease in both groups, with a higher peak on the 3rd postoperative day and\ndecreased on the 6th postoperative day, but the respiratory rate did not\nreturn to the preoperative values, Carvalho et al.[18] already mentioned above that they concluded in their studies\nthat the mean respiratory frequency had a significant increase between pre and\n2nd PO, 3rd PO, decreasing on the 4th postoperative\nday and increased again on the 5th postoperative day and decreased in\nhospital discharge, but patients did not return to the preoperative values.\nAccording to the results obtained in this study, we can affirm that there was no\nsignificant difference between the groups regarding the time variables: anesthesia,\nsurgery, perfusion, anoxia, mechanical ventilation, intubation. This suggests that the\ngroups were quite homogeneous. As for the time of postoperative hospital stay, there was\na decrease of approximately 25 hours, comparing patients who underwent physical therapy\nbefore surgery with patients who did not.\nIn contrast to the findings of this study, Patman et al.[20] performed a randomized study of 236 patients to\nevaluate the effects of physical therapy interventions performed early versus only\nstarted after extubation in patients undergoing cardiac surgery and no significant\ndifferences were found in the time of intubation, ICU stay and length of hospital stay\nbetween the groups.\nHowever, Celli et al.[21] conducted a\nstudy with 172 patients and showed a decrease in length of hospital stay in the group\nthat had received guidelines breathing exercises (9.6±3.2 days) compared to the control\ngroup (13±5 days).\nCorroborating these findings, as well as those obtained in the present study, Leguisamo\net al.[9] performed a prospective\nstudy to evaluate the effectiveness of a physiotherapy program start in the preoperative\nperiod in reducing the length of hospital stay and showed a significant reduction in\nlength of hospital stay for the intervention group compared to the control group\n(P<0.05).\nThese data suggest that physical therapy initiated before surgery can improve conditions\nof patients, reestablishing early respiratory pressures, reducing the length of stay and\ndecreased hospital costs for the period of hospitalization.\n Limitation of the study The major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.\nThe major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.","The major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.","The physical therapy has an important role in the preoperative period, restoring greater\nreadiness ventilatory parameters of patients undergoing coronary artery bypass grafting\nwith cardiopulmonary bypass, resulting in a decrease in length of hospital stay after\nsurgery."],"string":"[\n \"The cardiac surgery was always clothed with great interest, curiosity, and in some\\nmoments, mysticism, and fact understandable when we analyzed the noble importance of\\nthis component for the proper functioning of the human body[1].\\nIn recent decades the frequency of surgical procedures has progressively increased,\\namong them the myocardial revascularization (MR). Cardiovascular diseases are a serious\\npublic health problem in Brazil[2].\\nIn individuals over 70 years old it is estimated an incidence of 70% of coronary artery\\ndisease. As the age is a determinant of coronary atherosclerosis, a growing number of\\nelderly people will be subjected to MR and/or other therapeutic methods[3,4].\\nThe main risk factors that contribute to cardiac diseases are: smoking, high levels of\\nlow-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density\\nlipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,\\nfamily history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors\\nrelated to atherosclerosis and its clinical manifestations[4,5].\\nThe coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment\\nof ischemic heart disease that is manifested clinically by angina and acute myocardial\\ninfarction. The CABG surgery creates a new route for the blood flow and, is used for\\nmaking a diversion of blood to the portions of the coronary artery distal to the\\nobstructive atherosclerotic involvement[1,5].\\nWith the advancements in care of physical therapy in the preoperative period, surgical\\ntechniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,\\nanesthesia and intensive care in the post-operative period, there was a decrease in\\nmorbidity and mortality of MR which caused the surgical indication in groups of patients\\nincreasingly complex[6,7].\\nObserving the fact that they are common pulmonary complications of CABG with CPB was\\njustified to carry out this research, there is great importance in properly carrying out\\nevaluations, guidelines and breathing exercises in the period before surgery, and\\naddition, observe the influence and duration of physical therapy assistance in the\\npreoperative period and also its behavior in the postoperative period.\\n Objective To demonstrate the importance of the role of physical therapy in the preoperative\\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\\nsurgery.\\nTo demonstrate the importance of the role of physical therapy in the preoperative\\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\\nsurgery.\",\n \"To demonstrate the importance of the role of physical therapy in the preoperative\\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\\nsurgery.\",\n \"We performed a prospective clinical study, simple blind, in the period from July 2009 to\\nSeptember 2011 in the ward of cardiac and thoracic surgery at Hospital das Clínicas da\\nUniversidade Estadual Paulista (UNESP), Botucatu-SP. We evaluated 70 patients of both\\ngender, age ranging from 40 to 75 years, undergoing CABG with use of CPB, randomized by\\nmeans of a computerized software (STATTREK) and delivered in a sealed envelope to the\\nphysical therapist responsible for primary health care, subdividing into two groups:\\n- Group I (GI) - Thirty-five patients, undergoing physiotherapeutic intervention,\\nperformed under supervision, once a day, during the period that preceded the surgery.\\nThe protocol consisted of breathing exercises (breathing in time, deep breathing\\nfollowed by prolonged expiration, sustained maximal inspiration with apnea of 6 seconds,\\nand diaphragmatic breathing associated with the mobilization of the upper limbs) and\\nbreathing exercises with threshold - IMT® (Threshold - IMT®\\nInspiratory Muscle Trainer, HealthscanProducts Inc.) at an intensity of 40% of the\\ninitial MIP with three sets of ten repetitions, respecting two-minute intervals between\\neach series.\\n- Group II (GII) - thirty-five patients who did not undergo the physical therapy\\nprotocol during the preoperative period, receiving only guidelines ward routine.\\nWe included patients of both sexes, aged 40-75 years with coronary artery disease\\nadmitted for cardiac surgery with cardiopulmonary bypass (CPB) MR, who agreed to\\nparticipate in the study. We excluded patients who require the use of intra-aortic\\nballoon, carrying pulmonary pathology, musculoskeletal impairment and severe\\nneurological surgery performed without CPB, emergency surgery, homodynamic instability,\\nand any event that threatens the integrity of patient or the fidelity of the measures\\nanalyzed.\\n Delimitation All patients were evaluated by the same evaluator (preoperative period - PRE), third\\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\\nphysiotherapeutical treatment, according to their needs in the postoperative period\\nby physical therapy team in the hospital and only Group I received physiotherapy in\\nthe preoperative period.\\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\\nanesthesia time and the time that the patient remained with the endotracheal tube in\\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\\nICU stay (when the patient arrived in the intensive care unit until discharged to the\\nward bed) and length of stay in the OP (obtained from the time that the patient\\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\\nwere obtained by means of the daily developments of medical and nursing staff.\\nThe assessment of respiratory muscle strength was obtained by measurement of\\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\\nforceps according to the technique described by Nephew et al. (2008). The MIP\\nmeasurement was performed from residual volume, and MEP was obtained from total lung\\ncapacity. Three measurements were taken technically satisfactory, and if there were\\nmore than 20% difference between the measurements, a fourth and even fifth\\nmeasurement would be performed, being always considered the highest value obtained.\\nThe MIP was performed for evaluation of inspiratory muscle strength in three\\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\\nthe buccinator muscles during MIP maneuver[9].\\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\\nminute (cpm) and obtained by direct observation of respiratory cycles during the\\nevaluation of the minute volume[7].\\nAll patients were evaluated by the same evaluator (preoperative period - PRE), third\\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\\nphysiotherapeutical treatment, according to their needs in the postoperative period\\nby physical therapy team in the hospital and only Group I received physiotherapy in\\nthe preoperative period.\\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\\nanesthesia time and the time that the patient remained with the endotracheal tube in\\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\\nICU stay (when the patient arrived in the intensive care unit until discharged to the\\nward bed) and length of stay in the OP (obtained from the time that the patient\\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\\nwere obtained by means of the daily developments of medical and nursing staff.\\nThe assessment of respiratory muscle strength was obtained by measurement of\\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\\nforceps according to the technique described by Nephew et al. (2008). The MIP\\nmeasurement was performed from residual volume, and MEP was obtained from total lung\\ncapacity. Three measurements were taken technically satisfactory, and if there were\\nmore than 20% difference between the measurements, a fourth and even fifth\\nmeasurement would be performed, being always considered the highest value obtained.\\nThe MIP was performed for evaluation of inspiratory muscle strength in three\\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\\nthe buccinator muscles during MIP maneuver[9].\\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\\nminute (cpm) and obtained by direct observation of respiratory cycles during the\\nevaluation of the minute volume[7].\\n Procedures/treatment Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\\nof breathing exercises (breathing in time, deep breath and then exhaling long,\\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\\nassociated with mobilization of the upper limb, with three series out of ten for each\\nbreathing exercise) and breathing exercises with the device Threshold -\\nIMT®. We conducted three sets of ten repetitions with an interval of\\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\\nThe GII patients received only guidelines ward routine before surgery, but\\npostoperatively, both groups performed physical therapy as needed by staff\\nphysiotherapy service.\\nPatients allocated to the GI protocol underwent preoperative physiotherapy consisting\\nof breathing exercises (breathing in time, deep breath and then exhaling long,\\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\\nassociated with mobilization of the upper limb, with three series out of ten for each\\nbreathing exercise) and breathing exercises with the device Threshold -\\nIMT®. We conducted three sets of ten repetitions with an interval of\\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\\nThe GII patients received only guidelines ward routine before surgery, but\\npostoperatively, both groups performed physical therapy as needed by staff\\nphysiotherapy service.\\n Statistical Analysis The sample should be made with at least 26 patients who were randomly divided into\\ntwo study groups (with physical therapy preoperatively and control patients who did\\nnot receive preoperative physiotherapy). We considered the level of significance of\\n5% and a test power in the order of 80% for a minimum difference in the order of\\ntwo.\\nThe comparison between the groups for age and body mass index (BMI) was performed by\\nWilcoxon test for independent samples. The smoking variable was analyzed using\\nGoldman to compare proportions within and among multinomial populations. The\\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\\nhospital stay were obtained in minutes and analyzed by Student's t test or the\\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\\ntests for comparison of the moments in each group and Mann Whitney test to compare\\ngroups at each time, adopting a significance level of 5% probability of rejecting\\nnull hypothesis.\\nThis study was approved by the Research Ethics Committee of Universidade Estadual\\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\\nprotocol 3223) and all patients signed an informed consent form.\\nThe sample should be made with at least 26 patients who were randomly divided into\\ntwo study groups (with physical therapy preoperatively and control patients who did\\nnot receive preoperative physiotherapy). We considered the level of significance of\\n5% and a test power in the order of 80% for a minimum difference in the order of\\ntwo.\\nThe comparison between the groups for age and body mass index (BMI) was performed by\\nWilcoxon test for independent samples. The smoking variable was analyzed using\\nGoldman to compare proportions within and among multinomial populations. The\\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\\nhospital stay were obtained in minutes and analyzed by Student's t test or the\\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\\ntests for comparison of the moments in each group and Mann Whitney test to compare\\ngroups at each time, adopting a significance level of 5% probability of rejecting\\nnull hypothesis.\\nThis study was approved by the Research Ethics Committee of Universidade Estadual\\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\\nprotocol 3223) and all patients signed an informed consent form.\",\n \"All patients were evaluated by the same evaluator (preoperative period - PRE), third\\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\\nphysiotherapeutical treatment, according to their needs in the postoperative period\\nby physical therapy team in the hospital and only Group I received physiotherapy in\\nthe preoperative period.\\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\\nanesthesia time and the time that the patient remained with the endotracheal tube in\\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\\nICU stay (when the patient arrived in the intensive care unit until discharged to the\\nward bed) and length of stay in the OP (obtained from the time that the patient\\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\\nwere obtained by means of the daily developments of medical and nursing staff.\\nThe assessment of respiratory muscle strength was obtained by measurement of\\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\\nforceps according to the technique described by Nephew et al. (2008). The MIP\\nmeasurement was performed from residual volume, and MEP was obtained from total lung\\ncapacity. Three measurements were taken technically satisfactory, and if there were\\nmore than 20% difference between the measurements, a fourth and even fifth\\nmeasurement would be performed, being always considered the highest value obtained.\\nThe MIP was performed for evaluation of inspiratory muscle strength in three\\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\\nthe buccinator muscles during MIP maneuver[9].\\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\\nminute (cpm) and obtained by direct observation of respiratory cycles during the\\nevaluation of the minute volume[7].\",\n \"Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\\nof breathing exercises (breathing in time, deep breath and then exhaling long,\\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\\nassociated with mobilization of the upper limb, with three series out of ten for each\\nbreathing exercise) and breathing exercises with the device Threshold -\\nIMT®. We conducted three sets of ten repetitions with an interval of\\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\\nThe GII patients received only guidelines ward routine before surgery, but\\npostoperatively, both groups performed physical therapy as needed by staff\\nphysiotherapy service.\",\n \"The sample should be made with at least 26 patients who were randomly divided into\\ntwo study groups (with physical therapy preoperatively and control patients who did\\nnot receive preoperative physiotherapy). We considered the level of significance of\\n5% and a test power in the order of 80% for a minimum difference in the order of\\ntwo.\\nThe comparison between the groups for age and body mass index (BMI) was performed by\\nWilcoxon test for independent samples. The smoking variable was analyzed using\\nGoldman to compare proportions within and among multinomial populations. The\\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\\nhospital stay were obtained in minutes and analyzed by Student's t test or the\\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\\ntests for comparison of the moments in each group and Mann Whitney test to compare\\ngroups at each time, adopting a significance level of 5% probability of rejecting\\nnull hypothesis.\\nThis study was approved by the Research Ethics Committee of Universidade Estadual\\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\\nprotocol 3223) and all patients signed an informed consent form.\",\n \"Seventy patients were divided into two groups, Group I composed of 35 patients (12\\nfemale and 23 male) and Group II, with 35 patients (six female and 29 male), as\\nillustrated in Figure 1.\\nCharacterization of groups by gender\\nNo significant difference was noticed between groups for the variables age (GI 58.9±9.53\\nyears and GII 61.4±8.43 years; P=0.26) and body mass index (GI\\n26.8±3.96 kg/m2 and G II 26±3.86 kg/m2) (Table 1).\\nPatient characteristics regarding age and BMI.\\nBMI: body mass index; Wilcoxon test. * P<0.05\\nThe groups were homogenous concerning presence of active smoking, ex-smokers and\\nnonsmokers, as illustrated in Table 2.\\nNumber and proportion of patients in each group according to the presence of\\nactive smoking, ex-smokers and nonsmokers.\\nTwo proportions followed by the same lowercase letter do not differ in groups\\n(rows). Two proportions followed by the same capital letter do not differ in\\nthe types of smoking (P>0.05). Goldman test to compare proportions between\\nand within populations multinomina.\\nBetween the GI and GII, no significant difference regarding the proportion of\\nex-smokers, smokers and nonsmokers, in other words, the groups were homogeneous as\\ndemonstrated below (Table 2).\\n Maximum Inspiratory Pressure (MIP) The mean values the for maximum inspiratory pressures evaluated in the preoperative\\nperiod and 3PO showed no significant difference between groups\\n(P=0.276 and 0.065; respectively), however, we observed greater\\ninspiratory muscle strength for GI in relation to GII in 5PO\\n(P=0.001) (Figure 2).\\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\\noperation of each group analyzed (with and without physical therapy\\npreoperatively). Friedman test for comparison of moments in each group and Mann\\nWhitney test to compare the groups at each time, *P<0.05.\\nThe mean values the for maximum inspiratory pressures evaluated in the preoperative\\nperiod and 3PO showed no significant difference between groups\\n(P=0.276 and 0.065; respectively), however, we observed greater\\ninspiratory muscle strength for GI in relation to GII in 5PO\\n(P=0.001) (Figure 2).\\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\\noperation of each group analyzed (with and without physical therapy\\npreoperatively). Friedman test for comparison of moments in each group and Mann\\nWhitney test to compare the groups at each time, *P<0.05.\\n Maximum Expiratory Pressure (MEP) The mean value for the maximum expiratory pressure obtained in the preoperative\\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\\ncmH2O) for GII, not presenting significant difference between groups\\n(P=0.704). However, the values referring MEP measured in the 5PO\\nshowed significant difference between groups (P=0.001), as\\nillustrated in Figure 3.\\nMedian for maximum expiratory pressure (MEP) variable at different times of the\\noperation in the different groups. Friedman test for comparison of moments in\\neach group and Mann Whitney test to compare the groups at each time,\\n*P<0.05.\\nThe mean value for the maximum expiratory pressure obtained in the preoperative\\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\\ncmH2O) for GII, not presenting significant difference between groups\\n(P=0.704). However, the values referring MEP measured in the 5PO\\nshowed significant difference between groups (P=0.001), as\\nillustrated in Figure 3.\\nMedian for maximum expiratory pressure (MEP) variable at different times of the\\noperation in the different groups. Friedman test for comparison of moments in\\neach group and Mann Whitney test to compare the groups at each time,\\n*P<0.05.\\n Minute Volume (MV) The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\\nP=0.001). However, these values have not showed significative\\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\\nrespectively).\\nMedian minute volume (VM ) variable at different times of the operation of each\\ngroup analyzed. Friedman test for comparison of the moments in each group and\\nthe Mann Whitney test for comparison between groups at any time,\\n*P<0.05.\\nThe analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\\nP=0.001). However, these values have not showed significative\\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\\nrespectively).\\nMedian minute volume (VM ) variable at different times of the operation of each\\ngroup analyzed. Friedman test for comparison of the moments in each group and\\nthe Mann Whitney test for comparison between groups at any time,\\n*P<0.05.\\n Tidal Volume Figure 5 illustrates the analysis of variable\\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\\nvalues were not significantly different for the 3PO and 5 PO\\n(P=0.059 and P=0.549; respectively).\\nMedian tidal volume variable at different times for the group treated with\\npreoperative physiotherapy and without physical therapy preoperatively.\\nFriedman test for comparison of moments in each group and Mann Whitney test to\\ncompare the groups at each time, *P<0.05.\\nFigure 5 illustrates the analysis of variable\\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\\nvalues were not significantly different for the 3PO and 5 PO\\n(P=0.059 and P=0.549; respectively).\\nMedian tidal volume variable at different times for the group treated with\\npreoperative physiotherapy and without physical therapy preoperatively.\\nFriedman test for comparison of moments in each group and Mann Whitney test to\\ncompare the groups at each time, *P<0.05.\\n Respiratory rate (RR) The median refers to the respiratory rate obtained preoperatively for GI and GII\\nshowed no significant difference (16 cpm and 18 cpm, respectively,\\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\\nshowed no significant difference between groups (P=0.090 and\\nP=0.886, respectively), as shown in Figure 6.\\nMedian variable respiratory rate at different times of the operation of each\\ngroup analyzed (with and without physical therapy preoperatively). Friedman\\ntest for comparison of moments in each group and Mann Whitney test to compare\\nthe groups at each time, *P<0.05.\\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\\nhospital stay after surgery.\\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\\nstay did not significantly differ between groups, however, significant difference was\\nobserved for the length of stay variable in the postoperative period\\n(P=0.001), as shown in Table\\n3.\\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\\n5PO.\\nValues are shown as mean ± standard deviation or median (minimum and maximum\\nvalues).\\nMann and Whitney\\nStudent's t test.\\nSignificant difference between groups (P <0.05). ICU: intensive care\\nunit; PO: postoperative\\nThe median refers to the respiratory rate obtained preoperatively for GI and GII\\nshowed no significant difference (16 cpm and 18 cpm, respectively,\\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\\nshowed no significant difference between groups (P=0.090 and\\nP=0.886, respectively), as shown in Figure 6.\\nMedian variable respiratory rate at different times of the operation of each\\ngroup analyzed (with and without physical therapy preoperatively). Friedman\\ntest for comparison of moments in each group and Mann Whitney test to compare\\nthe groups at each time, *P<0.05.\\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\\nhospital stay after surgery.\\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\\nstay did not significantly differ between groups, however, significant difference was\\nobserved for the length of stay variable in the postoperative period\\n(P=0.001), as shown in Table\\n3.\\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\\n5PO.\\nValues are shown as mean ± standard deviation or median (minimum and maximum\\nvalues).\\nMann and Whitney\\nStudent's t test.\\nSignificant difference between groups (P <0.05). ICU: intensive care\\nunit; PO: postoperative\",\n \"The mean values the for maximum inspiratory pressures evaluated in the preoperative\\nperiod and 3PO showed no significant difference between groups\\n(P=0.276 and 0.065; respectively), however, we observed greater\\ninspiratory muscle strength for GI in relation to GII in 5PO\\n(P=0.001) (Figure 2).\\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\\noperation of each group analyzed (with and without physical therapy\\npreoperatively). Friedman test for comparison of moments in each group and Mann\\nWhitney test to compare the groups at each time, *P<0.05.\",\n \"The mean value for the maximum expiratory pressure obtained in the preoperative\\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\\ncmH2O) for GII, not presenting significant difference between groups\\n(P=0.704). However, the values referring MEP measured in the 5PO\\nshowed significant difference between groups (P=0.001), as\\nillustrated in Figure 3.\\nMedian for maximum expiratory pressure (MEP) variable at different times of the\\noperation in the different groups. Friedman test for comparison of moments in\\neach group and Mann Whitney test to compare the groups at each time,\\n*P<0.05.\",\n \"The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\\nP=0.001). However, these values have not showed significative\\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\\nrespectively).\\nMedian minute volume (VM ) variable at different times of the operation of each\\ngroup analyzed. Friedman test for comparison of the moments in each group and\\nthe Mann Whitney test for comparison between groups at any time,\\n*P<0.05.\",\n \"Figure 5 illustrates the analysis of variable\\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\\nvalues were not significantly different for the 3PO and 5 PO\\n(P=0.059 and P=0.549; respectively).\\nMedian tidal volume variable at different times for the group treated with\\npreoperative physiotherapy and without physical therapy preoperatively.\\nFriedman test for comparison of moments in each group and Mann Whitney test to\\ncompare the groups at each time, *P<0.05.\",\n \"The median refers to the respiratory rate obtained preoperatively for GI and GII\\nshowed no significant difference (16 cpm and 18 cpm, respectively,\\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\\nshowed no significant difference between groups (P=0.090 and\\nP=0.886, respectively), as shown in Figure 6.\\nMedian variable respiratory rate at different times of the operation of each\\ngroup analyzed (with and without physical therapy preoperatively). Friedman\\ntest for comparison of moments in each group and Mann Whitney test to compare\\nthe groups at each time, *P<0.05.\\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\\nhospital stay after surgery.\\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\\nstay did not significantly differ between groups, however, significant difference was\\nobserved for the length of stay variable in the postoperative period\\n(P=0.001), as shown in Table\\n3.\\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\\n5PO.\\nValues are shown as mean ± standard deviation or median (minimum and maximum\\nvalues).\\nMann and Whitney\\nStudent's t test.\\nSignificant difference between groups (P <0.05). ICU: intensive care\\nunit; PO: postoperative\",\n \"Studies by Feltrim et al.[10] show\\nthat performing preoperative physiotherapy is more effective in reducing respiratory\\ncomplications in patients with moderate or higher risk than those whose risk was\\nlow.\\nIn the present study, we observed no significant difference between the subjects of\\nGroup I and Group II as the characteristics (age, BMI, smoking and number of co\\nmorbidities), which suggests that the study subjects in both groups were\\nhomogeneous.\\nIn the study by Leguisamo et al.[9],\\nwith 86 patients undergoing CABG, divided into two groups: the intervention group (44\\npatients) who were assessed and received physiotherapeutic guidance at least 15 days\\nbefore surgery. The control group received routine care on the day of\\nhospitalization.\\nThe average MIP between groups showed neither significant difference in the preoperative\\nperiod nor in the 1PO and 6PO.\\nIn the present study it was observed that there was no significant difference between\\ngroups to measure MIP in the preoperative period, but now in times of 3PO and 5PO\\nsignificant difference between groups was found. Similarly, no significant difference in\\nthe amount of MEP preoperatively in the 3PO and between groups. However in 5PO, there\\nwas significant difference between the groups with the best values of the group that\\nperformed physical therapy before surgery.\\nBarros et al.[11] evaluated the MIP\\nand MEP in the preoperative period, the first day after surgery and in 38 patients\\nundergoing CABG with CPB who were divided into two groups: 23 patients in group I\\n(respiratory muscle training) and 15 in group II (control). Group I received\\nconventional physiotherapy over respiratory muscle training and Group II the\\nconventional physiotherapy. The authors found that the values obtained for MIP and MEP\\nin hospital discharge were higher in group I.\\nThere was no decrease in maximal respiratory pressures, transdiaphragmatic pressure and\\ndiaphragm pressure, indicating weakness of respiratory muscle strength[12,13].\\nAgreeing with the authors cited above, we observed in the present study, after cardiac\\nsurgery, MIP values in GI and GII which decreased in 3PO, however the value of MIP in\\n5PO increase was observed for GI values compared to those in PRE and 3PO since the GII\\nvalues remained stable in 3PO. The authors noted the drop in MEP in 3PO in both groups,\\nhowever, in 5PO MEP has increased in GI. Regarding PRE and 3PO in GII no return value\\nwas observed in PRE.\\nArcêncio et al.[12] conducted a study\\nwith 30 patients, aged 50 years-old and candidates for CABG and / or heart valve,\\ndividing into two groups. Intervention group comprised 15 patients who underwent at\\nleast a two weeks inspiratory muscle training using incentive spirometry (\\\"Threshold -\\nIMT®) with 40% charge of the MIP and control group with 15 patients who\\nreceived only general guidelines. The authors compared the spirometric values before and\\nafter training and showed no significant difference in the values of MIP and\\nMEP[14].\\nElias et al.[15] studied 42 patients\\naged 18 to 75 years-old with coronary artery disease, in the pre-and postoperative\\nperiod, divided into two groups. Intervention group received TMR with Threshold -\\nIMT® and control group received only guidelines. The TMR both pre and\\npost-operative period consisted of three sets of 10 inspiratory exercises once a day for\\nthree consecutive days. It was observed that after cardiac surgery there was a\\nsignificant reduction in MIP and MEP in both groups.\\nMorsch et al.[13] conducted a study to\\nevaluate 108 patients undergoing elective CABG surgery from April 2006 to February 2007\\n(preoperatively and postoperatively).\\nThe average values of MIP in the preoperative period were significantly decreased\\ncompared to 6 days after the surgery (P<0.001). The authors also\\nobserved a significant decrease in the values of MEP obtained on the 6th\\npostoperative day compared to the preoperative period (P<0.001).\\nPatients with respiratory muscle weakness have higher risk of postoperative pulmonary\\ncomplications. The respiratory muscle training in the pre-operative may prevent\\npulmonary complications in patients undergoing thoracic surgery[16]. There may be a reduction of all lung\\nvolumes resulting from factors such as diaphragmatic dysfunction, pain, absence of deep\\nbreaths, pulmonary changes and ribcage. The functional residual capacity (FRC) decreases\\nbecause of the reduction of both the residual volume (RV) and expiratory reserve volume.\\nThe ventilation is affected by VC reduction by approximately 20%, and the increase in\\nFR(22-24). The sum of these factors cause changes in the mechanics of\\nrespiration, which leads to a shallow breathing pattern of decreased lung\\nvolume[13].\\nPatients undergoing CABG develop mostly PO pulmonary dysfunction with a significant\\nreduction in lung volumes, impairments in respiratory function, decreased lung\\ncompliance and increased work of breathing. The reduction in lung volumes and capacities\\ncontributes to changes in gas exchange, resulting in hypoxemia[17].\\nIn this study, the VM showed significant difference regarding the groups I and II in the\\npreoperative period but not significant between groups in 3PO. Likewise, the mean value\\nof the VM for the GI 5PO, there was no significant difference in the GII. The mean value\\nof VC in PRE in GI was significant, as the mean value of the VC in GII patients, however\\nin GI and GII we observed in 5PO and 3PO that there was no significant difference. It\\nwas observed in GI at different times that the increase of VM will be very likely due to\\nincreased FR, since the current VC remained without many changes between different\\ntimes.\\nBarros et al.[11] demonstrated a\\nsignificant decrease in lung volumes in patients undergoing cardiac surgery in the\\npostoperative period.\\nCarvalho et al.[18] observed a\\ndecrease in the values of minute volume and tidal volume measured on the 2nd\\npostoperative day, however, these values showed gradual improvement returning to the\\nvalues obtained at baseline (preoperative period) at the time of hospital discharge.\\nIn a study, Guizilini et al.[19]\\nevaluated 30 patients with a mean age of 56 years and divided into two groups, group A\\n(n=15) without CPB and group B (n=15) with CPB. All patients underwent pulmonary\\nfunction. In both groups, there was a decrease in FVC until the fifth postoperative day\\n(P<0.05).\\nRegarding the respiratory rate in the present study, we can verify that there was an\\nincrease in both groups, with a higher peak on the 3rd postoperative day and\\ndecreased on the 6th postoperative day, but the respiratory rate did not\\nreturn to the preoperative values, Carvalho et al.[18] already mentioned above that they concluded in their studies\\nthat the mean respiratory frequency had a significant increase between pre and\\n2nd PO, 3rd PO, decreasing on the 4th postoperative\\nday and increased again on the 5th postoperative day and decreased in\\nhospital discharge, but patients did not return to the preoperative values.\\nAccording to the results obtained in this study, we can affirm that there was no\\nsignificant difference between the groups regarding the time variables: anesthesia,\\nsurgery, perfusion, anoxia, mechanical ventilation, intubation. This suggests that the\\ngroups were quite homogeneous. As for the time of postoperative hospital stay, there was\\na decrease of approximately 25 hours, comparing patients who underwent physical therapy\\nbefore surgery with patients who did not.\\nIn contrast to the findings of this study, Patman et al.[20] performed a randomized study of 236 patients to\\nevaluate the effects of physical therapy interventions performed early versus only\\nstarted after extubation in patients undergoing cardiac surgery and no significant\\ndifferences were found in the time of intubation, ICU stay and length of hospital stay\\nbetween the groups.\\nHowever, Celli et al.[21] conducted a\\nstudy with 172 patients and showed a decrease in length of hospital stay in the group\\nthat had received guidelines breathing exercises (9.6±3.2 days) compared to the control\\ngroup (13±5 days).\\nCorroborating these findings, as well as those obtained in the present study, Leguisamo\\net al.[9] performed a prospective\\nstudy to evaluate the effectiveness of a physiotherapy program start in the preoperative\\nperiod in reducing the length of hospital stay and showed a significant reduction in\\nlength of hospital stay for the intervention group compared to the control group\\n(P<0.05).\\nThese data suggest that physical therapy initiated before surgery can improve conditions\\nof patients, reestablishing early respiratory pressures, reducing the length of stay and\\ndecreased hospital costs for the period of hospitalization.\\n Limitation of the study The major limitation was the short period of time that patients received preoperative\\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\\nrelevance even in a short period, so the study may add knowledge of response to\\ntherapy performed during intraoperative CABG surgery.\\nNot performing spirometry can be mentioned as a limitation on the analysis of\\nventilation parameters, however, due to the conditions offered by our service, we\\ncould not perform this evaluation.\\nOn the other hand, the results showed that even with limited time monitoring in the\\npreoperative period and respiratory variables obtained only by respirometry and\\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\\npatients undergoing surgery CABG.\\nWe understand that the results may open up new avenues of future research related to\\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\\nfurther studies should be conducted using tools such as spirometry, to assess and\\nquantify the volume and lung capacity more accurately.\\nThe major limitation was the short period of time that patients received preoperative\\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\\nrelevance even in a short period, so the study may add knowledge of response to\\ntherapy performed during intraoperative CABG surgery.\\nNot performing spirometry can be mentioned as a limitation on the analysis of\\nventilation parameters, however, due to the conditions offered by our service, we\\ncould not perform this evaluation.\\nOn the other hand, the results showed that even with limited time monitoring in the\\npreoperative period and respiratory variables obtained only by respirometry and\\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\\npatients undergoing surgery CABG.\\nWe understand that the results may open up new avenues of future research related to\\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\\nfurther studies should be conducted using tools such as spirometry, to assess and\\nquantify the volume and lung capacity more accurately.\",\n \"The major limitation was the short period of time that patients received preoperative\\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\\nrelevance even in a short period, so the study may add knowledge of response to\\ntherapy performed during intraoperative CABG surgery.\\nNot performing spirometry can be mentioned as a limitation on the analysis of\\nventilation parameters, however, due to the conditions offered by our service, we\\ncould not perform this evaluation.\\nOn the other hand, the results showed that even with limited time monitoring in the\\npreoperative period and respiratory variables obtained only by respirometry and\\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\\npatients undergoing surgery CABG.\\nWe understand that the results may open up new avenues of future research related to\\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\\nfurther studies should be conducted using tools such as spirometry, to assess and\\nquantify the volume and lung capacity more accurately.\",\n \"The physical therapy has an important role in the preoperative period, restoring greater\\nreadiness ventilatory parameters of patients undergoing coronary artery bypass grafting\\nwith cardiopulmonary bypass, resulting in a decrease in length of hospital stay after\\nsurgery.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,"methods",null,null,null,"results",null,null,null,null,null,"discussion",null,"conclusions"],"string":"[\n \"intro\",\n null,\n \"methods\",\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["Thoracic Surgery","Breathing Exercises","Physical Therapy (Specialty)"],"string":"[\n \"Thoracic Surgery\",\n \"Breathing Exercises\",\n \"Physical Therapy (Specialty)\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nThe cardiac surgery was always clothed with great interest, curiosity, and in some\nmoments, mysticism, and fact understandable when we analyzed the noble importance of\nthis component for the proper functioning of the human body[1].\nIn recent decades the frequency of surgical procedures has progressively increased,\namong them the myocardial revascularization (MR). Cardiovascular diseases are a serious\npublic health problem in Brazil[2].\nIn individuals over 70 years old it is estimated an incidence of 70% of coronary artery\ndisease. As the age is a determinant of coronary atherosclerosis, a growing number of\nelderly people will be subjected to MR and/or other therapeutic methods[3,4].\nThe main risk factors that contribute to cardiac diseases are: smoking, high levels of\nlow-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density\nlipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,\nfamily history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors\nrelated to atherosclerosis and its clinical manifestations[4,5].\nThe coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment\nof ischemic heart disease that is manifested clinically by angina and acute myocardial\ninfarction. The CABG surgery creates a new route for the blood flow and, is used for\nmaking a diversion of blood to the portions of the coronary artery distal to the\nobstructive atherosclerotic involvement[1,5].\nWith the advancements in care of physical therapy in the preoperative period, surgical\ntechniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,\nanesthesia and intensive care in the post-operative period, there was a decrease in\nmorbidity and mortality of MR which caused the surgical indication in groups of patients\nincreasingly complex[6,7].\nObserving the fact that they are common pulmonary complications of CABG with CPB was\njustified to carry out this research, there is great importance in properly carrying out\nevaluations, guidelines and breathing exercises in the period before surgery, and\naddition, observe the influence and duration of physical therapy assistance in the\npreoperative period and also its behavior in the postoperative period.\n Objective To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\nTo demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\n\nObjective:\nTo demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\n\nMETHODS:\nWe performed a prospective clinical study, simple blind, in the period from July 2009 to\nSeptember 2011 in the ward of cardiac and thoracic surgery at Hospital das Clínicas da\nUniversidade Estadual Paulista (UNESP), Botucatu-SP. We evaluated 70 patients of both\ngender, age ranging from 40 to 75 years, undergoing CABG with use of CPB, randomized by\nmeans of a computerized software (STATTREK) and delivered in a sealed envelope to the\nphysical therapist responsible for primary health care, subdividing into two groups:\n- Group I (GI) - Thirty-five patients, undergoing physiotherapeutic intervention,\nperformed under supervision, once a day, during the period that preceded the surgery.\nThe protocol consisted of breathing exercises (breathing in time, deep breathing\nfollowed by prolonged expiration, sustained maximal inspiration with apnea of 6 seconds,\nand diaphragmatic breathing associated with the mobilization of the upper limbs) and\nbreathing exercises with threshold - IMT® (Threshold - IMT®\nInspiratory Muscle Trainer, HealthscanProducts Inc.) at an intensity of 40% of the\ninitial MIP with three sets of ten repetitions, respecting two-minute intervals between\neach series.\n- Group II (GII) - thirty-five patients who did not undergo the physical therapy\nprotocol during the preoperative period, receiving only guidelines ward routine.\nWe included patients of both sexes, aged 40-75 years with coronary artery disease\nadmitted for cardiac surgery with cardiopulmonary bypass (CPB) MR, who agreed to\nparticipate in the study. We excluded patients who require the use of intra-aortic\nballoon, carrying pulmonary pathology, musculoskeletal impairment and severe\nneurological surgery performed without CPB, emergency surgery, homodynamic instability,\nand any event that threatens the integrity of patient or the fidelity of the measures\nanalyzed.\n Delimitation All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\nAll patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\n Procedures/treatment Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\nPatients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\n Statistical Analysis The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.\nThe sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.\n\nDelimitation:\nAll patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\n\nProcedures/treatment:\nPatients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\n\nStatistical Analysis:\nThe sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.\n\nRESULTS:\nSeventy patients were divided into two groups, Group I composed of 35 patients (12\nfemale and 23 male) and Group II, with 35 patients (six female and 29 male), as\nillustrated in Figure 1.\nCharacterization of groups by gender\nNo significant difference was noticed between groups for the variables age (GI 58.9±9.53\nyears and GII 61.4±8.43 years; P=0.26) and body mass index (GI\n26.8±3.96 kg/m2 and G II 26±3.86 kg/m2) (Table 1).\nPatient characteristics regarding age and BMI.\nBMI: body mass index; Wilcoxon test. * P<0.05\nThe groups were homogenous concerning presence of active smoking, ex-smokers and\nnonsmokers, as illustrated in Table 2.\nNumber and proportion of patients in each group according to the presence of\nactive smoking, ex-smokers and nonsmokers.\nTwo proportions followed by the same lowercase letter do not differ in groups\n(rows). Two proportions followed by the same capital letter do not differ in\nthe types of smoking (P>0.05). Goldman test to compare proportions between\nand within populations multinomina.\nBetween the GI and GII, no significant difference regarding the proportion of\nex-smokers, smokers and nonsmokers, in other words, the groups were homogeneous as\ndemonstrated below (Table 2).\n Maximum Inspiratory Pressure (MIP) The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\nThe mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\n Maximum Expiratory Pressure (MEP) The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\nThe mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\n Minute Volume (MV) The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\nThe analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\n Tidal Volume Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\nFigure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\n Respiratory rate (RR) The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative\nThe median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative\n\nMaximum Inspiratory Pressure (MIP):\nThe mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\n\nMaximum Expiratory Pressure (MEP):\nThe mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\n\nMinute Volume (MV):\nThe analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\n\nTidal Volume:\nFigure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\n\nRespiratory rate (RR):\nThe median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative\n\nDISCUSSION:\nStudies by Feltrim et al.[10] show\nthat performing preoperative physiotherapy is more effective in reducing respiratory\ncomplications in patients with moderate or higher risk than those whose risk was\nlow.\nIn the present study, we observed no significant difference between the subjects of\nGroup I and Group II as the characteristics (age, BMI, smoking and number of co\nmorbidities), which suggests that the study subjects in both groups were\nhomogeneous.\nIn the study by Leguisamo et al.[9],\nwith 86 patients undergoing CABG, divided into two groups: the intervention group (44\npatients) who were assessed and received physiotherapeutic guidance at least 15 days\nbefore surgery. The control group received routine care on the day of\nhospitalization.\nThe average MIP between groups showed neither significant difference in the preoperative\nperiod nor in the 1PO and 6PO.\nIn the present study it was observed that there was no significant difference between\ngroups to measure MIP in the preoperative period, but now in times of 3PO and 5PO\nsignificant difference between groups was found. Similarly, no significant difference in\nthe amount of MEP preoperatively in the 3PO and between groups. However in 5PO, there\nwas significant difference between the groups with the best values of the group that\nperformed physical therapy before surgery.\nBarros et al.[11] evaluated the MIP\nand MEP in the preoperative period, the first day after surgery and in 38 patients\nundergoing CABG with CPB who were divided into two groups: 23 patients in group I\n(respiratory muscle training) and 15 in group II (control). Group I received\nconventional physiotherapy over respiratory muscle training and Group II the\nconventional physiotherapy. The authors found that the values obtained for MIP and MEP\nin hospital discharge were higher in group I.\nThere was no decrease in maximal respiratory pressures, transdiaphragmatic pressure and\ndiaphragm pressure, indicating weakness of respiratory muscle strength[12,13].\nAgreeing with the authors cited above, we observed in the present study, after cardiac\nsurgery, MIP values in GI and GII which decreased in 3PO, however the value of MIP in\n5PO increase was observed for GI values compared to those in PRE and 3PO since the GII\nvalues remained stable in 3PO. The authors noted the drop in MEP in 3PO in both groups,\nhowever, in 5PO MEP has increased in GI. Regarding PRE and 3PO in GII no return value\nwas observed in PRE.\nArcêncio et al.[12] conducted a study\nwith 30 patients, aged 50 years-old and candidates for CABG and / or heart valve,\ndividing into two groups. Intervention group comprised 15 patients who underwent at\nleast a two weeks inspiratory muscle training using incentive spirometry (\"Threshold -\nIMT®) with 40% charge of the MIP and control group with 15 patients who\nreceived only general guidelines. The authors compared the spirometric values before and\nafter training and showed no significant difference in the values of MIP and\nMEP[14].\nElias et al.[15] studied 42 patients\naged 18 to 75 years-old with coronary artery disease, in the pre-and postoperative\nperiod, divided into two groups. Intervention group received TMR with Threshold -\nIMT® and control group received only guidelines. The TMR both pre and\npost-operative period consisted of three sets of 10 inspiratory exercises once a day for\nthree consecutive days. It was observed that after cardiac surgery there was a\nsignificant reduction in MIP and MEP in both groups.\nMorsch et al.[13] conducted a study to\nevaluate 108 patients undergoing elective CABG surgery from April 2006 to February 2007\n(preoperatively and postoperatively).\nThe average values of MIP in the preoperative period were significantly decreased\ncompared to 6 days after the surgery (P<0.001). The authors also\nobserved a significant decrease in the values of MEP obtained on the 6th\npostoperative day compared to the preoperative period (P<0.001).\nPatients with respiratory muscle weakness have higher risk of postoperative pulmonary\ncomplications. The respiratory muscle training in the pre-operative may prevent\npulmonary complications in patients undergoing thoracic surgery[16]. There may be a reduction of all lung\nvolumes resulting from factors such as diaphragmatic dysfunction, pain, absence of deep\nbreaths, pulmonary changes and ribcage. The functional residual capacity (FRC) decreases\nbecause of the reduction of both the residual volume (RV) and expiratory reserve volume.\nThe ventilation is affected by VC reduction by approximately 20%, and the increase in\nFR(22-24). The sum of these factors cause changes in the mechanics of\nrespiration, which leads to a shallow breathing pattern of decreased lung\nvolume[13].\nPatients undergoing CABG develop mostly PO pulmonary dysfunction with a significant\nreduction in lung volumes, impairments in respiratory function, decreased lung\ncompliance and increased work of breathing. The reduction in lung volumes and capacities\ncontributes to changes in gas exchange, resulting in hypoxemia[17].\nIn this study, the VM showed significant difference regarding the groups I and II in the\npreoperative period but not significant between groups in 3PO. Likewise, the mean value\nof the VM for the GI 5PO, there was no significant difference in the GII. The mean value\nof VC in PRE in GI was significant, as the mean value of the VC in GII patients, however\nin GI and GII we observed in 5PO and 3PO that there was no significant difference. It\nwas observed in GI at different times that the increase of VM will be very likely due to\nincreased FR, since the current VC remained without many changes between different\ntimes.\nBarros et al.[11] demonstrated a\nsignificant decrease in lung volumes in patients undergoing cardiac surgery in the\npostoperative period.\nCarvalho et al.[18] observed a\ndecrease in the values of minute volume and tidal volume measured on the 2nd\npostoperative day, however, these values showed gradual improvement returning to the\nvalues obtained at baseline (preoperative period) at the time of hospital discharge.\nIn a study, Guizilini et al.[19]\nevaluated 30 patients with a mean age of 56 years and divided into two groups, group A\n(n=15) without CPB and group B (n=15) with CPB. All patients underwent pulmonary\nfunction. In both groups, there was a decrease in FVC until the fifth postoperative day\n(P<0.05).\nRegarding the respiratory rate in the present study, we can verify that there was an\nincrease in both groups, with a higher peak on the 3rd postoperative day and\ndecreased on the 6th postoperative day, but the respiratory rate did not\nreturn to the preoperative values, Carvalho et al.[18] already mentioned above that they concluded in their studies\nthat the mean respiratory frequency had a significant increase between pre and\n2nd PO, 3rd PO, decreasing on the 4th postoperative\nday and increased again on the 5th postoperative day and decreased in\nhospital discharge, but patients did not return to the preoperative values.\nAccording to the results obtained in this study, we can affirm that there was no\nsignificant difference between the groups regarding the time variables: anesthesia,\nsurgery, perfusion, anoxia, mechanical ventilation, intubation. This suggests that the\ngroups were quite homogeneous. As for the time of postoperative hospital stay, there was\na decrease of approximately 25 hours, comparing patients who underwent physical therapy\nbefore surgery with patients who did not.\nIn contrast to the findings of this study, Patman et al.[20] performed a randomized study of 236 patients to\nevaluate the effects of physical therapy interventions performed early versus only\nstarted after extubation in patients undergoing cardiac surgery and no significant\ndifferences were found in the time of intubation, ICU stay and length of hospital stay\nbetween the groups.\nHowever, Celli et al.[21] conducted a\nstudy with 172 patients and showed a decrease in length of hospital stay in the group\nthat had received guidelines breathing exercises (9.6±3.2 days) compared to the control\ngroup (13±5 days).\nCorroborating these findings, as well as those obtained in the present study, Leguisamo\net al.[9] performed a prospective\nstudy to evaluate the effectiveness of a physiotherapy program start in the preoperative\nperiod in reducing the length of hospital stay and showed a significant reduction in\nlength of hospital stay for the intervention group compared to the control group\n(P<0.05).\nThese data suggest that physical therapy initiated before surgery can improve conditions\nof patients, reestablishing early respiratory pressures, reducing the length of stay and\ndecreased hospital costs for the period of hospitalization.\n Limitation of the study The major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.\nThe major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.\n\nLimitation of the study:\nThe major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.\n\nCONCLUSIONS:\nThe physical therapy has an important role in the preoperative period, restoring greater\nreadiness ventilatory parameters of patients undergoing coronary artery bypass grafting\nwith cardiopulmonary bypass, resulting in a decrease in length of hospital stay after\nsurgery.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe frequency of surgical procedures has increased steadily in recent decades, including the myocardial revascularization.\n\nMethods:\nWe conducted a prospective study with patients undergoing myocardial revascularization, the Hospital das Clínicas da Universidade Estadual Paulista (UNESP)/Botucatu - SP. We evaluated 70 patients of both genders, aged between 40 and 75 years, subdivided into two groups: group I - 35 patients of both genders, who received a written protocol guidance, breathing exercises and respiratory muscle training in the preoperative period and group II - 35 patients of both genders, who received only orientation of the ward on the day of surgery. This study was approved by the Ethics Committee of UNESP / Botucatu - SP.\n\nResults:\nMaximal inspiratory pressure in third postoperative day and fifth postoperative day and significant difference between groups, being better for the intervention group. Expiratory pressure was significant in fifth postoperative day in the intervention group compared to controls. The difference of length of hospital stay in the postoperative was found between the groups with shorter hospital stay in the group receiving preoperative therapy.\n\nConclusions:\nPhysical therapy plays an important role in the preoperative period, so that individuals in the intervention group more readily restored the parameters evaluated before surgery, in addition, there was a decrease in the time of the postoperative hospital stay. Thus, it is thought the cost-effectiveness of a program of preoperative physiotherapy."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nThe cardiac surgery was always clothed with great interest, curiosity, and in some\nmoments, mysticism, and fact understandable when we analyzed the noble importance of\nthis component for the proper functioning of the human body[1].\nIn recent decades the frequency of surgical procedures has progressively increased,\namong them the myocardial revascularization (MR). Cardiovascular diseases are a serious\npublic health problem in Brazil[2].\nIn individuals over 70 years old it is estimated an incidence of 70% of coronary artery\ndisease. As the age is a determinant of coronary atherosclerosis, a growing number of\nelderly people will be subjected to MR and/or other therapeutic methods[3,4].\nThe main risk factors that contribute to cardiac diseases are: smoking, high levels of\nlow-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density\nlipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,\nfamily history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors\nrelated to atherosclerosis and its clinical manifestations[4,5].\nThe coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment\nof ischemic heart disease that is manifested clinically by angina and acute myocardial\ninfarction. The CABG surgery creates a new route for the blood flow and, is used for\nmaking a diversion of blood to the portions of the coronary artery distal to the\nobstructive atherosclerotic involvement[1,5].\nWith the advancements in care of physical therapy in the preoperative period, surgical\ntechniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,\nanesthesia and intensive care in the post-operative period, there was a decrease in\nmorbidity and mortality of MR which caused the surgical indication in groups of patients\nincreasingly complex[6,7].\nObserving the fact that they are common pulmonary complications of CABG with CPB was\njustified to carry out this research, there is great importance in properly carrying out\nevaluations, guidelines and breathing exercises in the period before surgery, and\naddition, observe the influence and duration of physical therapy assistance in the\npreoperative period and also its behavior in the postoperative period.\n Objective To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\nTo demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\n\nCONCLUSIONS:\nThe physical therapy has an important role in the preoperative period, restoring greater\nreadiness ventilatory parameters of patients undergoing coronary artery bypass grafting\nwith cardiopulmonary bypass, resulting in a decrease in length of hospital stay after\nsurgery."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe frequency of surgical procedures has increased steadily in recent decades, including the myocardial revascularization.\n\nMethods:\nWe conducted a prospective study with patients undergoing myocardial revascularization, the Hospital das Clínicas da Universidade Estadual Paulista (UNESP)/Botucatu - SP. We evaluated 70 patients of both genders, aged between 40 and 75 years, subdivided into two groups: group I - 35 patients of both genders, who received a written protocol guidance, breathing exercises and respiratory muscle training in the preoperative period and group II - 35 patients of both genders, who received only orientation of the ward on the day of surgery. This study was approved by the Ethics Committee of UNESP / Botucatu - SP.\n\nResults:\nMaximal inspiratory pressure in third postoperative day and fifth postoperative day and significant difference between groups, being better for the intervention group. Expiratory pressure was significant in fifth postoperative day in the intervention group compared to controls. The difference of length of hospital stay in the postoperative was found between the groups with shorter hospital stay in the group receiving preoperative therapy.\n\nConclusions:\nPhysical therapy plays an important role in the preoperative period, so that individuals in the intervention group more readily restored the parameters evaluated before surgery, in addition, there was a decrease in the time of the postoperative hospital stay. Thus, it is thought the cost-effectiveness of a program of preoperative physiotherapy."},"whole_article_text_length":{"kind":"number","value":8719,"string":"8,719"},"whole_article_abstract_length":{"kind":"number","value":265,"string":"265"},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["groups","surgery","time","patients","period","preoperative","group","obtained","test","significant"],"string":"[\n \"groups\",\n \"surgery\",\n \"time\",\n \"patients\",\n \"period\",\n \"preoperative\",\n \"group\",\n \"obtained\",\n \"test\",\n \"significant\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cholesterol | importance | surgery | surgical | cardiac | coronary | period | duration | mr | myocardial [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] breathing | patients | performed | patient | obtained | mip | time | minute | pre | threshold [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | test | significant | significant difference | difference | group | values | maximum | median | variable [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] bypass | coronary artery bypass grafting | grafting cardiopulmonary bypass resulting | undergoing coronary | role preoperative period restoring | grafting | grafting cardiopulmonary | grafting cardiopulmonary bypass | ventilatory parameters patients undergoing | undergoing coronary artery bypass [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | test | surgery | period | group | patients | significant | time | preoperative | difference [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | test | surgery | period | group | patients | significant | time | preoperative | difference [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] recent decades [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Estadual Paulista | UNESP)/Botucatu - SP ||| 70 | between 40 and 75 years | two | II - 35 | the day ||| the Ethics Committee of UNESP / Botucatu - SP [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] third postoperative day | fifth ||| fifth ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] recent decades ||| Estadual Paulista | UNESP)/Botucatu - SP ||| 70 | between 40 and 75 years | two | II - 35 | the day ||| the Ethics Committee of UNESP / Botucatu - SP ||| ||| third postoperative day | fifth ||| fifth ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] recent decades ||| Estadual Paulista | UNESP)/Botucatu - SP ||| 70 | between 40 and 75 years | two | II - 35 | the day ||| the Ethics Committee of UNESP / Botucatu - SP ||| ||| third postoperative day | fifth ||| fifth ||| ||| ||| [SUMMARY]"}}},{"rowIdx":285,"cells":{"title":{"kind":"string","value":"DNA Methylation status of Wnt antagonist SFRP5 can predict the response to the EGFR-tyrosine kinase inhibitor therapy in non-small cell lung cancer."},"pmid":{"kind":"string","value":"23009178"},"background_abstract":{"kind":"string","value":"It is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Tumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"We found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Patients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adaptor Proteins, Signal Transducing","Aged","Biomarkers, Pharmacological","Carcinoma, Non-Small-Cell Lung","DNA Methylation","Disease-Free Survival","ErbB Receptors","Eye Proteins","Female","Humans","Male","Membrane Proteins","Middle Aged","Mutation","Neoplasm Staging","Protein Kinase Inhibitors","Treatment Outcome","Wnt Signaling Pathway"],"string":"[\n \"Adaptor Proteins, Signal Transducing\",\n \"Aged\",\n \"Biomarkers, Pharmacological\",\n \"Carcinoma, Non-Small-Cell Lung\",\n \"DNA Methylation\",\n \"Disease-Free Survival\",\n \"ErbB Receptors\",\n \"Eye Proteins\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Membrane Proteins\",\n \"Middle Aged\",\n \"Mutation\",\n \"Neoplasm Staging\",\n \"Protein Kinase Inhibitors\",\n \"Treatment Outcome\",\n \"Wnt Signaling Pathway\"\n]"},"pmcid":{"kind":"string","value":"3524045"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Lung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Patients 155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n DNA extraction and methylation-specific PCR Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\nGenomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\n Mutation detection The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\nThe denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\n Statistical analysis All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\nAll data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Characteristics of study patients Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\nTable\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\n Epigenotype of Wnt antagonists in NSCLC Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\nGenomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\n Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\nThe RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\n Epigenotype of Wnt antagonists and progression-free survival (PFS) We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\nWe next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\n Epigenotype of Wnt antagonists and overall survival rate (OS) To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\nTo test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\n Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\nIn order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development\n[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC."},"other_sections_titles":{"kind":"list like","value":["Background","Patients","DNA extraction and methylation-specific PCR","Mutation detection","Statistical analysis","Characteristics of study patients","Epigenotype of Wnt antagonists in NSCLC","Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy","Epigenotype of Wnt antagonists and progression-free survival (PFS)","Epigenotype of Wnt antagonists and overall survival rate (OS)","Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy","Abbreviations","Competing interests","Authors’ contributions","Authors’ informations"],"string":"[\n \"Background\",\n \"Patients\",\n \"DNA extraction and methylation-specific PCR\",\n \"Mutation detection\",\n \"Statistical analysis\",\n \"Characteristics of study patients\",\n \"Epigenotype of Wnt antagonists in NSCLC\",\n \"Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy\",\n \"Epigenotype of Wnt antagonists and progression-free survival (PFS)\",\n \"Epigenotype of Wnt antagonists and overall survival rate (OS)\",\n \"Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Authors’ informations\"\n]"},"other_sections_texts":{"kind":"list like","value":["Lung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.","155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.","Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.","The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].","All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.","Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.","Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.","The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).","We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).","To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n","In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.","EGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.","The authors declare that they have no competing interests.","JZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.","Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22)."],"string":"[\n \"Lung cancer is the leading cause of cancer death worldwide\\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\\n[7-11].\\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\\n[10,16,17]. In addition, previous studies reported that both T790M mutation\\n[18] and c-MET amplification\\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.\",\n \"155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\",\n \"Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\",\n \"The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\\n[28].\",\n \"All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\",\n \"Table\\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\\nMethylation and mutation profile of NSCLC\\n*The frequency of this group is significantly higher than their counterparts.\",\n \"Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\\n2).\\nP value among methylated genes and EGFR mutation\\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \\n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\",\n \"The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\",\n \"We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \\n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \\n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\\n\\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), different genotype of \\n\\nEGFR \\n\\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\\n\\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\\nSimilar to the previous discovery\\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \\n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \\n2D).\",\n \"To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \\n3B), while the epigenotypes of SFRP5 (Figure \\n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \\n3C) were not associated with OS in our patients.\\n\\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), or different genotype of \\n\\nEGFR \\n\\n(C).\\n\",\n \"In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\",\n \"EGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.\",\n \"The authors declare that they have no competing interests.\",\n \"JZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.\",\n \"Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Patients","DNA extraction and methylation-specific PCR","Mutation detection","Statistical analysis","Results","Characteristics of study patients","Epigenotype of Wnt antagonists in NSCLC","Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy","Epigenotype of Wnt antagonists and progression-free survival (PFS)","Epigenotype of Wnt antagonists and overall survival rate (OS)","Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions","Authors’ informations","Supplementary Material"],"string":"[\n \"Background\",\n \"Methods\",\n \"Patients\",\n \"DNA extraction and methylation-specific PCR\",\n \"Mutation detection\",\n \"Statistical analysis\",\n \"Results\",\n \"Characteristics of study patients\",\n \"Epigenotype of Wnt antagonists in NSCLC\",\n \"Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy\",\n \"Epigenotype of Wnt antagonists and progression-free survival (PFS)\",\n \"Epigenotype of Wnt antagonists and overall survival rate (OS)\",\n \"Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Authors’ informations\",\n \"Supplementary Material\"\n]"},"all_sections_texts":{"kind":"list like","value":["Lung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists."," Patients 155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n DNA extraction and methylation-specific PCR Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\nGenomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\n Mutation detection The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\nThe denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\n Statistical analysis All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\nAll data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.","155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.","Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.","The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].","All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant."," Characteristics of study patients Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\nTable\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\n Epigenotype of Wnt antagonists in NSCLC Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\nGenomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\n Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\nThe RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\n Epigenotype of Wnt antagonists and progression-free survival (PFS) We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\nWe next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\n Epigenotype of Wnt antagonists and overall survival rate (OS) To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\nTo test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\n Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\nIn order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.","Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.","Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.","The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).","We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).","To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n","In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.","Recent studies have demonstrated that cancer is as much an epigenetic disease as it is a genetic disease (Iacobuzio-Donahue). Therefore, in addition to genetic alterations, changes in epigenetic features such as CpG DNA methylation status of specific gene loci also mark the progress of cancers. Our current study showed that methylation of Wnt antagonist SFRP5 gene before treatment, independent of the genotype of EGFR gene, correlated with decreased progression free survival rate in NSCLC patients in response to the EGFR-TKI therapy. To our knowledge, this is the first report indicating that DNA methylation at specific gene loci in patient may predict drug response to the EGFT-TKI therapy.\nBoth genetic and epigenetic risk factors for NSCLC have been studied extensively. Suzuki et al\n[23] has reported that methylation of the Wnt antagonist DKK3 correlated with low survival rate in NSCLC patients, despite of the different therapies patients received. However, in our study, we did not find significant difference in the EGFR-TKI responses between patient groups with or without methylated DKK3 (Additional file\n1: Figure S2 and S3). In contrast, our results suggested epigenotype of SFRP5 provide better prognostic estimation for the EGFR-TKI response, comparing to other Wnt antagonists.\nSFRP5 is a member of the SFRP protein family containing a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins. It acts as soluble antagonist of Wnt signaling and is highly expressed in the retinal pigment epithelium, and moderately expressed in the pancreas (\"Entrez Gene: SFRP5 secreted frizzled-related protein 5\"). Previous studies has identified association of SFRP5 promoter hypermethylation with Acute myeloid leukemia\n[29], ovarian cancer\n[30], gastric cancer\n[31], oral squamous cell carcinoma\n[32], pancreatic cancer\n[33] and breast cancer\n[34].\nWe found that hypermethylation of SFRP5 predicted worse outcomes of the EGFR-TKI therapy. Therefore, SFRP5 DNA methylation status may serve as a prognostic molecular marker for appropriately predicting whether NSCLC patients would benefit from the EGFR-TKI therapy. Especially, it is interesting that in the subgroup with adenocarcinoma and EGFR mutation, patients with sFRP5 methylation have a significantly shorter PFS than those without sFRP5 methylation, While in nonsmokers without EGFR mutation, patients without sFRP1 methylation have a longer PFS compared with patients with its methylation(9.7 ms vs 2.0 ms, p = 0.05). Based on these results, we can make a hypothesis that activation of Wnt signaling by antagonist methylation could confer tumors the characters of stem cell, which consequently causes tumors resistant to EGFR TKIs therapy by generating acquired resistance, such as MET amplification or changes of PTEN tumor suppressor activity and so on. Further study is needed to validate this hypothesis.","In conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development\n[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC.","EGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.","The authors declare that they have no competing interests.","JZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.","Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22).","Figure S1. Methylated and unmethyalted bands of Wnt antagonist genes and wild/mutant EGFR. S1: The example graphs of methylated and unmethyalted bands of Wnt antagonist genes (A) and EGFR wild (B) and mutation types (C, D) by methylation specific PCR and DHPLC respectively. Figure S2 PFS with different epigenotypes of Wnt antagonist genes. Figure2S A-F.Kaplan-Meier curves of comparing the progression free survival of patients with different epigenotypes of SFRP1(A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F). Figure S3 OS with different epigenotypes of Wnt antagonist genes. Figure3S A-F. Kaplan-Meier curves of comparing the overall survival of patients with different epigenotypes of SFRP1 (A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F).\nClick here for file"],"string":"[\n \"Lung cancer is the leading cause of cancer death worldwide\\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\\n[7-11].\\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\\n[10,16,17]. In addition, previous studies reported that both T790M mutation\\n[18] and c-MET amplification\\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.\",\n \" Patients 155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\\n155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\\n DNA extraction and methylation-specific PCR Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\\nGenomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\\n Mutation detection The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\\n[28].\\nThe denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\\n[28].\\n Statistical analysis All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\\nAll data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\",\n \"155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\",\n \"Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\",\n \"The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\\n[28].\",\n \"All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\",\n \" Characteristics of study patients Table\\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\\nMethylation and mutation profile of NSCLC\\n*The frequency of this group is significantly higher than their counterparts.\\nTable\\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\\nMethylation and mutation profile of NSCLC\\n*The frequency of this group is significantly higher than their counterparts.\\n Epigenotype of Wnt antagonists in NSCLC Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\\n2).\\nP value among methylated genes and EGFR mutation\\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \\n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\\nGenomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\\n2).\\nP value among methylated genes and EGFR mutation\\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \\n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\\n Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\\nThe RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\\n Epigenotype of Wnt antagonists and progression-free survival (PFS) We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \\n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \\n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\\n\\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), different genotype of \\n\\nEGFR \\n\\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\\n\\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\\nSimilar to the previous discovery\\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \\n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \\n2D).\\nWe next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \\n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \\n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\\n\\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), different genotype of \\n\\nEGFR \\n\\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\\n\\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\\nSimilar to the previous discovery\\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \\n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \\n2D).\\n Epigenotype of Wnt antagonists and overall survival rate (OS) To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \\n3B), while the epigenotypes of SFRP5 (Figure \\n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \\n3C) were not associated with OS in our patients.\\n\\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), or different genotype of \\n\\nEGFR \\n\\n(C).\\n\\nTo test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \\n3B), while the epigenotypes of SFRP5 (Figure \\n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \\n3C) were not associated with OS in our patients.\\n\\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), or different genotype of \\n\\nEGFR \\n\\n(C).\\n\\n Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\\nIn order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\",\n \"Table\\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\\nMethylation and mutation profile of NSCLC\\n*The frequency of this group is significantly higher than their counterparts.\",\n \"Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\\n2).\\nP value among methylated genes and EGFR mutation\\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \\n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\",\n \"The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\",\n \"We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \\n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \\n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\\n\\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), different genotype of \\n\\nEGFR \\n\\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\\n\\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\\nSimilar to the previous discovery\\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \\n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \\n2D).\",\n \"To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \\n3B), while the epigenotypes of SFRP5 (Figure \\n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \\n3C) were not associated with OS in our patients.\\n\\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \\n\\nSFRP5 \\n\\n(A), \\n\\nWIF1 \\n\\n(B), or different genotype of \\n\\nEGFR \\n\\n(C).\\n\",\n \"In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\",\n \"Recent studies have demonstrated that cancer is as much an epigenetic disease as it is a genetic disease (Iacobuzio-Donahue). Therefore, in addition to genetic alterations, changes in epigenetic features such as CpG DNA methylation status of specific gene loci also mark the progress of cancers. Our current study showed that methylation of Wnt antagonist SFRP5 gene before treatment, independent of the genotype of EGFR gene, correlated with decreased progression free survival rate in NSCLC patients in response to the EGFR-TKI therapy. To our knowledge, this is the first report indicating that DNA methylation at specific gene loci in patient may predict drug response to the EGFT-TKI therapy.\\nBoth genetic and epigenetic risk factors for NSCLC have been studied extensively. Suzuki et al\\n[23] has reported that methylation of the Wnt antagonist DKK3 correlated with low survival rate in NSCLC patients, despite of the different therapies patients received. However, in our study, we did not find significant difference in the EGFR-TKI responses between patient groups with or without methylated DKK3 (Additional file\\n1: Figure S2 and S3). In contrast, our results suggested epigenotype of SFRP5 provide better prognostic estimation for the EGFR-TKI response, comparing to other Wnt antagonists.\\nSFRP5 is a member of the SFRP protein family containing a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins. It acts as soluble antagonist of Wnt signaling and is highly expressed in the retinal pigment epithelium, and moderately expressed in the pancreas (\\\"Entrez Gene: SFRP5 secreted frizzled-related protein 5\\\"). Previous studies has identified association of SFRP5 promoter hypermethylation with Acute myeloid leukemia\\n[29], ovarian cancer\\n[30], gastric cancer\\n[31], oral squamous cell carcinoma\\n[32], pancreatic cancer\\n[33] and breast cancer\\n[34].\\nWe found that hypermethylation of SFRP5 predicted worse outcomes of the EGFR-TKI therapy. Therefore, SFRP5 DNA methylation status may serve as a prognostic molecular marker for appropriately predicting whether NSCLC patients would benefit from the EGFR-TKI therapy. Especially, it is interesting that in the subgroup with adenocarcinoma and EGFR mutation, patients with sFRP5 methylation have a significantly shorter PFS than those without sFRP5 methylation, While in nonsmokers without EGFR mutation, patients without sFRP1 methylation have a longer PFS compared with patients with its methylation(9.7 ms vs 2.0 ms, p = 0.05). Based on these results, we can make a hypothesis that activation of Wnt signaling by antagonist methylation could confer tumors the characters of stem cell, which consequently causes tumors resistant to EGFR TKIs therapy by generating acquired resistance, such as MET amplification or changes of PTEN tumor suppressor activity and so on. Further study is needed to validate this hypothesis.\",\n \"In conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development\\n[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC.\",\n \"EGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.\",\n \"The authors declare that they have no competing interests.\",\n \"JZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.\",\n \"Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22).\",\n \"Figure S1. Methylated and unmethyalted bands of Wnt antagonist genes and wild/mutant EGFR. S1: The example graphs of methylated and unmethyalted bands of Wnt antagonist genes (A) and EGFR wild (B) and mutation types (C, D) by methylation specific PCR and DHPLC respectively. Figure S2 PFS with different epigenotypes of Wnt antagonist genes. Figure2S A-F.Kaplan-Meier curves of comparing the progression free survival of patients with different epigenotypes of SFRP1(A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F). Figure S3 OS with different epigenotypes of Wnt antagonist genes. Figure3S A-F. Kaplan-Meier curves of comparing the overall survival of patients with different epigenotypes of SFRP1 (A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F).\\nClick here for file\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,"results",null,null,null,null,null,null,"discussion","conclusions",null,null,null,null,"supplementary-material"],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null,\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["DNA methylation","EGFR-TKI","Wnt antagonists","Non-small cell lung cancer"],"string":"[\n \"DNA methylation\",\n \"EGFR-TKI\",\n \"Wnt antagonists\",\n \"Non-small cell lung cancer\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nLung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.\n\nMethods:\n Patients 155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n DNA extraction and methylation-specific PCR Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\nGenomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\n Mutation detection The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\nThe denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\n Statistical analysis All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\nAll data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\n\nPatients:\n155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n\nDNA extraction and methylation-specific PCR:\nGenomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\n\nMutation detection:\nThe denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\n\nStatistical analysis:\nAll data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\n\nResults:\n Characteristics of study patients Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\nTable\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\n Epigenotype of Wnt antagonists in NSCLC Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\nGenomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\n Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\nThe RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\n Epigenotype of Wnt antagonists and progression-free survival (PFS) We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\nWe next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\n Epigenotype of Wnt antagonists and overall survival rate (OS) To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\nTo test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\n Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\nIn order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\n\nCharacteristics of study patients:\nTable\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\n\nEpigenotype of Wnt antagonists in NSCLC:\nGenomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\n\nEpigenotype of Wnt antagonist genes and clinical responses to TKI therapy:\nThe RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\n\nEpigenotype of Wnt antagonists and progression-free survival (PFS):\nWe next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\n\nEpigenotype of Wnt antagonists and overall survival rate (OS):\nTo test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\n\nCorrelation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy:\nIn order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\n\nDiscussion:\nRecent studies have demonstrated that cancer is as much an epigenetic disease as it is a genetic disease (Iacobuzio-Donahue). Therefore, in addition to genetic alterations, changes in epigenetic features such as CpG DNA methylation status of specific gene loci also mark the progress of cancers. Our current study showed that methylation of Wnt antagonist SFRP5 gene before treatment, independent of the genotype of EGFR gene, correlated with decreased progression free survival rate in NSCLC patients in response to the EGFR-TKI therapy. To our knowledge, this is the first report indicating that DNA methylation at specific gene loci in patient may predict drug response to the EGFT-TKI therapy.\nBoth genetic and epigenetic risk factors for NSCLC have been studied extensively. Suzuki et al\n[23] has reported that methylation of the Wnt antagonist DKK3 correlated with low survival rate in NSCLC patients, despite of the different therapies patients received. However, in our study, we did not find significant difference in the EGFR-TKI responses between patient groups with or without methylated DKK3 (Additional file\n1: Figure S2 and S3). In contrast, our results suggested epigenotype of SFRP5 provide better prognostic estimation for the EGFR-TKI response, comparing to other Wnt antagonists.\nSFRP5 is a member of the SFRP protein family containing a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins. It acts as soluble antagonist of Wnt signaling and is highly expressed in the retinal pigment epithelium, and moderately expressed in the pancreas (\"Entrez Gene: SFRP5 secreted frizzled-related protein 5\"). Previous studies has identified association of SFRP5 promoter hypermethylation with Acute myeloid leukemia\n[29], ovarian cancer\n[30], gastric cancer\n[31], oral squamous cell carcinoma\n[32], pancreatic cancer\n[33] and breast cancer\n[34].\nWe found that hypermethylation of SFRP5 predicted worse outcomes of the EGFR-TKI therapy. Therefore, SFRP5 DNA methylation status may serve as a prognostic molecular marker for appropriately predicting whether NSCLC patients would benefit from the EGFR-TKI therapy. Especially, it is interesting that in the subgroup with adenocarcinoma and EGFR mutation, patients with sFRP5 methylation have a significantly shorter PFS than those without sFRP5 methylation, While in nonsmokers without EGFR mutation, patients without sFRP1 methylation have a longer PFS compared with patients with its methylation(9.7 ms vs 2.0 ms, p = 0.05). Based on these results, we can make a hypothesis that activation of Wnt signaling by antagonist methylation could confer tumors the characters of stem cell, which consequently causes tumors resistant to EGFR TKIs therapy by generating acquired resistance, such as MET amplification or changes of PTEN tumor suppressor activity and so on. Further study is needed to validate this hypothesis.\n\nConclusions:\nIn conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development\n[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC.\n\nAbbreviations:\nEGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nJZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.\n\nAuthors’ informations:\nSupported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22).\n\nSupplementary Material:\nFigure S1. Methylated and unmethyalted bands of Wnt antagonist genes and wild/mutant EGFR. S1: The example graphs of methylated and unmethyalted bands of Wnt antagonist genes (A) and EGFR wild (B) and mutation types (C, D) by methylation specific PCR and DHPLC respectively. Figure S2 PFS with different epigenotypes of Wnt antagonist genes. Figure2S A-F.Kaplan-Meier curves of comparing the progression free survival of patients with different epigenotypes of SFRP1(A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F). Figure S3 OS with different epigenotypes of Wnt antagonist genes. Figure3S A-F. Kaplan-Meier curves of comparing the overall survival of patients with different epigenotypes of SFRP1 (A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F).\nClick here for file\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIt is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy.\n\nMethods:\nTumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC).\n\nResults:\nWe found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype.\n\nConclusions:\nPatients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy."},"background_conclusion_text":{"kind":"string","value":"Background:\nLung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.\n\nConclusions:\nIn conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development\n[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nIt is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy.\n\nMethods:\nTumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC).\n\nResults:\nWe found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype.\n\nConclusions:\nPatients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy."},"whole_article_text_length":{"kind":"number","value":10366,"string":"10,366"},"whole_article_abstract_length":{"kind":"number","value":266,"string":"266"},"num_sections":{"kind":"number","value":20,"string":"20"},"most_frequent_words":{"kind":"list like","value":["egfr","patients","methylation","wnt","sfrp5","tki","therapy","pfs","95","95 ci"],"string":"[\n \"egfr\",\n \"patients\",\n \"methylation\",\n \"wnt\",\n \"sfrp5\",\n \"tki\",\n \"therapy\",\n \"pfs\",\n \"95\",\n \"95 ci\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | lung | lung cancer | cancer | therapy | wnt | tki | signaling | egfr tki | tki therapy [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] unmethylatedf | dna | defined | disease | pcr | pairs | methylatedf | primer pairs | primer | specific [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ci | 95 ci | 95 | patients | egfr | sfrp5 | pfs | months | wnt | significantly [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] drugs | detection | dna methylation | dna | egfr tki | protein rna | sfrp5 independent | pfs long | conclusion study revealed sfrp5 | conclusion study revealed [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | wnt | methylation | tki | sfrp5 | 95 | 95 ci | ci | therapy [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] egfr | patients | wnt | methylation | tki | sfrp5 | 95 | 95 ci | ci | therapy [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] EGFR | EGFR ||| EGFR [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 155 | EGFR | Wnt | SFRP1 | SFRP5 | DKK3 | APC | PCR | MSP ||| EGFR | Denaturing High Performance Liquid Chromatography (DHPLC [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Wnt ||| EGFR | 0.005 | 0.011 ||| Wnt | EGFR ||| SFRP5 | SFRP5 | EGFR | 0.002 | EGFR [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] SFRP5 | EGFR [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] EGFR | EGFR ||| EGFR ||| 155 | EGFR | Wnt | SFRP1 | SFRP5 | DKK3 | APC | PCR | MSP ||| EGFR | Denaturing High Performance Liquid Chromatography (DHPLC ||| ||| Wnt ||| EGFR | 0.005 | 0.011 ||| Wnt | EGFR ||| SFRP5 | SFRP5 | EGFR | 0.002 | EGFR ||| SFRP5 | EGFR [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] EGFR | EGFR ||| EGFR ||| 155 | EGFR | Wnt | SFRP1 | SFRP5 | DKK3 | APC | PCR | MSP ||| EGFR | Denaturing High Performance Liquid Chromatography (DHPLC ||| ||| Wnt ||| EGFR | 0.005 | 0.011 ||| Wnt | EGFR ||| SFRP5 | SFRP5 | EGFR | 0.002 | EGFR ||| SFRP5 | EGFR [SUMMARY]"}}},{"rowIdx":286,"cells":{"title":{"kind":"string","value":"Factors Associated with Increased Alpha-Tocopherol Content in Milk in Response to Maternal Supplementation with 800 IU of Vitamin E."},"pmid":{"kind":"string","value":"31013594"},"background_abstract":{"kind":"string","value":"Vitamin E supplementation might represent an efficient strategy to increase the vitamin E content in milk. The present study aimed to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol content of milk and the factors associated with the increase in vitamin E."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Randomized clinical trial with 79 lactating women from Brazil, who were assigned to the control group, or to the supplemented group (800 IU of RRR-alpha-tocopherol). Milk and serum were collected between 30 and 90 days after delivery (collection 1), and on the next day (collection 2). Alpha-tocopherol was analyzed using high-performance liquid chromatography."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In the supplemented group, the alpha-tocopherol content in serum and milk increased after supplementation (p < 0.001). In the multivariate analysis, only alpha-tocopherol in milk (collection 1) was associated with the level of this vitamin in milk after supplementation (β = 0.927, p < 0.001), and binary logistic regression showed that the dietary intake was the only determinant for the greater effect of supplementation in milk."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The pre-existing vitamin level in milk and diet are determinants for the efficacy of supplementation in milk, suggesting that in populations with vitamin E deficiency, high-dose supplementation can be used to restore its level in milk."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Breast Feeding","Dietary Supplements","Female","Humans","Logistic Models","Maternal Nutritional Physiological Phenomena","Milk, Human","Vitamin E","Young Adult","alpha-Tocopherol"],"string":"[\n \"Adult\",\n \"Breast Feeding\",\n \"Dietary Supplements\",\n \"Female\",\n \"Humans\",\n \"Logistic Models\",\n \"Maternal Nutritional Physiological Phenomena\",\n \"Milk, Human\",\n \"Vitamin E\",\n \"Young Adult\",\n \"alpha-Tocopherol\"\n]"},"pmcid":{"kind":"string","value":"6520676"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Breast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].\nStudies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].\nOne strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].\nSingle-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.\nInterestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.\nThus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":" 3.1. General Characteristics of the Population The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\nThe socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\n 3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\nAt collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\n 3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\nIn the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively)."},"conclusions_title":{"kind":"string","value":"5. Conclusions"},"conclusions_text":{"kind":"string","value":"Vitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E."},"other_sections_titles":{"kind":"list like","value":["2. Materials and Methods","2.2. Data Collection","2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples","2.4. Dietary Intake of Vitamin E","2.5. Statistical Analysis","3.1. General Characteristics of the Population","3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk","3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation"],"string":"[\n \"2. Materials and Methods\",\n \"2.2. Data Collection\",\n \"2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples\",\n \"2.4. Dietary Intake of Vitamin E\",\n \"2.5. Statistical Analysis\",\n \"3.1. General Characteristics of the Population\",\n \"3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk\",\n \"3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation\"\n]"},"other_sections_texts":{"kind":"list like","value":[" 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.","A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.","The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].","Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.","Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.","The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).","At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.","In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively)."],"string":"[\n \" 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\",\n \"A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\",\n \"The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\",\n \"Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\",\n \"Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\",\n \"The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\",\n \"At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\",\n \"In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Materials and Methods","2.1. Participants and Intervention","2.2. Data Collection","2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples","2.4. Dietary Intake of Vitamin E","2.5. Statistical Analysis","3. Results","3.1. General Characteristics of the Population","3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk","3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation","4. Discussion","5. Conclusions"],"string":"[\n \"1. Introduction\",\n \"2. Materials and Methods\",\n \"2.1. Participants and Intervention\",\n \"2.2. Data Collection\",\n \"2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples\",\n \"2.4. Dietary Intake of Vitamin E\",\n \"2.5. Statistical Analysis\",\n \"3. Results\",\n \"3.1. General Characteristics of the Population\",\n \"3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk\",\n \"3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation\",\n \"4. Discussion\",\n \"5. Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Breast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].\nStudies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].\nOne strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].\nSingle-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.\nInterestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.\nThus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period."," 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.","The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).","A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.","The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].","Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.","Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05."," 3.1. General Characteristics of the Population The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\nThe socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\n 3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\nAt collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\n 3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\nIn the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).","The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).","At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.","In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).","Mature milk is the most stable stage of lactation, in which the content of alpha-tocopherol is not influenced by pregnancy-related factors, as occurs in the colostrum [2,7]. It should be emphasized that the mature milk presents a higher concentration of lipids and in contrast, there is a lower secretion of alpha-tocopherol, suggesting that there are distinct mechanisms involved in the transfer of this vitamin into breast milk [2,35,36].\nIn the present study, the lactating women had adequate vitamin E status, in accordance with other studies considering the same stage of lactation [8,21]. However, a low dietary intake of vitamin E was noted (Table 1), which could trigger the mobilization of alpha-tocopherol from maternal reserves, such as the adipose tissue, into breast milk [15,19]. This low consumption of vitamin E was also reported in other populations in Brazil, Greece and Poland [21,25,36], which suggests a frequent inadequacy in vitamin E consumption during lactation.\nEven in situations of inadequate consumption, the vitamin E concentration in milk was not influenced by diet and circulating maternal levels [8,20,21,37,38,39,40]. However, this present study was the first to identify a positive association between alpha-tocopherol levels in milk and ingested vitamin E (diet + supplementation) and with serum alpha-tocopherol (Figure 2d,c). This suggested that in high-consumption situations, the ingested and circulating maternal levels are the main factors responsible for the vitamin level in milk. It is likely that in situations of low vitamin E consumption (as found in the studies cited), milk vitamin E might originate from other sources, such as the body’s reserve [4], explaining the absence of a relationship between those variables.\nTo prevent of VED in infants, it is necessary for breast milk to contain adequate levels of vitamin E, so that children can obtain the benefits of the micronutrient, through the creation of vitamin reserves in the body and its antioxidant action [4,19]. Some studies that evaluated this vitamin in mature milk observed values below the nutritional requirements of infants [20,23,36,37], which suggested that maternal vitamin E supplementation could be an important strategy to increase milk vitamin contents [17,20,21].\nIn this study, supplementation with 800 IU of alpha-tocopherol caused a 183% increase in serum alpha-tocopherol, and a 124% increase in breast milk alpha-tocopherol (Table 2). Other clinical trials using a lower dose (400 IU alpha-tocopherol) found an increase of 60% to 80% in the vitamin content in milk after supplementation [18,20,21,22], showing a reduced effect compared with that shown in the present study. These findings suggested that the response to supplementation might be influenced by both the dosage used and the determinant factors. However, trials have not evaluated the factors associated with the different responses to large vitamin E doses [17,20], being important to understand how this vitamin is transferred into the mammary gland.\nWhen analyzing the factors associated with a better response in milk after supplementation, it is important to highlight that only the content of this vitamin in the basal milk (before supplementation) and the dietary intake were demonstrated to increase the levels of this micronutrient in milk (Table 3), which suggested that the higher the consumption of vitamin E and its levels in milk, the greater the transfer of alpha-tocopherol from the supplement to the mammary gland.\nAssessment of the profile of lactating women in the supplemented group, showed that the participants with the highest supplementation effect (quartiles 2–4) had a higher intake of calories, alpha-tocopherol and total fat (Table 4). These findings suggested that the amount of fat available in the diet may improve the bioavailability of the vitamin in the body, such as its absorption and distribution to tissues, and in this case, to the mammary gland, as reported in [41,42,43].\nSuch evidence also demonstrated that in mature milk, the Michaelis–Menten kinetic theory could not be applied, as it proposes that the transfer of vitamin to milk occurs through active transport, characterized by a saturation of the lipoprotein receptors in the breast tissue in situations of large contents of vitamin E, which would prevent the continuous transfer of the vitamin into the breast milk after supplementation [38,44]. Notably, in a study of dairy cows, Weiss and Wyatt (2003) [45] suggested that the ability of lipoproteins to carry alpha-tocopherol could determine the uptake of this vitamin by the breast tissue, and that the limiting factor for this mechanism would be the maximum content of vitamin E in the lipoproteins.\nTo further investigate this relationship between lipoproteins and vitamin E transport, we determined the circulating lipoproteins and the serum cholesterol and triglycerides profiles; however, no relation between them and the response to supplementation was found (Table 2). In fact, the mechanism of transport of vitamin E to the mammary gland is poorly understood [2,46,47]. Circulating lipoproteins are responsible for this transfer, with LDL being the main carrier [44,48]; however, transport may occur in the presence or absence of its receptors in the mammary gland [2,44]. Other receptors are found in breast tissue, such as Scavenger Receptor B-1 (SR-B1), which has binding sites for both LDL and HDL, and CD36, which has high affinity binding sites for HDL, LDL, and very low-density lipoprotein (VLDL) [2,48]. It has also been suggested the participation of lipoprotein lipase (LPL), which may show increased activity during lactation, contributes to the greater circulation of alpha-tocopherol and its uptake [49].\nThese findings provide important information to understand the mechanisms by which vitamin E is transferred into the mammary gland, demonstrating that, in situations of supplementation with 800 IU of vitamin E and its effect in mature milk, the better the vitamin E status (considering the milk and dietary intake), the more effective uptake into the mammary gland will be, regardless of receptor saturation. Further investigation into how this transport occurs is required, by means of in vitro and in vivo studies and using labeled isotopes of alpha-tocopherol, for example, to investigate its biotransformation. Notably, in populations with dietary inadequacy and low contents of vitamin E in milk, supplementation with higher doses of the vitamin, such as 800 IU alpha-tocopherol, might be required to obtain a more effective intervention. The analysis of a single dose during the day allowed us to investigate possible factors that could interfere with the response to supplementation; however, it is necessary to analyze how long the effect of this supplementation could be sustained, its contribution to maternal and infant nutritional status, and the use of smaller daily doses.\nTherefore, supplementation associated with an adequate intake of vitamin E is an effective strategy to increase vitamin E levels in breast milk and prevent cases of vitamin E deficiency in infants, especially premature infants [2,4,11].","Vitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E."],"string":"[\n \"Breast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].\\nStudies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].\\nOne strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].\\nSingle-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.\\nInterestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.\\nThus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period.\",\n \" 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\",\n \"The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\",\n \"A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\",\n \"The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\",\n \"Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\",\n \"Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\",\n \" 3.1. General Characteristics of the Population The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\\nThe socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\\n 3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\\nAt collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\\n 3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\\nIn the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\",\n \"The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\",\n \"At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\",\n \"In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\",\n \"Mature milk is the most stable stage of lactation, in which the content of alpha-tocopherol is not influenced by pregnancy-related factors, as occurs in the colostrum [2,7]. It should be emphasized that the mature milk presents a higher concentration of lipids and in contrast, there is a lower secretion of alpha-tocopherol, suggesting that there are distinct mechanisms involved in the transfer of this vitamin into breast milk [2,35,36].\\nIn the present study, the lactating women had adequate vitamin E status, in accordance with other studies considering the same stage of lactation [8,21]. However, a low dietary intake of vitamin E was noted (Table 1), which could trigger the mobilization of alpha-tocopherol from maternal reserves, such as the adipose tissue, into breast milk [15,19]. This low consumption of vitamin E was also reported in other populations in Brazil, Greece and Poland [21,25,36], which suggests a frequent inadequacy in vitamin E consumption during lactation.\\nEven in situations of inadequate consumption, the vitamin E concentration in milk was not influenced by diet and circulating maternal levels [8,20,21,37,38,39,40]. However, this present study was the first to identify a positive association between alpha-tocopherol levels in milk and ingested vitamin E (diet + supplementation) and with serum alpha-tocopherol (Figure 2d,c). This suggested that in high-consumption situations, the ingested and circulating maternal levels are the main factors responsible for the vitamin level in milk. It is likely that in situations of low vitamin E consumption (as found in the studies cited), milk vitamin E might originate from other sources, such as the body’s reserve [4], explaining the absence of a relationship between those variables.\\nTo prevent of VED in infants, it is necessary for breast milk to contain adequate levels of vitamin E, so that children can obtain the benefits of the micronutrient, through the creation of vitamin reserves in the body and its antioxidant action [4,19]. Some studies that evaluated this vitamin in mature milk observed values below the nutritional requirements of infants [20,23,36,37], which suggested that maternal vitamin E supplementation could be an important strategy to increase milk vitamin contents [17,20,21].\\nIn this study, supplementation with 800 IU of alpha-tocopherol caused a 183% increase in serum alpha-tocopherol, and a 124% increase in breast milk alpha-tocopherol (Table 2). Other clinical trials using a lower dose (400 IU alpha-tocopherol) found an increase of 60% to 80% in the vitamin content in milk after supplementation [18,20,21,22], showing a reduced effect compared with that shown in the present study. These findings suggested that the response to supplementation might be influenced by both the dosage used and the determinant factors. However, trials have not evaluated the factors associated with the different responses to large vitamin E doses [17,20], being important to understand how this vitamin is transferred into the mammary gland.\\nWhen analyzing the factors associated with a better response in milk after supplementation, it is important to highlight that only the content of this vitamin in the basal milk (before supplementation) and the dietary intake were demonstrated to increase the levels of this micronutrient in milk (Table 3), which suggested that the higher the consumption of vitamin E and its levels in milk, the greater the transfer of alpha-tocopherol from the supplement to the mammary gland.\\nAssessment of the profile of lactating women in the supplemented group, showed that the participants with the highest supplementation effect (quartiles 2–4) had a higher intake of calories, alpha-tocopherol and total fat (Table 4). These findings suggested that the amount of fat available in the diet may improve the bioavailability of the vitamin in the body, such as its absorption and distribution to tissues, and in this case, to the mammary gland, as reported in [41,42,43].\\nSuch evidence also demonstrated that in mature milk, the Michaelis–Menten kinetic theory could not be applied, as it proposes that the transfer of vitamin to milk occurs through active transport, characterized by a saturation of the lipoprotein receptors in the breast tissue in situations of large contents of vitamin E, which would prevent the continuous transfer of the vitamin into the breast milk after supplementation [38,44]. Notably, in a study of dairy cows, Weiss and Wyatt (2003) [45] suggested that the ability of lipoproteins to carry alpha-tocopherol could determine the uptake of this vitamin by the breast tissue, and that the limiting factor for this mechanism would be the maximum content of vitamin E in the lipoproteins.\\nTo further investigate this relationship between lipoproteins and vitamin E transport, we determined the circulating lipoproteins and the serum cholesterol and triglycerides profiles; however, no relation between them and the response to supplementation was found (Table 2). In fact, the mechanism of transport of vitamin E to the mammary gland is poorly understood [2,46,47]. Circulating lipoproteins are responsible for this transfer, with LDL being the main carrier [44,48]; however, transport may occur in the presence or absence of its receptors in the mammary gland [2,44]. Other receptors are found in breast tissue, such as Scavenger Receptor B-1 (SR-B1), which has binding sites for both LDL and HDL, and CD36, which has high affinity binding sites for HDL, LDL, and very low-density lipoprotein (VLDL) [2,48]. It has also been suggested the participation of lipoprotein lipase (LPL), which may show increased activity during lactation, contributes to the greater circulation of alpha-tocopherol and its uptake [49].\\nThese findings provide important information to understand the mechanisms by which vitamin E is transferred into the mammary gland, demonstrating that, in situations of supplementation with 800 IU of vitamin E and its effect in mature milk, the better the vitamin E status (considering the milk and dietary intake), the more effective uptake into the mammary gland will be, regardless of receptor saturation. Further investigation into how this transport occurs is required, by means of in vitro and in vivo studies and using labeled isotopes of alpha-tocopherol, for example, to investigate its biotransformation. Notably, in populations with dietary inadequacy and low contents of vitamin E in milk, supplementation with higher doses of the vitamin, such as 800 IU alpha-tocopherol, might be required to obtain a more effective intervention. The analysis of a single dose during the day allowed us to investigate possible factors that could interfere with the response to supplementation; however, it is necessary to analyze how long the effect of this supplementation could be sustained, its contribution to maternal and infant nutritional status, and the use of smaller daily doses.\\nTherefore, supplementation associated with an adequate intake of vitamin E is an effective strategy to increase vitamin E levels in breast milk and prevent cases of vitamin E deficiency in infants, especially premature infants [2,4,11].\",\n \"Vitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,"subjects",null,null,null,null,"results",null,null,null,"discussion","conclusions"],"string":"[\n \"intro\",\n null,\n \"subjects\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["clinical trial","lactation","infants","breastfeeding","lactating women"],"string":"[\n \"clinical trial\",\n \"lactation\",\n \"infants\",\n \"breastfeeding\",\n \"lactating women\"\n]"},"whole_article_text":{"kind":"string","value":"1. Introduction:\nBreast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].\nStudies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].\nOne strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].\nSingle-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.\nInterestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.\nThus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period.\n\n2. Materials and Methods:\n 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\n\n2.1. Participants and Intervention:\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\n\n2.2. Data Collection:\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\n\n2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples:\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\n\n2.4. Dietary Intake of Vitamin E:\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\n\n2.5. Statistical Analysis:\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\n\n3. Results:\n 3.1. General Characteristics of the Population The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\nThe socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\n 3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\nAt collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\n 3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\nIn the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\n\n3.1. General Characteristics of the Population:\nThe socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\n\n3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk:\nAt collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\n\n3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation:\nIn the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\n\n4. Discussion:\nMature milk is the most stable stage of lactation, in which the content of alpha-tocopherol is not influenced by pregnancy-related factors, as occurs in the colostrum [2,7]. It should be emphasized that the mature milk presents a higher concentration of lipids and in contrast, there is a lower secretion of alpha-tocopherol, suggesting that there are distinct mechanisms involved in the transfer of this vitamin into breast milk [2,35,36].\nIn the present study, the lactating women had adequate vitamin E status, in accordance with other studies considering the same stage of lactation [8,21]. However, a low dietary intake of vitamin E was noted (Table 1), which could trigger the mobilization of alpha-tocopherol from maternal reserves, such as the adipose tissue, into breast milk [15,19]. This low consumption of vitamin E was also reported in other populations in Brazil, Greece and Poland [21,25,36], which suggests a frequent inadequacy in vitamin E consumption during lactation.\nEven in situations of inadequate consumption, the vitamin E concentration in milk was not influenced by diet and circulating maternal levels [8,20,21,37,38,39,40]. However, this present study was the first to identify a positive association between alpha-tocopherol levels in milk and ingested vitamin E (diet + supplementation) and with serum alpha-tocopherol (Figure 2d,c). This suggested that in high-consumption situations, the ingested and circulating maternal levels are the main factors responsible for the vitamin level in milk. It is likely that in situations of low vitamin E consumption (as found in the studies cited), milk vitamin E might originate from other sources, such as the body’s reserve [4], explaining the absence of a relationship between those variables.\nTo prevent of VED in infants, it is necessary for breast milk to contain adequate levels of vitamin E, so that children can obtain the benefits of the micronutrient, through the creation of vitamin reserves in the body and its antioxidant action [4,19]. Some studies that evaluated this vitamin in mature milk observed values below the nutritional requirements of infants [20,23,36,37], which suggested that maternal vitamin E supplementation could be an important strategy to increase milk vitamin contents [17,20,21].\nIn this study, supplementation with 800 IU of alpha-tocopherol caused a 183% increase in serum alpha-tocopherol, and a 124% increase in breast milk alpha-tocopherol (Table 2). Other clinical trials using a lower dose (400 IU alpha-tocopherol) found an increase of 60% to 80% in the vitamin content in milk after supplementation [18,20,21,22], showing a reduced effect compared with that shown in the present study. These findings suggested that the response to supplementation might be influenced by both the dosage used and the determinant factors. However, trials have not evaluated the factors associated with the different responses to large vitamin E doses [17,20], being important to understand how this vitamin is transferred into the mammary gland.\nWhen analyzing the factors associated with a better response in milk after supplementation, it is important to highlight that only the content of this vitamin in the basal milk (before supplementation) and the dietary intake were demonstrated to increase the levels of this micronutrient in milk (Table 3), which suggested that the higher the consumption of vitamin E and its levels in milk, the greater the transfer of alpha-tocopherol from the supplement to the mammary gland.\nAssessment of the profile of lactating women in the supplemented group, showed that the participants with the highest supplementation effect (quartiles 2–4) had a higher intake of calories, alpha-tocopherol and total fat (Table 4). These findings suggested that the amount of fat available in the diet may improve the bioavailability of the vitamin in the body, such as its absorption and distribution to tissues, and in this case, to the mammary gland, as reported in [41,42,43].\nSuch evidence also demonstrated that in mature milk, the Michaelis–Menten kinetic theory could not be applied, as it proposes that the transfer of vitamin to milk occurs through active transport, characterized by a saturation of the lipoprotein receptors in the breast tissue in situations of large contents of vitamin E, which would prevent the continuous transfer of the vitamin into the breast milk after supplementation [38,44]. Notably, in a study of dairy cows, Weiss and Wyatt (2003) [45] suggested that the ability of lipoproteins to carry alpha-tocopherol could determine the uptake of this vitamin by the breast tissue, and that the limiting factor for this mechanism would be the maximum content of vitamin E in the lipoproteins.\nTo further investigate this relationship between lipoproteins and vitamin E transport, we determined the circulating lipoproteins and the serum cholesterol and triglycerides profiles; however, no relation between them and the response to supplementation was found (Table 2). In fact, the mechanism of transport of vitamin E to the mammary gland is poorly understood [2,46,47]. Circulating lipoproteins are responsible for this transfer, with LDL being the main carrier [44,48]; however, transport may occur in the presence or absence of its receptors in the mammary gland [2,44]. Other receptors are found in breast tissue, such as Scavenger Receptor B-1 (SR-B1), which has binding sites for both LDL and HDL, and CD36, which has high affinity binding sites for HDL, LDL, and very low-density lipoprotein (VLDL) [2,48]. It has also been suggested the participation of lipoprotein lipase (LPL), which may show increased activity during lactation, contributes to the greater circulation of alpha-tocopherol and its uptake [49].\nThese findings provide important information to understand the mechanisms by which vitamin E is transferred into the mammary gland, demonstrating that, in situations of supplementation with 800 IU of vitamin E and its effect in mature milk, the better the vitamin E status (considering the milk and dietary intake), the more effective uptake into the mammary gland will be, regardless of receptor saturation. Further investigation into how this transport occurs is required, by means of in vitro and in vivo studies and using labeled isotopes of alpha-tocopherol, for example, to investigate its biotransformation. Notably, in populations with dietary inadequacy and low contents of vitamin E in milk, supplementation with higher doses of the vitamin, such as 800 IU alpha-tocopherol, might be required to obtain a more effective intervention. The analysis of a single dose during the day allowed us to investigate possible factors that could interfere with the response to supplementation; however, it is necessary to analyze how long the effect of this supplementation could be sustained, its contribution to maternal and infant nutritional status, and the use of smaller daily doses.\nTherefore, supplementation associated with an adequate intake of vitamin E is an effective strategy to increase vitamin E levels in breast milk and prevent cases of vitamin E deficiency in infants, especially premature infants [2,4,11].\n\n5. Conclusions:\nVitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nVitamin E supplementation might represent an efficient strategy to increase the vitamin E content in milk. The present study aimed to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol content of milk and the factors associated with the increase in vitamin E.\n\nMethods:\nRandomized clinical trial with 79 lactating women from Brazil, who were assigned to the control group, or to the supplemented group (800 IU of RRR-alpha-tocopherol). Milk and serum were collected between 30 and 90 days after delivery (collection 1), and on the next day (collection 2). Alpha-tocopherol was analyzed using high-performance liquid chromatography.\n\nResults:\nIn the supplemented group, the alpha-tocopherol content in serum and milk increased after supplementation (p < 0.001). In the multivariate analysis, only alpha-tocopherol in milk (collection 1) was associated with the level of this vitamin in milk after supplementation (β = 0.927, p < 0.001), and binary logistic regression showed that the dietary intake was the only determinant for the greater effect of supplementation in milk.\n\nConclusions:\nThe pre-existing vitamin level in milk and diet are determinants for the efficacy of supplementation in milk, suggesting that in populations with vitamin E deficiency, high-dose supplementation can be used to restore its level in milk."},"background_conclusion_text":{"kind":"string","value":"1. Introduction:\nBreast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].\nStudies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].\nOne strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].\nSingle-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.\nInterestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.\nThus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period.\n\n5. Conclusions:\nVitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nVitamin E supplementation might represent an efficient strategy to increase the vitamin E content in milk. The present study aimed to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol content of milk and the factors associated with the increase in vitamin E.\n\nMethods:\nRandomized clinical trial with 79 lactating women from Brazil, who were assigned to the control group, or to the supplemented group (800 IU of RRR-alpha-tocopherol). Milk and serum were collected between 30 and 90 days after delivery (collection 1), and on the next day (collection 2). Alpha-tocopherol was analyzed using high-performance liquid chromatography.\n\nResults:\nIn the supplemented group, the alpha-tocopherol content in serum and milk increased after supplementation (p < 0.001). In the multivariate analysis, only alpha-tocopherol in milk (collection 1) was associated with the level of this vitamin in milk after supplementation (β = 0.927, p < 0.001), and binary logistic regression showed that the dietary intake was the only determinant for the greater effect of supplementation in milk.\n\nConclusions:\nThe pre-existing vitamin level in milk and diet are determinants for the efficacy of supplementation in milk, suggesting that in populations with vitamin E deficiency, high-dose supplementation can be used to restore its level in milk."},"whole_article_text_length":{"kind":"number","value":9199,"string":"9,199"},"whole_article_abstract_length":{"kind":"number","value":266,"string":"266"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["vitamin","milk","alpha","tocopherol","alpha tocopherol","supplementation","collection","group","serum","intake"],"string":"[\n \"vitamin\",\n \"milk\",\n \"alpha\",\n 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Alpha ||| ||| ||| 1 | 0.927 ||| [SUMMARY]"}}},{"rowIdx":287,"cells":{"title":{"kind":"string","value":"Mobile Phone Apps to Promote Weight Loss and Increase Physical Activity: A Systematic Review and Meta-Analysis."},"pmid":{"kind":"string","value":"26554314"},"background_abstract":{"kind":"string","value":"To our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We conducted a systematic review and meta-analysis of relevant studies identified by a search of PubMed, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), and Scopus from their inception through to August 2015. Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted data. We included all controlled studies that assessed a mobile phone app intervention with weight-related health measures (ie, body weight, body mass index, or waist circumference) or physical activity outcomes. Net change estimates comparing the intervention group with the control group were pooled across studies using random-effects models."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"We included 12 articles in this systematic review and meta-analysis. Compared with the control group, use of a mobile phone app was associated with significant changes in body weight (kg) and body mass index (kg/m(2)) of -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) and -0.43 kg/m(2) (95% CI -0.74 to -0.13; I2 = 50%), respectively. Moreover, a nonsignificant difference in physical activity was observed between the two groups (standardized mean difference 0.40, 95% CI -0.07 to 0.87; I2 = 93%). These findings were remarkably robust in the sensitivity analysis. No publication bias was shown."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Evidence from this study shows that mobile phone app-based interventions may be useful tools for weight loss."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Cell Phone","Humans","Mobile Applications","Motor Activity","Obesity","Weight Loss"],"string":"[\n \"Cell Phone\",\n \"Humans\",\n \"Mobile Applications\",\n \"Motor Activity\",\n \"Obesity\",\n \"Weight Loss\"\n]"},"pmcid":{"kind":"string","value":"4704965"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Overweight and obesity are a global public health issue and an important feature in discussions about strategies for primary and secondary health care. Developing since the 1960s and now gathering pace rapidly, the issue is contributing, together with population aging, to major increases in the prevalence of high blood pressure and cholesterol levels, type 2 diabetes, and cancers [1]. Mortality rates are increasing with increasing degrees of overweight, as measured by body mass index (BMI) [2].\nIn 2008, 35% of adults older than 20 years were overweight (BMI ≥ 25 kg/m2) and the worldwide prevalence of obesity (BMI ≥30 kg/m2) had nearly doubled since 1980, from 5% of men and 8% of women to 10% and 14%, respectively [2]. An estimated 205 million men and 297 million women were obese—a total of more than half a billion adults worldwide [3]. For these reasons, identifying effective interventions is an important component in public health efforts to curb obesity, but the most effective strategies for weight loss remain unclear.\nWith the extensive market penetration of mobile phones, the International Telecommunications Union (ITU) reports that as of 2015, there are more than 7 billion mobile cellular subscriptions worldwide, corresponding to a 97% penetration rate—defined by ITU as mobile cellular telephone subscribers per 100 inhabitants [4]. Advanced-feature mobile phones (those with computer operating systems) have broadened the functions of mobile phones considerably. Mobile phone apps meet a variety of user needs, and are designed and adapted for each type of mobile device; therefore, they are applicable in nearly all social and economic sectors and environments. At present, these apps, apart from their recreational function, are becoming instruments of patient education and support and are also helpful to health care professionals [5]. Nonetheless, the market for health care apps is very fragmented because many of them are very specific or directed at minority diseases or specialties. The world market for medical apps for mobile phones and tablets multiplied seven times over in 2011 alone, reaching a total of US $718 million according to a market analysis by the American firm research2guidance [6]. A recent analysis of app store catalogs identified more than 97,000 mHealth apps, most of them dealing with general health and physical fitness; in general, they facilitate the monitoring of various parameters by individual users and provide general information and support related to those topics [5]. Previous research has suggested that mobile apps may be beneficial in asthma control [7] and diabetes management [8,9].\nTo our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity. The objective of this study was to perform a systematic review and meta-analysis of published studies to evaluate the efficacy of interventions that included mobile phone apps compared with other interventions to reduce weight and increase physical activity in populations of children and adults."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Search Strategy We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\nWe conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\n Study Selection Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\nTwo members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\n Data Extraction and Quality Assessment Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\nTwo investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\n Statistical Methods For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\nFor each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration)."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Study Selection The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\nThe search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\n Meta-Analysis of Mobile App Intervention and Body Weight Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Body Mass Index Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Physical Activity Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Risk of Bias in Included Studies Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\nRandomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Search Strategy","Study Selection","Data Extraction and Quality Assessment","Statistical Methods","Study Selection","Meta-Analysis of Mobile App Intervention and Body Weight","Meta-Analysis of Mobile App Intervention and Body Mass Index","Meta-Analysis of Mobile App Intervention and Physical Activity","Risk of Bias in Included Studies"],"string":"[\n \"Search Strategy\",\n \"Study Selection\",\n \"Data Extraction and Quality Assessment\",\n \"Statistical Methods\",\n \"Study Selection\",\n \"Meta-Analysis of Mobile App Intervention and Body Weight\",\n \"Meta-Analysis of Mobile App Intervention and Body Mass Index\",\n \"Meta-Analysis of Mobile App Intervention and Physical Activity\",\n \"Risk of Bias in Included Studies\"\n]"},"other_sections_texts":{"kind":"list like","value":["We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.","Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.","Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.","For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).","The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.","Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study."],"string":"[\n \"We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\",\n \"Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\\nFlowchart for the selection of the articles in this meta-analysis.\",\n \"Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\",\n \"For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\\nSD2\\ndiff= SD2\\npre+ SD2\\npost- 2×ρ×SDpre×SDpost\\n\\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\",\n \"The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\\nCharacteristics of included clinical trials.\\n\\naSS: sample size.\\n\\nbCCS: case-control study.\\n\\ncN/A: not applicable.\\n\\ndBMI: body mass index.\\n\\neRCT: randomized controlled trial.\\n\\nfMCCT: matched case-control trial.\\n\\ngMPA: moderate physical activity.\\n\\nhVPA: vigorous physical activity.\\nCharacteristics of intervention types and description of apps.\\n\\naBMI: body mass index.\\n\\nbMPA: moderate physical activity.\\n\\ncVPA: vigorous physical activity.\\n\\ndSMS: short message service.\",\n \"Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Search Strategy","Study Selection","Data Extraction and Quality Assessment","Statistical Methods","Results","Study Selection","Meta-Analysis of Mobile App Intervention and Body Weight","Meta-Analysis of Mobile App Intervention and Body Mass Index","Meta-Analysis of Mobile App Intervention and Physical Activity","Risk of Bias in Included Studies","Discussion"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Search Strategy\",\n \"Study Selection\",\n \"Data Extraction and Quality Assessment\",\n \"Statistical Methods\",\n \"Results\",\n \"Study Selection\",\n \"Meta-Analysis of Mobile App Intervention and Body Weight\",\n \"Meta-Analysis of Mobile App Intervention and Body Mass Index\",\n \"Meta-Analysis of Mobile App Intervention and Physical Activity\",\n \"Risk of Bias in Included Studies\",\n \"Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Overweight and obesity are a global public health issue and an important feature in discussions about strategies for primary and secondary health care. Developing since the 1960s and now gathering pace rapidly, the issue is contributing, together with population aging, to major increases in the prevalence of high blood pressure and cholesterol levels, type 2 diabetes, and cancers [1]. Mortality rates are increasing with increasing degrees of overweight, as measured by body mass index (BMI) [2].\nIn 2008, 35% of adults older than 20 years were overweight (BMI ≥ 25 kg/m2) and the worldwide prevalence of obesity (BMI ≥30 kg/m2) had nearly doubled since 1980, from 5% of men and 8% of women to 10% and 14%, respectively [2]. An estimated 205 million men and 297 million women were obese—a total of more than half a billion adults worldwide [3]. For these reasons, identifying effective interventions is an important component in public health efforts to curb obesity, but the most effective strategies for weight loss remain unclear.\nWith the extensive market penetration of mobile phones, the International Telecommunications Union (ITU) reports that as of 2015, there are more than 7 billion mobile cellular subscriptions worldwide, corresponding to a 97% penetration rate—defined by ITU as mobile cellular telephone subscribers per 100 inhabitants [4]. Advanced-feature mobile phones (those with computer operating systems) have broadened the functions of mobile phones considerably. Mobile phone apps meet a variety of user needs, and are designed and adapted for each type of mobile device; therefore, they are applicable in nearly all social and economic sectors and environments. At present, these apps, apart from their recreational function, are becoming instruments of patient education and support and are also helpful to health care professionals [5]. Nonetheless, the market for health care apps is very fragmented because many of them are very specific or directed at minority diseases or specialties. The world market for medical apps for mobile phones and tablets multiplied seven times over in 2011 alone, reaching a total of US $718 million according to a market analysis by the American firm research2guidance [6]. A recent analysis of app store catalogs identified more than 97,000 mHealth apps, most of them dealing with general health and physical fitness; in general, they facilitate the monitoring of various parameters by individual users and provide general information and support related to those topics [5]. Previous research has suggested that mobile apps may be beneficial in asthma control [7] and diabetes management [8,9].\nTo our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity. The objective of this study was to perform a systematic review and meta-analysis of published studies to evaluate the efficacy of interventions that included mobile phone apps compared with other interventions to reduce weight and increase physical activity in populations of children and adults."," Search Strategy We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\nWe conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\n Study Selection Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\nTwo members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\n Data Extraction and Quality Assessment Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\nTwo investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\n Statistical Methods For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\nFor each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).","We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.","Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.","Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.","For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration)."," Study Selection The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\nThe search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\n Meta-Analysis of Mobile App Intervention and Body Weight Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Body Mass Index Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Physical Activity Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Risk of Bias in Included Studies Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\nRandomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.","The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.","Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.","Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.","The current meta-analysis suggested that mobile phone app interventions compared with various control interventions significantly reduced body weight by 1.04 kg, reduced BMI by 0.43 kg/m2, and nonsignificantly increased physical activity by an SMD of 0.40. Our findings were robust across sensitivity analyses. Although the mean reductions in body weight and BMI were modest, it would not be expected for a single change in weight-loss interventions, such as mobile phone apps, to cause clinically meaningful weight loss compared with other control interventions [25]. Many of the control group treatments were other interventions. This could dilute the analysis, as it is possible that in some of the studies the treatment group showed a significant change, while the control group also showed a similar significant result. In our sensitivity analyses, the results were not modified when we excluded one study that did not describe if the control group had received any intervention [13].\nSome of the most recognizable research in mobile interventions has focused on text-messaging interventions, or SMS. A previous meta-analysis [26] found that mobile phone interventions were associated with significant changes in body weight and BMI compared with the control group (-1.44 kg and -0.24 units, respectively). This meta-analysis included only mobile interventions based on contacts by SMS and multimedia message services (MMS). A previous systematic review found strong evidence from the included RCT that weight loss occurs in the short term because of mobile technology interventions [27]. Another systematic review that included seven articles demonstrated a beneficial impact of text messaging or a mobile app for reducing physical inactivity and/or overweight/obesity [28]. Finally, a recent systematic review found that most apps that are focused on weight loss have inconsistent outcomes [29].\nTo our knowledge, this study is the first meta-analysis to summarize the evidence to date regarding effects of mobile phone app interventions compared with various control interventions. We excluded from our meta-analysis interventions based only on text messaging and focused solely on mobile phone apps because text-message interventions do not utilize the full potential of mobile phone technologies. Well-designed apps expand the potential for technology-based health interventions to impact populations in ways that previously were not possible and cannot be achieved without the capabilities of mobile phone software. Therefore, the need to regulate this growing market is becoming a concern, with increased advertising claims about effectiveness and researchers emphasizing the need for studies that will contribute scientific evidence about the true impact of these types of apps. The portability of mobile phones enables users to have access 24 hours a day, making possible the long-term management and reinforcement of health behaviors through a variety of communications and apps. Fitness and weight-loss mobile phone apps allow for the tracking of diet, weight, and physical activity; making grocery and restaurant decisions; cooking healthy meals [28]; or gamification of the intervention. Moreover, participants do not need to carry an extra piece of purchased technology, such as a pedometer, to track physical activity.\nSeveral limitations have been noted. Only a small number of available studies assess the effectiveness of mobile phone apps in weight-loss programs, and they included small sample sizes and short follow-up periods. The use of apps for improving physical activity and reducing anthropometric measures is relatively new. More randomized controlled trials with larger sample sizes and longer follow-up periods are needed to determine the effectiveness of mobile apps in improving health outcomes.\nA recent study aimed to evaluate diet/nutrition and anthropometric apps based on incorporation of features consistent with theories of behavior change; all apps were found to be very low in theoretical content or use of theory to guide behavior change [28]. The studies included in this meta-analysis also varied in the content and theoretical basis of the intervention. Further investigation into the effective features of the mobile phone apps and the interventions’ consistencies with theories of behavior change was not possible; this should be considered an area for future research.\nThe risk of bias was high in most of the studies and future research should improve on several issues, such as the use of blinding or improving attrition rate. All studies, except for one [19], failed to conceal the blinding of participants and personnel, and only one study [24] blinded the research staff collecting the data on outcomes as to the allocation of participants. Given the nature of the intervention, blinding of participants and personnel is difficult. However, it is important to recognize the possible influence of patient and personnel expectations. Therefore, adoption of blinding techniques, such as the use of sham procedures, blinding participants to the study hypothesis, or using a blinded centralized assessment of primary outcomes, will improve the quality of the evidence [30].\nA large attrition rate was noted in some of the studies [17,20] included in this meta-analysis. High attrition rates are common in weight-loss interventions, and the reasons for this are likely complex and varied [31]. Attrition has an obvious impact on the validity of results obtained and can introduce bias; for example, those more motivated to reduce their weight or increase physical activity may remain in the trial. Moreover, several studies found that most participants rarely used the app after the first month of the study [20,23]. As with other weight-loss interventions, the most effective app may be one that can engage people for the longest period. It is known that adherence to self-monitoring of food intake is associated with twice as much weight loss as infrequent monitoring [32]. Without the participant’s active engagement, the app is not likely to be used and as a result will not be effective [28]. The most highly ranked engagement strategies identified are (in order of preference) ease of use, design aesthetic, feedback, function, ability to change design to suit own preference, tailored information, and unique mobile phone features [33]. Weight-loss apps may need to be substantially more engaging or less time-consuming to produce weight reduction in the average individual. It would be useful to design strategies to increase “app appeal” before implementing this type of intervention. Gamification of the app, financial incentives, or delivering the app in a setting of group competition could be important adjuncts to increase motivation to use the app and lose weight [20,34].\nTo our knowledge, this is the first meta-analysis to summarize the effectiveness of mobile apps designed to improve physical activity and reduce anthropometric measures. Our meta-analysis highlighted the need to perform larger, high-quality, randomized controlled clinical trials with longer follow-up. The number of available mobile phone apps is growing steadily, and mobile phones are constantly undergoing updates so the features have changed over time. Incorporating features consistent with theories of behavior change into health-related apps would be useful to improve weight-loss outcomes [35]. We searched several databases in order to avoid publication bias, which is a concern in meta-analyses that only include published studies. Using funnel plots in our meta-analysis made it possible to exclude publication bias with some confidence.\nAs the world’s understanding of health and how to empower individuals to take better care of their health changes, health professionals must treat this change as progress. However, we must ensure that the patient is using a mobile app with appropriate quality guarantees. This meta-analysis aimed to provide a rigorous, systematic, and quantitative review of the studies that have analyzed the effectiveness of mobile apps and attempted to measure their influence on lifestyle changes. Bombarded by information overload in all arenas, health professionals and managers have a need for the insights provided by a tool like the meta-analysis; this could help them make decisions and decide what direction we should be moving in our efforts to promote weight loss, increase physical activity, and confront the public health crisis presented by overweight and obesity.\nIn summary, the results from this meta-analysis demonstrated that interventions based on mobile phone apps are associated with more weight loss than other types of interventions. Furthermore, a nonsignificant increase in physical activity was detected. Evidence form this meta-analysis shows that mobile phone app-based intervention may be useful tools for weight loss."],"string":"[\n \"Overweight and obesity are a global public health issue and an important feature in discussions about strategies for primary and secondary health care. Developing since the 1960s and now gathering pace rapidly, the issue is contributing, together with population aging, to major increases in the prevalence of high blood pressure and cholesterol levels, type 2 diabetes, and cancers [1]. Mortality rates are increasing with increasing degrees of overweight, as measured by body mass index (BMI) [2].\\nIn 2008, 35% of adults older than 20 years were overweight (BMI ≥ 25 kg/m2) and the worldwide prevalence of obesity (BMI ≥30 kg/m2) had nearly doubled since 1980, from 5% of men and 8% of women to 10% and 14%, respectively [2]. An estimated 205 million men and 297 million women were obese—a total of more than half a billion adults worldwide [3]. For these reasons, identifying effective interventions is an important component in public health efforts to curb obesity, but the most effective strategies for weight loss remain unclear.\\nWith the extensive market penetration of mobile phones, the International Telecommunications Union (ITU) reports that as of 2015, there are more than 7 billion mobile cellular subscriptions worldwide, corresponding to a 97% penetration rate—defined by ITU as mobile cellular telephone subscribers per 100 inhabitants [4]. Advanced-feature mobile phones (those with computer operating systems) have broadened the functions of mobile phones considerably. Mobile phone apps meet a variety of user needs, and are designed and adapted for each type of mobile device; therefore, they are applicable in nearly all social and economic sectors and environments. At present, these apps, apart from their recreational function, are becoming instruments of patient education and support and are also helpful to health care professionals [5]. Nonetheless, the market for health care apps is very fragmented because many of them are very specific or directed at minority diseases or specialties. The world market for medical apps for mobile phones and tablets multiplied seven times over in 2011 alone, reaching a total of US $718 million according to a market analysis by the American firm research2guidance [6]. A recent analysis of app store catalogs identified more than 97,000 mHealth apps, most of them dealing with general health and physical fitness; in general, they facilitate the monitoring of various parameters by individual users and provide general information and support related to those topics [5]. Previous research has suggested that mobile apps may be beneficial in asthma control [7] and diabetes management [8,9].\\nTo our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity. The objective of this study was to perform a systematic review and meta-analysis of published studies to evaluate the efficacy of interventions that included mobile phone apps compared with other interventions to reduce weight and increase physical activity in populations of children and adults.\",\n \" Search Strategy We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\\nWe conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\\n Study Selection Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\\nFlowchart for the selection of the articles in this meta-analysis.\\nTwo members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\\nFlowchart for the selection of the articles in this meta-analysis.\\n Data Extraction and Quality Assessment Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\\nTwo investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\\n Statistical Methods For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\\nSD2\\ndiff= SD2\\npre+ SD2\\npost- 2×ρ×SDpre×SDpost\\n\\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\\nFor each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\\nSD2\\ndiff= SD2\\npre+ SD2\\npost- 2×ρ×SDpre×SDpost\\n\\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\",\n \"We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\",\n \"Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\\nFlowchart for the selection of the articles in this meta-analysis.\",\n \"Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\",\n \"For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\\nSD2\\ndiff= SD2\\npre+ SD2\\npost- 2×ρ×SDpre×SDpost\\n\\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\",\n \" Study Selection The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\\nCharacteristics of included clinical trials.\\n\\naSS: sample size.\\n\\nbCCS: case-control study.\\n\\ncN/A: not applicable.\\n\\ndBMI: body mass index.\\n\\neRCT: randomized controlled trial.\\n\\nfMCCT: matched case-control trial.\\n\\ngMPA: moderate physical activity.\\n\\nhVPA: vigorous physical activity.\\nCharacteristics of intervention types and description of apps.\\n\\naBMI: body mass index.\\n\\nbMPA: moderate physical activity.\\n\\ncVPA: vigorous physical activity.\\n\\ndSMS: short message service.\\nThe search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\\nCharacteristics of included clinical trials.\\n\\naSS: sample size.\\n\\nbCCS: case-control study.\\n\\ncN/A: not applicable.\\n\\ndBMI: body mass index.\\n\\neRCT: randomized controlled trial.\\n\\nfMCCT: matched case-control trial.\\n\\ngMPA: moderate physical activity.\\n\\nhVPA: vigorous physical activity.\\nCharacteristics of intervention types and description of apps.\\n\\naBMI: body mass index.\\n\\nbMPA: moderate physical activity.\\n\\ncVPA: vigorous physical activity.\\n\\ndSMS: short message service.\\n Meta-Analysis of Mobile App Intervention and Body Weight Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\nData from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\n Meta-Analysis of Mobile App Intervention and Body Mass Index Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\nData from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\n Meta-Analysis of Mobile App Intervention and Physical Activity Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\nData from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\\n Risk of Bias in Included Studies Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\\nRandomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\",\n \"The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\\nCharacteristics of included clinical trials.\\n\\naSS: sample size.\\n\\nbCCS: case-control study.\\n\\ncN/A: not applicable.\\n\\ndBMI: body mass index.\\n\\neRCT: randomized controlled trial.\\n\\nfMCCT: matched case-control trial.\\n\\ngMPA: moderate physical activity.\\n\\nhVPA: vigorous physical activity.\\nCharacteristics of intervention types and description of apps.\\n\\naBMI: body mass index.\\n\\nbMPA: moderate physical activity.\\n\\ncVPA: vigorous physical activity.\\n\\ndSMS: short message service.\",\n \"Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\",\n \"Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\",\n \"The current meta-analysis suggested that mobile phone app interventions compared with various control interventions significantly reduced body weight by 1.04 kg, reduced BMI by 0.43 kg/m2, and nonsignificantly increased physical activity by an SMD of 0.40. Our findings were robust across sensitivity analyses. Although the mean reductions in body weight and BMI were modest, it would not be expected for a single change in weight-loss interventions, such as mobile phone apps, to cause clinically meaningful weight loss compared with other control interventions [25]. Many of the control group treatments were other interventions. This could dilute the analysis, as it is possible that in some of the studies the treatment group showed a significant change, while the control group also showed a similar significant result. In our sensitivity analyses, the results were not modified when we excluded one study that did not describe if the control group had received any intervention [13].\\nSome of the most recognizable research in mobile interventions has focused on text-messaging interventions, or SMS. A previous meta-analysis [26] found that mobile phone interventions were associated with significant changes in body weight and BMI compared with the control group (-1.44 kg and -0.24 units, respectively). This meta-analysis included only mobile interventions based on contacts by SMS and multimedia message services (MMS). A previous systematic review found strong evidence from the included RCT that weight loss occurs in the short term because of mobile technology interventions [27]. Another systematic review that included seven articles demonstrated a beneficial impact of text messaging or a mobile app for reducing physical inactivity and/or overweight/obesity [28]. Finally, a recent systematic review found that most apps that are focused on weight loss have inconsistent outcomes [29].\\nTo our knowledge, this study is the first meta-analysis to summarize the evidence to date regarding effects of mobile phone app interventions compared with various control interventions. We excluded from our meta-analysis interventions based only on text messaging and focused solely on mobile phone apps because text-message interventions do not utilize the full potential of mobile phone technologies. Well-designed apps expand the potential for technology-based health interventions to impact populations in ways that previously were not possible and cannot be achieved without the capabilities of mobile phone software. Therefore, the need to regulate this growing market is becoming a concern, with increased advertising claims about effectiveness and researchers emphasizing the need for studies that will contribute scientific evidence about the true impact of these types of apps. The portability of mobile phones enables users to have access 24 hours a day, making possible the long-term management and reinforcement of health behaviors through a variety of communications and apps. Fitness and weight-loss mobile phone apps allow for the tracking of diet, weight, and physical activity; making grocery and restaurant decisions; cooking healthy meals [28]; or gamification of the intervention. Moreover, participants do not need to carry an extra piece of purchased technology, such as a pedometer, to track physical activity.\\nSeveral limitations have been noted. Only a small number of available studies assess the effectiveness of mobile phone apps in weight-loss programs, and they included small sample sizes and short follow-up periods. The use of apps for improving physical activity and reducing anthropometric measures is relatively new. More randomized controlled trials with larger sample sizes and longer follow-up periods are needed to determine the effectiveness of mobile apps in improving health outcomes.\\nA recent study aimed to evaluate diet/nutrition and anthropometric apps based on incorporation of features consistent with theories of behavior change; all apps were found to be very low in theoretical content or use of theory to guide behavior change [28]. The studies included in this meta-analysis also varied in the content and theoretical basis of the intervention. Further investigation into the effective features of the mobile phone apps and the interventions’ consistencies with theories of behavior change was not possible; this should be considered an area for future research.\\nThe risk of bias was high in most of the studies and future research should improve on several issues, such as the use of blinding or improving attrition rate. All studies, except for one [19], failed to conceal the blinding of participants and personnel, and only one study [24] blinded the research staff collecting the data on outcomes as to the allocation of participants. Given the nature of the intervention, blinding of participants and personnel is difficult. However, it is important to recognize the possible influence of patient and personnel expectations. Therefore, adoption of blinding techniques, such as the use of sham procedures, blinding participants to the study hypothesis, or using a blinded centralized assessment of primary outcomes, will improve the quality of the evidence [30].\\nA large attrition rate was noted in some of the studies [17,20] included in this meta-analysis. High attrition rates are common in weight-loss interventions, and the reasons for this are likely complex and varied [31]. Attrition has an obvious impact on the validity of results obtained and can introduce bias; for example, those more motivated to reduce their weight or increase physical activity may remain in the trial. Moreover, several studies found that most participants rarely used the app after the first month of the study [20,23]. As with other weight-loss interventions, the most effective app may be one that can engage people for the longest period. It is known that adherence to self-monitoring of food intake is associated with twice as much weight loss as infrequent monitoring [32]. Without the participant’s active engagement, the app is not likely to be used and as a result will not be effective [28]. The most highly ranked engagement strategies identified are (in order of preference) ease of use, design aesthetic, feedback, function, ability to change design to suit own preference, tailored information, and unique mobile phone features [33]. Weight-loss apps may need to be substantially more engaging or less time-consuming to produce weight reduction in the average individual. It would be useful to design strategies to increase “app appeal” before implementing this type of intervention. Gamification of the app, financial incentives, or delivering the app in a setting of group competition could be important adjuncts to increase motivation to use the app and lose weight [20,34].\\nTo our knowledge, this is the first meta-analysis to summarize the effectiveness of mobile apps designed to improve physical activity and reduce anthropometric measures. Our meta-analysis highlighted the need to perform larger, high-quality, randomized controlled clinical trials with longer follow-up. The number of available mobile phone apps is growing steadily, and mobile phones are constantly undergoing updates so the features have changed over time. Incorporating features consistent with theories of behavior change into health-related apps would be useful to improve weight-loss outcomes [35]. We searched several databases in order to avoid publication bias, which is a concern in meta-analyses that only include published studies. Using funnel plots in our meta-analysis made it possible to exclude publication bias with some confidence.\\nAs the world’s understanding of health and how to empower individuals to take better care of their health changes, health professionals must treat this change as progress. However, we must ensure that the patient is using a mobile app with appropriate quality guarantees. This meta-analysis aimed to provide a rigorous, systematic, and quantitative review of the studies that have analyzed the effectiveness of mobile apps and attempted to measure their influence on lifestyle changes. Bombarded by information overload in all arenas, health professionals and managers have a need for the insights provided by a tool like the meta-analysis; this could help them make decisions and decide what direction we should be moving in our efforts to promote weight loss, increase physical activity, and confront the public health crisis presented by overweight and obesity.\\nIn summary, the results from this meta-analysis demonstrated that interventions based on mobile phone apps are associated with more weight loss than other types of interventions. Furthermore, a nonsignificant increase in physical activity was detected. Evidence form this meta-analysis shows that mobile phone app-based intervention may be useful tools for weight loss.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["introduction","methods",null,null,null,null,"results",null,null,null,null,null,"discussion"],"string":"[\n \"introduction\",\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["mHealth","mobile phone","apps","obesity","physical activity","intervention"],"string":"[\n \"mHealth\",\n \"mobile phone\",\n \"apps\",\n \"obesity\",\n \"physical activity\",\n \"intervention\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nOverweight and obesity are a global public health issue and an important feature in discussions about strategies for primary and secondary health care. Developing since the 1960s and now gathering pace rapidly, the issue is contributing, together with population aging, to major increases in the prevalence of high blood pressure and cholesterol levels, type 2 diabetes, and cancers [1]. Mortality rates are increasing with increasing degrees of overweight, as measured by body mass index (BMI) [2].\nIn 2008, 35% of adults older than 20 years were overweight (BMI ≥ 25 kg/m2) and the worldwide prevalence of obesity (BMI ≥30 kg/m2) had nearly doubled since 1980, from 5% of men and 8% of women to 10% and 14%, respectively [2]. An estimated 205 million men and 297 million women were obese—a total of more than half a billion adults worldwide [3]. For these reasons, identifying effective interventions is an important component in public health efforts to curb obesity, but the most effective strategies for weight loss remain unclear.\nWith the extensive market penetration of mobile phones, the International Telecommunications Union (ITU) reports that as of 2015, there are more than 7 billion mobile cellular subscriptions worldwide, corresponding to a 97% penetration rate—defined by ITU as mobile cellular telephone subscribers per 100 inhabitants [4]. Advanced-feature mobile phones (those with computer operating systems) have broadened the functions of mobile phones considerably. Mobile phone apps meet a variety of user needs, and are designed and adapted for each type of mobile device; therefore, they are applicable in nearly all social and economic sectors and environments. At present, these apps, apart from their recreational function, are becoming instruments of patient education and support and are also helpful to health care professionals [5]. Nonetheless, the market for health care apps is very fragmented because many of them are very specific or directed at minority diseases or specialties. The world market for medical apps for mobile phones and tablets multiplied seven times over in 2011 alone, reaching a total of US $718 million according to a market analysis by the American firm research2guidance [6]. A recent analysis of app store catalogs identified more than 97,000 mHealth apps, most of them dealing with general health and physical fitness; in general, they facilitate the monitoring of various parameters by individual users and provide general information and support related to those topics [5]. Previous research has suggested that mobile apps may be beneficial in asthma control [7] and diabetes management [8,9].\nTo our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity. The objective of this study was to perform a systematic review and meta-analysis of published studies to evaluate the efficacy of interventions that included mobile phone apps compared with other interventions to reduce weight and increase physical activity in populations of children and adults.\n\nMethods:\n Search Strategy We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\nWe conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\n Study Selection Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\nTwo members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\n Data Extraction and Quality Assessment Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\nTwo investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\n Statistical Methods For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\nFor each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\n\nSearch Strategy:\nWe conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\n\nStudy Selection:\nTwo members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\n\nData Extraction and Quality Assessment:\nTwo investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\n\nStatistical Methods:\nFor each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\n\nResults:\n Study Selection The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\nThe search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\n Meta-Analysis of Mobile App Intervention and Body Weight Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Body Mass Index Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Physical Activity Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Risk of Bias in Included Studies Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\nRandomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\n\nStudy Selection:\nThe search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\n\nMeta-Analysis of Mobile App Intervention and Body Weight:\nData from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n\nMeta-Analysis of Mobile App Intervention and Body Mass Index:\nData from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n\nMeta-Analysis of Mobile App Intervention and Physical Activity:\nData from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n\nRisk of Bias in Included Studies:\nRandomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\n\nDiscussion:\nThe current meta-analysis suggested that mobile phone app interventions compared with various control interventions significantly reduced body weight by 1.04 kg, reduced BMI by 0.43 kg/m2, and nonsignificantly increased physical activity by an SMD of 0.40. Our findings were robust across sensitivity analyses. Although the mean reductions in body weight and BMI were modest, it would not be expected for a single change in weight-loss interventions, such as mobile phone apps, to cause clinically meaningful weight loss compared with other control interventions [25]. Many of the control group treatments were other interventions. This could dilute the analysis, as it is possible that in some of the studies the treatment group showed a significant change, while the control group also showed a similar significant result. In our sensitivity analyses, the results were not modified when we excluded one study that did not describe if the control group had received any intervention [13].\nSome of the most recognizable research in mobile interventions has focused on text-messaging interventions, or SMS. A previous meta-analysis [26] found that mobile phone interventions were associated with significant changes in body weight and BMI compared with the control group (-1.44 kg and -0.24 units, respectively). This meta-analysis included only mobile interventions based on contacts by SMS and multimedia message services (MMS). A previous systematic review found strong evidence from the included RCT that weight loss occurs in the short term because of mobile technology interventions [27]. Another systematic review that included seven articles demonstrated a beneficial impact of text messaging or a mobile app for reducing physical inactivity and/or overweight/obesity [28]. Finally, a recent systematic review found that most apps that are focused on weight loss have inconsistent outcomes [29].\nTo our knowledge, this study is the first meta-analysis to summarize the evidence to date regarding effects of mobile phone app interventions compared with various control interventions. We excluded from our meta-analysis interventions based only on text messaging and focused solely on mobile phone apps because text-message interventions do not utilize the full potential of mobile phone technologies. Well-designed apps expand the potential for technology-based health interventions to impact populations in ways that previously were not possible and cannot be achieved without the capabilities of mobile phone software. Therefore, the need to regulate this growing market is becoming a concern, with increased advertising claims about effectiveness and researchers emphasizing the need for studies that will contribute scientific evidence about the true impact of these types of apps. The portability of mobile phones enables users to have access 24 hours a day, making possible the long-term management and reinforcement of health behaviors through a variety of communications and apps. Fitness and weight-loss mobile phone apps allow for the tracking of diet, weight, and physical activity; making grocery and restaurant decisions; cooking healthy meals [28]; or gamification of the intervention. Moreover, participants do not need to carry an extra piece of purchased technology, such as a pedometer, to track physical activity.\nSeveral limitations have been noted. Only a small number of available studies assess the effectiveness of mobile phone apps in weight-loss programs, and they included small sample sizes and short follow-up periods. The use of apps for improving physical activity and reducing anthropometric measures is relatively new. More randomized controlled trials with larger sample sizes and longer follow-up periods are needed to determine the effectiveness of mobile apps in improving health outcomes.\nA recent study aimed to evaluate diet/nutrition and anthropometric apps based on incorporation of features consistent with theories of behavior change; all apps were found to be very low in theoretical content or use of theory to guide behavior change [28]. The studies included in this meta-analysis also varied in the content and theoretical basis of the intervention. Further investigation into the effective features of the mobile phone apps and the interventions’ consistencies with theories of behavior change was not possible; this should be considered an area for future research.\nThe risk of bias was high in most of the studies and future research should improve on several issues, such as the use of blinding or improving attrition rate. All studies, except for one [19], failed to conceal the blinding of participants and personnel, and only one study [24] blinded the research staff collecting the data on outcomes as to the allocation of participants. Given the nature of the intervention, blinding of participants and personnel is difficult. However, it is important to recognize the possible influence of patient and personnel expectations. Therefore, adoption of blinding techniques, such as the use of sham procedures, blinding participants to the study hypothesis, or using a blinded centralized assessment of primary outcomes, will improve the quality of the evidence [30].\nA large attrition rate was noted in some of the studies [17,20] included in this meta-analysis. High attrition rates are common in weight-loss interventions, and the reasons for this are likely complex and varied [31]. Attrition has an obvious impact on the validity of results obtained and can introduce bias; for example, those more motivated to reduce their weight or increase physical activity may remain in the trial. Moreover, several studies found that most participants rarely used the app after the first month of the study [20,23]. As with other weight-loss interventions, the most effective app may be one that can engage people for the longest period. It is known that adherence to self-monitoring of food intake is associated with twice as much weight loss as infrequent monitoring [32]. Without the participant’s active engagement, the app is not likely to be used and as a result will not be effective [28]. The most highly ranked engagement strategies identified are (in order of preference) ease of use, design aesthetic, feedback, function, ability to change design to suit own preference, tailored information, and unique mobile phone features [33]. Weight-loss apps may need to be substantially more engaging or less time-consuming to produce weight reduction in the average individual. It would be useful to design strategies to increase “app appeal” before implementing this type of intervention. Gamification of the app, financial incentives, or delivering the app in a setting of group competition could be important adjuncts to increase motivation to use the app and lose weight [20,34].\nTo our knowledge, this is the first meta-analysis to summarize the effectiveness of mobile apps designed to improve physical activity and reduce anthropometric measures. Our meta-analysis highlighted the need to perform larger, high-quality, randomized controlled clinical trials with longer follow-up. The number of available mobile phone apps is growing steadily, and mobile phones are constantly undergoing updates so the features have changed over time. Incorporating features consistent with theories of behavior change into health-related apps would be useful to improve weight-loss outcomes [35]. We searched several databases in order to avoid publication bias, which is a concern in meta-analyses that only include published studies. Using funnel plots in our meta-analysis made it possible to exclude publication bias with some confidence.\nAs the world’s understanding of health and how to empower individuals to take better care of their health changes, health professionals must treat this change as progress. However, we must ensure that the patient is using a mobile app with appropriate quality guarantees. This meta-analysis aimed to provide a rigorous, systematic, and quantitative review of the studies that have analyzed the effectiveness of mobile apps and attempted to measure their influence on lifestyle changes. Bombarded by information overload in all arenas, health professionals and managers have a need for the insights provided by a tool like the meta-analysis; this could help them make decisions and decide what direction we should be moving in our efforts to promote weight loss, increase physical activity, and confront the public health crisis presented by overweight and obesity.\nIn summary, the results from this meta-analysis demonstrated that interventions based on mobile phone apps are associated with more weight loss than other types of interventions. Furthermore, a nonsignificant increase in physical activity was detected. Evidence form this meta-analysis shows that mobile phone app-based intervention may be useful tools for weight loss.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nTo our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity.\n\nMethods:\nWe conducted a systematic review and meta-analysis of relevant studies identified by a search of PubMed, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), and Scopus from their inception through to August 2015. Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted data. We included all controlled studies that assessed a mobile phone app intervention with weight-related health measures (ie, body weight, body mass index, or waist circumference) or physical activity outcomes. Net change estimates comparing the intervention group with the control group were pooled across studies using random-effects models.\n\nResults:\nWe included 12 articles in this systematic review and meta-analysis. Compared with the control group, use of a mobile phone app was associated with significant changes in body weight (kg) and body mass index (kg/m(2)) of -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) and -0.43 kg/m(2) (95% CI -0.74 to -0.13; I2 = 50%), respectively. Moreover, a nonsignificant difference in physical activity was observed between the two groups (standardized mean difference 0.40, 95% CI -0.07 to 0.87; I2 = 93%). These findings were remarkably robust in the sensitivity analysis. No publication bias was shown.\n\nConclusions:\nEvidence from this study shows that mobile phone app-based interventions may be useful tools for weight loss."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":8752,"string":"8,752"},"whole_article_abstract_length":{"kind":"number","value":320,"string":"320"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["study","mobile","intervention","studies","weight","physical","analysis","activity","physical activity","control"],"string":"[\n \"study\",\n \"mobile\",\n \"intervention\",\n \"studies\",\n \"weight\",\n \"physical\",\n \"analysis\",\n \"activity\",\n \"physical activity\",\n \"control\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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| Humans | Mobile Applications | Motor Activity | Obesity | Weight Loss [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] study | mobile | intervention | studies | weight | physical | analysis | activity | physical activity | control 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[SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] intervention | sd | study | baseline | end | end intervention | weight | body weight | search | baseline end intervention [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] mobile | pooled | kg | change | intervention | study | trials | 95 | app | 13 [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mobile | study | intervention | physical | weight | studies | physical activity | activity | change | app [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] PubMed | Allied Health Literature (CINAHL | August 2015 ||| Two ||| ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 12 ||| kg/m(2 | -1.04 kg | 95% | CI | I2 | 41% | 95% | CI | I2 | 50% ||| two | 0.40 | 95% | CI | 0.87 | I2 | 93% ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| PubMed | Allied Health Literature (CINAHL | August 2015 ||| Two ||| ||| ||| ||| 12 ||| kg/m(2 | -1.04 kg | 95% | CI | I2 | 41% | 95% | CI | I2 | 50% ||| two | 0.40 | 95% | CI | 0.87 | I2 | 93% ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":288,"cells":{"title":{"kind":"string","value":"Association of simultaneously measured four-limb blood pressures with cardiovascular function: a cross-sectional study."},"pmid":{"kind":"string","value":"28155695"},"background_abstract":{"kind":"string","value":"Simultaneous measurement of four-limb blood pressures can improve the accuracy of cardiovascular disease diagnosis. This study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the non-invasive diagnostic method of cardiovascular disease in primary care."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled. Four-limb blood pressure measurements were simultaneously performed using a blood pressure and pulse monitor device in the supine position. Cardiac functional parameters were also measured by using a cardiac hemodynamic detector in the same position. Data were statistically analyzed with SPSS15.0."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The mean age of the 229 subjects was 60.56 ± 8.68 years. Cardiovascular functional parameters decreased with age and body mass index (BMI), only the total peripheral resistance (TPR) was in contrast. Age, BMI, left ankle diastolic pressure (LADP), high arm mean arterial pressure (HARMAP), left arm diastolic pressure (LARDP) and right ankle diastolic pressure (RADP) were significantly correlated with cardiovascular functional parameters. Cardiovascular functional parameters have significant differences with inter-arm difference in systolic blood pressure (SBP) between ≥10 and <10 mmHg, inter-ankle difference in SBP between ≥15 and ≥20 mmHg, inter-ankle difference in SBP between ≥15 and <10 mmHg and right ankle brachial index (RABI) between ≤0.9 and ≥1.0. After excluding 99 hypertensive patients, a part of cardiovascular functional parameters has still significant differences with inter-arm difference in SBP between ≥10 and ≥15 mmHg and RABI between ≤0.9 and ≥1.0."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Age, BMI, LADP, HARMAP, LARDP and RADP were the determinants of cardiovascular functional parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in SBP ≥10 mmHg, inter-ankle difference in SBP ≥15 mmHg and RABI ≤0.9, while these differences still existed after excluding 99 hypertensive patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the non-invasive diagnostic method of cardiovascular disease in primary care."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Ankle Brachial Index","Arterial Pressure","Blood Pressure","Blood Pressure Determination","Cardiovascular Diseases","Cardiovascular System","Computer Simulation","Cross-Sectional Studies","Female","Hemodynamics","Humans","Hypertension","Linear Models","Male","Middle Aged","Models, Statistical","Primary Health Care","Risk Assessment"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Ankle Brachial Index\",\n \"Arterial Pressure\",\n \"Blood Pressure\",\n \"Blood Pressure Determination\",\n \"Cardiovascular Diseases\",\n \"Cardiovascular System\",\n \"Computer Simulation\",\n \"Cross-Sectional Studies\",\n \"Female\",\n \"Hemodynamics\",\n \"Humans\",\n \"Hypertension\",\n \"Linear Models\",\n \"Male\",\n \"Middle Aged\",\n \"Models, Statistical\",\n \"Primary Health Care\",\n \"Risk Assessment\"\n]"},"pmcid":{"kind":"string","value":"5259996"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Baseline characteristics of subjects The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\nThe mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\n Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\nPearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\n Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\nMultiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\n Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\nIn additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\nSince cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"LADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care."},"other_sections_titles":{"kind":"list like","value":["Background","Subjects","Four-limb blood pressure measurements","Cardiovascular function measurements","Statistical analysis","Baseline characteristics of subjects","Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters","Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters","Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters","Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup"],"string":"[\n \"Background\",\n \"Subjects\",\n \"Four-limb blood pressure measurements\",\n \"Cardiovascular function measurements\",\n \"Statistical analysis\",\n \"Baseline characteristics of subjects\",\n \"Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters\",\n \"Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters\",\n \"Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters\",\n \"Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup\"\n]"},"other_sections_texts":{"kind":"list like","value":["Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.","This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.","Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.","Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.","Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.","The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants","Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.","Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters","In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences","Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0."],"string":"[\n \"Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.\",\n \"This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\",\n \"Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\",\n \"Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\n\\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\",\n \"Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\",\n \"The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\\n\\nBaseline characteristics of the study participants\",\n \"Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\",\n \"Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\\n\\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\",\n \"In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\n\\nThe distribution of four-limb blood pressure difference in the subjects\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\",\n \"Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Subjects","Four-limb blood pressure measurements","Cardiovascular function measurements","Statistical analysis","Results","Baseline characteristics of subjects","Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters","Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters","Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters","Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup","Discussion","Conclusion"],"string":"[\n \"Background\",\n \"Methods\",\n \"Subjects\",\n \"Four-limb blood pressure measurements\",\n \"Cardiovascular function measurements\",\n \"Statistical analysis\",\n \"Results\",\n \"Baseline characteristics of subjects\",\n \"Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters\",\n \"Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters\",\n \"Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters\",\n \"Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup\",\n \"Discussion\",\n \"Conclusion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care."," Subjects This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\nThis study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\n Four-limb blood pressure measurements Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\nFour-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\n Cardiovascular function measurements Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\nCardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\n Statistical analysis Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\nData were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.","This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.","Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.","Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.","Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05."," Baseline characteristics of subjects The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\nThe mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\n Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\nPearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\n Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\nMultiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\n Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\nIn additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\nSince cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.","The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants","Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.","Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters","In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences","Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.","In this cross-sectional study, by using a simultaneous measurement technique, the association between four-limb blood pressure and cardiovascular function and related risk factors was evaluated. Cardiovascular functional parameters illustrated the cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity. Thus, this study has more systematicness and pertinence than previous studies. This study suggested that cardiovascular functional parameters (CO, SV, CI, SVI, EF, IC, HI, EDV, LVEDP, SWI, AC and TPR) have all a significant difference with HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP, while there was a negative correlation between them except TPR. Meanwhile, the determinants of cardiac functional parameter were age, BMI, LADP, HARMAP LARDP, HARPPI ADBPD, RARSP, RADP and LARSP, while the TPR was positively correlated with age, BMI, LADP and HARMAP.\nPrevious studies demonstrated that population aging has become the uncontrolled risk factor of cardiovascular disease [16–18]. It is a physiology problem that the effect between blood pressure and cardiovascular function is mutual. The cardiovascular function decreased gradually with age, especially arteries can produce physiological degeneration. The thickening of vessels wall, the decrease or even rupture and calcification of elastic fibers, the increase of peripheral resistance, the decrease of blood vessel compliance, all these factors will result in the systolic blood pressure and diastolic blood pressure increasing with age. In addition, it is a fluid mechanics problem that different arteries away from heart have different resistance and pressure. According to the Poiseuille’s law, brachial artery resistance is relatively small and the blood pressure is low because heart is close to the upper limb, while the phenomenon of the ankle artery is in contrast. Hence blood pressures in four limbs are different. In addition, the distance from the heart affects pressure wave propagation and reflection because of the geometry tapper and elasticity tapper, and augments the pressure of peripheral vascular compared with that of upper limb.\nPrevious studies have reported that inter-arm or inter-ankle blood pressure difference predicted cardiovascular mortality [1, 2, 8–13, 15]. These results suggested that the simultaneous measurement of four-limb blood pressure is needed to improve diagnostic accuracy between cardiovascular disease and blood pressure difference [2–5]. The present study demonstrated the significant correlation between cardiovascular functional parameters and four-limb blood pressure, while cardiovascular functional parameters had no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI. However, the mean plots of cardiovascular functional parameters present obvious change trend in four-limb systolic blood pressure difference and diastolic blood pressure difference ≥10 or ≥15 mmHg. These results were different form previous studies. Verberk et al. [19] reported that the prevalence of inter-arm difference in systolic blood pressure of 10 mmHg or more was roughly doubled when diagnosis measurements method used a sequential approach, or used manual approach rather than automated measurements approach. Thus, simultaneous measurement of four-limb blood pressure and calculation of four-limb blood pressure difference may be helpful and necessary in evaluating the heart function and predicting patients with cardiovascular disease. Further study, the subgroup analysis showed that the predictive value of four limbs blood pressure for cardiovascular function. Subgroup analysis found that a part of cardiovascular functional parameter has still significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and with RABI between ≤0.9 and ≥1.0. However, the exact reason is unclear now, hypertension is the influence factors of cardiovascular function parameters, and four-limb blood pressure differences are also applicable to diagnose hypertensive patients. As mentioned above, four limbs blood pressures were not only influence factors of cardiovascular function parameters, but a predictor for cardiovascular disease, evaluating cardiovascular function parameters was important in clinical practice and epidemiological studies. In addition, the better way for cardiovascular function parameters was simultaneous blood pressures for four limbs.\nA clinical device, the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China), has been developed to automatically and simultaneously measure blood pressure in four limbs, and the measurement can be easily obtained. The similar device was reported by previous studies, for example, VP-1000 ABI—form device (Colin Co. Ltd., Komaki, Japan) and VP-1000 device (Omron, Kyoto, Japan) were used to automatically and simultaneously measure four limbs’ blood pressures. The validation in measurement technology of device (Boundless Horizon Company, Shandong, China) might be prone for measurement errors, especially in multinomial detection of cardiac function parameters rather than single detection. Although it could not give very precise measurements for the clinical value of cardiac function parameters, it can provide objective evaluation of health in the early stage of cardiovascular diseases. Thus, these devices can be applied for the epidemiological evaluation between four limbs’ blood pressures and cardiac function parameters.\nIn this study, by using a simultaneous and noninvasive measurement technique, we measured four limbs blood pressures. But this technique was not a popular one to measure blood pressure in daily clinical practice. Hence, although four limbs blood pressure with simultaneous measurement could improve the predictive value for cardiovascular disease, our results might be changed if daily clinical measurement was used. In addition, the subjects were mainly from retired people, whose health care consciousness is better than the serving officer. The factors of smoking, drinking, salting and movement have no significant difference in this study subjects. Hence, the clinical utility of this study may be limited in community people.","LADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care."],"string":"[\n \"Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.\",\n \" Subjects This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\\nThis study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\\n Four-limb blood pressure measurements Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\\nFour-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\\n Cardiovascular function measurements Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\n\\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\\nCardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\n\\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\\n Statistical analysis Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\\nData were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\",\n \"This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\",\n \"Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\",\n \"Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\n\\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\",\n \"Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\",\n \" Baseline characteristics of subjects The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\\n\\nBaseline characteristics of the study participants\\nThe mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\\n\\nBaseline characteristics of the study participants\\n Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\\nPearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\\n Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\\n\\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\\nMultiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\\n\\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\\n Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\n\\nThe distribution of four-limb blood pressure difference in the subjects\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\nIn additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\n\\nThe distribution of four-limb blood pressure difference in the subjects\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\n Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\\nSince cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\",\n \"The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\\n\\nBaseline characteristics of the study participants\",\n \"Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\",\n \"Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\\n\\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\",\n \"In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\\n\\nThe distribution of four-limb blood pressure difference in the subjects\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\",\n \"Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\",\n \"In this cross-sectional study, by using a simultaneous measurement technique, the association between four-limb blood pressure and cardiovascular function and related risk factors was evaluated. Cardiovascular functional parameters illustrated the cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity. Thus, this study has more systematicness and pertinence than previous studies. This study suggested that cardiovascular functional parameters (CO, SV, CI, SVI, EF, IC, HI, EDV, LVEDP, SWI, AC and TPR) have all a significant difference with HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP, while there was a negative correlation between them except TPR. Meanwhile, the determinants of cardiac functional parameter were age, BMI, LADP, HARMAP LARDP, HARPPI ADBPD, RARSP, RADP and LARSP, while the TPR was positively correlated with age, BMI, LADP and HARMAP.\\nPrevious studies demonstrated that population aging has become the uncontrolled risk factor of cardiovascular disease [16–18]. It is a physiology problem that the effect between blood pressure and cardiovascular function is mutual. The cardiovascular function decreased gradually with age, especially arteries can produce physiological degeneration. The thickening of vessels wall, the decrease or even rupture and calcification of elastic fibers, the increase of peripheral resistance, the decrease of blood vessel compliance, all these factors will result in the systolic blood pressure and diastolic blood pressure increasing with age. In addition, it is a fluid mechanics problem that different arteries away from heart have different resistance and pressure. According to the Poiseuille’s law, brachial artery resistance is relatively small and the blood pressure is low because heart is close to the upper limb, while the phenomenon of the ankle artery is in contrast. Hence blood pressures in four limbs are different. In addition, the distance from the heart affects pressure wave propagation and reflection because of the geometry tapper and elasticity tapper, and augments the pressure of peripheral vascular compared with that of upper limb.\\nPrevious studies have reported that inter-arm or inter-ankle blood pressure difference predicted cardiovascular mortality [1, 2, 8–13, 15]. These results suggested that the simultaneous measurement of four-limb blood pressure is needed to improve diagnostic accuracy between cardiovascular disease and blood pressure difference [2–5]. The present study demonstrated the significant correlation between cardiovascular functional parameters and four-limb blood pressure, while cardiovascular functional parameters had no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI. However, the mean plots of cardiovascular functional parameters present obvious change trend in four-limb systolic blood pressure difference and diastolic blood pressure difference ≥10 or ≥15 mmHg. These results were different form previous studies. Verberk et al. [19] reported that the prevalence of inter-arm difference in systolic blood pressure of 10 mmHg or more was roughly doubled when diagnosis measurements method used a sequential approach, or used manual approach rather than automated measurements approach. Thus, simultaneous measurement of four-limb blood pressure and calculation of four-limb blood pressure difference may be helpful and necessary in evaluating the heart function and predicting patients with cardiovascular disease. Further study, the subgroup analysis showed that the predictive value of four limbs blood pressure for cardiovascular function. Subgroup analysis found that a part of cardiovascular functional parameter has still significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and with RABI between ≤0.9 and ≥1.0. However, the exact reason is unclear now, hypertension is the influence factors of cardiovascular function parameters, and four-limb blood pressure differences are also applicable to diagnose hypertensive patients. As mentioned above, four limbs blood pressures were not only influence factors of cardiovascular function parameters, but a predictor for cardiovascular disease, evaluating cardiovascular function parameters was important in clinical practice and epidemiological studies. In addition, the better way for cardiovascular function parameters was simultaneous blood pressures for four limbs.\\nA clinical device, the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China), has been developed to automatically and simultaneously measure blood pressure in four limbs, and the measurement can be easily obtained. The similar device was reported by previous studies, for example, VP-1000 ABI—form device (Colin Co. Ltd., Komaki, Japan) and VP-1000 device (Omron, Kyoto, Japan) were used to automatically and simultaneously measure four limbs’ blood pressures. The validation in measurement technology of device (Boundless Horizon Company, Shandong, China) might be prone for measurement errors, especially in multinomial detection of cardiac function parameters rather than single detection. Although it could not give very precise measurements for the clinical value of cardiac function parameters, it can provide objective evaluation of health in the early stage of cardiovascular diseases. Thus, these devices can be applied for the epidemiological evaluation between four limbs’ blood pressures and cardiac function parameters.\\nIn this study, by using a simultaneous and noninvasive measurement technique, we measured four limbs blood pressures. But this technique was not a popular one to measure blood pressure in daily clinical practice. Hence, although four limbs blood pressure with simultaneous measurement could improve the predictive value for cardiovascular disease, our results might be changed if daily clinical measurement was used. In addition, the subjects were mainly from retired people, whose health care consciousness is better than the serving officer. The factors of smoking, drinking, salting and movement have no significant difference in this study subjects. Hence, the clinical utility of this study may be limited in community people.\",\n \"LADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"materials|methods",null,null,null,null,"results",null,null,null,null,null,"discussion","conclusion"],"string":"[\n null,\n \"materials|methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusion\"\n]"},"keywords":{"kind":"list like","value":["Cardiovascular function","Four limbs","Blood pressure difference","Simultaneous measurement"],"string":"[\n \"Cardiovascular function\",\n \"Four limbs\",\n \"Blood pressure difference\",\n \"Simultaneous measurement\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nAccurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.\n\nMethods:\n Subjects This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\nThis study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\n Four-limb blood pressure measurements Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\nFour-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\n Cardiovascular function measurements Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\nCardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\n Statistical analysis Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\nData were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\n\nSubjects:\nThis study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\n\nFour-limb blood pressure measurements:\nFour-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\n\nCardiovascular function measurements:\nCardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\n\nStatistical analysis:\nData were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\n\nResults:\n Baseline characteristics of subjects The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\nThe mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\n Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\nPearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\n Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\nMultiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\n Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\nIn additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\nSince cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\n\nBaseline characteristics of subjects:\nThe mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\n\nPearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters:\nPearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\n\nMultiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters:\nMultiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\n\nVariance analysis between four-limb blood pressure differences and cardiovascular functional parameters:\nIn additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nAnalysis between four-limb blood pressures and cardiovascular functional parameters in subgroup:\nSince cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\n\nDiscussion:\nIn this cross-sectional study, by using a simultaneous measurement technique, the association between four-limb blood pressure and cardiovascular function and related risk factors was evaluated. Cardiovascular functional parameters illustrated the cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity. Thus, this study has more systematicness and pertinence than previous studies. This study suggested that cardiovascular functional parameters (CO, SV, CI, SVI, EF, IC, HI, EDV, LVEDP, SWI, AC and TPR) have all a significant difference with HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP, while there was a negative correlation between them except TPR. Meanwhile, the determinants of cardiac functional parameter were age, BMI, LADP, HARMAP LARDP, HARPPI ADBPD, RARSP, RADP and LARSP, while the TPR was positively correlated with age, BMI, LADP and HARMAP.\nPrevious studies demonstrated that population aging has become the uncontrolled risk factor of cardiovascular disease [16–18]. It is a physiology problem that the effect between blood pressure and cardiovascular function is mutual. The cardiovascular function decreased gradually with age, especially arteries can produce physiological degeneration. The thickening of vessels wall, the decrease or even rupture and calcification of elastic fibers, the increase of peripheral resistance, the decrease of blood vessel compliance, all these factors will result in the systolic blood pressure and diastolic blood pressure increasing with age. In addition, it is a fluid mechanics problem that different arteries away from heart have different resistance and pressure. According to the Poiseuille’s law, brachial artery resistance is relatively small and the blood pressure is low because heart is close to the upper limb, while the phenomenon of the ankle artery is in contrast. Hence blood pressures in four limbs are different. In addition, the distance from the heart affects pressure wave propagation and reflection because of the geometry tapper and elasticity tapper, and augments the pressure of peripheral vascular compared with that of upper limb.\nPrevious studies have reported that inter-arm or inter-ankle blood pressure difference predicted cardiovascular mortality [1, 2, 8–13, 15]. These results suggested that the simultaneous measurement of four-limb blood pressure is needed to improve diagnostic accuracy between cardiovascular disease and blood pressure difference [2–5]. The present study demonstrated the significant correlation between cardiovascular functional parameters and four-limb blood pressure, while cardiovascular functional parameters had no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI. However, the mean plots of cardiovascular functional parameters present obvious change trend in four-limb systolic blood pressure difference and diastolic blood pressure difference ≥10 or ≥15 mmHg. These results were different form previous studies. Verberk et al. [19] reported that the prevalence of inter-arm difference in systolic blood pressure of 10 mmHg or more was roughly doubled when diagnosis measurements method used a sequential approach, or used manual approach rather than automated measurements approach. Thus, simultaneous measurement of four-limb blood pressure and calculation of four-limb blood pressure difference may be helpful and necessary in evaluating the heart function and predicting patients with cardiovascular disease. Further study, the subgroup analysis showed that the predictive value of four limbs blood pressure for cardiovascular function. Subgroup analysis found that a part of cardiovascular functional parameter has still significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and with RABI between ≤0.9 and ≥1.0. However, the exact reason is unclear now, hypertension is the influence factors of cardiovascular function parameters, and four-limb blood pressure differences are also applicable to diagnose hypertensive patients. As mentioned above, four limbs blood pressures were not only influence factors of cardiovascular function parameters, but a predictor for cardiovascular disease, evaluating cardiovascular function parameters was important in clinical practice and epidemiological studies. In addition, the better way for cardiovascular function parameters was simultaneous blood pressures for four limbs.\nA clinical device, the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China), has been developed to automatically and simultaneously measure blood pressure in four limbs, and the measurement can be easily obtained. The similar device was reported by previous studies, for example, VP-1000 ABI—form device (Colin Co. Ltd., Komaki, Japan) and VP-1000 device (Omron, Kyoto, Japan) were used to automatically and simultaneously measure four limbs’ blood pressures. The validation in measurement technology of device (Boundless Horizon Company, Shandong, China) might be prone for measurement errors, especially in multinomial detection of cardiac function parameters rather than single detection. Although it could not give very precise measurements for the clinical value of cardiac function parameters, it can provide objective evaluation of health in the early stage of cardiovascular diseases. Thus, these devices can be applied for the epidemiological evaluation between four limbs’ blood pressures and cardiac function parameters.\nIn this study, by using a simultaneous and noninvasive measurement technique, we measured four limbs blood pressures. But this technique was not a popular one to measure blood pressure in daily clinical practice. Hence, although four limbs blood pressure with simultaneous measurement could improve the predictive value for cardiovascular disease, our results might be changed if daily clinical measurement was used. In addition, the subjects were mainly from retired people, whose health care consciousness is better than the serving officer. The factors of smoking, drinking, salting and movement have no significant difference in this study subjects. Hence, the clinical utility of this study may be limited in community people.\n\nConclusion:\nLADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nSimultaneous measurement of four-limb blood pressures can improve the accuracy of cardiovascular disease diagnosis. This study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the non-invasive diagnostic method of cardiovascular disease in primary care.\n\nMethods:\n229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled. Four-limb blood pressure measurements were simultaneously performed using a blood pressure and pulse monitor device in the supine position. Cardiac functional parameters were also measured by using a cardiac hemodynamic detector in the same position. Data were statistically analyzed with SPSS15.0.\n\nResults:\nThe mean age of the 229 subjects was 60.56 ± 8.68 years. Cardiovascular functional parameters decreased with age and body mass index (BMI), only the total peripheral resistance (TPR) was in contrast. Age, BMI, left ankle diastolic pressure (LADP), high arm mean arterial pressure (HARMAP), left arm diastolic pressure (LARDP) and right ankle diastolic pressure (RADP) were significantly correlated with cardiovascular functional parameters. Cardiovascular functional parameters have significant differences with inter-arm difference in systolic blood pressure (SBP) between ≥10 and <10 mmHg, inter-ankle difference in SBP between ≥15 and ≥20 mmHg, inter-ankle difference in SBP between ≥15 and <10 mmHg and right ankle brachial index (RABI) between ≤0.9 and ≥1.0. After excluding 99 hypertensive patients, a part of cardiovascular functional parameters has still significant differences with inter-arm difference in SBP between ≥10 and ≥15 mmHg and RABI between ≤0.9 and ≥1.0.\n\nConclusions:\nAge, BMI, LADP, HARMAP, LARDP and RADP were the determinants of cardiovascular functional parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in SBP ≥10 mmHg, inter-ankle difference in SBP ≥15 mmHg and RABI ≤0.9, while these differences still existed after excluding 99 hypertensive patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the non-invasive diagnostic method of cardiovascular disease in primary care."},"background_conclusion_text":{"kind":"string","value":"Background:\nAccurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.\n\nConclusion:\nLADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nSimultaneous measurement of four-limb blood pressures can improve the accuracy of cardiovascular disease diagnosis. This study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the non-invasive diagnostic method of cardiovascular disease in primary care.\n\nMethods:\n229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled. Four-limb blood pressure measurements were simultaneously performed using a blood pressure and pulse monitor device in the supine position. Cardiac functional parameters were also measured by using a cardiac hemodynamic detector in the same position. Data were statistically analyzed with SPSS15.0.\n\nResults:\nThe mean age of the 229 subjects was 60.56 ± 8.68 years. Cardiovascular functional parameters decreased with age and body mass index (BMI), only the total peripheral resistance (TPR) was in contrast. Age, BMI, left ankle diastolic pressure (LADP), high arm mean arterial pressure (HARMAP), left arm diastolic pressure (LARDP) and right ankle diastolic pressure (RADP) were significantly correlated with cardiovascular functional parameters. Cardiovascular functional parameters have significant differences with inter-arm difference in systolic blood pressure (SBP) between ≥10 and <10 mmHg, inter-ankle difference in SBP between ≥15 and ≥20 mmHg, inter-ankle difference in SBP between ≥15 and <10 mmHg and right ankle brachial index (RABI) between ≤0.9 and ≥1.0. After excluding 99 hypertensive patients, a part of cardiovascular functional parameters has still significant differences with inter-arm difference in SBP between ≥10 and ≥15 mmHg and RABI between ≤0.9 and ≥1.0.\n\nConclusions:\nAge, BMI, LADP, HARMAP, LARDP and RADP were the determinants of cardiovascular functional parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in SBP ≥10 mmHg, inter-ankle difference in SBP ≥15 mmHg and RABI ≤0.9, while these differences still existed after excluding 99 hypertensive patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the non-invasive diagnostic method of cardiovascular disease in primary care."},"whole_article_text_length":{"kind":"number","value":13813,"string":"13,813"},"whole_article_abstract_length":{"kind":"number","value":427,"string":"427"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["pressure","blood","blood pressure","limb","limb blood","differences","limb blood pressure","parameters","mean","cardiovascular"],"string":"[\n \"pressure\",\n \"blood\",\n \"blood pressure\",\n \"limb\",\n \"limb blood\",\n \"differences\",\n \"limb blood pressure\",\n \"parameters\",\n \"mean\",\n \"cardiovascular\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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[SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] pressure | blood | blood pressure | parameters | cardiovascular | cardiac | differences | 000 | limb blood pressure | limb blood [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] four ||| four [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 229 | 60.56 | 8.68 years ||| BMI | TPR ||| BMI | RADP ||| SBP | 10 | SBP | SBP | 10 | RABI | ≥1.0 ||| 99 | SBP | RABI | ≥1.0 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] BMI | HARMAP | LARDP | RADP ||| SBP | SBP | RABI | 99 ||| four [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] four ||| four ||| 229 | 62 | 63.50 | 11.13 years | 167 | 59.47 | 7.33 years ||| Four ||| Cardiac ||| ||| ||| 229 | 60.56 | 8.68 years ||| BMI | TPR ||| BMI | RADP ||| SBP | 10 | SBP | SBP | 10 | RABI | ≥1.0 ||| 99 | SBP | RABI | ≥1.0 ||| BMI | HARMAP | LARDP | RADP ||| SBP | SBP | RABI | 99 ||| four [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] four ||| four ||| 229 | 62 | 63.50 | 11.13 years | 167 | 59.47 | 7.33 years ||| Four ||| Cardiac ||| ||| ||| 229 | 60.56 | 8.68 years ||| BMI | TPR ||| BMI | RADP ||| SBP | 10 | SBP | SBP | 10 | RABI | ≥1.0 ||| 99 | SBP | RABI | ≥1.0 ||| BMI | HARMAP | LARDP | RADP ||| SBP | SBP | RABI | 99 ||| four [SUMMARY]"}}},{"rowIdx":289,"cells":{"title":{"kind":"string","value":"Incidence of symptoms and accidents during baths and showers among the Japanese general public."},"pmid":{"kind":"string","value":"21478641"},"background_abstract":{"kind":"string","value":"Bathing is a deeply ingrained custom among Japanese; however, data on the incidence rate of symptoms and accidents during bathing have not yet been reported for the Japanese general public."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We conducted a population-based cross-sectional study of 617 Japanese adults who attended a specialized health checkup. Participants completed a self-administered questionnaire to assess weekly frequencies of bathtub bathing and showering and the frequency of symptoms/accidents (falling, loss of consciousness, and other) during these activities in the past year. We calculated the incidence rates of accidents per 10 000 baths/showers and 95% confidence intervals (CIs) and compared the clinical characteristics of participants who had symptoms/accidents with those who did not."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22-0.84) and 0.24 (95% CI: 0.04-1.37). Although these rates are low, there were 740 000 bathtub bathing-related accidents in Japan, due to the fact that bathing is an almost-daily habit. There was no significant difference in clinical characteristics between groups."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"We collected basic information on the incidence of bathing-related accidents in Japan. Falls and loss of consciousness during bathing or showering can potentially lead to a serious accident, so the general public should be educated about the possibility of such accidents during bathing."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Accidental Falls","Accidents, Home","Adult","Aged","Baths","Confidence Intervals","Cross-Sectional Studies","Female","Health Surveys","Humans","Incidence","Japan","Male","Middle Aged","Public Health","Self-Assessment","Surveys and Questionnaires"],"string":"[\n \"Accidental Falls\",\n \"Accidents, Home\",\n \"Adult\",\n \"Aged\",\n \"Baths\",\n \"Confidence Intervals\",\n \"Cross-Sectional Studies\",\n \"Female\",\n \"Health Surveys\",\n \"Humans\",\n \"Incidence\",\n \"Japan\",\n \"Male\",\n \"Middle Aged\",\n \"Public Health\",\n \"Self-Assessment\",\n \"Surveys and Questionnaires\"\n]"},"pmcid":{"kind":"string","value":"3899424"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"This cross-sectional study enrolled adults aged 40 to 74 years who underwent a specialized health checkup provided in October 2008 in the district of Kawane in Shimada city, Shizuoka. The investigation utilized self-administered questionnaires, measurement of height and weight, blood pressure examination, and blood testing for triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, L-aspartate aminotransferase, L-alanine aminotransferase, and hemoglobin A1c, based on a program standardized by the Japanese Ministry of Health, Labour and Welfare.9 From a total of 1319 eligible adults, 617 who actually completed the health checkup and gave consent participated in the study. Participants completed a self-administered questionnaire to assess the weekly frequencies of bathtub bathing and showering during summer (July through September) and winter (December through February). In addition, we investigated the frequency of symptoms/accidents (falling, loss of consciousness, pallor, nausea/vomiting, and other) during bathtub bathing and showering in the past year: the list of symptoms/accidents consisted of those frequently identified in previous research.7\nFirst, we calculated the weekly frequencies of bathtub bathing and showering by averaging the frequencies between the 2 seasons, after which we assessed the incidence of each type of accident during bathtub bathing and showering in the past year for all participants and in 2 groups divided by the median age of the participants (ie, 66 years). Next, based on the weekly frequencies of bathtub bathing and showering, we estimated the overall frequencies of bathtub bathing and showering during the past year for all participants. We then divided the total number of accidents by the annual frequencies of bathtub bathing and showering for all participants and calculated the incidence rates and 95% confidence intervals (CIs) of accidents per 10 000 baths/showers. We calculated 95% CIs using a binomial distribution according to Wilson’s method.10 Then, to estimate the annual frequencies of bathtub bathing and showering among Japanese aged 40 to 74 years, we used the 2008 Vital Statistics of Japan to determine how many Japanese were included in this age group.11 By multiplying these estimates by the incidence rates for accidents (which were obtained earlier), we estimated the total annual numbers of accidents that were associated with bathtub bathing and showering in Japanese aged 40 to 74 years.\nAfter dividing the participants into 2 groups according to their accident status, ie, cases with and without bath-related accidents, we used the t-test to compare mean values for age, body mass index, blood pressure, and blood chemistry findings and Fisher’s exact test to compare differences in sex, medication use, and past medical history.\nThe study protocol was approved by the institutional review board (IRB) of Hamamatsu University School of Medicine (No. 20-55). The nature of the study was explained to the participants using written statements approved by the IRB. Voluntary informed consent was obtained from all participants before beginning the study. Completion of the questionnaire was deemed as indicating consent to participate in the study. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (SPSS Japan Inc., version 17.0, Tokyo, Japan)."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"All 617 individuals (266 men, 351 women) who had completed the health checkup and were recruited for the study agreed to participate. The frequencies of bathtub bathing and showering were calculated based on summer and winter frequencies (mean ± SD) of 5.82 ± 2.09 and 1.29 ± 1.81 times per week, respectively (Table 1). The overall annual numbers of bathtub baths and showers were estimated based on the weekly frequencies and were approximately 190 000 and 41 000, respectively. Of the 617 individuals, 588 completed a self-administered questionnaire about accidents. Of these, 5 reported a total of 8 bathtub bathing-related accidents over the past year, including 5 cases of facial pallor, 2 cases of loss of consciousness, and 1 case of falling. In contrast, there was only 1 shower-related accident (loss of consciousness). The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22–0.84) and 0.24 (95% CI: 0.04–1.37), respectively. According to estimates based on the Japanese population of adults aged 40 to 74 years, people in this age group bathe a total of 1.7 × 1010 times per year and have a total of 740 000 (95% CI: 380 000 to 1 500 000) accidents. They shower 3.8 × 109 times per year and have a total of 93 000 (95% CI: 16 000 to 530 000) accidents.\nSD: standard deviation; CI: confidence interval.\nA comparison of the characteristics of participants who did and did not have bath-related accidents are shown in Table 2. There was no significant difference in the characteristics of the 5 participants who had accidents and those who did not.\nBMI: Body mass index; SBP: Systolic blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; AST: L-aspartate aminotransferase; ALT: L-alanine aminotransferase."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION"],"string":"[\n \"INTRODUCTION\"\n]"},"other_sections_texts":{"kind":"list like","value":["Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public."],"string":"[\n \"Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null],"string":"[\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","RESULTS","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"RESULTS\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public.","This cross-sectional study enrolled adults aged 40 to 74 years who underwent a specialized health checkup provided in October 2008 in the district of Kawane in Shimada city, Shizuoka. The investigation utilized self-administered questionnaires, measurement of height and weight, blood pressure examination, and blood testing for triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, L-aspartate aminotransferase, L-alanine aminotransferase, and hemoglobin A1c, based on a program standardized by the Japanese Ministry of Health, Labour and Welfare.9 From a total of 1319 eligible adults, 617 who actually completed the health checkup and gave consent participated in the study. Participants completed a self-administered questionnaire to assess the weekly frequencies of bathtub bathing and showering during summer (July through September) and winter (December through February). In addition, we investigated the frequency of symptoms/accidents (falling, loss of consciousness, pallor, nausea/vomiting, and other) during bathtub bathing and showering in the past year: the list of symptoms/accidents consisted of those frequently identified in previous research.7\nFirst, we calculated the weekly frequencies of bathtub bathing and showering by averaging the frequencies between the 2 seasons, after which we assessed the incidence of each type of accident during bathtub bathing and showering in the past year for all participants and in 2 groups divided by the median age of the participants (ie, 66 years). Next, based on the weekly frequencies of bathtub bathing and showering, we estimated the overall frequencies of bathtub bathing and showering during the past year for all participants. We then divided the total number of accidents by the annual frequencies of bathtub bathing and showering for all participants and calculated the incidence rates and 95% confidence intervals (CIs) of accidents per 10 000 baths/showers. We calculated 95% CIs using a binomial distribution according to Wilson’s method.10 Then, to estimate the annual frequencies of bathtub bathing and showering among Japanese aged 40 to 74 years, we used the 2008 Vital Statistics of Japan to determine how many Japanese were included in this age group.11 By multiplying these estimates by the incidence rates for accidents (which were obtained earlier), we estimated the total annual numbers of accidents that were associated with bathtub bathing and showering in Japanese aged 40 to 74 years.\nAfter dividing the participants into 2 groups according to their accident status, ie, cases with and without bath-related accidents, we used the t-test to compare mean values for age, body mass index, blood pressure, and blood chemistry findings and Fisher’s exact test to compare differences in sex, medication use, and past medical history.\nThe study protocol was approved by the institutional review board (IRB) of Hamamatsu University School of Medicine (No. 20-55). The nature of the study was explained to the participants using written statements approved by the IRB. Voluntary informed consent was obtained from all participants before beginning the study. Completion of the questionnaire was deemed as indicating consent to participate in the study. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (SPSS Japan Inc., version 17.0, Tokyo, Japan).","All 617 individuals (266 men, 351 women) who had completed the health checkup and were recruited for the study agreed to participate. The frequencies of bathtub bathing and showering were calculated based on summer and winter frequencies (mean ± SD) of 5.82 ± 2.09 and 1.29 ± 1.81 times per week, respectively (Table 1). The overall annual numbers of bathtub baths and showers were estimated based on the weekly frequencies and were approximately 190 000 and 41 000, respectively. Of the 617 individuals, 588 completed a self-administered questionnaire about accidents. Of these, 5 reported a total of 8 bathtub bathing-related accidents over the past year, including 5 cases of facial pallor, 2 cases of loss of consciousness, and 1 case of falling. In contrast, there was only 1 shower-related accident (loss of consciousness). The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22–0.84) and 0.24 (95% CI: 0.04–1.37), respectively. According to estimates based on the Japanese population of adults aged 40 to 74 years, people in this age group bathe a total of 1.7 × 1010 times per year and have a total of 740 000 (95% CI: 380 000 to 1 500 000) accidents. They shower 3.8 × 109 times per year and have a total of 93 000 (95% CI: 16 000 to 530 000) accidents.\nSD: standard deviation; CI: confidence interval.\nA comparison of the characteristics of participants who did and did not have bath-related accidents are shown in Table 2. There was no significant difference in the characteristics of the 5 participants who had accidents and those who did not.\nBMI: Body mass index; SBP: Systolic blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; AST: L-aspartate aminotransferase; ALT: L-alanine aminotransferase.","The present study is the first to ascertain the incidence rate of bathing-related accidents among the Japanese general public. The incidence rate in the present study was somewhat higher than that noted in an investigation of public welfare facilities for the aged (0.067 and 0.204 per 10 000 in-facility baths and home-visit baths, respectively).8 The somewhat lower incidence rate of accidents in bathing services provided by welfare facilities may be attributable to the following facts. First, in welfare services, a physical checkup is routinely performed before bathing, which may prevent accidents. Second, bathing in the presence of a caregiver may allow for early detection of potential accidents and serious symptoms. Third, the incidence rate was likely to have been underreported in the previous study,8 as it was based only on the objective findings of caregivers.\nIn the present study, the incidence rate of accidents during showering was lower than that during bathtub bathing. It has been suggested that, as compared with bathtub bathing, the lower water pressure used during showering imposes a lower burden on the body.12 However, as habitual bathing is reported to be associated with good self-rated health and sleep quality,13 it would not be wise to suggest that bathtub bathing is, in general, more hazardous to general health than showering.\nWhile the incidence rate of accidents in the present study is small, the total number of bathtub bathing-related accidents in Japan was as high as 740 000, because bathing is an almost-daily habit. Although no accidents resulted in death in this study, falls and loss of consciousness could potentially lead to a serious accident.14 One participant who reported falling in the bath lost consciousness twice. Therefore, the general public must be educated about accidents such as falls during bathing.\nThis study has several limitations. First, the use of self-report questionnaires may have caused underreporting due to obscured memory. Second, the sample size was relatively small, and the study was conducted within 1 geographic region. It is very difficult to use a small sample in a specific area to estimate the incidence for the general population. To determine the characteristics of the accident group, we analyzed the relationship between disease history and the results of the health checkup using additional data from the health checkup. However, there were no characteristic findings. Third, participants had all sought a health checkup, suggesting possible selection bias. A previous study reported high incidence rates of drowning among the very old.3 It is possible that, in the present study, recall bias explains why incidence rates were lower among the very old than among middle-aged adults. Although there were some limitations in the present report, it is the first to collect information on the incidence of bathing-related accidents among the Japanese general public."],"string":"[\n \"Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public.\",\n \"This cross-sectional study enrolled adults aged 40 to 74 years who underwent a specialized health checkup provided in October 2008 in the district of Kawane in Shimada city, Shizuoka. The investigation utilized self-administered questionnaires, measurement of height and weight, blood pressure examination, and blood testing for triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, L-aspartate aminotransferase, L-alanine aminotransferase, and hemoglobin A1c, based on a program standardized by the Japanese Ministry of Health, Labour and Welfare.9 From a total of 1319 eligible adults, 617 who actually completed the health checkup and gave consent participated in the study. Participants completed a self-administered questionnaire to assess the weekly frequencies of bathtub bathing and showering during summer (July through September) and winter (December through February). In addition, we investigated the frequency of symptoms/accidents (falling, loss of consciousness, pallor, nausea/vomiting, and other) during bathtub bathing and showering in the past year: the list of symptoms/accidents consisted of those frequently identified in previous research.7\\nFirst, we calculated the weekly frequencies of bathtub bathing and showering by averaging the frequencies between the 2 seasons, after which we assessed the incidence of each type of accident during bathtub bathing and showering in the past year for all participants and in 2 groups divided by the median age of the participants (ie, 66 years). Next, based on the weekly frequencies of bathtub bathing and showering, we estimated the overall frequencies of bathtub bathing and showering during the past year for all participants. We then divided the total number of accidents by the annual frequencies of bathtub bathing and showering for all participants and calculated the incidence rates and 95% confidence intervals (CIs) of accidents per 10 000 baths/showers. We calculated 95% CIs using a binomial distribution according to Wilson’s method.10 Then, to estimate the annual frequencies of bathtub bathing and showering among Japanese aged 40 to 74 years, we used the 2008 Vital Statistics of Japan to determine how many Japanese were included in this age group.11 By multiplying these estimates by the incidence rates for accidents (which were obtained earlier), we estimated the total annual numbers of accidents that were associated with bathtub bathing and showering in Japanese aged 40 to 74 years.\\nAfter dividing the participants into 2 groups according to their accident status, ie, cases with and without bath-related accidents, we used the t-test to compare mean values for age, body mass index, blood pressure, and blood chemistry findings and Fisher’s exact test to compare differences in sex, medication use, and past medical history.\\nThe study protocol was approved by the institutional review board (IRB) of Hamamatsu University School of Medicine (No. 20-55). The nature of the study was explained to the participants using written statements approved by the IRB. Voluntary informed consent was obtained from all participants before beginning the study. Completion of the questionnaire was deemed as indicating consent to participate in the study. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (SPSS Japan Inc., version 17.0, Tokyo, Japan).\",\n \"All 617 individuals (266 men, 351 women) who had completed the health checkup and were recruited for the study agreed to participate. The frequencies of bathtub bathing and showering were calculated based on summer and winter frequencies (mean ± SD) of 5.82 ± 2.09 and 1.29 ± 1.81 times per week, respectively (Table 1). The overall annual numbers of bathtub baths and showers were estimated based on the weekly frequencies and were approximately 190 000 and 41 000, respectively. Of the 617 individuals, 588 completed a self-administered questionnaire about accidents. Of these, 5 reported a total of 8 bathtub bathing-related accidents over the past year, including 5 cases of facial pallor, 2 cases of loss of consciousness, and 1 case of falling. In contrast, there was only 1 shower-related accident (loss of consciousness). The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22–0.84) and 0.24 (95% CI: 0.04–1.37), respectively. According to estimates based on the Japanese population of adults aged 40 to 74 years, people in this age group bathe a total of 1.7 × 1010 times per year and have a total of 740 000 (95% CI: 380 000 to 1 500 000) accidents. They shower 3.8 × 109 times per year and have a total of 93 000 (95% CI: 16 000 to 530 000) accidents.\\nSD: standard deviation; CI: confidence interval.\\nA comparison of the characteristics of participants who did and did not have bath-related accidents are shown in Table 2. There was no significant difference in the characteristics of the 5 participants who had accidents and those who did not.\\nBMI: Body mass index; SBP: Systolic blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; AST: L-aspartate aminotransferase; ALT: L-alanine aminotransferase.\",\n \"The present study is the first to ascertain the incidence rate of bathing-related accidents among the Japanese general public. The incidence rate in the present study was somewhat higher than that noted in an investigation of public welfare facilities for the aged (0.067 and 0.204 per 10 000 in-facility baths and home-visit baths, respectively).8 The somewhat lower incidence rate of accidents in bathing services provided by welfare facilities may be attributable to the following facts. First, in welfare services, a physical checkup is routinely performed before bathing, which may prevent accidents. Second, bathing in the presence of a caregiver may allow for early detection of potential accidents and serious symptoms. Third, the incidence rate was likely to have been underreported in the previous study,8 as it was based only on the objective findings of caregivers.\\nIn the present study, the incidence rate of accidents during showering was lower than that during bathtub bathing. It has been suggested that, as compared with bathtub bathing, the lower water pressure used during showering imposes a lower burden on the body.12 However, as habitual bathing is reported to be associated with good self-rated health and sleep quality,13 it would not be wise to suggest that bathtub bathing is, in general, more hazardous to general health than showering.\\nWhile the incidence rate of accidents in the present study is small, the total number of bathtub bathing-related accidents in Japan was as high as 740 000, because bathing is an almost-daily habit. Although no accidents resulted in death in this study, falls and loss of consciousness could potentially lead to a serious accident.14 One participant who reported falling in the bath lost consciousness twice. Therefore, the general public must be educated about accidents such as falls during bathing.\\nThis study has several limitations. First, the use of self-report questionnaires may have caused underreporting due to obscured memory. Second, the sample size was relatively small, and the study was conducted within 1 geographic region. It is very difficult to use a small sample in a specific area to estimate the incidence for the general population. To determine the characteristics of the accident group, we analyzed the relationship between disease history and the results of the health checkup using additional data from the health checkup. However, there were no characteristic findings. Third, participants had all sought a health checkup, suggesting possible selection bias. A previous study reported high incidence rates of drowning among the very old.3 It is possible that, in the present study, recall bias explains why incidence rates were lower among the very old than among middle-aged adults. Although there were some limitations in the present report, it is the first to collect information on the incidence of bathing-related accidents among the Japanese general public.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods","results","discussion"],"string":"[\n null,\n \"methods\",\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["baths","shower","incidence","accidents","population-based study"],"string":"[\n \"baths\",\n \"shower\",\n \"incidence\",\n \"accidents\",\n \"population-based study\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nBathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public.\n\nMETHODS:\nThis cross-sectional study enrolled adults aged 40 to 74 years who underwent a specialized health checkup provided in October 2008 in the district of Kawane in Shimada city, Shizuoka. The investigation utilized self-administered questionnaires, measurement of height and weight, blood pressure examination, and blood testing for triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, L-aspartate aminotransferase, L-alanine aminotransferase, and hemoglobin A1c, based on a program standardized by the Japanese Ministry of Health, Labour and Welfare.9 From a total of 1319 eligible adults, 617 who actually completed the health checkup and gave consent participated in the study. Participants completed a self-administered questionnaire to assess the weekly frequencies of bathtub bathing and showering during summer (July through September) and winter (December through February). In addition, we investigated the frequency of symptoms/accidents (falling, loss of consciousness, pallor, nausea/vomiting, and other) during bathtub bathing and showering in the past year: the list of symptoms/accidents consisted of those frequently identified in previous research.7\nFirst, we calculated the weekly frequencies of bathtub bathing and showering by averaging the frequencies between the 2 seasons, after which we assessed the incidence of each type of accident during bathtub bathing and showering in the past year for all participants and in 2 groups divided by the median age of the participants (ie, 66 years). Next, based on the weekly frequencies of bathtub bathing and showering, we estimated the overall frequencies of bathtub bathing and showering during the past year for all participants. We then divided the total number of accidents by the annual frequencies of bathtub bathing and showering for all participants and calculated the incidence rates and 95% confidence intervals (CIs) of accidents per 10 000 baths/showers. We calculated 95% CIs using a binomial distribution according to Wilson’s method.10 Then, to estimate the annual frequencies of bathtub bathing and showering among Japanese aged 40 to 74 years, we used the 2008 Vital Statistics of Japan to determine how many Japanese were included in this age group.11 By multiplying these estimates by the incidence rates for accidents (which were obtained earlier), we estimated the total annual numbers of accidents that were associated with bathtub bathing and showering in Japanese aged 40 to 74 years.\nAfter dividing the participants into 2 groups according to their accident status, ie, cases with and without bath-related accidents, we used the t-test to compare mean values for age, body mass index, blood pressure, and blood chemistry findings and Fisher’s exact test to compare differences in sex, medication use, and past medical history.\nThe study protocol was approved by the institutional review board (IRB) of Hamamatsu University School of Medicine (No. 20-55). The nature of the study was explained to the participants using written statements approved by the IRB. Voluntary informed consent was obtained from all participants before beginning the study. Completion of the questionnaire was deemed as indicating consent to participate in the study. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (SPSS Japan Inc., version 17.0, Tokyo, Japan).\n\nRESULTS:\nAll 617 individuals (266 men, 351 women) who had completed the health checkup and were recruited for the study agreed to participate. The frequencies of bathtub bathing and showering were calculated based on summer and winter frequencies (mean ± SD) of 5.82 ± 2.09 and 1.29 ± 1.81 times per week, respectively (Table 1). The overall annual numbers of bathtub baths and showers were estimated based on the weekly frequencies and were approximately 190 000 and 41 000, respectively. Of the 617 individuals, 588 completed a self-administered questionnaire about accidents. Of these, 5 reported a total of 8 bathtub bathing-related accidents over the past year, including 5 cases of facial pallor, 2 cases of loss of consciousness, and 1 case of falling. In contrast, there was only 1 shower-related accident (loss of consciousness). The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22–0.84) and 0.24 (95% CI: 0.04–1.37), respectively. According to estimates based on the Japanese population of adults aged 40 to 74 years, people in this age group bathe a total of 1.7 × 1010 times per year and have a total of 740 000 (95% CI: 380 000 to 1 500 000) accidents. They shower 3.8 × 109 times per year and have a total of 93 000 (95% CI: 16 000 to 530 000) accidents.\nSD: standard deviation; CI: confidence interval.\nA comparison of the characteristics of participants who did and did not have bath-related accidents are shown in Table 2. There was no significant difference in the characteristics of the 5 participants who had accidents and those who did not.\nBMI: Body mass index; SBP: Systolic blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; AST: L-aspartate aminotransferase; ALT: L-alanine aminotransferase.\n\nDISCUSSION:\nThe present study is the first to ascertain the incidence rate of bathing-related accidents among the Japanese general public. The incidence rate in the present study was somewhat higher than that noted in an investigation of public welfare facilities for the aged (0.067 and 0.204 per 10 000 in-facility baths and home-visit baths, respectively).8 The somewhat lower incidence rate of accidents in bathing services provided by welfare facilities may be attributable to the following facts. First, in welfare services, a physical checkup is routinely performed before bathing, which may prevent accidents. Second, bathing in the presence of a caregiver may allow for early detection of potential accidents and serious symptoms. Third, the incidence rate was likely to have been underreported in the previous study,8 as it was based only on the objective findings of caregivers.\nIn the present study, the incidence rate of accidents during showering was lower than that during bathtub bathing. It has been suggested that, as compared with bathtub bathing, the lower water pressure used during showering imposes a lower burden on the body.12 However, as habitual bathing is reported to be associated with good self-rated health and sleep quality,13 it would not be wise to suggest that bathtub bathing is, in general, more hazardous to general health than showering.\nWhile the incidence rate of accidents in the present study is small, the total number of bathtub bathing-related accidents in Japan was as high as 740 000, because bathing is an almost-daily habit. Although no accidents resulted in death in this study, falls and loss of consciousness could potentially lead to a serious accident.14 One participant who reported falling in the bath lost consciousness twice. Therefore, the general public must be educated about accidents such as falls during bathing.\nThis study has several limitations. First, the use of self-report questionnaires may have caused underreporting due to obscured memory. Second, the sample size was relatively small, and the study was conducted within 1 geographic region. It is very difficult to use a small sample in a specific area to estimate the incidence for the general population. To determine the characteristics of the accident group, we analyzed the relationship between disease history and the results of the health checkup using additional data from the health checkup. However, there were no characteristic findings. Third, participants had all sought a health checkup, suggesting possible selection bias. A previous study reported high incidence rates of drowning among the very old.3 It is possible that, in the present study, recall bias explains why incidence rates were lower among the very old than among middle-aged adults. Although there were some limitations in the present report, it is the first to collect information on the incidence of bathing-related accidents among the Japanese general public.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nBathing is a deeply ingrained custom among Japanese; however, data on the incidence rate of symptoms and accidents during bathing have not yet been reported for the Japanese general public.\n\nMethods:\nWe conducted a population-based cross-sectional study of 617 Japanese adults who attended a specialized health checkup. Participants completed a self-administered questionnaire to assess weekly frequencies of bathtub bathing and showering and the frequency of symptoms/accidents (falling, loss of consciousness, and other) during these activities in the past year. We calculated the incidence rates of accidents per 10 000 baths/showers and 95% confidence intervals (CIs) and compared the clinical characteristics of participants who had symptoms/accidents with those who did not.\n\nResults:\nThe incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22-0.84) and 0.24 (95% CI: 0.04-1.37). Although these rates are low, there were 740 000 bathtub bathing-related accidents in Japan, due to the fact that bathing is an almost-daily habit. There was no significant difference in clinical characteristics between groups.\n\nConclusions:\nWe collected basic information on the incidence of bathing-related accidents in Japan. Falls and loss of consciousness during bathing or showering can potentially lead to a serious accident, so the general public should be educated about the possibility of such accidents during bathing."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":1754,"string":"1,754"},"whole_article_abstract_length":{"kind":"number","value":274,"string":"274"},"num_sections":{"kind":"number","value":4,"string":"4"},"most_frequent_words":{"kind":"list like","value":["bathing","accidents","study","bathtub","incidence","bathtub bathing","showering","000","japanese","participants"],"string":"[\n \"bathing\",\n \"accidents\",\n \"study\",\n \"bathtub\",\n \"incidence\",\n \"bathtub bathing\",\n \"showering\",\n \"000\",\n \"japanese\",\n \"participants\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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[SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] baths | shower | incidence | accidents | population-based study [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | japanese | bathing related | incidence rate | rate | public | general public | general | related [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing showering | bathtub bathing showering | showering | bathtub bathing | frequencies | participants | frequencies bathtub bathing | frequencies bathtub bathing showering | frequencies bathtub | bathing [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 000 | ci | 95 ci | accidents | 95 | times | total | respectively | frequencies | bathtub [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | 000 | general | rate | incidence rate [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Japanese | Japanese [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 617 | Japanese ||| weekly | the past year ||| 10 000 | 95% [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 10 | 95% | 0.22-0.84 | 0.24 | 95% | CI | 0.04 ||| 740 | Japan ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Japanese | Japanese ||| 617 | Japanese ||| weekly | the past year ||| 10 000 | 95% ||| ||| 10 | 95% | 0.22-0.84 | 0.24 | 95% | CI | 0.04 ||| 740 | Japan ||| ||| Japan ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":290,"cells":{"title":{"kind":"string","value":"Effect of probiotics on length of hospitalization in mild acute pancreatitis: A randomized, double-blind, placebo-controlled trial."},"pmid":{"kind":"string","value":"33510561"},"background_abstract":{"kind":"string","value":"Acute pancreatitis is the leading cause of hospitalization for acute gastrointestinal disease worldwide. The effects of probiotics in mild acute pancreatitis have not been studied. We hypothesized that the administration of probiotics may accelerate the recovery of intestinal function and shorten the length of hospital stay (LOS) in patients with mild pancreatitis."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We conducted a double-blind randomized clinical trial to evaluate the effects of probiotics administered to patients with mild acute pancreatitis at a tertiary medical center. The patients were given probiotics capsules (a mixed preparation of Bacillus subtilis and Enterococcus faecium) or placebo. The primary study endpoint was the LOS. The secondary endpoints included time to abdominal pain relief, recurrent abdominal pain, and time to successful oral feeding."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A total of 128 patients were included, with 64 patients in each arm. The severity of illness and the etiological distribution of disease were similar in the two groups. There was a significant reduction in the LOS in the probiotics treatment group vs the placebo group (5.36 ± 0.15 vs 6.02 ± 0.17 d, P < 0.05). The probiotics group was associated with a shorter time to abdominal pain relief and time to successful oral feeding (P < 0.01 for both) than the placebo group. No statistical difference was found in recurrent abdominal pain between the two groups."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The study results showed that the administration of probiotics capsules is associated with a shorter duration of hospitalization in patients with mild acute pancreatitis."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Acute Disease","Double-Blind Method","Hospitalization","Humans","Pancreatitis","Probiotics","Treatment Outcome"],"string":"[\n \"Acute Disease\",\n \"Double-Blind Method\",\n \"Hospitalization\",\n \"Humans\",\n \"Pancreatitis\",\n \"Probiotics\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"7807297"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis."},"methods_title":{"kind":"string","value":"MATERIALS AND METHODS"},"methods_text":{"kind":"string","value":" Study design This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\nThis 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\n Inclusion and exclusion criteria The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\nThe inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\n Data collection We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \nWe extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \n Outcomes We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \nWe used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \n Sample size estimation According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\nAccording to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\n Statistical analysis Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\nContinuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States)."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"Probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Study design","Inclusion and exclusion criteria","Data collection","Outcomes","Sample size estimation","Statistical analysis","RESULTS","Participant characteristics ","Primary endpoint","Secondary end points","Safety and harm","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"Study design\",\n \"Inclusion and exclusion criteria\",\n \"Data collection\",\n \"Outcomes\",\n \"Sample size estimation\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Participant characteristics \",\n \"Primary endpoint\",\n \"Secondary end points\",\n \"Safety and harm\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]"},"other_sections_texts":{"kind":"list like","value":["Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.","This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.","The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.","We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. ","We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. ","According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.","Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States)."," Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups","We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.","The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.","The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups","The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups","The present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.","In conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases."],"string":"[\n \"Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.\",\n \"This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\",\n \"The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\",\n \"We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \",\n \"We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \",\n \"According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\",\n \"Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\",\n \" Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\",\n \"We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\",\n \"The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\",\n \"The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\",\n \"The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\",\n \"The present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \\nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.\",\n \"In conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Study design","Inclusion and exclusion criteria","Data collection","Outcomes","Sample size estimation","Statistical analysis","RESULTS","Participant characteristics ","Primary endpoint","Secondary end points","Safety and harm","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Study design\",\n \"Inclusion and exclusion criteria\",\n \"Data collection\",\n \"Outcomes\",\n \"Sample size estimation\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Participant characteristics \",\n \"Primary endpoint\",\n \"Secondary end points\",\n \"Safety and harm\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis."," Study design This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\nThis 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\n Inclusion and exclusion criteria The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\nThe inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\n Data collection We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \nWe extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \n Outcomes We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \nWe used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \n Sample size estimation According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\nAccording to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\n Statistical analysis Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\nContinuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).","This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.","The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.","We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. ","We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. ","According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.","Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States)."," Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups","We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.","The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.","The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups","The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups","The present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.","In conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases."],"string":"[\n \"Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.\",\n \" Study design This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\\nThis 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\\n Inclusion and exclusion criteria The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\\nThe inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\\n Data collection We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \\nWe extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \\n Outcomes We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \\nWe used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \\n Sample size estimation According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\\nAccording to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\\n Statistical analysis Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\\nContinuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\",\n \"This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\",\n \"The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\",\n \"We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \",\n \"We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \",\n \"According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\",\n \"Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\",\n \" Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\",\n \"We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \\nCONSORT diagram.\\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \\nBaseline characteristics of participants\\nBMI: Body mass index; CRP: C-reactive protein.\",\n \"The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\\nComparison of length of hospital stay between probiotics and placebo groups.\",\n \"The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\\nComparison of secondary endpoints between probiotics and placebo groups\",\n \"The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\\nComparison of side effects between probiotics and placebo groups\",\n \"The present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \\nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.\",\n \"In conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Acute pancreatitis","Probiotics","Length of hospitalization","Randomized study","Placebo","Mild"],"string":"[\n \"Acute pancreatitis\",\n \"Probiotics\",\n \"Length of hospitalization\",\n \"Randomized study\",\n \"Placebo\",\n \"Mild\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nAcute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.\n\nMATERIALS AND METHODS:\n Study design This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\nThis 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\n Inclusion and exclusion criteria The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\nThe inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\n Data collection We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \nWe extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \n Outcomes We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \nWe used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \n Sample size estimation According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\nAccording to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\n Statistical analysis Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\nContinuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\n\nStudy design:\nThis 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\n\nInclusion and exclusion criteria:\nThe inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\n\nData collection:\nWe extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \n\nOutcomes:\nWe used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \n\nSample size estimation:\nAccording to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\n\nStatistical analysis:\nContinuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\n\nRESULTS:\n Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\n\nParticipant characteristics :\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\n\nPrimary endpoint:\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\n\nSecondary end points:\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\n\nSafety and harm:\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\n\nDISCUSSION:\nThe present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.\n\nCONCLUSION:\nIn conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nAcute pancreatitis is the leading cause of hospitalization for acute gastrointestinal disease worldwide. The effects of probiotics in mild acute pancreatitis have not been studied. We hypothesized that the administration of probiotics may accelerate the recovery of intestinal function and shorten the length of hospital stay (LOS) in patients with mild pancreatitis.\n\nMethods:\nWe conducted a double-blind randomized clinical trial to evaluate the effects of probiotics administered to patients with mild acute pancreatitis at a tertiary medical center. The patients were given probiotics capsules (a mixed preparation of Bacillus subtilis and Enterococcus faecium) or placebo. The primary study endpoint was the LOS. The secondary endpoints included time to abdominal pain relief, recurrent abdominal pain, and time to successful oral feeding.\n\nResults:\nA total of 128 patients were included, with 64 patients in each arm. The severity of illness and the etiological distribution of disease were similar in the two groups. There was a significant reduction in the LOS in the probiotics treatment group vs the placebo group (5.36 ± 0.15 vs 6.02 ± 0.17 d, P < 0.05). The probiotics group was associated with a shorter time to abdominal pain relief and time to successful oral feeding (P < 0.01 for both) than the placebo group. No statistical difference was found in recurrent abdominal pain between the two groups.\n\nConclusions:\nThe study results showed that the administration of probiotics capsules is associated with a shorter duration of hospitalization in patients with mild acute pancreatitis."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nAcute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.\n\nCONCLUSION:\nProbiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nAcute pancreatitis is the leading cause of hospitalization for acute gastrointestinal disease worldwide. The effects of probiotics in mild acute pancreatitis have not been studied. We hypothesized that the administration of probiotics may accelerate the recovery of intestinal function and shorten the length of hospital stay (LOS) in patients with mild pancreatitis.\n\nMethods:\nWe conducted a double-blind randomized clinical trial to evaluate the effects of probiotics administered to patients with mild acute pancreatitis at a tertiary medical center. The patients were given probiotics capsules (a mixed preparation of Bacillus subtilis and Enterococcus faecium) or placebo. The primary study endpoint was the LOS. The secondary endpoints included time to abdominal pain relief, recurrent abdominal pain, and time to successful oral feeding.\n\nResults:\nA total of 128 patients were included, with 64 patients in each arm. The severity of illness and the etiological distribution of disease were similar in the two groups. There was a significant reduction in the LOS in the probiotics treatment group vs the placebo group (5.36 ± 0.15 vs 6.02 ± 0.17 d, P < 0.05). The probiotics group was associated with a shorter time to abdominal pain relief and time to successful oral feeding (P < 0.01 for both) than the placebo group. No statistical difference was found in recurrent abdominal pain between the two groups.\n\nConclusions:\nThe study results showed that the administration of probiotics capsules is associated with a shorter duration of hospitalization in patients with mild acute pancreatitis."},"whole_article_text_length":{"kind":"number","value":6334,"string":"6,334"},"whole_article_abstract_length":{"kind":"number","value":283,"string":"283"},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["probiotics","patients","abdominal","pancreatitis","study","group","pain","abdominal pain","placebo","acute"],"string":"[\n \"probiotics\",\n \"patients\",\n \"abdominal\",\n \"pancreatitis\",\n \"study\",\n \"group\",\n \"pain\",\n \"abdominal pain\",\n \"placebo\",\n \"acute\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Double-Blind Method | Hospitalization | Humans | Pancreatitis | Probiotics | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute Disease | Double-Blind Method | Hospitalization | Humans | Pancreatitis | Probiotics | Treatment Outcome 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[SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] acute | pancreatitis | acute pancreatitis | disease | probiotics | microbiota | intestinal | patients | mild | analysis [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] abdominal | abdominal pain | pain | patients | data | capsules | time | study | normally distributed | test [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] sample | probiotics | randomized | acute | acute pancreatitis | pancreatitis double blinded randomized | follow time short | time short study future | time short study | time short [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | abdominal | group | patients | pancreatitis | placebo | study | acute | pain | abdominal pain [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] probiotics | abdominal | group | patients | pancreatitis | placebo | study | acute | pain | abdominal pain [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] tertiary ||| Enterococcus ||| ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| tertiary ||| Enterococcus ||| ||| ||| ||| 128 | 64 ||| two ||| 5.36 ± | 0.15 | 6.02 | 0.17 ||| ||| two ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| tertiary ||| Enterococcus ||| ||| ||| ||| 128 | 64 ||| two ||| 5.36 ± | 0.15 | 6.02 | 0.17 ||| ||| two ||| [SUMMARY]"}}},{"rowIdx":291,"cells":{"title":{"kind":"string","value":"Extract of Corallodiscus flabellata attenuates renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS signaling pathway."},"pmid":{"kind":"string","value":"35227255"},"background_abstract":{"kind":"string","value":"Fibrosis is one of the most common pathological features of the aging process of the kidney, and fibrosis in aging kidneys also aggravates the process of chronic kidney disease (CKD). Corallodiscus flabellata B. L. Burtt (C. flabellata, CF) is a commonly used botanical drug in Chinese folklore. However, few studies have reported its pharmacological effects. This study aimed to explore the effect of CF ethanol extract on renal fibrosis in SAMP8 mice and identify potentially active compounds."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Senescence-accelerated mouse-prone 8 (SAMP8) were used as animal models, and different doses of CF were given by gavage for one month. To observe the degree of renal aging in mice using β-galactosidase staining. Masson staining and the expression levels of Col-I, α-SMA, and FN were used to evaluate the renal fibrosis in mice. The protein expression levels of Nrf2 pathway and Wnt/β-catenin/RAS pathway in the kidney were measured. And β-galactosidase (β-gal) induced NRK-52E cells as an in vitro model to screen the active components of CF."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The CF ethanol extract significantly inhibited the activity of renal β-galactosidase and the expression levels of Col-I, α-SMA, and FN in SAMP8 mice, and improved Masson staining in SAMP8 mice. CF remarkably reduced urinary protein, creatinine, urea nitrogen and serum levels of TNF-α and IL-1β in SAMP8 mice, and significantly increased the levels of SOD and GSH-Px. Moreover, CF activated the Nrf2 pathway and blocked the Wnt/β-catenin/RAS pathway in the kidneys of mice. Besides, 3,4-dihydroxyphenylethanol (SDC-0-14, 16) and (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (SDC-1-8) were isolated from CF, which reduced the senescence of NRK-52E cells, and maybe the active ingredients of CF playing the anti-aging role."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Our experiments illuminated that CF ethanol extract may ameliorate renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS pathway. And SDC-0-14,16 and SDC-1-8 may be the material basis for CF to exert anti-renal senescence-related effects."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Fibrosis","Mice","Plant Extracts","Renal Insufficiency, Chronic","Wnt Signaling Pathway","beta Catenin"],"string":"[\n \"Animals\",\n \"Fibrosis\",\n \"Mice\",\n \"Plant Extracts\",\n \"Renal Insufficiency, Chronic\",\n \"Wnt Signaling Pathway\",\n \"beta Catenin\"\n]"},"pmcid":{"kind":"string","value":"8887028"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"The global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.\nCF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" CF improved the aging and fibrosis of the kidneys in SAMP8 mice To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nTo investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\n CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nBesides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\n CF activated the Nrf2 pathway in the kidney of SAMP8 mice The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nThe expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\n CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nIn order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\n CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nD-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\n CF improved PCA analysis of kidney tissue in SAMP8 mice Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPrincipal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF."},"other_sections_titles":{"kind":"list like","value":["Collection and extraction of the plant material","Animals and administration","Sample collection","Cell culture and in vitro study","Senescence-associated β-galactosidase staining","Histological analysis","Biochemical measurements","Immunohistochemical analysis","Western blot analysis","UPLC-Q-TOF-MS analysis for kidney samples","Statistical analysis","CF improved the aging and fibrosis of the kidneys in SAMP8 mice","CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice","CF activated the Nrf2 pathway in the kidney of SAMP8 mice","CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice","CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal","CF improved PCA analysis of kidney tissue in SAMP8 mice",""],"string":"[\n \"Collection and extraction of the plant material\",\n \"Animals and administration\",\n \"Sample collection\",\n \"Cell culture and in vitro study\",\n \"Senescence-associated β-galactosidase staining\",\n \"Histological analysis\",\n \"Biochemical measurements\",\n \"Immunohistochemical analysis\",\n \"Western blot analysis\",\n \"UPLC-Q-TOF-MS analysis for kidney samples\",\n \"Statistical analysis\",\n \"CF improved the aging and fibrosis of the kidneys in SAMP8 mice\",\n \"CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice\",\n \"CF activated the Nrf2 pathway in the kidney of SAMP8 mice\",\n \"CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice\",\n \"CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal\",\n \"CF improved PCA analysis of kidney tissue in SAMP8 mice\",\n \"\"\n]"},"other_sections_texts":{"kind":"list like","value":["“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).","Six-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.","At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.","NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.","Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.","The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.","The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.","The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.","Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.","The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.","The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.","To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)","Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)","The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)","In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)","D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group","Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;","\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3."],"string":"[\n \"“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\",\n \"Six-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\",\n \"At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\",\n \"NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\",\n \"Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\",\n \"The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\",\n \"The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\",\n \"The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\",\n \"Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\",\n \"The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\",\n \"The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\",\n \"To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\\n\\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\",\n \"Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\\n\\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\",\n \"The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\\n\\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\",\n \"In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\\n\\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\",\n \"D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\\n\\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\",\n \"Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\\n\\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\",\n \"\\nAdditional file 1.\\nAdditional file 1.\\n\\nAdditional file 2.\\nAdditional file 2.\\n\\nAdditional file 3.\\nAdditional file 3.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and methods","Collection and extraction of the plant material","Animals and administration","Sample collection","Cell culture and in vitro study","Senescence-associated β-galactosidase staining","Histological analysis","Biochemical measurements","Immunohistochemical analysis","Western blot analysis","UPLC-Q-TOF-MS analysis for kidney samples","Statistical analysis","Results","CF improved the aging and fibrosis of the kidneys in SAMP8 mice","CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice","CF activated the Nrf2 pathway in the kidney of SAMP8 mice","CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice","CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal","CF improved PCA analysis of kidney tissue in SAMP8 mice","Discussion","Conclusions","Supplementary Information",""],"string":"[\n \"Introduction\",\n \"Materials and methods\",\n \"Collection and extraction of the plant material\",\n \"Animals and administration\",\n \"Sample collection\",\n \"Cell culture and in vitro study\",\n \"Senescence-associated β-galactosidase staining\",\n \"Histological analysis\",\n \"Biochemical measurements\",\n \"Immunohistochemical analysis\",\n \"Western blot analysis\",\n \"UPLC-Q-TOF-MS analysis for kidney samples\",\n \"Statistical analysis\",\n \"Results\",\n \"CF improved the aging and fibrosis of the kidneys in SAMP8 mice\",\n \"CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice\",\n \"CF activated the Nrf2 pathway in the kidney of SAMP8 mice\",\n \"CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice\",\n \"CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal\",\n \"CF improved PCA analysis of kidney tissue in SAMP8 mice\",\n \"Discussion\",\n \"Conclusions\",\n \"Supplementary Information\",\n \"\"\n]"},"all_sections_texts":{"kind":"list like","value":["The global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.\nCF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books."," Collection and extraction of the plant material “Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\n“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).","“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).","Six-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.","At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.","NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.","Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.","The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.","The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.","The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.","Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.","The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.","The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant."," CF improved the aging and fibrosis of the kidneys in SAMP8 mice To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nTo investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\n CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nBesides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\n CF activated the Nrf2 pathway in the kidney of SAMP8 mice The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nThe expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\n CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nIn order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\n CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nD-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\n CF improved PCA analysis of kidney tissue in SAMP8 mice Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPrincipal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;","To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)","Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)","The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)","In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)","D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group","Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;","Aging plays an important role in the progression of CKD [19]. As CKD fibrosis progresses, senescent cells express and secrete pro-fibrotic factors (TGF-β, CTGF, and so forth) and pro-inflammatory factors (IL-1β, IL-6, TNF-α, and so forth), which are senescence-associated secretory phenotype factors, thereby accelerating renal fibrosis [20, 21]. At present, traditional Chinese medicine has achieved good results in treating renal fibrosis with fewer side effects [22]. This study explored the interventional effects of CF extract on renal fibrosis in SAMP8 mice.\nThe mouse strain selected for this study was SAMP8, which was developed based on the lifespan, senescence, and pathological phenotypic grading scores of AKR/J mice and was an accelerated aging model solely of genetic origin [23, 24]. Studies have shown that the changes in the renal pathology of SAMP8 mice (9 months) include tubulointerstitial fibrosis and focal segmental glomerulosclerosis [25]. And it was found that renal fibrosis in SAMP8 mice was age-related [6]. Therefore, we tested the activity of β-galactosidase and the level of renal fibrosis in SAMP8 mice, and found that CF-L and CF-M improved kidney fibrosis and β-galactosidase activity in SAMP8 mice (Fig. 1). And we also tested the urine output of the mice. Consistent with previously published studies indicating that CF obtained by two different processes had diuretic effects [13], the extract significantly increased the urine volume of SAMP8 mice. Also, the present study showed that supplementation with CF improved the renal function, the activity of antioxidant enzymes, and levels of inflammatory factors in SAMP8 mice (Fig. 2).\nThe Keap1–Nrf2 system gained the attention of many scholars in recent years due to its antioxidant and anti-inflammatory properties. Its pharmacological potential in treating kidney diseases had been extensively studied in nonclinical and clinical studies [26]. Nrf2 is a master transcriptional regulator for genes related to redox status and antioxidant effects [27]. Studies have shown that phosphorylation is required for Nrf2 activation and target gene induction [28]. Activating Nrf2 improved CKD progression by preventing oxidative stress and maintaining cellular redox homeostasis [29]. As a key transcription factor, Nrf2 plays a crucial role in defense against oxidative stress by regulating its downstream antioxidants and detoxification enzymes [30]. Kim et al. reported that resveratrol, as a potent Nrf2 activator, ameliorated aging-related progressive renal injury [31]. In the present study, CF improved the attenuation of Nrf2 in the kidney of SAMP8 mice without affecting the expression level of Keap1, suggesting that CF crude extract might improve oxidative damage in the kidneys by activating the Nrf2 pathway (Fig. 3).\nRenal fibrosis is characterized by excessive extracellular matrix (ECM) deposition leading to the formation of scars in the renal parenchyma [32]. Transforming growth factor-β (TGF-β) is thought to be a key cytokine in fibroblast overactivation [33, 34]. Of course, targeting only the TGF-β signaling pathway is insufficient to reduce renal fibrosis. Some studies indicated that CTGF, Wnt/β-catenin, renin-angiotensin system, oxidative stress, and so forth, were implicated in renal fibrosis [35–37]. Wnt/β-catenin is an evolutionarily conserved signaling pathway involved in the regulation of tissue homeostasis, organ development, and injury repair [38]. Cisternas et al. discussed the pro-fibrotic effect of Wnt signaling in both skeletal muscle and kidney [39]. Mounting evidence established that the Wnt/β-catenin signaling pathway plays a crucial role in regulating the development and progression of renal fibrotic lesions following injury [40–42]. The Wnt/β-catenin signal is relatively silent in the kidneys of healthy adults and is activated once the kidneys are subjected to various kinds of damages [43]. In mammals, the Wnt family has at least 19 family members critical for kidney development. And at least 15 of these family members are differentially upregulated in the aging kidney, including Wnt4 [6]. The results of the present study showed that the expression level of Wnt4 protein in the kidneys of SAMP8 mice was significantly higher than that in SAMR1 mice, which was verified by both Western blot and immunohistochemical analyses (Fig. 4a, d and f). Coincidentally, the expression level of β-catenin protein was also upregulated. Also, both Wnt4 and β-catenin were expressed in renal tubular epithelial cells, as revealed by the immunohistochemical analysis (Fig. 4a and f). Wnt/β-catenin elicited renal fibrosis by inducing multiple fibrogenic genes such as RAS components, matrix metalloproteinase-7 (MMP-7), plasminogen activator inhibitor 1 (PAI-1), and Snail1 [37]. Zhou et al. described Wnt/β-catenin as the major upstream regulator, which controls the expression of all tested RAS components in the kidneys [44]. Zhou et al. used a 5/6 nephrectomy (5/6 NX) rat model to show that the expression levels of major components of RAS in the brain and kidneys, such as angiotensinogen, angiotensin-converting enzyme, and angiotensin II AT1-receptor, was significantly upregulated. The upregulated expression level was inhibited by a central blocker of Wnt, which was an adeno-associated virus vector overexpressing the DKK1 gene [45]. Similarly, the present study found that AGTR1 was still expressed in the renal tubular epithelium, and the expression levels of renin and AGTR1 in the kidney tissues of SAMP8 mice were significantly greater than those in SAMR1 mice (Fig. 4a and d g). These results indicated that the Wnt/β-catenin/RAS signaling pathway was activated in the kidneys of SAMP8 mice, and the CF effectively inhibited the activation of this pathway.\nCTGF exerts multiple biological functions, including promoting mitosis of chemotactic cells, inducing adhesion, and promoting cell proliferation and ECM synthesis [35, 46]. CCN2 (CTGF) modulated Wnt signaling by binding to low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to further mediate fibrosis [39, 47]. Other studies used gene silencing to discover and confirm that Nrf2 regulated the Wnt pathway by regulating the expression level of CTGF, affecting renal interstitial disease. Also, Nrf2 regulated the CTGF transcription level mainly via CTGF transcriptional regulator c-Fos [48]. The immunohistochemical analysis revealed that CTGF and p-c-Fos were expressed mainly in renal tubular epithelial cells, and p-c-Fos was also distributed in glomeruli. The quantitative analysis showed that the expression levels of both proteins was inhibited by CF (Figs. 3b and e and 4a and c). Finally, the PCA analysis was used to further verify that CF improved kidney damage in SAMP8 mice (Fig. 6). The present study provided evidence that the CF not only activated Nrf2 signaling but also relied on Nrf2 to balance oxidative stress and inflammation, inhibited Wnt/β-catenin/RAS signaling, and improved kidney aging and renal fibrogenesis. However, we also found inconsistent improvement in the effect of CF on Masson and SA-β-gal staining in this experiment. It is speculated that this may be due to the inconsistent progression of renal aging and renal fibrosis in SAMP8 mice. The degree of renal aging in the mice in this experiment may be more serious than fibrosis. Therefore, the ameliorating effect of CF-H on renal aging in SAMP8 mice was not as obvious as that of renal fibrosis.\nAs a reducing monosaccharide, D-galactose is widely used in various age-related diseases in vivo and in vitro. D-gal was found to cause senescence and injury in NRK-52E cells [49], induce senescence of human kidney proximal tubular epithelial cells (HKC-8 cells), and increase the expression levels of two renal fibrosis marker proteins FN and α-SMA [6]. In this study, D-gal was used to induce NRK-52E cells in vitro. Using MTT assay and β-galactosidase staining to detect the compounds isolated from CF, it was found that SDC-0-14,16 and SDC-1-8 enhanced the cell viability induced by D-gal and inhibited D-gal induced cell senescence (Fig. 5). These suggested that SDC-0-14,16 and SDC-1-8 may be the material basis for CF to delay kidney aging. SDC-1-8 is one of the phenylethanoid glycosides. phenylethanoid glycosides were found to extend the life span of Caenorhabditis Elegans [50], with anti-aging, [51] and neuroprotective effects [52–54]. These provide a direction for our follow-up study on the pharmacological effects of CF.","In conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF."," \nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.\n\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.","\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3."],"string":"[\n \"The global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.\\nCF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books.\",\n \" Collection and extraction of the plant material “Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\\n“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\",\n \"“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\",\n \"Six-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\",\n \"At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\",\n \"NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\",\n \"Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\",\n \"The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\",\n \"The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\",\n \"The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\",\n \"Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\",\n \"The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\",\n \"The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\",\n \" CF improved the aging and fibrosis of the kidneys in SAMP8 mice To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\\n\\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\nTo investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\\n\\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\n CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\\n\\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\nBesides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\\n\\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\n CF activated the Nrf2 pathway in the kidney of SAMP8 mice The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\\n\\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\nThe expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\\n\\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\n CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\\n\\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\nIn order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\\n\\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\n CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\\n\\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\nD-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\\n\\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\n CF improved PCA analysis of kidney tissue in SAMP8 mice Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\\n\\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\\nPrincipal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\\n\\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\",\n \"To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\\n\\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\",\n \"Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\\n\\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\",\n \"The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\\n\\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\",\n \"In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\\n\\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\",\n \"D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\\n\\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\",\n \"Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\\n\\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\",\n \"Aging plays an important role in the progression of CKD [19]. As CKD fibrosis progresses, senescent cells express and secrete pro-fibrotic factors (TGF-β, CTGF, and so forth) and pro-inflammatory factors (IL-1β, IL-6, TNF-α, and so forth), which are senescence-associated secretory phenotype factors, thereby accelerating renal fibrosis [20, 21]. At present, traditional Chinese medicine has achieved good results in treating renal fibrosis with fewer side effects [22]. This study explored the interventional effects of CF extract on renal fibrosis in SAMP8 mice.\\nThe mouse strain selected for this study was SAMP8, which was developed based on the lifespan, senescence, and pathological phenotypic grading scores of AKR/J mice and was an accelerated aging model solely of genetic origin [23, 24]. Studies have shown that the changes in the renal pathology of SAMP8 mice (9 months) include tubulointerstitial fibrosis and focal segmental glomerulosclerosis [25]. And it was found that renal fibrosis in SAMP8 mice was age-related [6]. Therefore, we tested the activity of β-galactosidase and the level of renal fibrosis in SAMP8 mice, and found that CF-L and CF-M improved kidney fibrosis and β-galactosidase activity in SAMP8 mice (Fig. 1). And we also tested the urine output of the mice. Consistent with previously published studies indicating that CF obtained by two different processes had diuretic effects [13], the extract significantly increased the urine volume of SAMP8 mice. Also, the present study showed that supplementation with CF improved the renal function, the activity of antioxidant enzymes, and levels of inflammatory factors in SAMP8 mice (Fig. 2).\\nThe Keap1–Nrf2 system gained the attention of many scholars in recent years due to its antioxidant and anti-inflammatory properties. Its pharmacological potential in treating kidney diseases had been extensively studied in nonclinical and clinical studies [26]. Nrf2 is a master transcriptional regulator for genes related to redox status and antioxidant effects [27]. Studies have shown that phosphorylation is required for Nrf2 activation and target gene induction [28]. Activating Nrf2 improved CKD progression by preventing oxidative stress and maintaining cellular redox homeostasis [29]. As a key transcription factor, Nrf2 plays a crucial role in defense against oxidative stress by regulating its downstream antioxidants and detoxification enzymes [30]. Kim et al. reported that resveratrol, as a potent Nrf2 activator, ameliorated aging-related progressive renal injury [31]. In the present study, CF improved the attenuation of Nrf2 in the kidney of SAMP8 mice without affecting the expression level of Keap1, suggesting that CF crude extract might improve oxidative damage in the kidneys by activating the Nrf2 pathway (Fig. 3).\\nRenal fibrosis is characterized by excessive extracellular matrix (ECM) deposition leading to the formation of scars in the renal parenchyma [32]. Transforming growth factor-β (TGF-β) is thought to be a key cytokine in fibroblast overactivation [33, 34]. Of course, targeting only the TGF-β signaling pathway is insufficient to reduce renal fibrosis. Some studies indicated that CTGF, Wnt/β-catenin, renin-angiotensin system, oxidative stress, and so forth, were implicated in renal fibrosis [35–37]. Wnt/β-catenin is an evolutionarily conserved signaling pathway involved in the regulation of tissue homeostasis, organ development, and injury repair [38]. Cisternas et al. discussed the pro-fibrotic effect of Wnt signaling in both skeletal muscle and kidney [39]. Mounting evidence established that the Wnt/β-catenin signaling pathway plays a crucial role in regulating the development and progression of renal fibrotic lesions following injury [40–42]. The Wnt/β-catenin signal is relatively silent in the kidneys of healthy adults and is activated once the kidneys are subjected to various kinds of damages [43]. In mammals, the Wnt family has at least 19 family members critical for kidney development. And at least 15 of these family members are differentially upregulated in the aging kidney, including Wnt4 [6]. The results of the present study showed that the expression level of Wnt4 protein in the kidneys of SAMP8 mice was significantly higher than that in SAMR1 mice, which was verified by both Western blot and immunohistochemical analyses (Fig. 4a, d and f). Coincidentally, the expression level of β-catenin protein was also upregulated. Also, both Wnt4 and β-catenin were expressed in renal tubular epithelial cells, as revealed by the immunohistochemical analysis (Fig. 4a and f). Wnt/β-catenin elicited renal fibrosis by inducing multiple fibrogenic genes such as RAS components, matrix metalloproteinase-7 (MMP-7), plasminogen activator inhibitor 1 (PAI-1), and Snail1 [37]. Zhou et al. described Wnt/β-catenin as the major upstream regulator, which controls the expression of all tested RAS components in the kidneys [44]. Zhou et al. used a 5/6 nephrectomy (5/6 NX) rat model to show that the expression levels of major components of RAS in the brain and kidneys, such as angiotensinogen, angiotensin-converting enzyme, and angiotensin II AT1-receptor, was significantly upregulated. The upregulated expression level was inhibited by a central blocker of Wnt, which was an adeno-associated virus vector overexpressing the DKK1 gene [45]. Similarly, the present study found that AGTR1 was still expressed in the renal tubular epithelium, and the expression levels of renin and AGTR1 in the kidney tissues of SAMP8 mice were significantly greater than those in SAMR1 mice (Fig. 4a and d g). These results indicated that the Wnt/β-catenin/RAS signaling pathway was activated in the kidneys of SAMP8 mice, and the CF effectively inhibited the activation of this pathway.\\nCTGF exerts multiple biological functions, including promoting mitosis of chemotactic cells, inducing adhesion, and promoting cell proliferation and ECM synthesis [35, 46]. CCN2 (CTGF) modulated Wnt signaling by binding to low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to further mediate fibrosis [39, 47]. Other studies used gene silencing to discover and confirm that Nrf2 regulated the Wnt pathway by regulating the expression level of CTGF, affecting renal interstitial disease. Also, Nrf2 regulated the CTGF transcription level mainly via CTGF transcriptional regulator c-Fos [48]. The immunohistochemical analysis revealed that CTGF and p-c-Fos were expressed mainly in renal tubular epithelial cells, and p-c-Fos was also distributed in glomeruli. The quantitative analysis showed that the expression levels of both proteins was inhibited by CF (Figs. 3b and e and 4a and c). Finally, the PCA analysis was used to further verify that CF improved kidney damage in SAMP8 mice (Fig. 6). The present study provided evidence that the CF not only activated Nrf2 signaling but also relied on Nrf2 to balance oxidative stress and inflammation, inhibited Wnt/β-catenin/RAS signaling, and improved kidney aging and renal fibrogenesis. However, we also found inconsistent improvement in the effect of CF on Masson and SA-β-gal staining in this experiment. It is speculated that this may be due to the inconsistent progression of renal aging and renal fibrosis in SAMP8 mice. The degree of renal aging in the mice in this experiment may be more serious than fibrosis. Therefore, the ameliorating effect of CF-H on renal aging in SAMP8 mice was not as obvious as that of renal fibrosis.\\nAs a reducing monosaccharide, D-galactose is widely used in various age-related diseases in vivo and in vitro. D-gal was found to cause senescence and injury in NRK-52E cells [49], induce senescence of human kidney proximal tubular epithelial cells (HKC-8 cells), and increase the expression levels of two renal fibrosis marker proteins FN and α-SMA [6]. In this study, D-gal was used to induce NRK-52E cells in vitro. Using MTT assay and β-galactosidase staining to detect the compounds isolated from CF, it was found that SDC-0-14,16 and SDC-1-8 enhanced the cell viability induced by D-gal and inhibited D-gal induced cell senescence (Fig. 5). These suggested that SDC-0-14,16 and SDC-1-8 may be the material basis for CF to delay kidney aging. SDC-1-8 is one of the phenylethanoid glycosides. phenylethanoid glycosides were found to extend the life span of Caenorhabditis Elegans [50], with anti-aging, [51] and neuroprotective effects [52–54]. These provide a direction for our follow-up study on the pharmacological effects of CF.\",\n \"In conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF.\",\n \" \\nAdditional file 1.\\nAdditional file 1.\\n\\nAdditional file 2.\\nAdditional file 2.\\n\\nAdditional file 3.\\nAdditional file 3.\\n\\nAdditional file 1.\\nAdditional file 1.\\n\\nAdditional file 2.\\nAdditional file 2.\\n\\nAdditional file 3.\\nAdditional file 3.\",\n \"\\nAdditional file 1.\\nAdditional file 1.\\n\\nAdditional file 2.\\nAdditional file 2.\\n\\nAdditional file 3.\\nAdditional file 3.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["introduction","materials|methods",null,null,null,null,null,null,null,null,null,null,null,"results",null,null,null,null,null,null,"discussion","conclusion","supplementary-material",null],"string":"[\n \"introduction\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusion\",\n \"supplementary-material\",\n null\n]"},"keywords":{"kind":"list like","value":["Aging","\nCorallodiscus flabellata\n","Kidney","Renal fibrosis","SAMP8","Senescence","Wnt/β-catenin/RAS"],"string":"[\n \"Aging\",\n \"\\nCorallodiscus flabellata\\n\",\n \"Kidney\",\n \"Renal fibrosis\",\n \"SAMP8\",\n \"Senescence\",\n \"Wnt/β-catenin/RAS\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nThe global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.\nCF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books.\n\nMaterials and methods:\n Collection and extraction of the plant material “Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\n“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\n\nCollection and extraction of the plant material:\n“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\n\nAnimals and administration:\nSix-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\n\nSample collection:\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\n\nCell culture and in vitro study:\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\n\nSenescence-associated β-galactosidase staining:\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\n\nHistological analysis:\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\n\nBiochemical measurements:\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\n\nImmunohistochemical analysis:\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\n\nWestern blot analysis:\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\n\nUPLC-Q-TOF-MS analysis for kidney samples:\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\n\nStatistical analysis:\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\n\nResults:\n CF improved the aging and fibrosis of the kidneys in SAMP8 mice To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nTo investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\n CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nBesides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\n CF activated the Nrf2 pathway in the kidney of SAMP8 mice The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nThe expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\n CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nIn order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\n CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nD-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\n CF improved PCA analysis of kidney tissue in SAMP8 mice Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPrincipal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\n\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice:\nTo investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\n\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice:\nBesides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\n\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice:\nThe expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\n\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice:\nIn order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\n\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal:\nD-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\n\nCF improved PCA analysis of kidney tissue in SAMP8 mice:\nPrincipal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\n\nDiscussion:\nAging plays an important role in the progression of CKD [19]. As CKD fibrosis progresses, senescent cells express and secrete pro-fibrotic factors (TGF-β, CTGF, and so forth) and pro-inflammatory factors (IL-1β, IL-6, TNF-α, and so forth), which are senescence-associated secretory phenotype factors, thereby accelerating renal fibrosis [20, 21]. At present, traditional Chinese medicine has achieved good results in treating renal fibrosis with fewer side effects [22]. This study explored the interventional effects of CF extract on renal fibrosis in SAMP8 mice.\nThe mouse strain selected for this study was SAMP8, which was developed based on the lifespan, senescence, and pathological phenotypic grading scores of AKR/J mice and was an accelerated aging model solely of genetic origin [23, 24]. Studies have shown that the changes in the renal pathology of SAMP8 mice (9 months) include tubulointerstitial fibrosis and focal segmental glomerulosclerosis [25]. And it was found that renal fibrosis in SAMP8 mice was age-related [6]. Therefore, we tested the activity of β-galactosidase and the level of renal fibrosis in SAMP8 mice, and found that CF-L and CF-M improved kidney fibrosis and β-galactosidase activity in SAMP8 mice (Fig. 1). And we also tested the urine output of the mice. Consistent with previously published studies indicating that CF obtained by two different processes had diuretic effects [13], the extract significantly increased the urine volume of SAMP8 mice. Also, the present study showed that supplementation with CF improved the renal function, the activity of antioxidant enzymes, and levels of inflammatory factors in SAMP8 mice (Fig. 2).\nThe Keap1–Nrf2 system gained the attention of many scholars in recent years due to its antioxidant and anti-inflammatory properties. Its pharmacological potential in treating kidney diseases had been extensively studied in nonclinical and clinical studies [26]. Nrf2 is a master transcriptional regulator for genes related to redox status and antioxidant effects [27]. Studies have shown that phosphorylation is required for Nrf2 activation and target gene induction [28]. Activating Nrf2 improved CKD progression by preventing oxidative stress and maintaining cellular redox homeostasis [29]. As a key transcription factor, Nrf2 plays a crucial role in defense against oxidative stress by regulating its downstream antioxidants and detoxification enzymes [30]. Kim et al. reported that resveratrol, as a potent Nrf2 activator, ameliorated aging-related progressive renal injury [31]. In the present study, CF improved the attenuation of Nrf2 in the kidney of SAMP8 mice without affecting the expression level of Keap1, suggesting that CF crude extract might improve oxidative damage in the kidneys by activating the Nrf2 pathway (Fig. 3).\nRenal fibrosis is characterized by excessive extracellular matrix (ECM) deposition leading to the formation of scars in the renal parenchyma [32]. Transforming growth factor-β (TGF-β) is thought to be a key cytokine in fibroblast overactivation [33, 34]. Of course, targeting only the TGF-β signaling pathway is insufficient to reduce renal fibrosis. Some studies indicated that CTGF, Wnt/β-catenin, renin-angiotensin system, oxidative stress, and so forth, were implicated in renal fibrosis [35–37]. Wnt/β-catenin is an evolutionarily conserved signaling pathway involved in the regulation of tissue homeostasis, organ development, and injury repair [38]. Cisternas et al. discussed the pro-fibrotic effect of Wnt signaling in both skeletal muscle and kidney [39]. Mounting evidence established that the Wnt/β-catenin signaling pathway plays a crucial role in regulating the development and progression of renal fibrotic lesions following injury [40–42]. The Wnt/β-catenin signal is relatively silent in the kidneys of healthy adults and is activated once the kidneys are subjected to various kinds of damages [43]. In mammals, the Wnt family has at least 19 family members critical for kidney development. And at least 15 of these family members are differentially upregulated in the aging kidney, including Wnt4 [6]. The results of the present study showed that the expression level of Wnt4 protein in the kidneys of SAMP8 mice was significantly higher than that in SAMR1 mice, which was verified by both Western blot and immunohistochemical analyses (Fig. 4a, d and f). Coincidentally, the expression level of β-catenin protein was also upregulated. Also, both Wnt4 and β-catenin were expressed in renal tubular epithelial cells, as revealed by the immunohistochemical analysis (Fig. 4a and f). Wnt/β-catenin elicited renal fibrosis by inducing multiple fibrogenic genes such as RAS components, matrix metalloproteinase-7 (MMP-7), plasminogen activator inhibitor 1 (PAI-1), and Snail1 [37]. Zhou et al. described Wnt/β-catenin as the major upstream regulator, which controls the expression of all tested RAS components in the kidneys [44]. Zhou et al. used a 5/6 nephrectomy (5/6 NX) rat model to show that the expression levels of major components of RAS in the brain and kidneys, such as angiotensinogen, angiotensin-converting enzyme, and angiotensin II AT1-receptor, was significantly upregulated. The upregulated expression level was inhibited by a central blocker of Wnt, which was an adeno-associated virus vector overexpressing the DKK1 gene [45]. Similarly, the present study found that AGTR1 was still expressed in the renal tubular epithelium, and the expression levels of renin and AGTR1 in the kidney tissues of SAMP8 mice were significantly greater than those in SAMR1 mice (Fig. 4a and d g). These results indicated that the Wnt/β-catenin/RAS signaling pathway was activated in the kidneys of SAMP8 mice, and the CF effectively inhibited the activation of this pathway.\nCTGF exerts multiple biological functions, including promoting mitosis of chemotactic cells, inducing adhesion, and promoting cell proliferation and ECM synthesis [35, 46]. CCN2 (CTGF) modulated Wnt signaling by binding to low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to further mediate fibrosis [39, 47]. Other studies used gene silencing to discover and confirm that Nrf2 regulated the Wnt pathway by regulating the expression level of CTGF, affecting renal interstitial disease. Also, Nrf2 regulated the CTGF transcription level mainly via CTGF transcriptional regulator c-Fos [48]. The immunohistochemical analysis revealed that CTGF and p-c-Fos were expressed mainly in renal tubular epithelial cells, and p-c-Fos was also distributed in glomeruli. The quantitative analysis showed that the expression levels of both proteins was inhibited by CF (Figs. 3b and e and 4a and c). Finally, the PCA analysis was used to further verify that CF improved kidney damage in SAMP8 mice (Fig. 6). The present study provided evidence that the CF not only activated Nrf2 signaling but also relied on Nrf2 to balance oxidative stress and inflammation, inhibited Wnt/β-catenin/RAS signaling, and improved kidney aging and renal fibrogenesis. However, we also found inconsistent improvement in the effect of CF on Masson and SA-β-gal staining in this experiment. It is speculated that this may be due to the inconsistent progression of renal aging and renal fibrosis in SAMP8 mice. The degree of renal aging in the mice in this experiment may be more serious than fibrosis. Therefore, the ameliorating effect of CF-H on renal aging in SAMP8 mice was not as obvious as that of renal fibrosis.\nAs a reducing monosaccharide, D-galactose is widely used in various age-related diseases in vivo and in vitro. D-gal was found to cause senescence and injury in NRK-52E cells [49], induce senescence of human kidney proximal tubular epithelial cells (HKC-8 cells), and increase the expression levels of two renal fibrosis marker proteins FN and α-SMA [6]. In this study, D-gal was used to induce NRK-52E cells in vitro. Using MTT assay and β-galactosidase staining to detect the compounds isolated from CF, it was found that SDC-0-14,16 and SDC-1-8 enhanced the cell viability induced by D-gal and inhibited D-gal induced cell senescence (Fig. 5). These suggested that SDC-0-14,16 and SDC-1-8 may be the material basis for CF to delay kidney aging. SDC-1-8 is one of the phenylethanoid glycosides. phenylethanoid glycosides were found to extend the life span of Caenorhabditis Elegans [50], with anti-aging, [51] and neuroprotective effects [52–54]. These provide a direction for our follow-up study on the pharmacological effects of CF.\n\nConclusions:\nIn conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF.\n\nSupplementary Information:\n \nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.\n\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.\n\n:\n\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nFibrosis is one of the most common pathological features of the aging process of the kidney, and fibrosis in aging kidneys also aggravates the process of chronic kidney disease (CKD). Corallodiscus flabellata B. L. Burtt (C. flabellata, CF) is a commonly used botanical drug in Chinese folklore. However, few studies have reported its pharmacological effects. This study aimed to explore the effect of CF ethanol extract on renal fibrosis in SAMP8 mice and identify potentially active compounds.\n\nMethods:\nSenescence-accelerated mouse-prone 8 (SAMP8) were used as animal models, and different doses of CF were given by gavage for one month. To observe the degree of renal aging in mice using β-galactosidase staining. Masson staining and the expression levels of Col-I, α-SMA, and FN were used to evaluate the renal fibrosis in mice. The protein expression levels of Nrf2 pathway and Wnt/β-catenin/RAS pathway in the kidney were measured. And β-galactosidase (β-gal) induced NRK-52E cells as an in vitro model to screen the active components of CF.\n\nResults:\nThe CF ethanol extract significantly inhibited the activity of renal β-galactosidase and the expression levels of Col-I, α-SMA, and FN in SAMP8 mice, and improved Masson staining in SAMP8 mice. CF remarkably reduced urinary protein, creatinine, urea nitrogen and serum levels of TNF-α and IL-1β in SAMP8 mice, and significantly increased the levels of SOD and GSH-Px. Moreover, CF activated the Nrf2 pathway and blocked the Wnt/β-catenin/RAS pathway in the kidneys of mice. Besides, 3,4-dihydroxyphenylethanol (SDC-0-14, 16) and (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (SDC-1-8) were isolated from CF, which reduced the senescence of NRK-52E cells, and maybe the active ingredients of CF playing the anti-aging role.\n\nConclusions:\nOur experiments illuminated that CF ethanol extract may ameliorate renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS pathway. And SDC-0-14,16 and SDC-1-8 may be the material basis for CF to exert anti-renal senescence-related effects."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nThe global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.\nCF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books.\n\nConclusions:\nIn conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nFibrosis is one of the most common pathological features of the aging process of the kidney, and fibrosis in aging kidneys also aggravates the process of chronic kidney disease (CKD). Corallodiscus flabellata B. L. Burtt (C. flabellata, CF) is a commonly used botanical drug in Chinese folklore. However, few studies have reported its pharmacological effects. This study aimed to explore the effect of CF ethanol extract on renal fibrosis in SAMP8 mice and identify potentially active compounds.\n\nMethods:\nSenescence-accelerated mouse-prone 8 (SAMP8) were used as animal models, and different doses of CF were given by gavage for one month. To observe the degree of renal aging in mice using β-galactosidase staining. Masson staining and the expression levels of Col-I, α-SMA, and FN were used to evaluate the renal fibrosis in mice. The protein expression levels of Nrf2 pathway and Wnt/β-catenin/RAS pathway in the kidney were measured. And β-galactosidase (β-gal) induced NRK-52E cells as an in vitro model to screen the active components of CF.\n\nResults:\nThe CF ethanol extract significantly inhibited the activity of renal β-galactosidase and the expression levels of Col-I, α-SMA, and FN in SAMP8 mice, and improved Masson staining in SAMP8 mice. CF remarkably reduced urinary protein, creatinine, urea nitrogen and serum levels of TNF-α and IL-1β in SAMP8 mice, and significantly increased the levels of SOD and GSH-Px. Moreover, CF activated the Nrf2 pathway and blocked the Wnt/β-catenin/RAS pathway in the kidneys of mice. Besides, 3,4-dihydroxyphenylethanol (SDC-0-14, 16) and (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (SDC-1-8) were isolated from CF, which reduced the senescence of NRK-52E cells, and maybe the active ingredients of CF playing the anti-aging role.\n\nConclusions:\nOur experiments illuminated that CF ethanol extract may ameliorate renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS pathway. And SDC-0-14,16 and SDC-1-8 may be the material basis for CF to exert anti-renal senescence-related effects."},"whole_article_text_length":{"kind":"number","value":17396,"string":"17,396"},"whole_article_abstract_length":{"kind":"number","value":442,"string":"442"},"num_sections":{"kind":"number","value":24,"string":"24"},"most_frequent_words":{"kind":"list like","value":["cf","mice","extract","kidney","samp8","ethanol","samp8 mice","treated","dose","ethanol extract"],"string":"[\n \"cf\",\n \"mice\",\n \"extract\",\n \"kidney\",\n \"samp8\",\n \"ethanol\",\n \"samp8 mice\",\n \"treated\",\n \"dose\",\n \"ethanol extract\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | \nCorallodiscus flabellata\n | Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | \nCorallodiscus flabellata\n | Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | \nCorallodiscus flabellata\n | Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | \nCorallodiscus flabellata\n | Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | \nCorallodiscus flabellata\n | Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] fibrosis | renal fibrosis | catenin | renal | ras | disease | lef | cf | wnt catenin | wnt [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | cf treated | dose cf ethanol | dose cf ethanol extract | cf ethanol extract | dose cf | ethanol extract | cf ethanol | dose [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ingredients | active | active ingredients | cf | pharmacological support treating | research active | experiments findings provided pharmacological | experiments findings provided | experiments findings | ingredients found [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | ethanol | extract | china | additional | additional file | file | kidney | samp8 [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cf | mice | ethanol | extract | china | additional | additional file | file | kidney | samp8 [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| B. L. Burtt | CF | Chinese ||| ||| CF | SAMP8 [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] CF | SAMP8 | Masson | SAMP8 ||| TNF | IL-1β | SAMP8 | SOD | GSH-Px ||| Nrf2 | Wnt ||| 3,4 | SDC-0-14 | 16 | 3,4 | SDC-1-8) | CF | NRK-52E | CF [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] CF | SAMP8 | Wnt ||| SDC-0-14,16 | SDC-1-8 | CF [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| B. L. Burtt | CF | Chinese ||| ||| CF | SAMP8 ||| CF | one month ||| ||| Masson ||| Nrf2 | Wnt ||| NRK-52E | CF ||| ||| CF | SAMP8 | Masson | SAMP8 ||| TNF | IL-1β | SAMP8 | SOD | GSH-Px ||| Nrf2 | Wnt ||| 3,4 | SDC-0-14 | 16 | 3,4 | SDC-1-8) | CF | NRK-52E | CF ||| CF | SAMP8 | Wnt ||| SDC-0-14,16 | SDC-1-8 | CF [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| B. L. Burtt | CF | Chinese ||| ||| CF | SAMP8 ||| CF | one month ||| ||| Masson ||| Nrf2 | Wnt ||| NRK-52E | CF ||| ||| CF | SAMP8 | Masson | SAMP8 ||| TNF | IL-1β | SAMP8 | SOD | GSH-Px ||| Nrf2 | Wnt ||| 3,4 | SDC-0-14 | 16 | 3,4 | SDC-1-8) | CF | NRK-52E | CF ||| CF | SAMP8 | Wnt ||| SDC-0-14,16 | SDC-1-8 | CF [SUMMARY]"}}},{"rowIdx":292,"cells":{"title":{"kind":"string","value":"The plant coumarins auraptene and lacinartin as potential multifunctional therapeutic agents for treating periodontal disease."},"pmid":{"kind":"string","value":"22742512"},"background_abstract":{"kind":"string","value":"Periodontal diseases are bacterial infections leading to chronic inflammation disorders that are frequently observed in adults. In the present study, we evaluated the effect of auraptene and lacinartin, two natural oxyprenylated coumarins, on the growth, adherence properties, and collagenase activity of Porphyromonas gingivalis. We also investigated the capacity of these compounds to reduce cytokine and matrix metalloproteinase (MMP) secretion by lipopolysaccharide (LPS)-stimulated macrophages and to inhibit MMP-9 activity."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Microplate dilution assays were performed to determine the effect of auraptene and lacinartin on P. gingivalis growth as well as biofilm formation stained with crystal violet. Adhesion of FITC-labeled P. gingivalis to oral epithelial cells was monitored by fluorometry. The effects of auraptene and lacinartin on LPS-induced cytokine and MMP secretion by macrophages were determined by immunological assays. Fluorogenic assays were used to evaluate the capacity of the two coumarins to inhibit the activity of P. gingivalis collagenase and MMP-9."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Only lacinartin completely inhibited P. gingivalis growth in a complex culture medium. However, under iron-limiting conditions, auraptene and lacinartin both inhibited the growth of P. gingivalis. Lacinartin also inhibited biofilm formation by P. gingivalis and promoted biofilm desorption. Both compounds prevented the adherence of P. gingivalis to oral epithelial cells, dose-dependently reduced the secretion of cytokines (IL-8 and TNF-α) and MMP-8 and MMP-9 by LPS-stimulated macrophages, and inhibited MMP-9 activity. Lacinartin also inhibited P. gingivalis collagenase activity."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"By acting on multiple targets, including pathogenic bacteria, tissue-destructive enzymes, and the host inflammatory response, auraptene and lacinartin may be promising natural compounds for preventing and treating periodontal diseases."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Bacterial Adhesion","Biofilms","Cell Line","Coumarins","Epithelial Cells","Humans","Macrophages","Periodontal Diseases","Plant Extracts","Porphyromonas gingivalis"],"string":"[\n \"Bacterial Adhesion\",\n \"Biofilms\",\n \"Cell Line\",\n \"Coumarins\",\n \"Epithelial Cells\",\n \"Humans\",\n \"Macrophages\",\n \"Periodontal Diseases\",\n \"Plant Extracts\",\n \"Porphyromonas gingivalis\"\n]"},"pmcid":{"kind":"string","value":"3489859"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Lacinartin (50 and 100 μg/ml) almost completely inhibited the growth of P. gingivalis in THB-HK (Figure.\n2B), while the highest concentration of auraptene tested (100 μg/ml) only reduced growth by 42% (Figure.\n2A). Bacterial growth was slightly lower in MBB-H (OD660 = 0.59), which represents an iron poor condition for P. gingivalis, than in THB-HK (OD660 = 0.78) (Figure.\n2). The lowest concentration of auraptene tested (12.5 μg/ml) inhibited growth by 58% in MBB-H, while such a concentration had no significant inhibitory effect in THB-HK (Figure.\n2A). Almost complete inhibition was observed at higher concentrations (50 and 100 μg/ml). As for auraptene, lacinartin seemed to be more effective for inhibiting growth of P. gingivalis in iron-limiting conditions.\n Effect of auraptene (A) and lacinartin (B) on the growth of P. gingivalis in a complex medium (THB-HK) and under iron-limiting conditions in MBB-H. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed using the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene had no obvious inhibitory effect on P. gingivalis biofilm formation or desorption (Figure.\n3). On the contrary, high concentrations (50 and 100 μg/ml) of auraptene appear to increase biofilm formation (Figure.\n3A). Lacinartin at 50 and 100 μg/ml inhibited biofilm formation by P. gingivalis by approximately 75% (Figure.\n3A). In addition, lacinartin (12.5-100 μg/ml) caused approximately one-third of the biofilm to desorb (Figure.\n3B).\n Effect of auraptene and lacinartin on P. gingivalis biofilm formation (A) and on desorption of a pre-formed P. gingivalis biofilm (B). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene and lacinartin both dose-dependently inhibited bacterial adhesion to oral epithelial cells (Figure.\n4). At the lowest concentration tested (12.5 μg/ml), auraptene and lacinartin reduced the adherence of P. gingivalis to epithelial cells by 33% and 43%, respectively, while 100 μg/ml of auraptene and lacinartin reduced adherence by 37% and 71%, respectively (Figure.\n4).\n Effect of auraptene and lacinartin on the adherence of P. gingivalis to human oral epithelial cells. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. A value of 100% was assigned to the control (no auraptene; no lacinartin). Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nThe highest non-cytotoxic concentrations of auraptene and lacinartin that can be used to evaluate their effect on the inflammatory response of a human macrophage model stimulated with LPS were 25 and 50 μg/ml, respectively (data not shown). Following a 2-h pretreatment of the model with auraptene (6.25, 12.5, and 25 μg/ml) or lacinartin (12.5, 25, and 50 μg/ml), macrophages were stimulated with LPS to induce an inflammatory response (cytokine and MMP secretion). Auraptene and lacinartin both had a significant inhibitory effect on IL-8 and TNF-α secretion. At the lowest concentration tested (6.25 μg/ml), auraptene reduced IL-8 and TNF-α secretion by 22% and 37%, respectively, compared to untreated cells, while at the highest concentration tested (25 μg/ml), it inhibited IL-8 and TNF-α secretion by 92% and 85%, respectively (Figure.\n5). As shown in Fig.\n5, the highest concentration of lacinartin tested (50 μg/ml) reduced IL-8 and TNF-α secretion by 95% and 99%, respectively, while 12.5 and 25 μg/ml of lacinartin increased the secretion of both cytokines, likely due to a synergistic effect of LPS and lacinartin, since lacinartin alone had no effect (data not shown). Auraptene and lacinartin both reduced MMP-8 and MMP-9 secretion, sometimes below basal levels (Figure.\n6). Auraptene reduced MMP-8 secretion by 63% (6.25 μg/ml), 69% (12.5 μg/ml), and 73% (25 μg/ml), while lacinartin reduced MMP-8 secretion by 79% (12.5 μg/ml), 83% (25 μg/ml), and 89% (50 μg/ml) (Figure.\n6A). At their highest concentrations tested, auraptene (25 μg/ml) reduced MMP-9 secretion by 76% (25 μg/ml) (Figure.\n6B), while lacinartin (50 μg/ml) reduced MMP-9 secretion by 82% (Figure.\n6B).\n Effect of auraptene and lacinartin on the secretion of IL-8 (A) and TNF-α (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control, †: p ≤ 0.05 vs. control without auraptene or lacinartin).\n Effect of auraptene and lacinartin on the secretion of MMP-8 (A) and MMP-9 (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control).\nAfter demonstrating that auraptene and lacinartin can decrease MMP secretion in a macrophage model, we evaluated their effect on proteinase activity. Both auraptene and lacinartin (12.5 μg/ml) reduced MMP-9 activity by 74% (Table\n1). Auraptene had no inhibitory effect on P. gingivalis collagenase activity while 12.5 μg/ml and 100 μg/ml of lacinartin reduced collagenase activity by 20% and 64%, respectively (Table\n1).\n\nEffect of auraptene and lacinartin on the activity of MMP-9 and \n\nP. gingivalis\n\n collagenase\n\n1Inhibitor of MMP-9: GM6001 (0.025 μM); inhibitor of P. gingivalis collagenase: leupeptin (1 μM)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity."},"other_sections_titles":{"kind":"list like","value":["Introduction","Compounds","Effect on Porphyromonas gingivalis growth","Effect on P. gingivalis biofilm formation/desorption","Effect on P. gingivalis adherence to oral epithelial cells","Anti-inflammatory properties in a macrophage model","Inhibition of MMP-9 and P. gingivalis collagenase activity","Statistical analysis","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Introduction\",\n \"Compounds\",\n \"Effect on Porphyromonas gingivalis growth\",\n \"Effect on P. gingivalis biofilm formation/desorption\",\n \"Effect on P. gingivalis adherence to oral epithelial cells\",\n \"Anti-inflammatory properties in a macrophage model\",\n \"Inhibition of MMP-9 and P. gingivalis collagenase activity\",\n \"Statistical analysis\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.","Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.","P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.","P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.","P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.","U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.","Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.","Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.","The authors declare that they have no competing interests.","All authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\n"],"string":"[\n \"Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\\n[6,7].\\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\\n[18,19] while little is known about lacinartin. \\n Chemical structures of auraptene (A) and lacinartin (B).\\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.\",\n \"Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\",\n \"P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\",\n \"P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\",\n \"P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\",\n \"U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\",\n \"Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\",\n \"Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\",\n \"The authors declare that they have no competing interests.\",\n \"All authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and methods","Compounds","Effect on Porphyromonas gingivalis growth","Effect on P. gingivalis biofilm formation/desorption","Effect on P. gingivalis adherence to oral epithelial cells","Anti-inflammatory properties in a macrophage model","Inhibition of MMP-9 and P. gingivalis collagenase activity","Statistical analysis","Results","Discussion","Conclusions","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Introduction\",\n \"Materials and methods\",\n \"Compounds\",\n \"Effect on Porphyromonas gingivalis growth\",\n \"Effect on P. gingivalis biofilm formation/desorption\",\n \"Effect on P. gingivalis adherence to oral epithelial cells\",\n \"Anti-inflammatory properties in a macrophage model\",\n \"Inhibition of MMP-9 and P. gingivalis collagenase activity\",\n \"Statistical analysis\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase."," Compounds Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\nAuraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\n Effect on Porphyromonas gingivalis growth P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\nP. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\n Effect on P. gingivalis biofilm formation/desorption P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\nP. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\n Effect on P. gingivalis adherence to oral epithelial cells P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\nP. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\n Anti-inflammatory properties in a macrophage model U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\nU937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\n Inhibition of MMP-9 and P. gingivalis collagenase activity Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\nHuman recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\n Statistical analysis Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\nResults are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.","Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.","P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.","P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.","P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.","U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.","Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.","Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.","Lacinartin (50 and 100 μg/ml) almost completely inhibited the growth of P. gingivalis in THB-HK (Figure.\n2B), while the highest concentration of auraptene tested (100 μg/ml) only reduced growth by 42% (Figure.\n2A). Bacterial growth was slightly lower in MBB-H (OD660 = 0.59), which represents an iron poor condition for P. gingivalis, than in THB-HK (OD660 = 0.78) (Figure.\n2). The lowest concentration of auraptene tested (12.5 μg/ml) inhibited growth by 58% in MBB-H, while such a concentration had no significant inhibitory effect in THB-HK (Figure.\n2A). Almost complete inhibition was observed at higher concentrations (50 and 100 μg/ml). As for auraptene, lacinartin seemed to be more effective for inhibiting growth of P. gingivalis in iron-limiting conditions.\n Effect of auraptene (A) and lacinartin (B) on the growth of P. gingivalis in a complex medium (THB-HK) and under iron-limiting conditions in MBB-H. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed using the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene had no obvious inhibitory effect on P. gingivalis biofilm formation or desorption (Figure.\n3). On the contrary, high concentrations (50 and 100 μg/ml) of auraptene appear to increase biofilm formation (Figure.\n3A). Lacinartin at 50 and 100 μg/ml inhibited biofilm formation by P. gingivalis by approximately 75% (Figure.\n3A). In addition, lacinartin (12.5-100 μg/ml) caused approximately one-third of the biofilm to desorb (Figure.\n3B).\n Effect of auraptene and lacinartin on P. gingivalis biofilm formation (A) and on desorption of a pre-formed P. gingivalis biofilm (B). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene and lacinartin both dose-dependently inhibited bacterial adhesion to oral epithelial cells (Figure.\n4). At the lowest concentration tested (12.5 μg/ml), auraptene and lacinartin reduced the adherence of P. gingivalis to epithelial cells by 33% and 43%, respectively, while 100 μg/ml of auraptene and lacinartin reduced adherence by 37% and 71%, respectively (Figure.\n4).\n Effect of auraptene and lacinartin on the adherence of P. gingivalis to human oral epithelial cells. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. A value of 100% was assigned to the control (no auraptene; no lacinartin). Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nThe highest non-cytotoxic concentrations of auraptene and lacinartin that can be used to evaluate their effect on the inflammatory response of a human macrophage model stimulated with LPS were 25 and 50 μg/ml, respectively (data not shown). Following a 2-h pretreatment of the model with auraptene (6.25, 12.5, and 25 μg/ml) or lacinartin (12.5, 25, and 50 μg/ml), macrophages were stimulated with LPS to induce an inflammatory response (cytokine and MMP secretion). Auraptene and lacinartin both had a significant inhibitory effect on IL-8 and TNF-α secretion. At the lowest concentration tested (6.25 μg/ml), auraptene reduced IL-8 and TNF-α secretion by 22% and 37%, respectively, compared to untreated cells, while at the highest concentration tested (25 μg/ml), it inhibited IL-8 and TNF-α secretion by 92% and 85%, respectively (Figure.\n5). As shown in Fig.\n5, the highest concentration of lacinartin tested (50 μg/ml) reduced IL-8 and TNF-α secretion by 95% and 99%, respectively, while 12.5 and 25 μg/ml of lacinartin increased the secretion of both cytokines, likely due to a synergistic effect of LPS and lacinartin, since lacinartin alone had no effect (data not shown). Auraptene and lacinartin both reduced MMP-8 and MMP-9 secretion, sometimes below basal levels (Figure.\n6). Auraptene reduced MMP-8 secretion by 63% (6.25 μg/ml), 69% (12.5 μg/ml), and 73% (25 μg/ml), while lacinartin reduced MMP-8 secretion by 79% (12.5 μg/ml), 83% (25 μg/ml), and 89% (50 μg/ml) (Figure.\n6A). At their highest concentrations tested, auraptene (25 μg/ml) reduced MMP-9 secretion by 76% (25 μg/ml) (Figure.\n6B), while lacinartin (50 μg/ml) reduced MMP-9 secretion by 82% (Figure.\n6B).\n Effect of auraptene and lacinartin on the secretion of IL-8 (A) and TNF-α (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control, †: p ≤ 0.05 vs. control without auraptene or lacinartin).\n Effect of auraptene and lacinartin on the secretion of MMP-8 (A) and MMP-9 (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control).\nAfter demonstrating that auraptene and lacinartin can decrease MMP secretion in a macrophage model, we evaluated their effect on proteinase activity. Both auraptene and lacinartin (12.5 μg/ml) reduced MMP-9 activity by 74% (Table\n1). Auraptene had no inhibitory effect on P. gingivalis collagenase activity while 12.5 μg/ml and 100 μg/ml of lacinartin reduced collagenase activity by 20% and 64%, respectively (Table\n1).\n\nEffect of auraptene and lacinartin on the activity of MMP-9 and \n\nP. gingivalis\n\n collagenase\n\n1Inhibitor of MMP-9: GM6001 (0.025 μM); inhibitor of P. gingivalis collagenase: leupeptin (1 μM).","Periodontal diseases are polymicrobial infections and are the most common chronic inflammatory disorders in adults\n[22]. Periodontitis is induced by a specific group of Gram-negative anaerobic bacteria and is the major cause of tooth loss in adults\n[23]. Over the past two decades, natural compounds with antibacterial and anti-inflammatory properties have received considerable attention as new therapeutic agents for the treatment of periodontal infections. In this study, we investigated the potential of auraptene and lacinartin for preventing and treating periodontal diseases.\nWe first showed that lacinartin and to a lesser extent auraptene reduced P. gingivalis growth. This is the first report indicating that lacinartin possesses anti-bacterial properties. Previous studies have shown that auraptene has antibacterial properties against Helicobacter pylori[24,25]. The exact mechanism by which lacinartin and auraptene inhibit bacterial growth is unknown. However, other natural coumarins (novobiocin and clorobiocin) inhibit deoxyribonuclease gyrase activity, which results in bacteria death\n[14,26]. In addition, we showed that auraptene and lacinartin inhibit growth more effectively under iron-limiting conditions, requiring much lower concentrations to significantly reduce the growth of P. gingivalis. Our results are in agreement with those of Mladnka et al.\n[27], who showed that coumarins possess iron-chelating properties. Additional studies are required to investigate interactions between iron and auraptene and lacinartin.\nWe also showed that lacinartin, but not auraptene, inhibits biofilm formation by P. gingivalis. Lacinartin also caused the desorption of a pre-formed P. gingivalis biofilm. To the best of our knowledge, this is the first report regarding the inhibitory effects of lacinartin on bacterial biofilms. Auraptene and lacinartin prevented the adherence of P. gingivalis to oral epithelial cells to a significant degree. Epithelial cells act as a physical barrier, and bacterial adherence to these host cells may be a critical step for the initiation of periodontal diseases\n[28]. Given its ability to reduce growth of P. gingivalis and its adherence to epithelial cells, lacinartin may be a promising therapeutic candidate through its action on different targets.\nPolyphenols reduce inflammatory mediator secretion and, as such, inflammation-mediated damage\n[29]. We showed that auraptene markedly reduces IL-8 and TNF-α secretion by LPS-stimulated macrophages. Our results are in agreement with those of Genovese et al., who reported that auraptene inhibits the release of TNF-α by RAW 264.7 macrophages\n[30]. To our knowledge, no one has investigated the anti-inflammatory properties of lacinartin. We showed that 12.5 μg/ml of lacinartin induced IL-8 and TNF-α secretion, likely due to a synergistic interaction between LPS and lacinartin. On the other hand, 50 μg/ml of lacinartin significantly inhibited IL-8 and TNF-α secretion. We also showed that auraptene and lacinartin reduced MMP-8 and MMP-9 secretion. These results are in agreement with those of a study by Epifano et al., who reported that auraptene inhibits MMP-7 secretion by HT-29 epithelial cells\n[18]. Since MMP release and cytokine secretion are associated with tooth-supporting tissue destruction, our results suggested that both compounds may contribute to reducing host cell damage, including bone resorption\n[5,31]. The mechanisms by which auraptene and lacinartin reduce inflammatory mediator secretion are unknown, but previous studies have shown that coumarins can block the activation of nuclear factor-κB and inhibit kinase pathways (Akt/PKB)\n[26]. Considering that gingival fibroblasts may also play a significant role in periodontal tissue destruction through cytokine-inducible MMP secretion, future studies should investigate the effects of auraptene and lacinartin on this cell type.\nWe further showed that auraptene and lacinartin reduce MMP-9 activity while only lacinartin inhibits P. gingivalis collagenase activity. Auraptene has previously been shown to inhibit MMP-7 activity\n[32]. These observations suggest that these coumarins may contribute to reducing tissue destruction.","In conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity.","The authors declare that they have no competing interests.","All authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\n"],"string":"[\n \"Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\\n[6,7].\\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\\n[18,19] while little is known about lacinartin. \\n Chemical structures of auraptene (A) and lacinartin (B).\\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.\",\n \" Compounds Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\\nAuraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\\n Effect on Porphyromonas gingivalis growth P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\\nP. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\\n Effect on P. gingivalis biofilm formation/desorption P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\\nP. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\\n Effect on P. gingivalis adherence to oral epithelial cells P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\\nP. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\\n Anti-inflammatory properties in a macrophage model U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\\nU937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\\n Inhibition of MMP-9 and P. gingivalis collagenase activity Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\\nHuman recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\\n Statistical analysis Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\\nResults are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\",\n \"Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\",\n \"P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\",\n \"P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\",\n \"P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\",\n \"U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\",\n \"Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\",\n \"Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\",\n \"Lacinartin (50 and 100 μg/ml) almost completely inhibited the growth of P. gingivalis in THB-HK (Figure.\\n2B), while the highest concentration of auraptene tested (100 μg/ml) only reduced growth by 42% (Figure.\\n2A). Bacterial growth was slightly lower in MBB-H (OD660 = 0.59), which represents an iron poor condition for P. gingivalis, than in THB-HK (OD660 = 0.78) (Figure.\\n2). The lowest concentration of auraptene tested (12.5 μg/ml) inhibited growth by 58% in MBB-H, while such a concentration had no significant inhibitory effect in THB-HK (Figure.\\n2A). Almost complete inhibition was observed at higher concentrations (50 and 100 μg/ml). As for auraptene, lacinartin seemed to be more effective for inhibiting growth of P. gingivalis in iron-limiting conditions.\\n Effect of auraptene (A) and lacinartin (B) on the growth of P. gingivalis in a complex medium (THB-HK) and under iron-limiting conditions in MBB-H. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed using the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\\nAuraptene had no obvious inhibitory effect on P. gingivalis biofilm formation or desorption (Figure.\\n3). On the contrary, high concentrations (50 and 100 μg/ml) of auraptene appear to increase biofilm formation (Figure.\\n3A). Lacinartin at 50 and 100 μg/ml inhibited biofilm formation by P. gingivalis by approximately 75% (Figure.\\n3A). In addition, lacinartin (12.5-100 μg/ml) caused approximately one-third of the biofilm to desorb (Figure.\\n3B).\\n Effect of auraptene and lacinartin on P. gingivalis biofilm formation (A) and on desorption of a pre-formed P. gingivalis biofilm (B). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\\nAuraptene and lacinartin both dose-dependently inhibited bacterial adhesion to oral epithelial cells (Figure.\\n4). At the lowest concentration tested (12.5 μg/ml), auraptene and lacinartin reduced the adherence of P. gingivalis to epithelial cells by 33% and 43%, respectively, while 100 μg/ml of auraptene and lacinartin reduced adherence by 37% and 71%, respectively (Figure.\\n4).\\n Effect of auraptene and lacinartin on the adherence of P. gingivalis to human oral epithelial cells. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. A value of 100% was assigned to the control (no auraptene; no lacinartin). Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\\nThe highest non-cytotoxic concentrations of auraptene and lacinartin that can be used to evaluate their effect on the inflammatory response of a human macrophage model stimulated with LPS were 25 and 50 μg/ml, respectively (data not shown). Following a 2-h pretreatment of the model with auraptene (6.25, 12.5, and 25 μg/ml) or lacinartin (12.5, 25, and 50 μg/ml), macrophages were stimulated with LPS to induce an inflammatory response (cytokine and MMP secretion). Auraptene and lacinartin both had a significant inhibitory effect on IL-8 and TNF-α secretion. At the lowest concentration tested (6.25 μg/ml), auraptene reduced IL-8 and TNF-α secretion by 22% and 37%, respectively, compared to untreated cells, while at the highest concentration tested (25 μg/ml), it inhibited IL-8 and TNF-α secretion by 92% and 85%, respectively (Figure.\\n5). As shown in Fig.\\n5, the highest concentration of lacinartin tested (50 μg/ml) reduced IL-8 and TNF-α secretion by 95% and 99%, respectively, while 12.5 and 25 μg/ml of lacinartin increased the secretion of both cytokines, likely due to a synergistic effect of LPS and lacinartin, since lacinartin alone had no effect (data not shown). Auraptene and lacinartin both reduced MMP-8 and MMP-9 secretion, sometimes below basal levels (Figure.\\n6). Auraptene reduced MMP-8 secretion by 63% (6.25 μg/ml), 69% (12.5 μg/ml), and 73% (25 μg/ml), while lacinartin reduced MMP-8 secretion by 79% (12.5 μg/ml), 83% (25 μg/ml), and 89% (50 μg/ml) (Figure.\\n6A). At their highest concentrations tested, auraptene (25 μg/ml) reduced MMP-9 secretion by 76% (25 μg/ml) (Figure.\\n6B), while lacinartin (50 μg/ml) reduced MMP-9 secretion by 82% (Figure.\\n6B).\\n Effect of auraptene and lacinartin on the secretion of IL-8 (A) and TNF-α (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control, †: p ≤ 0.05 vs. control without auraptene or lacinartin).\\n Effect of auraptene and lacinartin on the secretion of MMP-8 (A) and MMP-9 (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control).\\nAfter demonstrating that auraptene and lacinartin can decrease MMP secretion in a macrophage model, we evaluated their effect on proteinase activity. Both auraptene and lacinartin (12.5 μg/ml) reduced MMP-9 activity by 74% (Table\\n1). Auraptene had no inhibitory effect on P. gingivalis collagenase activity while 12.5 μg/ml and 100 μg/ml of lacinartin reduced collagenase activity by 20% and 64%, respectively (Table\\n1).\\n\\nEffect of auraptene and lacinartin on the activity of MMP-9 and \\n\\nP. gingivalis\\n\\n collagenase\\n\\n1Inhibitor of MMP-9: GM6001 (0.025 μM); inhibitor of P. gingivalis collagenase: leupeptin (1 μM).\",\n \"Periodontal diseases are polymicrobial infections and are the most common chronic inflammatory disorders in adults\\n[22]. Periodontitis is induced by a specific group of Gram-negative anaerobic bacteria and is the major cause of tooth loss in adults\\n[23]. Over the past two decades, natural compounds with antibacterial and anti-inflammatory properties have received considerable attention as new therapeutic agents for the treatment of periodontal infections. In this study, we investigated the potential of auraptene and lacinartin for preventing and treating periodontal diseases.\\nWe first showed that lacinartin and to a lesser extent auraptene reduced P. gingivalis growth. This is the first report indicating that lacinartin possesses anti-bacterial properties. Previous studies have shown that auraptene has antibacterial properties against Helicobacter pylori[24,25]. The exact mechanism by which lacinartin and auraptene inhibit bacterial growth is unknown. However, other natural coumarins (novobiocin and clorobiocin) inhibit deoxyribonuclease gyrase activity, which results in bacteria death\\n[14,26]. In addition, we showed that auraptene and lacinartin inhibit growth more effectively under iron-limiting conditions, requiring much lower concentrations to significantly reduce the growth of P. gingivalis. Our results are in agreement with those of Mladnka et al.\\n[27], who showed that coumarins possess iron-chelating properties. Additional studies are required to investigate interactions between iron and auraptene and lacinartin.\\nWe also showed that lacinartin, but not auraptene, inhibits biofilm formation by P. gingivalis. Lacinartin also caused the desorption of a pre-formed P. gingivalis biofilm. To the best of our knowledge, this is the first report regarding the inhibitory effects of lacinartin on bacterial biofilms. Auraptene and lacinartin prevented the adherence of P. gingivalis to oral epithelial cells to a significant degree. Epithelial cells act as a physical barrier, and bacterial adherence to these host cells may be a critical step for the initiation of periodontal diseases\\n[28]. Given its ability to reduce growth of P. gingivalis and its adherence to epithelial cells, lacinartin may be a promising therapeutic candidate through its action on different targets.\\nPolyphenols reduce inflammatory mediator secretion and, as such, inflammation-mediated damage\\n[29]. We showed that auraptene markedly reduces IL-8 and TNF-α secretion by LPS-stimulated macrophages. Our results are in agreement with those of Genovese et al., who reported that auraptene inhibits the release of TNF-α by RAW 264.7 macrophages\\n[30]. To our knowledge, no one has investigated the anti-inflammatory properties of lacinartin. We showed that 12.5 μg/ml of lacinartin induced IL-8 and TNF-α secretion, likely due to a synergistic interaction between LPS and lacinartin. On the other hand, 50 μg/ml of lacinartin significantly inhibited IL-8 and TNF-α secretion. We also showed that auraptene and lacinartin reduced MMP-8 and MMP-9 secretion. These results are in agreement with those of a study by Epifano et al., who reported that auraptene inhibits MMP-7 secretion by HT-29 epithelial cells\\n[18]. Since MMP release and cytokine secretion are associated with tooth-supporting tissue destruction, our results suggested that both compounds may contribute to reducing host cell damage, including bone resorption\\n[5,31]. The mechanisms by which auraptene and lacinartin reduce inflammatory mediator secretion are unknown, but previous studies have shown that coumarins can block the activation of nuclear factor-κB and inhibit kinase pathways (Akt/PKB)\\n[26]. Considering that gingival fibroblasts may also play a significant role in periodontal tissue destruction through cytokine-inducible MMP secretion, future studies should investigate the effects of auraptene and lacinartin on this cell type.\\nWe further showed that auraptene and lacinartin reduce MMP-9 activity while only lacinartin inhibits P. gingivalis collagenase activity. Auraptene has previously been shown to inhibit MMP-7 activity\\n[32]. These observations suggest that these coumarins may contribute to reducing tissue destruction.\",\n \"In conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity.\",\n \"The authors declare that they have no competing interests.\",\n \"All authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"materials|methods",null,null,null,null,null,null,null,"results","discussion","conclusions",null,null,null],"string":"[\n null,\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Coumarins","Auraptene","Lacinartin","Antibacterial","Anti-adherence","Anti-inflammatory"],"string":"[\n \"Coumarins\",\n \"Auraptene\",\n \"Lacinartin\",\n \"Antibacterial\",\n \"Anti-adherence\",\n \"Anti-inflammatory\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nPeriodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.\n\nMaterials and methods:\n Compounds Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\nAuraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\n Effect on Porphyromonas gingivalis growth P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\nP. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\n Effect on P. gingivalis biofilm formation/desorption P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\nP. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\n Effect on P. gingivalis adherence to oral epithelial cells P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\nP. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\n Anti-inflammatory properties in a macrophage model U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\nU937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\n Inhibition of MMP-9 and P. gingivalis collagenase activity Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\nHuman recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\n Statistical analysis Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\nResults are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\n\nCompounds:\nAuraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\n\nEffect on Porphyromonas gingivalis growth:\nP. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\n\nEffect on P. gingivalis biofilm formation/desorption:\nP. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\n\nEffect on P. gingivalis adherence to oral epithelial cells:\nP. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\n\nAnti-inflammatory properties in a macrophage model:\nU937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\n\nInhibition of MMP-9 and P. gingivalis collagenase activity:\nHuman recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\n\nStatistical analysis:\nResults are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\n\nResults:\nLacinartin (50 and 100 μg/ml) almost completely inhibited the growth of P. gingivalis in THB-HK (Figure.\n2B), while the highest concentration of auraptene tested (100 μg/ml) only reduced growth by 42% (Figure.\n2A). Bacterial growth was slightly lower in MBB-H (OD660 = 0.59), which represents an iron poor condition for P. gingivalis, than in THB-HK (OD660 = 0.78) (Figure.\n2). The lowest concentration of auraptene tested (12.5 μg/ml) inhibited growth by 58% in MBB-H, while such a concentration had no significant inhibitory effect in THB-HK (Figure.\n2A). Almost complete inhibition was observed at higher concentrations (50 and 100 μg/ml). As for auraptene, lacinartin seemed to be more effective for inhibiting growth of P. gingivalis in iron-limiting conditions.\n Effect of auraptene (A) and lacinartin (B) on the growth of P. gingivalis in a complex medium (THB-HK) and under iron-limiting conditions in MBB-H. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed using the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene had no obvious inhibitory effect on P. gingivalis biofilm formation or desorption (Figure.\n3). On the contrary, high concentrations (50 and 100 μg/ml) of auraptene appear to increase biofilm formation (Figure.\n3A). Lacinartin at 50 and 100 μg/ml inhibited biofilm formation by P. gingivalis by approximately 75% (Figure.\n3A). In addition, lacinartin (12.5-100 μg/ml) caused approximately one-third of the biofilm to desorb (Figure.\n3B).\n Effect of auraptene and lacinartin on P. gingivalis biofilm formation (A) and on desorption of a pre-formed P. gingivalis biofilm (B). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene and lacinartin both dose-dependently inhibited bacterial adhesion to oral epithelial cells (Figure.\n4). At the lowest concentration tested (12.5 μg/ml), auraptene and lacinartin reduced the adherence of P. gingivalis to epithelial cells by 33% and 43%, respectively, while 100 μg/ml of auraptene and lacinartin reduced adherence by 37% and 71%, respectively (Figure.\n4).\n Effect of auraptene and lacinartin on the adherence of P. gingivalis to human oral epithelial cells. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. A value of 100% was assigned to the control (no auraptene; no lacinartin). Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nThe highest non-cytotoxic concentrations of auraptene and lacinartin that can be used to evaluate their effect on the inflammatory response of a human macrophage model stimulated with LPS were 25 and 50 μg/ml, respectively (data not shown). Following a 2-h pretreatment of the model with auraptene (6.25, 12.5, and 25 μg/ml) or lacinartin (12.5, 25, and 50 μg/ml), macrophages were stimulated with LPS to induce an inflammatory response (cytokine and MMP secretion). Auraptene and lacinartin both had a significant inhibitory effect on IL-8 and TNF-α secretion. At the lowest concentration tested (6.25 μg/ml), auraptene reduced IL-8 and TNF-α secretion by 22% and 37%, respectively, compared to untreated cells, while at the highest concentration tested (25 μg/ml), it inhibited IL-8 and TNF-α secretion by 92% and 85%, respectively (Figure.\n5). As shown in Fig.\n5, the highest concentration of lacinartin tested (50 μg/ml) reduced IL-8 and TNF-α secretion by 95% and 99%, respectively, while 12.5 and 25 μg/ml of lacinartin increased the secretion of both cytokines, likely due to a synergistic effect of LPS and lacinartin, since lacinartin alone had no effect (data not shown). Auraptene and lacinartin both reduced MMP-8 and MMP-9 secretion, sometimes below basal levels (Figure.\n6). Auraptene reduced MMP-8 secretion by 63% (6.25 μg/ml), 69% (12.5 μg/ml), and 73% (25 μg/ml), while lacinartin reduced MMP-8 secretion by 79% (12.5 μg/ml), 83% (25 μg/ml), and 89% (50 μg/ml) (Figure.\n6A). At their highest concentrations tested, auraptene (25 μg/ml) reduced MMP-9 secretion by 76% (25 μg/ml) (Figure.\n6B), while lacinartin (50 μg/ml) reduced MMP-9 secretion by 82% (Figure.\n6B).\n Effect of auraptene and lacinartin on the secretion of IL-8 (A) and TNF-α (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control, †: p ≤ 0.05 vs. control without auraptene or lacinartin).\n Effect of auraptene and lacinartin on the secretion of MMP-8 (A) and MMP-9 (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control).\nAfter demonstrating that auraptene and lacinartin can decrease MMP secretion in a macrophage model, we evaluated their effect on proteinase activity. Both auraptene and lacinartin (12.5 μg/ml) reduced MMP-9 activity by 74% (Table\n1). Auraptene had no inhibitory effect on P. gingivalis collagenase activity while 12.5 μg/ml and 100 μg/ml of lacinartin reduced collagenase activity by 20% and 64%, respectively (Table\n1).\n\nEffect of auraptene and lacinartin on the activity of MMP-9 and \n\nP. gingivalis\n\n collagenase\n\n1Inhibitor of MMP-9: GM6001 (0.025 μM); inhibitor of P. gingivalis collagenase: leupeptin (1 μM).\n\nDiscussion:\nPeriodontal diseases are polymicrobial infections and are the most common chronic inflammatory disorders in adults\n[22]. Periodontitis is induced by a specific group of Gram-negative anaerobic bacteria and is the major cause of tooth loss in adults\n[23]. Over the past two decades, natural compounds with antibacterial and anti-inflammatory properties have received considerable attention as new therapeutic agents for the treatment of periodontal infections. In this study, we investigated the potential of auraptene and lacinartin for preventing and treating periodontal diseases.\nWe first showed that lacinartin and to a lesser extent auraptene reduced P. gingivalis growth. This is the first report indicating that lacinartin possesses anti-bacterial properties. Previous studies have shown that auraptene has antibacterial properties against Helicobacter pylori[24,25]. The exact mechanism by which lacinartin and auraptene inhibit bacterial growth is unknown. However, other natural coumarins (novobiocin and clorobiocin) inhibit deoxyribonuclease gyrase activity, which results in bacteria death\n[14,26]. In addition, we showed that auraptene and lacinartin inhibit growth more effectively under iron-limiting conditions, requiring much lower concentrations to significantly reduce the growth of P. gingivalis. Our results are in agreement with those of Mladnka et al.\n[27], who showed that coumarins possess iron-chelating properties. Additional studies are required to investigate interactions between iron and auraptene and lacinartin.\nWe also showed that lacinartin, but not auraptene, inhibits biofilm formation by P. gingivalis. Lacinartin also caused the desorption of a pre-formed P. gingivalis biofilm. To the best of our knowledge, this is the first report regarding the inhibitory effects of lacinartin on bacterial biofilms. Auraptene and lacinartin prevented the adherence of P. gingivalis to oral epithelial cells to a significant degree. Epithelial cells act as a physical barrier, and bacterial adherence to these host cells may be a critical step for the initiation of periodontal diseases\n[28]. Given its ability to reduce growth of P. gingivalis and its adherence to epithelial cells, lacinartin may be a promising therapeutic candidate through its action on different targets.\nPolyphenols reduce inflammatory mediator secretion and, as such, inflammation-mediated damage\n[29]. We showed that auraptene markedly reduces IL-8 and TNF-α secretion by LPS-stimulated macrophages. Our results are in agreement with those of Genovese et al., who reported that auraptene inhibits the release of TNF-α by RAW 264.7 macrophages\n[30]. To our knowledge, no one has investigated the anti-inflammatory properties of lacinartin. We showed that 12.5 μg/ml of lacinartin induced IL-8 and TNF-α secretion, likely due to a synergistic interaction between LPS and lacinartin. On the other hand, 50 μg/ml of lacinartin significantly inhibited IL-8 and TNF-α secretion. We also showed that auraptene and lacinartin reduced MMP-8 and MMP-9 secretion. These results are in agreement with those of a study by Epifano et al., who reported that auraptene inhibits MMP-7 secretion by HT-29 epithelial cells\n[18]. Since MMP release and cytokine secretion are associated with tooth-supporting tissue destruction, our results suggested that both compounds may contribute to reducing host cell damage, including bone resorption\n[5,31]. The mechanisms by which auraptene and lacinartin reduce inflammatory mediator secretion are unknown, but previous studies have shown that coumarins can block the activation of nuclear factor-κB and inhibit kinase pathways (Akt/PKB)\n[26]. Considering that gingival fibroblasts may also play a significant role in periodontal tissue destruction through cytokine-inducible MMP secretion, future studies should investigate the effects of auraptene and lacinartin on this cell type.\nWe further showed that auraptene and lacinartin reduce MMP-9 activity while only lacinartin inhibits P. gingivalis collagenase activity. Auraptene has previously been shown to inhibit MMP-7 activity\n[32]. These observations suggest that these coumarins may contribute to reducing tissue destruction.\n\nConclusions:\nIn conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nAll authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nPeriodontal diseases are bacterial infections leading to chronic inflammation disorders that are frequently observed in adults. In the present study, we evaluated the effect of auraptene and lacinartin, two natural oxyprenylated coumarins, on the growth, adherence properties, and collagenase activity of Porphyromonas gingivalis. We also investigated the capacity of these compounds to reduce cytokine and matrix metalloproteinase (MMP) secretion by lipopolysaccharide (LPS)-stimulated macrophages and to inhibit MMP-9 activity.\n\nMethods:\nMicroplate dilution assays were performed to determine the effect of auraptene and lacinartin on P. gingivalis growth as well as biofilm formation stained with crystal violet. Adhesion of FITC-labeled P. gingivalis to oral epithelial cells was monitored by fluorometry. The effects of auraptene and lacinartin on LPS-induced cytokine and MMP secretion by macrophages were determined by immunological assays. Fluorogenic assays were used to evaluate the capacity of the two coumarins to inhibit the activity of P. gingivalis collagenase and MMP-9.\n\nResults:\nOnly lacinartin completely inhibited P. gingivalis growth in a complex culture medium. However, under iron-limiting conditions, auraptene and lacinartin both inhibited the growth of P. gingivalis. Lacinartin also inhibited biofilm formation by P. gingivalis and promoted biofilm desorption. Both compounds prevented the adherence of P. gingivalis to oral epithelial cells, dose-dependently reduced the secretion of cytokines (IL-8 and TNF-α) and MMP-8 and MMP-9 by LPS-stimulated macrophages, and inhibited MMP-9 activity. Lacinartin also inhibited P. gingivalis collagenase activity.\n\nConclusions:\nBy acting on multiple targets, including pathogenic bacteria, tissue-destructive enzymes, and the host inflammatory response, auraptene and lacinartin may be promising natural compounds for preventing and treating periodontal diseases."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nPeriodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.\n\nConclusions:\nIn conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nPeriodontal diseases are bacterial infections leading to chronic inflammation disorders that are frequently observed in adults. In the present study, we evaluated the effect of auraptene and lacinartin, two natural oxyprenylated coumarins, on the growth, adherence properties, and collagenase activity of Porphyromonas gingivalis. We also investigated the capacity of these compounds to reduce cytokine and matrix metalloproteinase (MMP) secretion by lipopolysaccharide (LPS)-stimulated macrophages and to inhibit MMP-9 activity.\n\nMethods:\nMicroplate dilution assays were performed to determine the effect of auraptene and lacinartin on P. gingivalis growth as well as biofilm formation stained with crystal violet. Adhesion of FITC-labeled P. gingivalis to oral epithelial cells was monitored by fluorometry. The effects of auraptene and lacinartin on LPS-induced cytokine and MMP secretion by macrophages were determined by immunological assays. Fluorogenic assays were used to evaluate the capacity of the two coumarins to inhibit the activity of P. gingivalis collagenase and MMP-9.\n\nResults:\nOnly lacinartin completely inhibited P. gingivalis growth in a complex culture medium. However, under iron-limiting conditions, auraptene and lacinartin both inhibited the growth of P. gingivalis. Lacinartin also inhibited biofilm formation by P. gingivalis and promoted biofilm desorption. Both compounds prevented the adherence of P. gingivalis to oral epithelial cells, dose-dependently reduced the secretion of cytokines (IL-8 and TNF-α) and MMP-8 and MMP-9 by LPS-stimulated macrophages, and inhibited MMP-9 activity. Lacinartin also inhibited P. gingivalis collagenase activity.\n\nConclusions:\nBy acting on multiple targets, including pathogenic bacteria, tissue-destructive enzymes, and the host inflammatory response, auraptene and lacinartin may be promising natural compounds for preventing and treating periodontal diseases."},"whole_article_text_length":{"kind":"number","value":8512,"string":"8,512"},"whole_article_abstract_length":{"kind":"number","value":314,"string":"314"},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["lacinartin","auraptene","ml","auraptene lacinartin","gingivalis","μg","μg ml","100","cells","mmp"],"string":"[\n \"lacinartin\",\n \"auraptene\",\n \"ml\",\n \"auraptene lacinartin\",\n \"gingivalis\",\n \"μg\",\n \"μg ml\",\n \"100\",\n \"cells\",\n \"mmp\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] diseases | compounds | inflammatory | periodontal | known | immune | properties | anti | natural | polyphenols [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] μg | μg ml | ml | figure | lacinartin | secretion | auraptene | reduced | effect | 25 μg ml [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] anti | inflammatory | mechanisms | periodontal | periodontal diseases | diseases | anti inflammatory | protease | properties useful prevention treatment | properties useful prevention [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | 100 | μg | μg ml | authors | wells [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | 100 | μg | μg ml | authors | wells [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| lacinartin | two | Porphyromonas ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin ||| lacinartin ||| Lacinartin ||| TNF | MMP-8 | MMP-9 | LPS ||| Lacinartin [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] lacinartin [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| lacinartin | two | Porphyromonas ||| ||| ||| ||| lacinartin | LPS ||| two | MMP-9 ||| ||| lacinartin ||| Lacinartin ||| TNF | MMP-8 | MMP-9 | LPS ||| Lacinartin ||| lacinartin [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| lacinartin | two | Porphyromonas ||| ||| ||| ||| lacinartin | LPS ||| two | MMP-9 ||| ||| lacinartin ||| Lacinartin ||| TNF | MMP-8 | MMP-9 | LPS ||| Lacinartin ||| lacinartin [SUMMARY]"}}},{"rowIdx":293,"cells":{"title":{"kind":"string","value":"A pilot prospective study to evaluate whether the bladder morphology in cystography and/or urodynamic may help predict the response to botulinum toxin a injection in neurogenic bladder refractory to anticholinergics."},"pmid":{"kind":"string","value":"25123234"},"background_abstract":{"kind":"string","value":"We have observed different clinical responses to botulinum toxin A (BTX-A) in patients who had similar urodynamic parameters before the procedure. Furthermore, some bladders evaluated by cystography and cystoscopy during the procedure had different characteristics that could influence the outcome of the treatment. The aim of this study was to assess whether cystography and urodynamic parameters could help predict which patients with neurogenic detrusor overactivity (NDO) refractory to anticholinergics respond better to treatment with injection of BTX-A."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"In total, 34 patients with spinal cord injury were prospectively evaluated. All patients emptied their bladder by clean intermittent catheterization (CIC) and had incontinence and NDO, despite using 40 mg or more of intravesical oxybutynin and undergoing detrusor injection of BTX-A (300 IU). Pretreatment evaluation included urodynamic, and cystography. Follow-up consisted of urodynamic and ambulatory visits four months after treatment. The cystography parameters used were bladder shape, capacity and presence of diverticula. Urodynamic parameters used for assessment were maximum cystometric capacity (MCC), maximum detrusor pressure (MDP), compliance and reflex volume (RV)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"After injection of BTX-A, 70% of the patients had success, with 4 months or more of continence. Before the treatment, there were significant differences in most urodynamic parameters between those who responded successfully compared to those who did not. Patients who responded successfully had greater MCC (p = 0.019), higher RV (p = 0.041), and greater compliance (p = 0.043). There was no significant difference in the MDP (0.691). The cystography parameters were not significantly different between these groups bladder shape (p = 0.271), capacity (p > 0.720) and presence of diverticula (p > 0.999). Statistical analyses were performed using SPSS (version 20.0) and included Student's t-test for two paired samples and Fisher's exact test, with a significance threshold of 0.05."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"This study suggests that the cystography parameters evaluated cannot be used to help predict the response to injection of BTX-A in the treatment of refractory NDO. However, the urodynamic parameters were significantly different in patients who responded to the treatment, with the exception of the MDP."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Botulinum Toxins, Type A","Female","Humans","Male","Mandelic Acids","Middle Aged","Muscarinic Antagonists","Neuromuscular Agents","Pilot Projects","Prospective Studies","Radiography","Urinary Bladder","Urinary Bladder, Neurogenic","Urinary Bladder, Overactive","Urodynamics","Young Adult"],"string":"[\n \"Adult\",\n \"Botulinum Toxins, Type A\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Mandelic Acids\",\n \"Middle Aged\",\n \"Muscarinic Antagonists\",\n \"Neuromuscular Agents\",\n \"Pilot Projects\",\n \"Prospective Studies\",\n \"Radiography\",\n \"Urinary Bladder\",\n \"Urinary Bladder, Neurogenic\",\n \"Urinary Bladder, Overactive\",\n \"Urodynamics\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"4139716"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Thirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7 [8] for two dependent groups (matched pairs). Parameters used in the calculations were as follows: effect size, 0.50; significance probability, 0.05; and power test, 0.80. Of the 34 patients, 23 were male and 11 were female. All methods and definitions were based on the standardization of terminology of lower urinary tract function, as described by Abrams et al. [9]. The study was approved by the ethics committee of Sarah Hospital in Brasilia (CAAE 24188413.3.0000.0022). Informed consent was obtained from all patients participating in the study.The inclusion criteria for the injection of BTX-A were those patients who emptied their bladder by CIC and had urinary incontinence due to hyperactivity refractory to intravesical oxybutynin doses equal or greater than 40 mg. An evaluation was conducted before the procedure and included a clinical history, physical examination, ultrasonography of the kidneys and urinary tract, cystography and urodynamic tests (Multichannel Urodynamics - Medtronic Duet systems, version 8.20, Minneapolis). The following urodynamic parameters were measured: reflex volume (RV), maximum detrusor pressure (MDP), bladder compliance, and maximum cystometric capacity (MCC). For better assessment of compliance, only the study bladders with a capacity ≥ 150 ml were used and compliance was measured five minutes after the end of bladder filling to allow for stabilization of the detrusor pressure. During cystography, the bladder filling was stopped when the maximum capacity had been reached, urinary leakage started, or there was supra pubic discomfort. Outcome was assessed in relation to the bladder shape and capacity and the presence of diverticula. The shape of the bladder was characterized as either “rounded” or “pear-shaped” and/or “pine” (see Figure 1). The cystometric capacity was measured in milliliters. Diverticula were characterized as absent or present, with one group consisting of patients with a diverticula number <10 and another group with a diverticula number ≥ 10 (Figure 1).\nThe shape of the bladder (“rounded” shape, “pear-shaped” or “pine”).\nAll procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A (Westport Allergan Pharmaceuticals Ireland - Ireland) was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. [4]. Patients were instructed to continue using the anticholinergic medication and to gradually reduce the dose after the procedure, if it was successful and suspend its use if possible. The treatment was considered successful if the patient remained continent for four months or more, with complete absence of urinary leak, regardless of the use of the anticholinergic medication. Clinical and urodynamic control assessments were performed 4 months after treatment. Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test with a significance threshold of 0.05. Before each t-test, the hypothesis of equality of variances was checked using Levene’s test. Differences between the groups were compared with the Mann–Whitney U test for two independent samples."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Of the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366) (Table 1). There were significant differences in most of the pretreatment urodynamic parameters in patients with a successful response compared with those with an unsuccessful response. The MCC (p = 0.019), RV (0.041) and compliance (p = 0.043) were higher in patients with a successful response. The MDP was not significantly different between groups (p = 0.691) (Table 2).\nUrodynamic assessment before and after botulinum toxin injection\n*Paired, two-sided Student’s t-test.\nUrodynamic parameters before botulinum toxin injection\n*Mann–Whitney U Test for two independent samples.\nRegarding cystography, before the procedure, 24 patients (70%) had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) (Table 3).\nResults and cystography parameters\n*Mann–Whitney U Test for two independent samples.\nAfter 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 of these patients were able to decrease the dose of the anticholinergic drugs after the procedure (Table 5).\nAnticholinergic use after botulinum toxin injection (Total)\nAnticholinergic use after botulinum toxin injection (success)"},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"This study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work."},"other_sections_titles":{"kind":"list like","value":["Background","Abbreviations","Competing interests","Author’s contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Author’s contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.","NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.","The authors declare that they have no competing interests.","RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\n"],"string":"[\n \"Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.\",\n \"NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.\",\n \"The authors declare that they have no competing interests.\",\n \"RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Results","Discussion","Conclusion","Abbreviations","Competing interests","Author’s contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Results\",\n \"Discussion\",\n \"Conclusion\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Author’s contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.","Thirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7 [8] for two dependent groups (matched pairs). Parameters used in the calculations were as follows: effect size, 0.50; significance probability, 0.05; and power test, 0.80. Of the 34 patients, 23 were male and 11 were female. All methods and definitions were based on the standardization of terminology of lower urinary tract function, as described by Abrams et al. [9]. The study was approved by the ethics committee of Sarah Hospital in Brasilia (CAAE 24188413.3.0000.0022). Informed consent was obtained from all patients participating in the study.The inclusion criteria for the injection of BTX-A were those patients who emptied their bladder by CIC and had urinary incontinence due to hyperactivity refractory to intravesical oxybutynin doses equal or greater than 40 mg. An evaluation was conducted before the procedure and included a clinical history, physical examination, ultrasonography of the kidneys and urinary tract, cystography and urodynamic tests (Multichannel Urodynamics - Medtronic Duet systems, version 8.20, Minneapolis). The following urodynamic parameters were measured: reflex volume (RV), maximum detrusor pressure (MDP), bladder compliance, and maximum cystometric capacity (MCC). For better assessment of compliance, only the study bladders with a capacity ≥ 150 ml were used and compliance was measured five minutes after the end of bladder filling to allow for stabilization of the detrusor pressure. During cystography, the bladder filling was stopped when the maximum capacity had been reached, urinary leakage started, or there was supra pubic discomfort. Outcome was assessed in relation to the bladder shape and capacity and the presence of diverticula. The shape of the bladder was characterized as either “rounded” or “pear-shaped” and/or “pine” (see Figure 1). The cystometric capacity was measured in milliliters. Diverticula were characterized as absent or present, with one group consisting of patients with a diverticula number <10 and another group with a diverticula number ≥ 10 (Figure 1).\nThe shape of the bladder (“rounded” shape, “pear-shaped” or “pine”).\nAll procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A (Westport Allergan Pharmaceuticals Ireland - Ireland) was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. [4]. Patients were instructed to continue using the anticholinergic medication and to gradually reduce the dose after the procedure, if it was successful and suspend its use if possible. The treatment was considered successful if the patient remained continent for four months or more, with complete absence of urinary leak, regardless of the use of the anticholinergic medication. Clinical and urodynamic control assessments were performed 4 months after treatment. Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test with a significance threshold of 0.05. Before each t-test, the hypothesis of equality of variances was checked using Levene’s test. Differences between the groups were compared with the Mann–Whitney U test for two independent samples.","Of the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366) (Table 1). There were significant differences in most of the pretreatment urodynamic parameters in patients with a successful response compared with those with an unsuccessful response. The MCC (p = 0.019), RV (0.041) and compliance (p = 0.043) were higher in patients with a successful response. The MDP was not significantly different between groups (p = 0.691) (Table 2).\nUrodynamic assessment before and after botulinum toxin injection\n*Paired, two-sided Student’s t-test.\nUrodynamic parameters before botulinum toxin injection\n*Mann–Whitney U Test for two independent samples.\nRegarding cystography, before the procedure, 24 patients (70%) had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) (Table 3).\nResults and cystography parameters\n*Mann–Whitney U Test for two independent samples.\nAfter 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 of these patients were able to decrease the dose of the anticholinergic drugs after the procedure (Table 5).\nAnticholinergic use after botulinum toxin injection (Total)\nAnticholinergic use after botulinum toxin injection (success)","BTX-A injections into the detrusor muscle provide a clinically significant improvement in patients with NDO refractory to anticholinergics and are very well tolerated (5;6). In this study, continence was observed over a period of more than 4 months in 70% of the patients undergoing treatment with BTX-A. In studies with similar populations of patients, the percentage of patients with continence after injection of the toxin ranged from 42 to 87% [10]. Karsenty et al. reported that anticholinergic agents may be discontinued in 28% to 58% of patients after treatment with BTX-A and the dose can be substantially reduced in the remaining patients [10]. In this study, after 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) discontinued the use and nine (26.5%) did not change the dose of medication (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 patients were able to decrease the dose of anticholinergic drugs after the procedure (Table 5). We note that some of these patients did not reduce the dose because they were afraid that the urinary losses would return after being continent. According to previous studies, some factors that may be related to the efficacy of BTX-A injections into the detrusor muscle include, for example, whether the doses of anticholinergics used before the procedure were considered high (refractory bladder) [7], what the optimal dose of BTX-A is [11,12], the formulations used [13,14] and the injection technique [11]. These factors may explain some of the differences observed in the results across different studies. The results obtained in this study were similar to earlier studies. Among patients presenting with NDO that is refractory to anticholinergic agents, there was a percentage with unsuccessful results.\nAlthough it has been reported that alterations can occur in the detrusor muscle of the NDO [15,16], it was postulated that the morphology of the bladder (shape and presence of diverticula) could be a result of these alterations, and could, in turn, influence the response to BTX-A injections. These changes in bladder shape are most likely the result of smooth muscle hypertrophy and changes in the connective tissue matrix that do not respond to conservative treatment. However, the present study did not demonstrate that the cystography parameters could predict which cases would be more likely to have a better response to BTX-A injections.\nRegarding urodynamic parameters, we observed in this study that the bladders that had better compliance, greater capacity and increased reflex volume before treatment showed a better response to treatment. Furthermore, there was no significant change in compliance after surgery (p = 0.366), suggesting that compliance is related to alterations in the bladder wall, is unresponsive to drug therapy, and is, therefore, directly related to the treatment response. This is in contrast to a study conducted by Klaphajone J [17], in which a small number of patients did not show this relationship.","This study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work.","NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.","The authors declare that they have no competing interests.","RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\n"],"string":"[\n \"Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.\",\n \"Thirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7 [8] for two dependent groups (matched pairs). Parameters used in the calculations were as follows: effect size, 0.50; significance probability, 0.05; and power test, 0.80. Of the 34 patients, 23 were male and 11 were female. All methods and definitions were based on the standardization of terminology of lower urinary tract function, as described by Abrams et al. [9]. The study was approved by the ethics committee of Sarah Hospital in Brasilia (CAAE 24188413.3.0000.0022). Informed consent was obtained from all patients participating in the study.The inclusion criteria for the injection of BTX-A were those patients who emptied their bladder by CIC and had urinary incontinence due to hyperactivity refractory to intravesical oxybutynin doses equal or greater than 40 mg. An evaluation was conducted before the procedure and included a clinical history, physical examination, ultrasonography of the kidneys and urinary tract, cystography and urodynamic tests (Multichannel Urodynamics - Medtronic Duet systems, version 8.20, Minneapolis). The following urodynamic parameters were measured: reflex volume (RV), maximum detrusor pressure (MDP), bladder compliance, and maximum cystometric capacity (MCC). For better assessment of compliance, only the study bladders with a capacity ≥ 150 ml were used and compliance was measured five minutes after the end of bladder filling to allow for stabilization of the detrusor pressure. During cystography, the bladder filling was stopped when the maximum capacity had been reached, urinary leakage started, or there was supra pubic discomfort. Outcome was assessed in relation to the bladder shape and capacity and the presence of diverticula. The shape of the bladder was characterized as either “rounded” or “pear-shaped” and/or “pine” (see Figure 1). The cystometric capacity was measured in milliliters. Diverticula were characterized as absent or present, with one group consisting of patients with a diverticula number <10 and another group with a diverticula number ≥ 10 (Figure 1).\\nThe shape of the bladder (“rounded” shape, “pear-shaped” or “pine”).\\nAll procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A (Westport Allergan Pharmaceuticals Ireland - Ireland) was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. [4]. Patients were instructed to continue using the anticholinergic medication and to gradually reduce the dose after the procedure, if it was successful and suspend its use if possible. The treatment was considered successful if the patient remained continent for four months or more, with complete absence of urinary leak, regardless of the use of the anticholinergic medication. Clinical and urodynamic control assessments were performed 4 months after treatment. Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test with a significance threshold of 0.05. Before each t-test, the hypothesis of equality of variances was checked using Levene’s test. Differences between the groups were compared with the Mann–Whitney U test for two independent samples.\",\n \"Of the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366) (Table 1). There were significant differences in most of the pretreatment urodynamic parameters in patients with a successful response compared with those with an unsuccessful response. The MCC (p = 0.019), RV (0.041) and compliance (p = 0.043) were higher in patients with a successful response. The MDP was not significantly different between groups (p = 0.691) (Table 2).\\nUrodynamic assessment before and after botulinum toxin injection\\n*Paired, two-sided Student’s t-test.\\nUrodynamic parameters before botulinum toxin injection\\n*Mann–Whitney U Test for two independent samples.\\nRegarding cystography, before the procedure, 24 patients (70%) had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) (Table 3).\\nResults and cystography parameters\\n*Mann–Whitney U Test for two independent samples.\\nAfter 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 of these patients were able to decrease the dose of the anticholinergic drugs after the procedure (Table 5).\\nAnticholinergic use after botulinum toxin injection (Total)\\nAnticholinergic use after botulinum toxin injection (success)\",\n \"BTX-A injections into the detrusor muscle provide a clinically significant improvement in patients with NDO refractory to anticholinergics and are very well tolerated (5;6). In this study, continence was observed over a period of more than 4 months in 70% of the patients undergoing treatment with BTX-A. In studies with similar populations of patients, the percentage of patients with continence after injection of the toxin ranged from 42 to 87% [10]. Karsenty et al. reported that anticholinergic agents may be discontinued in 28% to 58% of patients after treatment with BTX-A and the dose can be substantially reduced in the remaining patients [10]. In this study, after 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) discontinued the use and nine (26.5%) did not change the dose of medication (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 patients were able to decrease the dose of anticholinergic drugs after the procedure (Table 5). We note that some of these patients did not reduce the dose because they were afraid that the urinary losses would return after being continent. According to previous studies, some factors that may be related to the efficacy of BTX-A injections into the detrusor muscle include, for example, whether the doses of anticholinergics used before the procedure were considered high (refractory bladder) [7], what the optimal dose of BTX-A is [11,12], the formulations used [13,14] and the injection technique [11]. These factors may explain some of the differences observed in the results across different studies. The results obtained in this study were similar to earlier studies. Among patients presenting with NDO that is refractory to anticholinergic agents, there was a percentage with unsuccessful results.\\nAlthough it has been reported that alterations can occur in the detrusor muscle of the NDO [15,16], it was postulated that the morphology of the bladder (shape and presence of diverticula) could be a result of these alterations, and could, in turn, influence the response to BTX-A injections. These changes in bladder shape are most likely the result of smooth muscle hypertrophy and changes in the connective tissue matrix that do not respond to conservative treatment. However, the present study did not demonstrate that the cystography parameters could predict which cases would be more likely to have a better response to BTX-A injections.\\nRegarding urodynamic parameters, we observed in this study that the bladders that had better compliance, greater capacity and increased reflex volume before treatment showed a better response to treatment. Furthermore, there was no significant change in compliance after surgery (p = 0.366), suggesting that compliance is related to alterations in the bladder wall, is unresponsive to drug therapy, and is, therefore, directly related to the treatment response. This is in contrast to a study conducted by Klaphajone J [17], in which a small number of patients did not show this relationship.\",\n \"This study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work.\",\n \"NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.\",\n \"The authors declare that they have no competing interests.\",\n \"RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods","results","discussion","conclusions",null,null,null,null],"string":"[\n null,\n \"methods\",\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Neurogenic detrusor overactivity","Botulinum toxin A","Cystography","Urodynamic","Neurogenic bladder"],"string":"[\n \"Neurogenic detrusor overactivity\",\n \"Botulinum toxin A\",\n \"Cystography\",\n \"Urodynamic\",\n \"Neurogenic bladder\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nBotulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.\n\nMethods:\nThirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7 [8] for two dependent groups (matched pairs). Parameters used in the calculations were as follows: effect size, 0.50; significance probability, 0.05; and power test, 0.80. Of the 34 patients, 23 were male and 11 were female. All methods and definitions were based on the standardization of terminology of lower urinary tract function, as described by Abrams et al. [9]. The study was approved by the ethics committee of Sarah Hospital in Brasilia (CAAE 24188413.3.0000.0022). Informed consent was obtained from all patients participating in the study.The inclusion criteria for the injection of BTX-A were those patients who emptied their bladder by CIC and had urinary incontinence due to hyperactivity refractory to intravesical oxybutynin doses equal or greater than 40 mg. An evaluation was conducted before the procedure and included a clinical history, physical examination, ultrasonography of the kidneys and urinary tract, cystography and urodynamic tests (Multichannel Urodynamics - Medtronic Duet systems, version 8.20, Minneapolis). The following urodynamic parameters were measured: reflex volume (RV), maximum detrusor pressure (MDP), bladder compliance, and maximum cystometric capacity (MCC). For better assessment of compliance, only the study bladders with a capacity ≥ 150 ml were used and compliance was measured five minutes after the end of bladder filling to allow for stabilization of the detrusor pressure. During cystography, the bladder filling was stopped when the maximum capacity had been reached, urinary leakage started, or there was supra pubic discomfort. Outcome was assessed in relation to the bladder shape and capacity and the presence of diverticula. The shape of the bladder was characterized as either “rounded” or “pear-shaped” and/or “pine” (see Figure 1). The cystometric capacity was measured in milliliters. Diverticula were characterized as absent or present, with one group consisting of patients with a diverticula number <10 and another group with a diverticula number ≥ 10 (Figure 1).\nThe shape of the bladder (“rounded” shape, “pear-shaped” or “pine”).\nAll procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A (Westport Allergan Pharmaceuticals Ireland - Ireland) was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. [4]. Patients were instructed to continue using the anticholinergic medication and to gradually reduce the dose after the procedure, if it was successful and suspend its use if possible. The treatment was considered successful if the patient remained continent for four months or more, with complete absence of urinary leak, regardless of the use of the anticholinergic medication. Clinical and urodynamic control assessments were performed 4 months after treatment. Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test with a significance threshold of 0.05. Before each t-test, the hypothesis of equality of variances was checked using Levene’s test. Differences between the groups were compared with the Mann–Whitney U test for two independent samples.\n\nResults:\nOf the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366) (Table 1). There were significant differences in most of the pretreatment urodynamic parameters in patients with a successful response compared with those with an unsuccessful response. The MCC (p = 0.019), RV (0.041) and compliance (p = 0.043) were higher in patients with a successful response. The MDP was not significantly different between groups (p = 0.691) (Table 2).\nUrodynamic assessment before and after botulinum toxin injection\n*Paired, two-sided Student’s t-test.\nUrodynamic parameters before botulinum toxin injection\n*Mann–Whitney U Test for two independent samples.\nRegarding cystography, before the procedure, 24 patients (70%) had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) (Table 3).\nResults and cystography parameters\n*Mann–Whitney U Test for two independent samples.\nAfter 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 of these patients were able to decrease the dose of the anticholinergic drugs after the procedure (Table 5).\nAnticholinergic use after botulinum toxin injection (Total)\nAnticholinergic use after botulinum toxin injection (success)\n\nDiscussion:\nBTX-A injections into the detrusor muscle provide a clinically significant improvement in patients with NDO refractory to anticholinergics and are very well tolerated (5;6). In this study, continence was observed over a period of more than 4 months in 70% of the patients undergoing treatment with BTX-A. In studies with similar populations of patients, the percentage of patients with continence after injection of the toxin ranged from 42 to 87% [10]. Karsenty et al. reported that anticholinergic agents may be discontinued in 28% to 58% of patients after treatment with BTX-A and the dose can be substantially reduced in the remaining patients [10]. In this study, after 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) discontinued the use and nine (26.5%) did not change the dose of medication (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 patients were able to decrease the dose of anticholinergic drugs after the procedure (Table 5). We note that some of these patients did not reduce the dose because they were afraid that the urinary losses would return after being continent. According to previous studies, some factors that may be related to the efficacy of BTX-A injections into the detrusor muscle include, for example, whether the doses of anticholinergics used before the procedure were considered high (refractory bladder) [7], what the optimal dose of BTX-A is [11,12], the formulations used [13,14] and the injection technique [11]. These factors may explain some of the differences observed in the results across different studies. The results obtained in this study were similar to earlier studies. Among patients presenting with NDO that is refractory to anticholinergic agents, there was a percentage with unsuccessful results.\nAlthough it has been reported that alterations can occur in the detrusor muscle of the NDO [15,16], it was postulated that the morphology of the bladder (shape and presence of diverticula) could be a result of these alterations, and could, in turn, influence the response to BTX-A injections. These changes in bladder shape are most likely the result of smooth muscle hypertrophy and changes in the connective tissue matrix that do not respond to conservative treatment. However, the present study did not demonstrate that the cystography parameters could predict which cases would be more likely to have a better response to BTX-A injections.\nRegarding urodynamic parameters, we observed in this study that the bladders that had better compliance, greater capacity and increased reflex volume before treatment showed a better response to treatment. Furthermore, there was no significant change in compliance after surgery (p = 0.366), suggesting that compliance is related to alterations in the bladder wall, is unresponsive to drug therapy, and is, therefore, directly related to the treatment response. This is in contrast to a study conducted by Klaphajone J [17], in which a small number of patients did not show this relationship.\n\nConclusion:\nThis study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work.\n\nAbbreviations:\nNDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthor’s contributions:\nRAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWe have observed different clinical responses to botulinum toxin A (BTX-A) in patients who had similar urodynamic parameters before the procedure. Furthermore, some bladders evaluated by cystography and cystoscopy during the procedure had different characteristics that could influence the outcome of the treatment. The aim of this study was to assess whether cystography and urodynamic parameters could help predict which patients with neurogenic detrusor overactivity (NDO) refractory to anticholinergics respond better to treatment with injection of BTX-A.\n\nMethods:\nIn total, 34 patients with spinal cord injury were prospectively evaluated. All patients emptied their bladder by clean intermittent catheterization (CIC) and had incontinence and NDO, despite using 40 mg or more of intravesical oxybutynin and undergoing detrusor injection of BTX-A (300 IU). Pretreatment evaluation included urodynamic, and cystography. Follow-up consisted of urodynamic and ambulatory visits four months after treatment. The cystography parameters used were bladder shape, capacity and presence of diverticula. Urodynamic parameters used for assessment were maximum cystometric capacity (MCC), maximum detrusor pressure (MDP), compliance and reflex volume (RV).\n\nResults:\nAfter injection of BTX-A, 70% of the patients had success, with 4 months or more of continence. Before the treatment, there were significant differences in most urodynamic parameters between those who responded successfully compared to those who did not. Patients who responded successfully had greater MCC (p = 0.019), higher RV (p = 0.041), and greater compliance (p = 0.043). There was no significant difference in the MDP (0.691). The cystography parameters were not significantly different between these groups bladder shape (p = 0.271), capacity (p > 0.720) and presence of diverticula (p > 0.999). Statistical analyses were performed using SPSS (version 20.0) and included Student's t-test for two paired samples and Fisher's exact test, with a significance threshold of 0.05.\n\nConclusions:\nThis study suggests that the cystography parameters evaluated cannot be used to help predict the response to injection of BTX-A in the treatment of refractory NDO. However, the urodynamic parameters were significantly different in patients who responded to the treatment, with the exception of the MDP."},"background_conclusion_text":{"kind":"string","value":"Background:\nBotulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.\n\nConclusion:\nThis study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nWe have observed different clinical responses to botulinum toxin A (BTX-A) in patients who had similar urodynamic parameters before the procedure. Furthermore, some bladders evaluated by cystography and cystoscopy during the procedure had different characteristics that could influence the outcome of the treatment. The aim of this study was to assess whether cystography and urodynamic parameters could help predict which patients with neurogenic detrusor overactivity (NDO) refractory to anticholinergics respond better to treatment with injection of BTX-A.\n\nMethods:\nIn total, 34 patients with spinal cord injury were prospectively evaluated. All patients emptied their bladder by clean intermittent catheterization (CIC) and had incontinence and NDO, despite using 40 mg or more of intravesical oxybutynin and undergoing detrusor injection of BTX-A (300 IU). Pretreatment evaluation included urodynamic, and cystography. Follow-up consisted of urodynamic and ambulatory visits four months after treatment. The cystography parameters used were bladder shape, capacity and presence of diverticula. Urodynamic parameters used for assessment were maximum cystometric capacity (MCC), maximum detrusor pressure (MDP), compliance and reflex volume (RV).\n\nResults:\nAfter injection of BTX-A, 70% of the patients had success, with 4 months or more of continence. Before the treatment, there were significant differences in most urodynamic parameters between those who responded successfully compared to those who did not. Patients who responded successfully had greater MCC (p = 0.019), higher RV (p = 0.041), and greater compliance (p = 0.043). There was no significant difference in the MDP (0.691). The cystography parameters were not significantly different between these groups bladder shape (p = 0.271), capacity (p > 0.720) and presence of diverticula (p > 0.999). Statistical analyses were performed using SPSS (version 20.0) and included Student's t-test for two paired samples and Fisher's exact test, with a significance threshold of 0.05.\n\nConclusions:\nThis study suggests that the cystography parameters evaluated cannot be used to help predict the response to injection of BTX-A in the treatment of refractory NDO. However, the urodynamic parameters were significantly different in patients who responded to the treatment, with the exception of the MDP."},"whole_article_text_length":{"kind":"number","value":2504,"string":"2,504"},"whole_article_abstract_length":{"kind":"number","value":430,"string":"430"},"num_sections":{"kind":"number","value":9,"string":"9"},"most_frequent_words":{"kind":"list like","value":["patients","btx","treatment","bladder","study","parameters","detrusor","urodynamic","procedure","capacity"],"string":"[\n \"patients\",\n \"btx\",\n \"treatment\",\n \"bladder\",\n \"study\",\n \"parameters\",\n \"detrusor\",\n \"urodynamic\",\n \"procedure\",\n \"capacity\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] btx | treatment | invasive | muscle | evaluated | detrusor | clinical | release | acetylcholine | serotypes [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | patients | bladder | performed | urinary | measured | ml | version | capacity | diverticula [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | table | toxin injection | years | botulinum toxin injection | response | months | test | 001 | mean [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] compliance showed | showed | greater | compliance | results | treatment | study | parameters | analysis appropriate clarify results | bladders higher cystometric capacity [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | study | authors | detrusor | bladder | authors declare competing interests | competing interests | declare competing interests [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | btx | treatment | study | authors | detrusor | bladder | authors declare competing interests | competing interests | declare competing interests [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| NDO | BTX-A. [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 34 ||| CIC | NDO | 40 | BTX-A ||| ||| four months ||| ||| MDP | RV [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] BTX-A | 70% | 4 months ||| ||| MCC | 0.019 | RV | 0.041 | 0.043 ||| MDP | 0.691 ||| 0.271 | 0.720 | 0.999 ||| SPSS | 20.0 | Student | two | Fisher | 0.05 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] BTX-A | NDO ||| MDP [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| NDO | BTX-A. Methods | 34 ||| CIC | NDO | 40 | BTX-A ||| ||| four months ||| ||| MDP | RV ||| ||| BTX-A | 70% | 4 months ||| ||| MCC | 0.019 | RV | 0.041 | 0.043 ||| MDP | 0.691 ||| 0.271 | 0.720 | 0.999 ||| SPSS | 20.0 | Student | two | Fisher | 0.05 ||| BTX-A | NDO ||| MDP [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| NDO | BTX-A. Methods | 34 ||| CIC | NDO | 40 | BTX-A ||| ||| four months ||| ||| MDP | RV ||| ||| BTX-A | 70% | 4 months ||| ||| MCC | 0.019 | RV | 0.041 | 0.043 ||| MDP | 0.691 ||| 0.271 | 0.720 | 0.999 ||| SPSS | 20.0 | Student | two | Fisher | 0.05 ||| BTX-A | NDO ||| MDP [SUMMARY]"}}},{"rowIdx":294,"cells":{"title":{"kind":"string","value":"Efficacy and safety of risedronate 150-mg once a month in the treatment of postmenopausal osteoporosis: 2-year data."},"pmid":{"kind":"string","value":"22752050"},"background_abstract":{"kind":"string","value":"Risedronate is effective in the treatment of postmenopausal osteoporosis in oral daily, weekly, or on two consecutive days per month doses. This 2-year randomized, double-blind, multicenter study assesses the efficacy and safety of a single risedronate 150-mg once-a-month oral dose compared with the 5-mg daily regimen."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"Women with postmenopausal osteoporosis were randomly assigned to receive risedronate 5-mg daily (n = 642) or 150-mg once a month (n = 650) for 2 years. Bone mineral density (BMD), bone turnover markers, new vertebral fractures, and adverse events were evaluated. The primary efficacy endpoint was the mean percent change from baseline in lumbar spine BMD after 1 year."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Four hundred ninety-eight subjects in the daily group (77.6 %) and 513 subjects in the once-a-month group (78.9 %) completed the study. After 24 months, the mean percent change in lumbar spine BMD was 3.9 % (95 % confidence interval [CI], 3.43 to 4.42 %) and 4.2 % (95 % CI, 3.68 to 4.65 %) in the daily and once-a-month groups, respectively. The once-a-month regimen was determined to be non-inferior to the daily regimen. The mean percent changes in BMD at the hip were similar in both dose groups, as were changes in biochemical markers of bone turnover. The incidence of adverse events, adverse events leading to withdrawal, and upper gastrointestinal tract adverse events were similar in the two treatment groups."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"After 2 years, treatment with risedronate 150-mg once a month provided similar efficacy and tolerability to daily dosing and provides an alternative for patients who prefer once-a-month oral dosing."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Administration, Oral","Aged","Biomarkers","Bone Density","Bone Density Conservation Agents","Double-Blind Method","Drug Administration Schedule","Etidronic Acid","Female","Femur","Humans","Lumbar Vertebrae","Middle Aged","Osteoporosis, Postmenopausal","Risedronic Acid","Treatment Outcome"],"string":"[\n \"Administration, Oral\",\n \"Aged\",\n \"Biomarkers\",\n \"Bone Density\",\n \"Bone Density Conservation Agents\",\n \"Double-Blind Method\",\n \"Drug Administration Schedule\",\n \"Etidronic Acid\",\n \"Female\",\n \"Femur\",\n \"Humans\",\n \"Lumbar Vertebrae\",\n \"Middle Aged\",\n \"Osteoporosis, Postmenopausal\",\n \"Risedronic Acid\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"3536944"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Risedronate is a pyridinyl bisphosphonate that has been shown in prospective studies to reduce the risk of vertebral, nonvertebral, and hip fractures [1–3]. Like other bisphosphonates, risedronate remains active on the surface of bone for long periods after dosing, providing the opportunity to develop a range of dosing schedules.\nThe original risedronate dosing regimen for postmenopausal osteoporosis was an oral dose of 5-mg daily [1–3]. It was later demonstrated that risedronate 35-mg once a week and 75-mg each day for two consecutive days a month provided similar efficacy and safety to the daily regimen [4, 5]. The efficacy and tolerability of risedronate once-a-month dosing (150-mg) was compared with risedronate daily dosing (5-mg) in women with osteoporosis with changes in lumbar spine bone mineral density (BMD) as the primary endpoint. After 1 year of treatment, published previously, the efficacy of risedronate 150-mg once-a-month regimen was non-inferior to the 5-mg daily regimen [6]. The once-a-month regimen also had a similar tolerability profile as the daily regimen after 1 year of treatment. This study continued for an additional year of treatment, and the results of the complete study over 2 years are presented here."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Subjects From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\nFrom the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\n Efficacy assessments The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\nThe within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n Safety assessments Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\nOverall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Study design","Patients","Treatments","Efficacy assessments","Safety assessments","Statistical analysis","Subjects","Efficacy assessments","Safety assessments"],"string":"[\n \"Study design\",\n \"Patients\",\n \"Treatments\",\n \"Efficacy assessments\",\n \"Safety assessments\",\n \"Statistical analysis\",\n \"Subjects\",\n \"Efficacy assessments\",\n \"Safety assessments\"\n]"},"other_sections_texts":{"kind":"list like","value":["This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.","Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].","Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.","Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.","Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).","The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.","From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density","The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)","Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups."],"string":"[\n \"This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\",\n \"Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\",\n \"Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\",\n \"Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\",\n \"Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\",\n \"The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\",\n \"From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\\n\\nDisposition of subjects. BMD bone mineral density\",\n \"The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\n\\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\n\\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\",\n \"Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\\nn (%)\\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\\n23 (3.6)25 (3.8)\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\nAE adverse event\\n\\nSummary of adverse events\\n\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\n\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\n\\nAE adverse event\\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and methods","Study design","Patients","Treatments","Efficacy assessments","Safety assessments","Statistical analysis","Results","Subjects","Efficacy assessments","Safety assessments","Discussion"],"string":"[\n \"Introduction\",\n \"Materials and methods\",\n \"Study design\",\n \"Patients\",\n \"Treatments\",\n \"Efficacy assessments\",\n \"Safety assessments\",\n \"Statistical analysis\",\n \"Results\",\n \"Subjects\",\n \"Efficacy assessments\",\n \"Safety assessments\",\n \"Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Risedronate is a pyridinyl bisphosphonate that has been shown in prospective studies to reduce the risk of vertebral, nonvertebral, and hip fractures [1–3]. Like other bisphosphonates, risedronate remains active on the surface of bone for long periods after dosing, providing the opportunity to develop a range of dosing schedules.\nThe original risedronate dosing regimen for postmenopausal osteoporosis was an oral dose of 5-mg daily [1–3]. It was later demonstrated that risedronate 35-mg once a week and 75-mg each day for two consecutive days a month provided similar efficacy and safety to the daily regimen [4, 5]. The efficacy and tolerability of risedronate once-a-month dosing (150-mg) was compared with risedronate daily dosing (5-mg) in women with osteoporosis with changes in lumbar spine bone mineral density (BMD) as the primary endpoint. After 1 year of treatment, published previously, the efficacy of risedronate 150-mg once-a-month regimen was non-inferior to the 5-mg daily regimen [6]. The once-a-month regimen also had a similar tolerability profile as the daily regimen after 1 year of treatment. This study continued for an additional year of treatment, and the results of the complete study over 2 years are presented here."," Study design This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\nThis randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\n Patients Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\nEligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\n Treatments Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\nSubjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\n Efficacy assessments Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\nDual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\n Safety assessments Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\nPhysical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\n Statistical analysis The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\nThe primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.","This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.","Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].","Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.","Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.","Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).","The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group."," Subjects From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\nFrom the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\n Efficacy assessments The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\nThe within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n Safety assessments Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\nOverall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.","From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density","The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)","Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.","Risedronate is a widely used osteoporosis treatment with proven vertebral and nonvertebral antifracture efficacy and a minimum wait of 30 min after dosing before eating or drinking anything other than water. A 5-mg daily regimen was developed originally, but less frequent dose regimens have now been developed. This study was a preplanned 2-year study comparing a dose of risedronate 150-mg once a month to the 5-mg daily dose. These 2-year data show that the 150-mg once-a-month dose continues to produce clinical effects that are similar to those seen with the 5-mg daily dose. Specifically, the mean percent change in lumbar spine BMD at 24 months in the monthly group was non-inferior to the mean percent change in lumbar spine BMD in the daily group. Changes in secondary efficacy parameters, including BMD at the hip, bone turnover markers at endpoint, and morphometric vertebral fractures, were also similar in both groups. Statistically significant differences between treatment groups were observed for all three bone turnover markers at month 3, but persisted at months 6 and 12 for CTX only. No statistically significant differences between groups were observed for any marker at month 24 or endpoint. That the CTX response did not differ between treatment groups at month 24 might be explained by the small number of subjects at month 24 that would limit statistical power to observe difference. It is not likely that these small differences between groups in bone turnover markers are clinically meaningful.\nThe risedronate 150-mg once-a-month dose was well tolerated over 2 years, with a safety profile similar to that seen with the 5-mg daily regimen. The low incidences of subjects with vertebral and nonvertebral clinical fractures were similar between groups and consistent with rates previously observed with the 5-mg daily dose [1–3].\nChange in BMD is an appropriate endpoint when evaluating a new dosing schedule of a bisphosphonate for which a fracture benefit has already been established. Similar non-inferiority trials have been conducted previously to evaluate new dosing regimens of oral bisphosphonates [4, 8, 9], and this approach has been accepted by both the US Food and Drug Administration and the European Medicines Agency [10] for approval of new regimens of established agents. The magnitude of BMD change associated with the vertebral and nonvertebral antifracture efficacy of risedronate has been established in multiple large studies that had fracture as the primary endpoint [1–3]. This study has demonstrated that the 150-mg once-a-month dose reduces bone turnover and increases BMD to a degree comparable to that observed with the 5-mg daily dose in these fracture studies.\nThe results of this study after 2 years are consistent with the findings at month 12 [6], demonstrating the persistent similarity between risedronate 150-mg once-a-month and the 5-mg daily dosing regimens. Additionally, these results are consistent with the favorable tolerability and efficacy profiles observed in large placebo-controlled clinical trials of the risedronate 5-mg daily regimen [1–3]. The findings are also consistent with previous studies of less frequent dosing with risedronate. Such studies showed that the treatment effects of risedronate 35-mg weekly and 75-mg on two consecutive days each month were similar to the effects of daily dosing [4, 5].\nRisedronate 150-mg once a month, taken for 2 years, is similar in efficacy and tolerability to the 5-mg daily dosing regimen that had been proven to reduce the incidence of vertebral and nonvertebral fractures. The addition of this dosing regimen to the therapeutic armamentarium will provide women with postmenopausal osteoporosis a full range of risedronate oral dosing options, from daily to weekly to monthly."],"string":"[\n \"Risedronate is a pyridinyl bisphosphonate that has been shown in prospective studies to reduce the risk of vertebral, nonvertebral, and hip fractures [1–3]. Like other bisphosphonates, risedronate remains active on the surface of bone for long periods after dosing, providing the opportunity to develop a range of dosing schedules.\\nThe original risedronate dosing regimen for postmenopausal osteoporosis was an oral dose of 5-mg daily [1–3]. It was later demonstrated that risedronate 35-mg once a week and 75-mg each day for two consecutive days a month provided similar efficacy and safety to the daily regimen [4, 5]. The efficacy and tolerability of risedronate once-a-month dosing (150-mg) was compared with risedronate daily dosing (5-mg) in women with osteoporosis with changes in lumbar spine bone mineral density (BMD) as the primary endpoint. After 1 year of treatment, published previously, the efficacy of risedronate 150-mg once-a-month regimen was non-inferior to the 5-mg daily regimen [6]. The once-a-month regimen also had a similar tolerability profile as the daily regimen after 1 year of treatment. This study continued for an additional year of treatment, and the results of the complete study over 2 years are presented here.\",\n \" Study design This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\\nThis randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\\n Patients Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\\nEligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\\n Treatments Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\\nSubjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\\n Efficacy assessments Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\\nDual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\\n Safety assessments Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\\nPhysical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\\n Statistical analysis The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\\nThe primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\",\n \"This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\",\n \"Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\",\n \"Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\",\n \"Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\",\n \"Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\",\n \"The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\",\n \" Subjects From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\\n\\nDisposition of subjects. BMD bone mineral density\\nFrom the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\\n\\nDisposition of subjects. BMD bone mineral density\\n Efficacy assessments The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\n\\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\n\\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\nThe within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\n\\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\n\\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\n Safety assessments Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\\nn (%)\\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\\n23 (3.6)25 (3.8)\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\nAE adverse event\\n\\nSummary of adverse events\\n\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\n\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\n\\nAE adverse event\\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\\nOverall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\\nn (%)\\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\\n23 (3.6)25 (3.8)\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\nAE adverse event\\n\\nSummary of adverse events\\n\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\n\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\n\\nAE adverse event\\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\",\n \"From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\\n\\nDisposition of subjects. BMD bone mineral density\",\n \"The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\n\\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\\n\\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\",\n \"Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\\nn (%)\\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\\n23 (3.6)25 (3.8)\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\nAE adverse event\\n\\nSummary of adverse events\\n\\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\\n\\nbIncludes benign and malignant neoplasms, polyps, and cysts\\n\\nAE adverse event\\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\",\n \"Risedronate is a widely used osteoporosis treatment with proven vertebral and nonvertebral antifracture efficacy and a minimum wait of 30 min after dosing before eating or drinking anything other than water. A 5-mg daily regimen was developed originally, but less frequent dose regimens have now been developed. This study was a preplanned 2-year study comparing a dose of risedronate 150-mg once a month to the 5-mg daily dose. These 2-year data show that the 150-mg once-a-month dose continues to produce clinical effects that are similar to those seen with the 5-mg daily dose. Specifically, the mean percent change in lumbar spine BMD at 24 months in the monthly group was non-inferior to the mean percent change in lumbar spine BMD in the daily group. Changes in secondary efficacy parameters, including BMD at the hip, bone turnover markers at endpoint, and morphometric vertebral fractures, were also similar in both groups. Statistically significant differences between treatment groups were observed for all three bone turnover markers at month 3, but persisted at months 6 and 12 for CTX only. No statistically significant differences between groups were observed for any marker at month 24 or endpoint. That the CTX response did not differ between treatment groups at month 24 might be explained by the small number of subjects at month 24 that would limit statistical power to observe difference. It is not likely that these small differences between groups in bone turnover markers are clinically meaningful.\\nThe risedronate 150-mg once-a-month dose was well tolerated over 2 years, with a safety profile similar to that seen with the 5-mg daily regimen. The low incidences of subjects with vertebral and nonvertebral clinical fractures were similar between groups and consistent with rates previously observed with the 5-mg daily dose [1–3].\\nChange in BMD is an appropriate endpoint when evaluating a new dosing schedule of a bisphosphonate for which a fracture benefit has already been established. Similar non-inferiority trials have been conducted previously to evaluate new dosing regimens of oral bisphosphonates [4, 8, 9], and this approach has been accepted by both the US Food and Drug Administration and the European Medicines Agency [10] for approval of new regimens of established agents. The magnitude of BMD change associated with the vertebral and nonvertebral antifracture efficacy of risedronate has been established in multiple large studies that had fracture as the primary endpoint [1–3]. This study has demonstrated that the 150-mg once-a-month dose reduces bone turnover and increases BMD to a degree comparable to that observed with the 5-mg daily dose in these fracture studies.\\nThe results of this study after 2 years are consistent with the findings at month 12 [6], demonstrating the persistent similarity between risedronate 150-mg once-a-month and the 5-mg daily dosing regimens. Additionally, these results are consistent with the favorable tolerability and efficacy profiles observed in large placebo-controlled clinical trials of the risedronate 5-mg daily regimen [1–3]. The findings are also consistent with previous studies of less frequent dosing with risedronate. Such studies showed that the treatment effects of risedronate 35-mg weekly and 75-mg on two consecutive days each month were similar to the effects of daily dosing [4, 5].\\nRisedronate 150-mg once a month, taken for 2 years, is similar in efficacy and tolerability to the 5-mg daily dosing regimen that had been proven to reduce the incidence of vertebral and nonvertebral fractures. The addition of this dosing regimen to the therapeutic armamentarium will provide women with postmenopausal osteoporosis a full range of risedronate oral dosing options, from daily to weekly to monthly.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["introduction","materials|methods",null,null,null,null,null,null,"results",null,null,null,"discussion"],"string":"[\n \"introduction\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Bone mineral density","Fracture risk","Monthly","Osteoporosis","Risedronate"],"string":"[\n \"Bone mineral density\",\n \"Fracture risk\",\n \"Monthly\",\n \"Osteoporosis\",\n \"Risedronate\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nRisedronate is a pyridinyl bisphosphonate that has been shown in prospective studies to reduce the risk of vertebral, nonvertebral, and hip fractures [1–3]. Like other bisphosphonates, risedronate remains active on the surface of bone for long periods after dosing, providing the opportunity to develop a range of dosing schedules.\nThe original risedronate dosing regimen for postmenopausal osteoporosis was an oral dose of 5-mg daily [1–3]. It was later demonstrated that risedronate 35-mg once a week and 75-mg each day for two consecutive days a month provided similar efficacy and safety to the daily regimen [4, 5]. The efficacy and tolerability of risedronate once-a-month dosing (150-mg) was compared with risedronate daily dosing (5-mg) in women with osteoporosis with changes in lumbar spine bone mineral density (BMD) as the primary endpoint. After 1 year of treatment, published previously, the efficacy of risedronate 150-mg once-a-month regimen was non-inferior to the 5-mg daily regimen [6]. The once-a-month regimen also had a similar tolerability profile as the daily regimen after 1 year of treatment. This study continued for an additional year of treatment, and the results of the complete study over 2 years are presented here.\n\nMaterials and methods:\n Study design This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\nThis randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\n Patients Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\nEligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\n Treatments Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\nSubjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\n Efficacy assessments Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\nDual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\n Safety assessments Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\nPhysical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\n Statistical analysis The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\nThe primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\n\nStudy design:\nThis randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\n\nPatients:\nEligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\n\nTreatments:\nSubjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\n\nEfficacy assessments:\nDual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\n\nSafety assessments:\nPhysical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\n\nStatistical analysis:\nThe primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\n\nResults:\n Subjects From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\nFrom the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\n Efficacy assessments The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\nThe within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n Safety assessments Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\nOverall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\n\nSubjects:\nFrom the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\n\nEfficacy assessments:\nThe within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nSafety assessments:\nOverall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\n\nDiscussion:\nRisedronate is a widely used osteoporosis treatment with proven vertebral and nonvertebral antifracture efficacy and a minimum wait of 30 min after dosing before eating or drinking anything other than water. A 5-mg daily regimen was developed originally, but less frequent dose regimens have now been developed. This study was a preplanned 2-year study comparing a dose of risedronate 150-mg once a month to the 5-mg daily dose. These 2-year data show that the 150-mg once-a-month dose continues to produce clinical effects that are similar to those seen with the 5-mg daily dose. Specifically, the mean percent change in lumbar spine BMD at 24 months in the monthly group was non-inferior to the mean percent change in lumbar spine BMD in the daily group. Changes in secondary efficacy parameters, including BMD at the hip, bone turnover markers at endpoint, and morphometric vertebral fractures, were also similar in both groups. Statistically significant differences between treatment groups were observed for all three bone turnover markers at month 3, but persisted at months 6 and 12 for CTX only. No statistically significant differences between groups were observed for any marker at month 24 or endpoint. That the CTX response did not differ between treatment groups at month 24 might be explained by the small number of subjects at month 24 that would limit statistical power to observe difference. It is not likely that these small differences between groups in bone turnover markers are clinically meaningful.\nThe risedronate 150-mg once-a-month dose was well tolerated over 2 years, with a safety profile similar to that seen with the 5-mg daily regimen. The low incidences of subjects with vertebral and nonvertebral clinical fractures were similar between groups and consistent with rates previously observed with the 5-mg daily dose [1–3].\nChange in BMD is an appropriate endpoint when evaluating a new dosing schedule of a bisphosphonate for which a fracture benefit has already been established. Similar non-inferiority trials have been conducted previously to evaluate new dosing regimens of oral bisphosphonates [4, 8, 9], and this approach has been accepted by both the US Food and Drug Administration and the European Medicines Agency [10] for approval of new regimens of established agents. The magnitude of BMD change associated with the vertebral and nonvertebral antifracture efficacy of risedronate has been established in multiple large studies that had fracture as the primary endpoint [1–3]. This study has demonstrated that the 150-mg once-a-month dose reduces bone turnover and increases BMD to a degree comparable to that observed with the 5-mg daily dose in these fracture studies.\nThe results of this study after 2 years are consistent with the findings at month 12 [6], demonstrating the persistent similarity between risedronate 150-mg once-a-month and the 5-mg daily dosing regimens. Additionally, these results are consistent with the favorable tolerability and efficacy profiles observed in large placebo-controlled clinical trials of the risedronate 5-mg daily regimen [1–3]. The findings are also consistent with previous studies of less frequent dosing with risedronate. Such studies showed that the treatment effects of risedronate 35-mg weekly and 75-mg on two consecutive days each month were similar to the effects of daily dosing [4, 5].\nRisedronate 150-mg once a month, taken for 2 years, is similar in efficacy and tolerability to the 5-mg daily dosing regimen that had been proven to reduce the incidence of vertebral and nonvertebral fractures. The addition of this dosing regimen to the therapeutic armamentarium will provide women with postmenopausal osteoporosis a full range of risedronate oral dosing options, from daily to weekly to monthly.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nRisedronate is effective in the treatment of postmenopausal osteoporosis in oral daily, weekly, or on two consecutive days per month doses. This 2-year randomized, double-blind, multicenter study assesses the efficacy and safety of a single risedronate 150-mg once-a-month oral dose compared with the 5-mg daily regimen.\n\nMethods:\nWomen with postmenopausal osteoporosis were randomly assigned to receive risedronate 5-mg daily (n = 642) or 150-mg once a month (n = 650) for 2 years. Bone mineral density (BMD), bone turnover markers, new vertebral fractures, and adverse events were evaluated. The primary efficacy endpoint was the mean percent change from baseline in lumbar spine BMD after 1 year.\n\nResults:\nFour hundred ninety-eight subjects in the daily group (77.6 %) and 513 subjects in the once-a-month group (78.9 %) completed the study. After 24 months, the mean percent change in lumbar spine BMD was 3.9 % (95 % confidence interval [CI], 3.43 to 4.42 %) and 4.2 % (95 % CI, 3.68 to 4.65 %) in the daily and once-a-month groups, respectively. The once-a-month regimen was determined to be non-inferior to the daily regimen. The mean percent changes in BMD at the hip were similar in both dose groups, as were changes in biochemical markers of bone turnover. The incidence of adverse events, adverse events leading to withdrawal, and upper gastrointestinal tract adverse events were similar in the two treatment groups.\n\nConclusions:\nAfter 2 years, treatment with risedronate 150-mg once a month provided similar efficacy and tolerability to daily dosing and provides an alternative for patients who prefer once-a-month oral dosing."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":10390,"string":"10,390"},"whole_article_abstract_length":{"kind":"number","value":365,"string":"365"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["month","mg","treatment","24","groups","daily","subjects","group","baseline","mg daily"],"string":"[\n \"month\",\n \"mg\",\n \"treatment\",\n \"24\",\n \"groups\",\n \"daily\",\n \"subjects\",\n \"group\",\n \"baseline\",\n \"mg daily\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Bone mineral density | Fracture risk | Monthly | Osteoporosis | Risedronate [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Bone mineral density | Fracture risk | Monthly | Osteoporosis | Risedronate [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Bone mineral density | Fracture risk | Monthly | Osteoporosis | Risedronate [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Oral | Aged | Biomarkers | Bone Density | Bone Density Conservation Agents | Double-Blind Method | Drug Administration Schedule | Etidronic Acid | Female | Femur | Humans | Lumbar Vertebrae | Middle Aged | Osteoporosis, Postmenopausal | Risedronic Acid | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Oral | Aged | Biomarkers | Bone Density | Bone Density Conservation Agents | Double-Blind Method | Drug Administration Schedule | Etidronic Acid | Female | Femur | Humans | Lumbar Vertebrae | Middle Aged | Osteoporosis, Postmenopausal | Risedronic Acid | Treatment Outcome [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Oral | Aged | Biomarkers | Bone Density | Bone Density Conservation Agents | Double-Blind Method | Drug Administration Schedule | Etidronic Acid | Female | Femur | Humans | Lumbar Vertebrae | Middle Aged | Osteoporosis, Postmenopausal | Risedronic Acid | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] month | mg | treatment | 24 | groups | daily | subjects | group | baseline | mg daily [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] month | mg | treatment | 24 | groups | daily | subjects | group | baseline | mg daily [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] month | mg | treatment | 24 | groups | daily | subjects | group | baseline | mg daily [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] regimen | risedronate | dosing | mg | year treatment | daily | efficacy | daily regimen | tolerability | year [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] mg | groups | month | group | treatment | adverse | treatment groups | significant | events | line [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mg | month | treatment | 24 | daily | subjects | groups | group | baseline | adverse [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] daily | two consecutive days per month ||| 2-year | 150 | 5 | daily [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Four hundred ninety-eight | daily | 77.6 % | 513 | 78.9 % ||| 24 months | the mean percent | BMD | 3.9 % | 95 % | CI | 3.43 | 4.42 % | 4.2 % | 95 % | CI | 3.68 | 4.65 % | daily ||| daily ||| The mean percent | BMD ||| two [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] daily | two consecutive days per month ||| 2-year | 150 | 5 | daily ||| 5 | daily | 642 | 150 | 650 | 2 years ||| BMD ||| the mean percent | BMD | 1 year ||| Four hundred ninety-eight | daily | 77.6 % | 513 | 78.9 % ||| 24 months | the mean percent | BMD | 3.9 % | 95 % | CI | 3.43 | 4.42 % | 4.2 % | 95 % | CI | 3.68 | 4.65 % | daily ||| daily ||| The mean percent | BMD ||| two ||| 2 years | 150 | daily [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":295,"cells":{"title":{"kind":"string","value":"Survival Trend of Tuberculosis Patients and Risk Factors Associated with Mortality and Developing Drug-Resistant Tuberculosis in Hospital Pulau Pinang, Malaysia: A Retrospective Study."},"pmid":{"kind":"string","value":"36412638"},"background_abstract":{"kind":"string","value":"Multidrug resistance TB (MDR-TB) has emerged as a public health issue worldwide, and the mortality rate is worrying. Therefore, this study was conducted to investigate the factors related to MDR-TB occurrence and the survival experience of TB patients."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A retrospective cohort study was conducted at Hospital Pulau Pinang in Malaysia. Medical records of active TB patients from 2014-2018 were reviewed. Cox regression was used to identify the factors associated with MDR-TB development and mortality among TB patients."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The patients had a mean age of 48.84 ± 16.713 years, and a majority of the Chinese race (46.4%). Out of 351 TB patients, 325 (92.6%) were drug-susceptible TB, and 26 (7.4%) were diagnosed with MDR-TB. Among drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, and 73 (22.5%) passed away. In multivariable Cox regression, drug addiction, levels of white blood cells, urea, platelets, and albumin were significantly associated with death. Relapsed TB, alcohol consumption, and being single were significant risk factors for MDR-TB development."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Patients achieved a success rate of 75.4%, which is encouraging but still far below the WHO target (at least an 85% success rate) and has room for further improvement."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Humans","Adult","Middle Aged","Aged","Retrospective Studies","Antitubercular Agents","Malaysia","Tuberculosis, Multidrug-Resistant","Risk Factors","Hospitals"],"string":"[\n \"Humans\",\n \"Adult\",\n \"Middle Aged\",\n \"Aged\",\n \"Retrospective Studies\",\n \"Antitubercular Agents\",\n \"Malaysia\",\n \"Tuberculosis, Multidrug-Resistant\",\n \"Risk Factors\",\n \"Hospitals\"\n]"},"pmcid":{"kind":"string","value":"9774739"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Tuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":" 3.1. Sociodemographic and Clinical Characteristics At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\nAt the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\n 3.2. Drug Resistant Patterns of TB Patients The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\nThe study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\n 3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\nOf 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\n 3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\nAmong the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10)."},"conclusions_title":{"kind":"string","value":"5. Conclusions"},"conclusions_text":{"kind":"string","value":"Eventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single."},"other_sections_titles":{"kind":"list like","value":["2. Materials and Methods","2.1. Study Design and Population","2.2. Diagnosis and Treatment","2.3. Data Collection","2.4. Statistical Analysis","3.1. Sociodemographic and Clinical Characteristics","3.2. Drug Resistant Patterns of TB Patients","3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB"],"string":"[\n \"2. Materials and Methods\",\n \"2.1. Study Design and Population\",\n \"2.2. Diagnosis and Treatment\",\n \"2.3. Data Collection\",\n \"2.4. Statistical Analysis\",\n \"3.1. Sociodemographic and Clinical Characteristics\",\n \"3.2. Drug Resistant Patterns of TB Patients\",\n \"3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB\"\n]"},"other_sections_texts":{"kind":"list like","value":[" 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. ","A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. ","Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. ","Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. ","To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. ","At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).","The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).","Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10)."],"string":"[\n \" 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \\nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \\n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \\nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \\n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \\nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \\n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \\nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \",\n \"A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \",\n \"Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \",\n \"Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \",\n \"To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \",\n \"At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\",\n \"The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\",\n \"Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,"subjects",null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n \"subjects\",\n null\n]"},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Materials and Methods","2.1. Study Design and Population","2.2. Diagnosis and Treatment","2.3. Data Collection","2.4. Statistical Analysis","3. Results","3.1. Sociodemographic and Clinical Characteristics","3.2. Drug Resistant Patterns of TB Patients","3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients","3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB","4. Discussion","5. Conclusions"],"string":"[\n \"1. Introduction\",\n \"2. Materials and Methods\",\n \"2.1. Study Design and Population\",\n \"2.2. Diagnosis and Treatment\",\n \"2.3. Data Collection\",\n \"2.4. Statistical Analysis\",\n \"3. Results\",\n \"3.1. Sociodemographic and Clinical Characteristics\",\n \"3.2. Drug Resistant Patterns of TB Patients\",\n \"3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients\",\n \"3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB\",\n \"4. Discussion\",\n \"5. Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Tuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients."," 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. ","A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. ","Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. ","Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. ","To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. "," 3.1. Sociodemographic and Clinical Characteristics At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\nAt the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\n 3.2. Drug Resistant Patterns of TB Patients The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\nThe study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\n 3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\nOf 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\n 3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\nAmong the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).","At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).","The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).","Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).","Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).","Our research looked into the factors that contribute to mortality among TB patients, as well as the factors associated with MDR-TB development. Since then, tuberculosis treatment has been problematic and difficult to apply properly because of long duration and compliance issues due to anti-tubercular drugs’ side effects. Some of the side effects are self-limiting, but others necessitate treatment discontinuation. Although rare, thrombocytopenia is one of the most common and serious life-threatening side effects. Thrombocytopenia is caused by the formation of antibodies that bind to platelets and initiate the complement cascade, causing these cells to lyse. These antibodies also suppress the production of platelets [8]. This study demonstrated that thrombocytopenia significantly increased the risk of mortality among TB patients. [9] reported a low platelet count as a significant risk factor for mortality.\nMortality among patients suffering from any infection has been linked to WBC count [10]. As a result, elevated WBC counts in TB patients may be related to the severity of lung involvement and provide an estimated risk of mortality [11]. During TB infection, the body produces leukocytes and macrophages as part of the immune mechanism to defend the body against invading Mycobacterium tuberculosis, which raises the total number of WBC [12]. The current study findings documented that every 103 cells/µL increment in WBC above the normal range increased the risk of death by 1.102. In line with our results, a study conducted in Malaysia pointed out that the risk of death increased by 1.12 times for every 103 cells per microliter unit increase in total WBC [13]. Other studies have indicated that WBC levels are a significant risk factor correlated with TB mortality [11,14]. Pneumonia ranked the second cause of death among the study population. The WBC count elevation has been reported as a predictor of the severity of pneumonia, one of the leading causes of death in TB patients [11].\nThe present findings indicated that the level of urea was a strong significant predictor of death. Increased levels of urea were found to be significantly associated with mortality [11]. We can relate elevated urea levels to kidney dysfunction, where renal clearance is reduced due to renal impairment/failure or chronic kidney disease (CKD). Urea levels may also increase in other conditions, such as upper GI bleeding [15]. As the prevalence of CKD in our cohort was 5.69%, where 20 patients developed CKD while receiving anti-tuberculosis treatment, further research is needed to determine the relationship between CKD and TB. Strategies for the prevention, detection, and treatment of TB-CKD cannot be refined without advanced understanding.\nHypoalbuminemia showed a statistically significant relationship with death; patients who had hypoalbuminemia were more likely to pass away than patients who did not. Matching our results, lower albumin levels showed an association with non-survivors (p< 0.001) compared to survivors. Low levels of albumin were found to be significantly correlated with the hazard of death [9,16]. Furthermore, hypoalbuminemia was considered a predictor of mortality in community-acquired pneumonia (CAP), which is not surprising considering that albumin is involved in inflammation and indicates malnutrition [17]. The initial value of serum albumin might also be a result of malnutrition or an underlying disease that can worsen the nutritional status of the patient [18]. As a result of malnutrition, deceased patients’ immune systems were suppressed and were therefore sicker upon initial presentation.\nAs for drug addiction characteristics, the findings of this study revealed that patients who had a history of addiction or were current addicts had a 1.895 times higher risk of death than those who had never abused drugs. In line with our findings, deceased patients were more likely to abuse heroin IV than living patients, and addiction was correlated with all causes of death in tuberculosis patients [19,20]. Supporting our results, a history of addiction was a statistically significant risk factor for death within the first year after diagnosis in Hong Kong, Russia, and Iran [21,22,23]. Poor adherence of drug users, default treatment, and poor access to health services can create challenges for the treatment of the drug addict population [24]. As drug users have a higher likelihood of default treatment than non-drug users, the increased hazard of death can be attributed to the increased prevalence of alcohol misuse, chronic viral hepatitis, and anti-tubercular drug-induced hepatotoxicity that injectable drug users exhibit [25,26].\nDrug resistance TB has emerged as a serious health issue undermining global efforts to control TB and reduce the spread of the disease, in addition to making TB treatment more complicated. Thus, we aimed to study the risk factors associated with the development of MDR-TB in TB patients. In the current study, relapsed TB cases were more likely to develop MDR-TB than newly registered cases that had never had exposure to anti-TB treatment. In agreement with our findings, a history of treatment with anti-tubercular drugs has been mentioned in the previous literature as a risk factor for MDR-TB development in common with other risk factors [27,28,29,30]. Relapsed TB cases were found to be more likely to achieve unsuccessful treatment outcomes [31,32]. As the history of being treated with anti-TB drugs raises the hazard of resistance to many drugs, more attention must be paid to identifying the underlying causes of treatment failure and default treatment, which may decrease the incidence of retreatment among tuberculosis patients [28]. The link between prior anti-TB exposure and the emergence of drug resistance could be due to the fact that prior anti-TB exposure suppresses only the growth of susceptible bacilli. On the other hand, it may allow for the multiplication of pre-existing drug-resistant mutants [33].\nOne of the sociodemographic characteristics related to tuberculosis is marital status. Our findings reported that single patients were at 6.817 times higher risk of developing MDR-TB than married patients, which was in agreement with other studies that revealed that single individuals were at higher risk of developing MDR-TB than married individuals [34,35]. Even though there is no biological relationship between the marital status of the patients and TB, when compared to married people, single people are more likely to be infected with tuberculosis or MDR-TB strains. Being at high risk can still be due to a lack of social support or participation in high-risk behaviors, such as alcohol and drug addiction [36].\nAside from the previously mentioned risk factors, alcohol consumption has been significantly associated with developing MDR-TB. Patients who had a history of drinking or were current drinkers had a 7.591 times higher risk of MDR-TB than those who had never drank. Drinkers had a higher risk of MDR-TB, according to the results of a study conducted in China and Uganda [37,38]. Alcohol use was a contributing factor in TB treatment interruption, which is the main factor in MDR-TB development [39]. Many explanations could stand behind the impact of alcohol consumption on MDR-TB. Poorer treatment outcomes, including default treatment, mortality, and treatment failure, have been observed in TB patients who have consumed alcohol and MDR-TB, while treatment failure has been identified as a significant risk factor for drug resistance [38]. In addition, the consequences of alcohol ingestion can result in liver damage, weakened immunity, and nutritional deficiency, contributing to sensitive and resistant TB infection [40,41,42].\nThe findings of the current study highlighted patients’ groups that are at high risk of achieving unsuccessful TB treatment outcomes. As MDR-TB has proven to be a major influencer on patients’ health and safety, more attention must be paid to the factors leading to relapse and overcoming them in order to minimize the risk of MDR-TB occurrence in TB patients. A must improve public awareness about the worsening effect of alcohol on TB treatment and the importance of psychological support from the patient’s family to adhere to TB treatment. Moreover, the government should implement more restricted rules to minimize the availability of drugs in the population hands and to increase the awareness of people regarding the hazard of drugs. Additionally, malnutrition status was a risk factor for death among TB patients, so monthly counseling and material support in the form of food is suggested.\nThe study has many limitations that must be recognized. This study targeted only patients registered with HPP, which may not cover the true proportion of the Malaysian population. Being a unicentral study and a convenient sample technique limits the generalization of the results to the entire Malaysian population. An unequal proportionality among several variables, including age, ethnicity, and gender, might influence the final results in comparison with randomized control trials (selection bias). Due to the retrospective nature of the present study, it was impossible to record certain sociodemographic characteristics of the patients who passed away and default treatment (information bias), which might enhance the findings of the study. It was difficult to set a control group that needed further clinical trials to support the study results. The aforementioned limitations might affect the extent to which the current findings are accurate and consistent and can be reproduced under the same methodology.","Eventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single."],"string":"[\n \"Tuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients.\",\n \" 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \\nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \\n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \\nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \\n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \\nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \\n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \\nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \",\n \"A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \",\n \"Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \\nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \\nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \\nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \\nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \\nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \",\n \"Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \\nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \",\n \"To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \",\n \" 3.1. Sociodemographic and Clinical Characteristics At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\\nAt the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\\n 3.2. Drug Resistant Patterns of TB Patients The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\\nThe study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\\n 3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\\nOf 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\\n 3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\\nAmong the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\",\n \"At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\",\n \"The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\",\n \"Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\",\n \"Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\",\n \"Our research looked into the factors that contribute to mortality among TB patients, as well as the factors associated with MDR-TB development. Since then, tuberculosis treatment has been problematic and difficult to apply properly because of long duration and compliance issues due to anti-tubercular drugs’ side effects. Some of the side effects are self-limiting, but others necessitate treatment discontinuation. Although rare, thrombocytopenia is one of the most common and serious life-threatening side effects. Thrombocytopenia is caused by the formation of antibodies that bind to platelets and initiate the complement cascade, causing these cells to lyse. These antibodies also suppress the production of platelets [8]. This study demonstrated that thrombocytopenia significantly increased the risk of mortality among TB patients. [9] reported a low platelet count as a significant risk factor for mortality.\\nMortality among patients suffering from any infection has been linked to WBC count [10]. As a result, elevated WBC counts in TB patients may be related to the severity of lung involvement and provide an estimated risk of mortality [11]. During TB infection, the body produces leukocytes and macrophages as part of the immune mechanism to defend the body against invading Mycobacterium tuberculosis, which raises the total number of WBC [12]. The current study findings documented that every 103 cells/µL increment in WBC above the normal range increased the risk of death by 1.102. In line with our results, a study conducted in Malaysia pointed out that the risk of death increased by 1.12 times for every 103 cells per microliter unit increase in total WBC [13]. Other studies have indicated that WBC levels are a significant risk factor correlated with TB mortality [11,14]. Pneumonia ranked the second cause of death among the study population. The WBC count elevation has been reported as a predictor of the severity of pneumonia, one of the leading causes of death in TB patients [11].\\nThe present findings indicated that the level of urea was a strong significant predictor of death. Increased levels of urea were found to be significantly associated with mortality [11]. We can relate elevated urea levels to kidney dysfunction, where renal clearance is reduced due to renal impairment/failure or chronic kidney disease (CKD). Urea levels may also increase in other conditions, such as upper GI bleeding [15]. As the prevalence of CKD in our cohort was 5.69%, where 20 patients developed CKD while receiving anti-tuberculosis treatment, further research is needed to determine the relationship between CKD and TB. Strategies for the prevention, detection, and treatment of TB-CKD cannot be refined without advanced understanding.\\nHypoalbuminemia showed a statistically significant relationship with death; patients who had hypoalbuminemia were more likely to pass away than patients who did not. Matching our results, lower albumin levels showed an association with non-survivors (p< 0.001) compared to survivors. Low levels of albumin were found to be significantly correlated with the hazard of death [9,16]. Furthermore, hypoalbuminemia was considered a predictor of mortality in community-acquired pneumonia (CAP), which is not surprising considering that albumin is involved in inflammation and indicates malnutrition [17]. The initial value of serum albumin might also be a result of malnutrition or an underlying disease that can worsen the nutritional status of the patient [18]. As a result of malnutrition, deceased patients’ immune systems were suppressed and were therefore sicker upon initial presentation.\\nAs for drug addiction characteristics, the findings of this study revealed that patients who had a history of addiction or were current addicts had a 1.895 times higher risk of death than those who had never abused drugs. In line with our findings, deceased patients were more likely to abuse heroin IV than living patients, and addiction was correlated with all causes of death in tuberculosis patients [19,20]. Supporting our results, a history of addiction was a statistically significant risk factor for death within the first year after diagnosis in Hong Kong, Russia, and Iran [21,22,23]. Poor adherence of drug users, default treatment, and poor access to health services can create challenges for the treatment of the drug addict population [24]. As drug users have a higher likelihood of default treatment than non-drug users, the increased hazard of death can be attributed to the increased prevalence of alcohol misuse, chronic viral hepatitis, and anti-tubercular drug-induced hepatotoxicity that injectable drug users exhibit [25,26].\\nDrug resistance TB has emerged as a serious health issue undermining global efforts to control TB and reduce the spread of the disease, in addition to making TB treatment more complicated. Thus, we aimed to study the risk factors associated with the development of MDR-TB in TB patients. In the current study, relapsed TB cases were more likely to develop MDR-TB than newly registered cases that had never had exposure to anti-TB treatment. In agreement with our findings, a history of treatment with anti-tubercular drugs has been mentioned in the previous literature as a risk factor for MDR-TB development in common with other risk factors [27,28,29,30]. Relapsed TB cases were found to be more likely to achieve unsuccessful treatment outcomes [31,32]. As the history of being treated with anti-TB drugs raises the hazard of resistance to many drugs, more attention must be paid to identifying the underlying causes of treatment failure and default treatment, which may decrease the incidence of retreatment among tuberculosis patients [28]. The link between prior anti-TB exposure and the emergence of drug resistance could be due to the fact that prior anti-TB exposure suppresses only the growth of susceptible bacilli. On the other hand, it may allow for the multiplication of pre-existing drug-resistant mutants [33].\\nOne of the sociodemographic characteristics related to tuberculosis is marital status. Our findings reported that single patients were at 6.817 times higher risk of developing MDR-TB than married patients, which was in agreement with other studies that revealed that single individuals were at higher risk of developing MDR-TB than married individuals [34,35]. Even though there is no biological relationship between the marital status of the patients and TB, when compared to married people, single people are more likely to be infected with tuberculosis or MDR-TB strains. Being at high risk can still be due to a lack of social support or participation in high-risk behaviors, such as alcohol and drug addiction [36].\\nAside from the previously mentioned risk factors, alcohol consumption has been significantly associated with developing MDR-TB. Patients who had a history of drinking or were current drinkers had a 7.591 times higher risk of MDR-TB than those who had never drank. Drinkers had a higher risk of MDR-TB, according to the results of a study conducted in China and Uganda [37,38]. Alcohol use was a contributing factor in TB treatment interruption, which is the main factor in MDR-TB development [39]. Many explanations could stand behind the impact of alcohol consumption on MDR-TB. Poorer treatment outcomes, including default treatment, mortality, and treatment failure, have been observed in TB patients who have consumed alcohol and MDR-TB, while treatment failure has been identified as a significant risk factor for drug resistance [38]. In addition, the consequences of alcohol ingestion can result in liver damage, weakened immunity, and nutritional deficiency, contributing to sensitive and resistant TB infection [40,41,42].\\nThe findings of the current study highlighted patients’ groups that are at high risk of achieving unsuccessful TB treatment outcomes. As MDR-TB has proven to be a major influencer on patients’ health and safety, more attention must be paid to the factors leading to relapse and overcoming them in order to minimize the risk of MDR-TB occurrence in TB patients. A must improve public awareness about the worsening effect of alcohol on TB treatment and the importance of psychological support from the patient’s family to adhere to TB treatment. Moreover, the government should implement more restricted rules to minimize the availability of drugs in the population hands and to increase the awareness of people regarding the hazard of drugs. Additionally, malnutrition status was a risk factor for death among TB patients, so monthly counseling and material support in the form of food is suggested.\\nThe study has many limitations that must be recognized. This study targeted only patients registered with HPP, which may not cover the true proportion of the Malaysian population. Being a unicentral study and a convenient sample technique limits the generalization of the results to the entire Malaysian population. An unequal proportionality among several variables, including age, ethnicity, and gender, might influence the final results in comparison with randomized control trials (selection bias). Due to the retrospective nature of the present study, it was impossible to record certain sociodemographic characteristics of the patients who passed away and default treatment (information bias), which might enhance the findings of the study. It was difficult to set a control group that needed further clinical trials to support the study results. The aforementioned limitations might affect the extent to which the current findings are accurate and consistent and can be reproduced under the same methodology.\",\n \"Eventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,null,null,null,null,"results",null,"subjects","subjects",null,"discussion","conclusions"],"string":"[\n \"intro\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n \"subjects\",\n \"subjects\",\n null,\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["multidrug-resistant","mortality","death","treatment outcomes","survival","unsuccessful treatment"],"string":"[\n \"multidrug-resistant\",\n \"mortality\",\n \"death\",\n \"treatment outcomes\",\n \"survival\",\n \"unsuccessful treatment\"\n]"},"whole_article_text":{"kind":"string","value":"1. Introduction:\nTuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients.\n\n2. Materials and Methods:\n 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \n\n2.1. Study Design and Population:\nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \n\n2.2. Diagnosis and Treatment:\nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \n\n2.3. Data Collection:\nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \n\n2.4. Statistical Analysis:\nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \n\n3. Results:\n 3.1. Sociodemographic and Clinical Characteristics At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\nAt the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\n 3.2. Drug Resistant Patterns of TB Patients The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\nThe study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\n 3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\nOf 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\n 3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\nAmong the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\n\n3.1. Sociodemographic and Clinical Characteristics:\nAt the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\n\n3.2. Drug Resistant Patterns of TB Patients:\nThe study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\n\n3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients:\nOf 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\n\n3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB:\nAmong the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\n\n4. Discussion:\nOur research looked into the factors that contribute to mortality among TB patients, as well as the factors associated with MDR-TB development. Since then, tuberculosis treatment has been problematic and difficult to apply properly because of long duration and compliance issues due to anti-tubercular drugs’ side effects. Some of the side effects are self-limiting, but others necessitate treatment discontinuation. Although rare, thrombocytopenia is one of the most common and serious life-threatening side effects. Thrombocytopenia is caused by the formation of antibodies that bind to platelets and initiate the complement cascade, causing these cells to lyse. These antibodies also suppress the production of platelets [8]. This study demonstrated that thrombocytopenia significantly increased the risk of mortality among TB patients. [9] reported a low platelet count as a significant risk factor for mortality.\nMortality among patients suffering from any infection has been linked to WBC count [10]. As a result, elevated WBC counts in TB patients may be related to the severity of lung involvement and provide an estimated risk of mortality [11]. During TB infection, the body produces leukocytes and macrophages as part of the immune mechanism to defend the body against invading Mycobacterium tuberculosis, which raises the total number of WBC [12]. The current study findings documented that every 103 cells/µL increment in WBC above the normal range increased the risk of death by 1.102. In line with our results, a study conducted in Malaysia pointed out that the risk of death increased by 1.12 times for every 103 cells per microliter unit increase in total WBC [13]. Other studies have indicated that WBC levels are a significant risk factor correlated with TB mortality [11,14]. Pneumonia ranked the second cause of death among the study population. The WBC count elevation has been reported as a predictor of the severity of pneumonia, one of the leading causes of death in TB patients [11].\nThe present findings indicated that the level of urea was a strong significant predictor of death. Increased levels of urea were found to be significantly associated with mortality [11]. We can relate elevated urea levels to kidney dysfunction, where renal clearance is reduced due to renal impairment/failure or chronic kidney disease (CKD). Urea levels may also increase in other conditions, such as upper GI bleeding [15]. As the prevalence of CKD in our cohort was 5.69%, where 20 patients developed CKD while receiving anti-tuberculosis treatment, further research is needed to determine the relationship between CKD and TB. Strategies for the prevention, detection, and treatment of TB-CKD cannot be refined without advanced understanding.\nHypoalbuminemia showed a statistically significant relationship with death; patients who had hypoalbuminemia were more likely to pass away than patients who did not. Matching our results, lower albumin levels showed an association with non-survivors (p< 0.001) compared to survivors. Low levels of albumin were found to be significantly correlated with the hazard of death [9,16]. Furthermore, hypoalbuminemia was considered a predictor of mortality in community-acquired pneumonia (CAP), which is not surprising considering that albumin is involved in inflammation and indicates malnutrition [17]. The initial value of serum albumin might also be a result of malnutrition or an underlying disease that can worsen the nutritional status of the patient [18]. As a result of malnutrition, deceased patients’ immune systems were suppressed and were therefore sicker upon initial presentation.\nAs for drug addiction characteristics, the findings of this study revealed that patients who had a history of addiction or were current addicts had a 1.895 times higher risk of death than those who had never abused drugs. In line with our findings, deceased patients were more likely to abuse heroin IV than living patients, and addiction was correlated with all causes of death in tuberculosis patients [19,20]. Supporting our results, a history of addiction was a statistically significant risk factor for death within the first year after diagnosis in Hong Kong, Russia, and Iran [21,22,23]. Poor adherence of drug users, default treatment, and poor access to health services can create challenges for the treatment of the drug addict population [24]. As drug users have a higher likelihood of default treatment than non-drug users, the increased hazard of death can be attributed to the increased prevalence of alcohol misuse, chronic viral hepatitis, and anti-tubercular drug-induced hepatotoxicity that injectable drug users exhibit [25,26].\nDrug resistance TB has emerged as a serious health issue undermining global efforts to control TB and reduce the spread of the disease, in addition to making TB treatment more complicated. Thus, we aimed to study the risk factors associated with the development of MDR-TB in TB patients. In the current study, relapsed TB cases were more likely to develop MDR-TB than newly registered cases that had never had exposure to anti-TB treatment. In agreement with our findings, a history of treatment with anti-tubercular drugs has been mentioned in the previous literature as a risk factor for MDR-TB development in common with other risk factors [27,28,29,30]. Relapsed TB cases were found to be more likely to achieve unsuccessful treatment outcomes [31,32]. As the history of being treated with anti-TB drugs raises the hazard of resistance to many drugs, more attention must be paid to identifying the underlying causes of treatment failure and default treatment, which may decrease the incidence of retreatment among tuberculosis patients [28]. The link between prior anti-TB exposure and the emergence of drug resistance could be due to the fact that prior anti-TB exposure suppresses only the growth of susceptible bacilli. On the other hand, it may allow for the multiplication of pre-existing drug-resistant mutants [33].\nOne of the sociodemographic characteristics related to tuberculosis is marital status. Our findings reported that single patients were at 6.817 times higher risk of developing MDR-TB than married patients, which was in agreement with other studies that revealed that single individuals were at higher risk of developing MDR-TB than married individuals [34,35]. Even though there is no biological relationship between the marital status of the patients and TB, when compared to married people, single people are more likely to be infected with tuberculosis or MDR-TB strains. Being at high risk can still be due to a lack of social support or participation in high-risk behaviors, such as alcohol and drug addiction [36].\nAside from the previously mentioned risk factors, alcohol consumption has been significantly associated with developing MDR-TB. Patients who had a history of drinking or were current drinkers had a 7.591 times higher risk of MDR-TB than those who had never drank. Drinkers had a higher risk of MDR-TB, according to the results of a study conducted in China and Uganda [37,38]. Alcohol use was a contributing factor in TB treatment interruption, which is the main factor in MDR-TB development [39]. Many explanations could stand behind the impact of alcohol consumption on MDR-TB. Poorer treatment outcomes, including default treatment, mortality, and treatment failure, have been observed in TB patients who have consumed alcohol and MDR-TB, while treatment failure has been identified as a significant risk factor for drug resistance [38]. In addition, the consequences of alcohol ingestion can result in liver damage, weakened immunity, and nutritional deficiency, contributing to sensitive and resistant TB infection [40,41,42].\nThe findings of the current study highlighted patients’ groups that are at high risk of achieving unsuccessful TB treatment outcomes. As MDR-TB has proven to be a major influencer on patients’ health and safety, more attention must be paid to the factors leading to relapse and overcoming them in order to minimize the risk of MDR-TB occurrence in TB patients. A must improve public awareness about the worsening effect of alcohol on TB treatment and the importance of psychological support from the patient’s family to adhere to TB treatment. Moreover, the government should implement more restricted rules to minimize the availability of drugs in the population hands and to increase the awareness of people regarding the hazard of drugs. Additionally, malnutrition status was a risk factor for death among TB patients, so monthly counseling and material support in the form of food is suggested.\nThe study has many limitations that must be recognized. This study targeted only patients registered with HPP, which may not cover the true proportion of the Malaysian population. Being a unicentral study and a convenient sample technique limits the generalization of the results to the entire Malaysian population. An unequal proportionality among several variables, including age, ethnicity, and gender, might influence the final results in comparison with randomized control trials (selection bias). Due to the retrospective nature of the present study, it was impossible to record certain sociodemographic characteristics of the patients who passed away and default treatment (information bias), which might enhance the findings of the study. It was difficult to set a control group that needed further clinical trials to support the study results. The aforementioned limitations might affect the extent to which the current findings are accurate and consistent and can be reproduced under the same methodology.\n\n5. Conclusions:\nEventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nMultidrug resistance TB (MDR-TB) has emerged as a public health issue worldwide, and the mortality rate is worrying. Therefore, this study was conducted to investigate the factors related to MDR-TB occurrence and the survival experience of TB patients.\n\nMethods:\nA retrospective cohort study was conducted at Hospital Pulau Pinang in Malaysia. Medical records of active TB patients from 2014-2018 were reviewed. Cox regression was used to identify the factors associated with MDR-TB development and mortality among TB patients.\n\nResults:\nThe patients had a mean age of 48.84 ± 16.713 years, and a majority of the Chinese race (46.4%). Out of 351 TB patients, 325 (92.6%) were drug-susceptible TB, and 26 (7.4%) were diagnosed with MDR-TB. Among drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, and 73 (22.5%) passed away. In multivariable Cox regression, drug addiction, levels of white blood cells, urea, platelets, and albumin were significantly associated with death. Relapsed TB, alcohol consumption, and being single were significant risk factors for MDR-TB development.\n\nConclusions:\nPatients achieved a success rate of 75.4%, which is encouraging but still far below the WHO target (at least an 85% success rate) and has room for further improvement."},"background_conclusion_text":{"kind":"string","value":"1. Introduction:\nTuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients.\n\n5. Conclusions:\nEventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nMultidrug resistance TB (MDR-TB) has emerged as a public health issue worldwide, and the mortality rate is worrying. Therefore, this study was conducted to investigate the factors related to MDR-TB occurrence and the survival experience of TB patients.\n\nMethods:\nA retrospective cohort study was conducted at Hospital Pulau Pinang in Malaysia. Medical records of active TB patients from 2014-2018 were reviewed. Cox regression was used to identify the factors associated with MDR-TB development and mortality among TB patients.\n\nResults:\nThe patients had a mean age of 48.84 ± 16.713 years, and a majority of the Chinese race (46.4%). Out of 351 TB patients, 325 (92.6%) were drug-susceptible TB, and 26 (7.4%) were diagnosed with MDR-TB. Among drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, and 73 (22.5%) passed away. In multivariable Cox regression, drug addiction, levels of white blood cells, urea, platelets, and albumin were significantly associated with death. Relapsed TB, alcohol consumption, and being single were significant risk factors for MDR-TB development.\n\nConclusions:\nPatients achieved a success rate of 75.4%, which is encouraging but still far below the WHO target (at least an 85% success rate) and has room for further improvement."},"whole_article_text_length":{"kind":"number","value":9383,"string":"9,383"},"whole_article_abstract_length":{"kind":"number","value":268,"string":"268"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["tb","patients","treatment","hr","ci","95 ci","95","mdr tb","mdr","drug"],"string":"[\n \"tb\",\n \"patients\",\n \"treatment\",\n \"hr\",\n \"ci\",\n \"95 ci\",\n \"95\",\n \"mdr tb\",\n \"mdr\",\n \"drug\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | mdr tb | mdr | 2019 | disease | people | 000 | million | mortality | 2020 [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 95 | 95 ci | ci | hr | table | tb | associated | risk | patients | drug [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | levels | addiction | eventually patients history addiction | patients drug addiction | related relapsed cases alcohol | tuberculosis developed | tuberculosis developed 26 | tuberculosis developed 26 351 | patients drug addiction white [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | 95 | ci | 95 ci | hr | treatment | mdr tb | mdr | drug [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] tb | patients | 95 | ci | 95 ci | hr | treatment | mdr tb | mdr | drug [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] TB ||| MDR | TB [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 16.713 years | Chinese | 46.4% ||| 351 | 325 | 92.6% | TB | 26 | 7.4% | MDR ||| 245 | 75.4% | 73 | 22.5% ||| ||| TB | MDR [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] 75.4% | WHO | at least an | 85% [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] TB ||| MDR | TB ||| Hospital Pulau Pinang | Malaysia ||| TB | 2014-2018 ||| MDR | TB ||| ||| 16.713 years | Chinese | 46.4% ||| 351 | 325 | 92.6% | TB | 26 | 7.4% | MDR ||| 245 | 75.4% | 73 | 22.5% ||| ||| TB | MDR ||| 75.4% | WHO | at least an | 85% [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] TB ||| MDR | TB ||| Hospital Pulau Pinang | Malaysia ||| TB | 2014-2018 ||| MDR | TB ||| ||| 16.713 years | Chinese | 46.4% ||| 351 | 325 | 92.6% | TB | 26 | 7.4% | MDR ||| 245 | 75.4% | 73 | 22.5% ||| ||| TB | MDR ||| 75.4% | WHO | at least an | 85% [SUMMARY]"}}},{"rowIdx":296,"cells":{"title":{"kind":"string","value":"Is the Association between Age and Fertility Problems Modified by Diet Quality? Findings from a National Study of Reproductive Age Women in Australia."},"pmid":{"kind":"string","value":"36297039"},"background_abstract":{"kind":"string","value":"Increasing age is a strong risk factor for infertility, and there is accumulating evidence of the importance of a healthier diet for fertility. Whether a healthier diet modifies the association between increasing age and infertility has not been investigated. This study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Data were from Surveys 3 and 5 of the 1973-1978 birth cohort of the Australian Longitudinal Study on Women's Health. Cross-sectional analysis with multivariable generalized linear models were used to examine the association between age and fertility status, adjusted for various confounders. Multiplicative and additive effect modification by diet quality was assessed, with additive effect modification evaluated with the relative risk for interaction (RERI)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In total, 3387 women were included from Survey 3 (age range 24-31 years) and 5614 women from Survey 5 (age range 30-38 years); 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems in the respective surveys. In Survey 3, compared to younger women with a good-quality diet, older women with a poor-quality diet had a 43% increased risk for fertility problems, with risk increasing after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62). In Survey 5 in younger women (<33.9 years), there was no association between diet quality and risk for infertility problems. The RERI (across different adjusted models) was between -0.08 (-0.70, 0.55) to -0.39 (-1.40, 0.62) in survey 3 and 0.07 (-0.17, 0.31) to 0.08 (-0.17, 0.32) in Survey 5."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"There is little evidence to suggest effect modification on the effect of age and fertility problems with diet quality."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Female","Humans","Aged","Young Adult","Adult","Infertility, Female","Longitudinal Studies","Cross-Sectional Studies","Australia","Fertility","Diet","Risk Factors"],"string":"[\n \"Female\",\n \"Humans\",\n \"Aged\",\n \"Young Adult\",\n \"Adult\",\n \"Infertility, Female\",\n \"Longitudinal Studies\",\n \"Cross-Sectional Studies\",\n \"Australia\",\n \"Fertility\",\n \"Diet\",\n \"Risk Factors\"\n]"},"pmcid":{"kind":"string","value":"9606952"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Infertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].\nIncreasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].\nAlthough increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].\nThere is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":" 3.1. Participants A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\nA total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\n 3.2. Effect Modification of Diet on Age and Fertility Problems Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\nTable 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\n 3.3. Dietary Change and Fertility Status Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\nTable 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5)."},"conclusions_title":{"kind":"string","value":"5. Conclusions"},"conclusions_text":{"kind":"string","value":"In conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age."},"other_sections_titles":{"kind":"list like","value":["2. Materials and Methods","2.1. Study Design","2.2. Study Population","2.3. Exposure Variables","2.4. Outcome Variable","2.5. Confounding Variables","2.6. Statistical Analysis","3.2. Effect Modification of Diet on Age and Fertility Problems","3.3. Dietary Change and Fertility Status"],"string":"[\n \"2. Materials and Methods\",\n \"2.1. Study Design\",\n \"2.2. Study Population\",\n \"2.3. Exposure Variables\",\n \"2.4. Outcome Variable\",\n \"2.5. Confounding Variables\",\n \"2.6. Statistical Analysis\",\n \"3.2. Effect Modification of Diet on Age and Fertility Problems\",\n \"3.3. Dietary Change and Fertility Status\"\n]"},"other_sections_texts":{"kind":"list like","value":[" 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).","Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.","This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).","The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.","Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.","A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].","Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).","Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.","Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5)."],"string":"[\n \" 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\",\n \"Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\",\n \"This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\",\n \"The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\",\n \"Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\",\n \"A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\",\n \"Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\",\n \"Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\",\n \"Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Materials and Methods","2.1. Study Design","2.2. Study Population","2.3. Exposure Variables","2.4. Outcome Variable","2.5. Confounding Variables","2.6. Statistical Analysis","3. Results","3.1. Participants","3.2. Effect Modification of Diet on Age and Fertility Problems","3.3. Dietary Change and Fertility Status","4. Discussion","5. Conclusions"],"string":"[\n \"1. Introduction\",\n \"2. Materials and Methods\",\n \"2.1. Study Design\",\n \"2.2. Study Population\",\n \"2.3. Exposure Variables\",\n \"2.4. Outcome Variable\",\n \"2.5. Confounding Variables\",\n \"2.6. Statistical Analysis\",\n \"3. Results\",\n \"3.1. Participants\",\n \"3.2. Effect Modification of Diet on Age and Fertility Problems\",\n \"3.3. Dietary Change and Fertility Status\",\n \"4. Discussion\",\n \"5. Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Infertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].\nIncreasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].\nAlthough increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].\nThere is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality."," 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).","Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.","This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).","The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.","Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.","A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].","Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA)."," 3.1. Participants A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\nA total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\n 3.2. Effect Modification of Diet on Age and Fertility Problems Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\nTable 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\n 3.3. Dietary Change and Fertility Status Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\nTable 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).","A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.","Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.","Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).","Among a large cohort of Australian women, we examined whether diet modified the relationship between age and infertility, and whether a change in diet impacts fertility. Our results do not support our hypothesis; that is, in both Survey 3 and in Survey 5, there was little evidence that diet quality modified the association between age and fertility problems. Although increasing age (from Survey 3 to Survey 5) was associated with an approximate 5% higher rate of fertility problems, a change in diet quality over this time was not associated with fertility problems at Survey 5.\nAmong Survey 3 participants who were aged between 24–31 years, independent associations were found, such that older woman (28.2 to 30.8 years) who had a poor-quality diet had higher risk for infertility problems compared to the younger women (24.4 to <28.2 years) with a good-quality diet. At Survey 5 when women were aged 30–38 years, there was no difference in risk for fertility problems by diet quality or age. This was an unexpected finding. At Survey 5, there was an approximate 10% higher percentage of women with a good-quality diet; thus, with increasing age, the importance of a healthier diet may be realised, irrespective of fertility status. Yet, the older women at Survey 5 did appear to drink slightly more alcohol and perform less metabolic minutes of exercise, which are both associated with higher likelihood for infertility [6,36]. Interestingly, the mean BMI among women with and without fertility problems was consistent over the 5-year period from Survey 3 to Survey 5, with the women with infertility problems maintaining the approximate two-unit higher BMI than the women without infertility problems. The combination of some infertility risk factors increasing over time, but others remaining consistent, may partly explain the lack of association in this slightly older age group of women. Nevertheless, our results might suggest a small window of opportunity in women aged 28–31 years to optimise their diet to support fertility, whereas after this age, the current data do not support this. Although many studies have reported on the independent effects of increasing age with infertility, and increasing evidence shows the importance of a healthy diet for reproductive health, qualitative data reveals that women do not have a clear understanding of the age at which fertility begins to decline [37]. There are also no specific recommendations for an appropriate diet to optimise fertility. The knowledge gaps and misconceptions surrounding reproductive health and nutrition, highlights a critical opportunity that is needed to support patient awareness, counselling to support dietary behaviours, and that involves pregnancy planning.\nOur findings provide little evidence of effect modification by diet at either survey. Whilst at Survey 3 there was a suggestion of a negative effect measure modification, such that the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet, the wide 95% CIs are consistent with positive effect modification by diet. At Survey 5, the RERI was positive, suggesting that the effect of age interacting with diet quality was higher than the sum of the independent effects of age and diet, but the wide confidence intervals are also consistent with positive effect modification. Thus, we cannot conclude that better quality diet could attenuate the decline in fertility with age over the observed periods. These findings suggest that the effect of a poor-quality diet and older age on fertility may not be as pronounced as expected, but also that a poor diet quality is not appreciably leading to reduced fertility in older women.\nWhilst our results are unexpected, they build the foundation for further studies to assess the interactions between age and diet quality. Although the additive effect modification RERI is the more relevant public health measure, and we would theorise that the targeted group for intervention would be older women with a poor-quality diet, in this instance, we cannot confidently draw this conclusion. Importantly, our results do not mean that a good-quality diet is adverse in any way but implies that a good-quality diet does not attenuate the relationship between age and infertility, and that the joint effect of the two exposures is less than their main effects combined. The indication of a positive RERI at Survey 5 supports the promotion of improved diet quality in older women.\nOverall diet quality of women was low in both surveys, which is consistent with previous studies reporting that a poor-quality diet is common amongst women of reproductive age [23,38,39,40,41]. Despite the slight increase in proportion of women having a good-quality diet from Survey 3 to Survey 5, there was no evidence that a change in diet, whether that was an improvement or worsening of diet quality over time, was associated with fertility problems. However, we acknowledge that this change in diet quality is very small and may be equivalent to only replacing white bread with brown (whole meal or multigrain) as the usual bread choice. In line with our findings, a review of studies assessing pregnancy intentions and diet or physical activity behaviours in the preconception and antenatal periods found that women intending to become pregnant do not report different preconception dietary or physical activity behaviours compared with women without pregnancy intentions [42]. This reflects the common barriers observed in women of reproductive age to achieve successful health behaviour change [43]. In our study, the lack of association between a change in diet and fertility may also relate to reverse causation. That is, following a diagnosis of fertility problems, women may alter their behaviours towards healthier choices, or their improvement in dietary intake may not be sufficient to alter the effect on fertility problems.\nTo the best of our knowledge, this is the first study to assess whether better diet quality can reduce the age-related decline in fertility. A strength of this study is the use of data from a nationally representative cohort of Australian women at two different time points, increasing the generalisability of our findings to all Australian women. We used robust statistical measures, including calculation of the RERI, whereby effect measure modification of diet on the additive scale provides a clear comparison of the effect of diet on the association between one of the major risk factors of fertility (i.e., age) and fertility problems among women. Some limitations are also worth mentioning. A validated, semi-quantitative FFQ was used to capture women’s dietary consumption, which was designed for use in the Australian community. Although the ARFS is an acceptable tool for assessing diet quality, for most foods the scoring was based on ‘at least once per week’. Additional response options might be helpful to quantify diet quality more accurately. While the analysis included a large cohort of women, it may not be sufficiently powered to identify interactions; thus, further work in this area, with larger populations and covering wider age ranges, is warranted. Lastly, the current study included self-reported fertility problems and dietary intakes that could be subject to recall bias.","In conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age."],"string":"[\n \"Infertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].\\nIncreasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].\\nAlthough increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].\\nThere is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality.\",\n \" 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\",\n \"Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\",\n \"This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\",\n \"The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\",\n \"Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\",\n \"A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\",\n \"Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\",\n \" 3.1. Participants A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\\nA total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\\n 3.2. Effect Modification of Diet on Age and Fertility Problems Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\\nTable 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\\n 3.3. Dietary Change and Fertility Status Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\\nTable 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\",\n \"A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\",\n \"Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\",\n \"Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\",\n \"Among a large cohort of Australian women, we examined whether diet modified the relationship between age and infertility, and whether a change in diet impacts fertility. Our results do not support our hypothesis; that is, in both Survey 3 and in Survey 5, there was little evidence that diet quality modified the association between age and fertility problems. Although increasing age (from Survey 3 to Survey 5) was associated with an approximate 5% higher rate of fertility problems, a change in diet quality over this time was not associated with fertility problems at Survey 5.\\nAmong Survey 3 participants who were aged between 24–31 years, independent associations were found, such that older woman (28.2 to 30.8 years) who had a poor-quality diet had higher risk for infertility problems compared to the younger women (24.4 to <28.2 years) with a good-quality diet. At Survey 5 when women were aged 30–38 years, there was no difference in risk for fertility problems by diet quality or age. This was an unexpected finding. At Survey 5, there was an approximate 10% higher percentage of women with a good-quality diet; thus, with increasing age, the importance of a healthier diet may be realised, irrespective of fertility status. Yet, the older women at Survey 5 did appear to drink slightly more alcohol and perform less metabolic minutes of exercise, which are both associated with higher likelihood for infertility [6,36]. Interestingly, the mean BMI among women with and without fertility problems was consistent over the 5-year period from Survey 3 to Survey 5, with the women with infertility problems maintaining the approximate two-unit higher BMI than the women without infertility problems. The combination of some infertility risk factors increasing over time, but others remaining consistent, may partly explain the lack of association in this slightly older age group of women. Nevertheless, our results might suggest a small window of opportunity in women aged 28–31 years to optimise their diet to support fertility, whereas after this age, the current data do not support this. Although many studies have reported on the independent effects of increasing age with infertility, and increasing evidence shows the importance of a healthy diet for reproductive health, qualitative data reveals that women do not have a clear understanding of the age at which fertility begins to decline [37]. There are also no specific recommendations for an appropriate diet to optimise fertility. The knowledge gaps and misconceptions surrounding reproductive health and nutrition, highlights a critical opportunity that is needed to support patient awareness, counselling to support dietary behaviours, and that involves pregnancy planning.\\nOur findings provide little evidence of effect modification by diet at either survey. Whilst at Survey 3 there was a suggestion of a negative effect measure modification, such that the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet, the wide 95% CIs are consistent with positive effect modification by diet. At Survey 5, the RERI was positive, suggesting that the effect of age interacting with diet quality was higher than the sum of the independent effects of age and diet, but the wide confidence intervals are also consistent with positive effect modification. Thus, we cannot conclude that better quality diet could attenuate the decline in fertility with age over the observed periods. These findings suggest that the effect of a poor-quality diet and older age on fertility may not be as pronounced as expected, but also that a poor diet quality is not appreciably leading to reduced fertility in older women.\\nWhilst our results are unexpected, they build the foundation for further studies to assess the interactions between age and diet quality. Although the additive effect modification RERI is the more relevant public health measure, and we would theorise that the targeted group for intervention would be older women with a poor-quality diet, in this instance, we cannot confidently draw this conclusion. Importantly, our results do not mean that a good-quality diet is adverse in any way but implies that a good-quality diet does not attenuate the relationship between age and infertility, and that the joint effect of the two exposures is less than their main effects combined. The indication of a positive RERI at Survey 5 supports the promotion of improved diet quality in older women.\\nOverall diet quality of women was low in both surveys, which is consistent with previous studies reporting that a poor-quality diet is common amongst women of reproductive age [23,38,39,40,41]. Despite the slight increase in proportion of women having a good-quality diet from Survey 3 to Survey 5, there was no evidence that a change in diet, whether that was an improvement or worsening of diet quality over time, was associated with fertility problems. However, we acknowledge that this change in diet quality is very small and may be equivalent to only replacing white bread with brown (whole meal or multigrain) as the usual bread choice. In line with our findings, a review of studies assessing pregnancy intentions and diet or physical activity behaviours in the preconception and antenatal periods found that women intending to become pregnant do not report different preconception dietary or physical activity behaviours compared with women without pregnancy intentions [42]. This reflects the common barriers observed in women of reproductive age to achieve successful health behaviour change [43]. In our study, the lack of association between a change in diet and fertility may also relate to reverse causation. That is, following a diagnosis of fertility problems, women may alter their behaviours towards healthier choices, or their improvement in dietary intake may not be sufficient to alter the effect on fertility problems.\\nTo the best of our knowledge, this is the first study to assess whether better diet quality can reduce the age-related decline in fertility. A strength of this study is the use of data from a nationally representative cohort of Australian women at two different time points, increasing the generalisability of our findings to all Australian women. We used robust statistical measures, including calculation of the RERI, whereby effect measure modification of diet on the additive scale provides a clear comparison of the effect of diet on the association between one of the major risk factors of fertility (i.e., age) and fertility problems among women. Some limitations are also worth mentioning. A validated, semi-quantitative FFQ was used to capture women’s dietary consumption, which was designed for use in the Australian community. Although the ARFS is an acceptable tool for assessing diet quality, for most foods the scoring was based on ‘at least once per week’. Additional response options might be helpful to quantify diet quality more accurately. While the analysis included a large cohort of women, it may not be sufficiently powered to identify interactions; thus, further work in this area, with larger populations and covering wider age ranges, is warranted. Lastly, the current study included self-reported fertility problems and dietary intakes that could be subject to recall bias.\",\n \"In conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,null,null,null,null,null,null,"results","subjects",null,null,"discussion","conclusions"],"string":"[\n \"intro\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"subjects\",\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["age","diet quality","reproductive age","Australia","survey","infertility","women"],"string":"[\n \"age\",\n \"diet quality\",\n \"reproductive age\",\n \"Australia\",\n \"survey\",\n \"infertility\",\n \"women\"\n]"},"whole_article_text":{"kind":"string","value":"1. Introduction:\nInfertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].\nIncreasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].\nAlthough increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].\nThere is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality.\n\n2. Materials and Methods:\n 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\n\n2.1. Study Design:\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\n\n2.2. Study Population:\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\n\n2.3. Exposure Variables:\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\n\n2.4. Outcome Variable:\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\n\n2.5. Confounding Variables:\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\n\n2.6. Statistical Analysis:\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\n\n3. Results:\n 3.1. Participants A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\nA total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\n 3.2. Effect Modification of Diet on Age and Fertility Problems Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\nTable 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\n 3.3. Dietary Change and Fertility Status Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\nTable 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\n\n3.1. Participants:\nA total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\n\n3.2. Effect Modification of Diet on Age and Fertility Problems:\nTable 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\n\n3.3. Dietary Change and Fertility Status:\nTable 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\n\n4. Discussion:\nAmong a large cohort of Australian women, we examined whether diet modified the relationship between age and infertility, and whether a change in diet impacts fertility. Our results do not support our hypothesis; that is, in both Survey 3 and in Survey 5, there was little evidence that diet quality modified the association between age and fertility problems. Although increasing age (from Survey 3 to Survey 5) was associated with an approximate 5% higher rate of fertility problems, a change in diet quality over this time was not associated with fertility problems at Survey 5.\nAmong Survey 3 participants who were aged between 24–31 years, independent associations were found, such that older woman (28.2 to 30.8 years) who had a poor-quality diet had higher risk for infertility problems compared to the younger women (24.4 to <28.2 years) with a good-quality diet. At Survey 5 when women were aged 30–38 years, there was no difference in risk for fertility problems by diet quality or age. This was an unexpected finding. At Survey 5, there was an approximate 10% higher percentage of women with a good-quality diet; thus, with increasing age, the importance of a healthier diet may be realised, irrespective of fertility status. Yet, the older women at Survey 5 did appear to drink slightly more alcohol and perform less metabolic minutes of exercise, which are both associated with higher likelihood for infertility [6,36]. Interestingly, the mean BMI among women with and without fertility problems was consistent over the 5-year period from Survey 3 to Survey 5, with the women with infertility problems maintaining the approximate two-unit higher BMI than the women without infertility problems. The combination of some infertility risk factors increasing over time, but others remaining consistent, may partly explain the lack of association in this slightly older age group of women. Nevertheless, our results might suggest a small window of opportunity in women aged 28–31 years to optimise their diet to support fertility, whereas after this age, the current data do not support this. Although many studies have reported on the independent effects of increasing age with infertility, and increasing evidence shows the importance of a healthy diet for reproductive health, qualitative data reveals that women do not have a clear understanding of the age at which fertility begins to decline [37]. There are also no specific recommendations for an appropriate diet to optimise fertility. The knowledge gaps and misconceptions surrounding reproductive health and nutrition, highlights a critical opportunity that is needed to support patient awareness, counselling to support dietary behaviours, and that involves pregnancy planning.\nOur findings provide little evidence of effect modification by diet at either survey. Whilst at Survey 3 there was a suggestion of a negative effect measure modification, such that the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet, the wide 95% CIs are consistent with positive effect modification by diet. At Survey 5, the RERI was positive, suggesting that the effect of age interacting with diet quality was higher than the sum of the independent effects of age and diet, but the wide confidence intervals are also consistent with positive effect modification. Thus, we cannot conclude that better quality diet could attenuate the decline in fertility with age over the observed periods. These findings suggest that the effect of a poor-quality diet and older age on fertility may not be as pronounced as expected, but also that a poor diet quality is not appreciably leading to reduced fertility in older women.\nWhilst our results are unexpected, they build the foundation for further studies to assess the interactions between age and diet quality. Although the additive effect modification RERI is the more relevant public health measure, and we would theorise that the targeted group for intervention would be older women with a poor-quality diet, in this instance, we cannot confidently draw this conclusion. Importantly, our results do not mean that a good-quality diet is adverse in any way but implies that a good-quality diet does not attenuate the relationship between age and infertility, and that the joint effect of the two exposures is less than their main effects combined. The indication of a positive RERI at Survey 5 supports the promotion of improved diet quality in older women.\nOverall diet quality of women was low in both surveys, which is consistent with previous studies reporting that a poor-quality diet is common amongst women of reproductive age [23,38,39,40,41]. Despite the slight increase in proportion of women having a good-quality diet from Survey 3 to Survey 5, there was no evidence that a change in diet, whether that was an improvement or worsening of diet quality over time, was associated with fertility problems. However, we acknowledge that this change in diet quality is very small and may be equivalent to only replacing white bread with brown (whole meal or multigrain) as the usual bread choice. In line with our findings, a review of studies assessing pregnancy intentions and diet or physical activity behaviours in the preconception and antenatal periods found that women intending to become pregnant do not report different preconception dietary or physical activity behaviours compared with women without pregnancy intentions [42]. This reflects the common barriers observed in women of reproductive age to achieve successful health behaviour change [43]. In our study, the lack of association between a change in diet and fertility may also relate to reverse causation. That is, following a diagnosis of fertility problems, women may alter their behaviours towards healthier choices, or their improvement in dietary intake may not be sufficient to alter the effect on fertility problems.\nTo the best of our knowledge, this is the first study to assess whether better diet quality can reduce the age-related decline in fertility. A strength of this study is the use of data from a nationally representative cohort of Australian women at two different time points, increasing the generalisability of our findings to all Australian women. We used robust statistical measures, including calculation of the RERI, whereby effect measure modification of diet on the additive scale provides a clear comparison of the effect of diet on the association between one of the major risk factors of fertility (i.e., age) and fertility problems among women. Some limitations are also worth mentioning. A validated, semi-quantitative FFQ was used to capture women’s dietary consumption, which was designed for use in the Australian community. Although the ARFS is an acceptable tool for assessing diet quality, for most foods the scoring was based on ‘at least once per week’. Additional response options might be helpful to quantify diet quality more accurately. While the analysis included a large cohort of women, it may not be sufficiently powered to identify interactions; thus, further work in this area, with larger populations and covering wider age ranges, is warranted. Lastly, the current study included self-reported fertility problems and dietary intakes that could be subject to recall bias.\n\n5. Conclusions:\nIn conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIncreasing age is a strong risk factor for infertility, and there is accumulating evidence of the importance of a healthier diet for fertility. Whether a healthier diet modifies the association between increasing age and infertility has not been investigated. This study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems.\n\nMethods:\nData were from Surveys 3 and 5 of the 1973-1978 birth cohort of the Australian Longitudinal Study on Women's Health. Cross-sectional analysis with multivariable generalized linear models were used to examine the association between age and fertility status, adjusted for various confounders. Multiplicative and additive effect modification by diet quality was assessed, with additive effect modification evaluated with the relative risk for interaction (RERI).\n\nResults:\nIn total, 3387 women were included from Survey 3 (age range 24-31 years) and 5614 women from Survey 5 (age range 30-38 years); 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems in the respective surveys. In Survey 3, compared to younger women with a good-quality diet, older women with a poor-quality diet had a 43% increased risk for fertility problems, with risk increasing after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62). In Survey 5 in younger women (<33.9 years), there was no association between diet quality and risk for infertility problems. The RERI (across different adjusted models) was between -0.08 (-0.70, 0.55) to -0.39 (-1.40, 0.62) in survey 3 and 0.07 (-0.17, 0.31) to 0.08 (-0.17, 0.32) in Survey 5.\n\nConclusions:\nThere is little evidence to suggest effect modification on the effect of age and fertility problems with diet quality."},"background_conclusion_text":{"kind":"string","value":"1. Introduction:\nInfertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].\nIncreasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].\nAlthough increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].\nThere is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality.\n\n5. Conclusions:\nIn conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nIncreasing age is a strong risk factor for infertility, and there is accumulating evidence of the importance of a healthier diet for fertility. Whether a healthier diet modifies the association between increasing age and infertility has not been investigated. This study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems.\n\nMethods:\nData were from Surveys 3 and 5 of the 1973-1978 birth cohort of the Australian Longitudinal Study on Women's Health. Cross-sectional analysis with multivariable generalized linear models were used to examine the association between age and fertility status, adjusted for various confounders. Multiplicative and additive effect modification by diet quality was assessed, with additive effect modification evaluated with the relative risk for interaction (RERI).\n\nResults:\nIn total, 3387 women were included from Survey 3 (age range 24-31 years) and 5614 women from Survey 5 (age range 30-38 years); 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems in the respective surveys. In Survey 3, compared to younger women with a good-quality diet, older women with a poor-quality diet had a 43% increased risk for fertility problems, with risk increasing after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62). In Survey 5 in younger women (<33.9 years), there was no association between diet quality and risk for infertility problems. The RERI (across different adjusted models) was between -0.08 (-0.70, 0.55) to -0.39 (-1.40, 0.62) in survey 3 and 0.07 (-0.17, 0.31) to 0.08 (-0.17, 0.32) in Survey 5.\n\nConclusions:\nThere is little evidence to suggest effect modification on the effect of age and fertility problems with diet quality."},"whole_article_text_length":{"kind":"number","value":9634,"string":"9,634"},"whole_article_abstract_length":{"kind":"number","value":386,"string":"386"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["diet","survey","women","fertility","quality","age","effect","problems","years","fertility problems"],"string":"[\n \"diet\",\n \"survey\",\n \"women\",\n \"fertility\",\n \"quality\",\n \"age\",\n \"effect\",\n \"problems\",\n \"years\",\n \"fertility problems\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] age | infertility | women | fertility | factor | diet | pregnancy | study | years | associated [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | women | fertility | fertility problems | survey | quality | effect | quality diet | problems | table [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] age | diet | findings | helpful | diet quality older | quality older | age range | older | older age | quality [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 3387 | 3 | 24-31 years | 5614 | Survey 5 | 30-38 years | 588 | 17.4% | 1321 | 23.4% ||| Survey 3 | 43% | BMI | 1.59 | 95% | CI | 1.07 | 2.37 | PCOS (RR: 1.74 | 95% | CI | 1.15 | 2.62 ||| Survey 5 ||| -0.70 | 0.55 | 0.62 | 3 | 0.31 | 0.08 | 0.32 | Survey 5 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| Surveys 3 and 5 | 1973-1978 | the Australian Longitudinal Study on Women's Health ||| linear ||| ||| ||| 3387 | 3 | 24-31 years | 5614 | Survey 5 | 30-38 years | 588 | 17.4% | 1321 | 23.4% ||| Survey 3 | 43% | BMI | 1.59 | 95% | CI | 1.07 | 2.37 | PCOS (RR: 1.74 | 95% | CI | 1.15 | 2.62 ||| Survey 5 ||| -0.70 | 0.55 | 0.62 | 3 | 0.31 | 0.08 | 0.32 | Survey 5 [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| Surveys 3 and 5 | 1973-1978 | the Australian Longitudinal Study on Women's Health ||| linear ||| ||| ||| 3387 | 3 | 24-31 years | 5614 | Survey 5 | 30-38 years | 588 | 17.4% | 1321 | 23.4% ||| Survey 3 | 43% | BMI | 1.59 | 95% | CI | 1.07 | 2.37 | PCOS (RR: 1.74 | 95% | CI | 1.15 | 2.62 ||| Survey 5 ||| -0.70 | 0.55 | 0.62 | 3 | 0.31 | 0.08 | 0.32 | Survey 5 [SUMMARY]"}}},{"rowIdx":297,"cells":{"title":{"kind":"string","value":"The I405V and Taq1B polymorphisms of the CETP gene differentially affect sub-clinical carotid atherosclerosis."},"pmid":{"kind":"string","value":"23039379"},"background_abstract":{"kind":"string","value":"Cholesteryl ester transfer protein (CETP) plays a major role in lipid metabolism, but studies on the association of CETP polymorphisms with risks of cardiovascular disease are inconsistent. This study investigated whether the CETP gene I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian population sample."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"The polymorphisms were analyzed using polymerase chain reaction in 207 adult volunteers. Serum lipid profiles, oxLDL Ab titers, C-reactive protein and tumor necrosis factor-α concentrations and CETP and phospholipid transfer protein (PLTP) activities were determined, and common carotid artery intima-media thickness (cIMT) was measured using ultrasonography."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"No differences in cIMT were observed between the presence or absence of the minor B2 and V alleles in either polymorphism. However, inverse correlations between mean cIMT and CETP activity in the presence of these polymorphisms were observed, and positive correlations of these polymorphisms with PLTP activity and oxLDL Ab titers were identified. Moreover, logistic multivariate analysis revealed that the presence of the B2 allele was associated with a 5.1-fold (CI 95%, OR: 1.26 - 21.06) increased risk for cIMT, which was equal and above the 66th percentile and positively interacted with age. However, no associations with the V allele or CETP and PLTP activities were observed."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"None of the studied parameters, including CETP activity, explained the different relationships between these polymorphisms and cIMT, suggesting that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Autoantibodies","Brazil","C-Reactive Protein","Carotid Artery Diseases","Carotid Intima-Media Thickness","Cholesterol Ester Transfer Proteins","Female","Gene Frequency","Humans","Lipids","Lipoproteins, LDL","Male","Middle Aged","Phospholipid Transfer Proteins","Polymorphism, Genetic","Tumor Necrosis Factor-alpha","Young Adult"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Autoantibodies\",\n \"Brazil\",\n \"C-Reactive Protein\",\n \"Carotid Artery Diseases\",\n \"Carotid Intima-Media Thickness\",\n \"Cholesterol Ester Transfer Proteins\",\n \"Female\",\n \"Gene Frequency\",\n \"Humans\",\n \"Lipids\",\n \"Lipoproteins, LDL\",\n \"Male\",\n \"Middle Aged\",\n \"Phospholipid Transfer Proteins\",\n \"Polymorphism, Genetic\",\n \"Tumor Necrosis Factor-alpha\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"3503625"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Study subjects A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nA total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\n Polymorphism detection Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\nGenomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\n Measurement of cIMT A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\nA single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\n Statistical analysis Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].\nMann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15]."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The frequencies of I405V and Taq1B genotypes in our population sample, represented by the presence (VV, IV) and absence (II) of the V allele and the presence (B2B2, B1B2) and absence (B1B1) of the B2 allele, are presented in Table \n1. The frequencies of the II, IV and VV genotypes were 28%, 53% and 19%, respectively, and the frequencies of the B1B1, B1B2 and B2B2 were 33%, 41% and 26%, respectively.\nBiochemical parameters in the presence and absence of minor alleles\nData are presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values: Mann–Whitney U test for comparisons between the presence and absence of the minor alleles (V and B2). Significant differences: p≤0.05 in bold. CETP (%) = activity of cholesterol ester transfer protein; HDL-C = high-density lipoprotein cholesterol; PLTP (%) = activity of phospholipid transfer protein; oxLDL Ab = antibodies against oxidized LDL titers; (OD)= optical density.\nAllele frequencies were consistent with Hardy–Weinberg equilibrium for the I405V polymorphism (p=0.36) but not the Taq1B (p=0.02), and the MAFs (minor allele frequency) were <45% and <46%, respectively. The linkage disequilibrium between the two polymorphisms was very weak, as evidenced by the D`=0.17.\nClinical and anthropometric characteristics and biochemical data of the investigated population are presented in Tables \n2 and\n1.\nClinical and anthropometric characteristics in the presence and absence of minor alleles\nData presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values = Mann–Whitney U test for comparisons between the presence and absence of the alleles (V and B2); gender comparisons, Chi-Square test. Significant differences: p≤0.05 in bold. BMI= Body Mass Index; WC= waist circumference; SBP= systolic blood pressure; DBP= diastolic blood pressure; cIMT= carotid intima-media thickness.\nTable \n3 presents the significant correlations between mean cIMT, oxLDL Ab titers and CETP and PLTP activities.\nSignificant correlations between cIMT, oxLDL Ab, CETP (%) and PLTP (%) in the presence of minor alleles\nr= Spearman´s coefficient; Significant differences: p≤0.05 in bold. cIMT= carotid intima-media thickness; oxLDL Ab= antibodies against oxidized LDL titers; (OD) = optical density; CETP(%) = activity of cholesteryl ester transfer protein; PLTP (%) = activity of phospholipid transfer protein.\nThe univariate analyses (Table \n4) indicated that the female gender, age, HDL-C, BMI and SBP were significantly associated (p value ≤0.05) with higher cIMT.\n\nUnivariate Logistic Regression analyses for higher cIMT (above 66 percentile) (\n\nn=\n\n118)\n\ncIMT below and above the 66th percentile, <0.795 (n=76) and >0.795 (n=42), respectively. (ref).=reference level. (cont).= continue variable. OR= Odds ratio for higher cIMT, above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for the Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism, V = minor allele of I405V polymorphism, HDL-C = high-density lipoprotein cholesterol, BMI: body mass index, SBP: systolic blood pressure.\nThe multivariate analysis (Table \n5) demonstrated that age and the presence of the B2 allele were positively related to higher cIMT. Individuals with increased risk exhibited the B2 allele (5.1-fold increase) and higher age (21.2% increase/each year of age).\n\nMultivariate Logistic Regression* analyses for higher cIMT (above 66\n\nth\n\npercentile) (\n\nn=\n\n114)\n\n*Stepwise criteria for selection of variables. cIMT below and above 66th percentile, <0.795 (n=73) and >0.795 (n=41), respectively. (ref).= reference level; (cont).=continue variable. OR= Odds ratio for higher cIMT above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism.\nA Chi-Square analysis indicated an interaction between the presence of the B2 allele and age. The combination between age, categorized in ≥ and <50 years (median value), and the presence or absence B2 allele formed 4 groups of individuals who were crossed with cIMTs above and below 0.795 mm (data not shown)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved."},"other_sections_titles":{"kind":"list like","value":["Background","Study subjects","Biochemical analysis","Polymorphism detection","Measurement of cIMT","Statistical analysis","Abbreviations","Misc","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Study subjects\",\n \"Biochemical analysis\",\n \"Polymorphism detection\",\n \"Measurement of cIMT\",\n \"Statistical analysis\",\n \"Abbreviations\",\n \"Misc\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.","A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].","The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].","Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.","A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.","Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].","CETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.","Eliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.","The authors state that they have no conflicts of interest.","ESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript."],"string":"[\n \"Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\\n[3].\\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\\n[5], and their association with subclinical CVD are less well characterized.\\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.\",\n \"A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\",\n \"The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\",\n \"Genomic DNA was extracted\\n[11] and amplified using polymerase chain reaction to identify the Taq1B\\n[12] and I405V\\n[13] polymorphisms.\",\n \"A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\",\n \"Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\\n[15].\",\n \"CETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.\",\n \"Eliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.\",\n \"The authors state that they have no conflicts of interest.\",\n \"ESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study subjects","Biochemical analysis","Polymorphism detection","Measurement of cIMT","Statistical analysis","Results","Discussion","Conclusions","Abbreviations","Misc","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study subjects\",\n \"Biochemical analysis\",\n \"Polymorphism detection\",\n \"Measurement of cIMT\",\n \"Statistical analysis\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Misc\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population."," Study subjects A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nA total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\n Polymorphism detection Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\nGenomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\n Measurement of cIMT A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\nA single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\n Statistical analysis Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].\nMann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].","A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].","The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].","Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.","A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.","Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].","The frequencies of I405V and Taq1B genotypes in our population sample, represented by the presence (VV, IV) and absence (II) of the V allele and the presence (B2B2, B1B2) and absence (B1B1) of the B2 allele, are presented in Table \n1. The frequencies of the II, IV and VV genotypes were 28%, 53% and 19%, respectively, and the frequencies of the B1B1, B1B2 and B2B2 were 33%, 41% and 26%, respectively.\nBiochemical parameters in the presence and absence of minor alleles\nData are presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values: Mann–Whitney U test for comparisons between the presence and absence of the minor alleles (V and B2). Significant differences: p≤0.05 in bold. CETP (%) = activity of cholesterol ester transfer protein; HDL-C = high-density lipoprotein cholesterol; PLTP (%) = activity of phospholipid transfer protein; oxLDL Ab = antibodies against oxidized LDL titers; (OD)= optical density.\nAllele frequencies were consistent with Hardy–Weinberg equilibrium for the I405V polymorphism (p=0.36) but not the Taq1B (p=0.02), and the MAFs (minor allele frequency) were <45% and <46%, respectively. The linkage disequilibrium between the two polymorphisms was very weak, as evidenced by the D`=0.17.\nClinical and anthropometric characteristics and biochemical data of the investigated population are presented in Tables \n2 and\n1.\nClinical and anthropometric characteristics in the presence and absence of minor alleles\nData presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values = Mann–Whitney U test for comparisons between the presence and absence of the alleles (V and B2); gender comparisons, Chi-Square test. Significant differences: p≤0.05 in bold. BMI= Body Mass Index; WC= waist circumference; SBP= systolic blood pressure; DBP= diastolic blood pressure; cIMT= carotid intima-media thickness.\nTable \n3 presents the significant correlations between mean cIMT, oxLDL Ab titers and CETP and PLTP activities.\nSignificant correlations between cIMT, oxLDL Ab, CETP (%) and PLTP (%) in the presence of minor alleles\nr= Spearman´s coefficient; Significant differences: p≤0.05 in bold. cIMT= carotid intima-media thickness; oxLDL Ab= antibodies against oxidized LDL titers; (OD) = optical density; CETP(%) = activity of cholesteryl ester transfer protein; PLTP (%) = activity of phospholipid transfer protein.\nThe univariate analyses (Table \n4) indicated that the female gender, age, HDL-C, BMI and SBP were significantly associated (p value ≤0.05) with higher cIMT.\n\nUnivariate Logistic Regression analyses for higher cIMT (above 66 percentile) (\n\nn=\n\n118)\n\ncIMT below and above the 66th percentile, <0.795 (n=76) and >0.795 (n=42), respectively. (ref).=reference level. (cont).= continue variable. OR= Odds ratio for higher cIMT, above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for the Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism, V = minor allele of I405V polymorphism, HDL-C = high-density lipoprotein cholesterol, BMI: body mass index, SBP: systolic blood pressure.\nThe multivariate analysis (Table \n5) demonstrated that age and the presence of the B2 allele were positively related to higher cIMT. Individuals with increased risk exhibited the B2 allele (5.1-fold increase) and higher age (21.2% increase/each year of age).\n\nMultivariate Logistic Regression* analyses for higher cIMT (above 66\n\nth\n\npercentile) (\n\nn=\n\n114)\n\n*Stepwise criteria for selection of variables. cIMT below and above 66th percentile, <0.795 (n=73) and >0.795 (n=41), respectively. (ref).= reference level; (cont).=continue variable. OR= Odds ratio for higher cIMT above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism.\nA Chi-Square analysis indicated an interaction between the presence of the B2 allele and age. The combination between age, categorized in ≥ and <50 years (median value), and the presence or absence B2 allele formed 4 groups of individuals who were crossed with cIMTs above and below 0.795 mm (data not shown).","This study investigated whether the I405V and Taq1B polymorphisms modify subclinical atherosclerosis in an asymptomatic population sample.\nPrevious studies present controversial data on the role of Taq1B and I405V in atherosclerosis and CVD. The B2 allele is associated with lower internal carotid artery IMT in men\n[16], but other studies do not identify associations between the Taq1B polymorphism and cIMT\n[17,18]. The present study demonstrated that cIMT did not differ between either allele of the polymorphisms. However, inverse correlations between cIMT and CETP activity were demonstrated in both polymorphisms (Table \n3), which suggest a potential atheroprotective role for CETP in these individuals. A potential anti-inflammatory role for CETP was demonstrated previously\n[19] wherein the induction of acute inflammation produced lower mortality due to sepsis and lower cytokine circulation in mice that express human CETP. Reductions in CETP concentrations in human patients with sepsis are more pronounced in non-survivors\n[20].\nWe identified positive correlations between cIMT and PLTP and oxLDL Ab in the presence of the V and B2 alleles. Previous studies have suggested that PLTP increases the risk of cardiovascular disease in humans\n[21], and the balance of evidence suggests a pro-atherogenic role for oxLDL Ab titers\n[22] in the development of atherosclerosis. These positive correlations and the lack of correlations of these variables in both common B1B1 and II allele homozygotes (CETP p≤0.318 and 0.091, oxLDL Ab p≤ 0.709 and 0.386 and PLTP p≤0.823 and 0.470, respectively) (data not shown) suggest a possible pro-atherogenic role of these markers associated with the polymorphisms. Our sample size was relatively small and different between groups. Therefore, further studies are underway in our lab to further confirm these correlations.\nThe presence of the V allele was associated with lower CETP (24%) and PLTP (29%) activities and higher HDL2-C (13%) concentrations. Patients with coronary disease typically exhibit fewer large HDL particles (HDL2), and the concentration of these large particles is a better predictor of future cardiovascular events than HDL-C concentration\n[23].\nLipoprotein (a) plasma levels were also significantly increased in the presence of the B2 allele. High levels of lipoprotein (a) are significantly associated with the presence, severity and extent of carotid atherosclerosis\n[24], but this increase was not observed as a possible pro-atherogenic factor.\nNo significant differences in CETP activity and HDL-C levels were observed in Taq1B polymorphism, despite a 15.4% reduction in CETP activity in presence of the B2 allele. These results are consistent with a previous study in another Brazilian population, in which no association between Taq1B genotypes and HDL-C levels was observed\n[25].\nA univariate analysis (Table \n4) followed by a multivariate logistic stepwise analysis (Table \n5) demonstrated that the presence of the B2 allele was independently associated with a mean 5.1-fold increased risk for higher cIMT and age interacted with the allele effect. The presence of the B2 allele in the ≥ 50 years age group exhibited the highest risk of higher cIMT in the Chi-Square analysis (data not shown).\nThe effects of the Taq1B variant on circulating plasma lipids may be related to other functional mutations within the CETP gene locus that are in LD\n[26], which may explain the association in our study. However, other factors than the CETP activity may exert different atherosclerotic repercussions in Taq1B and I405V polymorphisms. A multivariate analysis was performed to verify the independence of the associations between the qualitative binary variable cIMT above or below 66th percentile (0.795mm), CETP and PLTP plasma activities and the potential confounding factors, including gender, age, BMI, SBP and HDL-C. Only age exhibited a significant statistical association (OR: 1.247, CI 95%OR: 1.129 – 1.377), which was a strong determinant of cIMT (data not shown). The absence of interactions of other tested parameters with higher cIMT in this study suggests that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.\nOur results focused on several biomarkers that were assessed for the first time in the Brazilian population, and the results were partially based on the correlations between a relatively small numbers of individuals. These results merit further exploration due to the high frequency of the investigated polymorphisms in the present Brazilian sample. Identifying the impact of these polymorphisms is an ongoing aim of our research group.","The I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved.","CETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.","Eliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.","The authors state that they have no conflicts of interest.","ESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript."],"string":"[\n \"Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\\n[3].\\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\\n[5], and their association with subclinical CVD are less well characterized.\\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.\",\n \" Study subjects A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\nA total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\n Polymorphism detection Genomic DNA was extracted\\n[11] and amplified using polymerase chain reaction to identify the Taq1B\\n[12] and I405V\\n[13] polymorphisms.\\nGenomic DNA was extracted\\n[11] and amplified using polymerase chain reaction to identify the Taq1B\\n[12] and I405V\\n[13] polymorphisms.\\n Measurement of cIMT A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\\nA single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\\n Statistical analysis Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\\n[15].\\nMann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\\n[15].\",\n \"A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\",\n \"The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\\n[7].\\nCETP\\n[8] and phospholipid transfer protein (PLTP) activities\\n[9] in plasma were determined using radioassays with exogenous substrates.\\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\\n[10].\",\n \"Genomic DNA was extracted\\n[11] and amplified using polymerase chain reaction to identify the Taq1B\\n[12] and I405V\\n[13] polymorphisms.\",\n \"A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\",\n \"Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\\n[15].\",\n \"The frequencies of I405V and Taq1B genotypes in our population sample, represented by the presence (VV, IV) and absence (II) of the V allele and the presence (B2B2, B1B2) and absence (B1B1) of the B2 allele, are presented in Table \\n1. The frequencies of the II, IV and VV genotypes were 28%, 53% and 19%, respectively, and the frequencies of the B1B1, B1B2 and B2B2 were 33%, 41% and 26%, respectively.\\nBiochemical parameters in the presence and absence of minor alleles\\nData are presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values: Mann–Whitney U test for comparisons between the presence and absence of the minor alleles (V and B2). Significant differences: p≤0.05 in bold. CETP (%) = activity of cholesterol ester transfer protein; HDL-C = high-density lipoprotein cholesterol; PLTP (%) = activity of phospholipid transfer protein; oxLDL Ab = antibodies against oxidized LDL titers; (OD)= optical density.\\nAllele frequencies were consistent with Hardy–Weinberg equilibrium for the I405V polymorphism (p=0.36) but not the Taq1B (p=0.02), and the MAFs (minor allele frequency) were <45% and <46%, respectively. The linkage disequilibrium between the two polymorphisms was very weak, as evidenced by the D`=0.17.\\nClinical and anthropometric characteristics and biochemical data of the investigated population are presented in Tables \\n2 and\\n1.\\nClinical and anthropometric characteristics in the presence and absence of minor alleles\\nData presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values = Mann–Whitney U test for comparisons between the presence and absence of the alleles (V and B2); gender comparisons, Chi-Square test. Significant differences: p≤0.05 in bold. BMI= Body Mass Index; WC= waist circumference; SBP= systolic blood pressure; DBP= diastolic blood pressure; cIMT= carotid intima-media thickness.\\nTable \\n3 presents the significant correlations between mean cIMT, oxLDL Ab titers and CETP and PLTP activities.\\nSignificant correlations between cIMT, oxLDL Ab, CETP (%) and PLTP (%) in the presence of minor alleles\\nr= Spearman´s coefficient; Significant differences: p≤0.05 in bold. cIMT= carotid intima-media thickness; oxLDL Ab= antibodies against oxidized LDL titers; (OD) = optical density; CETP(%) = activity of cholesteryl ester transfer protein; PLTP (%) = activity of phospholipid transfer protein.\\nThe univariate analyses (Table \\n4) indicated that the female gender, age, HDL-C, BMI and SBP were significantly associated (p value ≤0.05) with higher cIMT.\\n\\nUnivariate Logistic Regression analyses for higher cIMT (above 66 percentile) (\\n\\nn=\\n\\n118)\\n\\ncIMT below and above the 66th percentile, <0.795 (n=76) and >0.795 (n=42), respectively. (ref).=reference level. (cont).= continue variable. OR= Odds ratio for higher cIMT, above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for the Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism, V = minor allele of I405V polymorphism, HDL-C = high-density lipoprotein cholesterol, BMI: body mass index, SBP: systolic blood pressure.\\nThe multivariate analysis (Table \\n5) demonstrated that age and the presence of the B2 allele were positively related to higher cIMT. Individuals with increased risk exhibited the B2 allele (5.1-fold increase) and higher age (21.2% increase/each year of age).\\n\\nMultivariate Logistic Regression* analyses for higher cIMT (above 66\\n\\nth\\n\\npercentile) (\\n\\nn=\\n\\n114)\\n\\n*Stepwise criteria for selection of variables. cIMT below and above 66th percentile, <0.795 (n=73) and >0.795 (n=41), respectively. (ref).= reference level; (cont).=continue variable. OR= Odds ratio for higher cIMT above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism.\\nA Chi-Square analysis indicated an interaction between the presence of the B2 allele and age. The combination between age, categorized in ≥ and <50 years (median value), and the presence or absence B2 allele formed 4 groups of individuals who were crossed with cIMTs above and below 0.795 mm (data not shown).\",\n \"This study investigated whether the I405V and Taq1B polymorphisms modify subclinical atherosclerosis in an asymptomatic population sample.\\nPrevious studies present controversial data on the role of Taq1B and I405V in atherosclerosis and CVD. The B2 allele is associated with lower internal carotid artery IMT in men\\n[16], but other studies do not identify associations between the Taq1B polymorphism and cIMT\\n[17,18]. The present study demonstrated that cIMT did not differ between either allele of the polymorphisms. However, inverse correlations between cIMT and CETP activity were demonstrated in both polymorphisms (Table \\n3), which suggest a potential atheroprotective role for CETP in these individuals. A potential anti-inflammatory role for CETP was demonstrated previously\\n[19] wherein the induction of acute inflammation produced lower mortality due to sepsis and lower cytokine circulation in mice that express human CETP. Reductions in CETP concentrations in human patients with sepsis are more pronounced in non-survivors\\n[20].\\nWe identified positive correlations between cIMT and PLTP and oxLDL Ab in the presence of the V and B2 alleles. Previous studies have suggested that PLTP increases the risk of cardiovascular disease in humans\\n[21], and the balance of evidence suggests a pro-atherogenic role for oxLDL Ab titers\\n[22] in the development of atherosclerosis. These positive correlations and the lack of correlations of these variables in both common B1B1 and II allele homozygotes (CETP p≤0.318 and 0.091, oxLDL Ab p≤ 0.709 and 0.386 and PLTP p≤0.823 and 0.470, respectively) (data not shown) suggest a possible pro-atherogenic role of these markers associated with the polymorphisms. Our sample size was relatively small and different between groups. Therefore, further studies are underway in our lab to further confirm these correlations.\\nThe presence of the V allele was associated with lower CETP (24%) and PLTP (29%) activities and higher HDL2-C (13%) concentrations. Patients with coronary disease typically exhibit fewer large HDL particles (HDL2), and the concentration of these large particles is a better predictor of future cardiovascular events than HDL-C concentration\\n[23].\\nLipoprotein (a) plasma levels were also significantly increased in the presence of the B2 allele. High levels of lipoprotein (a) are significantly associated with the presence, severity and extent of carotid atherosclerosis\\n[24], but this increase was not observed as a possible pro-atherogenic factor.\\nNo significant differences in CETP activity and HDL-C levels were observed in Taq1B polymorphism, despite a 15.4% reduction in CETP activity in presence of the B2 allele. These results are consistent with a previous study in another Brazilian population, in which no association between Taq1B genotypes and HDL-C levels was observed\\n[25].\\nA univariate analysis (Table \\n4) followed by a multivariate logistic stepwise analysis (Table \\n5) demonstrated that the presence of the B2 allele was independently associated with a mean 5.1-fold increased risk for higher cIMT and age interacted with the allele effect. The presence of the B2 allele in the ≥ 50 years age group exhibited the highest risk of higher cIMT in the Chi-Square analysis (data not shown).\\nThe effects of the Taq1B variant on circulating plasma lipids may be related to other functional mutations within the CETP gene locus that are in LD\\n[26], which may explain the association in our study. However, other factors than the CETP activity may exert different atherosclerotic repercussions in Taq1B and I405V polymorphisms. A multivariate analysis was performed to verify the independence of the associations between the qualitative binary variable cIMT above or below 66th percentile (0.795mm), CETP and PLTP plasma activities and the potential confounding factors, including gender, age, BMI, SBP and HDL-C. Only age exhibited a significant statistical association (OR: 1.247, CI 95%OR: 1.129 – 1.377), which was a strong determinant of cIMT (data not shown). The absence of interactions of other tested parameters with higher cIMT in this study suggests that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.\\nOur results focused on several biomarkers that were assessed for the first time in the Brazilian population, and the results were partially based on the correlations between a relatively small numbers of individuals. These results merit further exploration due to the high frequency of the investigated polymorphisms in the present Brazilian sample. Identifying the impact of these polymorphisms is an ongoing aim of our research group.\",\n \"The I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved.\",\n \"CETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.\",\n \"Eliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.\",\n \"The authors state that they have no conflicts of interest.\",\n \"ESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,"results","discussion","conclusions",null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Carotid atherosclerosis","Carotid intima-media thickness","Cholesteryl ester transfer protein","Genetic polymorphism"],"string":"[\n \"Carotid atherosclerosis\",\n \"Carotid intima-media thickness\",\n \"Cholesteryl ester transfer protein\",\n \"Genetic polymorphism\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nAtherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.\n\nMethods:\n Study subjects A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nA total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\n Polymorphism detection Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\nGenomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\n Measurement of cIMT A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\nA single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\n Statistical analysis Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].\nMann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].\n\nStudy subjects:\nA total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\n\nBiochemical analysis:\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\n\nPolymorphism detection:\nGenomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\n\nMeasurement of cIMT:\nA single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\n\nStatistical analysis:\nMann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].\n\nResults:\nThe frequencies of I405V and Taq1B genotypes in our population sample, represented by the presence (VV, IV) and absence (II) of the V allele and the presence (B2B2, B1B2) and absence (B1B1) of the B2 allele, are presented in Table \n1. The frequencies of the II, IV and VV genotypes were 28%, 53% and 19%, respectively, and the frequencies of the B1B1, B1B2 and B2B2 were 33%, 41% and 26%, respectively.\nBiochemical parameters in the presence and absence of minor alleles\nData are presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values: Mann–Whitney U test for comparisons between the presence and absence of the minor alleles (V and B2). Significant differences: p≤0.05 in bold. CETP (%) = activity of cholesterol ester transfer protein; HDL-C = high-density lipoprotein cholesterol; PLTP (%) = activity of phospholipid transfer protein; oxLDL Ab = antibodies against oxidized LDL titers; (OD)= optical density.\nAllele frequencies were consistent with Hardy–Weinberg equilibrium for the I405V polymorphism (p=0.36) but not the Taq1B (p=0.02), and the MAFs (minor allele frequency) were <45% and <46%, respectively. The linkage disequilibrium between the two polymorphisms was very weak, as evidenced by the D`=0.17.\nClinical and anthropometric characteristics and biochemical data of the investigated population are presented in Tables \n2 and\n1.\nClinical and anthropometric characteristics in the presence and absence of minor alleles\nData presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values = Mann–Whitney U test for comparisons between the presence and absence of the alleles (V and B2); gender comparisons, Chi-Square test. Significant differences: p≤0.05 in bold. BMI= Body Mass Index; WC= waist circumference; SBP= systolic blood pressure; DBP= diastolic blood pressure; cIMT= carotid intima-media thickness.\nTable \n3 presents the significant correlations between mean cIMT, oxLDL Ab titers and CETP and PLTP activities.\nSignificant correlations between cIMT, oxLDL Ab, CETP (%) and PLTP (%) in the presence of minor alleles\nr= Spearman´s coefficient; Significant differences: p≤0.05 in bold. cIMT= carotid intima-media thickness; oxLDL Ab= antibodies against oxidized LDL titers; (OD) = optical density; CETP(%) = activity of cholesteryl ester transfer protein; PLTP (%) = activity of phospholipid transfer protein.\nThe univariate analyses (Table \n4) indicated that the female gender, age, HDL-C, BMI and SBP were significantly associated (p value ≤0.05) with higher cIMT.\n\nUnivariate Logistic Regression analyses for higher cIMT (above 66 percentile) (\n\nn=\n\n118)\n\ncIMT below and above the 66th percentile, <0.795 (n=76) and >0.795 (n=42), respectively. (ref).=reference level. (cont).= continue variable. OR= Odds ratio for higher cIMT, above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for the Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism, V = minor allele of I405V polymorphism, HDL-C = high-density lipoprotein cholesterol, BMI: body mass index, SBP: systolic blood pressure.\nThe multivariate analysis (Table \n5) demonstrated that age and the presence of the B2 allele were positively related to higher cIMT. Individuals with increased risk exhibited the B2 allele (5.1-fold increase) and higher age (21.2% increase/each year of age).\n\nMultivariate Logistic Regression* analyses for higher cIMT (above 66\n\nth\n\npercentile) (\n\nn=\n\n114)\n\n*Stepwise criteria for selection of variables. cIMT below and above 66th percentile, <0.795 (n=73) and >0.795 (n=41), respectively. (ref).= reference level; (cont).=continue variable. OR= Odds ratio for higher cIMT above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism.\nA Chi-Square analysis indicated an interaction between the presence of the B2 allele and age. The combination between age, categorized in ≥ and <50 years (median value), and the presence or absence B2 allele formed 4 groups of individuals who were crossed with cIMTs above and below 0.795 mm (data not shown).\n\nDiscussion:\nThis study investigated whether the I405V and Taq1B polymorphisms modify subclinical atherosclerosis in an asymptomatic population sample.\nPrevious studies present controversial data on the role of Taq1B and I405V in atherosclerosis and CVD. The B2 allele is associated with lower internal carotid artery IMT in men\n[16], but other studies do not identify associations between the Taq1B polymorphism and cIMT\n[17,18]. The present study demonstrated that cIMT did not differ between either allele of the polymorphisms. However, inverse correlations between cIMT and CETP activity were demonstrated in both polymorphisms (Table \n3), which suggest a potential atheroprotective role for CETP in these individuals. A potential anti-inflammatory role for CETP was demonstrated previously\n[19] wherein the induction of acute inflammation produced lower mortality due to sepsis and lower cytokine circulation in mice that express human CETP. Reductions in CETP concentrations in human patients with sepsis are more pronounced in non-survivors\n[20].\nWe identified positive correlations between cIMT and PLTP and oxLDL Ab in the presence of the V and B2 alleles. Previous studies have suggested that PLTP increases the risk of cardiovascular disease in humans\n[21], and the balance of evidence suggests a pro-atherogenic role for oxLDL Ab titers\n[22] in the development of atherosclerosis. These positive correlations and the lack of correlations of these variables in both common B1B1 and II allele homozygotes (CETP p≤0.318 and 0.091, oxLDL Ab p≤ 0.709 and 0.386 and PLTP p≤0.823 and 0.470, respectively) (data not shown) suggest a possible pro-atherogenic role of these markers associated with the polymorphisms. Our sample size was relatively small and different between groups. Therefore, further studies are underway in our lab to further confirm these correlations.\nThe presence of the V allele was associated with lower CETP (24%) and PLTP (29%) activities and higher HDL2-C (13%) concentrations. Patients with coronary disease typically exhibit fewer large HDL particles (HDL2), and the concentration of these large particles is a better predictor of future cardiovascular events than HDL-C concentration\n[23].\nLipoprotein (a) plasma levels were also significantly increased in the presence of the B2 allele. High levels of lipoprotein (a) are significantly associated with the presence, severity and extent of carotid atherosclerosis\n[24], but this increase was not observed as a possible pro-atherogenic factor.\nNo significant differences in CETP activity and HDL-C levels were observed in Taq1B polymorphism, despite a 15.4% reduction in CETP activity in presence of the B2 allele. These results are consistent with a previous study in another Brazilian population, in which no association between Taq1B genotypes and HDL-C levels was observed\n[25].\nA univariate analysis (Table \n4) followed by a multivariate logistic stepwise analysis (Table \n5) demonstrated that the presence of the B2 allele was independently associated with a mean 5.1-fold increased risk for higher cIMT and age interacted with the allele effect. The presence of the B2 allele in the ≥ 50 years age group exhibited the highest risk of higher cIMT in the Chi-Square analysis (data not shown).\nThe effects of the Taq1B variant on circulating plasma lipids may be related to other functional mutations within the CETP gene locus that are in LD\n[26], which may explain the association in our study. However, other factors than the CETP activity may exert different atherosclerotic repercussions in Taq1B and I405V polymorphisms. A multivariate analysis was performed to verify the independence of the associations between the qualitative binary variable cIMT above or below 66th percentile (0.795mm), CETP and PLTP plasma activities and the potential confounding factors, including gender, age, BMI, SBP and HDL-C. Only age exhibited a significant statistical association (OR: 1.247, CI 95%OR: 1.129 – 1.377), which was a strong determinant of cIMT (data not shown). The absence of interactions of other tested parameters with higher cIMT in this study suggests that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.\nOur results focused on several biomarkers that were assessed for the first time in the Brazilian population, and the results were partially based on the correlations between a relatively small numbers of individuals. These results merit further exploration due to the high frequency of the investigated polymorphisms in the present Brazilian sample. Identifying the impact of these polymorphisms is an ongoing aim of our research group.\n\nConclusions:\nThe I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved.\n\nAbbreviations:\nCETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.\n\nMisc:\nEliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.\n\nCompeting interests:\nThe authors state that they have no conflicts of interest.\n\nAuthors’ contributions:\nESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nCholesteryl ester transfer protein (CETP) plays a major role in lipid metabolism, but studies on the association of CETP polymorphisms with risks of cardiovascular disease are inconsistent. This study investigated whether the CETP gene I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian population sample.\n\nMethods:\nThe polymorphisms were analyzed using polymerase chain reaction in 207 adult volunteers. Serum lipid profiles, oxLDL Ab titers, C-reactive protein and tumor necrosis factor-α concentrations and CETP and phospholipid transfer protein (PLTP) activities were determined, and common carotid artery intima-media thickness (cIMT) was measured using ultrasonography.\n\nResults:\nNo differences in cIMT were observed between the presence or absence of the minor B2 and V alleles in either polymorphism. However, inverse correlations between mean cIMT and CETP activity in the presence of these polymorphisms were observed, and positive correlations of these polymorphisms with PLTP activity and oxLDL Ab titers were identified. Moreover, logistic multivariate analysis revealed that the presence of the B2 allele was associated with a 5.1-fold (CI 95%, OR: 1.26 - 21.06) increased risk for cIMT, which was equal and above the 66th percentile and positively interacted with age. However, no associations with the V allele or CETP and PLTP activities were observed.\n\nConclusions:\nNone of the studied parameters, including CETP activity, explained the different relationships between these polymorphisms and cIMT, suggesting that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease."},"background_conclusion_text":{"kind":"string","value":"Background:\nAtherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.\n\nConclusions:\nThe I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nCholesteryl ester transfer protein (CETP) plays a major role in lipid metabolism, but studies on the association of CETP polymorphisms with risks of cardiovascular disease are inconsistent. This study investigated whether the CETP gene I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian population sample.\n\nMethods:\nThe polymorphisms were analyzed using polymerase chain reaction in 207 adult volunteers. Serum lipid profiles, oxLDL Ab titers, C-reactive protein and tumor necrosis factor-α concentrations and CETP and phospholipid transfer protein (PLTP) activities were determined, and common carotid artery intima-media thickness (cIMT) was measured using ultrasonography.\n\nResults:\nNo differences in cIMT were observed between the presence or absence of the minor B2 and V alleles in either polymorphism. However, inverse correlations between mean cIMT and CETP activity in the presence of these polymorphisms were observed, and positive correlations of these polymorphisms with PLTP activity and oxLDL Ab titers were identified. Moreover, logistic multivariate analysis revealed that the presence of the B2 allele was associated with a 5.1-fold (CI 95%, OR: 1.26 - 21.06) increased risk for cIMT, which was equal and above the 66th percentile and positively interacted with age. However, no associations with the V allele or CETP and PLTP activities were observed.\n\nConclusions:\nNone of the studied parameters, including CETP activity, explained the different relationships between these polymorphisms and cIMT, suggesting that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease."},"whole_article_text_length":{"kind":"number","value":5263,"string":"5,263"},"whole_article_abstract_length":{"kind":"number","value":290,"string":"290"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["cimt","allele","hdl","cetp","performed","tg","protein","cholesterol","b2","presence"],"string":"[\n \"cimt\",\n \"allele\",\n \"hdl\",\n \"cetp\",\n \"performed\",\n \"tg\",\n \"protein\",\n \"cholesterol\",\n \"b2\",\n \"presence\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] polymorphisms | cholesterol | association | atherosclerosis | subclinical | disease | cvd | i405v | taq1b | cetp [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] tg | performed | usa | mg dl | dl | plus | diagnostics | pap | obtained | nas [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | presence | significant | absence | 05 | minor | 795 | b2 | significant differences 05 bold [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] allele | factors | cimt | studied | associated | b2 allele | polymorphisms | b2 | explained different | explained different relationships [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | polymorphisms | performed | cholesterol | protein | tg | cetp | b2 | taq1b [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cimt | allele | polymorphisms | performed | cholesterol | protein | tg | cetp | b2 | taq1b [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| CETP | Brazilian [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 207 ||| Ab | CETP | PLTP [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| CETP | PLTP ||| ||| 5.1-fold | CI | 95% | 1.26 - 21.06 | 66th ||| CETP | PLTP [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] CETP [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Cholesteryl ||| CETP | Brazilian ||| 207 ||| Ab | CETP | PLTP ||| ||| CETP | PLTP ||| ||| 5.1-fold | CI | 95% | 1.26 - 21.06 | 66th ||| CETP | PLTP ||| CETP [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Cholesteryl ||| CETP | Brazilian ||| 207 ||| Ab | CETP | PLTP ||| ||| CETP | PLTP ||| ||| 5.1-fold | CI | 95% | 1.26 - 21.06 | 66th ||| CETP | PLTP ||| CETP [SUMMARY]"}}},{"rowIdx":298,"cells":{"title":{"kind":"string","value":"Subjective Evaluation of the Results of Injectable Hyaluronic Acid Fillers for the Face."},"pmid":{"kind":"string","value":"32021131"},"background_abstract":{"kind":"string","value":"Skin ageing is a physiological process, progressive and irreversible. Hyaluronic acid injection treatments are used to correct the signs of skin ageing."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Hyaluronic acid was implanted in the area of the cheek and the forehead aesthetic units in 57 women, aged 35-55 years. Apart from the clinical observation, self-assessment of the therapeutic results was conducted. The \"My skin\" questionnaire was used for subjective evaluation of the treatment results."},"methods_abstract_label":{"kind":"string","value":"MATERIAL AND METHODS"},"results_abstract":{"kind":"string","value":"Mean wrinkle score in the pre-menopausal group changed after the treatment, from 3.2±0.6 to 1.1±0.3 and from 3.2±0.6 to 0.8±0.6 for the forehead and the cheek esthetic units, respectively. In the post-menopausal group, the score decreased from 3.8±0.4 to 1.7± 0.7 and from 3.2±0.617 to 0.8± 0.6 for the forehead and the cheek esthetic units, respectively. The changes were age-dependent. Improved appearance of the facial skin - higher satisfaction with skin tone and scent - was reported after hyaluronic acid injections. Higher subjective perception of improvement corresponded to older age, irrespectively of the menopausal status. Correlations between age and the effect of the treatment on maintaining proper skin hydration as well as between improved appearance of the forehead area and feelings of autonomy and well-being were found."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Hyaluronic acid injections significantly improved the subjective perception and overall assessment of the scent and appearance of the facial skin."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Cosmetic Techniques","Face","Female","Humans","Hyaluronic Acid","Injections","Middle Aged","Patient Satisfaction","Rejuvenation","Skin Aging","Treatment Outcome","Viscoelastic Substances"],"string":"[\n \"Adult\",\n \"Cosmetic Techniques\",\n \"Face\",\n \"Female\",\n \"Humans\",\n \"Hyaluronic Acid\",\n \"Injections\",\n \"Middle Aged\",\n \"Patient Satisfaction\",\n \"Rejuvenation\",\n \"Skin Aging\",\n \"Treatment Outcome\",\n \"Viscoelastic Substances\"\n]"},"pmcid":{"kind":"string","value":"6968800"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"The skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.\nSkin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being."},"other_sections_titles":{"kind":"list like","value":["Objectives","Materials and Methods","Results","Discussion","Conclusions"],"string":"[\n \"Objectives\",\n \"Materials and Methods\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\"\n]"},"other_sections_texts":{"kind":"list like","value":["The aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.","A total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).","Mean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\n\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.","The condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47","HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being."],"string":"[\n \"The aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.\",\n \"A total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).\",\n \"Mean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\\n\\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.\",\n \"The condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47\",\n \"HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Objectives","Materials and Methods","Results","Discussion","Conclusions"],"string":"[\n \"Introduction\",\n \"Objectives\",\n \"Materials and Methods\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["The skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.\nSkin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.","The aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.","A total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).","Mean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\n\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.","The condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47","HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being."],"string":"[\n \"The skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.\\nSkin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.\",\n \"The aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.\",\n \"A total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).\",\n \"Mean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\\n\\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.\",\n \"The condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47\",\n \"HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,null,null,null,null],"string":"[\n \"intro\",\n null,\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["hyaluronic acid fillers","skin","women","subjective evaluation"],"string":"[\n \"hyaluronic acid fillers\",\n \"skin\",\n \"women\",\n \"subjective evaluation\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nThe skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.\nSkin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.\n\nObjectives:\nThe aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.\n\nMaterials and Methods:\nA total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).\n\nResults:\nMean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\n\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.\n\nDiscussion:\nThe condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47\n\nConclusions:\nHA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nSkin ageing is a physiological process, progressive and irreversible. Hyaluronic acid injection treatments are used to correct the signs of skin ageing.\n\nMethods:\nHyaluronic acid was implanted in the area of the cheek and the forehead aesthetic units in 57 women, aged 35-55 years. Apart from the clinical observation, self-assessment of the therapeutic results was conducted. The \"My skin\" questionnaire was used for subjective evaluation of the treatment results.\n\nResults:\nMean wrinkle score in the pre-menopausal group changed after the treatment, from 3.2±0.6 to 1.1±0.3 and from 3.2±0.6 to 0.8±0.6 for the forehead and the cheek esthetic units, respectively. In the post-menopausal group, the score decreased from 3.8±0.4 to 1.7± 0.7 and from 3.2±0.617 to 0.8± 0.6 for the forehead and the cheek esthetic units, respectively. The changes were age-dependent. Improved appearance of the facial skin - higher satisfaction with skin tone and scent - was reported after hyaluronic acid injections. Higher subjective perception of improvement corresponded to older age, irrespectively of the menopausal status. Correlations between age and the effect of the treatment on maintaining proper skin hydration as well as between improved appearance of the forehead area and feelings of autonomy and well-being were found.\n\nConclusions:\nHyaluronic acid injections significantly improved the subjective perception and overall assessment of the scent and appearance of the facial skin."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nThe skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.\nSkin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.\n\nConclusions:\nHA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nSkin ageing is a physiological process, progressive and irreversible. Hyaluronic acid injection treatments are used to correct the signs of skin ageing.\n\nMethods:\nHyaluronic acid was implanted in the area of the cheek and the forehead aesthetic units in 57 women, aged 35-55 years. Apart from the clinical observation, self-assessment of the therapeutic results was conducted. The \"My skin\" questionnaire was used for subjective evaluation of the treatment results.\n\nResults:\nMean wrinkle score in the pre-menopausal group changed after the treatment, from 3.2±0.6 to 1.1±0.3 and from 3.2±0.6 to 0.8±0.6 for the forehead and the cheek esthetic units, respectively. In the post-menopausal group, the score decreased from 3.8±0.4 to 1.7± 0.7 and from 3.2±0.617 to 0.8± 0.6 for the forehead and the cheek esthetic units, respectively. The changes were age-dependent. Improved appearance of the facial skin - higher satisfaction with skin tone and scent - was reported after hyaluronic acid injections. Higher subjective perception of improvement corresponded to older age, irrespectively of the menopausal status. Correlations between age and the effect of the treatment on maintaining proper skin hydration as well as between improved appearance of the forehead area and feelings of autonomy and well-being were found.\n\nConclusions:\nHyaluronic acid injections significantly improved the subjective perception and overall assessment of the scent and appearance of the facial skin."},"whole_article_text_length":{"kind":"number","value":3976,"string":"3,976"},"whole_article_abstract_length":{"kind":"number","value":264,"string":"264"},"num_sections":{"kind":"number","value":6,"string":"6"},"most_frequent_words":{"kind":"list like","value":["skin","appearance","treatment","age","menopausal","changes","ha","facial","skin appearance","significant"],"string":"[\n \"skin\",\n \"appearance\",\n \"treatment\",\n \"age\",\n \"menopausal\",\n \"changes\",\n \"ha\",\n \"facial\",\n \"skin appearance\",\n \"significant\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n \"test\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] test | test [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | changes | effectiveness | indicative | biophysical | body | number | associated | physiological | treatments [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ha injections connected | injections connected | evaluation | ha | positive | self | injections | evaluation skin | ha injections | connected [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | treatment | appearance | ha | age | menopausal | evaluation | changes | facial | haf [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] skin | treatment | appearance | ha | age | menopausal | evaluation | changes | facial | haf [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| 57 | 35-55 years ||| ||| ||| 3.2±0.6 | 1.1±0.3 | 3.2±0.6 ||| 3.8±0.4 | 0.7 | 0.8± 0.6 ||| ||| ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| 57 | 35-55 years ||| ||| ||| 3.2±0.6 | 1.1±0.3 | 3.2±0.6 ||| 3.8±0.4 | 0.7 | 0.8± 0.6 ||| ||| ||| ||| ||| [SUMMARY]"}}},{"rowIdx":299,"cells":{"title":{"kind":"string","value":"Function and regulation annotation of up-regulated long non-coding RNA LINC01234 in gastric cancer."},"pmid":{"kind":"string","value":"32011780"},"background_abstract":{"kind":"string","value":"Accumulated evidences indicate that long non-coding RNAs (lncRNAs) participate in many biological mechanisms. Moreover, it acts as an essential regulator in various human diseases such as gastric cancer (GC). Nevertheless, the comprehensive regulatory roles and clinical significance of most lncRNAs in GC are not fully understood."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"In this research, our aim was to investigate the underlying mechanism of lncRNA LINC01234 in GC. Firstly, the usage of qRT-PCR helped to establish expression pattern of LINC01234 in GC tissues. Following this, appropriate statistical tests were applied to analyze the relation between expression level and clinicopathological factors. Ultimately, potential functions and regulatory network of LINC01234 were concluded via GSEA and a series of bioinformatics tools or databases, respectively."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Consequently, at the end of research we found LINC01234 is up-regulated in GC tissues in comparison with adjacent normal tissues. Furthermore, its expression level is correlated with differentiation of patients with GC. It is also important to highlight bioinformatics analysis revealed that LINC01234 is involved in cancer-associated pathways such as cell cycle and mismatch repair. Also, regulatory network of LINC01234 presented a probability in the involvement of tumorigenesis through regulating cancer-associated genes."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Overall, our results suggested that LINC01234 may play a crucial role in GC."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adenocarcinoma","Aged","Biomarkers, Tumor","Female","Gene Expression Regulation, Neoplastic","Gene Regulatory Networks","Humans","Male","MicroRNAs","Middle Aged","RNA, Long Noncoding","Stomach Neoplasms","Up-Regulation"],"string":"[\n \"Adenocarcinoma\",\n \"Aged\",\n \"Biomarkers, Tumor\",\n \"Female\",\n \"Gene Expression Regulation, Neoplastic\",\n \"Gene Regulatory Networks\",\n \"Humans\",\n \"Male\",\n \"MicroRNAs\",\n \"Middle Aged\",\n \"RNA, Long Noncoding\",\n \"Stomach Neoplasms\",\n \"Up-Regulation\"\n]"},"pmcid":{"kind":"string","value":"7246363"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":" Experimental verification of LINC01234 up‐regulation in GC tissues Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\nFirstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\n Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\nIn the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\n Potential functions of LINC01234\n To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\nTo explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\n Regulatory network of LINC01234\n LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nLncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Differently expression analysis of lncRNAs in STAD from TCGA","Collection of GC samples and patients' clinical information","Total RNA extraction and qRT‐PCR","Gene set enrichment analysis of LINC01234\n","Construction of LINC01234 regulatory network","TF‐LINC01234 regulation","miRNA‐LINC01234 interactions","RBP‐LINC01234 interactions","Statistical analysis","Experimental verification of LINC01234 up‐regulation in GC tissues","Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients","Potential functions of LINC01234\n","Regulatory network of LINC01234\n","TF‐LINC01234 regulation","miRNA‐LINC01234 regulation","RBP‐LINC01234 regulation"],"string":"[\n \"INTRODUCTION\",\n \"Differently expression analysis of lncRNAs in STAD from TCGA\",\n \"Collection of GC samples and patients' clinical information\",\n \"Total RNA extraction and qRT‐PCR\",\n \"Gene set enrichment analysis of LINC01234\\n\",\n \"Construction of LINC01234 regulatory network\",\n \"TF‐LINC01234 regulation\",\n \"miRNA‐LINC01234 interactions\",\n \"RBP‐LINC01234 interactions\",\n \"Statistical analysis\",\n \"Experimental verification of LINC01234 up‐regulation in GC tissues\",\n \"Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients\",\n \"Potential functions of LINC01234\\n\",\n \"Regulatory network of LINC01234\\n\",\n \"TF‐LINC01234 regulation\",\n \"miRNA‐LINC01234 regulation\",\n \"RBP‐LINC01234 regulation\"\n]"},"other_sections_texts":{"kind":"list like","value":["Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.","Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.","Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.","The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.","By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant."," TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.","We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.","The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n","Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.","IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.","Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues","In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR","To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)","LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).","Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).","A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n","A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4)."],"string":"[\n \"Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.\",\n \"Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\",\n \"Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\",\n \"The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\",\n \"By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\",\n \" TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\",\n \"We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\",\n \"The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\",\n \"Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\",\n \"IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\",\n \"Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\",\n \"In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\\nAbbreviation: SE, standard error.\\nThe sample size is so small that the result may be unbelievable even though P < .05.\\n\\nP < .05.\\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\",\n \"To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\",\n \"LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\",\n \"Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\",\n \"A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\",\n \"A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Differently expression analysis of lncRNAs in STAD from TCGA","Collection of GC samples and patients' clinical information","Total RNA extraction and qRT‐PCR","Gene set enrichment analysis of LINC01234\n","Construction of LINC01234 regulatory network","TF‐LINC01234 regulation","miRNA‐LINC01234 interactions","RBP‐LINC01234 interactions","Statistical analysis","RESULTS","Experimental verification of LINC01234 up‐regulation in GC tissues","Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients","Potential functions of LINC01234\n","Regulatory network of LINC01234\n","TF‐LINC01234 regulation","miRNA‐LINC01234 regulation","RBP‐LINC01234 regulation","DISCUSSION","Supporting information"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Differently expression analysis of lncRNAs in STAD from TCGA\",\n \"Collection of GC samples and patients' clinical information\",\n \"Total RNA extraction and qRT‐PCR\",\n \"Gene set enrichment analysis of LINC01234\\n\",\n \"Construction of LINC01234 regulatory network\",\n \"TF‐LINC01234 regulation\",\n \"miRNA‐LINC01234 interactions\",\n \"RBP‐LINC01234 interactions\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Experimental verification of LINC01234 up‐regulation in GC tissues\",\n \"Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients\",\n \"Potential functions of LINC01234\\n\",\n \"Regulatory network of LINC01234\\n\",\n \"TF‐LINC01234 regulation\",\n \"miRNA‐LINC01234 regulation\",\n \"RBP‐LINC01234 regulation\",\n \"DISCUSSION\",\n \"Supporting information\"\n]"},"all_sections_texts":{"kind":"list like","value":["Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC."," Differently expression analysis of lncRNAs in STAD from TCGA Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\nFragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\n Collection of GC samples and patients' clinical information Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\nPaired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\n Total RNA extraction and qRT‐PCR The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\nThe methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\n Gene set enrichment analysis of LINC01234\n By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\nBy using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\n Construction of LINC01234 regulatory network TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n Statistical analysis IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\nIBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.","Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.","Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.","The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.","By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant."," TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.","We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.","The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n","Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.","IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances."," Experimental verification of LINC01234 up‐regulation in GC tissues Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\nFirstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\n Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\nIn the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\n Potential functions of LINC01234\n To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\nTo explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\n Regulatory network of LINC01234\n LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nLncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).","Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues","In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR","To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)","LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).","Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).","A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n","A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).","LncRNA is a type of non‐coding RNAs without the ability of encoding proteins; despite this, it has a regulating gene expression at chromatin modification, transcriptional, or post‐transcriptional levels.35 In addition, the polymorphisms in lncRNAs could be a risk of disease or cancer.36 In fact, an increasing number of lncRNAs have been identified to be related to numerous kinds of diseases,37 including GC. For example, through the expression analysis of metabolic pathway‐related lncRNAs and protein‐coding genes, a dozen of lncRNAs were functionally annotated and discovered to be important in GC.38\nDGCR9, another lncRNA up‐regulated in GC, was shown to promote the tumorigenesis of GC.\n39 Some of lncRNAs were even indeed considered as biomarkers for diagnosis or prognosis of GC. For instance, a metabolism‐related lncRNA, RP11‐555H23.1, was found to be a potential diagnostic biomarker in GC.40 Also, the expression level of H19 in plasma could be served as a biomarker for patients with GC.41 Due to the regulatory role of lncRNAs, exploring new lncRNA biomarkers can help to explain the initiation and progression mechanism of GC. Nevertheless, still there are many unknown lncRNAs in GC nowadays.\nThe previous study found LINC01234 is highly expressed in esophageal squamous cell carcinoma (ESCC). Also, it is one of the three lncRNAs that can be a signature to predict the survival time of ESCC patients accurately.42, 43, 44 Besides, it was likewise discovered to be up‐regulated in the GC in prior study.26 However, the functions and regulatory role of LINC01234 need to be studied.\nIn this research, we identified that LINC01234 was also highly expressed in GC tissues compared with adjacent non‐cancerous tissues. Later, we explored the associations between the expression level of LINC01234 and clinical features through which we found LINC01234 was correlated with differentiation of GC. LncRNAs usually interact with other kinds of molecules to involve in multiple biological processes. For example, the binding of lncRNA OLC8 and IL‐11 will impair the degradation of IL‐11 mRNAs to accelerate GC development.45 Besides, combination of lncRNA and its target may increase the diagnostic value of lncRNA. Just as it was found by previous report that the combined use of RP11‐19P22.6‐001 and its target NOS2 may be useful to diagnose patients with GC.46 Thus, we further explored the potential functions and regulatory network of LINC01234. We identified 218 relationships of LINC01234 in total. Among them, 17 associations including two pairs of TF regulation and 17 pairs of RBP interaction were found to be co‐expressed. One of 2 TFs, ELK1, is an important regulator and known to activate many lncRNAs including TRPM2‐AS, MIR100HG, and HOXA10‐AS in cancer until now,47, 48, 49 indicating ELK1 may induce expression of LINC01234 to promote tumor progression in GC too.\nIn conclusion, the results of this present study indicated that LINC01234 expression is linked with the diagnostics of patients with GC and similarly may be involved in differentiation in GC through cell cycle or other cancer‐related pathways."," \nClick here for additional data file.\n \nClick here for additional data file.\n \nClick here for additional data file.\n \nClick here for additional data file."],"string":"[\n \"Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.\",\n \" Differently expression analysis of lncRNAs in STAD from TCGA Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\\nFragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\\n Collection of GC samples and patients' clinical information Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\\nPaired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\\n Total RNA extraction and qRT‐PCR The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\\nThe methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\\n Gene set enrichment analysis of LINC01234\\n By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\\nBy using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\\n Construction of LINC01234 regulatory network TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\n TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\n Statistical analysis IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\\nIBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\",\n \"Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\",\n \"Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\",\n \"The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\",\n \"By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\",\n \" TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\",\n \"We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\",\n \"The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\\n\",\n \"Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\",\n \"IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\",\n \" Experimental verification of LINC01234 up‐regulation in GC tissues Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\\nFirstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\\n Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\\nAbbreviation: SE, standard error.\\nThe sample size is so small that the result may be unbelievable even though P < .05.\\n\\nP < .05.\\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\\nIn the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\\nAbbreviation: SE, standard error.\\nThe sample size is so small that the result may be unbelievable even though P < .05.\\n\\nP < .05.\\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\\n Potential functions of LINC01234\\n To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\\nTo explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\\n Regulatory network of LINC01234\\n LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\\nLncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\",\n \"Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\",\n \"In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\\nAbbreviation: SE, standard error.\\nThe sample size is so small that the result may be unbelievable even though P < .05.\\n\\nP < .05.\\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\",\n \"To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\",\n \"LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\",\n \"Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\",\n \"A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\\n\",\n \"A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\",\n \"LncRNA is a type of non‐coding RNAs without the ability of encoding proteins; despite this, it has a regulating gene expression at chromatin modification, transcriptional, or post‐transcriptional levels.35 In addition, the polymorphisms in lncRNAs could be a risk of disease or cancer.36 In fact, an increasing number of lncRNAs have been identified to be related to numerous kinds of diseases,37 including GC. For example, through the expression analysis of metabolic pathway‐related lncRNAs and protein‐coding genes, a dozen of lncRNAs were functionally annotated and discovered to be important in GC.38\\nDGCR9, another lncRNA up‐regulated in GC, was shown to promote the tumorigenesis of GC.\\n39 Some of lncRNAs were even indeed considered as biomarkers for diagnosis or prognosis of GC. For instance, a metabolism‐related lncRNA, RP11‐555H23.1, was found to be a potential diagnostic biomarker in GC.40 Also, the expression level of H19 in plasma could be served as a biomarker for patients with GC.41 Due to the regulatory role of lncRNAs, exploring new lncRNA biomarkers can help to explain the initiation and progression mechanism of GC. Nevertheless, still there are many unknown lncRNAs in GC nowadays.\\nThe previous study found LINC01234 is highly expressed in esophageal squamous cell carcinoma (ESCC). Also, it is one of the three lncRNAs that can be a signature to predict the survival time of ESCC patients accurately.42, 43, 44 Besides, it was likewise discovered to be up‐regulated in the GC in prior study.26 However, the functions and regulatory role of LINC01234 need to be studied.\\nIn this research, we identified that LINC01234 was also highly expressed in GC tissues compared with adjacent non‐cancerous tissues. Later, we explored the associations between the expression level of LINC01234 and clinical features through which we found LINC01234 was correlated with differentiation of GC. LncRNAs usually interact with other kinds of molecules to involve in multiple biological processes. For example, the binding of lncRNA OLC8 and IL‐11 will impair the degradation of IL‐11 mRNAs to accelerate GC development.45 Besides, combination of lncRNA and its target may increase the diagnostic value of lncRNA. Just as it was found by previous report that the combined use of RP11‐19P22.6‐001 and its target NOS2 may be useful to diagnose patients with GC.46 Thus, we further explored the potential functions and regulatory network of LINC01234. We identified 218 relationships of LINC01234 in total. Among them, 17 associations including two pairs of TF regulation and 17 pairs of RBP interaction were found to be co‐expressed. One of 2 TFs, ELK1, is an important regulator and known to activate many lncRNAs including TRPM2‐AS, MIR100HG, and HOXA10‐AS in cancer until now,47, 48, 49 indicating ELK1 may induce expression of LINC01234 to promote tumor progression in GC too.\\nIn conclusion, the results of this present study indicated that LINC01234 expression is linked with the diagnostics of patients with GC and similarly may be involved in differentiation in GC through cell cycle or other cancer‐related pathways.\",\n \" \\nClick here for additional data file.\\n \\nClick here for additional data file.\\n \\nClick here for additional data file.\\n \\nClick here for additional data file.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"materials-and-methods",null,null,null,null,null,null,null,null,null,"results",null,null,null,null,null,null,null,"discussion","supplementary-material"],"string":"[\n null,\n \"materials-and-methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["gastric cancer","\nLINC01234\n","long non‐coding RNA","regulatory network"],"string":"[\n \"gastric cancer\",\n \"\\nLINC01234\\n\",\n \"long non‐coding RNA\",\n \"regulatory network\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nGastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.\n\nMATERIALS AND METHODS:\n Differently expression analysis of lncRNAs in STAD from TCGA Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\nFragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\n Collection of GC samples and patients' clinical information Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\nPaired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\n Total RNA extraction and qRT‐PCR The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\nThe methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\n Gene set enrichment analysis of LINC01234\n By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\nBy using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\n Construction of LINC01234 regulatory network TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n Statistical analysis IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\nIBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\n\nDifferently expression analysis of lncRNAs in STAD from TCGA:\nFragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\n\nCollection of GC samples and patients' clinical information:\nPaired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\n\nTotal RNA extraction and qRT‐PCR:\nThe methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\n\nGene set enrichment analysis of LINC01234\n:\nBy using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\n\nConstruction of LINC01234 regulatory network:\n TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n\nTF‐LINC01234 regulation:\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n\nmiRNA‐LINC01234 interactions:\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n\nRBP‐LINC01234 interactions:\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n\nStatistical analysis:\nIBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\n\nRESULTS:\n Experimental verification of LINC01234 up‐regulation in GC tissues Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\nFirstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\n Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\nIn the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\n Potential functions of LINC01234\n To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\nTo explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\n Regulatory network of LINC01234\n LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nLncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\n\nExperimental verification of LINC01234 up‐regulation in GC tissues:\nFirstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\n\nAssociation analysis between expression level of LINC01234 and clinicopathological factors in GC patients:\nIn the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\n\nPotential functions of LINC01234\n:\nTo explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\n\nRegulatory network of LINC01234\n:\nLncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\n\nTF‐LINC01234 regulation:\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n\nmiRNA‐LINC01234 regulation:\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n\nRBP‐LINC01234 regulation:\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\n\nDISCUSSION:\nLncRNA is a type of non‐coding RNAs without the ability of encoding proteins; despite this, it has a regulating gene expression at chromatin modification, transcriptional, or post‐transcriptional levels.35 In addition, the polymorphisms in lncRNAs could be a risk of disease or cancer.36 In fact, an increasing number of lncRNAs have been identified to be related to numerous kinds of diseases,37 including GC. For example, through the expression analysis of metabolic pathway‐related lncRNAs and protein‐coding genes, a dozen of lncRNAs were functionally annotated and discovered to be important in GC.38\nDGCR9, another lncRNA up‐regulated in GC, was shown to promote the tumorigenesis of GC.\n39 Some of lncRNAs were even indeed considered as biomarkers for diagnosis or prognosis of GC. For instance, a metabolism‐related lncRNA, RP11‐555H23.1, was found to be a potential diagnostic biomarker in GC.40 Also, the expression level of H19 in plasma could be served as a biomarker for patients with GC.41 Due to the regulatory role of lncRNAs, exploring new lncRNA biomarkers can help to explain the initiation and progression mechanism of GC. Nevertheless, still there are many unknown lncRNAs in GC nowadays.\nThe previous study found LINC01234 is highly expressed in esophageal squamous cell carcinoma (ESCC). Also, it is one of the three lncRNAs that can be a signature to predict the survival time of ESCC patients accurately.42, 43, 44 Besides, it was likewise discovered to be up‐regulated in the GC in prior study.26 However, the functions and regulatory role of LINC01234 need to be studied.\nIn this research, we identified that LINC01234 was also highly expressed in GC tissues compared with adjacent non‐cancerous tissues. Later, we explored the associations between the expression level of LINC01234 and clinical features through which we found LINC01234 was correlated with differentiation of GC. LncRNAs usually interact with other kinds of molecules to involve in multiple biological processes. For example, the binding of lncRNA OLC8 and IL‐11 will impair the degradation of IL‐11 mRNAs to accelerate GC development.45 Besides, combination of lncRNA and its target may increase the diagnostic value of lncRNA. Just as it was found by previous report that the combined use of RP11‐19P22.6‐001 and its target NOS2 may be useful to diagnose patients with GC.46 Thus, we further explored the potential functions and regulatory network of LINC01234. We identified 218 relationships of LINC01234 in total. Among them, 17 associations including two pairs of TF regulation and 17 pairs of RBP interaction were found to be co‐expressed. One of 2 TFs, ELK1, is an important regulator and known to activate many lncRNAs including TRPM2‐AS, MIR100HG, and HOXA10‐AS in cancer until now,47, 48, 49 indicating ELK1 may induce expression of LINC01234 to promote tumor progression in GC too.\nIn conclusion, the results of this present study indicated that LINC01234 expression is linked with the diagnostics of patients with GC and similarly may be involved in differentiation in GC through cell cycle or other cancer‐related pathways.\n\nSupporting information:\n \nClick here for additional data file.\n \nClick here for additional data file.\n \nClick here for additional data file.\n \nClick here for additional data file.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nAccumulated evidences indicate that long non-coding RNAs (lncRNAs) participate in many biological mechanisms. Moreover, it acts as an essential regulator in various human diseases such as gastric cancer (GC). Nevertheless, the comprehensive regulatory roles and clinical significance of most lncRNAs in GC are not fully understood.\n\nMethods:\nIn this research, our aim was to investigate the underlying mechanism of lncRNA LINC01234 in GC. Firstly, the usage of qRT-PCR helped to establish expression pattern of LINC01234 in GC tissues. Following this, appropriate statistical tests were applied to analyze the relation between expression level and clinicopathological factors. Ultimately, potential functions and regulatory network of LINC01234 were concluded via GSEA and a series of bioinformatics tools or databases, respectively.\n\nResults:\nConsequently, at the end of research we found LINC01234 is up-regulated in GC tissues in comparison with adjacent normal tissues. Furthermore, its expression level is correlated with differentiation of patients with GC. It is also important to highlight bioinformatics analysis revealed that LINC01234 is involved in cancer-associated pathways such as cell cycle and mismatch repair. Also, regulatory network of LINC01234 presented a probability in the involvement of tumorigenesis through regulating cancer-associated genes.\n\nConclusions:\nOverall, our results suggested that LINC01234 may play a crucial role in GC."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":9795,"string":"9,795"},"whole_article_abstract_length":{"kind":"number","value":252,"string":"252"},"num_sections":{"kind":"number","value":21,"string":"21"},"most_frequent_words":{"kind":"list like","value":["gc","linc01234","expression","tissues","regulation","cell","stad","lncrnas","patients","cancer"],"string":"[\n \"gc\",\n \"linc01234\",\n \"expression\",\n \"tissues\",\n \"regulation\",\n \"cell\",\n \"stad\",\n \"lncrnas\",\n \"patients\",\n \"cancer\"\n]"},"keybert_topics":{"kind":"list like","value":["test","test"],"string":"[\n \"test\",\n 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Biatrial volume ratio predicts low voltage areas in atrial fibrillation.
|
34494677
|
Left atrial volume (LAV) and low voltage areas (LVAs) are acknowledged markers for worse rhythm outcome after ablation of atrial fibrillation (AF). Some studies reported the importance of increased right atrial volume (RAV) as a predictor for arrhythmia recurrences in AF patients.
|
BACKGROUND
|
Patients undergoing first AF ablation were included. LVAs were assessed peri-procedurally using high-density 3D maps and defined as <0.5 mV. All patients underwent pre-procedural cardiovascular magnetic resonance imaging. LAV (biplane) and RAV (monoplane 4-chamber) were assessed prior to ablation, and the LAV/RAV ratio was calculated.
|
METHODS
|
The study population included 189 patients (age mean 63 ± 10 years, 33% women, 57% persistent AF, 22% LVAs). There were 149 (79%) patients with LAV > RAV. In univariable analysis LAV > RAV was associated with LVAs (OR 6.803, 95%CI 1.395-26.514, p = .016). The association remained robust in multivariable model after adjustment for persistent AF, CHA2 DS2 -VASc score, and heart rate (OR 5.981, 95%CI 1.256-28.484, p = .025). Using receiver operator curve analysis, LAV > RAV (AUC 0.668, 95%CI 0.585-0.751, p = .001) was significant predictor for LVAs. In multivariable analysis, after adjustment for age, persistent AF, and renal function, RAV≥LAV was threefold higher in males (OR 3.040, 95%CI 1.050-8.802, p = .04).
|
RESULTS
|
LAV > RAV is useful for the prediction of electro-anatomical substrate in AF. LAV > RAV was associated with LVAs presence, while male sex remained associated with RAV≥LAV and less LVAs.
|
CONCLUSIONS
|
[
"Aged",
"Atrial Fibrillation",
"Catheter Ablation",
"Female",
"Heart Atria",
"Heart Rate",
"Humans",
"Male",
"Middle Aged",
"Recurrence"
] |
8571553
|
INTRODUCTION
|
Becoming a cornerstone therapy in many patients with atrial fibrillation (AF),
1
in some patients, catheter ablation with circumferential pulmonary vein isolation alone is not enough for sinus rhythm maintenance during follow‐up. The arrhythmia recurrences remain an important clinical challenge and require individualized AF treatment plan already before intervention. One major feature reflecting left atrial (LA) remodeling is peri‐procedural evidence of low voltage areas (LVAs).
2
At least 20%–25% of AF patients have significant LVAs in peri‐procedural mapping, which are an important characteristic of AF progression and treatment failure if not treated with additional ablation.
2
,
3
Therefore, assessment of LVAs presence before catheter ablation is an important task for the electrophysiologist allowing individually tailored AF ablation therapy.
The LA size is another important parameter of AF progression. The role of the LA volume (LAV) and LA function as surrogate parameters for higher AF burden
4
and LVAs presence
5
,
6
are well described. In addition, there is an association between atrial flutter—a right atrial (RA), and AF—a left atrial disease.
7
,
8
Several studies described an importance of RA assessment as a prognosis marker in heart failure, pulmonary hypertension, and chronic obstructive pulmonary disease.
9
,
10
,
11
Diastolic functional changes—as a preliminary stage for AF—appeared to occur earlier in the right chambers
12
suggesting that RA dilatation might be an early marker for atrial remodeling associated with AF initiation. Previous studies reported the importance of increased RA volume (RAV) as predictor for arrhythmia recurrences in AF patients.
13
,
14
It was hypothesized that RA is more prone to hemodynamic changes and is a more responsive marker of structural remodeling.
13
However, association between RAV and LVAs and the prediction capability for LAV/RAV ratio is unknown. Therefore, we aimed to investigate the indexed LAV/RAV ratio assessed in cardiovascular magnetic resonance (CMR) imaging and the association with LVAs in patients undergoing AF catheter ablation. We hypothesize that the biatrial ratio is an independent predictor for LVAs presence.
|
METHODS
|
The study population was described previously.
6
Briefly, patients presenting for catheter ablation due to symptomatic AF from October 2015 to April 2017 were included in the study. According to current guidelines, AF subtypes were defined as paroxysmal and persistent.
15
Patients with pregnancy, age <18 or >75 years, valvular AF (any valvulopathies >second degree), cancer, acute, or systemic inflammatory diseases, and acute hyperthyreotic state were excluded from the study. The study was approved by the local Ethical Committee (Medical Faculty, University of Leipzig), and patients provided written informed consent for participation.
Cardiovascular magnetic resonance Prior to AF catheter ablation, all patients underwent 1.5 T CMR (Ingenia, Philips Medical) for LA anatomy assessment as previously described.
5
Briefly, LAV was determined using a biplane model based on cine 4‐ and 2‐chamber views, and RAV using a monoplane model based on the cine 4‐chamber view. Both volumes were indexed to body surface areas, and the LAV/RAV ratio was calculated before ablation. We defined two subgroups according to the LAV/RAV ratio: (1) LAV is greater than RAV (LAV > RAV), and (2) RAV is equal or greater than LAV (RAV ≥ LAV).
Prior to AF catheter ablation, all patients underwent 1.5 T CMR (Ingenia, Philips Medical) for LA anatomy assessment as previously described.
5
Briefly, LAV was determined using a biplane model based on cine 4‐ and 2‐chamber views, and RAV using a monoplane model based on the cine 4‐chamber view. Both volumes were indexed to body surface areas, and the LAV/RAV ratio was calculated before ablation. We defined two subgroups according to the LAV/RAV ratio: (1) LAV is greater than RAV (LAV > RAV), and (2) RAV is equal or greater than LAV (RAV ≥ LAV).
Peri‐procedural LA mapping and AF ablation Transseptal access and catheter navigation were performed with a steerable sheath (Agilis, St. Jude Medical, St. Paul, MN). The electro‐anatomical mapping was performed in sinus rhythm as described previously.
5
In case of AF at the beginning of the procedure, the arrhythmia was terminated by electrical cardioversion and the mapping was performed in sinus rhythm.
Multielectrode spiral mapping catheters (Reflexion Spiral and Advisor, St Jude Medical [SJM], Saint Paul, MN in NavX Ensite procedures and Carto Lasso, Biosense Webster, Diamond Bar, CA in Carto3 procedures) were used to generate electro‐anatomical voltage maps of the LA. The cutoff value for LVAs was defined as bipolar signal amplitude <0.5 mV. Ectopic beats were excluded from the voltage map. The number of points obtained was >1000. In case of AF recurrence during electro‐anatomical mapping, only anatomical map was assessed. Then the electro‐anatomical mapping was completed in sinus rhythm after PVI, and if still needed, after further cardioversion.
All patients received circumferential ablation lines around the antrum of the ipsilateral pulmonary veins. The ablation catheters (irrigated tip catheter and power of 25–40 W) used in NavX Ensite procedures were TactiCath (St Jude Medical [SJM], Saint Paul, MN) and for Carto3 procedures SmartTouch Thermocool (Biosense Webster, Diamond Bar, CA). End point of catheter ablation was PV isolation, which was verified with a multipolar circular mapping catheter. Additional linear lesions were added between if LVAs were present in this area. To be considered as relevant the LVAs ought to consist of adjacent low‐amplitude mapping points, bearing certain additional characteristics such as fragmentation and duration. Finally, the relevance of the LVAs was evaluated by experienced operators based on the mapping point qualities as well as induction of extra‐PV macro‐reentry tachycardia, in which case additional linear ablation was performed. Patients with small/negligible LVAs dispersely distributed in LA and not suitable for ablation, who received the PVI only without additional ablation lines, were excluded.
Transseptal access and catheter navigation were performed with a steerable sheath (Agilis, St. Jude Medical, St. Paul, MN). The electro‐anatomical mapping was performed in sinus rhythm as described previously.
5
In case of AF at the beginning of the procedure, the arrhythmia was terminated by electrical cardioversion and the mapping was performed in sinus rhythm.
Multielectrode spiral mapping catheters (Reflexion Spiral and Advisor, St Jude Medical [SJM], Saint Paul, MN in NavX Ensite procedures and Carto Lasso, Biosense Webster, Diamond Bar, CA in Carto3 procedures) were used to generate electro‐anatomical voltage maps of the LA. The cutoff value for LVAs was defined as bipolar signal amplitude <0.5 mV. Ectopic beats were excluded from the voltage map. The number of points obtained was >1000. In case of AF recurrence during electro‐anatomical mapping, only anatomical map was assessed. Then the electro‐anatomical mapping was completed in sinus rhythm after PVI, and if still needed, after further cardioversion.
All patients received circumferential ablation lines around the antrum of the ipsilateral pulmonary veins. The ablation catheters (irrigated tip catheter and power of 25–40 W) used in NavX Ensite procedures were TactiCath (St Jude Medical [SJM], Saint Paul, MN) and for Carto3 procedures SmartTouch Thermocool (Biosense Webster, Diamond Bar, CA). End point of catheter ablation was PV isolation, which was verified with a multipolar circular mapping catheter. Additional linear lesions were added between if LVAs were present in this area. To be considered as relevant the LVAs ought to consist of adjacent low‐amplitude mapping points, bearing certain additional characteristics such as fragmentation and duration. Finally, the relevance of the LVAs was evaluated by experienced operators based on the mapping point qualities as well as induction of extra‐PV macro‐reentry tachycardia, in which case additional linear ablation was performed. Patients with small/negligible LVAs dispersely distributed in LA and not suitable for ablation, who received the PVI only without additional ablation lines, were excluded.
Statistical analysis Data are presented as mean and standard deviation for normally distributed or median (interquartile range, 25th and 75th percentiles) for skewed continuous variables, and as proportions for categorical variables. The differences between continuous values were assessed using an unpaired t‐test or the Mann–Whitney, and a χ
2 test for categorical variables.
Logistic regression analysis was used to identify factors associated with LVAs. We performed three analyses using logistic regression of LVAs presence (Model 1 – unadjusted analysis; Model 2 – adjusted for age and sex; and Model 3 – adjusted for persistent AF, heart rate, and CHA2DS2‐VASc score.
Receiver operating characteristic curves (ROC) were generated to analyze performance of the LAV/RAV ratio predicting LVAs, with the area under the curve (AUC) being equivalent to the c‐index for determining the predictive value for the parameters. Finally, we compared the c‐indices (i.e., areas under the ROC curves) of LAV > RAV and LAV using DeLong's method.
16
A p‐value <.05 was considered statistically significant. All analyses were performed with SPSS statistical software version 26 (SPSS Inc., Chicago).
Data are presented as mean and standard deviation for normally distributed or median (interquartile range, 25th and 75th percentiles) for skewed continuous variables, and as proportions for categorical variables. The differences between continuous values were assessed using an unpaired t‐test or the Mann–Whitney, and a χ
2 test for categorical variables.
Logistic regression analysis was used to identify factors associated with LVAs. We performed three analyses using logistic regression of LVAs presence (Model 1 – unadjusted analysis; Model 2 – adjusted for age and sex; and Model 3 – adjusted for persistent AF, heart rate, and CHA2DS2‐VASc score.
Receiver operating characteristic curves (ROC) were generated to analyze performance of the LAV/RAV ratio predicting LVAs, with the area under the curve (AUC) being equivalent to the c‐index for determining the predictive value for the parameters. Finally, we compared the c‐indices (i.e., areas under the ROC curves) of LAV > RAV and LAV using DeLong's method.
16
A p‐value <.05 was considered statistically significant. All analyses were performed with SPSS statistical software version 26 (SPSS Inc., Chicago).
|
RESULTS
|
Clinical characteristics of the study population The study population included 189 patients (mean 63 ± 10 years, 33% women, 57% persistent AF) undergoing their first AF catheter ablation. Clinical characteristics of the study population are summarized in Table 1. Forty‐one patients (22%) had LVAs in periprocedural mapping, which required additional substrate modification. We observed LAV > RAV in 149 (79%) patients (26% with LVAs), while 40 (21%) patients had RAV≥LAV (5% with LVAs). Patients with LAV > RAV were significantly older, more often females, had a lower estimated glomerular filtration rate, had more often hypertension and more often LVAs (Table 1).
Clinical characteristics of the study cohort
Abbreviations: AF, atrial fibrillation; BMI, body mass index; BSA, body surface area; eGFR, estimated glomerular filtration rate; LA, left atrial; LVAs, low voltage areas; RA, right atrial.
The study population included 189 patients (mean 63 ± 10 years, 33% women, 57% persistent AF) undergoing their first AF catheter ablation. Clinical characteristics of the study population are summarized in Table 1. Forty‐one patients (22%) had LVAs in periprocedural mapping, which required additional substrate modification. We observed LAV > RAV in 149 (79%) patients (26% with LVAs), while 40 (21%) patients had RAV≥LAV (5% with LVAs). Patients with LAV > RAV were significantly older, more often females, had a lower estimated glomerular filtration rate, had more often hypertension and more often LVAs (Table 1).
Clinical characteristics of the study cohort
Abbreviations: AF, atrial fibrillation; BMI, body mass index; BSA, body surface area; eGFR, estimated glomerular filtration rate; LA, left atrial; LVAs, low voltage areas; RA, right atrial.
Association of LAV/RAV ratio with LVAs
In univariable analysis logistic regression, LAV > RAV was associated with sixxfold risk of LVAs presence (OR 6.083, 95%CI 1.395–26.514, p = .016). In multivariable analysis, after adjustment for CHA2DS2‐VASc score, persistent AF, and heart rate, LAV > RAV remained significantly associated with LVAs (OR 5.981, 95%CI 1.256–28.484, p = .025) (Table 2). Using ROC analysis, LAV > RAV showed moderate prediction of LVAs presence (AUC 0.668, 95%CI 0.585–0.751, p = .001) (Figure 1). Comparing the LAV > RAV with LAV (AUC 0.724, 95% CI 0.639–0.809, p < .001) using DeLong's method, the difference between ROC curves was not significant (p = .353).
Prediction of LVAs using LAV/RAV and LA volume
Abbreviations: AF, atrial fibrillation; CI, confidence interval; LAV, left atrial volume; OR, odds ratio; RAV, right atrial volume.
Further adjusted for persistent AF; CHA2DS2‐VASc score and heart frequency.
Association between LVAs and LAV > RAV. AF, atrial fibrillation; AUC, area under the curve; CI, confidence interval; LAV, left atrial volume; RAV, right atrial volume
In univariable analysis logistic regression, LAV > RAV was associated with sixxfold risk of LVAs presence (OR 6.083, 95%CI 1.395–26.514, p = .016). In multivariable analysis, after adjustment for CHA2DS2‐VASc score, persistent AF, and heart rate, LAV > RAV remained significantly associated with LVAs (OR 5.981, 95%CI 1.256–28.484, p = .025) (Table 2). Using ROC analysis, LAV > RAV showed moderate prediction of LVAs presence (AUC 0.668, 95%CI 0.585–0.751, p = .001) (Figure 1). Comparing the LAV > RAV with LAV (AUC 0.724, 95% CI 0.639–0.809, p < .001) using DeLong's method, the difference between ROC curves was not significant (p = .353).
Prediction of LVAs using LAV/RAV and LA volume
Abbreviations: AF, atrial fibrillation; CI, confidence interval; LAV, left atrial volume; OR, odds ratio; RAV, right atrial volume.
Further adjusted for persistent AF; CHA2DS2‐VASc score and heart frequency.
Association between LVAs and LAV > RAV. AF, atrial fibrillation; AUC, area under the curve; CI, confidence interval; LAV, left atrial volume; RAV, right atrial volume
Clinical factors associated with RAV ≥ LAV
In univariable analysis, RAV ≥ LAV was associated with younger age (OR 0.946, 95% CI 0.914–0.978, p = .001), male sex (OR 4.462, 95% CI 1.652–12.046, p = .003), and renal function measured as glomerular filtration rate (OR per 1 ml/kg/1.73m2 1.041, 95% CI 1.016–1.066, p = .001, Table 3). In multivariable analysis, the RAV ≥ LAV was 3‐fold higher in males (OR 3.040, 95% CI 1.050–8.802, p = .04).
Clinical factors associated with RAV ≥ LAV
Abbreviations: AF, fibrillation; CI, confidence interval; eGFR, estimated glomerular filtration rate; OR, odds ratio.
Adjusted for age, sex, persistent AF, and eGFR.
In univariable analysis, RAV ≥ LAV was associated with younger age (OR 0.946, 95% CI 0.914–0.978, p = .001), male sex (OR 4.462, 95% CI 1.652–12.046, p = .003), and renal function measured as glomerular filtration rate (OR per 1 ml/kg/1.73m2 1.041, 95% CI 1.016–1.066, p = .001, Table 3). In multivariable analysis, the RAV ≥ LAV was 3‐fold higher in males (OR 3.040, 95% CI 1.050–8.802, p = .04).
Clinical factors associated with RAV ≥ LAV
Abbreviations: AF, fibrillation; CI, confidence interval; eGFR, estimated glomerular filtration rate; OR, odds ratio.
Adjusted for age, sex, persistent AF, and eGFR.
|
CONCLUSIONS
|
LAV/RAV ratio is useful parameter predicting electro‐anatomical substrate in AF. LAV > RAV was associated with sixfold risk for LVAs presence, while male sex was associated with RAV ≥ LAV and less LVAs.
|
[
"INTRODUCTION",
"Cardiovascular magnetic resonance",
"Peri‐procedural LA mapping and AF ablation",
"Statistical analysis",
"Clinical characteristics of the study population",
"Association of LAV/RAV ratio with LVAs\n",
"Clinical factors associated with RAV ≥ LAV\n",
"Biatrial ratio as parameter for AF progression and LVAs prediction",
"Clinical implications",
"Future directions",
"Limitations"
] |
[
"Becoming a cornerstone therapy in many patients with atrial fibrillation (AF),\n1\n in some patients, catheter ablation with circumferential pulmonary vein isolation alone is not enough for sinus rhythm maintenance during follow‐up. The arrhythmia recurrences remain an important clinical challenge and require individualized AF treatment plan already before intervention. One major feature reflecting left atrial (LA) remodeling is peri‐procedural evidence of low voltage areas (LVAs).\n2\n At least 20%–25% of AF patients have significant LVAs in peri‐procedural mapping, which are an important characteristic of AF progression and treatment failure if not treated with additional ablation.\n2\n, \n3\n Therefore, assessment of LVAs presence before catheter ablation is an important task for the electrophysiologist allowing individually tailored AF ablation therapy.\nThe LA size is another important parameter of AF progression. The role of the LA volume (LAV) and LA function as surrogate parameters for higher AF burden\n4\n and LVAs presence\n5\n, \n6\n are well described. In addition, there is an association between atrial flutter—a right atrial (RA), and AF—a left atrial disease.\n7\n, \n8\n Several studies described an importance of RA assessment as a prognosis marker in heart failure, pulmonary hypertension, and chronic obstructive pulmonary disease.\n9\n, \n10\n, \n11\n Diastolic functional changes—as a preliminary stage for AF—appeared to occur earlier in the right chambers\n12\n suggesting that RA dilatation might be an early marker for atrial remodeling associated with AF initiation. Previous studies reported the importance of increased RA volume (RAV) as predictor for arrhythmia recurrences in AF patients.\n13\n, \n14\n It was hypothesized that RA is more prone to hemodynamic changes and is a more responsive marker of structural remodeling.\n13\n However, association between RAV and LVAs and the prediction capability for LAV/RAV ratio is unknown. Therefore, we aimed to investigate the indexed LAV/RAV ratio assessed in cardiovascular magnetic resonance (CMR) imaging and the association with LVAs in patients undergoing AF catheter ablation. We hypothesize that the biatrial ratio is an independent predictor for LVAs presence.",
"Prior to AF catheter ablation, all patients underwent 1.5 T CMR (Ingenia, Philips Medical) for LA anatomy assessment as previously described.\n5\n Briefly, LAV was determined using a biplane model based on cine 4‐ and 2‐chamber views, and RAV using a monoplane model based on the cine 4‐chamber view. Both volumes were indexed to body surface areas, and the LAV/RAV ratio was calculated before ablation. We defined two subgroups according to the LAV/RAV ratio: (1) LAV is greater than RAV (LAV > RAV), and (2) RAV is equal or greater than LAV (RAV ≥ LAV).",
"Transseptal access and catheter navigation were performed with a steerable sheath (Agilis, St. Jude Medical, St. Paul, MN). The electro‐anatomical mapping was performed in sinus rhythm as described previously.\n5\n In case of AF at the beginning of the procedure, the arrhythmia was terminated by electrical cardioversion and the mapping was performed in sinus rhythm.\nMultielectrode spiral mapping catheters (Reflexion Spiral and Advisor, St Jude Medical [SJM], Saint Paul, MN in NavX Ensite procedures and Carto Lasso, Biosense Webster, Diamond Bar, CA in Carto3 procedures) were used to generate electro‐anatomical voltage maps of the LA. The cutoff value for LVAs was defined as bipolar signal amplitude <0.5 mV. Ectopic beats were excluded from the voltage map. The number of points obtained was >1000. In case of AF recurrence during electro‐anatomical mapping, only anatomical map was assessed. Then the electro‐anatomical mapping was completed in sinus rhythm after PVI, and if still needed, after further cardioversion.\nAll patients received circumferential ablation lines around the antrum of the ipsilateral pulmonary veins. The ablation catheters (irrigated tip catheter and power of 25–40 W) used in NavX Ensite procedures were TactiCath (St Jude Medical [SJM], Saint Paul, MN) and for Carto3 procedures SmartTouch Thermocool (Biosense Webster, Diamond Bar, CA). End point of catheter ablation was PV isolation, which was verified with a multipolar circular mapping catheter. Additional linear lesions were added between if LVAs were present in this area. To be considered as relevant the LVAs ought to consist of adjacent low‐amplitude mapping points, bearing certain additional characteristics such as fragmentation and duration. Finally, the relevance of the LVAs was evaluated by experienced operators based on the mapping point qualities as well as induction of extra‐PV macro‐reentry tachycardia, in which case additional linear ablation was performed. Patients with small/negligible LVAs dispersely distributed in LA and not suitable for ablation, who received the PVI only without additional ablation lines, were excluded.",
"Data are presented as mean and standard deviation for normally distributed or median (interquartile range, 25th and 75th percentiles) for skewed continuous variables, and as proportions for categorical variables. The differences between continuous values were assessed using an unpaired t‐test or the Mann–Whitney, and a χ\n2 test for categorical variables.\nLogistic regression analysis was used to identify factors associated with LVAs. We performed three analyses using logistic regression of LVAs presence (Model 1 – unadjusted analysis; Model 2 – adjusted for age and sex; and Model 3 – adjusted for persistent AF, heart rate, and CHA2DS2‐VASc score.\nReceiver operating characteristic curves (ROC) were generated to analyze performance of the LAV/RAV ratio predicting LVAs, with the area under the curve (AUC) being equivalent to the c‐index for determining the predictive value for the parameters. Finally, we compared the c‐indices (i.e., areas under the ROC curves) of LAV > RAV and LAV using DeLong's method.\n16\n A p‐value <.05 was considered statistically significant. All analyses were performed with SPSS statistical software version 26 (SPSS Inc., Chicago).",
"The study population included 189 patients (mean 63 ± 10 years, 33% women, 57% persistent AF) undergoing their first AF catheter ablation. Clinical characteristics of the study population are summarized in Table 1. Forty‐one patients (22%) had LVAs in periprocedural mapping, which required additional substrate modification. We observed LAV > RAV in 149 (79%) patients (26% with LVAs), while 40 (21%) patients had RAV≥LAV (5% with LVAs). Patients with LAV > RAV were significantly older, more often females, had a lower estimated glomerular filtration rate, had more often hypertension and more often LVAs (Table 1).\nClinical characteristics of the study cohort\nAbbreviations: AF, atrial fibrillation; BMI, body mass index; BSA, body surface area; eGFR, estimated glomerular filtration rate; LA, left atrial; LVAs, low voltage areas; RA, right atrial.",
"In univariable analysis logistic regression, LAV > RAV was associated with sixxfold risk of LVAs presence (OR 6.083, 95%CI 1.395–26.514, p = .016). In multivariable analysis, after adjustment for CHA2DS2‐VASc score, persistent AF, and heart rate, LAV > RAV remained significantly associated with LVAs (OR 5.981, 95%CI 1.256–28.484, p = .025) (Table 2). Using ROC analysis, LAV > RAV showed moderate prediction of LVAs presence (AUC 0.668, 95%CI 0.585–0.751, p = .001) (Figure 1). Comparing the LAV > RAV with LAV (AUC 0.724, 95% CI 0.639–0.809, p < .001) using DeLong's method, the difference between ROC curves was not significant (p = .353).\nPrediction of LVAs using LAV/RAV and LA volume\nAbbreviations: AF, atrial fibrillation; CI, confidence interval; LAV, left atrial volume; OR, odds ratio; RAV, right atrial volume.\nFurther adjusted for persistent AF; CHA2DS2‐VASc score and heart frequency.\nAssociation between LVAs and LAV > RAV. AF, atrial fibrillation; AUC, area under the curve; CI, confidence interval; LAV, left atrial volume; RAV, right atrial volume",
"In univariable analysis, RAV ≥ LAV was associated with younger age (OR 0.946, 95% CI 0.914–0.978, p = .001), male sex (OR 4.462, 95% CI 1.652–12.046, p = .003), and renal function measured as glomerular filtration rate (OR per 1 ml/kg/1.73m2 1.041, 95% CI 1.016–1.066, p = .001, Table 3). In multivariable analysis, the RAV ≥ LAV was 3‐fold higher in males (OR 3.040, 95% CI 1.050–8.802, p = .04).\nClinical factors associated with RAV ≥ LAV\nAbbreviations: AF, fibrillation; CI, confidence interval; eGFR, estimated glomerular filtration rate; OR, odds ratio.\nAdjusted for age, sex, persistent AF, and eGFR.",
"The role of RA size in AF pathogenesis is controversial. It had been reported that RAV and the RAV/LAV ratio were predictive for AF recurrence after PVI in patients with persistent AF, while LAV was not.\n14\n Another study confirmed these findings in AF patients after cardioversion.\n13\n Both studies suggested that AF is a biatrial disease, not being exclusively associated with isolated LA remodeling. However, other studies reported only moderate\n17\n or weak\n18\n prediction of AF recurrences using echocardiographic RA diameter. Our study contradicts previous results and shows that RAV ≥ LAV was associated with less LVAs presence, suggesting a rather minor role in LA remodeling. However, we found that males had threefold higher odds for RAV ≥ LAV and less LVAs. Taking into account that females are more prone to LVAs compared to male\n19\n and have more often‐unfavorable outcomes after catheter ablation, our findings are in line with previous research calling for an attention and an urgent need to address underrepresentation of females in clinical research.",
"LA diameter (LAD) is an acknowledged marker of advanced electro‐anatomical remodeling.\n2\n, \n20\n, \n21\n LA remodeling is associated with increased atrial volume, interstitial fibrosis, and increased myocardial stretch favoring AF sustainability.\n22\n Previously, we reported that besides anteroposterior LAD, the LAV assessed in CMR is a strong predictor for LVAs presence.\n6\n In current analysis we confirm the role of LA in AF pathogenesis showing that LAV > RAV was associated with sixfold risk for LVAs presence. Although LAV alone showed better predictive value than LAV > RAV (AUC 0.724 vs. 0.668), the difference between ROC curves was not significant. However, the risk of LVAs presence was more obvious using LAV/RAV ratio than LAV alone (OR 5.98 vs. 1.03). Our results indicate that the LA enlargement indexed for the RAV (as self‐reference for enlargement) as reflected with the LAV/RAV ratio is a helpful tool for LVAs prediction and for shaping an individualized AF management approach prior to AF catheter ablation.\nThe present findings add to our knowledge about the importance of side‐specific atrial remodeling. As previously described, LA remodeling is associated with later stages of AF progression resulting from risk factors like aging, hypertension, left ventricular (LV) diastolic dysfunction and an altered electromechanical activation.\n23\n, \n24\n, \n25\n, \n26\n LV stiffness results into higher LA pressure with reduced LA emptying and consequent atrial dilatation.\n23\n Described pathologic changes represent a common pathway associated with interatrial delay seen as biphasic P‐wave in ECG and caused by deterioration of the Bachmann bundle conduction, and finally impaired electromechanical LA activation.\n25\n These pathophysiologic changes contribute to advanced remodeling and wall deformation that has been associated with LVA.\n27\n Our study supplement these findings of side specific pathophysiological LA changes (especially in relation to RAV) and emphasize the need for accurate pre‐procedural LA assessment.\nIn contrast, pathophysiology of RA remodeling seems to be different. In patients without heart failure, volume and pressure overload in the RA is mainly associated with pulmonary resistance, valvular disease, and RV dysfunction.\n28\n, \n29\n, \n30\n Although AF may contribute to RA dilation as well,\n31\n AF triggers from the RA are rare\n32\n and RA ablation in AF patients has not shown any benefit for outcomes.\n33\n In our study, RAV > LAV was higher in males and was not associated with LVAs. An explanation for such sex‐specific difference remains unknown, but it is in accordance with large echocardiographic studies and has not been assigned any clinical significance.\n34\n, \n35\n, \n36\n\n",
"Our findings show a strong association between LVAs and LAV/RAV ratio. Despite previous data reporting a correlation between RAV ≥ LAV and AF recurrences, our results imply that increased RAV is not a suitable parameter for LVAs prediction and therefore not a marker for left atrial myopathy. This is in line with our previous studies that emphasized the importance of LV diastolic dysfunction, electro‐anatomical dysfunction for asymmetric LA remodeling, and ablation outcomes.\n23\n, \n24\n, \n25\n Future studies should thus focus on the LA size and its proportional enlargement in relation to the RA, when assessing patients prior to an AF ablation procedure.",
"Several limitations could impact interpretation of our results. First, the study is relatively small and included only 33% women. Therefore, statistical analysis for females is very likely underpowered. Similarly, we cannot exclude that male sex is a possible effect modifier analyzing an impact of sex for RAV ≥ LAV. Of note, the study included only patients of European ancestry from a localized area in Eastern Germany, limiting transferability of the results to other ethnic populations. Furthermore, although LVAs are one of parameters describing atrial myopathy, other parameters such as extrapulmonary substrate and triggers or typical atrial flutter, impairing AF ablation outcome, were not assessed. We did not consider arrhythmia recurrences as an outcome after AF catheter ablation due to several issues: (1) In our opinion LVAs are more robust outcome than arrhythmia recurrences, which occur within weeks/months after catheter ablation, while LVAs are measurable during the procedure; (2) Arrhythmia recurrences depend on many factors such as ablator experience, follow‐up strategy including antiarrhythmic drug prescription, follow‐up frequency, ECG monitoring; (3) In our study, patients did not have a continuous rhythm monitoring. Finally, specific LA fibrosis assessment using late gadolinium enhancement was not conducted in current study and should be addressed in future studies."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"METHODS",
"Cardiovascular magnetic resonance",
"Peri‐procedural LA mapping and AF ablation",
"Statistical analysis",
"RESULTS",
"Clinical characteristics of the study population",
"Association of LAV/RAV ratio with LVAs\n",
"Clinical factors associated with RAV ≥ LAV\n",
"DISCUSSION",
"Biatrial ratio as parameter for AF progression and LVAs prediction",
"Clinical implications",
"Future directions",
"Limitations",
"CONCLUSIONS",
"CONFLICT OF INTEREST"
] |
[
"Becoming a cornerstone therapy in many patients with atrial fibrillation (AF),\n1\n in some patients, catheter ablation with circumferential pulmonary vein isolation alone is not enough for sinus rhythm maintenance during follow‐up. The arrhythmia recurrences remain an important clinical challenge and require individualized AF treatment plan already before intervention. One major feature reflecting left atrial (LA) remodeling is peri‐procedural evidence of low voltage areas (LVAs).\n2\n At least 20%–25% of AF patients have significant LVAs in peri‐procedural mapping, which are an important characteristic of AF progression and treatment failure if not treated with additional ablation.\n2\n, \n3\n Therefore, assessment of LVAs presence before catheter ablation is an important task for the electrophysiologist allowing individually tailored AF ablation therapy.\nThe LA size is another important parameter of AF progression. The role of the LA volume (LAV) and LA function as surrogate parameters for higher AF burden\n4\n and LVAs presence\n5\n, \n6\n are well described. In addition, there is an association between atrial flutter—a right atrial (RA), and AF—a left atrial disease.\n7\n, \n8\n Several studies described an importance of RA assessment as a prognosis marker in heart failure, pulmonary hypertension, and chronic obstructive pulmonary disease.\n9\n, \n10\n, \n11\n Diastolic functional changes—as a preliminary stage for AF—appeared to occur earlier in the right chambers\n12\n suggesting that RA dilatation might be an early marker for atrial remodeling associated with AF initiation. Previous studies reported the importance of increased RA volume (RAV) as predictor for arrhythmia recurrences in AF patients.\n13\n, \n14\n It was hypothesized that RA is more prone to hemodynamic changes and is a more responsive marker of structural remodeling.\n13\n However, association between RAV and LVAs and the prediction capability for LAV/RAV ratio is unknown. Therefore, we aimed to investigate the indexed LAV/RAV ratio assessed in cardiovascular magnetic resonance (CMR) imaging and the association with LVAs in patients undergoing AF catheter ablation. We hypothesize that the biatrial ratio is an independent predictor for LVAs presence.",
"The study population was described previously.\n6\n Briefly, patients presenting for catheter ablation due to symptomatic AF from October 2015 to April 2017 were included in the study. According to current guidelines, AF subtypes were defined as paroxysmal and persistent.\n15\n Patients with pregnancy, age <18 or >75 years, valvular AF (any valvulopathies >second degree), cancer, acute, or systemic inflammatory diseases, and acute hyperthyreotic state were excluded from the study. The study was approved by the local Ethical Committee (Medical Faculty, University of Leipzig), and patients provided written informed consent for participation.\n Cardiovascular magnetic resonance Prior to AF catheter ablation, all patients underwent 1.5 T CMR (Ingenia, Philips Medical) for LA anatomy assessment as previously described.\n5\n Briefly, LAV was determined using a biplane model based on cine 4‐ and 2‐chamber views, and RAV using a monoplane model based on the cine 4‐chamber view. Both volumes were indexed to body surface areas, and the LAV/RAV ratio was calculated before ablation. We defined two subgroups according to the LAV/RAV ratio: (1) LAV is greater than RAV (LAV > RAV), and (2) RAV is equal or greater than LAV (RAV ≥ LAV).\nPrior to AF catheter ablation, all patients underwent 1.5 T CMR (Ingenia, Philips Medical) for LA anatomy assessment as previously described.\n5\n Briefly, LAV was determined using a biplane model based on cine 4‐ and 2‐chamber views, and RAV using a monoplane model based on the cine 4‐chamber view. Both volumes were indexed to body surface areas, and the LAV/RAV ratio was calculated before ablation. We defined two subgroups according to the LAV/RAV ratio: (1) LAV is greater than RAV (LAV > RAV), and (2) RAV is equal or greater than LAV (RAV ≥ LAV).\n Peri‐procedural LA mapping and AF ablation Transseptal access and catheter navigation were performed with a steerable sheath (Agilis, St. Jude Medical, St. Paul, MN). The electro‐anatomical mapping was performed in sinus rhythm as described previously.\n5\n In case of AF at the beginning of the procedure, the arrhythmia was terminated by electrical cardioversion and the mapping was performed in sinus rhythm.\nMultielectrode spiral mapping catheters (Reflexion Spiral and Advisor, St Jude Medical [SJM], Saint Paul, MN in NavX Ensite procedures and Carto Lasso, Biosense Webster, Diamond Bar, CA in Carto3 procedures) were used to generate electro‐anatomical voltage maps of the LA. The cutoff value for LVAs was defined as bipolar signal amplitude <0.5 mV. Ectopic beats were excluded from the voltage map. The number of points obtained was >1000. In case of AF recurrence during electro‐anatomical mapping, only anatomical map was assessed. Then the electro‐anatomical mapping was completed in sinus rhythm after PVI, and if still needed, after further cardioversion.\nAll patients received circumferential ablation lines around the antrum of the ipsilateral pulmonary veins. The ablation catheters (irrigated tip catheter and power of 25–40 W) used in NavX Ensite procedures were TactiCath (St Jude Medical [SJM], Saint Paul, MN) and for Carto3 procedures SmartTouch Thermocool (Biosense Webster, Diamond Bar, CA). End point of catheter ablation was PV isolation, which was verified with a multipolar circular mapping catheter. Additional linear lesions were added between if LVAs were present in this area. To be considered as relevant the LVAs ought to consist of adjacent low‐amplitude mapping points, bearing certain additional characteristics such as fragmentation and duration. Finally, the relevance of the LVAs was evaluated by experienced operators based on the mapping point qualities as well as induction of extra‐PV macro‐reentry tachycardia, in which case additional linear ablation was performed. Patients with small/negligible LVAs dispersely distributed in LA and not suitable for ablation, who received the PVI only without additional ablation lines, were excluded.\nTransseptal access and catheter navigation were performed with a steerable sheath (Agilis, St. Jude Medical, St. Paul, MN). The electro‐anatomical mapping was performed in sinus rhythm as described previously.\n5\n In case of AF at the beginning of the procedure, the arrhythmia was terminated by electrical cardioversion and the mapping was performed in sinus rhythm.\nMultielectrode spiral mapping catheters (Reflexion Spiral and Advisor, St Jude Medical [SJM], Saint Paul, MN in NavX Ensite procedures and Carto Lasso, Biosense Webster, Diamond Bar, CA in Carto3 procedures) were used to generate electro‐anatomical voltage maps of the LA. The cutoff value for LVAs was defined as bipolar signal amplitude <0.5 mV. Ectopic beats were excluded from the voltage map. The number of points obtained was >1000. In case of AF recurrence during electro‐anatomical mapping, only anatomical map was assessed. Then the electro‐anatomical mapping was completed in sinus rhythm after PVI, and if still needed, after further cardioversion.\nAll patients received circumferential ablation lines around the antrum of the ipsilateral pulmonary veins. The ablation catheters (irrigated tip catheter and power of 25–40 W) used in NavX Ensite procedures were TactiCath (St Jude Medical [SJM], Saint Paul, MN) and for Carto3 procedures SmartTouch Thermocool (Biosense Webster, Diamond Bar, CA). End point of catheter ablation was PV isolation, which was verified with a multipolar circular mapping catheter. Additional linear lesions were added between if LVAs were present in this area. To be considered as relevant the LVAs ought to consist of adjacent low‐amplitude mapping points, bearing certain additional characteristics such as fragmentation and duration. Finally, the relevance of the LVAs was evaluated by experienced operators based on the mapping point qualities as well as induction of extra‐PV macro‐reentry tachycardia, in which case additional linear ablation was performed. Patients with small/negligible LVAs dispersely distributed in LA and not suitable for ablation, who received the PVI only without additional ablation lines, were excluded.\n Statistical analysis Data are presented as mean and standard deviation for normally distributed or median (interquartile range, 25th and 75th percentiles) for skewed continuous variables, and as proportions for categorical variables. The differences between continuous values were assessed using an unpaired t‐test or the Mann–Whitney, and a χ\n2 test for categorical variables.\nLogistic regression analysis was used to identify factors associated with LVAs. We performed three analyses using logistic regression of LVAs presence (Model 1 – unadjusted analysis; Model 2 – adjusted for age and sex; and Model 3 – adjusted for persistent AF, heart rate, and CHA2DS2‐VASc score.\nReceiver operating characteristic curves (ROC) were generated to analyze performance of the LAV/RAV ratio predicting LVAs, with the area under the curve (AUC) being equivalent to the c‐index for determining the predictive value for the parameters. Finally, we compared the c‐indices (i.e., areas under the ROC curves) of LAV > RAV and LAV using DeLong's method.\n16\n A p‐value <.05 was considered statistically significant. All analyses were performed with SPSS statistical software version 26 (SPSS Inc., Chicago).\nData are presented as mean and standard deviation for normally distributed or median (interquartile range, 25th and 75th percentiles) for skewed continuous variables, and as proportions for categorical variables. The differences between continuous values were assessed using an unpaired t‐test or the Mann–Whitney, and a χ\n2 test for categorical variables.\nLogistic regression analysis was used to identify factors associated with LVAs. We performed three analyses using logistic regression of LVAs presence (Model 1 – unadjusted analysis; Model 2 – adjusted for age and sex; and Model 3 – adjusted for persistent AF, heart rate, and CHA2DS2‐VASc score.\nReceiver operating characteristic curves (ROC) were generated to analyze performance of the LAV/RAV ratio predicting LVAs, with the area under the curve (AUC) being equivalent to the c‐index for determining the predictive value for the parameters. Finally, we compared the c‐indices (i.e., areas under the ROC curves) of LAV > RAV and LAV using DeLong's method.\n16\n A p‐value <.05 was considered statistically significant. All analyses were performed with SPSS statistical software version 26 (SPSS Inc., Chicago).",
"Prior to AF catheter ablation, all patients underwent 1.5 T CMR (Ingenia, Philips Medical) for LA anatomy assessment as previously described.\n5\n Briefly, LAV was determined using a biplane model based on cine 4‐ and 2‐chamber views, and RAV using a monoplane model based on the cine 4‐chamber view. Both volumes were indexed to body surface areas, and the LAV/RAV ratio was calculated before ablation. We defined two subgroups according to the LAV/RAV ratio: (1) LAV is greater than RAV (LAV > RAV), and (2) RAV is equal or greater than LAV (RAV ≥ LAV).",
"Transseptal access and catheter navigation were performed with a steerable sheath (Agilis, St. Jude Medical, St. Paul, MN). The electro‐anatomical mapping was performed in sinus rhythm as described previously.\n5\n In case of AF at the beginning of the procedure, the arrhythmia was terminated by electrical cardioversion and the mapping was performed in sinus rhythm.\nMultielectrode spiral mapping catheters (Reflexion Spiral and Advisor, St Jude Medical [SJM], Saint Paul, MN in NavX Ensite procedures and Carto Lasso, Biosense Webster, Diamond Bar, CA in Carto3 procedures) were used to generate electro‐anatomical voltage maps of the LA. The cutoff value for LVAs was defined as bipolar signal amplitude <0.5 mV. Ectopic beats were excluded from the voltage map. The number of points obtained was >1000. In case of AF recurrence during electro‐anatomical mapping, only anatomical map was assessed. Then the electro‐anatomical mapping was completed in sinus rhythm after PVI, and if still needed, after further cardioversion.\nAll patients received circumferential ablation lines around the antrum of the ipsilateral pulmonary veins. The ablation catheters (irrigated tip catheter and power of 25–40 W) used in NavX Ensite procedures were TactiCath (St Jude Medical [SJM], Saint Paul, MN) and for Carto3 procedures SmartTouch Thermocool (Biosense Webster, Diamond Bar, CA). End point of catheter ablation was PV isolation, which was verified with a multipolar circular mapping catheter. Additional linear lesions were added between if LVAs were present in this area. To be considered as relevant the LVAs ought to consist of adjacent low‐amplitude mapping points, bearing certain additional characteristics such as fragmentation and duration. Finally, the relevance of the LVAs was evaluated by experienced operators based on the mapping point qualities as well as induction of extra‐PV macro‐reentry tachycardia, in which case additional linear ablation was performed. Patients with small/negligible LVAs dispersely distributed in LA and not suitable for ablation, who received the PVI only without additional ablation lines, were excluded.",
"Data are presented as mean and standard deviation for normally distributed or median (interquartile range, 25th and 75th percentiles) for skewed continuous variables, and as proportions for categorical variables. The differences between continuous values were assessed using an unpaired t‐test or the Mann–Whitney, and a χ\n2 test for categorical variables.\nLogistic regression analysis was used to identify factors associated with LVAs. We performed three analyses using logistic regression of LVAs presence (Model 1 – unadjusted analysis; Model 2 – adjusted for age and sex; and Model 3 – adjusted for persistent AF, heart rate, and CHA2DS2‐VASc score.\nReceiver operating characteristic curves (ROC) were generated to analyze performance of the LAV/RAV ratio predicting LVAs, with the area under the curve (AUC) being equivalent to the c‐index for determining the predictive value for the parameters. Finally, we compared the c‐indices (i.e., areas under the ROC curves) of LAV > RAV and LAV using DeLong's method.\n16\n A p‐value <.05 was considered statistically significant. All analyses were performed with SPSS statistical software version 26 (SPSS Inc., Chicago).",
" Clinical characteristics of the study population The study population included 189 patients (mean 63 ± 10 years, 33% women, 57% persistent AF) undergoing their first AF catheter ablation. Clinical characteristics of the study population are summarized in Table 1. Forty‐one patients (22%) had LVAs in periprocedural mapping, which required additional substrate modification. We observed LAV > RAV in 149 (79%) patients (26% with LVAs), while 40 (21%) patients had RAV≥LAV (5% with LVAs). Patients with LAV > RAV were significantly older, more often females, had a lower estimated glomerular filtration rate, had more often hypertension and more often LVAs (Table 1).\nClinical characteristics of the study cohort\nAbbreviations: AF, atrial fibrillation; BMI, body mass index; BSA, body surface area; eGFR, estimated glomerular filtration rate; LA, left atrial; LVAs, low voltage areas; RA, right atrial.\nThe study population included 189 patients (mean 63 ± 10 years, 33% women, 57% persistent AF) undergoing their first AF catheter ablation. Clinical characteristics of the study population are summarized in Table 1. Forty‐one patients (22%) had LVAs in periprocedural mapping, which required additional substrate modification. We observed LAV > RAV in 149 (79%) patients (26% with LVAs), while 40 (21%) patients had RAV≥LAV (5% with LVAs). Patients with LAV > RAV were significantly older, more often females, had a lower estimated glomerular filtration rate, had more often hypertension and more often LVAs (Table 1).\nClinical characteristics of the study cohort\nAbbreviations: AF, atrial fibrillation; BMI, body mass index; BSA, body surface area; eGFR, estimated glomerular filtration rate; LA, left atrial; LVAs, low voltage areas; RA, right atrial.\n Association of LAV/RAV ratio with LVAs\n In univariable analysis logistic regression, LAV > RAV was associated with sixxfold risk of LVAs presence (OR 6.083, 95%CI 1.395–26.514, p = .016). In multivariable analysis, after adjustment for CHA2DS2‐VASc score, persistent AF, and heart rate, LAV > RAV remained significantly associated with LVAs (OR 5.981, 95%CI 1.256–28.484, p = .025) (Table 2). Using ROC analysis, LAV > RAV showed moderate prediction of LVAs presence (AUC 0.668, 95%CI 0.585–0.751, p = .001) (Figure 1). Comparing the LAV > RAV with LAV (AUC 0.724, 95% CI 0.639–0.809, p < .001) using DeLong's method, the difference between ROC curves was not significant (p = .353).\nPrediction of LVAs using LAV/RAV and LA volume\nAbbreviations: AF, atrial fibrillation; CI, confidence interval; LAV, left atrial volume; OR, odds ratio; RAV, right atrial volume.\nFurther adjusted for persistent AF; CHA2DS2‐VASc score and heart frequency.\nAssociation between LVAs and LAV > RAV. AF, atrial fibrillation; AUC, area under the curve; CI, confidence interval; LAV, left atrial volume; RAV, right atrial volume\nIn univariable analysis logistic regression, LAV > RAV was associated with sixxfold risk of LVAs presence (OR 6.083, 95%CI 1.395–26.514, p = .016). In multivariable analysis, after adjustment for CHA2DS2‐VASc score, persistent AF, and heart rate, LAV > RAV remained significantly associated with LVAs (OR 5.981, 95%CI 1.256–28.484, p = .025) (Table 2). Using ROC analysis, LAV > RAV showed moderate prediction of LVAs presence (AUC 0.668, 95%CI 0.585–0.751, p = .001) (Figure 1). Comparing the LAV > RAV with LAV (AUC 0.724, 95% CI 0.639–0.809, p < .001) using DeLong's method, the difference between ROC curves was not significant (p = .353).\nPrediction of LVAs using LAV/RAV and LA volume\nAbbreviations: AF, atrial fibrillation; CI, confidence interval; LAV, left atrial volume; OR, odds ratio; RAV, right atrial volume.\nFurther adjusted for persistent AF; CHA2DS2‐VASc score and heart frequency.\nAssociation between LVAs and LAV > RAV. AF, atrial fibrillation; AUC, area under the curve; CI, confidence interval; LAV, left atrial volume; RAV, right atrial volume\n Clinical factors associated with RAV ≥ LAV\n In univariable analysis, RAV ≥ LAV was associated with younger age (OR 0.946, 95% CI 0.914–0.978, p = .001), male sex (OR 4.462, 95% CI 1.652–12.046, p = .003), and renal function measured as glomerular filtration rate (OR per 1 ml/kg/1.73m2 1.041, 95% CI 1.016–1.066, p = .001, Table 3). In multivariable analysis, the RAV ≥ LAV was 3‐fold higher in males (OR 3.040, 95% CI 1.050–8.802, p = .04).\nClinical factors associated with RAV ≥ LAV\nAbbreviations: AF, fibrillation; CI, confidence interval; eGFR, estimated glomerular filtration rate; OR, odds ratio.\nAdjusted for age, sex, persistent AF, and eGFR.\nIn univariable analysis, RAV ≥ LAV was associated with younger age (OR 0.946, 95% CI 0.914–0.978, p = .001), male sex (OR 4.462, 95% CI 1.652–12.046, p = .003), and renal function measured as glomerular filtration rate (OR per 1 ml/kg/1.73m2 1.041, 95% CI 1.016–1.066, p = .001, Table 3). In multivariable analysis, the RAV ≥ LAV was 3‐fold higher in males (OR 3.040, 95% CI 1.050–8.802, p = .04).\nClinical factors associated with RAV ≥ LAV\nAbbreviations: AF, fibrillation; CI, confidence interval; eGFR, estimated glomerular filtration rate; OR, odds ratio.\nAdjusted for age, sex, persistent AF, and eGFR.",
"The study population included 189 patients (mean 63 ± 10 years, 33% women, 57% persistent AF) undergoing their first AF catheter ablation. Clinical characteristics of the study population are summarized in Table 1. Forty‐one patients (22%) had LVAs in periprocedural mapping, which required additional substrate modification. We observed LAV > RAV in 149 (79%) patients (26% with LVAs), while 40 (21%) patients had RAV≥LAV (5% with LVAs). Patients with LAV > RAV were significantly older, more often females, had a lower estimated glomerular filtration rate, had more often hypertension and more often LVAs (Table 1).\nClinical characteristics of the study cohort\nAbbreviations: AF, atrial fibrillation; BMI, body mass index; BSA, body surface area; eGFR, estimated glomerular filtration rate; LA, left atrial; LVAs, low voltage areas; RA, right atrial.",
"In univariable analysis logistic regression, LAV > RAV was associated with sixxfold risk of LVAs presence (OR 6.083, 95%CI 1.395–26.514, p = .016). In multivariable analysis, after adjustment for CHA2DS2‐VASc score, persistent AF, and heart rate, LAV > RAV remained significantly associated with LVAs (OR 5.981, 95%CI 1.256–28.484, p = .025) (Table 2). Using ROC analysis, LAV > RAV showed moderate prediction of LVAs presence (AUC 0.668, 95%CI 0.585–0.751, p = .001) (Figure 1). Comparing the LAV > RAV with LAV (AUC 0.724, 95% CI 0.639–0.809, p < .001) using DeLong's method, the difference between ROC curves was not significant (p = .353).\nPrediction of LVAs using LAV/RAV and LA volume\nAbbreviations: AF, atrial fibrillation; CI, confidence interval; LAV, left atrial volume; OR, odds ratio; RAV, right atrial volume.\nFurther adjusted for persistent AF; CHA2DS2‐VASc score and heart frequency.\nAssociation between LVAs and LAV > RAV. AF, atrial fibrillation; AUC, area under the curve; CI, confidence interval; LAV, left atrial volume; RAV, right atrial volume",
"In univariable analysis, RAV ≥ LAV was associated with younger age (OR 0.946, 95% CI 0.914–0.978, p = .001), male sex (OR 4.462, 95% CI 1.652–12.046, p = .003), and renal function measured as glomerular filtration rate (OR per 1 ml/kg/1.73m2 1.041, 95% CI 1.016–1.066, p = .001, Table 3). In multivariable analysis, the RAV ≥ LAV was 3‐fold higher in males (OR 3.040, 95% CI 1.050–8.802, p = .04).\nClinical factors associated with RAV ≥ LAV\nAbbreviations: AF, fibrillation; CI, confidence interval; eGFR, estimated glomerular filtration rate; OR, odds ratio.\nAdjusted for age, sex, persistent AF, and eGFR.",
"In our study, we investigated the indexed LAV/RAV ratio and its predictive value on the pre‐procedural LVAs in patients undergoing AF catheter ablation (Figure 2). We found that LAV > RAV was associated with sixfold risk for LVAs presence. Also, LAV > RAV was less observed in males.\nStudy overview. Central figure describes the study population and main results. Isolated LA dilatation (LAV > RAV) was significantly associated with LVAs, while biatrial/isolated RA dilatation (RAV≥LAV) was associated with male sex. AF, atrial fibrillation; CI, confidence interval; CMR, cardiovascular magnetic resonance imaging; HR, heart rate; LA, left atrial; LAV, left atrial volume; LVAs, low voltage areas; RA, right atrial; RAV, right atrial volume; OR, odds ratio\n Biatrial ratio as parameter for AF progression and LVAs prediction The role of RA size in AF pathogenesis is controversial. It had been reported that RAV and the RAV/LAV ratio were predictive for AF recurrence after PVI in patients with persistent AF, while LAV was not.\n14\n Another study confirmed these findings in AF patients after cardioversion.\n13\n Both studies suggested that AF is a biatrial disease, not being exclusively associated with isolated LA remodeling. However, other studies reported only moderate\n17\n or weak\n18\n prediction of AF recurrences using echocardiographic RA diameter. Our study contradicts previous results and shows that RAV ≥ LAV was associated with less LVAs presence, suggesting a rather minor role in LA remodeling. However, we found that males had threefold higher odds for RAV ≥ LAV and less LVAs. Taking into account that females are more prone to LVAs compared to male\n19\n and have more often‐unfavorable outcomes after catheter ablation, our findings are in line with previous research calling for an attention and an urgent need to address underrepresentation of females in clinical research.\nThe role of RA size in AF pathogenesis is controversial. It had been reported that RAV and the RAV/LAV ratio were predictive for AF recurrence after PVI in patients with persistent AF, while LAV was not.\n14\n Another study confirmed these findings in AF patients after cardioversion.\n13\n Both studies suggested that AF is a biatrial disease, not being exclusively associated with isolated LA remodeling. However, other studies reported only moderate\n17\n or weak\n18\n prediction of AF recurrences using echocardiographic RA diameter. Our study contradicts previous results and shows that RAV ≥ LAV was associated with less LVAs presence, suggesting a rather minor role in LA remodeling. However, we found that males had threefold higher odds for RAV ≥ LAV and less LVAs. Taking into account that females are more prone to LVAs compared to male\n19\n and have more often‐unfavorable outcomes after catheter ablation, our findings are in line with previous research calling for an attention and an urgent need to address underrepresentation of females in clinical research.\n Clinical implications LA diameter (LAD) is an acknowledged marker of advanced electro‐anatomical remodeling.\n2\n, \n20\n, \n21\n LA remodeling is associated with increased atrial volume, interstitial fibrosis, and increased myocardial stretch favoring AF sustainability.\n22\n Previously, we reported that besides anteroposterior LAD, the LAV assessed in CMR is a strong predictor for LVAs presence.\n6\n In current analysis we confirm the role of LA in AF pathogenesis showing that LAV > RAV was associated with sixfold risk for LVAs presence. Although LAV alone showed better predictive value than LAV > RAV (AUC 0.724 vs. 0.668), the difference between ROC curves was not significant. However, the risk of LVAs presence was more obvious using LAV/RAV ratio than LAV alone (OR 5.98 vs. 1.03). Our results indicate that the LA enlargement indexed for the RAV (as self‐reference for enlargement) as reflected with the LAV/RAV ratio is a helpful tool for LVAs prediction and for shaping an individualized AF management approach prior to AF catheter ablation.\nThe present findings add to our knowledge about the importance of side‐specific atrial remodeling. As previously described, LA remodeling is associated with later stages of AF progression resulting from risk factors like aging, hypertension, left ventricular (LV) diastolic dysfunction and an altered electromechanical activation.\n23\n, \n24\n, \n25\n, \n26\n LV stiffness results into higher LA pressure with reduced LA emptying and consequent atrial dilatation.\n23\n Described pathologic changes represent a common pathway associated with interatrial delay seen as biphasic P‐wave in ECG and caused by deterioration of the Bachmann bundle conduction, and finally impaired electromechanical LA activation.\n25\n These pathophysiologic changes contribute to advanced remodeling and wall deformation that has been associated with LVA.\n27\n Our study supplement these findings of side specific pathophysiological LA changes (especially in relation to RAV) and emphasize the need for accurate pre‐procedural LA assessment.\nIn contrast, pathophysiology of RA remodeling seems to be different. In patients without heart failure, volume and pressure overload in the RA is mainly associated with pulmonary resistance, valvular disease, and RV dysfunction.\n28\n, \n29\n, \n30\n Although AF may contribute to RA dilation as well,\n31\n AF triggers from the RA are rare\n32\n and RA ablation in AF patients has not shown any benefit for outcomes.\n33\n In our study, RAV > LAV was higher in males and was not associated with LVAs. An explanation for such sex‐specific difference remains unknown, but it is in accordance with large echocardiographic studies and has not been assigned any clinical significance.\n34\n, \n35\n, \n36\n\n\nLA diameter (LAD) is an acknowledged marker of advanced electro‐anatomical remodeling.\n2\n, \n20\n, \n21\n LA remodeling is associated with increased atrial volume, interstitial fibrosis, and increased myocardial stretch favoring AF sustainability.\n22\n Previously, we reported that besides anteroposterior LAD, the LAV assessed in CMR is a strong predictor for LVAs presence.\n6\n In current analysis we confirm the role of LA in AF pathogenesis showing that LAV > RAV was associated with sixfold risk for LVAs presence. Although LAV alone showed better predictive value than LAV > RAV (AUC 0.724 vs. 0.668), the difference between ROC curves was not significant. However, the risk of LVAs presence was more obvious using LAV/RAV ratio than LAV alone (OR 5.98 vs. 1.03). Our results indicate that the LA enlargement indexed for the RAV (as self‐reference for enlargement) as reflected with the LAV/RAV ratio is a helpful tool for LVAs prediction and for shaping an individualized AF management approach prior to AF catheter ablation.\nThe present findings add to our knowledge about the importance of side‐specific atrial remodeling. As previously described, LA remodeling is associated with later stages of AF progression resulting from risk factors like aging, hypertension, left ventricular (LV) diastolic dysfunction and an altered electromechanical activation.\n23\n, \n24\n, \n25\n, \n26\n LV stiffness results into higher LA pressure with reduced LA emptying and consequent atrial dilatation.\n23\n Described pathologic changes represent a common pathway associated with interatrial delay seen as biphasic P‐wave in ECG and caused by deterioration of the Bachmann bundle conduction, and finally impaired electromechanical LA activation.\n25\n These pathophysiologic changes contribute to advanced remodeling and wall deformation that has been associated with LVA.\n27\n Our study supplement these findings of side specific pathophysiological LA changes (especially in relation to RAV) and emphasize the need for accurate pre‐procedural LA assessment.\nIn contrast, pathophysiology of RA remodeling seems to be different. In patients without heart failure, volume and pressure overload in the RA is mainly associated with pulmonary resistance, valvular disease, and RV dysfunction.\n28\n, \n29\n, \n30\n Although AF may contribute to RA dilation as well,\n31\n AF triggers from the RA are rare\n32\n and RA ablation in AF patients has not shown any benefit for outcomes.\n33\n In our study, RAV > LAV was higher in males and was not associated with LVAs. An explanation for such sex‐specific difference remains unknown, but it is in accordance with large echocardiographic studies and has not been assigned any clinical significance.\n34\n, \n35\n, \n36\n\n\n Future directions Our findings show a strong association between LVAs and LAV/RAV ratio. Despite previous data reporting a correlation between RAV ≥ LAV and AF recurrences, our results imply that increased RAV is not a suitable parameter for LVAs prediction and therefore not a marker for left atrial myopathy. This is in line with our previous studies that emphasized the importance of LV diastolic dysfunction, electro‐anatomical dysfunction for asymmetric LA remodeling, and ablation outcomes.\n23\n, \n24\n, \n25\n Future studies should thus focus on the LA size and its proportional enlargement in relation to the RA, when assessing patients prior to an AF ablation procedure.\nOur findings show a strong association between LVAs and LAV/RAV ratio. Despite previous data reporting a correlation between RAV ≥ LAV and AF recurrences, our results imply that increased RAV is not a suitable parameter for LVAs prediction and therefore not a marker for left atrial myopathy. This is in line with our previous studies that emphasized the importance of LV diastolic dysfunction, electro‐anatomical dysfunction for asymmetric LA remodeling, and ablation outcomes.\n23\n, \n24\n, \n25\n Future studies should thus focus on the LA size and its proportional enlargement in relation to the RA, when assessing patients prior to an AF ablation procedure.\n Limitations Several limitations could impact interpretation of our results. First, the study is relatively small and included only 33% women. Therefore, statistical analysis for females is very likely underpowered. Similarly, we cannot exclude that male sex is a possible effect modifier analyzing an impact of sex for RAV ≥ LAV. Of note, the study included only patients of European ancestry from a localized area in Eastern Germany, limiting transferability of the results to other ethnic populations. Furthermore, although LVAs are one of parameters describing atrial myopathy, other parameters such as extrapulmonary substrate and triggers or typical atrial flutter, impairing AF ablation outcome, were not assessed. We did not consider arrhythmia recurrences as an outcome after AF catheter ablation due to several issues: (1) In our opinion LVAs are more robust outcome than arrhythmia recurrences, which occur within weeks/months after catheter ablation, while LVAs are measurable during the procedure; (2) Arrhythmia recurrences depend on many factors such as ablator experience, follow‐up strategy including antiarrhythmic drug prescription, follow‐up frequency, ECG monitoring; (3) In our study, patients did not have a continuous rhythm monitoring. Finally, specific LA fibrosis assessment using late gadolinium enhancement was not conducted in current study and should be addressed in future studies.\nSeveral limitations could impact interpretation of our results. First, the study is relatively small and included only 33% women. Therefore, statistical analysis for females is very likely underpowered. Similarly, we cannot exclude that male sex is a possible effect modifier analyzing an impact of sex for RAV ≥ LAV. Of note, the study included only patients of European ancestry from a localized area in Eastern Germany, limiting transferability of the results to other ethnic populations. Furthermore, although LVAs are one of parameters describing atrial myopathy, other parameters such as extrapulmonary substrate and triggers or typical atrial flutter, impairing AF ablation outcome, were not assessed. We did not consider arrhythmia recurrences as an outcome after AF catheter ablation due to several issues: (1) In our opinion LVAs are more robust outcome than arrhythmia recurrences, which occur within weeks/months after catheter ablation, while LVAs are measurable during the procedure; (2) Arrhythmia recurrences depend on many factors such as ablator experience, follow‐up strategy including antiarrhythmic drug prescription, follow‐up frequency, ECG monitoring; (3) In our study, patients did not have a continuous rhythm monitoring. Finally, specific LA fibrosis assessment using late gadolinium enhancement was not conducted in current study and should be addressed in future studies.",
"The role of RA size in AF pathogenesis is controversial. It had been reported that RAV and the RAV/LAV ratio were predictive for AF recurrence after PVI in patients with persistent AF, while LAV was not.\n14\n Another study confirmed these findings in AF patients after cardioversion.\n13\n Both studies suggested that AF is a biatrial disease, not being exclusively associated with isolated LA remodeling. However, other studies reported only moderate\n17\n or weak\n18\n prediction of AF recurrences using echocardiographic RA diameter. Our study contradicts previous results and shows that RAV ≥ LAV was associated with less LVAs presence, suggesting a rather minor role in LA remodeling. However, we found that males had threefold higher odds for RAV ≥ LAV and less LVAs. Taking into account that females are more prone to LVAs compared to male\n19\n and have more often‐unfavorable outcomes after catheter ablation, our findings are in line with previous research calling for an attention and an urgent need to address underrepresentation of females in clinical research.",
"LA diameter (LAD) is an acknowledged marker of advanced electro‐anatomical remodeling.\n2\n, \n20\n, \n21\n LA remodeling is associated with increased atrial volume, interstitial fibrosis, and increased myocardial stretch favoring AF sustainability.\n22\n Previously, we reported that besides anteroposterior LAD, the LAV assessed in CMR is a strong predictor for LVAs presence.\n6\n In current analysis we confirm the role of LA in AF pathogenesis showing that LAV > RAV was associated with sixfold risk for LVAs presence. Although LAV alone showed better predictive value than LAV > RAV (AUC 0.724 vs. 0.668), the difference between ROC curves was not significant. However, the risk of LVAs presence was more obvious using LAV/RAV ratio than LAV alone (OR 5.98 vs. 1.03). Our results indicate that the LA enlargement indexed for the RAV (as self‐reference for enlargement) as reflected with the LAV/RAV ratio is a helpful tool for LVAs prediction and for shaping an individualized AF management approach prior to AF catheter ablation.\nThe present findings add to our knowledge about the importance of side‐specific atrial remodeling. As previously described, LA remodeling is associated with later stages of AF progression resulting from risk factors like aging, hypertension, left ventricular (LV) diastolic dysfunction and an altered electromechanical activation.\n23\n, \n24\n, \n25\n, \n26\n LV stiffness results into higher LA pressure with reduced LA emptying and consequent atrial dilatation.\n23\n Described pathologic changes represent a common pathway associated with interatrial delay seen as biphasic P‐wave in ECG and caused by deterioration of the Bachmann bundle conduction, and finally impaired electromechanical LA activation.\n25\n These pathophysiologic changes contribute to advanced remodeling and wall deformation that has been associated with LVA.\n27\n Our study supplement these findings of side specific pathophysiological LA changes (especially in relation to RAV) and emphasize the need for accurate pre‐procedural LA assessment.\nIn contrast, pathophysiology of RA remodeling seems to be different. In patients without heart failure, volume and pressure overload in the RA is mainly associated with pulmonary resistance, valvular disease, and RV dysfunction.\n28\n, \n29\n, \n30\n Although AF may contribute to RA dilation as well,\n31\n AF triggers from the RA are rare\n32\n and RA ablation in AF patients has not shown any benefit for outcomes.\n33\n In our study, RAV > LAV was higher in males and was not associated with LVAs. An explanation for such sex‐specific difference remains unknown, but it is in accordance with large echocardiographic studies and has not been assigned any clinical significance.\n34\n, \n35\n, \n36\n\n",
"Our findings show a strong association between LVAs and LAV/RAV ratio. Despite previous data reporting a correlation between RAV ≥ LAV and AF recurrences, our results imply that increased RAV is not a suitable parameter for LVAs prediction and therefore not a marker for left atrial myopathy. This is in line with our previous studies that emphasized the importance of LV diastolic dysfunction, electro‐anatomical dysfunction for asymmetric LA remodeling, and ablation outcomes.\n23\n, \n24\n, \n25\n Future studies should thus focus on the LA size and its proportional enlargement in relation to the RA, when assessing patients prior to an AF ablation procedure.",
"Several limitations could impact interpretation of our results. First, the study is relatively small and included only 33% women. Therefore, statistical analysis for females is very likely underpowered. Similarly, we cannot exclude that male sex is a possible effect modifier analyzing an impact of sex for RAV ≥ LAV. Of note, the study included only patients of European ancestry from a localized area in Eastern Germany, limiting transferability of the results to other ethnic populations. Furthermore, although LVAs are one of parameters describing atrial myopathy, other parameters such as extrapulmonary substrate and triggers or typical atrial flutter, impairing AF ablation outcome, were not assessed. We did not consider arrhythmia recurrences as an outcome after AF catheter ablation due to several issues: (1) In our opinion LVAs are more robust outcome than arrhythmia recurrences, which occur within weeks/months after catheter ablation, while LVAs are measurable during the procedure; (2) Arrhythmia recurrences depend on many factors such as ablator experience, follow‐up strategy including antiarrhythmic drug prescription, follow‐up frequency, ECG monitoring; (3) In our study, patients did not have a continuous rhythm monitoring. Finally, specific LA fibrosis assessment using late gadolinium enhancement was not conducted in current study and should be addressed in future studies.",
"LAV/RAV ratio is useful parameter predicting electro‐anatomical substrate in AF. LAV > RAV was associated with sixfold risk for LVAs presence, while male sex was associated with RAV ≥ LAV and less LVAs.",
"Philipp Sommer is in the advisory board for Abbott, Biosense Webster, Medtronic, und Boston Scientific."
] |
[
null,
"methods",
null,
null,
null,
"results",
null,
null,
null,
"discussion",
null,
null,
null,
null,
"conclusions",
"COI-statement"
] |
[
"atrial fibrillation",
"cardiac magnetic resonance",
"left atrial size",
"low voltage areas",
"right atrial volume"
] |
INTRODUCTION:
Becoming a cornerstone therapy in many patients with atrial fibrillation (AF),
1
in some patients, catheter ablation with circumferential pulmonary vein isolation alone is not enough for sinus rhythm maintenance during follow‐up. The arrhythmia recurrences remain an important clinical challenge and require individualized AF treatment plan already before intervention. One major feature reflecting left atrial (LA) remodeling is peri‐procedural evidence of low voltage areas (LVAs).
2
At least 20%–25% of AF patients have significant LVAs in peri‐procedural mapping, which are an important characteristic of AF progression and treatment failure if not treated with additional ablation.
2
,
3
Therefore, assessment of LVAs presence before catheter ablation is an important task for the electrophysiologist allowing individually tailored AF ablation therapy.
The LA size is another important parameter of AF progression. The role of the LA volume (LAV) and LA function as surrogate parameters for higher AF burden
4
and LVAs presence
5
,
6
are well described. In addition, there is an association between atrial flutter—a right atrial (RA), and AF—a left atrial disease.
7
,
8
Several studies described an importance of RA assessment as a prognosis marker in heart failure, pulmonary hypertension, and chronic obstructive pulmonary disease.
9
,
10
,
11
Diastolic functional changes—as a preliminary stage for AF—appeared to occur earlier in the right chambers
12
suggesting that RA dilatation might be an early marker for atrial remodeling associated with AF initiation. Previous studies reported the importance of increased RA volume (RAV) as predictor for arrhythmia recurrences in AF patients.
13
,
14
It was hypothesized that RA is more prone to hemodynamic changes and is a more responsive marker of structural remodeling.
13
However, association between RAV and LVAs and the prediction capability for LAV/RAV ratio is unknown. Therefore, we aimed to investigate the indexed LAV/RAV ratio assessed in cardiovascular magnetic resonance (CMR) imaging and the association with LVAs in patients undergoing AF catheter ablation. We hypothesize that the biatrial ratio is an independent predictor for LVAs presence.
METHODS:
The study population was described previously.
6
Briefly, patients presenting for catheter ablation due to symptomatic AF from October 2015 to April 2017 were included in the study. According to current guidelines, AF subtypes were defined as paroxysmal and persistent.
15
Patients with pregnancy, age <18 or >75 years, valvular AF (any valvulopathies >second degree), cancer, acute, or systemic inflammatory diseases, and acute hyperthyreotic state were excluded from the study. The study was approved by the local Ethical Committee (Medical Faculty, University of Leipzig), and patients provided written informed consent for participation.
Cardiovascular magnetic resonance Prior to AF catheter ablation, all patients underwent 1.5 T CMR (Ingenia, Philips Medical) for LA anatomy assessment as previously described.
5
Briefly, LAV was determined using a biplane model based on cine 4‐ and 2‐chamber views, and RAV using a monoplane model based on the cine 4‐chamber view. Both volumes were indexed to body surface areas, and the LAV/RAV ratio was calculated before ablation. We defined two subgroups according to the LAV/RAV ratio: (1) LAV is greater than RAV (LAV > RAV), and (2) RAV is equal or greater than LAV (RAV ≥ LAV).
Prior to AF catheter ablation, all patients underwent 1.5 T CMR (Ingenia, Philips Medical) for LA anatomy assessment as previously described.
5
Briefly, LAV was determined using a biplane model based on cine 4‐ and 2‐chamber views, and RAV using a monoplane model based on the cine 4‐chamber view. Both volumes were indexed to body surface areas, and the LAV/RAV ratio was calculated before ablation. We defined two subgroups according to the LAV/RAV ratio: (1) LAV is greater than RAV (LAV > RAV), and (2) RAV is equal or greater than LAV (RAV ≥ LAV).
Peri‐procedural LA mapping and AF ablation Transseptal access and catheter navigation were performed with a steerable sheath (Agilis, St. Jude Medical, St. Paul, MN). The electro‐anatomical mapping was performed in sinus rhythm as described previously.
5
In case of AF at the beginning of the procedure, the arrhythmia was terminated by electrical cardioversion and the mapping was performed in sinus rhythm.
Multielectrode spiral mapping catheters (Reflexion Spiral and Advisor, St Jude Medical [SJM], Saint Paul, MN in NavX Ensite procedures and Carto Lasso, Biosense Webster, Diamond Bar, CA in Carto3 procedures) were used to generate electro‐anatomical voltage maps of the LA. The cutoff value for LVAs was defined as bipolar signal amplitude <0.5 mV. Ectopic beats were excluded from the voltage map. The number of points obtained was >1000. In case of AF recurrence during electro‐anatomical mapping, only anatomical map was assessed. Then the electro‐anatomical mapping was completed in sinus rhythm after PVI, and if still needed, after further cardioversion.
All patients received circumferential ablation lines around the antrum of the ipsilateral pulmonary veins. The ablation catheters (irrigated tip catheter and power of 25–40 W) used in NavX Ensite procedures were TactiCath (St Jude Medical [SJM], Saint Paul, MN) and for Carto3 procedures SmartTouch Thermocool (Biosense Webster, Diamond Bar, CA). End point of catheter ablation was PV isolation, which was verified with a multipolar circular mapping catheter. Additional linear lesions were added between if LVAs were present in this area. To be considered as relevant the LVAs ought to consist of adjacent low‐amplitude mapping points, bearing certain additional characteristics such as fragmentation and duration. Finally, the relevance of the LVAs was evaluated by experienced operators based on the mapping point qualities as well as induction of extra‐PV macro‐reentry tachycardia, in which case additional linear ablation was performed. Patients with small/negligible LVAs dispersely distributed in LA and not suitable for ablation, who received the PVI only without additional ablation lines, were excluded.
Transseptal access and catheter navigation were performed with a steerable sheath (Agilis, St. Jude Medical, St. Paul, MN). The electro‐anatomical mapping was performed in sinus rhythm as described previously.
5
In case of AF at the beginning of the procedure, the arrhythmia was terminated by electrical cardioversion and the mapping was performed in sinus rhythm.
Multielectrode spiral mapping catheters (Reflexion Spiral and Advisor, St Jude Medical [SJM], Saint Paul, MN in NavX Ensite procedures and Carto Lasso, Biosense Webster, Diamond Bar, CA in Carto3 procedures) were used to generate electro‐anatomical voltage maps of the LA. The cutoff value for LVAs was defined as bipolar signal amplitude <0.5 mV. Ectopic beats were excluded from the voltage map. The number of points obtained was >1000. In case of AF recurrence during electro‐anatomical mapping, only anatomical map was assessed. Then the electro‐anatomical mapping was completed in sinus rhythm after PVI, and if still needed, after further cardioversion.
All patients received circumferential ablation lines around the antrum of the ipsilateral pulmonary veins. The ablation catheters (irrigated tip catheter and power of 25–40 W) used in NavX Ensite procedures were TactiCath (St Jude Medical [SJM], Saint Paul, MN) and for Carto3 procedures SmartTouch Thermocool (Biosense Webster, Diamond Bar, CA). End point of catheter ablation was PV isolation, which was verified with a multipolar circular mapping catheter. Additional linear lesions were added between if LVAs were present in this area. To be considered as relevant the LVAs ought to consist of adjacent low‐amplitude mapping points, bearing certain additional characteristics such as fragmentation and duration. Finally, the relevance of the LVAs was evaluated by experienced operators based on the mapping point qualities as well as induction of extra‐PV macro‐reentry tachycardia, in which case additional linear ablation was performed. Patients with small/negligible LVAs dispersely distributed in LA and not suitable for ablation, who received the PVI only without additional ablation lines, were excluded.
Statistical analysis Data are presented as mean and standard deviation for normally distributed or median (interquartile range, 25th and 75th percentiles) for skewed continuous variables, and as proportions for categorical variables. The differences between continuous values were assessed using an unpaired t‐test or the Mann–Whitney, and a χ
2 test for categorical variables.
Logistic regression analysis was used to identify factors associated with LVAs. We performed three analyses using logistic regression of LVAs presence (Model 1 – unadjusted analysis; Model 2 – adjusted for age and sex; and Model 3 – adjusted for persistent AF, heart rate, and CHA2DS2‐VASc score.
Receiver operating characteristic curves (ROC) were generated to analyze performance of the LAV/RAV ratio predicting LVAs, with the area under the curve (AUC) being equivalent to the c‐index for determining the predictive value for the parameters. Finally, we compared the c‐indices (i.e., areas under the ROC curves) of LAV > RAV and LAV using DeLong's method.
16
A p‐value <.05 was considered statistically significant. All analyses were performed with SPSS statistical software version 26 (SPSS Inc., Chicago).
Data are presented as mean and standard deviation for normally distributed or median (interquartile range, 25th and 75th percentiles) for skewed continuous variables, and as proportions for categorical variables. The differences between continuous values were assessed using an unpaired t‐test or the Mann–Whitney, and a χ
2 test for categorical variables.
Logistic regression analysis was used to identify factors associated with LVAs. We performed three analyses using logistic regression of LVAs presence (Model 1 – unadjusted analysis; Model 2 – adjusted for age and sex; and Model 3 – adjusted for persistent AF, heart rate, and CHA2DS2‐VASc score.
Receiver operating characteristic curves (ROC) were generated to analyze performance of the LAV/RAV ratio predicting LVAs, with the area under the curve (AUC) being equivalent to the c‐index for determining the predictive value for the parameters. Finally, we compared the c‐indices (i.e., areas under the ROC curves) of LAV > RAV and LAV using DeLong's method.
16
A p‐value <.05 was considered statistically significant. All analyses were performed with SPSS statistical software version 26 (SPSS Inc., Chicago).
Cardiovascular magnetic resonance:
Prior to AF catheter ablation, all patients underwent 1.5 T CMR (Ingenia, Philips Medical) for LA anatomy assessment as previously described.
5
Briefly, LAV was determined using a biplane model based on cine 4‐ and 2‐chamber views, and RAV using a monoplane model based on the cine 4‐chamber view. Both volumes were indexed to body surface areas, and the LAV/RAV ratio was calculated before ablation. We defined two subgroups according to the LAV/RAV ratio: (1) LAV is greater than RAV (LAV > RAV), and (2) RAV is equal or greater than LAV (RAV ≥ LAV).
Peri‐procedural LA mapping and AF ablation:
Transseptal access and catheter navigation were performed with a steerable sheath (Agilis, St. Jude Medical, St. Paul, MN). The electro‐anatomical mapping was performed in sinus rhythm as described previously.
5
In case of AF at the beginning of the procedure, the arrhythmia was terminated by electrical cardioversion and the mapping was performed in sinus rhythm.
Multielectrode spiral mapping catheters (Reflexion Spiral and Advisor, St Jude Medical [SJM], Saint Paul, MN in NavX Ensite procedures and Carto Lasso, Biosense Webster, Diamond Bar, CA in Carto3 procedures) were used to generate electro‐anatomical voltage maps of the LA. The cutoff value for LVAs was defined as bipolar signal amplitude <0.5 mV. Ectopic beats were excluded from the voltage map. The number of points obtained was >1000. In case of AF recurrence during electro‐anatomical mapping, only anatomical map was assessed. Then the electro‐anatomical mapping was completed in sinus rhythm after PVI, and if still needed, after further cardioversion.
All patients received circumferential ablation lines around the antrum of the ipsilateral pulmonary veins. The ablation catheters (irrigated tip catheter and power of 25–40 W) used in NavX Ensite procedures were TactiCath (St Jude Medical [SJM], Saint Paul, MN) and for Carto3 procedures SmartTouch Thermocool (Biosense Webster, Diamond Bar, CA). End point of catheter ablation was PV isolation, which was verified with a multipolar circular mapping catheter. Additional linear lesions were added between if LVAs were present in this area. To be considered as relevant the LVAs ought to consist of adjacent low‐amplitude mapping points, bearing certain additional characteristics such as fragmentation and duration. Finally, the relevance of the LVAs was evaluated by experienced operators based on the mapping point qualities as well as induction of extra‐PV macro‐reentry tachycardia, in which case additional linear ablation was performed. Patients with small/negligible LVAs dispersely distributed in LA and not suitable for ablation, who received the PVI only without additional ablation lines, were excluded.
Statistical analysis:
Data are presented as mean and standard deviation for normally distributed or median (interquartile range, 25th and 75th percentiles) for skewed continuous variables, and as proportions for categorical variables. The differences between continuous values were assessed using an unpaired t‐test or the Mann–Whitney, and a χ
2 test for categorical variables.
Logistic regression analysis was used to identify factors associated with LVAs. We performed three analyses using logistic regression of LVAs presence (Model 1 – unadjusted analysis; Model 2 – adjusted for age and sex; and Model 3 – adjusted for persistent AF, heart rate, and CHA2DS2‐VASc score.
Receiver operating characteristic curves (ROC) were generated to analyze performance of the LAV/RAV ratio predicting LVAs, with the area under the curve (AUC) being equivalent to the c‐index for determining the predictive value for the parameters. Finally, we compared the c‐indices (i.e., areas under the ROC curves) of LAV > RAV and LAV using DeLong's method.
16
A p‐value <.05 was considered statistically significant. All analyses were performed with SPSS statistical software version 26 (SPSS Inc., Chicago).
RESULTS:
Clinical characteristics of the study population The study population included 189 patients (mean 63 ± 10 years, 33% women, 57% persistent AF) undergoing their first AF catheter ablation. Clinical characteristics of the study population are summarized in Table 1. Forty‐one patients (22%) had LVAs in periprocedural mapping, which required additional substrate modification. We observed LAV > RAV in 149 (79%) patients (26% with LVAs), while 40 (21%) patients had RAV≥LAV (5% with LVAs). Patients with LAV > RAV were significantly older, more often females, had a lower estimated glomerular filtration rate, had more often hypertension and more often LVAs (Table 1).
Clinical characteristics of the study cohort
Abbreviations: AF, atrial fibrillation; BMI, body mass index; BSA, body surface area; eGFR, estimated glomerular filtration rate; LA, left atrial; LVAs, low voltage areas; RA, right atrial.
The study population included 189 patients (mean 63 ± 10 years, 33% women, 57% persistent AF) undergoing their first AF catheter ablation. Clinical characteristics of the study population are summarized in Table 1. Forty‐one patients (22%) had LVAs in periprocedural mapping, which required additional substrate modification. We observed LAV > RAV in 149 (79%) patients (26% with LVAs), while 40 (21%) patients had RAV≥LAV (5% with LVAs). Patients with LAV > RAV were significantly older, more often females, had a lower estimated glomerular filtration rate, had more often hypertension and more often LVAs (Table 1).
Clinical characteristics of the study cohort
Abbreviations: AF, atrial fibrillation; BMI, body mass index; BSA, body surface area; eGFR, estimated glomerular filtration rate; LA, left atrial; LVAs, low voltage areas; RA, right atrial.
Association of LAV/RAV ratio with LVAs
In univariable analysis logistic regression, LAV > RAV was associated with sixxfold risk of LVAs presence (OR 6.083, 95%CI 1.395–26.514, p = .016). In multivariable analysis, after adjustment for CHA2DS2‐VASc score, persistent AF, and heart rate, LAV > RAV remained significantly associated with LVAs (OR 5.981, 95%CI 1.256–28.484, p = .025) (Table 2). Using ROC analysis, LAV > RAV showed moderate prediction of LVAs presence (AUC 0.668, 95%CI 0.585–0.751, p = .001) (Figure 1). Comparing the LAV > RAV with LAV (AUC 0.724, 95% CI 0.639–0.809, p < .001) using DeLong's method, the difference between ROC curves was not significant (p = .353).
Prediction of LVAs using LAV/RAV and LA volume
Abbreviations: AF, atrial fibrillation; CI, confidence interval; LAV, left atrial volume; OR, odds ratio; RAV, right atrial volume.
Further adjusted for persistent AF; CHA2DS2‐VASc score and heart frequency.
Association between LVAs and LAV > RAV. AF, atrial fibrillation; AUC, area under the curve; CI, confidence interval; LAV, left atrial volume; RAV, right atrial volume
In univariable analysis logistic regression, LAV > RAV was associated with sixxfold risk of LVAs presence (OR 6.083, 95%CI 1.395–26.514, p = .016). In multivariable analysis, after adjustment for CHA2DS2‐VASc score, persistent AF, and heart rate, LAV > RAV remained significantly associated with LVAs (OR 5.981, 95%CI 1.256–28.484, p = .025) (Table 2). Using ROC analysis, LAV > RAV showed moderate prediction of LVAs presence (AUC 0.668, 95%CI 0.585–0.751, p = .001) (Figure 1). Comparing the LAV > RAV with LAV (AUC 0.724, 95% CI 0.639–0.809, p < .001) using DeLong's method, the difference between ROC curves was not significant (p = .353).
Prediction of LVAs using LAV/RAV and LA volume
Abbreviations: AF, atrial fibrillation; CI, confidence interval; LAV, left atrial volume; OR, odds ratio; RAV, right atrial volume.
Further adjusted for persistent AF; CHA2DS2‐VASc score and heart frequency.
Association between LVAs and LAV > RAV. AF, atrial fibrillation; AUC, area under the curve; CI, confidence interval; LAV, left atrial volume; RAV, right atrial volume
Clinical factors associated with RAV ≥ LAV
In univariable analysis, RAV ≥ LAV was associated with younger age (OR 0.946, 95% CI 0.914–0.978, p = .001), male sex (OR 4.462, 95% CI 1.652–12.046, p = .003), and renal function measured as glomerular filtration rate (OR per 1 ml/kg/1.73m2 1.041, 95% CI 1.016–1.066, p = .001, Table 3). In multivariable analysis, the RAV ≥ LAV was 3‐fold higher in males (OR 3.040, 95% CI 1.050–8.802, p = .04).
Clinical factors associated with RAV ≥ LAV
Abbreviations: AF, fibrillation; CI, confidence interval; eGFR, estimated glomerular filtration rate; OR, odds ratio.
Adjusted for age, sex, persistent AF, and eGFR.
In univariable analysis, RAV ≥ LAV was associated with younger age (OR 0.946, 95% CI 0.914–0.978, p = .001), male sex (OR 4.462, 95% CI 1.652–12.046, p = .003), and renal function measured as glomerular filtration rate (OR per 1 ml/kg/1.73m2 1.041, 95% CI 1.016–1.066, p = .001, Table 3). In multivariable analysis, the RAV ≥ LAV was 3‐fold higher in males (OR 3.040, 95% CI 1.050–8.802, p = .04).
Clinical factors associated with RAV ≥ LAV
Abbreviations: AF, fibrillation; CI, confidence interval; eGFR, estimated glomerular filtration rate; OR, odds ratio.
Adjusted for age, sex, persistent AF, and eGFR.
Clinical characteristics of the study population:
The study population included 189 patients (mean 63 ± 10 years, 33% women, 57% persistent AF) undergoing their first AF catheter ablation. Clinical characteristics of the study population are summarized in Table 1. Forty‐one patients (22%) had LVAs in periprocedural mapping, which required additional substrate modification. We observed LAV > RAV in 149 (79%) patients (26% with LVAs), while 40 (21%) patients had RAV≥LAV (5% with LVAs). Patients with LAV > RAV were significantly older, more often females, had a lower estimated glomerular filtration rate, had more often hypertension and more often LVAs (Table 1).
Clinical characteristics of the study cohort
Abbreviations: AF, atrial fibrillation; BMI, body mass index; BSA, body surface area; eGFR, estimated glomerular filtration rate; LA, left atrial; LVAs, low voltage areas; RA, right atrial.
Association of LAV/RAV ratio with LVAs
:
In univariable analysis logistic regression, LAV > RAV was associated with sixxfold risk of LVAs presence (OR 6.083, 95%CI 1.395–26.514, p = .016). In multivariable analysis, after adjustment for CHA2DS2‐VASc score, persistent AF, and heart rate, LAV > RAV remained significantly associated with LVAs (OR 5.981, 95%CI 1.256–28.484, p = .025) (Table 2). Using ROC analysis, LAV > RAV showed moderate prediction of LVAs presence (AUC 0.668, 95%CI 0.585–0.751, p = .001) (Figure 1). Comparing the LAV > RAV with LAV (AUC 0.724, 95% CI 0.639–0.809, p < .001) using DeLong's method, the difference between ROC curves was not significant (p = .353).
Prediction of LVAs using LAV/RAV and LA volume
Abbreviations: AF, atrial fibrillation; CI, confidence interval; LAV, left atrial volume; OR, odds ratio; RAV, right atrial volume.
Further adjusted for persistent AF; CHA2DS2‐VASc score and heart frequency.
Association between LVAs and LAV > RAV. AF, atrial fibrillation; AUC, area under the curve; CI, confidence interval; LAV, left atrial volume; RAV, right atrial volume
Clinical factors associated with RAV ≥ LAV
:
In univariable analysis, RAV ≥ LAV was associated with younger age (OR 0.946, 95% CI 0.914–0.978, p = .001), male sex (OR 4.462, 95% CI 1.652–12.046, p = .003), and renal function measured as glomerular filtration rate (OR per 1 ml/kg/1.73m2 1.041, 95% CI 1.016–1.066, p = .001, Table 3). In multivariable analysis, the RAV ≥ LAV was 3‐fold higher in males (OR 3.040, 95% CI 1.050–8.802, p = .04).
Clinical factors associated with RAV ≥ LAV
Abbreviations: AF, fibrillation; CI, confidence interval; eGFR, estimated glomerular filtration rate; OR, odds ratio.
Adjusted for age, sex, persistent AF, and eGFR.
DISCUSSION:
In our study, we investigated the indexed LAV/RAV ratio and its predictive value on the pre‐procedural LVAs in patients undergoing AF catheter ablation (Figure 2). We found that LAV > RAV was associated with sixfold risk for LVAs presence. Also, LAV > RAV was less observed in males.
Study overview. Central figure describes the study population and main results. Isolated LA dilatation (LAV > RAV) was significantly associated with LVAs, while biatrial/isolated RA dilatation (RAV≥LAV) was associated with male sex. AF, atrial fibrillation; CI, confidence interval; CMR, cardiovascular magnetic resonance imaging; HR, heart rate; LA, left atrial; LAV, left atrial volume; LVAs, low voltage areas; RA, right atrial; RAV, right atrial volume; OR, odds ratio
Biatrial ratio as parameter for AF progression and LVAs prediction The role of RA size in AF pathogenesis is controversial. It had been reported that RAV and the RAV/LAV ratio were predictive for AF recurrence after PVI in patients with persistent AF, while LAV was not.
14
Another study confirmed these findings in AF patients after cardioversion.
13
Both studies suggested that AF is a biatrial disease, not being exclusively associated with isolated LA remodeling. However, other studies reported only moderate
17
or weak
18
prediction of AF recurrences using echocardiographic RA diameter. Our study contradicts previous results and shows that RAV ≥ LAV was associated with less LVAs presence, suggesting a rather minor role in LA remodeling. However, we found that males had threefold higher odds for RAV ≥ LAV and less LVAs. Taking into account that females are more prone to LVAs compared to male
19
and have more often‐unfavorable outcomes after catheter ablation, our findings are in line with previous research calling for an attention and an urgent need to address underrepresentation of females in clinical research.
The role of RA size in AF pathogenesis is controversial. It had been reported that RAV and the RAV/LAV ratio were predictive for AF recurrence after PVI in patients with persistent AF, while LAV was not.
14
Another study confirmed these findings in AF patients after cardioversion.
13
Both studies suggested that AF is a biatrial disease, not being exclusively associated with isolated LA remodeling. However, other studies reported only moderate
17
or weak
18
prediction of AF recurrences using echocardiographic RA diameter. Our study contradicts previous results and shows that RAV ≥ LAV was associated with less LVAs presence, suggesting a rather minor role in LA remodeling. However, we found that males had threefold higher odds for RAV ≥ LAV and less LVAs. Taking into account that females are more prone to LVAs compared to male
19
and have more often‐unfavorable outcomes after catheter ablation, our findings are in line with previous research calling for an attention and an urgent need to address underrepresentation of females in clinical research.
Clinical implications LA diameter (LAD) is an acknowledged marker of advanced electro‐anatomical remodeling.
2
,
20
,
21
LA remodeling is associated with increased atrial volume, interstitial fibrosis, and increased myocardial stretch favoring AF sustainability.
22
Previously, we reported that besides anteroposterior LAD, the LAV assessed in CMR is a strong predictor for LVAs presence.
6
In current analysis we confirm the role of LA in AF pathogenesis showing that LAV > RAV was associated with sixfold risk for LVAs presence. Although LAV alone showed better predictive value than LAV > RAV (AUC 0.724 vs. 0.668), the difference between ROC curves was not significant. However, the risk of LVAs presence was more obvious using LAV/RAV ratio than LAV alone (OR 5.98 vs. 1.03). Our results indicate that the LA enlargement indexed for the RAV (as self‐reference for enlargement) as reflected with the LAV/RAV ratio is a helpful tool for LVAs prediction and for shaping an individualized AF management approach prior to AF catheter ablation.
The present findings add to our knowledge about the importance of side‐specific atrial remodeling. As previously described, LA remodeling is associated with later stages of AF progression resulting from risk factors like aging, hypertension, left ventricular (LV) diastolic dysfunction and an altered electromechanical activation.
23
,
24
,
25
,
26
LV stiffness results into higher LA pressure with reduced LA emptying and consequent atrial dilatation.
23
Described pathologic changes represent a common pathway associated with interatrial delay seen as biphasic P‐wave in ECG and caused by deterioration of the Bachmann bundle conduction, and finally impaired electromechanical LA activation.
25
These pathophysiologic changes contribute to advanced remodeling and wall deformation that has been associated with LVA.
27
Our study supplement these findings of side specific pathophysiological LA changes (especially in relation to RAV) and emphasize the need for accurate pre‐procedural LA assessment.
In contrast, pathophysiology of RA remodeling seems to be different. In patients without heart failure, volume and pressure overload in the RA is mainly associated with pulmonary resistance, valvular disease, and RV dysfunction.
28
,
29
,
30
Although AF may contribute to RA dilation as well,
31
AF triggers from the RA are rare
32
and RA ablation in AF patients has not shown any benefit for outcomes.
33
In our study, RAV > LAV was higher in males and was not associated with LVAs. An explanation for such sex‐specific difference remains unknown, but it is in accordance with large echocardiographic studies and has not been assigned any clinical significance.
34
,
35
,
36
LA diameter (LAD) is an acknowledged marker of advanced electro‐anatomical remodeling.
2
,
20
,
21
LA remodeling is associated with increased atrial volume, interstitial fibrosis, and increased myocardial stretch favoring AF sustainability.
22
Previously, we reported that besides anteroposterior LAD, the LAV assessed in CMR is a strong predictor for LVAs presence.
6
In current analysis we confirm the role of LA in AF pathogenesis showing that LAV > RAV was associated with sixfold risk for LVAs presence. Although LAV alone showed better predictive value than LAV > RAV (AUC 0.724 vs. 0.668), the difference between ROC curves was not significant. However, the risk of LVAs presence was more obvious using LAV/RAV ratio than LAV alone (OR 5.98 vs. 1.03). Our results indicate that the LA enlargement indexed for the RAV (as self‐reference for enlargement) as reflected with the LAV/RAV ratio is a helpful tool for LVAs prediction and for shaping an individualized AF management approach prior to AF catheter ablation.
The present findings add to our knowledge about the importance of side‐specific atrial remodeling. As previously described, LA remodeling is associated with later stages of AF progression resulting from risk factors like aging, hypertension, left ventricular (LV) diastolic dysfunction and an altered electromechanical activation.
23
,
24
,
25
,
26
LV stiffness results into higher LA pressure with reduced LA emptying and consequent atrial dilatation.
23
Described pathologic changes represent a common pathway associated with interatrial delay seen as biphasic P‐wave in ECG and caused by deterioration of the Bachmann bundle conduction, and finally impaired electromechanical LA activation.
25
These pathophysiologic changes contribute to advanced remodeling and wall deformation that has been associated with LVA.
27
Our study supplement these findings of side specific pathophysiological LA changes (especially in relation to RAV) and emphasize the need for accurate pre‐procedural LA assessment.
In contrast, pathophysiology of RA remodeling seems to be different. In patients without heart failure, volume and pressure overload in the RA is mainly associated with pulmonary resistance, valvular disease, and RV dysfunction.
28
,
29
,
30
Although AF may contribute to RA dilation as well,
31
AF triggers from the RA are rare
32
and RA ablation in AF patients has not shown any benefit for outcomes.
33
In our study, RAV > LAV was higher in males and was not associated with LVAs. An explanation for such sex‐specific difference remains unknown, but it is in accordance with large echocardiographic studies and has not been assigned any clinical significance.
34
,
35
,
36
Future directions Our findings show a strong association between LVAs and LAV/RAV ratio. Despite previous data reporting a correlation between RAV ≥ LAV and AF recurrences, our results imply that increased RAV is not a suitable parameter for LVAs prediction and therefore not a marker for left atrial myopathy. This is in line with our previous studies that emphasized the importance of LV diastolic dysfunction, electro‐anatomical dysfunction for asymmetric LA remodeling, and ablation outcomes.
23
,
24
,
25
Future studies should thus focus on the LA size and its proportional enlargement in relation to the RA, when assessing patients prior to an AF ablation procedure.
Our findings show a strong association between LVAs and LAV/RAV ratio. Despite previous data reporting a correlation between RAV ≥ LAV and AF recurrences, our results imply that increased RAV is not a suitable parameter for LVAs prediction and therefore not a marker for left atrial myopathy. This is in line with our previous studies that emphasized the importance of LV diastolic dysfunction, electro‐anatomical dysfunction for asymmetric LA remodeling, and ablation outcomes.
23
,
24
,
25
Future studies should thus focus on the LA size and its proportional enlargement in relation to the RA, when assessing patients prior to an AF ablation procedure.
Limitations Several limitations could impact interpretation of our results. First, the study is relatively small and included only 33% women. Therefore, statistical analysis for females is very likely underpowered. Similarly, we cannot exclude that male sex is a possible effect modifier analyzing an impact of sex for RAV ≥ LAV. Of note, the study included only patients of European ancestry from a localized area in Eastern Germany, limiting transferability of the results to other ethnic populations. Furthermore, although LVAs are one of parameters describing atrial myopathy, other parameters such as extrapulmonary substrate and triggers or typical atrial flutter, impairing AF ablation outcome, were not assessed. We did not consider arrhythmia recurrences as an outcome after AF catheter ablation due to several issues: (1) In our opinion LVAs are more robust outcome than arrhythmia recurrences, which occur within weeks/months after catheter ablation, while LVAs are measurable during the procedure; (2) Arrhythmia recurrences depend on many factors such as ablator experience, follow‐up strategy including antiarrhythmic drug prescription, follow‐up frequency, ECG monitoring; (3) In our study, patients did not have a continuous rhythm monitoring. Finally, specific LA fibrosis assessment using late gadolinium enhancement was not conducted in current study and should be addressed in future studies.
Several limitations could impact interpretation of our results. First, the study is relatively small and included only 33% women. Therefore, statistical analysis for females is very likely underpowered. Similarly, we cannot exclude that male sex is a possible effect modifier analyzing an impact of sex for RAV ≥ LAV. Of note, the study included only patients of European ancestry from a localized area in Eastern Germany, limiting transferability of the results to other ethnic populations. Furthermore, although LVAs are one of parameters describing atrial myopathy, other parameters such as extrapulmonary substrate and triggers or typical atrial flutter, impairing AF ablation outcome, were not assessed. We did not consider arrhythmia recurrences as an outcome after AF catheter ablation due to several issues: (1) In our opinion LVAs are more robust outcome than arrhythmia recurrences, which occur within weeks/months after catheter ablation, while LVAs are measurable during the procedure; (2) Arrhythmia recurrences depend on many factors such as ablator experience, follow‐up strategy including antiarrhythmic drug prescription, follow‐up frequency, ECG monitoring; (3) In our study, patients did not have a continuous rhythm monitoring. Finally, specific LA fibrosis assessment using late gadolinium enhancement was not conducted in current study and should be addressed in future studies.
Biatrial ratio as parameter for AF progression and LVAs prediction:
The role of RA size in AF pathogenesis is controversial. It had been reported that RAV and the RAV/LAV ratio were predictive for AF recurrence after PVI in patients with persistent AF, while LAV was not.
14
Another study confirmed these findings in AF patients after cardioversion.
13
Both studies suggested that AF is a biatrial disease, not being exclusively associated with isolated LA remodeling. However, other studies reported only moderate
17
or weak
18
prediction of AF recurrences using echocardiographic RA diameter. Our study contradicts previous results and shows that RAV ≥ LAV was associated with less LVAs presence, suggesting a rather minor role in LA remodeling. However, we found that males had threefold higher odds for RAV ≥ LAV and less LVAs. Taking into account that females are more prone to LVAs compared to male
19
and have more often‐unfavorable outcomes after catheter ablation, our findings are in line with previous research calling for an attention and an urgent need to address underrepresentation of females in clinical research.
Clinical implications:
LA diameter (LAD) is an acknowledged marker of advanced electro‐anatomical remodeling.
2
,
20
,
21
LA remodeling is associated with increased atrial volume, interstitial fibrosis, and increased myocardial stretch favoring AF sustainability.
22
Previously, we reported that besides anteroposterior LAD, the LAV assessed in CMR is a strong predictor for LVAs presence.
6
In current analysis we confirm the role of LA in AF pathogenesis showing that LAV > RAV was associated with sixfold risk for LVAs presence. Although LAV alone showed better predictive value than LAV > RAV (AUC 0.724 vs. 0.668), the difference between ROC curves was not significant. However, the risk of LVAs presence was more obvious using LAV/RAV ratio than LAV alone (OR 5.98 vs. 1.03). Our results indicate that the LA enlargement indexed for the RAV (as self‐reference for enlargement) as reflected with the LAV/RAV ratio is a helpful tool for LVAs prediction and for shaping an individualized AF management approach prior to AF catheter ablation.
The present findings add to our knowledge about the importance of side‐specific atrial remodeling. As previously described, LA remodeling is associated with later stages of AF progression resulting from risk factors like aging, hypertension, left ventricular (LV) diastolic dysfunction and an altered electromechanical activation.
23
,
24
,
25
,
26
LV stiffness results into higher LA pressure with reduced LA emptying and consequent atrial dilatation.
23
Described pathologic changes represent a common pathway associated with interatrial delay seen as biphasic P‐wave in ECG and caused by deterioration of the Bachmann bundle conduction, and finally impaired electromechanical LA activation.
25
These pathophysiologic changes contribute to advanced remodeling and wall deformation that has been associated with LVA.
27
Our study supplement these findings of side specific pathophysiological LA changes (especially in relation to RAV) and emphasize the need for accurate pre‐procedural LA assessment.
In contrast, pathophysiology of RA remodeling seems to be different. In patients without heart failure, volume and pressure overload in the RA is mainly associated with pulmonary resistance, valvular disease, and RV dysfunction.
28
,
29
,
30
Although AF may contribute to RA dilation as well,
31
AF triggers from the RA are rare
32
and RA ablation in AF patients has not shown any benefit for outcomes.
33
In our study, RAV > LAV was higher in males and was not associated with LVAs. An explanation for such sex‐specific difference remains unknown, but it is in accordance with large echocardiographic studies and has not been assigned any clinical significance.
34
,
35
,
36
Future directions:
Our findings show a strong association between LVAs and LAV/RAV ratio. Despite previous data reporting a correlation between RAV ≥ LAV and AF recurrences, our results imply that increased RAV is not a suitable parameter for LVAs prediction and therefore not a marker for left atrial myopathy. This is in line with our previous studies that emphasized the importance of LV diastolic dysfunction, electro‐anatomical dysfunction for asymmetric LA remodeling, and ablation outcomes.
23
,
24
,
25
Future studies should thus focus on the LA size and its proportional enlargement in relation to the RA, when assessing patients prior to an AF ablation procedure.
Limitations:
Several limitations could impact interpretation of our results. First, the study is relatively small and included only 33% women. Therefore, statistical analysis for females is very likely underpowered. Similarly, we cannot exclude that male sex is a possible effect modifier analyzing an impact of sex for RAV ≥ LAV. Of note, the study included only patients of European ancestry from a localized area in Eastern Germany, limiting transferability of the results to other ethnic populations. Furthermore, although LVAs are one of parameters describing atrial myopathy, other parameters such as extrapulmonary substrate and triggers or typical atrial flutter, impairing AF ablation outcome, were not assessed. We did not consider arrhythmia recurrences as an outcome after AF catheter ablation due to several issues: (1) In our opinion LVAs are more robust outcome than arrhythmia recurrences, which occur within weeks/months after catheter ablation, while LVAs are measurable during the procedure; (2) Arrhythmia recurrences depend on many factors such as ablator experience, follow‐up strategy including antiarrhythmic drug prescription, follow‐up frequency, ECG monitoring; (3) In our study, patients did not have a continuous rhythm monitoring. Finally, specific LA fibrosis assessment using late gadolinium enhancement was not conducted in current study and should be addressed in future studies.
CONCLUSIONS:
LAV/RAV ratio is useful parameter predicting electro‐anatomical substrate in AF. LAV > RAV was associated with sixfold risk for LVAs presence, while male sex was associated with RAV ≥ LAV and less LVAs.
CONFLICT OF INTEREST:
Philipp Sommer is in the advisory board for Abbott, Biosense Webster, Medtronic, und Boston Scientific.
|
Background:
Left atrial volume (LAV) and low voltage areas (LVAs) are acknowledged markers for worse rhythm outcome after ablation of atrial fibrillation (AF). Some studies reported the importance of increased right atrial volume (RAV) as a predictor for arrhythmia recurrences in AF patients.
Methods:
Patients undergoing first AF ablation were included. LVAs were assessed peri-procedurally using high-density 3D maps and defined as <0.5 mV. All patients underwent pre-procedural cardiovascular magnetic resonance imaging. LAV (biplane) and RAV (monoplane 4-chamber) were assessed prior to ablation, and the LAV/RAV ratio was calculated.
Results:
The study population included 189 patients (age mean 63 ± 10 years, 33% women, 57% persistent AF, 22% LVAs). There were 149 (79%) patients with LAV > RAV. In univariable analysis LAV > RAV was associated with LVAs (OR 6.803, 95%CI 1.395-26.514, p = .016). The association remained robust in multivariable model after adjustment for persistent AF, CHA2 DS2 -VASc score, and heart rate (OR 5.981, 95%CI 1.256-28.484, p = .025). Using receiver operator curve analysis, LAV > RAV (AUC 0.668, 95%CI 0.585-0.751, p = .001) was significant predictor for LVAs. In multivariable analysis, after adjustment for age, persistent AF, and renal function, RAV≥LAV was threefold higher in males (OR 3.040, 95%CI 1.050-8.802, p = .04).
Conclusions:
LAV > RAV is useful for the prediction of electro-anatomical substrate in AF. LAV > RAV was associated with LVAs presence, while male sex remained associated with RAV≥LAV and less LVAs.
|
INTRODUCTION:
Becoming a cornerstone therapy in many patients with atrial fibrillation (AF),
1
in some patients, catheter ablation with circumferential pulmonary vein isolation alone is not enough for sinus rhythm maintenance during follow‐up. The arrhythmia recurrences remain an important clinical challenge and require individualized AF treatment plan already before intervention. One major feature reflecting left atrial (LA) remodeling is peri‐procedural evidence of low voltage areas (LVAs).
2
At least 20%–25% of AF patients have significant LVAs in peri‐procedural mapping, which are an important characteristic of AF progression and treatment failure if not treated with additional ablation.
2
,
3
Therefore, assessment of LVAs presence before catheter ablation is an important task for the electrophysiologist allowing individually tailored AF ablation therapy.
The LA size is another important parameter of AF progression. The role of the LA volume (LAV) and LA function as surrogate parameters for higher AF burden
4
and LVAs presence
5
,
6
are well described. In addition, there is an association between atrial flutter—a right atrial (RA), and AF—a left atrial disease.
7
,
8
Several studies described an importance of RA assessment as a prognosis marker in heart failure, pulmonary hypertension, and chronic obstructive pulmonary disease.
9
,
10
,
11
Diastolic functional changes—as a preliminary stage for AF—appeared to occur earlier in the right chambers
12
suggesting that RA dilatation might be an early marker for atrial remodeling associated with AF initiation. Previous studies reported the importance of increased RA volume (RAV) as predictor for arrhythmia recurrences in AF patients.
13
,
14
It was hypothesized that RA is more prone to hemodynamic changes and is a more responsive marker of structural remodeling.
13
However, association between RAV and LVAs and the prediction capability for LAV/RAV ratio is unknown. Therefore, we aimed to investigate the indexed LAV/RAV ratio assessed in cardiovascular magnetic resonance (CMR) imaging and the association with LVAs in patients undergoing AF catheter ablation. We hypothesize that the biatrial ratio is an independent predictor for LVAs presence.
CONCLUSIONS:
LAV/RAV ratio is useful parameter predicting electro‐anatomical substrate in AF. LAV > RAV was associated with sixfold risk for LVAs presence, while male sex was associated with RAV ≥ LAV and less LVAs.
|
Background:
Left atrial volume (LAV) and low voltage areas (LVAs) are acknowledged markers for worse rhythm outcome after ablation of atrial fibrillation (AF). Some studies reported the importance of increased right atrial volume (RAV) as a predictor for arrhythmia recurrences in AF patients.
Methods:
Patients undergoing first AF ablation were included. LVAs were assessed peri-procedurally using high-density 3D maps and defined as <0.5 mV. All patients underwent pre-procedural cardiovascular magnetic resonance imaging. LAV (biplane) and RAV (monoplane 4-chamber) were assessed prior to ablation, and the LAV/RAV ratio was calculated.
Results:
The study population included 189 patients (age mean 63 ± 10 years, 33% women, 57% persistent AF, 22% LVAs). There were 149 (79%) patients with LAV > RAV. In univariable analysis LAV > RAV was associated with LVAs (OR 6.803, 95%CI 1.395-26.514, p = .016). The association remained robust in multivariable model after adjustment for persistent AF, CHA2 DS2 -VASc score, and heart rate (OR 5.981, 95%CI 1.256-28.484, p = .025). Using receiver operator curve analysis, LAV > RAV (AUC 0.668, 95%CI 0.585-0.751, p = .001) was significant predictor for LVAs. In multivariable analysis, after adjustment for age, persistent AF, and renal function, RAV≥LAV was threefold higher in males (OR 3.040, 95%CI 1.050-8.802, p = .04).
Conclusions:
LAV > RAV is useful for the prediction of electro-anatomical substrate in AF. LAV > RAV was associated with LVAs presence, while male sex remained associated with RAV≥LAV and less LVAs.
| 8,152 | 353 | 16 |
[
"lav",
"rav",
"af",
"lvas",
"la",
"lav rav",
"ablation",
"atrial",
"patients",
"associated"
] |
[
"test",
"test"
] |
[CONTENT] atrial fibrillation | cardiac magnetic resonance | left atrial size | low voltage areas | right atrial volume [SUMMARY]
|
[CONTENT] atrial fibrillation | cardiac magnetic resonance | left atrial size | low voltage areas | right atrial volume [SUMMARY]
|
[CONTENT] atrial fibrillation | cardiac magnetic resonance | left atrial size | low voltage areas | right atrial volume [SUMMARY]
|
[CONTENT] atrial fibrillation | cardiac magnetic resonance | left atrial size | low voltage areas | right atrial volume [SUMMARY]
|
[CONTENT] atrial fibrillation | cardiac magnetic resonance | left atrial size | low voltage areas | right atrial volume [SUMMARY]
|
[CONTENT] atrial fibrillation | cardiac magnetic resonance | left atrial size | low voltage areas | right atrial volume [SUMMARY]
|
[CONTENT] Aged | Atrial Fibrillation | Catheter Ablation | Female | Heart Atria | Heart Rate | Humans | Male | Middle Aged | Recurrence [SUMMARY]
|
[CONTENT] Aged | Atrial Fibrillation | Catheter Ablation | Female | Heart Atria | Heart Rate | Humans | Male | Middle Aged | Recurrence [SUMMARY]
|
[CONTENT] Aged | Atrial Fibrillation | Catheter Ablation | Female | Heart Atria | Heart Rate | Humans | Male | Middle Aged | Recurrence [SUMMARY]
|
[CONTENT] Aged | Atrial Fibrillation | Catheter Ablation | Female | Heart Atria | Heart Rate | Humans | Male | Middle Aged | Recurrence [SUMMARY]
|
[CONTENT] Aged | Atrial Fibrillation | Catheter Ablation | Female | Heart Atria | Heart Rate | Humans | Male | Middle Aged | Recurrence [SUMMARY]
|
[CONTENT] Aged | Atrial Fibrillation | Catheter Ablation | Female | Heart Atria | Heart Rate | Humans | Male | Middle Aged | Recurrence [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] lav | rav | af | lvas | la | lav rav | ablation | atrial | patients | associated [SUMMARY]
|
[CONTENT] lav | rav | af | lvas | la | lav rav | ablation | atrial | patients | associated [SUMMARY]
|
[CONTENT] lav | rav | af | lvas | la | lav rav | ablation | atrial | patients | associated [SUMMARY]
|
[CONTENT] lav | rav | af | lvas | la | lav rav | ablation | atrial | patients | associated [SUMMARY]
|
[CONTENT] lav | rav | af | lvas | la | lav rav | ablation | atrial | patients | associated [SUMMARY]
|
[CONTENT] lav | rav | af | lvas | la | lav rav | ablation | atrial | patients | associated [SUMMARY]
|
[CONTENT] af | important | atrial | ra | lvas | patients | ablation | marker | af patients | therapy [SUMMARY]
|
[CONTENT] mapping | performed | model | ablation | lav | medical | st | procedures | rav | lvas [SUMMARY]
|
[CONTENT] ci | 95 | 95 ci | lav | rav | atrial | lvas | lav rav | volume | filtration [SUMMARY]
|
[CONTENT] lav | rav | lvas presence male | ratio useful parameter predicting | ratio useful parameter | ratio useful | male sex associated rav | sex associated rav | sex associated | male sex associated [SUMMARY]
|
[CONTENT] lav | rav | lvas | af | lav rav | atrial | ci | ablation | la | patients [SUMMARY]
|
[CONTENT] lav | rav | lvas | af | lav rav | atrial | ci | ablation | la | patients [SUMMARY]
|
[CONTENT] ||| RAV [SUMMARY]
|
[CONTENT] ||| ||| RAV | monoplane 4-chamber | RAV [SUMMARY]
|
[CONTENT] 189 | 63 ± | 10 years | 33% | 57% | AF | 22% ||| 149 | 79% | LAV | RAV ||| RAV | 6.803 | 1.395 | .016 ||| 5.981 | 1.256 | .025 ||| LAV | RAV | AUC | 0.668 | 0.585-0.751 ||| 3.040 | 1.050-8.802 [SUMMARY]
|
[CONTENT] RAV | AF ||| RAV [SUMMARY]
|
[CONTENT] ||| RAV ||| ||| ||| RAV | monoplane 4-chamber | RAV ||| ||| 189 | 63 ± | 10 years | 33% | 57% | AF | 22% ||| 149 | 79% | LAV | RAV ||| RAV | 6.803 | 1.395 | .016 ||| 5.981 | 1.256 | .025 ||| LAV | RAV | AUC | 0.668 | 0.585-0.751 ||| 3.040 | 1.050-8.802 ||| RAV | AF ||| RAV [SUMMARY]
|
[CONTENT] ||| RAV ||| ||| ||| RAV | monoplane 4-chamber | RAV ||| ||| 189 | 63 ± | 10 years | 33% | 57% | AF | 22% ||| 149 | 79% | LAV | RAV ||| RAV | 6.803 | 1.395 | .016 ||| 5.981 | 1.256 | .025 ||| LAV | RAV | AUC | 0.668 | 0.585-0.751 ||| 3.040 | 1.050-8.802 ||| RAV | AF ||| RAV [SUMMARY]
|
||||||
Improving stability of elastic stable intramedullary nailing in a transverse midshaft femur fracture model: biomechanical analysis of using end caps or a third nail.
|
26109085
|
Elastic stable intramedullary nailing (ESIN) is accepted widely for treatment of diaphyseal femur fractures in children. However, complication rates of 10 to 50 % are described due to shortening or axial deviation, especially in older or heavier children. Biomechanical in vitro testing was performed to determine whether two modified osteosyntheses with end caps or a third nail could significantly improve the stability in comparison to classical elastic stable intramedullary nailing in a transverse femur fracture model.
|
BACKGROUND
|
We performed biomechanical testing in 24 synthetic adolescent femoral bone models (Sawbones®) with a transverse midshaft (diaphyseal) fracture. First, in all models, two nails were inserted in a C-shaped manner (2 × 3.5 mm steel nails, prebent), then eight osteosyntheses were modified by using end caps and another eight by adding a third nail from the antero-lateral (2.5-mm steel, not prebent). Testing was performed in four-point bending, torsion, and shifting under physiological 9° compression.
|
METHODS
|
The third nail from the lateral showed a significant positive influence on the stiffness in all four-point bendings as well as in internal rotation comparing to the classical 2C configuration: mean values were significantly higher anterior-posterior (1.04 vs. 0.52 Nm/mm, p < 0.001), posterior-anterior (0.85 vs. 0.43 Nm/mm, p < 0.001), lateral-medial (1.26 vs. 0.70 Nm/mm, p < 0.001), and medial-lateral (1.16 vs. 0.76 Nm/mm, p < 0.001) and during internal rotation (0.16 vs. 0.11 Nm/°, p < 0.001). The modification with end caps did not improve the stiffness in any direction.
|
RESULTS
|
The configuration with a third nail provided a significantly higher stiffness than the classical 2C configuration as well as the modification with end caps in this biomechanical model. This supports the ongoing transfer of the additional third nail into clinical practice to reduce the axial deviation occurring in clinical practice.
|
CONCLUSIONS
|
[
"Adolescent",
"Biomechanical Phenomena",
"Bone Nails",
"Elasticity",
"Equipment Design",
"Femoral Fractures",
"Fracture Fixation, Intramedullary",
"Humans"
] |
4528722
|
Introduction
|
Fractures of the femoral diaphysis are the second most frequent location of fractures affecting the lower extremity in children (20–26/100,000 children per year) [1, 2] and comprise 1 to 2 % of all fractures in children [3, 4]. More than two thirds occur in children older than 6 years of age [2, 5]. Following the guidelines of the German Society of Paediatric Surgery, children beyond the age of 3 years should be treated with elastic stable intramedullary nailing (ESIN osteosynthesis) even in complex fractures or children older than 12 years as long as sufficient stability can be achieved [6]. ESIN osteosynthesis is said to produce a rapid recovery and a faster reintegration of children and adolescents and lack possible negative effects of immobilisation compared to conservative treatment, especially in schoolchildren [7, 8]. Yet, clinical studies focused on complications following ESIN osteosyntheses revealed problem rates between 10 and 50 % [9–12]. Most complications were observed as a result of instability in complex fracture types and older children weighing more than 40 kg [9, 13, 14]. Because of these instabilities, other authors used additional immobilisation (e.g. application of a cast), additional screws, and an additional external fixation or recommended submuscular plating or external fixation [10, 15–17].
In finding ways to modify elastic stable intramedullary nailing to gain more stability, we first developed a validated adolescent femur spiral facture biomechanical in vitro setting [18]. With this, we found that prebending the nails more than 30° is an essential part of the method [19] and furthermore that steel nails improve stability in contrast to titanium nails [18], which might be the reason for fewer complications of steel nails in clinical practice [12]. Although we could not show any improvement with end caps in our validated spiral fracture biomechanical in vitro setting [20], there seemed to be an improvement in stability with the implementation of a third nail under some circumstances [21]. In contrast, Volpon and co-workers described during combined axial-bending tests the TEN + CAP combination to be 8.75 % stiffer than nails alone as well as during torsion tests a 14 % increased stiffness in (rare) distal femoral fractures [22]. Their data tend to be congruent with preliminary clinical results in few patients [23, 24]. Therefore, the aim of this study was to determine the influence of these two interesting and intensively discussed modifications with end caps and a third nail (end caps = 2CEC; third nail = 3E) to improve the stiffness of the classical C-shaped elastic stable intramedullary nailing osteosynthesis (2C) in displaced transverse femoral fractures in all possible stress planes.
| null | null |
Results
|
All results of the stiffness and the shifting during compression of the three different prebending configurations are shown in Tables 1, 2 and 3. Using end caps did not improve stability in any direction (Table 1). Furthermore, before adjusting the results with the Holm-Bonferroni method, the classical 2C configuration showed greater stiffness in the anterior-posterior and latero-medial as well as less shifting at the crista intertrochanterica in the 0° position. In contrast, the use of a third nail (3E) offered more stiffness in all four-point bendings as well as in the internal and external rotation in comparison to the classical 2C configuration and the modification with end caps (2CEC). Adjusting the results with the Holm-Bonferroni method, the significant differences for the 3C modification mentioned above could all be affirmed, except for the external rotation compared to the classical 2C configuration (Table 2). In comparing the shiftings in the 9° position, the modification with end caps had the highest shifting and was least stable (Table 3).Table 1Comparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the 2C configuration with end caps (2CEC). Lower changes in shifting tests = higher stiffness2C classical (2C)2C with end caps (2CEC)
P value(n = 8)(n = 7)Mean (SD) four-point bending (Nm/mm)Anterior-posterior0.52 (0.49)0.44 (0.11)0.04a
Posterior-anterior0.43 (0.11)0.48 (0.13)0.89Lateral-medial0.70 (0.16)0.60 (0.19)0.04a
Medial-lateral0.76 (0.27)0.73 (0.25)0.57Mean (SD) rotation (Nm/°)External rotation0.12 (0.04)0.10 (0.03)0.15Internal rotation0.11 (0.03)0.10 (0.03)0.09Mean (SD) 9° compression/shifting (mm)Shifting 9° trochanter major0.80 (0.65)1.77 (1.70)0.06Shifting 9° crista intertrochanterica4.56 (2.67)7.44 (2.48)0.01a
aAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmedTable 2Comparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness2C classical (2C)Third nail from lateral (3E)
P value(n = 8)(n = 8)Mean (SD) four-point bending (Nm/mm)Anterior-posterior0.52 (0.49)1.04 (0.37)<0.001Posterior-anterior0.43 (0.11)0.85 (0.30)<0.001Lateral-medial0.70 (0.16)1.26 (0.54)<0.001Medial-lateral0.76 (0.27)1.16 (0.33)<0.001Mean (SD) rotation (Nm/°)External rotation0.12 (0.04)0.14 (0.02)<0.01a
Internal rotation0.11 (0.03)0.16 (0.04)0.001Mean (SD) 9° compression/shifting (mm)Shifting 9° trochanter major0.80 (0.65)1.14 (1.41)0.98Shifting 9° crista intertrochanterica4.56 (2.67)4.97 (2.89)0.69
aAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmedTable 3Comparison between the stiffness of the osteosyntheses with the 2C configuration with end caps (2CEC) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness2C with end caps (2CEC)Third nail from lateral (3E)
P value(n = 7)(n = 8)Mean (SD) four-point bending (Nm/mm)Anterior-posterior0.44 (0.11)1.04 (0.37)<0.001Posterior-anterior0.48 (0.13)0.85 (0.30)<0.001Lateral-medial0.60 (0.19)1.26 (0.54)<0.001Medial-lateral0.73 (0.25)1.16 (0.33)<0.001Mean (SD) rotation (Nm/°)External rotation0.10 (0.03)0.14 (0.02)<0.001Internal rotation0.10 (0.03)0.16 (0.04)<0.001Mean (SD) 9° compression/shifting (mm)Shifting 9° trochanter major1.77 (1.70)1.14 (1.41)0.24Shifting 9° crista intertrochanterica7.44 (2.48)4.97 (2.89)<0.001Adjusting the results with the Holm-Bonferroni method, the significance of the differences could all be confirmed
Comparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the 2C configuration with end caps (2CEC). Lower changes in shifting tests = higher stiffness
aAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmed
Comparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness
aAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmed
Comparison between the stiffness of the osteosyntheses with the 2C configuration with end caps (2CEC) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness
Adjusting the results with the Holm-Bonferroni method, the significance of the differences could all be confirmed
|
Conclusions
|
The results support the modification of the classical two-C-shaped elastic osteosynthesis in femoral fractures with an additional third nail also in transverse fractures. As feasibility and short implantation time could already be shown in spiral fractures, this treatment improves stability and will help to reduce misalignment or revision surgery.
|
[] |
[] |
[] |
[
"Introduction",
"Materials and methods",
"Results",
"Discussion",
"Conclusions"
] |
[
"Fractures of the femoral diaphysis are the second most frequent location of fractures affecting the lower extremity in children (20–26/100,000 children per year) [1, 2] and comprise 1 to 2 % of all fractures in children [3, 4]. More than two thirds occur in children older than 6 years of age [2, 5]. Following the guidelines of the German Society of Paediatric Surgery, children beyond the age of 3 years should be treated with elastic stable intramedullary nailing (ESIN osteosynthesis) even in complex fractures or children older than 12 years as long as sufficient stability can be achieved [6]. ESIN osteosynthesis is said to produce a rapid recovery and a faster reintegration of children and adolescents and lack possible negative effects of immobilisation compared to conservative treatment, especially in schoolchildren [7, 8]. Yet, clinical studies focused on complications following ESIN osteosyntheses revealed problem rates between 10 and 50 % [9–12]. Most complications were observed as a result of instability in complex fracture types and older children weighing more than 40 kg [9, 13, 14]. Because of these instabilities, other authors used additional immobilisation (e.g. application of a cast), additional screws, and an additional external fixation or recommended submuscular plating or external fixation [10, 15–17].\nIn finding ways to modify elastic stable intramedullary nailing to gain more stability, we first developed a validated adolescent femur spiral facture biomechanical in vitro setting [18]. With this, we found that prebending the nails more than 30° is an essential part of the method [19] and furthermore that steel nails improve stability in contrast to titanium nails [18], which might be the reason for fewer complications of steel nails in clinical practice [12]. Although we could not show any improvement with end caps in our validated spiral fracture biomechanical in vitro setting [20], there seemed to be an improvement in stability with the implementation of a third nail under some circumstances [21]. In contrast, Volpon and co-workers described during combined axial-bending tests the TEN + CAP combination to be 8.75 % stiffer than nails alone as well as during torsion tests a 14 % increased stiffness in (rare) distal femoral fractures [22]. Their data tend to be congruent with preliminary clinical results in few patients [23, 24]. Therefore, the aim of this study was to determine the influence of these two interesting and intensively discussed modifications with end caps and a third nail (end caps = 2CEC; third nail = 3E) to improve the stiffness of the classical C-shaped elastic stable intramedullary nailing osteosynthesis (2C) in displaced transverse femoral fractures in all possible stress planes.",
"Biomechanical testing was performed using 24 synthetic adolescent-sized composite femoral models (fourth generation, Sawbones®, Malmö, Sweden). The whole setting followed in principle the standardised protocol of previous studies [18]. The femoral model measured 45 cm in length, with a central canal diameter of 10 mm. Each standard midshaft transverse fracture was sawed exactly in the middle of the distance between condyles and trochanter minor (AO paediatric comprehensive classification of long bone fractures: 32D41 [25]; LiLa classification for paediatric long bone fractures: 3.2.s.3.2. [26]) (Fig. 1). The fracture parameters were measured before use in the biomechanical model. The distal femoral entry portals (medial/lateral) were created by a 5-mm drill 2 cm proximal to the virtual physis (Fig. 2). In eight specimens, a further entry point for a third nail was drilled 2 cm cranial anterior of the lateral entry point (Fig. 3). All 24 specimens underwent retrograde elastic stable intramedullary nailing with two 3.5-mm steel nails (Santech Nord, Schneverdingen, Germany), equally prebent to 40°, by the same paediatric surgeon specialised in paediatric traumatology (MMK), with special emphasis on broad contact of the fragments of the transverse fracture. Due to the conception of the composite femur, the ends of the nails were just inferior to the greater trochanter (Fig. 4); fluoroscopic imaging confirmed the correct configuration and position.Fig. 1Sawing of a standard midshaft transverse fracture exactly in the middle of the distance between the condyles and trochanter minor (AO paediatric comprehensive classification of long bone fractures: 32D41 [25]; LiLa classification for paediatric long bone fractures: 3.2.s.3.2. [26])Fig. 2Template for the drilling of the distal femoral entry portals (medial/lateral)Fig. 3Insertion point for the third nail 2.0 cm cranial anterior of the lateral entry point, the so called “3E” modificationFig. 4X-ray of the proximal part of the Sawbone with a “3E” modification. Due to the conception of the composite femur, the ends of the nails were just inferior to the greater trochanter\nSawing of a standard midshaft transverse fracture exactly in the middle of the distance between the condyles and trochanter minor (AO paediatric comprehensive classification of long bone fractures: 32D41 [25]; LiLa classification for paediatric long bone fractures: 3.2.s.3.2. [26])\nTemplate for the drilling of the distal femoral entry portals (medial/lateral)\nInsertion point for the third nail 2.0 cm cranial anterior of the lateral entry point, the so called “3E” modification\nX-ray of the proximal part of the Sawbone with a “3E” modification. Due to the conception of the composite femur, the ends of the nails were just inferior to the greater trochanter\nThe 24 composite models were divided into three configuration groups:In the control group (n = 8), no further modifications were performed on the two retrograde elastic stable intramedullary nailing configurations (2C).In the second group (n = 8), two additional cylindric hollow-threaded end caps (green cap for 3.0- to 4.0-mm nail diameters; Synthes Company, Oberdorf, Switzerland) were placed over the external tips of the nails at the entry portals and then screwed into the bone cortex. This modification is further called “2CEC” (Fig. 5).Fig. 5X-ray of the distal part of the Sawbone with a “2CEC” modificationIn the third group (n = 8), a third nail (2.5 mm) was inserted over the third entry point from the antero-lateral without prebending, the so-called “3E”-modification (Fig. 3) [21].\nIn the control group (n = 8), no further modifications were performed on the two retrograde elastic stable intramedullary nailing configurations (2C).\nIn the second group (n = 8), two additional cylindric hollow-threaded end caps (green cap for 3.0- to 4.0-mm nail diameters; Synthes Company, Oberdorf, Switzerland) were placed over the external tips of the nails at the entry portals and then screwed into the bone cortex. This modification is further called “2CEC” (Fig. 5).Fig. 5X-ray of the distal part of the Sawbone with a “2CEC” modification\nX-ray of the distal part of the Sawbone with a “2CEC” modification\nIn the third group (n = 8), a third nail (2.5 mm) was inserted over the third entry point from the antero-lateral without prebending, the so-called “3E”-modification (Fig. 3) [21].\nTesting was done with a Zwick 1465 universal testing machine (UTM; Zwick GmbH & Co. KG, Ulm, Germany). Fixation of the head of the femur and the femoral condyles in the testing machine was achieved with custom-fit polymethylmethacrylate (Technovit 4006, Heraeus Kulzer, Wehrheim, Germany) moulds for both sides. Set-up followed the ASTM F383-73 and F1264-03 description [27, 28].\nInitially, the femur was positioned in a 0° position to test for construct stability with a compression load of up to 150 N to the femoral head with a speed of 0.05 mm/s.\nFour-point bending was measured with an incremental linear encoder (MS30-1-LD-2, Megatron, Putzbrunn, Germany) at midpoint of the two lower force bars with a maximum bending moment of 5 Nm (Fig. 6). Speed was set at 0.05 mm/s; maximum bending was 2 mm.Fig. 6Photograph showing the specimen undergoing the four-point bending test, by using the Zwick 1465 universal testing machine, with the bending measured with a linear encoder. (arrows up to down: application of load/Sawbone/sensing device/bearings for the Sawbones)\nPhotograph showing the specimen undergoing the four-point bending test, by using the Zwick 1465 universal testing machine, with the bending measured with a linear encoder. (arrows up to down: application of load/Sawbone/sensing device/bearings for the Sawbones)\nIn torsional testing, two angular encoders measured the torsion, and the femoral head area was gimbal-mounted. Speed was set at 20°/min; torsion was limited to 10°.\nFor shifting testing, the models were installed in a 9° position with a calibrated wedge. During physiological 9° compression, lateral/medial shifting was measured at the trochanter major, while ventral/dorsal shifting was measured at the crista intertrochanterica. A compressive load of up to 100 N was applied to the femoral head with a speed of 0.05 mm/s. In contrast to our spiral models, the reduction of the fracture gap in the 0 and 9° position was not measured due to direct contact of both fragments [18–20].\nThe course of the tests was equal to previously published studies: first, each specimen was placed in the machine for the four-point bending tests in a standardised order (anterior-posterior (AP), posterior-anterior (PA), lateral-medial (LM) and finally medial-lateral (ML)), followed by internal (IR) and external (ER) torsional tests and finally shifting tests in the 9° position. The first cycle of the tests was used as preconditioning; data for evaluation were collected from three subsequent cycles. After the last cycle of testing, all specimens were again tested with anterior-posterior bending to check for possible destructive changes that could have influenced the results. Thus, we could exclude destruction of the osteosyntheses and the specimen. Deformation of the UTM was determined up to 50 Nm in pretesting during the four-point bending and did not influence the results.\nData (bending moments in four-point bending, torsional stiffness in IR/ER and shifting in 9° position) were analysed with SPSS 18.0 (SPSS Inc., Chicago, USA). Distributions were first checked for normality (Shapiro-Wilk test); when significant discrepancy from a normal distribution occurred, a Mann-Whitney test was performed. When there was no significant discrepancy from normal distribution, the F-test and analysis of variance (ANOVA) were used. All values are presented as mean values. Significance level was set to P < 0.05. Due to multiple testing, the Holm-Bonferroni method was used for post hoc comparison.",
"All results of the stiffness and the shifting during compression of the three different prebending configurations are shown in Tables 1, 2 and 3. Using end caps did not improve stability in any direction (Table 1). Furthermore, before adjusting the results with the Holm-Bonferroni method, the classical 2C configuration showed greater stiffness in the anterior-posterior and latero-medial as well as less shifting at the crista intertrochanterica in the 0° position. In contrast, the use of a third nail (3E) offered more stiffness in all four-point bendings as well as in the internal and external rotation in comparison to the classical 2C configuration and the modification with end caps (2CEC). Adjusting the results with the Holm-Bonferroni method, the significant differences for the 3C modification mentioned above could all be affirmed, except for the external rotation compared to the classical 2C configuration (Table 2). In comparing the shiftings in the 9° position, the modification with end caps had the highest shifting and was least stable (Table 3).Table 1Comparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the 2C configuration with end caps (2CEC). Lower changes in shifting tests = higher stiffness2C classical (2C)2C with end caps (2CEC)\nP value(n = 8)(n = 7)Mean (SD) four-point bending (Nm/mm)Anterior-posterior0.52 (0.49)0.44 (0.11)0.04a\nPosterior-anterior0.43 (0.11)0.48 (0.13)0.89Lateral-medial0.70 (0.16)0.60 (0.19)0.04a\nMedial-lateral0.76 (0.27)0.73 (0.25)0.57Mean (SD) rotation (Nm/°)External rotation0.12 (0.04)0.10 (0.03)0.15Internal rotation0.11 (0.03)0.10 (0.03)0.09Mean (SD) 9° compression/shifting (mm)Shifting 9° trochanter major0.80 (0.65)1.77 (1.70)0.06Shifting 9° crista intertrochanterica4.56 (2.67)7.44 (2.48)0.01a\n\naAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmedTable 2Comparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness2C classical (2C)Third nail from lateral (3E)\nP value(n = 8)(n = 8)Mean (SD) four-point bending (Nm/mm)Anterior-posterior0.52 (0.49)1.04 (0.37)<0.001Posterior-anterior0.43 (0.11)0.85 (0.30)<0.001Lateral-medial0.70 (0.16)1.26 (0.54)<0.001Medial-lateral0.76 (0.27)1.16 (0.33)<0.001Mean (SD) rotation (Nm/°)External rotation0.12 (0.04)0.14 (0.02)<0.01a\nInternal rotation0.11 (0.03)0.16 (0.04)0.001Mean (SD) 9° compression/shifting (mm)Shifting 9° trochanter major0.80 (0.65)1.14 (1.41)0.98Shifting 9° crista intertrochanterica4.56 (2.67)4.97 (2.89)0.69\naAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmedTable 3Comparison between the stiffness of the osteosyntheses with the 2C configuration with end caps (2CEC) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness2C with end caps (2CEC)Third nail from lateral (3E)\nP value(n = 7)(n = 8)Mean (SD) four-point bending (Nm/mm)Anterior-posterior0.44 (0.11)1.04 (0.37)<0.001Posterior-anterior0.48 (0.13)0.85 (0.30)<0.001Lateral-medial0.60 (0.19)1.26 (0.54)<0.001Medial-lateral0.73 (0.25)1.16 (0.33)<0.001Mean (SD) rotation (Nm/°)External rotation0.10 (0.03)0.14 (0.02)<0.001Internal rotation0.10 (0.03)0.16 (0.04)<0.001Mean (SD) 9° compression/shifting (mm)Shifting 9° trochanter major1.77 (1.70)1.14 (1.41)0.24Shifting 9° crista intertrochanterica7.44 (2.48)4.97 (2.89)<0.001Adjusting the results with the Holm-Bonferroni method, the significance of the differences could all be confirmed\nComparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the 2C configuration with end caps (2CEC). Lower changes in shifting tests = higher stiffness\n\naAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmed\nComparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness\n\naAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmed\nComparison between the stiffness of the osteosyntheses with the 2C configuration with end caps (2CEC) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness\nAdjusting the results with the Holm-Bonferroni method, the significance of the differences could all be confirmed",
"Elastic stable intramedullary nailing (ESIN osteosynthesis) for displaced paediatric femoral fractures in childhood has gained wide acceptance [7, 29], and even older children and sometimes adolescents are treated this way in Europe [6, 10]. On the other hand, publications with special emphasis on problems of this technique revealed complication rates between 10 and 50 % especially—because of residual instability—in complex femoral shaft fractures and in older children or adolescents [9, 10, 12, 30]. To improve elastic stable intramedullary nailing and using stiffness as a marker for stability [31], biomechanical properties of retrograde C-shaped flexible intramedullary nailing are described in the literature [18, 19, 32–36]: Gwyn performed biomechanical testing with different fracture types in synthetic bone models using two titanium elastic nails of 4-mm diameter. The tests were limited to rotational forces, both external and internal, and showed that transverse fractures are as less stable as comminuted fractures with the classical 2 ESIN osteosynthesis [35]. This result was confirmed by Fricka [33]. Most of the other authors focussed on transverse fractures, too, but tested only one or two stress planes without any further explanations [33–36]. In contrast, we are certain that, considering the complex treatment problems, femur fractures require testing in all stress planes as in our previous in vitro settings [18–21]. With the aim of achieving further improvement in the ESIN technique, we tested two different modifications (end caps and a third nail), both established clinically in case series [21, 24] but not yet well analysed in a validated biomechanical model of a transverse femoral fracture. Only one study focussed on end caps in a transverse fracture model but in a very distal fracture type, which is a less frequent transverse shaft fracture. In focussing on the impact of end caps and a third nail to improve the stability in ESIN osteosynthesis in a transverse fracture model, this study revealed a benefit towards the configuration with a third nail providing a significantly higher stiffness than the classical 2C configuration. The configuration with a third nail was even stiffer than the modification with end caps which showed no statistical difference to the classical 2C configuration in this transverse biomechanical fracture model. In this biomechanical model, the insertion of the additional third nail revealed no difficulties if the first and second nails of the classical 2C configuration were placed in a technically correct way.\nThe ongoing transfer of the additional third nail into routine clinical practice in our hospital showed that one has to ensure that the third nail has the least length at the trochanter major and is only used if the first two nails are placed correctly. Then the additional operation time is below 10 min, and the complications due to insufficient stability are reduced [21].\nAs with every biomechanical study, this one suffers two limitations in comparison to clinical “reality”. The first one includes the use of a synthetic bone model that cannot precisely reproduce all in vivo conditions. However, the synthetic bone model has been used successfully in previous biomechanical studies [19, 37] and provides more consistency among specimens than cadaveric bones [38–41]. Also, surrounding tissue like periosteum and muscles were missing which might be an additional stabilising part of the elastic stable fixation method. As in other study groups [34–37], we used a “pure” model without further fixation devices. As reduction of paediatric femoral fractures is almost always performed in a closed manner, we have no precise data about the condition of the periosteum in the case of a fracture which could be used for a more realistic model.\nAnother limitation was due to the configuration [18, 37]: the ends of the nails could not be placed as proximal as intended in a real procedure. In our opinion, this limitation should be equalised as all three configurations were identically established. On the other hand, during the set-up, the focus was on a consistent surgical technique with an identical and reproducible set-up.\nImproper location of the nails or the bends in the nails creates an imbalance in the bending forces, resulting in an angular deformity. This serious technical mistake has been reported in the literature [8]. In our opinion, the proper configuration of the nails was achieved more precisely in the present study than in a real surgical situation.",
"The results support the modification of the classical two-C-shaped elastic osteosynthesis in femoral fractures with an additional third nail also in transverse fractures. As feasibility and short implantation time could already be shown in spiral fractures, this treatment improves stability and will help to reduce misalignment or revision surgery."
] |
[
"intro",
"materials|methods",
"results",
"discussion",
"conclusions"
] |
[
"Elastic stable intramedullary nailing",
"Biomechanical testing",
"Femur fracture",
"End caps",
"Third nail"
] |
Introduction:
Fractures of the femoral diaphysis are the second most frequent location of fractures affecting the lower extremity in children (20–26/100,000 children per year) [1, 2] and comprise 1 to 2 % of all fractures in children [3, 4]. More than two thirds occur in children older than 6 years of age [2, 5]. Following the guidelines of the German Society of Paediatric Surgery, children beyond the age of 3 years should be treated with elastic stable intramedullary nailing (ESIN osteosynthesis) even in complex fractures or children older than 12 years as long as sufficient stability can be achieved [6]. ESIN osteosynthesis is said to produce a rapid recovery and a faster reintegration of children and adolescents and lack possible negative effects of immobilisation compared to conservative treatment, especially in schoolchildren [7, 8]. Yet, clinical studies focused on complications following ESIN osteosyntheses revealed problem rates between 10 and 50 % [9–12]. Most complications were observed as a result of instability in complex fracture types and older children weighing more than 40 kg [9, 13, 14]. Because of these instabilities, other authors used additional immobilisation (e.g. application of a cast), additional screws, and an additional external fixation or recommended submuscular plating or external fixation [10, 15–17].
In finding ways to modify elastic stable intramedullary nailing to gain more stability, we first developed a validated adolescent femur spiral facture biomechanical in vitro setting [18]. With this, we found that prebending the nails more than 30° is an essential part of the method [19] and furthermore that steel nails improve stability in contrast to titanium nails [18], which might be the reason for fewer complications of steel nails in clinical practice [12]. Although we could not show any improvement with end caps in our validated spiral fracture biomechanical in vitro setting [20], there seemed to be an improvement in stability with the implementation of a third nail under some circumstances [21]. In contrast, Volpon and co-workers described during combined axial-bending tests the TEN + CAP combination to be 8.75 % stiffer than nails alone as well as during torsion tests a 14 % increased stiffness in (rare) distal femoral fractures [22]. Their data tend to be congruent with preliminary clinical results in few patients [23, 24]. Therefore, the aim of this study was to determine the influence of these two interesting and intensively discussed modifications with end caps and a third nail (end caps = 2CEC; third nail = 3E) to improve the stiffness of the classical C-shaped elastic stable intramedullary nailing osteosynthesis (2C) in displaced transverse femoral fractures in all possible stress planes.
Materials and methods:
Biomechanical testing was performed using 24 synthetic adolescent-sized composite femoral models (fourth generation, Sawbones®, Malmö, Sweden). The whole setting followed in principle the standardised protocol of previous studies [18]. The femoral model measured 45 cm in length, with a central canal diameter of 10 mm. Each standard midshaft transverse fracture was sawed exactly in the middle of the distance between condyles and trochanter minor (AO paediatric comprehensive classification of long bone fractures: 32D41 [25]; LiLa classification for paediatric long bone fractures: 3.2.s.3.2. [26]) (Fig. 1). The fracture parameters were measured before use in the biomechanical model. The distal femoral entry portals (medial/lateral) were created by a 5-mm drill 2 cm proximal to the virtual physis (Fig. 2). In eight specimens, a further entry point for a third nail was drilled 2 cm cranial anterior of the lateral entry point (Fig. 3). All 24 specimens underwent retrograde elastic stable intramedullary nailing with two 3.5-mm steel nails (Santech Nord, Schneverdingen, Germany), equally prebent to 40°, by the same paediatric surgeon specialised in paediatric traumatology (MMK), with special emphasis on broad contact of the fragments of the transverse fracture. Due to the conception of the composite femur, the ends of the nails were just inferior to the greater trochanter (Fig. 4); fluoroscopic imaging confirmed the correct configuration and position.Fig. 1Sawing of a standard midshaft transverse fracture exactly in the middle of the distance between the condyles and trochanter minor (AO paediatric comprehensive classification of long bone fractures: 32D41 [25]; LiLa classification for paediatric long bone fractures: 3.2.s.3.2. [26])Fig. 2Template for the drilling of the distal femoral entry portals (medial/lateral)Fig. 3Insertion point for the third nail 2.0 cm cranial anterior of the lateral entry point, the so called “3E” modificationFig. 4X-ray of the proximal part of the Sawbone with a “3E” modification. Due to the conception of the composite femur, the ends of the nails were just inferior to the greater trochanter
Sawing of a standard midshaft transverse fracture exactly in the middle of the distance between the condyles and trochanter minor (AO paediatric comprehensive classification of long bone fractures: 32D41 [25]; LiLa classification for paediatric long bone fractures: 3.2.s.3.2. [26])
Template for the drilling of the distal femoral entry portals (medial/lateral)
Insertion point for the third nail 2.0 cm cranial anterior of the lateral entry point, the so called “3E” modification
X-ray of the proximal part of the Sawbone with a “3E” modification. Due to the conception of the composite femur, the ends of the nails were just inferior to the greater trochanter
The 24 composite models were divided into three configuration groups:In the control group (n = 8), no further modifications were performed on the two retrograde elastic stable intramedullary nailing configurations (2C).In the second group (n = 8), two additional cylindric hollow-threaded end caps (green cap for 3.0- to 4.0-mm nail diameters; Synthes Company, Oberdorf, Switzerland) were placed over the external tips of the nails at the entry portals and then screwed into the bone cortex. This modification is further called “2CEC” (Fig. 5).Fig. 5X-ray of the distal part of the Sawbone with a “2CEC” modificationIn the third group (n = 8), a third nail (2.5 mm) was inserted over the third entry point from the antero-lateral without prebending, the so-called “3E”-modification (Fig. 3) [21].
In the control group (n = 8), no further modifications were performed on the two retrograde elastic stable intramedullary nailing configurations (2C).
In the second group (n = 8), two additional cylindric hollow-threaded end caps (green cap for 3.0- to 4.0-mm nail diameters; Synthes Company, Oberdorf, Switzerland) were placed over the external tips of the nails at the entry portals and then screwed into the bone cortex. This modification is further called “2CEC” (Fig. 5).Fig. 5X-ray of the distal part of the Sawbone with a “2CEC” modification
X-ray of the distal part of the Sawbone with a “2CEC” modification
In the third group (n = 8), a third nail (2.5 mm) was inserted over the third entry point from the antero-lateral without prebending, the so-called “3E”-modification (Fig. 3) [21].
Testing was done with a Zwick 1465 universal testing machine (UTM; Zwick GmbH & Co. KG, Ulm, Germany). Fixation of the head of the femur and the femoral condyles in the testing machine was achieved with custom-fit polymethylmethacrylate (Technovit 4006, Heraeus Kulzer, Wehrheim, Germany) moulds for both sides. Set-up followed the ASTM F383-73 and F1264-03 description [27, 28].
Initially, the femur was positioned in a 0° position to test for construct stability with a compression load of up to 150 N to the femoral head with a speed of 0.05 mm/s.
Four-point bending was measured with an incremental linear encoder (MS30-1-LD-2, Megatron, Putzbrunn, Germany) at midpoint of the two lower force bars with a maximum bending moment of 5 Nm (Fig. 6). Speed was set at 0.05 mm/s; maximum bending was 2 mm.Fig. 6Photograph showing the specimen undergoing the four-point bending test, by using the Zwick 1465 universal testing machine, with the bending measured with a linear encoder. (arrows up to down: application of load/Sawbone/sensing device/bearings for the Sawbones)
Photograph showing the specimen undergoing the four-point bending test, by using the Zwick 1465 universal testing machine, with the bending measured with a linear encoder. (arrows up to down: application of load/Sawbone/sensing device/bearings for the Sawbones)
In torsional testing, two angular encoders measured the torsion, and the femoral head area was gimbal-mounted. Speed was set at 20°/min; torsion was limited to 10°.
For shifting testing, the models were installed in a 9° position with a calibrated wedge. During physiological 9° compression, lateral/medial shifting was measured at the trochanter major, while ventral/dorsal shifting was measured at the crista intertrochanterica. A compressive load of up to 100 N was applied to the femoral head with a speed of 0.05 mm/s. In contrast to our spiral models, the reduction of the fracture gap in the 0 and 9° position was not measured due to direct contact of both fragments [18–20].
The course of the tests was equal to previously published studies: first, each specimen was placed in the machine for the four-point bending tests in a standardised order (anterior-posterior (AP), posterior-anterior (PA), lateral-medial (LM) and finally medial-lateral (ML)), followed by internal (IR) and external (ER) torsional tests and finally shifting tests in the 9° position. The first cycle of the tests was used as preconditioning; data for evaluation were collected from three subsequent cycles. After the last cycle of testing, all specimens were again tested with anterior-posterior bending to check for possible destructive changes that could have influenced the results. Thus, we could exclude destruction of the osteosyntheses and the specimen. Deformation of the UTM was determined up to 50 Nm in pretesting during the four-point bending and did not influence the results.
Data (bending moments in four-point bending, torsional stiffness in IR/ER and shifting in 9° position) were analysed with SPSS 18.0 (SPSS Inc., Chicago, USA). Distributions were first checked for normality (Shapiro-Wilk test); when significant discrepancy from a normal distribution occurred, a Mann-Whitney test was performed. When there was no significant discrepancy from normal distribution, the F-test and analysis of variance (ANOVA) were used. All values are presented as mean values. Significance level was set to P < 0.05. Due to multiple testing, the Holm-Bonferroni method was used for post hoc comparison.
Results:
All results of the stiffness and the shifting during compression of the three different prebending configurations are shown in Tables 1, 2 and 3. Using end caps did not improve stability in any direction (Table 1). Furthermore, before adjusting the results with the Holm-Bonferroni method, the classical 2C configuration showed greater stiffness in the anterior-posterior and latero-medial as well as less shifting at the crista intertrochanterica in the 0° position. In contrast, the use of a third nail (3E) offered more stiffness in all four-point bendings as well as in the internal and external rotation in comparison to the classical 2C configuration and the modification with end caps (2CEC). Adjusting the results with the Holm-Bonferroni method, the significant differences for the 3C modification mentioned above could all be affirmed, except for the external rotation compared to the classical 2C configuration (Table 2). In comparing the shiftings in the 9° position, the modification with end caps had the highest shifting and was least stable (Table 3).Table 1Comparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the 2C configuration with end caps (2CEC). Lower changes in shifting tests = higher stiffness2C classical (2C)2C with end caps (2CEC)
P value(n = 8)(n = 7)Mean (SD) four-point bending (Nm/mm)Anterior-posterior0.52 (0.49)0.44 (0.11)0.04a
Posterior-anterior0.43 (0.11)0.48 (0.13)0.89Lateral-medial0.70 (0.16)0.60 (0.19)0.04a
Medial-lateral0.76 (0.27)0.73 (0.25)0.57Mean (SD) rotation (Nm/°)External rotation0.12 (0.04)0.10 (0.03)0.15Internal rotation0.11 (0.03)0.10 (0.03)0.09Mean (SD) 9° compression/shifting (mm)Shifting 9° trochanter major0.80 (0.65)1.77 (1.70)0.06Shifting 9° crista intertrochanterica4.56 (2.67)7.44 (2.48)0.01a
aAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmedTable 2Comparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness2C classical (2C)Third nail from lateral (3E)
P value(n = 8)(n = 8)Mean (SD) four-point bending (Nm/mm)Anterior-posterior0.52 (0.49)1.04 (0.37)<0.001Posterior-anterior0.43 (0.11)0.85 (0.30)<0.001Lateral-medial0.70 (0.16)1.26 (0.54)<0.001Medial-lateral0.76 (0.27)1.16 (0.33)<0.001Mean (SD) rotation (Nm/°)External rotation0.12 (0.04)0.14 (0.02)<0.01a
Internal rotation0.11 (0.03)0.16 (0.04)0.001Mean (SD) 9° compression/shifting (mm)Shifting 9° trochanter major0.80 (0.65)1.14 (1.41)0.98Shifting 9° crista intertrochanterica4.56 (2.67)4.97 (2.89)0.69
aAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmedTable 3Comparison between the stiffness of the osteosyntheses with the 2C configuration with end caps (2CEC) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness2C with end caps (2CEC)Third nail from lateral (3E)
P value(n = 7)(n = 8)Mean (SD) four-point bending (Nm/mm)Anterior-posterior0.44 (0.11)1.04 (0.37)<0.001Posterior-anterior0.48 (0.13)0.85 (0.30)<0.001Lateral-medial0.60 (0.19)1.26 (0.54)<0.001Medial-lateral0.73 (0.25)1.16 (0.33)<0.001Mean (SD) rotation (Nm/°)External rotation0.10 (0.03)0.14 (0.02)<0.001Internal rotation0.10 (0.03)0.16 (0.04)<0.001Mean (SD) 9° compression/shifting (mm)Shifting 9° trochanter major1.77 (1.70)1.14 (1.41)0.24Shifting 9° crista intertrochanterica7.44 (2.48)4.97 (2.89)<0.001Adjusting the results with the Holm-Bonferroni method, the significance of the differences could all be confirmed
Comparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the 2C configuration with end caps (2CEC). Lower changes in shifting tests = higher stiffness
aAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmed
Comparison between the stiffness of the osteosyntheses with the 2C classical configuration (2C) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness
aAdjusting the results with the Holm-Bonferroni method, the significance of the differences could not be confirmed
Comparison between the stiffness of the osteosyntheses with the 2C configuration with end caps (2CEC) and the configuration with a third nail from lateral (3E). Lower changes in shifting tests = higher stiffness
Adjusting the results with the Holm-Bonferroni method, the significance of the differences could all be confirmed
Discussion:
Elastic stable intramedullary nailing (ESIN osteosynthesis) for displaced paediatric femoral fractures in childhood has gained wide acceptance [7, 29], and even older children and sometimes adolescents are treated this way in Europe [6, 10]. On the other hand, publications with special emphasis on problems of this technique revealed complication rates between 10 and 50 % especially—because of residual instability—in complex femoral shaft fractures and in older children or adolescents [9, 10, 12, 30]. To improve elastic stable intramedullary nailing and using stiffness as a marker for stability [31], biomechanical properties of retrograde C-shaped flexible intramedullary nailing are described in the literature [18, 19, 32–36]: Gwyn performed biomechanical testing with different fracture types in synthetic bone models using two titanium elastic nails of 4-mm diameter. The tests were limited to rotational forces, both external and internal, and showed that transverse fractures are as less stable as comminuted fractures with the classical 2 ESIN osteosynthesis [35]. This result was confirmed by Fricka [33]. Most of the other authors focussed on transverse fractures, too, but tested only one or two stress planes without any further explanations [33–36]. In contrast, we are certain that, considering the complex treatment problems, femur fractures require testing in all stress planes as in our previous in vitro settings [18–21]. With the aim of achieving further improvement in the ESIN technique, we tested two different modifications (end caps and a third nail), both established clinically in case series [21, 24] but not yet well analysed in a validated biomechanical model of a transverse femoral fracture. Only one study focussed on end caps in a transverse fracture model but in a very distal fracture type, which is a less frequent transverse shaft fracture. In focussing on the impact of end caps and a third nail to improve the stability in ESIN osteosynthesis in a transverse fracture model, this study revealed a benefit towards the configuration with a third nail providing a significantly higher stiffness than the classical 2C configuration. The configuration with a third nail was even stiffer than the modification with end caps which showed no statistical difference to the classical 2C configuration in this transverse biomechanical fracture model. In this biomechanical model, the insertion of the additional third nail revealed no difficulties if the first and second nails of the classical 2C configuration were placed in a technically correct way.
The ongoing transfer of the additional third nail into routine clinical practice in our hospital showed that one has to ensure that the third nail has the least length at the trochanter major and is only used if the first two nails are placed correctly. Then the additional operation time is below 10 min, and the complications due to insufficient stability are reduced [21].
As with every biomechanical study, this one suffers two limitations in comparison to clinical “reality”. The first one includes the use of a synthetic bone model that cannot precisely reproduce all in vivo conditions. However, the synthetic bone model has been used successfully in previous biomechanical studies [19, 37] and provides more consistency among specimens than cadaveric bones [38–41]. Also, surrounding tissue like periosteum and muscles were missing which might be an additional stabilising part of the elastic stable fixation method. As in other study groups [34–37], we used a “pure” model without further fixation devices. As reduction of paediatric femoral fractures is almost always performed in a closed manner, we have no precise data about the condition of the periosteum in the case of a fracture which could be used for a more realistic model.
Another limitation was due to the configuration [18, 37]: the ends of the nails could not be placed as proximal as intended in a real procedure. In our opinion, this limitation should be equalised as all three configurations were identically established. On the other hand, during the set-up, the focus was on a consistent surgical technique with an identical and reproducible set-up.
Improper location of the nails or the bends in the nails creates an imbalance in the bending forces, resulting in an angular deformity. This serious technical mistake has been reported in the literature [8]. In our opinion, the proper configuration of the nails was achieved more precisely in the present study than in a real surgical situation.
Conclusions:
The results support the modification of the classical two-C-shaped elastic osteosynthesis in femoral fractures with an additional third nail also in transverse fractures. As feasibility and short implantation time could already be shown in spiral fractures, this treatment improves stability and will help to reduce misalignment or revision surgery.
|
Background:
Elastic stable intramedullary nailing (ESIN) is accepted widely for treatment of diaphyseal femur fractures in children. However, complication rates of 10 to 50 % are described due to shortening or axial deviation, especially in older or heavier children. Biomechanical in vitro testing was performed to determine whether two modified osteosyntheses with end caps or a third nail could significantly improve the stability in comparison to classical elastic stable intramedullary nailing in a transverse femur fracture model.
Methods:
We performed biomechanical testing in 24 synthetic adolescent femoral bone models (Sawbones®) with a transverse midshaft (diaphyseal) fracture. First, in all models, two nails were inserted in a C-shaped manner (2 × 3.5 mm steel nails, prebent), then eight osteosyntheses were modified by using end caps and another eight by adding a third nail from the antero-lateral (2.5-mm steel, not prebent). Testing was performed in four-point bending, torsion, and shifting under physiological 9° compression.
Results:
The third nail from the lateral showed a significant positive influence on the stiffness in all four-point bendings as well as in internal rotation comparing to the classical 2C configuration: mean values were significantly higher anterior-posterior (1.04 vs. 0.52 Nm/mm, p < 0.001), posterior-anterior (0.85 vs. 0.43 Nm/mm, p < 0.001), lateral-medial (1.26 vs. 0.70 Nm/mm, p < 0.001), and medial-lateral (1.16 vs. 0.76 Nm/mm, p < 0.001) and during internal rotation (0.16 vs. 0.11 Nm/°, p < 0.001). The modification with end caps did not improve the stiffness in any direction.
Conclusions:
The configuration with a third nail provided a significantly higher stiffness than the classical 2C configuration as well as the modification with end caps in this biomechanical model. This supports the ongoing transfer of the additional third nail into clinical practice to reduce the axial deviation occurring in clinical practice.
|
Introduction:
Fractures of the femoral diaphysis are the second most frequent location of fractures affecting the lower extremity in children (20–26/100,000 children per year) [1, 2] and comprise 1 to 2 % of all fractures in children [3, 4]. More than two thirds occur in children older than 6 years of age [2, 5]. Following the guidelines of the German Society of Paediatric Surgery, children beyond the age of 3 years should be treated with elastic stable intramedullary nailing (ESIN osteosynthesis) even in complex fractures or children older than 12 years as long as sufficient stability can be achieved [6]. ESIN osteosynthesis is said to produce a rapid recovery and a faster reintegration of children and adolescents and lack possible negative effects of immobilisation compared to conservative treatment, especially in schoolchildren [7, 8]. Yet, clinical studies focused on complications following ESIN osteosyntheses revealed problem rates between 10 and 50 % [9–12]. Most complications were observed as a result of instability in complex fracture types and older children weighing more than 40 kg [9, 13, 14]. Because of these instabilities, other authors used additional immobilisation (e.g. application of a cast), additional screws, and an additional external fixation or recommended submuscular plating or external fixation [10, 15–17].
In finding ways to modify elastic stable intramedullary nailing to gain more stability, we first developed a validated adolescent femur spiral facture biomechanical in vitro setting [18]. With this, we found that prebending the nails more than 30° is an essential part of the method [19] and furthermore that steel nails improve stability in contrast to titanium nails [18], which might be the reason for fewer complications of steel nails in clinical practice [12]. Although we could not show any improvement with end caps in our validated spiral fracture biomechanical in vitro setting [20], there seemed to be an improvement in stability with the implementation of a third nail under some circumstances [21]. In contrast, Volpon and co-workers described during combined axial-bending tests the TEN + CAP combination to be 8.75 % stiffer than nails alone as well as during torsion tests a 14 % increased stiffness in (rare) distal femoral fractures [22]. Their data tend to be congruent with preliminary clinical results in few patients [23, 24]. Therefore, the aim of this study was to determine the influence of these two interesting and intensively discussed modifications with end caps and a third nail (end caps = 2CEC; third nail = 3E) to improve the stiffness of the classical C-shaped elastic stable intramedullary nailing osteosynthesis (2C) in displaced transverse femoral fractures in all possible stress planes.
Conclusions:
The results support the modification of the classical two-C-shaped elastic osteosynthesis in femoral fractures with an additional third nail also in transverse fractures. As feasibility and short implantation time could already be shown in spiral fractures, this treatment improves stability and will help to reduce misalignment or revision surgery.
|
Background:
Elastic stable intramedullary nailing (ESIN) is accepted widely for treatment of diaphyseal femur fractures in children. However, complication rates of 10 to 50 % are described due to shortening or axial deviation, especially in older or heavier children. Biomechanical in vitro testing was performed to determine whether two modified osteosyntheses with end caps or a third nail could significantly improve the stability in comparison to classical elastic stable intramedullary nailing in a transverse femur fracture model.
Methods:
We performed biomechanical testing in 24 synthetic adolescent femoral bone models (Sawbones®) with a transverse midshaft (diaphyseal) fracture. First, in all models, two nails were inserted in a C-shaped manner (2 × 3.5 mm steel nails, prebent), then eight osteosyntheses were modified by using end caps and another eight by adding a third nail from the antero-lateral (2.5-mm steel, not prebent). Testing was performed in four-point bending, torsion, and shifting under physiological 9° compression.
Results:
The third nail from the lateral showed a significant positive influence on the stiffness in all four-point bendings as well as in internal rotation comparing to the classical 2C configuration: mean values were significantly higher anterior-posterior (1.04 vs. 0.52 Nm/mm, p < 0.001), posterior-anterior (0.85 vs. 0.43 Nm/mm, p < 0.001), lateral-medial (1.26 vs. 0.70 Nm/mm, p < 0.001), and medial-lateral (1.16 vs. 0.76 Nm/mm, p < 0.001) and during internal rotation (0.16 vs. 0.11 Nm/°, p < 0.001). The modification with end caps did not improve the stiffness in any direction.
Conclusions:
The configuration with a third nail provided a significantly higher stiffness than the classical 2C configuration as well as the modification with end caps in this biomechanical model. This supports the ongoing transfer of the additional third nail into clinical practice to reduce the axial deviation occurring in clinical practice.
| 3,877 | 384 | 5 |
[
"nail",
"2c",
"configuration",
"fractures",
"shifting",
"mm",
"caps",
"point",
"nails",
"end caps"
] |
[
"test",
"test"
] | null |
[CONTENT] Elastic stable intramedullary nailing | Biomechanical testing | Femur fracture | End caps | Third nail [SUMMARY]
| null |
[CONTENT] Elastic stable intramedullary nailing | Biomechanical testing | Femur fracture | End caps | Third nail [SUMMARY]
|
[CONTENT] Elastic stable intramedullary nailing | Biomechanical testing | Femur fracture | End caps | Third nail [SUMMARY]
|
[CONTENT] Elastic stable intramedullary nailing | Biomechanical testing | Femur fracture | End caps | Third nail [SUMMARY]
|
[CONTENT] Elastic stable intramedullary nailing | Biomechanical testing | Femur fracture | End caps | Third nail [SUMMARY]
|
[CONTENT] Adolescent | Biomechanical Phenomena | Bone Nails | Elasticity | Equipment Design | Femoral Fractures | Fracture Fixation, Intramedullary | Humans [SUMMARY]
| null |
[CONTENT] Adolescent | Biomechanical Phenomena | Bone Nails | Elasticity | Equipment Design | Femoral Fractures | Fracture Fixation, Intramedullary | Humans [SUMMARY]
|
[CONTENT] Adolescent | Biomechanical Phenomena | Bone Nails | Elasticity | Equipment Design | Femoral Fractures | Fracture Fixation, Intramedullary | Humans [SUMMARY]
|
[CONTENT] Adolescent | Biomechanical Phenomena | Bone Nails | Elasticity | Equipment Design | Femoral Fractures | Fracture Fixation, Intramedullary | Humans [SUMMARY]
|
[CONTENT] Adolescent | Biomechanical Phenomena | Bone Nails | Elasticity | Equipment Design | Femoral Fractures | Fracture Fixation, Intramedullary | Humans [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] nail | 2c | configuration | fractures | shifting | mm | caps | point | nails | end caps [SUMMARY]
| null |
[CONTENT] nail | 2c | configuration | fractures | shifting | mm | caps | point | nails | end caps [SUMMARY]
|
[CONTENT] nail | 2c | configuration | fractures | shifting | mm | caps | point | nails | end caps [SUMMARY]
|
[CONTENT] nail | 2c | configuration | fractures | shifting | mm | caps | point | nails | end caps [SUMMARY]
|
[CONTENT] nail | 2c | configuration | fractures | shifting | mm | caps | point | nails | end caps [SUMMARY]
|
[CONTENT] children | fractures | nails | years | complications | clinical | older | esin | stable intramedullary | nailing [SUMMARY]
| null |
[CONTENT] shifting | 2c | configuration | sd | results holm | results holm bonferroni | results holm bonferroni method | 2c configuration | differences | stiffness [SUMMARY]
|
[CONTENT] fractures | elastic osteosynthesis | improves stability help reduce | improves stability help | improves stability | improves | shown spiral | shown spiral fractures | shown spiral fractures treatment | time shown spiral fractures [SUMMARY]
|
[CONTENT] fractures | configuration | nails | nail | children | femoral | shifting | fracture | 2c | classical [SUMMARY]
|
[CONTENT] fractures | configuration | nails | nail | children | femoral | shifting | fracture | 2c | classical [SUMMARY]
|
[CONTENT] ESIN ||| 10 to 50 % ||| two | third [SUMMARY]
| null |
[CONTENT] third | four | 2C | 1.04 | 0.52 | 0.85 | Nm/mm | 1.26 | 0.70 | Nm/mm | 1.16 | 0.76 | Nm/mm | 0.16 | 0.11 | p < 0.001 ||| [SUMMARY]
|
[CONTENT] third | 2C ||| third [SUMMARY]
|
[CONTENT] ESIN ||| 10 to 50 % ||| two | third ||| 24 ||| First | two | 2 | eight | another eight | third | 2.5-mm ||| four | 9 ||| ||| third | four | 2C | 1.04 | 0.52 | 0.85 | Nm/mm | 1.26 | 0.70 | Nm/mm | 1.16 | 0.76 | Nm/mm | 0.16 | 0.11 | p < 0.001 ||| ||| third | 2C ||| third [SUMMARY]
|
[CONTENT] ESIN ||| 10 to 50 % ||| two | third ||| 24 ||| First | two | 2 | eight | another eight | third | 2.5-mm ||| four | 9 ||| ||| third | four | 2C | 1.04 | 0.52 | 0.85 | Nm/mm | 1.26 | 0.70 | Nm/mm | 1.16 | 0.76 | Nm/mm | 0.16 | 0.11 | p < 0.001 ||| ||| third | 2C ||| third [SUMMARY]
|
|||||
Prevalence and predictors for spontaneous bacterial peritonitis in cirrhotic patients with ascites admitted at medical block in Korle-Bu Teaching Hospital, Ghana.
|
31384350
|
Spontaneous bacterial peritonitis (SBP) is one of the most common and life-threatening complications of patients with cirrhotic ascites. Recognition and prompt treatment of this condition is essential to prevent serious morbidity and mortality. This study aimed to determine the prevalence of SBP among in-patients with cirrhotic ascites attending our facility and to determine the clinical and laboratory parameters associated with SBP.
|
INTRODUCTION
|
A cross-sectional study was conducted involving one hundred and three (103) patients admitted at medical block in the Korle-Bu Teaching Hospital (KBTH) with cirrhotic ascites from 25th March, 2016 to 25th November, 2016. Demographic and clinical data were collected using a standardized questionnaire. Ascitic fluid culture and cell count were conducted. Positive ascitic fluid culture and/or ascitic polymorphonuclear leukocyte ≥ 250cells/mm3 were diagnostic for SBP.
|
METHODS
|
Of the 103 patients with cirrhotic ascites, the mean age was 43.5 ± 12.2 years. There were fifty eight (58) male patients. The prevalence of SBP was 25.24% (26/103). Majority, 5 (55.6%) of the bacteria isolated from ascitic fluid with SBP was Escherichia coli. Severe ascites and high INR were found to be independent predictors of SBP.
|
RESULTS
|
SBP is common among patients with cirrhotic ascites admitted at KBTH. Severe ascites and high INR were highly suggestive of SBP. Diagnostic paracentesis should be done immediately on admission to confirm the diagnosis irrespective of the clinical characteristics as part of baseline investigation.
|
CONCLUSION
|
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"Adolescent",
"Adult",
"Aged",
"Ascites",
"Ascitic Fluid",
"Bacterial Infections",
"Cross-Sectional Studies",
"Female",
"Ghana",
"Hospitals, Teaching",
"Humans",
"Liver Cirrhosis",
"Male",
"Middle Aged",
"Peritonitis",
"Prevalence",
"Young Adult"
] |
6658157
|
Introduction
|
Spontaneous bacterial peritonitis (SBP) is a common and serious infection occurring in patients with cirrhosis and ascites [1]. Numerous studies suggest that 10-30% of hospitalized patients and 3.5% of outpatients with cirrhosis and ascites have SBP, with in-hospital mortality ranging from 20-40% [2-5]. SBP may be the precipitating factor for the occurrence of kidney failure, hepatic encephalopathy, gastrointestinal bleeding, hypervolemic hyponatremia and development of acute on chronic liver failure, systemic sepsis and poor survival [6]. An increase in the permeability of the intestinal wall leads to translocation of bacteria and subsequent development of SBP [7, 8]. Intestinal bacterial overgrowth and uncontrolled bacterial growth in ascitic fluid then occur, as a result of an impaired host immune response [9, 10]. Factors associated with the risk of developing SBP in cirrhotic patients include upper gastrointestinal bleeding, poor liver function, low ascitic fluid protein levels, prior SBP and hospitalization [11, 12]. Bacteria most commonly isolated from ascitic fluid in patients with SBP are usually those of the normal intestinal flora [13, 14]. More than 92% of all cases are monomicrobial with aerobic gram negative bacilli being responsible for more than two thirds of cases [13]. Escherichia coli accounts for nearly half of these cases followed by Klebsiella species and other gram negative bacteria. Twenty-five percent of cases are caused by gram positive organisms with Streptococcus species being the most common [13]. The symptoms and signs of SBP are subtle compared with those of patients who have surgical peritonitis in the absence of ascites. SBP may be asymptomatic in about 10-32% of cases, particularly in outpatients [4, 14, 15]. Symptoms and signs patients with SBP normally present with include fever, abdominal pain, altered mental status, abdominal tenderness, diarrhoea, paralytic ileus, hypotension and hypothermia (17%) [16]. Early diagnosis and prompt management of SBP once it has developed and preventing infections in high risk groups by giving prophylactic antibiotics are measures that can reduce morbidity and mortality in patients with liver cirrhosis. Because of the significant risk of adverse outcomes related to SBP, identifying predisposing factors is of utmost urgency. There are no recent published studies that exclusively address the prevalence and predictors of SBP in a hospital population in Ghana. This study aims to determine the prevalence and predictors of SBP among in-patients with cirrhotic ascites attending a tertiary referral center in Accra, Ghana. This will help with identification of high risk patients for early and appropriate treatment to reduce morbidity and mortality.
|
Methods
|
A formal approval of this study was obtained from the Ethical and Protocol Committee of the University of Ghana School of Medicine and Dentistry. This study was conducted in accordance with the Helsinki Declaration. The research design was a cross -sectional hospital-based study, carried out at the Department of Medicine, Korle-Bu Teaching Hospital (KBTH), Accra, from 25th March, 2016 to 25th November, 2016. One hundred and three (103) patients with cirrhotic ascites admitted to the medical block of KBTH were consecutively recruited. All adult patients above 18 years with cirrhotic ascites who provided informed consent were included. Exclusion criteria were patients who had already been started on antibiotics at the time of recruitment or who had taken antibiotics up to 2 weeks preceding recruitment, as well as refusal of consent. Diagnosis of liver cirrhosis was made based on the clinical features, laboratory investigations and abdominal ultrasound findings suggestive of liver cirrhosis. Patients' medical records were reviewed to exclude those with cirrhotic ascites who were on antibiotics or had been on antibiotics in the preceding two weeks to the date of recruitment. Relevant history including alcohol use and clinical features of liver cirrhosis (spider angioma, palmar erythema, ascites, asterixis, hepatomegaly, splenomegaly and abdominal vein collaterals) were obtained. Ascites was graded as mild (detectable only on ultrasound), moderate (visible moderate symmetrical abdominal distension) or severe (marked abdominal distension). After thoroughly explaining the study to patients, individuals who gave informed consent were recruited and a questionnaire was administered (socio-demographic data and clinical history of the patients were obtained). A sample of 15mls of venous blood was taken for haematological, biochemical and serological investigations. Abdominal paracentesis was performed using an aseptic technique at the right or left iliac fossa, 3cm above and 3cm medial to the anterior superior iliac spine. Exactly 15mls of ascitic fluid was collected using a sterile syringe for culture, cell count and differentials, albumin and protein. Furthermore, an abdominal ultrasound scan was performed for all patients. The following details were recorded: maximum vertical span of the liver; nodularity of liver surface; spleen size (length of its longest axis); and presence of ascites.
Ascitic fluid work up: 10mls of ascitic fluid was inoculated into a blood culture bottle at the bed side and 5mls of ascitic fluid was inoculated into a sterile EDTA bottle for culture, cell count and differentials, albumin and protein. Ascitic fluid culture was performed by an experienced laboratory technologist by inoculating the ascitic fluid into Blood Agar and MacConckey Agar. Preliminary results were obtained after 48 hours, followed by conventional biochemical identification tests. SBP diagnosis was based on neutrophil count in ascitic fluid ≥ 250 cells/mm3 and/or positive ascitic fluid culture. If ascitic fluid cultures were positive and the neutrophils count was > 250cells/mm3, patients were diagnosed as having culture-positive neutrocytic ascites. If ascitic fluid cultures were negative in the presence of neutrophils > 250 cells/mm3, patients were characterized as having culture-negative neutrocytic ascites (CNNA). Patients with positive cultures on ascitic fluid but without neutrocytic ascites were classified as having monobacterial bacterascites (MNB).
Blood test: hematological, biochemical and serological workup included measurement of total serum bilirubin, serum albumin, alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), aspartate aminotransferase (AST), serum creatinine, serum sodium (Na+), serum potassium (K+), heamoglobin (HB), platelet count, white blood cells (WBC), and prothrombin time with international normalized ratio (INR). Serological work up included hepatitis B surface antigen (HBSAg) and anti-bodies to hepatitis C virus (anti-HCV Ab). Anti-nuclear antibodies (ANA), immune globin G (IgG) and anti-smooth muscle antibodies (ASMA) were done for some of the patients with suspicion of autoimmune hepatitis. Model for end Stage Liver Disease Sodium (MELD-Na+) score was calculated based on laboratory parameters (bilirubin, creatinine levels, sodium and INR) collected at admission and determined by using the UNOS Internet site MELD-Na+ calculator. Child-Pugh Score (CPS) was calculated based on grade of ascites and hepatic encephalopathy, serum albumin, bilirubin and INR collected at admission using QxMD calculator on the net.
Data analysis: data obtained were analyzed using STATA 15 statistical software. Descriptive statistics was run for all the variables. The prevalence of SBP and other categorical variables were expressed as proportions. Biochemical parameters and the clinical scoring systems (MELD-Na+ Score and CPS) were reported as Mean ± SD (normal data) and median (IQR) (non-normal data). The Student t-test or Wilcoxon rank sum test were used to test the difference in means. Chi-squared test and the Fishers exact tests were used to determine the association of categorical variables and SBP. Univariable and multivariable logistic regression models were used to determine the strength of association between CPS, MELD-Na+ score, laboratory and other clinical parameters to SBP. This was presented as crude and adjusted odds ratios on a 95% confidence interval. For all analysis, p-values < 0.05 were considered statistically significant.
|
Results
|
One hundred and three patients with cirrhotic ascites were recruited for the study with a mean age of 43.5 ± 12.2 years (age range 18 to 74) years. Fifty eight (58) (56.3%) patients were males and 44 (43.4%) were females with male to female ratio of 1.7:1. HBV infection was the cause of liver cirrhosis in 53.4% when acting alone and alcohol alone accounted for 21.4% of causes of liver cirrhosis. HCV infection was the cause in 8.9% of cases and HBV infection in combination with alcohol accounted for 6.9%. Autoimmune hepatitis, fatty liver disease and congenital atresia were uncommon causes (Table 1). The prevalence of SBP was 25.24% (95% CI=17.69-34.67). Of the 26 patients that had spontaneous bacterial peritonitis, culture positive SBP was present in 26.9% (9/26) while CNNA was found in 65.4% (17/26). The prevalence of MNB was 7.7% (2/26) in this study. The major isolates from the positive cultures were Escherichia coli (5/9, 55.6%) and Klebsiella spp. (2/9, 22.2%). Staphylococcus epidermidis and Corynebacterium spp. accounted for 1/9 (11.1%) each (Table 2). Results from the univariable logistic regression showed that patients who had SBP had increased odds of having severe ascites (cOR=4.20, 95% CI=1.52-11.66), fever (cOR=3.14, 95% CI=1.25-7.81), chills (cOR=4.42, 95% CI=1.61-10.55) and encephalopathy (cOR=3.03, 95% CI=1.16-7.92). There was a 4% reduced odds of SBP with increasing age (cOR=0.96, 95% CI=092- 0.99) (Table 3). The multiple logistic regression model was fitted whilst controlling for age, sex and the other variables in Table 3. The clinical feature significantly associated with SBP from the multivariable analysis was severe ascites (aOR=5.82, 95% CI=1.51-22.42) (Table 4). Laboratory parameters associated with SBP from the univariable analysis were INR (cOR=2.09, 95%CI=1.28-3.45), CPS (cOR=1.34, 95%CI=1.07-1.69) and MELD-Na+ score (cOR=1.09, 95%CI=1.03-1.16). The adjusted multivariable logistic regression model fitted using a stepwise approach (backward selection). The variables that remained in the final model were Fluid Albumin, Meld-Na+ Score and INR, though only INR (aOR=1.90, 95%CI=1.08-3.35) was a significant predictor of SBP (Table 5).
Causes of liver cirrhosis
Bacteria isolated from ascitic fluid
Bivariate analysis (Clinical and demographic parameters)
- p < 0.05 – statistically significant X2- Chi-squared value
- Mean (± SD)
Univariate and multiple logistic regression (socio-demographic and clinical)
- p < 0.05 – statistically significant
Univariate and stepwise multiple logistic regression
- p < 0.05 – statistically significant
|
Conclusion
|
The common causes of decompensated liver cirrhosis with ascites admitted at Korle-Bu Teaching Hospital were chronic HBV and chromic alcohol abuse. Spontaneous bacterial peritonitis was common among patients with cirrhotic ascites admitted at Korle-Bu Teaching Hospital. Fever, chills, hepatic encephalopathy, severe ascites, high INR, high MELD-Na+ and CPS were predictors of SBP but only severe ascites and high INR were independent predictors. Therefore diagnostic paracentesis should be done immediately for all patients on admission to confirm the diagnosis of SBP preferably before starting empiric antibiotic therapy especially in patients with severe ascites and high INR.
What is known about this topic There is diversity of clinical and laboratory parameters associated with the presence of SBP;
Upper gastrointestinal bleeding is a risk factor associated with the risk of developing SBP.
There is diversity of clinical and laboratory parameters associated with the presence of SBP;
Upper gastrointestinal bleeding is a risk factor associated with the risk of developing SBP.
What this study adds Only severe ascites and high INR were independent predictors associated with SBP;
Patient presented with upper gastrointestinal bleeding were not at high risk of developing SBP.
Only severe ascites and high INR were independent predictors associated with SBP;
Patient presented with upper gastrointestinal bleeding were not at high risk of developing SBP.
|
[
"What is known about this topic",
"What this study adds"
] |
[
"There is diversity of clinical and laboratory parameters associated with the presence of SBP;\nUpper gastrointestinal bleeding is a risk factor associated with the risk of developing SBP.",
"Only severe ascites and high INR were independent predictors associated with SBP;\nPatient presented with upper gastrointestinal bleeding were not at high risk of developing SBP."
] |
[
null,
null
] |
[
"Introduction",
"Methods",
"Results",
"Discussion",
"Conclusion",
"What is known about this topic",
"What this study adds",
"Competing interests"
] |
[
"Spontaneous bacterial peritonitis (SBP) is a common and serious infection occurring in patients with cirrhosis and ascites [1]. Numerous studies suggest that 10-30% of hospitalized patients and 3.5% of outpatients with cirrhosis and ascites have SBP, with in-hospital mortality ranging from 20-40% [2-5]. SBP may be the precipitating factor for the occurrence of kidney failure, hepatic encephalopathy, gastrointestinal bleeding, hypervolemic hyponatremia and development of acute on chronic liver failure, systemic sepsis and poor survival [6]. An increase in the permeability of the intestinal wall leads to translocation of bacteria and subsequent development of SBP [7, 8]. Intestinal bacterial overgrowth and uncontrolled bacterial growth in ascitic fluid then occur, as a result of an impaired host immune response [9, 10]. Factors associated with the risk of developing SBP in cirrhotic patients include upper gastrointestinal bleeding, poor liver function, low ascitic fluid protein levels, prior SBP and hospitalization [11, 12]. Bacteria most commonly isolated from ascitic fluid in patients with SBP are usually those of the normal intestinal flora [13, 14]. More than 92% of all cases are monomicrobial with aerobic gram negative bacilli being responsible for more than two thirds of cases [13]. Escherichia coli accounts for nearly half of these cases followed by Klebsiella species and other gram negative bacteria. Twenty-five percent of cases are caused by gram positive organisms with Streptococcus species being the most common [13]. The symptoms and signs of SBP are subtle compared with those of patients who have surgical peritonitis in the absence of ascites. SBP may be asymptomatic in about 10-32% of cases, particularly in outpatients [4, 14, 15]. Symptoms and signs patients with SBP normally present with include fever, abdominal pain, altered mental status, abdominal tenderness, diarrhoea, paralytic ileus, hypotension and hypothermia (17%) [16]. Early diagnosis and prompt management of SBP once it has developed and preventing infections in high risk groups by giving prophylactic antibiotics are measures that can reduce morbidity and mortality in patients with liver cirrhosis. Because of the significant risk of adverse outcomes related to SBP, identifying predisposing factors is of utmost urgency. There are no recent published studies that exclusively address the prevalence and predictors of SBP in a hospital population in Ghana. This study aims to determine the prevalence and predictors of SBP among in-patients with cirrhotic ascites attending a tertiary referral center in Accra, Ghana. This will help with identification of high risk patients for early and appropriate treatment to reduce morbidity and mortality.",
"A formal approval of this study was obtained from the Ethical and Protocol Committee of the University of Ghana School of Medicine and Dentistry. This study was conducted in accordance with the Helsinki Declaration. The research design was a cross -sectional hospital-based study, carried out at the Department of Medicine, Korle-Bu Teaching Hospital (KBTH), Accra, from 25th March, 2016 to 25th November, 2016. One hundred and three (103) patients with cirrhotic ascites admitted to the medical block of KBTH were consecutively recruited. All adult patients above 18 years with cirrhotic ascites who provided informed consent were included. Exclusion criteria were patients who had already been started on antibiotics at the time of recruitment or who had taken antibiotics up to 2 weeks preceding recruitment, as well as refusal of consent. Diagnosis of liver cirrhosis was made based on the clinical features, laboratory investigations and abdominal ultrasound findings suggestive of liver cirrhosis. Patients' medical records were reviewed to exclude those with cirrhotic ascites who were on antibiotics or had been on antibiotics in the preceding two weeks to the date of recruitment. Relevant history including alcohol use and clinical features of liver cirrhosis (spider angioma, palmar erythema, ascites, asterixis, hepatomegaly, splenomegaly and abdominal vein collaterals) were obtained. Ascites was graded as mild (detectable only on ultrasound), moderate (visible moderate symmetrical abdominal distension) or severe (marked abdominal distension). After thoroughly explaining the study to patients, individuals who gave informed consent were recruited and a questionnaire was administered (socio-demographic data and clinical history of the patients were obtained). A sample of 15mls of venous blood was taken for haematological, biochemical and serological investigations. Abdominal paracentesis was performed using an aseptic technique at the right or left iliac fossa, 3cm above and 3cm medial to the anterior superior iliac spine. Exactly 15mls of ascitic fluid was collected using a sterile syringe for culture, cell count and differentials, albumin and protein. Furthermore, an abdominal ultrasound scan was performed for all patients. The following details were recorded: maximum vertical span of the liver; nodularity of liver surface; spleen size (length of its longest axis); and presence of ascites.\nAscitic fluid work up: 10mls of ascitic fluid was inoculated into a blood culture bottle at the bed side and 5mls of ascitic fluid was inoculated into a sterile EDTA bottle for culture, cell count and differentials, albumin and protein. Ascitic fluid culture was performed by an experienced laboratory technologist by inoculating the ascitic fluid into Blood Agar and MacConckey Agar. Preliminary results were obtained after 48 hours, followed by conventional biochemical identification tests. SBP diagnosis was based on neutrophil count in ascitic fluid ≥ 250 cells/mm3 and/or positive ascitic fluid culture. If ascitic fluid cultures were positive and the neutrophils count was > 250cells/mm3, patients were diagnosed as having culture-positive neutrocytic ascites. If ascitic fluid cultures were negative in the presence of neutrophils > 250 cells/mm3, patients were characterized as having culture-negative neutrocytic ascites (CNNA). Patients with positive cultures on ascitic fluid but without neutrocytic ascites were classified as having monobacterial bacterascites (MNB).\nBlood test: hematological, biochemical and serological workup included measurement of total serum bilirubin, serum albumin, alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), aspartate aminotransferase (AST), serum creatinine, serum sodium (Na+), serum potassium (K+), heamoglobin (HB), platelet count, white blood cells (WBC), and prothrombin time with international normalized ratio (INR). Serological work up included hepatitis B surface antigen (HBSAg) and anti-bodies to hepatitis C virus (anti-HCV Ab). Anti-nuclear antibodies (ANA), immune globin G (IgG) and anti-smooth muscle antibodies (ASMA) were done for some of the patients with suspicion of autoimmune hepatitis. Model for end Stage Liver Disease Sodium (MELD-Na+) score was calculated based on laboratory parameters (bilirubin, creatinine levels, sodium and INR) collected at admission and determined by using the UNOS Internet site MELD-Na+ calculator. Child-Pugh Score (CPS) was calculated based on grade of ascites and hepatic encephalopathy, serum albumin, bilirubin and INR collected at admission using QxMD calculator on the net.\nData analysis: data obtained were analyzed using STATA 15 statistical software. Descriptive statistics was run for all the variables. The prevalence of SBP and other categorical variables were expressed as proportions. Biochemical parameters and the clinical scoring systems (MELD-Na+ Score and CPS) were reported as Mean ± SD (normal data) and median (IQR) (non-normal data). The Student t-test or Wilcoxon rank sum test were used to test the difference in means. Chi-squared test and the Fishers exact tests were used to determine the association of categorical variables and SBP. Univariable and multivariable logistic regression models were used to determine the strength of association between CPS, MELD-Na+ score, laboratory and other clinical parameters to SBP. This was presented as crude and adjusted odds ratios on a 95% confidence interval. For all analysis, p-values < 0.05 were considered statistically significant.",
"One hundred and three patients with cirrhotic ascites were recruited for the study with a mean age of 43.5 ± 12.2 years (age range 18 to 74) years. Fifty eight (58) (56.3%) patients were males and 44 (43.4%) were females with male to female ratio of 1.7:1. HBV infection was the cause of liver cirrhosis in 53.4% when acting alone and alcohol alone accounted for 21.4% of causes of liver cirrhosis. HCV infection was the cause in 8.9% of cases and HBV infection in combination with alcohol accounted for 6.9%. Autoimmune hepatitis, fatty liver disease and congenital atresia were uncommon causes (Table 1). The prevalence of SBP was 25.24% (95% CI=17.69-34.67). Of the 26 patients that had spontaneous bacterial peritonitis, culture positive SBP was present in 26.9% (9/26) while CNNA was found in 65.4% (17/26). The prevalence of MNB was 7.7% (2/26) in this study. The major isolates from the positive cultures were Escherichia coli (5/9, 55.6%) and Klebsiella spp. (2/9, 22.2%). Staphylococcus epidermidis and Corynebacterium spp. accounted for 1/9 (11.1%) each (Table 2). Results from the univariable logistic regression showed that patients who had SBP had increased odds of having severe ascites (cOR=4.20, 95% CI=1.52-11.66), fever (cOR=3.14, 95% CI=1.25-7.81), chills (cOR=4.42, 95% CI=1.61-10.55) and encephalopathy (cOR=3.03, 95% CI=1.16-7.92). There was a 4% reduced odds of SBP with increasing age (cOR=0.96, 95% CI=092- 0.99) (Table 3). The multiple logistic regression model was fitted whilst controlling for age, sex and the other variables in Table 3. The clinical feature significantly associated with SBP from the multivariable analysis was severe ascites (aOR=5.82, 95% CI=1.51-22.42) (Table 4). Laboratory parameters associated with SBP from the univariable analysis were INR (cOR=2.09, 95%CI=1.28-3.45), CPS (cOR=1.34, 95%CI=1.07-1.69) and MELD-Na+ score (cOR=1.09, 95%CI=1.03-1.16). The adjusted multivariable logistic regression model fitted using a stepwise approach (backward selection). The variables that remained in the final model were Fluid Albumin, Meld-Na+ Score and INR, though only INR (aOR=1.90, 95%CI=1.08-3.35) was a significant predictor of SBP (Table 5).\nCauses of liver cirrhosis\nBacteria isolated from ascitic fluid\nBivariate analysis (Clinical and demographic parameters)\n- p < 0.05 – statistically significant X2- Chi-squared value\n- Mean (± SD)\nUnivariate and multiple logistic regression (socio-demographic and clinical)\n- p < 0.05 – statistically significant\nUnivariate and stepwise multiple logistic regression\n- p < 0.05 – statistically significant",
"This study aimed to determine the prevalence, clinical and laboratory features predicting the presence of SBP among inpatients with cirrhotic ascites in a tertiary care center in Accra, Ghana. The prevalence of SBP in this study was 25.24%. This is similar compared to 10-30% found by most studies from the developed world [12,14, 17, 18] but lower than one reported by Oladimeji et al. [19]. This may be due to the severity of liver cirrhosis involved in the study. Oladimeji et al. [19] stated that almost all the patients recruited in their study were in Child's grade C compared with this study with majority of the participants in child's grade B and C The diversity of the clinical and laboratory parameters that is associated with the presence of SBP has been reported in various literatures. This justifies the indication for diagnostic paracentesis in all patients with decompensated cirrhosis with ascites admitted in the hospital. In a study by Evan's et al. [4] no clinical or laboratory parameters were found to be associated with the presence of SBP whiles Figueiredo et al. [20] identified serum albumin, complement C4 of ascitic fluid and upper gastrointestinal bleeding as independent predictors for the diagnosis of SBP. Hypoalbuminaemia, high MELD score, C-reactive protein, Child-Pugh stage C, low protein level in ascitic fluid, low prothrombin concentration, increased serum aspartate aminotransferase levels, high serum bilirubin, low platelet count, hepatic encephalopathy and abdominal pain have also been reported by other studies to predict SBP [21-24]. In this study fever, chills, hepatic encephalopathy, severe ascites, lower age group, high INR, high CPS and high MELD-Na+ predicted the presence of SBP. However, only severe ascites and high INR showed strong independent association with SBP from the multivariate analysis. Severe ascites and high INR are among five markers used to stage the severity of liver disease according to Child-Pugh rankings [25]. The higher the CPS, the greater the risk of SBP [5]. This helps to explain why 70% of cases of SBP are seen in patients with Child-Pugh class C cirrhosis [26]. In the current study, though the Child-Pugh score was not an independent predictor of SBP, but 80% of the patients with SBP were in Child-Pugh grade C.\nHBV (52.4%) was the major cause of liver cirrhosis in this study followed by alcohol (21.4%). This compared similarly with other studies done in Africa [27-29] but differed from reports from the western countries [30, 31]. The most common causes of liver cirrhosis globally are thought to be HBV, HCV and alcohol but the causes vary from country to country and from region to region. In countries where alcohol consumption is more common, alcohol is the commonest cause of liver cirrhosis and in countries where chronic HBV infection is endemic, HBV is the commonest cause of liver cirrhosis [32]. HBV infection is endemic in sub-Saharan Africa including Ghana and this makes it the major cause of cirrhosis in this study. Alcohol abuse was the second commonest cause of liver cirrhosis in this study which implies that alcohol is a significant cause of liver cirrhosis in patients attending clinic at KBTH. This is of public health concern; therefore society should be educated on the harmful effects of alcohol abuse on the liver. In this study E. coli (55.6%) was the most common organism isolated followed by Klebsiella spp (22.2%). The isolation of these organisms is consistent with studies conducted by Bhuva et al. [33] and Oladimeji et al. [19] which shows E. coli as the dominant bacteria cultured in patients with spontaneous bacterial peritonitis.",
"The common causes of decompensated liver cirrhosis with ascites admitted at Korle-Bu Teaching Hospital were chronic HBV and chromic alcohol abuse. Spontaneous bacterial peritonitis was common among patients with cirrhotic ascites admitted at Korle-Bu Teaching Hospital. Fever, chills, hepatic encephalopathy, severe ascites, high INR, high MELD-Na+ and CPS were predictors of SBP but only severe ascites and high INR were independent predictors. Therefore diagnostic paracentesis should be done immediately for all patients on admission to confirm the diagnosis of SBP preferably before starting empiric antibiotic therapy especially in patients with severe ascites and high INR.\n What is known about this topic There is diversity of clinical and laboratory parameters associated with the presence of SBP;\nUpper gastrointestinal bleeding is a risk factor associated with the risk of developing SBP.\nThere is diversity of clinical and laboratory parameters associated with the presence of SBP;\nUpper gastrointestinal bleeding is a risk factor associated with the risk of developing SBP.\n What this study adds Only severe ascites and high INR were independent predictors associated with SBP;\nPatient presented with upper gastrointestinal bleeding were not at high risk of developing SBP.\nOnly severe ascites and high INR were independent predictors associated with SBP;\nPatient presented with upper gastrointestinal bleeding were not at high risk of developing SBP.",
"There is diversity of clinical and laboratory parameters associated with the presence of SBP;\nUpper gastrointestinal bleeding is a risk factor associated with the risk of developing SBP.",
"Only severe ascites and high INR were independent predictors associated with SBP;\nPatient presented with upper gastrointestinal bleeding were not at high risk of developing SBP.",
"The authors declare no competing interests."
] |
[
"intro",
"methods",
"results",
"discussion",
"conclusions",
null,
null,
"COI-statement"
] |
[
"Liver cirrhosis",
"spontaneous bacterial peritonitis",
"ascites"
] |
Introduction:
Spontaneous bacterial peritonitis (SBP) is a common and serious infection occurring in patients with cirrhosis and ascites [1]. Numerous studies suggest that 10-30% of hospitalized patients and 3.5% of outpatients with cirrhosis and ascites have SBP, with in-hospital mortality ranging from 20-40% [2-5]. SBP may be the precipitating factor for the occurrence of kidney failure, hepatic encephalopathy, gastrointestinal bleeding, hypervolemic hyponatremia and development of acute on chronic liver failure, systemic sepsis and poor survival [6]. An increase in the permeability of the intestinal wall leads to translocation of bacteria and subsequent development of SBP [7, 8]. Intestinal bacterial overgrowth and uncontrolled bacterial growth in ascitic fluid then occur, as a result of an impaired host immune response [9, 10]. Factors associated with the risk of developing SBP in cirrhotic patients include upper gastrointestinal bleeding, poor liver function, low ascitic fluid protein levels, prior SBP and hospitalization [11, 12]. Bacteria most commonly isolated from ascitic fluid in patients with SBP are usually those of the normal intestinal flora [13, 14]. More than 92% of all cases are monomicrobial with aerobic gram negative bacilli being responsible for more than two thirds of cases [13]. Escherichia coli accounts for nearly half of these cases followed by Klebsiella species and other gram negative bacteria. Twenty-five percent of cases are caused by gram positive organisms with Streptococcus species being the most common [13]. The symptoms and signs of SBP are subtle compared with those of patients who have surgical peritonitis in the absence of ascites. SBP may be asymptomatic in about 10-32% of cases, particularly in outpatients [4, 14, 15]. Symptoms and signs patients with SBP normally present with include fever, abdominal pain, altered mental status, abdominal tenderness, diarrhoea, paralytic ileus, hypotension and hypothermia (17%) [16]. Early diagnosis and prompt management of SBP once it has developed and preventing infections in high risk groups by giving prophylactic antibiotics are measures that can reduce morbidity and mortality in patients with liver cirrhosis. Because of the significant risk of adverse outcomes related to SBP, identifying predisposing factors is of utmost urgency. There are no recent published studies that exclusively address the prevalence and predictors of SBP in a hospital population in Ghana. This study aims to determine the prevalence and predictors of SBP among in-patients with cirrhotic ascites attending a tertiary referral center in Accra, Ghana. This will help with identification of high risk patients for early and appropriate treatment to reduce morbidity and mortality.
Methods:
A formal approval of this study was obtained from the Ethical and Protocol Committee of the University of Ghana School of Medicine and Dentistry. This study was conducted in accordance with the Helsinki Declaration. The research design was a cross -sectional hospital-based study, carried out at the Department of Medicine, Korle-Bu Teaching Hospital (KBTH), Accra, from 25th March, 2016 to 25th November, 2016. One hundred and three (103) patients with cirrhotic ascites admitted to the medical block of KBTH were consecutively recruited. All adult patients above 18 years with cirrhotic ascites who provided informed consent were included. Exclusion criteria were patients who had already been started on antibiotics at the time of recruitment or who had taken antibiotics up to 2 weeks preceding recruitment, as well as refusal of consent. Diagnosis of liver cirrhosis was made based on the clinical features, laboratory investigations and abdominal ultrasound findings suggestive of liver cirrhosis. Patients' medical records were reviewed to exclude those with cirrhotic ascites who were on antibiotics or had been on antibiotics in the preceding two weeks to the date of recruitment. Relevant history including alcohol use and clinical features of liver cirrhosis (spider angioma, palmar erythema, ascites, asterixis, hepatomegaly, splenomegaly and abdominal vein collaterals) were obtained. Ascites was graded as mild (detectable only on ultrasound), moderate (visible moderate symmetrical abdominal distension) or severe (marked abdominal distension). After thoroughly explaining the study to patients, individuals who gave informed consent were recruited and a questionnaire was administered (socio-demographic data and clinical history of the patients were obtained). A sample of 15mls of venous blood was taken for haematological, biochemical and serological investigations. Abdominal paracentesis was performed using an aseptic technique at the right or left iliac fossa, 3cm above and 3cm medial to the anterior superior iliac spine. Exactly 15mls of ascitic fluid was collected using a sterile syringe for culture, cell count and differentials, albumin and protein. Furthermore, an abdominal ultrasound scan was performed for all patients. The following details were recorded: maximum vertical span of the liver; nodularity of liver surface; spleen size (length of its longest axis); and presence of ascites.
Ascitic fluid work up: 10mls of ascitic fluid was inoculated into a blood culture bottle at the bed side and 5mls of ascitic fluid was inoculated into a sterile EDTA bottle for culture, cell count and differentials, albumin and protein. Ascitic fluid culture was performed by an experienced laboratory technologist by inoculating the ascitic fluid into Blood Agar and MacConckey Agar. Preliminary results were obtained after 48 hours, followed by conventional biochemical identification tests. SBP diagnosis was based on neutrophil count in ascitic fluid ≥ 250 cells/mm3 and/or positive ascitic fluid culture. If ascitic fluid cultures were positive and the neutrophils count was > 250cells/mm3, patients were diagnosed as having culture-positive neutrocytic ascites. If ascitic fluid cultures were negative in the presence of neutrophils > 250 cells/mm3, patients were characterized as having culture-negative neutrocytic ascites (CNNA). Patients with positive cultures on ascitic fluid but without neutrocytic ascites were classified as having monobacterial bacterascites (MNB).
Blood test: hematological, biochemical and serological workup included measurement of total serum bilirubin, serum albumin, alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), aspartate aminotransferase (AST), serum creatinine, serum sodium (Na+), serum potassium (K+), heamoglobin (HB), platelet count, white blood cells (WBC), and prothrombin time with international normalized ratio (INR). Serological work up included hepatitis B surface antigen (HBSAg) and anti-bodies to hepatitis C virus (anti-HCV Ab). Anti-nuclear antibodies (ANA), immune globin G (IgG) and anti-smooth muscle antibodies (ASMA) were done for some of the patients with suspicion of autoimmune hepatitis. Model for end Stage Liver Disease Sodium (MELD-Na+) score was calculated based on laboratory parameters (bilirubin, creatinine levels, sodium and INR) collected at admission and determined by using the UNOS Internet site MELD-Na+ calculator. Child-Pugh Score (CPS) was calculated based on grade of ascites and hepatic encephalopathy, serum albumin, bilirubin and INR collected at admission using QxMD calculator on the net.
Data analysis: data obtained were analyzed using STATA 15 statistical software. Descriptive statistics was run for all the variables. The prevalence of SBP and other categorical variables were expressed as proportions. Biochemical parameters and the clinical scoring systems (MELD-Na+ Score and CPS) were reported as Mean ± SD (normal data) and median (IQR) (non-normal data). The Student t-test or Wilcoxon rank sum test were used to test the difference in means. Chi-squared test and the Fishers exact tests were used to determine the association of categorical variables and SBP. Univariable and multivariable logistic regression models were used to determine the strength of association between CPS, MELD-Na+ score, laboratory and other clinical parameters to SBP. This was presented as crude and adjusted odds ratios on a 95% confidence interval. For all analysis, p-values < 0.05 were considered statistically significant.
Results:
One hundred and three patients with cirrhotic ascites were recruited for the study with a mean age of 43.5 ± 12.2 years (age range 18 to 74) years. Fifty eight (58) (56.3%) patients were males and 44 (43.4%) were females with male to female ratio of 1.7:1. HBV infection was the cause of liver cirrhosis in 53.4% when acting alone and alcohol alone accounted for 21.4% of causes of liver cirrhosis. HCV infection was the cause in 8.9% of cases and HBV infection in combination with alcohol accounted for 6.9%. Autoimmune hepatitis, fatty liver disease and congenital atresia were uncommon causes (Table 1). The prevalence of SBP was 25.24% (95% CI=17.69-34.67). Of the 26 patients that had spontaneous bacterial peritonitis, culture positive SBP was present in 26.9% (9/26) while CNNA was found in 65.4% (17/26). The prevalence of MNB was 7.7% (2/26) in this study. The major isolates from the positive cultures were Escherichia coli (5/9, 55.6%) and Klebsiella spp. (2/9, 22.2%). Staphylococcus epidermidis and Corynebacterium spp. accounted for 1/9 (11.1%) each (Table 2). Results from the univariable logistic regression showed that patients who had SBP had increased odds of having severe ascites (cOR=4.20, 95% CI=1.52-11.66), fever (cOR=3.14, 95% CI=1.25-7.81), chills (cOR=4.42, 95% CI=1.61-10.55) and encephalopathy (cOR=3.03, 95% CI=1.16-7.92). There was a 4% reduced odds of SBP with increasing age (cOR=0.96, 95% CI=092- 0.99) (Table 3). The multiple logistic regression model was fitted whilst controlling for age, sex and the other variables in Table 3. The clinical feature significantly associated with SBP from the multivariable analysis was severe ascites (aOR=5.82, 95% CI=1.51-22.42) (Table 4). Laboratory parameters associated with SBP from the univariable analysis were INR (cOR=2.09, 95%CI=1.28-3.45), CPS (cOR=1.34, 95%CI=1.07-1.69) and MELD-Na+ score (cOR=1.09, 95%CI=1.03-1.16). The adjusted multivariable logistic regression model fitted using a stepwise approach (backward selection). The variables that remained in the final model were Fluid Albumin, Meld-Na+ Score and INR, though only INR (aOR=1.90, 95%CI=1.08-3.35) was a significant predictor of SBP (Table 5).
Causes of liver cirrhosis
Bacteria isolated from ascitic fluid
Bivariate analysis (Clinical and demographic parameters)
- p < 0.05 – statistically significant X2- Chi-squared value
- Mean (± SD)
Univariate and multiple logistic regression (socio-demographic and clinical)
- p < 0.05 – statistically significant
Univariate and stepwise multiple logistic regression
- p < 0.05 – statistically significant
Discussion:
This study aimed to determine the prevalence, clinical and laboratory features predicting the presence of SBP among inpatients with cirrhotic ascites in a tertiary care center in Accra, Ghana. The prevalence of SBP in this study was 25.24%. This is similar compared to 10-30% found by most studies from the developed world [12,14, 17, 18] but lower than one reported by Oladimeji et al. [19]. This may be due to the severity of liver cirrhosis involved in the study. Oladimeji et al. [19] stated that almost all the patients recruited in their study were in Child's grade C compared with this study with majority of the participants in child's grade B and C The diversity of the clinical and laboratory parameters that is associated with the presence of SBP has been reported in various literatures. This justifies the indication for diagnostic paracentesis in all patients with decompensated cirrhosis with ascites admitted in the hospital. In a study by Evan's et al. [4] no clinical or laboratory parameters were found to be associated with the presence of SBP whiles Figueiredo et al. [20] identified serum albumin, complement C4 of ascitic fluid and upper gastrointestinal bleeding as independent predictors for the diagnosis of SBP. Hypoalbuminaemia, high MELD score, C-reactive protein, Child-Pugh stage C, low protein level in ascitic fluid, low prothrombin concentration, increased serum aspartate aminotransferase levels, high serum bilirubin, low platelet count, hepatic encephalopathy and abdominal pain have also been reported by other studies to predict SBP [21-24]. In this study fever, chills, hepatic encephalopathy, severe ascites, lower age group, high INR, high CPS and high MELD-Na+ predicted the presence of SBP. However, only severe ascites and high INR showed strong independent association with SBP from the multivariate analysis. Severe ascites and high INR are among five markers used to stage the severity of liver disease according to Child-Pugh rankings [25]. The higher the CPS, the greater the risk of SBP [5]. This helps to explain why 70% of cases of SBP are seen in patients with Child-Pugh class C cirrhosis [26]. In the current study, though the Child-Pugh score was not an independent predictor of SBP, but 80% of the patients with SBP were in Child-Pugh grade C.
HBV (52.4%) was the major cause of liver cirrhosis in this study followed by alcohol (21.4%). This compared similarly with other studies done in Africa [27-29] but differed from reports from the western countries [30, 31]. The most common causes of liver cirrhosis globally are thought to be HBV, HCV and alcohol but the causes vary from country to country and from region to region. In countries where alcohol consumption is more common, alcohol is the commonest cause of liver cirrhosis and in countries where chronic HBV infection is endemic, HBV is the commonest cause of liver cirrhosis [32]. HBV infection is endemic in sub-Saharan Africa including Ghana and this makes it the major cause of cirrhosis in this study. Alcohol abuse was the second commonest cause of liver cirrhosis in this study which implies that alcohol is a significant cause of liver cirrhosis in patients attending clinic at KBTH. This is of public health concern; therefore society should be educated on the harmful effects of alcohol abuse on the liver. In this study E. coli (55.6%) was the most common organism isolated followed by Klebsiella spp (22.2%). The isolation of these organisms is consistent with studies conducted by Bhuva et al. [33] and Oladimeji et al. [19] which shows E. coli as the dominant bacteria cultured in patients with spontaneous bacterial peritonitis.
Conclusion:
The common causes of decompensated liver cirrhosis with ascites admitted at Korle-Bu Teaching Hospital were chronic HBV and chromic alcohol abuse. Spontaneous bacterial peritonitis was common among patients with cirrhotic ascites admitted at Korle-Bu Teaching Hospital. Fever, chills, hepatic encephalopathy, severe ascites, high INR, high MELD-Na+ and CPS were predictors of SBP but only severe ascites and high INR were independent predictors. Therefore diagnostic paracentesis should be done immediately for all patients on admission to confirm the diagnosis of SBP preferably before starting empiric antibiotic therapy especially in patients with severe ascites and high INR.
What is known about this topic There is diversity of clinical and laboratory parameters associated with the presence of SBP;
Upper gastrointestinal bleeding is a risk factor associated with the risk of developing SBP.
There is diversity of clinical and laboratory parameters associated with the presence of SBP;
Upper gastrointestinal bleeding is a risk factor associated with the risk of developing SBP.
What this study adds Only severe ascites and high INR were independent predictors associated with SBP;
Patient presented with upper gastrointestinal bleeding were not at high risk of developing SBP.
Only severe ascites and high INR were independent predictors associated with SBP;
Patient presented with upper gastrointestinal bleeding were not at high risk of developing SBP.
What is known about this topic:
There is diversity of clinical and laboratory parameters associated with the presence of SBP;
Upper gastrointestinal bleeding is a risk factor associated with the risk of developing SBP.
What this study adds:
Only severe ascites and high INR were independent predictors associated with SBP;
Patient presented with upper gastrointestinal bleeding were not at high risk of developing SBP.
Competing interests:
The authors declare no competing interests.
|
Background:
Spontaneous bacterial peritonitis (SBP) is one of the most common and life-threatening complications of patients with cirrhotic ascites. Recognition and prompt treatment of this condition is essential to prevent serious morbidity and mortality. This study aimed to determine the prevalence of SBP among in-patients with cirrhotic ascites attending our facility and to determine the clinical and laboratory parameters associated with SBP.
Methods:
A cross-sectional study was conducted involving one hundred and three (103) patients admitted at medical block in the Korle-Bu Teaching Hospital (KBTH) with cirrhotic ascites from 25th March, 2016 to 25th November, 2016. Demographic and clinical data were collected using a standardized questionnaire. Ascitic fluid culture and cell count were conducted. Positive ascitic fluid culture and/or ascitic polymorphonuclear leukocyte ≥ 250cells/mm3 were diagnostic for SBP.
Results:
Of the 103 patients with cirrhotic ascites, the mean age was 43.5 ± 12.2 years. There were fifty eight (58) male patients. The prevalence of SBP was 25.24% (26/103). Majority, 5 (55.6%) of the bacteria isolated from ascitic fluid with SBP was Escherichia coli. Severe ascites and high INR were found to be independent predictors of SBP.
Conclusions:
SBP is common among patients with cirrhotic ascites admitted at KBTH. Severe ascites and high INR were highly suggestive of SBP. Diagnostic paracentesis should be done immediately on admission to confirm the diagnosis irrespective of the clinical characteristics as part of baseline investigation.
|
Introduction:
Spontaneous bacterial peritonitis (SBP) is a common and serious infection occurring in patients with cirrhosis and ascites [1]. Numerous studies suggest that 10-30% of hospitalized patients and 3.5% of outpatients with cirrhosis and ascites have SBP, with in-hospital mortality ranging from 20-40% [2-5]. SBP may be the precipitating factor for the occurrence of kidney failure, hepatic encephalopathy, gastrointestinal bleeding, hypervolemic hyponatremia and development of acute on chronic liver failure, systemic sepsis and poor survival [6]. An increase in the permeability of the intestinal wall leads to translocation of bacteria and subsequent development of SBP [7, 8]. Intestinal bacterial overgrowth and uncontrolled bacterial growth in ascitic fluid then occur, as a result of an impaired host immune response [9, 10]. Factors associated with the risk of developing SBP in cirrhotic patients include upper gastrointestinal bleeding, poor liver function, low ascitic fluid protein levels, prior SBP and hospitalization [11, 12]. Bacteria most commonly isolated from ascitic fluid in patients with SBP are usually those of the normal intestinal flora [13, 14]. More than 92% of all cases are monomicrobial with aerobic gram negative bacilli being responsible for more than two thirds of cases [13]. Escherichia coli accounts for nearly half of these cases followed by Klebsiella species and other gram negative bacteria. Twenty-five percent of cases are caused by gram positive organisms with Streptococcus species being the most common [13]. The symptoms and signs of SBP are subtle compared with those of patients who have surgical peritonitis in the absence of ascites. SBP may be asymptomatic in about 10-32% of cases, particularly in outpatients [4, 14, 15]. Symptoms and signs patients with SBP normally present with include fever, abdominal pain, altered mental status, abdominal tenderness, diarrhoea, paralytic ileus, hypotension and hypothermia (17%) [16]. Early diagnosis and prompt management of SBP once it has developed and preventing infections in high risk groups by giving prophylactic antibiotics are measures that can reduce morbidity and mortality in patients with liver cirrhosis. Because of the significant risk of adverse outcomes related to SBP, identifying predisposing factors is of utmost urgency. There are no recent published studies that exclusively address the prevalence and predictors of SBP in a hospital population in Ghana. This study aims to determine the prevalence and predictors of SBP among in-patients with cirrhotic ascites attending a tertiary referral center in Accra, Ghana. This will help with identification of high risk patients for early and appropriate treatment to reduce morbidity and mortality.
Conclusion:
The common causes of decompensated liver cirrhosis with ascites admitted at Korle-Bu Teaching Hospital were chronic HBV and chromic alcohol abuse. Spontaneous bacterial peritonitis was common among patients with cirrhotic ascites admitted at Korle-Bu Teaching Hospital. Fever, chills, hepatic encephalopathy, severe ascites, high INR, high MELD-Na+ and CPS were predictors of SBP but only severe ascites and high INR were independent predictors. Therefore diagnostic paracentesis should be done immediately for all patients on admission to confirm the diagnosis of SBP preferably before starting empiric antibiotic therapy especially in patients with severe ascites and high INR.
What is known about this topic There is diversity of clinical and laboratory parameters associated with the presence of SBP;
Upper gastrointestinal bleeding is a risk factor associated with the risk of developing SBP.
There is diversity of clinical and laboratory parameters associated with the presence of SBP;
Upper gastrointestinal bleeding is a risk factor associated with the risk of developing SBP.
What this study adds Only severe ascites and high INR were independent predictors associated with SBP;
Patient presented with upper gastrointestinal bleeding were not at high risk of developing SBP.
Only severe ascites and high INR were independent predictors associated with SBP;
Patient presented with upper gastrointestinal bleeding were not at high risk of developing SBP.
|
Background:
Spontaneous bacterial peritonitis (SBP) is one of the most common and life-threatening complications of patients with cirrhotic ascites. Recognition and prompt treatment of this condition is essential to prevent serious morbidity and mortality. This study aimed to determine the prevalence of SBP among in-patients with cirrhotic ascites attending our facility and to determine the clinical and laboratory parameters associated with SBP.
Methods:
A cross-sectional study was conducted involving one hundred and three (103) patients admitted at medical block in the Korle-Bu Teaching Hospital (KBTH) with cirrhotic ascites from 25th March, 2016 to 25th November, 2016. Demographic and clinical data were collected using a standardized questionnaire. Ascitic fluid culture and cell count were conducted. Positive ascitic fluid culture and/or ascitic polymorphonuclear leukocyte ≥ 250cells/mm3 were diagnostic for SBP.
Results:
Of the 103 patients with cirrhotic ascites, the mean age was 43.5 ± 12.2 years. There were fifty eight (58) male patients. The prevalence of SBP was 25.24% (26/103). Majority, 5 (55.6%) of the bacteria isolated from ascitic fluid with SBP was Escherichia coli. Severe ascites and high INR were found to be independent predictors of SBP.
Conclusions:
SBP is common among patients with cirrhotic ascites admitted at KBTH. Severe ascites and high INR were highly suggestive of SBP. Diagnostic paracentesis should be done immediately on admission to confirm the diagnosis irrespective of the clinical characteristics as part of baseline investigation.
| 3,107 | 284 | 8 |
[
"sbp",
"patients",
"ascites",
"liver",
"study",
"cirrhosis",
"high",
"fluid",
"ascitic",
"ascitic fluid"
] |
[
"test",
"test"
] |
[CONTENT] Liver cirrhosis | spontaneous bacterial peritonitis | ascites [SUMMARY]
|
[CONTENT] Liver cirrhosis | spontaneous bacterial peritonitis | ascites [SUMMARY]
|
[CONTENT] Liver cirrhosis | spontaneous bacterial peritonitis | ascites [SUMMARY]
|
[CONTENT] Liver cirrhosis | spontaneous bacterial peritonitis | ascites [SUMMARY]
|
[CONTENT] Liver cirrhosis | spontaneous bacterial peritonitis | ascites [SUMMARY]
|
[CONTENT] Liver cirrhosis | spontaneous bacterial peritonitis | ascites [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Ascites | Ascitic Fluid | Bacterial Infections | Cross-Sectional Studies | Female | Ghana | Hospitals, Teaching | Humans | Liver Cirrhosis | Male | Middle Aged | Peritonitis | Prevalence | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Ascites | Ascitic Fluid | Bacterial Infections | Cross-Sectional Studies | Female | Ghana | Hospitals, Teaching | Humans | Liver Cirrhosis | Male | Middle Aged | Peritonitis | Prevalence | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Ascites | Ascitic Fluid | Bacterial Infections | Cross-Sectional Studies | Female | Ghana | Hospitals, Teaching | Humans | Liver Cirrhosis | Male | Middle Aged | Peritonitis | Prevalence | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Ascites | Ascitic Fluid | Bacterial Infections | Cross-Sectional Studies | Female | Ghana | Hospitals, Teaching | Humans | Liver Cirrhosis | Male | Middle Aged | Peritonitis | Prevalence | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Ascites | Ascitic Fluid | Bacterial Infections | Cross-Sectional Studies | Female | Ghana | Hospitals, Teaching | Humans | Liver Cirrhosis | Male | Middle Aged | Peritonitis | Prevalence | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Ascites | Ascitic Fluid | Bacterial Infections | Cross-Sectional Studies | Female | Ghana | Hospitals, Teaching | Humans | Liver Cirrhosis | Male | Middle Aged | Peritonitis | Prevalence | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] sbp | patients | ascites | liver | study | cirrhosis | high | fluid | ascitic | ascitic fluid [SUMMARY]
|
[CONTENT] sbp | patients | ascites | liver | study | cirrhosis | high | fluid | ascitic | ascitic fluid [SUMMARY]
|
[CONTENT] sbp | patients | ascites | liver | study | cirrhosis | high | fluid | ascitic | ascitic fluid [SUMMARY]
|
[CONTENT] sbp | patients | ascites | liver | study | cirrhosis | high | fluid | ascitic | ascitic fluid [SUMMARY]
|
[CONTENT] sbp | patients | ascites | liver | study | cirrhosis | high | fluid | ascitic | ascitic fluid [SUMMARY]
|
[CONTENT] sbp | patients | ascites | liver | study | cirrhosis | high | fluid | ascitic | ascitic fluid [SUMMARY]
|
[CONTENT] sbp | patients | cases | gram | intestinal | 13 | mortality | risk | bacteria | 10 [SUMMARY]
|
[CONTENT] ascitic | fluid | ascitic fluid | patients | culture | serum | data | obtained | based | test [SUMMARY]
|
[CONTENT] ci | 95 ci | 95 | cor | table | logistic regression | 26 | regression | logistic | age [SUMMARY]
|
[CONTENT] high | sbp | ascites high | high inr | ascites high inr | severe ascites high | severe ascites high inr | ascites | risk | severe ascites [SUMMARY]
|
[CONTENT] sbp | high | risk | patients | ascites | associated | developing | developing sbp | risk developing sbp | risk developing [SUMMARY]
|
[CONTENT] sbp | high | risk | patients | ascites | associated | developing | developing sbp | risk developing sbp | risk developing [SUMMARY]
|
[CONTENT] SBP ||| Recognition ||| SBP | SBP [SUMMARY]
|
[CONTENT] one hundred and three | 103 | the Korle-Bu Teaching Hospital | KBTH | 25th March, 2016 to 25th November, 2016 ||| ||| ||| ≥ ||| 250cells | SBP [SUMMARY]
|
[CONTENT] 103 | 43.5 | 12.2 years ||| fifty eight | 58 ||| SBP | 25.24% ||| 5 | 55.6% | SBP | Escherichia ||| INR | SBP [SUMMARY]
|
[CONTENT] SBP | KBTH ||| INR | SBP ||| [SUMMARY]
|
[CONTENT] SBP ||| Recognition ||| SBP | SBP | one hundred and three | 103 | the Korle-Bu Teaching Hospital | KBTH | 25th March, 2016 to 25th November, 2016 ||| ||| ||| ≥ ||| 250cells | SBP ||| 103 | 43.5 | 12.2 years ||| fifty eight | 58 ||| SBP | 25.24% ||| 5 | 55.6% | SBP | Escherichia ||| INR | SBP ||| SBP | KBTH ||| INR | SBP ||| [SUMMARY]
|
[CONTENT] SBP ||| Recognition ||| SBP | SBP | one hundred and three | 103 | the Korle-Bu Teaching Hospital | KBTH | 25th March, 2016 to 25th November, 2016 ||| ||| ||| ≥ ||| 250cells | SBP ||| 103 | 43.5 | 12.2 years ||| fifty eight | 58 ||| SBP | 25.24% ||| 5 | 55.6% | SBP | Escherichia ||| INR | SBP ||| SBP | KBTH ||| INR | SBP ||| [SUMMARY]
|
||||||
Hyberbaric oxygen increases atresia in normal & steroid induced PCO rat ovaries.
|
22309835
|
In this study, we investigated the effect of hyperbaric oxygen therapy (HBOT) on the morphology of estradiol valerate (EV) induced polycystic ovary (PCO) to find a new treatment modality for improvement of PCO.
|
BACKGROUND
|
The rats were divided into four groups. Group1, control; group 2, PCO group; group 3, PCO with HBOT group and group 4, normal ovary with HBOT. PCO was induced by a single intramuscular injection of 4 mg EV in adult cycling rats. Other rats with normal ovaries had oil injection as placebo. HBOT was applied to third and fourth groups for six weeks. Histopathologic evaluation of ovaries of all groups were performed & compared.
|
METHODS
|
Six weeks of HBOT was resulted in increase in follicular atresia, decrease in the number of primary, secondary, tertiary follicles and decrease in the number of fresh corpus luteum in normal rat ovary. HBOT on polycystic rat ovary, resulted in significant increase in atretic follicles which were already present.
|
RESULTS
|
HBOT of six weeks itself, changed ovarian morphology in favor of atresia both in PCO group and control group. This result of aggravated follicular atresia after HBOT on EV induced PCO may be due to long-term exposure in our protocol which with this state seems to be inapplicable in the improvement of PCO morphology.
|
CONCLUSIONS
|
[
"Animals",
"Estradiol",
"Female",
"Hyperbaric Oxygenation",
"Ovarian Follicle",
"Ovary",
"Polycystic Ovary Syndrome",
"Rats",
"Rats, Wistar"
] |
3395821
|
Background
|
Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women of reproductive age is a multifactorial metabolic disease associated with insulin resistance [1]. PCOS is a common condition with a range of clinical features. These reproductive features include anovulation, irregular menstrual cycles, clinical and biochemical hyperandrogenism and infertility. Metabolic features include increased risk factors for type 2 diabetes mellitus and cardiovascular disease and increase in the prevalence of the metabolic syndrome [1,2].
Previously, the diagnosis of PCOS was based on the National Institute of Health (NIH) criteria comprising biochemical or clinical hyperandrogenism and anovulatory irregular menstrual cycles with the exclusion of related reproductive disorders. In 2003, diagnostic guidelines for PCOS were expanded to the so-called Rotterdam criteria, now based on presentation with any two of the three criteria of hyperandrogenism, irregular anovulatory periods or polycystic ovaries on ultrasound [1,3]. The reported prevalence range of PCOS is between 2.2% to 26% [1,3,4]. The wide range of estimated PCOS prevalence can be explained by different recruitment processes of the study populations, selection biases, ethnic and racial variations in addition to, the criteria used for its definition and the screening methods used to identify each criteria; considering the Rotterdam versus NIH criteria increase the PCOS prevalence by 2 times. The latest study by Tehrani et al. demonstrated that the prevalence's of PCOS using the NIH definition were 8.5% [4].
The abnormalities detected in PCOS have been attributed to primary defects in the hypothalamic-pituitary-adrenal axis, the ovarian microenvironment, the adrenal gland and the insulin/insulin-like growth factor metabolic regulatory system [2,5]. There is evidence that both hypothalamic and pituitary mechanisms contribute to the gonadotropin dysfunction in PCOS.
The reproductive systems of human beings and other vertebrates are grossly similar [6]. Although the rat is a polytocous rodent, the female has a regular ovarian cyclicity of 4 or 5 days, with distinct proestrus, estrus, and diestrus phases. PCO can be experimentally produced in the rat, that species is a good model for studying the pathophysiology of human PCO by a single dose of the long-acting estrogen, estradiol valerate (EV) which leads to anovulatory state in 13-15 week-old rats [6,7].
The therapeutic application of hyperbaric oxygen (HBO) has been known over three decades in different fields of medicine. Hyperbaric oxygen therapy (HBOT) is defined by the Undersea and Hyperbaric Medical Society (UHMS) as a treatment in which a patient intermittently breathes 100% oxygen while the treatment chamber is pressurized to a pressure greater than sea level (1 atmosphere absolute, ATA). The therapeutic principle behind HBO stems from increasing the partial pressure of oxygen in the tissues of the body. The effects of HBO are based on the gas laws, and the physiological, biochemical effects of hyperoxia [8,9]. High tissue levels of oxygen stimulate angiogenesis by increasing the production of cytokines and many growth factors, especially VEGF. Because of these effects on angiogenesis and hyperoxia, it is a unique method used in the treatment of various illnesses and clinical conditions, such as carbon monoxide poisoning, decompression sickness, osteomyelitis, even diabetic foot and ischaemic wounds [9,10].
Besides, wound healing, HBOT has been used for many clinical treatments. Previous experiments have demonstrated that HBO improves survival in anesthetized rats during heatstroke by reducing multiorgan dysfunction [11]. Furthermore, microarray analysis of gene expression in rat cortical neurons exposed to HBO showed that 17 genes changed in response to all exposure conditions [12]. HBOT is also used for primary liver nonfunction as it stimulated hepatocytes to proliferate. Experimentally, HBO was found significantly effective in the survival of transplanted cells or tissues like liver, bone, thyroid, pancreatic islet cells [13]. Although a large proportion of ovarian follicles were lost during the initial ischemia after transplantation of ovaries, recently it was also found that HBO was also beneficial for the follicular survival of the transplanted mouse ovaries [14].
HBOT was also used in invitro-fertilisation (IVF) protocols in different studies for improving ovarian folicular stimulation and endometrial receptivity as an adjunct to infertility therapies with the hypothesis of increasing impaired follicular angiogenesis and oxygenation and increasing pregnancy implantation by improving endometrial receptivity by adequate vascularisation and oxygenation in unexplained infertility patients respectively [15,16]. Although new; these trials offer hopeful results for future infertility patients.
Principal mechanisms of HBO are based on intracellular generation of reactive species of oxygen and nitrogen by causing oxidative stress. Reactive oxygen species (ROS) are recognized to play a central role in cell signal transduction cascades or pathways, for a variety of growth factors, cytokines, and hormones. Scavenging antioxidants combat the overproduction of reactive species like vitamine E and C, glutation [10,17]. Recently, some new data about the role of oxidative stress and antioxidants in modulation of ovarian techal-interstitial cells have been enlightened. First, it was demonstrated that antioxidants inhibited proliferation of theca-interstitial cells then new findings added that antioxidants with distinctly different mechanisms of action induce a series of events consistent with the process of apoptosis in ovarian mesenchyme [18,19]. As PCOS is a condition associated with excessive growth and activity of theca-interstitial cells, these findings bring the question of the role of oxidative stress and antioxidants in PCO disease.
Treatment of human PCOS is symptomatic, lifestyle measures such as diet and exercise (or both) could play an important role in optimising healthy weight, improving underlying hormonal disturbances, prevention of future reproductive and metabolic complications and improving quality of life. Lifestyle interventions are recommended first-line in an international position statement on PCOS and present a cost effective initial treatment strategy compared to surgical and pharmacological options [20]. In the recent Cochrane review the positive effects of lifestyle treatment on anthropometric (abdominal adiposity, adiposity distribution), reproductive (biochemical and clinical hyperandrogenism) and metabolic features (markers of insulin resistance) were confirmed with medium or long term lifestyle measures [21].
In the present study, we tried to find a new, alternative treatment modality for PCO disease, considering the different effects of hyperoxia on different tissues, mentioned above. For this, we tested the hypothesis that 6 weeks of HBO administration on polycystic rat ovary would make an improvement or reversal of PCO state. This was carried out by studying the effect of HBO on ovarian morphology including different folicular stages.
|
Methods
|
Animals 80 young, cyclic female Wistar-Albino rats (250-300 g) were used for the experiment. All rats were provided by Experimental Medicine Research Center (DETAM) of Istanbul University and housed in the Animal Laboratory of the same centre. They were caged in a controlled environment of 22°C with 12 h light/dark cycles and a humidity range between 40 and 60%. Standard rat feed and reverse-osmosis-purified water were provided ad libitum. All rats were allowed to have 1 week of acclimation to this environment before the experiment. This study approved by 'Animal Studies Comittee' at DETAM Unite of Istanbul University, Istanbul and all investigations complied with the 1996 National Academy of Science's Guide for Care and Use of Laboratory. Eighty animals were divided into four experimental groups: group 1, control (n = 20); group 2, PCO only group (n = 20); group 3, PCO+HBO group (n = 20) and group 4, HBO only group (n = 20).
80 young, cyclic female Wistar-Albino rats (250-300 g) were used for the experiment. All rats were provided by Experimental Medicine Research Center (DETAM) of Istanbul University and housed in the Animal Laboratory of the same centre. They were caged in a controlled environment of 22°C with 12 h light/dark cycles and a humidity range between 40 and 60%. Standard rat feed and reverse-osmosis-purified water were provided ad libitum. All rats were allowed to have 1 week of acclimation to this environment before the experiment. This study approved by 'Animal Studies Comittee' at DETAM Unite of Istanbul University, Istanbul and all investigations complied with the 1996 National Academy of Science's Guide for Care and Use of Laboratory. Eighty animals were divided into four experimental groups: group 1, control (n = 20); group 2, PCO only group (n = 20); group 3, PCO+HBO group (n = 20) and group 4, HBO only group (n = 20).
PCO induction Young cyclic Wistar females were taken and had free access to pelleted rat food and water. All animals were displaying at least 2 consecutive normal 4-day estrous cycles were used in this study. Estrous cycles prior to and after treatment were monitored by daily examination of vaginal smears.
To induce a well defined PCO, 4 mg im injection of EV (Sigma, Riedeldehaen, Germany) was administered in 0.2 ml of sesame oil. Forthy rats those in the PCO groups received EV and the remaining rats (also in estrus) those in the oil groups were injected 0.2 ml oil. A duration of 6 weeks was chosen because a single injection of EV induces, after a lag period of 4-6 week, a chronic anovulatory PCO condition in adult rats [6,7].
Young cyclic Wistar females were taken and had free access to pelleted rat food and water. All animals were displaying at least 2 consecutive normal 4-day estrous cycles were used in this study. Estrous cycles prior to and after treatment were monitored by daily examination of vaginal smears.
To induce a well defined PCO, 4 mg im injection of EV (Sigma, Riedeldehaen, Germany) was administered in 0.2 ml of sesame oil. Forthy rats those in the PCO groups received EV and the remaining rats (also in estrus) those in the oil groups were injected 0.2 ml oil. A duration of 6 weeks was chosen because a single injection of EV induces, after a lag period of 4-6 week, a chronic anovulatory PCO condition in adult rats [6,7].
HBO After full PCO state was obtained (after six weeks from the EV or oil injection), the rats were allowed to take six weeks of HBOT. We have chosen 6 weeks of HBOT depending on protocols of Department of Underwater and Hyberbaric Oxygen Unite for chronic diseases protocol for rats.
Hyperbaric Chambers and standard protocols used for rats that is 2.5 atm pressure, totally 60 minutes once a day protocol is used [8,9]. The chamber was ventilated with 100% oxygen at a flow rate of 20 L/min to minimize CO2 accumulation. Rats were then slowly decompressed at a rate of 1 ATA/min at Istanbul University Underseas and Hyperbaric O2 Department.
The rats were decapitated after HBOT finished, independent of cycle day. Before decapitation, all rats were anaesthetised with an i.p. administration 50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi, Levent, Istanbul, Turkey) and 7 mg/kg Xylazine hydrochloric acid (Rompun, Bayer Sisli, Istanbul, Turkey)
After full PCO state was obtained (after six weeks from the EV or oil injection), the rats were allowed to take six weeks of HBOT. We have chosen 6 weeks of HBOT depending on protocols of Department of Underwater and Hyberbaric Oxygen Unite for chronic diseases protocol for rats.
Hyperbaric Chambers and standard protocols used for rats that is 2.5 atm pressure, totally 60 minutes once a day protocol is used [8,9]. The chamber was ventilated with 100% oxygen at a flow rate of 20 L/min to minimize CO2 accumulation. Rats were then slowly decompressed at a rate of 1 ATA/min at Istanbul University Underseas and Hyperbaric O2 Department.
The rats were decapitated after HBOT finished, independent of cycle day. Before decapitation, all rats were anaesthetised with an i.p. administration 50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi, Levent, Istanbul, Turkey) and 7 mg/kg Xylazine hydrochloric acid (Rompun, Bayer Sisli, Istanbul, Turkey)
Ovarian morphology and quantitative analysis of follicle populations Tissues After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.
Only the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.
Quantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.
After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.
Only the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.
Quantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.
Tissues After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.
Only the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.
Quantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.
After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.
Only the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.
Quantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.
Statistical analysis All statistical evaluations were performed using the software SPSS 15.0 (SPSS Inc., Chicago, USA). The effect of the follicle counts of the ovaries after EV injection and/or HBO on were evaluated using Kruskal Wallis test. p < 0.05 was considered statistically significant. After Kruskal Wallis, Mann Whitney-U test with Benforini adjustment was applied.
All statistical evaluations were performed using the software SPSS 15.0 (SPSS Inc., Chicago, USA). The effect of the follicle counts of the ovaries after EV injection and/or HBO on were evaluated using Kruskal Wallis test. p < 0.05 was considered statistically significant. After Kruskal Wallis, Mann Whitney-U test with Benforini adjustment was applied.
|
Results
|
In control group (group 1), the ovaries exhibited a typically normal appearance with follicles and corpora lutea in different stages of development and regression (Figure 1). The number of atresic follicles was significantly lower and the numbers of fresh corpora luteas, tertiary follicles, secondary follicles and primary follicles were significantly higher than other groups (Table 1 and Table 2).
Ovarian morphology in Control group.
Comparison of mean numbers of follicles
*p values of Kruskal-Wallis test
AF: atretic follicle, CL:fresh corpus luteum, TF: tertiary follicle, SF:secondary follicle, PF: primary follicle.
Comparison of p values of groups regarding follicle types
*p values of Mann Whitney-U test with Benforini adjustment for multiple comparisons.
NS: nonsignificant
AF:atretic follicle, CL:fresh corpus luteum, TF:tertiary follicle, SF:secondary follicle, PF:primary follicle
In PCO only group (group 2), the ovaries displayed typical PCO-like changes (Figure 2); mean numbers of tertiary, secondary and primary follicles were significantly lower compared with the control group (Table 1 and Table 2). Typically, the number of atretic follicles were significantly higher in the PCO only group compared to control and very few fresh corpora lutea was observed.
Cystic Follicles of Polycystic ovary (PCO) group.
In the PCO+HBO group (group 3), there were significantly higher atretic follicles (Figure 3) compared to the PCO only group (p = 0.037). Although number of atretic follicles in group 3 seems to be more than group 4, the difference was not statistically significant (Table 2).
Atretic and cystic follicles in PCO+HBO group.
In the only HBO group (Group 4), there were significantly higher atretic follicles compared to group 1 (p = 0,024). There were significantly lower fresh corporea lutea (p = 0,034) and healthy growing follicles (Table 2). Although the number of atretic follicles were seems to be higher (Figure 4), it did not differ significantly compared with group 3.
Marked atretic follicles in HBO group.
|
Conclusions
|
We conclude that in our experimental rat model, HBOT did not improve or reverse the PCO state in EV induced PCO rat ovaries, in contrast, after HBOT, follicular atresia increased significantly. With this protocol it seems unfavorable to administrate HBOT in PCO state. Further studies are needed to identify the accompanying factors of this result like exposure time or duration of therapy
|
[
"Background",
"Animals",
"PCO induction",
"HBO",
"Ovarian morphology and quantitative analysis of follicle populations",
"Tissues",
"Statistical analysis",
"Abbreviations",
"Competing interests",
"Authors' contributions"
] |
[
"Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women of reproductive age is a multifactorial metabolic disease associated with insulin resistance [1]. PCOS is a common condition with a range of clinical features. These reproductive features include anovulation, irregular menstrual cycles, clinical and biochemical hyperandrogenism and infertility. Metabolic features include increased risk factors for type 2 diabetes mellitus and cardiovascular disease and increase in the prevalence of the metabolic syndrome [1,2].\nPreviously, the diagnosis of PCOS was based on the National Institute of Health (NIH) criteria comprising biochemical or clinical hyperandrogenism and anovulatory irregular menstrual cycles with the exclusion of related reproductive disorders. In 2003, diagnostic guidelines for PCOS were expanded to the so-called Rotterdam criteria, now based on presentation with any two of the three criteria of hyperandrogenism, irregular anovulatory periods or polycystic ovaries on ultrasound [1,3]. The reported prevalence range of PCOS is between 2.2% to 26% [1,3,4]. The wide range of estimated PCOS prevalence can be explained by different recruitment processes of the study populations, selection biases, ethnic and racial variations in addition to, the criteria used for its definition and the screening methods used to identify each criteria; considering the Rotterdam versus NIH criteria increase the PCOS prevalence by 2 times. The latest study by Tehrani et al. demonstrated that the prevalence's of PCOS using the NIH definition were 8.5% [4].\nThe abnormalities detected in PCOS have been attributed to primary defects in the hypothalamic-pituitary-adrenal axis, the ovarian microenvironment, the adrenal gland and the insulin/insulin-like growth factor metabolic regulatory system [2,5]. There is evidence that both hypothalamic and pituitary mechanisms contribute to the gonadotropin dysfunction in PCOS.\nThe reproductive systems of human beings and other vertebrates are grossly similar [6]. Although the rat is a polytocous rodent, the female has a regular ovarian cyclicity of 4 or 5 days, with distinct proestrus, estrus, and diestrus phases. PCO can be experimentally produced in the rat, that species is a good model for studying the pathophysiology of human PCO by a single dose of the long-acting estrogen, estradiol valerate (EV) which leads to anovulatory state in 13-15 week-old rats [6,7].\nThe therapeutic application of hyperbaric oxygen (HBO) has been known over three decades in different fields of medicine. Hyperbaric oxygen therapy (HBOT) is defined by the Undersea and Hyperbaric Medical Society (UHMS) as a treatment in which a patient intermittently breathes 100% oxygen while the treatment chamber is pressurized to a pressure greater than sea level (1 atmosphere absolute, ATA). The therapeutic principle behind HBO stems from increasing the partial pressure of oxygen in the tissues of the body. The effects of HBO are based on the gas laws, and the physiological, biochemical effects of hyperoxia [8,9]. High tissue levels of oxygen stimulate angiogenesis by increasing the production of cytokines and many growth factors, especially VEGF. Because of these effects on angiogenesis and hyperoxia, it is a unique method used in the treatment of various illnesses and clinical conditions, such as carbon monoxide poisoning, decompression sickness, osteomyelitis, even diabetic foot and ischaemic wounds [9,10].\nBesides, wound healing, HBOT has been used for many clinical treatments. Previous experiments have demonstrated that HBO improves survival in anesthetized rats during heatstroke by reducing multiorgan dysfunction [11]. Furthermore, microarray analysis of gene expression in rat cortical neurons exposed to HBO showed that 17 genes changed in response to all exposure conditions [12]. HBOT is also used for primary liver nonfunction as it stimulated hepatocytes to proliferate. Experimentally, HBO was found significantly effective in the survival of transplanted cells or tissues like liver, bone, thyroid, pancreatic islet cells [13]. Although a large proportion of ovarian follicles were lost during the initial ischemia after transplantation of ovaries, recently it was also found that HBO was also beneficial for the follicular survival of the transplanted mouse ovaries [14].\nHBOT was also used in invitro-fertilisation (IVF) protocols in different studies for improving ovarian folicular stimulation and endometrial receptivity as an adjunct to infertility therapies with the hypothesis of increasing impaired follicular angiogenesis and oxygenation and increasing pregnancy implantation by improving endometrial receptivity by adequate vascularisation and oxygenation in unexplained infertility patients respectively [15,16]. Although new; these trials offer hopeful results for future infertility patients.\nPrincipal mechanisms of HBO are based on intracellular generation of reactive species of oxygen and nitrogen by causing oxidative stress. Reactive oxygen species (ROS) are recognized to play a central role in cell signal transduction cascades or pathways, for a variety of growth factors, cytokines, and hormones. Scavenging antioxidants combat the overproduction of reactive species like vitamine E and C, glutation [10,17]. Recently, some new data about the role of oxidative stress and antioxidants in modulation of ovarian techal-interstitial cells have been enlightened. First, it was demonstrated that antioxidants inhibited proliferation of theca-interstitial cells then new findings added that antioxidants with distinctly different mechanisms of action induce a series of events consistent with the process of apoptosis in ovarian mesenchyme [18,19]. As PCOS is a condition associated with excessive growth and activity of theca-interstitial cells, these findings bring the question of the role of oxidative stress and antioxidants in PCO disease.\nTreatment of human PCOS is symptomatic, lifestyle measures such as diet and exercise (or both) could play an important role in optimising healthy weight, improving underlying hormonal disturbances, prevention of future reproductive and metabolic complications and improving quality of life. Lifestyle interventions are recommended first-line in an international position statement on PCOS and present a cost effective initial treatment strategy compared to surgical and pharmacological options [20]. In the recent Cochrane review the positive effects of lifestyle treatment on anthropometric (abdominal adiposity, adiposity distribution), reproductive (biochemical and clinical hyperandrogenism) and metabolic features (markers of insulin resistance) were confirmed with medium or long term lifestyle measures [21].\nIn the present study, we tried to find a new, alternative treatment modality for PCO disease, considering the different effects of hyperoxia on different tissues, mentioned above. For this, we tested the hypothesis that 6 weeks of HBO administration on polycystic rat ovary would make an improvement or reversal of PCO state. This was carried out by studying the effect of HBO on ovarian morphology including different folicular stages.",
"80 young, cyclic female Wistar-Albino rats (250-300 g) were used for the experiment. All rats were provided by Experimental Medicine Research Center (DETAM) of Istanbul University and housed in the Animal Laboratory of the same centre. They were caged in a controlled environment of 22°C with 12 h light/dark cycles and a humidity range between 40 and 60%. Standard rat feed and reverse-osmosis-purified water were provided ad libitum. All rats were allowed to have 1 week of acclimation to this environment before the experiment. This study approved by 'Animal Studies Comittee' at DETAM Unite of Istanbul University, Istanbul and all investigations complied with the 1996 National Academy of Science's Guide for Care and Use of Laboratory. Eighty animals were divided into four experimental groups: group 1, control (n = 20); group 2, PCO only group (n = 20); group 3, PCO+HBO group (n = 20) and group 4, HBO only group (n = 20).",
"Young cyclic Wistar females were taken and had free access to pelleted rat food and water. All animals were displaying at least 2 consecutive normal 4-day estrous cycles were used in this study. Estrous cycles prior to and after treatment were monitored by daily examination of vaginal smears.\nTo induce a well defined PCO, 4 mg im injection of EV (Sigma, Riedeldehaen, Germany) was administered in 0.2 ml of sesame oil. Forthy rats those in the PCO groups received EV and the remaining rats (also in estrus) those in the oil groups were injected 0.2 ml oil. A duration of 6 weeks was chosen because a single injection of EV induces, after a lag period of 4-6 week, a chronic anovulatory PCO condition in adult rats [6,7].",
"After full PCO state was obtained (after six weeks from the EV or oil injection), the rats were allowed to take six weeks of HBOT. We have chosen 6 weeks of HBOT depending on protocols of Department of Underwater and Hyberbaric Oxygen Unite for chronic diseases protocol for rats.\nHyperbaric Chambers and standard protocols used for rats that is 2.5 atm pressure, totally 60 minutes once a day protocol is used [8,9]. The chamber was ventilated with 100% oxygen at a flow rate of 20 L/min to minimize CO2 accumulation. Rats were then slowly decompressed at a rate of 1 ATA/min at Istanbul University Underseas and Hyperbaric O2 Department.\nThe rats were decapitated after HBOT finished, independent of cycle day. Before decapitation, all rats were anaesthetised with an i.p. administration 50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi, Levent, Istanbul, Turkey) and 7 mg/kg Xylazine hydrochloric acid (Rompun, Bayer Sisli, Istanbul, Turkey)",
" Tissues After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.\nOnly the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.\nQuantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.\nAfter death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.\nOnly the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.\nQuantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.",
"After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.\nOnly the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.\nQuantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.",
"All statistical evaluations were performed using the software SPSS 15.0 (SPSS Inc., Chicago, USA). The effect of the follicle counts of the ovaries after EV injection and/or HBO on were evaluated using Kruskal Wallis test. p < 0.05 was considered statistically significant. After Kruskal Wallis, Mann Whitney-U test with Benforini adjustment was applied.",
"PCOS: Polycystic Ovary Syndrome; HBO: Hyperbaric Oxygen; HBOT: Hyberbaric Oxygen Therapy; UHMS: Undersea and Hyperbaric Medical Society; EV: Estradiol Valerate; ROS: Reactive Oxygen Species; NIH: National Institute of Health; AF: Atretic Follicle; CL: Fresh Corpus Luteum; TF: Tertiary Follicle; SF: Secondary Follicle; PF: Primary Follicle; VEGF: Vascular Endothelial Growth Factor; IVF: İn-Vitro Fertilization; ATA: Atmosphere Absolute.",
"The authors declare that they have no competing interests.",
"AA has designed the study, YA has dealt with statistics, FC has dealt with rats, DS has assessed the microscopy, AArslan and AST has given the HBOT, MD has reviewed the drafts, NG read & approved the final version of the manuscript. All authors read and approved the final manuscript."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Animals",
"PCO induction",
"HBO",
"Ovarian morphology and quantitative analysis of follicle populations",
"Tissues",
"Statistical analysis",
"Results",
"Discussion",
"Conclusions",
"Abbreviations",
"Competing interests",
"Authors' contributions"
] |
[
"Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women of reproductive age is a multifactorial metabolic disease associated with insulin resistance [1]. PCOS is a common condition with a range of clinical features. These reproductive features include anovulation, irregular menstrual cycles, clinical and biochemical hyperandrogenism and infertility. Metabolic features include increased risk factors for type 2 diabetes mellitus and cardiovascular disease and increase in the prevalence of the metabolic syndrome [1,2].\nPreviously, the diagnosis of PCOS was based on the National Institute of Health (NIH) criteria comprising biochemical or clinical hyperandrogenism and anovulatory irregular menstrual cycles with the exclusion of related reproductive disorders. In 2003, diagnostic guidelines for PCOS were expanded to the so-called Rotterdam criteria, now based on presentation with any two of the three criteria of hyperandrogenism, irregular anovulatory periods or polycystic ovaries on ultrasound [1,3]. The reported prevalence range of PCOS is between 2.2% to 26% [1,3,4]. The wide range of estimated PCOS prevalence can be explained by different recruitment processes of the study populations, selection biases, ethnic and racial variations in addition to, the criteria used for its definition and the screening methods used to identify each criteria; considering the Rotterdam versus NIH criteria increase the PCOS prevalence by 2 times. The latest study by Tehrani et al. demonstrated that the prevalence's of PCOS using the NIH definition were 8.5% [4].\nThe abnormalities detected in PCOS have been attributed to primary defects in the hypothalamic-pituitary-adrenal axis, the ovarian microenvironment, the adrenal gland and the insulin/insulin-like growth factor metabolic regulatory system [2,5]. There is evidence that both hypothalamic and pituitary mechanisms contribute to the gonadotropin dysfunction in PCOS.\nThe reproductive systems of human beings and other vertebrates are grossly similar [6]. Although the rat is a polytocous rodent, the female has a regular ovarian cyclicity of 4 or 5 days, with distinct proestrus, estrus, and diestrus phases. PCO can be experimentally produced in the rat, that species is a good model for studying the pathophysiology of human PCO by a single dose of the long-acting estrogen, estradiol valerate (EV) which leads to anovulatory state in 13-15 week-old rats [6,7].\nThe therapeutic application of hyperbaric oxygen (HBO) has been known over three decades in different fields of medicine. Hyperbaric oxygen therapy (HBOT) is defined by the Undersea and Hyperbaric Medical Society (UHMS) as a treatment in which a patient intermittently breathes 100% oxygen while the treatment chamber is pressurized to a pressure greater than sea level (1 atmosphere absolute, ATA). The therapeutic principle behind HBO stems from increasing the partial pressure of oxygen in the tissues of the body. The effects of HBO are based on the gas laws, and the physiological, biochemical effects of hyperoxia [8,9]. High tissue levels of oxygen stimulate angiogenesis by increasing the production of cytokines and many growth factors, especially VEGF. Because of these effects on angiogenesis and hyperoxia, it is a unique method used in the treatment of various illnesses and clinical conditions, such as carbon monoxide poisoning, decompression sickness, osteomyelitis, even diabetic foot and ischaemic wounds [9,10].\nBesides, wound healing, HBOT has been used for many clinical treatments. Previous experiments have demonstrated that HBO improves survival in anesthetized rats during heatstroke by reducing multiorgan dysfunction [11]. Furthermore, microarray analysis of gene expression in rat cortical neurons exposed to HBO showed that 17 genes changed in response to all exposure conditions [12]. HBOT is also used for primary liver nonfunction as it stimulated hepatocytes to proliferate. Experimentally, HBO was found significantly effective in the survival of transplanted cells or tissues like liver, bone, thyroid, pancreatic islet cells [13]. Although a large proportion of ovarian follicles were lost during the initial ischemia after transplantation of ovaries, recently it was also found that HBO was also beneficial for the follicular survival of the transplanted mouse ovaries [14].\nHBOT was also used in invitro-fertilisation (IVF) protocols in different studies for improving ovarian folicular stimulation and endometrial receptivity as an adjunct to infertility therapies with the hypothesis of increasing impaired follicular angiogenesis and oxygenation and increasing pregnancy implantation by improving endometrial receptivity by adequate vascularisation and oxygenation in unexplained infertility patients respectively [15,16]. Although new; these trials offer hopeful results for future infertility patients.\nPrincipal mechanisms of HBO are based on intracellular generation of reactive species of oxygen and nitrogen by causing oxidative stress. Reactive oxygen species (ROS) are recognized to play a central role in cell signal transduction cascades or pathways, for a variety of growth factors, cytokines, and hormones. Scavenging antioxidants combat the overproduction of reactive species like vitamine E and C, glutation [10,17]. Recently, some new data about the role of oxidative stress and antioxidants in modulation of ovarian techal-interstitial cells have been enlightened. First, it was demonstrated that antioxidants inhibited proliferation of theca-interstitial cells then new findings added that antioxidants with distinctly different mechanisms of action induce a series of events consistent with the process of apoptosis in ovarian mesenchyme [18,19]. As PCOS is a condition associated with excessive growth and activity of theca-interstitial cells, these findings bring the question of the role of oxidative stress and antioxidants in PCO disease.\nTreatment of human PCOS is symptomatic, lifestyle measures such as diet and exercise (or both) could play an important role in optimising healthy weight, improving underlying hormonal disturbances, prevention of future reproductive and metabolic complications and improving quality of life. Lifestyle interventions are recommended first-line in an international position statement on PCOS and present a cost effective initial treatment strategy compared to surgical and pharmacological options [20]. In the recent Cochrane review the positive effects of lifestyle treatment on anthropometric (abdominal adiposity, adiposity distribution), reproductive (biochemical and clinical hyperandrogenism) and metabolic features (markers of insulin resistance) were confirmed with medium or long term lifestyle measures [21].\nIn the present study, we tried to find a new, alternative treatment modality for PCO disease, considering the different effects of hyperoxia on different tissues, mentioned above. For this, we tested the hypothesis that 6 weeks of HBO administration on polycystic rat ovary would make an improvement or reversal of PCO state. This was carried out by studying the effect of HBO on ovarian morphology including different folicular stages.",
" Animals 80 young, cyclic female Wistar-Albino rats (250-300 g) were used for the experiment. All rats were provided by Experimental Medicine Research Center (DETAM) of Istanbul University and housed in the Animal Laboratory of the same centre. They were caged in a controlled environment of 22°C with 12 h light/dark cycles and a humidity range between 40 and 60%. Standard rat feed and reverse-osmosis-purified water were provided ad libitum. All rats were allowed to have 1 week of acclimation to this environment before the experiment. This study approved by 'Animal Studies Comittee' at DETAM Unite of Istanbul University, Istanbul and all investigations complied with the 1996 National Academy of Science's Guide for Care and Use of Laboratory. Eighty animals were divided into four experimental groups: group 1, control (n = 20); group 2, PCO only group (n = 20); group 3, PCO+HBO group (n = 20) and group 4, HBO only group (n = 20).\n80 young, cyclic female Wistar-Albino rats (250-300 g) were used for the experiment. All rats were provided by Experimental Medicine Research Center (DETAM) of Istanbul University and housed in the Animal Laboratory of the same centre. They were caged in a controlled environment of 22°C with 12 h light/dark cycles and a humidity range between 40 and 60%. Standard rat feed and reverse-osmosis-purified water were provided ad libitum. All rats were allowed to have 1 week of acclimation to this environment before the experiment. This study approved by 'Animal Studies Comittee' at DETAM Unite of Istanbul University, Istanbul and all investigations complied with the 1996 National Academy of Science's Guide for Care and Use of Laboratory. Eighty animals were divided into four experimental groups: group 1, control (n = 20); group 2, PCO only group (n = 20); group 3, PCO+HBO group (n = 20) and group 4, HBO only group (n = 20).\n PCO induction Young cyclic Wistar females were taken and had free access to pelleted rat food and water. All animals were displaying at least 2 consecutive normal 4-day estrous cycles were used in this study. Estrous cycles prior to and after treatment were monitored by daily examination of vaginal smears.\nTo induce a well defined PCO, 4 mg im injection of EV (Sigma, Riedeldehaen, Germany) was administered in 0.2 ml of sesame oil. Forthy rats those in the PCO groups received EV and the remaining rats (also in estrus) those in the oil groups were injected 0.2 ml oil. A duration of 6 weeks was chosen because a single injection of EV induces, after a lag period of 4-6 week, a chronic anovulatory PCO condition in adult rats [6,7].\nYoung cyclic Wistar females were taken and had free access to pelleted rat food and water. All animals were displaying at least 2 consecutive normal 4-day estrous cycles were used in this study. Estrous cycles prior to and after treatment were monitored by daily examination of vaginal smears.\nTo induce a well defined PCO, 4 mg im injection of EV (Sigma, Riedeldehaen, Germany) was administered in 0.2 ml of sesame oil. Forthy rats those in the PCO groups received EV and the remaining rats (also in estrus) those in the oil groups were injected 0.2 ml oil. A duration of 6 weeks was chosen because a single injection of EV induces, after a lag period of 4-6 week, a chronic anovulatory PCO condition in adult rats [6,7].\n HBO After full PCO state was obtained (after six weeks from the EV or oil injection), the rats were allowed to take six weeks of HBOT. We have chosen 6 weeks of HBOT depending on protocols of Department of Underwater and Hyberbaric Oxygen Unite for chronic diseases protocol for rats.\nHyperbaric Chambers and standard protocols used for rats that is 2.5 atm pressure, totally 60 minutes once a day protocol is used [8,9]. The chamber was ventilated with 100% oxygen at a flow rate of 20 L/min to minimize CO2 accumulation. Rats were then slowly decompressed at a rate of 1 ATA/min at Istanbul University Underseas and Hyperbaric O2 Department.\nThe rats were decapitated after HBOT finished, independent of cycle day. Before decapitation, all rats were anaesthetised with an i.p. administration 50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi, Levent, Istanbul, Turkey) and 7 mg/kg Xylazine hydrochloric acid (Rompun, Bayer Sisli, Istanbul, Turkey)\nAfter full PCO state was obtained (after six weeks from the EV or oil injection), the rats were allowed to take six weeks of HBOT. We have chosen 6 weeks of HBOT depending on protocols of Department of Underwater and Hyberbaric Oxygen Unite for chronic diseases protocol for rats.\nHyperbaric Chambers and standard protocols used for rats that is 2.5 atm pressure, totally 60 minutes once a day protocol is used [8,9]. The chamber was ventilated with 100% oxygen at a flow rate of 20 L/min to minimize CO2 accumulation. Rats were then slowly decompressed at a rate of 1 ATA/min at Istanbul University Underseas and Hyperbaric O2 Department.\nThe rats were decapitated after HBOT finished, independent of cycle day. Before decapitation, all rats were anaesthetised with an i.p. administration 50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi, Levent, Istanbul, Turkey) and 7 mg/kg Xylazine hydrochloric acid (Rompun, Bayer Sisli, Istanbul, Turkey)\n Ovarian morphology and quantitative analysis of follicle populations Tissues After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.\nOnly the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.\nQuantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.\nAfter death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.\nOnly the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.\nQuantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.\n Tissues After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.\nOnly the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.\nQuantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.\nAfter death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.\nOnly the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.\nQuantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.\n Statistical analysis All statistical evaluations were performed using the software SPSS 15.0 (SPSS Inc., Chicago, USA). The effect of the follicle counts of the ovaries after EV injection and/or HBO on were evaluated using Kruskal Wallis test. p < 0.05 was considered statistically significant. After Kruskal Wallis, Mann Whitney-U test with Benforini adjustment was applied.\nAll statistical evaluations were performed using the software SPSS 15.0 (SPSS Inc., Chicago, USA). The effect of the follicle counts of the ovaries after EV injection and/or HBO on were evaluated using Kruskal Wallis test. p < 0.05 was considered statistically significant. After Kruskal Wallis, Mann Whitney-U test with Benforini adjustment was applied.",
"80 young, cyclic female Wistar-Albino rats (250-300 g) were used for the experiment. All rats were provided by Experimental Medicine Research Center (DETAM) of Istanbul University and housed in the Animal Laboratory of the same centre. They were caged in a controlled environment of 22°C with 12 h light/dark cycles and a humidity range between 40 and 60%. Standard rat feed and reverse-osmosis-purified water were provided ad libitum. All rats were allowed to have 1 week of acclimation to this environment before the experiment. This study approved by 'Animal Studies Comittee' at DETAM Unite of Istanbul University, Istanbul and all investigations complied with the 1996 National Academy of Science's Guide for Care and Use of Laboratory. Eighty animals were divided into four experimental groups: group 1, control (n = 20); group 2, PCO only group (n = 20); group 3, PCO+HBO group (n = 20) and group 4, HBO only group (n = 20).",
"Young cyclic Wistar females were taken and had free access to pelleted rat food and water. All animals were displaying at least 2 consecutive normal 4-day estrous cycles were used in this study. Estrous cycles prior to and after treatment were monitored by daily examination of vaginal smears.\nTo induce a well defined PCO, 4 mg im injection of EV (Sigma, Riedeldehaen, Germany) was administered in 0.2 ml of sesame oil. Forthy rats those in the PCO groups received EV and the remaining rats (also in estrus) those in the oil groups were injected 0.2 ml oil. A duration of 6 weeks was chosen because a single injection of EV induces, after a lag period of 4-6 week, a chronic anovulatory PCO condition in adult rats [6,7].",
"After full PCO state was obtained (after six weeks from the EV or oil injection), the rats were allowed to take six weeks of HBOT. We have chosen 6 weeks of HBOT depending on protocols of Department of Underwater and Hyberbaric Oxygen Unite for chronic diseases protocol for rats.\nHyperbaric Chambers and standard protocols used for rats that is 2.5 atm pressure, totally 60 minutes once a day protocol is used [8,9]. The chamber was ventilated with 100% oxygen at a flow rate of 20 L/min to minimize CO2 accumulation. Rats were then slowly decompressed at a rate of 1 ATA/min at Istanbul University Underseas and Hyperbaric O2 Department.\nThe rats were decapitated after HBOT finished, independent of cycle day. Before decapitation, all rats were anaesthetised with an i.p. administration 50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi, Levent, Istanbul, Turkey) and 7 mg/kg Xylazine hydrochloric acid (Rompun, Bayer Sisli, Istanbul, Turkey)",
" Tissues After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.\nOnly the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.\nQuantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.\nAfter death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.\nOnly the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.\nQuantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.",
"After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.\nOnly the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.\nQuantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.",
"All statistical evaluations were performed using the software SPSS 15.0 (SPSS Inc., Chicago, USA). The effect of the follicle counts of the ovaries after EV injection and/or HBO on were evaluated using Kruskal Wallis test. p < 0.05 was considered statistically significant. After Kruskal Wallis, Mann Whitney-U test with Benforini adjustment was applied.",
"In control group (group 1), the ovaries exhibited a typically normal appearance with follicles and corpora lutea in different stages of development and regression (Figure 1). The number of atresic follicles was significantly lower and the numbers of fresh corpora luteas, tertiary follicles, secondary follicles and primary follicles were significantly higher than other groups (Table 1 and Table 2).\nOvarian morphology in Control group.\nComparison of mean numbers of follicles\n*p values of Kruskal-Wallis test\nAF: atretic follicle, CL:fresh corpus luteum, TF: tertiary follicle, SF:secondary follicle, PF: primary follicle.\nComparison of p values of groups regarding follicle types\n*p values of Mann Whitney-U test with Benforini adjustment for multiple comparisons.\nNS: nonsignificant\nAF:atretic follicle, CL:fresh corpus luteum, TF:tertiary follicle, SF:secondary follicle, PF:primary follicle\nIn PCO only group (group 2), the ovaries displayed typical PCO-like changes (Figure 2); mean numbers of tertiary, secondary and primary follicles were significantly lower compared with the control group (Table 1 and Table 2). Typically, the number of atretic follicles were significantly higher in the PCO only group compared to control and very few fresh corpora lutea was observed.\nCystic Follicles of Polycystic ovary (PCO) group.\nIn the PCO+HBO group (group 3), there were significantly higher atretic follicles (Figure 3) compared to the PCO only group (p = 0.037). Although number of atretic follicles in group 3 seems to be more than group 4, the difference was not statistically significant (Table 2).\nAtretic and cystic follicles in PCO+HBO group.\nIn the only HBO group (Group 4), there were significantly higher atretic follicles compared to group 1 (p = 0,024). There were significantly lower fresh corporea lutea (p = 0,034) and healthy growing follicles (Table 2). Although the number of atretic follicles were seems to be higher (Figure 4), it did not differ significantly compared with group 3.\nMarked atretic follicles in HBO group.",
"Cystic ovarian disease and/or PCOS are disorders of the reproduction that affect bovine, ovine, caprine and porcine species and even human beings [1,2]. Several experimental models for PCO have been developed in rats; of these EV cause a sudden appearance of PCO due to disturbances in metabolic and physiologic processes [6,7].\nMany studies were established such as electroacupuncture, hemohim, Korean red ginseng extract, herbal medicine, exercise assessing the effect of different therapies on EV induced PCOS rats [24-27]. Some of them found improvement in ovarian morphology of PCO state, and some of them did not. The difference of our study from those is that; they investigated the inhibition of PCO state starting therapies on the same day with EV injection, but we wanted to investigate the improvement in ovarian morphology of PCO state by starting HBOT after PCO morphology was fully established, rather than to investigate the inhibition of formation of PCO morphology.\nThe application of HBO ensures a normal supply of molecular oxygen to mitochondria, transported from the lungs to the endometrial cells or to ovarian tissue in a physically dissolved form in body fluids. Ultimately the increases in dissolved oxygen generated by HBO have several physiologic effects that can alter tissue responses to disease and injury [8,15]. In our study, after PCO formation, six weeks of HBO treatment, 60 minutes once a day protocol did not reverse but in contrast, aggravated the atresia of follicular growth in the rat ovaries, significantly. HBOT of healthy rat ovary increased the young follicular atresia and decreased the healthy growing follicles and also decreased the fresh corporea lutea significantly. The mean number healthy growing follicles were found lower than control and PCO groups but similar with PCO+HBO group. So this forced us to think that HBO administration created the same effect on ovarian histology like EV. Significantly, the mean number of atresia was higher in HBO+PCO group compared to PCO group, we think that the accelerated atresia is due to HBOT and HBO did not help to improve PCO state in the ovaries but inspite aggravated it. When we reviewed our results with rat studies about HBOT, we came to know that HBO administration also led to unwelcome side-effects, such as oxidative stress and/or oxygen toxicity, the formation of ROS can cause cellular damage through the oxidation of lipids, proteins and DNA [28]. One study assessing oxidative stress levels in rats following exposure to oxygen at 3 atm for 0-120 minutes exhibited a clear relationship of HBO-induced oxidative action to exposure time. This action was most pronounced from 90 to 120 minutes of exposure [29]. Another study on the effects of HBO in rats revealed that after 2 hours of HBO exposure at 3 ATA, levels of the oxidative stress markers elevated in lung, brain and erythrocytes [30]. Repetitive treatment with HBO was proved to cause oxidative stress in critical tissues including the rat brain, lymphocytes. Malondialdehyde levels and other oxidative stres markers were found to be significantly increased in the therapies after 20 day to 40 day session groups [31]. Although HBO-mediated free radicals were accepted to be responsible for the benefits of this therapeutic modality, especially in cases with prolonged exposure, possible injurious effects of supranormal values of bio-oxidative products need to be considered. Our application of 6 weeks HBO might be too much regarding exposure time and duration of it for PCO rats which is a multi-system disease that already carries underlying metabolic pathologies. Confirming this, Takahashi et al. found a biphasic effect of the generation of superoxide radicals on proliferation consistent with the concept that while moderate oxidative stress may induce cell growth, very high levels of ROS were cytotoxic and led to cell, tissue damage and apoptotic cell death [32]. Although we did not measure oxidative stress markers, all these aforementioned data may help to explain why we found aggrevation of atresia rather than reversal of it. As we did not measure the VEGF levels we can't declare clearly if modulated mechanisms of angiogenesis were causative.\nAlthough human PCO is not the same as rat model, the oxidative effects of HBO have been investigated both in animals and human [29,30]. The protocols are consistent with the principles of partial pressures reaching that tissue. Henry's law states that the amount of gas dissolved in a liquid or tissue is proportional to the partial pressure of that gas in contact with the liquid or tissue. This is the basis for increased tissue oxygen tensions with HBOT [8,9].\nThe clinically approved maximum pressure and duration of HBO exposure are 3 ATA and 120 minutes, respectively although the most commonly-used protocol for standard therapeutic purposes is slightly lower (1.8-2.8 ATA for 60-90 min) [9,28]. We applied chronic diseases standard HBO protocol (2.5 atm, 60 minute once a day) for our rat PCO model, considering the PCO as multi-systemic and chronic disease. Treatment recommendations mentioned in this paper are taken from the UHMS report and are evidence-based [8]. Protocols vary greatly, but the UHMS recommend treatments beginning at 2.0-2.5 ATA for up to 120 minutes at least once a day until no improvement in diseases are reached. When we reviewed rat HBO studies, we found there were different protocols like 2 atm, 2.8 atm/90 min for 5-40 days or 3 atm for up to 120 minutes, although guidelines vary depending on the injury [28-31]. It would be more favourable for our study if we could apply different pressures and different exposure time and analyze their effects. This will be our goal for future studies.\nThere is a substantial body of literature that has examined the adverse and beneficial effects of HBOT in animal models. As technology becomes more readily available to clinical practice and more clinical trials are performed to define its effectiveness, HBOT may be considered as an additional therapeutic option in many conditions. Recent studies assessed the role of HBOT in unsuccessful IVF protocols or endometrial implantation failures and found convincing results although the standard protocols are not yet established. We tried to find an alternative or adjunctive treatment modality for PCO. Depending on the tissue concentration it can either exert beneficial physiologic effects or damage cell structures. We suggest that our present HBO protocol is not applicable for treatment of PCO disease and that should be considered in infertility patients with PCO; but may be rearranged accordingly in rat PCO model which needs further studies.\nQuantifying the follicles may be time consuming and not an easy way of assessing ovarian histology, this is an experimental study and we wanted to assess the details of ovarian morphology other than ovulation [22,23].\nOur study is the first that HBO is used in EV induced PCO rats and control rats. Our study is privileged from others that, the long- term effect of HBO on PCOS was demonstrated. To our knowledge, there is no study in the literature evaluating the effect of HBOT on PCO.",
"We conclude that in our experimental rat model, HBOT did not improve or reverse the PCO state in EV induced PCO rat ovaries, in contrast, after HBOT, follicular atresia increased significantly. With this protocol it seems unfavorable to administrate HBOT in PCO state. Further studies are needed to identify the accompanying factors of this result like exposure time or duration of therapy",
"PCOS: Polycystic Ovary Syndrome; HBO: Hyperbaric Oxygen; HBOT: Hyberbaric Oxygen Therapy; UHMS: Undersea and Hyperbaric Medical Society; EV: Estradiol Valerate; ROS: Reactive Oxygen Species; NIH: National Institute of Health; AF: Atretic Follicle; CL: Fresh Corpus Luteum; TF: Tertiary Follicle; SF: Secondary Follicle; PF: Primary Follicle; VEGF: Vascular Endothelial Growth Factor; IVF: İn-Vitro Fertilization; ATA: Atmosphere Absolute.",
"The authors declare that they have no competing interests.",
"AA has designed the study, YA has dealt with statistics, FC has dealt with rats, DS has assessed the microscopy, AArslan and AST has given the HBOT, MD has reviewed the drafts, NG read & approved the final version of the manuscript. All authors read and approved the final manuscript."
] |
[
null,
"methods",
null,
null,
null,
null,
null,
null,
"results",
"discussion",
"conclusions",
null,
null,
null
] |
[
"Hyperbaric-oxygen",
"Polycystic-ovary",
"Rat",
"Estradiol valerate"
] |
Background:
Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women of reproductive age is a multifactorial metabolic disease associated with insulin resistance [1]. PCOS is a common condition with a range of clinical features. These reproductive features include anovulation, irregular menstrual cycles, clinical and biochemical hyperandrogenism and infertility. Metabolic features include increased risk factors for type 2 diabetes mellitus and cardiovascular disease and increase in the prevalence of the metabolic syndrome [1,2].
Previously, the diagnosis of PCOS was based on the National Institute of Health (NIH) criteria comprising biochemical or clinical hyperandrogenism and anovulatory irregular menstrual cycles with the exclusion of related reproductive disorders. In 2003, diagnostic guidelines for PCOS were expanded to the so-called Rotterdam criteria, now based on presentation with any two of the three criteria of hyperandrogenism, irregular anovulatory periods or polycystic ovaries on ultrasound [1,3]. The reported prevalence range of PCOS is between 2.2% to 26% [1,3,4]. The wide range of estimated PCOS prevalence can be explained by different recruitment processes of the study populations, selection biases, ethnic and racial variations in addition to, the criteria used for its definition and the screening methods used to identify each criteria; considering the Rotterdam versus NIH criteria increase the PCOS prevalence by 2 times. The latest study by Tehrani et al. demonstrated that the prevalence's of PCOS using the NIH definition were 8.5% [4].
The abnormalities detected in PCOS have been attributed to primary defects in the hypothalamic-pituitary-adrenal axis, the ovarian microenvironment, the adrenal gland and the insulin/insulin-like growth factor metabolic regulatory system [2,5]. There is evidence that both hypothalamic and pituitary mechanisms contribute to the gonadotropin dysfunction in PCOS.
The reproductive systems of human beings and other vertebrates are grossly similar [6]. Although the rat is a polytocous rodent, the female has a regular ovarian cyclicity of 4 or 5 days, with distinct proestrus, estrus, and diestrus phases. PCO can be experimentally produced in the rat, that species is a good model for studying the pathophysiology of human PCO by a single dose of the long-acting estrogen, estradiol valerate (EV) which leads to anovulatory state in 13-15 week-old rats [6,7].
The therapeutic application of hyperbaric oxygen (HBO) has been known over three decades in different fields of medicine. Hyperbaric oxygen therapy (HBOT) is defined by the Undersea and Hyperbaric Medical Society (UHMS) as a treatment in which a patient intermittently breathes 100% oxygen while the treatment chamber is pressurized to a pressure greater than sea level (1 atmosphere absolute, ATA). The therapeutic principle behind HBO stems from increasing the partial pressure of oxygen in the tissues of the body. The effects of HBO are based on the gas laws, and the physiological, biochemical effects of hyperoxia [8,9]. High tissue levels of oxygen stimulate angiogenesis by increasing the production of cytokines and many growth factors, especially VEGF. Because of these effects on angiogenesis and hyperoxia, it is a unique method used in the treatment of various illnesses and clinical conditions, such as carbon monoxide poisoning, decompression sickness, osteomyelitis, even diabetic foot and ischaemic wounds [9,10].
Besides, wound healing, HBOT has been used for many clinical treatments. Previous experiments have demonstrated that HBO improves survival in anesthetized rats during heatstroke by reducing multiorgan dysfunction [11]. Furthermore, microarray analysis of gene expression in rat cortical neurons exposed to HBO showed that 17 genes changed in response to all exposure conditions [12]. HBOT is also used for primary liver nonfunction as it stimulated hepatocytes to proliferate. Experimentally, HBO was found significantly effective in the survival of transplanted cells or tissues like liver, bone, thyroid, pancreatic islet cells [13]. Although a large proportion of ovarian follicles were lost during the initial ischemia after transplantation of ovaries, recently it was also found that HBO was also beneficial for the follicular survival of the transplanted mouse ovaries [14].
HBOT was also used in invitro-fertilisation (IVF) protocols in different studies for improving ovarian folicular stimulation and endometrial receptivity as an adjunct to infertility therapies with the hypothesis of increasing impaired follicular angiogenesis and oxygenation and increasing pregnancy implantation by improving endometrial receptivity by adequate vascularisation and oxygenation in unexplained infertility patients respectively [15,16]. Although new; these trials offer hopeful results for future infertility patients.
Principal mechanisms of HBO are based on intracellular generation of reactive species of oxygen and nitrogen by causing oxidative stress. Reactive oxygen species (ROS) are recognized to play a central role in cell signal transduction cascades or pathways, for a variety of growth factors, cytokines, and hormones. Scavenging antioxidants combat the overproduction of reactive species like vitamine E and C, glutation [10,17]. Recently, some new data about the role of oxidative stress and antioxidants in modulation of ovarian techal-interstitial cells have been enlightened. First, it was demonstrated that antioxidants inhibited proliferation of theca-interstitial cells then new findings added that antioxidants with distinctly different mechanisms of action induce a series of events consistent with the process of apoptosis in ovarian mesenchyme [18,19]. As PCOS is a condition associated with excessive growth and activity of theca-interstitial cells, these findings bring the question of the role of oxidative stress and antioxidants in PCO disease.
Treatment of human PCOS is symptomatic, lifestyle measures such as diet and exercise (or both) could play an important role in optimising healthy weight, improving underlying hormonal disturbances, prevention of future reproductive and metabolic complications and improving quality of life. Lifestyle interventions are recommended first-line in an international position statement on PCOS and present a cost effective initial treatment strategy compared to surgical and pharmacological options [20]. In the recent Cochrane review the positive effects of lifestyle treatment on anthropometric (abdominal adiposity, adiposity distribution), reproductive (biochemical and clinical hyperandrogenism) and metabolic features (markers of insulin resistance) were confirmed with medium or long term lifestyle measures [21].
In the present study, we tried to find a new, alternative treatment modality for PCO disease, considering the different effects of hyperoxia on different tissues, mentioned above. For this, we tested the hypothesis that 6 weeks of HBO administration on polycystic rat ovary would make an improvement or reversal of PCO state. This was carried out by studying the effect of HBO on ovarian morphology including different folicular stages.
Methods:
Animals 80 young, cyclic female Wistar-Albino rats (250-300 g) were used for the experiment. All rats were provided by Experimental Medicine Research Center (DETAM) of Istanbul University and housed in the Animal Laboratory of the same centre. They were caged in a controlled environment of 22°C with 12 h light/dark cycles and a humidity range between 40 and 60%. Standard rat feed and reverse-osmosis-purified water were provided ad libitum. All rats were allowed to have 1 week of acclimation to this environment before the experiment. This study approved by 'Animal Studies Comittee' at DETAM Unite of Istanbul University, Istanbul and all investigations complied with the 1996 National Academy of Science's Guide for Care and Use of Laboratory. Eighty animals were divided into four experimental groups: group 1, control (n = 20); group 2, PCO only group (n = 20); group 3, PCO+HBO group (n = 20) and group 4, HBO only group (n = 20).
80 young, cyclic female Wistar-Albino rats (250-300 g) were used for the experiment. All rats were provided by Experimental Medicine Research Center (DETAM) of Istanbul University and housed in the Animal Laboratory of the same centre. They were caged in a controlled environment of 22°C with 12 h light/dark cycles and a humidity range between 40 and 60%. Standard rat feed and reverse-osmosis-purified water were provided ad libitum. All rats were allowed to have 1 week of acclimation to this environment before the experiment. This study approved by 'Animal Studies Comittee' at DETAM Unite of Istanbul University, Istanbul and all investigations complied with the 1996 National Academy of Science's Guide for Care and Use of Laboratory. Eighty animals were divided into four experimental groups: group 1, control (n = 20); group 2, PCO only group (n = 20); group 3, PCO+HBO group (n = 20) and group 4, HBO only group (n = 20).
PCO induction Young cyclic Wistar females were taken and had free access to pelleted rat food and water. All animals were displaying at least 2 consecutive normal 4-day estrous cycles were used in this study. Estrous cycles prior to and after treatment were monitored by daily examination of vaginal smears.
To induce a well defined PCO, 4 mg im injection of EV (Sigma, Riedeldehaen, Germany) was administered in 0.2 ml of sesame oil. Forthy rats those in the PCO groups received EV and the remaining rats (also in estrus) those in the oil groups were injected 0.2 ml oil. A duration of 6 weeks was chosen because a single injection of EV induces, after a lag period of 4-6 week, a chronic anovulatory PCO condition in adult rats [6,7].
Young cyclic Wistar females were taken and had free access to pelleted rat food and water. All animals were displaying at least 2 consecutive normal 4-day estrous cycles were used in this study. Estrous cycles prior to and after treatment were monitored by daily examination of vaginal smears.
To induce a well defined PCO, 4 mg im injection of EV (Sigma, Riedeldehaen, Germany) was administered in 0.2 ml of sesame oil. Forthy rats those in the PCO groups received EV and the remaining rats (also in estrus) those in the oil groups were injected 0.2 ml oil. A duration of 6 weeks was chosen because a single injection of EV induces, after a lag period of 4-6 week, a chronic anovulatory PCO condition in adult rats [6,7].
HBO After full PCO state was obtained (after six weeks from the EV or oil injection), the rats were allowed to take six weeks of HBOT. We have chosen 6 weeks of HBOT depending on protocols of Department of Underwater and Hyberbaric Oxygen Unite for chronic diseases protocol for rats.
Hyperbaric Chambers and standard protocols used for rats that is 2.5 atm pressure, totally 60 minutes once a day protocol is used [8,9]. The chamber was ventilated with 100% oxygen at a flow rate of 20 L/min to minimize CO2 accumulation. Rats were then slowly decompressed at a rate of 1 ATA/min at Istanbul University Underseas and Hyperbaric O2 Department.
The rats were decapitated after HBOT finished, independent of cycle day. Before decapitation, all rats were anaesthetised with an i.p. administration 50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi, Levent, Istanbul, Turkey) and 7 mg/kg Xylazine hydrochloric acid (Rompun, Bayer Sisli, Istanbul, Turkey)
After full PCO state was obtained (after six weeks from the EV or oil injection), the rats were allowed to take six weeks of HBOT. We have chosen 6 weeks of HBOT depending on protocols of Department of Underwater and Hyberbaric Oxygen Unite for chronic diseases protocol for rats.
Hyperbaric Chambers and standard protocols used for rats that is 2.5 atm pressure, totally 60 minutes once a day protocol is used [8,9]. The chamber was ventilated with 100% oxygen at a flow rate of 20 L/min to minimize CO2 accumulation. Rats were then slowly decompressed at a rate of 1 ATA/min at Istanbul University Underseas and Hyperbaric O2 Department.
The rats were decapitated after HBOT finished, independent of cycle day. Before decapitation, all rats were anaesthetised with an i.p. administration 50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi, Levent, Istanbul, Turkey) and 7 mg/kg Xylazine hydrochloric acid (Rompun, Bayer Sisli, Istanbul, Turkey)
Ovarian morphology and quantitative analysis of follicle populations Tissues After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.
Only the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.
Quantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.
After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.
Only the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.
Quantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.
Tissues After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.
Only the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.
Quantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.
After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.
Only the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.
Quantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.
Statistical analysis All statistical evaluations were performed using the software SPSS 15.0 (SPSS Inc., Chicago, USA). The effect of the follicle counts of the ovaries after EV injection and/or HBO on were evaluated using Kruskal Wallis test. p < 0.05 was considered statistically significant. After Kruskal Wallis, Mann Whitney-U test with Benforini adjustment was applied.
All statistical evaluations were performed using the software SPSS 15.0 (SPSS Inc., Chicago, USA). The effect of the follicle counts of the ovaries after EV injection and/or HBO on were evaluated using Kruskal Wallis test. p < 0.05 was considered statistically significant. After Kruskal Wallis, Mann Whitney-U test with Benforini adjustment was applied.
Animals:
80 young, cyclic female Wistar-Albino rats (250-300 g) were used for the experiment. All rats were provided by Experimental Medicine Research Center (DETAM) of Istanbul University and housed in the Animal Laboratory of the same centre. They were caged in a controlled environment of 22°C with 12 h light/dark cycles and a humidity range between 40 and 60%. Standard rat feed and reverse-osmosis-purified water were provided ad libitum. All rats were allowed to have 1 week of acclimation to this environment before the experiment. This study approved by 'Animal Studies Comittee' at DETAM Unite of Istanbul University, Istanbul and all investigations complied with the 1996 National Academy of Science's Guide for Care and Use of Laboratory. Eighty animals were divided into four experimental groups: group 1, control (n = 20); group 2, PCO only group (n = 20); group 3, PCO+HBO group (n = 20) and group 4, HBO only group (n = 20).
PCO induction:
Young cyclic Wistar females were taken and had free access to pelleted rat food and water. All animals were displaying at least 2 consecutive normal 4-day estrous cycles were used in this study. Estrous cycles prior to and after treatment were monitored by daily examination of vaginal smears.
To induce a well defined PCO, 4 mg im injection of EV (Sigma, Riedeldehaen, Germany) was administered in 0.2 ml of sesame oil. Forthy rats those in the PCO groups received EV and the remaining rats (also in estrus) those in the oil groups were injected 0.2 ml oil. A duration of 6 weeks was chosen because a single injection of EV induces, after a lag period of 4-6 week, a chronic anovulatory PCO condition in adult rats [6,7].
HBO:
After full PCO state was obtained (after six weeks from the EV or oil injection), the rats were allowed to take six weeks of HBOT. We have chosen 6 weeks of HBOT depending on protocols of Department of Underwater and Hyberbaric Oxygen Unite for chronic diseases protocol for rats.
Hyperbaric Chambers and standard protocols used for rats that is 2.5 atm pressure, totally 60 minutes once a day protocol is used [8,9]. The chamber was ventilated with 100% oxygen at a flow rate of 20 L/min to minimize CO2 accumulation. Rats were then slowly decompressed at a rate of 1 ATA/min at Istanbul University Underseas and Hyperbaric O2 Department.
The rats were decapitated after HBOT finished, independent of cycle day. Before decapitation, all rats were anaesthetised with an i.p. administration 50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi, Levent, Istanbul, Turkey) and 7 mg/kg Xylazine hydrochloric acid (Rompun, Bayer Sisli, Istanbul, Turkey)
Ovarian morphology and quantitative analysis of follicle populations:
Tissues After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.
Only the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.
Quantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.
After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.
Only the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.
Quantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.
Tissues:
After death of one rat, rats were analysed, independent of cycle day when full characteristic changes of ovaries ensued, the ovaries were removed. Both ovaries were weighed and fixed in buffered 4% formaldehyde for at least 24 h. Thereafter, the samples were dehydrated and embedded in paraffin to await morphological analyses by the same author.
Only the number of follicles containing an oocyte with a nucleus were classified as healthy and counted. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. Primary and secondary follicles in each section were enumerated only if the nucleolus of the ovum was visible. The enumerated follicle was then classified as healthy or atretic. Primary follicles were described as those having an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Secondary follicles were described as follicles with two or more layers of cuboidal granulosa cells, but formation of a cavity is not evident. Tertiary or graafian follicles which was described as the presence of an antral cavity within the follicle.
Quantification analyses were performed using 8-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary [22,23]. The primary, secondary, tertiary antral and atretic follicles, fresh corpora lutea were each counted that stained with haematoxylin and eosin on each ovary and numbers were given per rat. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10× for others.
Statistical analysis:
All statistical evaluations were performed using the software SPSS 15.0 (SPSS Inc., Chicago, USA). The effect of the follicle counts of the ovaries after EV injection and/or HBO on were evaluated using Kruskal Wallis test. p < 0.05 was considered statistically significant. After Kruskal Wallis, Mann Whitney-U test with Benforini adjustment was applied.
Results:
In control group (group 1), the ovaries exhibited a typically normal appearance with follicles and corpora lutea in different stages of development and regression (Figure 1). The number of atresic follicles was significantly lower and the numbers of fresh corpora luteas, tertiary follicles, secondary follicles and primary follicles were significantly higher than other groups (Table 1 and Table 2).
Ovarian morphology in Control group.
Comparison of mean numbers of follicles
*p values of Kruskal-Wallis test
AF: atretic follicle, CL:fresh corpus luteum, TF: tertiary follicle, SF:secondary follicle, PF: primary follicle.
Comparison of p values of groups regarding follicle types
*p values of Mann Whitney-U test with Benforini adjustment for multiple comparisons.
NS: nonsignificant
AF:atretic follicle, CL:fresh corpus luteum, TF:tertiary follicle, SF:secondary follicle, PF:primary follicle
In PCO only group (group 2), the ovaries displayed typical PCO-like changes (Figure 2); mean numbers of tertiary, secondary and primary follicles were significantly lower compared with the control group (Table 1 and Table 2). Typically, the number of atretic follicles were significantly higher in the PCO only group compared to control and very few fresh corpora lutea was observed.
Cystic Follicles of Polycystic ovary (PCO) group.
In the PCO+HBO group (group 3), there were significantly higher atretic follicles (Figure 3) compared to the PCO only group (p = 0.037). Although number of atretic follicles in group 3 seems to be more than group 4, the difference was not statistically significant (Table 2).
Atretic and cystic follicles in PCO+HBO group.
In the only HBO group (Group 4), there were significantly higher atretic follicles compared to group 1 (p = 0,024). There were significantly lower fresh corporea lutea (p = 0,034) and healthy growing follicles (Table 2). Although the number of atretic follicles were seems to be higher (Figure 4), it did not differ significantly compared with group 3.
Marked atretic follicles in HBO group.
Discussion:
Cystic ovarian disease and/or PCOS are disorders of the reproduction that affect bovine, ovine, caprine and porcine species and even human beings [1,2]. Several experimental models for PCO have been developed in rats; of these EV cause a sudden appearance of PCO due to disturbances in metabolic and physiologic processes [6,7].
Many studies were established such as electroacupuncture, hemohim, Korean red ginseng extract, herbal medicine, exercise assessing the effect of different therapies on EV induced PCOS rats [24-27]. Some of them found improvement in ovarian morphology of PCO state, and some of them did not. The difference of our study from those is that; they investigated the inhibition of PCO state starting therapies on the same day with EV injection, but we wanted to investigate the improvement in ovarian morphology of PCO state by starting HBOT after PCO morphology was fully established, rather than to investigate the inhibition of formation of PCO morphology.
The application of HBO ensures a normal supply of molecular oxygen to mitochondria, transported from the lungs to the endometrial cells or to ovarian tissue in a physically dissolved form in body fluids. Ultimately the increases in dissolved oxygen generated by HBO have several physiologic effects that can alter tissue responses to disease and injury [8,15]. In our study, after PCO formation, six weeks of HBO treatment, 60 minutes once a day protocol did not reverse but in contrast, aggravated the atresia of follicular growth in the rat ovaries, significantly. HBOT of healthy rat ovary increased the young follicular atresia and decreased the healthy growing follicles and also decreased the fresh corporea lutea significantly. The mean number healthy growing follicles were found lower than control and PCO groups but similar with PCO+HBO group. So this forced us to think that HBO administration created the same effect on ovarian histology like EV. Significantly, the mean number of atresia was higher in HBO+PCO group compared to PCO group, we think that the accelerated atresia is due to HBOT and HBO did not help to improve PCO state in the ovaries but inspite aggravated it. When we reviewed our results with rat studies about HBOT, we came to know that HBO administration also led to unwelcome side-effects, such as oxidative stress and/or oxygen toxicity, the formation of ROS can cause cellular damage through the oxidation of lipids, proteins and DNA [28]. One study assessing oxidative stress levels in rats following exposure to oxygen at 3 atm for 0-120 minutes exhibited a clear relationship of HBO-induced oxidative action to exposure time. This action was most pronounced from 90 to 120 minutes of exposure [29]. Another study on the effects of HBO in rats revealed that after 2 hours of HBO exposure at 3 ATA, levels of the oxidative stress markers elevated in lung, brain and erythrocytes [30]. Repetitive treatment with HBO was proved to cause oxidative stress in critical tissues including the rat brain, lymphocytes. Malondialdehyde levels and other oxidative stres markers were found to be significantly increased in the therapies after 20 day to 40 day session groups [31]. Although HBO-mediated free radicals were accepted to be responsible for the benefits of this therapeutic modality, especially in cases with prolonged exposure, possible injurious effects of supranormal values of bio-oxidative products need to be considered. Our application of 6 weeks HBO might be too much regarding exposure time and duration of it for PCO rats which is a multi-system disease that already carries underlying metabolic pathologies. Confirming this, Takahashi et al. found a biphasic effect of the generation of superoxide radicals on proliferation consistent with the concept that while moderate oxidative stress may induce cell growth, very high levels of ROS were cytotoxic and led to cell, tissue damage and apoptotic cell death [32]. Although we did not measure oxidative stress markers, all these aforementioned data may help to explain why we found aggrevation of atresia rather than reversal of it. As we did not measure the VEGF levels we can't declare clearly if modulated mechanisms of angiogenesis were causative.
Although human PCO is not the same as rat model, the oxidative effects of HBO have been investigated both in animals and human [29,30]. The protocols are consistent with the principles of partial pressures reaching that tissue. Henry's law states that the amount of gas dissolved in a liquid or tissue is proportional to the partial pressure of that gas in contact with the liquid or tissue. This is the basis for increased tissue oxygen tensions with HBOT [8,9].
The clinically approved maximum pressure and duration of HBO exposure are 3 ATA and 120 minutes, respectively although the most commonly-used protocol for standard therapeutic purposes is slightly lower (1.8-2.8 ATA for 60-90 min) [9,28]. We applied chronic diseases standard HBO protocol (2.5 atm, 60 minute once a day) for our rat PCO model, considering the PCO as multi-systemic and chronic disease. Treatment recommendations mentioned in this paper are taken from the UHMS report and are evidence-based [8]. Protocols vary greatly, but the UHMS recommend treatments beginning at 2.0-2.5 ATA for up to 120 minutes at least once a day until no improvement in diseases are reached. When we reviewed rat HBO studies, we found there were different protocols like 2 atm, 2.8 atm/90 min for 5-40 days or 3 atm for up to 120 minutes, although guidelines vary depending on the injury [28-31]. It would be more favourable for our study if we could apply different pressures and different exposure time and analyze their effects. This will be our goal for future studies.
There is a substantial body of literature that has examined the adverse and beneficial effects of HBOT in animal models. As technology becomes more readily available to clinical practice and more clinical trials are performed to define its effectiveness, HBOT may be considered as an additional therapeutic option in many conditions. Recent studies assessed the role of HBOT in unsuccessful IVF protocols or endometrial implantation failures and found convincing results although the standard protocols are not yet established. We tried to find an alternative or adjunctive treatment modality for PCO. Depending on the tissue concentration it can either exert beneficial physiologic effects or damage cell structures. We suggest that our present HBO protocol is not applicable for treatment of PCO disease and that should be considered in infertility patients with PCO; but may be rearranged accordingly in rat PCO model which needs further studies.
Quantifying the follicles may be time consuming and not an easy way of assessing ovarian histology, this is an experimental study and we wanted to assess the details of ovarian morphology other than ovulation [22,23].
Our study is the first that HBO is used in EV induced PCO rats and control rats. Our study is privileged from others that, the long- term effect of HBO on PCOS was demonstrated. To our knowledge, there is no study in the literature evaluating the effect of HBOT on PCO.
Conclusions:
We conclude that in our experimental rat model, HBOT did not improve or reverse the PCO state in EV induced PCO rat ovaries, in contrast, after HBOT, follicular atresia increased significantly. With this protocol it seems unfavorable to administrate HBOT in PCO state. Further studies are needed to identify the accompanying factors of this result like exposure time or duration of therapy
Abbreviations:
PCOS: Polycystic Ovary Syndrome; HBO: Hyperbaric Oxygen; HBOT: Hyberbaric Oxygen Therapy; UHMS: Undersea and Hyperbaric Medical Society; EV: Estradiol Valerate; ROS: Reactive Oxygen Species; NIH: National Institute of Health; AF: Atretic Follicle; CL: Fresh Corpus Luteum; TF: Tertiary Follicle; SF: Secondary Follicle; PF: Primary Follicle; VEGF: Vascular Endothelial Growth Factor; IVF: İn-Vitro Fertilization; ATA: Atmosphere Absolute.
Competing interests:
The authors declare that they have no competing interests.
Authors' contributions:
AA has designed the study, YA has dealt with statistics, FC has dealt with rats, DS has assessed the microscopy, AArslan and AST has given the HBOT, MD has reviewed the drafts, NG read & approved the final version of the manuscript. All authors read and approved the final manuscript.
|
Background:
In this study, we investigated the effect of hyperbaric oxygen therapy (HBOT) on the morphology of estradiol valerate (EV) induced polycystic ovary (PCO) to find a new treatment modality for improvement of PCO.
Methods:
The rats were divided into four groups. Group1, control; group 2, PCO group; group 3, PCO with HBOT group and group 4, normal ovary with HBOT. PCO was induced by a single intramuscular injection of 4 mg EV in adult cycling rats. Other rats with normal ovaries had oil injection as placebo. HBOT was applied to third and fourth groups for six weeks. Histopathologic evaluation of ovaries of all groups were performed & compared.
Results:
Six weeks of HBOT was resulted in increase in follicular atresia, decrease in the number of primary, secondary, tertiary follicles and decrease in the number of fresh corpus luteum in normal rat ovary. HBOT on polycystic rat ovary, resulted in significant increase in atretic follicles which were already present.
Conclusions:
HBOT of six weeks itself, changed ovarian morphology in favor of atresia both in PCO group and control group. This result of aggravated follicular atresia after HBOT on EV induced PCO may be due to long-term exposure in our protocol which with this state seems to be inapplicable in the improvement of PCO morphology.
|
Background:
Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women of reproductive age is a multifactorial metabolic disease associated with insulin resistance [1]. PCOS is a common condition with a range of clinical features. These reproductive features include anovulation, irregular menstrual cycles, clinical and biochemical hyperandrogenism and infertility. Metabolic features include increased risk factors for type 2 diabetes mellitus and cardiovascular disease and increase in the prevalence of the metabolic syndrome [1,2].
Previously, the diagnosis of PCOS was based on the National Institute of Health (NIH) criteria comprising biochemical or clinical hyperandrogenism and anovulatory irregular menstrual cycles with the exclusion of related reproductive disorders. In 2003, diagnostic guidelines for PCOS were expanded to the so-called Rotterdam criteria, now based on presentation with any two of the three criteria of hyperandrogenism, irregular anovulatory periods or polycystic ovaries on ultrasound [1,3]. The reported prevalence range of PCOS is between 2.2% to 26% [1,3,4]. The wide range of estimated PCOS prevalence can be explained by different recruitment processes of the study populations, selection biases, ethnic and racial variations in addition to, the criteria used for its definition and the screening methods used to identify each criteria; considering the Rotterdam versus NIH criteria increase the PCOS prevalence by 2 times. The latest study by Tehrani et al. demonstrated that the prevalence's of PCOS using the NIH definition were 8.5% [4].
The abnormalities detected in PCOS have been attributed to primary defects in the hypothalamic-pituitary-adrenal axis, the ovarian microenvironment, the adrenal gland and the insulin/insulin-like growth factor metabolic regulatory system [2,5]. There is evidence that both hypothalamic and pituitary mechanisms contribute to the gonadotropin dysfunction in PCOS.
The reproductive systems of human beings and other vertebrates are grossly similar [6]. Although the rat is a polytocous rodent, the female has a regular ovarian cyclicity of 4 or 5 days, with distinct proestrus, estrus, and diestrus phases. PCO can be experimentally produced in the rat, that species is a good model for studying the pathophysiology of human PCO by a single dose of the long-acting estrogen, estradiol valerate (EV) which leads to anovulatory state in 13-15 week-old rats [6,7].
The therapeutic application of hyperbaric oxygen (HBO) has been known over three decades in different fields of medicine. Hyperbaric oxygen therapy (HBOT) is defined by the Undersea and Hyperbaric Medical Society (UHMS) as a treatment in which a patient intermittently breathes 100% oxygen while the treatment chamber is pressurized to a pressure greater than sea level (1 atmosphere absolute, ATA). The therapeutic principle behind HBO stems from increasing the partial pressure of oxygen in the tissues of the body. The effects of HBO are based on the gas laws, and the physiological, biochemical effects of hyperoxia [8,9]. High tissue levels of oxygen stimulate angiogenesis by increasing the production of cytokines and many growth factors, especially VEGF. Because of these effects on angiogenesis and hyperoxia, it is a unique method used in the treatment of various illnesses and clinical conditions, such as carbon monoxide poisoning, decompression sickness, osteomyelitis, even diabetic foot and ischaemic wounds [9,10].
Besides, wound healing, HBOT has been used for many clinical treatments. Previous experiments have demonstrated that HBO improves survival in anesthetized rats during heatstroke by reducing multiorgan dysfunction [11]. Furthermore, microarray analysis of gene expression in rat cortical neurons exposed to HBO showed that 17 genes changed in response to all exposure conditions [12]. HBOT is also used for primary liver nonfunction as it stimulated hepatocytes to proliferate. Experimentally, HBO was found significantly effective in the survival of transplanted cells or tissues like liver, bone, thyroid, pancreatic islet cells [13]. Although a large proportion of ovarian follicles were lost during the initial ischemia after transplantation of ovaries, recently it was also found that HBO was also beneficial for the follicular survival of the transplanted mouse ovaries [14].
HBOT was also used in invitro-fertilisation (IVF) protocols in different studies for improving ovarian folicular stimulation and endometrial receptivity as an adjunct to infertility therapies with the hypothesis of increasing impaired follicular angiogenesis and oxygenation and increasing pregnancy implantation by improving endometrial receptivity by adequate vascularisation and oxygenation in unexplained infertility patients respectively [15,16]. Although new; these trials offer hopeful results for future infertility patients.
Principal mechanisms of HBO are based on intracellular generation of reactive species of oxygen and nitrogen by causing oxidative stress. Reactive oxygen species (ROS) are recognized to play a central role in cell signal transduction cascades or pathways, for a variety of growth factors, cytokines, and hormones. Scavenging antioxidants combat the overproduction of reactive species like vitamine E and C, glutation [10,17]. Recently, some new data about the role of oxidative stress and antioxidants in modulation of ovarian techal-interstitial cells have been enlightened. First, it was demonstrated that antioxidants inhibited proliferation of theca-interstitial cells then new findings added that antioxidants with distinctly different mechanisms of action induce a series of events consistent with the process of apoptosis in ovarian mesenchyme [18,19]. As PCOS is a condition associated with excessive growth and activity of theca-interstitial cells, these findings bring the question of the role of oxidative stress and antioxidants in PCO disease.
Treatment of human PCOS is symptomatic, lifestyle measures such as diet and exercise (or both) could play an important role in optimising healthy weight, improving underlying hormonal disturbances, prevention of future reproductive and metabolic complications and improving quality of life. Lifestyle interventions are recommended first-line in an international position statement on PCOS and present a cost effective initial treatment strategy compared to surgical and pharmacological options [20]. In the recent Cochrane review the positive effects of lifestyle treatment on anthropometric (abdominal adiposity, adiposity distribution), reproductive (biochemical and clinical hyperandrogenism) and metabolic features (markers of insulin resistance) were confirmed with medium or long term lifestyle measures [21].
In the present study, we tried to find a new, alternative treatment modality for PCO disease, considering the different effects of hyperoxia on different tissues, mentioned above. For this, we tested the hypothesis that 6 weeks of HBO administration on polycystic rat ovary would make an improvement or reversal of PCO state. This was carried out by studying the effect of HBO on ovarian morphology including different folicular stages.
Conclusions:
We conclude that in our experimental rat model, HBOT did not improve or reverse the PCO state in EV induced PCO rat ovaries, in contrast, after HBOT, follicular atresia increased significantly. With this protocol it seems unfavorable to administrate HBOT in PCO state. Further studies are needed to identify the accompanying factors of this result like exposure time or duration of therapy
|
Background:
In this study, we investigated the effect of hyperbaric oxygen therapy (HBOT) on the morphology of estradiol valerate (EV) induced polycystic ovary (PCO) to find a new treatment modality for improvement of PCO.
Methods:
The rats were divided into four groups. Group1, control; group 2, PCO group; group 3, PCO with HBOT group and group 4, normal ovary with HBOT. PCO was induced by a single intramuscular injection of 4 mg EV in adult cycling rats. Other rats with normal ovaries had oil injection as placebo. HBOT was applied to third and fourth groups for six weeks. Histopathologic evaluation of ovaries of all groups were performed & compared.
Results:
Six weeks of HBOT was resulted in increase in follicular atresia, decrease in the number of primary, secondary, tertiary follicles and decrease in the number of fresh corpus luteum in normal rat ovary. HBOT on polycystic rat ovary, resulted in significant increase in atretic follicles which were already present.
Conclusions:
HBOT of six weeks itself, changed ovarian morphology in favor of atresia both in PCO group and control group. This result of aggravated follicular atresia after HBOT on EV induced PCO may be due to long-term exposure in our protocol which with this state seems to be inapplicable in the improvement of PCO morphology.
| 7,196 | 256 | 14 |
[
"follicles",
"pco",
"rats",
"hbo",
"group",
"follicle",
"primary",
"rat",
"ovaries",
"atretic"
] |
[
"test",
"test"
] |
[CONTENT] Hyperbaric-oxygen | Polycystic-ovary | Rat | Estradiol valerate [SUMMARY]
|
[CONTENT] Hyperbaric-oxygen | Polycystic-ovary | Rat | Estradiol valerate [SUMMARY]
|
[CONTENT] Hyperbaric-oxygen | Polycystic-ovary | Rat | Estradiol valerate [SUMMARY]
|
[CONTENT] Hyperbaric-oxygen | Polycystic-ovary | Rat | Estradiol valerate [SUMMARY]
|
[CONTENT] Hyperbaric-oxygen | Polycystic-ovary | Rat | Estradiol valerate [SUMMARY]
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[CONTENT] Hyperbaric-oxygen | Polycystic-ovary | Rat | Estradiol valerate [SUMMARY]
|
[CONTENT] Animals | Estradiol | Female | Hyperbaric Oxygenation | Ovarian Follicle | Ovary | Polycystic Ovary Syndrome | Rats | Rats, Wistar [SUMMARY]
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[CONTENT] Animals | Estradiol | Female | Hyperbaric Oxygenation | Ovarian Follicle | Ovary | Polycystic Ovary Syndrome | Rats | Rats, Wistar [SUMMARY]
|
[CONTENT] Animals | Estradiol | Female | Hyperbaric Oxygenation | Ovarian Follicle | Ovary | Polycystic Ovary Syndrome | Rats | Rats, Wistar [SUMMARY]
|
[CONTENT] Animals | Estradiol | Female | Hyperbaric Oxygenation | Ovarian Follicle | Ovary | Polycystic Ovary Syndrome | Rats | Rats, Wistar [SUMMARY]
|
[CONTENT] Animals | Estradiol | Female | Hyperbaric Oxygenation | Ovarian Follicle | Ovary | Polycystic Ovary Syndrome | Rats | Rats, Wistar [SUMMARY]
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[CONTENT] Animals | Estradiol | Female | Hyperbaric Oxygenation | Ovarian Follicle | Ovary | Polycystic Ovary Syndrome | Rats | Rats, Wistar [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] follicles | pco | rats | hbo | group | follicle | primary | rat | ovaries | atretic [SUMMARY]
|
[CONTENT] follicles | pco | rats | hbo | group | follicle | primary | rat | ovaries | atretic [SUMMARY]
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[CONTENT] follicles | pco | rats | hbo | group | follicle | primary | rat | ovaries | atretic [SUMMARY]
|
[CONTENT] follicles | pco | rats | hbo | group | follicle | primary | rat | ovaries | atretic [SUMMARY]
|
[CONTENT] follicles | pco | rats | hbo | group | follicle | primary | rat | ovaries | atretic [SUMMARY]
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[CONTENT] follicles | pco | rats | hbo | group | follicle | primary | rat | ovaries | atretic [SUMMARY]
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[CONTENT] pcos | reproductive | criteria | hbo | different | metabolic | clinical | antioxidants | prevalence | ovarian [SUMMARY]
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[CONTENT] follicles | rats | group | primary | classified | follicles described | granulosa | istanbul | described | follicle [SUMMARY]
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[CONTENT] group | follicles | table | atretic | significantly | follicle | group group | higher | atretic follicles | follicles significantly [SUMMARY]
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[CONTENT] hbot | pco | state | pco state | rat | significantly protocol unfavorable | hbot pco state | significantly protocol unfavorable administrate | identify accompanying factors result | identify accompanying factors [SUMMARY]
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[CONTENT] follicles | group | pco | follicle | rats | hbo | hbot | atretic | primary | rat [SUMMARY]
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[CONTENT] follicles | group | pco | follicle | rats | hbo | hbot | atretic | primary | rat [SUMMARY]
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[CONTENT] EV | PCO [SUMMARY]
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[CONTENT] four ||| 2 | PCO group | 3 | PCO | 4 | HBOT ||| PCO | 4 | EV ||| ||| third | fourth | six weeks ||| [SUMMARY]
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[CONTENT] Six weeks | HBOT | atresia ||| [SUMMARY]
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[CONTENT] six weeks | atresia | PCO ||| EV | PCO [SUMMARY]
|
[CONTENT] EV | PCO ||| four ||| 2 | PCO group | 3 | PCO | 4 | HBOT ||| PCO | 4 | EV ||| ||| third | fourth | six weeks ||| ||| Six weeks | HBOT | atresia ||| ||| six weeks | atresia | PCO ||| EV | PCO [SUMMARY]
|
[CONTENT] EV | PCO ||| four ||| 2 | PCO group | 3 | PCO | 4 | HBOT ||| PCO | 4 | EV ||| ||| third | fourth | six weeks ||| ||| Six weeks | HBOT | atresia ||| ||| six weeks | atresia | PCO ||| EV | PCO [SUMMARY]
|
||||||
Risk factors for HIV and STI diagnosis in a community-based HIV/STI testing and counselling site for men having sex with men (MSM) in a large German city in 2011-2012.
|
25582975
|
In recent years community-based voluntary counselling and testing sites (CB-VCT) for men having sex with men (MSM) have been established in larger cities in Germany to offer more opportunities for HIV testing. Increasingly, CB-VCTs also offer testing for other bacterial sexually transmitted infections. In Hamburg, tests in CB-VCTs are offered free and anonymously. Data on demographics and sexual risk behaviours are collected with a paper questionnaire.
|
BACKGROUND
|
Questionnaire data from the MSM CB-VCT in Hamburg were linked with serological test results for HIV and syphilis, and with rectal and pharyngeal swab results for gonorrhoea and chlamydia. MSM were defined as males reporting male sex partners. CB-VCT clients were characterized demographically, and associations between sexual behaviour variables and diagnosis of HIV and sexually transmitted infections (STI) were analysed by bivariate and multivariate logistic regression analysis.
|
METHODS
|
Among the male clients of the CB-VCT in 2011-2012 who were tested for HIV or any STI 1476 reported male sex partners. Unprotected anal intercourse (UAI) was reported as reason for testing by 61% of the clients. Forty-one of 1413 clients testing for HIV were tested positive (2.9%). Twenty-four of 1380 clients testing for syphilis required treatment (1.7%). Tests for simultaneous detection of N. gonorrhoea and Chlamydia trachomatis were conducted on 882 pharyngeal and 642 rectal swabs, revealing 58 (=6.6%) pharyngeal and 71 (=11.1%) rectal infections with one or both pathogens. In multivariate logistic regression analysis number of partners, UAI (OR=2.42) and relying on visual impression when selecting sex partners (OR = 2.92) were associated with increased risks for diagnosis of syphilis or a rectal STI. Syphilis or rectal STI diagnosis (OR=4.52) were associated with increased risk for HIV diagnosis.
|
RESULTS
|
The MSM CB-VCT in Hamburg reaches clients at high risk for HIV and STIs. The diagnosis of syphilis or a rectal STI was associated with increased odds of testing positive for HIV. Due to the high prevalence of curable bacterial STI among clients and because syphilis and rectal bacterial STI may facilitate HIV transmission, MSM asking for HIV tests in CB-VCTs should also be offered tests for other bacterial STIs.
|
CONCLUSIONS
|
[
"Adult",
"Chlamydia Infections",
"Cities",
"Community Health Services",
"Counseling",
"Germany",
"Gonorrhea",
"HIV Infections",
"Homosexuality, Male",
"Humans",
"Logistic Models",
"Male",
"Mass Screening",
"Middle Aged",
"Prevalence",
"Risk Factors",
"Sexual Behavior",
"Sexual Partners",
"Sexually Transmitted Diseases",
"Surveys and Questionnaires",
"Syphilis",
"Young Adult"
] |
4307229
|
Background
|
In recent years community-based voluntary counselling and testing sites (CB-VCT) [1] for HIV have been established for MSM in larger cities in Germany. One of these CB-VCTs is “Hein&Fiete” in the city of Hamburg. Hein&Fiete was founded in 1990 as an HIV prevention project for MSM. It includes a community drop-in centre with daily opening hours, providing information on gay life in Hamburg, meeting spaces for community groups, outreach prevention work for MSM, HIV and STI serological testing since 2004, and STI swab testing with nucleic acid amplification tests since 2011. The staff consists of approximately 85 volunteers and 5 full or part-time paid staff members. The work of Hein&Fiete, including free testing for HIV and STIs, is funded by the Federal state of Hamburg and by donations.
As low-threshold (anonymous, gay-friendly, low-cost) sexually transmitted infection (STI) screening opportunities for MSM are scarce in the German health care system [2], STI tests, particularly serological tests for syphilis and nucleic acid amplification tests for gonorrhoea and chlamydia infections, are increasingly offered by CB-VCTs. Since these tests can be provided free of charge, uptake of STI tests at Hein&Fiete is the highest among German CB-VCT. To facilitate the pre-test counselling session, clients of CB-VCT sites are invited to fill out an anonymous paper-based and standardized questionnaire on demographics, testing history, transmission risks, and risk management strategies. We analysed these data to identify current risk factors for a diagnosis of HIV and/or STI.
|
Methods
|
Study procedures Tests for HIV, HBV, HCV, syphilis, gonorrhoea and Chlamydia trachomatis were offered anonymously and free of charge. Decisions on which tests to take were based on pre-test needs counselling and the clients’ own choice. “Hein&Fiete” provides only testing and counselling and needs to refer clients with an infection diagnosis for further treatment to a private practice or hospital. Because most urethral infections with gonorrhoea and chlamydia in men are expected to be symptomatic, testing for urethral infections with these pathogens is not prioritized, while tests for the mostly asymptomatic pharyngeal and rectal infections are offered. Counsellors recommended pharyngeal and rectal swabs particularly to clients who reported higher numbers of sex partners.
Clients were invited to receive their test results two to five days after testing. In case of a positive test result, adequate therapy options were provided through referral to specialized MSM-friendly physicians.
Questionnaires and test results were entered in a Microsoft Access database by Hein&Fiete staff.
Data were fully anonymized and the anonymous code used at the CB-VCT was exchanged with another unrelated code. Therefore, ethical approval to conduct this analysis was waived.
Tests for HIV, HBV, HCV, syphilis, gonorrhoea and Chlamydia trachomatis were offered anonymously and free of charge. Decisions on which tests to take were based on pre-test needs counselling and the clients’ own choice. “Hein&Fiete” provides only testing and counselling and needs to refer clients with an infection diagnosis for further treatment to a private practice or hospital. Because most urethral infections with gonorrhoea and chlamydia in men are expected to be symptomatic, testing for urethral infections with these pathogens is not prioritized, while tests for the mostly asymptomatic pharyngeal and rectal infections are offered. Counsellors recommended pharyngeal and rectal swabs particularly to clients who reported higher numbers of sex partners.
Clients were invited to receive their test results two to five days after testing. In case of a positive test result, adequate therapy options were provided through referral to specialized MSM-friendly physicians.
Questionnaires and test results were entered in a Microsoft Access database by Hein&Fiete staff.
Data were fully anonymized and the anonymous code used at the CB-VCT was exchanged with another unrelated code. Therefore, ethical approval to conduct this analysis was waived.
Study sample Study population were all MSM clients attending the CB-VCT of “Hein&Fiete” who were tested for at least one STI (Chlamydia trachomatis, gonorrhoea, HIV, syphilis) and who completed the standardized questionnaire in 2011 and 2012. Men were defined as MSM if they reported at least one male sex partner in the previous 12 months.
Study population were all MSM clients attending the CB-VCT of “Hein&Fiete” who were tested for at least one STI (Chlamydia trachomatis, gonorrhoea, HIV, syphilis) and who completed the standardized questionnaire in 2011 and 2012. Men were defined as MSM if they reported at least one male sex partner in the previous 12 months.
Measurements Using a 25 item questionnaire, Hein&Fiete collected data on demographics (age, migration status, education), sexual orientation and partnerships, number and gender of sex partners in the previous 12 months, place where sex partners were met, use of drugs in combination with sex, testing history and test results for HIV and STIs, reasons for testing, condom use and reasons for not using condoms, and risk management strategies other than condom use.
Clients were tested for HIV with the Abbott Architect HIV Ag/Ab Combo assay, reactive samples were confirmed by Mikrogen, recomLine HIV-1 & HIV-2 IgG testing. For syphilis testing, samples were screened by Abbott Architect Syphilis TP Reagent, and confirmed by a quantitative TPPA test (MAST). Active infection was defined as either detection of anti-treponemal IgM (Mikrogen, recomWell Treponema IgM) or a positive test for Cardiolipin-KBR (Virion). Pharyngeal swabs taken by medical personnel and rectal swabs taken by the clients themselves were tested for gonorrhoea (Gc) and Chlamydia trachomatis (CT) with the BD ProbeTec™ ET System, which uses strand displacement amplification (SDA) for real-time detection of CT/GC.
Using a 25 item questionnaire, Hein&Fiete collected data on demographics (age, migration status, education), sexual orientation and partnerships, number and gender of sex partners in the previous 12 months, place where sex partners were met, use of drugs in combination with sex, testing history and test results for HIV and STIs, reasons for testing, condom use and reasons for not using condoms, and risk management strategies other than condom use.
Clients were tested for HIV with the Abbott Architect HIV Ag/Ab Combo assay, reactive samples were confirmed by Mikrogen, recomLine HIV-1 & HIV-2 IgG testing. For syphilis testing, samples were screened by Abbott Architect Syphilis TP Reagent, and confirmed by a quantitative TPPA test (MAST). Active infection was defined as either detection of anti-treponemal IgM (Mikrogen, recomWell Treponema IgM) or a positive test for Cardiolipin-KBR (Virion). Pharyngeal swabs taken by medical personnel and rectal swabs taken by the clients themselves were tested for gonorrhoea (Gc) and Chlamydia trachomatis (CT) with the BD ProbeTec™ ET System, which uses strand displacement amplification (SDA) for real-time detection of CT/GC.
Statistical analysis The data were analysed using SPSS© (Version 20, IBM Corp.). We used descriptive statistics, calculated bivariate correlations for reporting UAI, being diagnosed with a rectal STI or syphilis, or being diagnosed with HIV, with odds ratios, 95% confidence intervals (CI) and p-values, and constructed three models for multivariate logistic regression analyses to identify factors associated with reporting UAI (principal mode of HIV transmission among MSM), diagnosis of a rectal STI or syphilis (both cofactors for acquiring HIV), and factors associated with diagnosis of HIV. Meeting places were used as binary variables (yes/no). Age and partner number were included as categorical variables (<30 years, 30–44, >44; 0–2 partners, 3–5, 6–10, >10) in all three models (for the age grouping see [3]). Additional factors were only included if they had been significant or borderline significant (p < 0.10) in bivariate analysis. The method used was a conditional forward inclusion (p < 0.05) and backward elimination (p > 0.10) procedure. Reasons for not using condoms during a recent risk situation and risk management strategies other than condom use were not included in the multivariate model for UAI, only in the models for STI and HIV diagnosis.
The data were analysed using SPSS© (Version 20, IBM Corp.). We used descriptive statistics, calculated bivariate correlations for reporting UAI, being diagnosed with a rectal STI or syphilis, or being diagnosed with HIV, with odds ratios, 95% confidence intervals (CI) and p-values, and constructed three models for multivariate logistic regression analyses to identify factors associated with reporting UAI (principal mode of HIV transmission among MSM), diagnosis of a rectal STI or syphilis (both cofactors for acquiring HIV), and factors associated with diagnosis of HIV. Meeting places were used as binary variables (yes/no). Age and partner number were included as categorical variables (<30 years, 30–44, >44; 0–2 partners, 3–5, 6–10, >10) in all three models (for the age grouping see [3]). Additional factors were only included if they had been significant or borderline significant (p < 0.10) in bivariate analysis. The method used was a conditional forward inclusion (p < 0.05) and backward elimination (p > 0.10) procedure. Reasons for not using condoms during a recent risk situation and risk management strategies other than condom use were not included in the multivariate model for UAI, only in the models for STI and HIV diagnosis.
|
Results
|
A total of 1630 persons filled in a questionnaire at Hein&Fiete in 2011 or 2012. Of these, 1565 men reported on the gender of their sexual partners. There were 1506 men identifiable as MSM. Since not all men filling out the questionnaire were actually tested - some clients were only counselled and some were recommended to get tested later because reported risks were too recent - the final sample for this analysis consisted of 1476 men reporting male partners and having been tested for at least one infection. HIV was tested for most frequently (1413), followed by syphilis (1380), pharyngeal (882) and rectal (642) infections with gonorrhoea and chlamydia. According to self-reported previous test date and place, up to 295 men were tested at least twice at Hein&Fiete during the two years. Numbers and proportions testing positive for the respective infections are presented in Table 1.Table 1
HIV and STI test results of MSM clients, 2011-2012
Number of tests (any test N = 1476)#
Number (%) testing positive
Number tested at least twice in 2011-2012
Number (%) seroconverting/newly infected
HIV141341 (2.9)486*16 (3.3)Syphilis (active)138027 (2.0)271°3 (1.1)Syphilis (antibodies)1380131 (9.5)271n.a.Gonorrhoea rectal64223 (3.6)154°8 (5.2)Gonorrhoea pharyngeal88248 (5.4)212°16 (7.5)Chlamydia rectal64256 (8.7)154°14 (9.1)Chlamydia pharyngeal88215 (1.7)212°5 (2.4)Any rectal STI64271 (11.1)Any rectal STI or active syphilis140395 (6.8)# discrepancies of numbers compared with Figure 1 due to missing age data for 40 clients.n.a. =not applicable, because treponemal antibodies often persist after treatment. Among the clients reporting testing at least twice 28 (10.3%) had detectable treponemal antibodies. Since the result of the previous tests is unknown, new or re-infection rates could not be determined.*= 295 had been tested at Hein&Fiete, 191 had been tested elsewhere first and the second time at Hein&Fiete.°= clients had been tested at Hein&Fiete at least twice in 2011–2012, however it is unknown whether they had been tested twice for this STI or at this location.
HIV and STI test results of MSM clients, 2011-2012
# discrepancies of numbers compared with Figure 1 due to missing age data for 40 clients.
n.a. =not applicable, because treponemal antibodies often persist after treatment. Among the clients reporting testing at least twice 28 (10.3%) had detectable treponemal antibodies. Since the result of the previous tests is unknown, new or re-infection rates could not be determined.
*= 295 had been tested at Hein&Fiete, 191 had been tested elsewhere first and the second time at Hein&Fiete.
°= clients had been tested at Hein&Fiete at least twice in 2011–2012, however it is unknown whether they had been tested twice for this STI or at this location.
The clients having been tested more than once during the two-year observation period may impact the number and proportion of clients testing positive for treponemal antibodies. Based on antibody detection, the new or re-infection rate for syphilis between the repeated tests cannot be determined, and the same individuals may be counted twice or more.
The completeness of core variables in the MSM sample ranged from 82% (response to question on previous chlamydia testing) to 99.9% (sexual orientation). Information e.g. on risk behaviour (unprotected intercourse) and HIV test results were available for approximately 1300 clients, information on rectal STI and risk behaviour for approximately 600 clients.
The median age of the respondents was 36 years (interquartile range: 28–45 years). A migration background (parents or respondent himself immigrated to Germany) was reported by 25% of the respondents. A high school diploma or a higher degree of education was held by 69%. Twelve percent of the sample self-identified as bisexual, 87% as gay. Relationship status at the time of testing was reported as single by 53% of the respondents; another 22% reported being in an open and 25% reported being in a mutually exclusive relationship. A previous test for HIV was reported by 87% of the tested MSM, a previous syphilis test by 59%, 37% had been tested for gonorrhoea, and 31% for Chlamydia trachomatis (both not differentiated by location).
Overall, MSM clients reported a median of 6 and a mean of 11 sexual partners (standard deviation 18) in the previous twelve months. The pharyngeal swab subsample reported a median of 7 and a mean of 13 partners (standard deviation 21), the rectal swab subsample a median of 8 and a mean of 14 partners (standard deviation 24), reflecting the preferential recommendation to take swabs for clients with higher partner numbers.
Transmission risk behaviour, particularly in terms of HIV risks, was queried by a question asking for the reasons for testing. Unprotected anal intercourse (UAI) was reported by 61% of the clients as a reason for testing; 44% reported insertive UAI, and 35% reported receptive UAI. Unprotected oral intercourse with ejaculation into the mouth was reported as risk factor by 33% of MSM. Of note, a large proportion of clients who were diagnosed with a rectal STI did not report receptive UAI (27 out of 71 = 38%). The context in which risks were taken could be derived from questions on drug and alcohol use before or during sex (completed by 65%) and from a question on where most sex partners were met. Bivariate associations of these factors with UAI, STI and HIV diagnosis are shown in Table 2.Table 2
Results of Bi- und Multivariate analysis of associations between age, partner numbers, meeting places with sex partners, substance use during sex, biological cofactors and risk behaviours with the outcomes UAI, rectal STI and HIV diagnosis
Bivariate
Multivariate
Bivariate
Multivariate
Bivariate
Multivariate
UAI
STI rectal or syphilis
HIV
OR
95% CI
OR
95% CI
OR
95% CI
OR
95% CI
OR
95% CI
OR
95% CI
Age group
<301.54***1.18-1.961.67***1.26-2.231.78**1.12-2.861.060.49-2.3330-44 (ref.)111>44 years1.110.86-1.430.870.49-1.541.270.58-2.80
Partner number
0-2 (ref.)1113-51.150.80-1.642.710.76-9.661.410.48-4.146-101.180.83-1.706.10***1.82-20.416.13***1.79-20.90.820.25-2.72>101.59***1.12-2.255.06***1.51-16.915.27***1.55-18.00.910.30-2.83
Meeting sex partners
At friends1.200.93-1.471.310.85-2.020.47*0.21-1.06In bars1.020.81-1.280.950.60-1.490.840.42-1.69In gay bathhouses1.050.83-1.321.250.80-1.951.080.55-2.15In porn cinemas1.070.81-1.421.110.65-1.891.83*0.90-3.71In outside cruising sites1.020.71-1.470.660.28-1.531.100.39-3.13On highway resting areas1.290.67-2.461.510.53-4.330.900.12-6.74Online1.49***1.20-1.841.29**1.01-1.651.45*0.93-2.260.810.44-1.52At parties or discotheques1.41***1.12-1.780.940.60-1.480.610.29-1.29In public urinals1.550.73-3.261.340.40-4.480.970.96-0.98In fitness centres1.120.75-1.690.680.27-1.710.310.04-2.26
Substance use
Alcohol1.32***1.07-1.620.820.54-1.240.630.34-1.17Inhaled Amyl nitrite1.68***1.31-2.151.63***1.23-2.171.040.65-1.671.670.88-3.19Sildenafil1.53*0.96-2.431.220.55-2.721.200.36-3.95Cocaine, Speed1.65*0.93-2.912.36**1.09-5.151.210.28-5.12Cannabis1.50*0.97-2.301.230.58-2.621.040.32-3.42Ecstasy3.540.78-16.034.23*1.15-15.655.77**1.40-23.82.830.36-22.32GBL,GHB0.61**0.58-0.634.67*0.93-23.444.880.59-40.56
Cofactors, behaviours
Unprotected anal intercoursen.a.2.39***1.46-3.932.42***1.29-4.534.52***1.76-11.593.24* °STI co-diagnosisn.a.n.a.3.02**1.22-7.464.52***1.68-12.1Having sex with partners who appear healthyn.a.3.09***1.94-4.922.92***1.68-5.071.270.56-2.91Asking the insertive partner not to ejaculate insiden.a.2.11**1.16-3.864.34***2.11-8.894.44***1.75-11.3Asking the partner about HIV statusn.a.1.71**1.03-2.852.03*1.00-4.11Partner reluctant to use condomsn.a.1.300.72-2.342.68**1.32-5.45No condom availablen.a.2.81**1.46-5.413.10**1.26-7.60*p > 0.05 < =0.10; **p < =0.05 > 0.01; ***p < =0.01.n.a. = not applicable.°Results of Multivariate logistic regression only reported if p < =0.05 (except for UAI as risk factor for HIV diagnosis, p = 0.07).
Results of Bi- und Multivariate analysis of associations between age, partner numbers, meeting places with sex partners, substance use during sex, biological cofactors and risk behaviours with the outcomes UAI, rectal STI and HIV diagnosis
*p > 0.05 < =0.10; **p < =0.05 > 0.01; ***p < =0.01.
n.a. = not applicable.
°Results of Multivariate logistic regression only reported if p < =0.05 (except for UAI as risk factor for HIV diagnosis, p = 0.07).
Acute syphilis and HIV diagnoses were almost equally distributed across all age groups in the tested sample. MSM diagnosed with HIV were younger than MSM diagnosed with acute syphilis. Noticeably, while within age groups those men who were diagnosed with rectal or pharyngeal gonorrhoea or chlamydia reported higher numbers of partners, though the differences were statistically not significant, across the whole sample the median number of partners increased with age (from a median of 5 for age <30 years to a median of 10 for age 45–59), while the proportion of clients diagnosed with gonorrhoea and chlamydia decreased (Figure 1). Please note that numbers do not sum up to the numbers in Table 1 due to missing age data of 40 clients.Figure 1
Proportion of MSM testing positive for different STIs and HIV, by age groups.
Proportion of MSM testing positive for different STIs and HIV, by age groups.
Clients reporting a self-perceived risk situation as a reason for testing (compared to routine testing or testing when entering a new partnership) were significantly more likely to be diagnosed with HIV and STI (OR = 2.8; 95% CI 1.4-5.6). In bivariate analysis, reported reasons positively associated with risk situations were reluctance of the sex partner to use a condom and unavailability of condoms at a sexual encounter (see Table 2).
Among the risk management strategies besides condom use, asking the active partner to withdraw before ejaculation and asking the partner about his HIV status were positively associated with being diagnosed with a bacterial STI or with HIV (Table 2).
Reported UAI was strongly associated with an HIV diagnosis and less strongly associated with the diagnosis of a rectal STI or syphilis. The concomitant diagnosis of a rectal infection with gonorrhoea or chlamydia or a diagnosis of syphilis was strongly associated with an HIV diagnosis. For clients reporting an HIV positive partner the odds ratio for being diagnosed with HIV was 1.8 (95% CI 0.8-4.1; p = 0.175) compared to others, which did not reach statistical significance.
In multivariate logistic regression analyses including age and partner numbers (significantly associated with UAI and STI diagnoses in bivariate analysis) in addition to reported substance use and risk management strategies (the latter not included for UAI, but only for STI and HIV diagnosis outcomes) showing statistical significance in bivariate analysis, the following factors remained significantly associated (results see also Table 2)with UAI: age group, a high partner number, and use of inhaled amyl nitrite (poppers);with diagnosis of a rectal STI or syphilis: high partner numbers, reporting use of ecstasy when having sex, reporting UAI, and having sex only with partners who appear healthy;with HIV diagnosis: diagnosis of rectal gonorrhoea or chlamydia infection or diagnosis of syphilis; asking the active partner to withdraw before ejaculation. The odds ratio for being diagnosed with HIV when reporting UAI was 2.8, but this did not reach statistical significance (95% CI 0.8-10.1).
with UAI: age group, a high partner number, and use of inhaled amyl nitrite (poppers);
with diagnosis of a rectal STI or syphilis: high partner numbers, reporting use of ecstasy when having sex, reporting UAI, and having sex only with partners who appear healthy;
with HIV diagnosis: diagnosis of rectal gonorrhoea or chlamydia infection or diagnosis of syphilis; asking the active partner to withdraw before ejaculation. The odds ratio for being diagnosed with HIV when reporting UAI was 2.8, but this did not reach statistical significance (95% CI 0.8-10.1).
|
Conclusions
|
Our results emphasize the need to combine HIV and STI testing for MSM and the need for comprehensive screening for mostly asymptomatic rectal and pharyngeal infections [19]. Community-based VCTs can reach the MSM target group quite efficiently, and offering comprehensive HIV and STI testing in this setting is likely to be much more cost effective than relying on traditional health care settings. However, the CB-VCT approach for MSM is likely only feasible in larger cities with respective gay communities. Additional alternative approaches will be necessary to reach rural MSM, MSM less connected to the gay community, and MSM less willing to self-identify as gay. Behavioural data which may be useful to adapt and fine-tune prevention messages for MSM can also be collected in this setting. The data collected routinely before the counselling session appears quite useful for the analysis of risk behaviours and trends in risk management strategies, especially combined with testing data.
|
[
"Background",
"Study procedures",
"Study sample",
"Measurements",
"Statistical analysis"
] |
[
"In recent years community-based voluntary counselling and testing sites (CB-VCT) [1] for HIV have been established for MSM in larger cities in Germany. One of these CB-VCTs is “Hein&Fiete” in the city of Hamburg. Hein&Fiete was founded in 1990 as an HIV prevention project for MSM. It includes a community drop-in centre with daily opening hours, providing information on gay life in Hamburg, meeting spaces for community groups, outreach prevention work for MSM, HIV and STI serological testing since 2004, and STI swab testing with nucleic acid amplification tests since 2011. The staff consists of approximately 85 volunteers and 5 full or part-time paid staff members. The work of Hein&Fiete, including free testing for HIV and STIs, is funded by the Federal state of Hamburg and by donations.\nAs low-threshold (anonymous, gay-friendly, low-cost) sexually transmitted infection (STI) screening opportunities for MSM are scarce in the German health care system [2], STI tests, particularly serological tests for syphilis and nucleic acid amplification tests for gonorrhoea and chlamydia infections, are increasingly offered by CB-VCTs. Since these tests can be provided free of charge, uptake of STI tests at Hein&Fiete is the highest among German CB-VCT. To facilitate the pre-test counselling session, clients of CB-VCT sites are invited to fill out an anonymous paper-based and standardized questionnaire on demographics, testing history, transmission risks, and risk management strategies. We analysed these data to identify current risk factors for a diagnosis of HIV and/or STI.",
"Tests for HIV, HBV, HCV, syphilis, gonorrhoea and Chlamydia trachomatis were offered anonymously and free of charge. Decisions on which tests to take were based on pre-test needs counselling and the clients’ own choice. “Hein&Fiete” provides only testing and counselling and needs to refer clients with an infection diagnosis for further treatment to a private practice or hospital. Because most urethral infections with gonorrhoea and chlamydia in men are expected to be symptomatic, testing for urethral infections with these pathogens is not prioritized, while tests for the mostly asymptomatic pharyngeal and rectal infections are offered. Counsellors recommended pharyngeal and rectal swabs particularly to clients who reported higher numbers of sex partners.\nClients were invited to receive their test results two to five days after testing. In case of a positive test result, adequate therapy options were provided through referral to specialized MSM-friendly physicians.\nQuestionnaires and test results were entered in a Microsoft Access database by Hein&Fiete staff.\nData were fully anonymized and the anonymous code used at the CB-VCT was exchanged with another unrelated code. Therefore, ethical approval to conduct this analysis was waived.",
"Study population were all MSM clients attending the CB-VCT of “Hein&Fiete” who were tested for at least one STI (Chlamydia trachomatis, gonorrhoea, HIV, syphilis) and who completed the standardized questionnaire in 2011 and 2012. Men were defined as MSM if they reported at least one male sex partner in the previous 12 months.",
"Using a 25 item questionnaire, Hein&Fiete collected data on demographics (age, migration status, education), sexual orientation and partnerships, number and gender of sex partners in the previous 12 months, place where sex partners were met, use of drugs in combination with sex, testing history and test results for HIV and STIs, reasons for testing, condom use and reasons for not using condoms, and risk management strategies other than condom use.\nClients were tested for HIV with the Abbott Architect HIV Ag/Ab Combo assay, reactive samples were confirmed by Mikrogen, recomLine HIV-1 & HIV-2 IgG testing. For syphilis testing, samples were screened by Abbott Architect Syphilis TP Reagent, and confirmed by a quantitative TPPA test (MAST). Active infection was defined as either detection of anti-treponemal IgM (Mikrogen, recomWell Treponema IgM) or a positive test for Cardiolipin-KBR (Virion). Pharyngeal swabs taken by medical personnel and rectal swabs taken by the clients themselves were tested for gonorrhoea (Gc) and Chlamydia trachomatis (CT) with the BD ProbeTec™ ET System, which uses strand displacement amplification (SDA) for real-time detection of CT/GC.",
"The data were analysed using SPSS© (Version 20, IBM Corp.). We used descriptive statistics, calculated bivariate correlations for reporting UAI, being diagnosed with a rectal STI or syphilis, or being diagnosed with HIV, with odds ratios, 95% confidence intervals (CI) and p-values, and constructed three models for multivariate logistic regression analyses to identify factors associated with reporting UAI (principal mode of HIV transmission among MSM), diagnosis of a rectal STI or syphilis (both cofactors for acquiring HIV), and factors associated with diagnosis of HIV. Meeting places were used as binary variables (yes/no). Age and partner number were included as categorical variables (<30 years, 30–44, >44; 0–2 partners, 3–5, 6–10, >10) in all three models (for the age grouping see [3]). Additional factors were only included if they had been significant or borderline significant (p < 0.10) in bivariate analysis. The method used was a conditional forward inclusion (p < 0.05) and backward elimination (p > 0.10) procedure. Reasons for not using condoms during a recent risk situation and risk management strategies other than condom use were not included in the multivariate model for UAI, only in the models for STI and HIV diagnosis."
] |
[
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Study procedures",
"Study sample",
"Measurements",
"Statistical analysis",
"Results",
"Discussion",
"Conclusions"
] |
[
"In recent years community-based voluntary counselling and testing sites (CB-VCT) [1] for HIV have been established for MSM in larger cities in Germany. One of these CB-VCTs is “Hein&Fiete” in the city of Hamburg. Hein&Fiete was founded in 1990 as an HIV prevention project for MSM. It includes a community drop-in centre with daily opening hours, providing information on gay life in Hamburg, meeting spaces for community groups, outreach prevention work for MSM, HIV and STI serological testing since 2004, and STI swab testing with nucleic acid amplification tests since 2011. The staff consists of approximately 85 volunteers and 5 full or part-time paid staff members. The work of Hein&Fiete, including free testing for HIV and STIs, is funded by the Federal state of Hamburg and by donations.\nAs low-threshold (anonymous, gay-friendly, low-cost) sexually transmitted infection (STI) screening opportunities for MSM are scarce in the German health care system [2], STI tests, particularly serological tests for syphilis and nucleic acid amplification tests for gonorrhoea and chlamydia infections, are increasingly offered by CB-VCTs. Since these tests can be provided free of charge, uptake of STI tests at Hein&Fiete is the highest among German CB-VCT. To facilitate the pre-test counselling session, clients of CB-VCT sites are invited to fill out an anonymous paper-based and standardized questionnaire on demographics, testing history, transmission risks, and risk management strategies. We analysed these data to identify current risk factors for a diagnosis of HIV and/or STI.",
" Study procedures Tests for HIV, HBV, HCV, syphilis, gonorrhoea and Chlamydia trachomatis were offered anonymously and free of charge. Decisions on which tests to take were based on pre-test needs counselling and the clients’ own choice. “Hein&Fiete” provides only testing and counselling and needs to refer clients with an infection diagnosis for further treatment to a private practice or hospital. Because most urethral infections with gonorrhoea and chlamydia in men are expected to be symptomatic, testing for urethral infections with these pathogens is not prioritized, while tests for the mostly asymptomatic pharyngeal and rectal infections are offered. Counsellors recommended pharyngeal and rectal swabs particularly to clients who reported higher numbers of sex partners.\nClients were invited to receive their test results two to five days after testing. In case of a positive test result, adequate therapy options were provided through referral to specialized MSM-friendly physicians.\nQuestionnaires and test results were entered in a Microsoft Access database by Hein&Fiete staff.\nData were fully anonymized and the anonymous code used at the CB-VCT was exchanged with another unrelated code. Therefore, ethical approval to conduct this analysis was waived.\nTests for HIV, HBV, HCV, syphilis, gonorrhoea and Chlamydia trachomatis were offered anonymously and free of charge. Decisions on which tests to take were based on pre-test needs counselling and the clients’ own choice. “Hein&Fiete” provides only testing and counselling and needs to refer clients with an infection diagnosis for further treatment to a private practice or hospital. Because most urethral infections with gonorrhoea and chlamydia in men are expected to be symptomatic, testing for urethral infections with these pathogens is not prioritized, while tests for the mostly asymptomatic pharyngeal and rectal infections are offered. Counsellors recommended pharyngeal and rectal swabs particularly to clients who reported higher numbers of sex partners.\nClients were invited to receive their test results two to five days after testing. In case of a positive test result, adequate therapy options were provided through referral to specialized MSM-friendly physicians.\nQuestionnaires and test results were entered in a Microsoft Access database by Hein&Fiete staff.\nData were fully anonymized and the anonymous code used at the CB-VCT was exchanged with another unrelated code. Therefore, ethical approval to conduct this analysis was waived.\n Study sample Study population were all MSM clients attending the CB-VCT of “Hein&Fiete” who were tested for at least one STI (Chlamydia trachomatis, gonorrhoea, HIV, syphilis) and who completed the standardized questionnaire in 2011 and 2012. Men were defined as MSM if they reported at least one male sex partner in the previous 12 months.\nStudy population were all MSM clients attending the CB-VCT of “Hein&Fiete” who were tested for at least one STI (Chlamydia trachomatis, gonorrhoea, HIV, syphilis) and who completed the standardized questionnaire in 2011 and 2012. Men were defined as MSM if they reported at least one male sex partner in the previous 12 months.\n Measurements Using a 25 item questionnaire, Hein&Fiete collected data on demographics (age, migration status, education), sexual orientation and partnerships, number and gender of sex partners in the previous 12 months, place where sex partners were met, use of drugs in combination with sex, testing history and test results for HIV and STIs, reasons for testing, condom use and reasons for not using condoms, and risk management strategies other than condom use.\nClients were tested for HIV with the Abbott Architect HIV Ag/Ab Combo assay, reactive samples were confirmed by Mikrogen, recomLine HIV-1 & HIV-2 IgG testing. For syphilis testing, samples were screened by Abbott Architect Syphilis TP Reagent, and confirmed by a quantitative TPPA test (MAST). Active infection was defined as either detection of anti-treponemal IgM (Mikrogen, recomWell Treponema IgM) or a positive test for Cardiolipin-KBR (Virion). Pharyngeal swabs taken by medical personnel and rectal swabs taken by the clients themselves were tested for gonorrhoea (Gc) and Chlamydia trachomatis (CT) with the BD ProbeTec™ ET System, which uses strand displacement amplification (SDA) for real-time detection of CT/GC.\nUsing a 25 item questionnaire, Hein&Fiete collected data on demographics (age, migration status, education), sexual orientation and partnerships, number and gender of sex partners in the previous 12 months, place where sex partners were met, use of drugs in combination with sex, testing history and test results for HIV and STIs, reasons for testing, condom use and reasons for not using condoms, and risk management strategies other than condom use.\nClients were tested for HIV with the Abbott Architect HIV Ag/Ab Combo assay, reactive samples were confirmed by Mikrogen, recomLine HIV-1 & HIV-2 IgG testing. For syphilis testing, samples were screened by Abbott Architect Syphilis TP Reagent, and confirmed by a quantitative TPPA test (MAST). Active infection was defined as either detection of anti-treponemal IgM (Mikrogen, recomWell Treponema IgM) or a positive test for Cardiolipin-KBR (Virion). Pharyngeal swabs taken by medical personnel and rectal swabs taken by the clients themselves were tested for gonorrhoea (Gc) and Chlamydia trachomatis (CT) with the BD ProbeTec™ ET System, which uses strand displacement amplification (SDA) for real-time detection of CT/GC.\n Statistical analysis The data were analysed using SPSS© (Version 20, IBM Corp.). We used descriptive statistics, calculated bivariate correlations for reporting UAI, being diagnosed with a rectal STI or syphilis, or being diagnosed with HIV, with odds ratios, 95% confidence intervals (CI) and p-values, and constructed three models for multivariate logistic regression analyses to identify factors associated with reporting UAI (principal mode of HIV transmission among MSM), diagnosis of a rectal STI or syphilis (both cofactors for acquiring HIV), and factors associated with diagnosis of HIV. Meeting places were used as binary variables (yes/no). Age and partner number were included as categorical variables (<30 years, 30–44, >44; 0–2 partners, 3–5, 6–10, >10) in all three models (for the age grouping see [3]). Additional factors were only included if they had been significant or borderline significant (p < 0.10) in bivariate analysis. The method used was a conditional forward inclusion (p < 0.05) and backward elimination (p > 0.10) procedure. Reasons for not using condoms during a recent risk situation and risk management strategies other than condom use were not included in the multivariate model for UAI, only in the models for STI and HIV diagnosis.\nThe data were analysed using SPSS© (Version 20, IBM Corp.). We used descriptive statistics, calculated bivariate correlations for reporting UAI, being diagnosed with a rectal STI or syphilis, or being diagnosed with HIV, with odds ratios, 95% confidence intervals (CI) and p-values, and constructed three models for multivariate logistic regression analyses to identify factors associated with reporting UAI (principal mode of HIV transmission among MSM), diagnosis of a rectal STI or syphilis (both cofactors for acquiring HIV), and factors associated with diagnosis of HIV. Meeting places were used as binary variables (yes/no). Age and partner number were included as categorical variables (<30 years, 30–44, >44; 0–2 partners, 3–5, 6–10, >10) in all three models (for the age grouping see [3]). Additional factors were only included if they had been significant or borderline significant (p < 0.10) in bivariate analysis. The method used was a conditional forward inclusion (p < 0.05) and backward elimination (p > 0.10) procedure. Reasons for not using condoms during a recent risk situation and risk management strategies other than condom use were not included in the multivariate model for UAI, only in the models for STI and HIV diagnosis.",
"Tests for HIV, HBV, HCV, syphilis, gonorrhoea and Chlamydia trachomatis were offered anonymously and free of charge. Decisions on which tests to take were based on pre-test needs counselling and the clients’ own choice. “Hein&Fiete” provides only testing and counselling and needs to refer clients with an infection diagnosis for further treatment to a private practice or hospital. Because most urethral infections with gonorrhoea and chlamydia in men are expected to be symptomatic, testing for urethral infections with these pathogens is not prioritized, while tests for the mostly asymptomatic pharyngeal and rectal infections are offered. Counsellors recommended pharyngeal and rectal swabs particularly to clients who reported higher numbers of sex partners.\nClients were invited to receive their test results two to five days after testing. In case of a positive test result, adequate therapy options were provided through referral to specialized MSM-friendly physicians.\nQuestionnaires and test results were entered in a Microsoft Access database by Hein&Fiete staff.\nData were fully anonymized and the anonymous code used at the CB-VCT was exchanged with another unrelated code. Therefore, ethical approval to conduct this analysis was waived.",
"Study population were all MSM clients attending the CB-VCT of “Hein&Fiete” who were tested for at least one STI (Chlamydia trachomatis, gonorrhoea, HIV, syphilis) and who completed the standardized questionnaire in 2011 and 2012. Men were defined as MSM if they reported at least one male sex partner in the previous 12 months.",
"Using a 25 item questionnaire, Hein&Fiete collected data on demographics (age, migration status, education), sexual orientation and partnerships, number and gender of sex partners in the previous 12 months, place where sex partners were met, use of drugs in combination with sex, testing history and test results for HIV and STIs, reasons for testing, condom use and reasons for not using condoms, and risk management strategies other than condom use.\nClients were tested for HIV with the Abbott Architect HIV Ag/Ab Combo assay, reactive samples were confirmed by Mikrogen, recomLine HIV-1 & HIV-2 IgG testing. For syphilis testing, samples were screened by Abbott Architect Syphilis TP Reagent, and confirmed by a quantitative TPPA test (MAST). Active infection was defined as either detection of anti-treponemal IgM (Mikrogen, recomWell Treponema IgM) or a positive test for Cardiolipin-KBR (Virion). Pharyngeal swabs taken by medical personnel and rectal swabs taken by the clients themselves were tested for gonorrhoea (Gc) and Chlamydia trachomatis (CT) with the BD ProbeTec™ ET System, which uses strand displacement amplification (SDA) for real-time detection of CT/GC.",
"The data were analysed using SPSS© (Version 20, IBM Corp.). We used descriptive statistics, calculated bivariate correlations for reporting UAI, being diagnosed with a rectal STI or syphilis, or being diagnosed with HIV, with odds ratios, 95% confidence intervals (CI) and p-values, and constructed three models for multivariate logistic regression analyses to identify factors associated with reporting UAI (principal mode of HIV transmission among MSM), diagnosis of a rectal STI or syphilis (both cofactors for acquiring HIV), and factors associated with diagnosis of HIV. Meeting places were used as binary variables (yes/no). Age and partner number were included as categorical variables (<30 years, 30–44, >44; 0–2 partners, 3–5, 6–10, >10) in all three models (for the age grouping see [3]). Additional factors were only included if they had been significant or borderline significant (p < 0.10) in bivariate analysis. The method used was a conditional forward inclusion (p < 0.05) and backward elimination (p > 0.10) procedure. Reasons for not using condoms during a recent risk situation and risk management strategies other than condom use were not included in the multivariate model for UAI, only in the models for STI and HIV diagnosis.",
"A total of 1630 persons filled in a questionnaire at Hein&Fiete in 2011 or 2012. Of these, 1565 men reported on the gender of their sexual partners. There were 1506 men identifiable as MSM. Since not all men filling out the questionnaire were actually tested - some clients were only counselled and some were recommended to get tested later because reported risks were too recent - the final sample for this analysis consisted of 1476 men reporting male partners and having been tested for at least one infection. HIV was tested for most frequently (1413), followed by syphilis (1380), pharyngeal (882) and rectal (642) infections with gonorrhoea and chlamydia. According to self-reported previous test date and place, up to 295 men were tested at least twice at Hein&Fiete during the two years. Numbers and proportions testing positive for the respective infections are presented in Table 1.Table 1\nHIV and STI test results of MSM clients, 2011-2012\n\nNumber of tests (any test N = 1476)#\n\nNumber (%) testing positive\n\nNumber tested at least twice in 2011-2012\n\nNumber (%) seroconverting/newly infected\nHIV141341 (2.9)486*16 (3.3)Syphilis (active)138027 (2.0)271°3 (1.1)Syphilis (antibodies)1380131 (9.5)271n.a.Gonorrhoea rectal64223 (3.6)154°8 (5.2)Gonorrhoea pharyngeal88248 (5.4)212°16 (7.5)Chlamydia rectal64256 (8.7)154°14 (9.1)Chlamydia pharyngeal88215 (1.7)212°5 (2.4)Any rectal STI64271 (11.1)Any rectal STI or active syphilis140395 (6.8)# discrepancies of numbers compared with Figure 1 due to missing age data for 40 clients.n.a. =not applicable, because treponemal antibodies often persist after treatment. Among the clients reporting testing at least twice 28 (10.3%) had detectable treponemal antibodies. Since the result of the previous tests is unknown, new or re-infection rates could not be determined.*= 295 had been tested at Hein&Fiete, 191 had been tested elsewhere first and the second time at Hein&Fiete.°= clients had been tested at Hein&Fiete at least twice in 2011–2012, however it is unknown whether they had been tested twice for this STI or at this location.\n\nHIV and STI test results of MSM clients, 2011-2012\n\n# discrepancies of numbers compared with Figure 1 due to missing age data for 40 clients.\nn.a. =not applicable, because treponemal antibodies often persist after treatment. Among the clients reporting testing at least twice 28 (10.3%) had detectable treponemal antibodies. Since the result of the previous tests is unknown, new or re-infection rates could not be determined.\n*= 295 had been tested at Hein&Fiete, 191 had been tested elsewhere first and the second time at Hein&Fiete.\n°= clients had been tested at Hein&Fiete at least twice in 2011–2012, however it is unknown whether they had been tested twice for this STI or at this location.\nThe clients having been tested more than once during the two-year observation period may impact the number and proportion of clients testing positive for treponemal antibodies. Based on antibody detection, the new or re-infection rate for syphilis between the repeated tests cannot be determined, and the same individuals may be counted twice or more.\nThe completeness of core variables in the MSM sample ranged from 82% (response to question on previous chlamydia testing) to 99.9% (sexual orientation). Information e.g. on risk behaviour (unprotected intercourse) and HIV test results were available for approximately 1300 clients, information on rectal STI and risk behaviour for approximately 600 clients.\nThe median age of the respondents was 36 years (interquartile range: 28–45 years). A migration background (parents or respondent himself immigrated to Germany) was reported by 25% of the respondents. A high school diploma or a higher degree of education was held by 69%. Twelve percent of the sample self-identified as bisexual, 87% as gay. Relationship status at the time of testing was reported as single by 53% of the respondents; another 22% reported being in an open and 25% reported being in a mutually exclusive relationship. A previous test for HIV was reported by 87% of the tested MSM, a previous syphilis test by 59%, 37% had been tested for gonorrhoea, and 31% for Chlamydia trachomatis (both not differentiated by location).\nOverall, MSM clients reported a median of 6 and a mean of 11 sexual partners (standard deviation 18) in the previous twelve months. The pharyngeal swab subsample reported a median of 7 and a mean of 13 partners (standard deviation 21), the rectal swab subsample a median of 8 and a mean of 14 partners (standard deviation 24), reflecting the preferential recommendation to take swabs for clients with higher partner numbers.\nTransmission risk behaviour, particularly in terms of HIV risks, was queried by a question asking for the reasons for testing. Unprotected anal intercourse (UAI) was reported by 61% of the clients as a reason for testing; 44% reported insertive UAI, and 35% reported receptive UAI. Unprotected oral intercourse with ejaculation into the mouth was reported as risk factor by 33% of MSM. Of note, a large proportion of clients who were diagnosed with a rectal STI did not report receptive UAI (27 out of 71 = 38%). The context in which risks were taken could be derived from questions on drug and alcohol use before or during sex (completed by 65%) and from a question on where most sex partners were met. Bivariate associations of these factors with UAI, STI and HIV diagnosis are shown in Table 2.Table 2\nResults of Bi- und Multivariate analysis of associations between age, partner numbers, meeting places with sex partners, substance use during sex, biological cofactors and risk behaviours with the outcomes UAI, rectal STI and HIV diagnosis\n\nBivariate\n\nMultivariate\n\nBivariate\n\nMultivariate\n\nBivariate\n\nMultivariate\n\nUAI\n\nSTI rectal or syphilis\n\nHIV\n\nOR\n\n95% CI\n\nOR\n\n95% CI\n\nOR\n\n95% CI\n\nOR\n\n95% CI\n\nOR\n\n95% CI\n\nOR\n\n95% CI\n\nAge group\n<301.54***1.18-1.961.67***1.26-2.231.78**1.12-2.861.060.49-2.3330-44 (ref.)111>44 years1.110.86-1.430.870.49-1.541.270.58-2.80\nPartner number\n0-2 (ref.)1113-51.150.80-1.642.710.76-9.661.410.48-4.146-101.180.83-1.706.10***1.82-20.416.13***1.79-20.90.820.25-2.72>101.59***1.12-2.255.06***1.51-16.915.27***1.55-18.00.910.30-2.83\nMeeting sex partners\nAt friends1.200.93-1.471.310.85-2.020.47*0.21-1.06In bars1.020.81-1.280.950.60-1.490.840.42-1.69In gay bathhouses1.050.83-1.321.250.80-1.951.080.55-2.15In porn cinemas1.070.81-1.421.110.65-1.891.83*0.90-3.71In outside cruising sites1.020.71-1.470.660.28-1.531.100.39-3.13On highway resting areas1.290.67-2.461.510.53-4.330.900.12-6.74Online1.49***1.20-1.841.29**1.01-1.651.45*0.93-2.260.810.44-1.52At parties or discotheques1.41***1.12-1.780.940.60-1.480.610.29-1.29In public urinals1.550.73-3.261.340.40-4.480.970.96-0.98In fitness centres1.120.75-1.690.680.27-1.710.310.04-2.26\nSubstance use\nAlcohol1.32***1.07-1.620.820.54-1.240.630.34-1.17Inhaled Amyl nitrite1.68***1.31-2.151.63***1.23-2.171.040.65-1.671.670.88-3.19Sildenafil1.53*0.96-2.431.220.55-2.721.200.36-3.95Cocaine, Speed1.65*0.93-2.912.36**1.09-5.151.210.28-5.12Cannabis1.50*0.97-2.301.230.58-2.621.040.32-3.42Ecstasy3.540.78-16.034.23*1.15-15.655.77**1.40-23.82.830.36-22.32GBL,GHB0.61**0.58-0.634.67*0.93-23.444.880.59-40.56\nCofactors, behaviours\nUnprotected anal intercoursen.a.2.39***1.46-3.932.42***1.29-4.534.52***1.76-11.593.24* °STI co-diagnosisn.a.n.a.3.02**1.22-7.464.52***1.68-12.1Having sex with partners who appear healthyn.a.3.09***1.94-4.922.92***1.68-5.071.270.56-2.91Asking the insertive partner not to ejaculate insiden.a.2.11**1.16-3.864.34***2.11-8.894.44***1.75-11.3Asking the partner about HIV statusn.a.1.71**1.03-2.852.03*1.00-4.11Partner reluctant to use condomsn.a.1.300.72-2.342.68**1.32-5.45No condom availablen.a.2.81**1.46-5.413.10**1.26-7.60*p > 0.05 < =0.10; **p < =0.05 > 0.01; ***p < =0.01.n.a. = not applicable.°Results of Multivariate logistic regression only reported if p < =0.05 (except for UAI as risk factor for HIV diagnosis, p = 0.07).\n\nResults of Bi- und Multivariate analysis of associations between age, partner numbers, meeting places with sex partners, substance use during sex, biological cofactors and risk behaviours with the outcomes UAI, rectal STI and HIV diagnosis\n\n*p > 0.05 < =0.10; **p < =0.05 > 0.01; ***p < =0.01.\nn.a. = not applicable.\n°Results of Multivariate logistic regression only reported if p < =0.05 (except for UAI as risk factor for HIV diagnosis, p = 0.07).\nAcute syphilis and HIV diagnoses were almost equally distributed across all age groups in the tested sample. MSM diagnosed with HIV were younger than MSM diagnosed with acute syphilis. Noticeably, while within age groups those men who were diagnosed with rectal or pharyngeal gonorrhoea or chlamydia reported higher numbers of partners, though the differences were statistically not significant, across the whole sample the median number of partners increased with age (from a median of 5 for age <30 years to a median of 10 for age 45–59), while the proportion of clients diagnosed with gonorrhoea and chlamydia decreased (Figure 1). Please note that numbers do not sum up to the numbers in Table 1 due to missing age data of 40 clients.Figure 1\nProportion of MSM testing positive for different STIs and HIV, by age groups.\n\n\nProportion of MSM testing positive for different STIs and HIV, by age groups.\n\nClients reporting a self-perceived risk situation as a reason for testing (compared to routine testing or testing when entering a new partnership) were significantly more likely to be diagnosed with HIV and STI (OR = 2.8; 95% CI 1.4-5.6). In bivariate analysis, reported reasons positively associated with risk situations were reluctance of the sex partner to use a condom and unavailability of condoms at a sexual encounter (see Table 2).\nAmong the risk management strategies besides condom use, asking the active partner to withdraw before ejaculation and asking the partner about his HIV status were positively associated with being diagnosed with a bacterial STI or with HIV (Table 2).\nReported UAI was strongly associated with an HIV diagnosis and less strongly associated with the diagnosis of a rectal STI or syphilis. The concomitant diagnosis of a rectal infection with gonorrhoea or chlamydia or a diagnosis of syphilis was strongly associated with an HIV diagnosis. For clients reporting an HIV positive partner the odds ratio for being diagnosed with HIV was 1.8 (95% CI 0.8-4.1; p = 0.175) compared to others, which did not reach statistical significance.\nIn multivariate logistic regression analyses including age and partner numbers (significantly associated with UAI and STI diagnoses in bivariate analysis) in addition to reported substance use and risk management strategies (the latter not included for UAI, but only for STI and HIV diagnosis outcomes) showing statistical significance in bivariate analysis, the following factors remained significantly associated (results see also Table 2)with UAI: age group, a high partner number, and use of inhaled amyl nitrite (poppers);with diagnosis of a rectal STI or syphilis: high partner numbers, reporting use of ecstasy when having sex, reporting UAI, and having sex only with partners who appear healthy;with HIV diagnosis: diagnosis of rectal gonorrhoea or chlamydia infection or diagnosis of syphilis; asking the active partner to withdraw before ejaculation. The odds ratio for being diagnosed with HIV when reporting UAI was 2.8, but this did not reach statistical significance (95% CI 0.8-10.1).\nwith UAI: age group, a high partner number, and use of inhaled amyl nitrite (poppers);\nwith diagnosis of a rectal STI or syphilis: high partner numbers, reporting use of ecstasy when having sex, reporting UAI, and having sex only with partners who appear healthy;\nwith HIV diagnosis: diagnosis of rectal gonorrhoea or chlamydia infection or diagnosis of syphilis; asking the active partner to withdraw before ejaculation. The odds ratio for being diagnosed with HIV when reporting UAI was 2.8, but this did not reach statistical significance (95% CI 0.8-10.1).",
"The data show that the CB-VCT site in Hamburg reached MSM at high risk for HIV and bacterial STI. Though a cost-effectiveness analysis was not conducted, considering the high prevalence and low overhead costs of the CB-VCT site, testing at the CB-VCT is likely a very cost-effective way of reducing the proportion of undiagnosed HIV and STI infections in this population compared to untargeted screening in medical facilities and also compared to untargeted VCT sites [4-9]. However, well-educated and self-identified gay men were reached disproportionally: a high school diploma or a higher degree of education was held by 69%, compared to 35% in the German general population and 60% in a recent MSM online survey (“Gay men and AIDS 2013”- GMA 2013, unpublished observations). Eighty seven percent self-identified as gay, while large population-based surveys suggest a proportion of non-gay-identified MSM which is clearly higher than 13% [10]. Thus, to reach a broader spectrum of MSM, the current CB-VCT approach may need to be combined with other approaches to reach MSM who are less well educated and less out about their sexual preferences than the clients reached by Hein&Fiete.\nMany CB-VCT sites in Europe so far only offer HIV testing, sometimes in combination with syphilis screening. The Hamburg experience, finding higher prevalence for rectal and pharyngeal infections with gonorrhoea or chlamydia than for syphilis or HIV (though in a more preselected subsample) convincingly argues for the combination of HIV and STI testing for MSM. Our data show that a more comprehensive offer to screen for all relevant bacterial STIs is warranted for this population. While testing costs have not been an issue so far in Hamburg where tests are covered by public funds, nucleic acid amplification tests for gonorrhoea and chlamydia may increase testing costs considerably, particularly when all three possibly affected sites (urethra, pharynx, and rectum) are screened. One option to minimize costs might be the use of pooled combination tests, i.e. pooling pharyngeal, rectal and urethral swabs or a urine specimen for analysis and using combination tests for gonorrhoea and chlamydia [11]. Selective offering of tests according to reported sexual risks could be another option. However, a large proportion of clients who were diagnosed with a rectal STI did not report receptive UAI. Reasons for this may be responses biased by social desirability to underreport UAI in general as well as a lack of questions that would cover all possible modes of transmission of bacterial STI to the rectal mucosa beyond insertive UAI.\nWe found an interesting inverse association between median numbers of partners and the proportion of rectal and pharyngeal infections with gonorrhoea and chlamydia by age group: although MSM from older age groups reported higher median numbers of partners, they were less likely to be diagnosed with rectal or pharyngeal infections. Similar observations have already been reported by other authors [12,13]. However, within the same age groups MSM who were diagnosed with rectal or pharyngeal infections reported higher median numbers of partner than MSM who were not diagnosed with an infection. This is reminiscent of the age-dependence of chlamydia diagnoses in women, which might be explained by an evolving immunity to this pathogen by repeated exposures over time [14]. Other reasons for this inverse association could be differences in condom use or partner selection for anal intercourse without a condom by age group. However, lack of condom use would not explain the differences for pharyngeal infections, since condoms are very rarely used for oral intercourse regardless of age group.\nThe associations we found between risk management strategies aiming at identifying partners infected and diagnosed with HIV and outcomes such as UAI, rectal STI or HIV diagnosis may reflect how widespread these risk management strategies are, that they are ineffective for avoiding other STI, and that even their utility to avoid HIV infection may be very limited. A high proportion of MSM diagnosed with HIV receives antiretroviral treatment and may thus be only minimally infectious, while most men who are still infectious are not yet diagnosed and thus not identifiable by respective questions. Our finding of a non-significant association of testing HIV positive when a partner is known to be infected with HIV is in contrast to a very high hazard ratio for testing HIV positive when reporting unprotected anal intercourse with a known HIV positive partner in the United States-based EXPLORE study conducted from 1999 through 2003 [15]. The main explanation for this discrepant finding may be the increased treatment rate among MSM in Germany compared to the situation in the United States ten years ago.\nContrastingly, men who have multiple partners and rely predominantly on HIV serosorting instead of condom use for protection against HIV transmission may be more likely to meet partners using the same risk management strategy. Once HIV is present in sexual networks using this risk management strategy, it may spread quite efficiently, particularly because other bacterial STIs accumulating in these networks are further enhancing HIV transmission. Alternatively or additionally, rectal STIs may just be a marker for risk behaviours or networks with increased HIV transmission risks. Other STIs, particularly rectal STIs, have been reported to be associated with an increased risk of acquiring HIV, e.g. by a group from New York in a longitudinal study design [16].\nThe association of STI diagnosis (rectal STI or syphilis) with the risk management strategy ‘having sex only with partners who appear healthy’ in multivariate regression analysis is a reminder that infections with bacterial STI are even less visible than HIV infection. Relying on visual appearance is therefore particularly ineffective for avoiding STI infections. The strong association between HIV diagnosis and the risk management strategy of asking the active partner to withdraw before ejaculation is difficult to interpret: the format of the question leaves open whether this strategy refers to oral or anal intercourse or both. Since a causal association with HIV transmission is conceivable only for anal intercourse, this association might be confounded. Asking for withdrawal could just be a surrogate for other unmeasured characteristics or risk management strategies of men at increased risk for HIV.\nAnother interesting finding is the subtle discrepancy between meeting places as risk factors for STI and for HIV diagnosis in the bivariate analysis, considering only the results which reached borderline statistical significance. While discrepancies in statistical significance regarding risk factors may just be due to the lower numbers of HIV compared to STI diagnoses, it could also be an indication of subtle differences regarding HIV and STI transmission risks in different settings. High STI transmission risks would be expected in settings with low condom use due to high probability of HIV serostatus communication (e.g. when meeting partners online and in private settings [17]. High HIV transmission risk was associated with meeting partners at porn cinemas, which in Hamburg is probably the most popular sex-focused gay venue with only minimal serostatus communication.\nThere are of course several limitations to this investigation: the questionnaire was primarily designed to facilitate clients’ sexual-health counselling needs, not to systematically detect specific risk factors. Associations we detected, particularly between risk management strategies and STI and HIV diagnosis, may therefore be confounded by other, unmeasured factors. It is likely that some questions were answered with regard to recent situations where respondents assumed risks; however this does not necessarily mean that HIV or STIs were transmitted during these encounters. The use of a combined outcome (rectal STI or syphilis), in which the number of men tested for rectal STI was less than half of the number of men tested for syphilis weakens associations, because many rectal STIs may have remained undiagnosed. Using only rectal STI as outcome changes some of the associations in bivariate analysis, but has no substantial impact on the variables remaining significant in multivariate analysis (data not shown). A social desirability bias, particularly regarding the reporting of unsafe sexual practices and drug use, should be expected, since the responses were known to be discussed with a sexual-health counsellor. That UAI was not significantly associated with HIV diagnosis in the multivariate regression model may be an indication of UAI underreporting. Lastly, with its focus on common sexual practices among MSM, the scope of sexual practices that could possibly result or contribute to the transmission of bacteria to the rectum or pharynx was not fully explored [18].",
"Our results emphasize the need to combine HIV and STI testing for MSM and the need for comprehensive screening for mostly asymptomatic rectal and pharyngeal infections [19]. Community-based VCTs can reach the MSM target group quite efficiently, and offering comprehensive HIV and STI testing in this setting is likely to be much more cost effective than relying on traditional health care settings. However, the CB-VCT approach for MSM is likely only feasible in larger cities with respective gay communities. Additional alternative approaches will be necessary to reach rural MSM, MSM less connected to the gay community, and MSM less willing to self-identify as gay. Behavioural data which may be useful to adapt and fine-tune prevention messages for MSM can also be collected in this setting. The data collected routinely before the counselling session appears quite useful for the analysis of risk behaviours and trends in risk management strategies, especially combined with testing data."
] |
[
null,
"methods",
null,
null,
null,
null,
"results",
"discussion",
"conclusions"
] |
[
"HIV infection",
"Men who have sex with men-MSM",
"Sexually transmitted infections",
"Community based voluntary counselling and testing"
] |
Background:
In recent years community-based voluntary counselling and testing sites (CB-VCT) [1] for HIV have been established for MSM in larger cities in Germany. One of these CB-VCTs is “Hein&Fiete” in the city of Hamburg. Hein&Fiete was founded in 1990 as an HIV prevention project for MSM. It includes a community drop-in centre with daily opening hours, providing information on gay life in Hamburg, meeting spaces for community groups, outreach prevention work for MSM, HIV and STI serological testing since 2004, and STI swab testing with nucleic acid amplification tests since 2011. The staff consists of approximately 85 volunteers and 5 full or part-time paid staff members. The work of Hein&Fiete, including free testing for HIV and STIs, is funded by the Federal state of Hamburg and by donations.
As low-threshold (anonymous, gay-friendly, low-cost) sexually transmitted infection (STI) screening opportunities for MSM are scarce in the German health care system [2], STI tests, particularly serological tests for syphilis and nucleic acid amplification tests for gonorrhoea and chlamydia infections, are increasingly offered by CB-VCTs. Since these tests can be provided free of charge, uptake of STI tests at Hein&Fiete is the highest among German CB-VCT. To facilitate the pre-test counselling session, clients of CB-VCT sites are invited to fill out an anonymous paper-based and standardized questionnaire on demographics, testing history, transmission risks, and risk management strategies. We analysed these data to identify current risk factors for a diagnosis of HIV and/or STI.
Methods:
Study procedures Tests for HIV, HBV, HCV, syphilis, gonorrhoea and Chlamydia trachomatis were offered anonymously and free of charge. Decisions on which tests to take were based on pre-test needs counselling and the clients’ own choice. “Hein&Fiete” provides only testing and counselling and needs to refer clients with an infection diagnosis for further treatment to a private practice or hospital. Because most urethral infections with gonorrhoea and chlamydia in men are expected to be symptomatic, testing for urethral infections with these pathogens is not prioritized, while tests for the mostly asymptomatic pharyngeal and rectal infections are offered. Counsellors recommended pharyngeal and rectal swabs particularly to clients who reported higher numbers of sex partners.
Clients were invited to receive their test results two to five days after testing. In case of a positive test result, adequate therapy options were provided through referral to specialized MSM-friendly physicians.
Questionnaires and test results were entered in a Microsoft Access database by Hein&Fiete staff.
Data were fully anonymized and the anonymous code used at the CB-VCT was exchanged with another unrelated code. Therefore, ethical approval to conduct this analysis was waived.
Tests for HIV, HBV, HCV, syphilis, gonorrhoea and Chlamydia trachomatis were offered anonymously and free of charge. Decisions on which tests to take were based on pre-test needs counselling and the clients’ own choice. “Hein&Fiete” provides only testing and counselling and needs to refer clients with an infection diagnosis for further treatment to a private practice or hospital. Because most urethral infections with gonorrhoea and chlamydia in men are expected to be symptomatic, testing for urethral infections with these pathogens is not prioritized, while tests for the mostly asymptomatic pharyngeal and rectal infections are offered. Counsellors recommended pharyngeal and rectal swabs particularly to clients who reported higher numbers of sex partners.
Clients were invited to receive their test results two to five days after testing. In case of a positive test result, adequate therapy options were provided through referral to specialized MSM-friendly physicians.
Questionnaires and test results were entered in a Microsoft Access database by Hein&Fiete staff.
Data were fully anonymized and the anonymous code used at the CB-VCT was exchanged with another unrelated code. Therefore, ethical approval to conduct this analysis was waived.
Study sample Study population were all MSM clients attending the CB-VCT of “Hein&Fiete” who were tested for at least one STI (Chlamydia trachomatis, gonorrhoea, HIV, syphilis) and who completed the standardized questionnaire in 2011 and 2012. Men were defined as MSM if they reported at least one male sex partner in the previous 12 months.
Study population were all MSM clients attending the CB-VCT of “Hein&Fiete” who were tested for at least one STI (Chlamydia trachomatis, gonorrhoea, HIV, syphilis) and who completed the standardized questionnaire in 2011 and 2012. Men were defined as MSM if they reported at least one male sex partner in the previous 12 months.
Measurements Using a 25 item questionnaire, Hein&Fiete collected data on demographics (age, migration status, education), sexual orientation and partnerships, number and gender of sex partners in the previous 12 months, place where sex partners were met, use of drugs in combination with sex, testing history and test results for HIV and STIs, reasons for testing, condom use and reasons for not using condoms, and risk management strategies other than condom use.
Clients were tested for HIV with the Abbott Architect HIV Ag/Ab Combo assay, reactive samples were confirmed by Mikrogen, recomLine HIV-1 & HIV-2 IgG testing. For syphilis testing, samples were screened by Abbott Architect Syphilis TP Reagent, and confirmed by a quantitative TPPA test (MAST). Active infection was defined as either detection of anti-treponemal IgM (Mikrogen, recomWell Treponema IgM) or a positive test for Cardiolipin-KBR (Virion). Pharyngeal swabs taken by medical personnel and rectal swabs taken by the clients themselves were tested for gonorrhoea (Gc) and Chlamydia trachomatis (CT) with the BD ProbeTec™ ET System, which uses strand displacement amplification (SDA) for real-time detection of CT/GC.
Using a 25 item questionnaire, Hein&Fiete collected data on demographics (age, migration status, education), sexual orientation and partnerships, number and gender of sex partners in the previous 12 months, place where sex partners were met, use of drugs in combination with sex, testing history and test results for HIV and STIs, reasons for testing, condom use and reasons for not using condoms, and risk management strategies other than condom use.
Clients were tested for HIV with the Abbott Architect HIV Ag/Ab Combo assay, reactive samples were confirmed by Mikrogen, recomLine HIV-1 & HIV-2 IgG testing. For syphilis testing, samples were screened by Abbott Architect Syphilis TP Reagent, and confirmed by a quantitative TPPA test (MAST). Active infection was defined as either detection of anti-treponemal IgM (Mikrogen, recomWell Treponema IgM) or a positive test for Cardiolipin-KBR (Virion). Pharyngeal swabs taken by medical personnel and rectal swabs taken by the clients themselves were tested for gonorrhoea (Gc) and Chlamydia trachomatis (CT) with the BD ProbeTec™ ET System, which uses strand displacement amplification (SDA) for real-time detection of CT/GC.
Statistical analysis The data were analysed using SPSS© (Version 20, IBM Corp.). We used descriptive statistics, calculated bivariate correlations for reporting UAI, being diagnosed with a rectal STI or syphilis, or being diagnosed with HIV, with odds ratios, 95% confidence intervals (CI) and p-values, and constructed three models for multivariate logistic regression analyses to identify factors associated with reporting UAI (principal mode of HIV transmission among MSM), diagnosis of a rectal STI or syphilis (both cofactors for acquiring HIV), and factors associated with diagnosis of HIV. Meeting places were used as binary variables (yes/no). Age and partner number were included as categorical variables (<30 years, 30–44, >44; 0–2 partners, 3–5, 6–10, >10) in all three models (for the age grouping see [3]). Additional factors were only included if they had been significant or borderline significant (p < 0.10) in bivariate analysis. The method used was a conditional forward inclusion (p < 0.05) and backward elimination (p > 0.10) procedure. Reasons for not using condoms during a recent risk situation and risk management strategies other than condom use were not included in the multivariate model for UAI, only in the models for STI and HIV diagnosis.
The data were analysed using SPSS© (Version 20, IBM Corp.). We used descriptive statistics, calculated bivariate correlations for reporting UAI, being diagnosed with a rectal STI or syphilis, or being diagnosed with HIV, with odds ratios, 95% confidence intervals (CI) and p-values, and constructed three models for multivariate logistic regression analyses to identify factors associated with reporting UAI (principal mode of HIV transmission among MSM), diagnosis of a rectal STI or syphilis (both cofactors for acquiring HIV), and factors associated with diagnosis of HIV. Meeting places were used as binary variables (yes/no). Age and partner number were included as categorical variables (<30 years, 30–44, >44; 0–2 partners, 3–5, 6–10, >10) in all three models (for the age grouping see [3]). Additional factors were only included if they had been significant or borderline significant (p < 0.10) in bivariate analysis. The method used was a conditional forward inclusion (p < 0.05) and backward elimination (p > 0.10) procedure. Reasons for not using condoms during a recent risk situation and risk management strategies other than condom use were not included in the multivariate model for UAI, only in the models for STI and HIV diagnosis.
Study procedures:
Tests for HIV, HBV, HCV, syphilis, gonorrhoea and Chlamydia trachomatis were offered anonymously and free of charge. Decisions on which tests to take were based on pre-test needs counselling and the clients’ own choice. “Hein&Fiete” provides only testing and counselling and needs to refer clients with an infection diagnosis for further treatment to a private practice or hospital. Because most urethral infections with gonorrhoea and chlamydia in men are expected to be symptomatic, testing for urethral infections with these pathogens is not prioritized, while tests for the mostly asymptomatic pharyngeal and rectal infections are offered. Counsellors recommended pharyngeal and rectal swabs particularly to clients who reported higher numbers of sex partners.
Clients were invited to receive their test results two to five days after testing. In case of a positive test result, adequate therapy options were provided through referral to specialized MSM-friendly physicians.
Questionnaires and test results were entered in a Microsoft Access database by Hein&Fiete staff.
Data were fully anonymized and the anonymous code used at the CB-VCT was exchanged with another unrelated code. Therefore, ethical approval to conduct this analysis was waived.
Study sample:
Study population were all MSM clients attending the CB-VCT of “Hein&Fiete” who were tested for at least one STI (Chlamydia trachomatis, gonorrhoea, HIV, syphilis) and who completed the standardized questionnaire in 2011 and 2012. Men were defined as MSM if they reported at least one male sex partner in the previous 12 months.
Measurements:
Using a 25 item questionnaire, Hein&Fiete collected data on demographics (age, migration status, education), sexual orientation and partnerships, number and gender of sex partners in the previous 12 months, place where sex partners were met, use of drugs in combination with sex, testing history and test results for HIV and STIs, reasons for testing, condom use and reasons for not using condoms, and risk management strategies other than condom use.
Clients were tested for HIV with the Abbott Architect HIV Ag/Ab Combo assay, reactive samples were confirmed by Mikrogen, recomLine HIV-1 & HIV-2 IgG testing. For syphilis testing, samples were screened by Abbott Architect Syphilis TP Reagent, and confirmed by a quantitative TPPA test (MAST). Active infection was defined as either detection of anti-treponemal IgM (Mikrogen, recomWell Treponema IgM) or a positive test for Cardiolipin-KBR (Virion). Pharyngeal swabs taken by medical personnel and rectal swabs taken by the clients themselves were tested for gonorrhoea (Gc) and Chlamydia trachomatis (CT) with the BD ProbeTec™ ET System, which uses strand displacement amplification (SDA) for real-time detection of CT/GC.
Statistical analysis:
The data were analysed using SPSS© (Version 20, IBM Corp.). We used descriptive statistics, calculated bivariate correlations for reporting UAI, being diagnosed with a rectal STI or syphilis, or being diagnosed with HIV, with odds ratios, 95% confidence intervals (CI) and p-values, and constructed three models for multivariate logistic regression analyses to identify factors associated with reporting UAI (principal mode of HIV transmission among MSM), diagnosis of a rectal STI or syphilis (both cofactors for acquiring HIV), and factors associated with diagnosis of HIV. Meeting places were used as binary variables (yes/no). Age and partner number were included as categorical variables (<30 years, 30–44, >44; 0–2 partners, 3–5, 6–10, >10) in all three models (for the age grouping see [3]). Additional factors were only included if they had been significant or borderline significant (p < 0.10) in bivariate analysis. The method used was a conditional forward inclusion (p < 0.05) and backward elimination (p > 0.10) procedure. Reasons for not using condoms during a recent risk situation and risk management strategies other than condom use were not included in the multivariate model for UAI, only in the models for STI and HIV diagnosis.
Results:
A total of 1630 persons filled in a questionnaire at Hein&Fiete in 2011 or 2012. Of these, 1565 men reported on the gender of their sexual partners. There were 1506 men identifiable as MSM. Since not all men filling out the questionnaire were actually tested - some clients were only counselled and some were recommended to get tested later because reported risks were too recent - the final sample for this analysis consisted of 1476 men reporting male partners and having been tested for at least one infection. HIV was tested for most frequently (1413), followed by syphilis (1380), pharyngeal (882) and rectal (642) infections with gonorrhoea and chlamydia. According to self-reported previous test date and place, up to 295 men were tested at least twice at Hein&Fiete during the two years. Numbers and proportions testing positive for the respective infections are presented in Table 1.Table 1
HIV and STI test results of MSM clients, 2011-2012
Number of tests (any test N = 1476)#
Number (%) testing positive
Number tested at least twice in 2011-2012
Number (%) seroconverting/newly infected
HIV141341 (2.9)486*16 (3.3)Syphilis (active)138027 (2.0)271°3 (1.1)Syphilis (antibodies)1380131 (9.5)271n.a.Gonorrhoea rectal64223 (3.6)154°8 (5.2)Gonorrhoea pharyngeal88248 (5.4)212°16 (7.5)Chlamydia rectal64256 (8.7)154°14 (9.1)Chlamydia pharyngeal88215 (1.7)212°5 (2.4)Any rectal STI64271 (11.1)Any rectal STI or active syphilis140395 (6.8)# discrepancies of numbers compared with Figure 1 due to missing age data for 40 clients.n.a. =not applicable, because treponemal antibodies often persist after treatment. Among the clients reporting testing at least twice 28 (10.3%) had detectable treponemal antibodies. Since the result of the previous tests is unknown, new or re-infection rates could not be determined.*= 295 had been tested at Hein&Fiete, 191 had been tested elsewhere first and the second time at Hein&Fiete.°= clients had been tested at Hein&Fiete at least twice in 2011–2012, however it is unknown whether they had been tested twice for this STI or at this location.
HIV and STI test results of MSM clients, 2011-2012
# discrepancies of numbers compared with Figure 1 due to missing age data for 40 clients.
n.a. =not applicable, because treponemal antibodies often persist after treatment. Among the clients reporting testing at least twice 28 (10.3%) had detectable treponemal antibodies. Since the result of the previous tests is unknown, new or re-infection rates could not be determined.
*= 295 had been tested at Hein&Fiete, 191 had been tested elsewhere first and the second time at Hein&Fiete.
°= clients had been tested at Hein&Fiete at least twice in 2011–2012, however it is unknown whether they had been tested twice for this STI or at this location.
The clients having been tested more than once during the two-year observation period may impact the number and proportion of clients testing positive for treponemal antibodies. Based on antibody detection, the new or re-infection rate for syphilis between the repeated tests cannot be determined, and the same individuals may be counted twice or more.
The completeness of core variables in the MSM sample ranged from 82% (response to question on previous chlamydia testing) to 99.9% (sexual orientation). Information e.g. on risk behaviour (unprotected intercourse) and HIV test results were available for approximately 1300 clients, information on rectal STI and risk behaviour for approximately 600 clients.
The median age of the respondents was 36 years (interquartile range: 28–45 years). A migration background (parents or respondent himself immigrated to Germany) was reported by 25% of the respondents. A high school diploma or a higher degree of education was held by 69%. Twelve percent of the sample self-identified as bisexual, 87% as gay. Relationship status at the time of testing was reported as single by 53% of the respondents; another 22% reported being in an open and 25% reported being in a mutually exclusive relationship. A previous test for HIV was reported by 87% of the tested MSM, a previous syphilis test by 59%, 37% had been tested for gonorrhoea, and 31% for Chlamydia trachomatis (both not differentiated by location).
Overall, MSM clients reported a median of 6 and a mean of 11 sexual partners (standard deviation 18) in the previous twelve months. The pharyngeal swab subsample reported a median of 7 and a mean of 13 partners (standard deviation 21), the rectal swab subsample a median of 8 and a mean of 14 partners (standard deviation 24), reflecting the preferential recommendation to take swabs for clients with higher partner numbers.
Transmission risk behaviour, particularly in terms of HIV risks, was queried by a question asking for the reasons for testing. Unprotected anal intercourse (UAI) was reported by 61% of the clients as a reason for testing; 44% reported insertive UAI, and 35% reported receptive UAI. Unprotected oral intercourse with ejaculation into the mouth was reported as risk factor by 33% of MSM. Of note, a large proportion of clients who were diagnosed with a rectal STI did not report receptive UAI (27 out of 71 = 38%). The context in which risks were taken could be derived from questions on drug and alcohol use before or during sex (completed by 65%) and from a question on where most sex partners were met. Bivariate associations of these factors with UAI, STI and HIV diagnosis are shown in Table 2.Table 2
Results of Bi- und Multivariate analysis of associations between age, partner numbers, meeting places with sex partners, substance use during sex, biological cofactors and risk behaviours with the outcomes UAI, rectal STI and HIV diagnosis
Bivariate
Multivariate
Bivariate
Multivariate
Bivariate
Multivariate
UAI
STI rectal or syphilis
HIV
OR
95% CI
OR
95% CI
OR
95% CI
OR
95% CI
OR
95% CI
OR
95% CI
Age group
<301.54***1.18-1.961.67***1.26-2.231.78**1.12-2.861.060.49-2.3330-44 (ref.)111>44 years1.110.86-1.430.870.49-1.541.270.58-2.80
Partner number
0-2 (ref.)1113-51.150.80-1.642.710.76-9.661.410.48-4.146-101.180.83-1.706.10***1.82-20.416.13***1.79-20.90.820.25-2.72>101.59***1.12-2.255.06***1.51-16.915.27***1.55-18.00.910.30-2.83
Meeting sex partners
At friends1.200.93-1.471.310.85-2.020.47*0.21-1.06In bars1.020.81-1.280.950.60-1.490.840.42-1.69In gay bathhouses1.050.83-1.321.250.80-1.951.080.55-2.15In porn cinemas1.070.81-1.421.110.65-1.891.83*0.90-3.71In outside cruising sites1.020.71-1.470.660.28-1.531.100.39-3.13On highway resting areas1.290.67-2.461.510.53-4.330.900.12-6.74Online1.49***1.20-1.841.29**1.01-1.651.45*0.93-2.260.810.44-1.52At parties or discotheques1.41***1.12-1.780.940.60-1.480.610.29-1.29In public urinals1.550.73-3.261.340.40-4.480.970.96-0.98In fitness centres1.120.75-1.690.680.27-1.710.310.04-2.26
Substance use
Alcohol1.32***1.07-1.620.820.54-1.240.630.34-1.17Inhaled Amyl nitrite1.68***1.31-2.151.63***1.23-2.171.040.65-1.671.670.88-3.19Sildenafil1.53*0.96-2.431.220.55-2.721.200.36-3.95Cocaine, Speed1.65*0.93-2.912.36**1.09-5.151.210.28-5.12Cannabis1.50*0.97-2.301.230.58-2.621.040.32-3.42Ecstasy3.540.78-16.034.23*1.15-15.655.77**1.40-23.82.830.36-22.32GBL,GHB0.61**0.58-0.634.67*0.93-23.444.880.59-40.56
Cofactors, behaviours
Unprotected anal intercoursen.a.2.39***1.46-3.932.42***1.29-4.534.52***1.76-11.593.24* °STI co-diagnosisn.a.n.a.3.02**1.22-7.464.52***1.68-12.1Having sex with partners who appear healthyn.a.3.09***1.94-4.922.92***1.68-5.071.270.56-2.91Asking the insertive partner not to ejaculate insiden.a.2.11**1.16-3.864.34***2.11-8.894.44***1.75-11.3Asking the partner about HIV statusn.a.1.71**1.03-2.852.03*1.00-4.11Partner reluctant to use condomsn.a.1.300.72-2.342.68**1.32-5.45No condom availablen.a.2.81**1.46-5.413.10**1.26-7.60*p > 0.05 < =0.10; **p < =0.05 > 0.01; ***p < =0.01.n.a. = not applicable.°Results of Multivariate logistic regression only reported if p < =0.05 (except for UAI as risk factor for HIV diagnosis, p = 0.07).
Results of Bi- und Multivariate analysis of associations between age, partner numbers, meeting places with sex partners, substance use during sex, biological cofactors and risk behaviours with the outcomes UAI, rectal STI and HIV diagnosis
*p > 0.05 < =0.10; **p < =0.05 > 0.01; ***p < =0.01.
n.a. = not applicable.
°Results of Multivariate logistic regression only reported if p < =0.05 (except for UAI as risk factor for HIV diagnosis, p = 0.07).
Acute syphilis and HIV diagnoses were almost equally distributed across all age groups in the tested sample. MSM diagnosed with HIV were younger than MSM diagnosed with acute syphilis. Noticeably, while within age groups those men who were diagnosed with rectal or pharyngeal gonorrhoea or chlamydia reported higher numbers of partners, though the differences were statistically not significant, across the whole sample the median number of partners increased with age (from a median of 5 for age <30 years to a median of 10 for age 45–59), while the proportion of clients diagnosed with gonorrhoea and chlamydia decreased (Figure 1). Please note that numbers do not sum up to the numbers in Table 1 due to missing age data of 40 clients.Figure 1
Proportion of MSM testing positive for different STIs and HIV, by age groups.
Proportion of MSM testing positive for different STIs and HIV, by age groups.
Clients reporting a self-perceived risk situation as a reason for testing (compared to routine testing or testing when entering a new partnership) were significantly more likely to be diagnosed with HIV and STI (OR = 2.8; 95% CI 1.4-5.6). In bivariate analysis, reported reasons positively associated with risk situations were reluctance of the sex partner to use a condom and unavailability of condoms at a sexual encounter (see Table 2).
Among the risk management strategies besides condom use, asking the active partner to withdraw before ejaculation and asking the partner about his HIV status were positively associated with being diagnosed with a bacterial STI or with HIV (Table 2).
Reported UAI was strongly associated with an HIV diagnosis and less strongly associated with the diagnosis of a rectal STI or syphilis. The concomitant diagnosis of a rectal infection with gonorrhoea or chlamydia or a diagnosis of syphilis was strongly associated with an HIV diagnosis. For clients reporting an HIV positive partner the odds ratio for being diagnosed with HIV was 1.8 (95% CI 0.8-4.1; p = 0.175) compared to others, which did not reach statistical significance.
In multivariate logistic regression analyses including age and partner numbers (significantly associated with UAI and STI diagnoses in bivariate analysis) in addition to reported substance use and risk management strategies (the latter not included for UAI, but only for STI and HIV diagnosis outcomes) showing statistical significance in bivariate analysis, the following factors remained significantly associated (results see also Table 2)with UAI: age group, a high partner number, and use of inhaled amyl nitrite (poppers);with diagnosis of a rectal STI or syphilis: high partner numbers, reporting use of ecstasy when having sex, reporting UAI, and having sex only with partners who appear healthy;with HIV diagnosis: diagnosis of rectal gonorrhoea or chlamydia infection or diagnosis of syphilis; asking the active partner to withdraw before ejaculation. The odds ratio for being diagnosed with HIV when reporting UAI was 2.8, but this did not reach statistical significance (95% CI 0.8-10.1).
with UAI: age group, a high partner number, and use of inhaled amyl nitrite (poppers);
with diagnosis of a rectal STI or syphilis: high partner numbers, reporting use of ecstasy when having sex, reporting UAI, and having sex only with partners who appear healthy;
with HIV diagnosis: diagnosis of rectal gonorrhoea or chlamydia infection or diagnosis of syphilis; asking the active partner to withdraw before ejaculation. The odds ratio for being diagnosed with HIV when reporting UAI was 2.8, but this did not reach statistical significance (95% CI 0.8-10.1).
Discussion:
The data show that the CB-VCT site in Hamburg reached MSM at high risk for HIV and bacterial STI. Though a cost-effectiveness analysis was not conducted, considering the high prevalence and low overhead costs of the CB-VCT site, testing at the CB-VCT is likely a very cost-effective way of reducing the proportion of undiagnosed HIV and STI infections in this population compared to untargeted screening in medical facilities and also compared to untargeted VCT sites [4-9]. However, well-educated and self-identified gay men were reached disproportionally: a high school diploma or a higher degree of education was held by 69%, compared to 35% in the German general population and 60% in a recent MSM online survey (“Gay men and AIDS 2013”- GMA 2013, unpublished observations). Eighty seven percent self-identified as gay, while large population-based surveys suggest a proportion of non-gay-identified MSM which is clearly higher than 13% [10]. Thus, to reach a broader spectrum of MSM, the current CB-VCT approach may need to be combined with other approaches to reach MSM who are less well educated and less out about their sexual preferences than the clients reached by Hein&Fiete.
Many CB-VCT sites in Europe so far only offer HIV testing, sometimes in combination with syphilis screening. The Hamburg experience, finding higher prevalence for rectal and pharyngeal infections with gonorrhoea or chlamydia than for syphilis or HIV (though in a more preselected subsample) convincingly argues for the combination of HIV and STI testing for MSM. Our data show that a more comprehensive offer to screen for all relevant bacterial STIs is warranted for this population. While testing costs have not been an issue so far in Hamburg where tests are covered by public funds, nucleic acid amplification tests for gonorrhoea and chlamydia may increase testing costs considerably, particularly when all three possibly affected sites (urethra, pharynx, and rectum) are screened. One option to minimize costs might be the use of pooled combination tests, i.e. pooling pharyngeal, rectal and urethral swabs or a urine specimen for analysis and using combination tests for gonorrhoea and chlamydia [11]. Selective offering of tests according to reported sexual risks could be another option. However, a large proportion of clients who were diagnosed with a rectal STI did not report receptive UAI. Reasons for this may be responses biased by social desirability to underreport UAI in general as well as a lack of questions that would cover all possible modes of transmission of bacterial STI to the rectal mucosa beyond insertive UAI.
We found an interesting inverse association between median numbers of partners and the proportion of rectal and pharyngeal infections with gonorrhoea and chlamydia by age group: although MSM from older age groups reported higher median numbers of partners, they were less likely to be diagnosed with rectal or pharyngeal infections. Similar observations have already been reported by other authors [12,13]. However, within the same age groups MSM who were diagnosed with rectal or pharyngeal infections reported higher median numbers of partner than MSM who were not diagnosed with an infection. This is reminiscent of the age-dependence of chlamydia diagnoses in women, which might be explained by an evolving immunity to this pathogen by repeated exposures over time [14]. Other reasons for this inverse association could be differences in condom use or partner selection for anal intercourse without a condom by age group. However, lack of condom use would not explain the differences for pharyngeal infections, since condoms are very rarely used for oral intercourse regardless of age group.
The associations we found between risk management strategies aiming at identifying partners infected and diagnosed with HIV and outcomes such as UAI, rectal STI or HIV diagnosis may reflect how widespread these risk management strategies are, that they are ineffective for avoiding other STI, and that even their utility to avoid HIV infection may be very limited. A high proportion of MSM diagnosed with HIV receives antiretroviral treatment and may thus be only minimally infectious, while most men who are still infectious are not yet diagnosed and thus not identifiable by respective questions. Our finding of a non-significant association of testing HIV positive when a partner is known to be infected with HIV is in contrast to a very high hazard ratio for testing HIV positive when reporting unprotected anal intercourse with a known HIV positive partner in the United States-based EXPLORE study conducted from 1999 through 2003 [15]. The main explanation for this discrepant finding may be the increased treatment rate among MSM in Germany compared to the situation in the United States ten years ago.
Contrastingly, men who have multiple partners and rely predominantly on HIV serosorting instead of condom use for protection against HIV transmission may be more likely to meet partners using the same risk management strategy. Once HIV is present in sexual networks using this risk management strategy, it may spread quite efficiently, particularly because other bacterial STIs accumulating in these networks are further enhancing HIV transmission. Alternatively or additionally, rectal STIs may just be a marker for risk behaviours or networks with increased HIV transmission risks. Other STIs, particularly rectal STIs, have been reported to be associated with an increased risk of acquiring HIV, e.g. by a group from New York in a longitudinal study design [16].
The association of STI diagnosis (rectal STI or syphilis) with the risk management strategy ‘having sex only with partners who appear healthy’ in multivariate regression analysis is a reminder that infections with bacterial STI are even less visible than HIV infection. Relying on visual appearance is therefore particularly ineffective for avoiding STI infections. The strong association between HIV diagnosis and the risk management strategy of asking the active partner to withdraw before ejaculation is difficult to interpret: the format of the question leaves open whether this strategy refers to oral or anal intercourse or both. Since a causal association with HIV transmission is conceivable only for anal intercourse, this association might be confounded. Asking for withdrawal could just be a surrogate for other unmeasured characteristics or risk management strategies of men at increased risk for HIV.
Another interesting finding is the subtle discrepancy between meeting places as risk factors for STI and for HIV diagnosis in the bivariate analysis, considering only the results which reached borderline statistical significance. While discrepancies in statistical significance regarding risk factors may just be due to the lower numbers of HIV compared to STI diagnoses, it could also be an indication of subtle differences regarding HIV and STI transmission risks in different settings. High STI transmission risks would be expected in settings with low condom use due to high probability of HIV serostatus communication (e.g. when meeting partners online and in private settings [17]. High HIV transmission risk was associated with meeting partners at porn cinemas, which in Hamburg is probably the most popular sex-focused gay venue with only minimal serostatus communication.
There are of course several limitations to this investigation: the questionnaire was primarily designed to facilitate clients’ sexual-health counselling needs, not to systematically detect specific risk factors. Associations we detected, particularly between risk management strategies and STI and HIV diagnosis, may therefore be confounded by other, unmeasured factors. It is likely that some questions were answered with regard to recent situations where respondents assumed risks; however this does not necessarily mean that HIV or STIs were transmitted during these encounters. The use of a combined outcome (rectal STI or syphilis), in which the number of men tested for rectal STI was less than half of the number of men tested for syphilis weakens associations, because many rectal STIs may have remained undiagnosed. Using only rectal STI as outcome changes some of the associations in bivariate analysis, but has no substantial impact on the variables remaining significant in multivariate analysis (data not shown). A social desirability bias, particularly regarding the reporting of unsafe sexual practices and drug use, should be expected, since the responses were known to be discussed with a sexual-health counsellor. That UAI was not significantly associated with HIV diagnosis in the multivariate regression model may be an indication of UAI underreporting. Lastly, with its focus on common sexual practices among MSM, the scope of sexual practices that could possibly result or contribute to the transmission of bacteria to the rectum or pharynx was not fully explored [18].
Conclusions:
Our results emphasize the need to combine HIV and STI testing for MSM and the need for comprehensive screening for mostly asymptomatic rectal and pharyngeal infections [19]. Community-based VCTs can reach the MSM target group quite efficiently, and offering comprehensive HIV and STI testing in this setting is likely to be much more cost effective than relying on traditional health care settings. However, the CB-VCT approach for MSM is likely only feasible in larger cities with respective gay communities. Additional alternative approaches will be necessary to reach rural MSM, MSM less connected to the gay community, and MSM less willing to self-identify as gay. Behavioural data which may be useful to adapt and fine-tune prevention messages for MSM can also be collected in this setting. The data collected routinely before the counselling session appears quite useful for the analysis of risk behaviours and trends in risk management strategies, especially combined with testing data.
|
Background:
In recent years community-based voluntary counselling and testing sites (CB-VCT) for men having sex with men (MSM) have been established in larger cities in Germany to offer more opportunities for HIV testing. Increasingly, CB-VCTs also offer testing for other bacterial sexually transmitted infections. In Hamburg, tests in CB-VCTs are offered free and anonymously. Data on demographics and sexual risk behaviours are collected with a paper questionnaire.
Methods:
Questionnaire data from the MSM CB-VCT in Hamburg were linked with serological test results for HIV and syphilis, and with rectal and pharyngeal swab results for gonorrhoea and chlamydia. MSM were defined as males reporting male sex partners. CB-VCT clients were characterized demographically, and associations between sexual behaviour variables and diagnosis of HIV and sexually transmitted infections (STI) were analysed by bivariate and multivariate logistic regression analysis.
Results:
Among the male clients of the CB-VCT in 2011-2012 who were tested for HIV or any STI 1476 reported male sex partners. Unprotected anal intercourse (UAI) was reported as reason for testing by 61% of the clients. Forty-one of 1413 clients testing for HIV were tested positive (2.9%). Twenty-four of 1380 clients testing for syphilis required treatment (1.7%). Tests for simultaneous detection of N. gonorrhoea and Chlamydia trachomatis were conducted on 882 pharyngeal and 642 rectal swabs, revealing 58 (=6.6%) pharyngeal and 71 (=11.1%) rectal infections with one or both pathogens. In multivariate logistic regression analysis number of partners, UAI (OR=2.42) and relying on visual impression when selecting sex partners (OR = 2.92) were associated with increased risks for diagnosis of syphilis or a rectal STI. Syphilis or rectal STI diagnosis (OR=4.52) were associated with increased risk for HIV diagnosis.
Conclusions:
The MSM CB-VCT in Hamburg reaches clients at high risk for HIV and STIs. The diagnosis of syphilis or a rectal STI was associated with increased odds of testing positive for HIV. Due to the high prevalence of curable bacterial STI among clients and because syphilis and rectal bacterial STI may facilitate HIV transmission, MSM asking for HIV tests in CB-VCTs should also be offered tests for other bacterial STIs.
|
Background:
In recent years community-based voluntary counselling and testing sites (CB-VCT) [1] for HIV have been established for MSM in larger cities in Germany. One of these CB-VCTs is “Hein&Fiete” in the city of Hamburg. Hein&Fiete was founded in 1990 as an HIV prevention project for MSM. It includes a community drop-in centre with daily opening hours, providing information on gay life in Hamburg, meeting spaces for community groups, outreach prevention work for MSM, HIV and STI serological testing since 2004, and STI swab testing with nucleic acid amplification tests since 2011. The staff consists of approximately 85 volunteers and 5 full or part-time paid staff members. The work of Hein&Fiete, including free testing for HIV and STIs, is funded by the Federal state of Hamburg and by donations.
As low-threshold (anonymous, gay-friendly, low-cost) sexually transmitted infection (STI) screening opportunities for MSM are scarce in the German health care system [2], STI tests, particularly serological tests for syphilis and nucleic acid amplification tests for gonorrhoea and chlamydia infections, are increasingly offered by CB-VCTs. Since these tests can be provided free of charge, uptake of STI tests at Hein&Fiete is the highest among German CB-VCT. To facilitate the pre-test counselling session, clients of CB-VCT sites are invited to fill out an anonymous paper-based and standardized questionnaire on demographics, testing history, transmission risks, and risk management strategies. We analysed these data to identify current risk factors for a diagnosis of HIV and/or STI.
Conclusions:
Our results emphasize the need to combine HIV and STI testing for MSM and the need for comprehensive screening for mostly asymptomatic rectal and pharyngeal infections [19]. Community-based VCTs can reach the MSM target group quite efficiently, and offering comprehensive HIV and STI testing in this setting is likely to be much more cost effective than relying on traditional health care settings. However, the CB-VCT approach for MSM is likely only feasible in larger cities with respective gay communities. Additional alternative approaches will be necessary to reach rural MSM, MSM less connected to the gay community, and MSM less willing to self-identify as gay. Behavioural data which may be useful to adapt and fine-tune prevention messages for MSM can also be collected in this setting. The data collected routinely before the counselling session appears quite useful for the analysis of risk behaviours and trends in risk management strategies, especially combined with testing data.
|
Background:
In recent years community-based voluntary counselling and testing sites (CB-VCT) for men having sex with men (MSM) have been established in larger cities in Germany to offer more opportunities for HIV testing. Increasingly, CB-VCTs also offer testing for other bacterial sexually transmitted infections. In Hamburg, tests in CB-VCTs are offered free and anonymously. Data on demographics and sexual risk behaviours are collected with a paper questionnaire.
Methods:
Questionnaire data from the MSM CB-VCT in Hamburg were linked with serological test results for HIV and syphilis, and with rectal and pharyngeal swab results for gonorrhoea and chlamydia. MSM were defined as males reporting male sex partners. CB-VCT clients were characterized demographically, and associations between sexual behaviour variables and diagnosis of HIV and sexually transmitted infections (STI) were analysed by bivariate and multivariate logistic regression analysis.
Results:
Among the male clients of the CB-VCT in 2011-2012 who were tested for HIV or any STI 1476 reported male sex partners. Unprotected anal intercourse (UAI) was reported as reason for testing by 61% of the clients. Forty-one of 1413 clients testing for HIV were tested positive (2.9%). Twenty-four of 1380 clients testing for syphilis required treatment (1.7%). Tests for simultaneous detection of N. gonorrhoea and Chlamydia trachomatis were conducted on 882 pharyngeal and 642 rectal swabs, revealing 58 (=6.6%) pharyngeal and 71 (=11.1%) rectal infections with one or both pathogens. In multivariate logistic regression analysis number of partners, UAI (OR=2.42) and relying on visual impression when selecting sex partners (OR = 2.92) were associated with increased risks for diagnosis of syphilis or a rectal STI. Syphilis or rectal STI diagnosis (OR=4.52) were associated with increased risk for HIV diagnosis.
Conclusions:
The MSM CB-VCT in Hamburg reaches clients at high risk for HIV and STIs. The diagnosis of syphilis or a rectal STI was associated with increased odds of testing positive for HIV. Due to the high prevalence of curable bacterial STI among clients and because syphilis and rectal bacterial STI may facilitate HIV transmission, MSM asking for HIV tests in CB-VCTs should also be offered tests for other bacterial STIs.
| 6,706 | 433 | 9 |
[
"hiv",
"sti",
"testing",
"rectal",
"msm",
"clients",
"risk",
"diagnosis",
"syphilis",
"partners"
] |
[
"test",
"test"
] |
[CONTENT] HIV infection | Men who have sex with men-MSM | Sexually transmitted infections | Community based voluntary counselling and testing [SUMMARY]
|
[CONTENT] HIV infection | Men who have sex with men-MSM | Sexually transmitted infections | Community based voluntary counselling and testing [SUMMARY]
|
[CONTENT] HIV infection | Men who have sex with men-MSM | Sexually transmitted infections | Community based voluntary counselling and testing [SUMMARY]
|
[CONTENT] HIV infection | Men who have sex with men-MSM | Sexually transmitted infections | Community based voluntary counselling and testing [SUMMARY]
|
[CONTENT] HIV infection | Men who have sex with men-MSM | Sexually transmitted infections | Community based voluntary counselling and testing [SUMMARY]
|
[CONTENT] HIV infection | Men who have sex with men-MSM | Sexually transmitted infections | Community based voluntary counselling and testing [SUMMARY]
|
[CONTENT] Adult | Chlamydia Infections | Cities | Community Health Services | Counseling | Germany | Gonorrhea | HIV Infections | Homosexuality, Male | Humans | Logistic Models | Male | Mass Screening | Middle Aged | Prevalence | Risk Factors | Sexual Behavior | Sexual Partners | Sexually Transmitted Diseases | Surveys and Questionnaires | Syphilis | Young Adult [SUMMARY]
|
[CONTENT] Adult | Chlamydia Infections | Cities | Community Health Services | Counseling | Germany | Gonorrhea | HIV Infections | Homosexuality, Male | Humans | Logistic Models | Male | Mass Screening | Middle Aged | Prevalence | Risk Factors | Sexual Behavior | Sexual Partners | Sexually Transmitted Diseases | Surveys and Questionnaires | Syphilis | Young Adult [SUMMARY]
|
[CONTENT] Adult | Chlamydia Infections | Cities | Community Health Services | Counseling | Germany | Gonorrhea | HIV Infections | Homosexuality, Male | Humans | Logistic Models | Male | Mass Screening | Middle Aged | Prevalence | Risk Factors | Sexual Behavior | Sexual Partners | Sexually Transmitted Diseases | Surveys and Questionnaires | Syphilis | Young Adult [SUMMARY]
|
[CONTENT] Adult | Chlamydia Infections | Cities | Community Health Services | Counseling | Germany | Gonorrhea | HIV Infections | Homosexuality, Male | Humans | Logistic Models | Male | Mass Screening | Middle Aged | Prevalence | Risk Factors | Sexual Behavior | Sexual Partners | Sexually Transmitted Diseases | Surveys and Questionnaires | Syphilis | Young Adult [SUMMARY]
|
[CONTENT] Adult | Chlamydia Infections | Cities | Community Health Services | Counseling | Germany | Gonorrhea | HIV Infections | Homosexuality, Male | Humans | Logistic Models | Male | Mass Screening | Middle Aged | Prevalence | Risk Factors | Sexual Behavior | Sexual Partners | Sexually Transmitted Diseases | Surveys and Questionnaires | Syphilis | Young Adult [SUMMARY]
|
[CONTENT] Adult | Chlamydia Infections | Cities | Community Health Services | Counseling | Germany | Gonorrhea | HIV Infections | Homosexuality, Male | Humans | Logistic Models | Male | Mass Screening | Middle Aged | Prevalence | Risk Factors | Sexual Behavior | Sexual Partners | Sexually Transmitted Diseases | Surveys and Questionnaires | Syphilis | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] hiv | sti | testing | rectal | msm | clients | risk | diagnosis | syphilis | partners [SUMMARY]
|
[CONTENT] hiv | sti | testing | rectal | msm | clients | risk | diagnosis | syphilis | partners [SUMMARY]
|
[CONTENT] hiv | sti | testing | rectal | msm | clients | risk | diagnosis | syphilis | partners [SUMMARY]
|
[CONTENT] hiv | sti | testing | rectal | msm | clients | risk | diagnosis | syphilis | partners [SUMMARY]
|
[CONTENT] hiv | sti | testing | rectal | msm | clients | risk | diagnosis | syphilis | partners [SUMMARY]
|
[CONTENT] hiv | sti | testing | rectal | msm | clients | risk | diagnosis | syphilis | partners [SUMMARY]
|
[CONTENT] tests | sti | cb | hamburg | community | testing | serological | cb vcts | work | sti tests [SUMMARY]
|
[CONTENT] hiv | test | testing | clients | sex | 10 | syphilis | models | rectal | use [SUMMARY]
|
[CONTENT] uai | reported | hiv | tested | 95 ci | diagnosis | clients | age | partner | twice [SUMMARY]
|
[CONTENT] msm | setting | useful | gay | sti testing | hiv sti testing | comprehensive | community | need | reach [SUMMARY]
|
[CONTENT] hiv | sti | testing | msm | clients | rectal | test | risk | uai | tests [SUMMARY]
|
[CONTENT] hiv | sti | testing | msm | clients | rectal | test | risk | uai | tests [SUMMARY]
|
[CONTENT] recent years | CB-VCT | MSM | Germany ||| ||| Hamburg | CB-VCTs ||| [SUMMARY]
|
[CONTENT] the MSM CB-VCT | Hamburg ||| MSM ||| CB-VCT | STI [SUMMARY]
|
[CONTENT] the CB-VCT | 2011-2012 ||| UAI | 61% ||| Forty-one | 1413 | 2.9% ||| Twenty-four | 1380 | 1.7% ||| N. | Chlamydia | 882 | 642 | 58 | 6.6% | 71 | 11.1% | one ||| UAI | 2.92 | STI ||| Syphilis | STI [SUMMARY]
|
[CONTENT] Hamburg ||| STI ||| STI | STI | MSM | CB-VCTs [SUMMARY]
|
[CONTENT] recent years | CB-VCT | MSM | Germany ||| ||| Hamburg | CB-VCTs ||| ||| the MSM CB-VCT | Hamburg ||| MSM ||| CB-VCT | STI ||| ||| the CB-VCT | 2011-2012 ||| UAI | 61% ||| Forty-one | 1413 | 2.9% ||| Twenty-four | 1380 | 1.7% ||| N. | Chlamydia | 882 | 642 | 58 | 6.6% | 71 | 11.1% | one ||| UAI | 2.92 | STI ||| Syphilis | STI ||| Hamburg ||| STI ||| STI | STI | MSM | CB-VCTs [SUMMARY]
|
[CONTENT] recent years | CB-VCT | MSM | Germany ||| ||| Hamburg | CB-VCTs ||| ||| the MSM CB-VCT | Hamburg ||| MSM ||| CB-VCT | STI ||| ||| the CB-VCT | 2011-2012 ||| UAI | 61% ||| Forty-one | 1413 | 2.9% ||| Twenty-four | 1380 | 1.7% ||| N. | Chlamydia | 882 | 642 | 58 | 6.6% | 71 | 11.1% | one ||| UAI | 2.92 | STI ||| Syphilis | STI ||| Hamburg ||| STI ||| STI | STI | MSM | CB-VCTs [SUMMARY]
|
||||||
Synergistic Effect of Micro-Nano-Hybrid Surfaces and Sr Doping on the Osteogenic and Angiogenic Capacity of Hydroxyapatite Bioceramics Scaffolds.
|
35221685
|
The synergistic effect of chemical element doping and surface modification is considered a novel way to regulate cell biological responses and improve the osteoinductive ability of biomaterials.
|
BACKGROUND
|
Hydroxyapatite (HAp) bioceramics with micro-nano-hybrid (a mixture of microrods and nanorods) surfaces and different strontium (Sr) doping contents of 2.5, 5, 10, and 20% (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were prepared via a hydrothermal transformation method. The effect of Srx-mnHAp on osteogenesis and angiogenesis of bone marrow stromal cells (BMSCs) was evaluated in vitro, and the bioceramics scaffolds were further implanted into rat calvarial defects for the observation of bone regeneration in vivo.
|
METHODS
|
HAp bioceramics with micro-nano-hybrid surfaces (mnHAp) could facilitate cell spreading, proliferation ability, ALP activity, and gene expression of osteogenic and angiogenic factors, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. More importantly, Srx-mnHAp (x: 2.5, 5, 10 and 20%) further promoted cellular osteogenic activity, and Sr10-mnHAp possessed the best stimulatory effect. The results of calvarial defects revealed that Sr10-mnHAp could promote more bone and blood vessel regeneration, with mnHAp and HAp bioceramics (dense and flat surfaces) as compared.
|
RESULTS
|
The present study suggests that HAp bioceramics with micro-nano-hybrid surface and Sr doping had synergistic promotion effects on bone regeneration, which can be a promising material for bone defect repair.
|
CONCLUSION
|
[
"Animals",
"Bone Regeneration",
"Cell Differentiation",
"Cell Proliferation",
"Durapatite",
"Osteogenesis",
"Rats",
"Strontium",
"Tissue Scaffolds"
] |
8865905
|
Introduction
|
Bone transplantation is a crucial method to reconstruct bone defects caused by trauma, tumors, and other diseases, and autologous bone grafts remain the “gold standard” for bone transplantation. However, the limitation of insufficient bone mass and related postoperative complications have facilitated the development of substitute materials.1,2 Bioceramic materials are widely used as a physiological scaffold for bone defect repair because of its excellent biocompatibility and bioactivity, and are currently considered as the most promising alternative to autologous bone grafting. The porous structure of bioceramics facilitates the adhesion of osteoblasts, transport of nutrients, and speeds up the process of bone resorption and reconstruction,3 which makes it better for repairing large bone defects.4
The inorganic minerals in human bone tissue have a multilevel ordered structure and contain many microelements. From the viewpoint of bio mimics, we can tune the osteoinduction ability of materials by controlling the surface microstructure and element doping. The ideal scaffold materials for bone regeneration require an appropriate three-dimensional microporous structure for cell growth and nutrient metabolism.2,5 For bioceramics scaffolds, the pores are 150 ~ 500 μm in diameter and over 50% in porosity are appropriate to stimulate cell response, and nanostructured surfaces and high connectivity between pores is also important.1,6–8 The results of our recent study confirmed that the HAp bioceramics with nanostructured modified topography significantly enhanced cell viability, spreading, and osteogenic differentiation of BMSCs and bone formation, mineralization and vascularization in vivo.9 Moreover, suitable surface chemistry plays critical roles in cell viability, spreading, proliferation, and osteogenic differentiation.2,5 Biomaterials can be modified by the introduction of functional inorganic ions. Recently, more attention has been given to strontium (Sr), which is an important trace element in the human body, and 99% of Sr exists in bones. Sr can improve the mechanical properties and modify the bone balance toward osteosynthesis.10 Recent studies11 have found that Sr can enhance the proliferation of osteoblasts during bone metabolism. Yang et al12 suggested that the surface of Sr-doped hydroxyapatite can significantly increase the growth of osteoblasts. The results of Wang et al13 confirmed that the Sr-HAp coatings significantly promoted osteoblast attachment and proliferation as the Sr content increased. Recently, researchers focused on the preparation of strontium-substituted bioceramics and their application in bone regeneration.14–16 However, few studies have focused on the synergistic promotion of nanostructures and Sr doping on bone regeneration.
Therefore, based on the above research and our previous results,9,17 we proposed that the combination of nanostructure surfaces and Sr-doping might lead to synergistic enhancement of osteogenesis and angiogenesis.18,19 Herein, HAp bioceramics with micro-nano-hybrid surfaces and different strontium (Sr) doping contents of 2.5, 5, 10, and 20% (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were fabricated via the hydrothermal transformation method. The effect on cell adhesion, proliferation, and ALP activity and the expression of genes involved in osteogenesis and angiogenesis were investigated to determine the optimal Sr doping content, followed by calvarial defect experiments to evaluate bone regeneration and vascularization in vivo.
| null | null | null | null |
Conclusion
|
Surface structure and chemical composition are considered to be the key clues to regulate the biological response of biomaterials. Herein, HAp bioceramics with micro-nano-hybrid surface and different Sr doping contents (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were successfully fabricated via a hydrothermal transformation method using Srx-ɑ-TCP powder as a precursor without additives. The nanostructure surface can support cell adhesion, and Sr-doping could further enhance osteogenic differentiation with an optimal doping concentration of 10%. Finally, the calvarial defect model showed that the Sr10-mnHAp bioceramics scaffolds possessed better bone regeneration capacity. All of the results confirmed our hypothesis that micro-nano-hybrid surface and Sr doping have synergistic effects on bone regeneration and provided a new strategy for improving the osteoinduction ability of traditional bioceramics.
|
[
"Materials and Methods",
"Preparation of Micro-Nano-Hybrid HAp (mnHAp) and Sr-Doped mnHAp (Srx-mnHAp) Bioceramics",
"Samples Characterization",
"Cell Extraction and Culture",
"Cell Morphology and Adhesion",
"Cell Proliferation",
"ALP Staining and Activity",
"Quantitative Real-Time PCR (qRT-PCR) and Western Blot Experiment",
"Animal Experiment",
"Micro-CT Analyses",
"Histological Analyses",
"Statistical Analyses",
"Results",
"Characterization of the Samples",
"The Morphology of BMSCs",
"Cell Proliferation",
"ALP Activity Assay",
"qRT–PCR Assay and Western Blot Experiment",
"Micro-CT Measurement",
"Histological and Histomorphometric Analysis",
"Discussion",
"Conclusion"
] |
[
" Preparation of Micro-Nano-Hybrid HAp (mnHAp) and Sr-Doped mnHAp (Srx-mnHAp) Bioceramics The micro-nano-hybrid HAp (mnHAp) and Sr-doped mnHAp (Srx-mnHAp, x: 2.5, 5, 10 and 20%) bioceramics were prepared by the hydrothermal transformation method. First, ɑ-tricalcium phosphate (ɑ-TCP) and Sr-doped ɑ-TCP with different doping contents of 2.5, 5, 10, and 20% (Srx-ɑ-TCP, x: 2.5, 5, 10 and 20%) powders were synthesized by chemical precipitation.20 Then, the synthesized ɑ-TCP and Srx-ɑ-TCP powders were mixed with 7 wt% polyvinyl alcohol (PVA), dry-pressed into discs 10 mm in diameter and 2 mm in thickness under a pressure of 5 MPa, and further calcined at 1050 °C for 5 h to obtain ɑ-TCP and Srx-ɑ-TCP bioceramics, respectively.21 Finally, the mnHAp and Srx-mnHAp bioceramics were prepared using ɑ-TCP and Sr-ɑ-TCP discs as precursors via hydrothermal reaction in pH=7 aqueous solution at 180 °C for 72 h.17,20 In addition, HAp bioceramics with dense and flat surfaces were used as controls and labeled S0.9\nThe micro-nano-hybrid HAp (mnHAp) and Sr-doped mnHAp (Srx-mnHAp, x: 2.5, 5, 10 and 20%) bioceramics were prepared by the hydrothermal transformation method. First, ɑ-tricalcium phosphate (ɑ-TCP) and Sr-doped ɑ-TCP with different doping contents of 2.5, 5, 10, and 20% (Srx-ɑ-TCP, x: 2.5, 5, 10 and 20%) powders were synthesized by chemical precipitation.20 Then, the synthesized ɑ-TCP and Srx-ɑ-TCP powders were mixed with 7 wt% polyvinyl alcohol (PVA), dry-pressed into discs 10 mm in diameter and 2 mm in thickness under a pressure of 5 MPa, and further calcined at 1050 °C for 5 h to obtain ɑ-TCP and Srx-ɑ-TCP bioceramics, respectively.21 Finally, the mnHAp and Srx-mnHAp bioceramics were prepared using ɑ-TCP and Sr-ɑ-TCP discs as precursors via hydrothermal reaction in pH=7 aqueous solution at 180 °C for 72 h.17,20 In addition, HAp bioceramics with dense and flat surfaces were used as controls and labeled S0.9\n Samples Characterization HAp (S0), mnHAp (S3), and Srx-mnHAp (x: 2.5, 5, 10 and 20%) were incubated in 1 mL of α-Minimum Essential Medium (ɑ-MEM, Gibco, USA) for 1 and 4 d, respectively. The medium of each sample changed daily and the concentrations of Sr, Ca, and P ions released from HAp, mnHAp, and Srx-mnHAp at each timepoint were evaluated using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The crystal phase compositions of the samples were determined with XRD (Rigaku, Japan), and the surface microstructures were observed by SEM (JEOL, Japan).17\nHAp (S0), mnHAp (S3), and Srx-mnHAp (x: 2.5, 5, 10 and 20%) were incubated in 1 mL of α-Minimum Essential Medium (ɑ-MEM, Gibco, USA) for 1 and 4 d, respectively. The medium of each sample changed daily and the concentrations of Sr, Ca, and P ions released from HAp, mnHAp, and Srx-mnHAp at each timepoint were evaluated using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The crystal phase compositions of the samples were determined with XRD (Rigaku, Japan), and the surface microstructures were observed by SEM (JEOL, Japan).17\n Cell Extraction and Culture Bone marrow stem cells (BMSCs) were harvested as previously described.17 Briefly, Sprague–Dawley (SD) rats (4 weeks old) were selected and injected intraperitoneally with an overdose of pentobarbital sodium. The scalp was separated for exposure, and both femora were cut off. Fresh marrow was flushed out and cultured in ɑ-MEM (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin and streptomycin (Hyclone, USA). The culture medium was maintained at 37 °C in a 5% CO2 incubator and renewed 3 times a week. The cells were passaged after reaching 90% confluence, and passages 2–4 were used in the following experiment. All animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine and conducted in compliance with the guidelines of Institutional Animal Care and Use Committee of Shanghai Ninth People’s Hospital Affiliated to Shanghai JiaoTong University, School of Medicine [SYXK (Hu) 2016–0016].\nBone marrow stem cells (BMSCs) were harvested as previously described.17 Briefly, Sprague–Dawley (SD) rats (4 weeks old) were selected and injected intraperitoneally with an overdose of pentobarbital sodium. The scalp was separated for exposure, and both femora were cut off. Fresh marrow was flushed out and cultured in ɑ-MEM (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin and streptomycin (Hyclone, USA). The culture medium was maintained at 37 °C in a 5% CO2 incubator and renewed 3 times a week. The cells were passaged after reaching 90% confluence, and passages 2–4 were used in the following experiment. All animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine and conducted in compliance with the guidelines of Institutional Animal Care and Use Committee of Shanghai Ninth People’s Hospital Affiliated to Shanghai JiaoTong University, School of Medicine [SYXK (Hu) 2016–0016].\n Cell Morphology and Adhesion Phalloidin staining was performed to evaluate the morphology and spreading of the BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at an initial density of 1×104 cells per mL in 24-well plates. At 6 h after seeding, all specimens were fixed with 4% formaldehyde for 10 min, and then treated with 1% Triton X-100 for 5 min. Next, these specimens were incubated with phalloidin (Sigma, USA) for 30 min, followed by DAPI (Sigma, USA) for approximately 30s. Finally, actin cytoskeletons were observed by fluorescence microscopy (Leica, Germany).\nPhalloidin staining was performed to evaluate the morphology and spreading of the BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at an initial density of 1×104 cells per mL in 24-well plates. At 6 h after seeding, all specimens were fixed with 4% formaldehyde for 10 min, and then treated with 1% Triton X-100 for 5 min. Next, these specimens were incubated with phalloidin (Sigma, USA) for 30 min, followed by DAPI (Sigma, USA) for approximately 30s. Finally, actin cytoskeletons were observed by fluorescence microscopy (Leica, Germany).\n Cell Proliferation The proliferation of BMSCs in the samples was investigated by MTT assay, with a cell density of 1×104 cells per mL in 24-well plates. After culturing for 1, 4, and 7 d, the culture medium was replaced, and the BMSC-seeded bioceramics were rinsed and incubated with sterile MTT (Amresco, USA) at 37 °C for 4 h. DMSO solution (Sigma, USA) was then added and incubated for 10 min to dissolve formazan. The OD value was read at 490 nm by a microplate Reader (Biotek, USA). All experiments were repeated 3 times.\nThe proliferation of BMSCs in the samples was investigated by MTT assay, with a cell density of 1×104 cells per mL in 24-well plates. After culturing for 1, 4, and 7 d, the culture medium was replaced, and the BMSC-seeded bioceramics were rinsed and incubated with sterile MTT (Amresco, USA) at 37 °C for 4 h. DMSO solution (Sigma, USA) was then added and incubated for 10 min to dissolve formazan. The OD value was read at 490 nm by a microplate Reader (Biotek, USA). All experiments were repeated 3 times.\n ALP Staining and Activity BMSCs were cultured onto S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr samples at a cell density of 1×104 cells per mL in 24-well plates. After culturing for 10 d, ALP staining was performed with a BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). The ALP activity was then assessed at 4, 7, and 10 d.22 Briefly, all samples were lysed in 1% Triton X-100 for 30 min at 4 °C, and the cellular lysate of each well was collected and centrifuged at 4 °C (12,000 rpm × 10 min). Then, the OD value was determined at 520 nm with a microplate Reader (Bio-Tek, USA), and total cellular protein was determined by a BCA protein kit (Beyotime, China) at 562 nm. Finally, ALP activity was evaluated by normalizing to total protein content. All experiments were repeated 3 times.\nBMSCs were cultured onto S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr samples at a cell density of 1×104 cells per mL in 24-well plates. After culturing for 10 d, ALP staining was performed with a BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). The ALP activity was then assessed at 4, 7, and 10 d.22 Briefly, all samples were lysed in 1% Triton X-100 for 30 min at 4 °C, and the cellular lysate of each well was collected and centrifuged at 4 °C (12,000 rpm × 10 min). Then, the OD value was determined at 520 nm with a microplate Reader (Bio-Tek, USA), and total cellular protein was determined by a BCA protein kit (Beyotime, China) at 562 nm. Finally, ALP activity was evaluated by normalizing to total protein content. All experiments were repeated 3 times.\n Quantitative Real-Time PCR (qRT-PCR) and Western Blot Experiment qRT-PCR was carried out to assess the expression of osteogenic and angiogenic factors in BMSCs cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr. After culturing for 4 and 7 d, total RNA of the samples was isolated according to our previous study.21 Briefly, the cells cultured in the bioceramics were lysed in TRIzol Reagent (Life Technologies, USA) and reverse transcribed to cDNA by PrimeScriptTM RT reagent Kit (TaKaRa, Japan). The expression levels of osteogenesis- and angiogenesis-related genes were further evaluated by qRT-PCR analysis, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. GAPDH was selected as a housekeeping gene to normalize the expression level. The primer sequences selected are listed in Table 1. All experiments were repeated 3 times.Table 1Primer Sequences of the Selected GenesTarget GenePrimers (F = Forward; R = Reverse)Accession NumberProduct Size (bp)COL1F:5ʹCTGCCCAGAAGAATATGTATCACC3’R:5ʹGAAGCAAAGTTTCCTCCAAGACC3’NM_053304.1198BSPF: 5ʹAGAAAGAGCAGCACGGTTGAGT3’R: 5ʹGACCCTCGTAGCCTTCATAGCC3’NM_012587.2175BMP-2F: 5ʹTGGGTTTGTGGTGGAAGTGGC3’R: 5ʹTGGATGTCCTTTACCGTCGTG3’NM_017178.2154OPNF: 5ʹCCAAGCGTGGAAACACACAGCC3’R: 5ʹGGCTTTGGAACTCGCCTGACTG3’NM_012881.2165VEGFF: 5′GGCTCTGAAACCATGAACTTTCT3′R: 5′GCAGTAGCTGCGCTGGTAGAC3′NM_001110334.2165ANG-1F:5′GGACAGCAGGCAAACAGAGCAGC3′R: 5′CCACAGGCATCAAACCACCAACC3′NM_053546.2130GAPDHF: 5ʹCCTGCACCACCAACTGCTTA3’R: 5ʹGGCCATCCACAGTCTTCTGAG3’NM_017008.4120\n\nPrimer Sequences of the Selected Genes\nWestern blot experiment was applied to assess expression of BMP-2. BMSCs were cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr for 7 d. Total protein was extracted and centrifuged at 12,000 rpm for 15 minutes. Then, the samples were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 volts for 20 minutes and 120 volts for 50 minutes, and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The PVDF membranes were incubated with primary antibody (Abcam, UK) at 37 °C for 2h. After that, the membranes were incubated with HRP-conjugated secondary antibody (Abcam, UK) for 1 h at room temperature. The protein bands were visualized, and the images were captured by an automated luminescent image analysis system (Tanon, China).\nqRT-PCR was carried out to assess the expression of osteogenic and angiogenic factors in BMSCs cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr. After culturing for 4 and 7 d, total RNA of the samples was isolated according to our previous study.21 Briefly, the cells cultured in the bioceramics were lysed in TRIzol Reagent (Life Technologies, USA) and reverse transcribed to cDNA by PrimeScriptTM RT reagent Kit (TaKaRa, Japan). The expression levels of osteogenesis- and angiogenesis-related genes were further evaluated by qRT-PCR analysis, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. GAPDH was selected as a housekeeping gene to normalize the expression level. The primer sequences selected are listed in Table 1. All experiments were repeated 3 times.Table 1Primer Sequences of the Selected GenesTarget GenePrimers (F = Forward; R = Reverse)Accession NumberProduct Size (bp)COL1F:5ʹCTGCCCAGAAGAATATGTATCACC3’R:5ʹGAAGCAAAGTTTCCTCCAAGACC3’NM_053304.1198BSPF: 5ʹAGAAAGAGCAGCACGGTTGAGT3’R: 5ʹGACCCTCGTAGCCTTCATAGCC3’NM_012587.2175BMP-2F: 5ʹTGGGTTTGTGGTGGAAGTGGC3’R: 5ʹTGGATGTCCTTTACCGTCGTG3’NM_017178.2154OPNF: 5ʹCCAAGCGTGGAAACACACAGCC3’R: 5ʹGGCTTTGGAACTCGCCTGACTG3’NM_012881.2165VEGFF: 5′GGCTCTGAAACCATGAACTTTCT3′R: 5′GCAGTAGCTGCGCTGGTAGAC3′NM_001110334.2165ANG-1F:5′GGACAGCAGGCAAACAGAGCAGC3′R: 5′CCACAGGCATCAAACCACCAACC3′NM_053546.2130GAPDHF: 5ʹCCTGCACCACCAACTGCTTA3’R: 5ʹGGCCATCCACAGTCTTCTGAG3’NM_017008.4120\n\nPrimer Sequences of the Selected Genes\nWestern blot experiment was applied to assess expression of BMP-2. BMSCs were cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr for 7 d. Total protein was extracted and centrifuged at 12,000 rpm for 15 minutes. Then, the samples were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 volts for 20 minutes and 120 volts for 50 minutes, and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The PVDF membranes were incubated with primary antibody (Abcam, UK) at 37 °C for 2h. After that, the membranes were incubated with HRP-conjugated secondary antibody (Abcam, UK) for 1 h at room temperature. The protein bands were visualized, and the images were captured by an automated luminescent image analysis system (Tanon, China).\n Animal Experiment Calvarial defects were established in twelve 6-week-old SD rats as previously described.17 Animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine. All rats were randomly divided into the following 3 groups: S0, S3, and 10Sr (n = 4 for each group). Briefly, all rats were injected intraperitoneally with pentobarbital (3.5 mg/100 g). Under sterile conditions, a 2 cm longitudinal incision was made, and two symmetrical round defects (5 mm in diameter) were created using the implant drill at each rat. Subsequently, the HAp, mnHAp, and Sr10-mnHAp were placed into the defects, and the operation incision was carefully sutured with absorbable sutures.\nCalvarial defects were established in twelve 6-week-old SD rats as previously described.17 Animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine. All rats were randomly divided into the following 3 groups: S0, S3, and 10Sr (n = 4 for each group). Briefly, all rats were injected intraperitoneally with pentobarbital (3.5 mg/100 g). Under sterile conditions, a 2 cm longitudinal incision was made, and two symmetrical round defects (5 mm in diameter) were created using the implant drill at each rat. Subsequently, the HAp, mnHAp, and Sr10-mnHAp were placed into the defects, and the operation incision was carefully sutured with absorbable sutures.\n Micro-CT Analyses At the 8th week after implantation, all rats were sacrificed and perfused with Microfil (Flowtech, USA) to observe new blood vessels.23 The samples were decalcified using EDTA for 1 month and then scanned by microcomputed tomography (Scanco, Switzerland), the percentages of new blood vessels were quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA). For the observation of new bone, the specimens were fixed in 4% paraformaldehyde for 2 d and then transferred to 75% alcohol. Finally, the samples were scanned by microcomputed tomography. Bone volume fraction (BV/TV) and trabecular thickness (Tb. Th) were calculated with auxiliary software (Scanco, Switzerland).24\nAt the 8th week after implantation, all rats were sacrificed and perfused with Microfil (Flowtech, USA) to observe new blood vessels.23 The samples were decalcified using EDTA for 1 month and then scanned by microcomputed tomography (Scanco, Switzerland), the percentages of new blood vessels were quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA). For the observation of new bone, the specimens were fixed in 4% paraformaldehyde for 2 d and then transferred to 75% alcohol. Finally, the samples were scanned by microcomputed tomography. Bone volume fraction (BV/TV) and trabecular thickness (Tb. Th) were calculated with auxiliary software (Scanco, Switzerland).24\n Histological Analyses All specimens were decalcified with 10% EDTA, dehydrated in increased concentrations of ethanol (75% to 100%) and embedded in PMMA for 3 weeks. Tissue blocks were sectioned by a microtome (Leica, Germany) and further ground to approximately 40 μm. Next, the sections were subjected to Van Gieson staining for histological analysis, and the proportion of bone regeneration in the defect area was quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA).17,25\nAll specimens were decalcified with 10% EDTA, dehydrated in increased concentrations of ethanol (75% to 100%) and embedded in PMMA for 3 weeks. Tissue blocks were sectioned by a microtome (Leica, Germany) and further ground to approximately 40 μm. Next, the sections were subjected to Van Gieson staining for histological analysis, and the proportion of bone regeneration in the defect area was quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA).17,25\n Statistical Analyses All data are expressed as the means ± standard deviation (SD), and statistical analysis was performed using SPSS 17.0 software (SPSS Inc., USA). A p value <0.05 was considered statistically significant.\nAll data are expressed as the means ± standard deviation (SD), and statistical analysis was performed using SPSS 17.0 software (SPSS Inc., USA). A p value <0.05 was considered statistically significant.",
"The micro-nano-hybrid HAp (mnHAp) and Sr-doped mnHAp (Srx-mnHAp, x: 2.5, 5, 10 and 20%) bioceramics were prepared by the hydrothermal transformation method. First, ɑ-tricalcium phosphate (ɑ-TCP) and Sr-doped ɑ-TCP with different doping contents of 2.5, 5, 10, and 20% (Srx-ɑ-TCP, x: 2.5, 5, 10 and 20%) powders were synthesized by chemical precipitation.20 Then, the synthesized ɑ-TCP and Srx-ɑ-TCP powders were mixed with 7 wt% polyvinyl alcohol (PVA), dry-pressed into discs 10 mm in diameter and 2 mm in thickness under a pressure of 5 MPa, and further calcined at 1050 °C for 5 h to obtain ɑ-TCP and Srx-ɑ-TCP bioceramics, respectively.21 Finally, the mnHAp and Srx-mnHAp bioceramics were prepared using ɑ-TCP and Sr-ɑ-TCP discs as precursors via hydrothermal reaction in pH=7 aqueous solution at 180 °C for 72 h.17,20 In addition, HAp bioceramics with dense and flat surfaces were used as controls and labeled S0.9",
"HAp (S0), mnHAp (S3), and Srx-mnHAp (x: 2.5, 5, 10 and 20%) were incubated in 1 mL of α-Minimum Essential Medium (ɑ-MEM, Gibco, USA) for 1 and 4 d, respectively. The medium of each sample changed daily and the concentrations of Sr, Ca, and P ions released from HAp, mnHAp, and Srx-mnHAp at each timepoint were evaluated using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The crystal phase compositions of the samples were determined with XRD (Rigaku, Japan), and the surface microstructures were observed by SEM (JEOL, Japan).17",
"Bone marrow stem cells (BMSCs) were harvested as previously described.17 Briefly, Sprague–Dawley (SD) rats (4 weeks old) were selected and injected intraperitoneally with an overdose of pentobarbital sodium. The scalp was separated for exposure, and both femora were cut off. Fresh marrow was flushed out and cultured in ɑ-MEM (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin and streptomycin (Hyclone, USA). The culture medium was maintained at 37 °C in a 5% CO2 incubator and renewed 3 times a week. The cells were passaged after reaching 90% confluence, and passages 2–4 were used in the following experiment. All animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine and conducted in compliance with the guidelines of Institutional Animal Care and Use Committee of Shanghai Ninth People’s Hospital Affiliated to Shanghai JiaoTong University, School of Medicine [SYXK (Hu) 2016–0016].",
"Phalloidin staining was performed to evaluate the morphology and spreading of the BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at an initial density of 1×104 cells per mL in 24-well plates. At 6 h after seeding, all specimens were fixed with 4% formaldehyde for 10 min, and then treated with 1% Triton X-100 for 5 min. Next, these specimens were incubated with phalloidin (Sigma, USA) for 30 min, followed by DAPI (Sigma, USA) for approximately 30s. Finally, actin cytoskeletons were observed by fluorescence microscopy (Leica, Germany).",
"The proliferation of BMSCs in the samples was investigated by MTT assay, with a cell density of 1×104 cells per mL in 24-well plates. After culturing for 1, 4, and 7 d, the culture medium was replaced, and the BMSC-seeded bioceramics were rinsed and incubated with sterile MTT (Amresco, USA) at 37 °C for 4 h. DMSO solution (Sigma, USA) was then added and incubated for 10 min to dissolve formazan. The OD value was read at 490 nm by a microplate Reader (Biotek, USA). All experiments were repeated 3 times.",
"BMSCs were cultured onto S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr samples at a cell density of 1×104 cells per mL in 24-well plates. After culturing for 10 d, ALP staining was performed with a BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). The ALP activity was then assessed at 4, 7, and 10 d.22 Briefly, all samples were lysed in 1% Triton X-100 for 30 min at 4 °C, and the cellular lysate of each well was collected and centrifuged at 4 °C (12,000 rpm × 10 min). Then, the OD value was determined at 520 nm with a microplate Reader (Bio-Tek, USA), and total cellular protein was determined by a BCA protein kit (Beyotime, China) at 562 nm. Finally, ALP activity was evaluated by normalizing to total protein content. All experiments were repeated 3 times.",
"qRT-PCR was carried out to assess the expression of osteogenic and angiogenic factors in BMSCs cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr. After culturing for 4 and 7 d, total RNA of the samples was isolated according to our previous study.21 Briefly, the cells cultured in the bioceramics were lysed in TRIzol Reagent (Life Technologies, USA) and reverse transcribed to cDNA by PrimeScriptTM RT reagent Kit (TaKaRa, Japan). The expression levels of osteogenesis- and angiogenesis-related genes were further evaluated by qRT-PCR analysis, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. GAPDH was selected as a housekeeping gene to normalize the expression level. The primer sequences selected are listed in Table 1. All experiments were repeated 3 times.Table 1Primer Sequences of the Selected GenesTarget GenePrimers (F = Forward; R = Reverse)Accession NumberProduct Size (bp)COL1F:5ʹCTGCCCAGAAGAATATGTATCACC3’R:5ʹGAAGCAAAGTTTCCTCCAAGACC3’NM_053304.1198BSPF: 5ʹAGAAAGAGCAGCACGGTTGAGT3’R: 5ʹGACCCTCGTAGCCTTCATAGCC3’NM_012587.2175BMP-2F: 5ʹTGGGTTTGTGGTGGAAGTGGC3’R: 5ʹTGGATGTCCTTTACCGTCGTG3’NM_017178.2154OPNF: 5ʹCCAAGCGTGGAAACACACAGCC3’R: 5ʹGGCTTTGGAACTCGCCTGACTG3’NM_012881.2165VEGFF: 5′GGCTCTGAAACCATGAACTTTCT3′R: 5′GCAGTAGCTGCGCTGGTAGAC3′NM_001110334.2165ANG-1F:5′GGACAGCAGGCAAACAGAGCAGC3′R: 5′CCACAGGCATCAAACCACCAACC3′NM_053546.2130GAPDHF: 5ʹCCTGCACCACCAACTGCTTA3’R: 5ʹGGCCATCCACAGTCTTCTGAG3’NM_017008.4120\n\nPrimer Sequences of the Selected Genes\nWestern blot experiment was applied to assess expression of BMP-2. BMSCs were cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr for 7 d. Total protein was extracted and centrifuged at 12,000 rpm for 15 minutes. Then, the samples were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 volts for 20 minutes and 120 volts for 50 minutes, and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The PVDF membranes were incubated with primary antibody (Abcam, UK) at 37 °C for 2h. After that, the membranes were incubated with HRP-conjugated secondary antibody (Abcam, UK) for 1 h at room temperature. The protein bands were visualized, and the images were captured by an automated luminescent image analysis system (Tanon, China).",
"Calvarial defects were established in twelve 6-week-old SD rats as previously described.17 Animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine. All rats were randomly divided into the following 3 groups: S0, S3, and 10Sr (n = 4 for each group). Briefly, all rats were injected intraperitoneally with pentobarbital (3.5 mg/100 g). Under sterile conditions, a 2 cm longitudinal incision was made, and two symmetrical round defects (5 mm in diameter) were created using the implant drill at each rat. Subsequently, the HAp, mnHAp, and Sr10-mnHAp were placed into the defects, and the operation incision was carefully sutured with absorbable sutures.",
"At the 8th week after implantation, all rats were sacrificed and perfused with Microfil (Flowtech, USA) to observe new blood vessels.23 The samples were decalcified using EDTA for 1 month and then scanned by microcomputed tomography (Scanco, Switzerland), the percentages of new blood vessels were quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA). For the observation of new bone, the specimens were fixed in 4% paraformaldehyde for 2 d and then transferred to 75% alcohol. Finally, the samples were scanned by microcomputed tomography. Bone volume fraction (BV/TV) and trabecular thickness (Tb. Th) were calculated with auxiliary software (Scanco, Switzerland).24",
"All specimens were decalcified with 10% EDTA, dehydrated in increased concentrations of ethanol (75% to 100%) and embedded in PMMA for 3 weeks. Tissue blocks were sectioned by a microtome (Leica, Germany) and further ground to approximately 40 μm. Next, the sections were subjected to Van Gieson staining for histological analysis, and the proportion of bone regeneration in the defect area was quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA).17,25",
"All data are expressed as the means ± standard deviation (SD), and statistical analysis was performed using SPSS 17.0 software (SPSS Inc., USA). A p value <0.05 was considered statistically significant.",
" Characterization of the Samples XRD results of mnHAp and Srx-mnHAp (x: 2.5, 5, 10 and 20%) are shown in Figure 1. All the diffraction peaks confirmed that these samples were converted into HAp without any other phases after the hydrothermal transformation method. The obvious micro-nanorods topography surfaces of mnHAp (Figure 2B) and Srx-mnHAp (Figure 2C–F) could be observed by SEM, and Sr10-mnHAp possessed the densest rod-shaped structure (Figure 2E). The lengths of the microrods and nanorods were approximately 10~20 μm, while the diameters were approximately 1~4 μm and 80~120 nm, respectively. As a comparison, a dense and flat surface with a particle size of approximately 0.9 µm was observed on control sample S0 (Figure 2A).Figure 1XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).Figure 2SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nXRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).\nSEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nThe results of ICP-AES analysis revealed that the release of Sr ions could be detected in Srx-mnHAp bioceramics and showed an ascending trend with increasing Sr content at each timepoint (Figure 3C). No significant differences in the release amount of Ca and P could be found among HAp, mnHAp, and Srx-mnHAp (Figure 3A and B).Figure 3The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.\nThe release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.\nXRD results of mnHAp and Srx-mnHAp (x: 2.5, 5, 10 and 20%) are shown in Figure 1. All the diffraction peaks confirmed that these samples were converted into HAp without any other phases after the hydrothermal transformation method. The obvious micro-nanorods topography surfaces of mnHAp (Figure 2B) and Srx-mnHAp (Figure 2C–F) could be observed by SEM, and Sr10-mnHAp possessed the densest rod-shaped structure (Figure 2E). The lengths of the microrods and nanorods were approximately 10~20 μm, while the diameters were approximately 1~4 μm and 80~120 nm, respectively. As a comparison, a dense and flat surface with a particle size of approximately 0.9 µm was observed on control sample S0 (Figure 2A).Figure 1XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).Figure 2SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nXRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).\nSEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nThe results of ICP-AES analysis revealed that the release of Sr ions could be detected in Srx-mnHAp bioceramics and showed an ascending trend with increasing Sr content at each timepoint (Figure 3C). No significant differences in the release amount of Ca and P could be found among HAp, mnHAp, and Srx-mnHAp (Figure 3A and B).Figure 3The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.\nThe release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.\n The Morphology of BMSCs As shown in Figure 4, the actin cytoskeleton was stained red, and the nucleus was stained blue. After being cultured for 6 h, fluorescence microscopy images showed that the cells seeded in sample S0 spread slightly and almost had a round shape (Figure 4A1). Meanwhile, as a comparison, the cells seeded on the mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) exhibited better early cell attachment and demonstrated a typical polygonal morphology with apparent stretch tentacles. No significant differences in the cell morphology and quantity could be found with increasing Sr content. This experiment showed that the nanostructure surface could promote the early cell attachment of BMSCs instead of the concentrations of Sr ions.Figure 4Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.\nFluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.\nAs shown in Figure 4, the actin cytoskeleton was stained red, and the nucleus was stained blue. After being cultured for 6 h, fluorescence microscopy images showed that the cells seeded in sample S0 spread slightly and almost had a round shape (Figure 4A1). Meanwhile, as a comparison, the cells seeded on the mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) exhibited better early cell attachment and demonstrated a typical polygonal morphology with apparent stretch tentacles. No significant differences in the cell morphology and quantity could be found with increasing Sr content. This experiment showed that the nanostructure surface could promote the early cell attachment of BMSCs instead of the concentrations of Sr ions.Figure 4Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.\nFluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.\n Cell Proliferation The cells adhered to the HAp, mnHAp and Srx-mnHAp bioceramics proliferated through the MTT assay (Figure 5). The cells cultured on mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) displayed higher proliferation than control samples on days 4 and 7. (p<0.05) In addition, among the Srx-mnHAp with different Sr contents, proliferation significantly proceeded on Sr10-mnHAp and Sr20-mnHAp on days 4 and 7 (p<0.05).Figure 5MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nThe cells adhered to the HAp, mnHAp and Srx-mnHAp bioceramics proliferated through the MTT assay (Figure 5). The cells cultured on mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) displayed higher proliferation than control samples on days 4 and 7. (p<0.05) In addition, among the Srx-mnHAp with different Sr contents, proliferation significantly proceeded on Sr10-mnHAp and Sr20-mnHAp on days 4 and 7 (p<0.05).Figure 5MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\n ALP Activity Assay The cells cultured on mnHAp bioceramics possessed more intensive ALP staining, with control samples (dense and flat surface) compared (Figure 6A). Moreover, the staining became deeper with Sr doping (2.5Sr, 5Sr, 10Sr, and 20Sr). In particular, the deepest staining was found on the Sr10-mnHAp sample. The ALP semi-quantification assay on days 4, 7 and 10 further confirmed the staining results (Figure 6B). The ALP activity increased throughout the whole assay period, and the mnHAp bioceramics possessed higher cellular ALP activity. More importantly, the samples with Sr doping achieved better ALP activity on days 4 and 7 (p<0.05). When the experiment time was extended to 10 d, compared to mnHAp, only Sr10-mnHAp and Sr20-mnHAp further enhanced ALP activity.Figure 6ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nThe cells cultured on mnHAp bioceramics possessed more intensive ALP staining, with control samples (dense and flat surface) compared (Figure 6A). Moreover, the staining became deeper with Sr doping (2.5Sr, 5Sr, 10Sr, and 20Sr). In particular, the deepest staining was found on the Sr10-mnHAp sample. The ALP semi-quantification assay on days 4, 7 and 10 further confirmed the staining results (Figure 6B). The ALP activity increased throughout the whole assay period, and the mnHAp bioceramics possessed higher cellular ALP activity. More importantly, the samples with Sr doping achieved better ALP activity on days 4 and 7 (p<0.05). When the experiment time was extended to 10 d, compared to mnHAp, only Sr10-mnHAp and Sr20-mnHAp further enhanced ALP activity.Figure 6ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\n qRT–PCR Assay and Western Blot Experiment qRT–PCR was carried out to analyze the expression of osteogenesis and angiogenesis factors in the cells cultured on HAp, mnHAp and Srx-mnHAp at days 4 and 7. As shown in Figure 7, the mnHAp bioceramics (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) could strengthen the expression of COL1, BSP, BMP-2, OPN, VEGF and ANG-1 with sample S0 (dense and flat surface) compared at 4 and 7 d. Furthermore, Sr doping improved the mRNA levels of COL1, BSP, and OPN at 4 and 7 d and ANG-1 at 7 d. The western bolt result of day 7 confirmed the enhanced expression of BMP-2 in the Srx-mnHAp, especially in the Sr10-mnHAp. More importantly, the promotion effect of Sr doping occurred in a dose-dependent manner, and Sr10-mnHAp achieved the best stimulation ability.Figure 7qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nqRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nqRT–PCR was carried out to analyze the expression of osteogenesis and angiogenesis factors in the cells cultured on HAp, mnHAp and Srx-mnHAp at days 4 and 7. As shown in Figure 7, the mnHAp bioceramics (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) could strengthen the expression of COL1, BSP, BMP-2, OPN, VEGF and ANG-1 with sample S0 (dense and flat surface) compared at 4 and 7 d. Furthermore, Sr doping improved the mRNA levels of COL1, BSP, and OPN at 4 and 7 d and ANG-1 at 7 d. The western bolt result of day 7 confirmed the enhanced expression of BMP-2 in the Srx-mnHAp, especially in the Sr10-mnHAp. More importantly, the promotion effect of Sr doping occurred in a dose-dependent manner, and Sr10-mnHAp achieved the best stimulation ability.Figure 7qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nqRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\n Micro-CT Measurement Micro-CT scanning and analysis were performed to assess the bone regeneration of the calvarial defect areas. From Figure 8A, we can see that more bone formation in the surface and pores of the mnHAp bioceramics at the 8th week after implantation (p<0.05) and Sr10-mnHAp achieved the best repair effect with S0 and S3 compared (p<0.05). Morphometric analysis further proved that the BV/TV ratio (Figure 8B) and Tb. Th (Figure 8C) in mnHAp bioceramics were higher than that of pure HAp. Moreover, significant differences were found between mnHAp and Sr10-mnHAp (p<0.05). Figure 9 showed the angiographic results, more blood vessels were found in the implant site of the mnHAp bioceramics (p<0.05), and the Sr10-mnHAp groups possessed the most newly formed blood vessels. The results of quantitative analysis also confirmed that samples S3 and 10Sr possessed more blood vessels, and the differences between samples 10Sr and S3 were remarkable (p<0.05).Figure 8Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).Figure 9Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT scanning and analysis were performed to assess the bone regeneration of the calvarial defect areas. From Figure 8A, we can see that more bone formation in the surface and pores of the mnHAp bioceramics at the 8th week after implantation (p<0.05) and Sr10-mnHAp achieved the best repair effect with S0 and S3 compared (p<0.05). Morphometric analysis further proved that the BV/TV ratio (Figure 8B) and Tb. Th (Figure 8C) in mnHAp bioceramics were higher than that of pure HAp. Moreover, significant differences were found between mnHAp and Sr10-mnHAp (p<0.05). Figure 9 showed the angiographic results, more blood vessels were found in the implant site of the mnHAp bioceramics (p<0.05), and the Sr10-mnHAp groups possessed the most newly formed blood vessels. The results of quantitative analysis also confirmed that samples S3 and 10Sr possessed more blood vessels, and the differences between samples 10Sr and S3 were remarkable (p<0.05).Figure 8Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).Figure 9Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\n Histological and Histomorphometric Analysis Van Gieson staining was applied to observe bone regeneration in the surface and pores of the HAp bioceramics. As shown in Figure 10A, bone formation was only found on the edge of the defect in group S0, while groups S3 and 10Sr achieved more new bone formation from the margins to the Center, especially group 10Sr. As shown in Figure 10B, the results of bone histomorphometric analysis further confirmed that groups S3 and 10Sr possessed more newly formed bone areas. More importantly, remarkable differences could be found between groups S3 and 10Sr, which indicates that Sr10-mnHAp has the best effect on promoting bone formation (p<0.05).Figure 10Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nHistological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nVan Gieson staining was applied to observe bone regeneration in the surface and pores of the HAp bioceramics. As shown in Figure 10A, bone formation was only found on the edge of the defect in group S0, while groups S3 and 10Sr achieved more new bone formation from the margins to the Center, especially group 10Sr. As shown in Figure 10B, the results of bone histomorphometric analysis further confirmed that groups S3 and 10Sr possessed more newly formed bone areas. More importantly, remarkable differences could be found between groups S3 and 10Sr, which indicates that Sr10-mnHAp has the best effect on promoting bone formation (p<0.05).Figure 10Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nHistological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"XRD results of mnHAp and Srx-mnHAp (x: 2.5, 5, 10 and 20%) are shown in Figure 1. All the diffraction peaks confirmed that these samples were converted into HAp without any other phases after the hydrothermal transformation method. The obvious micro-nanorods topography surfaces of mnHAp (Figure 2B) and Srx-mnHAp (Figure 2C–F) could be observed by SEM, and Sr10-mnHAp possessed the densest rod-shaped structure (Figure 2E). The lengths of the microrods and nanorods were approximately 10~20 μm, while the diameters were approximately 1~4 μm and 80~120 nm, respectively. As a comparison, a dense and flat surface with a particle size of approximately 0.9 µm was observed on control sample S0 (Figure 2A).Figure 1XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).Figure 2SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nXRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).\nSEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nThe results of ICP-AES analysis revealed that the release of Sr ions could be detected in Srx-mnHAp bioceramics and showed an ascending trend with increasing Sr content at each timepoint (Figure 3C). No significant differences in the release amount of Ca and P could be found among HAp, mnHAp, and Srx-mnHAp (Figure 3A and B).Figure 3The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.\nThe release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.",
"As shown in Figure 4, the actin cytoskeleton was stained red, and the nucleus was stained blue. After being cultured for 6 h, fluorescence microscopy images showed that the cells seeded in sample S0 spread slightly and almost had a round shape (Figure 4A1). Meanwhile, as a comparison, the cells seeded on the mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) exhibited better early cell attachment and demonstrated a typical polygonal morphology with apparent stretch tentacles. No significant differences in the cell morphology and quantity could be found with increasing Sr content. This experiment showed that the nanostructure surface could promote the early cell attachment of BMSCs instead of the concentrations of Sr ions.Figure 4Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.\nFluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.",
"The cells adhered to the HAp, mnHAp and Srx-mnHAp bioceramics proliferated through the MTT assay (Figure 5). The cells cultured on mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) displayed higher proliferation than control samples on days 4 and 7. (p<0.05) In addition, among the Srx-mnHAp with different Sr contents, proliferation significantly proceeded on Sr10-mnHAp and Sr20-mnHAp on days 4 and 7 (p<0.05).Figure 5MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"The cells cultured on mnHAp bioceramics possessed more intensive ALP staining, with control samples (dense and flat surface) compared (Figure 6A). Moreover, the staining became deeper with Sr doping (2.5Sr, 5Sr, 10Sr, and 20Sr). In particular, the deepest staining was found on the Sr10-mnHAp sample. The ALP semi-quantification assay on days 4, 7 and 10 further confirmed the staining results (Figure 6B). The ALP activity increased throughout the whole assay period, and the mnHAp bioceramics possessed higher cellular ALP activity. More importantly, the samples with Sr doping achieved better ALP activity on days 4 and 7 (p<0.05). When the experiment time was extended to 10 d, compared to mnHAp, only Sr10-mnHAp and Sr20-mnHAp further enhanced ALP activity.Figure 6ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"qRT–PCR was carried out to analyze the expression of osteogenesis and angiogenesis factors in the cells cultured on HAp, mnHAp and Srx-mnHAp at days 4 and 7. As shown in Figure 7, the mnHAp bioceramics (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) could strengthen the expression of COL1, BSP, BMP-2, OPN, VEGF and ANG-1 with sample S0 (dense and flat surface) compared at 4 and 7 d. Furthermore, Sr doping improved the mRNA levels of COL1, BSP, and OPN at 4 and 7 d and ANG-1 at 7 d. The western bolt result of day 7 confirmed the enhanced expression of BMP-2 in the Srx-mnHAp, especially in the Sr10-mnHAp. More importantly, the promotion effect of Sr doping occurred in a dose-dependent manner, and Sr10-mnHAp achieved the best stimulation ability.Figure 7qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nqRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"Micro-CT scanning and analysis were performed to assess the bone regeneration of the calvarial defect areas. From Figure 8A, we can see that more bone formation in the surface and pores of the mnHAp bioceramics at the 8th week after implantation (p<0.05) and Sr10-mnHAp achieved the best repair effect with S0 and S3 compared (p<0.05). Morphometric analysis further proved that the BV/TV ratio (Figure 8B) and Tb. Th (Figure 8C) in mnHAp bioceramics were higher than that of pure HAp. Moreover, significant differences were found between mnHAp and Sr10-mnHAp (p<0.05). Figure 9 showed the angiographic results, more blood vessels were found in the implant site of the mnHAp bioceramics (p<0.05), and the Sr10-mnHAp groups possessed the most newly formed blood vessels. The results of quantitative analysis also confirmed that samples S3 and 10Sr possessed more blood vessels, and the differences between samples 10Sr and S3 were remarkable (p<0.05).Figure 8Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).Figure 9Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"Van Gieson staining was applied to observe bone regeneration in the surface and pores of the HAp bioceramics. As shown in Figure 10A, bone formation was only found on the edge of the defect in group S0, while groups S3 and 10Sr achieved more new bone formation from the margins to the Center, especially group 10Sr. As shown in Figure 10B, the results of bone histomorphometric analysis further confirmed that groups S3 and 10Sr possessed more newly formed bone areas. More importantly, remarkable differences could be found between groups S3 and 10Sr, which indicates that Sr10-mnHAp has the best effect on promoting bone formation (p<0.05).Figure 10Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nHistological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"The skeleton is an integral part of the human body and has a limited capacity to regenerate after large bone defects caused by acute injuries, fall fractures, or tumors. The repair of bone defects and bone regeneration remain challenging problems in clinical treatment.2 Currently, reconstruction of these skeletal defects is often achieved by autogenous and allogeneic bone transplantation, although they have drawbacks separately. The emergence and development of tissue engineering offers tremendous potential. HAp ceramics are a multifunctional biological material with good biocompatibility and bioactivity but are limited in Clinical application because of their insufficient osteoinduction ability.4 Osteoblast/material interactions play a fundamental role in the biological response of host cells and can be modified by the surface characteristics of bone grafts.26\nNumerous studies have shown that controlling the surface morphology and roughness of materials is a direct and effective strategy to improve biological properties, and surface topography can affect cell attachment and subsequent proliferation and differentiation.9,27–29 Our previous study demonstrated that the microstructure surface can support cell adhesion, which is critical for osteogenic proliferation and differentiation.9 Changing the chemical compositions of the surface by element doping can also improve the osteogenic properties of biomaterials.21 Trace elementSr has been applied to the preparation of bone tissue engineering scaffolds, which can promote bone regeneration and inhibit bone resorption.30 Previous studies showed that appropriate release of Sr ions could activate osteogenesis-related signal transduction pathways, stimulate osteogenesis gene expression and ultimately new bone formation in a dose-dependent manner.31,32 Most of the previous studies mainly focused on the effects and underlying mechanisms of surface modification or chemical composition changes on the biological characteristics. Whether osteogenic differentiation and angiogenesis could be promoted by topographic modification and element doping is still lacking and requires further evaluation.\nHerein, HAp bioceramics with micro-nano-hybrid surfaces and different Sr doping contents (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were synthesized using Srx-α-TCP as a precursor via hydrothermal transformation.20 The XRD patterns confirmed that all the samples were completely transformed into HAp, and the visible micro-nanorods structure on the surface of mnHAp and Srx-mnHAp could be observed by SEM. According to the ICP-AES analysis, the release of Sr ions could be detected in the Srx-mnHAp bioceramics, and the concentration was between 0.2 and 1.0 ug mL−1. Previous Research suggested that the suitable concentration of Sr ions varies from 0.2107 to 21.07 μg mL−1, which indicated that the release of Sr is in an appropriate range.33\nSubsequently, the effects of mnHAp and Srx-mnHAp bioceramics on BMSCs were evaluated in vitro. Previous studies have suggested that the physical properties of biomaterial scaffolds can affect adhesion, migration, and differentiation into osteoblasts.34–37 The cytoskeleton staining assay showed that the cells cultured on the mnHAp exhibited better cell attachment, with typical fibroblastic morphology. However, Sr doping did not significantly promote cell attachment or extension. It indicates that the nanostructure surface, instead of Sr ions, is the Key promotion factor of cellular responses in the early stages. ALP is considered to be an indicator of osteogenic differentiation and an important index of bone formation and turnover. BMP-2 is the strongest osteoinduction cytokine and can promote bone formation and growth. OCN is tightly related to the maturation of osteoblasts,38 and COL1 and BSP are vital genes involved in osteoblastic differentiation. VEGF is the most important factor in angiogenesis, and ANG-1 plays a basic role in promoting maturity and maintaining vascular stabilization. According to the results of MTT, ALP activity, and PCR assays, the mnHAp bioceramics could facilitate proliferation ability, ALP activity and mRNA expression of COL1, BSP, BMP-2, OPN, VEGF, and ANG-1, which is consistent with our previous results.22,39 More importantly, Sr doping furthers promoted cellular osteogenic activity, while Sr10-mnHAp possessed the best stimulatory effect. In addition, the results of calvarial defects confirmed that the mnHAp bioceramics could promote bone and blood vessel regeneration with the control samples (dense and flat surface). More importantly, Sr doping further promoted bone and blood vessel regeneration, while Sr10-mnHAp possessed the most stimulatory effect. All the above results proved our hypothesis that micro-nano-hybrid surface and Sr doping had synergistic promotion effects on bone regeneration, the Srx-mnHAp bioceramics can be a promising material for bone defect repair. Meanwhile, its multifunctional properties make it a good candidate for potential biomedical applications, including targeted drug delivery applications, and the stable and controlled release of drugs is worthy of further investigation.",
"Surface structure and chemical composition are considered to be the key clues to regulate the biological response of biomaterials. Herein, HAp bioceramics with micro-nano-hybrid surface and different Sr doping contents (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were successfully fabricated via a hydrothermal transformation method using Srx-ɑ-TCP powder as a precursor without additives. The nanostructure surface can support cell adhesion, and Sr-doping could further enhance osteogenic differentiation with an optimal doping concentration of 10%. Finally, the calvarial defect model showed that the Sr10-mnHAp bioceramics scaffolds possessed better bone regeneration capacity. All of the results confirmed our hypothesis that micro-nano-hybrid surface and Sr doping have synergistic effects on bone regeneration and provided a new strategy for improving the osteoinduction ability of traditional bioceramics."
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[
"Introduction",
"Materials and Methods",
"Preparation of Micro-Nano-Hybrid HAp (mnHAp) and Sr-Doped mnHAp (Srx-mnHAp) Bioceramics",
"Samples Characterization",
"Cell Extraction and Culture",
"Cell Morphology and Adhesion",
"Cell Proliferation",
"ALP Staining and Activity",
"Quantitative Real-Time PCR (qRT-PCR) and Western Blot Experiment",
"Animal Experiment",
"Micro-CT Analyses",
"Histological Analyses",
"Statistical Analyses",
"Results",
"Characterization of the Samples",
"The Morphology of BMSCs",
"Cell Proliferation",
"ALP Activity Assay",
"qRT–PCR Assay and Western Blot Experiment",
"Micro-CT Measurement",
"Histological and Histomorphometric Analysis",
"Discussion",
"Conclusion"
] |
[
"Bone transplantation is a crucial method to reconstruct bone defects caused by trauma, tumors, and other diseases, and autologous bone grafts remain the “gold standard” for bone transplantation. However, the limitation of insufficient bone mass and related postoperative complications have facilitated the development of substitute materials.1,2 Bioceramic materials are widely used as a physiological scaffold for bone defect repair because of its excellent biocompatibility and bioactivity, and are currently considered as the most promising alternative to autologous bone grafting. The porous structure of bioceramics facilitates the adhesion of osteoblasts, transport of nutrients, and speeds up the process of bone resorption and reconstruction,3 which makes it better for repairing large bone defects.4\nThe inorganic minerals in human bone tissue have a multilevel ordered structure and contain many microelements. From the viewpoint of bio mimics, we can tune the osteoinduction ability of materials by controlling the surface microstructure and element doping. The ideal scaffold materials for bone regeneration require an appropriate three-dimensional microporous structure for cell growth and nutrient metabolism.2,5 For bioceramics scaffolds, the pores are 150 ~ 500 μm in diameter and over 50% in porosity are appropriate to stimulate cell response, and nanostructured surfaces and high connectivity between pores is also important.1,6–8 The results of our recent study confirmed that the HAp bioceramics with nanostructured modified topography significantly enhanced cell viability, spreading, and osteogenic differentiation of BMSCs and bone formation, mineralization and vascularization in vivo.9 Moreover, suitable surface chemistry plays critical roles in cell viability, spreading, proliferation, and osteogenic differentiation.2,5 Biomaterials can be modified by the introduction of functional inorganic ions. Recently, more attention has been given to strontium (Sr), which is an important trace element in the human body, and 99% of Sr exists in bones. Sr can improve the mechanical properties and modify the bone balance toward osteosynthesis.10 Recent studies11 have found that Sr can enhance the proliferation of osteoblasts during bone metabolism. Yang et al12 suggested that the surface of Sr-doped hydroxyapatite can significantly increase the growth of osteoblasts. The results of Wang et al13 confirmed that the Sr-HAp coatings significantly promoted osteoblast attachment and proliferation as the Sr content increased. Recently, researchers focused on the preparation of strontium-substituted bioceramics and their application in bone regeneration.14–16 However, few studies have focused on the synergistic promotion of nanostructures and Sr doping on bone regeneration.\nTherefore, based on the above research and our previous results,9,17 we proposed that the combination of nanostructure surfaces and Sr-doping might lead to synergistic enhancement of osteogenesis and angiogenesis.18,19 Herein, HAp bioceramics with micro-nano-hybrid surfaces and different strontium (Sr) doping contents of 2.5, 5, 10, and 20% (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were fabricated via the hydrothermal transformation method. The effect on cell adhesion, proliferation, and ALP activity and the expression of genes involved in osteogenesis and angiogenesis were investigated to determine the optimal Sr doping content, followed by calvarial defect experiments to evaluate bone regeneration and vascularization in vivo.",
" Preparation of Micro-Nano-Hybrid HAp (mnHAp) and Sr-Doped mnHAp (Srx-mnHAp) Bioceramics The micro-nano-hybrid HAp (mnHAp) and Sr-doped mnHAp (Srx-mnHAp, x: 2.5, 5, 10 and 20%) bioceramics were prepared by the hydrothermal transformation method. First, ɑ-tricalcium phosphate (ɑ-TCP) and Sr-doped ɑ-TCP with different doping contents of 2.5, 5, 10, and 20% (Srx-ɑ-TCP, x: 2.5, 5, 10 and 20%) powders were synthesized by chemical precipitation.20 Then, the synthesized ɑ-TCP and Srx-ɑ-TCP powders were mixed with 7 wt% polyvinyl alcohol (PVA), dry-pressed into discs 10 mm in diameter and 2 mm in thickness under a pressure of 5 MPa, and further calcined at 1050 °C for 5 h to obtain ɑ-TCP and Srx-ɑ-TCP bioceramics, respectively.21 Finally, the mnHAp and Srx-mnHAp bioceramics were prepared using ɑ-TCP and Sr-ɑ-TCP discs as precursors via hydrothermal reaction in pH=7 aqueous solution at 180 °C for 72 h.17,20 In addition, HAp bioceramics with dense and flat surfaces were used as controls and labeled S0.9\nThe micro-nano-hybrid HAp (mnHAp) and Sr-doped mnHAp (Srx-mnHAp, x: 2.5, 5, 10 and 20%) bioceramics were prepared by the hydrothermal transformation method. First, ɑ-tricalcium phosphate (ɑ-TCP) and Sr-doped ɑ-TCP with different doping contents of 2.5, 5, 10, and 20% (Srx-ɑ-TCP, x: 2.5, 5, 10 and 20%) powders were synthesized by chemical precipitation.20 Then, the synthesized ɑ-TCP and Srx-ɑ-TCP powders were mixed with 7 wt% polyvinyl alcohol (PVA), dry-pressed into discs 10 mm in diameter and 2 mm in thickness under a pressure of 5 MPa, and further calcined at 1050 °C for 5 h to obtain ɑ-TCP and Srx-ɑ-TCP bioceramics, respectively.21 Finally, the mnHAp and Srx-mnHAp bioceramics were prepared using ɑ-TCP and Sr-ɑ-TCP discs as precursors via hydrothermal reaction in pH=7 aqueous solution at 180 °C for 72 h.17,20 In addition, HAp bioceramics with dense and flat surfaces were used as controls and labeled S0.9\n Samples Characterization HAp (S0), mnHAp (S3), and Srx-mnHAp (x: 2.5, 5, 10 and 20%) were incubated in 1 mL of α-Minimum Essential Medium (ɑ-MEM, Gibco, USA) for 1 and 4 d, respectively. The medium of each sample changed daily and the concentrations of Sr, Ca, and P ions released from HAp, mnHAp, and Srx-mnHAp at each timepoint were evaluated using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The crystal phase compositions of the samples were determined with XRD (Rigaku, Japan), and the surface microstructures were observed by SEM (JEOL, Japan).17\nHAp (S0), mnHAp (S3), and Srx-mnHAp (x: 2.5, 5, 10 and 20%) were incubated in 1 mL of α-Minimum Essential Medium (ɑ-MEM, Gibco, USA) for 1 and 4 d, respectively. The medium of each sample changed daily and the concentrations of Sr, Ca, and P ions released from HAp, mnHAp, and Srx-mnHAp at each timepoint were evaluated using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The crystal phase compositions of the samples were determined with XRD (Rigaku, Japan), and the surface microstructures were observed by SEM (JEOL, Japan).17\n Cell Extraction and Culture Bone marrow stem cells (BMSCs) were harvested as previously described.17 Briefly, Sprague–Dawley (SD) rats (4 weeks old) were selected and injected intraperitoneally with an overdose of pentobarbital sodium. The scalp was separated for exposure, and both femora were cut off. Fresh marrow was flushed out and cultured in ɑ-MEM (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin and streptomycin (Hyclone, USA). The culture medium was maintained at 37 °C in a 5% CO2 incubator and renewed 3 times a week. The cells were passaged after reaching 90% confluence, and passages 2–4 were used in the following experiment. All animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine and conducted in compliance with the guidelines of Institutional Animal Care and Use Committee of Shanghai Ninth People’s Hospital Affiliated to Shanghai JiaoTong University, School of Medicine [SYXK (Hu) 2016–0016].\nBone marrow stem cells (BMSCs) were harvested as previously described.17 Briefly, Sprague–Dawley (SD) rats (4 weeks old) were selected and injected intraperitoneally with an overdose of pentobarbital sodium. The scalp was separated for exposure, and both femora were cut off. Fresh marrow was flushed out and cultured in ɑ-MEM (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin and streptomycin (Hyclone, USA). The culture medium was maintained at 37 °C in a 5% CO2 incubator and renewed 3 times a week. The cells were passaged after reaching 90% confluence, and passages 2–4 were used in the following experiment. All animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine and conducted in compliance with the guidelines of Institutional Animal Care and Use Committee of Shanghai Ninth People’s Hospital Affiliated to Shanghai JiaoTong University, School of Medicine [SYXK (Hu) 2016–0016].\n Cell Morphology and Adhesion Phalloidin staining was performed to evaluate the morphology and spreading of the BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at an initial density of 1×104 cells per mL in 24-well plates. At 6 h after seeding, all specimens were fixed with 4% formaldehyde for 10 min, and then treated with 1% Triton X-100 for 5 min. Next, these specimens were incubated with phalloidin (Sigma, USA) for 30 min, followed by DAPI (Sigma, USA) for approximately 30s. Finally, actin cytoskeletons were observed by fluorescence microscopy (Leica, Germany).\nPhalloidin staining was performed to evaluate the morphology and spreading of the BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at an initial density of 1×104 cells per mL in 24-well plates. At 6 h after seeding, all specimens were fixed with 4% formaldehyde for 10 min, and then treated with 1% Triton X-100 for 5 min. Next, these specimens were incubated with phalloidin (Sigma, USA) for 30 min, followed by DAPI (Sigma, USA) for approximately 30s. Finally, actin cytoskeletons were observed by fluorescence microscopy (Leica, Germany).\n Cell Proliferation The proliferation of BMSCs in the samples was investigated by MTT assay, with a cell density of 1×104 cells per mL in 24-well plates. After culturing for 1, 4, and 7 d, the culture medium was replaced, and the BMSC-seeded bioceramics were rinsed and incubated with sterile MTT (Amresco, USA) at 37 °C for 4 h. DMSO solution (Sigma, USA) was then added and incubated for 10 min to dissolve formazan. The OD value was read at 490 nm by a microplate Reader (Biotek, USA). All experiments were repeated 3 times.\nThe proliferation of BMSCs in the samples was investigated by MTT assay, with a cell density of 1×104 cells per mL in 24-well plates. After culturing for 1, 4, and 7 d, the culture medium was replaced, and the BMSC-seeded bioceramics were rinsed and incubated with sterile MTT (Amresco, USA) at 37 °C for 4 h. DMSO solution (Sigma, USA) was then added and incubated for 10 min to dissolve formazan. The OD value was read at 490 nm by a microplate Reader (Biotek, USA). All experiments were repeated 3 times.\n ALP Staining and Activity BMSCs were cultured onto S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr samples at a cell density of 1×104 cells per mL in 24-well plates. After culturing for 10 d, ALP staining was performed with a BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). The ALP activity was then assessed at 4, 7, and 10 d.22 Briefly, all samples were lysed in 1% Triton X-100 for 30 min at 4 °C, and the cellular lysate of each well was collected and centrifuged at 4 °C (12,000 rpm × 10 min). Then, the OD value was determined at 520 nm with a microplate Reader (Bio-Tek, USA), and total cellular protein was determined by a BCA protein kit (Beyotime, China) at 562 nm. Finally, ALP activity was evaluated by normalizing to total protein content. All experiments were repeated 3 times.\nBMSCs were cultured onto S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr samples at a cell density of 1×104 cells per mL in 24-well plates. After culturing for 10 d, ALP staining was performed with a BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). The ALP activity was then assessed at 4, 7, and 10 d.22 Briefly, all samples were lysed in 1% Triton X-100 for 30 min at 4 °C, and the cellular lysate of each well was collected and centrifuged at 4 °C (12,000 rpm × 10 min). Then, the OD value was determined at 520 nm with a microplate Reader (Bio-Tek, USA), and total cellular protein was determined by a BCA protein kit (Beyotime, China) at 562 nm. Finally, ALP activity was evaluated by normalizing to total protein content. All experiments were repeated 3 times.\n Quantitative Real-Time PCR (qRT-PCR) and Western Blot Experiment qRT-PCR was carried out to assess the expression of osteogenic and angiogenic factors in BMSCs cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr. After culturing for 4 and 7 d, total RNA of the samples was isolated according to our previous study.21 Briefly, the cells cultured in the bioceramics were lysed in TRIzol Reagent (Life Technologies, USA) and reverse transcribed to cDNA by PrimeScriptTM RT reagent Kit (TaKaRa, Japan). The expression levels of osteogenesis- and angiogenesis-related genes were further evaluated by qRT-PCR analysis, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. GAPDH was selected as a housekeeping gene to normalize the expression level. The primer sequences selected are listed in Table 1. All experiments were repeated 3 times.Table 1Primer Sequences of the Selected GenesTarget GenePrimers (F = Forward; R = Reverse)Accession NumberProduct Size (bp)COL1F:5ʹCTGCCCAGAAGAATATGTATCACC3’R:5ʹGAAGCAAAGTTTCCTCCAAGACC3’NM_053304.1198BSPF: 5ʹAGAAAGAGCAGCACGGTTGAGT3’R: 5ʹGACCCTCGTAGCCTTCATAGCC3’NM_012587.2175BMP-2F: 5ʹTGGGTTTGTGGTGGAAGTGGC3’R: 5ʹTGGATGTCCTTTACCGTCGTG3’NM_017178.2154OPNF: 5ʹCCAAGCGTGGAAACACACAGCC3’R: 5ʹGGCTTTGGAACTCGCCTGACTG3’NM_012881.2165VEGFF: 5′GGCTCTGAAACCATGAACTTTCT3′R: 5′GCAGTAGCTGCGCTGGTAGAC3′NM_001110334.2165ANG-1F:5′GGACAGCAGGCAAACAGAGCAGC3′R: 5′CCACAGGCATCAAACCACCAACC3′NM_053546.2130GAPDHF: 5ʹCCTGCACCACCAACTGCTTA3’R: 5ʹGGCCATCCACAGTCTTCTGAG3’NM_017008.4120\n\nPrimer Sequences of the Selected Genes\nWestern blot experiment was applied to assess expression of BMP-2. BMSCs were cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr for 7 d. Total protein was extracted and centrifuged at 12,000 rpm for 15 minutes. Then, the samples were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 volts for 20 minutes and 120 volts for 50 minutes, and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The PVDF membranes were incubated with primary antibody (Abcam, UK) at 37 °C for 2h. After that, the membranes were incubated with HRP-conjugated secondary antibody (Abcam, UK) for 1 h at room temperature. The protein bands were visualized, and the images were captured by an automated luminescent image analysis system (Tanon, China).\nqRT-PCR was carried out to assess the expression of osteogenic and angiogenic factors in BMSCs cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr. After culturing for 4 and 7 d, total RNA of the samples was isolated according to our previous study.21 Briefly, the cells cultured in the bioceramics were lysed in TRIzol Reagent (Life Technologies, USA) and reverse transcribed to cDNA by PrimeScriptTM RT reagent Kit (TaKaRa, Japan). The expression levels of osteogenesis- and angiogenesis-related genes were further evaluated by qRT-PCR analysis, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. GAPDH was selected as a housekeeping gene to normalize the expression level. The primer sequences selected are listed in Table 1. All experiments were repeated 3 times.Table 1Primer Sequences of the Selected GenesTarget GenePrimers (F = Forward; R = Reverse)Accession NumberProduct Size (bp)COL1F:5ʹCTGCCCAGAAGAATATGTATCACC3’R:5ʹGAAGCAAAGTTTCCTCCAAGACC3’NM_053304.1198BSPF: 5ʹAGAAAGAGCAGCACGGTTGAGT3’R: 5ʹGACCCTCGTAGCCTTCATAGCC3’NM_012587.2175BMP-2F: 5ʹTGGGTTTGTGGTGGAAGTGGC3’R: 5ʹTGGATGTCCTTTACCGTCGTG3’NM_017178.2154OPNF: 5ʹCCAAGCGTGGAAACACACAGCC3’R: 5ʹGGCTTTGGAACTCGCCTGACTG3’NM_012881.2165VEGFF: 5′GGCTCTGAAACCATGAACTTTCT3′R: 5′GCAGTAGCTGCGCTGGTAGAC3′NM_001110334.2165ANG-1F:5′GGACAGCAGGCAAACAGAGCAGC3′R: 5′CCACAGGCATCAAACCACCAACC3′NM_053546.2130GAPDHF: 5ʹCCTGCACCACCAACTGCTTA3’R: 5ʹGGCCATCCACAGTCTTCTGAG3’NM_017008.4120\n\nPrimer Sequences of the Selected Genes\nWestern blot experiment was applied to assess expression of BMP-2. BMSCs were cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr for 7 d. Total protein was extracted and centrifuged at 12,000 rpm for 15 minutes. Then, the samples were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 volts for 20 minutes and 120 volts for 50 minutes, and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The PVDF membranes were incubated with primary antibody (Abcam, UK) at 37 °C for 2h. After that, the membranes were incubated with HRP-conjugated secondary antibody (Abcam, UK) for 1 h at room temperature. The protein bands were visualized, and the images were captured by an automated luminescent image analysis system (Tanon, China).\n Animal Experiment Calvarial defects were established in twelve 6-week-old SD rats as previously described.17 Animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine. All rats were randomly divided into the following 3 groups: S0, S3, and 10Sr (n = 4 for each group). Briefly, all rats were injected intraperitoneally with pentobarbital (3.5 mg/100 g). Under sterile conditions, a 2 cm longitudinal incision was made, and two symmetrical round defects (5 mm in diameter) were created using the implant drill at each rat. Subsequently, the HAp, mnHAp, and Sr10-mnHAp were placed into the defects, and the operation incision was carefully sutured with absorbable sutures.\nCalvarial defects were established in twelve 6-week-old SD rats as previously described.17 Animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine. All rats were randomly divided into the following 3 groups: S0, S3, and 10Sr (n = 4 for each group). Briefly, all rats were injected intraperitoneally with pentobarbital (3.5 mg/100 g). Under sterile conditions, a 2 cm longitudinal incision was made, and two symmetrical round defects (5 mm in diameter) were created using the implant drill at each rat. Subsequently, the HAp, mnHAp, and Sr10-mnHAp were placed into the defects, and the operation incision was carefully sutured with absorbable sutures.\n Micro-CT Analyses At the 8th week after implantation, all rats were sacrificed and perfused with Microfil (Flowtech, USA) to observe new blood vessels.23 The samples were decalcified using EDTA for 1 month and then scanned by microcomputed tomography (Scanco, Switzerland), the percentages of new blood vessels were quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA). For the observation of new bone, the specimens were fixed in 4% paraformaldehyde for 2 d and then transferred to 75% alcohol. Finally, the samples were scanned by microcomputed tomography. Bone volume fraction (BV/TV) and trabecular thickness (Tb. Th) were calculated with auxiliary software (Scanco, Switzerland).24\nAt the 8th week after implantation, all rats were sacrificed and perfused with Microfil (Flowtech, USA) to observe new blood vessels.23 The samples were decalcified using EDTA for 1 month and then scanned by microcomputed tomography (Scanco, Switzerland), the percentages of new blood vessels were quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA). For the observation of new bone, the specimens were fixed in 4% paraformaldehyde for 2 d and then transferred to 75% alcohol. Finally, the samples were scanned by microcomputed tomography. Bone volume fraction (BV/TV) and trabecular thickness (Tb. Th) were calculated with auxiliary software (Scanco, Switzerland).24\n Histological Analyses All specimens were decalcified with 10% EDTA, dehydrated in increased concentrations of ethanol (75% to 100%) and embedded in PMMA for 3 weeks. Tissue blocks were sectioned by a microtome (Leica, Germany) and further ground to approximately 40 μm. Next, the sections were subjected to Van Gieson staining for histological analysis, and the proportion of bone regeneration in the defect area was quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA).17,25\nAll specimens were decalcified with 10% EDTA, dehydrated in increased concentrations of ethanol (75% to 100%) and embedded in PMMA for 3 weeks. Tissue blocks were sectioned by a microtome (Leica, Germany) and further ground to approximately 40 μm. Next, the sections were subjected to Van Gieson staining for histological analysis, and the proportion of bone regeneration in the defect area was quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA).17,25\n Statistical Analyses All data are expressed as the means ± standard deviation (SD), and statistical analysis was performed using SPSS 17.0 software (SPSS Inc., USA). A p value <0.05 was considered statistically significant.\nAll data are expressed as the means ± standard deviation (SD), and statistical analysis was performed using SPSS 17.0 software (SPSS Inc., USA). A p value <0.05 was considered statistically significant.",
"The micro-nano-hybrid HAp (mnHAp) and Sr-doped mnHAp (Srx-mnHAp, x: 2.5, 5, 10 and 20%) bioceramics were prepared by the hydrothermal transformation method. First, ɑ-tricalcium phosphate (ɑ-TCP) and Sr-doped ɑ-TCP with different doping contents of 2.5, 5, 10, and 20% (Srx-ɑ-TCP, x: 2.5, 5, 10 and 20%) powders were synthesized by chemical precipitation.20 Then, the synthesized ɑ-TCP and Srx-ɑ-TCP powders were mixed with 7 wt% polyvinyl alcohol (PVA), dry-pressed into discs 10 mm in diameter and 2 mm in thickness under a pressure of 5 MPa, and further calcined at 1050 °C for 5 h to obtain ɑ-TCP and Srx-ɑ-TCP bioceramics, respectively.21 Finally, the mnHAp and Srx-mnHAp bioceramics were prepared using ɑ-TCP and Sr-ɑ-TCP discs as precursors via hydrothermal reaction in pH=7 aqueous solution at 180 °C for 72 h.17,20 In addition, HAp bioceramics with dense and flat surfaces were used as controls and labeled S0.9",
"HAp (S0), mnHAp (S3), and Srx-mnHAp (x: 2.5, 5, 10 and 20%) were incubated in 1 mL of α-Minimum Essential Medium (ɑ-MEM, Gibco, USA) for 1 and 4 d, respectively. The medium of each sample changed daily and the concentrations of Sr, Ca, and P ions released from HAp, mnHAp, and Srx-mnHAp at each timepoint were evaluated using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The crystal phase compositions of the samples were determined with XRD (Rigaku, Japan), and the surface microstructures were observed by SEM (JEOL, Japan).17",
"Bone marrow stem cells (BMSCs) were harvested as previously described.17 Briefly, Sprague–Dawley (SD) rats (4 weeks old) were selected and injected intraperitoneally with an overdose of pentobarbital sodium. The scalp was separated for exposure, and both femora were cut off. Fresh marrow was flushed out and cultured in ɑ-MEM (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin and streptomycin (Hyclone, USA). The culture medium was maintained at 37 °C in a 5% CO2 incubator and renewed 3 times a week. The cells were passaged after reaching 90% confluence, and passages 2–4 were used in the following experiment. All animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine and conducted in compliance with the guidelines of Institutional Animal Care and Use Committee of Shanghai Ninth People’s Hospital Affiliated to Shanghai JiaoTong University, School of Medicine [SYXK (Hu) 2016–0016].",
"Phalloidin staining was performed to evaluate the morphology and spreading of the BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at an initial density of 1×104 cells per mL in 24-well plates. At 6 h after seeding, all specimens were fixed with 4% formaldehyde for 10 min, and then treated with 1% Triton X-100 for 5 min. Next, these specimens were incubated with phalloidin (Sigma, USA) for 30 min, followed by DAPI (Sigma, USA) for approximately 30s. Finally, actin cytoskeletons were observed by fluorescence microscopy (Leica, Germany).",
"The proliferation of BMSCs in the samples was investigated by MTT assay, with a cell density of 1×104 cells per mL in 24-well plates. After culturing for 1, 4, and 7 d, the culture medium was replaced, and the BMSC-seeded bioceramics were rinsed and incubated with sterile MTT (Amresco, USA) at 37 °C for 4 h. DMSO solution (Sigma, USA) was then added and incubated for 10 min to dissolve formazan. The OD value was read at 490 nm by a microplate Reader (Biotek, USA). All experiments were repeated 3 times.",
"BMSCs were cultured onto S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr samples at a cell density of 1×104 cells per mL in 24-well plates. After culturing for 10 d, ALP staining was performed with a BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). The ALP activity was then assessed at 4, 7, and 10 d.22 Briefly, all samples were lysed in 1% Triton X-100 for 30 min at 4 °C, and the cellular lysate of each well was collected and centrifuged at 4 °C (12,000 rpm × 10 min). Then, the OD value was determined at 520 nm with a microplate Reader (Bio-Tek, USA), and total cellular protein was determined by a BCA protein kit (Beyotime, China) at 562 nm. Finally, ALP activity was evaluated by normalizing to total protein content. All experiments were repeated 3 times.",
"qRT-PCR was carried out to assess the expression of osteogenic and angiogenic factors in BMSCs cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr. After culturing for 4 and 7 d, total RNA of the samples was isolated according to our previous study.21 Briefly, the cells cultured in the bioceramics were lysed in TRIzol Reagent (Life Technologies, USA) and reverse transcribed to cDNA by PrimeScriptTM RT reagent Kit (TaKaRa, Japan). The expression levels of osteogenesis- and angiogenesis-related genes were further evaluated by qRT-PCR analysis, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. GAPDH was selected as a housekeeping gene to normalize the expression level. The primer sequences selected are listed in Table 1. All experiments were repeated 3 times.Table 1Primer Sequences of the Selected GenesTarget GenePrimers (F = Forward; R = Reverse)Accession NumberProduct Size (bp)COL1F:5ʹCTGCCCAGAAGAATATGTATCACC3’R:5ʹGAAGCAAAGTTTCCTCCAAGACC3’NM_053304.1198BSPF: 5ʹAGAAAGAGCAGCACGGTTGAGT3’R: 5ʹGACCCTCGTAGCCTTCATAGCC3’NM_012587.2175BMP-2F: 5ʹTGGGTTTGTGGTGGAAGTGGC3’R: 5ʹTGGATGTCCTTTACCGTCGTG3’NM_017178.2154OPNF: 5ʹCCAAGCGTGGAAACACACAGCC3’R: 5ʹGGCTTTGGAACTCGCCTGACTG3’NM_012881.2165VEGFF: 5′GGCTCTGAAACCATGAACTTTCT3′R: 5′GCAGTAGCTGCGCTGGTAGAC3′NM_001110334.2165ANG-1F:5′GGACAGCAGGCAAACAGAGCAGC3′R: 5′CCACAGGCATCAAACCACCAACC3′NM_053546.2130GAPDHF: 5ʹCCTGCACCACCAACTGCTTA3’R: 5ʹGGCCATCCACAGTCTTCTGAG3’NM_017008.4120\n\nPrimer Sequences of the Selected Genes\nWestern blot experiment was applied to assess expression of BMP-2. BMSCs were cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr for 7 d. Total protein was extracted and centrifuged at 12,000 rpm for 15 minutes. Then, the samples were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 volts for 20 minutes and 120 volts for 50 minutes, and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The PVDF membranes were incubated with primary antibody (Abcam, UK) at 37 °C for 2h. After that, the membranes were incubated with HRP-conjugated secondary antibody (Abcam, UK) for 1 h at room temperature. The protein bands were visualized, and the images were captured by an automated luminescent image analysis system (Tanon, China).",
"Calvarial defects were established in twelve 6-week-old SD rats as previously described.17 Animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine. All rats were randomly divided into the following 3 groups: S0, S3, and 10Sr (n = 4 for each group). Briefly, all rats were injected intraperitoneally with pentobarbital (3.5 mg/100 g). Under sterile conditions, a 2 cm longitudinal incision was made, and two symmetrical round defects (5 mm in diameter) were created using the implant drill at each rat. Subsequently, the HAp, mnHAp, and Sr10-mnHAp were placed into the defects, and the operation incision was carefully sutured with absorbable sutures.",
"At the 8th week after implantation, all rats were sacrificed and perfused with Microfil (Flowtech, USA) to observe new blood vessels.23 The samples were decalcified using EDTA for 1 month and then scanned by microcomputed tomography (Scanco, Switzerland), the percentages of new blood vessels were quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA). For the observation of new bone, the specimens were fixed in 4% paraformaldehyde for 2 d and then transferred to 75% alcohol. Finally, the samples were scanned by microcomputed tomography. Bone volume fraction (BV/TV) and trabecular thickness (Tb. Th) were calculated with auxiliary software (Scanco, Switzerland).24",
"All specimens were decalcified with 10% EDTA, dehydrated in increased concentrations of ethanol (75% to 100%) and embedded in PMMA for 3 weeks. Tissue blocks were sectioned by a microtome (Leica, Germany) and further ground to approximately 40 μm. Next, the sections were subjected to Van Gieson staining for histological analysis, and the proportion of bone regeneration in the defect area was quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA).17,25",
"All data are expressed as the means ± standard deviation (SD), and statistical analysis was performed using SPSS 17.0 software (SPSS Inc., USA). A p value <0.05 was considered statistically significant.",
" Characterization of the Samples XRD results of mnHAp and Srx-mnHAp (x: 2.5, 5, 10 and 20%) are shown in Figure 1. All the diffraction peaks confirmed that these samples were converted into HAp without any other phases after the hydrothermal transformation method. The obvious micro-nanorods topography surfaces of mnHAp (Figure 2B) and Srx-mnHAp (Figure 2C–F) could be observed by SEM, and Sr10-mnHAp possessed the densest rod-shaped structure (Figure 2E). The lengths of the microrods and nanorods were approximately 10~20 μm, while the diameters were approximately 1~4 μm and 80~120 nm, respectively. As a comparison, a dense and flat surface with a particle size of approximately 0.9 µm was observed on control sample S0 (Figure 2A).Figure 1XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).Figure 2SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nXRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).\nSEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nThe results of ICP-AES analysis revealed that the release of Sr ions could be detected in Srx-mnHAp bioceramics and showed an ascending trend with increasing Sr content at each timepoint (Figure 3C). No significant differences in the release amount of Ca and P could be found among HAp, mnHAp, and Srx-mnHAp (Figure 3A and B).Figure 3The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.\nThe release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.\nXRD results of mnHAp and Srx-mnHAp (x: 2.5, 5, 10 and 20%) are shown in Figure 1. All the diffraction peaks confirmed that these samples were converted into HAp without any other phases after the hydrothermal transformation method. The obvious micro-nanorods topography surfaces of mnHAp (Figure 2B) and Srx-mnHAp (Figure 2C–F) could be observed by SEM, and Sr10-mnHAp possessed the densest rod-shaped structure (Figure 2E). The lengths of the microrods and nanorods were approximately 10~20 μm, while the diameters were approximately 1~4 μm and 80~120 nm, respectively. As a comparison, a dense and flat surface with a particle size of approximately 0.9 µm was observed on control sample S0 (Figure 2A).Figure 1XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).Figure 2SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nXRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).\nSEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nThe results of ICP-AES analysis revealed that the release of Sr ions could be detected in Srx-mnHAp bioceramics and showed an ascending trend with increasing Sr content at each timepoint (Figure 3C). No significant differences in the release amount of Ca and P could be found among HAp, mnHAp, and Srx-mnHAp (Figure 3A and B).Figure 3The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.\nThe release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.\n The Morphology of BMSCs As shown in Figure 4, the actin cytoskeleton was stained red, and the nucleus was stained blue. After being cultured for 6 h, fluorescence microscopy images showed that the cells seeded in sample S0 spread slightly and almost had a round shape (Figure 4A1). Meanwhile, as a comparison, the cells seeded on the mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) exhibited better early cell attachment and demonstrated a typical polygonal morphology with apparent stretch tentacles. No significant differences in the cell morphology and quantity could be found with increasing Sr content. This experiment showed that the nanostructure surface could promote the early cell attachment of BMSCs instead of the concentrations of Sr ions.Figure 4Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.\nFluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.\nAs shown in Figure 4, the actin cytoskeleton was stained red, and the nucleus was stained blue. After being cultured for 6 h, fluorescence microscopy images showed that the cells seeded in sample S0 spread slightly and almost had a round shape (Figure 4A1). Meanwhile, as a comparison, the cells seeded on the mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) exhibited better early cell attachment and demonstrated a typical polygonal morphology with apparent stretch tentacles. No significant differences in the cell morphology and quantity could be found with increasing Sr content. This experiment showed that the nanostructure surface could promote the early cell attachment of BMSCs instead of the concentrations of Sr ions.Figure 4Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.\nFluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.\n Cell Proliferation The cells adhered to the HAp, mnHAp and Srx-mnHAp bioceramics proliferated through the MTT assay (Figure 5). The cells cultured on mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) displayed higher proliferation than control samples on days 4 and 7. (p<0.05) In addition, among the Srx-mnHAp with different Sr contents, proliferation significantly proceeded on Sr10-mnHAp and Sr20-mnHAp on days 4 and 7 (p<0.05).Figure 5MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nThe cells adhered to the HAp, mnHAp and Srx-mnHAp bioceramics proliferated through the MTT assay (Figure 5). The cells cultured on mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) displayed higher proliferation than control samples on days 4 and 7. (p<0.05) In addition, among the Srx-mnHAp with different Sr contents, proliferation significantly proceeded on Sr10-mnHAp and Sr20-mnHAp on days 4 and 7 (p<0.05).Figure 5MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\n ALP Activity Assay The cells cultured on mnHAp bioceramics possessed more intensive ALP staining, with control samples (dense and flat surface) compared (Figure 6A). Moreover, the staining became deeper with Sr doping (2.5Sr, 5Sr, 10Sr, and 20Sr). In particular, the deepest staining was found on the Sr10-mnHAp sample. The ALP semi-quantification assay on days 4, 7 and 10 further confirmed the staining results (Figure 6B). The ALP activity increased throughout the whole assay period, and the mnHAp bioceramics possessed higher cellular ALP activity. More importantly, the samples with Sr doping achieved better ALP activity on days 4 and 7 (p<0.05). When the experiment time was extended to 10 d, compared to mnHAp, only Sr10-mnHAp and Sr20-mnHAp further enhanced ALP activity.Figure 6ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nThe cells cultured on mnHAp bioceramics possessed more intensive ALP staining, with control samples (dense and flat surface) compared (Figure 6A). Moreover, the staining became deeper with Sr doping (2.5Sr, 5Sr, 10Sr, and 20Sr). In particular, the deepest staining was found on the Sr10-mnHAp sample. The ALP semi-quantification assay on days 4, 7 and 10 further confirmed the staining results (Figure 6B). The ALP activity increased throughout the whole assay period, and the mnHAp bioceramics possessed higher cellular ALP activity. More importantly, the samples with Sr doping achieved better ALP activity on days 4 and 7 (p<0.05). When the experiment time was extended to 10 d, compared to mnHAp, only Sr10-mnHAp and Sr20-mnHAp further enhanced ALP activity.Figure 6ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\n qRT–PCR Assay and Western Blot Experiment qRT–PCR was carried out to analyze the expression of osteogenesis and angiogenesis factors in the cells cultured on HAp, mnHAp and Srx-mnHAp at days 4 and 7. As shown in Figure 7, the mnHAp bioceramics (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) could strengthen the expression of COL1, BSP, BMP-2, OPN, VEGF and ANG-1 with sample S0 (dense and flat surface) compared at 4 and 7 d. Furthermore, Sr doping improved the mRNA levels of COL1, BSP, and OPN at 4 and 7 d and ANG-1 at 7 d. The western bolt result of day 7 confirmed the enhanced expression of BMP-2 in the Srx-mnHAp, especially in the Sr10-mnHAp. More importantly, the promotion effect of Sr doping occurred in a dose-dependent manner, and Sr10-mnHAp achieved the best stimulation ability.Figure 7qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nqRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nqRT–PCR was carried out to analyze the expression of osteogenesis and angiogenesis factors in the cells cultured on HAp, mnHAp and Srx-mnHAp at days 4 and 7. As shown in Figure 7, the mnHAp bioceramics (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) could strengthen the expression of COL1, BSP, BMP-2, OPN, VEGF and ANG-1 with sample S0 (dense and flat surface) compared at 4 and 7 d. Furthermore, Sr doping improved the mRNA levels of COL1, BSP, and OPN at 4 and 7 d and ANG-1 at 7 d. The western bolt result of day 7 confirmed the enhanced expression of BMP-2 in the Srx-mnHAp, especially in the Sr10-mnHAp. More importantly, the promotion effect of Sr doping occurred in a dose-dependent manner, and Sr10-mnHAp achieved the best stimulation ability.Figure 7qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nqRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\n Micro-CT Measurement Micro-CT scanning and analysis were performed to assess the bone regeneration of the calvarial defect areas. From Figure 8A, we can see that more bone formation in the surface and pores of the mnHAp bioceramics at the 8th week after implantation (p<0.05) and Sr10-mnHAp achieved the best repair effect with S0 and S3 compared (p<0.05). Morphometric analysis further proved that the BV/TV ratio (Figure 8B) and Tb. Th (Figure 8C) in mnHAp bioceramics were higher than that of pure HAp. Moreover, significant differences were found between mnHAp and Sr10-mnHAp (p<0.05). Figure 9 showed the angiographic results, more blood vessels were found in the implant site of the mnHAp bioceramics (p<0.05), and the Sr10-mnHAp groups possessed the most newly formed blood vessels. The results of quantitative analysis also confirmed that samples S3 and 10Sr possessed more blood vessels, and the differences between samples 10Sr and S3 were remarkable (p<0.05).Figure 8Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).Figure 9Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT scanning and analysis were performed to assess the bone regeneration of the calvarial defect areas. From Figure 8A, we can see that more bone formation in the surface and pores of the mnHAp bioceramics at the 8th week after implantation (p<0.05) and Sr10-mnHAp achieved the best repair effect with S0 and S3 compared (p<0.05). Morphometric analysis further proved that the BV/TV ratio (Figure 8B) and Tb. Th (Figure 8C) in mnHAp bioceramics were higher than that of pure HAp. Moreover, significant differences were found between mnHAp and Sr10-mnHAp (p<0.05). Figure 9 showed the angiographic results, more blood vessels were found in the implant site of the mnHAp bioceramics (p<0.05), and the Sr10-mnHAp groups possessed the most newly formed blood vessels. The results of quantitative analysis also confirmed that samples S3 and 10Sr possessed more blood vessels, and the differences between samples 10Sr and S3 were remarkable (p<0.05).Figure 8Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).Figure 9Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\n Histological and Histomorphometric Analysis Van Gieson staining was applied to observe bone regeneration in the surface and pores of the HAp bioceramics. As shown in Figure 10A, bone formation was only found on the edge of the defect in group S0, while groups S3 and 10Sr achieved more new bone formation from the margins to the Center, especially group 10Sr. As shown in Figure 10B, the results of bone histomorphometric analysis further confirmed that groups S3 and 10Sr possessed more newly formed bone areas. More importantly, remarkable differences could be found between groups S3 and 10Sr, which indicates that Sr10-mnHAp has the best effect on promoting bone formation (p<0.05).Figure 10Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nHistological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nVan Gieson staining was applied to observe bone regeneration in the surface and pores of the HAp bioceramics. As shown in Figure 10A, bone formation was only found on the edge of the defect in group S0, while groups S3 and 10Sr achieved more new bone formation from the margins to the Center, especially group 10Sr. As shown in Figure 10B, the results of bone histomorphometric analysis further confirmed that groups S3 and 10Sr possessed more newly formed bone areas. More importantly, remarkable differences could be found between groups S3 and 10Sr, which indicates that Sr10-mnHAp has the best effect on promoting bone formation (p<0.05).Figure 10Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nHistological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"XRD results of mnHAp and Srx-mnHAp (x: 2.5, 5, 10 and 20%) are shown in Figure 1. All the diffraction peaks confirmed that these samples were converted into HAp without any other phases after the hydrothermal transformation method. The obvious micro-nanorods topography surfaces of mnHAp (Figure 2B) and Srx-mnHAp (Figure 2C–F) could be observed by SEM, and Sr10-mnHAp possessed the densest rod-shaped structure (Figure 2E). The lengths of the microrods and nanorods were approximately 10~20 μm, while the diameters were approximately 1~4 μm and 80~120 nm, respectively. As a comparison, a dense and flat surface with a particle size of approximately 0.9 µm was observed on control sample S0 (Figure 2A).Figure 1XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).Figure 2SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nXRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).\nSEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.\nThe results of ICP-AES analysis revealed that the release of Sr ions could be detected in Srx-mnHAp bioceramics and showed an ascending trend with increasing Sr content at each timepoint (Figure 3C). No significant differences in the release amount of Ca and P could be found among HAp, mnHAp, and Srx-mnHAp (Figure 3A and B).Figure 3The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.\nThe release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.",
"As shown in Figure 4, the actin cytoskeleton was stained red, and the nucleus was stained blue. After being cultured for 6 h, fluorescence microscopy images showed that the cells seeded in sample S0 spread slightly and almost had a round shape (Figure 4A1). Meanwhile, as a comparison, the cells seeded on the mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) exhibited better early cell attachment and demonstrated a typical polygonal morphology with apparent stretch tentacles. No significant differences in the cell morphology and quantity could be found with increasing Sr content. This experiment showed that the nanostructure surface could promote the early cell attachment of BMSCs instead of the concentrations of Sr ions.Figure 4Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.\nFluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.",
"The cells adhered to the HAp, mnHAp and Srx-mnHAp bioceramics proliferated through the MTT assay (Figure 5). The cells cultured on mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) displayed higher proliferation than control samples on days 4 and 7. (p<0.05) In addition, among the Srx-mnHAp with different Sr contents, proliferation significantly proceeded on Sr10-mnHAp and Sr20-mnHAp on days 4 and 7 (p<0.05).Figure 5MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"The cells cultured on mnHAp bioceramics possessed more intensive ALP staining, with control samples (dense and flat surface) compared (Figure 6A). Moreover, the staining became deeper with Sr doping (2.5Sr, 5Sr, 10Sr, and 20Sr). In particular, the deepest staining was found on the Sr10-mnHAp sample. The ALP semi-quantification assay on days 4, 7 and 10 further confirmed the staining results (Figure 6B). The ALP activity increased throughout the whole assay period, and the mnHAp bioceramics possessed higher cellular ALP activity. More importantly, the samples with Sr doping achieved better ALP activity on days 4 and 7 (p<0.05). When the experiment time was extended to 10 d, compared to mnHAp, only Sr10-mnHAp and Sr20-mnHAp further enhanced ALP activity.Figure 6ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"qRT–PCR was carried out to analyze the expression of osteogenesis and angiogenesis factors in the cells cultured on HAp, mnHAp and Srx-mnHAp at days 4 and 7. As shown in Figure 7, the mnHAp bioceramics (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) could strengthen the expression of COL1, BSP, BMP-2, OPN, VEGF and ANG-1 with sample S0 (dense and flat surface) compared at 4 and 7 d. Furthermore, Sr doping improved the mRNA levels of COL1, BSP, and OPN at 4 and 7 d and ANG-1 at 7 d. The western bolt result of day 7 confirmed the enhanced expression of BMP-2 in the Srx-mnHAp, especially in the Sr10-mnHAp. More importantly, the promotion effect of Sr doping occurred in a dose-dependent manner, and Sr10-mnHAp achieved the best stimulation ability.Figure 7qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nqRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"Micro-CT scanning and analysis were performed to assess the bone regeneration of the calvarial defect areas. From Figure 8A, we can see that more bone formation in the surface and pores of the mnHAp bioceramics at the 8th week after implantation (p<0.05) and Sr10-mnHAp achieved the best repair effect with S0 and S3 compared (p<0.05). Morphometric analysis further proved that the BV/TV ratio (Figure 8B) and Tb. Th (Figure 8C) in mnHAp bioceramics were higher than that of pure HAp. Moreover, significant differences were found between mnHAp and Sr10-mnHAp (p<0.05). Figure 9 showed the angiographic results, more blood vessels were found in the implant site of the mnHAp bioceramics (p<0.05), and the Sr10-mnHAp groups possessed the most newly formed blood vessels. The results of quantitative analysis also confirmed that samples S3 and 10Sr possessed more blood vessels, and the differences between samples 10Sr and S3 were remarkable (p<0.05).Figure 8Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).Figure 9Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nMicro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"Van Gieson staining was applied to observe bone regeneration in the surface and pores of the HAp bioceramics. As shown in Figure 10A, bone formation was only found on the edge of the defect in group S0, while groups S3 and 10Sr achieved more new bone formation from the margins to the Center, especially group 10Sr. As shown in Figure 10B, the results of bone histomorphometric analysis further confirmed that groups S3 and 10Sr possessed more newly formed bone areas. More importantly, remarkable differences could be found between groups S3 and 10Sr, which indicates that Sr10-mnHAp has the best effect on promoting bone formation (p<0.05).Figure 10Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).\nHistological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).",
"The skeleton is an integral part of the human body and has a limited capacity to regenerate after large bone defects caused by acute injuries, fall fractures, or tumors. The repair of bone defects and bone regeneration remain challenging problems in clinical treatment.2 Currently, reconstruction of these skeletal defects is often achieved by autogenous and allogeneic bone transplantation, although they have drawbacks separately. The emergence and development of tissue engineering offers tremendous potential. HAp ceramics are a multifunctional biological material with good biocompatibility and bioactivity but are limited in Clinical application because of their insufficient osteoinduction ability.4 Osteoblast/material interactions play a fundamental role in the biological response of host cells and can be modified by the surface characteristics of bone grafts.26\nNumerous studies have shown that controlling the surface morphology and roughness of materials is a direct and effective strategy to improve biological properties, and surface topography can affect cell attachment and subsequent proliferation and differentiation.9,27–29 Our previous study demonstrated that the microstructure surface can support cell adhesion, which is critical for osteogenic proliferation and differentiation.9 Changing the chemical compositions of the surface by element doping can also improve the osteogenic properties of biomaterials.21 Trace elementSr has been applied to the preparation of bone tissue engineering scaffolds, which can promote bone regeneration and inhibit bone resorption.30 Previous studies showed that appropriate release of Sr ions could activate osteogenesis-related signal transduction pathways, stimulate osteogenesis gene expression and ultimately new bone formation in a dose-dependent manner.31,32 Most of the previous studies mainly focused on the effects and underlying mechanisms of surface modification or chemical composition changes on the biological characteristics. Whether osteogenic differentiation and angiogenesis could be promoted by topographic modification and element doping is still lacking and requires further evaluation.\nHerein, HAp bioceramics with micro-nano-hybrid surfaces and different Sr doping contents (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were synthesized using Srx-α-TCP as a precursor via hydrothermal transformation.20 The XRD patterns confirmed that all the samples were completely transformed into HAp, and the visible micro-nanorods structure on the surface of mnHAp and Srx-mnHAp could be observed by SEM. According to the ICP-AES analysis, the release of Sr ions could be detected in the Srx-mnHAp bioceramics, and the concentration was between 0.2 and 1.0 ug mL−1. Previous Research suggested that the suitable concentration of Sr ions varies from 0.2107 to 21.07 μg mL−1, which indicated that the release of Sr is in an appropriate range.33\nSubsequently, the effects of mnHAp and Srx-mnHAp bioceramics on BMSCs were evaluated in vitro. Previous studies have suggested that the physical properties of biomaterial scaffolds can affect adhesion, migration, and differentiation into osteoblasts.34–37 The cytoskeleton staining assay showed that the cells cultured on the mnHAp exhibited better cell attachment, with typical fibroblastic morphology. However, Sr doping did not significantly promote cell attachment or extension. It indicates that the nanostructure surface, instead of Sr ions, is the Key promotion factor of cellular responses in the early stages. ALP is considered to be an indicator of osteogenic differentiation and an important index of bone formation and turnover. BMP-2 is the strongest osteoinduction cytokine and can promote bone formation and growth. OCN is tightly related to the maturation of osteoblasts,38 and COL1 and BSP are vital genes involved in osteoblastic differentiation. VEGF is the most important factor in angiogenesis, and ANG-1 plays a basic role in promoting maturity and maintaining vascular stabilization. According to the results of MTT, ALP activity, and PCR assays, the mnHAp bioceramics could facilitate proliferation ability, ALP activity and mRNA expression of COL1, BSP, BMP-2, OPN, VEGF, and ANG-1, which is consistent with our previous results.22,39 More importantly, Sr doping furthers promoted cellular osteogenic activity, while Sr10-mnHAp possessed the best stimulatory effect. In addition, the results of calvarial defects confirmed that the mnHAp bioceramics could promote bone and blood vessel regeneration with the control samples (dense and flat surface). More importantly, Sr doping further promoted bone and blood vessel regeneration, while Sr10-mnHAp possessed the most stimulatory effect. All the above results proved our hypothesis that micro-nano-hybrid surface and Sr doping had synergistic promotion effects on bone regeneration, the Srx-mnHAp bioceramics can be a promising material for bone defect repair. Meanwhile, its multifunctional properties make it a good candidate for potential biomedical applications, including targeted drug delivery applications, and the stable and controlled release of drugs is worthy of further investigation.",
"Surface structure and chemical composition are considered to be the key clues to regulate the biological response of biomaterials. Herein, HAp bioceramics with micro-nano-hybrid surface and different Sr doping contents (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were successfully fabricated via a hydrothermal transformation method using Srx-ɑ-TCP powder as a precursor without additives. The nanostructure surface can support cell adhesion, and Sr-doping could further enhance osteogenic differentiation with an optimal doping concentration of 10%. Finally, the calvarial defect model showed that the Sr10-mnHAp bioceramics scaffolds possessed better bone regeneration capacity. All of the results confirmed our hypothesis that micro-nano-hybrid surface and Sr doping have synergistic effects on bone regeneration and provided a new strategy for improving the osteoinduction ability of traditional bioceramics."
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[
"bioceramics",
"scaffolds",
"surface modification",
"element doping",
"bone regeneration"
] |
Introduction:
Bone transplantation is a crucial method to reconstruct bone defects caused by trauma, tumors, and other diseases, and autologous bone grafts remain the “gold standard” for bone transplantation. However, the limitation of insufficient bone mass and related postoperative complications have facilitated the development of substitute materials.1,2 Bioceramic materials are widely used as a physiological scaffold for bone defect repair because of its excellent biocompatibility and bioactivity, and are currently considered as the most promising alternative to autologous bone grafting. The porous structure of bioceramics facilitates the adhesion of osteoblasts, transport of nutrients, and speeds up the process of bone resorption and reconstruction,3 which makes it better for repairing large bone defects.4
The inorganic minerals in human bone tissue have a multilevel ordered structure and contain many microelements. From the viewpoint of bio mimics, we can tune the osteoinduction ability of materials by controlling the surface microstructure and element doping. The ideal scaffold materials for bone regeneration require an appropriate three-dimensional microporous structure for cell growth and nutrient metabolism.2,5 For bioceramics scaffolds, the pores are 150 ~ 500 μm in diameter and over 50% in porosity are appropriate to stimulate cell response, and nanostructured surfaces and high connectivity between pores is also important.1,6–8 The results of our recent study confirmed that the HAp bioceramics with nanostructured modified topography significantly enhanced cell viability, spreading, and osteogenic differentiation of BMSCs and bone formation, mineralization and vascularization in vivo.9 Moreover, suitable surface chemistry plays critical roles in cell viability, spreading, proliferation, and osteogenic differentiation.2,5 Biomaterials can be modified by the introduction of functional inorganic ions. Recently, more attention has been given to strontium (Sr), which is an important trace element in the human body, and 99% of Sr exists in bones. Sr can improve the mechanical properties and modify the bone balance toward osteosynthesis.10 Recent studies11 have found that Sr can enhance the proliferation of osteoblasts during bone metabolism. Yang et al12 suggested that the surface of Sr-doped hydroxyapatite can significantly increase the growth of osteoblasts. The results of Wang et al13 confirmed that the Sr-HAp coatings significantly promoted osteoblast attachment and proliferation as the Sr content increased. Recently, researchers focused on the preparation of strontium-substituted bioceramics and their application in bone regeneration.14–16 However, few studies have focused on the synergistic promotion of nanostructures and Sr doping on bone regeneration.
Therefore, based on the above research and our previous results,9,17 we proposed that the combination of nanostructure surfaces and Sr-doping might lead to synergistic enhancement of osteogenesis and angiogenesis.18,19 Herein, HAp bioceramics with micro-nano-hybrid surfaces and different strontium (Sr) doping contents of 2.5, 5, 10, and 20% (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were fabricated via the hydrothermal transformation method. The effect on cell adhesion, proliferation, and ALP activity and the expression of genes involved in osteogenesis and angiogenesis were investigated to determine the optimal Sr doping content, followed by calvarial defect experiments to evaluate bone regeneration and vascularization in vivo.
Materials and Methods:
Preparation of Micro-Nano-Hybrid HAp (mnHAp) and Sr-Doped mnHAp (Srx-mnHAp) Bioceramics The micro-nano-hybrid HAp (mnHAp) and Sr-doped mnHAp (Srx-mnHAp, x: 2.5, 5, 10 and 20%) bioceramics were prepared by the hydrothermal transformation method. First, ɑ-tricalcium phosphate (ɑ-TCP) and Sr-doped ɑ-TCP with different doping contents of 2.5, 5, 10, and 20% (Srx-ɑ-TCP, x: 2.5, 5, 10 and 20%) powders were synthesized by chemical precipitation.20 Then, the synthesized ɑ-TCP and Srx-ɑ-TCP powders were mixed with 7 wt% polyvinyl alcohol (PVA), dry-pressed into discs 10 mm in diameter and 2 mm in thickness under a pressure of 5 MPa, and further calcined at 1050 °C for 5 h to obtain ɑ-TCP and Srx-ɑ-TCP bioceramics, respectively.21 Finally, the mnHAp and Srx-mnHAp bioceramics were prepared using ɑ-TCP and Sr-ɑ-TCP discs as precursors via hydrothermal reaction in pH=7 aqueous solution at 180 °C for 72 h.17,20 In addition, HAp bioceramics with dense and flat surfaces were used as controls and labeled S0.9
The micro-nano-hybrid HAp (mnHAp) and Sr-doped mnHAp (Srx-mnHAp, x: 2.5, 5, 10 and 20%) bioceramics were prepared by the hydrothermal transformation method. First, ɑ-tricalcium phosphate (ɑ-TCP) and Sr-doped ɑ-TCP with different doping contents of 2.5, 5, 10, and 20% (Srx-ɑ-TCP, x: 2.5, 5, 10 and 20%) powders were synthesized by chemical precipitation.20 Then, the synthesized ɑ-TCP and Srx-ɑ-TCP powders were mixed with 7 wt% polyvinyl alcohol (PVA), dry-pressed into discs 10 mm in diameter and 2 mm in thickness under a pressure of 5 MPa, and further calcined at 1050 °C for 5 h to obtain ɑ-TCP and Srx-ɑ-TCP bioceramics, respectively.21 Finally, the mnHAp and Srx-mnHAp bioceramics were prepared using ɑ-TCP and Sr-ɑ-TCP discs as precursors via hydrothermal reaction in pH=7 aqueous solution at 180 °C for 72 h.17,20 In addition, HAp bioceramics with dense and flat surfaces were used as controls and labeled S0.9
Samples Characterization HAp (S0), mnHAp (S3), and Srx-mnHAp (x: 2.5, 5, 10 and 20%) were incubated in 1 mL of α-Minimum Essential Medium (ɑ-MEM, Gibco, USA) for 1 and 4 d, respectively. The medium of each sample changed daily and the concentrations of Sr, Ca, and P ions released from HAp, mnHAp, and Srx-mnHAp at each timepoint were evaluated using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The crystal phase compositions of the samples were determined with XRD (Rigaku, Japan), and the surface microstructures were observed by SEM (JEOL, Japan).17
HAp (S0), mnHAp (S3), and Srx-mnHAp (x: 2.5, 5, 10 and 20%) were incubated in 1 mL of α-Minimum Essential Medium (ɑ-MEM, Gibco, USA) for 1 and 4 d, respectively. The medium of each sample changed daily and the concentrations of Sr, Ca, and P ions released from HAp, mnHAp, and Srx-mnHAp at each timepoint were evaluated using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The crystal phase compositions of the samples were determined with XRD (Rigaku, Japan), and the surface microstructures were observed by SEM (JEOL, Japan).17
Cell Extraction and Culture Bone marrow stem cells (BMSCs) were harvested as previously described.17 Briefly, Sprague–Dawley (SD) rats (4 weeks old) were selected and injected intraperitoneally with an overdose of pentobarbital sodium. The scalp was separated for exposure, and both femora were cut off. Fresh marrow was flushed out and cultured in ɑ-MEM (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin and streptomycin (Hyclone, USA). The culture medium was maintained at 37 °C in a 5% CO2 incubator and renewed 3 times a week. The cells were passaged after reaching 90% confluence, and passages 2–4 were used in the following experiment. All animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine and conducted in compliance with the guidelines of Institutional Animal Care and Use Committee of Shanghai Ninth People’s Hospital Affiliated to Shanghai JiaoTong University, School of Medicine [SYXK (Hu) 2016–0016].
Bone marrow stem cells (BMSCs) were harvested as previously described.17 Briefly, Sprague–Dawley (SD) rats (4 weeks old) were selected and injected intraperitoneally with an overdose of pentobarbital sodium. The scalp was separated for exposure, and both femora were cut off. Fresh marrow was flushed out and cultured in ɑ-MEM (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin and streptomycin (Hyclone, USA). The culture medium was maintained at 37 °C in a 5% CO2 incubator and renewed 3 times a week. The cells were passaged after reaching 90% confluence, and passages 2–4 were used in the following experiment. All animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine and conducted in compliance with the guidelines of Institutional Animal Care and Use Committee of Shanghai Ninth People’s Hospital Affiliated to Shanghai JiaoTong University, School of Medicine [SYXK (Hu) 2016–0016].
Cell Morphology and Adhesion Phalloidin staining was performed to evaluate the morphology and spreading of the BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at an initial density of 1×104 cells per mL in 24-well plates. At 6 h after seeding, all specimens were fixed with 4% formaldehyde for 10 min, and then treated with 1% Triton X-100 for 5 min. Next, these specimens were incubated with phalloidin (Sigma, USA) for 30 min, followed by DAPI (Sigma, USA) for approximately 30s. Finally, actin cytoskeletons were observed by fluorescence microscopy (Leica, Germany).
Phalloidin staining was performed to evaluate the morphology and spreading of the BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at an initial density of 1×104 cells per mL in 24-well plates. At 6 h after seeding, all specimens were fixed with 4% formaldehyde for 10 min, and then treated with 1% Triton X-100 for 5 min. Next, these specimens were incubated with phalloidin (Sigma, USA) for 30 min, followed by DAPI (Sigma, USA) for approximately 30s. Finally, actin cytoskeletons were observed by fluorescence microscopy (Leica, Germany).
Cell Proliferation The proliferation of BMSCs in the samples was investigated by MTT assay, with a cell density of 1×104 cells per mL in 24-well plates. After culturing for 1, 4, and 7 d, the culture medium was replaced, and the BMSC-seeded bioceramics were rinsed and incubated with sterile MTT (Amresco, USA) at 37 °C for 4 h. DMSO solution (Sigma, USA) was then added and incubated for 10 min to dissolve formazan. The OD value was read at 490 nm by a microplate Reader (Biotek, USA). All experiments were repeated 3 times.
The proliferation of BMSCs in the samples was investigated by MTT assay, with a cell density of 1×104 cells per mL in 24-well plates. After culturing for 1, 4, and 7 d, the culture medium was replaced, and the BMSC-seeded bioceramics were rinsed and incubated with sterile MTT (Amresco, USA) at 37 °C for 4 h. DMSO solution (Sigma, USA) was then added and incubated for 10 min to dissolve formazan. The OD value was read at 490 nm by a microplate Reader (Biotek, USA). All experiments were repeated 3 times.
ALP Staining and Activity BMSCs were cultured onto S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr samples at a cell density of 1×104 cells per mL in 24-well plates. After culturing for 10 d, ALP staining was performed with a BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). The ALP activity was then assessed at 4, 7, and 10 d.22 Briefly, all samples were lysed in 1% Triton X-100 for 30 min at 4 °C, and the cellular lysate of each well was collected and centrifuged at 4 °C (12,000 rpm × 10 min). Then, the OD value was determined at 520 nm with a microplate Reader (Bio-Tek, USA), and total cellular protein was determined by a BCA protein kit (Beyotime, China) at 562 nm. Finally, ALP activity was evaluated by normalizing to total protein content. All experiments were repeated 3 times.
BMSCs were cultured onto S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr samples at a cell density of 1×104 cells per mL in 24-well plates. After culturing for 10 d, ALP staining was performed with a BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). The ALP activity was then assessed at 4, 7, and 10 d.22 Briefly, all samples were lysed in 1% Triton X-100 for 30 min at 4 °C, and the cellular lysate of each well was collected and centrifuged at 4 °C (12,000 rpm × 10 min). Then, the OD value was determined at 520 nm with a microplate Reader (Bio-Tek, USA), and total cellular protein was determined by a BCA protein kit (Beyotime, China) at 562 nm. Finally, ALP activity was evaluated by normalizing to total protein content. All experiments were repeated 3 times.
Quantitative Real-Time PCR (qRT-PCR) and Western Blot Experiment qRT-PCR was carried out to assess the expression of osteogenic and angiogenic factors in BMSCs cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr. After culturing for 4 and 7 d, total RNA of the samples was isolated according to our previous study.21 Briefly, the cells cultured in the bioceramics were lysed in TRIzol Reagent (Life Technologies, USA) and reverse transcribed to cDNA by PrimeScriptTM RT reagent Kit (TaKaRa, Japan). The expression levels of osteogenesis- and angiogenesis-related genes were further evaluated by qRT-PCR analysis, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. GAPDH was selected as a housekeeping gene to normalize the expression level. The primer sequences selected are listed in Table 1. All experiments were repeated 3 times.Table 1Primer Sequences of the Selected GenesTarget GenePrimers (F = Forward; R = Reverse)Accession NumberProduct Size (bp)COL1F:5ʹCTGCCCAGAAGAATATGTATCACC3’R:5ʹGAAGCAAAGTTTCCTCCAAGACC3’NM_053304.1198BSPF: 5ʹAGAAAGAGCAGCACGGTTGAGT3’R: 5ʹGACCCTCGTAGCCTTCATAGCC3’NM_012587.2175BMP-2F: 5ʹTGGGTTTGTGGTGGAAGTGGC3’R: 5ʹTGGATGTCCTTTACCGTCGTG3’NM_017178.2154OPNF: 5ʹCCAAGCGTGGAAACACACAGCC3’R: 5ʹGGCTTTGGAACTCGCCTGACTG3’NM_012881.2165VEGFF: 5′GGCTCTGAAACCATGAACTTTCT3′R: 5′GCAGTAGCTGCGCTGGTAGAC3′NM_001110334.2165ANG-1F:5′GGACAGCAGGCAAACAGAGCAGC3′R: 5′CCACAGGCATCAAACCACCAACC3′NM_053546.2130GAPDHF: 5ʹCCTGCACCACCAACTGCTTA3’R: 5ʹGGCCATCCACAGTCTTCTGAG3’NM_017008.4120
Primer Sequences of the Selected Genes
Western blot experiment was applied to assess expression of BMP-2. BMSCs were cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr for 7 d. Total protein was extracted and centrifuged at 12,000 rpm for 15 minutes. Then, the samples were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 volts for 20 minutes and 120 volts for 50 minutes, and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The PVDF membranes were incubated with primary antibody (Abcam, UK) at 37 °C for 2h. After that, the membranes were incubated with HRP-conjugated secondary antibody (Abcam, UK) for 1 h at room temperature. The protein bands were visualized, and the images were captured by an automated luminescent image analysis system (Tanon, China).
qRT-PCR was carried out to assess the expression of osteogenic and angiogenic factors in BMSCs cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr. After culturing for 4 and 7 d, total RNA of the samples was isolated according to our previous study.21 Briefly, the cells cultured in the bioceramics were lysed in TRIzol Reagent (Life Technologies, USA) and reverse transcribed to cDNA by PrimeScriptTM RT reagent Kit (TaKaRa, Japan). The expression levels of osteogenesis- and angiogenesis-related genes were further evaluated by qRT-PCR analysis, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. GAPDH was selected as a housekeeping gene to normalize the expression level. The primer sequences selected are listed in Table 1. All experiments were repeated 3 times.Table 1Primer Sequences of the Selected GenesTarget GenePrimers (F = Forward; R = Reverse)Accession NumberProduct Size (bp)COL1F:5ʹCTGCCCAGAAGAATATGTATCACC3’R:5ʹGAAGCAAAGTTTCCTCCAAGACC3’NM_053304.1198BSPF: 5ʹAGAAAGAGCAGCACGGTTGAGT3’R: 5ʹGACCCTCGTAGCCTTCATAGCC3’NM_012587.2175BMP-2F: 5ʹTGGGTTTGTGGTGGAAGTGGC3’R: 5ʹTGGATGTCCTTTACCGTCGTG3’NM_017178.2154OPNF: 5ʹCCAAGCGTGGAAACACACAGCC3’R: 5ʹGGCTTTGGAACTCGCCTGACTG3’NM_012881.2165VEGFF: 5′GGCTCTGAAACCATGAACTTTCT3′R: 5′GCAGTAGCTGCGCTGGTAGAC3′NM_001110334.2165ANG-1F:5′GGACAGCAGGCAAACAGAGCAGC3′R: 5′CCACAGGCATCAAACCACCAACC3′NM_053546.2130GAPDHF: 5ʹCCTGCACCACCAACTGCTTA3’R: 5ʹGGCCATCCACAGTCTTCTGAG3’NM_017008.4120
Primer Sequences of the Selected Genes
Western blot experiment was applied to assess expression of BMP-2. BMSCs were cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr for 7 d. Total protein was extracted and centrifuged at 12,000 rpm for 15 minutes. Then, the samples were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 volts for 20 minutes and 120 volts for 50 minutes, and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The PVDF membranes were incubated with primary antibody (Abcam, UK) at 37 °C for 2h. After that, the membranes were incubated with HRP-conjugated secondary antibody (Abcam, UK) for 1 h at room temperature. The protein bands were visualized, and the images were captured by an automated luminescent image analysis system (Tanon, China).
Animal Experiment Calvarial defects were established in twelve 6-week-old SD rats as previously described.17 Animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine. All rats were randomly divided into the following 3 groups: S0, S3, and 10Sr (n = 4 for each group). Briefly, all rats were injected intraperitoneally with pentobarbital (3.5 mg/100 g). Under sterile conditions, a 2 cm longitudinal incision was made, and two symmetrical round defects (5 mm in diameter) were created using the implant drill at each rat. Subsequently, the HAp, mnHAp, and Sr10-mnHAp were placed into the defects, and the operation incision was carefully sutured with absorbable sutures.
Calvarial defects were established in twelve 6-week-old SD rats as previously described.17 Animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine. All rats were randomly divided into the following 3 groups: S0, S3, and 10Sr (n = 4 for each group). Briefly, all rats were injected intraperitoneally with pentobarbital (3.5 mg/100 g). Under sterile conditions, a 2 cm longitudinal incision was made, and two symmetrical round defects (5 mm in diameter) were created using the implant drill at each rat. Subsequently, the HAp, mnHAp, and Sr10-mnHAp were placed into the defects, and the operation incision was carefully sutured with absorbable sutures.
Micro-CT Analyses At the 8th week after implantation, all rats were sacrificed and perfused with Microfil (Flowtech, USA) to observe new blood vessels.23 The samples were decalcified using EDTA for 1 month and then scanned by microcomputed tomography (Scanco, Switzerland), the percentages of new blood vessels were quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA). For the observation of new bone, the specimens were fixed in 4% paraformaldehyde for 2 d and then transferred to 75% alcohol. Finally, the samples were scanned by microcomputed tomography. Bone volume fraction (BV/TV) and trabecular thickness (Tb. Th) were calculated with auxiliary software (Scanco, Switzerland).24
At the 8th week after implantation, all rats were sacrificed and perfused with Microfil (Flowtech, USA) to observe new blood vessels.23 The samples were decalcified using EDTA for 1 month and then scanned by microcomputed tomography (Scanco, Switzerland), the percentages of new blood vessels were quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA). For the observation of new bone, the specimens were fixed in 4% paraformaldehyde for 2 d and then transferred to 75% alcohol. Finally, the samples were scanned by microcomputed tomography. Bone volume fraction (BV/TV) and trabecular thickness (Tb. Th) were calculated with auxiliary software (Scanco, Switzerland).24
Histological Analyses All specimens were decalcified with 10% EDTA, dehydrated in increased concentrations of ethanol (75% to 100%) and embedded in PMMA for 3 weeks. Tissue blocks were sectioned by a microtome (Leica, Germany) and further ground to approximately 40 μm. Next, the sections were subjected to Van Gieson staining for histological analysis, and the proportion of bone regeneration in the defect area was quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA).17,25
All specimens were decalcified with 10% EDTA, dehydrated in increased concentrations of ethanol (75% to 100%) and embedded in PMMA for 3 weeks. Tissue blocks were sectioned by a microtome (Leica, Germany) and further ground to approximately 40 μm. Next, the sections were subjected to Van Gieson staining for histological analysis, and the proportion of bone regeneration in the defect area was quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA).17,25
Statistical Analyses All data are expressed as the means ± standard deviation (SD), and statistical analysis was performed using SPSS 17.0 software (SPSS Inc., USA). A p value <0.05 was considered statistically significant.
All data are expressed as the means ± standard deviation (SD), and statistical analysis was performed using SPSS 17.0 software (SPSS Inc., USA). A p value <0.05 was considered statistically significant.
Preparation of Micro-Nano-Hybrid HAp (mnHAp) and Sr-Doped mnHAp (Srx-mnHAp) Bioceramics:
The micro-nano-hybrid HAp (mnHAp) and Sr-doped mnHAp (Srx-mnHAp, x: 2.5, 5, 10 and 20%) bioceramics were prepared by the hydrothermal transformation method. First, ɑ-tricalcium phosphate (ɑ-TCP) and Sr-doped ɑ-TCP with different doping contents of 2.5, 5, 10, and 20% (Srx-ɑ-TCP, x: 2.5, 5, 10 and 20%) powders were synthesized by chemical precipitation.20 Then, the synthesized ɑ-TCP and Srx-ɑ-TCP powders were mixed with 7 wt% polyvinyl alcohol (PVA), dry-pressed into discs 10 mm in diameter and 2 mm in thickness under a pressure of 5 MPa, and further calcined at 1050 °C for 5 h to obtain ɑ-TCP and Srx-ɑ-TCP bioceramics, respectively.21 Finally, the mnHAp and Srx-mnHAp bioceramics were prepared using ɑ-TCP and Sr-ɑ-TCP discs as precursors via hydrothermal reaction in pH=7 aqueous solution at 180 °C for 72 h.17,20 In addition, HAp bioceramics with dense and flat surfaces were used as controls and labeled S0.9
Samples Characterization:
HAp (S0), mnHAp (S3), and Srx-mnHAp (x: 2.5, 5, 10 and 20%) were incubated in 1 mL of α-Minimum Essential Medium (ɑ-MEM, Gibco, USA) for 1 and 4 d, respectively. The medium of each sample changed daily and the concentrations of Sr, Ca, and P ions released from HAp, mnHAp, and Srx-mnHAp at each timepoint were evaluated using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The crystal phase compositions of the samples were determined with XRD (Rigaku, Japan), and the surface microstructures were observed by SEM (JEOL, Japan).17
Cell Extraction and Culture:
Bone marrow stem cells (BMSCs) were harvested as previously described.17 Briefly, Sprague–Dawley (SD) rats (4 weeks old) were selected and injected intraperitoneally with an overdose of pentobarbital sodium. The scalp was separated for exposure, and both femora were cut off. Fresh marrow was flushed out and cultured in ɑ-MEM (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin and streptomycin (Hyclone, USA). The culture medium was maintained at 37 °C in a 5% CO2 incubator and renewed 3 times a week. The cells were passaged after reaching 90% confluence, and passages 2–4 were used in the following experiment. All animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine and conducted in compliance with the guidelines of Institutional Animal Care and Use Committee of Shanghai Ninth People’s Hospital Affiliated to Shanghai JiaoTong University, School of Medicine [SYXK (Hu) 2016–0016].
Cell Morphology and Adhesion:
Phalloidin staining was performed to evaluate the morphology and spreading of the BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at an initial density of 1×104 cells per mL in 24-well plates. At 6 h after seeding, all specimens were fixed with 4% formaldehyde for 10 min, and then treated with 1% Triton X-100 for 5 min. Next, these specimens were incubated with phalloidin (Sigma, USA) for 30 min, followed by DAPI (Sigma, USA) for approximately 30s. Finally, actin cytoskeletons were observed by fluorescence microscopy (Leica, Germany).
Cell Proliferation:
The proliferation of BMSCs in the samples was investigated by MTT assay, with a cell density of 1×104 cells per mL in 24-well plates. After culturing for 1, 4, and 7 d, the culture medium was replaced, and the BMSC-seeded bioceramics were rinsed and incubated with sterile MTT (Amresco, USA) at 37 °C for 4 h. DMSO solution (Sigma, USA) was then added and incubated for 10 min to dissolve formazan. The OD value was read at 490 nm by a microplate Reader (Biotek, USA). All experiments were repeated 3 times.
ALP Staining and Activity:
BMSCs were cultured onto S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr samples at a cell density of 1×104 cells per mL in 24-well plates. After culturing for 10 d, ALP staining was performed with a BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). The ALP activity was then assessed at 4, 7, and 10 d.22 Briefly, all samples were lysed in 1% Triton X-100 for 30 min at 4 °C, and the cellular lysate of each well was collected and centrifuged at 4 °C (12,000 rpm × 10 min). Then, the OD value was determined at 520 nm with a microplate Reader (Bio-Tek, USA), and total cellular protein was determined by a BCA protein kit (Beyotime, China) at 562 nm. Finally, ALP activity was evaluated by normalizing to total protein content. All experiments were repeated 3 times.
Quantitative Real-Time PCR (qRT-PCR) and Western Blot Experiment:
qRT-PCR was carried out to assess the expression of osteogenic and angiogenic factors in BMSCs cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr. After culturing for 4 and 7 d, total RNA of the samples was isolated according to our previous study.21 Briefly, the cells cultured in the bioceramics were lysed in TRIzol Reagent (Life Technologies, USA) and reverse transcribed to cDNA by PrimeScriptTM RT reagent Kit (TaKaRa, Japan). The expression levels of osteogenesis- and angiogenesis-related genes were further evaluated by qRT-PCR analysis, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. GAPDH was selected as a housekeeping gene to normalize the expression level. The primer sequences selected are listed in Table 1. All experiments were repeated 3 times.Table 1Primer Sequences of the Selected GenesTarget GenePrimers (F = Forward; R = Reverse)Accession NumberProduct Size (bp)COL1F:5ʹCTGCCCAGAAGAATATGTATCACC3’R:5ʹGAAGCAAAGTTTCCTCCAAGACC3’NM_053304.1198BSPF: 5ʹAGAAAGAGCAGCACGGTTGAGT3’R: 5ʹGACCCTCGTAGCCTTCATAGCC3’NM_012587.2175BMP-2F: 5ʹTGGGTTTGTGGTGGAAGTGGC3’R: 5ʹTGGATGTCCTTTACCGTCGTG3’NM_017178.2154OPNF: 5ʹCCAAGCGTGGAAACACACAGCC3’R: 5ʹGGCTTTGGAACTCGCCTGACTG3’NM_012881.2165VEGFF: 5′GGCTCTGAAACCATGAACTTTCT3′R: 5′GCAGTAGCTGCGCTGGTAGAC3′NM_001110334.2165ANG-1F:5′GGACAGCAGGCAAACAGAGCAGC3′R: 5′CCACAGGCATCAAACCACCAACC3′NM_053546.2130GAPDHF: 5ʹCCTGCACCACCAACTGCTTA3’R: 5ʹGGCCATCCACAGTCTTCTGAG3’NM_017008.4120
Primer Sequences of the Selected Genes
Western blot experiment was applied to assess expression of BMP-2. BMSCs were cultured in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr for 7 d. Total protein was extracted and centrifuged at 12,000 rpm for 15 minutes. Then, the samples were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 volts for 20 minutes and 120 volts for 50 minutes, and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The PVDF membranes were incubated with primary antibody (Abcam, UK) at 37 °C for 2h. After that, the membranes were incubated with HRP-conjugated secondary antibody (Abcam, UK) for 1 h at room temperature. The protein bands were visualized, and the images were captured by an automated luminescent image analysis system (Tanon, China).
Animal Experiment:
Calvarial defects were established in twelve 6-week-old SD rats as previously described.17 Animal experiments and procedures received approval from the Animal Ethics Committee of the Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine. All rats were randomly divided into the following 3 groups: S0, S3, and 10Sr (n = 4 for each group). Briefly, all rats were injected intraperitoneally with pentobarbital (3.5 mg/100 g). Under sterile conditions, a 2 cm longitudinal incision was made, and two symmetrical round defects (5 mm in diameter) were created using the implant drill at each rat. Subsequently, the HAp, mnHAp, and Sr10-mnHAp were placed into the defects, and the operation incision was carefully sutured with absorbable sutures.
Micro-CT Analyses:
At the 8th week after implantation, all rats were sacrificed and perfused with Microfil (Flowtech, USA) to observe new blood vessels.23 The samples were decalcified using EDTA for 1 month and then scanned by microcomputed tomography (Scanco, Switzerland), the percentages of new blood vessels were quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA). For the observation of new bone, the specimens were fixed in 4% paraformaldehyde for 2 d and then transferred to 75% alcohol. Finally, the samples were scanned by microcomputed tomography. Bone volume fraction (BV/TV) and trabecular thickness (Tb. Th) were calculated with auxiliary software (Scanco, Switzerland).24
Histological Analyses:
All specimens were decalcified with 10% EDTA, dehydrated in increased concentrations of ethanol (75% to 100%) and embedded in PMMA for 3 weeks. Tissue blocks were sectioned by a microtome (Leica, Germany) and further ground to approximately 40 μm. Next, the sections were subjected to Van Gieson staining for histological analysis, and the proportion of bone regeneration in the defect area was quantitatively determined with Image-Pro 5.0 (Media Cybernetic, USA).17,25
Statistical Analyses:
All data are expressed as the means ± standard deviation (SD), and statistical analysis was performed using SPSS 17.0 software (SPSS Inc., USA). A p value <0.05 was considered statistically significant.
Results:
Characterization of the Samples XRD results of mnHAp and Srx-mnHAp (x: 2.5, 5, 10 and 20%) are shown in Figure 1. All the diffraction peaks confirmed that these samples were converted into HAp without any other phases after the hydrothermal transformation method. The obvious micro-nanorods topography surfaces of mnHAp (Figure 2B) and Srx-mnHAp (Figure 2C–F) could be observed by SEM, and Sr10-mnHAp possessed the densest rod-shaped structure (Figure 2E). The lengths of the microrods and nanorods were approximately 10~20 μm, while the diameters were approximately 1~4 μm and 80~120 nm, respectively. As a comparison, a dense and flat surface with a particle size of approximately 0.9 µm was observed on control sample S0 (Figure 2A).Figure 1XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).Figure 2SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.
XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).
SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.
The results of ICP-AES analysis revealed that the release of Sr ions could be detected in Srx-mnHAp bioceramics and showed an ascending trend with increasing Sr content at each timepoint (Figure 3C). No significant differences in the release amount of Ca and P could be found among HAp, mnHAp, and Srx-mnHAp (Figure 3A and B).Figure 3The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.
The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.
XRD results of mnHAp and Srx-mnHAp (x: 2.5, 5, 10 and 20%) are shown in Figure 1. All the diffraction peaks confirmed that these samples were converted into HAp without any other phases after the hydrothermal transformation method. The obvious micro-nanorods topography surfaces of mnHAp (Figure 2B) and Srx-mnHAp (Figure 2C–F) could be observed by SEM, and Sr10-mnHAp possessed the densest rod-shaped structure (Figure 2E). The lengths of the microrods and nanorods were approximately 10~20 μm, while the diameters were approximately 1~4 μm and 80~120 nm, respectively. As a comparison, a dense and flat surface with a particle size of approximately 0.9 µm was observed on control sample S0 (Figure 2A).Figure 1XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).Figure 2SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.
XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).
SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.
The results of ICP-AES analysis revealed that the release of Sr ions could be detected in Srx-mnHAp bioceramics and showed an ascending trend with increasing Sr content at each timepoint (Figure 3C). No significant differences in the release amount of Ca and P could be found among HAp, mnHAp, and Srx-mnHAp (Figure 3A and B).Figure 3The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.
The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.
The Morphology of BMSCs As shown in Figure 4, the actin cytoskeleton was stained red, and the nucleus was stained blue. After being cultured for 6 h, fluorescence microscopy images showed that the cells seeded in sample S0 spread slightly and almost had a round shape (Figure 4A1). Meanwhile, as a comparison, the cells seeded on the mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) exhibited better early cell attachment and demonstrated a typical polygonal morphology with apparent stretch tentacles. No significant differences in the cell morphology and quantity could be found with increasing Sr content. This experiment showed that the nanostructure surface could promote the early cell attachment of BMSCs instead of the concentrations of Sr ions.Figure 4Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.
Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.
As shown in Figure 4, the actin cytoskeleton was stained red, and the nucleus was stained blue. After being cultured for 6 h, fluorescence microscopy images showed that the cells seeded in sample S0 spread slightly and almost had a round shape (Figure 4A1). Meanwhile, as a comparison, the cells seeded on the mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) exhibited better early cell attachment and demonstrated a typical polygonal morphology with apparent stretch tentacles. No significant differences in the cell morphology and quantity could be found with increasing Sr content. This experiment showed that the nanostructure surface could promote the early cell attachment of BMSCs instead of the concentrations of Sr ions.Figure 4Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.
Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.
Cell Proliferation The cells adhered to the HAp, mnHAp and Srx-mnHAp bioceramics proliferated through the MTT assay (Figure 5). The cells cultured on mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) displayed higher proliferation than control samples on days 4 and 7. (p<0.05) In addition, among the Srx-mnHAp with different Sr contents, proliferation significantly proceeded on Sr10-mnHAp and Sr20-mnHAp on days 4 and 7 (p<0.05).Figure 5MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
The cells adhered to the HAp, mnHAp and Srx-mnHAp bioceramics proliferated through the MTT assay (Figure 5). The cells cultured on mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) displayed higher proliferation than control samples on days 4 and 7. (p<0.05) In addition, among the Srx-mnHAp with different Sr contents, proliferation significantly proceeded on Sr10-mnHAp and Sr20-mnHAp on days 4 and 7 (p<0.05).Figure 5MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
ALP Activity Assay The cells cultured on mnHAp bioceramics possessed more intensive ALP staining, with control samples (dense and flat surface) compared (Figure 6A). Moreover, the staining became deeper with Sr doping (2.5Sr, 5Sr, 10Sr, and 20Sr). In particular, the deepest staining was found on the Sr10-mnHAp sample. The ALP semi-quantification assay on days 4, 7 and 10 further confirmed the staining results (Figure 6B). The ALP activity increased throughout the whole assay period, and the mnHAp bioceramics possessed higher cellular ALP activity. More importantly, the samples with Sr doping achieved better ALP activity on days 4 and 7 (p<0.05). When the experiment time was extended to 10 d, compared to mnHAp, only Sr10-mnHAp and Sr20-mnHAp further enhanced ALP activity.Figure 6ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
The cells cultured on mnHAp bioceramics possessed more intensive ALP staining, with control samples (dense and flat surface) compared (Figure 6A). Moreover, the staining became deeper with Sr doping (2.5Sr, 5Sr, 10Sr, and 20Sr). In particular, the deepest staining was found on the Sr10-mnHAp sample. The ALP semi-quantification assay on days 4, 7 and 10 further confirmed the staining results (Figure 6B). The ALP activity increased throughout the whole assay period, and the mnHAp bioceramics possessed higher cellular ALP activity. More importantly, the samples with Sr doping achieved better ALP activity on days 4 and 7 (p<0.05). When the experiment time was extended to 10 d, compared to mnHAp, only Sr10-mnHAp and Sr20-mnHAp further enhanced ALP activity.Figure 6ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
qRT–PCR Assay and Western Blot Experiment qRT–PCR was carried out to analyze the expression of osteogenesis and angiogenesis factors in the cells cultured on HAp, mnHAp and Srx-mnHAp at days 4 and 7. As shown in Figure 7, the mnHAp bioceramics (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) could strengthen the expression of COL1, BSP, BMP-2, OPN, VEGF and ANG-1 with sample S0 (dense and flat surface) compared at 4 and 7 d. Furthermore, Sr doping improved the mRNA levels of COL1, BSP, and OPN at 4 and 7 d and ANG-1 at 7 d. The western bolt result of day 7 confirmed the enhanced expression of BMP-2 in the Srx-mnHAp, especially in the Sr10-mnHAp. More importantly, the promotion effect of Sr doping occurred in a dose-dependent manner, and Sr10-mnHAp achieved the best stimulation ability.Figure 7qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
qRT–PCR was carried out to analyze the expression of osteogenesis and angiogenesis factors in the cells cultured on HAp, mnHAp and Srx-mnHAp at days 4 and 7. As shown in Figure 7, the mnHAp bioceramics (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) could strengthen the expression of COL1, BSP, BMP-2, OPN, VEGF and ANG-1 with sample S0 (dense and flat surface) compared at 4 and 7 d. Furthermore, Sr doping improved the mRNA levels of COL1, BSP, and OPN at 4 and 7 d and ANG-1 at 7 d. The western bolt result of day 7 confirmed the enhanced expression of BMP-2 in the Srx-mnHAp, especially in the Sr10-mnHAp. More importantly, the promotion effect of Sr doping occurred in a dose-dependent manner, and Sr10-mnHAp achieved the best stimulation ability.Figure 7qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Micro-CT Measurement Micro-CT scanning and analysis were performed to assess the bone regeneration of the calvarial defect areas. From Figure 8A, we can see that more bone formation in the surface and pores of the mnHAp bioceramics at the 8th week after implantation (p<0.05) and Sr10-mnHAp achieved the best repair effect with S0 and S3 compared (p<0.05). Morphometric analysis further proved that the BV/TV ratio (Figure 8B) and Tb. Th (Figure 8C) in mnHAp bioceramics were higher than that of pure HAp. Moreover, significant differences were found between mnHAp and Sr10-mnHAp (p<0.05). Figure 9 showed the angiographic results, more blood vessels were found in the implant site of the mnHAp bioceramics (p<0.05), and the Sr10-mnHAp groups possessed the most newly formed blood vessels. The results of quantitative analysis also confirmed that samples S3 and 10Sr possessed more blood vessels, and the differences between samples 10Sr and S3 were remarkable (p<0.05).Figure 8Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).Figure 9Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Micro-CT scanning and analysis were performed to assess the bone regeneration of the calvarial defect areas. From Figure 8A, we can see that more bone formation in the surface and pores of the mnHAp bioceramics at the 8th week after implantation (p<0.05) and Sr10-mnHAp achieved the best repair effect with S0 and S3 compared (p<0.05). Morphometric analysis further proved that the BV/TV ratio (Figure 8B) and Tb. Th (Figure 8C) in mnHAp bioceramics were higher than that of pure HAp. Moreover, significant differences were found between mnHAp and Sr10-mnHAp (p<0.05). Figure 9 showed the angiographic results, more blood vessels were found in the implant site of the mnHAp bioceramics (p<0.05), and the Sr10-mnHAp groups possessed the most newly formed blood vessels. The results of quantitative analysis also confirmed that samples S3 and 10Sr possessed more blood vessels, and the differences between samples 10Sr and S3 were remarkable (p<0.05).Figure 8Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).Figure 9Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Histological and Histomorphometric Analysis Van Gieson staining was applied to observe bone regeneration in the surface and pores of the HAp bioceramics. As shown in Figure 10A, bone formation was only found on the edge of the defect in group S0, while groups S3 and 10Sr achieved more new bone formation from the margins to the Center, especially group 10Sr. As shown in Figure 10B, the results of bone histomorphometric analysis further confirmed that groups S3 and 10Sr possessed more newly formed bone areas. More importantly, remarkable differences could be found between groups S3 and 10Sr, which indicates that Sr10-mnHAp has the best effect on promoting bone formation (p<0.05).Figure 10Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Van Gieson staining was applied to observe bone regeneration in the surface and pores of the HAp bioceramics. As shown in Figure 10A, bone formation was only found on the edge of the defect in group S0, while groups S3 and 10Sr achieved more new bone formation from the margins to the Center, especially group 10Sr. As shown in Figure 10B, the results of bone histomorphometric analysis further confirmed that groups S3 and 10Sr possessed more newly formed bone areas. More importantly, remarkable differences could be found between groups S3 and 10Sr, which indicates that Sr10-mnHAp has the best effect on promoting bone formation (p<0.05).Figure 10Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Characterization of the Samples:
XRD results of mnHAp and Srx-mnHAp (x: 2.5, 5, 10 and 20%) are shown in Figure 1. All the diffraction peaks confirmed that these samples were converted into HAp without any other phases after the hydrothermal transformation method. The obvious micro-nanorods topography surfaces of mnHAp (Figure 2B) and Srx-mnHAp (Figure 2C–F) could be observed by SEM, and Sr10-mnHAp possessed the densest rod-shaped structure (Figure 2E). The lengths of the microrods and nanorods were approximately 10~20 μm, while the diameters were approximately 1~4 μm and 80~120 nm, respectively. As a comparison, a dense and flat surface with a particle size of approximately 0.9 µm was observed on control sample S0 (Figure 2A).Figure 1XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).Figure 2SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.
XRD results of mnHAp (S3) and Srx-mnHAp bioceramics (2.5Sr, 5Sr, 10Sr, 20Sr).
SEM micrographs of topographic surface for HAp (A), mnHAp (B), and Srx-mnHAp (C–F): (A) scale bar=500 nm, (B-F) scale bar=20 μm.
The results of ICP-AES analysis revealed that the release of Sr ions could be detected in Srx-mnHAp bioceramics and showed an ascending trend with increasing Sr content at each timepoint (Figure 3C). No significant differences in the release amount of Ca and P could be found among HAp, mnHAp, and Srx-mnHAp (Figure 3A and B).Figure 3The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.
The release of Ca (A), P (B), and Sr (C) ions at 1 and 4 d.
The Morphology of BMSCs:
As shown in Figure 4, the actin cytoskeleton was stained red, and the nucleus was stained blue. After being cultured for 6 h, fluorescence microscopy images showed that the cells seeded in sample S0 spread slightly and almost had a round shape (Figure 4A1). Meanwhile, as a comparison, the cells seeded on the mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) exhibited better early cell attachment and demonstrated a typical polygonal morphology with apparent stretch tentacles. No significant differences in the cell morphology and quantity could be found with increasing Sr content. This experiment showed that the nanostructure surface could promote the early cell attachment of BMSCs instead of the concentrations of Sr ions.Figure 4Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.
Fluorescence microscopy images of the cells in samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr after 6 h: red represents the actin cytoskeleton (A1–F1), blue represents the nucleus (A2–F2), and the merged images of the actin cytoskeleton and the nucleus (A3–F3): scale bar=50 μm.
Cell Proliferation:
The cells adhered to the HAp, mnHAp and Srx-mnHAp bioceramics proliferated through the MTT assay (Figure 5). The cells cultured on mnHAp (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) displayed higher proliferation than control samples on days 4 and 7. (p<0.05) In addition, among the Srx-mnHAp with different Sr contents, proliferation significantly proceeded on Sr10-mnHAp and Sr20-mnHAp on days 4 and 7 (p<0.05).Figure 5MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
MTT analysis of the cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr. (* indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
ALP Activity Assay:
The cells cultured on mnHAp bioceramics possessed more intensive ALP staining, with control samples (dense and flat surface) compared (Figure 6A). Moreover, the staining became deeper with Sr doping (2.5Sr, 5Sr, 10Sr, and 20Sr). In particular, the deepest staining was found on the Sr10-mnHAp sample. The ALP semi-quantification assay on days 4, 7 and 10 further confirmed the staining results (Figure 6B). The ALP activity increased throughout the whole assay period, and the mnHAp bioceramics possessed higher cellular ALP activity. More importantly, the samples with Sr doping achieved better ALP activity on days 4 and 7 (p<0.05). When the experiment time was extended to 10 d, compared to mnHAp, only Sr10-mnHAp and Sr20-mnHAp further enhanced ALP activity.Figure 6ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
ALP activity measurement: (A) ALP staining of BMSCs seeded onto samples S0, S3, 2.5Sr, 5Sr, 10Sr, and 20Sr at 10 d. (B) ALP quantitative analysis of cells seeded in samples S0, S3, 2.5Sr, 5Sr, 10Sr and 20Sr for 4, 7 and 10 d. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
qRT–PCR Assay and Western Blot Experiment:
qRT–PCR was carried out to analyze the expression of osteogenesis and angiogenesis factors in the cells cultured on HAp, mnHAp and Srx-mnHAp at days 4 and 7. As shown in Figure 7, the mnHAp bioceramics (S3, 2.5Sr, 5Sr, 10Sr, and 20Sr) could strengthen the expression of COL1, BSP, BMP-2, OPN, VEGF and ANG-1 with sample S0 (dense and flat surface) compared at 4 and 7 d. Furthermore, Sr doping improved the mRNA levels of COL1, BSP, and OPN at 4 and 7 d and ANG-1 at 7 d. The western bolt result of day 7 confirmed the enhanced expression of BMP-2 in the Srx-mnHAp, especially in the Sr10-mnHAp. More importantly, the promotion effect of Sr doping occurred in a dose-dependent manner, and Sr10-mnHAp achieved the best stimulation ability.Figure 7qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
qRT–PCR analysis of the expression of COL1 (A), BMP-2 (B), BSP (C), OPN (D), VEGF (E), and ANG-1 (F) and Western blot result of BMP-2 (G). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Micro-CT Measurement:
Micro-CT scanning and analysis were performed to assess the bone regeneration of the calvarial defect areas. From Figure 8A, we can see that more bone formation in the surface and pores of the mnHAp bioceramics at the 8th week after implantation (p<0.05) and Sr10-mnHAp achieved the best repair effect with S0 and S3 compared (p<0.05). Morphometric analysis further proved that the BV/TV ratio (Figure 8B) and Tb. Th (Figure 8C) in mnHAp bioceramics were higher than that of pure HAp. Moreover, significant differences were found between mnHAp and Sr10-mnHAp (p<0.05). Figure 9 showed the angiographic results, more blood vessels were found in the implant site of the mnHAp bioceramics (p<0.05), and the Sr10-mnHAp groups possessed the most newly formed blood vessels. The results of quantitative analysis also confirmed that samples S3 and 10Sr possessed more blood vessels, and the differences between samples 10Sr and S3 were remarkable (p<0.05).Figure 8Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).Figure 9Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Micro-CT assessment at 8 weeks after implantation. (A) 3D and 2D images of newly formed bone of groups S0, S3, and 10Sr. Quantitative measurement of the BV/TV ratio (B) and Tb. Th (C). (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Micro-CT angiography at the 8th week after implantation. (A) Images of new blood vessels in groups S0, S3, and 10Sr. (B) Quantitative measurement of the new blood vessel area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Histological and Histomorphometric Analysis:
Van Gieson staining was applied to observe bone regeneration in the surface and pores of the HAp bioceramics. As shown in Figure 10A, bone formation was only found on the edge of the defect in group S0, while groups S3 and 10Sr achieved more new bone formation from the margins to the Center, especially group 10Sr. As shown in Figure 10B, the results of bone histomorphometric analysis further confirmed that groups S3 and 10Sr possessed more newly formed bone areas. More importantly, remarkable differences could be found between groups S3 and 10Sr, which indicates that Sr10-mnHAp has the best effect on promoting bone formation (p<0.05).Figure 10Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Histological and histomorphometric analysis: (A) Images of Van Gieson’s staining of bone formation at the 8th week after implantation. (B) Quantitative measurement of the new bone area. (*indicates a significant difference with S0, Δ indicates a significant difference with S3, p<0.05).
Discussion:
The skeleton is an integral part of the human body and has a limited capacity to regenerate after large bone defects caused by acute injuries, fall fractures, or tumors. The repair of bone defects and bone regeneration remain challenging problems in clinical treatment.2 Currently, reconstruction of these skeletal defects is often achieved by autogenous and allogeneic bone transplantation, although they have drawbacks separately. The emergence and development of tissue engineering offers tremendous potential. HAp ceramics are a multifunctional biological material with good biocompatibility and bioactivity but are limited in Clinical application because of their insufficient osteoinduction ability.4 Osteoblast/material interactions play a fundamental role in the biological response of host cells and can be modified by the surface characteristics of bone grafts.26
Numerous studies have shown that controlling the surface morphology and roughness of materials is a direct and effective strategy to improve biological properties, and surface topography can affect cell attachment and subsequent proliferation and differentiation.9,27–29 Our previous study demonstrated that the microstructure surface can support cell adhesion, which is critical for osteogenic proliferation and differentiation.9 Changing the chemical compositions of the surface by element doping can also improve the osteogenic properties of biomaterials.21 Trace elementSr has been applied to the preparation of bone tissue engineering scaffolds, which can promote bone regeneration and inhibit bone resorption.30 Previous studies showed that appropriate release of Sr ions could activate osteogenesis-related signal transduction pathways, stimulate osteogenesis gene expression and ultimately new bone formation in a dose-dependent manner.31,32 Most of the previous studies mainly focused on the effects and underlying mechanisms of surface modification or chemical composition changes on the biological characteristics. Whether osteogenic differentiation and angiogenesis could be promoted by topographic modification and element doping is still lacking and requires further evaluation.
Herein, HAp bioceramics with micro-nano-hybrid surfaces and different Sr doping contents (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were synthesized using Srx-α-TCP as a precursor via hydrothermal transformation.20 The XRD patterns confirmed that all the samples were completely transformed into HAp, and the visible micro-nanorods structure on the surface of mnHAp and Srx-mnHAp could be observed by SEM. According to the ICP-AES analysis, the release of Sr ions could be detected in the Srx-mnHAp bioceramics, and the concentration was between 0.2 and 1.0 ug mL−1. Previous Research suggested that the suitable concentration of Sr ions varies from 0.2107 to 21.07 μg mL−1, which indicated that the release of Sr is in an appropriate range.33
Subsequently, the effects of mnHAp and Srx-mnHAp bioceramics on BMSCs were evaluated in vitro. Previous studies have suggested that the physical properties of biomaterial scaffolds can affect adhesion, migration, and differentiation into osteoblasts.34–37 The cytoskeleton staining assay showed that the cells cultured on the mnHAp exhibited better cell attachment, with typical fibroblastic morphology. However, Sr doping did not significantly promote cell attachment or extension. It indicates that the nanostructure surface, instead of Sr ions, is the Key promotion factor of cellular responses in the early stages. ALP is considered to be an indicator of osteogenic differentiation and an important index of bone formation and turnover. BMP-2 is the strongest osteoinduction cytokine and can promote bone formation and growth. OCN is tightly related to the maturation of osteoblasts,38 and COL1 and BSP are vital genes involved in osteoblastic differentiation. VEGF is the most important factor in angiogenesis, and ANG-1 plays a basic role in promoting maturity and maintaining vascular stabilization. According to the results of MTT, ALP activity, and PCR assays, the mnHAp bioceramics could facilitate proliferation ability, ALP activity and mRNA expression of COL1, BSP, BMP-2, OPN, VEGF, and ANG-1, which is consistent with our previous results.22,39 More importantly, Sr doping furthers promoted cellular osteogenic activity, while Sr10-mnHAp possessed the best stimulatory effect. In addition, the results of calvarial defects confirmed that the mnHAp bioceramics could promote bone and blood vessel regeneration with the control samples (dense and flat surface). More importantly, Sr doping further promoted bone and blood vessel regeneration, while Sr10-mnHAp possessed the most stimulatory effect. All the above results proved our hypothesis that micro-nano-hybrid surface and Sr doping had synergistic promotion effects on bone regeneration, the Srx-mnHAp bioceramics can be a promising material for bone defect repair. Meanwhile, its multifunctional properties make it a good candidate for potential biomedical applications, including targeted drug delivery applications, and the stable and controlled release of drugs is worthy of further investigation.
Conclusion:
Surface structure and chemical composition are considered to be the key clues to regulate the biological response of biomaterials. Herein, HAp bioceramics with micro-nano-hybrid surface and different Sr doping contents (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were successfully fabricated via a hydrothermal transformation method using Srx-ɑ-TCP powder as a precursor without additives. The nanostructure surface can support cell adhesion, and Sr-doping could further enhance osteogenic differentiation with an optimal doping concentration of 10%. Finally, the calvarial defect model showed that the Sr10-mnHAp bioceramics scaffolds possessed better bone regeneration capacity. All of the results confirmed our hypothesis that micro-nano-hybrid surface and Sr doping have synergistic effects on bone regeneration and provided a new strategy for improving the osteoinduction ability of traditional bioceramics.
|
Background:
The synergistic effect of chemical element doping and surface modification is considered a novel way to regulate cell biological responses and improve the osteoinductive ability of biomaterials.
Methods:
Hydroxyapatite (HAp) bioceramics with micro-nano-hybrid (a mixture of microrods and nanorods) surfaces and different strontium (Sr) doping contents of 2.5, 5, 10, and 20% (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were prepared via a hydrothermal transformation method. The effect of Srx-mnHAp on osteogenesis and angiogenesis of bone marrow stromal cells (BMSCs) was evaluated in vitro, and the bioceramics scaffolds were further implanted into rat calvarial defects for the observation of bone regeneration in vivo.
Results:
HAp bioceramics with micro-nano-hybrid surfaces (mnHAp) could facilitate cell spreading, proliferation ability, ALP activity, and gene expression of osteogenic and angiogenic factors, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. More importantly, Srx-mnHAp (x: 2.5, 5, 10 and 20%) further promoted cellular osteogenic activity, and Sr10-mnHAp possessed the best stimulatory effect. The results of calvarial defects revealed that Sr10-mnHAp could promote more bone and blood vessel regeneration, with mnHAp and HAp bioceramics (dense and flat surfaces) as compared.
Conclusions:
The present study suggests that HAp bioceramics with micro-nano-hybrid surface and Sr doping had synergistic promotion effects on bone regeneration, which can be a promising material for bone defect repair.
|
Introduction:
Bone transplantation is a crucial method to reconstruct bone defects caused by trauma, tumors, and other diseases, and autologous bone grafts remain the “gold standard” for bone transplantation. However, the limitation of insufficient bone mass and related postoperative complications have facilitated the development of substitute materials.1,2 Bioceramic materials are widely used as a physiological scaffold for bone defect repair because of its excellent biocompatibility and bioactivity, and are currently considered as the most promising alternative to autologous bone grafting. The porous structure of bioceramics facilitates the adhesion of osteoblasts, transport of nutrients, and speeds up the process of bone resorption and reconstruction,3 which makes it better for repairing large bone defects.4
The inorganic minerals in human bone tissue have a multilevel ordered structure and contain many microelements. From the viewpoint of bio mimics, we can tune the osteoinduction ability of materials by controlling the surface microstructure and element doping. The ideal scaffold materials for bone regeneration require an appropriate three-dimensional microporous structure for cell growth and nutrient metabolism.2,5 For bioceramics scaffolds, the pores are 150 ~ 500 μm in diameter and over 50% in porosity are appropriate to stimulate cell response, and nanostructured surfaces and high connectivity between pores is also important.1,6–8 The results of our recent study confirmed that the HAp bioceramics with nanostructured modified topography significantly enhanced cell viability, spreading, and osteogenic differentiation of BMSCs and bone formation, mineralization and vascularization in vivo.9 Moreover, suitable surface chemistry plays critical roles in cell viability, spreading, proliferation, and osteogenic differentiation.2,5 Biomaterials can be modified by the introduction of functional inorganic ions. Recently, more attention has been given to strontium (Sr), which is an important trace element in the human body, and 99% of Sr exists in bones. Sr can improve the mechanical properties and modify the bone balance toward osteosynthesis.10 Recent studies11 have found that Sr can enhance the proliferation of osteoblasts during bone metabolism. Yang et al12 suggested that the surface of Sr-doped hydroxyapatite can significantly increase the growth of osteoblasts. The results of Wang et al13 confirmed that the Sr-HAp coatings significantly promoted osteoblast attachment and proliferation as the Sr content increased. Recently, researchers focused on the preparation of strontium-substituted bioceramics and their application in bone regeneration.14–16 However, few studies have focused on the synergistic promotion of nanostructures and Sr doping on bone regeneration.
Therefore, based on the above research and our previous results,9,17 we proposed that the combination of nanostructure surfaces and Sr-doping might lead to synergistic enhancement of osteogenesis and angiogenesis.18,19 Herein, HAp bioceramics with micro-nano-hybrid surfaces and different strontium (Sr) doping contents of 2.5, 5, 10, and 20% (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were fabricated via the hydrothermal transformation method. The effect on cell adhesion, proliferation, and ALP activity and the expression of genes involved in osteogenesis and angiogenesis were investigated to determine the optimal Sr doping content, followed by calvarial defect experiments to evaluate bone regeneration and vascularization in vivo.
Conclusion:
Surface structure and chemical composition are considered to be the key clues to regulate the biological response of biomaterials. Herein, HAp bioceramics with micro-nano-hybrid surface and different Sr doping contents (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were successfully fabricated via a hydrothermal transformation method using Srx-ɑ-TCP powder as a precursor without additives. The nanostructure surface can support cell adhesion, and Sr-doping could further enhance osteogenic differentiation with an optimal doping concentration of 10%. Finally, the calvarial defect model showed that the Sr10-mnHAp bioceramics scaffolds possessed better bone regeneration capacity. All of the results confirmed our hypothesis that micro-nano-hybrid surface and Sr doping have synergistic effects on bone regeneration and provided a new strategy for improving the osteoinduction ability of traditional bioceramics.
|
Background:
The synergistic effect of chemical element doping and surface modification is considered a novel way to regulate cell biological responses and improve the osteoinductive ability of biomaterials.
Methods:
Hydroxyapatite (HAp) bioceramics with micro-nano-hybrid (a mixture of microrods and nanorods) surfaces and different strontium (Sr) doping contents of 2.5, 5, 10, and 20% (Srx-mnHAp, x: 2.5, 5, 10 and 20%) were prepared via a hydrothermal transformation method. The effect of Srx-mnHAp on osteogenesis and angiogenesis of bone marrow stromal cells (BMSCs) was evaluated in vitro, and the bioceramics scaffolds were further implanted into rat calvarial defects for the observation of bone regeneration in vivo.
Results:
HAp bioceramics with micro-nano-hybrid surfaces (mnHAp) could facilitate cell spreading, proliferation ability, ALP activity, and gene expression of osteogenic and angiogenic factors, including COL1, BSP, BMP-2, OPN, VEGF, and ANG-1. More importantly, Srx-mnHAp (x: 2.5, 5, 10 and 20%) further promoted cellular osteogenic activity, and Sr10-mnHAp possessed the best stimulatory effect. The results of calvarial defects revealed that Sr10-mnHAp could promote more bone and blood vessel regeneration, with mnHAp and HAp bioceramics (dense and flat surfaces) as compared.
Conclusions:
The present study suggests that HAp bioceramics with micro-nano-hybrid surface and Sr doping had synergistic promotion effects on bone regeneration, which can be a promising material for bone defect repair.
| 13,295 | 297 | 23 |
[
"mnhap",
"s3",
"s0",
"5sr",
"bone",
"10sr",
"figure",
"significant",
"samples",
"indicates"
] |
[
"test",
"test"
] | null | null |
[CONTENT] bioceramics | scaffolds | surface modification | element doping | bone regeneration [SUMMARY]
| null | null |
[CONTENT] bioceramics | scaffolds | surface modification | element doping | bone regeneration [SUMMARY]
|
[CONTENT] bioceramics | scaffolds | surface modification | element doping | bone regeneration [SUMMARY]
|
[CONTENT] bioceramics | scaffolds | surface modification | element doping | bone regeneration [SUMMARY]
|
[CONTENT] Animals | Bone Regeneration | Cell Differentiation | Cell Proliferation | Durapatite | Osteogenesis | Rats | Strontium | Tissue Scaffolds [SUMMARY]
| null | null |
[CONTENT] Animals | Bone Regeneration | Cell Differentiation | Cell Proliferation | Durapatite | Osteogenesis | Rats | Strontium | Tissue Scaffolds [SUMMARY]
|
[CONTENT] Animals | Bone Regeneration | Cell Differentiation | Cell Proliferation | Durapatite | Osteogenesis | Rats | Strontium | Tissue Scaffolds [SUMMARY]
|
[CONTENT] Animals | Bone Regeneration | Cell Differentiation | Cell Proliferation | Durapatite | Osteogenesis | Rats | Strontium | Tissue Scaffolds [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null | null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] mnhap | s3 | s0 | 5sr | bone | 10sr | figure | significant | samples | indicates [SUMMARY]
| null | null |
[CONTENT] mnhap | s3 | s0 | 5sr | bone | 10sr | figure | significant | samples | indicates [SUMMARY]
|
[CONTENT] mnhap | s3 | s0 | 5sr | bone | 10sr | figure | significant | samples | indicates [SUMMARY]
|
[CONTENT] mnhap | s3 | s0 | 5sr | bone | 10sr | figure | significant | samples | indicates [SUMMARY]
|
[CONTENT] bone | sr | materials | strontium | doping | cell | osteoblasts | sr doping | proliferation | bone regeneration [SUMMARY]
| null | null |
[CONTENT] doping | sr doping | surface | nano hybrid surface | hybrid surface | micro nano hybrid surface | sr | micro nano | nano hybrid | nano [SUMMARY]
|
[CONTENT] mnhap | 5sr | bone | s3 | figure | s0 | indicates significant | indicates significant difference | significant difference | difference [SUMMARY]
|
[CONTENT] mnhap | 5sr | bone | s3 | figure | s0 | indicates significant | indicates significant difference | significant difference | difference [SUMMARY]
|
[CONTENT] [SUMMARY]
| null | null |
[CONTENT] Sr [SUMMARY]
|
[CONTENT] ||| 2.5 | 5 | 10 | 20% | 2.5 | 5 | 10 | 20% ||| BMSCs ||| ALP | COL1 | BSP | BMP-2 | OPN | VEGF | ANG-1 ||| Srx-mnHAp | 2.5 | 5 | 10 | 20% ||| mnHAp ||| Sr [SUMMARY]
|
[CONTENT] ||| 2.5 | 5 | 10 | 20% | 2.5 | 5 | 10 | 20% ||| BMSCs ||| ALP | COL1 | BSP | BMP-2 | OPN | VEGF | ANG-1 ||| Srx-mnHAp | 2.5 | 5 | 10 | 20% ||| mnHAp ||| Sr [SUMMARY]
|
||||
The contribution of dance to daily physical activity among adolescent girls.
|
21816074
|
Structured physical activity (PA) programs are well positioned to promote PA among youth, however, little is known about these programs, particularly dance classes. The aims of this study were to: 1) describe PA levels of girls enrolled in dance classes, 2) determine the contribution of dance classes to total moderate-to-vigorous physical activity (MVPA), and 3) compare PA between days with a dance class (program days) and days without a dance class (non-program days).
|
BACKGROUND
|
Participants were 149 girls (11-18 years) enrolled in dance classes in 11 dance studios. Overall PA was assessed with accelerometry for 8 consecutive days, and girls reported when they attended dance classes during those days. The percent contribution of dance classes to total MVPA was calculated, and data were reduced to compare PA on program days to non-program days. Data were analyzed using mixed models, adjusting for total monitoring time.
|
METHODS
|
Girls engaged in 25.0 ± 0.9 minutes/day of MVPA. Dance classes contributed 28.7% (95% CI: 25.9%-31.6%) to girls' total MVPA. Girls accumulated more MVPA on program (28.7 ± 1.4 minutes/day) than non-program days (16.4 ± 1.5 minutes/day) (p < 0.001). Girls had less sedentary behavior on program (554.0 ± 8.1 minutes/day) than non-program days (600.2 ± 8.7 minutes/day) (p < 0.001).
|
RESULTS
|
Dance classes contributed a substantial proportion (29%) to girls' total MVPA, and girls accumulated 70% more MVPA and 8% less sedentary behavior on program days than on non-program days. Dance classes can make an important contribution to girls' total physical activity.
|
CONCLUSIONS
|
[
"Adolescent",
"Anthropometry",
"Child",
"Cross-Sectional Studies",
"Dancing",
"Female",
"Humans",
"Motor Activity",
"Sedentary Behavior"
] |
3162521
|
Background
|
Helping youth achieve the current physical activity guideline of at least 60 minutes of daily moderate-to-vigorous physical activity (MVPA) is a key public health objective for the 21st century [1]. Structured physical activity programs are major avenues for providing physical activity to youth, and as such, they are a recommended strategy for the promotion of physical activity [1-3]. Structured physical activity programs are organized activities that are typically planned and occur within a specific setting [4]. These programs include physical education classes, organized sports, activity classes or lessons, and after-school programs.
Although structured physical activity programs are well positioned to assist youth in meeting the physical activity guideline, little is known about these programs. Specifically, there is limited knowledge of the overall physical activity levels of program participants and the contribution of these programs to overall physical activity. In addition, very few structured physical activity programs have been studied previously using objective measurement of physical activity by accelerometry. Only one study [5] has used accelerometry to examine the contribution of structured physical activity programs to total daily physical activity. Wickel and Eisenmann [5] found that among 6- to 12-year-old boys, youth sport and physical education classes contributed approximately 23% and 11% to their daily MVPA, respectively.
Dance classes are an important example of structured physical activity programs, because dance is a highly prevalent type of physical activity among adolescent girls [6-8]. Given the steep decline in girls' physical activity levels during adolescence [9-13], there is a need to study dance participation in adolescent girls. Further, the overall physical activity levels of girls who participate in dance classes are unknown, as is the contribution of dance classes to girls' overall physical activity levels. Accordingly, the objectives of this study were 1) to describe the overall physical activity levels of girls who participate in structured dance classes, 2) to determine the contribution of structured dance classes to total light, moderate, vigorous, and MVPA, and 3) to compare physical activity between days with a dance class (program days) and days without a dance class (non-program days).
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Methods
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Study Design This study employed a cross-sectional design in describing overall physical activity levels in girls who participate in structured dance classes. Each aim was addressed using a distinct methodology and design. Data were collected in a sample of girls registered for lessons in dance studios in Columbia, South Carolina. To be eligible, a dance studio was required to offer at least one ballet, jazz, or tap class per week to students aged 11 years and older. This protocol was designed to measure girls' physical activity via accelerometry over an eight-day period, and to link those data with the structured dance classes each girl attended during that period. In addition, girls' self-report of their structured dance classes allowed for determination of a program day (day with a dance class) and a non-program day (day without a dance class), for the comparison of physical activity levels.
This study employed a cross-sectional design in describing overall physical activity levels in girls who participate in structured dance classes. Each aim was addressed using a distinct methodology and design. Data were collected in a sample of girls registered for lessons in dance studios in Columbia, South Carolina. To be eligible, a dance studio was required to offer at least one ballet, jazz, or tap class per week to students aged 11 years and older. This protocol was designed to measure girls' physical activity via accelerometry over an eight-day period, and to link those data with the structured dance classes each girl attended during that period. In addition, girls' self-report of their structured dance classes allowed for determination of a program day (day with a dance class) and a non-program day (day without a dance class), for the comparison of physical activity levels.
Participants Participants were girls (ages 11 to 18 years) enrolled in dance classes at dance studios in Columbia, South Carolina. Forty-three dance studios, defined as commercial facilities whose primary business is to provide dance instruction, were identified using local published and electronic telephone books. The director of each dance studio was contacted by telephone to determine the studio's eligibility status. Twenty-three dance studios were eligible and were invited to participate. Of the 23 eligible dance studios, 11 directors agreed to participate, and those directors provided the schedules of the ballet, jazz, and tap classes that were offered to students aged 11 years and older. In each of the 11 dance studios, one to four classes (based on the studio size and the styles of dance offered) were selected for measurement, from which girls were recruited. Classes were not selected randomly; in small studios where there was only one eligible class, that class was chosen. In larger studios, convenience samples were taken that allowed for a variety of dance styles across age ranges. Girls were eligible to participate if they were enrolled in these dance classes and met the age criteria, which was a total of 212 girls. Of these students, 149 (70.3%) agreed to participate. Written informed consent was provided by each student's parent or guardian and informed assent was given by each student (if < 18 years) prior to collection of data. The study was approved by the University of South Carolina Institutional Review Board.
Participants were girls (ages 11 to 18 years) enrolled in dance classes at dance studios in Columbia, South Carolina. Forty-three dance studios, defined as commercial facilities whose primary business is to provide dance instruction, were identified using local published and electronic telephone books. The director of each dance studio was contacted by telephone to determine the studio's eligibility status. Twenty-three dance studios were eligible and were invited to participate. Of the 23 eligible dance studios, 11 directors agreed to participate, and those directors provided the schedules of the ballet, jazz, and tap classes that were offered to students aged 11 years and older. In each of the 11 dance studios, one to four classes (based on the studio size and the styles of dance offered) were selected for measurement, from which girls were recruited. Classes were not selected randomly; in small studios where there was only one eligible class, that class was chosen. In larger studios, convenience samples were taken that allowed for a variety of dance styles across age ranges. Girls were eligible to participate if they were enrolled in these dance classes and met the age criteria, which was a total of 212 girls. Of these students, 149 (70.3%) agreed to participate. Written informed consent was provided by each student's parent or guardian and informed assent was given by each student (if < 18 years) prior to collection of data. The study was approved by the University of South Carolina Institutional Review Board.
Objective Measurement of Physical Activity ActiGraph accelerometers (Model 7164, Pensacola, FL) were used to measure time spent in sedentary behavior, light, moderate, and vigorous physical activity, and combined MVPA. The ActiGraph is a reliable [14] and valid method for assessing children's and adolescent's physical activity, both in laboratory and field settings [15,16]. The cut-points established by Treuth and colleagues for use in adolescent girls aged 13 to 14 years were used to determine intensity levels [17]. Accelerometers were initialized to save data in 30-second intervals, in accordance with the procedures of Treuth et al. [17]. Accordingly, intensities of physical activity were operationally defined as: sedentary (< 50 counts/30 seconds), light (51-1499 counts/30 seconds), moderate (1500-2600 counts/30 seconds), vigorous physical activity (> 2600 counts/30 seconds), and MVPA (≥ 1500 counts/30 seconds) [17].
Accelerometers provide activity intensity counts which are associated with the corresponding date and time stamp. However, accelerometers do not detect the type of activity performed at any given time. Therefore, to estimate the amount of physical activity in structured dance classes, it was necessary for girls to report the days and times (e.g., Wednesday 5:00 PM - 6:30 PM) they participated in structured dance classes during the week when the accelerometer was worn. Girls reported this information on a written survey.
ActiGraph accelerometers (Model 7164, Pensacola, FL) were used to measure time spent in sedentary behavior, light, moderate, and vigorous physical activity, and combined MVPA. The ActiGraph is a reliable [14] and valid method for assessing children's and adolescent's physical activity, both in laboratory and field settings [15,16]. The cut-points established by Treuth and colleagues for use in adolescent girls aged 13 to 14 years were used to determine intensity levels [17]. Accelerometers were initialized to save data in 30-second intervals, in accordance with the procedures of Treuth et al. [17]. Accordingly, intensities of physical activity were operationally defined as: sedentary (< 50 counts/30 seconds), light (51-1499 counts/30 seconds), moderate (1500-2600 counts/30 seconds), vigorous physical activity (> 2600 counts/30 seconds), and MVPA (≥ 1500 counts/30 seconds) [17].
Accelerometers provide activity intensity counts which are associated with the corresponding date and time stamp. However, accelerometers do not detect the type of activity performed at any given time. Therefore, to estimate the amount of physical activity in structured dance classes, it was necessary for girls to report the days and times (e.g., Wednesday 5:00 PM - 6:30 PM) they participated in structured dance classes during the week when the accelerometer was worn. Girls reported this information on a written survey.
Measurement Protocol Overall physical activity was assessed with accelerometry for eight days. A research assistant placed accelerometers on participants approximately 10 minutes before the beginning of the dance class. Accelerometers were attached to an elastic belt and worn over the right hip. Participants wore the accelerometers during the entire class and continued to wear them for the following seven days. They were given instructions to wear the accelerometer during all waking hours, except when swimming or bathing. Participants wore the accelerometers to the same dance class the following week, and the accelerometers were removed by the research assistant at the end of the class. Upon collection of the accelerometers, activity counts were downloaded and saved on a computer for data reduction and analysis. In addition to the accelerometer collection, girls completed a written survey in which they reported the days and times they attended structured dance classes during the past week.
Overall physical activity was assessed with accelerometry for eight days. A research assistant placed accelerometers on participants approximately 10 minutes before the beginning of the dance class. Accelerometers were attached to an elastic belt and worn over the right hip. Participants wore the accelerometers during the entire class and continued to wear them for the following seven days. They were given instructions to wear the accelerometer during all waking hours, except when swimming or bathing. Participants wore the accelerometers to the same dance class the following week, and the accelerometers were removed by the research assistant at the end of the class. Upon collection of the accelerometers, activity counts were downloaded and saved on a computer for data reduction and analysis. In addition to the accelerometer collection, girls completed a written survey in which they reported the days and times they attended structured dance classes during the past week.
Data Reduction Overall physical activity An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.
An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.
Contribution of dance classes to total light, moderate, vigorous, and MVPA Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.
Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.
Program days vs. non-program days Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.
Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.
Overall physical activity An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.
An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.
Contribution of dance classes to total light, moderate, vigorous, and MVPA Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.
Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.
Program days vs. non-program days Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.
Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.
Anthropometric Measures Height and weight measurements were conducted in a private setting. Height and weight were assessed objectively using a portable stadiometer measured to the nearest 0.1 cm (Shorr Productions; Olney, MD) and an electronic scale measured to the nearest 0.1 kg (model 770; Seca, Hamburg, Germany). The average of two measurements was used. Body mass index (BMI) was calculated and expressed as body mass (kg) divided by height (m2), and BMI was converted to BMI percentiles using the CDC Growth Charts [19].
Height and weight measurements were conducted in a private setting. Height and weight were assessed objectively using a portable stadiometer measured to the nearest 0.1 cm (Shorr Productions; Olney, MD) and an electronic scale measured to the nearest 0.1 kg (model 770; Seca, Hamburg, Germany). The average of two measurements was used. Body mass index (BMI) was calculated and expressed as body mass (kg) divided by height (m2), and BMI was converted to BMI percentiles using the CDC Growth Charts [19].
Additional Variables Participants' date of birth and race/ethnicity were reported by parents on the consent form. To describe the sample, participants self-reported information about their dance participation. This included the starting age of dance instruction, dance styles ever studied, number of dance classes taken per week, hours of rehearsal per week, and participation in competitive or company dance. The number of classes per week, rehearsal hours per week, and competitive or company dance were in reference to current participation (i.e., present time).
Participants' date of birth and race/ethnicity were reported by parents on the consent form. To describe the sample, participants self-reported information about their dance participation. This included the starting age of dance instruction, dance styles ever studied, number of dance classes taken per week, hours of rehearsal per week, and participation in competitive or company dance. The number of classes per week, rehearsal hours per week, and competitive or company dance were in reference to current participation (i.e., present time).
Statistical Analyses All data were analyzed using generalized linear mixed models (PROC MIXED). For the purpose of comparing program days and non-program days, least squares means were used, and models were adjusted for total monitoring time. Means and standard errors were calculated. SAS software (version 9.2; SAS Institute, Cary, NC) was used for all statistical analyses. Statistical significance was set at P < 0.05.
All data were analyzed using generalized linear mixed models (PROC MIXED). For the purpose of comparing program days and non-program days, least squares means were used, and models were adjusted for total monitoring time. Means and standard errors were calculated. SAS software (version 9.2; SAS Institute, Cary, NC) was used for all statistical analyses. Statistical significance was set at P < 0.05.
| null | null |
Conclusion
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JRO conceived of and designed the study, acquired the data, analyzed and interpreted the data, and drafted the manuscript. RRP provided critical input during data analysis and manuscript development. The co-authors (RRP and SPH) participated in the interpretation of data and critical revision for important intellectual content. All authors read and approved the final manuscript.
|
[
"Background",
"Study Design",
"Participants",
"Objective Measurement of Physical Activity",
"Measurement Protocol",
"Data Reduction",
"Overall physical activity",
"Contribution of dance classes to total light, moderate, vigorous, and MVPA",
"Program days vs. non-program days",
"Anthropometric Measures",
"Additional Variables",
"Statistical Analyses",
"Results",
"Overall physical activity",
"Contribution of dance classes to total light, moderate, vigorous, and MVPA",
"Program day vs. non-program day",
"Discussion",
"Conclusion"
] |
[
"Helping youth achieve the current physical activity guideline of at least 60 minutes of daily moderate-to-vigorous physical activity (MVPA) is a key public health objective for the 21st century [1]. Structured physical activity programs are major avenues for providing physical activity to youth, and as such, they are a recommended strategy for the promotion of physical activity [1-3]. Structured physical activity programs are organized activities that are typically planned and occur within a specific setting [4]. These programs include physical education classes, organized sports, activity classes or lessons, and after-school programs.\nAlthough structured physical activity programs are well positioned to assist youth in meeting the physical activity guideline, little is known about these programs. Specifically, there is limited knowledge of the overall physical activity levels of program participants and the contribution of these programs to overall physical activity. In addition, very few structured physical activity programs have been studied previously using objective measurement of physical activity by accelerometry. Only one study [5] has used accelerometry to examine the contribution of structured physical activity programs to total daily physical activity. Wickel and Eisenmann [5] found that among 6- to 12-year-old boys, youth sport and physical education classes contributed approximately 23% and 11% to their daily MVPA, respectively.\nDance classes are an important example of structured physical activity programs, because dance is a highly prevalent type of physical activity among adolescent girls [6-8]. Given the steep decline in girls' physical activity levels during adolescence [9-13], there is a need to study dance participation in adolescent girls. Further, the overall physical activity levels of girls who participate in dance classes are unknown, as is the contribution of dance classes to girls' overall physical activity levels. Accordingly, the objectives of this study were 1) to describe the overall physical activity levels of girls who participate in structured dance classes, 2) to determine the contribution of structured dance classes to total light, moderate, vigorous, and MVPA, and 3) to compare physical activity between days with a dance class (program days) and days without a dance class (non-program days).",
"This study employed a cross-sectional design in describing overall physical activity levels in girls who participate in structured dance classes. Each aim was addressed using a distinct methodology and design. Data were collected in a sample of girls registered for lessons in dance studios in Columbia, South Carolina. To be eligible, a dance studio was required to offer at least one ballet, jazz, or tap class per week to students aged 11 years and older. This protocol was designed to measure girls' physical activity via accelerometry over an eight-day period, and to link those data with the structured dance classes each girl attended during that period. In addition, girls' self-report of their structured dance classes allowed for determination of a program day (day with a dance class) and a non-program day (day without a dance class), for the comparison of physical activity levels.",
"Participants were girls (ages 11 to 18 years) enrolled in dance classes at dance studios in Columbia, South Carolina. Forty-three dance studios, defined as commercial facilities whose primary business is to provide dance instruction, were identified using local published and electronic telephone books. The director of each dance studio was contacted by telephone to determine the studio's eligibility status. Twenty-three dance studios were eligible and were invited to participate. Of the 23 eligible dance studios, 11 directors agreed to participate, and those directors provided the schedules of the ballet, jazz, and tap classes that were offered to students aged 11 years and older. In each of the 11 dance studios, one to four classes (based on the studio size and the styles of dance offered) were selected for measurement, from which girls were recruited. Classes were not selected randomly; in small studios where there was only one eligible class, that class was chosen. In larger studios, convenience samples were taken that allowed for a variety of dance styles across age ranges. Girls were eligible to participate if they were enrolled in these dance classes and met the age criteria, which was a total of 212 girls. Of these students, 149 (70.3%) agreed to participate. Written informed consent was provided by each student's parent or guardian and informed assent was given by each student (if < 18 years) prior to collection of data. The study was approved by the University of South Carolina Institutional Review Board.",
"ActiGraph accelerometers (Model 7164, Pensacola, FL) were used to measure time spent in sedentary behavior, light, moderate, and vigorous physical activity, and combined MVPA. The ActiGraph is a reliable [14] and valid method for assessing children's and adolescent's physical activity, both in laboratory and field settings [15,16]. The cut-points established by Treuth and colleagues for use in adolescent girls aged 13 to 14 years were used to determine intensity levels [17]. Accelerometers were initialized to save data in 30-second intervals, in accordance with the procedures of Treuth et al. [17]. Accordingly, intensities of physical activity were operationally defined as: sedentary (< 50 counts/30 seconds), light (51-1499 counts/30 seconds), moderate (1500-2600 counts/30 seconds), vigorous physical activity (> 2600 counts/30 seconds), and MVPA (≥ 1500 counts/30 seconds) [17].\nAccelerometers provide activity intensity counts which are associated with the corresponding date and time stamp. However, accelerometers do not detect the type of activity performed at any given time. Therefore, to estimate the amount of physical activity in structured dance classes, it was necessary for girls to report the days and times (e.g., Wednesday 5:00 PM - 6:30 PM) they participated in structured dance classes during the week when the accelerometer was worn. Girls reported this information on a written survey.",
"Overall physical activity was assessed with accelerometry for eight days. A research assistant placed accelerometers on participants approximately 10 minutes before the beginning of the dance class. Accelerometers were attached to an elastic belt and worn over the right hip. Participants wore the accelerometers during the entire class and continued to wear them for the following seven days. They were given instructions to wear the accelerometer during all waking hours, except when swimming or bathing. Participants wore the accelerometers to the same dance class the following week, and the accelerometers were removed by the research assistant at the end of the class. Upon collection of the accelerometers, activity counts were downloaded and saved on a computer for data reduction and analysis. In addition to the accelerometer collection, girls completed a written survey in which they reported the days and times they attended structured dance classes during the past week.",
" Overall physical activity An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.\nAn established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.\n Contribution of dance classes to total light, moderate, vigorous, and MVPA Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.\nSeveral steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.\n Program days vs. non-program days Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.\nData were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.",
"An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.",
"Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.",
"Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.",
"Height and weight measurements were conducted in a private setting. Height and weight were assessed objectively using a portable stadiometer measured to the nearest 0.1 cm (Shorr Productions; Olney, MD) and an electronic scale measured to the nearest 0.1 kg (model 770; Seca, Hamburg, Germany). The average of two measurements was used. Body mass index (BMI) was calculated and expressed as body mass (kg) divided by height (m2), and BMI was converted to BMI percentiles using the CDC Growth Charts [19].",
"Participants' date of birth and race/ethnicity were reported by parents on the consent form. To describe the sample, participants self-reported information about their dance participation. This included the starting age of dance instruction, dance styles ever studied, number of dance classes taken per week, hours of rehearsal per week, and participation in competitive or company dance. The number of classes per week, rehearsal hours per week, and competitive or company dance were in reference to current participation (i.e., present time).",
"All data were analyzed using generalized linear mixed models (PROC MIXED). For the purpose of comparing program days and non-program days, least squares means were used, and models were adjusted for total monitoring time. Means and standard errors were calculated. SAS software (version 9.2; SAS Institute, Cary, NC) was used for all statistical analyses. Statistical significance was set at P < 0.05.",
"A total of 149 girls participated in the study. Eleven girls were excluded due to accelerometer malfunctions, and four were excluded for incomplete data, leaving 134 included in the data analyses. Due to the three analytical methods, there were three analytic samples; the descriptive characteristics of the three samples are presented in Table 1. The mean age was 14.6 ± 1.9 years, and over 80% of the participants were Caucasian. There are many styles of dance, and girls reported participating in the following styles during the previous week: ballet, jazz, tap, contemporary, hip hop, theatrical jazz/musical theatre, lyrical, baton, character, belly dance and ballroom.\nDescriptive characteristics of girls enrolled in structured dance classes.\nSD, standard deviation; BMI, body mass index.\n‡ Sample 2: Girls with ≥ 3 weekdays and ≥ 1 weekend day.\n¶ Sample 3: Girls with ≥ 1 program and 1 non-program day.\n** Sample 1: Asian (n = 5), Hispanic (n = 1), Multi-racial/Other (n = 5), Not Reported (n = 4).\n† Dance styles ever studied: ballet, jazz, tap, contemporary, hip hop, theatrical jazz/musical theatre, lyrical, baton, character/folk, ballroom, African, Indian, Irish, social dance, clogging, and belly dance.\n* Referred to current participation (i.e., present time).\n§ The average dance class length was 71.1 ± 18.1 minutes.\n Overall physical activity The analysis sample for overall physical activity included 134 girls. The means for the overall physical activity intensities are presented in Table 2. Girls obtained an average of 25.0 minutes per day of MVPA, 271.1 minutes per day of light physical activity, and 535.0 minutes per day of sedentary behavior.\nOverall physical activity, minutes per day, among girls enrolled in dance classes, (n = 134).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.\nThe analysis sample for overall physical activity included 134 girls. The means for the overall physical activity intensities are presented in Table 2. Girls obtained an average of 25.0 minutes per day of MVPA, 271.1 minutes per day of light physical activity, and 535.0 minutes per day of sedentary behavior.\nOverall physical activity, minutes per day, among girls enrolled in dance classes, (n = 134).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.\n Contribution of dance classes to total light, moderate, vigorous, and MVPA A total of 33 girls were excluded from this analysis for not having the required amount of qualifying days, resulting in an analysis sample of 101 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall physical activity; however, excluded girls had fewer dance classes per week compared to others (4.9 ± 1.8 vs. 6.6 ± 2.7, P < .001). The average reported dance class length was 71.1 ± 18.1 minutes. During structured dance classes, girls accumulated an average of 39.4 minutes per day of light physical activity and 7.1 minutes per day of MVPA (Table 3). Dance classes contributed 15.4%, 26.7%, 39.6%, and 28.7% of the girls' total light, moderate, vigorous, and MVPA, respectively.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA (n = 101).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.\nA total of 33 girls were excluded from this analysis for not having the required amount of qualifying days, resulting in an analysis sample of 101 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall physical activity; however, excluded girls had fewer dance classes per week compared to others (4.9 ± 1.8 vs. 6.6 ± 2.7, P < .001). The average reported dance class length was 71.1 ± 18.1 minutes. During structured dance classes, girls accumulated an average of 39.4 minutes per day of light physical activity and 7.1 minutes per day of MVPA (Table 3). Dance classes contributed 15.4%, 26.7%, 39.6%, and 28.7% of the girls' total light, moderate, vigorous, and MVPA, respectively.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA (n = 101).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.\n Program day vs. non-program day To be included in this analysis, girls needed to have at least one program day and one non-program day. From the first sample of 134 girls, 13 girls were excluded because they had less than two eligible days, 10 girls were excluded because they did not have any program days (on the eligible days), and 35 girls were excluded because they did not have any non-program days (i.e., they attended dance class on every eligible day). Therefore, the sample consisted of 76 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall sedentary, light, or moderate physical activity; however, excluded girls had a slightly greater percentage of vigorous (1.0% ± 0.4% vs. 0.8% ± 0.5%, p = 0.01) and MVPA (3.3% ± 1.0% vs. 2.8% ± 1.3%, p = 0.03) compared to the other girls. The comparison of physical activity intensities between program days and non-program days are presented in Table 4. There were no significant differences in the total monitoring time between the two days (program day: 854.5 ± 12.0 minutes; non-program day: 880.0 ± 15.0 minutes; p = 0.13). After controlling for total monitoring time, girls accumulated significantly more minutes of MVPA on program days (28.7 ± 1.4 minutes per day) than non-program days (16.4 ± 1.5 minutes per day) (p < 0.001). Girls had significantly fewer minutes of sedentary behavior on program days (554.0 ± 8.1 minutes per day) than non-program days (600.2 ± 8.7 minutes per day) (p < 0.001). Girls engaged in significantly more light, moderate, and vigorous physical activity on program days than non-program days (p < 0.001).\nProgram days vs. non-program days, minutes/day (mean ± SE), (n = 76).†\nMVPA, moderate-to-vigorous physical activity; SE, standard error.\n† Adjusted for total monitoring time.\nIn addition, we analyzed the physical activity data from the girls with zero non-program days (i.e., they attended dance class on every eligible day). Overall, these girls obtained an average of 28.9 minutes per day of MVPA, 9.0 minutes per day of vigorous physical activity, and 292.5 minutes per day of light physical activity (Table 5). They engaged in more vigorous physical activity and MVPA per day than girls in Sample 1. During structured dance classes, these girls accumulated an average of 54.5 minutes per day of light physical activity and 9.4 minutes per day of MVPA (Table 6), which was significantly more than girls in Sample 1. Dance classes contributed 20.5%, 31.2%, 42.6%, and 33.1% of the girls' total light, moderate, vigorous, and MVPA, respectively. This sub-sample of girls had a higher percent contribution of dance classes to total light, moderate, vigorous, and MVPA than girls in Sample 1.\nOverall physical activity for girls with zero non-program days, minutes/day (mean ± SE), (n = 35).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA for girls with zero non-program days (n = 31).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.\nTo be included in this analysis, girls needed to have at least one program day and one non-program day. From the first sample of 134 girls, 13 girls were excluded because they had less than two eligible days, 10 girls were excluded because they did not have any program days (on the eligible days), and 35 girls were excluded because they did not have any non-program days (i.e., they attended dance class on every eligible day). Therefore, the sample consisted of 76 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall sedentary, light, or moderate physical activity; however, excluded girls had a slightly greater percentage of vigorous (1.0% ± 0.4% vs. 0.8% ± 0.5%, p = 0.01) and MVPA (3.3% ± 1.0% vs. 2.8% ± 1.3%, p = 0.03) compared to the other girls. The comparison of physical activity intensities between program days and non-program days are presented in Table 4. There were no significant differences in the total monitoring time between the two days (program day: 854.5 ± 12.0 minutes; non-program day: 880.0 ± 15.0 minutes; p = 0.13). After controlling for total monitoring time, girls accumulated significantly more minutes of MVPA on program days (28.7 ± 1.4 minutes per day) than non-program days (16.4 ± 1.5 minutes per day) (p < 0.001). Girls had significantly fewer minutes of sedentary behavior on program days (554.0 ± 8.1 minutes per day) than non-program days (600.2 ± 8.7 minutes per day) (p < 0.001). Girls engaged in significantly more light, moderate, and vigorous physical activity on program days than non-program days (p < 0.001).\nProgram days vs. non-program days, minutes/day (mean ± SE), (n = 76).†\nMVPA, moderate-to-vigorous physical activity; SE, standard error.\n† Adjusted for total monitoring time.\nIn addition, we analyzed the physical activity data from the girls with zero non-program days (i.e., they attended dance class on every eligible day). Overall, these girls obtained an average of 28.9 minutes per day of MVPA, 9.0 minutes per day of vigorous physical activity, and 292.5 minutes per day of light physical activity (Table 5). They engaged in more vigorous physical activity and MVPA per day than girls in Sample 1. During structured dance classes, these girls accumulated an average of 54.5 minutes per day of light physical activity and 9.4 minutes per day of MVPA (Table 6), which was significantly more than girls in Sample 1. Dance classes contributed 20.5%, 31.2%, 42.6%, and 33.1% of the girls' total light, moderate, vigorous, and MVPA, respectively. This sub-sample of girls had a higher percent contribution of dance classes to total light, moderate, vigorous, and MVPA than girls in Sample 1.\nOverall physical activity for girls with zero non-program days, minutes/day (mean ± SE), (n = 35).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA for girls with zero non-program days (n = 31).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.",
"The analysis sample for overall physical activity included 134 girls. The means for the overall physical activity intensities are presented in Table 2. Girls obtained an average of 25.0 minutes per day of MVPA, 271.1 minutes per day of light physical activity, and 535.0 minutes per day of sedentary behavior.\nOverall physical activity, minutes per day, among girls enrolled in dance classes, (n = 134).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.",
"A total of 33 girls were excluded from this analysis for not having the required amount of qualifying days, resulting in an analysis sample of 101 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall physical activity; however, excluded girls had fewer dance classes per week compared to others (4.9 ± 1.8 vs. 6.6 ± 2.7, P < .001). The average reported dance class length was 71.1 ± 18.1 minutes. During structured dance classes, girls accumulated an average of 39.4 minutes per day of light physical activity and 7.1 minutes per day of MVPA (Table 3). Dance classes contributed 15.4%, 26.7%, 39.6%, and 28.7% of the girls' total light, moderate, vigorous, and MVPA, respectively.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA (n = 101).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.",
"To be included in this analysis, girls needed to have at least one program day and one non-program day. From the first sample of 134 girls, 13 girls were excluded because they had less than two eligible days, 10 girls were excluded because they did not have any program days (on the eligible days), and 35 girls were excluded because they did not have any non-program days (i.e., they attended dance class on every eligible day). Therefore, the sample consisted of 76 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall sedentary, light, or moderate physical activity; however, excluded girls had a slightly greater percentage of vigorous (1.0% ± 0.4% vs. 0.8% ± 0.5%, p = 0.01) and MVPA (3.3% ± 1.0% vs. 2.8% ± 1.3%, p = 0.03) compared to the other girls. The comparison of physical activity intensities between program days and non-program days are presented in Table 4. There were no significant differences in the total monitoring time between the two days (program day: 854.5 ± 12.0 minutes; non-program day: 880.0 ± 15.0 minutes; p = 0.13). After controlling for total monitoring time, girls accumulated significantly more minutes of MVPA on program days (28.7 ± 1.4 minutes per day) than non-program days (16.4 ± 1.5 minutes per day) (p < 0.001). Girls had significantly fewer minutes of sedentary behavior on program days (554.0 ± 8.1 minutes per day) than non-program days (600.2 ± 8.7 minutes per day) (p < 0.001). Girls engaged in significantly more light, moderate, and vigorous physical activity on program days than non-program days (p < 0.001).\nProgram days vs. non-program days, minutes/day (mean ± SE), (n = 76).†\nMVPA, moderate-to-vigorous physical activity; SE, standard error.\n† Adjusted for total monitoring time.\nIn addition, we analyzed the physical activity data from the girls with zero non-program days (i.e., they attended dance class on every eligible day). Overall, these girls obtained an average of 28.9 minutes per day of MVPA, 9.0 minutes per day of vigorous physical activity, and 292.5 minutes per day of light physical activity (Table 5). They engaged in more vigorous physical activity and MVPA per day than girls in Sample 1. During structured dance classes, these girls accumulated an average of 54.5 minutes per day of light physical activity and 9.4 minutes per day of MVPA (Table 6), which was significantly more than girls in Sample 1. Dance classes contributed 20.5%, 31.2%, 42.6%, and 33.1% of the girls' total light, moderate, vigorous, and MVPA, respectively. This sub-sample of girls had a higher percent contribution of dance classes to total light, moderate, vigorous, and MVPA than girls in Sample 1.\nOverall physical activity for girls with zero non-program days, minutes/day (mean ± SE), (n = 35).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA for girls with zero non-program days (n = 31).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.",
"This was the first study to describe the physical activity levels of girls who were enrolled in structured dance classes using an objective measure of physical activity. Activity performed in structured dance classes accounted for a substantial proportion (29%) of their total weekly MVPA. Most notably, girls obtained 70% more total MVPA and 8% less sedentary behavior on program days (i.e., participation in dance class), than non-program days. Therefore, these novel findings provide strong evidence of the important contribution that dance classes can make to girls' total physical activity.\nThe present study used accelerometry to measure physical activity, which has been done very rarely to examine the contribution of structured physical activity programs to total physical activity. Although several previous studies [5,20-22] have used accelerometry to examine physical activity during structured physical activity programs, to the best of our knowledge, only one study [5] has used accelerometry to examine the contribution of structured physical activity programs to total physical activity. Organized sports and physical education contributed 23% and 11% to boys' total daily MVPA, respectively [5]. These are lower than the contribution of dance classes in the present study (29%). Other studies have examined the contribution of structured physical activity programs to total physical activity, but they utilized different methodologies to assess physical activity [23,24]. For example, Katzmarzyk and Malina administered a 3-day activity diary to 12- to 14-year-old sport participants and found that youth sport contributed to 60% of their daily moderate to vigorous energy expenditure [24]. Morgan et al. [23] used pedometers in 1st through 6th graders and reported that the least, moderately active, and most active children obtained approximately 20%, 9%, and 16% of their total steps per day during physical education classes. The differences in these estimates are most likely attributable to the differences in physical activity assessments. However, it is important to recognize the advantages of accelerometry over other physical activity measurements: its ability to measure objectively, assess intensity, and provide activity counts with corresponding date and time stamps. Nonetheless, the present study supports previous findings that structured physical activity programs provide youth with a substantial proportion of MVPA to total MVPA.\nA key finding of this study was that girls accumulated 70% more MVPA on program days (i.e., participation in dance class) than on non-program days. Therefore, the additional amount of MVPA obtained on program days was not maintained on non-program days. Further, girls in this study had 46.2 fewer minutes of sedentary behavior on program days compared to non-program days. These findings are consistent with those of Wickel and Eisenmann [5] who found that 6- to 12-year-old boys engaged in more MVPA and less sedentary behavior on a sport day compared to a non-sport day. The present findings are also similar to those of Dale et al. [25], who reported that when school physical activity opportunities were restricted, children did not compensate by increasing their activity during the after-school hours. These findings provide compelling evidence of the potential importance of dance classes for increasing MVPA and reducing sedentary behavior in adolescent girls.\nAlthough a substantial proportion of girls' total weekly MVPA was attributable to dance classes, girls in this study were observed to have physical activity levels approximately the same as girls in other studies that objectively measured physical activity. Girls in this study engaged in an average of 25 minutes of MVPA per day, which is consistent with sixth-grade girls in the Trial of Activity for Adolescent Girls (TAAG) study who accumulated 23.7 minutes of MVPA per day [26]. The physical activity estimates for TAAG were also reported by geographic location, including South Carolina, and that sub-sample of girls obtained 20.8 minutes of MVPA per day [26]. Both the current study and TAAG used the accelerometer cut-points developed by Treuth et al. [17]. The present findings are also similar to those of 12- to 15-year-old girls in the National Health and Nutrition Examination Survey (NHANES) who obtained 24.6 minutes of MVPA per day [27]. In contrast, the amount of daily MVPA of girls in this study was slightly higher than that of 12-year-old girls in the UK [28] and 16- to 19-year-old girls in NHANES [27] who engaged in 18.3 and 19.6 minutes of MVPA per day, respectively. The MVPA cut-point in the UK study [28] was higher than the current study, whereas the MVPA cut-point in NHANES [27] was lower than the current study, which could potentially explain the differences in estimates. It is also important to note that in this study, the standard deviation was 10.1 minutes of MVPA per day, which indicates high inter-individual variability, which is consistent with the findings of Pate et al. [26]. These findings together indicate the need to increase overall physical activity levels of adolescent girls, as they, on average, are not achieving the physical activity guideline of at least 60 minutes of daily MVPA [1].\nThis study has strengths and limitations that should be noted. An important strength of this study was the objective measurement of physical activity and sedentary behavior via accelerometry. A second strength was the inclusion of a variety of dance styles from 11 dance studios. Another strength was the ability to measure physical activity on both program and non-program days. Limitations were that the accelerometer cut-points were developed for 13- to 14-year-old girls and were applied to girls ages 11 to 18 years, and the cut-points were not specifically developed for dancing. This may have resulted in an underestimation of physical activity from arm movement and its associated energy expenditure. Another limitation was the self-report of dance class schedules, which may be subject to bias and recall limitations. Lastly, because data were collected in one metropolitan area and mostly with Caucasian girls, they may not be generalizable to other populations. However, the sample size allowed us to test multiple hypotheses.",
"In summary, our findings demonstrate the importance of dance classes to girls' physical activity levels. We found that dance classes contributed a substantial proportion (29%) of MVPA to girls' total weekly MVPA, and girls accumulated 70% more MVPA and 8% less sedentary behavior on dance class days than on non-dance class days. Thus, the additional minutes of MVPA on dance class days were not maintained on non-dance class days. Therefore, dance classes can play a critical role by providing health-enhancing physical activity to adolescent girls, and can assist them in meeting the current physical activity guideline."
] |
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[
"Background",
"Methods",
"Study Design",
"Participants",
"Objective Measurement of Physical Activity",
"Measurement Protocol",
"Data Reduction",
"Overall physical activity",
"Contribution of dance classes to total light, moderate, vigorous, and MVPA",
"Program days vs. non-program days",
"Anthropometric Measures",
"Additional Variables",
"Statistical Analyses",
"Results",
"Overall physical activity",
"Contribution of dance classes to total light, moderate, vigorous, and MVPA",
"Program day vs. non-program day",
"Discussion",
"Conclusion"
] |
[
"Helping youth achieve the current physical activity guideline of at least 60 minutes of daily moderate-to-vigorous physical activity (MVPA) is a key public health objective for the 21st century [1]. Structured physical activity programs are major avenues for providing physical activity to youth, and as such, they are a recommended strategy for the promotion of physical activity [1-3]. Structured physical activity programs are organized activities that are typically planned and occur within a specific setting [4]. These programs include physical education classes, organized sports, activity classes or lessons, and after-school programs.\nAlthough structured physical activity programs are well positioned to assist youth in meeting the physical activity guideline, little is known about these programs. Specifically, there is limited knowledge of the overall physical activity levels of program participants and the contribution of these programs to overall physical activity. In addition, very few structured physical activity programs have been studied previously using objective measurement of physical activity by accelerometry. Only one study [5] has used accelerometry to examine the contribution of structured physical activity programs to total daily physical activity. Wickel and Eisenmann [5] found that among 6- to 12-year-old boys, youth sport and physical education classes contributed approximately 23% and 11% to their daily MVPA, respectively.\nDance classes are an important example of structured physical activity programs, because dance is a highly prevalent type of physical activity among adolescent girls [6-8]. Given the steep decline in girls' physical activity levels during adolescence [9-13], there is a need to study dance participation in adolescent girls. Further, the overall physical activity levels of girls who participate in dance classes are unknown, as is the contribution of dance classes to girls' overall physical activity levels. Accordingly, the objectives of this study were 1) to describe the overall physical activity levels of girls who participate in structured dance classes, 2) to determine the contribution of structured dance classes to total light, moderate, vigorous, and MVPA, and 3) to compare physical activity between days with a dance class (program days) and days without a dance class (non-program days).",
" Study Design This study employed a cross-sectional design in describing overall physical activity levels in girls who participate in structured dance classes. Each aim was addressed using a distinct methodology and design. Data were collected in a sample of girls registered for lessons in dance studios in Columbia, South Carolina. To be eligible, a dance studio was required to offer at least one ballet, jazz, or tap class per week to students aged 11 years and older. This protocol was designed to measure girls' physical activity via accelerometry over an eight-day period, and to link those data with the structured dance classes each girl attended during that period. In addition, girls' self-report of their structured dance classes allowed for determination of a program day (day with a dance class) and a non-program day (day without a dance class), for the comparison of physical activity levels.\nThis study employed a cross-sectional design in describing overall physical activity levels in girls who participate in structured dance classes. Each aim was addressed using a distinct methodology and design. Data were collected in a sample of girls registered for lessons in dance studios in Columbia, South Carolina. To be eligible, a dance studio was required to offer at least one ballet, jazz, or tap class per week to students aged 11 years and older. This protocol was designed to measure girls' physical activity via accelerometry over an eight-day period, and to link those data with the structured dance classes each girl attended during that period. In addition, girls' self-report of their structured dance classes allowed for determination of a program day (day with a dance class) and a non-program day (day without a dance class), for the comparison of physical activity levels.\n Participants Participants were girls (ages 11 to 18 years) enrolled in dance classes at dance studios in Columbia, South Carolina. Forty-three dance studios, defined as commercial facilities whose primary business is to provide dance instruction, were identified using local published and electronic telephone books. The director of each dance studio was contacted by telephone to determine the studio's eligibility status. Twenty-three dance studios were eligible and were invited to participate. Of the 23 eligible dance studios, 11 directors agreed to participate, and those directors provided the schedules of the ballet, jazz, and tap classes that were offered to students aged 11 years and older. In each of the 11 dance studios, one to four classes (based on the studio size and the styles of dance offered) were selected for measurement, from which girls were recruited. Classes were not selected randomly; in small studios where there was only one eligible class, that class was chosen. In larger studios, convenience samples were taken that allowed for a variety of dance styles across age ranges. Girls were eligible to participate if they were enrolled in these dance classes and met the age criteria, which was a total of 212 girls. Of these students, 149 (70.3%) agreed to participate. Written informed consent was provided by each student's parent or guardian and informed assent was given by each student (if < 18 years) prior to collection of data. The study was approved by the University of South Carolina Institutional Review Board.\nParticipants were girls (ages 11 to 18 years) enrolled in dance classes at dance studios in Columbia, South Carolina. Forty-three dance studios, defined as commercial facilities whose primary business is to provide dance instruction, were identified using local published and electronic telephone books. The director of each dance studio was contacted by telephone to determine the studio's eligibility status. Twenty-three dance studios were eligible and were invited to participate. Of the 23 eligible dance studios, 11 directors agreed to participate, and those directors provided the schedules of the ballet, jazz, and tap classes that were offered to students aged 11 years and older. In each of the 11 dance studios, one to four classes (based on the studio size and the styles of dance offered) were selected for measurement, from which girls were recruited. Classes were not selected randomly; in small studios where there was only one eligible class, that class was chosen. In larger studios, convenience samples were taken that allowed for a variety of dance styles across age ranges. Girls were eligible to participate if they were enrolled in these dance classes and met the age criteria, which was a total of 212 girls. Of these students, 149 (70.3%) agreed to participate. Written informed consent was provided by each student's parent or guardian and informed assent was given by each student (if < 18 years) prior to collection of data. The study was approved by the University of South Carolina Institutional Review Board.\n Objective Measurement of Physical Activity ActiGraph accelerometers (Model 7164, Pensacola, FL) were used to measure time spent in sedentary behavior, light, moderate, and vigorous physical activity, and combined MVPA. The ActiGraph is a reliable [14] and valid method for assessing children's and adolescent's physical activity, both in laboratory and field settings [15,16]. The cut-points established by Treuth and colleagues for use in adolescent girls aged 13 to 14 years were used to determine intensity levels [17]. Accelerometers were initialized to save data in 30-second intervals, in accordance with the procedures of Treuth et al. [17]. Accordingly, intensities of physical activity were operationally defined as: sedentary (< 50 counts/30 seconds), light (51-1499 counts/30 seconds), moderate (1500-2600 counts/30 seconds), vigorous physical activity (> 2600 counts/30 seconds), and MVPA (≥ 1500 counts/30 seconds) [17].\nAccelerometers provide activity intensity counts which are associated with the corresponding date and time stamp. However, accelerometers do not detect the type of activity performed at any given time. Therefore, to estimate the amount of physical activity in structured dance classes, it was necessary for girls to report the days and times (e.g., Wednesday 5:00 PM - 6:30 PM) they participated in structured dance classes during the week when the accelerometer was worn. Girls reported this information on a written survey.\nActiGraph accelerometers (Model 7164, Pensacola, FL) were used to measure time spent in sedentary behavior, light, moderate, and vigorous physical activity, and combined MVPA. The ActiGraph is a reliable [14] and valid method for assessing children's and adolescent's physical activity, both in laboratory and field settings [15,16]. The cut-points established by Treuth and colleagues for use in adolescent girls aged 13 to 14 years were used to determine intensity levels [17]. Accelerometers were initialized to save data in 30-second intervals, in accordance with the procedures of Treuth et al. [17]. Accordingly, intensities of physical activity were operationally defined as: sedentary (< 50 counts/30 seconds), light (51-1499 counts/30 seconds), moderate (1500-2600 counts/30 seconds), vigorous physical activity (> 2600 counts/30 seconds), and MVPA (≥ 1500 counts/30 seconds) [17].\nAccelerometers provide activity intensity counts which are associated with the corresponding date and time stamp. However, accelerometers do not detect the type of activity performed at any given time. Therefore, to estimate the amount of physical activity in structured dance classes, it was necessary for girls to report the days and times (e.g., Wednesday 5:00 PM - 6:30 PM) they participated in structured dance classes during the week when the accelerometer was worn. Girls reported this information on a written survey.\n Measurement Protocol Overall physical activity was assessed with accelerometry for eight days. A research assistant placed accelerometers on participants approximately 10 minutes before the beginning of the dance class. Accelerometers were attached to an elastic belt and worn over the right hip. Participants wore the accelerometers during the entire class and continued to wear them for the following seven days. They were given instructions to wear the accelerometer during all waking hours, except when swimming or bathing. Participants wore the accelerometers to the same dance class the following week, and the accelerometers were removed by the research assistant at the end of the class. Upon collection of the accelerometers, activity counts were downloaded and saved on a computer for data reduction and analysis. In addition to the accelerometer collection, girls completed a written survey in which they reported the days and times they attended structured dance classes during the past week.\nOverall physical activity was assessed with accelerometry for eight days. A research assistant placed accelerometers on participants approximately 10 minutes before the beginning of the dance class. Accelerometers were attached to an elastic belt and worn over the right hip. Participants wore the accelerometers during the entire class and continued to wear them for the following seven days. They were given instructions to wear the accelerometer during all waking hours, except when swimming or bathing. Participants wore the accelerometers to the same dance class the following week, and the accelerometers were removed by the research assistant at the end of the class. Upon collection of the accelerometers, activity counts were downloaded and saved on a computer for data reduction and analysis. In addition to the accelerometer collection, girls completed a written survey in which they reported the days and times they attended structured dance classes during the past week.\n Data Reduction Overall physical activity An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.\nAn established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.\n Contribution of dance classes to total light, moderate, vigorous, and MVPA Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.\nSeveral steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.\n Program days vs. non-program days Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.\nData were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.\n Overall physical activity An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.\nAn established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.\n Contribution of dance classes to total light, moderate, vigorous, and MVPA Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.\nSeveral steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.\n Program days vs. non-program days Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.\nData were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.\n Anthropometric Measures Height and weight measurements were conducted in a private setting. Height and weight were assessed objectively using a portable stadiometer measured to the nearest 0.1 cm (Shorr Productions; Olney, MD) and an electronic scale measured to the nearest 0.1 kg (model 770; Seca, Hamburg, Germany). The average of two measurements was used. Body mass index (BMI) was calculated and expressed as body mass (kg) divided by height (m2), and BMI was converted to BMI percentiles using the CDC Growth Charts [19].\nHeight and weight measurements were conducted in a private setting. Height and weight were assessed objectively using a portable stadiometer measured to the nearest 0.1 cm (Shorr Productions; Olney, MD) and an electronic scale measured to the nearest 0.1 kg (model 770; Seca, Hamburg, Germany). The average of two measurements was used. Body mass index (BMI) was calculated and expressed as body mass (kg) divided by height (m2), and BMI was converted to BMI percentiles using the CDC Growth Charts [19].\n Additional Variables Participants' date of birth and race/ethnicity were reported by parents on the consent form. To describe the sample, participants self-reported information about their dance participation. This included the starting age of dance instruction, dance styles ever studied, number of dance classes taken per week, hours of rehearsal per week, and participation in competitive or company dance. The number of classes per week, rehearsal hours per week, and competitive or company dance were in reference to current participation (i.e., present time).\nParticipants' date of birth and race/ethnicity were reported by parents on the consent form. To describe the sample, participants self-reported information about their dance participation. This included the starting age of dance instruction, dance styles ever studied, number of dance classes taken per week, hours of rehearsal per week, and participation in competitive or company dance. The number of classes per week, rehearsal hours per week, and competitive or company dance were in reference to current participation (i.e., present time).\n Statistical Analyses All data were analyzed using generalized linear mixed models (PROC MIXED). For the purpose of comparing program days and non-program days, least squares means were used, and models were adjusted for total monitoring time. Means and standard errors were calculated. SAS software (version 9.2; SAS Institute, Cary, NC) was used for all statistical analyses. Statistical significance was set at P < 0.05.\nAll data were analyzed using generalized linear mixed models (PROC MIXED). For the purpose of comparing program days and non-program days, least squares means were used, and models were adjusted for total monitoring time. Means and standard errors were calculated. SAS software (version 9.2; SAS Institute, Cary, NC) was used for all statistical analyses. Statistical significance was set at P < 0.05.",
"This study employed a cross-sectional design in describing overall physical activity levels in girls who participate in structured dance classes. Each aim was addressed using a distinct methodology and design. Data were collected in a sample of girls registered for lessons in dance studios in Columbia, South Carolina. To be eligible, a dance studio was required to offer at least one ballet, jazz, or tap class per week to students aged 11 years and older. This protocol was designed to measure girls' physical activity via accelerometry over an eight-day period, and to link those data with the structured dance classes each girl attended during that period. In addition, girls' self-report of their structured dance classes allowed for determination of a program day (day with a dance class) and a non-program day (day without a dance class), for the comparison of physical activity levels.",
"Participants were girls (ages 11 to 18 years) enrolled in dance classes at dance studios in Columbia, South Carolina. Forty-three dance studios, defined as commercial facilities whose primary business is to provide dance instruction, were identified using local published and electronic telephone books. The director of each dance studio was contacted by telephone to determine the studio's eligibility status. Twenty-three dance studios were eligible and were invited to participate. Of the 23 eligible dance studios, 11 directors agreed to participate, and those directors provided the schedules of the ballet, jazz, and tap classes that were offered to students aged 11 years and older. In each of the 11 dance studios, one to four classes (based on the studio size and the styles of dance offered) were selected for measurement, from which girls were recruited. Classes were not selected randomly; in small studios where there was only one eligible class, that class was chosen. In larger studios, convenience samples were taken that allowed for a variety of dance styles across age ranges. Girls were eligible to participate if they were enrolled in these dance classes and met the age criteria, which was a total of 212 girls. Of these students, 149 (70.3%) agreed to participate. Written informed consent was provided by each student's parent or guardian and informed assent was given by each student (if < 18 years) prior to collection of data. The study was approved by the University of South Carolina Institutional Review Board.",
"ActiGraph accelerometers (Model 7164, Pensacola, FL) were used to measure time spent in sedentary behavior, light, moderate, and vigorous physical activity, and combined MVPA. The ActiGraph is a reliable [14] and valid method for assessing children's and adolescent's physical activity, both in laboratory and field settings [15,16]. The cut-points established by Treuth and colleagues for use in adolescent girls aged 13 to 14 years were used to determine intensity levels [17]. Accelerometers were initialized to save data in 30-second intervals, in accordance with the procedures of Treuth et al. [17]. Accordingly, intensities of physical activity were operationally defined as: sedentary (< 50 counts/30 seconds), light (51-1499 counts/30 seconds), moderate (1500-2600 counts/30 seconds), vigorous physical activity (> 2600 counts/30 seconds), and MVPA (≥ 1500 counts/30 seconds) [17].\nAccelerometers provide activity intensity counts which are associated with the corresponding date and time stamp. However, accelerometers do not detect the type of activity performed at any given time. Therefore, to estimate the amount of physical activity in structured dance classes, it was necessary for girls to report the days and times (e.g., Wednesday 5:00 PM - 6:30 PM) they participated in structured dance classes during the week when the accelerometer was worn. Girls reported this information on a written survey.",
"Overall physical activity was assessed with accelerometry for eight days. A research assistant placed accelerometers on participants approximately 10 minutes before the beginning of the dance class. Accelerometers were attached to an elastic belt and worn over the right hip. Participants wore the accelerometers during the entire class and continued to wear them for the following seven days. They were given instructions to wear the accelerometer during all waking hours, except when swimming or bathing. Participants wore the accelerometers to the same dance class the following week, and the accelerometers were removed by the research assistant at the end of the class. Upon collection of the accelerometers, activity counts were downloaded and saved on a computer for data reduction and analysis. In addition to the accelerometer collection, girls completed a written survey in which they reported the days and times they attended structured dance classes during the past week.",
" Overall physical activity An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.\nAn established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.\n Contribution of dance classes to total light, moderate, vigorous, and MVPA Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.\nSeveral steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.\n Program days vs. non-program days Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.\nData were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.",
"An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.",
"Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.",
"Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.",
"Height and weight measurements were conducted in a private setting. Height and weight were assessed objectively using a portable stadiometer measured to the nearest 0.1 cm (Shorr Productions; Olney, MD) and an electronic scale measured to the nearest 0.1 kg (model 770; Seca, Hamburg, Germany). The average of two measurements was used. Body mass index (BMI) was calculated and expressed as body mass (kg) divided by height (m2), and BMI was converted to BMI percentiles using the CDC Growth Charts [19].",
"Participants' date of birth and race/ethnicity were reported by parents on the consent form. To describe the sample, participants self-reported information about their dance participation. This included the starting age of dance instruction, dance styles ever studied, number of dance classes taken per week, hours of rehearsal per week, and participation in competitive or company dance. The number of classes per week, rehearsal hours per week, and competitive or company dance were in reference to current participation (i.e., present time).",
"All data were analyzed using generalized linear mixed models (PROC MIXED). For the purpose of comparing program days and non-program days, least squares means were used, and models were adjusted for total monitoring time. Means and standard errors were calculated. SAS software (version 9.2; SAS Institute, Cary, NC) was used for all statistical analyses. Statistical significance was set at P < 0.05.",
"A total of 149 girls participated in the study. Eleven girls were excluded due to accelerometer malfunctions, and four were excluded for incomplete data, leaving 134 included in the data analyses. Due to the three analytical methods, there were three analytic samples; the descriptive characteristics of the three samples are presented in Table 1. The mean age was 14.6 ± 1.9 years, and over 80% of the participants were Caucasian. There are many styles of dance, and girls reported participating in the following styles during the previous week: ballet, jazz, tap, contemporary, hip hop, theatrical jazz/musical theatre, lyrical, baton, character, belly dance and ballroom.\nDescriptive characteristics of girls enrolled in structured dance classes.\nSD, standard deviation; BMI, body mass index.\n‡ Sample 2: Girls with ≥ 3 weekdays and ≥ 1 weekend day.\n¶ Sample 3: Girls with ≥ 1 program and 1 non-program day.\n** Sample 1: Asian (n = 5), Hispanic (n = 1), Multi-racial/Other (n = 5), Not Reported (n = 4).\n† Dance styles ever studied: ballet, jazz, tap, contemporary, hip hop, theatrical jazz/musical theatre, lyrical, baton, character/folk, ballroom, African, Indian, Irish, social dance, clogging, and belly dance.\n* Referred to current participation (i.e., present time).\n§ The average dance class length was 71.1 ± 18.1 minutes.\n Overall physical activity The analysis sample for overall physical activity included 134 girls. The means for the overall physical activity intensities are presented in Table 2. Girls obtained an average of 25.0 minutes per day of MVPA, 271.1 minutes per day of light physical activity, and 535.0 minutes per day of sedentary behavior.\nOverall physical activity, minutes per day, among girls enrolled in dance classes, (n = 134).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.\nThe analysis sample for overall physical activity included 134 girls. The means for the overall physical activity intensities are presented in Table 2. Girls obtained an average of 25.0 minutes per day of MVPA, 271.1 minutes per day of light physical activity, and 535.0 minutes per day of sedentary behavior.\nOverall physical activity, minutes per day, among girls enrolled in dance classes, (n = 134).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.\n Contribution of dance classes to total light, moderate, vigorous, and MVPA A total of 33 girls were excluded from this analysis for not having the required amount of qualifying days, resulting in an analysis sample of 101 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall physical activity; however, excluded girls had fewer dance classes per week compared to others (4.9 ± 1.8 vs. 6.6 ± 2.7, P < .001). The average reported dance class length was 71.1 ± 18.1 minutes. During structured dance classes, girls accumulated an average of 39.4 minutes per day of light physical activity and 7.1 minutes per day of MVPA (Table 3). Dance classes contributed 15.4%, 26.7%, 39.6%, and 28.7% of the girls' total light, moderate, vigorous, and MVPA, respectively.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA (n = 101).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.\nA total of 33 girls were excluded from this analysis for not having the required amount of qualifying days, resulting in an analysis sample of 101 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall physical activity; however, excluded girls had fewer dance classes per week compared to others (4.9 ± 1.8 vs. 6.6 ± 2.7, P < .001). The average reported dance class length was 71.1 ± 18.1 minutes. During structured dance classes, girls accumulated an average of 39.4 minutes per day of light physical activity and 7.1 minutes per day of MVPA (Table 3). Dance classes contributed 15.4%, 26.7%, 39.6%, and 28.7% of the girls' total light, moderate, vigorous, and MVPA, respectively.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA (n = 101).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.\n Program day vs. non-program day To be included in this analysis, girls needed to have at least one program day and one non-program day. From the first sample of 134 girls, 13 girls were excluded because they had less than two eligible days, 10 girls were excluded because they did not have any program days (on the eligible days), and 35 girls were excluded because they did not have any non-program days (i.e., they attended dance class on every eligible day). Therefore, the sample consisted of 76 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall sedentary, light, or moderate physical activity; however, excluded girls had a slightly greater percentage of vigorous (1.0% ± 0.4% vs. 0.8% ± 0.5%, p = 0.01) and MVPA (3.3% ± 1.0% vs. 2.8% ± 1.3%, p = 0.03) compared to the other girls. The comparison of physical activity intensities between program days and non-program days are presented in Table 4. There were no significant differences in the total monitoring time between the two days (program day: 854.5 ± 12.0 minutes; non-program day: 880.0 ± 15.0 minutes; p = 0.13). After controlling for total monitoring time, girls accumulated significantly more minutes of MVPA on program days (28.7 ± 1.4 minutes per day) than non-program days (16.4 ± 1.5 minutes per day) (p < 0.001). Girls had significantly fewer minutes of sedentary behavior on program days (554.0 ± 8.1 minutes per day) than non-program days (600.2 ± 8.7 minutes per day) (p < 0.001). Girls engaged in significantly more light, moderate, and vigorous physical activity on program days than non-program days (p < 0.001).\nProgram days vs. non-program days, minutes/day (mean ± SE), (n = 76).†\nMVPA, moderate-to-vigorous physical activity; SE, standard error.\n† Adjusted for total monitoring time.\nIn addition, we analyzed the physical activity data from the girls with zero non-program days (i.e., they attended dance class on every eligible day). Overall, these girls obtained an average of 28.9 minutes per day of MVPA, 9.0 minutes per day of vigorous physical activity, and 292.5 minutes per day of light physical activity (Table 5). They engaged in more vigorous physical activity and MVPA per day than girls in Sample 1. During structured dance classes, these girls accumulated an average of 54.5 minutes per day of light physical activity and 9.4 minutes per day of MVPA (Table 6), which was significantly more than girls in Sample 1. Dance classes contributed 20.5%, 31.2%, 42.6%, and 33.1% of the girls' total light, moderate, vigorous, and MVPA, respectively. This sub-sample of girls had a higher percent contribution of dance classes to total light, moderate, vigorous, and MVPA than girls in Sample 1.\nOverall physical activity for girls with zero non-program days, minutes/day (mean ± SE), (n = 35).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA for girls with zero non-program days (n = 31).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.\nTo be included in this analysis, girls needed to have at least one program day and one non-program day. From the first sample of 134 girls, 13 girls were excluded because they had less than two eligible days, 10 girls were excluded because they did not have any program days (on the eligible days), and 35 girls were excluded because they did not have any non-program days (i.e., they attended dance class on every eligible day). Therefore, the sample consisted of 76 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall sedentary, light, or moderate physical activity; however, excluded girls had a slightly greater percentage of vigorous (1.0% ± 0.4% vs. 0.8% ± 0.5%, p = 0.01) and MVPA (3.3% ± 1.0% vs. 2.8% ± 1.3%, p = 0.03) compared to the other girls. The comparison of physical activity intensities between program days and non-program days are presented in Table 4. There were no significant differences in the total monitoring time between the two days (program day: 854.5 ± 12.0 minutes; non-program day: 880.0 ± 15.0 minutes; p = 0.13). After controlling for total monitoring time, girls accumulated significantly more minutes of MVPA on program days (28.7 ± 1.4 minutes per day) than non-program days (16.4 ± 1.5 minutes per day) (p < 0.001). Girls had significantly fewer minutes of sedentary behavior on program days (554.0 ± 8.1 minutes per day) than non-program days (600.2 ± 8.7 minutes per day) (p < 0.001). Girls engaged in significantly more light, moderate, and vigorous physical activity on program days than non-program days (p < 0.001).\nProgram days vs. non-program days, minutes/day (mean ± SE), (n = 76).†\nMVPA, moderate-to-vigorous physical activity; SE, standard error.\n† Adjusted for total monitoring time.\nIn addition, we analyzed the physical activity data from the girls with zero non-program days (i.e., they attended dance class on every eligible day). Overall, these girls obtained an average of 28.9 minutes per day of MVPA, 9.0 minutes per day of vigorous physical activity, and 292.5 minutes per day of light physical activity (Table 5). They engaged in more vigorous physical activity and MVPA per day than girls in Sample 1. During structured dance classes, these girls accumulated an average of 54.5 minutes per day of light physical activity and 9.4 minutes per day of MVPA (Table 6), which was significantly more than girls in Sample 1. Dance classes contributed 20.5%, 31.2%, 42.6%, and 33.1% of the girls' total light, moderate, vigorous, and MVPA, respectively. This sub-sample of girls had a higher percent contribution of dance classes to total light, moderate, vigorous, and MVPA than girls in Sample 1.\nOverall physical activity for girls with zero non-program days, minutes/day (mean ± SE), (n = 35).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA for girls with zero non-program days (n = 31).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.",
"The analysis sample for overall physical activity included 134 girls. The means for the overall physical activity intensities are presented in Table 2. Girls obtained an average of 25.0 minutes per day of MVPA, 271.1 minutes per day of light physical activity, and 535.0 minutes per day of sedentary behavior.\nOverall physical activity, minutes per day, among girls enrolled in dance classes, (n = 134).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.",
"A total of 33 girls were excluded from this analysis for not having the required amount of qualifying days, resulting in an analysis sample of 101 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall physical activity; however, excluded girls had fewer dance classes per week compared to others (4.9 ± 1.8 vs. 6.6 ± 2.7, P < .001). The average reported dance class length was 71.1 ± 18.1 minutes. During structured dance classes, girls accumulated an average of 39.4 minutes per day of light physical activity and 7.1 minutes per day of MVPA (Table 3). Dance classes contributed 15.4%, 26.7%, 39.6%, and 28.7% of the girls' total light, moderate, vigorous, and MVPA, respectively.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA (n = 101).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.",
"To be included in this analysis, girls needed to have at least one program day and one non-program day. From the first sample of 134 girls, 13 girls were excluded because they had less than two eligible days, 10 girls were excluded because they did not have any program days (on the eligible days), and 35 girls were excluded because they did not have any non-program days (i.e., they attended dance class on every eligible day). Therefore, the sample consisted of 76 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall sedentary, light, or moderate physical activity; however, excluded girls had a slightly greater percentage of vigorous (1.0% ± 0.4% vs. 0.8% ± 0.5%, p = 0.01) and MVPA (3.3% ± 1.0% vs. 2.8% ± 1.3%, p = 0.03) compared to the other girls. The comparison of physical activity intensities between program days and non-program days are presented in Table 4. There were no significant differences in the total monitoring time between the two days (program day: 854.5 ± 12.0 minutes; non-program day: 880.0 ± 15.0 minutes; p = 0.13). After controlling for total monitoring time, girls accumulated significantly more minutes of MVPA on program days (28.7 ± 1.4 minutes per day) than non-program days (16.4 ± 1.5 minutes per day) (p < 0.001). Girls had significantly fewer minutes of sedentary behavior on program days (554.0 ± 8.1 minutes per day) than non-program days (600.2 ± 8.7 minutes per day) (p < 0.001). Girls engaged in significantly more light, moderate, and vigorous physical activity on program days than non-program days (p < 0.001).\nProgram days vs. non-program days, minutes/day (mean ± SE), (n = 76).†\nMVPA, moderate-to-vigorous physical activity; SE, standard error.\n† Adjusted for total monitoring time.\nIn addition, we analyzed the physical activity data from the girls with zero non-program days (i.e., they attended dance class on every eligible day). Overall, these girls obtained an average of 28.9 minutes per day of MVPA, 9.0 minutes per day of vigorous physical activity, and 292.5 minutes per day of light physical activity (Table 5). They engaged in more vigorous physical activity and MVPA per day than girls in Sample 1. During structured dance classes, these girls accumulated an average of 54.5 minutes per day of light physical activity and 9.4 minutes per day of MVPA (Table 6), which was significantly more than girls in Sample 1. Dance classes contributed 20.5%, 31.2%, 42.6%, and 33.1% of the girls' total light, moderate, vigorous, and MVPA, respectively. This sub-sample of girls had a higher percent contribution of dance classes to total light, moderate, vigorous, and MVPA than girls in Sample 1.\nOverall physical activity for girls with zero non-program days, minutes/day (mean ± SE), (n = 35).\nMVPA, moderate-to-vigorous physical activity; SE, standard error;\nCI, confidence interval.\nContribution of dance classes to total light, moderate, and vigorous physical activity and MVPA for girls with zero non-program days (n = 31).\nLPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.",
"This was the first study to describe the physical activity levels of girls who were enrolled in structured dance classes using an objective measure of physical activity. Activity performed in structured dance classes accounted for a substantial proportion (29%) of their total weekly MVPA. Most notably, girls obtained 70% more total MVPA and 8% less sedentary behavior on program days (i.e., participation in dance class), than non-program days. Therefore, these novel findings provide strong evidence of the important contribution that dance classes can make to girls' total physical activity.\nThe present study used accelerometry to measure physical activity, which has been done very rarely to examine the contribution of structured physical activity programs to total physical activity. Although several previous studies [5,20-22] have used accelerometry to examine physical activity during structured physical activity programs, to the best of our knowledge, only one study [5] has used accelerometry to examine the contribution of structured physical activity programs to total physical activity. Organized sports and physical education contributed 23% and 11% to boys' total daily MVPA, respectively [5]. These are lower than the contribution of dance classes in the present study (29%). Other studies have examined the contribution of structured physical activity programs to total physical activity, but they utilized different methodologies to assess physical activity [23,24]. For example, Katzmarzyk and Malina administered a 3-day activity diary to 12- to 14-year-old sport participants and found that youth sport contributed to 60% of their daily moderate to vigorous energy expenditure [24]. Morgan et al. [23] used pedometers in 1st through 6th graders and reported that the least, moderately active, and most active children obtained approximately 20%, 9%, and 16% of their total steps per day during physical education classes. The differences in these estimates are most likely attributable to the differences in physical activity assessments. However, it is important to recognize the advantages of accelerometry over other physical activity measurements: its ability to measure objectively, assess intensity, and provide activity counts with corresponding date and time stamps. Nonetheless, the present study supports previous findings that structured physical activity programs provide youth with a substantial proportion of MVPA to total MVPA.\nA key finding of this study was that girls accumulated 70% more MVPA on program days (i.e., participation in dance class) than on non-program days. Therefore, the additional amount of MVPA obtained on program days was not maintained on non-program days. Further, girls in this study had 46.2 fewer minutes of sedentary behavior on program days compared to non-program days. These findings are consistent with those of Wickel and Eisenmann [5] who found that 6- to 12-year-old boys engaged in more MVPA and less sedentary behavior on a sport day compared to a non-sport day. The present findings are also similar to those of Dale et al. [25], who reported that when school physical activity opportunities were restricted, children did not compensate by increasing their activity during the after-school hours. These findings provide compelling evidence of the potential importance of dance classes for increasing MVPA and reducing sedentary behavior in adolescent girls.\nAlthough a substantial proportion of girls' total weekly MVPA was attributable to dance classes, girls in this study were observed to have physical activity levels approximately the same as girls in other studies that objectively measured physical activity. Girls in this study engaged in an average of 25 minutes of MVPA per day, which is consistent with sixth-grade girls in the Trial of Activity for Adolescent Girls (TAAG) study who accumulated 23.7 minutes of MVPA per day [26]. The physical activity estimates for TAAG were also reported by geographic location, including South Carolina, and that sub-sample of girls obtained 20.8 minutes of MVPA per day [26]. Both the current study and TAAG used the accelerometer cut-points developed by Treuth et al. [17]. The present findings are also similar to those of 12- to 15-year-old girls in the National Health and Nutrition Examination Survey (NHANES) who obtained 24.6 minutes of MVPA per day [27]. In contrast, the amount of daily MVPA of girls in this study was slightly higher than that of 12-year-old girls in the UK [28] and 16- to 19-year-old girls in NHANES [27] who engaged in 18.3 and 19.6 minutes of MVPA per day, respectively. The MVPA cut-point in the UK study [28] was higher than the current study, whereas the MVPA cut-point in NHANES [27] was lower than the current study, which could potentially explain the differences in estimates. It is also important to note that in this study, the standard deviation was 10.1 minutes of MVPA per day, which indicates high inter-individual variability, which is consistent with the findings of Pate et al. [26]. These findings together indicate the need to increase overall physical activity levels of adolescent girls, as they, on average, are not achieving the physical activity guideline of at least 60 minutes of daily MVPA [1].\nThis study has strengths and limitations that should be noted. An important strength of this study was the objective measurement of physical activity and sedentary behavior via accelerometry. A second strength was the inclusion of a variety of dance styles from 11 dance studios. Another strength was the ability to measure physical activity on both program and non-program days. Limitations were that the accelerometer cut-points were developed for 13- to 14-year-old girls and were applied to girls ages 11 to 18 years, and the cut-points were not specifically developed for dancing. This may have resulted in an underestimation of physical activity from arm movement and its associated energy expenditure. Another limitation was the self-report of dance class schedules, which may be subject to bias and recall limitations. Lastly, because data were collected in one metropolitan area and mostly with Caucasian girls, they may not be generalizable to other populations. However, the sample size allowed us to test multiple hypotheses.",
"In summary, our findings demonstrate the importance of dance classes to girls' physical activity levels. We found that dance classes contributed a substantial proportion (29%) of MVPA to girls' total weekly MVPA, and girls accumulated 70% more MVPA and 8% less sedentary behavior on dance class days than on non-dance class days. Thus, the additional minutes of MVPA on dance class days were not maintained on non-dance class days. Therefore, dance classes can play a critical role by providing health-enhancing physical activity to adolescent girls, and can assist them in meeting the current physical activity guideline."
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[
"accelerometer",
"children",
"moderate-to-vigorous physical activity",
"light activity",
"sedentary behavior"
] |
Background:
Helping youth achieve the current physical activity guideline of at least 60 minutes of daily moderate-to-vigorous physical activity (MVPA) is a key public health objective for the 21st century [1]. Structured physical activity programs are major avenues for providing physical activity to youth, and as such, they are a recommended strategy for the promotion of physical activity [1-3]. Structured physical activity programs are organized activities that are typically planned and occur within a specific setting [4]. These programs include physical education classes, organized sports, activity classes or lessons, and after-school programs.
Although structured physical activity programs are well positioned to assist youth in meeting the physical activity guideline, little is known about these programs. Specifically, there is limited knowledge of the overall physical activity levels of program participants and the contribution of these programs to overall physical activity. In addition, very few structured physical activity programs have been studied previously using objective measurement of physical activity by accelerometry. Only one study [5] has used accelerometry to examine the contribution of structured physical activity programs to total daily physical activity. Wickel and Eisenmann [5] found that among 6- to 12-year-old boys, youth sport and physical education classes contributed approximately 23% and 11% to their daily MVPA, respectively.
Dance classes are an important example of structured physical activity programs, because dance is a highly prevalent type of physical activity among adolescent girls [6-8]. Given the steep decline in girls' physical activity levels during adolescence [9-13], there is a need to study dance participation in adolescent girls. Further, the overall physical activity levels of girls who participate in dance classes are unknown, as is the contribution of dance classes to girls' overall physical activity levels. Accordingly, the objectives of this study were 1) to describe the overall physical activity levels of girls who participate in structured dance classes, 2) to determine the contribution of structured dance classes to total light, moderate, vigorous, and MVPA, and 3) to compare physical activity between days with a dance class (program days) and days without a dance class (non-program days).
Methods:
Study Design This study employed a cross-sectional design in describing overall physical activity levels in girls who participate in structured dance classes. Each aim was addressed using a distinct methodology and design. Data were collected in a sample of girls registered for lessons in dance studios in Columbia, South Carolina. To be eligible, a dance studio was required to offer at least one ballet, jazz, or tap class per week to students aged 11 years and older. This protocol was designed to measure girls' physical activity via accelerometry over an eight-day period, and to link those data with the structured dance classes each girl attended during that period. In addition, girls' self-report of their structured dance classes allowed for determination of a program day (day with a dance class) and a non-program day (day without a dance class), for the comparison of physical activity levels.
This study employed a cross-sectional design in describing overall physical activity levels in girls who participate in structured dance classes. Each aim was addressed using a distinct methodology and design. Data were collected in a sample of girls registered for lessons in dance studios in Columbia, South Carolina. To be eligible, a dance studio was required to offer at least one ballet, jazz, or tap class per week to students aged 11 years and older. This protocol was designed to measure girls' physical activity via accelerometry over an eight-day period, and to link those data with the structured dance classes each girl attended during that period. In addition, girls' self-report of their structured dance classes allowed for determination of a program day (day with a dance class) and a non-program day (day without a dance class), for the comparison of physical activity levels.
Participants Participants were girls (ages 11 to 18 years) enrolled in dance classes at dance studios in Columbia, South Carolina. Forty-three dance studios, defined as commercial facilities whose primary business is to provide dance instruction, were identified using local published and electronic telephone books. The director of each dance studio was contacted by telephone to determine the studio's eligibility status. Twenty-three dance studios were eligible and were invited to participate. Of the 23 eligible dance studios, 11 directors agreed to participate, and those directors provided the schedules of the ballet, jazz, and tap classes that were offered to students aged 11 years and older. In each of the 11 dance studios, one to four classes (based on the studio size and the styles of dance offered) were selected for measurement, from which girls were recruited. Classes were not selected randomly; in small studios where there was only one eligible class, that class was chosen. In larger studios, convenience samples were taken that allowed for a variety of dance styles across age ranges. Girls were eligible to participate if they were enrolled in these dance classes and met the age criteria, which was a total of 212 girls. Of these students, 149 (70.3%) agreed to participate. Written informed consent was provided by each student's parent or guardian and informed assent was given by each student (if < 18 years) prior to collection of data. The study was approved by the University of South Carolina Institutional Review Board.
Participants were girls (ages 11 to 18 years) enrolled in dance classes at dance studios in Columbia, South Carolina. Forty-three dance studios, defined as commercial facilities whose primary business is to provide dance instruction, were identified using local published and electronic telephone books. The director of each dance studio was contacted by telephone to determine the studio's eligibility status. Twenty-three dance studios were eligible and were invited to participate. Of the 23 eligible dance studios, 11 directors agreed to participate, and those directors provided the schedules of the ballet, jazz, and tap classes that were offered to students aged 11 years and older. In each of the 11 dance studios, one to four classes (based on the studio size and the styles of dance offered) were selected for measurement, from which girls were recruited. Classes were not selected randomly; in small studios where there was only one eligible class, that class was chosen. In larger studios, convenience samples were taken that allowed for a variety of dance styles across age ranges. Girls were eligible to participate if they were enrolled in these dance classes and met the age criteria, which was a total of 212 girls. Of these students, 149 (70.3%) agreed to participate. Written informed consent was provided by each student's parent or guardian and informed assent was given by each student (if < 18 years) prior to collection of data. The study was approved by the University of South Carolina Institutional Review Board.
Objective Measurement of Physical Activity ActiGraph accelerometers (Model 7164, Pensacola, FL) were used to measure time spent in sedentary behavior, light, moderate, and vigorous physical activity, and combined MVPA. The ActiGraph is a reliable [14] and valid method for assessing children's and adolescent's physical activity, both in laboratory and field settings [15,16]. The cut-points established by Treuth and colleagues for use in adolescent girls aged 13 to 14 years were used to determine intensity levels [17]. Accelerometers were initialized to save data in 30-second intervals, in accordance with the procedures of Treuth et al. [17]. Accordingly, intensities of physical activity were operationally defined as: sedentary (< 50 counts/30 seconds), light (51-1499 counts/30 seconds), moderate (1500-2600 counts/30 seconds), vigorous physical activity (> 2600 counts/30 seconds), and MVPA (≥ 1500 counts/30 seconds) [17].
Accelerometers provide activity intensity counts which are associated with the corresponding date and time stamp. However, accelerometers do not detect the type of activity performed at any given time. Therefore, to estimate the amount of physical activity in structured dance classes, it was necessary for girls to report the days and times (e.g., Wednesday 5:00 PM - 6:30 PM) they participated in structured dance classes during the week when the accelerometer was worn. Girls reported this information on a written survey.
ActiGraph accelerometers (Model 7164, Pensacola, FL) were used to measure time spent in sedentary behavior, light, moderate, and vigorous physical activity, and combined MVPA. The ActiGraph is a reliable [14] and valid method for assessing children's and adolescent's physical activity, both in laboratory and field settings [15,16]. The cut-points established by Treuth and colleagues for use in adolescent girls aged 13 to 14 years were used to determine intensity levels [17]. Accelerometers were initialized to save data in 30-second intervals, in accordance with the procedures of Treuth et al. [17]. Accordingly, intensities of physical activity were operationally defined as: sedentary (< 50 counts/30 seconds), light (51-1499 counts/30 seconds), moderate (1500-2600 counts/30 seconds), vigorous physical activity (> 2600 counts/30 seconds), and MVPA (≥ 1500 counts/30 seconds) [17].
Accelerometers provide activity intensity counts which are associated with the corresponding date and time stamp. However, accelerometers do not detect the type of activity performed at any given time. Therefore, to estimate the amount of physical activity in structured dance classes, it was necessary for girls to report the days and times (e.g., Wednesday 5:00 PM - 6:30 PM) they participated in structured dance classes during the week when the accelerometer was worn. Girls reported this information on a written survey.
Measurement Protocol Overall physical activity was assessed with accelerometry for eight days. A research assistant placed accelerometers on participants approximately 10 minutes before the beginning of the dance class. Accelerometers were attached to an elastic belt and worn over the right hip. Participants wore the accelerometers during the entire class and continued to wear them for the following seven days. They were given instructions to wear the accelerometer during all waking hours, except when swimming or bathing. Participants wore the accelerometers to the same dance class the following week, and the accelerometers were removed by the research assistant at the end of the class. Upon collection of the accelerometers, activity counts were downloaded and saved on a computer for data reduction and analysis. In addition to the accelerometer collection, girls completed a written survey in which they reported the days and times they attended structured dance classes during the past week.
Overall physical activity was assessed with accelerometry for eight days. A research assistant placed accelerometers on participants approximately 10 minutes before the beginning of the dance class. Accelerometers were attached to an elastic belt and worn over the right hip. Participants wore the accelerometers during the entire class and continued to wear them for the following seven days. They were given instructions to wear the accelerometer during all waking hours, except when swimming or bathing. Participants wore the accelerometers to the same dance class the following week, and the accelerometers were removed by the research assistant at the end of the class. Upon collection of the accelerometers, activity counts were downloaded and saved on a computer for data reduction and analysis. In addition to the accelerometer collection, girls completed a written survey in which they reported the days and times they attended structured dance classes during the past week.
Data Reduction Overall physical activity An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.
An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.
Contribution of dance classes to total light, moderate, vigorous, and MVPA Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.
Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.
Program days vs. non-program days Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.
Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.
Overall physical activity An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.
An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.
Contribution of dance classes to total light, moderate, vigorous, and MVPA Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.
Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.
Program days vs. non-program days Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.
Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.
Anthropometric Measures Height and weight measurements were conducted in a private setting. Height and weight were assessed objectively using a portable stadiometer measured to the nearest 0.1 cm (Shorr Productions; Olney, MD) and an electronic scale measured to the nearest 0.1 kg (model 770; Seca, Hamburg, Germany). The average of two measurements was used. Body mass index (BMI) was calculated and expressed as body mass (kg) divided by height (m2), and BMI was converted to BMI percentiles using the CDC Growth Charts [19].
Height and weight measurements were conducted in a private setting. Height and weight were assessed objectively using a portable stadiometer measured to the nearest 0.1 cm (Shorr Productions; Olney, MD) and an electronic scale measured to the nearest 0.1 kg (model 770; Seca, Hamburg, Germany). The average of two measurements was used. Body mass index (BMI) was calculated and expressed as body mass (kg) divided by height (m2), and BMI was converted to BMI percentiles using the CDC Growth Charts [19].
Additional Variables Participants' date of birth and race/ethnicity were reported by parents on the consent form. To describe the sample, participants self-reported information about their dance participation. This included the starting age of dance instruction, dance styles ever studied, number of dance classes taken per week, hours of rehearsal per week, and participation in competitive or company dance. The number of classes per week, rehearsal hours per week, and competitive or company dance were in reference to current participation (i.e., present time).
Participants' date of birth and race/ethnicity were reported by parents on the consent form. To describe the sample, participants self-reported information about their dance participation. This included the starting age of dance instruction, dance styles ever studied, number of dance classes taken per week, hours of rehearsal per week, and participation in competitive or company dance. The number of classes per week, rehearsal hours per week, and competitive or company dance were in reference to current participation (i.e., present time).
Statistical Analyses All data were analyzed using generalized linear mixed models (PROC MIXED). For the purpose of comparing program days and non-program days, least squares means were used, and models were adjusted for total monitoring time. Means and standard errors were calculated. SAS software (version 9.2; SAS Institute, Cary, NC) was used for all statistical analyses. Statistical significance was set at P < 0.05.
All data were analyzed using generalized linear mixed models (PROC MIXED). For the purpose of comparing program days and non-program days, least squares means were used, and models were adjusted for total monitoring time. Means and standard errors were calculated. SAS software (version 9.2; SAS Institute, Cary, NC) was used for all statistical analyses. Statistical significance was set at P < 0.05.
Study Design:
This study employed a cross-sectional design in describing overall physical activity levels in girls who participate in structured dance classes. Each aim was addressed using a distinct methodology and design. Data were collected in a sample of girls registered for lessons in dance studios in Columbia, South Carolina. To be eligible, a dance studio was required to offer at least one ballet, jazz, or tap class per week to students aged 11 years and older. This protocol was designed to measure girls' physical activity via accelerometry over an eight-day period, and to link those data with the structured dance classes each girl attended during that period. In addition, girls' self-report of their structured dance classes allowed for determination of a program day (day with a dance class) and a non-program day (day without a dance class), for the comparison of physical activity levels.
Participants:
Participants were girls (ages 11 to 18 years) enrolled in dance classes at dance studios in Columbia, South Carolina. Forty-three dance studios, defined as commercial facilities whose primary business is to provide dance instruction, were identified using local published and electronic telephone books. The director of each dance studio was contacted by telephone to determine the studio's eligibility status. Twenty-three dance studios were eligible and were invited to participate. Of the 23 eligible dance studios, 11 directors agreed to participate, and those directors provided the schedules of the ballet, jazz, and tap classes that were offered to students aged 11 years and older. In each of the 11 dance studios, one to four classes (based on the studio size and the styles of dance offered) were selected for measurement, from which girls were recruited. Classes were not selected randomly; in small studios where there was only one eligible class, that class was chosen. In larger studios, convenience samples were taken that allowed for a variety of dance styles across age ranges. Girls were eligible to participate if they were enrolled in these dance classes and met the age criteria, which was a total of 212 girls. Of these students, 149 (70.3%) agreed to participate. Written informed consent was provided by each student's parent or guardian and informed assent was given by each student (if < 18 years) prior to collection of data. The study was approved by the University of South Carolina Institutional Review Board.
Objective Measurement of Physical Activity:
ActiGraph accelerometers (Model 7164, Pensacola, FL) were used to measure time spent in sedentary behavior, light, moderate, and vigorous physical activity, and combined MVPA. The ActiGraph is a reliable [14] and valid method for assessing children's and adolescent's physical activity, both in laboratory and field settings [15,16]. The cut-points established by Treuth and colleagues for use in adolescent girls aged 13 to 14 years were used to determine intensity levels [17]. Accelerometers were initialized to save data in 30-second intervals, in accordance with the procedures of Treuth et al. [17]. Accordingly, intensities of physical activity were operationally defined as: sedentary (< 50 counts/30 seconds), light (51-1499 counts/30 seconds), moderate (1500-2600 counts/30 seconds), vigorous physical activity (> 2600 counts/30 seconds), and MVPA (≥ 1500 counts/30 seconds) [17].
Accelerometers provide activity intensity counts which are associated with the corresponding date and time stamp. However, accelerometers do not detect the type of activity performed at any given time. Therefore, to estimate the amount of physical activity in structured dance classes, it was necessary for girls to report the days and times (e.g., Wednesday 5:00 PM - 6:30 PM) they participated in structured dance classes during the week when the accelerometer was worn. Girls reported this information on a written survey.
Measurement Protocol:
Overall physical activity was assessed with accelerometry for eight days. A research assistant placed accelerometers on participants approximately 10 minutes before the beginning of the dance class. Accelerometers were attached to an elastic belt and worn over the right hip. Participants wore the accelerometers during the entire class and continued to wear them for the following seven days. They were given instructions to wear the accelerometer during all waking hours, except when swimming or bathing. Participants wore the accelerometers to the same dance class the following week, and the accelerometers were removed by the research assistant at the end of the class. Upon collection of the accelerometers, activity counts were downloaded and saved on a computer for data reduction and analysis. In addition to the accelerometer collection, girls completed a written survey in which they reported the days and times they attended structured dance classes during the past week.
Data Reduction:
Overall physical activity An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.
An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.
Contribution of dance classes to total light, moderate, vigorous, and MVPA Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.
Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.
Program days vs. non-program days Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.
Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.
Overall physical activity:
An established method of accelerometer data reduction is to concatenate data from the first day and last day of data collection when those days are the same day of the week [18]. In this study, the accelerometers were put on (day 1) and taken off (day 8) on the same day of the week, and for data reduction, a single day of data was created by combining the last part of day one and the first part of day eight. Periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. Missing accelerometer data on non-adherent days were replaced via imputation using the expectation maximization algorithm, based on the methods of Catellier et al. [18]. Data were also reduced without imputation; however, there were no differences between the mean physical activity levels, therefore, the imputed means are presented.
Contribution of dance classes to total light, moderate, vigorous, and MVPA:
Several steps were used to determine the contribution of dance classes to total light, moderate, vigorous, and MVPA. This analysis was conducted separately for light, moderate, vigorous, and MVPA, but for simplicity, only MVPA will be included in this section. First, total MVPA was calculated. As in the previous analysis, data from day one and day eight were combined, and periods of 60 minutes or more of zeros were considered to be non-wear time and were excluded from the analysis. Adherent days were those with a minimum of eight hours per weekday and six hours per weekend day of monitoring time. To be included in this analysis, girls were required to have a minimum of three weekdays and one weekend day. For each of these qualifying days, the amount of time (minutes) spent in MVPA was calculated. For each girl, MVPA was divided by the corresponding number of qualifying days to obtain the average minutes per day of total MVPA. Second, MVPA in dance classes was determined. For each of the qualifying days, accelerometer counts collected during the reported duration of each dance class were segmented from the raw accelerometer data file. The amount of time (minutes) spent in MVPA during dance classes was calculated. If a girl did not report participation in a dance class on a qualifying day, MVPA during dance class was zero. For each girl, MVPA in dance classes was divided by the corresponding number of qualifying days to obtain the average minutes per day of MVPA in dance classes. Third, the percent contribution of dance classes to total MVPA was calculated from minutes per day of MVPA in dance classes and minutes per day of total MVPA.
Program days vs. non-program days:
Data were also reduced to compare physical activity on program days to physical activity on non-program days. For this analysis, only weekdays with eight or more hours of monitoring time were included. Because the first day and last day of accelerometer wear (day one and day eight) were not full days of wear, these days were excluded. All eligible days were used in the analysis, with the minimum of one program day and one non-program day. The amount of time (minutes) spent in sedentary, light, moderate, vigorous, and MVPA were calculated separately for the program and the non-program days.
Anthropometric Measures:
Height and weight measurements were conducted in a private setting. Height and weight were assessed objectively using a portable stadiometer measured to the nearest 0.1 cm (Shorr Productions; Olney, MD) and an electronic scale measured to the nearest 0.1 kg (model 770; Seca, Hamburg, Germany). The average of two measurements was used. Body mass index (BMI) was calculated and expressed as body mass (kg) divided by height (m2), and BMI was converted to BMI percentiles using the CDC Growth Charts [19].
Additional Variables:
Participants' date of birth and race/ethnicity were reported by parents on the consent form. To describe the sample, participants self-reported information about their dance participation. This included the starting age of dance instruction, dance styles ever studied, number of dance classes taken per week, hours of rehearsal per week, and participation in competitive or company dance. The number of classes per week, rehearsal hours per week, and competitive or company dance were in reference to current participation (i.e., present time).
Statistical Analyses:
All data were analyzed using generalized linear mixed models (PROC MIXED). For the purpose of comparing program days and non-program days, least squares means were used, and models were adjusted for total monitoring time. Means and standard errors were calculated. SAS software (version 9.2; SAS Institute, Cary, NC) was used for all statistical analyses. Statistical significance was set at P < 0.05.
Results:
A total of 149 girls participated in the study. Eleven girls were excluded due to accelerometer malfunctions, and four were excluded for incomplete data, leaving 134 included in the data analyses. Due to the three analytical methods, there were three analytic samples; the descriptive characteristics of the three samples are presented in Table 1. The mean age was 14.6 ± 1.9 years, and over 80% of the participants were Caucasian. There are many styles of dance, and girls reported participating in the following styles during the previous week: ballet, jazz, tap, contemporary, hip hop, theatrical jazz/musical theatre, lyrical, baton, character, belly dance and ballroom.
Descriptive characteristics of girls enrolled in structured dance classes.
SD, standard deviation; BMI, body mass index.
‡ Sample 2: Girls with ≥ 3 weekdays and ≥ 1 weekend day.
¶ Sample 3: Girls with ≥ 1 program and 1 non-program day.
** Sample 1: Asian (n = 5), Hispanic (n = 1), Multi-racial/Other (n = 5), Not Reported (n = 4).
† Dance styles ever studied: ballet, jazz, tap, contemporary, hip hop, theatrical jazz/musical theatre, lyrical, baton, character/folk, ballroom, African, Indian, Irish, social dance, clogging, and belly dance.
* Referred to current participation (i.e., present time).
§ The average dance class length was 71.1 ± 18.1 minutes.
Overall physical activity The analysis sample for overall physical activity included 134 girls. The means for the overall physical activity intensities are presented in Table 2. Girls obtained an average of 25.0 minutes per day of MVPA, 271.1 minutes per day of light physical activity, and 535.0 minutes per day of sedentary behavior.
Overall physical activity, minutes per day, among girls enrolled in dance classes, (n = 134).
MVPA, moderate-to-vigorous physical activity; SE, standard error;
CI, confidence interval.
The analysis sample for overall physical activity included 134 girls. The means for the overall physical activity intensities are presented in Table 2. Girls obtained an average of 25.0 minutes per day of MVPA, 271.1 minutes per day of light physical activity, and 535.0 minutes per day of sedentary behavior.
Overall physical activity, minutes per day, among girls enrolled in dance classes, (n = 134).
MVPA, moderate-to-vigorous physical activity; SE, standard error;
CI, confidence interval.
Contribution of dance classes to total light, moderate, vigorous, and MVPA A total of 33 girls were excluded from this analysis for not having the required amount of qualifying days, resulting in an analysis sample of 101 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall physical activity; however, excluded girls had fewer dance classes per week compared to others (4.9 ± 1.8 vs. 6.6 ± 2.7, P < .001). The average reported dance class length was 71.1 ± 18.1 minutes. During structured dance classes, girls accumulated an average of 39.4 minutes per day of light physical activity and 7.1 minutes per day of MVPA (Table 3). Dance classes contributed 15.4%, 26.7%, 39.6%, and 28.7% of the girls' total light, moderate, vigorous, and MVPA, respectively.
Contribution of dance classes to total light, moderate, and vigorous physical activity and MVPA (n = 101).
LPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.
A total of 33 girls were excluded from this analysis for not having the required amount of qualifying days, resulting in an analysis sample of 101 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall physical activity; however, excluded girls had fewer dance classes per week compared to others (4.9 ± 1.8 vs. 6.6 ± 2.7, P < .001). The average reported dance class length was 71.1 ± 18.1 minutes. During structured dance classes, girls accumulated an average of 39.4 minutes per day of light physical activity and 7.1 minutes per day of MVPA (Table 3). Dance classes contributed 15.4%, 26.7%, 39.6%, and 28.7% of the girls' total light, moderate, vigorous, and MVPA, respectively.
Contribution of dance classes to total light, moderate, and vigorous physical activity and MVPA (n = 101).
LPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.
Program day vs. non-program day To be included in this analysis, girls needed to have at least one program day and one non-program day. From the first sample of 134 girls, 13 girls were excluded because they had less than two eligible days, 10 girls were excluded because they did not have any program days (on the eligible days), and 35 girls were excluded because they did not have any non-program days (i.e., they attended dance class on every eligible day). Therefore, the sample consisted of 76 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall sedentary, light, or moderate physical activity; however, excluded girls had a slightly greater percentage of vigorous (1.0% ± 0.4% vs. 0.8% ± 0.5%, p = 0.01) and MVPA (3.3% ± 1.0% vs. 2.8% ± 1.3%, p = 0.03) compared to the other girls. The comparison of physical activity intensities between program days and non-program days are presented in Table 4. There were no significant differences in the total monitoring time between the two days (program day: 854.5 ± 12.0 minutes; non-program day: 880.0 ± 15.0 minutes; p = 0.13). After controlling for total monitoring time, girls accumulated significantly more minutes of MVPA on program days (28.7 ± 1.4 minutes per day) than non-program days (16.4 ± 1.5 minutes per day) (p < 0.001). Girls had significantly fewer minutes of sedentary behavior on program days (554.0 ± 8.1 minutes per day) than non-program days (600.2 ± 8.7 minutes per day) (p < 0.001). Girls engaged in significantly more light, moderate, and vigorous physical activity on program days than non-program days (p < 0.001).
Program days vs. non-program days, minutes/day (mean ± SE), (n = 76).†
MVPA, moderate-to-vigorous physical activity; SE, standard error.
† Adjusted for total monitoring time.
In addition, we analyzed the physical activity data from the girls with zero non-program days (i.e., they attended dance class on every eligible day). Overall, these girls obtained an average of 28.9 minutes per day of MVPA, 9.0 minutes per day of vigorous physical activity, and 292.5 minutes per day of light physical activity (Table 5). They engaged in more vigorous physical activity and MVPA per day than girls in Sample 1. During structured dance classes, these girls accumulated an average of 54.5 minutes per day of light physical activity and 9.4 minutes per day of MVPA (Table 6), which was significantly more than girls in Sample 1. Dance classes contributed 20.5%, 31.2%, 42.6%, and 33.1% of the girls' total light, moderate, vigorous, and MVPA, respectively. This sub-sample of girls had a higher percent contribution of dance classes to total light, moderate, vigorous, and MVPA than girls in Sample 1.
Overall physical activity for girls with zero non-program days, minutes/day (mean ± SE), (n = 35).
MVPA, moderate-to-vigorous physical activity; SE, standard error;
CI, confidence interval.
Contribution of dance classes to total light, moderate, and vigorous physical activity and MVPA for girls with zero non-program days (n = 31).
LPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.
To be included in this analysis, girls needed to have at least one program day and one non-program day. From the first sample of 134 girls, 13 girls were excluded because they had less than two eligible days, 10 girls were excluded because they did not have any program days (on the eligible days), and 35 girls were excluded because they did not have any non-program days (i.e., they attended dance class on every eligible day). Therefore, the sample consisted of 76 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall sedentary, light, or moderate physical activity; however, excluded girls had a slightly greater percentage of vigorous (1.0% ± 0.4% vs. 0.8% ± 0.5%, p = 0.01) and MVPA (3.3% ± 1.0% vs. 2.8% ± 1.3%, p = 0.03) compared to the other girls. The comparison of physical activity intensities between program days and non-program days are presented in Table 4. There were no significant differences in the total monitoring time between the two days (program day: 854.5 ± 12.0 minutes; non-program day: 880.0 ± 15.0 minutes; p = 0.13). After controlling for total monitoring time, girls accumulated significantly more minutes of MVPA on program days (28.7 ± 1.4 minutes per day) than non-program days (16.4 ± 1.5 minutes per day) (p < 0.001). Girls had significantly fewer minutes of sedentary behavior on program days (554.0 ± 8.1 minutes per day) than non-program days (600.2 ± 8.7 minutes per day) (p < 0.001). Girls engaged in significantly more light, moderate, and vigorous physical activity on program days than non-program days (p < 0.001).
Program days vs. non-program days, minutes/day (mean ± SE), (n = 76).†
MVPA, moderate-to-vigorous physical activity; SE, standard error.
† Adjusted for total monitoring time.
In addition, we analyzed the physical activity data from the girls with zero non-program days (i.e., they attended dance class on every eligible day). Overall, these girls obtained an average of 28.9 minutes per day of MVPA, 9.0 minutes per day of vigorous physical activity, and 292.5 minutes per day of light physical activity (Table 5). They engaged in more vigorous physical activity and MVPA per day than girls in Sample 1. During structured dance classes, these girls accumulated an average of 54.5 minutes per day of light physical activity and 9.4 minutes per day of MVPA (Table 6), which was significantly more than girls in Sample 1. Dance classes contributed 20.5%, 31.2%, 42.6%, and 33.1% of the girls' total light, moderate, vigorous, and MVPA, respectively. This sub-sample of girls had a higher percent contribution of dance classes to total light, moderate, vigorous, and MVPA than girls in Sample 1.
Overall physical activity for girls with zero non-program days, minutes/day (mean ± SE), (n = 35).
MVPA, moderate-to-vigorous physical activity; SE, standard error;
CI, confidence interval.
Contribution of dance classes to total light, moderate, and vigorous physical activity and MVPA for girls with zero non-program days (n = 31).
LPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.
Overall physical activity:
The analysis sample for overall physical activity included 134 girls. The means for the overall physical activity intensities are presented in Table 2. Girls obtained an average of 25.0 minutes per day of MVPA, 271.1 minutes per day of light physical activity, and 535.0 minutes per day of sedentary behavior.
Overall physical activity, minutes per day, among girls enrolled in dance classes, (n = 134).
MVPA, moderate-to-vigorous physical activity; SE, standard error;
CI, confidence interval.
Contribution of dance classes to total light, moderate, vigorous, and MVPA:
A total of 33 girls were excluded from this analysis for not having the required amount of qualifying days, resulting in an analysis sample of 101 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall physical activity; however, excluded girls had fewer dance classes per week compared to others (4.9 ± 1.8 vs. 6.6 ± 2.7, P < .001). The average reported dance class length was 71.1 ± 18.1 minutes. During structured dance classes, girls accumulated an average of 39.4 minutes per day of light physical activity and 7.1 minutes per day of MVPA (Table 3). Dance classes contributed 15.4%, 26.7%, 39.6%, and 28.7% of the girls' total light, moderate, vigorous, and MVPA, respectively.
Contribution of dance classes to total light, moderate, and vigorous physical activity and MVPA (n = 101).
LPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.
Program day vs. non-program day:
To be included in this analysis, girls needed to have at least one program day and one non-program day. From the first sample of 134 girls, 13 girls were excluded because they had less than two eligible days, 10 girls were excluded because they did not have any program days (on the eligible days), and 35 girls were excluded because they did not have any non-program days (i.e., they attended dance class on every eligible day). Therefore, the sample consisted of 76 girls. Excluded girls did not differ from other girls for any of the demographic variables or overall sedentary, light, or moderate physical activity; however, excluded girls had a slightly greater percentage of vigorous (1.0% ± 0.4% vs. 0.8% ± 0.5%, p = 0.01) and MVPA (3.3% ± 1.0% vs. 2.8% ± 1.3%, p = 0.03) compared to the other girls. The comparison of physical activity intensities between program days and non-program days are presented in Table 4. There were no significant differences in the total monitoring time between the two days (program day: 854.5 ± 12.0 minutes; non-program day: 880.0 ± 15.0 minutes; p = 0.13). After controlling for total monitoring time, girls accumulated significantly more minutes of MVPA on program days (28.7 ± 1.4 minutes per day) than non-program days (16.4 ± 1.5 minutes per day) (p < 0.001). Girls had significantly fewer minutes of sedentary behavior on program days (554.0 ± 8.1 minutes per day) than non-program days (600.2 ± 8.7 minutes per day) (p < 0.001). Girls engaged in significantly more light, moderate, and vigorous physical activity on program days than non-program days (p < 0.001).
Program days vs. non-program days, minutes/day (mean ± SE), (n = 76).†
MVPA, moderate-to-vigorous physical activity; SE, standard error.
† Adjusted for total monitoring time.
In addition, we analyzed the physical activity data from the girls with zero non-program days (i.e., they attended dance class on every eligible day). Overall, these girls obtained an average of 28.9 minutes per day of MVPA, 9.0 minutes per day of vigorous physical activity, and 292.5 minutes per day of light physical activity (Table 5). They engaged in more vigorous physical activity and MVPA per day than girls in Sample 1. During structured dance classes, these girls accumulated an average of 54.5 minutes per day of light physical activity and 9.4 minutes per day of MVPA (Table 6), which was significantly more than girls in Sample 1. Dance classes contributed 20.5%, 31.2%, 42.6%, and 33.1% of the girls' total light, moderate, vigorous, and MVPA, respectively. This sub-sample of girls had a higher percent contribution of dance classes to total light, moderate, vigorous, and MVPA than girls in Sample 1.
Overall physical activity for girls with zero non-program days, minutes/day (mean ± SE), (n = 35).
MVPA, moderate-to-vigorous physical activity; SE, standard error;
CI, confidence interval.
Contribution of dance classes to total light, moderate, and vigorous physical activity and MVPA for girls with zero non-program days (n = 31).
LPA, light physical activity; MPA, moderate physical activity; VPA, vigorous physical activity; MVPA, moderate-to-vigorous physical activity; SE, standard error; CI, confidence interval.
Discussion:
This was the first study to describe the physical activity levels of girls who were enrolled in structured dance classes using an objective measure of physical activity. Activity performed in structured dance classes accounted for a substantial proportion (29%) of their total weekly MVPA. Most notably, girls obtained 70% more total MVPA and 8% less sedentary behavior on program days (i.e., participation in dance class), than non-program days. Therefore, these novel findings provide strong evidence of the important contribution that dance classes can make to girls' total physical activity.
The present study used accelerometry to measure physical activity, which has been done very rarely to examine the contribution of structured physical activity programs to total physical activity. Although several previous studies [5,20-22] have used accelerometry to examine physical activity during structured physical activity programs, to the best of our knowledge, only one study [5] has used accelerometry to examine the contribution of structured physical activity programs to total physical activity. Organized sports and physical education contributed 23% and 11% to boys' total daily MVPA, respectively [5]. These are lower than the contribution of dance classes in the present study (29%). Other studies have examined the contribution of structured physical activity programs to total physical activity, but they utilized different methodologies to assess physical activity [23,24]. For example, Katzmarzyk and Malina administered a 3-day activity diary to 12- to 14-year-old sport participants and found that youth sport contributed to 60% of their daily moderate to vigorous energy expenditure [24]. Morgan et al. [23] used pedometers in 1st through 6th graders and reported that the least, moderately active, and most active children obtained approximately 20%, 9%, and 16% of their total steps per day during physical education classes. The differences in these estimates are most likely attributable to the differences in physical activity assessments. However, it is important to recognize the advantages of accelerometry over other physical activity measurements: its ability to measure objectively, assess intensity, and provide activity counts with corresponding date and time stamps. Nonetheless, the present study supports previous findings that structured physical activity programs provide youth with a substantial proportion of MVPA to total MVPA.
A key finding of this study was that girls accumulated 70% more MVPA on program days (i.e., participation in dance class) than on non-program days. Therefore, the additional amount of MVPA obtained on program days was not maintained on non-program days. Further, girls in this study had 46.2 fewer minutes of sedentary behavior on program days compared to non-program days. These findings are consistent with those of Wickel and Eisenmann [5] who found that 6- to 12-year-old boys engaged in more MVPA and less sedentary behavior on a sport day compared to a non-sport day. The present findings are also similar to those of Dale et al. [25], who reported that when school physical activity opportunities were restricted, children did not compensate by increasing their activity during the after-school hours. These findings provide compelling evidence of the potential importance of dance classes for increasing MVPA and reducing sedentary behavior in adolescent girls.
Although a substantial proportion of girls' total weekly MVPA was attributable to dance classes, girls in this study were observed to have physical activity levels approximately the same as girls in other studies that objectively measured physical activity. Girls in this study engaged in an average of 25 minutes of MVPA per day, which is consistent with sixth-grade girls in the Trial of Activity for Adolescent Girls (TAAG) study who accumulated 23.7 minutes of MVPA per day [26]. The physical activity estimates for TAAG were also reported by geographic location, including South Carolina, and that sub-sample of girls obtained 20.8 minutes of MVPA per day [26]. Both the current study and TAAG used the accelerometer cut-points developed by Treuth et al. [17]. The present findings are also similar to those of 12- to 15-year-old girls in the National Health and Nutrition Examination Survey (NHANES) who obtained 24.6 minutes of MVPA per day [27]. In contrast, the amount of daily MVPA of girls in this study was slightly higher than that of 12-year-old girls in the UK [28] and 16- to 19-year-old girls in NHANES [27] who engaged in 18.3 and 19.6 minutes of MVPA per day, respectively. The MVPA cut-point in the UK study [28] was higher than the current study, whereas the MVPA cut-point in NHANES [27] was lower than the current study, which could potentially explain the differences in estimates. It is also important to note that in this study, the standard deviation was 10.1 minutes of MVPA per day, which indicates high inter-individual variability, which is consistent with the findings of Pate et al. [26]. These findings together indicate the need to increase overall physical activity levels of adolescent girls, as they, on average, are not achieving the physical activity guideline of at least 60 minutes of daily MVPA [1].
This study has strengths and limitations that should be noted. An important strength of this study was the objective measurement of physical activity and sedentary behavior via accelerometry. A second strength was the inclusion of a variety of dance styles from 11 dance studios. Another strength was the ability to measure physical activity on both program and non-program days. Limitations were that the accelerometer cut-points were developed for 13- to 14-year-old girls and were applied to girls ages 11 to 18 years, and the cut-points were not specifically developed for dancing. This may have resulted in an underestimation of physical activity from arm movement and its associated energy expenditure. Another limitation was the self-report of dance class schedules, which may be subject to bias and recall limitations. Lastly, because data were collected in one metropolitan area and mostly with Caucasian girls, they may not be generalizable to other populations. However, the sample size allowed us to test multiple hypotheses.
Conclusion:
In summary, our findings demonstrate the importance of dance classes to girls' physical activity levels. We found that dance classes contributed a substantial proportion (29%) of MVPA to girls' total weekly MVPA, and girls accumulated 70% more MVPA and 8% less sedentary behavior on dance class days than on non-dance class days. Thus, the additional minutes of MVPA on dance class days were not maintained on non-dance class days. Therefore, dance classes can play a critical role by providing health-enhancing physical activity to adolescent girls, and can assist them in meeting the current physical activity guideline.
|
Background:
Structured physical activity (PA) programs are well positioned to promote PA among youth, however, little is known about these programs, particularly dance classes. The aims of this study were to: 1) describe PA levels of girls enrolled in dance classes, 2) determine the contribution of dance classes to total moderate-to-vigorous physical activity (MVPA), and 3) compare PA between days with a dance class (program days) and days without a dance class (non-program days).
Methods:
Participants were 149 girls (11-18 years) enrolled in dance classes in 11 dance studios. Overall PA was assessed with accelerometry for 8 consecutive days, and girls reported when they attended dance classes during those days. The percent contribution of dance classes to total MVPA was calculated, and data were reduced to compare PA on program days to non-program days. Data were analyzed using mixed models, adjusting for total monitoring time.
Results:
Girls engaged in 25.0 ± 0.9 minutes/day of MVPA. Dance classes contributed 28.7% (95% CI: 25.9%-31.6%) to girls' total MVPA. Girls accumulated more MVPA on program (28.7 ± 1.4 minutes/day) than non-program days (16.4 ± 1.5 minutes/day) (p < 0.001). Girls had less sedentary behavior on program (554.0 ± 8.1 minutes/day) than non-program days (600.2 ± 8.7 minutes/day) (p < 0.001).
Conclusions:
Dance classes contributed a substantial proportion (29%) to girls' total MVPA, and girls accumulated 70% more MVPA and 8% less sedentary behavior on program days than on non-program days. Dance classes can make an important contribution to girls' total physical activity.
|
Background:
Helping youth achieve the current physical activity guideline of at least 60 minutes of daily moderate-to-vigorous physical activity (MVPA) is a key public health objective for the 21st century [1]. Structured physical activity programs are major avenues for providing physical activity to youth, and as such, they are a recommended strategy for the promotion of physical activity [1-3]. Structured physical activity programs are organized activities that are typically planned and occur within a specific setting [4]. These programs include physical education classes, organized sports, activity classes or lessons, and after-school programs.
Although structured physical activity programs are well positioned to assist youth in meeting the physical activity guideline, little is known about these programs. Specifically, there is limited knowledge of the overall physical activity levels of program participants and the contribution of these programs to overall physical activity. In addition, very few structured physical activity programs have been studied previously using objective measurement of physical activity by accelerometry. Only one study [5] has used accelerometry to examine the contribution of structured physical activity programs to total daily physical activity. Wickel and Eisenmann [5] found that among 6- to 12-year-old boys, youth sport and physical education classes contributed approximately 23% and 11% to their daily MVPA, respectively.
Dance classes are an important example of structured physical activity programs, because dance is a highly prevalent type of physical activity among adolescent girls [6-8]. Given the steep decline in girls' physical activity levels during adolescence [9-13], there is a need to study dance participation in adolescent girls. Further, the overall physical activity levels of girls who participate in dance classes are unknown, as is the contribution of dance classes to girls' overall physical activity levels. Accordingly, the objectives of this study were 1) to describe the overall physical activity levels of girls who participate in structured dance classes, 2) to determine the contribution of structured dance classes to total light, moderate, vigorous, and MVPA, and 3) to compare physical activity between days with a dance class (program days) and days without a dance class (non-program days).
Conclusion:
JRO conceived of and designed the study, acquired the data, analyzed and interpreted the data, and drafted the manuscript. RRP provided critical input during data analysis and manuscript development. The co-authors (RRP and SPH) participated in the interpretation of data and critical revision for important intellectual content. All authors read and approved the final manuscript.
|
Background:
Structured physical activity (PA) programs are well positioned to promote PA among youth, however, little is known about these programs, particularly dance classes. The aims of this study were to: 1) describe PA levels of girls enrolled in dance classes, 2) determine the contribution of dance classes to total moderate-to-vigorous physical activity (MVPA), and 3) compare PA between days with a dance class (program days) and days without a dance class (non-program days).
Methods:
Participants were 149 girls (11-18 years) enrolled in dance classes in 11 dance studios. Overall PA was assessed with accelerometry for 8 consecutive days, and girls reported when they attended dance classes during those days. The percent contribution of dance classes to total MVPA was calculated, and data were reduced to compare PA on program days to non-program days. Data were analyzed using mixed models, adjusting for total monitoring time.
Results:
Girls engaged in 25.0 ± 0.9 minutes/day of MVPA. Dance classes contributed 28.7% (95% CI: 25.9%-31.6%) to girls' total MVPA. Girls accumulated more MVPA on program (28.7 ± 1.4 minutes/day) than non-program days (16.4 ± 1.5 minutes/day) (p < 0.001). Girls had less sedentary behavior on program (554.0 ± 8.1 minutes/day) than non-program days (600.2 ± 8.7 minutes/day) (p < 0.001).
Conclusions:
Dance classes contributed a substantial proportion (29%) to girls' total MVPA, and girls accumulated 70% more MVPA and 8% less sedentary behavior on program days than on non-program days. Dance classes can make an important contribution to girls' total physical activity.
| 13,299 | 347 | 19 |
[
"day",
"dance",
"activity",
"mvpa",
"days",
"physical",
"physical activity",
"girls",
"minutes",
"classes"
] |
[
"test",
"test"
] | null |
[CONTENT] accelerometer | children | moderate-to-vigorous physical activity | light activity | sedentary behavior [SUMMARY]
|
[CONTENT] accelerometer | children | moderate-to-vigorous physical activity | light activity | sedentary behavior [SUMMARY]
| null |
[CONTENT] accelerometer | children | moderate-to-vigorous physical activity | light activity | sedentary behavior [SUMMARY]
|
[CONTENT] accelerometer | children | moderate-to-vigorous physical activity | light activity | sedentary behavior [SUMMARY]
|
[CONTENT] accelerometer | children | moderate-to-vigorous physical activity | light activity | sedentary behavior [SUMMARY]
|
[CONTENT] Adolescent | Anthropometry | Child | Cross-Sectional Studies | Dancing | Female | Humans | Motor Activity | Sedentary Behavior [SUMMARY]
|
[CONTENT] Adolescent | Anthropometry | Child | Cross-Sectional Studies | Dancing | Female | Humans | Motor Activity | Sedentary Behavior [SUMMARY]
| null |
[CONTENT] Adolescent | Anthropometry | Child | Cross-Sectional Studies | Dancing | Female | Humans | Motor Activity | Sedentary Behavior [SUMMARY]
|
[CONTENT] Adolescent | Anthropometry | Child | Cross-Sectional Studies | Dancing | Female | Humans | Motor Activity | Sedentary Behavior [SUMMARY]
|
[CONTENT] Adolescent | Anthropometry | Child | Cross-Sectional Studies | Dancing | Female | Humans | Motor Activity | Sedentary Behavior [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] day | dance | activity | mvpa | days | physical | physical activity | girls | minutes | classes [SUMMARY]
|
[CONTENT] day | dance | activity | mvpa | days | physical | physical activity | girls | minutes | classes [SUMMARY]
| null |
[CONTENT] day | dance | activity | mvpa | days | physical | physical activity | girls | minutes | classes [SUMMARY]
|
[CONTENT] day | dance | activity | mvpa | days | physical | physical activity | girls | minutes | classes [SUMMARY]
|
[CONTENT] day | dance | activity | mvpa | days | physical | physical activity | girls | minutes | classes [SUMMARY]
|
[CONTENT] physical | programs | activity | physical activity | structured physical activity | structured physical activity programs | activity programs | physical activity programs | structured physical | structured [SUMMARY]
|
[CONTENT] day | dance | mvpa | days | data | classes | time | program | dance classes | day day [SUMMARY]
| null |
[CONTENT] dance class days | class days | dance | non dance | non dance class | non dance class days | dance class | mvpa | class | days [SUMMARY]
|
[CONTENT] day | dance | activity | physical | physical activity | mvpa | days | girls | program | minutes [SUMMARY]
|
[CONTENT] day | dance | activity | physical | physical activity | mvpa | days | girls | program | minutes [SUMMARY]
|
[CONTENT] Structured ||| 1 | 2 | MVPA | 3 | between days [SUMMARY]
|
[CONTENT] 149 | 11-18 years | 11 ||| 8 consecutive days | those days ||| MVPA ||| [SUMMARY]
| null |
[CONTENT] 29% | MVPA | 70% | MVPA | 8% | days ||| [SUMMARY]
|
[CONTENT] ||| 1 | 2 | MVPA | 3 | between days ||| 149 | 11-18 years | 11 ||| 8 consecutive days | those days ||| MVPA ||| ||| ||| 25.0 ± | 0.9 minutes | MVPA ||| 28.7% | 95% | CI | 25.9%-31.6% | MVPA ||| MVPA | 28.7 ± | 1.4 minutes/day | 16.4 ± 1.5 minutes ||| 554.0 | 8.1 minutes/day | 600.2 ± | 8.7 minutes/day ||| 29% | MVPA | 70% | MVPA | 8% | days ||| [SUMMARY]
|
[CONTENT] ||| 1 | 2 | MVPA | 3 | between days ||| 149 | 11-18 years | 11 ||| 8 consecutive days | those days ||| MVPA ||| ||| ||| 25.0 ± | 0.9 minutes | MVPA ||| 28.7% | 95% | CI | 25.9%-31.6% | MVPA ||| MVPA | 28.7 ± | 1.4 minutes/day | 16.4 ± 1.5 minutes ||| 554.0 | 8.1 minutes/day | 600.2 ± | 8.7 minutes/day ||| 29% | MVPA | 70% | MVPA | 8% | days ||| [SUMMARY]
|
|||||
Establishment of an integrated model incorporating standardised uptake value and N-classification for predicting metastasis in nasopharyngeal carcinoma.
|
26871291
|
Previous studies reported a correlation between the maximum standardised uptake value (SUVmax) obtained by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) and distant metastasis in nasopharyngeal carcinoma (NPC). However, an integrated model incorporating SUVmax and anatomic staging for stratifying metastasis risk has not been reported.
|
BACKGROUND
|
Data from 449 patients with with histologically-confirmed, stage I-IVB NPC treated with radiotherapy or chemoradiotherapy were retrospectively analysed. A prognostic model for distant metastasis-free survival (DMFS) was derived by recursive partitioning analysis (RPA) combining independent predictors identified by multivariate analysis.
|
METHODS
|
The median SUVmax for primary tumour (SUV-T) and cervical lymph nodes (SUV-N) was 13.6 (range, 2.2 to 39.3) and 8.4 (range, 2.6 to 40.9), respectively. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005), SUV-N (HR, 2.688; 95%CI, 1.250-5.781; P = 0.011) and N-classification (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001) were identified as independent predictors for DMFS from multivariate analysis. Three valid risk groups were derived by RPA: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). The three risk groups contained 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Moreover, multivariate analysis confirmed the RPA-based prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95%CI, 1.975-4.835; P <0.001).
|
RESULTS
|
SUV-T, SUV-N and N-classification were identified as independent predictors for DMFS. An integrated RPA-based prognostic model for DMFS incorporating SUV-N and N-classification was proposed.
|
CONCLUSION
|
[
"Adult",
"Aged",
"Carcinoma",
"Carcinoma, Squamous Cell",
"Combined Modality Therapy",
"Female",
"Fluorodeoxyglucose F18",
"Follow-Up Studies",
"Humans",
"Lymphatic Metastasis",
"Male",
"Middle Aged",
"Models, Theoretical",
"Multimodal Imaging",
"Nasopharyngeal Carcinoma",
"Nasopharyngeal Neoplasms",
"Neoplasm Invasiveness",
"Neoplasm Staging",
"Positron-Emission Tomography",
"Prognosis",
"Radiopharmaceuticals",
"Retrospective Studies",
"Survival Rate",
"Tomography, X-Ray Computed",
"Young Adult"
] |
4924665
|
INTRODUCTION
|
Nasopharyngeal carcinoma (NPC) is particularly prevalent in southern China, Southeast Asia, North Africa, the Middle East, and Alaska [1]. Radiotherapy is the primary treatment used for non-disseminated NPC [2, 3]. With advances in imaging and radiation therapies, local-regional control has exceeded 90% [4]. However, 20-30% of NPC patients eventually develop distant metastasis [5–8], which accounts for the majority of failures [7, 8]. Effort should therefore be made to stratify patients into different groups based on the risk of metastasis to tailor individualized treatments and improve outcomes.
N-classification in the TNM staging system is a measure of the extent of node involvement, and is currently the most reliable tool for assessing metastasis risk in NPC [9, 10]. However, there is remaining room for improvement in the correlation between the N classification and metastasis [11, 12], perhaps because N-classification is based solely on anatomic extent and lacks non-anatomic information such as tumour physiology.
18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) imaging is used to probe glucose metabolism in tumour cells [13]. The maximal intensity of FDG uptake by the tumour (maximum standardized uptake value; SUVmax) is a valuable marker of tumour biological behaviour [13, 14] and a useful predictor of distant metastasis in NPC [15, 16]. However, an integrated model incorporating SUVmax and anatomic staging for stratifying metastasis risk has not been reported. Clinicians are therefore somewhat troubled as to how best to incorporate SUVmax into clinical decision-making. A valid approach for incorporating non-anatomic prognostic factors and anatomic staging into an integrated prognosis grouping was recently described [17], which significantly improved survival prediction compared with previous models. In the present study, we extended this approach by using recursive partitioning analysis (RPA) to develop an integrated prognostic model for metastasis that combines SUV parameters and N-classification.
|
PATIENTS AND METHODS
|
Patients This study was approved by the institutional review board, and the requirement to obtain informed consent was waived. From January 2010 to February 2012, 449 patients with stage I-IVB NPC treated at our institution received a positron emission tomography-computed tomography (PET-CT) examination before treatment followed by intensity-modulated radiotherapy (IMRT) with or without chemotherapy. All of the enrolled patients were of Chinese ethnicity. The median age was 46 years (range, 20- 77), with a male-to-female ratio of 3:1 (Table 2).
Abbreviations: NPC = nasopharyngeal carcinoma; T = tumour; N = node.
Pathological type according to the 2005 World Health Organization classification of tumours.
According to the 7th edition of the UICC/AJCC staging system.
All patients underwent a pretreatment evaluation that included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, and PET-CT. All patients were staged according to the 7th edition of the International Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) system [9].
This study was approved by the institutional review board, and the requirement to obtain informed consent was waived. From January 2010 to February 2012, 449 patients with stage I-IVB NPC treated at our institution received a positron emission tomography-computed tomography (PET-CT) examination before treatment followed by intensity-modulated radiotherapy (IMRT) with or without chemotherapy. All of the enrolled patients were of Chinese ethnicity. The median age was 46 years (range, 20- 77), with a male-to-female ratio of 3:1 (Table 2).
Abbreviations: NPC = nasopharyngeal carcinoma; T = tumour; N = node.
Pathological type according to the 2005 World Health Organization classification of tumours.
According to the 7th edition of the UICC/AJCC staging system.
All patients underwent a pretreatment evaluation that included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, and PET-CT. All patients were staged according to the 7th edition of the International Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) system [9].
PET/CT imaging Serum glucose levels were measured in all NPC patients, all of whom fasted for at least 6 h before PET/CT scans, and individuals with a fasting plasma glucose >200 mg/dl were excluded. PET/CT imaging was performed with a combination PET/CT scanner (Discovery ST 16; GE Healthcare, Little Chalfont, UK) according to published guidelines [23]. Helical CT was performed from the head to the proximal thigh before PET acquisition, according to a standardized protocol and were 45-60 min after injection of 5.55 MBq/kg FDG. PET images were reconstructed from CT data for attenuation correction using an ordered-subset expectation maximization iterative reconstruction algorithm. SUVmax was determined for each region of interest using the whole-body attenuation corrected image and the following formula: SUVmax = tissue concentration of 18F-FDG / injected dose / body weight.
Serum glucose levels were measured in all NPC patients, all of whom fasted for at least 6 h before PET/CT scans, and individuals with a fasting plasma glucose >200 mg/dl were excluded. PET/CT imaging was performed with a combination PET/CT scanner (Discovery ST 16; GE Healthcare, Little Chalfont, UK) according to published guidelines [23]. Helical CT was performed from the head to the proximal thigh before PET acquisition, according to a standardized protocol and were 45-60 min after injection of 5.55 MBq/kg FDG. PET images were reconstructed from CT data for attenuation correction using an ordered-subset expectation maximization iterative reconstruction algorithm. SUVmax was determined for each region of interest using the whole-body attenuation corrected image and the following formula: SUVmax = tissue concentration of 18F-FDG / injected dose / body weight.
Treatment The nasopharyngeal and neck tumour volumes of all patients were treated using radical radiotherapy based on IMRT for the entire treatment course. Institutional guidelines recommended radiotherapy only for stage I and concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy for stage II-IVB. In total, 92.8% (308/332) of patients with stage III-IVB disease received concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy. When possible, salvage treatments (intracavitary brachytherapy, surgery or chemotherapy) were provided and persistent disease or relapse was documented.
The nasopharyngeal and neck tumour volumes of all patients were treated using radical radiotherapy based on IMRT for the entire treatment course. Institutional guidelines recommended radiotherapy only for stage I and concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy for stage II-IVB. In total, 92.8% (308/332) of patients with stage III-IVB disease received concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy. When possible, salvage treatments (intracavitary brachytherapy, surgery or chemotherapy) were provided and persistent disease or relapse was documented.
Follow-up Patients were examined at least every three months during the first two years, and every six months during years 3-5 or until death. Evaluation during follow-up included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, chest radiography, abdominal sonography and a whole-body bone scan. Any residual disease found at the nasopharynx or cervical nodes within 6 months after completion of RT was regarded as local failure or regional failure, respectively. All distant metastases were diagnosed by clinical symptoms, physical examination, and imaging methods that included chest radiography, bone scan, MRI, CT, PET-CT, and abdominal sonography [24].
Patients were examined at least every three months during the first two years, and every six months during years 3-5 or until death. Evaluation during follow-up included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, chest radiography, abdominal sonography and a whole-body bone scan. Any residual disease found at the nasopharynx or cervical nodes within 6 months after completion of RT was regarded as local failure or regional failure, respectively. All distant metastases were diagnosed by clinical symptoms, physical examination, and imaging methods that included chest radiography, bone scan, MRI, CT, PET-CT, and abdominal sonography [24].
Statistical analysis Statistical analysis was performed using SPSS version 22.0 (IBM Corporation, Armonk, NY, USA), and distant metastasis-free survival (DMFS) was the defined outcome and was calculated from the first day of treatment to the first distant metastasis. The area under the receiver-operating characteristic (ROC) curve was used to select the optimal cut-off point for SUV-T and SUV-N by maximizing the conditional Youden score, based on the method described by Hanley [25] and Zweig [26]. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test [27]. Multivariate analysis based on the Cox proportional hazards model was used to calculate HRs and 95% confidence intervals (CIs), and to test the independent significance of different factors by backward elimination of insignificant variables [28] including host factors (sex, age), tumour factors (T-classification, N-classification), and therapeutic intervention (chemotherapy) as covariates.
Finally, we performed RPA for DMFS to derive prognostic groups that combined anatomic category with other survival predictors identified from multivariate analysis. The RPA algorithm is based on the optimized binary partition of predictors. The resultant subgroups were similar in terms of survival. All tests were two-sided, and P <0.05 was considered statistically significant.
Statistical analysis was performed using SPSS version 22.0 (IBM Corporation, Armonk, NY, USA), and distant metastasis-free survival (DMFS) was the defined outcome and was calculated from the first day of treatment to the first distant metastasis. The area under the receiver-operating characteristic (ROC) curve was used to select the optimal cut-off point for SUV-T and SUV-N by maximizing the conditional Youden score, based on the method described by Hanley [25] and Zweig [26]. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test [27]. Multivariate analysis based on the Cox proportional hazards model was used to calculate HRs and 95% confidence intervals (CIs), and to test the independent significance of different factors by backward elimination of insignificant variables [28] including host factors (sex, age), tumour factors (T-classification, N-classification), and therapeutic intervention (chemotherapy) as covariates.
Finally, we performed RPA for DMFS to derive prognostic groups that combined anatomic category with other survival predictors identified from multivariate analysis. The RPA algorithm is based on the optimized binary partition of predictors. The resultant subgroups were similar in terms of survival. All tests were two-sided, and P <0.05 was considered statistically significant.
| null | null |
DISCUSSION
|
Statistical analysis was performed using SPSS version 22.0 (IBM Corporation, Armonk, NY, USA), and distant metastasis-free survival (DMFS) was the defined outcome and was calculated from the first day of treatment to the first distant metastasis. The area under the receiver-operating characteristic (ROC) curve was used to select the optimal cut-off point for SUV-T and SUV-N by maximizing the conditional Youden score, based on the method described by Hanley [25] and Zweig [26]. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test [27]. Multivariate analysis based on the Cox proportional hazards model was used to calculate HRs and 95% confidence intervals (CIs), and to test the independent significance of different factors by backward elimination of insignificant variables [28] including host factors (sex, age), tumour factors (T-classification, N-classification), and therapeutic intervention (chemotherapy) as covariates.
Finally, we performed RPA for DMFS to derive prognostic groups that combined anatomic category with other survival predictors identified from multivariate analysis. The RPA algorithm is based on the optimized binary partition of predictors. The resultant subgroups were similar in terms of survival. All tests were two-sided, and P <0.05 was considered statistically significant.
|
[
"INTRODUCTION",
"RESULTS",
"Treatment failure and survival",
"Prognosis of different N subcategories",
"Prognostic value of SUV-T and SUV-N in NPC",
"RPA-based prognostic model for DMFS",
"DISCUSSION"
] |
[
"Nasopharyngeal carcinoma (NPC) is particularly prevalent in southern China, Southeast Asia, North Africa, the Middle East, and Alaska [1]. Radiotherapy is the primary treatment used for non-disseminated NPC [2, 3]. With advances in imaging and radiation therapies, local-regional control has exceeded 90% [4]. However, 20-30% of NPC patients eventually develop distant metastasis [5–8], which accounts for the majority of failures [7, 8]. Effort should therefore be made to stratify patients into different groups based on the risk of metastasis to tailor individualized treatments and improve outcomes.\nN-classification in the TNM staging system is a measure of the extent of node involvement, and is currently the most reliable tool for assessing metastasis risk in NPC [9, 10]. However, there is remaining room for improvement in the correlation between the N classification and metastasis [11, 12], perhaps because N-classification is based solely on anatomic extent and lacks non-anatomic information such as tumour physiology.\n18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) imaging is used to probe glucose metabolism in tumour cells [13]. The maximal intensity of FDG uptake by the tumour (maximum standardized uptake value; SUVmax) is a valuable marker of tumour biological behaviour [13, 14] and a useful predictor of distant metastasis in NPC [15, 16]. However, an integrated model incorporating SUVmax and anatomic staging for stratifying metastasis risk has not been reported. Clinicians are therefore somewhat troubled as to how best to incorporate SUVmax into clinical decision-making. A valid approach for incorporating non-anatomic prognostic factors and anatomic staging into an integrated prognosis grouping was recently described [17], which significantly improved survival prediction compared with previous models. In the present study, we extended this approach by using recursive partitioning analysis (RPA) to develop an integrated prognostic model for metastasis that combines SUV parameters and N-classification.",
" Treatment failure and survival The median follow-up time was 49.5 months (range, 3.37–67.9 months), and 385/402 (95.8%) of surviving patients were followed up for >3 years. A total of 84 patients experienced treatment failure, with 19/449 (4.2%), 21/449 (4.7%), and 53/449 (11.8%) developing local recurrence, regional recurrence, and distant metastases, respectively. 8/449 (1.8%) patients experienced both local-regional recurrence and distant metastases and 47/449 (10.5%) patients died- the causes of death were nasopharyngeal carcinoma (93.6%, 44/47), other diseases (2.1%, 1/47) and unknown causes (4.3%, 2/47). The 3-year distant metastasis-free survival, local relapse-free survival, regional relapse-free survival, disease-free survival and overall survival was 89.4%, 96.6%, 95.6%, 83.9% and 94.4%, respectively.\nThe median follow-up time was 49.5 months (range, 3.37–67.9 months), and 385/402 (95.8%) of surviving patients were followed up for >3 years. A total of 84 patients experienced treatment failure, with 19/449 (4.2%), 21/449 (4.7%), and 53/449 (11.8%) developing local recurrence, regional recurrence, and distant metastases, respectively. 8/449 (1.8%) patients experienced both local-regional recurrence and distant metastases and 47/449 (10.5%) patients died- the causes of death were nasopharyngeal carcinoma (93.6%, 44/47), other diseases (2.1%, 1/47) and unknown causes (4.3%, 2/47). The 3-year distant metastasis-free survival, local relapse-free survival, regional relapse-free survival, disease-free survival and overall survival was 89.4%, 96.6%, 95.6%, 83.9% and 94.4%, respectively.\n Prognosis of different N subcategories In total, 75 (16.7%), 251 (55.9%), 76 (16.9%), and 47 (10.5%) patients were classified as N0-3, respectively. The 3-year DMFS decreased only very slightly with increasingly higher N category (96.0%, 93.2%, 81.1% and 71.7%, P <0.001). However, no significant differences were observed between N0 and N1 (P = 0.202) and N2 and N3 (P = 0.188).\nIn total, 75 (16.7%), 251 (55.9%), 76 (16.9%), and 47 (10.5%) patients were classified as N0-3, respectively. The 3-year DMFS decreased only very slightly with increasingly higher N category (96.0%, 93.2%, 81.1% and 71.7%, P <0.001). However, no significant differences were observed between N0 and N1 (P = 0.202) and N2 and N3 (P = 0.188).\n Prognostic value of SUV-T and SUV-N in NPC The SUVmax for primary tumours ranged from 2.2 to 39.3 (median, 13.6), and the optimal cut-off SUV-T value for distant metastasis was 10.45. This value was selected to classify patients into SUV-Thigh (≥10.45) and SUV-Tlow (<10.45) groups. Kaplan-Meier survival curves for the two groups (Figure 1A) showed that 3-year DMFS rates for the SUV-Thigh group (86.2% vs. 97.0%, P = 0.002) were significantly lower than the corresponding rates for the SUV-Tlow group.\nA. SUV-T and B. SUV-N. Abbreviations: SUV-T = SUVmax of the primary tumour; SUV-N = SUVmax of cervical lymph nodes; 3-y = 3-year; DMFS = distant metastasis-free survival; SUVmax = maximum standardized uptake value.\nSUVmax for cervical lymph nodes ranged from 2.6 to 40.9 (median, 8.4), and the optimal cut-off SUV-N value for predicting distant metastasis was 6.65. This value was selected to classify patients into SUV-Nhigh (≥6.65) and SUV-Nlow (<6.65) groups. The 3-year DMFS rates for the SUV-N high group (83.6% vs. 96.9%, P <0.001) were significantly lower than the corresponding rates for the SUV-Nlow group (Figure 1B).\nMultivariate analysis was performed to adjust for confounding factors. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005) and SUV-N (HR, 2.688; 95% CI, 1.250-5.781; P = 0.011) were found to be independent prognostic factors for DMFS. Additionally, advanced N-classification (N2-3 vs. N0-1) was also associated with an increased risk of distant metastasis (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001).\nThe SUVmax for primary tumours ranged from 2.2 to 39.3 (median, 13.6), and the optimal cut-off SUV-T value for distant metastasis was 10.45. This value was selected to classify patients into SUV-Thigh (≥10.45) and SUV-Tlow (<10.45) groups. Kaplan-Meier survival curves for the two groups (Figure 1A) showed that 3-year DMFS rates for the SUV-Thigh group (86.2% vs. 97.0%, P = 0.002) were significantly lower than the corresponding rates for the SUV-Tlow group.\nA. SUV-T and B. SUV-N. Abbreviations: SUV-T = SUVmax of the primary tumour; SUV-N = SUVmax of cervical lymph nodes; 3-y = 3-year; DMFS = distant metastasis-free survival; SUVmax = maximum standardized uptake value.\nSUVmax for cervical lymph nodes ranged from 2.6 to 40.9 (median, 8.4), and the optimal cut-off SUV-N value for predicting distant metastasis was 6.65. This value was selected to classify patients into SUV-Nhigh (≥6.65) and SUV-Nlow (<6.65) groups. The 3-year DMFS rates for the SUV-N high group (83.6% vs. 96.9%, P <0.001) were significantly lower than the corresponding rates for the SUV-Nlow group (Figure 1B).\nMultivariate analysis was performed to adjust for confounding factors. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005) and SUV-N (HR, 2.688; 95% CI, 1.250-5.781; P = 0.011) were found to be independent prognostic factors for DMFS. Additionally, advanced N-classification (N2-3 vs. N0-1) was also associated with an increased risk of distant metastasis (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001).\n RPA-based prognostic model for DMFS We then used RPA to develop an integrated prognostic model based on the independent prognostic factors identified from multivariate analysis (SUV-T, SUV-N and N-classification). Three valid risk groups were derived: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). In total, 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients belonged to low, medium and high risk groups, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Significant differences were observed between the three groups (Figure 2). Multivariate analysis that included host factors (sex, age), tumour factors (T-classification, N-classification), therapeutic intervention (chemotherapy) and RPA-based grouping confirmed the prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95% CI, 1.975-4.835; P <0.001; Table 1).\nA. Prognostic model for DMFS using recursive partitioning analysis (RPA). B. Distant metastasis-free survival for derived prognostic groups. Abbreviations: 3-y = 3-year; DMFS = distant metastasis-free survival.\nAbbreviations: NPC = nasopharyngeal carcinoma; HR = hazard ratio; CI = confidence interval; T = tumour; N = node; RPA = recursive partitioning analysis.\nP-values were calculated using an adjusted Cox proportional hazards model.\nPathological type according to the 2005 World Health Organization classification of tumours.\nAccording to the 7th UICC/AJCC staging system.\nWe then used RPA to develop an integrated prognostic model based on the independent prognostic factors identified from multivariate analysis (SUV-T, SUV-N and N-classification). Three valid risk groups were derived: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). In total, 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients belonged to low, medium and high risk groups, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Significant differences were observed between the three groups (Figure 2). Multivariate analysis that included host factors (sex, age), tumour factors (T-classification, N-classification), therapeutic intervention (chemotherapy) and RPA-based grouping confirmed the prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95% CI, 1.975-4.835; P <0.001; Table 1).\nA. Prognostic model for DMFS using recursive partitioning analysis (RPA). B. Distant metastasis-free survival for derived prognostic groups. Abbreviations: 3-y = 3-year; DMFS = distant metastasis-free survival.\nAbbreviations: NPC = nasopharyngeal carcinoma; HR = hazard ratio; CI = confidence interval; T = tumour; N = node; RPA = recursive partitioning analysis.\nP-values were calculated using an adjusted Cox proportional hazards model.\nPathological type according to the 2005 World Health Organization classification of tumours.\nAccording to the 7th UICC/AJCC staging system.",
"The median follow-up time was 49.5 months (range, 3.37–67.9 months), and 385/402 (95.8%) of surviving patients were followed up for >3 years. A total of 84 patients experienced treatment failure, with 19/449 (4.2%), 21/449 (4.7%), and 53/449 (11.8%) developing local recurrence, regional recurrence, and distant metastases, respectively. 8/449 (1.8%) patients experienced both local-regional recurrence and distant metastases and 47/449 (10.5%) patients died- the causes of death were nasopharyngeal carcinoma (93.6%, 44/47), other diseases (2.1%, 1/47) and unknown causes (4.3%, 2/47). The 3-year distant metastasis-free survival, local relapse-free survival, regional relapse-free survival, disease-free survival and overall survival was 89.4%, 96.6%, 95.6%, 83.9% and 94.4%, respectively.",
"In total, 75 (16.7%), 251 (55.9%), 76 (16.9%), and 47 (10.5%) patients were classified as N0-3, respectively. The 3-year DMFS decreased only very slightly with increasingly higher N category (96.0%, 93.2%, 81.1% and 71.7%, P <0.001). However, no significant differences were observed between N0 and N1 (P = 0.202) and N2 and N3 (P = 0.188).",
"The SUVmax for primary tumours ranged from 2.2 to 39.3 (median, 13.6), and the optimal cut-off SUV-T value for distant metastasis was 10.45. This value was selected to classify patients into SUV-Thigh (≥10.45) and SUV-Tlow (<10.45) groups. Kaplan-Meier survival curves for the two groups (Figure 1A) showed that 3-year DMFS rates for the SUV-Thigh group (86.2% vs. 97.0%, P = 0.002) were significantly lower than the corresponding rates for the SUV-Tlow group.\nA. SUV-T and B. SUV-N. Abbreviations: SUV-T = SUVmax of the primary tumour; SUV-N = SUVmax of cervical lymph nodes; 3-y = 3-year; DMFS = distant metastasis-free survival; SUVmax = maximum standardized uptake value.\nSUVmax for cervical lymph nodes ranged from 2.6 to 40.9 (median, 8.4), and the optimal cut-off SUV-N value for predicting distant metastasis was 6.65. This value was selected to classify patients into SUV-Nhigh (≥6.65) and SUV-Nlow (<6.65) groups. The 3-year DMFS rates for the SUV-N high group (83.6% vs. 96.9%, P <0.001) were significantly lower than the corresponding rates for the SUV-Nlow group (Figure 1B).\nMultivariate analysis was performed to adjust for confounding factors. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005) and SUV-N (HR, 2.688; 95% CI, 1.250-5.781; P = 0.011) were found to be independent prognostic factors for DMFS. Additionally, advanced N-classification (N2-3 vs. N0-1) was also associated with an increased risk of distant metastasis (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001).",
"We then used RPA to develop an integrated prognostic model based on the independent prognostic factors identified from multivariate analysis (SUV-T, SUV-N and N-classification). Three valid risk groups were derived: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). In total, 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients belonged to low, medium and high risk groups, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Significant differences were observed between the three groups (Figure 2). Multivariate analysis that included host factors (sex, age), tumour factors (T-classification, N-classification), therapeutic intervention (chemotherapy) and RPA-based grouping confirmed the prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95% CI, 1.975-4.835; P <0.001; Table 1).\nA. Prognostic model for DMFS using recursive partitioning analysis (RPA). B. Distant metastasis-free survival for derived prognostic groups. Abbreviations: 3-y = 3-year; DMFS = distant metastasis-free survival.\nAbbreviations: NPC = nasopharyngeal carcinoma; HR = hazard ratio; CI = confidence interval; T = tumour; N = node; RPA = recursive partitioning analysis.\nP-values were calculated using an adjusted Cox proportional hazards model.\nPathological type according to the 2005 World Health Organization classification of tumours.\nAccording to the 7th UICC/AJCC staging system.",
"In this study, we firstly developed an integrated RPA-based prognostic model for DMFS that incorporated SUV-N and N-classification. Using multivariate analysis, the RPA-based prognostic grouping was the only significant indicator for DMFS.\nThe intensity of tumour FDG uptake is emerging as a valuable predictive factor of treatment outcome [18–20]. 18F-FDG uptake, measured by SUVmax, is correlated with the density and glucose metabolic rate of tumour cells. Tumours with a high pretreatment SUVmax are therefore likely to be dense and metabolically active, and are likely to have a poor prognosis [18]. Previous studies reported that the SUVmax of primary tumours or regional lymph nodes could predict distant failure in patients with NPC [15, 16], which is in accordance with our results.\nAnatomic disease extent reflecting disease burden was the original basis of stage grouping of cancers in the TNM classification [9]. However, more and more non-anatomic prognostic factors are emerging [21, 22]. Even though the UICC and AJCC have recognized that prognostic classifications should extend beyond anatomic parameters alone, a method incorporating non-anatomic prognostic factors that meets the needs of practitioners and researchers has not been reported. Incorporating selected non-anatomic factors into the anatomic classification system while maintaining the consistency and sustainability of the TNM framework is perhaps the biggest challenge.\nAn RPA-based prognostic grouping incorporating anatomic staging, age, and smoking pack-years for human papilloma virus–related oropharyngeal carcinomas has been recently reported, and this has significantly improved survival prediction [17]. In the present study, we have extended this system by integrating an RPA-based prognostic algorithm with SUV-T and N-classification for predicting distant metastasis. The resultant model identified three distinct risk groups: low risk (N0-1 disease + SUV-T <10.45), medium risk (N0-1 disease + SUV-T >10.45), and high risk (N2-3 disease). This RPA-based prognostic model generated a more balanced distribution and offered superior hazard discrimination compared to N-classification alone, and was confirmed to be the only significant prognostic indicator for DMFS in multivariate analysis.\nDespite the promising results, our study has some limitations. Firstly, the proposed model is derived from retrospective analysis of existing data from one institution, and a multi-institution study is needed to confirm our results. Secondly, pretreatment EBV DNA load has been demonstrated to be a valuable prognostic factor in NPC, but this data was only available for a few patients in our cohort and could not be incorporated in our model. Further studies are therefore needed to investigate whether adding EBV DNA data could further improve prediction of metastasis.\nIn conclusion, analysis of data from a large cohort of NPC patients allowed us to develop an integrated RPA-based prognostic model that incorporates SUV-N and N-classification. Our model performed better at predicting the likelihood of metastasis than previously reported models, and may prove useful for predicting distant metastasis and aiding treatment decisions in the clinic."
] |
[
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"RESULTS",
"Treatment failure and survival",
"Prognosis of different N subcategories",
"Prognostic value of SUV-T and SUV-N in NPC",
"RPA-based prognostic model for DMFS",
"DISCUSSION",
"PATIENTS AND METHODS"
] |
[
"Nasopharyngeal carcinoma (NPC) is particularly prevalent in southern China, Southeast Asia, North Africa, the Middle East, and Alaska [1]. Radiotherapy is the primary treatment used for non-disseminated NPC [2, 3]. With advances in imaging and radiation therapies, local-regional control has exceeded 90% [4]. However, 20-30% of NPC patients eventually develop distant metastasis [5–8], which accounts for the majority of failures [7, 8]. Effort should therefore be made to stratify patients into different groups based on the risk of metastasis to tailor individualized treatments and improve outcomes.\nN-classification in the TNM staging system is a measure of the extent of node involvement, and is currently the most reliable tool for assessing metastasis risk in NPC [9, 10]. However, there is remaining room for improvement in the correlation between the N classification and metastasis [11, 12], perhaps because N-classification is based solely on anatomic extent and lacks non-anatomic information such as tumour physiology.\n18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) imaging is used to probe glucose metabolism in tumour cells [13]. The maximal intensity of FDG uptake by the tumour (maximum standardized uptake value; SUVmax) is a valuable marker of tumour biological behaviour [13, 14] and a useful predictor of distant metastasis in NPC [15, 16]. However, an integrated model incorporating SUVmax and anatomic staging for stratifying metastasis risk has not been reported. Clinicians are therefore somewhat troubled as to how best to incorporate SUVmax into clinical decision-making. A valid approach for incorporating non-anatomic prognostic factors and anatomic staging into an integrated prognosis grouping was recently described [17], which significantly improved survival prediction compared with previous models. In the present study, we extended this approach by using recursive partitioning analysis (RPA) to develop an integrated prognostic model for metastasis that combines SUV parameters and N-classification.",
" Treatment failure and survival The median follow-up time was 49.5 months (range, 3.37–67.9 months), and 385/402 (95.8%) of surviving patients were followed up for >3 years. A total of 84 patients experienced treatment failure, with 19/449 (4.2%), 21/449 (4.7%), and 53/449 (11.8%) developing local recurrence, regional recurrence, and distant metastases, respectively. 8/449 (1.8%) patients experienced both local-regional recurrence and distant metastases and 47/449 (10.5%) patients died- the causes of death were nasopharyngeal carcinoma (93.6%, 44/47), other diseases (2.1%, 1/47) and unknown causes (4.3%, 2/47). The 3-year distant metastasis-free survival, local relapse-free survival, regional relapse-free survival, disease-free survival and overall survival was 89.4%, 96.6%, 95.6%, 83.9% and 94.4%, respectively.\nThe median follow-up time was 49.5 months (range, 3.37–67.9 months), and 385/402 (95.8%) of surviving patients were followed up for >3 years. A total of 84 patients experienced treatment failure, with 19/449 (4.2%), 21/449 (4.7%), and 53/449 (11.8%) developing local recurrence, regional recurrence, and distant metastases, respectively. 8/449 (1.8%) patients experienced both local-regional recurrence and distant metastases and 47/449 (10.5%) patients died- the causes of death were nasopharyngeal carcinoma (93.6%, 44/47), other diseases (2.1%, 1/47) and unknown causes (4.3%, 2/47). The 3-year distant metastasis-free survival, local relapse-free survival, regional relapse-free survival, disease-free survival and overall survival was 89.4%, 96.6%, 95.6%, 83.9% and 94.4%, respectively.\n Prognosis of different N subcategories In total, 75 (16.7%), 251 (55.9%), 76 (16.9%), and 47 (10.5%) patients were classified as N0-3, respectively. The 3-year DMFS decreased only very slightly with increasingly higher N category (96.0%, 93.2%, 81.1% and 71.7%, P <0.001). However, no significant differences were observed between N0 and N1 (P = 0.202) and N2 and N3 (P = 0.188).\nIn total, 75 (16.7%), 251 (55.9%), 76 (16.9%), and 47 (10.5%) patients were classified as N0-3, respectively. The 3-year DMFS decreased only very slightly with increasingly higher N category (96.0%, 93.2%, 81.1% and 71.7%, P <0.001). However, no significant differences were observed between N0 and N1 (P = 0.202) and N2 and N3 (P = 0.188).\n Prognostic value of SUV-T and SUV-N in NPC The SUVmax for primary tumours ranged from 2.2 to 39.3 (median, 13.6), and the optimal cut-off SUV-T value for distant metastasis was 10.45. This value was selected to classify patients into SUV-Thigh (≥10.45) and SUV-Tlow (<10.45) groups. Kaplan-Meier survival curves for the two groups (Figure 1A) showed that 3-year DMFS rates for the SUV-Thigh group (86.2% vs. 97.0%, P = 0.002) were significantly lower than the corresponding rates for the SUV-Tlow group.\nA. SUV-T and B. SUV-N. Abbreviations: SUV-T = SUVmax of the primary tumour; SUV-N = SUVmax of cervical lymph nodes; 3-y = 3-year; DMFS = distant metastasis-free survival; SUVmax = maximum standardized uptake value.\nSUVmax for cervical lymph nodes ranged from 2.6 to 40.9 (median, 8.4), and the optimal cut-off SUV-N value for predicting distant metastasis was 6.65. This value was selected to classify patients into SUV-Nhigh (≥6.65) and SUV-Nlow (<6.65) groups. The 3-year DMFS rates for the SUV-N high group (83.6% vs. 96.9%, P <0.001) were significantly lower than the corresponding rates for the SUV-Nlow group (Figure 1B).\nMultivariate analysis was performed to adjust for confounding factors. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005) and SUV-N (HR, 2.688; 95% CI, 1.250-5.781; P = 0.011) were found to be independent prognostic factors for DMFS. Additionally, advanced N-classification (N2-3 vs. N0-1) was also associated with an increased risk of distant metastasis (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001).\nThe SUVmax for primary tumours ranged from 2.2 to 39.3 (median, 13.6), and the optimal cut-off SUV-T value for distant metastasis was 10.45. This value was selected to classify patients into SUV-Thigh (≥10.45) and SUV-Tlow (<10.45) groups. Kaplan-Meier survival curves for the two groups (Figure 1A) showed that 3-year DMFS rates for the SUV-Thigh group (86.2% vs. 97.0%, P = 0.002) were significantly lower than the corresponding rates for the SUV-Tlow group.\nA. SUV-T and B. SUV-N. Abbreviations: SUV-T = SUVmax of the primary tumour; SUV-N = SUVmax of cervical lymph nodes; 3-y = 3-year; DMFS = distant metastasis-free survival; SUVmax = maximum standardized uptake value.\nSUVmax for cervical lymph nodes ranged from 2.6 to 40.9 (median, 8.4), and the optimal cut-off SUV-N value for predicting distant metastasis was 6.65. This value was selected to classify patients into SUV-Nhigh (≥6.65) and SUV-Nlow (<6.65) groups. The 3-year DMFS rates for the SUV-N high group (83.6% vs. 96.9%, P <0.001) were significantly lower than the corresponding rates for the SUV-Nlow group (Figure 1B).\nMultivariate analysis was performed to adjust for confounding factors. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005) and SUV-N (HR, 2.688; 95% CI, 1.250-5.781; P = 0.011) were found to be independent prognostic factors for DMFS. Additionally, advanced N-classification (N2-3 vs. N0-1) was also associated with an increased risk of distant metastasis (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001).\n RPA-based prognostic model for DMFS We then used RPA to develop an integrated prognostic model based on the independent prognostic factors identified from multivariate analysis (SUV-T, SUV-N and N-classification). Three valid risk groups were derived: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). In total, 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients belonged to low, medium and high risk groups, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Significant differences were observed between the three groups (Figure 2). Multivariate analysis that included host factors (sex, age), tumour factors (T-classification, N-classification), therapeutic intervention (chemotherapy) and RPA-based grouping confirmed the prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95% CI, 1.975-4.835; P <0.001; Table 1).\nA. Prognostic model for DMFS using recursive partitioning analysis (RPA). B. Distant metastasis-free survival for derived prognostic groups. Abbreviations: 3-y = 3-year; DMFS = distant metastasis-free survival.\nAbbreviations: NPC = nasopharyngeal carcinoma; HR = hazard ratio; CI = confidence interval; T = tumour; N = node; RPA = recursive partitioning analysis.\nP-values were calculated using an adjusted Cox proportional hazards model.\nPathological type according to the 2005 World Health Organization classification of tumours.\nAccording to the 7th UICC/AJCC staging system.\nWe then used RPA to develop an integrated prognostic model based on the independent prognostic factors identified from multivariate analysis (SUV-T, SUV-N and N-classification). Three valid risk groups were derived: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). In total, 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients belonged to low, medium and high risk groups, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Significant differences were observed between the three groups (Figure 2). Multivariate analysis that included host factors (sex, age), tumour factors (T-classification, N-classification), therapeutic intervention (chemotherapy) and RPA-based grouping confirmed the prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95% CI, 1.975-4.835; P <0.001; Table 1).\nA. Prognostic model for DMFS using recursive partitioning analysis (RPA). B. Distant metastasis-free survival for derived prognostic groups. Abbreviations: 3-y = 3-year; DMFS = distant metastasis-free survival.\nAbbreviations: NPC = nasopharyngeal carcinoma; HR = hazard ratio; CI = confidence interval; T = tumour; N = node; RPA = recursive partitioning analysis.\nP-values were calculated using an adjusted Cox proportional hazards model.\nPathological type according to the 2005 World Health Organization classification of tumours.\nAccording to the 7th UICC/AJCC staging system.",
"The median follow-up time was 49.5 months (range, 3.37–67.9 months), and 385/402 (95.8%) of surviving patients were followed up for >3 years. A total of 84 patients experienced treatment failure, with 19/449 (4.2%), 21/449 (4.7%), and 53/449 (11.8%) developing local recurrence, regional recurrence, and distant metastases, respectively. 8/449 (1.8%) patients experienced both local-regional recurrence and distant metastases and 47/449 (10.5%) patients died- the causes of death were nasopharyngeal carcinoma (93.6%, 44/47), other diseases (2.1%, 1/47) and unknown causes (4.3%, 2/47). The 3-year distant metastasis-free survival, local relapse-free survival, regional relapse-free survival, disease-free survival and overall survival was 89.4%, 96.6%, 95.6%, 83.9% and 94.4%, respectively.",
"In total, 75 (16.7%), 251 (55.9%), 76 (16.9%), and 47 (10.5%) patients were classified as N0-3, respectively. The 3-year DMFS decreased only very slightly with increasingly higher N category (96.0%, 93.2%, 81.1% and 71.7%, P <0.001). However, no significant differences were observed between N0 and N1 (P = 0.202) and N2 and N3 (P = 0.188).",
"The SUVmax for primary tumours ranged from 2.2 to 39.3 (median, 13.6), and the optimal cut-off SUV-T value for distant metastasis was 10.45. This value was selected to classify patients into SUV-Thigh (≥10.45) and SUV-Tlow (<10.45) groups. Kaplan-Meier survival curves for the two groups (Figure 1A) showed that 3-year DMFS rates for the SUV-Thigh group (86.2% vs. 97.0%, P = 0.002) were significantly lower than the corresponding rates for the SUV-Tlow group.\nA. SUV-T and B. SUV-N. Abbreviations: SUV-T = SUVmax of the primary tumour; SUV-N = SUVmax of cervical lymph nodes; 3-y = 3-year; DMFS = distant metastasis-free survival; SUVmax = maximum standardized uptake value.\nSUVmax for cervical lymph nodes ranged from 2.6 to 40.9 (median, 8.4), and the optimal cut-off SUV-N value for predicting distant metastasis was 6.65. This value was selected to classify patients into SUV-Nhigh (≥6.65) and SUV-Nlow (<6.65) groups. The 3-year DMFS rates for the SUV-N high group (83.6% vs. 96.9%, P <0.001) were significantly lower than the corresponding rates for the SUV-Nlow group (Figure 1B).\nMultivariate analysis was performed to adjust for confounding factors. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005) and SUV-N (HR, 2.688; 95% CI, 1.250-5.781; P = 0.011) were found to be independent prognostic factors for DMFS. Additionally, advanced N-classification (N2-3 vs. N0-1) was also associated with an increased risk of distant metastasis (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001).",
"We then used RPA to develop an integrated prognostic model based on the independent prognostic factors identified from multivariate analysis (SUV-T, SUV-N and N-classification). Three valid risk groups were derived: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). In total, 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients belonged to low, medium and high risk groups, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Significant differences were observed between the three groups (Figure 2). Multivariate analysis that included host factors (sex, age), tumour factors (T-classification, N-classification), therapeutic intervention (chemotherapy) and RPA-based grouping confirmed the prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95% CI, 1.975-4.835; P <0.001; Table 1).\nA. Prognostic model for DMFS using recursive partitioning analysis (RPA). B. Distant metastasis-free survival for derived prognostic groups. Abbreviations: 3-y = 3-year; DMFS = distant metastasis-free survival.\nAbbreviations: NPC = nasopharyngeal carcinoma; HR = hazard ratio; CI = confidence interval; T = tumour; N = node; RPA = recursive partitioning analysis.\nP-values were calculated using an adjusted Cox proportional hazards model.\nPathological type according to the 2005 World Health Organization classification of tumours.\nAccording to the 7th UICC/AJCC staging system.",
"In this study, we firstly developed an integrated RPA-based prognostic model for DMFS that incorporated SUV-N and N-classification. Using multivariate analysis, the RPA-based prognostic grouping was the only significant indicator for DMFS.\nThe intensity of tumour FDG uptake is emerging as a valuable predictive factor of treatment outcome [18–20]. 18F-FDG uptake, measured by SUVmax, is correlated with the density and glucose metabolic rate of tumour cells. Tumours with a high pretreatment SUVmax are therefore likely to be dense and metabolically active, and are likely to have a poor prognosis [18]. Previous studies reported that the SUVmax of primary tumours or regional lymph nodes could predict distant failure in patients with NPC [15, 16], which is in accordance with our results.\nAnatomic disease extent reflecting disease burden was the original basis of stage grouping of cancers in the TNM classification [9]. However, more and more non-anatomic prognostic factors are emerging [21, 22]. Even though the UICC and AJCC have recognized that prognostic classifications should extend beyond anatomic parameters alone, a method incorporating non-anatomic prognostic factors that meets the needs of practitioners and researchers has not been reported. Incorporating selected non-anatomic factors into the anatomic classification system while maintaining the consistency and sustainability of the TNM framework is perhaps the biggest challenge.\nAn RPA-based prognostic grouping incorporating anatomic staging, age, and smoking pack-years for human papilloma virus–related oropharyngeal carcinomas has been recently reported, and this has significantly improved survival prediction [17]. In the present study, we have extended this system by integrating an RPA-based prognostic algorithm with SUV-T and N-classification for predicting distant metastasis. The resultant model identified three distinct risk groups: low risk (N0-1 disease + SUV-T <10.45), medium risk (N0-1 disease + SUV-T >10.45), and high risk (N2-3 disease). This RPA-based prognostic model generated a more balanced distribution and offered superior hazard discrimination compared to N-classification alone, and was confirmed to be the only significant prognostic indicator for DMFS in multivariate analysis.\nDespite the promising results, our study has some limitations. Firstly, the proposed model is derived from retrospective analysis of existing data from one institution, and a multi-institution study is needed to confirm our results. Secondly, pretreatment EBV DNA load has been demonstrated to be a valuable prognostic factor in NPC, but this data was only available for a few patients in our cohort and could not be incorporated in our model. Further studies are therefore needed to investigate whether adding EBV DNA data could further improve prediction of metastasis.\nIn conclusion, analysis of data from a large cohort of NPC patients allowed us to develop an integrated RPA-based prognostic model that incorporates SUV-N and N-classification. Our model performed better at predicting the likelihood of metastasis than previously reported models, and may prove useful for predicting distant metastasis and aiding treatment decisions in the clinic.",
" Patients This study was approved by the institutional review board, and the requirement to obtain informed consent was waived. From January 2010 to February 2012, 449 patients with stage I-IVB NPC treated at our institution received a positron emission tomography-computed tomography (PET-CT) examination before treatment followed by intensity-modulated radiotherapy (IMRT) with or without chemotherapy. All of the enrolled patients were of Chinese ethnicity. The median age was 46 years (range, 20- 77), with a male-to-female ratio of 3:1 (Table 2).\nAbbreviations: NPC = nasopharyngeal carcinoma; T = tumour; N = node.\nPathological type according to the 2005 World Health Organization classification of tumours.\nAccording to the 7th edition of the UICC/AJCC staging system.\nAll patients underwent a pretreatment evaluation that included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, and PET-CT. All patients were staged according to the 7th edition of the International Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) system [9].\nThis study was approved by the institutional review board, and the requirement to obtain informed consent was waived. From January 2010 to February 2012, 449 patients with stage I-IVB NPC treated at our institution received a positron emission tomography-computed tomography (PET-CT) examination before treatment followed by intensity-modulated radiotherapy (IMRT) with or without chemotherapy. All of the enrolled patients were of Chinese ethnicity. The median age was 46 years (range, 20- 77), with a male-to-female ratio of 3:1 (Table 2).\nAbbreviations: NPC = nasopharyngeal carcinoma; T = tumour; N = node.\nPathological type according to the 2005 World Health Organization classification of tumours.\nAccording to the 7th edition of the UICC/AJCC staging system.\nAll patients underwent a pretreatment evaluation that included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, and PET-CT. All patients were staged according to the 7th edition of the International Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) system [9].\n PET/CT imaging Serum glucose levels were measured in all NPC patients, all of whom fasted for at least 6 h before PET/CT scans, and individuals with a fasting plasma glucose >200 mg/dl were excluded. PET/CT imaging was performed with a combination PET/CT scanner (Discovery ST 16; GE Healthcare, Little Chalfont, UK) according to published guidelines [23]. Helical CT was performed from the head to the proximal thigh before PET acquisition, according to a standardized protocol and were 45-60 min after injection of 5.55 MBq/kg FDG. PET images were reconstructed from CT data for attenuation correction using an ordered-subset expectation maximization iterative reconstruction algorithm. SUVmax was determined for each region of interest using the whole-body attenuation corrected image and the following formula: SUVmax = tissue concentration of 18F-FDG / injected dose / body weight.\nSerum glucose levels were measured in all NPC patients, all of whom fasted for at least 6 h before PET/CT scans, and individuals with a fasting plasma glucose >200 mg/dl were excluded. PET/CT imaging was performed with a combination PET/CT scanner (Discovery ST 16; GE Healthcare, Little Chalfont, UK) according to published guidelines [23]. Helical CT was performed from the head to the proximal thigh before PET acquisition, according to a standardized protocol and were 45-60 min after injection of 5.55 MBq/kg FDG. PET images were reconstructed from CT data for attenuation correction using an ordered-subset expectation maximization iterative reconstruction algorithm. SUVmax was determined for each region of interest using the whole-body attenuation corrected image and the following formula: SUVmax = tissue concentration of 18F-FDG / injected dose / body weight.\n Treatment The nasopharyngeal and neck tumour volumes of all patients were treated using radical radiotherapy based on IMRT for the entire treatment course. Institutional guidelines recommended radiotherapy only for stage I and concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy for stage II-IVB. In total, 92.8% (308/332) of patients with stage III-IVB disease received concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy. When possible, salvage treatments (intracavitary brachytherapy, surgery or chemotherapy) were provided and persistent disease or relapse was documented.\nThe nasopharyngeal and neck tumour volumes of all patients were treated using radical radiotherapy based on IMRT for the entire treatment course. Institutional guidelines recommended radiotherapy only for stage I and concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy for stage II-IVB. In total, 92.8% (308/332) of patients with stage III-IVB disease received concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy. When possible, salvage treatments (intracavitary brachytherapy, surgery or chemotherapy) were provided and persistent disease or relapse was documented.\n Follow-up Patients were examined at least every three months during the first two years, and every six months during years 3-5 or until death. Evaluation during follow-up included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, chest radiography, abdominal sonography and a whole-body bone scan. Any residual disease found at the nasopharynx or cervical nodes within 6 months after completion of RT was regarded as local failure or regional failure, respectively. All distant metastases were diagnosed by clinical symptoms, physical examination, and imaging methods that included chest radiography, bone scan, MRI, CT, PET-CT, and abdominal sonography [24].\nPatients were examined at least every three months during the first two years, and every six months during years 3-5 or until death. Evaluation during follow-up included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, chest radiography, abdominal sonography and a whole-body bone scan. Any residual disease found at the nasopharynx or cervical nodes within 6 months after completion of RT was regarded as local failure or regional failure, respectively. All distant metastases were diagnosed by clinical symptoms, physical examination, and imaging methods that included chest radiography, bone scan, MRI, CT, PET-CT, and abdominal sonography [24].\n Statistical analysis Statistical analysis was performed using SPSS version 22.0 (IBM Corporation, Armonk, NY, USA), and distant metastasis-free survival (DMFS) was the defined outcome and was calculated from the first day of treatment to the first distant metastasis. The area under the receiver-operating characteristic (ROC) curve was used to select the optimal cut-off point for SUV-T and SUV-N by maximizing the conditional Youden score, based on the method described by Hanley [25] and Zweig [26]. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test [27]. Multivariate analysis based on the Cox proportional hazards model was used to calculate HRs and 95% confidence intervals (CIs), and to test the independent significance of different factors by backward elimination of insignificant variables [28] including host factors (sex, age), tumour factors (T-classification, N-classification), and therapeutic intervention (chemotherapy) as covariates.\nFinally, we performed RPA for DMFS to derive prognostic groups that combined anatomic category with other survival predictors identified from multivariate analysis. The RPA algorithm is based on the optimized binary partition of predictors. The resultant subgroups were similar in terms of survival. All tests were two-sided, and P <0.05 was considered statistically significant.\nStatistical analysis was performed using SPSS version 22.0 (IBM Corporation, Armonk, NY, USA), and distant metastasis-free survival (DMFS) was the defined outcome and was calculated from the first day of treatment to the first distant metastasis. The area under the receiver-operating characteristic (ROC) curve was used to select the optimal cut-off point for SUV-T and SUV-N by maximizing the conditional Youden score, based on the method described by Hanley [25] and Zweig [26]. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test [27]. Multivariate analysis based on the Cox proportional hazards model was used to calculate HRs and 95% confidence intervals (CIs), and to test the independent significance of different factors by backward elimination of insignificant variables [28] including host factors (sex, age), tumour factors (T-classification, N-classification), and therapeutic intervention (chemotherapy) as covariates.\nFinally, we performed RPA for DMFS to derive prognostic groups that combined anatomic category with other survival predictors identified from multivariate analysis. The RPA algorithm is based on the optimized binary partition of predictors. The resultant subgroups were similar in terms of survival. All tests were two-sided, and P <0.05 was considered statistically significant."
] |
[
null,
null,
null,
null,
null,
null,
null,
"methods"
] |
[
"nasopharyngeal neoplasms",
"metastasis",
"TNM staging",
"maximum standardized uptake value",
"recursive partitioning analysis"
] |
INTRODUCTION:
Nasopharyngeal carcinoma (NPC) is particularly prevalent in southern China, Southeast Asia, North Africa, the Middle East, and Alaska [1]. Radiotherapy is the primary treatment used for non-disseminated NPC [2, 3]. With advances in imaging and radiation therapies, local-regional control has exceeded 90% [4]. However, 20-30% of NPC patients eventually develop distant metastasis [5–8], which accounts for the majority of failures [7, 8]. Effort should therefore be made to stratify patients into different groups based on the risk of metastasis to tailor individualized treatments and improve outcomes.
N-classification in the TNM staging system is a measure of the extent of node involvement, and is currently the most reliable tool for assessing metastasis risk in NPC [9, 10]. However, there is remaining room for improvement in the correlation between the N classification and metastasis [11, 12], perhaps because N-classification is based solely on anatomic extent and lacks non-anatomic information such as tumour physiology.
18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) imaging is used to probe glucose metabolism in tumour cells [13]. The maximal intensity of FDG uptake by the tumour (maximum standardized uptake value; SUVmax) is a valuable marker of tumour biological behaviour [13, 14] and a useful predictor of distant metastasis in NPC [15, 16]. However, an integrated model incorporating SUVmax and anatomic staging for stratifying metastasis risk has not been reported. Clinicians are therefore somewhat troubled as to how best to incorporate SUVmax into clinical decision-making. A valid approach for incorporating non-anatomic prognostic factors and anatomic staging into an integrated prognosis grouping was recently described [17], which significantly improved survival prediction compared with previous models. In the present study, we extended this approach by using recursive partitioning analysis (RPA) to develop an integrated prognostic model for metastasis that combines SUV parameters and N-classification.
RESULTS:
Treatment failure and survival The median follow-up time was 49.5 months (range, 3.37–67.9 months), and 385/402 (95.8%) of surviving patients were followed up for >3 years. A total of 84 patients experienced treatment failure, with 19/449 (4.2%), 21/449 (4.7%), and 53/449 (11.8%) developing local recurrence, regional recurrence, and distant metastases, respectively. 8/449 (1.8%) patients experienced both local-regional recurrence and distant metastases and 47/449 (10.5%) patients died- the causes of death were nasopharyngeal carcinoma (93.6%, 44/47), other diseases (2.1%, 1/47) and unknown causes (4.3%, 2/47). The 3-year distant metastasis-free survival, local relapse-free survival, regional relapse-free survival, disease-free survival and overall survival was 89.4%, 96.6%, 95.6%, 83.9% and 94.4%, respectively.
The median follow-up time was 49.5 months (range, 3.37–67.9 months), and 385/402 (95.8%) of surviving patients were followed up for >3 years. A total of 84 patients experienced treatment failure, with 19/449 (4.2%), 21/449 (4.7%), and 53/449 (11.8%) developing local recurrence, regional recurrence, and distant metastases, respectively. 8/449 (1.8%) patients experienced both local-regional recurrence and distant metastases and 47/449 (10.5%) patients died- the causes of death were nasopharyngeal carcinoma (93.6%, 44/47), other diseases (2.1%, 1/47) and unknown causes (4.3%, 2/47). The 3-year distant metastasis-free survival, local relapse-free survival, regional relapse-free survival, disease-free survival and overall survival was 89.4%, 96.6%, 95.6%, 83.9% and 94.4%, respectively.
Prognosis of different N subcategories In total, 75 (16.7%), 251 (55.9%), 76 (16.9%), and 47 (10.5%) patients were classified as N0-3, respectively. The 3-year DMFS decreased only very slightly with increasingly higher N category (96.0%, 93.2%, 81.1% and 71.7%, P <0.001). However, no significant differences were observed between N0 and N1 (P = 0.202) and N2 and N3 (P = 0.188).
In total, 75 (16.7%), 251 (55.9%), 76 (16.9%), and 47 (10.5%) patients were classified as N0-3, respectively. The 3-year DMFS decreased only very slightly with increasingly higher N category (96.0%, 93.2%, 81.1% and 71.7%, P <0.001). However, no significant differences were observed between N0 and N1 (P = 0.202) and N2 and N3 (P = 0.188).
Prognostic value of SUV-T and SUV-N in NPC The SUVmax for primary tumours ranged from 2.2 to 39.3 (median, 13.6), and the optimal cut-off SUV-T value for distant metastasis was 10.45. This value was selected to classify patients into SUV-Thigh (≥10.45) and SUV-Tlow (<10.45) groups. Kaplan-Meier survival curves for the two groups (Figure 1A) showed that 3-year DMFS rates for the SUV-Thigh group (86.2% vs. 97.0%, P = 0.002) were significantly lower than the corresponding rates for the SUV-Tlow group.
A. SUV-T and B. SUV-N. Abbreviations: SUV-T = SUVmax of the primary tumour; SUV-N = SUVmax of cervical lymph nodes; 3-y = 3-year; DMFS = distant metastasis-free survival; SUVmax = maximum standardized uptake value.
SUVmax for cervical lymph nodes ranged from 2.6 to 40.9 (median, 8.4), and the optimal cut-off SUV-N value for predicting distant metastasis was 6.65. This value was selected to classify patients into SUV-Nhigh (≥6.65) and SUV-Nlow (<6.65) groups. The 3-year DMFS rates for the SUV-N high group (83.6% vs. 96.9%, P <0.001) were significantly lower than the corresponding rates for the SUV-Nlow group (Figure 1B).
Multivariate analysis was performed to adjust for confounding factors. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005) and SUV-N (HR, 2.688; 95% CI, 1.250-5.781; P = 0.011) were found to be independent prognostic factors for DMFS. Additionally, advanced N-classification (N2-3 vs. N0-1) was also associated with an increased risk of distant metastasis (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001).
The SUVmax for primary tumours ranged from 2.2 to 39.3 (median, 13.6), and the optimal cut-off SUV-T value for distant metastasis was 10.45. This value was selected to classify patients into SUV-Thigh (≥10.45) and SUV-Tlow (<10.45) groups. Kaplan-Meier survival curves for the two groups (Figure 1A) showed that 3-year DMFS rates for the SUV-Thigh group (86.2% vs. 97.0%, P = 0.002) were significantly lower than the corresponding rates for the SUV-Tlow group.
A. SUV-T and B. SUV-N. Abbreviations: SUV-T = SUVmax of the primary tumour; SUV-N = SUVmax of cervical lymph nodes; 3-y = 3-year; DMFS = distant metastasis-free survival; SUVmax = maximum standardized uptake value.
SUVmax for cervical lymph nodes ranged from 2.6 to 40.9 (median, 8.4), and the optimal cut-off SUV-N value for predicting distant metastasis was 6.65. This value was selected to classify patients into SUV-Nhigh (≥6.65) and SUV-Nlow (<6.65) groups. The 3-year DMFS rates for the SUV-N high group (83.6% vs. 96.9%, P <0.001) were significantly lower than the corresponding rates for the SUV-Nlow group (Figure 1B).
Multivariate analysis was performed to adjust for confounding factors. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005) and SUV-N (HR, 2.688; 95% CI, 1.250-5.781; P = 0.011) were found to be independent prognostic factors for DMFS. Additionally, advanced N-classification (N2-3 vs. N0-1) was also associated with an increased risk of distant metastasis (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001).
RPA-based prognostic model for DMFS We then used RPA to develop an integrated prognostic model based on the independent prognostic factors identified from multivariate analysis (SUV-T, SUV-N and N-classification). Three valid risk groups were derived: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). In total, 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients belonged to low, medium and high risk groups, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Significant differences were observed between the three groups (Figure 2). Multivariate analysis that included host factors (sex, age), tumour factors (T-classification, N-classification), therapeutic intervention (chemotherapy) and RPA-based grouping confirmed the prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95% CI, 1.975-4.835; P <0.001; Table 1).
A. Prognostic model for DMFS using recursive partitioning analysis (RPA). B. Distant metastasis-free survival for derived prognostic groups. Abbreviations: 3-y = 3-year; DMFS = distant metastasis-free survival.
Abbreviations: NPC = nasopharyngeal carcinoma; HR = hazard ratio; CI = confidence interval; T = tumour; N = node; RPA = recursive partitioning analysis.
P-values were calculated using an adjusted Cox proportional hazards model.
Pathological type according to the 2005 World Health Organization classification of tumours.
According to the 7th UICC/AJCC staging system.
We then used RPA to develop an integrated prognostic model based on the independent prognostic factors identified from multivariate analysis (SUV-T, SUV-N and N-classification). Three valid risk groups were derived: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). In total, 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients belonged to low, medium and high risk groups, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Significant differences were observed between the three groups (Figure 2). Multivariate analysis that included host factors (sex, age), tumour factors (T-classification, N-classification), therapeutic intervention (chemotherapy) and RPA-based grouping confirmed the prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95% CI, 1.975-4.835; P <0.001; Table 1).
A. Prognostic model for DMFS using recursive partitioning analysis (RPA). B. Distant metastasis-free survival for derived prognostic groups. Abbreviations: 3-y = 3-year; DMFS = distant metastasis-free survival.
Abbreviations: NPC = nasopharyngeal carcinoma; HR = hazard ratio; CI = confidence interval; T = tumour; N = node; RPA = recursive partitioning analysis.
P-values were calculated using an adjusted Cox proportional hazards model.
Pathological type according to the 2005 World Health Organization classification of tumours.
According to the 7th UICC/AJCC staging system.
Treatment failure and survival:
The median follow-up time was 49.5 months (range, 3.37–67.9 months), and 385/402 (95.8%) of surviving patients were followed up for >3 years. A total of 84 patients experienced treatment failure, with 19/449 (4.2%), 21/449 (4.7%), and 53/449 (11.8%) developing local recurrence, regional recurrence, and distant metastases, respectively. 8/449 (1.8%) patients experienced both local-regional recurrence and distant metastases and 47/449 (10.5%) patients died- the causes of death were nasopharyngeal carcinoma (93.6%, 44/47), other diseases (2.1%, 1/47) and unknown causes (4.3%, 2/47). The 3-year distant metastasis-free survival, local relapse-free survival, regional relapse-free survival, disease-free survival and overall survival was 89.4%, 96.6%, 95.6%, 83.9% and 94.4%, respectively.
Prognosis of different N subcategories:
In total, 75 (16.7%), 251 (55.9%), 76 (16.9%), and 47 (10.5%) patients were classified as N0-3, respectively. The 3-year DMFS decreased only very slightly with increasingly higher N category (96.0%, 93.2%, 81.1% and 71.7%, P <0.001). However, no significant differences were observed between N0 and N1 (P = 0.202) and N2 and N3 (P = 0.188).
Prognostic value of SUV-T and SUV-N in NPC:
The SUVmax for primary tumours ranged from 2.2 to 39.3 (median, 13.6), and the optimal cut-off SUV-T value for distant metastasis was 10.45. This value was selected to classify patients into SUV-Thigh (≥10.45) and SUV-Tlow (<10.45) groups. Kaplan-Meier survival curves for the two groups (Figure 1A) showed that 3-year DMFS rates for the SUV-Thigh group (86.2% vs. 97.0%, P = 0.002) were significantly lower than the corresponding rates for the SUV-Tlow group.
A. SUV-T and B. SUV-N. Abbreviations: SUV-T = SUVmax of the primary tumour; SUV-N = SUVmax of cervical lymph nodes; 3-y = 3-year; DMFS = distant metastasis-free survival; SUVmax = maximum standardized uptake value.
SUVmax for cervical lymph nodes ranged from 2.6 to 40.9 (median, 8.4), and the optimal cut-off SUV-N value for predicting distant metastasis was 6.65. This value was selected to classify patients into SUV-Nhigh (≥6.65) and SUV-Nlow (<6.65) groups. The 3-year DMFS rates for the SUV-N high group (83.6% vs. 96.9%, P <0.001) were significantly lower than the corresponding rates for the SUV-Nlow group (Figure 1B).
Multivariate analysis was performed to adjust for confounding factors. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005) and SUV-N (HR, 2.688; 95% CI, 1.250-5.781; P = 0.011) were found to be independent prognostic factors for DMFS. Additionally, advanced N-classification (N2-3 vs. N0-1) was also associated with an increased risk of distant metastasis (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001).
RPA-based prognostic model for DMFS:
We then used RPA to develop an integrated prognostic model based on the independent prognostic factors identified from multivariate analysis (SUV-T, SUV-N and N-classification). Three valid risk groups were derived: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). In total, 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients belonged to low, medium and high risk groups, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Significant differences were observed between the three groups (Figure 2). Multivariate analysis that included host factors (sex, age), tumour factors (T-classification, N-classification), therapeutic intervention (chemotherapy) and RPA-based grouping confirmed the prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95% CI, 1.975-4.835; P <0.001; Table 1).
A. Prognostic model for DMFS using recursive partitioning analysis (RPA). B. Distant metastasis-free survival for derived prognostic groups. Abbreviations: 3-y = 3-year; DMFS = distant metastasis-free survival.
Abbreviations: NPC = nasopharyngeal carcinoma; HR = hazard ratio; CI = confidence interval; T = tumour; N = node; RPA = recursive partitioning analysis.
P-values were calculated using an adjusted Cox proportional hazards model.
Pathological type according to the 2005 World Health Organization classification of tumours.
According to the 7th UICC/AJCC staging system.
DISCUSSION:
In this study, we firstly developed an integrated RPA-based prognostic model for DMFS that incorporated SUV-N and N-classification. Using multivariate analysis, the RPA-based prognostic grouping was the only significant indicator for DMFS.
The intensity of tumour FDG uptake is emerging as a valuable predictive factor of treatment outcome [18–20]. 18F-FDG uptake, measured by SUVmax, is correlated with the density and glucose metabolic rate of tumour cells. Tumours with a high pretreatment SUVmax are therefore likely to be dense and metabolically active, and are likely to have a poor prognosis [18]. Previous studies reported that the SUVmax of primary tumours or regional lymph nodes could predict distant failure in patients with NPC [15, 16], which is in accordance with our results.
Anatomic disease extent reflecting disease burden was the original basis of stage grouping of cancers in the TNM classification [9]. However, more and more non-anatomic prognostic factors are emerging [21, 22]. Even though the UICC and AJCC have recognized that prognostic classifications should extend beyond anatomic parameters alone, a method incorporating non-anatomic prognostic factors that meets the needs of practitioners and researchers has not been reported. Incorporating selected non-anatomic factors into the anatomic classification system while maintaining the consistency and sustainability of the TNM framework is perhaps the biggest challenge.
An RPA-based prognostic grouping incorporating anatomic staging, age, and smoking pack-years for human papilloma virus–related oropharyngeal carcinomas has been recently reported, and this has significantly improved survival prediction [17]. In the present study, we have extended this system by integrating an RPA-based prognostic algorithm with SUV-T and N-classification for predicting distant metastasis. The resultant model identified three distinct risk groups: low risk (N0-1 disease + SUV-T <10.45), medium risk (N0-1 disease + SUV-T >10.45), and high risk (N2-3 disease). This RPA-based prognostic model generated a more balanced distribution and offered superior hazard discrimination compared to N-classification alone, and was confirmed to be the only significant prognostic indicator for DMFS in multivariate analysis.
Despite the promising results, our study has some limitations. Firstly, the proposed model is derived from retrospective analysis of existing data from one institution, and a multi-institution study is needed to confirm our results. Secondly, pretreatment EBV DNA load has been demonstrated to be a valuable prognostic factor in NPC, but this data was only available for a few patients in our cohort and could not be incorporated in our model. Further studies are therefore needed to investigate whether adding EBV DNA data could further improve prediction of metastasis.
In conclusion, analysis of data from a large cohort of NPC patients allowed us to develop an integrated RPA-based prognostic model that incorporates SUV-N and N-classification. Our model performed better at predicting the likelihood of metastasis than previously reported models, and may prove useful for predicting distant metastasis and aiding treatment decisions in the clinic.
PATIENTS AND METHODS:
Patients This study was approved by the institutional review board, and the requirement to obtain informed consent was waived. From January 2010 to February 2012, 449 patients with stage I-IVB NPC treated at our institution received a positron emission tomography-computed tomography (PET-CT) examination before treatment followed by intensity-modulated radiotherapy (IMRT) with or without chemotherapy. All of the enrolled patients were of Chinese ethnicity. The median age was 46 years (range, 20- 77), with a male-to-female ratio of 3:1 (Table 2).
Abbreviations: NPC = nasopharyngeal carcinoma; T = tumour; N = node.
Pathological type according to the 2005 World Health Organization classification of tumours.
According to the 7th edition of the UICC/AJCC staging system.
All patients underwent a pretreatment evaluation that included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, and PET-CT. All patients were staged according to the 7th edition of the International Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) system [9].
This study was approved by the institutional review board, and the requirement to obtain informed consent was waived. From January 2010 to February 2012, 449 patients with stage I-IVB NPC treated at our institution received a positron emission tomography-computed tomography (PET-CT) examination before treatment followed by intensity-modulated radiotherapy (IMRT) with or without chemotherapy. All of the enrolled patients were of Chinese ethnicity. The median age was 46 years (range, 20- 77), with a male-to-female ratio of 3:1 (Table 2).
Abbreviations: NPC = nasopharyngeal carcinoma; T = tumour; N = node.
Pathological type according to the 2005 World Health Organization classification of tumours.
According to the 7th edition of the UICC/AJCC staging system.
All patients underwent a pretreatment evaluation that included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, and PET-CT. All patients were staged according to the 7th edition of the International Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) system [9].
PET/CT imaging Serum glucose levels were measured in all NPC patients, all of whom fasted for at least 6 h before PET/CT scans, and individuals with a fasting plasma glucose >200 mg/dl were excluded. PET/CT imaging was performed with a combination PET/CT scanner (Discovery ST 16; GE Healthcare, Little Chalfont, UK) according to published guidelines [23]. Helical CT was performed from the head to the proximal thigh before PET acquisition, according to a standardized protocol and were 45-60 min after injection of 5.55 MBq/kg FDG. PET images were reconstructed from CT data for attenuation correction using an ordered-subset expectation maximization iterative reconstruction algorithm. SUVmax was determined for each region of interest using the whole-body attenuation corrected image and the following formula: SUVmax = tissue concentration of 18F-FDG / injected dose / body weight.
Serum glucose levels were measured in all NPC patients, all of whom fasted for at least 6 h before PET/CT scans, and individuals with a fasting plasma glucose >200 mg/dl were excluded. PET/CT imaging was performed with a combination PET/CT scanner (Discovery ST 16; GE Healthcare, Little Chalfont, UK) according to published guidelines [23]. Helical CT was performed from the head to the proximal thigh before PET acquisition, according to a standardized protocol and were 45-60 min after injection of 5.55 MBq/kg FDG. PET images were reconstructed from CT data for attenuation correction using an ordered-subset expectation maximization iterative reconstruction algorithm. SUVmax was determined for each region of interest using the whole-body attenuation corrected image and the following formula: SUVmax = tissue concentration of 18F-FDG / injected dose / body weight.
Treatment The nasopharyngeal and neck tumour volumes of all patients were treated using radical radiotherapy based on IMRT for the entire treatment course. Institutional guidelines recommended radiotherapy only for stage I and concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy for stage II-IVB. In total, 92.8% (308/332) of patients with stage III-IVB disease received concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy. When possible, salvage treatments (intracavitary brachytherapy, surgery or chemotherapy) were provided and persistent disease or relapse was documented.
The nasopharyngeal and neck tumour volumes of all patients were treated using radical radiotherapy based on IMRT for the entire treatment course. Institutional guidelines recommended radiotherapy only for stage I and concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy for stage II-IVB. In total, 92.8% (308/332) of patients with stage III-IVB disease received concurrent chemoradiotherapy ± neoadjuvant/adjuvant chemotherapy. When possible, salvage treatments (intracavitary brachytherapy, surgery or chemotherapy) were provided and persistent disease or relapse was documented.
Follow-up Patients were examined at least every three months during the first two years, and every six months during years 3-5 or until death. Evaluation during follow-up included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, chest radiography, abdominal sonography and a whole-body bone scan. Any residual disease found at the nasopharynx or cervical nodes within 6 months after completion of RT was regarded as local failure or regional failure, respectively. All distant metastases were diagnosed by clinical symptoms, physical examination, and imaging methods that included chest radiography, bone scan, MRI, CT, PET-CT, and abdominal sonography [24].
Patients were examined at least every three months during the first two years, and every six months during years 3-5 or until death. Evaluation during follow-up included a complete patient history, physical examination, haematology and biochemistry profiles, MRI of the neck and nasopharynx, chest radiography, abdominal sonography and a whole-body bone scan. Any residual disease found at the nasopharynx or cervical nodes within 6 months after completion of RT was regarded as local failure or regional failure, respectively. All distant metastases were diagnosed by clinical symptoms, physical examination, and imaging methods that included chest radiography, bone scan, MRI, CT, PET-CT, and abdominal sonography [24].
Statistical analysis Statistical analysis was performed using SPSS version 22.0 (IBM Corporation, Armonk, NY, USA), and distant metastasis-free survival (DMFS) was the defined outcome and was calculated from the first day of treatment to the first distant metastasis. The area under the receiver-operating characteristic (ROC) curve was used to select the optimal cut-off point for SUV-T and SUV-N by maximizing the conditional Youden score, based on the method described by Hanley [25] and Zweig [26]. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test [27]. Multivariate analysis based on the Cox proportional hazards model was used to calculate HRs and 95% confidence intervals (CIs), and to test the independent significance of different factors by backward elimination of insignificant variables [28] including host factors (sex, age), tumour factors (T-classification, N-classification), and therapeutic intervention (chemotherapy) as covariates.
Finally, we performed RPA for DMFS to derive prognostic groups that combined anatomic category with other survival predictors identified from multivariate analysis. The RPA algorithm is based on the optimized binary partition of predictors. The resultant subgroups were similar in terms of survival. All tests were two-sided, and P <0.05 was considered statistically significant.
Statistical analysis was performed using SPSS version 22.0 (IBM Corporation, Armonk, NY, USA), and distant metastasis-free survival (DMFS) was the defined outcome and was calculated from the first day of treatment to the first distant metastasis. The area under the receiver-operating characteristic (ROC) curve was used to select the optimal cut-off point for SUV-T and SUV-N by maximizing the conditional Youden score, based on the method described by Hanley [25] and Zweig [26]. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test [27]. Multivariate analysis based on the Cox proportional hazards model was used to calculate HRs and 95% confidence intervals (CIs), and to test the independent significance of different factors by backward elimination of insignificant variables [28] including host factors (sex, age), tumour factors (T-classification, N-classification), and therapeutic intervention (chemotherapy) as covariates.
Finally, we performed RPA for DMFS to derive prognostic groups that combined anatomic category with other survival predictors identified from multivariate analysis. The RPA algorithm is based on the optimized binary partition of predictors. The resultant subgroups were similar in terms of survival. All tests were two-sided, and P <0.05 was considered statistically significant.
|
Background:
Previous studies reported a correlation between the maximum standardised uptake value (SUVmax) obtained by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) and distant metastasis in nasopharyngeal carcinoma (NPC). However, an integrated model incorporating SUVmax and anatomic staging for stratifying metastasis risk has not been reported.
Methods:
Data from 449 patients with with histologically-confirmed, stage I-IVB NPC treated with radiotherapy or chemoradiotherapy were retrospectively analysed. A prognostic model for distant metastasis-free survival (DMFS) was derived by recursive partitioning analysis (RPA) combining independent predictors identified by multivariate analysis.
Results:
The median SUVmax for primary tumour (SUV-T) and cervical lymph nodes (SUV-N) was 13.6 (range, 2.2 to 39.3) and 8.4 (range, 2.6 to 40.9), respectively. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005), SUV-N (HR, 2.688; 95%CI, 1.250-5.781; P = 0.011) and N-classification (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001) were identified as independent predictors for DMFS from multivariate analysis. Three valid risk groups were derived by RPA: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). The three risk groups contained 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Moreover, multivariate analysis confirmed the RPA-based prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95%CI, 1.975-4.835; P <0.001).
Conclusions:
SUV-T, SUV-N and N-classification were identified as independent predictors for DMFS. An integrated RPA-based prognostic model for DMFS incorporating SUV-N and N-classification was proposed.
|
INTRODUCTION:
Nasopharyngeal carcinoma (NPC) is particularly prevalent in southern China, Southeast Asia, North Africa, the Middle East, and Alaska [1]. Radiotherapy is the primary treatment used for non-disseminated NPC [2, 3]. With advances in imaging and radiation therapies, local-regional control has exceeded 90% [4]. However, 20-30% of NPC patients eventually develop distant metastasis [5–8], which accounts for the majority of failures [7, 8]. Effort should therefore be made to stratify patients into different groups based on the risk of metastasis to tailor individualized treatments and improve outcomes.
N-classification in the TNM staging system is a measure of the extent of node involvement, and is currently the most reliable tool for assessing metastasis risk in NPC [9, 10]. However, there is remaining room for improvement in the correlation between the N classification and metastasis [11, 12], perhaps because N-classification is based solely on anatomic extent and lacks non-anatomic information such as tumour physiology.
18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) imaging is used to probe glucose metabolism in tumour cells [13]. The maximal intensity of FDG uptake by the tumour (maximum standardized uptake value; SUVmax) is a valuable marker of tumour biological behaviour [13, 14] and a useful predictor of distant metastasis in NPC [15, 16]. However, an integrated model incorporating SUVmax and anatomic staging for stratifying metastasis risk has not been reported. Clinicians are therefore somewhat troubled as to how best to incorporate SUVmax into clinical decision-making. A valid approach for incorporating non-anatomic prognostic factors and anatomic staging into an integrated prognosis grouping was recently described [17], which significantly improved survival prediction compared with previous models. In the present study, we extended this approach by using recursive partitioning analysis (RPA) to develop an integrated prognostic model for metastasis that combines SUV parameters and N-classification.
DISCUSSION:
Statistical analysis was performed using SPSS version 22.0 (IBM Corporation, Armonk, NY, USA), and distant metastasis-free survival (DMFS) was the defined outcome and was calculated from the first day of treatment to the first distant metastasis. The area under the receiver-operating characteristic (ROC) curve was used to select the optimal cut-off point for SUV-T and SUV-N by maximizing the conditional Youden score, based on the method described by Hanley [25] and Zweig [26]. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test [27]. Multivariate analysis based on the Cox proportional hazards model was used to calculate HRs and 95% confidence intervals (CIs), and to test the independent significance of different factors by backward elimination of insignificant variables [28] including host factors (sex, age), tumour factors (T-classification, N-classification), and therapeutic intervention (chemotherapy) as covariates.
Finally, we performed RPA for DMFS to derive prognostic groups that combined anatomic category with other survival predictors identified from multivariate analysis. The RPA algorithm is based on the optimized binary partition of predictors. The resultant subgroups were similar in terms of survival. All tests were two-sided, and P <0.05 was considered statistically significant.
|
Background:
Previous studies reported a correlation between the maximum standardised uptake value (SUVmax) obtained by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) and distant metastasis in nasopharyngeal carcinoma (NPC). However, an integrated model incorporating SUVmax and anatomic staging for stratifying metastasis risk has not been reported.
Methods:
Data from 449 patients with with histologically-confirmed, stage I-IVB NPC treated with radiotherapy or chemoradiotherapy were retrospectively analysed. A prognostic model for distant metastasis-free survival (DMFS) was derived by recursive partitioning analysis (RPA) combining independent predictors identified by multivariate analysis.
Results:
The median SUVmax for primary tumour (SUV-T) and cervical lymph nodes (SUV-N) was 13.6 (range, 2.2 to 39.3) and 8.4 (range, 2.6 to 40.9), respectively. SUV-T (HR, 3.396; 95% CI, 1.451-7.947; P = 0.005), SUV-N (HR, 2.688; 95%CI, 1.250-5.781; P = 0.011) and N-classification (HR, 2.570; 95%CI, 1.422-4.579; P = 0.001) were identified as independent predictors for DMFS from multivariate analysis. Three valid risk groups were derived by RPA: low risk (N0-1 + SUV-T <10.45), medium risk (N0-1 + SUV-T >10.45) and high risk (N2-3). The three risk groups contained 100 (22.3%), 226 (50.3%), and 123 (27.4%) patients, respectively, with corresponding 3-year DMFS rates of 99.0%, 91.5%, and 77.5% (P <0.001). Moreover, multivariate analysis confirmed the RPA-based prognostic grouping as the only significant prognostic indicator for DMFS (HR, 3.090; 95%CI, 1.975-4.835; P <0.001).
Conclusions:
SUV-T, SUV-N and N-classification were identified as independent predictors for DMFS. An integrated RPA-based prognostic model for DMFS incorporating SUV-N and N-classification was proposed.
| 5,711 | 406 | 8 |
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[CONTENT] nasopharyngeal neoplasms | metastasis | TNM staging | maximum standardized uptake value | recursive partitioning analysis [SUMMARY]
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[CONTENT] nasopharyngeal neoplasms | metastasis | TNM staging | maximum standardized uptake value | recursive partitioning analysis [SUMMARY]
| null |
[CONTENT] nasopharyngeal neoplasms | metastasis | TNM staging | maximum standardized uptake value | recursive partitioning analysis [SUMMARY]
|
[CONTENT] nasopharyngeal neoplasms | metastasis | TNM staging | maximum standardized uptake value | recursive partitioning analysis [SUMMARY]
|
[CONTENT] nasopharyngeal neoplasms | metastasis | TNM staging | maximum standardized uptake value | recursive partitioning analysis [SUMMARY]
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[CONTENT] Adult | Aged | Carcinoma | Carcinoma, Squamous Cell | Combined Modality Therapy | Female | Fluorodeoxyglucose F18 | Follow-Up Studies | Humans | Lymphatic Metastasis | Male | Middle Aged | Models, Theoretical | Multimodal Imaging | Nasopharyngeal Carcinoma | Nasopharyngeal Neoplasms | Neoplasm Invasiveness | Neoplasm Staging | Positron-Emission Tomography | Prognosis | Radiopharmaceuticals | Retrospective Studies | Survival Rate | Tomography, X-Ray Computed | Young Adult [SUMMARY]
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[CONTENT] Adult | Aged | Carcinoma | Carcinoma, Squamous Cell | Combined Modality Therapy | Female | Fluorodeoxyglucose F18 | Follow-Up Studies | Humans | Lymphatic Metastasis | Male | Middle Aged | Models, Theoretical | Multimodal Imaging | Nasopharyngeal Carcinoma | Nasopharyngeal Neoplasms | Neoplasm Invasiveness | Neoplasm Staging | Positron-Emission Tomography | Prognosis | Radiopharmaceuticals | Retrospective Studies | Survival Rate | Tomography, X-Ray Computed | Young Adult [SUMMARY]
| null |
[CONTENT] Adult | Aged | Carcinoma | Carcinoma, Squamous Cell | Combined Modality Therapy | Female | Fluorodeoxyglucose F18 | Follow-Up Studies | Humans | Lymphatic Metastasis | Male | Middle Aged | Models, Theoretical | Multimodal Imaging | Nasopharyngeal Carcinoma | Nasopharyngeal Neoplasms | Neoplasm Invasiveness | Neoplasm Staging | Positron-Emission Tomography | Prognosis | Radiopharmaceuticals | Retrospective Studies | Survival Rate | Tomography, X-Ray Computed | Young Adult [SUMMARY]
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[CONTENT] Adult | Aged | Carcinoma | Carcinoma, Squamous Cell | Combined Modality Therapy | Female | Fluorodeoxyglucose F18 | Follow-Up Studies | Humans | Lymphatic Metastasis | Male | Middle Aged | Models, Theoretical | Multimodal Imaging | Nasopharyngeal Carcinoma | Nasopharyngeal Neoplasms | Neoplasm Invasiveness | Neoplasm Staging | Positron-Emission Tomography | Prognosis | Radiopharmaceuticals | Retrospective Studies | Survival Rate | Tomography, X-Ray Computed | Young Adult [SUMMARY]
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[CONTENT] Adult | Aged | Carcinoma | Carcinoma, Squamous Cell | Combined Modality Therapy | Female | Fluorodeoxyglucose F18 | Follow-Up Studies | Humans | Lymphatic Metastasis | Male | Middle Aged | Models, Theoretical | Multimodal Imaging | Nasopharyngeal Carcinoma | Nasopharyngeal Neoplasms | Neoplasm Invasiveness | Neoplasm Staging | Positron-Emission Tomography | Prognosis | Radiopharmaceuticals | Retrospective Studies | Survival Rate | Tomography, X-Ray Computed | Young Adult [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] suv | patients | prognostic | survival | distant | metastasis | dmfs | classification | distant metastasis | analysis [SUMMARY]
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[CONTENT] suv | patients | prognostic | survival | distant | metastasis | dmfs | classification | distant metastasis | analysis [SUMMARY]
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[CONTENT] suv | patients | prognostic | survival | distant | metastasis | dmfs | classification | distant metastasis | analysis [SUMMARY]
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[CONTENT] suv | patients | prognostic | survival | distant | metastasis | dmfs | classification | distant metastasis | analysis [SUMMARY]
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[CONTENT] suv | patients | prognostic | survival | distant | metastasis | dmfs | classification | distant metastasis | analysis [SUMMARY]
|
[CONTENT] anatomic | metastasis | npc | non | metastasis risk | approach | classification | tumour | integrated | suvmax [SUMMARY]
|
[CONTENT] ct | pet | pet ct | examination | according | chemotherapy | patients | stage | physical examination | physical [SUMMARY]
| null |
[CONTENT] prognostic | anatomic | based prognostic | rpa based prognostic | rpa based | model | based | rpa | reported | data [SUMMARY]
|
[CONTENT] suv | prognostic | dmfs | patients | metastasis | distant | survival | classification | risk | 47 [SUMMARY]
|
[CONTENT] suv | prognostic | dmfs | patients | metastasis | distant | survival | classification | risk | 47 [SUMMARY]
|
[CONTENT] PET | NPC ||| SUVmax [SUMMARY]
|
[CONTENT] 449 | I-IVB NPC ||| DMFS | RPA [SUMMARY]
| null |
[CONTENT] DMFS ||| RPA | DMFS [SUMMARY]
|
[CONTENT] PET | NPC ||| SUVmax ||| 449 | I-IVB NPC ||| DMFS | RPA ||| 13.6 | 2.2 | 39.3 | 8.4 | 2.6 | 40.9 ||| 3.396 | 95% | CI | 1.451-7.947 | 0.005 | 2.688 | 1.250 ||| 0.011 | 2.570 | 95%CI | 1.422 | P = | 0.001 | DMFS ||| Three | RPA | 10.45 | N2-3 ||| three | 100 | 22.3% | 226 | 50.3% | 123 | 27.4% | 3-year | DMFS | 99.0% | 91.5% | 77.5% ||| RPA | DMFS | 3.090 | 95%CI | 1.975 | P <0.001 ||| DMFS ||| RPA | DMFS [SUMMARY]
|
[CONTENT] PET | NPC ||| SUVmax ||| 449 | I-IVB NPC ||| DMFS | RPA ||| 13.6 | 2.2 | 39.3 | 8.4 | 2.6 | 40.9 ||| 3.396 | 95% | CI | 1.451-7.947 | 0.005 | 2.688 | 1.250 ||| 0.011 | 2.570 | 95%CI | 1.422 | P = | 0.001 | DMFS ||| Three | RPA | 10.45 | N2-3 ||| three | 100 | 22.3% | 226 | 50.3% | 123 | 27.4% | 3-year | DMFS | 99.0% | 91.5% | 77.5% ||| RPA | DMFS | 3.090 | 95%CI | 1.975 | P <0.001 ||| DMFS ||| RPA | DMFS [SUMMARY]
|
|||||
Clinical-pathological characteristics and prognosis of a cohort of oesophageal cancer patients: a competing risks survival analysis.
|
25716135
|
To determine the clinical course, follow-up strategies, and survival of oesophageal cancer patients using a competing risks survival analysis.
|
BACKGROUND
|
We conducted a retrospective and prospective follow-up study. The study included 180 patients with a pathological diagnosis of oesophageal cancer in A Coruña, Spain, between 2003 and 2008. The Kaplan-Meier methodology and competing risks survival analysis were used to calculate the specific survival rate. The study was approved by the Ethics Review Board (code 2011/372, CEIC Galicia).
|
METHODS
|
The specific survival rate at the first, third, and fifth years was 40.2%, 18.1%, and 12.4%, respectively. Using the Kaplan-Meier methodology, the survival rate was slightly higher after the third year of follow-up. In the multivariate analysis, poor prognosis factors were female sex (hazard ratio [HR] 1.94; 95% confidence interval [CI], 1.24-3.03), Charlson's comorbidity index (HR 1.17; 95% CI, 1.02-1.33), and stage IV tumours (HR 1.70; 95% CI, 1.11-2.59). The probability of dying decreased with surgical and oncological treatment (chemotherapy and/or radiotherapy) (HR 0.23; 95% CI, 0.12-0.45). The number of hospital consultations per year during the follow-up period, from diagnosis to the appearance of a new event (local recurrences, newly appeared metastasis, and newly appeared neoplasias) did not affect the probability of survival (HR 1.03; 95% CI, 0.92-1.15).
|
RESULTS
|
The Kaplan-Meier methodology overestimates the survival rate in comparison to competing risks analysis. The variables associated with a poor prognosis are female sex, Charlson's comorbidity score and extensive tumour invasion. Type of follow-up strategy employed after diagnosis does not affect the prognosis of the disease.
|
CONCLUSIONS
|
[
"Aged",
"Esophageal Neoplasms",
"Female",
"Follow-Up Studies",
"Humans",
"Kaplan-Meier Estimate",
"Male",
"Middle Aged",
"Prognosis",
"Prospective Studies",
"Retrospective Studies",
"Risk Factors",
"Sex Distribution",
"Spain",
"Survival Rate"
] |
4341000
|
INTRODUCTION
|
Oesophageal cancer is the eighth-most common cancer worldwide.1 In the 27-member European Union, a total of 33 013 new cases were estimated in 2008 (age-standardised rate for world population [ASR{W}]: 3.5 per 100 000).1 In relation to the rest of Europe, Spain is at a midway point in terms of occurrence (ASR[W]: 2.8 per 100 000). Within Spain, this cancer occurs at a higher rate in the country’s northern regions than in southern ones.2
At the worldwide level, oesophageal cancer is the sixth-most frequent cause of death by cancer. In 2008, the global age-standardised rate of cancer-related mortality was 2.9 per 100 000 inhabitants in the European Union and 2.3 per 100 000 in Spain. The mortality rate, like the occurrence rate, is higher in Spain’s northern regions.3
According to the European Cancer Registry EUROCARE-4 study,4 the average European survival rate is close to 35% at one year and close to 10% at 5 years, and survival is strongly associated with the clinical stage of the tumour and the treatment the patient receives.5–7 There is little consensus when it comes to defining the follow-up protocols for these patients.8 Furthermore, to the best of our knowledge, there are no observational or experimental studies that have investigated the role of the different follow-up strategies on these patients’ prognosis.
With regard to analysing the survival rate, competing risk models are the most suitable for analysing the behaviour of subjects who may die for different reasons. However, when applied in the presence of competitive risks, the usual techniques for analysing the time until the event, such as the Kaplan-Meier methodology, produce biased results.9,10 By using specific techniques, it is possible to reduce bias so that results can be correctly interpreted.
Therefore, the geographic variability in the occurrence and mortality rates, the existence of different risk factors associated with the low survival rate for these kinds of tumours, and the lack of consensus with regard to optimum follow-up strategies justify the undertaking of this study. The aim of the study was to identify the epidemiology of oesophageal cancer in the area of A Coruña, Spain, the supportive process applied to these patients, and their prognosis, using the competing risks methodology.
|
METHODS
|
A total of 234 patients were included in the study, with anatomical-pathological confirmation of cancer of the oesophagus diagnosed at the University Hospital Complex in A Coruña, Spain between the 1st of January, 2003, and the 31st of December, 2008. A retrospective review of the patients’ clinical records was carried out together with a prospective follow-up until the 31st of January, 2012, in order to guarantee a minimum follow-up period of 3 years. The study excluded prevalent or recurring cases, subjects with multiple or metastatic cancers, or those that had been treated and/or diagnosed at other hospitals. After exclusions, the final study sample comprised 180 patients.
Measurements We collected information on the socio-demographic variables of the patient, their personal backgrounds, comorbidity variables using Charlson’s comorbidity index, the symptoms present at diagnosis, location of the tumour, histopathologic cell type, and tumour stage (TNM, seventh edition).11,12 As data on risk factors and symptoms were collected retrospectively from electronic hospital clinical records, we registered data that were indicated in the clinical records but could not quantify the frequency and amount of cigarette or alcohol consumption, nor the number of kilograms lost during the previous months before the diagnosis.
We also collected data on the treatment received (surgery, chemotherapy, and radiotherapy), as well as the visits and tests carried out during the follow-up period: consultations and hospital stays, endoscopies, computer-aided tomography scans, and thorax X-rays. Also, the presence of follow-up events, including newly appeared local recurrences, metastasis, and neoplasias, was studied. For all dead patients, cause of death was obtained from the Galician Mortality Registry (General Directory of Public Health, Xunta de Galicia, Spain), according to the 21 diagnostic categories of the 10th revision of the International Classification of Diseases.
We collected information on the socio-demographic variables of the patient, their personal backgrounds, comorbidity variables using Charlson’s comorbidity index, the symptoms present at diagnosis, location of the tumour, histopathologic cell type, and tumour stage (TNM, seventh edition).11,12 As data on risk factors and symptoms were collected retrospectively from electronic hospital clinical records, we registered data that were indicated in the clinical records but could not quantify the frequency and amount of cigarette or alcohol consumption, nor the number of kilograms lost during the previous months before the diagnosis.
We also collected data on the treatment received (surgery, chemotherapy, and radiotherapy), as well as the visits and tests carried out during the follow-up period: consultations and hospital stays, endoscopies, computer-aided tomography scans, and thorax X-rays. Also, the presence of follow-up events, including newly appeared local recurrences, metastasis, and neoplasias, was studied. For all dead patients, cause of death was obtained from the Galician Mortality Registry (General Directory of Public Health, Xunta de Galicia, Spain), according to the 21 diagnostic categories of the 10th revision of the International Classification of Diseases.
Sample size justification The study included a total of 180 patients. This sample size makes it possible to detect as significant a hazard ratio of 1.6 or more, with a prevalence of exposure of 50% and a censored data percentage of 20% (security: 95%; statistical power: 80%).
The study included a total of 180 patients. This sample size makes it possible to detect as significant a hazard ratio of 1.6 or more, with a prevalence of exposure of 50% and a censored data percentage of 20% (security: 95%; statistical power: 80%).
Statistical analysis A descriptive study was made of the variables that were obtained. The specific survival rate was calculated using the Kaplan-Meier methodology and competing risks survival analysis. The accumulated occurrence of dying as a result of oesophageal cancer during the follow-up period was estimated, considering death as a result of other causes as a competitive event, using the method proposed by Kalbfleisch and Prentice.13 The accumulated occurrence of death due to oesophageal cancer according to different characteristics was compared using the test proposed by Gray.14 Finally, in order to identify which characteristics were associated with the risk of dying as a result of oesophageal cancer, a multivariate analysis was carried out using the model proposed by Fine and Gray.15 All of the tests were carried out bilaterally, considering values of P < 0.05 as significant. The analyses were carried out using the programmes Epidat 3.1 (Xunta de Galicia, Santiago de Compostela, Spain), SPSS 19.0 (IBM Company, Chicago, IL, USA), and R 2.15.1 (Free Software Foundation, Boston, MA, USA).
A descriptive study was made of the variables that were obtained. The specific survival rate was calculated using the Kaplan-Meier methodology and competing risks survival analysis. The accumulated occurrence of dying as a result of oesophageal cancer during the follow-up period was estimated, considering death as a result of other causes as a competitive event, using the method proposed by Kalbfleisch and Prentice.13 The accumulated occurrence of death due to oesophageal cancer according to different characteristics was compared using the test proposed by Gray.14 Finally, in order to identify which characteristics were associated with the risk of dying as a result of oesophageal cancer, a multivariate analysis was carried out using the model proposed by Fine and Gray.15 All of the tests were carried out bilaterally, considering values of P < 0.05 as significant. The analyses were carried out using the programmes Epidat 3.1 (Xunta de Galicia, Santiago de Compostela, Spain), SPSS 19.0 (IBM Company, Chicago, IL, USA), and R 2.15.1 (Free Software Foundation, Boston, MA, USA).
Ethics The study was carried out according to the principles laid down in the Declaration of Helsinki and ensuring compliance with Spanish Decree 29/2009, which regulates the use of and access to electronic medical records. Confidentiality was maintained in accordance with the current Spanish Data Protection Law (15/1999). The study received written approval from the regional Ethics Committee for Clinical Research (code 2011/372 CEIC Galicia).
The study was carried out according to the principles laid down in the Declaration of Helsinki and ensuring compliance with Spanish Decree 29/2009, which regulates the use of and access to electronic medical records. Confidentiality was maintained in accordance with the current Spanish Data Protection Law (15/1999). The study received written approval from the regional Ethics Committee for Clinical Research (code 2011/372 CEIC Galicia).
|
RESULTS
|
Characteristics of the patients studied Table 1 shows the baseline characteristics, comorbidities, and cause of death of the patients. The median age was 64.5 years, 87.8% of the sample subjects were male, 58.6% had a body mass index (BMI) within normal range, and 11.4% were obese. The most frequent symptom reported was dysphagia (82.0%), followed by weight loss (49.4%). Regarding tumour stage, 46.8% of the tumours were moderately differentiated, 38.0% were poorly differentiated, and 28.9% had metastasis at the time of diagnosis (Table 2).
BMI, body mass index; CI, confidence interval; SD, standard deviation.
CI, confidence interval.
Table 1 shows the baseline characteristics, comorbidities, and cause of death of the patients. The median age was 64.5 years, 87.8% of the sample subjects were male, 58.6% had a body mass index (BMI) within normal range, and 11.4% were obese. The most frequent symptom reported was dysphagia (82.0%), followed by weight loss (49.4%). Regarding tumour stage, 46.8% of the tumours were moderately differentiated, 38.0% were poorly differentiated, and 28.9% had metastasis at the time of diagnosis (Table 2).
BMI, body mass index; CI, confidence interval; SD, standard deviation.
CI, confidence interval.
Treatment Treatments involved chemotherapy and/or radiotherapy exclusively in 36.1%, surgery as the sole treatment in 23.3%, and a combination of both in 19.4% (Table 2). In the case of patients who only received surgery, resection was carried out for curative purposes in 74%. Both chemotherapy and radiotherapy were mainly applied for palliative purposes.
Treatments involved chemotherapy and/or radiotherapy exclusively in 36.1%, surgery as the sole treatment in 23.3%, and a combination of both in 19.4% (Table 2). In the case of patients who only received surgery, resection was carried out for curative purposes in 74%. Both chemotherapy and radiotherapy were mainly applied for palliative purposes.
Prognosis The specific survival rate at 1, 3, and 5 years after diagnosis obtained with the Kaplan-Meier methodology was 39.9%, 19%, and 15%, respectively, while respective survival rates according to competing risks survival analysis were 40.2%, 18.1%, and 12.4% (Table 3).
CI, confidence interval; HR, hazard ratio; SD, standard deviation.
At 1 year from diagnosis, the probability of dying as a result of the cancer was 59.2%, with the probability of dying from other causes being 0.6% (Figure). At 5 years from diagnosis, the probability of dying as a result of the cancer rose to 83.4% and the probability of dying from other causes was 4.2%; therefore, the probability of survival was reduced to 12.4%.
The variables in the univariate analysis that were significantly associated with the probability of dying during the follow-up period were: gender, Charlson’s comorbidity index, presence of weight loss, histopathological cell type, tumour stage, and type of treatment (Table 3). The specific probability of dying was increased among females (hazard ratio [HR] 1.63; 95% CI, 1.00–2.64), those with a higher score on age-adjusted Charlson’s comorbidity index (HR 1.14; 95% CI, 1.05–1.23), and among those with weight loss on diagnosis (HR 1.68; 95% CI, 1.20–2.34). The histopathologic cell type with the highest mortality rate was adenocarcinoma (HR 1.68; 95% CI, 1.08–2.62). In turn, those with stage IV tumours had a higher mortality rate than those in earlier stages (0-III) (HR 2.38; 95% CI, 1.63–3.46).
The patients who had received some kind of treatment had a lower probability of dying. Those who had received a combination of surgical and oncological treatment (chemotherapy and/or radiotherapy) had the lowest probability of dying (HR 0.17; 95% CI, 0.10–0.27), followed by those who had only received surgical treatment (HR 0.22; 95% CI, 0.12–0.38).
No association was found between the probability of dying during the follow-up period and the following variables: year of diagnosis, age, BMI, personal background (smoking, regular alcohol consumption, gastro-oesophageal reflux, achalasia, and a family history of cancer), presence of dysphagia on diagnosis, or tumour location.
According to the final adjustment by multivariate competing risks analysis, we found that the variables with an independent effect to predict mortality are gender, Charlson’s comorbidity index, and tumour stage (Table 4). In order to adjust for age as a clinically relevant and confounding variable and avoid over-adjustment, we included in the model the crude Charlson’s comorbidity index. The specific probability of dying from oesophageal cancer was increased among females (HR 1.94; 95% CI, 1.24–3.03), those with a higher score on Charlson’s comorbidity index (HR 1.17; 95% CI, 1.02–1.33), and in those with stage IV tumours at the time of diagnosis (HR 1.70; 95% CI, 1.11–2.59). Having received some type of treatment improved the prognosis, with a greater impact in cases that received a combination of surgical and oncological treatment (HR 0.23; 95% CI, 0.12–0.45). Furthermore, no significant effect was found when the interaction between TNM classification and type of treatment was added to the multivariate model (P = 0.600).
AC, adenocarcinoma; B, regression coefficient; CI, confidence interval; HR, hazard ratio; SCC, squamous cell carcinoma; SE, standard error.
The specific survival rate at 1, 3, and 5 years after diagnosis obtained with the Kaplan-Meier methodology was 39.9%, 19%, and 15%, respectively, while respective survival rates according to competing risks survival analysis were 40.2%, 18.1%, and 12.4% (Table 3).
CI, confidence interval; HR, hazard ratio; SD, standard deviation.
At 1 year from diagnosis, the probability of dying as a result of the cancer was 59.2%, with the probability of dying from other causes being 0.6% (Figure). At 5 years from diagnosis, the probability of dying as a result of the cancer rose to 83.4% and the probability of dying from other causes was 4.2%; therefore, the probability of survival was reduced to 12.4%.
The variables in the univariate analysis that were significantly associated with the probability of dying during the follow-up period were: gender, Charlson’s comorbidity index, presence of weight loss, histopathological cell type, tumour stage, and type of treatment (Table 3). The specific probability of dying was increased among females (hazard ratio [HR] 1.63; 95% CI, 1.00–2.64), those with a higher score on age-adjusted Charlson’s comorbidity index (HR 1.14; 95% CI, 1.05–1.23), and among those with weight loss on diagnosis (HR 1.68; 95% CI, 1.20–2.34). The histopathologic cell type with the highest mortality rate was adenocarcinoma (HR 1.68; 95% CI, 1.08–2.62). In turn, those with stage IV tumours had a higher mortality rate than those in earlier stages (0-III) (HR 2.38; 95% CI, 1.63–3.46).
The patients who had received some kind of treatment had a lower probability of dying. Those who had received a combination of surgical and oncological treatment (chemotherapy and/or radiotherapy) had the lowest probability of dying (HR 0.17; 95% CI, 0.10–0.27), followed by those who had only received surgical treatment (HR 0.22; 95% CI, 0.12–0.38).
No association was found between the probability of dying during the follow-up period and the following variables: year of diagnosis, age, BMI, personal background (smoking, regular alcohol consumption, gastro-oesophageal reflux, achalasia, and a family history of cancer), presence of dysphagia on diagnosis, or tumour location.
According to the final adjustment by multivariate competing risks analysis, we found that the variables with an independent effect to predict mortality are gender, Charlson’s comorbidity index, and tumour stage (Table 4). In order to adjust for age as a clinically relevant and confounding variable and avoid over-adjustment, we included in the model the crude Charlson’s comorbidity index. The specific probability of dying from oesophageal cancer was increased among females (HR 1.94; 95% CI, 1.24–3.03), those with a higher score on Charlson’s comorbidity index (HR 1.17; 95% CI, 1.02–1.33), and in those with stage IV tumours at the time of diagnosis (HR 1.70; 95% CI, 1.11–2.59). Having received some type of treatment improved the prognosis, with a greater impact in cases that received a combination of surgical and oncological treatment (HR 0.23; 95% CI, 0.12–0.45). Furthermore, no significant effect was found when the interaction between TNM classification and type of treatment was added to the multivariate model (P = 0.600).
AC, adenocarcinoma; B, regression coefficient; CI, confidence interval; HR, hazard ratio; SCC, squamous cell carcinoma; SE, standard error.
Follow-up The total length of follow-up was 3221.8 months (268.5 years), with a mean of 9.4 months per patient. The most frequently detected events during follow-up were newly appeared metastasis (19.0%)—mainly located in the lungs (48.5%)—and local tumour recurrences (16.7%) (Table 5).
In those patients who underwent curative surgery, the probability of being diagnosed with a tumour recurrence (local tumour recurrence, distant recurrence, or both) during the first year after the surgery was 21.1%, while 25.5% of the patients died without presenting any recurrence. Therefore, 53.4% of the patients who underwent curative surgery were alive and remained disease-free 1 year after the surgery. Similarly, the probability of tumour recurrence 5 years after the surgery was 52.4%, while 29.6% of the patients died without presenting any recurrence. Therefore, only 18% of the patients were alive and disease-free 5 years after curative surgery.
Considering that the follow-up strategy is more intensive once an event occurs during the follow-up period, we studied the follow-up procedure carried out with patients who underwent curative surgery from the time of diagnosis until a new event occurred (local tumour recurrence, newly appeared metastasis, or newly appeared neoplasias) (Table 6). The mean number of hospital consultations per year of follow-up for these patients was 4.1 (standard deviation, 4.2) consultations/year, with a median of 3.1 consultations per year of follow-up (Table 6).
CAT, computer-aided tomography; IQR, inter-quartile range; SD, standard deviation.
When we take into account gender, age, Charlson’s comorbidity index, weight loss, histopathological cell type, TNM stage, treatment carried out, and number of consultations per follow-up year, in the interval between the diagnosis until an event occurs, we see that the consultations carried out during this period do not substantially alter the likelihood of survival (P = 0.640; HR 1.03; 95% CI, 0.92–1.15).
The total length of follow-up was 3221.8 months (268.5 years), with a mean of 9.4 months per patient. The most frequently detected events during follow-up were newly appeared metastasis (19.0%)—mainly located in the lungs (48.5%)—and local tumour recurrences (16.7%) (Table 5).
In those patients who underwent curative surgery, the probability of being diagnosed with a tumour recurrence (local tumour recurrence, distant recurrence, or both) during the first year after the surgery was 21.1%, while 25.5% of the patients died without presenting any recurrence. Therefore, 53.4% of the patients who underwent curative surgery were alive and remained disease-free 1 year after the surgery. Similarly, the probability of tumour recurrence 5 years after the surgery was 52.4%, while 29.6% of the patients died without presenting any recurrence. Therefore, only 18% of the patients were alive and disease-free 5 years after curative surgery.
Considering that the follow-up strategy is more intensive once an event occurs during the follow-up period, we studied the follow-up procedure carried out with patients who underwent curative surgery from the time of diagnosis until a new event occurred (local tumour recurrence, newly appeared metastasis, or newly appeared neoplasias) (Table 6). The mean number of hospital consultations per year of follow-up for these patients was 4.1 (standard deviation, 4.2) consultations/year, with a median of 3.1 consultations per year of follow-up (Table 6).
CAT, computer-aided tomography; IQR, inter-quartile range; SD, standard deviation.
When we take into account gender, age, Charlson’s comorbidity index, weight loss, histopathological cell type, TNM stage, treatment carried out, and number of consultations per follow-up year, in the interval between the diagnosis until an event occurs, we see that the consultations carried out during this period do not substantially alter the likelihood of survival (P = 0.640; HR 1.03; 95% CI, 0.92–1.15).
| null | null |
[
"Measurements",
"Sample size justification",
"Statistical analysis",
"Ethics",
"Characteristics of the patients studied",
"Treatment",
"Prognosis",
"Follow-up"
] |
[
"We collected information on the socio-demographic variables of the patient, their personal backgrounds, comorbidity variables using Charlson’s comorbidity index, the symptoms present at diagnosis, location of the tumour, histopathologic cell type, and tumour stage (TNM, seventh edition).11,12 As data on risk factors and symptoms were collected retrospectively from electronic hospital clinical records, we registered data that were indicated in the clinical records but could not quantify the frequency and amount of cigarette or alcohol consumption, nor the number of kilograms lost during the previous months before the diagnosis.\nWe also collected data on the treatment received (surgery, chemotherapy, and radiotherapy), as well as the visits and tests carried out during the follow-up period: consultations and hospital stays, endoscopies, computer-aided tomography scans, and thorax X-rays. Also, the presence of follow-up events, including newly appeared local recurrences, metastasis, and neoplasias, was studied. For all dead patients, cause of death was obtained from the Galician Mortality Registry (General Directory of Public Health, Xunta de Galicia, Spain), according to the 21 diagnostic categories of the 10th revision of the International Classification of Diseases.",
"The study included a total of 180 patients. This sample size makes it possible to detect as significant a hazard ratio of 1.6 or more, with a prevalence of exposure of 50% and a censored data percentage of 20% (security: 95%; statistical power: 80%).",
"A descriptive study was made of the variables that were obtained. The specific survival rate was calculated using the Kaplan-Meier methodology and competing risks survival analysis. The accumulated occurrence of dying as a result of oesophageal cancer during the follow-up period was estimated, considering death as a result of other causes as a competitive event, using the method proposed by Kalbfleisch and Prentice.13 The accumulated occurrence of death due to oesophageal cancer according to different characteristics was compared using the test proposed by Gray.14 Finally, in order to identify which characteristics were associated with the risk of dying as a result of oesophageal cancer, a multivariate analysis was carried out using the model proposed by Fine and Gray.15 All of the tests were carried out bilaterally, considering values of P < 0.05 as significant. The analyses were carried out using the programmes Epidat 3.1 (Xunta de Galicia, Santiago de Compostela, Spain), SPSS 19.0 (IBM Company, Chicago, IL, USA), and R 2.15.1 (Free Software Foundation, Boston, MA, USA).",
"The study was carried out according to the principles laid down in the Declaration of Helsinki and ensuring compliance with Spanish Decree 29/2009, which regulates the use of and access to electronic medical records. Confidentiality was maintained in accordance with the current Spanish Data Protection Law (15/1999). The study received written approval from the regional Ethics Committee for Clinical Research (code 2011/372 CEIC Galicia).",
"Table 1 shows the baseline characteristics, comorbidities, and cause of death of the patients. The median age was 64.5 years, 87.8% of the sample subjects were male, 58.6% had a body mass index (BMI) within normal range, and 11.4% were obese. The most frequent symptom reported was dysphagia (82.0%), followed by weight loss (49.4%). Regarding tumour stage, 46.8% of the tumours were moderately differentiated, 38.0% were poorly differentiated, and 28.9% had metastasis at the time of diagnosis (Table 2).\nBMI, body mass index; CI, confidence interval; SD, standard deviation.\nCI, confidence interval.",
"Treatments involved chemotherapy and/or radiotherapy exclusively in 36.1%, surgery as the sole treatment in 23.3%, and a combination of both in 19.4% (Table 2). In the case of patients who only received surgery, resection was carried out for curative purposes in 74%. Both chemotherapy and radiotherapy were mainly applied for palliative purposes.",
"The specific survival rate at 1, 3, and 5 years after diagnosis obtained with the Kaplan-Meier methodology was 39.9%, 19%, and 15%, respectively, while respective survival rates according to competing risks survival analysis were 40.2%, 18.1%, and 12.4% (Table 3).\nCI, confidence interval; HR, hazard ratio; SD, standard deviation.\nAt 1 year from diagnosis, the probability of dying as a result of the cancer was 59.2%, with the probability of dying from other causes being 0.6% (Figure). At 5 years from diagnosis, the probability of dying as a result of the cancer rose to 83.4% and the probability of dying from other causes was 4.2%; therefore, the probability of survival was reduced to 12.4%.\nThe variables in the univariate analysis that were significantly associated with the probability of dying during the follow-up period were: gender, Charlson’s comorbidity index, presence of weight loss, histopathological cell type, tumour stage, and type of treatment (Table 3). The specific probability of dying was increased among females (hazard ratio [HR] 1.63; 95% CI, 1.00–2.64), those with a higher score on age-adjusted Charlson’s comorbidity index (HR 1.14; 95% CI, 1.05–1.23), and among those with weight loss on diagnosis (HR 1.68; 95% CI, 1.20–2.34). The histopathologic cell type with the highest mortality rate was adenocarcinoma (HR 1.68; 95% CI, 1.08–2.62). In turn, those with stage IV tumours had a higher mortality rate than those in earlier stages (0-III) (HR 2.38; 95% CI, 1.63–3.46).\nThe patients who had received some kind of treatment had a lower probability of dying. Those who had received a combination of surgical and oncological treatment (chemotherapy and/or radiotherapy) had the lowest probability of dying (HR 0.17; 95% CI, 0.10–0.27), followed by those who had only received surgical treatment (HR 0.22; 95% CI, 0.12–0.38).\nNo association was found between the probability of dying during the follow-up period and the following variables: year of diagnosis, age, BMI, personal background (smoking, regular alcohol consumption, gastro-oesophageal reflux, achalasia, and a family history of cancer), presence of dysphagia on diagnosis, or tumour location.\nAccording to the final adjustment by multivariate competing risks analysis, we found that the variables with an independent effect to predict mortality are gender, Charlson’s comorbidity index, and tumour stage (Table 4). In order to adjust for age as a clinically relevant and confounding variable and avoid over-adjustment, we included in the model the crude Charlson’s comorbidity index. The specific probability of dying from oesophageal cancer was increased among females (HR 1.94; 95% CI, 1.24–3.03), those with a higher score on Charlson’s comorbidity index (HR 1.17; 95% CI, 1.02–1.33), and in those with stage IV tumours at the time of diagnosis (HR 1.70; 95% CI, 1.11–2.59). Having received some type of treatment improved the prognosis, with a greater impact in cases that received a combination of surgical and oncological treatment (HR 0.23; 95% CI, 0.12–0.45). Furthermore, no significant effect was found when the interaction between TNM classification and type of treatment was added to the multivariate model (P = 0.600).\nAC, adenocarcinoma; B, regression coefficient; CI, confidence interval; HR, hazard ratio; SCC, squamous cell carcinoma; SE, standard error.",
"The total length of follow-up was 3221.8 months (268.5 years), with a mean of 9.4 months per patient. The most frequently detected events during follow-up were newly appeared metastasis (19.0%)—mainly located in the lungs (48.5%)—and local tumour recurrences (16.7%) (Table 5).\nIn those patients who underwent curative surgery, the probability of being diagnosed with a tumour recurrence (local tumour recurrence, distant recurrence, or both) during the first year after the surgery was 21.1%, while 25.5% of the patients died without presenting any recurrence. Therefore, 53.4% of the patients who underwent curative surgery were alive and remained disease-free 1 year after the surgery. Similarly, the probability of tumour recurrence 5 years after the surgery was 52.4%, while 29.6% of the patients died without presenting any recurrence. Therefore, only 18% of the patients were alive and disease-free 5 years after curative surgery.\nConsidering that the follow-up strategy is more intensive once an event occurs during the follow-up period, we studied the follow-up procedure carried out with patients who underwent curative surgery from the time of diagnosis until a new event occurred (local tumour recurrence, newly appeared metastasis, or newly appeared neoplasias) (Table 6). The mean number of hospital consultations per year of follow-up for these patients was 4.1 (standard deviation, 4.2) consultations/year, with a median of 3.1 consultations per year of follow-up (Table 6).\nCAT, computer-aided tomography; IQR, inter-quartile range; SD, standard deviation.\nWhen we take into account gender, age, Charlson’s comorbidity index, weight loss, histopathological cell type, TNM stage, treatment carried out, and number of consultations per follow-up year, in the interval between the diagnosis until an event occurs, we see that the consultations carried out during this period do not substantially alter the likelihood of survival (P = 0.640; HR 1.03; 95% CI, 0.92–1.15)."
] |
[
null,
null,
null,
null,
null,
null,
null,
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] |
[
"INTRODUCTION",
"METHODS",
"Measurements",
"Sample size justification",
"Statistical analysis",
"Ethics",
"RESULTS",
"Characteristics of the patients studied",
"Treatment",
"Prognosis",
"Follow-up",
"DISCUSSION"
] |
[
"Oesophageal cancer is the eighth-most common cancer worldwide.1 In the 27-member European Union, a total of 33 013 new cases were estimated in 2008 (age-standardised rate for world population [ASR{W}]: 3.5 per 100 000).1 In relation to the rest of Europe, Spain is at a midway point in terms of occurrence (ASR[W]: 2.8 per 100 000). Within Spain, this cancer occurs at a higher rate in the country’s northern regions than in southern ones.2\nAt the worldwide level, oesophageal cancer is the sixth-most frequent cause of death by cancer. In 2008, the global age-standardised rate of cancer-related mortality was 2.9 per 100 000 inhabitants in the European Union and 2.3 per 100 000 in Spain. The mortality rate, like the occurrence rate, is higher in Spain’s northern regions.3\nAccording to the European Cancer Registry EUROCARE-4 study,4 the average European survival rate is close to 35% at one year and close to 10% at 5 years, and survival is strongly associated with the clinical stage of the tumour and the treatment the patient receives.5–7 There is little consensus when it comes to defining the follow-up protocols for these patients.8 Furthermore, to the best of our knowledge, there are no observational or experimental studies that have investigated the role of the different follow-up strategies on these patients’ prognosis.\nWith regard to analysing the survival rate, competing risk models are the most suitable for analysing the behaviour of subjects who may die for different reasons. However, when applied in the presence of competitive risks, the usual techniques for analysing the time until the event, such as the Kaplan-Meier methodology, produce biased results.9,10 By using specific techniques, it is possible to reduce bias so that results can be correctly interpreted.\nTherefore, the geographic variability in the occurrence and mortality rates, the existence of different risk factors associated with the low survival rate for these kinds of tumours, and the lack of consensus with regard to optimum follow-up strategies justify the undertaking of this study. The aim of the study was to identify the epidemiology of oesophageal cancer in the area of A Coruña, Spain, the supportive process applied to these patients, and their prognosis, using the competing risks methodology.",
"A total of 234 patients were included in the study, with anatomical-pathological confirmation of cancer of the oesophagus diagnosed at the University Hospital Complex in A Coruña, Spain between the 1st of January, 2003, and the 31st of December, 2008. A retrospective review of the patients’ clinical records was carried out together with a prospective follow-up until the 31st of January, 2012, in order to guarantee a minimum follow-up period of 3 years. The study excluded prevalent or recurring cases, subjects with multiple or metastatic cancers, or those that had been treated and/or diagnosed at other hospitals. After exclusions, the final study sample comprised 180 patients.\n Measurements We collected information on the socio-demographic variables of the patient, their personal backgrounds, comorbidity variables using Charlson’s comorbidity index, the symptoms present at diagnosis, location of the tumour, histopathologic cell type, and tumour stage (TNM, seventh edition).11,12 As data on risk factors and symptoms were collected retrospectively from electronic hospital clinical records, we registered data that were indicated in the clinical records but could not quantify the frequency and amount of cigarette or alcohol consumption, nor the number of kilograms lost during the previous months before the diagnosis.\nWe also collected data on the treatment received (surgery, chemotherapy, and radiotherapy), as well as the visits and tests carried out during the follow-up period: consultations and hospital stays, endoscopies, computer-aided tomography scans, and thorax X-rays. Also, the presence of follow-up events, including newly appeared local recurrences, metastasis, and neoplasias, was studied. For all dead patients, cause of death was obtained from the Galician Mortality Registry (General Directory of Public Health, Xunta de Galicia, Spain), according to the 21 diagnostic categories of the 10th revision of the International Classification of Diseases.\nWe collected information on the socio-demographic variables of the patient, their personal backgrounds, comorbidity variables using Charlson’s comorbidity index, the symptoms present at diagnosis, location of the tumour, histopathologic cell type, and tumour stage (TNM, seventh edition).11,12 As data on risk factors and symptoms were collected retrospectively from electronic hospital clinical records, we registered data that were indicated in the clinical records but could not quantify the frequency and amount of cigarette or alcohol consumption, nor the number of kilograms lost during the previous months before the diagnosis.\nWe also collected data on the treatment received (surgery, chemotherapy, and radiotherapy), as well as the visits and tests carried out during the follow-up period: consultations and hospital stays, endoscopies, computer-aided tomography scans, and thorax X-rays. Also, the presence of follow-up events, including newly appeared local recurrences, metastasis, and neoplasias, was studied. For all dead patients, cause of death was obtained from the Galician Mortality Registry (General Directory of Public Health, Xunta de Galicia, Spain), according to the 21 diagnostic categories of the 10th revision of the International Classification of Diseases.\n Sample size justification The study included a total of 180 patients. This sample size makes it possible to detect as significant a hazard ratio of 1.6 or more, with a prevalence of exposure of 50% and a censored data percentage of 20% (security: 95%; statistical power: 80%).\nThe study included a total of 180 patients. This sample size makes it possible to detect as significant a hazard ratio of 1.6 or more, with a prevalence of exposure of 50% and a censored data percentage of 20% (security: 95%; statistical power: 80%).\n Statistical analysis A descriptive study was made of the variables that were obtained. The specific survival rate was calculated using the Kaplan-Meier methodology and competing risks survival analysis. The accumulated occurrence of dying as a result of oesophageal cancer during the follow-up period was estimated, considering death as a result of other causes as a competitive event, using the method proposed by Kalbfleisch and Prentice.13 The accumulated occurrence of death due to oesophageal cancer according to different characteristics was compared using the test proposed by Gray.14 Finally, in order to identify which characteristics were associated with the risk of dying as a result of oesophageal cancer, a multivariate analysis was carried out using the model proposed by Fine and Gray.15 All of the tests were carried out bilaterally, considering values of P < 0.05 as significant. The analyses were carried out using the programmes Epidat 3.1 (Xunta de Galicia, Santiago de Compostela, Spain), SPSS 19.0 (IBM Company, Chicago, IL, USA), and R 2.15.1 (Free Software Foundation, Boston, MA, USA).\nA descriptive study was made of the variables that were obtained. The specific survival rate was calculated using the Kaplan-Meier methodology and competing risks survival analysis. The accumulated occurrence of dying as a result of oesophageal cancer during the follow-up period was estimated, considering death as a result of other causes as a competitive event, using the method proposed by Kalbfleisch and Prentice.13 The accumulated occurrence of death due to oesophageal cancer according to different characteristics was compared using the test proposed by Gray.14 Finally, in order to identify which characteristics were associated with the risk of dying as a result of oesophageal cancer, a multivariate analysis was carried out using the model proposed by Fine and Gray.15 All of the tests were carried out bilaterally, considering values of P < 0.05 as significant. The analyses were carried out using the programmes Epidat 3.1 (Xunta de Galicia, Santiago de Compostela, Spain), SPSS 19.0 (IBM Company, Chicago, IL, USA), and R 2.15.1 (Free Software Foundation, Boston, MA, USA).\n Ethics The study was carried out according to the principles laid down in the Declaration of Helsinki and ensuring compliance with Spanish Decree 29/2009, which regulates the use of and access to electronic medical records. Confidentiality was maintained in accordance with the current Spanish Data Protection Law (15/1999). The study received written approval from the regional Ethics Committee for Clinical Research (code 2011/372 CEIC Galicia).\nThe study was carried out according to the principles laid down in the Declaration of Helsinki and ensuring compliance with Spanish Decree 29/2009, which regulates the use of and access to electronic medical records. Confidentiality was maintained in accordance with the current Spanish Data Protection Law (15/1999). The study received written approval from the regional Ethics Committee for Clinical Research (code 2011/372 CEIC Galicia).",
"We collected information on the socio-demographic variables of the patient, their personal backgrounds, comorbidity variables using Charlson’s comorbidity index, the symptoms present at diagnosis, location of the tumour, histopathologic cell type, and tumour stage (TNM, seventh edition).11,12 As data on risk factors and symptoms were collected retrospectively from electronic hospital clinical records, we registered data that were indicated in the clinical records but could not quantify the frequency and amount of cigarette or alcohol consumption, nor the number of kilograms lost during the previous months before the diagnosis.\nWe also collected data on the treatment received (surgery, chemotherapy, and radiotherapy), as well as the visits and tests carried out during the follow-up period: consultations and hospital stays, endoscopies, computer-aided tomography scans, and thorax X-rays. Also, the presence of follow-up events, including newly appeared local recurrences, metastasis, and neoplasias, was studied. For all dead patients, cause of death was obtained from the Galician Mortality Registry (General Directory of Public Health, Xunta de Galicia, Spain), according to the 21 diagnostic categories of the 10th revision of the International Classification of Diseases.",
"The study included a total of 180 patients. This sample size makes it possible to detect as significant a hazard ratio of 1.6 or more, with a prevalence of exposure of 50% and a censored data percentage of 20% (security: 95%; statistical power: 80%).",
"A descriptive study was made of the variables that were obtained. The specific survival rate was calculated using the Kaplan-Meier methodology and competing risks survival analysis. The accumulated occurrence of dying as a result of oesophageal cancer during the follow-up period was estimated, considering death as a result of other causes as a competitive event, using the method proposed by Kalbfleisch and Prentice.13 The accumulated occurrence of death due to oesophageal cancer according to different characteristics was compared using the test proposed by Gray.14 Finally, in order to identify which characteristics were associated with the risk of dying as a result of oesophageal cancer, a multivariate analysis was carried out using the model proposed by Fine and Gray.15 All of the tests were carried out bilaterally, considering values of P < 0.05 as significant. The analyses were carried out using the programmes Epidat 3.1 (Xunta de Galicia, Santiago de Compostela, Spain), SPSS 19.0 (IBM Company, Chicago, IL, USA), and R 2.15.1 (Free Software Foundation, Boston, MA, USA).",
"The study was carried out according to the principles laid down in the Declaration of Helsinki and ensuring compliance with Spanish Decree 29/2009, which regulates the use of and access to electronic medical records. Confidentiality was maintained in accordance with the current Spanish Data Protection Law (15/1999). The study received written approval from the regional Ethics Committee for Clinical Research (code 2011/372 CEIC Galicia).",
" Characteristics of the patients studied Table 1 shows the baseline characteristics, comorbidities, and cause of death of the patients. The median age was 64.5 years, 87.8% of the sample subjects were male, 58.6% had a body mass index (BMI) within normal range, and 11.4% were obese. The most frequent symptom reported was dysphagia (82.0%), followed by weight loss (49.4%). Regarding tumour stage, 46.8% of the tumours were moderately differentiated, 38.0% were poorly differentiated, and 28.9% had metastasis at the time of diagnosis (Table 2).\nBMI, body mass index; CI, confidence interval; SD, standard deviation.\nCI, confidence interval.\nTable 1 shows the baseline characteristics, comorbidities, and cause of death of the patients. The median age was 64.5 years, 87.8% of the sample subjects were male, 58.6% had a body mass index (BMI) within normal range, and 11.4% were obese. The most frequent symptom reported was dysphagia (82.0%), followed by weight loss (49.4%). Regarding tumour stage, 46.8% of the tumours were moderately differentiated, 38.0% were poorly differentiated, and 28.9% had metastasis at the time of diagnosis (Table 2).\nBMI, body mass index; CI, confidence interval; SD, standard deviation.\nCI, confidence interval.\n Treatment Treatments involved chemotherapy and/or radiotherapy exclusively in 36.1%, surgery as the sole treatment in 23.3%, and a combination of both in 19.4% (Table 2). In the case of patients who only received surgery, resection was carried out for curative purposes in 74%. Both chemotherapy and radiotherapy were mainly applied for palliative purposes.\nTreatments involved chemotherapy and/or radiotherapy exclusively in 36.1%, surgery as the sole treatment in 23.3%, and a combination of both in 19.4% (Table 2). In the case of patients who only received surgery, resection was carried out for curative purposes in 74%. Both chemotherapy and radiotherapy were mainly applied for palliative purposes.\n Prognosis The specific survival rate at 1, 3, and 5 years after diagnosis obtained with the Kaplan-Meier methodology was 39.9%, 19%, and 15%, respectively, while respective survival rates according to competing risks survival analysis were 40.2%, 18.1%, and 12.4% (Table 3).\nCI, confidence interval; HR, hazard ratio; SD, standard deviation.\nAt 1 year from diagnosis, the probability of dying as a result of the cancer was 59.2%, with the probability of dying from other causes being 0.6% (Figure). At 5 years from diagnosis, the probability of dying as a result of the cancer rose to 83.4% and the probability of dying from other causes was 4.2%; therefore, the probability of survival was reduced to 12.4%.\nThe variables in the univariate analysis that were significantly associated with the probability of dying during the follow-up period were: gender, Charlson’s comorbidity index, presence of weight loss, histopathological cell type, tumour stage, and type of treatment (Table 3). The specific probability of dying was increased among females (hazard ratio [HR] 1.63; 95% CI, 1.00–2.64), those with a higher score on age-adjusted Charlson’s comorbidity index (HR 1.14; 95% CI, 1.05–1.23), and among those with weight loss on diagnosis (HR 1.68; 95% CI, 1.20–2.34). The histopathologic cell type with the highest mortality rate was adenocarcinoma (HR 1.68; 95% CI, 1.08–2.62). In turn, those with stage IV tumours had a higher mortality rate than those in earlier stages (0-III) (HR 2.38; 95% CI, 1.63–3.46).\nThe patients who had received some kind of treatment had a lower probability of dying. Those who had received a combination of surgical and oncological treatment (chemotherapy and/or radiotherapy) had the lowest probability of dying (HR 0.17; 95% CI, 0.10–0.27), followed by those who had only received surgical treatment (HR 0.22; 95% CI, 0.12–0.38).\nNo association was found between the probability of dying during the follow-up period and the following variables: year of diagnosis, age, BMI, personal background (smoking, regular alcohol consumption, gastro-oesophageal reflux, achalasia, and a family history of cancer), presence of dysphagia on diagnosis, or tumour location.\nAccording to the final adjustment by multivariate competing risks analysis, we found that the variables with an independent effect to predict mortality are gender, Charlson’s comorbidity index, and tumour stage (Table 4). In order to adjust for age as a clinically relevant and confounding variable and avoid over-adjustment, we included in the model the crude Charlson’s comorbidity index. The specific probability of dying from oesophageal cancer was increased among females (HR 1.94; 95% CI, 1.24–3.03), those with a higher score on Charlson’s comorbidity index (HR 1.17; 95% CI, 1.02–1.33), and in those with stage IV tumours at the time of diagnosis (HR 1.70; 95% CI, 1.11–2.59). Having received some type of treatment improved the prognosis, with a greater impact in cases that received a combination of surgical and oncological treatment (HR 0.23; 95% CI, 0.12–0.45). Furthermore, no significant effect was found when the interaction between TNM classification and type of treatment was added to the multivariate model (P = 0.600).\nAC, adenocarcinoma; B, regression coefficient; CI, confidence interval; HR, hazard ratio; SCC, squamous cell carcinoma; SE, standard error.\nThe specific survival rate at 1, 3, and 5 years after diagnosis obtained with the Kaplan-Meier methodology was 39.9%, 19%, and 15%, respectively, while respective survival rates according to competing risks survival analysis were 40.2%, 18.1%, and 12.4% (Table 3).\nCI, confidence interval; HR, hazard ratio; SD, standard deviation.\nAt 1 year from diagnosis, the probability of dying as a result of the cancer was 59.2%, with the probability of dying from other causes being 0.6% (Figure). At 5 years from diagnosis, the probability of dying as a result of the cancer rose to 83.4% and the probability of dying from other causes was 4.2%; therefore, the probability of survival was reduced to 12.4%.\nThe variables in the univariate analysis that were significantly associated with the probability of dying during the follow-up period were: gender, Charlson’s comorbidity index, presence of weight loss, histopathological cell type, tumour stage, and type of treatment (Table 3). The specific probability of dying was increased among females (hazard ratio [HR] 1.63; 95% CI, 1.00–2.64), those with a higher score on age-adjusted Charlson’s comorbidity index (HR 1.14; 95% CI, 1.05–1.23), and among those with weight loss on diagnosis (HR 1.68; 95% CI, 1.20–2.34). The histopathologic cell type with the highest mortality rate was adenocarcinoma (HR 1.68; 95% CI, 1.08–2.62). In turn, those with stage IV tumours had a higher mortality rate than those in earlier stages (0-III) (HR 2.38; 95% CI, 1.63–3.46).\nThe patients who had received some kind of treatment had a lower probability of dying. Those who had received a combination of surgical and oncological treatment (chemotherapy and/or radiotherapy) had the lowest probability of dying (HR 0.17; 95% CI, 0.10–0.27), followed by those who had only received surgical treatment (HR 0.22; 95% CI, 0.12–0.38).\nNo association was found between the probability of dying during the follow-up period and the following variables: year of diagnosis, age, BMI, personal background (smoking, regular alcohol consumption, gastro-oesophageal reflux, achalasia, and a family history of cancer), presence of dysphagia on diagnosis, or tumour location.\nAccording to the final adjustment by multivariate competing risks analysis, we found that the variables with an independent effect to predict mortality are gender, Charlson’s comorbidity index, and tumour stage (Table 4). In order to adjust for age as a clinically relevant and confounding variable and avoid over-adjustment, we included in the model the crude Charlson’s comorbidity index. The specific probability of dying from oesophageal cancer was increased among females (HR 1.94; 95% CI, 1.24–3.03), those with a higher score on Charlson’s comorbidity index (HR 1.17; 95% CI, 1.02–1.33), and in those with stage IV tumours at the time of diagnosis (HR 1.70; 95% CI, 1.11–2.59). Having received some type of treatment improved the prognosis, with a greater impact in cases that received a combination of surgical and oncological treatment (HR 0.23; 95% CI, 0.12–0.45). Furthermore, no significant effect was found when the interaction between TNM classification and type of treatment was added to the multivariate model (P = 0.600).\nAC, adenocarcinoma; B, regression coefficient; CI, confidence interval; HR, hazard ratio; SCC, squamous cell carcinoma; SE, standard error.\n Follow-up The total length of follow-up was 3221.8 months (268.5 years), with a mean of 9.4 months per patient. The most frequently detected events during follow-up were newly appeared metastasis (19.0%)—mainly located in the lungs (48.5%)—and local tumour recurrences (16.7%) (Table 5).\nIn those patients who underwent curative surgery, the probability of being diagnosed with a tumour recurrence (local tumour recurrence, distant recurrence, or both) during the first year after the surgery was 21.1%, while 25.5% of the patients died without presenting any recurrence. Therefore, 53.4% of the patients who underwent curative surgery were alive and remained disease-free 1 year after the surgery. Similarly, the probability of tumour recurrence 5 years after the surgery was 52.4%, while 29.6% of the patients died without presenting any recurrence. Therefore, only 18% of the patients were alive and disease-free 5 years after curative surgery.\nConsidering that the follow-up strategy is more intensive once an event occurs during the follow-up period, we studied the follow-up procedure carried out with patients who underwent curative surgery from the time of diagnosis until a new event occurred (local tumour recurrence, newly appeared metastasis, or newly appeared neoplasias) (Table 6). The mean number of hospital consultations per year of follow-up for these patients was 4.1 (standard deviation, 4.2) consultations/year, with a median of 3.1 consultations per year of follow-up (Table 6).\nCAT, computer-aided tomography; IQR, inter-quartile range; SD, standard deviation.\nWhen we take into account gender, age, Charlson’s comorbidity index, weight loss, histopathological cell type, TNM stage, treatment carried out, and number of consultations per follow-up year, in the interval between the diagnosis until an event occurs, we see that the consultations carried out during this period do not substantially alter the likelihood of survival (P = 0.640; HR 1.03; 95% CI, 0.92–1.15).\nThe total length of follow-up was 3221.8 months (268.5 years), with a mean of 9.4 months per patient. The most frequently detected events during follow-up were newly appeared metastasis (19.0%)—mainly located in the lungs (48.5%)—and local tumour recurrences (16.7%) (Table 5).\nIn those patients who underwent curative surgery, the probability of being diagnosed with a tumour recurrence (local tumour recurrence, distant recurrence, or both) during the first year after the surgery was 21.1%, while 25.5% of the patients died without presenting any recurrence. Therefore, 53.4% of the patients who underwent curative surgery were alive and remained disease-free 1 year after the surgery. Similarly, the probability of tumour recurrence 5 years after the surgery was 52.4%, while 29.6% of the patients died without presenting any recurrence. Therefore, only 18% of the patients were alive and disease-free 5 years after curative surgery.\nConsidering that the follow-up strategy is more intensive once an event occurs during the follow-up period, we studied the follow-up procedure carried out with patients who underwent curative surgery from the time of diagnosis until a new event occurred (local tumour recurrence, newly appeared metastasis, or newly appeared neoplasias) (Table 6). The mean number of hospital consultations per year of follow-up for these patients was 4.1 (standard deviation, 4.2) consultations/year, with a median of 3.1 consultations per year of follow-up (Table 6).\nCAT, computer-aided tomography; IQR, inter-quartile range; SD, standard deviation.\nWhen we take into account gender, age, Charlson’s comorbidity index, weight loss, histopathological cell type, TNM stage, treatment carried out, and number of consultations per follow-up year, in the interval between the diagnosis until an event occurs, we see that the consultations carried out during this period do not substantially alter the likelihood of survival (P = 0.640; HR 1.03; 95% CI, 0.92–1.15).",
"Table 1 shows the baseline characteristics, comorbidities, and cause of death of the patients. The median age was 64.5 years, 87.8% of the sample subjects were male, 58.6% had a body mass index (BMI) within normal range, and 11.4% were obese. The most frequent symptom reported was dysphagia (82.0%), followed by weight loss (49.4%). Regarding tumour stage, 46.8% of the tumours were moderately differentiated, 38.0% were poorly differentiated, and 28.9% had metastasis at the time of diagnosis (Table 2).\nBMI, body mass index; CI, confidence interval; SD, standard deviation.\nCI, confidence interval.",
"Treatments involved chemotherapy and/or radiotherapy exclusively in 36.1%, surgery as the sole treatment in 23.3%, and a combination of both in 19.4% (Table 2). In the case of patients who only received surgery, resection was carried out for curative purposes in 74%. Both chemotherapy and radiotherapy were mainly applied for palliative purposes.",
"The specific survival rate at 1, 3, and 5 years after diagnosis obtained with the Kaplan-Meier methodology was 39.9%, 19%, and 15%, respectively, while respective survival rates according to competing risks survival analysis were 40.2%, 18.1%, and 12.4% (Table 3).\nCI, confidence interval; HR, hazard ratio; SD, standard deviation.\nAt 1 year from diagnosis, the probability of dying as a result of the cancer was 59.2%, with the probability of dying from other causes being 0.6% (Figure). At 5 years from diagnosis, the probability of dying as a result of the cancer rose to 83.4% and the probability of dying from other causes was 4.2%; therefore, the probability of survival was reduced to 12.4%.\nThe variables in the univariate analysis that were significantly associated with the probability of dying during the follow-up period were: gender, Charlson’s comorbidity index, presence of weight loss, histopathological cell type, tumour stage, and type of treatment (Table 3). The specific probability of dying was increased among females (hazard ratio [HR] 1.63; 95% CI, 1.00–2.64), those with a higher score on age-adjusted Charlson’s comorbidity index (HR 1.14; 95% CI, 1.05–1.23), and among those with weight loss on diagnosis (HR 1.68; 95% CI, 1.20–2.34). The histopathologic cell type with the highest mortality rate was adenocarcinoma (HR 1.68; 95% CI, 1.08–2.62). In turn, those with stage IV tumours had a higher mortality rate than those in earlier stages (0-III) (HR 2.38; 95% CI, 1.63–3.46).\nThe patients who had received some kind of treatment had a lower probability of dying. Those who had received a combination of surgical and oncological treatment (chemotherapy and/or radiotherapy) had the lowest probability of dying (HR 0.17; 95% CI, 0.10–0.27), followed by those who had only received surgical treatment (HR 0.22; 95% CI, 0.12–0.38).\nNo association was found between the probability of dying during the follow-up period and the following variables: year of diagnosis, age, BMI, personal background (smoking, regular alcohol consumption, gastro-oesophageal reflux, achalasia, and a family history of cancer), presence of dysphagia on diagnosis, or tumour location.\nAccording to the final adjustment by multivariate competing risks analysis, we found that the variables with an independent effect to predict mortality are gender, Charlson’s comorbidity index, and tumour stage (Table 4). In order to adjust for age as a clinically relevant and confounding variable and avoid over-adjustment, we included in the model the crude Charlson’s comorbidity index. The specific probability of dying from oesophageal cancer was increased among females (HR 1.94; 95% CI, 1.24–3.03), those with a higher score on Charlson’s comorbidity index (HR 1.17; 95% CI, 1.02–1.33), and in those with stage IV tumours at the time of diagnosis (HR 1.70; 95% CI, 1.11–2.59). Having received some type of treatment improved the prognosis, with a greater impact in cases that received a combination of surgical and oncological treatment (HR 0.23; 95% CI, 0.12–0.45). Furthermore, no significant effect was found when the interaction between TNM classification and type of treatment was added to the multivariate model (P = 0.600).\nAC, adenocarcinoma; B, regression coefficient; CI, confidence interval; HR, hazard ratio; SCC, squamous cell carcinoma; SE, standard error.",
"The total length of follow-up was 3221.8 months (268.5 years), with a mean of 9.4 months per patient. The most frequently detected events during follow-up were newly appeared metastasis (19.0%)—mainly located in the lungs (48.5%)—and local tumour recurrences (16.7%) (Table 5).\nIn those patients who underwent curative surgery, the probability of being diagnosed with a tumour recurrence (local tumour recurrence, distant recurrence, or both) during the first year after the surgery was 21.1%, while 25.5% of the patients died without presenting any recurrence. Therefore, 53.4% of the patients who underwent curative surgery were alive and remained disease-free 1 year after the surgery. Similarly, the probability of tumour recurrence 5 years after the surgery was 52.4%, while 29.6% of the patients died without presenting any recurrence. Therefore, only 18% of the patients were alive and disease-free 5 years after curative surgery.\nConsidering that the follow-up strategy is more intensive once an event occurs during the follow-up period, we studied the follow-up procedure carried out with patients who underwent curative surgery from the time of diagnosis until a new event occurred (local tumour recurrence, newly appeared metastasis, or newly appeared neoplasias) (Table 6). The mean number of hospital consultations per year of follow-up for these patients was 4.1 (standard deviation, 4.2) consultations/year, with a median of 3.1 consultations per year of follow-up (Table 6).\nCAT, computer-aided tomography; IQR, inter-quartile range; SD, standard deviation.\nWhen we take into account gender, age, Charlson’s comorbidity index, weight loss, histopathological cell type, TNM stage, treatment carried out, and number of consultations per follow-up year, in the interval between the diagnosis until an event occurs, we see that the consultations carried out during this period do not substantially alter the likelihood of survival (P = 0.640; HR 1.03; 95% CI, 0.92–1.15).",
"In Spain, the occurrence of oesophageal cancer is at a midway point with regard to the rest of Europe.1 It is more frequent in men16–18 and usually appears between the ages of 55 and 70 years,6,19–21 and the most common symptom associated with its appearance is dysphagia.22 The results of the present study confirm these findings and are in line with those of other previously published series.19,20,23,24\nWith regard to the specific survival rate for these types of tumours, data published from the EUROCARE-44 study, which included 48 353 cases from 23 European countries, reveal a mean estimated survival rate of 35%, 14.1%, and 9.6% at the first, third, and fifth years of follow-up, respectively, highlighting the low survival rate associated with these tumours. These data also concur with those published in countries such as Canada25 or Iran,20 which reported 5-year survival rates of 8.8% and 12%, respectively. Series published in Europe, such as those in Sweden,6 England,26 and France27 found similar figures, with 5-year survival rates between 9.3%–13.1%, 3.2%–9.8%, and 9%–14%, respectively. In our study, the 5-year survival rate was 12.4%. It is important to note that we used the competing risks survival analysis in our study, which is suitable for analysing the behaviour of a person who may die as a result of different causes. Using the Kaplan-Meier methodology, the results obtained for our sample slightly overestimated the survival rate from the third year onwards. The patients in this cohort mainly die as a result of the illness in question, and so for this reason the differences found in the survival rate between the Kaplan-Meier methodology and competing risks survival analysis are very low. Despite this, the competing risks survival analysis method is the most suitable for analysing the specific survival rate in the presence of other causes of death. This overestimation of the survival rate using the Kaplan-Meier methodology in comparison to competing risks survival analysis has previously been described in the literature.9,10\nWith regard to the factors associated with survival, most studies have indicated that women have higher survival rates than men,5,19,25,27 although in some studies these differences were not significant.20 In a study of patients from the Donostia Hospital in the Basque Country, Spain,23 the mean survival rate in women was lower, as in our study, although the differences were not significant. In our study, we found that the patient’s number and severity of comorbidities, assessed using Charlson’s comorbidity index, is significantly associated with the likelihood of dying as a result of the tumour. In a recent study in the Netherlands,19 having comorbidities was associated with poor survival, as in our study, although no significant differences were found.\nAmongst the limitations to the study, we did not study any molecular marker that could play an important role in the prognosis of oesophageal cancer and which, in combination with other parameters such as tumour stage, would help to identify and predict the survival rate from these tumours, as well as to individually adapt the treatment to each patient, as recently described in the literature.28,29 Although our sample only consisted of 180 patients, we were able to compare the consistency of our results with larger series published internationally,5–7,20,23,24,30–32 in which the tumour stage and treatment received by the patient were the main prognostic factors for survival.\nThis study reveals how the different follow-up strategies used (visits and tests carried out) in patients where the intention is to apply curative treatment until a new event occurs do not alter their survival rate. This finding is in agreement with the lack of consensus at the international level8,33,34 in terms of defining the follow-up protocols for these types of tumours. Furthermore, we did not find any studies in the literature that describe the follow-up process applied to these patients and its possible effect on their survival rate. In The European Society for Medical Oncology’s guidelines for the diagnosis, treatment, and monitoring of oesophageal cancer for 2013,35 the group concluded that, with the exception of patients who could be candidates for rescue surgery after a failed endoscopic resection or definitive chemo-radiotherapy, there is no evidence that regular follow-up after the initial therapy impacts the final outcome.\nIn conclusion, the specific survival rate detected in our study was low and coincided with figures published at the international level. Our results confirm that studying the survival rate using the Kaplan-Meier methodology overestimates the survival rate in comparison to competing risks survival analysis. We also found that the different follow-up strategies used after diagnosing illness in patients who are surgically treated with the intention to cure do not alter the prognosis."
] |
[
"intro",
"methods",
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion"
] |
[
"oesophageal neoplasms",
"therapeutics",
"survival",
"follow-up studies"
] |
INTRODUCTION:
Oesophageal cancer is the eighth-most common cancer worldwide.1 In the 27-member European Union, a total of 33 013 new cases were estimated in 2008 (age-standardised rate for world population [ASR{W}]: 3.5 per 100 000).1 In relation to the rest of Europe, Spain is at a midway point in terms of occurrence (ASR[W]: 2.8 per 100 000). Within Spain, this cancer occurs at a higher rate in the country’s northern regions than in southern ones.2
At the worldwide level, oesophageal cancer is the sixth-most frequent cause of death by cancer. In 2008, the global age-standardised rate of cancer-related mortality was 2.9 per 100 000 inhabitants in the European Union and 2.3 per 100 000 in Spain. The mortality rate, like the occurrence rate, is higher in Spain’s northern regions.3
According to the European Cancer Registry EUROCARE-4 study,4 the average European survival rate is close to 35% at one year and close to 10% at 5 years, and survival is strongly associated with the clinical stage of the tumour and the treatment the patient receives.5–7 There is little consensus when it comes to defining the follow-up protocols for these patients.8 Furthermore, to the best of our knowledge, there are no observational or experimental studies that have investigated the role of the different follow-up strategies on these patients’ prognosis.
With regard to analysing the survival rate, competing risk models are the most suitable for analysing the behaviour of subjects who may die for different reasons. However, when applied in the presence of competitive risks, the usual techniques for analysing the time until the event, such as the Kaplan-Meier methodology, produce biased results.9,10 By using specific techniques, it is possible to reduce bias so that results can be correctly interpreted.
Therefore, the geographic variability in the occurrence and mortality rates, the existence of different risk factors associated with the low survival rate for these kinds of tumours, and the lack of consensus with regard to optimum follow-up strategies justify the undertaking of this study. The aim of the study was to identify the epidemiology of oesophageal cancer in the area of A Coruña, Spain, the supportive process applied to these patients, and their prognosis, using the competing risks methodology.
METHODS:
A total of 234 patients were included in the study, with anatomical-pathological confirmation of cancer of the oesophagus diagnosed at the University Hospital Complex in A Coruña, Spain between the 1st of January, 2003, and the 31st of December, 2008. A retrospective review of the patients’ clinical records was carried out together with a prospective follow-up until the 31st of January, 2012, in order to guarantee a minimum follow-up period of 3 years. The study excluded prevalent or recurring cases, subjects with multiple or metastatic cancers, or those that had been treated and/or diagnosed at other hospitals. After exclusions, the final study sample comprised 180 patients.
Measurements We collected information on the socio-demographic variables of the patient, their personal backgrounds, comorbidity variables using Charlson’s comorbidity index, the symptoms present at diagnosis, location of the tumour, histopathologic cell type, and tumour stage (TNM, seventh edition).11,12 As data on risk factors and symptoms were collected retrospectively from electronic hospital clinical records, we registered data that were indicated in the clinical records but could not quantify the frequency and amount of cigarette or alcohol consumption, nor the number of kilograms lost during the previous months before the diagnosis.
We also collected data on the treatment received (surgery, chemotherapy, and radiotherapy), as well as the visits and tests carried out during the follow-up period: consultations and hospital stays, endoscopies, computer-aided tomography scans, and thorax X-rays. Also, the presence of follow-up events, including newly appeared local recurrences, metastasis, and neoplasias, was studied. For all dead patients, cause of death was obtained from the Galician Mortality Registry (General Directory of Public Health, Xunta de Galicia, Spain), according to the 21 diagnostic categories of the 10th revision of the International Classification of Diseases.
We collected information on the socio-demographic variables of the patient, their personal backgrounds, comorbidity variables using Charlson’s comorbidity index, the symptoms present at diagnosis, location of the tumour, histopathologic cell type, and tumour stage (TNM, seventh edition).11,12 As data on risk factors and symptoms were collected retrospectively from electronic hospital clinical records, we registered data that were indicated in the clinical records but could not quantify the frequency and amount of cigarette or alcohol consumption, nor the number of kilograms lost during the previous months before the diagnosis.
We also collected data on the treatment received (surgery, chemotherapy, and radiotherapy), as well as the visits and tests carried out during the follow-up period: consultations and hospital stays, endoscopies, computer-aided tomography scans, and thorax X-rays. Also, the presence of follow-up events, including newly appeared local recurrences, metastasis, and neoplasias, was studied. For all dead patients, cause of death was obtained from the Galician Mortality Registry (General Directory of Public Health, Xunta de Galicia, Spain), according to the 21 diagnostic categories of the 10th revision of the International Classification of Diseases.
Sample size justification The study included a total of 180 patients. This sample size makes it possible to detect as significant a hazard ratio of 1.6 or more, with a prevalence of exposure of 50% and a censored data percentage of 20% (security: 95%; statistical power: 80%).
The study included a total of 180 patients. This sample size makes it possible to detect as significant a hazard ratio of 1.6 or more, with a prevalence of exposure of 50% and a censored data percentage of 20% (security: 95%; statistical power: 80%).
Statistical analysis A descriptive study was made of the variables that were obtained. The specific survival rate was calculated using the Kaplan-Meier methodology and competing risks survival analysis. The accumulated occurrence of dying as a result of oesophageal cancer during the follow-up period was estimated, considering death as a result of other causes as a competitive event, using the method proposed by Kalbfleisch and Prentice.13 The accumulated occurrence of death due to oesophageal cancer according to different characteristics was compared using the test proposed by Gray.14 Finally, in order to identify which characteristics were associated with the risk of dying as a result of oesophageal cancer, a multivariate analysis was carried out using the model proposed by Fine and Gray.15 All of the tests were carried out bilaterally, considering values of P < 0.05 as significant. The analyses were carried out using the programmes Epidat 3.1 (Xunta de Galicia, Santiago de Compostela, Spain), SPSS 19.0 (IBM Company, Chicago, IL, USA), and R 2.15.1 (Free Software Foundation, Boston, MA, USA).
A descriptive study was made of the variables that were obtained. The specific survival rate was calculated using the Kaplan-Meier methodology and competing risks survival analysis. The accumulated occurrence of dying as a result of oesophageal cancer during the follow-up period was estimated, considering death as a result of other causes as a competitive event, using the method proposed by Kalbfleisch and Prentice.13 The accumulated occurrence of death due to oesophageal cancer according to different characteristics was compared using the test proposed by Gray.14 Finally, in order to identify which characteristics were associated with the risk of dying as a result of oesophageal cancer, a multivariate analysis was carried out using the model proposed by Fine and Gray.15 All of the tests were carried out bilaterally, considering values of P < 0.05 as significant. The analyses were carried out using the programmes Epidat 3.1 (Xunta de Galicia, Santiago de Compostela, Spain), SPSS 19.0 (IBM Company, Chicago, IL, USA), and R 2.15.1 (Free Software Foundation, Boston, MA, USA).
Ethics The study was carried out according to the principles laid down in the Declaration of Helsinki and ensuring compliance with Spanish Decree 29/2009, which regulates the use of and access to electronic medical records. Confidentiality was maintained in accordance with the current Spanish Data Protection Law (15/1999). The study received written approval from the regional Ethics Committee for Clinical Research (code 2011/372 CEIC Galicia).
The study was carried out according to the principles laid down in the Declaration of Helsinki and ensuring compliance with Spanish Decree 29/2009, which regulates the use of and access to electronic medical records. Confidentiality was maintained in accordance with the current Spanish Data Protection Law (15/1999). The study received written approval from the regional Ethics Committee for Clinical Research (code 2011/372 CEIC Galicia).
Measurements:
We collected information on the socio-demographic variables of the patient, their personal backgrounds, comorbidity variables using Charlson’s comorbidity index, the symptoms present at diagnosis, location of the tumour, histopathologic cell type, and tumour stage (TNM, seventh edition).11,12 As data on risk factors and symptoms were collected retrospectively from electronic hospital clinical records, we registered data that were indicated in the clinical records but could not quantify the frequency and amount of cigarette or alcohol consumption, nor the number of kilograms lost during the previous months before the diagnosis.
We also collected data on the treatment received (surgery, chemotherapy, and radiotherapy), as well as the visits and tests carried out during the follow-up period: consultations and hospital stays, endoscopies, computer-aided tomography scans, and thorax X-rays. Also, the presence of follow-up events, including newly appeared local recurrences, metastasis, and neoplasias, was studied. For all dead patients, cause of death was obtained from the Galician Mortality Registry (General Directory of Public Health, Xunta de Galicia, Spain), according to the 21 diagnostic categories of the 10th revision of the International Classification of Diseases.
Sample size justification:
The study included a total of 180 patients. This sample size makes it possible to detect as significant a hazard ratio of 1.6 or more, with a prevalence of exposure of 50% and a censored data percentage of 20% (security: 95%; statistical power: 80%).
Statistical analysis:
A descriptive study was made of the variables that were obtained. The specific survival rate was calculated using the Kaplan-Meier methodology and competing risks survival analysis. The accumulated occurrence of dying as a result of oesophageal cancer during the follow-up period was estimated, considering death as a result of other causes as a competitive event, using the method proposed by Kalbfleisch and Prentice.13 The accumulated occurrence of death due to oesophageal cancer according to different characteristics was compared using the test proposed by Gray.14 Finally, in order to identify which characteristics were associated with the risk of dying as a result of oesophageal cancer, a multivariate analysis was carried out using the model proposed by Fine and Gray.15 All of the tests were carried out bilaterally, considering values of P < 0.05 as significant. The analyses were carried out using the programmes Epidat 3.1 (Xunta de Galicia, Santiago de Compostela, Spain), SPSS 19.0 (IBM Company, Chicago, IL, USA), and R 2.15.1 (Free Software Foundation, Boston, MA, USA).
Ethics:
The study was carried out according to the principles laid down in the Declaration of Helsinki and ensuring compliance with Spanish Decree 29/2009, which regulates the use of and access to electronic medical records. Confidentiality was maintained in accordance with the current Spanish Data Protection Law (15/1999). The study received written approval from the regional Ethics Committee for Clinical Research (code 2011/372 CEIC Galicia).
RESULTS:
Characteristics of the patients studied Table 1 shows the baseline characteristics, comorbidities, and cause of death of the patients. The median age was 64.5 years, 87.8% of the sample subjects were male, 58.6% had a body mass index (BMI) within normal range, and 11.4% were obese. The most frequent symptom reported was dysphagia (82.0%), followed by weight loss (49.4%). Regarding tumour stage, 46.8% of the tumours were moderately differentiated, 38.0% were poorly differentiated, and 28.9% had metastasis at the time of diagnosis (Table 2).
BMI, body mass index; CI, confidence interval; SD, standard deviation.
CI, confidence interval.
Table 1 shows the baseline characteristics, comorbidities, and cause of death of the patients. The median age was 64.5 years, 87.8% of the sample subjects were male, 58.6% had a body mass index (BMI) within normal range, and 11.4% were obese. The most frequent symptom reported was dysphagia (82.0%), followed by weight loss (49.4%). Regarding tumour stage, 46.8% of the tumours were moderately differentiated, 38.0% were poorly differentiated, and 28.9% had metastasis at the time of diagnosis (Table 2).
BMI, body mass index; CI, confidence interval; SD, standard deviation.
CI, confidence interval.
Treatment Treatments involved chemotherapy and/or radiotherapy exclusively in 36.1%, surgery as the sole treatment in 23.3%, and a combination of both in 19.4% (Table 2). In the case of patients who only received surgery, resection was carried out for curative purposes in 74%. Both chemotherapy and radiotherapy were mainly applied for palliative purposes.
Treatments involved chemotherapy and/or radiotherapy exclusively in 36.1%, surgery as the sole treatment in 23.3%, and a combination of both in 19.4% (Table 2). In the case of patients who only received surgery, resection was carried out for curative purposes in 74%. Both chemotherapy and radiotherapy were mainly applied for palliative purposes.
Prognosis The specific survival rate at 1, 3, and 5 years after diagnosis obtained with the Kaplan-Meier methodology was 39.9%, 19%, and 15%, respectively, while respective survival rates according to competing risks survival analysis were 40.2%, 18.1%, and 12.4% (Table 3).
CI, confidence interval; HR, hazard ratio; SD, standard deviation.
At 1 year from diagnosis, the probability of dying as a result of the cancer was 59.2%, with the probability of dying from other causes being 0.6% (Figure). At 5 years from diagnosis, the probability of dying as a result of the cancer rose to 83.4% and the probability of dying from other causes was 4.2%; therefore, the probability of survival was reduced to 12.4%.
The variables in the univariate analysis that were significantly associated with the probability of dying during the follow-up period were: gender, Charlson’s comorbidity index, presence of weight loss, histopathological cell type, tumour stage, and type of treatment (Table 3). The specific probability of dying was increased among females (hazard ratio [HR] 1.63; 95% CI, 1.00–2.64), those with a higher score on age-adjusted Charlson’s comorbidity index (HR 1.14; 95% CI, 1.05–1.23), and among those with weight loss on diagnosis (HR 1.68; 95% CI, 1.20–2.34). The histopathologic cell type with the highest mortality rate was adenocarcinoma (HR 1.68; 95% CI, 1.08–2.62). In turn, those with stage IV tumours had a higher mortality rate than those in earlier stages (0-III) (HR 2.38; 95% CI, 1.63–3.46).
The patients who had received some kind of treatment had a lower probability of dying. Those who had received a combination of surgical and oncological treatment (chemotherapy and/or radiotherapy) had the lowest probability of dying (HR 0.17; 95% CI, 0.10–0.27), followed by those who had only received surgical treatment (HR 0.22; 95% CI, 0.12–0.38).
No association was found between the probability of dying during the follow-up period and the following variables: year of diagnosis, age, BMI, personal background (smoking, regular alcohol consumption, gastro-oesophageal reflux, achalasia, and a family history of cancer), presence of dysphagia on diagnosis, or tumour location.
According to the final adjustment by multivariate competing risks analysis, we found that the variables with an independent effect to predict mortality are gender, Charlson’s comorbidity index, and tumour stage (Table 4). In order to adjust for age as a clinically relevant and confounding variable and avoid over-adjustment, we included in the model the crude Charlson’s comorbidity index. The specific probability of dying from oesophageal cancer was increased among females (HR 1.94; 95% CI, 1.24–3.03), those with a higher score on Charlson’s comorbidity index (HR 1.17; 95% CI, 1.02–1.33), and in those with stage IV tumours at the time of diagnosis (HR 1.70; 95% CI, 1.11–2.59). Having received some type of treatment improved the prognosis, with a greater impact in cases that received a combination of surgical and oncological treatment (HR 0.23; 95% CI, 0.12–0.45). Furthermore, no significant effect was found when the interaction between TNM classification and type of treatment was added to the multivariate model (P = 0.600).
AC, adenocarcinoma; B, regression coefficient; CI, confidence interval; HR, hazard ratio; SCC, squamous cell carcinoma; SE, standard error.
The specific survival rate at 1, 3, and 5 years after diagnosis obtained with the Kaplan-Meier methodology was 39.9%, 19%, and 15%, respectively, while respective survival rates according to competing risks survival analysis were 40.2%, 18.1%, and 12.4% (Table 3).
CI, confidence interval; HR, hazard ratio; SD, standard deviation.
At 1 year from diagnosis, the probability of dying as a result of the cancer was 59.2%, with the probability of dying from other causes being 0.6% (Figure). At 5 years from diagnosis, the probability of dying as a result of the cancer rose to 83.4% and the probability of dying from other causes was 4.2%; therefore, the probability of survival was reduced to 12.4%.
The variables in the univariate analysis that were significantly associated with the probability of dying during the follow-up period were: gender, Charlson’s comorbidity index, presence of weight loss, histopathological cell type, tumour stage, and type of treatment (Table 3). The specific probability of dying was increased among females (hazard ratio [HR] 1.63; 95% CI, 1.00–2.64), those with a higher score on age-adjusted Charlson’s comorbidity index (HR 1.14; 95% CI, 1.05–1.23), and among those with weight loss on diagnosis (HR 1.68; 95% CI, 1.20–2.34). The histopathologic cell type with the highest mortality rate was adenocarcinoma (HR 1.68; 95% CI, 1.08–2.62). In turn, those with stage IV tumours had a higher mortality rate than those in earlier stages (0-III) (HR 2.38; 95% CI, 1.63–3.46).
The patients who had received some kind of treatment had a lower probability of dying. Those who had received a combination of surgical and oncological treatment (chemotherapy and/or radiotherapy) had the lowest probability of dying (HR 0.17; 95% CI, 0.10–0.27), followed by those who had only received surgical treatment (HR 0.22; 95% CI, 0.12–0.38).
No association was found between the probability of dying during the follow-up period and the following variables: year of diagnosis, age, BMI, personal background (smoking, regular alcohol consumption, gastro-oesophageal reflux, achalasia, and a family history of cancer), presence of dysphagia on diagnosis, or tumour location.
According to the final adjustment by multivariate competing risks analysis, we found that the variables with an independent effect to predict mortality are gender, Charlson’s comorbidity index, and tumour stage (Table 4). In order to adjust for age as a clinically relevant and confounding variable and avoid over-adjustment, we included in the model the crude Charlson’s comorbidity index. The specific probability of dying from oesophageal cancer was increased among females (HR 1.94; 95% CI, 1.24–3.03), those with a higher score on Charlson’s comorbidity index (HR 1.17; 95% CI, 1.02–1.33), and in those with stage IV tumours at the time of diagnosis (HR 1.70; 95% CI, 1.11–2.59). Having received some type of treatment improved the prognosis, with a greater impact in cases that received a combination of surgical and oncological treatment (HR 0.23; 95% CI, 0.12–0.45). Furthermore, no significant effect was found when the interaction between TNM classification and type of treatment was added to the multivariate model (P = 0.600).
AC, adenocarcinoma; B, regression coefficient; CI, confidence interval; HR, hazard ratio; SCC, squamous cell carcinoma; SE, standard error.
Follow-up The total length of follow-up was 3221.8 months (268.5 years), with a mean of 9.4 months per patient. The most frequently detected events during follow-up were newly appeared metastasis (19.0%)—mainly located in the lungs (48.5%)—and local tumour recurrences (16.7%) (Table 5).
In those patients who underwent curative surgery, the probability of being diagnosed with a tumour recurrence (local tumour recurrence, distant recurrence, or both) during the first year after the surgery was 21.1%, while 25.5% of the patients died without presenting any recurrence. Therefore, 53.4% of the patients who underwent curative surgery were alive and remained disease-free 1 year after the surgery. Similarly, the probability of tumour recurrence 5 years after the surgery was 52.4%, while 29.6% of the patients died without presenting any recurrence. Therefore, only 18% of the patients were alive and disease-free 5 years after curative surgery.
Considering that the follow-up strategy is more intensive once an event occurs during the follow-up period, we studied the follow-up procedure carried out with patients who underwent curative surgery from the time of diagnosis until a new event occurred (local tumour recurrence, newly appeared metastasis, or newly appeared neoplasias) (Table 6). The mean number of hospital consultations per year of follow-up for these patients was 4.1 (standard deviation, 4.2) consultations/year, with a median of 3.1 consultations per year of follow-up (Table 6).
CAT, computer-aided tomography; IQR, inter-quartile range; SD, standard deviation.
When we take into account gender, age, Charlson’s comorbidity index, weight loss, histopathological cell type, TNM stage, treatment carried out, and number of consultations per follow-up year, in the interval between the diagnosis until an event occurs, we see that the consultations carried out during this period do not substantially alter the likelihood of survival (P = 0.640; HR 1.03; 95% CI, 0.92–1.15).
The total length of follow-up was 3221.8 months (268.5 years), with a mean of 9.4 months per patient. The most frequently detected events during follow-up were newly appeared metastasis (19.0%)—mainly located in the lungs (48.5%)—and local tumour recurrences (16.7%) (Table 5).
In those patients who underwent curative surgery, the probability of being diagnosed with a tumour recurrence (local tumour recurrence, distant recurrence, or both) during the first year after the surgery was 21.1%, while 25.5% of the patients died without presenting any recurrence. Therefore, 53.4% of the patients who underwent curative surgery were alive and remained disease-free 1 year after the surgery. Similarly, the probability of tumour recurrence 5 years after the surgery was 52.4%, while 29.6% of the patients died without presenting any recurrence. Therefore, only 18% of the patients were alive and disease-free 5 years after curative surgery.
Considering that the follow-up strategy is more intensive once an event occurs during the follow-up period, we studied the follow-up procedure carried out with patients who underwent curative surgery from the time of diagnosis until a new event occurred (local tumour recurrence, newly appeared metastasis, or newly appeared neoplasias) (Table 6). The mean number of hospital consultations per year of follow-up for these patients was 4.1 (standard deviation, 4.2) consultations/year, with a median of 3.1 consultations per year of follow-up (Table 6).
CAT, computer-aided tomography; IQR, inter-quartile range; SD, standard deviation.
When we take into account gender, age, Charlson’s comorbidity index, weight loss, histopathological cell type, TNM stage, treatment carried out, and number of consultations per follow-up year, in the interval between the diagnosis until an event occurs, we see that the consultations carried out during this period do not substantially alter the likelihood of survival (P = 0.640; HR 1.03; 95% CI, 0.92–1.15).
Characteristics of the patients studied:
Table 1 shows the baseline characteristics, comorbidities, and cause of death of the patients. The median age was 64.5 years, 87.8% of the sample subjects were male, 58.6% had a body mass index (BMI) within normal range, and 11.4% were obese. The most frequent symptom reported was dysphagia (82.0%), followed by weight loss (49.4%). Regarding tumour stage, 46.8% of the tumours were moderately differentiated, 38.0% were poorly differentiated, and 28.9% had metastasis at the time of diagnosis (Table 2).
BMI, body mass index; CI, confidence interval; SD, standard deviation.
CI, confidence interval.
Treatment:
Treatments involved chemotherapy and/or radiotherapy exclusively in 36.1%, surgery as the sole treatment in 23.3%, and a combination of both in 19.4% (Table 2). In the case of patients who only received surgery, resection was carried out for curative purposes in 74%. Both chemotherapy and radiotherapy were mainly applied for palliative purposes.
Prognosis:
The specific survival rate at 1, 3, and 5 years after diagnosis obtained with the Kaplan-Meier methodology was 39.9%, 19%, and 15%, respectively, while respective survival rates according to competing risks survival analysis were 40.2%, 18.1%, and 12.4% (Table 3).
CI, confidence interval; HR, hazard ratio; SD, standard deviation.
At 1 year from diagnosis, the probability of dying as a result of the cancer was 59.2%, with the probability of dying from other causes being 0.6% (Figure). At 5 years from diagnosis, the probability of dying as a result of the cancer rose to 83.4% and the probability of dying from other causes was 4.2%; therefore, the probability of survival was reduced to 12.4%.
The variables in the univariate analysis that were significantly associated with the probability of dying during the follow-up period were: gender, Charlson’s comorbidity index, presence of weight loss, histopathological cell type, tumour stage, and type of treatment (Table 3). The specific probability of dying was increased among females (hazard ratio [HR] 1.63; 95% CI, 1.00–2.64), those with a higher score on age-adjusted Charlson’s comorbidity index (HR 1.14; 95% CI, 1.05–1.23), and among those with weight loss on diagnosis (HR 1.68; 95% CI, 1.20–2.34). The histopathologic cell type with the highest mortality rate was adenocarcinoma (HR 1.68; 95% CI, 1.08–2.62). In turn, those with stage IV tumours had a higher mortality rate than those in earlier stages (0-III) (HR 2.38; 95% CI, 1.63–3.46).
The patients who had received some kind of treatment had a lower probability of dying. Those who had received a combination of surgical and oncological treatment (chemotherapy and/or radiotherapy) had the lowest probability of dying (HR 0.17; 95% CI, 0.10–0.27), followed by those who had only received surgical treatment (HR 0.22; 95% CI, 0.12–0.38).
No association was found between the probability of dying during the follow-up period and the following variables: year of diagnosis, age, BMI, personal background (smoking, regular alcohol consumption, gastro-oesophageal reflux, achalasia, and a family history of cancer), presence of dysphagia on diagnosis, or tumour location.
According to the final adjustment by multivariate competing risks analysis, we found that the variables with an independent effect to predict mortality are gender, Charlson’s comorbidity index, and tumour stage (Table 4). In order to adjust for age as a clinically relevant and confounding variable and avoid over-adjustment, we included in the model the crude Charlson’s comorbidity index. The specific probability of dying from oesophageal cancer was increased among females (HR 1.94; 95% CI, 1.24–3.03), those with a higher score on Charlson’s comorbidity index (HR 1.17; 95% CI, 1.02–1.33), and in those with stage IV tumours at the time of diagnosis (HR 1.70; 95% CI, 1.11–2.59). Having received some type of treatment improved the prognosis, with a greater impact in cases that received a combination of surgical and oncological treatment (HR 0.23; 95% CI, 0.12–0.45). Furthermore, no significant effect was found when the interaction between TNM classification and type of treatment was added to the multivariate model (P = 0.600).
AC, adenocarcinoma; B, regression coefficient; CI, confidence interval; HR, hazard ratio; SCC, squamous cell carcinoma; SE, standard error.
Follow-up:
The total length of follow-up was 3221.8 months (268.5 years), with a mean of 9.4 months per patient. The most frequently detected events during follow-up were newly appeared metastasis (19.0%)—mainly located in the lungs (48.5%)—and local tumour recurrences (16.7%) (Table 5).
In those patients who underwent curative surgery, the probability of being diagnosed with a tumour recurrence (local tumour recurrence, distant recurrence, or both) during the first year after the surgery was 21.1%, while 25.5% of the patients died without presenting any recurrence. Therefore, 53.4% of the patients who underwent curative surgery were alive and remained disease-free 1 year after the surgery. Similarly, the probability of tumour recurrence 5 years after the surgery was 52.4%, while 29.6% of the patients died without presenting any recurrence. Therefore, only 18% of the patients were alive and disease-free 5 years after curative surgery.
Considering that the follow-up strategy is more intensive once an event occurs during the follow-up period, we studied the follow-up procedure carried out with patients who underwent curative surgery from the time of diagnosis until a new event occurred (local tumour recurrence, newly appeared metastasis, or newly appeared neoplasias) (Table 6). The mean number of hospital consultations per year of follow-up for these patients was 4.1 (standard deviation, 4.2) consultations/year, with a median of 3.1 consultations per year of follow-up (Table 6).
CAT, computer-aided tomography; IQR, inter-quartile range; SD, standard deviation.
When we take into account gender, age, Charlson’s comorbidity index, weight loss, histopathological cell type, TNM stage, treatment carried out, and number of consultations per follow-up year, in the interval between the diagnosis until an event occurs, we see that the consultations carried out during this period do not substantially alter the likelihood of survival (P = 0.640; HR 1.03; 95% CI, 0.92–1.15).
DISCUSSION:
In Spain, the occurrence of oesophageal cancer is at a midway point with regard to the rest of Europe.1 It is more frequent in men16–18 and usually appears between the ages of 55 and 70 years,6,19–21 and the most common symptom associated with its appearance is dysphagia.22 The results of the present study confirm these findings and are in line with those of other previously published series.19,20,23,24
With regard to the specific survival rate for these types of tumours, data published from the EUROCARE-44 study, which included 48 353 cases from 23 European countries, reveal a mean estimated survival rate of 35%, 14.1%, and 9.6% at the first, third, and fifth years of follow-up, respectively, highlighting the low survival rate associated with these tumours. These data also concur with those published in countries such as Canada25 or Iran,20 which reported 5-year survival rates of 8.8% and 12%, respectively. Series published in Europe, such as those in Sweden,6 England,26 and France27 found similar figures, with 5-year survival rates between 9.3%–13.1%, 3.2%–9.8%, and 9%–14%, respectively. In our study, the 5-year survival rate was 12.4%. It is important to note that we used the competing risks survival analysis in our study, which is suitable for analysing the behaviour of a person who may die as a result of different causes. Using the Kaplan-Meier methodology, the results obtained for our sample slightly overestimated the survival rate from the third year onwards. The patients in this cohort mainly die as a result of the illness in question, and so for this reason the differences found in the survival rate between the Kaplan-Meier methodology and competing risks survival analysis are very low. Despite this, the competing risks survival analysis method is the most suitable for analysing the specific survival rate in the presence of other causes of death. This overestimation of the survival rate using the Kaplan-Meier methodology in comparison to competing risks survival analysis has previously been described in the literature.9,10
With regard to the factors associated with survival, most studies have indicated that women have higher survival rates than men,5,19,25,27 although in some studies these differences were not significant.20 In a study of patients from the Donostia Hospital in the Basque Country, Spain,23 the mean survival rate in women was lower, as in our study, although the differences were not significant. In our study, we found that the patient’s number and severity of comorbidities, assessed using Charlson’s comorbidity index, is significantly associated with the likelihood of dying as a result of the tumour. In a recent study in the Netherlands,19 having comorbidities was associated with poor survival, as in our study, although no significant differences were found.
Amongst the limitations to the study, we did not study any molecular marker that could play an important role in the prognosis of oesophageal cancer and which, in combination with other parameters such as tumour stage, would help to identify and predict the survival rate from these tumours, as well as to individually adapt the treatment to each patient, as recently described in the literature.28,29 Although our sample only consisted of 180 patients, we were able to compare the consistency of our results with larger series published internationally,5–7,20,23,24,30–32 in which the tumour stage and treatment received by the patient were the main prognostic factors for survival.
This study reveals how the different follow-up strategies used (visits and tests carried out) in patients where the intention is to apply curative treatment until a new event occurs do not alter their survival rate. This finding is in agreement with the lack of consensus at the international level8,33,34 in terms of defining the follow-up protocols for these types of tumours. Furthermore, we did not find any studies in the literature that describe the follow-up process applied to these patients and its possible effect on their survival rate. In The European Society for Medical Oncology’s guidelines for the diagnosis, treatment, and monitoring of oesophageal cancer for 2013,35 the group concluded that, with the exception of patients who could be candidates for rescue surgery after a failed endoscopic resection or definitive chemo-radiotherapy, there is no evidence that regular follow-up after the initial therapy impacts the final outcome.
In conclusion, the specific survival rate detected in our study was low and coincided with figures published at the international level. Our results confirm that studying the survival rate using the Kaplan-Meier methodology overestimates the survival rate in comparison to competing risks survival analysis. We also found that the different follow-up strategies used after diagnosing illness in patients who are surgically treated with the intention to cure do not alter the prognosis.
|
Background:
To determine the clinical course, follow-up strategies, and survival of oesophageal cancer patients using a competing risks survival analysis.
Methods:
We conducted a retrospective and prospective follow-up study. The study included 180 patients with a pathological diagnosis of oesophageal cancer in A Coruña, Spain, between 2003 and 2008. The Kaplan-Meier methodology and competing risks survival analysis were used to calculate the specific survival rate. The study was approved by the Ethics Review Board (code 2011/372, CEIC Galicia).
Results:
The specific survival rate at the first, third, and fifth years was 40.2%, 18.1%, and 12.4%, respectively. Using the Kaplan-Meier methodology, the survival rate was slightly higher after the third year of follow-up. In the multivariate analysis, poor prognosis factors were female sex (hazard ratio [HR] 1.94; 95% confidence interval [CI], 1.24-3.03), Charlson's comorbidity index (HR 1.17; 95% CI, 1.02-1.33), and stage IV tumours (HR 1.70; 95% CI, 1.11-2.59). The probability of dying decreased with surgical and oncological treatment (chemotherapy and/or radiotherapy) (HR 0.23; 95% CI, 0.12-0.45). The number of hospital consultations per year during the follow-up period, from diagnosis to the appearance of a new event (local recurrences, newly appeared metastasis, and newly appeared neoplasias) did not affect the probability of survival (HR 1.03; 95% CI, 0.92-1.15).
Conclusions:
The Kaplan-Meier methodology overestimates the survival rate in comparison to competing risks analysis. The variables associated with a poor prognosis are female sex, Charlson's comorbidity score and extensive tumour invasion. Type of follow-up strategy employed after diagnosis does not affect the prognosis of the disease.
| null | null | 7,038 | 361 | 12 |
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[
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[CONTENT] oesophageal neoplasms | therapeutics | survival | follow-up studies [SUMMARY]
|
[CONTENT] oesophageal neoplasms | therapeutics | survival | follow-up studies [SUMMARY]
|
[CONTENT] oesophageal neoplasms | therapeutics | survival | follow-up studies [SUMMARY]
| null |
[CONTENT] oesophageal neoplasms | therapeutics | survival | follow-up studies [SUMMARY]
| null |
[CONTENT] Aged | Esophageal Neoplasms | Female | Follow-Up Studies | Humans | Kaplan-Meier Estimate | Male | Middle Aged | Prognosis | Prospective Studies | Retrospective Studies | Risk Factors | Sex Distribution | Spain | Survival Rate [SUMMARY]
|
[CONTENT] Aged | Esophageal Neoplasms | Female | Follow-Up Studies | Humans | Kaplan-Meier Estimate | Male | Middle Aged | Prognosis | Prospective Studies | Retrospective Studies | Risk Factors | Sex Distribution | Spain | Survival Rate [SUMMARY]
|
[CONTENT] Aged | Esophageal Neoplasms | Female | Follow-Up Studies | Humans | Kaplan-Meier Estimate | Male | Middle Aged | Prognosis | Prospective Studies | Retrospective Studies | Risk Factors | Sex Distribution | Spain | Survival Rate [SUMMARY]
| null |
[CONTENT] Aged | Esophageal Neoplasms | Female | Follow-Up Studies | Humans | Kaplan-Meier Estimate | Male | Middle Aged | Prognosis | Prospective Studies | Retrospective Studies | Risk Factors | Sex Distribution | Spain | Survival Rate [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] follow | patients | survival | ci | hr | 95 | probability | dying | treatment | diagnosis [SUMMARY]
|
[CONTENT] follow | patients | survival | ci | hr | 95 | probability | dying | treatment | diagnosis [SUMMARY]
|
[CONTENT] follow | patients | survival | ci | hr | 95 | probability | dying | treatment | diagnosis [SUMMARY]
| null |
[CONTENT] follow | patients | survival | ci | hr | 95 | probability | dying | treatment | diagnosis [SUMMARY]
| null |
[CONTENT] rate | cancer | 100 | 100 000 | 000 | european | spain | analysing | survival | different [SUMMARY]
|
[CONTENT] study | data | records | carried | collected | proposed | clinical | de | clinical records | galicia [SUMMARY]
|
[CONTENT] ci | hr | probability | 95 ci | probability dying | 95 | dying | recurrence | table | diagnosis [SUMMARY]
| null |
[CONTENT] survival | ci | study | follow | patients | hr | rate | probability | cancer | surgery [SUMMARY]
| null |
[CONTENT] [SUMMARY]
|
[CONTENT] ||| 180 | Coruña | Spain | between 2003 and 2008 ||| ||| the Ethics Review Board | 2011/372 | CEIC Galicia [SUMMARY]
|
[CONTENT] first | third | fifth years | 40.2% | 18.1% | 12.4% ||| the third year ||| ||| 1.94 | 95% ||| CI | 1.24 | Charlson | 1.17 | 95% | CI | 1.02 | IV | 1.70 | 95% | CI | 1.11-2.59 ||| 0.23 | 95% | CI | 0.12-0.45 ||| 1.03 | 95% | CI | 0.92 [SUMMARY]
| null |
[CONTENT] ||| ||| 180 | Coruña | Spain | between 2003 and 2008 ||| ||| the Ethics Review Board | 2011/372 | CEIC Galicia ||| ||| first | third | fifth years | 40.2% | 18.1% | 12.4% ||| the third year ||| ||| 1.94 | 95% ||| CI | 1.24 | Charlson | 1.17 | 95% | CI | 1.02 | IV | 1.70 | 95% | CI | 1.11-2.59 ||| 0.23 | 95% | CI | 0.12-0.45 ||| 1.03 | 95% | CI | 0.92 ||| ||| Charlson ||| [SUMMARY]
| null |
||||
The longitudinal relationship of changes of adiposity to changes in pulmonary function and risk of asthma in a general adult population.
|
25532602
|
Adiposity has been linked to both higher risk of asthma and reduced lung function. The effects of adiposity on asthma may depend on both atopic status and gender, while the relationship is less clear with respect to lung function. This study aimed to explore longitudinal weight changes to changes in forced expiratory volume in first second (FEV1) and forced vital capacity (FVC), as well as to incident cases of asthma and wheezing, according to atopy and gender.
|
BACKGROUND
|
A general population sample aged 19-72 years was examined with the same methodology five years apart. Longitudinal changes in weight, body mass index, waist circumference, and fat percentage (bio-impedance) were analyzed with respect to changes of FEV1 and FVC (spirometry), and incidence of asthma and wheezing (questionnaire). Gender, atopy (serum specific IgE-positivity to inhalant allergens) and adipose tissue mass prior to adiposity changes were examined as potential effect modifiers.
|
METHODS
|
A total of 2,308 persons participated in both baseline and five-year follow-up examinations. Over the entire span of adiposity changes, adiposity gain was associated with decreasing levels of lung function, whereas adiposity loss was associated with increasing levels of lung function. All associations were dependent on gender (p-interactions < 0.0001). For one standard deviation weight gain or weight loss, FEV1 changed with (+/-)72 ml (66-78 ml) and FVC with (+/-)103 ml (94-112 ml) in males. In females FEV1 changed with (+/-) 27 ml (22-32 ml) and FVC with (+/-) 36 ml (28-44 ml). There were no changes in the FEV1/FVC-ratio. The effect of adiposity changes increased with the level of adipose tissue mass at the start of the study (baseline), thus, indicating an aggregate effect of the total adipose tissue mass. Atopy did not modify these associations. There were no statistically significant associations between changes in adiposity measures and risk of incident asthma or wheeze.
|
RESULTS
|
Over a five-year period, increasing adiposity was associated with decreasing lung function, whereas decreasing adiposity was associated with increasing lung function. This effect was significantly greater in males than in females and increased with pre-existing adiposity, but was independent of atopy.
|
CONCLUSIONS
|
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"Aged",
"Asthma",
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"Immunoglobulin E",
"Longitudinal Studies",
"Lung",
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"Middle Aged",
"Nitric Oxide",
"Obesity",
"Risk",
"Vital Capacity",
"Young Adult"
] |
4364582
|
Background
|
Adiposity has consistently been associated with asthma [1] and has also been associated with impaired lung function, e.g. as assessed by decreased levels of forced expiratory volume in first second (FEV1) and forced vital capacity (FVC) [2–6]. Thus, adiposity may influence the lungs in several ways, e.g. through inflammation leading to asthma-like, obstructive changes, or in terms of a mechanical impact on lung function (restrictive changes).
Adiposity increases the risk of asthma and may cause more severe symptoms along with a reduced response to medications [1, 7, 8]. Some studies have indicated that these effects are stronger in non-atopic individuals than atopic individuals [9–11], although contrary results also have been noted [12]. Further, adiposity has been reported to be more strongly associated with asthma types characterised by non-eosinophilic inflammation rather than by eosinophilic inflammation [13]. However, it has not been investigated whether adiposity also has differential effects on lung function in atopic and non-atopic individuals or in individuals with or without eosinophilic inflammation in the airways. Underlying pathological processes, such as eosinophilic or neutrophilic inflammation [14], could possibly modify the longitudinal relationship of adiposity with lung function.
Mechanically, adiposity may cause an extra load on the thoracic cage [15], increase the intra-abdominal pressure, and impede the movements of the diaphragm [16]. This has in smaller experimental settings been reported to decrease static lung volumes and lead to breathing at smaller tidal volumes [17]. Cross-sectional studies have suggested that these changes are stronger in persons with predominantly abdominal adiposity than in persons with predominantly general adiposity [18]. Yet, prospective population based studies are sparse and have assessed adiposity changes mainly by weight or BMI [2, 3, 19–22], except from one study that included waist and hip circumferences but not FVC [4]. Therefore, studies quantifying longitudinal changes of lung function with respect to different adiposity phenotypes may add further information about the longitudinal relationship between adiposity and lung function.
This prospective general population study aimed to investigate the association of longitudinal changes in weight, body mass index (BMI), waist circumference (WC), and fat percentage with longitudinal changes in FEV1 and FVC, and with concomitant incidence of asthma and wheezing. Further, we examined whether these associations were modified by gender, atopy, or eosinophilic inflammation of the lower airways as reflected by forced expiratory nitric oxide (FENO).
|
Methods
|
Study population The current study used already sampled data from the Health2006 study and the Health2006-follow-up study conducted five years apart. The participants in the baseline Health2006 cohort were drawn as a random sample from the background population aged 18–69 years, living in 11 municipalities in the south-western part of suburban Copenhagen. A total of 3471 individuals (44.7%) entered the study and participated in the health examination, which took place in 2006–08 (Health2006 baseline [23]). In 2011–12, participants in the baseline Health2006 were invited for a 5-year follow-up examination including essentially the same study protocol. A total of 3,405 were eligible for invitation (21 had emigrated and 45 died). A total of 2,308 (68.6%) agreed to participate and were re-examined between November 2011 and November 2012. Fourteen participants did not have valid measures of either FEV1 or FVC at either baseline or follow-up and were excluded from the study, which for the main analyses was based on 2,294 participants. All participants gave written informed consent before taking part in the study, which was approved by the ethics committee of the Capital Region of Denmark, Copenhagen (KA20060011).
The current study used already sampled data from the Health2006 study and the Health2006-follow-up study conducted five years apart. The participants in the baseline Health2006 cohort were drawn as a random sample from the background population aged 18–69 years, living in 11 municipalities in the south-western part of suburban Copenhagen. A total of 3471 individuals (44.7%) entered the study and participated in the health examination, which took place in 2006–08 (Health2006 baseline [23]). In 2011–12, participants in the baseline Health2006 were invited for a 5-year follow-up examination including essentially the same study protocol. A total of 3,405 were eligible for invitation (21 had emigrated and 45 died). A total of 2,308 (68.6%) agreed to participate and were re-examined between November 2011 and November 2012. Fourteen participants did not have valid measures of either FEV1 or FVC at either baseline or follow-up and were excluded from the study, which for the main analyses was based on 2,294 participants. All participants gave written informed consent before taking part in the study, which was approved by the ethics committee of the Capital Region of Denmark, Copenhagen (KA20060011).
Questionnaire data Information on socio-demographic variables, leisure time physical activity, smoking habits, chronic illnesses, respiratory symptoms, and medication were obtained from a questionnaire that was mailed to and answered by the participants before their visit to the research centre. Asthma was defined according to the ECRHS criteria [24] as a confirmatory answer to at least one of the following three questions ‘Have you been woken by an attack of shortness of breath at any time during the last 12 months?’, ‘Have you had an attack of asthma within the last 12 months?’ and ‘Are you currently taking any medication (including inhalers, aerosols or tablets) for asthma?’ Wheezing without a cold (in the following referred to as wheezing) was defined as a confirmatory answer to ‘Have you had wheezing or whistling in your chest at any time during the last 12 months?’ combined with a confirmatory answer to ‘If yes, have you had this wheezing or whistling when you did not have a cold?’. Smoking was recorded as pack years (one pack year equalling one pack of cigarettes per day for a year) and categorised in five levels: (1) never smokers, (2) 0- ≤ 10, (3) 10- ≤ 30, (4) 30- ≤ 50, or (5) more than 50 pack years but was also used as a continuous measure. Educational level was categorised as: (1) less than 2 years, (2) skilled worker, (3) less than 3 years, (4) 3–4 years, and (5) more than four years. All questions used in the present analyses were identical in the baseline and follow-up studies.
Information on socio-demographic variables, leisure time physical activity, smoking habits, chronic illnesses, respiratory symptoms, and medication were obtained from a questionnaire that was mailed to and answered by the participants before their visit to the research centre. Asthma was defined according to the ECRHS criteria [24] as a confirmatory answer to at least one of the following three questions ‘Have you been woken by an attack of shortness of breath at any time during the last 12 months?’, ‘Have you had an attack of asthma within the last 12 months?’ and ‘Are you currently taking any medication (including inhalers, aerosols or tablets) for asthma?’ Wheezing without a cold (in the following referred to as wheezing) was defined as a confirmatory answer to ‘Have you had wheezing or whistling in your chest at any time during the last 12 months?’ combined with a confirmatory answer to ‘If yes, have you had this wheezing or whistling when you did not have a cold?’. Smoking was recorded as pack years (one pack year equalling one pack of cigarettes per day for a year) and categorised in five levels: (1) never smokers, (2) 0- ≤ 10, (3) 10- ≤ 30, (4) 30- ≤ 50, or (5) more than 50 pack years but was also used as a continuous measure. Educational level was categorised as: (1) less than 2 years, (2) skilled worker, (3) less than 3 years, (4) 3–4 years, and (5) more than four years. All questions used in the present analyses were identical in the baseline and follow-up studies.
Physical measurements and assessment of lung function and FENO Anthropometric measures were obtained with participants in light clothing and without shoes. Height was measured to the nearest cm, weight to the nearest 0.1 kg, and body mass index (BMI) was calculated as kg/m2. Waist circumference (WC) was measured to the nearest cm using a tape measure midway between the lower rib margin and the iliac crest. Fat percentage was measured using a foot-to-foot Tanita Body Composition Analyzer (TBF-300, TANITA Corporation of America, Inc., Illinois, U.S.A.). Spirometry was performed according to American Thoracic Society and the European Respiratory Society (ATS/ERS) standard [25] using Spiro USB Spirometers (MicroMedical Limited, Rochester, Kent, UK). Spirometers were checked daily with a 3-liter calibrated syringe, and checked every six months with a decompression flow simulator [26]. FENO measurements were performed only at baseline using a NIOX MINO (Aerocrine AB, Stockholm, Sweden). A dynamic flow restrictor yielded a constant flow rate of 50 mL/s, in accordance with recommendations of the ATS/ERS guidelines for FENO measurement [27]. Nitric oxide concentrations between 5 and 300 ppb were measured and measurements below 5 ppb were set to zero by the NIOX MINO. Nitric oxide concentrations above 20 ppb were considered high.
Anthropometric measures were obtained with participants in light clothing and without shoes. Height was measured to the nearest cm, weight to the nearest 0.1 kg, and body mass index (BMI) was calculated as kg/m2. Waist circumference (WC) was measured to the nearest cm using a tape measure midway between the lower rib margin and the iliac crest. Fat percentage was measured using a foot-to-foot Tanita Body Composition Analyzer (TBF-300, TANITA Corporation of America, Inc., Illinois, U.S.A.). Spirometry was performed according to American Thoracic Society and the European Respiratory Society (ATS/ERS) standard [25] using Spiro USB Spirometers (MicroMedical Limited, Rochester, Kent, UK). Spirometers were checked daily with a 3-liter calibrated syringe, and checked every six months with a decompression flow simulator [26]. FENO measurements were performed only at baseline using a NIOX MINO (Aerocrine AB, Stockholm, Sweden). A dynamic flow restrictor yielded a constant flow rate of 50 mL/s, in accordance with recommendations of the ATS/ERS guidelines for FENO measurement [27]. Nitric oxide concentrations between 5 and 300 ppb were measured and measurements below 5 ppb were set to zero by the NIOX MINO. Nitric oxide concentrations above 20 ppb were considered high.
Biomarkers Baseline serum samples were analysed for serum specific IgE against the four most clinically important inhalant allergens in Denmark: birch, grass, cat, and the house dust mite Dermatophagoides pteronyssinus, using the ADVIA Centaur Specific IgE assay [28]. Atopy was defined as at least one positive test (≥0.35 kilo units/L) against specific IgE. Plasma glucose concentrations were analysed by a hexokinase/glucose-6-phosphate dehydrogenase assay (Roche Diagnostic, Germany), and insulin concentrations were measured by fluoroimmunoassay technique (Dako Diagnostics Ltd., UK). The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated as: fasting plasma glucose (mmol/L) x fasting serum insulin (mU/L)/22.5 and applied only to the non-diabetic individuals. High-density lipoprotein (HDL) and triglyceride concentrations were measured by standard enzymatic techniques (Roche Diagnostic, Germany).
Baseline serum samples were analysed for serum specific IgE against the four most clinically important inhalant allergens in Denmark: birch, grass, cat, and the house dust mite Dermatophagoides pteronyssinus, using the ADVIA Centaur Specific IgE assay [28]. Atopy was defined as at least one positive test (≥0.35 kilo units/L) against specific IgE. Plasma glucose concentrations were analysed by a hexokinase/glucose-6-phosphate dehydrogenase assay (Roche Diagnostic, Germany), and insulin concentrations were measured by fluoroimmunoassay technique (Dako Diagnostics Ltd., UK). The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated as: fasting plasma glucose (mmol/L) x fasting serum insulin (mU/L)/22.5 and applied only to the non-diabetic individuals. High-density lipoprotein (HDL) and triglyceride concentrations were measured by standard enzymatic techniques (Roche Diagnostic, Germany).
Statistics We used the R-statistical package, version 3.0.2 (http://www.r-project.org/) for all analyses. All p-values are two-tailed and statistical significance defined as p <0.05. P-values of likelihood ratio tests were used to test for significance of all multivariate analyses. We used linear regression and checked for equal residual variance across the full range of all exposure variables to check the linear model assumptions. We also tested possible non-linear associations by dividing the exposure variables in splines and by testing the quadratic term of the variables in the regression models. For our main analyses, we used absolute or percentage changes (between baseline and five-year follow up) of the adiposity measures to changes in FEV1 and FVC. The changes of FEV1 (or FVC) were modelled as five-year FEV1 (or FVC) adjusted for FEV1 (or FVC) at baseline. We also tested the ‘reverse’ association of baseline lung function measures to changes of adiposity measures. The adiposity measures were standardised by division of each measure by its own standard error to ease interpretation of the regression coefficients and to ease a comparison of the magnitude of effects between the different adiposity measures. In analyses of incidence of asthma and wheezing, logistic regression was used, similar model checks were applied, and the adiposity measures were categorised in four equally sized groups by quartiles of their respective distributions. Possible interactions were tested by adding an interaction term to the regression models, e.g. (adiposity measure*atopy) or (adiposity measure*gender). Finally, all analyses were repeated with s-Trig, s-HDL, and HOMA-IR included in the models to test whether these metabolic markers would attenuate the estimates found for adiposity. Sensitivity analyses included exclusion of the lower and upper 0.0025, 0.005, 0.01 fractiles of the adiposity measures, as well as separate analyses in non- and never smokers, and in younger or older (age categories were +/− 40, +/−45, +/−50 years) individuals.
Finally, we made a simulation-model based on randomly generated heights, BMI-levels, and levels of lung volume dependent on height. This model gives an example of how adipose tissue above a certain ‘person-dependent ideal’ would influence lung function (Additional file 1).
We used the R-statistical package, version 3.0.2 (http://www.r-project.org/) for all analyses. All p-values are two-tailed and statistical significance defined as p <0.05. P-values of likelihood ratio tests were used to test for significance of all multivariate analyses. We used linear regression and checked for equal residual variance across the full range of all exposure variables to check the linear model assumptions. We also tested possible non-linear associations by dividing the exposure variables in splines and by testing the quadratic term of the variables in the regression models. For our main analyses, we used absolute or percentage changes (between baseline and five-year follow up) of the adiposity measures to changes in FEV1 and FVC. The changes of FEV1 (or FVC) were modelled as five-year FEV1 (or FVC) adjusted for FEV1 (or FVC) at baseline. We also tested the ‘reverse’ association of baseline lung function measures to changes of adiposity measures. The adiposity measures were standardised by division of each measure by its own standard error to ease interpretation of the regression coefficients and to ease a comparison of the magnitude of effects between the different adiposity measures. In analyses of incidence of asthma and wheezing, logistic regression was used, similar model checks were applied, and the adiposity measures were categorised in four equally sized groups by quartiles of their respective distributions. Possible interactions were tested by adding an interaction term to the regression models, e.g. (adiposity measure*atopy) or (adiposity measure*gender). Finally, all analyses were repeated with s-Trig, s-HDL, and HOMA-IR included in the models to test whether these metabolic markers would attenuate the estimates found for adiposity. Sensitivity analyses included exclusion of the lower and upper 0.0025, 0.005, 0.01 fractiles of the adiposity measures, as well as separate analyses in non- and never smokers, and in younger or older (age categories were +/− 40, +/−45, +/−50 years) individuals.
Finally, we made a simulation-model based on randomly generated heights, BMI-levels, and levels of lung volume dependent on height. This model gives an example of how adipose tissue above a certain ‘person-dependent ideal’ would influence lung function (Additional file 1).
|
Results
|
The baseline characteristics of the study population are given in Table 1. All characteristics are listed separately for participants and non-participants in the follow-up study. Of note is that non-participants as compared to participants had significantly higher levels of adiposity measures and higher levels of FEV1 and FVC. However, the baseline associations of adipose tissue levels with asthma, wheezing, FEV1 or FVC were not significantly different between participants and non-participants, all p-values for interaction between participation and each adiposity measure were between 0.12 and 0.98. Sensitivity analyses did not change the pattern of results presented.Table 1
Baseline characteristics of the study population according to participation in the five-year follow-up study
Participants follow-upNon-participants follow-upN = 2294N = 1163MeanSdn missMeanSdn missAge (y)50.0212.4948.0213.98FEV1 (liter)3.160.7993.040.885FVC (liter)4.030.9693.891.065
N
% group
N
% group
Gender
Males104845.749542.6
Adiposity measures
18.5 > BMI (kg/m2)381.7272.3 18.5 < =BMI < 25 (kg/m2)109647.849142.2 25 < =BMI < 30 (kg/m2)83936.640835.1 30 < =BMI (kg/m2)31913.9223720.4 WC, normal§
174876.235950.7 WC, high§
54523.8234949.33
Serum lipids
HDL, normal§
203689.11099386.313 HDL, low§
24910.915813.7 Triglyc <150 mg/dl190183.288777.1 Triglyc 150–500 mg/dl23010.116114.0 Triglyc > = 500 mg/dl1546.7101038.913
Atopy, asthma, wheezing
Atopy*, not present174175.992479.4 Atopy*, present55324.123920.6 Asthma, not present187282.290678.9 Asthma, present40517.81724221.115 Wheezing, not present197486.791579.9 Wheezing, present30313.31723020.118
Lifestyle
Smoking, never102748.21841738.9 Smoking, 0- ≤ 10 pack years49223.125323.6 Smoking, >10 pack years61328.840337.689 Alcohol, no drinking22510.115914.3 Alcohol 1–14 units/week144865.071464.4 Alcohol >14 units/week55524.96623621.354 Education§, none23910.521518.8 Education§, 1 year/practical54323.927223.8 Education§§, 2–3 years94341.445039.3 Education§§, > = 4 years55124.21820818.218FEV1, forced expiratory volumes first second; FVC, forced vital capacity; BMI, body mass index; WC, waist circumference; Diabetes*, either self-reported diabetes or fasting p-glucose > =7.1 mmol/L; HDL, serum level of high-density lipoprotein; Triglyc, serum level of triglyceride; *Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; §above primary school, §§above high school.
Baseline characteristics of the study population according to participation in the five-year follow-up study
FEV1, forced expiratory volumes first second; FVC, forced vital capacity; BMI, body mass index; WC, waist circumference; Diabetes*, either self-reported diabetes or fasting p-glucose > =7.1 mmol/L; HDL, serum level of high-density lipoprotein; Triglyc, serum level of triglyceride; *Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; §above primary school, §§above high school.
From baseline to five-year follow-up, lung function was inversely associated with the level of adiposity as reflected by all four adiposity measures. In participants with weight gain, lung function declined; whereas in participants with weight loss, lung function increased to a similar extend (Figure 1 and Table 2). This pattern was observed in both males and females but the strength of these associations differed significantly between the two genders (Table 2): For all four adiposity measures, the estimated effect of adiposity changes on changes in FEV1 and FVC were approximately three times higher in males than in females in absolute numbers. The percentage changes were the double in males as compared to females. For instance, for every one standard deviation of weight change (5.4 kg) from baseline to follow-up, FEV1 either increased or decreased with approximately 72 ml (66-78 ml) in males and 26 ml (21-31 ml) in females, similarly FVC either increased or decreased with 111 ml (101-121 ml) in males and 35 ml (27-43 ml) in females. The metabolic markers, s-triglyceride, s-HDL, or HOMA-IR did not attenuate the effects of adiposity measures on changes in FEV1 and FVC (data not shown). Estimated mean declines for one year of increasing age were approximately 32.8 (31.3-34.4) ml FEV1 and 21.4 (19.0-23.9) ml FVC. In never-smokers, the corresponding estimates were 31.0 (28.7-33.3) ml FEV1and 21.0 (17.4-24.6) ml FVC. All age-estimates were essentially similar in males and females but were significantly smaller in younger than in older individuals.Figure 1
Changes of lung function according to weight changes. Five-year changes of FEV1 (upper panel) and FVC (lower panel) in quintiles of weight change in males (circles) and females (diamonds). Quintile one-two include individuals with weight loss, quintile four-five include individuals with weight gain. The middle quintile is used as reference. Analysis adjusted for age, tobacco use (pack years), and atopy.
Changes of lung function according to weight changes. Five-year changes of FEV1 (upper panel) and FVC (lower panel) in quintiles of weight change in males (circles) and females (diamonds). Quintile one-two include individuals with weight loss, quintile four-five include individuals with weight gain. The middle quintile is used as reference. Analysis adjusted for age, tobacco use (pack years), and atopy.
Five-year changes of FEV (ml) and FVC (ml) in males and females per one standard deviation (1 sd) increase of adiposity measure
Linear regression models fitted with interaction between the specific adiposity measure and gender. Adjusted for age, atopy, tobacco use (pack years). Est, beta-estimate; Se, standard error for Est; p, p-value; p-i, p-value for difference (interaction) between males and females; BMI, body mass index; WC, waist circumference; FatP, fat percentage.
The five-year adiposity changes had a differential impact on lung function changes according to baseline adiposity levels, as assessed as baseline BMI (Figure 2). Five-year adiposity changes were associated with dose-dependent greater declines of FEV1 and FVC in participants who were overweight and obese at baseline as compared to participants who were lean at baseline (Figure 2). Again, this effect was significantly different in males and females but the interaction of baseline-BMI*five-year changes of adiposity was only significant with respect to FVC (p = 0.02). Essentially similar results can be obtained by running our simulation example (Additional file 1). There were not any associations between changes of adiposity with changes of the FEV1/FVC-ratio between baseline and follow-up. For one standard deviation change of any adiposity measure, the FEV1/FVC-ratio changed between −0.0023 (fat percentage) to 0.00097 (BMI) in males (p-values 0.14-0.83) and this was not different in females (p for interactions 0.66-0.99).Figure 2
Changes of lung function according to weight changes and baseline adiposity. Five-year changes of FEV1 (upper panel) and FVC (lower panel) per one standard deviation increase of weight between baseline and follow-up according to baseline levels of BMI (X-axis) in males (circles) and females (diamonds), adjusted for age, atopy, tobacco use (pack years), and atopy.
Changes of lung function according to weight changes and baseline adiposity. Five-year changes of FEV1 (upper panel) and FVC (lower panel) per one standard deviation increase of weight between baseline and follow-up according to baseline levels of BMI (X-axis) in males (circles) and females (diamonds), adjusted for age, atopy, tobacco use (pack years), and atopy.
To further investigate the temporal relationship of associations between changes in adiposity and lung function, we performed ‘reverse’ analyses where baseline FEV1 and FVC were used as explanatory variables for subsequent adiposity changes. However, there were no such statistically significant associations. Thus, these results support that changes in adiposity have an influence on lung function, but lung function does not have a similar influence on changes in adiposity.
The inverse association of adiposity with lung function was confirmed in both persons with and without asthma at baseline (Table 3). The gender differences in lung function declines from baseline to follow-up were also independent of asthma status at baseline (Table 3). With respect to atopy and FENO -levels, there were essentially no differences in the associations of adiposity changes to changes of FEV1 and FVC between atopic and non-atopic individuals or between persons with or without elevated FENO-levels (Table 4). Analyses in never- and non-smokers gave similar results (data not shown).Table 3
Five-year changes of FEV1 (ml) and FVC (ml) in males and females per one standard deviation increase of BMI from baseline to follow-up, according to asthma status at baseline
MalesFemalesEstSepEstSep
FEV1
Asthma−96.416.7<.001−10.710.80.32Non-asthma−75.67.0<.001−29.15.2<.001
FVC
Asthma−109.724.4<.001−15.015.80.34Non-asthma−115.011.0<.001−37.38.1<.001All models fitted with BMI-gender interaction (all interactions significant, p ≤ .001) and adjusted for age, atopy, and smoking (pack years). BMI, body mass index; Est, beta-estimate; Se, standard error; ml, milliliter; p, p-value for Est.Table 4
Five-year changes of FEV (ml) and FVC (ml) in non-atopic and atopic individuals (upper panel), and in individuals with or without elevated FeNO-levels (lower panel), per one standard deviation increase of adiposity measures
Est (ml)Se (ml)PEst (ml)Se (ml)Pp-i
Non-atopic
Atopic
FEV1
Weight (1 sd)−46.04.2<.001−46.28.5<.001ns BMI (1 sd)−42.34.2<.001−47.08.6<.001ns WC (1 sd)−38.94.3<.001−43.18.3<.001ns FatP (1 sd)−30.64.3<.001−35.88.5<.001ns
FVC
Weight (1 sd)−65.56.5<.001−59.313.1<.001ns BMI (1 sd)−61.66.6<.001−54.813.2<.001ns WC (1 sd)−53.26.6<.001−48.612.8<.001ns Fat (1 sd)−32.36.7<.001−32.113.1<.02ns
Normal FeNO (<=20 ppb)
Elevated FeNO (>20 ppb)
FEV1
Weight (1 sd)−45.64.7<.001−48.77.6<.001ns BMI (1 sd)−43.44.7<.001−43.97.7<.001ns WC (1 sd)−43.34.8<.001−31.77.2<.001ns FatP (1 sd)−31.84.8<.001−24.37.4<.001ns
FVC
Weight (1 sd)−57.17.2<.001−85.212.6<.0010.04 BMI (1 sd)−54.57.2<.001−78.512.8<.001ns WC (1 sd)−48.17.5<.001−59.411.9<.001ns FatP (1 sd)−32.27.5<.001−28.112.4<.02nsLinear regression models fitted with interaction between adiposity measure and either atopy or elevated FeNO and adjusted for age, atopy, and smoking (pack years); p-i, p-values for interaction test. Est, beta-estimate; Se, standard error of Est; p, p-value; Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; FeNO, fractional expiratory nitric oxide;1 sd, one standard deviation; ns, non-significant; BMI, body mass index; WC, waist circumference; FatP, fat percentage.
Five-year changes of FEV1 (ml) and FVC (ml) in males and females per one standard deviation increase of BMI from baseline to follow-up, according to asthma status at baseline
All models fitted with BMI-gender interaction (all interactions significant, p ≤ .001) and adjusted for age, atopy, and smoking (pack years). BMI, body mass index; Est, beta-estimate; Se, standard error; ml, milliliter; p, p-value for Est.
Five-year changes of FEV (ml) and FVC (ml) in non-atopic and atopic individuals (upper panel), and in individuals with or without elevated FeNO-levels (lower panel), per one standard deviation increase of adiposity measures
Linear regression models fitted with interaction between adiposity measure and either atopy or elevated FeNO and adjusted for age, atopy, and smoking (pack years); p-i, p-values for interaction test. Est, beta-estimate; Se, standard error of Est; p, p-value; Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; FeNO, fractional expiratory nitric oxide;1 sd, one standard deviation; ns, non-significant; BMI, body mass index; WC, waist circumference; FatP, fat percentage.
Finally, we investigated whether baseline levels of adiposity measures or changes of adiposity levels from baseline to follow-up were associated with incident asthma and wheezing. The incidence of asthma and wheezing between baseline and follow-up was 7.0% (158/2265) and 5.9% (133/2263), respectively. There were no statistically significant associations of any adiposity measure (baseline or changes) with incident asthma but for changes of weight and BMI there was a positive association with wheezing (Table 5). Notably, the novel cases of asthma in our cohort could be categorised as ‘late-onset’ asthma because all participants were > =19 years at baseline. Restricting our analysis further to novel cases of asthma and wheezing in participants e.g. above > = 25 years or > = 40 years yielded similar results. There were no differences between atopic and non-atopic individuals or individuals with or without elevated FENO-levels with respect to incident asthma or wheezing (data not shown).Table 5
The association of changes of adiposity measures (quartiles) between baseline and five-year follow-up with risk of novel (incident) asthma and wheezing
Incident asthmaIncident wheezingNnn/NORCI1CI2nn/NORCI1CI2
Weight
Kg
q1<−1.93570325.7
ref
--264.7
ref
--q21.93 - <0.7573478.31.470.912.42274.70.890.481.63q30.7 - <3.2570325.70.950.561.62346.01.250.722.20q4>3.2576468.11.170.711.95468.11.570.932.70
BMI
Kg/m
2
q1<−0.57572366.4
ref
--254.5
ref
--q2−0.57 - <0.31572437.61.170.721.90274.81.020.561.86q30.31 - <1.216572366.41.000.611.65356.21.350.772.41q4>1.22573427.40.880.531.43467.11.690.992.94
WC
Cm
q1<−1.5564305.4
Ref
--274.9
ref
--q2−1.5 - <2576396.91.150.681.96254.40.850.461.57q32 - <5525458.71.620.982.71438.31.670.982.91q4>5626436.91.140.691.91386.21.270.742.22
FatP
Percentage points
q1<−1.40569325.7
ref
--315.0
ref
--q2−1.40 - <0.50570478.31.450.892.38346.01.130.661.96q30.50 - <2.402569427.51.240.752.06254.50.770.421.38q4>2.40570335.80.780.451.35407.11.200.712.05Number of participants in each group (N), number of novel cases (n) and age, gender, and tobacco (pack years) adjusted odds ratios (OR) with 95% confidence intervals (CI1-CI2)); q1-q4, quartiles 1-4; BMI, body mass index; WC, waist circumference; FatP, Fat percentage.
The association of changes of adiposity measures (quartiles) between baseline and five-year follow-up with risk of novel (incident) asthma and wheezing
Number of participants in each group (N), number of novel cases (n) and age, gender, and tobacco (pack years) adjusted odds ratios (OR) with 95% confidence intervals (CI1-CI2)); q1-q4, quartiles 1-4; BMI, body mass index; WC, waist circumference; FatP, Fat percentage.
|
Conclusion
|
In conclusion, changes of adiposity had a high, gender specific impact on FEV1 and FVC but not on the FEV1/FVC-ratio. Increasing adiposity was associated with lung function decline, whereas loss of adiposity was associated with increasing lung function. These associations were independent of atopy, FENO -levels, and metabolic markers, but were modified by the level of adipose tissue prior to the adiposity changes indicating the importance of aggregate adipose tissue mass. The adiposity changes leading to declines of FEV1 and FVC were not concomitantly associated with incidence of asthma or wheezing, however, a low number of novel cases of asthma and wheezing may have limited the power to detect associations with these outcomes.
|
[
"Background",
"Study population",
"Questionnaire data",
"Physical measurements and assessment of lung function and FENO",
"Biomarkers",
"Statistics",
""
] |
[
"Adiposity has consistently been associated with asthma [1] and has also been associated with impaired lung function, e.g. as assessed by decreased levels of forced expiratory volume in first second (FEV1) and forced vital capacity (FVC) [2–6]. Thus, adiposity may influence the lungs in several ways, e.g. through inflammation leading to asthma-like, obstructive changes, or in terms of a mechanical impact on lung function (restrictive changes).\nAdiposity increases the risk of asthma and may cause more severe symptoms along with a reduced response to medications [1, 7, 8]. Some studies have indicated that these effects are stronger in non-atopic individuals than atopic individuals [9–11], although contrary results also have been noted [12]. Further, adiposity has been reported to be more strongly associated with asthma types characterised by non-eosinophilic inflammation rather than by eosinophilic inflammation [13]. However, it has not been investigated whether adiposity also has differential effects on lung function in atopic and non-atopic individuals or in individuals with or without eosinophilic inflammation in the airways. Underlying pathological processes, such as eosinophilic or neutrophilic inflammation [14], could possibly modify the longitudinal relationship of adiposity with lung function.\nMechanically, adiposity may cause an extra load on the thoracic cage [15], increase the intra-abdominal pressure, and impede the movements of the diaphragm [16]. This has in smaller experimental settings been reported to decrease static lung volumes and lead to breathing at smaller tidal volumes [17]. Cross-sectional studies have suggested that these changes are stronger in persons with predominantly abdominal adiposity than in persons with predominantly general adiposity [18]. Yet, prospective population based studies are sparse and have assessed adiposity changes mainly by weight or BMI [2, 3, 19–22], except from one study that included waist and hip circumferences but not FVC [4]. Therefore, studies quantifying longitudinal changes of lung function with respect to different adiposity phenotypes may add further information about the longitudinal relationship between adiposity and lung function.\nThis prospective general population study aimed to investigate the association of longitudinal changes in weight, body mass index (BMI), waist circumference (WC), and fat percentage with longitudinal changes in FEV1 and FVC, and with concomitant incidence of asthma and wheezing. Further, we examined whether these associations were modified by gender, atopy, or eosinophilic inflammation of the lower airways as reflected by forced expiratory nitric oxide (FENO).",
"The current study used already sampled data from the Health2006 study and the Health2006-follow-up study conducted five years apart. The participants in the baseline Health2006 cohort were drawn as a random sample from the background population aged 18–69 years, living in 11 municipalities in the south-western part of suburban Copenhagen. A total of 3471 individuals (44.7%) entered the study and participated in the health examination, which took place in 2006–08 (Health2006 baseline [23]). In 2011–12, participants in the baseline Health2006 were invited for a 5-year follow-up examination including essentially the same study protocol. A total of 3,405 were eligible for invitation (21 had emigrated and 45 died). A total of 2,308 (68.6%) agreed to participate and were re-examined between November 2011 and November 2012. Fourteen participants did not have valid measures of either FEV1 or FVC at either baseline or follow-up and were excluded from the study, which for the main analyses was based on 2,294 participants. All participants gave written informed consent before taking part in the study, which was approved by the ethics committee of the Capital Region of Denmark, Copenhagen (KA20060011).",
"Information on socio-demographic variables, leisure time physical activity, smoking habits, chronic illnesses, respiratory symptoms, and medication were obtained from a questionnaire that was mailed to and answered by the participants before their visit to the research centre. Asthma was defined according to the ECRHS criteria [24] as a confirmatory answer to at least one of the following three questions ‘Have you been woken by an attack of shortness of breath at any time during the last 12 months?’, ‘Have you had an attack of asthma within the last 12 months?’ and ‘Are you currently taking any medication (including inhalers, aerosols or tablets) for asthma?’ Wheezing without a cold (in the following referred to as wheezing) was defined as a confirmatory answer to ‘Have you had wheezing or whistling in your chest at any time during the last 12 months?’ combined with a confirmatory answer to ‘If yes, have you had this wheezing or whistling when you did not have a cold?’. Smoking was recorded as pack years (one pack year equalling one pack of cigarettes per day for a year) and categorised in five levels: (1) never smokers, (2) 0- ≤ 10, (3) 10- ≤ 30, (4) 30- ≤ 50, or (5) more than 50 pack years but was also used as a continuous measure. Educational level was categorised as: (1) less than 2 years, (2) skilled worker, (3) less than 3 years, (4) 3–4 years, and (5) more than four years. All questions used in the present analyses were identical in the baseline and follow-up studies.",
"Anthropometric measures were obtained with participants in light clothing and without shoes. Height was measured to the nearest cm, weight to the nearest 0.1 kg, and body mass index (BMI) was calculated as kg/m2. Waist circumference (WC) was measured to the nearest cm using a tape measure midway between the lower rib margin and the iliac crest. Fat percentage was measured using a foot-to-foot Tanita Body Composition Analyzer (TBF-300, TANITA Corporation of America, Inc., Illinois, U.S.A.). Spirometry was performed according to American Thoracic Society and the European Respiratory Society (ATS/ERS) standard [25] using Spiro USB Spirometers (MicroMedical Limited, Rochester, Kent, UK). Spirometers were checked daily with a 3-liter calibrated syringe, and checked every six months with a decompression flow simulator [26]. FENO measurements were performed only at baseline using a NIOX MINO (Aerocrine AB, Stockholm, Sweden). A dynamic flow restrictor yielded a constant flow rate of 50 mL/s, in accordance with recommendations of the ATS/ERS guidelines for FENO measurement [27]. Nitric oxide concentrations between 5 and 300 ppb were measured and measurements below 5 ppb were set to zero by the NIOX MINO. Nitric oxide concentrations above 20 ppb were considered high.",
"Baseline serum samples were analysed for serum specific IgE against the four most clinically important inhalant allergens in Denmark: birch, grass, cat, and the house dust mite Dermatophagoides pteronyssinus, using the ADVIA Centaur Specific IgE assay [28]. Atopy was defined as at least one positive test (≥0.35 kilo units/L) against specific IgE. Plasma glucose concentrations were analysed by a hexokinase/glucose-6-phosphate dehydrogenase assay (Roche Diagnostic, Germany), and insulin concentrations were measured by fluoroimmunoassay technique (Dako Diagnostics Ltd., UK). The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated as: fasting plasma glucose (mmol/L) x fasting serum insulin (mU/L)/22.5 and applied only to the non-diabetic individuals. High-density lipoprotein (HDL) and triglyceride concentrations were measured by standard enzymatic techniques (Roche Diagnostic, Germany).",
"We used the R-statistical package, version 3.0.2 (http://www.r-project.org/) for all analyses. All p-values are two-tailed and statistical significance defined as p <0.05. P-values of likelihood ratio tests were used to test for significance of all multivariate analyses. We used linear regression and checked for equal residual variance across the full range of all exposure variables to check the linear model assumptions. We also tested possible non-linear associations by dividing the exposure variables in splines and by testing the quadratic term of the variables in the regression models. For our main analyses, we used absolute or percentage changes (between baseline and five-year follow up) of the adiposity measures to changes in FEV1 and FVC. The changes of FEV1 (or FVC) were modelled as five-year FEV1 (or FVC) adjusted for FEV1 (or FVC) at baseline. We also tested the ‘reverse’ association of baseline lung function measures to changes of adiposity measures. The adiposity measures were standardised by division of each measure by its own standard error to ease interpretation of the regression coefficients and to ease a comparison of the magnitude of effects between the different adiposity measures. In analyses of incidence of asthma and wheezing, logistic regression was used, similar model checks were applied, and the adiposity measures were categorised in four equally sized groups by quartiles of their respective distributions. Possible interactions were tested by adding an interaction term to the regression models, e.g. (adiposity measure*atopy) or (adiposity measure*gender). Finally, all analyses were repeated with s-Trig, s-HDL, and HOMA-IR included in the models to test whether these metabolic markers would attenuate the estimates found for adiposity. Sensitivity analyses included exclusion of the lower and upper 0.0025, 0.005, 0.01 fractiles of the adiposity measures, as well as separate analyses in non- and never smokers, and in younger or older (age categories were +/− 40, +/−45, +/−50 years) individuals.\nFinally, we made a simulation-model based on randomly generated heights, BMI-levels, and levels of lung volume dependent on height. This model gives an example of how adipose tissue above a certain ‘person-dependent ideal’ would influence lung function (Additional file 1).",
"Additional file 1:\nSimulation model of lung function depending on BMI.\n(DOCX 15 KB)\nAdditional file 2:\nOverview of longitudinal studies of the association between increasing adiposity and decreasing lung function conducted in general adult populations.\n(DOCX 24 KB)"
] |
[
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Study population",
"Questionnaire data",
"Physical measurements and assessment of lung function and FENO",
"Biomarkers",
"Statistics",
"Results",
"Discussion",
"Conclusion",
"Electronic supplementary material",
""
] |
[
"Adiposity has consistently been associated with asthma [1] and has also been associated with impaired lung function, e.g. as assessed by decreased levels of forced expiratory volume in first second (FEV1) and forced vital capacity (FVC) [2–6]. Thus, adiposity may influence the lungs in several ways, e.g. through inflammation leading to asthma-like, obstructive changes, or in terms of a mechanical impact on lung function (restrictive changes).\nAdiposity increases the risk of asthma and may cause more severe symptoms along with a reduced response to medications [1, 7, 8]. Some studies have indicated that these effects are stronger in non-atopic individuals than atopic individuals [9–11], although contrary results also have been noted [12]. Further, adiposity has been reported to be more strongly associated with asthma types characterised by non-eosinophilic inflammation rather than by eosinophilic inflammation [13]. However, it has not been investigated whether adiposity also has differential effects on lung function in atopic and non-atopic individuals or in individuals with or without eosinophilic inflammation in the airways. Underlying pathological processes, such as eosinophilic or neutrophilic inflammation [14], could possibly modify the longitudinal relationship of adiposity with lung function.\nMechanically, adiposity may cause an extra load on the thoracic cage [15], increase the intra-abdominal pressure, and impede the movements of the diaphragm [16]. This has in smaller experimental settings been reported to decrease static lung volumes and lead to breathing at smaller tidal volumes [17]. Cross-sectional studies have suggested that these changes are stronger in persons with predominantly abdominal adiposity than in persons with predominantly general adiposity [18]. Yet, prospective population based studies are sparse and have assessed adiposity changes mainly by weight or BMI [2, 3, 19–22], except from one study that included waist and hip circumferences but not FVC [4]. Therefore, studies quantifying longitudinal changes of lung function with respect to different adiposity phenotypes may add further information about the longitudinal relationship between adiposity and lung function.\nThis prospective general population study aimed to investigate the association of longitudinal changes in weight, body mass index (BMI), waist circumference (WC), and fat percentage with longitudinal changes in FEV1 and FVC, and with concomitant incidence of asthma and wheezing. Further, we examined whether these associations were modified by gender, atopy, or eosinophilic inflammation of the lower airways as reflected by forced expiratory nitric oxide (FENO).",
" Study population The current study used already sampled data from the Health2006 study and the Health2006-follow-up study conducted five years apart. The participants in the baseline Health2006 cohort were drawn as a random sample from the background population aged 18–69 years, living in 11 municipalities in the south-western part of suburban Copenhagen. A total of 3471 individuals (44.7%) entered the study and participated in the health examination, which took place in 2006–08 (Health2006 baseline [23]). In 2011–12, participants in the baseline Health2006 were invited for a 5-year follow-up examination including essentially the same study protocol. A total of 3,405 were eligible for invitation (21 had emigrated and 45 died). A total of 2,308 (68.6%) agreed to participate and were re-examined between November 2011 and November 2012. Fourteen participants did not have valid measures of either FEV1 or FVC at either baseline or follow-up and were excluded from the study, which for the main analyses was based on 2,294 participants. All participants gave written informed consent before taking part in the study, which was approved by the ethics committee of the Capital Region of Denmark, Copenhagen (KA20060011).\nThe current study used already sampled data from the Health2006 study and the Health2006-follow-up study conducted five years apart. The participants in the baseline Health2006 cohort were drawn as a random sample from the background population aged 18–69 years, living in 11 municipalities in the south-western part of suburban Copenhagen. A total of 3471 individuals (44.7%) entered the study and participated in the health examination, which took place in 2006–08 (Health2006 baseline [23]). In 2011–12, participants in the baseline Health2006 were invited for a 5-year follow-up examination including essentially the same study protocol. A total of 3,405 were eligible for invitation (21 had emigrated and 45 died). A total of 2,308 (68.6%) agreed to participate and were re-examined between November 2011 and November 2012. Fourteen participants did not have valid measures of either FEV1 or FVC at either baseline or follow-up and were excluded from the study, which for the main analyses was based on 2,294 participants. All participants gave written informed consent before taking part in the study, which was approved by the ethics committee of the Capital Region of Denmark, Copenhagen (KA20060011).\n Questionnaire data Information on socio-demographic variables, leisure time physical activity, smoking habits, chronic illnesses, respiratory symptoms, and medication were obtained from a questionnaire that was mailed to and answered by the participants before their visit to the research centre. Asthma was defined according to the ECRHS criteria [24] as a confirmatory answer to at least one of the following three questions ‘Have you been woken by an attack of shortness of breath at any time during the last 12 months?’, ‘Have you had an attack of asthma within the last 12 months?’ and ‘Are you currently taking any medication (including inhalers, aerosols or tablets) for asthma?’ Wheezing without a cold (in the following referred to as wheezing) was defined as a confirmatory answer to ‘Have you had wheezing or whistling in your chest at any time during the last 12 months?’ combined with a confirmatory answer to ‘If yes, have you had this wheezing or whistling when you did not have a cold?’. Smoking was recorded as pack years (one pack year equalling one pack of cigarettes per day for a year) and categorised in five levels: (1) never smokers, (2) 0- ≤ 10, (3) 10- ≤ 30, (4) 30- ≤ 50, or (5) more than 50 pack years but was also used as a continuous measure. Educational level was categorised as: (1) less than 2 years, (2) skilled worker, (3) less than 3 years, (4) 3–4 years, and (5) more than four years. All questions used in the present analyses were identical in the baseline and follow-up studies.\nInformation on socio-demographic variables, leisure time physical activity, smoking habits, chronic illnesses, respiratory symptoms, and medication were obtained from a questionnaire that was mailed to and answered by the participants before their visit to the research centre. Asthma was defined according to the ECRHS criteria [24] as a confirmatory answer to at least one of the following three questions ‘Have you been woken by an attack of shortness of breath at any time during the last 12 months?’, ‘Have you had an attack of asthma within the last 12 months?’ and ‘Are you currently taking any medication (including inhalers, aerosols or tablets) for asthma?’ Wheezing without a cold (in the following referred to as wheezing) was defined as a confirmatory answer to ‘Have you had wheezing or whistling in your chest at any time during the last 12 months?’ combined with a confirmatory answer to ‘If yes, have you had this wheezing or whistling when you did not have a cold?’. Smoking was recorded as pack years (one pack year equalling one pack of cigarettes per day for a year) and categorised in five levels: (1) never smokers, (2) 0- ≤ 10, (3) 10- ≤ 30, (4) 30- ≤ 50, or (5) more than 50 pack years but was also used as a continuous measure. Educational level was categorised as: (1) less than 2 years, (2) skilled worker, (3) less than 3 years, (4) 3–4 years, and (5) more than four years. All questions used in the present analyses were identical in the baseline and follow-up studies.\n Physical measurements and assessment of lung function and FENO Anthropometric measures were obtained with participants in light clothing and without shoes. Height was measured to the nearest cm, weight to the nearest 0.1 kg, and body mass index (BMI) was calculated as kg/m2. Waist circumference (WC) was measured to the nearest cm using a tape measure midway between the lower rib margin and the iliac crest. Fat percentage was measured using a foot-to-foot Tanita Body Composition Analyzer (TBF-300, TANITA Corporation of America, Inc., Illinois, U.S.A.). Spirometry was performed according to American Thoracic Society and the European Respiratory Society (ATS/ERS) standard [25] using Spiro USB Spirometers (MicroMedical Limited, Rochester, Kent, UK). Spirometers were checked daily with a 3-liter calibrated syringe, and checked every six months with a decompression flow simulator [26]. FENO measurements were performed only at baseline using a NIOX MINO (Aerocrine AB, Stockholm, Sweden). A dynamic flow restrictor yielded a constant flow rate of 50 mL/s, in accordance with recommendations of the ATS/ERS guidelines for FENO measurement [27]. Nitric oxide concentrations between 5 and 300 ppb were measured and measurements below 5 ppb were set to zero by the NIOX MINO. Nitric oxide concentrations above 20 ppb were considered high.\nAnthropometric measures were obtained with participants in light clothing and without shoes. Height was measured to the nearest cm, weight to the nearest 0.1 kg, and body mass index (BMI) was calculated as kg/m2. Waist circumference (WC) was measured to the nearest cm using a tape measure midway between the lower rib margin and the iliac crest. Fat percentage was measured using a foot-to-foot Tanita Body Composition Analyzer (TBF-300, TANITA Corporation of America, Inc., Illinois, U.S.A.). Spirometry was performed according to American Thoracic Society and the European Respiratory Society (ATS/ERS) standard [25] using Spiro USB Spirometers (MicroMedical Limited, Rochester, Kent, UK). Spirometers were checked daily with a 3-liter calibrated syringe, and checked every six months with a decompression flow simulator [26]. FENO measurements were performed only at baseline using a NIOX MINO (Aerocrine AB, Stockholm, Sweden). A dynamic flow restrictor yielded a constant flow rate of 50 mL/s, in accordance with recommendations of the ATS/ERS guidelines for FENO measurement [27]. Nitric oxide concentrations between 5 and 300 ppb were measured and measurements below 5 ppb were set to zero by the NIOX MINO. Nitric oxide concentrations above 20 ppb were considered high.\n Biomarkers Baseline serum samples were analysed for serum specific IgE against the four most clinically important inhalant allergens in Denmark: birch, grass, cat, and the house dust mite Dermatophagoides pteronyssinus, using the ADVIA Centaur Specific IgE assay [28]. Atopy was defined as at least one positive test (≥0.35 kilo units/L) against specific IgE. Plasma glucose concentrations were analysed by a hexokinase/glucose-6-phosphate dehydrogenase assay (Roche Diagnostic, Germany), and insulin concentrations were measured by fluoroimmunoassay technique (Dako Diagnostics Ltd., UK). The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated as: fasting plasma glucose (mmol/L) x fasting serum insulin (mU/L)/22.5 and applied only to the non-diabetic individuals. High-density lipoprotein (HDL) and triglyceride concentrations were measured by standard enzymatic techniques (Roche Diagnostic, Germany).\nBaseline serum samples were analysed for serum specific IgE against the four most clinically important inhalant allergens in Denmark: birch, grass, cat, and the house dust mite Dermatophagoides pteronyssinus, using the ADVIA Centaur Specific IgE assay [28]. Atopy was defined as at least one positive test (≥0.35 kilo units/L) against specific IgE. Plasma glucose concentrations were analysed by a hexokinase/glucose-6-phosphate dehydrogenase assay (Roche Diagnostic, Germany), and insulin concentrations were measured by fluoroimmunoassay technique (Dako Diagnostics Ltd., UK). The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated as: fasting plasma glucose (mmol/L) x fasting serum insulin (mU/L)/22.5 and applied only to the non-diabetic individuals. High-density lipoprotein (HDL) and triglyceride concentrations were measured by standard enzymatic techniques (Roche Diagnostic, Germany).\n Statistics We used the R-statistical package, version 3.0.2 (http://www.r-project.org/) for all analyses. All p-values are two-tailed and statistical significance defined as p <0.05. P-values of likelihood ratio tests were used to test for significance of all multivariate analyses. We used linear regression and checked for equal residual variance across the full range of all exposure variables to check the linear model assumptions. We also tested possible non-linear associations by dividing the exposure variables in splines and by testing the quadratic term of the variables in the regression models. For our main analyses, we used absolute or percentage changes (between baseline and five-year follow up) of the adiposity measures to changes in FEV1 and FVC. The changes of FEV1 (or FVC) were modelled as five-year FEV1 (or FVC) adjusted for FEV1 (or FVC) at baseline. We also tested the ‘reverse’ association of baseline lung function measures to changes of adiposity measures. The adiposity measures were standardised by division of each measure by its own standard error to ease interpretation of the regression coefficients and to ease a comparison of the magnitude of effects between the different adiposity measures. In analyses of incidence of asthma and wheezing, logistic regression was used, similar model checks were applied, and the adiposity measures were categorised in four equally sized groups by quartiles of their respective distributions. Possible interactions were tested by adding an interaction term to the regression models, e.g. (adiposity measure*atopy) or (adiposity measure*gender). Finally, all analyses were repeated with s-Trig, s-HDL, and HOMA-IR included in the models to test whether these metabolic markers would attenuate the estimates found for adiposity. Sensitivity analyses included exclusion of the lower and upper 0.0025, 0.005, 0.01 fractiles of the adiposity measures, as well as separate analyses in non- and never smokers, and in younger or older (age categories were +/− 40, +/−45, +/−50 years) individuals.\nFinally, we made a simulation-model based on randomly generated heights, BMI-levels, and levels of lung volume dependent on height. This model gives an example of how adipose tissue above a certain ‘person-dependent ideal’ would influence lung function (Additional file 1).\nWe used the R-statistical package, version 3.0.2 (http://www.r-project.org/) for all analyses. All p-values are two-tailed and statistical significance defined as p <0.05. P-values of likelihood ratio tests were used to test for significance of all multivariate analyses. We used linear regression and checked for equal residual variance across the full range of all exposure variables to check the linear model assumptions. We also tested possible non-linear associations by dividing the exposure variables in splines and by testing the quadratic term of the variables in the regression models. For our main analyses, we used absolute or percentage changes (between baseline and five-year follow up) of the adiposity measures to changes in FEV1 and FVC. The changes of FEV1 (or FVC) were modelled as five-year FEV1 (or FVC) adjusted for FEV1 (or FVC) at baseline. We also tested the ‘reverse’ association of baseline lung function measures to changes of adiposity measures. The adiposity measures were standardised by division of each measure by its own standard error to ease interpretation of the regression coefficients and to ease a comparison of the magnitude of effects between the different adiposity measures. In analyses of incidence of asthma and wheezing, logistic regression was used, similar model checks were applied, and the adiposity measures were categorised in four equally sized groups by quartiles of their respective distributions. Possible interactions were tested by adding an interaction term to the regression models, e.g. (adiposity measure*atopy) or (adiposity measure*gender). Finally, all analyses were repeated with s-Trig, s-HDL, and HOMA-IR included in the models to test whether these metabolic markers would attenuate the estimates found for adiposity. Sensitivity analyses included exclusion of the lower and upper 0.0025, 0.005, 0.01 fractiles of the adiposity measures, as well as separate analyses in non- and never smokers, and in younger or older (age categories were +/− 40, +/−45, +/−50 years) individuals.\nFinally, we made a simulation-model based on randomly generated heights, BMI-levels, and levels of lung volume dependent on height. This model gives an example of how adipose tissue above a certain ‘person-dependent ideal’ would influence lung function (Additional file 1).",
"The current study used already sampled data from the Health2006 study and the Health2006-follow-up study conducted five years apart. The participants in the baseline Health2006 cohort were drawn as a random sample from the background population aged 18–69 years, living in 11 municipalities in the south-western part of suburban Copenhagen. A total of 3471 individuals (44.7%) entered the study and participated in the health examination, which took place in 2006–08 (Health2006 baseline [23]). In 2011–12, participants in the baseline Health2006 were invited for a 5-year follow-up examination including essentially the same study protocol. A total of 3,405 were eligible for invitation (21 had emigrated and 45 died). A total of 2,308 (68.6%) agreed to participate and were re-examined between November 2011 and November 2012. Fourteen participants did not have valid measures of either FEV1 or FVC at either baseline or follow-up and were excluded from the study, which for the main analyses was based on 2,294 participants. All participants gave written informed consent before taking part in the study, which was approved by the ethics committee of the Capital Region of Denmark, Copenhagen (KA20060011).",
"Information on socio-demographic variables, leisure time physical activity, smoking habits, chronic illnesses, respiratory symptoms, and medication were obtained from a questionnaire that was mailed to and answered by the participants before their visit to the research centre. Asthma was defined according to the ECRHS criteria [24] as a confirmatory answer to at least one of the following three questions ‘Have you been woken by an attack of shortness of breath at any time during the last 12 months?’, ‘Have you had an attack of asthma within the last 12 months?’ and ‘Are you currently taking any medication (including inhalers, aerosols or tablets) for asthma?’ Wheezing without a cold (in the following referred to as wheezing) was defined as a confirmatory answer to ‘Have you had wheezing or whistling in your chest at any time during the last 12 months?’ combined with a confirmatory answer to ‘If yes, have you had this wheezing or whistling when you did not have a cold?’. Smoking was recorded as pack years (one pack year equalling one pack of cigarettes per day for a year) and categorised in five levels: (1) never smokers, (2) 0- ≤ 10, (3) 10- ≤ 30, (4) 30- ≤ 50, or (5) more than 50 pack years but was also used as a continuous measure. Educational level was categorised as: (1) less than 2 years, (2) skilled worker, (3) less than 3 years, (4) 3–4 years, and (5) more than four years. All questions used in the present analyses were identical in the baseline and follow-up studies.",
"Anthropometric measures were obtained with participants in light clothing and without shoes. Height was measured to the nearest cm, weight to the nearest 0.1 kg, and body mass index (BMI) was calculated as kg/m2. Waist circumference (WC) was measured to the nearest cm using a tape measure midway between the lower rib margin and the iliac crest. Fat percentage was measured using a foot-to-foot Tanita Body Composition Analyzer (TBF-300, TANITA Corporation of America, Inc., Illinois, U.S.A.). Spirometry was performed according to American Thoracic Society and the European Respiratory Society (ATS/ERS) standard [25] using Spiro USB Spirometers (MicroMedical Limited, Rochester, Kent, UK). Spirometers were checked daily with a 3-liter calibrated syringe, and checked every six months with a decompression flow simulator [26]. FENO measurements were performed only at baseline using a NIOX MINO (Aerocrine AB, Stockholm, Sweden). A dynamic flow restrictor yielded a constant flow rate of 50 mL/s, in accordance with recommendations of the ATS/ERS guidelines for FENO measurement [27]. Nitric oxide concentrations between 5 and 300 ppb were measured and measurements below 5 ppb were set to zero by the NIOX MINO. Nitric oxide concentrations above 20 ppb were considered high.",
"Baseline serum samples were analysed for serum specific IgE against the four most clinically important inhalant allergens in Denmark: birch, grass, cat, and the house dust mite Dermatophagoides pteronyssinus, using the ADVIA Centaur Specific IgE assay [28]. Atopy was defined as at least one positive test (≥0.35 kilo units/L) against specific IgE. Plasma glucose concentrations were analysed by a hexokinase/glucose-6-phosphate dehydrogenase assay (Roche Diagnostic, Germany), and insulin concentrations were measured by fluoroimmunoassay technique (Dako Diagnostics Ltd., UK). The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated as: fasting plasma glucose (mmol/L) x fasting serum insulin (mU/L)/22.5 and applied only to the non-diabetic individuals. High-density lipoprotein (HDL) and triglyceride concentrations were measured by standard enzymatic techniques (Roche Diagnostic, Germany).",
"We used the R-statistical package, version 3.0.2 (http://www.r-project.org/) for all analyses. All p-values are two-tailed and statistical significance defined as p <0.05. P-values of likelihood ratio tests were used to test for significance of all multivariate analyses. We used linear regression and checked for equal residual variance across the full range of all exposure variables to check the linear model assumptions. We also tested possible non-linear associations by dividing the exposure variables in splines and by testing the quadratic term of the variables in the regression models. For our main analyses, we used absolute or percentage changes (between baseline and five-year follow up) of the adiposity measures to changes in FEV1 and FVC. The changes of FEV1 (or FVC) were modelled as five-year FEV1 (or FVC) adjusted for FEV1 (or FVC) at baseline. We also tested the ‘reverse’ association of baseline lung function measures to changes of adiposity measures. The adiposity measures were standardised by division of each measure by its own standard error to ease interpretation of the regression coefficients and to ease a comparison of the magnitude of effects between the different adiposity measures. In analyses of incidence of asthma and wheezing, logistic regression was used, similar model checks were applied, and the adiposity measures were categorised in four equally sized groups by quartiles of their respective distributions. Possible interactions were tested by adding an interaction term to the regression models, e.g. (adiposity measure*atopy) or (adiposity measure*gender). Finally, all analyses were repeated with s-Trig, s-HDL, and HOMA-IR included in the models to test whether these metabolic markers would attenuate the estimates found for adiposity. Sensitivity analyses included exclusion of the lower and upper 0.0025, 0.005, 0.01 fractiles of the adiposity measures, as well as separate analyses in non- and never smokers, and in younger or older (age categories were +/− 40, +/−45, +/−50 years) individuals.\nFinally, we made a simulation-model based on randomly generated heights, BMI-levels, and levels of lung volume dependent on height. This model gives an example of how adipose tissue above a certain ‘person-dependent ideal’ would influence lung function (Additional file 1).",
"The baseline characteristics of the study population are given in Table 1. All characteristics are listed separately for participants and non-participants in the follow-up study. Of note is that non-participants as compared to participants had significantly higher levels of adiposity measures and higher levels of FEV1 and FVC. However, the baseline associations of adipose tissue levels with asthma, wheezing, FEV1 or FVC were not significantly different between participants and non-participants, all p-values for interaction between participation and each adiposity measure were between 0.12 and 0.98. Sensitivity analyses did not change the pattern of results presented.Table 1\nBaseline characteristics of the study population according to participation in the five-year follow-up study\nParticipants follow-upNon-participants follow-upN = 2294N = 1163MeanSdn missMeanSdn missAge (y)50.0212.4948.0213.98FEV1 (liter)3.160.7993.040.885FVC (liter)4.030.9693.891.065\nN\n\n% group\n\nN\n\n% group\n\nGender\n Males104845.749542.6\nAdiposity measures\n 18.5 > BMI (kg/m2)381.7272.3 18.5 < =BMI < 25 (kg/m2)109647.849142.2 25 < =BMI < 30 (kg/m2)83936.640835.1 30 < =BMI (kg/m2)31913.9223720.4 WC, normal§\n174876.235950.7 WC, high§\n54523.8234949.33\nSerum lipids\n HDL, normal§\n203689.11099386.313 HDL, low§\n24910.915813.7 Triglyc <150 mg/dl190183.288777.1 Triglyc 150–500 mg/dl23010.116114.0 Triglyc > = 500 mg/dl1546.7101038.913\nAtopy, asthma, wheezing\n Atopy*, not present174175.992479.4 Atopy*, present55324.123920.6 Asthma, not present187282.290678.9 Asthma, present40517.81724221.115 Wheezing, not present197486.791579.9 Wheezing, present30313.31723020.118\nLifestyle\n Smoking, never102748.21841738.9 Smoking, 0- ≤ 10 pack years49223.125323.6 Smoking, >10 pack years61328.840337.689 Alcohol, no drinking22510.115914.3 Alcohol 1–14 units/week144865.071464.4 Alcohol >14 units/week55524.96623621.354 Education§, none23910.521518.8 Education§, 1 year/practical54323.927223.8 Education§§, 2–3 years94341.445039.3 Education§§, > = 4 years55124.21820818.218FEV1, forced expiratory volumes first second; FVC, forced vital capacity; BMI, body mass index; WC, waist circumference; Diabetes*, either self-reported diabetes or fasting p-glucose > =7.1 mmol/L; HDL, serum level of high-density lipoprotein; Triglyc, serum level of triglyceride; *Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; §above primary school, §§above high school.\n\nBaseline characteristics of the study population according to participation in the five-year follow-up study\n\nFEV1, forced expiratory volumes first second; FVC, forced vital capacity; BMI, body mass index; WC, waist circumference; Diabetes*, either self-reported diabetes or fasting p-glucose > =7.1 mmol/L; HDL, serum level of high-density lipoprotein; Triglyc, serum level of triglyceride; *Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; §above primary school, §§above high school.\nFrom baseline to five-year follow-up, lung function was inversely associated with the level of adiposity as reflected by all four adiposity measures. In participants with weight gain, lung function declined; whereas in participants with weight loss, lung function increased to a similar extend (Figure 1 and Table 2). This pattern was observed in both males and females but the strength of these associations differed significantly between the two genders (Table 2): For all four adiposity measures, the estimated effect of adiposity changes on changes in FEV1 and FVC were approximately three times higher in males than in females in absolute numbers. The percentage changes were the double in males as compared to females. For instance, for every one standard deviation of weight change (5.4 kg) from baseline to follow-up, FEV1 either increased or decreased with approximately 72 ml (66-78 ml) in males and 26 ml (21-31 ml) in females, similarly FVC either increased or decreased with 111 ml (101-121 ml) in males and 35 ml (27-43 ml) in females. The metabolic markers, s-triglyceride, s-HDL, or HOMA-IR did not attenuate the effects of adiposity measures on changes in FEV1 and FVC (data not shown). Estimated mean declines for one year of increasing age were approximately 32.8 (31.3-34.4) ml FEV1 and 21.4 (19.0-23.9) ml FVC. In never-smokers, the corresponding estimates were 31.0 (28.7-33.3) ml FEV1and 21.0 (17.4-24.6) ml FVC. All age-estimates were essentially similar in males and females but were significantly smaller in younger than in older individuals.Figure 1\nChanges of lung function according to weight changes. Five-year changes of FEV1 (upper panel) and FVC (lower panel) in quintiles of weight change in males (circles) and females (diamonds). Quintile one-two include individuals with weight loss, quintile four-five include individuals with weight gain. The middle quintile is used as reference. Analysis adjusted for age, tobacco use (pack years), and atopy.\n\nChanges of lung function according to weight changes. Five-year changes of FEV1 (upper panel) and FVC (lower panel) in quintiles of weight change in males (circles) and females (diamonds). Quintile one-two include individuals with weight loss, quintile four-five include individuals with weight gain. The middle quintile is used as reference. Analysis adjusted for age, tobacco use (pack years), and atopy.\n\nFive-year changes of FEV (ml) and FVC (ml) in males and females per one standard deviation (1 sd) increase of adiposity measure\n\nLinear regression models fitted with interaction between the specific adiposity measure and gender. Adjusted for age, atopy, tobacco use (pack years). Est, beta-estimate; Se, standard error for Est; p, p-value; p-i, p-value for difference (interaction) between males and females; BMI, body mass index; WC, waist circumference; FatP, fat percentage.\nThe five-year adiposity changes had a differential impact on lung function changes according to baseline adiposity levels, as assessed as baseline BMI (Figure 2). Five-year adiposity changes were associated with dose-dependent greater declines of FEV1 and FVC in participants who were overweight and obese at baseline as compared to participants who were lean at baseline (Figure 2). Again, this effect was significantly different in males and females but the interaction of baseline-BMI*five-year changes of adiposity was only significant with respect to FVC (p = 0.02). Essentially similar results can be obtained by running our simulation example (Additional file 1). There were not any associations between changes of adiposity with changes of the FEV1/FVC-ratio between baseline and follow-up. For one standard deviation change of any adiposity measure, the FEV1/FVC-ratio changed between −0.0023 (fat percentage) to 0.00097 (BMI) in males (p-values 0.14-0.83) and this was not different in females (p for interactions 0.66-0.99).Figure 2\nChanges of lung function according to weight changes and baseline adiposity. Five-year changes of FEV1 (upper panel) and FVC (lower panel) per one standard deviation increase of weight between baseline and follow-up according to baseline levels of BMI (X-axis) in males (circles) and females (diamonds), adjusted for age, atopy, tobacco use (pack years), and atopy.\n\nChanges of lung function according to weight changes and baseline adiposity. Five-year changes of FEV1 (upper panel) and FVC (lower panel) per one standard deviation increase of weight between baseline and follow-up according to baseline levels of BMI (X-axis) in males (circles) and females (diamonds), adjusted for age, atopy, tobacco use (pack years), and atopy.\nTo further investigate the temporal relationship of associations between changes in adiposity and lung function, we performed ‘reverse’ analyses where baseline FEV1 and FVC were used as explanatory variables for subsequent adiposity changes. However, there were no such statistically significant associations. Thus, these results support that changes in adiposity have an influence on lung function, but lung function does not have a similar influence on changes in adiposity.\nThe inverse association of adiposity with lung function was confirmed in both persons with and without asthma at baseline (Table 3). The gender differences in lung function declines from baseline to follow-up were also independent of asthma status at baseline (Table 3). With respect to atopy and FENO -levels, there were essentially no differences in the associations of adiposity changes to changes of FEV1 and FVC between atopic and non-atopic individuals or between persons with or without elevated FENO-levels (Table 4). Analyses in never- and non-smokers gave similar results (data not shown).Table 3\nFive-year changes of FEV1 (ml) and FVC (ml) in males and females per one standard deviation increase of BMI from baseline to follow-up, according to asthma status at baseline\nMalesFemalesEstSepEstSep\nFEV1\nAsthma−96.416.7<.001−10.710.80.32Non-asthma−75.67.0<.001−29.15.2<.001\nFVC\nAsthma−109.724.4<.001−15.015.80.34Non-asthma−115.011.0<.001−37.38.1<.001All models fitted with BMI-gender interaction (all interactions significant, p ≤ .001) and adjusted for age, atopy, and smoking (pack years). BMI, body mass index; Est, beta-estimate; Se, standard error; ml, milliliter; p, p-value for Est.Table 4\nFive-year changes of FEV (ml) and FVC (ml) in non-atopic and atopic individuals (upper panel), and in individuals with or without elevated FeNO-levels (lower panel), per one standard deviation increase of adiposity measures\nEst (ml)Se (ml)PEst (ml)Se (ml)Pp-i\nNon-atopic\n\nAtopic\n\nFEV1\n Weight (1 sd)−46.04.2<.001−46.28.5<.001ns BMI (1 sd)−42.34.2<.001−47.08.6<.001ns WC (1 sd)−38.94.3<.001−43.18.3<.001ns FatP (1 sd)−30.64.3<.001−35.88.5<.001ns\nFVC\n Weight (1 sd)−65.56.5<.001−59.313.1<.001ns BMI (1 sd)−61.66.6<.001−54.813.2<.001ns WC (1 sd)−53.26.6<.001−48.612.8<.001ns Fat (1 sd)−32.36.7<.001−32.113.1<.02ns\nNormal FeNO (<=20 ppb)\n\nElevated FeNO (>20 ppb)\n\nFEV1\n Weight (1 sd)−45.64.7<.001−48.77.6<.001ns BMI (1 sd)−43.44.7<.001−43.97.7<.001ns WC (1 sd)−43.34.8<.001−31.77.2<.001ns FatP (1 sd)−31.84.8<.001−24.37.4<.001ns\nFVC\n Weight (1 sd)−57.17.2<.001−85.212.6<.0010.04 BMI (1 sd)−54.57.2<.001−78.512.8<.001ns WC (1 sd)−48.17.5<.001−59.411.9<.001ns FatP (1 sd)−32.27.5<.001−28.112.4<.02nsLinear regression models fitted with interaction between adiposity measure and either atopy or elevated FeNO and adjusted for age, atopy, and smoking (pack years); p-i, p-values for interaction test. Est, beta-estimate; Se, standard error of Est; p, p-value; Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; FeNO, fractional expiratory nitric oxide;1 sd, one standard deviation; ns, non-significant; BMI, body mass index; WC, waist circumference; FatP, fat percentage.\n\nFive-year changes of FEV1 (ml) and FVC (ml) in males and females per one standard deviation increase of BMI from baseline to follow-up, according to asthma status at baseline\n\nAll models fitted with BMI-gender interaction (all interactions significant, p ≤ .001) and adjusted for age, atopy, and smoking (pack years). BMI, body mass index; Est, beta-estimate; Se, standard error; ml, milliliter; p, p-value for Est.\n\nFive-year changes of FEV (ml) and FVC (ml) in non-atopic and atopic individuals (upper panel), and in individuals with or without elevated FeNO-levels (lower panel), per one standard deviation increase of adiposity measures\n\nLinear regression models fitted with interaction between adiposity measure and either atopy or elevated FeNO and adjusted for age, atopy, and smoking (pack years); p-i, p-values for interaction test. Est, beta-estimate; Se, standard error of Est; p, p-value; Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; FeNO, fractional expiratory nitric oxide;1 sd, one standard deviation; ns, non-significant; BMI, body mass index; WC, waist circumference; FatP, fat percentage.\nFinally, we investigated whether baseline levels of adiposity measures or changes of adiposity levels from baseline to follow-up were associated with incident asthma and wheezing. The incidence of asthma and wheezing between baseline and follow-up was 7.0% (158/2265) and 5.9% (133/2263), respectively. There were no statistically significant associations of any adiposity measure (baseline or changes) with incident asthma but for changes of weight and BMI there was a positive association with wheezing (Table 5). Notably, the novel cases of asthma in our cohort could be categorised as ‘late-onset’ asthma because all participants were > =19 years at baseline. Restricting our analysis further to novel cases of asthma and wheezing in participants e.g. above > = 25 years or > = 40 years yielded similar results. There were no differences between atopic and non-atopic individuals or individuals with or without elevated FENO-levels with respect to incident asthma or wheezing (data not shown).Table 5\nThe association of changes of adiposity measures (quartiles) between baseline and five-year follow-up with risk of novel (incident) asthma and wheezing\nIncident asthmaIncident wheezingNnn/NORCI1CI2nn/NORCI1CI2\nWeight\n\nKg\nq1<−1.93570325.7\nref\n--264.7\nref\n--q21.93 - <0.7573478.31.470.912.42274.70.890.481.63q30.7 - <3.2570325.70.950.561.62346.01.250.722.20q4>3.2576468.11.170.711.95468.11.570.932.70\nBMI\n\nKg/m\n2\nq1<−0.57572366.4\nref\n--254.5\nref\n--q2−0.57 - <0.31572437.61.170.721.90274.81.020.561.86q30.31 - <1.216572366.41.000.611.65356.21.350.772.41q4>1.22573427.40.880.531.43467.11.690.992.94\nWC\n\nCm\nq1<−1.5564305.4\nRef\n--274.9\nref\n--q2−1.5 - <2576396.91.150.681.96254.40.850.461.57q32 - <5525458.71.620.982.71438.31.670.982.91q4>5626436.91.140.691.91386.21.270.742.22\nFatP\n\nPercentage points\nq1<−1.40569325.7\nref\n--315.0\nref\n--q2−1.40 - <0.50570478.31.450.892.38346.01.130.661.96q30.50 - <2.402569427.51.240.752.06254.50.770.421.38q4>2.40570335.80.780.451.35407.11.200.712.05Number of participants in each group (N), number of novel cases (n) and age, gender, and tobacco (pack years) adjusted odds ratios (OR) with 95% confidence intervals (CI1-CI2)); q1-q4, quartiles 1-4; BMI, body mass index; WC, waist circumference; FatP, Fat percentage.\n\nThe association of changes of adiposity measures (quartiles) between baseline and five-year follow-up with risk of novel (incident) asthma and wheezing\n\nNumber of participants in each group (N), number of novel cases (n) and age, gender, and tobacco (pack years) adjusted odds ratios (OR) with 95% confidence intervals (CI1-CI2)); q1-q4, quartiles 1-4; BMI, body mass index; WC, waist circumference; FatP, Fat percentage.",
"We quantified the longitudinal relationship of several adiposity measures, including waist circumference and fat percentage, with lung function reflected by FEV1 and FVC. Thus, we confirmed changes of FEV1 and FVC per one kilo weight increase as summarised in Additional file 2 and extended that knowledge by reporting longitudinal estimates for abdominal adiposity (waist circumference) and fat percentage. We found that an increase in adiposity over five years was associated with declines in FEV1 and FVC, whereas a decrease in adiposity was associated with increases of FEV1 and FVC. These associations were more pronounced in males than in females but were independent of atopy and FENO -levels. Adiposity changes were not associated with changes in the FEV1/FVC-ratio and were not associated with incident asthma.\nAdipose tissue and especially upper body fat have in cross-sectional studies been shown to be associated with decreased static lung volumes as assessed by functional residual capacity and expiratory reserve volume [16, 17, 29]. As expiratory flow velocity is dependent on the degree of previous lung inflation due to the elastic properties of the lung [30], lower static lung volumes would subsequently impact on expiratory flows. Our results support such mechanical changes in a longitudinal setting. Firstly, the longitudinal adiposity changes were inversely associated with expiratory flow as reflected by FEV1. Secondly, pre-existing adiposity (at baseline) enhanced the negative impact of adiposity gain (Figure 2). Thus, the cumulative mass of adipose tissue was associated with reduced lung function as assessed by FEV1 and FVC. Adding that the FEV1/FVC-ratio remained unchanged, there was not a measurable increasing obstructive pattern with increasing adiposity. In comparison, inflammatory mechanisms would be expected to induce asthma-like, obstructive alterations in lung function [31].\nThe gender differences in the adiposity-lung function associations in this study were substantial, and may emphasise the mechanical aspects as males have more abdominal fat for the same degree of adiposity than females [32]. Similar conclusions have been put forward by authors in other studies [4, 33, 34] but supplementary explanations may exist. At least in asthma patients, recent cluster analyses have shown that a subgroup of non-adipose males have a high yearly loss of FEV1 that is not explained by presence or absence of atopy, the duration of disease etc. [35, 36]. This could indicate that another pathological mechanism towards declines of lung volumes occurs in those males, and this could also apply to males without asthma. Differences in sex hormonal levels, which are emphasised by decreasing gender differences in lung volume decline in elderly [3] is a possible explanation for an apparently different airway pathology in males and females. Other possibilities are different thresholds for the detrimental effects of pulmonary irritants, or differences in airway calibre between males and females [37], although none have been fully documented so far.\nAtopy or FENO-levels did not modify the effects of adiposity on lung function. Thus, an underlying atopic as compared to non-atopic condition or an underlying eosinophilic as compared to non-eosinophilic airway inflammation did not appear to enhance the inverse association of adiposity with lung function in our general population. It is possible that a characterisation of participants by e.g. eosinophils in sputum or blood would have yielded other results, as elevated FENO-levels only are indicative of eosinophil airway inflammation [38]. For instance, cluster analyses have indicated that some groups of asthma patients are characterised by eosinophil airway inflammation and low lung function [39]. Further, faster declines of lung volumes in non-atopic as compared to atopic asthma patients have also been reported [36, 40, 41]. However, our study was conducted in a general population and not in specific groups of patients. Thus, absence of effect modification by atopy or FENO-levels in our study appears to emphasise restrictive changes of lung function with increasing adiposity. However, our study was not powered to investigate effect modifications. Therefore, more studies are needed to confirm or refute the presence of effect modification by atopy or elevated FENO-levels.\nWe did not find an association between increasing adiposity and asthma incidence but for increasing weight and BMI there was a positive association with incident wheezing. This is in contrast with findings of most longitudinal studies, which have reported a positive association between increasing BMI and incidence of asthma [1]. However, the power to detect such differences in the present study was relatively low as indicated by relatively wide 95% confidence intervals. Possibly, testing the association of adiposity with bronchial hyperreactivity (BHR) would have yielded other results; in a previous study we found a positive association of adiposity with BHR [42], but such information was not available in the current study.\nThe strengths of the study include the prospective design, validated and similar methods for measurements of FEV1 and FVC at baseline and follow-up, assessment of atopy by serum specific IgE and airway eosinophil inflammation by FENO. The limitations include the lack of tests for variability of lung function to define asthma, lack of measures of lung volumes, and that the analyses are based on the assumption that increases or decreases of weight and BMI were due to adipose tissue and not muscle mass. Selection bias may have decreased the generalisability of the study and loss to follow-up influenced the reported associations. However, we found similar baseline associations of adipose tissue measures with all outcomes in participants and non-participants in the follow-up indicating that our results may apply to a general population.",
"In conclusion, changes of adiposity had a high, gender specific impact on FEV1 and FVC but not on the FEV1/FVC-ratio. Increasing adiposity was associated with lung function decline, whereas loss of adiposity was associated with increasing lung function. These associations were independent of atopy, FENO -levels, and metabolic markers, but were modified by the level of adipose tissue prior to the adiposity changes indicating the importance of aggregate adipose tissue mass. The adiposity changes leading to declines of FEV1 and FVC were not concomitantly associated with incidence of asthma or wheezing, however, a low number of novel cases of asthma and wheezing may have limited the power to detect associations with these outcomes.",
" Additional file 1:\nSimulation model of lung function depending on BMI.\n(DOCX 15 KB)\nAdditional file 2:\nOverview of longitudinal studies of the association between increasing adiposity and decreasing lung function conducted in general adult populations.\n(DOCX 24 KB)\nAdditional file 1:\nSimulation model of lung function depending on BMI.\n(DOCX 15 KB)\nAdditional file 2:\nOverview of longitudinal studies of the association between increasing adiposity and decreasing lung function conducted in general adult populations.\n(DOCX 24 KB)",
"Additional file 1:\nSimulation model of lung function depending on BMI.\n(DOCX 15 KB)\nAdditional file 2:\nOverview of longitudinal studies of the association between increasing adiposity and decreasing lung function conducted in general adult populations.\n(DOCX 24 KB)"
] |
[
null,
"methods",
null,
null,
null,
null,
null,
"results",
"discussion",
"conclusions",
"supplementary-material",
null
] |
[
"Adiposity",
"Asthma",
"Atopy",
"FENO",
"Longitudinal",
"Lung function",
"Obesity"
] |
Background:
Adiposity has consistently been associated with asthma [1] and has also been associated with impaired lung function, e.g. as assessed by decreased levels of forced expiratory volume in first second (FEV1) and forced vital capacity (FVC) [2–6]. Thus, adiposity may influence the lungs in several ways, e.g. through inflammation leading to asthma-like, obstructive changes, or in terms of a mechanical impact on lung function (restrictive changes).
Adiposity increases the risk of asthma and may cause more severe symptoms along with a reduced response to medications [1, 7, 8]. Some studies have indicated that these effects are stronger in non-atopic individuals than atopic individuals [9–11], although contrary results also have been noted [12]. Further, adiposity has been reported to be more strongly associated with asthma types characterised by non-eosinophilic inflammation rather than by eosinophilic inflammation [13]. However, it has not been investigated whether adiposity also has differential effects on lung function in atopic and non-atopic individuals or in individuals with or without eosinophilic inflammation in the airways. Underlying pathological processes, such as eosinophilic or neutrophilic inflammation [14], could possibly modify the longitudinal relationship of adiposity with lung function.
Mechanically, adiposity may cause an extra load on the thoracic cage [15], increase the intra-abdominal pressure, and impede the movements of the diaphragm [16]. This has in smaller experimental settings been reported to decrease static lung volumes and lead to breathing at smaller tidal volumes [17]. Cross-sectional studies have suggested that these changes are stronger in persons with predominantly abdominal adiposity than in persons with predominantly general adiposity [18]. Yet, prospective population based studies are sparse and have assessed adiposity changes mainly by weight or BMI [2, 3, 19–22], except from one study that included waist and hip circumferences but not FVC [4]. Therefore, studies quantifying longitudinal changes of lung function with respect to different adiposity phenotypes may add further information about the longitudinal relationship between adiposity and lung function.
This prospective general population study aimed to investigate the association of longitudinal changes in weight, body mass index (BMI), waist circumference (WC), and fat percentage with longitudinal changes in FEV1 and FVC, and with concomitant incidence of asthma and wheezing. Further, we examined whether these associations were modified by gender, atopy, or eosinophilic inflammation of the lower airways as reflected by forced expiratory nitric oxide (FENO).
Methods:
Study population The current study used already sampled data from the Health2006 study and the Health2006-follow-up study conducted five years apart. The participants in the baseline Health2006 cohort were drawn as a random sample from the background population aged 18–69 years, living in 11 municipalities in the south-western part of suburban Copenhagen. A total of 3471 individuals (44.7%) entered the study and participated in the health examination, which took place in 2006–08 (Health2006 baseline [23]). In 2011–12, participants in the baseline Health2006 were invited for a 5-year follow-up examination including essentially the same study protocol. A total of 3,405 were eligible for invitation (21 had emigrated and 45 died). A total of 2,308 (68.6%) agreed to participate and were re-examined between November 2011 and November 2012. Fourteen participants did not have valid measures of either FEV1 or FVC at either baseline or follow-up and were excluded from the study, which for the main analyses was based on 2,294 participants. All participants gave written informed consent before taking part in the study, which was approved by the ethics committee of the Capital Region of Denmark, Copenhagen (KA20060011).
The current study used already sampled data from the Health2006 study and the Health2006-follow-up study conducted five years apart. The participants in the baseline Health2006 cohort were drawn as a random sample from the background population aged 18–69 years, living in 11 municipalities in the south-western part of suburban Copenhagen. A total of 3471 individuals (44.7%) entered the study and participated in the health examination, which took place in 2006–08 (Health2006 baseline [23]). In 2011–12, participants in the baseline Health2006 were invited for a 5-year follow-up examination including essentially the same study protocol. A total of 3,405 were eligible for invitation (21 had emigrated and 45 died). A total of 2,308 (68.6%) agreed to participate and were re-examined between November 2011 and November 2012. Fourteen participants did not have valid measures of either FEV1 or FVC at either baseline or follow-up and were excluded from the study, which for the main analyses was based on 2,294 participants. All participants gave written informed consent before taking part in the study, which was approved by the ethics committee of the Capital Region of Denmark, Copenhagen (KA20060011).
Questionnaire data Information on socio-demographic variables, leisure time physical activity, smoking habits, chronic illnesses, respiratory symptoms, and medication were obtained from a questionnaire that was mailed to and answered by the participants before their visit to the research centre. Asthma was defined according to the ECRHS criteria [24] as a confirmatory answer to at least one of the following three questions ‘Have you been woken by an attack of shortness of breath at any time during the last 12 months?’, ‘Have you had an attack of asthma within the last 12 months?’ and ‘Are you currently taking any medication (including inhalers, aerosols or tablets) for asthma?’ Wheezing without a cold (in the following referred to as wheezing) was defined as a confirmatory answer to ‘Have you had wheezing or whistling in your chest at any time during the last 12 months?’ combined with a confirmatory answer to ‘If yes, have you had this wheezing or whistling when you did not have a cold?’. Smoking was recorded as pack years (one pack year equalling one pack of cigarettes per day for a year) and categorised in five levels: (1) never smokers, (2) 0- ≤ 10, (3) 10- ≤ 30, (4) 30- ≤ 50, or (5) more than 50 pack years but was also used as a continuous measure. Educational level was categorised as: (1) less than 2 years, (2) skilled worker, (3) less than 3 years, (4) 3–4 years, and (5) more than four years. All questions used in the present analyses were identical in the baseline and follow-up studies.
Information on socio-demographic variables, leisure time physical activity, smoking habits, chronic illnesses, respiratory symptoms, and medication were obtained from a questionnaire that was mailed to and answered by the participants before their visit to the research centre. Asthma was defined according to the ECRHS criteria [24] as a confirmatory answer to at least one of the following three questions ‘Have you been woken by an attack of shortness of breath at any time during the last 12 months?’, ‘Have you had an attack of asthma within the last 12 months?’ and ‘Are you currently taking any medication (including inhalers, aerosols or tablets) for asthma?’ Wheezing without a cold (in the following referred to as wheezing) was defined as a confirmatory answer to ‘Have you had wheezing or whistling in your chest at any time during the last 12 months?’ combined with a confirmatory answer to ‘If yes, have you had this wheezing or whistling when you did not have a cold?’. Smoking was recorded as pack years (one pack year equalling one pack of cigarettes per day for a year) and categorised in five levels: (1) never smokers, (2) 0- ≤ 10, (3) 10- ≤ 30, (4) 30- ≤ 50, or (5) more than 50 pack years but was also used as a continuous measure. Educational level was categorised as: (1) less than 2 years, (2) skilled worker, (3) less than 3 years, (4) 3–4 years, and (5) more than four years. All questions used in the present analyses were identical in the baseline and follow-up studies.
Physical measurements and assessment of lung function and FENO Anthropometric measures were obtained with participants in light clothing and without shoes. Height was measured to the nearest cm, weight to the nearest 0.1 kg, and body mass index (BMI) was calculated as kg/m2. Waist circumference (WC) was measured to the nearest cm using a tape measure midway between the lower rib margin and the iliac crest. Fat percentage was measured using a foot-to-foot Tanita Body Composition Analyzer (TBF-300, TANITA Corporation of America, Inc., Illinois, U.S.A.). Spirometry was performed according to American Thoracic Society and the European Respiratory Society (ATS/ERS) standard [25] using Spiro USB Spirometers (MicroMedical Limited, Rochester, Kent, UK). Spirometers were checked daily with a 3-liter calibrated syringe, and checked every six months with a decompression flow simulator [26]. FENO measurements were performed only at baseline using a NIOX MINO (Aerocrine AB, Stockholm, Sweden). A dynamic flow restrictor yielded a constant flow rate of 50 mL/s, in accordance with recommendations of the ATS/ERS guidelines for FENO measurement [27]. Nitric oxide concentrations between 5 and 300 ppb were measured and measurements below 5 ppb were set to zero by the NIOX MINO. Nitric oxide concentrations above 20 ppb were considered high.
Anthropometric measures were obtained with participants in light clothing and without shoes. Height was measured to the nearest cm, weight to the nearest 0.1 kg, and body mass index (BMI) was calculated as kg/m2. Waist circumference (WC) was measured to the nearest cm using a tape measure midway between the lower rib margin and the iliac crest. Fat percentage was measured using a foot-to-foot Tanita Body Composition Analyzer (TBF-300, TANITA Corporation of America, Inc., Illinois, U.S.A.). Spirometry was performed according to American Thoracic Society and the European Respiratory Society (ATS/ERS) standard [25] using Spiro USB Spirometers (MicroMedical Limited, Rochester, Kent, UK). Spirometers were checked daily with a 3-liter calibrated syringe, and checked every six months with a decompression flow simulator [26]. FENO measurements were performed only at baseline using a NIOX MINO (Aerocrine AB, Stockholm, Sweden). A dynamic flow restrictor yielded a constant flow rate of 50 mL/s, in accordance with recommendations of the ATS/ERS guidelines for FENO measurement [27]. Nitric oxide concentrations between 5 and 300 ppb were measured and measurements below 5 ppb were set to zero by the NIOX MINO. Nitric oxide concentrations above 20 ppb were considered high.
Biomarkers Baseline serum samples were analysed for serum specific IgE against the four most clinically important inhalant allergens in Denmark: birch, grass, cat, and the house dust mite Dermatophagoides pteronyssinus, using the ADVIA Centaur Specific IgE assay [28]. Atopy was defined as at least one positive test (≥0.35 kilo units/L) against specific IgE. Plasma glucose concentrations were analysed by a hexokinase/glucose-6-phosphate dehydrogenase assay (Roche Diagnostic, Germany), and insulin concentrations were measured by fluoroimmunoassay technique (Dako Diagnostics Ltd., UK). The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated as: fasting plasma glucose (mmol/L) x fasting serum insulin (mU/L)/22.5 and applied only to the non-diabetic individuals. High-density lipoprotein (HDL) and triglyceride concentrations were measured by standard enzymatic techniques (Roche Diagnostic, Germany).
Baseline serum samples were analysed for serum specific IgE against the four most clinically important inhalant allergens in Denmark: birch, grass, cat, and the house dust mite Dermatophagoides pteronyssinus, using the ADVIA Centaur Specific IgE assay [28]. Atopy was defined as at least one positive test (≥0.35 kilo units/L) against specific IgE. Plasma glucose concentrations were analysed by a hexokinase/glucose-6-phosphate dehydrogenase assay (Roche Diagnostic, Germany), and insulin concentrations were measured by fluoroimmunoassay technique (Dako Diagnostics Ltd., UK). The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated as: fasting plasma glucose (mmol/L) x fasting serum insulin (mU/L)/22.5 and applied only to the non-diabetic individuals. High-density lipoprotein (HDL) and triglyceride concentrations were measured by standard enzymatic techniques (Roche Diagnostic, Germany).
Statistics We used the R-statistical package, version 3.0.2 (http://www.r-project.org/) for all analyses. All p-values are two-tailed and statistical significance defined as p <0.05. P-values of likelihood ratio tests were used to test for significance of all multivariate analyses. We used linear regression and checked for equal residual variance across the full range of all exposure variables to check the linear model assumptions. We also tested possible non-linear associations by dividing the exposure variables in splines and by testing the quadratic term of the variables in the regression models. For our main analyses, we used absolute or percentage changes (between baseline and five-year follow up) of the adiposity measures to changes in FEV1 and FVC. The changes of FEV1 (or FVC) were modelled as five-year FEV1 (or FVC) adjusted for FEV1 (or FVC) at baseline. We also tested the ‘reverse’ association of baseline lung function measures to changes of adiposity measures. The adiposity measures were standardised by division of each measure by its own standard error to ease interpretation of the regression coefficients and to ease a comparison of the magnitude of effects between the different adiposity measures. In analyses of incidence of asthma and wheezing, logistic regression was used, similar model checks were applied, and the adiposity measures were categorised in four equally sized groups by quartiles of their respective distributions. Possible interactions were tested by adding an interaction term to the regression models, e.g. (adiposity measure*atopy) or (adiposity measure*gender). Finally, all analyses were repeated with s-Trig, s-HDL, and HOMA-IR included in the models to test whether these metabolic markers would attenuate the estimates found for adiposity. Sensitivity analyses included exclusion of the lower and upper 0.0025, 0.005, 0.01 fractiles of the adiposity measures, as well as separate analyses in non- and never smokers, and in younger or older (age categories were +/− 40, +/−45, +/−50 years) individuals.
Finally, we made a simulation-model based on randomly generated heights, BMI-levels, and levels of lung volume dependent on height. This model gives an example of how adipose tissue above a certain ‘person-dependent ideal’ would influence lung function (Additional file 1).
We used the R-statistical package, version 3.0.2 (http://www.r-project.org/) for all analyses. All p-values are two-tailed and statistical significance defined as p <0.05. P-values of likelihood ratio tests were used to test for significance of all multivariate analyses. We used linear regression and checked for equal residual variance across the full range of all exposure variables to check the linear model assumptions. We also tested possible non-linear associations by dividing the exposure variables in splines and by testing the quadratic term of the variables in the regression models. For our main analyses, we used absolute or percentage changes (between baseline and five-year follow up) of the adiposity measures to changes in FEV1 and FVC. The changes of FEV1 (or FVC) were modelled as five-year FEV1 (or FVC) adjusted for FEV1 (or FVC) at baseline. We also tested the ‘reverse’ association of baseline lung function measures to changes of adiposity measures. The adiposity measures were standardised by division of each measure by its own standard error to ease interpretation of the regression coefficients and to ease a comparison of the magnitude of effects between the different adiposity measures. In analyses of incidence of asthma and wheezing, logistic regression was used, similar model checks were applied, and the adiposity measures were categorised in four equally sized groups by quartiles of their respective distributions. Possible interactions were tested by adding an interaction term to the regression models, e.g. (adiposity measure*atopy) or (adiposity measure*gender). Finally, all analyses were repeated with s-Trig, s-HDL, and HOMA-IR included in the models to test whether these metabolic markers would attenuate the estimates found for adiposity. Sensitivity analyses included exclusion of the lower and upper 0.0025, 0.005, 0.01 fractiles of the adiposity measures, as well as separate analyses in non- and never smokers, and in younger or older (age categories were +/− 40, +/−45, +/−50 years) individuals.
Finally, we made a simulation-model based on randomly generated heights, BMI-levels, and levels of lung volume dependent on height. This model gives an example of how adipose tissue above a certain ‘person-dependent ideal’ would influence lung function (Additional file 1).
Study population:
The current study used already sampled data from the Health2006 study and the Health2006-follow-up study conducted five years apart. The participants in the baseline Health2006 cohort were drawn as a random sample from the background population aged 18–69 years, living in 11 municipalities in the south-western part of suburban Copenhagen. A total of 3471 individuals (44.7%) entered the study and participated in the health examination, which took place in 2006–08 (Health2006 baseline [23]). In 2011–12, participants in the baseline Health2006 were invited for a 5-year follow-up examination including essentially the same study protocol. A total of 3,405 were eligible for invitation (21 had emigrated and 45 died). A total of 2,308 (68.6%) agreed to participate and were re-examined between November 2011 and November 2012. Fourteen participants did not have valid measures of either FEV1 or FVC at either baseline or follow-up and were excluded from the study, which for the main analyses was based on 2,294 participants. All participants gave written informed consent before taking part in the study, which was approved by the ethics committee of the Capital Region of Denmark, Copenhagen (KA20060011).
Questionnaire data:
Information on socio-demographic variables, leisure time physical activity, smoking habits, chronic illnesses, respiratory symptoms, and medication were obtained from a questionnaire that was mailed to and answered by the participants before their visit to the research centre. Asthma was defined according to the ECRHS criteria [24] as a confirmatory answer to at least one of the following three questions ‘Have you been woken by an attack of shortness of breath at any time during the last 12 months?’, ‘Have you had an attack of asthma within the last 12 months?’ and ‘Are you currently taking any medication (including inhalers, aerosols or tablets) for asthma?’ Wheezing without a cold (in the following referred to as wheezing) was defined as a confirmatory answer to ‘Have you had wheezing or whistling in your chest at any time during the last 12 months?’ combined with a confirmatory answer to ‘If yes, have you had this wheezing or whistling when you did not have a cold?’. Smoking was recorded as pack years (one pack year equalling one pack of cigarettes per day for a year) and categorised in five levels: (1) never smokers, (2) 0- ≤ 10, (3) 10- ≤ 30, (4) 30- ≤ 50, or (5) more than 50 pack years but was also used as a continuous measure. Educational level was categorised as: (1) less than 2 years, (2) skilled worker, (3) less than 3 years, (4) 3–4 years, and (5) more than four years. All questions used in the present analyses were identical in the baseline and follow-up studies.
Physical measurements and assessment of lung function and FENO:
Anthropometric measures were obtained with participants in light clothing and without shoes. Height was measured to the nearest cm, weight to the nearest 0.1 kg, and body mass index (BMI) was calculated as kg/m2. Waist circumference (WC) was measured to the nearest cm using a tape measure midway between the lower rib margin and the iliac crest. Fat percentage was measured using a foot-to-foot Tanita Body Composition Analyzer (TBF-300, TANITA Corporation of America, Inc., Illinois, U.S.A.). Spirometry was performed according to American Thoracic Society and the European Respiratory Society (ATS/ERS) standard [25] using Spiro USB Spirometers (MicroMedical Limited, Rochester, Kent, UK). Spirometers were checked daily with a 3-liter calibrated syringe, and checked every six months with a decompression flow simulator [26]. FENO measurements were performed only at baseline using a NIOX MINO (Aerocrine AB, Stockholm, Sweden). A dynamic flow restrictor yielded a constant flow rate of 50 mL/s, in accordance with recommendations of the ATS/ERS guidelines for FENO measurement [27]. Nitric oxide concentrations between 5 and 300 ppb were measured and measurements below 5 ppb were set to zero by the NIOX MINO. Nitric oxide concentrations above 20 ppb were considered high.
Biomarkers:
Baseline serum samples were analysed for serum specific IgE against the four most clinically important inhalant allergens in Denmark: birch, grass, cat, and the house dust mite Dermatophagoides pteronyssinus, using the ADVIA Centaur Specific IgE assay [28]. Atopy was defined as at least one positive test (≥0.35 kilo units/L) against specific IgE. Plasma glucose concentrations were analysed by a hexokinase/glucose-6-phosphate dehydrogenase assay (Roche Diagnostic, Germany), and insulin concentrations were measured by fluoroimmunoassay technique (Dako Diagnostics Ltd., UK). The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated as: fasting plasma glucose (mmol/L) x fasting serum insulin (mU/L)/22.5 and applied only to the non-diabetic individuals. High-density lipoprotein (HDL) and triglyceride concentrations were measured by standard enzymatic techniques (Roche Diagnostic, Germany).
Statistics:
We used the R-statistical package, version 3.0.2 (http://www.r-project.org/) for all analyses. All p-values are two-tailed and statistical significance defined as p <0.05. P-values of likelihood ratio tests were used to test for significance of all multivariate analyses. We used linear regression and checked for equal residual variance across the full range of all exposure variables to check the linear model assumptions. We also tested possible non-linear associations by dividing the exposure variables in splines and by testing the quadratic term of the variables in the regression models. For our main analyses, we used absolute or percentage changes (between baseline and five-year follow up) of the adiposity measures to changes in FEV1 and FVC. The changes of FEV1 (or FVC) were modelled as five-year FEV1 (or FVC) adjusted for FEV1 (or FVC) at baseline. We also tested the ‘reverse’ association of baseline lung function measures to changes of adiposity measures. The adiposity measures were standardised by division of each measure by its own standard error to ease interpretation of the regression coefficients and to ease a comparison of the magnitude of effects between the different adiposity measures. In analyses of incidence of asthma and wheezing, logistic regression was used, similar model checks were applied, and the adiposity measures were categorised in four equally sized groups by quartiles of their respective distributions. Possible interactions were tested by adding an interaction term to the regression models, e.g. (adiposity measure*atopy) or (adiposity measure*gender). Finally, all analyses were repeated with s-Trig, s-HDL, and HOMA-IR included in the models to test whether these metabolic markers would attenuate the estimates found for adiposity. Sensitivity analyses included exclusion of the lower and upper 0.0025, 0.005, 0.01 fractiles of the adiposity measures, as well as separate analyses in non- and never smokers, and in younger or older (age categories were +/− 40, +/−45, +/−50 years) individuals.
Finally, we made a simulation-model based on randomly generated heights, BMI-levels, and levels of lung volume dependent on height. This model gives an example of how adipose tissue above a certain ‘person-dependent ideal’ would influence lung function (Additional file 1).
Results:
The baseline characteristics of the study population are given in Table 1. All characteristics are listed separately for participants and non-participants in the follow-up study. Of note is that non-participants as compared to participants had significantly higher levels of adiposity measures and higher levels of FEV1 and FVC. However, the baseline associations of adipose tissue levels with asthma, wheezing, FEV1 or FVC were not significantly different between participants and non-participants, all p-values for interaction between participation and each adiposity measure were between 0.12 and 0.98. Sensitivity analyses did not change the pattern of results presented.Table 1
Baseline characteristics of the study population according to participation in the five-year follow-up study
Participants follow-upNon-participants follow-upN = 2294N = 1163MeanSdn missMeanSdn missAge (y)50.0212.4948.0213.98FEV1 (liter)3.160.7993.040.885FVC (liter)4.030.9693.891.065
N
% group
N
% group
Gender
Males104845.749542.6
Adiposity measures
18.5 > BMI (kg/m2)381.7272.3 18.5 < =BMI < 25 (kg/m2)109647.849142.2 25 < =BMI < 30 (kg/m2)83936.640835.1 30 < =BMI (kg/m2)31913.9223720.4 WC, normal§
174876.235950.7 WC, high§
54523.8234949.33
Serum lipids
HDL, normal§
203689.11099386.313 HDL, low§
24910.915813.7 Triglyc <150 mg/dl190183.288777.1 Triglyc 150–500 mg/dl23010.116114.0 Triglyc > = 500 mg/dl1546.7101038.913
Atopy, asthma, wheezing
Atopy*, not present174175.992479.4 Atopy*, present55324.123920.6 Asthma, not present187282.290678.9 Asthma, present40517.81724221.115 Wheezing, not present197486.791579.9 Wheezing, present30313.31723020.118
Lifestyle
Smoking, never102748.21841738.9 Smoking, 0- ≤ 10 pack years49223.125323.6 Smoking, >10 pack years61328.840337.689 Alcohol, no drinking22510.115914.3 Alcohol 1–14 units/week144865.071464.4 Alcohol >14 units/week55524.96623621.354 Education§, none23910.521518.8 Education§, 1 year/practical54323.927223.8 Education§§, 2–3 years94341.445039.3 Education§§, > = 4 years55124.21820818.218FEV1, forced expiratory volumes first second; FVC, forced vital capacity; BMI, body mass index; WC, waist circumference; Diabetes*, either self-reported diabetes or fasting p-glucose > =7.1 mmol/L; HDL, serum level of high-density lipoprotein; Triglyc, serum level of triglyceride; *Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; §above primary school, §§above high school.
Baseline characteristics of the study population according to participation in the five-year follow-up study
FEV1, forced expiratory volumes first second; FVC, forced vital capacity; BMI, body mass index; WC, waist circumference; Diabetes*, either self-reported diabetes or fasting p-glucose > =7.1 mmol/L; HDL, serum level of high-density lipoprotein; Triglyc, serum level of triglyceride; *Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; §above primary school, §§above high school.
From baseline to five-year follow-up, lung function was inversely associated with the level of adiposity as reflected by all four adiposity measures. In participants with weight gain, lung function declined; whereas in participants with weight loss, lung function increased to a similar extend (Figure 1 and Table 2). This pattern was observed in both males and females but the strength of these associations differed significantly between the two genders (Table 2): For all four adiposity measures, the estimated effect of adiposity changes on changes in FEV1 and FVC were approximately three times higher in males than in females in absolute numbers. The percentage changes were the double in males as compared to females. For instance, for every one standard deviation of weight change (5.4 kg) from baseline to follow-up, FEV1 either increased or decreased with approximately 72 ml (66-78 ml) in males and 26 ml (21-31 ml) in females, similarly FVC either increased or decreased with 111 ml (101-121 ml) in males and 35 ml (27-43 ml) in females. The metabolic markers, s-triglyceride, s-HDL, or HOMA-IR did not attenuate the effects of adiposity measures on changes in FEV1 and FVC (data not shown). Estimated mean declines for one year of increasing age were approximately 32.8 (31.3-34.4) ml FEV1 and 21.4 (19.0-23.9) ml FVC. In never-smokers, the corresponding estimates were 31.0 (28.7-33.3) ml FEV1and 21.0 (17.4-24.6) ml FVC. All age-estimates were essentially similar in males and females but were significantly smaller in younger than in older individuals.Figure 1
Changes of lung function according to weight changes. Five-year changes of FEV1 (upper panel) and FVC (lower panel) in quintiles of weight change in males (circles) and females (diamonds). Quintile one-two include individuals with weight loss, quintile four-five include individuals with weight gain. The middle quintile is used as reference. Analysis adjusted for age, tobacco use (pack years), and atopy.
Changes of lung function according to weight changes. Five-year changes of FEV1 (upper panel) and FVC (lower panel) in quintiles of weight change in males (circles) and females (diamonds). Quintile one-two include individuals with weight loss, quintile four-five include individuals with weight gain. The middle quintile is used as reference. Analysis adjusted for age, tobacco use (pack years), and atopy.
Five-year changes of FEV (ml) and FVC (ml) in males and females per one standard deviation (1 sd) increase of adiposity measure
Linear regression models fitted with interaction between the specific adiposity measure and gender. Adjusted for age, atopy, tobacco use (pack years). Est, beta-estimate; Se, standard error for Est; p, p-value; p-i, p-value for difference (interaction) between males and females; BMI, body mass index; WC, waist circumference; FatP, fat percentage.
The five-year adiposity changes had a differential impact on lung function changes according to baseline adiposity levels, as assessed as baseline BMI (Figure 2). Five-year adiposity changes were associated with dose-dependent greater declines of FEV1 and FVC in participants who were overweight and obese at baseline as compared to participants who were lean at baseline (Figure 2). Again, this effect was significantly different in males and females but the interaction of baseline-BMI*five-year changes of adiposity was only significant with respect to FVC (p = 0.02). Essentially similar results can be obtained by running our simulation example (Additional file 1). There were not any associations between changes of adiposity with changes of the FEV1/FVC-ratio between baseline and follow-up. For one standard deviation change of any adiposity measure, the FEV1/FVC-ratio changed between −0.0023 (fat percentage) to 0.00097 (BMI) in males (p-values 0.14-0.83) and this was not different in females (p for interactions 0.66-0.99).Figure 2
Changes of lung function according to weight changes and baseline adiposity. Five-year changes of FEV1 (upper panel) and FVC (lower panel) per one standard deviation increase of weight between baseline and follow-up according to baseline levels of BMI (X-axis) in males (circles) and females (diamonds), adjusted for age, atopy, tobacco use (pack years), and atopy.
Changes of lung function according to weight changes and baseline adiposity. Five-year changes of FEV1 (upper panel) and FVC (lower panel) per one standard deviation increase of weight between baseline and follow-up according to baseline levels of BMI (X-axis) in males (circles) and females (diamonds), adjusted for age, atopy, tobacco use (pack years), and atopy.
To further investigate the temporal relationship of associations between changes in adiposity and lung function, we performed ‘reverse’ analyses where baseline FEV1 and FVC were used as explanatory variables for subsequent adiposity changes. However, there were no such statistically significant associations. Thus, these results support that changes in adiposity have an influence on lung function, but lung function does not have a similar influence on changes in adiposity.
The inverse association of adiposity with lung function was confirmed in both persons with and without asthma at baseline (Table 3). The gender differences in lung function declines from baseline to follow-up were also independent of asthma status at baseline (Table 3). With respect to atopy and FENO -levels, there were essentially no differences in the associations of adiposity changes to changes of FEV1 and FVC between atopic and non-atopic individuals or between persons with or without elevated FENO-levels (Table 4). Analyses in never- and non-smokers gave similar results (data not shown).Table 3
Five-year changes of FEV1 (ml) and FVC (ml) in males and females per one standard deviation increase of BMI from baseline to follow-up, according to asthma status at baseline
MalesFemalesEstSepEstSep
FEV1
Asthma−96.416.7<.001−10.710.80.32Non-asthma−75.67.0<.001−29.15.2<.001
FVC
Asthma−109.724.4<.001−15.015.80.34Non-asthma−115.011.0<.001−37.38.1<.001All models fitted with BMI-gender interaction (all interactions significant, p ≤ .001) and adjusted for age, atopy, and smoking (pack years). BMI, body mass index; Est, beta-estimate; Se, standard error; ml, milliliter; p, p-value for Est.Table 4
Five-year changes of FEV (ml) and FVC (ml) in non-atopic and atopic individuals (upper panel), and in individuals with or without elevated FeNO-levels (lower panel), per one standard deviation increase of adiposity measures
Est (ml)Se (ml)PEst (ml)Se (ml)Pp-i
Non-atopic
Atopic
FEV1
Weight (1 sd)−46.04.2<.001−46.28.5<.001ns BMI (1 sd)−42.34.2<.001−47.08.6<.001ns WC (1 sd)−38.94.3<.001−43.18.3<.001ns FatP (1 sd)−30.64.3<.001−35.88.5<.001ns
FVC
Weight (1 sd)−65.56.5<.001−59.313.1<.001ns BMI (1 sd)−61.66.6<.001−54.813.2<.001ns WC (1 sd)−53.26.6<.001−48.612.8<.001ns Fat (1 sd)−32.36.7<.001−32.113.1<.02ns
Normal FeNO (<=20 ppb)
Elevated FeNO (>20 ppb)
FEV1
Weight (1 sd)−45.64.7<.001−48.77.6<.001ns BMI (1 sd)−43.44.7<.001−43.97.7<.001ns WC (1 sd)−43.34.8<.001−31.77.2<.001ns FatP (1 sd)−31.84.8<.001−24.37.4<.001ns
FVC
Weight (1 sd)−57.17.2<.001−85.212.6<.0010.04 BMI (1 sd)−54.57.2<.001−78.512.8<.001ns WC (1 sd)−48.17.5<.001−59.411.9<.001ns FatP (1 sd)−32.27.5<.001−28.112.4<.02nsLinear regression models fitted with interaction between adiposity measure and either atopy or elevated FeNO and adjusted for age, atopy, and smoking (pack years); p-i, p-values for interaction test. Est, beta-estimate; Se, standard error of Est; p, p-value; Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; FeNO, fractional expiratory nitric oxide;1 sd, one standard deviation; ns, non-significant; BMI, body mass index; WC, waist circumference; FatP, fat percentage.
Five-year changes of FEV1 (ml) and FVC (ml) in males and females per one standard deviation increase of BMI from baseline to follow-up, according to asthma status at baseline
All models fitted with BMI-gender interaction (all interactions significant, p ≤ .001) and adjusted for age, atopy, and smoking (pack years). BMI, body mass index; Est, beta-estimate; Se, standard error; ml, milliliter; p, p-value for Est.
Five-year changes of FEV (ml) and FVC (ml) in non-atopic and atopic individuals (upper panel), and in individuals with or without elevated FeNO-levels (lower panel), per one standard deviation increase of adiposity measures
Linear regression models fitted with interaction between adiposity measure and either atopy or elevated FeNO and adjusted for age, atopy, and smoking (pack years); p-i, p-values for interaction test. Est, beta-estimate; Se, standard error of Est; p, p-value; Atopy defined as serum specific IgE positivity (> = .35 kU/l) to inhalant allergens; FeNO, fractional expiratory nitric oxide;1 sd, one standard deviation; ns, non-significant; BMI, body mass index; WC, waist circumference; FatP, fat percentage.
Finally, we investigated whether baseline levels of adiposity measures or changes of adiposity levels from baseline to follow-up were associated with incident asthma and wheezing. The incidence of asthma and wheezing between baseline and follow-up was 7.0% (158/2265) and 5.9% (133/2263), respectively. There were no statistically significant associations of any adiposity measure (baseline or changes) with incident asthma but for changes of weight and BMI there was a positive association with wheezing (Table 5). Notably, the novel cases of asthma in our cohort could be categorised as ‘late-onset’ asthma because all participants were > =19 years at baseline. Restricting our analysis further to novel cases of asthma and wheezing in participants e.g. above > = 25 years or > = 40 years yielded similar results. There were no differences between atopic and non-atopic individuals or individuals with or without elevated FENO-levels with respect to incident asthma or wheezing (data not shown).Table 5
The association of changes of adiposity measures (quartiles) between baseline and five-year follow-up with risk of novel (incident) asthma and wheezing
Incident asthmaIncident wheezingNnn/NORCI1CI2nn/NORCI1CI2
Weight
Kg
q1<−1.93570325.7
ref
--264.7
ref
--q21.93 - <0.7573478.31.470.912.42274.70.890.481.63q30.7 - <3.2570325.70.950.561.62346.01.250.722.20q4>3.2576468.11.170.711.95468.11.570.932.70
BMI
Kg/m
2
q1<−0.57572366.4
ref
--254.5
ref
--q2−0.57 - <0.31572437.61.170.721.90274.81.020.561.86q30.31 - <1.216572366.41.000.611.65356.21.350.772.41q4>1.22573427.40.880.531.43467.11.690.992.94
WC
Cm
q1<−1.5564305.4
Ref
--274.9
ref
--q2−1.5 - <2576396.91.150.681.96254.40.850.461.57q32 - <5525458.71.620.982.71438.31.670.982.91q4>5626436.91.140.691.91386.21.270.742.22
FatP
Percentage points
q1<−1.40569325.7
ref
--315.0
ref
--q2−1.40 - <0.50570478.31.450.892.38346.01.130.661.96q30.50 - <2.402569427.51.240.752.06254.50.770.421.38q4>2.40570335.80.780.451.35407.11.200.712.05Number of participants in each group (N), number of novel cases (n) and age, gender, and tobacco (pack years) adjusted odds ratios (OR) with 95% confidence intervals (CI1-CI2)); q1-q4, quartiles 1-4; BMI, body mass index; WC, waist circumference; FatP, Fat percentage.
The association of changes of adiposity measures (quartiles) between baseline and five-year follow-up with risk of novel (incident) asthma and wheezing
Number of participants in each group (N), number of novel cases (n) and age, gender, and tobacco (pack years) adjusted odds ratios (OR) with 95% confidence intervals (CI1-CI2)); q1-q4, quartiles 1-4; BMI, body mass index; WC, waist circumference; FatP, Fat percentage.
Discussion:
We quantified the longitudinal relationship of several adiposity measures, including waist circumference and fat percentage, with lung function reflected by FEV1 and FVC. Thus, we confirmed changes of FEV1 and FVC per one kilo weight increase as summarised in Additional file 2 and extended that knowledge by reporting longitudinal estimates for abdominal adiposity (waist circumference) and fat percentage. We found that an increase in adiposity over five years was associated with declines in FEV1 and FVC, whereas a decrease in adiposity was associated with increases of FEV1 and FVC. These associations were more pronounced in males than in females but were independent of atopy and FENO -levels. Adiposity changes were not associated with changes in the FEV1/FVC-ratio and were not associated with incident asthma.
Adipose tissue and especially upper body fat have in cross-sectional studies been shown to be associated with decreased static lung volumes as assessed by functional residual capacity and expiratory reserve volume [16, 17, 29]. As expiratory flow velocity is dependent on the degree of previous lung inflation due to the elastic properties of the lung [30], lower static lung volumes would subsequently impact on expiratory flows. Our results support such mechanical changes in a longitudinal setting. Firstly, the longitudinal adiposity changes were inversely associated with expiratory flow as reflected by FEV1. Secondly, pre-existing adiposity (at baseline) enhanced the negative impact of adiposity gain (Figure 2). Thus, the cumulative mass of adipose tissue was associated with reduced lung function as assessed by FEV1 and FVC. Adding that the FEV1/FVC-ratio remained unchanged, there was not a measurable increasing obstructive pattern with increasing adiposity. In comparison, inflammatory mechanisms would be expected to induce asthma-like, obstructive alterations in lung function [31].
The gender differences in the adiposity-lung function associations in this study were substantial, and may emphasise the mechanical aspects as males have more abdominal fat for the same degree of adiposity than females [32]. Similar conclusions have been put forward by authors in other studies [4, 33, 34] but supplementary explanations may exist. At least in asthma patients, recent cluster analyses have shown that a subgroup of non-adipose males have a high yearly loss of FEV1 that is not explained by presence or absence of atopy, the duration of disease etc. [35, 36]. This could indicate that another pathological mechanism towards declines of lung volumes occurs in those males, and this could also apply to males without asthma. Differences in sex hormonal levels, which are emphasised by decreasing gender differences in lung volume decline in elderly [3] is a possible explanation for an apparently different airway pathology in males and females. Other possibilities are different thresholds for the detrimental effects of pulmonary irritants, or differences in airway calibre between males and females [37], although none have been fully documented so far.
Atopy or FENO-levels did not modify the effects of adiposity on lung function. Thus, an underlying atopic as compared to non-atopic condition or an underlying eosinophilic as compared to non-eosinophilic airway inflammation did not appear to enhance the inverse association of adiposity with lung function in our general population. It is possible that a characterisation of participants by e.g. eosinophils in sputum or blood would have yielded other results, as elevated FENO-levels only are indicative of eosinophil airway inflammation [38]. For instance, cluster analyses have indicated that some groups of asthma patients are characterised by eosinophil airway inflammation and low lung function [39]. Further, faster declines of lung volumes in non-atopic as compared to atopic asthma patients have also been reported [36, 40, 41]. However, our study was conducted in a general population and not in specific groups of patients. Thus, absence of effect modification by atopy or FENO-levels in our study appears to emphasise restrictive changes of lung function with increasing adiposity. However, our study was not powered to investigate effect modifications. Therefore, more studies are needed to confirm or refute the presence of effect modification by atopy or elevated FENO-levels.
We did not find an association between increasing adiposity and asthma incidence but for increasing weight and BMI there was a positive association with incident wheezing. This is in contrast with findings of most longitudinal studies, which have reported a positive association between increasing BMI and incidence of asthma [1]. However, the power to detect such differences in the present study was relatively low as indicated by relatively wide 95% confidence intervals. Possibly, testing the association of adiposity with bronchial hyperreactivity (BHR) would have yielded other results; in a previous study we found a positive association of adiposity with BHR [42], but such information was not available in the current study.
The strengths of the study include the prospective design, validated and similar methods for measurements of FEV1 and FVC at baseline and follow-up, assessment of atopy by serum specific IgE and airway eosinophil inflammation by FENO. The limitations include the lack of tests for variability of lung function to define asthma, lack of measures of lung volumes, and that the analyses are based on the assumption that increases or decreases of weight and BMI were due to adipose tissue and not muscle mass. Selection bias may have decreased the generalisability of the study and loss to follow-up influenced the reported associations. However, we found similar baseline associations of adipose tissue measures with all outcomes in participants and non-participants in the follow-up indicating that our results may apply to a general population.
Conclusion:
In conclusion, changes of adiposity had a high, gender specific impact on FEV1 and FVC but not on the FEV1/FVC-ratio. Increasing adiposity was associated with lung function decline, whereas loss of adiposity was associated with increasing lung function. These associations were independent of atopy, FENO -levels, and metabolic markers, but were modified by the level of adipose tissue prior to the adiposity changes indicating the importance of aggregate adipose tissue mass. The adiposity changes leading to declines of FEV1 and FVC were not concomitantly associated with incidence of asthma or wheezing, however, a low number of novel cases of asthma and wheezing may have limited the power to detect associations with these outcomes.
Electronic supplementary material:
Additional file 1:
Simulation model of lung function depending on BMI.
(DOCX 15 KB)
Additional file 2:
Overview of longitudinal studies of the association between increasing adiposity and decreasing lung function conducted in general adult populations.
(DOCX 24 KB)
Additional file 1:
Simulation model of lung function depending on BMI.
(DOCX 15 KB)
Additional file 2:
Overview of longitudinal studies of the association between increasing adiposity and decreasing lung function conducted in general adult populations.
(DOCX 24 KB)
:
Additional file 1:
Simulation model of lung function depending on BMI.
(DOCX 15 KB)
Additional file 2:
Overview of longitudinal studies of the association between increasing adiposity and decreasing lung function conducted in general adult populations.
(DOCX 24 KB)
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Background:
Adiposity has been linked to both higher risk of asthma and reduced lung function. The effects of adiposity on asthma may depend on both atopic status and gender, while the relationship is less clear with respect to lung function. This study aimed to explore longitudinal weight changes to changes in forced expiratory volume in first second (FEV1) and forced vital capacity (FVC), as well as to incident cases of asthma and wheezing, according to atopy and gender.
Methods:
A general population sample aged 19-72 years was examined with the same methodology five years apart. Longitudinal changes in weight, body mass index, waist circumference, and fat percentage (bio-impedance) were analyzed with respect to changes of FEV1 and FVC (spirometry), and incidence of asthma and wheezing (questionnaire). Gender, atopy (serum specific IgE-positivity to inhalant allergens) and adipose tissue mass prior to adiposity changes were examined as potential effect modifiers.
Results:
A total of 2,308 persons participated in both baseline and five-year follow-up examinations. Over the entire span of adiposity changes, adiposity gain was associated with decreasing levels of lung function, whereas adiposity loss was associated with increasing levels of lung function. All associations were dependent on gender (p-interactions < 0.0001). For one standard deviation weight gain or weight loss, FEV1 changed with (+/-)72 ml (66-78 ml) and FVC with (+/-)103 ml (94-112 ml) in males. In females FEV1 changed with (+/-) 27 ml (22-32 ml) and FVC with (+/-) 36 ml (28-44 ml). There were no changes in the FEV1/FVC-ratio. The effect of adiposity changes increased with the level of adipose tissue mass at the start of the study (baseline), thus, indicating an aggregate effect of the total adipose tissue mass. Atopy did not modify these associations. There were no statistically significant associations between changes in adiposity measures and risk of incident asthma or wheeze.
Conclusions:
Over a five-year period, increasing adiposity was associated with decreasing lung function, whereas decreasing adiposity was associated with increasing lung function. This effect was significantly greater in males than in females and increased with pre-existing adiposity, but was independent of atopy.
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Background:
Adiposity has consistently been associated with asthma [1] and has also been associated with impaired lung function, e.g. as assessed by decreased levels of forced expiratory volume in first second (FEV1) and forced vital capacity (FVC) [2–6]. Thus, adiposity may influence the lungs in several ways, e.g. through inflammation leading to asthma-like, obstructive changes, or in terms of a mechanical impact on lung function (restrictive changes).
Adiposity increases the risk of asthma and may cause more severe symptoms along with a reduced response to medications [1, 7, 8]. Some studies have indicated that these effects are stronger in non-atopic individuals than atopic individuals [9–11], although contrary results also have been noted [12]. Further, adiposity has been reported to be more strongly associated with asthma types characterised by non-eosinophilic inflammation rather than by eosinophilic inflammation [13]. However, it has not been investigated whether adiposity also has differential effects on lung function in atopic and non-atopic individuals or in individuals with or without eosinophilic inflammation in the airways. Underlying pathological processes, such as eosinophilic or neutrophilic inflammation [14], could possibly modify the longitudinal relationship of adiposity with lung function.
Mechanically, adiposity may cause an extra load on the thoracic cage [15], increase the intra-abdominal pressure, and impede the movements of the diaphragm [16]. This has in smaller experimental settings been reported to decrease static lung volumes and lead to breathing at smaller tidal volumes [17]. Cross-sectional studies have suggested that these changes are stronger in persons with predominantly abdominal adiposity than in persons with predominantly general adiposity [18]. Yet, prospective population based studies are sparse and have assessed adiposity changes mainly by weight or BMI [2, 3, 19–22], except from one study that included waist and hip circumferences but not FVC [4]. Therefore, studies quantifying longitudinal changes of lung function with respect to different adiposity phenotypes may add further information about the longitudinal relationship between adiposity and lung function.
This prospective general population study aimed to investigate the association of longitudinal changes in weight, body mass index (BMI), waist circumference (WC), and fat percentage with longitudinal changes in FEV1 and FVC, and with concomitant incidence of asthma and wheezing. Further, we examined whether these associations were modified by gender, atopy, or eosinophilic inflammation of the lower airways as reflected by forced expiratory nitric oxide (FENO).
Conclusion:
In conclusion, changes of adiposity had a high, gender specific impact on FEV1 and FVC but not on the FEV1/FVC-ratio. Increasing adiposity was associated with lung function decline, whereas loss of adiposity was associated with increasing lung function. These associations were independent of atopy, FENO -levels, and metabolic markers, but were modified by the level of adipose tissue prior to the adiposity changes indicating the importance of aggregate adipose tissue mass. The adiposity changes leading to declines of FEV1 and FVC were not concomitantly associated with incidence of asthma or wheezing, however, a low number of novel cases of asthma and wheezing may have limited the power to detect associations with these outcomes.
|
Background:
Adiposity has been linked to both higher risk of asthma and reduced lung function. The effects of adiposity on asthma may depend on both atopic status and gender, while the relationship is less clear with respect to lung function. This study aimed to explore longitudinal weight changes to changes in forced expiratory volume in first second (FEV1) and forced vital capacity (FVC), as well as to incident cases of asthma and wheezing, according to atopy and gender.
Methods:
A general population sample aged 19-72 years was examined with the same methodology five years apart. Longitudinal changes in weight, body mass index, waist circumference, and fat percentage (bio-impedance) were analyzed with respect to changes of FEV1 and FVC (spirometry), and incidence of asthma and wheezing (questionnaire). Gender, atopy (serum specific IgE-positivity to inhalant allergens) and adipose tissue mass prior to adiposity changes were examined as potential effect modifiers.
Results:
A total of 2,308 persons participated in both baseline and five-year follow-up examinations. Over the entire span of adiposity changes, adiposity gain was associated with decreasing levels of lung function, whereas adiposity loss was associated with increasing levels of lung function. All associations were dependent on gender (p-interactions < 0.0001). For one standard deviation weight gain or weight loss, FEV1 changed with (+/-)72 ml (66-78 ml) and FVC with (+/-)103 ml (94-112 ml) in males. In females FEV1 changed with (+/-) 27 ml (22-32 ml) and FVC with (+/-) 36 ml (28-44 ml). There were no changes in the FEV1/FVC-ratio. The effect of adiposity changes increased with the level of adipose tissue mass at the start of the study (baseline), thus, indicating an aggregate effect of the total adipose tissue mass. Atopy did not modify these associations. There were no statistically significant associations between changes in adiposity measures and risk of incident asthma or wheeze.
Conclusions:
Over a five-year period, increasing adiposity was associated with decreasing lung function, whereas decreasing adiposity was associated with increasing lung function. This effect was significantly greater in males than in females and increased with pre-existing adiposity, but was independent of atopy.
| 9,100 | 454 | 12 |
[
"adiposity",
"changes",
"baseline",
"fvc",
"lung",
"fev1",
"asthma",
"lung function",
"function",
"bmi"
] |
[
"test",
"test"
] |
[CONTENT] Adiposity | Asthma | Atopy | FENO | Longitudinal | Lung function | Obesity [SUMMARY]
|
[CONTENT] Adiposity | Asthma | Atopy | FENO | Longitudinal | Lung function | Obesity [SUMMARY]
|
[CONTENT] Adiposity | Asthma | Atopy | FENO | Longitudinal | Lung function | Obesity [SUMMARY]
|
[CONTENT] Adiposity | Asthma | Atopy | FENO | Longitudinal | Lung function | Obesity [SUMMARY]
|
[CONTENT] Adiposity | Asthma | Atopy | FENO | Longitudinal | Lung function | Obesity [SUMMARY]
|
[CONTENT] Adiposity | Asthma | Atopy | FENO | Longitudinal | Lung function | Obesity [SUMMARY]
|
[CONTENT] Adiposity | Adult | Aged | Asthma | Body Composition | Body Mass Index | Breath Tests | Electric Impedance | Female | Forced Expiratory Volume | Humans | Hypersensitivity, Immediate | Immunoglobulin E | Longitudinal Studies | Lung | Male | Middle Aged | Nitric Oxide | Obesity | Risk | Vital Capacity | Young Adult [SUMMARY]
|
[CONTENT] Adiposity | Adult | Aged | Asthma | Body Composition | Body Mass Index | Breath Tests | Electric Impedance | Female | Forced Expiratory Volume | Humans | Hypersensitivity, Immediate | Immunoglobulin E | Longitudinal Studies | Lung | Male | Middle Aged | Nitric Oxide | Obesity | Risk | Vital Capacity | Young Adult [SUMMARY]
|
[CONTENT] Adiposity | Adult | Aged | Asthma | Body Composition | Body Mass Index | Breath Tests | Electric Impedance | Female | Forced Expiratory Volume | Humans | Hypersensitivity, Immediate | Immunoglobulin E | Longitudinal Studies | Lung | Male | Middle Aged | Nitric Oxide | Obesity | Risk | Vital Capacity | Young Adult [SUMMARY]
|
[CONTENT] Adiposity | Adult | Aged | Asthma | Body Composition | Body Mass Index | Breath Tests | Electric Impedance | Female | Forced Expiratory Volume | Humans | Hypersensitivity, Immediate | Immunoglobulin E | Longitudinal Studies | Lung | Male | Middle Aged | Nitric Oxide | Obesity | Risk | Vital Capacity | Young Adult [SUMMARY]
|
[CONTENT] Adiposity | Adult | Aged | Asthma | Body Composition | Body Mass Index | Breath Tests | Electric Impedance | Female | Forced Expiratory Volume | Humans | Hypersensitivity, Immediate | Immunoglobulin E | Longitudinal Studies | Lung | Male | Middle Aged | Nitric Oxide | Obesity | Risk | Vital Capacity | Young Adult [SUMMARY]
|
[CONTENT] Adiposity | Adult | Aged | Asthma | Body Composition | Body Mass Index | Breath Tests | Electric Impedance | Female | Forced Expiratory Volume | Humans | Hypersensitivity, Immediate | Immunoglobulin E | Longitudinal Studies | Lung | Male | Middle Aged | Nitric Oxide | Obesity | Risk | Vital Capacity | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] adiposity | changes | baseline | fvc | lung | fev1 | asthma | lung function | function | bmi [SUMMARY]
|
[CONTENT] adiposity | changes | baseline | fvc | lung | fev1 | asthma | lung function | function | bmi [SUMMARY]
|
[CONTENT] adiposity | changes | baseline | fvc | lung | fev1 | asthma | lung function | function | bmi [SUMMARY]
|
[CONTENT] adiposity | changes | baseline | fvc | lung | fev1 | asthma | lung function | function | bmi [SUMMARY]
|
[CONTENT] adiposity | changes | baseline | fvc | lung | fev1 | asthma | lung function | function | bmi [SUMMARY]
|
[CONTENT] adiposity | changes | baseline | fvc | lung | fev1 | asthma | lung function | function | bmi [SUMMARY]
|
[CONTENT] adiposity | inflammation | eosinophilic | eosinophilic inflammation | changes | longitudinal | lung | atopic | longitudinal changes | function [SUMMARY]
|
[CONTENT] analyses | measures | years | baseline | study | measured | health2006 | adiposity | adiposity measures | participants [SUMMARY]
|
[CONTENT] 001 | changes | ml | sd | adiposity | baseline | fvc | males | females | bmi [SUMMARY]
|
[CONTENT] adiposity | associated | adiposity associated | changes | fev1 | fvc | fev1 fvc | adiposity changes | adipose tissue | adipose [SUMMARY]
|
[CONTENT] adiposity | lung | changes | function | lung function | study | fvc | fev1 | baseline | asthma [SUMMARY]
|
[CONTENT] adiposity | lung | changes | function | lung function | study | fvc | fev1 | baseline | asthma [SUMMARY]
|
[CONTENT] ||| ||| first second | FEV1 [SUMMARY]
|
[CONTENT] 19-72 years | five years ||| FEV1 | FVC ||| [SUMMARY]
|
[CONTENT] 2,308 | five-year ||| ||| 0.0001 ||| one | FEV1 | FVC ||| ||| 27 ml | 22 | FVC | 36 ml | 28-44 ||| ||| ||| Atopy ||| [SUMMARY]
|
[CONTENT] five-year ||| [SUMMARY]
|
[CONTENT] ||| ||| first second | FEV1 ||| 19-72 years | five years ||| FEV1 | FVC ||| ||| 2,308 | five-year ||| ||| 0.0001 ||| one | FEV1 | FVC ||| ||| 27 ml | 22 | FVC | 36 ml | 28-44 ||| ||| ||| Atopy ||| ||| five-year ||| [SUMMARY]
|
[CONTENT] ||| ||| first second | FEV1 ||| 19-72 years | five years ||| FEV1 | FVC ||| ||| 2,308 | five-year ||| ||| 0.0001 ||| one | FEV1 | FVC ||| ||| 27 ml | 22 | FVC | 36 ml | 28-44 ||| ||| ||| Atopy ||| ||| five-year ||| [SUMMARY]
|
||||||
Ginsenoside Rg2 alleviates myocardial fibrosis by regulating TGF-β1/Smad signalling pathway.
|
33535854
|
Panax ginseng C.A. Meyer (Araliaceae) has cardioprotective effects. Ginsenosides are responsible for most of the pharmacological activities of ginseng.
|
CONTEXT
|
Male Wistar rats were divided into control, isoproterenol, ginsenoside Rg2 (5, 20 mg/kg) groups (n = 8). The rats were subcutaneously injected with isoproterenol (5 mg/kg) or normal saline (control group) once daily for 7 days. The animals were intragastrically treated with ginsenoside Rg2 or 0.5% CMC-Na (control and isoproterenol groups) daily for 28 days. At day 28, cardiac function, myocardial fibrosis, and TGF-β1/Smad signalling pathway were evaluated.
|
MATERIALS AND METHODS
|
Compared with myocardial ischaemic rats, ginsenoside Rg2 at doses of 5, 20 mg/kg abated partially the augment of LVEDP (8.9 ± 1.3 vs. 7.5 ± 0.7, 7.2 ± 1.0 mmHg) and the decreases of the LVSP (96.75 ± 13.2 vs. 118.3 ± 19.4, 124.3 ± 21.3 mmHg), the + dp/dt (2142.8 ± 309.3 vs. 2598.6 ± 404.0, 2661.5 ± 445.2 mmHg/s), and the -dp/dt (1996.3 ± 306.3 vs. 2476.6 ± 289.7, 2509.6 ± 353.1 mmHg/s). Ginsenoside Rg2 (9.2 ± 0.9%, 8.5 ± 0.8%) alleviated myocardial fibrosis when compared with the isoproterenol group (10.1 ± 1.0%), which was accompanied by suppressed TGF-β1/Smad signalling in heart tissues.
|
RESULTS
|
Ginsenosides from ginseng possess the property of alleviating myocardial fibrosis, improving cardiac function after myocardial ischaemia. Ginsenosides may be promising agents for improving the outcomes of patients with myocardial ischaemia.
|
CONCLUSIONS
|
[
"Animals",
"Cardiotonic Agents",
"Dose-Response Relationship, Drug",
"Fibrosis",
"Ginsenosides",
"Isoproterenol",
"Male",
"Myocardial Ischemia",
"Panax",
"Rats",
"Rats, Wistar",
"Signal Transduction",
"Smad Proteins",
"Transforming Growth Factor beta1"
] |
8871615
|
Introduction
|
Cardiovascular diseases (CVDs) are the primary cause of death worldwide (Hausenloy and Yellon 2013). Myocardial ischaemia, the most common CVDs, is caused by the reduction in coronary blood flow due to atherosclerotic coronary arterial obstruction. Researchers find that surgical approaches restoring blood flow of coronary arteries fail to improve cardiac function of patients with myocardial ischaemia (Ngu et al. 2018). On average, myocardial fibrosis will occur in 10% of patients. Myocardial fibrosis is the common cause of death after myocardial ischaemia, which accounts for a higher 5-year mortality (about 50%) (Benjamin et al. 2018). Myocardial fibrosis is characterised by the collagen formation, which leads to a chamber stiffness and an increased myocardial mass (Li et al. 2018a). Tremendous efforts have been made to develop therapeutics to attenuate myocardial fibrosis and improve cardiac function in patients of myocardial ischaemia. Traditional Chinese medicine (TCM) and small-molecule drugs are promising agents for preventing myocardial ischaemia-induced heart function impairment (Hashimoto et al. 2018).
In China, TCM has been used in treating CVDs for thousands of years. A great deal of interest has focussed on elucidating the mechanisms underlying the cardioprotective effects of the active ingredients of TCM (Fan et al. 2017). Panax ginseng C.A. Meyer (Araliaceae), a well-known TCM, is widely used for increasing vital energy and improving organ functions. Ginsenosides are the major active ingredients of ginseng. According to the structures of the extracted components of ginseng, ginsenosides are classified into three groups: the panaxadiol group including Rb1, Rb2, Rg3, and Rh2, the panaxatriol group including Re, Rh1, and Rg1, and oleanolic acid (Zhang et al. 2019). Recent work demonstrates that ginsenoside Rb1 can reduce the injury of myocardial ischaemia/reperfusion and then improve the heart function by inhibiting the apoptosis of cardiomyocytes (Li et al. 2020). Ginsenoside Rg1 exerts a protective effect against myocardial ischaemia-induced injury, which is associated with modulating energy metabolism and inhibiting myocardial apoptosis (Li et al. 2018b). Our previous studies indicate that ginsenoside Re treatment abrogates the myocardial fibrosis caused by myocardial ischaemia and then restores the heart function (Wang et al. 2019). Ginsenoside Rg2 is one of the cardinal components of ginseng. Ginsenoside Rg2 has a property of attenuating the neuronal damage induced by hypoxia in hippocampal neurons (Shuangyan et al. 2012). Ginsenoside Rg2 can also improve cerebral ischaemia-induced dementia through abating the apoptosis of neurons (Zhang et al. 2008). However, it is still not clear whether ginsenoside Rg2 has a protective effect against myocardial ischaemic injury. Therefore, this study investigates whether ginsenoside Rg2 could alleviate myocardial ischaemia-induced myocardial fibrosis and cardiac function impairment in rats.
| null | null |
Results
|
Effect of ginsenoside Rg2 on cardiac function and myocardial fibrosis in normal rats Compared with the control group, the LVSP, the LVEDP, the + dp/dt, and the -dp/dt of ginsenoside Rg2 group did not show significant changes (Figure 1, p > 0.05). Further histopathological examination demonstrated that myocardial fibrosis in the ginsenoside Rg2 group was the same as that of the control group (Figure 2, p > 0.05). These findings indicated that ginsenoside Rg2 alone had no effect on the cardiac function and the myocardial fibrosis in normal rats.
Effect of ginsenoside Rg2 on cardiac function in normal rats. Data are presented as the mean ± SD (n = 7–8).
Effect of ginsenoside Rg2 on myocardial fibrosis in normal rats. Data are presented as the mean ± SD (n = 3). A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.
Compared with the control group, the LVSP, the LVEDP, the + dp/dt, and the -dp/dt of ginsenoside Rg2 group did not show significant changes (Figure 1, p > 0.05). Further histopathological examination demonstrated that myocardial fibrosis in the ginsenoside Rg2 group was the same as that of the control group (Figure 2, p > 0.05). These findings indicated that ginsenoside Rg2 alone had no effect on the cardiac function and the myocardial fibrosis in normal rats.
Effect of ginsenoside Rg2 on cardiac function in normal rats. Data are presented as the mean ± SD (n = 7–8).
Effect of ginsenoside Rg2 on myocardial fibrosis in normal rats. Data are presented as the mean ± SD (n = 3). A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.
Effect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats The cardiac function of the rats was evaluated with a hemodynamic analysing system. The LVEDP of the rats in the isoproterenol group was significantly increased, as compared with the control group (p < 0.01). The LVSP, the + dp/dt, and the –dp/dt in the isoproterenol group were markedly decreased (p < 0.01). Compared with the isoproterenol group, LVEDP of the rats in the ginsenoside Rg2 groups was significantly decreased, whereas the LVSP, the + dp/dt, and the –dp/dt of the ginsenoside Rg2 groups were increased (p < 0.05 or p < 0.01). These results demonstrated that ginsenoside Rg2 has a property improving the cardiac function in myocardial ischaemic rats (Figure 3).
Effect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group.
The cardiac function of the rats was evaluated with a hemodynamic analysing system. The LVEDP of the rats in the isoproterenol group was significantly increased, as compared with the control group (p < 0.01). The LVSP, the + dp/dt, and the –dp/dt in the isoproterenol group were markedly decreased (p < 0.01). Compared with the isoproterenol group, LVEDP of the rats in the ginsenoside Rg2 groups was significantly decreased, whereas the LVSP, the + dp/dt, and the –dp/dt of the ginsenoside Rg2 groups were increased (p < 0.05 or p < 0.01). These results demonstrated that ginsenoside Rg2 has a property improving the cardiac function in myocardial ischaemic rats (Figure 3).
Effect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group.
Effect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats The gross morphological examination showed that hearts of the rats from the isoproterenol group were significantly larger than those from the control group (Figure 4(A)). In addition, heart weight and heart weight index were significantly increased in the isoproterenol group when compared to the control group (p < 0.01). Treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg abrogated partially myocardial ischaemia-induced increases of heart weight and heart weight index (p < 0.05 or p < 0.01, Figure 4(B–D)).
Effect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of the gross morphological hearts. B indicates bar graph of body weight. C indicates bar graph of heart weight. D indicates bar graph of heart weight index.
The gross morphological examination showed that hearts of the rats from the isoproterenol group were significantly larger than those from the control group (Figure 4(A)). In addition, heart weight and heart weight index were significantly increased in the isoproterenol group when compared to the control group (p < 0.01). Treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg abrogated partially myocardial ischaemia-induced increases of heart weight and heart weight index (p < 0.05 or p < 0.01, Figure 4(B–D)).
Effect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of the gross morphological hearts. B indicates bar graph of body weight. C indicates bar graph of heart weight. D indicates bar graph of heart weight index.
Effect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats The Sirius red staining showed that there was extensive collagen deposition in the border zone of the myocardial ischaemia in the myocardial ischaemic rats, whereas treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg markedly reduced contents of collagen deposition in myocardial ischaemic rats (Figure 5(A)). The red-stained area was calculated as a percentage of the total area with Image-Pro Plus software. In line with the observations mentioned above, the red-appearing collagens of the heart tissues in myocardial ischaemic animals were significantly augmented (p < 0.01). However, ginsenoside Rg2 treatment markedly decreased the contents of the collagen content in the heart tissues (p < 0.05 or p < 0.01, Figure 5(B)).
Effect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.
The Sirius red staining showed that there was extensive collagen deposition in the border zone of the myocardial ischaemia in the myocardial ischaemic rats, whereas treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg markedly reduced contents of collagen deposition in myocardial ischaemic rats (Figure 5(A)). The red-stained area was calculated as a percentage of the total area with Image-Pro Plus software. In line with the observations mentioned above, the red-appearing collagens of the heart tissues in myocardial ischaemic animals were significantly augmented (p < 0.01). However, ginsenoside Rg2 treatment markedly decreased the contents of the collagen content in the heart tissues (p < 0.05 or p < 0.01, Figure 5(B)).
Effect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.
Effect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats TGF-β1 leads to the production of collagens in myocardial fibroblasts. Smad3 is the downstream effectors of the TGF-β1 receptor. The effects of ginsenoside Rg2 on the TGF-β1/Smad signalling pathway in the heart tissues were investigated in the present study. The results showed that expression of TGF-β1, p-Smad3, collagen I and α-SMA were significantly up-regulated in the heart tissues of myocardial ischaemic rats (p < 0.05 or p < 0.01). However, treatment with ginsenoside Rg2 significantly decreased the expression of TGF-β1, p-Smad3, collagen I and α-SMA in the heart tissues (p < 0.05 or p < 0.01, Figure 6(A–D).
Effect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photograph and bar graph of TGF-β1. B indicates representative photograph and bar graph of p-Smad3. C indicates representative photograph and bar graph of collagen I. D indicates representative photograph and bar graph of α-SMA.
TGF-β1 leads to the production of collagens in myocardial fibroblasts. Smad3 is the downstream effectors of the TGF-β1 receptor. The effects of ginsenoside Rg2 on the TGF-β1/Smad signalling pathway in the heart tissues were investigated in the present study. The results showed that expression of TGF-β1, p-Smad3, collagen I and α-SMA were significantly up-regulated in the heart tissues of myocardial ischaemic rats (p < 0.05 or p < 0.01). However, treatment with ginsenoside Rg2 significantly decreased the expression of TGF-β1, p-Smad3, collagen I and α-SMA in the heart tissues (p < 0.05 or p < 0.01, Figure 6(A–D).
Effect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photograph and bar graph of TGF-β1. B indicates representative photograph and bar graph of p-Smad3. C indicates representative photograph and bar graph of collagen I. D indicates representative photograph and bar graph of α-SMA.
|
Conclusions
|
The present experiments demonstrated that ginsenoside Rg2 possesses the property of alleviating myocardial fibrosis and improving cardiac function by regulating the TGF-β1/Smad signalling pathway after myocardial ischaemia. This study provides insights into the mechanisms of ginseng in the clinical application for myocardial ischaemia.
|
[
"Ginsenoside Rg2",
"Animals",
"Experimental protocol",
"Cardiac function evaluation",
"Heart weight index assessment",
"Histopathological examination",
"Western blot",
"Statistical analysis",
"Effect of ginsenoside Rg2 on cardiac function and myocardial fibrosis in normal rats",
"Effect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats",
"Effect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats",
"Effect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats",
"Effect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats",
"Geolocation information"
] |
[
"Ginsenoside Rg2 extracted from ginseng cultivated in China was kindly provided by Professor Yifa Zhou (School of Life Sciences, Northeast Normal University, Changchun, China). The purity of ginsenoside Rg2 was >98.0%.",
"Male Wistar rats weighing 240–260 g were obtained from the Experiment Animal Centre of Jilin University (Changchun, China). Animals were housed on a 12 h light/dark cycle at a controlled temperature and humidity with unlimited access to food and water. The experiments were approved by the Ethics Committee of Jilin University (Approval number, JLU2018-03-0023) and were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (publication 86-23, revised in 1986).",
"Experiment 1: Rats were randomly divided into two groups (n = 8): a control group (1 rat was excluded because of anaesthetic overdose) and a ginsenoside Rg2 at dose of 20 mg/kg group. The rats of the control group were intragastrically administered with 0.5% CMC-Na daily for 28 days. The animals of the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 daily for 28 days. Experiment 2: Rats were randomly divided into four groups (n =8): a control group, an isoproterenol group and ginsenoside Rg2 (5, 20 mg/kg) groups. To induce myocardial fibrosis, the rats were injected subcutaneously with isoproterenol (Sigma-Aldrich, USA) at a dose of 5 mg/kg, once daily for 7 consecutive days (Zhang et al. 2014). Two hours later, the animals in the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 at doses of 5 or 20 mg/kg, daily for 28 days. The rats of the control and the isoproterenol groups were intragastrically administered with 0.5% CMC-Na daily for 28 days.",
"At day 28, the body weights of the rats were recorded. The rats were anaesthetised by an intraperitoneal injection with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). A 2 F polyethylene catheter was inserted into the left ventricle through the right common carotid artery. The catheter was connected with a hemodynamic analysing system (Model RM-6000, Nihon Kohden, Japan). Then, the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and positive (+dp/dt) and negative (-dp/dt) maximal values of the first derivative of the left ventricular pressure were evaluated.",
"The rats were sacrificed with overdoses of carbon dioxide after cardiac function evaluation. The hearts of the animals were collected and washed with PBS solution (4 °C). The aorta, atria, and adipose tissue were excised. Heart weights were weighed and heart weight index (mg/g) was calculated by dividing heart weight by body weight.",
"The hearts of the rats were fixed with 10% formalin for 24 h. The specimens were embedded with paraffin and then were cut into 5 μm thick sections. According to the instruction of the manufacturer, the heart sections were stained with a Sirius red staining kit (Solarbio Life Sciences Company, China). Then, the histopathological examination was conducted by an experienced observer who was blinded to the experimental design under an Olympus microscope (IX-70, Olympus Corp., Japan). Ten fields were randomly selected from five sections in each heart. The ratio of the interstitial fibrosis area in the hearts was analysed and calculated with NIH-Image software (National Institutes of Health, Bethesda, MD, U.S.A.). The myocardial fibrosis was presented by the calculation: (fibrotic tissue area)/(fibrotic tissue area + cardiomyocyte area) × 100%.",
"Western blot was performed as previously described (Ahn et al. 2019). The membranes were incubated with the primary antibodies: rabbit anti-TGF-β1 (1:1000, Proteintech, USA), rabbit anti-α-smooth muscle actin (α-SMA) (1:2000, ImmunoWay Biotechnology, USA), rabbit anti-collagen I (1:2000, Abcam, UK), rabbit anti-Smad3 (1:1500, Abcam, UK), rabbit anti-p-Smad3 (1:1500, Abcam, UK) or rabbit anti-β-actin (1:1500, Beyotime Institute of Biotechnology, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labelled secondary antibody (1:2000, Beyotime Institute of Biotechnology, China). The bands were visualised using the ECL detection reagents (Beyotime Institute of Biotechnology, China). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.",
"Data were presented as the mean ± SD and processed with SPSS 22.0. Statistical analysis among various groups was conducted by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. The p < 0.05 was considered as statistically significant.",
"Compared with the control group, the LVSP, the LVEDP, the + dp/dt, and the -dp/dt of ginsenoside Rg2 group did not show significant changes (Figure 1, p > 0.05). Further histopathological examination demonstrated that myocardial fibrosis in the ginsenoside Rg2 group was the same as that of the control group (Figure 2, p > 0.05). These findings indicated that ginsenoside Rg2 alone had no effect on the cardiac function and the myocardial fibrosis in normal rats.\nEffect of ginsenoside Rg2 on cardiac function in normal rats. Data are presented as the mean ± SD (n = 7–8).\nEffect of ginsenoside Rg2 on myocardial fibrosis in normal rats. Data are presented as the mean ± SD (n = 3). A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.",
"The cardiac function of the rats was evaluated with a hemodynamic analysing system. The LVEDP of the rats in the isoproterenol group was significantly increased, as compared with the control group (p < 0.01). The LVSP, the + dp/dt, and the –dp/dt in the isoproterenol group were markedly decreased (p < 0.01). Compared with the isoproterenol group, LVEDP of the rats in the ginsenoside Rg2 groups was significantly decreased, whereas the LVSP, the + dp/dt, and the –dp/dt of the ginsenoside Rg2 groups were increased (p < 0.05 or p < 0.01). These results demonstrated that ginsenoside Rg2 has a property improving the cardiac function in myocardial ischaemic rats (Figure 3).\nEffect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group.",
"The gross morphological examination showed that hearts of the rats from the isoproterenol group were significantly larger than those from the control group (Figure 4(A)). In addition, heart weight and heart weight index were significantly increased in the isoproterenol group when compared to the control group (p < 0.01). Treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg abrogated partially myocardial ischaemia-induced increases of heart weight and heart weight index (p < 0.05 or p < 0.01, Figure 4(B–D)).\nEffect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of the gross morphological hearts. B indicates bar graph of body weight. C indicates bar graph of heart weight. D indicates bar graph of heart weight index.",
"The Sirius red staining showed that there was extensive collagen deposition in the border zone of the myocardial ischaemia in the myocardial ischaemic rats, whereas treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg markedly reduced contents of collagen deposition in myocardial ischaemic rats (Figure 5(A)). The red-stained area was calculated as a percentage of the total area with Image-Pro Plus software. In line with the observations mentioned above, the red-appearing collagens of the heart tissues in myocardial ischaemic animals were significantly augmented (p < 0.01). However, ginsenoside Rg2 treatment markedly decreased the contents of the collagen content in the heart tissues (p < 0.05 or p < 0.01, Figure 5(B)).\nEffect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.",
"TGF-β1 leads to the production of collagens in myocardial fibroblasts. Smad3 is the downstream effectors of the TGF-β1 receptor. The effects of ginsenoside Rg2 on the TGF-β1/Smad signalling pathway in the heart tissues were investigated in the present study. The results showed that expression of TGF-β1, p-Smad3, collagen I and α-SMA were significantly up-regulated in the heart tissues of myocardial ischaemic rats (p < 0.05 or p < 0.01). However, treatment with ginsenoside Rg2 significantly decreased the expression of TGF-β1, p-Smad3, collagen I and α-SMA in the heart tissues (p < 0.05 or p < 0.01, Figure 6(A–D).\nEffect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photograph and bar graph of TGF-β1. B indicates representative photograph and bar graph of p-Smad3. C indicates representative photograph and bar graph of collagen I. D indicates representative photograph and bar graph of α-SMA.",
"Changchun is at 125.35 degrees East longitude and 43.88 degrees North latitude."
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[
"Introduction",
"Materials and methods",
"Ginsenoside Rg2",
"Animals",
"Experimental protocol",
"Cardiac function evaluation",
"Heart weight index assessment",
"Histopathological examination",
"Western blot",
"Statistical analysis",
"Results",
"Effect of ginsenoside Rg2 on cardiac function and myocardial fibrosis in normal rats",
"Effect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats",
"Effect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats",
"Effect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats",
"Effect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats",
"Discussion",
"Conclusions",
"Geolocation information"
] |
[
"Cardiovascular diseases (CVDs) are the primary cause of death worldwide (Hausenloy and Yellon 2013). Myocardial ischaemia, the most common CVDs, is caused by the reduction in coronary blood flow due to atherosclerotic coronary arterial obstruction. Researchers find that surgical approaches restoring blood flow of coronary arteries fail to improve cardiac function of patients with myocardial ischaemia (Ngu et al. 2018). On average, myocardial fibrosis will occur in 10% of patients. Myocardial fibrosis is the common cause of death after myocardial ischaemia, which accounts for a higher 5-year mortality (about 50%) (Benjamin et al. 2018). Myocardial fibrosis is characterised by the collagen formation, which leads to a chamber stiffness and an increased myocardial mass (Li et al. 2018a). Tremendous efforts have been made to develop therapeutics to attenuate myocardial fibrosis and improve cardiac function in patients of myocardial ischaemia. Traditional Chinese medicine (TCM) and small-molecule drugs are promising agents for preventing myocardial ischaemia-induced heart function impairment (Hashimoto et al. 2018).\nIn China, TCM has been used in treating CVDs for thousands of years. A great deal of interest has focussed on elucidating the mechanisms underlying the cardioprotective effects of the active ingredients of TCM (Fan et al. 2017). Panax ginseng C.A. Meyer (Araliaceae), a well-known TCM, is widely used for increasing vital energy and improving organ functions. Ginsenosides are the major active ingredients of ginseng. According to the structures of the extracted components of ginseng, ginsenosides are classified into three groups: the panaxadiol group including Rb1, Rb2, Rg3, and Rh2, the panaxatriol group including Re, Rh1, and Rg1, and oleanolic acid (Zhang et al. 2019). Recent work demonstrates that ginsenoside Rb1 can reduce the injury of myocardial ischaemia/reperfusion and then improve the heart function by inhibiting the apoptosis of cardiomyocytes (Li et al. 2020). Ginsenoside Rg1 exerts a protective effect against myocardial ischaemia-induced injury, which is associated with modulating energy metabolism and inhibiting myocardial apoptosis (Li et al. 2018b). Our previous studies indicate that ginsenoside Re treatment abrogates the myocardial fibrosis caused by myocardial ischaemia and then restores the heart function (Wang et al. 2019). Ginsenoside Rg2 is one of the cardinal components of ginseng. Ginsenoside Rg2 has a property of attenuating the neuronal damage induced by hypoxia in hippocampal neurons (Shuangyan et al. 2012). Ginsenoside Rg2 can also improve cerebral ischaemia-induced dementia through abating the apoptosis of neurons (Zhang et al. 2008). However, it is still not clear whether ginsenoside Rg2 has a protective effect against myocardial ischaemic injury. Therefore, this study investigates whether ginsenoside Rg2 could alleviate myocardial ischaemia-induced myocardial fibrosis and cardiac function impairment in rats.",
" Ginsenoside Rg2 Ginsenoside Rg2 extracted from ginseng cultivated in China was kindly provided by Professor Yifa Zhou (School of Life Sciences, Northeast Normal University, Changchun, China). The purity of ginsenoside Rg2 was >98.0%.\nGinsenoside Rg2 extracted from ginseng cultivated in China was kindly provided by Professor Yifa Zhou (School of Life Sciences, Northeast Normal University, Changchun, China). The purity of ginsenoside Rg2 was >98.0%.\n Animals Male Wistar rats weighing 240–260 g were obtained from the Experiment Animal Centre of Jilin University (Changchun, China). Animals were housed on a 12 h light/dark cycle at a controlled temperature and humidity with unlimited access to food and water. The experiments were approved by the Ethics Committee of Jilin University (Approval number, JLU2018-03-0023) and were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (publication 86-23, revised in 1986).\nMale Wistar rats weighing 240–260 g were obtained from the Experiment Animal Centre of Jilin University (Changchun, China). Animals were housed on a 12 h light/dark cycle at a controlled temperature and humidity with unlimited access to food and water. The experiments were approved by the Ethics Committee of Jilin University (Approval number, JLU2018-03-0023) and were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (publication 86-23, revised in 1986).\n Experimental protocol Experiment 1: Rats were randomly divided into two groups (n = 8): a control group (1 rat was excluded because of anaesthetic overdose) and a ginsenoside Rg2 at dose of 20 mg/kg group. The rats of the control group were intragastrically administered with 0.5% CMC-Na daily for 28 days. The animals of the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 daily for 28 days. Experiment 2: Rats were randomly divided into four groups (n =8): a control group, an isoproterenol group and ginsenoside Rg2 (5, 20 mg/kg) groups. To induce myocardial fibrosis, the rats were injected subcutaneously with isoproterenol (Sigma-Aldrich, USA) at a dose of 5 mg/kg, once daily for 7 consecutive days (Zhang et al. 2014). Two hours later, the animals in the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 at doses of 5 or 20 mg/kg, daily for 28 days. The rats of the control and the isoproterenol groups were intragastrically administered with 0.5% CMC-Na daily for 28 days.\nExperiment 1: Rats were randomly divided into two groups (n = 8): a control group (1 rat was excluded because of anaesthetic overdose) and a ginsenoside Rg2 at dose of 20 mg/kg group. The rats of the control group were intragastrically administered with 0.5% CMC-Na daily for 28 days. The animals of the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 daily for 28 days. Experiment 2: Rats were randomly divided into four groups (n =8): a control group, an isoproterenol group and ginsenoside Rg2 (5, 20 mg/kg) groups. To induce myocardial fibrosis, the rats were injected subcutaneously with isoproterenol (Sigma-Aldrich, USA) at a dose of 5 mg/kg, once daily for 7 consecutive days (Zhang et al. 2014). Two hours later, the animals in the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 at doses of 5 or 20 mg/kg, daily for 28 days. The rats of the control and the isoproterenol groups were intragastrically administered with 0.5% CMC-Na daily for 28 days.\n Cardiac function evaluation At day 28, the body weights of the rats were recorded. The rats were anaesthetised by an intraperitoneal injection with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). A 2 F polyethylene catheter was inserted into the left ventricle through the right common carotid artery. The catheter was connected with a hemodynamic analysing system (Model RM-6000, Nihon Kohden, Japan). Then, the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and positive (+dp/dt) and negative (-dp/dt) maximal values of the first derivative of the left ventricular pressure were evaluated.\nAt day 28, the body weights of the rats were recorded. The rats were anaesthetised by an intraperitoneal injection with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). A 2 F polyethylene catheter was inserted into the left ventricle through the right common carotid artery. The catheter was connected with a hemodynamic analysing system (Model RM-6000, Nihon Kohden, Japan). Then, the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and positive (+dp/dt) and negative (-dp/dt) maximal values of the first derivative of the left ventricular pressure were evaluated.\n Heart weight index assessment The rats were sacrificed with overdoses of carbon dioxide after cardiac function evaluation. The hearts of the animals were collected and washed with PBS solution (4 °C). The aorta, atria, and adipose tissue were excised. Heart weights were weighed and heart weight index (mg/g) was calculated by dividing heart weight by body weight.\nThe rats were sacrificed with overdoses of carbon dioxide after cardiac function evaluation. The hearts of the animals were collected and washed with PBS solution (4 °C). The aorta, atria, and adipose tissue were excised. Heart weights were weighed and heart weight index (mg/g) was calculated by dividing heart weight by body weight.\n Histopathological examination The hearts of the rats were fixed with 10% formalin for 24 h. The specimens were embedded with paraffin and then were cut into 5 μm thick sections. According to the instruction of the manufacturer, the heart sections were stained with a Sirius red staining kit (Solarbio Life Sciences Company, China). Then, the histopathological examination was conducted by an experienced observer who was blinded to the experimental design under an Olympus microscope (IX-70, Olympus Corp., Japan). Ten fields were randomly selected from five sections in each heart. The ratio of the interstitial fibrosis area in the hearts was analysed and calculated with NIH-Image software (National Institutes of Health, Bethesda, MD, U.S.A.). The myocardial fibrosis was presented by the calculation: (fibrotic tissue area)/(fibrotic tissue area + cardiomyocyte area) × 100%.\nThe hearts of the rats were fixed with 10% formalin for 24 h. The specimens were embedded with paraffin and then were cut into 5 μm thick sections. According to the instruction of the manufacturer, the heart sections were stained with a Sirius red staining kit (Solarbio Life Sciences Company, China). Then, the histopathological examination was conducted by an experienced observer who was blinded to the experimental design under an Olympus microscope (IX-70, Olympus Corp., Japan). Ten fields were randomly selected from five sections in each heart. The ratio of the interstitial fibrosis area in the hearts was analysed and calculated with NIH-Image software (National Institutes of Health, Bethesda, MD, U.S.A.). The myocardial fibrosis was presented by the calculation: (fibrotic tissue area)/(fibrotic tissue area + cardiomyocyte area) × 100%.\n Western blot Western blot was performed as previously described (Ahn et al. 2019). The membranes were incubated with the primary antibodies: rabbit anti-TGF-β1 (1:1000, Proteintech, USA), rabbit anti-α-smooth muscle actin (α-SMA) (1:2000, ImmunoWay Biotechnology, USA), rabbit anti-collagen I (1:2000, Abcam, UK), rabbit anti-Smad3 (1:1500, Abcam, UK), rabbit anti-p-Smad3 (1:1500, Abcam, UK) or rabbit anti-β-actin (1:1500, Beyotime Institute of Biotechnology, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labelled secondary antibody (1:2000, Beyotime Institute of Biotechnology, China). The bands were visualised using the ECL detection reagents (Beyotime Institute of Biotechnology, China). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.\nWestern blot was performed as previously described (Ahn et al. 2019). The membranes were incubated with the primary antibodies: rabbit anti-TGF-β1 (1:1000, Proteintech, USA), rabbit anti-α-smooth muscle actin (α-SMA) (1:2000, ImmunoWay Biotechnology, USA), rabbit anti-collagen I (1:2000, Abcam, UK), rabbit anti-Smad3 (1:1500, Abcam, UK), rabbit anti-p-Smad3 (1:1500, Abcam, UK) or rabbit anti-β-actin (1:1500, Beyotime Institute of Biotechnology, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labelled secondary antibody (1:2000, Beyotime Institute of Biotechnology, China). The bands were visualised using the ECL detection reagents (Beyotime Institute of Biotechnology, China). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.\n Statistical analysis Data were presented as the mean ± SD and processed with SPSS 22.0. Statistical analysis among various groups was conducted by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. The p < 0.05 was considered as statistically significant.\nData were presented as the mean ± SD and processed with SPSS 22.0. Statistical analysis among various groups was conducted by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. The p < 0.05 was considered as statistically significant.",
"Ginsenoside Rg2 extracted from ginseng cultivated in China was kindly provided by Professor Yifa Zhou (School of Life Sciences, Northeast Normal University, Changchun, China). The purity of ginsenoside Rg2 was >98.0%.",
"Male Wistar rats weighing 240–260 g were obtained from the Experiment Animal Centre of Jilin University (Changchun, China). Animals were housed on a 12 h light/dark cycle at a controlled temperature and humidity with unlimited access to food and water. The experiments were approved by the Ethics Committee of Jilin University (Approval number, JLU2018-03-0023) and were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (publication 86-23, revised in 1986).",
"Experiment 1: Rats were randomly divided into two groups (n = 8): a control group (1 rat was excluded because of anaesthetic overdose) and a ginsenoside Rg2 at dose of 20 mg/kg group. The rats of the control group were intragastrically administered with 0.5% CMC-Na daily for 28 days. The animals of the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 daily for 28 days. Experiment 2: Rats were randomly divided into four groups (n =8): a control group, an isoproterenol group and ginsenoside Rg2 (5, 20 mg/kg) groups. To induce myocardial fibrosis, the rats were injected subcutaneously with isoproterenol (Sigma-Aldrich, USA) at a dose of 5 mg/kg, once daily for 7 consecutive days (Zhang et al. 2014). Two hours later, the animals in the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 at doses of 5 or 20 mg/kg, daily for 28 days. The rats of the control and the isoproterenol groups were intragastrically administered with 0.5% CMC-Na daily for 28 days.",
"At day 28, the body weights of the rats were recorded. The rats were anaesthetised by an intraperitoneal injection with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). A 2 F polyethylene catheter was inserted into the left ventricle through the right common carotid artery. The catheter was connected with a hemodynamic analysing system (Model RM-6000, Nihon Kohden, Japan). Then, the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and positive (+dp/dt) and negative (-dp/dt) maximal values of the first derivative of the left ventricular pressure were evaluated.",
"The rats were sacrificed with overdoses of carbon dioxide after cardiac function evaluation. The hearts of the animals were collected and washed with PBS solution (4 °C). The aorta, atria, and adipose tissue were excised. Heart weights were weighed and heart weight index (mg/g) was calculated by dividing heart weight by body weight.",
"The hearts of the rats were fixed with 10% formalin for 24 h. The specimens were embedded with paraffin and then were cut into 5 μm thick sections. According to the instruction of the manufacturer, the heart sections were stained with a Sirius red staining kit (Solarbio Life Sciences Company, China). Then, the histopathological examination was conducted by an experienced observer who was blinded to the experimental design under an Olympus microscope (IX-70, Olympus Corp., Japan). Ten fields were randomly selected from five sections in each heart. The ratio of the interstitial fibrosis area in the hearts was analysed and calculated with NIH-Image software (National Institutes of Health, Bethesda, MD, U.S.A.). The myocardial fibrosis was presented by the calculation: (fibrotic tissue area)/(fibrotic tissue area + cardiomyocyte area) × 100%.",
"Western blot was performed as previously described (Ahn et al. 2019). The membranes were incubated with the primary antibodies: rabbit anti-TGF-β1 (1:1000, Proteintech, USA), rabbit anti-α-smooth muscle actin (α-SMA) (1:2000, ImmunoWay Biotechnology, USA), rabbit anti-collagen I (1:2000, Abcam, UK), rabbit anti-Smad3 (1:1500, Abcam, UK), rabbit anti-p-Smad3 (1:1500, Abcam, UK) or rabbit anti-β-actin (1:1500, Beyotime Institute of Biotechnology, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labelled secondary antibody (1:2000, Beyotime Institute of Biotechnology, China). The bands were visualised using the ECL detection reagents (Beyotime Institute of Biotechnology, China). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.",
"Data were presented as the mean ± SD and processed with SPSS 22.0. Statistical analysis among various groups was conducted by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. The p < 0.05 was considered as statistically significant.",
" Effect of ginsenoside Rg2 on cardiac function and myocardial fibrosis in normal rats Compared with the control group, the LVSP, the LVEDP, the + dp/dt, and the -dp/dt of ginsenoside Rg2 group did not show significant changes (Figure 1, p > 0.05). Further histopathological examination demonstrated that myocardial fibrosis in the ginsenoside Rg2 group was the same as that of the control group (Figure 2, p > 0.05). These findings indicated that ginsenoside Rg2 alone had no effect on the cardiac function and the myocardial fibrosis in normal rats.\nEffect of ginsenoside Rg2 on cardiac function in normal rats. Data are presented as the mean ± SD (n = 7–8).\nEffect of ginsenoside Rg2 on myocardial fibrosis in normal rats. Data are presented as the mean ± SD (n = 3). A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.\nCompared with the control group, the LVSP, the LVEDP, the + dp/dt, and the -dp/dt of ginsenoside Rg2 group did not show significant changes (Figure 1, p > 0.05). Further histopathological examination demonstrated that myocardial fibrosis in the ginsenoside Rg2 group was the same as that of the control group (Figure 2, p > 0.05). These findings indicated that ginsenoside Rg2 alone had no effect on the cardiac function and the myocardial fibrosis in normal rats.\nEffect of ginsenoside Rg2 on cardiac function in normal rats. Data are presented as the mean ± SD (n = 7–8).\nEffect of ginsenoside Rg2 on myocardial fibrosis in normal rats. Data are presented as the mean ± SD (n = 3). A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.\n Effect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats The cardiac function of the rats was evaluated with a hemodynamic analysing system. The LVEDP of the rats in the isoproterenol group was significantly increased, as compared with the control group (p < 0.01). The LVSP, the + dp/dt, and the –dp/dt in the isoproterenol group were markedly decreased (p < 0.01). Compared with the isoproterenol group, LVEDP of the rats in the ginsenoside Rg2 groups was significantly decreased, whereas the LVSP, the + dp/dt, and the –dp/dt of the ginsenoside Rg2 groups were increased (p < 0.05 or p < 0.01). These results demonstrated that ginsenoside Rg2 has a property improving the cardiac function in myocardial ischaemic rats (Figure 3).\nEffect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group.\nThe cardiac function of the rats was evaluated with a hemodynamic analysing system. The LVEDP of the rats in the isoproterenol group was significantly increased, as compared with the control group (p < 0.01). The LVSP, the + dp/dt, and the –dp/dt in the isoproterenol group were markedly decreased (p < 0.01). Compared with the isoproterenol group, LVEDP of the rats in the ginsenoside Rg2 groups was significantly decreased, whereas the LVSP, the + dp/dt, and the –dp/dt of the ginsenoside Rg2 groups were increased (p < 0.05 or p < 0.01). These results demonstrated that ginsenoside Rg2 has a property improving the cardiac function in myocardial ischaemic rats (Figure 3).\nEffect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group.\n Effect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats The gross morphological examination showed that hearts of the rats from the isoproterenol group were significantly larger than those from the control group (Figure 4(A)). In addition, heart weight and heart weight index were significantly increased in the isoproterenol group when compared to the control group (p < 0.01). Treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg abrogated partially myocardial ischaemia-induced increases of heart weight and heart weight index (p < 0.05 or p < 0.01, Figure 4(B–D)).\nEffect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of the gross morphological hearts. B indicates bar graph of body weight. C indicates bar graph of heart weight. D indicates bar graph of heart weight index.\nThe gross morphological examination showed that hearts of the rats from the isoproterenol group were significantly larger than those from the control group (Figure 4(A)). In addition, heart weight and heart weight index were significantly increased in the isoproterenol group when compared to the control group (p < 0.01). Treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg abrogated partially myocardial ischaemia-induced increases of heart weight and heart weight index (p < 0.05 or p < 0.01, Figure 4(B–D)).\nEffect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of the gross morphological hearts. B indicates bar graph of body weight. C indicates bar graph of heart weight. D indicates bar graph of heart weight index.\n Effect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats The Sirius red staining showed that there was extensive collagen deposition in the border zone of the myocardial ischaemia in the myocardial ischaemic rats, whereas treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg markedly reduced contents of collagen deposition in myocardial ischaemic rats (Figure 5(A)). The red-stained area was calculated as a percentage of the total area with Image-Pro Plus software. In line with the observations mentioned above, the red-appearing collagens of the heart tissues in myocardial ischaemic animals were significantly augmented (p < 0.01). However, ginsenoside Rg2 treatment markedly decreased the contents of the collagen content in the heart tissues (p < 0.05 or p < 0.01, Figure 5(B)).\nEffect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.\nThe Sirius red staining showed that there was extensive collagen deposition in the border zone of the myocardial ischaemia in the myocardial ischaemic rats, whereas treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg markedly reduced contents of collagen deposition in myocardial ischaemic rats (Figure 5(A)). The red-stained area was calculated as a percentage of the total area with Image-Pro Plus software. In line with the observations mentioned above, the red-appearing collagens of the heart tissues in myocardial ischaemic animals were significantly augmented (p < 0.01). However, ginsenoside Rg2 treatment markedly decreased the contents of the collagen content in the heart tissues (p < 0.05 or p < 0.01, Figure 5(B)).\nEffect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.\n Effect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats TGF-β1 leads to the production of collagens in myocardial fibroblasts. Smad3 is the downstream effectors of the TGF-β1 receptor. The effects of ginsenoside Rg2 on the TGF-β1/Smad signalling pathway in the heart tissues were investigated in the present study. The results showed that expression of TGF-β1, p-Smad3, collagen I and α-SMA were significantly up-regulated in the heart tissues of myocardial ischaemic rats (p < 0.05 or p < 0.01). However, treatment with ginsenoside Rg2 significantly decreased the expression of TGF-β1, p-Smad3, collagen I and α-SMA in the heart tissues (p < 0.05 or p < 0.01, Figure 6(A–D).\nEffect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photograph and bar graph of TGF-β1. B indicates representative photograph and bar graph of p-Smad3. C indicates representative photograph and bar graph of collagen I. D indicates representative photograph and bar graph of α-SMA.\nTGF-β1 leads to the production of collagens in myocardial fibroblasts. Smad3 is the downstream effectors of the TGF-β1 receptor. The effects of ginsenoside Rg2 on the TGF-β1/Smad signalling pathway in the heart tissues were investigated in the present study. The results showed that expression of TGF-β1, p-Smad3, collagen I and α-SMA were significantly up-regulated in the heart tissues of myocardial ischaemic rats (p < 0.05 or p < 0.01). However, treatment with ginsenoside Rg2 significantly decreased the expression of TGF-β1, p-Smad3, collagen I and α-SMA in the heart tissues (p < 0.05 or p < 0.01, Figure 6(A–D).\nEffect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photograph and bar graph of TGF-β1. B indicates representative photograph and bar graph of p-Smad3. C indicates representative photograph and bar graph of collagen I. D indicates representative photograph and bar graph of α-SMA.",
"Compared with the control group, the LVSP, the LVEDP, the + dp/dt, and the -dp/dt of ginsenoside Rg2 group did not show significant changes (Figure 1, p > 0.05). Further histopathological examination demonstrated that myocardial fibrosis in the ginsenoside Rg2 group was the same as that of the control group (Figure 2, p > 0.05). These findings indicated that ginsenoside Rg2 alone had no effect on the cardiac function and the myocardial fibrosis in normal rats.\nEffect of ginsenoside Rg2 on cardiac function in normal rats. Data are presented as the mean ± SD (n = 7–8).\nEffect of ginsenoside Rg2 on myocardial fibrosis in normal rats. Data are presented as the mean ± SD (n = 3). A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.",
"The cardiac function of the rats was evaluated with a hemodynamic analysing system. The LVEDP of the rats in the isoproterenol group was significantly increased, as compared with the control group (p < 0.01). The LVSP, the + dp/dt, and the –dp/dt in the isoproterenol group were markedly decreased (p < 0.01). Compared with the isoproterenol group, LVEDP of the rats in the ginsenoside Rg2 groups was significantly decreased, whereas the LVSP, the + dp/dt, and the –dp/dt of the ginsenoside Rg2 groups were increased (p < 0.05 or p < 0.01). These results demonstrated that ginsenoside Rg2 has a property improving the cardiac function in myocardial ischaemic rats (Figure 3).\nEffect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group.",
"The gross morphological examination showed that hearts of the rats from the isoproterenol group were significantly larger than those from the control group (Figure 4(A)). In addition, heart weight and heart weight index were significantly increased in the isoproterenol group when compared to the control group (p < 0.01). Treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg abrogated partially myocardial ischaemia-induced increases of heart weight and heart weight index (p < 0.05 or p < 0.01, Figure 4(B–D)).\nEffect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of the gross morphological hearts. B indicates bar graph of body weight. C indicates bar graph of heart weight. D indicates bar graph of heart weight index.",
"The Sirius red staining showed that there was extensive collagen deposition in the border zone of the myocardial ischaemia in the myocardial ischaemic rats, whereas treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg markedly reduced contents of collagen deposition in myocardial ischaemic rats (Figure 5(A)). The red-stained area was calculated as a percentage of the total area with Image-Pro Plus software. In line with the observations mentioned above, the red-appearing collagens of the heart tissues in myocardial ischaemic animals were significantly augmented (p < 0.01). However, ginsenoside Rg2 treatment markedly decreased the contents of the collagen content in the heart tissues (p < 0.05 or p < 0.01, Figure 5(B)).\nEffect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.",
"TGF-β1 leads to the production of collagens in myocardial fibroblasts. Smad3 is the downstream effectors of the TGF-β1 receptor. The effects of ginsenoside Rg2 on the TGF-β1/Smad signalling pathway in the heart tissues were investigated in the present study. The results showed that expression of TGF-β1, p-Smad3, collagen I and α-SMA were significantly up-regulated in the heart tissues of myocardial ischaemic rats (p < 0.05 or p < 0.01). However, treatment with ginsenoside Rg2 significantly decreased the expression of TGF-β1, p-Smad3, collagen I and α-SMA in the heart tissues (p < 0.05 or p < 0.01, Figure 6(A–D).\nEffect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photograph and bar graph of TGF-β1. B indicates representative photograph and bar graph of p-Smad3. C indicates representative photograph and bar graph of collagen I. D indicates representative photograph and bar graph of α-SMA.",
"Cardiomyocytes death during myocardial ischaemia leads to a multiphase reparative response. The damaged heart tissues are replaced by a fibrotic scar produced by myofibroblasts. The progression of ischaemic necrosis induces interstitial and perivascular fibrosis in heart ventricular wall. The interstitial fibrosis and focal replacement scars can prevent cardiac rupture which is a fatal complication of myocardial ischaemia. However, the progressive myocardial fibrosis is detrimental as it produces abnormalities in ventricular function and leads to heart failure (Prabhu and Frangogiannis 2016). Therefore, attenuating myocardial fibrosis would offer a favourable prognosis in patients with ischaemic heart disease. The present study demonstrated for the first time ginsenoside Rg2 alone did not induce myocardial fibrosis and had no adverse effect on cardiac function. Ginsenoside Rg2 showed a property of alleviating the myocardial fibrosis and improving the impairment of cardiac function by regulating the TGF-β1/Smad signalling pathway after myocardial ischaemia.\nMyocardial ischaemia causes the changes in the size, shape, and function of the heart and leads to cardiac failure. It is well known that the heart has no capacity of regeneration, and the lost cardiomyocytes will be replaced by a fibrotic scar (Laflamme and Murry 2005). The fibrotic process after myocardial ischaemia is classified into replacement and reactive fibrosis, both of which are associated with myofibroblasts (Gao et al. 2019). The characteristic of myofibroblasts is the expression of α-SMA. Therefore, the content of α-SMA is a commonly used to evaluate the myofibroblasts in heart tissues (Shinde et al. 2017). Collagen I is the primary structural protein in the heart interstitium and is synthesised by myofibroblasts (van den Borne et al. 2010). Myofibroblasts secrete a great deal of interstitial collagens after myocardial ischaemia, which is crucial for increasing heart weight and impairing cardiac function. The current study showed that cardiac function was impaired after myocardial ischaemia and there were significant depositions of collagen I in the heart tissues. However, treatment with ginsenoside Rg2 for 28 days improved cardiac function and decreased collagen I depositions, suggesting that ginsenoside Rg2 can ameliorate ischaemia-induced myocardial fibrosis.\nNumerous factors play a role in modulating the fibrosis induced by myocardial ischaemia. TGF-β1 is the cardinal pro-fibrotic growth factor involved in fibroblast activation and collagens production (Dobaczewski et al. 2011). The previous report demonstrates that TGF-β1 leads to the transdifferentiation of myofibroblast and therefore increases the synthesis of extracellular matrix protein (Dobaczewski et al. 2012). In hearts, TGF-β1 will be rapidly produced and released in response to ischaemia. TGF-β1 exerts its effects through binding to its receptor and induces phosphorylation of Smads. Smad3 is one of the major downstream regulators that are associated with TGF-β1-mediated myocardial fibrosis. The phosphorylation of Smad3 forms heteromeric complexes with Smad4 and then they translocate into the nucleus. In the nucleus, the complex regulates the expression of target genes related to fibrosis (e.g., collagen I) after binding to the Smad-binding element (Shi and Massagué 2003). In this study, we found that the expression of collagen I in the myocardial ischaemic rat hearts was increased. Also, expressions of TGF-β1 and p-Smad3 were augmented, suggesting that the TGF-β1/Smad3 signalling pathway was involved in the process of myocardial fibrosis. Consistent with our hypothesis, ginsenoside Rg2 reduced expressions of TGF-β1, p-Smad3, and collagen I. These findings suggested that ginsenoside Rg2 alleviates myocardial fibrosis and therefore improves the impairment of cardiac function by regulating TGF-β1/Smad signalling pathway after myocardial ischaemia.",
"The present experiments demonstrated that ginsenoside Rg2 possesses the property of alleviating myocardial fibrosis and improving cardiac function by regulating the TGF-β1/Smad signalling pathway after myocardial ischaemia. This study provides insights into the mechanisms of ginseng in the clinical application for myocardial ischaemia.",
"Changchun is at 125.35 degrees East longitude and 43.88 degrees North latitude."
] |
[
"intro",
"materials",
null,
null,
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
"discussion",
"conclusions",
null
] |
[
"Myocardial ischaemia",
"cardiac function",
"isoproterenol"
] |
Introduction:
Cardiovascular diseases (CVDs) are the primary cause of death worldwide (Hausenloy and Yellon 2013). Myocardial ischaemia, the most common CVDs, is caused by the reduction in coronary blood flow due to atherosclerotic coronary arterial obstruction. Researchers find that surgical approaches restoring blood flow of coronary arteries fail to improve cardiac function of patients with myocardial ischaemia (Ngu et al. 2018). On average, myocardial fibrosis will occur in 10% of patients. Myocardial fibrosis is the common cause of death after myocardial ischaemia, which accounts for a higher 5-year mortality (about 50%) (Benjamin et al. 2018). Myocardial fibrosis is characterised by the collagen formation, which leads to a chamber stiffness and an increased myocardial mass (Li et al. 2018a). Tremendous efforts have been made to develop therapeutics to attenuate myocardial fibrosis and improve cardiac function in patients of myocardial ischaemia. Traditional Chinese medicine (TCM) and small-molecule drugs are promising agents for preventing myocardial ischaemia-induced heart function impairment (Hashimoto et al. 2018).
In China, TCM has been used in treating CVDs for thousands of years. A great deal of interest has focussed on elucidating the mechanisms underlying the cardioprotective effects of the active ingredients of TCM (Fan et al. 2017). Panax ginseng C.A. Meyer (Araliaceae), a well-known TCM, is widely used for increasing vital energy and improving organ functions. Ginsenosides are the major active ingredients of ginseng. According to the structures of the extracted components of ginseng, ginsenosides are classified into three groups: the panaxadiol group including Rb1, Rb2, Rg3, and Rh2, the panaxatriol group including Re, Rh1, and Rg1, and oleanolic acid (Zhang et al. 2019). Recent work demonstrates that ginsenoside Rb1 can reduce the injury of myocardial ischaemia/reperfusion and then improve the heart function by inhibiting the apoptosis of cardiomyocytes (Li et al. 2020). Ginsenoside Rg1 exerts a protective effect against myocardial ischaemia-induced injury, which is associated with modulating energy metabolism and inhibiting myocardial apoptosis (Li et al. 2018b). Our previous studies indicate that ginsenoside Re treatment abrogates the myocardial fibrosis caused by myocardial ischaemia and then restores the heart function (Wang et al. 2019). Ginsenoside Rg2 is one of the cardinal components of ginseng. Ginsenoside Rg2 has a property of attenuating the neuronal damage induced by hypoxia in hippocampal neurons (Shuangyan et al. 2012). Ginsenoside Rg2 can also improve cerebral ischaemia-induced dementia through abating the apoptosis of neurons (Zhang et al. 2008). However, it is still not clear whether ginsenoside Rg2 has a protective effect against myocardial ischaemic injury. Therefore, this study investigates whether ginsenoside Rg2 could alleviate myocardial ischaemia-induced myocardial fibrosis and cardiac function impairment in rats.
Materials and methods:
Ginsenoside Rg2 Ginsenoside Rg2 extracted from ginseng cultivated in China was kindly provided by Professor Yifa Zhou (School of Life Sciences, Northeast Normal University, Changchun, China). The purity of ginsenoside Rg2 was >98.0%.
Ginsenoside Rg2 extracted from ginseng cultivated in China was kindly provided by Professor Yifa Zhou (School of Life Sciences, Northeast Normal University, Changchun, China). The purity of ginsenoside Rg2 was >98.0%.
Animals Male Wistar rats weighing 240–260 g were obtained from the Experiment Animal Centre of Jilin University (Changchun, China). Animals were housed on a 12 h light/dark cycle at a controlled temperature and humidity with unlimited access to food and water. The experiments were approved by the Ethics Committee of Jilin University (Approval number, JLU2018-03-0023) and were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (publication 86-23, revised in 1986).
Male Wistar rats weighing 240–260 g were obtained from the Experiment Animal Centre of Jilin University (Changchun, China). Animals were housed on a 12 h light/dark cycle at a controlled temperature and humidity with unlimited access to food and water. The experiments were approved by the Ethics Committee of Jilin University (Approval number, JLU2018-03-0023) and were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (publication 86-23, revised in 1986).
Experimental protocol Experiment 1: Rats were randomly divided into two groups (n = 8): a control group (1 rat was excluded because of anaesthetic overdose) and a ginsenoside Rg2 at dose of 20 mg/kg group. The rats of the control group were intragastrically administered with 0.5% CMC-Na daily for 28 days. The animals of the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 daily for 28 days. Experiment 2: Rats were randomly divided into four groups (n =8): a control group, an isoproterenol group and ginsenoside Rg2 (5, 20 mg/kg) groups. To induce myocardial fibrosis, the rats were injected subcutaneously with isoproterenol (Sigma-Aldrich, USA) at a dose of 5 mg/kg, once daily for 7 consecutive days (Zhang et al. 2014). Two hours later, the animals in the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 at doses of 5 or 20 mg/kg, daily for 28 days. The rats of the control and the isoproterenol groups were intragastrically administered with 0.5% CMC-Na daily for 28 days.
Experiment 1: Rats were randomly divided into two groups (n = 8): a control group (1 rat was excluded because of anaesthetic overdose) and a ginsenoside Rg2 at dose of 20 mg/kg group. The rats of the control group were intragastrically administered with 0.5% CMC-Na daily for 28 days. The animals of the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 daily for 28 days. Experiment 2: Rats were randomly divided into four groups (n =8): a control group, an isoproterenol group and ginsenoside Rg2 (5, 20 mg/kg) groups. To induce myocardial fibrosis, the rats were injected subcutaneously with isoproterenol (Sigma-Aldrich, USA) at a dose of 5 mg/kg, once daily for 7 consecutive days (Zhang et al. 2014). Two hours later, the animals in the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 at doses of 5 or 20 mg/kg, daily for 28 days. The rats of the control and the isoproterenol groups were intragastrically administered with 0.5% CMC-Na daily for 28 days.
Cardiac function evaluation At day 28, the body weights of the rats were recorded. The rats were anaesthetised by an intraperitoneal injection with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). A 2 F polyethylene catheter was inserted into the left ventricle through the right common carotid artery. The catheter was connected with a hemodynamic analysing system (Model RM-6000, Nihon Kohden, Japan). Then, the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and positive (+dp/dt) and negative (-dp/dt) maximal values of the first derivative of the left ventricular pressure were evaluated.
At day 28, the body weights of the rats were recorded. The rats were anaesthetised by an intraperitoneal injection with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). A 2 F polyethylene catheter was inserted into the left ventricle through the right common carotid artery. The catheter was connected with a hemodynamic analysing system (Model RM-6000, Nihon Kohden, Japan). Then, the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and positive (+dp/dt) and negative (-dp/dt) maximal values of the first derivative of the left ventricular pressure were evaluated.
Heart weight index assessment The rats were sacrificed with overdoses of carbon dioxide after cardiac function evaluation. The hearts of the animals were collected and washed with PBS solution (4 °C). The aorta, atria, and adipose tissue were excised. Heart weights were weighed and heart weight index (mg/g) was calculated by dividing heart weight by body weight.
The rats were sacrificed with overdoses of carbon dioxide after cardiac function evaluation. The hearts of the animals were collected and washed with PBS solution (4 °C). The aorta, atria, and adipose tissue were excised. Heart weights were weighed and heart weight index (mg/g) was calculated by dividing heart weight by body weight.
Histopathological examination The hearts of the rats were fixed with 10% formalin for 24 h. The specimens were embedded with paraffin and then were cut into 5 μm thick sections. According to the instruction of the manufacturer, the heart sections were stained with a Sirius red staining kit (Solarbio Life Sciences Company, China). Then, the histopathological examination was conducted by an experienced observer who was blinded to the experimental design under an Olympus microscope (IX-70, Olympus Corp., Japan). Ten fields were randomly selected from five sections in each heart. The ratio of the interstitial fibrosis area in the hearts was analysed and calculated with NIH-Image software (National Institutes of Health, Bethesda, MD, U.S.A.). The myocardial fibrosis was presented by the calculation: (fibrotic tissue area)/(fibrotic tissue area + cardiomyocyte area) × 100%.
The hearts of the rats were fixed with 10% formalin for 24 h. The specimens were embedded with paraffin and then were cut into 5 μm thick sections. According to the instruction of the manufacturer, the heart sections were stained with a Sirius red staining kit (Solarbio Life Sciences Company, China). Then, the histopathological examination was conducted by an experienced observer who was blinded to the experimental design under an Olympus microscope (IX-70, Olympus Corp., Japan). Ten fields were randomly selected from five sections in each heart. The ratio of the interstitial fibrosis area in the hearts was analysed and calculated with NIH-Image software (National Institutes of Health, Bethesda, MD, U.S.A.). The myocardial fibrosis was presented by the calculation: (fibrotic tissue area)/(fibrotic tissue area + cardiomyocyte area) × 100%.
Western blot Western blot was performed as previously described (Ahn et al. 2019). The membranes were incubated with the primary antibodies: rabbit anti-TGF-β1 (1:1000, Proteintech, USA), rabbit anti-α-smooth muscle actin (α-SMA) (1:2000, ImmunoWay Biotechnology, USA), rabbit anti-collagen I (1:2000, Abcam, UK), rabbit anti-Smad3 (1:1500, Abcam, UK), rabbit anti-p-Smad3 (1:1500, Abcam, UK) or rabbit anti-β-actin (1:1500, Beyotime Institute of Biotechnology, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labelled secondary antibody (1:2000, Beyotime Institute of Biotechnology, China). The bands were visualised using the ECL detection reagents (Beyotime Institute of Biotechnology, China). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.
Western blot was performed as previously described (Ahn et al. 2019). The membranes were incubated with the primary antibodies: rabbit anti-TGF-β1 (1:1000, Proteintech, USA), rabbit anti-α-smooth muscle actin (α-SMA) (1:2000, ImmunoWay Biotechnology, USA), rabbit anti-collagen I (1:2000, Abcam, UK), rabbit anti-Smad3 (1:1500, Abcam, UK), rabbit anti-p-Smad3 (1:1500, Abcam, UK) or rabbit anti-β-actin (1:1500, Beyotime Institute of Biotechnology, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labelled secondary antibody (1:2000, Beyotime Institute of Biotechnology, China). The bands were visualised using the ECL detection reagents (Beyotime Institute of Biotechnology, China). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.
Statistical analysis Data were presented as the mean ± SD and processed with SPSS 22.0. Statistical analysis among various groups was conducted by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. The p < 0.05 was considered as statistically significant.
Data were presented as the mean ± SD and processed with SPSS 22.0. Statistical analysis among various groups was conducted by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. The p < 0.05 was considered as statistically significant.
Ginsenoside Rg2:
Ginsenoside Rg2 extracted from ginseng cultivated in China was kindly provided by Professor Yifa Zhou (School of Life Sciences, Northeast Normal University, Changchun, China). The purity of ginsenoside Rg2 was >98.0%.
Animals:
Male Wistar rats weighing 240–260 g were obtained from the Experiment Animal Centre of Jilin University (Changchun, China). Animals were housed on a 12 h light/dark cycle at a controlled temperature and humidity with unlimited access to food and water. The experiments were approved by the Ethics Committee of Jilin University (Approval number, JLU2018-03-0023) and were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (publication 86-23, revised in 1986).
Experimental protocol:
Experiment 1: Rats were randomly divided into two groups (n = 8): a control group (1 rat was excluded because of anaesthetic overdose) and a ginsenoside Rg2 at dose of 20 mg/kg group. The rats of the control group were intragastrically administered with 0.5% CMC-Na daily for 28 days. The animals of the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 daily for 28 days. Experiment 2: Rats were randomly divided into four groups (n =8): a control group, an isoproterenol group and ginsenoside Rg2 (5, 20 mg/kg) groups. To induce myocardial fibrosis, the rats were injected subcutaneously with isoproterenol (Sigma-Aldrich, USA) at a dose of 5 mg/kg, once daily for 7 consecutive days (Zhang et al. 2014). Two hours later, the animals in the ginsenoside Rg2 groups were intragastrically treated with ginsenoside Rg2 at doses of 5 or 20 mg/kg, daily for 28 days. The rats of the control and the isoproterenol groups were intragastrically administered with 0.5% CMC-Na daily for 28 days.
Cardiac function evaluation:
At day 28, the body weights of the rats were recorded. The rats were anaesthetised by an intraperitoneal injection with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). A 2 F polyethylene catheter was inserted into the left ventricle through the right common carotid artery. The catheter was connected with a hemodynamic analysing system (Model RM-6000, Nihon Kohden, Japan). Then, the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and positive (+dp/dt) and negative (-dp/dt) maximal values of the first derivative of the left ventricular pressure were evaluated.
Heart weight index assessment:
The rats were sacrificed with overdoses of carbon dioxide after cardiac function evaluation. The hearts of the animals were collected and washed with PBS solution (4 °C). The aorta, atria, and adipose tissue were excised. Heart weights were weighed and heart weight index (mg/g) was calculated by dividing heart weight by body weight.
Histopathological examination:
The hearts of the rats were fixed with 10% formalin for 24 h. The specimens were embedded with paraffin and then were cut into 5 μm thick sections. According to the instruction of the manufacturer, the heart sections were stained with a Sirius red staining kit (Solarbio Life Sciences Company, China). Then, the histopathological examination was conducted by an experienced observer who was blinded to the experimental design under an Olympus microscope (IX-70, Olympus Corp., Japan). Ten fields were randomly selected from five sections in each heart. The ratio of the interstitial fibrosis area in the hearts was analysed and calculated with NIH-Image software (National Institutes of Health, Bethesda, MD, U.S.A.). The myocardial fibrosis was presented by the calculation: (fibrotic tissue area)/(fibrotic tissue area + cardiomyocyte area) × 100%.
Western blot:
Western blot was performed as previously described (Ahn et al. 2019). The membranes were incubated with the primary antibodies: rabbit anti-TGF-β1 (1:1000, Proteintech, USA), rabbit anti-α-smooth muscle actin (α-SMA) (1:2000, ImmunoWay Biotechnology, USA), rabbit anti-collagen I (1:2000, Abcam, UK), rabbit anti-Smad3 (1:1500, Abcam, UK), rabbit anti-p-Smad3 (1:1500, Abcam, UK) or rabbit anti-β-actin (1:1500, Beyotime Institute of Biotechnology, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labelled secondary antibody (1:2000, Beyotime Institute of Biotechnology, China). The bands were visualised using the ECL detection reagents (Beyotime Institute of Biotechnology, China). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.
Statistical analysis:
Data were presented as the mean ± SD and processed with SPSS 22.0. Statistical analysis among various groups was conducted by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. The p < 0.05 was considered as statistically significant.
Results:
Effect of ginsenoside Rg2 on cardiac function and myocardial fibrosis in normal rats Compared with the control group, the LVSP, the LVEDP, the + dp/dt, and the -dp/dt of ginsenoside Rg2 group did not show significant changes (Figure 1, p > 0.05). Further histopathological examination demonstrated that myocardial fibrosis in the ginsenoside Rg2 group was the same as that of the control group (Figure 2, p > 0.05). These findings indicated that ginsenoside Rg2 alone had no effect on the cardiac function and the myocardial fibrosis in normal rats.
Effect of ginsenoside Rg2 on cardiac function in normal rats. Data are presented as the mean ± SD (n = 7–8).
Effect of ginsenoside Rg2 on myocardial fibrosis in normal rats. Data are presented as the mean ± SD (n = 3). A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.
Compared with the control group, the LVSP, the LVEDP, the + dp/dt, and the -dp/dt of ginsenoside Rg2 group did not show significant changes (Figure 1, p > 0.05). Further histopathological examination demonstrated that myocardial fibrosis in the ginsenoside Rg2 group was the same as that of the control group (Figure 2, p > 0.05). These findings indicated that ginsenoside Rg2 alone had no effect on the cardiac function and the myocardial fibrosis in normal rats.
Effect of ginsenoside Rg2 on cardiac function in normal rats. Data are presented as the mean ± SD (n = 7–8).
Effect of ginsenoside Rg2 on myocardial fibrosis in normal rats. Data are presented as the mean ± SD (n = 3). A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.
Effect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats The cardiac function of the rats was evaluated with a hemodynamic analysing system. The LVEDP of the rats in the isoproterenol group was significantly increased, as compared with the control group (p < 0.01). The LVSP, the + dp/dt, and the –dp/dt in the isoproterenol group were markedly decreased (p < 0.01). Compared with the isoproterenol group, LVEDP of the rats in the ginsenoside Rg2 groups was significantly decreased, whereas the LVSP, the + dp/dt, and the –dp/dt of the ginsenoside Rg2 groups were increased (p < 0.05 or p < 0.01). These results demonstrated that ginsenoside Rg2 has a property improving the cardiac function in myocardial ischaemic rats (Figure 3).
Effect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group.
The cardiac function of the rats was evaluated with a hemodynamic analysing system. The LVEDP of the rats in the isoproterenol group was significantly increased, as compared with the control group (p < 0.01). The LVSP, the + dp/dt, and the –dp/dt in the isoproterenol group were markedly decreased (p < 0.01). Compared with the isoproterenol group, LVEDP of the rats in the ginsenoside Rg2 groups was significantly decreased, whereas the LVSP, the + dp/dt, and the –dp/dt of the ginsenoside Rg2 groups were increased (p < 0.05 or p < 0.01). These results demonstrated that ginsenoside Rg2 has a property improving the cardiac function in myocardial ischaemic rats (Figure 3).
Effect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group.
Effect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats The gross morphological examination showed that hearts of the rats from the isoproterenol group were significantly larger than those from the control group (Figure 4(A)). In addition, heart weight and heart weight index were significantly increased in the isoproterenol group when compared to the control group (p < 0.01). Treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg abrogated partially myocardial ischaemia-induced increases of heart weight and heart weight index (p < 0.05 or p < 0.01, Figure 4(B–D)).
Effect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of the gross morphological hearts. B indicates bar graph of body weight. C indicates bar graph of heart weight. D indicates bar graph of heart weight index.
The gross morphological examination showed that hearts of the rats from the isoproterenol group were significantly larger than those from the control group (Figure 4(A)). In addition, heart weight and heart weight index were significantly increased in the isoproterenol group when compared to the control group (p < 0.01). Treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg abrogated partially myocardial ischaemia-induced increases of heart weight and heart weight index (p < 0.05 or p < 0.01, Figure 4(B–D)).
Effect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of the gross morphological hearts. B indicates bar graph of body weight. C indicates bar graph of heart weight. D indicates bar graph of heart weight index.
Effect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats The Sirius red staining showed that there was extensive collagen deposition in the border zone of the myocardial ischaemia in the myocardial ischaemic rats, whereas treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg markedly reduced contents of collagen deposition in myocardial ischaemic rats (Figure 5(A)). The red-stained area was calculated as a percentage of the total area with Image-Pro Plus software. In line with the observations mentioned above, the red-appearing collagens of the heart tissues in myocardial ischaemic animals were significantly augmented (p < 0.01). However, ginsenoside Rg2 treatment markedly decreased the contents of the collagen content in the heart tissues (p < 0.05 or p < 0.01, Figure 5(B)).
Effect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.
The Sirius red staining showed that there was extensive collagen deposition in the border zone of the myocardial ischaemia in the myocardial ischaemic rats, whereas treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg markedly reduced contents of collagen deposition in myocardial ischaemic rats (Figure 5(A)). The red-stained area was calculated as a percentage of the total area with Image-Pro Plus software. In line with the observations mentioned above, the red-appearing collagens of the heart tissues in myocardial ischaemic animals were significantly augmented (p < 0.01). However, ginsenoside Rg2 treatment markedly decreased the contents of the collagen content in the heart tissues (p < 0.05 or p < 0.01, Figure 5(B)).
Effect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.
Effect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats TGF-β1 leads to the production of collagens in myocardial fibroblasts. Smad3 is the downstream effectors of the TGF-β1 receptor. The effects of ginsenoside Rg2 on the TGF-β1/Smad signalling pathway in the heart tissues were investigated in the present study. The results showed that expression of TGF-β1, p-Smad3, collagen I and α-SMA were significantly up-regulated in the heart tissues of myocardial ischaemic rats (p < 0.05 or p < 0.01). However, treatment with ginsenoside Rg2 significantly decreased the expression of TGF-β1, p-Smad3, collagen I and α-SMA in the heart tissues (p < 0.05 or p < 0.01, Figure 6(A–D).
Effect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photograph and bar graph of TGF-β1. B indicates representative photograph and bar graph of p-Smad3. C indicates representative photograph and bar graph of collagen I. D indicates representative photograph and bar graph of α-SMA.
TGF-β1 leads to the production of collagens in myocardial fibroblasts. Smad3 is the downstream effectors of the TGF-β1 receptor. The effects of ginsenoside Rg2 on the TGF-β1/Smad signalling pathway in the heart tissues were investigated in the present study. The results showed that expression of TGF-β1, p-Smad3, collagen I and α-SMA were significantly up-regulated in the heart tissues of myocardial ischaemic rats (p < 0.05 or p < 0.01). However, treatment with ginsenoside Rg2 significantly decreased the expression of TGF-β1, p-Smad3, collagen I and α-SMA in the heart tissues (p < 0.05 or p < 0.01, Figure 6(A–D).
Effect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photograph and bar graph of TGF-β1. B indicates representative photograph and bar graph of p-Smad3. C indicates representative photograph and bar graph of collagen I. D indicates representative photograph and bar graph of α-SMA.
Effect of ginsenoside Rg2 on cardiac function and myocardial fibrosis in normal rats:
Compared with the control group, the LVSP, the LVEDP, the + dp/dt, and the -dp/dt of ginsenoside Rg2 group did not show significant changes (Figure 1, p > 0.05). Further histopathological examination demonstrated that myocardial fibrosis in the ginsenoside Rg2 group was the same as that of the control group (Figure 2, p > 0.05). These findings indicated that ginsenoside Rg2 alone had no effect on the cardiac function and the myocardial fibrosis in normal rats.
Effect of ginsenoside Rg2 on cardiac function in normal rats. Data are presented as the mean ± SD (n = 7–8).
Effect of ginsenoside Rg2 on myocardial fibrosis in normal rats. Data are presented as the mean ± SD (n = 3). A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.
Effect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats:
The cardiac function of the rats was evaluated with a hemodynamic analysing system. The LVEDP of the rats in the isoproterenol group was significantly increased, as compared with the control group (p < 0.01). The LVSP, the + dp/dt, and the –dp/dt in the isoproterenol group were markedly decreased (p < 0.01). Compared with the isoproterenol group, LVEDP of the rats in the ginsenoside Rg2 groups was significantly decreased, whereas the LVSP, the + dp/dt, and the –dp/dt of the ginsenoside Rg2 groups were increased (p < 0.05 or p < 0.01). These results demonstrated that ginsenoside Rg2 has a property improving the cardiac function in myocardial ischaemic rats (Figure 3).
Effect of ginsenoside Rg2 on cardiac function in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group.
Effect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats:
The gross morphological examination showed that hearts of the rats from the isoproterenol group were significantly larger than those from the control group (Figure 4(A)). In addition, heart weight and heart weight index were significantly increased in the isoproterenol group when compared to the control group (p < 0.01). Treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg abrogated partially myocardial ischaemia-induced increases of heart weight and heart weight index (p < 0.05 or p < 0.01, Figure 4(B–D)).
Effect of ginsenoside Rg2 on cardiac hypertrophy in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 8). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of the gross morphological hearts. B indicates bar graph of body weight. C indicates bar graph of heart weight. D indicates bar graph of heart weight index.
Effect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats:
The Sirius red staining showed that there was extensive collagen deposition in the border zone of the myocardial ischaemia in the myocardial ischaemic rats, whereas treatment with ginsenoside Rg2 at doses of 5, 20 mg/kg markedly reduced contents of collagen deposition in myocardial ischaemic rats (Figure 5(A)). The red-stained area was calculated as a percentage of the total area with Image-Pro Plus software. In line with the observations mentioned above, the red-appearing collagens of the heart tissues in myocardial ischaemic animals were significantly augmented (p < 0.01). However, ginsenoside Rg2 treatment markedly decreased the contents of the collagen content in the heart tissues (p < 0.05 or p < 0.01, Figure 5(B)).
Effect of ginsenoside Rg2 on myocardial fibrosis in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photomicrographs of Sirius red staining of the hearts. B indicates bar graph of myocardial fibrosis.
Effect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats:
TGF-β1 leads to the production of collagens in myocardial fibroblasts. Smad3 is the downstream effectors of the TGF-β1 receptor. The effects of ginsenoside Rg2 on the TGF-β1/Smad signalling pathway in the heart tissues were investigated in the present study. The results showed that expression of TGF-β1, p-Smad3, collagen I and α-SMA were significantly up-regulated in the heart tissues of myocardial ischaemic rats (p < 0.05 or p < 0.01). However, treatment with ginsenoside Rg2 significantly decreased the expression of TGF-β1, p-Smad3, collagen I and α-SMA in the heart tissues (p < 0.05 or p < 0.01, Figure 6(A–D).
Effect of ginsenoside Rg2 on TGF-β1/Smad signalling pathway in myocardial ischaemic rats. Data are presented as the mean ± SD (n = 3). ##p < 0.01 compared with control group; *p < 0.05, **p < 0.01 compared with isoproterenol group. A indicates representative photograph and bar graph of TGF-β1. B indicates representative photograph and bar graph of p-Smad3. C indicates representative photograph and bar graph of collagen I. D indicates representative photograph and bar graph of α-SMA.
Discussion:
Cardiomyocytes death during myocardial ischaemia leads to a multiphase reparative response. The damaged heart tissues are replaced by a fibrotic scar produced by myofibroblasts. The progression of ischaemic necrosis induces interstitial and perivascular fibrosis in heart ventricular wall. The interstitial fibrosis and focal replacement scars can prevent cardiac rupture which is a fatal complication of myocardial ischaemia. However, the progressive myocardial fibrosis is detrimental as it produces abnormalities in ventricular function and leads to heart failure (Prabhu and Frangogiannis 2016). Therefore, attenuating myocardial fibrosis would offer a favourable prognosis in patients with ischaemic heart disease. The present study demonstrated for the first time ginsenoside Rg2 alone did not induce myocardial fibrosis and had no adverse effect on cardiac function. Ginsenoside Rg2 showed a property of alleviating the myocardial fibrosis and improving the impairment of cardiac function by regulating the TGF-β1/Smad signalling pathway after myocardial ischaemia.
Myocardial ischaemia causes the changes in the size, shape, and function of the heart and leads to cardiac failure. It is well known that the heart has no capacity of regeneration, and the lost cardiomyocytes will be replaced by a fibrotic scar (Laflamme and Murry 2005). The fibrotic process after myocardial ischaemia is classified into replacement and reactive fibrosis, both of which are associated with myofibroblasts (Gao et al. 2019). The characteristic of myofibroblasts is the expression of α-SMA. Therefore, the content of α-SMA is a commonly used to evaluate the myofibroblasts in heart tissues (Shinde et al. 2017). Collagen I is the primary structural protein in the heart interstitium and is synthesised by myofibroblasts (van den Borne et al. 2010). Myofibroblasts secrete a great deal of interstitial collagens after myocardial ischaemia, which is crucial for increasing heart weight and impairing cardiac function. The current study showed that cardiac function was impaired after myocardial ischaemia and there were significant depositions of collagen I in the heart tissues. However, treatment with ginsenoside Rg2 for 28 days improved cardiac function and decreased collagen I depositions, suggesting that ginsenoside Rg2 can ameliorate ischaemia-induced myocardial fibrosis.
Numerous factors play a role in modulating the fibrosis induced by myocardial ischaemia. TGF-β1 is the cardinal pro-fibrotic growth factor involved in fibroblast activation and collagens production (Dobaczewski et al. 2011). The previous report demonstrates that TGF-β1 leads to the transdifferentiation of myofibroblast and therefore increases the synthesis of extracellular matrix protein (Dobaczewski et al. 2012). In hearts, TGF-β1 will be rapidly produced and released in response to ischaemia. TGF-β1 exerts its effects through binding to its receptor and induces phosphorylation of Smads. Smad3 is one of the major downstream regulators that are associated with TGF-β1-mediated myocardial fibrosis. The phosphorylation of Smad3 forms heteromeric complexes with Smad4 and then they translocate into the nucleus. In the nucleus, the complex regulates the expression of target genes related to fibrosis (e.g., collagen I) after binding to the Smad-binding element (Shi and Massagué 2003). In this study, we found that the expression of collagen I in the myocardial ischaemic rat hearts was increased. Also, expressions of TGF-β1 and p-Smad3 were augmented, suggesting that the TGF-β1/Smad3 signalling pathway was involved in the process of myocardial fibrosis. Consistent with our hypothesis, ginsenoside Rg2 reduced expressions of TGF-β1, p-Smad3, and collagen I. These findings suggested that ginsenoside Rg2 alleviates myocardial fibrosis and therefore improves the impairment of cardiac function by regulating TGF-β1/Smad signalling pathway after myocardial ischaemia.
Conclusions:
The present experiments demonstrated that ginsenoside Rg2 possesses the property of alleviating myocardial fibrosis and improving cardiac function by regulating the TGF-β1/Smad signalling pathway after myocardial ischaemia. This study provides insights into the mechanisms of ginseng in the clinical application for myocardial ischaemia.
Geolocation information:
Changchun is at 125.35 degrees East longitude and 43.88 degrees North latitude.
|
Background:
Panax ginseng C.A. Meyer (Araliaceae) has cardioprotective effects. Ginsenosides are responsible for most of the pharmacological activities of ginseng.
Methods:
Male Wistar rats were divided into control, isoproterenol, ginsenoside Rg2 (5, 20 mg/kg) groups (n = 8). The rats were subcutaneously injected with isoproterenol (5 mg/kg) or normal saline (control group) once daily for 7 days. The animals were intragastrically treated with ginsenoside Rg2 or 0.5% CMC-Na (control and isoproterenol groups) daily for 28 days. At day 28, cardiac function, myocardial fibrosis, and TGF-β1/Smad signalling pathway were evaluated.
Results:
Compared with myocardial ischaemic rats, ginsenoside Rg2 at doses of 5, 20 mg/kg abated partially the augment of LVEDP (8.9 ± 1.3 vs. 7.5 ± 0.7, 7.2 ± 1.0 mmHg) and the decreases of the LVSP (96.75 ± 13.2 vs. 118.3 ± 19.4, 124.3 ± 21.3 mmHg), the + dp/dt (2142.8 ± 309.3 vs. 2598.6 ± 404.0, 2661.5 ± 445.2 mmHg/s), and the -dp/dt (1996.3 ± 306.3 vs. 2476.6 ± 289.7, 2509.6 ± 353.1 mmHg/s). Ginsenoside Rg2 (9.2 ± 0.9%, 8.5 ± 0.8%) alleviated myocardial fibrosis when compared with the isoproterenol group (10.1 ± 1.0%), which was accompanied by suppressed TGF-β1/Smad signalling in heart tissues.
Conclusions:
Ginsenosides from ginseng possess the property of alleviating myocardial fibrosis, improving cardiac function after myocardial ischaemia. Ginsenosides may be promising agents for improving the outcomes of patients with myocardial ischaemia.
|
Introduction:
Cardiovascular diseases (CVDs) are the primary cause of death worldwide (Hausenloy and Yellon 2013). Myocardial ischaemia, the most common CVDs, is caused by the reduction in coronary blood flow due to atherosclerotic coronary arterial obstruction. Researchers find that surgical approaches restoring blood flow of coronary arteries fail to improve cardiac function of patients with myocardial ischaemia (Ngu et al. 2018). On average, myocardial fibrosis will occur in 10% of patients. Myocardial fibrosis is the common cause of death after myocardial ischaemia, which accounts for a higher 5-year mortality (about 50%) (Benjamin et al. 2018). Myocardial fibrosis is characterised by the collagen formation, which leads to a chamber stiffness and an increased myocardial mass (Li et al. 2018a). Tremendous efforts have been made to develop therapeutics to attenuate myocardial fibrosis and improve cardiac function in patients of myocardial ischaemia. Traditional Chinese medicine (TCM) and small-molecule drugs are promising agents for preventing myocardial ischaemia-induced heart function impairment (Hashimoto et al. 2018).
In China, TCM has been used in treating CVDs for thousands of years. A great deal of interest has focussed on elucidating the mechanisms underlying the cardioprotective effects of the active ingredients of TCM (Fan et al. 2017). Panax ginseng C.A. Meyer (Araliaceae), a well-known TCM, is widely used for increasing vital energy and improving organ functions. Ginsenosides are the major active ingredients of ginseng. According to the structures of the extracted components of ginseng, ginsenosides are classified into three groups: the panaxadiol group including Rb1, Rb2, Rg3, and Rh2, the panaxatriol group including Re, Rh1, and Rg1, and oleanolic acid (Zhang et al. 2019). Recent work demonstrates that ginsenoside Rb1 can reduce the injury of myocardial ischaemia/reperfusion and then improve the heart function by inhibiting the apoptosis of cardiomyocytes (Li et al. 2020). Ginsenoside Rg1 exerts a protective effect against myocardial ischaemia-induced injury, which is associated with modulating energy metabolism and inhibiting myocardial apoptosis (Li et al. 2018b). Our previous studies indicate that ginsenoside Re treatment abrogates the myocardial fibrosis caused by myocardial ischaemia and then restores the heart function (Wang et al. 2019). Ginsenoside Rg2 is one of the cardinal components of ginseng. Ginsenoside Rg2 has a property of attenuating the neuronal damage induced by hypoxia in hippocampal neurons (Shuangyan et al. 2012). Ginsenoside Rg2 can also improve cerebral ischaemia-induced dementia through abating the apoptosis of neurons (Zhang et al. 2008). However, it is still not clear whether ginsenoside Rg2 has a protective effect against myocardial ischaemic injury. Therefore, this study investigates whether ginsenoside Rg2 could alleviate myocardial ischaemia-induced myocardial fibrosis and cardiac function impairment in rats.
Conclusions:
The present experiments demonstrated that ginsenoside Rg2 possesses the property of alleviating myocardial fibrosis and improving cardiac function by regulating the TGF-β1/Smad signalling pathway after myocardial ischaemia. This study provides insights into the mechanisms of ginseng in the clinical application for myocardial ischaemia.
|
Background:
Panax ginseng C.A. Meyer (Araliaceae) has cardioprotective effects. Ginsenosides are responsible for most of the pharmacological activities of ginseng.
Methods:
Male Wistar rats were divided into control, isoproterenol, ginsenoside Rg2 (5, 20 mg/kg) groups (n = 8). The rats were subcutaneously injected with isoproterenol (5 mg/kg) or normal saline (control group) once daily for 7 days. The animals were intragastrically treated with ginsenoside Rg2 or 0.5% CMC-Na (control and isoproterenol groups) daily for 28 days. At day 28, cardiac function, myocardial fibrosis, and TGF-β1/Smad signalling pathway were evaluated.
Results:
Compared with myocardial ischaemic rats, ginsenoside Rg2 at doses of 5, 20 mg/kg abated partially the augment of LVEDP (8.9 ± 1.3 vs. 7.5 ± 0.7, 7.2 ± 1.0 mmHg) and the decreases of the LVSP (96.75 ± 13.2 vs. 118.3 ± 19.4, 124.3 ± 21.3 mmHg), the + dp/dt (2142.8 ± 309.3 vs. 2598.6 ± 404.0, 2661.5 ± 445.2 mmHg/s), and the -dp/dt (1996.3 ± 306.3 vs. 2476.6 ± 289.7, 2509.6 ± 353.1 mmHg/s). Ginsenoside Rg2 (9.2 ± 0.9%, 8.5 ± 0.8%) alleviated myocardial fibrosis when compared with the isoproterenol group (10.1 ± 1.0%), which was accompanied by suppressed TGF-β1/Smad signalling in heart tissues.
Conclusions:
Ginsenosides from ginseng possess the property of alleviating myocardial fibrosis, improving cardiac function after myocardial ischaemia. Ginsenosides may be promising agents for improving the outcomes of patients with myocardial ischaemia.
| 7,660 | 358 | 19 |
[
"myocardial",
"ginsenoside",
"ginsenoside rg2",
"rg2",
"rats",
"group",
"heart",
"01",
"fibrosis",
"myocardial fibrosis"
] |
[
"test",
"test"
] | null |
[CONTENT] Myocardial ischaemia | cardiac function | isoproterenol [SUMMARY]
| null |
[CONTENT] Myocardial ischaemia | cardiac function | isoproterenol [SUMMARY]
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[CONTENT] Myocardial ischaemia | cardiac function | isoproterenol [SUMMARY]
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[CONTENT] Myocardial ischaemia | cardiac function | isoproterenol [SUMMARY]
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[CONTENT] Myocardial ischaemia | cardiac function | isoproterenol [SUMMARY]
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[CONTENT] Animals | Cardiotonic Agents | Dose-Response Relationship, Drug | Fibrosis | Ginsenosides | Isoproterenol | Male | Myocardial Ischemia | Panax | Rats | Rats, Wistar | Signal Transduction | Smad Proteins | Transforming Growth Factor beta1 [SUMMARY]
| null |
[CONTENT] Animals | Cardiotonic Agents | Dose-Response Relationship, Drug | Fibrosis | Ginsenosides | Isoproterenol | Male | Myocardial Ischemia | Panax | Rats | Rats, Wistar | Signal Transduction | Smad Proteins | Transforming Growth Factor beta1 [SUMMARY]
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[CONTENT] Animals | Cardiotonic Agents | Dose-Response Relationship, Drug | Fibrosis | Ginsenosides | Isoproterenol | Male | Myocardial Ischemia | Panax | Rats | Rats, Wistar | Signal Transduction | Smad Proteins | Transforming Growth Factor beta1 [SUMMARY]
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[CONTENT] Animals | Cardiotonic Agents | Dose-Response Relationship, Drug | Fibrosis | Ginsenosides | Isoproterenol | Male | Myocardial Ischemia | Panax | Rats | Rats, Wistar | Signal Transduction | Smad Proteins | Transforming Growth Factor beta1 [SUMMARY]
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[CONTENT] Animals | Cardiotonic Agents | Dose-Response Relationship, Drug | Fibrosis | Ginsenosides | Isoproterenol | Male | Myocardial Ischemia | Panax | Rats | Rats, Wistar | Signal Transduction | Smad Proteins | Transforming Growth Factor beta1 [SUMMARY]
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[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] myocardial | ginsenoside | ginsenoside rg2 | rg2 | rats | group | heart | 01 | fibrosis | myocardial fibrosis [SUMMARY]
| null |
[CONTENT] myocardial | ginsenoside | ginsenoside rg2 | rg2 | rats | group | heart | 01 | fibrosis | myocardial fibrosis [SUMMARY]
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[CONTENT] myocardial | ginsenoside | ginsenoside rg2 | rg2 | rats | group | heart | 01 | fibrosis | myocardial fibrosis [SUMMARY]
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[CONTENT] myocardial | ginsenoside | ginsenoside rg2 | rg2 | rats | group | heart | 01 | fibrosis | myocardial fibrosis [SUMMARY]
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[CONTENT] myocardial | ginsenoside | ginsenoside rg2 | rg2 | rats | group | heart | 01 | fibrosis | myocardial fibrosis [SUMMARY]
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[CONTENT] myocardial | ischaemia | myocardial ischaemia | tcm | improve | induced | ginsenoside | function | fibrosis | myocardial fibrosis [SUMMARY]
| null |
[CONTENT] 01 | group | myocardial | rg2 | ginsenoside | ginsenoside rg2 | indicates | compared | rats | ischaemic rats [SUMMARY]
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[CONTENT] myocardial | ischaemia | myocardial ischaemia | clinical application myocardial ischaemia | provides insights mechanisms | insights mechanisms ginseng | insights mechanisms ginseng clinical | application myocardial ischaemia | mechanisms ginseng | mechanisms ginseng clinical [SUMMARY]
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[CONTENT] myocardial | ginsenoside | rg2 | ginsenoside rg2 | group | heart | rats | 01 | fibrosis | weight [SUMMARY]
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[CONTENT] myocardial | ginsenoside | rg2 | ginsenoside rg2 | group | heart | rats | 01 | fibrosis | weight [SUMMARY]
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[CONTENT] C.A. Meyer | Araliaceae ||| [SUMMARY]
| null |
[CONTENT] 5 | 20 mg/kg | 8.9 ± | 1.3 | 7.5 ± | 0.7 ||| 7.2 ± | 1.0 | LVSP | 96.75 ± | 13.2 | 118.3 | 19.4 | 124.3 | 21.3 | 2142.8 ± | 309.3 | 2598.6 | 404.0 | 2661.5 | 445.2 | mmHg/s | 1996.3 ± | 306.3 | 2476.6 | 289.7 | 2509.6 ± | 353.1 ||| 9.2 ± | 0.9% | 8.5 ± | 0.8% | 10.1 ± | 1.0% [SUMMARY]
|
[CONTENT] ||| [SUMMARY]
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[CONTENT] C.A. Meyer | Araliaceae ||| ||| Male Wistar | 5 | 20 mg/kg | 8) ||| 5 mg/kg | 7 days ||| 0.5% | CMC-Na | daily | 28 days ||| day 28 ||| 5 | 20 mg/kg | 8.9 ± | 1.3 | 7.5 ± | 0.7 ||| 7.2 ± | 1.0 | LVSP | 96.75 ± | 13.2 | 118.3 | 19.4 | 124.3 | 21.3 | 2142.8 ± | 309.3 | 2598.6 | 404.0 | 2661.5 | 445.2 | mmHg/s | 1996.3 ± | 306.3 | 2476.6 | 289.7 | 2509.6 ± | 353.1 ||| 9.2 ± | 0.9% | 8.5 ± | 0.8% | 10.1 ± | 1.0% ||| ||| [SUMMARY]
|
[CONTENT] C.A. Meyer | Araliaceae ||| ||| Male Wistar | 5 | 20 mg/kg | 8) ||| 5 mg/kg | 7 days ||| 0.5% | CMC-Na | daily | 28 days ||| day 28 ||| 5 | 20 mg/kg | 8.9 ± | 1.3 | 7.5 ± | 0.7 ||| 7.2 ± | 1.0 | LVSP | 96.75 ± | 13.2 | 118.3 | 19.4 | 124.3 | 21.3 | 2142.8 ± | 309.3 | 2598.6 | 404.0 | 2661.5 | 445.2 | mmHg/s | 1996.3 ± | 306.3 | 2476.6 | 289.7 | 2509.6 ± | 353.1 ||| 9.2 ± | 0.9% | 8.5 ± | 0.8% | 10.1 ± | 1.0% ||| ||| [SUMMARY]
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The incremental treatment of ESRD: a low-protein diet combined with weekly hemodialysis may be beneficial for selected patients.
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25352299
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Infrequent dialysis, namely once-a-week session combined with very low-protein, low-phosphorus diet supplemented with ketoacids was reported as a useful treatment schedule for ESRD patients with markedly reduced residual renal function but preserved urine output. This study reports our findings from the application of a weekly dialysis schedule plus less severe protein restriction (standard low-protein low-phosphorus diet) in stage 5 CKD patients with consistent dietary discipline.
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BACKGROUND
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This is a multicenter, prospective controlled study, including 68 incident CKD patients followed in a pre-dialysis clinic with Glomerular Filtration Rate 5 to 10 ml/min/1.73/ m2 who became unstable on the only medical treatment. They were offered to begin a Combined Diet Dialysis Program (CDDP) or a standard thrice-a-week hemodialysis (THD): 38 patients joined the CDDP, whereas 30 patients chose THD. Patients were studied at baseline, 6 and 12 months; hospitalization and survival rate were followed-up for 24 months.
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METHODS
|
Volume output and residual renal function were maintained in the CDDP Group while those features dropped quickly in THD Group. Throughout the study, CDDP patients had a lower erythropoietin resistance index, lower β2 microglobulin levels and lower need for cinacalcet of phosphate binders than THD, and stable parameters of nutritional status. At 24 month follow-up, 39.4% of patients were still on CDDP; survival rates were 94.7% and 86.8% for CDDP and THD patients, respectively, but hospitalization rate was much higher in THD than in CDDP patients. The cost per patient per year resulted significantly lower in CDDP than in THD Group.
|
RESULTS
|
This study shows that a CDDP served to protect the residual renal function, to maintain urine volume output and to preserve a good nutritional status. CDDP also blunted the rapid β2 microglobulin increase and resulted in better control of anemia and calcium-phosphate abnormalities. CDDP was also associated with a lower hospitalization rate and reduced need of erythropoietin, as well as of drugs used for treatment of calcium-phosphate abnormalities, thus leading to a significant cost-saving. We concluded that in selected ESRD patients with preserved urine output attitude to protein restriction, CDDP may be a beneficial choice for an incremental hemodialysis program.
|
CONCLUSIONS
|
[
"Aged",
"Aged, 80 and over",
"Appointments and Schedules",
"Calcium",
"Combined Modality Therapy",
"Cost Savings",
"Diet, Protein-Restricted",
"Dietary Proteins",
"Female",
"Hospitalization",
"Humans",
"Hyperparathyroidism, Secondary",
"Kidney Failure, Chronic",
"Kidney Function Tests",
"Male",
"Middle Aged",
"Phosphorus",
"Phosphorus, Dietary",
"Prospective Studies",
"Renal Dialysis",
"Survival Rate",
"Treatment Outcome"
] |
4232716
|
Background
|
The Epidemiology of chronic kidney disease (CKD) is on an upward trend worldwide. Since the 1990s incidence has risen by approximately 40% per decade, with prevalence rates in the general adult population up to 13% in the USA and ranging 10.2-11.6% in Europe [1–4]. These figures indicate a CKD epidemic leading to an increased number of patients requiring dialysis and a heavy burden on public health resources.
Nutritional and pharmacological therapy constitute the basis for the prevention and treatment of signs and symptoms of CKD, and they serve to delay the commencement of dialysis. The appropriate time to start dialysis is still a matter of debate. In particular, dialysis is not superior that conservative treatment especially in the comorbid-elderly population. It is known that a standard thrice--weekly hemodialysis schedule invariably leads to rapid loss of the residual renal function, to psychological and socials drawbacks, and to high costs. Real incremental dialysis programs are implemented in peritoneal dialysis but not in the hemodialysis setting where only a twice -weekly schedule may be proposed prior to maintenance hemodialysis.
During the 1980’s and 1990’s, an Integrated Diet Dialysis Program (IDDP) was proposed as a therapeutic option for selected patients with markedly reduced residual renal function and it consisted of weekly hemodialysis session (HD) combined with nutritional therapy, namely a very low-protein diet supplemented with ketoacids [5–8]. Malnutrition risk and low compliance to the severe dietary restrictions represented major concerns that prevented a widespread application of this schedule. In the present study we propose a Combined Diet Dialysis Program (CDDP) which includes a less severe low protein (0.6 g/Kg/d) diet combined with once-weekly hemodialysis. The goal is to prolong a conservative approach and thus reduce the need for hemodialysis, thereby limiting the risk of malnutrition and of poor dietary adherence. This study aimed to assess the safety, benefits and drawbacks of the CDDP approach including nutritional status, residual renal function, morbidity, mortality and costs.
|
Methods
|
This is a multicenter, non-randomized, prospective controlled study, including stage V CKD patients followed in a pre-dialysis clinic who were not suitable for a peritoneal dialysis program.
Patients with GFR 5 to 10 ml/min who were approaching hemodialysis treatment and who had vascular access were included in the study. Global criteria of starting dialysis and then for entering the study were fluid retention, inadequate control of BUN or severe secondary hyperparathyroidism or hyperphosphatemia, reduced compliance to dietary treatment.
Patients with pericarditis, congestive heart failure, severe fluid retention, overt protein-energy wasting, hyperkalemia or severe metabolic acidosis with acute reduction of residual renal function or concurrent diseases were excluded, as well patients with unreliable discipline with regards to dietary restrictions were treated at once with maintenance hemodialysis and then excluded from the study. During the pre-dialysis phase, patients were followed in a tertiary care CKD clinic every two-three months (stage 4) or on a monthly basis (stage 5). Compliance to dietary and pharmacological treatment was acceptable, as assessed by urinary urea excretion and by dietary recall and interview.
Sixty-eight incident patients were recruited and all the patients were provided a functioning native artero-venous fistula or graft.
Underlying kidney diseases included mainly hypertension/vascular nephropathy, ADPKD. The patients were offered the choice to commence a weekly hemodialysis schedule plus low-protein diet on the non-dialysis day (namely, CDDP), or to begin a standard thrice- weekly hemodialysis (THD) schedule and free-choice diet. All the patients were informed and directly involved in the decision making process. Randomization was not applied since any dietary regimen requires adherence and motivation.
According to their own choice, 38 patients entered in the CDDP Group, whereas the remaining 30 patients formed the THD control group. The clinical and biochemical features of the two groups at baseline are reported in Table 1. At baseline, the prevalence of cardiovascular disease history was similar in CDDP (28.9%) and in THD (30%) group. Six type 2 diabetics were included in CDDP group and 13 in THD group.Table 1
Baseline characteristics of the studied groups
CDDP group (n = 38)THD group (n = 30)pMale/females25/1319/11Age, years64.5 ± 13.265.2 ± 11
0.82
Body weight (kg)65.5 ± 15.166.2 ± 11.9
0.73
BMI (kg/m2)23.7 ± 4.025.6 ± 4.13
0.03
Urine volume output (mL/24 h)1983 ± 6511472.6 ± 433
<0.001
GFR (mL/min × 1.73 mq b.s)7.8 ± 1.99.2 ± 4.2
<0.01
EPO (IU/kg/week)104 ± 108184 ± 84
<0.001
CRP <5 mg/dl, %8966.6
<0.01
iPTH, >300 ρg/mL, %31.550
<0.01
Charlson comorbidity index score5.5 ± 2.53.8 ± 2.5
0.004
Charlson comorbidity index score >4, %62.533.3
<0.01
Baseline characteristics of the studied groups
All the patients were studied at baseline, and after 6 and 12 months. The survival rate and hospitalization rate were followed up to 24 months.
Blood samples were collected from the arterial line before the start of the first dialysis of the week; 24h urines were collected during the day before the HD session. GFR was measured as the average of creatinine clearance and urea clearance, and expressed as ml/min*1.73 m2 body surface area [9]; eGFR was also estimated by MDRD formula [10]. Protein catabolic rate (PCR) was used as an indicator of dietary protein intake and calculated by the urea appearance method according to Maroni’s formula [11]. Maroni’s formula was used throughout the course of the CDDP and only at baseline time in patients in the THD Group. Within the THD Group, for patients with the significant loss of renal function and urine output volume we included the method of urea kinetics model for estimation of nPCR. Dialytic adequacy was expressed using eKt/V according to Daugirdas formula et al [12]. Body mass index was calculated by the body weight at the end of the dialysis session. The bioelectrical parameters, were measured with a single-frequency impedance analyzer (BIA 101 – Akern - Florence), 30’ after the end of the hemodialysis session.
Erythropoietin Resistance Index (ERI) was calculated as weekly EPO dosage (units)/body weight (Kg)/Hb (g/dl) [13].
Charlson’s comorbidity index (CCI) score, was calculated to evaluate comorbidity conditions [14].
Creatinine production, as a surrogate of muscle mass, was calculated by the sum of 24h urine creatinine excretion and metabolized creatinine. Metabolized creatinine was calculated from the product of serum creatinine and extrarenal Creatinine clearance (estimated as 0.038 L/kg BW/d) [7].
All the patients on CDDP underwent nutritional counseling by a renal dietician and were prescribed a low protein (0,6 g/kg b.w./d), low phosphorus (600–700 mg/d), low sodium diet with an energy intake of 30–35 kcal/kg b.w./d. Proteins were mostly from animal sources (0.4 g/Kg/b.w.), to cover the requirement of essential amino acids; no dairy or processed foods are included to the aim of limiting the sodium and phosphorus intake; [15] protein-free products are included to supply energy with negligible load of phosphorus, sodium, potassium and nitrogen [16]. The 60–62% of energy intake derived from carbohydrates (mostly represented by protein-free products), 30–32% from lipids and 8-10% from proteins. As average, the daily dietary plan consisted of 5–7 servings of protein free products, namely 2–3 serving for breakfast and snacks and 2 servings for both lunch and dinner; the patients were usually advised that they can add other servings, when required to satisfy the energy requirement. The servings of animal protein sources (meat, fish, egg white) were calculated for each patient in order to guarantee a daily total amount of 0.4 g protein per kg b.w. Two servings of vegetables and two of fruits were provided with specific suggestions for patients with high potassium levels as well as boiling was suggested to reduce to mineral content (potassium, sodium, calcium, and phosphorus) of food. Finally, 5–6 servings of fats (olive oil as first choice) were suggested.
This diet was prescribed for 6 days a week, while no dietary restrictions occurred on the dialysis day.
Patients in the THD control group followed an unrestricted protein diet, but they were given dietary counseling focused on preventing excess sodium, potassium, phosphorus and fluid intakes.
All the patients were prescribed 4-hour hemodialysis sessions with highly biocompatible synthetic membranes.
The study protocol was approved by the Cagliari Hospital Ethics Committee, and all the patients gave their written informed consent for participation in the study.
Statistical analysis Descriptive statistics is given as mean ± standard deviation. Normality tests were performed on all continuous variables measured throughout the study. The log-rank test was used for hypothesis testing. Time repeated measurements were analyzed using linear mixed models including treatment, time, and treatment by time interaction for all measured variables using ANOVA. Inter-group drug use was compared by means of the binomial test with Bonferroni’s correction for multiple comparisons.
Differences were considered as statistically significant when p <0.05.
Descriptive statistics is given as mean ± standard deviation. Normality tests were performed on all continuous variables measured throughout the study. The log-rank test was used for hypothesis testing. Time repeated measurements were analyzed using linear mixed models including treatment, time, and treatment by time interaction for all measured variables using ANOVA. Inter-group drug use was compared by means of the binomial test with Bonferroni’s correction for multiple comparisons.
Differences were considered as statistically significant when p <0.05.
|
Results
|
Patients who entered the CDDP showed several differences with respect to patients who commenced THD (Table 1). Despite a lower residual renal function (RRF), CDDP patients showed lower circulating levels of phosphate, BUN, PTH and higher albumin levels with respect to THD patients. 34 out of the 38 patients were still on CDDP at 12 months: 2 patients were shifted to a thrice- weekly dialysis schedule at 8 and 10 months, while 2 patients partially recovered their renal function and remained clinically stable with the low-protein diet and no need for dialysis.
During the study period, GFR was preserved in CDDP patients in comparison to THD group (Table 2). GFR loss progression was very low in the former (-0.13 ml/min/month) while it was faster in the latter (-1.53 ml/min/month). Similarly, in the CDDP group an effective urine volume output was maintained whereas it dramatically dropped in the THD patients (Figure 1a). As a consequence, the interdialytic weight gain in CDDP patients was limited to (800 ± 300 g) per week. The dosage of frusemide increased from 150 ± 154 to 273 ± 201 mg/day in the CDDP group, and from 332 ± 171 to 409 ± 205 mg/day (p =0.003) in the THD group. The increase of β2-microglobulin circulating levels was much lower in CDDP than in THD patients (+11.2% vs +34%, respectively) (Figure 1c).Table 2
Outcome of nutritional and functional parameters in both groups in 12 months
CDDP groupTHD groupBaseline (n = 38)6 months (n = 38)12 months (n = 34)Baseline (n = 30)6 months (n = 30)12 months (n = 29)
P
Dry weight (kg)
65 ± 15.0
63.9 ± 14.4
63.4 ± 14.7
66.2 ± 11.9
64.9 ± 11.5
65.5 ± 12.3
0.07
BMI (kg/m
2
)
23.7 ± 4.0
23.4 ± 3.7
23.0 ± 3.9
25.6 ± 4.13
25.1 ± 4.07
25.4 ± 4.2
0.07
Total body water (L)
44.4 ± 10.5
41 ± 9.6
43 ± 11
35.4 ± 6
33.5 ± 5.8
a
33.8 ± 5.9
a
0.02
Extracellular water (L)
20 ± 4.5
18.6 ± 4.3
19.7 ± 4
17.5 ± 3.2
16 ± 2.8
b
15.7 ± 2.4
b
0.04
Fat mass (kg)
11 ± 12.3
13 ± 11.8
11.4 ± 11
20 ± 9
21.4 ± 8.5
22.6 ± 9.4
0.03
Body cell mass (kg)
31 ± 9.6
28 ± 8.9
29.7 ± 10.3
22.5 ± 6.2
22.7 ± 5
22.7 ± 5.8
0.02
Phase angle, (°)
6.2 ± 1.3
6.2 ± 1.3
6.1 ± 1.4
5.0 ± 1.2
6 ± 0.9
5.8 ± 1.2
0.06
SBP (mmHg)
139 ± 18
132 ± 20
128 ± 15
b
136.8 ± 17.2
138.5 ± 18.5
139 ± 20.8
0.21
DBP (mmHg)
80 ± 12
73 ± 10
74 ± 9
c
71.6 ± 11.4
71.6 ± 10
72.9 ± 12.3
0.08
Serum creatinine (mg/dL)
6.4 ± 1.9
7.0 ± 2.5
7.8 ± 3.0
b
5.9 ± 1.8
7.8 ± 2.2
8.2 ± 2.4
b
0.05
B.U.N. (mg/dL)
68 ± 18
68 ± 16
70 ± 17
84.4 ± 18.5
77 ± 15.5
77 ± 22.9
0.003
GFR (mL/min)
7.8 ± 1.9
6.7 ± 2.3
6.3 ± 2.1
b
9.2 ± 4.2
-
-
-
eGFR (mL/min)
9.4 ± 2.9
8.4 ± 3.5
a
8.0 ± 3.4
b
10.3 ± 3.9
-
-
-
Total protein (g/dL)
6.7 ± 0.5
6.7 ± 0.5
6.8 ± 0.4
6.35 ± 0.52
6.4 ± 0.5
6.5 ± 0.43
0.004
Albumin (g/dL)
3.8 ± 0.4
3.9 ± 0.4
4.1 ± 0.4
b
3.6 ± 0.40
3.6 ± 0.37
3.7 ± 0.48
0.01
Transferrine (mg/dL)
219 ± 53
203 ± 72
222 ± 53
264 ± 94
249 ± 43
251 ± 61
0.001
C3 (mg/dL)
90 ± 20
91 ± 23
94 ± 23
99.6 ± 39.6
96 ± 35.7
95.9 ± 32
0.48
C4 (mg/dL)
24 ± 7
24 ± 8
26 ± 7
33 ± 28
29 ± 18
29 ± 30
0.26
Total IgG (mg/dL)
1599 ± 497
1590 ± 402
1543 ± 373
1440 ± 461
1482 ± 487
1409 ± 413
0.21
Lynphocytes/mm
3
1539 ± 651
1390 ± 831
1581 ± 537
1452 ± 811
1458 ± 622
1445 ± 580
0.38
Legend: changes versus baseline: ap < 0.05; bp < 0.01; c< 0.03; P: ANOVA significance for treatment by time interaction.Figure 1
Changes in urine volume (a), serum albumin (b), β2-microglobulin (c) and serum phosphorus (d) in CDDP Group (dotted Line) and THD Group (full line), at baseline, 6 and 12 months of follow-up.
Outcome of nutritional and functional parameters in both groups in 12 months
Legend: changes versus baseline: ap < 0.05; bp < 0.01; c< 0.03; P: ANOVA significance for treatment by time interaction.
Changes in urine volume (a), serum albumin (b), β2-microglobulin (c) and serum phosphorus (d) in CDDP Group (dotted Line) and THD Group (full line), at baseline, 6 and 12 months of follow-up.
No difference in arterial blood pressure values emerged between the two groups.
The main nutritional indicators, i.e. total protein, albumin and transferrin improved significantly in the CDDP group (Figure 1b, Table 2). Estimated dietary protein intake remained stable in the CDDP groups throughout the study (from 0.58 ± 0.2 to 0.57 ± 0.11 g/kg/day). This was also the case for THD patients (from 1.03 ± 0.1 to 1.04 ± 0.3 g/kg/day). No significative changes in body weight or Body Mass Index (BMI) were recorded in the two groups (Table 2).
A significant reduction in the ESAs requirement was observed in the CDDP group along with markedly lower ERI values (Table 3).Table 3
Biochemistry parameters in CDDP and THD groups at 6 and 12 months
CDDP groupTHD groupBaseline (n = 38)6 months (n = 38)12 months (n = 34)Baseline (n = 30)6 months (n = 30)12 months (n = 29)P
Triglycerides (mg/dL)
109 ± 36134 ± 92126 ± 50131 ± 72152 ± 67139 ± 52
0.03
Total Cholesterol (mg/dL)
164 ± 36168 ± 36164 ± 35165 ± 41168.5 ± 34170 ± 40
0.51
HDL Cholesterol (mg/dL)
44 ± 1447 ± 1348 ± 2044 ± 1245 ± 1344 ± 12
0.58
LDL Cholesterol (mg/dL)
101 ± 2993 ± 3493 ± 3195 ± 3293 ± 2997.8 ± 34
0.95
Cholinesterases (UI/mL)
5474 ± 13966073 ± 1474586 ± 16464968 ± 14885511 ± 16044673 ± 1450b
0.27
Uric acid (mg/dL)
7.3 ± 2.37.5 ± 2.47.1 ± 1.96.4 ± 1.8b
7.3 ± 1.27.2 ± 1.3b
0.60
Sodium (mmol/L)
139 ± 2.6137 ± 3.6137 ± 4.0138 ± 2.7138 ± 2.3134 ± 3.3
0.58
Potassium (mmol/L)
4.4 ± 0.64.3 ± 0.64.3 ± 0.74.3 ± 0,674.7 ± 0.74.8 ± 0.9b
0.07
Calcium (mg/dL)
9.2 ± 0.59.0 ± 0.59.1 ± 0.69.1 ± 0.69.1 ± 0.79.0 ± 0.5
0.51
Phosphatemia (mg/dL)
4.4 ± 0.94.6 ± 0.84.6 ± 0.85.1 ± 1.55.6 ± 1.15.2 ± 1.1
0.001
Bicarbonate (mEq/L)
22.0 ± 3.123.1 ± 3.323.2 ± 3.221.8 ± 4.221.9 ± 3.221.6 ± 2.7
0.17
β2-microglobulin (mg/dL)
14.2 ± 3.916.8 ± 5.716.0 ± 5.118.4 ± 11.631.0 ± 16.028.0 ± 11.4b
0.007
Hb (g/dl)
10.8 ± 0.111.5 ± 0.9511.5 ± 0.97a
10.5 ± 1.411.3 ± 0.911.2 ± 0.95a
0.31
EPO (IU/kg/week)104 ± 10869 ± 5960 ± 74b
184 ± 84172 ± 138204 ± 252
0.002
ERI (IU/kg/week)/Hb10 ± 116 ± 55 ± 7b
19 ± 1015 ± 1319 ± 23
<0.001
CRP (% patients) <5.0
8981.57966.683.376.6
<0.05
CRP (mg/dL)
3.3 ± 3.93.5 ± 3.33.2 ± 3.68.6 ± 9.74.9 ± 6.55.8 ± 5.8
<0.01
iPTH (ρg/mL) <150 (%)
39.552.65236.743.334.5
0.09
iPTH (ρg/mL) 150–300 (%)
29293013.32027.6
0.26
iPTH (ρg/mL) >300 (%)
31.5a
18.418a
5036.737.9
0.02
Changes versus baseline: ap < 0.01; bp < 0.03; P: ANOVA significance for treatment by time interaction.
Biochemistry parameters in CDDP and THD groups at 6 and 12 months
Changes versus baseline: ap < 0.01; bp < 0.03; P: ANOVA significance for treatment by time interaction.
Calcium phosphate metabolism parameters were stable and better controlled in the CDDP (Table 3, Figure 1d) group despite a significantly lower use of non-calcium containing phosphate-binders and cinacalcet and among all the drugs, only allopurinol use was reduced during THD (Table 4).Table 4
Pharmacologic treatments in CDDP and THD groups
CDDP GroupTHD groupP% of patientsBaseline12 monthsBaseline12 months
ACEi
34335048.3
n.s
ARBs
24263021
n.s
Calcium channel blockers
28.927.33017
n.s
Calcium carbonate
596076.672.4
n.s.
Sevelamer or Lanthanum carbonate
012a
041c
<0.03
Paricalcitol
012b
017.2b
n.s.
Cinacalcet
06b
024c
<0.04
Calcitriol
454246.745
n.s.
Kayexalate
21212038
n.s.
Allopurinol
48.65433.313.8a
0.01
Statins
373326.724
n.s
Polyunsaturated fats
1391017
n.s.
Legend: Changes versus baseline: ap<0.05; bp<0.01; c<0.03; P: ANOVA significance for treatment by time interaction.
Pharmacologic treatments in CDDP and THD groups
Legend: Changes versus baseline: ap<0.05; bp<0.01; c<0.03; P: ANOVA significance for treatment by time interaction.
Calcimimetics drugs are not delivered by Italian NHS during the pre-dialysis phase.
So their use began after commencing dialysis (even once-a-week): patients on THD showed a secondary hyperparathyroidism more severe than patients on CDDP , consequently the need of calcimimetics was higher in the former than in the latter.
No significant reduction in creatinine production was observed after 12 months of CDDP (16.8 ± 4.3 vs. 15.9 ± 3.9 mg/Kg/d).
Hospitalization Throughout the 24 month follow-up period, 3 CDDP patients were admitted to hospital for a total of 11 days (3.7 ± 1.5 days/patient) due to atrial fibrillation, acute bronchitis and acute cholecystitis. Instead 24 hospital admission were recorded in 15 patients of THD groups for a total of 147 days (6.1 ± 6.3 days/patient). Causes for admission were: set up of new vascular access (7), artero-venous fistula stenosis angioplasty (1), infection of central vein catheter (2), acute pulmonary edema (1), surgery for biological prosthetic valve (1), myocardial infarction (1), valvular and coronaropathy angioplasty (2), congestive heart failure (1), atrial fibrillation (2), hyperpyrexia (1), hypertensive crisis (1), hypoglycemic coma (1), abscess in thigh hematoma (1) and obstructive jaundice secondary to gallstones (2).
Throughout the 24 month follow-up period, 3 CDDP patients were admitted to hospital for a total of 11 days (3.7 ± 1.5 days/patient) due to atrial fibrillation, acute bronchitis and acute cholecystitis. Instead 24 hospital admission were recorded in 15 patients of THD groups for a total of 147 days (6.1 ± 6.3 days/patient). Causes for admission were: set up of new vascular access (7), artero-venous fistula stenosis angioplasty (1), infection of central vein catheter (2), acute pulmonary edema (1), surgery for biological prosthetic valve (1), myocardial infarction (1), valvular and coronaropathy angioplasty (2), congestive heart failure (1), atrial fibrillation (2), hyperpyrexia (1), hypertensive crisis (1), hypoglycemic coma (1), abscess in thigh hematoma (1) and obstructive jaundice secondary to gallstones (2).
Survival At the 24-month follow-up, no significant difference was detected between the CDDP and THD group survival rates: 94.7% and 86.8% respectively (Figure 2).Figure 2
Cumulative survival in CDDP group (Dotted line) and in THD Group (Full line).
Cumulative survival in CDDP group (Dotted line) and in THD Group (Full line).
Death in 3 CDDP patients was caused by cardiac events: 1 during the 13th, 1 during the 16th and 1 during the 18th month of CDDP. Four deaths occurred in the THD group: 1 in the 9th, 2 in the 13th and 1 in the 22nd month following start of dialysis. Causes included myocardial infarction and acute heart failure, sepsis and stroke.
At the 24-month follow-up, no significant difference was detected between the CDDP and THD group survival rates: 94.7% and 86.8% respectively (Figure 2).Figure 2
Cumulative survival in CDDP group (Dotted line) and in THD Group (Full line).
Cumulative survival in CDDP group (Dotted line) and in THD Group (Full line).
Death in 3 CDDP patients was caused by cardiac events: 1 during the 13th, 1 during the 16th and 1 during the 18th month of CDDP. Four deaths occurred in the THD group: 1 in the 9th, 2 in the 13th and 1 in the 22nd month following start of dialysis. Causes included myocardial infarction and acute heart failure, sepsis and stroke.
CDDP global outcome At 12 months 34 out of the 38 patients (89.5%) were on CDDP. At 24 months, 15 patients were still on CDDP (39.4%) since 1 patient received a kidney transplant, 3 patients had fatal myocardial infarction, whereas 17 patients have progressed to an incremental dialysis program (namely twice to thrice-weekly dialysis and free-protein diet) and 2 patients partially recovered their renal function. The main reasons for dropping out were loss of dietary adherence and/or reduction of residual renal function.
When compared to THD group, CDDP patients registered a lower number of hospitalizations and a lower need for expensive drugs. These findings, coupled with the 66% reduction in number of dialysis sessions (52 versus 156 sessions in THD Group in a year) allows to estimated that the cost of a CDDP patient is more than 60% less than that of a THD patient.
At 12 months 34 out of the 38 patients (89.5%) were on CDDP. At 24 months, 15 patients were still on CDDP (39.4%) since 1 patient received a kidney transplant, 3 patients had fatal myocardial infarction, whereas 17 patients have progressed to an incremental dialysis program (namely twice to thrice-weekly dialysis and free-protein diet) and 2 patients partially recovered their renal function. The main reasons for dropping out were loss of dietary adherence and/or reduction of residual renal function.
When compared to THD group, CDDP patients registered a lower number of hospitalizations and a lower need for expensive drugs. These findings, coupled with the 66% reduction in number of dialysis sessions (52 versus 156 sessions in THD Group in a year) allows to estimated that the cost of a CDDP patient is more than 60% less than that of a THD patient.
|
Conclusions
|
This study shows that in selected ESRD patients, a CDDP is able to protect the RRF, to maintain urine volume output, to preserve a good nutritional status , to blunt the rapid β2 microglobulin increase, and to allow better control of anemia and calcium-phosphate abnormalities. CDDP is also associated with a lower rate of hospitalization and reduced need of EPO and drugs used for the CKD-MBD treatment, thus leading to a significant cost-saving. As a whole, our findings suggest that CDDP could be a beneficial choice for selected collaborative patients who would otherwise be referred to thrice-weekly hemodialysis. CCDP should be considered as the first step of an incremental approach to hemodialysis treatment in motivated and selected ESRD patients, or anywhere dialysis facilities and resources are lacking.
|
[
"Background",
"Statistical analysis",
"Hospitalization",
"Survival",
"CDDP global outcome"
] |
[
"The Epidemiology of chronic kidney disease (CKD) is on an upward trend worldwide. Since the 1990s incidence has risen by approximately 40% per decade, with prevalence rates in the general adult population up to 13% in the USA and ranging 10.2-11.6% in Europe [1–4]. These figures indicate a CKD epidemic leading to an increased number of patients requiring dialysis and a heavy burden on public health resources.\nNutritional and pharmacological therapy constitute the basis for the prevention and treatment of signs and symptoms of CKD, and they serve to delay the commencement of dialysis. The appropriate time to start dialysis is still a matter of debate. In particular, dialysis is not superior that conservative treatment especially in the comorbid-elderly population. It is known that a standard thrice--weekly hemodialysis schedule invariably leads to rapid loss of the residual renal function, to psychological and socials drawbacks, and to high costs. Real incremental dialysis programs are implemented in peritoneal dialysis but not in the hemodialysis setting where only a twice -weekly schedule may be proposed prior to maintenance hemodialysis.\nDuring the 1980’s and 1990’s, an Integrated Diet Dialysis Program (IDDP) was proposed as a therapeutic option for selected patients with markedly reduced residual renal function and it consisted of weekly hemodialysis session (HD) combined with nutritional therapy, namely a very low-protein diet supplemented with ketoacids [5–8]. Malnutrition risk and low compliance to the severe dietary restrictions represented major concerns that prevented a widespread application of this schedule. In the present study we propose a Combined Diet Dialysis Program (CDDP) which includes a less severe low protein (0.6 g/Kg/d) diet combined with once-weekly hemodialysis. The goal is to prolong a conservative approach and thus reduce the need for hemodialysis, thereby limiting the risk of malnutrition and of poor dietary adherence. This study aimed to assess the safety, benefits and drawbacks of the CDDP approach including nutritional status, residual renal function, morbidity, mortality and costs.",
"Descriptive statistics is given as mean ± standard deviation. Normality tests were performed on all continuous variables measured throughout the study. The log-rank test was used for hypothesis testing. Time repeated measurements were analyzed using linear mixed models including treatment, time, and treatment by time interaction for all measured variables using ANOVA. Inter-group drug use was compared by means of the binomial test with Bonferroni’s correction for multiple comparisons.\nDifferences were considered as statistically significant when p <0.05.",
"Throughout the 24 month follow-up period, 3 CDDP patients were admitted to hospital for a total of 11 days (3.7 ± 1.5 days/patient) due to atrial fibrillation, acute bronchitis and acute cholecystitis. Instead 24 hospital admission were recorded in 15 patients of THD groups for a total of 147 days (6.1 ± 6.3 days/patient). Causes for admission were: set up of new vascular access (7), artero-venous fistula stenosis angioplasty (1), infection of central vein catheter (2), acute pulmonary edema (1), surgery for biological prosthetic valve (1), myocardial infarction (1), valvular and coronaropathy angioplasty (2), congestive heart failure (1), atrial fibrillation (2), hyperpyrexia (1), hypertensive crisis (1), hypoglycemic coma (1), abscess in thigh hematoma (1) and obstructive jaundice secondary to gallstones (2).",
"At the 24-month follow-up, no significant difference was detected between the CDDP and THD group survival rates: 94.7% and 86.8% respectively (Figure 2).Figure 2\nCumulative survival in CDDP group (Dotted line) and in THD Group (Full line).\n\n\nCumulative survival in CDDP group (Dotted line) and in THD Group (Full line).\n\nDeath in 3 CDDP patients was caused by cardiac events: 1 during the 13th, 1 during the 16th and 1 during the 18th month of CDDP. Four deaths occurred in the THD group: 1 in the 9th, 2 in the 13th and 1 in the 22nd month following start of dialysis. Causes included myocardial infarction and acute heart failure, sepsis and stroke.",
"At 12 months 34 out of the 38 patients (89.5%) were on CDDP. At 24 months, 15 patients were still on CDDP (39.4%) since 1 patient received a kidney transplant, 3 patients had fatal myocardial infarction, whereas 17 patients have progressed to an incremental dialysis program (namely twice to thrice-weekly dialysis and free-protein diet) and 2 patients partially recovered their renal function. The main reasons for dropping out were loss of dietary adherence and/or reduction of residual renal function.\nWhen compared to THD group, CDDP patients registered a lower number of hospitalizations and a lower need for expensive drugs. These findings, coupled with the 66% reduction in number of dialysis sessions (52 versus 156 sessions in THD Group in a year) allows to estimated that the cost of a CDDP patient is more than 60% less than that of a THD patient."
] |
[
null,
null,
null,
null,
null
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[
"Background",
"Methods",
"Statistical analysis",
"Results",
"Hospitalization",
"Survival",
"CDDP global outcome",
"Discussion",
"Conclusions"
] |
[
"The Epidemiology of chronic kidney disease (CKD) is on an upward trend worldwide. Since the 1990s incidence has risen by approximately 40% per decade, with prevalence rates in the general adult population up to 13% in the USA and ranging 10.2-11.6% in Europe [1–4]. These figures indicate a CKD epidemic leading to an increased number of patients requiring dialysis and a heavy burden on public health resources.\nNutritional and pharmacological therapy constitute the basis for the prevention and treatment of signs and symptoms of CKD, and they serve to delay the commencement of dialysis. The appropriate time to start dialysis is still a matter of debate. In particular, dialysis is not superior that conservative treatment especially in the comorbid-elderly population. It is known that a standard thrice--weekly hemodialysis schedule invariably leads to rapid loss of the residual renal function, to psychological and socials drawbacks, and to high costs. Real incremental dialysis programs are implemented in peritoneal dialysis but not in the hemodialysis setting where only a twice -weekly schedule may be proposed prior to maintenance hemodialysis.\nDuring the 1980’s and 1990’s, an Integrated Diet Dialysis Program (IDDP) was proposed as a therapeutic option for selected patients with markedly reduced residual renal function and it consisted of weekly hemodialysis session (HD) combined with nutritional therapy, namely a very low-protein diet supplemented with ketoacids [5–8]. Malnutrition risk and low compliance to the severe dietary restrictions represented major concerns that prevented a widespread application of this schedule. In the present study we propose a Combined Diet Dialysis Program (CDDP) which includes a less severe low protein (0.6 g/Kg/d) diet combined with once-weekly hemodialysis. The goal is to prolong a conservative approach and thus reduce the need for hemodialysis, thereby limiting the risk of malnutrition and of poor dietary adherence. This study aimed to assess the safety, benefits and drawbacks of the CDDP approach including nutritional status, residual renal function, morbidity, mortality and costs.",
"This is a multicenter, non-randomized, prospective controlled study, including stage V CKD patients followed in a pre-dialysis clinic who were not suitable for a peritoneal dialysis program.\nPatients with GFR 5 to 10 ml/min who were approaching hemodialysis treatment and who had vascular access were included in the study. Global criteria of starting dialysis and then for entering the study were fluid retention, inadequate control of BUN or severe secondary hyperparathyroidism or hyperphosphatemia, reduced compliance to dietary treatment.\nPatients with pericarditis, congestive heart failure, severe fluid retention, overt protein-energy wasting, hyperkalemia or severe metabolic acidosis with acute reduction of residual renal function or concurrent diseases were excluded, as well patients with unreliable discipline with regards to dietary restrictions were treated at once with maintenance hemodialysis and then excluded from the study. During the pre-dialysis phase, patients were followed in a tertiary care CKD clinic every two-three months (stage 4) or on a monthly basis (stage 5). Compliance to dietary and pharmacological treatment was acceptable, as assessed by urinary urea excretion and by dietary recall and interview.\nSixty-eight incident patients were recruited and all the patients were provided a functioning native artero-venous fistula or graft.\nUnderlying kidney diseases included mainly hypertension/vascular nephropathy, ADPKD. The patients were offered the choice to commence a weekly hemodialysis schedule plus low-protein diet on the non-dialysis day (namely, CDDP), or to begin a standard thrice- weekly hemodialysis (THD) schedule and free-choice diet. All the patients were informed and directly involved in the decision making process. Randomization was not applied since any dietary regimen requires adherence and motivation.\nAccording to their own choice, 38 patients entered in the CDDP Group, whereas the remaining 30 patients formed the THD control group. The clinical and biochemical features of the two groups at baseline are reported in Table 1. At baseline, the prevalence of cardiovascular disease history was similar in CDDP (28.9%) and in THD (30%) group. Six type 2 diabetics were included in CDDP group and 13 in THD group.Table 1\nBaseline characteristics of the studied groups\nCDDP group (n = 38)THD group (n = 30)pMale/females25/1319/11Age, years64.5 ± 13.265.2 ± 11\n0.82\nBody weight (kg)65.5 ± 15.166.2 ± 11.9\n0.73\nBMI (kg/m2)23.7 ± 4.025.6 ± 4.13\n0.03\nUrine volume output (mL/24 h)1983 ± 6511472.6 ± 433\n<0.001\nGFR (mL/min × 1.73 mq b.s)7.8 ± 1.99.2 ± 4.2\n<0.01\nEPO (IU/kg/week)104 ± 108184 ± 84\n<0.001\nCRP <5 mg/dl, %8966.6\n<0.01\niPTH, >300 ρg/mL, %31.550\n<0.01\nCharlson comorbidity index score5.5 ± 2.53.8 ± 2.5\n0.004\nCharlson comorbidity index score >4, %62.533.3\n<0.01\n\n\nBaseline characteristics of the studied groups\n\nAll the patients were studied at baseline, and after 6 and 12 months. The survival rate and hospitalization rate were followed up to 24 months.\nBlood samples were collected from the arterial line before the start of the first dialysis of the week; 24h urines were collected during the day before the HD session. GFR was measured as the average of creatinine clearance and urea clearance, and expressed as ml/min*1.73 m2 body surface area [9]; eGFR was also estimated by MDRD formula [10]. Protein catabolic rate (PCR) was used as an indicator of dietary protein intake and calculated by the urea appearance method according to Maroni’s formula [11]. Maroni’s formula was used throughout the course of the CDDP and only at baseline time in patients in the THD Group. Within the THD Group, for patients with the significant loss of renal function and urine output volume we included the method of urea kinetics model for estimation of nPCR. Dialytic adequacy was expressed using eKt/V according to Daugirdas formula et al [12]. Body mass index was calculated by the body weight at the end of the dialysis session. The bioelectrical parameters, were measured with a single-frequency impedance analyzer (BIA 101 – Akern - Florence), 30’ after the end of the hemodialysis session.\nErythropoietin Resistance Index (ERI) was calculated as weekly EPO dosage (units)/body weight (Kg)/Hb (g/dl) [13].\nCharlson’s comorbidity index (CCI) score, was calculated to evaluate comorbidity conditions [14].\nCreatinine production, as a surrogate of muscle mass, was calculated by the sum of 24h urine creatinine excretion and metabolized creatinine. Metabolized creatinine was calculated from the product of serum creatinine and extrarenal Creatinine clearance (estimated as 0.038 L/kg BW/d) [7].\nAll the patients on CDDP underwent nutritional counseling by a renal dietician and were prescribed a low protein (0,6 g/kg b.w./d), low phosphorus (600–700 mg/d), low sodium diet with an energy intake of 30–35 kcal/kg b.w./d. Proteins were mostly from animal sources (0.4 g/Kg/b.w.), to cover the requirement of essential amino acids; no dairy or processed foods are included to the aim of limiting the sodium and phosphorus intake; [15] protein-free products are included to supply energy with negligible load of phosphorus, sodium, potassium and nitrogen [16]. The 60–62% of energy intake derived from carbohydrates (mostly represented by protein-free products), 30–32% from lipids and 8-10% from proteins. As average, the daily dietary plan consisted of 5–7 servings of protein free products, namely 2–3 serving for breakfast and snacks and 2 servings for both lunch and dinner; the patients were usually advised that they can add other servings, when required to satisfy the energy requirement. The servings of animal protein sources (meat, fish, egg white) were calculated for each patient in order to guarantee a daily total amount of 0.4 g protein per kg b.w. Two servings of vegetables and two of fruits were provided with specific suggestions for patients with high potassium levels as well as boiling was suggested to reduce to mineral content (potassium, sodium, calcium, and phosphorus) of food. Finally, 5–6 servings of fats (olive oil as first choice) were suggested.\nThis diet was prescribed for 6 days a week, while no dietary restrictions occurred on the dialysis day.\nPatients in the THD control group followed an unrestricted protein diet, but they were given dietary counseling focused on preventing excess sodium, potassium, phosphorus and fluid intakes.\nAll the patients were prescribed 4-hour hemodialysis sessions with highly biocompatible synthetic membranes.\nThe study protocol was approved by the Cagliari Hospital Ethics Committee, and all the patients gave their written informed consent for participation in the study.\n Statistical analysis Descriptive statistics is given as mean ± standard deviation. Normality tests were performed on all continuous variables measured throughout the study. The log-rank test was used for hypothesis testing. Time repeated measurements were analyzed using linear mixed models including treatment, time, and treatment by time interaction for all measured variables using ANOVA. Inter-group drug use was compared by means of the binomial test with Bonferroni’s correction for multiple comparisons.\nDifferences were considered as statistically significant when p <0.05.\nDescriptive statistics is given as mean ± standard deviation. Normality tests were performed on all continuous variables measured throughout the study. The log-rank test was used for hypothesis testing. Time repeated measurements were analyzed using linear mixed models including treatment, time, and treatment by time interaction for all measured variables using ANOVA. Inter-group drug use was compared by means of the binomial test with Bonferroni’s correction for multiple comparisons.\nDifferences were considered as statistically significant when p <0.05.",
"Descriptive statistics is given as mean ± standard deviation. Normality tests were performed on all continuous variables measured throughout the study. The log-rank test was used for hypothesis testing. Time repeated measurements were analyzed using linear mixed models including treatment, time, and treatment by time interaction for all measured variables using ANOVA. Inter-group drug use was compared by means of the binomial test with Bonferroni’s correction for multiple comparisons.\nDifferences were considered as statistically significant when p <0.05.",
"Patients who entered the CDDP showed several differences with respect to patients who commenced THD (Table 1). Despite a lower residual renal function (RRF), CDDP patients showed lower circulating levels of phosphate, BUN, PTH and higher albumin levels with respect to THD patients. 34 out of the 38 patients were still on CDDP at 12 months: 2 patients were shifted to a thrice- weekly dialysis schedule at 8 and 10 months, while 2 patients partially recovered their renal function and remained clinically stable with the low-protein diet and no need for dialysis.\nDuring the study period, GFR was preserved in CDDP patients in comparison to THD group (Table 2). GFR loss progression was very low in the former (-0.13 ml/min/month) while it was faster in the latter (-1.53 ml/min/month). Similarly, in the CDDP group an effective urine volume output was maintained whereas it dramatically dropped in the THD patients (Figure 1a). As a consequence, the interdialytic weight gain in CDDP patients was limited to (800 ± 300 g) per week. The dosage of frusemide increased from 150 ± 154 to 273 ± 201 mg/day in the CDDP group, and from 332 ± 171 to 409 ± 205 mg/day (p =0.003) in the THD group. The increase of β2-microglobulin circulating levels was much lower in CDDP than in THD patients (+11.2% vs +34%, respectively) (Figure 1c).Table 2\nOutcome of nutritional and functional parameters in both groups in 12 months\nCDDP groupTHD groupBaseline (n = 38)6 months (n = 38)12 months (n = 34)Baseline (n = 30)6 months (n = 30)12 months (n = 29)\nP\n\nDry weight (kg)\n\n65 ± 15.0\n\n63.9 ± 14.4\n\n63.4 ± 14.7\n\n66.2 ± 11.9\n\n64.9 ± 11.5\n\n65.5 ± 12.3\n\n0.07\n\nBMI (kg/m\n2\n)\n\n23.7 ± 4.0\n\n23.4 ± 3.7\n\n23.0 ± 3.9\n\n25.6 ± 4.13\n\n25.1 ± 4.07\n\n25.4 ± 4.2\n\n0.07\n\nTotal body water (L)\n\n44.4 ± 10.5\n\n41 ± 9.6\n\n43 ± 11\n\n35.4 ± 6\n\n33.5 ± 5.8\na\n\n33.8 ± 5.9\na\n\n0.02\n\nExtracellular water (L)\n\n20 ± 4.5\n\n18.6 ± 4.3\n\n19.7 ± 4\n\n17.5 ± 3.2\n\n16 ± 2.8\nb\n\n15.7 ± 2.4\nb\n\n0.04\n\nFat mass (kg)\n\n11 ± 12.3\n\n13 ± 11.8\n\n11.4 ± 11\n\n20 ± 9\n\n21.4 ± 8.5\n\n22.6 ± 9.4\n\n0.03\n\nBody cell mass (kg)\n\n31 ± 9.6\n\n28 ± 8.9\n\n29.7 ± 10.3\n\n22.5 ± 6.2\n\n22.7 ± 5\n\n22.7 ± 5.8\n\n0.02\n\nPhase angle, (°)\n\n6.2 ± 1.3\n\n6.2 ± 1.3\n\n6.1 ± 1.4\n\n5.0 ± 1.2\n\n6 ± 0.9\n\n5.8 ± 1.2\n\n0.06\n\nSBP (mmHg)\n\n139 ± 18\n\n132 ± 20\n\n128 ± 15\nb\n\n136.8 ± 17.2\n\n138.5 ± 18.5\n\n139 ± 20.8\n\n0.21\n\nDBP (mmHg)\n\n80 ± 12\n\n73 ± 10\n\n74 ± 9\nc\n\n71.6 ± 11.4\n\n71.6 ± 10\n\n72.9 ± 12.3\n\n0.08\n\nSerum creatinine (mg/dL)\n\n6.4 ± 1.9\n\n7.0 ± 2.5\n\n7.8 ± 3.0\nb\n\n5.9 ± 1.8\n\n7.8 ± 2.2\n\n8.2 ± 2.4\nb\n\n0.05\n\nB.U.N. (mg/dL)\n\n68 ± 18\n\n68 ± 16\n\n70 ± 17\n\n84.4 ± 18.5\n\n77 ± 15.5\n\n77 ± 22.9\n\n0.003\n\nGFR (mL/min)\n\n7.8 ± 1.9\n\n6.7 ± 2.3\n\n6.3 ± 2.1\nb\n\n9.2 ± 4.2\n\n-\n\n-\n\n-\n\neGFR (mL/min)\n\n9.4 ± 2.9\n\n8.4 ± 3.5\na\n\n8.0 ± 3.4\nb\n\n10.3 ± 3.9\n\n-\n\n-\n\n-\n\nTotal protein (g/dL)\n\n6.7 ± 0.5\n\n6.7 ± 0.5\n\n6.8 ± 0.4\n\n6.35 ± 0.52\n\n6.4 ± 0.5\n\n6.5 ± 0.43\n\n0.004\n\nAlbumin (g/dL)\n\n3.8 ± 0.4\n\n3.9 ± 0.4\n\n4.1 ± 0.4\nb\n\n3.6 ± 0.40\n\n3.6 ± 0.37\n\n3.7 ± 0.48\n\n0.01\n\nTransferrine (mg/dL)\n\n219 ± 53\n\n203 ± 72\n\n222 ± 53\n\n264 ± 94\n\n249 ± 43\n\n251 ± 61\n\n0.001\n\nC3 (mg/dL)\n\n90 ± 20\n\n91 ± 23\n\n94 ± 23\n\n99.6 ± 39.6\n\n96 ± 35.7\n\n95.9 ± 32\n\n0.48\n\nC4 (mg/dL)\n\n24 ± 7\n\n24 ± 8\n\n26 ± 7\n\n33 ± 28\n\n29 ± 18\n\n29 ± 30\n\n0.26\n\nTotal IgG (mg/dL)\n\n1599 ± 497\n\n1590 ± 402\n\n1543 ± 373\n\n1440 ± 461\n\n1482 ± 487\n\n1409 ± 413\n\n0.21\n\nLynphocytes/mm\n3\n\n1539 ± 651\n\n1390 ± 831\n\n1581 ± 537\n\n1452 ± 811\n\n1458 ± 622\n\n1445 ± 580\n\n0.38\nLegend: changes versus baseline: ap < 0.05; bp < 0.01; c< 0.03; P: ANOVA significance for treatment by time interaction.Figure 1\nChanges in urine volume (a), serum albumin (b), β2-microglobulin (c) and serum phosphorus (d) in CDDP Group (dotted Line) and THD Group (full line), at baseline, 6 and 12 months of follow-up.\n\n\nOutcome of nutritional and functional parameters in both groups in 12 months\n\nLegend: changes versus baseline: ap < 0.05; bp < 0.01; c< 0.03; P: ANOVA significance for treatment by time interaction.\n\nChanges in urine volume (a), serum albumin (b), β2-microglobulin (c) and serum phosphorus (d) in CDDP Group (dotted Line) and THD Group (full line), at baseline, 6 and 12 months of follow-up.\n\nNo difference in arterial blood pressure values emerged between the two groups.\nThe main nutritional indicators, i.e. total protein, albumin and transferrin improved significantly in the CDDP group (Figure 1b, Table 2). Estimated dietary protein intake remained stable in the CDDP groups throughout the study (from 0.58 ± 0.2 to 0.57 ± 0.11 g/kg/day). This was also the case for THD patients (from 1.03 ± 0.1 to 1.04 ± 0.3 g/kg/day). No significative changes in body weight or Body Mass Index (BMI) were recorded in the two groups (Table 2).\nA significant reduction in the ESAs requirement was observed in the CDDP group along with markedly lower ERI values (Table 3).Table 3\nBiochemistry parameters in CDDP and THD groups at 6 and 12 months\nCDDP groupTHD groupBaseline (n = 38)6 months (n = 38)12 months (n = 34)Baseline (n = 30)6 months (n = 30)12 months (n = 29)P\nTriglycerides (mg/dL)\n109 ± 36134 ± 92126 ± 50131 ± 72152 ± 67139 ± 52\n0.03\n\nTotal Cholesterol (mg/dL)\n164 ± 36168 ± 36164 ± 35165 ± 41168.5 ± 34170 ± 40\n0.51\n\nHDL Cholesterol (mg/dL)\n44 ± 1447 ± 1348 ± 2044 ± 1245 ± 1344 ± 12\n0.58\n\nLDL Cholesterol (mg/dL)\n101 ± 2993 ± 3493 ± 3195 ± 3293 ± 2997.8 ± 34\n0.95\n\nCholinesterases (UI/mL)\n5474 ± 13966073 ± 1474586 ± 16464968 ± 14885511 ± 16044673 ± 1450b\n\n0.27\n\nUric acid (mg/dL)\n7.3 ± 2.37.5 ± 2.47.1 ± 1.96.4 ± 1.8b\n7.3 ± 1.27.2 ± 1.3b\n\n0.60\n\nSodium (mmol/L)\n139 ± 2.6137 ± 3.6137 ± 4.0138 ± 2.7138 ± 2.3134 ± 3.3\n0.58\n\nPotassium (mmol/L)\n4.4 ± 0.64.3 ± 0.64.3 ± 0.74.3 ± 0,674.7 ± 0.74.8 ± 0.9b\n\n0.07\n\nCalcium (mg/dL)\n9.2 ± 0.59.0 ± 0.59.1 ± 0.69.1 ± 0.69.1 ± 0.79.0 ± 0.5\n0.51\n\nPhosphatemia (mg/dL)\n4.4 ± 0.94.6 ± 0.84.6 ± 0.85.1 ± 1.55.6 ± 1.15.2 ± 1.1\n0.001\n\nBicarbonate (mEq/L)\n22.0 ± 3.123.1 ± 3.323.2 ± 3.221.8 ± 4.221.9 ± 3.221.6 ± 2.7\n0.17\n\nβ2-microglobulin (mg/dL)\n14.2 ± 3.916.8 ± 5.716.0 ± 5.118.4 ± 11.631.0 ± 16.028.0 ± 11.4b\n\n0.007\n\nHb (g/dl)\n10.8 ± 0.111.5 ± 0.9511.5 ± 0.97a\n10.5 ± 1.411.3 ± 0.911.2 ± 0.95a\n\n0.31\nEPO (IU/kg/week)104 ± 10869 ± 5960 ± 74b\n184 ± 84172 ± 138204 ± 252\n0.002\nERI (IU/kg/week)/Hb10 ± 116 ± 55 ± 7b\n19 ± 1015 ± 1319 ± 23\n<0.001\n\nCRP (% patients) <5.0\n8981.57966.683.376.6\n<0.05\n\nCRP (mg/dL)\n3.3 ± 3.93.5 ± 3.33.2 ± 3.68.6 ± 9.74.9 ± 6.55.8 ± 5.8\n<0.01\n\niPTH (ρg/mL) <150 (%)\n39.552.65236.743.334.5\n0.09\n\niPTH (ρg/mL) 150–300 (%)\n29293013.32027.6\n0.26\n\niPTH (ρg/mL) >300 (%)\n31.5a\n18.418a\n5036.737.9\n0.02\nChanges versus baseline: ap < 0.01; bp < 0.03; P: ANOVA significance for treatment by time interaction.\n\nBiochemistry parameters in CDDP and THD groups at 6 and 12 months\n\nChanges versus baseline: ap < 0.01; bp < 0.03; P: ANOVA significance for treatment by time interaction.\nCalcium phosphate metabolism parameters were stable and better controlled in the CDDP (Table 3, Figure 1d) group despite a significantly lower use of non-calcium containing phosphate-binders and cinacalcet and among all the drugs, only allopurinol use was reduced during THD (Table 4).Table 4\nPharmacologic treatments in CDDP and THD groups\nCDDP GroupTHD groupP% of patientsBaseline12 monthsBaseline12 months\nACEi\n34335048.3\nn.s\n\nARBs\n24263021\nn.s\n\nCalcium channel blockers\n28.927.33017\nn.s\n\nCalcium carbonate\n596076.672.4\nn.s.\n\nSevelamer or Lanthanum carbonate\n012a\n041c\n\n<0.03\n\nParicalcitol\n012b\n017.2b\n\nn.s.\n\nCinacalcet\n06b\n024c\n\n<0.04\n\nCalcitriol\n454246.745\nn.s.\n\nKayexalate\n21212038\nn.s.\n\nAllopurinol\n48.65433.313.8a\n\n0.01\n\nStatins\n373326.724\nn.s\n\nPolyunsaturated fats\n1391017\nn.s.\nLegend: Changes versus baseline: ap<0.05; bp<0.01; c<0.03; P: ANOVA significance for treatment by time interaction.\n\nPharmacologic treatments in CDDP and THD groups\n\nLegend: Changes versus baseline: ap<0.05; bp<0.01; c<0.03; P: ANOVA significance for treatment by time interaction.\nCalcimimetics drugs are not delivered by Italian NHS during the pre-dialysis phase.\nSo their use began after commencing dialysis (even once-a-week): patients on THD showed a secondary hyperparathyroidism more severe than patients on CDDP , consequently the need of calcimimetics was higher in the former than in the latter.\nNo significant reduction in creatinine production was observed after 12 months of CDDP (16.8 ± 4.3 vs. 15.9 ± 3.9 mg/Kg/d).\n Hospitalization Throughout the 24 month follow-up period, 3 CDDP patients were admitted to hospital for a total of 11 days (3.7 ± 1.5 days/patient) due to atrial fibrillation, acute bronchitis and acute cholecystitis. Instead 24 hospital admission were recorded in 15 patients of THD groups for a total of 147 days (6.1 ± 6.3 days/patient). Causes for admission were: set up of new vascular access (7), artero-venous fistula stenosis angioplasty (1), infection of central vein catheter (2), acute pulmonary edema (1), surgery for biological prosthetic valve (1), myocardial infarction (1), valvular and coronaropathy angioplasty (2), congestive heart failure (1), atrial fibrillation (2), hyperpyrexia (1), hypertensive crisis (1), hypoglycemic coma (1), abscess in thigh hematoma (1) and obstructive jaundice secondary to gallstones (2).\nThroughout the 24 month follow-up period, 3 CDDP patients were admitted to hospital for a total of 11 days (3.7 ± 1.5 days/patient) due to atrial fibrillation, acute bronchitis and acute cholecystitis. Instead 24 hospital admission were recorded in 15 patients of THD groups for a total of 147 days (6.1 ± 6.3 days/patient). Causes for admission were: set up of new vascular access (7), artero-venous fistula stenosis angioplasty (1), infection of central vein catheter (2), acute pulmonary edema (1), surgery for biological prosthetic valve (1), myocardial infarction (1), valvular and coronaropathy angioplasty (2), congestive heart failure (1), atrial fibrillation (2), hyperpyrexia (1), hypertensive crisis (1), hypoglycemic coma (1), abscess in thigh hematoma (1) and obstructive jaundice secondary to gallstones (2).\n Survival At the 24-month follow-up, no significant difference was detected between the CDDP and THD group survival rates: 94.7% and 86.8% respectively (Figure 2).Figure 2\nCumulative survival in CDDP group (Dotted line) and in THD Group (Full line).\n\n\nCumulative survival in CDDP group (Dotted line) and in THD Group (Full line).\n\nDeath in 3 CDDP patients was caused by cardiac events: 1 during the 13th, 1 during the 16th and 1 during the 18th month of CDDP. Four deaths occurred in the THD group: 1 in the 9th, 2 in the 13th and 1 in the 22nd month following start of dialysis. Causes included myocardial infarction and acute heart failure, sepsis and stroke.\nAt the 24-month follow-up, no significant difference was detected between the CDDP and THD group survival rates: 94.7% and 86.8% respectively (Figure 2).Figure 2\nCumulative survival in CDDP group (Dotted line) and in THD Group (Full line).\n\n\nCumulative survival in CDDP group (Dotted line) and in THD Group (Full line).\n\nDeath in 3 CDDP patients was caused by cardiac events: 1 during the 13th, 1 during the 16th and 1 during the 18th month of CDDP. Four deaths occurred in the THD group: 1 in the 9th, 2 in the 13th and 1 in the 22nd month following start of dialysis. Causes included myocardial infarction and acute heart failure, sepsis and stroke.\n CDDP global outcome At 12 months 34 out of the 38 patients (89.5%) were on CDDP. At 24 months, 15 patients were still on CDDP (39.4%) since 1 patient received a kidney transplant, 3 patients had fatal myocardial infarction, whereas 17 patients have progressed to an incremental dialysis program (namely twice to thrice-weekly dialysis and free-protein diet) and 2 patients partially recovered their renal function. The main reasons for dropping out were loss of dietary adherence and/or reduction of residual renal function.\nWhen compared to THD group, CDDP patients registered a lower number of hospitalizations and a lower need for expensive drugs. These findings, coupled with the 66% reduction in number of dialysis sessions (52 versus 156 sessions in THD Group in a year) allows to estimated that the cost of a CDDP patient is more than 60% less than that of a THD patient.\nAt 12 months 34 out of the 38 patients (89.5%) were on CDDP. At 24 months, 15 patients were still on CDDP (39.4%) since 1 patient received a kidney transplant, 3 patients had fatal myocardial infarction, whereas 17 patients have progressed to an incremental dialysis program (namely twice to thrice-weekly dialysis and free-protein diet) and 2 patients partially recovered their renal function. The main reasons for dropping out were loss of dietary adherence and/or reduction of residual renal function.\nWhen compared to THD group, CDDP patients registered a lower number of hospitalizations and a lower need for expensive drugs. These findings, coupled with the 66% reduction in number of dialysis sessions (52 versus 156 sessions in THD Group in a year) allows to estimated that the cost of a CDDP patient is more than 60% less than that of a THD patient.",
"Throughout the 24 month follow-up period, 3 CDDP patients were admitted to hospital for a total of 11 days (3.7 ± 1.5 days/patient) due to atrial fibrillation, acute bronchitis and acute cholecystitis. Instead 24 hospital admission were recorded in 15 patients of THD groups for a total of 147 days (6.1 ± 6.3 days/patient). Causes for admission were: set up of new vascular access (7), artero-venous fistula stenosis angioplasty (1), infection of central vein catheter (2), acute pulmonary edema (1), surgery for biological prosthetic valve (1), myocardial infarction (1), valvular and coronaropathy angioplasty (2), congestive heart failure (1), atrial fibrillation (2), hyperpyrexia (1), hypertensive crisis (1), hypoglycemic coma (1), abscess in thigh hematoma (1) and obstructive jaundice secondary to gallstones (2).",
"At the 24-month follow-up, no significant difference was detected between the CDDP and THD group survival rates: 94.7% and 86.8% respectively (Figure 2).Figure 2\nCumulative survival in CDDP group (Dotted line) and in THD Group (Full line).\n\n\nCumulative survival in CDDP group (Dotted line) and in THD Group (Full line).\n\nDeath in 3 CDDP patients was caused by cardiac events: 1 during the 13th, 1 during the 16th and 1 during the 18th month of CDDP. Four deaths occurred in the THD group: 1 in the 9th, 2 in the 13th and 1 in the 22nd month following start of dialysis. Causes included myocardial infarction and acute heart failure, sepsis and stroke.",
"At 12 months 34 out of the 38 patients (89.5%) were on CDDP. At 24 months, 15 patients were still on CDDP (39.4%) since 1 patient received a kidney transplant, 3 patients had fatal myocardial infarction, whereas 17 patients have progressed to an incremental dialysis program (namely twice to thrice-weekly dialysis and free-protein diet) and 2 patients partially recovered their renal function. The main reasons for dropping out were loss of dietary adherence and/or reduction of residual renal function.\nWhen compared to THD group, CDDP patients registered a lower number of hospitalizations and a lower need for expensive drugs. These findings, coupled with the 66% reduction in number of dialysis sessions (52 versus 156 sessions in THD Group in a year) allows to estimated that the cost of a CDDP patient is more than 60% less than that of a THD patient.",
"This study shows that in selected ESRD patients, a CDDP is able to protect the RRF, to maintain urine volume output, to preserve a good nutritional status, to blunt the rapid β2 microglobulin increase, and to allow better control of anemia and calcium-phosphate abnormalities. CDDP is also associated with a lower rate of hospitalization , reduced need of EPO and drugs used for the CKD-MBD treatment, thus leading to a significant cost-saving. CCDP may be considered as the first step of an incremental approach to hemodialysis treatment of ESRD.\nPatients who entered the CDDP showed a better control of phosphatemia, BUN, PTH levels and higher albumin levels despite a lower RRF. It suggests that patients who had maintained consistent adherence to dietary treatment chose the CDDP whereas THD was chosen by patients with poor compliance to dietary prescriptions. In fact, dietary discipline is the first pre-requisite for a safe and successful CDDP.\nIn the CDDP group an effective urine volume output was maintained whereas it dramatically dropped in the THD patients. As a consequence, the interdialytic weight gain in CDDP patients was quite small, thus allowing low intradialytic ultrafiltration volumes. In turn, this may contribute to further preservation of the residual renal function and to limit cardiovascular damage. When compared to THD, CDDP was not associated with greater mortality risk (Figure 2) and hospitalization rate was much lower.\nOne of the main goals of the CKD patients care is to preserve RRF as long as possible because it is of very favorable prognostic value. The majority of patients succeeded in sticking to CDDP for at least one year ensuring a good nutritional status and maintaining GFR (-1.56 ml/min/year); in stage 4–5 CKD Levin et al. reported a GFR loss of 2.6 ml/min/year [17].\nThree major studies investigated once a-weekly dialysis coupled with low protein regimens in ESRD patients [5–8]. Mitch et al. reported that combined diet-dialysis treatment resulted in a decrease in urea production with a positive nitrogen balance during interdialytic interval [5]. Patients studied by Giovannetti et al. showed no uremic symptoms even after years of IDDP despite an extremely low RRF, thus indicating that the nutritional therapy is a major factor preventing the onset of uremic symptoms [6]. The studies carried out by Locatelli et al. concluded that IDDP may be very important from a psychological and economic point of view, but concerns arose about compliance and long-term nutritional and depurative adequacy [7]. After a 4-year period they found a reduction of creatinine generation rate, possibly suggesting a reduction in muscle mass.\nThis was not confirmed in our patients where the CDDP differs from the similar strategies proposed in the 1980’s and 1990’s [6, 7]; the treatment is distinguished by less severe residual renal function from among patients, less severe protein restriction, and by closer nutritional monitoring. In addition, hemodialysis sessions were performed with high quality water and highly bio-compatible membranes [18–21].\nRetrospective and prospective studies failed to demonstrate any benefits, in terms of survival, for an early onset of dialysis. In 2010 the first randomized, controlled trial of early versus late initiation of dialysis reported that early access to dialysis (eGFR > 7 ml/min/1.73 m2) provided no statistically significant benefits in terms of patient survival [22].\nThe role played by RRF in the clearance of medium-sized molecules such as β2-microglobulin is well-known (Figure 1c). Accordingly, patients with a significant RRF manifest lower levels of β2-microglobulin. On commencing CDDP, maintenance of effective urine output volume, a better hyperparathyroidism control, and a good nutritional status may have produced a positive effect on erythropoiesis leading to better anemia correction with reduction of ERI [23–25]. Our findings are in keeping with those of Di Iorio et al. who demonstrated that a poorer response to EPO was related to secondary hyperparathyroidism [23]. At baseline, in 50% of THD patients PTH was >300 pg/ml with significantly higher serum phosphate levels and a significantly lower use of calcimimetic drugs compared to CDDP Group (Tables 3 and 4). During the study, CDDP patients had no need for additional dialysis sessions. In turn, an easy volumes balance with quite low interdialytic weight gains, and prevention of high ultrafiltration rates and of intradialytic hypotension, contribute to the protection of RRF during CDDP. Moreover, Fouque et al. showed that dietary treatment produced a positive effect in delaying the need of dialysis [26].\nIt is noteworthy that the hospitalization rate in CDDP was much lower than in THD Group. In the latter, the causes of admission were mainly vascular access complications and infection. In addition the overall 2 year-survival rate of CDDP was similar to that of THD patients. In fact, data from literature show that a conservative approach may be not inferior to dialysis in elderly ESRD patients, in terms of survival and/or quality of life [27–29].\nLastly, the CDDP treatment resulted in an approximate 50% savings on the cost of drugs and dialysis resources, including a lower number of dialysis hours/nursing and medical staff ( 66% less compared to thrice-weekly hemodialysis) and comorbid incidence in terms of days of hospitalization, as well as of indirect costs such as transport of patients to and from the dialysis unit. When compared to THD, the CDDP group showed a lower hospitalization rate, lower need for expensive drugs, and by definition a 66% lowering of number of dialysis sessions and related costs. CDDP patients may have the additional cost of the protein-free products. As a whole, CDDP seems to be a safe and effective exit strategy for lowering the cost of ESRD population, even if it can occur for a limited period of time and in selected ESRD patients.\nAnother advantage of CDDP is an improvement in patient’s acceptance and adaptation to renal replacement therapy. The patient feels less sick and less machine-dependent, resulting in an increased copying capacity (or psychological adaptation). Patients moreover tended to adapt better to low protein diets thanks to a more varied diet, as well as to address concerns about “dialysis-day”, which was also the day they were allowed to eat a “normal” diet.\nThe study has not had the opportunity to randomize and this may be seen as a limitation, but randomization was not feasible because one could not force patients to choose one method rather than the other. Patients were followed in pre-dialysis care: a multidisciplinary clinic (doctors, nurses, dietitians and psychologists) was able to provide the necessary education and choice of treatment modalities. This study aimed to reproduce the real world clinical practice where patient’s adherence is a crucial issue for the success of any treatment, especially if dietary in nature, where patients play an active role and must be involved in the decision-making process. Patients who entered the CDDP showed a lower residual renal function but better control of phosphatemia, BUN, PTH levels and higher albumin levels. This finding suggests that CDDP is suitable for patients who still have a positive inclination towards dietary manipulations. Moreover, together with a preserved urine output volume, good compliance to dietary prescriptions is mandatory and a pre-requisite for the success and safety of CDDP. Now it could be necessary to begin considering a tailored dialytic treatment and to evaluate the greater power of RRF. The incremental choice is considered a good option in peritoneal patients since the 2000’s [30, 31] thanks to RRF and it again underestimates for twice-weekly HD patients [32, 33]. Energy-adequate low-protein regimens have a fundamental role in controlling and maintaining a good metabolic status and in reducing GFR loss [26, 34].",
"This study shows that in selected ESRD patients, a CDDP is able to protect the RRF, to maintain urine volume output, to preserve a good nutritional status , to blunt the rapid β2 microglobulin increase, and to allow better control of anemia and calcium-phosphate abnormalities. CDDP is also associated with a lower rate of hospitalization and reduced need of EPO and drugs used for the CKD-MBD treatment, thus leading to a significant cost-saving. As a whole, our findings suggest that CDDP could be a beneficial choice for selected collaborative patients who would otherwise be referred to thrice-weekly hemodialysis. CCDP should be considered as the first step of an incremental approach to hemodialysis treatment in motivated and selected ESRD patients, or anywhere dialysis facilities and resources are lacking."
] |
[
null,
"methods",
null,
"results",
null,
null,
null,
"discussion",
"conclusions"
] |
[
"Nutrition",
"Low protein diet",
"Hemodialysis",
"CKD",
"ESRD",
"Incremental dialysis"
] |
Background:
The Epidemiology of chronic kidney disease (CKD) is on an upward trend worldwide. Since the 1990s incidence has risen by approximately 40% per decade, with prevalence rates in the general adult population up to 13% in the USA and ranging 10.2-11.6% in Europe [1–4]. These figures indicate a CKD epidemic leading to an increased number of patients requiring dialysis and a heavy burden on public health resources.
Nutritional and pharmacological therapy constitute the basis for the prevention and treatment of signs and symptoms of CKD, and they serve to delay the commencement of dialysis. The appropriate time to start dialysis is still a matter of debate. In particular, dialysis is not superior that conservative treatment especially in the comorbid-elderly population. It is known that a standard thrice--weekly hemodialysis schedule invariably leads to rapid loss of the residual renal function, to psychological and socials drawbacks, and to high costs. Real incremental dialysis programs are implemented in peritoneal dialysis but not in the hemodialysis setting where only a twice -weekly schedule may be proposed prior to maintenance hemodialysis.
During the 1980’s and 1990’s, an Integrated Diet Dialysis Program (IDDP) was proposed as a therapeutic option for selected patients with markedly reduced residual renal function and it consisted of weekly hemodialysis session (HD) combined with nutritional therapy, namely a very low-protein diet supplemented with ketoacids [5–8]. Malnutrition risk and low compliance to the severe dietary restrictions represented major concerns that prevented a widespread application of this schedule. In the present study we propose a Combined Diet Dialysis Program (CDDP) which includes a less severe low protein (0.6 g/Kg/d) diet combined with once-weekly hemodialysis. The goal is to prolong a conservative approach and thus reduce the need for hemodialysis, thereby limiting the risk of malnutrition and of poor dietary adherence. This study aimed to assess the safety, benefits and drawbacks of the CDDP approach including nutritional status, residual renal function, morbidity, mortality and costs.
Methods:
This is a multicenter, non-randomized, prospective controlled study, including stage V CKD patients followed in a pre-dialysis clinic who were not suitable for a peritoneal dialysis program.
Patients with GFR 5 to 10 ml/min who were approaching hemodialysis treatment and who had vascular access were included in the study. Global criteria of starting dialysis and then for entering the study were fluid retention, inadequate control of BUN or severe secondary hyperparathyroidism or hyperphosphatemia, reduced compliance to dietary treatment.
Patients with pericarditis, congestive heart failure, severe fluid retention, overt protein-energy wasting, hyperkalemia or severe metabolic acidosis with acute reduction of residual renal function or concurrent diseases were excluded, as well patients with unreliable discipline with regards to dietary restrictions were treated at once with maintenance hemodialysis and then excluded from the study. During the pre-dialysis phase, patients were followed in a tertiary care CKD clinic every two-three months (stage 4) or on a monthly basis (stage 5). Compliance to dietary and pharmacological treatment was acceptable, as assessed by urinary urea excretion and by dietary recall and interview.
Sixty-eight incident patients were recruited and all the patients were provided a functioning native artero-venous fistula or graft.
Underlying kidney diseases included mainly hypertension/vascular nephropathy, ADPKD. The patients were offered the choice to commence a weekly hemodialysis schedule plus low-protein diet on the non-dialysis day (namely, CDDP), or to begin a standard thrice- weekly hemodialysis (THD) schedule and free-choice diet. All the patients were informed and directly involved in the decision making process. Randomization was not applied since any dietary regimen requires adherence and motivation.
According to their own choice, 38 patients entered in the CDDP Group, whereas the remaining 30 patients formed the THD control group. The clinical and biochemical features of the two groups at baseline are reported in Table 1. At baseline, the prevalence of cardiovascular disease history was similar in CDDP (28.9%) and in THD (30%) group. Six type 2 diabetics were included in CDDP group and 13 in THD group.Table 1
Baseline characteristics of the studied groups
CDDP group (n = 38)THD group (n = 30)pMale/females25/1319/11Age, years64.5 ± 13.265.2 ± 11
0.82
Body weight (kg)65.5 ± 15.166.2 ± 11.9
0.73
BMI (kg/m2)23.7 ± 4.025.6 ± 4.13
0.03
Urine volume output (mL/24 h)1983 ± 6511472.6 ± 433
<0.001
GFR (mL/min × 1.73 mq b.s)7.8 ± 1.99.2 ± 4.2
<0.01
EPO (IU/kg/week)104 ± 108184 ± 84
<0.001
CRP <5 mg/dl, %8966.6
<0.01
iPTH, >300 ρg/mL, %31.550
<0.01
Charlson comorbidity index score5.5 ± 2.53.8 ± 2.5
0.004
Charlson comorbidity index score >4, %62.533.3
<0.01
Baseline characteristics of the studied groups
All the patients were studied at baseline, and after 6 and 12 months. The survival rate and hospitalization rate were followed up to 24 months.
Blood samples were collected from the arterial line before the start of the first dialysis of the week; 24h urines were collected during the day before the HD session. GFR was measured as the average of creatinine clearance and urea clearance, and expressed as ml/min*1.73 m2 body surface area [9]; eGFR was also estimated by MDRD formula [10]. Protein catabolic rate (PCR) was used as an indicator of dietary protein intake and calculated by the urea appearance method according to Maroni’s formula [11]. Maroni’s formula was used throughout the course of the CDDP and only at baseline time in patients in the THD Group. Within the THD Group, for patients with the significant loss of renal function and urine output volume we included the method of urea kinetics model for estimation of nPCR. Dialytic adequacy was expressed using eKt/V according to Daugirdas formula et al [12]. Body mass index was calculated by the body weight at the end of the dialysis session. The bioelectrical parameters, were measured with a single-frequency impedance analyzer (BIA 101 – Akern - Florence), 30’ after the end of the hemodialysis session.
Erythropoietin Resistance Index (ERI) was calculated as weekly EPO dosage (units)/body weight (Kg)/Hb (g/dl) [13].
Charlson’s comorbidity index (CCI) score, was calculated to evaluate comorbidity conditions [14].
Creatinine production, as a surrogate of muscle mass, was calculated by the sum of 24h urine creatinine excretion and metabolized creatinine. Metabolized creatinine was calculated from the product of serum creatinine and extrarenal Creatinine clearance (estimated as 0.038 L/kg BW/d) [7].
All the patients on CDDP underwent nutritional counseling by a renal dietician and were prescribed a low protein (0,6 g/kg b.w./d), low phosphorus (600–700 mg/d), low sodium diet with an energy intake of 30–35 kcal/kg b.w./d. Proteins were mostly from animal sources (0.4 g/Kg/b.w.), to cover the requirement of essential amino acids; no dairy or processed foods are included to the aim of limiting the sodium and phosphorus intake; [15] protein-free products are included to supply energy with negligible load of phosphorus, sodium, potassium and nitrogen [16]. The 60–62% of energy intake derived from carbohydrates (mostly represented by protein-free products), 30–32% from lipids and 8-10% from proteins. As average, the daily dietary plan consisted of 5–7 servings of protein free products, namely 2–3 serving for breakfast and snacks and 2 servings for both lunch and dinner; the patients were usually advised that they can add other servings, when required to satisfy the energy requirement. The servings of animal protein sources (meat, fish, egg white) were calculated for each patient in order to guarantee a daily total amount of 0.4 g protein per kg b.w. Two servings of vegetables and two of fruits were provided with specific suggestions for patients with high potassium levels as well as boiling was suggested to reduce to mineral content (potassium, sodium, calcium, and phosphorus) of food. Finally, 5–6 servings of fats (olive oil as first choice) were suggested.
This diet was prescribed for 6 days a week, while no dietary restrictions occurred on the dialysis day.
Patients in the THD control group followed an unrestricted protein diet, but they were given dietary counseling focused on preventing excess sodium, potassium, phosphorus and fluid intakes.
All the patients were prescribed 4-hour hemodialysis sessions with highly biocompatible synthetic membranes.
The study protocol was approved by the Cagliari Hospital Ethics Committee, and all the patients gave their written informed consent for participation in the study.
Statistical analysis Descriptive statistics is given as mean ± standard deviation. Normality tests were performed on all continuous variables measured throughout the study. The log-rank test was used for hypothesis testing. Time repeated measurements were analyzed using linear mixed models including treatment, time, and treatment by time interaction for all measured variables using ANOVA. Inter-group drug use was compared by means of the binomial test with Bonferroni’s correction for multiple comparisons.
Differences were considered as statistically significant when p <0.05.
Descriptive statistics is given as mean ± standard deviation. Normality tests were performed on all continuous variables measured throughout the study. The log-rank test was used for hypothesis testing. Time repeated measurements were analyzed using linear mixed models including treatment, time, and treatment by time interaction for all measured variables using ANOVA. Inter-group drug use was compared by means of the binomial test with Bonferroni’s correction for multiple comparisons.
Differences were considered as statistically significant when p <0.05.
Statistical analysis:
Descriptive statistics is given as mean ± standard deviation. Normality tests were performed on all continuous variables measured throughout the study. The log-rank test was used for hypothesis testing. Time repeated measurements were analyzed using linear mixed models including treatment, time, and treatment by time interaction for all measured variables using ANOVA. Inter-group drug use was compared by means of the binomial test with Bonferroni’s correction for multiple comparisons.
Differences were considered as statistically significant when p <0.05.
Results:
Patients who entered the CDDP showed several differences with respect to patients who commenced THD (Table 1). Despite a lower residual renal function (RRF), CDDP patients showed lower circulating levels of phosphate, BUN, PTH and higher albumin levels with respect to THD patients. 34 out of the 38 patients were still on CDDP at 12 months: 2 patients were shifted to a thrice- weekly dialysis schedule at 8 and 10 months, while 2 patients partially recovered their renal function and remained clinically stable with the low-protein diet and no need for dialysis.
During the study period, GFR was preserved in CDDP patients in comparison to THD group (Table 2). GFR loss progression was very low in the former (-0.13 ml/min/month) while it was faster in the latter (-1.53 ml/min/month). Similarly, in the CDDP group an effective urine volume output was maintained whereas it dramatically dropped in the THD patients (Figure 1a). As a consequence, the interdialytic weight gain in CDDP patients was limited to (800 ± 300 g) per week. The dosage of frusemide increased from 150 ± 154 to 273 ± 201 mg/day in the CDDP group, and from 332 ± 171 to 409 ± 205 mg/day (p =0.003) in the THD group. The increase of β2-microglobulin circulating levels was much lower in CDDP than in THD patients (+11.2% vs +34%, respectively) (Figure 1c).Table 2
Outcome of nutritional and functional parameters in both groups in 12 months
CDDP groupTHD groupBaseline (n = 38)6 months (n = 38)12 months (n = 34)Baseline (n = 30)6 months (n = 30)12 months (n = 29)
P
Dry weight (kg)
65 ± 15.0
63.9 ± 14.4
63.4 ± 14.7
66.2 ± 11.9
64.9 ± 11.5
65.5 ± 12.3
0.07
BMI (kg/m
2
)
23.7 ± 4.0
23.4 ± 3.7
23.0 ± 3.9
25.6 ± 4.13
25.1 ± 4.07
25.4 ± 4.2
0.07
Total body water (L)
44.4 ± 10.5
41 ± 9.6
43 ± 11
35.4 ± 6
33.5 ± 5.8
a
33.8 ± 5.9
a
0.02
Extracellular water (L)
20 ± 4.5
18.6 ± 4.3
19.7 ± 4
17.5 ± 3.2
16 ± 2.8
b
15.7 ± 2.4
b
0.04
Fat mass (kg)
11 ± 12.3
13 ± 11.8
11.4 ± 11
20 ± 9
21.4 ± 8.5
22.6 ± 9.4
0.03
Body cell mass (kg)
31 ± 9.6
28 ± 8.9
29.7 ± 10.3
22.5 ± 6.2
22.7 ± 5
22.7 ± 5.8
0.02
Phase angle, (°)
6.2 ± 1.3
6.2 ± 1.3
6.1 ± 1.4
5.0 ± 1.2
6 ± 0.9
5.8 ± 1.2
0.06
SBP (mmHg)
139 ± 18
132 ± 20
128 ± 15
b
136.8 ± 17.2
138.5 ± 18.5
139 ± 20.8
0.21
DBP (mmHg)
80 ± 12
73 ± 10
74 ± 9
c
71.6 ± 11.4
71.6 ± 10
72.9 ± 12.3
0.08
Serum creatinine (mg/dL)
6.4 ± 1.9
7.0 ± 2.5
7.8 ± 3.0
b
5.9 ± 1.8
7.8 ± 2.2
8.2 ± 2.4
b
0.05
B.U.N. (mg/dL)
68 ± 18
68 ± 16
70 ± 17
84.4 ± 18.5
77 ± 15.5
77 ± 22.9
0.003
GFR (mL/min)
7.8 ± 1.9
6.7 ± 2.3
6.3 ± 2.1
b
9.2 ± 4.2
-
-
-
eGFR (mL/min)
9.4 ± 2.9
8.4 ± 3.5
a
8.0 ± 3.4
b
10.3 ± 3.9
-
-
-
Total protein (g/dL)
6.7 ± 0.5
6.7 ± 0.5
6.8 ± 0.4
6.35 ± 0.52
6.4 ± 0.5
6.5 ± 0.43
0.004
Albumin (g/dL)
3.8 ± 0.4
3.9 ± 0.4
4.1 ± 0.4
b
3.6 ± 0.40
3.6 ± 0.37
3.7 ± 0.48
0.01
Transferrine (mg/dL)
219 ± 53
203 ± 72
222 ± 53
264 ± 94
249 ± 43
251 ± 61
0.001
C3 (mg/dL)
90 ± 20
91 ± 23
94 ± 23
99.6 ± 39.6
96 ± 35.7
95.9 ± 32
0.48
C4 (mg/dL)
24 ± 7
24 ± 8
26 ± 7
33 ± 28
29 ± 18
29 ± 30
0.26
Total IgG (mg/dL)
1599 ± 497
1590 ± 402
1543 ± 373
1440 ± 461
1482 ± 487
1409 ± 413
0.21
Lynphocytes/mm
3
1539 ± 651
1390 ± 831
1581 ± 537
1452 ± 811
1458 ± 622
1445 ± 580
0.38
Legend: changes versus baseline: ap < 0.05; bp < 0.01; c< 0.03; P: ANOVA significance for treatment by time interaction.Figure 1
Changes in urine volume (a), serum albumin (b), β2-microglobulin (c) and serum phosphorus (d) in CDDP Group (dotted Line) and THD Group (full line), at baseline, 6 and 12 months of follow-up.
Outcome of nutritional and functional parameters in both groups in 12 months
Legend: changes versus baseline: ap < 0.05; bp < 0.01; c< 0.03; P: ANOVA significance for treatment by time interaction.
Changes in urine volume (a), serum albumin (b), β2-microglobulin (c) and serum phosphorus (d) in CDDP Group (dotted Line) and THD Group (full line), at baseline, 6 and 12 months of follow-up.
No difference in arterial blood pressure values emerged between the two groups.
The main nutritional indicators, i.e. total protein, albumin and transferrin improved significantly in the CDDP group (Figure 1b, Table 2). Estimated dietary protein intake remained stable in the CDDP groups throughout the study (from 0.58 ± 0.2 to 0.57 ± 0.11 g/kg/day). This was also the case for THD patients (from 1.03 ± 0.1 to 1.04 ± 0.3 g/kg/day). No significative changes in body weight or Body Mass Index (BMI) were recorded in the two groups (Table 2).
A significant reduction in the ESAs requirement was observed in the CDDP group along with markedly lower ERI values (Table 3).Table 3
Biochemistry parameters in CDDP and THD groups at 6 and 12 months
CDDP groupTHD groupBaseline (n = 38)6 months (n = 38)12 months (n = 34)Baseline (n = 30)6 months (n = 30)12 months (n = 29)P
Triglycerides (mg/dL)
109 ± 36134 ± 92126 ± 50131 ± 72152 ± 67139 ± 52
0.03
Total Cholesterol (mg/dL)
164 ± 36168 ± 36164 ± 35165 ± 41168.5 ± 34170 ± 40
0.51
HDL Cholesterol (mg/dL)
44 ± 1447 ± 1348 ± 2044 ± 1245 ± 1344 ± 12
0.58
LDL Cholesterol (mg/dL)
101 ± 2993 ± 3493 ± 3195 ± 3293 ± 2997.8 ± 34
0.95
Cholinesterases (UI/mL)
5474 ± 13966073 ± 1474586 ± 16464968 ± 14885511 ± 16044673 ± 1450b
0.27
Uric acid (mg/dL)
7.3 ± 2.37.5 ± 2.47.1 ± 1.96.4 ± 1.8b
7.3 ± 1.27.2 ± 1.3b
0.60
Sodium (mmol/L)
139 ± 2.6137 ± 3.6137 ± 4.0138 ± 2.7138 ± 2.3134 ± 3.3
0.58
Potassium (mmol/L)
4.4 ± 0.64.3 ± 0.64.3 ± 0.74.3 ± 0,674.7 ± 0.74.8 ± 0.9b
0.07
Calcium (mg/dL)
9.2 ± 0.59.0 ± 0.59.1 ± 0.69.1 ± 0.69.1 ± 0.79.0 ± 0.5
0.51
Phosphatemia (mg/dL)
4.4 ± 0.94.6 ± 0.84.6 ± 0.85.1 ± 1.55.6 ± 1.15.2 ± 1.1
0.001
Bicarbonate (mEq/L)
22.0 ± 3.123.1 ± 3.323.2 ± 3.221.8 ± 4.221.9 ± 3.221.6 ± 2.7
0.17
β2-microglobulin (mg/dL)
14.2 ± 3.916.8 ± 5.716.0 ± 5.118.4 ± 11.631.0 ± 16.028.0 ± 11.4b
0.007
Hb (g/dl)
10.8 ± 0.111.5 ± 0.9511.5 ± 0.97a
10.5 ± 1.411.3 ± 0.911.2 ± 0.95a
0.31
EPO (IU/kg/week)104 ± 10869 ± 5960 ± 74b
184 ± 84172 ± 138204 ± 252
0.002
ERI (IU/kg/week)/Hb10 ± 116 ± 55 ± 7b
19 ± 1015 ± 1319 ± 23
<0.001
CRP (% patients) <5.0
8981.57966.683.376.6
<0.05
CRP (mg/dL)
3.3 ± 3.93.5 ± 3.33.2 ± 3.68.6 ± 9.74.9 ± 6.55.8 ± 5.8
<0.01
iPTH (ρg/mL) <150 (%)
39.552.65236.743.334.5
0.09
iPTH (ρg/mL) 150–300 (%)
29293013.32027.6
0.26
iPTH (ρg/mL) >300 (%)
31.5a
18.418a
5036.737.9
0.02
Changes versus baseline: ap < 0.01; bp < 0.03; P: ANOVA significance for treatment by time interaction.
Biochemistry parameters in CDDP and THD groups at 6 and 12 months
Changes versus baseline: ap < 0.01; bp < 0.03; P: ANOVA significance for treatment by time interaction.
Calcium phosphate metabolism parameters were stable and better controlled in the CDDP (Table 3, Figure 1d) group despite a significantly lower use of non-calcium containing phosphate-binders and cinacalcet and among all the drugs, only allopurinol use was reduced during THD (Table 4).Table 4
Pharmacologic treatments in CDDP and THD groups
CDDP GroupTHD groupP% of patientsBaseline12 monthsBaseline12 months
ACEi
34335048.3
n.s
ARBs
24263021
n.s
Calcium channel blockers
28.927.33017
n.s
Calcium carbonate
596076.672.4
n.s.
Sevelamer or Lanthanum carbonate
012a
041c
<0.03
Paricalcitol
012b
017.2b
n.s.
Cinacalcet
06b
024c
<0.04
Calcitriol
454246.745
n.s.
Kayexalate
21212038
n.s.
Allopurinol
48.65433.313.8a
0.01
Statins
373326.724
n.s
Polyunsaturated fats
1391017
n.s.
Legend: Changes versus baseline: ap<0.05; bp<0.01; c<0.03; P: ANOVA significance for treatment by time interaction.
Pharmacologic treatments in CDDP and THD groups
Legend: Changes versus baseline: ap<0.05; bp<0.01; c<0.03; P: ANOVA significance for treatment by time interaction.
Calcimimetics drugs are not delivered by Italian NHS during the pre-dialysis phase.
So their use began after commencing dialysis (even once-a-week): patients on THD showed a secondary hyperparathyroidism more severe than patients on CDDP , consequently the need of calcimimetics was higher in the former than in the latter.
No significant reduction in creatinine production was observed after 12 months of CDDP (16.8 ± 4.3 vs. 15.9 ± 3.9 mg/Kg/d).
Hospitalization Throughout the 24 month follow-up period, 3 CDDP patients were admitted to hospital for a total of 11 days (3.7 ± 1.5 days/patient) due to atrial fibrillation, acute bronchitis and acute cholecystitis. Instead 24 hospital admission were recorded in 15 patients of THD groups for a total of 147 days (6.1 ± 6.3 days/patient). Causes for admission were: set up of new vascular access (7), artero-venous fistula stenosis angioplasty (1), infection of central vein catheter (2), acute pulmonary edema (1), surgery for biological prosthetic valve (1), myocardial infarction (1), valvular and coronaropathy angioplasty (2), congestive heart failure (1), atrial fibrillation (2), hyperpyrexia (1), hypertensive crisis (1), hypoglycemic coma (1), abscess in thigh hematoma (1) and obstructive jaundice secondary to gallstones (2).
Throughout the 24 month follow-up period, 3 CDDP patients were admitted to hospital for a total of 11 days (3.7 ± 1.5 days/patient) due to atrial fibrillation, acute bronchitis and acute cholecystitis. Instead 24 hospital admission were recorded in 15 patients of THD groups for a total of 147 days (6.1 ± 6.3 days/patient). Causes for admission were: set up of new vascular access (7), artero-venous fistula stenosis angioplasty (1), infection of central vein catheter (2), acute pulmonary edema (1), surgery for biological prosthetic valve (1), myocardial infarction (1), valvular and coronaropathy angioplasty (2), congestive heart failure (1), atrial fibrillation (2), hyperpyrexia (1), hypertensive crisis (1), hypoglycemic coma (1), abscess in thigh hematoma (1) and obstructive jaundice secondary to gallstones (2).
Survival At the 24-month follow-up, no significant difference was detected between the CDDP and THD group survival rates: 94.7% and 86.8% respectively (Figure 2).Figure 2
Cumulative survival in CDDP group (Dotted line) and in THD Group (Full line).
Cumulative survival in CDDP group (Dotted line) and in THD Group (Full line).
Death in 3 CDDP patients was caused by cardiac events: 1 during the 13th, 1 during the 16th and 1 during the 18th month of CDDP. Four deaths occurred in the THD group: 1 in the 9th, 2 in the 13th and 1 in the 22nd month following start of dialysis. Causes included myocardial infarction and acute heart failure, sepsis and stroke.
At the 24-month follow-up, no significant difference was detected between the CDDP and THD group survival rates: 94.7% and 86.8% respectively (Figure 2).Figure 2
Cumulative survival in CDDP group (Dotted line) and in THD Group (Full line).
Cumulative survival in CDDP group (Dotted line) and in THD Group (Full line).
Death in 3 CDDP patients was caused by cardiac events: 1 during the 13th, 1 during the 16th and 1 during the 18th month of CDDP. Four deaths occurred in the THD group: 1 in the 9th, 2 in the 13th and 1 in the 22nd month following start of dialysis. Causes included myocardial infarction and acute heart failure, sepsis and stroke.
CDDP global outcome At 12 months 34 out of the 38 patients (89.5%) were on CDDP. At 24 months, 15 patients were still on CDDP (39.4%) since 1 patient received a kidney transplant, 3 patients had fatal myocardial infarction, whereas 17 patients have progressed to an incremental dialysis program (namely twice to thrice-weekly dialysis and free-protein diet) and 2 patients partially recovered their renal function. The main reasons for dropping out were loss of dietary adherence and/or reduction of residual renal function.
When compared to THD group, CDDP patients registered a lower number of hospitalizations and a lower need for expensive drugs. These findings, coupled with the 66% reduction in number of dialysis sessions (52 versus 156 sessions in THD Group in a year) allows to estimated that the cost of a CDDP patient is more than 60% less than that of a THD patient.
At 12 months 34 out of the 38 patients (89.5%) were on CDDP. At 24 months, 15 patients were still on CDDP (39.4%) since 1 patient received a kidney transplant, 3 patients had fatal myocardial infarction, whereas 17 patients have progressed to an incremental dialysis program (namely twice to thrice-weekly dialysis and free-protein diet) and 2 patients partially recovered their renal function. The main reasons for dropping out were loss of dietary adherence and/or reduction of residual renal function.
When compared to THD group, CDDP patients registered a lower number of hospitalizations and a lower need for expensive drugs. These findings, coupled with the 66% reduction in number of dialysis sessions (52 versus 156 sessions in THD Group in a year) allows to estimated that the cost of a CDDP patient is more than 60% less than that of a THD patient.
Hospitalization:
Throughout the 24 month follow-up period, 3 CDDP patients were admitted to hospital for a total of 11 days (3.7 ± 1.5 days/patient) due to atrial fibrillation, acute bronchitis and acute cholecystitis. Instead 24 hospital admission were recorded in 15 patients of THD groups for a total of 147 days (6.1 ± 6.3 days/patient). Causes for admission were: set up of new vascular access (7), artero-venous fistula stenosis angioplasty (1), infection of central vein catheter (2), acute pulmonary edema (1), surgery for biological prosthetic valve (1), myocardial infarction (1), valvular and coronaropathy angioplasty (2), congestive heart failure (1), atrial fibrillation (2), hyperpyrexia (1), hypertensive crisis (1), hypoglycemic coma (1), abscess in thigh hematoma (1) and obstructive jaundice secondary to gallstones (2).
Survival:
At the 24-month follow-up, no significant difference was detected between the CDDP and THD group survival rates: 94.7% and 86.8% respectively (Figure 2).Figure 2
Cumulative survival in CDDP group (Dotted line) and in THD Group (Full line).
Cumulative survival in CDDP group (Dotted line) and in THD Group (Full line).
Death in 3 CDDP patients was caused by cardiac events: 1 during the 13th, 1 during the 16th and 1 during the 18th month of CDDP. Four deaths occurred in the THD group: 1 in the 9th, 2 in the 13th and 1 in the 22nd month following start of dialysis. Causes included myocardial infarction and acute heart failure, sepsis and stroke.
CDDP global outcome:
At 12 months 34 out of the 38 patients (89.5%) were on CDDP. At 24 months, 15 patients were still on CDDP (39.4%) since 1 patient received a kidney transplant, 3 patients had fatal myocardial infarction, whereas 17 patients have progressed to an incremental dialysis program (namely twice to thrice-weekly dialysis and free-protein diet) and 2 patients partially recovered their renal function. The main reasons for dropping out were loss of dietary adherence and/or reduction of residual renal function.
When compared to THD group, CDDP patients registered a lower number of hospitalizations and a lower need for expensive drugs. These findings, coupled with the 66% reduction in number of dialysis sessions (52 versus 156 sessions in THD Group in a year) allows to estimated that the cost of a CDDP patient is more than 60% less than that of a THD patient.
Discussion:
This study shows that in selected ESRD patients, a CDDP is able to protect the RRF, to maintain urine volume output, to preserve a good nutritional status, to blunt the rapid β2 microglobulin increase, and to allow better control of anemia and calcium-phosphate abnormalities. CDDP is also associated with a lower rate of hospitalization , reduced need of EPO and drugs used for the CKD-MBD treatment, thus leading to a significant cost-saving. CCDP may be considered as the first step of an incremental approach to hemodialysis treatment of ESRD.
Patients who entered the CDDP showed a better control of phosphatemia, BUN, PTH levels and higher albumin levels despite a lower RRF. It suggests that patients who had maintained consistent adherence to dietary treatment chose the CDDP whereas THD was chosen by patients with poor compliance to dietary prescriptions. In fact, dietary discipline is the first pre-requisite for a safe and successful CDDP.
In the CDDP group an effective urine volume output was maintained whereas it dramatically dropped in the THD patients. As a consequence, the interdialytic weight gain in CDDP patients was quite small, thus allowing low intradialytic ultrafiltration volumes. In turn, this may contribute to further preservation of the residual renal function and to limit cardiovascular damage. When compared to THD, CDDP was not associated with greater mortality risk (Figure 2) and hospitalization rate was much lower.
One of the main goals of the CKD patients care is to preserve RRF as long as possible because it is of very favorable prognostic value. The majority of patients succeeded in sticking to CDDP for at least one year ensuring a good nutritional status and maintaining GFR (-1.56 ml/min/year); in stage 4–5 CKD Levin et al. reported a GFR loss of 2.6 ml/min/year [17].
Three major studies investigated once a-weekly dialysis coupled with low protein regimens in ESRD patients [5–8]. Mitch et al. reported that combined diet-dialysis treatment resulted in a decrease in urea production with a positive nitrogen balance during interdialytic interval [5]. Patients studied by Giovannetti et al. showed no uremic symptoms even after years of IDDP despite an extremely low RRF, thus indicating that the nutritional therapy is a major factor preventing the onset of uremic symptoms [6]. The studies carried out by Locatelli et al. concluded that IDDP may be very important from a psychological and economic point of view, but concerns arose about compliance and long-term nutritional and depurative adequacy [7]. After a 4-year period they found a reduction of creatinine generation rate, possibly suggesting a reduction in muscle mass.
This was not confirmed in our patients where the CDDP differs from the similar strategies proposed in the 1980’s and 1990’s [6, 7]; the treatment is distinguished by less severe residual renal function from among patients, less severe protein restriction, and by closer nutritional monitoring. In addition, hemodialysis sessions were performed with high quality water and highly bio-compatible membranes [18–21].
Retrospective and prospective studies failed to demonstrate any benefits, in terms of survival, for an early onset of dialysis. In 2010 the first randomized, controlled trial of early versus late initiation of dialysis reported that early access to dialysis (eGFR > 7 ml/min/1.73 m2) provided no statistically significant benefits in terms of patient survival [22].
The role played by RRF in the clearance of medium-sized molecules such as β2-microglobulin is well-known (Figure 1c). Accordingly, patients with a significant RRF manifest lower levels of β2-microglobulin. On commencing CDDP, maintenance of effective urine output volume, a better hyperparathyroidism control, and a good nutritional status may have produced a positive effect on erythropoiesis leading to better anemia correction with reduction of ERI [23–25]. Our findings are in keeping with those of Di Iorio et al. who demonstrated that a poorer response to EPO was related to secondary hyperparathyroidism [23]. At baseline, in 50% of THD patients PTH was >300 pg/ml with significantly higher serum phosphate levels and a significantly lower use of calcimimetic drugs compared to CDDP Group (Tables 3 and 4). During the study, CDDP patients had no need for additional dialysis sessions. In turn, an easy volumes balance with quite low interdialytic weight gains, and prevention of high ultrafiltration rates and of intradialytic hypotension, contribute to the protection of RRF during CDDP. Moreover, Fouque et al. showed that dietary treatment produced a positive effect in delaying the need of dialysis [26].
It is noteworthy that the hospitalization rate in CDDP was much lower than in THD Group. In the latter, the causes of admission were mainly vascular access complications and infection. In addition the overall 2 year-survival rate of CDDP was similar to that of THD patients. In fact, data from literature show that a conservative approach may be not inferior to dialysis in elderly ESRD patients, in terms of survival and/or quality of life [27–29].
Lastly, the CDDP treatment resulted in an approximate 50% savings on the cost of drugs and dialysis resources, including a lower number of dialysis hours/nursing and medical staff ( 66% less compared to thrice-weekly hemodialysis) and comorbid incidence in terms of days of hospitalization, as well as of indirect costs such as transport of patients to and from the dialysis unit. When compared to THD, the CDDP group showed a lower hospitalization rate, lower need for expensive drugs, and by definition a 66% lowering of number of dialysis sessions and related costs. CDDP patients may have the additional cost of the protein-free products. As a whole, CDDP seems to be a safe and effective exit strategy for lowering the cost of ESRD population, even if it can occur for a limited period of time and in selected ESRD patients.
Another advantage of CDDP is an improvement in patient’s acceptance and adaptation to renal replacement therapy. The patient feels less sick and less machine-dependent, resulting in an increased copying capacity (or psychological adaptation). Patients moreover tended to adapt better to low protein diets thanks to a more varied diet, as well as to address concerns about “dialysis-day”, which was also the day they were allowed to eat a “normal” diet.
The study has not had the opportunity to randomize and this may be seen as a limitation, but randomization was not feasible because one could not force patients to choose one method rather than the other. Patients were followed in pre-dialysis care: a multidisciplinary clinic (doctors, nurses, dietitians and psychologists) was able to provide the necessary education and choice of treatment modalities. This study aimed to reproduce the real world clinical practice where patient’s adherence is a crucial issue for the success of any treatment, especially if dietary in nature, where patients play an active role and must be involved in the decision-making process. Patients who entered the CDDP showed a lower residual renal function but better control of phosphatemia, BUN, PTH levels and higher albumin levels. This finding suggests that CDDP is suitable for patients who still have a positive inclination towards dietary manipulations. Moreover, together with a preserved urine output volume, good compliance to dietary prescriptions is mandatory and a pre-requisite for the success and safety of CDDP. Now it could be necessary to begin considering a tailored dialytic treatment and to evaluate the greater power of RRF. The incremental choice is considered a good option in peritoneal patients since the 2000’s [30, 31] thanks to RRF and it again underestimates for twice-weekly HD patients [32, 33]. Energy-adequate low-protein regimens have a fundamental role in controlling and maintaining a good metabolic status and in reducing GFR loss [26, 34].
Conclusions:
This study shows that in selected ESRD patients, a CDDP is able to protect the RRF, to maintain urine volume output, to preserve a good nutritional status , to blunt the rapid β2 microglobulin increase, and to allow better control of anemia and calcium-phosphate abnormalities. CDDP is also associated with a lower rate of hospitalization and reduced need of EPO and drugs used for the CKD-MBD treatment, thus leading to a significant cost-saving. As a whole, our findings suggest that CDDP could be a beneficial choice for selected collaborative patients who would otherwise be referred to thrice-weekly hemodialysis. CCDP should be considered as the first step of an incremental approach to hemodialysis treatment in motivated and selected ESRD patients, or anywhere dialysis facilities and resources are lacking.
|
Background:
Infrequent dialysis, namely once-a-week session combined with very low-protein, low-phosphorus diet supplemented with ketoacids was reported as a useful treatment schedule for ESRD patients with markedly reduced residual renal function but preserved urine output. This study reports our findings from the application of a weekly dialysis schedule plus less severe protein restriction (standard low-protein low-phosphorus diet) in stage 5 CKD patients with consistent dietary discipline.
Methods:
This is a multicenter, prospective controlled study, including 68 incident CKD patients followed in a pre-dialysis clinic with Glomerular Filtration Rate 5 to 10 ml/min/1.73/ m2 who became unstable on the only medical treatment. They were offered to begin a Combined Diet Dialysis Program (CDDP) or a standard thrice-a-week hemodialysis (THD): 38 patients joined the CDDP, whereas 30 patients chose THD. Patients were studied at baseline, 6 and 12 months; hospitalization and survival rate were followed-up for 24 months.
Results:
Volume output and residual renal function were maintained in the CDDP Group while those features dropped quickly in THD Group. Throughout the study, CDDP patients had a lower erythropoietin resistance index, lower β2 microglobulin levels and lower need for cinacalcet of phosphate binders than THD, and stable parameters of nutritional status. At 24 month follow-up, 39.4% of patients were still on CDDP; survival rates were 94.7% and 86.8% for CDDP and THD patients, respectively, but hospitalization rate was much higher in THD than in CDDP patients. The cost per patient per year resulted significantly lower in CDDP than in THD Group.
Conclusions:
This study shows that a CDDP served to protect the residual renal function, to maintain urine volume output and to preserve a good nutritional status. CDDP also blunted the rapid β2 microglobulin increase and resulted in better control of anemia and calcium-phosphate abnormalities. CDDP was also associated with a lower hospitalization rate and reduced need of erythropoietin, as well as of drugs used for treatment of calcium-phosphate abnormalities, thus leading to a significant cost-saving. We concluded that in selected ESRD patients with preserved urine output attitude to protein restriction, CDDP may be a beneficial choice for an incremental hemodialysis program.
|
Background:
The Epidemiology of chronic kidney disease (CKD) is on an upward trend worldwide. Since the 1990s incidence has risen by approximately 40% per decade, with prevalence rates in the general adult population up to 13% in the USA and ranging 10.2-11.6% in Europe [1–4]. These figures indicate a CKD epidemic leading to an increased number of patients requiring dialysis and a heavy burden on public health resources.
Nutritional and pharmacological therapy constitute the basis for the prevention and treatment of signs and symptoms of CKD, and they serve to delay the commencement of dialysis. The appropriate time to start dialysis is still a matter of debate. In particular, dialysis is not superior that conservative treatment especially in the comorbid-elderly population. It is known that a standard thrice--weekly hemodialysis schedule invariably leads to rapid loss of the residual renal function, to psychological and socials drawbacks, and to high costs. Real incremental dialysis programs are implemented in peritoneal dialysis but not in the hemodialysis setting where only a twice -weekly schedule may be proposed prior to maintenance hemodialysis.
During the 1980’s and 1990’s, an Integrated Diet Dialysis Program (IDDP) was proposed as a therapeutic option for selected patients with markedly reduced residual renal function and it consisted of weekly hemodialysis session (HD) combined with nutritional therapy, namely a very low-protein diet supplemented with ketoacids [5–8]. Malnutrition risk and low compliance to the severe dietary restrictions represented major concerns that prevented a widespread application of this schedule. In the present study we propose a Combined Diet Dialysis Program (CDDP) which includes a less severe low protein (0.6 g/Kg/d) diet combined with once-weekly hemodialysis. The goal is to prolong a conservative approach and thus reduce the need for hemodialysis, thereby limiting the risk of malnutrition and of poor dietary adherence. This study aimed to assess the safety, benefits and drawbacks of the CDDP approach including nutritional status, residual renal function, morbidity, mortality and costs.
Conclusions:
This study shows that in selected ESRD patients, a CDDP is able to protect the RRF, to maintain urine volume output, to preserve a good nutritional status , to blunt the rapid β2 microglobulin increase, and to allow better control of anemia and calcium-phosphate abnormalities. CDDP is also associated with a lower rate of hospitalization and reduced need of EPO and drugs used for the CKD-MBD treatment, thus leading to a significant cost-saving. As a whole, our findings suggest that CDDP could be a beneficial choice for selected collaborative patients who would otherwise be referred to thrice-weekly hemodialysis. CCDP should be considered as the first step of an incremental approach to hemodialysis treatment in motivated and selected ESRD patients, or anywhere dialysis facilities and resources are lacking.
|
Background:
Infrequent dialysis, namely once-a-week session combined with very low-protein, low-phosphorus diet supplemented with ketoacids was reported as a useful treatment schedule for ESRD patients with markedly reduced residual renal function but preserved urine output. This study reports our findings from the application of a weekly dialysis schedule plus less severe protein restriction (standard low-protein low-phosphorus diet) in stage 5 CKD patients with consistent dietary discipline.
Methods:
This is a multicenter, prospective controlled study, including 68 incident CKD patients followed in a pre-dialysis clinic with Glomerular Filtration Rate 5 to 10 ml/min/1.73/ m2 who became unstable on the only medical treatment. They were offered to begin a Combined Diet Dialysis Program (CDDP) or a standard thrice-a-week hemodialysis (THD): 38 patients joined the CDDP, whereas 30 patients chose THD. Patients were studied at baseline, 6 and 12 months; hospitalization and survival rate were followed-up for 24 months.
Results:
Volume output and residual renal function were maintained in the CDDP Group while those features dropped quickly in THD Group. Throughout the study, CDDP patients had a lower erythropoietin resistance index, lower β2 microglobulin levels and lower need for cinacalcet of phosphate binders than THD, and stable parameters of nutritional status. At 24 month follow-up, 39.4% of patients were still on CDDP; survival rates were 94.7% and 86.8% for CDDP and THD patients, respectively, but hospitalization rate was much higher in THD than in CDDP patients. The cost per patient per year resulted significantly lower in CDDP than in THD Group.
Conclusions:
This study shows that a CDDP served to protect the residual renal function, to maintain urine volume output and to preserve a good nutritional status. CDDP also blunted the rapid β2 microglobulin increase and resulted in better control of anemia and calcium-phosphate abnormalities. CDDP was also associated with a lower hospitalization rate and reduced need of erythropoietin, as well as of drugs used for treatment of calcium-phosphate abnormalities, thus leading to a significant cost-saving. We concluded that in selected ESRD patients with preserved urine output attitude to protein restriction, CDDP may be a beneficial choice for an incremental hemodialysis program.
| 7,662 | 430 | 9 |
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[CONTENT] Nutrition | Low protein diet | Hemodialysis | CKD | ESRD | Incremental dialysis [SUMMARY]
|
[CONTENT] Nutrition | Low protein diet | Hemodialysis | CKD | ESRD | Incremental dialysis [SUMMARY]
|
[CONTENT] Nutrition | Low protein diet | Hemodialysis | CKD | ESRD | Incremental dialysis [SUMMARY]
|
[CONTENT] Nutrition | Low protein diet | Hemodialysis | CKD | ESRD | Incremental dialysis [SUMMARY]
|
[CONTENT] Nutrition | Low protein diet | Hemodialysis | CKD | ESRD | Incremental dialysis [SUMMARY]
|
[CONTENT] Nutrition | Low protein diet | Hemodialysis | CKD | ESRD | Incremental dialysis [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Appointments and Schedules | Calcium | Combined Modality Therapy | Cost Savings | Diet, Protein-Restricted | Dietary Proteins | Female | Hospitalization | Humans | Hyperparathyroidism, Secondary | Kidney Failure, Chronic | Kidney Function Tests | Male | Middle Aged | Phosphorus | Phosphorus, Dietary | Prospective Studies | Renal Dialysis | Survival Rate | Treatment Outcome [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Appointments and Schedules | Calcium | Combined Modality Therapy | Cost Savings | Diet, Protein-Restricted | Dietary Proteins | Female | Hospitalization | Humans | Hyperparathyroidism, Secondary | Kidney Failure, Chronic | Kidney Function Tests | Male | Middle Aged | Phosphorus | Phosphorus, Dietary | Prospective Studies | Renal Dialysis | Survival Rate | Treatment Outcome [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Appointments and Schedules | Calcium | Combined Modality Therapy | Cost Savings | Diet, Protein-Restricted | Dietary Proteins | Female | Hospitalization | Humans | Hyperparathyroidism, Secondary | Kidney Failure, Chronic | Kidney Function Tests | Male | Middle Aged | Phosphorus | Phosphorus, Dietary | Prospective Studies | Renal Dialysis | Survival Rate | Treatment Outcome [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Appointments and Schedules | Calcium | Combined Modality Therapy | Cost Savings | Diet, Protein-Restricted | Dietary Proteins | Female | Hospitalization | Humans | Hyperparathyroidism, Secondary | Kidney Failure, Chronic | Kidney Function Tests | Male | Middle Aged | Phosphorus | Phosphorus, Dietary | Prospective Studies | Renal Dialysis | Survival Rate | Treatment Outcome [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Appointments and Schedules | Calcium | Combined Modality Therapy | Cost Savings | Diet, Protein-Restricted | Dietary Proteins | Female | Hospitalization | Humans | Hyperparathyroidism, Secondary | Kidney Failure, Chronic | Kidney Function Tests | Male | Middle Aged | Phosphorus | Phosphorus, Dietary | Prospective Studies | Renal Dialysis | Survival Rate | Treatment Outcome [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Appointments and Schedules | Calcium | Combined Modality Therapy | Cost Savings | Diet, Protein-Restricted | Dietary Proteins | Female | Hospitalization | Humans | Hyperparathyroidism, Secondary | Kidney Failure, Chronic | Kidney Function Tests | Male | Middle Aged | Phosphorus | Phosphorus, Dietary | Prospective Studies | Renal Dialysis | Survival Rate | Treatment Outcome [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] patients | cddp | thd | group | dialysis | treatment | months | thd group | protein | 12 [SUMMARY]
|
[CONTENT] patients | cddp | thd | group | dialysis | treatment | months | thd group | protein | 12 [SUMMARY]
|
[CONTENT] patients | cddp | thd | group | dialysis | treatment | months | thd group | protein | 12 [SUMMARY]
|
[CONTENT] patients | cddp | thd | group | dialysis | treatment | months | thd group | protein | 12 [SUMMARY]
|
[CONTENT] patients | cddp | thd | group | dialysis | treatment | months | thd group | protein | 12 [SUMMARY]
|
[CONTENT] patients | cddp | thd | group | dialysis | treatment | months | thd group | protein | 12 [SUMMARY]
|
[CONTENT] hemodialysis | dialysis | combined | diet | schedule | weekly | malnutrition | drawbacks | diet dialysis program | low [SUMMARY]
|
[CONTENT] patients | calculated | kg | protein | group | servings | dietary | creatinine | measured | included [SUMMARY]
|
[CONTENT] cddp | months | thd | dl | mg | group | patients | 12 | mg dl | 12 months [SUMMARY]
|
[CONTENT] selected | esrd patients | esrd | selected esrd | selected esrd patients | hemodialysis | patients | cddp | treatment | hemodialysis ccdp considered [SUMMARY]
|
[CONTENT] patients | cddp | group | thd | dialysis | treatment | thd group | hemodialysis | patient | lower [SUMMARY]
|
[CONTENT] patients | cddp | group | thd | dialysis | treatment | thd group | hemodialysis | patient | lower [SUMMARY]
|
[CONTENT] ESRD ||| weekly | 5 [SUMMARY]
|
[CONTENT] 68 | CKD | 10 ml | m2 ||| a Combined Diet Dialysis Program | THD | 38 | CDDP | 30 | THD ||| 6 and 12 months | 24 months [SUMMARY]
|
[CONTENT] the CDDP Group | THD Group ||| microglobulin | THD ||| 24 month | 39.4% | CDDP | 94.7% | 86.8% | CDDP | THD | THD ||| CDDP | THD Group [SUMMARY]
|
[CONTENT] CDDP ||| CDDP ||| CDDP ||| ESRD [SUMMARY]
|
[CONTENT] ESRD ||| weekly | 5 ||| 68 | CKD | 10 ml | m2 ||| a Combined Diet Dialysis Program | THD | 38 | CDDP | 30 | THD ||| 6 and 12 months | 24 months ||| the CDDP Group | THD Group ||| microglobulin | THD ||| 24 month | 39.4% | CDDP | 94.7% | 86.8% | CDDP | THD | THD ||| CDDP | THD Group ||| CDDP ||| CDDP ||| CDDP ||| ESRD [SUMMARY]
|
[CONTENT] ESRD ||| weekly | 5 ||| 68 | CKD | 10 ml | m2 ||| a Combined Diet Dialysis Program | THD | 38 | CDDP | 30 | THD ||| 6 and 12 months | 24 months ||| the CDDP Group | THD Group ||| microglobulin | THD ||| 24 month | 39.4% | CDDP | 94.7% | 86.8% | CDDP | THD | THD ||| CDDP | THD Group ||| CDDP ||| CDDP ||| CDDP ||| ESRD [SUMMARY]
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Lipopolysaccharide Stimulates Surfactant Protein-A in Human Renal Epithelial HK-2 Cells through Upregulating Toll-like Receptor 4 Dependent MEK1/2-ERK1/2-NF-κB Pathway.
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28485325
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Surfactant protein-A (SP-A) contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial (HK-2) cells can be stimulated by lipopolysaccharide (LPS). The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells.
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BACKGROUND
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Tetrazolium salt colorimetry (MTT) assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 (TLR4) messenger RNA (mRNA) expression, as well as phosphorylation of mitogen-activated/extracellular signal-regulated kinase (MEK) 1, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), and nuclear factor-kappa B (NF-κB) inhibitor-alpha (IkB-α). Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-κB in the cytoplasm and nucleus of HK-2 before LPS exposure.
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METHODS
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HK-2 cells exposed to 100 ng/ml of LPS for 1, 6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells (P = 0.16); however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased (P = 0.02). As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEK1, ERK1/2, and p38MAPK. In addition, levels of phosphorylated IκB-α and nuclear NF-κB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-κB expression were significantly inhibited by pretreatment with CLI-095.
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RESULTS
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The present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1-ERK1/2-NF-κB-dependent pathway.
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CONCLUSIONS
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[
"Cell Line",
"Cell Survival",
"Colorimetry",
"Humans",
"Kidney",
"Lipopolysaccharides",
"Mitogen-Activated Protein Kinase 1",
"Mitogen-Activated Protein Kinase 3",
"NF-kappa B",
"Pulmonary Surfactant-Associated Protein A",
"Sulfonamides",
"Tetrazolium Salts",
"Toll-Like Receptor 4"
] |
5443031
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Introduction
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Acute kidney injury (AKI) is always caused by sepsis and septic shock, the incidence of which is closely related to the severity of infection. About 19% of sepsis patients, 23% of severe sepsis patients, and 51% of patients with septic shock would accompany with AKI.[1] Clinical studies have showed that AKI may be an independent predictor of death, and the mortality of patients accompanied with AKI is as high as 74.5%.[2] There is growing evidence showing that 62.5% of sepsis in Chinese patients is caused by Gram-negative bacteria, and the lipopolysaccharide (LPS) on the bacterial cell wall is a major pathogenic factor of these Gram-negative bacteria.[3]
Surfactant protein-A (SP-A) was initially identified in the pulmonary epithelial cells. It is synthesized and secreted by alveolar Type II epithelial cells and encoded by two genes (SP-A1 and SP-A2). SP-A can reduce alveolar surface tension to keep the alveoli expanded.[4] In addition, it also plays important roles in the pulmonary immunity response and inflammatory regulation. Levels of tumor necrosis factor-alpha (TNF-α), pulmonary capillary permeability, and apoptotic cells in pulmonary were significantly increased in SP-A knockout mice caused by cisplatin-induced acute lung injury.[5] LPS-induced inflammation and apoptosis of pulmonary epithelial cells could be effectively depressed by intra-tracheal administration of exogenous SP-A.[6] Our previous study indicated that SP-A knockout mice exhibited more severe acute kidney injury (AKI) induced by sepsis.[7] Meanwhile, SP-A prevents renal tubular epithelial cell injury from sepsis through inhibiting nuclear factor-kappa B (NF-kB) activity to modulate TNF-α expression.[8] However, the underlying mechanisms of LPS-induced SP-A expression in renal tubular epithelial cells are still unclear.
Toll-like receptors (TLRs) are Type I transmembrane protein and composed of extracellular domain rich in leucine repeat sequence and intracellular domain. TLR4 expression has been found in the epithelium of multiple organs (such as alveolar epithelial cells and renal tubular epithelial cells).[9] LPS can bind to the TLR4/MD-2 complex on the membrane of renal tubular epithelial cells to activate intracellular signaling pathways.[10] Mitogen-activated protein kinase (MAPK) belongs to serine/threonine kinase extracellular signal-regulated kinase (ERK). MAPK family consists of ERK, p38MAPK, and C-Jun N-terminal kinase (JNK).[11] ERK, including ERK1 and ERK2, is crucial for the signal transmission from extracellular to nucleus.[12] It has been confirmed that ERK1/2 can be activated by mitogen-activated protein/ERK kinase (MEKs), and then may cause the translocation of cytoplasmic nuclear factor-κB (NF-κB) into nucleus, leading to NF-κB activation.[13] A variety of signaling pathways participated in TLR4 activation,[14] our previous study showed that the secretion of SP-A could be significantly induced by LPS in HK-2 cells,[15] but the possible mechanism is still unknown. In this study, we attempted to investigate the effects of LPS on the SP-A expression in human renal tubular epithelial (HK-2) cells and its potential mechanisms.
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Methods
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Cell culture and drug treatment Renal tubular epithelial cells (HK-2 cells, a proximal tubular cell line derived from normal kidney; CRL-2190) were purchased from ATCC and cultured at 37°C in a humidified atmosphere of 5% CO2 with Dulbecco's modified Eagle's medium (HyClone Pierce, USA) containing 10% fetal bovine serum (FBS; Invitrogen, USA) and 1% ampicillin. The culture medium was refreshed every 2 days until the cell confluence reached 70–80%. Cells were harvested after digestion with 0.25% trypsin/EDTA. The messenger RNA (mRNA) and protein expression were detected after LPS exposure for different durations. LPS extracted from serotype Escherichia coli 0111:B4 was purchased from Sigma (St. Louis, MO, USA). To prepare LPS suspension, LPS was dissolved in dimethyl sulfoxide (DMSO). Concentration of DMSO was <0.1% to reduce its toxicity to HK-2 cells. CLI-095 (Invitrogen, San Diego, CA, USA) is a cyclohexene derivative and an inhibitor of TLR4. CLI-095 was dissolved in DMOS at 10 μmol/l. Cells in control group were cultured in the medium only with DMSO at the same concentration.
Renal tubular epithelial cells (HK-2 cells, a proximal tubular cell line derived from normal kidney; CRL-2190) were purchased from ATCC and cultured at 37°C in a humidified atmosphere of 5% CO2 with Dulbecco's modified Eagle's medium (HyClone Pierce, USA) containing 10% fetal bovine serum (FBS; Invitrogen, USA) and 1% ampicillin. The culture medium was refreshed every 2 days until the cell confluence reached 70–80%. Cells were harvested after digestion with 0.25% trypsin/EDTA. The messenger RNA (mRNA) and protein expression were detected after LPS exposure for different durations. LPS extracted from serotype Escherichia coli 0111:B4 was purchased from Sigma (St. Louis, MO, USA). To prepare LPS suspension, LPS was dissolved in dimethyl sulfoxide (DMSO). Concentration of DMSO was <0.1% to reduce its toxicity to HK-2 cells. CLI-095 (Invitrogen, San Diego, CA, USA) is a cyclohexene derivative and an inhibitor of TLR4. CLI-095 was dissolved in DMOS at 10 μmol/l. Cells in control group were cultured in the medium only with DMSO at the same concentration.
Detection of cell viability Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described.[16] HK-2 cells were digested with 0.25% trypsin and then suspended in the medium containing 10% FBS at 5–8 × 104 cells/ml. Cells were cultured into 96-well plates (200 μl/well), followed by incubation for 4–5 h. After cells attached, 100 ng/ml of LPS was added to each well (100 μl/well) and incubated at 37°C with 5% CO2 for 0, 1, 6, and 12 h. Before the detection, 5 mg/ml MTT solution was added to each well (20 μl/well), and then incubated in dark for 4 h. The supernatant was removed, and 150 μl of DMSO was added to each well and incubated for 10 min to resolve the deposit. The absorbance was measured at 490 nm by MTT enzyme-linked immunometric meter (Bio Rad, USA). The cell viability was calculated according to the standard curve.
Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described.[16] HK-2 cells were digested with 0.25% trypsin and then suspended in the medium containing 10% FBS at 5–8 × 104 cells/ml. Cells were cultured into 96-well plates (200 μl/well), followed by incubation for 4–5 h. After cells attached, 100 ng/ml of LPS was added to each well (100 μl/well) and incubated at 37°C with 5% CO2 for 0, 1, 6, and 12 h. Before the detection, 5 mg/ml MTT solution was added to each well (20 μl/well), and then incubated in dark for 4 h. The supernatant was removed, and 150 μl of DMSO was added to each well and incubated for 10 min to resolve the deposit. The absorbance was measured at 490 nm by MTT enzyme-linked immunometric meter (Bio Rad, USA). The cell viability was calculated according to the standard curve.
Real-time polymerase chain reaction Cultured HK-2 cells were used for real-time polymerase chain reaction (PCR) analysis of SP-A and TLR4 mRNA. Total RNA was extracted from each sample using Trizol (Invitrogen, USA) following the manufacturer's instructions. Total RNA (1 μg) extracted from the tissue was used for the RT reaction (Takara, China), and then 2.5 μl of cDNA was used for amplification at a final volume of 25 μl according to the supplier's protocol (Fermentas, Germany). Then, the amplified PCR product was used for melting curve analysis, especially SP-A primer: 5’-TGA AAGGGAGTTCTAGCATCTCA CAGA-3’ and 5’-ACATATGCC TATGTA GGCCTGACT GAG-3’, TLR4 primer: 5’- TATCCAGAGCCGTTGGTGTATCT-3’, and 5’-AATGAAGATG ATGCCA GAGCG -3’, and β-actin primer: 5’-GTCTACATGTCTC GATCCCACTTA A -3’ and 5’-GGTCTTTCTCTCTCAT CGCGCTC-3’. The PCR was performed with 35 cycles of 94°C for 45 s, 60°C for 45 s (SP-A) or 55°C for 50 s (TLR4), and 72°C for 2 min. Each PCR product was subjected to electrophoresis on 2% agarose gel containing 0.1 μg/ml ethidium bromide. DNA bands in the agarose gel were photographed and quantified.
Cultured HK-2 cells were used for real-time polymerase chain reaction (PCR) analysis of SP-A and TLR4 mRNA. Total RNA was extracted from each sample using Trizol (Invitrogen, USA) following the manufacturer's instructions. Total RNA (1 μg) extracted from the tissue was used for the RT reaction (Takara, China), and then 2.5 μl of cDNA was used for amplification at a final volume of 25 μl according to the supplier's protocol (Fermentas, Germany). Then, the amplified PCR product was used for melting curve analysis, especially SP-A primer: 5’-TGA AAGGGAGTTCTAGCATCTCA CAGA-3’ and 5’-ACATATGCC TATGTA GGCCTGACT GAG-3’, TLR4 primer: 5’- TATCCAGAGCCGTTGGTGTATCT-3’, and 5’-AATGAAGATG ATGCCA GAGCG -3’, and β-actin primer: 5’-GTCTACATGTCTC GATCCCACTTA A -3’ and 5’-GGTCTTTCTCTCTCAT CGCGCTC-3’. The PCR was performed with 35 cycles of 94°C for 45 s, 60°C for 45 s (SP-A) or 55°C for 50 s (TLR4), and 72°C for 2 min. Each PCR product was subjected to electrophoresis on 2% agarose gel containing 0.1 μg/ml ethidium bromide. DNA bands in the agarose gel were photographed and quantified.
Extraction of nuclear proteins Nuclear components were extracted on ice following the method of Chiu et al. with a little bit improvement.[17] HK-2 cells were centrifuged, the deposit was collected and prepared in the 25 μl buffer A (10 mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 1. 5 mmol/L MgCl2; 1 mmol/L DTT; 5% glycerin; 0.2 mmol/L EDTA; 1 mmol/L PMSF; 3 mg/L aprotinin; 3 mg/L leupeptin; 2 mg/L pepstainA; 1% NP-40) for 10 min, and then centrifuged on ice (14,000 r/min) for 1 min; the deposit was prepared in 15 μl buffer B on ice (20 mmol/L HEPES, pH 7.9; 420 mmol/L NaCl; 1.5 mmol/L MgCl2; 0.2 mmol/L EDTA; 0.5 mmol/L DTT; 25% glycerin; 0.5 mmol/L PMSF; 5 mg/L aprotinin; 5 mg/L leupeptin; 3 mg/L pepstainA) for 30 min, and then centrifuged on ice for 15 min (14,000 r/min). Protein concentrations were quantified with a bicinchonic acid protein assay kit (Logan, Utah, Hyclone Pierce).
Nuclear components were extracted on ice following the method of Chiu et al. with a little bit improvement.[17] HK-2 cells were centrifuged, the deposit was collected and prepared in the 25 μl buffer A (10 mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 1. 5 mmol/L MgCl2; 1 mmol/L DTT; 5% glycerin; 0.2 mmol/L EDTA; 1 mmol/L PMSF; 3 mg/L aprotinin; 3 mg/L leupeptin; 2 mg/L pepstainA; 1% NP-40) for 10 min, and then centrifuged on ice (14,000 r/min) for 1 min; the deposit was prepared in 15 μl buffer B on ice (20 mmol/L HEPES, pH 7.9; 420 mmol/L NaCl; 1.5 mmol/L MgCl2; 0.2 mmol/L EDTA; 0.5 mmol/L DTT; 25% glycerin; 0.5 mmol/L PMSF; 5 mg/L aprotinin; 5 mg/L leupeptin; 3 mg/L pepstainA) for 30 min, and then centrifuged on ice for 15 min (14,000 r/min). Protein concentrations were quantified with a bicinchonic acid protein assay kit (Logan, Utah, Hyclone Pierce).
Western blotting Proteins from HK-2 cells were extracted using RIPA lysate (150 mmol/L sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], and 50 mM Tris at pH 8.0). The total protein concentrations were determined using the bicinchoninic acid protein assay (Logan, Utah, Hyclone Pierce). Total protein (50 μg) was resolved by reducing 12% SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically at 60 mA onto nitrocellulose membranes at 4°C overnight (Bio-Rad, USA). After the samples were blocked in 5% nonfat milk in Tris-buffered saline, immunoblotting was conducted using a primary antibody against SP-A at a dilution of 1:150 or TLR4 at a dilution of 1:500 (Abcam, Britain, ab22048) or NF-kB (Abcam, Britain, ab32536) at a dilution of 1:5000 or IkB-α (Abcam, Britain, ab133462) or MEK1 (Abcam, Britain, ab32091) at a dilution of 1:1000 and phosphorylated MEK1 (p-MEK1) (Abcam, Britain, ab96379) at a dilution of 1:800 or ERK2 (Abcam, Britain, ab32081) at a dilution of 1:1000 and phosphorylated ERK1/2 (Abcam, Britain, ab201015) at a dilution of 1:800 or P38MAPK at a dilution of 1:3000 (Abcam, Britain, ab170099) and phosphorylated P38MAPK (Abcam, Britain, ab47363) at a dilution of 1:500, and then secondary antibody conjugated with horseradish peroxidase. Cellular β-actin protein was immunodetected using a human monoclonal antibody against human β-actin (St. Louis, MO, USA) as the internal standard. Immunoproducts were detected using enhanced chemiluminescence peroxidase detection reagents (Amersham, Sweden).
Proteins from HK-2 cells were extracted using RIPA lysate (150 mmol/L sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], and 50 mM Tris at pH 8.0). The total protein concentrations were determined using the bicinchoninic acid protein assay (Logan, Utah, Hyclone Pierce). Total protein (50 μg) was resolved by reducing 12% SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically at 60 mA onto nitrocellulose membranes at 4°C overnight (Bio-Rad, USA). After the samples were blocked in 5% nonfat milk in Tris-buffered saline, immunoblotting was conducted using a primary antibody against SP-A at a dilution of 1:150 or TLR4 at a dilution of 1:500 (Abcam, Britain, ab22048) or NF-kB (Abcam, Britain, ab32536) at a dilution of 1:5000 or IkB-α (Abcam, Britain, ab133462) or MEK1 (Abcam, Britain, ab32091) at a dilution of 1:1000 and phosphorylated MEK1 (p-MEK1) (Abcam, Britain, ab96379) at a dilution of 1:800 or ERK2 (Abcam, Britain, ab32081) at a dilution of 1:1000 and phosphorylated ERK1/2 (Abcam, Britain, ab201015) at a dilution of 1:800 or P38MAPK at a dilution of 1:3000 (Abcam, Britain, ab170099) and phosphorylated P38MAPK (Abcam, Britain, ab47363) at a dilution of 1:500, and then secondary antibody conjugated with horseradish peroxidase. Cellular β-actin protein was immunodetected using a human monoclonal antibody against human β-actin (St. Louis, MO, USA) as the internal standard. Immunoproducts were detected using enhanced chemiluminescence peroxidase detection reagents (Amersham, Sweden).
Statistical analysis Data are expressed as mean ± standard error of mean. Results were analyzed by one-way ANOVA using the SPSS version 15.0 software (SPSS Inc., Chicago, Illinois, USA). P < 0.05 was considered to indicate statistical significance.
Data are expressed as mean ± standard error of mean. Results were analyzed by one-way ANOVA using the SPSS version 15.0 software (SPSS Inc., Chicago, Illinois, USA). P < 0.05 was considered to indicate statistical significance.
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Results
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Effect of lipopolysaccharide on the cell viability of HK-2 cells To evaluate the cytotoxic effect of LPS on cells, the cell viability of HK-2 cells was detected after LPS exposure. HK-2 cells were stimulated with 100 ng/ml of LPS for 0, 1, 6, and 12 h, and absorbance was measured at 490 nm. The absorbance did not show significant difference at different durations after LPS treatment [Figure 1, P > 0.05], suggesting that 100 ng/ml of LPS is not cytotoxic to HK-2 cells.
Effects of lipopolysaccharide on the cell viability of HK-2 cells. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 6, 12, and 24 h. Cell viability was assayed by MTT method. Optical density value of HK-2 cells for different durations was detected with colorimetric method. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).
To evaluate the cytotoxic effect of LPS on cells, the cell viability of HK-2 cells was detected after LPS exposure. HK-2 cells were stimulated with 100 ng/ml of LPS for 0, 1, 6, and 12 h, and absorbance was measured at 490 nm. The absorbance did not show significant difference at different durations after LPS treatment [Figure 1, P > 0.05], suggesting that 100 ng/ml of LPS is not cytotoxic to HK-2 cells.
Effects of lipopolysaccharide on the cell viability of HK-2 cells. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 6, 12, and 24 h. Cell viability was assayed by MTT method. Optical density value of HK-2 cells for different durations was detected with colorimetric method. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).
Effects of lipopolysaccharide on the messenger RNA and protein expression of surfactant protein-A in HK-2 cells Protein and mRNA expression of SP-A in HK-2 cells after LPS treatment were detected by Western blotting and real-time PCR, respectively. Cells were stimulated with LPS for 0, 1, 2, and 6 h, and then SP-A mRNA expression was detected; cells were treated with LPS for 0, 2, 6, and 12 h, and SP-A protein expression was assayed; and β-actin expression was detected as an internal standard. Expression of SP-A mRNA expression at 1, 2, and 6 h after LPS exposure was significantly increased by 1.5, 2.7, and 4.5 folds, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Moreover, the SP-A protein expression at 6 h after LPS treatment increased dramatically when compared with that at 0 h and 2 h [Figure 3, P < 0.05]. After LPS exposure for 12 h, the SP-A protein expression was markedly higher than that at 6 h. SP-A protein expression after LPS exposure for 6 h and 12 h was increased by 1.6 and 2.8 folds, respectively, as compared to that at 0 h. These findings suggested that LPS may upregulate SP-A expression at transcriptional level.
Expression of surfactant protein-A and toll-like receptor 4 mRNA in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to lipopolysaccharide for 0, 1, 2, and 6 h. β-actin mRNA was detected as the internal standard. Expression of surfactant protein-A and toll-like receptor 4 mRNA was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate that the value of surfactant protein-A and toll-like receptor 4 mRNA expression significantly (P < 0.05) differed from lipopolysaccharide exposure for 0 h, respectively. mRNA: Messenger RNA.
Expression of surfactant protein-A protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of surfactant protein-A protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Protein and mRNA expression of SP-A in HK-2 cells after LPS treatment were detected by Western blotting and real-time PCR, respectively. Cells were stimulated with LPS for 0, 1, 2, and 6 h, and then SP-A mRNA expression was detected; cells were treated with LPS for 0, 2, 6, and 12 h, and SP-A protein expression was assayed; and β-actin expression was detected as an internal standard. Expression of SP-A mRNA expression at 1, 2, and 6 h after LPS exposure was significantly increased by 1.5, 2.7, and 4.5 folds, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Moreover, the SP-A protein expression at 6 h after LPS treatment increased dramatically when compared with that at 0 h and 2 h [Figure 3, P < 0.05]. After LPS exposure for 12 h, the SP-A protein expression was markedly higher than that at 6 h. SP-A protein expression after LPS exposure for 6 h and 12 h was increased by 1.6 and 2.8 folds, respectively, as compared to that at 0 h. These findings suggested that LPS may upregulate SP-A expression at transcriptional level.
Expression of surfactant protein-A and toll-like receptor 4 mRNA in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to lipopolysaccharide for 0, 1, 2, and 6 h. β-actin mRNA was detected as the internal standard. Expression of surfactant protein-A and toll-like receptor 4 mRNA was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate that the value of surfactant protein-A and toll-like receptor 4 mRNA expression significantly (P < 0.05) differed from lipopolysaccharide exposure for 0 h, respectively. mRNA: Messenger RNA.
Expression of surfactant protein-A protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of surfactant protein-A protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Effects of lipopolysaccharide with or without CLI-095 on the nuclear translocation and activation of nuclear factor-κB in HK-2 cells To investigate the mechanism underlying LPS-induced SP-A expression in HK-2 cells, the expression of cytoplasmic and nuclear NF-κB as well as phosphorylated IκB-α was measured in HK-2 cells, and total NF-κB served as an internal standard. After LPS exposure for 0, 1, 2, and 6 h, the cytoplasmic NF-κB expression was decreased [Figure 4, P < 0.05], however the nuclear NF-κB expression and intracellular phosphorylated IκB-α expression were increased [Figure 5, P < 0.05]. The protein expression of nuclear factor-κB (NF-κB) at different time points was quantified, and results showed that the cytoplasmic NF-κB expression at 1, 2, and 6 h after LPS exposure was reduced by 32%, 48%, and 47%, respectively, but expressions of the nuclear NF-κB and phosphorylated IκB-α at 1, 2, and 6 h after LPS exposure were augmented by 117%, 155%, and 203% and by 167%, 320%, and 413%, respectively, as compared to those at 0 h. Moreover, the expression of the nuclear NF-κB and phosphorylated IκB-α was depressed by CLI-095 when compared with by LPS stimulation alone [Figures 4 and 5, P < 0.05].
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced nucleus factor-kB protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. Cytosolic (c) and nucleus (n) factor-kB were immunodetected. Total (t) nucleus factor-kB was detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced phosphorylated IkB-α protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. β-actin protein was detected as the internal standard. Expression of p-IkB-α was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of LPS exposure for 0 h.
To investigate the mechanism underlying LPS-induced SP-A expression in HK-2 cells, the expression of cytoplasmic and nuclear NF-κB as well as phosphorylated IκB-α was measured in HK-2 cells, and total NF-κB served as an internal standard. After LPS exposure for 0, 1, 2, and 6 h, the cytoplasmic NF-κB expression was decreased [Figure 4, P < 0.05], however the nuclear NF-κB expression and intracellular phosphorylated IκB-α expression were increased [Figure 5, P < 0.05]. The protein expression of nuclear factor-κB (NF-κB) at different time points was quantified, and results showed that the cytoplasmic NF-κB expression at 1, 2, and 6 h after LPS exposure was reduced by 32%, 48%, and 47%, respectively, but expressions of the nuclear NF-κB and phosphorylated IκB-α at 1, 2, and 6 h after LPS exposure were augmented by 117%, 155%, and 203% and by 167%, 320%, and 413%, respectively, as compared to those at 0 h. Moreover, the expression of the nuclear NF-κB and phosphorylated IκB-α was depressed by CLI-095 when compared with by LPS stimulation alone [Figures 4 and 5, P < 0.05].
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced nucleus factor-kB protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. Cytosolic (c) and nucleus (n) factor-kB were immunodetected. Total (t) nucleus factor-kB was detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced phosphorylated IkB-α protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. β-actin protein was detected as the internal standard. Expression of p-IkB-α was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of LPS exposure for 0 h.
Effects of lipopolysaccharide on the expression of toll-like receptor 4, phosphorylated p38 mitogen-activated protein kinase, and mitogen-activated/extracellular signal-regulated kinase 1 in HK-2 cells To elucidate the signaling pathway involved in LPS-induced NF-κB activation in HK-2 cells, the mRNA and protein expression of upstream transmembrane receptor TLR4 and protein expression of phosphorylated ERK1/2, phosphorylated p38MAPK, and p-MEK1 were assayed in HK-2 cells. With β-actin expression served as an internal standard, quantitative analysis showed that the levels of TLR4 mRNA after LPS exposure for 1, 2, and 6 h were increased by 111%, 221%, and 489%, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Meanwhile, the expression of TLR4 protein after LPS treatment for 1, 2, and 6 h was also increased [Figure 6, P < 0.05]. Expression of TLR4 protein at 2, 6, and 12 h was increased by 74%, 91%, and 154%, respectively, when compared with that at 0 h (P < 0.05). Phosphorylation of p38MAPK [Figure 7, P < 0.05] and MEK1 [Figure 8, P < 0.05] in HK-2 cells at 2, 6, and 12 h was increased by 75%, 130%, 215%, and 74%, 183%, 262%, respectively, as compared to that at 0 h. Levels of phosphorylated ERK1 in HK-2 cells were obviously raised after exposure to LPS for 2, 6, and 12 h by 62%, 162%, and 254%, respectively. Exposure of HK-2 cells to LPS for 2, 6, and 12 h significantly increased ERK2 phosphorylation by 77%, 239%, and 392%, respectively [Figure 9, P < 0.05].
Expression of toll-like receptor 4 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of toll-like receptor 4 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of P38MAPK protein in HK-2 cells induced by lipopolysaccharide exposure for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. P38MAPK protein was detected as the internal standard. Expression of p-P38MAPK protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of mitogen-activated/extracellular signal-regulated kinase 1 protein induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Mitogen-activated/extracellular signal-regulated kinase 1 protein was detected as the internal standard. Expression of phosphorylated mitogen-activated/extracellular signal-regulated kinase 1 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of ERK1/2 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. ERK2 protein was detected as the internal standard. Expression of p-ERK1/2 protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h. ERK: Extracellular signal-regulated kinase.
To elucidate the signaling pathway involved in LPS-induced NF-κB activation in HK-2 cells, the mRNA and protein expression of upstream transmembrane receptor TLR4 and protein expression of phosphorylated ERK1/2, phosphorylated p38MAPK, and p-MEK1 were assayed in HK-2 cells. With β-actin expression served as an internal standard, quantitative analysis showed that the levels of TLR4 mRNA after LPS exposure for 1, 2, and 6 h were increased by 111%, 221%, and 489%, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Meanwhile, the expression of TLR4 protein after LPS treatment for 1, 2, and 6 h was also increased [Figure 6, P < 0.05]. Expression of TLR4 protein at 2, 6, and 12 h was increased by 74%, 91%, and 154%, respectively, when compared with that at 0 h (P < 0.05). Phosphorylation of p38MAPK [Figure 7, P < 0.05] and MEK1 [Figure 8, P < 0.05] in HK-2 cells at 2, 6, and 12 h was increased by 75%, 130%, 215%, and 74%, 183%, 262%, respectively, as compared to that at 0 h. Levels of phosphorylated ERK1 in HK-2 cells were obviously raised after exposure to LPS for 2, 6, and 12 h by 62%, 162%, and 254%, respectively. Exposure of HK-2 cells to LPS for 2, 6, and 12 h significantly increased ERK2 phosphorylation by 77%, 239%, and 392%, respectively [Figure 9, P < 0.05].
Expression of toll-like receptor 4 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of toll-like receptor 4 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of P38MAPK protein in HK-2 cells induced by lipopolysaccharide exposure for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. P38MAPK protein was detected as the internal standard. Expression of p-P38MAPK protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of mitogen-activated/extracellular signal-regulated kinase 1 protein induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Mitogen-activated/extracellular signal-regulated kinase 1 protein was detected as the internal standard. Expression of phosphorylated mitogen-activated/extracellular signal-regulated kinase 1 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of ERK1/2 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. ERK2 protein was detected as the internal standard. Expression of p-ERK1/2 protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h. ERK: Extracellular signal-regulated kinase.
Effects of lipopolysaccharide with or without CLI-095 on the expression of toll-like receptor 4 and surfactant protein-A messenger RNA and protein in HK-2 cells We further explored the role of TLR4-dependent MEK1/2-ERK1/2-NF-κB signaling pathway in LPS-induced SP-A expression. HK-2 cells were pretreated with CLI-095 before LPS exposure. Induction of TLR4 and SP-A mRNA expression by LPS were analyzed by real-time PCR. After LPS administration, the mRNA expression of TLR4 and SP-A was significantly increased [Figure 9, P < 0.05]. Pretreatment of HK-2 cells with CLI-095, an inhibitor of TLR4, before LPS administration could markedly inhibit the mRNA and protein expression of TLR4 and SP-A [Figure 9, P < 0.05]. Expression of TLR4 and SP-A mRNA in HK-2 cells after LPS exposure for 6 h was augmented by 458% and 542%, respectively, when compared with that at 0 h. With CLI-095 pretreatment, mRNA expression of TLR4 and SP-A in HK-2 cells was decreased by 66% and 60%, respectively, when compared with that by LPS administration for 6 h alone [Figure 10, P < 0.05].
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced surfactant protein-A and toll-like receptor 4 messenger RNA expression. HK-2 cells were pretreated with CLI-095 for 1 h, and then exposed to CLI-095 and lipopolysaccharide for another 6 h. Levels of β-actin messenger RNA were detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate and significant (P < 0.05) difference between lipopolysaccharide and CLI-095 + lipopolysaccharide groups for surfactant protein-A and toll-like receptor 4 messenger RNA expression, respectively.
We further explored the role of TLR4-dependent MEK1/2-ERK1/2-NF-κB signaling pathway in LPS-induced SP-A expression. HK-2 cells were pretreated with CLI-095 before LPS exposure. Induction of TLR4 and SP-A mRNA expression by LPS were analyzed by real-time PCR. After LPS administration, the mRNA expression of TLR4 and SP-A was significantly increased [Figure 9, P < 0.05]. Pretreatment of HK-2 cells with CLI-095, an inhibitor of TLR4, before LPS administration could markedly inhibit the mRNA and protein expression of TLR4 and SP-A [Figure 9, P < 0.05]. Expression of TLR4 and SP-A mRNA in HK-2 cells after LPS exposure for 6 h was augmented by 458% and 542%, respectively, when compared with that at 0 h. With CLI-095 pretreatment, mRNA expression of TLR4 and SP-A in HK-2 cells was decreased by 66% and 60%, respectively, when compared with that by LPS administration for 6 h alone [Figure 10, P < 0.05].
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced surfactant protein-A and toll-like receptor 4 messenger RNA expression. HK-2 cells were pretreated with CLI-095 for 1 h, and then exposed to CLI-095 and lipopolysaccharide for another 6 h. Levels of β-actin messenger RNA were detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate and significant (P < 0.05) difference between lipopolysaccharide and CLI-095 + lipopolysaccharide groups for surfactant protein-A and toll-like receptor 4 messenger RNA expression, respectively.
| null | null |
[
"Cell culture and drug treatment",
"Detection of cell viability",
"Real-time polymerase chain reaction",
"Extraction of nuclear proteins",
"Western blotting",
"Statistical analysis",
"Effect of lipopolysaccharide on the cell viability of HK-2 cells",
"Effects of lipopolysaccharide on the messenger RNA and protein expression of surfactant protein-A in HK-2 cells",
"Effects of lipopolysaccharide with or without CLI-095 on the nuclear translocation and activation of nuclear factor-κB in HK-2 cells",
"Effects of lipopolysaccharide on the expression of toll-like receptor 4, phosphorylated p38 mitogen-activated protein kinase, and mitogen-activated/extracellular signal-regulated kinase 1 in HK-2 cells",
"Effects of lipopolysaccharide with or without CLI-095 on the expression of toll-like receptor 4 and surfactant protein-A messenger RNA and protein in HK-2 cells",
"Financial support and sponsorship"
] |
[
"Renal tubular epithelial cells (HK-2 cells, a proximal tubular cell line derived from normal kidney; CRL-2190) were purchased from ATCC and cultured at 37°C in a humidified atmosphere of 5% CO2 with Dulbecco's modified Eagle's medium (HyClone Pierce, USA) containing 10% fetal bovine serum (FBS; Invitrogen, USA) and 1% ampicillin. The culture medium was refreshed every 2 days until the cell confluence reached 70–80%. Cells were harvested after digestion with 0.25% trypsin/EDTA. The messenger RNA (mRNA) and protein expression were detected after LPS exposure for different durations. LPS extracted from serotype Escherichia coli 0111:B4 was purchased from Sigma (St. Louis, MO, USA). To prepare LPS suspension, LPS was dissolved in dimethyl sulfoxide (DMSO). Concentration of DMSO was <0.1% to reduce its toxicity to HK-2 cells. CLI-095 (Invitrogen, San Diego, CA, USA) is a cyclohexene derivative and an inhibitor of TLR4. CLI-095 was dissolved in DMOS at 10 μmol/l. Cells in control group were cultured in the medium only with DMSO at the same concentration.",
"Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described.[16] HK-2 cells were digested with 0.25% trypsin and then suspended in the medium containing 10% FBS at 5–8 × 104 cells/ml. Cells were cultured into 96-well plates (200 μl/well), followed by incubation for 4–5 h. After cells attached, 100 ng/ml of LPS was added to each well (100 μl/well) and incubated at 37°C with 5% CO2 for 0, 1, 6, and 12 h. Before the detection, 5 mg/ml MTT solution was added to each well (20 μl/well), and then incubated in dark for 4 h. The supernatant was removed, and 150 μl of DMSO was added to each well and incubated for 10 min to resolve the deposit. The absorbance was measured at 490 nm by MTT enzyme-linked immunometric meter (Bio Rad, USA). The cell viability was calculated according to the standard curve.",
"Cultured HK-2 cells were used for real-time polymerase chain reaction (PCR) analysis of SP-A and TLR4 mRNA. Total RNA was extracted from each sample using Trizol (Invitrogen, USA) following the manufacturer's instructions. Total RNA (1 μg) extracted from the tissue was used for the RT reaction (Takara, China), and then 2.5 μl of cDNA was used for amplification at a final volume of 25 μl according to the supplier's protocol (Fermentas, Germany). Then, the amplified PCR product was used for melting curve analysis, especially SP-A primer: 5’-TGA AAGGGAGTTCTAGCATCTCA CAGA-3’ and 5’-ACATATGCC TATGTA GGCCTGACT GAG-3’, TLR4 primer: 5’- TATCCAGAGCCGTTGGTGTATCT-3’, and 5’-AATGAAGATG ATGCCA GAGCG -3’, and β-actin primer: 5’-GTCTACATGTCTC GATCCCACTTA A -3’ and 5’-GGTCTTTCTCTCTCAT CGCGCTC-3’. The PCR was performed with 35 cycles of 94°C for 45 s, 60°C for 45 s (SP-A) or 55°C for 50 s (TLR4), and 72°C for 2 min. Each PCR product was subjected to electrophoresis on 2% agarose gel containing 0.1 μg/ml ethidium bromide. DNA bands in the agarose gel were photographed and quantified.",
"Nuclear components were extracted on ice following the method of Chiu et al. with a little bit improvement.[17] HK-2 cells were centrifuged, the deposit was collected and prepared in the 25 μl buffer A (10 mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 1. 5 mmol/L MgCl2; 1 mmol/L DTT; 5% glycerin; 0.2 mmol/L EDTA; 1 mmol/L PMSF; 3 mg/L aprotinin; 3 mg/L leupeptin; 2 mg/L pepstainA; 1% NP-40) for 10 min, and then centrifuged on ice (14,000 r/min) for 1 min; the deposit was prepared in 15 μl buffer B on ice (20 mmol/L HEPES, pH 7.9; 420 mmol/L NaCl; 1.5 mmol/L MgCl2; 0.2 mmol/L EDTA; 0.5 mmol/L DTT; 25% glycerin; 0.5 mmol/L PMSF; 5 mg/L aprotinin; 5 mg/L leupeptin; 3 mg/L pepstainA) for 30 min, and then centrifuged on ice for 15 min (14,000 r/min). Protein concentrations were quantified with a bicinchonic acid protein assay kit (Logan, Utah, Hyclone Pierce).",
"Proteins from HK-2 cells were extracted using RIPA lysate (150 mmol/L sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], and 50 mM Tris at pH 8.0). The total protein concentrations were determined using the bicinchoninic acid protein assay (Logan, Utah, Hyclone Pierce). Total protein (50 μg) was resolved by reducing 12% SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically at 60 mA onto nitrocellulose membranes at 4°C overnight (Bio-Rad, USA). After the samples were blocked in 5% nonfat milk in Tris-buffered saline, immunoblotting was conducted using a primary antibody against SP-A at a dilution of 1:150 or TLR4 at a dilution of 1:500 (Abcam, Britain, ab22048) or NF-kB (Abcam, Britain, ab32536) at a dilution of 1:5000 or IkB-α (Abcam, Britain, ab133462) or MEK1 (Abcam, Britain, ab32091) at a dilution of 1:1000 and phosphorylated MEK1 (p-MEK1) (Abcam, Britain, ab96379) at a dilution of 1:800 or ERK2 (Abcam, Britain, ab32081) at a dilution of 1:1000 and phosphorylated ERK1/2 (Abcam, Britain, ab201015) at a dilution of 1:800 or P38MAPK at a dilution of 1:3000 (Abcam, Britain, ab170099) and phosphorylated P38MAPK (Abcam, Britain, ab47363) at a dilution of 1:500, and then secondary antibody conjugated with horseradish peroxidase. Cellular β-actin protein was immunodetected using a human monoclonal antibody against human β-actin (St. Louis, MO, USA) as the internal standard. Immunoproducts were detected using enhanced chemiluminescence peroxidase detection reagents (Amersham, Sweden).",
"Data are expressed as mean ± standard error of mean. Results were analyzed by one-way ANOVA using the SPSS version 15.0 software (SPSS Inc., Chicago, Illinois, USA). P < 0.05 was considered to indicate statistical significance.",
"To evaluate the cytotoxic effect of LPS on cells, the cell viability of HK-2 cells was detected after LPS exposure. HK-2 cells were stimulated with 100 ng/ml of LPS for 0, 1, 6, and 12 h, and absorbance was measured at 490 nm. The absorbance did not show significant difference at different durations after LPS treatment [Figure 1, P > 0.05], suggesting that 100 ng/ml of LPS is not cytotoxic to HK-2 cells.\nEffects of lipopolysaccharide on the cell viability of HK-2 cells. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 6, 12, and 24 h. Cell viability was assayed by MTT method. Optical density value of HK-2 cells for different durations was detected with colorimetric method. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).",
"Protein and mRNA expression of SP-A in HK-2 cells after LPS treatment were detected by Western blotting and real-time PCR, respectively. Cells were stimulated with LPS for 0, 1, 2, and 6 h, and then SP-A mRNA expression was detected; cells were treated with LPS for 0, 2, 6, and 12 h, and SP-A protein expression was assayed; and β-actin expression was detected as an internal standard. Expression of SP-A mRNA expression at 1, 2, and 6 h after LPS exposure was significantly increased by 1.5, 2.7, and 4.5 folds, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Moreover, the SP-A protein expression at 6 h after LPS treatment increased dramatically when compared with that at 0 h and 2 h [Figure 3, P < 0.05]. After LPS exposure for 12 h, the SP-A protein expression was markedly higher than that at 6 h. SP-A protein expression after LPS exposure for 6 h and 12 h was increased by 1.6 and 2.8 folds, respectively, as compared to that at 0 h. These findings suggested that LPS may upregulate SP-A expression at transcriptional level.\nExpression of surfactant protein-A and toll-like receptor 4 mRNA in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to lipopolysaccharide for 0, 1, 2, and 6 h. β-actin mRNA was detected as the internal standard. Expression of surfactant protein-A and toll-like receptor 4 mRNA was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate that the value of surfactant protein-A and toll-like receptor 4 mRNA expression significantly (P < 0.05) differed from lipopolysaccharide exposure for 0 h, respectively. mRNA: Messenger RNA.\nExpression of surfactant protein-A protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of surfactant protein-A protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.",
"To investigate the mechanism underlying LPS-induced SP-A expression in HK-2 cells, the expression of cytoplasmic and nuclear NF-κB as well as phosphorylated IκB-α was measured in HK-2 cells, and total NF-κB served as an internal standard. After LPS exposure for 0, 1, 2, and 6 h, the cytoplasmic NF-κB expression was decreased [Figure 4, P < 0.05], however the nuclear NF-κB expression and intracellular phosphorylated IκB-α expression were increased [Figure 5, P < 0.05]. The protein expression of nuclear factor-κB (NF-κB) at different time points was quantified, and results showed that the cytoplasmic NF-κB expression at 1, 2, and 6 h after LPS exposure was reduced by 32%, 48%, and 47%, respectively, but expressions of the nuclear NF-κB and phosphorylated IκB-α at 1, 2, and 6 h after LPS exposure were augmented by 117%, 155%, and 203% and by 167%, 320%, and 413%, respectively, as compared to those at 0 h. Moreover, the expression of the nuclear NF-κB and phosphorylated IκB-α was depressed by CLI-095 when compared with by LPS stimulation alone [Figures 4 and 5, P < 0.05].\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced nucleus factor-kB protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. Cytosolic (c) and nucleus (n) factor-kB were immunodetected. Total (t) nucleus factor-kB was detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced phosphorylated IkB-α protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. β-actin protein was detected as the internal standard. Expression of p-IkB-α was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of LPS exposure for 0 h.",
"To elucidate the signaling pathway involved in LPS-induced NF-κB activation in HK-2 cells, the mRNA and protein expression of upstream transmembrane receptor TLR4 and protein expression of phosphorylated ERK1/2, phosphorylated p38MAPK, and p-MEK1 were assayed in HK-2 cells. With β-actin expression served as an internal standard, quantitative analysis showed that the levels of TLR4 mRNA after LPS exposure for 1, 2, and 6 h were increased by 111%, 221%, and 489%, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Meanwhile, the expression of TLR4 protein after LPS treatment for 1, 2, and 6 h was also increased [Figure 6, P < 0.05]. Expression of TLR4 protein at 2, 6, and 12 h was increased by 74%, 91%, and 154%, respectively, when compared with that at 0 h (P < 0.05). Phosphorylation of p38MAPK [Figure 7, P < 0.05] and MEK1 [Figure 8, P < 0.05] in HK-2 cells at 2, 6, and 12 h was increased by 75%, 130%, 215%, and 74%, 183%, 262%, respectively, as compared to that at 0 h. Levels of phosphorylated ERK1 in HK-2 cells were obviously raised after exposure to LPS for 2, 6, and 12 h by 62%, 162%, and 254%, respectively. Exposure of HK-2 cells to LPS for 2, 6, and 12 h significantly increased ERK2 phosphorylation by 77%, 239%, and 392%, respectively [Figure 9, P < 0.05].\nExpression of toll-like receptor 4 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of toll-like receptor 4 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of P38MAPK protein in HK-2 cells induced by lipopolysaccharide exposure for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. P38MAPK protein was detected as the internal standard. Expression of p-P38MAPK protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of mitogen-activated/extracellular signal-regulated kinase 1 protein induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Mitogen-activated/extracellular signal-regulated kinase 1 protein was detected as the internal standard. Expression of phosphorylated mitogen-activated/extracellular signal-regulated kinase 1 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of ERK1/2 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. ERK2 protein was detected as the internal standard. Expression of p-ERK1/2 protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h. ERK: Extracellular signal-regulated kinase.",
"We further explored the role of TLR4-dependent MEK1/2-ERK1/2-NF-κB signaling pathway in LPS-induced SP-A expression. HK-2 cells were pretreated with CLI-095 before LPS exposure. Induction of TLR4 and SP-A mRNA expression by LPS were analyzed by real-time PCR. After LPS administration, the mRNA expression of TLR4 and SP-A was significantly increased [Figure 9, P < 0.05]. Pretreatment of HK-2 cells with CLI-095, an inhibitor of TLR4, before LPS administration could markedly inhibit the mRNA and protein expression of TLR4 and SP-A [Figure 9, P < 0.05]. Expression of TLR4 and SP-A mRNA in HK-2 cells after LPS exposure for 6 h was augmented by 458% and 542%, respectively, when compared with that at 0 h. With CLI-095 pretreatment, mRNA expression of TLR4 and SP-A in HK-2 cells was decreased by 66% and 60%, respectively, when compared with that by LPS administration for 6 h alone [Figure 10, P < 0.05].\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced surfactant protein-A and toll-like receptor 4 messenger RNA expression. HK-2 cells were pretreated with CLI-095 for 1 h, and then exposed to CLI-095 and lipopolysaccharide for another 6 h. Levels of β-actin messenger RNA were detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate and significant (P < 0.05) difference between lipopolysaccharide and CLI-095 + lipopolysaccharide groups for surfactant protein-A and toll-like receptor 4 messenger RNA expression, respectively.",
"This study was supported by the National Natural Science Foundation of China (No. 81201457, No. 81173402, and No. 81571943) and Advanced Suitable Project of Shanghai Health System (No. 2013SY070)."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Introduction",
"Methods",
"Cell culture and drug treatment",
"Detection of cell viability",
"Real-time polymerase chain reaction",
"Extraction of nuclear proteins",
"Western blotting",
"Statistical analysis",
"Results",
"Effect of lipopolysaccharide on the cell viability of HK-2 cells",
"Effects of lipopolysaccharide on the messenger RNA and protein expression of surfactant protein-A in HK-2 cells",
"Effects of lipopolysaccharide with or without CLI-095 on the nuclear translocation and activation of nuclear factor-κB in HK-2 cells",
"Effects of lipopolysaccharide on the expression of toll-like receptor 4, phosphorylated p38 mitogen-activated protein kinase, and mitogen-activated/extracellular signal-regulated kinase 1 in HK-2 cells",
"Effects of lipopolysaccharide with or without CLI-095 on the expression of toll-like receptor 4 and surfactant protein-A messenger RNA and protein in HK-2 cells",
"Discussion",
"Financial support and sponsorship",
"Conflicts of interest"
] |
[
"Acute kidney injury (AKI) is always caused by sepsis and septic shock, the incidence of which is closely related to the severity of infection. About 19% of sepsis patients, 23% of severe sepsis patients, and 51% of patients with septic shock would accompany with AKI.[1] Clinical studies have showed that AKI may be an independent predictor of death, and the mortality of patients accompanied with AKI is as high as 74.5%.[2] There is growing evidence showing that 62.5% of sepsis in Chinese patients is caused by Gram-negative bacteria, and the lipopolysaccharide (LPS) on the bacterial cell wall is a major pathogenic factor of these Gram-negative bacteria.[3]\nSurfactant protein-A (SP-A) was initially identified in the pulmonary epithelial cells. It is synthesized and secreted by alveolar Type II epithelial cells and encoded by two genes (SP-A1 and SP-A2). SP-A can reduce alveolar surface tension to keep the alveoli expanded.[4] In addition, it also plays important roles in the pulmonary immunity response and inflammatory regulation. Levels of tumor necrosis factor-alpha (TNF-α), pulmonary capillary permeability, and apoptotic cells in pulmonary were significantly increased in SP-A knockout mice caused by cisplatin-induced acute lung injury.[5] LPS-induced inflammation and apoptosis of pulmonary epithelial cells could be effectively depressed by intra-tracheal administration of exogenous SP-A.[6] Our previous study indicated that SP-A knockout mice exhibited more severe acute kidney injury (AKI) induced by sepsis.[7] Meanwhile, SP-A prevents renal tubular epithelial cell injury from sepsis through inhibiting nuclear factor-kappa B (NF-kB) activity to modulate TNF-α expression.[8] However, the underlying mechanisms of LPS-induced SP-A expression in renal tubular epithelial cells are still unclear.\nToll-like receptors (TLRs) are Type I transmembrane protein and composed of extracellular domain rich in leucine repeat sequence and intracellular domain. TLR4 expression has been found in the epithelium of multiple organs (such as alveolar epithelial cells and renal tubular epithelial cells).[9] LPS can bind to the TLR4/MD-2 complex on the membrane of renal tubular epithelial cells to activate intracellular signaling pathways.[10] Mitogen-activated protein kinase (MAPK) belongs to serine/threonine kinase extracellular signal-regulated kinase (ERK). MAPK family consists of ERK, p38MAPK, and C-Jun N-terminal kinase (JNK).[11] ERK, including ERK1 and ERK2, is crucial for the signal transmission from extracellular to nucleus.[12] It has been confirmed that ERK1/2 can be activated by mitogen-activated protein/ERK kinase (MEKs), and then may cause the translocation of cytoplasmic nuclear factor-κB (NF-κB) into nucleus, leading to NF-κB activation.[13] A variety of signaling pathways participated in TLR4 activation,[14] our previous study showed that the secretion of SP-A could be significantly induced by LPS in HK-2 cells,[15] but the possible mechanism is still unknown. In this study, we attempted to investigate the effects of LPS on the SP-A expression in human renal tubular epithelial (HK-2) cells and its potential mechanisms.",
" Cell culture and drug treatment Renal tubular epithelial cells (HK-2 cells, a proximal tubular cell line derived from normal kidney; CRL-2190) were purchased from ATCC and cultured at 37°C in a humidified atmosphere of 5% CO2 with Dulbecco's modified Eagle's medium (HyClone Pierce, USA) containing 10% fetal bovine serum (FBS; Invitrogen, USA) and 1% ampicillin. The culture medium was refreshed every 2 days until the cell confluence reached 70–80%. Cells were harvested after digestion with 0.25% trypsin/EDTA. The messenger RNA (mRNA) and protein expression were detected after LPS exposure for different durations. LPS extracted from serotype Escherichia coli 0111:B4 was purchased from Sigma (St. Louis, MO, USA). To prepare LPS suspension, LPS was dissolved in dimethyl sulfoxide (DMSO). Concentration of DMSO was <0.1% to reduce its toxicity to HK-2 cells. CLI-095 (Invitrogen, San Diego, CA, USA) is a cyclohexene derivative and an inhibitor of TLR4. CLI-095 was dissolved in DMOS at 10 μmol/l. Cells in control group were cultured in the medium only with DMSO at the same concentration.\nRenal tubular epithelial cells (HK-2 cells, a proximal tubular cell line derived from normal kidney; CRL-2190) were purchased from ATCC and cultured at 37°C in a humidified atmosphere of 5% CO2 with Dulbecco's modified Eagle's medium (HyClone Pierce, USA) containing 10% fetal bovine serum (FBS; Invitrogen, USA) and 1% ampicillin. The culture medium was refreshed every 2 days until the cell confluence reached 70–80%. Cells were harvested after digestion with 0.25% trypsin/EDTA. The messenger RNA (mRNA) and protein expression were detected after LPS exposure for different durations. LPS extracted from serotype Escherichia coli 0111:B4 was purchased from Sigma (St. Louis, MO, USA). To prepare LPS suspension, LPS was dissolved in dimethyl sulfoxide (DMSO). Concentration of DMSO was <0.1% to reduce its toxicity to HK-2 cells. CLI-095 (Invitrogen, San Diego, CA, USA) is a cyclohexene derivative and an inhibitor of TLR4. CLI-095 was dissolved in DMOS at 10 μmol/l. Cells in control group were cultured in the medium only with DMSO at the same concentration.\n Detection of cell viability Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described.[16] HK-2 cells were digested with 0.25% trypsin and then suspended in the medium containing 10% FBS at 5–8 × 104 cells/ml. Cells were cultured into 96-well plates (200 μl/well), followed by incubation for 4–5 h. After cells attached, 100 ng/ml of LPS was added to each well (100 μl/well) and incubated at 37°C with 5% CO2 for 0, 1, 6, and 12 h. Before the detection, 5 mg/ml MTT solution was added to each well (20 μl/well), and then incubated in dark for 4 h. The supernatant was removed, and 150 μl of DMSO was added to each well and incubated for 10 min to resolve the deposit. The absorbance was measured at 490 nm by MTT enzyme-linked immunometric meter (Bio Rad, USA). The cell viability was calculated according to the standard curve.\nCell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described.[16] HK-2 cells were digested with 0.25% trypsin and then suspended in the medium containing 10% FBS at 5–8 × 104 cells/ml. Cells were cultured into 96-well plates (200 μl/well), followed by incubation for 4–5 h. After cells attached, 100 ng/ml of LPS was added to each well (100 μl/well) and incubated at 37°C with 5% CO2 for 0, 1, 6, and 12 h. Before the detection, 5 mg/ml MTT solution was added to each well (20 μl/well), and then incubated in dark for 4 h. The supernatant was removed, and 150 μl of DMSO was added to each well and incubated for 10 min to resolve the deposit. The absorbance was measured at 490 nm by MTT enzyme-linked immunometric meter (Bio Rad, USA). The cell viability was calculated according to the standard curve.\n Real-time polymerase chain reaction Cultured HK-2 cells were used for real-time polymerase chain reaction (PCR) analysis of SP-A and TLR4 mRNA. Total RNA was extracted from each sample using Trizol (Invitrogen, USA) following the manufacturer's instructions. Total RNA (1 μg) extracted from the tissue was used for the RT reaction (Takara, China), and then 2.5 μl of cDNA was used for amplification at a final volume of 25 μl according to the supplier's protocol (Fermentas, Germany). Then, the amplified PCR product was used for melting curve analysis, especially SP-A primer: 5’-TGA AAGGGAGTTCTAGCATCTCA CAGA-3’ and 5’-ACATATGCC TATGTA GGCCTGACT GAG-3’, TLR4 primer: 5’- TATCCAGAGCCGTTGGTGTATCT-3’, and 5’-AATGAAGATG ATGCCA GAGCG -3’, and β-actin primer: 5’-GTCTACATGTCTC GATCCCACTTA A -3’ and 5’-GGTCTTTCTCTCTCAT CGCGCTC-3’. The PCR was performed with 35 cycles of 94°C for 45 s, 60°C for 45 s (SP-A) or 55°C for 50 s (TLR4), and 72°C for 2 min. Each PCR product was subjected to electrophoresis on 2% agarose gel containing 0.1 μg/ml ethidium bromide. DNA bands in the agarose gel were photographed and quantified.\nCultured HK-2 cells were used for real-time polymerase chain reaction (PCR) analysis of SP-A and TLR4 mRNA. Total RNA was extracted from each sample using Trizol (Invitrogen, USA) following the manufacturer's instructions. Total RNA (1 μg) extracted from the tissue was used for the RT reaction (Takara, China), and then 2.5 μl of cDNA was used for amplification at a final volume of 25 μl according to the supplier's protocol (Fermentas, Germany). Then, the amplified PCR product was used for melting curve analysis, especially SP-A primer: 5’-TGA AAGGGAGTTCTAGCATCTCA CAGA-3’ and 5’-ACATATGCC TATGTA GGCCTGACT GAG-3’, TLR4 primer: 5’- TATCCAGAGCCGTTGGTGTATCT-3’, and 5’-AATGAAGATG ATGCCA GAGCG -3’, and β-actin primer: 5’-GTCTACATGTCTC GATCCCACTTA A -3’ and 5’-GGTCTTTCTCTCTCAT CGCGCTC-3’. The PCR was performed with 35 cycles of 94°C for 45 s, 60°C for 45 s (SP-A) or 55°C for 50 s (TLR4), and 72°C for 2 min. Each PCR product was subjected to electrophoresis on 2% agarose gel containing 0.1 μg/ml ethidium bromide. DNA bands in the agarose gel were photographed and quantified.\n Extraction of nuclear proteins Nuclear components were extracted on ice following the method of Chiu et al. with a little bit improvement.[17] HK-2 cells were centrifuged, the deposit was collected and prepared in the 25 μl buffer A (10 mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 1. 5 mmol/L MgCl2; 1 mmol/L DTT; 5% glycerin; 0.2 mmol/L EDTA; 1 mmol/L PMSF; 3 mg/L aprotinin; 3 mg/L leupeptin; 2 mg/L pepstainA; 1% NP-40) for 10 min, and then centrifuged on ice (14,000 r/min) for 1 min; the deposit was prepared in 15 μl buffer B on ice (20 mmol/L HEPES, pH 7.9; 420 mmol/L NaCl; 1.5 mmol/L MgCl2; 0.2 mmol/L EDTA; 0.5 mmol/L DTT; 25% glycerin; 0.5 mmol/L PMSF; 5 mg/L aprotinin; 5 mg/L leupeptin; 3 mg/L pepstainA) for 30 min, and then centrifuged on ice for 15 min (14,000 r/min). Protein concentrations were quantified with a bicinchonic acid protein assay kit (Logan, Utah, Hyclone Pierce).\nNuclear components were extracted on ice following the method of Chiu et al. with a little bit improvement.[17] HK-2 cells were centrifuged, the deposit was collected and prepared in the 25 μl buffer A (10 mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 1. 5 mmol/L MgCl2; 1 mmol/L DTT; 5% glycerin; 0.2 mmol/L EDTA; 1 mmol/L PMSF; 3 mg/L aprotinin; 3 mg/L leupeptin; 2 mg/L pepstainA; 1% NP-40) for 10 min, and then centrifuged on ice (14,000 r/min) for 1 min; the deposit was prepared in 15 μl buffer B on ice (20 mmol/L HEPES, pH 7.9; 420 mmol/L NaCl; 1.5 mmol/L MgCl2; 0.2 mmol/L EDTA; 0.5 mmol/L DTT; 25% glycerin; 0.5 mmol/L PMSF; 5 mg/L aprotinin; 5 mg/L leupeptin; 3 mg/L pepstainA) for 30 min, and then centrifuged on ice for 15 min (14,000 r/min). Protein concentrations were quantified with a bicinchonic acid protein assay kit (Logan, Utah, Hyclone Pierce).\n Western blotting Proteins from HK-2 cells were extracted using RIPA lysate (150 mmol/L sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], and 50 mM Tris at pH 8.0). The total protein concentrations were determined using the bicinchoninic acid protein assay (Logan, Utah, Hyclone Pierce). Total protein (50 μg) was resolved by reducing 12% SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically at 60 mA onto nitrocellulose membranes at 4°C overnight (Bio-Rad, USA). After the samples were blocked in 5% nonfat milk in Tris-buffered saline, immunoblotting was conducted using a primary antibody against SP-A at a dilution of 1:150 or TLR4 at a dilution of 1:500 (Abcam, Britain, ab22048) or NF-kB (Abcam, Britain, ab32536) at a dilution of 1:5000 or IkB-α (Abcam, Britain, ab133462) or MEK1 (Abcam, Britain, ab32091) at a dilution of 1:1000 and phosphorylated MEK1 (p-MEK1) (Abcam, Britain, ab96379) at a dilution of 1:800 or ERK2 (Abcam, Britain, ab32081) at a dilution of 1:1000 and phosphorylated ERK1/2 (Abcam, Britain, ab201015) at a dilution of 1:800 or P38MAPK at a dilution of 1:3000 (Abcam, Britain, ab170099) and phosphorylated P38MAPK (Abcam, Britain, ab47363) at a dilution of 1:500, and then secondary antibody conjugated with horseradish peroxidase. Cellular β-actin protein was immunodetected using a human monoclonal antibody against human β-actin (St. Louis, MO, USA) as the internal standard. Immunoproducts were detected using enhanced chemiluminescence peroxidase detection reagents (Amersham, Sweden).\nProteins from HK-2 cells were extracted using RIPA lysate (150 mmol/L sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], and 50 mM Tris at pH 8.0). The total protein concentrations were determined using the bicinchoninic acid protein assay (Logan, Utah, Hyclone Pierce). Total protein (50 μg) was resolved by reducing 12% SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically at 60 mA onto nitrocellulose membranes at 4°C overnight (Bio-Rad, USA). After the samples were blocked in 5% nonfat milk in Tris-buffered saline, immunoblotting was conducted using a primary antibody against SP-A at a dilution of 1:150 or TLR4 at a dilution of 1:500 (Abcam, Britain, ab22048) or NF-kB (Abcam, Britain, ab32536) at a dilution of 1:5000 or IkB-α (Abcam, Britain, ab133462) or MEK1 (Abcam, Britain, ab32091) at a dilution of 1:1000 and phosphorylated MEK1 (p-MEK1) (Abcam, Britain, ab96379) at a dilution of 1:800 or ERK2 (Abcam, Britain, ab32081) at a dilution of 1:1000 and phosphorylated ERK1/2 (Abcam, Britain, ab201015) at a dilution of 1:800 or P38MAPK at a dilution of 1:3000 (Abcam, Britain, ab170099) and phosphorylated P38MAPK (Abcam, Britain, ab47363) at a dilution of 1:500, and then secondary antibody conjugated with horseradish peroxidase. Cellular β-actin protein was immunodetected using a human monoclonal antibody against human β-actin (St. Louis, MO, USA) as the internal standard. Immunoproducts were detected using enhanced chemiluminescence peroxidase detection reagents (Amersham, Sweden).\n Statistical analysis Data are expressed as mean ± standard error of mean. Results were analyzed by one-way ANOVA using the SPSS version 15.0 software (SPSS Inc., Chicago, Illinois, USA). P < 0.05 was considered to indicate statistical significance.\nData are expressed as mean ± standard error of mean. Results were analyzed by one-way ANOVA using the SPSS version 15.0 software (SPSS Inc., Chicago, Illinois, USA). P < 0.05 was considered to indicate statistical significance.",
"Renal tubular epithelial cells (HK-2 cells, a proximal tubular cell line derived from normal kidney; CRL-2190) were purchased from ATCC and cultured at 37°C in a humidified atmosphere of 5% CO2 with Dulbecco's modified Eagle's medium (HyClone Pierce, USA) containing 10% fetal bovine serum (FBS; Invitrogen, USA) and 1% ampicillin. The culture medium was refreshed every 2 days until the cell confluence reached 70–80%. Cells were harvested after digestion with 0.25% trypsin/EDTA. The messenger RNA (mRNA) and protein expression were detected after LPS exposure for different durations. LPS extracted from serotype Escherichia coli 0111:B4 was purchased from Sigma (St. Louis, MO, USA). To prepare LPS suspension, LPS was dissolved in dimethyl sulfoxide (DMSO). Concentration of DMSO was <0.1% to reduce its toxicity to HK-2 cells. CLI-095 (Invitrogen, San Diego, CA, USA) is a cyclohexene derivative and an inhibitor of TLR4. CLI-095 was dissolved in DMOS at 10 μmol/l. Cells in control group were cultured in the medium only with DMSO at the same concentration.",
"Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described.[16] HK-2 cells were digested with 0.25% trypsin and then suspended in the medium containing 10% FBS at 5–8 × 104 cells/ml. Cells were cultured into 96-well plates (200 μl/well), followed by incubation for 4–5 h. After cells attached, 100 ng/ml of LPS was added to each well (100 μl/well) and incubated at 37°C with 5% CO2 for 0, 1, 6, and 12 h. Before the detection, 5 mg/ml MTT solution was added to each well (20 μl/well), and then incubated in dark for 4 h. The supernatant was removed, and 150 μl of DMSO was added to each well and incubated for 10 min to resolve the deposit. The absorbance was measured at 490 nm by MTT enzyme-linked immunometric meter (Bio Rad, USA). The cell viability was calculated according to the standard curve.",
"Cultured HK-2 cells were used for real-time polymerase chain reaction (PCR) analysis of SP-A and TLR4 mRNA. Total RNA was extracted from each sample using Trizol (Invitrogen, USA) following the manufacturer's instructions. Total RNA (1 μg) extracted from the tissue was used for the RT reaction (Takara, China), and then 2.5 μl of cDNA was used for amplification at a final volume of 25 μl according to the supplier's protocol (Fermentas, Germany). Then, the amplified PCR product was used for melting curve analysis, especially SP-A primer: 5’-TGA AAGGGAGTTCTAGCATCTCA CAGA-3’ and 5’-ACATATGCC TATGTA GGCCTGACT GAG-3’, TLR4 primer: 5’- TATCCAGAGCCGTTGGTGTATCT-3’, and 5’-AATGAAGATG ATGCCA GAGCG -3’, and β-actin primer: 5’-GTCTACATGTCTC GATCCCACTTA A -3’ and 5’-GGTCTTTCTCTCTCAT CGCGCTC-3’. The PCR was performed with 35 cycles of 94°C for 45 s, 60°C for 45 s (SP-A) or 55°C for 50 s (TLR4), and 72°C for 2 min. Each PCR product was subjected to electrophoresis on 2% agarose gel containing 0.1 μg/ml ethidium bromide. DNA bands in the agarose gel were photographed and quantified.",
"Nuclear components were extracted on ice following the method of Chiu et al. with a little bit improvement.[17] HK-2 cells were centrifuged, the deposit was collected and prepared in the 25 μl buffer A (10 mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 1. 5 mmol/L MgCl2; 1 mmol/L DTT; 5% glycerin; 0.2 mmol/L EDTA; 1 mmol/L PMSF; 3 mg/L aprotinin; 3 mg/L leupeptin; 2 mg/L pepstainA; 1% NP-40) for 10 min, and then centrifuged on ice (14,000 r/min) for 1 min; the deposit was prepared in 15 μl buffer B on ice (20 mmol/L HEPES, pH 7.9; 420 mmol/L NaCl; 1.5 mmol/L MgCl2; 0.2 mmol/L EDTA; 0.5 mmol/L DTT; 25% glycerin; 0.5 mmol/L PMSF; 5 mg/L aprotinin; 5 mg/L leupeptin; 3 mg/L pepstainA) for 30 min, and then centrifuged on ice for 15 min (14,000 r/min). Protein concentrations were quantified with a bicinchonic acid protein assay kit (Logan, Utah, Hyclone Pierce).",
"Proteins from HK-2 cells were extracted using RIPA lysate (150 mmol/L sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], and 50 mM Tris at pH 8.0). The total protein concentrations were determined using the bicinchoninic acid protein assay (Logan, Utah, Hyclone Pierce). Total protein (50 μg) was resolved by reducing 12% SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically at 60 mA onto nitrocellulose membranes at 4°C overnight (Bio-Rad, USA). After the samples were blocked in 5% nonfat milk in Tris-buffered saline, immunoblotting was conducted using a primary antibody against SP-A at a dilution of 1:150 or TLR4 at a dilution of 1:500 (Abcam, Britain, ab22048) or NF-kB (Abcam, Britain, ab32536) at a dilution of 1:5000 or IkB-α (Abcam, Britain, ab133462) or MEK1 (Abcam, Britain, ab32091) at a dilution of 1:1000 and phosphorylated MEK1 (p-MEK1) (Abcam, Britain, ab96379) at a dilution of 1:800 or ERK2 (Abcam, Britain, ab32081) at a dilution of 1:1000 and phosphorylated ERK1/2 (Abcam, Britain, ab201015) at a dilution of 1:800 or P38MAPK at a dilution of 1:3000 (Abcam, Britain, ab170099) and phosphorylated P38MAPK (Abcam, Britain, ab47363) at a dilution of 1:500, and then secondary antibody conjugated with horseradish peroxidase. Cellular β-actin protein was immunodetected using a human monoclonal antibody against human β-actin (St. Louis, MO, USA) as the internal standard. Immunoproducts were detected using enhanced chemiluminescence peroxidase detection reagents (Amersham, Sweden).",
"Data are expressed as mean ± standard error of mean. Results were analyzed by one-way ANOVA using the SPSS version 15.0 software (SPSS Inc., Chicago, Illinois, USA). P < 0.05 was considered to indicate statistical significance.",
" Effect of lipopolysaccharide on the cell viability of HK-2 cells To evaluate the cytotoxic effect of LPS on cells, the cell viability of HK-2 cells was detected after LPS exposure. HK-2 cells were stimulated with 100 ng/ml of LPS for 0, 1, 6, and 12 h, and absorbance was measured at 490 nm. The absorbance did not show significant difference at different durations after LPS treatment [Figure 1, P > 0.05], suggesting that 100 ng/ml of LPS is not cytotoxic to HK-2 cells.\nEffects of lipopolysaccharide on the cell viability of HK-2 cells. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 6, 12, and 24 h. Cell viability was assayed by MTT method. Optical density value of HK-2 cells for different durations was detected with colorimetric method. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).\nTo evaluate the cytotoxic effect of LPS on cells, the cell viability of HK-2 cells was detected after LPS exposure. HK-2 cells were stimulated with 100 ng/ml of LPS for 0, 1, 6, and 12 h, and absorbance was measured at 490 nm. The absorbance did not show significant difference at different durations after LPS treatment [Figure 1, P > 0.05], suggesting that 100 ng/ml of LPS is not cytotoxic to HK-2 cells.\nEffects of lipopolysaccharide on the cell viability of HK-2 cells. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 6, 12, and 24 h. Cell viability was assayed by MTT method. Optical density value of HK-2 cells for different durations was detected with colorimetric method. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).\n Effects of lipopolysaccharide on the messenger RNA and protein expression of surfactant protein-A in HK-2 cells Protein and mRNA expression of SP-A in HK-2 cells after LPS treatment were detected by Western blotting and real-time PCR, respectively. Cells were stimulated with LPS for 0, 1, 2, and 6 h, and then SP-A mRNA expression was detected; cells were treated with LPS for 0, 2, 6, and 12 h, and SP-A protein expression was assayed; and β-actin expression was detected as an internal standard. Expression of SP-A mRNA expression at 1, 2, and 6 h after LPS exposure was significantly increased by 1.5, 2.7, and 4.5 folds, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Moreover, the SP-A protein expression at 6 h after LPS treatment increased dramatically when compared with that at 0 h and 2 h [Figure 3, P < 0.05]. After LPS exposure for 12 h, the SP-A protein expression was markedly higher than that at 6 h. SP-A protein expression after LPS exposure for 6 h and 12 h was increased by 1.6 and 2.8 folds, respectively, as compared to that at 0 h. These findings suggested that LPS may upregulate SP-A expression at transcriptional level.\nExpression of surfactant protein-A and toll-like receptor 4 mRNA in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to lipopolysaccharide for 0, 1, 2, and 6 h. β-actin mRNA was detected as the internal standard. Expression of surfactant protein-A and toll-like receptor 4 mRNA was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate that the value of surfactant protein-A and toll-like receptor 4 mRNA expression significantly (P < 0.05) differed from lipopolysaccharide exposure for 0 h, respectively. mRNA: Messenger RNA.\nExpression of surfactant protein-A protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of surfactant protein-A protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nProtein and mRNA expression of SP-A in HK-2 cells after LPS treatment were detected by Western blotting and real-time PCR, respectively. Cells were stimulated with LPS for 0, 1, 2, and 6 h, and then SP-A mRNA expression was detected; cells were treated with LPS for 0, 2, 6, and 12 h, and SP-A protein expression was assayed; and β-actin expression was detected as an internal standard. Expression of SP-A mRNA expression at 1, 2, and 6 h after LPS exposure was significantly increased by 1.5, 2.7, and 4.5 folds, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Moreover, the SP-A protein expression at 6 h after LPS treatment increased dramatically when compared with that at 0 h and 2 h [Figure 3, P < 0.05]. After LPS exposure for 12 h, the SP-A protein expression was markedly higher than that at 6 h. SP-A protein expression after LPS exposure for 6 h and 12 h was increased by 1.6 and 2.8 folds, respectively, as compared to that at 0 h. These findings suggested that LPS may upregulate SP-A expression at transcriptional level.\nExpression of surfactant protein-A and toll-like receptor 4 mRNA in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to lipopolysaccharide for 0, 1, 2, and 6 h. β-actin mRNA was detected as the internal standard. Expression of surfactant protein-A and toll-like receptor 4 mRNA was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate that the value of surfactant protein-A and toll-like receptor 4 mRNA expression significantly (P < 0.05) differed from lipopolysaccharide exposure for 0 h, respectively. mRNA: Messenger RNA.\nExpression of surfactant protein-A protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of surfactant protein-A protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\n Effects of lipopolysaccharide with or without CLI-095 on the nuclear translocation and activation of nuclear factor-κB in HK-2 cells To investigate the mechanism underlying LPS-induced SP-A expression in HK-2 cells, the expression of cytoplasmic and nuclear NF-κB as well as phosphorylated IκB-α was measured in HK-2 cells, and total NF-κB served as an internal standard. After LPS exposure for 0, 1, 2, and 6 h, the cytoplasmic NF-κB expression was decreased [Figure 4, P < 0.05], however the nuclear NF-κB expression and intracellular phosphorylated IκB-α expression were increased [Figure 5, P < 0.05]. The protein expression of nuclear factor-κB (NF-κB) at different time points was quantified, and results showed that the cytoplasmic NF-κB expression at 1, 2, and 6 h after LPS exposure was reduced by 32%, 48%, and 47%, respectively, but expressions of the nuclear NF-κB and phosphorylated IκB-α at 1, 2, and 6 h after LPS exposure were augmented by 117%, 155%, and 203% and by 167%, 320%, and 413%, respectively, as compared to those at 0 h. Moreover, the expression of the nuclear NF-κB and phosphorylated IκB-α was depressed by CLI-095 when compared with by LPS stimulation alone [Figures 4 and 5, P < 0.05].\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced nucleus factor-kB protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. Cytosolic (c) and nucleus (n) factor-kB were immunodetected. Total (t) nucleus factor-kB was detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced phosphorylated IkB-α protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. β-actin protein was detected as the internal standard. Expression of p-IkB-α was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of LPS exposure for 0 h.\nTo investigate the mechanism underlying LPS-induced SP-A expression in HK-2 cells, the expression of cytoplasmic and nuclear NF-κB as well as phosphorylated IκB-α was measured in HK-2 cells, and total NF-κB served as an internal standard. After LPS exposure for 0, 1, 2, and 6 h, the cytoplasmic NF-κB expression was decreased [Figure 4, P < 0.05], however the nuclear NF-κB expression and intracellular phosphorylated IκB-α expression were increased [Figure 5, P < 0.05]. The protein expression of nuclear factor-κB (NF-κB) at different time points was quantified, and results showed that the cytoplasmic NF-κB expression at 1, 2, and 6 h after LPS exposure was reduced by 32%, 48%, and 47%, respectively, but expressions of the nuclear NF-κB and phosphorylated IκB-α at 1, 2, and 6 h after LPS exposure were augmented by 117%, 155%, and 203% and by 167%, 320%, and 413%, respectively, as compared to those at 0 h. Moreover, the expression of the nuclear NF-κB and phosphorylated IκB-α was depressed by CLI-095 when compared with by LPS stimulation alone [Figures 4 and 5, P < 0.05].\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced nucleus factor-kB protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. Cytosolic (c) and nucleus (n) factor-kB were immunodetected. Total (t) nucleus factor-kB was detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced phosphorylated IkB-α protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. β-actin protein was detected as the internal standard. Expression of p-IkB-α was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of LPS exposure for 0 h.\n Effects of lipopolysaccharide on the expression of toll-like receptor 4, phosphorylated p38 mitogen-activated protein kinase, and mitogen-activated/extracellular signal-regulated kinase 1 in HK-2 cells To elucidate the signaling pathway involved in LPS-induced NF-κB activation in HK-2 cells, the mRNA and protein expression of upstream transmembrane receptor TLR4 and protein expression of phosphorylated ERK1/2, phosphorylated p38MAPK, and p-MEK1 were assayed in HK-2 cells. With β-actin expression served as an internal standard, quantitative analysis showed that the levels of TLR4 mRNA after LPS exposure for 1, 2, and 6 h were increased by 111%, 221%, and 489%, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Meanwhile, the expression of TLR4 protein after LPS treatment for 1, 2, and 6 h was also increased [Figure 6, P < 0.05]. Expression of TLR4 protein at 2, 6, and 12 h was increased by 74%, 91%, and 154%, respectively, when compared with that at 0 h (P < 0.05). Phosphorylation of p38MAPK [Figure 7, P < 0.05] and MEK1 [Figure 8, P < 0.05] in HK-2 cells at 2, 6, and 12 h was increased by 75%, 130%, 215%, and 74%, 183%, 262%, respectively, as compared to that at 0 h. Levels of phosphorylated ERK1 in HK-2 cells were obviously raised after exposure to LPS for 2, 6, and 12 h by 62%, 162%, and 254%, respectively. Exposure of HK-2 cells to LPS for 2, 6, and 12 h significantly increased ERK2 phosphorylation by 77%, 239%, and 392%, respectively [Figure 9, P < 0.05].\nExpression of toll-like receptor 4 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of toll-like receptor 4 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of P38MAPK protein in HK-2 cells induced by lipopolysaccharide exposure for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. P38MAPK protein was detected as the internal standard. Expression of p-P38MAPK protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of mitogen-activated/extracellular signal-regulated kinase 1 protein induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Mitogen-activated/extracellular signal-regulated kinase 1 protein was detected as the internal standard. Expression of phosphorylated mitogen-activated/extracellular signal-regulated kinase 1 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of ERK1/2 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. ERK2 protein was detected as the internal standard. Expression of p-ERK1/2 protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h. ERK: Extracellular signal-regulated kinase.\nTo elucidate the signaling pathway involved in LPS-induced NF-κB activation in HK-2 cells, the mRNA and protein expression of upstream transmembrane receptor TLR4 and protein expression of phosphorylated ERK1/2, phosphorylated p38MAPK, and p-MEK1 were assayed in HK-2 cells. With β-actin expression served as an internal standard, quantitative analysis showed that the levels of TLR4 mRNA after LPS exposure for 1, 2, and 6 h were increased by 111%, 221%, and 489%, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Meanwhile, the expression of TLR4 protein after LPS treatment for 1, 2, and 6 h was also increased [Figure 6, P < 0.05]. Expression of TLR4 protein at 2, 6, and 12 h was increased by 74%, 91%, and 154%, respectively, when compared with that at 0 h (P < 0.05). Phosphorylation of p38MAPK [Figure 7, P < 0.05] and MEK1 [Figure 8, P < 0.05] in HK-2 cells at 2, 6, and 12 h was increased by 75%, 130%, 215%, and 74%, 183%, 262%, respectively, as compared to that at 0 h. Levels of phosphorylated ERK1 in HK-2 cells were obviously raised after exposure to LPS for 2, 6, and 12 h by 62%, 162%, and 254%, respectively. Exposure of HK-2 cells to LPS for 2, 6, and 12 h significantly increased ERK2 phosphorylation by 77%, 239%, and 392%, respectively [Figure 9, P < 0.05].\nExpression of toll-like receptor 4 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of toll-like receptor 4 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of P38MAPK protein in HK-2 cells induced by lipopolysaccharide exposure for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. P38MAPK protein was detected as the internal standard. Expression of p-P38MAPK protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of mitogen-activated/extracellular signal-regulated kinase 1 protein induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Mitogen-activated/extracellular signal-regulated kinase 1 protein was detected as the internal standard. Expression of phosphorylated mitogen-activated/extracellular signal-regulated kinase 1 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of ERK1/2 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. ERK2 protein was detected as the internal standard. Expression of p-ERK1/2 protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h. ERK: Extracellular signal-regulated kinase.\n Effects of lipopolysaccharide with or without CLI-095 on the expression of toll-like receptor 4 and surfactant protein-A messenger RNA and protein in HK-2 cells We further explored the role of TLR4-dependent MEK1/2-ERK1/2-NF-κB signaling pathway in LPS-induced SP-A expression. HK-2 cells were pretreated with CLI-095 before LPS exposure. Induction of TLR4 and SP-A mRNA expression by LPS were analyzed by real-time PCR. After LPS administration, the mRNA expression of TLR4 and SP-A was significantly increased [Figure 9, P < 0.05]. Pretreatment of HK-2 cells with CLI-095, an inhibitor of TLR4, before LPS administration could markedly inhibit the mRNA and protein expression of TLR4 and SP-A [Figure 9, P < 0.05]. Expression of TLR4 and SP-A mRNA in HK-2 cells after LPS exposure for 6 h was augmented by 458% and 542%, respectively, when compared with that at 0 h. With CLI-095 pretreatment, mRNA expression of TLR4 and SP-A in HK-2 cells was decreased by 66% and 60%, respectively, when compared with that by LPS administration for 6 h alone [Figure 10, P < 0.05].\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced surfactant protein-A and toll-like receptor 4 messenger RNA expression. HK-2 cells were pretreated with CLI-095 for 1 h, and then exposed to CLI-095 and lipopolysaccharide for another 6 h. Levels of β-actin messenger RNA were detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate and significant (P < 0.05) difference between lipopolysaccharide and CLI-095 + lipopolysaccharide groups for surfactant protein-A and toll-like receptor 4 messenger RNA expression, respectively.\nWe further explored the role of TLR4-dependent MEK1/2-ERK1/2-NF-κB signaling pathway in LPS-induced SP-A expression. HK-2 cells were pretreated with CLI-095 before LPS exposure. Induction of TLR4 and SP-A mRNA expression by LPS were analyzed by real-time PCR. After LPS administration, the mRNA expression of TLR4 and SP-A was significantly increased [Figure 9, P < 0.05]. Pretreatment of HK-2 cells with CLI-095, an inhibitor of TLR4, before LPS administration could markedly inhibit the mRNA and protein expression of TLR4 and SP-A [Figure 9, P < 0.05]. Expression of TLR4 and SP-A mRNA in HK-2 cells after LPS exposure for 6 h was augmented by 458% and 542%, respectively, when compared with that at 0 h. With CLI-095 pretreatment, mRNA expression of TLR4 and SP-A in HK-2 cells was decreased by 66% and 60%, respectively, when compared with that by LPS administration for 6 h alone [Figure 10, P < 0.05].\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced surfactant protein-A and toll-like receptor 4 messenger RNA expression. HK-2 cells were pretreated with CLI-095 for 1 h, and then exposed to CLI-095 and lipopolysaccharide for another 6 h. Levels of β-actin messenger RNA were detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate and significant (P < 0.05) difference between lipopolysaccharide and CLI-095 + lipopolysaccharide groups for surfactant protein-A and toll-like receptor 4 messenger RNA expression, respectively.",
"To evaluate the cytotoxic effect of LPS on cells, the cell viability of HK-2 cells was detected after LPS exposure. HK-2 cells were stimulated with 100 ng/ml of LPS for 0, 1, 6, and 12 h, and absorbance was measured at 490 nm. The absorbance did not show significant difference at different durations after LPS treatment [Figure 1, P > 0.05], suggesting that 100 ng/ml of LPS is not cytotoxic to HK-2 cells.\nEffects of lipopolysaccharide on the cell viability of HK-2 cells. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 6, 12, and 24 h. Cell viability was assayed by MTT method. Optical density value of HK-2 cells for different durations was detected with colorimetric method. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).",
"Protein and mRNA expression of SP-A in HK-2 cells after LPS treatment were detected by Western blotting and real-time PCR, respectively. Cells were stimulated with LPS for 0, 1, 2, and 6 h, and then SP-A mRNA expression was detected; cells were treated with LPS for 0, 2, 6, and 12 h, and SP-A protein expression was assayed; and β-actin expression was detected as an internal standard. Expression of SP-A mRNA expression at 1, 2, and 6 h after LPS exposure was significantly increased by 1.5, 2.7, and 4.5 folds, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Moreover, the SP-A protein expression at 6 h after LPS treatment increased dramatically when compared with that at 0 h and 2 h [Figure 3, P < 0.05]. After LPS exposure for 12 h, the SP-A protein expression was markedly higher than that at 6 h. SP-A protein expression after LPS exposure for 6 h and 12 h was increased by 1.6 and 2.8 folds, respectively, as compared to that at 0 h. These findings suggested that LPS may upregulate SP-A expression at transcriptional level.\nExpression of surfactant protein-A and toll-like receptor 4 mRNA in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to lipopolysaccharide for 0, 1, 2, and 6 h. β-actin mRNA was detected as the internal standard. Expression of surfactant protein-A and toll-like receptor 4 mRNA was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate that the value of surfactant protein-A and toll-like receptor 4 mRNA expression significantly (P < 0.05) differed from lipopolysaccharide exposure for 0 h, respectively. mRNA: Messenger RNA.\nExpression of surfactant protein-A protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of surfactant protein-A protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.",
"To investigate the mechanism underlying LPS-induced SP-A expression in HK-2 cells, the expression of cytoplasmic and nuclear NF-κB as well as phosphorylated IκB-α was measured in HK-2 cells, and total NF-κB served as an internal standard. After LPS exposure for 0, 1, 2, and 6 h, the cytoplasmic NF-κB expression was decreased [Figure 4, P < 0.05], however the nuclear NF-κB expression and intracellular phosphorylated IκB-α expression were increased [Figure 5, P < 0.05]. The protein expression of nuclear factor-κB (NF-κB) at different time points was quantified, and results showed that the cytoplasmic NF-κB expression at 1, 2, and 6 h after LPS exposure was reduced by 32%, 48%, and 47%, respectively, but expressions of the nuclear NF-κB and phosphorylated IκB-α at 1, 2, and 6 h after LPS exposure were augmented by 117%, 155%, and 203% and by 167%, 320%, and 413%, respectively, as compared to those at 0 h. Moreover, the expression of the nuclear NF-κB and phosphorylated IκB-α was depressed by CLI-095 when compared with by LPS stimulation alone [Figures 4 and 5, P < 0.05].\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced nucleus factor-kB protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. Cytosolic (c) and nucleus (n) factor-kB were immunodetected. Total (t) nucleus factor-kB was detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced phosphorylated IkB-α protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. β-actin protein was detected as the internal standard. Expression of p-IkB-α was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of LPS exposure for 0 h.",
"To elucidate the signaling pathway involved in LPS-induced NF-κB activation in HK-2 cells, the mRNA and protein expression of upstream transmembrane receptor TLR4 and protein expression of phosphorylated ERK1/2, phosphorylated p38MAPK, and p-MEK1 were assayed in HK-2 cells. With β-actin expression served as an internal standard, quantitative analysis showed that the levels of TLR4 mRNA after LPS exposure for 1, 2, and 6 h were increased by 111%, 221%, and 489%, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Meanwhile, the expression of TLR4 protein after LPS treatment for 1, 2, and 6 h was also increased [Figure 6, P < 0.05]. Expression of TLR4 protein at 2, 6, and 12 h was increased by 74%, 91%, and 154%, respectively, when compared with that at 0 h (P < 0.05). Phosphorylation of p38MAPK [Figure 7, P < 0.05] and MEK1 [Figure 8, P < 0.05] in HK-2 cells at 2, 6, and 12 h was increased by 75%, 130%, 215%, and 74%, 183%, 262%, respectively, as compared to that at 0 h. Levels of phosphorylated ERK1 in HK-2 cells were obviously raised after exposure to LPS for 2, 6, and 12 h by 62%, 162%, and 254%, respectively. Exposure of HK-2 cells to LPS for 2, 6, and 12 h significantly increased ERK2 phosphorylation by 77%, 239%, and 392%, respectively [Figure 9, P < 0.05].\nExpression of toll-like receptor 4 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of toll-like receptor 4 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of P38MAPK protein in HK-2 cells induced by lipopolysaccharide exposure for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. P38MAPK protein was detected as the internal standard. Expression of p-P38MAPK protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of mitogen-activated/extracellular signal-regulated kinase 1 protein induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Mitogen-activated/extracellular signal-regulated kinase 1 protein was detected as the internal standard. Expression of phosphorylated mitogen-activated/extracellular signal-regulated kinase 1 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.\nExpression of phosphorylation of ERK1/2 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. ERK2 protein was detected as the internal standard. Expression of p-ERK1/2 protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h. ERK: Extracellular signal-regulated kinase.",
"We further explored the role of TLR4-dependent MEK1/2-ERK1/2-NF-κB signaling pathway in LPS-induced SP-A expression. HK-2 cells were pretreated with CLI-095 before LPS exposure. Induction of TLR4 and SP-A mRNA expression by LPS were analyzed by real-time PCR. After LPS administration, the mRNA expression of TLR4 and SP-A was significantly increased [Figure 9, P < 0.05]. Pretreatment of HK-2 cells with CLI-095, an inhibitor of TLR4, before LPS administration could markedly inhibit the mRNA and protein expression of TLR4 and SP-A [Figure 9, P < 0.05]. Expression of TLR4 and SP-A mRNA in HK-2 cells after LPS exposure for 6 h was augmented by 458% and 542%, respectively, when compared with that at 0 h. With CLI-095 pretreatment, mRNA expression of TLR4 and SP-A in HK-2 cells was decreased by 66% and 60%, respectively, when compared with that by LPS administration for 6 h alone [Figure 10, P < 0.05].\nEffects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced surfactant protein-A and toll-like receptor 4 messenger RNA expression. HK-2 cells were pretreated with CLI-095 for 1 h, and then exposed to CLI-095 and lipopolysaccharide for another 6 h. Levels of β-actin messenger RNA were detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate and significant (P < 0.05) difference between lipopolysaccharide and CLI-095 + lipopolysaccharide groups for surfactant protein-A and toll-like receptor 4 messenger RNA expression, respectively.",
"The present study revealed that LPS can cause inflammatory responses in renal tubular epithelial HK-2 cells by means of enhancing SP-A mRNA and protein syntheses. Moreover, the signal-transducing mechanisms of LPS-induced regulation of SP-A expression arise through TLR4-dependent cascade phosphorylations of MEK1, ERK1/2, and p38MAPK, leading to the nuclear translocation and activation of NF-κB. Moreover, pretreatment with TLR4 inhibitor, CLI-095, may significantly decrease mRNA and protein expression of SP-A, depress IκB-α phosphorylation, and suppress NF-κB nuclear translocation. These findings indicate that LPS may induce SP-A synthesis through TLR4-mediated activation of MEK1-ERK1/2-NF-κB signaling pathway.\nLPS is a major pathogenic factor in septic shock and multiple organ dysfuction syndrome (MODS) caused by Gram-negative bacteria.[18] In vitro study indicated that the elevated SP-A expression in A549 cells could be induced by LPS in a time-dependent manner.[19] Our previous study showed that SP-A protein could be expressed and secreted by renal tubular epithelial cells.[15] SP-A may play important roles in anti-inflammatory and protective effects in both lung and kidney.[7] In the present study, 100 ng/ml of LPS was used to stimulate HK-2 cells (a human renal tubular epithelial cell line), however cytotoxic effects of LPS on HK-2 cells were not observed. In addition, expression of SP-A protein and mRNA in HK-2 cells was found to be increased in a time-dependent manner by LPS administration, moreover elevated mRNA expression of SP-A occurred earlier than the elevated protein expression. This indicated that LPS may influence the SP-A expression at transcriptional level.\nTLR4 is an transmembrane receptor involved in the innate immunity, which can recognize pathogen-associated molecular pattern (including LPS, a component of Gram-negative bacterial cell wall and part of Gram-positive bacteria) to modulate cytokine secretion in the immune response. Thus, the regulation of TLR4 expression may be involved in the innate immunity to pathogens.[20] Studies have showed that LPS may induce the aggregation of TLR4 on the THP1 cells to promote the transmission of extracellular signals into cells, leading to release of a large amount of pro-inflammatory cytokines such as TNF-α.[21] Differential TLR subtypes would be activated by LPS in different types of cells. LPS can significantly stimulate the TLR2 expression on A549 cells to activate downstream signaling pathway.[19] In the present study, HK-2 cells were stimulated by 100 ng/ml of LPS, significantly increased expression of TLR4 mRNA and protein in a time-dependent manner was observed, which indicated that TLR4 activation stimulated by LPS may be involved in the process of SP-A induction of HK-2 cells.\nNF-κB is a well-known transcription factor that regulates the expression of many inflammation-related genes in response to various infections.[22] When cells are stimulated, the NF-κB inhibitor-alpha (IκB) of NF-kB/IκB complex in the cytoplasm is phosphorylated and dissociated from this complex, and then NF-κB (p50 and p65) translocates from the cytoplasm into the nucleus, leading to NF-κB activation, which is related to MAPK pathway. Bacterial cell wall is able to activate NF-κB in the kidney in vivo, and the inhibition of NF-κB activity may ameliorate sepsis-induced AKI.[23] It has been confirmed in the present study that the expression of phosphorylated IkB-α and the nuclear NF-κB protein was significantly increased, but the expression of cytoplasmic NF-κB was decreased dramatically in HK-2 cells by LPS exposure, suggesting the translocation of NF-κB from the cytoplasm to the nucleus, and the subsequent activation of NF-κB in HK-2 cells would be regulated by LPS. There is growing evidence showing that LPS-induced NF-κB activation is related to TLR4-mediated MyD88-dependent signaling pathway.\nMAPK family consists of three protein kinases: ERK, p38MAPK, and c-JNK, which are the upstream kinases of NF-κB.[11] ERK1/2 activation may further activate NF-κB. The ERK1/2-induced activation of IκB-α kinase through phosphorylating two serine residuals at the N-terminal of IκB-α would lead to the degradation of IκB-α. Then, NF-κB translocates into the nucleus, which contributes to its activation.[24] NF-κB activation induced by LPS may be due to the phosphorylation of MAPKs. It has been validated in in vitro studies that the phosphorylation of MAPKs (including ERK1/2 and p38MAPK) in LPS-treated A549 cells was determined.[19] In addition, p38MAPK also plays an important role in the NF-κB activation signaling pathway.[25] In the present study, LPS time dependently increased the expression of phosphorylated ERK1/2 and p38MAPK in HK-2 cells. Thus, the phosphorylation of ERK1/2 and p38MAPK, two important members of MAPK family, may participate in NF-κB activation in HK-2 cells stimulated by LPS.\nIncreasing evidences indicate that ERK signaling pathway is related to the regulation of inflammation.[26] ERK activation is modulated by three different signaling pathways: Raf/MEK-dependent signaling pathway, non-PI3K/Raf-dependent signaling pathway, and unknown signaling pathway that can directly activate ERK protein kinase. MEK1 is an upstream protein kinase of ERK1/2, and the activation of MEK1 is associated with ERK1/2 phosphorylation.[27] Roles of MEK1 and ERK1/2 in SP-A expression induced by LPS were also determined in the previous study using alveolar epithelial A549 cells.[19] Our results exhibited that phosphorylation of ERK1/2 by LPS was due to the MEK1 phosphorylation. Thus, LPS-induced phosphorylation of MEK1 and ERK1/2 may be related to the subsequent NF-κB activation in HK-2 cells. Moreover, suppressing TLR4 expression with CLI-095 simultaneously inhibited LPS-induced NF-κB nuclear translocation and subsequent SP-A mRNA expression. Thus, our study indicated that the signal-transducing mechanisms of LPS-caused regulation of SP-A expression arise in renal tubular epithelial HK-2 cells through TLR4 activation and cascade phosphorylations of downstream kinase MEK1 and MAPK (ERK1/2 and p38MAPK). In succession, LPS induced NF-κB expression and translocation.\nHowever, there are certain limitations of the present study. HK-2 cells are not primary culture cells, and results in this study might be a little bit different from those in vivo. Thus, more translational studies are needed to explore the effect of LPS on the secretion of SP-A in human renal tubular epithelial cells in case of AKI.\n Financial support and sponsorship This study was supported by the National Natural Science Foundation of China (No. 81201457, No. 81173402, and No. 81571943) and Advanced Suitable Project of Shanghai Health System (No. 2013SY070).\nThis study was supported by the National Natural Science Foundation of China (No. 81201457, No. 81173402, and No. 81571943) and Advanced Suitable Project of Shanghai Health System (No. 2013SY070).\n Conflicts of interest There are no conflicts of interest.\nThere are no conflicts of interest.",
"This study was supported by the National Natural Science Foundation of China (No. 81201457, No. 81173402, and No. 81571943) and Advanced Suitable Project of Shanghai Health System (No. 2013SY070).",
"There are no conflicts of interest."
] |
[
"intro",
"methods",
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
"discussion",
null,
"COI-statement"
] |
[
"Acute Kidney Injury",
"HK-2 Cells",
"Lipopolysaccharide",
"Signal Pathway",
"Surfactant Protein-A"
] |
Introduction:
Acute kidney injury (AKI) is always caused by sepsis and septic shock, the incidence of which is closely related to the severity of infection. About 19% of sepsis patients, 23% of severe sepsis patients, and 51% of patients with septic shock would accompany with AKI.[1] Clinical studies have showed that AKI may be an independent predictor of death, and the mortality of patients accompanied with AKI is as high as 74.5%.[2] There is growing evidence showing that 62.5% of sepsis in Chinese patients is caused by Gram-negative bacteria, and the lipopolysaccharide (LPS) on the bacterial cell wall is a major pathogenic factor of these Gram-negative bacteria.[3]
Surfactant protein-A (SP-A) was initially identified in the pulmonary epithelial cells. It is synthesized and secreted by alveolar Type II epithelial cells and encoded by two genes (SP-A1 and SP-A2). SP-A can reduce alveolar surface tension to keep the alveoli expanded.[4] In addition, it also plays important roles in the pulmonary immunity response and inflammatory regulation. Levels of tumor necrosis factor-alpha (TNF-α), pulmonary capillary permeability, and apoptotic cells in pulmonary were significantly increased in SP-A knockout mice caused by cisplatin-induced acute lung injury.[5] LPS-induced inflammation and apoptosis of pulmonary epithelial cells could be effectively depressed by intra-tracheal administration of exogenous SP-A.[6] Our previous study indicated that SP-A knockout mice exhibited more severe acute kidney injury (AKI) induced by sepsis.[7] Meanwhile, SP-A prevents renal tubular epithelial cell injury from sepsis through inhibiting nuclear factor-kappa B (NF-kB) activity to modulate TNF-α expression.[8] However, the underlying mechanisms of LPS-induced SP-A expression in renal tubular epithelial cells are still unclear.
Toll-like receptors (TLRs) are Type I transmembrane protein and composed of extracellular domain rich in leucine repeat sequence and intracellular domain. TLR4 expression has been found in the epithelium of multiple organs (such as alveolar epithelial cells and renal tubular epithelial cells).[9] LPS can bind to the TLR4/MD-2 complex on the membrane of renal tubular epithelial cells to activate intracellular signaling pathways.[10] Mitogen-activated protein kinase (MAPK) belongs to serine/threonine kinase extracellular signal-regulated kinase (ERK). MAPK family consists of ERK, p38MAPK, and C-Jun N-terminal kinase (JNK).[11] ERK, including ERK1 and ERK2, is crucial for the signal transmission from extracellular to nucleus.[12] It has been confirmed that ERK1/2 can be activated by mitogen-activated protein/ERK kinase (MEKs), and then may cause the translocation of cytoplasmic nuclear factor-κB (NF-κB) into nucleus, leading to NF-κB activation.[13] A variety of signaling pathways participated in TLR4 activation,[14] our previous study showed that the secretion of SP-A could be significantly induced by LPS in HK-2 cells,[15] but the possible mechanism is still unknown. In this study, we attempted to investigate the effects of LPS on the SP-A expression in human renal tubular epithelial (HK-2) cells and its potential mechanisms.
Methods:
Cell culture and drug treatment Renal tubular epithelial cells (HK-2 cells, a proximal tubular cell line derived from normal kidney; CRL-2190) were purchased from ATCC and cultured at 37°C in a humidified atmosphere of 5% CO2 with Dulbecco's modified Eagle's medium (HyClone Pierce, USA) containing 10% fetal bovine serum (FBS; Invitrogen, USA) and 1% ampicillin. The culture medium was refreshed every 2 days until the cell confluence reached 70–80%. Cells were harvested after digestion with 0.25% trypsin/EDTA. The messenger RNA (mRNA) and protein expression were detected after LPS exposure for different durations. LPS extracted from serotype Escherichia coli 0111:B4 was purchased from Sigma (St. Louis, MO, USA). To prepare LPS suspension, LPS was dissolved in dimethyl sulfoxide (DMSO). Concentration of DMSO was <0.1% to reduce its toxicity to HK-2 cells. CLI-095 (Invitrogen, San Diego, CA, USA) is a cyclohexene derivative and an inhibitor of TLR4. CLI-095 was dissolved in DMOS at 10 μmol/l. Cells in control group were cultured in the medium only with DMSO at the same concentration.
Renal tubular epithelial cells (HK-2 cells, a proximal tubular cell line derived from normal kidney; CRL-2190) were purchased from ATCC and cultured at 37°C in a humidified atmosphere of 5% CO2 with Dulbecco's modified Eagle's medium (HyClone Pierce, USA) containing 10% fetal bovine serum (FBS; Invitrogen, USA) and 1% ampicillin. The culture medium was refreshed every 2 days until the cell confluence reached 70–80%. Cells were harvested after digestion with 0.25% trypsin/EDTA. The messenger RNA (mRNA) and protein expression were detected after LPS exposure for different durations. LPS extracted from serotype Escherichia coli 0111:B4 was purchased from Sigma (St. Louis, MO, USA). To prepare LPS suspension, LPS was dissolved in dimethyl sulfoxide (DMSO). Concentration of DMSO was <0.1% to reduce its toxicity to HK-2 cells. CLI-095 (Invitrogen, San Diego, CA, USA) is a cyclohexene derivative and an inhibitor of TLR4. CLI-095 was dissolved in DMOS at 10 μmol/l. Cells in control group were cultured in the medium only with DMSO at the same concentration.
Detection of cell viability Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described.[16] HK-2 cells were digested with 0.25% trypsin and then suspended in the medium containing 10% FBS at 5–8 × 104 cells/ml. Cells were cultured into 96-well plates (200 μl/well), followed by incubation for 4–5 h. After cells attached, 100 ng/ml of LPS was added to each well (100 μl/well) and incubated at 37°C with 5% CO2 for 0, 1, 6, and 12 h. Before the detection, 5 mg/ml MTT solution was added to each well (20 μl/well), and then incubated in dark for 4 h. The supernatant was removed, and 150 μl of DMSO was added to each well and incubated for 10 min to resolve the deposit. The absorbance was measured at 490 nm by MTT enzyme-linked immunometric meter (Bio Rad, USA). The cell viability was calculated according to the standard curve.
Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described.[16] HK-2 cells were digested with 0.25% trypsin and then suspended in the medium containing 10% FBS at 5–8 × 104 cells/ml. Cells were cultured into 96-well plates (200 μl/well), followed by incubation for 4–5 h. After cells attached, 100 ng/ml of LPS was added to each well (100 μl/well) and incubated at 37°C with 5% CO2 for 0, 1, 6, and 12 h. Before the detection, 5 mg/ml MTT solution was added to each well (20 μl/well), and then incubated in dark for 4 h. The supernatant was removed, and 150 μl of DMSO was added to each well and incubated for 10 min to resolve the deposit. The absorbance was measured at 490 nm by MTT enzyme-linked immunometric meter (Bio Rad, USA). The cell viability was calculated according to the standard curve.
Real-time polymerase chain reaction Cultured HK-2 cells were used for real-time polymerase chain reaction (PCR) analysis of SP-A and TLR4 mRNA. Total RNA was extracted from each sample using Trizol (Invitrogen, USA) following the manufacturer's instructions. Total RNA (1 μg) extracted from the tissue was used for the RT reaction (Takara, China), and then 2.5 μl of cDNA was used for amplification at a final volume of 25 μl according to the supplier's protocol (Fermentas, Germany). Then, the amplified PCR product was used for melting curve analysis, especially SP-A primer: 5’-TGA AAGGGAGTTCTAGCATCTCA CAGA-3’ and 5’-ACATATGCC TATGTA GGCCTGACT GAG-3’, TLR4 primer: 5’- TATCCAGAGCCGTTGGTGTATCT-3’, and 5’-AATGAAGATG ATGCCA GAGCG -3’, and β-actin primer: 5’-GTCTACATGTCTC GATCCCACTTA A -3’ and 5’-GGTCTTTCTCTCTCAT CGCGCTC-3’. The PCR was performed with 35 cycles of 94°C for 45 s, 60°C for 45 s (SP-A) or 55°C for 50 s (TLR4), and 72°C for 2 min. Each PCR product was subjected to electrophoresis on 2% agarose gel containing 0.1 μg/ml ethidium bromide. DNA bands in the agarose gel were photographed and quantified.
Cultured HK-2 cells were used for real-time polymerase chain reaction (PCR) analysis of SP-A and TLR4 mRNA. Total RNA was extracted from each sample using Trizol (Invitrogen, USA) following the manufacturer's instructions. Total RNA (1 μg) extracted from the tissue was used for the RT reaction (Takara, China), and then 2.5 μl of cDNA was used for amplification at a final volume of 25 μl according to the supplier's protocol (Fermentas, Germany). Then, the amplified PCR product was used for melting curve analysis, especially SP-A primer: 5’-TGA AAGGGAGTTCTAGCATCTCA CAGA-3’ and 5’-ACATATGCC TATGTA GGCCTGACT GAG-3’, TLR4 primer: 5’- TATCCAGAGCCGTTGGTGTATCT-3’, and 5’-AATGAAGATG ATGCCA GAGCG -3’, and β-actin primer: 5’-GTCTACATGTCTC GATCCCACTTA A -3’ and 5’-GGTCTTTCTCTCTCAT CGCGCTC-3’. The PCR was performed with 35 cycles of 94°C for 45 s, 60°C for 45 s (SP-A) or 55°C for 50 s (TLR4), and 72°C for 2 min. Each PCR product was subjected to electrophoresis on 2% agarose gel containing 0.1 μg/ml ethidium bromide. DNA bands in the agarose gel were photographed and quantified.
Extraction of nuclear proteins Nuclear components were extracted on ice following the method of Chiu et al. with a little bit improvement.[17] HK-2 cells were centrifuged, the deposit was collected and prepared in the 25 μl buffer A (10 mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 1. 5 mmol/L MgCl2; 1 mmol/L DTT; 5% glycerin; 0.2 mmol/L EDTA; 1 mmol/L PMSF; 3 mg/L aprotinin; 3 mg/L leupeptin; 2 mg/L pepstainA; 1% NP-40) for 10 min, and then centrifuged on ice (14,000 r/min) for 1 min; the deposit was prepared in 15 μl buffer B on ice (20 mmol/L HEPES, pH 7.9; 420 mmol/L NaCl; 1.5 mmol/L MgCl2; 0.2 mmol/L EDTA; 0.5 mmol/L DTT; 25% glycerin; 0.5 mmol/L PMSF; 5 mg/L aprotinin; 5 mg/L leupeptin; 3 mg/L pepstainA) for 30 min, and then centrifuged on ice for 15 min (14,000 r/min). Protein concentrations were quantified with a bicinchonic acid protein assay kit (Logan, Utah, Hyclone Pierce).
Nuclear components were extracted on ice following the method of Chiu et al. with a little bit improvement.[17] HK-2 cells were centrifuged, the deposit was collected and prepared in the 25 μl buffer A (10 mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 1. 5 mmol/L MgCl2; 1 mmol/L DTT; 5% glycerin; 0.2 mmol/L EDTA; 1 mmol/L PMSF; 3 mg/L aprotinin; 3 mg/L leupeptin; 2 mg/L pepstainA; 1% NP-40) for 10 min, and then centrifuged on ice (14,000 r/min) for 1 min; the deposit was prepared in 15 μl buffer B on ice (20 mmol/L HEPES, pH 7.9; 420 mmol/L NaCl; 1.5 mmol/L MgCl2; 0.2 mmol/L EDTA; 0.5 mmol/L DTT; 25% glycerin; 0.5 mmol/L PMSF; 5 mg/L aprotinin; 5 mg/L leupeptin; 3 mg/L pepstainA) for 30 min, and then centrifuged on ice for 15 min (14,000 r/min). Protein concentrations were quantified with a bicinchonic acid protein assay kit (Logan, Utah, Hyclone Pierce).
Western blotting Proteins from HK-2 cells were extracted using RIPA lysate (150 mmol/L sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], and 50 mM Tris at pH 8.0). The total protein concentrations were determined using the bicinchoninic acid protein assay (Logan, Utah, Hyclone Pierce). Total protein (50 μg) was resolved by reducing 12% SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically at 60 mA onto nitrocellulose membranes at 4°C overnight (Bio-Rad, USA). After the samples were blocked in 5% nonfat milk in Tris-buffered saline, immunoblotting was conducted using a primary antibody against SP-A at a dilution of 1:150 or TLR4 at a dilution of 1:500 (Abcam, Britain, ab22048) or NF-kB (Abcam, Britain, ab32536) at a dilution of 1:5000 or IkB-α (Abcam, Britain, ab133462) or MEK1 (Abcam, Britain, ab32091) at a dilution of 1:1000 and phosphorylated MEK1 (p-MEK1) (Abcam, Britain, ab96379) at a dilution of 1:800 or ERK2 (Abcam, Britain, ab32081) at a dilution of 1:1000 and phosphorylated ERK1/2 (Abcam, Britain, ab201015) at a dilution of 1:800 or P38MAPK at a dilution of 1:3000 (Abcam, Britain, ab170099) and phosphorylated P38MAPK (Abcam, Britain, ab47363) at a dilution of 1:500, and then secondary antibody conjugated with horseradish peroxidase. Cellular β-actin protein was immunodetected using a human monoclonal antibody against human β-actin (St. Louis, MO, USA) as the internal standard. Immunoproducts were detected using enhanced chemiluminescence peroxidase detection reagents (Amersham, Sweden).
Proteins from HK-2 cells were extracted using RIPA lysate (150 mmol/L sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], and 50 mM Tris at pH 8.0). The total protein concentrations were determined using the bicinchoninic acid protein assay (Logan, Utah, Hyclone Pierce). Total protein (50 μg) was resolved by reducing 12% SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically at 60 mA onto nitrocellulose membranes at 4°C overnight (Bio-Rad, USA). After the samples were blocked in 5% nonfat milk in Tris-buffered saline, immunoblotting was conducted using a primary antibody against SP-A at a dilution of 1:150 or TLR4 at a dilution of 1:500 (Abcam, Britain, ab22048) or NF-kB (Abcam, Britain, ab32536) at a dilution of 1:5000 or IkB-α (Abcam, Britain, ab133462) or MEK1 (Abcam, Britain, ab32091) at a dilution of 1:1000 and phosphorylated MEK1 (p-MEK1) (Abcam, Britain, ab96379) at a dilution of 1:800 or ERK2 (Abcam, Britain, ab32081) at a dilution of 1:1000 and phosphorylated ERK1/2 (Abcam, Britain, ab201015) at a dilution of 1:800 or P38MAPK at a dilution of 1:3000 (Abcam, Britain, ab170099) and phosphorylated P38MAPK (Abcam, Britain, ab47363) at a dilution of 1:500, and then secondary antibody conjugated with horseradish peroxidase. Cellular β-actin protein was immunodetected using a human monoclonal antibody against human β-actin (St. Louis, MO, USA) as the internal standard. Immunoproducts were detected using enhanced chemiluminescence peroxidase detection reagents (Amersham, Sweden).
Statistical analysis Data are expressed as mean ± standard error of mean. Results were analyzed by one-way ANOVA using the SPSS version 15.0 software (SPSS Inc., Chicago, Illinois, USA). P < 0.05 was considered to indicate statistical significance.
Data are expressed as mean ± standard error of mean. Results were analyzed by one-way ANOVA using the SPSS version 15.0 software (SPSS Inc., Chicago, Illinois, USA). P < 0.05 was considered to indicate statistical significance.
Cell culture and drug treatment:
Renal tubular epithelial cells (HK-2 cells, a proximal tubular cell line derived from normal kidney; CRL-2190) were purchased from ATCC and cultured at 37°C in a humidified atmosphere of 5% CO2 with Dulbecco's modified Eagle's medium (HyClone Pierce, USA) containing 10% fetal bovine serum (FBS; Invitrogen, USA) and 1% ampicillin. The culture medium was refreshed every 2 days until the cell confluence reached 70–80%. Cells were harvested after digestion with 0.25% trypsin/EDTA. The messenger RNA (mRNA) and protein expression were detected after LPS exposure for different durations. LPS extracted from serotype Escherichia coli 0111:B4 was purchased from Sigma (St. Louis, MO, USA). To prepare LPS suspension, LPS was dissolved in dimethyl sulfoxide (DMSO). Concentration of DMSO was <0.1% to reduce its toxicity to HK-2 cells. CLI-095 (Invitrogen, San Diego, CA, USA) is a cyclohexene derivative and an inhibitor of TLR4. CLI-095 was dissolved in DMOS at 10 μmol/l. Cells in control group were cultured in the medium only with DMSO at the same concentration.
Detection of cell viability:
Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described.[16] HK-2 cells were digested with 0.25% trypsin and then suspended in the medium containing 10% FBS at 5–8 × 104 cells/ml. Cells were cultured into 96-well plates (200 μl/well), followed by incubation for 4–5 h. After cells attached, 100 ng/ml of LPS was added to each well (100 μl/well) and incubated at 37°C with 5% CO2 for 0, 1, 6, and 12 h. Before the detection, 5 mg/ml MTT solution was added to each well (20 μl/well), and then incubated in dark for 4 h. The supernatant was removed, and 150 μl of DMSO was added to each well and incubated for 10 min to resolve the deposit. The absorbance was measured at 490 nm by MTT enzyme-linked immunometric meter (Bio Rad, USA). The cell viability was calculated according to the standard curve.
Real-time polymerase chain reaction:
Cultured HK-2 cells were used for real-time polymerase chain reaction (PCR) analysis of SP-A and TLR4 mRNA. Total RNA was extracted from each sample using Trizol (Invitrogen, USA) following the manufacturer's instructions. Total RNA (1 μg) extracted from the tissue was used for the RT reaction (Takara, China), and then 2.5 μl of cDNA was used for amplification at a final volume of 25 μl according to the supplier's protocol (Fermentas, Germany). Then, the amplified PCR product was used for melting curve analysis, especially SP-A primer: 5’-TGA AAGGGAGTTCTAGCATCTCA CAGA-3’ and 5’-ACATATGCC TATGTA GGCCTGACT GAG-3’, TLR4 primer: 5’- TATCCAGAGCCGTTGGTGTATCT-3’, and 5’-AATGAAGATG ATGCCA GAGCG -3’, and β-actin primer: 5’-GTCTACATGTCTC GATCCCACTTA A -3’ and 5’-GGTCTTTCTCTCTCAT CGCGCTC-3’. The PCR was performed with 35 cycles of 94°C for 45 s, 60°C for 45 s (SP-A) or 55°C for 50 s (TLR4), and 72°C for 2 min. Each PCR product was subjected to electrophoresis on 2% agarose gel containing 0.1 μg/ml ethidium bromide. DNA bands in the agarose gel were photographed and quantified.
Extraction of nuclear proteins:
Nuclear components were extracted on ice following the method of Chiu et al. with a little bit improvement.[17] HK-2 cells were centrifuged, the deposit was collected and prepared in the 25 μl buffer A (10 mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 1. 5 mmol/L MgCl2; 1 mmol/L DTT; 5% glycerin; 0.2 mmol/L EDTA; 1 mmol/L PMSF; 3 mg/L aprotinin; 3 mg/L leupeptin; 2 mg/L pepstainA; 1% NP-40) for 10 min, and then centrifuged on ice (14,000 r/min) for 1 min; the deposit was prepared in 15 μl buffer B on ice (20 mmol/L HEPES, pH 7.9; 420 mmol/L NaCl; 1.5 mmol/L MgCl2; 0.2 mmol/L EDTA; 0.5 mmol/L DTT; 25% glycerin; 0.5 mmol/L PMSF; 5 mg/L aprotinin; 5 mg/L leupeptin; 3 mg/L pepstainA) for 30 min, and then centrifuged on ice for 15 min (14,000 r/min). Protein concentrations were quantified with a bicinchonic acid protein assay kit (Logan, Utah, Hyclone Pierce).
Western blotting:
Proteins from HK-2 cells were extracted using RIPA lysate (150 mmol/L sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], and 50 mM Tris at pH 8.0). The total protein concentrations were determined using the bicinchoninic acid protein assay (Logan, Utah, Hyclone Pierce). Total protein (50 μg) was resolved by reducing 12% SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically at 60 mA onto nitrocellulose membranes at 4°C overnight (Bio-Rad, USA). After the samples were blocked in 5% nonfat milk in Tris-buffered saline, immunoblotting was conducted using a primary antibody against SP-A at a dilution of 1:150 or TLR4 at a dilution of 1:500 (Abcam, Britain, ab22048) or NF-kB (Abcam, Britain, ab32536) at a dilution of 1:5000 or IkB-α (Abcam, Britain, ab133462) or MEK1 (Abcam, Britain, ab32091) at a dilution of 1:1000 and phosphorylated MEK1 (p-MEK1) (Abcam, Britain, ab96379) at a dilution of 1:800 or ERK2 (Abcam, Britain, ab32081) at a dilution of 1:1000 and phosphorylated ERK1/2 (Abcam, Britain, ab201015) at a dilution of 1:800 or P38MAPK at a dilution of 1:3000 (Abcam, Britain, ab170099) and phosphorylated P38MAPK (Abcam, Britain, ab47363) at a dilution of 1:500, and then secondary antibody conjugated with horseradish peroxidase. Cellular β-actin protein was immunodetected using a human monoclonal antibody against human β-actin (St. Louis, MO, USA) as the internal standard. Immunoproducts were detected using enhanced chemiluminescence peroxidase detection reagents (Amersham, Sweden).
Statistical analysis:
Data are expressed as mean ± standard error of mean. Results were analyzed by one-way ANOVA using the SPSS version 15.0 software (SPSS Inc., Chicago, Illinois, USA). P < 0.05 was considered to indicate statistical significance.
Results:
Effect of lipopolysaccharide on the cell viability of HK-2 cells To evaluate the cytotoxic effect of LPS on cells, the cell viability of HK-2 cells was detected after LPS exposure. HK-2 cells were stimulated with 100 ng/ml of LPS for 0, 1, 6, and 12 h, and absorbance was measured at 490 nm. The absorbance did not show significant difference at different durations after LPS treatment [Figure 1, P > 0.05], suggesting that 100 ng/ml of LPS is not cytotoxic to HK-2 cells.
Effects of lipopolysaccharide on the cell viability of HK-2 cells. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 6, 12, and 24 h. Cell viability was assayed by MTT method. Optical density value of HK-2 cells for different durations was detected with colorimetric method. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).
To evaluate the cytotoxic effect of LPS on cells, the cell viability of HK-2 cells was detected after LPS exposure. HK-2 cells were stimulated with 100 ng/ml of LPS for 0, 1, 6, and 12 h, and absorbance was measured at 490 nm. The absorbance did not show significant difference at different durations after LPS treatment [Figure 1, P > 0.05], suggesting that 100 ng/ml of LPS is not cytotoxic to HK-2 cells.
Effects of lipopolysaccharide on the cell viability of HK-2 cells. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 6, 12, and 24 h. Cell viability was assayed by MTT method. Optical density value of HK-2 cells for different durations was detected with colorimetric method. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).
Effects of lipopolysaccharide on the messenger RNA and protein expression of surfactant protein-A in HK-2 cells Protein and mRNA expression of SP-A in HK-2 cells after LPS treatment were detected by Western blotting and real-time PCR, respectively. Cells were stimulated with LPS for 0, 1, 2, and 6 h, and then SP-A mRNA expression was detected; cells were treated with LPS for 0, 2, 6, and 12 h, and SP-A protein expression was assayed; and β-actin expression was detected as an internal standard. Expression of SP-A mRNA expression at 1, 2, and 6 h after LPS exposure was significantly increased by 1.5, 2.7, and 4.5 folds, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Moreover, the SP-A protein expression at 6 h after LPS treatment increased dramatically when compared with that at 0 h and 2 h [Figure 3, P < 0.05]. After LPS exposure for 12 h, the SP-A protein expression was markedly higher than that at 6 h. SP-A protein expression after LPS exposure for 6 h and 12 h was increased by 1.6 and 2.8 folds, respectively, as compared to that at 0 h. These findings suggested that LPS may upregulate SP-A expression at transcriptional level.
Expression of surfactant protein-A and toll-like receptor 4 mRNA in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to lipopolysaccharide for 0, 1, 2, and 6 h. β-actin mRNA was detected as the internal standard. Expression of surfactant protein-A and toll-like receptor 4 mRNA was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate that the value of surfactant protein-A and toll-like receptor 4 mRNA expression significantly (P < 0.05) differed from lipopolysaccharide exposure for 0 h, respectively. mRNA: Messenger RNA.
Expression of surfactant protein-A protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of surfactant protein-A protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Protein and mRNA expression of SP-A in HK-2 cells after LPS treatment were detected by Western blotting and real-time PCR, respectively. Cells were stimulated with LPS for 0, 1, 2, and 6 h, and then SP-A mRNA expression was detected; cells were treated with LPS for 0, 2, 6, and 12 h, and SP-A protein expression was assayed; and β-actin expression was detected as an internal standard. Expression of SP-A mRNA expression at 1, 2, and 6 h after LPS exposure was significantly increased by 1.5, 2.7, and 4.5 folds, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Moreover, the SP-A protein expression at 6 h after LPS treatment increased dramatically when compared with that at 0 h and 2 h [Figure 3, P < 0.05]. After LPS exposure for 12 h, the SP-A protein expression was markedly higher than that at 6 h. SP-A protein expression after LPS exposure for 6 h and 12 h was increased by 1.6 and 2.8 folds, respectively, as compared to that at 0 h. These findings suggested that LPS may upregulate SP-A expression at transcriptional level.
Expression of surfactant protein-A and toll-like receptor 4 mRNA in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to lipopolysaccharide for 0, 1, 2, and 6 h. β-actin mRNA was detected as the internal standard. Expression of surfactant protein-A and toll-like receptor 4 mRNA was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate that the value of surfactant protein-A and toll-like receptor 4 mRNA expression significantly (P < 0.05) differed from lipopolysaccharide exposure for 0 h, respectively. mRNA: Messenger RNA.
Expression of surfactant protein-A protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of surfactant protein-A protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Effects of lipopolysaccharide with or without CLI-095 on the nuclear translocation and activation of nuclear factor-κB in HK-2 cells To investigate the mechanism underlying LPS-induced SP-A expression in HK-2 cells, the expression of cytoplasmic and nuclear NF-κB as well as phosphorylated IκB-α was measured in HK-2 cells, and total NF-κB served as an internal standard. After LPS exposure for 0, 1, 2, and 6 h, the cytoplasmic NF-κB expression was decreased [Figure 4, P < 0.05], however the nuclear NF-κB expression and intracellular phosphorylated IκB-α expression were increased [Figure 5, P < 0.05]. The protein expression of nuclear factor-κB (NF-κB) at different time points was quantified, and results showed that the cytoplasmic NF-κB expression at 1, 2, and 6 h after LPS exposure was reduced by 32%, 48%, and 47%, respectively, but expressions of the nuclear NF-κB and phosphorylated IκB-α at 1, 2, and 6 h after LPS exposure were augmented by 117%, 155%, and 203% and by 167%, 320%, and 413%, respectively, as compared to those at 0 h. Moreover, the expression of the nuclear NF-κB and phosphorylated IκB-α was depressed by CLI-095 when compared with by LPS stimulation alone [Figures 4 and 5, P < 0.05].
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced nucleus factor-kB protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. Cytosolic (c) and nucleus (n) factor-kB were immunodetected. Total (t) nucleus factor-kB was detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced phosphorylated IkB-α protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. β-actin protein was detected as the internal standard. Expression of p-IkB-α was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of LPS exposure for 0 h.
To investigate the mechanism underlying LPS-induced SP-A expression in HK-2 cells, the expression of cytoplasmic and nuclear NF-κB as well as phosphorylated IκB-α was measured in HK-2 cells, and total NF-κB served as an internal standard. After LPS exposure for 0, 1, 2, and 6 h, the cytoplasmic NF-κB expression was decreased [Figure 4, P < 0.05], however the nuclear NF-κB expression and intracellular phosphorylated IκB-α expression were increased [Figure 5, P < 0.05]. The protein expression of nuclear factor-κB (NF-κB) at different time points was quantified, and results showed that the cytoplasmic NF-κB expression at 1, 2, and 6 h after LPS exposure was reduced by 32%, 48%, and 47%, respectively, but expressions of the nuclear NF-κB and phosphorylated IκB-α at 1, 2, and 6 h after LPS exposure were augmented by 117%, 155%, and 203% and by 167%, 320%, and 413%, respectively, as compared to those at 0 h. Moreover, the expression of the nuclear NF-κB and phosphorylated IκB-α was depressed by CLI-095 when compared with by LPS stimulation alone [Figures 4 and 5, P < 0.05].
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced nucleus factor-kB protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. Cytosolic (c) and nucleus (n) factor-kB were immunodetected. Total (t) nucleus factor-kB was detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced phosphorylated IkB-α protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. β-actin protein was detected as the internal standard. Expression of p-IkB-α was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of LPS exposure for 0 h.
Effects of lipopolysaccharide on the expression of toll-like receptor 4, phosphorylated p38 mitogen-activated protein kinase, and mitogen-activated/extracellular signal-regulated kinase 1 in HK-2 cells To elucidate the signaling pathway involved in LPS-induced NF-κB activation in HK-2 cells, the mRNA and protein expression of upstream transmembrane receptor TLR4 and protein expression of phosphorylated ERK1/2, phosphorylated p38MAPK, and p-MEK1 were assayed in HK-2 cells. With β-actin expression served as an internal standard, quantitative analysis showed that the levels of TLR4 mRNA after LPS exposure for 1, 2, and 6 h were increased by 111%, 221%, and 489%, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Meanwhile, the expression of TLR4 protein after LPS treatment for 1, 2, and 6 h was also increased [Figure 6, P < 0.05]. Expression of TLR4 protein at 2, 6, and 12 h was increased by 74%, 91%, and 154%, respectively, when compared with that at 0 h (P < 0.05). Phosphorylation of p38MAPK [Figure 7, P < 0.05] and MEK1 [Figure 8, P < 0.05] in HK-2 cells at 2, 6, and 12 h was increased by 75%, 130%, 215%, and 74%, 183%, 262%, respectively, as compared to that at 0 h. Levels of phosphorylated ERK1 in HK-2 cells were obviously raised after exposure to LPS for 2, 6, and 12 h by 62%, 162%, and 254%, respectively. Exposure of HK-2 cells to LPS for 2, 6, and 12 h significantly increased ERK2 phosphorylation by 77%, 239%, and 392%, respectively [Figure 9, P < 0.05].
Expression of toll-like receptor 4 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of toll-like receptor 4 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of P38MAPK protein in HK-2 cells induced by lipopolysaccharide exposure for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. P38MAPK protein was detected as the internal standard. Expression of p-P38MAPK protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of mitogen-activated/extracellular signal-regulated kinase 1 protein induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Mitogen-activated/extracellular signal-regulated kinase 1 protein was detected as the internal standard. Expression of phosphorylated mitogen-activated/extracellular signal-regulated kinase 1 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of ERK1/2 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. ERK2 protein was detected as the internal standard. Expression of p-ERK1/2 protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h. ERK: Extracellular signal-regulated kinase.
To elucidate the signaling pathway involved in LPS-induced NF-κB activation in HK-2 cells, the mRNA and protein expression of upstream transmembrane receptor TLR4 and protein expression of phosphorylated ERK1/2, phosphorylated p38MAPK, and p-MEK1 were assayed in HK-2 cells. With β-actin expression served as an internal standard, quantitative analysis showed that the levels of TLR4 mRNA after LPS exposure for 1, 2, and 6 h were increased by 111%, 221%, and 489%, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Meanwhile, the expression of TLR4 protein after LPS treatment for 1, 2, and 6 h was also increased [Figure 6, P < 0.05]. Expression of TLR4 protein at 2, 6, and 12 h was increased by 74%, 91%, and 154%, respectively, when compared with that at 0 h (P < 0.05). Phosphorylation of p38MAPK [Figure 7, P < 0.05] and MEK1 [Figure 8, P < 0.05] in HK-2 cells at 2, 6, and 12 h was increased by 75%, 130%, 215%, and 74%, 183%, 262%, respectively, as compared to that at 0 h. Levels of phosphorylated ERK1 in HK-2 cells were obviously raised after exposure to LPS for 2, 6, and 12 h by 62%, 162%, and 254%, respectively. Exposure of HK-2 cells to LPS for 2, 6, and 12 h significantly increased ERK2 phosphorylation by 77%, 239%, and 392%, respectively [Figure 9, P < 0.05].
Expression of toll-like receptor 4 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of toll-like receptor 4 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of P38MAPK protein in HK-2 cells induced by lipopolysaccharide exposure for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. P38MAPK protein was detected as the internal standard. Expression of p-P38MAPK protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of mitogen-activated/extracellular signal-regulated kinase 1 protein induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Mitogen-activated/extracellular signal-regulated kinase 1 protein was detected as the internal standard. Expression of phosphorylated mitogen-activated/extracellular signal-regulated kinase 1 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of ERK1/2 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. ERK2 protein was detected as the internal standard. Expression of p-ERK1/2 protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h. ERK: Extracellular signal-regulated kinase.
Effects of lipopolysaccharide with or without CLI-095 on the expression of toll-like receptor 4 and surfactant protein-A messenger RNA and protein in HK-2 cells We further explored the role of TLR4-dependent MEK1/2-ERK1/2-NF-κB signaling pathway in LPS-induced SP-A expression. HK-2 cells were pretreated with CLI-095 before LPS exposure. Induction of TLR4 and SP-A mRNA expression by LPS were analyzed by real-time PCR. After LPS administration, the mRNA expression of TLR4 and SP-A was significantly increased [Figure 9, P < 0.05]. Pretreatment of HK-2 cells with CLI-095, an inhibitor of TLR4, before LPS administration could markedly inhibit the mRNA and protein expression of TLR4 and SP-A [Figure 9, P < 0.05]. Expression of TLR4 and SP-A mRNA in HK-2 cells after LPS exposure for 6 h was augmented by 458% and 542%, respectively, when compared with that at 0 h. With CLI-095 pretreatment, mRNA expression of TLR4 and SP-A in HK-2 cells was decreased by 66% and 60%, respectively, when compared with that by LPS administration for 6 h alone [Figure 10, P < 0.05].
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced surfactant protein-A and toll-like receptor 4 messenger RNA expression. HK-2 cells were pretreated with CLI-095 for 1 h, and then exposed to CLI-095 and lipopolysaccharide for another 6 h. Levels of β-actin messenger RNA were detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate and significant (P < 0.05) difference between lipopolysaccharide and CLI-095 + lipopolysaccharide groups for surfactant protein-A and toll-like receptor 4 messenger RNA expression, respectively.
We further explored the role of TLR4-dependent MEK1/2-ERK1/2-NF-κB signaling pathway in LPS-induced SP-A expression. HK-2 cells were pretreated with CLI-095 before LPS exposure. Induction of TLR4 and SP-A mRNA expression by LPS were analyzed by real-time PCR. After LPS administration, the mRNA expression of TLR4 and SP-A was significantly increased [Figure 9, P < 0.05]. Pretreatment of HK-2 cells with CLI-095, an inhibitor of TLR4, before LPS administration could markedly inhibit the mRNA and protein expression of TLR4 and SP-A [Figure 9, P < 0.05]. Expression of TLR4 and SP-A mRNA in HK-2 cells after LPS exposure for 6 h was augmented by 458% and 542%, respectively, when compared with that at 0 h. With CLI-095 pretreatment, mRNA expression of TLR4 and SP-A in HK-2 cells was decreased by 66% and 60%, respectively, when compared with that by LPS administration for 6 h alone [Figure 10, P < 0.05].
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced surfactant protein-A and toll-like receptor 4 messenger RNA expression. HK-2 cells were pretreated with CLI-095 for 1 h, and then exposed to CLI-095 and lipopolysaccharide for another 6 h. Levels of β-actin messenger RNA were detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate and significant (P < 0.05) difference between lipopolysaccharide and CLI-095 + lipopolysaccharide groups for surfactant protein-A and toll-like receptor 4 messenger RNA expression, respectively.
Effect of lipopolysaccharide on the cell viability of HK-2 cells:
To evaluate the cytotoxic effect of LPS on cells, the cell viability of HK-2 cells was detected after LPS exposure. HK-2 cells were stimulated with 100 ng/ml of LPS for 0, 1, 6, and 12 h, and absorbance was measured at 490 nm. The absorbance did not show significant difference at different durations after LPS treatment [Figure 1, P > 0.05], suggesting that 100 ng/ml of LPS is not cytotoxic to HK-2 cells.
Effects of lipopolysaccharide on the cell viability of HK-2 cells. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 6, 12, and 24 h. Cell viability was assayed by MTT method. Optical density value of HK-2 cells for different durations was detected with colorimetric method. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).
Effects of lipopolysaccharide on the messenger RNA and protein expression of surfactant protein-A in HK-2 cells:
Protein and mRNA expression of SP-A in HK-2 cells after LPS treatment were detected by Western blotting and real-time PCR, respectively. Cells were stimulated with LPS for 0, 1, 2, and 6 h, and then SP-A mRNA expression was detected; cells were treated with LPS for 0, 2, 6, and 12 h, and SP-A protein expression was assayed; and β-actin expression was detected as an internal standard. Expression of SP-A mRNA expression at 1, 2, and 6 h after LPS exposure was significantly increased by 1.5, 2.7, and 4.5 folds, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Moreover, the SP-A protein expression at 6 h after LPS treatment increased dramatically when compared with that at 0 h and 2 h [Figure 3, P < 0.05]. After LPS exposure for 12 h, the SP-A protein expression was markedly higher than that at 6 h. SP-A protein expression after LPS exposure for 6 h and 12 h was increased by 1.6 and 2.8 folds, respectively, as compared to that at 0 h. These findings suggested that LPS may upregulate SP-A expression at transcriptional level.
Expression of surfactant protein-A and toll-like receptor 4 mRNA in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to lipopolysaccharide for 0, 1, 2, and 6 h. β-actin mRNA was detected as the internal standard. Expression of surfactant protein-A and toll-like receptor 4 mRNA was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate that the value of surfactant protein-A and toll-like receptor 4 mRNA expression significantly (P < 0.05) differed from lipopolysaccharide exposure for 0 h, respectively. mRNA: Messenger RNA.
Expression of surfactant protein-A protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of surfactant protein-A protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Effects of lipopolysaccharide with or without CLI-095 on the nuclear translocation and activation of nuclear factor-κB in HK-2 cells:
To investigate the mechanism underlying LPS-induced SP-A expression in HK-2 cells, the expression of cytoplasmic and nuclear NF-κB as well as phosphorylated IκB-α was measured in HK-2 cells, and total NF-κB served as an internal standard. After LPS exposure for 0, 1, 2, and 6 h, the cytoplasmic NF-κB expression was decreased [Figure 4, P < 0.05], however the nuclear NF-κB expression and intracellular phosphorylated IκB-α expression were increased [Figure 5, P < 0.05]. The protein expression of nuclear factor-κB (NF-κB) at different time points was quantified, and results showed that the cytoplasmic NF-κB expression at 1, 2, and 6 h after LPS exposure was reduced by 32%, 48%, and 47%, respectively, but expressions of the nuclear NF-κB and phosphorylated IκB-α at 1, 2, and 6 h after LPS exposure were augmented by 117%, 155%, and 203% and by 167%, 320%, and 413%, respectively, as compared to those at 0 h. Moreover, the expression of the nuclear NF-κB and phosphorylated IκB-α was depressed by CLI-095 when compared with by LPS stimulation alone [Figures 4 and 5, P < 0.05].
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced nucleus factor-kB protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. Cytosolic (c) and nucleus (n) factor-kB were immunodetected. Total (t) nucleus factor-kB was detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced phosphorylated IkB-α protein expression. HK-2 cells were exposed to lipopolysaccharide for 0, 2, 6, and 12 h and pretreated with CLI-095 for 1 h, and then exposed to a combination of CLI-095 and lipopolysaccharide for 6 h. β-actin protein was detected as the internal standard. Expression of p-IkB-α was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of LPS exposure for 0 h.
Effects of lipopolysaccharide on the expression of toll-like receptor 4, phosphorylated p38 mitogen-activated protein kinase, and mitogen-activated/extracellular signal-regulated kinase 1 in HK-2 cells:
To elucidate the signaling pathway involved in LPS-induced NF-κB activation in HK-2 cells, the mRNA and protein expression of upstream transmembrane receptor TLR4 and protein expression of phosphorylated ERK1/2, phosphorylated p38MAPK, and p-MEK1 were assayed in HK-2 cells. With β-actin expression served as an internal standard, quantitative analysis showed that the levels of TLR4 mRNA after LPS exposure for 1, 2, and 6 h were increased by 111%, 221%, and 489%, respectively, as compared to that at 0 h [Figure 2, P < 0.05]. Meanwhile, the expression of TLR4 protein after LPS treatment for 1, 2, and 6 h was also increased [Figure 6, P < 0.05]. Expression of TLR4 protein at 2, 6, and 12 h was increased by 74%, 91%, and 154%, respectively, when compared with that at 0 h (P < 0.05). Phosphorylation of p38MAPK [Figure 7, P < 0.05] and MEK1 [Figure 8, P < 0.05] in HK-2 cells at 2, 6, and 12 h was increased by 75%, 130%, 215%, and 74%, 183%, 262%, respectively, as compared to that at 0 h. Levels of phosphorylated ERK1 in HK-2 cells were obviously raised after exposure to LPS for 2, 6, and 12 h by 62%, 162%, and 254%, respectively. Exposure of HK-2 cells to LPS for 2, 6, and 12 h significantly increased ERK2 phosphorylation by 77%, 239%, and 392%, respectively [Figure 9, P < 0.05].
Expression of toll-like receptor 4 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. β-actin protein was detected as the internal standard. Expression of toll-like receptor 4 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of P38MAPK protein in HK-2 cells induced by lipopolysaccharide exposure for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. P38MAPK protein was detected as the internal standard. Expression of p-P38MAPK protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages).*: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of mitogen-activated/extracellular signal-regulated kinase 1 protein induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Mitogen-activated/extracellular signal-regulated kinase 1 protein was detected as the internal standard. Expression of phosphorylated mitogen-activated/extracellular signal-regulated kinase 1 protein of HK-2 was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h.
Expression of phosphorylation of ERK1/2 protein in HK-2 cells induced by lipopolysaccharide for different durations. HK-2 cells were exposed to 100 ng/ml of lipopolysaccharide for 0, 2, 6, and 12 h. Total protein was prepared for Western blot analysis. ERK2 protein was detected as the internal standard. Expression of p-ERK1/2 protein of HK-2 cells was quantified and then compared. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). *: The value significantly (P < 0.05) differed from that of lipopolysaccharide exposure for 0 h. ERK: Extracellular signal-regulated kinase.
Effects of lipopolysaccharide with or without CLI-095 on the expression of toll-like receptor 4 and surfactant protein-A messenger RNA and protein in HK-2 cells:
We further explored the role of TLR4-dependent MEK1/2-ERK1/2-NF-κB signaling pathway in LPS-induced SP-A expression. HK-2 cells were pretreated with CLI-095 before LPS exposure. Induction of TLR4 and SP-A mRNA expression by LPS were analyzed by real-time PCR. After LPS administration, the mRNA expression of TLR4 and SP-A was significantly increased [Figure 9, P < 0.05]. Pretreatment of HK-2 cells with CLI-095, an inhibitor of TLR4, before LPS administration could markedly inhibit the mRNA and protein expression of TLR4 and SP-A [Figure 9, P < 0.05]. Expression of TLR4 and SP-A mRNA in HK-2 cells after LPS exposure for 6 h was augmented by 458% and 542%, respectively, when compared with that at 0 h. With CLI-095 pretreatment, mRNA expression of TLR4 and SP-A in HK-2 cells was decreased by 66% and 60%, respectively, when compared with that by LPS administration for 6 h alone [Figure 10, P < 0.05].
Effects of lipopolysaccharide with or without CLI-095 on lipopolysaccharide-induced surfactant protein-A and toll-like receptor 4 messenger RNA expression. HK-2 cells were pretreated with CLI-095 for 1 h, and then exposed to CLI-095 and lipopolysaccharide for another 6 h. Levels of β-actin messenger RNA were detected as the internal standard. Each value represents the mean ± standard error of the mean (n = 5, samples were collected from five different passages). The symbols “*” and “†” indicate and significant (P < 0.05) difference between lipopolysaccharide and CLI-095 + lipopolysaccharide groups for surfactant protein-A and toll-like receptor 4 messenger RNA expression, respectively.
Discussion:
The present study revealed that LPS can cause inflammatory responses in renal tubular epithelial HK-2 cells by means of enhancing SP-A mRNA and protein syntheses. Moreover, the signal-transducing mechanisms of LPS-induced regulation of SP-A expression arise through TLR4-dependent cascade phosphorylations of MEK1, ERK1/2, and p38MAPK, leading to the nuclear translocation and activation of NF-κB. Moreover, pretreatment with TLR4 inhibitor, CLI-095, may significantly decrease mRNA and protein expression of SP-A, depress IκB-α phosphorylation, and suppress NF-κB nuclear translocation. These findings indicate that LPS may induce SP-A synthesis through TLR4-mediated activation of MEK1-ERK1/2-NF-κB signaling pathway.
LPS is a major pathogenic factor in septic shock and multiple organ dysfuction syndrome (MODS) caused by Gram-negative bacteria.[18] In vitro study indicated that the elevated SP-A expression in A549 cells could be induced by LPS in a time-dependent manner.[19] Our previous study showed that SP-A protein could be expressed and secreted by renal tubular epithelial cells.[15] SP-A may play important roles in anti-inflammatory and protective effects in both lung and kidney.[7] In the present study, 100 ng/ml of LPS was used to stimulate HK-2 cells (a human renal tubular epithelial cell line), however cytotoxic effects of LPS on HK-2 cells were not observed. In addition, expression of SP-A protein and mRNA in HK-2 cells was found to be increased in a time-dependent manner by LPS administration, moreover elevated mRNA expression of SP-A occurred earlier than the elevated protein expression. This indicated that LPS may influence the SP-A expression at transcriptional level.
TLR4 is an transmembrane receptor involved in the innate immunity, which can recognize pathogen-associated molecular pattern (including LPS, a component of Gram-negative bacterial cell wall and part of Gram-positive bacteria) to modulate cytokine secretion in the immune response. Thus, the regulation of TLR4 expression may be involved in the innate immunity to pathogens.[20] Studies have showed that LPS may induce the aggregation of TLR4 on the THP1 cells to promote the transmission of extracellular signals into cells, leading to release of a large amount of pro-inflammatory cytokines such as TNF-α.[21] Differential TLR subtypes would be activated by LPS in different types of cells. LPS can significantly stimulate the TLR2 expression on A549 cells to activate downstream signaling pathway.[19] In the present study, HK-2 cells were stimulated by 100 ng/ml of LPS, significantly increased expression of TLR4 mRNA and protein in a time-dependent manner was observed, which indicated that TLR4 activation stimulated by LPS may be involved in the process of SP-A induction of HK-2 cells.
NF-κB is a well-known transcription factor that regulates the expression of many inflammation-related genes in response to various infections.[22] When cells are stimulated, the NF-κB inhibitor-alpha (IκB) of NF-kB/IκB complex in the cytoplasm is phosphorylated and dissociated from this complex, and then NF-κB (p50 and p65) translocates from the cytoplasm into the nucleus, leading to NF-κB activation, which is related to MAPK pathway. Bacterial cell wall is able to activate NF-κB in the kidney in vivo, and the inhibition of NF-κB activity may ameliorate sepsis-induced AKI.[23] It has been confirmed in the present study that the expression of phosphorylated IkB-α and the nuclear NF-κB protein was significantly increased, but the expression of cytoplasmic NF-κB was decreased dramatically in HK-2 cells by LPS exposure, suggesting the translocation of NF-κB from the cytoplasm to the nucleus, and the subsequent activation of NF-κB in HK-2 cells would be regulated by LPS. There is growing evidence showing that LPS-induced NF-κB activation is related to TLR4-mediated MyD88-dependent signaling pathway.
MAPK family consists of three protein kinases: ERK, p38MAPK, and c-JNK, which are the upstream kinases of NF-κB.[11] ERK1/2 activation may further activate NF-κB. The ERK1/2-induced activation of IκB-α kinase through phosphorylating two serine residuals at the N-terminal of IκB-α would lead to the degradation of IκB-α. Then, NF-κB translocates into the nucleus, which contributes to its activation.[24] NF-κB activation induced by LPS may be due to the phosphorylation of MAPKs. It has been validated in in vitro studies that the phosphorylation of MAPKs (including ERK1/2 and p38MAPK) in LPS-treated A549 cells was determined.[19] In addition, p38MAPK also plays an important role in the NF-κB activation signaling pathway.[25] In the present study, LPS time dependently increased the expression of phosphorylated ERK1/2 and p38MAPK in HK-2 cells. Thus, the phosphorylation of ERK1/2 and p38MAPK, two important members of MAPK family, may participate in NF-κB activation in HK-2 cells stimulated by LPS.
Increasing evidences indicate that ERK signaling pathway is related to the regulation of inflammation.[26] ERK activation is modulated by three different signaling pathways: Raf/MEK-dependent signaling pathway, non-PI3K/Raf-dependent signaling pathway, and unknown signaling pathway that can directly activate ERK protein kinase. MEK1 is an upstream protein kinase of ERK1/2, and the activation of MEK1 is associated with ERK1/2 phosphorylation.[27] Roles of MEK1 and ERK1/2 in SP-A expression induced by LPS were also determined in the previous study using alveolar epithelial A549 cells.[19] Our results exhibited that phosphorylation of ERK1/2 by LPS was due to the MEK1 phosphorylation. Thus, LPS-induced phosphorylation of MEK1 and ERK1/2 may be related to the subsequent NF-κB activation in HK-2 cells. Moreover, suppressing TLR4 expression with CLI-095 simultaneously inhibited LPS-induced NF-κB nuclear translocation and subsequent SP-A mRNA expression. Thus, our study indicated that the signal-transducing mechanisms of LPS-caused regulation of SP-A expression arise in renal tubular epithelial HK-2 cells through TLR4 activation and cascade phosphorylations of downstream kinase MEK1 and MAPK (ERK1/2 and p38MAPK). In succession, LPS induced NF-κB expression and translocation.
However, there are certain limitations of the present study. HK-2 cells are not primary culture cells, and results in this study might be a little bit different from those in vivo. Thus, more translational studies are needed to explore the effect of LPS on the secretion of SP-A in human renal tubular epithelial cells in case of AKI.
Financial support and sponsorship This study was supported by the National Natural Science Foundation of China (No. 81201457, No. 81173402, and No. 81571943) and Advanced Suitable Project of Shanghai Health System (No. 2013SY070).
This study was supported by the National Natural Science Foundation of China (No. 81201457, No. 81173402, and No. 81571943) and Advanced Suitable Project of Shanghai Health System (No. 2013SY070).
Conflicts of interest There are no conflicts of interest.
There are no conflicts of interest.
Financial support and sponsorship:
This study was supported by the National Natural Science Foundation of China (No. 81201457, No. 81173402, and No. 81571943) and Advanced Suitable Project of Shanghai Health System (No. 2013SY070).
Conflicts of interest:
There are no conflicts of interest.
|
Background:
Surfactant protein-A (SP-A) contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial (HK-2) cells can be stimulated by lipopolysaccharide (LPS). The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells.
Methods:
Tetrazolium salt colorimetry (MTT) assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 (TLR4) messenger RNA (mRNA) expression, as well as phosphorylation of mitogen-activated/extracellular signal-regulated kinase (MEK) 1, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), and nuclear factor-kappa B (NF-κB) inhibitor-alpha (IkB-α). Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-κB in the cytoplasm and nucleus of HK-2 before LPS exposure.
Results:
HK-2 cells exposed to 100 ng/ml of LPS for 1, 6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells (P = 0.16); however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased (P = 0.02). As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEK1, ERK1/2, and p38MAPK. In addition, levels of phosphorylated IκB-α and nuclear NF-κB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-κB expression were significantly inhibited by pretreatment with CLI-095.
Conclusions:
The present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1-ERK1/2-NF-κB-dependent pathway.
| null | null | 13,029 | 431 | 17 |
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[CONTENT] Acute Kidney Injury | HK-2 Cells | Lipopolysaccharide | Signal Pathway | Surfactant Protein-A [SUMMARY]
|
[CONTENT] Acute Kidney Injury | HK-2 Cells | Lipopolysaccharide | Signal Pathway | Surfactant Protein-A [SUMMARY]
|
[CONTENT] Acute Kidney Injury | HK-2 Cells | Lipopolysaccharide | Signal Pathway | Surfactant Protein-A [SUMMARY]
| null |
[CONTENT] Acute Kidney Injury | HK-2 Cells | Lipopolysaccharide | Signal Pathway | Surfactant Protein-A [SUMMARY]
| null |
[CONTENT] Cell Line | Cell Survival | Colorimetry | Humans | Kidney | Lipopolysaccharides | Mitogen-Activated Protein Kinase 1 | Mitogen-Activated Protein Kinase 3 | NF-kappa B | Pulmonary Surfactant-Associated Protein A | Sulfonamides | Tetrazolium Salts | Toll-Like Receptor 4 [SUMMARY]
|
[CONTENT] Cell Line | Cell Survival | Colorimetry | Humans | Kidney | Lipopolysaccharides | Mitogen-Activated Protein Kinase 1 | Mitogen-Activated Protein Kinase 3 | NF-kappa B | Pulmonary Surfactant-Associated Protein A | Sulfonamides | Tetrazolium Salts | Toll-Like Receptor 4 [SUMMARY]
|
[CONTENT] Cell Line | Cell Survival | Colorimetry | Humans | Kidney | Lipopolysaccharides | Mitogen-Activated Protein Kinase 1 | Mitogen-Activated Protein Kinase 3 | NF-kappa B | Pulmonary Surfactant-Associated Protein A | Sulfonamides | Tetrazolium Salts | Toll-Like Receptor 4 [SUMMARY]
| null |
[CONTENT] Cell Line | Cell Survival | Colorimetry | Humans | Kidney | Lipopolysaccharides | Mitogen-Activated Protein Kinase 1 | Mitogen-Activated Protein Kinase 3 | NF-kappa B | Pulmonary Surfactant-Associated Protein A | Sulfonamides | Tetrazolium Salts | Toll-Like Receptor 4 [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] cells | expression | protein | hk | hk cells | lps | lipopolysaccharide | sp | standard | 05 [SUMMARY]
|
[CONTENT] cells | expression | protein | hk | hk cells | lps | lipopolysaccharide | sp | standard | 05 [SUMMARY]
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[CONTENT] cells | expression | protein | hk | hk cells | lps | lipopolysaccharide | sp | standard | 05 [SUMMARY]
| null |
[CONTENT] cells | expression | protein | hk | hk cells | lps | lipopolysaccharide | sp | standard | 05 [SUMMARY]
| null |
[CONTENT] epithelial | sp | sepsis | patients | pulmonary | epithelial cells | aki | injury | cells | tubular [SUMMARY]
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[CONTENT] mmol | abcam | britain | dilution | abcam britain | μl | min | mg | usa | cells [SUMMARY]
|
[CONTENT] expression | lipopolysaccharide | protein | cells | hk | lps | hk cells | 05 | value | compared [SUMMARY]
| null |
[CONTENT] expression | cells | lps | protein | hk | lipopolysaccharide | hk cells | sp | mmol | interest [SUMMARY]
| null |
[CONTENT] ||| SP-A | LPS ||| LPS [SUMMARY]
|
[CONTENT] MTT | LPS ||| 100 ng/ml of LPS | LPS | SP-A | 4 | TLR4 | RNA | 1 | 1/2 | NF-κB ||| SP-A | TLR4 | NF-κB | LPS [SUMMARY]
|
[CONTENT] 100 ng/ml of LPS | 1 | 6 | 24 | 100 ng/ml LPS | 0.16 | SP-A mRNA | 0.02 ||| LPS | TLR4 ||| LPS | MEK1, ERK1/2 ||| NF-κB | LPS | 2 | LPS | NF-κB [SUMMARY]
| null |
[CONTENT] ||| SP-A | LPS ||| LPS ||| MTT | LPS ||| 100 ng/ml of LPS | LPS | SP-A | 4 | TLR4 | RNA | 1 | 1/2 | NF-κB ||| SP-A | TLR4 | NF-κB | LPS ||| 100 ng/ml of LPS | 1 | 6 | 24 | 100 ng/ml LPS | 0.16 | SP-A mRNA | 0.02 ||| LPS | TLR4 ||| LPS | MEK1, ERK1/2 ||| NF-κB | LPS | 2 | LPS | NF-κB ||| LPS | MEK1-ERK1/2-NF-κB-dependent [SUMMARY]
| null |
||||
Teaching of evidence-based medicine to medical students in Mexico: a randomized controlled trial.
|
23131115
|
Evidence-Based Medicine (EBM) is an important competency for the healthcare professional. Experimental evidence of EBM educational interventions from rigorous research studies is limited. The main objective of this study was to assess EBM learning (knowledge, attitudes and self-reported skills) in undergraduate medical students with a randomized controlled trial.
|
BACKGROUND
|
The educational intervention was a one-semester EBM course in the 5th year of a public medical school in Mexico. The study design was an experimental parallel group randomized controlled trial for the main outcome measures in the 5th year class (M5 EBM vs. M5 non-EBM groups), and quasi-experimental with static-groups comparisons for the 4th year (M4, not yet exposed) and 6th year (M6, exposed 6 months to a year earlier) groups. EBM attitudes, knowledge and self-reported skills were measured using Taylor's questionnaire and a summative exam which comprised of a 100-item multiple-choice question (MCQ) test.
|
METHODS
|
289 Medical students were assessed: M5 EBM=48, M5 non-EBM=47, M4=87, and M6=107. There was a higher reported use of the Cochrane Library and secondary journals in the intervention group (M5 vs. M5 non-EBM). Critical appraisal skills and attitude scores were higher in the intervention group (M5) and in the group of students exposed to EBM instruction during the previous year (M6). The knowledge level was higher after the intervention in the M5 EBM group compared to the M5 non-EBM group (p<0.001, Cohen's d=0.88 with Taylor's instrument and 3.54 with the 100-item MCQ test). M6 Students that received the intervention in the previous year had a knowledge score higher than the M4 and M5 non-EBM groups, but lower than the M5 EBM group.
|
RESULTS
|
Formal medical student training in EBM produced higher scores in attitudes, knowledge and self-reported critical appraisal skills compared with a randomized control group. Data from the concurrent groups add validity evidence to the study, but rigorous follow-up needs to be done to document retention of EBM abilities.
|
CONCLUSIONS
|
[
"Aerospace Medicine",
"Clinical Competence",
"Cross-Over Studies",
"Curriculum",
"Developing Countries",
"Education, Medical, Undergraduate",
"Educational Measurement",
"Evidence-Based Medicine",
"Female",
"Health Knowledge, Attitudes, Practice",
"Humans",
"Internship and Residency",
"Male",
"Mexico",
"Military Medicine",
"Schools, Medical",
"Surveys and Questionnaires",
"Young Adult"
] |
3511203
|
Background
|
Evidence-based medicine (EBM) has been defined as “the integration of the best research evidence with our clinical expertise and our patient’s unique values and circumstances”, and it has emerged as a core competency necessary for all healthcare professionals [1-3]. Its fundamental principles are: translation of uncertainty to an answerable clinical question, systematic retrieval of the best evidence available, critical appraisal for validity, relevance and applicability, use of results in practice and evaluation of its performance by the healthcare provider [4].
Several organizations, including the Institute of Medicine in the United States and the World Federation for Medical Education, have advocated the implementation of EBM educational interventions in medical under and postgraduate training [2,5].
The concepts related to EBM and its educational implications have disseminated rapidly in the last decade, and this change needs to be accompanied with strong educational research to document its effectiveness. The challenges of teaching EBM and the paucity of rigorous educational research publications have prompted some medical educators to question the evidence of EBM teaching effectiveness [6]. Nonetheless, the foundations of EBM that support clinical decision making are intuitively attractive to many clinicians and educators, since it integrates the educational process with clinical practice [4].
The quality of the evidence about EBM education is heterogeneous, as has been described in several editorials, narrative and systematic reviews [7-11]. The majority of reviews have included mostly studies in postgraduate health professionals, and some have included studies in both post and undergraduate students. Green reviewed 18 reports, mostly resident-directed small-group seminars with the objective of improving critical appraisal skills [12]. The most commonly used outcome measure was a multiple-choice exam, and 72% used a traditional journal club format as teaching strategy. Only seven of the 18 studies included in Green’s review analyzed the effectiveness of the intervention, five of these had some type of control group and only one was a randomized study. Just two studies used an outcome measure that had validity evidence, and measurement of change in behavior used only self-report in all five papers. The impact of the intervention was focused mainly on critical appraisal, and ranged from no effect to 23% absolute increase in scores [12].
The Cochrane Collaboration systematic review on the subject of teaching critical appraisal skills in health care, which excluded medical students, found three studies that met stringent pre-specified methodological criteria. These articles reported statistically significant improvements in participants' knowledge in domains of critical appraisal in two of the three studies [9]. Another systematic review by Coomarasamy focused on postgraduate clinicians, and found significant effects of EBM educational interventions in knowledge, and more limited in attitudes, skills and behavior [10,11].
Despite the increasing number of medical school and postgraduate programs that have introduced EBM in their curricula, most of the information about it has been reported as observational data and descriptive studies in the medical literature, or as unpublished observations that are disseminated in medical meetings or informal venues. There are few randomized controlled educational trials about EBM training effectiveness, and the majority have been done in residents or practicing physicians [9-14].
Undergraduate medical students can be a receptive population to EBM concepts, and they will be the practicing clinicians and clinical teachers in the future. There are several published studies that describe medical schools’ experiences introducing EBM in their curriculum and teaching these concepts to undergraduates, with variable outcomes [15-19]. This curricular change has not occurred in many of their developing country counterparts, with few published reports of the implementation of EBM curricula in these settings [20-23]. There is a need to implement EBM educational interventions in developing countries medical schools’ curricula, and to assess their impact with appropriate educational research designs.
The purpose of this study was to assess the educational effectiveness (attitudes, knowledge and skills) of an EBM course in undergraduate medical students.
|
Methods
|
Setting The Mexican Army medical school trains career physicians for the national military healthcare system, and is located in Mexico City. It has a six year program, with a traditional curriculum: two years of basic sciences, three years of clinical sciences, and the sixth year is an internship period in the hospital. The school is a public institution funded by the federal government. Each yearly class is composed of about one hundred students, mostly middle- or low-socioeconomic class Hispanics.
The Mexican Army medical school trains career physicians for the national military healthcare system, and is located in Mexico City. It has a six year program, with a traditional curriculum: two years of basic sciences, three years of clinical sciences, and the sixth year is an internship period in the hospital. The school is a public institution funded by the federal government. Each yearly class is composed of about one hundred students, mostly middle- or low-socioeconomic class Hispanics.
Overall study design and participants Main outcomes and subjects The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].
Flow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.
The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].
Flow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.
Simultaneous validation Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).
The outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.
Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).
The outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.
Main outcomes and subjects The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].
Flow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.
The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].
Flow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.
Simultaneous validation Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).
The outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.
Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).
The outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.
Intervention The educational intervention was a one semester EBM course formally included in the medical school curriculum, with 14 two-hour weekly sessions. The course faculty were six professors trained in EBM teaching, all board-certified physicians with clinical practice, one of them with a postgraduate degree in health professions education and faculty development training in EBM education at McMaster University Faculty of Health Sciences in Canada. The course faculty had more than six years of experience teaching EBM to undergraduate medical students, residents of several specialties, and providing faculty development EBM workshops to teachers of several medical specialties. The EBM course teachers were not involved in the training of the 4th year students, but they participated in the training of the 6th year interns. The EBM program was linked with the internship program and the residency programs in the hospital, through the medical school curricular committee and the University Postgraduate Studies Division.
The course instructional strategies included large-group interactive sessions, small-group problem-solving activities, individual and group assignments, and informatics laboratory sessions. Traditional EBM resources were used as course bibliography, including Straus’ book [1] and an EBM text in Spanish written by the course professors [28]. The content and learning objectives of the course are outlined below.
1. Clinical decision making in medicine
• List and define the main difficulties for objective decision making in medicine as defined by Eddy
• Describe the components of a decision in medicine as defined by Eddy
• Apply the concepts of anatomy of a decision as defined by Eddy in the analysis of a clinical problem
2. Uncertainty and probability in medicine
• Define the concepts of uncertainty, probability and odds
• Understand the relevance of uncertainty in clinical practice
• Understand the limitations of personal experience in the estimation of probability, as related to diagnosis
• Define the heuristics used in medicine (representativeness, availability, anchor and adjustment) and list the cognitive errors a clinician can make when misapplying them
• Apply the concepts of heuristics in new clinical problems, and discuss the effects of their inappropriate use
3. Bayes’ theorem
• Define Bayes’ theorem
• Define pre-test and post-test probability
• Define the concepts of diagnostic and therapeutic threshold
• Explain the utility of Bayes' theorem in clinical medicine, mainly in diagnosis
• List the limitations of Bayes' theorem in clinical practice
• Apply Fagan’s nomogram to use Bayes' theorem in a diagnostic problem
• Apply the concepts of diagnostic and therapeutic threshold to a clinical problem
4. Principles of Evidence Based Medicine
• Describe the history and origin of EBM
• Define the concept of EBM
• List the five steps of EBM, and apply them in a clinical problem
• Explain the importance of EBM in clinical practice
5. Reflective medical practice
• Define the concept of reflection and reflective practitioner
• Define reflection-in-action and reflection-on-action
• Apply these concepts in a clinical scenario
6. Clinicians’ information needs
• Understand the magnitude of physician information needs
• Understand the literature that describe how clinicians underestimate their information needs
• Define the percentage of occasions when clinicians recognize and act upon perceived information needs
7. Clinical questions
• Define the concepts of background and foreground questions
• Understand the advantages of structuring questions generated during clinical work
• List the four components of a foreground clinical question (PICO)
• Apply these concepts in developing questions from clinical problems
• List the types of clinical questions (diagnosis, therapy, prognosis, harm, etiology)
8. Sources of biomedical information
• List the different sources of biomedical information available
• Identify the advantages and disadvantages of each source (textbooks, paper and electronic journals, original research papers)
• Understand the origin, development, cost, and availability of sources of information
9. The Cochrane Collaboration
• Describe the history and origin of the Cochrane Collaboration (CC)
• List the components of the Cochrane Library, and the sources where it’s available
• Understand the mission, logistics and work of the CC
• Perform effective searches for systematic reviews on the Cochrane Library
• Understand the advantages and limitations of the CC
• Use the Cochrane Library to solve a clinical problem
10. Search strategies to find the best medical scientific evidence
• List the main medical databases, and identify their relevance and location
• Describe the history of Medline
• Define MeSH terms, Boolean operators, search engine
• Design search strategies to find valid evidence
• Use PubMed Clinical Queries
• Perform effective searches of scientifically valid papers using PubMed, Cochrane Library, OVID Core Medical Library
• Understand the advantages and disadvantages of searching the different electronic medical databases and the Internet general purpose searching engines
11. Critical appraisal of the medical literature: Users’ Guides to the Medical Literature
• Describe the origin and history of the Users’ Guides series to appraise the medical literature
• List and understand the different hierarchies of evidence, study designs, grades of evidence
• Understand the relevance of using the original medical literature to solve clinical problems
• List and understand the three different steps to appraise a research article: internal validity, magnitude of the results and external validity
12. How to appraise an article about therapy
• Describe the criteria for internal validity of a therapy article
• Define randomized controlled trial, bias and random error, allocation concealment, double-blind, intention-to-treat analysis, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, p values, power and sample size, type I and II errors
• Understand the importance of all the previously defined concepts to apply in a therapy article
• Calculate OR, RR, RRR, ARR and NNT from a published therapy article
• Use a therapy article to solve a clinical problem
• Understand the concepts of external validity of a research paper, related to therapy
13. How to appraise an article about a diagnostic test, the Rational Clinical Examination Series
• Describe the criteria for internal validity of a diagnostic test article
• Define pre-test and post-test probability, sensitivity, specificity, likelihood ratios, positive and negative predictive value, accuracy
• Understand the importance of all the previously defined concepts to apply a diagnosis article
• Calculate sensitivity, specificity, likelihood ratios from a published diagnosis article
• Use a diagnosis article to solve a clinical problem
• Understand the concepts of external validity of a research paper, related to diagnosis
• Describe the origin and evolution of the Rational Clinical Examination JAMA series
• Use a JAMA Rational Clinical Examination paper to solve a clinical problem
14. How to appraise a Systematic Review or Meta-analysis
• Define meta-analysis, systematic review (qualitative and quantitative)
• Describe the advantages and limitations of systematic reviews and meta-analysis
• Describe the criteria for internal validity of a systematic review article
• Define bias and random error, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, forest plot, effect size
• Understand the importance of all the previously defined concepts applied to a systematic review article
• Calculate OR, RR, RRR, ARR and NNT from a published systematic review article
• Use a systematic review article to solve a clinical problem
• Understand the concepts of external validity of a systematic review
15. Clinical practice guidelines
• Define clinical practice guidelines
• Describe the sequence of developing an evidence-based clinical practice guideline
• Understand the advantages and limitations of a clinical guideline
• Describe and understand the internal validity requirements of a clinical guideline article
• List the available resources for clinical guidelines
• Use a clinical practice guideline to solve a clinical problem
The educational intervention was a one semester EBM course formally included in the medical school curriculum, with 14 two-hour weekly sessions. The course faculty were six professors trained in EBM teaching, all board-certified physicians with clinical practice, one of them with a postgraduate degree in health professions education and faculty development training in EBM education at McMaster University Faculty of Health Sciences in Canada. The course faculty had more than six years of experience teaching EBM to undergraduate medical students, residents of several specialties, and providing faculty development EBM workshops to teachers of several medical specialties. The EBM course teachers were not involved in the training of the 4th year students, but they participated in the training of the 6th year interns. The EBM program was linked with the internship program and the residency programs in the hospital, through the medical school curricular committee and the University Postgraduate Studies Division.
The course instructional strategies included large-group interactive sessions, small-group problem-solving activities, individual and group assignments, and informatics laboratory sessions. Traditional EBM resources were used as course bibliography, including Straus’ book [1] and an EBM text in Spanish written by the course professors [28]. The content and learning objectives of the course are outlined below.
1. Clinical decision making in medicine
• List and define the main difficulties for objective decision making in medicine as defined by Eddy
• Describe the components of a decision in medicine as defined by Eddy
• Apply the concepts of anatomy of a decision as defined by Eddy in the analysis of a clinical problem
2. Uncertainty and probability in medicine
• Define the concepts of uncertainty, probability and odds
• Understand the relevance of uncertainty in clinical practice
• Understand the limitations of personal experience in the estimation of probability, as related to diagnosis
• Define the heuristics used in medicine (representativeness, availability, anchor and adjustment) and list the cognitive errors a clinician can make when misapplying them
• Apply the concepts of heuristics in new clinical problems, and discuss the effects of their inappropriate use
3. Bayes’ theorem
• Define Bayes’ theorem
• Define pre-test and post-test probability
• Define the concepts of diagnostic and therapeutic threshold
• Explain the utility of Bayes' theorem in clinical medicine, mainly in diagnosis
• List the limitations of Bayes' theorem in clinical practice
• Apply Fagan’s nomogram to use Bayes' theorem in a diagnostic problem
• Apply the concepts of diagnostic and therapeutic threshold to a clinical problem
4. Principles of Evidence Based Medicine
• Describe the history and origin of EBM
• Define the concept of EBM
• List the five steps of EBM, and apply them in a clinical problem
• Explain the importance of EBM in clinical practice
5. Reflective medical practice
• Define the concept of reflection and reflective practitioner
• Define reflection-in-action and reflection-on-action
• Apply these concepts in a clinical scenario
6. Clinicians’ information needs
• Understand the magnitude of physician information needs
• Understand the literature that describe how clinicians underestimate their information needs
• Define the percentage of occasions when clinicians recognize and act upon perceived information needs
7. Clinical questions
• Define the concepts of background and foreground questions
• Understand the advantages of structuring questions generated during clinical work
• List the four components of a foreground clinical question (PICO)
• Apply these concepts in developing questions from clinical problems
• List the types of clinical questions (diagnosis, therapy, prognosis, harm, etiology)
8. Sources of biomedical information
• List the different sources of biomedical information available
• Identify the advantages and disadvantages of each source (textbooks, paper and electronic journals, original research papers)
• Understand the origin, development, cost, and availability of sources of information
9. The Cochrane Collaboration
• Describe the history and origin of the Cochrane Collaboration (CC)
• List the components of the Cochrane Library, and the sources where it’s available
• Understand the mission, logistics and work of the CC
• Perform effective searches for systematic reviews on the Cochrane Library
• Understand the advantages and limitations of the CC
• Use the Cochrane Library to solve a clinical problem
10. Search strategies to find the best medical scientific evidence
• List the main medical databases, and identify their relevance and location
• Describe the history of Medline
• Define MeSH terms, Boolean operators, search engine
• Design search strategies to find valid evidence
• Use PubMed Clinical Queries
• Perform effective searches of scientifically valid papers using PubMed, Cochrane Library, OVID Core Medical Library
• Understand the advantages and disadvantages of searching the different electronic medical databases and the Internet general purpose searching engines
11. Critical appraisal of the medical literature: Users’ Guides to the Medical Literature
• Describe the origin and history of the Users’ Guides series to appraise the medical literature
• List and understand the different hierarchies of evidence, study designs, grades of evidence
• Understand the relevance of using the original medical literature to solve clinical problems
• List and understand the three different steps to appraise a research article: internal validity, magnitude of the results and external validity
12. How to appraise an article about therapy
• Describe the criteria for internal validity of a therapy article
• Define randomized controlled trial, bias and random error, allocation concealment, double-blind, intention-to-treat analysis, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, p values, power and sample size, type I and II errors
• Understand the importance of all the previously defined concepts to apply in a therapy article
• Calculate OR, RR, RRR, ARR and NNT from a published therapy article
• Use a therapy article to solve a clinical problem
• Understand the concepts of external validity of a research paper, related to therapy
13. How to appraise an article about a diagnostic test, the Rational Clinical Examination Series
• Describe the criteria for internal validity of a diagnostic test article
• Define pre-test and post-test probability, sensitivity, specificity, likelihood ratios, positive and negative predictive value, accuracy
• Understand the importance of all the previously defined concepts to apply a diagnosis article
• Calculate sensitivity, specificity, likelihood ratios from a published diagnosis article
• Use a diagnosis article to solve a clinical problem
• Understand the concepts of external validity of a research paper, related to diagnosis
• Describe the origin and evolution of the Rational Clinical Examination JAMA series
• Use a JAMA Rational Clinical Examination paper to solve a clinical problem
14. How to appraise a Systematic Review or Meta-analysis
• Define meta-analysis, systematic review (qualitative and quantitative)
• Describe the advantages and limitations of systematic reviews and meta-analysis
• Describe the criteria for internal validity of a systematic review article
• Define bias and random error, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, forest plot, effect size
• Understand the importance of all the previously defined concepts applied to a systematic review article
• Calculate OR, RR, RRR, ARR and NNT from a published systematic review article
• Use a systematic review article to solve a clinical problem
• Understand the concepts of external validity of a systematic review
15. Clinical practice guidelines
• Define clinical practice guidelines
• Describe the sequence of developing an evidence-based clinical practice guideline
• Understand the advantages and limitations of a clinical guideline
• Describe and understand the internal validity requirements of a clinical guideline article
• List the available resources for clinical guidelines
• Use a clinical practice guideline to solve a clinical problem
Outcomes and Instrumentation The assessed outcomes were attitudes, knowledge and skills related to EBM. Two instruments were used: Taylor’s questionnaire, a published instrument designed to evaluate the effectiveness of evidence-based medicine teaching [29] and a 100 multiple-choice question test developed specifically for this study.
Taylor’s instrument was categorized as a level 1 instrument in a systematic review of tools to evaluate EBM education, since it has reasonable psychometric properties, has been evaluated for validity from at least three sources of evidence, and is recommended for use in the summative evaluation of individual trainees [30]. The instrument includes items to assess critical appraisal skills, use of evidence behaviors, knowledge and attitudes regarding evidence-based clinical practice [29]. The attitude portion of the questionnaire includes statements related to the use of scientific evidence using a Likert scale. Each statement is scored on a five point scale, responses are added to obtain a total attitude score, and the range of scores is 7 to 35. To determine an overall score for the confidence in critical appraisal skills section, six statements were scored using a scale where “Very confident” was assigned a score of 5, “Not at all confident” a score of 1, and “Don’t know” a score of 0. The scores of the six questions were added, providing a global critical appraisal skills confidence score, where 5 indicated “little or no confidence” and 30 indicated “complete confidence”.
The knowledge part of the questionnaire includes six multiple true-false questions, each with three items, using ‘true’, ‘false’ or ‘don’t know’ response categories. Correct responses to the knowledge questions have a score of 1, incorrect responses are negatively scored (−1) to try to prevent guessing, and the ‘don’t know’ response has a score of 0. The knowledge scores were added in an overall knowledge score, with a possible range of −18 to +18. In a previous paper, we translated the questionnaire to Spanish with the author’s permission, and verified it with backtranslation [21].
The other instrument used was the final summative test of the Evidence-Based Medicine Course. This instrument was developed, administered, scored, and analyzed following the 12 steps for effective test development described by Downing [31]. Item analysis was performed on a pilot application of the test with ITEMAN for Windows (Assessment Systems Corporation, St. Paul, MN), and the information obtained was used to improve the instrument for this study, choosing the better-performing items and preserving content validity. The pilot application of the original 140-items EBM test was done in 57 examinees, and had a Cronbach’s alpha of 0.82. Using the item analysis information 100 multiple-choice questions (MCQ) were selected by the test developers for the final version of the instrument.
The instruments were applied to the students on three consecutive weeks. The students had up to three hours to answer the test and the questionnaire, to minimize the risk of a speeded examination. Taylor’s questionnaires data sheets were captured in a Microsoft Excel spreadsheet. Op-scan answer sheets for item analysis were used for the EBM MCQ test.
The assessed outcomes were attitudes, knowledge and skills related to EBM. Two instruments were used: Taylor’s questionnaire, a published instrument designed to evaluate the effectiveness of evidence-based medicine teaching [29] and a 100 multiple-choice question test developed specifically for this study.
Taylor’s instrument was categorized as a level 1 instrument in a systematic review of tools to evaluate EBM education, since it has reasonable psychometric properties, has been evaluated for validity from at least three sources of evidence, and is recommended for use in the summative evaluation of individual trainees [30]. The instrument includes items to assess critical appraisal skills, use of evidence behaviors, knowledge and attitudes regarding evidence-based clinical practice [29]. The attitude portion of the questionnaire includes statements related to the use of scientific evidence using a Likert scale. Each statement is scored on a five point scale, responses are added to obtain a total attitude score, and the range of scores is 7 to 35. To determine an overall score for the confidence in critical appraisal skills section, six statements were scored using a scale where “Very confident” was assigned a score of 5, “Not at all confident” a score of 1, and “Don’t know” a score of 0. The scores of the six questions were added, providing a global critical appraisal skills confidence score, where 5 indicated “little or no confidence” and 30 indicated “complete confidence”.
The knowledge part of the questionnaire includes six multiple true-false questions, each with three items, using ‘true’, ‘false’ or ‘don’t know’ response categories. Correct responses to the knowledge questions have a score of 1, incorrect responses are negatively scored (−1) to try to prevent guessing, and the ‘don’t know’ response has a score of 0. The knowledge scores were added in an overall knowledge score, with a possible range of −18 to +18. In a previous paper, we translated the questionnaire to Spanish with the author’s permission, and verified it with backtranslation [21].
The other instrument used was the final summative test of the Evidence-Based Medicine Course. This instrument was developed, administered, scored, and analyzed following the 12 steps for effective test development described by Downing [31]. Item analysis was performed on a pilot application of the test with ITEMAN for Windows (Assessment Systems Corporation, St. Paul, MN), and the information obtained was used to improve the instrument for this study, choosing the better-performing items and preserving content validity. The pilot application of the original 140-items EBM test was done in 57 examinees, and had a Cronbach’s alpha of 0.82. Using the item analysis information 100 multiple-choice questions (MCQ) were selected by the test developers for the final version of the instrument.
The instruments were applied to the students on three consecutive weeks. The students had up to three hours to answer the test and the questionnaire, to minimize the risk of a speeded examination. Taylor’s questionnaires data sheets were captured in a Microsoft Excel spreadsheet. Op-scan answer sheets for item analysis were used for the EBM MCQ test.
Statistical analysis The piloting of the EBM MCQ test provided preliminary data for differences and standard deviation, and sample size calculation was performed for the primary hypothesis of knowledge increase with a power of 0.90 (beta error of 0.10), two-sided alpha error of 0.05. After a thorough review of the published studies that included magnitude of EBM knowledge differences in undergraduate medical students, and careful consideration by the course faculty of the smallest meaningful difference (SMD) in this parameter, it was estimated that a difference of 10 questions between the intervention group and the control group would be reasonable. Using this estimate, about 31 students per group would be necessary to detect an effect size of 0.5 or larger.
SPSS for Windows 15.0 and Instat 3.0 for the Macintosh were used for data analysis. The comparison of the use of evidence items in Taylor’s questionnaire between M5 and M5 non-EBM students was done with the non-parametric Mann–Whitney U test. The attitude and critical appraisal confidence scores measured with Taylor’s instrument were compared among groups using the Kruskal-Wallis with Dunn’s multiple comparison test. The groups’ knowledge test scores with both instruments were compared with one-way analysis of variance, with planned comparisons. A p-value of less than 0.05 was considered statistically significant. Cohen’s d with pooled standard deviations was calculated as a measure of effect size for the critical appraisal skills, attitude and knowledge scores among groups [32]. Item analysis of the EBM Test data was performed with ITEMAN for Windows 3.2, (Assessment Systems Corporation, St. Paul, MN http://www.assess.com).
The piloting of the EBM MCQ test provided preliminary data for differences and standard deviation, and sample size calculation was performed for the primary hypothesis of knowledge increase with a power of 0.90 (beta error of 0.10), two-sided alpha error of 0.05. After a thorough review of the published studies that included magnitude of EBM knowledge differences in undergraduate medical students, and careful consideration by the course faculty of the smallest meaningful difference (SMD) in this parameter, it was estimated that a difference of 10 questions between the intervention group and the control group would be reasonable. Using this estimate, about 31 students per group would be necessary to detect an effect size of 0.5 or larger.
SPSS for Windows 15.0 and Instat 3.0 for the Macintosh were used for data analysis. The comparison of the use of evidence items in Taylor’s questionnaire between M5 and M5 non-EBM students was done with the non-parametric Mann–Whitney U test. The attitude and critical appraisal confidence scores measured with Taylor’s instrument were compared among groups using the Kruskal-Wallis with Dunn’s multiple comparison test. The groups’ knowledge test scores with both instruments were compared with one-way analysis of variance, with planned comparisons. A p-value of less than 0.05 was considered statistically significant. Cohen’s d with pooled standard deviations was calculated as a measure of effect size for the critical appraisal skills, attitude and knowledge scores among groups [32]. Item analysis of the EBM Test data was performed with ITEMAN for Windows 3.2, (Assessment Systems Corporation, St. Paul, MN http://www.assess.com).
Ethical aspects The instruments did not have individual student identifiers, to eliminate the risk of potential harm to the participants. This study was reviewed by the Institutional Review Board of the Office for the Protection of Research Subjects of the University of Illinois at Chicago, and the Research Committee of the Mexican Army Medical School, and was considered to be in the exempt category for individual written informed consent.
The instruments did not have individual student identifiers, to eliminate the risk of potential harm to the participants. This study was reviewed by the Institutional Review Board of the Office for the Protection of Research Subjects of the University of Illinois at Chicago, and the Research Committee of the Mexican Army Medical School, and was considered to be in the exempt category for individual written informed consent.
|
Results
|
Subjects The flow diagram of the study participants throughout the trial is outlined in Figure 1. A total of 289 medical students were assessed. One student from the M5 non-EBM group was sick on the assessment day. Five subjects in the M4 non-EBM and 7 subjects in the M6 EBM groups didn't participate because they were on clinical duties on the testing day.
The students’ age (mean±SD) per group was: M4= 21.5±1.8, M5 EBM=22.8±2.0, M5 non-EBM=22.4±2.2 and M6=23.5±1.9 years. The groups’ gender composition was similar, with a predominance of women over men (about 60/40).
The flow diagram of the study participants throughout the trial is outlined in Figure 1. A total of 289 medical students were assessed. One student from the M5 non-EBM group was sick on the assessment day. Five subjects in the M4 non-EBM and 7 subjects in the M6 EBM groups didn't participate because they were on clinical duties on the testing day.
The students’ age (mean±SD) per group was: M4= 21.5±1.8, M5 EBM=22.8±2.0, M5 non-EBM=22.4±2.2 and M6=23.5±1.9 years. The groups’ gender composition was similar, with a predominance of women over men (about 60/40).
Use of the evidence The use of scientific evidence explored in the first section of Taylor’s questionnaire, includes two main questions: “What type of resources do you use to keep up to date?” and “What type of resources do you use to solve a specific health care problem?” The answers by group and type of resource are presented in Figures 2 and 3.
Use of evidence to keep up to date. Distribution of answers to the question: "what type of resources do you use to keep up to date?" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). *** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.
Use of evidence to solve a health problem. Distribution of answers to the question: "what type of resources do you use to solve a specific health problem?" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). * =P<0.01 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.*** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.
In the use of information resources to keep up to date and to solve a specific health care problem, the pattern of responses was the same. The answers were similar among the four student groups regarding the use of review articles, original research journals, textbooks, Internet resources and teachers, but there were statistically significant differences in the use of secondary journals (e.g. American Journal of Physicians Journal Club) and the Cochrane Library. The experimental group (M5 EBM) had a higher reported use of original research articles to solve a specific health problem than the randomized comparison group (M5 non-EBM) (P<0.01). The M5 EBM and M6 groups reported a higher use of secondary journals than the M4 and the M5 non-EBM groups, and a similar pattern of response was found in the use of the Cochrane Library (P<0.001) (Figures 2 and 3).
The use of scientific evidence explored in the first section of Taylor’s questionnaire, includes two main questions: “What type of resources do you use to keep up to date?” and “What type of resources do you use to solve a specific health care problem?” The answers by group and type of resource are presented in Figures 2 and 3.
Use of evidence to keep up to date. Distribution of answers to the question: "what type of resources do you use to keep up to date?" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). *** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.
Use of evidence to solve a health problem. Distribution of answers to the question: "what type of resources do you use to solve a specific health problem?" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). * =P<0.01 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.*** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.
In the use of information resources to keep up to date and to solve a specific health care problem, the pattern of responses was the same. The answers were similar among the four student groups regarding the use of review articles, original research journals, textbooks, Internet resources and teachers, but there were statistically significant differences in the use of secondary journals (e.g. American Journal of Physicians Journal Club) and the Cochrane Library. The experimental group (M5 EBM) had a higher reported use of original research articles to solve a specific health problem than the randomized comparison group (M5 non-EBM) (P<0.01). The M5 EBM and M6 groups reported a higher use of secondary journals than the M4 and the M5 non-EBM groups, and a similar pattern of response was found in the use of the Cochrane Library (P<0.001) (Figures 2 and 3).
Confidence in critical appraisal skills There was a higher confidence level of critical appraisal skills in all items in this section of Taylor’s instrument (assessing study design, evaluating bias, evaluating statistical tests), in the intervention group (P<0.001). The critical appraisal confidence global scores for the different study groups were as follows: M4=11.7±6.3 (mean±SD), M5 non-EBM=8.4±5.7, M5 EBM=17.1±3.6 and M6= 16.8±4.9. The summary data for each group is shown in Figure 4, where the experimental group (M5 EBM) had higher scores than the randomized control group (M5 non-EBM) and the M4 comparison group (P<0.001). The M4 score was slightly higher than the M5 non-EBM group (P<0.05), and the M6 group had higher scores than M4 and M5 non-EBM (P<0.001).
Critical appraisal skills scores. Critical appraisal confidence scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
There was a higher confidence level of critical appraisal skills in all items in this section of Taylor’s instrument (assessing study design, evaluating bias, evaluating statistical tests), in the intervention group (P<0.001). The critical appraisal confidence global scores for the different study groups were as follows: M4=11.7±6.3 (mean±SD), M5 non-EBM=8.4±5.7, M5 EBM=17.1±3.6 and M6= 16.8±4.9. The summary data for each group is shown in Figure 4, where the experimental group (M5 EBM) had higher scores than the randomized control group (M5 non-EBM) and the M4 comparison group (P<0.001). The M4 score was slightly higher than the M5 non-EBM group (P<0.05), and the M6 group had higher scores than M4 and M5 non-EBM (P<0.001).
Critical appraisal skills scores. Critical appraisal confidence scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Attitudes The EBM attitude scores measured with Taylor’s questionnaire are shown in Figure 5. The scores were similar between the groups that didn’t receive the EBM educational intervention, the M4 group had a score of 24.5±5.2 (mean±SD), and the M5 non-EBM group had 24.0±5.0 (P>0.05). The M5 EBM group had an attitude score of 28.7±2.2, higher than the M4 and M5 non-EBM groups (P<0.001). The M6 students had an attitude score of 26.7±3.6, higher than the control groups and lower than the M5 EBM group (P<0.05). Cohen’s d effect size for the comparison of M5 EBM vs. M5 non-EBM was 1.21 (Table 1).
Attitude scores. Attitude scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Effect size (Cohen’s “
d
”) in critical appraisal confidence, attitude and knowledge scores when comparing the different medical student groups
The EBM attitude scores measured with Taylor’s questionnaire are shown in Figure 5. The scores were similar between the groups that didn’t receive the EBM educational intervention, the M4 group had a score of 24.5±5.2 (mean±SD), and the M5 non-EBM group had 24.0±5.0 (P>0.05). The M5 EBM group had an attitude score of 28.7±2.2, higher than the M4 and M5 non-EBM groups (P<0.001). The M6 students had an attitude score of 26.7±3.6, higher than the control groups and lower than the M5 EBM group (P<0.05). Cohen’s d effect size for the comparison of M5 EBM vs. M5 non-EBM was 1.21 (Table 1).
Attitude scores. Attitude scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Effect size (Cohen’s “
d
”) in critical appraisal confidence, attitude and knowledge scores when comparing the different medical student groups
Knowledge scores with Taylor’s instrument The results of the knowledge score measured with Taylor’s questionnaire are shown in Figure 6. The scores were similar between non-EBM groups, M4=1.06±3.16 (mean±SD), and M5 non-EBM=1.13±3.27 (P=0.91). The M5 EBM intervention group had a knowledge score of 4.21±3.73, higher than those of M4 and M5 non-EBM. The planned contrast in the main comparison between M5 EBM and M5 non-EBM showed that the intervention group had a higher knowledge score than the randomized control group (P<0.001). The M6 group had a knowledge score of 2.44±3.77, higher than both control groups, (P<0.01), but lower than M5 EBM (P<0.01). The effect size measured with Cohen’s d for the knowledge score main comparison of M5 EBM vs. M5 non-EBM was 0.88 (Table 1).
Knowledge scores with Taylor’s instrument. Knowledge scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
The results of the knowledge score measured with Taylor’s questionnaire are shown in Figure 6. The scores were similar between non-EBM groups, M4=1.06±3.16 (mean±SD), and M5 non-EBM=1.13±3.27 (P=0.91). The M5 EBM intervention group had a knowledge score of 4.21±3.73, higher than those of M4 and M5 non-EBM. The planned contrast in the main comparison between M5 EBM and M5 non-EBM showed that the intervention group had a higher knowledge score than the randomized control group (P<0.001). The M6 group had a knowledge score of 2.44±3.77, higher than both control groups, (P<0.01), but lower than M5 EBM (P<0.01). The effect size measured with Cohen’s d for the knowledge score main comparison of M5 EBM vs. M5 non-EBM was 0.88 (Table 1).
Knowledge scores with Taylor’s instrument. Knowledge scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Knowledge scores with EBM summative MCQ test The results of the 100-item MCQ EBM knowledge test are presented as percent-correct scores (Figure 7). The reliability of the test with Cronbach’s alpha was 0.72 in the M5 EBM group, and 0.83 in the M6 group. The scores were similar between non-EBM groups, M4=30.6±5.6 (mean±SD), and M5 non-EBM=32.6±6.6 (P=0.18). The M5 EBM group had a test score of 58.5±7.9, higher than M4 and M5 non-EBM. The planned contrast between M5 EBM and M5 non-EBM found that the educational intervention group had a higher knowledge score (P<0.001). M6 had a knowledge score of 41.0±10.9, higher than the control groups (P<0.001), but lower than M5 EBM (P<0.001). The effect size with Cohen’s d for the knowledge score main outcome comparison of M5 EBM vs. M5 non-EBM was 3.54 (Table 1).
Knowledge scores EBM test. Knowledge scores in the different groups of medical students, measured with the 100 multiple-choice questions EBM test. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
The results of the 100-item MCQ EBM knowledge test are presented as percent-correct scores (Figure 7). The reliability of the test with Cronbach’s alpha was 0.72 in the M5 EBM group, and 0.83 in the M6 group. The scores were similar between non-EBM groups, M4=30.6±5.6 (mean±SD), and M5 non-EBM=32.6±6.6 (P=0.18). The M5 EBM group had a test score of 58.5±7.9, higher than M4 and M5 non-EBM. The planned contrast between M5 EBM and M5 non-EBM found that the educational intervention group had a higher knowledge score (P<0.001). M6 had a knowledge score of 41.0±10.9, higher than the control groups (P<0.001), but lower than M5 EBM (P<0.001). The effect size with Cohen’s d for the knowledge score main outcome comparison of M5 EBM vs. M5 non-EBM was 3.54 (Table 1).
Knowledge scores EBM test. Knowledge scores in the different groups of medical students, measured with the 100 multiple-choice questions EBM test. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
|
Conclusions
|
Our study has implications for the design, implementation and assessment of EBM educational interventions in developing countries. Firstly, it shows that EBM courses can be successfully implemented and embedded in a medical school’s curriculum. Secondly, it provides evidence that the course can improve knowledge, attitudes, critical appraisal confidence, and self-reported skills and behaviours about EBM and its related concepts, although the amount of knowledge that changes with time is still uncertain. And thirdly, it attests to the fact that using international test development standards can contribute to the development of a reliable instrument with evidence of construct validity for the measurement of EBM knowledge acquisition. The study findings contributed to the quality improvement process in the medical school, and provided data to be used in the planning and implementation of subsequent EBM courses. Educational planning will address its clinical links and vertical/horizontal integration with the rest of the curriculum (explicit and hidden), and more studies with rigorous follow-up should be undertaken to identify EBM competencies retention in the long-term. Published models and recommendations to increase the depth and duration of EBM learning should be taken into account when initiating educational interventions of this nature [47,48].
|
[
"Background",
"Setting",
"Overall study design and participants",
"Main outcomes and subjects",
"Simultaneous validation",
"Intervention",
"Outcomes and Instrumentation",
"Statistical analysis",
"Ethical aspects",
"Subjects",
"Use of the evidence",
"Confidence in critical appraisal skills",
"Attitudes",
"Knowledge scores with Taylor’s instrument",
"Knowledge scores with EBM summative MCQ test",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Evidence-based medicine (EBM) has been defined as “the integration of the best research evidence with our clinical expertise and our patient’s unique values and circumstances”, and it has emerged as a core competency necessary for all healthcare professionals [1-3]. Its fundamental principles are: translation of uncertainty to an answerable clinical question, systematic retrieval of the best evidence available, critical appraisal for validity, relevance and applicability, use of results in practice and evaluation of its performance by the healthcare provider [4].\nSeveral organizations, including the Institute of Medicine in the United States and the World Federation for Medical Education, have advocated the implementation of EBM educational interventions in medical under and postgraduate training [2,5].\nThe concepts related to EBM and its educational implications have disseminated rapidly in the last decade, and this change needs to be accompanied with strong educational research to document its effectiveness. The challenges of teaching EBM and the paucity of rigorous educational research publications have prompted some medical educators to question the evidence of EBM teaching effectiveness [6]. Nonetheless, the foundations of EBM that support clinical decision making are intuitively attractive to many clinicians and educators, since it integrates the educational process with clinical practice [4].\nThe quality of the evidence about EBM education is heterogeneous, as has been described in several editorials, narrative and systematic reviews [7-11]. The majority of reviews have included mostly studies in postgraduate health professionals, and some have included studies in both post and undergraduate students. Green reviewed 18 reports, mostly resident-directed small-group seminars with the objective of improving critical appraisal skills [12]. The most commonly used outcome measure was a multiple-choice exam, and 72% used a traditional journal club format as teaching strategy. Only seven of the 18 studies included in Green’s review analyzed the effectiveness of the intervention, five of these had some type of control group and only one was a randomized study. Just two studies used an outcome measure that had validity evidence, and measurement of change in behavior used only self-report in all five papers. The impact of the intervention was focused mainly on critical appraisal, and ranged from no effect to 23% absolute increase in scores [12].\nThe Cochrane Collaboration systematic review on the subject of teaching critical appraisal skills in health care, which excluded medical students, found three studies that met stringent pre-specified methodological criteria. These articles reported statistically significant improvements in participants' knowledge in domains of critical appraisal in two of the three studies [9]. Another systematic review by Coomarasamy focused on postgraduate clinicians, and found significant effects of EBM educational interventions in knowledge, and more limited in attitudes, skills and behavior [10,11].\nDespite the increasing number of medical school and postgraduate programs that have introduced EBM in their curricula, most of the information about it has been reported as observational data and descriptive studies in the medical literature, or as unpublished observations that are disseminated in medical meetings or informal venues. There are few randomized controlled educational trials about EBM training effectiveness, and the majority have been done in residents or practicing physicians [9-14].\nUndergraduate medical students can be a receptive population to EBM concepts, and they will be the practicing clinicians and clinical teachers in the future. There are several published studies that describe medical schools’ experiences introducing EBM in their curriculum and teaching these concepts to undergraduates, with variable outcomes [15-19]. This curricular change has not occurred in many of their developing country counterparts, with few published reports of the implementation of EBM curricula in these settings [20-23]. There is a need to implement EBM educational interventions in developing countries medical schools’ curricula, and to assess their impact with appropriate educational research designs.\nThe purpose of this study was to assess the educational effectiveness (attitudes, knowledge and skills) of an EBM course in undergraduate medical students.",
"The Mexican Army medical school trains career physicians for the national military healthcare system, and is located in Mexico City. It has a six year program, with a traditional curriculum: two years of basic sciences, three years of clinical sciences, and the sixth year is an internship period in the hospital. The school is a public institution funded by the federal government. Each yearly class is composed of about one hundred students, mostly middle- or low-socioeconomic class Hispanics.",
" Main outcomes and subjects The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].\nFlow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.\nThe core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].\nFlow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.\n Simultaneous validation Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).\nThe outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.\nQuasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).\nThe outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.",
"The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].\nFlow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.",
"Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).\nThe outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.",
"The educational intervention was a one semester EBM course formally included in the medical school curriculum, with 14 two-hour weekly sessions. The course faculty were six professors trained in EBM teaching, all board-certified physicians with clinical practice, one of them with a postgraduate degree in health professions education and faculty development training in EBM education at McMaster University Faculty of Health Sciences in Canada. The course faculty had more than six years of experience teaching EBM to undergraduate medical students, residents of several specialties, and providing faculty development EBM workshops to teachers of several medical specialties. The EBM course teachers were not involved in the training of the 4th year students, but they participated in the training of the 6th year interns. The EBM program was linked with the internship program and the residency programs in the hospital, through the medical school curricular committee and the University Postgraduate Studies Division.\nThe course instructional strategies included large-group interactive sessions, small-group problem-solving activities, individual and group assignments, and informatics laboratory sessions. Traditional EBM resources were used as course bibliography, including Straus’ book [1] and an EBM text in Spanish written by the course professors [28]. The content and learning objectives of the course are outlined below.\n1. Clinical decision making in medicine\n • List and define the main difficulties for objective decision making in medicine as defined by Eddy\n • Describe the components of a decision in medicine as defined by Eddy\n • Apply the concepts of anatomy of a decision as defined by Eddy in the analysis of a clinical problem\n2. Uncertainty and probability in medicine\n • Define the concepts of uncertainty, probability and odds\n • Understand the relevance of uncertainty in clinical practice\n • Understand the limitations of personal experience in the estimation of probability, as related to diagnosis\n • Define the heuristics used in medicine (representativeness, availability, anchor and adjustment) and list the cognitive errors a clinician can make when misapplying them\n • Apply the concepts of heuristics in new clinical problems, and discuss the effects of their inappropriate use\n3. Bayes’ theorem\n • Define Bayes’ theorem\n • Define pre-test and post-test probability\n • Define the concepts of diagnostic and therapeutic threshold\n • Explain the utility of Bayes' theorem in clinical medicine, mainly in diagnosis\n • List the limitations of Bayes' theorem in clinical practice\n • Apply Fagan’s nomogram to use Bayes' theorem in a diagnostic problem\n • Apply the concepts of diagnostic and therapeutic threshold to a clinical problem\n4. Principles of Evidence Based Medicine\n • Describe the history and origin of EBM\n • Define the concept of EBM\n • List the five steps of EBM, and apply them in a clinical problem\n • Explain the importance of EBM in clinical practice\n5. Reflective medical practice\n • Define the concept of reflection and reflective practitioner\n • Define reflection-in-action and reflection-on-action\n • Apply these concepts in a clinical scenario\n6. Clinicians’ information needs\n • Understand the magnitude of physician information needs\n • Understand the literature that describe how clinicians underestimate their information needs\n • Define the percentage of occasions when clinicians recognize and act upon perceived information needs\n7. Clinical questions\n • Define the concepts of background and foreground questions\n • Understand the advantages of structuring questions generated during clinical work\n • List the four components of a foreground clinical question (PICO)\n • Apply these concepts in developing questions from clinical problems\n • List the types of clinical questions (diagnosis, therapy, prognosis, harm, etiology)\n8. Sources of biomedical information\n • List the different sources of biomedical information available\n • Identify the advantages and disadvantages of each source (textbooks, paper and electronic journals, original research papers)\n • Understand the origin, development, cost, and availability of sources of information\n9. The Cochrane Collaboration\n • Describe the history and origin of the Cochrane Collaboration (CC)\n • List the components of the Cochrane Library, and the sources where it’s available\n • Understand the mission, logistics and work of the CC\n • Perform effective searches for systematic reviews on the Cochrane Library\n • Understand the advantages and limitations of the CC\n • Use the Cochrane Library to solve a clinical problem\n10. Search strategies to find the best medical scientific evidence\n • List the main medical databases, and identify their relevance and location\n • Describe the history of Medline\n • Define MeSH terms, Boolean operators, search engine\n • Design search strategies to find valid evidence\n • Use PubMed Clinical Queries\n • Perform effective searches of scientifically valid papers using PubMed, Cochrane Library, OVID Core Medical Library\n • Understand the advantages and disadvantages of searching the different electronic medical databases and the Internet general purpose searching engines\n11. Critical appraisal of the medical literature: Users’ Guides to the Medical Literature\n • Describe the origin and history of the Users’ Guides series to appraise the medical literature\n • List and understand the different hierarchies of evidence, study designs, grades of evidence\n • Understand the relevance of using the original medical literature to solve clinical problems\n • List and understand the three different steps to appraise a research article: internal validity, magnitude of the results and external validity\n12. How to appraise an article about therapy\n • Describe the criteria for internal validity of a therapy article\n • Define randomized controlled trial, bias and random error, allocation concealment, double-blind, intention-to-treat analysis, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, p values, power and sample size, type I and II errors\n • Understand the importance of all the previously defined concepts to apply in a therapy article\n • Calculate OR, RR, RRR, ARR and NNT from a published therapy article\n • Use a therapy article to solve a clinical problem\n • Understand the concepts of external validity of a research paper, related to therapy\n13. How to appraise an article about a diagnostic test, the Rational Clinical Examination Series\n • Describe the criteria for internal validity of a diagnostic test article\n • Define pre-test and post-test probability, sensitivity, specificity, likelihood ratios, positive and negative predictive value, accuracy\n • Understand the importance of all the previously defined concepts to apply a diagnosis article\n • Calculate sensitivity, specificity, likelihood ratios from a published diagnosis article\n • Use a diagnosis article to solve a clinical problem\n • Understand the concepts of external validity of a research paper, related to diagnosis\n • Describe the origin and evolution of the Rational Clinical Examination JAMA series\n • Use a JAMA Rational Clinical Examination paper to solve a clinical problem\n14. How to appraise a Systematic Review or Meta-analysis\n • Define meta-analysis, systematic review (qualitative and quantitative)\n • Describe the advantages and limitations of systematic reviews and meta-analysis\n • Describe the criteria for internal validity of a systematic review article\n • Define bias and random error, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, forest plot, effect size\n • Understand the importance of all the previously defined concepts applied to a systematic review article\n • Calculate OR, RR, RRR, ARR and NNT from a published systematic review article\n • Use a systematic review article to solve a clinical problem\n • Understand the concepts of external validity of a systematic review\n15. Clinical practice guidelines\n • Define clinical practice guidelines\n • Describe the sequence of developing an evidence-based clinical practice guideline\n • Understand the advantages and limitations of a clinical guideline\n • Describe and understand the internal validity requirements of a clinical guideline article\n • List the available resources for clinical guidelines\n • Use a clinical practice guideline to solve a clinical problem",
"The assessed outcomes were attitudes, knowledge and skills related to EBM. Two instruments were used: Taylor’s questionnaire, a published instrument designed to evaluate the effectiveness of evidence-based medicine teaching [29] and a 100 multiple-choice question test developed specifically for this study.\nTaylor’s instrument was categorized as a level 1 instrument in a systematic review of tools to evaluate EBM education, since it has reasonable psychometric properties, has been evaluated for validity from at least three sources of evidence, and is recommended for use in the summative evaluation of individual trainees [30]. The instrument includes items to assess critical appraisal skills, use of evidence behaviors, knowledge and attitudes regarding evidence-based clinical practice [29]. The attitude portion of the questionnaire includes statements related to the use of scientific evidence using a Likert scale. Each statement is scored on a five point scale, responses are added to obtain a total attitude score, and the range of scores is 7 to 35. To determine an overall score for the confidence in critical appraisal skills section, six statements were scored using a scale where “Very confident” was assigned a score of 5, “Not at all confident” a score of 1, and “Don’t know” a score of 0. The scores of the six questions were added, providing a global critical appraisal skills confidence score, where 5 indicated “little or no confidence” and 30 indicated “complete confidence”.\nThe knowledge part of the questionnaire includes six multiple true-false questions, each with three items, using ‘true’, ‘false’ or ‘don’t know’ response categories. Correct responses to the knowledge questions have a score of 1, incorrect responses are negatively scored (−1) to try to prevent guessing, and the ‘don’t know’ response has a score of 0. The knowledge scores were added in an overall knowledge score, with a possible range of −18 to +18. In a previous paper, we translated the questionnaire to Spanish with the author’s permission, and verified it with backtranslation [21].\nThe other instrument used was the final summative test of the Evidence-Based Medicine Course. This instrument was developed, administered, scored, and analyzed following the 12 steps for effective test development described by Downing [31]. Item analysis was performed on a pilot application of the test with ITEMAN for Windows (Assessment Systems Corporation, St. Paul, MN), and the information obtained was used to improve the instrument for this study, choosing the better-performing items and preserving content validity. The pilot application of the original 140-items EBM test was done in 57 examinees, and had a Cronbach’s alpha of 0.82. Using the item analysis information 100 multiple-choice questions (MCQ) were selected by the test developers for the final version of the instrument.\nThe instruments were applied to the students on three consecutive weeks. The students had up to three hours to answer the test and the questionnaire, to minimize the risk of a speeded examination. Taylor’s questionnaires data sheets were captured in a Microsoft Excel spreadsheet. Op-scan answer sheets for item analysis were used for the EBM MCQ test.",
"The piloting of the EBM MCQ test provided preliminary data for differences and standard deviation, and sample size calculation was performed for the primary hypothesis of knowledge increase with a power of 0.90 (beta error of 0.10), two-sided alpha error of 0.05. After a thorough review of the published studies that included magnitude of EBM knowledge differences in undergraduate medical students, and careful consideration by the course faculty of the smallest meaningful difference (SMD) in this parameter, it was estimated that a difference of 10 questions between the intervention group and the control group would be reasonable. Using this estimate, about 31 students per group would be necessary to detect an effect size of 0.5 or larger.\nSPSS for Windows 15.0 and Instat 3.0 for the Macintosh were used for data analysis. The comparison of the use of evidence items in Taylor’s questionnaire between M5 and M5 non-EBM students was done with the non-parametric Mann–Whitney U test. The attitude and critical appraisal confidence scores measured with Taylor’s instrument were compared among groups using the Kruskal-Wallis with Dunn’s multiple comparison test. The groups’ knowledge test scores with both instruments were compared with one-way analysis of variance, with planned comparisons. A p-value of less than 0.05 was considered statistically significant. Cohen’s d with pooled standard deviations was calculated as a measure of effect size for the critical appraisal skills, attitude and knowledge scores among groups [32]. Item analysis of the EBM Test data was performed with ITEMAN for Windows 3.2, (Assessment Systems Corporation, St. Paul, MN http://www.assess.com).",
"The instruments did not have individual student identifiers, to eliminate the risk of potential harm to the participants. This study was reviewed by the Institutional Review Board of the Office for the Protection of Research Subjects of the University of Illinois at Chicago, and the Research Committee of the Mexican Army Medical School, and was considered to be in the exempt category for individual written informed consent.",
"The flow diagram of the study participants throughout the trial is outlined in Figure 1. A total of 289 medical students were assessed. One student from the M5 non-EBM group was sick on the assessment day. Five subjects in the M4 non-EBM and 7 subjects in the M6 EBM groups didn't participate because they were on clinical duties on the testing day.\nThe students’ age (mean±SD) per group was: M4= 21.5±1.8, M5 EBM=22.8±2.0, M5 non-EBM=22.4±2.2 and M6=23.5±1.9 years. The groups’ gender composition was similar, with a predominance of women over men (about 60/40).",
"The use of scientific evidence explored in the first section of Taylor’s questionnaire, includes two main questions: “What type of resources do you use to keep up to date?” and “What type of resources do you use to solve a specific health care problem?” The answers by group and type of resource are presented in Figures 2 and 3.\nUse of evidence to keep up to date. Distribution of answers to the question: \"what type of resources do you use to keep up to date?\" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). *** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.\nUse of evidence to solve a health problem. Distribution of answers to the question: \"what type of resources do you use to solve a specific health problem?\" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). * =P<0.01 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.*** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.\nIn the use of information resources to keep up to date and to solve a specific health care problem, the pattern of responses was the same. The answers were similar among the four student groups regarding the use of review articles, original research journals, textbooks, Internet resources and teachers, but there were statistically significant differences in the use of secondary journals (e.g. American Journal of Physicians Journal Club) and the Cochrane Library. The experimental group (M5 EBM) had a higher reported use of original research articles to solve a specific health problem than the randomized comparison group (M5 non-EBM) (P<0.01). The M5 EBM and M6 groups reported a higher use of secondary journals than the M4 and the M5 non-EBM groups, and a similar pattern of response was found in the use of the Cochrane Library (P<0.001) (Figures 2 and 3).",
"There was a higher confidence level of critical appraisal skills in all items in this section of Taylor’s instrument (assessing study design, evaluating bias, evaluating statistical tests), in the intervention group (P<0.001). The critical appraisal confidence global scores for the different study groups were as follows: M4=11.7±6.3 (mean±SD), M5 non-EBM=8.4±5.7, M5 EBM=17.1±3.6 and M6= 16.8±4.9. The summary data for each group is shown in Figure 4, where the experimental group (M5 EBM) had higher scores than the randomized control group (M5 non-EBM) and the M4 comparison group (P<0.001). The M4 score was slightly higher than the M5 non-EBM group (P<0.05), and the M6 group had higher scores than M4 and M5 non-EBM (P<0.001).\nCritical appraisal skills scores. Critical appraisal confidence scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).",
"The EBM attitude scores measured with Taylor’s questionnaire are shown in Figure 5. The scores were similar between the groups that didn’t receive the EBM educational intervention, the M4 group had a score of 24.5±5.2 (mean±SD), and the M5 non-EBM group had 24.0±5.0 (P>0.05). The M5 EBM group had an attitude score of 28.7±2.2, higher than the M4 and M5 non-EBM groups (P<0.001). The M6 students had an attitude score of 26.7±3.6, higher than the control groups and lower than the M5 EBM group (P<0.05). Cohen’s d effect size for the comparison of M5 EBM vs. M5 non-EBM was 1.21 (Table 1).\nAttitude scores. Attitude scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).\n\nEffect size (Cohen’s “\n\nd\n\n”) in critical appraisal confidence, attitude and knowledge scores when comparing the different medical student groups\n",
"The results of the knowledge score measured with Taylor’s questionnaire are shown in Figure 6. The scores were similar between non-EBM groups, M4=1.06±3.16 (mean±SD), and M5 non-EBM=1.13±3.27 (P=0.91). The M5 EBM intervention group had a knowledge score of 4.21±3.73, higher than those of M4 and M5 non-EBM. The planned contrast in the main comparison between M5 EBM and M5 non-EBM showed that the intervention group had a higher knowledge score than the randomized control group (P<0.001). The M6 group had a knowledge score of 2.44±3.77, higher than both control groups, (P<0.01), but lower than M5 EBM (P<0.01). The effect size measured with Cohen’s d for the knowledge score main comparison of M5 EBM vs. M5 non-EBM was 0.88 (Table 1).\nKnowledge scores with Taylor’s instrument. Knowledge scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).",
"The results of the 100-item MCQ EBM knowledge test are presented as percent-correct scores (Figure 7). The reliability of the test with Cronbach’s alpha was 0.72 in the M5 EBM group, and 0.83 in the M6 group. The scores were similar between non-EBM groups, M4=30.6±5.6 (mean±SD), and M5 non-EBM=32.6±6.6 (P=0.18). The M5 EBM group had a test score of 58.5±7.9, higher than M4 and M5 non-EBM. The planned contrast between M5 EBM and M5 non-EBM found that the educational intervention group had a higher knowledge score (P<0.001). M6 had a knowledge score of 41.0±10.9, higher than the control groups (P<0.001), but lower than M5 EBM (P<0.001). The effect size with Cohen’s d for the knowledge score main outcome comparison of M5 EBM vs. M5 non-EBM was 3.54 (Table 1).\nKnowledge scores EBM test. Knowledge scores in the different groups of medical students, measured with the 100 multiple-choice questions EBM test. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).",
"The authors declare that they have no competing interests.",
"MS, LK and SM planned, designed and implemented the EBM course and the summative test, and applied the assessment instruments. MS, SD and AS participated in the design of the study and the statistical analysis. MS drafted the initial version of the manuscript. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6920/12/107/prepub\n"
] |
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[
"Background",
"Methods",
"Setting",
"Overall study design and participants",
"Main outcomes and subjects",
"Simultaneous validation",
"Intervention",
"Outcomes and Instrumentation",
"Statistical analysis",
"Ethical aspects",
"Results",
"Subjects",
"Use of the evidence",
"Confidence in critical appraisal skills",
"Attitudes",
"Knowledge scores with Taylor’s instrument",
"Knowledge scores with EBM summative MCQ test",
"Discussion",
"Conclusions",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Evidence-based medicine (EBM) has been defined as “the integration of the best research evidence with our clinical expertise and our patient’s unique values and circumstances”, and it has emerged as a core competency necessary for all healthcare professionals [1-3]. Its fundamental principles are: translation of uncertainty to an answerable clinical question, systematic retrieval of the best evidence available, critical appraisal for validity, relevance and applicability, use of results in practice and evaluation of its performance by the healthcare provider [4].\nSeveral organizations, including the Institute of Medicine in the United States and the World Federation for Medical Education, have advocated the implementation of EBM educational interventions in medical under and postgraduate training [2,5].\nThe concepts related to EBM and its educational implications have disseminated rapidly in the last decade, and this change needs to be accompanied with strong educational research to document its effectiveness. The challenges of teaching EBM and the paucity of rigorous educational research publications have prompted some medical educators to question the evidence of EBM teaching effectiveness [6]. Nonetheless, the foundations of EBM that support clinical decision making are intuitively attractive to many clinicians and educators, since it integrates the educational process with clinical practice [4].\nThe quality of the evidence about EBM education is heterogeneous, as has been described in several editorials, narrative and systematic reviews [7-11]. The majority of reviews have included mostly studies in postgraduate health professionals, and some have included studies in both post and undergraduate students. Green reviewed 18 reports, mostly resident-directed small-group seminars with the objective of improving critical appraisal skills [12]. The most commonly used outcome measure was a multiple-choice exam, and 72% used a traditional journal club format as teaching strategy. Only seven of the 18 studies included in Green’s review analyzed the effectiveness of the intervention, five of these had some type of control group and only one was a randomized study. Just two studies used an outcome measure that had validity evidence, and measurement of change in behavior used only self-report in all five papers. The impact of the intervention was focused mainly on critical appraisal, and ranged from no effect to 23% absolute increase in scores [12].\nThe Cochrane Collaboration systematic review on the subject of teaching critical appraisal skills in health care, which excluded medical students, found three studies that met stringent pre-specified methodological criteria. These articles reported statistically significant improvements in participants' knowledge in domains of critical appraisal in two of the three studies [9]. Another systematic review by Coomarasamy focused on postgraduate clinicians, and found significant effects of EBM educational interventions in knowledge, and more limited in attitudes, skills and behavior [10,11].\nDespite the increasing number of medical school and postgraduate programs that have introduced EBM in their curricula, most of the information about it has been reported as observational data and descriptive studies in the medical literature, or as unpublished observations that are disseminated in medical meetings or informal venues. There are few randomized controlled educational trials about EBM training effectiveness, and the majority have been done in residents or practicing physicians [9-14].\nUndergraduate medical students can be a receptive population to EBM concepts, and they will be the practicing clinicians and clinical teachers in the future. There are several published studies that describe medical schools’ experiences introducing EBM in their curriculum and teaching these concepts to undergraduates, with variable outcomes [15-19]. This curricular change has not occurred in many of their developing country counterparts, with few published reports of the implementation of EBM curricula in these settings [20-23]. There is a need to implement EBM educational interventions in developing countries medical schools’ curricula, and to assess their impact with appropriate educational research designs.\nThe purpose of this study was to assess the educational effectiveness (attitudes, knowledge and skills) of an EBM course in undergraduate medical students.",
" Setting The Mexican Army medical school trains career physicians for the national military healthcare system, and is located in Mexico City. It has a six year program, with a traditional curriculum: two years of basic sciences, three years of clinical sciences, and the sixth year is an internship period in the hospital. The school is a public institution funded by the federal government. Each yearly class is composed of about one hundred students, mostly middle- or low-socioeconomic class Hispanics.\nThe Mexican Army medical school trains career physicians for the national military healthcare system, and is located in Mexico City. It has a six year program, with a traditional curriculum: two years of basic sciences, three years of clinical sciences, and the sixth year is an internship period in the hospital. The school is a public institution funded by the federal government. Each yearly class is composed of about one hundred students, mostly middle- or low-socioeconomic class Hispanics.\n Overall study design and participants Main outcomes and subjects The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].\nFlow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.\nThe core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].\nFlow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.\n Simultaneous validation Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).\nThe outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.\nQuasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).\nThe outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.\n Main outcomes and subjects The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].\nFlow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.\nThe core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].\nFlow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.\n Simultaneous validation Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).\nThe outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.\nQuasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).\nThe outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.\n Intervention The educational intervention was a one semester EBM course formally included in the medical school curriculum, with 14 two-hour weekly sessions. The course faculty were six professors trained in EBM teaching, all board-certified physicians with clinical practice, one of them with a postgraduate degree in health professions education and faculty development training in EBM education at McMaster University Faculty of Health Sciences in Canada. The course faculty had more than six years of experience teaching EBM to undergraduate medical students, residents of several specialties, and providing faculty development EBM workshops to teachers of several medical specialties. The EBM course teachers were not involved in the training of the 4th year students, but they participated in the training of the 6th year interns. The EBM program was linked with the internship program and the residency programs in the hospital, through the medical school curricular committee and the University Postgraduate Studies Division.\nThe course instructional strategies included large-group interactive sessions, small-group problem-solving activities, individual and group assignments, and informatics laboratory sessions. Traditional EBM resources were used as course bibliography, including Straus’ book [1] and an EBM text in Spanish written by the course professors [28]. The content and learning objectives of the course are outlined below.\n1. Clinical decision making in medicine\n • List and define the main difficulties for objective decision making in medicine as defined by Eddy\n • Describe the components of a decision in medicine as defined by Eddy\n • Apply the concepts of anatomy of a decision as defined by Eddy in the analysis of a clinical problem\n2. Uncertainty and probability in medicine\n • Define the concepts of uncertainty, probability and odds\n • Understand the relevance of uncertainty in clinical practice\n • Understand the limitations of personal experience in the estimation of probability, as related to diagnosis\n • Define the heuristics used in medicine (representativeness, availability, anchor and adjustment) and list the cognitive errors a clinician can make when misapplying them\n • Apply the concepts of heuristics in new clinical problems, and discuss the effects of their inappropriate use\n3. Bayes’ theorem\n • Define Bayes’ theorem\n • Define pre-test and post-test probability\n • Define the concepts of diagnostic and therapeutic threshold\n • Explain the utility of Bayes' theorem in clinical medicine, mainly in diagnosis\n • List the limitations of Bayes' theorem in clinical practice\n • Apply Fagan’s nomogram to use Bayes' theorem in a diagnostic problem\n • Apply the concepts of diagnostic and therapeutic threshold to a clinical problem\n4. Principles of Evidence Based Medicine\n • Describe the history and origin of EBM\n • Define the concept of EBM\n • List the five steps of EBM, and apply them in a clinical problem\n • Explain the importance of EBM in clinical practice\n5. Reflective medical practice\n • Define the concept of reflection and reflective practitioner\n • Define reflection-in-action and reflection-on-action\n • Apply these concepts in a clinical scenario\n6. Clinicians’ information needs\n • Understand the magnitude of physician information needs\n • Understand the literature that describe how clinicians underestimate their information needs\n • Define the percentage of occasions when clinicians recognize and act upon perceived information needs\n7. Clinical questions\n • Define the concepts of background and foreground questions\n • Understand the advantages of structuring questions generated during clinical work\n • List the four components of a foreground clinical question (PICO)\n • Apply these concepts in developing questions from clinical problems\n • List the types of clinical questions (diagnosis, therapy, prognosis, harm, etiology)\n8. Sources of biomedical information\n • List the different sources of biomedical information available\n • Identify the advantages and disadvantages of each source (textbooks, paper and electronic journals, original research papers)\n • Understand the origin, development, cost, and availability of sources of information\n9. The Cochrane Collaboration\n • Describe the history and origin of the Cochrane Collaboration (CC)\n • List the components of the Cochrane Library, and the sources where it’s available\n • Understand the mission, logistics and work of the CC\n • Perform effective searches for systematic reviews on the Cochrane Library\n • Understand the advantages and limitations of the CC\n • Use the Cochrane Library to solve a clinical problem\n10. Search strategies to find the best medical scientific evidence\n • List the main medical databases, and identify their relevance and location\n • Describe the history of Medline\n • Define MeSH terms, Boolean operators, search engine\n • Design search strategies to find valid evidence\n • Use PubMed Clinical Queries\n • Perform effective searches of scientifically valid papers using PubMed, Cochrane Library, OVID Core Medical Library\n • Understand the advantages and disadvantages of searching the different electronic medical databases and the Internet general purpose searching engines\n11. Critical appraisal of the medical literature: Users’ Guides to the Medical Literature\n • Describe the origin and history of the Users’ Guides series to appraise the medical literature\n • List and understand the different hierarchies of evidence, study designs, grades of evidence\n • Understand the relevance of using the original medical literature to solve clinical problems\n • List and understand the three different steps to appraise a research article: internal validity, magnitude of the results and external validity\n12. How to appraise an article about therapy\n • Describe the criteria for internal validity of a therapy article\n • Define randomized controlled trial, bias and random error, allocation concealment, double-blind, intention-to-treat analysis, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, p values, power and sample size, type I and II errors\n • Understand the importance of all the previously defined concepts to apply in a therapy article\n • Calculate OR, RR, RRR, ARR and NNT from a published therapy article\n • Use a therapy article to solve a clinical problem\n • Understand the concepts of external validity of a research paper, related to therapy\n13. How to appraise an article about a diagnostic test, the Rational Clinical Examination Series\n • Describe the criteria for internal validity of a diagnostic test article\n • Define pre-test and post-test probability, sensitivity, specificity, likelihood ratios, positive and negative predictive value, accuracy\n • Understand the importance of all the previously defined concepts to apply a diagnosis article\n • Calculate sensitivity, specificity, likelihood ratios from a published diagnosis article\n • Use a diagnosis article to solve a clinical problem\n • Understand the concepts of external validity of a research paper, related to diagnosis\n • Describe the origin and evolution of the Rational Clinical Examination JAMA series\n • Use a JAMA Rational Clinical Examination paper to solve a clinical problem\n14. How to appraise a Systematic Review or Meta-analysis\n • Define meta-analysis, systematic review (qualitative and quantitative)\n • Describe the advantages and limitations of systematic reviews and meta-analysis\n • Describe the criteria for internal validity of a systematic review article\n • Define bias and random error, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, forest plot, effect size\n • Understand the importance of all the previously defined concepts applied to a systematic review article\n • Calculate OR, RR, RRR, ARR and NNT from a published systematic review article\n • Use a systematic review article to solve a clinical problem\n • Understand the concepts of external validity of a systematic review\n15. Clinical practice guidelines\n • Define clinical practice guidelines\n • Describe the sequence of developing an evidence-based clinical practice guideline\n • Understand the advantages and limitations of a clinical guideline\n • Describe and understand the internal validity requirements of a clinical guideline article\n • List the available resources for clinical guidelines\n • Use a clinical practice guideline to solve a clinical problem\nThe educational intervention was a one semester EBM course formally included in the medical school curriculum, with 14 two-hour weekly sessions. The course faculty were six professors trained in EBM teaching, all board-certified physicians with clinical practice, one of them with a postgraduate degree in health professions education and faculty development training in EBM education at McMaster University Faculty of Health Sciences in Canada. The course faculty had more than six years of experience teaching EBM to undergraduate medical students, residents of several specialties, and providing faculty development EBM workshops to teachers of several medical specialties. The EBM course teachers were not involved in the training of the 4th year students, but they participated in the training of the 6th year interns. The EBM program was linked with the internship program and the residency programs in the hospital, through the medical school curricular committee and the University Postgraduate Studies Division.\nThe course instructional strategies included large-group interactive sessions, small-group problem-solving activities, individual and group assignments, and informatics laboratory sessions. Traditional EBM resources were used as course bibliography, including Straus’ book [1] and an EBM text in Spanish written by the course professors [28]. The content and learning objectives of the course are outlined below.\n1. Clinical decision making in medicine\n • List and define the main difficulties for objective decision making in medicine as defined by Eddy\n • Describe the components of a decision in medicine as defined by Eddy\n • Apply the concepts of anatomy of a decision as defined by Eddy in the analysis of a clinical problem\n2. Uncertainty and probability in medicine\n • Define the concepts of uncertainty, probability and odds\n • Understand the relevance of uncertainty in clinical practice\n • Understand the limitations of personal experience in the estimation of probability, as related to diagnosis\n • Define the heuristics used in medicine (representativeness, availability, anchor and adjustment) and list the cognitive errors a clinician can make when misapplying them\n • Apply the concepts of heuristics in new clinical problems, and discuss the effects of their inappropriate use\n3. Bayes’ theorem\n • Define Bayes’ theorem\n • Define pre-test and post-test probability\n • Define the concepts of diagnostic and therapeutic threshold\n • Explain the utility of Bayes' theorem in clinical medicine, mainly in diagnosis\n • List the limitations of Bayes' theorem in clinical practice\n • Apply Fagan’s nomogram to use Bayes' theorem in a diagnostic problem\n • Apply the concepts of diagnostic and therapeutic threshold to a clinical problem\n4. Principles of Evidence Based Medicine\n • Describe the history and origin of EBM\n • Define the concept of EBM\n • List the five steps of EBM, and apply them in a clinical problem\n • Explain the importance of EBM in clinical practice\n5. Reflective medical practice\n • Define the concept of reflection and reflective practitioner\n • Define reflection-in-action and reflection-on-action\n • Apply these concepts in a clinical scenario\n6. Clinicians’ information needs\n • Understand the magnitude of physician information needs\n • Understand the literature that describe how clinicians underestimate their information needs\n • Define the percentage of occasions when clinicians recognize and act upon perceived information needs\n7. Clinical questions\n • Define the concepts of background and foreground questions\n • Understand the advantages of structuring questions generated during clinical work\n • List the four components of a foreground clinical question (PICO)\n • Apply these concepts in developing questions from clinical problems\n • List the types of clinical questions (diagnosis, therapy, prognosis, harm, etiology)\n8. Sources of biomedical information\n • List the different sources of biomedical information available\n • Identify the advantages and disadvantages of each source (textbooks, paper and electronic journals, original research papers)\n • Understand the origin, development, cost, and availability of sources of information\n9. The Cochrane Collaboration\n • Describe the history and origin of the Cochrane Collaboration (CC)\n • List the components of the Cochrane Library, and the sources where it’s available\n • Understand the mission, logistics and work of the CC\n • Perform effective searches for systematic reviews on the Cochrane Library\n • Understand the advantages and limitations of the CC\n • Use the Cochrane Library to solve a clinical problem\n10. Search strategies to find the best medical scientific evidence\n • List the main medical databases, and identify their relevance and location\n • Describe the history of Medline\n • Define MeSH terms, Boolean operators, search engine\n • Design search strategies to find valid evidence\n • Use PubMed Clinical Queries\n • Perform effective searches of scientifically valid papers using PubMed, Cochrane Library, OVID Core Medical Library\n • Understand the advantages and disadvantages of searching the different electronic medical databases and the Internet general purpose searching engines\n11. Critical appraisal of the medical literature: Users’ Guides to the Medical Literature\n • Describe the origin and history of the Users’ Guides series to appraise the medical literature\n • List and understand the different hierarchies of evidence, study designs, grades of evidence\n • Understand the relevance of using the original medical literature to solve clinical problems\n • List and understand the three different steps to appraise a research article: internal validity, magnitude of the results and external validity\n12. How to appraise an article about therapy\n • Describe the criteria for internal validity of a therapy article\n • Define randomized controlled trial, bias and random error, allocation concealment, double-blind, intention-to-treat analysis, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, p values, power and sample size, type I and II errors\n • Understand the importance of all the previously defined concepts to apply in a therapy article\n • Calculate OR, RR, RRR, ARR and NNT from a published therapy article\n • Use a therapy article to solve a clinical problem\n • Understand the concepts of external validity of a research paper, related to therapy\n13. How to appraise an article about a diagnostic test, the Rational Clinical Examination Series\n • Describe the criteria for internal validity of a diagnostic test article\n • Define pre-test and post-test probability, sensitivity, specificity, likelihood ratios, positive and negative predictive value, accuracy\n • Understand the importance of all the previously defined concepts to apply a diagnosis article\n • Calculate sensitivity, specificity, likelihood ratios from a published diagnosis article\n • Use a diagnosis article to solve a clinical problem\n • Understand the concepts of external validity of a research paper, related to diagnosis\n • Describe the origin and evolution of the Rational Clinical Examination JAMA series\n • Use a JAMA Rational Clinical Examination paper to solve a clinical problem\n14. How to appraise a Systematic Review or Meta-analysis\n • Define meta-analysis, systematic review (qualitative and quantitative)\n • Describe the advantages and limitations of systematic reviews and meta-analysis\n • Describe the criteria for internal validity of a systematic review article\n • Define bias and random error, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, forest plot, effect size\n • Understand the importance of all the previously defined concepts applied to a systematic review article\n • Calculate OR, RR, RRR, ARR and NNT from a published systematic review article\n • Use a systematic review article to solve a clinical problem\n • Understand the concepts of external validity of a systematic review\n15. Clinical practice guidelines\n • Define clinical practice guidelines\n • Describe the sequence of developing an evidence-based clinical practice guideline\n • Understand the advantages and limitations of a clinical guideline\n • Describe and understand the internal validity requirements of a clinical guideline article\n • List the available resources for clinical guidelines\n • Use a clinical practice guideline to solve a clinical problem\n Outcomes and Instrumentation The assessed outcomes were attitudes, knowledge and skills related to EBM. Two instruments were used: Taylor’s questionnaire, a published instrument designed to evaluate the effectiveness of evidence-based medicine teaching [29] and a 100 multiple-choice question test developed specifically for this study.\nTaylor’s instrument was categorized as a level 1 instrument in a systematic review of tools to evaluate EBM education, since it has reasonable psychometric properties, has been evaluated for validity from at least three sources of evidence, and is recommended for use in the summative evaluation of individual trainees [30]. The instrument includes items to assess critical appraisal skills, use of evidence behaviors, knowledge and attitudes regarding evidence-based clinical practice [29]. The attitude portion of the questionnaire includes statements related to the use of scientific evidence using a Likert scale. Each statement is scored on a five point scale, responses are added to obtain a total attitude score, and the range of scores is 7 to 35. To determine an overall score for the confidence in critical appraisal skills section, six statements were scored using a scale where “Very confident” was assigned a score of 5, “Not at all confident” a score of 1, and “Don’t know” a score of 0. The scores of the six questions were added, providing a global critical appraisal skills confidence score, where 5 indicated “little or no confidence” and 30 indicated “complete confidence”.\nThe knowledge part of the questionnaire includes six multiple true-false questions, each with three items, using ‘true’, ‘false’ or ‘don’t know’ response categories. Correct responses to the knowledge questions have a score of 1, incorrect responses are negatively scored (−1) to try to prevent guessing, and the ‘don’t know’ response has a score of 0. The knowledge scores were added in an overall knowledge score, with a possible range of −18 to +18. In a previous paper, we translated the questionnaire to Spanish with the author’s permission, and verified it with backtranslation [21].\nThe other instrument used was the final summative test of the Evidence-Based Medicine Course. This instrument was developed, administered, scored, and analyzed following the 12 steps for effective test development described by Downing [31]. Item analysis was performed on a pilot application of the test with ITEMAN for Windows (Assessment Systems Corporation, St. Paul, MN), and the information obtained was used to improve the instrument for this study, choosing the better-performing items and preserving content validity. The pilot application of the original 140-items EBM test was done in 57 examinees, and had a Cronbach’s alpha of 0.82. Using the item analysis information 100 multiple-choice questions (MCQ) were selected by the test developers for the final version of the instrument.\nThe instruments were applied to the students on three consecutive weeks. The students had up to three hours to answer the test and the questionnaire, to minimize the risk of a speeded examination. Taylor’s questionnaires data sheets were captured in a Microsoft Excel spreadsheet. Op-scan answer sheets for item analysis were used for the EBM MCQ test.\nThe assessed outcomes were attitudes, knowledge and skills related to EBM. Two instruments were used: Taylor’s questionnaire, a published instrument designed to evaluate the effectiveness of evidence-based medicine teaching [29] and a 100 multiple-choice question test developed specifically for this study.\nTaylor’s instrument was categorized as a level 1 instrument in a systematic review of tools to evaluate EBM education, since it has reasonable psychometric properties, has been evaluated for validity from at least three sources of evidence, and is recommended for use in the summative evaluation of individual trainees [30]. The instrument includes items to assess critical appraisal skills, use of evidence behaviors, knowledge and attitudes regarding evidence-based clinical practice [29]. The attitude portion of the questionnaire includes statements related to the use of scientific evidence using a Likert scale. Each statement is scored on a five point scale, responses are added to obtain a total attitude score, and the range of scores is 7 to 35. To determine an overall score for the confidence in critical appraisal skills section, six statements were scored using a scale where “Very confident” was assigned a score of 5, “Not at all confident” a score of 1, and “Don’t know” a score of 0. The scores of the six questions were added, providing a global critical appraisal skills confidence score, where 5 indicated “little or no confidence” and 30 indicated “complete confidence”.\nThe knowledge part of the questionnaire includes six multiple true-false questions, each with three items, using ‘true’, ‘false’ or ‘don’t know’ response categories. Correct responses to the knowledge questions have a score of 1, incorrect responses are negatively scored (−1) to try to prevent guessing, and the ‘don’t know’ response has a score of 0. The knowledge scores were added in an overall knowledge score, with a possible range of −18 to +18. In a previous paper, we translated the questionnaire to Spanish with the author’s permission, and verified it with backtranslation [21].\nThe other instrument used was the final summative test of the Evidence-Based Medicine Course. This instrument was developed, administered, scored, and analyzed following the 12 steps for effective test development described by Downing [31]. Item analysis was performed on a pilot application of the test with ITEMAN for Windows (Assessment Systems Corporation, St. Paul, MN), and the information obtained was used to improve the instrument for this study, choosing the better-performing items and preserving content validity. The pilot application of the original 140-items EBM test was done in 57 examinees, and had a Cronbach’s alpha of 0.82. Using the item analysis information 100 multiple-choice questions (MCQ) were selected by the test developers for the final version of the instrument.\nThe instruments were applied to the students on three consecutive weeks. The students had up to three hours to answer the test and the questionnaire, to minimize the risk of a speeded examination. Taylor’s questionnaires data sheets were captured in a Microsoft Excel spreadsheet. Op-scan answer sheets for item analysis were used for the EBM MCQ test.\n Statistical analysis The piloting of the EBM MCQ test provided preliminary data for differences and standard deviation, and sample size calculation was performed for the primary hypothesis of knowledge increase with a power of 0.90 (beta error of 0.10), two-sided alpha error of 0.05. After a thorough review of the published studies that included magnitude of EBM knowledge differences in undergraduate medical students, and careful consideration by the course faculty of the smallest meaningful difference (SMD) in this parameter, it was estimated that a difference of 10 questions between the intervention group and the control group would be reasonable. Using this estimate, about 31 students per group would be necessary to detect an effect size of 0.5 or larger.\nSPSS for Windows 15.0 and Instat 3.0 for the Macintosh were used for data analysis. The comparison of the use of evidence items in Taylor’s questionnaire between M5 and M5 non-EBM students was done with the non-parametric Mann–Whitney U test. The attitude and critical appraisal confidence scores measured with Taylor’s instrument were compared among groups using the Kruskal-Wallis with Dunn’s multiple comparison test. The groups’ knowledge test scores with both instruments were compared with one-way analysis of variance, with planned comparisons. A p-value of less than 0.05 was considered statistically significant. Cohen’s d with pooled standard deviations was calculated as a measure of effect size for the critical appraisal skills, attitude and knowledge scores among groups [32]. Item analysis of the EBM Test data was performed with ITEMAN for Windows 3.2, (Assessment Systems Corporation, St. Paul, MN http://www.assess.com).\nThe piloting of the EBM MCQ test provided preliminary data for differences and standard deviation, and sample size calculation was performed for the primary hypothesis of knowledge increase with a power of 0.90 (beta error of 0.10), two-sided alpha error of 0.05. After a thorough review of the published studies that included magnitude of EBM knowledge differences in undergraduate medical students, and careful consideration by the course faculty of the smallest meaningful difference (SMD) in this parameter, it was estimated that a difference of 10 questions between the intervention group and the control group would be reasonable. Using this estimate, about 31 students per group would be necessary to detect an effect size of 0.5 or larger.\nSPSS for Windows 15.0 and Instat 3.0 for the Macintosh were used for data analysis. The comparison of the use of evidence items in Taylor’s questionnaire between M5 and M5 non-EBM students was done with the non-parametric Mann–Whitney U test. The attitude and critical appraisal confidence scores measured with Taylor’s instrument were compared among groups using the Kruskal-Wallis with Dunn’s multiple comparison test. The groups’ knowledge test scores with both instruments were compared with one-way analysis of variance, with planned comparisons. A p-value of less than 0.05 was considered statistically significant. Cohen’s d with pooled standard deviations was calculated as a measure of effect size for the critical appraisal skills, attitude and knowledge scores among groups [32]. Item analysis of the EBM Test data was performed with ITEMAN for Windows 3.2, (Assessment Systems Corporation, St. Paul, MN http://www.assess.com).\n Ethical aspects The instruments did not have individual student identifiers, to eliminate the risk of potential harm to the participants. This study was reviewed by the Institutional Review Board of the Office for the Protection of Research Subjects of the University of Illinois at Chicago, and the Research Committee of the Mexican Army Medical School, and was considered to be in the exempt category for individual written informed consent.\nThe instruments did not have individual student identifiers, to eliminate the risk of potential harm to the participants. This study was reviewed by the Institutional Review Board of the Office for the Protection of Research Subjects of the University of Illinois at Chicago, and the Research Committee of the Mexican Army Medical School, and was considered to be in the exempt category for individual written informed consent.",
"The Mexican Army medical school trains career physicians for the national military healthcare system, and is located in Mexico City. It has a six year program, with a traditional curriculum: two years of basic sciences, three years of clinical sciences, and the sixth year is an internship period in the hospital. The school is a public institution funded by the federal government. Each yearly class is composed of about one hundred students, mostly middle- or low-socioeconomic class Hispanics.",
" Main outcomes and subjects The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].\nFlow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.\nThe core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].\nFlow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.\n Simultaneous validation Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).\nThe outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.\nQuasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).\nThe outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.",
"The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].\nFlow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.",
"Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).\nThe outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.",
"The educational intervention was a one semester EBM course formally included in the medical school curriculum, with 14 two-hour weekly sessions. The course faculty were six professors trained in EBM teaching, all board-certified physicians with clinical practice, one of them with a postgraduate degree in health professions education and faculty development training in EBM education at McMaster University Faculty of Health Sciences in Canada. The course faculty had more than six years of experience teaching EBM to undergraduate medical students, residents of several specialties, and providing faculty development EBM workshops to teachers of several medical specialties. The EBM course teachers were not involved in the training of the 4th year students, but they participated in the training of the 6th year interns. The EBM program was linked with the internship program and the residency programs in the hospital, through the medical school curricular committee and the University Postgraduate Studies Division.\nThe course instructional strategies included large-group interactive sessions, small-group problem-solving activities, individual and group assignments, and informatics laboratory sessions. Traditional EBM resources were used as course bibliography, including Straus’ book [1] and an EBM text in Spanish written by the course professors [28]. The content and learning objectives of the course are outlined below.\n1. Clinical decision making in medicine\n • List and define the main difficulties for objective decision making in medicine as defined by Eddy\n • Describe the components of a decision in medicine as defined by Eddy\n • Apply the concepts of anatomy of a decision as defined by Eddy in the analysis of a clinical problem\n2. Uncertainty and probability in medicine\n • Define the concepts of uncertainty, probability and odds\n • Understand the relevance of uncertainty in clinical practice\n • Understand the limitations of personal experience in the estimation of probability, as related to diagnosis\n • Define the heuristics used in medicine (representativeness, availability, anchor and adjustment) and list the cognitive errors a clinician can make when misapplying them\n • Apply the concepts of heuristics in new clinical problems, and discuss the effects of their inappropriate use\n3. Bayes’ theorem\n • Define Bayes’ theorem\n • Define pre-test and post-test probability\n • Define the concepts of diagnostic and therapeutic threshold\n • Explain the utility of Bayes' theorem in clinical medicine, mainly in diagnosis\n • List the limitations of Bayes' theorem in clinical practice\n • Apply Fagan’s nomogram to use Bayes' theorem in a diagnostic problem\n • Apply the concepts of diagnostic and therapeutic threshold to a clinical problem\n4. Principles of Evidence Based Medicine\n • Describe the history and origin of EBM\n • Define the concept of EBM\n • List the five steps of EBM, and apply them in a clinical problem\n • Explain the importance of EBM in clinical practice\n5. Reflective medical practice\n • Define the concept of reflection and reflective practitioner\n • Define reflection-in-action and reflection-on-action\n • Apply these concepts in a clinical scenario\n6. Clinicians’ information needs\n • Understand the magnitude of physician information needs\n • Understand the literature that describe how clinicians underestimate their information needs\n • Define the percentage of occasions when clinicians recognize and act upon perceived information needs\n7. Clinical questions\n • Define the concepts of background and foreground questions\n • Understand the advantages of structuring questions generated during clinical work\n • List the four components of a foreground clinical question (PICO)\n • Apply these concepts in developing questions from clinical problems\n • List the types of clinical questions (diagnosis, therapy, prognosis, harm, etiology)\n8. Sources of biomedical information\n • List the different sources of biomedical information available\n • Identify the advantages and disadvantages of each source (textbooks, paper and electronic journals, original research papers)\n • Understand the origin, development, cost, and availability of sources of information\n9. The Cochrane Collaboration\n • Describe the history and origin of the Cochrane Collaboration (CC)\n • List the components of the Cochrane Library, and the sources where it’s available\n • Understand the mission, logistics and work of the CC\n • Perform effective searches for systematic reviews on the Cochrane Library\n • Understand the advantages and limitations of the CC\n • Use the Cochrane Library to solve a clinical problem\n10. Search strategies to find the best medical scientific evidence\n • List the main medical databases, and identify their relevance and location\n • Describe the history of Medline\n • Define MeSH terms, Boolean operators, search engine\n • Design search strategies to find valid evidence\n • Use PubMed Clinical Queries\n • Perform effective searches of scientifically valid papers using PubMed, Cochrane Library, OVID Core Medical Library\n • Understand the advantages and disadvantages of searching the different electronic medical databases and the Internet general purpose searching engines\n11. Critical appraisal of the medical literature: Users’ Guides to the Medical Literature\n • Describe the origin and history of the Users’ Guides series to appraise the medical literature\n • List and understand the different hierarchies of evidence, study designs, grades of evidence\n • Understand the relevance of using the original medical literature to solve clinical problems\n • List and understand the three different steps to appraise a research article: internal validity, magnitude of the results and external validity\n12. How to appraise an article about therapy\n • Describe the criteria for internal validity of a therapy article\n • Define randomized controlled trial, bias and random error, allocation concealment, double-blind, intention-to-treat analysis, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, p values, power and sample size, type I and II errors\n • Understand the importance of all the previously defined concepts to apply in a therapy article\n • Calculate OR, RR, RRR, ARR and NNT from a published therapy article\n • Use a therapy article to solve a clinical problem\n • Understand the concepts of external validity of a research paper, related to therapy\n13. How to appraise an article about a diagnostic test, the Rational Clinical Examination Series\n • Describe the criteria for internal validity of a diagnostic test article\n • Define pre-test and post-test probability, sensitivity, specificity, likelihood ratios, positive and negative predictive value, accuracy\n • Understand the importance of all the previously defined concepts to apply a diagnosis article\n • Calculate sensitivity, specificity, likelihood ratios from a published diagnosis article\n • Use a diagnosis article to solve a clinical problem\n • Understand the concepts of external validity of a research paper, related to diagnosis\n • Describe the origin and evolution of the Rational Clinical Examination JAMA series\n • Use a JAMA Rational Clinical Examination paper to solve a clinical problem\n14. How to appraise a Systematic Review or Meta-analysis\n • Define meta-analysis, systematic review (qualitative and quantitative)\n • Describe the advantages and limitations of systematic reviews and meta-analysis\n • Describe the criteria for internal validity of a systematic review article\n • Define bias and random error, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, forest plot, effect size\n • Understand the importance of all the previously defined concepts applied to a systematic review article\n • Calculate OR, RR, RRR, ARR and NNT from a published systematic review article\n • Use a systematic review article to solve a clinical problem\n • Understand the concepts of external validity of a systematic review\n15. Clinical practice guidelines\n • Define clinical practice guidelines\n • Describe the sequence of developing an evidence-based clinical practice guideline\n • Understand the advantages and limitations of a clinical guideline\n • Describe and understand the internal validity requirements of a clinical guideline article\n • List the available resources for clinical guidelines\n • Use a clinical practice guideline to solve a clinical problem",
"The assessed outcomes were attitudes, knowledge and skills related to EBM. Two instruments were used: Taylor’s questionnaire, a published instrument designed to evaluate the effectiveness of evidence-based medicine teaching [29] and a 100 multiple-choice question test developed specifically for this study.\nTaylor’s instrument was categorized as a level 1 instrument in a systematic review of tools to evaluate EBM education, since it has reasonable psychometric properties, has been evaluated for validity from at least three sources of evidence, and is recommended for use in the summative evaluation of individual trainees [30]. The instrument includes items to assess critical appraisal skills, use of evidence behaviors, knowledge and attitudes regarding evidence-based clinical practice [29]. The attitude portion of the questionnaire includes statements related to the use of scientific evidence using a Likert scale. Each statement is scored on a five point scale, responses are added to obtain a total attitude score, and the range of scores is 7 to 35. To determine an overall score for the confidence in critical appraisal skills section, six statements were scored using a scale where “Very confident” was assigned a score of 5, “Not at all confident” a score of 1, and “Don’t know” a score of 0. The scores of the six questions were added, providing a global critical appraisal skills confidence score, where 5 indicated “little or no confidence” and 30 indicated “complete confidence”.\nThe knowledge part of the questionnaire includes six multiple true-false questions, each with three items, using ‘true’, ‘false’ or ‘don’t know’ response categories. Correct responses to the knowledge questions have a score of 1, incorrect responses are negatively scored (−1) to try to prevent guessing, and the ‘don’t know’ response has a score of 0. The knowledge scores were added in an overall knowledge score, with a possible range of −18 to +18. In a previous paper, we translated the questionnaire to Spanish with the author’s permission, and verified it with backtranslation [21].\nThe other instrument used was the final summative test of the Evidence-Based Medicine Course. This instrument was developed, administered, scored, and analyzed following the 12 steps for effective test development described by Downing [31]. Item analysis was performed on a pilot application of the test with ITEMAN for Windows (Assessment Systems Corporation, St. Paul, MN), and the information obtained was used to improve the instrument for this study, choosing the better-performing items and preserving content validity. The pilot application of the original 140-items EBM test was done in 57 examinees, and had a Cronbach’s alpha of 0.82. Using the item analysis information 100 multiple-choice questions (MCQ) were selected by the test developers for the final version of the instrument.\nThe instruments were applied to the students on three consecutive weeks. The students had up to three hours to answer the test and the questionnaire, to minimize the risk of a speeded examination. Taylor’s questionnaires data sheets were captured in a Microsoft Excel spreadsheet. Op-scan answer sheets for item analysis were used for the EBM MCQ test.",
"The piloting of the EBM MCQ test provided preliminary data for differences and standard deviation, and sample size calculation was performed for the primary hypothesis of knowledge increase with a power of 0.90 (beta error of 0.10), two-sided alpha error of 0.05. After a thorough review of the published studies that included magnitude of EBM knowledge differences in undergraduate medical students, and careful consideration by the course faculty of the smallest meaningful difference (SMD) in this parameter, it was estimated that a difference of 10 questions between the intervention group and the control group would be reasonable. Using this estimate, about 31 students per group would be necessary to detect an effect size of 0.5 or larger.\nSPSS for Windows 15.0 and Instat 3.0 for the Macintosh were used for data analysis. The comparison of the use of evidence items in Taylor’s questionnaire between M5 and M5 non-EBM students was done with the non-parametric Mann–Whitney U test. The attitude and critical appraisal confidence scores measured with Taylor’s instrument were compared among groups using the Kruskal-Wallis with Dunn’s multiple comparison test. The groups’ knowledge test scores with both instruments were compared with one-way analysis of variance, with planned comparisons. A p-value of less than 0.05 was considered statistically significant. Cohen’s d with pooled standard deviations was calculated as a measure of effect size for the critical appraisal skills, attitude and knowledge scores among groups [32]. Item analysis of the EBM Test data was performed with ITEMAN for Windows 3.2, (Assessment Systems Corporation, St. Paul, MN http://www.assess.com).",
"The instruments did not have individual student identifiers, to eliminate the risk of potential harm to the participants. This study was reviewed by the Institutional Review Board of the Office for the Protection of Research Subjects of the University of Illinois at Chicago, and the Research Committee of the Mexican Army Medical School, and was considered to be in the exempt category for individual written informed consent.",
" Subjects The flow diagram of the study participants throughout the trial is outlined in Figure 1. A total of 289 medical students were assessed. One student from the M5 non-EBM group was sick on the assessment day. Five subjects in the M4 non-EBM and 7 subjects in the M6 EBM groups didn't participate because they were on clinical duties on the testing day.\nThe students’ age (mean±SD) per group was: M4= 21.5±1.8, M5 EBM=22.8±2.0, M5 non-EBM=22.4±2.2 and M6=23.5±1.9 years. The groups’ gender composition was similar, with a predominance of women over men (about 60/40).\nThe flow diagram of the study participants throughout the trial is outlined in Figure 1. A total of 289 medical students were assessed. One student from the M5 non-EBM group was sick on the assessment day. Five subjects in the M4 non-EBM and 7 subjects in the M6 EBM groups didn't participate because they were on clinical duties on the testing day.\nThe students’ age (mean±SD) per group was: M4= 21.5±1.8, M5 EBM=22.8±2.0, M5 non-EBM=22.4±2.2 and M6=23.5±1.9 years. The groups’ gender composition was similar, with a predominance of women over men (about 60/40).\n Use of the evidence The use of scientific evidence explored in the first section of Taylor’s questionnaire, includes two main questions: “What type of resources do you use to keep up to date?” and “What type of resources do you use to solve a specific health care problem?” The answers by group and type of resource are presented in Figures 2 and 3.\nUse of evidence to keep up to date. Distribution of answers to the question: \"what type of resources do you use to keep up to date?\" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). *** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.\nUse of evidence to solve a health problem. Distribution of answers to the question: \"what type of resources do you use to solve a specific health problem?\" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). * =P<0.01 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.*** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.\nIn the use of information resources to keep up to date and to solve a specific health care problem, the pattern of responses was the same. The answers were similar among the four student groups regarding the use of review articles, original research journals, textbooks, Internet resources and teachers, but there were statistically significant differences in the use of secondary journals (e.g. American Journal of Physicians Journal Club) and the Cochrane Library. The experimental group (M5 EBM) had a higher reported use of original research articles to solve a specific health problem than the randomized comparison group (M5 non-EBM) (P<0.01). The M5 EBM and M6 groups reported a higher use of secondary journals than the M4 and the M5 non-EBM groups, and a similar pattern of response was found in the use of the Cochrane Library (P<0.001) (Figures 2 and 3).\nThe use of scientific evidence explored in the first section of Taylor’s questionnaire, includes two main questions: “What type of resources do you use to keep up to date?” and “What type of resources do you use to solve a specific health care problem?” The answers by group and type of resource are presented in Figures 2 and 3.\nUse of evidence to keep up to date. Distribution of answers to the question: \"what type of resources do you use to keep up to date?\" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). *** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.\nUse of evidence to solve a health problem. Distribution of answers to the question: \"what type of resources do you use to solve a specific health problem?\" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). * =P<0.01 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.*** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.\nIn the use of information resources to keep up to date and to solve a specific health care problem, the pattern of responses was the same. The answers were similar among the four student groups regarding the use of review articles, original research journals, textbooks, Internet resources and teachers, but there were statistically significant differences in the use of secondary journals (e.g. American Journal of Physicians Journal Club) and the Cochrane Library. The experimental group (M5 EBM) had a higher reported use of original research articles to solve a specific health problem than the randomized comparison group (M5 non-EBM) (P<0.01). The M5 EBM and M6 groups reported a higher use of secondary journals than the M4 and the M5 non-EBM groups, and a similar pattern of response was found in the use of the Cochrane Library (P<0.001) (Figures 2 and 3).\n Confidence in critical appraisal skills There was a higher confidence level of critical appraisal skills in all items in this section of Taylor’s instrument (assessing study design, evaluating bias, evaluating statistical tests), in the intervention group (P<0.001). The critical appraisal confidence global scores for the different study groups were as follows: M4=11.7±6.3 (mean±SD), M5 non-EBM=8.4±5.7, M5 EBM=17.1±3.6 and M6= 16.8±4.9. The summary data for each group is shown in Figure 4, where the experimental group (M5 EBM) had higher scores than the randomized control group (M5 non-EBM) and the M4 comparison group (P<0.001). The M4 score was slightly higher than the M5 non-EBM group (P<0.05), and the M6 group had higher scores than M4 and M5 non-EBM (P<0.001).\nCritical appraisal skills scores. Critical appraisal confidence scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).\nThere was a higher confidence level of critical appraisal skills in all items in this section of Taylor’s instrument (assessing study design, evaluating bias, evaluating statistical tests), in the intervention group (P<0.001). The critical appraisal confidence global scores for the different study groups were as follows: M4=11.7±6.3 (mean±SD), M5 non-EBM=8.4±5.7, M5 EBM=17.1±3.6 and M6= 16.8±4.9. The summary data for each group is shown in Figure 4, where the experimental group (M5 EBM) had higher scores than the randomized control group (M5 non-EBM) and the M4 comparison group (P<0.001). The M4 score was slightly higher than the M5 non-EBM group (P<0.05), and the M6 group had higher scores than M4 and M5 non-EBM (P<0.001).\nCritical appraisal skills scores. Critical appraisal confidence scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).\n Attitudes The EBM attitude scores measured with Taylor’s questionnaire are shown in Figure 5. The scores were similar between the groups that didn’t receive the EBM educational intervention, the M4 group had a score of 24.5±5.2 (mean±SD), and the M5 non-EBM group had 24.0±5.0 (P>0.05). The M5 EBM group had an attitude score of 28.7±2.2, higher than the M4 and M5 non-EBM groups (P<0.001). The M6 students had an attitude score of 26.7±3.6, higher than the control groups and lower than the M5 EBM group (P<0.05). Cohen’s d effect size for the comparison of M5 EBM vs. M5 non-EBM was 1.21 (Table 1).\nAttitude scores. Attitude scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).\n\nEffect size (Cohen’s “\n\nd\n\n”) in critical appraisal confidence, attitude and knowledge scores when comparing the different medical student groups\n\nThe EBM attitude scores measured with Taylor’s questionnaire are shown in Figure 5. The scores were similar between the groups that didn’t receive the EBM educational intervention, the M4 group had a score of 24.5±5.2 (mean±SD), and the M5 non-EBM group had 24.0±5.0 (P>0.05). The M5 EBM group had an attitude score of 28.7±2.2, higher than the M4 and M5 non-EBM groups (P<0.001). The M6 students had an attitude score of 26.7±3.6, higher than the control groups and lower than the M5 EBM group (P<0.05). Cohen’s d effect size for the comparison of M5 EBM vs. M5 non-EBM was 1.21 (Table 1).\nAttitude scores. Attitude scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).\n\nEffect size (Cohen’s “\n\nd\n\n”) in critical appraisal confidence, attitude and knowledge scores when comparing the different medical student groups\n\n Knowledge scores with Taylor’s instrument The results of the knowledge score measured with Taylor’s questionnaire are shown in Figure 6. The scores were similar between non-EBM groups, M4=1.06±3.16 (mean±SD), and M5 non-EBM=1.13±3.27 (P=0.91). The M5 EBM intervention group had a knowledge score of 4.21±3.73, higher than those of M4 and M5 non-EBM. The planned contrast in the main comparison between M5 EBM and M5 non-EBM showed that the intervention group had a higher knowledge score than the randomized control group (P<0.001). The M6 group had a knowledge score of 2.44±3.77, higher than both control groups, (P<0.01), but lower than M5 EBM (P<0.01). The effect size measured with Cohen’s d for the knowledge score main comparison of M5 EBM vs. M5 non-EBM was 0.88 (Table 1).\nKnowledge scores with Taylor’s instrument. Knowledge scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).\nThe results of the knowledge score measured with Taylor’s questionnaire are shown in Figure 6. The scores were similar between non-EBM groups, M4=1.06±3.16 (mean±SD), and M5 non-EBM=1.13±3.27 (P=0.91). The M5 EBM intervention group had a knowledge score of 4.21±3.73, higher than those of M4 and M5 non-EBM. The planned contrast in the main comparison between M5 EBM and M5 non-EBM showed that the intervention group had a higher knowledge score than the randomized control group (P<0.001). The M6 group had a knowledge score of 2.44±3.77, higher than both control groups, (P<0.01), but lower than M5 EBM (P<0.01). The effect size measured with Cohen’s d for the knowledge score main comparison of M5 EBM vs. M5 non-EBM was 0.88 (Table 1).\nKnowledge scores with Taylor’s instrument. Knowledge scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).\n Knowledge scores with EBM summative MCQ test The results of the 100-item MCQ EBM knowledge test are presented as percent-correct scores (Figure 7). The reliability of the test with Cronbach’s alpha was 0.72 in the M5 EBM group, and 0.83 in the M6 group. The scores were similar between non-EBM groups, M4=30.6±5.6 (mean±SD), and M5 non-EBM=32.6±6.6 (P=0.18). The M5 EBM group had a test score of 58.5±7.9, higher than M4 and M5 non-EBM. The planned contrast between M5 EBM and M5 non-EBM found that the educational intervention group had a higher knowledge score (P<0.001). M6 had a knowledge score of 41.0±10.9, higher than the control groups (P<0.001), but lower than M5 EBM (P<0.001). The effect size with Cohen’s d for the knowledge score main outcome comparison of M5 EBM vs. M5 non-EBM was 3.54 (Table 1).\nKnowledge scores EBM test. Knowledge scores in the different groups of medical students, measured with the 100 multiple-choice questions EBM test. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).\nThe results of the 100-item MCQ EBM knowledge test are presented as percent-correct scores (Figure 7). The reliability of the test with Cronbach’s alpha was 0.72 in the M5 EBM group, and 0.83 in the M6 group. The scores were similar between non-EBM groups, M4=30.6±5.6 (mean±SD), and M5 non-EBM=32.6±6.6 (P=0.18). The M5 EBM group had a test score of 58.5±7.9, higher than M4 and M5 non-EBM. The planned contrast between M5 EBM and M5 non-EBM found that the educational intervention group had a higher knowledge score (P<0.001). M6 had a knowledge score of 41.0±10.9, higher than the control groups (P<0.001), but lower than M5 EBM (P<0.001). The effect size with Cohen’s d for the knowledge score main outcome comparison of M5 EBM vs. M5 non-EBM was 3.54 (Table 1).\nKnowledge scores EBM test. Knowledge scores in the different groups of medical students, measured with the 100 multiple-choice questions EBM test. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).",
"The flow diagram of the study participants throughout the trial is outlined in Figure 1. A total of 289 medical students were assessed. One student from the M5 non-EBM group was sick on the assessment day. Five subjects in the M4 non-EBM and 7 subjects in the M6 EBM groups didn't participate because they were on clinical duties on the testing day.\nThe students’ age (mean±SD) per group was: M4= 21.5±1.8, M5 EBM=22.8±2.0, M5 non-EBM=22.4±2.2 and M6=23.5±1.9 years. The groups’ gender composition was similar, with a predominance of women over men (about 60/40).",
"The use of scientific evidence explored in the first section of Taylor’s questionnaire, includes two main questions: “What type of resources do you use to keep up to date?” and “What type of resources do you use to solve a specific health care problem?” The answers by group and type of resource are presented in Figures 2 and 3.\nUse of evidence to keep up to date. Distribution of answers to the question: \"what type of resources do you use to keep up to date?\" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). *** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.\nUse of evidence to solve a health problem. Distribution of answers to the question: \"what type of resources do you use to solve a specific health problem?\" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). * =P<0.01 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.*** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.\nIn the use of information resources to keep up to date and to solve a specific health care problem, the pattern of responses was the same. The answers were similar among the four student groups regarding the use of review articles, original research journals, textbooks, Internet resources and teachers, but there were statistically significant differences in the use of secondary journals (e.g. American Journal of Physicians Journal Club) and the Cochrane Library. The experimental group (M5 EBM) had a higher reported use of original research articles to solve a specific health problem than the randomized comparison group (M5 non-EBM) (P<0.01). The M5 EBM and M6 groups reported a higher use of secondary journals than the M4 and the M5 non-EBM groups, and a similar pattern of response was found in the use of the Cochrane Library (P<0.001) (Figures 2 and 3).",
"There was a higher confidence level of critical appraisal skills in all items in this section of Taylor’s instrument (assessing study design, evaluating bias, evaluating statistical tests), in the intervention group (P<0.001). The critical appraisal confidence global scores for the different study groups were as follows: M4=11.7±6.3 (mean±SD), M5 non-EBM=8.4±5.7, M5 EBM=17.1±3.6 and M6= 16.8±4.9. The summary data for each group is shown in Figure 4, where the experimental group (M5 EBM) had higher scores than the randomized control group (M5 non-EBM) and the M4 comparison group (P<0.001). The M4 score was slightly higher than the M5 non-EBM group (P<0.05), and the M6 group had higher scores than M4 and M5 non-EBM (P<0.001).\nCritical appraisal skills scores. Critical appraisal confidence scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).",
"The EBM attitude scores measured with Taylor’s questionnaire are shown in Figure 5. The scores were similar between the groups that didn’t receive the EBM educational intervention, the M4 group had a score of 24.5±5.2 (mean±SD), and the M5 non-EBM group had 24.0±5.0 (P>0.05). The M5 EBM group had an attitude score of 28.7±2.2, higher than the M4 and M5 non-EBM groups (P<0.001). The M6 students had an attitude score of 26.7±3.6, higher than the control groups and lower than the M5 EBM group (P<0.05). Cohen’s d effect size for the comparison of M5 EBM vs. M5 non-EBM was 1.21 (Table 1).\nAttitude scores. Attitude scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).\n\nEffect size (Cohen’s “\n\nd\n\n”) in critical appraisal confidence, attitude and knowledge scores when comparing the different medical student groups\n",
"The results of the knowledge score measured with Taylor’s questionnaire are shown in Figure 6. The scores were similar between non-EBM groups, M4=1.06±3.16 (mean±SD), and M5 non-EBM=1.13±3.27 (P=0.91). The M5 EBM intervention group had a knowledge score of 4.21±3.73, higher than those of M4 and M5 non-EBM. The planned contrast in the main comparison between M5 EBM and M5 non-EBM showed that the intervention group had a higher knowledge score than the randomized control group (P<0.001). The M6 group had a knowledge score of 2.44±3.77, higher than both control groups, (P<0.01), but lower than M5 EBM (P<0.01). The effect size measured with Cohen’s d for the knowledge score main comparison of M5 EBM vs. M5 non-EBM was 0.88 (Table 1).\nKnowledge scores with Taylor’s instrument. Knowledge scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).",
"The results of the 100-item MCQ EBM knowledge test are presented as percent-correct scores (Figure 7). The reliability of the test with Cronbach’s alpha was 0.72 in the M5 EBM group, and 0.83 in the M6 group. The scores were similar between non-EBM groups, M4=30.6±5.6 (mean±SD), and M5 non-EBM=32.6±6.6 (P=0.18). The M5 EBM group had a test score of 58.5±7.9, higher than M4 and M5 non-EBM. The planned contrast between M5 EBM and M5 non-EBM found that the educational intervention group had a higher knowledge score (P<0.001). M6 had a knowledge score of 41.0±10.9, higher than the control groups (P<0.001), but lower than M5 EBM (P<0.001). The effect size with Cohen’s d for the knowledge score main outcome comparison of M5 EBM vs. M5 non-EBM was 3.54 (Table 1).\nKnowledge scores EBM test. Knowledge scores in the different groups of medical students, measured with the 100 multiple-choice questions EBM test. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).",
"This research study presents experimental evidence that an EBM educational intervention in medical students increases attitudes, knowledge and self-reported critical appraisal skills, in the setting of a developing country medical school.\nThe research design was a parallel-group randomized control trial, with a quasi-experimental static-groups comparison, to take advantage of a strong study design and its implications in terms of internal validity and the causal inferences that can be made of the results [24,25,33]. Recent studies and systematic reviews suggest that well-planned and educationally sound EBM interventions can have a reasonable impact on the abilities of the individuals that undergo these educational experiences [9,14,34].\nThere are not many published randomized controlled trials that study the impact of EBM education and very few from developing countries [9-12,14]. Some of the randomized trials did not find an effect of EBM educational interventions, which point to the need of continuing research in this area [35-37].\nIn the present study the educational intervention was one semester long, it was mandatory, and had a summative test, all these factors probably contribute to the magnitude of the findings in the randomized comparison. Almost all published studies have used only one assessment instrument, while our study used two evaluation tools, a published questionnaire with validity evidence designed to measure the effectiveness of evidence-based practice teaching, and an ad hoc objective test developed for the course summative assessment [29,30]. This characteristic of our study design provided an opportunity to concurrently validate an already published instrument and a new objective test developed specifically for our course, contributing to the body of literature supporting the validity of Taylor’s instrument.\nWe found an increase in critical appraisal skills, and in the positive attitude to evidence-based practice. These findings are similar to Ghali et al. [16], with a higher reported use of secondary journals and Cochrane Library systematic reviews. It is important to recognize that these are self-reports, the actual behaviour of the students in the use of these resources in their daily routines wasn’t directly measured.\nIn our study the answers to two questions related to the use of evidence (to keep up to date and to solve clinical problems) had a similar pattern of responses to our previous paper, as measured with Taylor’s questionnaire [21]. There was a higher reported use of the Cochrane Library and secondary journals in both items in the M5 intervention group, and a higher use of original research papers to solve a healthcare problem. It is apparent that all the students use frequently textbooks, Internet resources, teachers and residents as sources of information in health care, as previously reported [21]. These resources are readily available, and culturally accepted in the daily practice of medicine.\nThe use of the Cochrane Library and secondary journals was higher in our intervention group, which suggests that these resources were virtually unknown to the students before the course and that its reported use probably increased as a result of the educational intervention. Even though these EBM information resources have been extensively used in developed countries in the last decades, developing countries have been slower in adopting them as formal information elements, probably because of a lack of availability and misunderstanding of their potential use [38,39]. The Cochrane Library has been translated to Spanish by the Iberoamerican Cochrane Network, as the Cochrane Library Plus (http://cochrane.bvsalud.org), which should improve the availability and use of this resource in Spanish-speaking countries.\nThis study found that the EBM intervention improved the confidence of medical students regarding several aspects of critical appraisal skills, as well as statistical concepts relevant to the correct interpretation of published research findings. An interesting aspect of these results is that the medical students who weren’t exposed to the EBM course (M4 and M5 non-EBM), already had courses on Biostatistics and Scientific Methodology and nonetheless had lower scores in this outcome. Probably those courses didn’t have a substantial impact or it was short-lived. Other explanations could be that the previous courses on related subjects were given by non-clinicians and/or basic research scientists with no clinical orientation, having a minor effect on the EBM outcomes. The increase in critical appraisal skills is in agreement with several published reports of EBM in undergraduate students [15,16]. Other studies haven’t found a significant improvement in critical appraisal skills, probably due to several factors inherent to the complexity of educational research interventions in healthcare settings [35-37]. In our study the effect size immediately after the course in critical appraisal skills score was higher than 1.0, which can be interpreted as large using Cohen's classification [32]. A similar effect size was found when comparing the students that had the EBM course six months to one year before with the control group (Table 1).\nIt is important to recognize that self-perceived skills can overestimate true competence and performance, so these findings may not reflect the real critical appraisal and statistics skills of the medical students, although confidence in a skill is an important component of the performance spectrum [40,41].\nThe overall attitude score findings in our study are congruent with several published papers, showing an increase immediately after the course of about 17-20% [16,21,23,42]. The 6th year students attitude score was higher than the control group and the 4th year students, which suggests that the attitude change can still occur from six months to a year after the course. Our previous study found very similar attitude score values measured with the same instrument, which adds reproducibility evidence to the use of Taylor’s instrument for measurement of EBM attitude in our population of students [21]. It is noteworthy that some studies, including randomized controlled trials of EBM teaching, didn’t find a change in attitudes, probably due to the shorter duration of the workshops and related activities [36,37].\nA major challenge of assessing EBM teaching is to demonstrate an increase in the “knowledge” of evidence-based clinical practice, since several disciplines intersect in the optimal use of scientific evidence (research methodology, biomedical informatics, biostatistics, clinical epidemiology) which integrate a large body of knowledge and facts. In this investigation, large effect sizes in the main randomized comparison (M5 vs. M5-nonEBM) were found in the EBM knowledge scores measured with Taylor’s questionnaire and the EBM MCQ test. The knowledge increase after the course was about 73% higher than the control group when measured with Taylor’s instrument, and 25.9% when measured with the EBM test. These increases can be interpreted as large when expressed as effect sizes using Cohen's classification, 0.88 and 3.54 respectively [32]. The fact that the changes were apparent when measured with two different instruments, adds validity evidence to the conclusion that the EBM course significantly improved the students’ knowledge base about EBM and its related concepts.\nThe EBM knowledge level was similar in the M4 and M5 non-EBM groups, which strongly suggests that the amount of EBM knowledge without a specific educational intervention is minimal even in the senior years of our medical school, and that there was no maturation threat to internal validity.\nThe significantly lower EBM knowledge scores in 6th year students, in the time period of six months to a year after a similar intervention, suggests the possibility of knowledge decay, with decreasing amount of knowledge as time passes, unless continuous learning and practice occurs [43]. This difference in knowledge could be explained by the fact that our 6th year measure was done in a different group of students, not the randomized 5th year class, so it may not represent a true measure of knowledge decay but a difference in students' ability and it is uncertain how this would impact their use of EBM in clinical practice.\nOther published randomized controlled trials of EBM educational intervention have produced conflicting results regarding knowledge change, with some of them showing minimal or no differences after the intervention [35-37] whereas others have found knowledge score increases of 36 to 58% [42,44]. These differences are probably due to the different nature of the educational interventions, their duration and the educational context (e.g. mandatory course). The use of effect size indices like Cohen’s d in EBM educational research publications could help visualize in a more standardized fashion the magnitude of the differences among studies, and promote reflection about the potential educational significance of the findings [45,46].\nA limitation of the study is that it does not measure the actual competence and performance of EBM-related skills in a real clinical setting. Another potential limitation is related to the generalizability of the study, since the medical school has some particular characteristics because of its military nature, which could limit extrapolation to other medical schools. As with any implementation of a new course in a medical school, there was an intense interest from the course instructors to develop and implement as effective an educational intervention as possible, so there could be a tendency for confirmation bias. This can be expected in an education experimental study, where it is not possible to blind either the instructors or the students to the educational intervention. The data analysis was blinded in an attempt to decrease this bias. Another possible source of bias could be the Hawthorne effect, since students in the randomized intervention group were aware that they were being assessed on the course effectiveness, differently from the students that had the regular course previously [25].",
"Our study has implications for the design, implementation and assessment of EBM educational interventions in developing countries. Firstly, it shows that EBM courses can be successfully implemented and embedded in a medical school’s curriculum. Secondly, it provides evidence that the course can improve knowledge, attitudes, critical appraisal confidence, and self-reported skills and behaviours about EBM and its related concepts, although the amount of knowledge that changes with time is still uncertain. And thirdly, it attests to the fact that using international test development standards can contribute to the development of a reliable instrument with evidence of construct validity for the measurement of EBM knowledge acquisition. The study findings contributed to the quality improvement process in the medical school, and provided data to be used in the planning and implementation of subsequent EBM courses. Educational planning will address its clinical links and vertical/horizontal integration with the rest of the curriculum (explicit and hidden), and more studies with rigorous follow-up should be undertaken to identify EBM competencies retention in the long-term. Published models and recommendations to increase the depth and duration of EBM learning should be taken into account when initiating educational interventions of this nature [47,48].",
"The authors declare that they have no competing interests.",
"MS, LK and SM planned, designed and implemented the EBM course and the summative test, and applied the assessment instruments. MS, SD and AS participated in the design of the study and the statistical analysis. MS drafted the initial version of the manuscript. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6920/12/107/prepub\n"
] |
[
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"methods",
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null,
null,
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"results",
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"discussion",
"conclusions",
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] |
[
"Evidence-based medicine",
"Undergraduate medical education",
"Curriculum development",
"Educational assessment",
"Critical appraisal skills"
] |
Background:
Evidence-based medicine (EBM) has been defined as “the integration of the best research evidence with our clinical expertise and our patient’s unique values and circumstances”, and it has emerged as a core competency necessary for all healthcare professionals [1-3]. Its fundamental principles are: translation of uncertainty to an answerable clinical question, systematic retrieval of the best evidence available, critical appraisal for validity, relevance and applicability, use of results in practice and evaluation of its performance by the healthcare provider [4].
Several organizations, including the Institute of Medicine in the United States and the World Federation for Medical Education, have advocated the implementation of EBM educational interventions in medical under and postgraduate training [2,5].
The concepts related to EBM and its educational implications have disseminated rapidly in the last decade, and this change needs to be accompanied with strong educational research to document its effectiveness. The challenges of teaching EBM and the paucity of rigorous educational research publications have prompted some medical educators to question the evidence of EBM teaching effectiveness [6]. Nonetheless, the foundations of EBM that support clinical decision making are intuitively attractive to many clinicians and educators, since it integrates the educational process with clinical practice [4].
The quality of the evidence about EBM education is heterogeneous, as has been described in several editorials, narrative and systematic reviews [7-11]. The majority of reviews have included mostly studies in postgraduate health professionals, and some have included studies in both post and undergraduate students. Green reviewed 18 reports, mostly resident-directed small-group seminars with the objective of improving critical appraisal skills [12]. The most commonly used outcome measure was a multiple-choice exam, and 72% used a traditional journal club format as teaching strategy. Only seven of the 18 studies included in Green’s review analyzed the effectiveness of the intervention, five of these had some type of control group and only one was a randomized study. Just two studies used an outcome measure that had validity evidence, and measurement of change in behavior used only self-report in all five papers. The impact of the intervention was focused mainly on critical appraisal, and ranged from no effect to 23% absolute increase in scores [12].
The Cochrane Collaboration systematic review on the subject of teaching critical appraisal skills in health care, which excluded medical students, found three studies that met stringent pre-specified methodological criteria. These articles reported statistically significant improvements in participants' knowledge in domains of critical appraisal in two of the three studies [9]. Another systematic review by Coomarasamy focused on postgraduate clinicians, and found significant effects of EBM educational interventions in knowledge, and more limited in attitudes, skills and behavior [10,11].
Despite the increasing number of medical school and postgraduate programs that have introduced EBM in their curricula, most of the information about it has been reported as observational data and descriptive studies in the medical literature, or as unpublished observations that are disseminated in medical meetings or informal venues. There are few randomized controlled educational trials about EBM training effectiveness, and the majority have been done in residents or practicing physicians [9-14].
Undergraduate medical students can be a receptive population to EBM concepts, and they will be the practicing clinicians and clinical teachers in the future. There are several published studies that describe medical schools’ experiences introducing EBM in their curriculum and teaching these concepts to undergraduates, with variable outcomes [15-19]. This curricular change has not occurred in many of their developing country counterparts, with few published reports of the implementation of EBM curricula in these settings [20-23]. There is a need to implement EBM educational interventions in developing countries medical schools’ curricula, and to assess their impact with appropriate educational research designs.
The purpose of this study was to assess the educational effectiveness (attitudes, knowledge and skills) of an EBM course in undergraduate medical students.
Methods:
Setting The Mexican Army medical school trains career physicians for the national military healthcare system, and is located in Mexico City. It has a six year program, with a traditional curriculum: two years of basic sciences, three years of clinical sciences, and the sixth year is an internship period in the hospital. The school is a public institution funded by the federal government. Each yearly class is composed of about one hundred students, mostly middle- or low-socioeconomic class Hispanics.
The Mexican Army medical school trains career physicians for the national military healthcare system, and is located in Mexico City. It has a six year program, with a traditional curriculum: two years of basic sciences, three years of clinical sciences, and the sixth year is an internship period in the hospital. The school is a public institution funded by the federal government. Each yearly class is composed of about one hundred students, mostly middle- or low-socioeconomic class Hispanics.
Overall study design and participants Main outcomes and subjects The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].
Flow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.
The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].
Flow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.
Simultaneous validation Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).
The outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.
Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).
The outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.
Main outcomes and subjects The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].
Flow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.
The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].
Flow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.
Simultaneous validation Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).
The outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.
Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).
The outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.
Intervention The educational intervention was a one semester EBM course formally included in the medical school curriculum, with 14 two-hour weekly sessions. The course faculty were six professors trained in EBM teaching, all board-certified physicians with clinical practice, one of them with a postgraduate degree in health professions education and faculty development training in EBM education at McMaster University Faculty of Health Sciences in Canada. The course faculty had more than six years of experience teaching EBM to undergraduate medical students, residents of several specialties, and providing faculty development EBM workshops to teachers of several medical specialties. The EBM course teachers were not involved in the training of the 4th year students, but they participated in the training of the 6th year interns. The EBM program was linked with the internship program and the residency programs in the hospital, through the medical school curricular committee and the University Postgraduate Studies Division.
The course instructional strategies included large-group interactive sessions, small-group problem-solving activities, individual and group assignments, and informatics laboratory sessions. Traditional EBM resources were used as course bibliography, including Straus’ book [1] and an EBM text in Spanish written by the course professors [28]. The content and learning objectives of the course are outlined below.
1. Clinical decision making in medicine
• List and define the main difficulties for objective decision making in medicine as defined by Eddy
• Describe the components of a decision in medicine as defined by Eddy
• Apply the concepts of anatomy of a decision as defined by Eddy in the analysis of a clinical problem
2. Uncertainty and probability in medicine
• Define the concepts of uncertainty, probability and odds
• Understand the relevance of uncertainty in clinical practice
• Understand the limitations of personal experience in the estimation of probability, as related to diagnosis
• Define the heuristics used in medicine (representativeness, availability, anchor and adjustment) and list the cognitive errors a clinician can make when misapplying them
• Apply the concepts of heuristics in new clinical problems, and discuss the effects of their inappropriate use
3. Bayes’ theorem
• Define Bayes’ theorem
• Define pre-test and post-test probability
• Define the concepts of diagnostic and therapeutic threshold
• Explain the utility of Bayes' theorem in clinical medicine, mainly in diagnosis
• List the limitations of Bayes' theorem in clinical practice
• Apply Fagan’s nomogram to use Bayes' theorem in a diagnostic problem
• Apply the concepts of diagnostic and therapeutic threshold to a clinical problem
4. Principles of Evidence Based Medicine
• Describe the history and origin of EBM
• Define the concept of EBM
• List the five steps of EBM, and apply them in a clinical problem
• Explain the importance of EBM in clinical practice
5. Reflective medical practice
• Define the concept of reflection and reflective practitioner
• Define reflection-in-action and reflection-on-action
• Apply these concepts in a clinical scenario
6. Clinicians’ information needs
• Understand the magnitude of physician information needs
• Understand the literature that describe how clinicians underestimate their information needs
• Define the percentage of occasions when clinicians recognize and act upon perceived information needs
7. Clinical questions
• Define the concepts of background and foreground questions
• Understand the advantages of structuring questions generated during clinical work
• List the four components of a foreground clinical question (PICO)
• Apply these concepts in developing questions from clinical problems
• List the types of clinical questions (diagnosis, therapy, prognosis, harm, etiology)
8. Sources of biomedical information
• List the different sources of biomedical information available
• Identify the advantages and disadvantages of each source (textbooks, paper and electronic journals, original research papers)
• Understand the origin, development, cost, and availability of sources of information
9. The Cochrane Collaboration
• Describe the history and origin of the Cochrane Collaboration (CC)
• List the components of the Cochrane Library, and the sources where it’s available
• Understand the mission, logistics and work of the CC
• Perform effective searches for systematic reviews on the Cochrane Library
• Understand the advantages and limitations of the CC
• Use the Cochrane Library to solve a clinical problem
10. Search strategies to find the best medical scientific evidence
• List the main medical databases, and identify their relevance and location
• Describe the history of Medline
• Define MeSH terms, Boolean operators, search engine
• Design search strategies to find valid evidence
• Use PubMed Clinical Queries
• Perform effective searches of scientifically valid papers using PubMed, Cochrane Library, OVID Core Medical Library
• Understand the advantages and disadvantages of searching the different electronic medical databases and the Internet general purpose searching engines
11. Critical appraisal of the medical literature: Users’ Guides to the Medical Literature
• Describe the origin and history of the Users’ Guides series to appraise the medical literature
• List and understand the different hierarchies of evidence, study designs, grades of evidence
• Understand the relevance of using the original medical literature to solve clinical problems
• List and understand the three different steps to appraise a research article: internal validity, magnitude of the results and external validity
12. How to appraise an article about therapy
• Describe the criteria for internal validity of a therapy article
• Define randomized controlled trial, bias and random error, allocation concealment, double-blind, intention-to-treat analysis, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, p values, power and sample size, type I and II errors
• Understand the importance of all the previously defined concepts to apply in a therapy article
• Calculate OR, RR, RRR, ARR and NNT from a published therapy article
• Use a therapy article to solve a clinical problem
• Understand the concepts of external validity of a research paper, related to therapy
13. How to appraise an article about a diagnostic test, the Rational Clinical Examination Series
• Describe the criteria for internal validity of a diagnostic test article
• Define pre-test and post-test probability, sensitivity, specificity, likelihood ratios, positive and negative predictive value, accuracy
• Understand the importance of all the previously defined concepts to apply a diagnosis article
• Calculate sensitivity, specificity, likelihood ratios from a published diagnosis article
• Use a diagnosis article to solve a clinical problem
• Understand the concepts of external validity of a research paper, related to diagnosis
• Describe the origin and evolution of the Rational Clinical Examination JAMA series
• Use a JAMA Rational Clinical Examination paper to solve a clinical problem
14. How to appraise a Systematic Review or Meta-analysis
• Define meta-analysis, systematic review (qualitative and quantitative)
• Describe the advantages and limitations of systematic reviews and meta-analysis
• Describe the criteria for internal validity of a systematic review article
• Define bias and random error, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, forest plot, effect size
• Understand the importance of all the previously defined concepts applied to a systematic review article
• Calculate OR, RR, RRR, ARR and NNT from a published systematic review article
• Use a systematic review article to solve a clinical problem
• Understand the concepts of external validity of a systematic review
15. Clinical practice guidelines
• Define clinical practice guidelines
• Describe the sequence of developing an evidence-based clinical practice guideline
• Understand the advantages and limitations of a clinical guideline
• Describe and understand the internal validity requirements of a clinical guideline article
• List the available resources for clinical guidelines
• Use a clinical practice guideline to solve a clinical problem
The educational intervention was a one semester EBM course formally included in the medical school curriculum, with 14 two-hour weekly sessions. The course faculty were six professors trained in EBM teaching, all board-certified physicians with clinical practice, one of them with a postgraduate degree in health professions education and faculty development training in EBM education at McMaster University Faculty of Health Sciences in Canada. The course faculty had more than six years of experience teaching EBM to undergraduate medical students, residents of several specialties, and providing faculty development EBM workshops to teachers of several medical specialties. The EBM course teachers were not involved in the training of the 4th year students, but they participated in the training of the 6th year interns. The EBM program was linked with the internship program and the residency programs in the hospital, through the medical school curricular committee and the University Postgraduate Studies Division.
The course instructional strategies included large-group interactive sessions, small-group problem-solving activities, individual and group assignments, and informatics laboratory sessions. Traditional EBM resources were used as course bibliography, including Straus’ book [1] and an EBM text in Spanish written by the course professors [28]. The content and learning objectives of the course are outlined below.
1. Clinical decision making in medicine
• List and define the main difficulties for objective decision making in medicine as defined by Eddy
• Describe the components of a decision in medicine as defined by Eddy
• Apply the concepts of anatomy of a decision as defined by Eddy in the analysis of a clinical problem
2. Uncertainty and probability in medicine
• Define the concepts of uncertainty, probability and odds
• Understand the relevance of uncertainty in clinical practice
• Understand the limitations of personal experience in the estimation of probability, as related to diagnosis
• Define the heuristics used in medicine (representativeness, availability, anchor and adjustment) and list the cognitive errors a clinician can make when misapplying them
• Apply the concepts of heuristics in new clinical problems, and discuss the effects of their inappropriate use
3. Bayes’ theorem
• Define Bayes’ theorem
• Define pre-test and post-test probability
• Define the concepts of diagnostic and therapeutic threshold
• Explain the utility of Bayes' theorem in clinical medicine, mainly in diagnosis
• List the limitations of Bayes' theorem in clinical practice
• Apply Fagan’s nomogram to use Bayes' theorem in a diagnostic problem
• Apply the concepts of diagnostic and therapeutic threshold to a clinical problem
4. Principles of Evidence Based Medicine
• Describe the history and origin of EBM
• Define the concept of EBM
• List the five steps of EBM, and apply them in a clinical problem
• Explain the importance of EBM in clinical practice
5. Reflective medical practice
• Define the concept of reflection and reflective practitioner
• Define reflection-in-action and reflection-on-action
• Apply these concepts in a clinical scenario
6. Clinicians’ information needs
• Understand the magnitude of physician information needs
• Understand the literature that describe how clinicians underestimate their information needs
• Define the percentage of occasions when clinicians recognize and act upon perceived information needs
7. Clinical questions
• Define the concepts of background and foreground questions
• Understand the advantages of structuring questions generated during clinical work
• List the four components of a foreground clinical question (PICO)
• Apply these concepts in developing questions from clinical problems
• List the types of clinical questions (diagnosis, therapy, prognosis, harm, etiology)
8. Sources of biomedical information
• List the different sources of biomedical information available
• Identify the advantages and disadvantages of each source (textbooks, paper and electronic journals, original research papers)
• Understand the origin, development, cost, and availability of sources of information
9. The Cochrane Collaboration
• Describe the history and origin of the Cochrane Collaboration (CC)
• List the components of the Cochrane Library, and the sources where it’s available
• Understand the mission, logistics and work of the CC
• Perform effective searches for systematic reviews on the Cochrane Library
• Understand the advantages and limitations of the CC
• Use the Cochrane Library to solve a clinical problem
10. Search strategies to find the best medical scientific evidence
• List the main medical databases, and identify their relevance and location
• Describe the history of Medline
• Define MeSH terms, Boolean operators, search engine
• Design search strategies to find valid evidence
• Use PubMed Clinical Queries
• Perform effective searches of scientifically valid papers using PubMed, Cochrane Library, OVID Core Medical Library
• Understand the advantages and disadvantages of searching the different electronic medical databases and the Internet general purpose searching engines
11. Critical appraisal of the medical literature: Users’ Guides to the Medical Literature
• Describe the origin and history of the Users’ Guides series to appraise the medical literature
• List and understand the different hierarchies of evidence, study designs, grades of evidence
• Understand the relevance of using the original medical literature to solve clinical problems
• List and understand the three different steps to appraise a research article: internal validity, magnitude of the results and external validity
12. How to appraise an article about therapy
• Describe the criteria for internal validity of a therapy article
• Define randomized controlled trial, bias and random error, allocation concealment, double-blind, intention-to-treat analysis, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, p values, power and sample size, type I and II errors
• Understand the importance of all the previously defined concepts to apply in a therapy article
• Calculate OR, RR, RRR, ARR and NNT from a published therapy article
• Use a therapy article to solve a clinical problem
• Understand the concepts of external validity of a research paper, related to therapy
13. How to appraise an article about a diagnostic test, the Rational Clinical Examination Series
• Describe the criteria for internal validity of a diagnostic test article
• Define pre-test and post-test probability, sensitivity, specificity, likelihood ratios, positive and negative predictive value, accuracy
• Understand the importance of all the previously defined concepts to apply a diagnosis article
• Calculate sensitivity, specificity, likelihood ratios from a published diagnosis article
• Use a diagnosis article to solve a clinical problem
• Understand the concepts of external validity of a research paper, related to diagnosis
• Describe the origin and evolution of the Rational Clinical Examination JAMA series
• Use a JAMA Rational Clinical Examination paper to solve a clinical problem
14. How to appraise a Systematic Review or Meta-analysis
• Define meta-analysis, systematic review (qualitative and quantitative)
• Describe the advantages and limitations of systematic reviews and meta-analysis
• Describe the criteria for internal validity of a systematic review article
• Define bias and random error, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, forest plot, effect size
• Understand the importance of all the previously defined concepts applied to a systematic review article
• Calculate OR, RR, RRR, ARR and NNT from a published systematic review article
• Use a systematic review article to solve a clinical problem
• Understand the concepts of external validity of a systematic review
15. Clinical practice guidelines
• Define clinical practice guidelines
• Describe the sequence of developing an evidence-based clinical practice guideline
• Understand the advantages and limitations of a clinical guideline
• Describe and understand the internal validity requirements of a clinical guideline article
• List the available resources for clinical guidelines
• Use a clinical practice guideline to solve a clinical problem
Outcomes and Instrumentation The assessed outcomes were attitudes, knowledge and skills related to EBM. Two instruments were used: Taylor’s questionnaire, a published instrument designed to evaluate the effectiveness of evidence-based medicine teaching [29] and a 100 multiple-choice question test developed specifically for this study.
Taylor’s instrument was categorized as a level 1 instrument in a systematic review of tools to evaluate EBM education, since it has reasonable psychometric properties, has been evaluated for validity from at least three sources of evidence, and is recommended for use in the summative evaluation of individual trainees [30]. The instrument includes items to assess critical appraisal skills, use of evidence behaviors, knowledge and attitudes regarding evidence-based clinical practice [29]. The attitude portion of the questionnaire includes statements related to the use of scientific evidence using a Likert scale. Each statement is scored on a five point scale, responses are added to obtain a total attitude score, and the range of scores is 7 to 35. To determine an overall score for the confidence in critical appraisal skills section, six statements were scored using a scale where “Very confident” was assigned a score of 5, “Not at all confident” a score of 1, and “Don’t know” a score of 0. The scores of the six questions were added, providing a global critical appraisal skills confidence score, where 5 indicated “little or no confidence” and 30 indicated “complete confidence”.
The knowledge part of the questionnaire includes six multiple true-false questions, each with three items, using ‘true’, ‘false’ or ‘don’t know’ response categories. Correct responses to the knowledge questions have a score of 1, incorrect responses are negatively scored (−1) to try to prevent guessing, and the ‘don’t know’ response has a score of 0. The knowledge scores were added in an overall knowledge score, with a possible range of −18 to +18. In a previous paper, we translated the questionnaire to Spanish with the author’s permission, and verified it with backtranslation [21].
The other instrument used was the final summative test of the Evidence-Based Medicine Course. This instrument was developed, administered, scored, and analyzed following the 12 steps for effective test development described by Downing [31]. Item analysis was performed on a pilot application of the test with ITEMAN for Windows (Assessment Systems Corporation, St. Paul, MN), and the information obtained was used to improve the instrument for this study, choosing the better-performing items and preserving content validity. The pilot application of the original 140-items EBM test was done in 57 examinees, and had a Cronbach’s alpha of 0.82. Using the item analysis information 100 multiple-choice questions (MCQ) were selected by the test developers for the final version of the instrument.
The instruments were applied to the students on three consecutive weeks. The students had up to three hours to answer the test and the questionnaire, to minimize the risk of a speeded examination. Taylor’s questionnaires data sheets were captured in a Microsoft Excel spreadsheet. Op-scan answer sheets for item analysis were used for the EBM MCQ test.
The assessed outcomes were attitudes, knowledge and skills related to EBM. Two instruments were used: Taylor’s questionnaire, a published instrument designed to evaluate the effectiveness of evidence-based medicine teaching [29] and a 100 multiple-choice question test developed specifically for this study.
Taylor’s instrument was categorized as a level 1 instrument in a systematic review of tools to evaluate EBM education, since it has reasonable psychometric properties, has been evaluated for validity from at least three sources of evidence, and is recommended for use in the summative evaluation of individual trainees [30]. The instrument includes items to assess critical appraisal skills, use of evidence behaviors, knowledge and attitudes regarding evidence-based clinical practice [29]. The attitude portion of the questionnaire includes statements related to the use of scientific evidence using a Likert scale. Each statement is scored on a five point scale, responses are added to obtain a total attitude score, and the range of scores is 7 to 35. To determine an overall score for the confidence in critical appraisal skills section, six statements were scored using a scale where “Very confident” was assigned a score of 5, “Not at all confident” a score of 1, and “Don’t know” a score of 0. The scores of the six questions were added, providing a global critical appraisal skills confidence score, where 5 indicated “little or no confidence” and 30 indicated “complete confidence”.
The knowledge part of the questionnaire includes six multiple true-false questions, each with three items, using ‘true’, ‘false’ or ‘don’t know’ response categories. Correct responses to the knowledge questions have a score of 1, incorrect responses are negatively scored (−1) to try to prevent guessing, and the ‘don’t know’ response has a score of 0. The knowledge scores were added in an overall knowledge score, with a possible range of −18 to +18. In a previous paper, we translated the questionnaire to Spanish with the author’s permission, and verified it with backtranslation [21].
The other instrument used was the final summative test of the Evidence-Based Medicine Course. This instrument was developed, administered, scored, and analyzed following the 12 steps for effective test development described by Downing [31]. Item analysis was performed on a pilot application of the test with ITEMAN for Windows (Assessment Systems Corporation, St. Paul, MN), and the information obtained was used to improve the instrument for this study, choosing the better-performing items and preserving content validity. The pilot application of the original 140-items EBM test was done in 57 examinees, and had a Cronbach’s alpha of 0.82. Using the item analysis information 100 multiple-choice questions (MCQ) were selected by the test developers for the final version of the instrument.
The instruments were applied to the students on three consecutive weeks. The students had up to three hours to answer the test and the questionnaire, to minimize the risk of a speeded examination. Taylor’s questionnaires data sheets were captured in a Microsoft Excel spreadsheet. Op-scan answer sheets for item analysis were used for the EBM MCQ test.
Statistical analysis The piloting of the EBM MCQ test provided preliminary data for differences and standard deviation, and sample size calculation was performed for the primary hypothesis of knowledge increase with a power of 0.90 (beta error of 0.10), two-sided alpha error of 0.05. After a thorough review of the published studies that included magnitude of EBM knowledge differences in undergraduate medical students, and careful consideration by the course faculty of the smallest meaningful difference (SMD) in this parameter, it was estimated that a difference of 10 questions between the intervention group and the control group would be reasonable. Using this estimate, about 31 students per group would be necessary to detect an effect size of 0.5 or larger.
SPSS for Windows 15.0 and Instat 3.0 for the Macintosh were used for data analysis. The comparison of the use of evidence items in Taylor’s questionnaire between M5 and M5 non-EBM students was done with the non-parametric Mann–Whitney U test. The attitude and critical appraisal confidence scores measured with Taylor’s instrument were compared among groups using the Kruskal-Wallis with Dunn’s multiple comparison test. The groups’ knowledge test scores with both instruments were compared with one-way analysis of variance, with planned comparisons. A p-value of less than 0.05 was considered statistically significant. Cohen’s d with pooled standard deviations was calculated as a measure of effect size for the critical appraisal skills, attitude and knowledge scores among groups [32]. Item analysis of the EBM Test data was performed with ITEMAN for Windows 3.2, (Assessment Systems Corporation, St. Paul, MN http://www.assess.com).
The piloting of the EBM MCQ test provided preliminary data for differences and standard deviation, and sample size calculation was performed for the primary hypothesis of knowledge increase with a power of 0.90 (beta error of 0.10), two-sided alpha error of 0.05. After a thorough review of the published studies that included magnitude of EBM knowledge differences in undergraduate medical students, and careful consideration by the course faculty of the smallest meaningful difference (SMD) in this parameter, it was estimated that a difference of 10 questions between the intervention group and the control group would be reasonable. Using this estimate, about 31 students per group would be necessary to detect an effect size of 0.5 or larger.
SPSS for Windows 15.0 and Instat 3.0 for the Macintosh were used for data analysis. The comparison of the use of evidence items in Taylor’s questionnaire between M5 and M5 non-EBM students was done with the non-parametric Mann–Whitney U test. The attitude and critical appraisal confidence scores measured with Taylor’s instrument were compared among groups using the Kruskal-Wallis with Dunn’s multiple comparison test. The groups’ knowledge test scores with both instruments were compared with one-way analysis of variance, with planned comparisons. A p-value of less than 0.05 was considered statistically significant. Cohen’s d with pooled standard deviations was calculated as a measure of effect size for the critical appraisal skills, attitude and knowledge scores among groups [32]. Item analysis of the EBM Test data was performed with ITEMAN for Windows 3.2, (Assessment Systems Corporation, St. Paul, MN http://www.assess.com).
Ethical aspects The instruments did not have individual student identifiers, to eliminate the risk of potential harm to the participants. This study was reviewed by the Institutional Review Board of the Office for the Protection of Research Subjects of the University of Illinois at Chicago, and the Research Committee of the Mexican Army Medical School, and was considered to be in the exempt category for individual written informed consent.
The instruments did not have individual student identifiers, to eliminate the risk of potential harm to the participants. This study was reviewed by the Institutional Review Board of the Office for the Protection of Research Subjects of the University of Illinois at Chicago, and the Research Committee of the Mexican Army Medical School, and was considered to be in the exempt category for individual written informed consent.
Setting:
The Mexican Army medical school trains career physicians for the national military healthcare system, and is located in Mexico City. It has a six year program, with a traditional curriculum: two years of basic sciences, three years of clinical sciences, and the sixth year is an internship period in the hospital. The school is a public institution funded by the federal government. Each yearly class is composed of about one hundred students, mostly middle- or low-socioeconomic class Hispanics.
Overall study design and participants:
Main outcomes and subjects The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].
Flow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.
The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].
Flow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.
Simultaneous validation Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).
The outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.
Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).
The outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.
Main outcomes and subjects:
The core portion of the study was a randomized post-test only control group design, for the main outcomes: attitudes, knowledge and skills in EBM. Fifth year medical students were randomized in two groups, one of which was subjected to the educational intervention during the first semester of the academic year (M5 EBM), while the other half (M5 non-EBM) had an Aviation Medicine course (Figure 1). The rest of the 5th year curriculum was similar in that semester. In the second semester the control group had the EBM course and the intervention group had the Aviation Medicine course. The randomization was done by the medical school with a computer generated list, using the block randomization method with blocks of two to ensure equal sample sizes [24].
Flow diagram of study participants. Flow diagram summarizing the groups of medical students and the progress of their participation in the study. M4 non-EBM=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6 EBM=6th year students exposed to the evidence-based medicine course during the year prior to assessment.
Simultaneous validation:
Quasi-experimental static-groups comparisons were added besides the randomized trial, with a more junior group of 4th year students not yet exposed to the EBM intervention (M4 non-EBM) and a more senior group in 6th year that had the EBM course during the previous year (M6 EBM). The 4th year students had courses on Medical Informatics, Statistics, Research Methodology and Epidemiology, which are taught by information technology professionals, statisticians, epidemiologists and basic-science researchers, most of them with no clinical background. The 6th year students were in the hospital internship and all of them had the EBM course during the previous year (half of them six months and half one year before the evaluation). These comparison groups were included to acquire more information from our population in concurrent groups and increase the validity of the study, addressing the history, maturation and contamination threats to validity and exploring the potential EBM knowledge in more senior students [25-27] (Figure 1).
The outcomes were measured in all groups at the end of the first semester of the academic year, after the EBM course ended. All the fifth, fourth and sixth year students were asked to participate in the study, about one hundred students per class.
Intervention:
The educational intervention was a one semester EBM course formally included in the medical school curriculum, with 14 two-hour weekly sessions. The course faculty were six professors trained in EBM teaching, all board-certified physicians with clinical practice, one of them with a postgraduate degree in health professions education and faculty development training in EBM education at McMaster University Faculty of Health Sciences in Canada. The course faculty had more than six years of experience teaching EBM to undergraduate medical students, residents of several specialties, and providing faculty development EBM workshops to teachers of several medical specialties. The EBM course teachers were not involved in the training of the 4th year students, but they participated in the training of the 6th year interns. The EBM program was linked with the internship program and the residency programs in the hospital, through the medical school curricular committee and the University Postgraduate Studies Division.
The course instructional strategies included large-group interactive sessions, small-group problem-solving activities, individual and group assignments, and informatics laboratory sessions. Traditional EBM resources were used as course bibliography, including Straus’ book [1] and an EBM text in Spanish written by the course professors [28]. The content and learning objectives of the course are outlined below.
1. Clinical decision making in medicine
• List and define the main difficulties for objective decision making in medicine as defined by Eddy
• Describe the components of a decision in medicine as defined by Eddy
• Apply the concepts of anatomy of a decision as defined by Eddy in the analysis of a clinical problem
2. Uncertainty and probability in medicine
• Define the concepts of uncertainty, probability and odds
• Understand the relevance of uncertainty in clinical practice
• Understand the limitations of personal experience in the estimation of probability, as related to diagnosis
• Define the heuristics used in medicine (representativeness, availability, anchor and adjustment) and list the cognitive errors a clinician can make when misapplying them
• Apply the concepts of heuristics in new clinical problems, and discuss the effects of their inappropriate use
3. Bayes’ theorem
• Define Bayes’ theorem
• Define pre-test and post-test probability
• Define the concepts of diagnostic and therapeutic threshold
• Explain the utility of Bayes' theorem in clinical medicine, mainly in diagnosis
• List the limitations of Bayes' theorem in clinical practice
• Apply Fagan’s nomogram to use Bayes' theorem in a diagnostic problem
• Apply the concepts of diagnostic and therapeutic threshold to a clinical problem
4. Principles of Evidence Based Medicine
• Describe the history and origin of EBM
• Define the concept of EBM
• List the five steps of EBM, and apply them in a clinical problem
• Explain the importance of EBM in clinical practice
5. Reflective medical practice
• Define the concept of reflection and reflective practitioner
• Define reflection-in-action and reflection-on-action
• Apply these concepts in a clinical scenario
6. Clinicians’ information needs
• Understand the magnitude of physician information needs
• Understand the literature that describe how clinicians underestimate their information needs
• Define the percentage of occasions when clinicians recognize and act upon perceived information needs
7. Clinical questions
• Define the concepts of background and foreground questions
• Understand the advantages of structuring questions generated during clinical work
• List the four components of a foreground clinical question (PICO)
• Apply these concepts in developing questions from clinical problems
• List the types of clinical questions (diagnosis, therapy, prognosis, harm, etiology)
8. Sources of biomedical information
• List the different sources of biomedical information available
• Identify the advantages and disadvantages of each source (textbooks, paper and electronic journals, original research papers)
• Understand the origin, development, cost, and availability of sources of information
9. The Cochrane Collaboration
• Describe the history and origin of the Cochrane Collaboration (CC)
• List the components of the Cochrane Library, and the sources where it’s available
• Understand the mission, logistics and work of the CC
• Perform effective searches for systematic reviews on the Cochrane Library
• Understand the advantages and limitations of the CC
• Use the Cochrane Library to solve a clinical problem
10. Search strategies to find the best medical scientific evidence
• List the main medical databases, and identify their relevance and location
• Describe the history of Medline
• Define MeSH terms, Boolean operators, search engine
• Design search strategies to find valid evidence
• Use PubMed Clinical Queries
• Perform effective searches of scientifically valid papers using PubMed, Cochrane Library, OVID Core Medical Library
• Understand the advantages and disadvantages of searching the different electronic medical databases and the Internet general purpose searching engines
11. Critical appraisal of the medical literature: Users’ Guides to the Medical Literature
• Describe the origin and history of the Users’ Guides series to appraise the medical literature
• List and understand the different hierarchies of evidence, study designs, grades of evidence
• Understand the relevance of using the original medical literature to solve clinical problems
• List and understand the three different steps to appraise a research article: internal validity, magnitude of the results and external validity
12. How to appraise an article about therapy
• Describe the criteria for internal validity of a therapy article
• Define randomized controlled trial, bias and random error, allocation concealment, double-blind, intention-to-treat analysis, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, p values, power and sample size, type I and II errors
• Understand the importance of all the previously defined concepts to apply in a therapy article
• Calculate OR, RR, RRR, ARR and NNT from a published therapy article
• Use a therapy article to solve a clinical problem
• Understand the concepts of external validity of a research paper, related to therapy
13. How to appraise an article about a diagnostic test, the Rational Clinical Examination Series
• Describe the criteria for internal validity of a diagnostic test article
• Define pre-test and post-test probability, sensitivity, specificity, likelihood ratios, positive and negative predictive value, accuracy
• Understand the importance of all the previously defined concepts to apply a diagnosis article
• Calculate sensitivity, specificity, likelihood ratios from a published diagnosis article
• Use a diagnosis article to solve a clinical problem
• Understand the concepts of external validity of a research paper, related to diagnosis
• Describe the origin and evolution of the Rational Clinical Examination JAMA series
• Use a JAMA Rational Clinical Examination paper to solve a clinical problem
14. How to appraise a Systematic Review or Meta-analysis
• Define meta-analysis, systematic review (qualitative and quantitative)
• Describe the advantages and limitations of systematic reviews and meta-analysis
• Describe the criteria for internal validity of a systematic review article
• Define bias and random error, odds ratio, relative risk, relative risk reduction, absolute risk reduction, number needed to treat, confidence intervals, forest plot, effect size
• Understand the importance of all the previously defined concepts applied to a systematic review article
• Calculate OR, RR, RRR, ARR and NNT from a published systematic review article
• Use a systematic review article to solve a clinical problem
• Understand the concepts of external validity of a systematic review
15. Clinical practice guidelines
• Define clinical practice guidelines
• Describe the sequence of developing an evidence-based clinical practice guideline
• Understand the advantages and limitations of a clinical guideline
• Describe and understand the internal validity requirements of a clinical guideline article
• List the available resources for clinical guidelines
• Use a clinical practice guideline to solve a clinical problem
Outcomes and Instrumentation:
The assessed outcomes were attitudes, knowledge and skills related to EBM. Two instruments were used: Taylor’s questionnaire, a published instrument designed to evaluate the effectiveness of evidence-based medicine teaching [29] and a 100 multiple-choice question test developed specifically for this study.
Taylor’s instrument was categorized as a level 1 instrument in a systematic review of tools to evaluate EBM education, since it has reasonable psychometric properties, has been evaluated for validity from at least three sources of evidence, and is recommended for use in the summative evaluation of individual trainees [30]. The instrument includes items to assess critical appraisal skills, use of evidence behaviors, knowledge and attitudes regarding evidence-based clinical practice [29]. The attitude portion of the questionnaire includes statements related to the use of scientific evidence using a Likert scale. Each statement is scored on a five point scale, responses are added to obtain a total attitude score, and the range of scores is 7 to 35. To determine an overall score for the confidence in critical appraisal skills section, six statements were scored using a scale where “Very confident” was assigned a score of 5, “Not at all confident” a score of 1, and “Don’t know” a score of 0. The scores of the six questions were added, providing a global critical appraisal skills confidence score, where 5 indicated “little or no confidence” and 30 indicated “complete confidence”.
The knowledge part of the questionnaire includes six multiple true-false questions, each with three items, using ‘true’, ‘false’ or ‘don’t know’ response categories. Correct responses to the knowledge questions have a score of 1, incorrect responses are negatively scored (−1) to try to prevent guessing, and the ‘don’t know’ response has a score of 0. The knowledge scores were added in an overall knowledge score, with a possible range of −18 to +18. In a previous paper, we translated the questionnaire to Spanish with the author’s permission, and verified it with backtranslation [21].
The other instrument used was the final summative test of the Evidence-Based Medicine Course. This instrument was developed, administered, scored, and analyzed following the 12 steps for effective test development described by Downing [31]. Item analysis was performed on a pilot application of the test with ITEMAN for Windows (Assessment Systems Corporation, St. Paul, MN), and the information obtained was used to improve the instrument for this study, choosing the better-performing items and preserving content validity. The pilot application of the original 140-items EBM test was done in 57 examinees, and had a Cronbach’s alpha of 0.82. Using the item analysis information 100 multiple-choice questions (MCQ) were selected by the test developers for the final version of the instrument.
The instruments were applied to the students on three consecutive weeks. The students had up to three hours to answer the test and the questionnaire, to minimize the risk of a speeded examination. Taylor’s questionnaires data sheets were captured in a Microsoft Excel spreadsheet. Op-scan answer sheets for item analysis were used for the EBM MCQ test.
Statistical analysis:
The piloting of the EBM MCQ test provided preliminary data for differences and standard deviation, and sample size calculation was performed for the primary hypothesis of knowledge increase with a power of 0.90 (beta error of 0.10), two-sided alpha error of 0.05. After a thorough review of the published studies that included magnitude of EBM knowledge differences in undergraduate medical students, and careful consideration by the course faculty of the smallest meaningful difference (SMD) in this parameter, it was estimated that a difference of 10 questions between the intervention group and the control group would be reasonable. Using this estimate, about 31 students per group would be necessary to detect an effect size of 0.5 or larger.
SPSS for Windows 15.0 and Instat 3.0 for the Macintosh were used for data analysis. The comparison of the use of evidence items in Taylor’s questionnaire between M5 and M5 non-EBM students was done with the non-parametric Mann–Whitney U test. The attitude and critical appraisal confidence scores measured with Taylor’s instrument were compared among groups using the Kruskal-Wallis with Dunn’s multiple comparison test. The groups’ knowledge test scores with both instruments were compared with one-way analysis of variance, with planned comparisons. A p-value of less than 0.05 was considered statistically significant. Cohen’s d with pooled standard deviations was calculated as a measure of effect size for the critical appraisal skills, attitude and knowledge scores among groups [32]. Item analysis of the EBM Test data was performed with ITEMAN for Windows 3.2, (Assessment Systems Corporation, St. Paul, MN http://www.assess.com).
Ethical aspects:
The instruments did not have individual student identifiers, to eliminate the risk of potential harm to the participants. This study was reviewed by the Institutional Review Board of the Office for the Protection of Research Subjects of the University of Illinois at Chicago, and the Research Committee of the Mexican Army Medical School, and was considered to be in the exempt category for individual written informed consent.
Results:
Subjects The flow diagram of the study participants throughout the trial is outlined in Figure 1. A total of 289 medical students were assessed. One student from the M5 non-EBM group was sick on the assessment day. Five subjects in the M4 non-EBM and 7 subjects in the M6 EBM groups didn't participate because they were on clinical duties on the testing day.
The students’ age (mean±SD) per group was: M4= 21.5±1.8, M5 EBM=22.8±2.0, M5 non-EBM=22.4±2.2 and M6=23.5±1.9 years. The groups’ gender composition was similar, with a predominance of women over men (about 60/40).
The flow diagram of the study participants throughout the trial is outlined in Figure 1. A total of 289 medical students were assessed. One student from the M5 non-EBM group was sick on the assessment day. Five subjects in the M4 non-EBM and 7 subjects in the M6 EBM groups didn't participate because they were on clinical duties on the testing day.
The students’ age (mean±SD) per group was: M4= 21.5±1.8, M5 EBM=22.8±2.0, M5 non-EBM=22.4±2.2 and M6=23.5±1.9 years. The groups’ gender composition was similar, with a predominance of women over men (about 60/40).
Use of the evidence The use of scientific evidence explored in the first section of Taylor’s questionnaire, includes two main questions: “What type of resources do you use to keep up to date?” and “What type of resources do you use to solve a specific health care problem?” The answers by group and type of resource are presented in Figures 2 and 3.
Use of evidence to keep up to date. Distribution of answers to the question: "what type of resources do you use to keep up to date?" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). *** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.
Use of evidence to solve a health problem. Distribution of answers to the question: "what type of resources do you use to solve a specific health problem?" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). * =P<0.01 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.*** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.
In the use of information resources to keep up to date and to solve a specific health care problem, the pattern of responses was the same. The answers were similar among the four student groups regarding the use of review articles, original research journals, textbooks, Internet resources and teachers, but there were statistically significant differences in the use of secondary journals (e.g. American Journal of Physicians Journal Club) and the Cochrane Library. The experimental group (M5 EBM) had a higher reported use of original research articles to solve a specific health problem than the randomized comparison group (M5 non-EBM) (P<0.01). The M5 EBM and M6 groups reported a higher use of secondary journals than the M4 and the M5 non-EBM groups, and a similar pattern of response was found in the use of the Cochrane Library (P<0.001) (Figures 2 and 3).
The use of scientific evidence explored in the first section of Taylor’s questionnaire, includes two main questions: “What type of resources do you use to keep up to date?” and “What type of resources do you use to solve a specific health care problem?” The answers by group and type of resource are presented in Figures 2 and 3.
Use of evidence to keep up to date. Distribution of answers to the question: "what type of resources do you use to keep up to date?" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). *** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.
Use of evidence to solve a health problem. Distribution of answers to the question: "what type of resources do you use to solve a specific health problem?" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). * =P<0.01 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.*** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.
In the use of information resources to keep up to date and to solve a specific health care problem, the pattern of responses was the same. The answers were similar among the four student groups regarding the use of review articles, original research journals, textbooks, Internet resources and teachers, but there were statistically significant differences in the use of secondary journals (e.g. American Journal of Physicians Journal Club) and the Cochrane Library. The experimental group (M5 EBM) had a higher reported use of original research articles to solve a specific health problem than the randomized comparison group (M5 non-EBM) (P<0.01). The M5 EBM and M6 groups reported a higher use of secondary journals than the M4 and the M5 non-EBM groups, and a similar pattern of response was found in the use of the Cochrane Library (P<0.001) (Figures 2 and 3).
Confidence in critical appraisal skills There was a higher confidence level of critical appraisal skills in all items in this section of Taylor’s instrument (assessing study design, evaluating bias, evaluating statistical tests), in the intervention group (P<0.001). The critical appraisal confidence global scores for the different study groups were as follows: M4=11.7±6.3 (mean±SD), M5 non-EBM=8.4±5.7, M5 EBM=17.1±3.6 and M6= 16.8±4.9. The summary data for each group is shown in Figure 4, where the experimental group (M5 EBM) had higher scores than the randomized control group (M5 non-EBM) and the M4 comparison group (P<0.001). The M4 score was slightly higher than the M5 non-EBM group (P<0.05), and the M6 group had higher scores than M4 and M5 non-EBM (P<0.001).
Critical appraisal skills scores. Critical appraisal confidence scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
There was a higher confidence level of critical appraisal skills in all items in this section of Taylor’s instrument (assessing study design, evaluating bias, evaluating statistical tests), in the intervention group (P<0.001). The critical appraisal confidence global scores for the different study groups were as follows: M4=11.7±6.3 (mean±SD), M5 non-EBM=8.4±5.7, M5 EBM=17.1±3.6 and M6= 16.8±4.9. The summary data for each group is shown in Figure 4, where the experimental group (M5 EBM) had higher scores than the randomized control group (M5 non-EBM) and the M4 comparison group (P<0.001). The M4 score was slightly higher than the M5 non-EBM group (P<0.05), and the M6 group had higher scores than M4 and M5 non-EBM (P<0.001).
Critical appraisal skills scores. Critical appraisal confidence scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Attitudes The EBM attitude scores measured with Taylor’s questionnaire are shown in Figure 5. The scores were similar between the groups that didn’t receive the EBM educational intervention, the M4 group had a score of 24.5±5.2 (mean±SD), and the M5 non-EBM group had 24.0±5.0 (P>0.05). The M5 EBM group had an attitude score of 28.7±2.2, higher than the M4 and M5 non-EBM groups (P<0.001). The M6 students had an attitude score of 26.7±3.6, higher than the control groups and lower than the M5 EBM group (P<0.05). Cohen’s d effect size for the comparison of M5 EBM vs. M5 non-EBM was 1.21 (Table 1).
Attitude scores. Attitude scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Effect size (Cohen’s “
d
”) in critical appraisal confidence, attitude and knowledge scores when comparing the different medical student groups
The EBM attitude scores measured with Taylor’s questionnaire are shown in Figure 5. The scores were similar between the groups that didn’t receive the EBM educational intervention, the M4 group had a score of 24.5±5.2 (mean±SD), and the M5 non-EBM group had 24.0±5.0 (P>0.05). The M5 EBM group had an attitude score of 28.7±2.2, higher than the M4 and M5 non-EBM groups (P<0.001). The M6 students had an attitude score of 26.7±3.6, higher than the control groups and lower than the M5 EBM group (P<0.05). Cohen’s d effect size for the comparison of M5 EBM vs. M5 non-EBM was 1.21 (Table 1).
Attitude scores. Attitude scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Effect size (Cohen’s “
d
”) in critical appraisal confidence, attitude and knowledge scores when comparing the different medical student groups
Knowledge scores with Taylor’s instrument The results of the knowledge score measured with Taylor’s questionnaire are shown in Figure 6. The scores were similar between non-EBM groups, M4=1.06±3.16 (mean±SD), and M5 non-EBM=1.13±3.27 (P=0.91). The M5 EBM intervention group had a knowledge score of 4.21±3.73, higher than those of M4 and M5 non-EBM. The planned contrast in the main comparison between M5 EBM and M5 non-EBM showed that the intervention group had a higher knowledge score than the randomized control group (P<0.001). The M6 group had a knowledge score of 2.44±3.77, higher than both control groups, (P<0.01), but lower than M5 EBM (P<0.01). The effect size measured with Cohen’s d for the knowledge score main comparison of M5 EBM vs. M5 non-EBM was 0.88 (Table 1).
Knowledge scores with Taylor’s instrument. Knowledge scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
The results of the knowledge score measured with Taylor’s questionnaire are shown in Figure 6. The scores were similar between non-EBM groups, M4=1.06±3.16 (mean±SD), and M5 non-EBM=1.13±3.27 (P=0.91). The M5 EBM intervention group had a knowledge score of 4.21±3.73, higher than those of M4 and M5 non-EBM. The planned contrast in the main comparison between M5 EBM and M5 non-EBM showed that the intervention group had a higher knowledge score than the randomized control group (P<0.001). The M6 group had a knowledge score of 2.44±3.77, higher than both control groups, (P<0.01), but lower than M5 EBM (P<0.01). The effect size measured with Cohen’s d for the knowledge score main comparison of M5 EBM vs. M5 non-EBM was 0.88 (Table 1).
Knowledge scores with Taylor’s instrument. Knowledge scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Knowledge scores with EBM summative MCQ test The results of the 100-item MCQ EBM knowledge test are presented as percent-correct scores (Figure 7). The reliability of the test with Cronbach’s alpha was 0.72 in the M5 EBM group, and 0.83 in the M6 group. The scores were similar between non-EBM groups, M4=30.6±5.6 (mean±SD), and M5 non-EBM=32.6±6.6 (P=0.18). The M5 EBM group had a test score of 58.5±7.9, higher than M4 and M5 non-EBM. The planned contrast between M5 EBM and M5 non-EBM found that the educational intervention group had a higher knowledge score (P<0.001). M6 had a knowledge score of 41.0±10.9, higher than the control groups (P<0.001), but lower than M5 EBM (P<0.001). The effect size with Cohen’s d for the knowledge score main outcome comparison of M5 EBM vs. M5 non-EBM was 3.54 (Table 1).
Knowledge scores EBM test. Knowledge scores in the different groups of medical students, measured with the 100 multiple-choice questions EBM test. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
The results of the 100-item MCQ EBM knowledge test are presented as percent-correct scores (Figure 7). The reliability of the test with Cronbach’s alpha was 0.72 in the M5 EBM group, and 0.83 in the M6 group. The scores were similar between non-EBM groups, M4=30.6±5.6 (mean±SD), and M5 non-EBM=32.6±6.6 (P=0.18). The M5 EBM group had a test score of 58.5±7.9, higher than M4 and M5 non-EBM. The planned contrast between M5 EBM and M5 non-EBM found that the educational intervention group had a higher knowledge score (P<0.001). M6 had a knowledge score of 41.0±10.9, higher than the control groups (P<0.001), but lower than M5 EBM (P<0.001). The effect size with Cohen’s d for the knowledge score main outcome comparison of M5 EBM vs. M5 non-EBM was 3.54 (Table 1).
Knowledge scores EBM test. Knowledge scores in the different groups of medical students, measured with the 100 multiple-choice questions EBM test. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Subjects:
The flow diagram of the study participants throughout the trial is outlined in Figure 1. A total of 289 medical students were assessed. One student from the M5 non-EBM group was sick on the assessment day. Five subjects in the M4 non-EBM and 7 subjects in the M6 EBM groups didn't participate because they were on clinical duties on the testing day.
The students’ age (mean±SD) per group was: M4= 21.5±1.8, M5 EBM=22.8±2.0, M5 non-EBM=22.4±2.2 and M6=23.5±1.9 years. The groups’ gender composition was similar, with a predominance of women over men (about 60/40).
Use of the evidence:
The use of scientific evidence explored in the first section of Taylor’s questionnaire, includes two main questions: “What type of resources do you use to keep up to date?” and “What type of resources do you use to solve a specific health care problem?” The answers by group and type of resource are presented in Figures 2 and 3.
Use of evidence to keep up to date. Distribution of answers to the question: "what type of resources do you use to keep up to date?" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). *** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.
Use of evidence to solve a health problem. Distribution of answers to the question: "what type of resources do you use to solve a specific health problem?" in the different medical student groups. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; EBM=Evidence-Based Medicine; ACPJC=American College of Physicians Journal Club). * =P<0.01 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.*** = P<0.001 Mann–Whitney U test for the M5 vs. M5 non-EBM comparison.
In the use of information resources to keep up to date and to solve a specific health care problem, the pattern of responses was the same. The answers were similar among the four student groups regarding the use of review articles, original research journals, textbooks, Internet resources and teachers, but there were statistically significant differences in the use of secondary journals (e.g. American Journal of Physicians Journal Club) and the Cochrane Library. The experimental group (M5 EBM) had a higher reported use of original research articles to solve a specific health problem than the randomized comparison group (M5 non-EBM) (P<0.01). The M5 EBM and M6 groups reported a higher use of secondary journals than the M4 and the M5 non-EBM groups, and a similar pattern of response was found in the use of the Cochrane Library (P<0.001) (Figures 2 and 3).
Confidence in critical appraisal skills:
There was a higher confidence level of critical appraisal skills in all items in this section of Taylor’s instrument (assessing study design, evaluating bias, evaluating statistical tests), in the intervention group (P<0.001). The critical appraisal confidence global scores for the different study groups were as follows: M4=11.7±6.3 (mean±SD), M5 non-EBM=8.4±5.7, M5 EBM=17.1±3.6 and M6= 16.8±4.9. The summary data for each group is shown in Figure 4, where the experimental group (M5 EBM) had higher scores than the randomized control group (M5 non-EBM) and the M4 comparison group (P<0.001). The M4 score was slightly higher than the M5 non-EBM group (P<0.05), and the M6 group had higher scores than M4 and M5 non-EBM (P<0.001).
Critical appraisal skills scores. Critical appraisal confidence scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Attitudes:
The EBM attitude scores measured with Taylor’s questionnaire are shown in Figure 5. The scores were similar between the groups that didn’t receive the EBM educational intervention, the M4 group had a score of 24.5±5.2 (mean±SD), and the M5 non-EBM group had 24.0±5.0 (P>0.05). The M5 EBM group had an attitude score of 28.7±2.2, higher than the M4 and M5 non-EBM groups (P<0.001). The M6 students had an attitude score of 26.7±3.6, higher than the control groups and lower than the M5 EBM group (P<0.05). Cohen’s d effect size for the comparison of M5 EBM vs. M5 non-EBM was 1.21 (Table 1).
Attitude scores. Attitude scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Effect size (Cohen’s “
d
”) in critical appraisal confidence, attitude and knowledge scores when comparing the different medical student groups
Knowledge scores with Taylor’s instrument:
The results of the knowledge score measured with Taylor’s questionnaire are shown in Figure 6. The scores were similar between non-EBM groups, M4=1.06±3.16 (mean±SD), and M5 non-EBM=1.13±3.27 (P=0.91). The M5 EBM intervention group had a knowledge score of 4.21±3.73, higher than those of M4 and M5 non-EBM. The planned contrast in the main comparison between M5 EBM and M5 non-EBM showed that the intervention group had a higher knowledge score than the randomized control group (P<0.001). The M6 group had a knowledge score of 2.44±3.77, higher than both control groups, (P<0.01), but lower than M5 EBM (P<0.01). The effect size measured with Cohen’s d for the knowledge score main comparison of M5 EBM vs. M5 non-EBM was 0.88 (Table 1).
Knowledge scores with Taylor’s instrument. Knowledge scores in the different groups of medical students, measured with Taylor’s questionnaire. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Knowledge scores with EBM summative MCQ test:
The results of the 100-item MCQ EBM knowledge test are presented as percent-correct scores (Figure 7). The reliability of the test with Cronbach’s alpha was 0.72 in the M5 EBM group, and 0.83 in the M6 group. The scores were similar between non-EBM groups, M4=30.6±5.6 (mean±SD), and M5 non-EBM=32.6±6.6 (P=0.18). The M5 EBM group had a test score of 58.5±7.9, higher than M4 and M5 non-EBM. The planned contrast between M5 EBM and M5 non-EBM found that the educational intervention group had a higher knowledge score (P<0.001). M6 had a knowledge score of 41.0±10.9, higher than the control groups (P<0.001), but lower than M5 EBM (P<0.001). The effect size with Cohen’s d for the knowledge score main outcome comparison of M5 EBM vs. M5 non-EBM was 3.54 (Table 1).
Knowledge scores EBM test. Knowledge scores in the different groups of medical students, measured with the 100 multiple-choice questions EBM test. (M4=4th year students with no evidence-based medicine training; M5 EBM and M5 non-EBM=5th year medical students with and without the evidence-based medicine course; M6=6th year students exposed to the evidence-based medicine course during the year prior to assessment; CI=confidence interval).
Discussion:
This research study presents experimental evidence that an EBM educational intervention in medical students increases attitudes, knowledge and self-reported critical appraisal skills, in the setting of a developing country medical school.
The research design was a parallel-group randomized control trial, with a quasi-experimental static-groups comparison, to take advantage of a strong study design and its implications in terms of internal validity and the causal inferences that can be made of the results [24,25,33]. Recent studies and systematic reviews suggest that well-planned and educationally sound EBM interventions can have a reasonable impact on the abilities of the individuals that undergo these educational experiences [9,14,34].
There are not many published randomized controlled trials that study the impact of EBM education and very few from developing countries [9-12,14]. Some of the randomized trials did not find an effect of EBM educational interventions, which point to the need of continuing research in this area [35-37].
In the present study the educational intervention was one semester long, it was mandatory, and had a summative test, all these factors probably contribute to the magnitude of the findings in the randomized comparison. Almost all published studies have used only one assessment instrument, while our study used two evaluation tools, a published questionnaire with validity evidence designed to measure the effectiveness of evidence-based practice teaching, and an ad hoc objective test developed for the course summative assessment [29,30]. This characteristic of our study design provided an opportunity to concurrently validate an already published instrument and a new objective test developed specifically for our course, contributing to the body of literature supporting the validity of Taylor’s instrument.
We found an increase in critical appraisal skills, and in the positive attitude to evidence-based practice. These findings are similar to Ghali et al. [16], with a higher reported use of secondary journals and Cochrane Library systematic reviews. It is important to recognize that these are self-reports, the actual behaviour of the students in the use of these resources in their daily routines wasn’t directly measured.
In our study the answers to two questions related to the use of evidence (to keep up to date and to solve clinical problems) had a similar pattern of responses to our previous paper, as measured with Taylor’s questionnaire [21]. There was a higher reported use of the Cochrane Library and secondary journals in both items in the M5 intervention group, and a higher use of original research papers to solve a healthcare problem. It is apparent that all the students use frequently textbooks, Internet resources, teachers and residents as sources of information in health care, as previously reported [21]. These resources are readily available, and culturally accepted in the daily practice of medicine.
The use of the Cochrane Library and secondary journals was higher in our intervention group, which suggests that these resources were virtually unknown to the students before the course and that its reported use probably increased as a result of the educational intervention. Even though these EBM information resources have been extensively used in developed countries in the last decades, developing countries have been slower in adopting them as formal information elements, probably because of a lack of availability and misunderstanding of their potential use [38,39]. The Cochrane Library has been translated to Spanish by the Iberoamerican Cochrane Network, as the Cochrane Library Plus (http://cochrane.bvsalud.org), which should improve the availability and use of this resource in Spanish-speaking countries.
This study found that the EBM intervention improved the confidence of medical students regarding several aspects of critical appraisal skills, as well as statistical concepts relevant to the correct interpretation of published research findings. An interesting aspect of these results is that the medical students who weren’t exposed to the EBM course (M4 and M5 non-EBM), already had courses on Biostatistics and Scientific Methodology and nonetheless had lower scores in this outcome. Probably those courses didn’t have a substantial impact or it was short-lived. Other explanations could be that the previous courses on related subjects were given by non-clinicians and/or basic research scientists with no clinical orientation, having a minor effect on the EBM outcomes. The increase in critical appraisal skills is in agreement with several published reports of EBM in undergraduate students [15,16]. Other studies haven’t found a significant improvement in critical appraisal skills, probably due to several factors inherent to the complexity of educational research interventions in healthcare settings [35-37]. In our study the effect size immediately after the course in critical appraisal skills score was higher than 1.0, which can be interpreted as large using Cohen's classification [32]. A similar effect size was found when comparing the students that had the EBM course six months to one year before with the control group (Table 1).
It is important to recognize that self-perceived skills can overestimate true competence and performance, so these findings may not reflect the real critical appraisal and statistics skills of the medical students, although confidence in a skill is an important component of the performance spectrum [40,41].
The overall attitude score findings in our study are congruent with several published papers, showing an increase immediately after the course of about 17-20% [16,21,23,42]. The 6th year students attitude score was higher than the control group and the 4th year students, which suggests that the attitude change can still occur from six months to a year after the course. Our previous study found very similar attitude score values measured with the same instrument, which adds reproducibility evidence to the use of Taylor’s instrument for measurement of EBM attitude in our population of students [21]. It is noteworthy that some studies, including randomized controlled trials of EBM teaching, didn’t find a change in attitudes, probably due to the shorter duration of the workshops and related activities [36,37].
A major challenge of assessing EBM teaching is to demonstrate an increase in the “knowledge” of evidence-based clinical practice, since several disciplines intersect in the optimal use of scientific evidence (research methodology, biomedical informatics, biostatistics, clinical epidemiology) which integrate a large body of knowledge and facts. In this investigation, large effect sizes in the main randomized comparison (M5 vs. M5-nonEBM) were found in the EBM knowledge scores measured with Taylor’s questionnaire and the EBM MCQ test. The knowledge increase after the course was about 73% higher than the control group when measured with Taylor’s instrument, and 25.9% when measured with the EBM test. These increases can be interpreted as large when expressed as effect sizes using Cohen's classification, 0.88 and 3.54 respectively [32]. The fact that the changes were apparent when measured with two different instruments, adds validity evidence to the conclusion that the EBM course significantly improved the students’ knowledge base about EBM and its related concepts.
The EBM knowledge level was similar in the M4 and M5 non-EBM groups, which strongly suggests that the amount of EBM knowledge without a specific educational intervention is minimal even in the senior years of our medical school, and that there was no maturation threat to internal validity.
The significantly lower EBM knowledge scores in 6th year students, in the time period of six months to a year after a similar intervention, suggests the possibility of knowledge decay, with decreasing amount of knowledge as time passes, unless continuous learning and practice occurs [43]. This difference in knowledge could be explained by the fact that our 6th year measure was done in a different group of students, not the randomized 5th year class, so it may not represent a true measure of knowledge decay but a difference in students' ability and it is uncertain how this would impact their use of EBM in clinical practice.
Other published randomized controlled trials of EBM educational intervention have produced conflicting results regarding knowledge change, with some of them showing minimal or no differences after the intervention [35-37] whereas others have found knowledge score increases of 36 to 58% [42,44]. These differences are probably due to the different nature of the educational interventions, their duration and the educational context (e.g. mandatory course). The use of effect size indices like Cohen’s d in EBM educational research publications could help visualize in a more standardized fashion the magnitude of the differences among studies, and promote reflection about the potential educational significance of the findings [45,46].
A limitation of the study is that it does not measure the actual competence and performance of EBM-related skills in a real clinical setting. Another potential limitation is related to the generalizability of the study, since the medical school has some particular characteristics because of its military nature, which could limit extrapolation to other medical schools. As with any implementation of a new course in a medical school, there was an intense interest from the course instructors to develop and implement as effective an educational intervention as possible, so there could be a tendency for confirmation bias. This can be expected in an education experimental study, where it is not possible to blind either the instructors or the students to the educational intervention. The data analysis was blinded in an attempt to decrease this bias. Another possible source of bias could be the Hawthorne effect, since students in the randomized intervention group were aware that they were being assessed on the course effectiveness, differently from the students that had the regular course previously [25].
Conclusions:
Our study has implications for the design, implementation and assessment of EBM educational interventions in developing countries. Firstly, it shows that EBM courses can be successfully implemented and embedded in a medical school’s curriculum. Secondly, it provides evidence that the course can improve knowledge, attitudes, critical appraisal confidence, and self-reported skills and behaviours about EBM and its related concepts, although the amount of knowledge that changes with time is still uncertain. And thirdly, it attests to the fact that using international test development standards can contribute to the development of a reliable instrument with evidence of construct validity for the measurement of EBM knowledge acquisition. The study findings contributed to the quality improvement process in the medical school, and provided data to be used in the planning and implementation of subsequent EBM courses. Educational planning will address its clinical links and vertical/horizontal integration with the rest of the curriculum (explicit and hidden), and more studies with rigorous follow-up should be undertaken to identify EBM competencies retention in the long-term. Published models and recommendations to increase the depth and duration of EBM learning should be taken into account when initiating educational interventions of this nature [47,48].
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
MS, LK and SM planned, designed and implemented the EBM course and the summative test, and applied the assessment instruments. MS, SD and AS participated in the design of the study and the statistical analysis. MS drafted the initial version of the manuscript. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1472-6920/12/107/prepub
|
Background:
Evidence-Based Medicine (EBM) is an important competency for the healthcare professional. Experimental evidence of EBM educational interventions from rigorous research studies is limited. The main objective of this study was to assess EBM learning (knowledge, attitudes and self-reported skills) in undergraduate medical students with a randomized controlled trial.
Methods:
The educational intervention was a one-semester EBM course in the 5th year of a public medical school in Mexico. The study design was an experimental parallel group randomized controlled trial for the main outcome measures in the 5th year class (M5 EBM vs. M5 non-EBM groups), and quasi-experimental with static-groups comparisons for the 4th year (M4, not yet exposed) and 6th year (M6, exposed 6 months to a year earlier) groups. EBM attitudes, knowledge and self-reported skills were measured using Taylor's questionnaire and a summative exam which comprised of a 100-item multiple-choice question (MCQ) test.
Results:
289 Medical students were assessed: M5 EBM=48, M5 non-EBM=47, M4=87, and M6=107. There was a higher reported use of the Cochrane Library and secondary journals in the intervention group (M5 vs. M5 non-EBM). Critical appraisal skills and attitude scores were higher in the intervention group (M5) and in the group of students exposed to EBM instruction during the previous year (M6). The knowledge level was higher after the intervention in the M5 EBM group compared to the M5 non-EBM group (p<0.001, Cohen's d=0.88 with Taylor's instrument and 3.54 with the 100-item MCQ test). M6 Students that received the intervention in the previous year had a knowledge score higher than the M4 and M5 non-EBM groups, but lower than the M5 EBM group.
Conclusions:
Formal medical student training in EBM produced higher scores in attitudes, knowledge and self-reported critical appraisal skills compared with a randomized control group. Data from the concurrent groups add validity evidence to the study, but rigorous follow-up needs to be done to document retention of EBM abilities.
|
Background:
Evidence-based medicine (EBM) has been defined as “the integration of the best research evidence with our clinical expertise and our patient’s unique values and circumstances”, and it has emerged as a core competency necessary for all healthcare professionals [1-3]. Its fundamental principles are: translation of uncertainty to an answerable clinical question, systematic retrieval of the best evidence available, critical appraisal for validity, relevance and applicability, use of results in practice and evaluation of its performance by the healthcare provider [4].
Several organizations, including the Institute of Medicine in the United States and the World Federation for Medical Education, have advocated the implementation of EBM educational interventions in medical under and postgraduate training [2,5].
The concepts related to EBM and its educational implications have disseminated rapidly in the last decade, and this change needs to be accompanied with strong educational research to document its effectiveness. The challenges of teaching EBM and the paucity of rigorous educational research publications have prompted some medical educators to question the evidence of EBM teaching effectiveness [6]. Nonetheless, the foundations of EBM that support clinical decision making are intuitively attractive to many clinicians and educators, since it integrates the educational process with clinical practice [4].
The quality of the evidence about EBM education is heterogeneous, as has been described in several editorials, narrative and systematic reviews [7-11]. The majority of reviews have included mostly studies in postgraduate health professionals, and some have included studies in both post and undergraduate students. Green reviewed 18 reports, mostly resident-directed small-group seminars with the objective of improving critical appraisal skills [12]. The most commonly used outcome measure was a multiple-choice exam, and 72% used a traditional journal club format as teaching strategy. Only seven of the 18 studies included in Green’s review analyzed the effectiveness of the intervention, five of these had some type of control group and only one was a randomized study. Just two studies used an outcome measure that had validity evidence, and measurement of change in behavior used only self-report in all five papers. The impact of the intervention was focused mainly on critical appraisal, and ranged from no effect to 23% absolute increase in scores [12].
The Cochrane Collaboration systematic review on the subject of teaching critical appraisal skills in health care, which excluded medical students, found three studies that met stringent pre-specified methodological criteria. These articles reported statistically significant improvements in participants' knowledge in domains of critical appraisal in two of the three studies [9]. Another systematic review by Coomarasamy focused on postgraduate clinicians, and found significant effects of EBM educational interventions in knowledge, and more limited in attitudes, skills and behavior [10,11].
Despite the increasing number of medical school and postgraduate programs that have introduced EBM in their curricula, most of the information about it has been reported as observational data and descriptive studies in the medical literature, or as unpublished observations that are disseminated in medical meetings or informal venues. There are few randomized controlled educational trials about EBM training effectiveness, and the majority have been done in residents or practicing physicians [9-14].
Undergraduate medical students can be a receptive population to EBM concepts, and they will be the practicing clinicians and clinical teachers in the future. There are several published studies that describe medical schools’ experiences introducing EBM in their curriculum and teaching these concepts to undergraduates, with variable outcomes [15-19]. This curricular change has not occurred in many of their developing country counterparts, with few published reports of the implementation of EBM curricula in these settings [20-23]. There is a need to implement EBM educational interventions in developing countries medical schools’ curricula, and to assess their impact with appropriate educational research designs.
The purpose of this study was to assess the educational effectiveness (attitudes, knowledge and skills) of an EBM course in undergraduate medical students.
Conclusions:
Our study has implications for the design, implementation and assessment of EBM educational interventions in developing countries. Firstly, it shows that EBM courses can be successfully implemented and embedded in a medical school’s curriculum. Secondly, it provides evidence that the course can improve knowledge, attitudes, critical appraisal confidence, and self-reported skills and behaviours about EBM and its related concepts, although the amount of knowledge that changes with time is still uncertain. And thirdly, it attests to the fact that using international test development standards can contribute to the development of a reliable instrument with evidence of construct validity for the measurement of EBM knowledge acquisition. The study findings contributed to the quality improvement process in the medical school, and provided data to be used in the planning and implementation of subsequent EBM courses. Educational planning will address its clinical links and vertical/horizontal integration with the rest of the curriculum (explicit and hidden), and more studies with rigorous follow-up should be undertaken to identify EBM competencies retention in the long-term. Published models and recommendations to increase the depth and duration of EBM learning should be taken into account when initiating educational interventions of this nature [47,48].
|
Background:
Evidence-Based Medicine (EBM) is an important competency for the healthcare professional. Experimental evidence of EBM educational interventions from rigorous research studies is limited. The main objective of this study was to assess EBM learning (knowledge, attitudes and self-reported skills) in undergraduate medical students with a randomized controlled trial.
Methods:
The educational intervention was a one-semester EBM course in the 5th year of a public medical school in Mexico. The study design was an experimental parallel group randomized controlled trial for the main outcome measures in the 5th year class (M5 EBM vs. M5 non-EBM groups), and quasi-experimental with static-groups comparisons for the 4th year (M4, not yet exposed) and 6th year (M6, exposed 6 months to a year earlier) groups. EBM attitudes, knowledge and self-reported skills were measured using Taylor's questionnaire and a summative exam which comprised of a 100-item multiple-choice question (MCQ) test.
Results:
289 Medical students were assessed: M5 EBM=48, M5 non-EBM=47, M4=87, and M6=107. There was a higher reported use of the Cochrane Library and secondary journals in the intervention group (M5 vs. M5 non-EBM). Critical appraisal skills and attitude scores were higher in the intervention group (M5) and in the group of students exposed to EBM instruction during the previous year (M6). The knowledge level was higher after the intervention in the M5 EBM group compared to the M5 non-EBM group (p<0.001, Cohen's d=0.88 with Taylor's instrument and 3.54 with the 100-item MCQ test). M6 Students that received the intervention in the previous year had a knowledge score higher than the M4 and M5 non-EBM groups, but lower than the M5 EBM group.
Conclusions:
Formal medical student training in EBM produced higher scores in attitudes, knowledge and self-reported critical appraisal skills compared with a randomized control group. Data from the concurrent groups add validity evidence to the study, but rigorous follow-up needs to be done to document retention of EBM abilities.
| 19,140 | 408 | 22 |
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"year",
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"students",
"evidence",
"medical",
"course",
"group",
"clinical",
"non"
] |
[
"test",
"test"
] |
[CONTENT] Evidence-based medicine | Undergraduate medical education | Curriculum development | Educational assessment | Critical appraisal skills [SUMMARY]
|
[CONTENT] Evidence-based medicine | Undergraduate medical education | Curriculum development | Educational assessment | Critical appraisal skills [SUMMARY]
|
[CONTENT] Evidence-based medicine | Undergraduate medical education | Curriculum development | Educational assessment | Critical appraisal skills [SUMMARY]
|
[CONTENT] Evidence-based medicine | Undergraduate medical education | Curriculum development | Educational assessment | Critical appraisal skills [SUMMARY]
|
[CONTENT] Evidence-based medicine | Undergraduate medical education | Curriculum development | Educational assessment | Critical appraisal skills [SUMMARY]
|
[CONTENT] Evidence-based medicine | Undergraduate medical education | Curriculum development | Educational assessment | Critical appraisal skills [SUMMARY]
|
[CONTENT] Aerospace Medicine | Clinical Competence | Cross-Over Studies | Curriculum | Developing Countries | Education, Medical, Undergraduate | Educational Measurement | Evidence-Based Medicine | Female | Health Knowledge, Attitudes, Practice | Humans | Internship and Residency | Male | Mexico | Military Medicine | Schools, Medical | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] Aerospace Medicine | Clinical Competence | Cross-Over Studies | Curriculum | Developing Countries | Education, Medical, Undergraduate | Educational Measurement | Evidence-Based Medicine | Female | Health Knowledge, Attitudes, Practice | Humans | Internship and Residency | Male | Mexico | Military Medicine | Schools, Medical | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] Aerospace Medicine | Clinical Competence | Cross-Over Studies | Curriculum | Developing Countries | Education, Medical, Undergraduate | Educational Measurement | Evidence-Based Medicine | Female | Health Knowledge, Attitudes, Practice | Humans | Internship and Residency | Male | Mexico | Military Medicine | Schools, Medical | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] Aerospace Medicine | Clinical Competence | Cross-Over Studies | Curriculum | Developing Countries | Education, Medical, Undergraduate | Educational Measurement | Evidence-Based Medicine | Female | Health Knowledge, Attitudes, Practice | Humans | Internship and Residency | Male | Mexico | Military Medicine | Schools, Medical | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] Aerospace Medicine | Clinical Competence | Cross-Over Studies | Curriculum | Developing Countries | Education, Medical, Undergraduate | Educational Measurement | Evidence-Based Medicine | Female | Health Knowledge, Attitudes, Practice | Humans | Internship and Residency | Male | Mexico | Military Medicine | Schools, Medical | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] Aerospace Medicine | Clinical Competence | Cross-Over Studies | Curriculum | Developing Countries | Education, Medical, Undergraduate | Educational Measurement | Evidence-Based Medicine | Female | Health Knowledge, Attitudes, Practice | Humans | Internship and Residency | Male | Mexico | Military Medicine | Schools, Medical | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] ebm | year | m5 | students | evidence | medical | course | group | clinical | non [SUMMARY]
|
[CONTENT] ebm | year | m5 | students | evidence | medical | course | group | clinical | non [SUMMARY]
|
[CONTENT] ebm | year | m5 | students | evidence | medical | course | group | clinical | non [SUMMARY]
|
[CONTENT] ebm | year | m5 | students | evidence | medical | course | group | clinical | non [SUMMARY]
|
[CONTENT] ebm | year | m5 | students | evidence | medical | course | group | clinical | non [SUMMARY]
|
[CONTENT] ebm | year | m5 | students | evidence | medical | course | group | clinical | non [SUMMARY]
|
[CONTENT] ebm | studies | educational | medical | effectiveness | teaching | postgraduate | curricula | ebm educational | systematic [SUMMARY]
|
[CONTENT] clinical | ebm | understand | year | define | article | students | describe | list | test [SUMMARY]
|
[CONTENT] m5 | ebm | non ebm | non | m5 non ebm | m5 non | m5 ebm | year | group | evidence [SUMMARY]
|
[CONTENT] ebm | planning | ebm courses | interventions | implementation | educational interventions | educational | development | courses | knowledge [SUMMARY]
|
[CONTENT] ebm | m5 | year | students | non | non ebm | group | evidence | groups | m5 non ebm [SUMMARY]
|
[CONTENT] ebm | m5 | year | students | non | non ebm | group | evidence | groups | m5 non ebm [SUMMARY]
|
[CONTENT] EBM ||| EBM ||| EBM [SUMMARY]
|
[CONTENT] one | EBM | the 5th year | Mexico ||| 5th | EBM | the 4th year | M4 | 6th year | M6 | 6 months to a year earlier ||| EBM | Taylor | 100 [SUMMARY]
|
[CONTENT] 289 | M4=87 ||| the Cochrane Library ||| EBM | the previous year ||| EBM | the M5 non-EBM | Cohen | Taylor | 3.54 | 100 ||| the previous year | M4 | EBM [SUMMARY]
|
[CONTENT] EBM ||| EBM [SUMMARY]
|
[CONTENT] EBM ||| EBM ||| EBM ||| one | EBM | the 5th year | Mexico ||| 5th | EBM | the 4th year | M4 | 6th year | M6 | 6 months to a year earlier ||| EBM | Taylor | 100 ||| 289 | M4=87 ||| the Cochrane Library ||| EBM | the previous year ||| EBM | the M5 non-EBM | Cohen | Taylor | 3.54 | 100 ||| the previous year | M4 | EBM ||| EBM ||| EBM [SUMMARY]
|
[CONTENT] EBM ||| EBM ||| EBM ||| one | EBM | the 5th year | Mexico ||| 5th | EBM | the 4th year | M4 | 6th year | M6 | 6 months to a year earlier ||| EBM | Taylor | 100 ||| 289 | M4=87 ||| the Cochrane Library ||| EBM | the previous year ||| EBM | the M5 non-EBM | Cohen | Taylor | 3.54 | 100 ||| the previous year | M4 | EBM ||| EBM ||| EBM [SUMMARY]
|
||||||
Evaluation of maternal preferences for neonatal male circumcision in Enugu Nigeria.
|
35017375
|
Although circumcision in male neonates is one of the most common procedures performed in neonatal surgery, mothers' preferences concerning the aspects of circumcision are not well-known. Since mother is the likely parent to present child for circumcision, her preferences should be given adequate consideration.
|
BACKGROUND
|
A cross-sectional study where questionnaire was distributed by the researchers to consenting pregnant women attending antenatal clinics in two teaching hospitals in Enugu. Data analysis was performed using the SPSS. The results presented as means, percentages and tables. Test for significance was done using the Chi-square test.
|
METHODOLOGY
|
Four hundred and sixty-one pregnant women participated in the study. Ninety-five percent (438/461) wanted circumcision and 83.5% (385/461) wanted it on or before the 8th day of life. The reasons were cultural/religious in 69% (302/447). Fifty-four percent (250/461) had no preferences as to methods, but for those who had, Plastibell was most preferred method in 28% (129/461) while 76% (235/309) preferred circumcision to be done in hospital. In 49.2% (227/461) preferred personnel were nurses but 79.6% (367/461) wanted doctors to attend to post-circumcision complications. In 79.2% (365/461), mothers will not insist on the use of anaesthesia for circumcision. Mothers with circumcised husbands were significantly more willing to circumcise a male child (P = 0.0018). Higher educational status of mother was significantly related to willingness to insist on the use of anaesthesia (P = 0.046) and use of analgesics after circumcision (P = 0.001).
|
RESULTS
|
Most mothers prefer neonatal male circumcision by nurses, while preferring doctors for post-circumcision complications. These choices are not affected by parents' educational status. Mothers with circumcised husbands accepted circumcision more than those with uncircumcised husbands. Higher maternal education encourages anaesthesia during circumcision and post-circumcision analgesia.
|
CONCLUSIONS
|
[
"Child",
"Circumcision, Male",
"Cross-Sectional Studies",
"Female",
"Humans",
"Infant, Newborn",
"Male",
"Nigeria",
"Parents",
"Pregnancy",
"Surveys and Questionnaires"
] |
8809468
|
INTRODUCTION
|
Circumcision is a common surgical procedure performed on male neonates.[12] In many parts of Africa, it is performed mainly for cultural and religious reasons.[345]
Although the mother is the more likely parent to present a male child for circumcision[6] and in many cases takes the final decision on circumcision of her male neonate,[78] there is a paucity of data concerning women's opinion about neonatal male circumcision in some other climes[9] and in our environment.
Circumcisers may include health-care workers (HCWs) (doctors and nurses) and non-orthodox practitioners, although in the many parts of Africa, most circumcisions are now performed in the hospitals.[91011] The choice of circumcisers vary among parents[3] and from one geographical location to another.[131011] Among the HCWs who circumcise, post-circumcision complications tend to occur more following circumcision by nurses than doctors.[11121314] Despite this, nurses are lead circumcisers in the many parts of the world.[11121516]
It is, therefore, important to determine maternal opinions and preferences as to acceptance or otherwise of circumcision, preferred methods, preferred circumciser, preferred age at circumcision, preferred personnel to handle complications and use or non-use of anaesthesia and post-circumcision analgesia.
Similar to some other studies elsewhere in the world,[91517] we set out to study the preferences of mothers in Enugu, Nigeria as regards the various aspects of neonatal male circumcision using a survey of pregnant mothers attending antenatal clinics.
| null | null |
RESULTS
|
Four hundred and sixty-one women with a mean age of 31.50 ± 4.95 years participated in the study. Most women wanted circumcision of their neonates if males (438/461, 95%) on or before the 8th day of life (385/461, 83.5%) [Table 1]. Their reasons for accepting circumcision were religiocultural in 69% of cases (302/447). In 54.2% (250/461) of cases, mothers did not have any preference for circumcision method but for those that had, Plastibell was the most preferred method (129/461, 28%) [Table 1] and preferred circumcision venue is the hospital (235/309, 76.1%). The preferred personnel to circumcise is the nurse (227/461, 49.2%) and this did not depend on educational status of mothers (P = 0.8) or fathers (P = 0.59). However, 79.6% (367/461) wanted doctors to attend to their child in case of post-circumcision complications [Table 1]. Seventy-nine percent (365/461) will not insist on the use of anaesthesia during circumcision. Sixty-six percent (303/461) of mothers had tertiary education [Table 2] and higher educational status of mother was significantly related to willingness to insist on the use of anaesthesia for circumcision (P = 0.046) and use of analgesics after circumcision (P = 0.001). Ninety-five percent (429/450) of the husbands were circumcised [Table 3] and the circumcision status of father was significantly related to maternal willingness to circumcise a male child (P = 0.0018). There is, however, no significant relationship between mothers’ marital status (P = 0.973), husband's highest educational level (P = 0.598), mothers’ highest educational level (P = 0.629), maternal age (P = 0.984), husband's age (P = 1.00), number of male children (P = 0.712), tribe (P = 0.972), parity (0.953) and maternal willingness to circumcise male neonate.
Circumcision preferences of mothers concerning their male child
Sociodemographic characteristics of mothers
Sociodemographic characteristics of husband
|
CONCLUSIONS
|
Most mothers in Enugu want male circumcision in the neonatal period and will prefer nurses to circumcise their male neonates, but doctors to handle post-circumcision complications. This choice is not affected by educational status of mothers. Mothers whose husbands are circumcised accepted circumcision more readily than those whose husbands were not. Maternal education encourages the use of anaesthesia for circumcision and post-circumcision analgesia.
Recommendations We recommend that training/certification of circumcisers, especially nurses, be done before they are allowed to carry out circumcision. Furthermore, training of nurses and doctors in circumcision and doctors in managing post-circumcision complications should be encouraged. Furthermore, we advocate increasing awareness of women on the use of anaesthesia during circumcision and post-circumcision analgesia. In addition, the hospital environment should be made more friendly and services affordable to encourage good patronage for circumcision services and ensure good outcome.
We recommend that training/certification of circumcisers, especially nurses, be done before they are allowed to carry out circumcision. Furthermore, training of nurses and doctors in circumcision and doctors in managing post-circumcision complications should be encouraged. Furthermore, we advocate increasing awareness of women on the use of anaesthesia during circumcision and post-circumcision analgesia. In addition, the hospital environment should be made more friendly and services affordable to encourage good patronage for circumcision services and ensure good outcome.
Financial support and sponsorship Nil.
Nil.
Conflicts of interest There are no conflicts of interest.
There are no conflicts of interest.
|
[
"Recommendations",
"Financial support and sponsorship"
] |
[
"We recommend that training/certification of circumcisers, especially nurses, be done before they are allowed to carry out circumcision. Furthermore, training of nurses and doctors in circumcision and doctors in managing post-circumcision complications should be encouraged. Furthermore, we advocate increasing awareness of women on the use of anaesthesia during circumcision and post-circumcision analgesia. In addition, the hospital environment should be made more friendly and services affordable to encourage good patronage for circumcision services and ensure good outcome.",
"Nil."
] |
[
null,
null
] |
[
"INTRODUCTION",
"METHODOLOGY",
"RESULTS",
"DISCUSSION",
"CONCLUSIONS",
"Recommendations",
"Financial support and sponsorship",
"Conflicts of interest"
] |
[
"Circumcision is a common surgical procedure performed on male neonates.[12] In many parts of Africa, it is performed mainly for cultural and religious reasons.[345]\nAlthough the mother is the more likely parent to present a male child for circumcision[6] and in many cases takes the final decision on circumcision of her male neonate,[78] there is a paucity of data concerning women's opinion about neonatal male circumcision in some other climes[9] and in our environment.\nCircumcisers may include health-care workers (HCWs) (doctors and nurses) and non-orthodox practitioners, although in the many parts of Africa, most circumcisions are now performed in the hospitals.[91011] The choice of circumcisers vary among parents[3] and from one geographical location to another.[131011] Among the HCWs who circumcise, post-circumcision complications tend to occur more following circumcision by nurses than doctors.[11121314] Despite this, nurses are lead circumcisers in the many parts of the world.[11121516]\nIt is, therefore, important to determine maternal opinions and preferences as to acceptance or otherwise of circumcision, preferred methods, preferred circumciser, preferred age at circumcision, preferred personnel to handle complications and use or non-use of anaesthesia and post-circumcision analgesia.\nSimilar to some other studies elsewhere in the world,[91517] we set out to study the preferences of mothers in Enugu, Nigeria as regards the various aspects of neonatal male circumcision using a survey of pregnant mothers attending antenatal clinics.",
"This is a cross-sectional study where copies of questionnaire were distributed to consenting pregnant mothers attending antenatal clinics in two teaching hospitals in Enugu, South-East Nigeria (University of Nigeria Teaching Hospital Ituku/Ozalla and Enugu State University Teaching Hospital) between 1st June and 31st November 2018. There was no pre-existing validated questionnaire available for assessing maternal circumcision preferences; hence, a new invalidated questionnaire was designed by the corresponding author following review of literature and valuable inputs from other authors. Copies of questionnaire were distributed to consenting pregnant mothers attending antenatal clinic in any of the two teaching hospitals by the authors and other doctors trained by them. We ensured that no one completed the questionnaire more than once. The copies of completed questionnaire were collected immediately at the antenatal clinic. Ethical clearance for the study was sought for and obtained from the Health Research and Ethics Committee of the University of Nigeria Teaching Hospital. Individual consent was implied when a prospective mother accepted, completed and returned the questionnaire. Those that declined consent were excluded from the study and not captured but were not stigmatised in any way. The data entry and analysis were performed using the Statistical Package for the Social Sciences software version 20 (SPSS Inc., Chicago, Illinois, USA) and results presented as means, percentages and tables. Test for significant relationships was done using the Chi-square test, and a P < 0.05 was deemed significant.",
"Four hundred and sixty-one women with a mean age of 31.50 ± 4.95 years participated in the study. Most women wanted circumcision of their neonates if males (438/461, 95%) on or before the 8th day of life (385/461, 83.5%) [Table 1]. Their reasons for accepting circumcision were religiocultural in 69% of cases (302/447). In 54.2% (250/461) of cases, mothers did not have any preference for circumcision method but for those that had, Plastibell was the most preferred method (129/461, 28%) [Table 1] and preferred circumcision venue is the hospital (235/309, 76.1%). The preferred personnel to circumcise is the nurse (227/461, 49.2%) and this did not depend on educational status of mothers (P = 0.8) or fathers (P = 0.59). However, 79.6% (367/461) wanted doctors to attend to their child in case of post-circumcision complications [Table 1]. Seventy-nine percent (365/461) will not insist on the use of anaesthesia during circumcision. Sixty-six percent (303/461) of mothers had tertiary education [Table 2] and higher educational status of mother was significantly related to willingness to insist on the use of anaesthesia for circumcision (P = 0.046) and use of analgesics after circumcision (P = 0.001). Ninety-five percent (429/450) of the husbands were circumcised [Table 3] and the circumcision status of father was significantly related to maternal willingness to circumcise a male child (P = 0.0018). There is, however, no significant relationship between mothers’ marital status (P = 0.973), husband's highest educational level (P = 0.598), mothers’ highest educational level (P = 0.629), maternal age (P = 0.984), husband's age (P = 1.00), number of male children (P = 0.712), tribe (P = 0.972), parity (0.953) and maternal willingness to circumcise male neonate.\nCircumcision preferences of mothers concerning their male child\nSociodemographic characteristics of mothers\nSociodemographic characteristics of husband",
"In this survey, 95% of the women who responded wanted circumcision for their newborn males [Table 1]. This shows a high acceptance rate for neonatal circumcision in our environment. This is higher than circumcision acceptance rates of 82.9% in a survey by Phili and Karim.[17] and 83.7% in a survey by Ikwegbue et al.[9] Circumcision rates from another study in Nigeria were 87% by Okeke et al.,[11] whereas it is 93.3% in a study in the USA by El Bcheraoui et al.[16] and almost 100% in a study by Ben Chaim et al. in Israel.[18]\nMost of the women in the current survey wanted male circumcision done on or before the 8th day of life [Table 1]. In another survey by Özveren,[8] most mothers also wanted circumcision in the early neonatal period, whereas most circumcisions were done mainly in the neonatal period in many other studies.[1341018] Due to the fact that the medical benefits of male circumcision in the neonatal period outweigh the risks[2] and neonatal circumcision is cost-effective,[119] this overwhelming maternal preference for early neonatal circumcision is justified and should be encouraged. Furthermore, compared to circumcisions performed in older males, neonatal and early infant circumcisions have lower complication rates when performed by trained professionals in the clinical settings.[1819] In some other studies, however, most circumcisions were done beyond infancy in later childhood.[142021]\nIndications for neonatal circumcisions in this study were mainly cultural and religious reasons [Table 1], and these are also the main considerations for circumcision in some other studies.[3452021] However, in a survey by Özveren,[8] the major indications were mainly medical and hygiene and in another study by Rediger and Muller[15] in Canada, achieving good hygiene was rated high as an indication for circumcision.\nIn the current study, majority of mothers had no preferences as regard to methods of circumcision [Table 1]. This may suggest that many mothers trust the circumciser’s choice of method. In those that had preference, plastibell is the most desired method as also was the most common method in other studies.[35]\nA larger proportion of mothers in this study will prefer circumcision to be done in the hospital as also observed in other studies.[391011] In this study, the most desired circumcisers were the nurses. This may be influenced by the fact that in one of the teaching hospitals nurses were the main circumcisers, although in the other hospital, routine neonatal circumcision services were not offered. Nurses were also the main circumcisers in some other studies,[101114] although in other studies, most circumcisions were done by doctors.[134720] These differences may suggest regional variations in the preferences pointing to the fact that identifying the preferred circumcisers in the different geographical locations and ensuring their adequate training in circumcision is important.\nNurses are the preferred circumcisers in the current study despite the fact that post-circumcision complications tend to be more following circumcisions performed by nurses[1011121322] and less when performed by medical professionals.[14] Since mothers in our environment still preferred nurses to circumcise their children in spite of the fact that complications are more with nurses, we suggest that to help reduce complication rates and respect the mothers’ circumciser preferences in our environment, better training of nurses for circumcision should be done as it has been shown in other climes that with adequate training nurses can perform circumcisions with low complication rates.[232425] Furthermore, American Academy of Paediatrics (AAP) task force on circumcision guidelines[2] shows that untrained circumcisers have more complications than well-trained circumcisers, regardless of whether the former are physicians, nurses or traditional religious providers. Ben Chaim in Israel[18] found no significant differences in post-circumcision complications following neonatal circumcisions performed by medical personnels and “mohels”(traditional circumcisers trained as professionals for the procedure of circumcision). Appiah et al.[10] in Ghana found knowledge and practice gaps among circumcisers and suggested that trainings be organised for all providers, especially nurses to reduce the incidence of circumcision-related injuries. In a meta-analysis, Weiss et al.[26] found that several studies stressed the importance of training and experience of the provider of circumcision services.\nConversely, most preferred personnel for managing post-circumcision complications in this survey are the doctors. It may be possible that mothers prefer doctors to manage post-circumcision complications because they think that the preferred initial circumciser, who are mainly nurses, have failed, hence the need to choose a supposedly more qualified personnel. It may also be that they believe that doctors are better trained to handle complications. Despite this burden of responsibility for managing post circumcision complications placed on doctors, it has been reported that training of doctors in circumcision is suboptimal in our environment.[27] As a corollary training in the management of post-circumcision complications may also be inadequate. Hence, we highly recommend adequate training of nurses to reduce post-circumcision complications and upgrading the skills of doctors in managing post-circumcision complications.\nUnlike in other studies where a large number of circumcisions were still done by non-orthodox circumcisers,[1821] many mothers in this survey did not desire the services of non-orthodox circumcisers such as traditional birth attendants (TBAs). Therefore, efforts may not be wasted training TBAs for circumcision in our environment.\nMost mothers, from this survey, will not insist on the use of anaesthesia for circumcision. Among those who responded that they will insist on use of anaesthesia, mothers with higher highest educational level are more likely to insist on the use of anaesthesia for circumcision than those with lower highest educational qualifications. It has been suggested by some reports that non-use of anaesthesia may increase complication rates[1322] and that anaesthesia for circumcision is appropriate. Despite this, most circumcisers use no anaesthesia or inadequate anaesthesia during circumcision[28] despite the fact that anaesthesia reduces pain score and is encouraged.[29] Mothers are therefore encouraged to request for the use of anaesthesia during male neonatal circumcision.\nThe maternal willingness to circumcise a child is significantly related to circumcision status of the husband. Despite the fact that it was mothers’ preference being assessed, their husbands’ circumcision status was a very important factor affecting their choice of circumcision for their male neonate. This may be due to their acceptance and satisfaction with the appearance of a circumcised male phallus when compared with uncircumcised one. Circumcision status of father was also an important factor influencing choice of circumcision in another study by Rediger and Muller.[15]\nAs also noted in some other studies,[1330] majority of the women in this survey will not use analgesics following circumcision, although the use of analgesics after circumcision was more likely in women with tertiary education than those without tertiary education. However, AAP[2] encouraged that adequate analgesia should be provided whenever newborn circumcision is carried out.\nThis study is limited by the fact that it was done only in teaching hospitals which may not be a true representation of the general population. It may have been more appropriate to use only pregnant women whose child (ren) have been circumcised instead of using all pregnant women including those with no experience of circumcision. Preferences of other key players in circumcision decision-making such as child’s father and grandmother were not studied. Furthermore, those that declined consent (non-responders) were not captured, and hence, it is difficult to calculate the response rate.",
"Most mothers in Enugu want male circumcision in the neonatal period and will prefer nurses to circumcise their male neonates, but doctors to handle post-circumcision complications. This choice is not affected by educational status of mothers. Mothers whose husbands are circumcised accepted circumcision more readily than those whose husbands were not. Maternal education encourages the use of anaesthesia for circumcision and post-circumcision analgesia.\n Recommendations We recommend that training/certification of circumcisers, especially nurses, be done before they are allowed to carry out circumcision. Furthermore, training of nurses and doctors in circumcision and doctors in managing post-circumcision complications should be encouraged. Furthermore, we advocate increasing awareness of women on the use of anaesthesia during circumcision and post-circumcision analgesia. In addition, the hospital environment should be made more friendly and services affordable to encourage good patronage for circumcision services and ensure good outcome.\nWe recommend that training/certification of circumcisers, especially nurses, be done before they are allowed to carry out circumcision. Furthermore, training of nurses and doctors in circumcision and doctors in managing post-circumcision complications should be encouraged. Furthermore, we advocate increasing awareness of women on the use of anaesthesia during circumcision and post-circumcision analgesia. In addition, the hospital environment should be made more friendly and services affordable to encourage good patronage for circumcision services and ensure good outcome.\n Financial support and sponsorship Nil.\nNil.\n Conflicts of interest There are no conflicts of interest.\nThere are no conflicts of interest.",
"We recommend that training/certification of circumcisers, especially nurses, be done before they are allowed to carry out circumcision. Furthermore, training of nurses and doctors in circumcision and doctors in managing post-circumcision complications should be encouraged. Furthermore, we advocate increasing awareness of women on the use of anaesthesia during circumcision and post-circumcision analgesia. In addition, the hospital environment should be made more friendly and services affordable to encourage good patronage for circumcision services and ensure good outcome.",
"Nil.",
"There are no conflicts of interest."
] |
[
"intro",
"method",
"results",
"discussion",
"conclusion",
null,
null,
"COI-statement"
] |
[
"Enugu Nigeria",
"evaluation",
"male circumcision",
"maternal preferences"
] |
INTRODUCTION:
Circumcision is a common surgical procedure performed on male neonates.[12] In many parts of Africa, it is performed mainly for cultural and religious reasons.[345]
Although the mother is the more likely parent to present a male child for circumcision[6] and in many cases takes the final decision on circumcision of her male neonate,[78] there is a paucity of data concerning women's opinion about neonatal male circumcision in some other climes[9] and in our environment.
Circumcisers may include health-care workers (HCWs) (doctors and nurses) and non-orthodox practitioners, although in the many parts of Africa, most circumcisions are now performed in the hospitals.[91011] The choice of circumcisers vary among parents[3] and from one geographical location to another.[131011] Among the HCWs who circumcise, post-circumcision complications tend to occur more following circumcision by nurses than doctors.[11121314] Despite this, nurses are lead circumcisers in the many parts of the world.[11121516]
It is, therefore, important to determine maternal opinions and preferences as to acceptance or otherwise of circumcision, preferred methods, preferred circumciser, preferred age at circumcision, preferred personnel to handle complications and use or non-use of anaesthesia and post-circumcision analgesia.
Similar to some other studies elsewhere in the world,[91517] we set out to study the preferences of mothers in Enugu, Nigeria as regards the various aspects of neonatal male circumcision using a survey of pregnant mothers attending antenatal clinics.
METHODOLOGY:
This is a cross-sectional study where copies of questionnaire were distributed to consenting pregnant mothers attending antenatal clinics in two teaching hospitals in Enugu, South-East Nigeria (University of Nigeria Teaching Hospital Ituku/Ozalla and Enugu State University Teaching Hospital) between 1st June and 31st November 2018. There was no pre-existing validated questionnaire available for assessing maternal circumcision preferences; hence, a new invalidated questionnaire was designed by the corresponding author following review of literature and valuable inputs from other authors. Copies of questionnaire were distributed to consenting pregnant mothers attending antenatal clinic in any of the two teaching hospitals by the authors and other doctors trained by them. We ensured that no one completed the questionnaire more than once. The copies of completed questionnaire were collected immediately at the antenatal clinic. Ethical clearance for the study was sought for and obtained from the Health Research and Ethics Committee of the University of Nigeria Teaching Hospital. Individual consent was implied when a prospective mother accepted, completed and returned the questionnaire. Those that declined consent were excluded from the study and not captured but were not stigmatised in any way. The data entry and analysis were performed using the Statistical Package for the Social Sciences software version 20 (SPSS Inc., Chicago, Illinois, USA) and results presented as means, percentages and tables. Test for significant relationships was done using the Chi-square test, and a P < 0.05 was deemed significant.
RESULTS:
Four hundred and sixty-one women with a mean age of 31.50 ± 4.95 years participated in the study. Most women wanted circumcision of their neonates if males (438/461, 95%) on or before the 8th day of life (385/461, 83.5%) [Table 1]. Their reasons for accepting circumcision were religiocultural in 69% of cases (302/447). In 54.2% (250/461) of cases, mothers did not have any preference for circumcision method but for those that had, Plastibell was the most preferred method (129/461, 28%) [Table 1] and preferred circumcision venue is the hospital (235/309, 76.1%). The preferred personnel to circumcise is the nurse (227/461, 49.2%) and this did not depend on educational status of mothers (P = 0.8) or fathers (P = 0.59). However, 79.6% (367/461) wanted doctors to attend to their child in case of post-circumcision complications [Table 1]. Seventy-nine percent (365/461) will not insist on the use of anaesthesia during circumcision. Sixty-six percent (303/461) of mothers had tertiary education [Table 2] and higher educational status of mother was significantly related to willingness to insist on the use of anaesthesia for circumcision (P = 0.046) and use of analgesics after circumcision (P = 0.001). Ninety-five percent (429/450) of the husbands were circumcised [Table 3] and the circumcision status of father was significantly related to maternal willingness to circumcise a male child (P = 0.0018). There is, however, no significant relationship between mothers’ marital status (P = 0.973), husband's highest educational level (P = 0.598), mothers’ highest educational level (P = 0.629), maternal age (P = 0.984), husband's age (P = 1.00), number of male children (P = 0.712), tribe (P = 0.972), parity (0.953) and maternal willingness to circumcise male neonate.
Circumcision preferences of mothers concerning their male child
Sociodemographic characteristics of mothers
Sociodemographic characteristics of husband
DISCUSSION:
In this survey, 95% of the women who responded wanted circumcision for their newborn males [Table 1]. This shows a high acceptance rate for neonatal circumcision in our environment. This is higher than circumcision acceptance rates of 82.9% in a survey by Phili and Karim.[17] and 83.7% in a survey by Ikwegbue et al.[9] Circumcision rates from another study in Nigeria were 87% by Okeke et al.,[11] whereas it is 93.3% in a study in the USA by El Bcheraoui et al.[16] and almost 100% in a study by Ben Chaim et al. in Israel.[18]
Most of the women in the current survey wanted male circumcision done on or before the 8th day of life [Table 1]. In another survey by Özveren,[8] most mothers also wanted circumcision in the early neonatal period, whereas most circumcisions were done mainly in the neonatal period in many other studies.[1341018] Due to the fact that the medical benefits of male circumcision in the neonatal period outweigh the risks[2] and neonatal circumcision is cost-effective,[119] this overwhelming maternal preference for early neonatal circumcision is justified and should be encouraged. Furthermore, compared to circumcisions performed in older males, neonatal and early infant circumcisions have lower complication rates when performed by trained professionals in the clinical settings.[1819] In some other studies, however, most circumcisions were done beyond infancy in later childhood.[142021]
Indications for neonatal circumcisions in this study were mainly cultural and religious reasons [Table 1], and these are also the main considerations for circumcision in some other studies.[3452021] However, in a survey by Özveren,[8] the major indications were mainly medical and hygiene and in another study by Rediger and Muller[15] in Canada, achieving good hygiene was rated high as an indication for circumcision.
In the current study, majority of mothers had no preferences as regard to methods of circumcision [Table 1]. This may suggest that many mothers trust the circumciser’s choice of method. In those that had preference, plastibell is the most desired method as also was the most common method in other studies.[35]
A larger proportion of mothers in this study will prefer circumcision to be done in the hospital as also observed in other studies.[391011] In this study, the most desired circumcisers were the nurses. This may be influenced by the fact that in one of the teaching hospitals nurses were the main circumcisers, although in the other hospital, routine neonatal circumcision services were not offered. Nurses were also the main circumcisers in some other studies,[101114] although in other studies, most circumcisions were done by doctors.[134720] These differences may suggest regional variations in the preferences pointing to the fact that identifying the preferred circumcisers in the different geographical locations and ensuring their adequate training in circumcision is important.
Nurses are the preferred circumcisers in the current study despite the fact that post-circumcision complications tend to be more following circumcisions performed by nurses[1011121322] and less when performed by medical professionals.[14] Since mothers in our environment still preferred nurses to circumcise their children in spite of the fact that complications are more with nurses, we suggest that to help reduce complication rates and respect the mothers’ circumciser preferences in our environment, better training of nurses for circumcision should be done as it has been shown in other climes that with adequate training nurses can perform circumcisions with low complication rates.[232425] Furthermore, American Academy of Paediatrics (AAP) task force on circumcision guidelines[2] shows that untrained circumcisers have more complications than well-trained circumcisers, regardless of whether the former are physicians, nurses or traditional religious providers. Ben Chaim in Israel[18] found no significant differences in post-circumcision complications following neonatal circumcisions performed by medical personnels and “mohels”(traditional circumcisers trained as professionals for the procedure of circumcision). Appiah et al.[10] in Ghana found knowledge and practice gaps among circumcisers and suggested that trainings be organised for all providers, especially nurses to reduce the incidence of circumcision-related injuries. In a meta-analysis, Weiss et al.[26] found that several studies stressed the importance of training and experience of the provider of circumcision services.
Conversely, most preferred personnel for managing post-circumcision complications in this survey are the doctors. It may be possible that mothers prefer doctors to manage post-circumcision complications because they think that the preferred initial circumciser, who are mainly nurses, have failed, hence the need to choose a supposedly more qualified personnel. It may also be that they believe that doctors are better trained to handle complications. Despite this burden of responsibility for managing post circumcision complications placed on doctors, it has been reported that training of doctors in circumcision is suboptimal in our environment.[27] As a corollary training in the management of post-circumcision complications may also be inadequate. Hence, we highly recommend adequate training of nurses to reduce post-circumcision complications and upgrading the skills of doctors in managing post-circumcision complications.
Unlike in other studies where a large number of circumcisions were still done by non-orthodox circumcisers,[1821] many mothers in this survey did not desire the services of non-orthodox circumcisers such as traditional birth attendants (TBAs). Therefore, efforts may not be wasted training TBAs for circumcision in our environment.
Most mothers, from this survey, will not insist on the use of anaesthesia for circumcision. Among those who responded that they will insist on use of anaesthesia, mothers with higher highest educational level are more likely to insist on the use of anaesthesia for circumcision than those with lower highest educational qualifications. It has been suggested by some reports that non-use of anaesthesia may increase complication rates[1322] and that anaesthesia for circumcision is appropriate. Despite this, most circumcisers use no anaesthesia or inadequate anaesthesia during circumcision[28] despite the fact that anaesthesia reduces pain score and is encouraged.[29] Mothers are therefore encouraged to request for the use of anaesthesia during male neonatal circumcision.
The maternal willingness to circumcise a child is significantly related to circumcision status of the husband. Despite the fact that it was mothers’ preference being assessed, their husbands’ circumcision status was a very important factor affecting their choice of circumcision for their male neonate. This may be due to their acceptance and satisfaction with the appearance of a circumcised male phallus when compared with uncircumcised one. Circumcision status of father was also an important factor influencing choice of circumcision in another study by Rediger and Muller.[15]
As also noted in some other studies,[1330] majority of the women in this survey will not use analgesics following circumcision, although the use of analgesics after circumcision was more likely in women with tertiary education than those without tertiary education. However, AAP[2] encouraged that adequate analgesia should be provided whenever newborn circumcision is carried out.
This study is limited by the fact that it was done only in teaching hospitals which may not be a true representation of the general population. It may have been more appropriate to use only pregnant women whose child (ren) have been circumcised instead of using all pregnant women including those with no experience of circumcision. Preferences of other key players in circumcision decision-making such as child’s father and grandmother were not studied. Furthermore, those that declined consent (non-responders) were not captured, and hence, it is difficult to calculate the response rate.
CONCLUSIONS:
Most mothers in Enugu want male circumcision in the neonatal period and will prefer nurses to circumcise their male neonates, but doctors to handle post-circumcision complications. This choice is not affected by educational status of mothers. Mothers whose husbands are circumcised accepted circumcision more readily than those whose husbands were not. Maternal education encourages the use of anaesthesia for circumcision and post-circumcision analgesia.
Recommendations We recommend that training/certification of circumcisers, especially nurses, be done before they are allowed to carry out circumcision. Furthermore, training of nurses and doctors in circumcision and doctors in managing post-circumcision complications should be encouraged. Furthermore, we advocate increasing awareness of women on the use of anaesthesia during circumcision and post-circumcision analgesia. In addition, the hospital environment should be made more friendly and services affordable to encourage good patronage for circumcision services and ensure good outcome.
We recommend that training/certification of circumcisers, especially nurses, be done before they are allowed to carry out circumcision. Furthermore, training of nurses and doctors in circumcision and doctors in managing post-circumcision complications should be encouraged. Furthermore, we advocate increasing awareness of women on the use of anaesthesia during circumcision and post-circumcision analgesia. In addition, the hospital environment should be made more friendly and services affordable to encourage good patronage for circumcision services and ensure good outcome.
Financial support and sponsorship Nil.
Nil.
Conflicts of interest There are no conflicts of interest.
There are no conflicts of interest.
Recommendations:
We recommend that training/certification of circumcisers, especially nurses, be done before they are allowed to carry out circumcision. Furthermore, training of nurses and doctors in circumcision and doctors in managing post-circumcision complications should be encouraged. Furthermore, we advocate increasing awareness of women on the use of anaesthesia during circumcision and post-circumcision analgesia. In addition, the hospital environment should be made more friendly and services affordable to encourage good patronage for circumcision services and ensure good outcome.
Financial support and sponsorship:
Nil.
Conflicts of interest:
There are no conflicts of interest.
|
Background:
Although circumcision in male neonates is one of the most common procedures performed in neonatal surgery, mothers' preferences concerning the aspects of circumcision are not well-known. Since mother is the likely parent to present child for circumcision, her preferences should be given adequate consideration.
Methods:
A cross-sectional study where questionnaire was distributed by the researchers to consenting pregnant women attending antenatal clinics in two teaching hospitals in Enugu. Data analysis was performed using the SPSS. The results presented as means, percentages and tables. Test for significance was done using the Chi-square test.
Results:
Four hundred and sixty-one pregnant women participated in the study. Ninety-five percent (438/461) wanted circumcision and 83.5% (385/461) wanted it on or before the 8th day of life. The reasons were cultural/religious in 69% (302/447). Fifty-four percent (250/461) had no preferences as to methods, but for those who had, Plastibell was most preferred method in 28% (129/461) while 76% (235/309) preferred circumcision to be done in hospital. In 49.2% (227/461) preferred personnel were nurses but 79.6% (367/461) wanted doctors to attend to post-circumcision complications. In 79.2% (365/461), mothers will not insist on the use of anaesthesia for circumcision. Mothers with circumcised husbands were significantly more willing to circumcise a male child (P = 0.0018). Higher educational status of mother was significantly related to willingness to insist on the use of anaesthesia (P = 0.046) and use of analgesics after circumcision (P = 0.001).
Conclusions:
Most mothers prefer neonatal male circumcision by nurses, while preferring doctors for post-circumcision complications. These choices are not affected by parents' educational status. Mothers with circumcised husbands accepted circumcision more than those with uncircumcised husbands. Higher maternal education encourages anaesthesia during circumcision and post-circumcision analgesia.
|
INTRODUCTION:
Circumcision is a common surgical procedure performed on male neonates.[12] In many parts of Africa, it is performed mainly for cultural and religious reasons.[345]
Although the mother is the more likely parent to present a male child for circumcision[6] and in many cases takes the final decision on circumcision of her male neonate,[78] there is a paucity of data concerning women's opinion about neonatal male circumcision in some other climes[9] and in our environment.
Circumcisers may include health-care workers (HCWs) (doctors and nurses) and non-orthodox practitioners, although in the many parts of Africa, most circumcisions are now performed in the hospitals.[91011] The choice of circumcisers vary among parents[3] and from one geographical location to another.[131011] Among the HCWs who circumcise, post-circumcision complications tend to occur more following circumcision by nurses than doctors.[11121314] Despite this, nurses are lead circumcisers in the many parts of the world.[11121516]
It is, therefore, important to determine maternal opinions and preferences as to acceptance or otherwise of circumcision, preferred methods, preferred circumciser, preferred age at circumcision, preferred personnel to handle complications and use or non-use of anaesthesia and post-circumcision analgesia.
Similar to some other studies elsewhere in the world,[91517] we set out to study the preferences of mothers in Enugu, Nigeria as regards the various aspects of neonatal male circumcision using a survey of pregnant mothers attending antenatal clinics.
CONCLUSIONS:
Most mothers in Enugu want male circumcision in the neonatal period and will prefer nurses to circumcise their male neonates, but doctors to handle post-circumcision complications. This choice is not affected by educational status of mothers. Mothers whose husbands are circumcised accepted circumcision more readily than those whose husbands were not. Maternal education encourages the use of anaesthesia for circumcision and post-circumcision analgesia.
Recommendations We recommend that training/certification of circumcisers, especially nurses, be done before they are allowed to carry out circumcision. Furthermore, training of nurses and doctors in circumcision and doctors in managing post-circumcision complications should be encouraged. Furthermore, we advocate increasing awareness of women on the use of anaesthesia during circumcision and post-circumcision analgesia. In addition, the hospital environment should be made more friendly and services affordable to encourage good patronage for circumcision services and ensure good outcome.
We recommend that training/certification of circumcisers, especially nurses, be done before they are allowed to carry out circumcision. Furthermore, training of nurses and doctors in circumcision and doctors in managing post-circumcision complications should be encouraged. Furthermore, we advocate increasing awareness of women on the use of anaesthesia during circumcision and post-circumcision analgesia. In addition, the hospital environment should be made more friendly and services affordable to encourage good patronage for circumcision services and ensure good outcome.
Financial support and sponsorship Nil.
Nil.
Conflicts of interest There are no conflicts of interest.
There are no conflicts of interest.
|
Background:
Although circumcision in male neonates is one of the most common procedures performed in neonatal surgery, mothers' preferences concerning the aspects of circumcision are not well-known. Since mother is the likely parent to present child for circumcision, her preferences should be given adequate consideration.
Methods:
A cross-sectional study where questionnaire was distributed by the researchers to consenting pregnant women attending antenatal clinics in two teaching hospitals in Enugu. Data analysis was performed using the SPSS. The results presented as means, percentages and tables. Test for significance was done using the Chi-square test.
Results:
Four hundred and sixty-one pregnant women participated in the study. Ninety-five percent (438/461) wanted circumcision and 83.5% (385/461) wanted it on or before the 8th day of life. The reasons were cultural/religious in 69% (302/447). Fifty-four percent (250/461) had no preferences as to methods, but for those who had, Plastibell was most preferred method in 28% (129/461) while 76% (235/309) preferred circumcision to be done in hospital. In 49.2% (227/461) preferred personnel were nurses but 79.6% (367/461) wanted doctors to attend to post-circumcision complications. In 79.2% (365/461), mothers will not insist on the use of anaesthesia for circumcision. Mothers with circumcised husbands were significantly more willing to circumcise a male child (P = 0.0018). Higher educational status of mother was significantly related to willingness to insist on the use of anaesthesia (P = 0.046) and use of analgesics after circumcision (P = 0.001).
Conclusions:
Most mothers prefer neonatal male circumcision by nurses, while preferring doctors for post-circumcision complications. These choices are not affected by parents' educational status. Mothers with circumcised husbands accepted circumcision more than those with uncircumcised husbands. Higher maternal education encourages anaesthesia during circumcision and post-circumcision analgesia.
| 2,737 | 372 | 8 |
[
"circumcision",
"mothers",
"nurses",
"post circumcision",
"post",
"circumcisers",
"use",
"complications",
"doctors",
"male"
] |
[
"test",
"test"
] | null |
[CONTENT] Enugu Nigeria | evaluation | male circumcision | maternal preferences [SUMMARY]
| null |
[CONTENT] Enugu Nigeria | evaluation | male circumcision | maternal preferences [SUMMARY]
|
[CONTENT] Enugu Nigeria | evaluation | male circumcision | maternal preferences [SUMMARY]
|
[CONTENT] Enugu Nigeria | evaluation | male circumcision | maternal preferences [SUMMARY]
|
[CONTENT] Enugu Nigeria | evaluation | male circumcision | maternal preferences [SUMMARY]
|
[CONTENT] Child | Circumcision, Male | Cross-Sectional Studies | Female | Humans | Infant, Newborn | Male | Nigeria | Parents | Pregnancy | Surveys and Questionnaires [SUMMARY]
| null |
[CONTENT] Child | Circumcision, Male | Cross-Sectional Studies | Female | Humans | Infant, Newborn | Male | Nigeria | Parents | Pregnancy | Surveys and Questionnaires [SUMMARY]
|
[CONTENT] Child | Circumcision, Male | Cross-Sectional Studies | Female | Humans | Infant, Newborn | Male | Nigeria | Parents | Pregnancy | Surveys and Questionnaires [SUMMARY]
|
[CONTENT] Child | Circumcision, Male | Cross-Sectional Studies | Female | Humans | Infant, Newborn | Male | Nigeria | Parents | Pregnancy | Surveys and Questionnaires [SUMMARY]
|
[CONTENT] Child | Circumcision, Male | Cross-Sectional Studies | Female | Humans | Infant, Newborn | Male | Nigeria | Parents | Pregnancy | Surveys and Questionnaires [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] circumcision | mothers | nurses | post circumcision | post | circumcisers | use | complications | doctors | male [SUMMARY]
| null |
[CONTENT] circumcision | mothers | nurses | post circumcision | post | circumcisers | use | complications | doctors | male [SUMMARY]
|
[CONTENT] circumcision | mothers | nurses | post circumcision | post | circumcisers | use | complications | doctors | male [SUMMARY]
|
[CONTENT] circumcision | mothers | nurses | post circumcision | post | circumcisers | use | complications | doctors | male [SUMMARY]
|
[CONTENT] circumcision | mothers | nurses | post circumcision | post | circumcisers | use | complications | doctors | male [SUMMARY]
|
[CONTENT] circumcision | male | parts | preferred | performed | hcws | circumcision preferred | parts africa | neonatal male circumcision | neonatal male [SUMMARY]
| null |
[CONTENT] 461 | circumcision | table | mothers | percent | status | educational | male | willingness | age [SUMMARY]
|
[CONTENT] circumcision | post circumcision | post | nurses | services | training | good | furthermore | circumcision post | anaesthesia circumcision post circumcision [SUMMARY]
|
[CONTENT] circumcision | nil | conflicts interest | interest | conflicts | nurses | mothers | post circumcision | post | training [SUMMARY]
|
[CONTENT] circumcision | nil | conflicts interest | interest | conflicts | nurses | mothers | post circumcision | post | training [SUMMARY]
|
[CONTENT] ||| [SUMMARY]
| null |
[CONTENT] Four hundred and sixty-one ||| Ninety-five percent | 438/461 | 83.5% | 385/461 | the 8th day ||| 69% | 302/447 ||| Fifty-four percent | 250/461 | Plastibell | 28% | 129/461 | 76% | 235/309 ||| 49.2% | 227/461 | 79.6% | 367/461 ||| 79.2% | anaesthesia ||| 0.0018 ||| anaesthesia | 0.046 | 0.001 [SUMMARY]
|
[CONTENT] ||| ||| ||| anaesthesia [SUMMARY]
|
[CONTENT] ||| ||| two | Enugu ||| SPSS ||| ||| ||| Four hundred and sixty-one ||| Ninety-five percent | 438/461 | 83.5% | 385/461 | the 8th day ||| 69% | 302/447 ||| Fifty-four percent | 250/461 | Plastibell | 28% | 129/461 | 76% | 235/309 ||| 49.2% | 227/461 | 79.6% | 367/461 ||| 79.2% | anaesthesia ||| 0.0018 ||| anaesthesia | 0.046 | 0.001 ||| ||| ||| ||| anaesthesia [SUMMARY]
|
[CONTENT] ||| ||| two | Enugu ||| SPSS ||| ||| ||| Four hundred and sixty-one ||| Ninety-five percent | 438/461 | 83.5% | 385/461 | the 8th day ||| 69% | 302/447 ||| Fifty-four percent | 250/461 | Plastibell | 28% | 129/461 | 76% | 235/309 ||| 49.2% | 227/461 | 79.6% | 367/461 ||| 79.2% | anaesthesia ||| 0.0018 ||| anaesthesia | 0.046 | 0.001 ||| ||| ||| ||| anaesthesia [SUMMARY]
|
|||||
The effect of polyvinylpyrrolidone-sodium hyaluronate gel (Gelclair) on oral microbial colonization and pain control compared with other rinsing solutions in patients with oral mucositis after allogeneic stem cells transplantation.
|
21959611
|
Gelclair is an oral lubricating gel used in the management of oral mucositis (OM). We evaluated its efficacy, tolerance and impact on oral cavity microbial colonization in patients with OM after allogeneic hematopoietic stem cells transplantation.
|
BACKGROUND
|
Gelclair was administered in a group of 22 patients with active OM. A control group of 15 patients used other rinsing solutions (chlorhexidine, benzydamine, salvia). Tests with oral cavity swabs for microbiology analysis were performed once a week.
|
MATERIAL/METHOD
|
The characteristics of OM in both groups were comparable, and rinsing solutions had satisfactory tolerability. There was no difference in the median improvement of oral intake and OM-related pain relief, which was assessed mostly as "slight effect". In the Gelclair group, the effect duration was longer (median 3 [0-5] vs. 1 [0-3] hours, p = 0.001). There was significant increase of Enterococcus faecalis and Candida sp. colonization of the oral cavity over the course of the hospitalization and significantly reduced incidence of such colonization in patients with OM in the Gelclair group: 1/22 (5%) vs. 6/15 (40%), p = 0.01. In vitro tests showed inhibited growth of Enterococcus faecalis and Candida sp. colonies within the area of the Gelclair application.
|
RESULTS
|
Gelclair may be individually helpful in the management of OM and pain in patients after allogeneic stem cells transplantation. Its use did not lead to worsened oral bacterial and yeast colonization and probably even helped to protect mucosa from Enterococcus and Candida sp. Further studies based on larger cohorts are needed.
|
CONCLUSIONS
|
[
"Benzydamine",
"Candida",
"Chlorhexidine",
"Drug Combinations",
"Enterococcus faecalis",
"Facial Pain",
"Humans",
"Hyaluronic Acid",
"Povidone",
"Prospective Studies",
"Stem Cell Transplantation",
"Stomatitis",
"Time Factors",
"Transplantation, Homologous"
] |
3539478
|
Background
|
Gelclair (Helsinn Healthcare SA, Switzerland) is a concentrated oral gel containing polyvinylpyrrolidone and sodium hyaluronate. It forms an adherent barrier and layer covering oral mucosa lesions, thus protecting sensitive nerve endings and lubricating the tissue. Several studies have suggested that it can help to manage pain and potentially can improve the ability to eat and drink in patients with oral mucositis (OM) after intensive chemotherapy or radiotherapy [1–5].
OM is a significant medical and nursing problem in high-dose chemotherapy and stem cells transplantation settings. This oral mucosa damage and injury is associated with pain and reduced oral intake. Since infections may also play a role in the pathophysiology of OM, antimicrobial mouthwashes such as chlorhexidine, benzydamine, povidone-iodine, and various local antibiotics/antimycotics are widely used in the nursing care and management of OM. However, there is little evidence supporting the use of these agents in the prophylaxis and treatment of this complication [6,7]. On the other hand, they are important in the treatment of oral bacterial and fungal infections [8,9]. Oral cavity cooling (cryotherapy) provided during high-dose melphalan bolus or short infusion administration contributes to local lower blood circulation and cytotoxic drug exposure and can significantly reduce OM in melphalan-containing protocols [10,11]. Effective analgesic therapy is necessary in patients suffering from this painful complication. Patient-controlled analgesia with morphine is a treatment of choice according to MASCC (Multinational Association of Supportive Care in Cancer), NCCN (National Comprehensive Cancer Network) and ESMO (European Society for Medical Oncology) recommendations [12–16].
Our observational study aimed to evaluate safety of Gelclair use with respect to the extent of bacterial and yeast colonization within the oral cavity, and to verify its clinical efficacy and tolerance in patients after allogeneic stem cells transplantation.
| null | null |
Results
|
A total of 22 patients were enrolled into OM treatment with Gelclair and 15 patients into the Control group using standard oral solutions with chlorhexidine (8/15, 53%), benzydamine (6/15, 40%) or salvia (1/15, 7%). Characteristics of the groups are shown in Table 1.
There was no difference in the median value of tolerability of the rinses in the Gelclair vs. Control group: 2 (1–5) vs. 2 (1–3), p=0.304. The individual tolerance in the Gelclair group in details was: 1 – tolerable without any problems in 9%, 2 – satisfactory in 73%, 3 – indifferent in 9%, 4 – unsatisfactory in 4.5% and 5 – intolerable in 4.5% patients. The individual tolerance in the Control group was: 1 – tolerable without any problems in 33%, 2 – satisfactory in 40% and 3 – indifferent in 27% patients.
Regarding the improvement of oral intake and OM pain relief after oral rinsing, there was no difference in the median value of improvement intensity observed between the Gelclair and the Control groups: 3 (2–4) vs. 3 (2–4) and 3 (2–5) vs. 3 (1–4) VAS score, p=0.381 and 0.190 (3 = slight effect). The median value of duration of pain relief was significantly longer in the Gelclair group: 3 (0–5) vs. 1 (0–3) hours, p=0.001. Significantly more patients on systemic analgesic opioid treatment were in the Gelclair group: 15/22 (68%) vs. 7/15 (46%), p=0.03. The analgesic medication administered in the Gelclair group was: buprenorphine 35 ug/hour transdermal patch in 13/22 (59%), buprenorphine 52.5 ug/hour transdermal patch in 1/22 (4.5%) and tramadol 4.1 mg/hour I.V. in 1/22 (4.5%) patients. The analgesic treatment administered in the Control group was: buprenorphine 35 ug/hour transdermal patch in 5/15 (33%), buprenorphine 52.5 ug/hour transdermal patch in 2/15 (13%) patients.
Oral microbiology swabs In the whole group of 37 patients, bacterial or yeast pathogens were detected in 6 (16%) patients in oral cavity swabs during the pre-OM phase, and in 18 (49%) in the post-OM phase (p=0.0024). During the OM phase, significantly fewer pathogens were found in the Gelclair compared to the Control group: 1/22 (5%) vs. 6/15 (40%), p=0.01. After the OM resolution and Gelclair use termination (the post-OM phase), there was a significant increase of pathogen colonization observed in the Gelclair group in comparison with the previous condition in this group: 12/22 (55%) vs. 1/22 (5%), p=0.0006. Negative microbial swab results were obtained in the Gelclair group only during the OM phase and in 2 patients later on. No difference was observed in patients using either chlorhexidine or benzydamine solutions. For more details see Table 2.
In the whole group of 37 patients, bacterial or yeast pathogens were detected in 6 (16%) patients in oral cavity swabs during the pre-OM phase, and in 18 (49%) in the post-OM phase (p=0.0024). During the OM phase, significantly fewer pathogens were found in the Gelclair compared to the Control group: 1/22 (5%) vs. 6/15 (40%), p=0.01. After the OM resolution and Gelclair use termination (the post-OM phase), there was a significant increase of pathogen colonization observed in the Gelclair group in comparison with the previous condition in this group: 12/22 (55%) vs. 1/22 (5%), p=0.0006. Negative microbial swab results were obtained in the Gelclair group only during the OM phase and in 2 patients later on. No difference was observed in patients using either chlorhexidine or benzydamine solutions. For more details see Table 2.
In vitro Gelclair inhibition test There was evident growth inhibition of the Enterococcus faecalis and Candida colonies within the area of Gelclair application (Figures 1 and 2).
There was evident growth inhibition of the Enterococcus faecalis and Candida colonies within the area of Gelclair application (Figures 1 and 2).
|
Conclusions
|
The results of this observation suggest that Gelclair may be individually helpful in the management of OM and pains in some patients after allogeneic stem cells transplantation. The use of Gelclair did not lead to worsened local bacterial and yeast colonization in the oral cavity and probably even helped to protect mucosa from Enterococcus and Candida colonization. Gelclair use appears to be safe in patients after allogeneic stem cells transplantation. Further observations and analysis based on larger cohorts of patients are needed.
|
[
"Background",
"The monitoring, assessment and definitions",
"Statistics",
"Oral microbiology swabs",
"In vitro Gelclair inhibition test"
] |
[
"Gelclair (Helsinn Healthcare SA, Switzerland) is a concentrated oral gel containing polyvinylpyrrolidone and sodium hyaluronate. It forms an adherent barrier and layer covering oral mucosa lesions, thus protecting sensitive nerve endings and lubricating the tissue. Several studies have suggested that it can help to manage pain and potentially can improve the ability to eat and drink in patients with oral mucositis (OM) after intensive chemotherapy or radiotherapy [1–5].\nOM is a significant medical and nursing problem in high-dose chemotherapy and stem cells transplantation settings. This oral mucosa damage and injury is associated with pain and reduced oral intake. Since infections may also play a role in the pathophysiology of OM, antimicrobial mouthwashes such as chlorhexidine, benzydamine, povidone-iodine, and various local antibiotics/antimycotics are widely used in the nursing care and management of OM. However, there is little evidence supporting the use of these agents in the prophylaxis and treatment of this complication [6,7]. On the other hand, they are important in the treatment of oral bacterial and fungal infections [8,9]. Oral cavity cooling (cryotherapy) provided during high-dose melphalan bolus or short infusion administration contributes to local lower blood circulation and cytotoxic drug exposure and can significantly reduce OM in melphalan-containing protocols [10,11]. Effective analgesic therapy is necessary in patients suffering from this painful complication. Patient-controlled analgesia with morphine is a treatment of choice according to MASCC (Multinational Association of Supportive Care in Cancer), NCCN (National Comprehensive Cancer Network) and ESMO (European Society for Medical Oncology) recommendations [12–16].\nOur observational study aimed to evaluate safety of Gelclair use with respect to the extent of bacterial and yeast colonization within the oral cavity, and to verify its clinical efficacy and tolerance in patients after allogeneic stem cells transplantation.",
"OM was assessed daily using the WHO grading 0–4 (0 = absent; 1 = pain and erythema; 2 = ulcers, patient can swallow solid food; 3 = ulcers, patient cannot swallow solid food; 4 = mucositis to the extent that alimentation is not possible). The tolerability of oral rinses was evaluated daily by the patients, using the Visual Analog Scale (VAS) scoring: 1–5 (1 = tolerable without any problems, 2 = satisfactory, 3 = indifferent, 4 = unsatisfactory, 5 = intolerable). The OM pain reduction and food intake improvement were assessed daily by the patients using the VAS scoring: 1–5 (1 = excellent, 2 = good, 3 = slight effect, 4 = almost no effect, 5 = no effect at all).\nTests with oral cavity swabs for microbiology analysis were performed once a week in the morning — on the admission to the transplantation unit (pre-OM phase), during active OM (OM-phase) and after the OM resolution (post-OM phase). The smears comprised buccal, palatal and sublingual mucosa.\nIn vitro Gelclair inhibition test was performed using the Enterococcus faecalis, Candida albicans, Candida parapsilosis and Candida krusei strains suspensions inoculated separately onto Müller-Hinton agar and 3 drops of Gelclair were added on the inoculated area. The plate samples were incubated in stable temperature 37°C for 24 hour (48 hours in Candida krusei sample). All the microbiology samples were processed and cultivated under controlled laboratory conditions in the institutional Department of Microbiology.\nColonies of generally physiological oral bacteria (Streptococcus viridans or Neisseria spec. or Staphylococcus coagulase-negative) were considered potentially pathogenic because of the significant immunodeficiency of transplanted patients.",
"Basic statistical univariate analyses were performed using statistical software (GraphPad InStat, GraphPad Software) with the Fisher’s exact test and Unpaired T test. The “p” values <0.05 were considered as statistically significant differences.",
"In the whole group of 37 patients, bacterial or yeast pathogens were detected in 6 (16%) patients in oral cavity swabs during the pre-OM phase, and in 18 (49%) in the post-OM phase (p=0.0024). During the OM phase, significantly fewer pathogens were found in the Gelclair compared to the Control group: 1/22 (5%) vs. 6/15 (40%), p=0.01. After the OM resolution and Gelclair use termination (the post-OM phase), there was a significant increase of pathogen colonization observed in the Gelclair group in comparison with the previous condition in this group: 12/22 (55%) vs. 1/22 (5%), p=0.0006. Negative microbial swab results were obtained in the Gelclair group only during the OM phase and in 2 patients later on. No difference was observed in patients using either chlorhexidine or benzydamine solutions. For more details see Table 2.",
"There was evident growth inhibition of the Enterococcus faecalis and Candida colonies within the area of Gelclair application (Figures 1 and 2)."
] |
[
null,
null,
null,
null,
null
] |
[
"Background",
"Material and Methods",
"The monitoring, assessment and definitions",
"Statistics",
"Results",
"Oral microbiology swabs",
"In vitro Gelclair inhibition test",
"Discussion",
"Conclusions"
] |
[
"Gelclair (Helsinn Healthcare SA, Switzerland) is a concentrated oral gel containing polyvinylpyrrolidone and sodium hyaluronate. It forms an adherent barrier and layer covering oral mucosa lesions, thus protecting sensitive nerve endings and lubricating the tissue. Several studies have suggested that it can help to manage pain and potentially can improve the ability to eat and drink in patients with oral mucositis (OM) after intensive chemotherapy or radiotherapy [1–5].\nOM is a significant medical and nursing problem in high-dose chemotherapy and stem cells transplantation settings. This oral mucosa damage and injury is associated with pain and reduced oral intake. Since infections may also play a role in the pathophysiology of OM, antimicrobial mouthwashes such as chlorhexidine, benzydamine, povidone-iodine, and various local antibiotics/antimycotics are widely used in the nursing care and management of OM. However, there is little evidence supporting the use of these agents in the prophylaxis and treatment of this complication [6,7]. On the other hand, they are important in the treatment of oral bacterial and fungal infections [8,9]. Oral cavity cooling (cryotherapy) provided during high-dose melphalan bolus or short infusion administration contributes to local lower blood circulation and cytotoxic drug exposure and can significantly reduce OM in melphalan-containing protocols [10,11]. Effective analgesic therapy is necessary in patients suffering from this painful complication. Patient-controlled analgesia with morphine is a treatment of choice according to MASCC (Multinational Association of Supportive Care in Cancer), NCCN (National Comprehensive Cancer Network) and ESMO (European Society for Medical Oncology) recommendations [12–16].\nOur observational study aimed to evaluate safety of Gelclair use with respect to the extent of bacterial and yeast colonization within the oral cavity, and to verify its clinical efficacy and tolerance in patients after allogeneic stem cells transplantation.",
"We conducted a single-centre prospective and observational study in adult patients with oral mucositis developed after allogeneic hematopoietic stem cells transplantation (HSCT) in 2008–2009. The HSCT conditioning regimens were BuCY2 (busulphan total dose 16 mg/kg, cyclophosphamide total dose 120 mg/kg) or FLU/MEL (fludarabine total dose 120 mg/m2, melphalan 140 mg/m2). Patients were given the standard systemic antimycotic, antibacterial and antiviral prophylaxis (fluconazole, chinolons, acyclovir).\nThe patients signed informed consent. As Gelclair had already been implemented in the standard nursing care for several months at our institution, no Ethics Committee approval was necessary for this study. The study was non-sponsored.\nThe oral cavity nursing started on the first day of the conditioning chemotherapy administration and covered the whole inpatient stay. Prior to OM development, the patients were allowed to use chlorhexidine, benzydamine or salvia solutions for regular oral rinses. On the day of first signs of OM development (WHO criteria), the first 22 patients were consecutively assigned to the Gelclair treatment (Gelclair group) and afterwards the other 15 consecutive patients were assigned to carry on using the original oral rinses (Control group). After the patients recovered from OM (post-OM phase), all enrolled patients either carried on (Control group) or returned back (Gelclair group) to the standard oral care with benzydamine, chlorhexidine or salvia solutions. The oral rinses were recommended to be used at least 3 times a day and the Gelclair was used in concordance with the product brochure (Gelclair package insert, Helsinn Healthcare SA, Switzerland, 2006) and specific web pages instructions at www.gelclair.com.\n The monitoring, assessment and definitions OM was assessed daily using the WHO grading 0–4 (0 = absent; 1 = pain and erythema; 2 = ulcers, patient can swallow solid food; 3 = ulcers, patient cannot swallow solid food; 4 = mucositis to the extent that alimentation is not possible). The tolerability of oral rinses was evaluated daily by the patients, using the Visual Analog Scale (VAS) scoring: 1–5 (1 = tolerable without any problems, 2 = satisfactory, 3 = indifferent, 4 = unsatisfactory, 5 = intolerable). The OM pain reduction and food intake improvement were assessed daily by the patients using the VAS scoring: 1–5 (1 = excellent, 2 = good, 3 = slight effect, 4 = almost no effect, 5 = no effect at all).\nTests with oral cavity swabs for microbiology analysis were performed once a week in the morning — on the admission to the transplantation unit (pre-OM phase), during active OM (OM-phase) and after the OM resolution (post-OM phase). The smears comprised buccal, palatal and sublingual mucosa.\nIn vitro Gelclair inhibition test was performed using the Enterococcus faecalis, Candida albicans, Candida parapsilosis and Candida krusei strains suspensions inoculated separately onto Müller-Hinton agar and 3 drops of Gelclair were added on the inoculated area. The plate samples were incubated in stable temperature 37°C for 24 hour (48 hours in Candida krusei sample). All the microbiology samples were processed and cultivated under controlled laboratory conditions in the institutional Department of Microbiology.\nColonies of generally physiological oral bacteria (Streptococcus viridans or Neisseria spec. or Staphylococcus coagulase-negative) were considered potentially pathogenic because of the significant immunodeficiency of transplanted patients.\nOM was assessed daily using the WHO grading 0–4 (0 = absent; 1 = pain and erythema; 2 = ulcers, patient can swallow solid food; 3 = ulcers, patient cannot swallow solid food; 4 = mucositis to the extent that alimentation is not possible). The tolerability of oral rinses was evaluated daily by the patients, using the Visual Analog Scale (VAS) scoring: 1–5 (1 = tolerable without any problems, 2 = satisfactory, 3 = indifferent, 4 = unsatisfactory, 5 = intolerable). The OM pain reduction and food intake improvement were assessed daily by the patients using the VAS scoring: 1–5 (1 = excellent, 2 = good, 3 = slight effect, 4 = almost no effect, 5 = no effect at all).\nTests with oral cavity swabs for microbiology analysis were performed once a week in the morning — on the admission to the transplantation unit (pre-OM phase), during active OM (OM-phase) and after the OM resolution (post-OM phase). The smears comprised buccal, palatal and sublingual mucosa.\nIn vitro Gelclair inhibition test was performed using the Enterococcus faecalis, Candida albicans, Candida parapsilosis and Candida krusei strains suspensions inoculated separately onto Müller-Hinton agar and 3 drops of Gelclair were added on the inoculated area. The plate samples were incubated in stable temperature 37°C for 24 hour (48 hours in Candida krusei sample). All the microbiology samples were processed and cultivated under controlled laboratory conditions in the institutional Department of Microbiology.\nColonies of generally physiological oral bacteria (Streptococcus viridans or Neisseria spec. or Staphylococcus coagulase-negative) were considered potentially pathogenic because of the significant immunodeficiency of transplanted patients.\n Statistics Basic statistical univariate analyses were performed using statistical software (GraphPad InStat, GraphPad Software) with the Fisher’s exact test and Unpaired T test. The “p” values <0.05 were considered as statistically significant differences.\nBasic statistical univariate analyses were performed using statistical software (GraphPad InStat, GraphPad Software) with the Fisher’s exact test and Unpaired T test. The “p” values <0.05 were considered as statistically significant differences.",
"OM was assessed daily using the WHO grading 0–4 (0 = absent; 1 = pain and erythema; 2 = ulcers, patient can swallow solid food; 3 = ulcers, patient cannot swallow solid food; 4 = mucositis to the extent that alimentation is not possible). The tolerability of oral rinses was evaluated daily by the patients, using the Visual Analog Scale (VAS) scoring: 1–5 (1 = tolerable without any problems, 2 = satisfactory, 3 = indifferent, 4 = unsatisfactory, 5 = intolerable). The OM pain reduction and food intake improvement were assessed daily by the patients using the VAS scoring: 1–5 (1 = excellent, 2 = good, 3 = slight effect, 4 = almost no effect, 5 = no effect at all).\nTests with oral cavity swabs for microbiology analysis were performed once a week in the morning — on the admission to the transplantation unit (pre-OM phase), during active OM (OM-phase) and after the OM resolution (post-OM phase). The smears comprised buccal, palatal and sublingual mucosa.\nIn vitro Gelclair inhibition test was performed using the Enterococcus faecalis, Candida albicans, Candida parapsilosis and Candida krusei strains suspensions inoculated separately onto Müller-Hinton agar and 3 drops of Gelclair were added on the inoculated area. The plate samples were incubated in stable temperature 37°C for 24 hour (48 hours in Candida krusei sample). All the microbiology samples were processed and cultivated under controlled laboratory conditions in the institutional Department of Microbiology.\nColonies of generally physiological oral bacteria (Streptococcus viridans or Neisseria spec. or Staphylococcus coagulase-negative) were considered potentially pathogenic because of the significant immunodeficiency of transplanted patients.",
"Basic statistical univariate analyses were performed using statistical software (GraphPad InStat, GraphPad Software) with the Fisher’s exact test and Unpaired T test. The “p” values <0.05 were considered as statistically significant differences.",
"A total of 22 patients were enrolled into OM treatment with Gelclair and 15 patients into the Control group using standard oral solutions with chlorhexidine (8/15, 53%), benzydamine (6/15, 40%) or salvia (1/15, 7%). Characteristics of the groups are shown in Table 1.\nThere was no difference in the median value of tolerability of the rinses in the Gelclair vs. Control group: 2 (1–5) vs. 2 (1–3), p=0.304. The individual tolerance in the Gelclair group in details was: 1 – tolerable without any problems in 9%, 2 – satisfactory in 73%, 3 – indifferent in 9%, 4 – unsatisfactory in 4.5% and 5 – intolerable in 4.5% patients. The individual tolerance in the Control group was: 1 – tolerable without any problems in 33%, 2 – satisfactory in 40% and 3 – indifferent in 27% patients.\nRegarding the improvement of oral intake and OM pain relief after oral rinsing, there was no difference in the median value of improvement intensity observed between the Gelclair and the Control groups: 3 (2–4) vs. 3 (2–4) and 3 (2–5) vs. 3 (1–4) VAS score, p=0.381 and 0.190 (3 = slight effect). The median value of duration of pain relief was significantly longer in the Gelclair group: 3 (0–5) vs. 1 (0–3) hours, p=0.001. Significantly more patients on systemic analgesic opioid treatment were in the Gelclair group: 15/22 (68%) vs. 7/15 (46%), p=0.03. The analgesic medication administered in the Gelclair group was: buprenorphine 35 ug/hour transdermal patch in 13/22 (59%), buprenorphine 52.5 ug/hour transdermal patch in 1/22 (4.5%) and tramadol 4.1 mg/hour I.V. in 1/22 (4.5%) patients. The analgesic treatment administered in the Control group was: buprenorphine 35 ug/hour transdermal patch in 5/15 (33%), buprenorphine 52.5 ug/hour transdermal patch in 2/15 (13%) patients.\n Oral microbiology swabs In the whole group of 37 patients, bacterial or yeast pathogens were detected in 6 (16%) patients in oral cavity swabs during the pre-OM phase, and in 18 (49%) in the post-OM phase (p=0.0024). During the OM phase, significantly fewer pathogens were found in the Gelclair compared to the Control group: 1/22 (5%) vs. 6/15 (40%), p=0.01. After the OM resolution and Gelclair use termination (the post-OM phase), there was a significant increase of pathogen colonization observed in the Gelclair group in comparison with the previous condition in this group: 12/22 (55%) vs. 1/22 (5%), p=0.0006. Negative microbial swab results were obtained in the Gelclair group only during the OM phase and in 2 patients later on. No difference was observed in patients using either chlorhexidine or benzydamine solutions. For more details see Table 2.\nIn the whole group of 37 patients, bacterial or yeast pathogens were detected in 6 (16%) patients in oral cavity swabs during the pre-OM phase, and in 18 (49%) in the post-OM phase (p=0.0024). During the OM phase, significantly fewer pathogens were found in the Gelclair compared to the Control group: 1/22 (5%) vs. 6/15 (40%), p=0.01. After the OM resolution and Gelclair use termination (the post-OM phase), there was a significant increase of pathogen colonization observed in the Gelclair group in comparison with the previous condition in this group: 12/22 (55%) vs. 1/22 (5%), p=0.0006. Negative microbial swab results were obtained in the Gelclair group only during the OM phase and in 2 patients later on. No difference was observed in patients using either chlorhexidine or benzydamine solutions. For more details see Table 2.\n In vitro Gelclair inhibition test There was evident growth inhibition of the Enterococcus faecalis and Candida colonies within the area of Gelclair application (Figures 1 and 2).\nThere was evident growth inhibition of the Enterococcus faecalis and Candida colonies within the area of Gelclair application (Figures 1 and 2).",
"In the whole group of 37 patients, bacterial or yeast pathogens were detected in 6 (16%) patients in oral cavity swabs during the pre-OM phase, and in 18 (49%) in the post-OM phase (p=0.0024). During the OM phase, significantly fewer pathogens were found in the Gelclair compared to the Control group: 1/22 (5%) vs. 6/15 (40%), p=0.01. After the OM resolution and Gelclair use termination (the post-OM phase), there was a significant increase of pathogen colonization observed in the Gelclair group in comparison with the previous condition in this group: 12/22 (55%) vs. 1/22 (5%), p=0.0006. Negative microbial swab results were obtained in the Gelclair group only during the OM phase and in 2 patients later on. No difference was observed in patients using either chlorhexidine or benzydamine solutions. For more details see Table 2.",
"There was evident growth inhibition of the Enterococcus faecalis and Candida colonies within the area of Gelclair application (Figures 1 and 2).",
"Gelclair oral gel containing polyvinylpyrrolidone and sodium hyaluronate has been used in the management of oral mucositis, stomatitis and various ulcerative oral conditions with some positive results, suggesting that it can help to reduce pain and improve the ability to eat and drink [2–4]. In a small group of patients with radiotherapy-induced OM, there was also short-term pain relief observed initially after the use of Gelclair; however, there was no improvement in capacity of oral intake [1]. To date there has been limited experience with the use of Gelclair in allogeneic stem cells transplantation patients and there is no available information about Gelclair’s impact on oral cavity microbial colonization. Based on the fact mentioned above, we decided to conduct this study.\nGelclair was administered in the group of 22 patients in order to treat an active oral mucositis since the first symptoms appeared until OM was resolved. Fifteen other patients were in the control group and carried on with the standard oral care with solutions containing chlorhexidine, benzydamine or salvia. As our patients were allowed to freely select one of the 3 solutions (according to their individual preference and taste), there was in general a good tolerability of these solutions and none of control group patients considered them as “unsatisfactory” or “intolerable”, which happened in the Gelclair group, where one of the patients even refused to carry on using the gel due to individual intolerance. The tolerability of Gelclair, however, was in general rather good in the majority of cases. Thus, the results of oral rinse tolerability must be considered in a larger context.\nAs for the OM pain relief and oral intake improvement, those issues were assessed as “light” in both study groups, with somewhat longer duration in the Gelclair group (median duration of 3 (0–5) vs. 1 (0–3) hours, p=0.001). It is necessary to mention the possible impact of systemic analgesic medication administered in order to reduce the intensity OM pain in the majority of patients. However, to get the most reliable data possible, the patients were individually and specifically asked to focus only on actual local analgesic effect of the rinses applied.\nThe characteristics of OM in both study groups were comparable. The median value for duration of the complication in both study groups was statistically comparable, as well as the median of OM maximal intensity. It is impossible to objectively compare differences of OM in patients after the FLU/MEL or BuCY2 conditioning regimens because of the small numbers of patients and selection bias – patients without OM were not enrolled into the study.\nAlthough there were differences in age and diagnoses variety between the study groups, in our opinion this had no significant impact on the study results.\nVery interesting and somewhat surprising were the results of microbial tests with oral cavity swabs, as there has not been any information yet on antimicrobial effect of Gelclair available, and Gelclair, in fact, does not contain any specific antimicrobial agent. Firstly, we observed a significant increase of pathogens colonization (predominantly Enterococcus faecalis and Candida sp.) of the oral cavity over the course of the hospitalization. Secondly, there was a significantly reduced incidence of such pathogen colonization, and even microbial negativity, in patients with active OM (OM-phase) in the Gelclair group. The difference observed was remarkable and significant compared to the Control group colonization, and compared to the post-OM phase within the group of patients with original Gelclair treatment (that means significant increase of pathogens in the post-OM phase when Gelclair use was discontinued). Based on these clinically positive results, we decided to test the possible inhibitory effect of Gelclair in vitro. The tests performed on Müller-Hinton agar showed inhibited growth of Enterococcus faecalis and Candida sp. colonies within the area of Gelclair application. These results, however, should be considered only as informative, because the methodology used for the testing was not based on a standardized protocol and had no specific certification or validation. We did not use latex agglutination assay for detection of circulating Candida antigen [17].",
"The results of this observation suggest that Gelclair may be individually helpful in the management of OM and pains in some patients after allogeneic stem cells transplantation. The use of Gelclair did not lead to worsened local bacterial and yeast colonization in the oral cavity and probably even helped to protect mucosa from Enterococcus and Candida colonization. Gelclair use appears to be safe in patients after allogeneic stem cells transplantation. Further observations and analysis based on larger cohorts of patients are needed."
] |
[
null,
"materials|methods",
null,
null,
"results",
null,
null,
"discussion",
"conclusions"
] |
[
"Gelclair",
"oral mucositis",
"pain",
"nursing",
"\nEnterococcus\n",
"\nCandida\n",
"microbiology"
] |
Background:
Gelclair (Helsinn Healthcare SA, Switzerland) is a concentrated oral gel containing polyvinylpyrrolidone and sodium hyaluronate. It forms an adherent barrier and layer covering oral mucosa lesions, thus protecting sensitive nerve endings and lubricating the tissue. Several studies have suggested that it can help to manage pain and potentially can improve the ability to eat and drink in patients with oral mucositis (OM) after intensive chemotherapy or radiotherapy [1–5].
OM is a significant medical and nursing problem in high-dose chemotherapy and stem cells transplantation settings. This oral mucosa damage and injury is associated with pain and reduced oral intake. Since infections may also play a role in the pathophysiology of OM, antimicrobial mouthwashes such as chlorhexidine, benzydamine, povidone-iodine, and various local antibiotics/antimycotics are widely used in the nursing care and management of OM. However, there is little evidence supporting the use of these agents in the prophylaxis and treatment of this complication [6,7]. On the other hand, they are important in the treatment of oral bacterial and fungal infections [8,9]. Oral cavity cooling (cryotherapy) provided during high-dose melphalan bolus or short infusion administration contributes to local lower blood circulation and cytotoxic drug exposure and can significantly reduce OM in melphalan-containing protocols [10,11]. Effective analgesic therapy is necessary in patients suffering from this painful complication. Patient-controlled analgesia with morphine is a treatment of choice according to MASCC (Multinational Association of Supportive Care in Cancer), NCCN (National Comprehensive Cancer Network) and ESMO (European Society for Medical Oncology) recommendations [12–16].
Our observational study aimed to evaluate safety of Gelclair use with respect to the extent of bacterial and yeast colonization within the oral cavity, and to verify its clinical efficacy and tolerance in patients after allogeneic stem cells transplantation.
Material and Methods:
We conducted a single-centre prospective and observational study in adult patients with oral mucositis developed after allogeneic hematopoietic stem cells transplantation (HSCT) in 2008–2009. The HSCT conditioning regimens were BuCY2 (busulphan total dose 16 mg/kg, cyclophosphamide total dose 120 mg/kg) or FLU/MEL (fludarabine total dose 120 mg/m2, melphalan 140 mg/m2). Patients were given the standard systemic antimycotic, antibacterial and antiviral prophylaxis (fluconazole, chinolons, acyclovir).
The patients signed informed consent. As Gelclair had already been implemented in the standard nursing care for several months at our institution, no Ethics Committee approval was necessary for this study. The study was non-sponsored.
The oral cavity nursing started on the first day of the conditioning chemotherapy administration and covered the whole inpatient stay. Prior to OM development, the patients were allowed to use chlorhexidine, benzydamine or salvia solutions for regular oral rinses. On the day of first signs of OM development (WHO criteria), the first 22 patients were consecutively assigned to the Gelclair treatment (Gelclair group) and afterwards the other 15 consecutive patients were assigned to carry on using the original oral rinses (Control group). After the patients recovered from OM (post-OM phase), all enrolled patients either carried on (Control group) or returned back (Gelclair group) to the standard oral care with benzydamine, chlorhexidine or salvia solutions. The oral rinses were recommended to be used at least 3 times a day and the Gelclair was used in concordance with the product brochure (Gelclair package insert, Helsinn Healthcare SA, Switzerland, 2006) and specific web pages instructions at www.gelclair.com.
The monitoring, assessment and definitions OM was assessed daily using the WHO grading 0–4 (0 = absent; 1 = pain and erythema; 2 = ulcers, patient can swallow solid food; 3 = ulcers, patient cannot swallow solid food; 4 = mucositis to the extent that alimentation is not possible). The tolerability of oral rinses was evaluated daily by the patients, using the Visual Analog Scale (VAS) scoring: 1–5 (1 = tolerable without any problems, 2 = satisfactory, 3 = indifferent, 4 = unsatisfactory, 5 = intolerable). The OM pain reduction and food intake improvement were assessed daily by the patients using the VAS scoring: 1–5 (1 = excellent, 2 = good, 3 = slight effect, 4 = almost no effect, 5 = no effect at all).
Tests with oral cavity swabs for microbiology analysis were performed once a week in the morning — on the admission to the transplantation unit (pre-OM phase), during active OM (OM-phase) and after the OM resolution (post-OM phase). The smears comprised buccal, palatal and sublingual mucosa.
In vitro Gelclair inhibition test was performed using the Enterococcus faecalis, Candida albicans, Candida parapsilosis and Candida krusei strains suspensions inoculated separately onto Müller-Hinton agar and 3 drops of Gelclair were added on the inoculated area. The plate samples were incubated in stable temperature 37°C for 24 hour (48 hours in Candida krusei sample). All the microbiology samples were processed and cultivated under controlled laboratory conditions in the institutional Department of Microbiology.
Colonies of generally physiological oral bacteria (Streptococcus viridans or Neisseria spec. or Staphylococcus coagulase-negative) were considered potentially pathogenic because of the significant immunodeficiency of transplanted patients.
OM was assessed daily using the WHO grading 0–4 (0 = absent; 1 = pain and erythema; 2 = ulcers, patient can swallow solid food; 3 = ulcers, patient cannot swallow solid food; 4 = mucositis to the extent that alimentation is not possible). The tolerability of oral rinses was evaluated daily by the patients, using the Visual Analog Scale (VAS) scoring: 1–5 (1 = tolerable without any problems, 2 = satisfactory, 3 = indifferent, 4 = unsatisfactory, 5 = intolerable). The OM pain reduction and food intake improvement were assessed daily by the patients using the VAS scoring: 1–5 (1 = excellent, 2 = good, 3 = slight effect, 4 = almost no effect, 5 = no effect at all).
Tests with oral cavity swabs for microbiology analysis were performed once a week in the morning — on the admission to the transplantation unit (pre-OM phase), during active OM (OM-phase) and after the OM resolution (post-OM phase). The smears comprised buccal, palatal and sublingual mucosa.
In vitro Gelclair inhibition test was performed using the Enterococcus faecalis, Candida albicans, Candida parapsilosis and Candida krusei strains suspensions inoculated separately onto Müller-Hinton agar and 3 drops of Gelclair were added on the inoculated area. The plate samples were incubated in stable temperature 37°C for 24 hour (48 hours in Candida krusei sample). All the microbiology samples were processed and cultivated under controlled laboratory conditions in the institutional Department of Microbiology.
Colonies of generally physiological oral bacteria (Streptococcus viridans or Neisseria spec. or Staphylococcus coagulase-negative) were considered potentially pathogenic because of the significant immunodeficiency of transplanted patients.
Statistics Basic statistical univariate analyses were performed using statistical software (GraphPad InStat, GraphPad Software) with the Fisher’s exact test and Unpaired T test. The “p” values <0.05 were considered as statistically significant differences.
Basic statistical univariate analyses were performed using statistical software (GraphPad InStat, GraphPad Software) with the Fisher’s exact test and Unpaired T test. The “p” values <0.05 were considered as statistically significant differences.
The monitoring, assessment and definitions:
OM was assessed daily using the WHO grading 0–4 (0 = absent; 1 = pain and erythema; 2 = ulcers, patient can swallow solid food; 3 = ulcers, patient cannot swallow solid food; 4 = mucositis to the extent that alimentation is not possible). The tolerability of oral rinses was evaluated daily by the patients, using the Visual Analog Scale (VAS) scoring: 1–5 (1 = tolerable without any problems, 2 = satisfactory, 3 = indifferent, 4 = unsatisfactory, 5 = intolerable). The OM pain reduction and food intake improvement were assessed daily by the patients using the VAS scoring: 1–5 (1 = excellent, 2 = good, 3 = slight effect, 4 = almost no effect, 5 = no effect at all).
Tests with oral cavity swabs for microbiology analysis were performed once a week in the morning — on the admission to the transplantation unit (pre-OM phase), during active OM (OM-phase) and after the OM resolution (post-OM phase). The smears comprised buccal, palatal and sublingual mucosa.
In vitro Gelclair inhibition test was performed using the Enterococcus faecalis, Candida albicans, Candida parapsilosis and Candida krusei strains suspensions inoculated separately onto Müller-Hinton agar and 3 drops of Gelclair were added on the inoculated area. The plate samples were incubated in stable temperature 37°C for 24 hour (48 hours in Candida krusei sample). All the microbiology samples were processed and cultivated under controlled laboratory conditions in the institutional Department of Microbiology.
Colonies of generally physiological oral bacteria (Streptococcus viridans or Neisseria spec. or Staphylococcus coagulase-negative) were considered potentially pathogenic because of the significant immunodeficiency of transplanted patients.
Statistics:
Basic statistical univariate analyses were performed using statistical software (GraphPad InStat, GraphPad Software) with the Fisher’s exact test and Unpaired T test. The “p” values <0.05 were considered as statistically significant differences.
Results:
A total of 22 patients were enrolled into OM treatment with Gelclair and 15 patients into the Control group using standard oral solutions with chlorhexidine (8/15, 53%), benzydamine (6/15, 40%) or salvia (1/15, 7%). Characteristics of the groups are shown in Table 1.
There was no difference in the median value of tolerability of the rinses in the Gelclair vs. Control group: 2 (1–5) vs. 2 (1–3), p=0.304. The individual tolerance in the Gelclair group in details was: 1 – tolerable without any problems in 9%, 2 – satisfactory in 73%, 3 – indifferent in 9%, 4 – unsatisfactory in 4.5% and 5 – intolerable in 4.5% patients. The individual tolerance in the Control group was: 1 – tolerable without any problems in 33%, 2 – satisfactory in 40% and 3 – indifferent in 27% patients.
Regarding the improvement of oral intake and OM pain relief after oral rinsing, there was no difference in the median value of improvement intensity observed between the Gelclair and the Control groups: 3 (2–4) vs. 3 (2–4) and 3 (2–5) vs. 3 (1–4) VAS score, p=0.381 and 0.190 (3 = slight effect). The median value of duration of pain relief was significantly longer in the Gelclair group: 3 (0–5) vs. 1 (0–3) hours, p=0.001. Significantly more patients on systemic analgesic opioid treatment were in the Gelclair group: 15/22 (68%) vs. 7/15 (46%), p=0.03. The analgesic medication administered in the Gelclair group was: buprenorphine 35 ug/hour transdermal patch in 13/22 (59%), buprenorphine 52.5 ug/hour transdermal patch in 1/22 (4.5%) and tramadol 4.1 mg/hour I.V. in 1/22 (4.5%) patients. The analgesic treatment administered in the Control group was: buprenorphine 35 ug/hour transdermal patch in 5/15 (33%), buprenorphine 52.5 ug/hour transdermal patch in 2/15 (13%) patients.
Oral microbiology swabs In the whole group of 37 patients, bacterial or yeast pathogens were detected in 6 (16%) patients in oral cavity swabs during the pre-OM phase, and in 18 (49%) in the post-OM phase (p=0.0024). During the OM phase, significantly fewer pathogens were found in the Gelclair compared to the Control group: 1/22 (5%) vs. 6/15 (40%), p=0.01. After the OM resolution and Gelclair use termination (the post-OM phase), there was a significant increase of pathogen colonization observed in the Gelclair group in comparison with the previous condition in this group: 12/22 (55%) vs. 1/22 (5%), p=0.0006. Negative microbial swab results were obtained in the Gelclair group only during the OM phase and in 2 patients later on. No difference was observed in patients using either chlorhexidine or benzydamine solutions. For more details see Table 2.
In the whole group of 37 patients, bacterial or yeast pathogens were detected in 6 (16%) patients in oral cavity swabs during the pre-OM phase, and in 18 (49%) in the post-OM phase (p=0.0024). During the OM phase, significantly fewer pathogens were found in the Gelclair compared to the Control group: 1/22 (5%) vs. 6/15 (40%), p=0.01. After the OM resolution and Gelclair use termination (the post-OM phase), there was a significant increase of pathogen colonization observed in the Gelclair group in comparison with the previous condition in this group: 12/22 (55%) vs. 1/22 (5%), p=0.0006. Negative microbial swab results were obtained in the Gelclair group only during the OM phase and in 2 patients later on. No difference was observed in patients using either chlorhexidine or benzydamine solutions. For more details see Table 2.
In vitro Gelclair inhibition test There was evident growth inhibition of the Enterococcus faecalis and Candida colonies within the area of Gelclair application (Figures 1 and 2).
There was evident growth inhibition of the Enterococcus faecalis and Candida colonies within the area of Gelclair application (Figures 1 and 2).
Oral microbiology swabs:
In the whole group of 37 patients, bacterial or yeast pathogens were detected in 6 (16%) patients in oral cavity swabs during the pre-OM phase, and in 18 (49%) in the post-OM phase (p=0.0024). During the OM phase, significantly fewer pathogens were found in the Gelclair compared to the Control group: 1/22 (5%) vs. 6/15 (40%), p=0.01. After the OM resolution and Gelclair use termination (the post-OM phase), there was a significant increase of pathogen colonization observed in the Gelclair group in comparison with the previous condition in this group: 12/22 (55%) vs. 1/22 (5%), p=0.0006. Negative microbial swab results were obtained in the Gelclair group only during the OM phase and in 2 patients later on. No difference was observed in patients using either chlorhexidine or benzydamine solutions. For more details see Table 2.
In vitro Gelclair inhibition test:
There was evident growth inhibition of the Enterococcus faecalis and Candida colonies within the area of Gelclair application (Figures 1 and 2).
Discussion:
Gelclair oral gel containing polyvinylpyrrolidone and sodium hyaluronate has been used in the management of oral mucositis, stomatitis and various ulcerative oral conditions with some positive results, suggesting that it can help to reduce pain and improve the ability to eat and drink [2–4]. In a small group of patients with radiotherapy-induced OM, there was also short-term pain relief observed initially after the use of Gelclair; however, there was no improvement in capacity of oral intake [1]. To date there has been limited experience with the use of Gelclair in allogeneic stem cells transplantation patients and there is no available information about Gelclair’s impact on oral cavity microbial colonization. Based on the fact mentioned above, we decided to conduct this study.
Gelclair was administered in the group of 22 patients in order to treat an active oral mucositis since the first symptoms appeared until OM was resolved. Fifteen other patients were in the control group and carried on with the standard oral care with solutions containing chlorhexidine, benzydamine or salvia. As our patients were allowed to freely select one of the 3 solutions (according to their individual preference and taste), there was in general a good tolerability of these solutions and none of control group patients considered them as “unsatisfactory” or “intolerable”, which happened in the Gelclair group, where one of the patients even refused to carry on using the gel due to individual intolerance. The tolerability of Gelclair, however, was in general rather good in the majority of cases. Thus, the results of oral rinse tolerability must be considered in a larger context.
As for the OM pain relief and oral intake improvement, those issues were assessed as “light” in both study groups, with somewhat longer duration in the Gelclair group (median duration of 3 (0–5) vs. 1 (0–3) hours, p=0.001). It is necessary to mention the possible impact of systemic analgesic medication administered in order to reduce the intensity OM pain in the majority of patients. However, to get the most reliable data possible, the patients were individually and specifically asked to focus only on actual local analgesic effect of the rinses applied.
The characteristics of OM in both study groups were comparable. The median value for duration of the complication in both study groups was statistically comparable, as well as the median of OM maximal intensity. It is impossible to objectively compare differences of OM in patients after the FLU/MEL or BuCY2 conditioning regimens because of the small numbers of patients and selection bias – patients without OM were not enrolled into the study.
Although there were differences in age and diagnoses variety between the study groups, in our opinion this had no significant impact on the study results.
Very interesting and somewhat surprising were the results of microbial tests with oral cavity swabs, as there has not been any information yet on antimicrobial effect of Gelclair available, and Gelclair, in fact, does not contain any specific antimicrobial agent. Firstly, we observed a significant increase of pathogens colonization (predominantly Enterococcus faecalis and Candida sp.) of the oral cavity over the course of the hospitalization. Secondly, there was a significantly reduced incidence of such pathogen colonization, and even microbial negativity, in patients with active OM (OM-phase) in the Gelclair group. The difference observed was remarkable and significant compared to the Control group colonization, and compared to the post-OM phase within the group of patients with original Gelclair treatment (that means significant increase of pathogens in the post-OM phase when Gelclair use was discontinued). Based on these clinically positive results, we decided to test the possible inhibitory effect of Gelclair in vitro. The tests performed on Müller-Hinton agar showed inhibited growth of Enterococcus faecalis and Candida sp. colonies within the area of Gelclair application. These results, however, should be considered only as informative, because the methodology used for the testing was not based on a standardized protocol and had no specific certification or validation. We did not use latex agglutination assay for detection of circulating Candida antigen [17].
Conclusions:
The results of this observation suggest that Gelclair may be individually helpful in the management of OM and pains in some patients after allogeneic stem cells transplantation. The use of Gelclair did not lead to worsened local bacterial and yeast colonization in the oral cavity and probably even helped to protect mucosa from Enterococcus and Candida colonization. Gelclair use appears to be safe in patients after allogeneic stem cells transplantation. Further observations and analysis based on larger cohorts of patients are needed.
|
Background:
Gelclair is an oral lubricating gel used in the management of oral mucositis (OM). We evaluated its efficacy, tolerance and impact on oral cavity microbial colonization in patients with OM after allogeneic hematopoietic stem cells transplantation.
Methods:
Gelclair was administered in a group of 22 patients with active OM. A control group of 15 patients used other rinsing solutions (chlorhexidine, benzydamine, salvia). Tests with oral cavity swabs for microbiology analysis were performed once a week.
Results:
The characteristics of OM in both groups were comparable, and rinsing solutions had satisfactory tolerability. There was no difference in the median improvement of oral intake and OM-related pain relief, which was assessed mostly as "slight effect". In the Gelclair group, the effect duration was longer (median 3 [0-5] vs. 1 [0-3] hours, p = 0.001). There was significant increase of Enterococcus faecalis and Candida sp. colonization of the oral cavity over the course of the hospitalization and significantly reduced incidence of such colonization in patients with OM in the Gelclair group: 1/22 (5%) vs. 6/15 (40%), p = 0.01. In vitro tests showed inhibited growth of Enterococcus faecalis and Candida sp. colonies within the area of the Gelclair application.
Conclusions:
Gelclair may be individually helpful in the management of OM and pain in patients after allogeneic stem cells transplantation. Its use did not lead to worsened oral bacterial and yeast colonization and probably even helped to protect mucosa from Enterococcus and Candida sp. Further studies based on larger cohorts are needed.
|
Background:
Gelclair (Helsinn Healthcare SA, Switzerland) is a concentrated oral gel containing polyvinylpyrrolidone and sodium hyaluronate. It forms an adherent barrier and layer covering oral mucosa lesions, thus protecting sensitive nerve endings and lubricating the tissue. Several studies have suggested that it can help to manage pain and potentially can improve the ability to eat and drink in patients with oral mucositis (OM) after intensive chemotherapy or radiotherapy [1–5].
OM is a significant medical and nursing problem in high-dose chemotherapy and stem cells transplantation settings. This oral mucosa damage and injury is associated with pain and reduced oral intake. Since infections may also play a role in the pathophysiology of OM, antimicrobial mouthwashes such as chlorhexidine, benzydamine, povidone-iodine, and various local antibiotics/antimycotics are widely used in the nursing care and management of OM. However, there is little evidence supporting the use of these agents in the prophylaxis and treatment of this complication [6,7]. On the other hand, they are important in the treatment of oral bacterial and fungal infections [8,9]. Oral cavity cooling (cryotherapy) provided during high-dose melphalan bolus or short infusion administration contributes to local lower blood circulation and cytotoxic drug exposure and can significantly reduce OM in melphalan-containing protocols [10,11]. Effective analgesic therapy is necessary in patients suffering from this painful complication. Patient-controlled analgesia with morphine is a treatment of choice according to MASCC (Multinational Association of Supportive Care in Cancer), NCCN (National Comprehensive Cancer Network) and ESMO (European Society for Medical Oncology) recommendations [12–16].
Our observational study aimed to evaluate safety of Gelclair use with respect to the extent of bacterial and yeast colonization within the oral cavity, and to verify its clinical efficacy and tolerance in patients after allogeneic stem cells transplantation.
Conclusions:
The results of this observation suggest that Gelclair may be individually helpful in the management of OM and pains in some patients after allogeneic stem cells transplantation. The use of Gelclair did not lead to worsened local bacterial and yeast colonization in the oral cavity and probably even helped to protect mucosa from Enterococcus and Candida colonization. Gelclair use appears to be safe in patients after allogeneic stem cells transplantation. Further observations and analysis based on larger cohorts of patients are needed.
|
Background:
Gelclair is an oral lubricating gel used in the management of oral mucositis (OM). We evaluated its efficacy, tolerance and impact on oral cavity microbial colonization in patients with OM after allogeneic hematopoietic stem cells transplantation.
Methods:
Gelclair was administered in a group of 22 patients with active OM. A control group of 15 patients used other rinsing solutions (chlorhexidine, benzydamine, salvia). Tests with oral cavity swabs for microbiology analysis were performed once a week.
Results:
The characteristics of OM in both groups were comparable, and rinsing solutions had satisfactory tolerability. There was no difference in the median improvement of oral intake and OM-related pain relief, which was assessed mostly as "slight effect". In the Gelclair group, the effect duration was longer (median 3 [0-5] vs. 1 [0-3] hours, p = 0.001). There was significant increase of Enterococcus faecalis and Candida sp. colonization of the oral cavity over the course of the hospitalization and significantly reduced incidence of such colonization in patients with OM in the Gelclair group: 1/22 (5%) vs. 6/15 (40%), p = 0.01. In vitro tests showed inhibited growth of Enterococcus faecalis and Candida sp. colonies within the area of the Gelclair application.
Conclusions:
Gelclair may be individually helpful in the management of OM and pain in patients after allogeneic stem cells transplantation. Its use did not lead to worsened oral bacterial and yeast colonization and probably even helped to protect mucosa from Enterococcus and Candida sp. Further studies based on larger cohorts are needed.
| 3,703 | 308 | 9 |
[
"om",
"gelclair",
"patients",
"oral",
"group",
"phase",
"om phase",
"candida",
"22",
"gelclair group"
] |
[
"test",
"test"
] | null |
[CONTENT] Gelclair | oral mucositis | pain | nursing |
Enterococcus
|
Candida
| microbiology [SUMMARY]
| null |
[CONTENT] Gelclair | oral mucositis | pain | nursing |
Enterococcus
|
Candida
| microbiology [SUMMARY]
|
[CONTENT] Gelclair | oral mucositis | pain | nursing |
Enterococcus
|
Candida
| microbiology [SUMMARY]
|
[CONTENT] Gelclair | oral mucositis | pain | nursing |
Enterococcus
|
Candida
| microbiology [SUMMARY]
|
[CONTENT] Gelclair | oral mucositis | pain | nursing |
Enterococcus
|
Candida
| microbiology [SUMMARY]
|
[CONTENT] Benzydamine | Candida | Chlorhexidine | Drug Combinations | Enterococcus faecalis | Facial Pain | Humans | Hyaluronic Acid | Povidone | Prospective Studies | Stem Cell Transplantation | Stomatitis | Time Factors | Transplantation, Homologous [SUMMARY]
| null |
[CONTENT] Benzydamine | Candida | Chlorhexidine | Drug Combinations | Enterococcus faecalis | Facial Pain | Humans | Hyaluronic Acid | Povidone | Prospective Studies | Stem Cell Transplantation | Stomatitis | Time Factors | Transplantation, Homologous [SUMMARY]
|
[CONTENT] Benzydamine | Candida | Chlorhexidine | Drug Combinations | Enterococcus faecalis | Facial Pain | Humans | Hyaluronic Acid | Povidone | Prospective Studies | Stem Cell Transplantation | Stomatitis | Time Factors | Transplantation, Homologous [SUMMARY]
|
[CONTENT] Benzydamine | Candida | Chlorhexidine | Drug Combinations | Enterococcus faecalis | Facial Pain | Humans | Hyaluronic Acid | Povidone | Prospective Studies | Stem Cell Transplantation | Stomatitis | Time Factors | Transplantation, Homologous [SUMMARY]
|
[CONTENT] Benzydamine | Candida | Chlorhexidine | Drug Combinations | Enterococcus faecalis | Facial Pain | Humans | Hyaluronic Acid | Povidone | Prospective Studies | Stem Cell Transplantation | Stomatitis | Time Factors | Transplantation, Homologous [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] om | gelclair | patients | oral | group | phase | om phase | candida | 22 | gelclair group [SUMMARY]
| null |
[CONTENT] om | gelclair | patients | oral | group | phase | om phase | candida | 22 | gelclair group [SUMMARY]
|
[CONTENT] om | gelclair | patients | oral | group | phase | om phase | candida | 22 | gelclair group [SUMMARY]
|
[CONTENT] om | gelclair | patients | oral | group | phase | om phase | candida | 22 | gelclair group [SUMMARY]
|
[CONTENT] om | gelclair | patients | oral | group | phase | om phase | candida | 22 | gelclair group [SUMMARY]
|
[CONTENT] oral | om | medical | oral mucosa | high dose | high | infections | cancer | treatment | melphalan [SUMMARY]
| null |
[CONTENT] group | gelclair | 15 | vs | 22 | patients | om | phase | om phase | gelclair group [SUMMARY]
|
[CONTENT] patients allogeneic stem | patients allogeneic stem cells | patients allogeneic | allogeneic stem | allogeneic stem cells transplantation | allogeneic stem cells | patients | stem cells transplantation | cells transplantation | stem [SUMMARY]
|
[CONTENT] om | patients | gelclair | group | oral | phase | om phase | candida | 22 | graphpad [SUMMARY]
|
[CONTENT] om | patients | gelclair | group | oral | phase | om phase | candida | 22 | graphpad [SUMMARY]
|
[CONTENT] ||| OM [SUMMARY]
| null |
[CONTENT] ||| ||| Gelclair | 3 ||| 0 | 1 ||| 0-3 | 0.001 ||| Candida sp | Gelclair | 1/22 | 5% | 6/15 | 40% | 0.01 ||| Candida sp | colonies | Gelclair [SUMMARY]
|
[CONTENT] ||| Enterococcus ||| [SUMMARY]
|
[CONTENT] ||| OM ||| 22 ||| 15 ||| ||| ||| ||| Gelclair | 3 ||| 0 | 1 ||| 0-3 | 0.001 ||| Candida sp | Gelclair | 1/22 | 5% | 6/15 | 40% | 0.01 ||| Candida sp | colonies | Gelclair ||| ||| Enterococcus ||| [SUMMARY]
|
[CONTENT] ||| OM ||| 22 ||| 15 ||| ||| ||| ||| Gelclair | 3 ||| 0 | 1 ||| 0-3 | 0.001 ||| Candida sp | Gelclair | 1/22 | 5% | 6/15 | 40% | 0.01 ||| Candida sp | colonies | Gelclair ||| ||| Enterococcus ||| [SUMMARY]
|
|||||
Perorally active nanomicellar formulation of quercetin in the treatment of lung cancer.
|
22334787
|
Realizing the therapeutic benefits of quercetin is mostly hampered by its low water solubility and poor absorption. In light of the advantages of nanovehicles in the delivery of flavanoids, we aimed to deliver quercetin perorally with nanomicelles made from the diblock copolymer, polyethylene glycol (PEG)-derivatized phosphatidylethanolamine (PE).
|
BACKGROUND
|
Quercetin-loaded nanomicelles were prepared by using the film casting method, and were evaluated in terms of drug incorporation efficiency, micelle size, interaction with Caco-2 cells, and anticancer activity in the A549 lung cancer cell line and murine xenograft model.
|
METHODS
|
The incorporation efficiency into the nanomicelles was ≥88.9% when the content of quercetin was up to 4% w/w, with sizes of 15.4-18.5 nm and polydispersity indices of <0.250. Solubilization of quercetin by the nanomicelles increased its aqueous concentration by 110-fold. The quercetin nanomicelles were stable when tested in simulated gastric (pH 1.2) and intestinal (pH 7.4) fluids, and were non-toxic to the Caco-2 cells as reflected by reversible reduction in transepithelial electrical resistance and ≤25% lactose dehydrogenase release. The anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation when tested using the A549 cancer cell line and murine xenograft model. The nanomicellar quercetin formulation was well tolerated by the tumor-bearing animals, with no significant weight loss observed at the end of the 10-week study period.
|
RESULTS
|
A stable PEG-PE nanomicellar formulation of quercetin was developed with enhanced peroral anticancer activity and no apparent toxicity to the intestinal epithelium.
|
CONCLUSION
|
[
"Administration, Oral",
"Analysis of Variance",
"Animals",
"Antineoplastic Agents",
"Antioxidants",
"Caco-2 Cells",
"Cell Line, Tumor",
"Cell Survival",
"Drug Carriers",
"Drug Stability",
"Female",
"Humans",
"Hydrogen-Ion Concentration",
"Lung Neoplasms",
"Mice",
"Micelles",
"Nanoparticles",
"Particle Size",
"Phosphatidylethanolamines",
"Polyethylene Glycols",
"Quercetin",
"Xenograft Model Antitumor Assays"
] |
3278229
|
Introduction
|
Cancer is a public health problem that affects many countries in the world. Globally, lung cancer is the leading cause of cancer death. In the treatment of advanced lung cancer, chemotherapy remains the mainstay; however, only modest increase in survival has been observed at the cost of significant toxicity to patients.1 It is imperative to search for new therapeutics with improved efficacy and safety profiles. In the anticancer drug discovery and development process, compounds with the highest anticancer activities often have bulky hydrophobic groups within their chemical structures, rendering them water-insoluble.2 Low water solubility not only gives rise to formulation difficulties but also serious therapeutic challenges. Administering the poorly soluble drug candidate intravenously might result in serious complications such as embolism and respiratory system failure due to drug precipitation,3 whereas poor absorption would result from extravascular dosing.4
The family of flavonoid compounds is one such example which has been shown to be promising for the wide applicability in the prevention and therapy of cancer, neurodegenerative, and cardiovascular diseases. Yet, the major hurdle in their clinical usage is poor water solubility, thus affecting their peroral biological activity.5 Among this family of agents, quercetin has been studied extensively for its anticancer activity, whereby it could inhibit a number of signal transduction targets important to cancer cell survival, proliferation, and metastasis.6,7 Quercetin progressed to early-stage clinical trials as an anticancer agent more than a decade ago; nevertheless, its administration required the use of solvents such as dimethylsulfoxide or ethanol.8 Chemical modifications have been attempted to improve quercetin solubility but could result in a loss of drug potency.9 Thus, alternative strategies based on the use of nanotechnologies have been undertaken to improve the water solubility and/or the bioavailability of quercetin so as to improve its biological activity.5 While most of the nanovehicles are designed with compositions sufficiently stable for intravenous administration, which is the most conventional route for cancer chemotherapy, orally active cancer chemotherapeutics have emerged as an attractive treatment modality because of patient preference, convenience in administration, and applicability in outpatient setting.10 A few studies have shown promising peroral antioxidant activity of quercetin when it was delivered by nanoscaled delivery systems,11,12 and recently, a lipid-based, submicron (~400 nm) vesicular complex of quercetin has been developed with improved peroral anticancer activity as compared to a peroral quercetin suspension.13
In light of the advantages of peroral anticancer drug delivery and recent developments in the use of nanovehicles for the delivery of flavonoids,5 we have attempted to deliver quercetin perorally through the use of polyethylene glycol (PEG)-derivatized phosphatidylethanolamine (PE) as the diblock copolymer to form a self-assembled nanomicellar delivery system, and have compared the anticancer activity of this nanomicellar formulation with a peroral suspension of quercetin. The research on PEG-PE nanomicellar systems, pioneered by Torchilin and Alkan-Onyuksel,14–16 has contributed greatly to the intravenous delivery of water-insoluble agents. For instance, improved therapeutic efficacy of the intravenously administered PEG-lipid micelle formulation of paclitaxel has been demonstrated in tumor-bearing mice.15,16 The potential of PEG-lipid nanomicelles for the peroral administration of water-insoluble compounds has yet to be demonstrated in animal models. Here, we have shown that the peroral anticancer activity of quercetin could be significantly increased when delivered in PEG-PE nanomicellar systems in the A549 human lung tumor xenograft model.
|
Animal efficacy study
|
The animal study was conducted in the facilities of the British Columbia Cancer Research Center, with methodologies approved by the Institutional Animal Care Committee of the University of British Columbia, Vancouver, BC, Canada. The mice were cared for and used in accordance with the Canadian Council on Animal Care Guidelines. All mice used were between 20–22 g, and were housed in micro-isolator cages and given free access to food and water. Tumors were established in female Rag-2M mice by a single subcutaneous injection of 5 × 106 A549 cells (in 50 μL) in the lower back area. Tumor growth was monitored by caliper measurements along the length and width. Tumor volumes were calculated by the following formula: Tumor volume = 0.5 × (length × width2). When the tumor size reached 50–100 mm3 (3 weeks post-tumor cell inoculation), mice were randomized into three study groups of six animals per group: (1) untreated control, (2) peroral quercetin suspension (vehicle = 25% v/v ethanol in water), and (3) peroral nanomicellar quercetin. The dose of quercetin was 30 mg/kg, following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. Tumor size and body weight of the mice was monitored thrice weekly. Animals whose tumor size reached 1000 mm3, developed ulcerations, or displayed a weight loss of more than 5% were euthanized.
|
Results
|
Formulating quercetin into PEG-lipid nanomicelles The commonly used PEG-lipid conjugate, DSPE-PEG2000, was combined with quercetin in various weight proportions (1%–5% w/w of quercetin in DSPE-PEG2000). As shown in Table 1, the incorporation efficiencies of quercetin into the DSPE-PEG2000 nanomicelles were found to be 88.9% and above when the content of quercetin was up to 4% w/w. At 5% w/w, the incorporation efficiency was reduced to 79.3%. The size of the drug-loaded nanomicelles ranged from 15.4 to 18.5 nm (Table 1), and the polydispersity indices were <0.250 for all the nanomicellar formulations. The zeta potentials of the empty and drug-loaded nanomicelles were −11.8 ± 0.7 mV and −14.8 ± 0.7 mV, respectively. The highest concentration of nanomicellar quercetin in Hepes-buffered saline that could be achieved without precipitation was 3 mg/mL. This represents a 110-fold increase in the aqueous concentration of quercetin, calculated based on the aqueous solubility of free quercetin of 80 μM.17,18 Furthermore, nanomicellar quercetin could be processed into a powder by freeze-drying, without the need of a cryoprotectant or lyoprotectant. Reconstitution of the freeze-dried, nanomicellar quercetin with water did not result in a change in size or quercetin content (data not shown). Hence, subsequent evaluation of the nanomicellar formulation was based on the reconstituted nanomicellar quercetin.
The commonly used PEG-lipid conjugate, DSPE-PEG2000, was combined with quercetin in various weight proportions (1%–5% w/w of quercetin in DSPE-PEG2000). As shown in Table 1, the incorporation efficiencies of quercetin into the DSPE-PEG2000 nanomicelles were found to be 88.9% and above when the content of quercetin was up to 4% w/w. At 5% w/w, the incorporation efficiency was reduced to 79.3%. The size of the drug-loaded nanomicelles ranged from 15.4 to 18.5 nm (Table 1), and the polydispersity indices were <0.250 for all the nanomicellar formulations. The zeta potentials of the empty and drug-loaded nanomicelles were −11.8 ± 0.7 mV and −14.8 ± 0.7 mV, respectively. The highest concentration of nanomicellar quercetin in Hepes-buffered saline that could be achieved without precipitation was 3 mg/mL. This represents a 110-fold increase in the aqueous concentration of quercetin, calculated based on the aqueous solubility of free quercetin of 80 μM.17,18 Furthermore, nanomicellar quercetin could be processed into a powder by freeze-drying, without the need of a cryoprotectant or lyoprotectant. Reconstitution of the freeze-dried, nanomicellar quercetin with water did not result in a change in size or quercetin content (data not shown). Hence, subsequent evaluation of the nanomicellar formulation was based on the reconstituted nanomicellar quercetin.
In vitro cytotoxicity of nanomicellar quercetin in the A549 lung cancer cell line The cytotoxicity of nanomicellar quercetin was evaluated in the A549 human lung cancer cell line. The highest concentration of quercetin evaluated was 100 μM, as drug precipitation was observed at higher drug concentrations. As shown in Figure 1, the anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation, as determined by the MTT viability assay. Of note, non-drug loaded DSPE-PEG2000 nanomicelles did not cause significant toxicity to the A549 lung cancer cells up to a concentration of 1 mM, with cell viability of >85%. To ensure that the cytotoxic effect was not cell line specific, the MDA-MB-231 human breast cancer cell line was treated with free and nanomicellar quercetin. As shown in Suppl Data Figure 1, a similar trend could be observed whereby the activity of quercetin could be significantly increased when formulated into the nanomicelles.
The cytotoxicity of nanomicellar quercetin was evaluated in the A549 human lung cancer cell line. The highest concentration of quercetin evaluated was 100 μM, as drug precipitation was observed at higher drug concentrations. As shown in Figure 1, the anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation, as determined by the MTT viability assay. Of note, non-drug loaded DSPE-PEG2000 nanomicelles did not cause significant toxicity to the A549 lung cancer cells up to a concentration of 1 mM, with cell viability of >85%. To ensure that the cytotoxic effect was not cell line specific, the MDA-MB-231 human breast cancer cell line was treated with free and nanomicellar quercetin. As shown in Suppl Data Figure 1, a similar trend could be observed whereby the activity of quercetin could be significantly increased when formulated into the nanomicelles.
Stability of nanomicellar quercetin in simulated physiological fluids Since the nanomicelles are to be administered perorally, stability in the fluids of the gastrointestinal tract has to be demonstrated. The stability of nanomicellar quercetin was evaluated over time in simulated gastric fluid (SGF, 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2) and simulated intestinal fluid (SIF, 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0). The quercetin nanomicelles were found to be stable, without significant change in size or drug precipitation (Figure 2). To investigate the amount of quercetin retained in the nanomicelles, the micelle samples were dialyzed against 1:5000 (v/v) SGF or SIF at 37°C using dialysis cassettes. Approximately 70%–75% of incorporated quercetin was retained in the nanomicelles in the respective simulated fluids (Figure 2).
Since the nanomicelles are to be administered perorally, stability in the fluids of the gastrointestinal tract has to be demonstrated. The stability of nanomicellar quercetin was evaluated over time in simulated gastric fluid (SGF, 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2) and simulated intestinal fluid (SIF, 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0). The quercetin nanomicelles were found to be stable, without significant change in size or drug precipitation (Figure 2). To investigate the amount of quercetin retained in the nanomicelles, the micelle samples were dialyzed against 1:5000 (v/v) SGF or SIF at 37°C using dialysis cassettes. Approximately 70%–75% of incorporated quercetin was retained in the nanomicelles in the respective simulated fluids (Figure 2).
Interaction of nanomicellar quercetin with Caco-2 cell monolayer The well-established Caco-2 cell monolayer was employed as the in vitro model for gastrointestinal epithelium, and the cellular accumulation of the PEG-lipid nanomicelles was assessed by confocal microscopy. To facilitate visualization, the nanomicelles were spiked with 1 mol% DSPE-PEG2000-N-carboxyfluorescein. The Caco-2 cells were exposed to the fluorescence-labeled nanomicelles for up to 24 hours at 37°C, and cellular accumulation of the nanomicelles increased over time (Figure 3A). Furthermore, the accumulation of the nanomicelles was independent of P-gp activity and endocytic processes, which was supported by results summarized in Suppl Data Figure 2. Cellular accumulation of the carboxyfluorescein-tagged nanomicelles occurred even in the presence of the P-gp inhibitor cyclosporine A or in the presence of endocytic inhibitors (hyperosmotic sucrose of 0.45 M or Brefeldin A) or a change of incubation temperature from 37°C to 4°C. Collectively, these data suggest that the nanomicelles are likely to accumulate inside Caco-2 cells by passive diffusion.
One possible route to facilitate drug absorption through the intestinal epithelium is by disrupting the tight junctions and thus making paracellular transport of drug molecules possible, which could be reflected by a decrease in the TEER.19 Nevertheless, it is also of equal importance that the disruption of the epithelial tight junctions is only transient, as irreversible disruption could represent potential toxicity to the intestinal epithelium. As such, differentiated Caco-2 cell monolayers grown in transwell inserts were exposed to free or nanomicellar quercetin for 24 hours, and TEER measurements were carried out over a time course of 96 hours. As shown in Figure 3B, Caco-2 cell monolayer treated with free quercetin had small yet statistically significant increase in TEER after 48 hours of exposure that gradually decreased back to control level (t = 0 hours). This observation is in line with the protective effect of quercetin on the intestinal barrier function, which acts by promoting the assembly and expression of tight junction proteins.20 In contrast, significant reduction in TEER was observed in Caco-2 cell monolayer treated with nanomicellar quercetin during the first 48 hours. Of note, the reduction in TEER in the nanomicellar quercetin treatment group could be gradually increased back to control level (t = 0 hours), indicating the reversibility of the treatment.
To further investigate the potential toxicity of the nanomicellar quercetin formulation, release of LDH was determined in differentiated Caco-2 cell monolayer upon 24-hour treatment with free or nanomicellar quercetin (Figure 4). Treatment with free quercetin was non-toxic over the concentration range tested, with ~10% LDH released relative to triton-X control. In contrast, nanomicellar quercetin treatment yielded a small but significant increase in LDH release of ~25% relative to triton-X control. Empty, non-drug loaded nanomicelles were evaluated at concentrations up to 1 mM and were found to be relatively non-toxic to the Caco-2 cell monolayer, with viability of ~80% at 1 mM (Suppl Data, Figure 3).
The well-established Caco-2 cell monolayer was employed as the in vitro model for gastrointestinal epithelium, and the cellular accumulation of the PEG-lipid nanomicelles was assessed by confocal microscopy. To facilitate visualization, the nanomicelles were spiked with 1 mol% DSPE-PEG2000-N-carboxyfluorescein. The Caco-2 cells were exposed to the fluorescence-labeled nanomicelles for up to 24 hours at 37°C, and cellular accumulation of the nanomicelles increased over time (Figure 3A). Furthermore, the accumulation of the nanomicelles was independent of P-gp activity and endocytic processes, which was supported by results summarized in Suppl Data Figure 2. Cellular accumulation of the carboxyfluorescein-tagged nanomicelles occurred even in the presence of the P-gp inhibitor cyclosporine A or in the presence of endocytic inhibitors (hyperosmotic sucrose of 0.45 M or Brefeldin A) or a change of incubation temperature from 37°C to 4°C. Collectively, these data suggest that the nanomicelles are likely to accumulate inside Caco-2 cells by passive diffusion.
One possible route to facilitate drug absorption through the intestinal epithelium is by disrupting the tight junctions and thus making paracellular transport of drug molecules possible, which could be reflected by a decrease in the TEER.19 Nevertheless, it is also of equal importance that the disruption of the epithelial tight junctions is only transient, as irreversible disruption could represent potential toxicity to the intestinal epithelium. As such, differentiated Caco-2 cell monolayers grown in transwell inserts were exposed to free or nanomicellar quercetin for 24 hours, and TEER measurements were carried out over a time course of 96 hours. As shown in Figure 3B, Caco-2 cell monolayer treated with free quercetin had small yet statistically significant increase in TEER after 48 hours of exposure that gradually decreased back to control level (t = 0 hours). This observation is in line with the protective effect of quercetin on the intestinal barrier function, which acts by promoting the assembly and expression of tight junction proteins.20 In contrast, significant reduction in TEER was observed in Caco-2 cell monolayer treated with nanomicellar quercetin during the first 48 hours. Of note, the reduction in TEER in the nanomicellar quercetin treatment group could be gradually increased back to control level (t = 0 hours), indicating the reversibility of the treatment.
To further investigate the potential toxicity of the nanomicellar quercetin formulation, release of LDH was determined in differentiated Caco-2 cell monolayer upon 24-hour treatment with free or nanomicellar quercetin (Figure 4). Treatment with free quercetin was non-toxic over the concentration range tested, with ~10% LDH released relative to triton-X control. In contrast, nanomicellar quercetin treatment yielded a small but significant increase in LDH release of ~25% relative to triton-X control. Empty, non-drug loaded nanomicelles were evaluated at concentrations up to 1 mM and were found to be relatively non-toxic to the Caco-2 cell monolayer, with viability of ~80% at 1 mM (Suppl Data, Figure 3).
In vivo efficacy of nanomicellar quercetin in human A549 lung tumor xenograft model Based on the in vitro evaluation of the nanomicellar quercetin formulation which provided promising results, the biological activity of the peroral formulation was further investigated in the A549 lung tumor xenograft model. Quercetin was administered to the tumor-bearing mice via oral gavage either in the nanomicellar form or in a 25% v/v ethanol-based suspension, with a dose of 30 mg/kg following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. The results demonstrated that the anti-tumor activity of quercetin was significantly increased when administered as the peroral nanomicelles compared to control and peroral quercetin suspension (Figure 5A). Specifically, tumor growth inhibition at 10-week post-tumor inoculation was 83% for peroral quercetin suspension and 48% for peroral nanomicellar formulation. The two quercetin formulations were well tolerated by the animals, as reflected by minimal changes in the body weight of the mice (Figure 5B). No unusual observations were made upon necropsies of the animals from all three study groups.
Based on the in vitro evaluation of the nanomicellar quercetin formulation which provided promising results, the biological activity of the peroral formulation was further investigated in the A549 lung tumor xenograft model. Quercetin was administered to the tumor-bearing mice via oral gavage either in the nanomicellar form or in a 25% v/v ethanol-based suspension, with a dose of 30 mg/kg following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. The results demonstrated that the anti-tumor activity of quercetin was significantly increased when administered as the peroral nanomicelles compared to control and peroral quercetin suspension (Figure 5A). Specifically, tumor growth inhibition at 10-week post-tumor inoculation was 83% for peroral quercetin suspension and 48% for peroral nanomicellar formulation. The two quercetin formulations were well tolerated by the animals, as reflected by minimal changes in the body weight of the mice (Figure 5B). No unusual observations were made upon necropsies of the animals from all three study groups.
| null | null |
[
"Introduction",
"Preparation of nanomicellar quercetin",
"Stability of nanomicellar quercetin in simulated physiological fluids",
"Cell culture",
"Accumulation of nanomicelles in Caco-2 cells",
"MTT viability assay and lactose dehydrogenase (LDH) release assay",
"Transepithelial electrical resistance (TEER) measurement in Caco-2 cells",
"Statistics",
"Formulating quercetin into PEG-lipid nanomicelles",
"In vitro cytotoxicity of nanomicellar quercetin in the A549 lung cancer cell line",
"Stability of nanomicellar quercetin in simulated physiological fluids",
"Interaction of nanomicellar quercetin with Caco-2 cell monolayer",
"In vivo efficacy of nanomicellar quercetin in human A549 lung tumor xenograft model"
] |
[
"Cancer is a public health problem that affects many countries in the world. Globally, lung cancer is the leading cause of cancer death. In the treatment of advanced lung cancer, chemotherapy remains the mainstay; however, only modest increase in survival has been observed at the cost of significant toxicity to patients.1 It is imperative to search for new therapeutics with improved efficacy and safety profiles. In the anticancer drug discovery and development process, compounds with the highest anticancer activities often have bulky hydrophobic groups within their chemical structures, rendering them water-insoluble.2 Low water solubility not only gives rise to formulation difficulties but also serious therapeutic challenges. Administering the poorly soluble drug candidate intravenously might result in serious complications such as embolism and respiratory system failure due to drug precipitation,3 whereas poor absorption would result from extravascular dosing.4\nThe family of flavonoid compounds is one such example which has been shown to be promising for the wide applicability in the prevention and therapy of cancer, neurodegenerative, and cardiovascular diseases. Yet, the major hurdle in their clinical usage is poor water solubility, thus affecting their peroral biological activity.5 Among this family of agents, quercetin has been studied extensively for its anticancer activity, whereby it could inhibit a number of signal transduction targets important to cancer cell survival, proliferation, and metastasis.6,7 Quercetin progressed to early-stage clinical trials as an anticancer agent more than a decade ago; nevertheless, its administration required the use of solvents such as dimethylsulfoxide or ethanol.8 Chemical modifications have been attempted to improve quercetin solubility but could result in a loss of drug potency.9 Thus, alternative strategies based on the use of nanotechnologies have been undertaken to improve the water solubility and/or the bioavailability of quercetin so as to improve its biological activity.5 While most of the nanovehicles are designed with compositions sufficiently stable for intravenous administration, which is the most conventional route for cancer chemotherapy, orally active cancer chemotherapeutics have emerged as an attractive treatment modality because of patient preference, convenience in administration, and applicability in outpatient setting.10 A few studies have shown promising peroral antioxidant activity of quercetin when it was delivered by nanoscaled delivery systems,11,12 and recently, a lipid-based, submicron (~400 nm) vesicular complex of quercetin has been developed with improved peroral anticancer activity as compared to a peroral quercetin suspension.13\nIn light of the advantages of peroral anticancer drug delivery and recent developments in the use of nanovehicles for the delivery of flavonoids,5 we have attempted to deliver quercetin perorally through the use of polyethylene glycol (PEG)-derivatized phosphatidylethanolamine (PE) as the diblock copolymer to form a self-assembled nanomicellar delivery system, and have compared the anticancer activity of this nanomicellar formulation with a peroral suspension of quercetin. The research on PEG-PE nanomicellar systems, pioneered by Torchilin and Alkan-Onyuksel,14–16 has contributed greatly to the intravenous delivery of water-insoluble agents. For instance, improved therapeutic efficacy of the intravenously administered PEG-lipid micelle formulation of paclitaxel has been demonstrated in tumor-bearing mice.15,16 The potential of PEG-lipid nanomicelles for the peroral administration of water-insoluble compounds has yet to be demonstrated in animal models. Here, we have shown that the peroral anticancer activity of quercetin could be significantly increased when delivered in PEG-PE nanomicellar systems in the A549 human lung tumor xenograft model.",
"The quercetin nanomicelles were prepared as follows: briefly, DSPE-PEG2000 was dissolved in chloroform and quercetin in ethanol, the two organic solutions were mixed together according to the various weight proportions of drug and PEG-lipid. Subsequently, the organic phase was evaporated off under a stream of nitrogen gas to obtain a dried thin film. The film was further placed under vacuum for at least 3 hours to remove the residual solvent. Hank’s balanced salt solution or phosphate-buffered saline (PBS) was used to hydrate the thin film to form the nanomicelles. For fluorescence-labeled nanomicelles, 1% of FITC-DSPE-PEG2000 was mixed with 99% of DSPE-PEG2000 (by mole) in chloroform, followed by evaporation and hydration as described above. Unincorporated quercetin was removed by centrifugation at 13,000 rpm for 10 minutes followed by filtration through a 0.2-μm filter. The resultant nanomicelle samples were frozen at −86°C for 8 hours (ULT Freezer; Thermo Electron Corporation Forma, Waltham, MA,) and subsequently lyophilized at −50°C to −55°C for 48 hours at 5 Pa (Alpha 2–4 LDplus Freeze Dryer; Christ, Osterode, Germany). No secondary drying was performed. Lyophilized samples were stored at 4°C ± 2°C until further evaluation, and subsequently were reconstituted to original volume using milli-Q water as and when required. Drug concentration was determined by reading absorbance at 376 nm after mixing the micelle samples in ethanol, using a standard curve constructed with quercetin dissolved in ethanol. The size of the nanomicelles was determined by dynamic light scattering (Malvern Zetasizer 3000HS; Malvern, Worcestershire, UK).",
"The stability of quercetin nanomicelles were evaluated using two different aqueous media. The composition of the simulated gastric fluid (SGF) was as follows: 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2, and that of the simulated intestinal fluid (SIF) was as follows: 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0. The nanomicellar quercetin formulation was dialyzed in 1:5000 v/v in the respective simulated fluids for the specified duration. Subsequently, the formulation was analyzed for size and drug content as described above.",
"Cells were grown in humidified atmosphere of 5% CO2 at 37°C, with media changed every 3–4 days. Caco-2 cells were maintained in Eagle’s minimal essential medium (EMEM) supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Stock cultures were grown in T-75 flasks. Upon 80%–90% confluency, cells were spilt using 2.5% trypsin containing 1.0 mM EDTA. All cell culture media and reagents were obtained from Gibco. A549 and MDA-MB-231 cells were maintained in RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Upon 80%–90% confluency, cells were spilt using 0.25% trypsin.",
"Caco-2 cells were seeded in 4-well Lab-tek chamber glass slides from Nalgene Nunc Inc (Naperville, IL) at a density of 7.2 × 104 cells/well and cultured for 21 days.\nSubsequently, FITC-labeled nanomicelles were added to the cells and incubated for the specified duration at 37°C. After incubation, cells were washed three times with ice-cold PBS before viewing under a Carl Zeiss fluorescence confocal microscope (Oberkochen, Germany) using 40× objective.",
"The plating densities for A549 cells, MDA-MB-231 cells, and Caco-2 cells were 5.0 × 103 cells/well, 3.0 × 103 cells/well, and 1.33 × 104 cells/well, respectively, in the 96-well plates. A549 and MDA-MB-231 cells were incubated for 24 hours to allow attachment to plate before experimentation, while Caco-2 cells were incubated for 21 days to allow differentiation and tight junction formation. The medium was aspirated and replaced with serum-supplemented medium containing serial dilutions of quercetin in free (dissolved in ethanol) or nanomicellar form.\nFor MTT viability assay, the cells were treated at 37°C for the specified duration, and at the end, MTT was added to each well after medium removal and PBS rinsing to remove residual drug. Following a 4-hour incubation at 37°C, the well content was aspirated and 150 μL DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured with a Tecan SpectraFluorPlus reader (Tecan, Mannedorf, Switzerland). Cell viability was calculated as follows:\nwhere Abstest, Absbackground, Absvehicle represent the absorbance readings from the quercetin-treated wells, the medium-only wells, and the vehicle control wells, respectively.\nFor LDH release assay, treatment groups, and conditions were similar to those for MTT viability assay, except that the culture medium was collected at the end of treatment for LDH detection using Cytotox-One Homogenous Membrane Integrity Assay Kit (Promega Corporation, Fitchburg, WI) following the manufacturer’s instructions. Fluorescence was measured using the Tecan SpectraFluorPlus plate reader with an excitation wavelength of 560 nm and emission wavelength of 590 nm. Cytotoxicity was calculated as follow:\nwhere Ftest, Ftriton-X and Fbackground represent the fluorescence readings from the test condition (CNM or vehicle alone), triton-X 0.2%, and background.",
"Cells were seeded at a density of 1.2 × 104 cells/well in collagen- coated filter membrane polycarbonate transwell inserts (12 mm, 1-μm pore size, 0.3 cm2 growth area) obtained from BD Biocoat (Erembodegem, Belgium) and incubated at 37°C for 21 days. On day 21, the baseline TEER values of Caco-2 cells were measured using Millicell ERS-2 Epithelial Volt-Ohm Meter, and inserts that record > 600 Ωcm2 were used for experiments. The chamber was washed twice with Hank’s balanced salt solution before the addition of drug treatment. The cells were treated for 24 hours at 37°C, and TEER values were recorded every 24 hours over the study duration of 96 hours.",
"Where appropriate, results are expressed as mean ± SEM of at least three independent experiments. Statistical significance was assessed using one-way ANOVA followed by Fisher’s least significant difference (LSD) test for multiple comparisons. A P value of <0.05 was considered to be statistically significant. All statistical analyses were carried out using SPSS software (v 19.0; IBM, Armonk, NY).",
"The commonly used PEG-lipid conjugate, DSPE-PEG2000, was combined with quercetin in various weight proportions (1%–5% w/w of quercetin in DSPE-PEG2000). As shown in Table 1, the incorporation efficiencies of quercetin into the DSPE-PEG2000 nanomicelles were found to be 88.9% and above when the content of quercetin was up to 4% w/w. At 5% w/w, the incorporation efficiency was reduced to 79.3%. The size of the drug-loaded nanomicelles ranged from 15.4 to 18.5 nm (Table 1), and the polydispersity indices were <0.250 for all the nanomicellar formulations. The zeta potentials of the empty and drug-loaded nanomicelles were −11.8 ± 0.7 mV and −14.8 ± 0.7 mV, respectively. The highest concentration of nanomicellar quercetin in Hepes-buffered saline that could be achieved without precipitation was 3 mg/mL. This represents a 110-fold increase in the aqueous concentration of quercetin, calculated based on the aqueous solubility of free quercetin of 80 μM.17,18 Furthermore, nanomicellar quercetin could be processed into a powder by freeze-drying, without the need of a cryoprotectant or lyoprotectant. Reconstitution of the freeze-dried, nanomicellar quercetin with water did not result in a change in size or quercetin content (data not shown). Hence, subsequent evaluation of the nanomicellar formulation was based on the reconstituted nanomicellar quercetin.",
"The cytotoxicity of nanomicellar quercetin was evaluated in the A549 human lung cancer cell line. The highest concentration of quercetin evaluated was 100 μM, as drug precipitation was observed at higher drug concentrations. As shown in Figure 1, the anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation, as determined by the MTT viability assay. Of note, non-drug loaded DSPE-PEG2000 nanomicelles did not cause significant toxicity to the A549 lung cancer cells up to a concentration of 1 mM, with cell viability of >85%. To ensure that the cytotoxic effect was not cell line specific, the MDA-MB-231 human breast cancer cell line was treated with free and nanomicellar quercetin. As shown in Suppl Data Figure 1, a similar trend could be observed whereby the activity of quercetin could be significantly increased when formulated into the nanomicelles.",
"Since the nanomicelles are to be administered perorally, stability in the fluids of the gastrointestinal tract has to be demonstrated. The stability of nanomicellar quercetin was evaluated over time in simulated gastric fluid (SGF, 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2) and simulated intestinal fluid (SIF, 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0). The quercetin nanomicelles were found to be stable, without significant change in size or drug precipitation (Figure 2). To investigate the amount of quercetin retained in the nanomicelles, the micelle samples were dialyzed against 1:5000 (v/v) SGF or SIF at 37°C using dialysis cassettes. Approximately 70%–75% of incorporated quercetin was retained in the nanomicelles in the respective simulated fluids (Figure 2).",
"The well-established Caco-2 cell monolayer was employed as the in vitro model for gastrointestinal epithelium, and the cellular accumulation of the PEG-lipid nanomicelles was assessed by confocal microscopy. To facilitate visualization, the nanomicelles were spiked with 1 mol% DSPE-PEG2000-N-carboxyfluorescein. The Caco-2 cells were exposed to the fluorescence-labeled nanomicelles for up to 24 hours at 37°C, and cellular accumulation of the nanomicelles increased over time (Figure 3A). Furthermore, the accumulation of the nanomicelles was independent of P-gp activity and endocytic processes, which was supported by results summarized in Suppl Data Figure 2. Cellular accumulation of the carboxyfluorescein-tagged nanomicelles occurred even in the presence of the P-gp inhibitor cyclosporine A or in the presence of endocytic inhibitors (hyperosmotic sucrose of 0.45 M or Brefeldin A) or a change of incubation temperature from 37°C to 4°C. Collectively, these data suggest that the nanomicelles are likely to accumulate inside Caco-2 cells by passive diffusion.\nOne possible route to facilitate drug absorption through the intestinal epithelium is by disrupting the tight junctions and thus making paracellular transport of drug molecules possible, which could be reflected by a decrease in the TEER.19 Nevertheless, it is also of equal importance that the disruption of the epithelial tight junctions is only transient, as irreversible disruption could represent potential toxicity to the intestinal epithelium. As such, differentiated Caco-2 cell monolayers grown in transwell inserts were exposed to free or nanomicellar quercetin for 24 hours, and TEER measurements were carried out over a time course of 96 hours. As shown in Figure 3B, Caco-2 cell monolayer treated with free quercetin had small yet statistically significant increase in TEER after 48 hours of exposure that gradually decreased back to control level (t = 0 hours). This observation is in line with the protective effect of quercetin on the intestinal barrier function, which acts by promoting the assembly and expression of tight junction proteins.20 In contrast, significant reduction in TEER was observed in Caco-2 cell monolayer treated with nanomicellar quercetin during the first 48 hours. Of note, the reduction in TEER in the nanomicellar quercetin treatment group could be gradually increased back to control level (t = 0 hours), indicating the reversibility of the treatment.\nTo further investigate the potential toxicity of the nanomicellar quercetin formulation, release of LDH was determined in differentiated Caco-2 cell monolayer upon 24-hour treatment with free or nanomicellar quercetin (Figure 4). Treatment with free quercetin was non-toxic over the concentration range tested, with ~10% LDH released relative to triton-X control. In contrast, nanomicellar quercetin treatment yielded a small but significant increase in LDH release of ~25% relative to triton-X control. Empty, non-drug loaded nanomicelles were evaluated at concentrations up to 1 mM and were found to be relatively non-toxic to the Caco-2 cell monolayer, with viability of ~80% at 1 mM (Suppl Data, Figure 3).",
"Based on the in vitro evaluation of the nanomicellar quercetin formulation which provided promising results, the biological activity of the peroral formulation was further investigated in the A549 lung tumor xenograft model. Quercetin was administered to the tumor-bearing mice via oral gavage either in the nanomicellar form or in a 25% v/v ethanol-based suspension, with a dose of 30 mg/kg following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. The results demonstrated that the anti-tumor activity of quercetin was significantly increased when administered as the peroral nanomicelles compared to control and peroral quercetin suspension (Figure 5A). Specifically, tumor growth inhibition at 10-week post-tumor inoculation was 83% for peroral quercetin suspension and 48% for peroral nanomicellar formulation. The two quercetin formulations were well tolerated by the animals, as reflected by minimal changes in the body weight of the mice (Figure 5B). No unusual observations were made upon necropsies of the animals from all three study groups."
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[
"Introduction",
"Materials and methods",
"Materials",
"Preparation of nanomicellar quercetin",
"Stability of nanomicellar quercetin in simulated physiological fluids",
"Cell culture",
"Accumulation of nanomicelles in Caco-2 cells",
"MTT viability assay and lactose dehydrogenase (LDH) release assay",
"Transepithelial electrical resistance (TEER) measurement in Caco-2 cells",
"Animal efficacy study",
"Statistics",
"Results",
"Formulating quercetin into PEG-lipid nanomicelles",
"In vitro cytotoxicity of nanomicellar quercetin in the A549 lung cancer cell line",
"Stability of nanomicellar quercetin in simulated physiological fluids",
"Interaction of nanomicellar quercetin with Caco-2 cell monolayer",
"In vivo efficacy of nanomicellar quercetin in human A549 lung tumor xenograft model",
"Discussion",
"Supplementary data"
] |
[
"Cancer is a public health problem that affects many countries in the world. Globally, lung cancer is the leading cause of cancer death. In the treatment of advanced lung cancer, chemotherapy remains the mainstay; however, only modest increase in survival has been observed at the cost of significant toxicity to patients.1 It is imperative to search for new therapeutics with improved efficacy and safety profiles. In the anticancer drug discovery and development process, compounds with the highest anticancer activities often have bulky hydrophobic groups within their chemical structures, rendering them water-insoluble.2 Low water solubility not only gives rise to formulation difficulties but also serious therapeutic challenges. Administering the poorly soluble drug candidate intravenously might result in serious complications such as embolism and respiratory system failure due to drug precipitation,3 whereas poor absorption would result from extravascular dosing.4\nThe family of flavonoid compounds is one such example which has been shown to be promising for the wide applicability in the prevention and therapy of cancer, neurodegenerative, and cardiovascular diseases. Yet, the major hurdle in their clinical usage is poor water solubility, thus affecting their peroral biological activity.5 Among this family of agents, quercetin has been studied extensively for its anticancer activity, whereby it could inhibit a number of signal transduction targets important to cancer cell survival, proliferation, and metastasis.6,7 Quercetin progressed to early-stage clinical trials as an anticancer agent more than a decade ago; nevertheless, its administration required the use of solvents such as dimethylsulfoxide or ethanol.8 Chemical modifications have been attempted to improve quercetin solubility but could result in a loss of drug potency.9 Thus, alternative strategies based on the use of nanotechnologies have been undertaken to improve the water solubility and/or the bioavailability of quercetin so as to improve its biological activity.5 While most of the nanovehicles are designed with compositions sufficiently stable for intravenous administration, which is the most conventional route for cancer chemotherapy, orally active cancer chemotherapeutics have emerged as an attractive treatment modality because of patient preference, convenience in administration, and applicability in outpatient setting.10 A few studies have shown promising peroral antioxidant activity of quercetin when it was delivered by nanoscaled delivery systems,11,12 and recently, a lipid-based, submicron (~400 nm) vesicular complex of quercetin has been developed with improved peroral anticancer activity as compared to a peroral quercetin suspension.13\nIn light of the advantages of peroral anticancer drug delivery and recent developments in the use of nanovehicles for the delivery of flavonoids,5 we have attempted to deliver quercetin perorally through the use of polyethylene glycol (PEG)-derivatized phosphatidylethanolamine (PE) as the diblock copolymer to form a self-assembled nanomicellar delivery system, and have compared the anticancer activity of this nanomicellar formulation with a peroral suspension of quercetin. The research on PEG-PE nanomicellar systems, pioneered by Torchilin and Alkan-Onyuksel,14–16 has contributed greatly to the intravenous delivery of water-insoluble agents. For instance, improved therapeutic efficacy of the intravenously administered PEG-lipid micelle formulation of paclitaxel has been demonstrated in tumor-bearing mice.15,16 The potential of PEG-lipid nanomicelles for the peroral administration of water-insoluble compounds has yet to be demonstrated in animal models. Here, we have shown that the peroral anticancer activity of quercetin could be significantly increased when delivered in PEG-PE nanomicellar systems in the A549 human lung tumor xenograft model.",
" Materials 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [poly(ethyleneglycol)-2000-N′-carboxyfluorescein] (FITC-DSPE-PEG2000) were purchased from Avanti-Polar-Lipids (Alabaster, AL). Cell culture media and supplements were from Gibco (Grand Island, NY). All other chemicals were purchased from Sigma-Aldrich Inc (St Louis, MO). A549, MDA-MB-231, and Caco-2 cell lines were purchased from the American Type Culture Collection (Manassas, VA).\n1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [poly(ethyleneglycol)-2000-N′-carboxyfluorescein] (FITC-DSPE-PEG2000) were purchased from Avanti-Polar-Lipids (Alabaster, AL). Cell culture media and supplements were from Gibco (Grand Island, NY). All other chemicals were purchased from Sigma-Aldrich Inc (St Louis, MO). A549, MDA-MB-231, and Caco-2 cell lines were purchased from the American Type Culture Collection (Manassas, VA).\n Preparation of nanomicellar quercetin The quercetin nanomicelles were prepared as follows: briefly, DSPE-PEG2000 was dissolved in chloroform and quercetin in ethanol, the two organic solutions were mixed together according to the various weight proportions of drug and PEG-lipid. Subsequently, the organic phase was evaporated off under a stream of nitrogen gas to obtain a dried thin film. The film was further placed under vacuum for at least 3 hours to remove the residual solvent. Hank’s balanced salt solution or phosphate-buffered saline (PBS) was used to hydrate the thin film to form the nanomicelles. For fluorescence-labeled nanomicelles, 1% of FITC-DSPE-PEG2000 was mixed with 99% of DSPE-PEG2000 (by mole) in chloroform, followed by evaporation and hydration as described above. Unincorporated quercetin was removed by centrifugation at 13,000 rpm for 10 minutes followed by filtration through a 0.2-μm filter. The resultant nanomicelle samples were frozen at −86°C for 8 hours (ULT Freezer; Thermo Electron Corporation Forma, Waltham, MA,) and subsequently lyophilized at −50°C to −55°C for 48 hours at 5 Pa (Alpha 2–4 LDplus Freeze Dryer; Christ, Osterode, Germany). No secondary drying was performed. Lyophilized samples were stored at 4°C ± 2°C until further evaluation, and subsequently were reconstituted to original volume using milli-Q water as and when required. Drug concentration was determined by reading absorbance at 376 nm after mixing the micelle samples in ethanol, using a standard curve constructed with quercetin dissolved in ethanol. The size of the nanomicelles was determined by dynamic light scattering (Malvern Zetasizer 3000HS; Malvern, Worcestershire, UK).\nThe quercetin nanomicelles were prepared as follows: briefly, DSPE-PEG2000 was dissolved in chloroform and quercetin in ethanol, the two organic solutions were mixed together according to the various weight proportions of drug and PEG-lipid. Subsequently, the organic phase was evaporated off under a stream of nitrogen gas to obtain a dried thin film. The film was further placed under vacuum for at least 3 hours to remove the residual solvent. Hank’s balanced salt solution or phosphate-buffered saline (PBS) was used to hydrate the thin film to form the nanomicelles. For fluorescence-labeled nanomicelles, 1% of FITC-DSPE-PEG2000 was mixed with 99% of DSPE-PEG2000 (by mole) in chloroform, followed by evaporation and hydration as described above. Unincorporated quercetin was removed by centrifugation at 13,000 rpm for 10 minutes followed by filtration through a 0.2-μm filter. The resultant nanomicelle samples were frozen at −86°C for 8 hours (ULT Freezer; Thermo Electron Corporation Forma, Waltham, MA,) and subsequently lyophilized at −50°C to −55°C for 48 hours at 5 Pa (Alpha 2–4 LDplus Freeze Dryer; Christ, Osterode, Germany). No secondary drying was performed. Lyophilized samples were stored at 4°C ± 2°C until further evaluation, and subsequently were reconstituted to original volume using milli-Q water as and when required. Drug concentration was determined by reading absorbance at 376 nm after mixing the micelle samples in ethanol, using a standard curve constructed with quercetin dissolved in ethanol. The size of the nanomicelles was determined by dynamic light scattering (Malvern Zetasizer 3000HS; Malvern, Worcestershire, UK).\n Stability of nanomicellar quercetin in simulated physiological fluids The stability of quercetin nanomicelles were evaluated using two different aqueous media. The composition of the simulated gastric fluid (SGF) was as follows: 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2, and that of the simulated intestinal fluid (SIF) was as follows: 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0. The nanomicellar quercetin formulation was dialyzed in 1:5000 v/v in the respective simulated fluids for the specified duration. Subsequently, the formulation was analyzed for size and drug content as described above.\nThe stability of quercetin nanomicelles were evaluated using two different aqueous media. The composition of the simulated gastric fluid (SGF) was as follows: 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2, and that of the simulated intestinal fluid (SIF) was as follows: 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0. The nanomicellar quercetin formulation was dialyzed in 1:5000 v/v in the respective simulated fluids for the specified duration. Subsequently, the formulation was analyzed for size and drug content as described above.\n Cell culture Cells were grown in humidified atmosphere of 5% CO2 at 37°C, with media changed every 3–4 days. Caco-2 cells were maintained in Eagle’s minimal essential medium (EMEM) supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Stock cultures were grown in T-75 flasks. Upon 80%–90% confluency, cells were spilt using 2.5% trypsin containing 1.0 mM EDTA. All cell culture media and reagents were obtained from Gibco. A549 and MDA-MB-231 cells were maintained in RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Upon 80%–90% confluency, cells were spilt using 0.25% trypsin.\nCells were grown in humidified atmosphere of 5% CO2 at 37°C, with media changed every 3–4 days. Caco-2 cells were maintained in Eagle’s minimal essential medium (EMEM) supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Stock cultures were grown in T-75 flasks. Upon 80%–90% confluency, cells were spilt using 2.5% trypsin containing 1.0 mM EDTA. All cell culture media and reagents were obtained from Gibco. A549 and MDA-MB-231 cells were maintained in RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Upon 80%–90% confluency, cells were spilt using 0.25% trypsin.\n Accumulation of nanomicelles in Caco-2 cells Caco-2 cells were seeded in 4-well Lab-tek chamber glass slides from Nalgene Nunc Inc (Naperville, IL) at a density of 7.2 × 104 cells/well and cultured for 21 days.\nSubsequently, FITC-labeled nanomicelles were added to the cells and incubated for the specified duration at 37°C. After incubation, cells were washed three times with ice-cold PBS before viewing under a Carl Zeiss fluorescence confocal microscope (Oberkochen, Germany) using 40× objective.\nCaco-2 cells were seeded in 4-well Lab-tek chamber glass slides from Nalgene Nunc Inc (Naperville, IL) at a density of 7.2 × 104 cells/well and cultured for 21 days.\nSubsequently, FITC-labeled nanomicelles were added to the cells and incubated for the specified duration at 37°C. After incubation, cells were washed three times with ice-cold PBS before viewing under a Carl Zeiss fluorescence confocal microscope (Oberkochen, Germany) using 40× objective.\n MTT viability assay and lactose dehydrogenase (LDH) release assay The plating densities for A549 cells, MDA-MB-231 cells, and Caco-2 cells were 5.0 × 103 cells/well, 3.0 × 103 cells/well, and 1.33 × 104 cells/well, respectively, in the 96-well plates. A549 and MDA-MB-231 cells were incubated for 24 hours to allow attachment to plate before experimentation, while Caco-2 cells were incubated for 21 days to allow differentiation and tight junction formation. The medium was aspirated and replaced with serum-supplemented medium containing serial dilutions of quercetin in free (dissolved in ethanol) or nanomicellar form.\nFor MTT viability assay, the cells were treated at 37°C for the specified duration, and at the end, MTT was added to each well after medium removal and PBS rinsing to remove residual drug. Following a 4-hour incubation at 37°C, the well content was aspirated and 150 μL DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured with a Tecan SpectraFluorPlus reader (Tecan, Mannedorf, Switzerland). Cell viability was calculated as follows:\nwhere Abstest, Absbackground, Absvehicle represent the absorbance readings from the quercetin-treated wells, the medium-only wells, and the vehicle control wells, respectively.\nFor LDH release assay, treatment groups, and conditions were similar to those for MTT viability assay, except that the culture medium was collected at the end of treatment for LDH detection using Cytotox-One Homogenous Membrane Integrity Assay Kit (Promega Corporation, Fitchburg, WI) following the manufacturer’s instructions. Fluorescence was measured using the Tecan SpectraFluorPlus plate reader with an excitation wavelength of 560 nm and emission wavelength of 590 nm. Cytotoxicity was calculated as follow:\nwhere Ftest, Ftriton-X and Fbackground represent the fluorescence readings from the test condition (CNM or vehicle alone), triton-X 0.2%, and background.\nThe plating densities for A549 cells, MDA-MB-231 cells, and Caco-2 cells were 5.0 × 103 cells/well, 3.0 × 103 cells/well, and 1.33 × 104 cells/well, respectively, in the 96-well plates. A549 and MDA-MB-231 cells were incubated for 24 hours to allow attachment to plate before experimentation, while Caco-2 cells were incubated for 21 days to allow differentiation and tight junction formation. The medium was aspirated and replaced with serum-supplemented medium containing serial dilutions of quercetin in free (dissolved in ethanol) or nanomicellar form.\nFor MTT viability assay, the cells were treated at 37°C for the specified duration, and at the end, MTT was added to each well after medium removal and PBS rinsing to remove residual drug. Following a 4-hour incubation at 37°C, the well content was aspirated and 150 μL DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured with a Tecan SpectraFluorPlus reader (Tecan, Mannedorf, Switzerland). Cell viability was calculated as follows:\nwhere Abstest, Absbackground, Absvehicle represent the absorbance readings from the quercetin-treated wells, the medium-only wells, and the vehicle control wells, respectively.\nFor LDH release assay, treatment groups, and conditions were similar to those for MTT viability assay, except that the culture medium was collected at the end of treatment for LDH detection using Cytotox-One Homogenous Membrane Integrity Assay Kit (Promega Corporation, Fitchburg, WI) following the manufacturer’s instructions. Fluorescence was measured using the Tecan SpectraFluorPlus plate reader with an excitation wavelength of 560 nm and emission wavelength of 590 nm. Cytotoxicity was calculated as follow:\nwhere Ftest, Ftriton-X and Fbackground represent the fluorescence readings from the test condition (CNM or vehicle alone), triton-X 0.2%, and background.\n Transepithelial electrical resistance (TEER) measurement in Caco-2 cells Cells were seeded at a density of 1.2 × 104 cells/well in collagen- coated filter membrane polycarbonate transwell inserts (12 mm, 1-μm pore size, 0.3 cm2 growth area) obtained from BD Biocoat (Erembodegem, Belgium) and incubated at 37°C for 21 days. On day 21, the baseline TEER values of Caco-2 cells were measured using Millicell ERS-2 Epithelial Volt-Ohm Meter, and inserts that record > 600 Ωcm2 were used for experiments. The chamber was washed twice with Hank’s balanced salt solution before the addition of drug treatment. The cells were treated for 24 hours at 37°C, and TEER values were recorded every 24 hours over the study duration of 96 hours.\nCells were seeded at a density of 1.2 × 104 cells/well in collagen- coated filter membrane polycarbonate transwell inserts (12 mm, 1-μm pore size, 0.3 cm2 growth area) obtained from BD Biocoat (Erembodegem, Belgium) and incubated at 37°C for 21 days. On day 21, the baseline TEER values of Caco-2 cells were measured using Millicell ERS-2 Epithelial Volt-Ohm Meter, and inserts that record > 600 Ωcm2 were used for experiments. The chamber was washed twice with Hank’s balanced salt solution before the addition of drug treatment. The cells were treated for 24 hours at 37°C, and TEER values were recorded every 24 hours over the study duration of 96 hours.\n Animal efficacy study The animal study was conducted in the facilities of the British Columbia Cancer Research Center, with methodologies approved by the Institutional Animal Care Committee of the University of British Columbia, Vancouver, BC, Canada. The mice were cared for and used in accordance with the Canadian Council on Animal Care Guidelines. All mice used were between 20–22 g, and were housed in micro-isolator cages and given free access to food and water. Tumors were established in female Rag-2M mice by a single subcutaneous injection of 5 × 106 A549 cells (in 50 μL) in the lower back area. Tumor growth was monitored by caliper measurements along the length and width. Tumor volumes were calculated by the following formula: Tumor volume = 0.5 × (length × width2). When the tumor size reached 50–100 mm3 (3 weeks post-tumor cell inoculation), mice were randomized into three study groups of six animals per group: (1) untreated control, (2) peroral quercetin suspension (vehicle = 25% v/v ethanol in water), and (3) peroral nanomicellar quercetin. The dose of quercetin was 30 mg/kg, following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. Tumor size and body weight of the mice was monitored thrice weekly. Animals whose tumor size reached 1000 mm3, developed ulcerations, or displayed a weight loss of more than 5% were euthanized.\nThe animal study was conducted in the facilities of the British Columbia Cancer Research Center, with methodologies approved by the Institutional Animal Care Committee of the University of British Columbia, Vancouver, BC, Canada. The mice were cared for and used in accordance with the Canadian Council on Animal Care Guidelines. All mice used were between 20–22 g, and were housed in micro-isolator cages and given free access to food and water. Tumors were established in female Rag-2M mice by a single subcutaneous injection of 5 × 106 A549 cells (in 50 μL) in the lower back area. Tumor growth was monitored by caliper measurements along the length and width. Tumor volumes were calculated by the following formula: Tumor volume = 0.5 × (length × width2). When the tumor size reached 50–100 mm3 (3 weeks post-tumor cell inoculation), mice were randomized into three study groups of six animals per group: (1) untreated control, (2) peroral quercetin suspension (vehicle = 25% v/v ethanol in water), and (3) peroral nanomicellar quercetin. The dose of quercetin was 30 mg/kg, following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. Tumor size and body weight of the mice was monitored thrice weekly. Animals whose tumor size reached 1000 mm3, developed ulcerations, or displayed a weight loss of more than 5% were euthanized.\n Statistics Where appropriate, results are expressed as mean ± SEM of at least three independent experiments. Statistical significance was assessed using one-way ANOVA followed by Fisher’s least significant difference (LSD) test for multiple comparisons. A P value of <0.05 was considered to be statistically significant. All statistical analyses were carried out using SPSS software (v 19.0; IBM, Armonk, NY).\nWhere appropriate, results are expressed as mean ± SEM of at least three independent experiments. Statistical significance was assessed using one-way ANOVA followed by Fisher’s least significant difference (LSD) test for multiple comparisons. A P value of <0.05 was considered to be statistically significant. All statistical analyses were carried out using SPSS software (v 19.0; IBM, Armonk, NY).",
"1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [poly(ethyleneglycol)-2000-N′-carboxyfluorescein] (FITC-DSPE-PEG2000) were purchased from Avanti-Polar-Lipids (Alabaster, AL). Cell culture media and supplements were from Gibco (Grand Island, NY). All other chemicals were purchased from Sigma-Aldrich Inc (St Louis, MO). A549, MDA-MB-231, and Caco-2 cell lines were purchased from the American Type Culture Collection (Manassas, VA).",
"The quercetin nanomicelles were prepared as follows: briefly, DSPE-PEG2000 was dissolved in chloroform and quercetin in ethanol, the two organic solutions were mixed together according to the various weight proportions of drug and PEG-lipid. Subsequently, the organic phase was evaporated off under a stream of nitrogen gas to obtain a dried thin film. The film was further placed under vacuum for at least 3 hours to remove the residual solvent. Hank’s balanced salt solution or phosphate-buffered saline (PBS) was used to hydrate the thin film to form the nanomicelles. For fluorescence-labeled nanomicelles, 1% of FITC-DSPE-PEG2000 was mixed with 99% of DSPE-PEG2000 (by mole) in chloroform, followed by evaporation and hydration as described above. Unincorporated quercetin was removed by centrifugation at 13,000 rpm for 10 minutes followed by filtration through a 0.2-μm filter. The resultant nanomicelle samples were frozen at −86°C for 8 hours (ULT Freezer; Thermo Electron Corporation Forma, Waltham, MA,) and subsequently lyophilized at −50°C to −55°C for 48 hours at 5 Pa (Alpha 2–4 LDplus Freeze Dryer; Christ, Osterode, Germany). No secondary drying was performed. Lyophilized samples were stored at 4°C ± 2°C until further evaluation, and subsequently were reconstituted to original volume using milli-Q water as and when required. Drug concentration was determined by reading absorbance at 376 nm after mixing the micelle samples in ethanol, using a standard curve constructed with quercetin dissolved in ethanol. The size of the nanomicelles was determined by dynamic light scattering (Malvern Zetasizer 3000HS; Malvern, Worcestershire, UK).",
"The stability of quercetin nanomicelles were evaluated using two different aqueous media. The composition of the simulated gastric fluid (SGF) was as follows: 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2, and that of the simulated intestinal fluid (SIF) was as follows: 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0. The nanomicellar quercetin formulation was dialyzed in 1:5000 v/v in the respective simulated fluids for the specified duration. Subsequently, the formulation was analyzed for size and drug content as described above.",
"Cells were grown in humidified atmosphere of 5% CO2 at 37°C, with media changed every 3–4 days. Caco-2 cells were maintained in Eagle’s minimal essential medium (EMEM) supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Stock cultures were grown in T-75 flasks. Upon 80%–90% confluency, cells were spilt using 2.5% trypsin containing 1.0 mM EDTA. All cell culture media and reagents were obtained from Gibco. A549 and MDA-MB-231 cells were maintained in RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Upon 80%–90% confluency, cells were spilt using 0.25% trypsin.",
"Caco-2 cells were seeded in 4-well Lab-tek chamber glass slides from Nalgene Nunc Inc (Naperville, IL) at a density of 7.2 × 104 cells/well and cultured for 21 days.\nSubsequently, FITC-labeled nanomicelles were added to the cells and incubated for the specified duration at 37°C. After incubation, cells were washed three times with ice-cold PBS before viewing under a Carl Zeiss fluorescence confocal microscope (Oberkochen, Germany) using 40× objective.",
"The plating densities for A549 cells, MDA-MB-231 cells, and Caco-2 cells were 5.0 × 103 cells/well, 3.0 × 103 cells/well, and 1.33 × 104 cells/well, respectively, in the 96-well plates. A549 and MDA-MB-231 cells were incubated for 24 hours to allow attachment to plate before experimentation, while Caco-2 cells were incubated for 21 days to allow differentiation and tight junction formation. The medium was aspirated and replaced with serum-supplemented medium containing serial dilutions of quercetin in free (dissolved in ethanol) or nanomicellar form.\nFor MTT viability assay, the cells were treated at 37°C for the specified duration, and at the end, MTT was added to each well after medium removal and PBS rinsing to remove residual drug. Following a 4-hour incubation at 37°C, the well content was aspirated and 150 μL DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured with a Tecan SpectraFluorPlus reader (Tecan, Mannedorf, Switzerland). Cell viability was calculated as follows:\nwhere Abstest, Absbackground, Absvehicle represent the absorbance readings from the quercetin-treated wells, the medium-only wells, and the vehicle control wells, respectively.\nFor LDH release assay, treatment groups, and conditions were similar to those for MTT viability assay, except that the culture medium was collected at the end of treatment for LDH detection using Cytotox-One Homogenous Membrane Integrity Assay Kit (Promega Corporation, Fitchburg, WI) following the manufacturer’s instructions. Fluorescence was measured using the Tecan SpectraFluorPlus plate reader with an excitation wavelength of 560 nm and emission wavelength of 590 nm. Cytotoxicity was calculated as follow:\nwhere Ftest, Ftriton-X and Fbackground represent the fluorescence readings from the test condition (CNM or vehicle alone), triton-X 0.2%, and background.",
"Cells were seeded at a density of 1.2 × 104 cells/well in collagen- coated filter membrane polycarbonate transwell inserts (12 mm, 1-μm pore size, 0.3 cm2 growth area) obtained from BD Biocoat (Erembodegem, Belgium) and incubated at 37°C for 21 days. On day 21, the baseline TEER values of Caco-2 cells were measured using Millicell ERS-2 Epithelial Volt-Ohm Meter, and inserts that record > 600 Ωcm2 were used for experiments. The chamber was washed twice with Hank’s balanced salt solution before the addition of drug treatment. The cells were treated for 24 hours at 37°C, and TEER values were recorded every 24 hours over the study duration of 96 hours.",
"The animal study was conducted in the facilities of the British Columbia Cancer Research Center, with methodologies approved by the Institutional Animal Care Committee of the University of British Columbia, Vancouver, BC, Canada. The mice were cared for and used in accordance with the Canadian Council on Animal Care Guidelines. All mice used were between 20–22 g, and were housed in micro-isolator cages and given free access to food and water. Tumors were established in female Rag-2M mice by a single subcutaneous injection of 5 × 106 A549 cells (in 50 μL) in the lower back area. Tumor growth was monitored by caliper measurements along the length and width. Tumor volumes were calculated by the following formula: Tumor volume = 0.5 × (length × width2). When the tumor size reached 50–100 mm3 (3 weeks post-tumor cell inoculation), mice were randomized into three study groups of six animals per group: (1) untreated control, (2) peroral quercetin suspension (vehicle = 25% v/v ethanol in water), and (3) peroral nanomicellar quercetin. The dose of quercetin was 30 mg/kg, following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. Tumor size and body weight of the mice was monitored thrice weekly. Animals whose tumor size reached 1000 mm3, developed ulcerations, or displayed a weight loss of more than 5% were euthanized.",
"Where appropriate, results are expressed as mean ± SEM of at least three independent experiments. Statistical significance was assessed using one-way ANOVA followed by Fisher’s least significant difference (LSD) test for multiple comparisons. A P value of <0.05 was considered to be statistically significant. All statistical analyses were carried out using SPSS software (v 19.0; IBM, Armonk, NY).",
" Formulating quercetin into PEG-lipid nanomicelles The commonly used PEG-lipid conjugate, DSPE-PEG2000, was combined with quercetin in various weight proportions (1%–5% w/w of quercetin in DSPE-PEG2000). As shown in Table 1, the incorporation efficiencies of quercetin into the DSPE-PEG2000 nanomicelles were found to be 88.9% and above when the content of quercetin was up to 4% w/w. At 5% w/w, the incorporation efficiency was reduced to 79.3%. The size of the drug-loaded nanomicelles ranged from 15.4 to 18.5 nm (Table 1), and the polydispersity indices were <0.250 for all the nanomicellar formulations. The zeta potentials of the empty and drug-loaded nanomicelles were −11.8 ± 0.7 mV and −14.8 ± 0.7 mV, respectively. The highest concentration of nanomicellar quercetin in Hepes-buffered saline that could be achieved without precipitation was 3 mg/mL. This represents a 110-fold increase in the aqueous concentration of quercetin, calculated based on the aqueous solubility of free quercetin of 80 μM.17,18 Furthermore, nanomicellar quercetin could be processed into a powder by freeze-drying, without the need of a cryoprotectant or lyoprotectant. Reconstitution of the freeze-dried, nanomicellar quercetin with water did not result in a change in size or quercetin content (data not shown). Hence, subsequent evaluation of the nanomicellar formulation was based on the reconstituted nanomicellar quercetin.\nThe commonly used PEG-lipid conjugate, DSPE-PEG2000, was combined with quercetin in various weight proportions (1%–5% w/w of quercetin in DSPE-PEG2000). As shown in Table 1, the incorporation efficiencies of quercetin into the DSPE-PEG2000 nanomicelles were found to be 88.9% and above when the content of quercetin was up to 4% w/w. At 5% w/w, the incorporation efficiency was reduced to 79.3%. The size of the drug-loaded nanomicelles ranged from 15.4 to 18.5 nm (Table 1), and the polydispersity indices were <0.250 for all the nanomicellar formulations. The zeta potentials of the empty and drug-loaded nanomicelles were −11.8 ± 0.7 mV and −14.8 ± 0.7 mV, respectively. The highest concentration of nanomicellar quercetin in Hepes-buffered saline that could be achieved without precipitation was 3 mg/mL. This represents a 110-fold increase in the aqueous concentration of quercetin, calculated based on the aqueous solubility of free quercetin of 80 μM.17,18 Furthermore, nanomicellar quercetin could be processed into a powder by freeze-drying, without the need of a cryoprotectant or lyoprotectant. Reconstitution of the freeze-dried, nanomicellar quercetin with water did not result in a change in size or quercetin content (data not shown). Hence, subsequent evaluation of the nanomicellar formulation was based on the reconstituted nanomicellar quercetin.\n In vitro cytotoxicity of nanomicellar quercetin in the A549 lung cancer cell line The cytotoxicity of nanomicellar quercetin was evaluated in the A549 human lung cancer cell line. The highest concentration of quercetin evaluated was 100 μM, as drug precipitation was observed at higher drug concentrations. As shown in Figure 1, the anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation, as determined by the MTT viability assay. Of note, non-drug loaded DSPE-PEG2000 nanomicelles did not cause significant toxicity to the A549 lung cancer cells up to a concentration of 1 mM, with cell viability of >85%. To ensure that the cytotoxic effect was not cell line specific, the MDA-MB-231 human breast cancer cell line was treated with free and nanomicellar quercetin. As shown in Suppl Data Figure 1, a similar trend could be observed whereby the activity of quercetin could be significantly increased when formulated into the nanomicelles.\nThe cytotoxicity of nanomicellar quercetin was evaluated in the A549 human lung cancer cell line. The highest concentration of quercetin evaluated was 100 μM, as drug precipitation was observed at higher drug concentrations. As shown in Figure 1, the anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation, as determined by the MTT viability assay. Of note, non-drug loaded DSPE-PEG2000 nanomicelles did not cause significant toxicity to the A549 lung cancer cells up to a concentration of 1 mM, with cell viability of >85%. To ensure that the cytotoxic effect was not cell line specific, the MDA-MB-231 human breast cancer cell line was treated with free and nanomicellar quercetin. As shown in Suppl Data Figure 1, a similar trend could be observed whereby the activity of quercetin could be significantly increased when formulated into the nanomicelles.\n Stability of nanomicellar quercetin in simulated physiological fluids Since the nanomicelles are to be administered perorally, stability in the fluids of the gastrointestinal tract has to be demonstrated. The stability of nanomicellar quercetin was evaluated over time in simulated gastric fluid (SGF, 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2) and simulated intestinal fluid (SIF, 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0). The quercetin nanomicelles were found to be stable, without significant change in size or drug precipitation (Figure 2). To investigate the amount of quercetin retained in the nanomicelles, the micelle samples were dialyzed against 1:5000 (v/v) SGF or SIF at 37°C using dialysis cassettes. Approximately 70%–75% of incorporated quercetin was retained in the nanomicelles in the respective simulated fluids (Figure 2).\nSince the nanomicelles are to be administered perorally, stability in the fluids of the gastrointestinal tract has to be demonstrated. The stability of nanomicellar quercetin was evaluated over time in simulated gastric fluid (SGF, 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2) and simulated intestinal fluid (SIF, 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0). The quercetin nanomicelles were found to be stable, without significant change in size or drug precipitation (Figure 2). To investigate the amount of quercetin retained in the nanomicelles, the micelle samples were dialyzed against 1:5000 (v/v) SGF or SIF at 37°C using dialysis cassettes. Approximately 70%–75% of incorporated quercetin was retained in the nanomicelles in the respective simulated fluids (Figure 2).\n Interaction of nanomicellar quercetin with Caco-2 cell monolayer The well-established Caco-2 cell monolayer was employed as the in vitro model for gastrointestinal epithelium, and the cellular accumulation of the PEG-lipid nanomicelles was assessed by confocal microscopy. To facilitate visualization, the nanomicelles were spiked with 1 mol% DSPE-PEG2000-N-carboxyfluorescein. The Caco-2 cells were exposed to the fluorescence-labeled nanomicelles for up to 24 hours at 37°C, and cellular accumulation of the nanomicelles increased over time (Figure 3A). Furthermore, the accumulation of the nanomicelles was independent of P-gp activity and endocytic processes, which was supported by results summarized in Suppl Data Figure 2. Cellular accumulation of the carboxyfluorescein-tagged nanomicelles occurred even in the presence of the P-gp inhibitor cyclosporine A or in the presence of endocytic inhibitors (hyperosmotic sucrose of 0.45 M or Brefeldin A) or a change of incubation temperature from 37°C to 4°C. Collectively, these data suggest that the nanomicelles are likely to accumulate inside Caco-2 cells by passive diffusion.\nOne possible route to facilitate drug absorption through the intestinal epithelium is by disrupting the tight junctions and thus making paracellular transport of drug molecules possible, which could be reflected by a decrease in the TEER.19 Nevertheless, it is also of equal importance that the disruption of the epithelial tight junctions is only transient, as irreversible disruption could represent potential toxicity to the intestinal epithelium. As such, differentiated Caco-2 cell monolayers grown in transwell inserts were exposed to free or nanomicellar quercetin for 24 hours, and TEER measurements were carried out over a time course of 96 hours. As shown in Figure 3B, Caco-2 cell monolayer treated with free quercetin had small yet statistically significant increase in TEER after 48 hours of exposure that gradually decreased back to control level (t = 0 hours). This observation is in line with the protective effect of quercetin on the intestinal barrier function, which acts by promoting the assembly and expression of tight junction proteins.20 In contrast, significant reduction in TEER was observed in Caco-2 cell monolayer treated with nanomicellar quercetin during the first 48 hours. Of note, the reduction in TEER in the nanomicellar quercetin treatment group could be gradually increased back to control level (t = 0 hours), indicating the reversibility of the treatment.\nTo further investigate the potential toxicity of the nanomicellar quercetin formulation, release of LDH was determined in differentiated Caco-2 cell monolayer upon 24-hour treatment with free or nanomicellar quercetin (Figure 4). Treatment with free quercetin was non-toxic over the concentration range tested, with ~10% LDH released relative to triton-X control. In contrast, nanomicellar quercetin treatment yielded a small but significant increase in LDH release of ~25% relative to triton-X control. Empty, non-drug loaded nanomicelles were evaluated at concentrations up to 1 mM and were found to be relatively non-toxic to the Caco-2 cell monolayer, with viability of ~80% at 1 mM (Suppl Data, Figure 3).\nThe well-established Caco-2 cell monolayer was employed as the in vitro model for gastrointestinal epithelium, and the cellular accumulation of the PEG-lipid nanomicelles was assessed by confocal microscopy. To facilitate visualization, the nanomicelles were spiked with 1 mol% DSPE-PEG2000-N-carboxyfluorescein. The Caco-2 cells were exposed to the fluorescence-labeled nanomicelles for up to 24 hours at 37°C, and cellular accumulation of the nanomicelles increased over time (Figure 3A). Furthermore, the accumulation of the nanomicelles was independent of P-gp activity and endocytic processes, which was supported by results summarized in Suppl Data Figure 2. Cellular accumulation of the carboxyfluorescein-tagged nanomicelles occurred even in the presence of the P-gp inhibitor cyclosporine A or in the presence of endocytic inhibitors (hyperosmotic sucrose of 0.45 M or Brefeldin A) or a change of incubation temperature from 37°C to 4°C. Collectively, these data suggest that the nanomicelles are likely to accumulate inside Caco-2 cells by passive diffusion.\nOne possible route to facilitate drug absorption through the intestinal epithelium is by disrupting the tight junctions and thus making paracellular transport of drug molecules possible, which could be reflected by a decrease in the TEER.19 Nevertheless, it is also of equal importance that the disruption of the epithelial tight junctions is only transient, as irreversible disruption could represent potential toxicity to the intestinal epithelium. As such, differentiated Caco-2 cell monolayers grown in transwell inserts were exposed to free or nanomicellar quercetin for 24 hours, and TEER measurements were carried out over a time course of 96 hours. As shown in Figure 3B, Caco-2 cell monolayer treated with free quercetin had small yet statistically significant increase in TEER after 48 hours of exposure that gradually decreased back to control level (t = 0 hours). This observation is in line with the protective effect of quercetin on the intestinal barrier function, which acts by promoting the assembly and expression of tight junction proteins.20 In contrast, significant reduction in TEER was observed in Caco-2 cell monolayer treated with nanomicellar quercetin during the first 48 hours. Of note, the reduction in TEER in the nanomicellar quercetin treatment group could be gradually increased back to control level (t = 0 hours), indicating the reversibility of the treatment.\nTo further investigate the potential toxicity of the nanomicellar quercetin formulation, release of LDH was determined in differentiated Caco-2 cell monolayer upon 24-hour treatment with free or nanomicellar quercetin (Figure 4). Treatment with free quercetin was non-toxic over the concentration range tested, with ~10% LDH released relative to triton-X control. In contrast, nanomicellar quercetin treatment yielded a small but significant increase in LDH release of ~25% relative to triton-X control. Empty, non-drug loaded nanomicelles were evaluated at concentrations up to 1 mM and were found to be relatively non-toxic to the Caco-2 cell monolayer, with viability of ~80% at 1 mM (Suppl Data, Figure 3).\n In vivo efficacy of nanomicellar quercetin in human A549 lung tumor xenograft model Based on the in vitro evaluation of the nanomicellar quercetin formulation which provided promising results, the biological activity of the peroral formulation was further investigated in the A549 lung tumor xenograft model. Quercetin was administered to the tumor-bearing mice via oral gavage either in the nanomicellar form or in a 25% v/v ethanol-based suspension, with a dose of 30 mg/kg following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. The results demonstrated that the anti-tumor activity of quercetin was significantly increased when administered as the peroral nanomicelles compared to control and peroral quercetin suspension (Figure 5A). Specifically, tumor growth inhibition at 10-week post-tumor inoculation was 83% for peroral quercetin suspension and 48% for peroral nanomicellar formulation. The two quercetin formulations were well tolerated by the animals, as reflected by minimal changes in the body weight of the mice (Figure 5B). No unusual observations were made upon necropsies of the animals from all three study groups.\nBased on the in vitro evaluation of the nanomicellar quercetin formulation which provided promising results, the biological activity of the peroral formulation was further investigated in the A549 lung tumor xenograft model. Quercetin was administered to the tumor-bearing mice via oral gavage either in the nanomicellar form or in a 25% v/v ethanol-based suspension, with a dose of 30 mg/kg following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. The results demonstrated that the anti-tumor activity of quercetin was significantly increased when administered as the peroral nanomicelles compared to control and peroral quercetin suspension (Figure 5A). Specifically, tumor growth inhibition at 10-week post-tumor inoculation was 83% for peroral quercetin suspension and 48% for peroral nanomicellar formulation. The two quercetin formulations were well tolerated by the animals, as reflected by minimal changes in the body weight of the mice (Figure 5B). No unusual observations were made upon necropsies of the animals from all three study groups.",
"The commonly used PEG-lipid conjugate, DSPE-PEG2000, was combined with quercetin in various weight proportions (1%–5% w/w of quercetin in DSPE-PEG2000). As shown in Table 1, the incorporation efficiencies of quercetin into the DSPE-PEG2000 nanomicelles were found to be 88.9% and above when the content of quercetin was up to 4% w/w. At 5% w/w, the incorporation efficiency was reduced to 79.3%. The size of the drug-loaded nanomicelles ranged from 15.4 to 18.5 nm (Table 1), and the polydispersity indices were <0.250 for all the nanomicellar formulations. The zeta potentials of the empty and drug-loaded nanomicelles were −11.8 ± 0.7 mV and −14.8 ± 0.7 mV, respectively. The highest concentration of nanomicellar quercetin in Hepes-buffered saline that could be achieved without precipitation was 3 mg/mL. This represents a 110-fold increase in the aqueous concentration of quercetin, calculated based on the aqueous solubility of free quercetin of 80 μM.17,18 Furthermore, nanomicellar quercetin could be processed into a powder by freeze-drying, without the need of a cryoprotectant or lyoprotectant. Reconstitution of the freeze-dried, nanomicellar quercetin with water did not result in a change in size or quercetin content (data not shown). Hence, subsequent evaluation of the nanomicellar formulation was based on the reconstituted nanomicellar quercetin.",
"The cytotoxicity of nanomicellar quercetin was evaluated in the A549 human lung cancer cell line. The highest concentration of quercetin evaluated was 100 μM, as drug precipitation was observed at higher drug concentrations. As shown in Figure 1, the anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation, as determined by the MTT viability assay. Of note, non-drug loaded DSPE-PEG2000 nanomicelles did not cause significant toxicity to the A549 lung cancer cells up to a concentration of 1 mM, with cell viability of >85%. To ensure that the cytotoxic effect was not cell line specific, the MDA-MB-231 human breast cancer cell line was treated with free and nanomicellar quercetin. As shown in Suppl Data Figure 1, a similar trend could be observed whereby the activity of quercetin could be significantly increased when formulated into the nanomicelles.",
"Since the nanomicelles are to be administered perorally, stability in the fluids of the gastrointestinal tract has to be demonstrated. The stability of nanomicellar quercetin was evaluated over time in simulated gastric fluid (SGF, 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2) and simulated intestinal fluid (SIF, 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0). The quercetin nanomicelles were found to be stable, without significant change in size or drug precipitation (Figure 2). To investigate the amount of quercetin retained in the nanomicelles, the micelle samples were dialyzed against 1:5000 (v/v) SGF or SIF at 37°C using dialysis cassettes. Approximately 70%–75% of incorporated quercetin was retained in the nanomicelles in the respective simulated fluids (Figure 2).",
"The well-established Caco-2 cell monolayer was employed as the in vitro model for gastrointestinal epithelium, and the cellular accumulation of the PEG-lipid nanomicelles was assessed by confocal microscopy. To facilitate visualization, the nanomicelles were spiked with 1 mol% DSPE-PEG2000-N-carboxyfluorescein. The Caco-2 cells were exposed to the fluorescence-labeled nanomicelles for up to 24 hours at 37°C, and cellular accumulation of the nanomicelles increased over time (Figure 3A). Furthermore, the accumulation of the nanomicelles was independent of P-gp activity and endocytic processes, which was supported by results summarized in Suppl Data Figure 2. Cellular accumulation of the carboxyfluorescein-tagged nanomicelles occurred even in the presence of the P-gp inhibitor cyclosporine A or in the presence of endocytic inhibitors (hyperosmotic sucrose of 0.45 M or Brefeldin A) or a change of incubation temperature from 37°C to 4°C. Collectively, these data suggest that the nanomicelles are likely to accumulate inside Caco-2 cells by passive diffusion.\nOne possible route to facilitate drug absorption through the intestinal epithelium is by disrupting the tight junctions and thus making paracellular transport of drug molecules possible, which could be reflected by a decrease in the TEER.19 Nevertheless, it is also of equal importance that the disruption of the epithelial tight junctions is only transient, as irreversible disruption could represent potential toxicity to the intestinal epithelium. As such, differentiated Caco-2 cell monolayers grown in transwell inserts were exposed to free or nanomicellar quercetin for 24 hours, and TEER measurements were carried out over a time course of 96 hours. As shown in Figure 3B, Caco-2 cell monolayer treated with free quercetin had small yet statistically significant increase in TEER after 48 hours of exposure that gradually decreased back to control level (t = 0 hours). This observation is in line with the protective effect of quercetin on the intestinal barrier function, which acts by promoting the assembly and expression of tight junction proteins.20 In contrast, significant reduction in TEER was observed in Caco-2 cell monolayer treated with nanomicellar quercetin during the first 48 hours. Of note, the reduction in TEER in the nanomicellar quercetin treatment group could be gradually increased back to control level (t = 0 hours), indicating the reversibility of the treatment.\nTo further investigate the potential toxicity of the nanomicellar quercetin formulation, release of LDH was determined in differentiated Caco-2 cell monolayer upon 24-hour treatment with free or nanomicellar quercetin (Figure 4). Treatment with free quercetin was non-toxic over the concentration range tested, with ~10% LDH released relative to triton-X control. In contrast, nanomicellar quercetin treatment yielded a small but significant increase in LDH release of ~25% relative to triton-X control. Empty, non-drug loaded nanomicelles were evaluated at concentrations up to 1 mM and were found to be relatively non-toxic to the Caco-2 cell monolayer, with viability of ~80% at 1 mM (Suppl Data, Figure 3).",
"Based on the in vitro evaluation of the nanomicellar quercetin formulation which provided promising results, the biological activity of the peroral formulation was further investigated in the A549 lung tumor xenograft model. Quercetin was administered to the tumor-bearing mice via oral gavage either in the nanomicellar form or in a 25% v/v ethanol-based suspension, with a dose of 30 mg/kg following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. The results demonstrated that the anti-tumor activity of quercetin was significantly increased when administered as the peroral nanomicelles compared to control and peroral quercetin suspension (Figure 5A). Specifically, tumor growth inhibition at 10-week post-tumor inoculation was 83% for peroral quercetin suspension and 48% for peroral nanomicellar formulation. The two quercetin formulations were well tolerated by the animals, as reflected by minimal changes in the body weight of the mice (Figure 5B). No unusual observations were made upon necropsies of the animals from all three study groups.",
"Quercetin, a flavanol that is found in certain vegetables, fruits, and tea, has been shown to have a range of biochemical and biological activities that could have the potential to translate to therapeutic benefits. In addition to its antioxidant and anti-inflammatory effects, quercetin as a tyrosine kinase inhibitor8 is able to modulate multiple signaling pathways that would have considerable implications in the therapeutic and prophylactic values for diseases such as cancer. However, realizing the biological benefits of quercetin in the clinical setting is mostly hampered by its low water solubility and poor absorption into the body. For instance, the baseline plasma concentration of quercetin from dietary intake is 50–80 nM,21 and even with supplementation at 1 g/day for 28 days, the plasma concentration is ~1.5 μM,22 which is far below the concentration that is necessary to bring about the intended pharmacological effects of quercetin (such as the concentrations needed for anticancer effect presented in Figure 1). Thus, it is imperative to develop appropriate vehicles for quercetin, and it is the intention of our current study to develop a peroral nanomicellar system that could potentially improve its anticancer activity, which has not been extensively studied.\nSince the quercetin-loaded DSPE-PEG2000 nanomicelles are intended for peroral application, the nanomicelles should be able to withstand the harsh environment of the digestive tract such as the drastic changes in pH, and at the same time, be relatively non-toxic to the intestinal epithelium. As demonstrated in Figure 2, the quercetin nanomicelles were relatively stable at pH 1.2 and pH 7, with no significant changes in size and ~30% release of quercetin. These findings are in good agreement with a previous study by Dabholkar et al which reported no significant change in the size of mixed micelles made of DSPE-PEG2000 and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) incubated in acidic (pH 1.2) and neutral (pH 7.4) conditions.23 In terms of toxicity to the intestinal epithelium, although the incorporation of quercetin into the nanomicelles has slightly increased the cytotoxicity, as reflected by the data in Figure 4, this level of cytotoxicity (~25% as determined by LDH release) was comparable to those reported for TPGS and its analogs which are PEG-derivatives well established for their ability to modulate peroral drug absorption. The Caco-2 cytotoxicity of the TPGS analogs was reported to range from 20%–40%, as determined by LDH release.24 Furthermore, quercetin-loaded nanomicelles did not induce irreversible reduction in the TEER of the Caco-2 epithelium (Figure 3B). This finding, together with those in Figure 4 and Suppl Data Figure 3, showed that the quercetin-loaded nanomicelles were relatively non-toxic, which is in line with another study reporting that empty or meso-tetraphenyl porphine loaded DSPE-PEG2000 micelles were minimally toxic to Caco-2 cells.25\nThe therapeutic benef its of perorally administered quercetin is limited by poor absorption into the body that could be attributed to a number of reasons, including its chemical structure, low water solubility, and extensive metabolism and degradation in the gastrointestinal tract.26 Of interest, quercetin has been shown to protect the intestinal epithelial barrier function by promoting the expression of a number of tight junction proteins and increase TEER of Caco-2 monolayer,27 implying that quercetin may limit its own absorption into the body by reducing paracellular permeability. Our current DSPE-PEG2000-based peroral nanomicellar formulation could increase the anticancer activity of quercetin by two possible mechanisms with supportive data: (1) increase in the aqueous concentration of quercetin (by 110-fold to 3 mg/mL) through solubilization in the nanomicelles, and (2) increase in permeability through modulation of the epithelial tight junctions as reflected by the transient decrease in TEER. At the molecular level, quercetin is able to increase the distribution of tight junction proteins, ZO-2, occludin, and claudin-1, to the actin cytoskeleton fraction, as well as increase the expression of claudin-4 in cytoskeleton fraction and in whole cell.27 From Figure 3B, it can be seen that empty nanomicelles could reduce TEER reversibly, and it is possible that the empty micelles could disrupt the assembly of the tight junction complex through the partition of DSPE-PEG2000 into the cell membrane bilayer.\nNevertheless, other possible mechanisms that could contribute to the increased activity of quercetin by the nanomicellar peroral formulation could not be ruled out. One such mechanism is the potential ability of the nanomicelles to protect quercetin from the enzymatic and degradative action in the intestinal tract. The metabolism of quercetin is complex, involving the formation of multiple metabolites by different pathways.28 Thus, future investigations on the full pharmacokinetic behavior of the nanomicellar formulation with metabolite profiling are necessary to gain insights if the nanomicelles could indeed protect quercetin from enzymatic metabolism in vivo. Currently, we are establishing mass spectroscopy-based analytical method to characterize the metabonomics of quercetin to address the question. Another possible mechanism is the increased mucoadhesion of the nanomicelles to the intestinal mucosa that could be provided by the PEG shell of the nanomicelles.29 Furthermore, it has been suggested that nanovehicles made of lipids could be taken up by lymphoid tissues in the intestinal tract which in turn could circumvent first pass metabolism.13\nIt is encouraging that the nanomicellar peroral formulation could enhance the anticancer activity of quercetin at a dose of 30 mg/kg. It has been reported that a dose of 3 g/kg, which is 100-fold higher than ours, did not cause any toxic or harmful effect to mice.30 Thus, future studies could titrate our current dose to higher levels, with the dosing frequency adjusted to daily dosing. The dose titration study could be further coupled to the examination of the correlation between tyrosine kinase inhibition and tumor growth inhibition, which could yield interesting pharmacodynamic data on this nanomicellar peroral formulation. Further evaluation could also include the potential of this nanomicellar peroral formulation as a chemotherapeutic and chemopreventive agent in animal models.",
"MTT viability of free and nanomicellar quercetin in MDA-MB-231 human breast cancer cell line upon 72 hours of exposure. The concentrations of empty micelle were 96 μM and 192 μM for 30 μM and 60 μM nanomicellar quercetin, respectively.\nNotes: Data represent mean ± SEM from three independent experiments. *P < 0.05 as compared to free quercetin group.\nCellular accumulation of FITC-labeled nanomicelles over 24 hours of incubation under various conditions: (A) at 4°C, (B) at 37°C, (C) without cyclosporine A, (D) with cyclosporine A, (E) without 0.45 M sucrose, (F) with 0.45 M sucrose, (G) without brefeldine A, (H) with brefeldine A.\nNotes: Representative images from three independent studies are shown.\nMTT viability of (A) empty, non-drug loaded nanomicelles, (B) quercetin-loaded nanomicelles (showing micelle concentrations), and (C) free quercetin in Caco-2 cell monolayer upon 24 h of exposure."
] |
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[
"quercetin",
"polymeric micelles",
"PEG-PE",
"peroral drug delivery",
"lung cancer"
] |
Introduction:
Cancer is a public health problem that affects many countries in the world. Globally, lung cancer is the leading cause of cancer death. In the treatment of advanced lung cancer, chemotherapy remains the mainstay; however, only modest increase in survival has been observed at the cost of significant toxicity to patients.1 It is imperative to search for new therapeutics with improved efficacy and safety profiles. In the anticancer drug discovery and development process, compounds with the highest anticancer activities often have bulky hydrophobic groups within their chemical structures, rendering them water-insoluble.2 Low water solubility not only gives rise to formulation difficulties but also serious therapeutic challenges. Administering the poorly soluble drug candidate intravenously might result in serious complications such as embolism and respiratory system failure due to drug precipitation,3 whereas poor absorption would result from extravascular dosing.4
The family of flavonoid compounds is one such example which has been shown to be promising for the wide applicability in the prevention and therapy of cancer, neurodegenerative, and cardiovascular diseases. Yet, the major hurdle in their clinical usage is poor water solubility, thus affecting their peroral biological activity.5 Among this family of agents, quercetin has been studied extensively for its anticancer activity, whereby it could inhibit a number of signal transduction targets important to cancer cell survival, proliferation, and metastasis.6,7 Quercetin progressed to early-stage clinical trials as an anticancer agent more than a decade ago; nevertheless, its administration required the use of solvents such as dimethylsulfoxide or ethanol.8 Chemical modifications have been attempted to improve quercetin solubility but could result in a loss of drug potency.9 Thus, alternative strategies based on the use of nanotechnologies have been undertaken to improve the water solubility and/or the bioavailability of quercetin so as to improve its biological activity.5 While most of the nanovehicles are designed with compositions sufficiently stable for intravenous administration, which is the most conventional route for cancer chemotherapy, orally active cancer chemotherapeutics have emerged as an attractive treatment modality because of patient preference, convenience in administration, and applicability in outpatient setting.10 A few studies have shown promising peroral antioxidant activity of quercetin when it was delivered by nanoscaled delivery systems,11,12 and recently, a lipid-based, submicron (~400 nm) vesicular complex of quercetin has been developed with improved peroral anticancer activity as compared to a peroral quercetin suspension.13
In light of the advantages of peroral anticancer drug delivery and recent developments in the use of nanovehicles for the delivery of flavonoids,5 we have attempted to deliver quercetin perorally through the use of polyethylene glycol (PEG)-derivatized phosphatidylethanolamine (PE) as the diblock copolymer to form a self-assembled nanomicellar delivery system, and have compared the anticancer activity of this nanomicellar formulation with a peroral suspension of quercetin. The research on PEG-PE nanomicellar systems, pioneered by Torchilin and Alkan-Onyuksel,14–16 has contributed greatly to the intravenous delivery of water-insoluble agents. For instance, improved therapeutic efficacy of the intravenously administered PEG-lipid micelle formulation of paclitaxel has been demonstrated in tumor-bearing mice.15,16 The potential of PEG-lipid nanomicelles for the peroral administration of water-insoluble compounds has yet to be demonstrated in animal models. Here, we have shown that the peroral anticancer activity of quercetin could be significantly increased when delivered in PEG-PE nanomicellar systems in the A549 human lung tumor xenograft model.
Materials and methods:
Materials 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [poly(ethyleneglycol)-2000-N′-carboxyfluorescein] (FITC-DSPE-PEG2000) were purchased from Avanti-Polar-Lipids (Alabaster, AL). Cell culture media and supplements were from Gibco (Grand Island, NY). All other chemicals were purchased from Sigma-Aldrich Inc (St Louis, MO). A549, MDA-MB-231, and Caco-2 cell lines were purchased from the American Type Culture Collection (Manassas, VA).
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [poly(ethyleneglycol)-2000-N′-carboxyfluorescein] (FITC-DSPE-PEG2000) were purchased from Avanti-Polar-Lipids (Alabaster, AL). Cell culture media and supplements were from Gibco (Grand Island, NY). All other chemicals were purchased from Sigma-Aldrich Inc (St Louis, MO). A549, MDA-MB-231, and Caco-2 cell lines were purchased from the American Type Culture Collection (Manassas, VA).
Preparation of nanomicellar quercetin The quercetin nanomicelles were prepared as follows: briefly, DSPE-PEG2000 was dissolved in chloroform and quercetin in ethanol, the two organic solutions were mixed together according to the various weight proportions of drug and PEG-lipid. Subsequently, the organic phase was evaporated off under a stream of nitrogen gas to obtain a dried thin film. The film was further placed under vacuum for at least 3 hours to remove the residual solvent. Hank’s balanced salt solution or phosphate-buffered saline (PBS) was used to hydrate the thin film to form the nanomicelles. For fluorescence-labeled nanomicelles, 1% of FITC-DSPE-PEG2000 was mixed with 99% of DSPE-PEG2000 (by mole) in chloroform, followed by evaporation and hydration as described above. Unincorporated quercetin was removed by centrifugation at 13,000 rpm for 10 minutes followed by filtration through a 0.2-μm filter. The resultant nanomicelle samples were frozen at −86°C for 8 hours (ULT Freezer; Thermo Electron Corporation Forma, Waltham, MA,) and subsequently lyophilized at −50°C to −55°C for 48 hours at 5 Pa (Alpha 2–4 LDplus Freeze Dryer; Christ, Osterode, Germany). No secondary drying was performed. Lyophilized samples were stored at 4°C ± 2°C until further evaluation, and subsequently were reconstituted to original volume using milli-Q water as and when required. Drug concentration was determined by reading absorbance at 376 nm after mixing the micelle samples in ethanol, using a standard curve constructed with quercetin dissolved in ethanol. The size of the nanomicelles was determined by dynamic light scattering (Malvern Zetasizer 3000HS; Malvern, Worcestershire, UK).
The quercetin nanomicelles were prepared as follows: briefly, DSPE-PEG2000 was dissolved in chloroform and quercetin in ethanol, the two organic solutions were mixed together according to the various weight proportions of drug and PEG-lipid. Subsequently, the organic phase was evaporated off under a stream of nitrogen gas to obtain a dried thin film. The film was further placed under vacuum for at least 3 hours to remove the residual solvent. Hank’s balanced salt solution or phosphate-buffered saline (PBS) was used to hydrate the thin film to form the nanomicelles. For fluorescence-labeled nanomicelles, 1% of FITC-DSPE-PEG2000 was mixed with 99% of DSPE-PEG2000 (by mole) in chloroform, followed by evaporation and hydration as described above. Unincorporated quercetin was removed by centrifugation at 13,000 rpm for 10 minutes followed by filtration through a 0.2-μm filter. The resultant nanomicelle samples were frozen at −86°C for 8 hours (ULT Freezer; Thermo Electron Corporation Forma, Waltham, MA,) and subsequently lyophilized at −50°C to −55°C for 48 hours at 5 Pa (Alpha 2–4 LDplus Freeze Dryer; Christ, Osterode, Germany). No secondary drying was performed. Lyophilized samples were stored at 4°C ± 2°C until further evaluation, and subsequently were reconstituted to original volume using milli-Q water as and when required. Drug concentration was determined by reading absorbance at 376 nm after mixing the micelle samples in ethanol, using a standard curve constructed with quercetin dissolved in ethanol. The size of the nanomicelles was determined by dynamic light scattering (Malvern Zetasizer 3000HS; Malvern, Worcestershire, UK).
Stability of nanomicellar quercetin in simulated physiological fluids The stability of quercetin nanomicelles were evaluated using two different aqueous media. The composition of the simulated gastric fluid (SGF) was as follows: 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2, and that of the simulated intestinal fluid (SIF) was as follows: 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0. The nanomicellar quercetin formulation was dialyzed in 1:5000 v/v in the respective simulated fluids for the specified duration. Subsequently, the formulation was analyzed for size and drug content as described above.
The stability of quercetin nanomicelles were evaluated using two different aqueous media. The composition of the simulated gastric fluid (SGF) was as follows: 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2, and that of the simulated intestinal fluid (SIF) was as follows: 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0. The nanomicellar quercetin formulation was dialyzed in 1:5000 v/v in the respective simulated fluids for the specified duration. Subsequently, the formulation was analyzed for size and drug content as described above.
Cell culture Cells were grown in humidified atmosphere of 5% CO2 at 37°C, with media changed every 3–4 days. Caco-2 cells were maintained in Eagle’s minimal essential medium (EMEM) supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Stock cultures were grown in T-75 flasks. Upon 80%–90% confluency, cells were spilt using 2.5% trypsin containing 1.0 mM EDTA. All cell culture media and reagents were obtained from Gibco. A549 and MDA-MB-231 cells were maintained in RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Upon 80%–90% confluency, cells were spilt using 0.25% trypsin.
Cells were grown in humidified atmosphere of 5% CO2 at 37°C, with media changed every 3–4 days. Caco-2 cells were maintained in Eagle’s minimal essential medium (EMEM) supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Stock cultures were grown in T-75 flasks. Upon 80%–90% confluency, cells were spilt using 2.5% trypsin containing 1.0 mM EDTA. All cell culture media and reagents were obtained from Gibco. A549 and MDA-MB-231 cells were maintained in RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Upon 80%–90% confluency, cells were spilt using 0.25% trypsin.
Accumulation of nanomicelles in Caco-2 cells Caco-2 cells were seeded in 4-well Lab-tek chamber glass slides from Nalgene Nunc Inc (Naperville, IL) at a density of 7.2 × 104 cells/well and cultured for 21 days.
Subsequently, FITC-labeled nanomicelles were added to the cells and incubated for the specified duration at 37°C. After incubation, cells were washed three times with ice-cold PBS before viewing under a Carl Zeiss fluorescence confocal microscope (Oberkochen, Germany) using 40× objective.
Caco-2 cells were seeded in 4-well Lab-tek chamber glass slides from Nalgene Nunc Inc (Naperville, IL) at a density of 7.2 × 104 cells/well and cultured for 21 days.
Subsequently, FITC-labeled nanomicelles were added to the cells and incubated for the specified duration at 37°C. After incubation, cells were washed three times with ice-cold PBS before viewing under a Carl Zeiss fluorescence confocal microscope (Oberkochen, Germany) using 40× objective.
MTT viability assay and lactose dehydrogenase (LDH) release assay The plating densities for A549 cells, MDA-MB-231 cells, and Caco-2 cells were 5.0 × 103 cells/well, 3.0 × 103 cells/well, and 1.33 × 104 cells/well, respectively, in the 96-well plates. A549 and MDA-MB-231 cells were incubated for 24 hours to allow attachment to plate before experimentation, while Caco-2 cells were incubated for 21 days to allow differentiation and tight junction formation. The medium was aspirated and replaced with serum-supplemented medium containing serial dilutions of quercetin in free (dissolved in ethanol) or nanomicellar form.
For MTT viability assay, the cells were treated at 37°C for the specified duration, and at the end, MTT was added to each well after medium removal and PBS rinsing to remove residual drug. Following a 4-hour incubation at 37°C, the well content was aspirated and 150 μL DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured with a Tecan SpectraFluorPlus reader (Tecan, Mannedorf, Switzerland). Cell viability was calculated as follows:
where Abstest, Absbackground, Absvehicle represent the absorbance readings from the quercetin-treated wells, the medium-only wells, and the vehicle control wells, respectively.
For LDH release assay, treatment groups, and conditions were similar to those for MTT viability assay, except that the culture medium was collected at the end of treatment for LDH detection using Cytotox-One Homogenous Membrane Integrity Assay Kit (Promega Corporation, Fitchburg, WI) following the manufacturer’s instructions. Fluorescence was measured using the Tecan SpectraFluorPlus plate reader with an excitation wavelength of 560 nm and emission wavelength of 590 nm. Cytotoxicity was calculated as follow:
where Ftest, Ftriton-X and Fbackground represent the fluorescence readings from the test condition (CNM or vehicle alone), triton-X 0.2%, and background.
The plating densities for A549 cells, MDA-MB-231 cells, and Caco-2 cells were 5.0 × 103 cells/well, 3.0 × 103 cells/well, and 1.33 × 104 cells/well, respectively, in the 96-well plates. A549 and MDA-MB-231 cells were incubated for 24 hours to allow attachment to plate before experimentation, while Caco-2 cells were incubated for 21 days to allow differentiation and tight junction formation. The medium was aspirated and replaced with serum-supplemented medium containing serial dilutions of quercetin in free (dissolved in ethanol) or nanomicellar form.
For MTT viability assay, the cells were treated at 37°C for the specified duration, and at the end, MTT was added to each well after medium removal and PBS rinsing to remove residual drug. Following a 4-hour incubation at 37°C, the well content was aspirated and 150 μL DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured with a Tecan SpectraFluorPlus reader (Tecan, Mannedorf, Switzerland). Cell viability was calculated as follows:
where Abstest, Absbackground, Absvehicle represent the absorbance readings from the quercetin-treated wells, the medium-only wells, and the vehicle control wells, respectively.
For LDH release assay, treatment groups, and conditions were similar to those for MTT viability assay, except that the culture medium was collected at the end of treatment for LDH detection using Cytotox-One Homogenous Membrane Integrity Assay Kit (Promega Corporation, Fitchburg, WI) following the manufacturer’s instructions. Fluorescence was measured using the Tecan SpectraFluorPlus plate reader with an excitation wavelength of 560 nm and emission wavelength of 590 nm. Cytotoxicity was calculated as follow:
where Ftest, Ftriton-X and Fbackground represent the fluorescence readings from the test condition (CNM or vehicle alone), triton-X 0.2%, and background.
Transepithelial electrical resistance (TEER) measurement in Caco-2 cells Cells were seeded at a density of 1.2 × 104 cells/well in collagen- coated filter membrane polycarbonate transwell inserts (12 mm, 1-μm pore size, 0.3 cm2 growth area) obtained from BD Biocoat (Erembodegem, Belgium) and incubated at 37°C for 21 days. On day 21, the baseline TEER values of Caco-2 cells were measured using Millicell ERS-2 Epithelial Volt-Ohm Meter, and inserts that record > 600 Ωcm2 were used for experiments. The chamber was washed twice with Hank’s balanced salt solution before the addition of drug treatment. The cells were treated for 24 hours at 37°C, and TEER values were recorded every 24 hours over the study duration of 96 hours.
Cells were seeded at a density of 1.2 × 104 cells/well in collagen- coated filter membrane polycarbonate transwell inserts (12 mm, 1-μm pore size, 0.3 cm2 growth area) obtained from BD Biocoat (Erembodegem, Belgium) and incubated at 37°C for 21 days. On day 21, the baseline TEER values of Caco-2 cells were measured using Millicell ERS-2 Epithelial Volt-Ohm Meter, and inserts that record > 600 Ωcm2 were used for experiments. The chamber was washed twice with Hank’s balanced salt solution before the addition of drug treatment. The cells were treated for 24 hours at 37°C, and TEER values were recorded every 24 hours over the study duration of 96 hours.
Animal efficacy study The animal study was conducted in the facilities of the British Columbia Cancer Research Center, with methodologies approved by the Institutional Animal Care Committee of the University of British Columbia, Vancouver, BC, Canada. The mice were cared for and used in accordance with the Canadian Council on Animal Care Guidelines. All mice used were between 20–22 g, and were housed in micro-isolator cages and given free access to food and water. Tumors were established in female Rag-2M mice by a single subcutaneous injection of 5 × 106 A549 cells (in 50 μL) in the lower back area. Tumor growth was monitored by caliper measurements along the length and width. Tumor volumes were calculated by the following formula: Tumor volume = 0.5 × (length × width2). When the tumor size reached 50–100 mm3 (3 weeks post-tumor cell inoculation), mice were randomized into three study groups of six animals per group: (1) untreated control, (2) peroral quercetin suspension (vehicle = 25% v/v ethanol in water), and (3) peroral nanomicellar quercetin. The dose of quercetin was 30 mg/kg, following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. Tumor size and body weight of the mice was monitored thrice weekly. Animals whose tumor size reached 1000 mm3, developed ulcerations, or displayed a weight loss of more than 5% were euthanized.
The animal study was conducted in the facilities of the British Columbia Cancer Research Center, with methodologies approved by the Institutional Animal Care Committee of the University of British Columbia, Vancouver, BC, Canada. The mice were cared for and used in accordance with the Canadian Council on Animal Care Guidelines. All mice used were between 20–22 g, and were housed in micro-isolator cages and given free access to food and water. Tumors were established in female Rag-2M mice by a single subcutaneous injection of 5 × 106 A549 cells (in 50 μL) in the lower back area. Tumor growth was monitored by caliper measurements along the length and width. Tumor volumes were calculated by the following formula: Tumor volume = 0.5 × (length × width2). When the tumor size reached 50–100 mm3 (3 weeks post-tumor cell inoculation), mice were randomized into three study groups of six animals per group: (1) untreated control, (2) peroral quercetin suspension (vehicle = 25% v/v ethanol in water), and (3) peroral nanomicellar quercetin. The dose of quercetin was 30 mg/kg, following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. Tumor size and body weight of the mice was monitored thrice weekly. Animals whose tumor size reached 1000 mm3, developed ulcerations, or displayed a weight loss of more than 5% were euthanized.
Statistics Where appropriate, results are expressed as mean ± SEM of at least three independent experiments. Statistical significance was assessed using one-way ANOVA followed by Fisher’s least significant difference (LSD) test for multiple comparisons. A P value of <0.05 was considered to be statistically significant. All statistical analyses were carried out using SPSS software (v 19.0; IBM, Armonk, NY).
Where appropriate, results are expressed as mean ± SEM of at least three independent experiments. Statistical significance was assessed using one-way ANOVA followed by Fisher’s least significant difference (LSD) test for multiple comparisons. A P value of <0.05 was considered to be statistically significant. All statistical analyses were carried out using SPSS software (v 19.0; IBM, Armonk, NY).
Materials:
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [poly(ethyleneglycol)-2000-N′-carboxyfluorescein] (FITC-DSPE-PEG2000) were purchased from Avanti-Polar-Lipids (Alabaster, AL). Cell culture media and supplements were from Gibco (Grand Island, NY). All other chemicals were purchased from Sigma-Aldrich Inc (St Louis, MO). A549, MDA-MB-231, and Caco-2 cell lines were purchased from the American Type Culture Collection (Manassas, VA).
Preparation of nanomicellar quercetin:
The quercetin nanomicelles were prepared as follows: briefly, DSPE-PEG2000 was dissolved in chloroform and quercetin in ethanol, the two organic solutions were mixed together according to the various weight proportions of drug and PEG-lipid. Subsequently, the organic phase was evaporated off under a stream of nitrogen gas to obtain a dried thin film. The film was further placed under vacuum for at least 3 hours to remove the residual solvent. Hank’s balanced salt solution or phosphate-buffered saline (PBS) was used to hydrate the thin film to form the nanomicelles. For fluorescence-labeled nanomicelles, 1% of FITC-DSPE-PEG2000 was mixed with 99% of DSPE-PEG2000 (by mole) in chloroform, followed by evaporation and hydration as described above. Unincorporated quercetin was removed by centrifugation at 13,000 rpm for 10 minutes followed by filtration through a 0.2-μm filter. The resultant nanomicelle samples were frozen at −86°C for 8 hours (ULT Freezer; Thermo Electron Corporation Forma, Waltham, MA,) and subsequently lyophilized at −50°C to −55°C for 48 hours at 5 Pa (Alpha 2–4 LDplus Freeze Dryer; Christ, Osterode, Germany). No secondary drying was performed. Lyophilized samples were stored at 4°C ± 2°C until further evaluation, and subsequently were reconstituted to original volume using milli-Q water as and when required. Drug concentration was determined by reading absorbance at 376 nm after mixing the micelle samples in ethanol, using a standard curve constructed with quercetin dissolved in ethanol. The size of the nanomicelles was determined by dynamic light scattering (Malvern Zetasizer 3000HS; Malvern, Worcestershire, UK).
Stability of nanomicellar quercetin in simulated physiological fluids:
The stability of quercetin nanomicelles were evaluated using two different aqueous media. The composition of the simulated gastric fluid (SGF) was as follows: 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2, and that of the simulated intestinal fluid (SIF) was as follows: 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0. The nanomicellar quercetin formulation was dialyzed in 1:5000 v/v in the respective simulated fluids for the specified duration. Subsequently, the formulation was analyzed for size and drug content as described above.
Cell culture:
Cells were grown in humidified atmosphere of 5% CO2 at 37°C, with media changed every 3–4 days. Caco-2 cells were maintained in Eagle’s minimal essential medium (EMEM) supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Stock cultures were grown in T-75 flasks. Upon 80%–90% confluency, cells were spilt using 2.5% trypsin containing 1.0 mM EDTA. All cell culture media and reagents were obtained from Gibco. A549 and MDA-MB-231 cells were maintained in RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. Upon 80%–90% confluency, cells were spilt using 0.25% trypsin.
Accumulation of nanomicelles in Caco-2 cells:
Caco-2 cells were seeded in 4-well Lab-tek chamber glass slides from Nalgene Nunc Inc (Naperville, IL) at a density of 7.2 × 104 cells/well and cultured for 21 days.
Subsequently, FITC-labeled nanomicelles were added to the cells and incubated for the specified duration at 37°C. After incubation, cells were washed three times with ice-cold PBS before viewing under a Carl Zeiss fluorescence confocal microscope (Oberkochen, Germany) using 40× objective.
MTT viability assay and lactose dehydrogenase (LDH) release assay:
The plating densities for A549 cells, MDA-MB-231 cells, and Caco-2 cells were 5.0 × 103 cells/well, 3.0 × 103 cells/well, and 1.33 × 104 cells/well, respectively, in the 96-well plates. A549 and MDA-MB-231 cells were incubated for 24 hours to allow attachment to plate before experimentation, while Caco-2 cells were incubated for 21 days to allow differentiation and tight junction formation. The medium was aspirated and replaced with serum-supplemented medium containing serial dilutions of quercetin in free (dissolved in ethanol) or nanomicellar form.
For MTT viability assay, the cells were treated at 37°C for the specified duration, and at the end, MTT was added to each well after medium removal and PBS rinsing to remove residual drug. Following a 4-hour incubation at 37°C, the well content was aspirated and 150 μL DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured with a Tecan SpectraFluorPlus reader (Tecan, Mannedorf, Switzerland). Cell viability was calculated as follows:
where Abstest, Absbackground, Absvehicle represent the absorbance readings from the quercetin-treated wells, the medium-only wells, and the vehicle control wells, respectively.
For LDH release assay, treatment groups, and conditions were similar to those for MTT viability assay, except that the culture medium was collected at the end of treatment for LDH detection using Cytotox-One Homogenous Membrane Integrity Assay Kit (Promega Corporation, Fitchburg, WI) following the manufacturer’s instructions. Fluorescence was measured using the Tecan SpectraFluorPlus plate reader with an excitation wavelength of 560 nm and emission wavelength of 590 nm. Cytotoxicity was calculated as follow:
where Ftest, Ftriton-X and Fbackground represent the fluorescence readings from the test condition (CNM or vehicle alone), triton-X 0.2%, and background.
Transepithelial electrical resistance (TEER) measurement in Caco-2 cells:
Cells were seeded at a density of 1.2 × 104 cells/well in collagen- coated filter membrane polycarbonate transwell inserts (12 mm, 1-μm pore size, 0.3 cm2 growth area) obtained from BD Biocoat (Erembodegem, Belgium) and incubated at 37°C for 21 days. On day 21, the baseline TEER values of Caco-2 cells were measured using Millicell ERS-2 Epithelial Volt-Ohm Meter, and inserts that record > 600 Ωcm2 were used for experiments. The chamber was washed twice with Hank’s balanced salt solution before the addition of drug treatment. The cells were treated for 24 hours at 37°C, and TEER values were recorded every 24 hours over the study duration of 96 hours.
Animal efficacy study:
The animal study was conducted in the facilities of the British Columbia Cancer Research Center, with methodologies approved by the Institutional Animal Care Committee of the University of British Columbia, Vancouver, BC, Canada. The mice were cared for and used in accordance with the Canadian Council on Animal Care Guidelines. All mice used were between 20–22 g, and were housed in micro-isolator cages and given free access to food and water. Tumors were established in female Rag-2M mice by a single subcutaneous injection of 5 × 106 A549 cells (in 50 μL) in the lower back area. Tumor growth was monitored by caliper measurements along the length and width. Tumor volumes were calculated by the following formula: Tumor volume = 0.5 × (length × width2). When the tumor size reached 50–100 mm3 (3 weeks post-tumor cell inoculation), mice were randomized into three study groups of six animals per group: (1) untreated control, (2) peroral quercetin suspension (vehicle = 25% v/v ethanol in water), and (3) peroral nanomicellar quercetin. The dose of quercetin was 30 mg/kg, following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. Tumor size and body weight of the mice was monitored thrice weekly. Animals whose tumor size reached 1000 mm3, developed ulcerations, or displayed a weight loss of more than 5% were euthanized.
Statistics:
Where appropriate, results are expressed as mean ± SEM of at least three independent experiments. Statistical significance was assessed using one-way ANOVA followed by Fisher’s least significant difference (LSD) test for multiple comparisons. A P value of <0.05 was considered to be statistically significant. All statistical analyses were carried out using SPSS software (v 19.0; IBM, Armonk, NY).
Results:
Formulating quercetin into PEG-lipid nanomicelles The commonly used PEG-lipid conjugate, DSPE-PEG2000, was combined with quercetin in various weight proportions (1%–5% w/w of quercetin in DSPE-PEG2000). As shown in Table 1, the incorporation efficiencies of quercetin into the DSPE-PEG2000 nanomicelles were found to be 88.9% and above when the content of quercetin was up to 4% w/w. At 5% w/w, the incorporation efficiency was reduced to 79.3%. The size of the drug-loaded nanomicelles ranged from 15.4 to 18.5 nm (Table 1), and the polydispersity indices were <0.250 for all the nanomicellar formulations. The zeta potentials of the empty and drug-loaded nanomicelles were −11.8 ± 0.7 mV and −14.8 ± 0.7 mV, respectively. The highest concentration of nanomicellar quercetin in Hepes-buffered saline that could be achieved without precipitation was 3 mg/mL. This represents a 110-fold increase in the aqueous concentration of quercetin, calculated based on the aqueous solubility of free quercetin of 80 μM.17,18 Furthermore, nanomicellar quercetin could be processed into a powder by freeze-drying, without the need of a cryoprotectant or lyoprotectant. Reconstitution of the freeze-dried, nanomicellar quercetin with water did not result in a change in size or quercetin content (data not shown). Hence, subsequent evaluation of the nanomicellar formulation was based on the reconstituted nanomicellar quercetin.
The commonly used PEG-lipid conjugate, DSPE-PEG2000, was combined with quercetin in various weight proportions (1%–5% w/w of quercetin in DSPE-PEG2000). As shown in Table 1, the incorporation efficiencies of quercetin into the DSPE-PEG2000 nanomicelles were found to be 88.9% and above when the content of quercetin was up to 4% w/w. At 5% w/w, the incorporation efficiency was reduced to 79.3%. The size of the drug-loaded nanomicelles ranged from 15.4 to 18.5 nm (Table 1), and the polydispersity indices were <0.250 for all the nanomicellar formulations. The zeta potentials of the empty and drug-loaded nanomicelles were −11.8 ± 0.7 mV and −14.8 ± 0.7 mV, respectively. The highest concentration of nanomicellar quercetin in Hepes-buffered saline that could be achieved without precipitation was 3 mg/mL. This represents a 110-fold increase in the aqueous concentration of quercetin, calculated based on the aqueous solubility of free quercetin of 80 μM.17,18 Furthermore, nanomicellar quercetin could be processed into a powder by freeze-drying, without the need of a cryoprotectant or lyoprotectant. Reconstitution of the freeze-dried, nanomicellar quercetin with water did not result in a change in size or quercetin content (data not shown). Hence, subsequent evaluation of the nanomicellar formulation was based on the reconstituted nanomicellar quercetin.
In vitro cytotoxicity of nanomicellar quercetin in the A549 lung cancer cell line The cytotoxicity of nanomicellar quercetin was evaluated in the A549 human lung cancer cell line. The highest concentration of quercetin evaluated was 100 μM, as drug precipitation was observed at higher drug concentrations. As shown in Figure 1, the anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation, as determined by the MTT viability assay. Of note, non-drug loaded DSPE-PEG2000 nanomicelles did not cause significant toxicity to the A549 lung cancer cells up to a concentration of 1 mM, with cell viability of >85%. To ensure that the cytotoxic effect was not cell line specific, the MDA-MB-231 human breast cancer cell line was treated with free and nanomicellar quercetin. As shown in Suppl Data Figure 1, a similar trend could be observed whereby the activity of quercetin could be significantly increased when formulated into the nanomicelles.
The cytotoxicity of nanomicellar quercetin was evaluated in the A549 human lung cancer cell line. The highest concentration of quercetin evaluated was 100 μM, as drug precipitation was observed at higher drug concentrations. As shown in Figure 1, the anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation, as determined by the MTT viability assay. Of note, non-drug loaded DSPE-PEG2000 nanomicelles did not cause significant toxicity to the A549 lung cancer cells up to a concentration of 1 mM, with cell viability of >85%. To ensure that the cytotoxic effect was not cell line specific, the MDA-MB-231 human breast cancer cell line was treated with free and nanomicellar quercetin. As shown in Suppl Data Figure 1, a similar trend could be observed whereby the activity of quercetin could be significantly increased when formulated into the nanomicelles.
Stability of nanomicellar quercetin in simulated physiological fluids Since the nanomicelles are to be administered perorally, stability in the fluids of the gastrointestinal tract has to be demonstrated. The stability of nanomicellar quercetin was evaluated over time in simulated gastric fluid (SGF, 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2) and simulated intestinal fluid (SIF, 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0). The quercetin nanomicelles were found to be stable, without significant change in size or drug precipitation (Figure 2). To investigate the amount of quercetin retained in the nanomicelles, the micelle samples were dialyzed against 1:5000 (v/v) SGF or SIF at 37°C using dialysis cassettes. Approximately 70%–75% of incorporated quercetin was retained in the nanomicelles in the respective simulated fluids (Figure 2).
Since the nanomicelles are to be administered perorally, stability in the fluids of the gastrointestinal tract has to be demonstrated. The stability of nanomicellar quercetin was evaluated over time in simulated gastric fluid (SGF, 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2) and simulated intestinal fluid (SIF, 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0). The quercetin nanomicelles were found to be stable, without significant change in size or drug precipitation (Figure 2). To investigate the amount of quercetin retained in the nanomicelles, the micelle samples were dialyzed against 1:5000 (v/v) SGF or SIF at 37°C using dialysis cassettes. Approximately 70%–75% of incorporated quercetin was retained in the nanomicelles in the respective simulated fluids (Figure 2).
Interaction of nanomicellar quercetin with Caco-2 cell monolayer The well-established Caco-2 cell monolayer was employed as the in vitro model for gastrointestinal epithelium, and the cellular accumulation of the PEG-lipid nanomicelles was assessed by confocal microscopy. To facilitate visualization, the nanomicelles were spiked with 1 mol% DSPE-PEG2000-N-carboxyfluorescein. The Caco-2 cells were exposed to the fluorescence-labeled nanomicelles for up to 24 hours at 37°C, and cellular accumulation of the nanomicelles increased over time (Figure 3A). Furthermore, the accumulation of the nanomicelles was independent of P-gp activity and endocytic processes, which was supported by results summarized in Suppl Data Figure 2. Cellular accumulation of the carboxyfluorescein-tagged nanomicelles occurred even in the presence of the P-gp inhibitor cyclosporine A or in the presence of endocytic inhibitors (hyperosmotic sucrose of 0.45 M or Brefeldin A) or a change of incubation temperature from 37°C to 4°C. Collectively, these data suggest that the nanomicelles are likely to accumulate inside Caco-2 cells by passive diffusion.
One possible route to facilitate drug absorption through the intestinal epithelium is by disrupting the tight junctions and thus making paracellular transport of drug molecules possible, which could be reflected by a decrease in the TEER.19 Nevertheless, it is also of equal importance that the disruption of the epithelial tight junctions is only transient, as irreversible disruption could represent potential toxicity to the intestinal epithelium. As such, differentiated Caco-2 cell monolayers grown in transwell inserts were exposed to free or nanomicellar quercetin for 24 hours, and TEER measurements were carried out over a time course of 96 hours. As shown in Figure 3B, Caco-2 cell monolayer treated with free quercetin had small yet statistically significant increase in TEER after 48 hours of exposure that gradually decreased back to control level (t = 0 hours). This observation is in line with the protective effect of quercetin on the intestinal barrier function, which acts by promoting the assembly and expression of tight junction proteins.20 In contrast, significant reduction in TEER was observed in Caco-2 cell monolayer treated with nanomicellar quercetin during the first 48 hours. Of note, the reduction in TEER in the nanomicellar quercetin treatment group could be gradually increased back to control level (t = 0 hours), indicating the reversibility of the treatment.
To further investigate the potential toxicity of the nanomicellar quercetin formulation, release of LDH was determined in differentiated Caco-2 cell monolayer upon 24-hour treatment with free or nanomicellar quercetin (Figure 4). Treatment with free quercetin was non-toxic over the concentration range tested, with ~10% LDH released relative to triton-X control. In contrast, nanomicellar quercetin treatment yielded a small but significant increase in LDH release of ~25% relative to triton-X control. Empty, non-drug loaded nanomicelles were evaluated at concentrations up to 1 mM and were found to be relatively non-toxic to the Caco-2 cell monolayer, with viability of ~80% at 1 mM (Suppl Data, Figure 3).
The well-established Caco-2 cell monolayer was employed as the in vitro model for gastrointestinal epithelium, and the cellular accumulation of the PEG-lipid nanomicelles was assessed by confocal microscopy. To facilitate visualization, the nanomicelles were spiked with 1 mol% DSPE-PEG2000-N-carboxyfluorescein. The Caco-2 cells were exposed to the fluorescence-labeled nanomicelles for up to 24 hours at 37°C, and cellular accumulation of the nanomicelles increased over time (Figure 3A). Furthermore, the accumulation of the nanomicelles was independent of P-gp activity and endocytic processes, which was supported by results summarized in Suppl Data Figure 2. Cellular accumulation of the carboxyfluorescein-tagged nanomicelles occurred even in the presence of the P-gp inhibitor cyclosporine A or in the presence of endocytic inhibitors (hyperosmotic sucrose of 0.45 M or Brefeldin A) or a change of incubation temperature from 37°C to 4°C. Collectively, these data suggest that the nanomicelles are likely to accumulate inside Caco-2 cells by passive diffusion.
One possible route to facilitate drug absorption through the intestinal epithelium is by disrupting the tight junctions and thus making paracellular transport of drug molecules possible, which could be reflected by a decrease in the TEER.19 Nevertheless, it is also of equal importance that the disruption of the epithelial tight junctions is only transient, as irreversible disruption could represent potential toxicity to the intestinal epithelium. As such, differentiated Caco-2 cell monolayers grown in transwell inserts were exposed to free or nanomicellar quercetin for 24 hours, and TEER measurements were carried out over a time course of 96 hours. As shown in Figure 3B, Caco-2 cell monolayer treated with free quercetin had small yet statistically significant increase in TEER after 48 hours of exposure that gradually decreased back to control level (t = 0 hours). This observation is in line with the protective effect of quercetin on the intestinal barrier function, which acts by promoting the assembly and expression of tight junction proteins.20 In contrast, significant reduction in TEER was observed in Caco-2 cell monolayer treated with nanomicellar quercetin during the first 48 hours. Of note, the reduction in TEER in the nanomicellar quercetin treatment group could be gradually increased back to control level (t = 0 hours), indicating the reversibility of the treatment.
To further investigate the potential toxicity of the nanomicellar quercetin formulation, release of LDH was determined in differentiated Caco-2 cell monolayer upon 24-hour treatment with free or nanomicellar quercetin (Figure 4). Treatment with free quercetin was non-toxic over the concentration range tested, with ~10% LDH released relative to triton-X control. In contrast, nanomicellar quercetin treatment yielded a small but significant increase in LDH release of ~25% relative to triton-X control. Empty, non-drug loaded nanomicelles were evaluated at concentrations up to 1 mM and were found to be relatively non-toxic to the Caco-2 cell monolayer, with viability of ~80% at 1 mM (Suppl Data, Figure 3).
In vivo efficacy of nanomicellar quercetin in human A549 lung tumor xenograft model Based on the in vitro evaluation of the nanomicellar quercetin formulation which provided promising results, the biological activity of the peroral formulation was further investigated in the A549 lung tumor xenograft model. Quercetin was administered to the tumor-bearing mice via oral gavage either in the nanomicellar form or in a 25% v/v ethanol-based suspension, with a dose of 30 mg/kg following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. The results demonstrated that the anti-tumor activity of quercetin was significantly increased when administered as the peroral nanomicelles compared to control and peroral quercetin suspension (Figure 5A). Specifically, tumor growth inhibition at 10-week post-tumor inoculation was 83% for peroral quercetin suspension and 48% for peroral nanomicellar formulation. The two quercetin formulations were well tolerated by the animals, as reflected by minimal changes in the body weight of the mice (Figure 5B). No unusual observations were made upon necropsies of the animals from all three study groups.
Based on the in vitro evaluation of the nanomicellar quercetin formulation which provided promising results, the biological activity of the peroral formulation was further investigated in the A549 lung tumor xenograft model. Quercetin was administered to the tumor-bearing mice via oral gavage either in the nanomicellar form or in a 25% v/v ethanol-based suspension, with a dose of 30 mg/kg following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. The results demonstrated that the anti-tumor activity of quercetin was significantly increased when administered as the peroral nanomicelles compared to control and peroral quercetin suspension (Figure 5A). Specifically, tumor growth inhibition at 10-week post-tumor inoculation was 83% for peroral quercetin suspension and 48% for peroral nanomicellar formulation. The two quercetin formulations were well tolerated by the animals, as reflected by minimal changes in the body weight of the mice (Figure 5B). No unusual observations were made upon necropsies of the animals from all three study groups.
Formulating quercetin into PEG-lipid nanomicelles:
The commonly used PEG-lipid conjugate, DSPE-PEG2000, was combined with quercetin in various weight proportions (1%–5% w/w of quercetin in DSPE-PEG2000). As shown in Table 1, the incorporation efficiencies of quercetin into the DSPE-PEG2000 nanomicelles were found to be 88.9% and above when the content of quercetin was up to 4% w/w. At 5% w/w, the incorporation efficiency was reduced to 79.3%. The size of the drug-loaded nanomicelles ranged from 15.4 to 18.5 nm (Table 1), and the polydispersity indices were <0.250 for all the nanomicellar formulations. The zeta potentials of the empty and drug-loaded nanomicelles were −11.8 ± 0.7 mV and −14.8 ± 0.7 mV, respectively. The highest concentration of nanomicellar quercetin in Hepes-buffered saline that could be achieved without precipitation was 3 mg/mL. This represents a 110-fold increase in the aqueous concentration of quercetin, calculated based on the aqueous solubility of free quercetin of 80 μM.17,18 Furthermore, nanomicellar quercetin could be processed into a powder by freeze-drying, without the need of a cryoprotectant or lyoprotectant. Reconstitution of the freeze-dried, nanomicellar quercetin with water did not result in a change in size or quercetin content (data not shown). Hence, subsequent evaluation of the nanomicellar formulation was based on the reconstituted nanomicellar quercetin.
In vitro cytotoxicity of nanomicellar quercetin in the A549 lung cancer cell line:
The cytotoxicity of nanomicellar quercetin was evaluated in the A549 human lung cancer cell line. The highest concentration of quercetin evaluated was 100 μM, as drug precipitation was observed at higher drug concentrations. As shown in Figure 1, the anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation, as determined by the MTT viability assay. Of note, non-drug loaded DSPE-PEG2000 nanomicelles did not cause significant toxicity to the A549 lung cancer cells up to a concentration of 1 mM, with cell viability of >85%. To ensure that the cytotoxic effect was not cell line specific, the MDA-MB-231 human breast cancer cell line was treated with free and nanomicellar quercetin. As shown in Suppl Data Figure 1, a similar trend could be observed whereby the activity of quercetin could be significantly increased when formulated into the nanomicelles.
Stability of nanomicellar quercetin in simulated physiological fluids:
Since the nanomicelles are to be administered perorally, stability in the fluids of the gastrointestinal tract has to be demonstrated. The stability of nanomicellar quercetin was evaluated over time in simulated gastric fluid (SGF, 0.2% w/v NaCl in 0.7% v/v HCl, pH 1.2) and simulated intestinal fluid (SIF, 0.05 M potassium dihydrogen phosphate/0.02 M sodium hydroxide, pH 7.0). The quercetin nanomicelles were found to be stable, without significant change in size or drug precipitation (Figure 2). To investigate the amount of quercetin retained in the nanomicelles, the micelle samples were dialyzed against 1:5000 (v/v) SGF or SIF at 37°C using dialysis cassettes. Approximately 70%–75% of incorporated quercetin was retained in the nanomicelles in the respective simulated fluids (Figure 2).
Interaction of nanomicellar quercetin with Caco-2 cell monolayer:
The well-established Caco-2 cell monolayer was employed as the in vitro model for gastrointestinal epithelium, and the cellular accumulation of the PEG-lipid nanomicelles was assessed by confocal microscopy. To facilitate visualization, the nanomicelles were spiked with 1 mol% DSPE-PEG2000-N-carboxyfluorescein. The Caco-2 cells were exposed to the fluorescence-labeled nanomicelles for up to 24 hours at 37°C, and cellular accumulation of the nanomicelles increased over time (Figure 3A). Furthermore, the accumulation of the nanomicelles was independent of P-gp activity and endocytic processes, which was supported by results summarized in Suppl Data Figure 2. Cellular accumulation of the carboxyfluorescein-tagged nanomicelles occurred even in the presence of the P-gp inhibitor cyclosporine A or in the presence of endocytic inhibitors (hyperosmotic sucrose of 0.45 M or Brefeldin A) or a change of incubation temperature from 37°C to 4°C. Collectively, these data suggest that the nanomicelles are likely to accumulate inside Caco-2 cells by passive diffusion.
One possible route to facilitate drug absorption through the intestinal epithelium is by disrupting the tight junctions and thus making paracellular transport of drug molecules possible, which could be reflected by a decrease in the TEER.19 Nevertheless, it is also of equal importance that the disruption of the epithelial tight junctions is only transient, as irreversible disruption could represent potential toxicity to the intestinal epithelium. As such, differentiated Caco-2 cell monolayers grown in transwell inserts were exposed to free or nanomicellar quercetin for 24 hours, and TEER measurements were carried out over a time course of 96 hours. As shown in Figure 3B, Caco-2 cell monolayer treated with free quercetin had small yet statistically significant increase in TEER after 48 hours of exposure that gradually decreased back to control level (t = 0 hours). This observation is in line with the protective effect of quercetin on the intestinal barrier function, which acts by promoting the assembly and expression of tight junction proteins.20 In contrast, significant reduction in TEER was observed in Caco-2 cell monolayer treated with nanomicellar quercetin during the first 48 hours. Of note, the reduction in TEER in the nanomicellar quercetin treatment group could be gradually increased back to control level (t = 0 hours), indicating the reversibility of the treatment.
To further investigate the potential toxicity of the nanomicellar quercetin formulation, release of LDH was determined in differentiated Caco-2 cell monolayer upon 24-hour treatment with free or nanomicellar quercetin (Figure 4). Treatment with free quercetin was non-toxic over the concentration range tested, with ~10% LDH released relative to triton-X control. In contrast, nanomicellar quercetin treatment yielded a small but significant increase in LDH release of ~25% relative to triton-X control. Empty, non-drug loaded nanomicelles were evaluated at concentrations up to 1 mM and were found to be relatively non-toxic to the Caco-2 cell monolayer, with viability of ~80% at 1 mM (Suppl Data, Figure 3).
In vivo efficacy of nanomicellar quercetin in human A549 lung tumor xenograft model:
Based on the in vitro evaluation of the nanomicellar quercetin formulation which provided promising results, the biological activity of the peroral formulation was further investigated in the A549 lung tumor xenograft model. Quercetin was administered to the tumor-bearing mice via oral gavage either in the nanomicellar form or in a 25% v/v ethanol-based suspension, with a dose of 30 mg/kg following a schedule of three times per week (Monday/Wednesday/Friday) for 3 weeks. The results demonstrated that the anti-tumor activity of quercetin was significantly increased when administered as the peroral nanomicelles compared to control and peroral quercetin suspension (Figure 5A). Specifically, tumor growth inhibition at 10-week post-tumor inoculation was 83% for peroral quercetin suspension and 48% for peroral nanomicellar formulation. The two quercetin formulations were well tolerated by the animals, as reflected by minimal changes in the body weight of the mice (Figure 5B). No unusual observations were made upon necropsies of the animals from all three study groups.
Discussion:
Quercetin, a flavanol that is found in certain vegetables, fruits, and tea, has been shown to have a range of biochemical and biological activities that could have the potential to translate to therapeutic benefits. In addition to its antioxidant and anti-inflammatory effects, quercetin as a tyrosine kinase inhibitor8 is able to modulate multiple signaling pathways that would have considerable implications in the therapeutic and prophylactic values for diseases such as cancer. However, realizing the biological benefits of quercetin in the clinical setting is mostly hampered by its low water solubility and poor absorption into the body. For instance, the baseline plasma concentration of quercetin from dietary intake is 50–80 nM,21 and even with supplementation at 1 g/day for 28 days, the plasma concentration is ~1.5 μM,22 which is far below the concentration that is necessary to bring about the intended pharmacological effects of quercetin (such as the concentrations needed for anticancer effect presented in Figure 1). Thus, it is imperative to develop appropriate vehicles for quercetin, and it is the intention of our current study to develop a peroral nanomicellar system that could potentially improve its anticancer activity, which has not been extensively studied.
Since the quercetin-loaded DSPE-PEG2000 nanomicelles are intended for peroral application, the nanomicelles should be able to withstand the harsh environment of the digestive tract such as the drastic changes in pH, and at the same time, be relatively non-toxic to the intestinal epithelium. As demonstrated in Figure 2, the quercetin nanomicelles were relatively stable at pH 1.2 and pH 7, with no significant changes in size and ~30% release of quercetin. These findings are in good agreement with a previous study by Dabholkar et al which reported no significant change in the size of mixed micelles made of DSPE-PEG2000 and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) incubated in acidic (pH 1.2) and neutral (pH 7.4) conditions.23 In terms of toxicity to the intestinal epithelium, although the incorporation of quercetin into the nanomicelles has slightly increased the cytotoxicity, as reflected by the data in Figure 4, this level of cytotoxicity (~25% as determined by LDH release) was comparable to those reported for TPGS and its analogs which are PEG-derivatives well established for their ability to modulate peroral drug absorption. The Caco-2 cytotoxicity of the TPGS analogs was reported to range from 20%–40%, as determined by LDH release.24 Furthermore, quercetin-loaded nanomicelles did not induce irreversible reduction in the TEER of the Caco-2 epithelium (Figure 3B). This finding, together with those in Figure 4 and Suppl Data Figure 3, showed that the quercetin-loaded nanomicelles were relatively non-toxic, which is in line with another study reporting that empty or meso-tetraphenyl porphine loaded DSPE-PEG2000 micelles were minimally toxic to Caco-2 cells.25
The therapeutic benef its of perorally administered quercetin is limited by poor absorption into the body that could be attributed to a number of reasons, including its chemical structure, low water solubility, and extensive metabolism and degradation in the gastrointestinal tract.26 Of interest, quercetin has been shown to protect the intestinal epithelial barrier function by promoting the expression of a number of tight junction proteins and increase TEER of Caco-2 monolayer,27 implying that quercetin may limit its own absorption into the body by reducing paracellular permeability. Our current DSPE-PEG2000-based peroral nanomicellar formulation could increase the anticancer activity of quercetin by two possible mechanisms with supportive data: (1) increase in the aqueous concentration of quercetin (by 110-fold to 3 mg/mL) through solubilization in the nanomicelles, and (2) increase in permeability through modulation of the epithelial tight junctions as reflected by the transient decrease in TEER. At the molecular level, quercetin is able to increase the distribution of tight junction proteins, ZO-2, occludin, and claudin-1, to the actin cytoskeleton fraction, as well as increase the expression of claudin-4 in cytoskeleton fraction and in whole cell.27 From Figure 3B, it can be seen that empty nanomicelles could reduce TEER reversibly, and it is possible that the empty micelles could disrupt the assembly of the tight junction complex through the partition of DSPE-PEG2000 into the cell membrane bilayer.
Nevertheless, other possible mechanisms that could contribute to the increased activity of quercetin by the nanomicellar peroral formulation could not be ruled out. One such mechanism is the potential ability of the nanomicelles to protect quercetin from the enzymatic and degradative action in the intestinal tract. The metabolism of quercetin is complex, involving the formation of multiple metabolites by different pathways.28 Thus, future investigations on the full pharmacokinetic behavior of the nanomicellar formulation with metabolite profiling are necessary to gain insights if the nanomicelles could indeed protect quercetin from enzymatic metabolism in vivo. Currently, we are establishing mass spectroscopy-based analytical method to characterize the metabonomics of quercetin to address the question. Another possible mechanism is the increased mucoadhesion of the nanomicelles to the intestinal mucosa that could be provided by the PEG shell of the nanomicelles.29 Furthermore, it has been suggested that nanovehicles made of lipids could be taken up by lymphoid tissues in the intestinal tract which in turn could circumvent first pass metabolism.13
It is encouraging that the nanomicellar peroral formulation could enhance the anticancer activity of quercetin at a dose of 30 mg/kg. It has been reported that a dose of 3 g/kg, which is 100-fold higher than ours, did not cause any toxic or harmful effect to mice.30 Thus, future studies could titrate our current dose to higher levels, with the dosing frequency adjusted to daily dosing. The dose titration study could be further coupled to the examination of the correlation between tyrosine kinase inhibition and tumor growth inhibition, which could yield interesting pharmacodynamic data on this nanomicellar peroral formulation. Further evaluation could also include the potential of this nanomicellar peroral formulation as a chemotherapeutic and chemopreventive agent in animal models.
Supplementary data:
MTT viability of free and nanomicellar quercetin in MDA-MB-231 human breast cancer cell line upon 72 hours of exposure. The concentrations of empty micelle were 96 μM and 192 μM for 30 μM and 60 μM nanomicellar quercetin, respectively.
Notes: Data represent mean ± SEM from three independent experiments. *P < 0.05 as compared to free quercetin group.
Cellular accumulation of FITC-labeled nanomicelles over 24 hours of incubation under various conditions: (A) at 4°C, (B) at 37°C, (C) without cyclosporine A, (D) with cyclosporine A, (E) without 0.45 M sucrose, (F) with 0.45 M sucrose, (G) without brefeldine A, (H) with brefeldine A.
Notes: Representative images from three independent studies are shown.
MTT viability of (A) empty, non-drug loaded nanomicelles, (B) quercetin-loaded nanomicelles (showing micelle concentrations), and (C) free quercetin in Caco-2 cell monolayer upon 24 h of exposure.
|
Background:
Realizing the therapeutic benefits of quercetin is mostly hampered by its low water solubility and poor absorption. In light of the advantages of nanovehicles in the delivery of flavanoids, we aimed to deliver quercetin perorally with nanomicelles made from the diblock copolymer, polyethylene glycol (PEG)-derivatized phosphatidylethanolamine (PE).
Methods:
Quercetin-loaded nanomicelles were prepared by using the film casting method, and were evaluated in terms of drug incorporation efficiency, micelle size, interaction with Caco-2 cells, and anticancer activity in the A549 lung cancer cell line and murine xenograft model.
Results:
The incorporation efficiency into the nanomicelles was ≥88.9% when the content of quercetin was up to 4% w/w, with sizes of 15.4-18.5 nm and polydispersity indices of <0.250. Solubilization of quercetin by the nanomicelles increased its aqueous concentration by 110-fold. The quercetin nanomicelles were stable when tested in simulated gastric (pH 1.2) and intestinal (pH 7.4) fluids, and were non-toxic to the Caco-2 cells as reflected by reversible reduction in transepithelial electrical resistance and ≤25% lactose dehydrogenase release. The anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation when tested using the A549 cancer cell line and murine xenograft model. The nanomicellar quercetin formulation was well tolerated by the tumor-bearing animals, with no significant weight loss observed at the end of the 10-week study period.
Conclusions:
A stable PEG-PE nanomicellar formulation of quercetin was developed with enhanced peroral anticancer activity and no apparent toxicity to the intestinal epithelium.
| null | null | 11,026 | 300 | 19 |
[
"quercetin",
"nanomicelles",
"nanomicellar",
"cells",
"nanomicellar quercetin",
"cell",
"caco",
"drug",
"hours",
"tumor"
] |
[
"test",
"test"
] | null | null |
[CONTENT] quercetin | polymeric micelles | PEG-PE | peroral drug delivery | lung cancer [SUMMARY]
|
[CONTENT] quercetin | polymeric micelles | PEG-PE | peroral drug delivery | lung cancer [SUMMARY]
|
[CONTENT] quercetin | polymeric micelles | PEG-PE | peroral drug delivery | lung cancer [SUMMARY]
| null |
[CONTENT] quercetin | polymeric micelles | PEG-PE | peroral drug delivery | lung cancer [SUMMARY]
| null |
[CONTENT] Administration, Oral | Analysis of Variance | Animals | Antineoplastic Agents | Antioxidants | Caco-2 Cells | Cell Line, Tumor | Cell Survival | Drug Carriers | Drug Stability | Female | Humans | Hydrogen-Ion Concentration | Lung Neoplasms | Mice | Micelles | Nanoparticles | Particle Size | Phosphatidylethanolamines | Polyethylene Glycols | Quercetin | Xenograft Model Antitumor Assays [SUMMARY]
|
[CONTENT] Administration, Oral | Analysis of Variance | Animals | Antineoplastic Agents | Antioxidants | Caco-2 Cells | Cell Line, Tumor | Cell Survival | Drug Carriers | Drug Stability | Female | Humans | Hydrogen-Ion Concentration | Lung Neoplasms | Mice | Micelles | Nanoparticles | Particle Size | Phosphatidylethanolamines | Polyethylene Glycols | Quercetin | Xenograft Model Antitumor Assays [SUMMARY]
|
[CONTENT] Administration, Oral | Analysis of Variance | Animals | Antineoplastic Agents | Antioxidants | Caco-2 Cells | Cell Line, Tumor | Cell Survival | Drug Carriers | Drug Stability | Female | Humans | Hydrogen-Ion Concentration | Lung Neoplasms | Mice | Micelles | Nanoparticles | Particle Size | Phosphatidylethanolamines | Polyethylene Glycols | Quercetin | Xenograft Model Antitumor Assays [SUMMARY]
| null |
[CONTENT] Administration, Oral | Analysis of Variance | Animals | Antineoplastic Agents | Antioxidants | Caco-2 Cells | Cell Line, Tumor | Cell Survival | Drug Carriers | Drug Stability | Female | Humans | Hydrogen-Ion Concentration | Lung Neoplasms | Mice | Micelles | Nanoparticles | Particle Size | Phosphatidylethanolamines | Polyethylene Glycols | Quercetin | Xenograft Model Antitumor Assays [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] quercetin | nanomicelles | nanomicellar | cells | nanomicellar quercetin | cell | caco | drug | hours | tumor [SUMMARY]
|
[CONTENT] quercetin | nanomicelles | nanomicellar | cells | nanomicellar quercetin | cell | caco | drug | hours | tumor [SUMMARY]
|
[CONTENT] quercetin | nanomicelles | nanomicellar | cells | nanomicellar quercetin | cell | caco | drug | hours | tumor [SUMMARY]
| null |
[CONTENT] quercetin | nanomicelles | nanomicellar | cells | nanomicellar quercetin | cell | caco | drug | hours | tumor [SUMMARY]
| null |
[CONTENT] anticancer | delivery | peroral | cancer | activity | use | administration | quercetin | water | peroral anticancer [SUMMARY]
|
[CONTENT] tumor | mice | tumor size | animal | care | size reached | animal care | monitored | reached | columbia [SUMMARY]
|
[CONTENT] quercetin | nanomicellar | nanomicellar quercetin | nanomicelles | figure | cell | cell monolayer | caco cell monolayer | caco cell | caco [SUMMARY]
| null |
[CONTENT] quercetin | cells | nanomicelles | nanomicellar | nanomicellar quercetin | tumor | hours | cell | figure | peroral [SUMMARY]
| null |
[CONTENT] quercetin ||| quercetin [SUMMARY]
|
[CONTENT] Quercetin | Caco-2 | A549 [SUMMARY]
|
[CONTENT] quercetin | up to 4% | 15.4 | 0.250 ||| 110-fold ||| Caco-2 ||| quercetin | A549 ||| the end of the | 10-week [SUMMARY]
| null |
[CONTENT] quercetin ||| quercetin ||| Caco-2 | A549 ||| quercetin | up to 4% | 15.4 | 0.250 ||| 110-fold ||| Caco-2 ||| quercetin | A549 ||| the end of the | 10-week ||| quercetin [SUMMARY]
| null |
||||
Rs12976445 Polymorphism is Associated with Risk of Diabetic Nephropathy Through Modulating Expression of MicroRNA-125 and Interleukin-6R.
|
26563755
|
Diabetic nephropathy (DN) is one of the most significant long-term complications of diabetes mellitus (DM), and it is a primary risk factor for end-stage renal disease. MicroRNAs (miRNAs) play important roles in regulating the expression of genes, including interleukin-6R (IL-6R), which has been reported to be involved in the development of DNDN. The aim of this study was to identify the dysregulation of miRNA and its target responsible for the development of DN in DM.
|
BACKGROUND
|
We collected the kidney tissues from patients with DN (N=36) and control patients (N=28), and performed real-time PCR and Western blot analysis to determine the expression of IL-6R. Computational analysis and luciferase assay were used to identify the miRNA that regulates IL-6R. To explore the association between rs12976445 polymorphism and risk of DN, we enrolled 594 DM patients with (N=282) or without DN (N=312), and studied the association between a variant in miR-125a and risk of DN in DM.
|
MATERIAL AND METHODS
|
The expression of IL-6R was barely detected in the control groups, while in the DN group, the IL-6R was clearly detectable. Next, miR-125a was identified as a regulator of IL-6R by using informatics analysis and luciferase assay. A single-nucleotide polymorphism (rs12976445) in pri-miR-125a has been shown to compromise the mature processing of miR-125a, and we showed that the expression levels of miR-125a was comparable between individuals carrying TT and CT, and when combined into 1 group, the miR-125a expression was approximately 3 times lower than in the CC group. We found significant differences regarding rs12976445 genotype distribution between the DN and the control (OR=1.45, 95% C.I.=1.02-2.08, p<0.05) with the possible confounding factors adjusted for by using logistic regression analysis.
|
RESULTS
|
We identified miR-125a as a direct regulator of IL-6R, and the genotype of rs12976445 might be a novel predictor of the development of DN in DM.
|
CONCLUSIONS
|
[
"Adult",
"Aged",
"Case-Control Studies",
"Diabetic Nephropathies",
"Female",
"Genetic Association Studies",
"Genetic Predisposition to Disease",
"Humans",
"Kidney Failure, Chronic",
"Male",
"MicroRNAs",
"Middle Aged",
"Polymorphism, Single Nucleotide",
"Real-Time Polymerase Chain Reaction",
"Receptors, Interleukin-6",
"Risk Factors"
] |
4648103
|
Background
|
Diabetic nephropathy (DN) is one of the most important long-term complications of diabetes, and it is the primary cause of end-stage renal disease and deaths in diabetic patients [1]. The major clinical characteristics of DN includes progressively decreasing glomerular filtration rate (GFR) and persistent albuminuria, with macroalbuminuria (>300mg/day) indicating DN progression and microalbuminuria (30 to 300 mg of albumin in urine per day) representing early DN [2]. The main pathological characteristics of DN are expansion and hypertrophy in the tubular compartments and glomerular mesangium, as well as buildup of extracellular matrix (ECM) proteins and dysfunction of podocytes. There are several mechanisms, such as protein kinase C, advanced glycation end-products, hyperglycemia, oxidative stress, poly (ADP-ribose) polymerase activation, and inflammation, which are thought to account for the development and pathogenesis of DN [3]. Unfortunately, the hidden molecular pathogenesis is not completely identified.
Interleukin 6 (IL-6) is a multi-potent cytokine; its main function appears to induce proliferation and differentiation of B lymphocyte and acute inflammatory responses. IL-6 has been shown to enhance the differentiation and growth of a wide range of cells, including renal mesangial cells [4]. The aberrantly enhanced proliferation of renal mesangial cells may lead to basement membrane thickening and mesangial matrix expansion, which is a major histological characteristic of diabetic nephropathy [5–7]. Actually, thickening of basement membrane and mesangial proliferation can be observed in early-stage nephropathy [8]. IL-6 may be very important in mesangial proliferation in patients who have glomerular renal disease [4,9]. IL-6 acts on a receptor that consists of 2 subunits, gp130 and IL-6 receptor (IL-6R). First, IL-6 binds to the IL-6R, and this complex subsequently binds to the gp130 subunit [10,11]. Elevated concentrations of IL-6R have been observed in both DN [12] and type II diabetes mellitus (DM) [13].
MicroRNAs (miRNAs) consist of short non-coding RNAs that regulate physiological and pathological processes by suppressing expression of target gene via blocking protein translation or promoting degradation of mRNA. Recently, studies have discovered key properties for specific miRNAs in a battery of biological and cellular processes, such as proliferation, development, and differentiation [14]. Although the role of miRNAs has been identified in the pathogenesis of many human diseases, their function in diabetes and DN is less understood. Earlier reports have shown that single-nucleotide polymorphisms (SNPs) in miRNA genes may interfere with the interaction between miRNAs and mRNAs of target genes, or compromise the processing of mature miRNA [15–18], which may contribute to susceptibility to certain human diseases [19,20].
In this study, we confirmed the differential expression of IL-6R in DN and identified miR-125a as a regulator of IL-6R. It has been previously shown that a SNP (rs12976445) is present in the pre-miR-125a gene, and compromises the mature processing of the miRNA, which leads to an increased risk of autoimmune thyroid conditions [21] and repeated pregnancy loss [22]. Based on the evidence mentioned above, we studied the association between rs12976445 polymorphism and risk of DN in the patients with DM.
|
Statistical analysis
|
A goodness-of-fit χ2 test in controls was used to assess Hardy-Weinberg equilibrium (HWE) for the genotype. The differences in distributions of demographic characteristics between cases and controls were determined using Pearson’s Tχ2 test. The strength of correlation between the polymorphism and DN risk odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined by univariate logistic regression model. After adjustment for age, sex, smoking status, duration of DM, or DN (as described in Table 1), a multivariate logistic regression model was used for the calculation of adjusted ORs and 95% CIs. Statistical significance was set at P<0.05 that was 2-sided, and SPSS 19.0 (SPSS, Chicago, IL) was used to perform all statistical analyses.
|
Results
|
In this study, we collected the kidney tissues from the patients with DN (N=36) and the control patients (N=28), and performed realtime PCR and Western blot analysis to determine the expression of IL-6R that had been previously reported to be functionally involved in the development of diabetic nephropathy [23]. As expected, the expression of IL-6R was barely detected in the controls groups, while in the DN groups, the IL-6R was clearly detectable (Figure 1A). Similarly, the mRNA expression level of IL-6R was substantially upregulated in the tissue samples from DN patients compared with the controls (Figure 1B).
To identify the potential regulator of IL-6R in the kidneys, we searched the miRNA database (www.mirdb.org) and identify miR-125a as a possible regulator of IL-6R with 2 putative binding sites (91–99 bp and 621–627 bp) within the 3′UTR of the gene. To test it, a luciferase assay reporter system was set up by amplifying and inserting the 3′UTR of IL-6R into pGL3 that contained downstream firefly luciferase. To identify the real binding site responsible for the interaction, the 2 putative binding sites were mutated individually (mutant-1 and mutant-2) and combinatively (mutant-3) as described in Figure 2. Next, the constructs carrying either wild-type or the mutated 3′UTR of IL-6R were transfected into the cultured mesangial cells that overexpressed miR-125a or the scramble control sequence. As presented in Figure 2, we found that the luciferase activity was significantly decreased in the cells transfected with miR-125a mimics and wild-type, mutant-1, and mutant-2 IL-6R 3′UTR, whereas the luciferase activity was similar between the cells transfected with mutant-3 IL-6R 3′UTR and the control (Figure 3), suggesting that either of the binding sites was sufficient for a full interaction between miR-125a and IL-6R.
To further verify the regulatory association between miR-125a and IL-6R, we examined the expression of miR-125a in the above-mentioned DN patients and controls, and we showed that the expression of miR-125a was significantly downregulated in DN patients compared with the controls (Figure 4A). We then regrouped the samples based the rs12976445 polymorphism genotypes, and we found that 35, 24, and 5 participants were genotyped as CC, CT, and TT, respectively. As shown in Figure 4B, the expression levels of miR-125a was comparable between TT and CT, so we merged those 2 groups into 1, which was significantly lower than the CC group.
To further validate IL-6R as a target of miR-125a, we examined the endogenous expression in mesangial cells transfected with miR-125a and IL-6R specific siRNA by using Western blot and real-time PCR analysis. We found that the protein expression level of IL-6R was significantly downregulated by introduction of miR-125a mimics and anti-IL-6R siRNA (Figure 5A); and, consistent with this, the mRNA of IL-6R was also substantially lower in the cells transfected with miR-125a mimics and anti-IL-6R siRNA compared with the control (Figure 5B). To test the effect of downregulation of IL-6R on the proliferation of mesangial cells, we evaluated the viability of the cells transfected with miR-125a and IL-6R specific siRNA. As shown in Figure 6, the viability of the cells transfected with miR-125a and IL-6R specific siRNA were significantly lower than that of the cells transfected with scramble control.
To explore the association between rs12976445 polymorphism and risk of DN, we enrolled 594 DM patients with (N=282) or without DN (N=312). The distributions of rs12976445 polymorphism were in Hardy-Weinberg equilibrium among the case group and the controls. As the expression levels of miR-125a was similar between TT and CT, and genotype frequency is low, we combined the 2 genotype groups together in comparison with the wild-type group (CC). The demographic and clinicopathological parameters in the 2 groups are presented in Table 1. As shown in Table 2, significant differences were noted regarding genotype distribution of rs12976445 between the DN and the controls (OR=1.45, 95% C.I.=1.02–2.08, p<0.05) with the possible confounding factors adjusted for by using logistic regression analysis.
|
Conclusions
|
Our results showed that miR-125a is a key player in the development of DN via targeting IL-6R, and underexpression of miR-125a promoted proliferation of mesangial cells by releasing the inhibition on IL-6R. Downregulation of miR-125a could be, at least partially, attributed to the presence of rs12976445 polymorphism, which compromises the mature processing of the miRNA, demonstrating that miR-125a is a promising novel diagnostic and therapeutic target for DN.
|
[
"Background",
"Preparation of human kidney sample",
"Real-time PCR analysis",
"Rs12976445 polymorphism genotyping",
"Cell culture",
"Transient transfection",
"In vitro cell proliferation assay",
"Luciferase reporter gene assay",
"Western blot"
] |
[
"Diabetic nephropathy (DN) is one of the most important long-term complications of diabetes, and it is the primary cause of end-stage renal disease and deaths in diabetic patients [1]. The major clinical characteristics of DN includes progressively decreasing glomerular filtration rate (GFR) and persistent albuminuria, with macroalbuminuria (>300mg/day) indicating DN progression and microalbuminuria (30 to 300 mg of albumin in urine per day) representing early DN [2]. The main pathological characteristics of DN are expansion and hypertrophy in the tubular compartments and glomerular mesangium, as well as buildup of extracellular matrix (ECM) proteins and dysfunction of podocytes. There are several mechanisms, such as protein kinase C, advanced glycation end-products, hyperglycemia, oxidative stress, poly (ADP-ribose) polymerase activation, and inflammation, which are thought to account for the development and pathogenesis of DN [3]. Unfortunately, the hidden molecular pathogenesis is not completely identified.\nInterleukin 6 (IL-6) is a multi-potent cytokine; its main function appears to induce proliferation and differentiation of B lymphocyte and acute inflammatory responses. IL-6 has been shown to enhance the differentiation and growth of a wide range of cells, including renal mesangial cells [4]. The aberrantly enhanced proliferation of renal mesangial cells may lead to basement membrane thickening and mesangial matrix expansion, which is a major histological characteristic of diabetic nephropathy [5–7]. Actually, thickening of basement membrane and mesangial proliferation can be observed in early-stage nephropathy [8]. IL-6 may be very important in mesangial proliferation in patients who have glomerular renal disease [4,9]. IL-6 acts on a receptor that consists of 2 subunits, gp130 and IL-6 receptor (IL-6R). First, IL-6 binds to the IL-6R, and this complex subsequently binds to the gp130 subunit [10,11]. Elevated concentrations of IL-6R have been observed in both DN [12] and type II diabetes mellitus (DM) [13].\nMicroRNAs (miRNAs) consist of short non-coding RNAs that regulate physiological and pathological processes by suppressing expression of target gene via blocking protein translation or promoting degradation of mRNA. Recently, studies have discovered key properties for specific miRNAs in a battery of biological and cellular processes, such as proliferation, development, and differentiation [14]. Although the role of miRNAs has been identified in the pathogenesis of many human diseases, their function in diabetes and DN is less understood. Earlier reports have shown that single-nucleotide polymorphisms (SNPs) in miRNA genes may interfere with the interaction between miRNAs and mRNAs of target genes, or compromise the processing of mature miRNA [15–18], which may contribute to susceptibility to certain human diseases [19,20].\nIn this study, we confirmed the differential expression of IL-6R in DN and identified miR-125a as a regulator of IL-6R. It has been previously shown that a SNP (rs12976445) is present in the pre-miR-125a gene, and compromises the mature processing of the miRNA, which leads to an increased risk of autoimmune thyroid conditions [21] and repeated pregnancy loss [22]. Based on the evidence mentioned above, we studied the association between rs12976445 polymorphism and risk of DN in the patients with DM.",
"Renal biopsies were performed to collect renal tissue samples from 36 DM patients who had DN, and 28 controls in our hospital. The controls included in this study were the patients confirmed with renal carcinoma who underwent nephrectomy and did not have diabetes, hypertension, and other co-existing conditions. After all participants signed informed consent, renal biopsies (DN patients) or nephrectomy surgery (controls, mainly kidney cortexes) were performed to collect kidney tissues for real-time polymerase chain reaction (PCR) and Western blot analysis. In addition, peripheral blood samples were collected from 594 DM patients with (N=282) or without (N=312) DN. The demographic and clinicopathological features are described in Table 1. Written consent was obtained from each participant, and the approval of this study was obtained from the Ethics Committee on Human Research of The People ‘s Hospital in Hanzhong (Hanzhong, China).",
"Total RNA was isolated from renal tissue samples or the cultured cells by using TRIzol (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) was then synthesized by using SuperScript III (Invitrogen, Carlsbad, CA). The expression of miR-125a and IL-6R mRNA was measured in the human tissue samples and cultured cells. An ABI PRISM7500 system (Applied Biosystems, Foster City, CA) was used to perform a quantitative PCR. Each experiment was repeated 3 times.",
"DNA extraction kit (Sangong, Shanghai, China) was used to extract genomic DNA from the peripheral blood samples. NanoDrop 1000 Spec-trophotometer (Thermo Fisher Scientific, DE) was used to determine the quantity and quality of the isolated genomic DNA. Next, the target chromosome segment was PCR-amplified using the primer set: 5′-CCA TCG TGT GGG TCT CAA G-3′ and 5′-TCT TTC ACA GTG GAT CCT CTG AC-3′. Subsequently, the genotype of rs12976445 polymorphism was determined by using direct Sanger sequencing.",
"Human glomerular mesangial IP15 cell line were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and were placed in a humidified incubator at 37°C with an atmosphere of 95% air and 5% CO2. All experiments used cells at passages from 3 and 8.",
"Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was used to perform the transient transfections as specified in the manufacturer’s protocol. miRNA oligonucleotide was transfected for function loss experiment as per a designated protocol. In brief, cells were plated in 6-well plates and grew to 80% confluency. Next, the culture media was supplemented with miR-125a mimics, IL-6R specific siRNA or a matched miRNA negative control (Genepharma, Shanghai, China) at a final concentration of 50 nM. At 6 h after transfection, the medium was replenished with RPMI-1640.",
"The colorimetric procedure was performed to assess cell viability by using the Cell Counting Kit-8 (Dojindo, Shanghai, China), following the manufacturer’s instruction. The absorbance was measured at 450 nm using a microplate reader. Five duplicate wells were determined for all experiments for each group.",
"Human genomic DNA was used for amplification of full-length 3′-UTR of IL-6D by PCR and the clone into luciferase reporter vector (Promega, Madison, WI). The Quick Change XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to introduce the mutations into the putative binding sites at 91–99 bp and 621–627 bp of the 3′UTR of IL-6R. The reporter constructs and miR-negative control or miR-125a mimic were used for co-transfection of renal mesangial cells. The cells were incubated for 24 h, and then the activities of luciferase were determined in a luminometer using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) as per the manufacturer’s instructions.",
"RIPA buffer was used to lyse the human tissues or cells, and total cellular protein was centrifuged and obtained. The concentration was determined by the BCA method. After separation by SDS-PAGE, proteins were transferred to PVDF membranes (0.45 mm, Millipore), and then wash with TBST, blockage with 5% skim milk powder dissolved in TBST for 1 h, the PVDF membranes were cultured with primary antibodies against IL-6R or β-actin (Santa Cruz, CA, USA) (Dilution 1:1500 for both primary antibodies) at 4°C overnight with dilutions as per the instruction of the manufacturer. The internal reference was β-actin. After washing with TBST, the PVDF membranes were cultured with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at 37°C. Chemiluminescence (ECL) reagent (Pierce, Thermo Scientific, Rockford, IL,) was used to detect the protein bands."
] |
[
null,
null,
"methods",
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Material and Methods",
"Preparation of human kidney sample",
"Real-time PCR analysis",
"Rs12976445 polymorphism genotyping",
"Cell culture",
"Transient transfection",
"In vitro cell proliferation assay",
"Luciferase reporter gene assay",
"Western blot",
"Statistical analysis",
"Results",
"Discussion",
"Conclusions"
] |
[
"Diabetic nephropathy (DN) is one of the most important long-term complications of diabetes, and it is the primary cause of end-stage renal disease and deaths in diabetic patients [1]. The major clinical characteristics of DN includes progressively decreasing glomerular filtration rate (GFR) and persistent albuminuria, with macroalbuminuria (>300mg/day) indicating DN progression and microalbuminuria (30 to 300 mg of albumin in urine per day) representing early DN [2]. The main pathological characteristics of DN are expansion and hypertrophy in the tubular compartments and glomerular mesangium, as well as buildup of extracellular matrix (ECM) proteins and dysfunction of podocytes. There are several mechanisms, such as protein kinase C, advanced glycation end-products, hyperglycemia, oxidative stress, poly (ADP-ribose) polymerase activation, and inflammation, which are thought to account for the development and pathogenesis of DN [3]. Unfortunately, the hidden molecular pathogenesis is not completely identified.\nInterleukin 6 (IL-6) is a multi-potent cytokine; its main function appears to induce proliferation and differentiation of B lymphocyte and acute inflammatory responses. IL-6 has been shown to enhance the differentiation and growth of a wide range of cells, including renal mesangial cells [4]. The aberrantly enhanced proliferation of renal mesangial cells may lead to basement membrane thickening and mesangial matrix expansion, which is a major histological characteristic of diabetic nephropathy [5–7]. Actually, thickening of basement membrane and mesangial proliferation can be observed in early-stage nephropathy [8]. IL-6 may be very important in mesangial proliferation in patients who have glomerular renal disease [4,9]. IL-6 acts on a receptor that consists of 2 subunits, gp130 and IL-6 receptor (IL-6R). First, IL-6 binds to the IL-6R, and this complex subsequently binds to the gp130 subunit [10,11]. Elevated concentrations of IL-6R have been observed in both DN [12] and type II diabetes mellitus (DM) [13].\nMicroRNAs (miRNAs) consist of short non-coding RNAs that regulate physiological and pathological processes by suppressing expression of target gene via blocking protein translation or promoting degradation of mRNA. Recently, studies have discovered key properties for specific miRNAs in a battery of biological and cellular processes, such as proliferation, development, and differentiation [14]. Although the role of miRNAs has been identified in the pathogenesis of many human diseases, their function in diabetes and DN is less understood. Earlier reports have shown that single-nucleotide polymorphisms (SNPs) in miRNA genes may interfere with the interaction between miRNAs and mRNAs of target genes, or compromise the processing of mature miRNA [15–18], which may contribute to susceptibility to certain human diseases [19,20].\nIn this study, we confirmed the differential expression of IL-6R in DN and identified miR-125a as a regulator of IL-6R. It has been previously shown that a SNP (rs12976445) is present in the pre-miR-125a gene, and compromises the mature processing of the miRNA, which leads to an increased risk of autoimmune thyroid conditions [21] and repeated pregnancy loss [22]. Based on the evidence mentioned above, we studied the association between rs12976445 polymorphism and risk of DN in the patients with DM.",
" Preparation of human kidney sample Renal biopsies were performed to collect renal tissue samples from 36 DM patients who had DN, and 28 controls in our hospital. The controls included in this study were the patients confirmed with renal carcinoma who underwent nephrectomy and did not have diabetes, hypertension, and other co-existing conditions. After all participants signed informed consent, renal biopsies (DN patients) or nephrectomy surgery (controls, mainly kidney cortexes) were performed to collect kidney tissues for real-time polymerase chain reaction (PCR) and Western blot analysis. In addition, peripheral blood samples were collected from 594 DM patients with (N=282) or without (N=312) DN. The demographic and clinicopathological features are described in Table 1. Written consent was obtained from each participant, and the approval of this study was obtained from the Ethics Committee on Human Research of The People ‘s Hospital in Hanzhong (Hanzhong, China).\nRenal biopsies were performed to collect renal tissue samples from 36 DM patients who had DN, and 28 controls in our hospital. The controls included in this study were the patients confirmed with renal carcinoma who underwent nephrectomy and did not have diabetes, hypertension, and other co-existing conditions. After all participants signed informed consent, renal biopsies (DN patients) or nephrectomy surgery (controls, mainly kidney cortexes) were performed to collect kidney tissues for real-time polymerase chain reaction (PCR) and Western blot analysis. In addition, peripheral blood samples were collected from 594 DM patients with (N=282) or without (N=312) DN. The demographic and clinicopathological features are described in Table 1. Written consent was obtained from each participant, and the approval of this study was obtained from the Ethics Committee on Human Research of The People ‘s Hospital in Hanzhong (Hanzhong, China).\n Real-time PCR analysis Total RNA was isolated from renal tissue samples or the cultured cells by using TRIzol (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) was then synthesized by using SuperScript III (Invitrogen, Carlsbad, CA). The expression of miR-125a and IL-6R mRNA was measured in the human tissue samples and cultured cells. An ABI PRISM7500 system (Applied Biosystems, Foster City, CA) was used to perform a quantitative PCR. Each experiment was repeated 3 times.\nTotal RNA was isolated from renal tissue samples or the cultured cells by using TRIzol (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) was then synthesized by using SuperScript III (Invitrogen, Carlsbad, CA). The expression of miR-125a and IL-6R mRNA was measured in the human tissue samples and cultured cells. An ABI PRISM7500 system (Applied Biosystems, Foster City, CA) was used to perform a quantitative PCR. Each experiment was repeated 3 times.\n Rs12976445 polymorphism genotyping DNA extraction kit (Sangong, Shanghai, China) was used to extract genomic DNA from the peripheral blood samples. NanoDrop 1000 Spec-trophotometer (Thermo Fisher Scientific, DE) was used to determine the quantity and quality of the isolated genomic DNA. Next, the target chromosome segment was PCR-amplified using the primer set: 5′-CCA TCG TGT GGG TCT CAA G-3′ and 5′-TCT TTC ACA GTG GAT CCT CTG AC-3′. Subsequently, the genotype of rs12976445 polymorphism was determined by using direct Sanger sequencing.\nDNA extraction kit (Sangong, Shanghai, China) was used to extract genomic DNA from the peripheral blood samples. NanoDrop 1000 Spec-trophotometer (Thermo Fisher Scientific, DE) was used to determine the quantity and quality of the isolated genomic DNA. Next, the target chromosome segment was PCR-amplified using the primer set: 5′-CCA TCG TGT GGG TCT CAA G-3′ and 5′-TCT TTC ACA GTG GAT CCT CTG AC-3′. Subsequently, the genotype of rs12976445 polymorphism was determined by using direct Sanger sequencing.\n Cell culture Human glomerular mesangial IP15 cell line were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and were placed in a humidified incubator at 37°C with an atmosphere of 95% air and 5% CO2. All experiments used cells at passages from 3 and 8.\nHuman glomerular mesangial IP15 cell line were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and were placed in a humidified incubator at 37°C with an atmosphere of 95% air and 5% CO2. All experiments used cells at passages from 3 and 8.\n Transient transfection Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was used to perform the transient transfections as specified in the manufacturer’s protocol. miRNA oligonucleotide was transfected for function loss experiment as per a designated protocol. In brief, cells were plated in 6-well plates and grew to 80% confluency. Next, the culture media was supplemented with miR-125a mimics, IL-6R specific siRNA or a matched miRNA negative control (Genepharma, Shanghai, China) at a final concentration of 50 nM. At 6 h after transfection, the medium was replenished with RPMI-1640.\nLipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was used to perform the transient transfections as specified in the manufacturer’s protocol. miRNA oligonucleotide was transfected for function loss experiment as per a designated protocol. In brief, cells were plated in 6-well plates and grew to 80% confluency. Next, the culture media was supplemented with miR-125a mimics, IL-6R specific siRNA or a matched miRNA negative control (Genepharma, Shanghai, China) at a final concentration of 50 nM. At 6 h after transfection, the medium was replenished with RPMI-1640.\n In vitro cell proliferation assay The colorimetric procedure was performed to assess cell viability by using the Cell Counting Kit-8 (Dojindo, Shanghai, China), following the manufacturer’s instruction. The absorbance was measured at 450 nm using a microplate reader. Five duplicate wells were determined for all experiments for each group.\nThe colorimetric procedure was performed to assess cell viability by using the Cell Counting Kit-8 (Dojindo, Shanghai, China), following the manufacturer’s instruction. The absorbance was measured at 450 nm using a microplate reader. Five duplicate wells were determined for all experiments for each group.\n Luciferase reporter gene assay Human genomic DNA was used for amplification of full-length 3′-UTR of IL-6D by PCR and the clone into luciferase reporter vector (Promega, Madison, WI). The Quick Change XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to introduce the mutations into the putative binding sites at 91–99 bp and 621–627 bp of the 3′UTR of IL-6R. The reporter constructs and miR-negative control or miR-125a mimic were used for co-transfection of renal mesangial cells. The cells were incubated for 24 h, and then the activities of luciferase were determined in a luminometer using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) as per the manufacturer’s instructions.\nHuman genomic DNA was used for amplification of full-length 3′-UTR of IL-6D by PCR and the clone into luciferase reporter vector (Promega, Madison, WI). The Quick Change XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to introduce the mutations into the putative binding sites at 91–99 bp and 621–627 bp of the 3′UTR of IL-6R. The reporter constructs and miR-negative control or miR-125a mimic were used for co-transfection of renal mesangial cells. The cells were incubated for 24 h, and then the activities of luciferase were determined in a luminometer using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) as per the manufacturer’s instructions.\n Western blot RIPA buffer was used to lyse the human tissues or cells, and total cellular protein was centrifuged and obtained. The concentration was determined by the BCA method. After separation by SDS-PAGE, proteins were transferred to PVDF membranes (0.45 mm, Millipore), and then wash with TBST, blockage with 5% skim milk powder dissolved in TBST for 1 h, the PVDF membranes were cultured with primary antibodies against IL-6R or β-actin (Santa Cruz, CA, USA) (Dilution 1:1500 for both primary antibodies) at 4°C overnight with dilutions as per the instruction of the manufacturer. The internal reference was β-actin. After washing with TBST, the PVDF membranes were cultured with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at 37°C. Chemiluminescence (ECL) reagent (Pierce, Thermo Scientific, Rockford, IL,) was used to detect the protein bands.\nRIPA buffer was used to lyse the human tissues or cells, and total cellular protein was centrifuged and obtained. The concentration was determined by the BCA method. After separation by SDS-PAGE, proteins were transferred to PVDF membranes (0.45 mm, Millipore), and then wash with TBST, blockage with 5% skim milk powder dissolved in TBST for 1 h, the PVDF membranes were cultured with primary antibodies against IL-6R or β-actin (Santa Cruz, CA, USA) (Dilution 1:1500 for both primary antibodies) at 4°C overnight with dilutions as per the instruction of the manufacturer. The internal reference was β-actin. After washing with TBST, the PVDF membranes were cultured with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at 37°C. Chemiluminescence (ECL) reagent (Pierce, Thermo Scientific, Rockford, IL,) was used to detect the protein bands.\n Statistical analysis A goodness-of-fit χ2 test in controls was used to assess Hardy-Weinberg equilibrium (HWE) for the genotype. The differences in distributions of demographic characteristics between cases and controls were determined using Pearson’s Tχ2 test. The strength of correlation between the polymorphism and DN risk odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined by univariate logistic regression model. After adjustment for age, sex, smoking status, duration of DM, or DN (as described in Table 1), a multivariate logistic regression model was used for the calculation of adjusted ORs and 95% CIs. Statistical significance was set at P<0.05 that was 2-sided, and SPSS 19.0 (SPSS, Chicago, IL) was used to perform all statistical analyses.\nA goodness-of-fit χ2 test in controls was used to assess Hardy-Weinberg equilibrium (HWE) for the genotype. The differences in distributions of demographic characteristics between cases and controls were determined using Pearson’s Tχ2 test. The strength of correlation between the polymorphism and DN risk odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined by univariate logistic regression model. After adjustment for age, sex, smoking status, duration of DM, or DN (as described in Table 1), a multivariate logistic regression model was used for the calculation of adjusted ORs and 95% CIs. Statistical significance was set at P<0.05 that was 2-sided, and SPSS 19.0 (SPSS, Chicago, IL) was used to perform all statistical analyses.",
"Renal biopsies were performed to collect renal tissue samples from 36 DM patients who had DN, and 28 controls in our hospital. The controls included in this study were the patients confirmed with renal carcinoma who underwent nephrectomy and did not have diabetes, hypertension, and other co-existing conditions. After all participants signed informed consent, renal biopsies (DN patients) or nephrectomy surgery (controls, mainly kidney cortexes) were performed to collect kidney tissues for real-time polymerase chain reaction (PCR) and Western blot analysis. In addition, peripheral blood samples were collected from 594 DM patients with (N=282) or without (N=312) DN. The demographic and clinicopathological features are described in Table 1. Written consent was obtained from each participant, and the approval of this study was obtained from the Ethics Committee on Human Research of The People ‘s Hospital in Hanzhong (Hanzhong, China).",
"Total RNA was isolated from renal tissue samples or the cultured cells by using TRIzol (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) was then synthesized by using SuperScript III (Invitrogen, Carlsbad, CA). The expression of miR-125a and IL-6R mRNA was measured in the human tissue samples and cultured cells. An ABI PRISM7500 system (Applied Biosystems, Foster City, CA) was used to perform a quantitative PCR. Each experiment was repeated 3 times.",
"DNA extraction kit (Sangong, Shanghai, China) was used to extract genomic DNA from the peripheral blood samples. NanoDrop 1000 Spec-trophotometer (Thermo Fisher Scientific, DE) was used to determine the quantity and quality of the isolated genomic DNA. Next, the target chromosome segment was PCR-amplified using the primer set: 5′-CCA TCG TGT GGG TCT CAA G-3′ and 5′-TCT TTC ACA GTG GAT CCT CTG AC-3′. Subsequently, the genotype of rs12976445 polymorphism was determined by using direct Sanger sequencing.",
"Human glomerular mesangial IP15 cell line were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and were placed in a humidified incubator at 37°C with an atmosphere of 95% air and 5% CO2. All experiments used cells at passages from 3 and 8.",
"Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was used to perform the transient transfections as specified in the manufacturer’s protocol. miRNA oligonucleotide was transfected for function loss experiment as per a designated protocol. In brief, cells were plated in 6-well plates and grew to 80% confluency. Next, the culture media was supplemented with miR-125a mimics, IL-6R specific siRNA or a matched miRNA negative control (Genepharma, Shanghai, China) at a final concentration of 50 nM. At 6 h after transfection, the medium was replenished with RPMI-1640.",
"The colorimetric procedure was performed to assess cell viability by using the Cell Counting Kit-8 (Dojindo, Shanghai, China), following the manufacturer’s instruction. The absorbance was measured at 450 nm using a microplate reader. Five duplicate wells were determined for all experiments for each group.",
"Human genomic DNA was used for amplification of full-length 3′-UTR of IL-6D by PCR and the clone into luciferase reporter vector (Promega, Madison, WI). The Quick Change XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to introduce the mutations into the putative binding sites at 91–99 bp and 621–627 bp of the 3′UTR of IL-6R. The reporter constructs and miR-negative control or miR-125a mimic were used for co-transfection of renal mesangial cells. The cells were incubated for 24 h, and then the activities of luciferase were determined in a luminometer using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) as per the manufacturer’s instructions.",
"RIPA buffer was used to lyse the human tissues or cells, and total cellular protein was centrifuged and obtained. The concentration was determined by the BCA method. After separation by SDS-PAGE, proteins were transferred to PVDF membranes (0.45 mm, Millipore), and then wash with TBST, blockage with 5% skim milk powder dissolved in TBST for 1 h, the PVDF membranes were cultured with primary antibodies against IL-6R or β-actin (Santa Cruz, CA, USA) (Dilution 1:1500 for both primary antibodies) at 4°C overnight with dilutions as per the instruction of the manufacturer. The internal reference was β-actin. After washing with TBST, the PVDF membranes were cultured with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at 37°C. Chemiluminescence (ECL) reagent (Pierce, Thermo Scientific, Rockford, IL,) was used to detect the protein bands.",
"A goodness-of-fit χ2 test in controls was used to assess Hardy-Weinberg equilibrium (HWE) for the genotype. The differences in distributions of demographic characteristics between cases and controls were determined using Pearson’s Tχ2 test. The strength of correlation between the polymorphism and DN risk odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined by univariate logistic regression model. After adjustment for age, sex, smoking status, duration of DM, or DN (as described in Table 1), a multivariate logistic regression model was used for the calculation of adjusted ORs and 95% CIs. Statistical significance was set at P<0.05 that was 2-sided, and SPSS 19.0 (SPSS, Chicago, IL) was used to perform all statistical analyses.",
"In this study, we collected the kidney tissues from the patients with DN (N=36) and the control patients (N=28), and performed realtime PCR and Western blot analysis to determine the expression of IL-6R that had been previously reported to be functionally involved in the development of diabetic nephropathy [23]. As expected, the expression of IL-6R was barely detected in the controls groups, while in the DN groups, the IL-6R was clearly detectable (Figure 1A). Similarly, the mRNA expression level of IL-6R was substantially upregulated in the tissue samples from DN patients compared with the controls (Figure 1B).\nTo identify the potential regulator of IL-6R in the kidneys, we searched the miRNA database (www.mirdb.org) and identify miR-125a as a possible regulator of IL-6R with 2 putative binding sites (91–99 bp and 621–627 bp) within the 3′UTR of the gene. To test it, a luciferase assay reporter system was set up by amplifying and inserting the 3′UTR of IL-6R into pGL3 that contained downstream firefly luciferase. To identify the real binding site responsible for the interaction, the 2 putative binding sites were mutated individually (mutant-1 and mutant-2) and combinatively (mutant-3) as described in Figure 2. Next, the constructs carrying either wild-type or the mutated 3′UTR of IL-6R were transfected into the cultured mesangial cells that overexpressed miR-125a or the scramble control sequence. As presented in Figure 2, we found that the luciferase activity was significantly decreased in the cells transfected with miR-125a mimics and wild-type, mutant-1, and mutant-2 IL-6R 3′UTR, whereas the luciferase activity was similar between the cells transfected with mutant-3 IL-6R 3′UTR and the control (Figure 3), suggesting that either of the binding sites was sufficient for a full interaction between miR-125a and IL-6R.\nTo further verify the regulatory association between miR-125a and IL-6R, we examined the expression of miR-125a in the above-mentioned DN patients and controls, and we showed that the expression of miR-125a was significantly downregulated in DN patients compared with the controls (Figure 4A). We then regrouped the samples based the rs12976445 polymorphism genotypes, and we found that 35, 24, and 5 participants were genotyped as CC, CT, and TT, respectively. As shown in Figure 4B, the expression levels of miR-125a was comparable between TT and CT, so we merged those 2 groups into 1, which was significantly lower than the CC group.\nTo further validate IL-6R as a target of miR-125a, we examined the endogenous expression in mesangial cells transfected with miR-125a and IL-6R specific siRNA by using Western blot and real-time PCR analysis. We found that the protein expression level of IL-6R was significantly downregulated by introduction of miR-125a mimics and anti-IL-6R siRNA (Figure 5A); and, consistent with this, the mRNA of IL-6R was also substantially lower in the cells transfected with miR-125a mimics and anti-IL-6R siRNA compared with the control (Figure 5B). To test the effect of downregulation of IL-6R on the proliferation of mesangial cells, we evaluated the viability of the cells transfected with miR-125a and IL-6R specific siRNA. As shown in Figure 6, the viability of the cells transfected with miR-125a and IL-6R specific siRNA were significantly lower than that of the cells transfected with scramble control.\nTo explore the association between rs12976445 polymorphism and risk of DN, we enrolled 594 DM patients with (N=282) or without DN (N=312). The distributions of rs12976445 polymorphism were in Hardy-Weinberg equilibrium among the case group and the controls. As the expression levels of miR-125a was similar between TT and CT, and genotype frequency is low, we combined the 2 genotype groups together in comparison with the wild-type group (CC). The demographic and clinicopathological parameters in the 2 groups are presented in Table 1. As shown in Table 2, significant differences were noted regarding genotype distribution of rs12976445 between the DN and the controls (OR=1.45, 95% C.I.=1.02–2.08, p<0.05) with the possible confounding factors adjusted for by using logistic regression analysis.",
"A major complication of DM is DN. A study reported that either environmental or genetic factors may cause various pathophysiological cascades correlated with development of DN [24]. In the past decade, studies have suggested that inflammation plays an important role in the occurrence and progression of such kidney injury [24,25]. Researchers first found inflammatory cytokines was associated with the pathogenesis of DN in 1991 [26]. Later, studies have shown that intrinsic renal cells (glomerular, mesangial, tubular epithelial, and endothelial cells) have the ability to synthesize proinflammatory cytokines [27]. As a major modulator of inflammation, Interleukin-6 (IL-6) is elevated in DM patients with DN, and is also presented at increased concentrations in patients with pronounced proteinuria when compared to patients with microalbuminuria [28]. The experimental animal models of DN showed over-activation of IL-6 signaling pathway in the kidney of the patients with DN [29]. In this study, we performed real-time PCR and Western blot analysis to determine the expression of IL-6R in the kidney tissues from the patients with DN and the control patients, and we found that the expression of IL-6R was barely detected in the controls groups, while in the DN groups, the IL-6R was clearly detectable. Similarly, the mRNA expression level of IL-6R was substantially upregulated in the tissue samples from DN patients compared with the controls. To identify the potential regulator of IL-6R in the kidneys, we searched the miRNA database online and identified miR-125a as a possible regulator of IL-6R, with 2 putative binding sites (91–99 bp and 621–627 bp) within the 3′UTR of the gene. To identify the real binding site responsible for the interaction, the 2 putative binding sites were mutated individually (mutant-1 and mutant-2) and combinatively (mutant-3), and we found that the luciferase activity was significantly decreased in the cells transfected with miR-125a mimics and wild-type, mutant-1, and mutant-2 IL-6R 3′UTR, whereas the luciferase activity was similar between the cells transfected with mutant-3 IL-6R 3′UTR and the controls (Figure 3), indicating that either of the binding sites was sufficient for a full interaction between miR-125a and IL-6R. To further validate IL-6R as a target of miR-125a, we examined the endogenous expression in mesangial cells transfected with miR-125a and IL-6R specific siRNA by using Western blot and real-time PCR analysis, and we found that protein and mRNA expression level of IL-6R was significantly downregulated by introduction of miR-125a mimics and anti-IL-6R siRNA.\nSuzuki et al. [30] investigated renal samples from patients with DN and observed over-activation of the IL-6/IL-6R signaling pathway in interstitium, mesangium, tubules, and infiltrating cells, which might be responsible for the abnormally enhanced proliferation of mesangial cells [31]. In line with this, it has also been shown that stimulation of the IL-6/IL-6R signaling pathway promoted proliferation of mesangial cells and fibronectin, which subsequently increased the permeability of glomerular endothelial cell [24,25]. To test the effect of downregulation of IL-6R on the proliferation of mesangial cells, we evaluated the viability of the cells transfected with miR-125a and IL-6R specific siRNA, and we found that the viability of the cells transfected with miR-125a and IL-6R specific siRNA were significantly lower than that of the cells transfected with scramble control.\nMiR-125a, located at chromosome 19q13.41, has been reported to be involved in pathogenesis of many human diseases as well as development of organs [32,33]. Accumulating studies have suggested that miR-125a is associated with the pathogenesis of human cancers, such as lung, gastric, and breast cancers [32,34–36]. miR-125a may act as either an oncogene or a tumor suppressor, depending on the cellular context. For instance, the downregulation of miR-125a was observed in some cancers, such as leukemia [32] and lung cancer [35,36], where the increased expression suppressed the proliferation of cancer cells and promoted apoptosis. Recent evidence showed that mature miRNA processing and mature miR-125a expression may be disrupted by the T allele of rs12976445, resulting in augmented production of target genes, including LIFR and ERBB2 [22, 38]. It has been previously shown that a SNP (rs12976445) is present in the pre-miR-125a gene, and it compromises the mature processing of the miRNA, which leads to an increased risk of autoimmune thyroid conditions [21] and repeated pregnancy loss [22]. In this study, we studied the association between rs12976445 polymorphism and risk of DN in a total of 594 DM patients with (N=282) or without DN (N=312), and we found that rs12976445 was significantly associated with DN (OR=1.45, 95% C.I.=1.02–2.08, p<0.05) after the possible confounding factors were adjusted for by using logistic regression analysis.",
"Our results showed that miR-125a is a key player in the development of DN via targeting IL-6R, and underexpression of miR-125a promoted proliferation of mesangial cells by releasing the inhibition on IL-6R. Downregulation of miR-125a could be, at least partially, attributed to the presence of rs12976445 polymorphism, which compromises the mature processing of the miRNA, demonstrating that miR-125a is a promising novel diagnostic and therapeutic target for DN."
] |
[
null,
"materials|methods",
null,
"methods",
null,
null,
null,
null,
null,
null,
"methods",
"results",
"discussion",
"conclusions"
] |
[
"Cytokine Receptor gp130",
"Diabetic Nephropathies",
"MicroRNAs"
] |
Background:
Diabetic nephropathy (DN) is one of the most important long-term complications of diabetes, and it is the primary cause of end-stage renal disease and deaths in diabetic patients [1]. The major clinical characteristics of DN includes progressively decreasing glomerular filtration rate (GFR) and persistent albuminuria, with macroalbuminuria (>300mg/day) indicating DN progression and microalbuminuria (30 to 300 mg of albumin in urine per day) representing early DN [2]. The main pathological characteristics of DN are expansion and hypertrophy in the tubular compartments and glomerular mesangium, as well as buildup of extracellular matrix (ECM) proteins and dysfunction of podocytes. There are several mechanisms, such as protein kinase C, advanced glycation end-products, hyperglycemia, oxidative stress, poly (ADP-ribose) polymerase activation, and inflammation, which are thought to account for the development and pathogenesis of DN [3]. Unfortunately, the hidden molecular pathogenesis is not completely identified.
Interleukin 6 (IL-6) is a multi-potent cytokine; its main function appears to induce proliferation and differentiation of B lymphocyte and acute inflammatory responses. IL-6 has been shown to enhance the differentiation and growth of a wide range of cells, including renal mesangial cells [4]. The aberrantly enhanced proliferation of renal mesangial cells may lead to basement membrane thickening and mesangial matrix expansion, which is a major histological characteristic of diabetic nephropathy [5–7]. Actually, thickening of basement membrane and mesangial proliferation can be observed in early-stage nephropathy [8]. IL-6 may be very important in mesangial proliferation in patients who have glomerular renal disease [4,9]. IL-6 acts on a receptor that consists of 2 subunits, gp130 and IL-6 receptor (IL-6R). First, IL-6 binds to the IL-6R, and this complex subsequently binds to the gp130 subunit [10,11]. Elevated concentrations of IL-6R have been observed in both DN [12] and type II diabetes mellitus (DM) [13].
MicroRNAs (miRNAs) consist of short non-coding RNAs that regulate physiological and pathological processes by suppressing expression of target gene via blocking protein translation or promoting degradation of mRNA. Recently, studies have discovered key properties for specific miRNAs in a battery of biological and cellular processes, such as proliferation, development, and differentiation [14]. Although the role of miRNAs has been identified in the pathogenesis of many human diseases, their function in diabetes and DN is less understood. Earlier reports have shown that single-nucleotide polymorphisms (SNPs) in miRNA genes may interfere with the interaction between miRNAs and mRNAs of target genes, or compromise the processing of mature miRNA [15–18], which may contribute to susceptibility to certain human diseases [19,20].
In this study, we confirmed the differential expression of IL-6R in DN and identified miR-125a as a regulator of IL-6R. It has been previously shown that a SNP (rs12976445) is present in the pre-miR-125a gene, and compromises the mature processing of the miRNA, which leads to an increased risk of autoimmune thyroid conditions [21] and repeated pregnancy loss [22]. Based on the evidence mentioned above, we studied the association between rs12976445 polymorphism and risk of DN in the patients with DM.
Material and Methods:
Preparation of human kidney sample Renal biopsies were performed to collect renal tissue samples from 36 DM patients who had DN, and 28 controls in our hospital. The controls included in this study were the patients confirmed with renal carcinoma who underwent nephrectomy and did not have diabetes, hypertension, and other co-existing conditions. After all participants signed informed consent, renal biopsies (DN patients) or nephrectomy surgery (controls, mainly kidney cortexes) were performed to collect kidney tissues for real-time polymerase chain reaction (PCR) and Western blot analysis. In addition, peripheral blood samples were collected from 594 DM patients with (N=282) or without (N=312) DN. The demographic and clinicopathological features are described in Table 1. Written consent was obtained from each participant, and the approval of this study was obtained from the Ethics Committee on Human Research of The People ‘s Hospital in Hanzhong (Hanzhong, China).
Renal biopsies were performed to collect renal tissue samples from 36 DM patients who had DN, and 28 controls in our hospital. The controls included in this study were the patients confirmed with renal carcinoma who underwent nephrectomy and did not have diabetes, hypertension, and other co-existing conditions. After all participants signed informed consent, renal biopsies (DN patients) or nephrectomy surgery (controls, mainly kidney cortexes) were performed to collect kidney tissues for real-time polymerase chain reaction (PCR) and Western blot analysis. In addition, peripheral blood samples were collected from 594 DM patients with (N=282) or without (N=312) DN. The demographic and clinicopathological features are described in Table 1. Written consent was obtained from each participant, and the approval of this study was obtained from the Ethics Committee on Human Research of The People ‘s Hospital in Hanzhong (Hanzhong, China).
Real-time PCR analysis Total RNA was isolated from renal tissue samples or the cultured cells by using TRIzol (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) was then synthesized by using SuperScript III (Invitrogen, Carlsbad, CA). The expression of miR-125a and IL-6R mRNA was measured in the human tissue samples and cultured cells. An ABI PRISM7500 system (Applied Biosystems, Foster City, CA) was used to perform a quantitative PCR. Each experiment was repeated 3 times.
Total RNA was isolated from renal tissue samples or the cultured cells by using TRIzol (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) was then synthesized by using SuperScript III (Invitrogen, Carlsbad, CA). The expression of miR-125a and IL-6R mRNA was measured in the human tissue samples and cultured cells. An ABI PRISM7500 system (Applied Biosystems, Foster City, CA) was used to perform a quantitative PCR. Each experiment was repeated 3 times.
Rs12976445 polymorphism genotyping DNA extraction kit (Sangong, Shanghai, China) was used to extract genomic DNA from the peripheral blood samples. NanoDrop 1000 Spec-trophotometer (Thermo Fisher Scientific, DE) was used to determine the quantity and quality of the isolated genomic DNA. Next, the target chromosome segment was PCR-amplified using the primer set: 5′-CCA TCG TGT GGG TCT CAA G-3′ and 5′-TCT TTC ACA GTG GAT CCT CTG AC-3′. Subsequently, the genotype of rs12976445 polymorphism was determined by using direct Sanger sequencing.
DNA extraction kit (Sangong, Shanghai, China) was used to extract genomic DNA from the peripheral blood samples. NanoDrop 1000 Spec-trophotometer (Thermo Fisher Scientific, DE) was used to determine the quantity and quality of the isolated genomic DNA. Next, the target chromosome segment was PCR-amplified using the primer set: 5′-CCA TCG TGT GGG TCT CAA G-3′ and 5′-TCT TTC ACA GTG GAT CCT CTG AC-3′. Subsequently, the genotype of rs12976445 polymorphism was determined by using direct Sanger sequencing.
Cell culture Human glomerular mesangial IP15 cell line were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and were placed in a humidified incubator at 37°C with an atmosphere of 95% air and 5% CO2. All experiments used cells at passages from 3 and 8.
Human glomerular mesangial IP15 cell line were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and were placed in a humidified incubator at 37°C with an atmosphere of 95% air and 5% CO2. All experiments used cells at passages from 3 and 8.
Transient transfection Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was used to perform the transient transfections as specified in the manufacturer’s protocol. miRNA oligonucleotide was transfected for function loss experiment as per a designated protocol. In brief, cells were plated in 6-well plates and grew to 80% confluency. Next, the culture media was supplemented with miR-125a mimics, IL-6R specific siRNA or a matched miRNA negative control (Genepharma, Shanghai, China) at a final concentration of 50 nM. At 6 h after transfection, the medium was replenished with RPMI-1640.
Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was used to perform the transient transfections as specified in the manufacturer’s protocol. miRNA oligonucleotide was transfected for function loss experiment as per a designated protocol. In brief, cells were plated in 6-well plates and grew to 80% confluency. Next, the culture media was supplemented with miR-125a mimics, IL-6R specific siRNA or a matched miRNA negative control (Genepharma, Shanghai, China) at a final concentration of 50 nM. At 6 h after transfection, the medium was replenished with RPMI-1640.
In vitro cell proliferation assay The colorimetric procedure was performed to assess cell viability by using the Cell Counting Kit-8 (Dojindo, Shanghai, China), following the manufacturer’s instruction. The absorbance was measured at 450 nm using a microplate reader. Five duplicate wells were determined for all experiments for each group.
The colorimetric procedure was performed to assess cell viability by using the Cell Counting Kit-8 (Dojindo, Shanghai, China), following the manufacturer’s instruction. The absorbance was measured at 450 nm using a microplate reader. Five duplicate wells were determined for all experiments for each group.
Luciferase reporter gene assay Human genomic DNA was used for amplification of full-length 3′-UTR of IL-6D by PCR and the clone into luciferase reporter vector (Promega, Madison, WI). The Quick Change XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to introduce the mutations into the putative binding sites at 91–99 bp and 621–627 bp of the 3′UTR of IL-6R. The reporter constructs and miR-negative control or miR-125a mimic were used for co-transfection of renal mesangial cells. The cells were incubated for 24 h, and then the activities of luciferase were determined in a luminometer using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) as per the manufacturer’s instructions.
Human genomic DNA was used for amplification of full-length 3′-UTR of IL-6D by PCR and the clone into luciferase reporter vector (Promega, Madison, WI). The Quick Change XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to introduce the mutations into the putative binding sites at 91–99 bp and 621–627 bp of the 3′UTR of IL-6R. The reporter constructs and miR-negative control or miR-125a mimic were used for co-transfection of renal mesangial cells. The cells were incubated for 24 h, and then the activities of luciferase were determined in a luminometer using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) as per the manufacturer’s instructions.
Western blot RIPA buffer was used to lyse the human tissues or cells, and total cellular protein was centrifuged and obtained. The concentration was determined by the BCA method. After separation by SDS-PAGE, proteins were transferred to PVDF membranes (0.45 mm, Millipore), and then wash with TBST, blockage with 5% skim milk powder dissolved in TBST for 1 h, the PVDF membranes were cultured with primary antibodies against IL-6R or β-actin (Santa Cruz, CA, USA) (Dilution 1:1500 for both primary antibodies) at 4°C overnight with dilutions as per the instruction of the manufacturer. The internal reference was β-actin. After washing with TBST, the PVDF membranes were cultured with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at 37°C. Chemiluminescence (ECL) reagent (Pierce, Thermo Scientific, Rockford, IL,) was used to detect the protein bands.
RIPA buffer was used to lyse the human tissues or cells, and total cellular protein was centrifuged and obtained. The concentration was determined by the BCA method. After separation by SDS-PAGE, proteins were transferred to PVDF membranes (0.45 mm, Millipore), and then wash with TBST, blockage with 5% skim milk powder dissolved in TBST for 1 h, the PVDF membranes were cultured with primary antibodies against IL-6R or β-actin (Santa Cruz, CA, USA) (Dilution 1:1500 for both primary antibodies) at 4°C overnight with dilutions as per the instruction of the manufacturer. The internal reference was β-actin. After washing with TBST, the PVDF membranes were cultured with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at 37°C. Chemiluminescence (ECL) reagent (Pierce, Thermo Scientific, Rockford, IL,) was used to detect the protein bands.
Statistical analysis A goodness-of-fit χ2 test in controls was used to assess Hardy-Weinberg equilibrium (HWE) for the genotype. The differences in distributions of demographic characteristics between cases and controls were determined using Pearson’s Tχ2 test. The strength of correlation between the polymorphism and DN risk odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined by univariate logistic regression model. After adjustment for age, sex, smoking status, duration of DM, or DN (as described in Table 1), a multivariate logistic regression model was used for the calculation of adjusted ORs and 95% CIs. Statistical significance was set at P<0.05 that was 2-sided, and SPSS 19.0 (SPSS, Chicago, IL) was used to perform all statistical analyses.
A goodness-of-fit χ2 test in controls was used to assess Hardy-Weinberg equilibrium (HWE) for the genotype. The differences in distributions of demographic characteristics between cases and controls were determined using Pearson’s Tχ2 test. The strength of correlation between the polymorphism and DN risk odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined by univariate logistic regression model. After adjustment for age, sex, smoking status, duration of DM, or DN (as described in Table 1), a multivariate logistic regression model was used for the calculation of adjusted ORs and 95% CIs. Statistical significance was set at P<0.05 that was 2-sided, and SPSS 19.0 (SPSS, Chicago, IL) was used to perform all statistical analyses.
Preparation of human kidney sample:
Renal biopsies were performed to collect renal tissue samples from 36 DM patients who had DN, and 28 controls in our hospital. The controls included in this study were the patients confirmed with renal carcinoma who underwent nephrectomy and did not have diabetes, hypertension, and other co-existing conditions. After all participants signed informed consent, renal biopsies (DN patients) or nephrectomy surgery (controls, mainly kidney cortexes) were performed to collect kidney tissues for real-time polymerase chain reaction (PCR) and Western blot analysis. In addition, peripheral blood samples were collected from 594 DM patients with (N=282) or without (N=312) DN. The demographic and clinicopathological features are described in Table 1. Written consent was obtained from each participant, and the approval of this study was obtained from the Ethics Committee on Human Research of The People ‘s Hospital in Hanzhong (Hanzhong, China).
Real-time PCR analysis:
Total RNA was isolated from renal tissue samples or the cultured cells by using TRIzol (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) was then synthesized by using SuperScript III (Invitrogen, Carlsbad, CA). The expression of miR-125a and IL-6R mRNA was measured in the human tissue samples and cultured cells. An ABI PRISM7500 system (Applied Biosystems, Foster City, CA) was used to perform a quantitative PCR. Each experiment was repeated 3 times.
Rs12976445 polymorphism genotyping:
DNA extraction kit (Sangong, Shanghai, China) was used to extract genomic DNA from the peripheral blood samples. NanoDrop 1000 Spec-trophotometer (Thermo Fisher Scientific, DE) was used to determine the quantity and quality of the isolated genomic DNA. Next, the target chromosome segment was PCR-amplified using the primer set: 5′-CCA TCG TGT GGG TCT CAA G-3′ and 5′-TCT TTC ACA GTG GAT CCT CTG AC-3′. Subsequently, the genotype of rs12976445 polymorphism was determined by using direct Sanger sequencing.
Cell culture:
Human glomerular mesangial IP15 cell line were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and were placed in a humidified incubator at 37°C with an atmosphere of 95% air and 5% CO2. All experiments used cells at passages from 3 and 8.
Transient transfection:
Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was used to perform the transient transfections as specified in the manufacturer’s protocol. miRNA oligonucleotide was transfected for function loss experiment as per a designated protocol. In brief, cells were plated in 6-well plates and grew to 80% confluency. Next, the culture media was supplemented with miR-125a mimics, IL-6R specific siRNA or a matched miRNA negative control (Genepharma, Shanghai, China) at a final concentration of 50 nM. At 6 h after transfection, the medium was replenished with RPMI-1640.
In vitro cell proliferation assay:
The colorimetric procedure was performed to assess cell viability by using the Cell Counting Kit-8 (Dojindo, Shanghai, China), following the manufacturer’s instruction. The absorbance was measured at 450 nm using a microplate reader. Five duplicate wells were determined for all experiments for each group.
Luciferase reporter gene assay:
Human genomic DNA was used for amplification of full-length 3′-UTR of IL-6D by PCR and the clone into luciferase reporter vector (Promega, Madison, WI). The Quick Change XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to introduce the mutations into the putative binding sites at 91–99 bp and 621–627 bp of the 3′UTR of IL-6R. The reporter constructs and miR-negative control or miR-125a mimic were used for co-transfection of renal mesangial cells. The cells were incubated for 24 h, and then the activities of luciferase were determined in a luminometer using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) as per the manufacturer’s instructions.
Western blot:
RIPA buffer was used to lyse the human tissues or cells, and total cellular protein was centrifuged and obtained. The concentration was determined by the BCA method. After separation by SDS-PAGE, proteins were transferred to PVDF membranes (0.45 mm, Millipore), and then wash with TBST, blockage with 5% skim milk powder dissolved in TBST for 1 h, the PVDF membranes were cultured with primary antibodies against IL-6R or β-actin (Santa Cruz, CA, USA) (Dilution 1:1500 for both primary antibodies) at 4°C overnight with dilutions as per the instruction of the manufacturer. The internal reference was β-actin. After washing with TBST, the PVDF membranes were cultured with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at 37°C. Chemiluminescence (ECL) reagent (Pierce, Thermo Scientific, Rockford, IL,) was used to detect the protein bands.
Statistical analysis:
A goodness-of-fit χ2 test in controls was used to assess Hardy-Weinberg equilibrium (HWE) for the genotype. The differences in distributions of demographic characteristics between cases and controls were determined using Pearson’s Tχ2 test. The strength of correlation between the polymorphism and DN risk odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined by univariate logistic regression model. After adjustment for age, sex, smoking status, duration of DM, or DN (as described in Table 1), a multivariate logistic regression model was used for the calculation of adjusted ORs and 95% CIs. Statistical significance was set at P<0.05 that was 2-sided, and SPSS 19.0 (SPSS, Chicago, IL) was used to perform all statistical analyses.
Results:
In this study, we collected the kidney tissues from the patients with DN (N=36) and the control patients (N=28), and performed realtime PCR and Western blot analysis to determine the expression of IL-6R that had been previously reported to be functionally involved in the development of diabetic nephropathy [23]. As expected, the expression of IL-6R was barely detected in the controls groups, while in the DN groups, the IL-6R was clearly detectable (Figure 1A). Similarly, the mRNA expression level of IL-6R was substantially upregulated in the tissue samples from DN patients compared with the controls (Figure 1B).
To identify the potential regulator of IL-6R in the kidneys, we searched the miRNA database (www.mirdb.org) and identify miR-125a as a possible regulator of IL-6R with 2 putative binding sites (91–99 bp and 621–627 bp) within the 3′UTR of the gene. To test it, a luciferase assay reporter system was set up by amplifying and inserting the 3′UTR of IL-6R into pGL3 that contained downstream firefly luciferase. To identify the real binding site responsible for the interaction, the 2 putative binding sites were mutated individually (mutant-1 and mutant-2) and combinatively (mutant-3) as described in Figure 2. Next, the constructs carrying either wild-type or the mutated 3′UTR of IL-6R were transfected into the cultured mesangial cells that overexpressed miR-125a or the scramble control sequence. As presented in Figure 2, we found that the luciferase activity was significantly decreased in the cells transfected with miR-125a mimics and wild-type, mutant-1, and mutant-2 IL-6R 3′UTR, whereas the luciferase activity was similar between the cells transfected with mutant-3 IL-6R 3′UTR and the control (Figure 3), suggesting that either of the binding sites was sufficient for a full interaction between miR-125a and IL-6R.
To further verify the regulatory association between miR-125a and IL-6R, we examined the expression of miR-125a in the above-mentioned DN patients and controls, and we showed that the expression of miR-125a was significantly downregulated in DN patients compared with the controls (Figure 4A). We then regrouped the samples based the rs12976445 polymorphism genotypes, and we found that 35, 24, and 5 participants were genotyped as CC, CT, and TT, respectively. As shown in Figure 4B, the expression levels of miR-125a was comparable between TT and CT, so we merged those 2 groups into 1, which was significantly lower than the CC group.
To further validate IL-6R as a target of miR-125a, we examined the endogenous expression in mesangial cells transfected with miR-125a and IL-6R specific siRNA by using Western blot and real-time PCR analysis. We found that the protein expression level of IL-6R was significantly downregulated by introduction of miR-125a mimics and anti-IL-6R siRNA (Figure 5A); and, consistent with this, the mRNA of IL-6R was also substantially lower in the cells transfected with miR-125a mimics and anti-IL-6R siRNA compared with the control (Figure 5B). To test the effect of downregulation of IL-6R on the proliferation of mesangial cells, we evaluated the viability of the cells transfected with miR-125a and IL-6R specific siRNA. As shown in Figure 6, the viability of the cells transfected with miR-125a and IL-6R specific siRNA were significantly lower than that of the cells transfected with scramble control.
To explore the association between rs12976445 polymorphism and risk of DN, we enrolled 594 DM patients with (N=282) or without DN (N=312). The distributions of rs12976445 polymorphism were in Hardy-Weinberg equilibrium among the case group and the controls. As the expression levels of miR-125a was similar between TT and CT, and genotype frequency is low, we combined the 2 genotype groups together in comparison with the wild-type group (CC). The demographic and clinicopathological parameters in the 2 groups are presented in Table 1. As shown in Table 2, significant differences were noted regarding genotype distribution of rs12976445 between the DN and the controls (OR=1.45, 95% C.I.=1.02–2.08, p<0.05) with the possible confounding factors adjusted for by using logistic regression analysis.
Discussion:
A major complication of DM is DN. A study reported that either environmental or genetic factors may cause various pathophysiological cascades correlated with development of DN [24]. In the past decade, studies have suggested that inflammation plays an important role in the occurrence and progression of such kidney injury [24,25]. Researchers first found inflammatory cytokines was associated with the pathogenesis of DN in 1991 [26]. Later, studies have shown that intrinsic renal cells (glomerular, mesangial, tubular epithelial, and endothelial cells) have the ability to synthesize proinflammatory cytokines [27]. As a major modulator of inflammation, Interleukin-6 (IL-6) is elevated in DM patients with DN, and is also presented at increased concentrations in patients with pronounced proteinuria when compared to patients with microalbuminuria [28]. The experimental animal models of DN showed over-activation of IL-6 signaling pathway in the kidney of the patients with DN [29]. In this study, we performed real-time PCR and Western blot analysis to determine the expression of IL-6R in the kidney tissues from the patients with DN and the control patients, and we found that the expression of IL-6R was barely detected in the controls groups, while in the DN groups, the IL-6R was clearly detectable. Similarly, the mRNA expression level of IL-6R was substantially upregulated in the tissue samples from DN patients compared with the controls. To identify the potential regulator of IL-6R in the kidneys, we searched the miRNA database online and identified miR-125a as a possible regulator of IL-6R, with 2 putative binding sites (91–99 bp and 621–627 bp) within the 3′UTR of the gene. To identify the real binding site responsible for the interaction, the 2 putative binding sites were mutated individually (mutant-1 and mutant-2) and combinatively (mutant-3), and we found that the luciferase activity was significantly decreased in the cells transfected with miR-125a mimics and wild-type, mutant-1, and mutant-2 IL-6R 3′UTR, whereas the luciferase activity was similar between the cells transfected with mutant-3 IL-6R 3′UTR and the controls (Figure 3), indicating that either of the binding sites was sufficient for a full interaction between miR-125a and IL-6R. To further validate IL-6R as a target of miR-125a, we examined the endogenous expression in mesangial cells transfected with miR-125a and IL-6R specific siRNA by using Western blot and real-time PCR analysis, and we found that protein and mRNA expression level of IL-6R was significantly downregulated by introduction of miR-125a mimics and anti-IL-6R siRNA.
Suzuki et al. [30] investigated renal samples from patients with DN and observed over-activation of the IL-6/IL-6R signaling pathway in interstitium, mesangium, tubules, and infiltrating cells, which might be responsible for the abnormally enhanced proliferation of mesangial cells [31]. In line with this, it has also been shown that stimulation of the IL-6/IL-6R signaling pathway promoted proliferation of mesangial cells and fibronectin, which subsequently increased the permeability of glomerular endothelial cell [24,25]. To test the effect of downregulation of IL-6R on the proliferation of mesangial cells, we evaluated the viability of the cells transfected with miR-125a and IL-6R specific siRNA, and we found that the viability of the cells transfected with miR-125a and IL-6R specific siRNA were significantly lower than that of the cells transfected with scramble control.
MiR-125a, located at chromosome 19q13.41, has been reported to be involved in pathogenesis of many human diseases as well as development of organs [32,33]. Accumulating studies have suggested that miR-125a is associated with the pathogenesis of human cancers, such as lung, gastric, and breast cancers [32,34–36]. miR-125a may act as either an oncogene or a tumor suppressor, depending on the cellular context. For instance, the downregulation of miR-125a was observed in some cancers, such as leukemia [32] and lung cancer [35,36], where the increased expression suppressed the proliferation of cancer cells and promoted apoptosis. Recent evidence showed that mature miRNA processing and mature miR-125a expression may be disrupted by the T allele of rs12976445, resulting in augmented production of target genes, including LIFR and ERBB2 [22, 38]. It has been previously shown that a SNP (rs12976445) is present in the pre-miR-125a gene, and it compromises the mature processing of the miRNA, which leads to an increased risk of autoimmune thyroid conditions [21] and repeated pregnancy loss [22]. In this study, we studied the association between rs12976445 polymorphism and risk of DN in a total of 594 DM patients with (N=282) or without DN (N=312), and we found that rs12976445 was significantly associated with DN (OR=1.45, 95% C.I.=1.02–2.08, p<0.05) after the possible confounding factors were adjusted for by using logistic regression analysis.
Conclusions:
Our results showed that miR-125a is a key player in the development of DN via targeting IL-6R, and underexpression of miR-125a promoted proliferation of mesangial cells by releasing the inhibition on IL-6R. Downregulation of miR-125a could be, at least partially, attributed to the presence of rs12976445 polymorphism, which compromises the mature processing of the miRNA, demonstrating that miR-125a is a promising novel diagnostic and therapeutic target for DN.
|
Background:
Diabetic nephropathy (DN) is one of the most significant long-term complications of diabetes mellitus (DM), and it is a primary risk factor for end-stage renal disease. MicroRNAs (miRNAs) play important roles in regulating the expression of genes, including interleukin-6R (IL-6R), which has been reported to be involved in the development of DNDN. The aim of this study was to identify the dysregulation of miRNA and its target responsible for the development of DN in DM.
Methods:
We collected the kidney tissues from patients with DN (N=36) and control patients (N=28), and performed real-time PCR and Western blot analysis to determine the expression of IL-6R. Computational analysis and luciferase assay were used to identify the miRNA that regulates IL-6R. To explore the association between rs12976445 polymorphism and risk of DN, we enrolled 594 DM patients with (N=282) or without DN (N=312), and studied the association between a variant in miR-125a and risk of DN in DM.
Results:
The expression of IL-6R was barely detected in the control groups, while in the DN group, the IL-6R was clearly detectable. Next, miR-125a was identified as a regulator of IL-6R by using informatics analysis and luciferase assay. A single-nucleotide polymorphism (rs12976445) in pri-miR-125a has been shown to compromise the mature processing of miR-125a, and we showed that the expression levels of miR-125a was comparable between individuals carrying TT and CT, and when combined into 1 group, the miR-125a expression was approximately 3 times lower than in the CC group. We found significant differences regarding rs12976445 genotype distribution between the DN and the control (OR=1.45, 95% C.I.=1.02-2.08, p<0.05) with the possible confounding factors adjusted for by using logistic regression analysis.
Conclusions:
We identified miR-125a as a direct regulator of IL-6R, and the genotype of rs12976445 might be a novel predictor of the development of DN in DM.
|
Background:
Diabetic nephropathy (DN) is one of the most important long-term complications of diabetes, and it is the primary cause of end-stage renal disease and deaths in diabetic patients [1]. The major clinical characteristics of DN includes progressively decreasing glomerular filtration rate (GFR) and persistent albuminuria, with macroalbuminuria (>300mg/day) indicating DN progression and microalbuminuria (30 to 300 mg of albumin in urine per day) representing early DN [2]. The main pathological characteristics of DN are expansion and hypertrophy in the tubular compartments and glomerular mesangium, as well as buildup of extracellular matrix (ECM) proteins and dysfunction of podocytes. There are several mechanisms, such as protein kinase C, advanced glycation end-products, hyperglycemia, oxidative stress, poly (ADP-ribose) polymerase activation, and inflammation, which are thought to account for the development and pathogenesis of DN [3]. Unfortunately, the hidden molecular pathogenesis is not completely identified.
Interleukin 6 (IL-6) is a multi-potent cytokine; its main function appears to induce proliferation and differentiation of B lymphocyte and acute inflammatory responses. IL-6 has been shown to enhance the differentiation and growth of a wide range of cells, including renal mesangial cells [4]. The aberrantly enhanced proliferation of renal mesangial cells may lead to basement membrane thickening and mesangial matrix expansion, which is a major histological characteristic of diabetic nephropathy [5–7]. Actually, thickening of basement membrane and mesangial proliferation can be observed in early-stage nephropathy [8]. IL-6 may be very important in mesangial proliferation in patients who have glomerular renal disease [4,9]. IL-6 acts on a receptor that consists of 2 subunits, gp130 and IL-6 receptor (IL-6R). First, IL-6 binds to the IL-6R, and this complex subsequently binds to the gp130 subunit [10,11]. Elevated concentrations of IL-6R have been observed in both DN [12] and type II diabetes mellitus (DM) [13].
MicroRNAs (miRNAs) consist of short non-coding RNAs that regulate physiological and pathological processes by suppressing expression of target gene via blocking protein translation or promoting degradation of mRNA. Recently, studies have discovered key properties for specific miRNAs in a battery of biological and cellular processes, such as proliferation, development, and differentiation [14]. Although the role of miRNAs has been identified in the pathogenesis of many human diseases, their function in diabetes and DN is less understood. Earlier reports have shown that single-nucleotide polymorphisms (SNPs) in miRNA genes may interfere with the interaction between miRNAs and mRNAs of target genes, or compromise the processing of mature miRNA [15–18], which may contribute to susceptibility to certain human diseases [19,20].
In this study, we confirmed the differential expression of IL-6R in DN and identified miR-125a as a regulator of IL-6R. It has been previously shown that a SNP (rs12976445) is present in the pre-miR-125a gene, and compromises the mature processing of the miRNA, which leads to an increased risk of autoimmune thyroid conditions [21] and repeated pregnancy loss [22]. Based on the evidence mentioned above, we studied the association between rs12976445 polymorphism and risk of DN in the patients with DM.
Conclusions:
Our results showed that miR-125a is a key player in the development of DN via targeting IL-6R, and underexpression of miR-125a promoted proliferation of mesangial cells by releasing the inhibition on IL-6R. Downregulation of miR-125a could be, at least partially, attributed to the presence of rs12976445 polymorphism, which compromises the mature processing of the miRNA, demonstrating that miR-125a is a promising novel diagnostic and therapeutic target for DN.
|
Background:
Diabetic nephropathy (DN) is one of the most significant long-term complications of diabetes mellitus (DM), and it is a primary risk factor for end-stage renal disease. MicroRNAs (miRNAs) play important roles in regulating the expression of genes, including interleukin-6R (IL-6R), which has been reported to be involved in the development of DNDN. The aim of this study was to identify the dysregulation of miRNA and its target responsible for the development of DN in DM.
Methods:
We collected the kidney tissues from patients with DN (N=36) and control patients (N=28), and performed real-time PCR and Western blot analysis to determine the expression of IL-6R. Computational analysis and luciferase assay were used to identify the miRNA that regulates IL-6R. To explore the association between rs12976445 polymorphism and risk of DN, we enrolled 594 DM patients with (N=282) or without DN (N=312), and studied the association between a variant in miR-125a and risk of DN in DM.
Results:
The expression of IL-6R was barely detected in the control groups, while in the DN group, the IL-6R was clearly detectable. Next, miR-125a was identified as a regulator of IL-6R by using informatics analysis and luciferase assay. A single-nucleotide polymorphism (rs12976445) in pri-miR-125a has been shown to compromise the mature processing of miR-125a, and we showed that the expression levels of miR-125a was comparable between individuals carrying TT and CT, and when combined into 1 group, the miR-125a expression was approximately 3 times lower than in the CC group. We found significant differences regarding rs12976445 genotype distribution between the DN and the control (OR=1.45, 95% C.I.=1.02-2.08, p<0.05) with the possible confounding factors adjusted for by using logistic regression analysis.
Conclusions:
We identified miR-125a as a direct regulator of IL-6R, and the genotype of rs12976445 might be a novel predictor of the development of DN in DM.
| 5,579 | 371 | 14 |
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[CONTENT] Cytokine Receptor gp130 | Diabetic Nephropathies | MicroRNAs [SUMMARY]
|
[CONTENT] Cytokine Receptor gp130 | Diabetic Nephropathies | MicroRNAs [SUMMARY]
|
[CONTENT] Cytokine Receptor gp130 | Diabetic Nephropathies | MicroRNAs [SUMMARY]
|
[CONTENT] Cytokine Receptor gp130 | Diabetic Nephropathies | MicroRNAs [SUMMARY]
|
[CONTENT] Cytokine Receptor gp130 | Diabetic Nephropathies | MicroRNAs [SUMMARY]
|
[CONTENT] Cytokine Receptor gp130 | Diabetic Nephropathies | MicroRNAs [SUMMARY]
|
[CONTENT] Adult | Aged | Case-Control Studies | Diabetic Nephropathies | Female | Genetic Association Studies | Genetic Predisposition to Disease | Humans | Kidney Failure, Chronic | Male | MicroRNAs | Middle Aged | Polymorphism, Single Nucleotide | Real-Time Polymerase Chain Reaction | Receptors, Interleukin-6 | Risk Factors [SUMMARY]
|
[CONTENT] Adult | Aged | Case-Control Studies | Diabetic Nephropathies | Female | Genetic Association Studies | Genetic Predisposition to Disease | Humans | Kidney Failure, Chronic | Male | MicroRNAs | Middle Aged | Polymorphism, Single Nucleotide | Real-Time Polymerase Chain Reaction | Receptors, Interleukin-6 | Risk Factors [SUMMARY]
|
[CONTENT] Adult | Aged | Case-Control Studies | Diabetic Nephropathies | Female | Genetic Association Studies | Genetic Predisposition to Disease | Humans | Kidney Failure, Chronic | Male | MicroRNAs | Middle Aged | Polymorphism, Single Nucleotide | Real-Time Polymerase Chain Reaction | Receptors, Interleukin-6 | Risk Factors [SUMMARY]
|
[CONTENT] Adult | Aged | Case-Control Studies | Diabetic Nephropathies | Female | Genetic Association Studies | Genetic Predisposition to Disease | Humans | Kidney Failure, Chronic | Male | MicroRNAs | Middle Aged | Polymorphism, Single Nucleotide | Real-Time Polymerase Chain Reaction | Receptors, Interleukin-6 | Risk Factors [SUMMARY]
|
[CONTENT] Adult | Aged | Case-Control Studies | Diabetic Nephropathies | Female | Genetic Association Studies | Genetic Predisposition to Disease | Humans | Kidney Failure, Chronic | Male | MicroRNAs | Middle Aged | Polymorphism, Single Nucleotide | Real-Time Polymerase Chain Reaction | Receptors, Interleukin-6 | Risk Factors [SUMMARY]
|
[CONTENT] Adult | Aged | Case-Control Studies | Diabetic Nephropathies | Female | Genetic Association Studies | Genetic Predisposition to Disease | Humans | Kidney Failure, Chronic | Male | MicroRNAs | Middle Aged | Polymorphism, Single Nucleotide | Real-Time Polymerase Chain Reaction | Receptors, Interleukin-6 | Risk Factors [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
|
[CONTENT] il | 6r | il 6r | cells | dn | mir | 125a | mir 125a | patients | renal [SUMMARY]
|
[CONTENT] il | 6r | il 6r | cells | dn | mir | 125a | mir 125a | patients | renal [SUMMARY]
|
[CONTENT] il | 6r | il 6r | cells | dn | mir | 125a | mir 125a | patients | renal [SUMMARY]
|
[CONTENT] il | 6r | il 6r | cells | dn | mir | 125a | mir 125a | patients | renal [SUMMARY]
|
[CONTENT] il | 6r | il 6r | cells | dn | mir | 125a | mir 125a | patients | renal [SUMMARY]
|
[CONTENT] il | 6r | il 6r | cells | dn | mir | 125a | mir 125a | patients | renal [SUMMARY]
|
[CONTENT] dn | il | mirnas | proliferation | differentiation | mesangial | identified | diabetic | pathogenesis | nephropathy [SUMMARY]
|
[CONTENT] 95 | 95 cis | statistical | ors | ors 95 | cis | regression model | logistic regression model | model | spss [SUMMARY]
|
[CONTENT] 6r | il 6r | il | figure | 125a | mir 125a | mir | cells transfected | expression | transfected [SUMMARY]
|
[CONTENT] 125a | mir | mir 125a | dn | mesangial cells releasing | diagnostic therapeutic target dn | targeting il | targeting il 6r | targeting il 6r underexpression | diagnostic therapeutic [SUMMARY]
|
[CONTENT] il | dn | 6r | il 6r | mir | mir 125a | 125a | cells | patients | renal [SUMMARY]
|
[CONTENT] il | dn | 6r | il 6r | mir | mir 125a | 125a | cells | patients | renal [SUMMARY]
|
[CONTENT] ||| IL-6R | DNDN ||| DM [SUMMARY]
|
[CONTENT] PCR | Western ||| 594 | N=282 | DM [SUMMARY]
|
[CONTENT] IL-6R | IL-6R ||| IL-6R ||| TT | CT | 1 | 3 | CC ||| 95% | 2.08 | p<0.05 [SUMMARY]
|
[CONTENT] IL-6R | DM [SUMMARY]
|
[CONTENT] ||| IL-6R | DNDN ||| DM ||| PCR | Western ||| 594 | N=282 | DM ||| IL-6R | IL-6R ||| IL-6R ||| TT | CT | 1 | 3 | CC ||| 95% | 2.08 | p<0.05 ||| IL-6R | DM [SUMMARY]
|
[CONTENT] ||| IL-6R | DNDN ||| DM ||| PCR | Western ||| 594 | N=282 | DM ||| IL-6R | IL-6R ||| IL-6R ||| TT | CT | 1 | 3 | CC ||| 95% | 2.08 | p<0.05 ||| IL-6R | DM [SUMMARY]
|
||||||
Imaging of pancreatic metastases from renal cell carcinoma.
|
25609358
|
To describe the main imaging characteristics of pancreatic metastases from renal cell carcinoma (RCC) with particular attention to CT features, underlining possible criteria for a differential diagnosis.
|
BACKGROUND
|
15 patients have been included in this study. 14 patients underwent multislice CT with triphasic acquisition (unenhanced, pancreatic parenchymal and portal venous phases). In 9 cases a delayed phase (120 sec) was also acquired. 5 patients underwent MRI, before and after administration of gadolinium.
|
METHODS
|
The mean time interval between nephrectomy and recurrence was 7.5 years (range 1-17 years). On CT metastases avidly enhanced in the parenchymal phase and then demonstrated a significant wash-out, approaching isodensity to the normal pancreatic parenchyma in the portal phase. In the portal phase 20 of the 25 lesions found in the arterial phase were recognizable. On non-enhanced scans, only 13 of the 25 lesions were detected.
|
RESULTS
|
Renal Cell Carcinomas require a prolonged CT or MRI follow-up.
|
CONCLUSION
|
[
"Adult",
"Aged",
"Aged, 80 and over",
"Carcinoma, Renal Cell",
"Female",
"Humans",
"Kidney Neoplasms",
"Magnetic Resonance Imaging",
"Male",
"Middle Aged",
"Pancreatic Neoplasms",
"Retrospective Studies",
"Tomography, X-Ray Computed"
] |
4212532
|
Background
|
Metastatic lesions in the pancreas are uncommon and account for 2% to 5% of all pancreatic malignancies [1-3]. However, pancreas is a possible site for metastases from renal cell carcinoma (RCC), not unfrequently being the only metastatic site [4].
Very delayed metastases, i.e. later than ten years after tumor nephrectomy, occur in more than 10% of patients [5].
Metastases are only rarely symptomatic in the early phases, with symptoms including abdominal pain, back pain, gastrointestinal bleeding due to duodenal infiltration, obstructive jaundice, weight loss, pancreatitis, and diabetes.
Retrospective series showed that when there is no evidence of metastatic spread in other organs, pancreatic metastatic lesions from RCC are a favorable indication for a radical surgery, offering good chances of long-term survival [6-8]. In a recent study Karam JA et al. reported that metastasectomy is feasible with acceptable morbidity in a cohort of select patients with a limited tumor burden after targeted therapy [9]. Moreover, it has been demonstrated that tumor burden characteristics are associated with clinical outcome in patients with metastasis from RCC treated with vascular endothelial growth factor-targeted therapy such as sunitinib [10].
For these reasons, it has been suggested that patients with a history of RCC should be monitored in order to detect early recurrences. Efficacy and modalities of post-operative radiological follow-up are still debated.
In this article, we describe the main imaging features of pancreatic metastases from RCC on the basis of a retrospective review of fifteen cases from our archives and a review of the literature.
|
Methods
|
A retrospective search for patients with a diagnosis of pancreatic metastases from RCC obtained between February 2004 and June 2010 at the University Hospital of Padova was performed.
Fifteen cases were identified and their clinical records reviewed. Diagnosis was based on medical history, imaging and histological examination of surgical specimens.
The group of patients comprised six women and nine men, aged between 39 and 82, with a mean age of 63.5 years.
Fourteen out of fifteen patients underwent multislice CT, using 16- and 64-slice scanners (Emotion 16 and Somatom Sensation 64; Siemens, Erlangen, Germany). An initial non-enhanced acquisition was performed, followed by a breath-hold pancreatic parenchymal phase acquisition at 30-40 sec after injection and a portal venous phase scan, obtained 60–70 seconds after injection. In 9 cases a delayed phase of 120 sec was also acquired, even if for purposes different from the characterization of the pancreatic tumor. The intravenous administration of 150-200 mL of non-ionic contrast material (Omnipaque 350 – GE Healthcare – USA) was given using an automatic injector at a rate of 3.5-5 mL/sec.
Four-millimeter axial images, as well as coronal and sagittal reformatted images, were sent to the picture archiving and communication system.
Five patients underwent an abdominal 1.5 Tesla MRI scan (Magnetom Espree, Siemens, Erlangen, Germany) (one only MRI, four CT plus MRI) before and after i.v administration of gadolinium. Our protocol included axial GRE T1 in-phase/out-of-phase images (TR of 129 ms, TE respectively of 4.87 and 2.38 ms; field of view 380 mm; slice thick-ness, 4 mm), TSE T2 weighted images (TR, 3700 ms; TE, 92 ms; field of view 380 mm; slice thick-ness, 4 mm), axial and coronal HASTE T2-weighed images with and without fat saturation (TR, 1500 ms; TE, 90 ms; field of view, 380 mm; slice thick-ness, 4-6 mm), and diffusion-weighted (b 50-400-800 s/mm2) images.
After administration of contrast agent axial GRE T1-weighted images (TR, 5.07 ms; TE, 2.38 ms, field of view 400 mm; slice thick-ness, 4 mm) were obtained at 30, 70 and 120 seconds.
Images were evaluated by three radiologists.
Fourteen patients underwent surgery, while in one patient the diagnosis was autoptical. Both typical resections (pancreatoduodenectomy, distal splenopancreatectomy, total pancreatectomy) and atypical resections (enucleation, middle pancreatectomy, spleen-preserving distal pancreatectomy) were performed. Histological examination of the resected specimens showed metastases from renal cell carcinoma in all of these cases. All patients were treated according to the Helsinki declaration ethical principles. We obtained our hospital’s institutional review board approval to conduct a retrospective review of the patients’ medical and imaging records.
|
Results
|
A total of 26 metastatic lesions were identified in 15 patients: 19 lesions were detected with CT, 1 with MRI and 6 with both CT and MRI (Tables 1 and 2).
CT characteristics and distribution of pancreatic metastases
MRI characteristics and distribution of pancreatic metastases
Disease-free time interval between primary treatment and recurrence ranged from 1 to 17 years (mean 7.5 years).
In 11 patients the pancreas was the only metastatic site. 3 patients also presented with lung metastases, in one case associated with liver metastases. 1 patient had a concomitant thyroid metastatic lesion.
Solitary lesions were found in 7 patients (Figure 1), while 8 patients presented with multiple metastases (Figure 2). The lesions were distributed throughout the pancreas without notable predilection for a particular part of the gland. No patients presented a diffuse involvement of the pancreas.
Solitary lesions. Two examples of solitary spherical tumors of the pancreatic tail, respectively on contrast-enhanced CT (A) and MRI (B) images, with the typical appearance of a predominantly solid, well-defined mass with smooth borders.
Multiple lesions. An arterial phase acquisition on CT shows two synchronous metastases as inhomogeneously enhancing nodular masses in the pancreatic body and tail.
The metastatic foci usually appeared well-defined, often with discrete margins. 21 were round or ovoid with smooth borders, 5 were lobular. They ranged in size from 0.7 cm to 4.0 cm (median diameter was 1.5 cm).
In 2 patients there was evident obstruction of the main pancreatic duct with dilatation of the duct upstream from the obstruction. 4 cases presented biliary tree dilatation.
No patients demonstrated involvement of a neighbouring extra-pancreatic artery; even if venous involvement is often more difficult to assess, in our series the superior mesenteric and portal veins appeared unaffected.
On contrast-enhanced CT, metastases avidly enhanced in the arterial or pancreatic parenchymal phase and demonstrated a significant wash-out on portal and delayed phase images, performed after 120 seconds (Figure 3). Nodules smaller than 2-2.5 cm in diameter showed homogeneous enhancement, while larger masses had an heterogeneous enhancement, with central hypodense areas probably due to necrosis.
Vascularity pattern. Axial CT scans obtained unenhanced (A) and during arterial (B), portal (C) and delayed (D) phases of contrast enhancement show a large, solitary round metastasis of the pancreatic head. Note that lesion is best seen in the arterial phase and less well seen in portal and delayed phases.
On MRI, the lesions showed signal intensity lower than normal pancreatic tissue on pre-contrast T1-weighted images, both on in-phase and out-of-phase acquisitions (Figure 4). They conversely presented an heterogeneous or moderately hyperintense signal on T2-weighted images with or without fat-saturation, including diffusion-weighted sequences.
In-phase, out-of-phase and diffusion-weighted images. Lesions with signal intensity lower than normal pancreatic tissue on in-phase (A) and out-of-phase images (B). They showed hyperintense signal on T2-weighted (C) and diffusion-weighted sequences obtained at b values of 400 (D).
After the administration of gadolinium, nodules smaller than 2-2.5 cm in diameter showed homogeneous enhancement, while larger masses presented heterogeneous enhancement with central hypodense areas, probably due to internal necrosis.
Only in 1 patient we found two metastatic foci that resulted almost isointense to the surrounding pancreatic parenchyma, from which they were separated by a thin hypointense rim. These lesions also showed loss of signal intensity on DWI with high b-values, while the other lesions in our series showed increased signal intensity with higher b-values (Figure 5).
Arterial phase and diffusion-weighted images. Lesions almost isointense to the surrounding pancreatic parenchyma on arterial phase acquisition, circumscribed by a thin hypointense rim (A). They show lower signal intensity on DWI sequences with the increasing of b-values (B, C, D).
|
Conclusion
|
Pancreatic metastases may present many years after the resection of the primary renal cell carcinoma.
An early arterial phase or a pancreatic parenchymal phase are mandatory both on CT and MRI for patients with a history of RCC, despite the reason why the patient is undergoing the exam or the time passed after surgery.
With regard to the differential diagnosis, endocrine pancreatic tumors are the most challenging ones, as the other primary pancreatic tumors tend to be hypovascular.
However, clinical history remains extremely helpful. A hypervascular pancreatic tumor in a patient with a history of renal cancer should be considered a metastasis from renal cancer until proven otherwise.
|
[
"Background",
"Competing interests",
"Authors’ contributions"
] |
[
"Metastatic lesions in the pancreas are uncommon and account for 2% to 5% of all pancreatic malignancies [1-3]. However, pancreas is a possible site for metastases from renal cell carcinoma (RCC), not unfrequently being the only metastatic site [4].\nVery delayed metastases, i.e. later than ten years after tumor nephrectomy, occur in more than 10% of patients [5].\nMetastases are only rarely symptomatic in the early phases, with symptoms including abdominal pain, back pain, gastrointestinal bleeding due to duodenal infiltration, obstructive jaundice, weight loss, pancreatitis, and diabetes.\nRetrospective series showed that when there is no evidence of metastatic spread in other organs, pancreatic metastatic lesions from RCC are a favorable indication for a radical surgery, offering good chances of long-term survival [6-8]. In a recent study Karam JA et al. reported that metastasectomy is feasible with acceptable morbidity in a cohort of select patients with a limited tumor burden after targeted therapy [9]. Moreover, it has been demonstrated that tumor burden characteristics are associated with clinical outcome in patients with metastasis from RCC treated with vascular endothelial growth factor-targeted therapy such as sunitinib [10].\nFor these reasons, it has been suggested that patients with a history of RCC should be monitored in order to detect early recurrences. Efficacy and modalities of post-operative radiological follow-up are still debated.\nIn this article, we describe the main imaging features of pancreatic metastases from RCC on the basis of a retrospective review of fifteen cases from our archives and a review of the literature.",
"The authors declared that they have no competing interests.",
"VM has been involved in planning the study, in acquisition of data and radiological examinations. He has also performed the statistical analysis. PG drafted the manuscript and has made substantial contributions to conception and design. PR has participated in the design of the study helped to draft the manuscript. PC has followed patients for surgical and clinical aspects, giving important contributions to interpretation of data. PF have given final approval of the version to be published. All authors read and approved the final manuscript."
] |
[
null,
null,
null
] |
[
"Background",
"Methods",
"Results",
"Discussion",
"Conclusion",
"Competing interests",
"Authors’ contributions"
] |
[
"Metastatic lesions in the pancreas are uncommon and account for 2% to 5% of all pancreatic malignancies [1-3]. However, pancreas is a possible site for metastases from renal cell carcinoma (RCC), not unfrequently being the only metastatic site [4].\nVery delayed metastases, i.e. later than ten years after tumor nephrectomy, occur in more than 10% of patients [5].\nMetastases are only rarely symptomatic in the early phases, with symptoms including abdominal pain, back pain, gastrointestinal bleeding due to duodenal infiltration, obstructive jaundice, weight loss, pancreatitis, and diabetes.\nRetrospective series showed that when there is no evidence of metastatic spread in other organs, pancreatic metastatic lesions from RCC are a favorable indication for a radical surgery, offering good chances of long-term survival [6-8]. In a recent study Karam JA et al. reported that metastasectomy is feasible with acceptable morbidity in a cohort of select patients with a limited tumor burden after targeted therapy [9]. Moreover, it has been demonstrated that tumor burden characteristics are associated with clinical outcome in patients with metastasis from RCC treated with vascular endothelial growth factor-targeted therapy such as sunitinib [10].\nFor these reasons, it has been suggested that patients with a history of RCC should be monitored in order to detect early recurrences. Efficacy and modalities of post-operative radiological follow-up are still debated.\nIn this article, we describe the main imaging features of pancreatic metastases from RCC on the basis of a retrospective review of fifteen cases from our archives and a review of the literature.",
"A retrospective search for patients with a diagnosis of pancreatic metastases from RCC obtained between February 2004 and June 2010 at the University Hospital of Padova was performed.\nFifteen cases were identified and their clinical records reviewed. Diagnosis was based on medical history, imaging and histological examination of surgical specimens.\nThe group of patients comprised six women and nine men, aged between 39 and 82, with a mean age of 63.5 years.\nFourteen out of fifteen patients underwent multislice CT, using 16- and 64-slice scanners (Emotion 16 and Somatom Sensation 64; Siemens, Erlangen, Germany). An initial non-enhanced acquisition was performed, followed by a breath-hold pancreatic parenchymal phase acquisition at 30-40 sec after injection and a portal venous phase scan, obtained 60–70 seconds after injection. In 9 cases a delayed phase of 120 sec was also acquired, even if for purposes different from the characterization of the pancreatic tumor. The intravenous administration of 150-200 mL of non-ionic contrast material (Omnipaque 350 – GE Healthcare – USA) was given using an automatic injector at a rate of 3.5-5 mL/sec.\nFour-millimeter axial images, as well as coronal and sagittal reformatted images, were sent to the picture archiving and communication system.\nFive patients underwent an abdominal 1.5 Tesla MRI scan (Magnetom Espree, Siemens, Erlangen, Germany) (one only MRI, four CT plus MRI) before and after i.v administration of gadolinium. Our protocol included axial GRE T1 in-phase/out-of-phase images (TR of 129 ms, TE respectively of 4.87 and 2.38 ms; field of view 380 mm; slice thick-ness, 4 mm), TSE T2 weighted images (TR, 3700 ms; TE, 92 ms; field of view 380 mm; slice thick-ness, 4 mm), axial and coronal HASTE T2-weighed images with and without fat saturation (TR, 1500 ms; TE, 90 ms; field of view, 380 mm; slice thick-ness, 4-6 mm), and diffusion-weighted (b 50-400-800 s/mm2) images.\nAfter administration of contrast agent axial GRE T1-weighted images (TR, 5.07 ms; TE, 2.38 ms, field of view 400 mm; slice thick-ness, 4 mm) were obtained at 30, 70 and 120 seconds.\nImages were evaluated by three radiologists.\nFourteen patients underwent surgery, while in one patient the diagnosis was autoptical. Both typical resections (pancreatoduodenectomy, distal splenopancreatectomy, total pancreatectomy) and atypical resections (enucleation, middle pancreatectomy, spleen-preserving distal pancreatectomy) were performed. Histological examination of the resected specimens showed metastases from renal cell carcinoma in all of these cases. All patients were treated according to the Helsinki declaration ethical principles. We obtained our hospital’s institutional review board approval to conduct a retrospective review of the patients’ medical and imaging records.",
"A total of 26 metastatic lesions were identified in 15 patients: 19 lesions were detected with CT, 1 with MRI and 6 with both CT and MRI (Tables 1 and 2).\nCT characteristics and distribution of pancreatic metastases\nMRI characteristics and distribution of pancreatic metastases\nDisease-free time interval between primary treatment and recurrence ranged from 1 to 17 years (mean 7.5 years).\nIn 11 patients the pancreas was the only metastatic site. 3 patients also presented with lung metastases, in one case associated with liver metastases. 1 patient had a concomitant thyroid metastatic lesion.\nSolitary lesions were found in 7 patients (Figure 1), while 8 patients presented with multiple metastases (Figure 2). The lesions were distributed throughout the pancreas without notable predilection for a particular part of the gland. No patients presented a diffuse involvement of the pancreas.\nSolitary lesions. Two examples of solitary spherical tumors of the pancreatic tail, respectively on contrast-enhanced CT (A) and MRI (B) images, with the typical appearance of a predominantly solid, well-defined mass with smooth borders.\nMultiple lesions. An arterial phase acquisition on CT shows two synchronous metastases as inhomogeneously enhancing nodular masses in the pancreatic body and tail.\nThe metastatic foci usually appeared well-defined, often with discrete margins. 21 were round or ovoid with smooth borders, 5 were lobular. They ranged in size from 0.7 cm to 4.0 cm (median diameter was 1.5 cm).\nIn 2 patients there was evident obstruction of the main pancreatic duct with dilatation of the duct upstream from the obstruction. 4 cases presented biliary tree dilatation.\nNo patients demonstrated involvement of a neighbouring extra-pancreatic artery; even if venous involvement is often more difficult to assess, in our series the superior mesenteric and portal veins appeared unaffected.\nOn contrast-enhanced CT, metastases avidly enhanced in the arterial or pancreatic parenchymal phase and demonstrated a significant wash-out on portal and delayed phase images, performed after 120 seconds (Figure 3). Nodules smaller than 2-2.5 cm in diameter showed homogeneous enhancement, while larger masses had an heterogeneous enhancement, with central hypodense areas probably due to necrosis.\nVascularity pattern. Axial CT scans obtained unenhanced (A) and during arterial (B), portal (C) and delayed (D) phases of contrast enhancement show a large, solitary round metastasis of the pancreatic head. Note that lesion is best seen in the arterial phase and less well seen in portal and delayed phases.\nOn MRI, the lesions showed signal intensity lower than normal pancreatic tissue on pre-contrast T1-weighted images, both on in-phase and out-of-phase acquisitions (Figure 4). They conversely presented an heterogeneous or moderately hyperintense signal on T2-weighted images with or without fat-saturation, including diffusion-weighted sequences.\nIn-phase, out-of-phase and diffusion-weighted images. Lesions with signal intensity lower than normal pancreatic tissue on in-phase (A) and out-of-phase images (B). They showed hyperintense signal on T2-weighted (C) and diffusion-weighted sequences obtained at b values of 400 (D).\nAfter the administration of gadolinium, nodules smaller than 2-2.5 cm in diameter showed homogeneous enhancement, while larger masses presented heterogeneous enhancement with central hypodense areas, probably due to internal necrosis.\nOnly in 1 patient we found two metastatic foci that resulted almost isointense to the surrounding pancreatic parenchyma, from which they were separated by a thin hypointense rim. These lesions also showed loss of signal intensity on DWI with high b-values, while the other lesions in our series showed increased signal intensity with higher b-values (Figure 5).\nArterial phase and diffusion-weighted images. Lesions almost isointense to the surrounding pancreatic parenchyma on arterial phase acquisition, circumscribed by a thin hypointense rim (A). They show lower signal intensity on DWI sequences with the increasing of b-values (B, C, D).",
"Metastases from renal cell carcinoma (RCC) may be found at the time of primary tumour diagnosis or, more frequently, during follow-up after surgery [5,7,11]. A report showed that approximately 10% of 10-year survivors after tumor nephrectomy had late recurrences from RCC [5].\nNevertheless, pancreatic metastases from RCC are unusual. In autopsy series of patients with RCC, the reported incidence ranged from 1 to 3% [12].\nIf pancreatic metastases are discovered at the follow-up, pancreas not unfrequently is the only metastatic site and relatively often has a solitary metastasis. In fact, RCC has been reported to be the most common primary tumor leading to solitary pancreatic metastasis [13]. No relationship has yet been found between the site of an isolated pancreatic metastasis and the site of the primary renal cell carcinoma [14].\nHowever, multifocality of pancreatic metastases from RCC is not unusual, ranging from 20% to 45% [3,15]. It does not necessarily relate to a worse outcome and it does not represent a contraindication to surgery.\nDespite the uni- or multifocality, when there are no detectable metastases in other organs there is a favorable indication for a radical surgery, with a reported mean survival of 4 years [3]. In fact surgery is considered the gold standard therapy for localized disease.\nIn addition, the introduction of targeted therapy, including inhibitors of vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR), has dramatically changed the outcome of patients with metastatic renal cell cancer, which was typically a chemoresistant disease [16].\nFor all these reasons, it is crucial to achieve an early radiological diagnosis of recurrence.\nIn our series, both on CT and MRI the metastatic foci appeared as round or ovoid masses, mostly well-delineated and with smooth borders.\nAs reported in the literature, metastases resulted isodense or hypodense in comparison to normal parenchyma on unenhanced CT, avidly enhanced in the pancreatic late arterial phase and demonstrated a significant wash-out, in particular on delayed phase images.\nIn the delayed phase lesions appeared sometimes hard to recognize, approaching isodensity with the surrounding pancreatic parenchyma: acquiring an arterial phase is therefore mandatory whenever we are dealing with a patient with history of RCC, whatever the clinical indication for imaging [2,4,12].\nUpon our knowledge, there are few reports in the literature concerning MRI imaging features of pancreatic metastases from RCC.\nIn our experience, metastases showed low signal intensity compared with the normal pancreatic tissue on pre-contrast T1-weighted images, and moderately hyperintense signal on T2- and T2 fat-sat images. Lesions larger than 2 cm often had heterogeneous signal both on T1- and T2-weighted sequences, due to necrosis [17].\nAfter the administration of gadolinium, metastases presented enhancement features similar to those described with CT. An early and homogeneous enhancement was usually seen in smaller metastases, while larger lesions showed a thick rim of enhancement.\nIn one patient, two intrapancreatic lesions appeared almost isointense to the surrounding pancreatic parenchyma on dynamic contrast enhanced MRI. This behavior, perhaps, might be related to a higher necrotic content of the lesions, as suggested also by the lowering of their signal intensity on DWI acquisitions with the increasing of the b-values, while the other metastatic lesions in our series showed a persistent elevated signal.\nThe non-enhanced features described above, and the early enhancement after contrast medium injection with both CT and MRI are characteristics that can also be found in the presence of endocrine pancreatic tumors, while primary adenocarcinomas tend to be hypovascular [18].\nIt is really hard to differentiate RCC metastases from endocrine tumors of the pancreas.\nSymptoms of pancreatic metastases such as abdominal pain, jaundice, weight loss, and steatorrhea may be caused by tumoral invasion of the choledochus or main pancreatic duct (Figure 6). Pancreatic endocrine tumors usually do not invade these structures: this could help in the differentiation between pancreatic metastasis from RCC and endocrine tumor [7,19].\nObstruction of the common bile duct with upstream dilatation of the biliary tree (A); distal dilatation of the main pancreatic duct (B).\nMoreover, a pancreatic endocrine tumor will cause endocrine symptoms if it is a functional tumor.\nEven if octreotide or somatostatin receptor scintigraphy is helpful in the identification of neuroendocrine tumors, some metastatic lesions may also express somatostatin receptors, showing an intense uptake of octreotide at scintigraphy [20]. This should be kept in mind as it could mislead the diagnosis. On the other hand, it might result useful for therapeutic purposes.\nConcerning primary adenocarcinomas of the pancreas, the fundamental clue for the differential diagnosis with RCC metastases consists in the pattern of enhancement after contrast medium, as described above. Also multifocality within the pancreas, relatively common in the presence of recurrence, is not characteristic of primary pancreatic carcinoma.\nAnother point of differentiation could be the evidence of encasement or infiltration of the peripancreatic arteries and veins. In our series no patients with metastases from RCC showed involvement of these structures.\nHowever, the clinical history remains always the first thing to consider. An hypervascular pancreatic tumor in a patient with a previously diagnosed renal cancer should be considered a metastasis from renal cancer until proven otherwise.",
"Pancreatic metastases may present many years after the resection of the primary renal cell carcinoma.\nAn early arterial phase or a pancreatic parenchymal phase are mandatory both on CT and MRI for patients with a history of RCC, despite the reason why the patient is undergoing the exam or the time passed after surgery.\nWith regard to the differential diagnosis, endocrine pancreatic tumors are the most challenging ones, as the other primary pancreatic tumors tend to be hypovascular.\nHowever, clinical history remains extremely helpful. A hypervascular pancreatic tumor in a patient with a history of renal cancer should be considered a metastasis from renal cancer until proven otherwise.",
"The authors declared that they have no competing interests.",
"VM has been involved in planning the study, in acquisition of data and radiological examinations. He has also performed the statistical analysis. PG drafted the manuscript and has made substantial contributions to conception and design. PR has participated in the design of the study helped to draft the manuscript. PC has followed patients for surgical and clinical aspects, giving important contributions to interpretation of data. PF have given final approval of the version to be published. All authors read and approved the final manuscript."
] |
[
null,
"methods",
"results",
"discussion",
"conclusions",
null,
null
] |
[
"Metastases",
"Pancreas",
"Renal cell carcinoma",
"Computed tomography",
"Magnetic resonance imaging"
] |
Background:
Metastatic lesions in the pancreas are uncommon and account for 2% to 5% of all pancreatic malignancies [1-3]. However, pancreas is a possible site for metastases from renal cell carcinoma (RCC), not unfrequently being the only metastatic site [4].
Very delayed metastases, i.e. later than ten years after tumor nephrectomy, occur in more than 10% of patients [5].
Metastases are only rarely symptomatic in the early phases, with symptoms including abdominal pain, back pain, gastrointestinal bleeding due to duodenal infiltration, obstructive jaundice, weight loss, pancreatitis, and diabetes.
Retrospective series showed that when there is no evidence of metastatic spread in other organs, pancreatic metastatic lesions from RCC are a favorable indication for a radical surgery, offering good chances of long-term survival [6-8]. In a recent study Karam JA et al. reported that metastasectomy is feasible with acceptable morbidity in a cohort of select patients with a limited tumor burden after targeted therapy [9]. Moreover, it has been demonstrated that tumor burden characteristics are associated with clinical outcome in patients with metastasis from RCC treated with vascular endothelial growth factor-targeted therapy such as sunitinib [10].
For these reasons, it has been suggested that patients with a history of RCC should be monitored in order to detect early recurrences. Efficacy and modalities of post-operative radiological follow-up are still debated.
In this article, we describe the main imaging features of pancreatic metastases from RCC on the basis of a retrospective review of fifteen cases from our archives and a review of the literature.
Methods:
A retrospective search for patients with a diagnosis of pancreatic metastases from RCC obtained between February 2004 and June 2010 at the University Hospital of Padova was performed.
Fifteen cases were identified and their clinical records reviewed. Diagnosis was based on medical history, imaging and histological examination of surgical specimens.
The group of patients comprised six women and nine men, aged between 39 and 82, with a mean age of 63.5 years.
Fourteen out of fifteen patients underwent multislice CT, using 16- and 64-slice scanners (Emotion 16 and Somatom Sensation 64; Siemens, Erlangen, Germany). An initial non-enhanced acquisition was performed, followed by a breath-hold pancreatic parenchymal phase acquisition at 30-40 sec after injection and a portal venous phase scan, obtained 60–70 seconds after injection. In 9 cases a delayed phase of 120 sec was also acquired, even if for purposes different from the characterization of the pancreatic tumor. The intravenous administration of 150-200 mL of non-ionic contrast material (Omnipaque 350 – GE Healthcare – USA) was given using an automatic injector at a rate of 3.5-5 mL/sec.
Four-millimeter axial images, as well as coronal and sagittal reformatted images, were sent to the picture archiving and communication system.
Five patients underwent an abdominal 1.5 Tesla MRI scan (Magnetom Espree, Siemens, Erlangen, Germany) (one only MRI, four CT plus MRI) before and after i.v administration of gadolinium. Our protocol included axial GRE T1 in-phase/out-of-phase images (TR of 129 ms, TE respectively of 4.87 and 2.38 ms; field of view 380 mm; slice thick-ness, 4 mm), TSE T2 weighted images (TR, 3700 ms; TE, 92 ms; field of view 380 mm; slice thick-ness, 4 mm), axial and coronal HASTE T2-weighed images with and without fat saturation (TR, 1500 ms; TE, 90 ms; field of view, 380 mm; slice thick-ness, 4-6 mm), and diffusion-weighted (b 50-400-800 s/mm2) images.
After administration of contrast agent axial GRE T1-weighted images (TR, 5.07 ms; TE, 2.38 ms, field of view 400 mm; slice thick-ness, 4 mm) were obtained at 30, 70 and 120 seconds.
Images were evaluated by three radiologists.
Fourteen patients underwent surgery, while in one patient the diagnosis was autoptical. Both typical resections (pancreatoduodenectomy, distal splenopancreatectomy, total pancreatectomy) and atypical resections (enucleation, middle pancreatectomy, spleen-preserving distal pancreatectomy) were performed. Histological examination of the resected specimens showed metastases from renal cell carcinoma in all of these cases. All patients were treated according to the Helsinki declaration ethical principles. We obtained our hospital’s institutional review board approval to conduct a retrospective review of the patients’ medical and imaging records.
Results:
A total of 26 metastatic lesions were identified in 15 patients: 19 lesions were detected with CT, 1 with MRI and 6 with both CT and MRI (Tables 1 and 2).
CT characteristics and distribution of pancreatic metastases
MRI characteristics and distribution of pancreatic metastases
Disease-free time interval between primary treatment and recurrence ranged from 1 to 17 years (mean 7.5 years).
In 11 patients the pancreas was the only metastatic site. 3 patients also presented with lung metastases, in one case associated with liver metastases. 1 patient had a concomitant thyroid metastatic lesion.
Solitary lesions were found in 7 patients (Figure 1), while 8 patients presented with multiple metastases (Figure 2). The lesions were distributed throughout the pancreas without notable predilection for a particular part of the gland. No patients presented a diffuse involvement of the pancreas.
Solitary lesions. Two examples of solitary spherical tumors of the pancreatic tail, respectively on contrast-enhanced CT (A) and MRI (B) images, with the typical appearance of a predominantly solid, well-defined mass with smooth borders.
Multiple lesions. An arterial phase acquisition on CT shows two synchronous metastases as inhomogeneously enhancing nodular masses in the pancreatic body and tail.
The metastatic foci usually appeared well-defined, often with discrete margins. 21 were round or ovoid with smooth borders, 5 were lobular. They ranged in size from 0.7 cm to 4.0 cm (median diameter was 1.5 cm).
In 2 patients there was evident obstruction of the main pancreatic duct with dilatation of the duct upstream from the obstruction. 4 cases presented biliary tree dilatation.
No patients demonstrated involvement of a neighbouring extra-pancreatic artery; even if venous involvement is often more difficult to assess, in our series the superior mesenteric and portal veins appeared unaffected.
On contrast-enhanced CT, metastases avidly enhanced in the arterial or pancreatic parenchymal phase and demonstrated a significant wash-out on portal and delayed phase images, performed after 120 seconds (Figure 3). Nodules smaller than 2-2.5 cm in diameter showed homogeneous enhancement, while larger masses had an heterogeneous enhancement, with central hypodense areas probably due to necrosis.
Vascularity pattern. Axial CT scans obtained unenhanced (A) and during arterial (B), portal (C) and delayed (D) phases of contrast enhancement show a large, solitary round metastasis of the pancreatic head. Note that lesion is best seen in the arterial phase and less well seen in portal and delayed phases.
On MRI, the lesions showed signal intensity lower than normal pancreatic tissue on pre-contrast T1-weighted images, both on in-phase and out-of-phase acquisitions (Figure 4). They conversely presented an heterogeneous or moderately hyperintense signal on T2-weighted images with or without fat-saturation, including diffusion-weighted sequences.
In-phase, out-of-phase and diffusion-weighted images. Lesions with signal intensity lower than normal pancreatic tissue on in-phase (A) and out-of-phase images (B). They showed hyperintense signal on T2-weighted (C) and diffusion-weighted sequences obtained at b values of 400 (D).
After the administration of gadolinium, nodules smaller than 2-2.5 cm in diameter showed homogeneous enhancement, while larger masses presented heterogeneous enhancement with central hypodense areas, probably due to internal necrosis.
Only in 1 patient we found two metastatic foci that resulted almost isointense to the surrounding pancreatic parenchyma, from which they were separated by a thin hypointense rim. These lesions also showed loss of signal intensity on DWI with high b-values, while the other lesions in our series showed increased signal intensity with higher b-values (Figure 5).
Arterial phase and diffusion-weighted images. Lesions almost isointense to the surrounding pancreatic parenchyma on arterial phase acquisition, circumscribed by a thin hypointense rim (A). They show lower signal intensity on DWI sequences with the increasing of b-values (B, C, D).
Discussion:
Metastases from renal cell carcinoma (RCC) may be found at the time of primary tumour diagnosis or, more frequently, during follow-up after surgery [5,7,11]. A report showed that approximately 10% of 10-year survivors after tumor nephrectomy had late recurrences from RCC [5].
Nevertheless, pancreatic metastases from RCC are unusual. In autopsy series of patients with RCC, the reported incidence ranged from 1 to 3% [12].
If pancreatic metastases are discovered at the follow-up, pancreas not unfrequently is the only metastatic site and relatively often has a solitary metastasis. In fact, RCC has been reported to be the most common primary tumor leading to solitary pancreatic metastasis [13]. No relationship has yet been found between the site of an isolated pancreatic metastasis and the site of the primary renal cell carcinoma [14].
However, multifocality of pancreatic metastases from RCC is not unusual, ranging from 20% to 45% [3,15]. It does not necessarily relate to a worse outcome and it does not represent a contraindication to surgery.
Despite the uni- or multifocality, when there are no detectable metastases in other organs there is a favorable indication for a radical surgery, with a reported mean survival of 4 years [3]. In fact surgery is considered the gold standard therapy for localized disease.
In addition, the introduction of targeted therapy, including inhibitors of vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR), has dramatically changed the outcome of patients with metastatic renal cell cancer, which was typically a chemoresistant disease [16].
For all these reasons, it is crucial to achieve an early radiological diagnosis of recurrence.
In our series, both on CT and MRI the metastatic foci appeared as round or ovoid masses, mostly well-delineated and with smooth borders.
As reported in the literature, metastases resulted isodense or hypodense in comparison to normal parenchyma on unenhanced CT, avidly enhanced in the pancreatic late arterial phase and demonstrated a significant wash-out, in particular on delayed phase images.
In the delayed phase lesions appeared sometimes hard to recognize, approaching isodensity with the surrounding pancreatic parenchyma: acquiring an arterial phase is therefore mandatory whenever we are dealing with a patient with history of RCC, whatever the clinical indication for imaging [2,4,12].
Upon our knowledge, there are few reports in the literature concerning MRI imaging features of pancreatic metastases from RCC.
In our experience, metastases showed low signal intensity compared with the normal pancreatic tissue on pre-contrast T1-weighted images, and moderately hyperintense signal on T2- and T2 fat-sat images. Lesions larger than 2 cm often had heterogeneous signal both on T1- and T2-weighted sequences, due to necrosis [17].
After the administration of gadolinium, metastases presented enhancement features similar to those described with CT. An early and homogeneous enhancement was usually seen in smaller metastases, while larger lesions showed a thick rim of enhancement.
In one patient, two intrapancreatic lesions appeared almost isointense to the surrounding pancreatic parenchyma on dynamic contrast enhanced MRI. This behavior, perhaps, might be related to a higher necrotic content of the lesions, as suggested also by the lowering of their signal intensity on DWI acquisitions with the increasing of the b-values, while the other metastatic lesions in our series showed a persistent elevated signal.
The non-enhanced features described above, and the early enhancement after contrast medium injection with both CT and MRI are characteristics that can also be found in the presence of endocrine pancreatic tumors, while primary adenocarcinomas tend to be hypovascular [18].
It is really hard to differentiate RCC metastases from endocrine tumors of the pancreas.
Symptoms of pancreatic metastases such as abdominal pain, jaundice, weight loss, and steatorrhea may be caused by tumoral invasion of the choledochus or main pancreatic duct (Figure 6). Pancreatic endocrine tumors usually do not invade these structures: this could help in the differentiation between pancreatic metastasis from RCC and endocrine tumor [7,19].
Obstruction of the common bile duct with upstream dilatation of the biliary tree (A); distal dilatation of the main pancreatic duct (B).
Moreover, a pancreatic endocrine tumor will cause endocrine symptoms if it is a functional tumor.
Even if octreotide or somatostatin receptor scintigraphy is helpful in the identification of neuroendocrine tumors, some metastatic lesions may also express somatostatin receptors, showing an intense uptake of octreotide at scintigraphy [20]. This should be kept in mind as it could mislead the diagnosis. On the other hand, it might result useful for therapeutic purposes.
Concerning primary adenocarcinomas of the pancreas, the fundamental clue for the differential diagnosis with RCC metastases consists in the pattern of enhancement after contrast medium, as described above. Also multifocality within the pancreas, relatively common in the presence of recurrence, is not characteristic of primary pancreatic carcinoma.
Another point of differentiation could be the evidence of encasement or infiltration of the peripancreatic arteries and veins. In our series no patients with metastases from RCC showed involvement of these structures.
However, the clinical history remains always the first thing to consider. An hypervascular pancreatic tumor in a patient with a previously diagnosed renal cancer should be considered a metastasis from renal cancer until proven otherwise.
Conclusion:
Pancreatic metastases may present many years after the resection of the primary renal cell carcinoma.
An early arterial phase or a pancreatic parenchymal phase are mandatory both on CT and MRI for patients with a history of RCC, despite the reason why the patient is undergoing the exam or the time passed after surgery.
With regard to the differential diagnosis, endocrine pancreatic tumors are the most challenging ones, as the other primary pancreatic tumors tend to be hypovascular.
However, clinical history remains extremely helpful. A hypervascular pancreatic tumor in a patient with a history of renal cancer should be considered a metastasis from renal cancer until proven otherwise.
Competing interests:
The authors declared that they have no competing interests.
Authors’ contributions:
VM has been involved in planning the study, in acquisition of data and radiological examinations. He has also performed the statistical analysis. PG drafted the manuscript and has made substantial contributions to conception and design. PR has participated in the design of the study helped to draft the manuscript. PC has followed patients for surgical and clinical aspects, giving important contributions to interpretation of data. PF have given final approval of the version to be published. All authors read and approved the final manuscript.
|
Background:
To describe the main imaging characteristics of pancreatic metastases from renal cell carcinoma (RCC) with particular attention to CT features, underlining possible criteria for a differential diagnosis.
Methods:
15 patients have been included in this study. 14 patients underwent multislice CT with triphasic acquisition (unenhanced, pancreatic parenchymal and portal venous phases). In 9 cases a delayed phase (120 sec) was also acquired. 5 patients underwent MRI, before and after administration of gadolinium.
Results:
The mean time interval between nephrectomy and recurrence was 7.5 years (range 1-17 years). On CT metastases avidly enhanced in the parenchymal phase and then demonstrated a significant wash-out, approaching isodensity to the normal pancreatic parenchyma in the portal phase. In the portal phase 20 of the 25 lesions found in the arterial phase were recognizable. On non-enhanced scans, only 13 of the 25 lesions were detected.
Conclusions:
Renal Cell Carcinomas require a prolonged CT or MRI follow-up.
|
Background:
Metastatic lesions in the pancreas are uncommon and account for 2% to 5% of all pancreatic malignancies [1-3]. However, pancreas is a possible site for metastases from renal cell carcinoma (RCC), not unfrequently being the only metastatic site [4].
Very delayed metastases, i.e. later than ten years after tumor nephrectomy, occur in more than 10% of patients [5].
Metastases are only rarely symptomatic in the early phases, with symptoms including abdominal pain, back pain, gastrointestinal bleeding due to duodenal infiltration, obstructive jaundice, weight loss, pancreatitis, and diabetes.
Retrospective series showed that when there is no evidence of metastatic spread in other organs, pancreatic metastatic lesions from RCC are a favorable indication for a radical surgery, offering good chances of long-term survival [6-8]. In a recent study Karam JA et al. reported that metastasectomy is feasible with acceptable morbidity in a cohort of select patients with a limited tumor burden after targeted therapy [9]. Moreover, it has been demonstrated that tumor burden characteristics are associated with clinical outcome in patients with metastasis from RCC treated with vascular endothelial growth factor-targeted therapy such as sunitinib [10].
For these reasons, it has been suggested that patients with a history of RCC should be monitored in order to detect early recurrences. Efficacy and modalities of post-operative radiological follow-up are still debated.
In this article, we describe the main imaging features of pancreatic metastases from RCC on the basis of a retrospective review of fifteen cases from our archives and a review of the literature.
Conclusion:
Pancreatic metastases may present many years after the resection of the primary renal cell carcinoma.
An early arterial phase or a pancreatic parenchymal phase are mandatory both on CT and MRI for patients with a history of RCC, despite the reason why the patient is undergoing the exam or the time passed after surgery.
With regard to the differential diagnosis, endocrine pancreatic tumors are the most challenging ones, as the other primary pancreatic tumors tend to be hypovascular.
However, clinical history remains extremely helpful. A hypervascular pancreatic tumor in a patient with a history of renal cancer should be considered a metastasis from renal cancer until proven otherwise.
|
Background:
To describe the main imaging characteristics of pancreatic metastases from renal cell carcinoma (RCC) with particular attention to CT features, underlining possible criteria for a differential diagnosis.
Methods:
15 patients have been included in this study. 14 patients underwent multislice CT with triphasic acquisition (unenhanced, pancreatic parenchymal and portal venous phases). In 9 cases a delayed phase (120 sec) was also acquired. 5 patients underwent MRI, before and after administration of gadolinium.
Results:
The mean time interval between nephrectomy and recurrence was 7.5 years (range 1-17 years). On CT metastases avidly enhanced in the parenchymal phase and then demonstrated a significant wash-out, approaching isodensity to the normal pancreatic parenchyma in the portal phase. In the portal phase 20 of the 25 lesions found in the arterial phase were recognizable. On non-enhanced scans, only 13 of the 25 lesions were detected.
Conclusions:
Renal Cell Carcinomas require a prolonged CT or MRI follow-up.
| 2,974 | 194 | 7 |
[
"pancreatic",
"metastases",
"patients",
"phase",
"lesions",
"rcc",
"images",
"ct",
"metastatic",
"showed"
] |
[
"test",
"test"
] |
[CONTENT] Metastases | Pancreas | Renal cell carcinoma | Computed tomography | Magnetic resonance imaging [SUMMARY]
|
[CONTENT] Metastases | Pancreas | Renal cell carcinoma | Computed tomography | Magnetic resonance imaging [SUMMARY]
|
[CONTENT] Metastases | Pancreas | Renal cell carcinoma | Computed tomography | Magnetic resonance imaging [SUMMARY]
|
[CONTENT] Metastases | Pancreas | Renal cell carcinoma | Computed tomography | Magnetic resonance imaging [SUMMARY]
|
[CONTENT] Metastases | Pancreas | Renal cell carcinoma | Computed tomography | Magnetic resonance imaging [SUMMARY]
|
[CONTENT] Metastases | Pancreas | Renal cell carcinoma | Computed tomography | Magnetic resonance imaging [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma, Renal Cell | Female | Humans | Kidney Neoplasms | Magnetic Resonance Imaging | Male | Middle Aged | Pancreatic Neoplasms | Retrospective Studies | Tomography, X-Ray Computed [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma, Renal Cell | Female | Humans | Kidney Neoplasms | Magnetic Resonance Imaging | Male | Middle Aged | Pancreatic Neoplasms | Retrospective Studies | Tomography, X-Ray Computed [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma, Renal Cell | Female | Humans | Kidney Neoplasms | Magnetic Resonance Imaging | Male | Middle Aged | Pancreatic Neoplasms | Retrospective Studies | Tomography, X-Ray Computed [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma, Renal Cell | Female | Humans | Kidney Neoplasms | Magnetic Resonance Imaging | Male | Middle Aged | Pancreatic Neoplasms | Retrospective Studies | Tomography, X-Ray Computed [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma, Renal Cell | Female | Humans | Kidney Neoplasms | Magnetic Resonance Imaging | Male | Middle Aged | Pancreatic Neoplasms | Retrospective Studies | Tomography, X-Ray Computed [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma, Renal Cell | Female | Humans | Kidney Neoplasms | Magnetic Resonance Imaging | Male | Middle Aged | Pancreatic Neoplasms | Retrospective Studies | Tomography, X-Ray Computed [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] pancreatic | metastases | patients | phase | lesions | rcc | images | ct | metastatic | showed [SUMMARY]
|
[CONTENT] pancreatic | metastases | patients | phase | lesions | rcc | images | ct | metastatic | showed [SUMMARY]
|
[CONTENT] pancreatic | metastases | patients | phase | lesions | rcc | images | ct | metastatic | showed [SUMMARY]
|
[CONTENT] pancreatic | metastases | patients | phase | lesions | rcc | images | ct | metastatic | showed [SUMMARY]
|
[CONTENT] pancreatic | metastases | patients | phase | lesions | rcc | images | ct | metastatic | showed [SUMMARY]
|
[CONTENT] pancreatic | metastases | patients | phase | lesions | rcc | images | ct | metastatic | showed [SUMMARY]
|
[CONTENT] rcc | metastatic | metastases | tumor burden | burden | patients | tumor | therapy | targeted therapy | targeted [SUMMARY]
|
[CONTENT] ms | mm | images | slice | ms te | ness | slice thick ness | field view | slice thick ness mm | mm slice [SUMMARY]
|
[CONTENT] lesions | phase | pancreatic | signal | presented | weighted | images | ct | arterial | intensity [SUMMARY]
|
[CONTENT] pancreatic | history | renal | renal cancer | cancer | pancreatic tumors | tumors | primary | patient | phase [SUMMARY]
|
[CONTENT] pancreatic | metastases | phase | rcc | patients | lesions | authors | interests | competing | declared competing interests [SUMMARY]
|
[CONTENT] pancreatic | metastases | phase | rcc | patients | lesions | authors | interests | competing | declared competing interests [SUMMARY]
|
[CONTENT] RCC | CT [SUMMARY]
|
[CONTENT] 15 ||| 14 ||| 9 | 120 | sec ||| 5 [SUMMARY]
|
[CONTENT] 7.5 years | 1-17 years ||| CT ||| 20 | 25 ||| only 13 | 25 [SUMMARY]
|
[CONTENT] CT [SUMMARY]
|
[CONTENT] RCC | CT ||| 15 ||| 14 ||| 9 | 120 | sec ||| 5 ||| ||| 7.5 years | 1-17 years ||| CT ||| 20 | 25 ||| only 13 | 25 ||| CT [SUMMARY]
|
[CONTENT] RCC | CT ||| 15 ||| 14 ||| 9 | 120 | sec ||| 5 ||| ||| 7.5 years | 1-17 years ||| CT ||| 20 | 25 ||| only 13 | 25 ||| CT [SUMMARY]
|
||||||
Analysis of serum metabolic profile by ultra-performance liquid chromatography-mass spectrometry for biomarkers discovery: application in a pilot study to discriminate patients with tuberculosis.
|
25591556
|
Tuberculosis (TB) is a chronic wasting inflammatory disease characterized by multisystem involvement, which can cause metabolic derangements in afflicted patients. Metabolic signatures have been exploited in the study of several diseases. However, the serum that is successfully used in TB diagnosis on the basis of metabolic profiling is not by much.
|
BACKGROUND
|
Orthogonal partial least-squares discriminant analysis was capable of distinguishing TB patients from both healthy subjects and patients with conditions other than TB. Therefore, TB-specific metabolic profiling was established. Clusters of potential biomarkers for differentiating TB active from non-TB diseases were identified using Mann-Whitney U-test. Multiple logistic regression analysis of metabolites was calculated to determine the suitable biomarker group that allows the efficient differentiation of patients with TB active from the control subjects.
|
METHODS
|
From among 271 participants, 12 metabolites were found to contribute to the distinction between the TB active group and the control groups. These metabolites were mainly involved in the metabolic pathways of the following three biomolecules: Fatty acids, amino acids, and lipids. The receiver operating characteristic curves of 3D, 7D, and 11D-phytanic acid, behenic acid, and threoninyl-γ-glutamate exhibited excellent efficiency with area under the curve (AUC) values of 0.904 (95% confidence interval [CI]: 0863-0.944), 0.93 (95% CI: 0.893-0.966), and 0.964 (95% CI: 00.941-0.988), respectively. The largest and smallest resulting AUCs were 0.964 and 0.720, indicating that these biomarkers may be involved in the disease mechanisms. The combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate was used to represent the most suitable biomarker group for the differentiation of patients with TB active from the control subjects, with an AUC value of 0.991.
|
RESULTS
|
The metabolic analysis results identified new serum biomarkers that can distinguish TB from non-TB diseases. The metabolomics-based analysis provides specific insights into the biology of TB and may offer new avenues for TB diagnosis.
|
CONCLUSION
|
[
"Adult",
"Aged",
"Biomarkers",
"Chromatography, High Pressure Liquid",
"Female",
"Humans",
"Male",
"Mass Spectrometry",
"Middle Aged",
"Tuberculosis"
] |
4837832
|
INTRODUCTION
|
Metabolomics is the quantitative measurement of the dynamic multiparametric metabolic responses of living systems to pathophysiological stimuli or genetic modifications.[1] Metabolites can be viewed as a close recapitulation of disease phenotypes, and they may represent the ongoing pathogenesis in an organism to a greater extent than changes in gene expression.[2] Consequently, it has deepened our understanding of the biological mechanisms involved in several noninfectious diseases and provided a platform for the identification of new biomarkers.[3] Metabolic signatures have been exploited in the study of several diseases, such as Alzheimer's disease,[4] Parkinson's disease,[5] myocardial ischemia,[6] hypertension,[7] cancer,[8] and diabetes.[9101112] However, this powerful analytical tool has not been widely applied to infectious diseases for the development of diagnostic biomarkers.[131415]
Tuberculosis (TB) is a major worldwide health problem, with a global estimate of 9.4 million incidences in 2010 (range; 8.9 × 106–9.9 × 106). Of the more than 2 billion people infected with Mycobacterium tuberculosis (Mtb) globally, more than one-tenth are likely to develop active TB during their lifetime. In many regions, notably in developing countries with high TB incidences, diagnosis is neither sensitive nor specific, and an estimated 40% of TB patients fail to be correctly diagnosed.[16] In recent years, advancement in the field of metabolomics has mainly been applied to metabolic profiling about TB drug metabolism, rather than diagnosis of TB.[1718192021] However, the serum that is successfully used in TB diagnosis on the basis of metabolic profiling is not by much.[2223] A previous study by Weiner et al. using gas chromatography-mass spectrometry (MS) showed differences in metabolic profiles among the uninfected individuals, individuals with latent (inactive) TB, and patients with active TB.[24] In a recent study, nuclear magnetic resonance spectroscopy was used to characterize the metabolism of the host during Mtb infection.[25] However, relatively few methods are considered to be capable of distinguishing between active TB and diseases other than TB, such as lung cancer, pneumonia, and so forth. It can be expected that several of the observed changes are the result of a general inflammatory process rather than a specific response to TB. In this study, 120 patients with active TB and 251 controls who were either healthy or had diseases other than TB (non-TB group) were enrolled. Using ultra performance liquid chromatography-MS (UPLC-MS), we investigated the feasibility of identifying small molecule biochemical profiles in serum for gaining novel biological insights into the mechanisms underlying TB. The unique feature of this study was to employ the largest number of patients included in any study of a similar nature to date (120 with active TB and 251 controls). Multivariate statistical analysis was implemented to discriminate patients with active TB from the control subjects on the basis of their metabolic profiles. We identified 12 distinct biomarkers, including clusters that could be categorized as amino acids, fatty acids, lysophosphatidylcholine (lysoPC), and terpenoid compounds. Finally, these new metabolite markers, which can distinguish TB from non-TB diseases, led to a number of hypotheses. Our results provide specific insights into the biology of TB and may offer new avenues for TB diagnosis or therapy.
|
METHODS
|
Chemicals Acetonitrile was purchased from Merck and Co. (Merck KGaA, Germany) and formic acid was obtained from Shanghai Nanxiang People Chemical Factory (China). All other solvents were of high-performance liquid chromatography grade. Reference standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ultrapure water was prepared by Milli-Q system (Millipore Co., USA).
Acetonitrile was purchased from Merck and Co. (Merck KGaA, Germany) and formic acid was obtained from Shanghai Nanxiang People Chemical Factory (China). All other solvents were of high-performance liquid chromatography grade. Reference standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ultrapure water was prepared by Milli-Q system (Millipore Co., USA).
Serum sample collection Serum samples were collected at Tianjin Haihe Hospital from 105 healthy individuals who visited the hospital for medical check-up, 146 patients with lung diseases that were due to non-TB conditions, and 120 patients with clinical signs of TB [Table 1]. TB patients had the following symptoms: Cough for more than 2 weeks and at least two additional symptoms (hemoptysis [coughing up blood], breathing difficulty, fever, night sweats, weight loss, chest pain, or fatigue). All patients were diagnosed based on chest X-rays and sputum samples obtained from each TB patient were analyzed by Ziehl-Neelsen staining and mycobacterium growth indicator tube culture. Active TB was diagnosed when (1) Mtb was cultured, (2) a caseating granuloma was found in the lung tissue by transthoracic needle biopsy and showed an appropriate response to treatment; or (3) clinical findings were compatible with TB, no clinical improvement was seen following treatment with empirical antibiotics, and treatment with anti-TB medication resulted in clinical and radiological improvement. The 251 controls without comorbidities were matched to the TB group with respect to age and sex. Whole-blood samples were drawn from a peripheral vein between 7:00 am and 9:00 am. Sera from patients and healthy volunteers were acquired from ethylenediaminetetraacetic acid-preserved whole blood samples following centrifugation and were stored at −80°C until analysis. At the time of sample collection, none of the patients were receiving treatment. Before the UPLC-MS analysis, serum samples were defrosted at room temperature for <20 minutes and 200 μl aliquots were combined with 300 μl of saline (0.9% NaCl in 20% D2O/80% H2O) and then centrifuged at 12,000 ×g for 5 minutes. A volume of 500 μl aliquot of the supernatant was pipetted into a 5 mm tube, and samples were stored at −80°C until further use.
Characteristics of TB patients and the control groups
There was no significant difference in demographic data between TB patients and the controls. *Data are presented as mean ± SD. SD: Standard deviation; TB: Tuberculosis.
Serum samples were collected at Tianjin Haihe Hospital from 105 healthy individuals who visited the hospital for medical check-up, 146 patients with lung diseases that were due to non-TB conditions, and 120 patients with clinical signs of TB [Table 1]. TB patients had the following symptoms: Cough for more than 2 weeks and at least two additional symptoms (hemoptysis [coughing up blood], breathing difficulty, fever, night sweats, weight loss, chest pain, or fatigue). All patients were diagnosed based on chest X-rays and sputum samples obtained from each TB patient were analyzed by Ziehl-Neelsen staining and mycobacterium growth indicator tube culture. Active TB was diagnosed when (1) Mtb was cultured, (2) a caseating granuloma was found in the lung tissue by transthoracic needle biopsy and showed an appropriate response to treatment; or (3) clinical findings were compatible with TB, no clinical improvement was seen following treatment with empirical antibiotics, and treatment with anti-TB medication resulted in clinical and radiological improvement. The 251 controls without comorbidities were matched to the TB group with respect to age and sex. Whole-blood samples were drawn from a peripheral vein between 7:00 am and 9:00 am. Sera from patients and healthy volunteers were acquired from ethylenediaminetetraacetic acid-preserved whole blood samples following centrifugation and were stored at −80°C until analysis. At the time of sample collection, none of the patients were receiving treatment. Before the UPLC-MS analysis, serum samples were defrosted at room temperature for <20 minutes and 200 μl aliquots were combined with 300 μl of saline (0.9% NaCl in 20% D2O/80% H2O) and then centrifuged at 12,000 ×g for 5 minutes. A volume of 500 μl aliquot of the supernatant was pipetted into a 5 mm tube, and samples were stored at −80°C until further use.
Characteristics of TB patients and the control groups
There was no significant difference in demographic data between TB patients and the controls. *Data are presented as mean ± SD. SD: Standard deviation; TB: Tuberculosis.
Sample extraction Before further metabolomics analysis, samples were defrosted at room temperature for <20 minutes. Briefly, 400 μl of acetonitrile was added to the samples in a 4:1 (v/v) ratio. These sample mixtures were then homogenized by shaking them for 30 seconds and centrifuged at 15,000 ×g for 20 minutes at −4°C. The supernatant was collected and divided into three fractions: One for analysis by UPLC-MS, one for quality control (QC) analysis, and one was stored for further analysis (if necessary). A 500 μl aliquot of the supernatant was pipetted into a 5 mm test tube and then loaded into the UPLC-MS/MS system for analysis.
Before further metabolomics analysis, samples were defrosted at room temperature for <20 minutes. Briefly, 400 μl of acetonitrile was added to the samples in a 4:1 (v/v) ratio. These sample mixtures were then homogenized by shaking them for 30 seconds and centrifuged at 15,000 ×g for 20 minutes at −4°C. The supernatant was collected and divided into three fractions: One for analysis by UPLC-MS, one for quality control (QC) analysis, and one was stored for further analysis (if necessary). A 500 μl aliquot of the supernatant was pipetted into a 5 mm test tube and then loaded into the UPLC-MS/MS system for analysis.
Instruments and conditions In the first part of the study, samples were measured with a nano liquid chromatography system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap™ mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific). Samples were loaded onto a trap column (PepMap C18, 300 μm internal diameter [ID], 5 mm length, 5 μm particle size, 100 Å pore size; Thermo Fisher Scientific), then washed and desalted for 15 minutes using 0.1% trifluoroacetic acid in water as the loading solvent. Next, the trap column was switched in-line with the analytical column (PepMap C18, 75 μm ID, 250 mm length, 3 μm particle size, 100 Å pore size; Thermo Fisher Scientific) and peptides were eluted with the following binary gradient: Starting with 100% solvent A and B, where solvent A consisted of 2% acetonitrile and 0.1% formic acid in water, and solvent B consisted of 80% acetonitrile and 0.08% formic acid in water. The column flow rate was set at 200 nl/min. For electro-spray ionization (ESI), nano ESI emitters (New Objective, Woburn, MA, USA) were used, and a spray voltage of 4.5 kV was applied. For MS detection, a data-dependent acquisition method was used: High-resolution survey scan from 50 to 1000 (m\z). Orbitrap full scan spectra and ion trap MS/MS fragmentation spectra were acquired partially simultaneously.
In the first part of the study, samples were measured with a nano liquid chromatography system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap™ mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific). Samples were loaded onto a trap column (PepMap C18, 300 μm internal diameter [ID], 5 mm length, 5 μm particle size, 100 Å pore size; Thermo Fisher Scientific), then washed and desalted for 15 minutes using 0.1% trifluoroacetic acid in water as the loading solvent. Next, the trap column was switched in-line with the analytical column (PepMap C18, 75 μm ID, 250 mm length, 3 μm particle size, 100 Å pore size; Thermo Fisher Scientific) and peptides were eluted with the following binary gradient: Starting with 100% solvent A and B, where solvent A consisted of 2% acetonitrile and 0.1% formic acid in water, and solvent B consisted of 80% acetonitrile and 0.08% formic acid in water. The column flow rate was set at 200 nl/min. For electro-spray ionization (ESI), nano ESI emitters (New Objective, Woburn, MA, USA) were used, and a spray voltage of 4.5 kV was applied. For MS detection, a data-dependent acquisition method was used: High-resolution survey scan from 50 to 1000 (m\z). Orbitrap full scan spectra and ion trap MS/MS fragmentation spectra were acquired partially simultaneously.
Quality control solution composition To observe the stability of the machine, QC solution was formulated from the mixed supernatants of all of the samples. The mixed samples solution was divided into 31 aliquots. Ten copies of QC solution were detected continuously before analyzing, and then the rest of copies were randomly inserted between each of the 10 sample runs [Figure 1]. The order in which samples were analyzed was randomized using the Excel 2013 software (Microsoft, USA), and a posttest blank sample was run after each of the 10 samples to avoid cross-contamination.
Quality control (QC) map. ▲: QC point. First ten QC points were run to and then the rest of the QC points were interleaved between the ten test samples. None of the QC points produced results outside of the control range.
To observe the stability of the machine, QC solution was formulated from the mixed supernatants of all of the samples. The mixed samples solution was divided into 31 aliquots. Ten copies of QC solution were detected continuously before analyzing, and then the rest of copies were randomly inserted between each of the 10 sample runs [Figure 1]. The order in which samples were analyzed was randomized using the Excel 2013 software (Microsoft, USA), and a posttest blank sample was run after each of the 10 samples to avoid cross-contamination.
Quality control (QC) map. ▲: QC point. First ten QC points were run to and then the rest of the QC points were interleaved between the ten test samples. None of the QC points produced results outside of the control range.
Compound identification, quantification, and data curation The UPLC-MS raw data were converted and processed by using MZmine 2.10 (http://www.biomedcentral.com/1471-2105/11/395). Briefly, chromatograms were built and peaks were recognized using the local minimum search function, and the ion intensities, matching m/z, and retention time was grouped into peak lists. Later, these peak lists were exported individually and imported into MetaboAnalyst 2.0 (http://nar.oxfordjournals.org/content/early/2012/05/01/nar.gks374.full). The peaks were aligned and normalized to the sum of all detected peaks. The processed and normalized data were imported into SIMCA-P (Umetrics, Umeε, Sweden) for multivariate statistical analysis. To distinguish TB from the controls, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed. Based on the OPLS-DA model, the specific metabolites were determined by applying Mann–Whitney U-test (SPSS 15.0, IBM, NY, USA) with P value threshold of 0.05. For each biomarker, a receiver operating characteristic (ROC) curve was generated. The area under curve (AUC) value and 95% confidence interval (CI) were calculated to determine the specificity and sensitivity of TB. To increase the diagnostic accuracy of combined changes in serum metabolites levels, multiple logistic regression analysis was carried out.
The UPLC-MS raw data were converted and processed by using MZmine 2.10 (http://www.biomedcentral.com/1471-2105/11/395). Briefly, chromatograms were built and peaks were recognized using the local minimum search function, and the ion intensities, matching m/z, and retention time was grouped into peak lists. Later, these peak lists were exported individually and imported into MetaboAnalyst 2.0 (http://nar.oxfordjournals.org/content/early/2012/05/01/nar.gks374.full). The peaks were aligned and normalized to the sum of all detected peaks. The processed and normalized data were imported into SIMCA-P (Umetrics, Umeε, Sweden) for multivariate statistical analysis. To distinguish TB from the controls, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed. Based on the OPLS-DA model, the specific metabolites were determined by applying Mann–Whitney U-test (SPSS 15.0, IBM, NY, USA) with P value threshold of 0.05. For each biomarker, a receiver operating characteristic (ROC) curve was generated. The area under curve (AUC) value and 95% confidence interval (CI) were calculated to determine the specificity and sensitivity of TB. To increase the diagnostic accuracy of combined changes in serum metabolites levels, multiple logistic regression analysis was carried out.
|
RESULTS
|
Baseline characteristics Table 1 lists the baseline characteristics of enrolled patients and the controls. The active TB patients were 48.32 ± 18.15 (mean ± standard deviation) years old on average and consisted of 65 males and 55 females. The healthy controls were 42.71 ± 15.31 years old and consisted of 56 males and 49 females. The non-TB controls were 50.35 ± 17.41 years old and consisted of 85 males and 61 females. Of the 146 patients with non-TB controls, 51 patients (60.1%) had lung cancer, comprising the cancer subgroup (L); 45 patients (39.9%) made up the pneumonia subgroup (P); 28 patients were in the chronic obstructive pulmonary disease (COPD) subgroup (C); and 22 patients comprised the bronchiectasis subgroup (B).
Table 1 lists the baseline characteristics of enrolled patients and the controls. The active TB patients were 48.32 ± 18.15 (mean ± standard deviation) years old on average and consisted of 65 males and 55 females. The healthy controls were 42.71 ± 15.31 years old and consisted of 56 males and 49 females. The non-TB controls were 50.35 ± 17.41 years old and consisted of 85 males and 61 females. Of the 146 patients with non-TB controls, 51 patients (60.1%) had lung cancer, comprising the cancer subgroup (L); 45 patients (39.9%) made up the pneumonia subgroup (P); 28 patients were in the chronic obstructive pulmonary disease (COPD) subgroup (C); and 22 patients comprised the bronchiectasis subgroup (B).
Ultra performance liquid chromatography-mass spectrometry platform performance Representative UPLC-MS chromatograms of the serum samples of the patients with active and the subjects with the healthy and non-TB controls were shown in Figure 2. The peaks were very well resolved and were evenly dispersed across the entire retention time domain, showing the high quality of the raw data. There were many significant differences in the areas and heights of peaks among groups, as a matter of fact, it is inferred that differences in peaks resulted from metabolic derangements along with diseases.
High performance liquid chromatography-mass spectrometry chromatograms of the serum samples of patients from the tuberculosis (TB) group, the healthy group, and the four subgroups of patients with non-TB diseases. Abscissa: Retention time. Ordinate: Relative abundance. The numbers on the point of the peaks indicate the retention time of each substance. T indicates TB group; N indicates healthy control; P indicates pulmonitis subgroup; L indicates lung cancer subgroup; C indicates chronic obstructive pulmonary disease subgroup; B indicates bronchiectasis subgroup.
Representative UPLC-MS chromatograms of the serum samples of the patients with active and the subjects with the healthy and non-TB controls were shown in Figure 2. The peaks were very well resolved and were evenly dispersed across the entire retention time domain, showing the high quality of the raw data. There were many significant differences in the areas and heights of peaks among groups, as a matter of fact, it is inferred that differences in peaks resulted from metabolic derangements along with diseases.
High performance liquid chromatography-mass spectrometry chromatograms of the serum samples of patients from the tuberculosis (TB) group, the healthy group, and the four subgroups of patients with non-TB diseases. Abscissa: Retention time. Ordinate: Relative abundance. The numbers on the point of the peaks indicate the retention time of each substance. T indicates TB group; N indicates healthy control; P indicates pulmonitis subgroup; L indicates lung cancer subgroup; C indicates chronic obstructive pulmonary disease subgroup; B indicates bronchiectasis subgroup.
Differentiation between tuberculosis patients and controls, and among the subgroups based on OPLS-DA As mentioned above, the overall peak profiles of the three groups looked quite different, which suggested that these profiles could be used to discriminate TB from the controls. For the holistic treatment of these data, multivariate analysis was used to identify the metabolomic differences between the groups. For data reduction and pattern recognition (PR), a series of PR methods were applied using SIMCA-P 12.1 software (Umetrics, Sweden). Principal component analysis (PCA) was initially applied to the data to visualize inherent clustering between the three groups. PCA involves the transformation of a multidimensional set of possibly correlated variables into two linearly uncorrelated dimensions. This model explained an estimated 41.4% of the original data (R2 = 0.414 and Q2 = 0.334) [Figure 3]. However, in addition to the effects of the diseases on the metabolome, there were other factors that are known to contribute to differences of endogenous metabolites, such as age and diet.
Principal component analysis (PCA) model. PCA scores plots of the SIMCA-P + 12.0.1.0 generated data, showing tuberculosis (TB) patients versus healthy controls and the non-TB group collected serum samples before the removal of ‘noise’ and interfering compounds from the dataset. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.
Therefore, orthogonal signal correction technology was used to filter out unrelated variable information and retain related variables. Hence, OPLS-DA model was introduced, which is used to determine the maximum separation between different kinds of samples according to the sample classification information. The OPLS-DA model was used to identify biomarkers that accounted for the differences between the three groups, and it clearly distinguished between the TB group and the two control groups (non-TB disease subgroups were combined) [Figure 4]. The results showed that 82.1% of samples were consistent with the discrimination of the model, and the predictive ability of the model was 58.2% (Q2Y = 0.582). These findings indicate that the OPLS-DA model may pave the way for the diagnosis of TB and permit differentiation between other kinds of related diseases.
(a) OPLS-DA two-dimensional model. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup. (b) OPLS-DA three-dimensional model. OPLS-DA scores plot discriminating serum samples of tuberculosis (TB) patients, healthy control and non-TB group based on the metabolite profiling data. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.
As mentioned above, the overall peak profiles of the three groups looked quite different, which suggested that these profiles could be used to discriminate TB from the controls. For the holistic treatment of these data, multivariate analysis was used to identify the metabolomic differences between the groups. For data reduction and pattern recognition (PR), a series of PR methods were applied using SIMCA-P 12.1 software (Umetrics, Sweden). Principal component analysis (PCA) was initially applied to the data to visualize inherent clustering between the three groups. PCA involves the transformation of a multidimensional set of possibly correlated variables into two linearly uncorrelated dimensions. This model explained an estimated 41.4% of the original data (R2 = 0.414 and Q2 = 0.334) [Figure 3]. However, in addition to the effects of the diseases on the metabolome, there were other factors that are known to contribute to differences of endogenous metabolites, such as age and diet.
Principal component analysis (PCA) model. PCA scores plots of the SIMCA-P + 12.0.1.0 generated data, showing tuberculosis (TB) patients versus healthy controls and the non-TB group collected serum samples before the removal of ‘noise’ and interfering compounds from the dataset. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.
Therefore, orthogonal signal correction technology was used to filter out unrelated variable information and retain related variables. Hence, OPLS-DA model was introduced, which is used to determine the maximum separation between different kinds of samples according to the sample classification information. The OPLS-DA model was used to identify biomarkers that accounted for the differences between the three groups, and it clearly distinguished between the TB group and the two control groups (non-TB disease subgroups were combined) [Figure 4]. The results showed that 82.1% of samples were consistent with the discrimination of the model, and the predictive ability of the model was 58.2% (Q2Y = 0.582). These findings indicate that the OPLS-DA model may pave the way for the diagnosis of TB and permit differentiation between other kinds of related diseases.
(a) OPLS-DA two-dimensional model. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup. (b) OPLS-DA three-dimensional model. OPLS-DA scores plot discriminating serum samples of tuberculosis (TB) patients, healthy control and non-TB group based on the metabolite profiling data. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.
Identification of tuberculosis-specific metabolites The successful use of UPLC-MS metabolomics analysis and the OPLS-DA model described above to distinguish between the TB group and control groups led us to search for the specific metabolites that contributed to the metabolomic differences. Based on the OPLS-DA model, the signals that were highly correlated and had high signal-to-noise ratio values were selected. The metabolites of >400 small molecules in the sera of patients in the three groups were explored. The molecules responsible for these signals were identified and differences in the abundance of these small molecules were determined by applying Mann–Whitney U-test for each of the three possible comparisons, using a P value threshold of 0.05. Twenty-seven metabolites were detected at significantly different levels between the active TB group and the control groups. Of those metabolites identified to differ significantly between groups, 12 metabolites [Table 2 and Figure 6], were clustered in the fatty acid, phospholipids, amino acids, and terpenoid compounds metabolite sets. To confirm that the three groups differed in terms of the serum levels of these metabolites, a heat map, a graphical representation of data where the individual values are represented as colors, was drawn [Figure 5]. Heat maps are 2D displays of the measured experimental values in the data matrix. The relatively high abundance of any specific metabolite is represented by yellow-colored squares (pixels) and a low abundance is represented by orange-colored squares.
Details of the 12 metabolites best describing the variation between patients with active TB and the controls
*Molecular weight (m/z) is denoted by its monoisotopic mass, †The chemical formulas were predicted based on accurate mass by using the molecular formula generator algorithm of Mass Frontier 6.0 software (Thermo Fisher Scientific), ‡The trend of marker levels in the active TB group. ↑ and ↓ indicate increased and decreased levels, respectively, compared with the healthy group. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine.
Heat map based on the differences in the abundance of small metabolic compounds among sera from the tuberculosis (TB) group, healthy group and non-TB group. Yellow color indicates higher abundance of metabolites; Orange color indicates lower abundance of metabolites.
Relative abundances of metabolites in tuberculosis patients (T), healthy controls (O) and nontuberculosis patients (N). The metabolites changed in relative abundance between groups were: Palmitic acid, LysoPC (16:0), LysoPC (18:0), phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, quinolinic acid, presqualene diphosphate, LysoPC (P-18:1 (9Z)), and LysoPC (P-16:0). Black lines indicates sample means. Asterisks indicate significant differences between 2 groups (results from t-tests corrected for multiple testing; *P < 0.05; **P < 0.01).
The results showed that the three groups were significantly different in terms of the abundances of these biomarker metabolites in the patient sera.
The successful use of UPLC-MS metabolomics analysis and the OPLS-DA model described above to distinguish between the TB group and control groups led us to search for the specific metabolites that contributed to the metabolomic differences. Based on the OPLS-DA model, the signals that were highly correlated and had high signal-to-noise ratio values were selected. The metabolites of >400 small molecules in the sera of patients in the three groups were explored. The molecules responsible for these signals were identified and differences in the abundance of these small molecules were determined by applying Mann–Whitney U-test for each of the three possible comparisons, using a P value threshold of 0.05. Twenty-seven metabolites were detected at significantly different levels between the active TB group and the control groups. Of those metabolites identified to differ significantly between groups, 12 metabolites [Table 2 and Figure 6], were clustered in the fatty acid, phospholipids, amino acids, and terpenoid compounds metabolite sets. To confirm that the three groups differed in terms of the serum levels of these metabolites, a heat map, a graphical representation of data where the individual values are represented as colors, was drawn [Figure 5]. Heat maps are 2D displays of the measured experimental values in the data matrix. The relatively high abundance of any specific metabolite is represented by yellow-colored squares (pixels) and a low abundance is represented by orange-colored squares.
Details of the 12 metabolites best describing the variation between patients with active TB and the controls
*Molecular weight (m/z) is denoted by its monoisotopic mass, †The chemical formulas were predicted based on accurate mass by using the molecular formula generator algorithm of Mass Frontier 6.0 software (Thermo Fisher Scientific), ‡The trend of marker levels in the active TB group. ↑ and ↓ indicate increased and decreased levels, respectively, compared with the healthy group. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine.
Heat map based on the differences in the abundance of small metabolic compounds among sera from the tuberculosis (TB) group, healthy group and non-TB group. Yellow color indicates higher abundance of metabolites; Orange color indicates lower abundance of metabolites.
Relative abundances of metabolites in tuberculosis patients (T), healthy controls (O) and nontuberculosis patients (N). The metabolites changed in relative abundance between groups were: Palmitic acid, LysoPC (16:0), LysoPC (18:0), phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, quinolinic acid, presqualene diphosphate, LysoPC (P-18:1 (9Z)), and LysoPC (P-16:0). Black lines indicates sample means. Asterisks indicate significant differences between 2 groups (results from t-tests corrected for multiple testing; *P < 0.05; **P < 0.01).
The results showed that the three groups were significantly different in terms of the abundances of these biomarker metabolites in the patient sera.
Differences in small metabolites can be used as specific and sensitive biosignatures of tuberculosis status To investigate whether the characteristics of the metabolites that significantly differed among the three groups could be efficiently exploited for building a sensitive biosignature of TB status, ROC curves, which have been conventionally used to evaluate diagnostic performance in clinical research, were calculated. In the specific metabolites that were decreased in active TB patients, the ROC curves of 3D, 7D, 11D-phytanic acid, behenic acid and threoninyl-γ-glutamate exhibited excellent efficiency with a AUC values of 0.904 (95% CI: 0863–0.944), 0.93 (95% CI: 0.893–0.966) and 0.964 (95% CI: 00.941–0.988), respectively [Table 3 and Figure 7a]. Kynurenine, quinolinic acid (QUIN), and presqualene diphosphate (PSDP) showed significant up-regulation in patients with active TB (P < 0.05). The AUC values for these metabolites were more than 0.8 [Table 3 and Figure 7b], showing moderate performance of diagnostic value. The largest and smallest resulting AUC values were 0.964 and 0.720 [Table 3, Figure 7a and b], which indicated that these biomarkers may potentially be involved in the disease mechanisms. Using multiple logistic regression analysis of these 12 metabolites, the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PSDP was used to represent a suitable biomarker group that allowed efficient differentiation of active TB from the controls. The resulting ROC curve of the biomarker combination had an AUC value of 0.991 (95% CI: 0.982–1.000) [Table 3 and Figure 7c], which reflects strong significant difference between active TB and the control patient groups.
The ROC curves of the 12 metabolites found to represent potential biomarkers of active TB
*Decreased metabolites, †Increased metabolites, ‡The biomarker combination, §Under the nonparametric assumption, ||Null hypothesis: True area = 0.5. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; ROC: Receiver operating characteristic.
(a) The receiver operating characteristic (ROC) curve of metabolites that were decreased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly decreased in the active TB group compared with controls. The ROC curves of each metabolite that was decreased in concentration in the TB group sera showed a moderate distinguishing efficiency. (b) The receiver operating characteristic (ROC) curve of metabolites that were increased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly increased in the active TB group compared with controls. The ROC curves of each metabolite that was increased in concentration in the TB group sera showed a moderate distinguishing efficiency. (c) The receiver operating characteristic (ROC) curve of the biomarker combination identified as a putative serum signature of active tuberculosis (TB). The ROC curve of the combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate showed excellent distinguishing efficiency between patients with active TB and controls.
To investigate whether the characteristics of the metabolites that significantly differed among the three groups could be efficiently exploited for building a sensitive biosignature of TB status, ROC curves, which have been conventionally used to evaluate diagnostic performance in clinical research, were calculated. In the specific metabolites that were decreased in active TB patients, the ROC curves of 3D, 7D, 11D-phytanic acid, behenic acid and threoninyl-γ-glutamate exhibited excellent efficiency with a AUC values of 0.904 (95% CI: 0863–0.944), 0.93 (95% CI: 0.893–0.966) and 0.964 (95% CI: 00.941–0.988), respectively [Table 3 and Figure 7a]. Kynurenine, quinolinic acid (QUIN), and presqualene diphosphate (PSDP) showed significant up-regulation in patients with active TB (P < 0.05). The AUC values for these metabolites were more than 0.8 [Table 3 and Figure 7b], showing moderate performance of diagnostic value. The largest and smallest resulting AUC values were 0.964 and 0.720 [Table 3, Figure 7a and b], which indicated that these biomarkers may potentially be involved in the disease mechanisms. Using multiple logistic regression analysis of these 12 metabolites, the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PSDP was used to represent a suitable biomarker group that allowed efficient differentiation of active TB from the controls. The resulting ROC curve of the biomarker combination had an AUC value of 0.991 (95% CI: 0.982–1.000) [Table 3 and Figure 7c], which reflects strong significant difference between active TB and the control patient groups.
The ROC curves of the 12 metabolites found to represent potential biomarkers of active TB
*Decreased metabolites, †Increased metabolites, ‡The biomarker combination, §Under the nonparametric assumption, ||Null hypothesis: True area = 0.5. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; ROC: Receiver operating characteristic.
(a) The receiver operating characteristic (ROC) curve of metabolites that were decreased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly decreased in the active TB group compared with controls. The ROC curves of each metabolite that was decreased in concentration in the TB group sera showed a moderate distinguishing efficiency. (b) The receiver operating characteristic (ROC) curve of metabolites that were increased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly increased in the active TB group compared with controls. The ROC curves of each metabolite that was increased in concentration in the TB group sera showed a moderate distinguishing efficiency. (c) The receiver operating characteristic (ROC) curve of the biomarker combination identified as a putative serum signature of active tuberculosis (TB). The ROC curve of the combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate showed excellent distinguishing efficiency between patients with active TB and controls.
Comparison between patients with active tuberculosis and nontuberculosis disease subgroups based on serum metabolic profiling To determine whether our metabolomic approach could be used to make a distinction between the active TB and non-TB groups, Mann–Whitney U-test was applied for comparison between the active TB group and the subgroups of non-TB. The findings indicated that the UPLC-MS analysis of serum may help in the diagnosis of TB and non-TB as remarked above. Finally, the specific markers between active TB and the subgroups were explored as below [Table 4]:
P values for comparisons between the active TB group and each subgroup of the Non-TB group
*P < 0.05, †P < 0.01. Application Mann-whitney U-test in TB group and each subgroup. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; COPD: Chronic obstructive pulmonary disease; LC: Liquid chromatography.
The serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroupsLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).
The serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)
Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)
Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)
The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroups
LysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).
To determine whether our metabolomic approach could be used to make a distinction between the active TB and non-TB groups, Mann–Whitney U-test was applied for comparison between the active TB group and the subgroups of non-TB. The findings indicated that the UPLC-MS analysis of serum may help in the diagnosis of TB and non-TB as remarked above. Finally, the specific markers between active TB and the subgroups were explored as below [Table 4]:
P values for comparisons between the active TB group and each subgroup of the Non-TB group
*P < 0.05, †P < 0.01. Application Mann-whitney U-test in TB group and each subgroup. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; COPD: Chronic obstructive pulmonary disease; LC: Liquid chromatography.
The serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroupsLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).
The serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)
Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)
Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)
The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroups
LysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).
|
CONCLUSIONS
|
Metabolic profiling approaches based on UPLC-MS were successfully used to distinguish TB patients from the controls and establish a TB-specific metabolite profiling. Twelve metabolites were identified to be significantly different among patient groups, with moderate performance of diagnostic value indicating that these biomarkers may potentially be involved in disease mechanisms. Most of the identified metabolites were mainly involved in fatty acid, amino acid, and lipid metabolism pathways, leading to a number of new hypotheses to better clarify the reasons for their differential abundance in active TB, including:
The decrease of palmitic acid, 3D, 7D, 11D-phytanic acid and behenic acid supported that the fatty acid was preferentially utilized by Mtb, and these metabolites may be one of the results of fatty acid consumptionTerpenoid compounds may have an important role in the processes of infection and host resistance to TB infectionMtb infection may produce enhanced activity of the TRP -kynurenine pathway, and kynurenine and QUIN, as the TRP downstream metabolites, may affect the immune function of patients with TBThe lower levels of lysoPCs observed in the active TB group may accelerate Mtb infection by reducing the generation of NO, which is produced by activated macrophagesMultiple logistic regression analysis was performed in the 12 metabolites identified as a signature of active TB, and the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PDSP was calculated to represent the best diagnostic value with the AUC value of 0.991 (95% CI: 0.982–1.000). Thus, this combination of metabolites may prove to be a metabolic profile that can be used for the diagnosis of TB.
The decrease of palmitic acid, 3D, 7D, 11D-phytanic acid and behenic acid supported that the fatty acid was preferentially utilized by Mtb, and these metabolites may be one of the results of fatty acid consumption
Terpenoid compounds may have an important role in the processes of infection and host resistance to TB infection
Mtb infection may produce enhanced activity of the TRP -kynurenine pathway, and kynurenine and QUIN, as the TRP downstream metabolites, may affect the immune function of patients with TB
The lower levels of lysoPCs observed in the active TB group may accelerate Mtb infection by reducing the generation of NO, which is produced by activated macrophages
Multiple logistic regression analysis was performed in the 12 metabolites identified as a signature of active TB, and the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PDSP was calculated to represent the best diagnostic value with the AUC value of 0.991 (95% CI: 0.982–1.000). Thus, this combination of metabolites may prove to be a metabolic profile that can be used for the diagnosis of TB.
|
[
"Chemicals",
"Serum sample collection",
"Sample extraction",
"Instruments and conditions",
"Quality control solution composition",
"Compound identification, quantification, and data curation",
"Baseline characteristics",
"Ultra performance liquid chromatography-mass spectrometry platform performance",
"Differentiation between tuberculosis patients and controls, and among the subgroups based on OPLS-DA",
"Identification of tuberculosis-specific metabolites",
"Differences in small metabolites can be used as specific and sensitive biosignatures of tuberculosis status",
"Comparison between patients with active tuberculosis and nontuberculosis disease subgroups based on serum metabolic profiling",
"Fatty acids",
"Lysophosphatidylcholines",
"Amino acids",
"Terpenoid compounds"
] |
[
"Acetonitrile was purchased from Merck and Co. (Merck KGaA, Germany) and formic acid was obtained from Shanghai Nanxiang People Chemical Factory (China). All other solvents were of high-performance liquid chromatography grade. Reference standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ultrapure water was prepared by Milli-Q system (Millipore Co., USA).",
"Serum samples were collected at Tianjin Haihe Hospital from 105 healthy individuals who visited the hospital for medical check-up, 146 patients with lung diseases that were due to non-TB conditions, and 120 patients with clinical signs of TB [Table 1]. TB patients had the following symptoms: Cough for more than 2 weeks and at least two additional symptoms (hemoptysis [coughing up blood], breathing difficulty, fever, night sweats, weight loss, chest pain, or fatigue). All patients were diagnosed based on chest X-rays and sputum samples obtained from each TB patient were analyzed by Ziehl-Neelsen staining and mycobacterium growth indicator tube culture. Active TB was diagnosed when (1) Mtb was cultured, (2) a caseating granuloma was found in the lung tissue by transthoracic needle biopsy and showed an appropriate response to treatment; or (3) clinical findings were compatible with TB, no clinical improvement was seen following treatment with empirical antibiotics, and treatment with anti-TB medication resulted in clinical and radiological improvement. The 251 controls without comorbidities were matched to the TB group with respect to age and sex. Whole-blood samples were drawn from a peripheral vein between 7:00 am and 9:00 am. Sera from patients and healthy volunteers were acquired from ethylenediaminetetraacetic acid-preserved whole blood samples following centrifugation and were stored at −80°C until analysis. At the time of sample collection, none of the patients were receiving treatment. Before the UPLC-MS analysis, serum samples were defrosted at room temperature for <20 minutes and 200 μl aliquots were combined with 300 μl of saline (0.9% NaCl in 20% D2O/80% H2O) and then centrifuged at 12,000 ×g for 5 minutes. A volume of 500 μl aliquot of the supernatant was pipetted into a 5 mm tube, and samples were stored at −80°C until further use.\nCharacteristics of TB patients and the control groups\nThere was no significant difference in demographic data between TB patients and the controls. *Data are presented as mean ± SD. SD: Standard deviation; TB: Tuberculosis.",
"Before further metabolomics analysis, samples were defrosted at room temperature for <20 minutes. Briefly, 400 μl of acetonitrile was added to the samples in a 4:1 (v/v) ratio. These sample mixtures were then homogenized by shaking them for 30 seconds and centrifuged at 15,000 ×g for 20 minutes at −4°C. The supernatant was collected and divided into three fractions: One for analysis by UPLC-MS, one for quality control (QC) analysis, and one was stored for further analysis (if necessary). A 500 μl aliquot of the supernatant was pipetted into a 5 mm test tube and then loaded into the UPLC-MS/MS system for analysis.",
"In the first part of the study, samples were measured with a nano liquid chromatography system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap™ mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific). Samples were loaded onto a trap column (PepMap C18, 300 μm internal diameter [ID], 5 mm length, 5 μm particle size, 100 Å pore size; Thermo Fisher Scientific), then washed and desalted for 15 minutes using 0.1% trifluoroacetic acid in water as the loading solvent. Next, the trap column was switched in-line with the analytical column (PepMap C18, 75 μm ID, 250 mm length, 3 μm particle size, 100 Å pore size; Thermo Fisher Scientific) and peptides were eluted with the following binary gradient: Starting with 100% solvent A and B, where solvent A consisted of 2% acetonitrile and 0.1% formic acid in water, and solvent B consisted of 80% acetonitrile and 0.08% formic acid in water. The column flow rate was set at 200 nl/min. For electro-spray ionization (ESI), nano ESI emitters (New Objective, Woburn, MA, USA) were used, and a spray voltage of 4.5 kV was applied. For MS detection, a data-dependent acquisition method was used: High-resolution survey scan from 50 to 1000 (m\\z). Orbitrap full scan spectra and ion trap MS/MS fragmentation spectra were acquired partially simultaneously.",
"To observe the stability of the machine, QC solution was formulated from the mixed supernatants of all of the samples. The mixed samples solution was divided into 31 aliquots. Ten copies of QC solution were detected continuously before analyzing, and then the rest of copies were randomly inserted between each of the 10 sample runs [Figure 1]. The order in which samples were analyzed was randomized using the Excel 2013 software (Microsoft, USA), and a posttest blank sample was run after each of the 10 samples to avoid cross-contamination.\nQuality control (QC) map. ▲: QC point. First ten QC points were run to and then the rest of the QC points were interleaved between the ten test samples. None of the QC points produced results outside of the control range.",
"The UPLC-MS raw data were converted and processed by using MZmine 2.10 (http://www.biomedcentral.com/1471-2105/11/395). Briefly, chromatograms were built and peaks were recognized using the local minimum search function, and the ion intensities, matching m/z, and retention time was grouped into peak lists. Later, these peak lists were exported individually and imported into MetaboAnalyst 2.0 (http://nar.oxfordjournals.org/content/early/2012/05/01/nar.gks374.full). The peaks were aligned and normalized to the sum of all detected peaks. The processed and normalized data were imported into SIMCA-P (Umetrics, Umeε, Sweden) for multivariate statistical analysis. To distinguish TB from the controls, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed. Based on the OPLS-DA model, the specific metabolites were determined by applying Mann–Whitney U-test (SPSS 15.0, IBM, NY, USA) with P value threshold of 0.05. For each biomarker, a receiver operating characteristic (ROC) curve was generated. The area under curve (AUC) value and 95% confidence interval (CI) were calculated to determine the specificity and sensitivity of TB. To increase the diagnostic accuracy of combined changes in serum metabolites levels, multiple logistic regression analysis was carried out.",
"Table 1 lists the baseline characteristics of enrolled patients and the controls. The active TB patients were 48.32 ± 18.15 (mean ± standard deviation) years old on average and consisted of 65 males and 55 females. The healthy controls were 42.71 ± 15.31 years old and consisted of 56 males and 49 females. The non-TB controls were 50.35 ± 17.41 years old and consisted of 85 males and 61 females. Of the 146 patients with non-TB controls, 51 patients (60.1%) had lung cancer, comprising the cancer subgroup (L); 45 patients (39.9%) made up the pneumonia subgroup (P); 28 patients were in the chronic obstructive pulmonary disease (COPD) subgroup (C); and 22 patients comprised the bronchiectasis subgroup (B).",
"Representative UPLC-MS chromatograms of the serum samples of the patients with active and the subjects with the healthy and non-TB controls were shown in Figure 2. The peaks were very well resolved and were evenly dispersed across the entire retention time domain, showing the high quality of the raw data. There were many significant differences in the areas and heights of peaks among groups, as a matter of fact, it is inferred that differences in peaks resulted from metabolic derangements along with diseases.\nHigh performance liquid chromatography-mass spectrometry chromatograms of the serum samples of patients from the tuberculosis (TB) group, the healthy group, and the four subgroups of patients with non-TB diseases. Abscissa: Retention time. Ordinate: Relative abundance. The numbers on the point of the peaks indicate the retention time of each substance. T indicates TB group; N indicates healthy control; P indicates pulmonitis subgroup; L indicates lung cancer subgroup; C indicates chronic obstructive pulmonary disease subgroup; B indicates bronchiectasis subgroup.",
"As mentioned above, the overall peak profiles of the three groups looked quite different, which suggested that these profiles could be used to discriminate TB from the controls. For the holistic treatment of these data, multivariate analysis was used to identify the metabolomic differences between the groups. For data reduction and pattern recognition (PR), a series of PR methods were applied using SIMCA-P 12.1 software (Umetrics, Sweden). Principal component analysis (PCA) was initially applied to the data to visualize inherent clustering between the three groups. PCA involves the transformation of a multidimensional set of possibly correlated variables into two linearly uncorrelated dimensions. This model explained an estimated 41.4% of the original data (R2 = 0.414 and Q2 = 0.334) [Figure 3]. However, in addition to the effects of the diseases on the metabolome, there were other factors that are known to contribute to differences of endogenous metabolites, such as age and diet.\nPrincipal component analysis (PCA) model. PCA scores plots of the SIMCA-P + 12.0.1.0 generated data, showing tuberculosis (TB) patients versus healthy controls and the non-TB group collected serum samples before the removal of ‘noise’ and interfering compounds from the dataset. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.\nTherefore, orthogonal signal correction technology was used to filter out unrelated variable information and retain related variables. Hence, OPLS-DA model was introduced, which is used to determine the maximum separation between different kinds of samples according to the sample classification information. The OPLS-DA model was used to identify biomarkers that accounted for the differences between the three groups, and it clearly distinguished between the TB group and the two control groups (non-TB disease subgroups were combined) [Figure 4]. The results showed that 82.1% of samples were consistent with the discrimination of the model, and the predictive ability of the model was 58.2% (Q2Y = 0.582). These findings indicate that the OPLS-DA model may pave the way for the diagnosis of TB and permit differentiation between other kinds of related diseases.\n(a) OPLS-DA two-dimensional model. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup. (b) OPLS-DA three-dimensional model. OPLS-DA scores plot discriminating serum samples of tuberculosis (TB) patients, healthy control and non-TB group based on the metabolite profiling data. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.",
"The successful use of UPLC-MS metabolomics analysis and the OPLS-DA model described above to distinguish between the TB group and control groups led us to search for the specific metabolites that contributed to the metabolomic differences. Based on the OPLS-DA model, the signals that were highly correlated and had high signal-to-noise ratio values were selected. The metabolites of >400 small molecules in the sera of patients in the three groups were explored. The molecules responsible for these signals were identified and differences in the abundance of these small molecules were determined by applying Mann–Whitney U-test for each of the three possible comparisons, using a P value threshold of 0.05. Twenty-seven metabolites were detected at significantly different levels between the active TB group and the control groups. Of those metabolites identified to differ significantly between groups, 12 metabolites [Table 2 and Figure 6], were clustered in the fatty acid, phospholipids, amino acids, and terpenoid compounds metabolite sets. To confirm that the three groups differed in terms of the serum levels of these metabolites, a heat map, a graphical representation of data where the individual values are represented as colors, was drawn [Figure 5]. Heat maps are 2D displays of the measured experimental values in the data matrix. The relatively high abundance of any specific metabolite is represented by yellow-colored squares (pixels) and a low abundance is represented by orange-colored squares.\nDetails of the 12 metabolites best describing the variation between patients with active TB and the controls\n*Molecular weight (m/z) is denoted by its monoisotopic mass, †The chemical formulas were predicted based on accurate mass by using the molecular formula generator algorithm of Mass Frontier 6.0 software (Thermo Fisher Scientific), ‡The trend of marker levels in the active TB group. ↑ and ↓ indicate increased and decreased levels, respectively, compared with the healthy group. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine.\nHeat map based on the differences in the abundance of small metabolic compounds among sera from the tuberculosis (TB) group, healthy group and non-TB group. Yellow color indicates higher abundance of metabolites; Orange color indicates lower abundance of metabolites.\nRelative abundances of metabolites in tuberculosis patients (T), healthy controls (O) and nontuberculosis patients (N). The metabolites changed in relative abundance between groups were: Palmitic acid, LysoPC (16:0), LysoPC (18:0), phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, quinolinic acid, presqualene diphosphate, LysoPC (P-18:1 (9Z)), and LysoPC (P-16:0). Black lines indicates sample means. Asterisks indicate significant differences between 2 groups (results from t-tests corrected for multiple testing; *P < 0.05; **P < 0.01).\nThe results showed that the three groups were significantly different in terms of the abundances of these biomarker metabolites in the patient sera.",
"To investigate whether the characteristics of the metabolites that significantly differed among the three groups could be efficiently exploited for building a sensitive biosignature of TB status, ROC curves, which have been conventionally used to evaluate diagnostic performance in clinical research, were calculated. In the specific metabolites that were decreased in active TB patients, the ROC curves of 3D, 7D, 11D-phytanic acid, behenic acid and threoninyl-γ-glutamate exhibited excellent efficiency with a AUC values of 0.904 (95% CI: 0863–0.944), 0.93 (95% CI: 0.893–0.966) and 0.964 (95% CI: 00.941–0.988), respectively [Table 3 and Figure 7a]. Kynurenine, quinolinic acid (QUIN), and presqualene diphosphate (PSDP) showed significant up-regulation in patients with active TB (P < 0.05). The AUC values for these metabolites were more than 0.8 [Table 3 and Figure 7b], showing moderate performance of diagnostic value. The largest and smallest resulting AUC values were 0.964 and 0.720 [Table 3, Figure 7a and b], which indicated that these biomarkers may potentially be involved in the disease mechanisms. Using multiple logistic regression analysis of these 12 metabolites, the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PSDP was used to represent a suitable biomarker group that allowed efficient differentiation of active TB from the controls. The resulting ROC curve of the biomarker combination had an AUC value of 0.991 (95% CI: 0.982–1.000) [Table 3 and Figure 7c], which reflects strong significant difference between active TB and the control patient groups.\nThe ROC curves of the 12 metabolites found to represent potential biomarkers of active TB\n*Decreased metabolites, †Increased metabolites, ‡The biomarker combination, §Under the nonparametric assumption, ||Null hypothesis: True area = 0.5. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; ROC: Receiver operating characteristic.\n(a) The receiver operating characteristic (ROC) curve of metabolites that were decreased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly decreased in the active TB group compared with controls. The ROC curves of each metabolite that was decreased in concentration in the TB group sera showed a moderate distinguishing efficiency. (b) The receiver operating characteristic (ROC) curve of metabolites that were increased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly increased in the active TB group compared with controls. The ROC curves of each metabolite that was increased in concentration in the TB group sera showed a moderate distinguishing efficiency. (c) The receiver operating characteristic (ROC) curve of the biomarker combination identified as a putative serum signature of active tuberculosis (TB). The ROC curve of the combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate showed excellent distinguishing efficiency between patients with active TB and controls.",
"To determine whether our metabolomic approach could be used to make a distinction between the active TB and non-TB groups, Mann–Whitney U-test was applied for comparison between the active TB group and the subgroups of non-TB. The findings indicated that the UPLC-MS analysis of serum may help in the diagnosis of TB and non-TB as remarked above. Finally, the specific markers between active TB and the subgroups were explored as below [Table 4]:\nP values for comparisons between the active TB group and each subgroup of the Non-TB group\n*P < 0.05, †P < 0.01. Application Mann-whitney U-test in TB group and each subgroup. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; COPD: Chronic obstructive pulmonary disease; LC: Liquid chromatography.\n\nThe serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroupsLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).\n\nThe serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)\nPalmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)\nPalmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)\nThe serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroups\nLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).",
"It is universally acknowledged that Mtb preferentially relies on fatty acid metabolism to maintain chronic infection.[26] When there is persistent infection in the lung tissue, the fatty acids, which are metabolized in two ways (in β-oxidation decomposition and the glyoxylate cycle) may be a source of carbon and energy of Mtb.[27] In this study, palmitic acid, phytanic acid and behenic acid, which decreased significantly in the sera of TB patients compared with the healthy group, bronchiectasis, and COPD subgroups, may be among the products of the fatty acid consumption. In addition, palmitic acid, as the most abundant free fatty acid in the human body, can induce inhibition of the electron transport chain of macrophage, which, in turn, reduces adenosine triphosphate production in the mitochondria and stimulates the activity of the mitochondrial apoptotic pathway. Another study that further supports our findings was that the palmitic acid, as one of the active compounds from the plant extraction, has some toxicity toward mycobacteria, but it is not highly toxic.[28]\nPhytanic acid, a 20-carbon branched chain fatty acid, can be used as the substrate of CYP124A1,[29] which is a heme-containing enzyme that belongs to the cytochrome p450 superfamily. When CYP124A1 was combined with the substrate of phytanic acid, the molecular conformation of CYP124A1 changed in favor of lipid oxidation, to play a better role in the important biological functions of Mtb.[30] Meanwhile, phytanic acid, as a substrate of CYP124A1, was consumed.\nIn our study, behenic acid was detected in a lower concentration in the TB group than the healthy and non-TB groups, except for the pneumonia subgroup. Behenic acid has been identified as a fatty acid that increases cholesterol in humans.[31] Cholesterol has a significant role in the development of TB, because it is necessary for the good functioning of macrophages and lymphocytes.[32] In the macrophage cell membrane, cholesterol directly participates in the function of phagocytizing Mtb, while, in the lymphocyte membrane, it is involved in the differentiation and proliferation of cytotoxic cells. Apart from this, cholesterol also has the functions of activating lymphocytes, CD4+ T cells, CD8+ T cells, and γ-T cells, and promoting the release of interferon and tumor necrosis factor-α, all of which effects are effective in killing Mtb. A previous study showed that TB patients universally had a lowered concentration of total serum cholesterol.[33] Due to this, the decreased concentration of behenic acid, as a cholesterol-raising fatty acid, in sera of TB patients may further explain the hypocholesteremia status in TB.[34]",
"Lysophosphatidylcholine (P-18:1(9Z)), lysoPC (P-16:0), lysoPC (16:0), and lysoPC (18:0) formed one cluster of metabolites that were present at lower levels in the active TB group and significantly differed between the healthy and non-TB groups. LysoPCs impair nitric oxide (NO) production and endothelium-dependent vasorelaxation. Moreover, lysoPC levels have been shown to be negatively correlated with NO levels.[36] Other research has pointed out that the generation of NO by activated macrophages could control mycobacterial infection in the murine system.[35] A low lysoPC level may accelerate Mtb infection by reducing the generation of NO. Additionally, another finding that TB can induce macrophage apoptosis by inhibition of phospholipase A2[373839] could also explain the low concentrations of lysoPCs in patients with active TB, a finding that is consistent with the previous report by Weiner et al.[24]",
"Our data showed an increase in the levels of two amino acids (kynurenine and QUIN), and a decrease in threoninyl-γ-glutamate in sera from TB patients relative to those of healthy controls, suggesting alterations in protein metabolism during active TB. Amino acid metabolism is complicated, involving a large number of metabolites. Gluconeogenesis, proteolysis, and oxidative catabolism contribute to amino acid balance. Tryptophan (TRP) is an essential amino acid that has various important biological functions. Kynurenine, as the downstream metabolite of TRP, can be accumulated by enhancing the activity of indoleamine 2, 3-dioxygenase-1 (IDO-1), which is widely known as the rate-limiting enzyme in TRP metabolism.[40] In addition, kynurenine can regulate human T cells, which are well known as the main anti-TB immunological cell type.\nQuinolinic acid, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines. These findings support the concept that the TRP-kynurenine pathway can influence immune functions through the effects of TRP depletion and the accumulation of kynurenine and QUIN, suggesting that the TRP degradation pathway controlled by IDO-1 is involved in the pathogenesis of pulmonary TB.[41] QUIN, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines.[42] In addition, imbalances in TRP metabolism have been linked to cancer-related immune escape and implicated in lung cancer.[43]\nThreoninyl-γ-glutamate exhibited the most excellent efficiency with an AUC value of 0.964 (95% CI: 00.941–0.988) [Table 3 and Figure 7a], is a dipeptide composed of threonine and γ-glutamate. Some dipeptides are known to have physiological or cell-signaling effects, although most are simply short-lived intermediates on their way to specific amino acid degradation pathways following further proteolysis. This dipeptide has not yet been identified in human tissues or biofluids; hence, it is classified as an “expected” metabolite.",
"One of the most prominent clusters of metabolites in our study represented terpenoid compounds, including PSDP and phytal. The latter compound, which to date has never been identified or associated with TB, belongs to the family of diterpenes. However, it is reported that elisapterosin B, one kind of diterpenes extracted from plants, could inhibit the growth of Mtb.[44] Thus, the relationship between phytal and disease as well as its involvement in disease mechanisms is open to further study.\nPresqualene diphosphate, an intermediate in the biosynthesis of terpenoids, was found at higher levels in the active TB group and significantly differed between the active TB and cancer subgroups by an order of magnitude. PSDP exerts an intercellular signal for the down-regulation of superoxide in neutrophils,[45] which is widely known to play a central role in host defense, inflammation, and tissue damage.[46] PSDP, an endogenous PI3K inhibitor, directly inhibited recombinant human p110-pI3K activity, which can activate polymorphonuclear neutrophils (PMN). PMN is the primary initial immune effectors of acute inflammation, and potentially PMN products released into surrounding tissues contribute to lung and respiratory injury. A negative correlation between PSDP and PMN was observed. The observed increase in the serum concentration of PSDP may be an indicator of TB inflammatory progression. PSDP, after the initial acute inflammatory state, may act to limit tissue injury by inhibiting phospholipase D, phosphoinositol-3 kinase, and superoxide anion generation, thus providing protection for the host tissues. In addition, PSDP, a well-recognized intermediate of cholesterol biosynthesis, exists in immune effector cells and is a potential regulator of the cellular response in host defense. The increased PSDP in TB patients may be a response to Mtb in host defense, which could also explain the result of this study."
] |
[
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[
"INTRODUCTION",
"METHODS",
"Chemicals",
"Serum sample collection",
"Sample extraction",
"Instruments and conditions",
"Quality control solution composition",
"Compound identification, quantification, and data curation",
"RESULTS",
"Baseline characteristics",
"Ultra performance liquid chromatography-mass spectrometry platform performance",
"Differentiation between tuberculosis patients and controls, and among the subgroups based on OPLS-DA",
"Identification of tuberculosis-specific metabolites",
"Differences in small metabolites can be used as specific and sensitive biosignatures of tuberculosis status",
"Comparison between patients with active tuberculosis and nontuberculosis disease subgroups based on serum metabolic profiling",
"DISCUSSION",
"Fatty acids",
"Lysophosphatidylcholines",
"Amino acids",
"Terpenoid compounds",
"CONCLUSIONS"
] |
[
"Metabolomics is the quantitative measurement of the dynamic multiparametric metabolic responses of living systems to pathophysiological stimuli or genetic modifications.[1] Metabolites can be viewed as a close recapitulation of disease phenotypes, and they may represent the ongoing pathogenesis in an organism to a greater extent than changes in gene expression.[2] Consequently, it has deepened our understanding of the biological mechanisms involved in several noninfectious diseases and provided a platform for the identification of new biomarkers.[3] Metabolic signatures have been exploited in the study of several diseases, such as Alzheimer's disease,[4] Parkinson's disease,[5] myocardial ischemia,[6] hypertension,[7] cancer,[8] and diabetes.[9101112] However, this powerful analytical tool has not been widely applied to infectious diseases for the development of diagnostic biomarkers.[131415]\nTuberculosis (TB) is a major worldwide health problem, with a global estimate of 9.4 million incidences in 2010 (range; 8.9 × 106–9.9 × 106). Of the more than 2 billion people infected with Mycobacterium tuberculosis (Mtb) globally, more than one-tenth are likely to develop active TB during their lifetime. In many regions, notably in developing countries with high TB incidences, diagnosis is neither sensitive nor specific, and an estimated 40% of TB patients fail to be correctly diagnosed.[16] In recent years, advancement in the field of metabolomics has mainly been applied to metabolic profiling about TB drug metabolism, rather than diagnosis of TB.[1718192021] However, the serum that is successfully used in TB diagnosis on the basis of metabolic profiling is not by much.[2223] A previous study by Weiner et al. using gas chromatography-mass spectrometry (MS) showed differences in metabolic profiles among the uninfected individuals, individuals with latent (inactive) TB, and patients with active TB.[24] In a recent study, nuclear magnetic resonance spectroscopy was used to characterize the metabolism of the host during Mtb infection.[25] However, relatively few methods are considered to be capable of distinguishing between active TB and diseases other than TB, such as lung cancer, pneumonia, and so forth. It can be expected that several of the observed changes are the result of a general inflammatory process rather than a specific response to TB. In this study, 120 patients with active TB and 251 controls who were either healthy or had diseases other than TB (non-TB group) were enrolled. Using ultra performance liquid chromatography-MS (UPLC-MS), we investigated the feasibility of identifying small molecule biochemical profiles in serum for gaining novel biological insights into the mechanisms underlying TB. The unique feature of this study was to employ the largest number of patients included in any study of a similar nature to date (120 with active TB and 251 controls). Multivariate statistical analysis was implemented to discriminate patients with active TB from the control subjects on the basis of their metabolic profiles. We identified 12 distinct biomarkers, including clusters that could be categorized as amino acids, fatty acids, lysophosphatidylcholine (lysoPC), and terpenoid compounds. Finally, these new metabolite markers, which can distinguish TB from non-TB diseases, led to a number of hypotheses. Our results provide specific insights into the biology of TB and may offer new avenues for TB diagnosis or therapy.",
" Chemicals Acetonitrile was purchased from Merck and Co. (Merck KGaA, Germany) and formic acid was obtained from Shanghai Nanxiang People Chemical Factory (China). All other solvents were of high-performance liquid chromatography grade. Reference standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ultrapure water was prepared by Milli-Q system (Millipore Co., USA).\nAcetonitrile was purchased from Merck and Co. (Merck KGaA, Germany) and formic acid was obtained from Shanghai Nanxiang People Chemical Factory (China). All other solvents were of high-performance liquid chromatography grade. Reference standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ultrapure water was prepared by Milli-Q system (Millipore Co., USA).\n Serum sample collection Serum samples were collected at Tianjin Haihe Hospital from 105 healthy individuals who visited the hospital for medical check-up, 146 patients with lung diseases that were due to non-TB conditions, and 120 patients with clinical signs of TB [Table 1]. TB patients had the following symptoms: Cough for more than 2 weeks and at least two additional symptoms (hemoptysis [coughing up blood], breathing difficulty, fever, night sweats, weight loss, chest pain, or fatigue). All patients were diagnosed based on chest X-rays and sputum samples obtained from each TB patient were analyzed by Ziehl-Neelsen staining and mycobacterium growth indicator tube culture. Active TB was diagnosed when (1) Mtb was cultured, (2) a caseating granuloma was found in the lung tissue by transthoracic needle biopsy and showed an appropriate response to treatment; or (3) clinical findings were compatible with TB, no clinical improvement was seen following treatment with empirical antibiotics, and treatment with anti-TB medication resulted in clinical and radiological improvement. The 251 controls without comorbidities were matched to the TB group with respect to age and sex. Whole-blood samples were drawn from a peripheral vein between 7:00 am and 9:00 am. Sera from patients and healthy volunteers were acquired from ethylenediaminetetraacetic acid-preserved whole blood samples following centrifugation and were stored at −80°C until analysis. At the time of sample collection, none of the patients were receiving treatment. Before the UPLC-MS analysis, serum samples were defrosted at room temperature for <20 minutes and 200 μl aliquots were combined with 300 μl of saline (0.9% NaCl in 20% D2O/80% H2O) and then centrifuged at 12,000 ×g for 5 minutes. A volume of 500 μl aliquot of the supernatant was pipetted into a 5 mm tube, and samples were stored at −80°C until further use.\nCharacteristics of TB patients and the control groups\nThere was no significant difference in demographic data between TB patients and the controls. *Data are presented as mean ± SD. SD: Standard deviation; TB: Tuberculosis.\nSerum samples were collected at Tianjin Haihe Hospital from 105 healthy individuals who visited the hospital for medical check-up, 146 patients with lung diseases that were due to non-TB conditions, and 120 patients with clinical signs of TB [Table 1]. TB patients had the following symptoms: Cough for more than 2 weeks and at least two additional symptoms (hemoptysis [coughing up blood], breathing difficulty, fever, night sweats, weight loss, chest pain, or fatigue). All patients were diagnosed based on chest X-rays and sputum samples obtained from each TB patient were analyzed by Ziehl-Neelsen staining and mycobacterium growth indicator tube culture. Active TB was diagnosed when (1) Mtb was cultured, (2) a caseating granuloma was found in the lung tissue by transthoracic needle biopsy and showed an appropriate response to treatment; or (3) clinical findings were compatible with TB, no clinical improvement was seen following treatment with empirical antibiotics, and treatment with anti-TB medication resulted in clinical and radiological improvement. The 251 controls without comorbidities were matched to the TB group with respect to age and sex. Whole-blood samples were drawn from a peripheral vein between 7:00 am and 9:00 am. Sera from patients and healthy volunteers were acquired from ethylenediaminetetraacetic acid-preserved whole blood samples following centrifugation and were stored at −80°C until analysis. At the time of sample collection, none of the patients were receiving treatment. Before the UPLC-MS analysis, serum samples were defrosted at room temperature for <20 minutes and 200 μl aliquots were combined with 300 μl of saline (0.9% NaCl in 20% D2O/80% H2O) and then centrifuged at 12,000 ×g for 5 minutes. A volume of 500 μl aliquot of the supernatant was pipetted into a 5 mm tube, and samples were stored at −80°C until further use.\nCharacteristics of TB patients and the control groups\nThere was no significant difference in demographic data between TB patients and the controls. *Data are presented as mean ± SD. SD: Standard deviation; TB: Tuberculosis.\n Sample extraction Before further metabolomics analysis, samples were defrosted at room temperature for <20 minutes. Briefly, 400 μl of acetonitrile was added to the samples in a 4:1 (v/v) ratio. These sample mixtures were then homogenized by shaking them for 30 seconds and centrifuged at 15,000 ×g for 20 minutes at −4°C. The supernatant was collected and divided into three fractions: One for analysis by UPLC-MS, one for quality control (QC) analysis, and one was stored for further analysis (if necessary). A 500 μl aliquot of the supernatant was pipetted into a 5 mm test tube and then loaded into the UPLC-MS/MS system for analysis.\nBefore further metabolomics analysis, samples were defrosted at room temperature for <20 minutes. Briefly, 400 μl of acetonitrile was added to the samples in a 4:1 (v/v) ratio. These sample mixtures were then homogenized by shaking them for 30 seconds and centrifuged at 15,000 ×g for 20 minutes at −4°C. The supernatant was collected and divided into three fractions: One for analysis by UPLC-MS, one for quality control (QC) analysis, and one was stored for further analysis (if necessary). A 500 μl aliquot of the supernatant was pipetted into a 5 mm test tube and then loaded into the UPLC-MS/MS system for analysis.\n Instruments and conditions In the first part of the study, samples were measured with a nano liquid chromatography system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap™ mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific). Samples were loaded onto a trap column (PepMap C18, 300 μm internal diameter [ID], 5 mm length, 5 μm particle size, 100 Å pore size; Thermo Fisher Scientific), then washed and desalted for 15 minutes using 0.1% trifluoroacetic acid in water as the loading solvent. Next, the trap column was switched in-line with the analytical column (PepMap C18, 75 μm ID, 250 mm length, 3 μm particle size, 100 Å pore size; Thermo Fisher Scientific) and peptides were eluted with the following binary gradient: Starting with 100% solvent A and B, where solvent A consisted of 2% acetonitrile and 0.1% formic acid in water, and solvent B consisted of 80% acetonitrile and 0.08% formic acid in water. The column flow rate was set at 200 nl/min. For electro-spray ionization (ESI), nano ESI emitters (New Objective, Woburn, MA, USA) were used, and a spray voltage of 4.5 kV was applied. For MS detection, a data-dependent acquisition method was used: High-resolution survey scan from 50 to 1000 (m\\z). Orbitrap full scan spectra and ion trap MS/MS fragmentation spectra were acquired partially simultaneously.\nIn the first part of the study, samples were measured with a nano liquid chromatography system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap™ mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific). Samples were loaded onto a trap column (PepMap C18, 300 μm internal diameter [ID], 5 mm length, 5 μm particle size, 100 Å pore size; Thermo Fisher Scientific), then washed and desalted for 15 minutes using 0.1% trifluoroacetic acid in water as the loading solvent. Next, the trap column was switched in-line with the analytical column (PepMap C18, 75 μm ID, 250 mm length, 3 μm particle size, 100 Å pore size; Thermo Fisher Scientific) and peptides were eluted with the following binary gradient: Starting with 100% solvent A and B, where solvent A consisted of 2% acetonitrile and 0.1% formic acid in water, and solvent B consisted of 80% acetonitrile and 0.08% formic acid in water. The column flow rate was set at 200 nl/min. For electro-spray ionization (ESI), nano ESI emitters (New Objective, Woburn, MA, USA) were used, and a spray voltage of 4.5 kV was applied. For MS detection, a data-dependent acquisition method was used: High-resolution survey scan from 50 to 1000 (m\\z). Orbitrap full scan spectra and ion trap MS/MS fragmentation spectra were acquired partially simultaneously.\n Quality control solution composition To observe the stability of the machine, QC solution was formulated from the mixed supernatants of all of the samples. The mixed samples solution was divided into 31 aliquots. Ten copies of QC solution were detected continuously before analyzing, and then the rest of copies were randomly inserted between each of the 10 sample runs [Figure 1]. The order in which samples were analyzed was randomized using the Excel 2013 software (Microsoft, USA), and a posttest blank sample was run after each of the 10 samples to avoid cross-contamination.\nQuality control (QC) map. ▲: QC point. First ten QC points were run to and then the rest of the QC points were interleaved between the ten test samples. None of the QC points produced results outside of the control range.\nTo observe the stability of the machine, QC solution was formulated from the mixed supernatants of all of the samples. The mixed samples solution was divided into 31 aliquots. Ten copies of QC solution were detected continuously before analyzing, and then the rest of copies were randomly inserted between each of the 10 sample runs [Figure 1]. The order in which samples were analyzed was randomized using the Excel 2013 software (Microsoft, USA), and a posttest blank sample was run after each of the 10 samples to avoid cross-contamination.\nQuality control (QC) map. ▲: QC point. First ten QC points were run to and then the rest of the QC points were interleaved between the ten test samples. None of the QC points produced results outside of the control range.\n Compound identification, quantification, and data curation The UPLC-MS raw data were converted and processed by using MZmine 2.10 (http://www.biomedcentral.com/1471-2105/11/395). Briefly, chromatograms were built and peaks were recognized using the local minimum search function, and the ion intensities, matching m/z, and retention time was grouped into peak lists. Later, these peak lists were exported individually and imported into MetaboAnalyst 2.0 (http://nar.oxfordjournals.org/content/early/2012/05/01/nar.gks374.full). The peaks were aligned and normalized to the sum of all detected peaks. The processed and normalized data were imported into SIMCA-P (Umetrics, Umeε, Sweden) for multivariate statistical analysis. To distinguish TB from the controls, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed. Based on the OPLS-DA model, the specific metabolites were determined by applying Mann–Whitney U-test (SPSS 15.0, IBM, NY, USA) with P value threshold of 0.05. For each biomarker, a receiver operating characteristic (ROC) curve was generated. The area under curve (AUC) value and 95% confidence interval (CI) were calculated to determine the specificity and sensitivity of TB. To increase the diagnostic accuracy of combined changes in serum metabolites levels, multiple logistic regression analysis was carried out.\nThe UPLC-MS raw data were converted and processed by using MZmine 2.10 (http://www.biomedcentral.com/1471-2105/11/395). Briefly, chromatograms were built and peaks were recognized using the local minimum search function, and the ion intensities, matching m/z, and retention time was grouped into peak lists. Later, these peak lists were exported individually and imported into MetaboAnalyst 2.0 (http://nar.oxfordjournals.org/content/early/2012/05/01/nar.gks374.full). The peaks were aligned and normalized to the sum of all detected peaks. The processed and normalized data were imported into SIMCA-P (Umetrics, Umeε, Sweden) for multivariate statistical analysis. To distinguish TB from the controls, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed. Based on the OPLS-DA model, the specific metabolites were determined by applying Mann–Whitney U-test (SPSS 15.0, IBM, NY, USA) with P value threshold of 0.05. For each biomarker, a receiver operating characteristic (ROC) curve was generated. The area under curve (AUC) value and 95% confidence interval (CI) were calculated to determine the specificity and sensitivity of TB. To increase the diagnostic accuracy of combined changes in serum metabolites levels, multiple logistic regression analysis was carried out.",
"Acetonitrile was purchased from Merck and Co. (Merck KGaA, Germany) and formic acid was obtained from Shanghai Nanxiang People Chemical Factory (China). All other solvents were of high-performance liquid chromatography grade. Reference standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ultrapure water was prepared by Milli-Q system (Millipore Co., USA).",
"Serum samples were collected at Tianjin Haihe Hospital from 105 healthy individuals who visited the hospital for medical check-up, 146 patients with lung diseases that were due to non-TB conditions, and 120 patients with clinical signs of TB [Table 1]. TB patients had the following symptoms: Cough for more than 2 weeks and at least two additional symptoms (hemoptysis [coughing up blood], breathing difficulty, fever, night sweats, weight loss, chest pain, or fatigue). All patients were diagnosed based on chest X-rays and sputum samples obtained from each TB patient were analyzed by Ziehl-Neelsen staining and mycobacterium growth indicator tube culture. Active TB was diagnosed when (1) Mtb was cultured, (2) a caseating granuloma was found in the lung tissue by transthoracic needle biopsy and showed an appropriate response to treatment; or (3) clinical findings were compatible with TB, no clinical improvement was seen following treatment with empirical antibiotics, and treatment with anti-TB medication resulted in clinical and radiological improvement. The 251 controls without comorbidities were matched to the TB group with respect to age and sex. Whole-blood samples were drawn from a peripheral vein between 7:00 am and 9:00 am. Sera from patients and healthy volunteers were acquired from ethylenediaminetetraacetic acid-preserved whole blood samples following centrifugation and were stored at −80°C until analysis. At the time of sample collection, none of the patients were receiving treatment. Before the UPLC-MS analysis, serum samples were defrosted at room temperature for <20 minutes and 200 μl aliquots were combined with 300 μl of saline (0.9% NaCl in 20% D2O/80% H2O) and then centrifuged at 12,000 ×g for 5 minutes. A volume of 500 μl aliquot of the supernatant was pipetted into a 5 mm tube, and samples were stored at −80°C until further use.\nCharacteristics of TB patients and the control groups\nThere was no significant difference in demographic data between TB patients and the controls. *Data are presented as mean ± SD. SD: Standard deviation; TB: Tuberculosis.",
"Before further metabolomics analysis, samples were defrosted at room temperature for <20 minutes. Briefly, 400 μl of acetonitrile was added to the samples in a 4:1 (v/v) ratio. These sample mixtures were then homogenized by shaking them for 30 seconds and centrifuged at 15,000 ×g for 20 minutes at −4°C. The supernatant was collected and divided into three fractions: One for analysis by UPLC-MS, one for quality control (QC) analysis, and one was stored for further analysis (if necessary). A 500 μl aliquot of the supernatant was pipetted into a 5 mm test tube and then loaded into the UPLC-MS/MS system for analysis.",
"In the first part of the study, samples were measured with a nano liquid chromatography system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap™ mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific). Samples were loaded onto a trap column (PepMap C18, 300 μm internal diameter [ID], 5 mm length, 5 μm particle size, 100 Å pore size; Thermo Fisher Scientific), then washed and desalted for 15 minutes using 0.1% trifluoroacetic acid in water as the loading solvent. Next, the trap column was switched in-line with the analytical column (PepMap C18, 75 μm ID, 250 mm length, 3 μm particle size, 100 Å pore size; Thermo Fisher Scientific) and peptides were eluted with the following binary gradient: Starting with 100% solvent A and B, where solvent A consisted of 2% acetonitrile and 0.1% formic acid in water, and solvent B consisted of 80% acetonitrile and 0.08% formic acid in water. The column flow rate was set at 200 nl/min. For electro-spray ionization (ESI), nano ESI emitters (New Objective, Woburn, MA, USA) were used, and a spray voltage of 4.5 kV was applied. For MS detection, a data-dependent acquisition method was used: High-resolution survey scan from 50 to 1000 (m\\z). Orbitrap full scan spectra and ion trap MS/MS fragmentation spectra were acquired partially simultaneously.",
"To observe the stability of the machine, QC solution was formulated from the mixed supernatants of all of the samples. The mixed samples solution was divided into 31 aliquots. Ten copies of QC solution were detected continuously before analyzing, and then the rest of copies were randomly inserted between each of the 10 sample runs [Figure 1]. The order in which samples were analyzed was randomized using the Excel 2013 software (Microsoft, USA), and a posttest blank sample was run after each of the 10 samples to avoid cross-contamination.\nQuality control (QC) map. ▲: QC point. First ten QC points were run to and then the rest of the QC points were interleaved between the ten test samples. None of the QC points produced results outside of the control range.",
"The UPLC-MS raw data were converted and processed by using MZmine 2.10 (http://www.biomedcentral.com/1471-2105/11/395). Briefly, chromatograms were built and peaks were recognized using the local minimum search function, and the ion intensities, matching m/z, and retention time was grouped into peak lists. Later, these peak lists were exported individually and imported into MetaboAnalyst 2.0 (http://nar.oxfordjournals.org/content/early/2012/05/01/nar.gks374.full). The peaks were aligned and normalized to the sum of all detected peaks. The processed and normalized data were imported into SIMCA-P (Umetrics, Umeε, Sweden) for multivariate statistical analysis. To distinguish TB from the controls, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed. Based on the OPLS-DA model, the specific metabolites were determined by applying Mann–Whitney U-test (SPSS 15.0, IBM, NY, USA) with P value threshold of 0.05. For each biomarker, a receiver operating characteristic (ROC) curve was generated. The area under curve (AUC) value and 95% confidence interval (CI) were calculated to determine the specificity and sensitivity of TB. To increase the diagnostic accuracy of combined changes in serum metabolites levels, multiple logistic regression analysis was carried out.",
" Baseline characteristics Table 1 lists the baseline characteristics of enrolled patients and the controls. The active TB patients were 48.32 ± 18.15 (mean ± standard deviation) years old on average and consisted of 65 males and 55 females. The healthy controls were 42.71 ± 15.31 years old and consisted of 56 males and 49 females. The non-TB controls were 50.35 ± 17.41 years old and consisted of 85 males and 61 females. Of the 146 patients with non-TB controls, 51 patients (60.1%) had lung cancer, comprising the cancer subgroup (L); 45 patients (39.9%) made up the pneumonia subgroup (P); 28 patients were in the chronic obstructive pulmonary disease (COPD) subgroup (C); and 22 patients comprised the bronchiectasis subgroup (B).\nTable 1 lists the baseline characteristics of enrolled patients and the controls. The active TB patients were 48.32 ± 18.15 (mean ± standard deviation) years old on average and consisted of 65 males and 55 females. The healthy controls were 42.71 ± 15.31 years old and consisted of 56 males and 49 females. The non-TB controls were 50.35 ± 17.41 years old and consisted of 85 males and 61 females. Of the 146 patients with non-TB controls, 51 patients (60.1%) had lung cancer, comprising the cancer subgroup (L); 45 patients (39.9%) made up the pneumonia subgroup (P); 28 patients were in the chronic obstructive pulmonary disease (COPD) subgroup (C); and 22 patients comprised the bronchiectasis subgroup (B).\n Ultra performance liquid chromatography-mass spectrometry platform performance Representative UPLC-MS chromatograms of the serum samples of the patients with active and the subjects with the healthy and non-TB controls were shown in Figure 2. The peaks were very well resolved and were evenly dispersed across the entire retention time domain, showing the high quality of the raw data. There were many significant differences in the areas and heights of peaks among groups, as a matter of fact, it is inferred that differences in peaks resulted from metabolic derangements along with diseases.\nHigh performance liquid chromatography-mass spectrometry chromatograms of the serum samples of patients from the tuberculosis (TB) group, the healthy group, and the four subgroups of patients with non-TB diseases. Abscissa: Retention time. Ordinate: Relative abundance. The numbers on the point of the peaks indicate the retention time of each substance. T indicates TB group; N indicates healthy control; P indicates pulmonitis subgroup; L indicates lung cancer subgroup; C indicates chronic obstructive pulmonary disease subgroup; B indicates bronchiectasis subgroup.\nRepresentative UPLC-MS chromatograms of the serum samples of the patients with active and the subjects with the healthy and non-TB controls were shown in Figure 2. The peaks were very well resolved and were evenly dispersed across the entire retention time domain, showing the high quality of the raw data. There were many significant differences in the areas and heights of peaks among groups, as a matter of fact, it is inferred that differences in peaks resulted from metabolic derangements along with diseases.\nHigh performance liquid chromatography-mass spectrometry chromatograms of the serum samples of patients from the tuberculosis (TB) group, the healthy group, and the four subgroups of patients with non-TB diseases. Abscissa: Retention time. Ordinate: Relative abundance. The numbers on the point of the peaks indicate the retention time of each substance. T indicates TB group; N indicates healthy control; P indicates pulmonitis subgroup; L indicates lung cancer subgroup; C indicates chronic obstructive pulmonary disease subgroup; B indicates bronchiectasis subgroup.\n Differentiation between tuberculosis patients and controls, and among the subgroups based on OPLS-DA As mentioned above, the overall peak profiles of the three groups looked quite different, which suggested that these profiles could be used to discriminate TB from the controls. For the holistic treatment of these data, multivariate analysis was used to identify the metabolomic differences between the groups. For data reduction and pattern recognition (PR), a series of PR methods were applied using SIMCA-P 12.1 software (Umetrics, Sweden). Principal component analysis (PCA) was initially applied to the data to visualize inherent clustering between the three groups. PCA involves the transformation of a multidimensional set of possibly correlated variables into two linearly uncorrelated dimensions. This model explained an estimated 41.4% of the original data (R2 = 0.414 and Q2 = 0.334) [Figure 3]. However, in addition to the effects of the diseases on the metabolome, there were other factors that are known to contribute to differences of endogenous metabolites, such as age and diet.\nPrincipal component analysis (PCA) model. PCA scores plots of the SIMCA-P + 12.0.1.0 generated data, showing tuberculosis (TB) patients versus healthy controls and the non-TB group collected serum samples before the removal of ‘noise’ and interfering compounds from the dataset. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.\nTherefore, orthogonal signal correction technology was used to filter out unrelated variable information and retain related variables. Hence, OPLS-DA model was introduced, which is used to determine the maximum separation between different kinds of samples according to the sample classification information. The OPLS-DA model was used to identify biomarkers that accounted for the differences between the three groups, and it clearly distinguished between the TB group and the two control groups (non-TB disease subgroups were combined) [Figure 4]. The results showed that 82.1% of samples were consistent with the discrimination of the model, and the predictive ability of the model was 58.2% (Q2Y = 0.582). These findings indicate that the OPLS-DA model may pave the way for the diagnosis of TB and permit differentiation between other kinds of related diseases.\n(a) OPLS-DA two-dimensional model. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup. (b) OPLS-DA three-dimensional model. OPLS-DA scores plot discriminating serum samples of tuberculosis (TB) patients, healthy control and non-TB group based on the metabolite profiling data. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.\nAs mentioned above, the overall peak profiles of the three groups looked quite different, which suggested that these profiles could be used to discriminate TB from the controls. For the holistic treatment of these data, multivariate analysis was used to identify the metabolomic differences between the groups. For data reduction and pattern recognition (PR), a series of PR methods were applied using SIMCA-P 12.1 software (Umetrics, Sweden). Principal component analysis (PCA) was initially applied to the data to visualize inherent clustering between the three groups. PCA involves the transformation of a multidimensional set of possibly correlated variables into two linearly uncorrelated dimensions. This model explained an estimated 41.4% of the original data (R2 = 0.414 and Q2 = 0.334) [Figure 3]. However, in addition to the effects of the diseases on the metabolome, there were other factors that are known to contribute to differences of endogenous metabolites, such as age and diet.\nPrincipal component analysis (PCA) model. PCA scores plots of the SIMCA-P + 12.0.1.0 generated data, showing tuberculosis (TB) patients versus healthy controls and the non-TB group collected serum samples before the removal of ‘noise’ and interfering compounds from the dataset. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.\nTherefore, orthogonal signal correction technology was used to filter out unrelated variable information and retain related variables. Hence, OPLS-DA model was introduced, which is used to determine the maximum separation between different kinds of samples according to the sample classification information. The OPLS-DA model was used to identify biomarkers that accounted for the differences between the three groups, and it clearly distinguished between the TB group and the two control groups (non-TB disease subgroups were combined) [Figure 4]. The results showed that 82.1% of samples were consistent with the discrimination of the model, and the predictive ability of the model was 58.2% (Q2Y = 0.582). These findings indicate that the OPLS-DA model may pave the way for the diagnosis of TB and permit differentiation between other kinds of related diseases.\n(a) OPLS-DA two-dimensional model. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup. (b) OPLS-DA three-dimensional model. OPLS-DA scores plot discriminating serum samples of tuberculosis (TB) patients, healthy control and non-TB group based on the metabolite profiling data. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.\n Identification of tuberculosis-specific metabolites The successful use of UPLC-MS metabolomics analysis and the OPLS-DA model described above to distinguish between the TB group and control groups led us to search for the specific metabolites that contributed to the metabolomic differences. Based on the OPLS-DA model, the signals that were highly correlated and had high signal-to-noise ratio values were selected. The metabolites of >400 small molecules in the sera of patients in the three groups were explored. The molecules responsible for these signals were identified and differences in the abundance of these small molecules were determined by applying Mann–Whitney U-test for each of the three possible comparisons, using a P value threshold of 0.05. Twenty-seven metabolites were detected at significantly different levels between the active TB group and the control groups. Of those metabolites identified to differ significantly between groups, 12 metabolites [Table 2 and Figure 6], were clustered in the fatty acid, phospholipids, amino acids, and terpenoid compounds metabolite sets. To confirm that the three groups differed in terms of the serum levels of these metabolites, a heat map, a graphical representation of data where the individual values are represented as colors, was drawn [Figure 5]. Heat maps are 2D displays of the measured experimental values in the data matrix. The relatively high abundance of any specific metabolite is represented by yellow-colored squares (pixels) and a low abundance is represented by orange-colored squares.\nDetails of the 12 metabolites best describing the variation between patients with active TB and the controls\n*Molecular weight (m/z) is denoted by its monoisotopic mass, †The chemical formulas were predicted based on accurate mass by using the molecular formula generator algorithm of Mass Frontier 6.0 software (Thermo Fisher Scientific), ‡The trend of marker levels in the active TB group. ↑ and ↓ indicate increased and decreased levels, respectively, compared with the healthy group. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine.\nHeat map based on the differences in the abundance of small metabolic compounds among sera from the tuberculosis (TB) group, healthy group and non-TB group. Yellow color indicates higher abundance of metabolites; Orange color indicates lower abundance of metabolites.\nRelative abundances of metabolites in tuberculosis patients (T), healthy controls (O) and nontuberculosis patients (N). The metabolites changed in relative abundance between groups were: Palmitic acid, LysoPC (16:0), LysoPC (18:0), phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, quinolinic acid, presqualene diphosphate, LysoPC (P-18:1 (9Z)), and LysoPC (P-16:0). Black lines indicates sample means. Asterisks indicate significant differences between 2 groups (results from t-tests corrected for multiple testing; *P < 0.05; **P < 0.01).\nThe results showed that the three groups were significantly different in terms of the abundances of these biomarker metabolites in the patient sera.\nThe successful use of UPLC-MS metabolomics analysis and the OPLS-DA model described above to distinguish between the TB group and control groups led us to search for the specific metabolites that contributed to the metabolomic differences. Based on the OPLS-DA model, the signals that were highly correlated and had high signal-to-noise ratio values were selected. The metabolites of >400 small molecules in the sera of patients in the three groups were explored. The molecules responsible for these signals were identified and differences in the abundance of these small molecules were determined by applying Mann–Whitney U-test for each of the three possible comparisons, using a P value threshold of 0.05. Twenty-seven metabolites were detected at significantly different levels between the active TB group and the control groups. Of those metabolites identified to differ significantly between groups, 12 metabolites [Table 2 and Figure 6], were clustered in the fatty acid, phospholipids, amino acids, and terpenoid compounds metabolite sets. To confirm that the three groups differed in terms of the serum levels of these metabolites, a heat map, a graphical representation of data where the individual values are represented as colors, was drawn [Figure 5]. Heat maps are 2D displays of the measured experimental values in the data matrix. The relatively high abundance of any specific metabolite is represented by yellow-colored squares (pixels) and a low abundance is represented by orange-colored squares.\nDetails of the 12 metabolites best describing the variation between patients with active TB and the controls\n*Molecular weight (m/z) is denoted by its monoisotopic mass, †The chemical formulas were predicted based on accurate mass by using the molecular formula generator algorithm of Mass Frontier 6.0 software (Thermo Fisher Scientific), ‡The trend of marker levels in the active TB group. ↑ and ↓ indicate increased and decreased levels, respectively, compared with the healthy group. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine.\nHeat map based on the differences in the abundance of small metabolic compounds among sera from the tuberculosis (TB) group, healthy group and non-TB group. Yellow color indicates higher abundance of metabolites; Orange color indicates lower abundance of metabolites.\nRelative abundances of metabolites in tuberculosis patients (T), healthy controls (O) and nontuberculosis patients (N). The metabolites changed in relative abundance between groups were: Palmitic acid, LysoPC (16:0), LysoPC (18:0), phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, quinolinic acid, presqualene diphosphate, LysoPC (P-18:1 (9Z)), and LysoPC (P-16:0). Black lines indicates sample means. Asterisks indicate significant differences between 2 groups (results from t-tests corrected for multiple testing; *P < 0.05; **P < 0.01).\nThe results showed that the three groups were significantly different in terms of the abundances of these biomarker metabolites in the patient sera.\n Differences in small metabolites can be used as specific and sensitive biosignatures of tuberculosis status To investigate whether the characteristics of the metabolites that significantly differed among the three groups could be efficiently exploited for building a sensitive biosignature of TB status, ROC curves, which have been conventionally used to evaluate diagnostic performance in clinical research, were calculated. In the specific metabolites that were decreased in active TB patients, the ROC curves of 3D, 7D, 11D-phytanic acid, behenic acid and threoninyl-γ-glutamate exhibited excellent efficiency with a AUC values of 0.904 (95% CI: 0863–0.944), 0.93 (95% CI: 0.893–0.966) and 0.964 (95% CI: 00.941–0.988), respectively [Table 3 and Figure 7a]. Kynurenine, quinolinic acid (QUIN), and presqualene diphosphate (PSDP) showed significant up-regulation in patients with active TB (P < 0.05). The AUC values for these metabolites were more than 0.8 [Table 3 and Figure 7b], showing moderate performance of diagnostic value. The largest and smallest resulting AUC values were 0.964 and 0.720 [Table 3, Figure 7a and b], which indicated that these biomarkers may potentially be involved in the disease mechanisms. Using multiple logistic regression analysis of these 12 metabolites, the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PSDP was used to represent a suitable biomarker group that allowed efficient differentiation of active TB from the controls. The resulting ROC curve of the biomarker combination had an AUC value of 0.991 (95% CI: 0.982–1.000) [Table 3 and Figure 7c], which reflects strong significant difference between active TB and the control patient groups.\nThe ROC curves of the 12 metabolites found to represent potential biomarkers of active TB\n*Decreased metabolites, †Increased metabolites, ‡The biomarker combination, §Under the nonparametric assumption, ||Null hypothesis: True area = 0.5. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; ROC: Receiver operating characteristic.\n(a) The receiver operating characteristic (ROC) curve of metabolites that were decreased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly decreased in the active TB group compared with controls. The ROC curves of each metabolite that was decreased in concentration in the TB group sera showed a moderate distinguishing efficiency. (b) The receiver operating characteristic (ROC) curve of metabolites that were increased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly increased in the active TB group compared with controls. The ROC curves of each metabolite that was increased in concentration in the TB group sera showed a moderate distinguishing efficiency. (c) The receiver operating characteristic (ROC) curve of the biomarker combination identified as a putative serum signature of active tuberculosis (TB). The ROC curve of the combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate showed excellent distinguishing efficiency between patients with active TB and controls.\nTo investigate whether the characteristics of the metabolites that significantly differed among the three groups could be efficiently exploited for building a sensitive biosignature of TB status, ROC curves, which have been conventionally used to evaluate diagnostic performance in clinical research, were calculated. In the specific metabolites that were decreased in active TB patients, the ROC curves of 3D, 7D, 11D-phytanic acid, behenic acid and threoninyl-γ-glutamate exhibited excellent efficiency with a AUC values of 0.904 (95% CI: 0863–0.944), 0.93 (95% CI: 0.893–0.966) and 0.964 (95% CI: 00.941–0.988), respectively [Table 3 and Figure 7a]. Kynurenine, quinolinic acid (QUIN), and presqualene diphosphate (PSDP) showed significant up-regulation in patients with active TB (P < 0.05). The AUC values for these metabolites were more than 0.8 [Table 3 and Figure 7b], showing moderate performance of diagnostic value. The largest and smallest resulting AUC values were 0.964 and 0.720 [Table 3, Figure 7a and b], which indicated that these biomarkers may potentially be involved in the disease mechanisms. Using multiple logistic regression analysis of these 12 metabolites, the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PSDP was used to represent a suitable biomarker group that allowed efficient differentiation of active TB from the controls. The resulting ROC curve of the biomarker combination had an AUC value of 0.991 (95% CI: 0.982–1.000) [Table 3 and Figure 7c], which reflects strong significant difference between active TB and the control patient groups.\nThe ROC curves of the 12 metabolites found to represent potential biomarkers of active TB\n*Decreased metabolites, †Increased metabolites, ‡The biomarker combination, §Under the nonparametric assumption, ||Null hypothesis: True area = 0.5. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; ROC: Receiver operating characteristic.\n(a) The receiver operating characteristic (ROC) curve of metabolites that were decreased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly decreased in the active TB group compared with controls. The ROC curves of each metabolite that was decreased in concentration in the TB group sera showed a moderate distinguishing efficiency. (b) The receiver operating characteristic (ROC) curve of metabolites that were increased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly increased in the active TB group compared with controls. The ROC curves of each metabolite that was increased in concentration in the TB group sera showed a moderate distinguishing efficiency. (c) The receiver operating characteristic (ROC) curve of the biomarker combination identified as a putative serum signature of active tuberculosis (TB). The ROC curve of the combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate showed excellent distinguishing efficiency between patients with active TB and controls.\n Comparison between patients with active tuberculosis and nontuberculosis disease subgroups based on serum metabolic profiling To determine whether our metabolomic approach could be used to make a distinction between the active TB and non-TB groups, Mann–Whitney U-test was applied for comparison between the active TB group and the subgroups of non-TB. The findings indicated that the UPLC-MS analysis of serum may help in the diagnosis of TB and non-TB as remarked above. Finally, the specific markers between active TB and the subgroups were explored as below [Table 4]:\nP values for comparisons between the active TB group and each subgroup of the Non-TB group\n*P < 0.05, †P < 0.01. Application Mann-whitney U-test in TB group and each subgroup. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; COPD: Chronic obstructive pulmonary disease; LC: Liquid chromatography.\n\nThe serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroupsLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).\n\nThe serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)\nPalmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)\nPalmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)\nThe serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroups\nLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).\nTo determine whether our metabolomic approach could be used to make a distinction between the active TB and non-TB groups, Mann–Whitney U-test was applied for comparison between the active TB group and the subgroups of non-TB. The findings indicated that the UPLC-MS analysis of serum may help in the diagnosis of TB and non-TB as remarked above. Finally, the specific markers between active TB and the subgroups were explored as below [Table 4]:\nP values for comparisons between the active TB group and each subgroup of the Non-TB group\n*P < 0.05, †P < 0.01. Application Mann-whitney U-test in TB group and each subgroup. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; COPD: Chronic obstructive pulmonary disease; LC: Liquid chromatography.\n\nThe serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroupsLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).\n\nThe serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)\nPalmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)\nPalmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)\nThe serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroups\nLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).",
"Table 1 lists the baseline characteristics of enrolled patients and the controls. The active TB patients were 48.32 ± 18.15 (mean ± standard deviation) years old on average and consisted of 65 males and 55 females. The healthy controls were 42.71 ± 15.31 years old and consisted of 56 males and 49 females. The non-TB controls were 50.35 ± 17.41 years old and consisted of 85 males and 61 females. Of the 146 patients with non-TB controls, 51 patients (60.1%) had lung cancer, comprising the cancer subgroup (L); 45 patients (39.9%) made up the pneumonia subgroup (P); 28 patients were in the chronic obstructive pulmonary disease (COPD) subgroup (C); and 22 patients comprised the bronchiectasis subgroup (B).",
"Representative UPLC-MS chromatograms of the serum samples of the patients with active and the subjects with the healthy and non-TB controls were shown in Figure 2. The peaks were very well resolved and were evenly dispersed across the entire retention time domain, showing the high quality of the raw data. There were many significant differences in the areas and heights of peaks among groups, as a matter of fact, it is inferred that differences in peaks resulted from metabolic derangements along with diseases.\nHigh performance liquid chromatography-mass spectrometry chromatograms of the serum samples of patients from the tuberculosis (TB) group, the healthy group, and the four subgroups of patients with non-TB diseases. Abscissa: Retention time. Ordinate: Relative abundance. The numbers on the point of the peaks indicate the retention time of each substance. T indicates TB group; N indicates healthy control; P indicates pulmonitis subgroup; L indicates lung cancer subgroup; C indicates chronic obstructive pulmonary disease subgroup; B indicates bronchiectasis subgroup.",
"As mentioned above, the overall peak profiles of the three groups looked quite different, which suggested that these profiles could be used to discriminate TB from the controls. For the holistic treatment of these data, multivariate analysis was used to identify the metabolomic differences between the groups. For data reduction and pattern recognition (PR), a series of PR methods were applied using SIMCA-P 12.1 software (Umetrics, Sweden). Principal component analysis (PCA) was initially applied to the data to visualize inherent clustering between the three groups. PCA involves the transformation of a multidimensional set of possibly correlated variables into two linearly uncorrelated dimensions. This model explained an estimated 41.4% of the original data (R2 = 0.414 and Q2 = 0.334) [Figure 3]. However, in addition to the effects of the diseases on the metabolome, there were other factors that are known to contribute to differences of endogenous metabolites, such as age and diet.\nPrincipal component analysis (PCA) model. PCA scores plots of the SIMCA-P + 12.0.1.0 generated data, showing tuberculosis (TB) patients versus healthy controls and the non-TB group collected serum samples before the removal of ‘noise’ and interfering compounds from the dataset. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.\nTherefore, orthogonal signal correction technology was used to filter out unrelated variable information and retain related variables. Hence, OPLS-DA model was introduced, which is used to determine the maximum separation between different kinds of samples according to the sample classification information. The OPLS-DA model was used to identify biomarkers that accounted for the differences between the three groups, and it clearly distinguished between the TB group and the two control groups (non-TB disease subgroups were combined) [Figure 4]. The results showed that 82.1% of samples were consistent with the discrimination of the model, and the predictive ability of the model was 58.2% (Q2Y = 0.582). These findings indicate that the OPLS-DA model may pave the way for the diagnosis of TB and permit differentiation between other kinds of related diseases.\n(a) OPLS-DA two-dimensional model. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup. (b) OPLS-DA three-dimensional model. OPLS-DA scores plot discriminating serum samples of tuberculosis (TB) patients, healthy control and non-TB group based on the metabolite profiling data. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.",
"The successful use of UPLC-MS metabolomics analysis and the OPLS-DA model described above to distinguish between the TB group and control groups led us to search for the specific metabolites that contributed to the metabolomic differences. Based on the OPLS-DA model, the signals that were highly correlated and had high signal-to-noise ratio values were selected. The metabolites of >400 small molecules in the sera of patients in the three groups were explored. The molecules responsible for these signals were identified and differences in the abundance of these small molecules were determined by applying Mann–Whitney U-test for each of the three possible comparisons, using a P value threshold of 0.05. Twenty-seven metabolites were detected at significantly different levels between the active TB group and the control groups. Of those metabolites identified to differ significantly between groups, 12 metabolites [Table 2 and Figure 6], were clustered in the fatty acid, phospholipids, amino acids, and terpenoid compounds metabolite sets. To confirm that the three groups differed in terms of the serum levels of these metabolites, a heat map, a graphical representation of data where the individual values are represented as colors, was drawn [Figure 5]. Heat maps are 2D displays of the measured experimental values in the data matrix. The relatively high abundance of any specific metabolite is represented by yellow-colored squares (pixels) and a low abundance is represented by orange-colored squares.\nDetails of the 12 metabolites best describing the variation between patients with active TB and the controls\n*Molecular weight (m/z) is denoted by its monoisotopic mass, †The chemical formulas were predicted based on accurate mass by using the molecular formula generator algorithm of Mass Frontier 6.0 software (Thermo Fisher Scientific), ‡The trend of marker levels in the active TB group. ↑ and ↓ indicate increased and decreased levels, respectively, compared with the healthy group. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine.\nHeat map based on the differences in the abundance of small metabolic compounds among sera from the tuberculosis (TB) group, healthy group and non-TB group. Yellow color indicates higher abundance of metabolites; Orange color indicates lower abundance of metabolites.\nRelative abundances of metabolites in tuberculosis patients (T), healthy controls (O) and nontuberculosis patients (N). The metabolites changed in relative abundance between groups were: Palmitic acid, LysoPC (16:0), LysoPC (18:0), phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, quinolinic acid, presqualene diphosphate, LysoPC (P-18:1 (9Z)), and LysoPC (P-16:0). Black lines indicates sample means. Asterisks indicate significant differences between 2 groups (results from t-tests corrected for multiple testing; *P < 0.05; **P < 0.01).\nThe results showed that the three groups were significantly different in terms of the abundances of these biomarker metabolites in the patient sera.",
"To investigate whether the characteristics of the metabolites that significantly differed among the three groups could be efficiently exploited for building a sensitive biosignature of TB status, ROC curves, which have been conventionally used to evaluate diagnostic performance in clinical research, were calculated. In the specific metabolites that were decreased in active TB patients, the ROC curves of 3D, 7D, 11D-phytanic acid, behenic acid and threoninyl-γ-glutamate exhibited excellent efficiency with a AUC values of 0.904 (95% CI: 0863–0.944), 0.93 (95% CI: 0.893–0.966) and 0.964 (95% CI: 00.941–0.988), respectively [Table 3 and Figure 7a]. Kynurenine, quinolinic acid (QUIN), and presqualene diphosphate (PSDP) showed significant up-regulation in patients with active TB (P < 0.05). The AUC values for these metabolites were more than 0.8 [Table 3 and Figure 7b], showing moderate performance of diagnostic value. The largest and smallest resulting AUC values were 0.964 and 0.720 [Table 3, Figure 7a and b], which indicated that these biomarkers may potentially be involved in the disease mechanisms. Using multiple logistic regression analysis of these 12 metabolites, the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PSDP was used to represent a suitable biomarker group that allowed efficient differentiation of active TB from the controls. The resulting ROC curve of the biomarker combination had an AUC value of 0.991 (95% CI: 0.982–1.000) [Table 3 and Figure 7c], which reflects strong significant difference between active TB and the control patient groups.\nThe ROC curves of the 12 metabolites found to represent potential biomarkers of active TB\n*Decreased metabolites, †Increased metabolites, ‡The biomarker combination, §Under the nonparametric assumption, ||Null hypothesis: True area = 0.5. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; ROC: Receiver operating characteristic.\n(a) The receiver operating characteristic (ROC) curve of metabolites that were decreased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly decreased in the active TB group compared with controls. The ROC curves of each metabolite that was decreased in concentration in the TB group sera showed a moderate distinguishing efficiency. (b) The receiver operating characteristic (ROC) curve of metabolites that were increased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly increased in the active TB group compared with controls. The ROC curves of each metabolite that was increased in concentration in the TB group sera showed a moderate distinguishing efficiency. (c) The receiver operating characteristic (ROC) curve of the biomarker combination identified as a putative serum signature of active tuberculosis (TB). The ROC curve of the combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate showed excellent distinguishing efficiency between patients with active TB and controls.",
"To determine whether our metabolomic approach could be used to make a distinction between the active TB and non-TB groups, Mann–Whitney U-test was applied for comparison between the active TB group and the subgroups of non-TB. The findings indicated that the UPLC-MS analysis of serum may help in the diagnosis of TB and non-TB as remarked above. Finally, the specific markers between active TB and the subgroups were explored as below [Table 4]:\nP values for comparisons between the active TB group and each subgroup of the Non-TB group\n*P < 0.05, †P < 0.01. Application Mann-whitney U-test in TB group and each subgroup. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; COPD: Chronic obstructive pulmonary disease; LC: Liquid chromatography.\n\nThe serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroupsLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).\n\nThe serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)\nPalmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)\nPalmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)\nThe serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroups\nLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).",
"The present study used UPLC-MS to profile active TB and build a statistical model that enabled the identification of biomarkers for disease diagnosis based on metabolomic research. Findings indicated that 12 metabolites were unambiguously altered in serum of active TB patients as compared with two other groups (healthy controls and non-TB disease patients) [Table 2]. In the following discussion, we consider the biological relevance of these prominent metabolites and their potential biosignatures for the diagnosis of active TB.\n Fatty acids It is universally acknowledged that Mtb preferentially relies on fatty acid metabolism to maintain chronic infection.[26] When there is persistent infection in the lung tissue, the fatty acids, which are metabolized in two ways (in β-oxidation decomposition and the glyoxylate cycle) may be a source of carbon and energy of Mtb.[27] In this study, palmitic acid, phytanic acid and behenic acid, which decreased significantly in the sera of TB patients compared with the healthy group, bronchiectasis, and COPD subgroups, may be among the products of the fatty acid consumption. In addition, palmitic acid, as the most abundant free fatty acid in the human body, can induce inhibition of the electron transport chain of macrophage, which, in turn, reduces adenosine triphosphate production in the mitochondria and stimulates the activity of the mitochondrial apoptotic pathway. Another study that further supports our findings was that the palmitic acid, as one of the active compounds from the plant extraction, has some toxicity toward mycobacteria, but it is not highly toxic.[28]\nPhytanic acid, a 20-carbon branched chain fatty acid, can be used as the substrate of CYP124A1,[29] which is a heme-containing enzyme that belongs to the cytochrome p450 superfamily. When CYP124A1 was combined with the substrate of phytanic acid, the molecular conformation of CYP124A1 changed in favor of lipid oxidation, to play a better role in the important biological functions of Mtb.[30] Meanwhile, phytanic acid, as a substrate of CYP124A1, was consumed.\nIn our study, behenic acid was detected in a lower concentration in the TB group than the healthy and non-TB groups, except for the pneumonia subgroup. Behenic acid has been identified as a fatty acid that increases cholesterol in humans.[31] Cholesterol has a significant role in the development of TB, because it is necessary for the good functioning of macrophages and lymphocytes.[32] In the macrophage cell membrane, cholesterol directly participates in the function of phagocytizing Mtb, while, in the lymphocyte membrane, it is involved in the differentiation and proliferation of cytotoxic cells. Apart from this, cholesterol also has the functions of activating lymphocytes, CD4+ T cells, CD8+ T cells, and γ-T cells, and promoting the release of interferon and tumor necrosis factor-α, all of which effects are effective in killing Mtb. A previous study showed that TB patients universally had a lowered concentration of total serum cholesterol.[33] Due to this, the decreased concentration of behenic acid, as a cholesterol-raising fatty acid, in sera of TB patients may further explain the hypocholesteremia status in TB.[34]\nIt is universally acknowledged that Mtb preferentially relies on fatty acid metabolism to maintain chronic infection.[26] When there is persistent infection in the lung tissue, the fatty acids, which are metabolized in two ways (in β-oxidation decomposition and the glyoxylate cycle) may be a source of carbon and energy of Mtb.[27] In this study, palmitic acid, phytanic acid and behenic acid, which decreased significantly in the sera of TB patients compared with the healthy group, bronchiectasis, and COPD subgroups, may be among the products of the fatty acid consumption. In addition, palmitic acid, as the most abundant free fatty acid in the human body, can induce inhibition of the electron transport chain of macrophage, which, in turn, reduces adenosine triphosphate production in the mitochondria and stimulates the activity of the mitochondrial apoptotic pathway. Another study that further supports our findings was that the palmitic acid, as one of the active compounds from the plant extraction, has some toxicity toward mycobacteria, but it is not highly toxic.[28]\nPhytanic acid, a 20-carbon branched chain fatty acid, can be used as the substrate of CYP124A1,[29] which is a heme-containing enzyme that belongs to the cytochrome p450 superfamily. When CYP124A1 was combined with the substrate of phytanic acid, the molecular conformation of CYP124A1 changed in favor of lipid oxidation, to play a better role in the important biological functions of Mtb.[30] Meanwhile, phytanic acid, as a substrate of CYP124A1, was consumed.\nIn our study, behenic acid was detected in a lower concentration in the TB group than the healthy and non-TB groups, except for the pneumonia subgroup. Behenic acid has been identified as a fatty acid that increases cholesterol in humans.[31] Cholesterol has a significant role in the development of TB, because it is necessary for the good functioning of macrophages and lymphocytes.[32] In the macrophage cell membrane, cholesterol directly participates in the function of phagocytizing Mtb, while, in the lymphocyte membrane, it is involved in the differentiation and proliferation of cytotoxic cells. Apart from this, cholesterol also has the functions of activating lymphocytes, CD4+ T cells, CD8+ T cells, and γ-T cells, and promoting the release of interferon and tumor necrosis factor-α, all of which effects are effective in killing Mtb. A previous study showed that TB patients universally had a lowered concentration of total serum cholesterol.[33] Due to this, the decreased concentration of behenic acid, as a cholesterol-raising fatty acid, in sera of TB patients may further explain the hypocholesteremia status in TB.[34]\n Lysophosphatidylcholines Lysophosphatidylcholine (P-18:1(9Z)), lysoPC (P-16:0), lysoPC (16:0), and lysoPC (18:0) formed one cluster of metabolites that were present at lower levels in the active TB group and significantly differed between the healthy and non-TB groups. LysoPCs impair nitric oxide (NO) production and endothelium-dependent vasorelaxation. Moreover, lysoPC levels have been shown to be negatively correlated with NO levels.[36] Other research has pointed out that the generation of NO by activated macrophages could control mycobacterial infection in the murine system.[35] A low lysoPC level may accelerate Mtb infection by reducing the generation of NO. Additionally, another finding that TB can induce macrophage apoptosis by inhibition of phospholipase A2[373839] could also explain the low concentrations of lysoPCs in patients with active TB, a finding that is consistent with the previous report by Weiner et al.[24]\nLysophosphatidylcholine (P-18:1(9Z)), lysoPC (P-16:0), lysoPC (16:0), and lysoPC (18:0) formed one cluster of metabolites that were present at lower levels in the active TB group and significantly differed between the healthy and non-TB groups. LysoPCs impair nitric oxide (NO) production and endothelium-dependent vasorelaxation. Moreover, lysoPC levels have been shown to be negatively correlated with NO levels.[36] Other research has pointed out that the generation of NO by activated macrophages could control mycobacterial infection in the murine system.[35] A low lysoPC level may accelerate Mtb infection by reducing the generation of NO. Additionally, another finding that TB can induce macrophage apoptosis by inhibition of phospholipase A2[373839] could also explain the low concentrations of lysoPCs in patients with active TB, a finding that is consistent with the previous report by Weiner et al.[24]\n Amino acids Our data showed an increase in the levels of two amino acids (kynurenine and QUIN), and a decrease in threoninyl-γ-glutamate in sera from TB patients relative to those of healthy controls, suggesting alterations in protein metabolism during active TB. Amino acid metabolism is complicated, involving a large number of metabolites. Gluconeogenesis, proteolysis, and oxidative catabolism contribute to amino acid balance. Tryptophan (TRP) is an essential amino acid that has various important biological functions. Kynurenine, as the downstream metabolite of TRP, can be accumulated by enhancing the activity of indoleamine 2, 3-dioxygenase-1 (IDO-1), which is widely known as the rate-limiting enzyme in TRP metabolism.[40] In addition, kynurenine can regulate human T cells, which are well known as the main anti-TB immunological cell type.\nQuinolinic acid, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines. These findings support the concept that the TRP-kynurenine pathway can influence immune functions through the effects of TRP depletion and the accumulation of kynurenine and QUIN, suggesting that the TRP degradation pathway controlled by IDO-1 is involved in the pathogenesis of pulmonary TB.[41] QUIN, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines.[42] In addition, imbalances in TRP metabolism have been linked to cancer-related immune escape and implicated in lung cancer.[43]\nThreoninyl-γ-glutamate exhibited the most excellent efficiency with an AUC value of 0.964 (95% CI: 00.941–0.988) [Table 3 and Figure 7a], is a dipeptide composed of threonine and γ-glutamate. Some dipeptides are known to have physiological or cell-signaling effects, although most are simply short-lived intermediates on their way to specific amino acid degradation pathways following further proteolysis. This dipeptide has not yet been identified in human tissues or biofluids; hence, it is classified as an “expected” metabolite.\nOur data showed an increase in the levels of two amino acids (kynurenine and QUIN), and a decrease in threoninyl-γ-glutamate in sera from TB patients relative to those of healthy controls, suggesting alterations in protein metabolism during active TB. Amino acid metabolism is complicated, involving a large number of metabolites. Gluconeogenesis, proteolysis, and oxidative catabolism contribute to amino acid balance. Tryptophan (TRP) is an essential amino acid that has various important biological functions. Kynurenine, as the downstream metabolite of TRP, can be accumulated by enhancing the activity of indoleamine 2, 3-dioxygenase-1 (IDO-1), which is widely known as the rate-limiting enzyme in TRP metabolism.[40] In addition, kynurenine can regulate human T cells, which are well known as the main anti-TB immunological cell type.\nQuinolinic acid, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines. These findings support the concept that the TRP-kynurenine pathway can influence immune functions through the effects of TRP depletion and the accumulation of kynurenine and QUIN, suggesting that the TRP degradation pathway controlled by IDO-1 is involved in the pathogenesis of pulmonary TB.[41] QUIN, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines.[42] In addition, imbalances in TRP metabolism have been linked to cancer-related immune escape and implicated in lung cancer.[43]\nThreoninyl-γ-glutamate exhibited the most excellent efficiency with an AUC value of 0.964 (95% CI: 00.941–0.988) [Table 3 and Figure 7a], is a dipeptide composed of threonine and γ-glutamate. Some dipeptides are known to have physiological or cell-signaling effects, although most are simply short-lived intermediates on their way to specific amino acid degradation pathways following further proteolysis. This dipeptide has not yet been identified in human tissues or biofluids; hence, it is classified as an “expected” metabolite.\n Terpenoid compounds One of the most prominent clusters of metabolites in our study represented terpenoid compounds, including PSDP and phytal. The latter compound, which to date has never been identified or associated with TB, belongs to the family of diterpenes. However, it is reported that elisapterosin B, one kind of diterpenes extracted from plants, could inhibit the growth of Mtb.[44] Thus, the relationship between phytal and disease as well as its involvement in disease mechanisms is open to further study.\nPresqualene diphosphate, an intermediate in the biosynthesis of terpenoids, was found at higher levels in the active TB group and significantly differed between the active TB and cancer subgroups by an order of magnitude. PSDP exerts an intercellular signal for the down-regulation of superoxide in neutrophils,[45] which is widely known to play a central role in host defense, inflammation, and tissue damage.[46] PSDP, an endogenous PI3K inhibitor, directly inhibited recombinant human p110-pI3K activity, which can activate polymorphonuclear neutrophils (PMN). PMN is the primary initial immune effectors of acute inflammation, and potentially PMN products released into surrounding tissues contribute to lung and respiratory injury. A negative correlation between PSDP and PMN was observed. The observed increase in the serum concentration of PSDP may be an indicator of TB inflammatory progression. PSDP, after the initial acute inflammatory state, may act to limit tissue injury by inhibiting phospholipase D, phosphoinositol-3 kinase, and superoxide anion generation, thus providing protection for the host tissues. In addition, PSDP, a well-recognized intermediate of cholesterol biosynthesis, exists in immune effector cells and is a potential regulator of the cellular response in host defense. The increased PSDP in TB patients may be a response to Mtb in host defense, which could also explain the result of this study.\nOne of the most prominent clusters of metabolites in our study represented terpenoid compounds, including PSDP and phytal. The latter compound, which to date has never been identified or associated with TB, belongs to the family of diterpenes. However, it is reported that elisapterosin B, one kind of diterpenes extracted from plants, could inhibit the growth of Mtb.[44] Thus, the relationship between phytal and disease as well as its involvement in disease mechanisms is open to further study.\nPresqualene diphosphate, an intermediate in the biosynthesis of terpenoids, was found at higher levels in the active TB group and significantly differed between the active TB and cancer subgroups by an order of magnitude. PSDP exerts an intercellular signal for the down-regulation of superoxide in neutrophils,[45] which is widely known to play a central role in host defense, inflammation, and tissue damage.[46] PSDP, an endogenous PI3K inhibitor, directly inhibited recombinant human p110-pI3K activity, which can activate polymorphonuclear neutrophils (PMN). PMN is the primary initial immune effectors of acute inflammation, and potentially PMN products released into surrounding tissues contribute to lung and respiratory injury. A negative correlation between PSDP and PMN was observed. The observed increase in the serum concentration of PSDP may be an indicator of TB inflammatory progression. PSDP, after the initial acute inflammatory state, may act to limit tissue injury by inhibiting phospholipase D, phosphoinositol-3 kinase, and superoxide anion generation, thus providing protection for the host tissues. In addition, PSDP, a well-recognized intermediate of cholesterol biosynthesis, exists in immune effector cells and is a potential regulator of the cellular response in host defense. The increased PSDP in TB patients may be a response to Mtb in host defense, which could also explain the result of this study.",
"It is universally acknowledged that Mtb preferentially relies on fatty acid metabolism to maintain chronic infection.[26] When there is persistent infection in the lung tissue, the fatty acids, which are metabolized in two ways (in β-oxidation decomposition and the glyoxylate cycle) may be a source of carbon and energy of Mtb.[27] In this study, palmitic acid, phytanic acid and behenic acid, which decreased significantly in the sera of TB patients compared with the healthy group, bronchiectasis, and COPD subgroups, may be among the products of the fatty acid consumption. In addition, palmitic acid, as the most abundant free fatty acid in the human body, can induce inhibition of the electron transport chain of macrophage, which, in turn, reduces adenosine triphosphate production in the mitochondria and stimulates the activity of the mitochondrial apoptotic pathway. Another study that further supports our findings was that the palmitic acid, as one of the active compounds from the plant extraction, has some toxicity toward mycobacteria, but it is not highly toxic.[28]\nPhytanic acid, a 20-carbon branched chain fatty acid, can be used as the substrate of CYP124A1,[29] which is a heme-containing enzyme that belongs to the cytochrome p450 superfamily. When CYP124A1 was combined with the substrate of phytanic acid, the molecular conformation of CYP124A1 changed in favor of lipid oxidation, to play a better role in the important biological functions of Mtb.[30] Meanwhile, phytanic acid, as a substrate of CYP124A1, was consumed.\nIn our study, behenic acid was detected in a lower concentration in the TB group than the healthy and non-TB groups, except for the pneumonia subgroup. Behenic acid has been identified as a fatty acid that increases cholesterol in humans.[31] Cholesterol has a significant role in the development of TB, because it is necessary for the good functioning of macrophages and lymphocytes.[32] In the macrophage cell membrane, cholesterol directly participates in the function of phagocytizing Mtb, while, in the lymphocyte membrane, it is involved in the differentiation and proliferation of cytotoxic cells. Apart from this, cholesterol also has the functions of activating lymphocytes, CD4+ T cells, CD8+ T cells, and γ-T cells, and promoting the release of interferon and tumor necrosis factor-α, all of which effects are effective in killing Mtb. A previous study showed that TB patients universally had a lowered concentration of total serum cholesterol.[33] Due to this, the decreased concentration of behenic acid, as a cholesterol-raising fatty acid, in sera of TB patients may further explain the hypocholesteremia status in TB.[34]",
"Lysophosphatidylcholine (P-18:1(9Z)), lysoPC (P-16:0), lysoPC (16:0), and lysoPC (18:0) formed one cluster of metabolites that were present at lower levels in the active TB group and significantly differed between the healthy and non-TB groups. LysoPCs impair nitric oxide (NO) production and endothelium-dependent vasorelaxation. Moreover, lysoPC levels have been shown to be negatively correlated with NO levels.[36] Other research has pointed out that the generation of NO by activated macrophages could control mycobacterial infection in the murine system.[35] A low lysoPC level may accelerate Mtb infection by reducing the generation of NO. Additionally, another finding that TB can induce macrophage apoptosis by inhibition of phospholipase A2[373839] could also explain the low concentrations of lysoPCs in patients with active TB, a finding that is consistent with the previous report by Weiner et al.[24]",
"Our data showed an increase in the levels of two amino acids (kynurenine and QUIN), and a decrease in threoninyl-γ-glutamate in sera from TB patients relative to those of healthy controls, suggesting alterations in protein metabolism during active TB. Amino acid metabolism is complicated, involving a large number of metabolites. Gluconeogenesis, proteolysis, and oxidative catabolism contribute to amino acid balance. Tryptophan (TRP) is an essential amino acid that has various important biological functions. Kynurenine, as the downstream metabolite of TRP, can be accumulated by enhancing the activity of indoleamine 2, 3-dioxygenase-1 (IDO-1), which is widely known as the rate-limiting enzyme in TRP metabolism.[40] In addition, kynurenine can regulate human T cells, which are well known as the main anti-TB immunological cell type.\nQuinolinic acid, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines. These findings support the concept that the TRP-kynurenine pathway can influence immune functions through the effects of TRP depletion and the accumulation of kynurenine and QUIN, suggesting that the TRP degradation pathway controlled by IDO-1 is involved in the pathogenesis of pulmonary TB.[41] QUIN, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines.[42] In addition, imbalances in TRP metabolism have been linked to cancer-related immune escape and implicated in lung cancer.[43]\nThreoninyl-γ-glutamate exhibited the most excellent efficiency with an AUC value of 0.964 (95% CI: 00.941–0.988) [Table 3 and Figure 7a], is a dipeptide composed of threonine and γ-glutamate. Some dipeptides are known to have physiological or cell-signaling effects, although most are simply short-lived intermediates on their way to specific amino acid degradation pathways following further proteolysis. This dipeptide has not yet been identified in human tissues or biofluids; hence, it is classified as an “expected” metabolite.",
"One of the most prominent clusters of metabolites in our study represented terpenoid compounds, including PSDP and phytal. The latter compound, which to date has never been identified or associated with TB, belongs to the family of diterpenes. However, it is reported that elisapterosin B, one kind of diterpenes extracted from plants, could inhibit the growth of Mtb.[44] Thus, the relationship between phytal and disease as well as its involvement in disease mechanisms is open to further study.\nPresqualene diphosphate, an intermediate in the biosynthesis of terpenoids, was found at higher levels in the active TB group and significantly differed between the active TB and cancer subgroups by an order of magnitude. PSDP exerts an intercellular signal for the down-regulation of superoxide in neutrophils,[45] which is widely known to play a central role in host defense, inflammation, and tissue damage.[46] PSDP, an endogenous PI3K inhibitor, directly inhibited recombinant human p110-pI3K activity, which can activate polymorphonuclear neutrophils (PMN). PMN is the primary initial immune effectors of acute inflammation, and potentially PMN products released into surrounding tissues contribute to lung and respiratory injury. A negative correlation between PSDP and PMN was observed. The observed increase in the serum concentration of PSDP may be an indicator of TB inflammatory progression. PSDP, after the initial acute inflammatory state, may act to limit tissue injury by inhibiting phospholipase D, phosphoinositol-3 kinase, and superoxide anion generation, thus providing protection for the host tissues. In addition, PSDP, a well-recognized intermediate of cholesterol biosynthesis, exists in immune effector cells and is a potential regulator of the cellular response in host defense. The increased PSDP in TB patients may be a response to Mtb in host defense, which could also explain the result of this study.",
"Metabolic profiling approaches based on UPLC-MS were successfully used to distinguish TB patients from the controls and establish a TB-specific metabolite profiling. Twelve metabolites were identified to be significantly different among patient groups, with moderate performance of diagnostic value indicating that these biomarkers may potentially be involved in disease mechanisms. Most of the identified metabolites were mainly involved in fatty acid, amino acid, and lipid metabolism pathways, leading to a number of new hypotheses to better clarify the reasons for their differential abundance in active TB, including:\n\nThe decrease of palmitic acid, 3D, 7D, 11D-phytanic acid and behenic acid supported that the fatty acid was preferentially utilized by Mtb, and these metabolites may be one of the results of fatty acid consumptionTerpenoid compounds may have an important role in the processes of infection and host resistance to TB infectionMtb infection may produce enhanced activity of the TRP -kynurenine pathway, and kynurenine and QUIN, as the TRP downstream metabolites, may affect the immune function of patients with TBThe lower levels of lysoPCs observed in the active TB group may accelerate Mtb infection by reducing the generation of NO, which is produced by activated macrophagesMultiple logistic regression analysis was performed in the 12 metabolites identified as a signature of active TB, and the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PDSP was calculated to represent the best diagnostic value with the AUC value of 0.991 (95% CI: 0.982–1.000). Thus, this combination of metabolites may prove to be a metabolic profile that can be used for the diagnosis of TB.\n\nThe decrease of palmitic acid, 3D, 7D, 11D-phytanic acid and behenic acid supported that the fatty acid was preferentially utilized by Mtb, and these metabolites may be one of the results of fatty acid consumption\nTerpenoid compounds may have an important role in the processes of infection and host resistance to TB infection\nMtb infection may produce enhanced activity of the TRP -kynurenine pathway, and kynurenine and QUIN, as the TRP downstream metabolites, may affect the immune function of patients with TB\nThe lower levels of lysoPCs observed in the active TB group may accelerate Mtb infection by reducing the generation of NO, which is produced by activated macrophages\nMultiple logistic regression analysis was performed in the 12 metabolites identified as a signature of active TB, and the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PDSP was calculated to represent the best diagnostic value with the AUC value of 0.991 (95% CI: 0.982–1.000). Thus, this combination of metabolites may prove to be a metabolic profile that can be used for the diagnosis of TB."
] |
[
"intro",
"methods",
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
null,
"discussion",
null,
null,
null,
null,
"conclusions"
] |
[
"Metabolites",
"Orthogonal Partial Least-squares Discriminant Analysis",
"Serum",
"Tuberculosis",
"Ultra-performance Liquid Chromatography-mass Spectrometry"
] |
INTRODUCTION:
Metabolomics is the quantitative measurement of the dynamic multiparametric metabolic responses of living systems to pathophysiological stimuli or genetic modifications.[1] Metabolites can be viewed as a close recapitulation of disease phenotypes, and they may represent the ongoing pathogenesis in an organism to a greater extent than changes in gene expression.[2] Consequently, it has deepened our understanding of the biological mechanisms involved in several noninfectious diseases and provided a platform for the identification of new biomarkers.[3] Metabolic signatures have been exploited in the study of several diseases, such as Alzheimer's disease,[4] Parkinson's disease,[5] myocardial ischemia,[6] hypertension,[7] cancer,[8] and diabetes.[9101112] However, this powerful analytical tool has not been widely applied to infectious diseases for the development of diagnostic biomarkers.[131415]
Tuberculosis (TB) is a major worldwide health problem, with a global estimate of 9.4 million incidences in 2010 (range; 8.9 × 106–9.9 × 106). Of the more than 2 billion people infected with Mycobacterium tuberculosis (Mtb) globally, more than one-tenth are likely to develop active TB during their lifetime. In many regions, notably in developing countries with high TB incidences, diagnosis is neither sensitive nor specific, and an estimated 40% of TB patients fail to be correctly diagnosed.[16] In recent years, advancement in the field of metabolomics has mainly been applied to metabolic profiling about TB drug metabolism, rather than diagnosis of TB.[1718192021] However, the serum that is successfully used in TB diagnosis on the basis of metabolic profiling is not by much.[2223] A previous study by Weiner et al. using gas chromatography-mass spectrometry (MS) showed differences in metabolic profiles among the uninfected individuals, individuals with latent (inactive) TB, and patients with active TB.[24] In a recent study, nuclear magnetic resonance spectroscopy was used to characterize the metabolism of the host during Mtb infection.[25] However, relatively few methods are considered to be capable of distinguishing between active TB and diseases other than TB, such as lung cancer, pneumonia, and so forth. It can be expected that several of the observed changes are the result of a general inflammatory process rather than a specific response to TB. In this study, 120 patients with active TB and 251 controls who were either healthy or had diseases other than TB (non-TB group) were enrolled. Using ultra performance liquid chromatography-MS (UPLC-MS), we investigated the feasibility of identifying small molecule biochemical profiles in serum for gaining novel biological insights into the mechanisms underlying TB. The unique feature of this study was to employ the largest number of patients included in any study of a similar nature to date (120 with active TB and 251 controls). Multivariate statistical analysis was implemented to discriminate patients with active TB from the control subjects on the basis of their metabolic profiles. We identified 12 distinct biomarkers, including clusters that could be categorized as amino acids, fatty acids, lysophosphatidylcholine (lysoPC), and terpenoid compounds. Finally, these new metabolite markers, which can distinguish TB from non-TB diseases, led to a number of hypotheses. Our results provide specific insights into the biology of TB and may offer new avenues for TB diagnosis or therapy.
METHODS:
Chemicals Acetonitrile was purchased from Merck and Co. (Merck KGaA, Germany) and formic acid was obtained from Shanghai Nanxiang People Chemical Factory (China). All other solvents were of high-performance liquid chromatography grade. Reference standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ultrapure water was prepared by Milli-Q system (Millipore Co., USA).
Acetonitrile was purchased from Merck and Co. (Merck KGaA, Germany) and formic acid was obtained from Shanghai Nanxiang People Chemical Factory (China). All other solvents were of high-performance liquid chromatography grade. Reference standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ultrapure water was prepared by Milli-Q system (Millipore Co., USA).
Serum sample collection Serum samples were collected at Tianjin Haihe Hospital from 105 healthy individuals who visited the hospital for medical check-up, 146 patients with lung diseases that were due to non-TB conditions, and 120 patients with clinical signs of TB [Table 1]. TB patients had the following symptoms: Cough for more than 2 weeks and at least two additional symptoms (hemoptysis [coughing up blood], breathing difficulty, fever, night sweats, weight loss, chest pain, or fatigue). All patients were diagnosed based on chest X-rays and sputum samples obtained from each TB patient were analyzed by Ziehl-Neelsen staining and mycobacterium growth indicator tube culture. Active TB was diagnosed when (1) Mtb was cultured, (2) a caseating granuloma was found in the lung tissue by transthoracic needle biopsy and showed an appropriate response to treatment; or (3) clinical findings were compatible with TB, no clinical improvement was seen following treatment with empirical antibiotics, and treatment with anti-TB medication resulted in clinical and radiological improvement. The 251 controls without comorbidities were matched to the TB group with respect to age and sex. Whole-blood samples were drawn from a peripheral vein between 7:00 am and 9:00 am. Sera from patients and healthy volunteers were acquired from ethylenediaminetetraacetic acid-preserved whole blood samples following centrifugation and were stored at −80°C until analysis. At the time of sample collection, none of the patients were receiving treatment. Before the UPLC-MS analysis, serum samples were defrosted at room temperature for <20 minutes and 200 μl aliquots were combined with 300 μl of saline (0.9% NaCl in 20% D2O/80% H2O) and then centrifuged at 12,000 ×g for 5 minutes. A volume of 500 μl aliquot of the supernatant was pipetted into a 5 mm tube, and samples were stored at −80°C until further use.
Characteristics of TB patients and the control groups
There was no significant difference in demographic data between TB patients and the controls. *Data are presented as mean ± SD. SD: Standard deviation; TB: Tuberculosis.
Serum samples were collected at Tianjin Haihe Hospital from 105 healthy individuals who visited the hospital for medical check-up, 146 patients with lung diseases that were due to non-TB conditions, and 120 patients with clinical signs of TB [Table 1]. TB patients had the following symptoms: Cough for more than 2 weeks and at least two additional symptoms (hemoptysis [coughing up blood], breathing difficulty, fever, night sweats, weight loss, chest pain, or fatigue). All patients were diagnosed based on chest X-rays and sputum samples obtained from each TB patient were analyzed by Ziehl-Neelsen staining and mycobacterium growth indicator tube culture. Active TB was diagnosed when (1) Mtb was cultured, (2) a caseating granuloma was found in the lung tissue by transthoracic needle biopsy and showed an appropriate response to treatment; or (3) clinical findings were compatible with TB, no clinical improvement was seen following treatment with empirical antibiotics, and treatment with anti-TB medication resulted in clinical and radiological improvement. The 251 controls without comorbidities were matched to the TB group with respect to age and sex. Whole-blood samples were drawn from a peripheral vein between 7:00 am and 9:00 am. Sera from patients and healthy volunteers were acquired from ethylenediaminetetraacetic acid-preserved whole blood samples following centrifugation and were stored at −80°C until analysis. At the time of sample collection, none of the patients were receiving treatment. Before the UPLC-MS analysis, serum samples were defrosted at room temperature for <20 minutes and 200 μl aliquots were combined with 300 μl of saline (0.9% NaCl in 20% D2O/80% H2O) and then centrifuged at 12,000 ×g for 5 minutes. A volume of 500 μl aliquot of the supernatant was pipetted into a 5 mm tube, and samples were stored at −80°C until further use.
Characteristics of TB patients and the control groups
There was no significant difference in demographic data between TB patients and the controls. *Data are presented as mean ± SD. SD: Standard deviation; TB: Tuberculosis.
Sample extraction Before further metabolomics analysis, samples were defrosted at room temperature for <20 minutes. Briefly, 400 μl of acetonitrile was added to the samples in a 4:1 (v/v) ratio. These sample mixtures were then homogenized by shaking them for 30 seconds and centrifuged at 15,000 ×g for 20 minutes at −4°C. The supernatant was collected and divided into three fractions: One for analysis by UPLC-MS, one for quality control (QC) analysis, and one was stored for further analysis (if necessary). A 500 μl aliquot of the supernatant was pipetted into a 5 mm test tube and then loaded into the UPLC-MS/MS system for analysis.
Before further metabolomics analysis, samples were defrosted at room temperature for <20 minutes. Briefly, 400 μl of acetonitrile was added to the samples in a 4:1 (v/v) ratio. These sample mixtures were then homogenized by shaking them for 30 seconds and centrifuged at 15,000 ×g for 20 minutes at −4°C. The supernatant was collected and divided into three fractions: One for analysis by UPLC-MS, one for quality control (QC) analysis, and one was stored for further analysis (if necessary). A 500 μl aliquot of the supernatant was pipetted into a 5 mm test tube and then loaded into the UPLC-MS/MS system for analysis.
Instruments and conditions In the first part of the study, samples were measured with a nano liquid chromatography system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap™ mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific). Samples were loaded onto a trap column (PepMap C18, 300 μm internal diameter [ID], 5 mm length, 5 μm particle size, 100 Å pore size; Thermo Fisher Scientific), then washed and desalted for 15 minutes using 0.1% trifluoroacetic acid in water as the loading solvent. Next, the trap column was switched in-line with the analytical column (PepMap C18, 75 μm ID, 250 mm length, 3 μm particle size, 100 Å pore size; Thermo Fisher Scientific) and peptides were eluted with the following binary gradient: Starting with 100% solvent A and B, where solvent A consisted of 2% acetonitrile and 0.1% formic acid in water, and solvent B consisted of 80% acetonitrile and 0.08% formic acid in water. The column flow rate was set at 200 nl/min. For electro-spray ionization (ESI), nano ESI emitters (New Objective, Woburn, MA, USA) were used, and a spray voltage of 4.5 kV was applied. For MS detection, a data-dependent acquisition method was used: High-resolution survey scan from 50 to 1000 (m\z). Orbitrap full scan spectra and ion trap MS/MS fragmentation spectra were acquired partially simultaneously.
In the first part of the study, samples were measured with a nano liquid chromatography system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap™ mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific). Samples were loaded onto a trap column (PepMap C18, 300 μm internal diameter [ID], 5 mm length, 5 μm particle size, 100 Å pore size; Thermo Fisher Scientific), then washed and desalted for 15 minutes using 0.1% trifluoroacetic acid in water as the loading solvent. Next, the trap column was switched in-line with the analytical column (PepMap C18, 75 μm ID, 250 mm length, 3 μm particle size, 100 Å pore size; Thermo Fisher Scientific) and peptides were eluted with the following binary gradient: Starting with 100% solvent A and B, where solvent A consisted of 2% acetonitrile and 0.1% formic acid in water, and solvent B consisted of 80% acetonitrile and 0.08% formic acid in water. The column flow rate was set at 200 nl/min. For electro-spray ionization (ESI), nano ESI emitters (New Objective, Woburn, MA, USA) were used, and a spray voltage of 4.5 kV was applied. For MS detection, a data-dependent acquisition method was used: High-resolution survey scan from 50 to 1000 (m\z). Orbitrap full scan spectra and ion trap MS/MS fragmentation spectra were acquired partially simultaneously.
Quality control solution composition To observe the stability of the machine, QC solution was formulated from the mixed supernatants of all of the samples. The mixed samples solution was divided into 31 aliquots. Ten copies of QC solution were detected continuously before analyzing, and then the rest of copies were randomly inserted between each of the 10 sample runs [Figure 1]. The order in which samples were analyzed was randomized using the Excel 2013 software (Microsoft, USA), and a posttest blank sample was run after each of the 10 samples to avoid cross-contamination.
Quality control (QC) map. ▲: QC point. First ten QC points were run to and then the rest of the QC points were interleaved between the ten test samples. None of the QC points produced results outside of the control range.
To observe the stability of the machine, QC solution was formulated from the mixed supernatants of all of the samples. The mixed samples solution was divided into 31 aliquots. Ten copies of QC solution were detected continuously before analyzing, and then the rest of copies were randomly inserted between each of the 10 sample runs [Figure 1]. The order in which samples were analyzed was randomized using the Excel 2013 software (Microsoft, USA), and a posttest blank sample was run after each of the 10 samples to avoid cross-contamination.
Quality control (QC) map. ▲: QC point. First ten QC points were run to and then the rest of the QC points were interleaved between the ten test samples. None of the QC points produced results outside of the control range.
Compound identification, quantification, and data curation The UPLC-MS raw data were converted and processed by using MZmine 2.10 (http://www.biomedcentral.com/1471-2105/11/395). Briefly, chromatograms were built and peaks were recognized using the local minimum search function, and the ion intensities, matching m/z, and retention time was grouped into peak lists. Later, these peak lists were exported individually and imported into MetaboAnalyst 2.0 (http://nar.oxfordjournals.org/content/early/2012/05/01/nar.gks374.full). The peaks were aligned and normalized to the sum of all detected peaks. The processed and normalized data were imported into SIMCA-P (Umetrics, Umeε, Sweden) for multivariate statistical analysis. To distinguish TB from the controls, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed. Based on the OPLS-DA model, the specific metabolites were determined by applying Mann–Whitney U-test (SPSS 15.0, IBM, NY, USA) with P value threshold of 0.05. For each biomarker, a receiver operating characteristic (ROC) curve was generated. The area under curve (AUC) value and 95% confidence interval (CI) were calculated to determine the specificity and sensitivity of TB. To increase the diagnostic accuracy of combined changes in serum metabolites levels, multiple logistic regression analysis was carried out.
The UPLC-MS raw data were converted and processed by using MZmine 2.10 (http://www.biomedcentral.com/1471-2105/11/395). Briefly, chromatograms were built and peaks were recognized using the local minimum search function, and the ion intensities, matching m/z, and retention time was grouped into peak lists. Later, these peak lists were exported individually and imported into MetaboAnalyst 2.0 (http://nar.oxfordjournals.org/content/early/2012/05/01/nar.gks374.full). The peaks were aligned and normalized to the sum of all detected peaks. The processed and normalized data were imported into SIMCA-P (Umetrics, Umeε, Sweden) for multivariate statistical analysis. To distinguish TB from the controls, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed. Based on the OPLS-DA model, the specific metabolites were determined by applying Mann–Whitney U-test (SPSS 15.0, IBM, NY, USA) with P value threshold of 0.05. For each biomarker, a receiver operating characteristic (ROC) curve was generated. The area under curve (AUC) value and 95% confidence interval (CI) were calculated to determine the specificity and sensitivity of TB. To increase the diagnostic accuracy of combined changes in serum metabolites levels, multiple logistic regression analysis was carried out.
Chemicals:
Acetonitrile was purchased from Merck and Co. (Merck KGaA, Germany) and formic acid was obtained from Shanghai Nanxiang People Chemical Factory (China). All other solvents were of high-performance liquid chromatography grade. Reference standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ultrapure water was prepared by Milli-Q system (Millipore Co., USA).
Serum sample collection:
Serum samples were collected at Tianjin Haihe Hospital from 105 healthy individuals who visited the hospital for medical check-up, 146 patients with lung diseases that were due to non-TB conditions, and 120 patients with clinical signs of TB [Table 1]. TB patients had the following symptoms: Cough for more than 2 weeks and at least two additional symptoms (hemoptysis [coughing up blood], breathing difficulty, fever, night sweats, weight loss, chest pain, or fatigue). All patients were diagnosed based on chest X-rays and sputum samples obtained from each TB patient were analyzed by Ziehl-Neelsen staining and mycobacterium growth indicator tube culture. Active TB was diagnosed when (1) Mtb was cultured, (2) a caseating granuloma was found in the lung tissue by transthoracic needle biopsy and showed an appropriate response to treatment; or (3) clinical findings were compatible with TB, no clinical improvement was seen following treatment with empirical antibiotics, and treatment with anti-TB medication resulted in clinical and radiological improvement. The 251 controls without comorbidities were matched to the TB group with respect to age and sex. Whole-blood samples were drawn from a peripheral vein between 7:00 am and 9:00 am. Sera from patients and healthy volunteers were acquired from ethylenediaminetetraacetic acid-preserved whole blood samples following centrifugation and were stored at −80°C until analysis. At the time of sample collection, none of the patients were receiving treatment. Before the UPLC-MS analysis, serum samples were defrosted at room temperature for <20 minutes and 200 μl aliquots were combined with 300 μl of saline (0.9% NaCl in 20% D2O/80% H2O) and then centrifuged at 12,000 ×g for 5 minutes. A volume of 500 μl aliquot of the supernatant was pipetted into a 5 mm tube, and samples were stored at −80°C until further use.
Characteristics of TB patients and the control groups
There was no significant difference in demographic data between TB patients and the controls. *Data are presented as mean ± SD. SD: Standard deviation; TB: Tuberculosis.
Sample extraction:
Before further metabolomics analysis, samples were defrosted at room temperature for <20 minutes. Briefly, 400 μl of acetonitrile was added to the samples in a 4:1 (v/v) ratio. These sample mixtures were then homogenized by shaking them for 30 seconds and centrifuged at 15,000 ×g for 20 minutes at −4°C. The supernatant was collected and divided into three fractions: One for analysis by UPLC-MS, one for quality control (QC) analysis, and one was stored for further analysis (if necessary). A 500 μl aliquot of the supernatant was pipetted into a 5 mm test tube and then loaded into the UPLC-MS/MS system for analysis.
Instruments and conditions:
In the first part of the study, samples were measured with a nano liquid chromatography system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap™ mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific). Samples were loaded onto a trap column (PepMap C18, 300 μm internal diameter [ID], 5 mm length, 5 μm particle size, 100 Å pore size; Thermo Fisher Scientific), then washed and desalted for 15 minutes using 0.1% trifluoroacetic acid in water as the loading solvent. Next, the trap column was switched in-line with the analytical column (PepMap C18, 75 μm ID, 250 mm length, 3 μm particle size, 100 Å pore size; Thermo Fisher Scientific) and peptides were eluted with the following binary gradient: Starting with 100% solvent A and B, where solvent A consisted of 2% acetonitrile and 0.1% formic acid in water, and solvent B consisted of 80% acetonitrile and 0.08% formic acid in water. The column flow rate was set at 200 nl/min. For electro-spray ionization (ESI), nano ESI emitters (New Objective, Woburn, MA, USA) were used, and a spray voltage of 4.5 kV was applied. For MS detection, a data-dependent acquisition method was used: High-resolution survey scan from 50 to 1000 (m\z). Orbitrap full scan spectra and ion trap MS/MS fragmentation spectra were acquired partially simultaneously.
Quality control solution composition:
To observe the stability of the machine, QC solution was formulated from the mixed supernatants of all of the samples. The mixed samples solution was divided into 31 aliquots. Ten copies of QC solution were detected continuously before analyzing, and then the rest of copies were randomly inserted between each of the 10 sample runs [Figure 1]. The order in which samples were analyzed was randomized using the Excel 2013 software (Microsoft, USA), and a posttest blank sample was run after each of the 10 samples to avoid cross-contamination.
Quality control (QC) map. ▲: QC point. First ten QC points were run to and then the rest of the QC points were interleaved between the ten test samples. None of the QC points produced results outside of the control range.
Compound identification, quantification, and data curation:
The UPLC-MS raw data were converted and processed by using MZmine 2.10 (http://www.biomedcentral.com/1471-2105/11/395). Briefly, chromatograms were built and peaks were recognized using the local minimum search function, and the ion intensities, matching m/z, and retention time was grouped into peak lists. Later, these peak lists were exported individually and imported into MetaboAnalyst 2.0 (http://nar.oxfordjournals.org/content/early/2012/05/01/nar.gks374.full). The peaks were aligned and normalized to the sum of all detected peaks. The processed and normalized data were imported into SIMCA-P (Umetrics, Umeε, Sweden) for multivariate statistical analysis. To distinguish TB from the controls, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed. Based on the OPLS-DA model, the specific metabolites were determined by applying Mann–Whitney U-test (SPSS 15.0, IBM, NY, USA) with P value threshold of 0.05. For each biomarker, a receiver operating characteristic (ROC) curve was generated. The area under curve (AUC) value and 95% confidence interval (CI) were calculated to determine the specificity and sensitivity of TB. To increase the diagnostic accuracy of combined changes in serum metabolites levels, multiple logistic regression analysis was carried out.
RESULTS:
Baseline characteristics Table 1 lists the baseline characteristics of enrolled patients and the controls. The active TB patients were 48.32 ± 18.15 (mean ± standard deviation) years old on average and consisted of 65 males and 55 females. The healthy controls were 42.71 ± 15.31 years old and consisted of 56 males and 49 females. The non-TB controls were 50.35 ± 17.41 years old and consisted of 85 males and 61 females. Of the 146 patients with non-TB controls, 51 patients (60.1%) had lung cancer, comprising the cancer subgroup (L); 45 patients (39.9%) made up the pneumonia subgroup (P); 28 patients were in the chronic obstructive pulmonary disease (COPD) subgroup (C); and 22 patients comprised the bronchiectasis subgroup (B).
Table 1 lists the baseline characteristics of enrolled patients and the controls. The active TB patients were 48.32 ± 18.15 (mean ± standard deviation) years old on average and consisted of 65 males and 55 females. The healthy controls were 42.71 ± 15.31 years old and consisted of 56 males and 49 females. The non-TB controls were 50.35 ± 17.41 years old and consisted of 85 males and 61 females. Of the 146 patients with non-TB controls, 51 patients (60.1%) had lung cancer, comprising the cancer subgroup (L); 45 patients (39.9%) made up the pneumonia subgroup (P); 28 patients were in the chronic obstructive pulmonary disease (COPD) subgroup (C); and 22 patients comprised the bronchiectasis subgroup (B).
Ultra performance liquid chromatography-mass spectrometry platform performance Representative UPLC-MS chromatograms of the serum samples of the patients with active and the subjects with the healthy and non-TB controls were shown in Figure 2. The peaks were very well resolved and were evenly dispersed across the entire retention time domain, showing the high quality of the raw data. There were many significant differences in the areas and heights of peaks among groups, as a matter of fact, it is inferred that differences in peaks resulted from metabolic derangements along with diseases.
High performance liquid chromatography-mass spectrometry chromatograms of the serum samples of patients from the tuberculosis (TB) group, the healthy group, and the four subgroups of patients with non-TB diseases. Abscissa: Retention time. Ordinate: Relative abundance. The numbers on the point of the peaks indicate the retention time of each substance. T indicates TB group; N indicates healthy control; P indicates pulmonitis subgroup; L indicates lung cancer subgroup; C indicates chronic obstructive pulmonary disease subgroup; B indicates bronchiectasis subgroup.
Representative UPLC-MS chromatograms of the serum samples of the patients with active and the subjects with the healthy and non-TB controls were shown in Figure 2. The peaks were very well resolved and were evenly dispersed across the entire retention time domain, showing the high quality of the raw data. There were many significant differences in the areas and heights of peaks among groups, as a matter of fact, it is inferred that differences in peaks resulted from metabolic derangements along with diseases.
High performance liquid chromatography-mass spectrometry chromatograms of the serum samples of patients from the tuberculosis (TB) group, the healthy group, and the four subgroups of patients with non-TB diseases. Abscissa: Retention time. Ordinate: Relative abundance. The numbers on the point of the peaks indicate the retention time of each substance. T indicates TB group; N indicates healthy control; P indicates pulmonitis subgroup; L indicates lung cancer subgroup; C indicates chronic obstructive pulmonary disease subgroup; B indicates bronchiectasis subgroup.
Differentiation between tuberculosis patients and controls, and among the subgroups based on OPLS-DA As mentioned above, the overall peak profiles of the three groups looked quite different, which suggested that these profiles could be used to discriminate TB from the controls. For the holistic treatment of these data, multivariate analysis was used to identify the metabolomic differences between the groups. For data reduction and pattern recognition (PR), a series of PR methods were applied using SIMCA-P 12.1 software (Umetrics, Sweden). Principal component analysis (PCA) was initially applied to the data to visualize inherent clustering between the three groups. PCA involves the transformation of a multidimensional set of possibly correlated variables into two linearly uncorrelated dimensions. This model explained an estimated 41.4% of the original data (R2 = 0.414 and Q2 = 0.334) [Figure 3]. However, in addition to the effects of the diseases on the metabolome, there were other factors that are known to contribute to differences of endogenous metabolites, such as age and diet.
Principal component analysis (PCA) model. PCA scores plots of the SIMCA-P + 12.0.1.0 generated data, showing tuberculosis (TB) patients versus healthy controls and the non-TB group collected serum samples before the removal of ‘noise’ and interfering compounds from the dataset. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.
Therefore, orthogonal signal correction technology was used to filter out unrelated variable information and retain related variables. Hence, OPLS-DA model was introduced, which is used to determine the maximum separation between different kinds of samples according to the sample classification information. The OPLS-DA model was used to identify biomarkers that accounted for the differences between the three groups, and it clearly distinguished between the TB group and the two control groups (non-TB disease subgroups were combined) [Figure 4]. The results showed that 82.1% of samples were consistent with the discrimination of the model, and the predictive ability of the model was 58.2% (Q2Y = 0.582). These findings indicate that the OPLS-DA model may pave the way for the diagnosis of TB and permit differentiation between other kinds of related diseases.
(a) OPLS-DA two-dimensional model. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup. (b) OPLS-DA three-dimensional model. OPLS-DA scores plot discriminating serum samples of tuberculosis (TB) patients, healthy control and non-TB group based on the metabolite profiling data. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.
As mentioned above, the overall peak profiles of the three groups looked quite different, which suggested that these profiles could be used to discriminate TB from the controls. For the holistic treatment of these data, multivariate analysis was used to identify the metabolomic differences between the groups. For data reduction and pattern recognition (PR), a series of PR methods were applied using SIMCA-P 12.1 software (Umetrics, Sweden). Principal component analysis (PCA) was initially applied to the data to visualize inherent clustering between the three groups. PCA involves the transformation of a multidimensional set of possibly correlated variables into two linearly uncorrelated dimensions. This model explained an estimated 41.4% of the original data (R2 = 0.414 and Q2 = 0.334) [Figure 3]. However, in addition to the effects of the diseases on the metabolome, there were other factors that are known to contribute to differences of endogenous metabolites, such as age and diet.
Principal component analysis (PCA) model. PCA scores plots of the SIMCA-P + 12.0.1.0 generated data, showing tuberculosis (TB) patients versus healthy controls and the non-TB group collected serum samples before the removal of ‘noise’ and interfering compounds from the dataset. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.
Therefore, orthogonal signal correction technology was used to filter out unrelated variable information and retain related variables. Hence, OPLS-DA model was introduced, which is used to determine the maximum separation between different kinds of samples according to the sample classification information. The OPLS-DA model was used to identify biomarkers that accounted for the differences between the three groups, and it clearly distinguished between the TB group and the two control groups (non-TB disease subgroups were combined) [Figure 4]. The results showed that 82.1% of samples were consistent with the discrimination of the model, and the predictive ability of the model was 58.2% (Q2Y = 0.582). These findings indicate that the OPLS-DA model may pave the way for the diagnosis of TB and permit differentiation between other kinds of related diseases.
(a) OPLS-DA two-dimensional model. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup. (b) OPLS-DA three-dimensional model. OPLS-DA scores plot discriminating serum samples of tuberculosis (TB) patients, healthy control and non-TB group based on the metabolite profiling data. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.
Identification of tuberculosis-specific metabolites The successful use of UPLC-MS metabolomics analysis and the OPLS-DA model described above to distinguish between the TB group and control groups led us to search for the specific metabolites that contributed to the metabolomic differences. Based on the OPLS-DA model, the signals that were highly correlated and had high signal-to-noise ratio values were selected. The metabolites of >400 small molecules in the sera of patients in the three groups were explored. The molecules responsible for these signals were identified and differences in the abundance of these small molecules were determined by applying Mann–Whitney U-test for each of the three possible comparisons, using a P value threshold of 0.05. Twenty-seven metabolites were detected at significantly different levels between the active TB group and the control groups. Of those metabolites identified to differ significantly between groups, 12 metabolites [Table 2 and Figure 6], were clustered in the fatty acid, phospholipids, amino acids, and terpenoid compounds metabolite sets. To confirm that the three groups differed in terms of the serum levels of these metabolites, a heat map, a graphical representation of data where the individual values are represented as colors, was drawn [Figure 5]. Heat maps are 2D displays of the measured experimental values in the data matrix. The relatively high abundance of any specific metabolite is represented by yellow-colored squares (pixels) and a low abundance is represented by orange-colored squares.
Details of the 12 metabolites best describing the variation between patients with active TB and the controls
*Molecular weight (m/z) is denoted by its monoisotopic mass, †The chemical formulas were predicted based on accurate mass by using the molecular formula generator algorithm of Mass Frontier 6.0 software (Thermo Fisher Scientific), ‡The trend of marker levels in the active TB group. ↑ and ↓ indicate increased and decreased levels, respectively, compared with the healthy group. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine.
Heat map based on the differences in the abundance of small metabolic compounds among sera from the tuberculosis (TB) group, healthy group and non-TB group. Yellow color indicates higher abundance of metabolites; Orange color indicates lower abundance of metabolites.
Relative abundances of metabolites in tuberculosis patients (T), healthy controls (O) and nontuberculosis patients (N). The metabolites changed in relative abundance between groups were: Palmitic acid, LysoPC (16:0), LysoPC (18:0), phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, quinolinic acid, presqualene diphosphate, LysoPC (P-18:1 (9Z)), and LysoPC (P-16:0). Black lines indicates sample means. Asterisks indicate significant differences between 2 groups (results from t-tests corrected for multiple testing; *P < 0.05; **P < 0.01).
The results showed that the three groups were significantly different in terms of the abundances of these biomarker metabolites in the patient sera.
The successful use of UPLC-MS metabolomics analysis and the OPLS-DA model described above to distinguish between the TB group and control groups led us to search for the specific metabolites that contributed to the metabolomic differences. Based on the OPLS-DA model, the signals that were highly correlated and had high signal-to-noise ratio values were selected. The metabolites of >400 small molecules in the sera of patients in the three groups were explored. The molecules responsible for these signals were identified and differences in the abundance of these small molecules were determined by applying Mann–Whitney U-test for each of the three possible comparisons, using a P value threshold of 0.05. Twenty-seven metabolites were detected at significantly different levels between the active TB group and the control groups. Of those metabolites identified to differ significantly between groups, 12 metabolites [Table 2 and Figure 6], were clustered in the fatty acid, phospholipids, amino acids, and terpenoid compounds metabolite sets. To confirm that the three groups differed in terms of the serum levels of these metabolites, a heat map, a graphical representation of data where the individual values are represented as colors, was drawn [Figure 5]. Heat maps are 2D displays of the measured experimental values in the data matrix. The relatively high abundance of any specific metabolite is represented by yellow-colored squares (pixels) and a low abundance is represented by orange-colored squares.
Details of the 12 metabolites best describing the variation between patients with active TB and the controls
*Molecular weight (m/z) is denoted by its monoisotopic mass, †The chemical formulas were predicted based on accurate mass by using the molecular formula generator algorithm of Mass Frontier 6.0 software (Thermo Fisher Scientific), ‡The trend of marker levels in the active TB group. ↑ and ↓ indicate increased and decreased levels, respectively, compared with the healthy group. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine.
Heat map based on the differences in the abundance of small metabolic compounds among sera from the tuberculosis (TB) group, healthy group and non-TB group. Yellow color indicates higher abundance of metabolites; Orange color indicates lower abundance of metabolites.
Relative abundances of metabolites in tuberculosis patients (T), healthy controls (O) and nontuberculosis patients (N). The metabolites changed in relative abundance between groups were: Palmitic acid, LysoPC (16:0), LysoPC (18:0), phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, quinolinic acid, presqualene diphosphate, LysoPC (P-18:1 (9Z)), and LysoPC (P-16:0). Black lines indicates sample means. Asterisks indicate significant differences between 2 groups (results from t-tests corrected for multiple testing; *P < 0.05; **P < 0.01).
The results showed that the three groups were significantly different in terms of the abundances of these biomarker metabolites in the patient sera.
Differences in small metabolites can be used as specific and sensitive biosignatures of tuberculosis status To investigate whether the characteristics of the metabolites that significantly differed among the three groups could be efficiently exploited for building a sensitive biosignature of TB status, ROC curves, which have been conventionally used to evaluate diagnostic performance in clinical research, were calculated. In the specific metabolites that were decreased in active TB patients, the ROC curves of 3D, 7D, 11D-phytanic acid, behenic acid and threoninyl-γ-glutamate exhibited excellent efficiency with a AUC values of 0.904 (95% CI: 0863–0.944), 0.93 (95% CI: 0.893–0.966) and 0.964 (95% CI: 00.941–0.988), respectively [Table 3 and Figure 7a]. Kynurenine, quinolinic acid (QUIN), and presqualene diphosphate (PSDP) showed significant up-regulation in patients with active TB (P < 0.05). The AUC values for these metabolites were more than 0.8 [Table 3 and Figure 7b], showing moderate performance of diagnostic value. The largest and smallest resulting AUC values were 0.964 and 0.720 [Table 3, Figure 7a and b], which indicated that these biomarkers may potentially be involved in the disease mechanisms. Using multiple logistic regression analysis of these 12 metabolites, the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PSDP was used to represent a suitable biomarker group that allowed efficient differentiation of active TB from the controls. The resulting ROC curve of the biomarker combination had an AUC value of 0.991 (95% CI: 0.982–1.000) [Table 3 and Figure 7c], which reflects strong significant difference between active TB and the control patient groups.
The ROC curves of the 12 metabolites found to represent potential biomarkers of active TB
*Decreased metabolites, †Increased metabolites, ‡The biomarker combination, §Under the nonparametric assumption, ||Null hypothesis: True area = 0.5. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; ROC: Receiver operating characteristic.
(a) The receiver operating characteristic (ROC) curve of metabolites that were decreased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly decreased in the active TB group compared with controls. The ROC curves of each metabolite that was decreased in concentration in the TB group sera showed a moderate distinguishing efficiency. (b) The receiver operating characteristic (ROC) curve of metabolites that were increased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly increased in the active TB group compared with controls. The ROC curves of each metabolite that was increased in concentration in the TB group sera showed a moderate distinguishing efficiency. (c) The receiver operating characteristic (ROC) curve of the biomarker combination identified as a putative serum signature of active tuberculosis (TB). The ROC curve of the combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate showed excellent distinguishing efficiency between patients with active TB and controls.
To investigate whether the characteristics of the metabolites that significantly differed among the three groups could be efficiently exploited for building a sensitive biosignature of TB status, ROC curves, which have been conventionally used to evaluate diagnostic performance in clinical research, were calculated. In the specific metabolites that were decreased in active TB patients, the ROC curves of 3D, 7D, 11D-phytanic acid, behenic acid and threoninyl-γ-glutamate exhibited excellent efficiency with a AUC values of 0.904 (95% CI: 0863–0.944), 0.93 (95% CI: 0.893–0.966) and 0.964 (95% CI: 00.941–0.988), respectively [Table 3 and Figure 7a]. Kynurenine, quinolinic acid (QUIN), and presqualene diphosphate (PSDP) showed significant up-regulation in patients with active TB (P < 0.05). The AUC values for these metabolites were more than 0.8 [Table 3 and Figure 7b], showing moderate performance of diagnostic value. The largest and smallest resulting AUC values were 0.964 and 0.720 [Table 3, Figure 7a and b], which indicated that these biomarkers may potentially be involved in the disease mechanisms. Using multiple logistic regression analysis of these 12 metabolites, the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PSDP was used to represent a suitable biomarker group that allowed efficient differentiation of active TB from the controls. The resulting ROC curve of the biomarker combination had an AUC value of 0.991 (95% CI: 0.982–1.000) [Table 3 and Figure 7c], which reflects strong significant difference between active TB and the control patient groups.
The ROC curves of the 12 metabolites found to represent potential biomarkers of active TB
*Decreased metabolites, †Increased metabolites, ‡The biomarker combination, §Under the nonparametric assumption, ||Null hypothesis: True area = 0.5. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; ROC: Receiver operating characteristic.
(a) The receiver operating characteristic (ROC) curve of metabolites that were decreased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly decreased in the active TB group compared with controls. The ROC curves of each metabolite that was decreased in concentration in the TB group sera showed a moderate distinguishing efficiency. (b) The receiver operating characteristic (ROC) curve of metabolites that were increased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly increased in the active TB group compared with controls. The ROC curves of each metabolite that was increased in concentration in the TB group sera showed a moderate distinguishing efficiency. (c) The receiver operating characteristic (ROC) curve of the biomarker combination identified as a putative serum signature of active tuberculosis (TB). The ROC curve of the combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate showed excellent distinguishing efficiency between patients with active TB and controls.
Comparison between patients with active tuberculosis and nontuberculosis disease subgroups based on serum metabolic profiling To determine whether our metabolomic approach could be used to make a distinction between the active TB and non-TB groups, Mann–Whitney U-test was applied for comparison between the active TB group and the subgroups of non-TB. The findings indicated that the UPLC-MS analysis of serum may help in the diagnosis of TB and non-TB as remarked above. Finally, the specific markers between active TB and the subgroups were explored as below [Table 4]:
P values for comparisons between the active TB group and each subgroup of the Non-TB group
*P < 0.05, †P < 0.01. Application Mann-whitney U-test in TB group and each subgroup. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; COPD: Chronic obstructive pulmonary disease; LC: Liquid chromatography.
The serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroupsLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).
The serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)
Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)
Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)
The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroups
LysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).
To determine whether our metabolomic approach could be used to make a distinction between the active TB and non-TB groups, Mann–Whitney U-test was applied for comparison between the active TB group and the subgroups of non-TB. The findings indicated that the UPLC-MS analysis of serum may help in the diagnosis of TB and non-TB as remarked above. Finally, the specific markers between active TB and the subgroups were explored as below [Table 4]:
P values for comparisons between the active TB group and each subgroup of the Non-TB group
*P < 0.05, †P < 0.01. Application Mann-whitney U-test in TB group and each subgroup. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; COPD: Chronic obstructive pulmonary disease; LC: Liquid chromatography.
The serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroupsLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).
The serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)
Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)
Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)
The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroups
LysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).
Baseline characteristics:
Table 1 lists the baseline characteristics of enrolled patients and the controls. The active TB patients were 48.32 ± 18.15 (mean ± standard deviation) years old on average and consisted of 65 males and 55 females. The healthy controls were 42.71 ± 15.31 years old and consisted of 56 males and 49 females. The non-TB controls were 50.35 ± 17.41 years old and consisted of 85 males and 61 females. Of the 146 patients with non-TB controls, 51 patients (60.1%) had lung cancer, comprising the cancer subgroup (L); 45 patients (39.9%) made up the pneumonia subgroup (P); 28 patients were in the chronic obstructive pulmonary disease (COPD) subgroup (C); and 22 patients comprised the bronchiectasis subgroup (B).
Ultra performance liquid chromatography-mass spectrometry platform performance:
Representative UPLC-MS chromatograms of the serum samples of the patients with active and the subjects with the healthy and non-TB controls were shown in Figure 2. The peaks were very well resolved and were evenly dispersed across the entire retention time domain, showing the high quality of the raw data. There were many significant differences in the areas and heights of peaks among groups, as a matter of fact, it is inferred that differences in peaks resulted from metabolic derangements along with diseases.
High performance liquid chromatography-mass spectrometry chromatograms of the serum samples of patients from the tuberculosis (TB) group, the healthy group, and the four subgroups of patients with non-TB diseases. Abscissa: Retention time. Ordinate: Relative abundance. The numbers on the point of the peaks indicate the retention time of each substance. T indicates TB group; N indicates healthy control; P indicates pulmonitis subgroup; L indicates lung cancer subgroup; C indicates chronic obstructive pulmonary disease subgroup; B indicates bronchiectasis subgroup.
Differentiation between tuberculosis patients and controls, and among the subgroups based on OPLS-DA:
As mentioned above, the overall peak profiles of the three groups looked quite different, which suggested that these profiles could be used to discriminate TB from the controls. For the holistic treatment of these data, multivariate analysis was used to identify the metabolomic differences between the groups. For data reduction and pattern recognition (PR), a series of PR methods were applied using SIMCA-P 12.1 software (Umetrics, Sweden). Principal component analysis (PCA) was initially applied to the data to visualize inherent clustering between the three groups. PCA involves the transformation of a multidimensional set of possibly correlated variables into two linearly uncorrelated dimensions. This model explained an estimated 41.4% of the original data (R2 = 0.414 and Q2 = 0.334) [Figure 3]. However, in addition to the effects of the diseases on the metabolome, there were other factors that are known to contribute to differences of endogenous metabolites, such as age and diet.
Principal component analysis (PCA) model. PCA scores plots of the SIMCA-P + 12.0.1.0 generated data, showing tuberculosis (TB) patients versus healthy controls and the non-TB group collected serum samples before the removal of ‘noise’ and interfering compounds from the dataset. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.
Therefore, orthogonal signal correction technology was used to filter out unrelated variable information and retain related variables. Hence, OPLS-DA model was introduced, which is used to determine the maximum separation between different kinds of samples according to the sample classification information. The OPLS-DA model was used to identify biomarkers that accounted for the differences between the three groups, and it clearly distinguished between the TB group and the two control groups (non-TB disease subgroups were combined) [Figure 4]. The results showed that 82.1% of samples were consistent with the discrimination of the model, and the predictive ability of the model was 58.2% (Q2Y = 0.582). These findings indicate that the OPLS-DA model may pave the way for the diagnosis of TB and permit differentiation between other kinds of related diseases.
(a) OPLS-DA two-dimensional model. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup. (b) OPLS-DA three-dimensional model. OPLS-DA scores plot discriminating serum samples of tuberculosis (TB) patients, healthy control and non-TB group based on the metabolite profiling data. : TB group; : Healthy control; : Pulmonitis subgroup; : Lung cancer subgroup; : COPD subgroup; : Bronchiectasis subgroup.
Identification of tuberculosis-specific metabolites:
The successful use of UPLC-MS metabolomics analysis and the OPLS-DA model described above to distinguish between the TB group and control groups led us to search for the specific metabolites that contributed to the metabolomic differences. Based on the OPLS-DA model, the signals that were highly correlated and had high signal-to-noise ratio values were selected. The metabolites of >400 small molecules in the sera of patients in the three groups were explored. The molecules responsible for these signals were identified and differences in the abundance of these small molecules were determined by applying Mann–Whitney U-test for each of the three possible comparisons, using a P value threshold of 0.05. Twenty-seven metabolites were detected at significantly different levels between the active TB group and the control groups. Of those metabolites identified to differ significantly between groups, 12 metabolites [Table 2 and Figure 6], were clustered in the fatty acid, phospholipids, amino acids, and terpenoid compounds metabolite sets. To confirm that the three groups differed in terms of the serum levels of these metabolites, a heat map, a graphical representation of data where the individual values are represented as colors, was drawn [Figure 5]. Heat maps are 2D displays of the measured experimental values in the data matrix. The relatively high abundance of any specific metabolite is represented by yellow-colored squares (pixels) and a low abundance is represented by orange-colored squares.
Details of the 12 metabolites best describing the variation between patients with active TB and the controls
*Molecular weight (m/z) is denoted by its monoisotopic mass, †The chemical formulas were predicted based on accurate mass by using the molecular formula generator algorithm of Mass Frontier 6.0 software (Thermo Fisher Scientific), ‡The trend of marker levels in the active TB group. ↑ and ↓ indicate increased and decreased levels, respectively, compared with the healthy group. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine.
Heat map based on the differences in the abundance of small metabolic compounds among sera from the tuberculosis (TB) group, healthy group and non-TB group. Yellow color indicates higher abundance of metabolites; Orange color indicates lower abundance of metabolites.
Relative abundances of metabolites in tuberculosis patients (T), healthy controls (O) and nontuberculosis patients (N). The metabolites changed in relative abundance between groups were: Palmitic acid, LysoPC (16:0), LysoPC (18:0), phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, quinolinic acid, presqualene diphosphate, LysoPC (P-18:1 (9Z)), and LysoPC (P-16:0). Black lines indicates sample means. Asterisks indicate significant differences between 2 groups (results from t-tests corrected for multiple testing; *P < 0.05; **P < 0.01).
The results showed that the three groups were significantly different in terms of the abundances of these biomarker metabolites in the patient sera.
Differences in small metabolites can be used as specific and sensitive biosignatures of tuberculosis status:
To investigate whether the characteristics of the metabolites that significantly differed among the three groups could be efficiently exploited for building a sensitive biosignature of TB status, ROC curves, which have been conventionally used to evaluate diagnostic performance in clinical research, were calculated. In the specific metabolites that were decreased in active TB patients, the ROC curves of 3D, 7D, 11D-phytanic acid, behenic acid and threoninyl-γ-glutamate exhibited excellent efficiency with a AUC values of 0.904 (95% CI: 0863–0.944), 0.93 (95% CI: 0.893–0.966) and 0.964 (95% CI: 00.941–0.988), respectively [Table 3 and Figure 7a]. Kynurenine, quinolinic acid (QUIN), and presqualene diphosphate (PSDP) showed significant up-regulation in patients with active TB (P < 0.05). The AUC values for these metabolites were more than 0.8 [Table 3 and Figure 7b], showing moderate performance of diagnostic value. The largest and smallest resulting AUC values were 0.964 and 0.720 [Table 3, Figure 7a and b], which indicated that these biomarkers may potentially be involved in the disease mechanisms. Using multiple logistic regression analysis of these 12 metabolites, the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PSDP was used to represent a suitable biomarker group that allowed efficient differentiation of active TB from the controls. The resulting ROC curve of the biomarker combination had an AUC value of 0.991 (95% CI: 0.982–1.000) [Table 3 and Figure 7c], which reflects strong significant difference between active TB and the control patient groups.
The ROC curves of the 12 metabolites found to represent potential biomarkers of active TB
*Decreased metabolites, †Increased metabolites, ‡The biomarker combination, §Under the nonparametric assumption, ||Null hypothesis: True area = 0.5. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; ROC: Receiver operating characteristic.
(a) The receiver operating characteristic (ROC) curve of metabolites that were decreased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly decreased in the active TB group compared with controls. The ROC curves of each metabolite that was decreased in concentration in the TB group sera showed a moderate distinguishing efficiency. (b) The receiver operating characteristic (ROC) curve of metabolites that were increased in the active tuberculosis (TB) group compared with controls. ROC curves of metabolites for which the serum concentrations were significantly increased in the active TB group compared with controls. The ROC curves of each metabolite that was increased in concentration in the TB group sera showed a moderate distinguishing efficiency. (c) The receiver operating characteristic (ROC) curve of the biomarker combination identified as a putative serum signature of active tuberculosis (TB). The ROC curve of the combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate showed excellent distinguishing efficiency between patients with active TB and controls.
Comparison between patients with active tuberculosis and nontuberculosis disease subgroups based on serum metabolic profiling:
To determine whether our metabolomic approach could be used to make a distinction between the active TB and non-TB groups, Mann–Whitney U-test was applied for comparison between the active TB group and the subgroups of non-TB. The findings indicated that the UPLC-MS analysis of serum may help in the diagnosis of TB and non-TB as remarked above. Finally, the specific markers between active TB and the subgroups were explored as below [Table 4]:
P values for comparisons between the active TB group and each subgroup of the Non-TB group
*P < 0.05, †P < 0.01. Application Mann-whitney U-test in TB group and each subgroup. TB: Tuberculosis; LysoPC: Lysophosphatidylcholine; COPD: Chronic obstructive pulmonary disease; LC: Liquid chromatography.
The serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroupsLysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).
The serum levels of PSDP, lysoPCs, 3D, 7D, 11D-phytanic acid, behenic acid, hypoglycin B, phytal and threoninyl-γ-glutamate were significantly different between the active TB and lung cancer subgroups (Π < 0.05)
Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, lysoPC (P-16:0), and kynurenine were more effective in discriminating active TB from bronchiectasis (Π < 0.05)
Palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, behenic acid, phytal, threoninyl-γ-glutamate, kynurenine, and lysoPC (P-16:0) were expected to become more effective in diagnosis and differentiation of active TB from COPD (Π < 0.05)
The serum levels of palmitic acid, lysoPC (16:0), 3D, 7D, 11D-phytanic acid, and kynurenine were significantly different between the active TB and pneumonia subgroups
LysoPC (16:0) was the only single metabolite to significantly distinguish active TB from the diseases other than TB (Π < 0.05).
DISCUSSION:
The present study used UPLC-MS to profile active TB and build a statistical model that enabled the identification of biomarkers for disease diagnosis based on metabolomic research. Findings indicated that 12 metabolites were unambiguously altered in serum of active TB patients as compared with two other groups (healthy controls and non-TB disease patients) [Table 2]. In the following discussion, we consider the biological relevance of these prominent metabolites and their potential biosignatures for the diagnosis of active TB.
Fatty acids It is universally acknowledged that Mtb preferentially relies on fatty acid metabolism to maintain chronic infection.[26] When there is persistent infection in the lung tissue, the fatty acids, which are metabolized in two ways (in β-oxidation decomposition and the glyoxylate cycle) may be a source of carbon and energy of Mtb.[27] In this study, palmitic acid, phytanic acid and behenic acid, which decreased significantly in the sera of TB patients compared with the healthy group, bronchiectasis, and COPD subgroups, may be among the products of the fatty acid consumption. In addition, palmitic acid, as the most abundant free fatty acid in the human body, can induce inhibition of the electron transport chain of macrophage, which, in turn, reduces adenosine triphosphate production in the mitochondria and stimulates the activity of the mitochondrial apoptotic pathway. Another study that further supports our findings was that the palmitic acid, as one of the active compounds from the plant extraction, has some toxicity toward mycobacteria, but it is not highly toxic.[28]
Phytanic acid, a 20-carbon branched chain fatty acid, can be used as the substrate of CYP124A1,[29] which is a heme-containing enzyme that belongs to the cytochrome p450 superfamily. When CYP124A1 was combined with the substrate of phytanic acid, the molecular conformation of CYP124A1 changed in favor of lipid oxidation, to play a better role in the important biological functions of Mtb.[30] Meanwhile, phytanic acid, as a substrate of CYP124A1, was consumed.
In our study, behenic acid was detected in a lower concentration in the TB group than the healthy and non-TB groups, except for the pneumonia subgroup. Behenic acid has been identified as a fatty acid that increases cholesterol in humans.[31] Cholesterol has a significant role in the development of TB, because it is necessary for the good functioning of macrophages and lymphocytes.[32] In the macrophage cell membrane, cholesterol directly participates in the function of phagocytizing Mtb, while, in the lymphocyte membrane, it is involved in the differentiation and proliferation of cytotoxic cells. Apart from this, cholesterol also has the functions of activating lymphocytes, CD4+ T cells, CD8+ T cells, and γ-T cells, and promoting the release of interferon and tumor necrosis factor-α, all of which effects are effective in killing Mtb. A previous study showed that TB patients universally had a lowered concentration of total serum cholesterol.[33] Due to this, the decreased concentration of behenic acid, as a cholesterol-raising fatty acid, in sera of TB patients may further explain the hypocholesteremia status in TB.[34]
It is universally acknowledged that Mtb preferentially relies on fatty acid metabolism to maintain chronic infection.[26] When there is persistent infection in the lung tissue, the fatty acids, which are metabolized in two ways (in β-oxidation decomposition and the glyoxylate cycle) may be a source of carbon and energy of Mtb.[27] In this study, palmitic acid, phytanic acid and behenic acid, which decreased significantly in the sera of TB patients compared with the healthy group, bronchiectasis, and COPD subgroups, may be among the products of the fatty acid consumption. In addition, palmitic acid, as the most abundant free fatty acid in the human body, can induce inhibition of the electron transport chain of macrophage, which, in turn, reduces adenosine triphosphate production in the mitochondria and stimulates the activity of the mitochondrial apoptotic pathway. Another study that further supports our findings was that the palmitic acid, as one of the active compounds from the plant extraction, has some toxicity toward mycobacteria, but it is not highly toxic.[28]
Phytanic acid, a 20-carbon branched chain fatty acid, can be used as the substrate of CYP124A1,[29] which is a heme-containing enzyme that belongs to the cytochrome p450 superfamily. When CYP124A1 was combined with the substrate of phytanic acid, the molecular conformation of CYP124A1 changed in favor of lipid oxidation, to play a better role in the important biological functions of Mtb.[30] Meanwhile, phytanic acid, as a substrate of CYP124A1, was consumed.
In our study, behenic acid was detected in a lower concentration in the TB group than the healthy and non-TB groups, except for the pneumonia subgroup. Behenic acid has been identified as a fatty acid that increases cholesterol in humans.[31] Cholesterol has a significant role in the development of TB, because it is necessary for the good functioning of macrophages and lymphocytes.[32] In the macrophage cell membrane, cholesterol directly participates in the function of phagocytizing Mtb, while, in the lymphocyte membrane, it is involved in the differentiation and proliferation of cytotoxic cells. Apart from this, cholesterol also has the functions of activating lymphocytes, CD4+ T cells, CD8+ T cells, and γ-T cells, and promoting the release of interferon and tumor necrosis factor-α, all of which effects are effective in killing Mtb. A previous study showed that TB patients universally had a lowered concentration of total serum cholesterol.[33] Due to this, the decreased concentration of behenic acid, as a cholesterol-raising fatty acid, in sera of TB patients may further explain the hypocholesteremia status in TB.[34]
Lysophosphatidylcholines Lysophosphatidylcholine (P-18:1(9Z)), lysoPC (P-16:0), lysoPC (16:0), and lysoPC (18:0) formed one cluster of metabolites that were present at lower levels in the active TB group and significantly differed between the healthy and non-TB groups. LysoPCs impair nitric oxide (NO) production and endothelium-dependent vasorelaxation. Moreover, lysoPC levels have been shown to be negatively correlated with NO levels.[36] Other research has pointed out that the generation of NO by activated macrophages could control mycobacterial infection in the murine system.[35] A low lysoPC level may accelerate Mtb infection by reducing the generation of NO. Additionally, another finding that TB can induce macrophage apoptosis by inhibition of phospholipase A2[373839] could also explain the low concentrations of lysoPCs in patients with active TB, a finding that is consistent with the previous report by Weiner et al.[24]
Lysophosphatidylcholine (P-18:1(9Z)), lysoPC (P-16:0), lysoPC (16:0), and lysoPC (18:0) formed one cluster of metabolites that were present at lower levels in the active TB group and significantly differed between the healthy and non-TB groups. LysoPCs impair nitric oxide (NO) production and endothelium-dependent vasorelaxation. Moreover, lysoPC levels have been shown to be negatively correlated with NO levels.[36] Other research has pointed out that the generation of NO by activated macrophages could control mycobacterial infection in the murine system.[35] A low lysoPC level may accelerate Mtb infection by reducing the generation of NO. Additionally, another finding that TB can induce macrophage apoptosis by inhibition of phospholipase A2[373839] could also explain the low concentrations of lysoPCs in patients with active TB, a finding that is consistent with the previous report by Weiner et al.[24]
Amino acids Our data showed an increase in the levels of two amino acids (kynurenine and QUIN), and a decrease in threoninyl-γ-glutamate in sera from TB patients relative to those of healthy controls, suggesting alterations in protein metabolism during active TB. Amino acid metabolism is complicated, involving a large number of metabolites. Gluconeogenesis, proteolysis, and oxidative catabolism contribute to amino acid balance. Tryptophan (TRP) is an essential amino acid that has various important biological functions. Kynurenine, as the downstream metabolite of TRP, can be accumulated by enhancing the activity of indoleamine 2, 3-dioxygenase-1 (IDO-1), which is widely known as the rate-limiting enzyme in TRP metabolism.[40] In addition, kynurenine can regulate human T cells, which are well known as the main anti-TB immunological cell type.
Quinolinic acid, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines. These findings support the concept that the TRP-kynurenine pathway can influence immune functions through the effects of TRP depletion and the accumulation of kynurenine and QUIN, suggesting that the TRP degradation pathway controlled by IDO-1 is involved in the pathogenesis of pulmonary TB.[41] QUIN, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines.[42] In addition, imbalances in TRP metabolism have been linked to cancer-related immune escape and implicated in lung cancer.[43]
Threoninyl-γ-glutamate exhibited the most excellent efficiency with an AUC value of 0.964 (95% CI: 00.941–0.988) [Table 3 and Figure 7a], is a dipeptide composed of threonine and γ-glutamate. Some dipeptides are known to have physiological or cell-signaling effects, although most are simply short-lived intermediates on their way to specific amino acid degradation pathways following further proteolysis. This dipeptide has not yet been identified in human tissues or biofluids; hence, it is classified as an “expected” metabolite.
Our data showed an increase in the levels of two amino acids (kynurenine and QUIN), and a decrease in threoninyl-γ-glutamate in sera from TB patients relative to those of healthy controls, suggesting alterations in protein metabolism during active TB. Amino acid metabolism is complicated, involving a large number of metabolites. Gluconeogenesis, proteolysis, and oxidative catabolism contribute to amino acid balance. Tryptophan (TRP) is an essential amino acid that has various important biological functions. Kynurenine, as the downstream metabolite of TRP, can be accumulated by enhancing the activity of indoleamine 2, 3-dioxygenase-1 (IDO-1), which is widely known as the rate-limiting enzyme in TRP metabolism.[40] In addition, kynurenine can regulate human T cells, which are well known as the main anti-TB immunological cell type.
Quinolinic acid, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines. These findings support the concept that the TRP-kynurenine pathway can influence immune functions through the effects of TRP depletion and the accumulation of kynurenine and QUIN, suggesting that the TRP degradation pathway controlled by IDO-1 is involved in the pathogenesis of pulmonary TB.[41] QUIN, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines.[42] In addition, imbalances in TRP metabolism have been linked to cancer-related immune escape and implicated in lung cancer.[43]
Threoninyl-γ-glutamate exhibited the most excellent efficiency with an AUC value of 0.964 (95% CI: 00.941–0.988) [Table 3 and Figure 7a], is a dipeptide composed of threonine and γ-glutamate. Some dipeptides are known to have physiological or cell-signaling effects, although most are simply short-lived intermediates on their way to specific amino acid degradation pathways following further proteolysis. This dipeptide has not yet been identified in human tissues or biofluids; hence, it is classified as an “expected” metabolite.
Terpenoid compounds One of the most prominent clusters of metabolites in our study represented terpenoid compounds, including PSDP and phytal. The latter compound, which to date has never been identified or associated with TB, belongs to the family of diterpenes. However, it is reported that elisapterosin B, one kind of diterpenes extracted from plants, could inhibit the growth of Mtb.[44] Thus, the relationship between phytal and disease as well as its involvement in disease mechanisms is open to further study.
Presqualene diphosphate, an intermediate in the biosynthesis of terpenoids, was found at higher levels in the active TB group and significantly differed between the active TB and cancer subgroups by an order of magnitude. PSDP exerts an intercellular signal for the down-regulation of superoxide in neutrophils,[45] which is widely known to play a central role in host defense, inflammation, and tissue damage.[46] PSDP, an endogenous PI3K inhibitor, directly inhibited recombinant human p110-pI3K activity, which can activate polymorphonuclear neutrophils (PMN). PMN is the primary initial immune effectors of acute inflammation, and potentially PMN products released into surrounding tissues contribute to lung and respiratory injury. A negative correlation between PSDP and PMN was observed. The observed increase in the serum concentration of PSDP may be an indicator of TB inflammatory progression. PSDP, after the initial acute inflammatory state, may act to limit tissue injury by inhibiting phospholipase D, phosphoinositol-3 kinase, and superoxide anion generation, thus providing protection for the host tissues. In addition, PSDP, a well-recognized intermediate of cholesterol biosynthesis, exists in immune effector cells and is a potential regulator of the cellular response in host defense. The increased PSDP in TB patients may be a response to Mtb in host defense, which could also explain the result of this study.
One of the most prominent clusters of metabolites in our study represented terpenoid compounds, including PSDP and phytal. The latter compound, which to date has never been identified or associated with TB, belongs to the family of diterpenes. However, it is reported that elisapterosin B, one kind of diterpenes extracted from plants, could inhibit the growth of Mtb.[44] Thus, the relationship between phytal and disease as well as its involvement in disease mechanisms is open to further study.
Presqualene diphosphate, an intermediate in the biosynthesis of terpenoids, was found at higher levels in the active TB group and significantly differed between the active TB and cancer subgroups by an order of magnitude. PSDP exerts an intercellular signal for the down-regulation of superoxide in neutrophils,[45] which is widely known to play a central role in host defense, inflammation, and tissue damage.[46] PSDP, an endogenous PI3K inhibitor, directly inhibited recombinant human p110-pI3K activity, which can activate polymorphonuclear neutrophils (PMN). PMN is the primary initial immune effectors of acute inflammation, and potentially PMN products released into surrounding tissues contribute to lung and respiratory injury. A negative correlation between PSDP and PMN was observed. The observed increase in the serum concentration of PSDP may be an indicator of TB inflammatory progression. PSDP, after the initial acute inflammatory state, may act to limit tissue injury by inhibiting phospholipase D, phosphoinositol-3 kinase, and superoxide anion generation, thus providing protection for the host tissues. In addition, PSDP, a well-recognized intermediate of cholesterol biosynthesis, exists in immune effector cells and is a potential regulator of the cellular response in host defense. The increased PSDP in TB patients may be a response to Mtb in host defense, which could also explain the result of this study.
Fatty acids:
It is universally acknowledged that Mtb preferentially relies on fatty acid metabolism to maintain chronic infection.[26] When there is persistent infection in the lung tissue, the fatty acids, which are metabolized in two ways (in β-oxidation decomposition and the glyoxylate cycle) may be a source of carbon and energy of Mtb.[27] In this study, palmitic acid, phytanic acid and behenic acid, which decreased significantly in the sera of TB patients compared with the healthy group, bronchiectasis, and COPD subgroups, may be among the products of the fatty acid consumption. In addition, palmitic acid, as the most abundant free fatty acid in the human body, can induce inhibition of the electron transport chain of macrophage, which, in turn, reduces adenosine triphosphate production in the mitochondria and stimulates the activity of the mitochondrial apoptotic pathway. Another study that further supports our findings was that the palmitic acid, as one of the active compounds from the plant extraction, has some toxicity toward mycobacteria, but it is not highly toxic.[28]
Phytanic acid, a 20-carbon branched chain fatty acid, can be used as the substrate of CYP124A1,[29] which is a heme-containing enzyme that belongs to the cytochrome p450 superfamily. When CYP124A1 was combined with the substrate of phytanic acid, the molecular conformation of CYP124A1 changed in favor of lipid oxidation, to play a better role in the important biological functions of Mtb.[30] Meanwhile, phytanic acid, as a substrate of CYP124A1, was consumed.
In our study, behenic acid was detected in a lower concentration in the TB group than the healthy and non-TB groups, except for the pneumonia subgroup. Behenic acid has been identified as a fatty acid that increases cholesterol in humans.[31] Cholesterol has a significant role in the development of TB, because it is necessary for the good functioning of macrophages and lymphocytes.[32] In the macrophage cell membrane, cholesterol directly participates in the function of phagocytizing Mtb, while, in the lymphocyte membrane, it is involved in the differentiation and proliferation of cytotoxic cells. Apart from this, cholesterol also has the functions of activating lymphocytes, CD4+ T cells, CD8+ T cells, and γ-T cells, and promoting the release of interferon and tumor necrosis factor-α, all of which effects are effective in killing Mtb. A previous study showed that TB patients universally had a lowered concentration of total serum cholesterol.[33] Due to this, the decreased concentration of behenic acid, as a cholesterol-raising fatty acid, in sera of TB patients may further explain the hypocholesteremia status in TB.[34]
Lysophosphatidylcholines:
Lysophosphatidylcholine (P-18:1(9Z)), lysoPC (P-16:0), lysoPC (16:0), and lysoPC (18:0) formed one cluster of metabolites that were present at lower levels in the active TB group and significantly differed between the healthy and non-TB groups. LysoPCs impair nitric oxide (NO) production and endothelium-dependent vasorelaxation. Moreover, lysoPC levels have been shown to be negatively correlated with NO levels.[36] Other research has pointed out that the generation of NO by activated macrophages could control mycobacterial infection in the murine system.[35] A low lysoPC level may accelerate Mtb infection by reducing the generation of NO. Additionally, another finding that TB can induce macrophage apoptosis by inhibition of phospholipase A2[373839] could also explain the low concentrations of lysoPCs in patients with active TB, a finding that is consistent with the previous report by Weiner et al.[24]
Amino acids:
Our data showed an increase in the levels of two amino acids (kynurenine and QUIN), and a decrease in threoninyl-γ-glutamate in sera from TB patients relative to those of healthy controls, suggesting alterations in protein metabolism during active TB. Amino acid metabolism is complicated, involving a large number of metabolites. Gluconeogenesis, proteolysis, and oxidative catabolism contribute to amino acid balance. Tryptophan (TRP) is an essential amino acid that has various important biological functions. Kynurenine, as the downstream metabolite of TRP, can be accumulated by enhancing the activity of indoleamine 2, 3-dioxygenase-1 (IDO-1), which is widely known as the rate-limiting enzyme in TRP metabolism.[40] In addition, kynurenine can regulate human T cells, which are well known as the main anti-TB immunological cell type.
Quinolinic acid, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines. These findings support the concept that the TRP-kynurenine pathway can influence immune functions through the effects of TRP depletion and the accumulation of kynurenine and QUIN, suggesting that the TRP degradation pathway controlled by IDO-1 is involved in the pathogenesis of pulmonary TB.[41] QUIN, a neuroactive metabolite of the kynurenine pathway, can induce the expression of several proinflammatory cytokines and chemokines.[42] In addition, imbalances in TRP metabolism have been linked to cancer-related immune escape and implicated in lung cancer.[43]
Threoninyl-γ-glutamate exhibited the most excellent efficiency with an AUC value of 0.964 (95% CI: 00.941–0.988) [Table 3 and Figure 7a], is a dipeptide composed of threonine and γ-glutamate. Some dipeptides are known to have physiological or cell-signaling effects, although most are simply short-lived intermediates on their way to specific amino acid degradation pathways following further proteolysis. This dipeptide has not yet been identified in human tissues or biofluids; hence, it is classified as an “expected” metabolite.
Terpenoid compounds:
One of the most prominent clusters of metabolites in our study represented terpenoid compounds, including PSDP and phytal. The latter compound, which to date has never been identified or associated with TB, belongs to the family of diterpenes. However, it is reported that elisapterosin B, one kind of diterpenes extracted from plants, could inhibit the growth of Mtb.[44] Thus, the relationship between phytal and disease as well as its involvement in disease mechanisms is open to further study.
Presqualene diphosphate, an intermediate in the biosynthesis of terpenoids, was found at higher levels in the active TB group and significantly differed between the active TB and cancer subgroups by an order of magnitude. PSDP exerts an intercellular signal for the down-regulation of superoxide in neutrophils,[45] which is widely known to play a central role in host defense, inflammation, and tissue damage.[46] PSDP, an endogenous PI3K inhibitor, directly inhibited recombinant human p110-pI3K activity, which can activate polymorphonuclear neutrophils (PMN). PMN is the primary initial immune effectors of acute inflammation, and potentially PMN products released into surrounding tissues contribute to lung and respiratory injury. A negative correlation between PSDP and PMN was observed. The observed increase in the serum concentration of PSDP may be an indicator of TB inflammatory progression. PSDP, after the initial acute inflammatory state, may act to limit tissue injury by inhibiting phospholipase D, phosphoinositol-3 kinase, and superoxide anion generation, thus providing protection for the host tissues. In addition, PSDP, a well-recognized intermediate of cholesterol biosynthesis, exists in immune effector cells and is a potential regulator of the cellular response in host defense. The increased PSDP in TB patients may be a response to Mtb in host defense, which could also explain the result of this study.
CONCLUSIONS:
Metabolic profiling approaches based on UPLC-MS were successfully used to distinguish TB patients from the controls and establish a TB-specific metabolite profiling. Twelve metabolites were identified to be significantly different among patient groups, with moderate performance of diagnostic value indicating that these biomarkers may potentially be involved in disease mechanisms. Most of the identified metabolites were mainly involved in fatty acid, amino acid, and lipid metabolism pathways, leading to a number of new hypotheses to better clarify the reasons for their differential abundance in active TB, including:
The decrease of palmitic acid, 3D, 7D, 11D-phytanic acid and behenic acid supported that the fatty acid was preferentially utilized by Mtb, and these metabolites may be one of the results of fatty acid consumptionTerpenoid compounds may have an important role in the processes of infection and host resistance to TB infectionMtb infection may produce enhanced activity of the TRP -kynurenine pathway, and kynurenine and QUIN, as the TRP downstream metabolites, may affect the immune function of patients with TBThe lower levels of lysoPCs observed in the active TB group may accelerate Mtb infection by reducing the generation of NO, which is produced by activated macrophagesMultiple logistic regression analysis was performed in the 12 metabolites identified as a signature of active TB, and the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PDSP was calculated to represent the best diagnostic value with the AUC value of 0.991 (95% CI: 0.982–1.000). Thus, this combination of metabolites may prove to be a metabolic profile that can be used for the diagnosis of TB.
The decrease of palmitic acid, 3D, 7D, 11D-phytanic acid and behenic acid supported that the fatty acid was preferentially utilized by Mtb, and these metabolites may be one of the results of fatty acid consumption
Terpenoid compounds may have an important role in the processes of infection and host resistance to TB infection
Mtb infection may produce enhanced activity of the TRP -kynurenine pathway, and kynurenine and QUIN, as the TRP downstream metabolites, may affect the immune function of patients with TB
The lower levels of lysoPCs observed in the active TB group may accelerate Mtb infection by reducing the generation of NO, which is produced by activated macrophages
Multiple logistic regression analysis was performed in the 12 metabolites identified as a signature of active TB, and the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PDSP was calculated to represent the best diagnostic value with the AUC value of 0.991 (95% CI: 0.982–1.000). Thus, this combination of metabolites may prove to be a metabolic profile that can be used for the diagnosis of TB.
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Background:
Tuberculosis (TB) is a chronic wasting inflammatory disease characterized by multisystem involvement, which can cause metabolic derangements in afflicted patients. Metabolic signatures have been exploited in the study of several diseases. However, the serum that is successfully used in TB diagnosis on the basis of metabolic profiling is not by much.
Methods:
Orthogonal partial least-squares discriminant analysis was capable of distinguishing TB patients from both healthy subjects and patients with conditions other than TB. Therefore, TB-specific metabolic profiling was established. Clusters of potential biomarkers for differentiating TB active from non-TB diseases were identified using Mann-Whitney U-test. Multiple logistic regression analysis of metabolites was calculated to determine the suitable biomarker group that allows the efficient differentiation of patients with TB active from the control subjects.
Results:
From among 271 participants, 12 metabolites were found to contribute to the distinction between the TB active group and the control groups. These metabolites were mainly involved in the metabolic pathways of the following three biomolecules: Fatty acids, amino acids, and lipids. The receiver operating characteristic curves of 3D, 7D, and 11D-phytanic acid, behenic acid, and threoninyl-γ-glutamate exhibited excellent efficiency with area under the curve (AUC) values of 0.904 (95% confidence interval [CI]: 0863-0.944), 0.93 (95% CI: 0.893-0.966), and 0.964 (95% CI: 00.941-0.988), respectively. The largest and smallest resulting AUCs were 0.964 and 0.720, indicating that these biomarkers may be involved in the disease mechanisms. The combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate was used to represent the most suitable biomarker group for the differentiation of patients with TB active from the control subjects, with an AUC value of 0.991.
Conclusions:
The metabolic analysis results identified new serum biomarkers that can distinguish TB from non-TB diseases. The metabolomics-based analysis provides specific insights into the biology of TB and may offer new avenues for TB diagnosis.
|
INTRODUCTION:
Metabolomics is the quantitative measurement of the dynamic multiparametric metabolic responses of living systems to pathophysiological stimuli or genetic modifications.[1] Metabolites can be viewed as a close recapitulation of disease phenotypes, and they may represent the ongoing pathogenesis in an organism to a greater extent than changes in gene expression.[2] Consequently, it has deepened our understanding of the biological mechanisms involved in several noninfectious diseases and provided a platform for the identification of new biomarkers.[3] Metabolic signatures have been exploited in the study of several diseases, such as Alzheimer's disease,[4] Parkinson's disease,[5] myocardial ischemia,[6] hypertension,[7] cancer,[8] and diabetes.[9101112] However, this powerful analytical tool has not been widely applied to infectious diseases for the development of diagnostic biomarkers.[131415]
Tuberculosis (TB) is a major worldwide health problem, with a global estimate of 9.4 million incidences in 2010 (range; 8.9 × 106–9.9 × 106). Of the more than 2 billion people infected with Mycobacterium tuberculosis (Mtb) globally, more than one-tenth are likely to develop active TB during their lifetime. In many regions, notably in developing countries with high TB incidences, diagnosis is neither sensitive nor specific, and an estimated 40% of TB patients fail to be correctly diagnosed.[16] In recent years, advancement in the field of metabolomics has mainly been applied to metabolic profiling about TB drug metabolism, rather than diagnosis of TB.[1718192021] However, the serum that is successfully used in TB diagnosis on the basis of metabolic profiling is not by much.[2223] A previous study by Weiner et al. using gas chromatography-mass spectrometry (MS) showed differences in metabolic profiles among the uninfected individuals, individuals with latent (inactive) TB, and patients with active TB.[24] In a recent study, nuclear magnetic resonance spectroscopy was used to characterize the metabolism of the host during Mtb infection.[25] However, relatively few methods are considered to be capable of distinguishing between active TB and diseases other than TB, such as lung cancer, pneumonia, and so forth. It can be expected that several of the observed changes are the result of a general inflammatory process rather than a specific response to TB. In this study, 120 patients with active TB and 251 controls who were either healthy or had diseases other than TB (non-TB group) were enrolled. Using ultra performance liquid chromatography-MS (UPLC-MS), we investigated the feasibility of identifying small molecule biochemical profiles in serum for gaining novel biological insights into the mechanisms underlying TB. The unique feature of this study was to employ the largest number of patients included in any study of a similar nature to date (120 with active TB and 251 controls). Multivariate statistical analysis was implemented to discriminate patients with active TB from the control subjects on the basis of their metabolic profiles. We identified 12 distinct biomarkers, including clusters that could be categorized as amino acids, fatty acids, lysophosphatidylcholine (lysoPC), and terpenoid compounds. Finally, these new metabolite markers, which can distinguish TB from non-TB diseases, led to a number of hypotheses. Our results provide specific insights into the biology of TB and may offer new avenues for TB diagnosis or therapy.
CONCLUSIONS:
Metabolic profiling approaches based on UPLC-MS were successfully used to distinguish TB patients from the controls and establish a TB-specific metabolite profiling. Twelve metabolites were identified to be significantly different among patient groups, with moderate performance of diagnostic value indicating that these biomarkers may potentially be involved in disease mechanisms. Most of the identified metabolites were mainly involved in fatty acid, amino acid, and lipid metabolism pathways, leading to a number of new hypotheses to better clarify the reasons for their differential abundance in active TB, including:
The decrease of palmitic acid, 3D, 7D, 11D-phytanic acid and behenic acid supported that the fatty acid was preferentially utilized by Mtb, and these metabolites may be one of the results of fatty acid consumptionTerpenoid compounds may have an important role in the processes of infection and host resistance to TB infectionMtb infection may produce enhanced activity of the TRP -kynurenine pathway, and kynurenine and QUIN, as the TRP downstream metabolites, may affect the immune function of patients with TBThe lower levels of lysoPCs observed in the active TB group may accelerate Mtb infection by reducing the generation of NO, which is produced by activated macrophagesMultiple logistic regression analysis was performed in the 12 metabolites identified as a signature of active TB, and the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PDSP was calculated to represent the best diagnostic value with the AUC value of 0.991 (95% CI: 0.982–1.000). Thus, this combination of metabolites may prove to be a metabolic profile that can be used for the diagnosis of TB.
The decrease of palmitic acid, 3D, 7D, 11D-phytanic acid and behenic acid supported that the fatty acid was preferentially utilized by Mtb, and these metabolites may be one of the results of fatty acid consumption
Terpenoid compounds may have an important role in the processes of infection and host resistance to TB infection
Mtb infection may produce enhanced activity of the TRP -kynurenine pathway, and kynurenine and QUIN, as the TRP downstream metabolites, may affect the immune function of patients with TB
The lower levels of lysoPCs observed in the active TB group may accelerate Mtb infection by reducing the generation of NO, which is produced by activated macrophages
Multiple logistic regression analysis was performed in the 12 metabolites identified as a signature of active TB, and the combination of lysoPC (18:0), behenic acid, threoninyl-γ-glutamate, and PDSP was calculated to represent the best diagnostic value with the AUC value of 0.991 (95% CI: 0.982–1.000). Thus, this combination of metabolites may prove to be a metabolic profile that can be used for the diagnosis of TB.
|
Background:
Tuberculosis (TB) is a chronic wasting inflammatory disease characterized by multisystem involvement, which can cause metabolic derangements in afflicted patients. Metabolic signatures have been exploited in the study of several diseases. However, the serum that is successfully used in TB diagnosis on the basis of metabolic profiling is not by much.
Methods:
Orthogonal partial least-squares discriminant analysis was capable of distinguishing TB patients from both healthy subjects and patients with conditions other than TB. Therefore, TB-specific metabolic profiling was established. Clusters of potential biomarkers for differentiating TB active from non-TB diseases were identified using Mann-Whitney U-test. Multiple logistic regression analysis of metabolites was calculated to determine the suitable biomarker group that allows the efficient differentiation of patients with TB active from the control subjects.
Results:
From among 271 participants, 12 metabolites were found to contribute to the distinction between the TB active group and the control groups. These metabolites were mainly involved in the metabolic pathways of the following three biomolecules: Fatty acids, amino acids, and lipids. The receiver operating characteristic curves of 3D, 7D, and 11D-phytanic acid, behenic acid, and threoninyl-γ-glutamate exhibited excellent efficiency with area under the curve (AUC) values of 0.904 (95% confidence interval [CI]: 0863-0.944), 0.93 (95% CI: 0.893-0.966), and 0.964 (95% CI: 00.941-0.988), respectively. The largest and smallest resulting AUCs were 0.964 and 0.720, indicating that these biomarkers may be involved in the disease mechanisms. The combination of lysophosphatidylcholine (18:0), behenic acid, threoninyl-γ-glutamate, and presqualene diphosphate was used to represent the most suitable biomarker group for the differentiation of patients with TB active from the control subjects, with an AUC value of 0.991.
Conclusions:
The metabolic analysis results identified new serum biomarkers that can distinguish TB from non-TB diseases. The metabolomics-based analysis provides specific insights into the biology of TB and may offer new avenues for TB diagnosis.
| 17,017 | 398 | 21 |
[
"tb",
"acid",
"active",
"patients",
"active tb",
"metabolites",
"group",
"tb group",
"lysopc",
"subgroup"
] |
[
"test",
"test"
] |
[CONTENT] Metabolites | Orthogonal Partial Least-squares Discriminant Analysis | Serum | Tuberculosis | Ultra-performance Liquid Chromatography-mass Spectrometry [SUMMARY]
|
[CONTENT] Metabolites | Orthogonal Partial Least-squares Discriminant Analysis | Serum | Tuberculosis | Ultra-performance Liquid Chromatography-mass Spectrometry [SUMMARY]
|
[CONTENT] Metabolites | Orthogonal Partial Least-squares Discriminant Analysis | Serum | Tuberculosis | Ultra-performance Liquid Chromatography-mass Spectrometry [SUMMARY]
|
[CONTENT] Metabolites | Orthogonal Partial Least-squares Discriminant Analysis | Serum | Tuberculosis | Ultra-performance Liquid Chromatography-mass Spectrometry [SUMMARY]
|
[CONTENT] Metabolites | Orthogonal Partial Least-squares Discriminant Analysis | Serum | Tuberculosis | Ultra-performance Liquid Chromatography-mass Spectrometry [SUMMARY]
|
[CONTENT] Metabolites | Orthogonal Partial Least-squares Discriminant Analysis | Serum | Tuberculosis | Ultra-performance Liquid Chromatography-mass Spectrometry [SUMMARY]
|
[CONTENT] Adult | Aged | Biomarkers | Chromatography, High Pressure Liquid | Female | Humans | Male | Mass Spectrometry | Middle Aged | Tuberculosis [SUMMARY]
|
[CONTENT] Adult | Aged | Biomarkers | Chromatography, High Pressure Liquid | Female | Humans | Male | Mass Spectrometry | Middle Aged | Tuberculosis [SUMMARY]
|
[CONTENT] Adult | Aged | Biomarkers | Chromatography, High Pressure Liquid | Female | Humans | Male | Mass Spectrometry | Middle Aged | Tuberculosis [SUMMARY]
|
[CONTENT] Adult | Aged | Biomarkers | Chromatography, High Pressure Liquid | Female | Humans | Male | Mass Spectrometry | Middle Aged | Tuberculosis [SUMMARY]
|
[CONTENT] Adult | Aged | Biomarkers | Chromatography, High Pressure Liquid | Female | Humans | Male | Mass Spectrometry | Middle Aged | Tuberculosis [SUMMARY]
|
[CONTENT] Adult | Aged | Biomarkers | Chromatography, High Pressure Liquid | Female | Humans | Male | Mass Spectrometry | Middle Aged | Tuberculosis [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] tb | acid | active | patients | active tb | metabolites | group | tb group | lysopc | subgroup [SUMMARY]
|
[CONTENT] tb | acid | active | patients | active tb | metabolites | group | tb group | lysopc | subgroup [SUMMARY]
|
[CONTENT] tb | acid | active | patients | active tb | metabolites | group | tb group | lysopc | subgroup [SUMMARY]
|
[CONTENT] tb | acid | active | patients | active tb | metabolites | group | tb group | lysopc | subgroup [SUMMARY]
|
[CONTENT] tb | acid | active | patients | active tb | metabolites | group | tb group | lysopc | subgroup [SUMMARY]
|
[CONTENT] tb | acid | active | patients | active tb | metabolites | group | tb group | lysopc | subgroup [SUMMARY]
|
[CONTENT] tb | metabolic | study | diseases | active tb | diagnosis | profiles | active | new | basis metabolic [SUMMARY]
|
[CONTENT] samples | qc | analysis | tb | μl | minutes | ms | trap | column | μm [SUMMARY]
|
[CONTENT] tb | acid | subgroup | active | metabolites | active tb | group | lysopc | roc | tb group [SUMMARY]
|
[CONTENT] acid | infection | metabolites | tb | fatty acid | combination | trp | fatty | value | mtb [SUMMARY]
|
[CONTENT] tb | acid | patients | samples | active | metabolites | active tb | subgroup | lysopc | group [SUMMARY]
|
[CONTENT] tb | acid | patients | samples | active | metabolites | active tb | subgroup | lysopc | group [SUMMARY]
|
[CONTENT] TB ||| Metabolic ||| [SUMMARY]
|
[CONTENT] TB ||| ||| TB | Mann-Whitney U-test ||| TB [SUMMARY]
|
[CONTENT] 271 | 12 | TB ||| three ||| 0.904 | 95% | CI | 0863 | 0.93 | 95% | CI | 0.893-0.966 | 0.964 | 95% | CI ||| 0.964 | 0.720 ||| 18:0 | TB | 0.991 [SUMMARY]
|
[CONTENT] TB ||| TB [SUMMARY]
|
[CONTENT] TB ||| Metabolic ||| ||| TB ||| ||| TB | Mann-Whitney U-test ||| TB ||| 271 | 12 | TB ||| three ||| 0.904 | 95% | CI | 0863 | 0.93 | 95% | CI | 0.893-0.966 | 0.964 | 95% | CI ||| 0.964 | 0.720 ||| 18:0 | TB | 0.991 ||| TB ||| TB [SUMMARY]
|
[CONTENT] TB ||| Metabolic ||| ||| TB ||| ||| TB | Mann-Whitney U-test ||| TB ||| 271 | 12 | TB ||| three ||| 0.904 | 95% | CI | 0863 | 0.93 | 95% | CI | 0.893-0.966 | 0.964 | 95% | CI ||| 0.964 | 0.720 ||| 18:0 | TB | 0.991 ||| TB ||| TB [SUMMARY]
|
||||||
Diffusion weighted imaging and blood oxygen level-dependent MR imaging of kidneys in patients with lupus nephritis.
|
25342208
|
Lupus nephritis (LN) is one of most common secondary glomerulonephritis. There is no ideal method to simultaneously assess renal structure and function in patients with LN. The aim of this study is to investigate the utility of diffusion weighted imaging (DWI) and blood oxygen level-dependent (BOLD) MR imaging in the assessment of renal involvement and pathological changes in patients with LN.
|
BACKGROUND
|
Sixty-five patients with LN and 16 healthy volunteers underwent coronal echo-planar DWI and BOLD MR imaging of the kidneys. The apparent diffusion coefficient (ADC) and R2* values of the kidneys were calculated with b values of 0 and 500 s/mm(2). The relationship between the renal injury variables and the ADCs or R2* values were evaluated. And 16 of 65 patients with LN underwent a repeated evaluation after the induction treatment for 9 to 12 months.
|
METHODS
|
The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10(-3) mm(2)/s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than that in volunteers. In patients with LN, the mean ADC values were correlated with eGFR (r = 0.510, p < 0.01). There was a negative correlation between the mean ADC values and renal pathology chronicity indexes (r = -0.249, p < 0.05), the R2* values of the renal medulla and proteinuria (r = -0.244, p < 0.05), and the degree of tubulointerstitial lesions (r = -0.242, p < 0.05). The ADC and R2* values of kidneys were significantly higher than those of pre-treatment in complete remission patients.
|
RESULTS
|
DWI and BOLD MR imaging of kidneys may be used to noninvasively monitor the disease activity and evaluate therapeutic efficacy in lupus nephritis.
|
CONCLUSIONS
|
[
"Adult",
"Case-Control Studies",
"Diffusion Magnetic Resonance Imaging",
"Female",
"Humans",
"Kidney",
"Kidney Function Tests",
"Lupus Nephritis",
"Male",
"Middle Aged",
"Oxygen",
"Young Adult"
] |
4221678
|
Background
|
Systemic lupus erythematosus (SLE) is an autoimmune disease with variable presentations, course and prognosis. Approximately half a million people in Europe and seventy per 100 000 people in China suffer from SLE. Renal involvement is one of the major determinants of the outcome in patients with SLE [1]. Lupus nephritis (LN) is one of the most common secondary glomerulonephritis in China. Renal interstitial infiltrates, tubular necrosis and interstitial fibrosis can be found in 60-70% patients with LN. Interstitial lesions are often associated with glomerular function, and may play a pivotal role on progression and prognosis of lupus nephritis [2-4]. Nowadays there is no ideal method to simultaneously assess renal structure and function in patients with LN. Moreover, effective means to evaluate tubulointerstitial hypoxia state is lack as well. Therefore, it’s difficult to dynamically monitor the disease progression in these patients.
Functional magnetic resonance (MR) imaging techniques such as diffusion-weighted imaging (DWI) and blood oxygen level-dependent (BOLD) imaging have shown considerable value in the evaluation of renal function in health and renal diseases [5-7]. The aim of this study is to investigate the feasibility of functional renal MR imaging in the assessment of renal functional and structural changes in patients with lupus nephritis.
| null | null |
Results
|
The mean ADC and R2* values of kidneys in patients with LN were lower than that in healthy volunteers The mean ADC values of kidneys in healthy volunteers were 2.52 ± 0.17 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 12.63 ± 1.40/sec and 18.14 ± 2.51/sec respectively. The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than the ADC and R2* values of kidneys in healthy volunteers (p = 0.048, p = 0.045 and p = 0.008, respectively) (Table 1). There was no significant difference of the ADC and R2* values between bilateral kidneys either in volunteers or in patients with LN. Moreover, the ADC and R2* values were much lower in patients with severe lupus nephritis (Figures 1 and 2).Table 1
The mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers
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ADC (×10
−3
mm
2
/ s)
R2* of cortex (1/sec)
R2*of medulla (1/sec)
Normal kidneys
2.52 ± 0.1712.63 ± 1.4018.14 ± 2.51
Kidneys with LN
2.40 ± 0.2511.03 ± 1.6014.05 ± 3.38
P value
0.0480.0450.008ADC: apparent diffusion coefficient; LN: lupus nephritis.Figure 1
Renal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.Figure 2
Renal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.
The mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers
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ADC: apparent diffusion coefficient; LN: lupus nephritis.
Renal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.
Renal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.
The mean ADC values of kidneys in healthy volunteers were 2.52 ± 0.17 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 12.63 ± 1.40/sec and 18.14 ± 2.51/sec respectively. The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than the ADC and R2* values of kidneys in healthy volunteers (p = 0.048, p = 0.045 and p = 0.008, respectively) (Table 1). There was no significant difference of the ADC and R2* values between bilateral kidneys either in volunteers or in patients with LN. Moreover, the ADC and R2* values were much lower in patients with severe lupus nephritis (Figures 1 and 2).Table 1
The mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers
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ADC (×10
−3
mm
2
/ s)
R2* of cortex (1/sec)
R2*of medulla (1/sec)
Normal kidneys
2.52 ± 0.1712.63 ± 1.4018.14 ± 2.51
Kidneys with LN
2.40 ± 0.2511.03 ± 1.6014.05 ± 3.38
P value
0.0480.0450.008ADC: apparent diffusion coefficient; LN: lupus nephritis.Figure 1
Renal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.Figure 2
Renal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.
The mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers
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ADC: apparent diffusion coefficient; LN: lupus nephritis.
Renal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.
Renal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.
The correlation between imaging values and renal function or pathological changes in patients with LN In the patients with LN, the mean ADC values were correlated with glomerular filtration rate estimated by abbreviated MDRD equation (eGFR) (r = 0.510, p < 0.01) (Figure 3). There was a negative correlation between the mean ADC values and pathology chronicity indexes (r = −0.249, p < 0.05). Moreover, the R2* values of the renal medulla were negatively correlated with 24 hours proteinuria (r = −0.244, p < 0.05), the degree of tubulointerstitial lesions (r = −0.242, p < 0.05).Figure 3
The relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).
The relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).
In the patients with LN, the mean ADC values were correlated with glomerular filtration rate estimated by abbreviated MDRD equation (eGFR) (r = 0.510, p < 0.01) (Figure 3). There was a negative correlation between the mean ADC values and pathology chronicity indexes (r = −0.249, p < 0.05). Moreover, the R2* values of the renal medulla were negatively correlated with 24 hours proteinuria (r = −0.244, p < 0.05), the degree of tubulointerstitial lesions (r = −0.242, p < 0.05).Figure 3
The relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).
The relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).
Comparison of the mean ADC and R2* values of kidneys in two groups of LN Of the 65 patients with LN, 6 were class I or II, 59 were class III, IV, V or combination. Fifty-nine patients were divided into 2 groups: Group A including 31 patients with LN class III, IV or V, and Group B including 28 patients with LN class V + III or V + IV. Urine N-acetyl-β-D-glucosaminidase (NAG) level in Group A was much lower than that in Group B (17.28 ± 11.23 vs 27.69 ± 13.24 U/L, p = 0.0037). There were 2 patients with abnormal tubular acidification, and 5 patients with impaired urinary concentrating function in Group A, while there being 9 patients with abnormal tubular acidification and 10 patients with impaired urinary concentrating function in Group B. The mean ADC values in Group A were higher than Group B ((2.50 ± 0.26) × 10−3 mm2/s vs (2.36 ± 0.21) × 10−3 mm2/s,p < 0.05). The mean R2* values of cortex in Group A were higher than Group B (11.70 ± 2.24/sec vs 10.57 ± 1.04/sec, p < 0.05), while mean R2* values of medulla in Group A being similar with that in Group B (13.99 ± 3.31/sec vs 13.78 ± 3.67/sec,p > 0.05) (Table 2).Table 2
Comparison of the mean ADC and R2* values of kidneys in two groups of LN
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n
Mean ADC (×10
−3
mm
2
/ s)
R2* values of cortex (1/sec)
R2* values of medulla (1/sec)
Group A
312.50 ± 0.2611.70 ± 2.2413.99 ± 3.31
Group B
282.36 ± 0.21a
10.57 ± 1.04a
13.78 ± 3.67Compared with group A, ap < 0.05.Group A = class III, IV or V; Group B = class V + III or V + IV.
Comparison of the mean ADC and R2* values of kidneys in two groups of LN
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Compared with group A, ap < 0.05.
Group A = class III, IV or V; Group B = class V + III or V + IV.
Of the 65 patients with LN, 6 were class I or II, 59 were class III, IV, V or combination. Fifty-nine patients were divided into 2 groups: Group A including 31 patients with LN class III, IV or V, and Group B including 28 patients with LN class V + III or V + IV. Urine N-acetyl-β-D-glucosaminidase (NAG) level in Group A was much lower than that in Group B (17.28 ± 11.23 vs 27.69 ± 13.24 U/L, p = 0.0037). There were 2 patients with abnormal tubular acidification, and 5 patients with impaired urinary concentrating function in Group A, while there being 9 patients with abnormal tubular acidification and 10 patients with impaired urinary concentrating function in Group B. The mean ADC values in Group A were higher than Group B ((2.50 ± 0.26) × 10−3 mm2/s vs (2.36 ± 0.21) × 10−3 mm2/s,p < 0.05). The mean R2* values of cortex in Group A were higher than Group B (11.70 ± 2.24/sec vs 10.57 ± 1.04/sec, p < 0.05), while mean R2* values of medulla in Group A being similar with that in Group B (13.99 ± 3.31/sec vs 13.78 ± 3.67/sec,p > 0.05) (Table 2).Table 2
Comparison of the mean ADC and R2* values of kidneys in two groups of LN
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n
Mean ADC (×10
−3
mm
2
/ s)
R2* values of cortex (1/sec)
R2* values of medulla (1/sec)
Group A
312.50 ± 0.2611.70 ± 2.2413.99 ± 3.31
Group B
282.36 ± 0.21a
10.57 ± 1.04a
13.78 ± 3.67Compared with group A, ap < 0.05.Group A = class III, IV or V; Group B = class V + III or V + IV.
Comparison of the mean ADC and R2* values of kidneys in two groups of LN
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Compared with group A, ap < 0.05.
Group A = class III, IV or V; Group B = class V + III or V + IV.
The changes in ADC and R2* values of kidneys after the induction treatment in patients with LN Among 16 patients who repeated MR scan after prednisone combined with immunosuppressant treatment in 9 to 12 months, 12 patients got complete remission (CR) and 4 had no response (NR). Complete remission was defined as urinary protein excretion that was less than 0.3 g/24 h, with normal urine sediment, and serum albumin concentration of greater than 35 g/L, and stabilization (±15%) or improvement in serum creatinine. The ADC and R2* values of kidneys were significantly higher than pre-treatment in CR patients (all p < 0.05), while the ADC and R2* values being lower than pre-treatment in NR patients with no statistical significance (p > 0.05) (Table 3).Table 3
Comparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment
ADC (×10
−3
mm
2
/s)
R2* of cortex (1/sec)
R2* of medulla (1/sec)
Group CR
Before
2.41 ± 0.1610.25 ± 1.1913.04 ± 3.15
After
2.58 ± 0.14a
11.77 ± 1.11a
16.94 ± 3.50a
Group NR
Before
2.43 ± 0.1210.84 ± 1.9613.25 ± 0.41
After
2.40 ± 0.2310.13 ± 1.6413.12 ± 0.47
ap < 0.05.ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.
Comparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment
ap < 0.05.
ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.
Among 16 patients who repeated MR scan after prednisone combined with immunosuppressant treatment in 9 to 12 months, 12 patients got complete remission (CR) and 4 had no response (NR). Complete remission was defined as urinary protein excretion that was less than 0.3 g/24 h, with normal urine sediment, and serum albumin concentration of greater than 35 g/L, and stabilization (±15%) or improvement in serum creatinine. The ADC and R2* values of kidneys were significantly higher than pre-treatment in CR patients (all p < 0.05), while the ADC and R2* values being lower than pre-treatment in NR patients with no statistical significance (p > 0.05) (Table 3).Table 3
Comparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment
ADC (×10
−3
mm
2
/s)
R2* of cortex (1/sec)
R2* of medulla (1/sec)
Group CR
Before
2.41 ± 0.1610.25 ± 1.1913.04 ± 3.15
After
2.58 ± 0.14a
11.77 ± 1.11a
16.94 ± 3.50a
Group NR
Before
2.43 ± 0.1210.84 ± 1.9613.25 ± 0.41
After
2.40 ± 0.2310.13 ± 1.6413.12 ± 0.47
ap < 0.05.ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.
Comparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment
ap < 0.05.
ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.
|
Conclusions
|
In summary, diffusion-weighted imaging and blood oxygen level-dependent MR imaging are novel functional MRI techniques with repeatable nature that have potential to assess glomerular filtration, parenchymal oxygenation and renal pathological changes. This pilot study indicated that DWI and BOLD MR imaging might be used to noninvasively monitor disease activity and evaluate therapeutic efficacy in lupus nephritis. Further studies involve larger group of patients with lupus nephritis are necessary.
|
[
"Study population",
"MR imaging",
"Imaging analysis",
"Statistical analysis",
"The mean ADC and R2* values of kidneys in patients with LN were lower than that in healthy volunteers",
"The correlation between imaging values and renal function or pathological changes in patients with LN",
"Comparison of the mean ADC and R2* values of kidneys in two groups of LN",
"The changes in ADC and R2* values of kidneys after the induction treatment in patients with LN"
] |
[
"Sixty-five patients with lupus nephritis, diagnosed according to clinical manifestation, laboratory findings and renal histopathological changes, had been followed up since their admission in our department during the recent two years. They all met the diagnosis criteria of systemic lupus erythematosus defined by the American Rheumatism Association in 1997. All the patients were included after signing informed consent. There were 10 male and 55 female patients, with mean age of 34 ± 12 years old. Sixteen healthy volunteers, 2 men and 14 women with mean age of 35 ± 11 years old, had no history of primary or secondary renal diseases.",
"All the LN patients and healthy volunteers underwent coronal echo-planar DW and BOLD MR imaging of the kidneys with a single breath-hold time. And 16 of the 65 patients with LN received functional MR imaging of kidneys again after 9 to 12 months induction treatment with immunosuppression. All of them fasted for 4–8 hours before the examination. MR imaging was performed with a 1.5 T MR imager (GE Twin-Speed, General Electric Medical Systems, Milwaukee, WI). DW MR imaging was performed with a spin echo-echo planar imaging (SE-EPI) sequence, and BOLD MRI was performed with a multiple gradient-recalled-echo (mGRE) sequence. The following parameters were used for DWI: 11 sections (section thickness, 5 mm; intersection gap, 1 mm); repetition time (TR), 2000 ms; echo time (TE), 52.9 ms; NEX, 2; field of view, 380 × 380 mm; matrix, 128 × 128; acquisition time, 16 s. The diffusion gradient b values of 0 and 500 s/mm2 were used. The following parameters were used for BOLD MRI: TR/TE, 110 ms/2.2-27.5 ms; flip angle, 60°; matrix, 132x128; section thickness, 5 mm; intersection gap, 1 mm. Three coronal sections were obtained for each kidney and eight T2*-weighted images were acquired for each section within one 16-second breath hold.",
"The apparent diffusion coefficient (ADC) and R2* values of the kidneys were calculated on a GE workstation (Sun Microsystems, ADW4.2) with Functool 2 image analysis software. In the transverse and coronal ADC map, freehand regions of interest (ROIs) were placed on the parenchyma of the kidneys, and fat within renal sinus were excluded. Three such ROIs were placed—one each in the upper pole, inter-polar region, and lower pole—and the mean of these three values were calculated.\nR2* values of renal cortex and medulla were measured on T2* maps. The size of ROI was set to reduce the noise and the influence of partial volume effects. Avoiding visible vessels, ROIs were placed manually on the clear corticomedullary differentiation image. Three ROIs were placed on each side of renal cortex and medulla, and the mean of R2* values were calculated.\nThe relationship between the renal injury variables and the ADCs or R2* values were evaluated. The renal function was estimated using the abbreviated modification of diet in renal disease (MDRD) study equation. Renal tubulointerstitial lesions were scored with semi quantitative method: no lesion was 0, mild lesion (≤25%) was 1, moderate lesion (25% ~ 50%) was 2, and severe lesion (≥50%) was 3. There were 0–9 scores for tubulointerstitial lesions including 0–3 for tubular atrophy, 0–3 for interstitial fibrosis, 0–3 for interstitial inflammatory cells infiltration. The renal pathology activity index (AI) and chronicity index (CI) were evaluated in patients with LN.",
"The SPSS software was used (version 13.0; SPSS; Chicago, Illinois, USA). All results were expressed as mean ± SD for normally distributed data, median and range for skewed data and frequency (%) for categorical data. The distribution of clinical and laboratory attributes among the groups were evaluated by t test. The Pearson correlation analysis was used to evaluate the relation between the clinical or pathological variables and the ADCs or R2* values. P < 0.05 was considered as statistically significant.",
"The mean ADC values of kidneys in healthy volunteers were 2.52 ± 0.17 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 12.63 ± 1.40/sec and 18.14 ± 2.51/sec respectively. The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than the ADC and R2* values of kidneys in healthy volunteers (p = 0.048, p = 0.045 and p = 0.008, respectively) (Table 1). There was no significant difference of the ADC and R2* values between bilateral kidneys either in volunteers or in patients with LN. Moreover, the ADC and R2* values were much lower in patients with severe lupus nephritis (Figures 1 and 2).Table 1\nThe mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nADC (×10\n−3 \nmm\n2\n/ s)\n\nR2* of cortex (1/sec)\n\nR2*of medulla (1/sec)\n\nNormal kidneys\n2.52 ± 0.1712.63 ± 1.4018.14 ± 2.51\nKidneys with LN\n2.40 ± 0.2511.03 ± 1.6014.05 ± 3.38\nP value\n0.0480.0450.008ADC: apparent diffusion coefficient; LN: lupus nephritis.Figure 1\nRenal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.Figure 2\nRenal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.\n\nThe mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nADC: apparent diffusion coefficient; LN: lupus nephritis.\n\nRenal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.\n\nRenal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.",
"In the patients with LN, the mean ADC values were correlated with glomerular filtration rate estimated by abbreviated MDRD equation (eGFR) (r = 0.510, p < 0.01) (Figure 3). There was a negative correlation between the mean ADC values and pathology chronicity indexes (r = −0.249, p < 0.05). Moreover, the R2* values of the renal medulla were negatively correlated with 24 hours proteinuria (r = −0.244, p < 0.05), the degree of tubulointerstitial lesions (r = −0.242, p < 0.05).Figure 3\nThe relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).\n\nThe relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).",
"Of the 65 patients with LN, 6 were class I or II, 59 were class III, IV, V or combination. Fifty-nine patients were divided into 2 groups: Group A including 31 patients with LN class III, IV or V, and Group B including 28 patients with LN class V + III or V + IV. Urine N-acetyl-β-D-glucosaminidase (NAG) level in Group A was much lower than that in Group B (17.28 ± 11.23 vs 27.69 ± 13.24 U/L, p = 0.0037). There were 2 patients with abnormal tubular acidification, and 5 patients with impaired urinary concentrating function in Group A, while there being 9 patients with abnormal tubular acidification and 10 patients with impaired urinary concentrating function in Group B. The mean ADC values in Group A were higher than Group B ((2.50 ± 0.26) × 10−3 mm2/s vs (2.36 ± 0.21) × 10−3 mm2/s,p < 0.05). The mean R2* values of cortex in Group A were higher than Group B (11.70 ± 2.24/sec vs 10.57 ± 1.04/sec, p < 0.05), while mean R2* values of medulla in Group A being similar with that in Group B (13.99 ± 3.31/sec vs 13.78 ± 3.67/sec,p > 0.05) (Table 2).Table 2\nComparison of the mean ADC and R2* values of kidneys in two groups of LN\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nn\n\nMean ADC (×10\n−3 \nmm\n2\n/ s)\n\nR2* values of cortex (1/sec)\n\nR2* values of medulla (1/sec)\n\nGroup A\n312.50 ± 0.2611.70 ± 2.2413.99 ± 3.31\nGroup B\n282.36 ± 0.21a\n10.57 ± 1.04a\n13.78 ± 3.67Compared with group A, ap < 0.05.Group A = class III, IV or V; Group B = class V + III or V + IV.\n\nComparison of the mean ADC and R2* values of kidneys in two groups of LN\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nCompared with group A, ap < 0.05.\nGroup A = class III, IV or V; Group B = class V + III or V + IV.",
"Among 16 patients who repeated MR scan after prednisone combined with immunosuppressant treatment in 9 to 12 months, 12 patients got complete remission (CR) and 4 had no response (NR). Complete remission was defined as urinary protein excretion that was less than 0.3 g/24 h, with normal urine sediment, and serum albumin concentration of greater than 35 g/L, and stabilization (±15%) or improvement in serum creatinine. The ADC and R2* values of kidneys were significantly higher than pre-treatment in CR patients (all p < 0.05), while the ADC and R2* values being lower than pre-treatment in NR patients with no statistical significance (p > 0.05) (Table 3).Table 3\nComparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment\n\nADC (×10\n−3 \nmm\n2\n/s)\n\nR2* of cortex (1/sec)\n\nR2* of medulla (1/sec)\n\nGroup CR\n\nBefore\n2.41 ± 0.1610.25 ± 1.1913.04 ± 3.15\nAfter\n2.58 ± 0.14a\n11.77 ± 1.11a\n16.94 ± 3.50a\n\nGroup NR\n\nBefore\n2.43 ± 0.1210.84 ± 1.9613.25 ± 0.41\nAfter\n2.40 ± 0.2310.13 ± 1.6413.12 ± 0.47\nap < 0.05.ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.\n\nComparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment\n\n\nap < 0.05.\nADC: apparent diffusion coefficient; CR: complete remission; NR: no response."
] |
[
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Study population",
"MR imaging",
"Imaging analysis",
"Statistical analysis",
"Results",
"The mean ADC and R2* values of kidneys in patients with LN were lower than that in healthy volunteers",
"The correlation between imaging values and renal function or pathological changes in patients with LN",
"Comparison of the mean ADC and R2* values of kidneys in two groups of LN",
"The changes in ADC and R2* values of kidneys after the induction treatment in patients with LN",
"Discussion",
"Conclusions"
] |
[
"Systemic lupus erythematosus (SLE) is an autoimmune disease with variable presentations, course and prognosis. Approximately half a million people in Europe and seventy per 100 000 people in China suffer from SLE. Renal involvement is one of the major determinants of the outcome in patients with SLE [1]. Lupus nephritis (LN) is one of the most common secondary glomerulonephritis in China. Renal interstitial infiltrates, tubular necrosis and interstitial fibrosis can be found in 60-70% patients with LN. Interstitial lesions are often associated with glomerular function, and may play a pivotal role on progression and prognosis of lupus nephritis [2-4]. Nowadays there is no ideal method to simultaneously assess renal structure and function in patients with LN. Moreover, effective means to evaluate tubulointerstitial hypoxia state is lack as well. Therefore, it’s difficult to dynamically monitor the disease progression in these patients.\nFunctional magnetic resonance (MR) imaging techniques such as diffusion-weighted imaging (DWI) and blood oxygen level-dependent (BOLD) imaging have shown considerable value in the evaluation of renal function in health and renal diseases [5-7]. The aim of this study is to investigate the feasibility of functional renal MR imaging in the assessment of renal functional and structural changes in patients with lupus nephritis.",
" Study population Sixty-five patients with lupus nephritis, diagnosed according to clinical manifestation, laboratory findings and renal histopathological changes, had been followed up since their admission in our department during the recent two years. They all met the diagnosis criteria of systemic lupus erythematosus defined by the American Rheumatism Association in 1997. All the patients were included after signing informed consent. There were 10 male and 55 female patients, with mean age of 34 ± 12 years old. Sixteen healthy volunteers, 2 men and 14 women with mean age of 35 ± 11 years old, had no history of primary or secondary renal diseases.\nSixty-five patients with lupus nephritis, diagnosed according to clinical manifestation, laboratory findings and renal histopathological changes, had been followed up since their admission in our department during the recent two years. They all met the diagnosis criteria of systemic lupus erythematosus defined by the American Rheumatism Association in 1997. All the patients were included after signing informed consent. There were 10 male and 55 female patients, with mean age of 34 ± 12 years old. Sixteen healthy volunteers, 2 men and 14 women with mean age of 35 ± 11 years old, had no history of primary or secondary renal diseases.\n MR imaging All the LN patients and healthy volunteers underwent coronal echo-planar DW and BOLD MR imaging of the kidneys with a single breath-hold time. And 16 of the 65 patients with LN received functional MR imaging of kidneys again after 9 to 12 months induction treatment with immunosuppression. All of them fasted for 4–8 hours before the examination. MR imaging was performed with a 1.5 T MR imager (GE Twin-Speed, General Electric Medical Systems, Milwaukee, WI). DW MR imaging was performed with a spin echo-echo planar imaging (SE-EPI) sequence, and BOLD MRI was performed with a multiple gradient-recalled-echo (mGRE) sequence. The following parameters were used for DWI: 11 sections (section thickness, 5 mm; intersection gap, 1 mm); repetition time (TR), 2000 ms; echo time (TE), 52.9 ms; NEX, 2; field of view, 380 × 380 mm; matrix, 128 × 128; acquisition time, 16 s. The diffusion gradient b values of 0 and 500 s/mm2 were used. The following parameters were used for BOLD MRI: TR/TE, 110 ms/2.2-27.5 ms; flip angle, 60°; matrix, 132x128; section thickness, 5 mm; intersection gap, 1 mm. Three coronal sections were obtained for each kidney and eight T2*-weighted images were acquired for each section within one 16-second breath hold.\nAll the LN patients and healthy volunteers underwent coronal echo-planar DW and BOLD MR imaging of the kidneys with a single breath-hold time. And 16 of the 65 patients with LN received functional MR imaging of kidneys again after 9 to 12 months induction treatment with immunosuppression. All of them fasted for 4–8 hours before the examination. MR imaging was performed with a 1.5 T MR imager (GE Twin-Speed, General Electric Medical Systems, Milwaukee, WI). DW MR imaging was performed with a spin echo-echo planar imaging (SE-EPI) sequence, and BOLD MRI was performed with a multiple gradient-recalled-echo (mGRE) sequence. The following parameters were used for DWI: 11 sections (section thickness, 5 mm; intersection gap, 1 mm); repetition time (TR), 2000 ms; echo time (TE), 52.9 ms; NEX, 2; field of view, 380 × 380 mm; matrix, 128 × 128; acquisition time, 16 s. The diffusion gradient b values of 0 and 500 s/mm2 were used. The following parameters were used for BOLD MRI: TR/TE, 110 ms/2.2-27.5 ms; flip angle, 60°; matrix, 132x128; section thickness, 5 mm; intersection gap, 1 mm. Three coronal sections were obtained for each kidney and eight T2*-weighted images were acquired for each section within one 16-second breath hold.\n Imaging analysis The apparent diffusion coefficient (ADC) and R2* values of the kidneys were calculated on a GE workstation (Sun Microsystems, ADW4.2) with Functool 2 image analysis software. In the transverse and coronal ADC map, freehand regions of interest (ROIs) were placed on the parenchyma of the kidneys, and fat within renal sinus were excluded. Three such ROIs were placed—one each in the upper pole, inter-polar region, and lower pole—and the mean of these three values were calculated.\nR2* values of renal cortex and medulla were measured on T2* maps. The size of ROI was set to reduce the noise and the influence of partial volume effects. Avoiding visible vessels, ROIs were placed manually on the clear corticomedullary differentiation image. Three ROIs were placed on each side of renal cortex and medulla, and the mean of R2* values were calculated.\nThe relationship between the renal injury variables and the ADCs or R2* values were evaluated. The renal function was estimated using the abbreviated modification of diet in renal disease (MDRD) study equation. Renal tubulointerstitial lesions were scored with semi quantitative method: no lesion was 0, mild lesion (≤25%) was 1, moderate lesion (25% ~ 50%) was 2, and severe lesion (≥50%) was 3. There were 0–9 scores for tubulointerstitial lesions including 0–3 for tubular atrophy, 0–3 for interstitial fibrosis, 0–3 for interstitial inflammatory cells infiltration. The renal pathology activity index (AI) and chronicity index (CI) were evaluated in patients with LN.\nThe apparent diffusion coefficient (ADC) and R2* values of the kidneys were calculated on a GE workstation (Sun Microsystems, ADW4.2) with Functool 2 image analysis software. In the transverse and coronal ADC map, freehand regions of interest (ROIs) were placed on the parenchyma of the kidneys, and fat within renal sinus were excluded. Three such ROIs were placed—one each in the upper pole, inter-polar region, and lower pole—and the mean of these three values were calculated.\nR2* values of renal cortex and medulla were measured on T2* maps. The size of ROI was set to reduce the noise and the influence of partial volume effects. Avoiding visible vessels, ROIs were placed manually on the clear corticomedullary differentiation image. Three ROIs were placed on each side of renal cortex and medulla, and the mean of R2* values were calculated.\nThe relationship between the renal injury variables and the ADCs or R2* values were evaluated. The renal function was estimated using the abbreviated modification of diet in renal disease (MDRD) study equation. Renal tubulointerstitial lesions were scored with semi quantitative method: no lesion was 0, mild lesion (≤25%) was 1, moderate lesion (25% ~ 50%) was 2, and severe lesion (≥50%) was 3. There were 0–9 scores for tubulointerstitial lesions including 0–3 for tubular atrophy, 0–3 for interstitial fibrosis, 0–3 for interstitial inflammatory cells infiltration. The renal pathology activity index (AI) and chronicity index (CI) were evaluated in patients with LN.\n Statistical analysis The SPSS software was used (version 13.0; SPSS; Chicago, Illinois, USA). All results were expressed as mean ± SD for normally distributed data, median and range for skewed data and frequency (%) for categorical data. The distribution of clinical and laboratory attributes among the groups were evaluated by t test. The Pearson correlation analysis was used to evaluate the relation between the clinical or pathological variables and the ADCs or R2* values. P < 0.05 was considered as statistically significant.\nThe SPSS software was used (version 13.0; SPSS; Chicago, Illinois, USA). All results were expressed as mean ± SD for normally distributed data, median and range for skewed data and frequency (%) for categorical data. The distribution of clinical and laboratory attributes among the groups were evaluated by t test. The Pearson correlation analysis was used to evaluate the relation between the clinical or pathological variables and the ADCs or R2* values. P < 0.05 was considered as statistically significant.",
"Sixty-five patients with lupus nephritis, diagnosed according to clinical manifestation, laboratory findings and renal histopathological changes, had been followed up since their admission in our department during the recent two years. They all met the diagnosis criteria of systemic lupus erythematosus defined by the American Rheumatism Association in 1997. All the patients were included after signing informed consent. There were 10 male and 55 female patients, with mean age of 34 ± 12 years old. Sixteen healthy volunteers, 2 men and 14 women with mean age of 35 ± 11 years old, had no history of primary or secondary renal diseases.",
"All the LN patients and healthy volunteers underwent coronal echo-planar DW and BOLD MR imaging of the kidneys with a single breath-hold time. And 16 of the 65 patients with LN received functional MR imaging of kidneys again after 9 to 12 months induction treatment with immunosuppression. All of them fasted for 4–8 hours before the examination. MR imaging was performed with a 1.5 T MR imager (GE Twin-Speed, General Electric Medical Systems, Milwaukee, WI). DW MR imaging was performed with a spin echo-echo planar imaging (SE-EPI) sequence, and BOLD MRI was performed with a multiple gradient-recalled-echo (mGRE) sequence. The following parameters were used for DWI: 11 sections (section thickness, 5 mm; intersection gap, 1 mm); repetition time (TR), 2000 ms; echo time (TE), 52.9 ms; NEX, 2; field of view, 380 × 380 mm; matrix, 128 × 128; acquisition time, 16 s. The diffusion gradient b values of 0 and 500 s/mm2 were used. The following parameters were used for BOLD MRI: TR/TE, 110 ms/2.2-27.5 ms; flip angle, 60°; matrix, 132x128; section thickness, 5 mm; intersection gap, 1 mm. Three coronal sections were obtained for each kidney and eight T2*-weighted images were acquired for each section within one 16-second breath hold.",
"The apparent diffusion coefficient (ADC) and R2* values of the kidneys were calculated on a GE workstation (Sun Microsystems, ADW4.2) with Functool 2 image analysis software. In the transverse and coronal ADC map, freehand regions of interest (ROIs) were placed on the parenchyma of the kidneys, and fat within renal sinus were excluded. Three such ROIs were placed—one each in the upper pole, inter-polar region, and lower pole—and the mean of these three values were calculated.\nR2* values of renal cortex and medulla were measured on T2* maps. The size of ROI was set to reduce the noise and the influence of partial volume effects. Avoiding visible vessels, ROIs were placed manually on the clear corticomedullary differentiation image. Three ROIs were placed on each side of renal cortex and medulla, and the mean of R2* values were calculated.\nThe relationship between the renal injury variables and the ADCs or R2* values were evaluated. The renal function was estimated using the abbreviated modification of diet in renal disease (MDRD) study equation. Renal tubulointerstitial lesions were scored with semi quantitative method: no lesion was 0, mild lesion (≤25%) was 1, moderate lesion (25% ~ 50%) was 2, and severe lesion (≥50%) was 3. There were 0–9 scores for tubulointerstitial lesions including 0–3 for tubular atrophy, 0–3 for interstitial fibrosis, 0–3 for interstitial inflammatory cells infiltration. The renal pathology activity index (AI) and chronicity index (CI) were evaluated in patients with LN.",
"The SPSS software was used (version 13.0; SPSS; Chicago, Illinois, USA). All results were expressed as mean ± SD for normally distributed data, median and range for skewed data and frequency (%) for categorical data. The distribution of clinical and laboratory attributes among the groups were evaluated by t test. The Pearson correlation analysis was used to evaluate the relation between the clinical or pathological variables and the ADCs or R2* values. P < 0.05 was considered as statistically significant.",
" The mean ADC and R2* values of kidneys in patients with LN were lower than that in healthy volunteers The mean ADC values of kidneys in healthy volunteers were 2.52 ± 0.17 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 12.63 ± 1.40/sec and 18.14 ± 2.51/sec respectively. The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than the ADC and R2* values of kidneys in healthy volunteers (p = 0.048, p = 0.045 and p = 0.008, respectively) (Table 1). There was no significant difference of the ADC and R2* values between bilateral kidneys either in volunteers or in patients with LN. Moreover, the ADC and R2* values were much lower in patients with severe lupus nephritis (Figures 1 and 2).Table 1\nThe mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nADC (×10\n−3 \nmm\n2\n/ s)\n\nR2* of cortex (1/sec)\n\nR2*of medulla (1/sec)\n\nNormal kidneys\n2.52 ± 0.1712.63 ± 1.4018.14 ± 2.51\nKidneys with LN\n2.40 ± 0.2511.03 ± 1.6014.05 ± 3.38\nP value\n0.0480.0450.008ADC: apparent diffusion coefficient; LN: lupus nephritis.Figure 1\nRenal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.Figure 2\nRenal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.\n\nThe mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nADC: apparent diffusion coefficient; LN: lupus nephritis.\n\nRenal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.\n\nRenal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.\nThe mean ADC values of kidneys in healthy volunteers were 2.52 ± 0.17 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 12.63 ± 1.40/sec and 18.14 ± 2.51/sec respectively. The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than the ADC and R2* values of kidneys in healthy volunteers (p = 0.048, p = 0.045 and p = 0.008, respectively) (Table 1). There was no significant difference of the ADC and R2* values between bilateral kidneys either in volunteers or in patients with LN. Moreover, the ADC and R2* values were much lower in patients with severe lupus nephritis (Figures 1 and 2).Table 1\nThe mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nADC (×10\n−3 \nmm\n2\n/ s)\n\nR2* of cortex (1/sec)\n\nR2*of medulla (1/sec)\n\nNormal kidneys\n2.52 ± 0.1712.63 ± 1.4018.14 ± 2.51\nKidneys with LN\n2.40 ± 0.2511.03 ± 1.6014.05 ± 3.38\nP value\n0.0480.0450.008ADC: apparent diffusion coefficient; LN: lupus nephritis.Figure 1\nRenal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.Figure 2\nRenal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.\n\nThe mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nADC: apparent diffusion coefficient; LN: lupus nephritis.\n\nRenal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.\n\nRenal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.\n The correlation between imaging values and renal function or pathological changes in patients with LN In the patients with LN, the mean ADC values were correlated with glomerular filtration rate estimated by abbreviated MDRD equation (eGFR) (r = 0.510, p < 0.01) (Figure 3). There was a negative correlation between the mean ADC values and pathology chronicity indexes (r = −0.249, p < 0.05). Moreover, the R2* values of the renal medulla were negatively correlated with 24 hours proteinuria (r = −0.244, p < 0.05), the degree of tubulointerstitial lesions (r = −0.242, p < 0.05).Figure 3\nThe relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).\n\nThe relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).\nIn the patients with LN, the mean ADC values were correlated with glomerular filtration rate estimated by abbreviated MDRD equation (eGFR) (r = 0.510, p < 0.01) (Figure 3). There was a negative correlation between the mean ADC values and pathology chronicity indexes (r = −0.249, p < 0.05). Moreover, the R2* values of the renal medulla were negatively correlated with 24 hours proteinuria (r = −0.244, p < 0.05), the degree of tubulointerstitial lesions (r = −0.242, p < 0.05).Figure 3\nThe relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).\n\nThe relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).\n Comparison of the mean ADC and R2* values of kidneys in two groups of LN Of the 65 patients with LN, 6 were class I or II, 59 were class III, IV, V or combination. Fifty-nine patients were divided into 2 groups: Group A including 31 patients with LN class III, IV or V, and Group B including 28 patients with LN class V + III or V + IV. Urine N-acetyl-β-D-glucosaminidase (NAG) level in Group A was much lower than that in Group B (17.28 ± 11.23 vs 27.69 ± 13.24 U/L, p = 0.0037). There were 2 patients with abnormal tubular acidification, and 5 patients with impaired urinary concentrating function in Group A, while there being 9 patients with abnormal tubular acidification and 10 patients with impaired urinary concentrating function in Group B. The mean ADC values in Group A were higher than Group B ((2.50 ± 0.26) × 10−3 mm2/s vs (2.36 ± 0.21) × 10−3 mm2/s,p < 0.05). The mean R2* values of cortex in Group A were higher than Group B (11.70 ± 2.24/sec vs 10.57 ± 1.04/sec, p < 0.05), while mean R2* values of medulla in Group A being similar with that in Group B (13.99 ± 3.31/sec vs 13.78 ± 3.67/sec,p > 0.05) (Table 2).Table 2\nComparison of the mean ADC and R2* values of kidneys in two groups of LN\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nn\n\nMean ADC (×10\n−3 \nmm\n2\n/ s)\n\nR2* values of cortex (1/sec)\n\nR2* values of medulla (1/sec)\n\nGroup A\n312.50 ± 0.2611.70 ± 2.2413.99 ± 3.31\nGroup B\n282.36 ± 0.21a\n10.57 ± 1.04a\n13.78 ± 3.67Compared with group A, ap < 0.05.Group A = class III, IV or V; Group B = class V + III or V + IV.\n\nComparison of the mean ADC and R2* values of kidneys in two groups of LN\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nCompared with group A, ap < 0.05.\nGroup A = class III, IV or V; Group B = class V + III or V + IV.\nOf the 65 patients with LN, 6 were class I or II, 59 were class III, IV, V or combination. Fifty-nine patients were divided into 2 groups: Group A including 31 patients with LN class III, IV or V, and Group B including 28 patients with LN class V + III or V + IV. Urine N-acetyl-β-D-glucosaminidase (NAG) level in Group A was much lower than that in Group B (17.28 ± 11.23 vs 27.69 ± 13.24 U/L, p = 0.0037). There were 2 patients with abnormal tubular acidification, and 5 patients with impaired urinary concentrating function in Group A, while there being 9 patients with abnormal tubular acidification and 10 patients with impaired urinary concentrating function in Group B. The mean ADC values in Group A were higher than Group B ((2.50 ± 0.26) × 10−3 mm2/s vs (2.36 ± 0.21) × 10−3 mm2/s,p < 0.05). The mean R2* values of cortex in Group A were higher than Group B (11.70 ± 2.24/sec vs 10.57 ± 1.04/sec, p < 0.05), while mean R2* values of medulla in Group A being similar with that in Group B (13.99 ± 3.31/sec vs 13.78 ± 3.67/sec,p > 0.05) (Table 2).Table 2\nComparison of the mean ADC and R2* values of kidneys in two groups of LN\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nn\n\nMean ADC (×10\n−3 \nmm\n2\n/ s)\n\nR2* values of cortex (1/sec)\n\nR2* values of medulla (1/sec)\n\nGroup A\n312.50 ± 0.2611.70 ± 2.2413.99 ± 3.31\nGroup B\n282.36 ± 0.21a\n10.57 ± 1.04a\n13.78 ± 3.67Compared with group A, ap < 0.05.Group A = class III, IV or V; Group B = class V + III or V + IV.\n\nComparison of the mean ADC and R2* values of kidneys in two groups of LN\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nCompared with group A, ap < 0.05.\nGroup A = class III, IV or V; Group B = class V + III or V + IV.\n The changes in ADC and R2* values of kidneys after the induction treatment in patients with LN Among 16 patients who repeated MR scan after prednisone combined with immunosuppressant treatment in 9 to 12 months, 12 patients got complete remission (CR) and 4 had no response (NR). Complete remission was defined as urinary protein excretion that was less than 0.3 g/24 h, with normal urine sediment, and serum albumin concentration of greater than 35 g/L, and stabilization (±15%) or improvement in serum creatinine. The ADC and R2* values of kidneys were significantly higher than pre-treatment in CR patients (all p < 0.05), while the ADC and R2* values being lower than pre-treatment in NR patients with no statistical significance (p > 0.05) (Table 3).Table 3\nComparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment\n\nADC (×10\n−3 \nmm\n2\n/s)\n\nR2* of cortex (1/sec)\n\nR2* of medulla (1/sec)\n\nGroup CR\n\nBefore\n2.41 ± 0.1610.25 ± 1.1913.04 ± 3.15\nAfter\n2.58 ± 0.14a\n11.77 ± 1.11a\n16.94 ± 3.50a\n\nGroup NR\n\nBefore\n2.43 ± 0.1210.84 ± 1.9613.25 ± 0.41\nAfter\n2.40 ± 0.2310.13 ± 1.6413.12 ± 0.47\nap < 0.05.ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.\n\nComparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment\n\n\nap < 0.05.\nADC: apparent diffusion coefficient; CR: complete remission; NR: no response.\nAmong 16 patients who repeated MR scan after prednisone combined with immunosuppressant treatment in 9 to 12 months, 12 patients got complete remission (CR) and 4 had no response (NR). Complete remission was defined as urinary protein excretion that was less than 0.3 g/24 h, with normal urine sediment, and serum albumin concentration of greater than 35 g/L, and stabilization (±15%) or improvement in serum creatinine. The ADC and R2* values of kidneys were significantly higher than pre-treatment in CR patients (all p < 0.05), while the ADC and R2* values being lower than pre-treatment in NR patients with no statistical significance (p > 0.05) (Table 3).Table 3\nComparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment\n\nADC (×10\n−3 \nmm\n2\n/s)\n\nR2* of cortex (1/sec)\n\nR2* of medulla (1/sec)\n\nGroup CR\n\nBefore\n2.41 ± 0.1610.25 ± 1.1913.04 ± 3.15\nAfter\n2.58 ± 0.14a\n11.77 ± 1.11a\n16.94 ± 3.50a\n\nGroup NR\n\nBefore\n2.43 ± 0.1210.84 ± 1.9613.25 ± 0.41\nAfter\n2.40 ± 0.2310.13 ± 1.6413.12 ± 0.47\nap < 0.05.ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.\n\nComparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment\n\n\nap < 0.05.\nADC: apparent diffusion coefficient; CR: complete remission; NR: no response.",
"The mean ADC values of kidneys in healthy volunteers were 2.52 ± 0.17 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 12.63 ± 1.40/sec and 18.14 ± 2.51/sec respectively. The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than the ADC and R2* values of kidneys in healthy volunteers (p = 0.048, p = 0.045 and p = 0.008, respectively) (Table 1). There was no significant difference of the ADC and R2* values between bilateral kidneys either in volunteers or in patients with LN. Moreover, the ADC and R2* values were much lower in patients with severe lupus nephritis (Figures 1 and 2).Table 1\nThe mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nADC (×10\n−3 \nmm\n2\n/ s)\n\nR2* of cortex (1/sec)\n\nR2*of medulla (1/sec)\n\nNormal kidneys\n2.52 ± 0.1712.63 ± 1.4018.14 ± 2.51\nKidneys with LN\n2.40 ± 0.2511.03 ± 1.6014.05 ± 3.38\nP value\n0.0480.0450.008ADC: apparent diffusion coefficient; LN: lupus nephritis.Figure 1\nRenal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.Figure 2\nRenal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.\n\nThe mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nADC: apparent diffusion coefficient; LN: lupus nephritis.\n\nRenal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.\n\nRenal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.",
"In the patients with LN, the mean ADC values were correlated with glomerular filtration rate estimated by abbreviated MDRD equation (eGFR) (r = 0.510, p < 0.01) (Figure 3). There was a negative correlation between the mean ADC values and pathology chronicity indexes (r = −0.249, p < 0.05). Moreover, the R2* values of the renal medulla were negatively correlated with 24 hours proteinuria (r = −0.244, p < 0.05), the degree of tubulointerstitial lesions (r = −0.242, p < 0.05).Figure 3\nThe relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).\n\nThe relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).",
"Of the 65 patients with LN, 6 were class I or II, 59 were class III, IV, V or combination. Fifty-nine patients were divided into 2 groups: Group A including 31 patients with LN class III, IV or V, and Group B including 28 patients with LN class V + III or V + IV. Urine N-acetyl-β-D-glucosaminidase (NAG) level in Group A was much lower than that in Group B (17.28 ± 11.23 vs 27.69 ± 13.24 U/L, p = 0.0037). There were 2 patients with abnormal tubular acidification, and 5 patients with impaired urinary concentrating function in Group A, while there being 9 patients with abnormal tubular acidification and 10 patients with impaired urinary concentrating function in Group B. The mean ADC values in Group A were higher than Group B ((2.50 ± 0.26) × 10−3 mm2/s vs (2.36 ± 0.21) × 10−3 mm2/s,p < 0.05). The mean R2* values of cortex in Group A were higher than Group B (11.70 ± 2.24/sec vs 10.57 ± 1.04/sec, p < 0.05), while mean R2* values of medulla in Group A being similar with that in Group B (13.99 ± 3.31/sec vs 13.78 ± 3.67/sec,p > 0.05) (Table 2).Table 2\nComparison of the mean ADC and R2* values of kidneys in two groups of LN\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nn\n\nMean ADC (×10\n−3 \nmm\n2\n/ s)\n\nR2* values of cortex (1/sec)\n\nR2* values of medulla (1/sec)\n\nGroup A\n312.50 ± 0.2611.70 ± 2.2413.99 ± 3.31\nGroup B\n282.36 ± 0.21a\n10.57 ± 1.04a\n13.78 ± 3.67Compared with group A, ap < 0.05.Group A = class III, IV or V; Group B = class V + III or V + IV.\n\nComparison of the mean ADC and R2* values of kidneys in two groups of LN\n\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\left(\\overline{\\boldsymbol{x}} \\pm \\kern0.5em \\boldsymbol{s}\\right) $$\\end{document}x¯±s\n\nCompared with group A, ap < 0.05.\nGroup A = class III, IV or V; Group B = class V + III or V + IV.",
"Among 16 patients who repeated MR scan after prednisone combined with immunosuppressant treatment in 9 to 12 months, 12 patients got complete remission (CR) and 4 had no response (NR). Complete remission was defined as urinary protein excretion that was less than 0.3 g/24 h, with normal urine sediment, and serum albumin concentration of greater than 35 g/L, and stabilization (±15%) or improvement in serum creatinine. The ADC and R2* values of kidneys were significantly higher than pre-treatment in CR patients (all p < 0.05), while the ADC and R2* values being lower than pre-treatment in NR patients with no statistical significance (p > 0.05) (Table 3).Table 3\nComparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment\n\nADC (×10\n−3 \nmm\n2\n/s)\n\nR2* of cortex (1/sec)\n\nR2* of medulla (1/sec)\n\nGroup CR\n\nBefore\n2.41 ± 0.1610.25 ± 1.1913.04 ± 3.15\nAfter\n2.58 ± 0.14a\n11.77 ± 1.11a\n16.94 ± 3.50a\n\nGroup NR\n\nBefore\n2.43 ± 0.1210.84 ± 1.9613.25 ± 0.41\nAfter\n2.40 ± 0.2310.13 ± 1.6413.12 ± 0.47\nap < 0.05.ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.\n\nComparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment\n\n\nap < 0.05.\nADC: apparent diffusion coefficient; CR: complete remission; NR: no response.",
"The current evaluation of renal lesions in patients with lupus nephritis mainly depends on renal biopsy. Moreover, renal biopsy usually has to be repeated due to the possibility of changes in pathological patterns in LN. Although renal biopsy can provide the pathological information directly, it is not ideal for follow-up of treatment effects. Since it is invasive, SLE patients tend to have a great risk of bleeding due to coagulant function abnormality, or some patients are intolerable for renal biopsies if their kidneys are atrophy. Ultrasonography, commonly used in clinical practice, is simple and noninvasive. However, the accuracy of ultrasonic inspection is unstable depending on the skill of inspectors and body fat mass in patients. Scintigraphy is regarded as the gold standard for evaluating glomerular filtration rate (GFR), but the radioactive pollution, special equipment and other factors limit its clinical use scope [8]. Therefore, it seems critical to find a noninvasive and effective modality to dynamically evaluate renal function and pathological changes in patients with LN.\nBecause of its great promise in assessing physiological and pathophysiological parameters in vivo, functional magnetic resonance imaging has emerged as a useful technique to investigate organ functional characteristics beyond morphologic changes [9,10]. Kidneys are located at retro peritoneum and less affected by breath, with plenty of blood supply, high water content in tissues, and urine dilution and concentration function, that make kidneys become one of the most ideal organs for functional MR imaging [11,12]. DWI is a MR modality using strong bipolar gradients to create a sensitivity of the signal to the thermally induced Brownian motion of water molecules in tissue. Although the etiologies of chronic kidney diseases are different, their physiological and pathophysiological changes are usually common, including reduced blood supply of the kidneys, glomerulosclerosis, tubular atrophy and interstitial fibrosis, inducing abnormal diffusion of water molecules within renal tissue, which will contribute to ADC changes in diffusion-weighted imaging [13]. Diffusion of water molecules and perfusion of tissues can be affected by various renal lesions such as inflammation, necrosis, edema and fibrosis. The ADC (related only with water diffusion capacity) and perfusion scores (related to perfusion in the microvasculature) measured by DWI of multiple b-values can provide information concerning the characteristics of renal lesions and renal filtration function [14]. Chronic hypoxic injury in tubulointerstitium may be the common pathway of various chronic kidney diseases progressing to end stage renal disease (ESRD). BOLD MR is used for noninvasive assessment of the intrarenal oxygen content, based on paramagnetic properties of deoxyhemoglobin [12]. The change of R2* values can be found in the early stage of kidney damage in case of renal artery stenosis, unilateral ureteral obstruction, diabetes, hypertension and rejection after kidney transplantation [15,16].\nTogao et al. [17,18] reported that DWI can reflect and monitor the progression of renal fibrosis in mice with unilateral ureteral obstruction, and ADC values were well associated with the changes in renal microstructure. It was found in our previous study [19] that DWI can be helpful to detect the early stage renal failure of CKD. We also found a linear correlation between the ADC values and the stages of CKD. Moreover, the ADC values reflected the unilateral renal function, while renal DWI was closely associated with renal tubulointerstitial lesions, especially tubular atrophy.\nKaradeli et al. [20] found significant difference between the ADClow values and the urine protein in patients with SLE, but no significant correlation between GFRs and ADCs. The limitation of their study was the small number of patients and mild SLE (only 10 cases with LN). To the best of our knowledge, our investigation is the first pilot study to evaluate and re-evaluate the renal functional and pathological changes before and after induction treatment in patients with LN by MR functional imaging. We found that renal ADC values and R2* values of cortex and medulla in LN were significantly lower than those in normal kidneys, and these values were significantly higher than before in complete remission patients (all p < 0.05). The results indicated that MR functional imaging had emerging as a new noninvasive method in the evaluation of renal prognosis and therapeutic effects.\nThere was no significant difference of the ADC values and R2* values of cortex or medulla between bilateral kidneys, reflecting the characteristics of diffuse lesions in LN. We analyzed the correlation of the mean ADC values of bilateral kidneys and eGFR which represents the overall filtration function of bilateral kidneys in patients with LN. It was found that the mean ADC values of kidneys were significantly correlated to eGFR in this study. The mechanism might be: Glomerular sclerosis, interstitial fibrosis and tubular atrophy leads to the decline in glomerular filtration rate, and the limitation in the intercellular movement of water molecules, which causing the abnormal diffusion of water molecules within renal tissues, and then the decrease in renal ADC values. It also can explain why there was a negative correlation between the mean ADC values and pathology chronicity indexes in LN.\nMoreover, the R2* values of the renal medulla were negatively correlated with 24 hours proteinuria, the degree of tubulointerstitial lesions. In active lupus nephritis, glomerular cells proliferation or crescent formation, interstitial inflammatory cells infiltration, epithelial and endothelial immune complex deposition is common. Inflammatory factors and immune injury may lead to damage of microvasculature in glomeruli and surrounding renal tubules, which contribute to hemodynamic disturbance and hypoxia within kidneys. Chronic hypoxic injury may further induce glomerular sclerosis, interstitial fibrosis and tubular atrophy, which result in lower metabolism and reduction of oxygen consumption within kidneys, and finally cause a decrease in R2* values. Mixed membranous and proliferative LN (class V + III and V + IV) are usually known with more serious clinical presentation and more severe tubulointerstitial lesions than pure membranous LN (class V) or pure proliferative LN (class III and IV) [21]. In this study, R2* values of cortex in class V + III and V + IV were much lower than that in class III, IV or V, suggesting abnormal microcirculation and chronic hypoxia injury within kidneys with LN (V + III)/(V + IV). These findings should be considered preliminary.",
"In summary, diffusion-weighted imaging and blood oxygen level-dependent MR imaging are novel functional MRI techniques with repeatable nature that have potential to assess glomerular filtration, parenchymal oxygenation and renal pathological changes. This pilot study indicated that DWI and BOLD MR imaging might be used to noninvasively monitor disease activity and evaluate therapeutic efficacy in lupus nephritis. Further studies involve larger group of patients with lupus nephritis are necessary."
] |
[
"introduction",
"materials|methods",
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
"conclusion"
] |
[
"Lupus nephritis",
"Functional MR imaging",
"Renal function",
"Pathological changes"
] |
Background:
Systemic lupus erythematosus (SLE) is an autoimmune disease with variable presentations, course and prognosis. Approximately half a million people in Europe and seventy per 100 000 people in China suffer from SLE. Renal involvement is one of the major determinants of the outcome in patients with SLE [1]. Lupus nephritis (LN) is one of the most common secondary glomerulonephritis in China. Renal interstitial infiltrates, tubular necrosis and interstitial fibrosis can be found in 60-70% patients with LN. Interstitial lesions are often associated with glomerular function, and may play a pivotal role on progression and prognosis of lupus nephritis [2-4]. Nowadays there is no ideal method to simultaneously assess renal structure and function in patients with LN. Moreover, effective means to evaluate tubulointerstitial hypoxia state is lack as well. Therefore, it’s difficult to dynamically monitor the disease progression in these patients.
Functional magnetic resonance (MR) imaging techniques such as diffusion-weighted imaging (DWI) and blood oxygen level-dependent (BOLD) imaging have shown considerable value in the evaluation of renal function in health and renal diseases [5-7]. The aim of this study is to investigate the feasibility of functional renal MR imaging in the assessment of renal functional and structural changes in patients with lupus nephritis.
Methods:
Study population Sixty-five patients with lupus nephritis, diagnosed according to clinical manifestation, laboratory findings and renal histopathological changes, had been followed up since their admission in our department during the recent two years. They all met the diagnosis criteria of systemic lupus erythematosus defined by the American Rheumatism Association in 1997. All the patients were included after signing informed consent. There were 10 male and 55 female patients, with mean age of 34 ± 12 years old. Sixteen healthy volunteers, 2 men and 14 women with mean age of 35 ± 11 years old, had no history of primary or secondary renal diseases.
Sixty-five patients with lupus nephritis, diagnosed according to clinical manifestation, laboratory findings and renal histopathological changes, had been followed up since their admission in our department during the recent two years. They all met the diagnosis criteria of systemic lupus erythematosus defined by the American Rheumatism Association in 1997. All the patients were included after signing informed consent. There were 10 male and 55 female patients, with mean age of 34 ± 12 years old. Sixteen healthy volunteers, 2 men and 14 women with mean age of 35 ± 11 years old, had no history of primary or secondary renal diseases.
MR imaging All the LN patients and healthy volunteers underwent coronal echo-planar DW and BOLD MR imaging of the kidneys with a single breath-hold time. And 16 of the 65 patients with LN received functional MR imaging of kidneys again after 9 to 12 months induction treatment with immunosuppression. All of them fasted for 4–8 hours before the examination. MR imaging was performed with a 1.5 T MR imager (GE Twin-Speed, General Electric Medical Systems, Milwaukee, WI). DW MR imaging was performed with a spin echo-echo planar imaging (SE-EPI) sequence, and BOLD MRI was performed with a multiple gradient-recalled-echo (mGRE) sequence. The following parameters were used for DWI: 11 sections (section thickness, 5 mm; intersection gap, 1 mm); repetition time (TR), 2000 ms; echo time (TE), 52.9 ms; NEX, 2; field of view, 380 × 380 mm; matrix, 128 × 128; acquisition time, 16 s. The diffusion gradient b values of 0 and 500 s/mm2 were used. The following parameters were used for BOLD MRI: TR/TE, 110 ms/2.2-27.5 ms; flip angle, 60°; matrix, 132x128; section thickness, 5 mm; intersection gap, 1 mm. Three coronal sections were obtained for each kidney and eight T2*-weighted images were acquired for each section within one 16-second breath hold.
All the LN patients and healthy volunteers underwent coronal echo-planar DW and BOLD MR imaging of the kidneys with a single breath-hold time. And 16 of the 65 patients with LN received functional MR imaging of kidneys again after 9 to 12 months induction treatment with immunosuppression. All of them fasted for 4–8 hours before the examination. MR imaging was performed with a 1.5 T MR imager (GE Twin-Speed, General Electric Medical Systems, Milwaukee, WI). DW MR imaging was performed with a spin echo-echo planar imaging (SE-EPI) sequence, and BOLD MRI was performed with a multiple gradient-recalled-echo (mGRE) sequence. The following parameters were used for DWI: 11 sections (section thickness, 5 mm; intersection gap, 1 mm); repetition time (TR), 2000 ms; echo time (TE), 52.9 ms; NEX, 2; field of view, 380 × 380 mm; matrix, 128 × 128; acquisition time, 16 s. The diffusion gradient b values of 0 and 500 s/mm2 were used. The following parameters were used for BOLD MRI: TR/TE, 110 ms/2.2-27.5 ms; flip angle, 60°; matrix, 132x128; section thickness, 5 mm; intersection gap, 1 mm. Three coronal sections were obtained for each kidney and eight T2*-weighted images were acquired for each section within one 16-second breath hold.
Imaging analysis The apparent diffusion coefficient (ADC) and R2* values of the kidneys were calculated on a GE workstation (Sun Microsystems, ADW4.2) with Functool 2 image analysis software. In the transverse and coronal ADC map, freehand regions of interest (ROIs) were placed on the parenchyma of the kidneys, and fat within renal sinus were excluded. Three such ROIs were placed—one each in the upper pole, inter-polar region, and lower pole—and the mean of these three values were calculated.
R2* values of renal cortex and medulla were measured on T2* maps. The size of ROI was set to reduce the noise and the influence of partial volume effects. Avoiding visible vessels, ROIs were placed manually on the clear corticomedullary differentiation image. Three ROIs were placed on each side of renal cortex and medulla, and the mean of R2* values were calculated.
The relationship between the renal injury variables and the ADCs or R2* values were evaluated. The renal function was estimated using the abbreviated modification of diet in renal disease (MDRD) study equation. Renal tubulointerstitial lesions were scored with semi quantitative method: no lesion was 0, mild lesion (≤25%) was 1, moderate lesion (25% ~ 50%) was 2, and severe lesion (≥50%) was 3. There were 0–9 scores for tubulointerstitial lesions including 0–3 for tubular atrophy, 0–3 for interstitial fibrosis, 0–3 for interstitial inflammatory cells infiltration. The renal pathology activity index (AI) and chronicity index (CI) were evaluated in patients with LN.
The apparent diffusion coefficient (ADC) and R2* values of the kidneys were calculated on a GE workstation (Sun Microsystems, ADW4.2) with Functool 2 image analysis software. In the transverse and coronal ADC map, freehand regions of interest (ROIs) were placed on the parenchyma of the kidneys, and fat within renal sinus were excluded. Three such ROIs were placed—one each in the upper pole, inter-polar region, and lower pole—and the mean of these three values were calculated.
R2* values of renal cortex and medulla were measured on T2* maps. The size of ROI was set to reduce the noise and the influence of partial volume effects. Avoiding visible vessels, ROIs were placed manually on the clear corticomedullary differentiation image. Three ROIs were placed on each side of renal cortex and medulla, and the mean of R2* values were calculated.
The relationship between the renal injury variables and the ADCs or R2* values were evaluated. The renal function was estimated using the abbreviated modification of diet in renal disease (MDRD) study equation. Renal tubulointerstitial lesions were scored with semi quantitative method: no lesion was 0, mild lesion (≤25%) was 1, moderate lesion (25% ~ 50%) was 2, and severe lesion (≥50%) was 3. There were 0–9 scores for tubulointerstitial lesions including 0–3 for tubular atrophy, 0–3 for interstitial fibrosis, 0–3 for interstitial inflammatory cells infiltration. The renal pathology activity index (AI) and chronicity index (CI) were evaluated in patients with LN.
Statistical analysis The SPSS software was used (version 13.0; SPSS; Chicago, Illinois, USA). All results were expressed as mean ± SD for normally distributed data, median and range for skewed data and frequency (%) for categorical data. The distribution of clinical and laboratory attributes among the groups were evaluated by t test. The Pearson correlation analysis was used to evaluate the relation between the clinical or pathological variables and the ADCs or R2* values. P < 0.05 was considered as statistically significant.
The SPSS software was used (version 13.0; SPSS; Chicago, Illinois, USA). All results were expressed as mean ± SD for normally distributed data, median and range for skewed data and frequency (%) for categorical data. The distribution of clinical and laboratory attributes among the groups were evaluated by t test. The Pearson correlation analysis was used to evaluate the relation between the clinical or pathological variables and the ADCs or R2* values. P < 0.05 was considered as statistically significant.
Study population:
Sixty-five patients with lupus nephritis, diagnosed according to clinical manifestation, laboratory findings and renal histopathological changes, had been followed up since their admission in our department during the recent two years. They all met the diagnosis criteria of systemic lupus erythematosus defined by the American Rheumatism Association in 1997. All the patients were included after signing informed consent. There were 10 male and 55 female patients, with mean age of 34 ± 12 years old. Sixteen healthy volunteers, 2 men and 14 women with mean age of 35 ± 11 years old, had no history of primary or secondary renal diseases.
MR imaging:
All the LN patients and healthy volunteers underwent coronal echo-planar DW and BOLD MR imaging of the kidneys with a single breath-hold time. And 16 of the 65 patients with LN received functional MR imaging of kidneys again after 9 to 12 months induction treatment with immunosuppression. All of them fasted for 4–8 hours before the examination. MR imaging was performed with a 1.5 T MR imager (GE Twin-Speed, General Electric Medical Systems, Milwaukee, WI). DW MR imaging was performed with a spin echo-echo planar imaging (SE-EPI) sequence, and BOLD MRI was performed with a multiple gradient-recalled-echo (mGRE) sequence. The following parameters were used for DWI: 11 sections (section thickness, 5 mm; intersection gap, 1 mm); repetition time (TR), 2000 ms; echo time (TE), 52.9 ms; NEX, 2; field of view, 380 × 380 mm; matrix, 128 × 128; acquisition time, 16 s. The diffusion gradient b values of 0 and 500 s/mm2 were used. The following parameters were used for BOLD MRI: TR/TE, 110 ms/2.2-27.5 ms; flip angle, 60°; matrix, 132x128; section thickness, 5 mm; intersection gap, 1 mm. Three coronal sections were obtained for each kidney and eight T2*-weighted images were acquired for each section within one 16-second breath hold.
Imaging analysis:
The apparent diffusion coefficient (ADC) and R2* values of the kidneys were calculated on a GE workstation (Sun Microsystems, ADW4.2) with Functool 2 image analysis software. In the transverse and coronal ADC map, freehand regions of interest (ROIs) were placed on the parenchyma of the kidneys, and fat within renal sinus were excluded. Three such ROIs were placed—one each in the upper pole, inter-polar region, and lower pole—and the mean of these three values were calculated.
R2* values of renal cortex and medulla were measured on T2* maps. The size of ROI was set to reduce the noise and the influence of partial volume effects. Avoiding visible vessels, ROIs were placed manually on the clear corticomedullary differentiation image. Three ROIs were placed on each side of renal cortex and medulla, and the mean of R2* values were calculated.
The relationship between the renal injury variables and the ADCs or R2* values were evaluated. The renal function was estimated using the abbreviated modification of diet in renal disease (MDRD) study equation. Renal tubulointerstitial lesions were scored with semi quantitative method: no lesion was 0, mild lesion (≤25%) was 1, moderate lesion (25% ~ 50%) was 2, and severe lesion (≥50%) was 3. There were 0–9 scores for tubulointerstitial lesions including 0–3 for tubular atrophy, 0–3 for interstitial fibrosis, 0–3 for interstitial inflammatory cells infiltration. The renal pathology activity index (AI) and chronicity index (CI) were evaluated in patients with LN.
Statistical analysis:
The SPSS software was used (version 13.0; SPSS; Chicago, Illinois, USA). All results were expressed as mean ± SD for normally distributed data, median and range for skewed data and frequency (%) for categorical data. The distribution of clinical and laboratory attributes among the groups were evaluated by t test. The Pearson correlation analysis was used to evaluate the relation between the clinical or pathological variables and the ADCs or R2* values. P < 0.05 was considered as statistically significant.
Results:
The mean ADC and R2* values of kidneys in patients with LN were lower than that in healthy volunteers The mean ADC values of kidneys in healthy volunteers were 2.52 ± 0.17 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 12.63 ± 1.40/sec and 18.14 ± 2.51/sec respectively. The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than the ADC and R2* values of kidneys in healthy volunteers (p = 0.048, p = 0.045 and p = 0.008, respectively) (Table 1). There was no significant difference of the ADC and R2* values between bilateral kidneys either in volunteers or in patients with LN. Moreover, the ADC and R2* values were much lower in patients with severe lupus nephritis (Figures 1 and 2).Table 1
The mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers
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\begin{document}$$ \left(\overline{\boldsymbol{x}} \pm \kern0.5em \boldsymbol{s}\right) $$\end{document}x¯±s
ADC (×10
−3
mm
2
/ s)
R2* of cortex (1/sec)
R2*of medulla (1/sec)
Normal kidneys
2.52 ± 0.1712.63 ± 1.4018.14 ± 2.51
Kidneys with LN
2.40 ± 0.2511.03 ± 1.6014.05 ± 3.38
P value
0.0480.0450.008ADC: apparent diffusion coefficient; LN: lupus nephritis.Figure 1
Renal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.Figure 2
Renal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.
The mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers
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ADC: apparent diffusion coefficient; LN: lupus nephritis.
Renal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.
Renal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.
The mean ADC values of kidneys in healthy volunteers were 2.52 ± 0.17 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 12.63 ± 1.40/sec and 18.14 ± 2.51/sec respectively. The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than the ADC and R2* values of kidneys in healthy volunteers (p = 0.048, p = 0.045 and p = 0.008, respectively) (Table 1). There was no significant difference of the ADC and R2* values between bilateral kidneys either in volunteers or in patients with LN. Moreover, the ADC and R2* values were much lower in patients with severe lupus nephritis (Figures 1 and 2).Table 1
The mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers
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ADC (×10
−3
mm
2
/ s)
R2* of cortex (1/sec)
R2*of medulla (1/sec)
Normal kidneys
2.52 ± 0.1712.63 ± 1.4018.14 ± 2.51
Kidneys with LN
2.40 ± 0.2511.03 ± 1.6014.05 ± 3.38
P value
0.0480.0450.008ADC: apparent diffusion coefficient; LN: lupus nephritis.Figure 1
Renal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.Figure 2
Renal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.
The mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers
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ADC: apparent diffusion coefficient; LN: lupus nephritis.
Renal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.
Renal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.
The correlation between imaging values and renal function or pathological changes in patients with LN In the patients with LN, the mean ADC values were correlated with glomerular filtration rate estimated by abbreviated MDRD equation (eGFR) (r = 0.510, p < 0.01) (Figure 3). There was a negative correlation between the mean ADC values and pathology chronicity indexes (r = −0.249, p < 0.05). Moreover, the R2* values of the renal medulla were negatively correlated with 24 hours proteinuria (r = −0.244, p < 0.05), the degree of tubulointerstitial lesions (r = −0.242, p < 0.05).Figure 3
The relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).
The relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).
In the patients with LN, the mean ADC values were correlated with glomerular filtration rate estimated by abbreviated MDRD equation (eGFR) (r = 0.510, p < 0.01) (Figure 3). There was a negative correlation between the mean ADC values and pathology chronicity indexes (r = −0.249, p < 0.05). Moreover, the R2* values of the renal medulla were negatively correlated with 24 hours proteinuria (r = −0.244, p < 0.05), the degree of tubulointerstitial lesions (r = −0.242, p < 0.05).Figure 3
The relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).
The relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).
Comparison of the mean ADC and R2* values of kidneys in two groups of LN Of the 65 patients with LN, 6 were class I or II, 59 were class III, IV, V or combination. Fifty-nine patients were divided into 2 groups: Group A including 31 patients with LN class III, IV or V, and Group B including 28 patients with LN class V + III or V + IV. Urine N-acetyl-β-D-glucosaminidase (NAG) level in Group A was much lower than that in Group B (17.28 ± 11.23 vs 27.69 ± 13.24 U/L, p = 0.0037). There were 2 patients with abnormal tubular acidification, and 5 patients with impaired urinary concentrating function in Group A, while there being 9 patients with abnormal tubular acidification and 10 patients with impaired urinary concentrating function in Group B. The mean ADC values in Group A were higher than Group B ((2.50 ± 0.26) × 10−3 mm2/s vs (2.36 ± 0.21) × 10−3 mm2/s,p < 0.05). The mean R2* values of cortex in Group A were higher than Group B (11.70 ± 2.24/sec vs 10.57 ± 1.04/sec, p < 0.05), while mean R2* values of medulla in Group A being similar with that in Group B (13.99 ± 3.31/sec vs 13.78 ± 3.67/sec,p > 0.05) (Table 2).Table 2
Comparison of the mean ADC and R2* values of kidneys in two groups of LN
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n
Mean ADC (×10
−3
mm
2
/ s)
R2* values of cortex (1/sec)
R2* values of medulla (1/sec)
Group A
312.50 ± 0.2611.70 ± 2.2413.99 ± 3.31
Group B
282.36 ± 0.21a
10.57 ± 1.04a
13.78 ± 3.67Compared with group A, ap < 0.05.Group A = class III, IV or V; Group B = class V + III or V + IV.
Comparison of the mean ADC and R2* values of kidneys in two groups of LN
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Compared with group A, ap < 0.05.
Group A = class III, IV or V; Group B = class V + III or V + IV.
Of the 65 patients with LN, 6 were class I or II, 59 were class III, IV, V or combination. Fifty-nine patients were divided into 2 groups: Group A including 31 patients with LN class III, IV or V, and Group B including 28 patients with LN class V + III or V + IV. Urine N-acetyl-β-D-glucosaminidase (NAG) level in Group A was much lower than that in Group B (17.28 ± 11.23 vs 27.69 ± 13.24 U/L, p = 0.0037). There were 2 patients with abnormal tubular acidification, and 5 patients with impaired urinary concentrating function in Group A, while there being 9 patients with abnormal tubular acidification and 10 patients with impaired urinary concentrating function in Group B. The mean ADC values in Group A were higher than Group B ((2.50 ± 0.26) × 10−3 mm2/s vs (2.36 ± 0.21) × 10−3 mm2/s,p < 0.05). The mean R2* values of cortex in Group A were higher than Group B (11.70 ± 2.24/sec vs 10.57 ± 1.04/sec, p < 0.05), while mean R2* values of medulla in Group A being similar with that in Group B (13.99 ± 3.31/sec vs 13.78 ± 3.67/sec,p > 0.05) (Table 2).Table 2
Comparison of the mean ADC and R2* values of kidneys in two groups of LN
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n
Mean ADC (×10
−3
mm
2
/ s)
R2* values of cortex (1/sec)
R2* values of medulla (1/sec)
Group A
312.50 ± 0.2611.70 ± 2.2413.99 ± 3.31
Group B
282.36 ± 0.21a
10.57 ± 1.04a
13.78 ± 3.67Compared with group A, ap < 0.05.Group A = class III, IV or V; Group B = class V + III or V + IV.
Comparison of the mean ADC and R2* values of kidneys in two groups of LN
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Compared with group A, ap < 0.05.
Group A = class III, IV or V; Group B = class V + III or V + IV.
The changes in ADC and R2* values of kidneys after the induction treatment in patients with LN Among 16 patients who repeated MR scan after prednisone combined with immunosuppressant treatment in 9 to 12 months, 12 patients got complete remission (CR) and 4 had no response (NR). Complete remission was defined as urinary protein excretion that was less than 0.3 g/24 h, with normal urine sediment, and serum albumin concentration of greater than 35 g/L, and stabilization (±15%) or improvement in serum creatinine. The ADC and R2* values of kidneys were significantly higher than pre-treatment in CR patients (all p < 0.05), while the ADC and R2* values being lower than pre-treatment in NR patients with no statistical significance (p > 0.05) (Table 3).Table 3
Comparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment
ADC (×10
−3
mm
2
/s)
R2* of cortex (1/sec)
R2* of medulla (1/sec)
Group CR
Before
2.41 ± 0.1610.25 ± 1.1913.04 ± 3.15
After
2.58 ± 0.14a
11.77 ± 1.11a
16.94 ± 3.50a
Group NR
Before
2.43 ± 0.1210.84 ± 1.9613.25 ± 0.41
After
2.40 ± 0.2310.13 ± 1.6413.12 ± 0.47
ap < 0.05.ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.
Comparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment
ap < 0.05.
ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.
Among 16 patients who repeated MR scan after prednisone combined with immunosuppressant treatment in 9 to 12 months, 12 patients got complete remission (CR) and 4 had no response (NR). Complete remission was defined as urinary protein excretion that was less than 0.3 g/24 h, with normal urine sediment, and serum albumin concentration of greater than 35 g/L, and stabilization (±15%) or improvement in serum creatinine. The ADC and R2* values of kidneys were significantly higher than pre-treatment in CR patients (all p < 0.05), while the ADC and R2* values being lower than pre-treatment in NR patients with no statistical significance (p > 0.05) (Table 3).Table 3
Comparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment
ADC (×10
−3
mm
2
/s)
R2* of cortex (1/sec)
R2* of medulla (1/sec)
Group CR
Before
2.41 ± 0.1610.25 ± 1.1913.04 ± 3.15
After
2.58 ± 0.14a
11.77 ± 1.11a
16.94 ± 3.50a
Group NR
Before
2.43 ± 0.1210.84 ± 1.9613.25 ± 0.41
After
2.40 ± 0.2310.13 ± 1.6413.12 ± 0.47
ap < 0.05.ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.
Comparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment
ap < 0.05.
ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.
The mean ADC and R2* values of kidneys in patients with LN were lower than that in healthy volunteers:
The mean ADC values of kidneys in healthy volunteers were 2.52 ± 0.17 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 12.63 ± 1.40/sec and 18.14 ± 2.51/sec respectively. The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10−3 mm2/ s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than the ADC and R2* values of kidneys in healthy volunteers (p = 0.048, p = 0.045 and p = 0.008, respectively) (Table 1). There was no significant difference of the ADC and R2* values between bilateral kidneys either in volunteers or in patients with LN. Moreover, the ADC and R2* values were much lower in patients with severe lupus nephritis (Figures 1 and 2).Table 1
The mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers
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ADC (×10
−3
mm
2
/ s)
R2* of cortex (1/sec)
R2*of medulla (1/sec)
Normal kidneys
2.52 ± 0.1712.63 ± 1.4018.14 ± 2.51
Kidneys with LN
2.40 ± 0.2511.03 ± 1.6014.05 ± 3.38
P value
0.0480.0450.008ADC: apparent diffusion coefficient; LN: lupus nephritis.Figure 1
Renal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.Figure 2
Renal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.
The mean ADC and R2* values of kidneys in patients with LN compared with healthy volunteers
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ADC: apparent diffusion coefficient; LN: lupus nephritis.
Renal functional MR imaging of Patient A with LN-IV(S)A. (a) DWI imaging. (b) T2* imaging. The proteinuria was 1.3 g/24 h and serum creatinine was 52 μmol/L, the renal pathology AI was 12 and CI was 0. Renal BOLD images showed a clear demarcation between cortex and medulla. The ADC value of left kidney was 2.81 x10−3 mm2/s and that of right kidney was 2.78 x10−3 mm2/s, the R2* values were similar to normal kidneys.
Renal functional MR imaging of Patient B with LN-IV(G)A/C + V. (a) DWI imaging. (b) T2* imaging. The proteinuria was 6.1 g/24 h and serum creatinine was 422 μmol/L, the renal pathology AI was 19 and CI was 4. The differentiation between cortex and medulla became less apparent in T2* imaging. The ADC value of left kidney was 2.14 x10−3 mm2/s and that of right kidney was 2.20 x10−3 mm2/s, the R2* values of cortex and medulla were 10.79/s and 8.67/s respectively.
The correlation between imaging values and renal function or pathological changes in patients with LN:
In the patients with LN, the mean ADC values were correlated with glomerular filtration rate estimated by abbreviated MDRD equation (eGFR) (r = 0.510, p < 0.01) (Figure 3). There was a negative correlation between the mean ADC values and pathology chronicity indexes (r = −0.249, p < 0.05). Moreover, the R2* values of the renal medulla were negatively correlated with 24 hours proteinuria (r = −0.244, p < 0.05), the degree of tubulointerstitial lesions (r = −0.242, p < 0.05).Figure 3
The relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).
The relationship of mean ADC and eGFR. In the patients with LN, the mean ADC values were significantly correlated with eGFR (r = 0.510, p < 0.01).
Comparison of the mean ADC and R2* values of kidneys in two groups of LN:
Of the 65 patients with LN, 6 were class I or II, 59 were class III, IV, V or combination. Fifty-nine patients were divided into 2 groups: Group A including 31 patients with LN class III, IV or V, and Group B including 28 patients with LN class V + III or V + IV. Urine N-acetyl-β-D-glucosaminidase (NAG) level in Group A was much lower than that in Group B (17.28 ± 11.23 vs 27.69 ± 13.24 U/L, p = 0.0037). There were 2 patients with abnormal tubular acidification, and 5 patients with impaired urinary concentrating function in Group A, while there being 9 patients with abnormal tubular acidification and 10 patients with impaired urinary concentrating function in Group B. The mean ADC values in Group A were higher than Group B ((2.50 ± 0.26) × 10−3 mm2/s vs (2.36 ± 0.21) × 10−3 mm2/s,p < 0.05). The mean R2* values of cortex in Group A were higher than Group B (11.70 ± 2.24/sec vs 10.57 ± 1.04/sec, p < 0.05), while mean R2* values of medulla in Group A being similar with that in Group B (13.99 ± 3.31/sec vs 13.78 ± 3.67/sec,p > 0.05) (Table 2).Table 2
Comparison of the mean ADC and R2* values of kidneys in two groups of LN
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n
Mean ADC (×10
−3
mm
2
/ s)
R2* values of cortex (1/sec)
R2* values of medulla (1/sec)
Group A
312.50 ± 0.2611.70 ± 2.2413.99 ± 3.31
Group B
282.36 ± 0.21a
10.57 ± 1.04a
13.78 ± 3.67Compared with group A, ap < 0.05.Group A = class III, IV or V; Group B = class V + III or V + IV.
Comparison of the mean ADC and R2* values of kidneys in two groups of LN
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Compared with group A, ap < 0.05.
Group A = class III, IV or V; Group B = class V + III or V + IV.
The changes in ADC and R2* values of kidneys after the induction treatment in patients with LN:
Among 16 patients who repeated MR scan after prednisone combined with immunosuppressant treatment in 9 to 12 months, 12 patients got complete remission (CR) and 4 had no response (NR). Complete remission was defined as urinary protein excretion that was less than 0.3 g/24 h, with normal urine sediment, and serum albumin concentration of greater than 35 g/L, and stabilization (±15%) or improvement in serum creatinine. The ADC and R2* values of kidneys were significantly higher than pre-treatment in CR patients (all p < 0.05), while the ADC and R2* values being lower than pre-treatment in NR patients with no statistical significance (p > 0.05) (Table 3).Table 3
Comparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment
ADC (×10
−3
mm
2
/s)
R2* of cortex (1/sec)
R2* of medulla (1/sec)
Group CR
Before
2.41 ± 0.1610.25 ± 1.1913.04 ± 3.15
After
2.58 ± 0.14a
11.77 ± 1.11a
16.94 ± 3.50a
Group NR
Before
2.43 ± 0.1210.84 ± 1.9613.25 ± 0.41
After
2.40 ± 0.2310.13 ± 1.6413.12 ± 0.47
ap < 0.05.ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.
Comparison of the mean ADC and R2* values of kidneys in patients with LN before and after induction treatment
ap < 0.05.
ADC: apparent diffusion coefficient; CR: complete remission; NR: no response.
Discussion:
The current evaluation of renal lesions in patients with lupus nephritis mainly depends on renal biopsy. Moreover, renal biopsy usually has to be repeated due to the possibility of changes in pathological patterns in LN. Although renal biopsy can provide the pathological information directly, it is not ideal for follow-up of treatment effects. Since it is invasive, SLE patients tend to have a great risk of bleeding due to coagulant function abnormality, or some patients are intolerable for renal biopsies if their kidneys are atrophy. Ultrasonography, commonly used in clinical practice, is simple and noninvasive. However, the accuracy of ultrasonic inspection is unstable depending on the skill of inspectors and body fat mass in patients. Scintigraphy is regarded as the gold standard for evaluating glomerular filtration rate (GFR), but the radioactive pollution, special equipment and other factors limit its clinical use scope [8]. Therefore, it seems critical to find a noninvasive and effective modality to dynamically evaluate renal function and pathological changes in patients with LN.
Because of its great promise in assessing physiological and pathophysiological parameters in vivo, functional magnetic resonance imaging has emerged as a useful technique to investigate organ functional characteristics beyond morphologic changes [9,10]. Kidneys are located at retro peritoneum and less affected by breath, with plenty of blood supply, high water content in tissues, and urine dilution and concentration function, that make kidneys become one of the most ideal organs for functional MR imaging [11,12]. DWI is a MR modality using strong bipolar gradients to create a sensitivity of the signal to the thermally induced Brownian motion of water molecules in tissue. Although the etiologies of chronic kidney diseases are different, their physiological and pathophysiological changes are usually common, including reduced blood supply of the kidneys, glomerulosclerosis, tubular atrophy and interstitial fibrosis, inducing abnormal diffusion of water molecules within renal tissue, which will contribute to ADC changes in diffusion-weighted imaging [13]. Diffusion of water molecules and perfusion of tissues can be affected by various renal lesions such as inflammation, necrosis, edema and fibrosis. The ADC (related only with water diffusion capacity) and perfusion scores (related to perfusion in the microvasculature) measured by DWI of multiple b-values can provide information concerning the characteristics of renal lesions and renal filtration function [14]. Chronic hypoxic injury in tubulointerstitium may be the common pathway of various chronic kidney diseases progressing to end stage renal disease (ESRD). BOLD MR is used for noninvasive assessment of the intrarenal oxygen content, based on paramagnetic properties of deoxyhemoglobin [12]. The change of R2* values can be found in the early stage of kidney damage in case of renal artery stenosis, unilateral ureteral obstruction, diabetes, hypertension and rejection after kidney transplantation [15,16].
Togao et al. [17,18] reported that DWI can reflect and monitor the progression of renal fibrosis in mice with unilateral ureteral obstruction, and ADC values were well associated with the changes in renal microstructure. It was found in our previous study [19] that DWI can be helpful to detect the early stage renal failure of CKD. We also found a linear correlation between the ADC values and the stages of CKD. Moreover, the ADC values reflected the unilateral renal function, while renal DWI was closely associated with renal tubulointerstitial lesions, especially tubular atrophy.
Karadeli et al. [20] found significant difference between the ADClow values and the urine protein in patients with SLE, but no significant correlation between GFRs and ADCs. The limitation of their study was the small number of patients and mild SLE (only 10 cases with LN). To the best of our knowledge, our investigation is the first pilot study to evaluate and re-evaluate the renal functional and pathological changes before and after induction treatment in patients with LN by MR functional imaging. We found that renal ADC values and R2* values of cortex and medulla in LN were significantly lower than those in normal kidneys, and these values were significantly higher than before in complete remission patients (all p < 0.05). The results indicated that MR functional imaging had emerging as a new noninvasive method in the evaluation of renal prognosis and therapeutic effects.
There was no significant difference of the ADC values and R2* values of cortex or medulla between bilateral kidneys, reflecting the characteristics of diffuse lesions in LN. We analyzed the correlation of the mean ADC values of bilateral kidneys and eGFR which represents the overall filtration function of bilateral kidneys in patients with LN. It was found that the mean ADC values of kidneys were significantly correlated to eGFR in this study. The mechanism might be: Glomerular sclerosis, interstitial fibrosis and tubular atrophy leads to the decline in glomerular filtration rate, and the limitation in the intercellular movement of water molecules, which causing the abnormal diffusion of water molecules within renal tissues, and then the decrease in renal ADC values. It also can explain why there was a negative correlation between the mean ADC values and pathology chronicity indexes in LN.
Moreover, the R2* values of the renal medulla were negatively correlated with 24 hours proteinuria, the degree of tubulointerstitial lesions. In active lupus nephritis, glomerular cells proliferation or crescent formation, interstitial inflammatory cells infiltration, epithelial and endothelial immune complex deposition is common. Inflammatory factors and immune injury may lead to damage of microvasculature in glomeruli and surrounding renal tubules, which contribute to hemodynamic disturbance and hypoxia within kidneys. Chronic hypoxic injury may further induce glomerular sclerosis, interstitial fibrosis and tubular atrophy, which result in lower metabolism and reduction of oxygen consumption within kidneys, and finally cause a decrease in R2* values. Mixed membranous and proliferative LN (class V + III and V + IV) are usually known with more serious clinical presentation and more severe tubulointerstitial lesions than pure membranous LN (class V) or pure proliferative LN (class III and IV) [21]. In this study, R2* values of cortex in class V + III and V + IV were much lower than that in class III, IV or V, suggesting abnormal microcirculation and chronic hypoxia injury within kidneys with LN (V + III)/(V + IV). These findings should be considered preliminary.
Conclusions:
In summary, diffusion-weighted imaging and blood oxygen level-dependent MR imaging are novel functional MRI techniques with repeatable nature that have potential to assess glomerular filtration, parenchymal oxygenation and renal pathological changes. This pilot study indicated that DWI and BOLD MR imaging might be used to noninvasively monitor disease activity and evaluate therapeutic efficacy in lupus nephritis. Further studies involve larger group of patients with lupus nephritis are necessary.
|
Background:
Lupus nephritis (LN) is one of most common secondary glomerulonephritis. There is no ideal method to simultaneously assess renal structure and function in patients with LN. The aim of this study is to investigate the utility of diffusion weighted imaging (DWI) and blood oxygen level-dependent (BOLD) MR imaging in the assessment of renal involvement and pathological changes in patients with LN.
Methods:
Sixty-five patients with LN and 16 healthy volunteers underwent coronal echo-planar DWI and BOLD MR imaging of the kidneys. The apparent diffusion coefficient (ADC) and R2* values of the kidneys were calculated with b values of 0 and 500 s/mm(2). The relationship between the renal injury variables and the ADCs or R2* values were evaluated. And 16 of 65 patients with LN underwent a repeated evaluation after the induction treatment for 9 to 12 months.
Results:
The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10(-3) mm(2)/s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than that in volunteers. In patients with LN, the mean ADC values were correlated with eGFR (r = 0.510, p < 0.01). There was a negative correlation between the mean ADC values and renal pathology chronicity indexes (r = -0.249, p < 0.05), the R2* values of the renal medulla and proteinuria (r = -0.244, p < 0.05), and the degree of tubulointerstitial lesions (r = -0.242, p < 0.05). The ADC and R2* values of kidneys were significantly higher than those of pre-treatment in complete remission patients.
Conclusions:
DWI and BOLD MR imaging of kidneys may be used to noninvasively monitor the disease activity and evaluate therapeutic efficacy in lupus nephritis.
|
Background:
Systemic lupus erythematosus (SLE) is an autoimmune disease with variable presentations, course and prognosis. Approximately half a million people in Europe and seventy per 100 000 people in China suffer from SLE. Renal involvement is one of the major determinants of the outcome in patients with SLE [1]. Lupus nephritis (LN) is one of the most common secondary glomerulonephritis in China. Renal interstitial infiltrates, tubular necrosis and interstitial fibrosis can be found in 60-70% patients with LN. Interstitial lesions are often associated with glomerular function, and may play a pivotal role on progression and prognosis of lupus nephritis [2-4]. Nowadays there is no ideal method to simultaneously assess renal structure and function in patients with LN. Moreover, effective means to evaluate tubulointerstitial hypoxia state is lack as well. Therefore, it’s difficult to dynamically monitor the disease progression in these patients.
Functional magnetic resonance (MR) imaging techniques such as diffusion-weighted imaging (DWI) and blood oxygen level-dependent (BOLD) imaging have shown considerable value in the evaluation of renal function in health and renal diseases [5-7]. The aim of this study is to investigate the feasibility of functional renal MR imaging in the assessment of renal functional and structural changes in patients with lupus nephritis.
Conclusions:
In summary, diffusion-weighted imaging and blood oxygen level-dependent MR imaging are novel functional MRI techniques with repeatable nature that have potential to assess glomerular filtration, parenchymal oxygenation and renal pathological changes. This pilot study indicated that DWI and BOLD MR imaging might be used to noninvasively monitor disease activity and evaluate therapeutic efficacy in lupus nephritis. Further studies involve larger group of patients with lupus nephritis are necessary.
|
Background:
Lupus nephritis (LN) is one of most common secondary glomerulonephritis. There is no ideal method to simultaneously assess renal structure and function in patients with LN. The aim of this study is to investigate the utility of diffusion weighted imaging (DWI) and blood oxygen level-dependent (BOLD) MR imaging in the assessment of renal involvement and pathological changes in patients with LN.
Methods:
Sixty-five patients with LN and 16 healthy volunteers underwent coronal echo-planar DWI and BOLD MR imaging of the kidneys. The apparent diffusion coefficient (ADC) and R2* values of the kidneys were calculated with b values of 0 and 500 s/mm(2). The relationship between the renal injury variables and the ADCs or R2* values were evaluated. And 16 of 65 patients with LN underwent a repeated evaluation after the induction treatment for 9 to 12 months.
Results:
The mean ADC values of kidneys in patients with LN were 2.40 ± 0.25 × 10(-3) mm(2)/s, the mean R2* values of the renal cortex and medulla were 11.03 ± 1.60/sec and 14.05 ± 3.38/sec respectively, which were all significantly lower than that in volunteers. In patients with LN, the mean ADC values were correlated with eGFR (r = 0.510, p < 0.01). There was a negative correlation between the mean ADC values and renal pathology chronicity indexes (r = -0.249, p < 0.05), the R2* values of the renal medulla and proteinuria (r = -0.244, p < 0.05), and the degree of tubulointerstitial lesions (r = -0.242, p < 0.05). The ADC and R2* values of kidneys were significantly higher than those of pre-treatment in complete remission patients.
Conclusions:
DWI and BOLD MR imaging of kidneys may be used to noninvasively monitor the disease activity and evaluate therapeutic efficacy in lupus nephritis.
| 10,036 | 364 | 13 |
[
"values",
"adc",
"patients",
"r2",
"renal",
"ln",
"r2 values",
"usepackage",
"mean",
"kidneys"
] |
[
"test",
"test"
] | null |
[CONTENT] Lupus nephritis | Functional MR imaging | Renal function | Pathological changes [SUMMARY]
| null |
[CONTENT] Lupus nephritis | Functional MR imaging | Renal function | Pathological changes [SUMMARY]
|
[CONTENT] Lupus nephritis | Functional MR imaging | Renal function | Pathological changes [SUMMARY]
|
[CONTENT] Lupus nephritis | Functional MR imaging | Renal function | Pathological changes [SUMMARY]
|
[CONTENT] Lupus nephritis | Functional MR imaging | Renal function | Pathological changes [SUMMARY]
|
[CONTENT] Adult | Case-Control Studies | Diffusion Magnetic Resonance Imaging | Female | Humans | Kidney | Kidney Function Tests | Lupus Nephritis | Male | Middle Aged | Oxygen | Young Adult [SUMMARY]
| null |
[CONTENT] Adult | Case-Control Studies | Diffusion Magnetic Resonance Imaging | Female | Humans | Kidney | Kidney Function Tests | Lupus Nephritis | Male | Middle Aged | Oxygen | Young Adult [SUMMARY]
|
[CONTENT] Adult | Case-Control Studies | Diffusion Magnetic Resonance Imaging | Female | Humans | Kidney | Kidney Function Tests | Lupus Nephritis | Male | Middle Aged | Oxygen | Young Adult [SUMMARY]
|
[CONTENT] Adult | Case-Control Studies | Diffusion Magnetic Resonance Imaging | Female | Humans | Kidney | Kidney Function Tests | Lupus Nephritis | Male | Middle Aged | Oxygen | Young Adult [SUMMARY]
|
[CONTENT] Adult | Case-Control Studies | Diffusion Magnetic Resonance Imaging | Female | Humans | Kidney | Kidney Function Tests | Lupus Nephritis | Male | Middle Aged | Oxygen | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] values | adc | patients | r2 | renal | ln | r2 values | usepackage | mean | kidneys [SUMMARY]
| null |
[CONTENT] values | adc | patients | r2 | renal | ln | r2 values | usepackage | mean | kidneys [SUMMARY]
|
[CONTENT] values | adc | patients | r2 | renal | ln | r2 values | usepackage | mean | kidneys [SUMMARY]
|
[CONTENT] values | adc | patients | r2 | renal | ln | r2 values | usepackage | mean | kidneys [SUMMARY]
|
[CONTENT] values | adc | patients | r2 | renal | ln | r2 values | usepackage | mean | kidneys [SUMMARY]
|
[CONTENT] renal | sle | imaging | lupus | interstitial | people | china | patients | prognosis | progression [SUMMARY]
| null |
[CONTENT] usepackage | adc | group | values | r2 | r2 values | mean adc | ln | sec | mean [SUMMARY]
|
[CONTENT] imaging | lupus | lupus nephritis | mr imaging | nephritis | mri techniques repeatable nature | novel functional mri techniques | summary | summary diffusion | summary diffusion weighted [SUMMARY]
|
[CONTENT] renal | values | adc | patients | usepackage | r2 | imaging | group | r2 values | mean [SUMMARY]
|
[CONTENT] renal | values | adc | patients | usepackage | r2 | imaging | group | r2 values | mean [SUMMARY]
|
[CONTENT] LN ||| LN ||| DWI ||| BOLD | LN [SUMMARY]
| null |
[CONTENT] LN | 2.40 | 0.25 × 10(-3 | mm(2)/s | R2 | 11.03 | 1.60 | sec | 14.05 | 3.38 | sec ||| LN | eGFR | 0.510 ||| proteinuria (r = -0.244 ||| ADC | R2 [SUMMARY]
|
[CONTENT] DWI | BOLD [SUMMARY]
|
[CONTENT] LN ||| LN ||| DWI ||| BOLD | LN ||| Sixty-five | LN | 16 | DWI | BOLD ||| R2 | 0 | 500 ||| R2 ||| 16 | 65 | LN | 9 to 12 months ||| ||| LN | 2.40 | 0.25 × 10(-3 | mm(2)/s | R2 | 11.03 | 1.60 | sec | 14.05 | 3.38 | sec ||| LN | eGFR | 0.510 ||| proteinuria (r = -0.244 ||| ADC | R2 ||| DWI | BOLD [SUMMARY]
|
[CONTENT] LN ||| LN ||| DWI ||| BOLD | LN ||| Sixty-five | LN | 16 | DWI | BOLD ||| R2 | 0 | 500 ||| R2 ||| 16 | 65 | LN | 9 to 12 months ||| ||| LN | 2.40 | 0.25 × 10(-3 | mm(2)/s | R2 | 11.03 | 1.60 | sec | 14.05 | 3.38 | sec ||| LN | eGFR | 0.510 ||| proteinuria (r = -0.244 ||| ADC | R2 ||| DWI | BOLD [SUMMARY]
|
|||||
Reported orofacial adverse effects of COVID-19 vaccines: The knowns and the unknowns.
|
33527524
|
Adverse events associated with vaccine administration can manifest in the oral cavity and orofacial region. Hence, the aim of this study was to compare the orofacial adverse effects of two recently authorised COVID-19 vaccines, namely BNT162b2 and mRNA-1273.
|
INTRODUCTION
|
Publicly available data on BNT162b2 and mRNA-1273 vaccines were accessed from the relevant regulatory authorities in the United States, Canada, European Union and United Kingdom. Both patient/recipient information and healthcare professional fact sheets for each of these drugs were manually searched to find their orofacial adverse effects.
|
METHODS
|
Adverse events affecting the orofacial region were reported for both vaccines. These were rare and included acute peripheral facial paralysis (Bell's palsy), facial swelling, and swelling of the lips, face or tongue associated with anaphylaxis. There was heterogeneity in the acknowledgement of vaccine-related adverse events in North America compared with Europe.
|
RESULTS
|
Globally, there are inconsistencies in the description of adverse effects presenting in the orofacial region of the COVID-19 vaccines BNT162b2 and mRNA-1273. We believe that awareness of these orofacial manifestations will improve recognition, management and reporting of vaccine-related adverse effects.
|
CONCLUSION
|
[
"BNT162 Vaccine",
"COVID-19",
"COVID-19 Vaccines",
"Europe",
"Humans",
"SARS-CoV-2",
"United Kingdom",
"United States"
] |
8013400
|
INTRODUCTION
|
Adverse drug reactions (ADRs) and medication‐related events are potentially life‐threatening consequences of the use of medicines, including vaccines. By and large, the benefits of vaccination clearly outweigh the risks—vaccines prevent between 2 and 3 million deaths from infectious diseases every year.
1
It is important, however, that adverse events are recognised and managed in a timely fashion to minimise the possible harm. To this aim, dentists and other health professionals should be prepared to detect orofacial ADRs as being drug‐induced.
Over the past decade, the scientific community and the vaccine industry have been asked to respond urgently to epidemics of H1N1 influenza, Ebola, Zika viruses, and this has resulted in an acceleration of vaccine‐development programmes worldwide.
2
Recently, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), a virus that causes coronavirus disease 2019 (COVID‐19), has emerged as a highly pathogenic agent and has spread globally. The development of effective and safe vaccines against this virus has been extremely fast.
3
Of the many candidates, currently two RNA‐based COVID‐19 vaccines have been granted emergency use and marketing authorisation by the relevant regulatory agencies in North America and Europe and are being used worldwide. A third vaccine (AZD1222, Oxford–AstraZeneca) has currently been approved for use in the UK. Initial efficacy and safety data for both BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines have been published
4
,
5
and product monographs including relevant information from the trials are available.
As vaccination campaigns kick off worldwide, and with several billion doses predicted to be administered in the near future,
6
it is likely that a sizeable number of adverse events will be observed. Hence, the aim of this study was to research and compare the reported orofacial adverse effects of two COVID‐19 vaccines. Dentists’ knowledge of these orofacial manifestations will improve recognition, management and reporting of vaccine‐related adverse effects.
| null | null |
RESULTS
|
Terminology Information leaflets intended for use of non‐healthcare professionals were referred to as Fact sheet for the recipients and caregivers (US), Patient Medication Information (Canada), Information for the user (EU) and Information for UK recipients (UK). Product Information for healthcare professionals was identified as Fact sheet for vaccination providers (US), Health Professional Information (Canada), Summary of product characteristics (EU) and Information for Healthcare Professionals (UK) (Table S1).
Information leaflets intended for use of non‐healthcare professionals were referred to as Fact sheet for the recipients and caregivers (US), Patient Medication Information (Canada), Information for the user (EU) and Information for UK recipients (UK). Product Information for healthcare professionals was identified as Fact sheet for vaccination providers (US), Health Professional Information (Canada), Summary of product characteristics (EU) and Information for Healthcare Professionals (UK) (Table S1).
Differences in reporting orofacial adverse effects between countries Both vaccines were associated with potential orofacial adverse effects. Rare (up to 1 in 1000 people) but serious allergic reactions causing swelling of the face, lips or tongue were reported (Tables 1 and 2). Description of localised orofacial adverse effects including facial drooping (Bell's palsy) was found, however these were not disclosed in the information for patients in US and Canada (Table 1). Swelling of the face in patients who have had facial cosmetic injections occurred exclusively with the mRNA‐1273 vaccine but was only reported in the product information available in EU and UK.
List of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to patients/users
Swelling of your lips, face, or throat
Rare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)
Very rare: swelling of the face or tongue
Rare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)
Severe allergic reaction (unknown frequency) was reported without explicitly referring to orofacial manifestations.
List of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to healthcare providers
a
Severe allergic reactions were reported for all drugs without explicitly reporting orofacial symptoms such as swelling of the face or tongue.
Both vaccines were associated with potential orofacial adverse effects. Rare (up to 1 in 1000 people) but serious allergic reactions causing swelling of the face, lips or tongue were reported (Tables 1 and 2). Description of localised orofacial adverse effects including facial drooping (Bell's palsy) was found, however these were not disclosed in the information for patients in US and Canada (Table 1). Swelling of the face in patients who have had facial cosmetic injections occurred exclusively with the mRNA‐1273 vaccine but was only reported in the product information available in EU and UK.
List of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to patients/users
Swelling of your lips, face, or throat
Rare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)
Very rare: swelling of the face or tongue
Rare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)
Severe allergic reaction (unknown frequency) was reported without explicitly referring to orofacial manifestations.
List of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to healthcare providers
a
Severe allergic reactions were reported for all drugs without explicitly reporting orofacial symptoms such as swelling of the face or tongue.
Consistency of product information for users and healthcare professionals For EU and UK consumers, temporary one sided facial drooping/acute peripheral facial paralysis (Bell's palsy) and facial swelling were reported in the product information to both patients (Table 1) and healthcare professionals (Table 2). Conversely, US and Canada vaccine fact sheets for patients did not report these side effects. Bell's palsy was also not acknowledged in the Health Professional Information of Canada.
For EU and UK consumers, temporary one sided facial drooping/acute peripheral facial paralysis (Bell's palsy) and facial swelling were reported in the product information to both patients (Table 1) and healthcare professionals (Table 2). Conversely, US and Canada vaccine fact sheets for patients did not report these side effects. Bell's palsy was also not acknowledged in the Health Professional Information of Canada.
| null | null |
[
"INTRODUCTION",
"Terminology",
"Differences in reporting orofacial adverse effects between countries",
"Consistency of product information for users and healthcare professionals",
"AUTHOR CONTRIBUTION",
"PEER REVIEW",
"PEER REVIEW"
] |
[
"Adverse drug reactions (ADRs) and medication‐related events are potentially life‐threatening consequences of the use of medicines, including vaccines. By and large, the benefits of vaccination clearly outweigh the risks—vaccines prevent between 2 and 3 million deaths from infectious diseases every year.\n1\n It is important, however, that adverse events are recognised and managed in a timely fashion to minimise the possible harm. To this aim, dentists and other health professionals should be prepared to detect orofacial ADRs as being drug‐induced.\nOver the past decade, the scientific community and the vaccine industry have been asked to respond urgently to epidemics of H1N1 influenza, Ebola, Zika viruses, and this has resulted in an acceleration of vaccine‐development programmes worldwide.\n2\n Recently, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), a virus that causes coronavirus disease 2019 (COVID‐19), has emerged as a highly pathogenic agent and has spread globally. The development of effective and safe vaccines against this virus has been extremely fast.\n3\n Of the many candidates, currently two RNA‐based COVID‐19 vaccines have been granted emergency use and marketing authorisation by the relevant regulatory agencies in North America and Europe and are being used worldwide. A third vaccine (AZD1222, Oxford–AstraZeneca) has currently been approved for use in the UK. Initial efficacy and safety data for both BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines have been published\n4\n, \n5\n and product monographs including relevant information from the trials are available.\nAs vaccination campaigns kick off worldwide, and with several billion doses predicted to be administered in the near future,\n6\n it is likely that a sizeable number of adverse events will be observed. Hence, the aim of this study was to research and compare the reported orofacial adverse effects of two COVID‐19 vaccines. Dentists’ knowledge of these orofacial manifestations will improve recognition, management and reporting of vaccine‐related adverse effects.",
"Information leaflets intended for use of non‐healthcare professionals were referred to as Fact sheet for the recipients and caregivers (US), Patient Medication Information (Canada), Information for the user (EU) and Information for UK recipients (UK). Product Information for healthcare professionals was identified as Fact sheet for vaccination providers (US), Health Professional Information (Canada), Summary of product characteristics (EU) and Information for Healthcare Professionals (UK) (Table S1).",
"Both vaccines were associated with potential orofacial adverse effects. Rare (up to 1 in 1000 people) but serious allergic reactions causing swelling of the face, lips or tongue were reported (Tables 1 and 2). Description of localised orofacial adverse effects including facial drooping (Bell's palsy) was found, however these were not disclosed in the information for patients in US and Canada (Table 1). Swelling of the face in patients who have had facial cosmetic injections occurred exclusively with the mRNA‐1273 vaccine but was only reported in the product information available in EU and UK.\nList of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to patients/users\nSwelling of your lips, face, or throat\nRare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)\nVery rare: swelling of the face or tongue\nRare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)\nSevere allergic reaction (unknown frequency) was reported without explicitly referring to orofacial manifestations.\nList of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to healthcare providers\na\n\n\nSevere allergic reactions were reported for all drugs without explicitly reporting orofacial symptoms such as swelling of the face or tongue.",
"For EU and UK consumers, temporary one sided facial drooping/acute peripheral facial paralysis (Bell's palsy) and facial swelling were reported in the product information to both patients (Table 1) and healthcare professionals (Table 2). Conversely, US and Canada vaccine fact sheets for patients did not report these side effects. Bell's palsy was also not acknowledged in the Health Professional Information of Canada.",
"Nicola Cirillo, conceptualization; data curation; formal analysis; investigation; methodology; writing‐original draft.",
" PEER REVIEW The peer review history for this article is available at https://publons.com/publon/10.1111/jop.13165.\nThe peer review history for this article is available at https://publons.com/publon/10.1111/jop.13165.",
"The peer review history for this article is available at https://publons.com/publon/10.1111/jop.13165."
] |
[
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"MATERIALS AND METHODS",
"RESULTS",
"Terminology",
"Differences in reporting orofacial adverse effects between countries",
"Consistency of product information for users and healthcare professionals",
"DISCUSSION",
"AUTHOR CONTRIBUTION",
"PEER REVIEW",
"PEER REVIEW",
"Supporting information"
] |
[
"Adverse drug reactions (ADRs) and medication‐related events are potentially life‐threatening consequences of the use of medicines, including vaccines. By and large, the benefits of vaccination clearly outweigh the risks—vaccines prevent between 2 and 3 million deaths from infectious diseases every year.\n1\n It is important, however, that adverse events are recognised and managed in a timely fashion to minimise the possible harm. To this aim, dentists and other health professionals should be prepared to detect orofacial ADRs as being drug‐induced.\nOver the past decade, the scientific community and the vaccine industry have been asked to respond urgently to epidemics of H1N1 influenza, Ebola, Zika viruses, and this has resulted in an acceleration of vaccine‐development programmes worldwide.\n2\n Recently, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), a virus that causes coronavirus disease 2019 (COVID‐19), has emerged as a highly pathogenic agent and has spread globally. The development of effective and safe vaccines against this virus has been extremely fast.\n3\n Of the many candidates, currently two RNA‐based COVID‐19 vaccines have been granted emergency use and marketing authorisation by the relevant regulatory agencies in North America and Europe and are being used worldwide. A third vaccine (AZD1222, Oxford–AstraZeneca) has currently been approved for use in the UK. Initial efficacy and safety data for both BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines have been published\n4\n, \n5\n and product monographs including relevant information from the trials are available.\nAs vaccination campaigns kick off worldwide, and with several billion doses predicted to be administered in the near future,\n6\n it is likely that a sizeable number of adverse events will be observed. Hence, the aim of this study was to research and compare the reported orofacial adverse effects of two COVID‐19 vaccines. Dentists’ knowledge of these orofacial manifestations will improve recognition, management and reporting of vaccine‐related adverse effects.",
"Product monographs, Product Information (PI) and Consumer Medicine Information (CMI) for the BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines were accessed from the following regulatory authorities: US Food and Drug Administration (FDA), Health Canada, European Medicines Agency (EMA)/European Commission and UK’s Medicines and Healthcare products Regulatory Agency (MHRA). Details and relevant websites are reported in Table S1. Reported side/adverse/undesirable effects concerning the orofacial region were extracted and tabulated.",
" Terminology Information leaflets intended for use of non‐healthcare professionals were referred to as Fact sheet for the recipients and caregivers (US), Patient Medication Information (Canada), Information for the user (EU) and Information for UK recipients (UK). Product Information for healthcare professionals was identified as Fact sheet for vaccination providers (US), Health Professional Information (Canada), Summary of product characteristics (EU) and Information for Healthcare Professionals (UK) (Table S1).\nInformation leaflets intended for use of non‐healthcare professionals were referred to as Fact sheet for the recipients and caregivers (US), Patient Medication Information (Canada), Information for the user (EU) and Information for UK recipients (UK). Product Information for healthcare professionals was identified as Fact sheet for vaccination providers (US), Health Professional Information (Canada), Summary of product characteristics (EU) and Information for Healthcare Professionals (UK) (Table S1).\n Differences in reporting orofacial adverse effects between countries Both vaccines were associated with potential orofacial adverse effects. Rare (up to 1 in 1000 people) but serious allergic reactions causing swelling of the face, lips or tongue were reported (Tables 1 and 2). Description of localised orofacial adverse effects including facial drooping (Bell's palsy) was found, however these were not disclosed in the information for patients in US and Canada (Table 1). Swelling of the face in patients who have had facial cosmetic injections occurred exclusively with the mRNA‐1273 vaccine but was only reported in the product information available in EU and UK.\nList of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to patients/users\nSwelling of your lips, face, or throat\nRare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)\nVery rare: swelling of the face or tongue\nRare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)\nSevere allergic reaction (unknown frequency) was reported without explicitly referring to orofacial manifestations.\nList of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to healthcare providers\na\n\n\nSevere allergic reactions were reported for all drugs without explicitly reporting orofacial symptoms such as swelling of the face or tongue.\nBoth vaccines were associated with potential orofacial adverse effects. Rare (up to 1 in 1000 people) but serious allergic reactions causing swelling of the face, lips or tongue were reported (Tables 1 and 2). Description of localised orofacial adverse effects including facial drooping (Bell's palsy) was found, however these were not disclosed in the information for patients in US and Canada (Table 1). Swelling of the face in patients who have had facial cosmetic injections occurred exclusively with the mRNA‐1273 vaccine but was only reported in the product information available in EU and UK.\nList of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to patients/users\nSwelling of your lips, face, or throat\nRare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)\nVery rare: swelling of the face or tongue\nRare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)\nSevere allergic reaction (unknown frequency) was reported without explicitly referring to orofacial manifestations.\nList of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to healthcare providers\na\n\n\nSevere allergic reactions were reported for all drugs without explicitly reporting orofacial symptoms such as swelling of the face or tongue.\n Consistency of product information for users and healthcare professionals For EU and UK consumers, temporary one sided facial drooping/acute peripheral facial paralysis (Bell's palsy) and facial swelling were reported in the product information to both patients (Table 1) and healthcare professionals (Table 2). Conversely, US and Canada vaccine fact sheets for patients did not report these side effects. Bell's palsy was also not acknowledged in the Health Professional Information of Canada.\nFor EU and UK consumers, temporary one sided facial drooping/acute peripheral facial paralysis (Bell's palsy) and facial swelling were reported in the product information to both patients (Table 1) and healthcare professionals (Table 2). Conversely, US and Canada vaccine fact sheets for patients did not report these side effects. Bell's palsy was also not acknowledged in the Health Professional Information of Canada.",
"Information leaflets intended for use of non‐healthcare professionals were referred to as Fact sheet for the recipients and caregivers (US), Patient Medication Information (Canada), Information for the user (EU) and Information for UK recipients (UK). Product Information for healthcare professionals was identified as Fact sheet for vaccination providers (US), Health Professional Information (Canada), Summary of product characteristics (EU) and Information for Healthcare Professionals (UK) (Table S1).",
"Both vaccines were associated with potential orofacial adverse effects. Rare (up to 1 in 1000 people) but serious allergic reactions causing swelling of the face, lips or tongue were reported (Tables 1 and 2). Description of localised orofacial adverse effects including facial drooping (Bell's palsy) was found, however these were not disclosed in the information for patients in US and Canada (Table 1). Swelling of the face in patients who have had facial cosmetic injections occurred exclusively with the mRNA‐1273 vaccine but was only reported in the product information available in EU and UK.\nList of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to patients/users\nSwelling of your lips, face, or throat\nRare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)\nVery rare: swelling of the face or tongue\nRare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)\nSevere allergic reaction (unknown frequency) was reported without explicitly referring to orofacial manifestations.\nList of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to healthcare providers\na\n\n\nSevere allergic reactions were reported for all drugs without explicitly reporting orofacial symptoms such as swelling of the face or tongue.",
"For EU and UK consumers, temporary one sided facial drooping/acute peripheral facial paralysis (Bell's palsy) and facial swelling were reported in the product information to both patients (Table 1) and healthcare professionals (Table 2). Conversely, US and Canada vaccine fact sheets for patients did not report these side effects. Bell's palsy was also not acknowledged in the Health Professional Information of Canada.",
"Our study shows that COVID‐19 vaccines have possible, albeit rare, orofacial side effects including Bell's palsy, facial swelling, and swelling of the lips, face or tongue associated with anaphylaxis. There appear to be inconsistencies in the description of these effects in the information provided to patients and healthcare professionals. For example, patient information leaflets in North America do not report orofacial side effects except those related to allergic reactions.\nIn addition to systemic ADRs with orofacial manifestations such as anaphylaxis, both vaccines were associated with acute peripheral facial paralysis. Specifically, of the 73 799 volunteers (36 901 effectively receiving at least one vaccine dose) taking part in the two large phase 3 vaccine trials,\n4\n, \n5\n eight cases of suspect Bell's palsy were reported, with a total of seven in the groups receiving a vaccine and one in placebo groups. The FDA concluded that currently available information was insufficient to determine a causal relationship with the vaccine because the cases in the vaccine groups did not differ from the frequency that is expected in the general population.\n7\n This may explain why this adverse effect was not reported in the information to users in the US.\nThere were two serious adverse events of facial swelling occurring only in the mRNA‐1273 vaccine recipients with a history of injection of dermatological fillers. The onset of swelling was reported 1 and 2 days after vaccine injection and was likely related to vaccination according to MHRA (UK).\n8\n However, no mention was made of this reaction in package leaflets in the US and Canada.\nIn general, we found that description of ADRs was more difficult to find in the US versions of product information sheets. Conversely, information for healthcare professionals in Europe and the UK reported a tabulated list of adverse reactions and their frequency, and were classified according to MedDRA system organ class.\nUnlike previous studies,\n9\n we assessed orofacial drug‐related adverse effects as reported both in the registered drug company full product information/monograph as well as in the package leaflet containing patient/consumer information. We believe that this is a more comprehensive approach and, in agreement with this view, our study found that some information was not explicitly disclosed to users. It is to note that diseases were included in our list only when the orofacial manifestations were explicitly stated, therefore, it is likely that the occurrence of oral ADRs was underreported. For example, common ADRs such as muscle pain, joint pain and enlarged lymph nodes may have manifested in the head and neck region and hence were not captured in the available reports.\nIn conclusion, we found that both BNT162b2 and mRNA‐1273 COVID‐19 vaccines are associated with orofacial ADRs and that there is heterogeneity in the description of these adverse effects worldwide.",
"Nicola Cirillo, conceptualization; data curation; formal analysis; investigation; methodology; writing‐original draft.",
" PEER REVIEW The peer review history for this article is available at https://publons.com/publon/10.1111/jop.13165.\nThe peer review history for this article is available at https://publons.com/publon/10.1111/jop.13165.",
"The peer review history for this article is available at https://publons.com/publon/10.1111/jop.13165.",
"Table S1\nClick here for additional data file."
] |
[
null,
"materials-and-methods",
"results",
null,
null,
null,
"discussion",
null,
null,
null,
"supplementary-material"
] |
[
"Bell's palsy",
"BNT162b2",
"COVID‐19",
"mRNA‐1273",
"side effects",
"Vaccine"
] |
INTRODUCTION:
Adverse drug reactions (ADRs) and medication‐related events are potentially life‐threatening consequences of the use of medicines, including vaccines. By and large, the benefits of vaccination clearly outweigh the risks—vaccines prevent between 2 and 3 million deaths from infectious diseases every year.
1
It is important, however, that adverse events are recognised and managed in a timely fashion to minimise the possible harm. To this aim, dentists and other health professionals should be prepared to detect orofacial ADRs as being drug‐induced.
Over the past decade, the scientific community and the vaccine industry have been asked to respond urgently to epidemics of H1N1 influenza, Ebola, Zika viruses, and this has resulted in an acceleration of vaccine‐development programmes worldwide.
2
Recently, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), a virus that causes coronavirus disease 2019 (COVID‐19), has emerged as a highly pathogenic agent and has spread globally. The development of effective and safe vaccines against this virus has been extremely fast.
3
Of the many candidates, currently two RNA‐based COVID‐19 vaccines have been granted emergency use and marketing authorisation by the relevant regulatory agencies in North America and Europe and are being used worldwide. A third vaccine (AZD1222, Oxford–AstraZeneca) has currently been approved for use in the UK. Initial efficacy and safety data for both BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines have been published
4
,
5
and product monographs including relevant information from the trials are available.
As vaccination campaigns kick off worldwide, and with several billion doses predicted to be administered in the near future,
6
it is likely that a sizeable number of adverse events will be observed. Hence, the aim of this study was to research and compare the reported orofacial adverse effects of two COVID‐19 vaccines. Dentists’ knowledge of these orofacial manifestations will improve recognition, management and reporting of vaccine‐related adverse effects.
MATERIALS AND METHODS:
Product monographs, Product Information (PI) and Consumer Medicine Information (CMI) for the BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines were accessed from the following regulatory authorities: US Food and Drug Administration (FDA), Health Canada, European Medicines Agency (EMA)/European Commission and UK’s Medicines and Healthcare products Regulatory Agency (MHRA). Details and relevant websites are reported in Table S1. Reported side/adverse/undesirable effects concerning the orofacial region were extracted and tabulated.
RESULTS:
Terminology Information leaflets intended for use of non‐healthcare professionals were referred to as Fact sheet for the recipients and caregivers (US), Patient Medication Information (Canada), Information for the user (EU) and Information for UK recipients (UK). Product Information for healthcare professionals was identified as Fact sheet for vaccination providers (US), Health Professional Information (Canada), Summary of product characteristics (EU) and Information for Healthcare Professionals (UK) (Table S1).
Information leaflets intended for use of non‐healthcare professionals were referred to as Fact sheet for the recipients and caregivers (US), Patient Medication Information (Canada), Information for the user (EU) and Information for UK recipients (UK). Product Information for healthcare professionals was identified as Fact sheet for vaccination providers (US), Health Professional Information (Canada), Summary of product characteristics (EU) and Information for Healthcare Professionals (UK) (Table S1).
Differences in reporting orofacial adverse effects between countries Both vaccines were associated with potential orofacial adverse effects. Rare (up to 1 in 1000 people) but serious allergic reactions causing swelling of the face, lips or tongue were reported (Tables 1 and 2). Description of localised orofacial adverse effects including facial drooping (Bell's palsy) was found, however these were not disclosed in the information for patients in US and Canada (Table 1). Swelling of the face in patients who have had facial cosmetic injections occurred exclusively with the mRNA‐1273 vaccine but was only reported in the product information available in EU and UK.
List of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to patients/users
Swelling of your lips, face, or throat
Rare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)
Very rare: swelling of the face or tongue
Rare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)
Severe allergic reaction (unknown frequency) was reported without explicitly referring to orofacial manifestations.
List of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to healthcare providers
a
Severe allergic reactions were reported for all drugs without explicitly reporting orofacial symptoms such as swelling of the face or tongue.
Both vaccines were associated with potential orofacial adverse effects. Rare (up to 1 in 1000 people) but serious allergic reactions causing swelling of the face, lips or tongue were reported (Tables 1 and 2). Description of localised orofacial adverse effects including facial drooping (Bell's palsy) was found, however these were not disclosed in the information for patients in US and Canada (Table 1). Swelling of the face in patients who have had facial cosmetic injections occurred exclusively with the mRNA‐1273 vaccine but was only reported in the product information available in EU and UK.
List of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to patients/users
Swelling of your lips, face, or throat
Rare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)
Very rare: swelling of the face or tongue
Rare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)
Severe allergic reaction (unknown frequency) was reported without explicitly referring to orofacial manifestations.
List of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to healthcare providers
a
Severe allergic reactions were reported for all drugs without explicitly reporting orofacial symptoms such as swelling of the face or tongue.
Consistency of product information for users and healthcare professionals For EU and UK consumers, temporary one sided facial drooping/acute peripheral facial paralysis (Bell's palsy) and facial swelling were reported in the product information to both patients (Table 1) and healthcare professionals (Table 2). Conversely, US and Canada vaccine fact sheets for patients did not report these side effects. Bell's palsy was also not acknowledged in the Health Professional Information of Canada.
For EU and UK consumers, temporary one sided facial drooping/acute peripheral facial paralysis (Bell's palsy) and facial swelling were reported in the product information to both patients (Table 1) and healthcare professionals (Table 2). Conversely, US and Canada vaccine fact sheets for patients did not report these side effects. Bell's palsy was also not acknowledged in the Health Professional Information of Canada.
Terminology:
Information leaflets intended for use of non‐healthcare professionals were referred to as Fact sheet for the recipients and caregivers (US), Patient Medication Information (Canada), Information for the user (EU) and Information for UK recipients (UK). Product Information for healthcare professionals was identified as Fact sheet for vaccination providers (US), Health Professional Information (Canada), Summary of product characteristics (EU) and Information for Healthcare Professionals (UK) (Table S1).
Differences in reporting orofacial adverse effects between countries:
Both vaccines were associated with potential orofacial adverse effects. Rare (up to 1 in 1000 people) but serious allergic reactions causing swelling of the face, lips or tongue were reported (Tables 1 and 2). Description of localised orofacial adverse effects including facial drooping (Bell's palsy) was found, however these were not disclosed in the information for patients in US and Canada (Table 1). Swelling of the face in patients who have had facial cosmetic injections occurred exclusively with the mRNA‐1273 vaccine but was only reported in the product information available in EU and UK.
List of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to patients/users
Swelling of your lips, face, or throat
Rare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)
Very rare: swelling of the face or tongue
Rare: temporary one sided facial drooping (Bell's palsy); swelling of the face (in patients who have had facial cosmetic injections)
Severe allergic reaction (unknown frequency) was reported without explicitly referring to orofacial manifestations.
List of orofacial adverse effects of BNT162b2 (Pfizer‐BioNTech) and mRNA‐1273 (Moderna) vaccines as reported in the information to healthcare providers
a
Severe allergic reactions were reported for all drugs without explicitly reporting orofacial symptoms such as swelling of the face or tongue.
Consistency of product information for users and healthcare professionals:
For EU and UK consumers, temporary one sided facial drooping/acute peripheral facial paralysis (Bell's palsy) and facial swelling were reported in the product information to both patients (Table 1) and healthcare professionals (Table 2). Conversely, US and Canada vaccine fact sheets for patients did not report these side effects. Bell's palsy was also not acknowledged in the Health Professional Information of Canada.
DISCUSSION:
Our study shows that COVID‐19 vaccines have possible, albeit rare, orofacial side effects including Bell's palsy, facial swelling, and swelling of the lips, face or tongue associated with anaphylaxis. There appear to be inconsistencies in the description of these effects in the information provided to patients and healthcare professionals. For example, patient information leaflets in North America do not report orofacial side effects except those related to allergic reactions.
In addition to systemic ADRs with orofacial manifestations such as anaphylaxis, both vaccines were associated with acute peripheral facial paralysis. Specifically, of the 73 799 volunteers (36 901 effectively receiving at least one vaccine dose) taking part in the two large phase 3 vaccine trials,
4
,
5
eight cases of suspect Bell's palsy were reported, with a total of seven in the groups receiving a vaccine and one in placebo groups. The FDA concluded that currently available information was insufficient to determine a causal relationship with the vaccine because the cases in the vaccine groups did not differ from the frequency that is expected in the general population.
7
This may explain why this adverse effect was not reported in the information to users in the US.
There were two serious adverse events of facial swelling occurring only in the mRNA‐1273 vaccine recipients with a history of injection of dermatological fillers. The onset of swelling was reported 1 and 2 days after vaccine injection and was likely related to vaccination according to MHRA (UK).
8
However, no mention was made of this reaction in package leaflets in the US and Canada.
In general, we found that description of ADRs was more difficult to find in the US versions of product information sheets. Conversely, information for healthcare professionals in Europe and the UK reported a tabulated list of adverse reactions and their frequency, and were classified according to MedDRA system organ class.
Unlike previous studies,
9
we assessed orofacial drug‐related adverse effects as reported both in the registered drug company full product information/monograph as well as in the package leaflet containing patient/consumer information. We believe that this is a more comprehensive approach and, in agreement with this view, our study found that some information was not explicitly disclosed to users. It is to note that diseases were included in our list only when the orofacial manifestations were explicitly stated, therefore, it is likely that the occurrence of oral ADRs was underreported. For example, common ADRs such as muscle pain, joint pain and enlarged lymph nodes may have manifested in the head and neck region and hence were not captured in the available reports.
In conclusion, we found that both BNT162b2 and mRNA‐1273 COVID‐19 vaccines are associated with orofacial ADRs and that there is heterogeneity in the description of these adverse effects worldwide.
AUTHOR CONTRIBUTION:
Nicola Cirillo, conceptualization; data curation; formal analysis; investigation; methodology; writing‐original draft.
PEER REVIEW:
PEER REVIEW The peer review history for this article is available at https://publons.com/publon/10.1111/jop.13165.
The peer review history for this article is available at https://publons.com/publon/10.1111/jop.13165.
PEER REVIEW:
The peer review history for this article is available at https://publons.com/publon/10.1111/jop.13165.
Supporting information:
Table S1
Click here for additional data file.
|
Background:
Adverse events associated with vaccine administration can manifest in the oral cavity and orofacial region. Hence, the aim of this study was to compare the orofacial adverse effects of two recently authorised COVID-19 vaccines, namely BNT162b2 and mRNA-1273.
Methods:
Publicly available data on BNT162b2 and mRNA-1273 vaccines were accessed from the relevant regulatory authorities in the United States, Canada, European Union and United Kingdom. Both patient/recipient information and healthcare professional fact sheets for each of these drugs were manually searched to find their orofacial adverse effects.
Results:
Adverse events affecting the orofacial region were reported for both vaccines. These were rare and included acute peripheral facial paralysis (Bell's palsy), facial swelling, and swelling of the lips, face or tongue associated with anaphylaxis. There was heterogeneity in the acknowledgement of vaccine-related adverse events in North America compared with Europe.
Conclusions:
Globally, there are inconsistencies in the description of adverse effects presenting in the orofacial region of the COVID-19 vaccines BNT162b2 and mRNA-1273. We believe that awareness of these orofacial manifestations will improve recognition, management and reporting of vaccine-related adverse effects.
| null | null | 2,496 | 218 | 11 |
[
"information",
"orofacial",
"facial",
"reported",
"swelling",
"effects",
"adverse",
"patients",
"face",
"healthcare"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] Bell's palsy | BNT162b2 | COVID‐19 | mRNA‐1273 | side effects | Vaccine [SUMMARY]
| null |
[CONTENT] Bell's palsy | BNT162b2 | COVID‐19 | mRNA‐1273 | side effects | Vaccine [SUMMARY]
| null |
[CONTENT] Bell's palsy | BNT162b2 | COVID‐19 | mRNA‐1273 | side effects | Vaccine [SUMMARY]
| null |
[CONTENT] BNT162 Vaccine | COVID-19 | COVID-19 Vaccines | Europe | Humans | SARS-CoV-2 | United Kingdom | United States [SUMMARY]
| null |
[CONTENT] BNT162 Vaccine | COVID-19 | COVID-19 Vaccines | Europe | Humans | SARS-CoV-2 | United Kingdom | United States [SUMMARY]
| null |
[CONTENT] BNT162 Vaccine | COVID-19 | COVID-19 Vaccines | Europe | Humans | SARS-CoV-2 | United Kingdom | United States [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] information | orofacial | facial | reported | swelling | effects | adverse | patients | face | healthcare [SUMMARY]
| null |
[CONTENT] information | orofacial | facial | reported | swelling | effects | adverse | patients | face | healthcare [SUMMARY]
| null |
[CONTENT] information | orofacial | facial | reported | swelling | effects | adverse | patients | face | healthcare [SUMMARY]
| null |
[CONTENT] vaccines | adverse | worldwide | events | covid | covid 19 | 19 | vaccine | use | aim [SUMMARY]
| null |
[CONTENT] information | facial | swelling | face | swelling face | patients | orofacial | reported | orofacial adverse | orofacial adverse effects [SUMMARY]
| null |
[CONTENT] information | facial | swelling | reported | orofacial | patients | adverse | table | effects | face [SUMMARY]
| null |
[CONTENT] ||| two | COVID-19 | BNT162b2 | mRNA-1273 [SUMMARY]
| null |
[CONTENT] ||| Bell ||| North America | Europe [SUMMARY]
| null |
[CONTENT] ||| two | COVID-19 | BNT162b2 | mRNA-1273 ||| BNT162b2 | the United States | Canada | European Union | United Kingdom ||| ||| ||| ||| Bell ||| North America | Europe ||| COVID-19 | mRNA-1273 ||| [SUMMARY]
| null |
|||
Anthropometrical measurements and maternal visceral fat during first half of pregnancy: a cross-sectional survey.
|
32993577
|
Determining anthropometric measures that indicate different fat deposits can be useful to predict metabolic risk and set specific treatment goals, reducing negative consequences for maternal and fetal health. In cases where pre-gestational weight measure and subsequent body mass index (BMI) values cannot be determined, other anthropometric measurements may be ideal for measuring the nutritional status of pregnant women, especially in low- and middle-income countries. This study aims to identify which anthropometric measurements correlate better with the maternal fat deposits measured by ultrasound.
|
BACKGROUND
|
A cross-sectional study was conducted with pregnant women from the city of Porto Alegre (city), capital of Rio Grande do Sul (state), southern Brazil, from October 2016 until January 2018. Anthropometrical variables (weight, height, mid-upper arm circumference [MUAC], circumferences of calf and neck and triceps skinfolds [TSF] and subscapular skinfolds [SBSF]), and ultrasound variables (visceral adipose tissue [VAT] and total adipose tissue [TAT]) were collected. To verify the correlation of anthropometric and ultrasound measurements, a non-adjusted and adjusted Spearman correlation was used. The study was approved by the ethics committees.
|
METHODS
|
The age median of the 149 pregnant women was 25 years [21-31], pre-pregnancy BMI was 26.22 kg/m² [22.16-31.21] and gestational age was 16.2 weeks [13.05-18.10]. The best measurements correlated with VAT and TAT were MUAC and SBSF, both of which showed a higher correlation than pre-pregnancy BMI.
|
RESULTS
|
It is possible to provide a practical and reliable estimate of VAT and TAT from the anthropometric evaluation (MUAC or SBSF) that is low cost, efficient and replicable in an outpatient clinic environment, especially in low- and middle-income countries.
|
CONCLUSIONS
|
[
"Adult",
"Body Weights and Measures",
"Correlation of Data",
"Cross-Sectional Studies",
"Female",
"Humans",
"Intra-Abdominal Fat",
"Organ Size",
"Pregnancy",
"Pregnancy Trimester, First",
"Pregnancy Trimester, Second",
"Ultrasonography, Prenatal",
"Young Adult"
] |
7526141
|
Background
|
Physiological adaptations during pregnancy are caused in order to ensure an adequate supply of nutrients to the fetus [1]. Among these adaptations, the accumulation of fat in different deposits is associated with metabolic consequences for the gestational environment. The mechanisms responsible for the structural and functional differences specific to adipose tissue deposits are still being investigated [2]. However, it is known that visceral fat is strongly associated with increased metabolic diseases [3]. The investigation of measures that assist in the identification of different fat deposits can be useful to predict risk and set specific treatment goals, reducing negative consequences for fetal and maternal health. The prenatal care is an opportune time, as it is characterized by a preventive practice recommended during pregnancy that provides a perspective for important healthcare functions, including health promotion, screening and diagnosis, and disease prevention [4].
For the mother’s anthropometry, the pre-pregnancy body mass index (BMI) is considered a reflection of maternal nutritional status before pregnancy, while gestational weight gain is the aggregate change of the mother’s, child’s and placental mass in the physiologic state [5, 6]. Utilizing BMI as a measurement of health during pregnancy can have limitations, primarily due to pregnancy-associated weight gain and oedema, as well as late booking into antenatal care in a population-level setting [6]. The assessment of the amount of maternal fat deposits during pregnancy is limited by the inability to use ionizing radiation in computerized tomography and dual-energy x-ray absorptiometry, high cost and maintenance of nuclear magnetic resonance and low accuracy of bioelectric impedance analysis. Thereafter, the most commonly used method to measure maternal body composition changes in pregnancy is anthropometry, particularly the use of skinfolds and circumferences [1, 7].
The use of ultrasound to measure the distribution of maternal visceral adipose tissue (VAT) and total adipose tissue (TAT) is becoming useful because it is widely available in hospitals to predict a higher risk of preeclampsia [8], insulin resistance and metabolic diseases [9, 10], premature birth [8] and average birth weight [11]. It is worth mentioning that ultrasound is an easy, quick, safe, non-invasive, precise and reliable method to identify patients with adverse metabolic profiles [12]. However, due to the easiness of execution and low cost, anthropometrical measurements, like mid-upper arm circumference (MUAC), may become an alternative to the use of ultrasound devices [6, 13–15].
There were no found studies that determined the predictive capabilities of anthropometric measures alternative to pre-pregnancy BMI in relation to a reference method, such as VAT and TAT obtained by ultrasound. The aim of this study is to identify which anthropometric measurements correlate better with the maternal fat deposits measured by ultrasound.
| null | null |
Results
|
The sample was comprised of 149 pregnant women up to 20 weeks of pregnancy. Nineteen participants were not included in the correlation analysis of the study outcomes due to the decision not to include cases that had some unanswered adjustment variable. Participants were on median 25 years of age [21–31], mostly Caucasian (54.8%, n = 80), had a median pre-pregnancy BMI of 26.22 kg/m² [22.16–31.21], 58.33% (n = 84) with pre-pregnancy BMI classified as overweight or obesity, often had two past pregnancies [1 - 3] and a gestational age average of 16.2 weeks [13.05–18.10]. The anthropometric measurements showed that the MUAC median was 31.0 cm [28.0–35.0], calf perimeter median was 37.0 cm [35.0–41.0] and neck perimeter was 34.0 cm [32.0–36.0]. TSF and SBSF showed median of 31.5 mm [25.0–40.5] and 30.0 mm [21.0–40.5], respectively. The median VAT was 41.7 mm [34.5–52.8] and TAT was 61.1 mm [50.72–71.42], as shown in Table 1.
Table 1Maternal demographic, gestational and clinical characteristicsVariablesn (%)Median (IQ)Age (years)14925.00 [21.00–31.00]Number of pregnancies14302 [01–03]Gestational age (weeks)14916.20 [13.05–18.10]RaceWhiteBlackDark-skinned14680 (54.80)42 (28.80)24 (16.40)Pre-pregnancy body mass index (kg/m²)14426.22 [22.16–31.21]Underweight3 (2.08)Adequate57 (39.58)Overweight37 (25.70)Obesity47 (32.64)Arm circumference (cm)14731.00 [28.00–35.00]Neck circumference (cm)14734.00 [32.00–36.00]Calf circumference (cm)14637.00 [35.00–41.00]Triceps Skinfold (mm)14731.50 [25.00–40.50]Subscapular Skinfold (mm)14730.00 [21.00–40.50]Central Visceral Fat (mm)14941.70 [34.50–52.80]Total Central Fat (mm)14261.20 [50.65–71.75]Descriptive table with medians [interquartile interval] and frequency (%)Totals may not add up to 149 because of missing values
Maternal demographic, gestational and clinical characteristics
Race
White
Black
Dark-skinned
146
80 (54.80)
42 (28.80)
24 (16.40)
Descriptive table with medians [interquartile interval] and frequency (%)
Totals may not add up to 149 because of missing values
Table 2 shows the non-adjusted correlation across anthropometrical measurements and pre-pregnancy BMI values with ultrasound measurements of the maternal abdomen. We can observe that all body perimeters and skin folds showed to be statistically correlated to VAT and TAT. When analyzed individually, calf and neck perimeters and TSF indicate weaker correlations to detect VAT and TAT, when compared to pre-pregnancy BMI. However, MUAC and SBSF presented greater correlations with VAT and TAT, when compared to pre-pregnancy BMI.
Table 2Non-adjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomenVariablesMid-upper arm circumference (cm)Calf circumference (cm)Neck circumference (cm)Triceps Skinfold (mm)Subscapular Skinfold(mm)Pre-pregnancy BMI (Kg/m²)Central Visceral Fat(mm)ρ0.603*0.498*0.500*0.541*0.597*0.546*n147146147147147144Total CentralFat (mm)ρ0.792*0.677*0.656*0.698*0.740*0.725*n140139140140140137BMI Body mass indexSpearman correlation; *p value < 0.05Totals may not add up to 149 because of missing values
Non-adjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomen
Central Visceral Fat
(mm)
Total Central
Fat (mm)
BMI Body mass index
Spearman correlation; *p value < 0.05
Totals may not add up to 149 because of missing values
Table 3 presents the adjusted correlation between anthropometric measurements, pre-pregnancy BMI with ultrasound measurements of the maternal abdomen. It is worth mentioning that even with the adjustment for variables associated to maternal adipose accumulation during pregnancy (i.e., number of pregnancies, maternal age and gestational age), the measurements of MUAC, TSF and SBSF maintained statistical significance (p < 0.05), showing values higher than pre-pregnancy BMI.
Table 3Adjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomenVariablesControl VariablesMid-upper arm circumference (cm)Triceps Skinfold (mm)Subscapular Skinfold (mm)Pre-pregnancy BMI (Kg/m²)Central Visceral Fat(mm)Coef. ρn = 130Without adjustment0.603*0.541*0.597*0.546*Number of pregnancies+ Maternal age (years)+ Gestational age (weeks)0.598*0.598*0.602*0.543*Total Central Fat(mm)Coef. ρn = 130Without adjustment0.792*0.698*0.740*0.725*Number of pregnancies+ Maternal age (years)+ Gestational age (weeks)0.7520.7120.7190.691*BMI Body mass indexSpearman correlation adjusted to number of children, maternal age and gestational age. *p value < 0.05Totals may not add up to 149 because of missing values
Adjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomen
Coef. ρ
n = 130
Number of pregnancies
+ Maternal age (years)
+ Gestational age (weeks)
Coef. ρ
n = 130
Number of pregnancies
+ Maternal age (years)
+ Gestational age (weeks)
BMI Body mass index
Spearman correlation adjusted to number of children, maternal age and gestational age. *p value < 0.05
Totals may not add up to 149 because of missing values
|
Conclusions
|
The study found that the anthropometric measurements most correlated with VAT and TAT were MUAC and SBSF, both of which had a higher correlation than pre-pregnancy BMI. It is possible to provide a practical, reliable and low-cost clinical estimate of ultrasound measurements of maternal central fat, which will help to identify women at high metabolic risk during pregnancy, based on efficient and replicable anthropometrical examination. In situations where pre-gestational weight measurement and subsequent BMI values cannot be obtained, the anthropometric measurements, MUAC and SBSF are useful and may be ideal for measuring the nutritional status of pregnant women, especially in low- and middle-income countries.
|
[
"Background",
"Methods",
"Design",
"Participants",
"Measures",
"Statistical analysis",
"Ethical Aspects",
"Strengths and limitations"
] |
[
"Physiological adaptations during pregnancy are caused in order to ensure an adequate supply of nutrients to the fetus [1]. Among these adaptations, the accumulation of fat in different deposits is associated with metabolic consequences for the gestational environment. The mechanisms responsible for the structural and functional differences specific to adipose tissue deposits are still being investigated [2]. However, it is known that visceral fat is strongly associated with increased metabolic diseases [3]. The investigation of measures that assist in the identification of different fat deposits can be useful to predict risk and set specific treatment goals, reducing negative consequences for fetal and maternal health. The prenatal care is an opportune time, as it is characterized by a preventive practice recommended during pregnancy that provides a perspective for important healthcare functions, including health promotion, screening and diagnosis, and disease prevention [4].\nFor the mother’s anthropometry, the pre-pregnancy body mass index (BMI) is considered a reflection of maternal nutritional status before pregnancy, while gestational weight gain is the aggregate change of the mother’s, child’s and placental mass in the physiologic state [5, 6]. Utilizing BMI as a measurement of health during pregnancy can have limitations, primarily due to pregnancy-associated weight gain and oedema, as well as late booking into antenatal care in a population-level setting [6]. The assessment of the amount of maternal fat deposits during pregnancy is limited by the inability to use ionizing radiation in computerized tomography and dual-energy x-ray absorptiometry, high cost and maintenance of nuclear magnetic resonance and low accuracy of bioelectric impedance analysis. Thereafter, the most commonly used method to measure maternal body composition changes in pregnancy is anthropometry, particularly the use of skinfolds and circumferences [1, 7].\nThe use of ultrasound to measure the distribution of maternal visceral adipose tissue (VAT) and total adipose tissue (TAT) is becoming useful because it is widely available in hospitals to predict a higher risk of preeclampsia [8], insulin resistance and metabolic diseases [9, 10], premature birth [8] and average birth weight [11]. It is worth mentioning that ultrasound is an easy, quick, safe, non-invasive, precise and reliable method to identify patients with adverse metabolic profiles [12]. However, due to the easiness of execution and low cost, anthropometrical measurements, like mid-upper arm circumference (MUAC), may become an alternative to the use of ultrasound devices [6, 13–15].\nThere were no found studies that determined the predictive capabilities of anthropometric measures alternative to pre-pregnancy BMI in relation to a reference method, such as VAT and TAT obtained by ultrasound. The aim of this study is to identify which anthropometric measurements correlate better with the maternal fat deposits measured by ultrasound.",
" Design The cross-sectional study recruited patients from 2016 to 2018 at the Ultrasound Department of Murialdo Health Center School that provides services of fetal medicine to Brazil’s Unified Health System at the city of Porto Alegre, Rio Grande do Sul, Brazil.\nThe cross-sectional study recruited patients from 2016 to 2018 at the Ultrasound Department of Murialdo Health Center School that provides services of fetal medicine to Brazil’s Unified Health System at the city of Porto Alegre, Rio Grande do Sul, Brazil.\n Participants For this study, single pregnancies, below twenty gestational weeks, with no evident fetal malformations, with no scars in the abdominal cavity or in sites to use adipometer that hide the measurements and scheduled for routine appointments in Brazil’s Unified Health System were included. The pregnant women who met the inclusion criteria were invited to participate and, after informed consent, were included with the completion of the maternal, clinical and epidemiological questionnaire.\nFor this study, single pregnancies, below twenty gestational weeks, with no evident fetal malformations, with no scars in the abdominal cavity or in sites to use adipometer that hide the measurements and scheduled for routine appointments in Brazil’s Unified Health System were included. The pregnant women who met the inclusion criteria were invited to participate and, after informed consent, were included with the completion of the maternal, clinical and epidemiological questionnaire.\n Measures The maternal anthropometric evaluation included assessment of anthropometric measures (weight and height) and evaluation of body composition. The participants were encouraged to use minimal clothing and no shoes and accessories like watches, bracelets and earrings. The body weight was measured in kilograms with portable electronic digital scale Marte® LC200-PP (São Paulo, São Paulo, Brazil) accurate to 50 g. The height was measured in meters with extensible portable stadiometer Alturexata® (Belo Horizonte, Minas Gerais, Brazil). Maternal pre-pregnancy weight was collected from the prenatal pregnant chart and confirmed by the maternal report and, when there was no record, in the BMI referring to the first trimester of pregnancy. The pre-pregnancy BMI (in kg/m²) was calculated through the formula, current weight divided by the current height squared. The classification used was pre-pregnancy BMI in underweight (BMI < 18.50 kg/m²), adequate weight (BMI between 18.50 and 24.99 kg/m²), overweight (BMI between 25.00 and 29.99 kg/m²) and obese (BMI ≥ 30.00 kg/m²), according to categories defined by World Health Organization [16].\nPerimeters were measured with an anthropometric tape on the right trunk, arm and leg. Calf perimeter was measured at the greater circumference. The neck perimeter was measured at the midpoint between the clavicle bone and chin. MUAC was measured at the midpoint between the acromion and olecranon bones. Triceps (TSF) and subscapular skinfolds (SBSF) were evaluated using a caliper (Lange®). The TSF was measured at the same levels as those of arm perimeter. The SBSF was measured two centimeters below the lower angle of the scapula bone. Anthropometric data were measured in duplicate by nutritionists, considering the arithmetic mean value among the measurements.\nMeasurement of maternal abdominal fat space was done with ultrasound device Toshiba Xario XG® with a 3.5 mHz multi-frequency convex probe placed above the maternal umbilical scar, with low pressure, and automatic calipers were positioned from anterior aortic wall to linea alba measuring maternal abdominal depth. Two measurements were performed by only one specialist medical researcher, first during maternal inspiration and after during maternal expiration. The arithmetic mean of measurements was used for this research. The measurement of maternal subcutaneous adipose tissue (SAT) was made in the same position as that of VAT measurement, with the automatic caliper positioned from linea alba to dermal edge on the surface of the maternal abdomen. The sum of VAT and SAT was used to estimate the total adipose tissue (TAT) during the evaluation.\nThe maternal anthropometric evaluation included assessment of anthropometric measures (weight and height) and evaluation of body composition. The participants were encouraged to use minimal clothing and no shoes and accessories like watches, bracelets and earrings. The body weight was measured in kilograms with portable electronic digital scale Marte® LC200-PP (São Paulo, São Paulo, Brazil) accurate to 50 g. The height was measured in meters with extensible portable stadiometer Alturexata® (Belo Horizonte, Minas Gerais, Brazil). Maternal pre-pregnancy weight was collected from the prenatal pregnant chart and confirmed by the maternal report and, when there was no record, in the BMI referring to the first trimester of pregnancy. The pre-pregnancy BMI (in kg/m²) was calculated through the formula, current weight divided by the current height squared. The classification used was pre-pregnancy BMI in underweight (BMI < 18.50 kg/m²), adequate weight (BMI between 18.50 and 24.99 kg/m²), overweight (BMI between 25.00 and 29.99 kg/m²) and obese (BMI ≥ 30.00 kg/m²), according to categories defined by World Health Organization [16].\nPerimeters were measured with an anthropometric tape on the right trunk, arm and leg. Calf perimeter was measured at the greater circumference. The neck perimeter was measured at the midpoint between the clavicle bone and chin. MUAC was measured at the midpoint between the acromion and olecranon bones. Triceps (TSF) and subscapular skinfolds (SBSF) were evaluated using a caliper (Lange®). The TSF was measured at the same levels as those of arm perimeter. The SBSF was measured two centimeters below the lower angle of the scapula bone. Anthropometric data were measured in duplicate by nutritionists, considering the arithmetic mean value among the measurements.\nMeasurement of maternal abdominal fat space was done with ultrasound device Toshiba Xario XG® with a 3.5 mHz multi-frequency convex probe placed above the maternal umbilical scar, with low pressure, and automatic calipers were positioned from anterior aortic wall to linea alba measuring maternal abdominal depth. Two measurements were performed by only one specialist medical researcher, first during maternal inspiration and after during maternal expiration. The arithmetic mean of measurements was used for this research. The measurement of maternal subcutaneous adipose tissue (SAT) was made in the same position as that of VAT measurement, with the automatic caliper positioned from linea alba to dermal edge on the surface of the maternal abdomen. The sum of VAT and SAT was used to estimate the total adipose tissue (TAT) during the evaluation.\n Statistical analysis Statistical analyses were performed through Statistical Package for Social Sciences (SPSS) 21.0. The level of significance was set at p < 0.05. Clinical, epidemiological and ultrasonographic data were presented as quantitative and categorical variables. The test of normality of variables distribution was made. Quantitative variables with asymmetric distributions were described as median and interquartile range. Categorical variables were reported as absolute frequency and percentage. To perform the associations between anthropometrical measurements and VAT and TAT measurements, non-adjusted Spearman correlation (ρ) was used. After that, Spearman correlation was adjusted for factors associated with maternal adipose accumulation during pregnancy: number of pregnancies, pregnant age and gestational age.\nStatistical analyses were performed through Statistical Package for Social Sciences (SPSS) 21.0. The level of significance was set at p < 0.05. Clinical, epidemiological and ultrasonographic data were presented as quantitative and categorical variables. The test of normality of variables distribution was made. Quantitative variables with asymmetric distributions were described as median and interquartile range. Categorical variables were reported as absolute frequency and percentage. To perform the associations between anthropometrical measurements and VAT and TAT measurements, non-adjusted Spearman correlation (ρ) was used. After that, Spearman correlation was adjusted for factors associated with maternal adipose accumulation during pregnancy: number of pregnancies, pregnant age and gestational age.\n Ethical Aspects The study was approved by the Research Ethics Committees of the Health Department of Porto Alegre under number 2.132.090 and in the Presidente Vargas Hospital under number 1.758.959. Written informed consent was obtained from participants.\nThe study was approved by the Research Ethics Committees of the Health Department of Porto Alegre under number 2.132.090 and in the Presidente Vargas Hospital under number 1.758.959. Written informed consent was obtained from participants.",
"The cross-sectional study recruited patients from 2016 to 2018 at the Ultrasound Department of Murialdo Health Center School that provides services of fetal medicine to Brazil’s Unified Health System at the city of Porto Alegre, Rio Grande do Sul, Brazil.",
"For this study, single pregnancies, below twenty gestational weeks, with no evident fetal malformations, with no scars in the abdominal cavity or in sites to use adipometer that hide the measurements and scheduled for routine appointments in Brazil’s Unified Health System were included. The pregnant women who met the inclusion criteria were invited to participate and, after informed consent, were included with the completion of the maternal, clinical and epidemiological questionnaire.",
"The maternal anthropometric evaluation included assessment of anthropometric measures (weight and height) and evaluation of body composition. The participants were encouraged to use minimal clothing and no shoes and accessories like watches, bracelets and earrings. The body weight was measured in kilograms with portable electronic digital scale Marte® LC200-PP (São Paulo, São Paulo, Brazil) accurate to 50 g. The height was measured in meters with extensible portable stadiometer Alturexata® (Belo Horizonte, Minas Gerais, Brazil). Maternal pre-pregnancy weight was collected from the prenatal pregnant chart and confirmed by the maternal report and, when there was no record, in the BMI referring to the first trimester of pregnancy. The pre-pregnancy BMI (in kg/m²) was calculated through the formula, current weight divided by the current height squared. The classification used was pre-pregnancy BMI in underweight (BMI < 18.50 kg/m²), adequate weight (BMI between 18.50 and 24.99 kg/m²), overweight (BMI between 25.00 and 29.99 kg/m²) and obese (BMI ≥ 30.00 kg/m²), according to categories defined by World Health Organization [16].\nPerimeters were measured with an anthropometric tape on the right trunk, arm and leg. Calf perimeter was measured at the greater circumference. The neck perimeter was measured at the midpoint between the clavicle bone and chin. MUAC was measured at the midpoint between the acromion and olecranon bones. Triceps (TSF) and subscapular skinfolds (SBSF) were evaluated using a caliper (Lange®). The TSF was measured at the same levels as those of arm perimeter. The SBSF was measured two centimeters below the lower angle of the scapula bone. Anthropometric data were measured in duplicate by nutritionists, considering the arithmetic mean value among the measurements.\nMeasurement of maternal abdominal fat space was done with ultrasound device Toshiba Xario XG® with a 3.5 mHz multi-frequency convex probe placed above the maternal umbilical scar, with low pressure, and automatic calipers were positioned from anterior aortic wall to linea alba measuring maternal abdominal depth. Two measurements were performed by only one specialist medical researcher, first during maternal inspiration and after during maternal expiration. The arithmetic mean of measurements was used for this research. The measurement of maternal subcutaneous adipose tissue (SAT) was made in the same position as that of VAT measurement, with the automatic caliper positioned from linea alba to dermal edge on the surface of the maternal abdomen. The sum of VAT and SAT was used to estimate the total adipose tissue (TAT) during the evaluation.",
"Statistical analyses were performed through Statistical Package for Social Sciences (SPSS) 21.0. The level of significance was set at p < 0.05. Clinical, epidemiological and ultrasonographic data were presented as quantitative and categorical variables. The test of normality of variables distribution was made. Quantitative variables with asymmetric distributions were described as median and interquartile range. Categorical variables were reported as absolute frequency and percentage. To perform the associations between anthropometrical measurements and VAT and TAT measurements, non-adjusted Spearman correlation (ρ) was used. After that, Spearman correlation was adjusted for factors associated with maternal adipose accumulation during pregnancy: number of pregnancies, pregnant age and gestational age.",
"The study was approved by the Research Ethics Committees of the Health Department of Porto Alegre under number 2.132.090 and in the Presidente Vargas Hospital under number 1.758.959. Written informed consent was obtained from participants.",
"The study was conducted in a low risk primary healthcare setting that did not provide additional intervention to the sample. The data were obtained from routine follow-up of patients, which suggests that the findings can be easily transferred to the clinical practice. The most important aspect of the research was that the study’s population was comprised of pregnant women where the anthropometrical measurements can be become tools for decision making in clinical practice both in low and high obstetric risk environments. The low number of researchers involved in data collection minimizes possible mistakes of measurement. Throughout the study, one researcher was in charge of ultrasound collections and two researchers were in charge of the general questionnaire and nutritional assessment. A limitation of the study is the cross-sectional design that prevents the verification of pregnancy outcomes among women with large amounts of central fat due to the absence of blood sampling that diagnoses metabolic risk in pregnancy. Another limitation of the study includes the self-reporting of pre-pregnancy weight, as it may have been affected by recall bias. However, several studies demonstrated that the use of self-reported weight in pregnant women and young adults is valid [28, 29]."
] |
[
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Design",
"Participants",
"Measures",
"Statistical analysis",
"Ethical Aspects",
"Results",
"Discussion",
"Strengths and limitations",
"Conclusions"
] |
[
"Physiological adaptations during pregnancy are caused in order to ensure an adequate supply of nutrients to the fetus [1]. Among these adaptations, the accumulation of fat in different deposits is associated with metabolic consequences for the gestational environment. The mechanisms responsible for the structural and functional differences specific to adipose tissue deposits are still being investigated [2]. However, it is known that visceral fat is strongly associated with increased metabolic diseases [3]. The investigation of measures that assist in the identification of different fat deposits can be useful to predict risk and set specific treatment goals, reducing negative consequences for fetal and maternal health. The prenatal care is an opportune time, as it is characterized by a preventive practice recommended during pregnancy that provides a perspective for important healthcare functions, including health promotion, screening and diagnosis, and disease prevention [4].\nFor the mother’s anthropometry, the pre-pregnancy body mass index (BMI) is considered a reflection of maternal nutritional status before pregnancy, while gestational weight gain is the aggregate change of the mother’s, child’s and placental mass in the physiologic state [5, 6]. Utilizing BMI as a measurement of health during pregnancy can have limitations, primarily due to pregnancy-associated weight gain and oedema, as well as late booking into antenatal care in a population-level setting [6]. The assessment of the amount of maternal fat deposits during pregnancy is limited by the inability to use ionizing radiation in computerized tomography and dual-energy x-ray absorptiometry, high cost and maintenance of nuclear magnetic resonance and low accuracy of bioelectric impedance analysis. Thereafter, the most commonly used method to measure maternal body composition changes in pregnancy is anthropometry, particularly the use of skinfolds and circumferences [1, 7].\nThe use of ultrasound to measure the distribution of maternal visceral adipose tissue (VAT) and total adipose tissue (TAT) is becoming useful because it is widely available in hospitals to predict a higher risk of preeclampsia [8], insulin resistance and metabolic diseases [9, 10], premature birth [8] and average birth weight [11]. It is worth mentioning that ultrasound is an easy, quick, safe, non-invasive, precise and reliable method to identify patients with adverse metabolic profiles [12]. However, due to the easiness of execution and low cost, anthropometrical measurements, like mid-upper arm circumference (MUAC), may become an alternative to the use of ultrasound devices [6, 13–15].\nThere were no found studies that determined the predictive capabilities of anthropometric measures alternative to pre-pregnancy BMI in relation to a reference method, such as VAT and TAT obtained by ultrasound. The aim of this study is to identify which anthropometric measurements correlate better with the maternal fat deposits measured by ultrasound.",
" Design The cross-sectional study recruited patients from 2016 to 2018 at the Ultrasound Department of Murialdo Health Center School that provides services of fetal medicine to Brazil’s Unified Health System at the city of Porto Alegre, Rio Grande do Sul, Brazil.\nThe cross-sectional study recruited patients from 2016 to 2018 at the Ultrasound Department of Murialdo Health Center School that provides services of fetal medicine to Brazil’s Unified Health System at the city of Porto Alegre, Rio Grande do Sul, Brazil.\n Participants For this study, single pregnancies, below twenty gestational weeks, with no evident fetal malformations, with no scars in the abdominal cavity or in sites to use adipometer that hide the measurements and scheduled for routine appointments in Brazil’s Unified Health System were included. The pregnant women who met the inclusion criteria were invited to participate and, after informed consent, were included with the completion of the maternal, clinical and epidemiological questionnaire.\nFor this study, single pregnancies, below twenty gestational weeks, with no evident fetal malformations, with no scars in the abdominal cavity or in sites to use adipometer that hide the measurements and scheduled for routine appointments in Brazil’s Unified Health System were included. The pregnant women who met the inclusion criteria were invited to participate and, after informed consent, were included with the completion of the maternal, clinical and epidemiological questionnaire.\n Measures The maternal anthropometric evaluation included assessment of anthropometric measures (weight and height) and evaluation of body composition. The participants were encouraged to use minimal clothing and no shoes and accessories like watches, bracelets and earrings. The body weight was measured in kilograms with portable electronic digital scale Marte® LC200-PP (São Paulo, São Paulo, Brazil) accurate to 50 g. The height was measured in meters with extensible portable stadiometer Alturexata® (Belo Horizonte, Minas Gerais, Brazil). Maternal pre-pregnancy weight was collected from the prenatal pregnant chart and confirmed by the maternal report and, when there was no record, in the BMI referring to the first trimester of pregnancy. The pre-pregnancy BMI (in kg/m²) was calculated through the formula, current weight divided by the current height squared. The classification used was pre-pregnancy BMI in underweight (BMI < 18.50 kg/m²), adequate weight (BMI between 18.50 and 24.99 kg/m²), overweight (BMI between 25.00 and 29.99 kg/m²) and obese (BMI ≥ 30.00 kg/m²), according to categories defined by World Health Organization [16].\nPerimeters were measured with an anthropometric tape on the right trunk, arm and leg. Calf perimeter was measured at the greater circumference. The neck perimeter was measured at the midpoint between the clavicle bone and chin. MUAC was measured at the midpoint between the acromion and olecranon bones. Triceps (TSF) and subscapular skinfolds (SBSF) were evaluated using a caliper (Lange®). The TSF was measured at the same levels as those of arm perimeter. The SBSF was measured two centimeters below the lower angle of the scapula bone. Anthropometric data were measured in duplicate by nutritionists, considering the arithmetic mean value among the measurements.\nMeasurement of maternal abdominal fat space was done with ultrasound device Toshiba Xario XG® with a 3.5 mHz multi-frequency convex probe placed above the maternal umbilical scar, with low pressure, and automatic calipers were positioned from anterior aortic wall to linea alba measuring maternal abdominal depth. Two measurements were performed by only one specialist medical researcher, first during maternal inspiration and after during maternal expiration. The arithmetic mean of measurements was used for this research. The measurement of maternal subcutaneous adipose tissue (SAT) was made in the same position as that of VAT measurement, with the automatic caliper positioned from linea alba to dermal edge on the surface of the maternal abdomen. The sum of VAT and SAT was used to estimate the total adipose tissue (TAT) during the evaluation.\nThe maternal anthropometric evaluation included assessment of anthropometric measures (weight and height) and evaluation of body composition. The participants were encouraged to use minimal clothing and no shoes and accessories like watches, bracelets and earrings. The body weight was measured in kilograms with portable electronic digital scale Marte® LC200-PP (São Paulo, São Paulo, Brazil) accurate to 50 g. The height was measured in meters with extensible portable stadiometer Alturexata® (Belo Horizonte, Minas Gerais, Brazil). Maternal pre-pregnancy weight was collected from the prenatal pregnant chart and confirmed by the maternal report and, when there was no record, in the BMI referring to the first trimester of pregnancy. The pre-pregnancy BMI (in kg/m²) was calculated through the formula, current weight divided by the current height squared. The classification used was pre-pregnancy BMI in underweight (BMI < 18.50 kg/m²), adequate weight (BMI between 18.50 and 24.99 kg/m²), overweight (BMI between 25.00 and 29.99 kg/m²) and obese (BMI ≥ 30.00 kg/m²), according to categories defined by World Health Organization [16].\nPerimeters were measured with an anthropometric tape on the right trunk, arm and leg. Calf perimeter was measured at the greater circumference. The neck perimeter was measured at the midpoint between the clavicle bone and chin. MUAC was measured at the midpoint between the acromion and olecranon bones. Triceps (TSF) and subscapular skinfolds (SBSF) were evaluated using a caliper (Lange®). The TSF was measured at the same levels as those of arm perimeter. The SBSF was measured two centimeters below the lower angle of the scapula bone. Anthropometric data were measured in duplicate by nutritionists, considering the arithmetic mean value among the measurements.\nMeasurement of maternal abdominal fat space was done with ultrasound device Toshiba Xario XG® with a 3.5 mHz multi-frequency convex probe placed above the maternal umbilical scar, with low pressure, and automatic calipers were positioned from anterior aortic wall to linea alba measuring maternal abdominal depth. Two measurements were performed by only one specialist medical researcher, first during maternal inspiration and after during maternal expiration. The arithmetic mean of measurements was used for this research. The measurement of maternal subcutaneous adipose tissue (SAT) was made in the same position as that of VAT measurement, with the automatic caliper positioned from linea alba to dermal edge on the surface of the maternal abdomen. The sum of VAT and SAT was used to estimate the total adipose tissue (TAT) during the evaluation.\n Statistical analysis Statistical analyses were performed through Statistical Package for Social Sciences (SPSS) 21.0. The level of significance was set at p < 0.05. Clinical, epidemiological and ultrasonographic data were presented as quantitative and categorical variables. The test of normality of variables distribution was made. Quantitative variables with asymmetric distributions were described as median and interquartile range. Categorical variables were reported as absolute frequency and percentage. To perform the associations between anthropometrical measurements and VAT and TAT measurements, non-adjusted Spearman correlation (ρ) was used. After that, Spearman correlation was adjusted for factors associated with maternal adipose accumulation during pregnancy: number of pregnancies, pregnant age and gestational age.\nStatistical analyses were performed through Statistical Package for Social Sciences (SPSS) 21.0. The level of significance was set at p < 0.05. Clinical, epidemiological and ultrasonographic data were presented as quantitative and categorical variables. The test of normality of variables distribution was made. Quantitative variables with asymmetric distributions were described as median and interquartile range. Categorical variables were reported as absolute frequency and percentage. To perform the associations between anthropometrical measurements and VAT and TAT measurements, non-adjusted Spearman correlation (ρ) was used. After that, Spearman correlation was adjusted for factors associated with maternal adipose accumulation during pregnancy: number of pregnancies, pregnant age and gestational age.\n Ethical Aspects The study was approved by the Research Ethics Committees of the Health Department of Porto Alegre under number 2.132.090 and in the Presidente Vargas Hospital under number 1.758.959. Written informed consent was obtained from participants.\nThe study was approved by the Research Ethics Committees of the Health Department of Porto Alegre under number 2.132.090 and in the Presidente Vargas Hospital under number 1.758.959. Written informed consent was obtained from participants.",
"The cross-sectional study recruited patients from 2016 to 2018 at the Ultrasound Department of Murialdo Health Center School that provides services of fetal medicine to Brazil’s Unified Health System at the city of Porto Alegre, Rio Grande do Sul, Brazil.",
"For this study, single pregnancies, below twenty gestational weeks, with no evident fetal malformations, with no scars in the abdominal cavity or in sites to use adipometer that hide the measurements and scheduled for routine appointments in Brazil’s Unified Health System were included. The pregnant women who met the inclusion criteria were invited to participate and, after informed consent, were included with the completion of the maternal, clinical and epidemiological questionnaire.",
"The maternal anthropometric evaluation included assessment of anthropometric measures (weight and height) and evaluation of body composition. The participants were encouraged to use minimal clothing and no shoes and accessories like watches, bracelets and earrings. The body weight was measured in kilograms with portable electronic digital scale Marte® LC200-PP (São Paulo, São Paulo, Brazil) accurate to 50 g. The height was measured in meters with extensible portable stadiometer Alturexata® (Belo Horizonte, Minas Gerais, Brazil). Maternal pre-pregnancy weight was collected from the prenatal pregnant chart and confirmed by the maternal report and, when there was no record, in the BMI referring to the first trimester of pregnancy. The pre-pregnancy BMI (in kg/m²) was calculated through the formula, current weight divided by the current height squared. The classification used was pre-pregnancy BMI in underweight (BMI < 18.50 kg/m²), adequate weight (BMI between 18.50 and 24.99 kg/m²), overweight (BMI between 25.00 and 29.99 kg/m²) and obese (BMI ≥ 30.00 kg/m²), according to categories defined by World Health Organization [16].\nPerimeters were measured with an anthropometric tape on the right trunk, arm and leg. Calf perimeter was measured at the greater circumference. The neck perimeter was measured at the midpoint between the clavicle bone and chin. MUAC was measured at the midpoint between the acromion and olecranon bones. Triceps (TSF) and subscapular skinfolds (SBSF) were evaluated using a caliper (Lange®). The TSF was measured at the same levels as those of arm perimeter. The SBSF was measured two centimeters below the lower angle of the scapula bone. Anthropometric data were measured in duplicate by nutritionists, considering the arithmetic mean value among the measurements.\nMeasurement of maternal abdominal fat space was done with ultrasound device Toshiba Xario XG® with a 3.5 mHz multi-frequency convex probe placed above the maternal umbilical scar, with low pressure, and automatic calipers were positioned from anterior aortic wall to linea alba measuring maternal abdominal depth. Two measurements were performed by only one specialist medical researcher, first during maternal inspiration and after during maternal expiration. The arithmetic mean of measurements was used for this research. The measurement of maternal subcutaneous adipose tissue (SAT) was made in the same position as that of VAT measurement, with the automatic caliper positioned from linea alba to dermal edge on the surface of the maternal abdomen. The sum of VAT and SAT was used to estimate the total adipose tissue (TAT) during the evaluation.",
"Statistical analyses were performed through Statistical Package for Social Sciences (SPSS) 21.0. The level of significance was set at p < 0.05. Clinical, epidemiological and ultrasonographic data were presented as quantitative and categorical variables. The test of normality of variables distribution was made. Quantitative variables with asymmetric distributions were described as median and interquartile range. Categorical variables were reported as absolute frequency and percentage. To perform the associations between anthropometrical measurements and VAT and TAT measurements, non-adjusted Spearman correlation (ρ) was used. After that, Spearman correlation was adjusted for factors associated with maternal adipose accumulation during pregnancy: number of pregnancies, pregnant age and gestational age.",
"The study was approved by the Research Ethics Committees of the Health Department of Porto Alegre under number 2.132.090 and in the Presidente Vargas Hospital under number 1.758.959. Written informed consent was obtained from participants.",
"The sample was comprised of 149 pregnant women up to 20 weeks of pregnancy. Nineteen participants were not included in the correlation analysis of the study outcomes due to the decision not to include cases that had some unanswered adjustment variable. Participants were on median 25 years of age [21–31], mostly Caucasian (54.8%, n = 80), had a median pre-pregnancy BMI of 26.22 kg/m² [22.16–31.21], 58.33% (n = 84) with pre-pregnancy BMI classified as overweight or obesity, often had two past pregnancies [1 - 3] and a gestational age average of 16.2 weeks [13.05–18.10]. The anthropometric measurements showed that the MUAC median was 31.0 cm [28.0–35.0], calf perimeter median was 37.0 cm [35.0–41.0] and neck perimeter was 34.0 cm [32.0–36.0]. TSF and SBSF showed median of 31.5 mm [25.0–40.5] and 30.0 mm [21.0–40.5], respectively. The median VAT was 41.7 mm [34.5–52.8] and TAT was 61.1 mm [50.72–71.42], as shown in Table 1.\nTable 1Maternal demographic, gestational and clinical characteristicsVariablesn (%)Median (IQ)Age (years)14925.00 [21.00–31.00]Number of pregnancies14302 [01–03]Gestational age (weeks)14916.20 [13.05–18.10]RaceWhiteBlackDark-skinned14680 (54.80)42 (28.80)24 (16.40)Pre-pregnancy body mass index (kg/m²)14426.22 [22.16–31.21]Underweight3 (2.08)Adequate57 (39.58)Overweight37 (25.70)Obesity47 (32.64)Arm circumference (cm)14731.00 [28.00–35.00]Neck circumference (cm)14734.00 [32.00–36.00]Calf circumference (cm)14637.00 [35.00–41.00]Triceps Skinfold (mm)14731.50 [25.00–40.50]Subscapular Skinfold (mm)14730.00 [21.00–40.50]Central Visceral Fat (mm)14941.70 [34.50–52.80]Total Central Fat (mm)14261.20 [50.65–71.75]Descriptive table with medians [interquartile interval] and frequency (%)Totals may not add up to 149 because of missing values\nMaternal demographic, gestational and clinical characteristics\nRace\nWhite\nBlack\nDark-skinned\n146\n80 (54.80)\n42 (28.80)\n24 (16.40)\nDescriptive table with medians [interquartile interval] and frequency (%)\nTotals may not add up to 149 because of missing values\nTable 2 shows the non-adjusted correlation across anthropometrical measurements and pre-pregnancy BMI values with ultrasound measurements of the maternal abdomen. We can observe that all body perimeters and skin folds showed to be statistically correlated to VAT and TAT. When analyzed individually, calf and neck perimeters and TSF indicate weaker correlations to detect VAT and TAT, when compared to pre-pregnancy BMI. However, MUAC and SBSF presented greater correlations with VAT and TAT, when compared to pre-pregnancy BMI.\nTable 2Non-adjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomenVariablesMid-upper arm circumference (cm)Calf circumference (cm)Neck circumference (cm)Triceps Skinfold (mm)Subscapular Skinfold(mm)Pre-pregnancy BMI (Kg/m²)Central Visceral Fat(mm)ρ0.603*0.498*0.500*0.541*0.597*0.546*n147146147147147144Total CentralFat (mm)ρ0.792*0.677*0.656*0.698*0.740*0.725*n140139140140140137BMI Body mass indexSpearman correlation; *p value < 0.05Totals may not add up to 149 because of missing values\nNon-adjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomen\nCentral Visceral Fat\n(mm)\nTotal Central\nFat (mm)\nBMI Body mass index\nSpearman correlation; *p value < 0.05\nTotals may not add up to 149 because of missing values\nTable 3 presents the adjusted correlation between anthropometric measurements, pre-pregnancy BMI with ultrasound measurements of the maternal abdomen. It is worth mentioning that even with the adjustment for variables associated to maternal adipose accumulation during pregnancy (i.e., number of pregnancies, maternal age and gestational age), the measurements of MUAC, TSF and SBSF maintained statistical significance (p < 0.05), showing values higher than pre-pregnancy BMI.\nTable 3Adjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomenVariablesControl VariablesMid-upper arm circumference (cm)Triceps Skinfold (mm)Subscapular Skinfold (mm)Pre-pregnancy BMI (Kg/m²)Central Visceral Fat(mm)Coef. ρn = 130Without adjustment0.603*0.541*0.597*0.546*Number of pregnancies+ Maternal age (years)+ Gestational age (weeks)0.598*0.598*0.602*0.543*Total Central Fat(mm)Coef. ρn = 130Without adjustment0.792*0.698*0.740*0.725*Number of pregnancies+ Maternal age (years)+ Gestational age (weeks)0.7520.7120.7190.691*BMI Body mass indexSpearman correlation adjusted to number of children, maternal age and gestational age. *p value < 0.05Totals may not add up to 149 because of missing values\nAdjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomen\nCoef. ρ\nn = 130\nNumber of pregnancies\n+ Maternal age (years)\n+ Gestational age (weeks)\nCoef. ρ\nn = 130\nNumber of pregnancies\n+ Maternal age (years)\n+ Gestational age (weeks)\nBMI Body mass index\nSpearman correlation adjusted to number of children, maternal age and gestational age. *p value < 0.05\nTotals may not add up to 149 because of missing values",
"The study found that the MUAC and SBSF measures presented greater correlations with VAT and TAT during the first 20 weeks of pregnancy, with a higher correlation than the pre-pregnancy BMI value. It is important to emphasize that these anthropometric measurements are considered low cost, efficient and replicable in in under-resourced settings.\nThese findings are particularly valuable in cases where pregnancy discovered late or if the individual does not accurately remember their pre-gestational weight in clinical practice, making it difficult to correctly estimate pre-pregnancy BMI. Currently, pre-pregnancy BMI is the anthropometrical indicator of the nutritional state most used as a metabolic risk marker, because women who were overweight or obese are at an elevated relative risk of preeclampsia [17, 18], cesarean section delivery [17], gestational diabetes [18], increasing the relative risk of intrauterine death [18] and more likely to be macrosomic [17]. However, this index is limited with regard to the differentiation of adipose content [1], particularly in the central region, the focus of the present study. Furthermore, despite the ease of measuring BMI, it has low predictive precision for abnormal pregnancy results; therefore, new diagnostic modalities can improve these scores, as demonstrated by Bourdages et al. (2018) and Souza et al. (2016), where the increase in visceral adipose tissue, identified during the first trimester, was associated with a greater chance of developing gestational diabetes mellitus [9, 19].\nResearch indicates that the use of circumferences and skinfolds to determine maternal nutritional state during the first weeks of pregnancy can facilitate metabolic risk detection [1, 7]. A study made in Nigeria with 578 pregnant women showed that MUAC has a strong positive correlation with maternal weight and could be used to identify obesity in women regardless of stage of pregnancy. The authors found that MUAC values of 33 cm might be reliable cut off points for diagnoses of obesity throughout pregnancy [15]. Another study in Central Malaysia with 498 pregnant women found that MUAC was inversely associated with an inadequate rate of gestational weight gain, as compared to normal gestational weight gain [13]. Besides that, a cross-sectional study conducted in South Africa with 164 women showed a strong correlation between MUAC and pre-pregnancy BMI in pregnant women up to 30 weeks’ gestation. The authors found that the MUAC cut-offs for obesity and malnutrition were calculated as 30.57 cm and 22.8 cm, respectively [6].\nAlong with the use of MUAC to determine maternal metabolic risk, SBSF proved useful in detecting low weight newborns in a prospective study conducted in Argentina with 488 pregnant women. The authors found that a low increase of skinfolds during pregnancy can indicate low birthweight, demonstrating significant consequences to the offspring’s health [20, 21].\nOn the other hand, the measurement of the different compartments of abdominal fat via ultrasound provides an adequate estimate of central adiposity [22]; however, the assessment of maternal central fat is not routinely performed in obstetric ultrasounds. The risk of adverse conditions caused by an excess of fat, particularly visceral fat, to the pregnant woman and fetus health is clearly consolidated in the literature [8, 10, 17, 23, 24], therefore, thus the precision and cost appraisal of different fat compartments is highly important to the population [25].\nAligned with the Brazilian Ministry of Health recommendations on resolute prenatal care [26] and the isolated capacity of MUAC and SBSF to detect the increase in amounts of maternal central fat, the inclusion of clinical anthropometry during the first 20 weeks of pregnancy can contribute to accurate maternal metabolic risk prediction. Thus, their complementary use in clinical practice is justified, as well as their possible inclusion in protocols for nutritional assessment during pregnancy. MUAC in particular can act as an alternative tool in screening for patients with metabolic risk in developing countries where monitoring of weight gain is not feasible due to limitations involving equipment (adipometer, for example), team or prenatal coverage.\nIt is well known that the early diagnosis of metabolic risk during the first half of pregnancy allows for the early implementation of preventive and therapeutic measures, resulting in improved maternal and fetal health due to immediate action following diagnosis [27]. On the other hand, cases where the maternal central fat estimate is appropriate, according to the suggested anthropometry, even among obese women, the high cost of following patients without metabolic risk and high risk prenatal could be avoided.\n Strengths and limitations The study was conducted in a low risk primary healthcare setting that did not provide additional intervention to the sample. The data were obtained from routine follow-up of patients, which suggests that the findings can be easily transferred to the clinical practice. The most important aspect of the research was that the study’s population was comprised of pregnant women where the anthropometrical measurements can be become tools for decision making in clinical practice both in low and high obstetric risk environments. The low number of researchers involved in data collection minimizes possible mistakes of measurement. Throughout the study, one researcher was in charge of ultrasound collections and two researchers were in charge of the general questionnaire and nutritional assessment. A limitation of the study is the cross-sectional design that prevents the verification of pregnancy outcomes among women with large amounts of central fat due to the absence of blood sampling that diagnoses metabolic risk in pregnancy. Another limitation of the study includes the self-reporting of pre-pregnancy weight, as it may have been affected by recall bias. However, several studies demonstrated that the use of self-reported weight in pregnant women and young adults is valid [28, 29].\nThe study was conducted in a low risk primary healthcare setting that did not provide additional intervention to the sample. The data were obtained from routine follow-up of patients, which suggests that the findings can be easily transferred to the clinical practice. The most important aspect of the research was that the study’s population was comprised of pregnant women where the anthropometrical measurements can be become tools for decision making in clinical practice both in low and high obstetric risk environments. The low number of researchers involved in data collection minimizes possible mistakes of measurement. Throughout the study, one researcher was in charge of ultrasound collections and two researchers were in charge of the general questionnaire and nutritional assessment. A limitation of the study is the cross-sectional design that prevents the verification of pregnancy outcomes among women with large amounts of central fat due to the absence of blood sampling that diagnoses metabolic risk in pregnancy. Another limitation of the study includes the self-reporting of pre-pregnancy weight, as it may have been affected by recall bias. However, several studies demonstrated that the use of self-reported weight in pregnant women and young adults is valid [28, 29].",
"The study was conducted in a low risk primary healthcare setting that did not provide additional intervention to the sample. The data were obtained from routine follow-up of patients, which suggests that the findings can be easily transferred to the clinical practice. The most important aspect of the research was that the study’s population was comprised of pregnant women where the anthropometrical measurements can be become tools for decision making in clinical practice both in low and high obstetric risk environments. The low number of researchers involved in data collection minimizes possible mistakes of measurement. Throughout the study, one researcher was in charge of ultrasound collections and two researchers were in charge of the general questionnaire and nutritional assessment. A limitation of the study is the cross-sectional design that prevents the verification of pregnancy outcomes among women with large amounts of central fat due to the absence of blood sampling that diagnoses metabolic risk in pregnancy. Another limitation of the study includes the self-reporting of pre-pregnancy weight, as it may have been affected by recall bias. However, several studies demonstrated that the use of self-reported weight in pregnant women and young adults is valid [28, 29].",
"The study found that the anthropometric measurements most correlated with VAT and TAT were MUAC and SBSF, both of which had a higher correlation than pre-pregnancy BMI. It is possible to provide a practical, reliable and low-cost clinical estimate of ultrasound measurements of maternal central fat, which will help to identify women at high metabolic risk during pregnancy, based on efficient and replicable anthropometrical examination. In situations where pre-gestational weight measurement and subsequent BMI values cannot be obtained, the anthropometric measurements, MUAC and SBSF are useful and may be ideal for measuring the nutritional status of pregnant women, especially in low- and middle-income countries."
] |
[
null,
null,
null,
null,
null,
null,
null,
"results",
"discussion",
null,
"conclusion"
] |
[
"Anthropometry",
"Pregnant women",
"Mid-upper arm circumference",
"Body mass index"
] |
Background:
Physiological adaptations during pregnancy are caused in order to ensure an adequate supply of nutrients to the fetus [1]. Among these adaptations, the accumulation of fat in different deposits is associated with metabolic consequences for the gestational environment. The mechanisms responsible for the structural and functional differences specific to adipose tissue deposits are still being investigated [2]. However, it is known that visceral fat is strongly associated with increased metabolic diseases [3]. The investigation of measures that assist in the identification of different fat deposits can be useful to predict risk and set specific treatment goals, reducing negative consequences for fetal and maternal health. The prenatal care is an opportune time, as it is characterized by a preventive practice recommended during pregnancy that provides a perspective for important healthcare functions, including health promotion, screening and diagnosis, and disease prevention [4].
For the mother’s anthropometry, the pre-pregnancy body mass index (BMI) is considered a reflection of maternal nutritional status before pregnancy, while gestational weight gain is the aggregate change of the mother’s, child’s and placental mass in the physiologic state [5, 6]. Utilizing BMI as a measurement of health during pregnancy can have limitations, primarily due to pregnancy-associated weight gain and oedema, as well as late booking into antenatal care in a population-level setting [6]. The assessment of the amount of maternal fat deposits during pregnancy is limited by the inability to use ionizing radiation in computerized tomography and dual-energy x-ray absorptiometry, high cost and maintenance of nuclear magnetic resonance and low accuracy of bioelectric impedance analysis. Thereafter, the most commonly used method to measure maternal body composition changes in pregnancy is anthropometry, particularly the use of skinfolds and circumferences [1, 7].
The use of ultrasound to measure the distribution of maternal visceral adipose tissue (VAT) and total adipose tissue (TAT) is becoming useful because it is widely available in hospitals to predict a higher risk of preeclampsia [8], insulin resistance and metabolic diseases [9, 10], premature birth [8] and average birth weight [11]. It is worth mentioning that ultrasound is an easy, quick, safe, non-invasive, precise and reliable method to identify patients with adverse metabolic profiles [12]. However, due to the easiness of execution and low cost, anthropometrical measurements, like mid-upper arm circumference (MUAC), may become an alternative to the use of ultrasound devices [6, 13–15].
There were no found studies that determined the predictive capabilities of anthropometric measures alternative to pre-pregnancy BMI in relation to a reference method, such as VAT and TAT obtained by ultrasound. The aim of this study is to identify which anthropometric measurements correlate better with the maternal fat deposits measured by ultrasound.
Methods:
Design The cross-sectional study recruited patients from 2016 to 2018 at the Ultrasound Department of Murialdo Health Center School that provides services of fetal medicine to Brazil’s Unified Health System at the city of Porto Alegre, Rio Grande do Sul, Brazil.
The cross-sectional study recruited patients from 2016 to 2018 at the Ultrasound Department of Murialdo Health Center School that provides services of fetal medicine to Brazil’s Unified Health System at the city of Porto Alegre, Rio Grande do Sul, Brazil.
Participants For this study, single pregnancies, below twenty gestational weeks, with no evident fetal malformations, with no scars in the abdominal cavity or in sites to use adipometer that hide the measurements and scheduled for routine appointments in Brazil’s Unified Health System were included. The pregnant women who met the inclusion criteria were invited to participate and, after informed consent, were included with the completion of the maternal, clinical and epidemiological questionnaire.
For this study, single pregnancies, below twenty gestational weeks, with no evident fetal malformations, with no scars in the abdominal cavity or in sites to use adipometer that hide the measurements and scheduled for routine appointments in Brazil’s Unified Health System were included. The pregnant women who met the inclusion criteria were invited to participate and, after informed consent, were included with the completion of the maternal, clinical and epidemiological questionnaire.
Measures The maternal anthropometric evaluation included assessment of anthropometric measures (weight and height) and evaluation of body composition. The participants were encouraged to use minimal clothing and no shoes and accessories like watches, bracelets and earrings. The body weight was measured in kilograms with portable electronic digital scale Marte® LC200-PP (São Paulo, São Paulo, Brazil) accurate to 50 g. The height was measured in meters with extensible portable stadiometer Alturexata® (Belo Horizonte, Minas Gerais, Brazil). Maternal pre-pregnancy weight was collected from the prenatal pregnant chart and confirmed by the maternal report and, when there was no record, in the BMI referring to the first trimester of pregnancy. The pre-pregnancy BMI (in kg/m²) was calculated through the formula, current weight divided by the current height squared. The classification used was pre-pregnancy BMI in underweight (BMI < 18.50 kg/m²), adequate weight (BMI between 18.50 and 24.99 kg/m²), overweight (BMI between 25.00 and 29.99 kg/m²) and obese (BMI ≥ 30.00 kg/m²), according to categories defined by World Health Organization [16].
Perimeters were measured with an anthropometric tape on the right trunk, arm and leg. Calf perimeter was measured at the greater circumference. The neck perimeter was measured at the midpoint between the clavicle bone and chin. MUAC was measured at the midpoint between the acromion and olecranon bones. Triceps (TSF) and subscapular skinfolds (SBSF) were evaluated using a caliper (Lange®). The TSF was measured at the same levels as those of arm perimeter. The SBSF was measured two centimeters below the lower angle of the scapula bone. Anthropometric data were measured in duplicate by nutritionists, considering the arithmetic mean value among the measurements.
Measurement of maternal abdominal fat space was done with ultrasound device Toshiba Xario XG® with a 3.5 mHz multi-frequency convex probe placed above the maternal umbilical scar, with low pressure, and automatic calipers were positioned from anterior aortic wall to linea alba measuring maternal abdominal depth. Two measurements were performed by only one specialist medical researcher, first during maternal inspiration and after during maternal expiration. The arithmetic mean of measurements was used for this research. The measurement of maternal subcutaneous adipose tissue (SAT) was made in the same position as that of VAT measurement, with the automatic caliper positioned from linea alba to dermal edge on the surface of the maternal abdomen. The sum of VAT and SAT was used to estimate the total adipose tissue (TAT) during the evaluation.
The maternal anthropometric evaluation included assessment of anthropometric measures (weight and height) and evaluation of body composition. The participants were encouraged to use minimal clothing and no shoes and accessories like watches, bracelets and earrings. The body weight was measured in kilograms with portable electronic digital scale Marte® LC200-PP (São Paulo, São Paulo, Brazil) accurate to 50 g. The height was measured in meters with extensible portable stadiometer Alturexata® (Belo Horizonte, Minas Gerais, Brazil). Maternal pre-pregnancy weight was collected from the prenatal pregnant chart and confirmed by the maternal report and, when there was no record, in the BMI referring to the first trimester of pregnancy. The pre-pregnancy BMI (in kg/m²) was calculated through the formula, current weight divided by the current height squared. The classification used was pre-pregnancy BMI in underweight (BMI < 18.50 kg/m²), adequate weight (BMI between 18.50 and 24.99 kg/m²), overweight (BMI between 25.00 and 29.99 kg/m²) and obese (BMI ≥ 30.00 kg/m²), according to categories defined by World Health Organization [16].
Perimeters were measured with an anthropometric tape on the right trunk, arm and leg. Calf perimeter was measured at the greater circumference. The neck perimeter was measured at the midpoint between the clavicle bone and chin. MUAC was measured at the midpoint between the acromion and olecranon bones. Triceps (TSF) and subscapular skinfolds (SBSF) were evaluated using a caliper (Lange®). The TSF was measured at the same levels as those of arm perimeter. The SBSF was measured two centimeters below the lower angle of the scapula bone. Anthropometric data were measured in duplicate by nutritionists, considering the arithmetic mean value among the measurements.
Measurement of maternal abdominal fat space was done with ultrasound device Toshiba Xario XG® with a 3.5 mHz multi-frequency convex probe placed above the maternal umbilical scar, with low pressure, and automatic calipers were positioned from anterior aortic wall to linea alba measuring maternal abdominal depth. Two measurements were performed by only one specialist medical researcher, first during maternal inspiration and after during maternal expiration. The arithmetic mean of measurements was used for this research. The measurement of maternal subcutaneous adipose tissue (SAT) was made in the same position as that of VAT measurement, with the automatic caliper positioned from linea alba to dermal edge on the surface of the maternal abdomen. The sum of VAT and SAT was used to estimate the total adipose tissue (TAT) during the evaluation.
Statistical analysis Statistical analyses were performed through Statistical Package for Social Sciences (SPSS) 21.0. The level of significance was set at p < 0.05. Clinical, epidemiological and ultrasonographic data were presented as quantitative and categorical variables. The test of normality of variables distribution was made. Quantitative variables with asymmetric distributions were described as median and interquartile range. Categorical variables were reported as absolute frequency and percentage. To perform the associations between anthropometrical measurements and VAT and TAT measurements, non-adjusted Spearman correlation (ρ) was used. After that, Spearman correlation was adjusted for factors associated with maternal adipose accumulation during pregnancy: number of pregnancies, pregnant age and gestational age.
Statistical analyses were performed through Statistical Package for Social Sciences (SPSS) 21.0. The level of significance was set at p < 0.05. Clinical, epidemiological and ultrasonographic data were presented as quantitative and categorical variables. The test of normality of variables distribution was made. Quantitative variables with asymmetric distributions were described as median and interquartile range. Categorical variables were reported as absolute frequency and percentage. To perform the associations between anthropometrical measurements and VAT and TAT measurements, non-adjusted Spearman correlation (ρ) was used. After that, Spearman correlation was adjusted for factors associated with maternal adipose accumulation during pregnancy: number of pregnancies, pregnant age and gestational age.
Ethical Aspects The study was approved by the Research Ethics Committees of the Health Department of Porto Alegre under number 2.132.090 and in the Presidente Vargas Hospital under number 1.758.959. Written informed consent was obtained from participants.
The study was approved by the Research Ethics Committees of the Health Department of Porto Alegre under number 2.132.090 and in the Presidente Vargas Hospital under number 1.758.959. Written informed consent was obtained from participants.
Design:
The cross-sectional study recruited patients from 2016 to 2018 at the Ultrasound Department of Murialdo Health Center School that provides services of fetal medicine to Brazil’s Unified Health System at the city of Porto Alegre, Rio Grande do Sul, Brazil.
Participants:
For this study, single pregnancies, below twenty gestational weeks, with no evident fetal malformations, with no scars in the abdominal cavity or in sites to use adipometer that hide the measurements and scheduled for routine appointments in Brazil’s Unified Health System were included. The pregnant women who met the inclusion criteria were invited to participate and, after informed consent, were included with the completion of the maternal, clinical and epidemiological questionnaire.
Measures:
The maternal anthropometric evaluation included assessment of anthropometric measures (weight and height) and evaluation of body composition. The participants were encouraged to use minimal clothing and no shoes and accessories like watches, bracelets and earrings. The body weight was measured in kilograms with portable electronic digital scale Marte® LC200-PP (São Paulo, São Paulo, Brazil) accurate to 50 g. The height was measured in meters with extensible portable stadiometer Alturexata® (Belo Horizonte, Minas Gerais, Brazil). Maternal pre-pregnancy weight was collected from the prenatal pregnant chart and confirmed by the maternal report and, when there was no record, in the BMI referring to the first trimester of pregnancy. The pre-pregnancy BMI (in kg/m²) was calculated through the formula, current weight divided by the current height squared. The classification used was pre-pregnancy BMI in underweight (BMI < 18.50 kg/m²), adequate weight (BMI between 18.50 and 24.99 kg/m²), overweight (BMI between 25.00 and 29.99 kg/m²) and obese (BMI ≥ 30.00 kg/m²), according to categories defined by World Health Organization [16].
Perimeters were measured with an anthropometric tape on the right trunk, arm and leg. Calf perimeter was measured at the greater circumference. The neck perimeter was measured at the midpoint between the clavicle bone and chin. MUAC was measured at the midpoint between the acromion and olecranon bones. Triceps (TSF) and subscapular skinfolds (SBSF) were evaluated using a caliper (Lange®). The TSF was measured at the same levels as those of arm perimeter. The SBSF was measured two centimeters below the lower angle of the scapula bone. Anthropometric data were measured in duplicate by nutritionists, considering the arithmetic mean value among the measurements.
Measurement of maternal abdominal fat space was done with ultrasound device Toshiba Xario XG® with a 3.5 mHz multi-frequency convex probe placed above the maternal umbilical scar, with low pressure, and automatic calipers were positioned from anterior aortic wall to linea alba measuring maternal abdominal depth. Two measurements were performed by only one specialist medical researcher, first during maternal inspiration and after during maternal expiration. The arithmetic mean of measurements was used for this research. The measurement of maternal subcutaneous adipose tissue (SAT) was made in the same position as that of VAT measurement, with the automatic caliper positioned from linea alba to dermal edge on the surface of the maternal abdomen. The sum of VAT and SAT was used to estimate the total adipose tissue (TAT) during the evaluation.
Statistical analysis:
Statistical analyses were performed through Statistical Package for Social Sciences (SPSS) 21.0. The level of significance was set at p < 0.05. Clinical, epidemiological and ultrasonographic data were presented as quantitative and categorical variables. The test of normality of variables distribution was made. Quantitative variables with asymmetric distributions were described as median and interquartile range. Categorical variables were reported as absolute frequency and percentage. To perform the associations between anthropometrical measurements and VAT and TAT measurements, non-adjusted Spearman correlation (ρ) was used. After that, Spearman correlation was adjusted for factors associated with maternal adipose accumulation during pregnancy: number of pregnancies, pregnant age and gestational age.
Ethical Aspects:
The study was approved by the Research Ethics Committees of the Health Department of Porto Alegre under number 2.132.090 and in the Presidente Vargas Hospital under number 1.758.959. Written informed consent was obtained from participants.
Results:
The sample was comprised of 149 pregnant women up to 20 weeks of pregnancy. Nineteen participants were not included in the correlation analysis of the study outcomes due to the decision not to include cases that had some unanswered adjustment variable. Participants were on median 25 years of age [21–31], mostly Caucasian (54.8%, n = 80), had a median pre-pregnancy BMI of 26.22 kg/m² [22.16–31.21], 58.33% (n = 84) with pre-pregnancy BMI classified as overweight or obesity, often had two past pregnancies [1 - 3] and a gestational age average of 16.2 weeks [13.05–18.10]. The anthropometric measurements showed that the MUAC median was 31.0 cm [28.0–35.0], calf perimeter median was 37.0 cm [35.0–41.0] and neck perimeter was 34.0 cm [32.0–36.0]. TSF and SBSF showed median of 31.5 mm [25.0–40.5] and 30.0 mm [21.0–40.5], respectively. The median VAT was 41.7 mm [34.5–52.8] and TAT was 61.1 mm [50.72–71.42], as shown in Table 1.
Table 1Maternal demographic, gestational and clinical characteristicsVariablesn (%)Median (IQ)Age (years)14925.00 [21.00–31.00]Number of pregnancies14302 [01–03]Gestational age (weeks)14916.20 [13.05–18.10]RaceWhiteBlackDark-skinned14680 (54.80)42 (28.80)24 (16.40)Pre-pregnancy body mass index (kg/m²)14426.22 [22.16–31.21]Underweight3 (2.08)Adequate57 (39.58)Overweight37 (25.70)Obesity47 (32.64)Arm circumference (cm)14731.00 [28.00–35.00]Neck circumference (cm)14734.00 [32.00–36.00]Calf circumference (cm)14637.00 [35.00–41.00]Triceps Skinfold (mm)14731.50 [25.00–40.50]Subscapular Skinfold (mm)14730.00 [21.00–40.50]Central Visceral Fat (mm)14941.70 [34.50–52.80]Total Central Fat (mm)14261.20 [50.65–71.75]Descriptive table with medians [interquartile interval] and frequency (%)Totals may not add up to 149 because of missing values
Maternal demographic, gestational and clinical characteristics
Race
White
Black
Dark-skinned
146
80 (54.80)
42 (28.80)
24 (16.40)
Descriptive table with medians [interquartile interval] and frequency (%)
Totals may not add up to 149 because of missing values
Table 2 shows the non-adjusted correlation across anthropometrical measurements and pre-pregnancy BMI values with ultrasound measurements of the maternal abdomen. We can observe that all body perimeters and skin folds showed to be statistically correlated to VAT and TAT. When analyzed individually, calf and neck perimeters and TSF indicate weaker correlations to detect VAT and TAT, when compared to pre-pregnancy BMI. However, MUAC and SBSF presented greater correlations with VAT and TAT, when compared to pre-pregnancy BMI.
Table 2Non-adjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomenVariablesMid-upper arm circumference (cm)Calf circumference (cm)Neck circumference (cm)Triceps Skinfold (mm)Subscapular Skinfold(mm)Pre-pregnancy BMI (Kg/m²)Central Visceral Fat(mm)ρ0.603*0.498*0.500*0.541*0.597*0.546*n147146147147147144Total CentralFat (mm)ρ0.792*0.677*0.656*0.698*0.740*0.725*n140139140140140137BMI Body mass indexSpearman correlation; *p value < 0.05Totals may not add up to 149 because of missing values
Non-adjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomen
Central Visceral Fat
(mm)
Total Central
Fat (mm)
BMI Body mass index
Spearman correlation; *p value < 0.05
Totals may not add up to 149 because of missing values
Table 3 presents the adjusted correlation between anthropometric measurements, pre-pregnancy BMI with ultrasound measurements of the maternal abdomen. It is worth mentioning that even with the adjustment for variables associated to maternal adipose accumulation during pregnancy (i.e., number of pregnancies, maternal age and gestational age), the measurements of MUAC, TSF and SBSF maintained statistical significance (p < 0.05), showing values higher than pre-pregnancy BMI.
Table 3Adjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomenVariablesControl VariablesMid-upper arm circumference (cm)Triceps Skinfold (mm)Subscapular Skinfold (mm)Pre-pregnancy BMI (Kg/m²)Central Visceral Fat(mm)Coef. ρn = 130Without adjustment0.603*0.541*0.597*0.546*Number of pregnancies+ Maternal age (years)+ Gestational age (weeks)0.598*0.598*0.602*0.543*Total Central Fat(mm)Coef. ρn = 130Without adjustment0.792*0.698*0.740*0.725*Number of pregnancies+ Maternal age (years)+ Gestational age (weeks)0.7520.7120.7190.691*BMI Body mass indexSpearman correlation adjusted to number of children, maternal age and gestational age. *p value < 0.05Totals may not add up to 149 because of missing values
Adjusted correlation of anthropometrical measurements and pre-pregnancy body mass index values with ultrasound measurements of maternal abdomen
Coef. ρ
n = 130
Number of pregnancies
+ Maternal age (years)
+ Gestational age (weeks)
Coef. ρ
n = 130
Number of pregnancies
+ Maternal age (years)
+ Gestational age (weeks)
BMI Body mass index
Spearman correlation adjusted to number of children, maternal age and gestational age. *p value < 0.05
Totals may not add up to 149 because of missing values
Discussion:
The study found that the MUAC and SBSF measures presented greater correlations with VAT and TAT during the first 20 weeks of pregnancy, with a higher correlation than the pre-pregnancy BMI value. It is important to emphasize that these anthropometric measurements are considered low cost, efficient and replicable in in under-resourced settings.
These findings are particularly valuable in cases where pregnancy discovered late or if the individual does not accurately remember their pre-gestational weight in clinical practice, making it difficult to correctly estimate pre-pregnancy BMI. Currently, pre-pregnancy BMI is the anthropometrical indicator of the nutritional state most used as a metabolic risk marker, because women who were overweight or obese are at an elevated relative risk of preeclampsia [17, 18], cesarean section delivery [17], gestational diabetes [18], increasing the relative risk of intrauterine death [18] and more likely to be macrosomic [17]. However, this index is limited with regard to the differentiation of adipose content [1], particularly in the central region, the focus of the present study. Furthermore, despite the ease of measuring BMI, it has low predictive precision for abnormal pregnancy results; therefore, new diagnostic modalities can improve these scores, as demonstrated by Bourdages et al. (2018) and Souza et al. (2016), where the increase in visceral adipose tissue, identified during the first trimester, was associated with a greater chance of developing gestational diabetes mellitus [9, 19].
Research indicates that the use of circumferences and skinfolds to determine maternal nutritional state during the first weeks of pregnancy can facilitate metabolic risk detection [1, 7]. A study made in Nigeria with 578 pregnant women showed that MUAC has a strong positive correlation with maternal weight and could be used to identify obesity in women regardless of stage of pregnancy. The authors found that MUAC values of 33 cm might be reliable cut off points for diagnoses of obesity throughout pregnancy [15]. Another study in Central Malaysia with 498 pregnant women found that MUAC was inversely associated with an inadequate rate of gestational weight gain, as compared to normal gestational weight gain [13]. Besides that, a cross-sectional study conducted in South Africa with 164 women showed a strong correlation between MUAC and pre-pregnancy BMI in pregnant women up to 30 weeks’ gestation. The authors found that the MUAC cut-offs for obesity and malnutrition were calculated as 30.57 cm and 22.8 cm, respectively [6].
Along with the use of MUAC to determine maternal metabolic risk, SBSF proved useful in detecting low weight newborns in a prospective study conducted in Argentina with 488 pregnant women. The authors found that a low increase of skinfolds during pregnancy can indicate low birthweight, demonstrating significant consequences to the offspring’s health [20, 21].
On the other hand, the measurement of the different compartments of abdominal fat via ultrasound provides an adequate estimate of central adiposity [22]; however, the assessment of maternal central fat is not routinely performed in obstetric ultrasounds. The risk of adverse conditions caused by an excess of fat, particularly visceral fat, to the pregnant woman and fetus health is clearly consolidated in the literature [8, 10, 17, 23, 24], therefore, thus the precision and cost appraisal of different fat compartments is highly important to the population [25].
Aligned with the Brazilian Ministry of Health recommendations on resolute prenatal care [26] and the isolated capacity of MUAC and SBSF to detect the increase in amounts of maternal central fat, the inclusion of clinical anthropometry during the first 20 weeks of pregnancy can contribute to accurate maternal metabolic risk prediction. Thus, their complementary use in clinical practice is justified, as well as their possible inclusion in protocols for nutritional assessment during pregnancy. MUAC in particular can act as an alternative tool in screening for patients with metabolic risk in developing countries where monitoring of weight gain is not feasible due to limitations involving equipment (adipometer, for example), team or prenatal coverage.
It is well known that the early diagnosis of metabolic risk during the first half of pregnancy allows for the early implementation of preventive and therapeutic measures, resulting in improved maternal and fetal health due to immediate action following diagnosis [27]. On the other hand, cases where the maternal central fat estimate is appropriate, according to the suggested anthropometry, even among obese women, the high cost of following patients without metabolic risk and high risk prenatal could be avoided.
Strengths and limitations The study was conducted in a low risk primary healthcare setting that did not provide additional intervention to the sample. The data were obtained from routine follow-up of patients, which suggests that the findings can be easily transferred to the clinical practice. The most important aspect of the research was that the study’s population was comprised of pregnant women where the anthropometrical measurements can be become tools for decision making in clinical practice both in low and high obstetric risk environments. The low number of researchers involved in data collection minimizes possible mistakes of measurement. Throughout the study, one researcher was in charge of ultrasound collections and two researchers were in charge of the general questionnaire and nutritional assessment. A limitation of the study is the cross-sectional design that prevents the verification of pregnancy outcomes among women with large amounts of central fat due to the absence of blood sampling that diagnoses metabolic risk in pregnancy. Another limitation of the study includes the self-reporting of pre-pregnancy weight, as it may have been affected by recall bias. However, several studies demonstrated that the use of self-reported weight in pregnant women and young adults is valid [28, 29].
The study was conducted in a low risk primary healthcare setting that did not provide additional intervention to the sample. The data were obtained from routine follow-up of patients, which suggests that the findings can be easily transferred to the clinical practice. The most important aspect of the research was that the study’s population was comprised of pregnant women where the anthropometrical measurements can be become tools for decision making in clinical practice both in low and high obstetric risk environments. The low number of researchers involved in data collection minimizes possible mistakes of measurement. Throughout the study, one researcher was in charge of ultrasound collections and two researchers were in charge of the general questionnaire and nutritional assessment. A limitation of the study is the cross-sectional design that prevents the verification of pregnancy outcomes among women with large amounts of central fat due to the absence of blood sampling that diagnoses metabolic risk in pregnancy. Another limitation of the study includes the self-reporting of pre-pregnancy weight, as it may have been affected by recall bias. However, several studies demonstrated that the use of self-reported weight in pregnant women and young adults is valid [28, 29].
Strengths and limitations:
The study was conducted in a low risk primary healthcare setting that did not provide additional intervention to the sample. The data were obtained from routine follow-up of patients, which suggests that the findings can be easily transferred to the clinical practice. The most important aspect of the research was that the study’s population was comprised of pregnant women where the anthropometrical measurements can be become tools for decision making in clinical practice both in low and high obstetric risk environments. The low number of researchers involved in data collection minimizes possible mistakes of measurement. Throughout the study, one researcher was in charge of ultrasound collections and two researchers were in charge of the general questionnaire and nutritional assessment. A limitation of the study is the cross-sectional design that prevents the verification of pregnancy outcomes among women with large amounts of central fat due to the absence of blood sampling that diagnoses metabolic risk in pregnancy. Another limitation of the study includes the self-reporting of pre-pregnancy weight, as it may have been affected by recall bias. However, several studies demonstrated that the use of self-reported weight in pregnant women and young adults is valid [28, 29].
Conclusions:
The study found that the anthropometric measurements most correlated with VAT and TAT were MUAC and SBSF, both of which had a higher correlation than pre-pregnancy BMI. It is possible to provide a practical, reliable and low-cost clinical estimate of ultrasound measurements of maternal central fat, which will help to identify women at high metabolic risk during pregnancy, based on efficient and replicable anthropometrical examination. In situations where pre-gestational weight measurement and subsequent BMI values cannot be obtained, the anthropometric measurements, MUAC and SBSF are useful and may be ideal for measuring the nutritional status of pregnant women, especially in low- and middle-income countries.
|
Background:
Determining anthropometric measures that indicate different fat deposits can be useful to predict metabolic risk and set specific treatment goals, reducing negative consequences for maternal and fetal health. In cases where pre-gestational weight measure and subsequent body mass index (BMI) values cannot be determined, other anthropometric measurements may be ideal for measuring the nutritional status of pregnant women, especially in low- and middle-income countries. This study aims to identify which anthropometric measurements correlate better with the maternal fat deposits measured by ultrasound.
Methods:
A cross-sectional study was conducted with pregnant women from the city of Porto Alegre (city), capital of Rio Grande do Sul (state), southern Brazil, from October 2016 until January 2018. Anthropometrical variables (weight, height, mid-upper arm circumference [MUAC], circumferences of calf and neck and triceps skinfolds [TSF] and subscapular skinfolds [SBSF]), and ultrasound variables (visceral adipose tissue [VAT] and total adipose tissue [TAT]) were collected. To verify the correlation of anthropometric and ultrasound measurements, a non-adjusted and adjusted Spearman correlation was used. The study was approved by the ethics committees.
Results:
The age median of the 149 pregnant women was 25 years [21-31], pre-pregnancy BMI was 26.22 kg/m² [22.16-31.21] and gestational age was 16.2 weeks [13.05-18.10]. The best measurements correlated with VAT and TAT were MUAC and SBSF, both of which showed a higher correlation than pre-pregnancy BMI.
Conclusions:
It is possible to provide a practical and reliable estimate of VAT and TAT from the anthropometric evaluation (MUAC or SBSF) that is low cost, efficient and replicable in an outpatient clinic environment, especially in low- and middle-income countries.
|
Background:
Physiological adaptations during pregnancy are caused in order to ensure an adequate supply of nutrients to the fetus [1]. Among these adaptations, the accumulation of fat in different deposits is associated with metabolic consequences for the gestational environment. The mechanisms responsible for the structural and functional differences specific to adipose tissue deposits are still being investigated [2]. However, it is known that visceral fat is strongly associated with increased metabolic diseases [3]. The investigation of measures that assist in the identification of different fat deposits can be useful to predict risk and set specific treatment goals, reducing negative consequences for fetal and maternal health. The prenatal care is an opportune time, as it is characterized by a preventive practice recommended during pregnancy that provides a perspective for important healthcare functions, including health promotion, screening and diagnosis, and disease prevention [4].
For the mother’s anthropometry, the pre-pregnancy body mass index (BMI) is considered a reflection of maternal nutritional status before pregnancy, while gestational weight gain is the aggregate change of the mother’s, child’s and placental mass in the physiologic state [5, 6]. Utilizing BMI as a measurement of health during pregnancy can have limitations, primarily due to pregnancy-associated weight gain and oedema, as well as late booking into antenatal care in a population-level setting [6]. The assessment of the amount of maternal fat deposits during pregnancy is limited by the inability to use ionizing radiation in computerized tomography and dual-energy x-ray absorptiometry, high cost and maintenance of nuclear magnetic resonance and low accuracy of bioelectric impedance analysis. Thereafter, the most commonly used method to measure maternal body composition changes in pregnancy is anthropometry, particularly the use of skinfolds and circumferences [1, 7].
The use of ultrasound to measure the distribution of maternal visceral adipose tissue (VAT) and total adipose tissue (TAT) is becoming useful because it is widely available in hospitals to predict a higher risk of preeclampsia [8], insulin resistance and metabolic diseases [9, 10], premature birth [8] and average birth weight [11]. It is worth mentioning that ultrasound is an easy, quick, safe, non-invasive, precise and reliable method to identify patients with adverse metabolic profiles [12]. However, due to the easiness of execution and low cost, anthropometrical measurements, like mid-upper arm circumference (MUAC), may become an alternative to the use of ultrasound devices [6, 13–15].
There were no found studies that determined the predictive capabilities of anthropometric measures alternative to pre-pregnancy BMI in relation to a reference method, such as VAT and TAT obtained by ultrasound. The aim of this study is to identify which anthropometric measurements correlate better with the maternal fat deposits measured by ultrasound.
Conclusions:
The study found that the anthropometric measurements most correlated with VAT and TAT were MUAC and SBSF, both of which had a higher correlation than pre-pregnancy BMI. It is possible to provide a practical, reliable and low-cost clinical estimate of ultrasound measurements of maternal central fat, which will help to identify women at high metabolic risk during pregnancy, based on efficient and replicable anthropometrical examination. In situations where pre-gestational weight measurement and subsequent BMI values cannot be obtained, the anthropometric measurements, MUAC and SBSF are useful and may be ideal for measuring the nutritional status of pregnant women, especially in low- and middle-income countries.
|
Background:
Determining anthropometric measures that indicate different fat deposits can be useful to predict metabolic risk and set specific treatment goals, reducing negative consequences for maternal and fetal health. In cases where pre-gestational weight measure and subsequent body mass index (BMI) values cannot be determined, other anthropometric measurements may be ideal for measuring the nutritional status of pregnant women, especially in low- and middle-income countries. This study aims to identify which anthropometric measurements correlate better with the maternal fat deposits measured by ultrasound.
Methods:
A cross-sectional study was conducted with pregnant women from the city of Porto Alegre (city), capital of Rio Grande do Sul (state), southern Brazil, from October 2016 until January 2018. Anthropometrical variables (weight, height, mid-upper arm circumference [MUAC], circumferences of calf and neck and triceps skinfolds [TSF] and subscapular skinfolds [SBSF]), and ultrasound variables (visceral adipose tissue [VAT] and total adipose tissue [TAT]) were collected. To verify the correlation of anthropometric and ultrasound measurements, a non-adjusted and adjusted Spearman correlation was used. The study was approved by the ethics committees.
Results:
The age median of the 149 pregnant women was 25 years [21-31], pre-pregnancy BMI was 26.22 kg/m² [22.16-31.21] and gestational age was 16.2 weeks [13.05-18.10]. The best measurements correlated with VAT and TAT were MUAC and SBSF, both of which showed a higher correlation than pre-pregnancy BMI.
Conclusions:
It is possible to provide a practical and reliable estimate of VAT and TAT from the anthropometric evaluation (MUAC or SBSF) that is low cost, efficient and replicable in an outpatient clinic environment, especially in low- and middle-income countries.
| 5,609 | 353 | 11 |
[
"maternal",
"pregnancy",
"bmi",
"measurements",
"pre",
"pre pregnancy",
"study",
"weight",
"measured",
"fat"
] |
[
"test",
"test"
] | null |
[CONTENT] Anthropometry | Pregnant women | Mid-upper arm circumference | Body mass index [SUMMARY]
| null |
[CONTENT] Anthropometry | Pregnant women | Mid-upper arm circumference | Body mass index [SUMMARY]
|
[CONTENT] Anthropometry | Pregnant women | Mid-upper arm circumference | Body mass index [SUMMARY]
|
[CONTENT] Anthropometry | Pregnant women | Mid-upper arm circumference | Body mass index [SUMMARY]
|
[CONTENT] Anthropometry | Pregnant women | Mid-upper arm circumference | Body mass index [SUMMARY]
|
[CONTENT] Adult | Body Weights and Measures | Correlation of Data | Cross-Sectional Studies | Female | Humans | Intra-Abdominal Fat | Organ Size | Pregnancy | Pregnancy Trimester, First | Pregnancy Trimester, Second | Ultrasonography, Prenatal | Young Adult [SUMMARY]
| null |
[CONTENT] Adult | Body Weights and Measures | Correlation of Data | Cross-Sectional Studies | Female | Humans | Intra-Abdominal Fat | Organ Size | Pregnancy | Pregnancy Trimester, First | Pregnancy Trimester, Second | Ultrasonography, Prenatal | Young Adult [SUMMARY]
|
[CONTENT] Adult | Body Weights and Measures | Correlation of Data | Cross-Sectional Studies | Female | Humans | Intra-Abdominal Fat | Organ Size | Pregnancy | Pregnancy Trimester, First | Pregnancy Trimester, Second | Ultrasonography, Prenatal | Young Adult [SUMMARY]
|
[CONTENT] Adult | Body Weights and Measures | Correlation of Data | Cross-Sectional Studies | Female | Humans | Intra-Abdominal Fat | Organ Size | Pregnancy | Pregnancy Trimester, First | Pregnancy Trimester, Second | Ultrasonography, Prenatal | Young Adult [SUMMARY]
|
[CONTENT] Adult | Body Weights and Measures | Correlation of Data | Cross-Sectional Studies | Female | Humans | Intra-Abdominal Fat | Organ Size | Pregnancy | Pregnancy Trimester, First | Pregnancy Trimester, Second | Ultrasonography, Prenatal | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] maternal | pregnancy | bmi | measurements | pre | pre pregnancy | study | weight | measured | fat [SUMMARY]
| null |
[CONTENT] maternal | pregnancy | bmi | measurements | pre | pre pregnancy | study | weight | measured | fat [SUMMARY]
|
[CONTENT] maternal | pregnancy | bmi | measurements | pre | pre pregnancy | study | weight | measured | fat [SUMMARY]
|
[CONTENT] maternal | pregnancy | bmi | measurements | pre | pre pregnancy | study | weight | measured | fat [SUMMARY]
|
[CONTENT] maternal | pregnancy | bmi | measurements | pre | pre pregnancy | study | weight | measured | fat [SUMMARY]
|
[CONTENT] deposits | pregnancy | method | fat deposits | maternal | metabolic | fat | ultrasound | use | tissue [SUMMARY]
| null |
[CONTENT] mm | age | 00 | values | cm | table | body mass | mass | pregnancy | pre [SUMMARY]
|
[CONTENT] muac sbsf | anthropometric measurements | measurements | sbsf | women | low | muac | anthropometric | bmi | pre [SUMMARY]
|
[CONTENT] pregnancy | maternal | bmi | study | measurements | measured | weight | health | risk | women [SUMMARY]
|
[CONTENT] pregnancy | maternal | bmi | study | measurements | measured | weight | health | risk | women [SUMMARY]
|
[CONTENT] ||| BMI ||| [SUMMARY]
| null |
[CONTENT] 149 | 25 years ||| 21-31 | BMI | 26.22 kg/m² ||| 22.16 | 16.2 weeks ||| 13.05-18.10 ||| VAT | TAT | MUAC | SBSF | BMI [SUMMARY]
|
[CONTENT] VAT | TAT | MUAC | SBSF [SUMMARY]
|
[CONTENT] ||| BMI ||| ||| Porto Alegre | Rio Grande | Brazil | October 2016 | January 2018 ||| VAT ||| Spearman ||| ||| ||| 149 | 25 years ||| 21-31 | BMI | 26.22 kg/m² ||| 22.16 | 16.2 weeks ||| 13.05-18.10 ||| VAT | TAT | MUAC | SBSF | BMI ||| VAT | TAT | MUAC | SBSF [SUMMARY]
|
[CONTENT] ||| BMI ||| ||| Porto Alegre | Rio Grande | Brazil | October 2016 | January 2018 ||| VAT ||| Spearman ||| ||| ||| 149 | 25 years ||| 21-31 | BMI | 26.22 kg/m² ||| 22.16 | 16.2 weeks ||| 13.05-18.10 ||| VAT | TAT | MUAC | SBSF | BMI ||| VAT | TAT | MUAC | SBSF [SUMMARY]
|
|||||
Low expression of microRNA-204 (miR-204) is associated with poor clinical outcome of acute myeloid leukemia (AML) patients.
|
26126974
|
Acute myeloid leukemia (AML) is a heterogeneous neoplasm of the bone marrow with poor prognosis. In clinical practice new prognostic factors are still needed. MicroRNAs (miRs), small endogenous noncoding RNAs, play an essential role in the development and progression of acute leukemia. The aim of the study was to evaluate miR-204 expression in patients with AML at diagnosis and after induction chemotherapy, in comparison to healthy controls. We also investigated, if miR-204 expression correlates with clinical features of AML patients.
|
BACKGROUND
|
miR-204 expression has been analyzed using RT-PCR in 95 bone marrow specimens from newly diagnosed AML patients in comparison to 20 healthy subject.
|
METHODS
|
We showed down-regulated miR-204 expression in AML patients, which was associated with shorter patients' survival. Higher expression of miR-204 in patients after induction therapy was correlated with complete remission achieving.
|
RESULTS
|
We showed low miR-204 expression in AML and found it to be an independent prognostic factor in this patient population.
|
CONCLUSIONS
|
[
"Adult",
"Aged",
"Aged, 80 and over",
"Female",
"Humans",
"Leukemia, Myeloid, Acute",
"Male",
"MicroRNAs",
"Middle Aged",
"Prognosis",
"Survival Analysis",
"Treatment Outcome",
"Young Adult"
] |
4508825
|
Introduction
|
Acute myeloid leukemia (AML) in adults is a hematological malignancy with proliferation of myeloblasts in the bone marrow. Population of AML patients has heterogenous clinical course and different prognosis. Although dynamic progress on the field of pathogenesis and development of this blood cancer has been made, it is still difficult to predict clinical outcome and response to therapy of AML patients. Genetic and molecular markers are used in every day practice, but new predictors are needed, for better patients’ classification and therapy planning [1, 2].
microRNA (miRs) are small non-coding RNAs, which play an important role in neoplastic transformation. miRs act by influencing posttranscriptional gene expression, cell development, differentiation, proliferation and apoptosis [3–8].
microRNA-204 (miR-204) role has been investigated and described in few solid tumors, particularly pancreatic and colorectal cancer, where it was found to be associated with process of autophagy [3, 9]. But there is no data about miR-204 role in acute myeloid leukemia. The purpose of this study was to evaluate miR-204 expression in AML patients in relation to clinical factors, survival and comparison to healthy subjects.
| null | null |
Results
|
We compared AML samples to healthy controls and found significantly lower miR-204 expression in AML patients (p < 0.05). There were no differences in miR-204 expression between male and female patients.
After successful induction chemotherapy expression of miR-204 significantly increased (median 0.443606; p = 0.000590). Patients with increased miR-204 expression after induction therapy had higher chance for remission (p = 0.01438). 83 % of pts with CR after induction therapy had high miR-204 expression and 60 % of patients who did not respond to therapy had low miR-204 expression (Fig. 1). At the moment of diagnosis, Mean miR-204 expression in group who achieved CR was significantly lower than in the group which did not achieve response.Fig. 1Correlation between miR-204 expression after chemotherapy and remission
Correlation between miR-204 expression after chemotherapy and remission
In patients with increase of miR-204 expression after chemotherapy time to relapse was longer (median 13 months) than in patients with decreased miR-204 expression (median 4.5 months), p = 0.003347, Fig. 2.Fig. 2miR-204 expression after chemotherapy and time to relapse
miR-204 expression after chemotherapy and time to relapse
miR-204 expression level also influenced patients’ outcome (median value was used as a cut-off). Higher miR-204 expression before therapy was associated with longer survival (Fig. 3).Fig. 3Predicted overall survival depending on miR-204 expression before chemotherapy
Predicted overall survival depending on miR-204 expression before chemotherapy
| null | null |
[
"Patients characteristic",
"Treatment schedules",
"Isolation and expression analysis of microRNAs",
"Statistical analysis"
] |
[
"The study included 95 patients (aged 60.2 ± 15.0, 22–90, Male = 61 %) with newly diagnosed AML. Samples of the bone marrow for miR-204 expression analysis were collected before start of chemotherapy and repeated after completed induction chemotherapy (in 40 patients). Patients were treated in the Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation of Wroclaw Medical University, Wroclaw, Poland. A control group of 20 healthy subjects was also taken into account (aged 64.2 ± 10.5, 39–80, Male = 65 %). According to AML FAB classification, 7 patients had AML M0, 34 had M1, 29 had M2, 14 had M4 and 11 had M5. There were 73 patients with primary leukemia and 22 patients with leukemia secondary to myelodysplastic or myeloproliferative syndrome. Summary of patients’ characteristics is presented in Table 1.Table 1Clinical characteristics of patients with AMLCharacteristicCasesSex Male56 Female39Age (years) Range22-90 Median61FAB subtype M07 M1/M263 M4/M525WBC (G/L) Range0.2-295 Median14HGB g% Range5.8-13.1 Median9.3PLT (G/L) Range2-310 Median65Lactate dehydrogenase (LDH) U/l Range108-4565 Median340Blasts in bone marrow <50 %35 ≥50 %60Cytogenetics Farorable5 Intermediate39 Unfavorable51Chemotherapy Intensive56 Low dose27 Best supportive care12Molecular testsTotal 60 patients AML/ETO (positive/negative)4/56 CBFb-MYH11 (positive/negative)2/58 NPM1 (positive/negative)7/53 FLT3/ITD (positive/negative)13/47Complete remission Yes (total)51 Yes (after 1st line therapy)36 No44Duration of remission (months) Range2-54 Median20Time to relapse (months) Range3-23 Median12Survival (months) Range0-55 Median3\nClinical characteristics of patients with AML\n Treatment schedules Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).\nResearch was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.\nFifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).\nResearch was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.",
"Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).\nResearch was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.",
"Bone marrow mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. Total RNA and microRNA were extracted from collected AML mononuclear cells using mirVana™ miRNA Isolation Kit (Ambion) according to the protocol of the manufacturer. Then 5 μl total miRNA was used as a template into synthesis of cDNA using TaqMan MicroRNA Trasncription Reaction Kit (Applied Biosystems) and 3 μl specific miRNA primers from the TaqMan MicroRNA Assays (Applied Biosystems). Individual reaction was carried out in 15 μl total volume in thermal condition: 16 °C for 30 min, 42° for 30 min, 85 °C for 5 min. TaqMan MicroRNA Assays for miR-204 (hsa-miR-204), and RNU48 were used. The expression level of each microRNA was measured in relative real-time PCR method using TaqMan Gene Expression Assays and TaqMan Fast Universal PCR Master Mix (Applied Biosytems). All reactions were done in triplicate in a total volume of 20 μl on 96-well plates. The real-time PCR was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems) under thermal cycling conditions: 20 s at 95 °C and 40 cycles of 1 s at 95 °C and 20 s at 60 °C. For quantification, the samples were normalized against the expression of RNU48 (internal control). Relative quantification factors (RQ) for the examined miRs were calculated using ΔΔCT method.",
"The differences in means of gene expressions between the study and the control patients were estimated using t-Student’s test (for independent samples). To examine the time it takes for death and remission to occur, a Cox’s regression was applied [11]. The difference between the gene expressions before and after treatment was estimated using robust regression and multivariate approach [12]. The computation was performed in R software [13] and based on the simulation technique known as Gibbs sampling in WinBUGS platform [14]. Kaplan-Meier survival curves were used to determine any significant relationship between miR-204 expression and clinical outcome. Results were considered statistically significant when p was <0.05."
] |
[
null,
null,
null,
null
] |
[
"Introduction",
"Material and methods",
"Patients characteristic",
"Treatment schedules",
"Isolation and expression analysis of microRNAs",
"Statistical analysis",
"Results",
"Discussion"
] |
[
"Acute myeloid leukemia (AML) in adults is a hematological malignancy with proliferation of myeloblasts in the bone marrow. Population of AML patients has heterogenous clinical course and different prognosis. Although dynamic progress on the field of pathogenesis and development of this blood cancer has been made, it is still difficult to predict clinical outcome and response to therapy of AML patients. Genetic and molecular markers are used in every day practice, but new predictors are needed, for better patients’ classification and therapy planning [1, 2].\nmicroRNA (miRs) are small non-coding RNAs, which play an important role in neoplastic transformation. miRs act by influencing posttranscriptional gene expression, cell development, differentiation, proliferation and apoptosis [3–8].\nmicroRNA-204 (miR-204) role has been investigated and described in few solid tumors, particularly pancreatic and colorectal cancer, where it was found to be associated with process of autophagy [3, 9]. But there is no data about miR-204 role in acute myeloid leukemia. The purpose of this study was to evaluate miR-204 expression in AML patients in relation to clinical factors, survival and comparison to healthy subjects.",
" Patients characteristic The study included 95 patients (aged 60.2 ± 15.0, 22–90, Male = 61 %) with newly diagnosed AML. Samples of the bone marrow for miR-204 expression analysis were collected before start of chemotherapy and repeated after completed induction chemotherapy (in 40 patients). Patients were treated in the Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation of Wroclaw Medical University, Wroclaw, Poland. A control group of 20 healthy subjects was also taken into account (aged 64.2 ± 10.5, 39–80, Male = 65 %). According to AML FAB classification, 7 patients had AML M0, 34 had M1, 29 had M2, 14 had M4 and 11 had M5. There were 73 patients with primary leukemia and 22 patients with leukemia secondary to myelodysplastic or myeloproliferative syndrome. Summary of patients’ characteristics is presented in Table 1.Table 1Clinical characteristics of patients with AMLCharacteristicCasesSex Male56 Female39Age (years) Range22-90 Median61FAB subtype M07 M1/M263 M4/M525WBC (G/L) Range0.2-295 Median14HGB g% Range5.8-13.1 Median9.3PLT (G/L) Range2-310 Median65Lactate dehydrogenase (LDH) U/l Range108-4565 Median340Blasts in bone marrow <50 %35 ≥50 %60Cytogenetics Farorable5 Intermediate39 Unfavorable51Chemotherapy Intensive56 Low dose27 Best supportive care12Molecular testsTotal 60 patients AML/ETO (positive/negative)4/56 CBFb-MYH11 (positive/negative)2/58 NPM1 (positive/negative)7/53 FLT3/ITD (positive/negative)13/47Complete remission Yes (total)51 Yes (after 1st line therapy)36 No44Duration of remission (months) Range2-54 Median20Time to relapse (months) Range3-23 Median12Survival (months) Range0-55 Median3\nClinical characteristics of patients with AML\n Treatment schedules Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).\nResearch was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.\nFifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).\nResearch was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.\nThe study included 95 patients (aged 60.2 ± 15.0, 22–90, Male = 61 %) with newly diagnosed AML. Samples of the bone marrow for miR-204 expression analysis were collected before start of chemotherapy and repeated after completed induction chemotherapy (in 40 patients). Patients were treated in the Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation of Wroclaw Medical University, Wroclaw, Poland. A control group of 20 healthy subjects was also taken into account (aged 64.2 ± 10.5, 39–80, Male = 65 %). According to AML FAB classification, 7 patients had AML M0, 34 had M1, 29 had M2, 14 had M4 and 11 had M5. There were 73 patients with primary leukemia and 22 patients with leukemia secondary to myelodysplastic or myeloproliferative syndrome. Summary of patients’ characteristics is presented in Table 1.Table 1Clinical characteristics of patients with AMLCharacteristicCasesSex Male56 Female39Age (years) Range22-90 Median61FAB subtype M07 M1/M263 M4/M525WBC (G/L) Range0.2-295 Median14HGB g% Range5.8-13.1 Median9.3PLT (G/L) Range2-310 Median65Lactate dehydrogenase (LDH) U/l Range108-4565 Median340Blasts in bone marrow <50 %35 ≥50 %60Cytogenetics Farorable5 Intermediate39 Unfavorable51Chemotherapy Intensive56 Low dose27 Best supportive care12Molecular testsTotal 60 patients AML/ETO (positive/negative)4/56 CBFb-MYH11 (positive/negative)2/58 NPM1 (positive/negative)7/53 FLT3/ITD (positive/negative)13/47Complete remission Yes (total)51 Yes (after 1st line therapy)36 No44Duration of remission (months) Range2-54 Median20Time to relapse (months) Range3-23 Median12Survival (months) Range0-55 Median3\nClinical characteristics of patients with AML\n Treatment schedules Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).\nResearch was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.\nFifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).\nResearch was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.\n Isolation and expression analysis of microRNAs Bone marrow mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. Total RNA and microRNA were extracted from collected AML mononuclear cells using mirVana™ miRNA Isolation Kit (Ambion) according to the protocol of the manufacturer. Then 5 μl total miRNA was used as a template into synthesis of cDNA using TaqMan MicroRNA Trasncription Reaction Kit (Applied Biosystems) and 3 μl specific miRNA primers from the TaqMan MicroRNA Assays (Applied Biosystems). Individual reaction was carried out in 15 μl total volume in thermal condition: 16 °C for 30 min, 42° for 30 min, 85 °C for 5 min. TaqMan MicroRNA Assays for miR-204 (hsa-miR-204), and RNU48 were used. The expression level of each microRNA was measured in relative real-time PCR method using TaqMan Gene Expression Assays and TaqMan Fast Universal PCR Master Mix (Applied Biosytems). All reactions were done in triplicate in a total volume of 20 μl on 96-well plates. The real-time PCR was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems) under thermal cycling conditions: 20 s at 95 °C and 40 cycles of 1 s at 95 °C and 20 s at 60 °C. For quantification, the samples were normalized against the expression of RNU48 (internal control). Relative quantification factors (RQ) for the examined miRs were calculated using ΔΔCT method.\nBone marrow mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. Total RNA and microRNA were extracted from collected AML mononuclear cells using mirVana™ miRNA Isolation Kit (Ambion) according to the protocol of the manufacturer. Then 5 μl total miRNA was used as a template into synthesis of cDNA using TaqMan MicroRNA Trasncription Reaction Kit (Applied Biosystems) and 3 μl specific miRNA primers from the TaqMan MicroRNA Assays (Applied Biosystems). Individual reaction was carried out in 15 μl total volume in thermal condition: 16 °C for 30 min, 42° for 30 min, 85 °C for 5 min. TaqMan MicroRNA Assays for miR-204 (hsa-miR-204), and RNU48 were used. The expression level of each microRNA was measured in relative real-time PCR method using TaqMan Gene Expression Assays and TaqMan Fast Universal PCR Master Mix (Applied Biosytems). All reactions were done in triplicate in a total volume of 20 μl on 96-well plates. The real-time PCR was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems) under thermal cycling conditions: 20 s at 95 °C and 40 cycles of 1 s at 95 °C and 20 s at 60 °C. For quantification, the samples were normalized against the expression of RNU48 (internal control). Relative quantification factors (RQ) for the examined miRs were calculated using ΔΔCT method.\n Statistical analysis The differences in means of gene expressions between the study and the control patients were estimated using t-Student’s test (for independent samples). To examine the time it takes for death and remission to occur, a Cox’s regression was applied [11]. The difference between the gene expressions before and after treatment was estimated using robust regression and multivariate approach [12]. The computation was performed in R software [13] and based on the simulation technique known as Gibbs sampling in WinBUGS platform [14]. Kaplan-Meier survival curves were used to determine any significant relationship between miR-204 expression and clinical outcome. Results were considered statistically significant when p was <0.05.\nThe differences in means of gene expressions between the study and the control patients were estimated using t-Student’s test (for independent samples). To examine the time it takes for death and remission to occur, a Cox’s regression was applied [11]. The difference between the gene expressions before and after treatment was estimated using robust regression and multivariate approach [12]. The computation was performed in R software [13] and based on the simulation technique known as Gibbs sampling in WinBUGS platform [14]. Kaplan-Meier survival curves were used to determine any significant relationship between miR-204 expression and clinical outcome. Results were considered statistically significant when p was <0.05.",
"The study included 95 patients (aged 60.2 ± 15.0, 22–90, Male = 61 %) with newly diagnosed AML. Samples of the bone marrow for miR-204 expression analysis were collected before start of chemotherapy and repeated after completed induction chemotherapy (in 40 patients). Patients were treated in the Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation of Wroclaw Medical University, Wroclaw, Poland. A control group of 20 healthy subjects was also taken into account (aged 64.2 ± 10.5, 39–80, Male = 65 %). According to AML FAB classification, 7 patients had AML M0, 34 had M1, 29 had M2, 14 had M4 and 11 had M5. There were 73 patients with primary leukemia and 22 patients with leukemia secondary to myelodysplastic or myeloproliferative syndrome. Summary of patients’ characteristics is presented in Table 1.Table 1Clinical characteristics of patients with AMLCharacteristicCasesSex Male56 Female39Age (years) Range22-90 Median61FAB subtype M07 M1/M263 M4/M525WBC (G/L) Range0.2-295 Median14HGB g% Range5.8-13.1 Median9.3PLT (G/L) Range2-310 Median65Lactate dehydrogenase (LDH) U/l Range108-4565 Median340Blasts in bone marrow <50 %35 ≥50 %60Cytogenetics Farorable5 Intermediate39 Unfavorable51Chemotherapy Intensive56 Low dose27 Best supportive care12Molecular testsTotal 60 patients AML/ETO (positive/negative)4/56 CBFb-MYH11 (positive/negative)2/58 NPM1 (positive/negative)7/53 FLT3/ITD (positive/negative)13/47Complete remission Yes (total)51 Yes (after 1st line therapy)36 No44Duration of remission (months) Range2-54 Median20Time to relapse (months) Range3-23 Median12Survival (months) Range0-55 Median3\nClinical characteristics of patients with AML\n Treatment schedules Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).\nResearch was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.\nFifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).\nResearch was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.",
"Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).\nResearch was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.",
"Bone marrow mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. Total RNA and microRNA were extracted from collected AML mononuclear cells using mirVana™ miRNA Isolation Kit (Ambion) according to the protocol of the manufacturer. Then 5 μl total miRNA was used as a template into synthesis of cDNA using TaqMan MicroRNA Trasncription Reaction Kit (Applied Biosystems) and 3 μl specific miRNA primers from the TaqMan MicroRNA Assays (Applied Biosystems). Individual reaction was carried out in 15 μl total volume in thermal condition: 16 °C for 30 min, 42° for 30 min, 85 °C for 5 min. TaqMan MicroRNA Assays for miR-204 (hsa-miR-204), and RNU48 were used. The expression level of each microRNA was measured in relative real-time PCR method using TaqMan Gene Expression Assays and TaqMan Fast Universal PCR Master Mix (Applied Biosytems). All reactions were done in triplicate in a total volume of 20 μl on 96-well plates. The real-time PCR was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems) under thermal cycling conditions: 20 s at 95 °C and 40 cycles of 1 s at 95 °C and 20 s at 60 °C. For quantification, the samples were normalized against the expression of RNU48 (internal control). Relative quantification factors (RQ) for the examined miRs were calculated using ΔΔCT method.",
"The differences in means of gene expressions between the study and the control patients were estimated using t-Student’s test (for independent samples). To examine the time it takes for death and remission to occur, a Cox’s regression was applied [11]. The difference between the gene expressions before and after treatment was estimated using robust regression and multivariate approach [12]. The computation was performed in R software [13] and based on the simulation technique known as Gibbs sampling in WinBUGS platform [14]. Kaplan-Meier survival curves were used to determine any significant relationship between miR-204 expression and clinical outcome. Results were considered statistically significant when p was <0.05.",
"We compared AML samples to healthy controls and found significantly lower miR-204 expression in AML patients (p < 0.05). There were no differences in miR-204 expression between male and female patients.\nAfter successful induction chemotherapy expression of miR-204 significantly increased (median 0.443606; p = 0.000590). Patients with increased miR-204 expression after induction therapy had higher chance for remission (p = 0.01438). 83 % of pts with CR after induction therapy had high miR-204 expression and 60 % of patients who did not respond to therapy had low miR-204 expression (Fig. 1). At the moment of diagnosis, Mean miR-204 expression in group who achieved CR was significantly lower than in the group which did not achieve response.Fig. 1Correlation between miR-204 expression after chemotherapy and remission\nCorrelation between miR-204 expression after chemotherapy and remission\nIn patients with increase of miR-204 expression after chemotherapy time to relapse was longer (median 13 months) than in patients with decreased miR-204 expression (median 4.5 months), p = 0.003347, Fig. 2.Fig. 2miR-204 expression after chemotherapy and time to relapse\nmiR-204 expression after chemotherapy and time to relapse\nmiR-204 expression level also influenced patients’ outcome (median value was used as a cut-off). Higher miR-204 expression before therapy was associated with longer survival (Fig. 3).Fig. 3Predicted overall survival depending on miR-204 expression before chemotherapy\nPredicted overall survival depending on miR-204 expression before chemotherapy",
"Small non-coding microRNAs affect process of formation, development, proliferation and apoptosis of normal and malignant cells in human body. Their role has been proved in many cancers, including leukemia [4, 15–17]. Pathways regulation based on microRNA expression is still unknown. Some miRs may act as tumor suppressors and others as oncogenes [18, 19]. Recently, many miRs have been investigated as prognostic and predictive markers. miR-204 expression and its role has been studied in some solid tumors: pancreatic, gastric, prostate [3, 9, 20], in which it acts by down-regulation of Bcl-2 and has tumor suppressor role. miR-204 also influences NTRK2 gene expression in neuroblastoma cancer [21] and FOXC1 gene in invasive endometrial cancer [22].\nIn our study we showed down-regulated expression of miR-204 in acute myeloid leukemia patients comparing to healthy controls which is in line with observation made by Garzon et al. [23]. Authors found lower miR-204 expression in nucleophosmin positive AML patients and assumed hypothesis, that miR-204 targeted HOXA10 and MEIS1. Those two genes perturb myeloid differentiation and can lead to AML. Ying et al. in their study revealed that loss of miR-204 promotes cancer cell migration through increased expression of brain derived neurotrophic factor or its TrkB receptor [24]. In gastric cancer loss of miR-204 expression was associated with poor outcome, because it caused increase of antiapoptotic protein Bcl-2. In the light of these results Chen et al. demonstrated that miR-204 was down-regulated and its overexpression leaded to loss of cancer cell viability in pancreatic cancer [3]. Authors also showed that miR-204 regulates expression of Mcl-1 (Myeloid cell leukemia) by direct binding to 3′UTR [3]. Overexpressed Mcl-1 is associated with cell survival, while its downregulation leads to cell death. Increased miR-204 negatively regulated Mcl-1. Those observations could explain results of our study. We also analyzed correlations between miR-204 at diagnosis and after chemotherapy and clinical outcome. After effective induction chemotherapy we observed increased miR-204 expression and this change correlated positively with chance for remission achieving. Supposedly higher miR-204 expression could induce leukemic cell deaths and leaded to disease remission. On the other side, patients who had higher miR-204 expression at diagnosis had more favorable clinical outcome than others. Similar role of miR-204 has been also proved in gliomas, where low miR-204 expression leaded to a stem cell-like phenotype, and its overexpression resulted in reduced tumorigenicity and loss of stemness transcription factor SOX4 [24]. In contrast were results by Sümbül et al., who detected high miR-204 expression in colorectal cancer in comparison to healthy population. This finding was not related to any clinicopathological parameters nor survival [9]. Authors explained discrepant findings by the role of increased miR-204 in autophagy and apoptosis.\nConcluding, as to our knowledge, we found for the first time down-regulated miR-204 expression in acute myeloid leukemia patients with its implication to disease prognosis. Functionality of miR-204 acting as a tumor suppressor makes this new molecule an useful biomarker in cancer diagnosis and management. Further investigation on miR-204 regulation and its target genes should be performed."
] |
[
"introduction",
"materials|methods",
null,
null,
null,
null,
"results",
"discussion"
] |
[
"miR-204",
"Acute myeloid leukemia",
"Expression",
"Survival",
"Prognosis"
] |
Introduction:
Acute myeloid leukemia (AML) in adults is a hematological malignancy with proliferation of myeloblasts in the bone marrow. Population of AML patients has heterogenous clinical course and different prognosis. Although dynamic progress on the field of pathogenesis and development of this blood cancer has been made, it is still difficult to predict clinical outcome and response to therapy of AML patients. Genetic and molecular markers are used in every day practice, but new predictors are needed, for better patients’ classification and therapy planning [1, 2].
microRNA (miRs) are small non-coding RNAs, which play an important role in neoplastic transformation. miRs act by influencing posttranscriptional gene expression, cell development, differentiation, proliferation and apoptosis [3–8].
microRNA-204 (miR-204) role has been investigated and described in few solid tumors, particularly pancreatic and colorectal cancer, where it was found to be associated with process of autophagy [3, 9]. But there is no data about miR-204 role in acute myeloid leukemia. The purpose of this study was to evaluate miR-204 expression in AML patients in relation to clinical factors, survival and comparison to healthy subjects.
Material and methods:
Patients characteristic The study included 95 patients (aged 60.2 ± 15.0, 22–90, Male = 61 %) with newly diagnosed AML. Samples of the bone marrow for miR-204 expression analysis were collected before start of chemotherapy and repeated after completed induction chemotherapy (in 40 patients). Patients were treated in the Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation of Wroclaw Medical University, Wroclaw, Poland. A control group of 20 healthy subjects was also taken into account (aged 64.2 ± 10.5, 39–80, Male = 65 %). According to AML FAB classification, 7 patients had AML M0, 34 had M1, 29 had M2, 14 had M4 and 11 had M5. There were 73 patients with primary leukemia and 22 patients with leukemia secondary to myelodysplastic or myeloproliferative syndrome. Summary of patients’ characteristics is presented in Table 1.Table 1Clinical characteristics of patients with AMLCharacteristicCasesSex Male56 Female39Age (years) Range22-90 Median61FAB subtype M07 M1/M263 M4/M525WBC (G/L) Range0.2-295 Median14HGB g% Range5.8-13.1 Median9.3PLT (G/L) Range2-310 Median65Lactate dehydrogenase (LDH) U/l Range108-4565 Median340Blasts in bone marrow <50 %35 ≥50 %60Cytogenetics Farorable5 Intermediate39 Unfavorable51Chemotherapy Intensive56 Low dose27 Best supportive care12Molecular testsTotal 60 patients AML/ETO (positive/negative)4/56 CBFb-MYH11 (positive/negative)2/58 NPM1 (positive/negative)7/53 FLT3/ITD (positive/negative)13/47Complete remission Yes (total)51 Yes (after 1st line therapy)36 No44Duration of remission (months) Range2-54 Median20Time to relapse (months) Range3-23 Median12Survival (months) Range0-55 Median3
Clinical characteristics of patients with AML
Treatment schedules Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).
Research was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.
Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).
Research was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.
The study included 95 patients (aged 60.2 ± 15.0, 22–90, Male = 61 %) with newly diagnosed AML. Samples of the bone marrow for miR-204 expression analysis were collected before start of chemotherapy and repeated after completed induction chemotherapy (in 40 patients). Patients were treated in the Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation of Wroclaw Medical University, Wroclaw, Poland. A control group of 20 healthy subjects was also taken into account (aged 64.2 ± 10.5, 39–80, Male = 65 %). According to AML FAB classification, 7 patients had AML M0, 34 had M1, 29 had M2, 14 had M4 and 11 had M5. There were 73 patients with primary leukemia and 22 patients with leukemia secondary to myelodysplastic or myeloproliferative syndrome. Summary of patients’ characteristics is presented in Table 1.Table 1Clinical characteristics of patients with AMLCharacteristicCasesSex Male56 Female39Age (years) Range22-90 Median61FAB subtype M07 M1/M263 M4/M525WBC (G/L) Range0.2-295 Median14HGB g% Range5.8-13.1 Median9.3PLT (G/L) Range2-310 Median65Lactate dehydrogenase (LDH) U/l Range108-4565 Median340Blasts in bone marrow <50 %35 ≥50 %60Cytogenetics Farorable5 Intermediate39 Unfavorable51Chemotherapy Intensive56 Low dose27 Best supportive care12Molecular testsTotal 60 patients AML/ETO (positive/negative)4/56 CBFb-MYH11 (positive/negative)2/58 NPM1 (positive/negative)7/53 FLT3/ITD (positive/negative)13/47Complete remission Yes (total)51 Yes (after 1st line therapy)36 No44Duration of remission (months) Range2-54 Median20Time to relapse (months) Range3-23 Median12Survival (months) Range0-55 Median3
Clinical characteristics of patients with AML
Treatment schedules Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).
Research was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.
Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).
Research was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.
Isolation and expression analysis of microRNAs Bone marrow mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. Total RNA and microRNA were extracted from collected AML mononuclear cells using mirVana™ miRNA Isolation Kit (Ambion) according to the protocol of the manufacturer. Then 5 μl total miRNA was used as a template into synthesis of cDNA using TaqMan MicroRNA Trasncription Reaction Kit (Applied Biosystems) and 3 μl specific miRNA primers from the TaqMan MicroRNA Assays (Applied Biosystems). Individual reaction was carried out in 15 μl total volume in thermal condition: 16 °C for 30 min, 42° for 30 min, 85 °C for 5 min. TaqMan MicroRNA Assays for miR-204 (hsa-miR-204), and RNU48 were used. The expression level of each microRNA was measured in relative real-time PCR method using TaqMan Gene Expression Assays and TaqMan Fast Universal PCR Master Mix (Applied Biosytems). All reactions were done in triplicate in a total volume of 20 μl on 96-well plates. The real-time PCR was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems) under thermal cycling conditions: 20 s at 95 °C and 40 cycles of 1 s at 95 °C and 20 s at 60 °C. For quantification, the samples were normalized against the expression of RNU48 (internal control). Relative quantification factors (RQ) for the examined miRs were calculated using ΔΔCT method.
Bone marrow mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. Total RNA and microRNA were extracted from collected AML mononuclear cells using mirVana™ miRNA Isolation Kit (Ambion) according to the protocol of the manufacturer. Then 5 μl total miRNA was used as a template into synthesis of cDNA using TaqMan MicroRNA Trasncription Reaction Kit (Applied Biosystems) and 3 μl specific miRNA primers from the TaqMan MicroRNA Assays (Applied Biosystems). Individual reaction was carried out in 15 μl total volume in thermal condition: 16 °C for 30 min, 42° for 30 min, 85 °C for 5 min. TaqMan MicroRNA Assays for miR-204 (hsa-miR-204), and RNU48 were used. The expression level of each microRNA was measured in relative real-time PCR method using TaqMan Gene Expression Assays and TaqMan Fast Universal PCR Master Mix (Applied Biosytems). All reactions were done in triplicate in a total volume of 20 μl on 96-well plates. The real-time PCR was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems) under thermal cycling conditions: 20 s at 95 °C and 40 cycles of 1 s at 95 °C and 20 s at 60 °C. For quantification, the samples were normalized against the expression of RNU48 (internal control). Relative quantification factors (RQ) for the examined miRs were calculated using ΔΔCT method.
Statistical analysis The differences in means of gene expressions between the study and the control patients were estimated using t-Student’s test (for independent samples). To examine the time it takes for death and remission to occur, a Cox’s regression was applied [11]. The difference between the gene expressions before and after treatment was estimated using robust regression and multivariate approach [12]. The computation was performed in R software [13] and based on the simulation technique known as Gibbs sampling in WinBUGS platform [14]. Kaplan-Meier survival curves were used to determine any significant relationship between miR-204 expression and clinical outcome. Results were considered statistically significant when p was <0.05.
The differences in means of gene expressions between the study and the control patients were estimated using t-Student’s test (for independent samples). To examine the time it takes for death and remission to occur, a Cox’s regression was applied [11]. The difference between the gene expressions before and after treatment was estimated using robust regression and multivariate approach [12]. The computation was performed in R software [13] and based on the simulation technique known as Gibbs sampling in WinBUGS platform [14]. Kaplan-Meier survival curves were used to determine any significant relationship between miR-204 expression and clinical outcome. Results were considered statistically significant when p was <0.05.
Patients characteristic:
The study included 95 patients (aged 60.2 ± 15.0, 22–90, Male = 61 %) with newly diagnosed AML. Samples of the bone marrow for miR-204 expression analysis were collected before start of chemotherapy and repeated after completed induction chemotherapy (in 40 patients). Patients were treated in the Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation of Wroclaw Medical University, Wroclaw, Poland. A control group of 20 healthy subjects was also taken into account (aged 64.2 ± 10.5, 39–80, Male = 65 %). According to AML FAB classification, 7 patients had AML M0, 34 had M1, 29 had M2, 14 had M4 and 11 had M5. There were 73 patients with primary leukemia and 22 patients with leukemia secondary to myelodysplastic or myeloproliferative syndrome. Summary of patients’ characteristics is presented in Table 1.Table 1Clinical characteristics of patients with AMLCharacteristicCasesSex Male56 Female39Age (years) Range22-90 Median61FAB subtype M07 M1/M263 M4/M525WBC (G/L) Range0.2-295 Median14HGB g% Range5.8-13.1 Median9.3PLT (G/L) Range2-310 Median65Lactate dehydrogenase (LDH) U/l Range108-4565 Median340Blasts in bone marrow <50 %35 ≥50 %60Cytogenetics Farorable5 Intermediate39 Unfavorable51Chemotherapy Intensive56 Low dose27 Best supportive care12Molecular testsTotal 60 patients AML/ETO (positive/negative)4/56 CBFb-MYH11 (positive/negative)2/58 NPM1 (positive/negative)7/53 FLT3/ITD (positive/negative)13/47Complete remission Yes (total)51 Yes (after 1st line therapy)36 No44Duration of remission (months) Range2-54 Median20Time to relapse (months) Range3-23 Median12Survival (months) Range0-55 Median3
Clinical characteristics of patients with AML
Treatment schedules Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).
Research was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.
Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).
Research was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.
Treatment schedules:
Fifty six patients were treated with standard induction intensive chemotherapy (daunorubicin plus cytarabine 3 + 7), 27 received low dose chemotherapy (low dose cytarabine or azacitidine) and 12 best supportive care only. After completion of induction therapy response to treatment was evaluated. CR was defined by Cheson criteria. [10]. 40 bone marrow samples were re-evaluated for miR-204 expression after chemotherapy. Patients were followed up for median 21 month (range 1–40 months).
Research was carried out in compliance with the Helsinki Declaration. For the study approval of Bioethical Committee of Wroclaw Medical University was obtained. Written informed consent for study was obtained from all the participants.
Isolation and expression analysis of microRNAs:
Bone marrow mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. Total RNA and microRNA were extracted from collected AML mononuclear cells using mirVana™ miRNA Isolation Kit (Ambion) according to the protocol of the manufacturer. Then 5 μl total miRNA was used as a template into synthesis of cDNA using TaqMan MicroRNA Trasncription Reaction Kit (Applied Biosystems) and 3 μl specific miRNA primers from the TaqMan MicroRNA Assays (Applied Biosystems). Individual reaction was carried out in 15 μl total volume in thermal condition: 16 °C for 30 min, 42° for 30 min, 85 °C for 5 min. TaqMan MicroRNA Assays for miR-204 (hsa-miR-204), and RNU48 were used. The expression level of each microRNA was measured in relative real-time PCR method using TaqMan Gene Expression Assays and TaqMan Fast Universal PCR Master Mix (Applied Biosytems). All reactions were done in triplicate in a total volume of 20 μl on 96-well plates. The real-time PCR was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems) under thermal cycling conditions: 20 s at 95 °C and 40 cycles of 1 s at 95 °C and 20 s at 60 °C. For quantification, the samples were normalized against the expression of RNU48 (internal control). Relative quantification factors (RQ) for the examined miRs were calculated using ΔΔCT method.
Statistical analysis:
The differences in means of gene expressions between the study and the control patients were estimated using t-Student’s test (for independent samples). To examine the time it takes for death and remission to occur, a Cox’s regression was applied [11]. The difference between the gene expressions before and after treatment was estimated using robust regression and multivariate approach [12]. The computation was performed in R software [13] and based on the simulation technique known as Gibbs sampling in WinBUGS platform [14]. Kaplan-Meier survival curves were used to determine any significant relationship between miR-204 expression and clinical outcome. Results were considered statistically significant when p was <0.05.
Results:
We compared AML samples to healthy controls and found significantly lower miR-204 expression in AML patients (p < 0.05). There were no differences in miR-204 expression between male and female patients.
After successful induction chemotherapy expression of miR-204 significantly increased (median 0.443606; p = 0.000590). Patients with increased miR-204 expression after induction therapy had higher chance for remission (p = 0.01438). 83 % of pts with CR after induction therapy had high miR-204 expression and 60 % of patients who did not respond to therapy had low miR-204 expression (Fig. 1). At the moment of diagnosis, Mean miR-204 expression in group who achieved CR was significantly lower than in the group which did not achieve response.Fig. 1Correlation between miR-204 expression after chemotherapy and remission
Correlation between miR-204 expression after chemotherapy and remission
In patients with increase of miR-204 expression after chemotherapy time to relapse was longer (median 13 months) than in patients with decreased miR-204 expression (median 4.5 months), p = 0.003347, Fig. 2.Fig. 2miR-204 expression after chemotherapy and time to relapse
miR-204 expression after chemotherapy and time to relapse
miR-204 expression level also influenced patients’ outcome (median value was used as a cut-off). Higher miR-204 expression before therapy was associated with longer survival (Fig. 3).Fig. 3Predicted overall survival depending on miR-204 expression before chemotherapy
Predicted overall survival depending on miR-204 expression before chemotherapy
Discussion:
Small non-coding microRNAs affect process of formation, development, proliferation and apoptosis of normal and malignant cells in human body. Their role has been proved in many cancers, including leukemia [4, 15–17]. Pathways regulation based on microRNA expression is still unknown. Some miRs may act as tumor suppressors and others as oncogenes [18, 19]. Recently, many miRs have been investigated as prognostic and predictive markers. miR-204 expression and its role has been studied in some solid tumors: pancreatic, gastric, prostate [3, 9, 20], in which it acts by down-regulation of Bcl-2 and has tumor suppressor role. miR-204 also influences NTRK2 gene expression in neuroblastoma cancer [21] and FOXC1 gene in invasive endometrial cancer [22].
In our study we showed down-regulated expression of miR-204 in acute myeloid leukemia patients comparing to healthy controls which is in line with observation made by Garzon et al. [23]. Authors found lower miR-204 expression in nucleophosmin positive AML patients and assumed hypothesis, that miR-204 targeted HOXA10 and MEIS1. Those two genes perturb myeloid differentiation and can lead to AML. Ying et al. in their study revealed that loss of miR-204 promotes cancer cell migration through increased expression of brain derived neurotrophic factor or its TrkB receptor [24]. In gastric cancer loss of miR-204 expression was associated with poor outcome, because it caused increase of antiapoptotic protein Bcl-2. In the light of these results Chen et al. demonstrated that miR-204 was down-regulated and its overexpression leaded to loss of cancer cell viability in pancreatic cancer [3]. Authors also showed that miR-204 regulates expression of Mcl-1 (Myeloid cell leukemia) by direct binding to 3′UTR [3]. Overexpressed Mcl-1 is associated with cell survival, while its downregulation leads to cell death. Increased miR-204 negatively regulated Mcl-1. Those observations could explain results of our study. We also analyzed correlations between miR-204 at diagnosis and after chemotherapy and clinical outcome. After effective induction chemotherapy we observed increased miR-204 expression and this change correlated positively with chance for remission achieving. Supposedly higher miR-204 expression could induce leukemic cell deaths and leaded to disease remission. On the other side, patients who had higher miR-204 expression at diagnosis had more favorable clinical outcome than others. Similar role of miR-204 has been also proved in gliomas, where low miR-204 expression leaded to a stem cell-like phenotype, and its overexpression resulted in reduced tumorigenicity and loss of stemness transcription factor SOX4 [24]. In contrast were results by Sümbül et al., who detected high miR-204 expression in colorectal cancer in comparison to healthy population. This finding was not related to any clinicopathological parameters nor survival [9]. Authors explained discrepant findings by the role of increased miR-204 in autophagy and apoptosis.
Concluding, as to our knowledge, we found for the first time down-regulated miR-204 expression in acute myeloid leukemia patients with its implication to disease prognosis. Functionality of miR-204 acting as a tumor suppressor makes this new molecule an useful biomarker in cancer diagnosis and management. Further investigation on miR-204 regulation and its target genes should be performed.
|
Background:
Acute myeloid leukemia (AML) is a heterogeneous neoplasm of the bone marrow with poor prognosis. In clinical practice new prognostic factors are still needed. MicroRNAs (miRs), small endogenous noncoding RNAs, play an essential role in the development and progression of acute leukemia. The aim of the study was to evaluate miR-204 expression in patients with AML at diagnosis and after induction chemotherapy, in comparison to healthy controls. We also investigated, if miR-204 expression correlates with clinical features of AML patients.
Methods:
miR-204 expression has been analyzed using RT-PCR in 95 bone marrow specimens from newly diagnosed AML patients in comparison to 20 healthy subject.
Results:
We showed down-regulated miR-204 expression in AML patients, which was associated with shorter patients' survival. Higher expression of miR-204 in patients after induction therapy was correlated with complete remission achieving.
Conclusions:
We showed low miR-204 expression in AML and found it to be an independent prognostic factor in this patient population.
| null | null | 4,367 | 191 | 8 |
[
"patients",
"204",
"mir",
"mir 204",
"expression",
"204 expression",
"mir 204 expression",
"chemotherapy",
"aml",
"study"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] miR-204 | Acute myeloid leukemia | Expression | Survival | Prognosis [SUMMARY]
| null |
[CONTENT] miR-204 | Acute myeloid leukemia | Expression | Survival | Prognosis [SUMMARY]
| null |
[CONTENT] miR-204 | Acute myeloid leukemia | Expression | Survival | Prognosis [SUMMARY]
| null |
[CONTENT] Adult | Aged | Aged, 80 and over | Female | Humans | Leukemia, Myeloid, Acute | Male | MicroRNAs | Middle Aged | Prognosis | Survival Analysis | Treatment Outcome | Young Adult [SUMMARY]
| null |
[CONTENT] Adult | Aged | Aged, 80 and over | Female | Humans | Leukemia, Myeloid, Acute | Male | MicroRNAs | Middle Aged | Prognosis | Survival Analysis | Treatment Outcome | Young Adult [SUMMARY]
| null |
[CONTENT] Adult | Aged | Aged, 80 and over | Female | Humans | Leukemia, Myeloid, Acute | Male | MicroRNAs | Middle Aged | Prognosis | Survival Analysis | Treatment Outcome | Young Adult [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] patients | 204 | mir | mir 204 | expression | 204 expression | mir 204 expression | chemotherapy | aml | study [SUMMARY]
| null |
[CONTENT] patients | 204 | mir | mir 204 | expression | 204 expression | mir 204 expression | chemotherapy | aml | study [SUMMARY]
| null |
[CONTENT] patients | 204 | mir | mir 204 | expression | 204 expression | mir 204 expression | chemotherapy | aml | study [SUMMARY]
| null |
[CONTENT] role | aml patients | mir 204 role | 204 role | aml | patients | clinical | cancer | acute myeloid | myeloid [SUMMARY]
| null |
[CONTENT] 204 expression | expression | 204 | mir 204 expression | mir 204 | mir | fig | chemotherapy | 204 expression chemotherapy | expression chemotherapy [SUMMARY]
| null |
[CONTENT] 204 | patients | mir 204 | mir | expression | chemotherapy | 204 expression | mir 204 expression | study | aml [SUMMARY]
| null |
[CONTENT] AML ||| ||| ||| AML ||| AML [SUMMARY]
| null |
[CONTENT] AML ||| [SUMMARY]
| null |
[CONTENT] AML ||| ||| ||| AML ||| AML ||| RT-PCR | 95 | AML | 20 ||| ||| AML ||| ||| AML [SUMMARY]
| null |
|||
Genetic testing
|
31341368
|
Carcinoembryonic antigen (CEA) and cytology in pancreatic cystic fluid are suboptimal for evaluation of pancreatic cystic neoplasms. Genetic testing and microforceps biopsy are promising tools for pre-operative diagnostic improvement but comparative performance of both methods is unknown.
|
BACKGROUND
|
We performed a literature search in Medline, Scopus, and Web of Science for studies evaluating genetic testing of cystic fluid and microforceps biopsy of pancreatic cysts, with endoscopic ultrasound with fine-needle aspiration (EUS-FNA) prior to surgery and surgical pathology as reference standard for diagnosis. We evaluated the diagnostic accuracy for: 1- benign cysts; 2- mucinous low-risk cysts; 3- high-risk cysts, and the diagnostic yield and rate of correctly identified cysts with microforceps biopsy and molecular analysis. We also assessed publication bias, heterogeneity, and study quality.
|
METHODS
|
Eight studies, including 1206 patients, of which 203 (17%) referred for surgery who met the inclusion criteria were analyzed in the systematic review, and seven studies were included in the meta-analysis. Genetic testing and microforceps biopsies were identical for diagnosis of benign cysts. Molecular analysis was superior for diagnosis of both low and high-risk mucinous cysts, with sensitivities of 0.89 (95%CI: 0.79-0.95) and 0.57 (95%CI: 0.42-0.71), specificities of 0.88 (95%CI: 0.75-0.95) and 0.88 (95%CI: 0.80-0.93) and AUC of 0.9555 and 0.92, respectively. The diagnostic yield was higher in microforceps biopsies than in genetic analysis (0.73 vs 0.54, respectively) but the rates of correctly identified cysts were identical (0.73 with 95%CI: 0.62-0.82 vs 0.71 with 95%CI: 0.49-0.86, respectively).
|
RESULTS
|
Genetic testing and microforceps biopsies are useful second tests, with identical results in benign pancreatic cysts. Genetic analysis performs better for low- and high-risk cysts but has lower diagnostic yield.
|
CONCLUSION
|
[
"Cyst Fluid",
"Diagnosis, Differential",
"Endoscopic Ultrasound-Guided Fine Needle Aspiration",
"Genetic Testing",
"Humans",
"Pancreas",
"Pancreatic Cyst",
"Pancreatic Neoplasms",
"Preoperative Period",
"Sensitivity and Specificity"
] |
6639554
|
INTRODUCTION
|
Pancreatic cystic neoplasms (PCNs) are on the rise in clinics due to an ageing population and the increase in routine use of high-quality abdominal imaging[1]. PCNs are generally classified into two main groups: mucinous cystic neoplasms (MCNs) and non-mucinous cystic neoplasms (NMCN). MCNs include intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystadenomas, which are precursor lesions of pancreatic carcinoma, and may be low-risk (pre-malignant with low or intermediate-grade atypia) or high-risk: pre-malignant with high-grade atypia (HGA) or malignant, including adenocarcinomas secondarily cystic. NMCNs include serous cystadenomas and inflammatory cysts (pseudocysts), mostly benign cysts, but may include some rare lesions, considered high-risk as cystic neuroendocrine tumors (cNETs), and acinar cell cystadenomas (ACCs). The heterogeneity in malignant potential, increased frequency, and significant morbidity and mortality of surgical treatment, makes pre-operative diagnosis of PCNs essential for management. The treatment options for PCNs encompass surgery or conservative surveillance for MCNs, according to malignancy risk, or no further evaluation for most NMCNs.
The differentiation between MCNs and NMCNs is critical, because a misdiagnosis of a MCN can lead to a missed opportunity to treat pancreatic cancer in an early stage and a misdiagnosis of NMCN can result in unnecessary surgery or surveillance with associated morbidity, costs, and negative impact on quality of life.
Currently, morphologic characterization of PCNs and pancreatic cystic fluid (PCF) analysis for carcinoembryonic Antigen (CEA) and cytology are central in diagnosis. A CEA level ≥ 192 ng/mL is the most accurate diagnostic test for MCNs and cytology is highly specific for malignancy[2], but with suboptimal results in large studies with surgical pathology as the gold standard[3]. In fact, a significant part of these lesions remains indeterminate and incorrect pre-operative diagnosis occurs in one third of patients[4,5], making new reliable diagnostic tools urgently needed.
In the last decade numerous studies have shown that genetic analysis of aspirates obtained by EUS-FNA provided a better characterization of PCNs than CEA and cytology[6-14]. Next-generation sequencing (NGS) is a very sensitive technique for detection of genetic mutations that allows the rapid detection of mutations in pre-defined panels of cancer genes, even in samples with limited DNA content, such as PCF. NGS requires storage, infrastructure, data processing, and expert personnel. Moreover, to be cost-effective, large numbers of samples need to be processed, making it applicable only in large centralized laboratories. These reasons make the implementation of NGS in clinical practice still a matter of debate.
The clinical need of better diagnostic tests in PCNs has recently led to the development of a through-the-needle miniature biopsy device for use during EUS-FNA[15,16]. The Moray micro forceps biopsy (MFB) device (US Endoscopy, Mentor, Ohio) is disposable and can pass through a standard 19-gauge EUS-FNA needle that is already used routinely. It allows tissue sampling from the cyst wall, septa or mural nodules and the obtention of a histological evaluation of the epithelial architecture and subepithelial stroma[17]. Adding to the high technical success and excellent safety profile[18,19], the new device has shown to improve the diagnostic accuracy of specific cyst subtypes[20,21]. Another major advantage of MFB is the simultaneous tissue sampling and PCF acquisition, with just an additional histologic analysis that follows standard definitions and is already routine in clinics.
The aim of this systematic review and meta-analysis is to evaluate the diagnostic performance of molecular analysis (MA) and MFB and find the most robust additional diagnostic technique in PCNs, in the pre-operative setting.
|
MATERIALS AND METHODS
|
This systematic review and meta-analysis is conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis of Diagnostic Test Accuracy Studies, the PRISMA-DTA Statement[22], and the protocol is registered at PROSPERO (CRD42018111910).
Literature search and study selection A comprehensive search of databases, including Medline, Scopus, and Web of Science, for the past 8 years (January 1st, 2010 to July 31st, 2018) and restricted to human studies was performed. No language restrictions were applied. The following search terms were used in two independent searches: “pancreas”, “cyst”, “molecular”, “analysis”; and “micro”, “forceps”, “microforceps”, “biopsy”. A search of related articles was performed, adding additional studies. Duplicate articles, reviews, trials including other kinds of neoplasms, and trials with molecular markers not compliant with the defined inclusion criteria were removed. The references of all selected studies were hand-searched for additional articles.
Inclusion criteria: Published studies were included in the meta-analysis if they analyzed: (1) Patients with symptomatic or incidental pancreatic cysts with a definitive surgical pathology diagnosis; (2) Genetic mutations performed with high sensitive techniques, such as NGS in PCF obtained by EUS-FNA prior to surgery; (3) At least four genetic mutations, including KRAS, GNAS, VHL, and at least another genetic mutation representative of aggressive neoplasms (PIK3CA, TP53, SMAD4, PTEN, CDKN2A); (4) PCNs evaluated by EUS-FNA with MFB for diagnosis; and (5) Surgical pathology specimens with available data.
Exclusion criteria: (1) Studies of MA with fewer than the four genetic mutations previously defined; (2) Studies involving solid pancreatic lesions; (3) Studies using PCF not obtained by EUS-FNA; (4) Reviews, case reports, case series with fewer than five patients, letters to editor, exploratory studies, and papers published only in abstract form; (5) Studies with cytology and clinical surveillance as standard of diagnosis. Two authors (SF and AL) independently judged study eligibility and disagreements were resolved by consensus.
Histological criteria: We classified the PCNs of the included studies into three main groups: (1) High-risk cysts (adenocarcinoma or high grade dysplasia in IPMNs and MCNs, secondarily cystic adenocarcinomas, cNETs, and ACCs; (2) Low-risk mucinous cysts (IPMNs and MCNs with intermediate or low-grade dysplasia); and (3) Benign cysts (SCAs, pseudocysts, and other rare cysts (RCs) included in some articles, as retention cysts, lymphoepithelial cysts, epidermoid cysts, squamoid cysts).
Tests under investigation: The index tests were: (1) MA of PCF; and (2) MFB of PCNs, including cyst wall, septs, and nodules. A diagnosis of cNET or ACC does not warrant a malignancy diagnosis, but surgery is recommended in surgically fit patients. Due to a recommendation of identical treatment to malignant and mucinous high-risk cysts, for the purpose of analysis in this study, each one of these diagnoses was classified as a high-risk cyst.
A comprehensive search of databases, including Medline, Scopus, and Web of Science, for the past 8 years (January 1st, 2010 to July 31st, 2018) and restricted to human studies was performed. No language restrictions were applied. The following search terms were used in two independent searches: “pancreas”, “cyst”, “molecular”, “analysis”; and “micro”, “forceps”, “microforceps”, “biopsy”. A search of related articles was performed, adding additional studies. Duplicate articles, reviews, trials including other kinds of neoplasms, and trials with molecular markers not compliant with the defined inclusion criteria were removed. The references of all selected studies were hand-searched for additional articles.
Inclusion criteria: Published studies were included in the meta-analysis if they analyzed: (1) Patients with symptomatic or incidental pancreatic cysts with a definitive surgical pathology diagnosis; (2) Genetic mutations performed with high sensitive techniques, such as NGS in PCF obtained by EUS-FNA prior to surgery; (3) At least four genetic mutations, including KRAS, GNAS, VHL, and at least another genetic mutation representative of aggressive neoplasms (PIK3CA, TP53, SMAD4, PTEN, CDKN2A); (4) PCNs evaluated by EUS-FNA with MFB for diagnosis; and (5) Surgical pathology specimens with available data.
Exclusion criteria: (1) Studies of MA with fewer than the four genetic mutations previously defined; (2) Studies involving solid pancreatic lesions; (3) Studies using PCF not obtained by EUS-FNA; (4) Reviews, case reports, case series with fewer than five patients, letters to editor, exploratory studies, and papers published only in abstract form; (5) Studies with cytology and clinical surveillance as standard of diagnosis. Two authors (SF and AL) independently judged study eligibility and disagreements were resolved by consensus.
Histological criteria: We classified the PCNs of the included studies into three main groups: (1) High-risk cysts (adenocarcinoma or high grade dysplasia in IPMNs and MCNs, secondarily cystic adenocarcinomas, cNETs, and ACCs; (2) Low-risk mucinous cysts (IPMNs and MCNs with intermediate or low-grade dysplasia); and (3) Benign cysts (SCAs, pseudocysts, and other rare cysts (RCs) included in some articles, as retention cysts, lymphoepithelial cysts, epidermoid cysts, squamoid cysts).
Tests under investigation: The index tests were: (1) MA of PCF; and (2) MFB of PCNs, including cyst wall, septs, and nodules. A diagnosis of cNET or ACC does not warrant a malignancy diagnosis, but surgery is recommended in surgically fit patients. Due to a recommendation of identical treatment to malignant and mucinous high-risk cysts, for the purpose of analysis in this study, each one of these diagnoses was classified as a high-risk cyst.
Data extraction After study selection, two authors (SF and AL) extracted and registered the data from each study onto a standardized worksheet. Disagreements were discussed and reviewed by a third author (LP). The data retrieved were: first author, publication year, study period and design (prospective or retrospective), reference for diagnosis, sample size (all patients included in the study), technical success, adverse events, diagnostic yield, surgical cohort (number of patients with a surgical pathology specimen), cyst size, cyst location, specific cyst types, number of high-risk cysts, mucinous low-risk and benign cysts diagnosed by MA and MFB comparing to surgical pathology specimens. In the MFB studies, technical success was defined as the ability to puncture the cysts and perform the biopsies; and the diagnostic yield was defined as the ratio between the number of patients included in the study and the patients in whom enough material allowed the acquisition of a histopathologic diagnosis. In the MA group, diagnostic yield was defined as a ratio between the number of patients included in the study and the number of patients with DNA available to perform molecular analysis in PCF.
After study selection, two authors (SF and AL) extracted and registered the data from each study onto a standardized worksheet. Disagreements were discussed and reviewed by a third author (LP). The data retrieved were: first author, publication year, study period and design (prospective or retrospective), reference for diagnosis, sample size (all patients included in the study), technical success, adverse events, diagnostic yield, surgical cohort (number of patients with a surgical pathology specimen), cyst size, cyst location, specific cyst types, number of high-risk cysts, mucinous low-risk and benign cysts diagnosed by MA and MFB comparing to surgical pathology specimens. In the MFB studies, technical success was defined as the ability to puncture the cysts and perform the biopsies; and the diagnostic yield was defined as the ratio between the number of patients included in the study and the patients in whom enough material allowed the acquisition of a histopathologic diagnosis. In the MA group, diagnostic yield was defined as a ratio between the number of patients included in the study and the number of patients with DNA available to perform molecular analysis in PCF.
Outcomes The primary outcomes of this study were the data to obtain the accuracies of MA and MFB for the diagnosis of PCNs, including high-risk cysts, mucinous low-risk cysts, and benign cysts. Secondary outcomes were the diagnostic yield of genetic testing and MFB and the number of cysts correctly identified for each of the tests studied.
The primary outcomes of this study were the data to obtain the accuracies of MA and MFB for the diagnosis of PCNs, including high-risk cysts, mucinous low-risk cysts, and benign cysts. Secondary outcomes were the diagnostic yield of genetic testing and MFB and the number of cysts correctly identified for each of the tests studied.
Quality analysis Methodological quality of included primary studies was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23]. The PRISMA-DTA Statement recommendations were used for reporting this systematic review[22,24].
Methodological quality of included primary studies was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23]. The PRISMA-DTA Statement recommendations were used for reporting this systematic review[22,24].
Statistical analysis and data synthesis The reference standard was a surgical pathology specimen that allowed the classification of PCNs into three defined groups of diagnosis: high-risk cysts, mucinous low-risk cysts, and benign cysts. This resulted in a two-by-three table with correct and incorrect test results in each of the three referenced groups, for each of the tests analyzed, MA and histology were obtained by MFB.
To calculate tests’ accuracy and to reflect on the categories that are useful in clinical practice and that guide management, we constructed two-by-two tables, considering three definitions of “relevant” cysts: (1) High-risk cysts – proven malignant cysts, IPMNs, and MCNs with HGA, cNETs, ACCs; Non-High-risk cysts – all cysts except those proven to be high-risk. (2) Low-risk mucinous cysts – proven mucinous low-risk cysts; High-risk cysts – all except those proven to be mucinous low-risk or benign. And (3) Non-benign cysts – all cysts except those proven to be benign; Benign cysts – proven benign cysts.
The ability of the tests to discriminate “relevant” and “non-relevant” cysts using the three definitions of “relevant cysts” was evaluated and the accuracy of the two tests was compared.
The data of the two-by-two tables were used to calculate sensitivity and specificity for each study. We present individual study results graphically by plotting the estimates of sensitivity and specificity (and their 95% confidence intervals (CI)) in both forest plots and on the summary receiver operating characteristic (sROC) curve plots. The area under the curve (AUC) is equal to 1 for a perfect test and 0.5 for a completely uninformative test. The AUC is equal to the probability that if a pair of relevant and non-relevant cysts is selected at random, the relevant cyst will have a higher test result than the non-relevant cyst. Pooled estimates of the sensitivity and specificity were obtained by the DerSimonian-Laird method (random effect model) to incorporate variation among studies, when data are heterogeneous. Otherwise, we used the Mantel-Haenszel method (fixed effect model).
Heterogeneity was investigated in the first instance through visual examination of forest plots of sensitivities and specificities and through visual examination of the ROC plot of the raw data. Last, we used statistical tests, including chi-square and Cochran-Q to evaluate if the differences across the studies were greater than expected by chance alone. A low P value suggests presence of heterogeneity. In addition to these statistics we used the statistic I2 of Higgins, which has been proposed as a measure to quantify the amount of heterogeneity[25,26]. The scale of I2 has a range of 0 to 100% and values on the order of 25%, 50% and 75% are considered low, moderate, and high heterogeneity, respectively.
Another goal of this work was to obtain, for each of the tests, the correctly identified cyst rate and the diagnostic yield in predicting a histopathologic diagnosis.
We used Comprehensive Meta-Analysis software (Version 2.0) for assessment of diagnostic yield of the tests and Meta-DiSc (version 1.4 – Meta-Analysis of Diagnostic and screening tests[27] ) to obtain the accuracy of each of the tests.
The reference standard was a surgical pathology specimen that allowed the classification of PCNs into three defined groups of diagnosis: high-risk cysts, mucinous low-risk cysts, and benign cysts. This resulted in a two-by-three table with correct and incorrect test results in each of the three referenced groups, for each of the tests analyzed, MA and histology were obtained by MFB.
To calculate tests’ accuracy and to reflect on the categories that are useful in clinical practice and that guide management, we constructed two-by-two tables, considering three definitions of “relevant” cysts: (1) High-risk cysts – proven malignant cysts, IPMNs, and MCNs with HGA, cNETs, ACCs; Non-High-risk cysts – all cysts except those proven to be high-risk. (2) Low-risk mucinous cysts – proven mucinous low-risk cysts; High-risk cysts – all except those proven to be mucinous low-risk or benign. And (3) Non-benign cysts – all cysts except those proven to be benign; Benign cysts – proven benign cysts.
The ability of the tests to discriminate “relevant” and “non-relevant” cysts using the three definitions of “relevant cysts” was evaluated and the accuracy of the two tests was compared.
The data of the two-by-two tables were used to calculate sensitivity and specificity for each study. We present individual study results graphically by plotting the estimates of sensitivity and specificity (and their 95% confidence intervals (CI)) in both forest plots and on the summary receiver operating characteristic (sROC) curve plots. The area under the curve (AUC) is equal to 1 for a perfect test and 0.5 for a completely uninformative test. The AUC is equal to the probability that if a pair of relevant and non-relevant cysts is selected at random, the relevant cyst will have a higher test result than the non-relevant cyst. Pooled estimates of the sensitivity and specificity were obtained by the DerSimonian-Laird method (random effect model) to incorporate variation among studies, when data are heterogeneous. Otherwise, we used the Mantel-Haenszel method (fixed effect model).
Heterogeneity was investigated in the first instance through visual examination of forest plots of sensitivities and specificities and through visual examination of the ROC plot of the raw data. Last, we used statistical tests, including chi-square and Cochran-Q to evaluate if the differences across the studies were greater than expected by chance alone. A low P value suggests presence of heterogeneity. In addition to these statistics we used the statistic I2 of Higgins, which has been proposed as a measure to quantify the amount of heterogeneity[25,26]. The scale of I2 has a range of 0 to 100% and values on the order of 25%, 50% and 75% are considered low, moderate, and high heterogeneity, respectively.
Another goal of this work was to obtain, for each of the tests, the correctly identified cyst rate and the diagnostic yield in predicting a histopathologic diagnosis.
We used Comprehensive Meta-Analysis software (Version 2.0) for assessment of diagnostic yield of the tests and Meta-DiSc (version 1.4 – Meta-Analysis of Diagnostic and screening tests[27] ) to obtain the accuracy of each of the tests.
| null | null |
CONCLUSION
|
For the present time, MA and MFB can only be recommended as complementary or as second line tests in case CEA and cytology of PCF are non-diagnostic. In the future, for MA to become relevant in routine clinical care, its role must be confirmed, in order to become a first line test with clinical impact in cyst diagnosis, prognosis, and patient management. MA, both in PCF and peripheral blood, for multiple simultaneous biomarkers and non-invasive diagnosis and risk stratification would be valuable. If MFB proves in larger studies to be safe and to allow a correct diagnosis of pancreatic cysts, it may be immediately implemented in clinics. MFB may be especially useful for benign lesions, for which both surgery and surveillance are unnecessary, with uncertain diagnosis due to current diagnostic limitations. For both tests, larger validation studies are missing.
|
[
"INTRODUCTION",
"Literature search and study selection",
"Data extraction",
"Outcomes",
"Quality analysis",
"Statistical analysis and data synthesis",
"RESULTS",
"Systematic Review",
"Meta-analysis",
"DISCUSSION",
"Strengths and limitations",
"Future perspectives",
"CONCLUSION"
] |
[
"Pancreatic cystic neoplasms (PCNs) are on the rise in clinics due to an ageing population and the increase in routine use of high-quality abdominal imaging[1]. PCNs are generally classified into two main groups: mucinous cystic neoplasms (MCNs) and non-mucinous cystic neoplasms (NMCN). MCNs include intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystadenomas, which are precursor lesions of pancreatic carcinoma, and may be low-risk (pre-malignant with low or intermediate-grade atypia) or high-risk: pre-malignant with high-grade atypia (HGA) or malignant, including adenocarcinomas secondarily cystic. NMCNs include serous cystadenomas and inflammatory cysts (pseudocysts), mostly benign cysts, but may include some rare lesions, considered high-risk as cystic neuroendocrine tumors (cNETs), and acinar cell cystadenomas (ACCs). The heterogeneity in malignant potential, increased frequency, and significant morbidity and mortality of surgical treatment, makes pre-operative diagnosis of PCNs essential for management. The treatment options for PCNs encompass surgery or conservative surveillance for MCNs, according to malignancy risk, or no further evaluation for most NMCNs.\nThe differentiation between MCNs and NMCNs is critical, because a misdiagnosis of a MCN can lead to a missed opportunity to treat pancreatic cancer in an early stage and a misdiagnosis of NMCN can result in unnecessary surgery or surveillance with associated morbidity, costs, and negative impact on quality of life.\nCurrently, morphologic characterization of PCNs and pancreatic cystic fluid (PCF) analysis for carcinoembryonic Antigen (CEA) and cytology are central in diagnosis. A CEA level ≥ 192 ng/mL is the most accurate diagnostic test for MCNs and cytology is highly specific for malignancy[2], but with suboptimal results in large studies with surgical pathology as the gold standard[3]. In fact, a significant part of these lesions remains indeterminate and incorrect pre-operative diagnosis occurs in one third of patients[4,5], making new reliable diagnostic tools urgently needed.\nIn the last decade numerous studies have shown that genetic analysis of aspirates obtained by EUS-FNA provided a better characterization of PCNs than CEA and cytology[6-14]. Next-generation sequencing (NGS) is a very sensitive technique for detection of genetic mutations that allows the rapid detection of mutations in pre-defined panels of cancer genes, even in samples with limited DNA content, such as PCF. NGS requires storage, infrastructure, data processing, and expert personnel. Moreover, to be cost-effective, large numbers of samples need to be processed, making it applicable only in large centralized laboratories. These reasons make the implementation of NGS in clinical practice still a matter of debate.\nThe clinical need of better diagnostic tests in PCNs has recently led to the development of a through-the-needle miniature biopsy device for use during EUS-FNA[15,16]. The Moray micro forceps biopsy (MFB) device (US Endoscopy, Mentor, Ohio) is disposable and can pass through a standard 19-gauge EUS-FNA needle that is already used routinely. It allows tissue sampling from the cyst wall, septa or mural nodules and the obtention of a histological evaluation of the epithelial architecture and subepithelial stroma[17]. Adding to the high technical success and excellent safety profile[18,19], the new device has shown to improve the diagnostic accuracy of specific cyst subtypes[20,21]. Another major advantage of MFB is the simultaneous tissue sampling and PCF acquisition, with just an additional histologic analysis that follows standard definitions and is already routine in clinics.\nThe aim of this systematic review and meta-analysis is to evaluate the diagnostic performance of molecular analysis (MA) and MFB and find the most robust additional diagnostic technique in PCNs, in the pre-operative setting.",
"A comprehensive search of databases, including Medline, Scopus, and Web of Science, for the past 8 years (January 1st, 2010 to July 31st, 2018) and restricted to human studies was performed. No language restrictions were applied. The following search terms were used in two independent searches: “pancreas”, “cyst”, “molecular”, “analysis”; and “micro”, “forceps”, “microforceps”, “biopsy”. A search of related articles was performed, adding additional studies. Duplicate articles, reviews, trials including other kinds of neoplasms, and trials with molecular markers not compliant with the defined inclusion criteria were removed. The references of all selected studies were hand-searched for additional articles.\nInclusion criteria: Published studies were included in the meta-analysis if they analyzed: (1) Patients with symptomatic or incidental pancreatic cysts with a definitive surgical pathology diagnosis; (2) Genetic mutations performed with high sensitive techniques, such as NGS in PCF obtained by EUS-FNA prior to surgery; (3) At least four genetic mutations, including KRAS, GNAS, VHL, and at least another genetic mutation representative of aggressive neoplasms (PIK3CA, TP53, SMAD4, PTEN, CDKN2A); (4) PCNs evaluated by EUS-FNA with MFB for diagnosis; and (5) Surgical pathology specimens with available data.\nExclusion criteria: (1) Studies of MA with fewer than the four genetic mutations previously defined; (2) Studies involving solid pancreatic lesions; (3) Studies using PCF not obtained by EUS-FNA; (4) Reviews, case reports, case series with fewer than five patients, letters to editor, exploratory studies, and papers published only in abstract form; (5) Studies with cytology and clinical surveillance as standard of diagnosis. Two authors (SF and AL) independently judged study eligibility and disagreements were resolved by consensus.\nHistological criteria: We classified the PCNs of the included studies into three main groups: (1) High-risk cysts (adenocarcinoma or high grade dysplasia in IPMNs and MCNs, secondarily cystic adenocarcinomas, cNETs, and ACCs; (2) Low-risk mucinous cysts (IPMNs and MCNs with intermediate or low-grade dysplasia); and (3) Benign cysts (SCAs, pseudocysts, and other rare cysts (RCs) included in some articles, as retention cysts, lymphoepithelial cysts, epidermoid cysts, squamoid cysts).\nTests under investigation: The index tests were: (1) MA of PCF; and (2) MFB of PCNs, including cyst wall, septs, and nodules. A diagnosis of cNET or ACC does not warrant a malignancy diagnosis, but surgery is recommended in surgically fit patients. Due to a recommendation of identical treatment to malignant and mucinous high-risk cysts, for the purpose of analysis in this study, each one of these diagnoses was classified as a high-risk cyst.",
"After study selection, two authors (SF and AL) extracted and registered the data from each study onto a standardized worksheet. Disagreements were discussed and reviewed by a third author (LP). The data retrieved were: first author, publication year, study period and design (prospective or retrospective), reference for diagnosis, sample size (all patients included in the study), technical success, adverse events, diagnostic yield, surgical cohort (number of patients with a surgical pathology specimen), cyst size, cyst location, specific cyst types, number of high-risk cysts, mucinous low-risk and benign cysts diagnosed by MA and MFB comparing to surgical pathology specimens. In the MFB studies, technical success was defined as the ability to puncture the cysts and perform the biopsies; and the diagnostic yield was defined as the ratio between the number of patients included in the study and the patients in whom enough material allowed the acquisition of a histopathologic diagnosis. In the MA group, diagnostic yield was defined as a ratio between the number of patients included in the study and the number of patients with DNA available to perform molecular analysis in PCF.",
"The primary outcomes of this study were the data to obtain the accuracies of MA and MFB for the diagnosis of PCNs, including high-risk cysts, mucinous low-risk cysts, and benign cysts. Secondary outcomes were the diagnostic yield of genetic testing and MFB and the number of cysts correctly identified for each of the tests studied.",
"Methodological quality of included primary studies was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23]. The PRISMA-DTA Statement recommendations were used for reporting this systematic review[22,24].",
"The reference standard was a surgical pathology specimen that allowed the classification of PCNs into three defined groups of diagnosis: high-risk cysts, mucinous low-risk cysts, and benign cysts. This resulted in a two-by-three table with correct and incorrect test results in each of the three referenced groups, for each of the tests analyzed, MA and histology were obtained by MFB.\nTo calculate tests’ accuracy and to reflect on the categories that are useful in clinical practice and that guide management, we constructed two-by-two tables, considering three definitions of “relevant” cysts: (1) High-risk cysts – proven malignant cysts, IPMNs, and MCNs with HGA, cNETs, ACCs; Non-High-risk cysts – all cysts except those proven to be high-risk. (2) Low-risk mucinous cysts – proven mucinous low-risk cysts; High-risk cysts – all except those proven to be mucinous low-risk or benign. And (3) Non-benign cysts – all cysts except those proven to be benign; Benign cysts – proven benign cysts.\nThe ability of the tests to discriminate “relevant” and “non-relevant” cysts using the three definitions of “relevant cysts” was evaluated and the accuracy of the two tests was compared.\nThe data of the two-by-two tables were used to calculate sensitivity and specificity for each study. We present individual study results graphically by plotting the estimates of sensitivity and specificity (and their 95% confidence intervals (CI)) in both forest plots and on the summary receiver operating characteristic (sROC) curve plots. The area under the curve (AUC) is equal to 1 for a perfect test and 0.5 for a completely uninformative test. The AUC is equal to the probability that if a pair of relevant and non-relevant cysts is selected at random, the relevant cyst will have a higher test result than the non-relevant cyst. Pooled estimates of the sensitivity and specificity were obtained by the DerSimonian-Laird method (random effect model) to incorporate variation among studies, when data are heterogeneous. Otherwise, we used the Mantel-Haenszel method (fixed effect model).\nHeterogeneity was investigated in the first instance through visual examination of forest plots of sensitivities and specificities and through visual examination of the ROC plot of the raw data. Last, we used statistical tests, including chi-square and Cochran-Q to evaluate if the differences across the studies were greater than expected by chance alone. A low P value suggests presence of heterogeneity. In addition to these statistics we used the statistic I2 of Higgins, which has been proposed as a measure to quantify the amount of heterogeneity[25,26]. The scale of I2 has a range of 0 to 100% and values on the order of 25%, 50% and 75% are considered low, moderate, and high heterogeneity, respectively.\nAnother goal of this work was to obtain, for each of the tests, the correctly identified cyst rate and the diagnostic yield in predicting a histopathologic diagnosis.\nWe used Comprehensive Meta-Analysis software (Version 2.0) for assessment of diagnostic yield of the tests and Meta-DiSc (version 1.4 – Meta-Analysis of Diagnostic and screening tests[27] ) to obtain the accuracy of each of the tests.",
" Systematic Review Our search revealed 16 study titles and abstracts for MFB and 264 titles for MA. In Figure 1A and B are described the selection process of the articles included in this study. After all steps, eight studies were considered suitable for qualitative and seven for quantitative analysis. We excluded 20 full-text articles after review, because they were case series of two patients[16] (n = 1), exploratory or pilot studies[28,29] (n = 2), no information of mutation status was available[30] (n = 1), pancreatic cystic fluid was obtained during surgery[31] (n = 1), insufficient or absent data of cysts with surgical pathology diagnoses[12,32,33] (n = 3), and mutations only of KRAS and/or GNAS[14,34-44] (n = 12).\nFlowchart with identification of eligible studies. A: Molecular analysis; B: Microforceps biopsy.\nOf the eight studies that met the inclusion criteria, design was retrospective in six and prospective in two, all were published from 2015 to 2018. These eight studies included a total of 1206 patients, of which 203 (17%) underwent surgical resection and a surgical pathology specimen was available as reference standard and included in the analysis. We excluded all patients with cytology and clinical follow-up data, but for whom a surgical pathology specimen was not available. The characteristics of the studies, surgical pathology diagnoses, and MA and MFB results are presented in Tables 1[32,45-47] and 2[18-21].\nCharacteristics of studies of molecular analysis included in the analysis\n113 samples of 105 patients; \nTP53, PIK3CA, PTEN; \n1 ACC; \nincludes: ACC, retention cyst, lymphoepithelial cyst, epidermoid cyst, and squamoid cyst. MA: Molecular analysis; NA: Non-available data; ACC: Acinar cell cyst; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.\nQuality assessment and publication bias: Methodological quality of primary studies included was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23], which evaluates the quality of articles for systematic reviews of diagnostic accuracy studies in four domains, including patient selection, index test, reference standard, and flow and timing, for risk of bias and applicability concerns. Results are presented in Figure 2, which was sketched with templates available at www.quadas.org. The studies included in this review all showed a “low-risk” classification as the index tests (MA and MFB) and the reference standard (surgical pathology specimen) were reliable and mentioned in all studies. However, a “high-risk” of selection bias was demonstrated in patient selection (neither random nor sequential patients included in several studies) and in flow and timing because only a small proportion of the patients evaluated in all studies, except one, were included in the analysis. In fact, most patients were excluded in all studies as the inclusion criteria requiring surgical pathology as diagnostic reference were not met. Applicability concerns in patient selection were also significant in all studies, because the subgroup of PCNs referred for surgery is more often malignant than PCNs on surveillance, which would also be targeted with this review. Because of this bias, there may be an overestimation of both the sensitivity of the index tests, due to a more severe spectrum of PCNs that are referred for surgery, and the positive predictive value (PPV) for diagnosis of high-risk cysts, due to an increased prevalence of malignant cysts in a surgical cohort of PCNs.\nQuality assessment of the studies using QUADAS-2. A: Tabular presentation of risk bias for each study; B: Graphical display of bias.\nOur search revealed 16 study titles and abstracts for MFB and 264 titles for MA. In Figure 1A and B are described the selection process of the articles included in this study. After all steps, eight studies were considered suitable for qualitative and seven for quantitative analysis. We excluded 20 full-text articles after review, because they were case series of two patients[16] (n = 1), exploratory or pilot studies[28,29] (n = 2), no information of mutation status was available[30] (n = 1), pancreatic cystic fluid was obtained during surgery[31] (n = 1), insufficient or absent data of cysts with surgical pathology diagnoses[12,32,33] (n = 3), and mutations only of KRAS and/or GNAS[14,34-44] (n = 12).\nFlowchart with identification of eligible studies. A: Molecular analysis; B: Microforceps biopsy.\nOf the eight studies that met the inclusion criteria, design was retrospective in six and prospective in two, all were published from 2015 to 2018. These eight studies included a total of 1206 patients, of which 203 (17%) underwent surgical resection and a surgical pathology specimen was available as reference standard and included in the analysis. We excluded all patients with cytology and clinical follow-up data, but for whom a surgical pathology specimen was not available. The characteristics of the studies, surgical pathology diagnoses, and MA and MFB results are presented in Tables 1[32,45-47] and 2[18-21].\nCharacteristics of studies of molecular analysis included in the analysis\n113 samples of 105 patients; \nTP53, PIK3CA, PTEN; \n1 ACC; \nincludes: ACC, retention cyst, lymphoepithelial cyst, epidermoid cyst, and squamoid cyst. MA: Molecular analysis; NA: Non-available data; ACC: Acinar cell cyst; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.\nQuality assessment and publication bias: Methodological quality of primary studies included was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23], which evaluates the quality of articles for systematic reviews of diagnostic accuracy studies in four domains, including patient selection, index test, reference standard, and flow and timing, for risk of bias and applicability concerns. Results are presented in Figure 2, which was sketched with templates available at www.quadas.org. The studies included in this review all showed a “low-risk” classification as the index tests (MA and MFB) and the reference standard (surgical pathology specimen) were reliable and mentioned in all studies. However, a “high-risk” of selection bias was demonstrated in patient selection (neither random nor sequential patients included in several studies) and in flow and timing because only a small proportion of the patients evaluated in all studies, except one, were included in the analysis. In fact, most patients were excluded in all studies as the inclusion criteria requiring surgical pathology as diagnostic reference were not met. Applicability concerns in patient selection were also significant in all studies, because the subgroup of PCNs referred for surgery is more often malignant than PCNs on surveillance, which would also be targeted with this review. Because of this bias, there may be an overestimation of both the sensitivity of the index tests, due to a more severe spectrum of PCNs that are referred for surgery, and the positive predictive value (PPV) for diagnosis of high-risk cysts, due to an increased prevalence of malignant cysts in a surgical cohort of PCNs.\nQuality assessment of the studies using QUADAS-2. A: Tabular presentation of risk bias for each study; B: Graphical display of bias.\n Meta-analysis Molecular analysis: Four articles were included in the meta-analysis for diagnostic accuracy of MA. For each of the three definitions of relevant cyst, forest plots of sensitivity and specificity with heterogeneous denoted are shown in Figure 3.\nForest plots of the studies included for molecular analysis. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95% CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nThe three criteria to define “relevant cysts” resulted in a different range of the specificity and sensitivity of the studies included as shown in Figure 3. For diagnosis of the subgroup with high-risk and low-risk mucinous cysts that require intervention (either surgery or surveillance) comparing to benign cysts the pooled sensitivity was 0.75 (95%CI: 0.66-0.83) and the pooled specificity was 0.72 (95%CI: 0.56-0.85) for MA. In the subgroup of high-risk cysts that require surgery, comparing to other cysts requiring conservative management, the sensitivity was 0.57 (95%CI: 0.42-0.71) with a specificity of 0.88 (95%CI: 0.80-0.93). In the subgroup of low-risk mucinous cysts comparing to high-risk, the pooled sensitivity was 0.89 (95%CI: 0.79-0.95) and the pooled specificity was 0.88 (95%CI: 0.75-0.95).\nFigure 4 displays the sROC curves of MA, showing the sensitivity of the individual articles mapped on the vertical scale, 1-specificity on the horizontal scale, with the summary (sensitivity, 1-specificity) point marked, as well as the summary ROC curve and the confidence region for the summary (sensitivity, 1-specificity) points. The area under the sROC curve was 0.7706 (SE: 0.0927) in non-benign cysts, 0.9248 (SE: 0.0691) in high-risk cysts, and 0.9555 (SE: 0.0293) in mucinous low-risk cysts. The results of the studies had greater variation in non-benign cysts as shown by the wide confidence region.\nSummary receiver operating characteristics plots. ROC: Receiver operating characteristic curve; AUC: Area under the curve; SE: Standard error.\nIn the four studies, 566 patients had DNA available to perform MA in PCF. Pooled analysis (Figure 5) showed a diagnostic yield of 54.3% (95%CI: 49.8%-58.7%; I2 = 39.605%; test for heterogeneity P = 0.174).\nForest plots of molecular analysis and microforceps biopsies on the secondary outcomes of this meta-analysis.\nBy considering the classification of cysts by specific type (IPMNs, MCNs, cNETs, SCAs, pseudocysts, ACCs, and other RCs), MA identified correctly 73.1% of cysts (95%CI: 61.6%-82.2%; I2 = 37.381%; test for heterogeneity P = 0.203) (Figure 5).\nMicro forceps biopsy: Four articles were included in the meta-analysis for diagnostic accuracy of histology obtained using MFB. Figure 6 shows the forest plots of sensitivity and specificity for the three subgroups of relevant cysts. The forest plots for MFB show variable specificities within the papers, from 0 to 1, which can be due to the small numbers of patients with the target condition in some studies.\nForest plots of the included studies for microforceps biopsies. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95%CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nFor each of the three subgroups there exists a low heterogeneity in sensitivity (I2 = 0%, I2 = 21.4%, I2 = 0%) and specificity (I2 = 0%, I2 = 0%, I2 = 21.4%), therefore fixed effect models were used. As presented in Figure 6, in the first subgroup the pooled sensitivity was 0.73 (95%CI: 0.50-0.89) and the pooled specificity was 0.88 (95%CI: 0.28-1.00). In the second subgroup sensitivity was 0.81 (95%CI: 0.46-0.98) with a specificity of 0.77 (95%CI: 0.50-0.94) and in the last subgroup the pooled sensitivity was 0.64 (95%CI: 0.33-0.88) and the pooled specificity was 0.81 (95%CI: 0.46-0.98).\nThe results were plotted as a symmetrical sROC curve (Figure 4). The area under the sROC curve was 0.7640 (SE: 0.1261) in the first subgroup, 0.8154 (SE: 0.098) in the second subgroup, and 0.7509 (SE: 0.1277) in the last subgroup.\nBy pooling the data of the four studies that investigated the use of MFB to obtain a histopathologic diagnosis, we obtained a diagnostic yield of 73.1% (95%CI: 61.4%-82.2%; I2 = 47.774%; test for heterogeneity P = 0.125) (Figure 5).\nBy considering the outcome “specific cyst type” diagnosis, MFB correctly identified 70.7% of the cysts (95%CI: 49.4%-85.6%; I2 = 0%; test for heterogeneity P = 0.056) (Figure 5).\nMolecular analysis: Four articles were included in the meta-analysis for diagnostic accuracy of MA. For each of the three definitions of relevant cyst, forest plots of sensitivity and specificity with heterogeneous denoted are shown in Figure 3.\nForest plots of the studies included for molecular analysis. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95% CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nThe three criteria to define “relevant cysts” resulted in a different range of the specificity and sensitivity of the studies included as shown in Figure 3. For diagnosis of the subgroup with high-risk and low-risk mucinous cysts that require intervention (either surgery or surveillance) comparing to benign cysts the pooled sensitivity was 0.75 (95%CI: 0.66-0.83) and the pooled specificity was 0.72 (95%CI: 0.56-0.85) for MA. In the subgroup of high-risk cysts that require surgery, comparing to other cysts requiring conservative management, the sensitivity was 0.57 (95%CI: 0.42-0.71) with a specificity of 0.88 (95%CI: 0.80-0.93). In the subgroup of low-risk mucinous cysts comparing to high-risk, the pooled sensitivity was 0.89 (95%CI: 0.79-0.95) and the pooled specificity was 0.88 (95%CI: 0.75-0.95).\nFigure 4 displays the sROC curves of MA, showing the sensitivity of the individual articles mapped on the vertical scale, 1-specificity on the horizontal scale, with the summary (sensitivity, 1-specificity) point marked, as well as the summary ROC curve and the confidence region for the summary (sensitivity, 1-specificity) points. The area under the sROC curve was 0.7706 (SE: 0.0927) in non-benign cysts, 0.9248 (SE: 0.0691) in high-risk cysts, and 0.9555 (SE: 0.0293) in mucinous low-risk cysts. The results of the studies had greater variation in non-benign cysts as shown by the wide confidence region.\nSummary receiver operating characteristics plots. ROC: Receiver operating characteristic curve; AUC: Area under the curve; SE: Standard error.\nIn the four studies, 566 patients had DNA available to perform MA in PCF. Pooled analysis (Figure 5) showed a diagnostic yield of 54.3% (95%CI: 49.8%-58.7%; I2 = 39.605%; test for heterogeneity P = 0.174).\nForest plots of molecular analysis and microforceps biopsies on the secondary outcomes of this meta-analysis.\nBy considering the classification of cysts by specific type (IPMNs, MCNs, cNETs, SCAs, pseudocysts, ACCs, and other RCs), MA identified correctly 73.1% of cysts (95%CI: 61.6%-82.2%; I2 = 37.381%; test for heterogeneity P = 0.203) (Figure 5).\nMicro forceps biopsy: Four articles were included in the meta-analysis for diagnostic accuracy of histology obtained using MFB. Figure 6 shows the forest plots of sensitivity and specificity for the three subgroups of relevant cysts. The forest plots for MFB show variable specificities within the papers, from 0 to 1, which can be due to the small numbers of patients with the target condition in some studies.\nForest plots of the included studies for microforceps biopsies. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95%CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nFor each of the three subgroups there exists a low heterogeneity in sensitivity (I2 = 0%, I2 = 21.4%, I2 = 0%) and specificity (I2 = 0%, I2 = 0%, I2 = 21.4%), therefore fixed effect models were used. As presented in Figure 6, in the first subgroup the pooled sensitivity was 0.73 (95%CI: 0.50-0.89) and the pooled specificity was 0.88 (95%CI: 0.28-1.00). In the second subgroup sensitivity was 0.81 (95%CI: 0.46-0.98) with a specificity of 0.77 (95%CI: 0.50-0.94) and in the last subgroup the pooled sensitivity was 0.64 (95%CI: 0.33-0.88) and the pooled specificity was 0.81 (95%CI: 0.46-0.98).\nThe results were plotted as a symmetrical sROC curve (Figure 4). The area under the sROC curve was 0.7640 (SE: 0.1261) in the first subgroup, 0.8154 (SE: 0.098) in the second subgroup, and 0.7509 (SE: 0.1277) in the last subgroup.\nBy pooling the data of the four studies that investigated the use of MFB to obtain a histopathologic diagnosis, we obtained a diagnostic yield of 73.1% (95%CI: 61.4%-82.2%; I2 = 47.774%; test for heterogeneity P = 0.125) (Figure 5).\nBy considering the outcome “specific cyst type” diagnosis, MFB correctly identified 70.7% of the cysts (95%CI: 49.4%-85.6%; I2 = 0%; test for heterogeneity P = 0.056) (Figure 5).",
"Our search revealed 16 study titles and abstracts for MFB and 264 titles for MA. In Figure 1A and B are described the selection process of the articles included in this study. After all steps, eight studies were considered suitable for qualitative and seven for quantitative analysis. We excluded 20 full-text articles after review, because they were case series of two patients[16] (n = 1), exploratory or pilot studies[28,29] (n = 2), no information of mutation status was available[30] (n = 1), pancreatic cystic fluid was obtained during surgery[31] (n = 1), insufficient or absent data of cysts with surgical pathology diagnoses[12,32,33] (n = 3), and mutations only of KRAS and/or GNAS[14,34-44] (n = 12).\nFlowchart with identification of eligible studies. A: Molecular analysis; B: Microforceps biopsy.\nOf the eight studies that met the inclusion criteria, design was retrospective in six and prospective in two, all were published from 2015 to 2018. These eight studies included a total of 1206 patients, of which 203 (17%) underwent surgical resection and a surgical pathology specimen was available as reference standard and included in the analysis. We excluded all patients with cytology and clinical follow-up data, but for whom a surgical pathology specimen was not available. The characteristics of the studies, surgical pathology diagnoses, and MA and MFB results are presented in Tables 1[32,45-47] and 2[18-21].\nCharacteristics of studies of molecular analysis included in the analysis\n113 samples of 105 patients; \nTP53, PIK3CA, PTEN; \n1 ACC; \nincludes: ACC, retention cyst, lymphoepithelial cyst, epidermoid cyst, and squamoid cyst. MA: Molecular analysis; NA: Non-available data; ACC: Acinar cell cyst; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.\nQuality assessment and publication bias: Methodological quality of primary studies included was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23], which evaluates the quality of articles for systematic reviews of diagnostic accuracy studies in four domains, including patient selection, index test, reference standard, and flow and timing, for risk of bias and applicability concerns. Results are presented in Figure 2, which was sketched with templates available at www.quadas.org. The studies included in this review all showed a “low-risk” classification as the index tests (MA and MFB) and the reference standard (surgical pathology specimen) were reliable and mentioned in all studies. However, a “high-risk” of selection bias was demonstrated in patient selection (neither random nor sequential patients included in several studies) and in flow and timing because only a small proportion of the patients evaluated in all studies, except one, were included in the analysis. In fact, most patients were excluded in all studies as the inclusion criteria requiring surgical pathology as diagnostic reference were not met. Applicability concerns in patient selection were also significant in all studies, because the subgroup of PCNs referred for surgery is more often malignant than PCNs on surveillance, which would also be targeted with this review. Because of this bias, there may be an overestimation of both the sensitivity of the index tests, due to a more severe spectrum of PCNs that are referred for surgery, and the positive predictive value (PPV) for diagnosis of high-risk cysts, due to an increased prevalence of malignant cysts in a surgical cohort of PCNs.\nQuality assessment of the studies using QUADAS-2. A: Tabular presentation of risk bias for each study; B: Graphical display of bias.",
"Molecular analysis: Four articles were included in the meta-analysis for diagnostic accuracy of MA. For each of the three definitions of relevant cyst, forest plots of sensitivity and specificity with heterogeneous denoted are shown in Figure 3.\nForest plots of the studies included for molecular analysis. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95% CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nThe three criteria to define “relevant cysts” resulted in a different range of the specificity and sensitivity of the studies included as shown in Figure 3. For diagnosis of the subgroup with high-risk and low-risk mucinous cysts that require intervention (either surgery or surveillance) comparing to benign cysts the pooled sensitivity was 0.75 (95%CI: 0.66-0.83) and the pooled specificity was 0.72 (95%CI: 0.56-0.85) for MA. In the subgroup of high-risk cysts that require surgery, comparing to other cysts requiring conservative management, the sensitivity was 0.57 (95%CI: 0.42-0.71) with a specificity of 0.88 (95%CI: 0.80-0.93). In the subgroup of low-risk mucinous cysts comparing to high-risk, the pooled sensitivity was 0.89 (95%CI: 0.79-0.95) and the pooled specificity was 0.88 (95%CI: 0.75-0.95).\nFigure 4 displays the sROC curves of MA, showing the sensitivity of the individual articles mapped on the vertical scale, 1-specificity on the horizontal scale, with the summary (sensitivity, 1-specificity) point marked, as well as the summary ROC curve and the confidence region for the summary (sensitivity, 1-specificity) points. The area under the sROC curve was 0.7706 (SE: 0.0927) in non-benign cysts, 0.9248 (SE: 0.0691) in high-risk cysts, and 0.9555 (SE: 0.0293) in mucinous low-risk cysts. The results of the studies had greater variation in non-benign cysts as shown by the wide confidence region.\nSummary receiver operating characteristics plots. ROC: Receiver operating characteristic curve; AUC: Area under the curve; SE: Standard error.\nIn the four studies, 566 patients had DNA available to perform MA in PCF. Pooled analysis (Figure 5) showed a diagnostic yield of 54.3% (95%CI: 49.8%-58.7%; I2 = 39.605%; test for heterogeneity P = 0.174).\nForest plots of molecular analysis and microforceps biopsies on the secondary outcomes of this meta-analysis.\nBy considering the classification of cysts by specific type (IPMNs, MCNs, cNETs, SCAs, pseudocysts, ACCs, and other RCs), MA identified correctly 73.1% of cysts (95%CI: 61.6%-82.2%; I2 = 37.381%; test for heterogeneity P = 0.203) (Figure 5).\nMicro forceps biopsy: Four articles were included in the meta-analysis for diagnostic accuracy of histology obtained using MFB. Figure 6 shows the forest plots of sensitivity and specificity for the three subgroups of relevant cysts. The forest plots for MFB show variable specificities within the papers, from 0 to 1, which can be due to the small numbers of patients with the target condition in some studies.\nForest plots of the included studies for microforceps biopsies. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95%CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nFor each of the three subgroups there exists a low heterogeneity in sensitivity (I2 = 0%, I2 = 21.4%, I2 = 0%) and specificity (I2 = 0%, I2 = 0%, I2 = 21.4%), therefore fixed effect models were used. As presented in Figure 6, in the first subgroup the pooled sensitivity was 0.73 (95%CI: 0.50-0.89) and the pooled specificity was 0.88 (95%CI: 0.28-1.00). In the second subgroup sensitivity was 0.81 (95%CI: 0.46-0.98) with a specificity of 0.77 (95%CI: 0.50-0.94) and in the last subgroup the pooled sensitivity was 0.64 (95%CI: 0.33-0.88) and the pooled specificity was 0.81 (95%CI: 0.46-0.98).\nThe results were plotted as a symmetrical sROC curve (Figure 4). The area under the sROC curve was 0.7640 (SE: 0.1261) in the first subgroup, 0.8154 (SE: 0.098) in the second subgroup, and 0.7509 (SE: 0.1277) in the last subgroup.\nBy pooling the data of the four studies that investigated the use of MFB to obtain a histopathologic diagnosis, we obtained a diagnostic yield of 73.1% (95%CI: 61.4%-82.2%; I2 = 47.774%; test for heterogeneity P = 0.125) (Figure 5).\nBy considering the outcome “specific cyst type” diagnosis, MFB correctly identified 70.7% of the cysts (95%CI: 49.4%-85.6%; I2 = 0%; test for heterogeneity P = 0.056) (Figure 5).",
"In this meta-analysis we analyzed two different but promising tests to diagnose PCNs – molecular analysis and microforceps biopsy. To our knowledge this is the first study of this nature, and it included 1206 patients with PCNs of which 1058 underwent MA and 148 MFB. All patients had the index tests performed in PCF obtained pre-operatively, exclusively with NGS for MA and the Moray micro forceps biopsy device (US Endoscopy, Mentor, Ohio) used for MFB. We analyzed 203 cysts, 178 evaluated with MA and 25 with MFB, all referred for surgery, and with a surgical pathology specimen used as reference standard for diagnosis.\nIn this comparative analysis we included all studies, without restriction to si-multaneous evaluation of both tests, because only one of such studies has been published[20]. This study, which includes 48 patients but only 10 surgical pathology specimens, showed identical results for MA and MFB in low-risk and high-risk cyst diagnosis, but higher specific cyst type diagnosis for MFB.\nThe data from the seven studies included in the meta-analysis, although with limited number of patients, particularly for MFB, suggests that MA is more accurate than MFB for diagnosis of PCNs, including high-risk and low-risk lesions. MA has superior accuracy to discriminate high-risk cysts from other PCNs and low-risk from high-risk neoplastic cysts. MA performance was considered excellent with AUC values of 0.92 and of 0.96 for high-risk and low-risk neoplastic lesions, respectively, as compared to MFB, which showed a fair or good performance, with an AUC of 0.81 and 0.75, respectively for the same lesions (Figure 4). The specificity of MA is good (0.88) but it has a low sensitivity (only 0.57) for high-risk cysts. This may be explained by technical issues, by low prevalence of relevant genetic mutations in malignant PCNs, or by mutations not included in the current NGS panels. The sensitivity and specificity are high (0.89 and 0.88, respectively) for MA when comparing low-risk to high-risk cysts, which reflects the genetic nature of pancreatic carcinogenesis with cumulative mutations from benign to malignant cysts[48].\nFor discriminating benign cysts from both low-risk and high-risk cysts, the performance of MA and MFB was identical and fair according to AUC values of 0.77 and 0.76, respectively. This non-superiority of MA in the diagnosis of benign cysts in this meta-analysis may be due to technique-inherent issues and/or under-representation of benign cysts in surgical series. In fact, “no genetic mutation” is considered a false negative result in most benign rare cysts, but some of these lesions (retention cysts, etc.) have no diagnostic genetic mutations. On the contrary, the most frequent benign cysts, SCAs, harbor a VHL mutation, exclusively present in these benign lesions and allowing for discarding a malignant lesion. In the MA studies, one third of rare benign cysts were classified as false negative results, due to absence of characteristic mutations (Table 1). Another example of PCN that is not amenable to a MA diagnosis with current genetic panels is cNET, also reducing the accuracy of MA for diagnosis of high-risk cysts. The sensitivities were identical for MA and MFB (0.75 and 0.72), but the latter had higher specificity (0.73 and 0.88, respectively). Limited tissue sampling with MFB can explain the reduced sensitivity with robust specificity. As MA depends on denuded DNA in suspension in PCF, no sampling error is expected, which may explain its greater accuracy in neoplastic cysts, comparing to MFB.\nConcerning secondary outcomes, even with the limitations of tissue sampling inherent to MFB, this meta-analysis showed that the diagnostic yield of MFB was superior to MA with rates of correctly identified cyst identical with MA and MFB (Tables 1 and 2). In fact, the definition of diagnostic yield, which for MA was “detection of genetic mutations”, may have led to a falsely low value due to the presence of some rare types of benign cysts (retention cysts, lymphoepithelial cysts, epidermoid cysts, squamous cysts in two studies[46,47]) that have no characteristic diagnostic genetic mutations.\nCharacteristics of studies of microforceps biopsies included in the analysis\n1 nondiagnostic on MFB; \n2 nondiagnostic on MFB; \n1 false negative IPMN associated invasive carcinoma; \n2 cases with a suspicion of pseudocyst. MFB: Microforceps biopsy; NA: Non-available data; ND: Nondiagnostic; ACC: Acinar cystadenoma; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.\nIn clinical practice, patient symptoms, cyst imaging features, CEA, and cytology of PCF are required for diagnosis and decision for either treatment or surveillance according to cyst types[49]. PCF analysis, including CEA to distinguish mucinous from non-mucinous cysts and cytology to select those that harbor HGA or early pancreatic carcinoma and require surgical treatment, have suboptimal accuracies[3], due to scant cellularity and limited PCF volume. In this context, additional diagnostic tests are necessary to improve cyst classification and refine clinical decision. DNA markers require limited amounts of PCF, increasing the diagnostic yield[32,45,50,51], but with considerable technical complexity and costs. In fact, in routine clinical practice a major pitfall for PCNs diagnosis is the limited volume of PCF obtained, precluding routine pre-operative testing. As DNA analysis requires less volume of PCF, it may become an alternative test in these circumstances. This major advantage of molecular analysis was not possible to evaluate in this meta-analysis, because the volume of cystic fluid obtained in pancreatic cysts was not available in most studies analyzed.\nAs MA continues to evolve, questions remain about its accuracy, how it influences patient management, and in what order the analysis should be performed to better support clinical decisions. Previous studies[49] have shown that DNA testing combined with clinical features increased PCNs diagnosis compared to either alone. With multiple recent advances in biomarkers, molecular genetics will probably prove to be useful in the management of PCNs[52]. In a previous meta-analysis, pre-operative cytology of PCNs has shown low sensitivity for diagnosis[53], endorsing additional tests to improve diagnosis. Another meta-analysis of diagnostic accuracy of EUS-FNA with CEA and cytology analysis in differentiating mucinous cysts has demonstrated to be accurate to confirm the diagnosis but performed poorly in excluding it[54]. The role of KRAS as individual screening test has been analyzed before[55] with poor accuracy and added benefit coming from a combined approach with cytology. A recently published meta-analysis supporting KRAS, GNAS, and RNF43 mutations as diagnostic markers of IPMNs[56] used different methods for mutation detection, different tumor materials, and clinicopathologic data as reference standard for diagnosis, which may limit its clinical application in evaluation of PCNs with mutational analysis performed only in PCF.\nIn this scenario, new markers are needed for PCNs stratification, and in our meta-analysis both MA and MFB have acceptable diagnostic accuracies. The two largest studies of MA[46,47] showed higher accuracy for diagnosis, which underscores the role of technical aspects of PCF collection, storage, and laboratory analysis for improved accuracy with this technique.\nOn the other hand, MFB provides tissue fragments for routine histological evaluation, without additional PCF required other than for standard analysis. The technical feasibility of through-the-needle microforceps biopsies revealed to be excellent, even in cysts located in the pancreatic head, despite the required 19-gauge caliber of the EUS-FNA needle. Another potential advantage of MFB is to allow the diagnosis of histologic subtypes of IPMNs, which can potentially be used for risk stratification[57], but still requires further validation.\n Strengths and limitations We applied strict exclusion criteria, with all analyzed patients having a surgical pathology specimen as the reference standard for diagnosis, because histopathology is the gold standard for diagnosis of neoplasia. Another major strength of this meta-analysis is having identical lesions (size and location) analyzed in both groups. These important strengths provide a more realistic accuracy estimate of the tests evaluated. In previous studies of cytology including both surgical pathology and clinical follow-up[54] as reference standard, pooled sensitivities were 12% higher than in studies with exclusive surgical pathology[55] as reference standard in the diagnosis of mucinous cysts, with test accuracy overestimation. Finally, the pooled results have low heterogeneity.\nThe quality of a systematic review depends on the quality of studies included, and our quality assessment of patient selection regarding the risk of bias and applicability was high. As sensitivity and specificity are sensitive to study design and influenced by the spectrum of disease, sample collection, and processing, there may be a risk of bias and the results, although correct, their interpretation may be inaccurate. Moreover, there was incomplete reporting in one primary study, having no separate information on specific cyst type, mucinous or malignant cyst diagnosis[32], and the study was excluded from quantitative analysis. Although one study was excluded from the meta-analysis, MA with three studies included more patients (953, of whom only 153 in the surgical cohort) than the group of MFB with four studies but fewer patients (148, with only 25 in the surgical cohort). This can represent a surgical selection bias for both tests studied. Moreover, MFB studies were all retrospective, with small sample size, without pathology diagnosis for most benign and pre-malignant cysts, and non-consecutive patients that were selected on endoscopist discretion, which may have led to bias. Another limitation is the time between the index tests and the reference standard, because the final diagnosis could have been made at different time intervals from the tests. If the time between index tests and reference standard is too long, the true disease status of the patient may have changed by the time the reference standard was assessed. Aditionally, the different number of malignant cysts per study, particularly in the MA group, may have led to part of the heterogeneity in sensitivity and specificity. Finally, as MA does not increase the risks of standard EUS-FNA (the analysis is performed in remnant cystic fluid after standard diagnosis) we did not perform a safety analysis of MFB, but the four studies analyzed described only rare non-severe adverse events.\nWe applied strict exclusion criteria, with all analyzed patients having a surgical pathology specimen as the reference standard for diagnosis, because histopathology is the gold standard for diagnosis of neoplasia. Another major strength of this meta-analysis is having identical lesions (size and location) analyzed in both groups. These important strengths provide a more realistic accuracy estimate of the tests evaluated. In previous studies of cytology including both surgical pathology and clinical follow-up[54] as reference standard, pooled sensitivities were 12% higher than in studies with exclusive surgical pathology[55] as reference standard in the diagnosis of mucinous cysts, with test accuracy overestimation. Finally, the pooled results have low heterogeneity.\nThe quality of a systematic review depends on the quality of studies included, and our quality assessment of patient selection regarding the risk of bias and applicability was high. As sensitivity and specificity are sensitive to study design and influenced by the spectrum of disease, sample collection, and processing, there may be a risk of bias and the results, although correct, their interpretation may be inaccurate. Moreover, there was incomplete reporting in one primary study, having no separate information on specific cyst type, mucinous or malignant cyst diagnosis[32], and the study was excluded from quantitative analysis. Although one study was excluded from the meta-analysis, MA with three studies included more patients (953, of whom only 153 in the surgical cohort) than the group of MFB with four studies but fewer patients (148, with only 25 in the surgical cohort). This can represent a surgical selection bias for both tests studied. Moreover, MFB studies were all retrospective, with small sample size, without pathology diagnosis for most benign and pre-malignant cysts, and non-consecutive patients that were selected on endoscopist discretion, which may have led to bias. Another limitation is the time between the index tests and the reference standard, because the final diagnosis could have been made at different time intervals from the tests. If the time between index tests and reference standard is too long, the true disease status of the patient may have changed by the time the reference standard was assessed. Aditionally, the different number of malignant cysts per study, particularly in the MA group, may have led to part of the heterogeneity in sensitivity and specificity. Finally, as MA does not increase the risks of standard EUS-FNA (the analysis is performed in remnant cystic fluid after standard diagnosis) we did not perform a safety analysis of MFB, but the four studies analyzed described only rare non-severe adverse events.\n Future perspectives With the increasing diagnosis of asymptomatic PCNs, most with potential for malignancy, there is a growing need to find accurate and affordable tests for diagnosis. The goal of management of patients with pancreatic cysts is to detect and resect cysts before progression of malignancy, while avoiding unnecessary follow-up procedures in benign cysts and surgery in low-risk PCNs.\nBiomarkers of malignancy are promising, but clinicians should be aware of their current diagnostic performance limitations and type of lesions identified. In addition to significant costs, logistic difficulties in preserving material for future molecular analysis in busy general hospitals, and the technical complexity of the test, the generalized use of MA seems difficult in clinical practice. On the other hand, if MFB proves in larger studies to be safe and to allow tissue acquisition and gives the histological criteria needed for a correct diagnosis of PCNs, it may be immediately implemented in clinics, because the endoscopic procedure is standard, and histology is already a widespread procedure in clinics. MFB may be especially useful for benign lesions, for which both surgery and surveillance are unnecessary, representing a considerable burden in pancreas clinics due to current diagnostic limitations[58].\nFor MA to become relevant in routine clinical care in the future, its role in early cancer diagnosis and its prognostic value in PCNs requiring periodic surveillance must be confirmed. Also, for successful massive implementation, it is required to develop as an universal, highly accurate, first line test with clinical impact in cyst diagnosis, prognosis, and patient management. MA, both in PCF and peripheral blood, for standard analysis of multiple simultaneous biomarkers, allowing non-invasive diagnosis and risk stratification of these lesions[59] would be valuable. For the present time, MA and MFB can only be recommended as complementary or as second line tests in case CEA and cytology of PCF are non-diagnostic. For both tests, large multicenter validation studies are still missing.\nWith the increasing diagnosis of asymptomatic PCNs, most with potential for malignancy, there is a growing need to find accurate and affordable tests for diagnosis. The goal of management of patients with pancreatic cysts is to detect and resect cysts before progression of malignancy, while avoiding unnecessary follow-up procedures in benign cysts and surgery in low-risk PCNs.\nBiomarkers of malignancy are promising, but clinicians should be aware of their current diagnostic performance limitations and type of lesions identified. In addition to significant costs, logistic difficulties in preserving material for future molecular analysis in busy general hospitals, and the technical complexity of the test, the generalized use of MA seems difficult in clinical practice. On the other hand, if MFB proves in larger studies to be safe and to allow tissue acquisition and gives the histological criteria needed for a correct diagnosis of PCNs, it may be immediately implemented in clinics, because the endoscopic procedure is standard, and histology is already a widespread procedure in clinics. MFB may be especially useful for benign lesions, for which both surgery and surveillance are unnecessary, representing a considerable burden in pancreas clinics due to current diagnostic limitations[58].\nFor MA to become relevant in routine clinical care in the future, its role in early cancer diagnosis and its prognostic value in PCNs requiring periodic surveillance must be confirmed. Also, for successful massive implementation, it is required to develop as an universal, highly accurate, first line test with clinical impact in cyst diagnosis, prognosis, and patient management. MA, both in PCF and peripheral blood, for standard analysis of multiple simultaneous biomarkers, allowing non-invasive diagnosis and risk stratification of these lesions[59] would be valuable. For the present time, MA and MFB can only be recommended as complementary or as second line tests in case CEA and cytology of PCF are non-diagnostic. For both tests, large multicenter validation studies are still missing.",
"We applied strict exclusion criteria, with all analyzed patients having a surgical pathology specimen as the reference standard for diagnosis, because histopathology is the gold standard for diagnosis of neoplasia. Another major strength of this meta-analysis is having identical lesions (size and location) analyzed in both groups. These important strengths provide a more realistic accuracy estimate of the tests evaluated. In previous studies of cytology including both surgical pathology and clinical follow-up[54] as reference standard, pooled sensitivities were 12% higher than in studies with exclusive surgical pathology[55] as reference standard in the diagnosis of mucinous cysts, with test accuracy overestimation. Finally, the pooled results have low heterogeneity.\nThe quality of a systematic review depends on the quality of studies included, and our quality assessment of patient selection regarding the risk of bias and applicability was high. As sensitivity and specificity are sensitive to study design and influenced by the spectrum of disease, sample collection, and processing, there may be a risk of bias and the results, although correct, their interpretation may be inaccurate. Moreover, there was incomplete reporting in one primary study, having no separate information on specific cyst type, mucinous or malignant cyst diagnosis[32], and the study was excluded from quantitative analysis. Although one study was excluded from the meta-analysis, MA with three studies included more patients (953, of whom only 153 in the surgical cohort) than the group of MFB with four studies but fewer patients (148, with only 25 in the surgical cohort). This can represent a surgical selection bias for both tests studied. Moreover, MFB studies were all retrospective, with small sample size, without pathology diagnosis for most benign and pre-malignant cysts, and non-consecutive patients that were selected on endoscopist discretion, which may have led to bias. Another limitation is the time between the index tests and the reference standard, because the final diagnosis could have been made at different time intervals from the tests. If the time between index tests and reference standard is too long, the true disease status of the patient may have changed by the time the reference standard was assessed. Aditionally, the different number of malignant cysts per study, particularly in the MA group, may have led to part of the heterogeneity in sensitivity and specificity. Finally, as MA does not increase the risks of standard EUS-FNA (the analysis is performed in remnant cystic fluid after standard diagnosis) we did not perform a safety analysis of MFB, but the four studies analyzed described only rare non-severe adverse events.",
"With the increasing diagnosis of asymptomatic PCNs, most with potential for malignancy, there is a growing need to find accurate and affordable tests for diagnosis. The goal of management of patients with pancreatic cysts is to detect and resect cysts before progression of malignancy, while avoiding unnecessary follow-up procedures in benign cysts and surgery in low-risk PCNs.\nBiomarkers of malignancy are promising, but clinicians should be aware of their current diagnostic performance limitations and type of lesions identified. In addition to significant costs, logistic difficulties in preserving material for future molecular analysis in busy general hospitals, and the technical complexity of the test, the generalized use of MA seems difficult in clinical practice. On the other hand, if MFB proves in larger studies to be safe and to allow tissue acquisition and gives the histological criteria needed for a correct diagnosis of PCNs, it may be immediately implemented in clinics, because the endoscopic procedure is standard, and histology is already a widespread procedure in clinics. MFB may be especially useful for benign lesions, for which both surgery and surveillance are unnecessary, representing a considerable burden in pancreas clinics due to current diagnostic limitations[58].\nFor MA to become relevant in routine clinical care in the future, its role in early cancer diagnosis and its prognostic value in PCNs requiring periodic surveillance must be confirmed. Also, for successful massive implementation, it is required to develop as an universal, highly accurate, first line test with clinical impact in cyst diagnosis, prognosis, and patient management. MA, both in PCF and peripheral blood, for standard analysis of multiple simultaneous biomarkers, allowing non-invasive diagnosis and risk stratification of these lesions[59] would be valuable. For the present time, MA and MFB can only be recommended as complementary or as second line tests in case CEA and cytology of PCF are non-diagnostic. For both tests, large multicenter validation studies are still missing.",
"Our study confirms the diagnostic value of both MA and MFB, with higher diagnostic accuracy of MA than MFB for both low-risk and high-risk mucinous cysts. Genetic analysis should not be replaced by MFB in this context. Clinicians should be aware of the higher accuracy of MA for the diagnosis of malignant and high-risk cysts."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"MATERIALS AND METHODS",
"Literature search and study selection",
"Data extraction",
"Outcomes",
"Quality analysis",
"Statistical analysis and data synthesis",
"RESULTS",
"Systematic Review",
"Meta-analysis",
"DISCUSSION",
"Strengths and limitations",
"Future perspectives",
"CONCLUSION"
] |
[
"Pancreatic cystic neoplasms (PCNs) are on the rise in clinics due to an ageing population and the increase in routine use of high-quality abdominal imaging[1]. PCNs are generally classified into two main groups: mucinous cystic neoplasms (MCNs) and non-mucinous cystic neoplasms (NMCN). MCNs include intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystadenomas, which are precursor lesions of pancreatic carcinoma, and may be low-risk (pre-malignant with low or intermediate-grade atypia) or high-risk: pre-malignant with high-grade atypia (HGA) or malignant, including adenocarcinomas secondarily cystic. NMCNs include serous cystadenomas and inflammatory cysts (pseudocysts), mostly benign cysts, but may include some rare lesions, considered high-risk as cystic neuroendocrine tumors (cNETs), and acinar cell cystadenomas (ACCs). The heterogeneity in malignant potential, increased frequency, and significant morbidity and mortality of surgical treatment, makes pre-operative diagnosis of PCNs essential for management. The treatment options for PCNs encompass surgery or conservative surveillance for MCNs, according to malignancy risk, or no further evaluation for most NMCNs.\nThe differentiation between MCNs and NMCNs is critical, because a misdiagnosis of a MCN can lead to a missed opportunity to treat pancreatic cancer in an early stage and a misdiagnosis of NMCN can result in unnecessary surgery or surveillance with associated morbidity, costs, and negative impact on quality of life.\nCurrently, morphologic characterization of PCNs and pancreatic cystic fluid (PCF) analysis for carcinoembryonic Antigen (CEA) and cytology are central in diagnosis. A CEA level ≥ 192 ng/mL is the most accurate diagnostic test for MCNs and cytology is highly specific for malignancy[2], but with suboptimal results in large studies with surgical pathology as the gold standard[3]. In fact, a significant part of these lesions remains indeterminate and incorrect pre-operative diagnosis occurs in one third of patients[4,5], making new reliable diagnostic tools urgently needed.\nIn the last decade numerous studies have shown that genetic analysis of aspirates obtained by EUS-FNA provided a better characterization of PCNs than CEA and cytology[6-14]. Next-generation sequencing (NGS) is a very sensitive technique for detection of genetic mutations that allows the rapid detection of mutations in pre-defined panels of cancer genes, even in samples with limited DNA content, such as PCF. NGS requires storage, infrastructure, data processing, and expert personnel. Moreover, to be cost-effective, large numbers of samples need to be processed, making it applicable only in large centralized laboratories. These reasons make the implementation of NGS in clinical practice still a matter of debate.\nThe clinical need of better diagnostic tests in PCNs has recently led to the development of a through-the-needle miniature biopsy device for use during EUS-FNA[15,16]. The Moray micro forceps biopsy (MFB) device (US Endoscopy, Mentor, Ohio) is disposable and can pass through a standard 19-gauge EUS-FNA needle that is already used routinely. It allows tissue sampling from the cyst wall, septa or mural nodules and the obtention of a histological evaluation of the epithelial architecture and subepithelial stroma[17]. Adding to the high technical success and excellent safety profile[18,19], the new device has shown to improve the diagnostic accuracy of specific cyst subtypes[20,21]. Another major advantage of MFB is the simultaneous tissue sampling and PCF acquisition, with just an additional histologic analysis that follows standard definitions and is already routine in clinics.\nThe aim of this systematic review and meta-analysis is to evaluate the diagnostic performance of molecular analysis (MA) and MFB and find the most robust additional diagnostic technique in PCNs, in the pre-operative setting.",
"This systematic review and meta-analysis is conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis of Diagnostic Test Accuracy Studies, the PRISMA-DTA Statement[22], and the protocol is registered at PROSPERO (CRD42018111910).\n Literature search and study selection A comprehensive search of databases, including Medline, Scopus, and Web of Science, for the past 8 years (January 1st, 2010 to July 31st, 2018) and restricted to human studies was performed. No language restrictions were applied. The following search terms were used in two independent searches: “pancreas”, “cyst”, “molecular”, “analysis”; and “micro”, “forceps”, “microforceps”, “biopsy”. A search of related articles was performed, adding additional studies. Duplicate articles, reviews, trials including other kinds of neoplasms, and trials with molecular markers not compliant with the defined inclusion criteria were removed. The references of all selected studies were hand-searched for additional articles.\nInclusion criteria: Published studies were included in the meta-analysis if they analyzed: (1) Patients with symptomatic or incidental pancreatic cysts with a definitive surgical pathology diagnosis; (2) Genetic mutations performed with high sensitive techniques, such as NGS in PCF obtained by EUS-FNA prior to surgery; (3) At least four genetic mutations, including KRAS, GNAS, VHL, and at least another genetic mutation representative of aggressive neoplasms (PIK3CA, TP53, SMAD4, PTEN, CDKN2A); (4) PCNs evaluated by EUS-FNA with MFB for diagnosis; and (5) Surgical pathology specimens with available data.\nExclusion criteria: (1) Studies of MA with fewer than the four genetic mutations previously defined; (2) Studies involving solid pancreatic lesions; (3) Studies using PCF not obtained by EUS-FNA; (4) Reviews, case reports, case series with fewer than five patients, letters to editor, exploratory studies, and papers published only in abstract form; (5) Studies with cytology and clinical surveillance as standard of diagnosis. Two authors (SF and AL) independently judged study eligibility and disagreements were resolved by consensus.\nHistological criteria: We classified the PCNs of the included studies into three main groups: (1) High-risk cysts (adenocarcinoma or high grade dysplasia in IPMNs and MCNs, secondarily cystic adenocarcinomas, cNETs, and ACCs; (2) Low-risk mucinous cysts (IPMNs and MCNs with intermediate or low-grade dysplasia); and (3) Benign cysts (SCAs, pseudocysts, and other rare cysts (RCs) included in some articles, as retention cysts, lymphoepithelial cysts, epidermoid cysts, squamoid cysts).\nTests under investigation: The index tests were: (1) MA of PCF; and (2) MFB of PCNs, including cyst wall, septs, and nodules. A diagnosis of cNET or ACC does not warrant a malignancy diagnosis, but surgery is recommended in surgically fit patients. Due to a recommendation of identical treatment to malignant and mucinous high-risk cysts, for the purpose of analysis in this study, each one of these diagnoses was classified as a high-risk cyst.\nA comprehensive search of databases, including Medline, Scopus, and Web of Science, for the past 8 years (January 1st, 2010 to July 31st, 2018) and restricted to human studies was performed. No language restrictions were applied. The following search terms were used in two independent searches: “pancreas”, “cyst”, “molecular”, “analysis”; and “micro”, “forceps”, “microforceps”, “biopsy”. A search of related articles was performed, adding additional studies. Duplicate articles, reviews, trials including other kinds of neoplasms, and trials with molecular markers not compliant with the defined inclusion criteria were removed. The references of all selected studies were hand-searched for additional articles.\nInclusion criteria: Published studies were included in the meta-analysis if they analyzed: (1) Patients with symptomatic or incidental pancreatic cysts with a definitive surgical pathology diagnosis; (2) Genetic mutations performed with high sensitive techniques, such as NGS in PCF obtained by EUS-FNA prior to surgery; (3) At least four genetic mutations, including KRAS, GNAS, VHL, and at least another genetic mutation representative of aggressive neoplasms (PIK3CA, TP53, SMAD4, PTEN, CDKN2A); (4) PCNs evaluated by EUS-FNA with MFB for diagnosis; and (5) Surgical pathology specimens with available data.\nExclusion criteria: (1) Studies of MA with fewer than the four genetic mutations previously defined; (2) Studies involving solid pancreatic lesions; (3) Studies using PCF not obtained by EUS-FNA; (4) Reviews, case reports, case series with fewer than five patients, letters to editor, exploratory studies, and papers published only in abstract form; (5) Studies with cytology and clinical surveillance as standard of diagnosis. Two authors (SF and AL) independently judged study eligibility and disagreements were resolved by consensus.\nHistological criteria: We classified the PCNs of the included studies into three main groups: (1) High-risk cysts (adenocarcinoma or high grade dysplasia in IPMNs and MCNs, secondarily cystic adenocarcinomas, cNETs, and ACCs; (2) Low-risk mucinous cysts (IPMNs and MCNs with intermediate or low-grade dysplasia); and (3) Benign cysts (SCAs, pseudocysts, and other rare cysts (RCs) included in some articles, as retention cysts, lymphoepithelial cysts, epidermoid cysts, squamoid cysts).\nTests under investigation: The index tests were: (1) MA of PCF; and (2) MFB of PCNs, including cyst wall, septs, and nodules. A diagnosis of cNET or ACC does not warrant a malignancy diagnosis, but surgery is recommended in surgically fit patients. Due to a recommendation of identical treatment to malignant and mucinous high-risk cysts, for the purpose of analysis in this study, each one of these diagnoses was classified as a high-risk cyst.\n Data extraction After study selection, two authors (SF and AL) extracted and registered the data from each study onto a standardized worksheet. Disagreements were discussed and reviewed by a third author (LP). The data retrieved were: first author, publication year, study period and design (prospective or retrospective), reference for diagnosis, sample size (all patients included in the study), technical success, adverse events, diagnostic yield, surgical cohort (number of patients with a surgical pathology specimen), cyst size, cyst location, specific cyst types, number of high-risk cysts, mucinous low-risk and benign cysts diagnosed by MA and MFB comparing to surgical pathology specimens. In the MFB studies, technical success was defined as the ability to puncture the cysts and perform the biopsies; and the diagnostic yield was defined as the ratio between the number of patients included in the study and the patients in whom enough material allowed the acquisition of a histopathologic diagnosis. In the MA group, diagnostic yield was defined as a ratio between the number of patients included in the study and the number of patients with DNA available to perform molecular analysis in PCF.\nAfter study selection, two authors (SF and AL) extracted and registered the data from each study onto a standardized worksheet. Disagreements were discussed and reviewed by a third author (LP). The data retrieved were: first author, publication year, study period and design (prospective or retrospective), reference for diagnosis, sample size (all patients included in the study), technical success, adverse events, diagnostic yield, surgical cohort (number of patients with a surgical pathology specimen), cyst size, cyst location, specific cyst types, number of high-risk cysts, mucinous low-risk and benign cysts diagnosed by MA and MFB comparing to surgical pathology specimens. In the MFB studies, technical success was defined as the ability to puncture the cysts and perform the biopsies; and the diagnostic yield was defined as the ratio between the number of patients included in the study and the patients in whom enough material allowed the acquisition of a histopathologic diagnosis. In the MA group, diagnostic yield was defined as a ratio between the number of patients included in the study and the number of patients with DNA available to perform molecular analysis in PCF.\n Outcomes The primary outcomes of this study were the data to obtain the accuracies of MA and MFB for the diagnosis of PCNs, including high-risk cysts, mucinous low-risk cysts, and benign cysts. Secondary outcomes were the diagnostic yield of genetic testing and MFB and the number of cysts correctly identified for each of the tests studied.\nThe primary outcomes of this study were the data to obtain the accuracies of MA and MFB for the diagnosis of PCNs, including high-risk cysts, mucinous low-risk cysts, and benign cysts. Secondary outcomes were the diagnostic yield of genetic testing and MFB and the number of cysts correctly identified for each of the tests studied.\n Quality analysis Methodological quality of included primary studies was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23]. The PRISMA-DTA Statement recommendations were used for reporting this systematic review[22,24].\nMethodological quality of included primary studies was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23]. The PRISMA-DTA Statement recommendations were used for reporting this systematic review[22,24].\n Statistical analysis and data synthesis The reference standard was a surgical pathology specimen that allowed the classification of PCNs into three defined groups of diagnosis: high-risk cysts, mucinous low-risk cysts, and benign cysts. This resulted in a two-by-three table with correct and incorrect test results in each of the three referenced groups, for each of the tests analyzed, MA and histology were obtained by MFB.\nTo calculate tests’ accuracy and to reflect on the categories that are useful in clinical practice and that guide management, we constructed two-by-two tables, considering three definitions of “relevant” cysts: (1) High-risk cysts – proven malignant cysts, IPMNs, and MCNs with HGA, cNETs, ACCs; Non-High-risk cysts – all cysts except those proven to be high-risk. (2) Low-risk mucinous cysts – proven mucinous low-risk cysts; High-risk cysts – all except those proven to be mucinous low-risk or benign. And (3) Non-benign cysts – all cysts except those proven to be benign; Benign cysts – proven benign cysts.\nThe ability of the tests to discriminate “relevant” and “non-relevant” cysts using the three definitions of “relevant cysts” was evaluated and the accuracy of the two tests was compared.\nThe data of the two-by-two tables were used to calculate sensitivity and specificity for each study. We present individual study results graphically by plotting the estimates of sensitivity and specificity (and their 95% confidence intervals (CI)) in both forest plots and on the summary receiver operating characteristic (sROC) curve plots. The area under the curve (AUC) is equal to 1 for a perfect test and 0.5 for a completely uninformative test. The AUC is equal to the probability that if a pair of relevant and non-relevant cysts is selected at random, the relevant cyst will have a higher test result than the non-relevant cyst. Pooled estimates of the sensitivity and specificity were obtained by the DerSimonian-Laird method (random effect model) to incorporate variation among studies, when data are heterogeneous. Otherwise, we used the Mantel-Haenszel method (fixed effect model).\nHeterogeneity was investigated in the first instance through visual examination of forest plots of sensitivities and specificities and through visual examination of the ROC plot of the raw data. Last, we used statistical tests, including chi-square and Cochran-Q to evaluate if the differences across the studies were greater than expected by chance alone. A low P value suggests presence of heterogeneity. In addition to these statistics we used the statistic I2 of Higgins, which has been proposed as a measure to quantify the amount of heterogeneity[25,26]. The scale of I2 has a range of 0 to 100% and values on the order of 25%, 50% and 75% are considered low, moderate, and high heterogeneity, respectively.\nAnother goal of this work was to obtain, for each of the tests, the correctly identified cyst rate and the diagnostic yield in predicting a histopathologic diagnosis.\nWe used Comprehensive Meta-Analysis software (Version 2.0) for assessment of diagnostic yield of the tests and Meta-DiSc (version 1.4 – Meta-Analysis of Diagnostic and screening tests[27] ) to obtain the accuracy of each of the tests.\nThe reference standard was a surgical pathology specimen that allowed the classification of PCNs into three defined groups of diagnosis: high-risk cysts, mucinous low-risk cysts, and benign cysts. This resulted in a two-by-three table with correct and incorrect test results in each of the three referenced groups, for each of the tests analyzed, MA and histology were obtained by MFB.\nTo calculate tests’ accuracy and to reflect on the categories that are useful in clinical practice and that guide management, we constructed two-by-two tables, considering three definitions of “relevant” cysts: (1) High-risk cysts – proven malignant cysts, IPMNs, and MCNs with HGA, cNETs, ACCs; Non-High-risk cysts – all cysts except those proven to be high-risk. (2) Low-risk mucinous cysts – proven mucinous low-risk cysts; High-risk cysts – all except those proven to be mucinous low-risk or benign. And (3) Non-benign cysts – all cysts except those proven to be benign; Benign cysts – proven benign cysts.\nThe ability of the tests to discriminate “relevant” and “non-relevant” cysts using the three definitions of “relevant cysts” was evaluated and the accuracy of the two tests was compared.\nThe data of the two-by-two tables were used to calculate sensitivity and specificity for each study. We present individual study results graphically by plotting the estimates of sensitivity and specificity (and their 95% confidence intervals (CI)) in both forest plots and on the summary receiver operating characteristic (sROC) curve plots. The area under the curve (AUC) is equal to 1 for a perfect test and 0.5 for a completely uninformative test. The AUC is equal to the probability that if a pair of relevant and non-relevant cysts is selected at random, the relevant cyst will have a higher test result than the non-relevant cyst. Pooled estimates of the sensitivity and specificity were obtained by the DerSimonian-Laird method (random effect model) to incorporate variation among studies, when data are heterogeneous. Otherwise, we used the Mantel-Haenszel method (fixed effect model).\nHeterogeneity was investigated in the first instance through visual examination of forest plots of sensitivities and specificities and through visual examination of the ROC plot of the raw data. Last, we used statistical tests, including chi-square and Cochran-Q to evaluate if the differences across the studies were greater than expected by chance alone. A low P value suggests presence of heterogeneity. In addition to these statistics we used the statistic I2 of Higgins, which has been proposed as a measure to quantify the amount of heterogeneity[25,26]. The scale of I2 has a range of 0 to 100% and values on the order of 25%, 50% and 75% are considered low, moderate, and high heterogeneity, respectively.\nAnother goal of this work was to obtain, for each of the tests, the correctly identified cyst rate and the diagnostic yield in predicting a histopathologic diagnosis.\nWe used Comprehensive Meta-Analysis software (Version 2.0) for assessment of diagnostic yield of the tests and Meta-DiSc (version 1.4 – Meta-Analysis of Diagnostic and screening tests[27] ) to obtain the accuracy of each of the tests.",
"A comprehensive search of databases, including Medline, Scopus, and Web of Science, for the past 8 years (January 1st, 2010 to July 31st, 2018) and restricted to human studies was performed. No language restrictions were applied. The following search terms were used in two independent searches: “pancreas”, “cyst”, “molecular”, “analysis”; and “micro”, “forceps”, “microforceps”, “biopsy”. A search of related articles was performed, adding additional studies. Duplicate articles, reviews, trials including other kinds of neoplasms, and trials with molecular markers not compliant with the defined inclusion criteria were removed. The references of all selected studies were hand-searched for additional articles.\nInclusion criteria: Published studies were included in the meta-analysis if they analyzed: (1) Patients with symptomatic or incidental pancreatic cysts with a definitive surgical pathology diagnosis; (2) Genetic mutations performed with high sensitive techniques, such as NGS in PCF obtained by EUS-FNA prior to surgery; (3) At least four genetic mutations, including KRAS, GNAS, VHL, and at least another genetic mutation representative of aggressive neoplasms (PIK3CA, TP53, SMAD4, PTEN, CDKN2A); (4) PCNs evaluated by EUS-FNA with MFB for diagnosis; and (5) Surgical pathology specimens with available data.\nExclusion criteria: (1) Studies of MA with fewer than the four genetic mutations previously defined; (2) Studies involving solid pancreatic lesions; (3) Studies using PCF not obtained by EUS-FNA; (4) Reviews, case reports, case series with fewer than five patients, letters to editor, exploratory studies, and papers published only in abstract form; (5) Studies with cytology and clinical surveillance as standard of diagnosis. Two authors (SF and AL) independently judged study eligibility and disagreements were resolved by consensus.\nHistological criteria: We classified the PCNs of the included studies into three main groups: (1) High-risk cysts (adenocarcinoma or high grade dysplasia in IPMNs and MCNs, secondarily cystic adenocarcinomas, cNETs, and ACCs; (2) Low-risk mucinous cysts (IPMNs and MCNs with intermediate or low-grade dysplasia); and (3) Benign cysts (SCAs, pseudocysts, and other rare cysts (RCs) included in some articles, as retention cysts, lymphoepithelial cysts, epidermoid cysts, squamoid cysts).\nTests under investigation: The index tests were: (1) MA of PCF; and (2) MFB of PCNs, including cyst wall, septs, and nodules. A diagnosis of cNET or ACC does not warrant a malignancy diagnosis, but surgery is recommended in surgically fit patients. Due to a recommendation of identical treatment to malignant and mucinous high-risk cysts, for the purpose of analysis in this study, each one of these diagnoses was classified as a high-risk cyst.",
"After study selection, two authors (SF and AL) extracted and registered the data from each study onto a standardized worksheet. Disagreements were discussed and reviewed by a third author (LP). The data retrieved were: first author, publication year, study period and design (prospective or retrospective), reference for diagnosis, sample size (all patients included in the study), technical success, adverse events, diagnostic yield, surgical cohort (number of patients with a surgical pathology specimen), cyst size, cyst location, specific cyst types, number of high-risk cysts, mucinous low-risk and benign cysts diagnosed by MA and MFB comparing to surgical pathology specimens. In the MFB studies, technical success was defined as the ability to puncture the cysts and perform the biopsies; and the diagnostic yield was defined as the ratio between the number of patients included in the study and the patients in whom enough material allowed the acquisition of a histopathologic diagnosis. In the MA group, diagnostic yield was defined as a ratio between the number of patients included in the study and the number of patients with DNA available to perform molecular analysis in PCF.",
"The primary outcomes of this study were the data to obtain the accuracies of MA and MFB for the diagnosis of PCNs, including high-risk cysts, mucinous low-risk cysts, and benign cysts. Secondary outcomes were the diagnostic yield of genetic testing and MFB and the number of cysts correctly identified for each of the tests studied.",
"Methodological quality of included primary studies was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23]. The PRISMA-DTA Statement recommendations were used for reporting this systematic review[22,24].",
"The reference standard was a surgical pathology specimen that allowed the classification of PCNs into three defined groups of diagnosis: high-risk cysts, mucinous low-risk cysts, and benign cysts. This resulted in a two-by-three table with correct and incorrect test results in each of the three referenced groups, for each of the tests analyzed, MA and histology were obtained by MFB.\nTo calculate tests’ accuracy and to reflect on the categories that are useful in clinical practice and that guide management, we constructed two-by-two tables, considering three definitions of “relevant” cysts: (1) High-risk cysts – proven malignant cysts, IPMNs, and MCNs with HGA, cNETs, ACCs; Non-High-risk cysts – all cysts except those proven to be high-risk. (2) Low-risk mucinous cysts – proven mucinous low-risk cysts; High-risk cysts – all except those proven to be mucinous low-risk or benign. And (3) Non-benign cysts – all cysts except those proven to be benign; Benign cysts – proven benign cysts.\nThe ability of the tests to discriminate “relevant” and “non-relevant” cysts using the three definitions of “relevant cysts” was evaluated and the accuracy of the two tests was compared.\nThe data of the two-by-two tables were used to calculate sensitivity and specificity for each study. We present individual study results graphically by plotting the estimates of sensitivity and specificity (and their 95% confidence intervals (CI)) in both forest plots and on the summary receiver operating characteristic (sROC) curve plots. The area under the curve (AUC) is equal to 1 for a perfect test and 0.5 for a completely uninformative test. The AUC is equal to the probability that if a pair of relevant and non-relevant cysts is selected at random, the relevant cyst will have a higher test result than the non-relevant cyst. Pooled estimates of the sensitivity and specificity were obtained by the DerSimonian-Laird method (random effect model) to incorporate variation among studies, when data are heterogeneous. Otherwise, we used the Mantel-Haenszel method (fixed effect model).\nHeterogeneity was investigated in the first instance through visual examination of forest plots of sensitivities and specificities and through visual examination of the ROC plot of the raw data. Last, we used statistical tests, including chi-square and Cochran-Q to evaluate if the differences across the studies were greater than expected by chance alone. A low P value suggests presence of heterogeneity. In addition to these statistics we used the statistic I2 of Higgins, which has been proposed as a measure to quantify the amount of heterogeneity[25,26]. The scale of I2 has a range of 0 to 100% and values on the order of 25%, 50% and 75% are considered low, moderate, and high heterogeneity, respectively.\nAnother goal of this work was to obtain, for each of the tests, the correctly identified cyst rate and the diagnostic yield in predicting a histopathologic diagnosis.\nWe used Comprehensive Meta-Analysis software (Version 2.0) for assessment of diagnostic yield of the tests and Meta-DiSc (version 1.4 – Meta-Analysis of Diagnostic and screening tests[27] ) to obtain the accuracy of each of the tests.",
" Systematic Review Our search revealed 16 study titles and abstracts for MFB and 264 titles for MA. In Figure 1A and B are described the selection process of the articles included in this study. After all steps, eight studies were considered suitable for qualitative and seven for quantitative analysis. We excluded 20 full-text articles after review, because they were case series of two patients[16] (n = 1), exploratory or pilot studies[28,29] (n = 2), no information of mutation status was available[30] (n = 1), pancreatic cystic fluid was obtained during surgery[31] (n = 1), insufficient or absent data of cysts with surgical pathology diagnoses[12,32,33] (n = 3), and mutations only of KRAS and/or GNAS[14,34-44] (n = 12).\nFlowchart with identification of eligible studies. A: Molecular analysis; B: Microforceps biopsy.\nOf the eight studies that met the inclusion criteria, design was retrospective in six and prospective in two, all were published from 2015 to 2018. These eight studies included a total of 1206 patients, of which 203 (17%) underwent surgical resection and a surgical pathology specimen was available as reference standard and included in the analysis. We excluded all patients with cytology and clinical follow-up data, but for whom a surgical pathology specimen was not available. The characteristics of the studies, surgical pathology diagnoses, and MA and MFB results are presented in Tables 1[32,45-47] and 2[18-21].\nCharacteristics of studies of molecular analysis included in the analysis\n113 samples of 105 patients; \nTP53, PIK3CA, PTEN; \n1 ACC; \nincludes: ACC, retention cyst, lymphoepithelial cyst, epidermoid cyst, and squamoid cyst. MA: Molecular analysis; NA: Non-available data; ACC: Acinar cell cyst; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.\nQuality assessment and publication bias: Methodological quality of primary studies included was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23], which evaluates the quality of articles for systematic reviews of diagnostic accuracy studies in four domains, including patient selection, index test, reference standard, and flow and timing, for risk of bias and applicability concerns. Results are presented in Figure 2, which was sketched with templates available at www.quadas.org. The studies included in this review all showed a “low-risk” classification as the index tests (MA and MFB) and the reference standard (surgical pathology specimen) were reliable and mentioned in all studies. However, a “high-risk” of selection bias was demonstrated in patient selection (neither random nor sequential patients included in several studies) and in flow and timing because only a small proportion of the patients evaluated in all studies, except one, were included in the analysis. In fact, most patients were excluded in all studies as the inclusion criteria requiring surgical pathology as diagnostic reference were not met. Applicability concerns in patient selection were also significant in all studies, because the subgroup of PCNs referred for surgery is more often malignant than PCNs on surveillance, which would also be targeted with this review. Because of this bias, there may be an overestimation of both the sensitivity of the index tests, due to a more severe spectrum of PCNs that are referred for surgery, and the positive predictive value (PPV) for diagnosis of high-risk cysts, due to an increased prevalence of malignant cysts in a surgical cohort of PCNs.\nQuality assessment of the studies using QUADAS-2. A: Tabular presentation of risk bias for each study; B: Graphical display of bias.\nOur search revealed 16 study titles and abstracts for MFB and 264 titles for MA. In Figure 1A and B are described the selection process of the articles included in this study. After all steps, eight studies were considered suitable for qualitative and seven for quantitative analysis. We excluded 20 full-text articles after review, because they were case series of two patients[16] (n = 1), exploratory or pilot studies[28,29] (n = 2), no information of mutation status was available[30] (n = 1), pancreatic cystic fluid was obtained during surgery[31] (n = 1), insufficient or absent data of cysts with surgical pathology diagnoses[12,32,33] (n = 3), and mutations only of KRAS and/or GNAS[14,34-44] (n = 12).\nFlowchart with identification of eligible studies. A: Molecular analysis; B: Microforceps biopsy.\nOf the eight studies that met the inclusion criteria, design was retrospective in six and prospective in two, all were published from 2015 to 2018. These eight studies included a total of 1206 patients, of which 203 (17%) underwent surgical resection and a surgical pathology specimen was available as reference standard and included in the analysis. We excluded all patients with cytology and clinical follow-up data, but for whom a surgical pathology specimen was not available. The characteristics of the studies, surgical pathology diagnoses, and MA and MFB results are presented in Tables 1[32,45-47] and 2[18-21].\nCharacteristics of studies of molecular analysis included in the analysis\n113 samples of 105 patients; \nTP53, PIK3CA, PTEN; \n1 ACC; \nincludes: ACC, retention cyst, lymphoepithelial cyst, epidermoid cyst, and squamoid cyst. MA: Molecular analysis; NA: Non-available data; ACC: Acinar cell cyst; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.\nQuality assessment and publication bias: Methodological quality of primary studies included was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23], which evaluates the quality of articles for systematic reviews of diagnostic accuracy studies in four domains, including patient selection, index test, reference standard, and flow and timing, for risk of bias and applicability concerns. Results are presented in Figure 2, which was sketched with templates available at www.quadas.org. The studies included in this review all showed a “low-risk” classification as the index tests (MA and MFB) and the reference standard (surgical pathology specimen) were reliable and mentioned in all studies. However, a “high-risk” of selection bias was demonstrated in patient selection (neither random nor sequential patients included in several studies) and in flow and timing because only a small proportion of the patients evaluated in all studies, except one, were included in the analysis. In fact, most patients were excluded in all studies as the inclusion criteria requiring surgical pathology as diagnostic reference were not met. Applicability concerns in patient selection were also significant in all studies, because the subgroup of PCNs referred for surgery is more often malignant than PCNs on surveillance, which would also be targeted with this review. Because of this bias, there may be an overestimation of both the sensitivity of the index tests, due to a more severe spectrum of PCNs that are referred for surgery, and the positive predictive value (PPV) for diagnosis of high-risk cysts, due to an increased prevalence of malignant cysts in a surgical cohort of PCNs.\nQuality assessment of the studies using QUADAS-2. A: Tabular presentation of risk bias for each study; B: Graphical display of bias.\n Meta-analysis Molecular analysis: Four articles were included in the meta-analysis for diagnostic accuracy of MA. For each of the three definitions of relevant cyst, forest plots of sensitivity and specificity with heterogeneous denoted are shown in Figure 3.\nForest plots of the studies included for molecular analysis. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95% CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nThe three criteria to define “relevant cysts” resulted in a different range of the specificity and sensitivity of the studies included as shown in Figure 3. For diagnosis of the subgroup with high-risk and low-risk mucinous cysts that require intervention (either surgery or surveillance) comparing to benign cysts the pooled sensitivity was 0.75 (95%CI: 0.66-0.83) and the pooled specificity was 0.72 (95%CI: 0.56-0.85) for MA. In the subgroup of high-risk cysts that require surgery, comparing to other cysts requiring conservative management, the sensitivity was 0.57 (95%CI: 0.42-0.71) with a specificity of 0.88 (95%CI: 0.80-0.93). In the subgroup of low-risk mucinous cysts comparing to high-risk, the pooled sensitivity was 0.89 (95%CI: 0.79-0.95) and the pooled specificity was 0.88 (95%CI: 0.75-0.95).\nFigure 4 displays the sROC curves of MA, showing the sensitivity of the individual articles mapped on the vertical scale, 1-specificity on the horizontal scale, with the summary (sensitivity, 1-specificity) point marked, as well as the summary ROC curve and the confidence region for the summary (sensitivity, 1-specificity) points. The area under the sROC curve was 0.7706 (SE: 0.0927) in non-benign cysts, 0.9248 (SE: 0.0691) in high-risk cysts, and 0.9555 (SE: 0.0293) in mucinous low-risk cysts. The results of the studies had greater variation in non-benign cysts as shown by the wide confidence region.\nSummary receiver operating characteristics plots. ROC: Receiver operating characteristic curve; AUC: Area under the curve; SE: Standard error.\nIn the four studies, 566 patients had DNA available to perform MA in PCF. Pooled analysis (Figure 5) showed a diagnostic yield of 54.3% (95%CI: 49.8%-58.7%; I2 = 39.605%; test for heterogeneity P = 0.174).\nForest plots of molecular analysis and microforceps biopsies on the secondary outcomes of this meta-analysis.\nBy considering the classification of cysts by specific type (IPMNs, MCNs, cNETs, SCAs, pseudocysts, ACCs, and other RCs), MA identified correctly 73.1% of cysts (95%CI: 61.6%-82.2%; I2 = 37.381%; test for heterogeneity P = 0.203) (Figure 5).\nMicro forceps biopsy: Four articles were included in the meta-analysis for diagnostic accuracy of histology obtained using MFB. Figure 6 shows the forest plots of sensitivity and specificity for the three subgroups of relevant cysts. The forest plots for MFB show variable specificities within the papers, from 0 to 1, which can be due to the small numbers of patients with the target condition in some studies.\nForest plots of the included studies for microforceps biopsies. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95%CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nFor each of the three subgroups there exists a low heterogeneity in sensitivity (I2 = 0%, I2 = 21.4%, I2 = 0%) and specificity (I2 = 0%, I2 = 0%, I2 = 21.4%), therefore fixed effect models were used. As presented in Figure 6, in the first subgroup the pooled sensitivity was 0.73 (95%CI: 0.50-0.89) and the pooled specificity was 0.88 (95%CI: 0.28-1.00). In the second subgroup sensitivity was 0.81 (95%CI: 0.46-0.98) with a specificity of 0.77 (95%CI: 0.50-0.94) and in the last subgroup the pooled sensitivity was 0.64 (95%CI: 0.33-0.88) and the pooled specificity was 0.81 (95%CI: 0.46-0.98).\nThe results were plotted as a symmetrical sROC curve (Figure 4). The area under the sROC curve was 0.7640 (SE: 0.1261) in the first subgroup, 0.8154 (SE: 0.098) in the second subgroup, and 0.7509 (SE: 0.1277) in the last subgroup.\nBy pooling the data of the four studies that investigated the use of MFB to obtain a histopathologic diagnosis, we obtained a diagnostic yield of 73.1% (95%CI: 61.4%-82.2%; I2 = 47.774%; test for heterogeneity P = 0.125) (Figure 5).\nBy considering the outcome “specific cyst type” diagnosis, MFB correctly identified 70.7% of the cysts (95%CI: 49.4%-85.6%; I2 = 0%; test for heterogeneity P = 0.056) (Figure 5).\nMolecular analysis: Four articles were included in the meta-analysis for diagnostic accuracy of MA. For each of the three definitions of relevant cyst, forest plots of sensitivity and specificity with heterogeneous denoted are shown in Figure 3.\nForest plots of the studies included for molecular analysis. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95% CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nThe three criteria to define “relevant cysts” resulted in a different range of the specificity and sensitivity of the studies included as shown in Figure 3. For diagnosis of the subgroup with high-risk and low-risk mucinous cysts that require intervention (either surgery or surveillance) comparing to benign cysts the pooled sensitivity was 0.75 (95%CI: 0.66-0.83) and the pooled specificity was 0.72 (95%CI: 0.56-0.85) for MA. In the subgroup of high-risk cysts that require surgery, comparing to other cysts requiring conservative management, the sensitivity was 0.57 (95%CI: 0.42-0.71) with a specificity of 0.88 (95%CI: 0.80-0.93). In the subgroup of low-risk mucinous cysts comparing to high-risk, the pooled sensitivity was 0.89 (95%CI: 0.79-0.95) and the pooled specificity was 0.88 (95%CI: 0.75-0.95).\nFigure 4 displays the sROC curves of MA, showing the sensitivity of the individual articles mapped on the vertical scale, 1-specificity on the horizontal scale, with the summary (sensitivity, 1-specificity) point marked, as well as the summary ROC curve and the confidence region for the summary (sensitivity, 1-specificity) points. The area under the sROC curve was 0.7706 (SE: 0.0927) in non-benign cysts, 0.9248 (SE: 0.0691) in high-risk cysts, and 0.9555 (SE: 0.0293) in mucinous low-risk cysts. The results of the studies had greater variation in non-benign cysts as shown by the wide confidence region.\nSummary receiver operating characteristics plots. ROC: Receiver operating characteristic curve; AUC: Area under the curve; SE: Standard error.\nIn the four studies, 566 patients had DNA available to perform MA in PCF. Pooled analysis (Figure 5) showed a diagnostic yield of 54.3% (95%CI: 49.8%-58.7%; I2 = 39.605%; test for heterogeneity P = 0.174).\nForest plots of molecular analysis and microforceps biopsies on the secondary outcomes of this meta-analysis.\nBy considering the classification of cysts by specific type (IPMNs, MCNs, cNETs, SCAs, pseudocysts, ACCs, and other RCs), MA identified correctly 73.1% of cysts (95%CI: 61.6%-82.2%; I2 = 37.381%; test for heterogeneity P = 0.203) (Figure 5).\nMicro forceps biopsy: Four articles were included in the meta-analysis for diagnostic accuracy of histology obtained using MFB. Figure 6 shows the forest plots of sensitivity and specificity for the three subgroups of relevant cysts. The forest plots for MFB show variable specificities within the papers, from 0 to 1, which can be due to the small numbers of patients with the target condition in some studies.\nForest plots of the included studies for microforceps biopsies. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95%CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nFor each of the three subgroups there exists a low heterogeneity in sensitivity (I2 = 0%, I2 = 21.4%, I2 = 0%) and specificity (I2 = 0%, I2 = 0%, I2 = 21.4%), therefore fixed effect models were used. As presented in Figure 6, in the first subgroup the pooled sensitivity was 0.73 (95%CI: 0.50-0.89) and the pooled specificity was 0.88 (95%CI: 0.28-1.00). In the second subgroup sensitivity was 0.81 (95%CI: 0.46-0.98) with a specificity of 0.77 (95%CI: 0.50-0.94) and in the last subgroup the pooled sensitivity was 0.64 (95%CI: 0.33-0.88) and the pooled specificity was 0.81 (95%CI: 0.46-0.98).\nThe results were plotted as a symmetrical sROC curve (Figure 4). The area under the sROC curve was 0.7640 (SE: 0.1261) in the first subgroup, 0.8154 (SE: 0.098) in the second subgroup, and 0.7509 (SE: 0.1277) in the last subgroup.\nBy pooling the data of the four studies that investigated the use of MFB to obtain a histopathologic diagnosis, we obtained a diagnostic yield of 73.1% (95%CI: 61.4%-82.2%; I2 = 47.774%; test for heterogeneity P = 0.125) (Figure 5).\nBy considering the outcome “specific cyst type” diagnosis, MFB correctly identified 70.7% of the cysts (95%CI: 49.4%-85.6%; I2 = 0%; test for heterogeneity P = 0.056) (Figure 5).",
"Our search revealed 16 study titles and abstracts for MFB and 264 titles for MA. In Figure 1A and B are described the selection process of the articles included in this study. After all steps, eight studies were considered suitable for qualitative and seven for quantitative analysis. We excluded 20 full-text articles after review, because they were case series of two patients[16] (n = 1), exploratory or pilot studies[28,29] (n = 2), no information of mutation status was available[30] (n = 1), pancreatic cystic fluid was obtained during surgery[31] (n = 1), insufficient or absent data of cysts with surgical pathology diagnoses[12,32,33] (n = 3), and mutations only of KRAS and/or GNAS[14,34-44] (n = 12).\nFlowchart with identification of eligible studies. A: Molecular analysis; B: Microforceps biopsy.\nOf the eight studies that met the inclusion criteria, design was retrospective in six and prospective in two, all were published from 2015 to 2018. These eight studies included a total of 1206 patients, of which 203 (17%) underwent surgical resection and a surgical pathology specimen was available as reference standard and included in the analysis. We excluded all patients with cytology and clinical follow-up data, but for whom a surgical pathology specimen was not available. The characteristics of the studies, surgical pathology diagnoses, and MA and MFB results are presented in Tables 1[32,45-47] and 2[18-21].\nCharacteristics of studies of molecular analysis included in the analysis\n113 samples of 105 patients; \nTP53, PIK3CA, PTEN; \n1 ACC; \nincludes: ACC, retention cyst, lymphoepithelial cyst, epidermoid cyst, and squamoid cyst. MA: Molecular analysis; NA: Non-available data; ACC: Acinar cell cyst; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.\nQuality assessment and publication bias: Methodological quality of primary studies included was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23], which evaluates the quality of articles for systematic reviews of diagnostic accuracy studies in four domains, including patient selection, index test, reference standard, and flow and timing, for risk of bias and applicability concerns. Results are presented in Figure 2, which was sketched with templates available at www.quadas.org. The studies included in this review all showed a “low-risk” classification as the index tests (MA and MFB) and the reference standard (surgical pathology specimen) were reliable and mentioned in all studies. However, a “high-risk” of selection bias was demonstrated in patient selection (neither random nor sequential patients included in several studies) and in flow and timing because only a small proportion of the patients evaluated in all studies, except one, were included in the analysis. In fact, most patients were excluded in all studies as the inclusion criteria requiring surgical pathology as diagnostic reference were not met. Applicability concerns in patient selection were also significant in all studies, because the subgroup of PCNs referred for surgery is more often malignant than PCNs on surveillance, which would also be targeted with this review. Because of this bias, there may be an overestimation of both the sensitivity of the index tests, due to a more severe spectrum of PCNs that are referred for surgery, and the positive predictive value (PPV) for diagnosis of high-risk cysts, due to an increased prevalence of malignant cysts in a surgical cohort of PCNs.\nQuality assessment of the studies using QUADAS-2. A: Tabular presentation of risk bias for each study; B: Graphical display of bias.",
"Molecular analysis: Four articles were included in the meta-analysis for diagnostic accuracy of MA. For each of the three definitions of relevant cyst, forest plots of sensitivity and specificity with heterogeneous denoted are shown in Figure 3.\nForest plots of the studies included for molecular analysis. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95% CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nThe three criteria to define “relevant cysts” resulted in a different range of the specificity and sensitivity of the studies included as shown in Figure 3. For diagnosis of the subgroup with high-risk and low-risk mucinous cysts that require intervention (either surgery or surveillance) comparing to benign cysts the pooled sensitivity was 0.75 (95%CI: 0.66-0.83) and the pooled specificity was 0.72 (95%CI: 0.56-0.85) for MA. In the subgroup of high-risk cysts that require surgery, comparing to other cysts requiring conservative management, the sensitivity was 0.57 (95%CI: 0.42-0.71) with a specificity of 0.88 (95%CI: 0.80-0.93). In the subgroup of low-risk mucinous cysts comparing to high-risk, the pooled sensitivity was 0.89 (95%CI: 0.79-0.95) and the pooled specificity was 0.88 (95%CI: 0.75-0.95).\nFigure 4 displays the sROC curves of MA, showing the sensitivity of the individual articles mapped on the vertical scale, 1-specificity on the horizontal scale, with the summary (sensitivity, 1-specificity) point marked, as well as the summary ROC curve and the confidence region for the summary (sensitivity, 1-specificity) points. The area under the sROC curve was 0.7706 (SE: 0.0927) in non-benign cysts, 0.9248 (SE: 0.0691) in high-risk cysts, and 0.9555 (SE: 0.0293) in mucinous low-risk cysts. The results of the studies had greater variation in non-benign cysts as shown by the wide confidence region.\nSummary receiver operating characteristics plots. ROC: Receiver operating characteristic curve; AUC: Area under the curve; SE: Standard error.\nIn the four studies, 566 patients had DNA available to perform MA in PCF. Pooled analysis (Figure 5) showed a diagnostic yield of 54.3% (95%CI: 49.8%-58.7%; I2 = 39.605%; test for heterogeneity P = 0.174).\nForest plots of molecular analysis and microforceps biopsies on the secondary outcomes of this meta-analysis.\nBy considering the classification of cysts by specific type (IPMNs, MCNs, cNETs, SCAs, pseudocysts, ACCs, and other RCs), MA identified correctly 73.1% of cysts (95%CI: 61.6%-82.2%; I2 = 37.381%; test for heterogeneity P = 0.203) (Figure 5).\nMicro forceps biopsy: Four articles were included in the meta-analysis for diagnostic accuracy of histology obtained using MFB. Figure 6 shows the forest plots of sensitivity and specificity for the three subgroups of relevant cysts. The forest plots for MFB show variable specificities within the papers, from 0 to 1, which can be due to the small numbers of patients with the target condition in some studies.\nForest plots of the included studies for microforceps biopsies. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95%CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.\nFor each of the three subgroups there exists a low heterogeneity in sensitivity (I2 = 0%, I2 = 21.4%, I2 = 0%) and specificity (I2 = 0%, I2 = 0%, I2 = 21.4%), therefore fixed effect models were used. As presented in Figure 6, in the first subgroup the pooled sensitivity was 0.73 (95%CI: 0.50-0.89) and the pooled specificity was 0.88 (95%CI: 0.28-1.00). In the second subgroup sensitivity was 0.81 (95%CI: 0.46-0.98) with a specificity of 0.77 (95%CI: 0.50-0.94) and in the last subgroup the pooled sensitivity was 0.64 (95%CI: 0.33-0.88) and the pooled specificity was 0.81 (95%CI: 0.46-0.98).\nThe results were plotted as a symmetrical sROC curve (Figure 4). The area under the sROC curve was 0.7640 (SE: 0.1261) in the first subgroup, 0.8154 (SE: 0.098) in the second subgroup, and 0.7509 (SE: 0.1277) in the last subgroup.\nBy pooling the data of the four studies that investigated the use of MFB to obtain a histopathologic diagnosis, we obtained a diagnostic yield of 73.1% (95%CI: 61.4%-82.2%; I2 = 47.774%; test for heterogeneity P = 0.125) (Figure 5).\nBy considering the outcome “specific cyst type” diagnosis, MFB correctly identified 70.7% of the cysts (95%CI: 49.4%-85.6%; I2 = 0%; test for heterogeneity P = 0.056) (Figure 5).",
"In this meta-analysis we analyzed two different but promising tests to diagnose PCNs – molecular analysis and microforceps biopsy. To our knowledge this is the first study of this nature, and it included 1206 patients with PCNs of which 1058 underwent MA and 148 MFB. All patients had the index tests performed in PCF obtained pre-operatively, exclusively with NGS for MA and the Moray micro forceps biopsy device (US Endoscopy, Mentor, Ohio) used for MFB. We analyzed 203 cysts, 178 evaluated with MA and 25 with MFB, all referred for surgery, and with a surgical pathology specimen used as reference standard for diagnosis.\nIn this comparative analysis we included all studies, without restriction to si-multaneous evaluation of both tests, because only one of such studies has been published[20]. This study, which includes 48 patients but only 10 surgical pathology specimens, showed identical results for MA and MFB in low-risk and high-risk cyst diagnosis, but higher specific cyst type diagnosis for MFB.\nThe data from the seven studies included in the meta-analysis, although with limited number of patients, particularly for MFB, suggests that MA is more accurate than MFB for diagnosis of PCNs, including high-risk and low-risk lesions. MA has superior accuracy to discriminate high-risk cysts from other PCNs and low-risk from high-risk neoplastic cysts. MA performance was considered excellent with AUC values of 0.92 and of 0.96 for high-risk and low-risk neoplastic lesions, respectively, as compared to MFB, which showed a fair or good performance, with an AUC of 0.81 and 0.75, respectively for the same lesions (Figure 4). The specificity of MA is good (0.88) but it has a low sensitivity (only 0.57) for high-risk cysts. This may be explained by technical issues, by low prevalence of relevant genetic mutations in malignant PCNs, or by mutations not included in the current NGS panels. The sensitivity and specificity are high (0.89 and 0.88, respectively) for MA when comparing low-risk to high-risk cysts, which reflects the genetic nature of pancreatic carcinogenesis with cumulative mutations from benign to malignant cysts[48].\nFor discriminating benign cysts from both low-risk and high-risk cysts, the performance of MA and MFB was identical and fair according to AUC values of 0.77 and 0.76, respectively. This non-superiority of MA in the diagnosis of benign cysts in this meta-analysis may be due to technique-inherent issues and/or under-representation of benign cysts in surgical series. In fact, “no genetic mutation” is considered a false negative result in most benign rare cysts, but some of these lesions (retention cysts, etc.) have no diagnostic genetic mutations. On the contrary, the most frequent benign cysts, SCAs, harbor a VHL mutation, exclusively present in these benign lesions and allowing for discarding a malignant lesion. In the MA studies, one third of rare benign cysts were classified as false negative results, due to absence of characteristic mutations (Table 1). Another example of PCN that is not amenable to a MA diagnosis with current genetic panels is cNET, also reducing the accuracy of MA for diagnosis of high-risk cysts. The sensitivities were identical for MA and MFB (0.75 and 0.72), but the latter had higher specificity (0.73 and 0.88, respectively). Limited tissue sampling with MFB can explain the reduced sensitivity with robust specificity. As MA depends on denuded DNA in suspension in PCF, no sampling error is expected, which may explain its greater accuracy in neoplastic cysts, comparing to MFB.\nConcerning secondary outcomes, even with the limitations of tissue sampling inherent to MFB, this meta-analysis showed that the diagnostic yield of MFB was superior to MA with rates of correctly identified cyst identical with MA and MFB (Tables 1 and 2). In fact, the definition of diagnostic yield, which for MA was “detection of genetic mutations”, may have led to a falsely low value due to the presence of some rare types of benign cysts (retention cysts, lymphoepithelial cysts, epidermoid cysts, squamous cysts in two studies[46,47]) that have no characteristic diagnostic genetic mutations.\nCharacteristics of studies of microforceps biopsies included in the analysis\n1 nondiagnostic on MFB; \n2 nondiagnostic on MFB; \n1 false negative IPMN associated invasive carcinoma; \n2 cases with a suspicion of pseudocyst. MFB: Microforceps biopsy; NA: Non-available data; ND: Nondiagnostic; ACC: Acinar cystadenoma; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.\nIn clinical practice, patient symptoms, cyst imaging features, CEA, and cytology of PCF are required for diagnosis and decision for either treatment or surveillance according to cyst types[49]. PCF analysis, including CEA to distinguish mucinous from non-mucinous cysts and cytology to select those that harbor HGA or early pancreatic carcinoma and require surgical treatment, have suboptimal accuracies[3], due to scant cellularity and limited PCF volume. In this context, additional diagnostic tests are necessary to improve cyst classification and refine clinical decision. DNA markers require limited amounts of PCF, increasing the diagnostic yield[32,45,50,51], but with considerable technical complexity and costs. In fact, in routine clinical practice a major pitfall for PCNs diagnosis is the limited volume of PCF obtained, precluding routine pre-operative testing. As DNA analysis requires less volume of PCF, it may become an alternative test in these circumstances. This major advantage of molecular analysis was not possible to evaluate in this meta-analysis, because the volume of cystic fluid obtained in pancreatic cysts was not available in most studies analyzed.\nAs MA continues to evolve, questions remain about its accuracy, how it influences patient management, and in what order the analysis should be performed to better support clinical decisions. Previous studies[49] have shown that DNA testing combined with clinical features increased PCNs diagnosis compared to either alone. With multiple recent advances in biomarkers, molecular genetics will probably prove to be useful in the management of PCNs[52]. In a previous meta-analysis, pre-operative cytology of PCNs has shown low sensitivity for diagnosis[53], endorsing additional tests to improve diagnosis. Another meta-analysis of diagnostic accuracy of EUS-FNA with CEA and cytology analysis in differentiating mucinous cysts has demonstrated to be accurate to confirm the diagnosis but performed poorly in excluding it[54]. The role of KRAS as individual screening test has been analyzed before[55] with poor accuracy and added benefit coming from a combined approach with cytology. A recently published meta-analysis supporting KRAS, GNAS, and RNF43 mutations as diagnostic markers of IPMNs[56] used different methods for mutation detection, different tumor materials, and clinicopathologic data as reference standard for diagnosis, which may limit its clinical application in evaluation of PCNs with mutational analysis performed only in PCF.\nIn this scenario, new markers are needed for PCNs stratification, and in our meta-analysis both MA and MFB have acceptable diagnostic accuracies. The two largest studies of MA[46,47] showed higher accuracy for diagnosis, which underscores the role of technical aspects of PCF collection, storage, and laboratory analysis for improved accuracy with this technique.\nOn the other hand, MFB provides tissue fragments for routine histological evaluation, without additional PCF required other than for standard analysis. The technical feasibility of through-the-needle microforceps biopsies revealed to be excellent, even in cysts located in the pancreatic head, despite the required 19-gauge caliber of the EUS-FNA needle. Another potential advantage of MFB is to allow the diagnosis of histologic subtypes of IPMNs, which can potentially be used for risk stratification[57], but still requires further validation.\n Strengths and limitations We applied strict exclusion criteria, with all analyzed patients having a surgical pathology specimen as the reference standard for diagnosis, because histopathology is the gold standard for diagnosis of neoplasia. Another major strength of this meta-analysis is having identical lesions (size and location) analyzed in both groups. These important strengths provide a more realistic accuracy estimate of the tests evaluated. In previous studies of cytology including both surgical pathology and clinical follow-up[54] as reference standard, pooled sensitivities were 12% higher than in studies with exclusive surgical pathology[55] as reference standard in the diagnosis of mucinous cysts, with test accuracy overestimation. Finally, the pooled results have low heterogeneity.\nThe quality of a systematic review depends on the quality of studies included, and our quality assessment of patient selection regarding the risk of bias and applicability was high. As sensitivity and specificity are sensitive to study design and influenced by the spectrum of disease, sample collection, and processing, there may be a risk of bias and the results, although correct, their interpretation may be inaccurate. Moreover, there was incomplete reporting in one primary study, having no separate information on specific cyst type, mucinous or malignant cyst diagnosis[32], and the study was excluded from quantitative analysis. Although one study was excluded from the meta-analysis, MA with three studies included more patients (953, of whom only 153 in the surgical cohort) than the group of MFB with four studies but fewer patients (148, with only 25 in the surgical cohort). This can represent a surgical selection bias for both tests studied. Moreover, MFB studies were all retrospective, with small sample size, without pathology diagnosis for most benign and pre-malignant cysts, and non-consecutive patients that were selected on endoscopist discretion, which may have led to bias. Another limitation is the time between the index tests and the reference standard, because the final diagnosis could have been made at different time intervals from the tests. If the time between index tests and reference standard is too long, the true disease status of the patient may have changed by the time the reference standard was assessed. Aditionally, the different number of malignant cysts per study, particularly in the MA group, may have led to part of the heterogeneity in sensitivity and specificity. Finally, as MA does not increase the risks of standard EUS-FNA (the analysis is performed in remnant cystic fluid after standard diagnosis) we did not perform a safety analysis of MFB, but the four studies analyzed described only rare non-severe adverse events.\nWe applied strict exclusion criteria, with all analyzed patients having a surgical pathology specimen as the reference standard for diagnosis, because histopathology is the gold standard for diagnosis of neoplasia. Another major strength of this meta-analysis is having identical lesions (size and location) analyzed in both groups. These important strengths provide a more realistic accuracy estimate of the tests evaluated. In previous studies of cytology including both surgical pathology and clinical follow-up[54] as reference standard, pooled sensitivities were 12% higher than in studies with exclusive surgical pathology[55] as reference standard in the diagnosis of mucinous cysts, with test accuracy overestimation. Finally, the pooled results have low heterogeneity.\nThe quality of a systematic review depends on the quality of studies included, and our quality assessment of patient selection regarding the risk of bias and applicability was high. As sensitivity and specificity are sensitive to study design and influenced by the spectrum of disease, sample collection, and processing, there may be a risk of bias and the results, although correct, their interpretation may be inaccurate. Moreover, there was incomplete reporting in one primary study, having no separate information on specific cyst type, mucinous or malignant cyst diagnosis[32], and the study was excluded from quantitative analysis. Although one study was excluded from the meta-analysis, MA with three studies included more patients (953, of whom only 153 in the surgical cohort) than the group of MFB with four studies but fewer patients (148, with only 25 in the surgical cohort). This can represent a surgical selection bias for both tests studied. Moreover, MFB studies were all retrospective, with small sample size, without pathology diagnosis for most benign and pre-malignant cysts, and non-consecutive patients that were selected on endoscopist discretion, which may have led to bias. Another limitation is the time between the index tests and the reference standard, because the final diagnosis could have been made at different time intervals from the tests. If the time between index tests and reference standard is too long, the true disease status of the patient may have changed by the time the reference standard was assessed. Aditionally, the different number of malignant cysts per study, particularly in the MA group, may have led to part of the heterogeneity in sensitivity and specificity. Finally, as MA does not increase the risks of standard EUS-FNA (the analysis is performed in remnant cystic fluid after standard diagnosis) we did not perform a safety analysis of MFB, but the four studies analyzed described only rare non-severe adverse events.\n Future perspectives With the increasing diagnosis of asymptomatic PCNs, most with potential for malignancy, there is a growing need to find accurate and affordable tests for diagnosis. The goal of management of patients with pancreatic cysts is to detect and resect cysts before progression of malignancy, while avoiding unnecessary follow-up procedures in benign cysts and surgery in low-risk PCNs.\nBiomarkers of malignancy are promising, but clinicians should be aware of their current diagnostic performance limitations and type of lesions identified. In addition to significant costs, logistic difficulties in preserving material for future molecular analysis in busy general hospitals, and the technical complexity of the test, the generalized use of MA seems difficult in clinical practice. On the other hand, if MFB proves in larger studies to be safe and to allow tissue acquisition and gives the histological criteria needed for a correct diagnosis of PCNs, it may be immediately implemented in clinics, because the endoscopic procedure is standard, and histology is already a widespread procedure in clinics. MFB may be especially useful for benign lesions, for which both surgery and surveillance are unnecessary, representing a considerable burden in pancreas clinics due to current diagnostic limitations[58].\nFor MA to become relevant in routine clinical care in the future, its role in early cancer diagnosis and its prognostic value in PCNs requiring periodic surveillance must be confirmed. Also, for successful massive implementation, it is required to develop as an universal, highly accurate, first line test with clinical impact in cyst diagnosis, prognosis, and patient management. MA, both in PCF and peripheral blood, for standard analysis of multiple simultaneous biomarkers, allowing non-invasive diagnosis and risk stratification of these lesions[59] would be valuable. For the present time, MA and MFB can only be recommended as complementary or as second line tests in case CEA and cytology of PCF are non-diagnostic. For both tests, large multicenter validation studies are still missing.\nWith the increasing diagnosis of asymptomatic PCNs, most with potential for malignancy, there is a growing need to find accurate and affordable tests for diagnosis. The goal of management of patients with pancreatic cysts is to detect and resect cysts before progression of malignancy, while avoiding unnecessary follow-up procedures in benign cysts and surgery in low-risk PCNs.\nBiomarkers of malignancy are promising, but clinicians should be aware of their current diagnostic performance limitations and type of lesions identified. In addition to significant costs, logistic difficulties in preserving material for future molecular analysis in busy general hospitals, and the technical complexity of the test, the generalized use of MA seems difficult in clinical practice. On the other hand, if MFB proves in larger studies to be safe and to allow tissue acquisition and gives the histological criteria needed for a correct diagnosis of PCNs, it may be immediately implemented in clinics, because the endoscopic procedure is standard, and histology is already a widespread procedure in clinics. MFB may be especially useful for benign lesions, for which both surgery and surveillance are unnecessary, representing a considerable burden in pancreas clinics due to current diagnostic limitations[58].\nFor MA to become relevant in routine clinical care in the future, its role in early cancer diagnosis and its prognostic value in PCNs requiring periodic surveillance must be confirmed. Also, for successful massive implementation, it is required to develop as an universal, highly accurate, first line test with clinical impact in cyst diagnosis, prognosis, and patient management. MA, both in PCF and peripheral blood, for standard analysis of multiple simultaneous biomarkers, allowing non-invasive diagnosis and risk stratification of these lesions[59] would be valuable. For the present time, MA and MFB can only be recommended as complementary or as second line tests in case CEA and cytology of PCF are non-diagnostic. For both tests, large multicenter validation studies are still missing.",
"We applied strict exclusion criteria, with all analyzed patients having a surgical pathology specimen as the reference standard for diagnosis, because histopathology is the gold standard for diagnosis of neoplasia. Another major strength of this meta-analysis is having identical lesions (size and location) analyzed in both groups. These important strengths provide a more realistic accuracy estimate of the tests evaluated. In previous studies of cytology including both surgical pathology and clinical follow-up[54] as reference standard, pooled sensitivities were 12% higher than in studies with exclusive surgical pathology[55] as reference standard in the diagnosis of mucinous cysts, with test accuracy overestimation. Finally, the pooled results have low heterogeneity.\nThe quality of a systematic review depends on the quality of studies included, and our quality assessment of patient selection regarding the risk of bias and applicability was high. As sensitivity and specificity are sensitive to study design and influenced by the spectrum of disease, sample collection, and processing, there may be a risk of bias and the results, although correct, their interpretation may be inaccurate. Moreover, there was incomplete reporting in one primary study, having no separate information on specific cyst type, mucinous or malignant cyst diagnosis[32], and the study was excluded from quantitative analysis. Although one study was excluded from the meta-analysis, MA with three studies included more patients (953, of whom only 153 in the surgical cohort) than the group of MFB with four studies but fewer patients (148, with only 25 in the surgical cohort). This can represent a surgical selection bias for both tests studied. Moreover, MFB studies were all retrospective, with small sample size, without pathology diagnosis for most benign and pre-malignant cysts, and non-consecutive patients that were selected on endoscopist discretion, which may have led to bias. Another limitation is the time between the index tests and the reference standard, because the final diagnosis could have been made at different time intervals from the tests. If the time between index tests and reference standard is too long, the true disease status of the patient may have changed by the time the reference standard was assessed. Aditionally, the different number of malignant cysts per study, particularly in the MA group, may have led to part of the heterogeneity in sensitivity and specificity. Finally, as MA does not increase the risks of standard EUS-FNA (the analysis is performed in remnant cystic fluid after standard diagnosis) we did not perform a safety analysis of MFB, but the four studies analyzed described only rare non-severe adverse events.",
"With the increasing diagnosis of asymptomatic PCNs, most with potential for malignancy, there is a growing need to find accurate and affordable tests for diagnosis. The goal of management of patients with pancreatic cysts is to detect and resect cysts before progression of malignancy, while avoiding unnecessary follow-up procedures in benign cysts and surgery in low-risk PCNs.\nBiomarkers of malignancy are promising, but clinicians should be aware of their current diagnostic performance limitations and type of lesions identified. In addition to significant costs, logistic difficulties in preserving material for future molecular analysis in busy general hospitals, and the technical complexity of the test, the generalized use of MA seems difficult in clinical practice. On the other hand, if MFB proves in larger studies to be safe and to allow tissue acquisition and gives the histological criteria needed for a correct diagnosis of PCNs, it may be immediately implemented in clinics, because the endoscopic procedure is standard, and histology is already a widespread procedure in clinics. MFB may be especially useful for benign lesions, for which both surgery and surveillance are unnecessary, representing a considerable burden in pancreas clinics due to current diagnostic limitations[58].\nFor MA to become relevant in routine clinical care in the future, its role in early cancer diagnosis and its prognostic value in PCNs requiring periodic surveillance must be confirmed. Also, for successful massive implementation, it is required to develop as an universal, highly accurate, first line test with clinical impact in cyst diagnosis, prognosis, and patient management. MA, both in PCF and peripheral blood, for standard analysis of multiple simultaneous biomarkers, allowing non-invasive diagnosis and risk stratification of these lesions[59] would be valuable. For the present time, MA and MFB can only be recommended as complementary or as second line tests in case CEA and cytology of PCF are non-diagnostic. For both tests, large multicenter validation studies are still missing.",
"Our study confirms the diagnostic value of both MA and MFB, with higher diagnostic accuracy of MA than MFB for both low-risk and high-risk mucinous cysts. Genetic analysis should not be replaced by MFB in this context. Clinicians should be aware of the higher accuracy of MA for the diagnosis of malignant and high-risk cysts."
] |
[
null,
"methods",
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Pancreatic cysts",
"Endoscopic ultrasound",
"Endoscopic ultrasound with fine-needle aspiration",
"Genetic testing",
"Microforceps biopsy",
"Molecular analysis",
"KRAS",
"Carcinoembryonic antigen",
"Cytology"
] |
INTRODUCTION:
Pancreatic cystic neoplasms (PCNs) are on the rise in clinics due to an ageing population and the increase in routine use of high-quality abdominal imaging[1]. PCNs are generally classified into two main groups: mucinous cystic neoplasms (MCNs) and non-mucinous cystic neoplasms (NMCN). MCNs include intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystadenomas, which are precursor lesions of pancreatic carcinoma, and may be low-risk (pre-malignant with low or intermediate-grade atypia) or high-risk: pre-malignant with high-grade atypia (HGA) or malignant, including adenocarcinomas secondarily cystic. NMCNs include serous cystadenomas and inflammatory cysts (pseudocysts), mostly benign cysts, but may include some rare lesions, considered high-risk as cystic neuroendocrine tumors (cNETs), and acinar cell cystadenomas (ACCs). The heterogeneity in malignant potential, increased frequency, and significant morbidity and mortality of surgical treatment, makes pre-operative diagnosis of PCNs essential for management. The treatment options for PCNs encompass surgery or conservative surveillance for MCNs, according to malignancy risk, or no further evaluation for most NMCNs.
The differentiation between MCNs and NMCNs is critical, because a misdiagnosis of a MCN can lead to a missed opportunity to treat pancreatic cancer in an early stage and a misdiagnosis of NMCN can result in unnecessary surgery or surveillance with associated morbidity, costs, and negative impact on quality of life.
Currently, morphologic characterization of PCNs and pancreatic cystic fluid (PCF) analysis for carcinoembryonic Antigen (CEA) and cytology are central in diagnosis. A CEA level ≥ 192 ng/mL is the most accurate diagnostic test for MCNs and cytology is highly specific for malignancy[2], but with suboptimal results in large studies with surgical pathology as the gold standard[3]. In fact, a significant part of these lesions remains indeterminate and incorrect pre-operative diagnosis occurs in one third of patients[4,5], making new reliable diagnostic tools urgently needed.
In the last decade numerous studies have shown that genetic analysis of aspirates obtained by EUS-FNA provided a better characterization of PCNs than CEA and cytology[6-14]. Next-generation sequencing (NGS) is a very sensitive technique for detection of genetic mutations that allows the rapid detection of mutations in pre-defined panels of cancer genes, even in samples with limited DNA content, such as PCF. NGS requires storage, infrastructure, data processing, and expert personnel. Moreover, to be cost-effective, large numbers of samples need to be processed, making it applicable only in large centralized laboratories. These reasons make the implementation of NGS in clinical practice still a matter of debate.
The clinical need of better diagnostic tests in PCNs has recently led to the development of a through-the-needle miniature biopsy device for use during EUS-FNA[15,16]. The Moray micro forceps biopsy (MFB) device (US Endoscopy, Mentor, Ohio) is disposable and can pass through a standard 19-gauge EUS-FNA needle that is already used routinely. It allows tissue sampling from the cyst wall, septa or mural nodules and the obtention of a histological evaluation of the epithelial architecture and subepithelial stroma[17]. Adding to the high technical success and excellent safety profile[18,19], the new device has shown to improve the diagnostic accuracy of specific cyst subtypes[20,21]. Another major advantage of MFB is the simultaneous tissue sampling and PCF acquisition, with just an additional histologic analysis that follows standard definitions and is already routine in clinics.
The aim of this systematic review and meta-analysis is to evaluate the diagnostic performance of molecular analysis (MA) and MFB and find the most robust additional diagnostic technique in PCNs, in the pre-operative setting.
MATERIALS AND METHODS:
This systematic review and meta-analysis is conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis of Diagnostic Test Accuracy Studies, the PRISMA-DTA Statement[22], and the protocol is registered at PROSPERO (CRD42018111910).
Literature search and study selection A comprehensive search of databases, including Medline, Scopus, and Web of Science, for the past 8 years (January 1st, 2010 to July 31st, 2018) and restricted to human studies was performed. No language restrictions were applied. The following search terms were used in two independent searches: “pancreas”, “cyst”, “molecular”, “analysis”; and “micro”, “forceps”, “microforceps”, “biopsy”. A search of related articles was performed, adding additional studies. Duplicate articles, reviews, trials including other kinds of neoplasms, and trials with molecular markers not compliant with the defined inclusion criteria were removed. The references of all selected studies were hand-searched for additional articles.
Inclusion criteria: Published studies were included in the meta-analysis if they analyzed: (1) Patients with symptomatic or incidental pancreatic cysts with a definitive surgical pathology diagnosis; (2) Genetic mutations performed with high sensitive techniques, such as NGS in PCF obtained by EUS-FNA prior to surgery; (3) At least four genetic mutations, including KRAS, GNAS, VHL, and at least another genetic mutation representative of aggressive neoplasms (PIK3CA, TP53, SMAD4, PTEN, CDKN2A); (4) PCNs evaluated by EUS-FNA with MFB for diagnosis; and (5) Surgical pathology specimens with available data.
Exclusion criteria: (1) Studies of MA with fewer than the four genetic mutations previously defined; (2) Studies involving solid pancreatic lesions; (3) Studies using PCF not obtained by EUS-FNA; (4) Reviews, case reports, case series with fewer than five patients, letters to editor, exploratory studies, and papers published only in abstract form; (5) Studies with cytology and clinical surveillance as standard of diagnosis. Two authors (SF and AL) independently judged study eligibility and disagreements were resolved by consensus.
Histological criteria: We classified the PCNs of the included studies into three main groups: (1) High-risk cysts (adenocarcinoma or high grade dysplasia in IPMNs and MCNs, secondarily cystic adenocarcinomas, cNETs, and ACCs; (2) Low-risk mucinous cysts (IPMNs and MCNs with intermediate or low-grade dysplasia); and (3) Benign cysts (SCAs, pseudocysts, and other rare cysts (RCs) included in some articles, as retention cysts, lymphoepithelial cysts, epidermoid cysts, squamoid cysts).
Tests under investigation: The index tests were: (1) MA of PCF; and (2) MFB of PCNs, including cyst wall, septs, and nodules. A diagnosis of cNET or ACC does not warrant a malignancy diagnosis, but surgery is recommended in surgically fit patients. Due to a recommendation of identical treatment to malignant and mucinous high-risk cysts, for the purpose of analysis in this study, each one of these diagnoses was classified as a high-risk cyst.
A comprehensive search of databases, including Medline, Scopus, and Web of Science, for the past 8 years (January 1st, 2010 to July 31st, 2018) and restricted to human studies was performed. No language restrictions were applied. The following search terms were used in two independent searches: “pancreas”, “cyst”, “molecular”, “analysis”; and “micro”, “forceps”, “microforceps”, “biopsy”. A search of related articles was performed, adding additional studies. Duplicate articles, reviews, trials including other kinds of neoplasms, and trials with molecular markers not compliant with the defined inclusion criteria were removed. The references of all selected studies were hand-searched for additional articles.
Inclusion criteria: Published studies were included in the meta-analysis if they analyzed: (1) Patients with symptomatic or incidental pancreatic cysts with a definitive surgical pathology diagnosis; (2) Genetic mutations performed with high sensitive techniques, such as NGS in PCF obtained by EUS-FNA prior to surgery; (3) At least four genetic mutations, including KRAS, GNAS, VHL, and at least another genetic mutation representative of aggressive neoplasms (PIK3CA, TP53, SMAD4, PTEN, CDKN2A); (4) PCNs evaluated by EUS-FNA with MFB for diagnosis; and (5) Surgical pathology specimens with available data.
Exclusion criteria: (1) Studies of MA with fewer than the four genetic mutations previously defined; (2) Studies involving solid pancreatic lesions; (3) Studies using PCF not obtained by EUS-FNA; (4) Reviews, case reports, case series with fewer than five patients, letters to editor, exploratory studies, and papers published only in abstract form; (5) Studies with cytology and clinical surveillance as standard of diagnosis. Two authors (SF and AL) independently judged study eligibility and disagreements were resolved by consensus.
Histological criteria: We classified the PCNs of the included studies into three main groups: (1) High-risk cysts (adenocarcinoma or high grade dysplasia in IPMNs and MCNs, secondarily cystic adenocarcinomas, cNETs, and ACCs; (2) Low-risk mucinous cysts (IPMNs and MCNs with intermediate or low-grade dysplasia); and (3) Benign cysts (SCAs, pseudocysts, and other rare cysts (RCs) included in some articles, as retention cysts, lymphoepithelial cysts, epidermoid cysts, squamoid cysts).
Tests under investigation: The index tests were: (1) MA of PCF; and (2) MFB of PCNs, including cyst wall, septs, and nodules. A diagnosis of cNET or ACC does not warrant a malignancy diagnosis, but surgery is recommended in surgically fit patients. Due to a recommendation of identical treatment to malignant and mucinous high-risk cysts, for the purpose of analysis in this study, each one of these diagnoses was classified as a high-risk cyst.
Data extraction After study selection, two authors (SF and AL) extracted and registered the data from each study onto a standardized worksheet. Disagreements were discussed and reviewed by a third author (LP). The data retrieved were: first author, publication year, study period and design (prospective or retrospective), reference for diagnosis, sample size (all patients included in the study), technical success, adverse events, diagnostic yield, surgical cohort (number of patients with a surgical pathology specimen), cyst size, cyst location, specific cyst types, number of high-risk cysts, mucinous low-risk and benign cysts diagnosed by MA and MFB comparing to surgical pathology specimens. In the MFB studies, technical success was defined as the ability to puncture the cysts and perform the biopsies; and the diagnostic yield was defined as the ratio between the number of patients included in the study and the patients in whom enough material allowed the acquisition of a histopathologic diagnosis. In the MA group, diagnostic yield was defined as a ratio between the number of patients included in the study and the number of patients with DNA available to perform molecular analysis in PCF.
After study selection, two authors (SF and AL) extracted and registered the data from each study onto a standardized worksheet. Disagreements were discussed and reviewed by a third author (LP). The data retrieved were: first author, publication year, study period and design (prospective or retrospective), reference for diagnosis, sample size (all patients included in the study), technical success, adverse events, diagnostic yield, surgical cohort (number of patients with a surgical pathology specimen), cyst size, cyst location, specific cyst types, number of high-risk cysts, mucinous low-risk and benign cysts diagnosed by MA and MFB comparing to surgical pathology specimens. In the MFB studies, technical success was defined as the ability to puncture the cysts and perform the biopsies; and the diagnostic yield was defined as the ratio between the number of patients included in the study and the patients in whom enough material allowed the acquisition of a histopathologic diagnosis. In the MA group, diagnostic yield was defined as a ratio between the number of patients included in the study and the number of patients with DNA available to perform molecular analysis in PCF.
Outcomes The primary outcomes of this study were the data to obtain the accuracies of MA and MFB for the diagnosis of PCNs, including high-risk cysts, mucinous low-risk cysts, and benign cysts. Secondary outcomes were the diagnostic yield of genetic testing and MFB and the number of cysts correctly identified for each of the tests studied.
The primary outcomes of this study were the data to obtain the accuracies of MA and MFB for the diagnosis of PCNs, including high-risk cysts, mucinous low-risk cysts, and benign cysts. Secondary outcomes were the diagnostic yield of genetic testing and MFB and the number of cysts correctly identified for each of the tests studied.
Quality analysis Methodological quality of included primary studies was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23]. The PRISMA-DTA Statement recommendations were used for reporting this systematic review[22,24].
Methodological quality of included primary studies was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23]. The PRISMA-DTA Statement recommendations were used for reporting this systematic review[22,24].
Statistical analysis and data synthesis The reference standard was a surgical pathology specimen that allowed the classification of PCNs into three defined groups of diagnosis: high-risk cysts, mucinous low-risk cysts, and benign cysts. This resulted in a two-by-three table with correct and incorrect test results in each of the three referenced groups, for each of the tests analyzed, MA and histology were obtained by MFB.
To calculate tests’ accuracy and to reflect on the categories that are useful in clinical practice and that guide management, we constructed two-by-two tables, considering three definitions of “relevant” cysts: (1) High-risk cysts – proven malignant cysts, IPMNs, and MCNs with HGA, cNETs, ACCs; Non-High-risk cysts – all cysts except those proven to be high-risk. (2) Low-risk mucinous cysts – proven mucinous low-risk cysts; High-risk cysts – all except those proven to be mucinous low-risk or benign. And (3) Non-benign cysts – all cysts except those proven to be benign; Benign cysts – proven benign cysts.
The ability of the tests to discriminate “relevant” and “non-relevant” cysts using the three definitions of “relevant cysts” was evaluated and the accuracy of the two tests was compared.
The data of the two-by-two tables were used to calculate sensitivity and specificity for each study. We present individual study results graphically by plotting the estimates of sensitivity and specificity (and their 95% confidence intervals (CI)) in both forest plots and on the summary receiver operating characteristic (sROC) curve plots. The area under the curve (AUC) is equal to 1 for a perfect test and 0.5 for a completely uninformative test. The AUC is equal to the probability that if a pair of relevant and non-relevant cysts is selected at random, the relevant cyst will have a higher test result than the non-relevant cyst. Pooled estimates of the sensitivity and specificity were obtained by the DerSimonian-Laird method (random effect model) to incorporate variation among studies, when data are heterogeneous. Otherwise, we used the Mantel-Haenszel method (fixed effect model).
Heterogeneity was investigated in the first instance through visual examination of forest plots of sensitivities and specificities and through visual examination of the ROC plot of the raw data. Last, we used statistical tests, including chi-square and Cochran-Q to evaluate if the differences across the studies were greater than expected by chance alone. A low P value suggests presence of heterogeneity. In addition to these statistics we used the statistic I2 of Higgins, which has been proposed as a measure to quantify the amount of heterogeneity[25,26]. The scale of I2 has a range of 0 to 100% and values on the order of 25%, 50% and 75% are considered low, moderate, and high heterogeneity, respectively.
Another goal of this work was to obtain, for each of the tests, the correctly identified cyst rate and the diagnostic yield in predicting a histopathologic diagnosis.
We used Comprehensive Meta-Analysis software (Version 2.0) for assessment of diagnostic yield of the tests and Meta-DiSc (version 1.4 – Meta-Analysis of Diagnostic and screening tests[27] ) to obtain the accuracy of each of the tests.
The reference standard was a surgical pathology specimen that allowed the classification of PCNs into three defined groups of diagnosis: high-risk cysts, mucinous low-risk cysts, and benign cysts. This resulted in a two-by-three table with correct and incorrect test results in each of the three referenced groups, for each of the tests analyzed, MA and histology were obtained by MFB.
To calculate tests’ accuracy and to reflect on the categories that are useful in clinical practice and that guide management, we constructed two-by-two tables, considering three definitions of “relevant” cysts: (1) High-risk cysts – proven malignant cysts, IPMNs, and MCNs with HGA, cNETs, ACCs; Non-High-risk cysts – all cysts except those proven to be high-risk. (2) Low-risk mucinous cysts – proven mucinous low-risk cysts; High-risk cysts – all except those proven to be mucinous low-risk or benign. And (3) Non-benign cysts – all cysts except those proven to be benign; Benign cysts – proven benign cysts.
The ability of the tests to discriminate “relevant” and “non-relevant” cysts using the three definitions of “relevant cysts” was evaluated and the accuracy of the two tests was compared.
The data of the two-by-two tables were used to calculate sensitivity and specificity for each study. We present individual study results graphically by plotting the estimates of sensitivity and specificity (and their 95% confidence intervals (CI)) in both forest plots and on the summary receiver operating characteristic (sROC) curve plots. The area under the curve (AUC) is equal to 1 for a perfect test and 0.5 for a completely uninformative test. The AUC is equal to the probability that if a pair of relevant and non-relevant cysts is selected at random, the relevant cyst will have a higher test result than the non-relevant cyst. Pooled estimates of the sensitivity and specificity were obtained by the DerSimonian-Laird method (random effect model) to incorporate variation among studies, when data are heterogeneous. Otherwise, we used the Mantel-Haenszel method (fixed effect model).
Heterogeneity was investigated in the first instance through visual examination of forest plots of sensitivities and specificities and through visual examination of the ROC plot of the raw data. Last, we used statistical tests, including chi-square and Cochran-Q to evaluate if the differences across the studies were greater than expected by chance alone. A low P value suggests presence of heterogeneity. In addition to these statistics we used the statistic I2 of Higgins, which has been proposed as a measure to quantify the amount of heterogeneity[25,26]. The scale of I2 has a range of 0 to 100% and values on the order of 25%, 50% and 75% are considered low, moderate, and high heterogeneity, respectively.
Another goal of this work was to obtain, for each of the tests, the correctly identified cyst rate and the diagnostic yield in predicting a histopathologic diagnosis.
We used Comprehensive Meta-Analysis software (Version 2.0) for assessment of diagnostic yield of the tests and Meta-DiSc (version 1.4 – Meta-Analysis of Diagnostic and screening tests[27] ) to obtain the accuracy of each of the tests.
Literature search and study selection:
A comprehensive search of databases, including Medline, Scopus, and Web of Science, for the past 8 years (January 1st, 2010 to July 31st, 2018) and restricted to human studies was performed. No language restrictions were applied. The following search terms were used in two independent searches: “pancreas”, “cyst”, “molecular”, “analysis”; and “micro”, “forceps”, “microforceps”, “biopsy”. A search of related articles was performed, adding additional studies. Duplicate articles, reviews, trials including other kinds of neoplasms, and trials with molecular markers not compliant with the defined inclusion criteria were removed. The references of all selected studies were hand-searched for additional articles.
Inclusion criteria: Published studies were included in the meta-analysis if they analyzed: (1) Patients with symptomatic or incidental pancreatic cysts with a definitive surgical pathology diagnosis; (2) Genetic mutations performed with high sensitive techniques, such as NGS in PCF obtained by EUS-FNA prior to surgery; (3) At least four genetic mutations, including KRAS, GNAS, VHL, and at least another genetic mutation representative of aggressive neoplasms (PIK3CA, TP53, SMAD4, PTEN, CDKN2A); (4) PCNs evaluated by EUS-FNA with MFB for diagnosis; and (5) Surgical pathology specimens with available data.
Exclusion criteria: (1) Studies of MA with fewer than the four genetic mutations previously defined; (2) Studies involving solid pancreatic lesions; (3) Studies using PCF not obtained by EUS-FNA; (4) Reviews, case reports, case series with fewer than five patients, letters to editor, exploratory studies, and papers published only in abstract form; (5) Studies with cytology and clinical surveillance as standard of diagnosis. Two authors (SF and AL) independently judged study eligibility and disagreements were resolved by consensus.
Histological criteria: We classified the PCNs of the included studies into three main groups: (1) High-risk cysts (adenocarcinoma or high grade dysplasia in IPMNs and MCNs, secondarily cystic adenocarcinomas, cNETs, and ACCs; (2) Low-risk mucinous cysts (IPMNs and MCNs with intermediate or low-grade dysplasia); and (3) Benign cysts (SCAs, pseudocysts, and other rare cysts (RCs) included in some articles, as retention cysts, lymphoepithelial cysts, epidermoid cysts, squamoid cysts).
Tests under investigation: The index tests were: (1) MA of PCF; and (2) MFB of PCNs, including cyst wall, septs, and nodules. A diagnosis of cNET or ACC does not warrant a malignancy diagnosis, but surgery is recommended in surgically fit patients. Due to a recommendation of identical treatment to malignant and mucinous high-risk cysts, for the purpose of analysis in this study, each one of these diagnoses was classified as a high-risk cyst.
Data extraction:
After study selection, two authors (SF and AL) extracted and registered the data from each study onto a standardized worksheet. Disagreements were discussed and reviewed by a third author (LP). The data retrieved were: first author, publication year, study period and design (prospective or retrospective), reference for diagnosis, sample size (all patients included in the study), technical success, adverse events, diagnostic yield, surgical cohort (number of patients with a surgical pathology specimen), cyst size, cyst location, specific cyst types, number of high-risk cysts, mucinous low-risk and benign cysts diagnosed by MA and MFB comparing to surgical pathology specimens. In the MFB studies, technical success was defined as the ability to puncture the cysts and perform the biopsies; and the diagnostic yield was defined as the ratio between the number of patients included in the study and the patients in whom enough material allowed the acquisition of a histopathologic diagnosis. In the MA group, diagnostic yield was defined as a ratio between the number of patients included in the study and the number of patients with DNA available to perform molecular analysis in PCF.
Outcomes:
The primary outcomes of this study were the data to obtain the accuracies of MA and MFB for the diagnosis of PCNs, including high-risk cysts, mucinous low-risk cysts, and benign cysts. Secondary outcomes were the diagnostic yield of genetic testing and MFB and the number of cysts correctly identified for each of the tests studied.
Quality analysis:
Methodological quality of included primary studies was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23]. The PRISMA-DTA Statement recommendations were used for reporting this systematic review[22,24].
Statistical analysis and data synthesis:
The reference standard was a surgical pathology specimen that allowed the classification of PCNs into three defined groups of diagnosis: high-risk cysts, mucinous low-risk cysts, and benign cysts. This resulted in a two-by-three table with correct and incorrect test results in each of the three referenced groups, for each of the tests analyzed, MA and histology were obtained by MFB.
To calculate tests’ accuracy and to reflect on the categories that are useful in clinical practice and that guide management, we constructed two-by-two tables, considering three definitions of “relevant” cysts: (1) High-risk cysts – proven malignant cysts, IPMNs, and MCNs with HGA, cNETs, ACCs; Non-High-risk cysts – all cysts except those proven to be high-risk. (2) Low-risk mucinous cysts – proven mucinous low-risk cysts; High-risk cysts – all except those proven to be mucinous low-risk or benign. And (3) Non-benign cysts – all cysts except those proven to be benign; Benign cysts – proven benign cysts.
The ability of the tests to discriminate “relevant” and “non-relevant” cysts using the three definitions of “relevant cysts” was evaluated and the accuracy of the two tests was compared.
The data of the two-by-two tables were used to calculate sensitivity and specificity for each study. We present individual study results graphically by plotting the estimates of sensitivity and specificity (and their 95% confidence intervals (CI)) in both forest plots and on the summary receiver operating characteristic (sROC) curve plots. The area under the curve (AUC) is equal to 1 for a perfect test and 0.5 for a completely uninformative test. The AUC is equal to the probability that if a pair of relevant and non-relevant cysts is selected at random, the relevant cyst will have a higher test result than the non-relevant cyst. Pooled estimates of the sensitivity and specificity were obtained by the DerSimonian-Laird method (random effect model) to incorporate variation among studies, when data are heterogeneous. Otherwise, we used the Mantel-Haenszel method (fixed effect model).
Heterogeneity was investigated in the first instance through visual examination of forest plots of sensitivities and specificities and through visual examination of the ROC plot of the raw data. Last, we used statistical tests, including chi-square and Cochran-Q to evaluate if the differences across the studies were greater than expected by chance alone. A low P value suggests presence of heterogeneity. In addition to these statistics we used the statistic I2 of Higgins, which has been proposed as a measure to quantify the amount of heterogeneity[25,26]. The scale of I2 has a range of 0 to 100% and values on the order of 25%, 50% and 75% are considered low, moderate, and high heterogeneity, respectively.
Another goal of this work was to obtain, for each of the tests, the correctly identified cyst rate and the diagnostic yield in predicting a histopathologic diagnosis.
We used Comprehensive Meta-Analysis software (Version 2.0) for assessment of diagnostic yield of the tests and Meta-DiSc (version 1.4 – Meta-Analysis of Diagnostic and screening tests[27] ) to obtain the accuracy of each of the tests.
RESULTS:
Systematic Review Our search revealed 16 study titles and abstracts for MFB and 264 titles for MA. In Figure 1A and B are described the selection process of the articles included in this study. After all steps, eight studies were considered suitable for qualitative and seven for quantitative analysis. We excluded 20 full-text articles after review, because they were case series of two patients[16] (n = 1), exploratory or pilot studies[28,29] (n = 2), no information of mutation status was available[30] (n = 1), pancreatic cystic fluid was obtained during surgery[31] (n = 1), insufficient or absent data of cysts with surgical pathology diagnoses[12,32,33] (n = 3), and mutations only of KRAS and/or GNAS[14,34-44] (n = 12).
Flowchart with identification of eligible studies. A: Molecular analysis; B: Microforceps biopsy.
Of the eight studies that met the inclusion criteria, design was retrospective in six and prospective in two, all were published from 2015 to 2018. These eight studies included a total of 1206 patients, of which 203 (17%) underwent surgical resection and a surgical pathology specimen was available as reference standard and included in the analysis. We excluded all patients with cytology and clinical follow-up data, but for whom a surgical pathology specimen was not available. The characteristics of the studies, surgical pathology diagnoses, and MA and MFB results are presented in Tables 1[32,45-47] and 2[18-21].
Characteristics of studies of molecular analysis included in the analysis
113 samples of 105 patients;
TP53, PIK3CA, PTEN;
1 ACC;
includes: ACC, retention cyst, lymphoepithelial cyst, epidermoid cyst, and squamoid cyst. MA: Molecular analysis; NA: Non-available data; ACC: Acinar cell cyst; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.
Quality assessment and publication bias: Methodological quality of primary studies included was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23], which evaluates the quality of articles for systematic reviews of diagnostic accuracy studies in four domains, including patient selection, index test, reference standard, and flow and timing, for risk of bias and applicability concerns. Results are presented in Figure 2, which was sketched with templates available at www.quadas.org. The studies included in this review all showed a “low-risk” classification as the index tests (MA and MFB) and the reference standard (surgical pathology specimen) were reliable and mentioned in all studies. However, a “high-risk” of selection bias was demonstrated in patient selection (neither random nor sequential patients included in several studies) and in flow and timing because only a small proportion of the patients evaluated in all studies, except one, were included in the analysis. In fact, most patients were excluded in all studies as the inclusion criteria requiring surgical pathology as diagnostic reference were not met. Applicability concerns in patient selection were also significant in all studies, because the subgroup of PCNs referred for surgery is more often malignant than PCNs on surveillance, which would also be targeted with this review. Because of this bias, there may be an overestimation of both the sensitivity of the index tests, due to a more severe spectrum of PCNs that are referred for surgery, and the positive predictive value (PPV) for diagnosis of high-risk cysts, due to an increased prevalence of malignant cysts in a surgical cohort of PCNs.
Quality assessment of the studies using QUADAS-2. A: Tabular presentation of risk bias for each study; B: Graphical display of bias.
Our search revealed 16 study titles and abstracts for MFB and 264 titles for MA. In Figure 1A and B are described the selection process of the articles included in this study. After all steps, eight studies were considered suitable for qualitative and seven for quantitative analysis. We excluded 20 full-text articles after review, because they were case series of two patients[16] (n = 1), exploratory or pilot studies[28,29] (n = 2), no information of mutation status was available[30] (n = 1), pancreatic cystic fluid was obtained during surgery[31] (n = 1), insufficient or absent data of cysts with surgical pathology diagnoses[12,32,33] (n = 3), and mutations only of KRAS and/or GNAS[14,34-44] (n = 12).
Flowchart with identification of eligible studies. A: Molecular analysis; B: Microforceps biopsy.
Of the eight studies that met the inclusion criteria, design was retrospective in six and prospective in two, all were published from 2015 to 2018. These eight studies included a total of 1206 patients, of which 203 (17%) underwent surgical resection and a surgical pathology specimen was available as reference standard and included in the analysis. We excluded all patients with cytology and clinical follow-up data, but for whom a surgical pathology specimen was not available. The characteristics of the studies, surgical pathology diagnoses, and MA and MFB results are presented in Tables 1[32,45-47] and 2[18-21].
Characteristics of studies of molecular analysis included in the analysis
113 samples of 105 patients;
TP53, PIK3CA, PTEN;
1 ACC;
includes: ACC, retention cyst, lymphoepithelial cyst, epidermoid cyst, and squamoid cyst. MA: Molecular analysis; NA: Non-available data; ACC: Acinar cell cyst; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.
Quality assessment and publication bias: Methodological quality of primary studies included was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23], which evaluates the quality of articles for systematic reviews of diagnostic accuracy studies in four domains, including patient selection, index test, reference standard, and flow and timing, for risk of bias and applicability concerns. Results are presented in Figure 2, which was sketched with templates available at www.quadas.org. The studies included in this review all showed a “low-risk” classification as the index tests (MA and MFB) and the reference standard (surgical pathology specimen) were reliable and mentioned in all studies. However, a “high-risk” of selection bias was demonstrated in patient selection (neither random nor sequential patients included in several studies) and in flow and timing because only a small proportion of the patients evaluated in all studies, except one, were included in the analysis. In fact, most patients were excluded in all studies as the inclusion criteria requiring surgical pathology as diagnostic reference were not met. Applicability concerns in patient selection were also significant in all studies, because the subgroup of PCNs referred for surgery is more often malignant than PCNs on surveillance, which would also be targeted with this review. Because of this bias, there may be an overestimation of both the sensitivity of the index tests, due to a more severe spectrum of PCNs that are referred for surgery, and the positive predictive value (PPV) for diagnosis of high-risk cysts, due to an increased prevalence of malignant cysts in a surgical cohort of PCNs.
Quality assessment of the studies using QUADAS-2. A: Tabular presentation of risk bias for each study; B: Graphical display of bias.
Meta-analysis Molecular analysis: Four articles were included in the meta-analysis for diagnostic accuracy of MA. For each of the three definitions of relevant cyst, forest plots of sensitivity and specificity with heterogeneous denoted are shown in Figure 3.
Forest plots of the studies included for molecular analysis. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95% CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.
The three criteria to define “relevant cysts” resulted in a different range of the specificity and sensitivity of the studies included as shown in Figure 3. For diagnosis of the subgroup with high-risk and low-risk mucinous cysts that require intervention (either surgery or surveillance) comparing to benign cysts the pooled sensitivity was 0.75 (95%CI: 0.66-0.83) and the pooled specificity was 0.72 (95%CI: 0.56-0.85) for MA. In the subgroup of high-risk cysts that require surgery, comparing to other cysts requiring conservative management, the sensitivity was 0.57 (95%CI: 0.42-0.71) with a specificity of 0.88 (95%CI: 0.80-0.93). In the subgroup of low-risk mucinous cysts comparing to high-risk, the pooled sensitivity was 0.89 (95%CI: 0.79-0.95) and the pooled specificity was 0.88 (95%CI: 0.75-0.95).
Figure 4 displays the sROC curves of MA, showing the sensitivity of the individual articles mapped on the vertical scale, 1-specificity on the horizontal scale, with the summary (sensitivity, 1-specificity) point marked, as well as the summary ROC curve and the confidence region for the summary (sensitivity, 1-specificity) points. The area under the sROC curve was 0.7706 (SE: 0.0927) in non-benign cysts, 0.9248 (SE: 0.0691) in high-risk cysts, and 0.9555 (SE: 0.0293) in mucinous low-risk cysts. The results of the studies had greater variation in non-benign cysts as shown by the wide confidence region.
Summary receiver operating characteristics plots. ROC: Receiver operating characteristic curve; AUC: Area under the curve; SE: Standard error.
In the four studies, 566 patients had DNA available to perform MA in PCF. Pooled analysis (Figure 5) showed a diagnostic yield of 54.3% (95%CI: 49.8%-58.7%; I2 = 39.605%; test for heterogeneity P = 0.174).
Forest plots of molecular analysis and microforceps biopsies on the secondary outcomes of this meta-analysis.
By considering the classification of cysts by specific type (IPMNs, MCNs, cNETs, SCAs, pseudocysts, ACCs, and other RCs), MA identified correctly 73.1% of cysts (95%CI: 61.6%-82.2%; I2 = 37.381%; test for heterogeneity P = 0.203) (Figure 5).
Micro forceps biopsy: Four articles were included in the meta-analysis for diagnostic accuracy of histology obtained using MFB. Figure 6 shows the forest plots of sensitivity and specificity for the three subgroups of relevant cysts. The forest plots for MFB show variable specificities within the papers, from 0 to 1, which can be due to the small numbers of patients with the target condition in some studies.
Forest plots of the included studies for microforceps biopsies. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95%CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.
For each of the three subgroups there exists a low heterogeneity in sensitivity (I2 = 0%, I2 = 21.4%, I2 = 0%) and specificity (I2 = 0%, I2 = 0%, I2 = 21.4%), therefore fixed effect models were used. As presented in Figure 6, in the first subgroup the pooled sensitivity was 0.73 (95%CI: 0.50-0.89) and the pooled specificity was 0.88 (95%CI: 0.28-1.00). In the second subgroup sensitivity was 0.81 (95%CI: 0.46-0.98) with a specificity of 0.77 (95%CI: 0.50-0.94) and in the last subgroup the pooled sensitivity was 0.64 (95%CI: 0.33-0.88) and the pooled specificity was 0.81 (95%CI: 0.46-0.98).
The results were plotted as a symmetrical sROC curve (Figure 4). The area under the sROC curve was 0.7640 (SE: 0.1261) in the first subgroup, 0.8154 (SE: 0.098) in the second subgroup, and 0.7509 (SE: 0.1277) in the last subgroup.
By pooling the data of the four studies that investigated the use of MFB to obtain a histopathologic diagnosis, we obtained a diagnostic yield of 73.1% (95%CI: 61.4%-82.2%; I2 = 47.774%; test for heterogeneity P = 0.125) (Figure 5).
By considering the outcome “specific cyst type” diagnosis, MFB correctly identified 70.7% of the cysts (95%CI: 49.4%-85.6%; I2 = 0%; test for heterogeneity P = 0.056) (Figure 5).
Molecular analysis: Four articles were included in the meta-analysis for diagnostic accuracy of MA. For each of the three definitions of relevant cyst, forest plots of sensitivity and specificity with heterogeneous denoted are shown in Figure 3.
Forest plots of the studies included for molecular analysis. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95% CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.
The three criteria to define “relevant cysts” resulted in a different range of the specificity and sensitivity of the studies included as shown in Figure 3. For diagnosis of the subgroup with high-risk and low-risk mucinous cysts that require intervention (either surgery or surveillance) comparing to benign cysts the pooled sensitivity was 0.75 (95%CI: 0.66-0.83) and the pooled specificity was 0.72 (95%CI: 0.56-0.85) for MA. In the subgroup of high-risk cysts that require surgery, comparing to other cysts requiring conservative management, the sensitivity was 0.57 (95%CI: 0.42-0.71) with a specificity of 0.88 (95%CI: 0.80-0.93). In the subgroup of low-risk mucinous cysts comparing to high-risk, the pooled sensitivity was 0.89 (95%CI: 0.79-0.95) and the pooled specificity was 0.88 (95%CI: 0.75-0.95).
Figure 4 displays the sROC curves of MA, showing the sensitivity of the individual articles mapped on the vertical scale, 1-specificity on the horizontal scale, with the summary (sensitivity, 1-specificity) point marked, as well as the summary ROC curve and the confidence region for the summary (sensitivity, 1-specificity) points. The area under the sROC curve was 0.7706 (SE: 0.0927) in non-benign cysts, 0.9248 (SE: 0.0691) in high-risk cysts, and 0.9555 (SE: 0.0293) in mucinous low-risk cysts. The results of the studies had greater variation in non-benign cysts as shown by the wide confidence region.
Summary receiver operating characteristics plots. ROC: Receiver operating characteristic curve; AUC: Area under the curve; SE: Standard error.
In the four studies, 566 patients had DNA available to perform MA in PCF. Pooled analysis (Figure 5) showed a diagnostic yield of 54.3% (95%CI: 49.8%-58.7%; I2 = 39.605%; test for heterogeneity P = 0.174).
Forest plots of molecular analysis and microforceps biopsies on the secondary outcomes of this meta-analysis.
By considering the classification of cysts by specific type (IPMNs, MCNs, cNETs, SCAs, pseudocysts, ACCs, and other RCs), MA identified correctly 73.1% of cysts (95%CI: 61.6%-82.2%; I2 = 37.381%; test for heterogeneity P = 0.203) (Figure 5).
Micro forceps biopsy: Four articles were included in the meta-analysis for diagnostic accuracy of histology obtained using MFB. Figure 6 shows the forest plots of sensitivity and specificity for the three subgroups of relevant cysts. The forest plots for MFB show variable specificities within the papers, from 0 to 1, which can be due to the small numbers of patients with the target condition in some studies.
Forest plots of the included studies for microforceps biopsies. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95%CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.
For each of the three subgroups there exists a low heterogeneity in sensitivity (I2 = 0%, I2 = 21.4%, I2 = 0%) and specificity (I2 = 0%, I2 = 0%, I2 = 21.4%), therefore fixed effect models were used. As presented in Figure 6, in the first subgroup the pooled sensitivity was 0.73 (95%CI: 0.50-0.89) and the pooled specificity was 0.88 (95%CI: 0.28-1.00). In the second subgroup sensitivity was 0.81 (95%CI: 0.46-0.98) with a specificity of 0.77 (95%CI: 0.50-0.94) and in the last subgroup the pooled sensitivity was 0.64 (95%CI: 0.33-0.88) and the pooled specificity was 0.81 (95%CI: 0.46-0.98).
The results were plotted as a symmetrical sROC curve (Figure 4). The area under the sROC curve was 0.7640 (SE: 0.1261) in the first subgroup, 0.8154 (SE: 0.098) in the second subgroup, and 0.7509 (SE: 0.1277) in the last subgroup.
By pooling the data of the four studies that investigated the use of MFB to obtain a histopathologic diagnosis, we obtained a diagnostic yield of 73.1% (95%CI: 61.4%-82.2%; I2 = 47.774%; test for heterogeneity P = 0.125) (Figure 5).
By considering the outcome “specific cyst type” diagnosis, MFB correctly identified 70.7% of the cysts (95%CI: 49.4%-85.6%; I2 = 0%; test for heterogeneity P = 0.056) (Figure 5).
Systematic Review:
Our search revealed 16 study titles and abstracts for MFB and 264 titles for MA. In Figure 1A and B are described the selection process of the articles included in this study. After all steps, eight studies were considered suitable for qualitative and seven for quantitative analysis. We excluded 20 full-text articles after review, because they were case series of two patients[16] (n = 1), exploratory or pilot studies[28,29] (n = 2), no information of mutation status was available[30] (n = 1), pancreatic cystic fluid was obtained during surgery[31] (n = 1), insufficient or absent data of cysts with surgical pathology diagnoses[12,32,33] (n = 3), and mutations only of KRAS and/or GNAS[14,34-44] (n = 12).
Flowchart with identification of eligible studies. A: Molecular analysis; B: Microforceps biopsy.
Of the eight studies that met the inclusion criteria, design was retrospective in six and prospective in two, all were published from 2015 to 2018. These eight studies included a total of 1206 patients, of which 203 (17%) underwent surgical resection and a surgical pathology specimen was available as reference standard and included in the analysis. We excluded all patients with cytology and clinical follow-up data, but for whom a surgical pathology specimen was not available. The characteristics of the studies, surgical pathology diagnoses, and MA and MFB results are presented in Tables 1[32,45-47] and 2[18-21].
Characteristics of studies of molecular analysis included in the analysis
113 samples of 105 patients;
TP53, PIK3CA, PTEN;
1 ACC;
includes: ACC, retention cyst, lymphoepithelial cyst, epidermoid cyst, and squamoid cyst. MA: Molecular analysis; NA: Non-available data; ACC: Acinar cell cyst; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.
Quality assessment and publication bias: Methodological quality of primary studies included was assessed by two authors (SF and AL) using the modified QUADAS-2 tool[23], which evaluates the quality of articles for systematic reviews of diagnostic accuracy studies in four domains, including patient selection, index test, reference standard, and flow and timing, for risk of bias and applicability concerns. Results are presented in Figure 2, which was sketched with templates available at www.quadas.org. The studies included in this review all showed a “low-risk” classification as the index tests (MA and MFB) and the reference standard (surgical pathology specimen) were reliable and mentioned in all studies. However, a “high-risk” of selection bias was demonstrated in patient selection (neither random nor sequential patients included in several studies) and in flow and timing because only a small proportion of the patients evaluated in all studies, except one, were included in the analysis. In fact, most patients were excluded in all studies as the inclusion criteria requiring surgical pathology as diagnostic reference were not met. Applicability concerns in patient selection were also significant in all studies, because the subgroup of PCNs referred for surgery is more often malignant than PCNs on surveillance, which would also be targeted with this review. Because of this bias, there may be an overestimation of both the sensitivity of the index tests, due to a more severe spectrum of PCNs that are referred for surgery, and the positive predictive value (PPV) for diagnosis of high-risk cysts, due to an increased prevalence of malignant cysts in a surgical cohort of PCNs.
Quality assessment of the studies using QUADAS-2. A: Tabular presentation of risk bias for each study; B: Graphical display of bias.
Meta-analysis:
Molecular analysis: Four articles were included in the meta-analysis for diagnostic accuracy of MA. For each of the three definitions of relevant cyst, forest plots of sensitivity and specificity with heterogeneous denoted are shown in Figure 3.
Forest plots of the studies included for molecular analysis. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95% CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.
The three criteria to define “relevant cysts” resulted in a different range of the specificity and sensitivity of the studies included as shown in Figure 3. For diagnosis of the subgroup with high-risk and low-risk mucinous cysts that require intervention (either surgery or surveillance) comparing to benign cysts the pooled sensitivity was 0.75 (95%CI: 0.66-0.83) and the pooled specificity was 0.72 (95%CI: 0.56-0.85) for MA. In the subgroup of high-risk cysts that require surgery, comparing to other cysts requiring conservative management, the sensitivity was 0.57 (95%CI: 0.42-0.71) with a specificity of 0.88 (95%CI: 0.80-0.93). In the subgroup of low-risk mucinous cysts comparing to high-risk, the pooled sensitivity was 0.89 (95%CI: 0.79-0.95) and the pooled specificity was 0.88 (95%CI: 0.75-0.95).
Figure 4 displays the sROC curves of MA, showing the sensitivity of the individual articles mapped on the vertical scale, 1-specificity on the horizontal scale, with the summary (sensitivity, 1-specificity) point marked, as well as the summary ROC curve and the confidence region for the summary (sensitivity, 1-specificity) points. The area under the sROC curve was 0.7706 (SE: 0.0927) in non-benign cysts, 0.9248 (SE: 0.0691) in high-risk cysts, and 0.9555 (SE: 0.0293) in mucinous low-risk cysts. The results of the studies had greater variation in non-benign cysts as shown by the wide confidence region.
Summary receiver operating characteristics plots. ROC: Receiver operating characteristic curve; AUC: Area under the curve; SE: Standard error.
In the four studies, 566 patients had DNA available to perform MA in PCF. Pooled analysis (Figure 5) showed a diagnostic yield of 54.3% (95%CI: 49.8%-58.7%; I2 = 39.605%; test for heterogeneity P = 0.174).
Forest plots of molecular analysis and microforceps biopsies on the secondary outcomes of this meta-analysis.
By considering the classification of cysts by specific type (IPMNs, MCNs, cNETs, SCAs, pseudocysts, ACCs, and other RCs), MA identified correctly 73.1% of cysts (95%CI: 61.6%-82.2%; I2 = 37.381%; test for heterogeneity P = 0.203) (Figure 5).
Micro forceps biopsy: Four articles were included in the meta-analysis for diagnostic accuracy of histology obtained using MFB. Figure 6 shows the forest plots of sensitivity and specificity for the three subgroups of relevant cysts. The forest plots for MFB show variable specificities within the papers, from 0 to 1, which can be due to the small numbers of patients with the target condition in some studies.
Forest plots of the included studies for microforceps biopsies. In parentheses are the 95% confidence intervals (CI) of the sensitivity and specificity. The figure shows the estimated sensitivity and specificity of the study (red circle) and its 95%CI (blue horizontal line). The area of the circle reflects the weight that the study contributes to the meta-analysis.
For each of the three subgroups there exists a low heterogeneity in sensitivity (I2 = 0%, I2 = 21.4%, I2 = 0%) and specificity (I2 = 0%, I2 = 0%, I2 = 21.4%), therefore fixed effect models were used. As presented in Figure 6, in the first subgroup the pooled sensitivity was 0.73 (95%CI: 0.50-0.89) and the pooled specificity was 0.88 (95%CI: 0.28-1.00). In the second subgroup sensitivity was 0.81 (95%CI: 0.46-0.98) with a specificity of 0.77 (95%CI: 0.50-0.94) and in the last subgroup the pooled sensitivity was 0.64 (95%CI: 0.33-0.88) and the pooled specificity was 0.81 (95%CI: 0.46-0.98).
The results were plotted as a symmetrical sROC curve (Figure 4). The area under the sROC curve was 0.7640 (SE: 0.1261) in the first subgroup, 0.8154 (SE: 0.098) in the second subgroup, and 0.7509 (SE: 0.1277) in the last subgroup.
By pooling the data of the four studies that investigated the use of MFB to obtain a histopathologic diagnosis, we obtained a diagnostic yield of 73.1% (95%CI: 61.4%-82.2%; I2 = 47.774%; test for heterogeneity P = 0.125) (Figure 5).
By considering the outcome “specific cyst type” diagnosis, MFB correctly identified 70.7% of the cysts (95%CI: 49.4%-85.6%; I2 = 0%; test for heterogeneity P = 0.056) (Figure 5).
DISCUSSION:
In this meta-analysis we analyzed two different but promising tests to diagnose PCNs – molecular analysis and microforceps biopsy. To our knowledge this is the first study of this nature, and it included 1206 patients with PCNs of which 1058 underwent MA and 148 MFB. All patients had the index tests performed in PCF obtained pre-operatively, exclusively with NGS for MA and the Moray micro forceps biopsy device (US Endoscopy, Mentor, Ohio) used for MFB. We analyzed 203 cysts, 178 evaluated with MA and 25 with MFB, all referred for surgery, and with a surgical pathology specimen used as reference standard for diagnosis.
In this comparative analysis we included all studies, without restriction to si-multaneous evaluation of both tests, because only one of such studies has been published[20]. This study, which includes 48 patients but only 10 surgical pathology specimens, showed identical results for MA and MFB in low-risk and high-risk cyst diagnosis, but higher specific cyst type diagnosis for MFB.
The data from the seven studies included in the meta-analysis, although with limited number of patients, particularly for MFB, suggests that MA is more accurate than MFB for diagnosis of PCNs, including high-risk and low-risk lesions. MA has superior accuracy to discriminate high-risk cysts from other PCNs and low-risk from high-risk neoplastic cysts. MA performance was considered excellent with AUC values of 0.92 and of 0.96 for high-risk and low-risk neoplastic lesions, respectively, as compared to MFB, which showed a fair or good performance, with an AUC of 0.81 and 0.75, respectively for the same lesions (Figure 4). The specificity of MA is good (0.88) but it has a low sensitivity (only 0.57) for high-risk cysts. This may be explained by technical issues, by low prevalence of relevant genetic mutations in malignant PCNs, or by mutations not included in the current NGS panels. The sensitivity and specificity are high (0.89 and 0.88, respectively) for MA when comparing low-risk to high-risk cysts, which reflects the genetic nature of pancreatic carcinogenesis with cumulative mutations from benign to malignant cysts[48].
For discriminating benign cysts from both low-risk and high-risk cysts, the performance of MA and MFB was identical and fair according to AUC values of 0.77 and 0.76, respectively. This non-superiority of MA in the diagnosis of benign cysts in this meta-analysis may be due to technique-inherent issues and/or under-representation of benign cysts in surgical series. In fact, “no genetic mutation” is considered a false negative result in most benign rare cysts, but some of these lesions (retention cysts, etc.) have no diagnostic genetic mutations. On the contrary, the most frequent benign cysts, SCAs, harbor a VHL mutation, exclusively present in these benign lesions and allowing for discarding a malignant lesion. In the MA studies, one third of rare benign cysts were classified as false negative results, due to absence of characteristic mutations (Table 1). Another example of PCN that is not amenable to a MA diagnosis with current genetic panels is cNET, also reducing the accuracy of MA for diagnosis of high-risk cysts. The sensitivities were identical for MA and MFB (0.75 and 0.72), but the latter had higher specificity (0.73 and 0.88, respectively). Limited tissue sampling with MFB can explain the reduced sensitivity with robust specificity. As MA depends on denuded DNA in suspension in PCF, no sampling error is expected, which may explain its greater accuracy in neoplastic cysts, comparing to MFB.
Concerning secondary outcomes, even with the limitations of tissue sampling inherent to MFB, this meta-analysis showed that the diagnostic yield of MFB was superior to MA with rates of correctly identified cyst identical with MA and MFB (Tables 1 and 2). In fact, the definition of diagnostic yield, which for MA was “detection of genetic mutations”, may have led to a falsely low value due to the presence of some rare types of benign cysts (retention cysts, lymphoepithelial cysts, epidermoid cysts, squamous cysts in two studies[46,47]) that have no characteristic diagnostic genetic mutations.
Characteristics of studies of microforceps biopsies included in the analysis
1 nondiagnostic on MFB;
2 nondiagnostic on MFB;
1 false negative IPMN associated invasive carcinoma;
2 cases with a suspicion of pseudocyst. MFB: Microforceps biopsy; NA: Non-available data; ND: Nondiagnostic; ACC: Acinar cystadenoma; H/B/T: Head body and tail of the pancreas; ADC: Adenocarcinoma; HG: High-grade; LG: Low-grade; NET: Neuroendocrine tumor.
In clinical practice, patient symptoms, cyst imaging features, CEA, and cytology of PCF are required for diagnosis and decision for either treatment or surveillance according to cyst types[49]. PCF analysis, including CEA to distinguish mucinous from non-mucinous cysts and cytology to select those that harbor HGA or early pancreatic carcinoma and require surgical treatment, have suboptimal accuracies[3], due to scant cellularity and limited PCF volume. In this context, additional diagnostic tests are necessary to improve cyst classification and refine clinical decision. DNA markers require limited amounts of PCF, increasing the diagnostic yield[32,45,50,51], but with considerable technical complexity and costs. In fact, in routine clinical practice a major pitfall for PCNs diagnosis is the limited volume of PCF obtained, precluding routine pre-operative testing. As DNA analysis requires less volume of PCF, it may become an alternative test in these circumstances. This major advantage of molecular analysis was not possible to evaluate in this meta-analysis, because the volume of cystic fluid obtained in pancreatic cysts was not available in most studies analyzed.
As MA continues to evolve, questions remain about its accuracy, how it influences patient management, and in what order the analysis should be performed to better support clinical decisions. Previous studies[49] have shown that DNA testing combined with clinical features increased PCNs diagnosis compared to either alone. With multiple recent advances in biomarkers, molecular genetics will probably prove to be useful in the management of PCNs[52]. In a previous meta-analysis, pre-operative cytology of PCNs has shown low sensitivity for diagnosis[53], endorsing additional tests to improve diagnosis. Another meta-analysis of diagnostic accuracy of EUS-FNA with CEA and cytology analysis in differentiating mucinous cysts has demonstrated to be accurate to confirm the diagnosis but performed poorly in excluding it[54]. The role of KRAS as individual screening test has been analyzed before[55] with poor accuracy and added benefit coming from a combined approach with cytology. A recently published meta-analysis supporting KRAS, GNAS, and RNF43 mutations as diagnostic markers of IPMNs[56] used different methods for mutation detection, different tumor materials, and clinicopathologic data as reference standard for diagnosis, which may limit its clinical application in evaluation of PCNs with mutational analysis performed only in PCF.
In this scenario, new markers are needed for PCNs stratification, and in our meta-analysis both MA and MFB have acceptable diagnostic accuracies. The two largest studies of MA[46,47] showed higher accuracy for diagnosis, which underscores the role of technical aspects of PCF collection, storage, and laboratory analysis for improved accuracy with this technique.
On the other hand, MFB provides tissue fragments for routine histological evaluation, without additional PCF required other than for standard analysis. The technical feasibility of through-the-needle microforceps biopsies revealed to be excellent, even in cysts located in the pancreatic head, despite the required 19-gauge caliber of the EUS-FNA needle. Another potential advantage of MFB is to allow the diagnosis of histologic subtypes of IPMNs, which can potentially be used for risk stratification[57], but still requires further validation.
Strengths and limitations We applied strict exclusion criteria, with all analyzed patients having a surgical pathology specimen as the reference standard for diagnosis, because histopathology is the gold standard for diagnosis of neoplasia. Another major strength of this meta-analysis is having identical lesions (size and location) analyzed in both groups. These important strengths provide a more realistic accuracy estimate of the tests evaluated. In previous studies of cytology including both surgical pathology and clinical follow-up[54] as reference standard, pooled sensitivities were 12% higher than in studies with exclusive surgical pathology[55] as reference standard in the diagnosis of mucinous cysts, with test accuracy overestimation. Finally, the pooled results have low heterogeneity.
The quality of a systematic review depends on the quality of studies included, and our quality assessment of patient selection regarding the risk of bias and applicability was high. As sensitivity and specificity are sensitive to study design and influenced by the spectrum of disease, sample collection, and processing, there may be a risk of bias and the results, although correct, their interpretation may be inaccurate. Moreover, there was incomplete reporting in one primary study, having no separate information on specific cyst type, mucinous or malignant cyst diagnosis[32], and the study was excluded from quantitative analysis. Although one study was excluded from the meta-analysis, MA with three studies included more patients (953, of whom only 153 in the surgical cohort) than the group of MFB with four studies but fewer patients (148, with only 25 in the surgical cohort). This can represent a surgical selection bias for both tests studied. Moreover, MFB studies were all retrospective, with small sample size, without pathology diagnosis for most benign and pre-malignant cysts, and non-consecutive patients that were selected on endoscopist discretion, which may have led to bias. Another limitation is the time between the index tests and the reference standard, because the final diagnosis could have been made at different time intervals from the tests. If the time between index tests and reference standard is too long, the true disease status of the patient may have changed by the time the reference standard was assessed. Aditionally, the different number of malignant cysts per study, particularly in the MA group, may have led to part of the heterogeneity in sensitivity and specificity. Finally, as MA does not increase the risks of standard EUS-FNA (the analysis is performed in remnant cystic fluid after standard diagnosis) we did not perform a safety analysis of MFB, but the four studies analyzed described only rare non-severe adverse events.
We applied strict exclusion criteria, with all analyzed patients having a surgical pathology specimen as the reference standard for diagnosis, because histopathology is the gold standard for diagnosis of neoplasia. Another major strength of this meta-analysis is having identical lesions (size and location) analyzed in both groups. These important strengths provide a more realistic accuracy estimate of the tests evaluated. In previous studies of cytology including both surgical pathology and clinical follow-up[54] as reference standard, pooled sensitivities were 12% higher than in studies with exclusive surgical pathology[55] as reference standard in the diagnosis of mucinous cysts, with test accuracy overestimation. Finally, the pooled results have low heterogeneity.
The quality of a systematic review depends on the quality of studies included, and our quality assessment of patient selection regarding the risk of bias and applicability was high. As sensitivity and specificity are sensitive to study design and influenced by the spectrum of disease, sample collection, and processing, there may be a risk of bias and the results, although correct, their interpretation may be inaccurate. Moreover, there was incomplete reporting in one primary study, having no separate information on specific cyst type, mucinous or malignant cyst diagnosis[32], and the study was excluded from quantitative analysis. Although one study was excluded from the meta-analysis, MA with three studies included more patients (953, of whom only 153 in the surgical cohort) than the group of MFB with four studies but fewer patients (148, with only 25 in the surgical cohort). This can represent a surgical selection bias for both tests studied. Moreover, MFB studies were all retrospective, with small sample size, without pathology diagnosis for most benign and pre-malignant cysts, and non-consecutive patients that were selected on endoscopist discretion, which may have led to bias. Another limitation is the time between the index tests and the reference standard, because the final diagnosis could have been made at different time intervals from the tests. If the time between index tests and reference standard is too long, the true disease status of the patient may have changed by the time the reference standard was assessed. Aditionally, the different number of malignant cysts per study, particularly in the MA group, may have led to part of the heterogeneity in sensitivity and specificity. Finally, as MA does not increase the risks of standard EUS-FNA (the analysis is performed in remnant cystic fluid after standard diagnosis) we did not perform a safety analysis of MFB, but the four studies analyzed described only rare non-severe adverse events.
Future perspectives With the increasing diagnosis of asymptomatic PCNs, most with potential for malignancy, there is a growing need to find accurate and affordable tests for diagnosis. The goal of management of patients with pancreatic cysts is to detect and resect cysts before progression of malignancy, while avoiding unnecessary follow-up procedures in benign cysts and surgery in low-risk PCNs.
Biomarkers of malignancy are promising, but clinicians should be aware of their current diagnostic performance limitations and type of lesions identified. In addition to significant costs, logistic difficulties in preserving material for future molecular analysis in busy general hospitals, and the technical complexity of the test, the generalized use of MA seems difficult in clinical practice. On the other hand, if MFB proves in larger studies to be safe and to allow tissue acquisition and gives the histological criteria needed for a correct diagnosis of PCNs, it may be immediately implemented in clinics, because the endoscopic procedure is standard, and histology is already a widespread procedure in clinics. MFB may be especially useful for benign lesions, for which both surgery and surveillance are unnecessary, representing a considerable burden in pancreas clinics due to current diagnostic limitations[58].
For MA to become relevant in routine clinical care in the future, its role in early cancer diagnosis and its prognostic value in PCNs requiring periodic surveillance must be confirmed. Also, for successful massive implementation, it is required to develop as an universal, highly accurate, first line test with clinical impact in cyst diagnosis, prognosis, and patient management. MA, both in PCF and peripheral blood, for standard analysis of multiple simultaneous biomarkers, allowing non-invasive diagnosis and risk stratification of these lesions[59] would be valuable. For the present time, MA and MFB can only be recommended as complementary or as second line tests in case CEA and cytology of PCF are non-diagnostic. For both tests, large multicenter validation studies are still missing.
With the increasing diagnosis of asymptomatic PCNs, most with potential for malignancy, there is a growing need to find accurate and affordable tests for diagnosis. The goal of management of patients with pancreatic cysts is to detect and resect cysts before progression of malignancy, while avoiding unnecessary follow-up procedures in benign cysts and surgery in low-risk PCNs.
Biomarkers of malignancy are promising, but clinicians should be aware of their current diagnostic performance limitations and type of lesions identified. In addition to significant costs, logistic difficulties in preserving material for future molecular analysis in busy general hospitals, and the technical complexity of the test, the generalized use of MA seems difficult in clinical practice. On the other hand, if MFB proves in larger studies to be safe and to allow tissue acquisition and gives the histological criteria needed for a correct diagnosis of PCNs, it may be immediately implemented in clinics, because the endoscopic procedure is standard, and histology is already a widespread procedure in clinics. MFB may be especially useful for benign lesions, for which both surgery and surveillance are unnecessary, representing a considerable burden in pancreas clinics due to current diagnostic limitations[58].
For MA to become relevant in routine clinical care in the future, its role in early cancer diagnosis and its prognostic value in PCNs requiring periodic surveillance must be confirmed. Also, for successful massive implementation, it is required to develop as an universal, highly accurate, first line test with clinical impact in cyst diagnosis, prognosis, and patient management. MA, both in PCF and peripheral blood, for standard analysis of multiple simultaneous biomarkers, allowing non-invasive diagnosis and risk stratification of these lesions[59] would be valuable. For the present time, MA and MFB can only be recommended as complementary or as second line tests in case CEA and cytology of PCF are non-diagnostic. For both tests, large multicenter validation studies are still missing.
Strengths and limitations:
We applied strict exclusion criteria, with all analyzed patients having a surgical pathology specimen as the reference standard for diagnosis, because histopathology is the gold standard for diagnosis of neoplasia. Another major strength of this meta-analysis is having identical lesions (size and location) analyzed in both groups. These important strengths provide a more realistic accuracy estimate of the tests evaluated. In previous studies of cytology including both surgical pathology and clinical follow-up[54] as reference standard, pooled sensitivities were 12% higher than in studies with exclusive surgical pathology[55] as reference standard in the diagnosis of mucinous cysts, with test accuracy overestimation. Finally, the pooled results have low heterogeneity.
The quality of a systematic review depends on the quality of studies included, and our quality assessment of patient selection regarding the risk of bias and applicability was high. As sensitivity and specificity are sensitive to study design and influenced by the spectrum of disease, sample collection, and processing, there may be a risk of bias and the results, although correct, their interpretation may be inaccurate. Moreover, there was incomplete reporting in one primary study, having no separate information on specific cyst type, mucinous or malignant cyst diagnosis[32], and the study was excluded from quantitative analysis. Although one study was excluded from the meta-analysis, MA with three studies included more patients (953, of whom only 153 in the surgical cohort) than the group of MFB with four studies but fewer patients (148, with only 25 in the surgical cohort). This can represent a surgical selection bias for both tests studied. Moreover, MFB studies were all retrospective, with small sample size, without pathology diagnosis for most benign and pre-malignant cysts, and non-consecutive patients that were selected on endoscopist discretion, which may have led to bias. Another limitation is the time between the index tests and the reference standard, because the final diagnosis could have been made at different time intervals from the tests. If the time between index tests and reference standard is too long, the true disease status of the patient may have changed by the time the reference standard was assessed. Aditionally, the different number of malignant cysts per study, particularly in the MA group, may have led to part of the heterogeneity in sensitivity and specificity. Finally, as MA does not increase the risks of standard EUS-FNA (the analysis is performed in remnant cystic fluid after standard diagnosis) we did not perform a safety analysis of MFB, but the four studies analyzed described only rare non-severe adverse events.
Future perspectives:
With the increasing diagnosis of asymptomatic PCNs, most with potential for malignancy, there is a growing need to find accurate and affordable tests for diagnosis. The goal of management of patients with pancreatic cysts is to detect and resect cysts before progression of malignancy, while avoiding unnecessary follow-up procedures in benign cysts and surgery in low-risk PCNs.
Biomarkers of malignancy are promising, but clinicians should be aware of their current diagnostic performance limitations and type of lesions identified. In addition to significant costs, logistic difficulties in preserving material for future molecular analysis in busy general hospitals, and the technical complexity of the test, the generalized use of MA seems difficult in clinical practice. On the other hand, if MFB proves in larger studies to be safe and to allow tissue acquisition and gives the histological criteria needed for a correct diagnosis of PCNs, it may be immediately implemented in clinics, because the endoscopic procedure is standard, and histology is already a widespread procedure in clinics. MFB may be especially useful for benign lesions, for which both surgery and surveillance are unnecessary, representing a considerable burden in pancreas clinics due to current diagnostic limitations[58].
For MA to become relevant in routine clinical care in the future, its role in early cancer diagnosis and its prognostic value in PCNs requiring periodic surveillance must be confirmed. Also, for successful massive implementation, it is required to develop as an universal, highly accurate, first line test with clinical impact in cyst diagnosis, prognosis, and patient management. MA, both in PCF and peripheral blood, for standard analysis of multiple simultaneous biomarkers, allowing non-invasive diagnosis and risk stratification of these lesions[59] would be valuable. For the present time, MA and MFB can only be recommended as complementary or as second line tests in case CEA and cytology of PCF are non-diagnostic. For both tests, large multicenter validation studies are still missing.
CONCLUSION:
Our study confirms the diagnostic value of both MA and MFB, with higher diagnostic accuracy of MA than MFB for both low-risk and high-risk mucinous cysts. Genetic analysis should not be replaced by MFB in this context. Clinicians should be aware of the higher accuracy of MA for the diagnosis of malignant and high-risk cysts.
|
Background:
Carcinoembryonic antigen (CEA) and cytology in pancreatic cystic fluid are suboptimal for evaluation of pancreatic cystic neoplasms. Genetic testing and microforceps biopsy are promising tools for pre-operative diagnostic improvement but comparative performance of both methods is unknown.
Methods:
We performed a literature search in Medline, Scopus, and Web of Science for studies evaluating genetic testing of cystic fluid and microforceps biopsy of pancreatic cysts, with endoscopic ultrasound with fine-needle aspiration (EUS-FNA) prior to surgery and surgical pathology as reference standard for diagnosis. We evaluated the diagnostic accuracy for: 1- benign cysts; 2- mucinous low-risk cysts; 3- high-risk cysts, and the diagnostic yield and rate of correctly identified cysts with microforceps biopsy and molecular analysis. We also assessed publication bias, heterogeneity, and study quality.
Results:
Eight studies, including 1206 patients, of which 203 (17%) referred for surgery who met the inclusion criteria were analyzed in the systematic review, and seven studies were included in the meta-analysis. Genetic testing and microforceps biopsies were identical for diagnosis of benign cysts. Molecular analysis was superior for diagnosis of both low and high-risk mucinous cysts, with sensitivities of 0.89 (95%CI: 0.79-0.95) and 0.57 (95%CI: 0.42-0.71), specificities of 0.88 (95%CI: 0.75-0.95) and 0.88 (95%CI: 0.80-0.93) and AUC of 0.9555 and 0.92, respectively. The diagnostic yield was higher in microforceps biopsies than in genetic analysis (0.73 vs 0.54, respectively) but the rates of correctly identified cysts were identical (0.73 with 95%CI: 0.62-0.82 vs 0.71 with 95%CI: 0.49-0.86, respectively).
Conclusions:
Genetic testing and microforceps biopsies are useful second tests, with identical results in benign pancreatic cysts. Genetic analysis performs better for low- and high-risk cysts but has lower diagnostic yield.
|
INTRODUCTION:
Pancreatic cystic neoplasms (PCNs) are on the rise in clinics due to an ageing population and the increase in routine use of high-quality abdominal imaging[1]. PCNs are generally classified into two main groups: mucinous cystic neoplasms (MCNs) and non-mucinous cystic neoplasms (NMCN). MCNs include intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystadenomas, which are precursor lesions of pancreatic carcinoma, and may be low-risk (pre-malignant with low or intermediate-grade atypia) or high-risk: pre-malignant with high-grade atypia (HGA) or malignant, including adenocarcinomas secondarily cystic. NMCNs include serous cystadenomas and inflammatory cysts (pseudocysts), mostly benign cysts, but may include some rare lesions, considered high-risk as cystic neuroendocrine tumors (cNETs), and acinar cell cystadenomas (ACCs). The heterogeneity in malignant potential, increased frequency, and significant morbidity and mortality of surgical treatment, makes pre-operative diagnosis of PCNs essential for management. The treatment options for PCNs encompass surgery or conservative surveillance for MCNs, according to malignancy risk, or no further evaluation for most NMCNs.
The differentiation between MCNs and NMCNs is critical, because a misdiagnosis of a MCN can lead to a missed opportunity to treat pancreatic cancer in an early stage and a misdiagnosis of NMCN can result in unnecessary surgery or surveillance with associated morbidity, costs, and negative impact on quality of life.
Currently, morphologic characterization of PCNs and pancreatic cystic fluid (PCF) analysis for carcinoembryonic Antigen (CEA) and cytology are central in diagnosis. A CEA level ≥ 192 ng/mL is the most accurate diagnostic test for MCNs and cytology is highly specific for malignancy[2], but with suboptimal results in large studies with surgical pathology as the gold standard[3]. In fact, a significant part of these lesions remains indeterminate and incorrect pre-operative diagnosis occurs in one third of patients[4,5], making new reliable diagnostic tools urgently needed.
In the last decade numerous studies have shown that genetic analysis of aspirates obtained by EUS-FNA provided a better characterization of PCNs than CEA and cytology[6-14]. Next-generation sequencing (NGS) is a very sensitive technique for detection of genetic mutations that allows the rapid detection of mutations in pre-defined panels of cancer genes, even in samples with limited DNA content, such as PCF. NGS requires storage, infrastructure, data processing, and expert personnel. Moreover, to be cost-effective, large numbers of samples need to be processed, making it applicable only in large centralized laboratories. These reasons make the implementation of NGS in clinical practice still a matter of debate.
The clinical need of better diagnostic tests in PCNs has recently led to the development of a through-the-needle miniature biopsy device for use during EUS-FNA[15,16]. The Moray micro forceps biopsy (MFB) device (US Endoscopy, Mentor, Ohio) is disposable and can pass through a standard 19-gauge EUS-FNA needle that is already used routinely. It allows tissue sampling from the cyst wall, septa or mural nodules and the obtention of a histological evaluation of the epithelial architecture and subepithelial stroma[17]. Adding to the high technical success and excellent safety profile[18,19], the new device has shown to improve the diagnostic accuracy of specific cyst subtypes[20,21]. Another major advantage of MFB is the simultaneous tissue sampling and PCF acquisition, with just an additional histologic analysis that follows standard definitions and is already routine in clinics.
The aim of this systematic review and meta-analysis is to evaluate the diagnostic performance of molecular analysis (MA) and MFB and find the most robust additional diagnostic technique in PCNs, in the pre-operative setting.
CONCLUSION:
For the present time, MA and MFB can only be recommended as complementary or as second line tests in case CEA and cytology of PCF are non-diagnostic. In the future, for MA to become relevant in routine clinical care, its role must be confirmed, in order to become a first line test with clinical impact in cyst diagnosis, prognosis, and patient management. MA, both in PCF and peripheral blood, for multiple simultaneous biomarkers and non-invasive diagnosis and risk stratification would be valuable. If MFB proves in larger studies to be safe and to allow a correct diagnosis of pancreatic cysts, it may be immediately implemented in clinics. MFB may be especially useful for benign lesions, for which both surgery and surveillance are unnecessary, with uncertain diagnosis due to current diagnostic limitations. For both tests, larger validation studies are missing.
|
Background:
Carcinoembryonic antigen (CEA) and cytology in pancreatic cystic fluid are suboptimal for evaluation of pancreatic cystic neoplasms. Genetic testing and microforceps biopsy are promising tools for pre-operative diagnostic improvement but comparative performance of both methods is unknown.
Methods:
We performed a literature search in Medline, Scopus, and Web of Science for studies evaluating genetic testing of cystic fluid and microforceps biopsy of pancreatic cysts, with endoscopic ultrasound with fine-needle aspiration (EUS-FNA) prior to surgery and surgical pathology as reference standard for diagnosis. We evaluated the diagnostic accuracy for: 1- benign cysts; 2- mucinous low-risk cysts; 3- high-risk cysts, and the diagnostic yield and rate of correctly identified cysts with microforceps biopsy and molecular analysis. We also assessed publication bias, heterogeneity, and study quality.
Results:
Eight studies, including 1206 patients, of which 203 (17%) referred for surgery who met the inclusion criteria were analyzed in the systematic review, and seven studies were included in the meta-analysis. Genetic testing and microforceps biopsies were identical for diagnosis of benign cysts. Molecular analysis was superior for diagnosis of both low and high-risk mucinous cysts, with sensitivities of 0.89 (95%CI: 0.79-0.95) and 0.57 (95%CI: 0.42-0.71), specificities of 0.88 (95%CI: 0.75-0.95) and 0.88 (95%CI: 0.80-0.93) and AUC of 0.9555 and 0.92, respectively. The diagnostic yield was higher in microforceps biopsies than in genetic analysis (0.73 vs 0.54, respectively) but the rates of correctly identified cysts were identical (0.73 with 95%CI: 0.62-0.82 vs 0.71 with 95%CI: 0.49-0.86, respectively).
Conclusions:
Genetic testing and microforceps biopsies are useful second tests, with identical results in benign pancreatic cysts. Genetic analysis performs better for low- and high-risk cysts but has lower diagnostic yield.
| 14,799 | 366 | 14 |
[
"cysts",
"studies",
"risk",
"analysis",
"diagnosis",
"ma",
"mfb",
"high",
"study",
"patients"
] |
[
"test",
"test"
] | null |
[CONTENT] Pancreatic cysts | Endoscopic ultrasound | Endoscopic ultrasound with fine-needle aspiration | Genetic testing | Microforceps biopsy | Molecular analysis | KRAS | Carcinoembryonic antigen | Cytology [SUMMARY]
|
[CONTENT] Pancreatic cysts | Endoscopic ultrasound | Endoscopic ultrasound with fine-needle aspiration | Genetic testing | Microforceps biopsy | Molecular analysis | KRAS | Carcinoembryonic antigen | Cytology [SUMMARY]
| null |
[CONTENT] Pancreatic cysts | Endoscopic ultrasound | Endoscopic ultrasound with fine-needle aspiration | Genetic testing | Microforceps biopsy | Molecular analysis | KRAS | Carcinoembryonic antigen | Cytology [SUMMARY]
|
[CONTENT] Pancreatic cysts | Endoscopic ultrasound | Endoscopic ultrasound with fine-needle aspiration | Genetic testing | Microforceps biopsy | Molecular analysis | KRAS | Carcinoembryonic antigen | Cytology [SUMMARY]
|
[CONTENT] Pancreatic cysts | Endoscopic ultrasound | Endoscopic ultrasound with fine-needle aspiration | Genetic testing | Microforceps biopsy | Molecular analysis | KRAS | Carcinoembryonic antigen | Cytology [SUMMARY]
|
[CONTENT] Cyst Fluid | Diagnosis, Differential | Endoscopic Ultrasound-Guided Fine Needle Aspiration | Genetic Testing | Humans | Pancreas | Pancreatic Cyst | Pancreatic Neoplasms | Preoperative Period | Sensitivity and Specificity [SUMMARY]
|
[CONTENT] Cyst Fluid | Diagnosis, Differential | Endoscopic Ultrasound-Guided Fine Needle Aspiration | Genetic Testing | Humans | Pancreas | Pancreatic Cyst | Pancreatic Neoplasms | Preoperative Period | Sensitivity and Specificity [SUMMARY]
| null |
[CONTENT] Cyst Fluid | Diagnosis, Differential | Endoscopic Ultrasound-Guided Fine Needle Aspiration | Genetic Testing | Humans | Pancreas | Pancreatic Cyst | Pancreatic Neoplasms | Preoperative Period | Sensitivity and Specificity [SUMMARY]
|
[CONTENT] Cyst Fluid | Diagnosis, Differential | Endoscopic Ultrasound-Guided Fine Needle Aspiration | Genetic Testing | Humans | Pancreas | Pancreatic Cyst | Pancreatic Neoplasms | Preoperative Period | Sensitivity and Specificity [SUMMARY]
|
[CONTENT] Cyst Fluid | Diagnosis, Differential | Endoscopic Ultrasound-Guided Fine Needle Aspiration | Genetic Testing | Humans | Pancreas | Pancreatic Cyst | Pancreatic Neoplasms | Preoperative Period | Sensitivity and Specificity [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] cysts | studies | risk | analysis | diagnosis | ma | mfb | high | study | patients [SUMMARY]
|
[CONTENT] cysts | studies | risk | analysis | diagnosis | ma | mfb | high | study | patients [SUMMARY]
| null |
[CONTENT] cysts | studies | risk | analysis | diagnosis | ma | mfb | high | study | patients [SUMMARY]
|
[CONTENT] cysts | studies | risk | analysis | diagnosis | ma | mfb | high | study | patients [SUMMARY]
|
[CONTENT] cysts | studies | risk | analysis | diagnosis | ma | mfb | high | study | patients [SUMMARY]
|
[CONTENT] pre | pcns | cystic | neoplasms | cystic neoplasms | cystadenomas | nmcns | include | mcns | operative [SUMMARY]
|
[CONTENT] cysts | risk | studies | tests | proven | cysts proven | high | risk cysts | relevant | study [SUMMARY]
| null |
[CONTENT] accuracy ma | higher | ma | risk | mfb | ma diagnosis malignant | diagnosis malignant | diagnosis malignant high | value ma mfb | ma diagnosis malignant high [SUMMARY]
|
[CONTENT] cysts | studies | risk | analysis | diagnosis | mfb | ma | study | high | patients [SUMMARY]
|
[CONTENT] cysts | studies | risk | analysis | diagnosis | mfb | ma | study | high | patients [SUMMARY]
|
[CONTENT] CEA ||| [SUMMARY]
|
[CONTENT] Medline | EUS-FNA ||| ||| ||| [SUMMARY]
| null |
[CONTENT] second ||| [SUMMARY]
|
[CONTENT] CEA ||| ||| Medline | EUS-FNA ||| ||| ||| ||| Eight | 1206 | 203 | 17% | seven ||| ||| 0.89 | 0.79-0.95 | 0.57 | 0.42-0.71 | 0.88 | 0.75-0.95 | 0.88 | 0.80-0.93 | 0.9555 | 0.92 ||| 0.73 | 0.54 | 0.73 | 0.62-0.82 | 0.71 | 0.49 ||| second ||| [SUMMARY]
|
[CONTENT] CEA ||| ||| Medline | EUS-FNA ||| ||| ||| ||| Eight | 1206 | 203 | 17% | seven ||| ||| 0.89 | 0.79-0.95 | 0.57 | 0.42-0.71 | 0.88 | 0.75-0.95 | 0.88 | 0.80-0.93 | 0.9555 | 0.92 ||| 0.73 | 0.54 | 0.73 | 0.62-0.82 | 0.71 | 0.49 ||| second ||| [SUMMARY]
|
|||||
Intramuscular Corticosteroid Therapy in the Treatment of Alopecia Areata: A Time-to-Event Analysis.
|
35027820
|
Intramuscular corticosteroids (IMC) have gained popularity for the treatment of severe alopecia areata (AA) in recent years; however, evidence on their efficacy and safety is still limited.
|
INTRODUCTION
|
Time-to-event analysis was performed on patients with severe, extensive, or rapidly progressive AA receiving IMC. The IMC regimen comprised triamcinolone acetonide 20-40 mg/mL injected every 4-6 weeks. The evaluated outcomes included initial (25% regrowth), significant (75% regrowth), and complete hair regrowth (100% regrowth). Relapse and adverse events were also noted. Factors associated with treatment outcomes and relapse were analyzed using the Cox proportional hazards model.
|
METHODS
|
A total of 101 patients were eligible for analysis. Significant hair regrowth was obtained in 80.2% of the patients (n = 81), in a median time of 3.4 months (95% confidence interval [CI] = 2.9-4.4). Complete hair regrowth was achieved in 48.5% of the subjects (n = 49), and relapse was observed in 47.5% (n = 48). Acneiform eruption was the most common adverse effect. Multivariable analysis revealed that nail involvement was a negative predictor of significant hair regrowth (adjusted hazard ratio [HR] = 0.04, 95% CI = 0.01-0.55; P = 0.015), whereas duration of AA longer than 6 months was associated with disease recurrence (adjusted HR = 4.02, 95% CI = 1.52-4.66; P = 0.005).
|
RESULTS
|
This study demonstrated the efficacy and safety of IMC in the treatment of severe or active AA; however, the relapse rate remained relatively high after discontinuation of the therapy. Nail involvement was a negative predictor of significant hair regrowth, while disease duration longer than 6 months predicted AA relapse.
|
CONCLUSION
|
[
"Adolescent",
"Adrenal Cortex Hormones",
"Adult",
"Aged",
"Alopecia Areata",
"Child",
"Child, Preschool",
"Female",
"Humans",
"Injections, Intramuscular",
"Male",
"Middle Aged",
"Retrospective Studies"
] |
8752075
|
Introduction
|
Alopecia areata (AA) is a non-scarring hair loss disorder that affects the scalp and hair-bearing areas.1 Variations on the clinical presentation of AA have been observed, ranging from small, well-defined circular or oval patches of hair loss to diffuse alopecia and complete scalp (alopecia totalis, AT) or total body hair loss (alopecia universalis, AU).2–5 Nail abnormalities, most frequently nail pitting, are occasionally present. Patients with AA often experience major psychological impacts due to the unpredictable clinical course and variable treatment response.6 Various immune-related comorbidities such as autoimmune thyroid diseases (AITD), vitiligo, psoriasis, and atopic diseases have been linked to AA.7–13
The exact etiopathogenesis of AA is unclear, though it is believed to be a cell-mediated autoimmune disease of the hair follicle. Autoreactive T-cell lymphocytes and several cytokines mediate hair follicular destruction and negatively affect the hair growth cycle.14–16 Despite the absence of curative/preventive treatments and FDA-approved regimens for AA, therapeutic approaches have been established through treatment guidelines. Topical and intralesional (IL) corticosteroids, topical minoxidil, and topical anthralin are recommended for AA patients with <50% scalp involvement, whereas in those with ≥50% hair loss, topical contact immunotherapy and systemic corticosteroids are advised.17–23
Corticosteroids, the main treatment option for AA, can be administered in different forms depending on the disease severity.24 Medium- to high-potency topical and IL corticosteroids are indicated as monotherapy or combined therapy in limited and/or slowly progressive AA,25–27 while systemic corticosteroids (eg, oral, intravenous, and intramuscular), are preferred in extensive and/or rapidly progressive disease.17
Among systemic modalities, intramuscular corticosteroids (IMC) may be most beneficial, as its monthly or bimonthly regimen increases patients’ compliance and treatment adherence.28 Moreover, IMC could minimize adverse effects from slowly releasing continuous and small amounts of medication.28 Previous studies of IMC showed a satisfactory outcome in severe and refractory AA, with response rates between 63% and 72.7%.29–31 Although IMC is often the preferred treatment for AA, there are limited data regarding its effectiveness, safety, dosing, and optimal duration. Thus, we conducted a retrospective cohort study based on our 20-year experience to evaluate the efficacy, relapse, and tolerability, as well as factors associated with treatment outcomes of IMC in patients with AA.
| null | null | null | null |
Conclusion
|
Our time-to-event analysis demonstrated the efficacy and safety of intramuscular administration of corticosteroids for the treatment of AA. IMC therapy showed high rates of significant hair regrowth, especially in patients with severe or active AA; however, approximately half of those experienced relapse. Nail involvement was negatively correlated with cosmetically acceptable hair regrowth, while disease duration >6 months was significantly associated with increased risk of relapse. Most importantly, the use of IMC depends on indication and perceived potential benefits and risks; therefore, physicians should be cautious when prescribing this treatment.
|
[
"Materials and Methods",
"Study Design and Population",
"Data Collection and Clinical Outcomes",
"Statistical Analysis",
"Results",
"Demographics",
"Overall Response",
"Initial Hair Regrowth",
"Significant Hair Regrowth",
"Factors Associated with Significant Hair Regrowth",
"Complete Hair Regrowth",
"Relapse and Its Associated Factors",
"Adverse Events",
"Discussion",
"Conclusion"
] |
[
" Study Design and Population This was a retrospective cohort study conducted at the Dermatology Clinic of Ramathibodi Hospital, Bangkok, Thailand. The study was approved by the Mahidol University Review Board for Ethics in Human Research (MURA2020/1282) and was performed in accordance with the principles of Declaration of Helsinki. The requirement for informed consent was waived, and the data were anonymized before analysis.\nWe reviewed the medical records of patients clinically and/or histopathologically diagnosed with AA between January 2000 and December 2019. Individuals with AA receiving an IMC treatment regimen were included in the study. Our IMC protocol was triamcinolone acetonide 20–40 mg/mL injected every 4–6 weeks. The treatment was indicated in patients with severe (≥50% scalp involvement), extensive (AT, AU, and ≥50% scalp involvement with body hair loss), or rapidly progressive AA (accelerated hair loss spreading ≥5% of scalp area per week for >2 consecutive weeks with positive hair-pull test results at the periphery of alopecic patch or across the entire scalp), and IMC was performed until patients achieved significant hair regrowth. Patients who did not achieve significant hair regrowth after six IMC injections were switched to other appropriate therapies to minimize adrenocortical alteration.32 We excluded participants treated with systemic immunosuppressive agents or topical contact immunotherapy within 6 months after discontinuation of the IMC protocol; therefore, those who underwent topical treatment (ie, topical corticosteroids and/or topical minoxidil) remained in our cohort. Patients with incomplete medical records, those who received other AA treatments during IMC therapy, those with other hair and scalp disorders, and those with systemic diseases or using medications affecting the hair growth cycle were also excluded.\nThis was a retrospective cohort study conducted at the Dermatology Clinic of Ramathibodi Hospital, Bangkok, Thailand. The study was approved by the Mahidol University Review Board for Ethics in Human Research (MURA2020/1282) and was performed in accordance with the principles of Declaration of Helsinki. The requirement for informed consent was waived, and the data were anonymized before analysis.\nWe reviewed the medical records of patients clinically and/or histopathologically diagnosed with AA between January 2000 and December 2019. Individuals with AA receiving an IMC treatment regimen were included in the study. Our IMC protocol was triamcinolone acetonide 20–40 mg/mL injected every 4–6 weeks. The treatment was indicated in patients with severe (≥50% scalp involvement), extensive (AT, AU, and ≥50% scalp involvement with body hair loss), or rapidly progressive AA (accelerated hair loss spreading ≥5% of scalp area per week for >2 consecutive weeks with positive hair-pull test results at the periphery of alopecic patch or across the entire scalp), and IMC was performed until patients achieved significant hair regrowth. Patients who did not achieve significant hair regrowth after six IMC injections were switched to other appropriate therapies to minimize adrenocortical alteration.32 We excluded participants treated with systemic immunosuppressive agents or topical contact immunotherapy within 6 months after discontinuation of the IMC protocol; therefore, those who underwent topical treatment (ie, topical corticosteroids and/or topical minoxidil) remained in our cohort. Patients with incomplete medical records, those who received other AA treatments during IMC therapy, those with other hair and scalp disorders, and those with systemic diseases or using medications affecting the hair growth cycle were also excluded.\n Data Collection and Clinical Outcomes Baseline demographics, clinical characteristics, duration of disease, AA subtypes (ie, patch, multiple patches, AT/AU, and ophiasis), body hair involvement, nail abnormalities, and family history of AA were collected. AA severity was evaluated using the Severity of Alopecia Tool (SALT) score. Details regarding IMC regimen were obtained, including dosage, duration, and clinical outcomes. Efficacy and tolerability assessment were performed at baseline and at every follow-up visit. The percentage of scalp area coverage by terminal hair regrowth from baseline was used to evaluate treatment response. Clinical endpoints included initial, significant, and complete hair regrowth, treatment failure, and relapse. Initial hair regrowth (noticeable) was defined as 25% regrowth, while significant hair regrowth (cosmetically acceptable) was defined as 75% regrowth. Patients with 100% regrowth were classified as having complete (full) hair regrowth. Treatment failure was defined as <10% hair regrowth after 6 months of IMC therapy. Relapse was defined as any new appearance of AA during follow-up visits. The cumulative incidence of significant hair regrowth was set as the outcome of interest in the time-to-event analysis. Furthermore, the median time and median cumulative corticosteroid dosage to achieve each clinical endpoint were evaluated. Relevant factors affecting significant hair regrowth and relapse were also analyzed.\nBaseline demographics, clinical characteristics, duration of disease, AA subtypes (ie, patch, multiple patches, AT/AU, and ophiasis), body hair involvement, nail abnormalities, and family history of AA were collected. AA severity was evaluated using the Severity of Alopecia Tool (SALT) score. Details regarding IMC regimen were obtained, including dosage, duration, and clinical outcomes. Efficacy and tolerability assessment were performed at baseline and at every follow-up visit. The percentage of scalp area coverage by terminal hair regrowth from baseline was used to evaluate treatment response. Clinical endpoints included initial, significant, and complete hair regrowth, treatment failure, and relapse. Initial hair regrowth (noticeable) was defined as 25% regrowth, while significant hair regrowth (cosmetically acceptable) was defined as 75% regrowth. Patients with 100% regrowth were classified as having complete (full) hair regrowth. Treatment failure was defined as <10% hair regrowth after 6 months of IMC therapy. Relapse was defined as any new appearance of AA during follow-up visits. The cumulative incidence of significant hair regrowth was set as the outcome of interest in the time-to-event analysis. Furthermore, the median time and median cumulative corticosteroid dosage to achieve each clinical endpoint were evaluated. Relevant factors affecting significant hair regrowth and relapse were also analyzed.\n Statistical Analysis Statistical analyses were performed using Stata 14.1 (Stata Corp, College Station, TX, USA). To detect a modestly sized hazard ratio (HR), the sample size was calculated based on data from a previous study regarding the IMC treatment of patients with AA.31 The minimum number of subjects required to achieve a statistical significance level of 5% and a power of 80% was 26. Continuous variables were reported as mean ± standard deviation or median (interquartile range, IQR). Categorical variables were expressed as number or proportion. Survival analysis was performed using the Kaplan–Meier method to estimate outcome rate, median time, and median cumulative corticosteroids to achieve endpoints. The log rank test was used to compare survival distributions between variables. Factors affecting clinical outcomes were assessed using univariable and multivariable stepwise analyses under the Cox proportional hazards regression model and expressed as HRs with 95% confidence intervals (CI). All analyses were two-tailed, and P-values <0.05 were considered statistically significant.\nStatistical analyses were performed using Stata 14.1 (Stata Corp, College Station, TX, USA). To detect a modestly sized hazard ratio (HR), the sample size was calculated based on data from a previous study regarding the IMC treatment of patients with AA.31 The minimum number of subjects required to achieve a statistical significance level of 5% and a power of 80% was 26. Continuous variables were reported as mean ± standard deviation or median (interquartile range, IQR). Categorical variables were expressed as number or proportion. Survival analysis was performed using the Kaplan–Meier method to estimate outcome rate, median time, and median cumulative corticosteroids to achieve endpoints. The log rank test was used to compare survival distributions between variables. Factors affecting clinical outcomes were assessed using univariable and multivariable stepwise analyses under the Cox proportional hazards regression model and expressed as HRs with 95% confidence intervals (CI). All analyses were two-tailed, and P-values <0.05 were considered statistically significant.",
"This was a retrospective cohort study conducted at the Dermatology Clinic of Ramathibodi Hospital, Bangkok, Thailand. The study was approved by the Mahidol University Review Board for Ethics in Human Research (MURA2020/1282) and was performed in accordance with the principles of Declaration of Helsinki. The requirement for informed consent was waived, and the data were anonymized before analysis.\nWe reviewed the medical records of patients clinically and/or histopathologically diagnosed with AA between January 2000 and December 2019. Individuals with AA receiving an IMC treatment regimen were included in the study. Our IMC protocol was triamcinolone acetonide 20–40 mg/mL injected every 4–6 weeks. The treatment was indicated in patients with severe (≥50% scalp involvement), extensive (AT, AU, and ≥50% scalp involvement with body hair loss), or rapidly progressive AA (accelerated hair loss spreading ≥5% of scalp area per week for >2 consecutive weeks with positive hair-pull test results at the periphery of alopecic patch or across the entire scalp), and IMC was performed until patients achieved significant hair regrowth. Patients who did not achieve significant hair regrowth after six IMC injections were switched to other appropriate therapies to minimize adrenocortical alteration.32 We excluded participants treated with systemic immunosuppressive agents or topical contact immunotherapy within 6 months after discontinuation of the IMC protocol; therefore, those who underwent topical treatment (ie, topical corticosteroids and/or topical minoxidil) remained in our cohort. Patients with incomplete medical records, those who received other AA treatments during IMC therapy, those with other hair and scalp disorders, and those with systemic diseases or using medications affecting the hair growth cycle were also excluded.",
"Baseline demographics, clinical characteristics, duration of disease, AA subtypes (ie, patch, multiple patches, AT/AU, and ophiasis), body hair involvement, nail abnormalities, and family history of AA were collected. AA severity was evaluated using the Severity of Alopecia Tool (SALT) score. Details regarding IMC regimen were obtained, including dosage, duration, and clinical outcomes. Efficacy and tolerability assessment were performed at baseline and at every follow-up visit. The percentage of scalp area coverage by terminal hair regrowth from baseline was used to evaluate treatment response. Clinical endpoints included initial, significant, and complete hair regrowth, treatment failure, and relapse. Initial hair regrowth (noticeable) was defined as 25% regrowth, while significant hair regrowth (cosmetically acceptable) was defined as 75% regrowth. Patients with 100% regrowth were classified as having complete (full) hair regrowth. Treatment failure was defined as <10% hair regrowth after 6 months of IMC therapy. Relapse was defined as any new appearance of AA during follow-up visits. The cumulative incidence of significant hair regrowth was set as the outcome of interest in the time-to-event analysis. Furthermore, the median time and median cumulative corticosteroid dosage to achieve each clinical endpoint were evaluated. Relevant factors affecting significant hair regrowth and relapse were also analyzed.",
"Statistical analyses were performed using Stata 14.1 (Stata Corp, College Station, TX, USA). To detect a modestly sized hazard ratio (HR), the sample size was calculated based on data from a previous study regarding the IMC treatment of patients with AA.31 The minimum number of subjects required to achieve a statistical significance level of 5% and a power of 80% was 26. Continuous variables were reported as mean ± standard deviation or median (interquartile range, IQR). Categorical variables were expressed as number or proportion. Survival analysis was performed using the Kaplan–Meier method to estimate outcome rate, median time, and median cumulative corticosteroids to achieve endpoints. The log rank test was used to compare survival distributions between variables. Factors affecting clinical outcomes were assessed using univariable and multivariable stepwise analyses under the Cox proportional hazards regression model and expressed as HRs with 95% confidence intervals (CI). All analyses were two-tailed, and P-values <0.05 were considered statistically significant.",
" Demographics A total of 126 patients with AA treated with IMC were initially included. Twenty-five patients were excluded due to coexisting diseases or concomitant use of other medications affecting the hair growth cycle (n = 16), incomplete medical records (n = 5), or because they received systemic treatment after the IMC protocol (n = 4). The majority of the remaining 101 patients were female (n = 70, 69.3%) with a female to male ratio of 2.26:1. The age range of AA onset was 5 to 68 years (mean = 40.4 ± 12.6 years), and the duration of disease varied from 1 week to 11 months (median = 2 [1–4] months). The most common AA subtype was multiple patches (n = 46, 45.5%), followed by patch (n = 20, 19.8%), diffuse (n = 16, 15.8%), AT (n = 9, 8.9%), AU (n = 5, 5.0%), and ophiasis (n = 5, 5.0%). The estimated baseline SALT score was 0–25%, 26–50%, 51–75%, or 76–100% in 11 (10.9%), 16 (15.8%), 60 (59.4%), and 14 patients (13.9%), respectively. Body hair involvement was found in 5.9% (n = 6), and nail abnormalities were observed in 8.9% (n = 9) of the patients. Six patients (5.9%) had coexisting AITD, 2 (1.9%) were atopic, and 2 (1.9%) had a family member with AA. All demographics are summarized in Table 1.Table 1Demographic and Clinical Characteristics of Participants with Alopecia AreataVariablesN = 101Sex, n (%) ● Male31 (30.7) ● Female70 (69.3)Age at AA onset, year, mean ± SD40.4 ± 12.6Age at AA onset, year, n (%) ● ≤40 years50 (49.5) ● >40 years51 (50.5)Duration of disease, month, median (IQR)2 (1–4)Duration of disease, n (%) ● ≤6 months77 (76.2) ● >6 months24 (23.8)AA subtype, n (%) ● Patch AA20 (19.8) ● Multiple AA46 (45.5) ● Diffuse AA16 (15.8) ● AT9 (8.9) ● AU5 (5.0) ● Ophiasis5 (5.0)Severity of AA (SALT score), n (%) ● 0–25%11 (10.9) ● 26–50%16 (15.8) ● 51–75%60 (59.4) ● 76–100%14 (13.9)Body hair involvement, n (%)6 (5.9)Nail involvement, n (%)9 (8.9)Comorbidity, n (%) ● Atopy2 (1.9) ● Autoimmune thyroid diseases6 (5.9)Family history of AA, n (%)2 (1.9)Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.\n\nDemographic and Clinical Characteristics of Participants with Alopecia Areata\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.\nA total of 126 patients with AA treated with IMC were initially included. Twenty-five patients were excluded due to coexisting diseases or concomitant use of other medications affecting the hair growth cycle (n = 16), incomplete medical records (n = 5), or because they received systemic treatment after the IMC protocol (n = 4). The majority of the remaining 101 patients were female (n = 70, 69.3%) with a female to male ratio of 2.26:1. The age range of AA onset was 5 to 68 years (mean = 40.4 ± 12.6 years), and the duration of disease varied from 1 week to 11 months (median = 2 [1–4] months). The most common AA subtype was multiple patches (n = 46, 45.5%), followed by patch (n = 20, 19.8%), diffuse (n = 16, 15.8%), AT (n = 9, 8.9%), AU (n = 5, 5.0%), and ophiasis (n = 5, 5.0%). The estimated baseline SALT score was 0–25%, 26–50%, 51–75%, or 76–100% in 11 (10.9%), 16 (15.8%), 60 (59.4%), and 14 patients (13.9%), respectively. Body hair involvement was found in 5.9% (n = 6), and nail abnormalities were observed in 8.9% (n = 9) of the patients. Six patients (5.9%) had coexisting AITD, 2 (1.9%) were atopic, and 2 (1.9%) had a family member with AA. All demographics are summarized in Table 1.Table 1Demographic and Clinical Characteristics of Participants with Alopecia AreataVariablesN = 101Sex, n (%) ● Male31 (30.7) ● Female70 (69.3)Age at AA onset, year, mean ± SD40.4 ± 12.6Age at AA onset, year, n (%) ● ≤40 years50 (49.5) ● >40 years51 (50.5)Duration of disease, month, median (IQR)2 (1–4)Duration of disease, n (%) ● ≤6 months77 (76.2) ● >6 months24 (23.8)AA subtype, n (%) ● Patch AA20 (19.8) ● Multiple AA46 (45.5) ● Diffuse AA16 (15.8) ● AT9 (8.9) ● AU5 (5.0) ● Ophiasis5 (5.0)Severity of AA (SALT score), n (%) ● 0–25%11 (10.9) ● 26–50%16 (15.8) ● 51–75%60 (59.4) ● 76–100%14 (13.9)Body hair involvement, n (%)6 (5.9)Nail involvement, n (%)9 (8.9)Comorbidity, n (%) ● Atopy2 (1.9) ● Autoimmune thyroid diseases6 (5.9)Family history of AA, n (%)2 (1.9)Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.\n\nDemographic and Clinical Characteristics of Participants with Alopecia Areata\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.\n Overall Response The median overall follow-up time for this cohort was 20 (6–38) months. Patients received 1 to 6 IMC injections (mean = 3.12 ± 0.9) per treatment course. Ninety-eight patients (97.1%) showed improvement, whereas 3 patients (2.9%) showed treatment failure and were further treated by topical contact immunotherapy at approximately 9 months (36 weeks in 2 patients and 37 weeks in another one) after completion of IMC therapy. The mean duration of IMC therapy was 4.2 ± 1.1 months, with a range of 1 to 6 months. Treatment responses are summarized in Table 2.Table 2Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia AreataTreatment OutcomesN = 101Treatment failure (<10% regrowth) ● Incidence, n (%)3 (2.9)Initial hair regrowth (25% regrowth) ● Cumulative incidence, n (%)86 (85.1) ● Incidence rate, person-year (CI)64.0 (51.9–79.3) ● Median time to achieve endpoint, month (CI)0.9 (0.8–1.2) ● Median cumulative dose to achieve endpoint, mg100 ● Cumulative dose to achieve endpoint, mg, median (IQR)80 (20–160)Significant hair regrowth (75% regrowth) ● Cumulative incidence, n (%)81 (80.2) ● Incidence rate, person-year (CI)17.0 (13.6–21.7) ● Median time to achieve endpoint, month (CI)3.4 (2.9–4.4) ● Median cumulative dose to achieve endpoint, mg200 ● Cumulative dose to achieve endpoint, mg, median (IQR)120 (40–200)Complete hair regrowth (100% regrowth) ● Cumulative incidence, n (%)49 (48.5) ● Incidence rate, person-year (CI)8.0 (6.2–10.9) ● Median time to achieve endpoint, month (CI)6.1 (5.1–8.1) ● Cumulative dose to achieve endpoint, mg, median (IQR)160 (60–240)Relapse ● Cumulative incidence, n (%)48 (47.5) ● Incidence rate, person-year (CI)5.5 (4.2–7.3) ● Median time to relapse, month (CI)11.3 (8.6–15.4)Abbreviations: CI, confidence interval; IQR, interquartile range.\n\nTreatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia Areata\nAbbreviations: CI, confidence interval; IQR, interquartile range.\nThe median overall follow-up time for this cohort was 20 (6–38) months. Patients received 1 to 6 IMC injections (mean = 3.12 ± 0.9) per treatment course. Ninety-eight patients (97.1%) showed improvement, whereas 3 patients (2.9%) showed treatment failure and were further treated by topical contact immunotherapy at approximately 9 months (36 weeks in 2 patients and 37 weeks in another one) after completion of IMC therapy. The mean duration of IMC therapy was 4.2 ± 1.1 months, with a range of 1 to 6 months. Treatment responses are summarized in Table 2.Table 2Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia AreataTreatment OutcomesN = 101Treatment failure (<10% regrowth) ● Incidence, n (%)3 (2.9)Initial hair regrowth (25% regrowth) ● Cumulative incidence, n (%)86 (85.1) ● Incidence rate, person-year (CI)64.0 (51.9–79.3) ● Median time to achieve endpoint, month (CI)0.9 (0.8–1.2) ● Median cumulative dose to achieve endpoint, mg100 ● Cumulative dose to achieve endpoint, mg, median (IQR)80 (20–160)Significant hair regrowth (75% regrowth) ● Cumulative incidence, n (%)81 (80.2) ● Incidence rate, person-year (CI)17.0 (13.6–21.7) ● Median time to achieve endpoint, month (CI)3.4 (2.9–4.4) ● Median cumulative dose to achieve endpoint, mg200 ● Cumulative dose to achieve endpoint, mg, median (IQR)120 (40–200)Complete hair regrowth (100% regrowth) ● Cumulative incidence, n (%)49 (48.5) ● Incidence rate, person-year (CI)8.0 (6.2–10.9) ● Median time to achieve endpoint, month (CI)6.1 (5.1–8.1) ● Cumulative dose to achieve endpoint, mg, median (IQR)160 (60–240)Relapse ● Cumulative incidence, n (%)48 (47.5) ● Incidence rate, person-year (CI)5.5 (4.2–7.3) ● Median time to relapse, month (CI)11.3 (8.6–15.4)Abbreviations: CI, confidence interval; IQR, interquartile range.\n\nTreatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia Areata\nAbbreviations: CI, confidence interval; IQR, interquartile range.\n Initial Hair Regrowth According to survival analysis, the cumulative incidence of initial hair regrowth after IMC therapy was 85.1%, with an incidence rate of 64.0 (95% CI = 51.9–79.3) person-years. The median time to achieve initial hair regrowth was 0.9 (95% CI = 0.8–1.2) months, and the average cumulative dose of IMC to achieve initial hair regrowth was 80 (20–160) mg.\nAccording to survival analysis, the cumulative incidence of initial hair regrowth after IMC therapy was 85.1%, with an incidence rate of 64.0 (95% CI = 51.9–79.3) person-years. The median time to achieve initial hair regrowth was 0.9 (95% CI = 0.8–1.2) months, and the average cumulative dose of IMC to achieve initial hair regrowth was 80 (20–160) mg.\n Significant Hair Regrowth Significant hair regrowth was indicated as the primary outcome and its cumulative incidence in our cohort was 80.2%, with an incidence rate of 17.0 (95% CI = 13.6–21.7) person-years. The median time required to achieve significant hair regrowth was 3.4 (95% CI = 2.9–4.4) months. According to the Kaplan–Meier curve, there was a lag of approximately 1 month from initiation of therapy to the first patients demonstrating significant hair regrowth. Following this initial event, significant hair regrowth was observed in 41.9% of patients at 3 months, 75.9% at 6 months, 81.0% at 9 months, and 86.5% at 12 months. The residual probabilities of significant regrowth as a function of the time after initiation of IMC are also shown in Figure 1A. The average cumulative dose of IMC to achieve significant hair regrowth was 120 (40–200) mg, while a median cumulative dose that 50% of patients attained significant hair regrowth was 200 mg (Figure 1B).Figure 1Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nKaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nThe baseline SALT score was identified as a negative factor for IMC treatment response in a previous study.31 To determine the effect of this variable on significant hair regrowth, a stratified Kaplan–Meier analysis was performed. Nine patients (81.8%) with a score of 0–25% obtained significant hair regrowth by a median time of 3.5 months; for the score range 26–50%, 12 (75.0%) patients achieved significant regrowth by 4.8 months; for the score range 51–75%, 48 (80.0%) experienced significant regrowth by 3.1 months; and for the score range 76–100%, 12 (85.7%) ultimately obtained significant regrowth at 3.2 months. However, the log rank test failed to demonstrate statistically significant differences among the four groups (P = 0.287; Figure 1C).\nSignificant hair regrowth was indicated as the primary outcome and its cumulative incidence in our cohort was 80.2%, with an incidence rate of 17.0 (95% CI = 13.6–21.7) person-years. The median time required to achieve significant hair regrowth was 3.4 (95% CI = 2.9–4.4) months. According to the Kaplan–Meier curve, there was a lag of approximately 1 month from initiation of therapy to the first patients demonstrating significant hair regrowth. Following this initial event, significant hair regrowth was observed in 41.9% of patients at 3 months, 75.9% at 6 months, 81.0% at 9 months, and 86.5% at 12 months. The residual probabilities of significant regrowth as a function of the time after initiation of IMC are also shown in Figure 1A. The average cumulative dose of IMC to achieve significant hair regrowth was 120 (40–200) mg, while a median cumulative dose that 50% of patients attained significant hair regrowth was 200 mg (Figure 1B).Figure 1Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nKaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nThe baseline SALT score was identified as a negative factor for IMC treatment response in a previous study.31 To determine the effect of this variable on significant hair regrowth, a stratified Kaplan–Meier analysis was performed. Nine patients (81.8%) with a score of 0–25% obtained significant hair regrowth by a median time of 3.5 months; for the score range 26–50%, 12 (75.0%) patients achieved significant regrowth by 4.8 months; for the score range 51–75%, 48 (80.0%) experienced significant regrowth by 3.1 months; and for the score range 76–100%, 12 (85.7%) ultimately obtained significant regrowth at 3.2 months. However, the log rank test failed to demonstrate statistically significant differences among the four groups (P = 0.287; Figure 1C).\n Factors Associated with Significant Hair Regrowth The Cox proportional hazards model was used to determine the factors associated with significant hair regrowth (Table 3). Univariable analysis revealed that the variables associated with significant hair regrowth were female sex (HR = 2.39, 95% CI = 1.09–4.61; P = 0.035), age at AA onset >40 years (HR = 2.45, 95% CI = 1.49–3.69; P = 0.046), and nail abnormalities (HR = 0.26, 95% CI = 0.08–0.69; P = 0.036). However, when age at AA onset was analyzed as a continuous variable, no association was found (HR = 1.02, 95% CI = 0.89–1.06; P = 0.423). After adjusting for all confounding factors in a multivariable model, nail involvement was the only independent factor negatively affecting the probability of significant hair regrowth (adjusted HR = 0.04, 95% CI = 0.01–0.55; P = 0.015).Table 3Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale2.39 (1.09–4.61)0.035*1.13 (0.81–5.91)0.086Age at AA onset1.02 (0.89–1.06)0.423Age group at AA onset ● ≤40 yearsReference ● >40 years2.45 (1.49–3.69)0.046*1.68 (0.92–3.42)0.187Duration of disease >6 months0.63 (0.25–1.49)0.141AA subtype ● Patch AAReference ● Multiple AA1.34 (0.25–2.92)0.182 ● Diffuse AA2.63 (0.99–4.61)0.062 ● AT0.82 (0.29–2.85)0.845 ● AU0.16 (0.09–3.15)0.753 ● Ophiasis0.76 (0.09–2.92)0.911Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.56 (0.32–2.09)0.186 ● 51–75%0.64 (0.31–2.67)0.823 ● 76–100%1.49 (0.81–3.68)0.595Body hair involvement0.67 (0.03–4.28)0.745Nail involvement0.26 (0.08–0.69)0.036*0.04 (0.01–0.55)0.015*Atopy0.68 (0.13–5.16)0.621Autoimmune thyroid diseases1.91 (0.83–3.28)0.142Family history of AA0.96 (0.41–3.56)0.363Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\nThe Cox proportional hazards model was used to determine the factors associated with significant hair regrowth (Table 3). Univariable analysis revealed that the variables associated with significant hair regrowth were female sex (HR = 2.39, 95% CI = 1.09–4.61; P = 0.035), age at AA onset >40 years (HR = 2.45, 95% CI = 1.49–3.69; P = 0.046), and nail abnormalities (HR = 0.26, 95% CI = 0.08–0.69; P = 0.036). However, when age at AA onset was analyzed as a continuous variable, no association was found (HR = 1.02, 95% CI = 0.89–1.06; P = 0.423). After adjusting for all confounding factors in a multivariable model, nail involvement was the only independent factor negatively affecting the probability of significant hair regrowth (adjusted HR = 0.04, 95% CI = 0.01–0.55; P = 0.015).Table 3Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale2.39 (1.09–4.61)0.035*1.13 (0.81–5.91)0.086Age at AA onset1.02 (0.89–1.06)0.423Age group at AA onset ● ≤40 yearsReference ● >40 years2.45 (1.49–3.69)0.046*1.68 (0.92–3.42)0.187Duration of disease >6 months0.63 (0.25–1.49)0.141AA subtype ● Patch AAReference ● Multiple AA1.34 (0.25–2.92)0.182 ● Diffuse AA2.63 (0.99–4.61)0.062 ● AT0.82 (0.29–2.85)0.845 ● AU0.16 (0.09–3.15)0.753 ● Ophiasis0.76 (0.09–2.92)0.911Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.56 (0.32–2.09)0.186 ● 51–75%0.64 (0.31–2.67)0.823 ● 76–100%1.49 (0.81–3.68)0.595Body hair involvement0.67 (0.03–4.28)0.745Nail involvement0.26 (0.08–0.69)0.036*0.04 (0.01–0.55)0.015*Atopy0.68 (0.13–5.16)0.621Autoimmune thyroid diseases1.91 (0.83–3.28)0.142Family history of AA0.96 (0.41–3.56)0.363Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n Complete Hair Regrowth Regarding complete hair regrowth, the cumulative incidence was 48.5%, with an incidence rate of 8.0 (95% CI = 6.2–10.9) person-years. The median time and average cumulative dose of IMC to achieve complete hair regrowth were 6.1 (95% CI = 5.1–8.1) months and 160 (60–240) mg, respectively.\nRegarding complete hair regrowth, the cumulative incidence was 48.5%, with an incidence rate of 8.0 (95% CI = 6.2–10.9) person-years. The median time and average cumulative dose of IMC to achieve complete hair regrowth were 6.1 (95% CI = 5.1–8.1) months and 160 (60–240) mg, respectively.\n Relapse and Its Associated Factors Following 51 months of follow-up, the overall cumulative relapse rate after achievement of clinical hair regrowth with IMC was 47.5% (n = 48; Figure 1D). The incidence rate of AA relapse was 5.5 (95% CI = 4.2–7.3) person-years, and the median time to relapse was 11.3 (95% CI = 8.6–15.4) months. Treatment in patients with recurrence included combination therapy with IL corticosteroids and topical minoxidil (n = 21), topical contact immunotherapy (n = 10), methotrexate (n = 9), IMC (n = 4), and IL corticosteroids (n = 4). All therapies were prescribed at >6 months after discontinuation of the IMC regimen.\nThe risk of AA relapse was determined using the Cox proportional hazards model. Univariable analysis revealed that coexisting AITD (HR = 2.39, 95% CI = 1.42–5.14; P = 0.041) and disease duration >6 months (HR = 3.12, 95% CI = 1.36–4.94; P = 0.016) were associated with a higher probability of disease recurrence. However, multivariable analysis showed that only AA duration >6 months (adjusted HR = 4.02, 95% CI = 1.52–4.66; P = 0.005) independently increased the probability of AA relapse (Table 4).Table 4Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale1.15 (0.64–1.69)0.536Age at AA onset1.01 (0.99–1.06)0.104Age group at AA onset ● ≤40 yearsReference ● >40 years1.55 (0.78–2.56)0.151Duration of disease >6 months3.12 (1.36–4.94)0.016*4.02 (1.52–4.66)0.005*AA subtype ● Patch AAReference ● Multiple AA0.94 (0.66–2.67)0.723 ● Diffuse AA0.85 (0.39–3.62)0.264 ● AT3.12 (0.79–5.64)0.459 ● AU2.92 (0.59–3.95)0.644 ● Ophiasis1.49 (0.08–3.12)0.814Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.91 (0.72–2.19)0.135 ● 51–75%1.14 (0.51–2.67)0.718 ● 76–100%0.69 (0.29–1.89)0.653Body hair involvement1.05 (0.29–3.18)0.425Nail involvement2.78 (0.98–4.09)0.812Atopy1.34 (0.63–3.89)0.291Autoimmune thyroid diseases2.39 (1.42–5.14)0.041*2.71 (0.82–4.65)0.069Family history of AA1.76 (0.87–2.88)0.635Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\nFollowing 51 months of follow-up, the overall cumulative relapse rate after achievement of clinical hair regrowth with IMC was 47.5% (n = 48; Figure 1D). The incidence rate of AA relapse was 5.5 (95% CI = 4.2–7.3) person-years, and the median time to relapse was 11.3 (95% CI = 8.6–15.4) months. Treatment in patients with recurrence included combination therapy with IL corticosteroids and topical minoxidil (n = 21), topical contact immunotherapy (n = 10), methotrexate (n = 9), IMC (n = 4), and IL corticosteroids (n = 4). All therapies were prescribed at >6 months after discontinuation of the IMC regimen.\nThe risk of AA relapse was determined using the Cox proportional hazards model. Univariable analysis revealed that coexisting AITD (HR = 2.39, 95% CI = 1.42–5.14; P = 0.041) and disease duration >6 months (HR = 3.12, 95% CI = 1.36–4.94; P = 0.016) were associated with a higher probability of disease recurrence. However, multivariable analysis showed that only AA duration >6 months (adjusted HR = 4.02, 95% CI = 1.52–4.66; P = 0.005) independently increased the probability of AA relapse (Table 4).Table 4Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale1.15 (0.64–1.69)0.536Age at AA onset1.01 (0.99–1.06)0.104Age group at AA onset ● ≤40 yearsReference ● >40 years1.55 (0.78–2.56)0.151Duration of disease >6 months3.12 (1.36–4.94)0.016*4.02 (1.52–4.66)0.005*AA subtype ● Patch AAReference ● Multiple AA0.94 (0.66–2.67)0.723 ● Diffuse AA0.85 (0.39–3.62)0.264 ● AT3.12 (0.79–5.64)0.459 ● AU2.92 (0.59–3.95)0.644 ● Ophiasis1.49 (0.08–3.12)0.814Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.91 (0.72–2.19)0.135 ● 51–75%1.14 (0.51–2.67)0.718 ● 76–100%0.69 (0.29–1.89)0.653Body hair involvement1.05 (0.29–3.18)0.425Nail involvement2.78 (0.98–4.09)0.812Atopy1.34 (0.63–3.89)0.291Autoimmune thyroid diseases2.39 (1.42–5.14)0.041*2.71 (0.82–4.65)0.069Family history of AA1.76 (0.87–2.88)0.635Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n Adverse Events Adverse events were experienced by 18.8% of the patients (n = 19) and included acneiform eruption (n = 16, 15.8%), abnormal menstrual cycle (n = 6, 5.9%), weight gain (n = 3, 2.9%), and sleep disturbance (n = 1, 0.9%). Acneiform eruption was managed with topical therapy, while the other side effects resolved spontaneously after discontinuing IMC. The presence of any of these adverse events did not cause dropout from therapy.\nAdverse events were experienced by 18.8% of the patients (n = 19) and included acneiform eruption (n = 16, 15.8%), abnormal menstrual cycle (n = 6, 5.9%), weight gain (n = 3, 2.9%), and sleep disturbance (n = 1, 0.9%). Acneiform eruption was managed with topical therapy, while the other side effects resolved spontaneously after discontinuing IMC. The presence of any of these adverse events did not cause dropout from therapy.",
"A total of 126 patients with AA treated with IMC were initially included. Twenty-five patients were excluded due to coexisting diseases or concomitant use of other medications affecting the hair growth cycle (n = 16), incomplete medical records (n = 5), or because they received systemic treatment after the IMC protocol (n = 4). The majority of the remaining 101 patients were female (n = 70, 69.3%) with a female to male ratio of 2.26:1. The age range of AA onset was 5 to 68 years (mean = 40.4 ± 12.6 years), and the duration of disease varied from 1 week to 11 months (median = 2 [1–4] months). The most common AA subtype was multiple patches (n = 46, 45.5%), followed by patch (n = 20, 19.8%), diffuse (n = 16, 15.8%), AT (n = 9, 8.9%), AU (n = 5, 5.0%), and ophiasis (n = 5, 5.0%). The estimated baseline SALT score was 0–25%, 26–50%, 51–75%, or 76–100% in 11 (10.9%), 16 (15.8%), 60 (59.4%), and 14 patients (13.9%), respectively. Body hair involvement was found in 5.9% (n = 6), and nail abnormalities were observed in 8.9% (n = 9) of the patients. Six patients (5.9%) had coexisting AITD, 2 (1.9%) were atopic, and 2 (1.9%) had a family member with AA. All demographics are summarized in Table 1.Table 1Demographic and Clinical Characteristics of Participants with Alopecia AreataVariablesN = 101Sex, n (%) ● Male31 (30.7) ● Female70 (69.3)Age at AA onset, year, mean ± SD40.4 ± 12.6Age at AA onset, year, n (%) ● ≤40 years50 (49.5) ● >40 years51 (50.5)Duration of disease, month, median (IQR)2 (1–4)Duration of disease, n (%) ● ≤6 months77 (76.2) ● >6 months24 (23.8)AA subtype, n (%) ● Patch AA20 (19.8) ● Multiple AA46 (45.5) ● Diffuse AA16 (15.8) ● AT9 (8.9) ● AU5 (5.0) ● Ophiasis5 (5.0)Severity of AA (SALT score), n (%) ● 0–25%11 (10.9) ● 26–50%16 (15.8) ● 51–75%60 (59.4) ● 76–100%14 (13.9)Body hair involvement, n (%)6 (5.9)Nail involvement, n (%)9 (8.9)Comorbidity, n (%) ● Atopy2 (1.9) ● Autoimmune thyroid diseases6 (5.9)Family history of AA, n (%)2 (1.9)Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.\n\nDemographic and Clinical Characteristics of Participants with Alopecia Areata\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.",
"The median overall follow-up time for this cohort was 20 (6–38) months. Patients received 1 to 6 IMC injections (mean = 3.12 ± 0.9) per treatment course. Ninety-eight patients (97.1%) showed improvement, whereas 3 patients (2.9%) showed treatment failure and were further treated by topical contact immunotherapy at approximately 9 months (36 weeks in 2 patients and 37 weeks in another one) after completion of IMC therapy. The mean duration of IMC therapy was 4.2 ± 1.1 months, with a range of 1 to 6 months. Treatment responses are summarized in Table 2.Table 2Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia AreataTreatment OutcomesN = 101Treatment failure (<10% regrowth) ● Incidence, n (%)3 (2.9)Initial hair regrowth (25% regrowth) ● Cumulative incidence, n (%)86 (85.1) ● Incidence rate, person-year (CI)64.0 (51.9–79.3) ● Median time to achieve endpoint, month (CI)0.9 (0.8–1.2) ● Median cumulative dose to achieve endpoint, mg100 ● Cumulative dose to achieve endpoint, mg, median (IQR)80 (20–160)Significant hair regrowth (75% regrowth) ● Cumulative incidence, n (%)81 (80.2) ● Incidence rate, person-year (CI)17.0 (13.6–21.7) ● Median time to achieve endpoint, month (CI)3.4 (2.9–4.4) ● Median cumulative dose to achieve endpoint, mg200 ● Cumulative dose to achieve endpoint, mg, median (IQR)120 (40–200)Complete hair regrowth (100% regrowth) ● Cumulative incidence, n (%)49 (48.5) ● Incidence rate, person-year (CI)8.0 (6.2–10.9) ● Median time to achieve endpoint, month (CI)6.1 (5.1–8.1) ● Cumulative dose to achieve endpoint, mg, median (IQR)160 (60–240)Relapse ● Cumulative incidence, n (%)48 (47.5) ● Incidence rate, person-year (CI)5.5 (4.2–7.3) ● Median time to relapse, month (CI)11.3 (8.6–15.4)Abbreviations: CI, confidence interval; IQR, interquartile range.\n\nTreatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia Areata\nAbbreviations: CI, confidence interval; IQR, interquartile range.",
"According to survival analysis, the cumulative incidence of initial hair regrowth after IMC therapy was 85.1%, with an incidence rate of 64.0 (95% CI = 51.9–79.3) person-years. The median time to achieve initial hair regrowth was 0.9 (95% CI = 0.8–1.2) months, and the average cumulative dose of IMC to achieve initial hair regrowth was 80 (20–160) mg.",
"Significant hair regrowth was indicated as the primary outcome and its cumulative incidence in our cohort was 80.2%, with an incidence rate of 17.0 (95% CI = 13.6–21.7) person-years. The median time required to achieve significant hair regrowth was 3.4 (95% CI = 2.9–4.4) months. According to the Kaplan–Meier curve, there was a lag of approximately 1 month from initiation of therapy to the first patients demonstrating significant hair regrowth. Following this initial event, significant hair regrowth was observed in 41.9% of patients at 3 months, 75.9% at 6 months, 81.0% at 9 months, and 86.5% at 12 months. The residual probabilities of significant regrowth as a function of the time after initiation of IMC are also shown in Figure 1A. The average cumulative dose of IMC to achieve significant hair regrowth was 120 (40–200) mg, while a median cumulative dose that 50% of patients attained significant hair regrowth was 200 mg (Figure 1B).Figure 1Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nKaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nThe baseline SALT score was identified as a negative factor for IMC treatment response in a previous study.31 To determine the effect of this variable on significant hair regrowth, a stratified Kaplan–Meier analysis was performed. Nine patients (81.8%) with a score of 0–25% obtained significant hair regrowth by a median time of 3.5 months; for the score range 26–50%, 12 (75.0%) patients achieved significant regrowth by 4.8 months; for the score range 51–75%, 48 (80.0%) experienced significant regrowth by 3.1 months; and for the score range 76–100%, 12 (85.7%) ultimately obtained significant regrowth at 3.2 months. However, the log rank test failed to demonstrate statistically significant differences among the four groups (P = 0.287; Figure 1C).",
"The Cox proportional hazards model was used to determine the factors associated with significant hair regrowth (Table 3). Univariable analysis revealed that the variables associated with significant hair regrowth were female sex (HR = 2.39, 95% CI = 1.09–4.61; P = 0.035), age at AA onset >40 years (HR = 2.45, 95% CI = 1.49–3.69; P = 0.046), and nail abnormalities (HR = 0.26, 95% CI = 0.08–0.69; P = 0.036). However, when age at AA onset was analyzed as a continuous variable, no association was found (HR = 1.02, 95% CI = 0.89–1.06; P = 0.423). After adjusting for all confounding factors in a multivariable model, nail involvement was the only independent factor negatively affecting the probability of significant hair regrowth (adjusted HR = 0.04, 95% CI = 0.01–0.55; P = 0.015).Table 3Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale2.39 (1.09–4.61)0.035*1.13 (0.81–5.91)0.086Age at AA onset1.02 (0.89–1.06)0.423Age group at AA onset ● ≤40 yearsReference ● >40 years2.45 (1.49–3.69)0.046*1.68 (0.92–3.42)0.187Duration of disease >6 months0.63 (0.25–1.49)0.141AA subtype ● Patch AAReference ● Multiple AA1.34 (0.25–2.92)0.182 ● Diffuse AA2.63 (0.99–4.61)0.062 ● AT0.82 (0.29–2.85)0.845 ● AU0.16 (0.09–3.15)0.753 ● Ophiasis0.76 (0.09–2.92)0.911Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.56 (0.32–2.09)0.186 ● 51–75%0.64 (0.31–2.67)0.823 ● 76–100%1.49 (0.81–3.68)0.595Body hair involvement0.67 (0.03–4.28)0.745Nail involvement0.26 (0.08–0.69)0.036*0.04 (0.01–0.55)0.015*Atopy0.68 (0.13–5.16)0.621Autoimmune thyroid diseases1.91 (0.83–3.28)0.142Family history of AA0.96 (0.41–3.56)0.363Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.",
"Regarding complete hair regrowth, the cumulative incidence was 48.5%, with an incidence rate of 8.0 (95% CI = 6.2–10.9) person-years. The median time and average cumulative dose of IMC to achieve complete hair regrowth were 6.1 (95% CI = 5.1–8.1) months and 160 (60–240) mg, respectively.",
"Following 51 months of follow-up, the overall cumulative relapse rate after achievement of clinical hair regrowth with IMC was 47.5% (n = 48; Figure 1D). The incidence rate of AA relapse was 5.5 (95% CI = 4.2–7.3) person-years, and the median time to relapse was 11.3 (95% CI = 8.6–15.4) months. Treatment in patients with recurrence included combination therapy with IL corticosteroids and topical minoxidil (n = 21), topical contact immunotherapy (n = 10), methotrexate (n = 9), IMC (n = 4), and IL corticosteroids (n = 4). All therapies were prescribed at >6 months after discontinuation of the IMC regimen.\nThe risk of AA relapse was determined using the Cox proportional hazards model. Univariable analysis revealed that coexisting AITD (HR = 2.39, 95% CI = 1.42–5.14; P = 0.041) and disease duration >6 months (HR = 3.12, 95% CI = 1.36–4.94; P = 0.016) were associated with a higher probability of disease recurrence. However, multivariable analysis showed that only AA duration >6 months (adjusted HR = 4.02, 95% CI = 1.52–4.66; P = 0.005) independently increased the probability of AA relapse (Table 4).Table 4Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale1.15 (0.64–1.69)0.536Age at AA onset1.01 (0.99–1.06)0.104Age group at AA onset ● ≤40 yearsReference ● >40 years1.55 (0.78–2.56)0.151Duration of disease >6 months3.12 (1.36–4.94)0.016*4.02 (1.52–4.66)0.005*AA subtype ● Patch AAReference ● Multiple AA0.94 (0.66–2.67)0.723 ● Diffuse AA0.85 (0.39–3.62)0.264 ● AT3.12 (0.79–5.64)0.459 ● AU2.92 (0.59–3.95)0.644 ● Ophiasis1.49 (0.08–3.12)0.814Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.91 (0.72–2.19)0.135 ● 51–75%1.14 (0.51–2.67)0.718 ● 76–100%0.69 (0.29–1.89)0.653Body hair involvement1.05 (0.29–3.18)0.425Nail involvement2.78 (0.98–4.09)0.812Atopy1.34 (0.63–3.89)0.291Autoimmune thyroid diseases2.39 (1.42–5.14)0.041*2.71 (0.82–4.65)0.069Family history of AA1.76 (0.87–2.88)0.635Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.",
"Adverse events were experienced by 18.8% of the patients (n = 19) and included acneiform eruption (n = 16, 15.8%), abnormal menstrual cycle (n = 6, 5.9%), weight gain (n = 3, 2.9%), and sleep disturbance (n = 1, 0.9%). Acneiform eruption was managed with topical therapy, while the other side effects resolved spontaneously after discontinuing IMC. The presence of any of these adverse events did not cause dropout from therapy.",
"Although IMC therapy has shown to be effective in the treatment of several dermatological conditions and gained popularity for AA treatment in recent years, information regarding the use of IMC for AA is limited in the literature.32 In this study, we modeled the therapeutic endpoints of the IMC regimen according to multiple variables in patients with AA. To the best of our knowledge, this is the first study to describe the efficacy, cumulative effective dose, and factors associated with treatment outcomes in patients with AA receiving IMC based on a time-to-event analysis. The primary endpoint, defined as significant hair regrowth, had a rate of 80.2%, with a median time of approximately 3–4 months. Nail involvement was demonstrated to be a negative predictor of significant hair regrowth. Relapse was found in 47.5% of the patients, with disease duration >6 months being a risk factor for AA recurrence.\nThe application of systemic corticosteroids for the treatment of AA was first introduced by Dillaha et al in 1952, with remarkable therapeutic effects.33 Since then, the efficacy of various regimens such as oral, intravenous, and intramuscular administration has been investigated. Oral corticosteroid pulse therapy is generally the most convenient method and has been well studied.34 However, poor medication compliance and adherence may lead to reduced treatment efficacy and substantial worsening of the disease. The therapeutic effect of intravenous pulse corticosteroids has also been confirmed. However, the overall adverse events of intramuscular and oral corticosteroids are relatively high, approaching 41% and 30%, respectively, compared to that of 10% from corticosteroid pulse therapy.29 Hypothalamic–pituitary–adrenal (HPA) axis suppression was reported in 23%, 67%, and 7% of intramuscular, oral, and pulse corticosteroids, respectively.29 Intramuscular injections may offer an advantage over the two methods. IMC maximizes treatment efficacy by providing rapid onset of action comparable to the oral form, and by increasing patients’ compliance and adherence owing to the monthly or bimonthly injection regimen.28 It also minimizes adverse effects due to its pharmacokinetic properties, with a gradual and steady release of corticosteroids over time.28\nOur study demonstrated the efficacy of IMC for the treatment of severe, acute, or rapidly progressive AA, as shown by the 80.2% cumulative incidence of significant hair regrowth rate in a median time of 3.4 months. Michalowski and Kuczyfiska reported a 72.7% response rate in 11 cases of extensive AA (AT/AU) receiving 40–80 mg of IMC therapy, initially once a week, then over a gradually lengthened interval up to 6 weeks.31 A retrospective study using IMC 40 mg every 4 weeks for a maximum of 6 months revealed a 63% response rate in refractory AA.30 Administration of IMC 40 mg monthly for 6 months, followed by 40 mg every 1.5 months for 1 year, had significantly superior efficacy, with a 74% response rate, to oral corticosteroid pulse therapy.29 However, our results could not be directly compared to those of earlier studies because of the difference in the severity of AA among the cohorts. The lower percentage of recalcitrant AA cases included in our study compared with previous ones could explain the higher response rate.\nRegarding other corticosteroid injection protocols for AA, IL injection of 2.5–10 mg/mL triamcinolone acetonide at four–to-six-week intervals is commonly used in mild forms of AA (ie, patch AA or SALT score <25%) with a response rate between 64% and 97%.23 For severe AA, intravenous injection of 500 mg methylprednisolone for three consecutive days at four-week intervals for three months has a response rate of approximately 80%.23 However, compared to IL injection, the dosage for intramuscular and intravenous injections is higher and may lead to potential adverse effects; therefore, they should be restricted to those with severe AA and administered with caution.\nIdentifying the factors affecting treatment outcomes of AA is essential for therapeutic success; nevertheless, specific predictors for IMC have been rarely reported. Seo et al indicated that the baseline SALT score was negatively correlated with hair regrowth response to IMC.30 Our stratified analysis using the Kaplan–Meier method did not support this finding, as no association was found between the extent of scalp involvement and treatment response. Earlier age at AA onset, longer disease duration, and nail involvement were identified as indicators of poor prognosis.35–38 A recent meta-analysis demonstrated four negative predictors for treatment response, namely SALT score ≥50, disease duration ≥1 year, atopic diseases, and nail abnormalities.35 Our analysis using a Cox proportional hazards model showed a lack of correlation between significant hair regrowth and SALT score, AA duration, or presence of atopy, but confirmed its negative association with nail abnormalities. This finding could be explained by the characteristics of the enrolled subjects in our cohort, namely the predominance of female patients presenting with rapidly progressive scalp hair loss >50% for <6 months. The clinical characteristics of our patients were compatible to those of patients with acute diffuse and total alopecia, a distinctive AA subtype, which shows a favorable treatment response to systemic corticosteroid therapy.39,40\nEmerging evidence suggests that approximately half the patients with AA treated with IMC experience disease recurrence. Seo et al demonstrated a 47.1% relapse rate during a follow-up period of 14.2 months.30 Kurosawa et al reported a comparable relapse rate of 46% after 6 months of treatment.29 Our result was consistent with previous studies, revealing recurrence in approximately half of patients with a median time of 11.3 months. Furthermore, disease duration >6 months was identified as a predictor of AA relapse. Longer AA duration could indicate disease chronicity, and physicians should remain vigilant for AA relapse despite complete IMC therapy. Therefore, clinicians should consider the use of post-IMC treatments such as application of 2% topical minoxidil three times daily, which was reported to limit post-steroid-treatment hair loss.41 For AA patients with chronic relapsing disease, further treatments after initial systemic corticosteroid therapy are challenging owing to a lack of high-quality randomized controlled trials supporting the efficacy and safety of other therapeutic modalities. However, a shared decision-making approach between the physician and patient is crucial as it would ensure that patients have a realistic expectation of treatment outcomes. Topical contact immunotherapy is currently considered the best modality for long-term therapy of patients with recalcitrant AA and is the first line treatment for these cases in our hair clinic.19\nHPA axis suppression is considered a major side effect of IMC therapy.32 Previous studies demonstrated adrenocortical alteration in 23% of subjects receiving intramuscular injection.29 Other adverse effects of IMC include abdominal discomfort, dysmenorrhea, and acne.29,30 The incidence of adverse events among our AA patients was low, and acneiform eruption was the most common; no serious side effects were reported. However, HPA axis suppression in our subjects may be underestimated due to a lack of data regarding adrenocortical evaluation in our follow-up protocol.\nA major strength of our study is the use of the time-to-event approach to characterize the therapeutic outcomes of patients with AA receiving IMC during the entire follow-up time, including censored observations. This method considers all available treatment and follow-up data, providing more precise and meaningful estimates of overall efficacy and tolerability, and better outcome prediction. The limitations of this study are its retrospective design involving a single tertiary center. These factors might lead to the unavailability of some data and limit the generalizability of the results. Our results may also be limited in their applicability to patients without severe and/or rapidly progressive AA. Additionally, it is difficult to compare our findings to those of previous studies, owing to differences in the definitions of treatment responses and in methodologies. Further large, multicenter randomized controlled trials evaluating the efficacy and safety of IMC therapy are required to overcome these limitations.",
"Our time-to-event analysis demonstrated the efficacy and safety of intramuscular administration of corticosteroids for the treatment of AA. IMC therapy showed high rates of significant hair regrowth, especially in patients with severe or active AA; however, approximately half of those experienced relapse. Nail involvement was negatively correlated with cosmetically acceptable hair regrowth, while disease duration >6 months was significantly associated with increased risk of relapse. Most importantly, the use of IMC depends on indication and perceived potential benefits and risks; therefore, physicians should be cautious when prescribing this treatment."
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[
"Introduction",
"Materials and Methods",
"Study Design and Population",
"Data Collection and Clinical Outcomes",
"Statistical Analysis",
"Results",
"Demographics",
"Overall Response",
"Initial Hair Regrowth",
"Significant Hair Regrowth",
"Factors Associated with Significant Hair Regrowth",
"Complete Hair Regrowth",
"Relapse and Its Associated Factors",
"Adverse Events",
"Discussion",
"Conclusion"
] |
[
"Alopecia areata (AA) is a non-scarring hair loss disorder that affects the scalp and hair-bearing areas.1 Variations on the clinical presentation of AA have been observed, ranging from small, well-defined circular or oval patches of hair loss to diffuse alopecia and complete scalp (alopecia totalis, AT) or total body hair loss (alopecia universalis, AU).2–5 Nail abnormalities, most frequently nail pitting, are occasionally present. Patients with AA often experience major psychological impacts due to the unpredictable clinical course and variable treatment response.6 Various immune-related comorbidities such as autoimmune thyroid diseases (AITD), vitiligo, psoriasis, and atopic diseases have been linked to AA.7–13\nThe exact etiopathogenesis of AA is unclear, though it is believed to be a cell-mediated autoimmune disease of the hair follicle. Autoreactive T-cell lymphocytes and several cytokines mediate hair follicular destruction and negatively affect the hair growth cycle.14–16 Despite the absence of curative/preventive treatments and FDA-approved regimens for AA, therapeutic approaches have been established through treatment guidelines. Topical and intralesional (IL) corticosteroids, topical minoxidil, and topical anthralin are recommended for AA patients with <50% scalp involvement, whereas in those with ≥50% hair loss, topical contact immunotherapy and systemic corticosteroids are advised.17–23\nCorticosteroids, the main treatment option for AA, can be administered in different forms depending on the disease severity.24 Medium- to high-potency topical and IL corticosteroids are indicated as monotherapy or combined therapy in limited and/or slowly progressive AA,25–27 while systemic corticosteroids (eg, oral, intravenous, and intramuscular), are preferred in extensive and/or rapidly progressive disease.17\nAmong systemic modalities, intramuscular corticosteroids (IMC) may be most beneficial, as its monthly or bimonthly regimen increases patients’ compliance and treatment adherence.28 Moreover, IMC could minimize adverse effects from slowly releasing continuous and small amounts of medication.28 Previous studies of IMC showed a satisfactory outcome in severe and refractory AA, with response rates between 63% and 72.7%.29–31 Although IMC is often the preferred treatment for AA, there are limited data regarding its effectiveness, safety, dosing, and optimal duration. Thus, we conducted a retrospective cohort study based on our 20-year experience to evaluate the efficacy, relapse, and tolerability, as well as factors associated with treatment outcomes of IMC in patients with AA.",
" Study Design and Population This was a retrospective cohort study conducted at the Dermatology Clinic of Ramathibodi Hospital, Bangkok, Thailand. The study was approved by the Mahidol University Review Board for Ethics in Human Research (MURA2020/1282) and was performed in accordance with the principles of Declaration of Helsinki. The requirement for informed consent was waived, and the data were anonymized before analysis.\nWe reviewed the medical records of patients clinically and/or histopathologically diagnosed with AA between January 2000 and December 2019. Individuals with AA receiving an IMC treatment regimen were included in the study. Our IMC protocol was triamcinolone acetonide 20–40 mg/mL injected every 4–6 weeks. The treatment was indicated in patients with severe (≥50% scalp involvement), extensive (AT, AU, and ≥50% scalp involvement with body hair loss), or rapidly progressive AA (accelerated hair loss spreading ≥5% of scalp area per week for >2 consecutive weeks with positive hair-pull test results at the periphery of alopecic patch or across the entire scalp), and IMC was performed until patients achieved significant hair regrowth. Patients who did not achieve significant hair regrowth after six IMC injections were switched to other appropriate therapies to minimize adrenocortical alteration.32 We excluded participants treated with systemic immunosuppressive agents or topical contact immunotherapy within 6 months after discontinuation of the IMC protocol; therefore, those who underwent topical treatment (ie, topical corticosteroids and/or topical minoxidil) remained in our cohort. Patients with incomplete medical records, those who received other AA treatments during IMC therapy, those with other hair and scalp disorders, and those with systemic diseases or using medications affecting the hair growth cycle were also excluded.\nThis was a retrospective cohort study conducted at the Dermatology Clinic of Ramathibodi Hospital, Bangkok, Thailand. The study was approved by the Mahidol University Review Board for Ethics in Human Research (MURA2020/1282) and was performed in accordance with the principles of Declaration of Helsinki. The requirement for informed consent was waived, and the data were anonymized before analysis.\nWe reviewed the medical records of patients clinically and/or histopathologically diagnosed with AA between January 2000 and December 2019. Individuals with AA receiving an IMC treatment regimen were included in the study. Our IMC protocol was triamcinolone acetonide 20–40 mg/mL injected every 4–6 weeks. The treatment was indicated in patients with severe (≥50% scalp involvement), extensive (AT, AU, and ≥50% scalp involvement with body hair loss), or rapidly progressive AA (accelerated hair loss spreading ≥5% of scalp area per week for >2 consecutive weeks with positive hair-pull test results at the periphery of alopecic patch or across the entire scalp), and IMC was performed until patients achieved significant hair regrowth. Patients who did not achieve significant hair regrowth after six IMC injections were switched to other appropriate therapies to minimize adrenocortical alteration.32 We excluded participants treated with systemic immunosuppressive agents or topical contact immunotherapy within 6 months after discontinuation of the IMC protocol; therefore, those who underwent topical treatment (ie, topical corticosteroids and/or topical minoxidil) remained in our cohort. Patients with incomplete medical records, those who received other AA treatments during IMC therapy, those with other hair and scalp disorders, and those with systemic diseases or using medications affecting the hair growth cycle were also excluded.\n Data Collection and Clinical Outcomes Baseline demographics, clinical characteristics, duration of disease, AA subtypes (ie, patch, multiple patches, AT/AU, and ophiasis), body hair involvement, nail abnormalities, and family history of AA were collected. AA severity was evaluated using the Severity of Alopecia Tool (SALT) score. Details regarding IMC regimen were obtained, including dosage, duration, and clinical outcomes. Efficacy and tolerability assessment were performed at baseline and at every follow-up visit. The percentage of scalp area coverage by terminal hair regrowth from baseline was used to evaluate treatment response. Clinical endpoints included initial, significant, and complete hair regrowth, treatment failure, and relapse. Initial hair regrowth (noticeable) was defined as 25% regrowth, while significant hair regrowth (cosmetically acceptable) was defined as 75% regrowth. Patients with 100% regrowth were classified as having complete (full) hair regrowth. Treatment failure was defined as <10% hair regrowth after 6 months of IMC therapy. Relapse was defined as any new appearance of AA during follow-up visits. The cumulative incidence of significant hair regrowth was set as the outcome of interest in the time-to-event analysis. Furthermore, the median time and median cumulative corticosteroid dosage to achieve each clinical endpoint were evaluated. Relevant factors affecting significant hair regrowth and relapse were also analyzed.\nBaseline demographics, clinical characteristics, duration of disease, AA subtypes (ie, patch, multiple patches, AT/AU, and ophiasis), body hair involvement, nail abnormalities, and family history of AA were collected. AA severity was evaluated using the Severity of Alopecia Tool (SALT) score. Details regarding IMC regimen were obtained, including dosage, duration, and clinical outcomes. Efficacy and tolerability assessment were performed at baseline and at every follow-up visit. The percentage of scalp area coverage by terminal hair regrowth from baseline was used to evaluate treatment response. Clinical endpoints included initial, significant, and complete hair regrowth, treatment failure, and relapse. Initial hair regrowth (noticeable) was defined as 25% regrowth, while significant hair regrowth (cosmetically acceptable) was defined as 75% regrowth. Patients with 100% regrowth were classified as having complete (full) hair regrowth. Treatment failure was defined as <10% hair regrowth after 6 months of IMC therapy. Relapse was defined as any new appearance of AA during follow-up visits. The cumulative incidence of significant hair regrowth was set as the outcome of interest in the time-to-event analysis. Furthermore, the median time and median cumulative corticosteroid dosage to achieve each clinical endpoint were evaluated. Relevant factors affecting significant hair regrowth and relapse were also analyzed.\n Statistical Analysis Statistical analyses were performed using Stata 14.1 (Stata Corp, College Station, TX, USA). To detect a modestly sized hazard ratio (HR), the sample size was calculated based on data from a previous study regarding the IMC treatment of patients with AA.31 The minimum number of subjects required to achieve a statistical significance level of 5% and a power of 80% was 26. Continuous variables were reported as mean ± standard deviation or median (interquartile range, IQR). Categorical variables were expressed as number or proportion. Survival analysis was performed using the Kaplan–Meier method to estimate outcome rate, median time, and median cumulative corticosteroids to achieve endpoints. The log rank test was used to compare survival distributions between variables. Factors affecting clinical outcomes were assessed using univariable and multivariable stepwise analyses under the Cox proportional hazards regression model and expressed as HRs with 95% confidence intervals (CI). All analyses were two-tailed, and P-values <0.05 were considered statistically significant.\nStatistical analyses were performed using Stata 14.1 (Stata Corp, College Station, TX, USA). To detect a modestly sized hazard ratio (HR), the sample size was calculated based on data from a previous study regarding the IMC treatment of patients with AA.31 The minimum number of subjects required to achieve a statistical significance level of 5% and a power of 80% was 26. Continuous variables were reported as mean ± standard deviation or median (interquartile range, IQR). Categorical variables were expressed as number or proportion. Survival analysis was performed using the Kaplan–Meier method to estimate outcome rate, median time, and median cumulative corticosteroids to achieve endpoints. The log rank test was used to compare survival distributions between variables. Factors affecting clinical outcomes were assessed using univariable and multivariable stepwise analyses under the Cox proportional hazards regression model and expressed as HRs with 95% confidence intervals (CI). All analyses were two-tailed, and P-values <0.05 were considered statistically significant.",
"This was a retrospective cohort study conducted at the Dermatology Clinic of Ramathibodi Hospital, Bangkok, Thailand. The study was approved by the Mahidol University Review Board for Ethics in Human Research (MURA2020/1282) and was performed in accordance with the principles of Declaration of Helsinki. The requirement for informed consent was waived, and the data were anonymized before analysis.\nWe reviewed the medical records of patients clinically and/or histopathologically diagnosed with AA between January 2000 and December 2019. Individuals with AA receiving an IMC treatment regimen were included in the study. Our IMC protocol was triamcinolone acetonide 20–40 mg/mL injected every 4–6 weeks. The treatment was indicated in patients with severe (≥50% scalp involvement), extensive (AT, AU, and ≥50% scalp involvement with body hair loss), or rapidly progressive AA (accelerated hair loss spreading ≥5% of scalp area per week for >2 consecutive weeks with positive hair-pull test results at the periphery of alopecic patch or across the entire scalp), and IMC was performed until patients achieved significant hair regrowth. Patients who did not achieve significant hair regrowth after six IMC injections were switched to other appropriate therapies to minimize adrenocortical alteration.32 We excluded participants treated with systemic immunosuppressive agents or topical contact immunotherapy within 6 months after discontinuation of the IMC protocol; therefore, those who underwent topical treatment (ie, topical corticosteroids and/or topical minoxidil) remained in our cohort. Patients with incomplete medical records, those who received other AA treatments during IMC therapy, those with other hair and scalp disorders, and those with systemic diseases or using medications affecting the hair growth cycle were also excluded.",
"Baseline demographics, clinical characteristics, duration of disease, AA subtypes (ie, patch, multiple patches, AT/AU, and ophiasis), body hair involvement, nail abnormalities, and family history of AA were collected. AA severity was evaluated using the Severity of Alopecia Tool (SALT) score. Details regarding IMC regimen were obtained, including dosage, duration, and clinical outcomes. Efficacy and tolerability assessment were performed at baseline and at every follow-up visit. The percentage of scalp area coverage by terminal hair regrowth from baseline was used to evaluate treatment response. Clinical endpoints included initial, significant, and complete hair regrowth, treatment failure, and relapse. Initial hair regrowth (noticeable) was defined as 25% regrowth, while significant hair regrowth (cosmetically acceptable) was defined as 75% regrowth. Patients with 100% regrowth were classified as having complete (full) hair regrowth. Treatment failure was defined as <10% hair regrowth after 6 months of IMC therapy. Relapse was defined as any new appearance of AA during follow-up visits. The cumulative incidence of significant hair regrowth was set as the outcome of interest in the time-to-event analysis. Furthermore, the median time and median cumulative corticosteroid dosage to achieve each clinical endpoint were evaluated. Relevant factors affecting significant hair regrowth and relapse were also analyzed.",
"Statistical analyses were performed using Stata 14.1 (Stata Corp, College Station, TX, USA). To detect a modestly sized hazard ratio (HR), the sample size was calculated based on data from a previous study regarding the IMC treatment of patients with AA.31 The minimum number of subjects required to achieve a statistical significance level of 5% and a power of 80% was 26. Continuous variables were reported as mean ± standard deviation or median (interquartile range, IQR). Categorical variables were expressed as number or proportion. Survival analysis was performed using the Kaplan–Meier method to estimate outcome rate, median time, and median cumulative corticosteroids to achieve endpoints. The log rank test was used to compare survival distributions between variables. Factors affecting clinical outcomes were assessed using univariable and multivariable stepwise analyses under the Cox proportional hazards regression model and expressed as HRs with 95% confidence intervals (CI). All analyses were two-tailed, and P-values <0.05 were considered statistically significant.",
" Demographics A total of 126 patients with AA treated with IMC were initially included. Twenty-five patients were excluded due to coexisting diseases or concomitant use of other medications affecting the hair growth cycle (n = 16), incomplete medical records (n = 5), or because they received systemic treatment after the IMC protocol (n = 4). The majority of the remaining 101 patients were female (n = 70, 69.3%) with a female to male ratio of 2.26:1. The age range of AA onset was 5 to 68 years (mean = 40.4 ± 12.6 years), and the duration of disease varied from 1 week to 11 months (median = 2 [1–4] months). The most common AA subtype was multiple patches (n = 46, 45.5%), followed by patch (n = 20, 19.8%), diffuse (n = 16, 15.8%), AT (n = 9, 8.9%), AU (n = 5, 5.0%), and ophiasis (n = 5, 5.0%). The estimated baseline SALT score was 0–25%, 26–50%, 51–75%, or 76–100% in 11 (10.9%), 16 (15.8%), 60 (59.4%), and 14 patients (13.9%), respectively. Body hair involvement was found in 5.9% (n = 6), and nail abnormalities were observed in 8.9% (n = 9) of the patients. Six patients (5.9%) had coexisting AITD, 2 (1.9%) were atopic, and 2 (1.9%) had a family member with AA. All demographics are summarized in Table 1.Table 1Demographic and Clinical Characteristics of Participants with Alopecia AreataVariablesN = 101Sex, n (%) ● Male31 (30.7) ● Female70 (69.3)Age at AA onset, year, mean ± SD40.4 ± 12.6Age at AA onset, year, n (%) ● ≤40 years50 (49.5) ● >40 years51 (50.5)Duration of disease, month, median (IQR)2 (1–4)Duration of disease, n (%) ● ≤6 months77 (76.2) ● >6 months24 (23.8)AA subtype, n (%) ● Patch AA20 (19.8) ● Multiple AA46 (45.5) ● Diffuse AA16 (15.8) ● AT9 (8.9) ● AU5 (5.0) ● Ophiasis5 (5.0)Severity of AA (SALT score), n (%) ● 0–25%11 (10.9) ● 26–50%16 (15.8) ● 51–75%60 (59.4) ● 76–100%14 (13.9)Body hair involvement, n (%)6 (5.9)Nail involvement, n (%)9 (8.9)Comorbidity, n (%) ● Atopy2 (1.9) ● Autoimmune thyroid diseases6 (5.9)Family history of AA, n (%)2 (1.9)Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.\n\nDemographic and Clinical Characteristics of Participants with Alopecia Areata\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.\nA total of 126 patients with AA treated with IMC were initially included. Twenty-five patients were excluded due to coexisting diseases or concomitant use of other medications affecting the hair growth cycle (n = 16), incomplete medical records (n = 5), or because they received systemic treatment after the IMC protocol (n = 4). The majority of the remaining 101 patients were female (n = 70, 69.3%) with a female to male ratio of 2.26:1. The age range of AA onset was 5 to 68 years (mean = 40.4 ± 12.6 years), and the duration of disease varied from 1 week to 11 months (median = 2 [1–4] months). The most common AA subtype was multiple patches (n = 46, 45.5%), followed by patch (n = 20, 19.8%), diffuse (n = 16, 15.8%), AT (n = 9, 8.9%), AU (n = 5, 5.0%), and ophiasis (n = 5, 5.0%). The estimated baseline SALT score was 0–25%, 26–50%, 51–75%, or 76–100% in 11 (10.9%), 16 (15.8%), 60 (59.4%), and 14 patients (13.9%), respectively. Body hair involvement was found in 5.9% (n = 6), and nail abnormalities were observed in 8.9% (n = 9) of the patients. Six patients (5.9%) had coexisting AITD, 2 (1.9%) were atopic, and 2 (1.9%) had a family member with AA. All demographics are summarized in Table 1.Table 1Demographic and Clinical Characteristics of Participants with Alopecia AreataVariablesN = 101Sex, n (%) ● Male31 (30.7) ● Female70 (69.3)Age at AA onset, year, mean ± SD40.4 ± 12.6Age at AA onset, year, n (%) ● ≤40 years50 (49.5) ● >40 years51 (50.5)Duration of disease, month, median (IQR)2 (1–4)Duration of disease, n (%) ● ≤6 months77 (76.2) ● >6 months24 (23.8)AA subtype, n (%) ● Patch AA20 (19.8) ● Multiple AA46 (45.5) ● Diffuse AA16 (15.8) ● AT9 (8.9) ● AU5 (5.0) ● Ophiasis5 (5.0)Severity of AA (SALT score), n (%) ● 0–25%11 (10.9) ● 26–50%16 (15.8) ● 51–75%60 (59.4) ● 76–100%14 (13.9)Body hair involvement, n (%)6 (5.9)Nail involvement, n (%)9 (8.9)Comorbidity, n (%) ● Atopy2 (1.9) ● Autoimmune thyroid diseases6 (5.9)Family history of AA, n (%)2 (1.9)Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.\n\nDemographic and Clinical Characteristics of Participants with Alopecia Areata\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.\n Overall Response The median overall follow-up time for this cohort was 20 (6–38) months. Patients received 1 to 6 IMC injections (mean = 3.12 ± 0.9) per treatment course. Ninety-eight patients (97.1%) showed improvement, whereas 3 patients (2.9%) showed treatment failure and were further treated by topical contact immunotherapy at approximately 9 months (36 weeks in 2 patients and 37 weeks in another one) after completion of IMC therapy. The mean duration of IMC therapy was 4.2 ± 1.1 months, with a range of 1 to 6 months. Treatment responses are summarized in Table 2.Table 2Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia AreataTreatment OutcomesN = 101Treatment failure (<10% regrowth) ● Incidence, n (%)3 (2.9)Initial hair regrowth (25% regrowth) ● Cumulative incidence, n (%)86 (85.1) ● Incidence rate, person-year (CI)64.0 (51.9–79.3) ● Median time to achieve endpoint, month (CI)0.9 (0.8–1.2) ● Median cumulative dose to achieve endpoint, mg100 ● Cumulative dose to achieve endpoint, mg, median (IQR)80 (20–160)Significant hair regrowth (75% regrowth) ● Cumulative incidence, n (%)81 (80.2) ● Incidence rate, person-year (CI)17.0 (13.6–21.7) ● Median time to achieve endpoint, month (CI)3.4 (2.9–4.4) ● Median cumulative dose to achieve endpoint, mg200 ● Cumulative dose to achieve endpoint, mg, median (IQR)120 (40–200)Complete hair regrowth (100% regrowth) ● Cumulative incidence, n (%)49 (48.5) ● Incidence rate, person-year (CI)8.0 (6.2–10.9) ● Median time to achieve endpoint, month (CI)6.1 (5.1–8.1) ● Cumulative dose to achieve endpoint, mg, median (IQR)160 (60–240)Relapse ● Cumulative incidence, n (%)48 (47.5) ● Incidence rate, person-year (CI)5.5 (4.2–7.3) ● Median time to relapse, month (CI)11.3 (8.6–15.4)Abbreviations: CI, confidence interval; IQR, interquartile range.\n\nTreatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia Areata\nAbbreviations: CI, confidence interval; IQR, interquartile range.\nThe median overall follow-up time for this cohort was 20 (6–38) months. Patients received 1 to 6 IMC injections (mean = 3.12 ± 0.9) per treatment course. Ninety-eight patients (97.1%) showed improvement, whereas 3 patients (2.9%) showed treatment failure and were further treated by topical contact immunotherapy at approximately 9 months (36 weeks in 2 patients and 37 weeks in another one) after completion of IMC therapy. The mean duration of IMC therapy was 4.2 ± 1.1 months, with a range of 1 to 6 months. Treatment responses are summarized in Table 2.Table 2Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia AreataTreatment OutcomesN = 101Treatment failure (<10% regrowth) ● Incidence, n (%)3 (2.9)Initial hair regrowth (25% regrowth) ● Cumulative incidence, n (%)86 (85.1) ● Incidence rate, person-year (CI)64.0 (51.9–79.3) ● Median time to achieve endpoint, month (CI)0.9 (0.8–1.2) ● Median cumulative dose to achieve endpoint, mg100 ● Cumulative dose to achieve endpoint, mg, median (IQR)80 (20–160)Significant hair regrowth (75% regrowth) ● Cumulative incidence, n (%)81 (80.2) ● Incidence rate, person-year (CI)17.0 (13.6–21.7) ● Median time to achieve endpoint, month (CI)3.4 (2.9–4.4) ● Median cumulative dose to achieve endpoint, mg200 ● Cumulative dose to achieve endpoint, mg, median (IQR)120 (40–200)Complete hair regrowth (100% regrowth) ● Cumulative incidence, n (%)49 (48.5) ● Incidence rate, person-year (CI)8.0 (6.2–10.9) ● Median time to achieve endpoint, month (CI)6.1 (5.1–8.1) ● Cumulative dose to achieve endpoint, mg, median (IQR)160 (60–240)Relapse ● Cumulative incidence, n (%)48 (47.5) ● Incidence rate, person-year (CI)5.5 (4.2–7.3) ● Median time to relapse, month (CI)11.3 (8.6–15.4)Abbreviations: CI, confidence interval; IQR, interquartile range.\n\nTreatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia Areata\nAbbreviations: CI, confidence interval; IQR, interquartile range.\n Initial Hair Regrowth According to survival analysis, the cumulative incidence of initial hair regrowth after IMC therapy was 85.1%, with an incidence rate of 64.0 (95% CI = 51.9–79.3) person-years. The median time to achieve initial hair regrowth was 0.9 (95% CI = 0.8–1.2) months, and the average cumulative dose of IMC to achieve initial hair regrowth was 80 (20–160) mg.\nAccording to survival analysis, the cumulative incidence of initial hair regrowth after IMC therapy was 85.1%, with an incidence rate of 64.0 (95% CI = 51.9–79.3) person-years. The median time to achieve initial hair regrowth was 0.9 (95% CI = 0.8–1.2) months, and the average cumulative dose of IMC to achieve initial hair regrowth was 80 (20–160) mg.\n Significant Hair Regrowth Significant hair regrowth was indicated as the primary outcome and its cumulative incidence in our cohort was 80.2%, with an incidence rate of 17.0 (95% CI = 13.6–21.7) person-years. The median time required to achieve significant hair regrowth was 3.4 (95% CI = 2.9–4.4) months. According to the Kaplan–Meier curve, there was a lag of approximately 1 month from initiation of therapy to the first patients demonstrating significant hair regrowth. Following this initial event, significant hair regrowth was observed in 41.9% of patients at 3 months, 75.9% at 6 months, 81.0% at 9 months, and 86.5% at 12 months. The residual probabilities of significant regrowth as a function of the time after initiation of IMC are also shown in Figure 1A. The average cumulative dose of IMC to achieve significant hair regrowth was 120 (40–200) mg, while a median cumulative dose that 50% of patients attained significant hair regrowth was 200 mg (Figure 1B).Figure 1Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nKaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nThe baseline SALT score was identified as a negative factor for IMC treatment response in a previous study.31 To determine the effect of this variable on significant hair regrowth, a stratified Kaplan–Meier analysis was performed. Nine patients (81.8%) with a score of 0–25% obtained significant hair regrowth by a median time of 3.5 months; for the score range 26–50%, 12 (75.0%) patients achieved significant regrowth by 4.8 months; for the score range 51–75%, 48 (80.0%) experienced significant regrowth by 3.1 months; and for the score range 76–100%, 12 (85.7%) ultimately obtained significant regrowth at 3.2 months. However, the log rank test failed to demonstrate statistically significant differences among the four groups (P = 0.287; Figure 1C).\nSignificant hair regrowth was indicated as the primary outcome and its cumulative incidence in our cohort was 80.2%, with an incidence rate of 17.0 (95% CI = 13.6–21.7) person-years. The median time required to achieve significant hair regrowth was 3.4 (95% CI = 2.9–4.4) months. According to the Kaplan–Meier curve, there was a lag of approximately 1 month from initiation of therapy to the first patients demonstrating significant hair regrowth. Following this initial event, significant hair regrowth was observed in 41.9% of patients at 3 months, 75.9% at 6 months, 81.0% at 9 months, and 86.5% at 12 months. The residual probabilities of significant regrowth as a function of the time after initiation of IMC are also shown in Figure 1A. The average cumulative dose of IMC to achieve significant hair regrowth was 120 (40–200) mg, while a median cumulative dose that 50% of patients attained significant hair regrowth was 200 mg (Figure 1B).Figure 1Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nKaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nThe baseline SALT score was identified as a negative factor for IMC treatment response in a previous study.31 To determine the effect of this variable on significant hair regrowth, a stratified Kaplan–Meier analysis was performed. Nine patients (81.8%) with a score of 0–25% obtained significant hair regrowth by a median time of 3.5 months; for the score range 26–50%, 12 (75.0%) patients achieved significant regrowth by 4.8 months; for the score range 51–75%, 48 (80.0%) experienced significant regrowth by 3.1 months; and for the score range 76–100%, 12 (85.7%) ultimately obtained significant regrowth at 3.2 months. However, the log rank test failed to demonstrate statistically significant differences among the four groups (P = 0.287; Figure 1C).\n Factors Associated with Significant Hair Regrowth The Cox proportional hazards model was used to determine the factors associated with significant hair regrowth (Table 3). Univariable analysis revealed that the variables associated with significant hair regrowth were female sex (HR = 2.39, 95% CI = 1.09–4.61; P = 0.035), age at AA onset >40 years (HR = 2.45, 95% CI = 1.49–3.69; P = 0.046), and nail abnormalities (HR = 0.26, 95% CI = 0.08–0.69; P = 0.036). However, when age at AA onset was analyzed as a continuous variable, no association was found (HR = 1.02, 95% CI = 0.89–1.06; P = 0.423). After adjusting for all confounding factors in a multivariable model, nail involvement was the only independent factor negatively affecting the probability of significant hair regrowth (adjusted HR = 0.04, 95% CI = 0.01–0.55; P = 0.015).Table 3Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale2.39 (1.09–4.61)0.035*1.13 (0.81–5.91)0.086Age at AA onset1.02 (0.89–1.06)0.423Age group at AA onset ● ≤40 yearsReference ● >40 years2.45 (1.49–3.69)0.046*1.68 (0.92–3.42)0.187Duration of disease >6 months0.63 (0.25–1.49)0.141AA subtype ● Patch AAReference ● Multiple AA1.34 (0.25–2.92)0.182 ● Diffuse AA2.63 (0.99–4.61)0.062 ● AT0.82 (0.29–2.85)0.845 ● AU0.16 (0.09–3.15)0.753 ● Ophiasis0.76 (0.09–2.92)0.911Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.56 (0.32–2.09)0.186 ● 51–75%0.64 (0.31–2.67)0.823 ● 76–100%1.49 (0.81–3.68)0.595Body hair involvement0.67 (0.03–4.28)0.745Nail involvement0.26 (0.08–0.69)0.036*0.04 (0.01–0.55)0.015*Atopy0.68 (0.13–5.16)0.621Autoimmune thyroid diseases1.91 (0.83–3.28)0.142Family history of AA0.96 (0.41–3.56)0.363Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\nThe Cox proportional hazards model was used to determine the factors associated with significant hair regrowth (Table 3). Univariable analysis revealed that the variables associated with significant hair regrowth were female sex (HR = 2.39, 95% CI = 1.09–4.61; P = 0.035), age at AA onset >40 years (HR = 2.45, 95% CI = 1.49–3.69; P = 0.046), and nail abnormalities (HR = 0.26, 95% CI = 0.08–0.69; P = 0.036). However, when age at AA onset was analyzed as a continuous variable, no association was found (HR = 1.02, 95% CI = 0.89–1.06; P = 0.423). After adjusting for all confounding factors in a multivariable model, nail involvement was the only independent factor negatively affecting the probability of significant hair regrowth (adjusted HR = 0.04, 95% CI = 0.01–0.55; P = 0.015).Table 3Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale2.39 (1.09–4.61)0.035*1.13 (0.81–5.91)0.086Age at AA onset1.02 (0.89–1.06)0.423Age group at AA onset ● ≤40 yearsReference ● >40 years2.45 (1.49–3.69)0.046*1.68 (0.92–3.42)0.187Duration of disease >6 months0.63 (0.25–1.49)0.141AA subtype ● Patch AAReference ● Multiple AA1.34 (0.25–2.92)0.182 ● Diffuse AA2.63 (0.99–4.61)0.062 ● AT0.82 (0.29–2.85)0.845 ● AU0.16 (0.09–3.15)0.753 ● Ophiasis0.76 (0.09–2.92)0.911Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.56 (0.32–2.09)0.186 ● 51–75%0.64 (0.31–2.67)0.823 ● 76–100%1.49 (0.81–3.68)0.595Body hair involvement0.67 (0.03–4.28)0.745Nail involvement0.26 (0.08–0.69)0.036*0.04 (0.01–0.55)0.015*Atopy0.68 (0.13–5.16)0.621Autoimmune thyroid diseases1.91 (0.83–3.28)0.142Family history of AA0.96 (0.41–3.56)0.363Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n Complete Hair Regrowth Regarding complete hair regrowth, the cumulative incidence was 48.5%, with an incidence rate of 8.0 (95% CI = 6.2–10.9) person-years. The median time and average cumulative dose of IMC to achieve complete hair regrowth were 6.1 (95% CI = 5.1–8.1) months and 160 (60–240) mg, respectively.\nRegarding complete hair regrowth, the cumulative incidence was 48.5%, with an incidence rate of 8.0 (95% CI = 6.2–10.9) person-years. The median time and average cumulative dose of IMC to achieve complete hair regrowth were 6.1 (95% CI = 5.1–8.1) months and 160 (60–240) mg, respectively.\n Relapse and Its Associated Factors Following 51 months of follow-up, the overall cumulative relapse rate after achievement of clinical hair regrowth with IMC was 47.5% (n = 48; Figure 1D). The incidence rate of AA relapse was 5.5 (95% CI = 4.2–7.3) person-years, and the median time to relapse was 11.3 (95% CI = 8.6–15.4) months. Treatment in patients with recurrence included combination therapy with IL corticosteroids and topical minoxidil (n = 21), topical contact immunotherapy (n = 10), methotrexate (n = 9), IMC (n = 4), and IL corticosteroids (n = 4). All therapies were prescribed at >6 months after discontinuation of the IMC regimen.\nThe risk of AA relapse was determined using the Cox proportional hazards model. Univariable analysis revealed that coexisting AITD (HR = 2.39, 95% CI = 1.42–5.14; P = 0.041) and disease duration >6 months (HR = 3.12, 95% CI = 1.36–4.94; P = 0.016) were associated with a higher probability of disease recurrence. However, multivariable analysis showed that only AA duration >6 months (adjusted HR = 4.02, 95% CI = 1.52–4.66; P = 0.005) independently increased the probability of AA relapse (Table 4).Table 4Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale1.15 (0.64–1.69)0.536Age at AA onset1.01 (0.99–1.06)0.104Age group at AA onset ● ≤40 yearsReference ● >40 years1.55 (0.78–2.56)0.151Duration of disease >6 months3.12 (1.36–4.94)0.016*4.02 (1.52–4.66)0.005*AA subtype ● Patch AAReference ● Multiple AA0.94 (0.66–2.67)0.723 ● Diffuse AA0.85 (0.39–3.62)0.264 ● AT3.12 (0.79–5.64)0.459 ● AU2.92 (0.59–3.95)0.644 ● Ophiasis1.49 (0.08–3.12)0.814Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.91 (0.72–2.19)0.135 ● 51–75%1.14 (0.51–2.67)0.718 ● 76–100%0.69 (0.29–1.89)0.653Body hair involvement1.05 (0.29–3.18)0.425Nail involvement2.78 (0.98–4.09)0.812Atopy1.34 (0.63–3.89)0.291Autoimmune thyroid diseases2.39 (1.42–5.14)0.041*2.71 (0.82–4.65)0.069Family history of AA1.76 (0.87–2.88)0.635Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\nFollowing 51 months of follow-up, the overall cumulative relapse rate after achievement of clinical hair regrowth with IMC was 47.5% (n = 48; Figure 1D). The incidence rate of AA relapse was 5.5 (95% CI = 4.2–7.3) person-years, and the median time to relapse was 11.3 (95% CI = 8.6–15.4) months. Treatment in patients with recurrence included combination therapy with IL corticosteroids and topical minoxidil (n = 21), topical contact immunotherapy (n = 10), methotrexate (n = 9), IMC (n = 4), and IL corticosteroids (n = 4). All therapies were prescribed at >6 months after discontinuation of the IMC regimen.\nThe risk of AA relapse was determined using the Cox proportional hazards model. Univariable analysis revealed that coexisting AITD (HR = 2.39, 95% CI = 1.42–5.14; P = 0.041) and disease duration >6 months (HR = 3.12, 95% CI = 1.36–4.94; P = 0.016) were associated with a higher probability of disease recurrence. However, multivariable analysis showed that only AA duration >6 months (adjusted HR = 4.02, 95% CI = 1.52–4.66; P = 0.005) independently increased the probability of AA relapse (Table 4).Table 4Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale1.15 (0.64–1.69)0.536Age at AA onset1.01 (0.99–1.06)0.104Age group at AA onset ● ≤40 yearsReference ● >40 years1.55 (0.78–2.56)0.151Duration of disease >6 months3.12 (1.36–4.94)0.016*4.02 (1.52–4.66)0.005*AA subtype ● Patch AAReference ● Multiple AA0.94 (0.66–2.67)0.723 ● Diffuse AA0.85 (0.39–3.62)0.264 ● AT3.12 (0.79–5.64)0.459 ● AU2.92 (0.59–3.95)0.644 ● Ophiasis1.49 (0.08–3.12)0.814Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.91 (0.72–2.19)0.135 ● 51–75%1.14 (0.51–2.67)0.718 ● 76–100%0.69 (0.29–1.89)0.653Body hair involvement1.05 (0.29–3.18)0.425Nail involvement2.78 (0.98–4.09)0.812Atopy1.34 (0.63–3.89)0.291Autoimmune thyroid diseases2.39 (1.42–5.14)0.041*2.71 (0.82–4.65)0.069Family history of AA1.76 (0.87–2.88)0.635Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n Adverse Events Adverse events were experienced by 18.8% of the patients (n = 19) and included acneiform eruption (n = 16, 15.8%), abnormal menstrual cycle (n = 6, 5.9%), weight gain (n = 3, 2.9%), and sleep disturbance (n = 1, 0.9%). Acneiform eruption was managed with topical therapy, while the other side effects resolved spontaneously after discontinuing IMC. The presence of any of these adverse events did not cause dropout from therapy.\nAdverse events were experienced by 18.8% of the patients (n = 19) and included acneiform eruption (n = 16, 15.8%), abnormal menstrual cycle (n = 6, 5.9%), weight gain (n = 3, 2.9%), and sleep disturbance (n = 1, 0.9%). Acneiform eruption was managed with topical therapy, while the other side effects resolved spontaneously after discontinuing IMC. The presence of any of these adverse events did not cause dropout from therapy.",
"A total of 126 patients with AA treated with IMC were initially included. Twenty-five patients were excluded due to coexisting diseases or concomitant use of other medications affecting the hair growth cycle (n = 16), incomplete medical records (n = 5), or because they received systemic treatment after the IMC protocol (n = 4). The majority of the remaining 101 patients were female (n = 70, 69.3%) with a female to male ratio of 2.26:1. The age range of AA onset was 5 to 68 years (mean = 40.4 ± 12.6 years), and the duration of disease varied from 1 week to 11 months (median = 2 [1–4] months). The most common AA subtype was multiple patches (n = 46, 45.5%), followed by patch (n = 20, 19.8%), diffuse (n = 16, 15.8%), AT (n = 9, 8.9%), AU (n = 5, 5.0%), and ophiasis (n = 5, 5.0%). The estimated baseline SALT score was 0–25%, 26–50%, 51–75%, or 76–100% in 11 (10.9%), 16 (15.8%), 60 (59.4%), and 14 patients (13.9%), respectively. Body hair involvement was found in 5.9% (n = 6), and nail abnormalities were observed in 8.9% (n = 9) of the patients. Six patients (5.9%) had coexisting AITD, 2 (1.9%) were atopic, and 2 (1.9%) had a family member with AA. All demographics are summarized in Table 1.Table 1Demographic and Clinical Characteristics of Participants with Alopecia AreataVariablesN = 101Sex, n (%) ● Male31 (30.7) ● Female70 (69.3)Age at AA onset, year, mean ± SD40.4 ± 12.6Age at AA onset, year, n (%) ● ≤40 years50 (49.5) ● >40 years51 (50.5)Duration of disease, month, median (IQR)2 (1–4)Duration of disease, n (%) ● ≤6 months77 (76.2) ● >6 months24 (23.8)AA subtype, n (%) ● Patch AA20 (19.8) ● Multiple AA46 (45.5) ● Diffuse AA16 (15.8) ● AT9 (8.9) ● AU5 (5.0) ● Ophiasis5 (5.0)Severity of AA (SALT score), n (%) ● 0–25%11 (10.9) ● 26–50%16 (15.8) ● 51–75%60 (59.4) ● 76–100%14 (13.9)Body hair involvement, n (%)6 (5.9)Nail involvement, n (%)9 (8.9)Comorbidity, n (%) ● Atopy2 (1.9) ● Autoimmune thyroid diseases6 (5.9)Family history of AA, n (%)2 (1.9)Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.\n\nDemographic and Clinical Characteristics of Participants with Alopecia Areata\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.",
"The median overall follow-up time for this cohort was 20 (6–38) months. Patients received 1 to 6 IMC injections (mean = 3.12 ± 0.9) per treatment course. Ninety-eight patients (97.1%) showed improvement, whereas 3 patients (2.9%) showed treatment failure and were further treated by topical contact immunotherapy at approximately 9 months (36 weeks in 2 patients and 37 weeks in another one) after completion of IMC therapy. The mean duration of IMC therapy was 4.2 ± 1.1 months, with a range of 1 to 6 months. Treatment responses are summarized in Table 2.Table 2Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia AreataTreatment OutcomesN = 101Treatment failure (<10% regrowth) ● Incidence, n (%)3 (2.9)Initial hair regrowth (25% regrowth) ● Cumulative incidence, n (%)86 (85.1) ● Incidence rate, person-year (CI)64.0 (51.9–79.3) ● Median time to achieve endpoint, month (CI)0.9 (0.8–1.2) ● Median cumulative dose to achieve endpoint, mg100 ● Cumulative dose to achieve endpoint, mg, median (IQR)80 (20–160)Significant hair regrowth (75% regrowth) ● Cumulative incidence, n (%)81 (80.2) ● Incidence rate, person-year (CI)17.0 (13.6–21.7) ● Median time to achieve endpoint, month (CI)3.4 (2.9–4.4) ● Median cumulative dose to achieve endpoint, mg200 ● Cumulative dose to achieve endpoint, mg, median (IQR)120 (40–200)Complete hair regrowth (100% regrowth) ● Cumulative incidence, n (%)49 (48.5) ● Incidence rate, person-year (CI)8.0 (6.2–10.9) ● Median time to achieve endpoint, month (CI)6.1 (5.1–8.1) ● Cumulative dose to achieve endpoint, mg, median (IQR)160 (60–240)Relapse ● Cumulative incidence, n (%)48 (47.5) ● Incidence rate, person-year (CI)5.5 (4.2–7.3) ● Median time to relapse, month (CI)11.3 (8.6–15.4)Abbreviations: CI, confidence interval; IQR, interquartile range.\n\nTreatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia Areata\nAbbreviations: CI, confidence interval; IQR, interquartile range.",
"According to survival analysis, the cumulative incidence of initial hair regrowth after IMC therapy was 85.1%, with an incidence rate of 64.0 (95% CI = 51.9–79.3) person-years. The median time to achieve initial hair regrowth was 0.9 (95% CI = 0.8–1.2) months, and the average cumulative dose of IMC to achieve initial hair regrowth was 80 (20–160) mg.",
"Significant hair regrowth was indicated as the primary outcome and its cumulative incidence in our cohort was 80.2%, with an incidence rate of 17.0 (95% CI = 13.6–21.7) person-years. The median time required to achieve significant hair regrowth was 3.4 (95% CI = 2.9–4.4) months. According to the Kaplan–Meier curve, there was a lag of approximately 1 month from initiation of therapy to the first patients demonstrating significant hair regrowth. Following this initial event, significant hair regrowth was observed in 41.9% of patients at 3 months, 75.9% at 6 months, 81.0% at 9 months, and 86.5% at 12 months. The residual probabilities of significant regrowth as a function of the time after initiation of IMC are also shown in Figure 1A. The average cumulative dose of IMC to achieve significant hair regrowth was 120 (40–200) mg, while a median cumulative dose that 50% of patients attained significant hair regrowth was 200 mg (Figure 1B).Figure 1Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nKaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.\nThe baseline SALT score was identified as a negative factor for IMC treatment response in a previous study.31 To determine the effect of this variable on significant hair regrowth, a stratified Kaplan–Meier analysis was performed. Nine patients (81.8%) with a score of 0–25% obtained significant hair regrowth by a median time of 3.5 months; for the score range 26–50%, 12 (75.0%) patients achieved significant regrowth by 4.8 months; for the score range 51–75%, 48 (80.0%) experienced significant regrowth by 3.1 months; and for the score range 76–100%, 12 (85.7%) ultimately obtained significant regrowth at 3.2 months. However, the log rank test failed to demonstrate statistically significant differences among the four groups (P = 0.287; Figure 1C).",
"The Cox proportional hazards model was used to determine the factors associated with significant hair regrowth (Table 3). Univariable analysis revealed that the variables associated with significant hair regrowth were female sex (HR = 2.39, 95% CI = 1.09–4.61; P = 0.035), age at AA onset >40 years (HR = 2.45, 95% CI = 1.49–3.69; P = 0.046), and nail abnormalities (HR = 0.26, 95% CI = 0.08–0.69; P = 0.036). However, when age at AA onset was analyzed as a continuous variable, no association was found (HR = 1.02, 95% CI = 0.89–1.06; P = 0.423). After adjusting for all confounding factors in a multivariable model, nail involvement was the only independent factor negatively affecting the probability of significant hair regrowth (adjusted HR = 0.04, 95% CI = 0.01–0.55; P = 0.015).Table 3Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale2.39 (1.09–4.61)0.035*1.13 (0.81–5.91)0.086Age at AA onset1.02 (0.89–1.06)0.423Age group at AA onset ● ≤40 yearsReference ● >40 years2.45 (1.49–3.69)0.046*1.68 (0.92–3.42)0.187Duration of disease >6 months0.63 (0.25–1.49)0.141AA subtype ● Patch AAReference ● Multiple AA1.34 (0.25–2.92)0.182 ● Diffuse AA2.63 (0.99–4.61)0.062 ● AT0.82 (0.29–2.85)0.845 ● AU0.16 (0.09–3.15)0.753 ● Ophiasis0.76 (0.09–2.92)0.911Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.56 (0.32–2.09)0.186 ● 51–75%0.64 (0.31–2.67)0.823 ● 76–100%1.49 (0.81–3.68)0.595Body hair involvement0.67 (0.03–4.28)0.745Nail involvement0.26 (0.08–0.69)0.036*0.04 (0.01–0.55)0.015*Atopy0.68 (0.13–5.16)0.621Autoimmune thyroid diseases1.91 (0.83–3.28)0.142Family history of AA0.96 (0.41–3.56)0.363Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.",
"Regarding complete hair regrowth, the cumulative incidence was 48.5%, with an incidence rate of 8.0 (95% CI = 6.2–10.9) person-years. The median time and average cumulative dose of IMC to achieve complete hair regrowth were 6.1 (95% CI = 5.1–8.1) months and 160 (60–240) mg, respectively.",
"Following 51 months of follow-up, the overall cumulative relapse rate after achievement of clinical hair regrowth with IMC was 47.5% (n = 48; Figure 1D). The incidence rate of AA relapse was 5.5 (95% CI = 4.2–7.3) person-years, and the median time to relapse was 11.3 (95% CI = 8.6–15.4) months. Treatment in patients with recurrence included combination therapy with IL corticosteroids and topical minoxidil (n = 21), topical contact immunotherapy (n = 10), methotrexate (n = 9), IMC (n = 4), and IL corticosteroids (n = 4). All therapies were prescribed at >6 months after discontinuation of the IMC regimen.\nThe risk of AA relapse was determined using the Cox proportional hazards model. Univariable analysis revealed that coexisting AITD (HR = 2.39, 95% CI = 1.42–5.14; P = 0.041) and disease duration >6 months (HR = 3.12, 95% CI = 1.36–4.94; P = 0.016) were associated with a higher probability of disease recurrence. However, multivariable analysis showed that only AA duration >6 months (adjusted HR = 4.02, 95% CI = 1.52–4.66; P = 0.005) independently increased the probability of AA relapse (Table 4).Table 4Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale1.15 (0.64–1.69)0.536Age at AA onset1.01 (0.99–1.06)0.104Age group at AA onset ● ≤40 yearsReference ● >40 years1.55 (0.78–2.56)0.151Duration of disease >6 months3.12 (1.36–4.94)0.016*4.02 (1.52–4.66)0.005*AA subtype ● Patch AAReference ● Multiple AA0.94 (0.66–2.67)0.723 ● Diffuse AA0.85 (0.39–3.62)0.264 ● AT3.12 (0.79–5.64)0.459 ● AU2.92 (0.59–3.95)0.644 ● Ophiasis1.49 (0.08–3.12)0.814Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.91 (0.72–2.19)0.135 ● 51–75%1.14 (0.51–2.67)0.718 ● 76–100%0.69 (0.29–1.89)0.653Body hair involvement1.05 (0.29–3.18)0.425Nail involvement2.78 (0.98–4.09)0.812Atopy1.34 (0.63–3.89)0.291Autoimmune thyroid diseases2.39 (1.42–5.14)0.041*2.71 (0.82–4.65)0.069Family history of AA1.76 (0.87–2.88)0.635Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.\n\nUnivariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata\nNote: *Statistically significant.\nAbbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.",
"Adverse events were experienced by 18.8% of the patients (n = 19) and included acneiform eruption (n = 16, 15.8%), abnormal menstrual cycle (n = 6, 5.9%), weight gain (n = 3, 2.9%), and sleep disturbance (n = 1, 0.9%). Acneiform eruption was managed with topical therapy, while the other side effects resolved spontaneously after discontinuing IMC. The presence of any of these adverse events did not cause dropout from therapy.",
"Although IMC therapy has shown to be effective in the treatment of several dermatological conditions and gained popularity for AA treatment in recent years, information regarding the use of IMC for AA is limited in the literature.32 In this study, we modeled the therapeutic endpoints of the IMC regimen according to multiple variables in patients with AA. To the best of our knowledge, this is the first study to describe the efficacy, cumulative effective dose, and factors associated with treatment outcomes in patients with AA receiving IMC based on a time-to-event analysis. The primary endpoint, defined as significant hair regrowth, had a rate of 80.2%, with a median time of approximately 3–4 months. Nail involvement was demonstrated to be a negative predictor of significant hair regrowth. Relapse was found in 47.5% of the patients, with disease duration >6 months being a risk factor for AA recurrence.\nThe application of systemic corticosteroids for the treatment of AA was first introduced by Dillaha et al in 1952, with remarkable therapeutic effects.33 Since then, the efficacy of various regimens such as oral, intravenous, and intramuscular administration has been investigated. Oral corticosteroid pulse therapy is generally the most convenient method and has been well studied.34 However, poor medication compliance and adherence may lead to reduced treatment efficacy and substantial worsening of the disease. The therapeutic effect of intravenous pulse corticosteroids has also been confirmed. However, the overall adverse events of intramuscular and oral corticosteroids are relatively high, approaching 41% and 30%, respectively, compared to that of 10% from corticosteroid pulse therapy.29 Hypothalamic–pituitary–adrenal (HPA) axis suppression was reported in 23%, 67%, and 7% of intramuscular, oral, and pulse corticosteroids, respectively.29 Intramuscular injections may offer an advantage over the two methods. IMC maximizes treatment efficacy by providing rapid onset of action comparable to the oral form, and by increasing patients’ compliance and adherence owing to the monthly or bimonthly injection regimen.28 It also minimizes adverse effects due to its pharmacokinetic properties, with a gradual and steady release of corticosteroids over time.28\nOur study demonstrated the efficacy of IMC for the treatment of severe, acute, or rapidly progressive AA, as shown by the 80.2% cumulative incidence of significant hair regrowth rate in a median time of 3.4 months. Michalowski and Kuczyfiska reported a 72.7% response rate in 11 cases of extensive AA (AT/AU) receiving 40–80 mg of IMC therapy, initially once a week, then over a gradually lengthened interval up to 6 weeks.31 A retrospective study using IMC 40 mg every 4 weeks for a maximum of 6 months revealed a 63% response rate in refractory AA.30 Administration of IMC 40 mg monthly for 6 months, followed by 40 mg every 1.5 months for 1 year, had significantly superior efficacy, with a 74% response rate, to oral corticosteroid pulse therapy.29 However, our results could not be directly compared to those of earlier studies because of the difference in the severity of AA among the cohorts. The lower percentage of recalcitrant AA cases included in our study compared with previous ones could explain the higher response rate.\nRegarding other corticosteroid injection protocols for AA, IL injection of 2.5–10 mg/mL triamcinolone acetonide at four–to-six-week intervals is commonly used in mild forms of AA (ie, patch AA or SALT score <25%) with a response rate between 64% and 97%.23 For severe AA, intravenous injection of 500 mg methylprednisolone for three consecutive days at four-week intervals for three months has a response rate of approximately 80%.23 However, compared to IL injection, the dosage for intramuscular and intravenous injections is higher and may lead to potential adverse effects; therefore, they should be restricted to those with severe AA and administered with caution.\nIdentifying the factors affecting treatment outcomes of AA is essential for therapeutic success; nevertheless, specific predictors for IMC have been rarely reported. Seo et al indicated that the baseline SALT score was negatively correlated with hair regrowth response to IMC.30 Our stratified analysis using the Kaplan–Meier method did not support this finding, as no association was found between the extent of scalp involvement and treatment response. Earlier age at AA onset, longer disease duration, and nail involvement were identified as indicators of poor prognosis.35–38 A recent meta-analysis demonstrated four negative predictors for treatment response, namely SALT score ≥50, disease duration ≥1 year, atopic diseases, and nail abnormalities.35 Our analysis using a Cox proportional hazards model showed a lack of correlation between significant hair regrowth and SALT score, AA duration, or presence of atopy, but confirmed its negative association with nail abnormalities. This finding could be explained by the characteristics of the enrolled subjects in our cohort, namely the predominance of female patients presenting with rapidly progressive scalp hair loss >50% for <6 months. The clinical characteristics of our patients were compatible to those of patients with acute diffuse and total alopecia, a distinctive AA subtype, which shows a favorable treatment response to systemic corticosteroid therapy.39,40\nEmerging evidence suggests that approximately half the patients with AA treated with IMC experience disease recurrence. Seo et al demonstrated a 47.1% relapse rate during a follow-up period of 14.2 months.30 Kurosawa et al reported a comparable relapse rate of 46% after 6 months of treatment.29 Our result was consistent with previous studies, revealing recurrence in approximately half of patients with a median time of 11.3 months. Furthermore, disease duration >6 months was identified as a predictor of AA relapse. Longer AA duration could indicate disease chronicity, and physicians should remain vigilant for AA relapse despite complete IMC therapy. Therefore, clinicians should consider the use of post-IMC treatments such as application of 2% topical minoxidil three times daily, which was reported to limit post-steroid-treatment hair loss.41 For AA patients with chronic relapsing disease, further treatments after initial systemic corticosteroid therapy are challenging owing to a lack of high-quality randomized controlled trials supporting the efficacy and safety of other therapeutic modalities. However, a shared decision-making approach between the physician and patient is crucial as it would ensure that patients have a realistic expectation of treatment outcomes. Topical contact immunotherapy is currently considered the best modality for long-term therapy of patients with recalcitrant AA and is the first line treatment for these cases in our hair clinic.19\nHPA axis suppression is considered a major side effect of IMC therapy.32 Previous studies demonstrated adrenocortical alteration in 23% of subjects receiving intramuscular injection.29 Other adverse effects of IMC include abdominal discomfort, dysmenorrhea, and acne.29,30 The incidence of adverse events among our AA patients was low, and acneiform eruption was the most common; no serious side effects were reported. However, HPA axis suppression in our subjects may be underestimated due to a lack of data regarding adrenocortical evaluation in our follow-up protocol.\nA major strength of our study is the use of the time-to-event approach to characterize the therapeutic outcomes of patients with AA receiving IMC during the entire follow-up time, including censored observations. This method considers all available treatment and follow-up data, providing more precise and meaningful estimates of overall efficacy and tolerability, and better outcome prediction. The limitations of this study are its retrospective design involving a single tertiary center. These factors might lead to the unavailability of some data and limit the generalizability of the results. Our results may also be limited in their applicability to patients without severe and/or rapidly progressive AA. Additionally, it is difficult to compare our findings to those of previous studies, owing to differences in the definitions of treatment responses and in methodologies. Further large, multicenter randomized controlled trials evaluating the efficacy and safety of IMC therapy are required to overcome these limitations.",
"Our time-to-event analysis demonstrated the efficacy and safety of intramuscular administration of corticosteroids for the treatment of AA. IMC therapy showed high rates of significant hair regrowth, especially in patients with severe or active AA; however, approximately half of those experienced relapse. Nail involvement was negatively correlated with cosmetically acceptable hair regrowth, while disease duration >6 months was significantly associated with increased risk of relapse. Most importantly, the use of IMC depends on indication and perceived potential benefits and risks; therefore, physicians should be cautious when prescribing this treatment."
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"acute diffuse and total alopecia",
"alopecia totalis",
"alopecia universalis",
"hair loss",
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"systemic"
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Introduction:
Alopecia areata (AA) is a non-scarring hair loss disorder that affects the scalp and hair-bearing areas.1 Variations on the clinical presentation of AA have been observed, ranging from small, well-defined circular or oval patches of hair loss to diffuse alopecia and complete scalp (alopecia totalis, AT) or total body hair loss (alopecia universalis, AU).2–5 Nail abnormalities, most frequently nail pitting, are occasionally present. Patients with AA often experience major psychological impacts due to the unpredictable clinical course and variable treatment response.6 Various immune-related comorbidities such as autoimmune thyroid diseases (AITD), vitiligo, psoriasis, and atopic diseases have been linked to AA.7–13
The exact etiopathogenesis of AA is unclear, though it is believed to be a cell-mediated autoimmune disease of the hair follicle. Autoreactive T-cell lymphocytes and several cytokines mediate hair follicular destruction and negatively affect the hair growth cycle.14–16 Despite the absence of curative/preventive treatments and FDA-approved regimens for AA, therapeutic approaches have been established through treatment guidelines. Topical and intralesional (IL) corticosteroids, topical minoxidil, and topical anthralin are recommended for AA patients with <50% scalp involvement, whereas in those with ≥50% hair loss, topical contact immunotherapy and systemic corticosteroids are advised.17–23
Corticosteroids, the main treatment option for AA, can be administered in different forms depending on the disease severity.24 Medium- to high-potency topical and IL corticosteroids are indicated as monotherapy or combined therapy in limited and/or slowly progressive AA,25–27 while systemic corticosteroids (eg, oral, intravenous, and intramuscular), are preferred in extensive and/or rapidly progressive disease.17
Among systemic modalities, intramuscular corticosteroids (IMC) may be most beneficial, as its monthly or bimonthly regimen increases patients’ compliance and treatment adherence.28 Moreover, IMC could minimize adverse effects from slowly releasing continuous and small amounts of medication.28 Previous studies of IMC showed a satisfactory outcome in severe and refractory AA, with response rates between 63% and 72.7%.29–31 Although IMC is often the preferred treatment for AA, there are limited data regarding its effectiveness, safety, dosing, and optimal duration. Thus, we conducted a retrospective cohort study based on our 20-year experience to evaluate the efficacy, relapse, and tolerability, as well as factors associated with treatment outcomes of IMC in patients with AA.
Materials and Methods:
Study Design and Population This was a retrospective cohort study conducted at the Dermatology Clinic of Ramathibodi Hospital, Bangkok, Thailand. The study was approved by the Mahidol University Review Board for Ethics in Human Research (MURA2020/1282) and was performed in accordance with the principles of Declaration of Helsinki. The requirement for informed consent was waived, and the data were anonymized before analysis.
We reviewed the medical records of patients clinically and/or histopathologically diagnosed with AA between January 2000 and December 2019. Individuals with AA receiving an IMC treatment regimen were included in the study. Our IMC protocol was triamcinolone acetonide 20–40 mg/mL injected every 4–6 weeks. The treatment was indicated in patients with severe (≥50% scalp involvement), extensive (AT, AU, and ≥50% scalp involvement with body hair loss), or rapidly progressive AA (accelerated hair loss spreading ≥5% of scalp area per week for >2 consecutive weeks with positive hair-pull test results at the periphery of alopecic patch or across the entire scalp), and IMC was performed until patients achieved significant hair regrowth. Patients who did not achieve significant hair regrowth after six IMC injections were switched to other appropriate therapies to minimize adrenocortical alteration.32 We excluded participants treated with systemic immunosuppressive agents or topical contact immunotherapy within 6 months after discontinuation of the IMC protocol; therefore, those who underwent topical treatment (ie, topical corticosteroids and/or topical minoxidil) remained in our cohort. Patients with incomplete medical records, those who received other AA treatments during IMC therapy, those with other hair and scalp disorders, and those with systemic diseases or using medications affecting the hair growth cycle were also excluded.
This was a retrospective cohort study conducted at the Dermatology Clinic of Ramathibodi Hospital, Bangkok, Thailand. The study was approved by the Mahidol University Review Board for Ethics in Human Research (MURA2020/1282) and was performed in accordance with the principles of Declaration of Helsinki. The requirement for informed consent was waived, and the data were anonymized before analysis.
We reviewed the medical records of patients clinically and/or histopathologically diagnosed with AA between January 2000 and December 2019. Individuals with AA receiving an IMC treatment regimen were included in the study. Our IMC protocol was triamcinolone acetonide 20–40 mg/mL injected every 4–6 weeks. The treatment was indicated in patients with severe (≥50% scalp involvement), extensive (AT, AU, and ≥50% scalp involvement with body hair loss), or rapidly progressive AA (accelerated hair loss spreading ≥5% of scalp area per week for >2 consecutive weeks with positive hair-pull test results at the periphery of alopecic patch or across the entire scalp), and IMC was performed until patients achieved significant hair regrowth. Patients who did not achieve significant hair regrowth after six IMC injections were switched to other appropriate therapies to minimize adrenocortical alteration.32 We excluded participants treated with systemic immunosuppressive agents or topical contact immunotherapy within 6 months after discontinuation of the IMC protocol; therefore, those who underwent topical treatment (ie, topical corticosteroids and/or topical minoxidil) remained in our cohort. Patients with incomplete medical records, those who received other AA treatments during IMC therapy, those with other hair and scalp disorders, and those with systemic diseases or using medications affecting the hair growth cycle were also excluded.
Data Collection and Clinical Outcomes Baseline demographics, clinical characteristics, duration of disease, AA subtypes (ie, patch, multiple patches, AT/AU, and ophiasis), body hair involvement, nail abnormalities, and family history of AA were collected. AA severity was evaluated using the Severity of Alopecia Tool (SALT) score. Details regarding IMC regimen were obtained, including dosage, duration, and clinical outcomes. Efficacy and tolerability assessment were performed at baseline and at every follow-up visit. The percentage of scalp area coverage by terminal hair regrowth from baseline was used to evaluate treatment response. Clinical endpoints included initial, significant, and complete hair regrowth, treatment failure, and relapse. Initial hair regrowth (noticeable) was defined as 25% regrowth, while significant hair regrowth (cosmetically acceptable) was defined as 75% regrowth. Patients with 100% regrowth were classified as having complete (full) hair regrowth. Treatment failure was defined as <10% hair regrowth after 6 months of IMC therapy. Relapse was defined as any new appearance of AA during follow-up visits. The cumulative incidence of significant hair regrowth was set as the outcome of interest in the time-to-event analysis. Furthermore, the median time and median cumulative corticosteroid dosage to achieve each clinical endpoint were evaluated. Relevant factors affecting significant hair regrowth and relapse were also analyzed.
Baseline demographics, clinical characteristics, duration of disease, AA subtypes (ie, patch, multiple patches, AT/AU, and ophiasis), body hair involvement, nail abnormalities, and family history of AA were collected. AA severity was evaluated using the Severity of Alopecia Tool (SALT) score. Details regarding IMC regimen were obtained, including dosage, duration, and clinical outcomes. Efficacy and tolerability assessment were performed at baseline and at every follow-up visit. The percentage of scalp area coverage by terminal hair regrowth from baseline was used to evaluate treatment response. Clinical endpoints included initial, significant, and complete hair regrowth, treatment failure, and relapse. Initial hair regrowth (noticeable) was defined as 25% regrowth, while significant hair regrowth (cosmetically acceptable) was defined as 75% regrowth. Patients with 100% regrowth were classified as having complete (full) hair regrowth. Treatment failure was defined as <10% hair regrowth after 6 months of IMC therapy. Relapse was defined as any new appearance of AA during follow-up visits. The cumulative incidence of significant hair regrowth was set as the outcome of interest in the time-to-event analysis. Furthermore, the median time and median cumulative corticosteroid dosage to achieve each clinical endpoint were evaluated. Relevant factors affecting significant hair regrowth and relapse were also analyzed.
Statistical Analysis Statistical analyses were performed using Stata 14.1 (Stata Corp, College Station, TX, USA). To detect a modestly sized hazard ratio (HR), the sample size was calculated based on data from a previous study regarding the IMC treatment of patients with AA.31 The minimum number of subjects required to achieve a statistical significance level of 5% and a power of 80% was 26. Continuous variables were reported as mean ± standard deviation or median (interquartile range, IQR). Categorical variables were expressed as number or proportion. Survival analysis was performed using the Kaplan–Meier method to estimate outcome rate, median time, and median cumulative corticosteroids to achieve endpoints. The log rank test was used to compare survival distributions between variables. Factors affecting clinical outcomes were assessed using univariable and multivariable stepwise analyses under the Cox proportional hazards regression model and expressed as HRs with 95% confidence intervals (CI). All analyses were two-tailed, and P-values <0.05 were considered statistically significant.
Statistical analyses were performed using Stata 14.1 (Stata Corp, College Station, TX, USA). To detect a modestly sized hazard ratio (HR), the sample size was calculated based on data from a previous study regarding the IMC treatment of patients with AA.31 The minimum number of subjects required to achieve a statistical significance level of 5% and a power of 80% was 26. Continuous variables were reported as mean ± standard deviation or median (interquartile range, IQR). Categorical variables were expressed as number or proportion. Survival analysis was performed using the Kaplan–Meier method to estimate outcome rate, median time, and median cumulative corticosteroids to achieve endpoints. The log rank test was used to compare survival distributions between variables. Factors affecting clinical outcomes were assessed using univariable and multivariable stepwise analyses under the Cox proportional hazards regression model and expressed as HRs with 95% confidence intervals (CI). All analyses were two-tailed, and P-values <0.05 were considered statistically significant.
Study Design and Population:
This was a retrospective cohort study conducted at the Dermatology Clinic of Ramathibodi Hospital, Bangkok, Thailand. The study was approved by the Mahidol University Review Board for Ethics in Human Research (MURA2020/1282) and was performed in accordance with the principles of Declaration of Helsinki. The requirement for informed consent was waived, and the data were anonymized before analysis.
We reviewed the medical records of patients clinically and/or histopathologically diagnosed with AA between January 2000 and December 2019. Individuals with AA receiving an IMC treatment regimen were included in the study. Our IMC protocol was triamcinolone acetonide 20–40 mg/mL injected every 4–6 weeks. The treatment was indicated in patients with severe (≥50% scalp involvement), extensive (AT, AU, and ≥50% scalp involvement with body hair loss), or rapidly progressive AA (accelerated hair loss spreading ≥5% of scalp area per week for >2 consecutive weeks with positive hair-pull test results at the periphery of alopecic patch or across the entire scalp), and IMC was performed until patients achieved significant hair regrowth. Patients who did not achieve significant hair regrowth after six IMC injections were switched to other appropriate therapies to minimize adrenocortical alteration.32 We excluded participants treated with systemic immunosuppressive agents or topical contact immunotherapy within 6 months after discontinuation of the IMC protocol; therefore, those who underwent topical treatment (ie, topical corticosteroids and/or topical minoxidil) remained in our cohort. Patients with incomplete medical records, those who received other AA treatments during IMC therapy, those with other hair and scalp disorders, and those with systemic diseases or using medications affecting the hair growth cycle were also excluded.
Data Collection and Clinical Outcomes:
Baseline demographics, clinical characteristics, duration of disease, AA subtypes (ie, patch, multiple patches, AT/AU, and ophiasis), body hair involvement, nail abnormalities, and family history of AA were collected. AA severity was evaluated using the Severity of Alopecia Tool (SALT) score. Details regarding IMC regimen were obtained, including dosage, duration, and clinical outcomes. Efficacy and tolerability assessment were performed at baseline and at every follow-up visit. The percentage of scalp area coverage by terminal hair regrowth from baseline was used to evaluate treatment response. Clinical endpoints included initial, significant, and complete hair regrowth, treatment failure, and relapse. Initial hair regrowth (noticeable) was defined as 25% regrowth, while significant hair regrowth (cosmetically acceptable) was defined as 75% regrowth. Patients with 100% regrowth were classified as having complete (full) hair regrowth. Treatment failure was defined as <10% hair regrowth after 6 months of IMC therapy. Relapse was defined as any new appearance of AA during follow-up visits. The cumulative incidence of significant hair regrowth was set as the outcome of interest in the time-to-event analysis. Furthermore, the median time and median cumulative corticosteroid dosage to achieve each clinical endpoint were evaluated. Relevant factors affecting significant hair regrowth and relapse were also analyzed.
Statistical Analysis:
Statistical analyses were performed using Stata 14.1 (Stata Corp, College Station, TX, USA). To detect a modestly sized hazard ratio (HR), the sample size was calculated based on data from a previous study regarding the IMC treatment of patients with AA.31 The minimum number of subjects required to achieve a statistical significance level of 5% and a power of 80% was 26. Continuous variables were reported as mean ± standard deviation or median (interquartile range, IQR). Categorical variables were expressed as number or proportion. Survival analysis was performed using the Kaplan–Meier method to estimate outcome rate, median time, and median cumulative corticosteroids to achieve endpoints. The log rank test was used to compare survival distributions between variables. Factors affecting clinical outcomes were assessed using univariable and multivariable stepwise analyses under the Cox proportional hazards regression model and expressed as HRs with 95% confidence intervals (CI). All analyses were two-tailed, and P-values <0.05 were considered statistically significant.
Results:
Demographics A total of 126 patients with AA treated with IMC were initially included. Twenty-five patients were excluded due to coexisting diseases or concomitant use of other medications affecting the hair growth cycle (n = 16), incomplete medical records (n = 5), or because they received systemic treatment after the IMC protocol (n = 4). The majority of the remaining 101 patients were female (n = 70, 69.3%) with a female to male ratio of 2.26:1. The age range of AA onset was 5 to 68 years (mean = 40.4 ± 12.6 years), and the duration of disease varied from 1 week to 11 months (median = 2 [1–4] months). The most common AA subtype was multiple patches (n = 46, 45.5%), followed by patch (n = 20, 19.8%), diffuse (n = 16, 15.8%), AT (n = 9, 8.9%), AU (n = 5, 5.0%), and ophiasis (n = 5, 5.0%). The estimated baseline SALT score was 0–25%, 26–50%, 51–75%, or 76–100% in 11 (10.9%), 16 (15.8%), 60 (59.4%), and 14 patients (13.9%), respectively. Body hair involvement was found in 5.9% (n = 6), and nail abnormalities were observed in 8.9% (n = 9) of the patients. Six patients (5.9%) had coexisting AITD, 2 (1.9%) were atopic, and 2 (1.9%) had a family member with AA. All demographics are summarized in Table 1.Table 1Demographic and Clinical Characteristics of Participants with Alopecia AreataVariablesN = 101Sex, n (%) ● Male31 (30.7) ● Female70 (69.3)Age at AA onset, year, mean ± SD40.4 ± 12.6Age at AA onset, year, n (%) ● ≤40 years50 (49.5) ● >40 years51 (50.5)Duration of disease, month, median (IQR)2 (1–4)Duration of disease, n (%) ● ≤6 months77 (76.2) ● >6 months24 (23.8)AA subtype, n (%) ● Patch AA20 (19.8) ● Multiple AA46 (45.5) ● Diffuse AA16 (15.8) ● AT9 (8.9) ● AU5 (5.0) ● Ophiasis5 (5.0)Severity of AA (SALT score), n (%) ● 0–25%11 (10.9) ● 26–50%16 (15.8) ● 51–75%60 (59.4) ● 76–100%14 (13.9)Body hair involvement, n (%)6 (5.9)Nail involvement, n (%)9 (8.9)Comorbidity, n (%) ● Atopy2 (1.9) ● Autoimmune thyroid diseases6 (5.9)Family history of AA, n (%)2 (1.9)Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.
Demographic and Clinical Characteristics of Participants with Alopecia Areata
Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.
A total of 126 patients with AA treated with IMC were initially included. Twenty-five patients were excluded due to coexisting diseases or concomitant use of other medications affecting the hair growth cycle (n = 16), incomplete medical records (n = 5), or because they received systemic treatment after the IMC protocol (n = 4). The majority of the remaining 101 patients were female (n = 70, 69.3%) with a female to male ratio of 2.26:1. The age range of AA onset was 5 to 68 years (mean = 40.4 ± 12.6 years), and the duration of disease varied from 1 week to 11 months (median = 2 [1–4] months). The most common AA subtype was multiple patches (n = 46, 45.5%), followed by patch (n = 20, 19.8%), diffuse (n = 16, 15.8%), AT (n = 9, 8.9%), AU (n = 5, 5.0%), and ophiasis (n = 5, 5.0%). The estimated baseline SALT score was 0–25%, 26–50%, 51–75%, or 76–100% in 11 (10.9%), 16 (15.8%), 60 (59.4%), and 14 patients (13.9%), respectively. Body hair involvement was found in 5.9% (n = 6), and nail abnormalities were observed in 8.9% (n = 9) of the patients. Six patients (5.9%) had coexisting AITD, 2 (1.9%) were atopic, and 2 (1.9%) had a family member with AA. All demographics are summarized in Table 1.Table 1Demographic and Clinical Characteristics of Participants with Alopecia AreataVariablesN = 101Sex, n (%) ● Male31 (30.7) ● Female70 (69.3)Age at AA onset, year, mean ± SD40.4 ± 12.6Age at AA onset, year, n (%) ● ≤40 years50 (49.5) ● >40 years51 (50.5)Duration of disease, month, median (IQR)2 (1–4)Duration of disease, n (%) ● ≤6 months77 (76.2) ● >6 months24 (23.8)AA subtype, n (%) ● Patch AA20 (19.8) ● Multiple AA46 (45.5) ● Diffuse AA16 (15.8) ● AT9 (8.9) ● AU5 (5.0) ● Ophiasis5 (5.0)Severity of AA (SALT score), n (%) ● 0–25%11 (10.9) ● 26–50%16 (15.8) ● 51–75%60 (59.4) ● 76–100%14 (13.9)Body hair involvement, n (%)6 (5.9)Nail involvement, n (%)9 (8.9)Comorbidity, n (%) ● Atopy2 (1.9) ● Autoimmune thyroid diseases6 (5.9)Family history of AA, n (%)2 (1.9)Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.
Demographic and Clinical Characteristics of Participants with Alopecia Areata
Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.
Overall Response The median overall follow-up time for this cohort was 20 (6–38) months. Patients received 1 to 6 IMC injections (mean = 3.12 ± 0.9) per treatment course. Ninety-eight patients (97.1%) showed improvement, whereas 3 patients (2.9%) showed treatment failure and were further treated by topical contact immunotherapy at approximately 9 months (36 weeks in 2 patients and 37 weeks in another one) after completion of IMC therapy. The mean duration of IMC therapy was 4.2 ± 1.1 months, with a range of 1 to 6 months. Treatment responses are summarized in Table 2.Table 2Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia AreataTreatment OutcomesN = 101Treatment failure (<10% regrowth) ● Incidence, n (%)3 (2.9)Initial hair regrowth (25% regrowth) ● Cumulative incidence, n (%)86 (85.1) ● Incidence rate, person-year (CI)64.0 (51.9–79.3) ● Median time to achieve endpoint, month (CI)0.9 (0.8–1.2) ● Median cumulative dose to achieve endpoint, mg100 ● Cumulative dose to achieve endpoint, mg, median (IQR)80 (20–160)Significant hair regrowth (75% regrowth) ● Cumulative incidence, n (%)81 (80.2) ● Incidence rate, person-year (CI)17.0 (13.6–21.7) ● Median time to achieve endpoint, month (CI)3.4 (2.9–4.4) ● Median cumulative dose to achieve endpoint, mg200 ● Cumulative dose to achieve endpoint, mg, median (IQR)120 (40–200)Complete hair regrowth (100% regrowth) ● Cumulative incidence, n (%)49 (48.5) ● Incidence rate, person-year (CI)8.0 (6.2–10.9) ● Median time to achieve endpoint, month (CI)6.1 (5.1–8.1) ● Cumulative dose to achieve endpoint, mg, median (IQR)160 (60–240)Relapse ● Cumulative incidence, n (%)48 (47.5) ● Incidence rate, person-year (CI)5.5 (4.2–7.3) ● Median time to relapse, month (CI)11.3 (8.6–15.4)Abbreviations: CI, confidence interval; IQR, interquartile range.
Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia Areata
Abbreviations: CI, confidence interval; IQR, interquartile range.
The median overall follow-up time for this cohort was 20 (6–38) months. Patients received 1 to 6 IMC injections (mean = 3.12 ± 0.9) per treatment course. Ninety-eight patients (97.1%) showed improvement, whereas 3 patients (2.9%) showed treatment failure and were further treated by topical contact immunotherapy at approximately 9 months (36 weeks in 2 patients and 37 weeks in another one) after completion of IMC therapy. The mean duration of IMC therapy was 4.2 ± 1.1 months, with a range of 1 to 6 months. Treatment responses are summarized in Table 2.Table 2Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia AreataTreatment OutcomesN = 101Treatment failure (<10% regrowth) ● Incidence, n (%)3 (2.9)Initial hair regrowth (25% regrowth) ● Cumulative incidence, n (%)86 (85.1) ● Incidence rate, person-year (CI)64.0 (51.9–79.3) ● Median time to achieve endpoint, month (CI)0.9 (0.8–1.2) ● Median cumulative dose to achieve endpoint, mg100 ● Cumulative dose to achieve endpoint, mg, median (IQR)80 (20–160)Significant hair regrowth (75% regrowth) ● Cumulative incidence, n (%)81 (80.2) ● Incidence rate, person-year (CI)17.0 (13.6–21.7) ● Median time to achieve endpoint, month (CI)3.4 (2.9–4.4) ● Median cumulative dose to achieve endpoint, mg200 ● Cumulative dose to achieve endpoint, mg, median (IQR)120 (40–200)Complete hair regrowth (100% regrowth) ● Cumulative incidence, n (%)49 (48.5) ● Incidence rate, person-year (CI)8.0 (6.2–10.9) ● Median time to achieve endpoint, month (CI)6.1 (5.1–8.1) ● Cumulative dose to achieve endpoint, mg, median (IQR)160 (60–240)Relapse ● Cumulative incidence, n (%)48 (47.5) ● Incidence rate, person-year (CI)5.5 (4.2–7.3) ● Median time to relapse, month (CI)11.3 (8.6–15.4)Abbreviations: CI, confidence interval; IQR, interquartile range.
Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia Areata
Abbreviations: CI, confidence interval; IQR, interquartile range.
Initial Hair Regrowth According to survival analysis, the cumulative incidence of initial hair regrowth after IMC therapy was 85.1%, with an incidence rate of 64.0 (95% CI = 51.9–79.3) person-years. The median time to achieve initial hair regrowth was 0.9 (95% CI = 0.8–1.2) months, and the average cumulative dose of IMC to achieve initial hair regrowth was 80 (20–160) mg.
According to survival analysis, the cumulative incidence of initial hair regrowth after IMC therapy was 85.1%, with an incidence rate of 64.0 (95% CI = 51.9–79.3) person-years. The median time to achieve initial hair regrowth was 0.9 (95% CI = 0.8–1.2) months, and the average cumulative dose of IMC to achieve initial hair regrowth was 80 (20–160) mg.
Significant Hair Regrowth Significant hair regrowth was indicated as the primary outcome and its cumulative incidence in our cohort was 80.2%, with an incidence rate of 17.0 (95% CI = 13.6–21.7) person-years. The median time required to achieve significant hair regrowth was 3.4 (95% CI = 2.9–4.4) months. According to the Kaplan–Meier curve, there was a lag of approximately 1 month from initiation of therapy to the first patients demonstrating significant hair regrowth. Following this initial event, significant hair regrowth was observed in 41.9% of patients at 3 months, 75.9% at 6 months, 81.0% at 9 months, and 86.5% at 12 months. The residual probabilities of significant regrowth as a function of the time after initiation of IMC are also shown in Figure 1A. The average cumulative dose of IMC to achieve significant hair regrowth was 120 (40–200) mg, while a median cumulative dose that 50% of patients attained significant hair regrowth was 200 mg (Figure 1B).Figure 1Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.
Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.
The baseline SALT score was identified as a negative factor for IMC treatment response in a previous study.31 To determine the effect of this variable on significant hair regrowth, a stratified Kaplan–Meier analysis was performed. Nine patients (81.8%) with a score of 0–25% obtained significant hair regrowth by a median time of 3.5 months; for the score range 26–50%, 12 (75.0%) patients achieved significant regrowth by 4.8 months; for the score range 51–75%, 48 (80.0%) experienced significant regrowth by 3.1 months; and for the score range 76–100%, 12 (85.7%) ultimately obtained significant regrowth at 3.2 months. However, the log rank test failed to demonstrate statistically significant differences among the four groups (P = 0.287; Figure 1C).
Significant hair regrowth was indicated as the primary outcome and its cumulative incidence in our cohort was 80.2%, with an incidence rate of 17.0 (95% CI = 13.6–21.7) person-years. The median time required to achieve significant hair regrowth was 3.4 (95% CI = 2.9–4.4) months. According to the Kaplan–Meier curve, there was a lag of approximately 1 month from initiation of therapy to the first patients demonstrating significant hair regrowth. Following this initial event, significant hair regrowth was observed in 41.9% of patients at 3 months, 75.9% at 6 months, 81.0% at 9 months, and 86.5% at 12 months. The residual probabilities of significant regrowth as a function of the time after initiation of IMC are also shown in Figure 1A. The average cumulative dose of IMC to achieve significant hair regrowth was 120 (40–200) mg, while a median cumulative dose that 50% of patients attained significant hair regrowth was 200 mg (Figure 1B).Figure 1Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.
Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.
The baseline SALT score was identified as a negative factor for IMC treatment response in a previous study.31 To determine the effect of this variable on significant hair regrowth, a stratified Kaplan–Meier analysis was performed. Nine patients (81.8%) with a score of 0–25% obtained significant hair regrowth by a median time of 3.5 months; for the score range 26–50%, 12 (75.0%) patients achieved significant regrowth by 4.8 months; for the score range 51–75%, 48 (80.0%) experienced significant regrowth by 3.1 months; and for the score range 76–100%, 12 (85.7%) ultimately obtained significant regrowth at 3.2 months. However, the log rank test failed to demonstrate statistically significant differences among the four groups (P = 0.287; Figure 1C).
Factors Associated with Significant Hair Regrowth The Cox proportional hazards model was used to determine the factors associated with significant hair regrowth (Table 3). Univariable analysis revealed that the variables associated with significant hair regrowth were female sex (HR = 2.39, 95% CI = 1.09–4.61; P = 0.035), age at AA onset >40 years (HR = 2.45, 95% CI = 1.49–3.69; P = 0.046), and nail abnormalities (HR = 0.26, 95% CI = 0.08–0.69; P = 0.036). However, when age at AA onset was analyzed as a continuous variable, no association was found (HR = 1.02, 95% CI = 0.89–1.06; P = 0.423). After adjusting for all confounding factors in a multivariable model, nail involvement was the only independent factor negatively affecting the probability of significant hair regrowth (adjusted HR = 0.04, 95% CI = 0.01–0.55; P = 0.015).Table 3Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale2.39 (1.09–4.61)0.035*1.13 (0.81–5.91)0.086Age at AA onset1.02 (0.89–1.06)0.423Age group at AA onset ● ≤40 yearsReference ● >40 years2.45 (1.49–3.69)0.046*1.68 (0.92–3.42)0.187Duration of disease >6 months0.63 (0.25–1.49)0.141AA subtype ● Patch AAReference ● Multiple AA1.34 (0.25–2.92)0.182 ● Diffuse AA2.63 (0.99–4.61)0.062 ● AT0.82 (0.29–2.85)0.845 ● AU0.16 (0.09–3.15)0.753 ● Ophiasis0.76 (0.09–2.92)0.911Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.56 (0.32–2.09)0.186 ● 51–75%0.64 (0.31–2.67)0.823 ● 76–100%1.49 (0.81–3.68)0.595Body hair involvement0.67 (0.03–4.28)0.745Nail involvement0.26 (0.08–0.69)0.036*0.04 (0.01–0.55)0.015*Atopy0.68 (0.13–5.16)0.621Autoimmune thyroid diseases1.91 (0.83–3.28)0.142Family history of AA0.96 (0.41–3.56)0.363Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata
Note: *Statistically significant.
Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
The Cox proportional hazards model was used to determine the factors associated with significant hair regrowth (Table 3). Univariable analysis revealed that the variables associated with significant hair regrowth were female sex (HR = 2.39, 95% CI = 1.09–4.61; P = 0.035), age at AA onset >40 years (HR = 2.45, 95% CI = 1.49–3.69; P = 0.046), and nail abnormalities (HR = 0.26, 95% CI = 0.08–0.69; P = 0.036). However, when age at AA onset was analyzed as a continuous variable, no association was found (HR = 1.02, 95% CI = 0.89–1.06; P = 0.423). After adjusting for all confounding factors in a multivariable model, nail involvement was the only independent factor negatively affecting the probability of significant hair regrowth (adjusted HR = 0.04, 95% CI = 0.01–0.55; P = 0.015).Table 3Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale2.39 (1.09–4.61)0.035*1.13 (0.81–5.91)0.086Age at AA onset1.02 (0.89–1.06)0.423Age group at AA onset ● ≤40 yearsReference ● >40 years2.45 (1.49–3.69)0.046*1.68 (0.92–3.42)0.187Duration of disease >6 months0.63 (0.25–1.49)0.141AA subtype ● Patch AAReference ● Multiple AA1.34 (0.25–2.92)0.182 ● Diffuse AA2.63 (0.99–4.61)0.062 ● AT0.82 (0.29–2.85)0.845 ● AU0.16 (0.09–3.15)0.753 ● Ophiasis0.76 (0.09–2.92)0.911Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.56 (0.32–2.09)0.186 ● 51–75%0.64 (0.31–2.67)0.823 ● 76–100%1.49 (0.81–3.68)0.595Body hair involvement0.67 (0.03–4.28)0.745Nail involvement0.26 (0.08–0.69)0.036*0.04 (0.01–0.55)0.015*Atopy0.68 (0.13–5.16)0.621Autoimmune thyroid diseases1.91 (0.83–3.28)0.142Family history of AA0.96 (0.41–3.56)0.363Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata
Note: *Statistically significant.
Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
Complete Hair Regrowth Regarding complete hair regrowth, the cumulative incidence was 48.5%, with an incidence rate of 8.0 (95% CI = 6.2–10.9) person-years. The median time and average cumulative dose of IMC to achieve complete hair regrowth were 6.1 (95% CI = 5.1–8.1) months and 160 (60–240) mg, respectively.
Regarding complete hair regrowth, the cumulative incidence was 48.5%, with an incidence rate of 8.0 (95% CI = 6.2–10.9) person-years. The median time and average cumulative dose of IMC to achieve complete hair regrowth were 6.1 (95% CI = 5.1–8.1) months and 160 (60–240) mg, respectively.
Relapse and Its Associated Factors Following 51 months of follow-up, the overall cumulative relapse rate after achievement of clinical hair regrowth with IMC was 47.5% (n = 48; Figure 1D). The incidence rate of AA relapse was 5.5 (95% CI = 4.2–7.3) person-years, and the median time to relapse was 11.3 (95% CI = 8.6–15.4) months. Treatment in patients with recurrence included combination therapy with IL corticosteroids and topical minoxidil (n = 21), topical contact immunotherapy (n = 10), methotrexate (n = 9), IMC (n = 4), and IL corticosteroids (n = 4). All therapies were prescribed at >6 months after discontinuation of the IMC regimen.
The risk of AA relapse was determined using the Cox proportional hazards model. Univariable analysis revealed that coexisting AITD (HR = 2.39, 95% CI = 1.42–5.14; P = 0.041) and disease duration >6 months (HR = 3.12, 95% CI = 1.36–4.94; P = 0.016) were associated with a higher probability of disease recurrence. However, multivariable analysis showed that only AA duration >6 months (adjusted HR = 4.02, 95% CI = 1.52–4.66; P = 0.005) independently increased the probability of AA relapse (Table 4).Table 4Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale1.15 (0.64–1.69)0.536Age at AA onset1.01 (0.99–1.06)0.104Age group at AA onset ● ≤40 yearsReference ● >40 years1.55 (0.78–2.56)0.151Duration of disease >6 months3.12 (1.36–4.94)0.016*4.02 (1.52–4.66)0.005*AA subtype ● Patch AAReference ● Multiple AA0.94 (0.66–2.67)0.723 ● Diffuse AA0.85 (0.39–3.62)0.264 ● AT3.12 (0.79–5.64)0.459 ● AU2.92 (0.59–3.95)0.644 ● Ophiasis1.49 (0.08–3.12)0.814Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.91 (0.72–2.19)0.135 ● 51–75%1.14 (0.51–2.67)0.718 ● 76–100%0.69 (0.29–1.89)0.653Body hair involvement1.05 (0.29–3.18)0.425Nail involvement2.78 (0.98–4.09)0.812Atopy1.34 (0.63–3.89)0.291Autoimmune thyroid diseases2.39 (1.42–5.14)0.041*2.71 (0.82–4.65)0.069Family history of AA1.76 (0.87–2.88)0.635Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata
Note: *Statistically significant.
Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
Following 51 months of follow-up, the overall cumulative relapse rate after achievement of clinical hair regrowth with IMC was 47.5% (n = 48; Figure 1D). The incidence rate of AA relapse was 5.5 (95% CI = 4.2–7.3) person-years, and the median time to relapse was 11.3 (95% CI = 8.6–15.4) months. Treatment in patients with recurrence included combination therapy with IL corticosteroids and topical minoxidil (n = 21), topical contact immunotherapy (n = 10), methotrexate (n = 9), IMC (n = 4), and IL corticosteroids (n = 4). All therapies were prescribed at >6 months after discontinuation of the IMC regimen.
The risk of AA relapse was determined using the Cox proportional hazards model. Univariable analysis revealed that coexisting AITD (HR = 2.39, 95% CI = 1.42–5.14; P = 0.041) and disease duration >6 months (HR = 3.12, 95% CI = 1.36–4.94; P = 0.016) were associated with a higher probability of disease recurrence. However, multivariable analysis showed that only AA duration >6 months (adjusted HR = 4.02, 95% CI = 1.52–4.66; P = 0.005) independently increased the probability of AA relapse (Table 4).Table 4Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale1.15 (0.64–1.69)0.536Age at AA onset1.01 (0.99–1.06)0.104Age group at AA onset ● ≤40 yearsReference ● >40 years1.55 (0.78–2.56)0.151Duration of disease >6 months3.12 (1.36–4.94)0.016*4.02 (1.52–4.66)0.005*AA subtype ● Patch AAReference ● Multiple AA0.94 (0.66–2.67)0.723 ● Diffuse AA0.85 (0.39–3.62)0.264 ● AT3.12 (0.79–5.64)0.459 ● AU2.92 (0.59–3.95)0.644 ● Ophiasis1.49 (0.08–3.12)0.814Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.91 (0.72–2.19)0.135 ● 51–75%1.14 (0.51–2.67)0.718 ● 76–100%0.69 (0.29–1.89)0.653Body hair involvement1.05 (0.29–3.18)0.425Nail involvement2.78 (0.98–4.09)0.812Atopy1.34 (0.63–3.89)0.291Autoimmune thyroid diseases2.39 (1.42–5.14)0.041*2.71 (0.82–4.65)0.069Family history of AA1.76 (0.87–2.88)0.635Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata
Note: *Statistically significant.
Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
Adverse Events Adverse events were experienced by 18.8% of the patients (n = 19) and included acneiform eruption (n = 16, 15.8%), abnormal menstrual cycle (n = 6, 5.9%), weight gain (n = 3, 2.9%), and sleep disturbance (n = 1, 0.9%). Acneiform eruption was managed with topical therapy, while the other side effects resolved spontaneously after discontinuing IMC. The presence of any of these adverse events did not cause dropout from therapy.
Adverse events were experienced by 18.8% of the patients (n = 19) and included acneiform eruption (n = 16, 15.8%), abnormal menstrual cycle (n = 6, 5.9%), weight gain (n = 3, 2.9%), and sleep disturbance (n = 1, 0.9%). Acneiform eruption was managed with topical therapy, while the other side effects resolved spontaneously after discontinuing IMC. The presence of any of these adverse events did not cause dropout from therapy.
Demographics:
A total of 126 patients with AA treated with IMC were initially included. Twenty-five patients were excluded due to coexisting diseases or concomitant use of other medications affecting the hair growth cycle (n = 16), incomplete medical records (n = 5), or because they received systemic treatment after the IMC protocol (n = 4). The majority of the remaining 101 patients were female (n = 70, 69.3%) with a female to male ratio of 2.26:1. The age range of AA onset was 5 to 68 years (mean = 40.4 ± 12.6 years), and the duration of disease varied from 1 week to 11 months (median = 2 [1–4] months). The most common AA subtype was multiple patches (n = 46, 45.5%), followed by patch (n = 20, 19.8%), diffuse (n = 16, 15.8%), AT (n = 9, 8.9%), AU (n = 5, 5.0%), and ophiasis (n = 5, 5.0%). The estimated baseline SALT score was 0–25%, 26–50%, 51–75%, or 76–100% in 11 (10.9%), 16 (15.8%), 60 (59.4%), and 14 patients (13.9%), respectively. Body hair involvement was found in 5.9% (n = 6), and nail abnormalities were observed in 8.9% (n = 9) of the patients. Six patients (5.9%) had coexisting AITD, 2 (1.9%) were atopic, and 2 (1.9%) had a family member with AA. All demographics are summarized in Table 1.Table 1Demographic and Clinical Characteristics of Participants with Alopecia AreataVariablesN = 101Sex, n (%) ● Male31 (30.7) ● Female70 (69.3)Age at AA onset, year, mean ± SD40.4 ± 12.6Age at AA onset, year, n (%) ● ≤40 years50 (49.5) ● >40 years51 (50.5)Duration of disease, month, median (IQR)2 (1–4)Duration of disease, n (%) ● ≤6 months77 (76.2) ● >6 months24 (23.8)AA subtype, n (%) ● Patch AA20 (19.8) ● Multiple AA46 (45.5) ● Diffuse AA16 (15.8) ● AT9 (8.9) ● AU5 (5.0) ● Ophiasis5 (5.0)Severity of AA (SALT score), n (%) ● 0–25%11 (10.9) ● 26–50%16 (15.8) ● 51–75%60 (59.4) ● 76–100%14 (13.9)Body hair involvement, n (%)6 (5.9)Nail involvement, n (%)9 (8.9)Comorbidity, n (%) ● Atopy2 (1.9) ● Autoimmune thyroid diseases6 (5.9)Family history of AA, n (%)2 (1.9)Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.
Demographic and Clinical Characteristics of Participants with Alopecia Areata
Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; IQR, interquartile range; SALT, Severity of Alopecia Tool; SD, standard deviation.
Overall Response:
The median overall follow-up time for this cohort was 20 (6–38) months. Patients received 1 to 6 IMC injections (mean = 3.12 ± 0.9) per treatment course. Ninety-eight patients (97.1%) showed improvement, whereas 3 patients (2.9%) showed treatment failure and were further treated by topical contact immunotherapy at approximately 9 months (36 weeks in 2 patients and 37 weeks in another one) after completion of IMC therapy. The mean duration of IMC therapy was 4.2 ± 1.1 months, with a range of 1 to 6 months. Treatment responses are summarized in Table 2.Table 2Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia AreataTreatment OutcomesN = 101Treatment failure (<10% regrowth) ● Incidence, n (%)3 (2.9)Initial hair regrowth (25% regrowth) ● Cumulative incidence, n (%)86 (85.1) ● Incidence rate, person-year (CI)64.0 (51.9–79.3) ● Median time to achieve endpoint, month (CI)0.9 (0.8–1.2) ● Median cumulative dose to achieve endpoint, mg100 ● Cumulative dose to achieve endpoint, mg, median (IQR)80 (20–160)Significant hair regrowth (75% regrowth) ● Cumulative incidence, n (%)81 (80.2) ● Incidence rate, person-year (CI)17.0 (13.6–21.7) ● Median time to achieve endpoint, month (CI)3.4 (2.9–4.4) ● Median cumulative dose to achieve endpoint, mg200 ● Cumulative dose to achieve endpoint, mg, median (IQR)120 (40–200)Complete hair regrowth (100% regrowth) ● Cumulative incidence, n (%)49 (48.5) ● Incidence rate, person-year (CI)8.0 (6.2–10.9) ● Median time to achieve endpoint, month (CI)6.1 (5.1–8.1) ● Cumulative dose to achieve endpoint, mg, median (IQR)160 (60–240)Relapse ● Cumulative incidence, n (%)48 (47.5) ● Incidence rate, person-year (CI)5.5 (4.2–7.3) ● Median time to relapse, month (CI)11.3 (8.6–15.4)Abbreviations: CI, confidence interval; IQR, interquartile range.
Treatment Outcomes of Intramuscular Corticosteroid Therapy in Participants with Alopecia Areata
Abbreviations: CI, confidence interval; IQR, interquartile range.
Initial Hair Regrowth:
According to survival analysis, the cumulative incidence of initial hair regrowth after IMC therapy was 85.1%, with an incidence rate of 64.0 (95% CI = 51.9–79.3) person-years. The median time to achieve initial hair regrowth was 0.9 (95% CI = 0.8–1.2) months, and the average cumulative dose of IMC to achieve initial hair regrowth was 80 (20–160) mg.
Significant Hair Regrowth:
Significant hair regrowth was indicated as the primary outcome and its cumulative incidence in our cohort was 80.2%, with an incidence rate of 17.0 (95% CI = 13.6–21.7) person-years. The median time required to achieve significant hair regrowth was 3.4 (95% CI = 2.9–4.4) months. According to the Kaplan–Meier curve, there was a lag of approximately 1 month from initiation of therapy to the first patients demonstrating significant hair regrowth. Following this initial event, significant hair regrowth was observed in 41.9% of patients at 3 months, 75.9% at 6 months, 81.0% at 9 months, and 86.5% at 12 months. The residual probabilities of significant regrowth as a function of the time after initiation of IMC are also shown in Figure 1A. The average cumulative dose of IMC to achieve significant hair regrowth was 120 (40–200) mg, while a median cumulative dose that 50% of patients attained significant hair regrowth was 200 mg (Figure 1B).Figure 1Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.
Kaplan–Meier curve demonstrating (A) the cumulative incidence of patients achieving significant hair regrowth (75% regrowth); (B) the cumulative corticosteroid dose of patients achieving significant hair regrowth; (C) the cumulative incidence of patients achieving significant hair regrowth of four groups according to baseline Severity of Alopecia Tool (SALT) score; and (D) the cumulative incidence of patients with relapse.
The baseline SALT score was identified as a negative factor for IMC treatment response in a previous study.31 To determine the effect of this variable on significant hair regrowth, a stratified Kaplan–Meier analysis was performed. Nine patients (81.8%) with a score of 0–25% obtained significant hair regrowth by a median time of 3.5 months; for the score range 26–50%, 12 (75.0%) patients achieved significant regrowth by 4.8 months; for the score range 51–75%, 48 (80.0%) experienced significant regrowth by 3.1 months; and for the score range 76–100%, 12 (85.7%) ultimately obtained significant regrowth at 3.2 months. However, the log rank test failed to demonstrate statistically significant differences among the four groups (P = 0.287; Figure 1C).
Factors Associated with Significant Hair Regrowth:
The Cox proportional hazards model was used to determine the factors associated with significant hair regrowth (Table 3). Univariable analysis revealed that the variables associated with significant hair regrowth were female sex (HR = 2.39, 95% CI = 1.09–4.61; P = 0.035), age at AA onset >40 years (HR = 2.45, 95% CI = 1.49–3.69; P = 0.046), and nail abnormalities (HR = 0.26, 95% CI = 0.08–0.69; P = 0.036). However, when age at AA onset was analyzed as a continuous variable, no association was found (HR = 1.02, 95% CI = 0.89–1.06; P = 0.423). After adjusting for all confounding factors in a multivariable model, nail involvement was the only independent factor negatively affecting the probability of significant hair regrowth (adjusted HR = 0.04, 95% CI = 0.01–0.55; P = 0.015).Table 3Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale2.39 (1.09–4.61)0.035*1.13 (0.81–5.91)0.086Age at AA onset1.02 (0.89–1.06)0.423Age group at AA onset ● ≤40 yearsReference ● >40 years2.45 (1.49–3.69)0.046*1.68 (0.92–3.42)0.187Duration of disease >6 months0.63 (0.25–1.49)0.141AA subtype ● Patch AAReference ● Multiple AA1.34 (0.25–2.92)0.182 ● Diffuse AA2.63 (0.99–4.61)0.062 ● AT0.82 (0.29–2.85)0.845 ● AU0.16 (0.09–3.15)0.753 ● Ophiasis0.76 (0.09–2.92)0.911Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.56 (0.32–2.09)0.186 ● 51–75%0.64 (0.31–2.67)0.823 ● 76–100%1.49 (0.81–3.68)0.595Body hair involvement0.67 (0.03–4.28)0.745Nail involvement0.26 (0.08–0.69)0.036*0.04 (0.01–0.55)0.015*Atopy0.68 (0.13–5.16)0.621Autoimmune thyroid diseases1.91 (0.83–3.28)0.142Family history of AA0.96 (0.41–3.56)0.363Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
Univariate and Multivariable Analyses for the Factor Associated with Significant Hair Regrowth After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata
Note: *Statistically significant.
Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
Complete Hair Regrowth:
Regarding complete hair regrowth, the cumulative incidence was 48.5%, with an incidence rate of 8.0 (95% CI = 6.2–10.9) person-years. The median time and average cumulative dose of IMC to achieve complete hair regrowth were 6.1 (95% CI = 5.1–8.1) months and 160 (60–240) mg, respectively.
Relapse and Its Associated Factors:
Following 51 months of follow-up, the overall cumulative relapse rate after achievement of clinical hair regrowth with IMC was 47.5% (n = 48; Figure 1D). The incidence rate of AA relapse was 5.5 (95% CI = 4.2–7.3) person-years, and the median time to relapse was 11.3 (95% CI = 8.6–15.4) months. Treatment in patients with recurrence included combination therapy with IL corticosteroids and topical minoxidil (n = 21), topical contact immunotherapy (n = 10), methotrexate (n = 9), IMC (n = 4), and IL corticosteroids (n = 4). All therapies were prescribed at >6 months after discontinuation of the IMC regimen.
The risk of AA relapse was determined using the Cox proportional hazards model. Univariable analysis revealed that coexisting AITD (HR = 2.39, 95% CI = 1.42–5.14; P = 0.041) and disease duration >6 months (HR = 3.12, 95% CI = 1.36–4.94; P = 0.016) were associated with a higher probability of disease recurrence. However, multivariable analysis showed that only AA duration >6 months (adjusted HR = 4.02, 95% CI = 1.52–4.66; P = 0.005) independently increased the probability of AA relapse (Table 4).Table 4Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia AreataVariablesHR (95% CI)P-valueAdjusted HR (95% CI)P-valueFemale1.15 (0.64–1.69)0.536Age at AA onset1.01 (0.99–1.06)0.104Age group at AA onset ● ≤40 yearsReference ● >40 years1.55 (0.78–2.56)0.151Duration of disease >6 months3.12 (1.36–4.94)0.016*4.02 (1.52–4.66)0.005*AA subtype ● Patch AAReference ● Multiple AA0.94 (0.66–2.67)0.723 ● Diffuse AA0.85 (0.39–3.62)0.264 ● AT3.12 (0.79–5.64)0.459 ● AU2.92 (0.59–3.95)0.644 ● Ophiasis1.49 (0.08–3.12)0.814Severity of AA (SALT score) ● 0–25%Reference ● 26–50%0.91 (0.72–2.19)0.135 ● 51–75%1.14 (0.51–2.67)0.718 ● 76–100%0.69 (0.29–1.89)0.653Body hair involvement1.05 (0.29–3.18)0.425Nail involvement2.78 (0.98–4.09)0.812Atopy1.34 (0.63–3.89)0.291Autoimmune thyroid diseases2.39 (1.42–5.14)0.041*2.71 (0.82–4.65)0.069Family history of AA1.76 (0.87–2.88)0.635Note: *Statistically significant.Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
Univariate and Multivariable Analyses for the Factor Associated with Relapse After Intramuscular Corticosteroid Therapy in Patients with Alopecia Areata
Note: *Statistically significant.
Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; CI, confidence interval; HR, hazard ratio; SALT, Severity of Alopecia Tool.
Adverse Events:
Adverse events were experienced by 18.8% of the patients (n = 19) and included acneiform eruption (n = 16, 15.8%), abnormal menstrual cycle (n = 6, 5.9%), weight gain (n = 3, 2.9%), and sleep disturbance (n = 1, 0.9%). Acneiform eruption was managed with topical therapy, while the other side effects resolved spontaneously after discontinuing IMC. The presence of any of these adverse events did not cause dropout from therapy.
Discussion:
Although IMC therapy has shown to be effective in the treatment of several dermatological conditions and gained popularity for AA treatment in recent years, information regarding the use of IMC for AA is limited in the literature.32 In this study, we modeled the therapeutic endpoints of the IMC regimen according to multiple variables in patients with AA. To the best of our knowledge, this is the first study to describe the efficacy, cumulative effective dose, and factors associated with treatment outcomes in patients with AA receiving IMC based on a time-to-event analysis. The primary endpoint, defined as significant hair regrowth, had a rate of 80.2%, with a median time of approximately 3–4 months. Nail involvement was demonstrated to be a negative predictor of significant hair regrowth. Relapse was found in 47.5% of the patients, with disease duration >6 months being a risk factor for AA recurrence.
The application of systemic corticosteroids for the treatment of AA was first introduced by Dillaha et al in 1952, with remarkable therapeutic effects.33 Since then, the efficacy of various regimens such as oral, intravenous, and intramuscular administration has been investigated. Oral corticosteroid pulse therapy is generally the most convenient method and has been well studied.34 However, poor medication compliance and adherence may lead to reduced treatment efficacy and substantial worsening of the disease. The therapeutic effect of intravenous pulse corticosteroids has also been confirmed. However, the overall adverse events of intramuscular and oral corticosteroids are relatively high, approaching 41% and 30%, respectively, compared to that of 10% from corticosteroid pulse therapy.29 Hypothalamic–pituitary–adrenal (HPA) axis suppression was reported in 23%, 67%, and 7% of intramuscular, oral, and pulse corticosteroids, respectively.29 Intramuscular injections may offer an advantage over the two methods. IMC maximizes treatment efficacy by providing rapid onset of action comparable to the oral form, and by increasing patients’ compliance and adherence owing to the monthly or bimonthly injection regimen.28 It also minimizes adverse effects due to its pharmacokinetic properties, with a gradual and steady release of corticosteroids over time.28
Our study demonstrated the efficacy of IMC for the treatment of severe, acute, or rapidly progressive AA, as shown by the 80.2% cumulative incidence of significant hair regrowth rate in a median time of 3.4 months. Michalowski and Kuczyfiska reported a 72.7% response rate in 11 cases of extensive AA (AT/AU) receiving 40–80 mg of IMC therapy, initially once a week, then over a gradually lengthened interval up to 6 weeks.31 A retrospective study using IMC 40 mg every 4 weeks for a maximum of 6 months revealed a 63% response rate in refractory AA.30 Administration of IMC 40 mg monthly for 6 months, followed by 40 mg every 1.5 months for 1 year, had significantly superior efficacy, with a 74% response rate, to oral corticosteroid pulse therapy.29 However, our results could not be directly compared to those of earlier studies because of the difference in the severity of AA among the cohorts. The lower percentage of recalcitrant AA cases included in our study compared with previous ones could explain the higher response rate.
Regarding other corticosteroid injection protocols for AA, IL injection of 2.5–10 mg/mL triamcinolone acetonide at four–to-six-week intervals is commonly used in mild forms of AA (ie, patch AA or SALT score <25%) with a response rate between 64% and 97%.23 For severe AA, intravenous injection of 500 mg methylprednisolone for three consecutive days at four-week intervals for three months has a response rate of approximately 80%.23 However, compared to IL injection, the dosage for intramuscular and intravenous injections is higher and may lead to potential adverse effects; therefore, they should be restricted to those with severe AA and administered with caution.
Identifying the factors affecting treatment outcomes of AA is essential for therapeutic success; nevertheless, specific predictors for IMC have been rarely reported. Seo et al indicated that the baseline SALT score was negatively correlated with hair regrowth response to IMC.30 Our stratified analysis using the Kaplan–Meier method did not support this finding, as no association was found between the extent of scalp involvement and treatment response. Earlier age at AA onset, longer disease duration, and nail involvement were identified as indicators of poor prognosis.35–38 A recent meta-analysis demonstrated four negative predictors for treatment response, namely SALT score ≥50, disease duration ≥1 year, atopic diseases, and nail abnormalities.35 Our analysis using a Cox proportional hazards model showed a lack of correlation between significant hair regrowth and SALT score, AA duration, or presence of atopy, but confirmed its negative association with nail abnormalities. This finding could be explained by the characteristics of the enrolled subjects in our cohort, namely the predominance of female patients presenting with rapidly progressive scalp hair loss >50% for <6 months. The clinical characteristics of our patients were compatible to those of patients with acute diffuse and total alopecia, a distinctive AA subtype, which shows a favorable treatment response to systemic corticosteroid therapy.39,40
Emerging evidence suggests that approximately half the patients with AA treated with IMC experience disease recurrence. Seo et al demonstrated a 47.1% relapse rate during a follow-up period of 14.2 months.30 Kurosawa et al reported a comparable relapse rate of 46% after 6 months of treatment.29 Our result was consistent with previous studies, revealing recurrence in approximately half of patients with a median time of 11.3 months. Furthermore, disease duration >6 months was identified as a predictor of AA relapse. Longer AA duration could indicate disease chronicity, and physicians should remain vigilant for AA relapse despite complete IMC therapy. Therefore, clinicians should consider the use of post-IMC treatments such as application of 2% topical minoxidil three times daily, which was reported to limit post-steroid-treatment hair loss.41 For AA patients with chronic relapsing disease, further treatments after initial systemic corticosteroid therapy are challenging owing to a lack of high-quality randomized controlled trials supporting the efficacy and safety of other therapeutic modalities. However, a shared decision-making approach between the physician and patient is crucial as it would ensure that patients have a realistic expectation of treatment outcomes. Topical contact immunotherapy is currently considered the best modality for long-term therapy of patients with recalcitrant AA and is the first line treatment for these cases in our hair clinic.19
HPA axis suppression is considered a major side effect of IMC therapy.32 Previous studies demonstrated adrenocortical alteration in 23% of subjects receiving intramuscular injection.29 Other adverse effects of IMC include abdominal discomfort, dysmenorrhea, and acne.29,30 The incidence of adverse events among our AA patients was low, and acneiform eruption was the most common; no serious side effects were reported. However, HPA axis suppression in our subjects may be underestimated due to a lack of data regarding adrenocortical evaluation in our follow-up protocol.
A major strength of our study is the use of the time-to-event approach to characterize the therapeutic outcomes of patients with AA receiving IMC during the entire follow-up time, including censored observations. This method considers all available treatment and follow-up data, providing more precise and meaningful estimates of overall efficacy and tolerability, and better outcome prediction. The limitations of this study are its retrospective design involving a single tertiary center. These factors might lead to the unavailability of some data and limit the generalizability of the results. Our results may also be limited in their applicability to patients without severe and/or rapidly progressive AA. Additionally, it is difficult to compare our findings to those of previous studies, owing to differences in the definitions of treatment responses and in methodologies. Further large, multicenter randomized controlled trials evaluating the efficacy and safety of IMC therapy are required to overcome these limitations.
Conclusion:
Our time-to-event analysis demonstrated the efficacy and safety of intramuscular administration of corticosteroids for the treatment of AA. IMC therapy showed high rates of significant hair regrowth, especially in patients with severe or active AA; however, approximately half of those experienced relapse. Nail involvement was negatively correlated with cosmetically acceptable hair regrowth, while disease duration >6 months was significantly associated with increased risk of relapse. Most importantly, the use of IMC depends on indication and perceived potential benefits and risks; therefore, physicians should be cautious when prescribing this treatment.
|
Background:
Intramuscular corticosteroids (IMC) have gained popularity for the treatment of severe alopecia areata (AA) in recent years; however, evidence on their efficacy and safety is still limited.
Methods:
Time-to-event analysis was performed on patients with severe, extensive, or rapidly progressive AA receiving IMC. The IMC regimen comprised triamcinolone acetonide 20-40 mg/mL injected every 4-6 weeks. The evaluated outcomes included initial (25% regrowth), significant (75% regrowth), and complete hair regrowth (100% regrowth). Relapse and adverse events were also noted. Factors associated with treatment outcomes and relapse were analyzed using the Cox proportional hazards model.
Results:
A total of 101 patients were eligible for analysis. Significant hair regrowth was obtained in 80.2% of the patients (n = 81), in a median time of 3.4 months (95% confidence interval [CI] = 2.9-4.4). Complete hair regrowth was achieved in 48.5% of the subjects (n = 49), and relapse was observed in 47.5% (n = 48). Acneiform eruption was the most common adverse effect. Multivariable analysis revealed that nail involvement was a negative predictor of significant hair regrowth (adjusted hazard ratio [HR] = 0.04, 95% CI = 0.01-0.55; P = 0.015), whereas duration of AA longer than 6 months was associated with disease recurrence (adjusted HR = 4.02, 95% CI = 1.52-4.66; P = 0.005).
Conclusions:
This study demonstrated the efficacy and safety of IMC in the treatment of severe or active AA; however, the relapse rate remained relatively high after discontinuation of the therapy. Nail involvement was a negative predictor of significant hair regrowth, while disease duration longer than 6 months predicted AA relapse.
|
Introduction:
Alopecia areata (AA) is a non-scarring hair loss disorder that affects the scalp and hair-bearing areas.1 Variations on the clinical presentation of AA have been observed, ranging from small, well-defined circular or oval patches of hair loss to diffuse alopecia and complete scalp (alopecia totalis, AT) or total body hair loss (alopecia universalis, AU).2–5 Nail abnormalities, most frequently nail pitting, are occasionally present. Patients with AA often experience major psychological impacts due to the unpredictable clinical course and variable treatment response.6 Various immune-related comorbidities such as autoimmune thyroid diseases (AITD), vitiligo, psoriasis, and atopic diseases have been linked to AA.7–13
The exact etiopathogenesis of AA is unclear, though it is believed to be a cell-mediated autoimmune disease of the hair follicle. Autoreactive T-cell lymphocytes and several cytokines mediate hair follicular destruction and negatively affect the hair growth cycle.14–16 Despite the absence of curative/preventive treatments and FDA-approved regimens for AA, therapeutic approaches have been established through treatment guidelines. Topical and intralesional (IL) corticosteroids, topical minoxidil, and topical anthralin are recommended for AA patients with <50% scalp involvement, whereas in those with ≥50% hair loss, topical contact immunotherapy and systemic corticosteroids are advised.17–23
Corticosteroids, the main treatment option for AA, can be administered in different forms depending on the disease severity.24 Medium- to high-potency topical and IL corticosteroids are indicated as monotherapy or combined therapy in limited and/or slowly progressive AA,25–27 while systemic corticosteroids (eg, oral, intravenous, and intramuscular), are preferred in extensive and/or rapidly progressive disease.17
Among systemic modalities, intramuscular corticosteroids (IMC) may be most beneficial, as its monthly or bimonthly regimen increases patients’ compliance and treatment adherence.28 Moreover, IMC could minimize adverse effects from slowly releasing continuous and small amounts of medication.28 Previous studies of IMC showed a satisfactory outcome in severe and refractory AA, with response rates between 63% and 72.7%.29–31 Although IMC is often the preferred treatment for AA, there are limited data regarding its effectiveness, safety, dosing, and optimal duration. Thus, we conducted a retrospective cohort study based on our 20-year experience to evaluate the efficacy, relapse, and tolerability, as well as factors associated with treatment outcomes of IMC in patients with AA.
Conclusion:
Our time-to-event analysis demonstrated the efficacy and safety of intramuscular administration of corticosteroids for the treatment of AA. IMC therapy showed high rates of significant hair regrowth, especially in patients with severe or active AA; however, approximately half of those experienced relapse. Nail involvement was negatively correlated with cosmetically acceptable hair regrowth, while disease duration >6 months was significantly associated with increased risk of relapse. Most importantly, the use of IMC depends on indication and perceived potential benefits and risks; therefore, physicians should be cautious when prescribing this treatment.
|
Background:
Intramuscular corticosteroids (IMC) have gained popularity for the treatment of severe alopecia areata (AA) in recent years; however, evidence on their efficacy and safety is still limited.
Methods:
Time-to-event analysis was performed on patients with severe, extensive, or rapidly progressive AA receiving IMC. The IMC regimen comprised triamcinolone acetonide 20-40 mg/mL injected every 4-6 weeks. The evaluated outcomes included initial (25% regrowth), significant (75% regrowth), and complete hair regrowth (100% regrowth). Relapse and adverse events were also noted. Factors associated with treatment outcomes and relapse were analyzed using the Cox proportional hazards model.
Results:
A total of 101 patients were eligible for analysis. Significant hair regrowth was obtained in 80.2% of the patients (n = 81), in a median time of 3.4 months (95% confidence interval [CI] = 2.9-4.4). Complete hair regrowth was achieved in 48.5% of the subjects (n = 49), and relapse was observed in 47.5% (n = 48). Acneiform eruption was the most common adverse effect. Multivariable analysis revealed that nail involvement was a negative predictor of significant hair regrowth (adjusted hazard ratio [HR] = 0.04, 95% CI = 0.01-0.55; P = 0.015), whereas duration of AA longer than 6 months was associated with disease recurrence (adjusted HR = 4.02, 95% CI = 1.52-4.66; P = 0.005).
Conclusions:
This study demonstrated the efficacy and safety of IMC in the treatment of severe or active AA; however, the relapse rate remained relatively high after discontinuation of the therapy. Nail involvement was a negative predictor of significant hair regrowth, while disease duration longer than 6 months predicted AA relapse.
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[CONTENT] acute diffuse and total alopecia | alopecia totalis | alopecia universalis | hair loss | injection | systemic [SUMMARY]
| null | null |
[CONTENT] acute diffuse and total alopecia | alopecia totalis | alopecia universalis | hair loss | injection | systemic [SUMMARY]
|
[CONTENT] acute diffuse and total alopecia | alopecia totalis | alopecia universalis | hair loss | injection | systemic [SUMMARY]
|
[CONTENT] acute diffuse and total alopecia | alopecia totalis | alopecia universalis | hair loss | injection | systemic [SUMMARY]
|
[CONTENT] Adolescent | Adrenal Cortex Hormones | Adult | Aged | Alopecia Areata | Child | Child, Preschool | Female | Humans | Injections, Intramuscular | Male | Middle Aged | Retrospective Studies [SUMMARY]
| null | null |
[CONTENT] Adolescent | Adrenal Cortex Hormones | Adult | Aged | Alopecia Areata | Child | Child, Preschool | Female | Humans | Injections, Intramuscular | Male | Middle Aged | Retrospective Studies [SUMMARY]
|
[CONTENT] Adolescent | Adrenal Cortex Hormones | Adult | Aged | Alopecia Areata | Child | Child, Preschool | Female | Humans | Injections, Intramuscular | Male | Middle Aged | Retrospective Studies [SUMMARY]
|
[CONTENT] Adolescent | Adrenal Cortex Hormones | Adult | Aged | Alopecia Areata | Child | Child, Preschool | Female | Humans | Injections, Intramuscular | Male | Middle Aged | Retrospective Studies [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null | null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] hair | regrowth | aa | hair regrowth | patients | significant | alopecia | ci | imc | cumulative [SUMMARY]
| null | null |
[CONTENT] hair | regrowth | aa | hair regrowth | patients | significant | alopecia | ci | imc | cumulative [SUMMARY]
|
[CONTENT] hair | regrowth | aa | hair regrowth | patients | significant | alopecia | ci | imc | cumulative [SUMMARY]
|
[CONTENT] hair | regrowth | aa | hair regrowth | patients | significant | alopecia | ci | imc | cumulative [SUMMARY]
|
[CONTENT] aa | corticosteroids | hair | loss | hair loss | topical | treatment | slowly | preferred | small [SUMMARY]
| null | null |
[CONTENT] use imc depends | approximately half experienced | especially patients severe | especially patients | especially | acceptable hair | acceptable hair regrowth | acceptable hair regrowth disease | imc therapy showed | safety intramuscular administration corticosteroids [SUMMARY]
|
[CONTENT] regrowth | aa | hair | hair regrowth | ci | patients | significant | alopecia | imc | cumulative [SUMMARY]
|
[CONTENT] regrowth | aa | hair | hair regrowth | ci | patients | significant | alopecia | imc | cumulative [SUMMARY]
|
[CONTENT] IMC | recent years [SUMMARY]
| null | null |
[CONTENT] IMC ||| 6 months [SUMMARY]
|
[CONTENT] IMC | recent years ||| IMC ||| IMC | 20-40 | 4-6 weeks ||| 25% | 75% | 100% ||| ||| Cox ||| 101 ||| 80.2% | 81 | 3.4 months | 95% ||| CI | 2.9-4.4 ||| 48.5% | 49 | 47.5% | 48 ||| ||| 0.04 | 95% | 0.01 | 0.015 | 6 months | 4.02 | 95% | CI | 1.52 | 0.005 ||| IMC ||| 6 months [SUMMARY]
|
[CONTENT] IMC | recent years ||| IMC ||| IMC | 20-40 | 4-6 weeks ||| 25% | 75% | 100% ||| ||| Cox ||| 101 ||| 80.2% | 81 | 3.4 months | 95% ||| CI | 2.9-4.4 ||| 48.5% | 49 | 47.5% | 48 ||| ||| 0.04 | 95% | 0.01 | 0.015 | 6 months | 4.02 | 95% | CI | 1.52 | 0.005 ||| IMC ||| 6 months [SUMMARY]
|
||||
Assessment of Health-Related Quality of Life, Medication Adherence, and Prevalence of Depression in Kidney Failure Patients.
|
36429988
|
Kidney failure is a global health problem with a worldwide mean prevalence rate of 13.4%. Kidney failure remains symptomless during most of the early stages until symptoms appear in the advanced stages. Kidney failure is associated with a decrease in health-related quality of life (HRQOL), deterioration in physical and mental health, and an increased risk of cardiovascular morbidity and mortality. This study aimed to evaluate the factors associated with decreased HRQOL and other factors affecting the overall health of patients. Another objective was to measure how medication adherence and depression could affect the overall HRQOL in patients with kidney failure.
|
BACKGROUND
|
The study used a prospective follow-up mix methodology approach with six-month follow-ups of patients. The participants included in the study population were those with chronic kidney disease grade 4 and kidney failure. Pre-validated and translated questionnaires (Kidney Disease Quality of Life-Short Form, Hamilton Depression Rating Scale Urdu Version, and Morisky Lewis Greens Adherence Scale) and assessment tools were used to collect data.
|
METHODOLOGY
|
This study recruited 314 patients after an initial assessment based on inclusion criteria. The mean age of the study population was 54.64 ± 15.33 years. There was a 47.6% male and a 52.4% female population. Hypertension and diabetes mellitus remained the most predominant comorbid condition, affecting 64.2% and 74.6% of the population, respectively. The study suggested a significant (p < 0.05) deterioration in the mental health composite score with worsening laboratory variables, particularly hematological and iron studies. Demographic variables significantly impact medication adherence. HRQOL was found to be deteriorating with a significant impact on mental health compared to physical health.
|
RESULTS
|
Patients on maintenance dialysis for kidney failure have a significant burden of physical and mental symptoms, depression, and low HRQOL. Given the substantial and well-known declines in physical and psychological well-being among kidney failure patients receiving hemodialysis, the findings of this research imply that these areas related to health should receive special attention in the growing and expanding population of kidney failure patients.
|
CONCLUSIONS
|
[
"Humans",
"Male",
"Female",
"Adult",
"Middle Aged",
"Aged",
"Quality of Life",
"Prevalence",
"Depression",
"Prospective Studies",
"Medication Adherence",
"Renal Insufficiency, Chronic"
] |
9690334
|
1. Introduction
|
Chronic kidney failure is a public health problem worldwide and can lead to conditions such as kidney failure and cardiovascular problems [1]. In different regions of the world, kidney disease has already reached an epidemic rate in people aged 20 years or above who are the most affected by this disease. The total number of men and women with kidney failure was 225.7 million and 271.8 million, respectively, by 2010 [2]. According to new research, the global prevalence of kidney failure was 9.1% in 2017 (697.5 million cases). Women and girls had a higher global majority of kidney failure than men and boys (9.5%) (7.3%) [3]. About a third of all kidney failure cases were in China (132.3 million) and India (115.1 million), with 10 countries having more than 10 million cases and 79 countries having more than 1 million cases [3,4].
Considering the high incidence of kidney failure and the challenges posed by the patients undergoing dialysis, rehabilitating one’s life is crucial. Patients suffer from different emotional and physical challenges during kidney failure [5]. Survival is not only the goal of placing the patient on a strenuous dialysis procedure but also improving the patient’s health-related quality of life (HRQOL). Understanding the deterioration of HRQOL is the most crucial aspect that can affect burden and mortality in these patients [6,7]. In patients with chronic kidney disease (CKD), including those with end-stage kidney disease (ESKD), monitoring the functional status and subjective state of well-being of a patient as they relate to a health condition, collectively known as HRQOL measurements, is of particular importance.
HRQOL has been significantly affected among patients with advanced chronic kidney disease and kidney failure. The fact that patients with CKD of various racial and ethnic backgrounds have varying HRQOL scores confirms the need to individualise the notion of HRQOL to evaluate critical aspects of life in patients and incorporate these domains into an all-encompassing plan of care. The urgency of HRQOL measurement and the need to advance our multidimensional tools with technology and a more patient-centered view of HRQOL is highlighted by these recent findings [8,9].
In dialysis patient populations, low HRQOL scores measured utilizing the Medical Outcomes Study Short Form-36 (SF-36) were associated with hospitalization and death in studies [10,11,12,13]. There were substantial HRQOL studies conducted before in patients with kidney failure. However, little has been done in Pakistan. Walters et al. assessed HRQOL at the beginning of dialysis therapy and found that in patients who began hemodialysis therapy (HD), HRQOL scores were significantly lower than in established long-term HD patients [14,15,16,17,18,19].
The Dialysis Outcomes and Practice Patterns Study (DOPPS) found a strong link between depressive symptoms and mortality and hospitalization rates [20]. On the contrary, the Choices for Healthy Outcomes in Caring for End-Stage Renal Disease (CHOICE) study found a link between high depressive symptoms and an increased risk of cardiovascular events [21]. However, this link between depression and clinical outcomes has not been thoroughly investigated in the early stages of kidney failure. A recent study by Wirkner et al. investigated an association between comorbid conditions and diabetes. The study concluded that advanced stages of CKD have significantly affected the overall HRQOL of the patients. Patients with kidney failure and undergoing dialysis were also found to have lower physical composite scores [22].
Therefore, it is crucial to assess all such parameters while dealing with a patient’s critical condition with kidney failure. This study was carried out to emphasize the use of tools to measure the HRQOL of patients and treatment outcomes [23,24,25,26]. Patient perceptions and feelings regarding their functionality and well-being are captured through patient-centered outcomes, such as HRQOL. In patients with CKD grade 4, outcome indicators are fundamental because they guide treatment objectives and strategies. However, there is little information on patient-centered outcomes in humans, especially in people who do not receive kidney replacement therapy and have CKD grade 4. More critically, there is a dearth of research in this patient population on the association between HRQOL and drug-related characteristics such as medication burden and adherence [27,28].
Previous studies have found that drug adherence rates range from 33.0 to 87.7%. Seng et al. investigated medication adherence in kidney failure. A pooled medication adherence rate of 67.4% (95% confidence interval (CI) 61.4–73.3%) was found in a meta-analysis of 54,652 patients. Between prospective and retrospective studies, the prevalence of adherence to medication among patients with dialysis kidney failure was similar (68.8%; 95% CI 61.1–76.6%) vs. (65.8%; 95% CI 57.0–74.6%) [28,29,30]. According to the study reported in Pakistan, 74% of patients with kidney failure experienced pruritus, negatively impacting their quality of life. Poor quality of life affects patients with mild to severe CKD-related pruritus. The hemodialysis patients had a lower HRQOL score as their pruritus intensity increased [31].
In Pakistan, kidney failure is the most common cause of morbidity and mortality [32]. According to the 2016 Pakistan National Kidney Federation Registry, 5935 patients were admitted to different dialysis units across the country, totaling 891 hemodialysis machines [33]. The data available have certain limitations as many large dialysis centers are not contributing their data to the registry.
There is a significant upsurge in the prevalence of kidney failure worldwide, particularly in low- to middle-income countries [34]. It is essential to evaluate the factor that could impact the quality of treatment and design a patient support program to increase patients’ involvement in the self-management of the disease condition. This study aimed to evaluate the factors associated with reduced HRQOL and other factors that affect the overall health of patients. Another objective was to measure how medication adherence and depression could impact the overall HRQOL of patients with kidney failure.
| null | null | null | null |
5. Conclusions
|
The patients on maintenance dialysis for kidney failure have significant burdens of physical and mental symptoms, depression, and low HRQOL. Given the substantial and well-known declines in physical and psychological well-being among patients with kidney failure receiving hemodialysis, findings drawn in this research imply that these health-related areas should receive special attention in the vast and expanding population of patients with kidney failure.
|
[
"2. Materials and Methods",
"2.1. Study Design",
"2.2. Inclusion and Exclusion Criteria",
"2.3. Tools",
"2.4. Sociodemographic Data Sheet",
"2.5. Laboratory Investigation Assessment Datasheet",
"2.6. Assessment of Depression, HRQOL, and Medication Adherence",
"2.7. Kidney Disease Quality of Life-Short Form (KDQOL-SF-36)",
"2.8. Hamilton Depression Rating Scale Urdu Version (HAM-D-U)",
"2.9. Morisky Levine Greens Adherence Scales (MLGS)",
"2.10. Study Flow",
"2.11. Ethical Approval and Consent to Participate",
"2.12. Statistical Analysis",
"3. Result",
"3.1. Patient Characteristics",
"3.2. Laboratory Parameters",
"3.3. Depression",
"3.4. Medication Adherence",
"3.5. HRQOL",
"3.6. Medication Adherence and HRQOL",
"3.7. Depression and HRQOL"
] |
[
" 2.1. Study Design This multicenter prospective cohort follow-up study was carried out in Rawalpindi and Islamabad districts. The study was conducted from April 2019 to February 2020. The patients were recruited from the Nephrology Unit of 3 major tertiary care hospitals, i.e., Federal Government Polyclinic Hospital, Islamabad, Pakistan Institute of Medical Sciences, Islamabad, and District Headquarter Hospital Rawalpindi, Pakistan.\nThis multicenter prospective cohort follow-up study was carried out in Rawalpindi and Islamabad districts. The study was conducted from April 2019 to February 2020. The patients were recruited from the Nephrology Unit of 3 major tertiary care hospitals, i.e., Federal Government Polyclinic Hospital, Islamabad, Pakistan Institute of Medical Sciences, Islamabad, and District Headquarter Hospital Rawalpindi, Pakistan.\n 2.2. Inclusion and Exclusion Criteria A total of 400 patients were recruited for the study. After initial screening, 314 participants met the eligibility criteria for the study. All patients aged >18 years with an eGFR value of <30 mL/min/1.73 m2 diagnosed by a physician as a patient having CKD grade 4 and kidney failure grade 5 were enrolled in the study. All patients diagnosed with kidney failure were graded based on their history and investigation conducted in the hospital’s medical unit. According to hospital guidelines, CKD-EPI was used to estimate the eGFR; however, to reduce factors such as weight and body mass index, Cockcroft–Gault formulae were used to determine the eGFR level of the participants. The final diagnosis to determine the grades of kidney failure was made using multiple creatinine assessments. As per the guidelines provided by the study centers, they used Cockcroft–Gault formulae to give better risk stratification in cardiovascular events [35].\nParticipants attended a baseline clinic after being referred by their physicians. All patients were informed about the purpose of the study and written consent was obtained during the patient recruitment phase. We excluded patients diagnosed with malignancy, hospitalized patients, and patients who did not have complete laboratory investigations available at the time of recruitment. We also excluded patients with cognitive disabilities or any other mental illness who could not correctly respond to our questionnaire or other limitations that hindered their participation in this study.\nA total of 400 patients were recruited for the study. After initial screening, 314 participants met the eligibility criteria for the study. All patients aged >18 years with an eGFR value of <30 mL/min/1.73 m2 diagnosed by a physician as a patient having CKD grade 4 and kidney failure grade 5 were enrolled in the study. All patients diagnosed with kidney failure were graded based on their history and investigation conducted in the hospital’s medical unit. According to hospital guidelines, CKD-EPI was used to estimate the eGFR; however, to reduce factors such as weight and body mass index, Cockcroft–Gault formulae were used to determine the eGFR level of the participants. The final diagnosis to determine the grades of kidney failure was made using multiple creatinine assessments. As per the guidelines provided by the study centers, they used Cockcroft–Gault formulae to give better risk stratification in cardiovascular events [35].\nParticipants attended a baseline clinic after being referred by their physicians. All patients were informed about the purpose of the study and written consent was obtained during the patient recruitment phase. We excluded patients diagnosed with malignancy, hospitalized patients, and patients who did not have complete laboratory investigations available at the time of recruitment. We also excluded patients with cognitive disabilities or any other mental illness who could not correctly respond to our questionnaire or other limitations that hindered their participation in this study.\n 2.3. Tools The following tools were used to achieve the study’s goal.\nThe following tools were used to achieve the study’s goal.\n 2.4. Sociodemographic Data Sheet It was designed by the study’s principal investigators to assess the relevant socio-demographic variables, e.g., age, gender, education status, duration of kidney failure treatment, and comorbidity condition.\nIt was designed by the study’s principal investigators to assess the relevant socio-demographic variables, e.g., age, gender, education status, duration of kidney failure treatment, and comorbidity condition.\n 2.5. Laboratory Investigation Assessment Datasheet During the baseline visit, the serum potassium, calcium and phosphorus, hemoglobin, and urea levels was collected from medical records. The same laboratory investigation was also assessed after the completion of the follow-up visit at six months’ duration.\nDuring the baseline visit, the serum potassium, calcium and phosphorus, hemoglobin, and urea levels was collected from medical records. The same laboratory investigation was also assessed after the completion of the follow-up visit at six months’ duration.\n 2.6. Assessment of Depression, HRQOL, and Medication Adherence The study included different Urdu pre-validated and translated questionnaires to achieve the study’s objectives. All these questionnaires have been validated for use among the Pakistani population. Therefore, no further validation test was measured during the study [20,21,22,23].\nThe study included different Urdu pre-validated and translated questionnaires to achieve the study’s objectives. All these questionnaires have been validated for use among the Pakistani population. Therefore, no further validation test was measured during the study [20,21,22,23].\n 2.7. Kidney Disease Quality of Life-Short Form (KDQOL-SF-36) The Urdu version of the KDQOL-SF-36 is reliable and validated for assessing HRQOL in kidney disease patients on dialysis in Pakistan [36]. The KDQOL- SF-36 consists of Physical Health Composite Summary (PCS), Mental Health Composite Summary (MCS), and Kidney Disease Composite Summary (KDCS) domains. These can be measured using the HRQOL scores for physical functioning, physical disabilities, pain, emotional wellness, social functioning, and energy. The KDQOL-SF-36 has five measures, including two essential HRQOL scales (12 items) from the SF-12 version 1 and three kidney-specific scales (24 items total). In the general population, the SF-12 PCS and MCS are assessed using a T-score methodology (mean = 50, SD = 10), with higher scores indicating improved HRQOL.\nThe Urdu version of the KDQOL-SF-36 is reliable and validated for assessing HRQOL in kidney disease patients on dialysis in Pakistan [36]. The KDQOL- SF-36 consists of Physical Health Composite Summary (PCS), Mental Health Composite Summary (MCS), and Kidney Disease Composite Summary (KDCS) domains. These can be measured using the HRQOL scores for physical functioning, physical disabilities, pain, emotional wellness, social functioning, and energy. The KDQOL-SF-36 has five measures, including two essential HRQOL scales (12 items) from the SF-12 version 1 and three kidney-specific scales (24 items total). In the general population, the SF-12 PCS and MCS are assessed using a T-score methodology (mean = 50, SD = 10), with higher scores indicating improved HRQOL.\n 2.8. Hamilton Depression Rating Scale Urdu Version (HAM-D-U) The Hamilton Depression Rating Scale was used in patients who speak Urdu. The HAM-D-U is an effective tool used for determining the magnitude of depression. Internal accuracy (Cronbach alpha 0.71), test–retest reliability, and inter-rater reliability were reasonably good for the Urdu version of the HAM-D (HAM-D-U) [37]. A HAM-D-U score of 0–7 is regarded as normal (or in clinical remission), but a score of 20 or more is deemed severe (showing at least moderate severity).\nHAM-D-U scores are interpreted as follows:No depression at all: <8Mild (subthreshold): 8–13Moderate (mild): 14–18Severe (moderate): 19–22Very severe (severe): >23\nNo depression at all: <8\nMild (subthreshold): 8–13\nModerate (mild): 14–18\nSevere (moderate): 19–22\nVery severe (severe): >23\nThe Hamilton Depression Rating Scale was used in patients who speak Urdu. The HAM-D-U is an effective tool used for determining the magnitude of depression. Internal accuracy (Cronbach alpha 0.71), test–retest reliability, and inter-rater reliability were reasonably good for the Urdu version of the HAM-D (HAM-D-U) [37]. A HAM-D-U score of 0–7 is regarded as normal (or in clinical remission), but a score of 20 or more is deemed severe (showing at least moderate severity).\nHAM-D-U scores are interpreted as follows:No depression at all: <8Mild (subthreshold): 8–13Moderate (mild): 14–18Severe (moderate): 19–22Very severe (severe): >23\nNo depression at all: <8\nMild (subthreshold): 8–13\nModerate (mild): 14–18\nSevere (moderate): 19–22\nVery severe (severe): >23\n 2.9. Morisky Levine Greens Adherence Scales (MLGS) The Morisky Levine Green adherence questionnaire (MLGS) was used to evaluate medication adherence. It is a four-item questionnaire with high reliability and validity that has proven to be particularly useful in chronic conditions such as hypertension, kidney disease, and other cardiovascular diseases. It assesses deliberate and accidental adherence using criteria such as forgetfulness, carelessness, and stopping the drug while feeling better and worse. Each question in the survey was answered with a 0 or 1 response. The total score of the four elements represents the degree of prescription adherence. Medication adherence is indicated by a score of 4, while a score of less than 4 indicates non-adherence. The questionnaire was translated into Urdu using the standard forward–backwards process, yielding a Cronbach alpha of 0.570, within the appropriate range (0.45–0.9) [38,39].\nThe Morisky Levine Green adherence questionnaire (MLGS) was used to evaluate medication adherence. It is a four-item questionnaire with high reliability and validity that has proven to be particularly useful in chronic conditions such as hypertension, kidney disease, and other cardiovascular diseases. It assesses deliberate and accidental adherence using criteria such as forgetfulness, carelessness, and stopping the drug while feeling better and worse. Each question in the survey was answered with a 0 or 1 response. The total score of the four elements represents the degree of prescription adherence. Medication adherence is indicated by a score of 4, while a score of less than 4 indicates non-adherence. The questionnaire was translated into Urdu using the standard forward–backwards process, yielding a Cronbach alpha of 0.570, within the appropriate range (0.45–0.9) [38,39].\n 2.10. Study Flow The tools were administered in the following manner:\nAll questionnaires were administered to patients using a self-reporting approach. In order to validate the responses of the patients, researchers conducted random interviews with the patients during their dialysis procedure. The details schematic flow of patient enrollment, data collection and questionnaire administration are displayed Figure 1 and Figure 2.\nThe tools were administered in the following manner:\nAll questionnaires were administered to patients using a self-reporting approach. In order to validate the responses of the patients, researchers conducted random interviews with the patients during their dialysis procedure. The details schematic flow of patient enrollment, data collection and questionnaire administration are displayed Figure 1 and Figure 2.\n 2.11. Ethical Approval and Consent to Participate The Bio-Ethics Committee (BEC) of Quaid-i-Azam University Islamabad permitted this prospective observational study in Tertiary Care Hospitals (BEC-FBS-QAU2017-13). The study was also approved by the Ethical Review Board of Shaheed Zulfikar Ali Bhutto Medical University (1-1/2015/ERB/SZABMU). Informed written consent was taken from all the participants included in this study. The study was conducted per the Declaration of Helsinki and reported according to “The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” guidelines.\nThe Bio-Ethics Committee (BEC) of Quaid-i-Azam University Islamabad permitted this prospective observational study in Tertiary Care Hospitals (BEC-FBS-QAU2017-13). The study was also approved by the Ethical Review Board of Shaheed Zulfikar Ali Bhutto Medical University (1-1/2015/ERB/SZABMU). Informed written consent was taken from all the participants included in this study. The study was conducted per the Declaration of Helsinki and reported according to “The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” guidelines.\n 2.12. Statistical Analysis Primary data were entered into Microsoft Excel 2010, and anonymous patient coding was used to generate secondary data. The data were analysed using the Statistical Package for Social Sciences (SPSS® IBM version 20, Armonk, NY, USA) program. The KDQOL SFTM1.3 scoring program v.2.0 (Santa Monica, CA, USA) was used to compute and calculate PCS and MCS. Continuous variables were expressed in terms of means and standard deviations, whereas categorical variables were expressed in frequencies and percentages. To summarize sample characteristics, descriptive statistics were used.\nMoreover, normality tests were assessed with the Shapiro–Wilk test. Data had normal distribution; therefore, continuous data’s mean and standard deviation have been presented. Categorical variables were presented as numbers and percentages. The Mann–Whitney U test was used to compare the depression and HRQOL domains. A p-value of <0.05 was considered statistically significant, and a p-value of < 0.01 was considered highly significant. The multivariate analysis was also performed using Bonferroni Correction. Binary logistic regression was used to examine factors related to medication non-adherence and depression, and effect sizes were reported using odds ratios (ORs) and 95% confidence intervals (CIs). The study variable includes dependent variables (KDQOL Domain and Scale, MCS, PCS, medication adherence, and depression) and independent variables (sociodemographic parameters, laboratory parameters).\nPrimary data were entered into Microsoft Excel 2010, and anonymous patient coding was used to generate secondary data. The data were analysed using the Statistical Package for Social Sciences (SPSS® IBM version 20, Armonk, NY, USA) program. The KDQOL SFTM1.3 scoring program v.2.0 (Santa Monica, CA, USA) was used to compute and calculate PCS and MCS. Continuous variables were expressed in terms of means and standard deviations, whereas categorical variables were expressed in frequencies and percentages. To summarize sample characteristics, descriptive statistics were used.\nMoreover, normality tests were assessed with the Shapiro–Wilk test. Data had normal distribution; therefore, continuous data’s mean and standard deviation have been presented. Categorical variables were presented as numbers and percentages. The Mann–Whitney U test was used to compare the depression and HRQOL domains. A p-value of <0.05 was considered statistically significant, and a p-value of < 0.01 was considered highly significant. The multivariate analysis was also performed using Bonferroni Correction. Binary logistic regression was used to examine factors related to medication non-adherence and depression, and effect sizes were reported using odds ratios (ORs) and 95% confidence intervals (CIs). The study variable includes dependent variables (KDQOL Domain and Scale, MCS, PCS, medication adherence, and depression) and independent variables (sociodemographic parameters, laboratory parameters).",
"This multicenter prospective cohort follow-up study was carried out in Rawalpindi and Islamabad districts. The study was conducted from April 2019 to February 2020. The patients were recruited from the Nephrology Unit of 3 major tertiary care hospitals, i.e., Federal Government Polyclinic Hospital, Islamabad, Pakistan Institute of Medical Sciences, Islamabad, and District Headquarter Hospital Rawalpindi, Pakistan.",
"A total of 400 patients were recruited for the study. After initial screening, 314 participants met the eligibility criteria for the study. All patients aged >18 years with an eGFR value of <30 mL/min/1.73 m2 diagnosed by a physician as a patient having CKD grade 4 and kidney failure grade 5 were enrolled in the study. All patients diagnosed with kidney failure were graded based on their history and investigation conducted in the hospital’s medical unit. According to hospital guidelines, CKD-EPI was used to estimate the eGFR; however, to reduce factors such as weight and body mass index, Cockcroft–Gault formulae were used to determine the eGFR level of the participants. The final diagnosis to determine the grades of kidney failure was made using multiple creatinine assessments. As per the guidelines provided by the study centers, they used Cockcroft–Gault formulae to give better risk stratification in cardiovascular events [35].\nParticipants attended a baseline clinic after being referred by their physicians. All patients were informed about the purpose of the study and written consent was obtained during the patient recruitment phase. We excluded patients diagnosed with malignancy, hospitalized patients, and patients who did not have complete laboratory investigations available at the time of recruitment. We also excluded patients with cognitive disabilities or any other mental illness who could not correctly respond to our questionnaire or other limitations that hindered their participation in this study.",
"The following tools were used to achieve the study’s goal.",
"It was designed by the study’s principal investigators to assess the relevant socio-demographic variables, e.g., age, gender, education status, duration of kidney failure treatment, and comorbidity condition.",
"During the baseline visit, the serum potassium, calcium and phosphorus, hemoglobin, and urea levels was collected from medical records. The same laboratory investigation was also assessed after the completion of the follow-up visit at six months’ duration.",
"The study included different Urdu pre-validated and translated questionnaires to achieve the study’s objectives. All these questionnaires have been validated for use among the Pakistani population. Therefore, no further validation test was measured during the study [20,21,22,23].",
"The Urdu version of the KDQOL-SF-36 is reliable and validated for assessing HRQOL in kidney disease patients on dialysis in Pakistan [36]. The KDQOL- SF-36 consists of Physical Health Composite Summary (PCS), Mental Health Composite Summary (MCS), and Kidney Disease Composite Summary (KDCS) domains. These can be measured using the HRQOL scores for physical functioning, physical disabilities, pain, emotional wellness, social functioning, and energy. The KDQOL-SF-36 has five measures, including two essential HRQOL scales (12 items) from the SF-12 version 1 and three kidney-specific scales (24 items total). In the general population, the SF-12 PCS and MCS are assessed using a T-score methodology (mean = 50, SD = 10), with higher scores indicating improved HRQOL.",
"The Hamilton Depression Rating Scale was used in patients who speak Urdu. The HAM-D-U is an effective tool used for determining the magnitude of depression. Internal accuracy (Cronbach alpha 0.71), test–retest reliability, and inter-rater reliability were reasonably good for the Urdu version of the HAM-D (HAM-D-U) [37]. A HAM-D-U score of 0–7 is regarded as normal (or in clinical remission), but a score of 20 or more is deemed severe (showing at least moderate severity).\nHAM-D-U scores are interpreted as follows:No depression at all: <8Mild (subthreshold): 8–13Moderate (mild): 14–18Severe (moderate): 19–22Very severe (severe): >23\nNo depression at all: <8\nMild (subthreshold): 8–13\nModerate (mild): 14–18\nSevere (moderate): 19–22\nVery severe (severe): >23",
"The Morisky Levine Green adherence questionnaire (MLGS) was used to evaluate medication adherence. It is a four-item questionnaire with high reliability and validity that has proven to be particularly useful in chronic conditions such as hypertension, kidney disease, and other cardiovascular diseases. It assesses deliberate and accidental adherence using criteria such as forgetfulness, carelessness, and stopping the drug while feeling better and worse. Each question in the survey was answered with a 0 or 1 response. The total score of the four elements represents the degree of prescription adherence. Medication adherence is indicated by a score of 4, while a score of less than 4 indicates non-adherence. The questionnaire was translated into Urdu using the standard forward–backwards process, yielding a Cronbach alpha of 0.570, within the appropriate range (0.45–0.9) [38,39].",
"The tools were administered in the following manner:\nAll questionnaires were administered to patients using a self-reporting approach. In order to validate the responses of the patients, researchers conducted random interviews with the patients during their dialysis procedure. The details schematic flow of patient enrollment, data collection and questionnaire administration are displayed Figure 1 and Figure 2.",
"The Bio-Ethics Committee (BEC) of Quaid-i-Azam University Islamabad permitted this prospective observational study in Tertiary Care Hospitals (BEC-FBS-QAU2017-13). The study was also approved by the Ethical Review Board of Shaheed Zulfikar Ali Bhutto Medical University (1-1/2015/ERB/SZABMU). Informed written consent was taken from all the participants included in this study. The study was conducted per the Declaration of Helsinki and reported according to “The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” guidelines.",
"Primary data were entered into Microsoft Excel 2010, and anonymous patient coding was used to generate secondary data. The data were analysed using the Statistical Package for Social Sciences (SPSS® IBM version 20, Armonk, NY, USA) program. The KDQOL SFTM1.3 scoring program v.2.0 (Santa Monica, CA, USA) was used to compute and calculate PCS and MCS. Continuous variables were expressed in terms of means and standard deviations, whereas categorical variables were expressed in frequencies and percentages. To summarize sample characteristics, descriptive statistics were used.\nMoreover, normality tests were assessed with the Shapiro–Wilk test. Data had normal distribution; therefore, continuous data’s mean and standard deviation have been presented. Categorical variables were presented as numbers and percentages. The Mann–Whitney U test was used to compare the depression and HRQOL domains. A p-value of <0.05 was considered statistically significant, and a p-value of < 0.01 was considered highly significant. The multivariate analysis was also performed using Bonferroni Correction. Binary logistic regression was used to examine factors related to medication non-adherence and depression, and effect sizes were reported using odds ratios (ORs) and 95% confidence intervals (CIs). The study variable includes dependent variables (KDQOL Domain and Scale, MCS, PCS, medication adherence, and depression) and independent variables (sociodemographic parameters, laboratory parameters).",
" 3.1. Patient Characteristics During the study recruitment period, 400 patients were enrolled at the treatment sites. Eighty-six patients did not meet the eligibility criteria and were excluded. In total, 314 patients participated in the study and completed KDQOL, HAM-D-U, and MLGS. Figure 2 shows the schematic flow of the study. Of the total 314 patients, 39.8% (n = 125) were CKD grade 4, 9.2% (n = 29) with grade 5 not on hemodialysis yet, and 50.9% (n = 160) were at kidney failure grade 5 on hemodialysis.\nThe demographic characteristics of the study participants are shown in Table 1. The mean age of the participants was 54.64 ± 15.33. The majority of the survey participants were women, with a significant proportion of the study population from the age group 41–60 years. The number of people with BMI in the normal range was 53.0%. The study population selected through convenience sampling comprised different levels of disease, treatment approaches, and durations.\nThe comorbidity conditions among the study populations are depicted in Figure 3. Among the population with CKD, diabetes and hypertension were the most prominent underlying comorbid conditions, i.e., 62% and 55%, respectively. Among the study population, 19% had various etiological factors, e.g., smoking, and illicit drug use. Most patients had a family history of kidney disease and a strong history of heart disease. A large group of people also had unknown etiology and represented 30% of the study population.\nDuring the study recruitment period, 400 patients were enrolled at the treatment sites. Eighty-six patients did not meet the eligibility criteria and were excluded. In total, 314 patients participated in the study and completed KDQOL, HAM-D-U, and MLGS. Figure 2 shows the schematic flow of the study. Of the total 314 patients, 39.8% (n = 125) were CKD grade 4, 9.2% (n = 29) with grade 5 not on hemodialysis yet, and 50.9% (n = 160) were at kidney failure grade 5 on hemodialysis.\nThe demographic characteristics of the study participants are shown in Table 1. The mean age of the participants was 54.64 ± 15.33. The majority of the survey participants were women, with a significant proportion of the study population from the age group 41–60 years. The number of people with BMI in the normal range was 53.0%. The study population selected through convenience sampling comprised different levels of disease, treatment approaches, and durations.\nThe comorbidity conditions among the study populations are depicted in Figure 3. Among the population with CKD, diabetes and hypertension were the most prominent underlying comorbid conditions, i.e., 62% and 55%, respectively. Among the study population, 19% had various etiological factors, e.g., smoking, and illicit drug use. Most patients had a family history of kidney disease and a strong history of heart disease. A large group of people also had unknown etiology and represented 30% of the study population.\n 3.2. Laboratory Parameters There were abnormalities in laboratory parameters, and their impact was also analysed in the overall HRQOL of patients. Statistical analysis suggested that hematological and serum iron studies were laboratory parameters that significantly affected the mental composite score (p-Value < 0.05). Contrary to this, the physical composite score was greatly affected by parameters such as hematological serum iron and renal function concentration in the blood, as shown in Table 2.\nThere were abnormalities in laboratory parameters, and their impact was also analysed in the overall HRQOL of patients. Statistical analysis suggested that hematological and serum iron studies were laboratory parameters that significantly affected the mental composite score (p-Value < 0.05). Contrary to this, the physical composite score was greatly affected by parameters such as hematological serum iron and renal function concentration in the blood, as shown in Table 2.\n 3.3. Depression In the study, the 21-item Hamilton Depression Rating Scale (HAM-D-U) was used to determine the study population’s pre-study and post-study analysis of depression levels.\nIt was observed that throughout treatment, there was no educational or counselling support provided to any of the patients. Analysis of the depression data was given by 314 patients in the pre-assessment questionnaire and 244 in the post-assessment questionnaire. A total of 45 patients refused to fill out the post-study HAM-D-U.\nThe frequency distribution of the depression assessment at baseline found that there was a significantly high depression level in both kidney failure populations (Figure 4). There were only a few cases with a normal-to-moderate level of depression.\nUpon the follow-up visit, it was observed that there was a significant increase in depression levels among the study population. Respondents indicated a higher level of depression in both the study populations compared to their previous state. However, further analysis with the Pearson Chi-square test found no significant difference between the baseline and post-study depression levels (p-value > 0.05).\nIn the study, the 21-item Hamilton Depression Rating Scale (HAM-D-U) was used to determine the study population’s pre-study and post-study analysis of depression levels.\nIt was observed that throughout treatment, there was no educational or counselling support provided to any of the patients. Analysis of the depression data was given by 314 patients in the pre-assessment questionnaire and 244 in the post-assessment questionnaire. A total of 45 patients refused to fill out the post-study HAM-D-U.\nThe frequency distribution of the depression assessment at baseline found that there was a significantly high depression level in both kidney failure populations (Figure 4). There were only a few cases with a normal-to-moderate level of depression.\nUpon the follow-up visit, it was observed that there was a significant increase in depression levels among the study population. Respondents indicated a higher level of depression in both the study populations compared to their previous state. However, further analysis with the Pearson Chi-square test found no significant difference between the baseline and post-study depression levels (p-value > 0.05).\n 3.4. Medication Adherence Around 38.8% (n = 122) of the 314 participants scored 4 and had high adherence, while the majority (61.2%, n = 192) belonged to a non-adherent population with a score of less than 4. In addition, the findings revealed no statistically significant relationship between adherence and gender, age, marital status, education level, smoking status or the number of medications (p > 0.05).\nAround 38.8% (n = 122) of the 314 participants scored 4 and had high adherence, while the majority (61.2%, n = 192) belonged to a non-adherent population with a score of less than 4. In addition, the findings revealed no statistically significant relationship between adherence and gender, age, marital status, education level, smoking status or the number of medications (p > 0.05).\n 3.5. HRQOL A total of 314 patients completed the KDQOL-SF-36 questionnaire during a baseline visit. As shown in Table 3, even though patients with grade 4 had a lower level of the physical score as compared to those having grade 5, there were no significant differences seen in the SF-36 PCS scale (31.28 ± 7.91 vs. 36.28 ± 8.41, p = 0.68). However, the overall mental health composite measured by the MCS at baseline was significantly lower for the grade 4 population than the grade 5 population, i.e., 36.66 ± 6.57 and 48.66 ± 5.44, respectively (p-value < 0.05).\nFor the KDQOL-SF-36 domain, the Mental Health Composite was the factor that affected the study population the most, which showed a statistically significant level in both the study groups (p-value < 0.05). The other major KDQOL-SF-36 domains that impacted both populations were Physical Functioning, Role Limitations-Physical, and Emotional well-being, as well as energy and fatigue evident from Table 3.\nIn the follow-up visit, 244 patients agreed to respond to the questionnaires. In the post-study, 35 patients with grade 4 and 10 patients with grade 5 did not respond. In the post-analysis, a marked deterioration was seen in KDQOL-SF-36 domains and scale parameters among patients with advanced kidney failure. Grade 4 patients scored significantly lower than grade 5 in the Problem’s list/Symptom, Burden of Kidney Disease, and Mental Health composite domains (p < 0.05). In the KDQOL-SF-36 scales, there was significant deterioration in Physical function, Emotional well-being, and Energy level in patients with grade 5 (p-value < 0.05).\nA total of 314 patients completed the KDQOL-SF-36 questionnaire during a baseline visit. As shown in Table 3, even though patients with grade 4 had a lower level of the physical score as compared to those having grade 5, there were no significant differences seen in the SF-36 PCS scale (31.28 ± 7.91 vs. 36.28 ± 8.41, p = 0.68). However, the overall mental health composite measured by the MCS at baseline was significantly lower for the grade 4 population than the grade 5 population, i.e., 36.66 ± 6.57 and 48.66 ± 5.44, respectively (p-value < 0.05).\nFor the KDQOL-SF-36 domain, the Mental Health Composite was the factor that affected the study population the most, which showed a statistically significant level in both the study groups (p-value < 0.05). The other major KDQOL-SF-36 domains that impacted both populations were Physical Functioning, Role Limitations-Physical, and Emotional well-being, as well as energy and fatigue evident from Table 3.\nIn the follow-up visit, 244 patients agreed to respond to the questionnaires. In the post-study, 35 patients with grade 4 and 10 patients with grade 5 did not respond. In the post-analysis, a marked deterioration was seen in KDQOL-SF-36 domains and scale parameters among patients with advanced kidney failure. Grade 4 patients scored significantly lower than grade 5 in the Problem’s list/Symptom, Burden of Kidney Disease, and Mental Health composite domains (p < 0.05). In the KDQOL-SF-36 scales, there was significant deterioration in Physical function, Emotional well-being, and Energy level in patients with grade 5 (p-value < 0.05).\n 3.6. Medication Adherence and HRQOL The PCS of the HRQOL improved over time in adherent individuals and declined in non-adherent individuals. Additionally, after controlling for age, gender, and baseline eGFR, medication non-adherence demonstrated a significant negative correlation with a change in the PCS of the HRQOL, as evident from Table 4. After adjusting for the same covariates, there was no significant correlation between medication non-adherence and a change in the MCS of the HRQOL.\nThe PCS of the HRQOL improved over time in adherent individuals and declined in non-adherent individuals. Additionally, after controlling for age, gender, and baseline eGFR, medication non-adherence demonstrated a significant negative correlation with a change in the PCS of the HRQOL, as evident from Table 4. After adjusting for the same covariates, there was no significant correlation between medication non-adherence and a change in the MCS of the HRQOL.\n 3.7. Depression and HRQOL Depressive symptoms were inversely correlated with PCS and MCS scores in the unadjusted longitudinal analysis. Depressive symptoms were independently reduced following multivariable adjustment and negatively correlated with the PCS and MCS scores, as shown in Table 5. When depressive symptoms were considered a dichotomous variable in unadjusted analyses, moderate-to-severe depression symptoms presented negative correlations between the PCS and MCS. In multivariable analyses, the PCS and MCS scores were independently and negatively related to the prevalence of moderate-to-severe depressive symptoms.\nDepressive symptoms were inversely correlated with PCS and MCS scores in the unadjusted longitudinal analysis. Depressive symptoms were independently reduced following multivariable adjustment and negatively correlated with the PCS and MCS scores, as shown in Table 5. When depressive symptoms were considered a dichotomous variable in unadjusted analyses, moderate-to-severe depression symptoms presented negative correlations between the PCS and MCS. In multivariable analyses, the PCS and MCS scores were independently and negatively related to the prevalence of moderate-to-severe depressive symptoms.",
"During the study recruitment period, 400 patients were enrolled at the treatment sites. Eighty-six patients did not meet the eligibility criteria and were excluded. In total, 314 patients participated in the study and completed KDQOL, HAM-D-U, and MLGS. Figure 2 shows the schematic flow of the study. Of the total 314 patients, 39.8% (n = 125) were CKD grade 4, 9.2% (n = 29) with grade 5 not on hemodialysis yet, and 50.9% (n = 160) were at kidney failure grade 5 on hemodialysis.\nThe demographic characteristics of the study participants are shown in Table 1. The mean age of the participants was 54.64 ± 15.33. The majority of the survey participants were women, with a significant proportion of the study population from the age group 41–60 years. The number of people with BMI in the normal range was 53.0%. The study population selected through convenience sampling comprised different levels of disease, treatment approaches, and durations.\nThe comorbidity conditions among the study populations are depicted in Figure 3. Among the population with CKD, diabetes and hypertension were the most prominent underlying comorbid conditions, i.e., 62% and 55%, respectively. Among the study population, 19% had various etiological factors, e.g., smoking, and illicit drug use. Most patients had a family history of kidney disease and a strong history of heart disease. A large group of people also had unknown etiology and represented 30% of the study population.",
"There were abnormalities in laboratory parameters, and their impact was also analysed in the overall HRQOL of patients. Statistical analysis suggested that hematological and serum iron studies were laboratory parameters that significantly affected the mental composite score (p-Value < 0.05). Contrary to this, the physical composite score was greatly affected by parameters such as hematological serum iron and renal function concentration in the blood, as shown in Table 2.",
"In the study, the 21-item Hamilton Depression Rating Scale (HAM-D-U) was used to determine the study population’s pre-study and post-study analysis of depression levels.\nIt was observed that throughout treatment, there was no educational or counselling support provided to any of the patients. Analysis of the depression data was given by 314 patients in the pre-assessment questionnaire and 244 in the post-assessment questionnaire. A total of 45 patients refused to fill out the post-study HAM-D-U.\nThe frequency distribution of the depression assessment at baseline found that there was a significantly high depression level in both kidney failure populations (Figure 4). There were only a few cases with a normal-to-moderate level of depression.\nUpon the follow-up visit, it was observed that there was a significant increase in depression levels among the study population. Respondents indicated a higher level of depression in both the study populations compared to their previous state. However, further analysis with the Pearson Chi-square test found no significant difference between the baseline and post-study depression levels (p-value > 0.05).",
"Around 38.8% (n = 122) of the 314 participants scored 4 and had high adherence, while the majority (61.2%, n = 192) belonged to a non-adherent population with a score of less than 4. In addition, the findings revealed no statistically significant relationship between adherence and gender, age, marital status, education level, smoking status or the number of medications (p > 0.05).",
"A total of 314 patients completed the KDQOL-SF-36 questionnaire during a baseline visit. As shown in Table 3, even though patients with grade 4 had a lower level of the physical score as compared to those having grade 5, there were no significant differences seen in the SF-36 PCS scale (31.28 ± 7.91 vs. 36.28 ± 8.41, p = 0.68). However, the overall mental health composite measured by the MCS at baseline was significantly lower for the grade 4 population than the grade 5 population, i.e., 36.66 ± 6.57 and 48.66 ± 5.44, respectively (p-value < 0.05).\nFor the KDQOL-SF-36 domain, the Mental Health Composite was the factor that affected the study population the most, which showed a statistically significant level in both the study groups (p-value < 0.05). The other major KDQOL-SF-36 domains that impacted both populations were Physical Functioning, Role Limitations-Physical, and Emotional well-being, as well as energy and fatigue evident from Table 3.\nIn the follow-up visit, 244 patients agreed to respond to the questionnaires. In the post-study, 35 patients with grade 4 and 10 patients with grade 5 did not respond. In the post-analysis, a marked deterioration was seen in KDQOL-SF-36 domains and scale parameters among patients with advanced kidney failure. Grade 4 patients scored significantly lower than grade 5 in the Problem’s list/Symptom, Burden of Kidney Disease, and Mental Health composite domains (p < 0.05). In the KDQOL-SF-36 scales, there was significant deterioration in Physical function, Emotional well-being, and Energy level in patients with grade 5 (p-value < 0.05).",
"The PCS of the HRQOL improved over time in adherent individuals and declined in non-adherent individuals. Additionally, after controlling for age, gender, and baseline eGFR, medication non-adherence demonstrated a significant negative correlation with a change in the PCS of the HRQOL, as evident from Table 4. After adjusting for the same covariates, there was no significant correlation between medication non-adherence and a change in the MCS of the HRQOL.",
"Depressive symptoms were inversely correlated with PCS and MCS scores in the unadjusted longitudinal analysis. Depressive symptoms were independently reduced following multivariable adjustment and negatively correlated with the PCS and MCS scores, as shown in Table 5. When depressive symptoms were considered a dichotomous variable in unadjusted analyses, moderate-to-severe depression symptoms presented negative correlations between the PCS and MCS. In multivariable analyses, the PCS and MCS scores were independently and negatively related to the prevalence of moderate-to-severe depressive symptoms."
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[
"1. Introduction",
"2. Materials and Methods",
"2.1. Study Design",
"2.2. Inclusion and Exclusion Criteria",
"2.3. Tools",
"2.4. Sociodemographic Data Sheet",
"2.5. Laboratory Investigation Assessment Datasheet",
"2.6. Assessment of Depression, HRQOL, and Medication Adherence",
"2.7. Kidney Disease Quality of Life-Short Form (KDQOL-SF-36)",
"2.8. Hamilton Depression Rating Scale Urdu Version (HAM-D-U)",
"2.9. Morisky Levine Greens Adherence Scales (MLGS)",
"2.10. Study Flow",
"2.11. Ethical Approval and Consent to Participate",
"2.12. Statistical Analysis",
"3. Result",
"3.1. Patient Characteristics",
"3.2. Laboratory Parameters",
"3.3. Depression",
"3.4. Medication Adherence",
"3.5. HRQOL",
"3.6. Medication Adherence and HRQOL",
"3.7. Depression and HRQOL",
"4. Discussion",
"5. Conclusions"
] |
[
"Chronic kidney failure is a public health problem worldwide and can lead to conditions such as kidney failure and cardiovascular problems [1]. In different regions of the world, kidney disease has already reached an epidemic rate in people aged 20 years or above who are the most affected by this disease. The total number of men and women with kidney failure was 225.7 million and 271.8 million, respectively, by 2010 [2]. According to new research, the global prevalence of kidney failure was 9.1% in 2017 (697.5 million cases). Women and girls had a higher global majority of kidney failure than men and boys (9.5%) (7.3%) [3]. About a third of all kidney failure cases were in China (132.3 million) and India (115.1 million), with 10 countries having more than 10 million cases and 79 countries having more than 1 million cases [3,4].\nConsidering the high incidence of kidney failure and the challenges posed by the patients undergoing dialysis, rehabilitating one’s life is crucial. Patients suffer from different emotional and physical challenges during kidney failure [5]. Survival is not only the goal of placing the patient on a strenuous dialysis procedure but also improving the patient’s health-related quality of life (HRQOL). Understanding the deterioration of HRQOL is the most crucial aspect that can affect burden and mortality in these patients [6,7]. In patients with chronic kidney disease (CKD), including those with end-stage kidney disease (ESKD), monitoring the functional status and subjective state of well-being of a patient as they relate to a health condition, collectively known as HRQOL measurements, is of particular importance.\nHRQOL has been significantly affected among patients with advanced chronic kidney disease and kidney failure. The fact that patients with CKD of various racial and ethnic backgrounds have varying HRQOL scores confirms the need to individualise the notion of HRQOL to evaluate critical aspects of life in patients and incorporate these domains into an all-encompassing plan of care. The urgency of HRQOL measurement and the need to advance our multidimensional tools with technology and a more patient-centered view of HRQOL is highlighted by these recent findings [8,9].\nIn dialysis patient populations, low HRQOL scores measured utilizing the Medical Outcomes Study Short Form-36 (SF-36) were associated with hospitalization and death in studies [10,11,12,13]. There were substantial HRQOL studies conducted before in patients with kidney failure. However, little has been done in Pakistan. Walters et al. assessed HRQOL at the beginning of dialysis therapy and found that in patients who began hemodialysis therapy (HD), HRQOL scores were significantly lower than in established long-term HD patients [14,15,16,17,18,19].\nThe Dialysis Outcomes and Practice Patterns Study (DOPPS) found a strong link between depressive symptoms and mortality and hospitalization rates [20]. On the contrary, the Choices for Healthy Outcomes in Caring for End-Stage Renal Disease (CHOICE) study found a link between high depressive symptoms and an increased risk of cardiovascular events [21]. However, this link between depression and clinical outcomes has not been thoroughly investigated in the early stages of kidney failure. A recent study by Wirkner et al. investigated an association between comorbid conditions and diabetes. The study concluded that advanced stages of CKD have significantly affected the overall HRQOL of the patients. Patients with kidney failure and undergoing dialysis were also found to have lower physical composite scores [22].\nTherefore, it is crucial to assess all such parameters while dealing with a patient’s critical condition with kidney failure. This study was carried out to emphasize the use of tools to measure the HRQOL of patients and treatment outcomes [23,24,25,26]. Patient perceptions and feelings regarding their functionality and well-being are captured through patient-centered outcomes, such as HRQOL. In patients with CKD grade 4, outcome indicators are fundamental because they guide treatment objectives and strategies. However, there is little information on patient-centered outcomes in humans, especially in people who do not receive kidney replacement therapy and have CKD grade 4. More critically, there is a dearth of research in this patient population on the association between HRQOL and drug-related characteristics such as medication burden and adherence [27,28].\nPrevious studies have found that drug adherence rates range from 33.0 to 87.7%. Seng et al. investigated medication adherence in kidney failure. A pooled medication adherence rate of 67.4% (95% confidence interval (CI) 61.4–73.3%) was found in a meta-analysis of 54,652 patients. Between prospective and retrospective studies, the prevalence of adherence to medication among patients with dialysis kidney failure was similar (68.8%; 95% CI 61.1–76.6%) vs. (65.8%; 95% CI 57.0–74.6%) [28,29,30]. According to the study reported in Pakistan, 74% of patients with kidney failure experienced pruritus, negatively impacting their quality of life. Poor quality of life affects patients with mild to severe CKD-related pruritus. The hemodialysis patients had a lower HRQOL score as their pruritus intensity increased [31].\nIn Pakistan, kidney failure is the most common cause of morbidity and mortality [32]. According to the 2016 Pakistan National Kidney Federation Registry, 5935 patients were admitted to different dialysis units across the country, totaling 891 hemodialysis machines [33]. The data available have certain limitations as many large dialysis centers are not contributing their data to the registry.\nThere is a significant upsurge in the prevalence of kidney failure worldwide, particularly in low- to middle-income countries [34]. It is essential to evaluate the factor that could impact the quality of treatment and design a patient support program to increase patients’ involvement in the self-management of the disease condition. This study aimed to evaluate the factors associated with reduced HRQOL and other factors that affect the overall health of patients. Another objective was to measure how medication adherence and depression could impact the overall HRQOL of patients with kidney failure.",
" 2.1. Study Design This multicenter prospective cohort follow-up study was carried out in Rawalpindi and Islamabad districts. The study was conducted from April 2019 to February 2020. The patients were recruited from the Nephrology Unit of 3 major tertiary care hospitals, i.e., Federal Government Polyclinic Hospital, Islamabad, Pakistan Institute of Medical Sciences, Islamabad, and District Headquarter Hospital Rawalpindi, Pakistan.\nThis multicenter prospective cohort follow-up study was carried out in Rawalpindi and Islamabad districts. The study was conducted from April 2019 to February 2020. The patients were recruited from the Nephrology Unit of 3 major tertiary care hospitals, i.e., Federal Government Polyclinic Hospital, Islamabad, Pakistan Institute of Medical Sciences, Islamabad, and District Headquarter Hospital Rawalpindi, Pakistan.\n 2.2. Inclusion and Exclusion Criteria A total of 400 patients were recruited for the study. After initial screening, 314 participants met the eligibility criteria for the study. All patients aged >18 years with an eGFR value of <30 mL/min/1.73 m2 diagnosed by a physician as a patient having CKD grade 4 and kidney failure grade 5 were enrolled in the study. All patients diagnosed with kidney failure were graded based on their history and investigation conducted in the hospital’s medical unit. According to hospital guidelines, CKD-EPI was used to estimate the eGFR; however, to reduce factors such as weight and body mass index, Cockcroft–Gault formulae were used to determine the eGFR level of the participants. The final diagnosis to determine the grades of kidney failure was made using multiple creatinine assessments. As per the guidelines provided by the study centers, they used Cockcroft–Gault formulae to give better risk stratification in cardiovascular events [35].\nParticipants attended a baseline clinic after being referred by their physicians. All patients were informed about the purpose of the study and written consent was obtained during the patient recruitment phase. We excluded patients diagnosed with malignancy, hospitalized patients, and patients who did not have complete laboratory investigations available at the time of recruitment. We also excluded patients with cognitive disabilities or any other mental illness who could not correctly respond to our questionnaire or other limitations that hindered their participation in this study.\nA total of 400 patients were recruited for the study. After initial screening, 314 participants met the eligibility criteria for the study. All patients aged >18 years with an eGFR value of <30 mL/min/1.73 m2 diagnosed by a physician as a patient having CKD grade 4 and kidney failure grade 5 were enrolled in the study. All patients diagnosed with kidney failure were graded based on their history and investigation conducted in the hospital’s medical unit. According to hospital guidelines, CKD-EPI was used to estimate the eGFR; however, to reduce factors such as weight and body mass index, Cockcroft–Gault formulae were used to determine the eGFR level of the participants. The final diagnosis to determine the grades of kidney failure was made using multiple creatinine assessments. As per the guidelines provided by the study centers, they used Cockcroft–Gault formulae to give better risk stratification in cardiovascular events [35].\nParticipants attended a baseline clinic after being referred by their physicians. All patients were informed about the purpose of the study and written consent was obtained during the patient recruitment phase. We excluded patients diagnosed with malignancy, hospitalized patients, and patients who did not have complete laboratory investigations available at the time of recruitment. We also excluded patients with cognitive disabilities or any other mental illness who could not correctly respond to our questionnaire or other limitations that hindered their participation in this study.\n 2.3. Tools The following tools were used to achieve the study’s goal.\nThe following tools were used to achieve the study’s goal.\n 2.4. Sociodemographic Data Sheet It was designed by the study’s principal investigators to assess the relevant socio-demographic variables, e.g., age, gender, education status, duration of kidney failure treatment, and comorbidity condition.\nIt was designed by the study’s principal investigators to assess the relevant socio-demographic variables, e.g., age, gender, education status, duration of kidney failure treatment, and comorbidity condition.\n 2.5. Laboratory Investigation Assessment Datasheet During the baseline visit, the serum potassium, calcium and phosphorus, hemoglobin, and urea levels was collected from medical records. The same laboratory investigation was also assessed after the completion of the follow-up visit at six months’ duration.\nDuring the baseline visit, the serum potassium, calcium and phosphorus, hemoglobin, and urea levels was collected from medical records. The same laboratory investigation was also assessed after the completion of the follow-up visit at six months’ duration.\n 2.6. Assessment of Depression, HRQOL, and Medication Adherence The study included different Urdu pre-validated and translated questionnaires to achieve the study’s objectives. All these questionnaires have been validated for use among the Pakistani population. Therefore, no further validation test was measured during the study [20,21,22,23].\nThe study included different Urdu pre-validated and translated questionnaires to achieve the study’s objectives. All these questionnaires have been validated for use among the Pakistani population. Therefore, no further validation test was measured during the study [20,21,22,23].\n 2.7. Kidney Disease Quality of Life-Short Form (KDQOL-SF-36) The Urdu version of the KDQOL-SF-36 is reliable and validated for assessing HRQOL in kidney disease patients on dialysis in Pakistan [36]. The KDQOL- SF-36 consists of Physical Health Composite Summary (PCS), Mental Health Composite Summary (MCS), and Kidney Disease Composite Summary (KDCS) domains. These can be measured using the HRQOL scores for physical functioning, physical disabilities, pain, emotional wellness, social functioning, and energy. The KDQOL-SF-36 has five measures, including two essential HRQOL scales (12 items) from the SF-12 version 1 and three kidney-specific scales (24 items total). In the general population, the SF-12 PCS and MCS are assessed using a T-score methodology (mean = 50, SD = 10), with higher scores indicating improved HRQOL.\nThe Urdu version of the KDQOL-SF-36 is reliable and validated for assessing HRQOL in kidney disease patients on dialysis in Pakistan [36]. The KDQOL- SF-36 consists of Physical Health Composite Summary (PCS), Mental Health Composite Summary (MCS), and Kidney Disease Composite Summary (KDCS) domains. These can be measured using the HRQOL scores for physical functioning, physical disabilities, pain, emotional wellness, social functioning, and energy. The KDQOL-SF-36 has five measures, including two essential HRQOL scales (12 items) from the SF-12 version 1 and three kidney-specific scales (24 items total). In the general population, the SF-12 PCS and MCS are assessed using a T-score methodology (mean = 50, SD = 10), with higher scores indicating improved HRQOL.\n 2.8. Hamilton Depression Rating Scale Urdu Version (HAM-D-U) The Hamilton Depression Rating Scale was used in patients who speak Urdu. The HAM-D-U is an effective tool used for determining the magnitude of depression. Internal accuracy (Cronbach alpha 0.71), test–retest reliability, and inter-rater reliability were reasonably good for the Urdu version of the HAM-D (HAM-D-U) [37]. A HAM-D-U score of 0–7 is regarded as normal (or in clinical remission), but a score of 20 or more is deemed severe (showing at least moderate severity).\nHAM-D-U scores are interpreted as follows:No depression at all: <8Mild (subthreshold): 8–13Moderate (mild): 14–18Severe (moderate): 19–22Very severe (severe): >23\nNo depression at all: <8\nMild (subthreshold): 8–13\nModerate (mild): 14–18\nSevere (moderate): 19–22\nVery severe (severe): >23\nThe Hamilton Depression Rating Scale was used in patients who speak Urdu. The HAM-D-U is an effective tool used for determining the magnitude of depression. Internal accuracy (Cronbach alpha 0.71), test–retest reliability, and inter-rater reliability were reasonably good for the Urdu version of the HAM-D (HAM-D-U) [37]. A HAM-D-U score of 0–7 is regarded as normal (or in clinical remission), but a score of 20 or more is deemed severe (showing at least moderate severity).\nHAM-D-U scores are interpreted as follows:No depression at all: <8Mild (subthreshold): 8–13Moderate (mild): 14–18Severe (moderate): 19–22Very severe (severe): >23\nNo depression at all: <8\nMild (subthreshold): 8–13\nModerate (mild): 14–18\nSevere (moderate): 19–22\nVery severe (severe): >23\n 2.9. Morisky Levine Greens Adherence Scales (MLGS) The Morisky Levine Green adherence questionnaire (MLGS) was used to evaluate medication adherence. It is a four-item questionnaire with high reliability and validity that has proven to be particularly useful in chronic conditions such as hypertension, kidney disease, and other cardiovascular diseases. It assesses deliberate and accidental adherence using criteria such as forgetfulness, carelessness, and stopping the drug while feeling better and worse. Each question in the survey was answered with a 0 or 1 response. The total score of the four elements represents the degree of prescription adherence. Medication adherence is indicated by a score of 4, while a score of less than 4 indicates non-adherence. The questionnaire was translated into Urdu using the standard forward–backwards process, yielding a Cronbach alpha of 0.570, within the appropriate range (0.45–0.9) [38,39].\nThe Morisky Levine Green adherence questionnaire (MLGS) was used to evaluate medication adherence. It is a four-item questionnaire with high reliability and validity that has proven to be particularly useful in chronic conditions such as hypertension, kidney disease, and other cardiovascular diseases. It assesses deliberate and accidental adherence using criteria such as forgetfulness, carelessness, and stopping the drug while feeling better and worse. Each question in the survey was answered with a 0 or 1 response. The total score of the four elements represents the degree of prescription adherence. Medication adherence is indicated by a score of 4, while a score of less than 4 indicates non-adherence. The questionnaire was translated into Urdu using the standard forward–backwards process, yielding a Cronbach alpha of 0.570, within the appropriate range (0.45–0.9) [38,39].\n 2.10. Study Flow The tools were administered in the following manner:\nAll questionnaires were administered to patients using a self-reporting approach. In order to validate the responses of the patients, researchers conducted random interviews with the patients during their dialysis procedure. The details schematic flow of patient enrollment, data collection and questionnaire administration are displayed Figure 1 and Figure 2.\nThe tools were administered in the following manner:\nAll questionnaires were administered to patients using a self-reporting approach. In order to validate the responses of the patients, researchers conducted random interviews with the patients during their dialysis procedure. The details schematic flow of patient enrollment, data collection and questionnaire administration are displayed Figure 1 and Figure 2.\n 2.11. Ethical Approval and Consent to Participate The Bio-Ethics Committee (BEC) of Quaid-i-Azam University Islamabad permitted this prospective observational study in Tertiary Care Hospitals (BEC-FBS-QAU2017-13). The study was also approved by the Ethical Review Board of Shaheed Zulfikar Ali Bhutto Medical University (1-1/2015/ERB/SZABMU). Informed written consent was taken from all the participants included in this study. The study was conducted per the Declaration of Helsinki and reported according to “The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” guidelines.\nThe Bio-Ethics Committee (BEC) of Quaid-i-Azam University Islamabad permitted this prospective observational study in Tertiary Care Hospitals (BEC-FBS-QAU2017-13). The study was also approved by the Ethical Review Board of Shaheed Zulfikar Ali Bhutto Medical University (1-1/2015/ERB/SZABMU). Informed written consent was taken from all the participants included in this study. The study was conducted per the Declaration of Helsinki and reported according to “The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” guidelines.\n 2.12. Statistical Analysis Primary data were entered into Microsoft Excel 2010, and anonymous patient coding was used to generate secondary data. The data were analysed using the Statistical Package for Social Sciences (SPSS® IBM version 20, Armonk, NY, USA) program. The KDQOL SFTM1.3 scoring program v.2.0 (Santa Monica, CA, USA) was used to compute and calculate PCS and MCS. Continuous variables were expressed in terms of means and standard deviations, whereas categorical variables were expressed in frequencies and percentages. To summarize sample characteristics, descriptive statistics were used.\nMoreover, normality tests were assessed with the Shapiro–Wilk test. Data had normal distribution; therefore, continuous data’s mean and standard deviation have been presented. Categorical variables were presented as numbers and percentages. The Mann–Whitney U test was used to compare the depression and HRQOL domains. A p-value of <0.05 was considered statistically significant, and a p-value of < 0.01 was considered highly significant. The multivariate analysis was also performed using Bonferroni Correction. Binary logistic regression was used to examine factors related to medication non-adherence and depression, and effect sizes were reported using odds ratios (ORs) and 95% confidence intervals (CIs). The study variable includes dependent variables (KDQOL Domain and Scale, MCS, PCS, medication adherence, and depression) and independent variables (sociodemographic parameters, laboratory parameters).\nPrimary data were entered into Microsoft Excel 2010, and anonymous patient coding was used to generate secondary data. The data were analysed using the Statistical Package for Social Sciences (SPSS® IBM version 20, Armonk, NY, USA) program. The KDQOL SFTM1.3 scoring program v.2.0 (Santa Monica, CA, USA) was used to compute and calculate PCS and MCS. Continuous variables were expressed in terms of means and standard deviations, whereas categorical variables were expressed in frequencies and percentages. To summarize sample characteristics, descriptive statistics were used.\nMoreover, normality tests were assessed with the Shapiro–Wilk test. Data had normal distribution; therefore, continuous data’s mean and standard deviation have been presented. Categorical variables were presented as numbers and percentages. The Mann–Whitney U test was used to compare the depression and HRQOL domains. A p-value of <0.05 was considered statistically significant, and a p-value of < 0.01 was considered highly significant. The multivariate analysis was also performed using Bonferroni Correction. Binary logistic regression was used to examine factors related to medication non-adherence and depression, and effect sizes were reported using odds ratios (ORs) and 95% confidence intervals (CIs). The study variable includes dependent variables (KDQOL Domain and Scale, MCS, PCS, medication adherence, and depression) and independent variables (sociodemographic parameters, laboratory parameters).",
"This multicenter prospective cohort follow-up study was carried out in Rawalpindi and Islamabad districts. The study was conducted from April 2019 to February 2020. The patients were recruited from the Nephrology Unit of 3 major tertiary care hospitals, i.e., Federal Government Polyclinic Hospital, Islamabad, Pakistan Institute of Medical Sciences, Islamabad, and District Headquarter Hospital Rawalpindi, Pakistan.",
"A total of 400 patients were recruited for the study. After initial screening, 314 participants met the eligibility criteria for the study. All patients aged >18 years with an eGFR value of <30 mL/min/1.73 m2 diagnosed by a physician as a patient having CKD grade 4 and kidney failure grade 5 were enrolled in the study. All patients diagnosed with kidney failure were graded based on their history and investigation conducted in the hospital’s medical unit. According to hospital guidelines, CKD-EPI was used to estimate the eGFR; however, to reduce factors such as weight and body mass index, Cockcroft–Gault formulae were used to determine the eGFR level of the participants. The final diagnosis to determine the grades of kidney failure was made using multiple creatinine assessments. As per the guidelines provided by the study centers, they used Cockcroft–Gault formulae to give better risk stratification in cardiovascular events [35].\nParticipants attended a baseline clinic after being referred by their physicians. All patients were informed about the purpose of the study and written consent was obtained during the patient recruitment phase. We excluded patients diagnosed with malignancy, hospitalized patients, and patients who did not have complete laboratory investigations available at the time of recruitment. We also excluded patients with cognitive disabilities or any other mental illness who could not correctly respond to our questionnaire or other limitations that hindered their participation in this study.",
"The following tools were used to achieve the study’s goal.",
"It was designed by the study’s principal investigators to assess the relevant socio-demographic variables, e.g., age, gender, education status, duration of kidney failure treatment, and comorbidity condition.",
"During the baseline visit, the serum potassium, calcium and phosphorus, hemoglobin, and urea levels was collected from medical records. The same laboratory investigation was also assessed after the completion of the follow-up visit at six months’ duration.",
"The study included different Urdu pre-validated and translated questionnaires to achieve the study’s objectives. All these questionnaires have been validated for use among the Pakistani population. Therefore, no further validation test was measured during the study [20,21,22,23].",
"The Urdu version of the KDQOL-SF-36 is reliable and validated for assessing HRQOL in kidney disease patients on dialysis in Pakistan [36]. The KDQOL- SF-36 consists of Physical Health Composite Summary (PCS), Mental Health Composite Summary (MCS), and Kidney Disease Composite Summary (KDCS) domains. These can be measured using the HRQOL scores for physical functioning, physical disabilities, pain, emotional wellness, social functioning, and energy. The KDQOL-SF-36 has five measures, including two essential HRQOL scales (12 items) from the SF-12 version 1 and three kidney-specific scales (24 items total). In the general population, the SF-12 PCS and MCS are assessed using a T-score methodology (mean = 50, SD = 10), with higher scores indicating improved HRQOL.",
"The Hamilton Depression Rating Scale was used in patients who speak Urdu. The HAM-D-U is an effective tool used for determining the magnitude of depression. Internal accuracy (Cronbach alpha 0.71), test–retest reliability, and inter-rater reliability were reasonably good for the Urdu version of the HAM-D (HAM-D-U) [37]. A HAM-D-U score of 0–7 is regarded as normal (or in clinical remission), but a score of 20 or more is deemed severe (showing at least moderate severity).\nHAM-D-U scores are interpreted as follows:No depression at all: <8Mild (subthreshold): 8–13Moderate (mild): 14–18Severe (moderate): 19–22Very severe (severe): >23\nNo depression at all: <8\nMild (subthreshold): 8–13\nModerate (mild): 14–18\nSevere (moderate): 19–22\nVery severe (severe): >23",
"The Morisky Levine Green adherence questionnaire (MLGS) was used to evaluate medication adherence. It is a four-item questionnaire with high reliability and validity that has proven to be particularly useful in chronic conditions such as hypertension, kidney disease, and other cardiovascular diseases. It assesses deliberate and accidental adherence using criteria such as forgetfulness, carelessness, and stopping the drug while feeling better and worse. Each question in the survey was answered with a 0 or 1 response. The total score of the four elements represents the degree of prescription adherence. Medication adherence is indicated by a score of 4, while a score of less than 4 indicates non-adherence. The questionnaire was translated into Urdu using the standard forward–backwards process, yielding a Cronbach alpha of 0.570, within the appropriate range (0.45–0.9) [38,39].",
"The tools were administered in the following manner:\nAll questionnaires were administered to patients using a self-reporting approach. In order to validate the responses of the patients, researchers conducted random interviews with the patients during their dialysis procedure. The details schematic flow of patient enrollment, data collection and questionnaire administration are displayed Figure 1 and Figure 2.",
"The Bio-Ethics Committee (BEC) of Quaid-i-Azam University Islamabad permitted this prospective observational study in Tertiary Care Hospitals (BEC-FBS-QAU2017-13). The study was also approved by the Ethical Review Board of Shaheed Zulfikar Ali Bhutto Medical University (1-1/2015/ERB/SZABMU). Informed written consent was taken from all the participants included in this study. The study was conducted per the Declaration of Helsinki and reported according to “The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” guidelines.",
"Primary data were entered into Microsoft Excel 2010, and anonymous patient coding was used to generate secondary data. The data were analysed using the Statistical Package for Social Sciences (SPSS® IBM version 20, Armonk, NY, USA) program. The KDQOL SFTM1.3 scoring program v.2.0 (Santa Monica, CA, USA) was used to compute and calculate PCS and MCS. Continuous variables were expressed in terms of means and standard deviations, whereas categorical variables were expressed in frequencies and percentages. To summarize sample characteristics, descriptive statistics were used.\nMoreover, normality tests were assessed with the Shapiro–Wilk test. Data had normal distribution; therefore, continuous data’s mean and standard deviation have been presented. Categorical variables were presented as numbers and percentages. The Mann–Whitney U test was used to compare the depression and HRQOL domains. A p-value of <0.05 was considered statistically significant, and a p-value of < 0.01 was considered highly significant. The multivariate analysis was also performed using Bonferroni Correction. Binary logistic regression was used to examine factors related to medication non-adherence and depression, and effect sizes were reported using odds ratios (ORs) and 95% confidence intervals (CIs). The study variable includes dependent variables (KDQOL Domain and Scale, MCS, PCS, medication adherence, and depression) and independent variables (sociodemographic parameters, laboratory parameters).",
" 3.1. Patient Characteristics During the study recruitment period, 400 patients were enrolled at the treatment sites. Eighty-six patients did not meet the eligibility criteria and were excluded. In total, 314 patients participated in the study and completed KDQOL, HAM-D-U, and MLGS. Figure 2 shows the schematic flow of the study. Of the total 314 patients, 39.8% (n = 125) were CKD grade 4, 9.2% (n = 29) with grade 5 not on hemodialysis yet, and 50.9% (n = 160) were at kidney failure grade 5 on hemodialysis.\nThe demographic characteristics of the study participants are shown in Table 1. The mean age of the participants was 54.64 ± 15.33. The majority of the survey participants were women, with a significant proportion of the study population from the age group 41–60 years. The number of people with BMI in the normal range was 53.0%. The study population selected through convenience sampling comprised different levels of disease, treatment approaches, and durations.\nThe comorbidity conditions among the study populations are depicted in Figure 3. Among the population with CKD, diabetes and hypertension were the most prominent underlying comorbid conditions, i.e., 62% and 55%, respectively. Among the study population, 19% had various etiological factors, e.g., smoking, and illicit drug use. Most patients had a family history of kidney disease and a strong history of heart disease. A large group of people also had unknown etiology and represented 30% of the study population.\nDuring the study recruitment period, 400 patients were enrolled at the treatment sites. Eighty-six patients did not meet the eligibility criteria and were excluded. In total, 314 patients participated in the study and completed KDQOL, HAM-D-U, and MLGS. Figure 2 shows the schematic flow of the study. Of the total 314 patients, 39.8% (n = 125) were CKD grade 4, 9.2% (n = 29) with grade 5 not on hemodialysis yet, and 50.9% (n = 160) were at kidney failure grade 5 on hemodialysis.\nThe demographic characteristics of the study participants are shown in Table 1. The mean age of the participants was 54.64 ± 15.33. The majority of the survey participants were women, with a significant proportion of the study population from the age group 41–60 years. The number of people with BMI in the normal range was 53.0%. The study population selected through convenience sampling comprised different levels of disease, treatment approaches, and durations.\nThe comorbidity conditions among the study populations are depicted in Figure 3. Among the population with CKD, diabetes and hypertension were the most prominent underlying comorbid conditions, i.e., 62% and 55%, respectively. Among the study population, 19% had various etiological factors, e.g., smoking, and illicit drug use. Most patients had a family history of kidney disease and a strong history of heart disease. A large group of people also had unknown etiology and represented 30% of the study population.\n 3.2. Laboratory Parameters There were abnormalities in laboratory parameters, and their impact was also analysed in the overall HRQOL of patients. Statistical analysis suggested that hematological and serum iron studies were laboratory parameters that significantly affected the mental composite score (p-Value < 0.05). Contrary to this, the physical composite score was greatly affected by parameters such as hematological serum iron and renal function concentration in the blood, as shown in Table 2.\nThere were abnormalities in laboratory parameters, and their impact was also analysed in the overall HRQOL of patients. Statistical analysis suggested that hematological and serum iron studies were laboratory parameters that significantly affected the mental composite score (p-Value < 0.05). Contrary to this, the physical composite score was greatly affected by parameters such as hematological serum iron and renal function concentration in the blood, as shown in Table 2.\n 3.3. Depression In the study, the 21-item Hamilton Depression Rating Scale (HAM-D-U) was used to determine the study population’s pre-study and post-study analysis of depression levels.\nIt was observed that throughout treatment, there was no educational or counselling support provided to any of the patients. Analysis of the depression data was given by 314 patients in the pre-assessment questionnaire and 244 in the post-assessment questionnaire. A total of 45 patients refused to fill out the post-study HAM-D-U.\nThe frequency distribution of the depression assessment at baseline found that there was a significantly high depression level in both kidney failure populations (Figure 4). There were only a few cases with a normal-to-moderate level of depression.\nUpon the follow-up visit, it was observed that there was a significant increase in depression levels among the study population. Respondents indicated a higher level of depression in both the study populations compared to their previous state. However, further analysis with the Pearson Chi-square test found no significant difference between the baseline and post-study depression levels (p-value > 0.05).\nIn the study, the 21-item Hamilton Depression Rating Scale (HAM-D-U) was used to determine the study population’s pre-study and post-study analysis of depression levels.\nIt was observed that throughout treatment, there was no educational or counselling support provided to any of the patients. Analysis of the depression data was given by 314 patients in the pre-assessment questionnaire and 244 in the post-assessment questionnaire. A total of 45 patients refused to fill out the post-study HAM-D-U.\nThe frequency distribution of the depression assessment at baseline found that there was a significantly high depression level in both kidney failure populations (Figure 4). There were only a few cases with a normal-to-moderate level of depression.\nUpon the follow-up visit, it was observed that there was a significant increase in depression levels among the study population. Respondents indicated a higher level of depression in both the study populations compared to their previous state. However, further analysis with the Pearson Chi-square test found no significant difference between the baseline and post-study depression levels (p-value > 0.05).\n 3.4. Medication Adherence Around 38.8% (n = 122) of the 314 participants scored 4 and had high adherence, while the majority (61.2%, n = 192) belonged to a non-adherent population with a score of less than 4. In addition, the findings revealed no statistically significant relationship between adherence and gender, age, marital status, education level, smoking status or the number of medications (p > 0.05).\nAround 38.8% (n = 122) of the 314 participants scored 4 and had high adherence, while the majority (61.2%, n = 192) belonged to a non-adherent population with a score of less than 4. In addition, the findings revealed no statistically significant relationship between adherence and gender, age, marital status, education level, smoking status or the number of medications (p > 0.05).\n 3.5. HRQOL A total of 314 patients completed the KDQOL-SF-36 questionnaire during a baseline visit. As shown in Table 3, even though patients with grade 4 had a lower level of the physical score as compared to those having grade 5, there were no significant differences seen in the SF-36 PCS scale (31.28 ± 7.91 vs. 36.28 ± 8.41, p = 0.68). However, the overall mental health composite measured by the MCS at baseline was significantly lower for the grade 4 population than the grade 5 population, i.e., 36.66 ± 6.57 and 48.66 ± 5.44, respectively (p-value < 0.05).\nFor the KDQOL-SF-36 domain, the Mental Health Composite was the factor that affected the study population the most, which showed a statistically significant level in both the study groups (p-value < 0.05). The other major KDQOL-SF-36 domains that impacted both populations were Physical Functioning, Role Limitations-Physical, and Emotional well-being, as well as energy and fatigue evident from Table 3.\nIn the follow-up visit, 244 patients agreed to respond to the questionnaires. In the post-study, 35 patients with grade 4 and 10 patients with grade 5 did not respond. In the post-analysis, a marked deterioration was seen in KDQOL-SF-36 domains and scale parameters among patients with advanced kidney failure. Grade 4 patients scored significantly lower than grade 5 in the Problem’s list/Symptom, Burden of Kidney Disease, and Mental Health composite domains (p < 0.05). In the KDQOL-SF-36 scales, there was significant deterioration in Physical function, Emotional well-being, and Energy level in patients with grade 5 (p-value < 0.05).\nA total of 314 patients completed the KDQOL-SF-36 questionnaire during a baseline visit. As shown in Table 3, even though patients with grade 4 had a lower level of the physical score as compared to those having grade 5, there were no significant differences seen in the SF-36 PCS scale (31.28 ± 7.91 vs. 36.28 ± 8.41, p = 0.68). However, the overall mental health composite measured by the MCS at baseline was significantly lower for the grade 4 population than the grade 5 population, i.e., 36.66 ± 6.57 and 48.66 ± 5.44, respectively (p-value < 0.05).\nFor the KDQOL-SF-36 domain, the Mental Health Composite was the factor that affected the study population the most, which showed a statistically significant level in both the study groups (p-value < 0.05). The other major KDQOL-SF-36 domains that impacted both populations were Physical Functioning, Role Limitations-Physical, and Emotional well-being, as well as energy and fatigue evident from Table 3.\nIn the follow-up visit, 244 patients agreed to respond to the questionnaires. In the post-study, 35 patients with grade 4 and 10 patients with grade 5 did not respond. In the post-analysis, a marked deterioration was seen in KDQOL-SF-36 domains and scale parameters among patients with advanced kidney failure. Grade 4 patients scored significantly lower than grade 5 in the Problem’s list/Symptom, Burden of Kidney Disease, and Mental Health composite domains (p < 0.05). In the KDQOL-SF-36 scales, there was significant deterioration in Physical function, Emotional well-being, and Energy level in patients with grade 5 (p-value < 0.05).\n 3.6. Medication Adherence and HRQOL The PCS of the HRQOL improved over time in adherent individuals and declined in non-adherent individuals. Additionally, after controlling for age, gender, and baseline eGFR, medication non-adherence demonstrated a significant negative correlation with a change in the PCS of the HRQOL, as evident from Table 4. After adjusting for the same covariates, there was no significant correlation between medication non-adherence and a change in the MCS of the HRQOL.\nThe PCS of the HRQOL improved over time in adherent individuals and declined in non-adherent individuals. Additionally, after controlling for age, gender, and baseline eGFR, medication non-adherence demonstrated a significant negative correlation with a change in the PCS of the HRQOL, as evident from Table 4. After adjusting for the same covariates, there was no significant correlation between medication non-adherence and a change in the MCS of the HRQOL.\n 3.7. Depression and HRQOL Depressive symptoms were inversely correlated with PCS and MCS scores in the unadjusted longitudinal analysis. Depressive symptoms were independently reduced following multivariable adjustment and negatively correlated with the PCS and MCS scores, as shown in Table 5. When depressive symptoms were considered a dichotomous variable in unadjusted analyses, moderate-to-severe depression symptoms presented negative correlations between the PCS and MCS. In multivariable analyses, the PCS and MCS scores were independently and negatively related to the prevalence of moderate-to-severe depressive symptoms.\nDepressive symptoms were inversely correlated with PCS and MCS scores in the unadjusted longitudinal analysis. Depressive symptoms were independently reduced following multivariable adjustment and negatively correlated with the PCS and MCS scores, as shown in Table 5. When depressive symptoms were considered a dichotomous variable in unadjusted analyses, moderate-to-severe depression symptoms presented negative correlations between the PCS and MCS. In multivariable analyses, the PCS and MCS scores were independently and negatively related to the prevalence of moderate-to-severe depressive symptoms.",
"During the study recruitment period, 400 patients were enrolled at the treatment sites. Eighty-six patients did not meet the eligibility criteria and were excluded. In total, 314 patients participated in the study and completed KDQOL, HAM-D-U, and MLGS. Figure 2 shows the schematic flow of the study. Of the total 314 patients, 39.8% (n = 125) were CKD grade 4, 9.2% (n = 29) with grade 5 not on hemodialysis yet, and 50.9% (n = 160) were at kidney failure grade 5 on hemodialysis.\nThe demographic characteristics of the study participants are shown in Table 1. The mean age of the participants was 54.64 ± 15.33. The majority of the survey participants were women, with a significant proportion of the study population from the age group 41–60 years. The number of people with BMI in the normal range was 53.0%. The study population selected through convenience sampling comprised different levels of disease, treatment approaches, and durations.\nThe comorbidity conditions among the study populations are depicted in Figure 3. Among the population with CKD, diabetes and hypertension were the most prominent underlying comorbid conditions, i.e., 62% and 55%, respectively. Among the study population, 19% had various etiological factors, e.g., smoking, and illicit drug use. Most patients had a family history of kidney disease and a strong history of heart disease. A large group of people also had unknown etiology and represented 30% of the study population.",
"There were abnormalities in laboratory parameters, and their impact was also analysed in the overall HRQOL of patients. Statistical analysis suggested that hematological and serum iron studies were laboratory parameters that significantly affected the mental composite score (p-Value < 0.05). Contrary to this, the physical composite score was greatly affected by parameters such as hematological serum iron and renal function concentration in the blood, as shown in Table 2.",
"In the study, the 21-item Hamilton Depression Rating Scale (HAM-D-U) was used to determine the study population’s pre-study and post-study analysis of depression levels.\nIt was observed that throughout treatment, there was no educational or counselling support provided to any of the patients. Analysis of the depression data was given by 314 patients in the pre-assessment questionnaire and 244 in the post-assessment questionnaire. A total of 45 patients refused to fill out the post-study HAM-D-U.\nThe frequency distribution of the depression assessment at baseline found that there was a significantly high depression level in both kidney failure populations (Figure 4). There were only a few cases with a normal-to-moderate level of depression.\nUpon the follow-up visit, it was observed that there was a significant increase in depression levels among the study population. Respondents indicated a higher level of depression in both the study populations compared to their previous state. However, further analysis with the Pearson Chi-square test found no significant difference between the baseline and post-study depression levels (p-value > 0.05).",
"Around 38.8% (n = 122) of the 314 participants scored 4 and had high adherence, while the majority (61.2%, n = 192) belonged to a non-adherent population with a score of less than 4. In addition, the findings revealed no statistically significant relationship between adherence and gender, age, marital status, education level, smoking status or the number of medications (p > 0.05).",
"A total of 314 patients completed the KDQOL-SF-36 questionnaire during a baseline visit. As shown in Table 3, even though patients with grade 4 had a lower level of the physical score as compared to those having grade 5, there were no significant differences seen in the SF-36 PCS scale (31.28 ± 7.91 vs. 36.28 ± 8.41, p = 0.68). However, the overall mental health composite measured by the MCS at baseline was significantly lower for the grade 4 population than the grade 5 population, i.e., 36.66 ± 6.57 and 48.66 ± 5.44, respectively (p-value < 0.05).\nFor the KDQOL-SF-36 domain, the Mental Health Composite was the factor that affected the study population the most, which showed a statistically significant level in both the study groups (p-value < 0.05). The other major KDQOL-SF-36 domains that impacted both populations were Physical Functioning, Role Limitations-Physical, and Emotional well-being, as well as energy and fatigue evident from Table 3.\nIn the follow-up visit, 244 patients agreed to respond to the questionnaires. In the post-study, 35 patients with grade 4 and 10 patients with grade 5 did not respond. In the post-analysis, a marked deterioration was seen in KDQOL-SF-36 domains and scale parameters among patients with advanced kidney failure. Grade 4 patients scored significantly lower than grade 5 in the Problem’s list/Symptom, Burden of Kidney Disease, and Mental Health composite domains (p < 0.05). In the KDQOL-SF-36 scales, there was significant deterioration in Physical function, Emotional well-being, and Energy level in patients with grade 5 (p-value < 0.05).",
"The PCS of the HRQOL improved over time in adherent individuals and declined in non-adherent individuals. Additionally, after controlling for age, gender, and baseline eGFR, medication non-adherence demonstrated a significant negative correlation with a change in the PCS of the HRQOL, as evident from Table 4. After adjusting for the same covariates, there was no significant correlation between medication non-adherence and a change in the MCS of the HRQOL.",
"Depressive symptoms were inversely correlated with PCS and MCS scores in the unadjusted longitudinal analysis. Depressive symptoms were independently reduced following multivariable adjustment and negatively correlated with the PCS and MCS scores, as shown in Table 5. When depressive symptoms were considered a dichotomous variable in unadjusted analyses, moderate-to-severe depression symptoms presented negative correlations between the PCS and MCS. In multivariable analyses, the PCS and MCS scores were independently and negatively related to the prevalence of moderate-to-severe depressive symptoms.",
"The study was conducted in two major cities in Pakistan and is the first-ever follow-up study investigating the point of concern for patients with kidney failure in grade 5 and CKD grade 4. According to the results of this study, targeted patients with kidney failure grade 5 who are not on chronic renal replacement therapy have a similar overall burden of symptoms and depression and a poor HRQOL. The results derived from this research have significant clinical implications for both patients and care providers.\nDespite studies showing that patients with kidney failure grade 4 have poor physical and psychosocial health, the clinical, medication, and patient-related factors that cause symptoms, depression, and poor HRQOL in the targeted patient population are unknown. Patients tend to develop symptoms, depression, and poor HRQOL if they lose much weight without losing kidney function. Therefore, it is critical to determine whether this is due to metabolic disturbances, retained uremic contaminants, comorbid medical conditions, anxiety about kidney failure, the possible need for renal replacement therapy in the future or other causes to administer adequate care. Due to the use of different tools for detailed analysis, the results derived in this study have significant implications for kidney failure patients.\nNonadherence to medication did not affect HRQOL at baseline but caused a gradual reduction in physical HRQOL (SF36-PCS) scores, although the mental HRQOL of all individuals decreased significantly during the follow-up period. Studies have shown that baseline eGFR levels influence physical deterioration in HRQOL [40]. However, we could not discover an association between initial eGFR and alterations in HRQOL over time in the current study.\nIt was also observed that the physical and mental health composite tends to deteriorate in patients with kidney failure. Previous studies reported that patients with moderate-to-progressing kidney disease had a lower PCS than MCS [27]. A previous study on kidney failure grade 1–4 patients reported that the PCS and MCS scores were 32.1 ± 8.1 and 40.6 ± 11.1, respectively. In the current study, the severity of the disease, particularly in individuals with kidney failure, had a considerable impact on HRQOL. This link has also been discovered in other investigations where HRQOL scores are compromised in patients with intermediate kidney failure and deteriorate over time in dialysis patients [9,41].\nA study by Ajeebi and his colleagues showed that patients with advanced kidney failure could likely be unaware of the effects of chronic renal replacement therapy on their physical and mental health [42]. Following this school of thought, it is evident that kidney failure patients need chronic dialysis for a drastic lifestyle change [43]. Based on the current study findings, there is evidence that patients with kidney failure experienced a significant difference in their physical and psychosocial well-being during the transition period of the disease.\nHowever, in the case of patients with kidney failure, complaints of sleep problems, muscle cramps, dry mouth, and lightheadedness are the most frequent. Although these discrepancies did not reach statistical significance after several comparisons, the biologi-cal plausibility of such differences warrants further investigation. In light of the results, it is evident that patients with kidney failure have lower scores on the SF-36 physical func-tion subscale. However, there were no variations in PCS scores due to this observation. As per the study referred to, this provides preliminary information on the subdomains of HRQOL that may differ between these two populations [24]. In a study conducted on the patients undergoing kidney transplantation and those on hemodialysis or peritoneal dialysis, the findings provided evidence that there was a significant improvement in HRQOL after one year of kidney replacement and dialysis [44]. The comparison between the current results and the existing literature provides evidence that patients with kidney failure are equally treated for health issues, including anemia, depression, sleeplessness, and bone and muscle diseases, that lead to the deterioration of their quality of life. Therefore, in the context of grade 5 kidney failure, patients should be informed about the unpredictability of their disease as it is difficult to anticipate the disease trajectory based on known predictive factors of mortality and the advantages and disadvantages of dialysis treatments. According to a study by Nataatmadja et al. (2021), of 3000 patients with an average age of 73 years, 60% regretted starting dialysis rather than having opted for conservative treatment [4]. As far as possible, the decision to initiate dialysis in patients with grade 5 kidney failure must be made personally between the patient, family members, the attending physician, and the referring nephrologist.\nNumerous studies have shown that dialysis has a detrimental effect on depression and that patients who experience severe depression are more likely to die. According to 15 large-scale investigations, there is an important link between depression and mortality among dialysis patients. Several longitudinal studies that evaluated the recurrent measurement of depression found that patients receiving dialysis therapy had significantly greater mortality risks when depressive symptoms were present. According to studies, early dialysis therapy initiation is associated with depression [45,46,47].\nThe general demographic features of our sample of grade 4 kidney failure were close to those of the United States population of grade 4 chronic kidney disease. On the contrary, the study’s kidney failure cohort had a higher proportion of men than the total population of kidney failure patients. The result supported the study mentioned above, as it was discovered that the depression rate was observed to worsen with time; a linear increase was identified from the start of the study until the second administration of the questionnaire 6 months later. Possible causes for this finding are likely to be lifelong dialysis therapy with at least 3 dialysis operations per week, patients taking too many medications at once, the economic strain on patients and their families, and changed familial and social ties. In a group of chronic HD patients, depression symptoms increased linearly and a link was discovered between poor sleep quality, unemployment, pruritus, hypoalbuminemia, diabetes, and depressive symptoms [48].\nThe findings of the current study and the prior research provided evidence that patients suffering from kidney failure are likely to develop mental health issues that the prolonged effects of illness could cause.\nChronic renal diseases are frequent in the population but often remain challenging to demonstrate clinically, as their symptoms are crude [49]. It is thus not uncommon for patients to develop end-stage renal failure, having presented very few symptoms or only vague and not very specific symptoms (fatigue, inappetence, etc.). In extensive population studies, it was found that only 23% of people with kidney failure were aware of their diagnosis [50]. According to the survey by Live and Zhang (2019), kidney failure is, in most cases, secondary to hypertension, diabetes, immunological disease, etc. Nevertheless, there are several kidney diseases that, although rare, could be diagnosed early, thanks to typical clinical and biological signs (red flags). However, even these diseases are often underdiagnosed, further prolonging the diagnostic error of these patients and their early management [51,52,53].\nIn addition, the treatment of hemodialysis patients includes all possible measures to reduce the impact of symptoms encountered in kidney failure [48]. These symptoms are pain, pruritus, nausea, headache, fatigue, anxiety, and depression. A total of 80% of patients suffered from chronic kidney pain and went to dialysis due to underlying mental health complications, especially depression. It has been stated in the study of Duane et al. that diseases affecting the kidneys are frequent but remain asymptomatic for a long time, leading to a diagnostic delay with consequences for the management of the patient [53]. However, convincing warning signs, whether cutaneous, articular or related to electrolyte abnormalities, should put the chip in the ear and guide the practitioner to the renal origin of the pathology. Therefore, certain rare diseases affecting the kidneys are discussed here based on clinical vignettes, emphasizing the biological and clinical signs that lead to their diagnosis [54].\nMoreover, mental health depends on individual, genetic, and hormonal factors, but also relational, community, and societal factors. Patients of kidney failure of all ages suffer from certain mental disorders more frequently, with more comorbidities. It also impacts their physical health and family and social balance. Specificities exist at the clinical level, as well as concerning the use of psychotropic drugs, their side effects, and adherence to care. The primary care physician’s role is essential for preventing and detecting mental disorders in kidney failure patients for their psychological support, guidance, and follow-up [41,55].\nThe current study has some limitations. The study only has a 6-month follow-up period; the findings should be interpreted cautiously. Although the variables have been adjusted for changes in physical activity, the possibility of residual confounders cannot be ruled out as one of the typical limitations of all cohort studies. For example, the health consciousness of an individual eating healthier also makes other more beneficial lifestyle changes that the questionnaire could not completely capture. A multicenter investigation with a large sample size and an extended follow-up period is required to corroborate the findings of the current study. Furthermore, future research in this domain needs to consider the comparison of mentioned health-related domains in a much more comprehensive sample of patients with kidney failure, including those with severe comorbid illnesses. In this study, which investigated several variables associated with low HRQOL in patients with kidney failure, depression significantly predicted a lower HRQOL. Therefore, our study emphasizes the potential need for randomized controlled trials in future research to determine the temporal relationship between low HRQOL and depression in kidney failure patients and to further assess the impact of early identification of depression and appropriate treatment of depression on the health outcomes of these patients. Despite these drawbacks, our study’s strength is that we performed thorough analyses to look at factors associated with HRQOL in patients with kidney failure, including medication adherence, depression, and modifiable and nonmodifiable clinical features.",
"The patients on maintenance dialysis for kidney failure have significant burdens of physical and mental symptoms, depression, and low HRQOL. Given the substantial and well-known declines in physical and psychological well-being among patients with kidney failure receiving hemodialysis, findings drawn in this research imply that these health-related areas should receive special attention in the vast and expanding population of patients with kidney failure."
] |
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"discussion",
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[
"kidney failure",
"health-related quality of life (HRQOL)",
"medication adherence",
"hemodialysis",
"depression"
] |
1. Introduction:
Chronic kidney failure is a public health problem worldwide and can lead to conditions such as kidney failure and cardiovascular problems [1]. In different regions of the world, kidney disease has already reached an epidemic rate in people aged 20 years or above who are the most affected by this disease. The total number of men and women with kidney failure was 225.7 million and 271.8 million, respectively, by 2010 [2]. According to new research, the global prevalence of kidney failure was 9.1% in 2017 (697.5 million cases). Women and girls had a higher global majority of kidney failure than men and boys (9.5%) (7.3%) [3]. About a third of all kidney failure cases were in China (132.3 million) and India (115.1 million), with 10 countries having more than 10 million cases and 79 countries having more than 1 million cases [3,4].
Considering the high incidence of kidney failure and the challenges posed by the patients undergoing dialysis, rehabilitating one’s life is crucial. Patients suffer from different emotional and physical challenges during kidney failure [5]. Survival is not only the goal of placing the patient on a strenuous dialysis procedure but also improving the patient’s health-related quality of life (HRQOL). Understanding the deterioration of HRQOL is the most crucial aspect that can affect burden and mortality in these patients [6,7]. In patients with chronic kidney disease (CKD), including those with end-stage kidney disease (ESKD), monitoring the functional status and subjective state of well-being of a patient as they relate to a health condition, collectively known as HRQOL measurements, is of particular importance.
HRQOL has been significantly affected among patients with advanced chronic kidney disease and kidney failure. The fact that patients with CKD of various racial and ethnic backgrounds have varying HRQOL scores confirms the need to individualise the notion of HRQOL to evaluate critical aspects of life in patients and incorporate these domains into an all-encompassing plan of care. The urgency of HRQOL measurement and the need to advance our multidimensional tools with technology and a more patient-centered view of HRQOL is highlighted by these recent findings [8,9].
In dialysis patient populations, low HRQOL scores measured utilizing the Medical Outcomes Study Short Form-36 (SF-36) were associated with hospitalization and death in studies [10,11,12,13]. There were substantial HRQOL studies conducted before in patients with kidney failure. However, little has been done in Pakistan. Walters et al. assessed HRQOL at the beginning of dialysis therapy and found that in patients who began hemodialysis therapy (HD), HRQOL scores were significantly lower than in established long-term HD patients [14,15,16,17,18,19].
The Dialysis Outcomes and Practice Patterns Study (DOPPS) found a strong link between depressive symptoms and mortality and hospitalization rates [20]. On the contrary, the Choices for Healthy Outcomes in Caring for End-Stage Renal Disease (CHOICE) study found a link between high depressive symptoms and an increased risk of cardiovascular events [21]. However, this link between depression and clinical outcomes has not been thoroughly investigated in the early stages of kidney failure. A recent study by Wirkner et al. investigated an association between comorbid conditions and diabetes. The study concluded that advanced stages of CKD have significantly affected the overall HRQOL of the patients. Patients with kidney failure and undergoing dialysis were also found to have lower physical composite scores [22].
Therefore, it is crucial to assess all such parameters while dealing with a patient’s critical condition with kidney failure. This study was carried out to emphasize the use of tools to measure the HRQOL of patients and treatment outcomes [23,24,25,26]. Patient perceptions and feelings regarding their functionality and well-being are captured through patient-centered outcomes, such as HRQOL. In patients with CKD grade 4, outcome indicators are fundamental because they guide treatment objectives and strategies. However, there is little information on patient-centered outcomes in humans, especially in people who do not receive kidney replacement therapy and have CKD grade 4. More critically, there is a dearth of research in this patient population on the association between HRQOL and drug-related characteristics such as medication burden and adherence [27,28].
Previous studies have found that drug adherence rates range from 33.0 to 87.7%. Seng et al. investigated medication adherence in kidney failure. A pooled medication adherence rate of 67.4% (95% confidence interval (CI) 61.4–73.3%) was found in a meta-analysis of 54,652 patients. Between prospective and retrospective studies, the prevalence of adherence to medication among patients with dialysis kidney failure was similar (68.8%; 95% CI 61.1–76.6%) vs. (65.8%; 95% CI 57.0–74.6%) [28,29,30]. According to the study reported in Pakistan, 74% of patients with kidney failure experienced pruritus, negatively impacting their quality of life. Poor quality of life affects patients with mild to severe CKD-related pruritus. The hemodialysis patients had a lower HRQOL score as their pruritus intensity increased [31].
In Pakistan, kidney failure is the most common cause of morbidity and mortality [32]. According to the 2016 Pakistan National Kidney Federation Registry, 5935 patients were admitted to different dialysis units across the country, totaling 891 hemodialysis machines [33]. The data available have certain limitations as many large dialysis centers are not contributing their data to the registry.
There is a significant upsurge in the prevalence of kidney failure worldwide, particularly in low- to middle-income countries [34]. It is essential to evaluate the factor that could impact the quality of treatment and design a patient support program to increase patients’ involvement in the self-management of the disease condition. This study aimed to evaluate the factors associated with reduced HRQOL and other factors that affect the overall health of patients. Another objective was to measure how medication adherence and depression could impact the overall HRQOL of patients with kidney failure.
2. Materials and Methods:
2.1. Study Design This multicenter prospective cohort follow-up study was carried out in Rawalpindi and Islamabad districts. The study was conducted from April 2019 to February 2020. The patients were recruited from the Nephrology Unit of 3 major tertiary care hospitals, i.e., Federal Government Polyclinic Hospital, Islamabad, Pakistan Institute of Medical Sciences, Islamabad, and District Headquarter Hospital Rawalpindi, Pakistan.
This multicenter prospective cohort follow-up study was carried out in Rawalpindi and Islamabad districts. The study was conducted from April 2019 to February 2020. The patients were recruited from the Nephrology Unit of 3 major tertiary care hospitals, i.e., Federal Government Polyclinic Hospital, Islamabad, Pakistan Institute of Medical Sciences, Islamabad, and District Headquarter Hospital Rawalpindi, Pakistan.
2.2. Inclusion and Exclusion Criteria A total of 400 patients were recruited for the study. After initial screening, 314 participants met the eligibility criteria for the study. All patients aged >18 years with an eGFR value of <30 mL/min/1.73 m2 diagnosed by a physician as a patient having CKD grade 4 and kidney failure grade 5 were enrolled in the study. All patients diagnosed with kidney failure were graded based on their history and investigation conducted in the hospital’s medical unit. According to hospital guidelines, CKD-EPI was used to estimate the eGFR; however, to reduce factors such as weight and body mass index, Cockcroft–Gault formulae were used to determine the eGFR level of the participants. The final diagnosis to determine the grades of kidney failure was made using multiple creatinine assessments. As per the guidelines provided by the study centers, they used Cockcroft–Gault formulae to give better risk stratification in cardiovascular events [35].
Participants attended a baseline clinic after being referred by their physicians. All patients were informed about the purpose of the study and written consent was obtained during the patient recruitment phase. We excluded patients diagnosed with malignancy, hospitalized patients, and patients who did not have complete laboratory investigations available at the time of recruitment. We also excluded patients with cognitive disabilities or any other mental illness who could not correctly respond to our questionnaire or other limitations that hindered their participation in this study.
A total of 400 patients were recruited for the study. After initial screening, 314 participants met the eligibility criteria for the study. All patients aged >18 years with an eGFR value of <30 mL/min/1.73 m2 diagnosed by a physician as a patient having CKD grade 4 and kidney failure grade 5 were enrolled in the study. All patients diagnosed with kidney failure were graded based on their history and investigation conducted in the hospital’s medical unit. According to hospital guidelines, CKD-EPI was used to estimate the eGFR; however, to reduce factors such as weight and body mass index, Cockcroft–Gault formulae were used to determine the eGFR level of the participants. The final diagnosis to determine the grades of kidney failure was made using multiple creatinine assessments. As per the guidelines provided by the study centers, they used Cockcroft–Gault formulae to give better risk stratification in cardiovascular events [35].
Participants attended a baseline clinic after being referred by their physicians. All patients were informed about the purpose of the study and written consent was obtained during the patient recruitment phase. We excluded patients diagnosed with malignancy, hospitalized patients, and patients who did not have complete laboratory investigations available at the time of recruitment. We also excluded patients with cognitive disabilities or any other mental illness who could not correctly respond to our questionnaire or other limitations that hindered their participation in this study.
2.3. Tools The following tools were used to achieve the study’s goal.
The following tools were used to achieve the study’s goal.
2.4. Sociodemographic Data Sheet It was designed by the study’s principal investigators to assess the relevant socio-demographic variables, e.g., age, gender, education status, duration of kidney failure treatment, and comorbidity condition.
It was designed by the study’s principal investigators to assess the relevant socio-demographic variables, e.g., age, gender, education status, duration of kidney failure treatment, and comorbidity condition.
2.5. Laboratory Investigation Assessment Datasheet During the baseline visit, the serum potassium, calcium and phosphorus, hemoglobin, and urea levels was collected from medical records. The same laboratory investigation was also assessed after the completion of the follow-up visit at six months’ duration.
During the baseline visit, the serum potassium, calcium and phosphorus, hemoglobin, and urea levels was collected from medical records. The same laboratory investigation was also assessed after the completion of the follow-up visit at six months’ duration.
2.6. Assessment of Depression, HRQOL, and Medication Adherence The study included different Urdu pre-validated and translated questionnaires to achieve the study’s objectives. All these questionnaires have been validated for use among the Pakistani population. Therefore, no further validation test was measured during the study [20,21,22,23].
The study included different Urdu pre-validated and translated questionnaires to achieve the study’s objectives. All these questionnaires have been validated for use among the Pakistani population. Therefore, no further validation test was measured during the study [20,21,22,23].
2.7. Kidney Disease Quality of Life-Short Form (KDQOL-SF-36) The Urdu version of the KDQOL-SF-36 is reliable and validated for assessing HRQOL in kidney disease patients on dialysis in Pakistan [36]. The KDQOL- SF-36 consists of Physical Health Composite Summary (PCS), Mental Health Composite Summary (MCS), and Kidney Disease Composite Summary (KDCS) domains. These can be measured using the HRQOL scores for physical functioning, physical disabilities, pain, emotional wellness, social functioning, and energy. The KDQOL-SF-36 has five measures, including two essential HRQOL scales (12 items) from the SF-12 version 1 and three kidney-specific scales (24 items total). In the general population, the SF-12 PCS and MCS are assessed using a T-score methodology (mean = 50, SD = 10), with higher scores indicating improved HRQOL.
The Urdu version of the KDQOL-SF-36 is reliable and validated for assessing HRQOL in kidney disease patients on dialysis in Pakistan [36]. The KDQOL- SF-36 consists of Physical Health Composite Summary (PCS), Mental Health Composite Summary (MCS), and Kidney Disease Composite Summary (KDCS) domains. These can be measured using the HRQOL scores for physical functioning, physical disabilities, pain, emotional wellness, social functioning, and energy. The KDQOL-SF-36 has five measures, including two essential HRQOL scales (12 items) from the SF-12 version 1 and three kidney-specific scales (24 items total). In the general population, the SF-12 PCS and MCS are assessed using a T-score methodology (mean = 50, SD = 10), with higher scores indicating improved HRQOL.
2.8. Hamilton Depression Rating Scale Urdu Version (HAM-D-U) The Hamilton Depression Rating Scale was used in patients who speak Urdu. The HAM-D-U is an effective tool used for determining the magnitude of depression. Internal accuracy (Cronbach alpha 0.71), test–retest reliability, and inter-rater reliability were reasonably good for the Urdu version of the HAM-D (HAM-D-U) [37]. A HAM-D-U score of 0–7 is regarded as normal (or in clinical remission), but a score of 20 or more is deemed severe (showing at least moderate severity).
HAM-D-U scores are interpreted as follows:No depression at all: <8Mild (subthreshold): 8–13Moderate (mild): 14–18Severe (moderate): 19–22Very severe (severe): >23
No depression at all: <8
Mild (subthreshold): 8–13
Moderate (mild): 14–18
Severe (moderate): 19–22
Very severe (severe): >23
The Hamilton Depression Rating Scale was used in patients who speak Urdu. The HAM-D-U is an effective tool used for determining the magnitude of depression. Internal accuracy (Cronbach alpha 0.71), test–retest reliability, and inter-rater reliability were reasonably good for the Urdu version of the HAM-D (HAM-D-U) [37]. A HAM-D-U score of 0–7 is regarded as normal (or in clinical remission), but a score of 20 or more is deemed severe (showing at least moderate severity).
HAM-D-U scores are interpreted as follows:No depression at all: <8Mild (subthreshold): 8–13Moderate (mild): 14–18Severe (moderate): 19–22Very severe (severe): >23
No depression at all: <8
Mild (subthreshold): 8–13
Moderate (mild): 14–18
Severe (moderate): 19–22
Very severe (severe): >23
2.9. Morisky Levine Greens Adherence Scales (MLGS) The Morisky Levine Green adherence questionnaire (MLGS) was used to evaluate medication adherence. It is a four-item questionnaire with high reliability and validity that has proven to be particularly useful in chronic conditions such as hypertension, kidney disease, and other cardiovascular diseases. It assesses deliberate and accidental adherence using criteria such as forgetfulness, carelessness, and stopping the drug while feeling better and worse. Each question in the survey was answered with a 0 or 1 response. The total score of the four elements represents the degree of prescription adherence. Medication adherence is indicated by a score of 4, while a score of less than 4 indicates non-adherence. The questionnaire was translated into Urdu using the standard forward–backwards process, yielding a Cronbach alpha of 0.570, within the appropriate range (0.45–0.9) [38,39].
The Morisky Levine Green adherence questionnaire (MLGS) was used to evaluate medication adherence. It is a four-item questionnaire with high reliability and validity that has proven to be particularly useful in chronic conditions such as hypertension, kidney disease, and other cardiovascular diseases. It assesses deliberate and accidental adherence using criteria such as forgetfulness, carelessness, and stopping the drug while feeling better and worse. Each question in the survey was answered with a 0 or 1 response. The total score of the four elements represents the degree of prescription adherence. Medication adherence is indicated by a score of 4, while a score of less than 4 indicates non-adherence. The questionnaire was translated into Urdu using the standard forward–backwards process, yielding a Cronbach alpha of 0.570, within the appropriate range (0.45–0.9) [38,39].
2.10. Study Flow The tools were administered in the following manner:
All questionnaires were administered to patients using a self-reporting approach. In order to validate the responses of the patients, researchers conducted random interviews with the patients during their dialysis procedure. The details schematic flow of patient enrollment, data collection and questionnaire administration are displayed Figure 1 and Figure 2.
The tools were administered in the following manner:
All questionnaires were administered to patients using a self-reporting approach. In order to validate the responses of the patients, researchers conducted random interviews with the patients during their dialysis procedure. The details schematic flow of patient enrollment, data collection and questionnaire administration are displayed Figure 1 and Figure 2.
2.11. Ethical Approval and Consent to Participate The Bio-Ethics Committee (BEC) of Quaid-i-Azam University Islamabad permitted this prospective observational study in Tertiary Care Hospitals (BEC-FBS-QAU2017-13). The study was also approved by the Ethical Review Board of Shaheed Zulfikar Ali Bhutto Medical University (1-1/2015/ERB/SZABMU). Informed written consent was taken from all the participants included in this study. The study was conducted per the Declaration of Helsinki and reported according to “The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” guidelines.
The Bio-Ethics Committee (BEC) of Quaid-i-Azam University Islamabad permitted this prospective observational study in Tertiary Care Hospitals (BEC-FBS-QAU2017-13). The study was also approved by the Ethical Review Board of Shaheed Zulfikar Ali Bhutto Medical University (1-1/2015/ERB/SZABMU). Informed written consent was taken from all the participants included in this study. The study was conducted per the Declaration of Helsinki and reported according to “The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” guidelines.
2.12. Statistical Analysis Primary data were entered into Microsoft Excel 2010, and anonymous patient coding was used to generate secondary data. The data were analysed using the Statistical Package for Social Sciences (SPSS® IBM version 20, Armonk, NY, USA) program. The KDQOL SFTM1.3 scoring program v.2.0 (Santa Monica, CA, USA) was used to compute and calculate PCS and MCS. Continuous variables were expressed in terms of means and standard deviations, whereas categorical variables were expressed in frequencies and percentages. To summarize sample characteristics, descriptive statistics were used.
Moreover, normality tests were assessed with the Shapiro–Wilk test. Data had normal distribution; therefore, continuous data’s mean and standard deviation have been presented. Categorical variables were presented as numbers and percentages. The Mann–Whitney U test was used to compare the depression and HRQOL domains. A p-value of <0.05 was considered statistically significant, and a p-value of < 0.01 was considered highly significant. The multivariate analysis was also performed using Bonferroni Correction. Binary logistic regression was used to examine factors related to medication non-adherence and depression, and effect sizes were reported using odds ratios (ORs) and 95% confidence intervals (CIs). The study variable includes dependent variables (KDQOL Domain and Scale, MCS, PCS, medication adherence, and depression) and independent variables (sociodemographic parameters, laboratory parameters).
Primary data were entered into Microsoft Excel 2010, and anonymous patient coding was used to generate secondary data. The data were analysed using the Statistical Package for Social Sciences (SPSS® IBM version 20, Armonk, NY, USA) program. The KDQOL SFTM1.3 scoring program v.2.0 (Santa Monica, CA, USA) was used to compute and calculate PCS and MCS. Continuous variables were expressed in terms of means and standard deviations, whereas categorical variables were expressed in frequencies and percentages. To summarize sample characteristics, descriptive statistics were used.
Moreover, normality tests were assessed with the Shapiro–Wilk test. Data had normal distribution; therefore, continuous data’s mean and standard deviation have been presented. Categorical variables were presented as numbers and percentages. The Mann–Whitney U test was used to compare the depression and HRQOL domains. A p-value of <0.05 was considered statistically significant, and a p-value of < 0.01 was considered highly significant. The multivariate analysis was also performed using Bonferroni Correction. Binary logistic regression was used to examine factors related to medication non-adherence and depression, and effect sizes were reported using odds ratios (ORs) and 95% confidence intervals (CIs). The study variable includes dependent variables (KDQOL Domain and Scale, MCS, PCS, medication adherence, and depression) and independent variables (sociodemographic parameters, laboratory parameters).
2.1. Study Design:
This multicenter prospective cohort follow-up study was carried out in Rawalpindi and Islamabad districts. The study was conducted from April 2019 to February 2020. The patients were recruited from the Nephrology Unit of 3 major tertiary care hospitals, i.e., Federal Government Polyclinic Hospital, Islamabad, Pakistan Institute of Medical Sciences, Islamabad, and District Headquarter Hospital Rawalpindi, Pakistan.
2.2. Inclusion and Exclusion Criteria:
A total of 400 patients were recruited for the study. After initial screening, 314 participants met the eligibility criteria for the study. All patients aged >18 years with an eGFR value of <30 mL/min/1.73 m2 diagnosed by a physician as a patient having CKD grade 4 and kidney failure grade 5 were enrolled in the study. All patients diagnosed with kidney failure were graded based on their history and investigation conducted in the hospital’s medical unit. According to hospital guidelines, CKD-EPI was used to estimate the eGFR; however, to reduce factors such as weight and body mass index, Cockcroft–Gault formulae were used to determine the eGFR level of the participants. The final diagnosis to determine the grades of kidney failure was made using multiple creatinine assessments. As per the guidelines provided by the study centers, they used Cockcroft–Gault formulae to give better risk stratification in cardiovascular events [35].
Participants attended a baseline clinic after being referred by their physicians. All patients were informed about the purpose of the study and written consent was obtained during the patient recruitment phase. We excluded patients diagnosed with malignancy, hospitalized patients, and patients who did not have complete laboratory investigations available at the time of recruitment. We also excluded patients with cognitive disabilities or any other mental illness who could not correctly respond to our questionnaire or other limitations that hindered their participation in this study.
2.3. Tools:
The following tools were used to achieve the study’s goal.
2.4. Sociodemographic Data Sheet:
It was designed by the study’s principal investigators to assess the relevant socio-demographic variables, e.g., age, gender, education status, duration of kidney failure treatment, and comorbidity condition.
2.5. Laboratory Investigation Assessment Datasheet:
During the baseline visit, the serum potassium, calcium and phosphorus, hemoglobin, and urea levels was collected from medical records. The same laboratory investigation was also assessed after the completion of the follow-up visit at six months’ duration.
2.6. Assessment of Depression, HRQOL, and Medication Adherence:
The study included different Urdu pre-validated and translated questionnaires to achieve the study’s objectives. All these questionnaires have been validated for use among the Pakistani population. Therefore, no further validation test was measured during the study [20,21,22,23].
2.7. Kidney Disease Quality of Life-Short Form (KDQOL-SF-36):
The Urdu version of the KDQOL-SF-36 is reliable and validated for assessing HRQOL in kidney disease patients on dialysis in Pakistan [36]. The KDQOL- SF-36 consists of Physical Health Composite Summary (PCS), Mental Health Composite Summary (MCS), and Kidney Disease Composite Summary (KDCS) domains. These can be measured using the HRQOL scores for physical functioning, physical disabilities, pain, emotional wellness, social functioning, and energy. The KDQOL-SF-36 has five measures, including two essential HRQOL scales (12 items) from the SF-12 version 1 and three kidney-specific scales (24 items total). In the general population, the SF-12 PCS and MCS are assessed using a T-score methodology (mean = 50, SD = 10), with higher scores indicating improved HRQOL.
2.8. Hamilton Depression Rating Scale Urdu Version (HAM-D-U):
The Hamilton Depression Rating Scale was used in patients who speak Urdu. The HAM-D-U is an effective tool used for determining the magnitude of depression. Internal accuracy (Cronbach alpha 0.71), test–retest reliability, and inter-rater reliability were reasonably good for the Urdu version of the HAM-D (HAM-D-U) [37]. A HAM-D-U score of 0–7 is regarded as normal (or in clinical remission), but a score of 20 or more is deemed severe (showing at least moderate severity).
HAM-D-U scores are interpreted as follows:No depression at all: <8Mild (subthreshold): 8–13Moderate (mild): 14–18Severe (moderate): 19–22Very severe (severe): >23
No depression at all: <8
Mild (subthreshold): 8–13
Moderate (mild): 14–18
Severe (moderate): 19–22
Very severe (severe): >23
2.9. Morisky Levine Greens Adherence Scales (MLGS):
The Morisky Levine Green adherence questionnaire (MLGS) was used to evaluate medication adherence. It is a four-item questionnaire with high reliability and validity that has proven to be particularly useful in chronic conditions such as hypertension, kidney disease, and other cardiovascular diseases. It assesses deliberate and accidental adherence using criteria such as forgetfulness, carelessness, and stopping the drug while feeling better and worse. Each question in the survey was answered with a 0 or 1 response. The total score of the four elements represents the degree of prescription adherence. Medication adherence is indicated by a score of 4, while a score of less than 4 indicates non-adherence. The questionnaire was translated into Urdu using the standard forward–backwards process, yielding a Cronbach alpha of 0.570, within the appropriate range (0.45–0.9) [38,39].
2.10. Study Flow:
The tools were administered in the following manner:
All questionnaires were administered to patients using a self-reporting approach. In order to validate the responses of the patients, researchers conducted random interviews with the patients during their dialysis procedure. The details schematic flow of patient enrollment, data collection and questionnaire administration are displayed Figure 1 and Figure 2.
2.11. Ethical Approval and Consent to Participate:
The Bio-Ethics Committee (BEC) of Quaid-i-Azam University Islamabad permitted this prospective observational study in Tertiary Care Hospitals (BEC-FBS-QAU2017-13). The study was also approved by the Ethical Review Board of Shaheed Zulfikar Ali Bhutto Medical University (1-1/2015/ERB/SZABMU). Informed written consent was taken from all the participants included in this study. The study was conducted per the Declaration of Helsinki and reported according to “The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” guidelines.
2.12. Statistical Analysis:
Primary data were entered into Microsoft Excel 2010, and anonymous patient coding was used to generate secondary data. The data were analysed using the Statistical Package for Social Sciences (SPSS® IBM version 20, Armonk, NY, USA) program. The KDQOL SFTM1.3 scoring program v.2.0 (Santa Monica, CA, USA) was used to compute and calculate PCS and MCS. Continuous variables were expressed in terms of means and standard deviations, whereas categorical variables were expressed in frequencies and percentages. To summarize sample characteristics, descriptive statistics were used.
Moreover, normality tests were assessed with the Shapiro–Wilk test. Data had normal distribution; therefore, continuous data’s mean and standard deviation have been presented. Categorical variables were presented as numbers and percentages. The Mann–Whitney U test was used to compare the depression and HRQOL domains. A p-value of <0.05 was considered statistically significant, and a p-value of < 0.01 was considered highly significant. The multivariate analysis was also performed using Bonferroni Correction. Binary logistic regression was used to examine factors related to medication non-adherence and depression, and effect sizes were reported using odds ratios (ORs) and 95% confidence intervals (CIs). The study variable includes dependent variables (KDQOL Domain and Scale, MCS, PCS, medication adherence, and depression) and independent variables (sociodemographic parameters, laboratory parameters).
3. Result:
3.1. Patient Characteristics During the study recruitment period, 400 patients were enrolled at the treatment sites. Eighty-six patients did not meet the eligibility criteria and were excluded. In total, 314 patients participated in the study and completed KDQOL, HAM-D-U, and MLGS. Figure 2 shows the schematic flow of the study. Of the total 314 patients, 39.8% (n = 125) were CKD grade 4, 9.2% (n = 29) with grade 5 not on hemodialysis yet, and 50.9% (n = 160) were at kidney failure grade 5 on hemodialysis.
The demographic characteristics of the study participants are shown in Table 1. The mean age of the participants was 54.64 ± 15.33. The majority of the survey participants were women, with a significant proportion of the study population from the age group 41–60 years. The number of people with BMI in the normal range was 53.0%. The study population selected through convenience sampling comprised different levels of disease, treatment approaches, and durations.
The comorbidity conditions among the study populations are depicted in Figure 3. Among the population with CKD, diabetes and hypertension were the most prominent underlying comorbid conditions, i.e., 62% and 55%, respectively. Among the study population, 19% had various etiological factors, e.g., smoking, and illicit drug use. Most patients had a family history of kidney disease and a strong history of heart disease. A large group of people also had unknown etiology and represented 30% of the study population.
During the study recruitment period, 400 patients were enrolled at the treatment sites. Eighty-six patients did not meet the eligibility criteria and were excluded. In total, 314 patients participated in the study and completed KDQOL, HAM-D-U, and MLGS. Figure 2 shows the schematic flow of the study. Of the total 314 patients, 39.8% (n = 125) were CKD grade 4, 9.2% (n = 29) with grade 5 not on hemodialysis yet, and 50.9% (n = 160) were at kidney failure grade 5 on hemodialysis.
The demographic characteristics of the study participants are shown in Table 1. The mean age of the participants was 54.64 ± 15.33. The majority of the survey participants were women, with a significant proportion of the study population from the age group 41–60 years. The number of people with BMI in the normal range was 53.0%. The study population selected through convenience sampling comprised different levels of disease, treatment approaches, and durations.
The comorbidity conditions among the study populations are depicted in Figure 3. Among the population with CKD, diabetes and hypertension were the most prominent underlying comorbid conditions, i.e., 62% and 55%, respectively. Among the study population, 19% had various etiological factors, e.g., smoking, and illicit drug use. Most patients had a family history of kidney disease and a strong history of heart disease. A large group of people also had unknown etiology and represented 30% of the study population.
3.2. Laboratory Parameters There were abnormalities in laboratory parameters, and their impact was also analysed in the overall HRQOL of patients. Statistical analysis suggested that hematological and serum iron studies were laboratory parameters that significantly affected the mental composite score (p-Value < 0.05). Contrary to this, the physical composite score was greatly affected by parameters such as hematological serum iron and renal function concentration in the blood, as shown in Table 2.
There were abnormalities in laboratory parameters, and their impact was also analysed in the overall HRQOL of patients. Statistical analysis suggested that hematological and serum iron studies were laboratory parameters that significantly affected the mental composite score (p-Value < 0.05). Contrary to this, the physical composite score was greatly affected by parameters such as hematological serum iron and renal function concentration in the blood, as shown in Table 2.
3.3. Depression In the study, the 21-item Hamilton Depression Rating Scale (HAM-D-U) was used to determine the study population’s pre-study and post-study analysis of depression levels.
It was observed that throughout treatment, there was no educational or counselling support provided to any of the patients. Analysis of the depression data was given by 314 patients in the pre-assessment questionnaire and 244 in the post-assessment questionnaire. A total of 45 patients refused to fill out the post-study HAM-D-U.
The frequency distribution of the depression assessment at baseline found that there was a significantly high depression level in both kidney failure populations (Figure 4). There were only a few cases with a normal-to-moderate level of depression.
Upon the follow-up visit, it was observed that there was a significant increase in depression levels among the study population. Respondents indicated a higher level of depression in both the study populations compared to their previous state. However, further analysis with the Pearson Chi-square test found no significant difference between the baseline and post-study depression levels (p-value > 0.05).
In the study, the 21-item Hamilton Depression Rating Scale (HAM-D-U) was used to determine the study population’s pre-study and post-study analysis of depression levels.
It was observed that throughout treatment, there was no educational or counselling support provided to any of the patients. Analysis of the depression data was given by 314 patients in the pre-assessment questionnaire and 244 in the post-assessment questionnaire. A total of 45 patients refused to fill out the post-study HAM-D-U.
The frequency distribution of the depression assessment at baseline found that there was a significantly high depression level in both kidney failure populations (Figure 4). There were only a few cases with a normal-to-moderate level of depression.
Upon the follow-up visit, it was observed that there was a significant increase in depression levels among the study population. Respondents indicated a higher level of depression in both the study populations compared to their previous state. However, further analysis with the Pearson Chi-square test found no significant difference between the baseline and post-study depression levels (p-value > 0.05).
3.4. Medication Adherence Around 38.8% (n = 122) of the 314 participants scored 4 and had high adherence, while the majority (61.2%, n = 192) belonged to a non-adherent population with a score of less than 4. In addition, the findings revealed no statistically significant relationship between adherence and gender, age, marital status, education level, smoking status or the number of medications (p > 0.05).
Around 38.8% (n = 122) of the 314 participants scored 4 and had high adherence, while the majority (61.2%, n = 192) belonged to a non-adherent population with a score of less than 4. In addition, the findings revealed no statistically significant relationship between adherence and gender, age, marital status, education level, smoking status or the number of medications (p > 0.05).
3.5. HRQOL A total of 314 patients completed the KDQOL-SF-36 questionnaire during a baseline visit. As shown in Table 3, even though patients with grade 4 had a lower level of the physical score as compared to those having grade 5, there were no significant differences seen in the SF-36 PCS scale (31.28 ± 7.91 vs. 36.28 ± 8.41, p = 0.68). However, the overall mental health composite measured by the MCS at baseline was significantly lower for the grade 4 population than the grade 5 population, i.e., 36.66 ± 6.57 and 48.66 ± 5.44, respectively (p-value < 0.05).
For the KDQOL-SF-36 domain, the Mental Health Composite was the factor that affected the study population the most, which showed a statistically significant level in both the study groups (p-value < 0.05). The other major KDQOL-SF-36 domains that impacted both populations were Physical Functioning, Role Limitations-Physical, and Emotional well-being, as well as energy and fatigue evident from Table 3.
In the follow-up visit, 244 patients agreed to respond to the questionnaires. In the post-study, 35 patients with grade 4 and 10 patients with grade 5 did not respond. In the post-analysis, a marked deterioration was seen in KDQOL-SF-36 domains and scale parameters among patients with advanced kidney failure. Grade 4 patients scored significantly lower than grade 5 in the Problem’s list/Symptom, Burden of Kidney Disease, and Mental Health composite domains (p < 0.05). In the KDQOL-SF-36 scales, there was significant deterioration in Physical function, Emotional well-being, and Energy level in patients with grade 5 (p-value < 0.05).
A total of 314 patients completed the KDQOL-SF-36 questionnaire during a baseline visit. As shown in Table 3, even though patients with grade 4 had a lower level of the physical score as compared to those having grade 5, there were no significant differences seen in the SF-36 PCS scale (31.28 ± 7.91 vs. 36.28 ± 8.41, p = 0.68). However, the overall mental health composite measured by the MCS at baseline was significantly lower for the grade 4 population than the grade 5 population, i.e., 36.66 ± 6.57 and 48.66 ± 5.44, respectively (p-value < 0.05).
For the KDQOL-SF-36 domain, the Mental Health Composite was the factor that affected the study population the most, which showed a statistically significant level in both the study groups (p-value < 0.05). The other major KDQOL-SF-36 domains that impacted both populations were Physical Functioning, Role Limitations-Physical, and Emotional well-being, as well as energy and fatigue evident from Table 3.
In the follow-up visit, 244 patients agreed to respond to the questionnaires. In the post-study, 35 patients with grade 4 and 10 patients with grade 5 did not respond. In the post-analysis, a marked deterioration was seen in KDQOL-SF-36 domains and scale parameters among patients with advanced kidney failure. Grade 4 patients scored significantly lower than grade 5 in the Problem’s list/Symptom, Burden of Kidney Disease, and Mental Health composite domains (p < 0.05). In the KDQOL-SF-36 scales, there was significant deterioration in Physical function, Emotional well-being, and Energy level in patients with grade 5 (p-value < 0.05).
3.6. Medication Adherence and HRQOL The PCS of the HRQOL improved over time in adherent individuals and declined in non-adherent individuals. Additionally, after controlling for age, gender, and baseline eGFR, medication non-adherence demonstrated a significant negative correlation with a change in the PCS of the HRQOL, as evident from Table 4. After adjusting for the same covariates, there was no significant correlation between medication non-adherence and a change in the MCS of the HRQOL.
The PCS of the HRQOL improved over time in adherent individuals and declined in non-adherent individuals. Additionally, after controlling for age, gender, and baseline eGFR, medication non-adherence demonstrated a significant negative correlation with a change in the PCS of the HRQOL, as evident from Table 4. After adjusting for the same covariates, there was no significant correlation between medication non-adherence and a change in the MCS of the HRQOL.
3.7. Depression and HRQOL Depressive symptoms were inversely correlated with PCS and MCS scores in the unadjusted longitudinal analysis. Depressive symptoms were independently reduced following multivariable adjustment and negatively correlated with the PCS and MCS scores, as shown in Table 5. When depressive symptoms were considered a dichotomous variable in unadjusted analyses, moderate-to-severe depression symptoms presented negative correlations between the PCS and MCS. In multivariable analyses, the PCS and MCS scores were independently and negatively related to the prevalence of moderate-to-severe depressive symptoms.
Depressive symptoms were inversely correlated with PCS and MCS scores in the unadjusted longitudinal analysis. Depressive symptoms were independently reduced following multivariable adjustment and negatively correlated with the PCS and MCS scores, as shown in Table 5. When depressive symptoms were considered a dichotomous variable in unadjusted analyses, moderate-to-severe depression symptoms presented negative correlations between the PCS and MCS. In multivariable analyses, the PCS and MCS scores were independently and negatively related to the prevalence of moderate-to-severe depressive symptoms.
3.1. Patient Characteristics:
During the study recruitment period, 400 patients were enrolled at the treatment sites. Eighty-six patients did not meet the eligibility criteria and were excluded. In total, 314 patients participated in the study and completed KDQOL, HAM-D-U, and MLGS. Figure 2 shows the schematic flow of the study. Of the total 314 patients, 39.8% (n = 125) were CKD grade 4, 9.2% (n = 29) with grade 5 not on hemodialysis yet, and 50.9% (n = 160) were at kidney failure grade 5 on hemodialysis.
The demographic characteristics of the study participants are shown in Table 1. The mean age of the participants was 54.64 ± 15.33. The majority of the survey participants were women, with a significant proportion of the study population from the age group 41–60 years. The number of people with BMI in the normal range was 53.0%. The study population selected through convenience sampling comprised different levels of disease, treatment approaches, and durations.
The comorbidity conditions among the study populations are depicted in Figure 3. Among the population with CKD, diabetes and hypertension were the most prominent underlying comorbid conditions, i.e., 62% and 55%, respectively. Among the study population, 19% had various etiological factors, e.g., smoking, and illicit drug use. Most patients had a family history of kidney disease and a strong history of heart disease. A large group of people also had unknown etiology and represented 30% of the study population.
3.2. Laboratory Parameters:
There were abnormalities in laboratory parameters, and their impact was also analysed in the overall HRQOL of patients. Statistical analysis suggested that hematological and serum iron studies were laboratory parameters that significantly affected the mental composite score (p-Value < 0.05). Contrary to this, the physical composite score was greatly affected by parameters such as hematological serum iron and renal function concentration in the blood, as shown in Table 2.
3.3. Depression:
In the study, the 21-item Hamilton Depression Rating Scale (HAM-D-U) was used to determine the study population’s pre-study and post-study analysis of depression levels.
It was observed that throughout treatment, there was no educational or counselling support provided to any of the patients. Analysis of the depression data was given by 314 patients in the pre-assessment questionnaire and 244 in the post-assessment questionnaire. A total of 45 patients refused to fill out the post-study HAM-D-U.
The frequency distribution of the depression assessment at baseline found that there was a significantly high depression level in both kidney failure populations (Figure 4). There were only a few cases with a normal-to-moderate level of depression.
Upon the follow-up visit, it was observed that there was a significant increase in depression levels among the study population. Respondents indicated a higher level of depression in both the study populations compared to their previous state. However, further analysis with the Pearson Chi-square test found no significant difference between the baseline and post-study depression levels (p-value > 0.05).
3.4. Medication Adherence:
Around 38.8% (n = 122) of the 314 participants scored 4 and had high adherence, while the majority (61.2%, n = 192) belonged to a non-adherent population with a score of less than 4. In addition, the findings revealed no statistically significant relationship between adherence and gender, age, marital status, education level, smoking status or the number of medications (p > 0.05).
3.5. HRQOL:
A total of 314 patients completed the KDQOL-SF-36 questionnaire during a baseline visit. As shown in Table 3, even though patients with grade 4 had a lower level of the physical score as compared to those having grade 5, there were no significant differences seen in the SF-36 PCS scale (31.28 ± 7.91 vs. 36.28 ± 8.41, p = 0.68). However, the overall mental health composite measured by the MCS at baseline was significantly lower for the grade 4 population than the grade 5 population, i.e., 36.66 ± 6.57 and 48.66 ± 5.44, respectively (p-value < 0.05).
For the KDQOL-SF-36 domain, the Mental Health Composite was the factor that affected the study population the most, which showed a statistically significant level in both the study groups (p-value < 0.05). The other major KDQOL-SF-36 domains that impacted both populations were Physical Functioning, Role Limitations-Physical, and Emotional well-being, as well as energy and fatigue evident from Table 3.
In the follow-up visit, 244 patients agreed to respond to the questionnaires. In the post-study, 35 patients with grade 4 and 10 patients with grade 5 did not respond. In the post-analysis, a marked deterioration was seen in KDQOL-SF-36 domains and scale parameters among patients with advanced kidney failure. Grade 4 patients scored significantly lower than grade 5 in the Problem’s list/Symptom, Burden of Kidney Disease, and Mental Health composite domains (p < 0.05). In the KDQOL-SF-36 scales, there was significant deterioration in Physical function, Emotional well-being, and Energy level in patients with grade 5 (p-value < 0.05).
3.6. Medication Adherence and HRQOL:
The PCS of the HRQOL improved over time in adherent individuals and declined in non-adherent individuals. Additionally, after controlling for age, gender, and baseline eGFR, medication non-adherence demonstrated a significant negative correlation with a change in the PCS of the HRQOL, as evident from Table 4. After adjusting for the same covariates, there was no significant correlation between medication non-adherence and a change in the MCS of the HRQOL.
3.7. Depression and HRQOL:
Depressive symptoms were inversely correlated with PCS and MCS scores in the unadjusted longitudinal analysis. Depressive symptoms were independently reduced following multivariable adjustment and negatively correlated with the PCS and MCS scores, as shown in Table 5. When depressive symptoms were considered a dichotomous variable in unadjusted analyses, moderate-to-severe depression symptoms presented negative correlations between the PCS and MCS. In multivariable analyses, the PCS and MCS scores were independently and negatively related to the prevalence of moderate-to-severe depressive symptoms.
4. Discussion:
The study was conducted in two major cities in Pakistan and is the first-ever follow-up study investigating the point of concern for patients with kidney failure in grade 5 and CKD grade 4. According to the results of this study, targeted patients with kidney failure grade 5 who are not on chronic renal replacement therapy have a similar overall burden of symptoms and depression and a poor HRQOL. The results derived from this research have significant clinical implications for both patients and care providers.
Despite studies showing that patients with kidney failure grade 4 have poor physical and psychosocial health, the clinical, medication, and patient-related factors that cause symptoms, depression, and poor HRQOL in the targeted patient population are unknown. Patients tend to develop symptoms, depression, and poor HRQOL if they lose much weight without losing kidney function. Therefore, it is critical to determine whether this is due to metabolic disturbances, retained uremic contaminants, comorbid medical conditions, anxiety about kidney failure, the possible need for renal replacement therapy in the future or other causes to administer adequate care. Due to the use of different tools for detailed analysis, the results derived in this study have significant implications for kidney failure patients.
Nonadherence to medication did not affect HRQOL at baseline but caused a gradual reduction in physical HRQOL (SF36-PCS) scores, although the mental HRQOL of all individuals decreased significantly during the follow-up period. Studies have shown that baseline eGFR levels influence physical deterioration in HRQOL [40]. However, we could not discover an association between initial eGFR and alterations in HRQOL over time in the current study.
It was also observed that the physical and mental health composite tends to deteriorate in patients with kidney failure. Previous studies reported that patients with moderate-to-progressing kidney disease had a lower PCS than MCS [27]. A previous study on kidney failure grade 1–4 patients reported that the PCS and MCS scores were 32.1 ± 8.1 and 40.6 ± 11.1, respectively. In the current study, the severity of the disease, particularly in individuals with kidney failure, had a considerable impact on HRQOL. This link has also been discovered in other investigations where HRQOL scores are compromised in patients with intermediate kidney failure and deteriorate over time in dialysis patients [9,41].
A study by Ajeebi and his colleagues showed that patients with advanced kidney failure could likely be unaware of the effects of chronic renal replacement therapy on their physical and mental health [42]. Following this school of thought, it is evident that kidney failure patients need chronic dialysis for a drastic lifestyle change [43]. Based on the current study findings, there is evidence that patients with kidney failure experienced a significant difference in their physical and psychosocial well-being during the transition period of the disease.
However, in the case of patients with kidney failure, complaints of sleep problems, muscle cramps, dry mouth, and lightheadedness are the most frequent. Although these discrepancies did not reach statistical significance after several comparisons, the biologi-cal plausibility of such differences warrants further investigation. In light of the results, it is evident that patients with kidney failure have lower scores on the SF-36 physical func-tion subscale. However, there were no variations in PCS scores due to this observation. As per the study referred to, this provides preliminary information on the subdomains of HRQOL that may differ between these two populations [24]. In a study conducted on the patients undergoing kidney transplantation and those on hemodialysis or peritoneal dialysis, the findings provided evidence that there was a significant improvement in HRQOL after one year of kidney replacement and dialysis [44]. The comparison between the current results and the existing literature provides evidence that patients with kidney failure are equally treated for health issues, including anemia, depression, sleeplessness, and bone and muscle diseases, that lead to the deterioration of their quality of life. Therefore, in the context of grade 5 kidney failure, patients should be informed about the unpredictability of their disease as it is difficult to anticipate the disease trajectory based on known predictive factors of mortality and the advantages and disadvantages of dialysis treatments. According to a study by Nataatmadja et al. (2021), of 3000 patients with an average age of 73 years, 60% regretted starting dialysis rather than having opted for conservative treatment [4]. As far as possible, the decision to initiate dialysis in patients with grade 5 kidney failure must be made personally between the patient, family members, the attending physician, and the referring nephrologist.
Numerous studies have shown that dialysis has a detrimental effect on depression and that patients who experience severe depression are more likely to die. According to 15 large-scale investigations, there is an important link between depression and mortality among dialysis patients. Several longitudinal studies that evaluated the recurrent measurement of depression found that patients receiving dialysis therapy had significantly greater mortality risks when depressive symptoms were present. According to studies, early dialysis therapy initiation is associated with depression [45,46,47].
The general demographic features of our sample of grade 4 kidney failure were close to those of the United States population of grade 4 chronic kidney disease. On the contrary, the study’s kidney failure cohort had a higher proportion of men than the total population of kidney failure patients. The result supported the study mentioned above, as it was discovered that the depression rate was observed to worsen with time; a linear increase was identified from the start of the study until the second administration of the questionnaire 6 months later. Possible causes for this finding are likely to be lifelong dialysis therapy with at least 3 dialysis operations per week, patients taking too many medications at once, the economic strain on patients and their families, and changed familial and social ties. In a group of chronic HD patients, depression symptoms increased linearly and a link was discovered between poor sleep quality, unemployment, pruritus, hypoalbuminemia, diabetes, and depressive symptoms [48].
The findings of the current study and the prior research provided evidence that patients suffering from kidney failure are likely to develop mental health issues that the prolonged effects of illness could cause.
Chronic renal diseases are frequent in the population but often remain challenging to demonstrate clinically, as their symptoms are crude [49]. It is thus not uncommon for patients to develop end-stage renal failure, having presented very few symptoms or only vague and not very specific symptoms (fatigue, inappetence, etc.). In extensive population studies, it was found that only 23% of people with kidney failure were aware of their diagnosis [50]. According to the survey by Live and Zhang (2019), kidney failure is, in most cases, secondary to hypertension, diabetes, immunological disease, etc. Nevertheless, there are several kidney diseases that, although rare, could be diagnosed early, thanks to typical clinical and biological signs (red flags). However, even these diseases are often underdiagnosed, further prolonging the diagnostic error of these patients and their early management [51,52,53].
In addition, the treatment of hemodialysis patients includes all possible measures to reduce the impact of symptoms encountered in kidney failure [48]. These symptoms are pain, pruritus, nausea, headache, fatigue, anxiety, and depression. A total of 80% of patients suffered from chronic kidney pain and went to dialysis due to underlying mental health complications, especially depression. It has been stated in the study of Duane et al. that diseases affecting the kidneys are frequent but remain asymptomatic for a long time, leading to a diagnostic delay with consequences for the management of the patient [53]. However, convincing warning signs, whether cutaneous, articular or related to electrolyte abnormalities, should put the chip in the ear and guide the practitioner to the renal origin of the pathology. Therefore, certain rare diseases affecting the kidneys are discussed here based on clinical vignettes, emphasizing the biological and clinical signs that lead to their diagnosis [54].
Moreover, mental health depends on individual, genetic, and hormonal factors, but also relational, community, and societal factors. Patients of kidney failure of all ages suffer from certain mental disorders more frequently, with more comorbidities. It also impacts their physical health and family and social balance. Specificities exist at the clinical level, as well as concerning the use of psychotropic drugs, their side effects, and adherence to care. The primary care physician’s role is essential for preventing and detecting mental disorders in kidney failure patients for their psychological support, guidance, and follow-up [41,55].
The current study has some limitations. The study only has a 6-month follow-up period; the findings should be interpreted cautiously. Although the variables have been adjusted for changes in physical activity, the possibility of residual confounders cannot be ruled out as one of the typical limitations of all cohort studies. For example, the health consciousness of an individual eating healthier also makes other more beneficial lifestyle changes that the questionnaire could not completely capture. A multicenter investigation with a large sample size and an extended follow-up period is required to corroborate the findings of the current study. Furthermore, future research in this domain needs to consider the comparison of mentioned health-related domains in a much more comprehensive sample of patients with kidney failure, including those with severe comorbid illnesses. In this study, which investigated several variables associated with low HRQOL in patients with kidney failure, depression significantly predicted a lower HRQOL. Therefore, our study emphasizes the potential need for randomized controlled trials in future research to determine the temporal relationship between low HRQOL and depression in kidney failure patients and to further assess the impact of early identification of depression and appropriate treatment of depression on the health outcomes of these patients. Despite these drawbacks, our study’s strength is that we performed thorough analyses to look at factors associated with HRQOL in patients with kidney failure, including medication adherence, depression, and modifiable and nonmodifiable clinical features.
5. Conclusions:
The patients on maintenance dialysis for kidney failure have significant burdens of physical and mental symptoms, depression, and low HRQOL. Given the substantial and well-known declines in physical and psychological well-being among patients with kidney failure receiving hemodialysis, findings drawn in this research imply that these health-related areas should receive special attention in the vast and expanding population of patients with kidney failure.
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Background:
Kidney failure is a global health problem with a worldwide mean prevalence rate of 13.4%. Kidney failure remains symptomless during most of the early stages until symptoms appear in the advanced stages. Kidney failure is associated with a decrease in health-related quality of life (HRQOL), deterioration in physical and mental health, and an increased risk of cardiovascular morbidity and mortality. This study aimed to evaluate the factors associated with decreased HRQOL and other factors affecting the overall health of patients. Another objective was to measure how medication adherence and depression could affect the overall HRQOL in patients with kidney failure.
Methods:
The study used a prospective follow-up mix methodology approach with six-month follow-ups of patients. The participants included in the study population were those with chronic kidney disease grade 4 and kidney failure. Pre-validated and translated questionnaires (Kidney Disease Quality of Life-Short Form, Hamilton Depression Rating Scale Urdu Version, and Morisky Lewis Greens Adherence Scale) and assessment tools were used to collect data.
Results:
This study recruited 314 patients after an initial assessment based on inclusion criteria. The mean age of the study population was 54.64 ± 15.33 years. There was a 47.6% male and a 52.4% female population. Hypertension and diabetes mellitus remained the most predominant comorbid condition, affecting 64.2% and 74.6% of the population, respectively. The study suggested a significant (p < 0.05) deterioration in the mental health composite score with worsening laboratory variables, particularly hematological and iron studies. Demographic variables significantly impact medication adherence. HRQOL was found to be deteriorating with a significant impact on mental health compared to physical health.
Conclusions:
Patients on maintenance dialysis for kidney failure have a significant burden of physical and mental symptoms, depression, and low HRQOL. Given the substantial and well-known declines in physical and psychological well-being among kidney failure patients receiving hemodialysis, the findings of this research imply that these areas related to health should receive special attention in the growing and expanding population of kidney failure patients.
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1. Introduction:
Chronic kidney failure is a public health problem worldwide and can lead to conditions such as kidney failure and cardiovascular problems [1]. In different regions of the world, kidney disease has already reached an epidemic rate in people aged 20 years or above who are the most affected by this disease. The total number of men and women with kidney failure was 225.7 million and 271.8 million, respectively, by 2010 [2]. According to new research, the global prevalence of kidney failure was 9.1% in 2017 (697.5 million cases). Women and girls had a higher global majority of kidney failure than men and boys (9.5%) (7.3%) [3]. About a third of all kidney failure cases were in China (132.3 million) and India (115.1 million), with 10 countries having more than 10 million cases and 79 countries having more than 1 million cases [3,4].
Considering the high incidence of kidney failure and the challenges posed by the patients undergoing dialysis, rehabilitating one’s life is crucial. Patients suffer from different emotional and physical challenges during kidney failure [5]. Survival is not only the goal of placing the patient on a strenuous dialysis procedure but also improving the patient’s health-related quality of life (HRQOL). Understanding the deterioration of HRQOL is the most crucial aspect that can affect burden and mortality in these patients [6,7]. In patients with chronic kidney disease (CKD), including those with end-stage kidney disease (ESKD), monitoring the functional status and subjective state of well-being of a patient as they relate to a health condition, collectively known as HRQOL measurements, is of particular importance.
HRQOL has been significantly affected among patients with advanced chronic kidney disease and kidney failure. The fact that patients with CKD of various racial and ethnic backgrounds have varying HRQOL scores confirms the need to individualise the notion of HRQOL to evaluate critical aspects of life in patients and incorporate these domains into an all-encompassing plan of care. The urgency of HRQOL measurement and the need to advance our multidimensional tools with technology and a more patient-centered view of HRQOL is highlighted by these recent findings [8,9].
In dialysis patient populations, low HRQOL scores measured utilizing the Medical Outcomes Study Short Form-36 (SF-36) were associated with hospitalization and death in studies [10,11,12,13]. There were substantial HRQOL studies conducted before in patients with kidney failure. However, little has been done in Pakistan. Walters et al. assessed HRQOL at the beginning of dialysis therapy and found that in patients who began hemodialysis therapy (HD), HRQOL scores were significantly lower than in established long-term HD patients [14,15,16,17,18,19].
The Dialysis Outcomes and Practice Patterns Study (DOPPS) found a strong link between depressive symptoms and mortality and hospitalization rates [20]. On the contrary, the Choices for Healthy Outcomes in Caring for End-Stage Renal Disease (CHOICE) study found a link between high depressive symptoms and an increased risk of cardiovascular events [21]. However, this link between depression and clinical outcomes has not been thoroughly investigated in the early stages of kidney failure. A recent study by Wirkner et al. investigated an association between comorbid conditions and diabetes. The study concluded that advanced stages of CKD have significantly affected the overall HRQOL of the patients. Patients with kidney failure and undergoing dialysis were also found to have lower physical composite scores [22].
Therefore, it is crucial to assess all such parameters while dealing with a patient’s critical condition with kidney failure. This study was carried out to emphasize the use of tools to measure the HRQOL of patients and treatment outcomes [23,24,25,26]. Patient perceptions and feelings regarding their functionality and well-being are captured through patient-centered outcomes, such as HRQOL. In patients with CKD grade 4, outcome indicators are fundamental because they guide treatment objectives and strategies. However, there is little information on patient-centered outcomes in humans, especially in people who do not receive kidney replacement therapy and have CKD grade 4. More critically, there is a dearth of research in this patient population on the association between HRQOL and drug-related characteristics such as medication burden and adherence [27,28].
Previous studies have found that drug adherence rates range from 33.0 to 87.7%. Seng et al. investigated medication adherence in kidney failure. A pooled medication adherence rate of 67.4% (95% confidence interval (CI) 61.4–73.3%) was found in a meta-analysis of 54,652 patients. Between prospective and retrospective studies, the prevalence of adherence to medication among patients with dialysis kidney failure was similar (68.8%; 95% CI 61.1–76.6%) vs. (65.8%; 95% CI 57.0–74.6%) [28,29,30]. According to the study reported in Pakistan, 74% of patients with kidney failure experienced pruritus, negatively impacting their quality of life. Poor quality of life affects patients with mild to severe CKD-related pruritus. The hemodialysis patients had a lower HRQOL score as their pruritus intensity increased [31].
In Pakistan, kidney failure is the most common cause of morbidity and mortality [32]. According to the 2016 Pakistan National Kidney Federation Registry, 5935 patients were admitted to different dialysis units across the country, totaling 891 hemodialysis machines [33]. The data available have certain limitations as many large dialysis centers are not contributing their data to the registry.
There is a significant upsurge in the prevalence of kidney failure worldwide, particularly in low- to middle-income countries [34]. It is essential to evaluate the factor that could impact the quality of treatment and design a patient support program to increase patients’ involvement in the self-management of the disease condition. This study aimed to evaluate the factors associated with reduced HRQOL and other factors that affect the overall health of patients. Another objective was to measure how medication adherence and depression could impact the overall HRQOL of patients with kidney failure.
5. Conclusions:
The patients on maintenance dialysis for kidney failure have significant burdens of physical and mental symptoms, depression, and low HRQOL. Given the substantial and well-known declines in physical and psychological well-being among patients with kidney failure receiving hemodialysis, findings drawn in this research imply that these health-related areas should receive special attention in the vast and expanding population of patients with kidney failure.
|
Background:
Kidney failure is a global health problem with a worldwide mean prevalence rate of 13.4%. Kidney failure remains symptomless during most of the early stages until symptoms appear in the advanced stages. Kidney failure is associated with a decrease in health-related quality of life (HRQOL), deterioration in physical and mental health, and an increased risk of cardiovascular morbidity and mortality. This study aimed to evaluate the factors associated with decreased HRQOL and other factors affecting the overall health of patients. Another objective was to measure how medication adherence and depression could affect the overall HRQOL in patients with kidney failure.
Methods:
The study used a prospective follow-up mix methodology approach with six-month follow-ups of patients. The participants included in the study population were those with chronic kidney disease grade 4 and kidney failure. Pre-validated and translated questionnaires (Kidney Disease Quality of Life-Short Form, Hamilton Depression Rating Scale Urdu Version, and Morisky Lewis Greens Adherence Scale) and assessment tools were used to collect data.
Results:
This study recruited 314 patients after an initial assessment based on inclusion criteria. The mean age of the study population was 54.64 ± 15.33 years. There was a 47.6% male and a 52.4% female population. Hypertension and diabetes mellitus remained the most predominant comorbid condition, affecting 64.2% and 74.6% of the population, respectively. The study suggested a significant (p < 0.05) deterioration in the mental health composite score with worsening laboratory variables, particularly hematological and iron studies. Demographic variables significantly impact medication adherence. HRQOL was found to be deteriorating with a significant impact on mental health compared to physical health.
Conclusions:
Patients on maintenance dialysis for kidney failure have a significant burden of physical and mental symptoms, depression, and low HRQOL. Given the substantial and well-known declines in physical and psychological well-being among kidney failure patients receiving hemodialysis, the findings of this research imply that these areas related to health should receive special attention in the growing and expanding population of kidney failure patients.
| 11,245 | 396 | 24 |
[
"patients",
"study",
"kidney",
"depression",
"failure",
"kidney failure",
"hrqol",
"grade",
"adherence",
"population"
] |
[
"test",
"test"
] | null | null |
[CONTENT] kidney failure | health-related quality of life (HRQOL) | medication adherence | hemodialysis | depression [SUMMARY]
| null | null |
[CONTENT] kidney failure | health-related quality of life (HRQOL) | medication adherence | hemodialysis | depression [SUMMARY]
|
[CONTENT] kidney failure | health-related quality of life (HRQOL) | medication adherence | hemodialysis | depression [SUMMARY]
|
[CONTENT] kidney failure | health-related quality of life (HRQOL) | medication adherence | hemodialysis | depression [SUMMARY]
|
[CONTENT] Humans | Male | Female | Adult | Middle Aged | Aged | Quality of Life | Prevalence | Depression | Prospective Studies | Medication Adherence | Renal Insufficiency, Chronic [SUMMARY]
| null | null |
[CONTENT] Humans | Male | Female | Adult | Middle Aged | Aged | Quality of Life | Prevalence | Depression | Prospective Studies | Medication Adherence | Renal Insufficiency, Chronic [SUMMARY]
|
[CONTENT] Humans | Male | Female | Adult | Middle Aged | Aged | Quality of Life | Prevalence | Depression | Prospective Studies | Medication Adherence | Renal Insufficiency, Chronic [SUMMARY]
|
[CONTENT] Humans | Male | Female | Adult | Middle Aged | Aged | Quality of Life | Prevalence | Depression | Prospective Studies | Medication Adherence | Renal Insufficiency, Chronic [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null | null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] patients | study | kidney | depression | failure | kidney failure | hrqol | grade | adherence | population [SUMMARY]
| null | null |
[CONTENT] patients | study | kidney | depression | failure | kidney failure | hrqol | grade | adherence | population [SUMMARY]
|
[CONTENT] patients | study | kidney | depression | failure | kidney failure | hrqol | grade | adherence | population [SUMMARY]
|
[CONTENT] patients | study | kidney | depression | failure | kidney failure | hrqol | grade | adherence | population [SUMMARY]
|
[CONTENT] kidney | hrqol | patients | kidney failure | failure | million | patient | outcomes | dialysis | found [SUMMARY]
| null | null |
[CONTENT] patients kidney | patients kidney failure | kidney failure | failure | kidney | patients | physical | receiving hemodialysis | receive special attention vast | imply [SUMMARY]
|
[CONTENT] study | patients | kidney | depression | hrqol | failure | kidney failure | adherence | grade | population [SUMMARY]
|
[CONTENT] study | patients | kidney | depression | hrqol | failure | kidney failure | adherence | grade | population [SUMMARY]
|
[CONTENT] Kidney | 13.4% ||| Kidney ||| Kidney ||| HRQOL ||| HRQOL [SUMMARY]
| null | null |
[CONTENT] HRQOL ||| [SUMMARY]
|
[CONTENT] Kidney | 13.4% ||| Kidney ||| Kidney ||| HRQOL ||| HRQOL ||| six-month ||| 4 ||| Kidney Disease Quality of Life-Short Form | Hamilton Depression | Morisky Lewis | Adherence Scale ||| ||| 314 ||| 54.64 | 15.33 years ||| 47.6% | 52.4% ||| 64.2% | 74.6% ||| p < | 0.05 ||| ||| ||| HRQOL ||| [SUMMARY]
|
[CONTENT] Kidney | 13.4% ||| Kidney ||| Kidney ||| HRQOL ||| HRQOL ||| six-month ||| 4 ||| Kidney Disease Quality of Life-Short Form | Hamilton Depression | Morisky Lewis | Adherence Scale ||| ||| 314 ||| 54.64 | 15.33 years ||| 47.6% | 52.4% ||| 64.2% | 74.6% ||| p < | 0.05 ||| ||| ||| HRQOL ||| [SUMMARY]
|
||||
Clonal spread of carbapenem non-susceptible Acinetobacter baumannii in an intensive care unit in a teaching hospital in China.
|
23130340
|
This study was aimed to investigate the genetic diversity and antibiotic resistance profile of the nosocomial infection agent Acinetobacter baumannii from a medical intensive care unit (ICU) in a teaching hospital in Suzhou, China.
|
BACKGROUND
|
The genetic relationship among A. baumannii isolates in an ICU was investigated using multilocus sequence typing (MLST). The antibiotic resistance pattern was determined by performing an antibiotic susceptible test, which included an agar dilution method and an E-test method. Resistant determinants, e.g., carbapenemase genes, metallo-β-lactamases, and class 1 integron, were analyzed by specific PCR and DNA sequencing.
|
METHODS
|
In the present study, 33 non-duplicate isolates were identified as 5 existing sequence types (STs) (ST92, ST75, ST112, ST145, and ST345) and 1 new sequence type STn, which has a G-A mutation at nt268 on ropD40 of ST251. These results reveal limited diversity in carbapenem non-susceptible A. baumannii (CNSAb) isolates in our ICU, which are comprised of only 2 distinct STs, with ST92 and ST75 clustering into a clonal complex (CC) 92. Most CNSAb isolates (94.4%, 17/18) harbored the OXA-23 gene, while no carbapenem-susceptible A. baumannii (CSAb) isolates harbored it. In addition, 66.7% (22/33) isolates were positive for class 1 integrase, and gene cassette analysis showed there are 3 gene arrays among them, i.e., aacA4-catB8-aadA1 (77.3%, 17/22), aacA4 (22.7%, 5/22), and aacC1-orfX-orfX'-aadA1 (4.5%, 1/22).
|
RESULTS
|
When all these data are combined, the antibiotic resistance and wide distribution of CNSAb isolates in our ICU are probably caused by expansion of the CC92 clone.
|
CONCLUSIONS
|
[
"Acinetobacter baumannii",
"Anti-Bacterial Agents",
"Bacterial Proteins",
"Carbapenems",
"China",
"Drug Resistance, Bacterial",
"Hospitals, Teaching",
"Humans",
"Integrases",
"Intensive Care Units",
"Microbial Sensitivity Tests",
"Mutation",
"Polymerase Chain Reaction",
"Sequence Analysis, DNA",
"beta-Lactamases"
] |
3486935
|
INTRODUCTION
|
Acinetobacter baumannii is an opportunistic human pathogen associated with an increasing incidence of nosocomial infections, including bacteremia, urinary tract infections, and ventilator-associated pneumonia [1-3]. Most A. baumannii isolates have a multidrug resistant phenotype (MDRAb) with an increasing prevalence in the intensive care unit (ICU) [2, 4]. Although carbapenem is a common clinical choice to treat MDRAb infections, the resistance rate has increased dramatically over the past decade. Carbapenem resistance is caused by the production of carbapenemases, including class D β-lactamases (OXA-type carbapenemases) and class B metallo-β-lactamases (MBLs) as previously described [5-8]. The OXA-type carbapenemases are comprised of 4 kinds of genes: blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and an intrinsic blaOXA-51-like type as previously reported [9, 10]. In addition, ISAba1, an insertion sequence (IS), was found in A. baumannii isolates, which can enhance the expression of OXA-type carbapenemase genes and mobilize these genes among the strains [11].
Integron is a widely studied vehicle for gene capture, which is highly related to antibiotic resistant gene dissemination [12, 13]. Among MDRAb isolates, the class 1 integron is commonly detected, which infers a high association with carbapenem resistance. The integron structure contains an integrase, followed by an attI site for the integration of cassettes and the recognition of integrase, and a promoter to drive its expression. It can appear on a plasmid or chromosome, and can be classified into different types (class 1, class 2, and class 3 integron) on the basis of their integrases. The most classical integron, class 1 integron, has 2 conserved terminal sequences. Therefore, the primer set (5'-conserved sequence [CS] and 3'-CS), which targets 2 terminal sequences in the integron, is commonly used for integron cassette analysis.
Multi locus sequence typing (MLST) is an unambiguous typing method for identifying accurate and portable nucleotide sequences of internal fragments of multiple housekeeping genes. Recently, some studies [14-16] revealed that the clonal complex (CC) 92 in the carbapenem non-susceptible A. baumannii (CNSAb) isolate has a worldwide dissemination. The ST92 A. baumannii isolate is the largest clone and is predicted to be the founder of this clonal complex. Molecular epidemiological surveillance not only provides a better understanding of nosocomial infections in certain hospitals, but also provides a chance to understand the route of global dissemination. However, the genetic diversity and molecular characteristics of A. baumannii in our region remain unclear. Therefore, in order to better understand the epidemiological characteristics of A. baumannii in the ICU and the relationship of carbapenem resistance with integrons, in this study, we performed MLST typing and a molecular investigation of antibiotic resistance determinants in A. baumannii isolates collected in our medical ICU.
|
METHODS
|
1. Bacterial strains and antimicrobial susceptibility test In total, 33 non-duplicated clinical A. baumannii isolates were collected by standard isolation methods between January and December 2009 in our medical ICU. All isolates were identified by conventional methods and further identified by 16S-23S rRNA gene spacer region analysis as described previously [17]. All of these isolates were stored at -80℃ in brain-heart infusion broth containing 50% (v/v) glycerol before inclusion in this study.
Antimicrobial susceptibility of isolates to cefepime, ceftazidime, piperacillin, amikacin, gentamicin, ciprofloxacin, ampicillin/sulbactam, and piperacillin/tazobactam were determined by using the disc diffusion method. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by using the agar dilution method, and that of colistin was determined by using the E-test (AB bioMérieux, Marcy-l'Etoile, France). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for the antimicrobial susceptibility test. The results were interpreted according to the manufacturer's instructions and CLSI guidelines [18].
In total, 33 non-duplicated clinical A. baumannii isolates were collected by standard isolation methods between January and December 2009 in our medical ICU. All isolates were identified by conventional methods and further identified by 16S-23S rRNA gene spacer region analysis as described previously [17]. All of these isolates were stored at -80℃ in brain-heart infusion broth containing 50% (v/v) glycerol before inclusion in this study.
Antimicrobial susceptibility of isolates to cefepime, ceftazidime, piperacillin, amikacin, gentamicin, ciprofloxacin, ampicillin/sulbactam, and piperacillin/tazobactam were determined by using the disc diffusion method. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by using the agar dilution method, and that of colistin was determined by using the E-test (AB bioMérieux, Marcy-l'Etoile, France). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for the antimicrobial susceptibility test. The results were interpreted according to the manufacturer's instructions and CLSI guidelines [18].
2. Carbapenemase genes and class 1 integron analysis Genomic DNA from A. baumannii was extracted using a genomic DNA extraction Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. MBL-producing isolates were screened using the imipenem-EDTA double-disk synergy test [19]. Metallo-β-lactamase (IPM4)-producing A. baumannii was used as a positive control. The genes encoding carbapenemases blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like were analyzed by PCR using primers as described previously [5-8]. The location of blaOXA-23-like and blaOXA-51-like with an insert element (ISAba1) was screened by PCR [11].
Integron analysis was conducted by using PCR with the class 1 integrase (int1 gene) as the targeting gene, as previously described [12]. Gene cassettes in class 1 integrase-positive isolates were performed by amplifying the sequence from the 5'-CS to the 3'-CS region [12]. The PCR products were purified and sequenced, either directly by using the PCR product, or by cloning into a pGEM®-T vector (Promega, Madison, WI, USA). All sequencing reactions were performed using an ABI prism sequencer 3730 (Applied Biosystems, Foster City, CA, USA), and the sequence data were analyzed by using BLAST at the NCBI website (http://www.ncbi.nlm.nih.gov).
Genomic DNA from A. baumannii was extracted using a genomic DNA extraction Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. MBL-producing isolates were screened using the imipenem-EDTA double-disk synergy test [19]. Metallo-β-lactamase (IPM4)-producing A. baumannii was used as a positive control. The genes encoding carbapenemases blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like were analyzed by PCR using primers as described previously [5-8]. The location of blaOXA-23-like and blaOXA-51-like with an insert element (ISAba1) was screened by PCR [11].
Integron analysis was conducted by using PCR with the class 1 integrase (int1 gene) as the targeting gene, as previously described [12]. Gene cassettes in class 1 integrase-positive isolates were performed by amplifying the sequence from the 5'-CS to the 3'-CS region [12]. The PCR products were purified and sequenced, either directly by using the PCR product, or by cloning into a pGEM®-T vector (Promega, Madison, WI, USA). All sequencing reactions were performed using an ABI prism sequencer 3730 (Applied Biosystems, Foster City, CA, USA), and the sequence data were analyzed by using BLAST at the NCBI website (http://www.ncbi.nlm.nih.gov).
3. MLST MLST was performed on A. baumannii as described by Bartual et al. [20]. Briefly, 7 housekeeping genes, i.e., gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, were amplified, followed by sequencing on the ABI prism sequencer 3730 (Applied Biosystems). The sequences of these 7 housekeeping genes were analyzed using the Pubmlst database (http://pubmlst.org/abaumannii/). The sequence type (ST) was designated according to the allelic profiles in the database. The eBURST algorithm (version 3; http://eburst.mlst.net/) was used to assign STs to CCs and to assess the genetic relationship with definition of the groups sharing alleles at ≥6 of 7 loci [21]. The CC comprises a founding ST as a common ancestor and several other closely related STs descending from the predicted founding genotype.
MLST was performed on A. baumannii as described by Bartual et al. [20]. Briefly, 7 housekeeping genes, i.e., gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, were amplified, followed by sequencing on the ABI prism sequencer 3730 (Applied Biosystems). The sequences of these 7 housekeeping genes were analyzed using the Pubmlst database (http://pubmlst.org/abaumannii/). The sequence type (ST) was designated according to the allelic profiles in the database. The eBURST algorithm (version 3; http://eburst.mlst.net/) was used to assign STs to CCs and to assess the genetic relationship with definition of the groups sharing alleles at ≥6 of 7 loci [21]. The CC comprises a founding ST as a common ancestor and several other closely related STs descending from the predicted founding genotype.
4. Statistical analysis Resistance rates for each antibiotic were compared between CNSAb and carbapenem-susceptible A. baumannii (CSAb), ST92-CNSAb and ST92-CSAb, and the int1-positive group and int1-negative group, using Fisher's exact test. A P value <0.05 was considered as significant difference.
Resistance rates for each antibiotic were compared between CNSAb and carbapenem-susceptible A. baumannii (CSAb), ST92-CNSAb and ST92-CSAb, and the int1-positive group and int1-negative group, using Fisher's exact test. A P value <0.05 was considered as significant difference.
|
RESULTS
|
1. Characteristics of A. baumannii isolates In this study, 33 non-duplicate A. baumannii isolates were isolated from patients hospitalized in our medical ICU. All of these isolates were confirmed by 16S-23S rRNA internal spacer analysis as previously described [17]. Among these isolates (26 from lower respiratory tract, 4 from urinary tract, and 2 from blood, and 1 from pus), 54.5% (18/33) isolates were resistant to either imipenem, meropenem, or both. The resistance rate of colistin was 3% (1/33), which coincides with the observation that colistin is the most effective antibiotic to treat A. baumannii infections in our area. From a nationwide surveillance conducted during 2007, the resistance rates of imipenem and meropenem in China were calculated to be 40% and 35%, respectively [22]. However, from a new surveillance report generated in 2010, the prevalence of CNSAb isolates was dramatically increased to 80% [23]. Therefore, the CNSAb isolate is becoming a major concern as a nosocomial infection in China.
In this study, 33 non-duplicate A. baumannii isolates were isolated from patients hospitalized in our medical ICU. All of these isolates were confirmed by 16S-23S rRNA internal spacer analysis as previously described [17]. Among these isolates (26 from lower respiratory tract, 4 from urinary tract, and 2 from blood, and 1 from pus), 54.5% (18/33) isolates were resistant to either imipenem, meropenem, or both. The resistance rate of colistin was 3% (1/33), which coincides with the observation that colistin is the most effective antibiotic to treat A. baumannii infections in our area. From a nationwide surveillance conducted during 2007, the resistance rates of imipenem and meropenem in China were calculated to be 40% and 35%, respectively [22]. However, from a new surveillance report generated in 2010, the prevalence of CNSAb isolates was dramatically increased to 80% [23]. Therefore, the CNSAb isolate is becoming a major concern as a nosocomial infection in China.
2. MLST and clonal relationship of A. baumannii isolates According to the MLST, 33 A. baumannii isolates can be grouped into 5 existing STs (ST92, 1-3-3-2-2-7-3; ST75, 1-3-3-2-2-11-3; ST112, 1-12-56-36-4-61-26; ST145, 21-35-2-28-1-52-4; ST345, 46-12-110-1-16-141-50) and 1 new ST, which has a G-A mutation at nt268 on the rpoD40 loci of ST251(32-48-54-35-1-11-40) (Table 1). All CNSAb isolates comprised of ST92 and ST75 only, accounting for 77.8% (14/18) and 22.2% (4/18), respectively.
Clonal relation analysis showed that both ST92 and ST75 belong to the CC92, which is a globally disseminated CC for A. baumannii (Fig. 1). This result suggests that CC92 is the major clone that spreads in the ICU of our hospital. eBURST analysis [21] showed that ST92 is the founder of CC92. Previous studies [14, 15, 24] showed that several STs exist, e.g., ST75, ST88, ST90, ST136, ST137, and ST138, which are SLVs of the founder ST92. However, we still found other types, e.g., ST381 and ST375, which are double locus variants (DLVs) of ST92.
According to the MLST, 33 A. baumannii isolates can be grouped into 5 existing STs (ST92, 1-3-3-2-2-7-3; ST75, 1-3-3-2-2-11-3; ST112, 1-12-56-36-4-61-26; ST145, 21-35-2-28-1-52-4; ST345, 46-12-110-1-16-141-50) and 1 new ST, which has a G-A mutation at nt268 on the rpoD40 loci of ST251(32-48-54-35-1-11-40) (Table 1). All CNSAb isolates comprised of ST92 and ST75 only, accounting for 77.8% (14/18) and 22.2% (4/18), respectively.
Clonal relation analysis showed that both ST92 and ST75 belong to the CC92, which is a globally disseminated CC for A. baumannii (Fig. 1). This result suggests that CC92 is the major clone that spreads in the ICU of our hospital. eBURST analysis [21] showed that ST92 is the founder of CC92. Previous studies [14, 15, 24] showed that several STs exist, e.g., ST75, ST88, ST90, ST136, ST137, and ST138, which are SLVs of the founder ST92. However, we still found other types, e.g., ST381 and ST375, which are double locus variants (DLVs) of ST92.
3. Genetic determinants in CNSAb isolates No blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-24-like, and blaOXA-58-like genes were observed among these isolates, and no MBLs producing these isolates were detected by the EDTA double-disk synergy test. Most (94.4%, 17/18) ST92-CNSAb isolates harbor the blaOXA-23 gene, as shown in Table 2. Interestingly, there was 1 ST92-CSAb isolate that could be detected as being positive for the blaOXA-23 gene. However, ISAba1
blaOXA-23 gene mapping showed that no ISAba1 gene could be detected in this isolate. Therefore, this result confirmed the importance of the ISAba1 gene in A. baumannii isolates, which exhibit the carbapenem resistance phenotype. ISAba1 was detected in most (88.2%, 15/17) blaOXA-23-positive isolates, which are located upstream of blaOXA-23. The blaOXA-51-like gene was detected in all isolates in this study, which is consistent with the blaOXA-51-like gene as an intrinsic carbapenemase. However, there were no ISAba1 genes located with the blaOXA-51-like gene as determined by PCR mapping in CSAb isolates. All blaOXA-51-like genes were confirmed as blaOXA-66 genes by sequencing of CNSAb isolates.
Integron analysis revealed that all CNSAb isolates have a class 1 integrase (Table 2). When comparing the antibiotic resistance profiles (Table 1), most antibiotics showed resistance rates that were much higher in the int1-positive isolates than in the negative ones. Integron gene cassette analysis showed that there were 3 distinct antibiotic gene arrays, i.e., aacA4-catB8-aadA1 (77.3%, 17/22; Genebank accession no. EU340419), aacA4 (22.7%, 5/22; Genebank accession no. EU523056), and aacC1-orfX-orfX'-aadA1 (4.5%, 1/22; Genebank accession no. AY307113). Functional analysis showed that these genes were only associated with aminoglycoside and chloramphenicol resistance, which are not related to carbapenem resistance. It seems that carbapenem-resistant genes might not be transmitted via integron structures in our area.
No blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-24-like, and blaOXA-58-like genes were observed among these isolates, and no MBLs producing these isolates were detected by the EDTA double-disk synergy test. Most (94.4%, 17/18) ST92-CNSAb isolates harbor the blaOXA-23 gene, as shown in Table 2. Interestingly, there was 1 ST92-CSAb isolate that could be detected as being positive for the blaOXA-23 gene. However, ISAba1
blaOXA-23 gene mapping showed that no ISAba1 gene could be detected in this isolate. Therefore, this result confirmed the importance of the ISAba1 gene in A. baumannii isolates, which exhibit the carbapenem resistance phenotype. ISAba1 was detected in most (88.2%, 15/17) blaOXA-23-positive isolates, which are located upstream of blaOXA-23. The blaOXA-51-like gene was detected in all isolates in this study, which is consistent with the blaOXA-51-like gene as an intrinsic carbapenemase. However, there were no ISAba1 genes located with the blaOXA-51-like gene as determined by PCR mapping in CSAb isolates. All blaOXA-51-like genes were confirmed as blaOXA-66 genes by sequencing of CNSAb isolates.
Integron analysis revealed that all CNSAb isolates have a class 1 integrase (Table 2). When comparing the antibiotic resistance profiles (Table 1), most antibiotics showed resistance rates that were much higher in the int1-positive isolates than in the negative ones. Integron gene cassette analysis showed that there were 3 distinct antibiotic gene arrays, i.e., aacA4-catB8-aadA1 (77.3%, 17/22; Genebank accession no. EU340419), aacA4 (22.7%, 5/22; Genebank accession no. EU523056), and aacC1-orfX-orfX'-aadA1 (4.5%, 1/22; Genebank accession no. AY307113). Functional analysis showed that these genes were only associated with aminoglycoside and chloramphenicol resistance, which are not related to carbapenem resistance. It seems that carbapenem-resistant genes might not be transmitted via integron structures in our area.
| null | null |
[
"INTRODUCTION",
"1. Bacterial strains and antimicrobial susceptibility test",
"2. Carbapenemase genes and class 1 integron analysis",
"3. MLST",
"4. Statistical analysis",
"1. Characteristics of A. baumannii isolates",
"2. MLST and clonal relationship of A. baumannii isolates",
"3. Genetic determinants in CNSAb isolates"
] |
[
"Acinetobacter baumannii is an opportunistic human pathogen associated with an increasing incidence of nosocomial infections, including bacteremia, urinary tract infections, and ventilator-associated pneumonia [1-3]. Most A. baumannii isolates have a multidrug resistant phenotype (MDRAb) with an increasing prevalence in the intensive care unit (ICU) [2, 4]. Although carbapenem is a common clinical choice to treat MDRAb infections, the resistance rate has increased dramatically over the past decade. Carbapenem resistance is caused by the production of carbapenemases, including class D β-lactamases (OXA-type carbapenemases) and class B metallo-β-lactamases (MBLs) as previously described [5-8]. The OXA-type carbapenemases are comprised of 4 kinds of genes: blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and an intrinsic blaOXA-51-like type as previously reported [9, 10]. In addition, ISAba1, an insertion sequence (IS), was found in A. baumannii isolates, which can enhance the expression of OXA-type carbapenemase genes and mobilize these genes among the strains [11].\nIntegron is a widely studied vehicle for gene capture, which is highly related to antibiotic resistant gene dissemination [12, 13]. Among MDRAb isolates, the class 1 integron is commonly detected, which infers a high association with carbapenem resistance. The integron structure contains an integrase, followed by an attI site for the integration of cassettes and the recognition of integrase, and a promoter to drive its expression. It can appear on a plasmid or chromosome, and can be classified into different types (class 1, class 2, and class 3 integron) on the basis of their integrases. The most classical integron, class 1 integron, has 2 conserved terminal sequences. Therefore, the primer set (5'-conserved sequence [CS] and 3'-CS), which targets 2 terminal sequences in the integron, is commonly used for integron cassette analysis.\nMulti locus sequence typing (MLST) is an unambiguous typing method for identifying accurate and portable nucleotide sequences of internal fragments of multiple housekeeping genes. Recently, some studies [14-16] revealed that the clonal complex (CC) 92 in the carbapenem non-susceptible A. baumannii (CNSAb) isolate has a worldwide dissemination. The ST92 A. baumannii isolate is the largest clone and is predicted to be the founder of this clonal complex. Molecular epidemiological surveillance not only provides a better understanding of nosocomial infections in certain hospitals, but also provides a chance to understand the route of global dissemination. However, the genetic diversity and molecular characteristics of A. baumannii in our region remain unclear. Therefore, in order to better understand the epidemiological characteristics of A. baumannii in the ICU and the relationship of carbapenem resistance with integrons, in this study, we performed MLST typing and a molecular investigation of antibiotic resistance determinants in A. baumannii isolates collected in our medical ICU.",
"In total, 33 non-duplicated clinical A. baumannii isolates were collected by standard isolation methods between January and December 2009 in our medical ICU. All isolates were identified by conventional methods and further identified by 16S-23S rRNA gene spacer region analysis as described previously [17]. All of these isolates were stored at -80℃ in brain-heart infusion broth containing 50% (v/v) glycerol before inclusion in this study.\nAntimicrobial susceptibility of isolates to cefepime, ceftazidime, piperacillin, amikacin, gentamicin, ciprofloxacin, ampicillin/sulbactam, and piperacillin/tazobactam were determined by using the disc diffusion method. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by using the agar dilution method, and that of colistin was determined by using the E-test (AB bioMérieux, Marcy-l'Etoile, France). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for the antimicrobial susceptibility test. The results were interpreted according to the manufacturer's instructions and CLSI guidelines [18].",
"Genomic DNA from A. baumannii was extracted using a genomic DNA extraction Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. MBL-producing isolates were screened using the imipenem-EDTA double-disk synergy test [19]. Metallo-β-lactamase (IPM4)-producing A. baumannii was used as a positive control. The genes encoding carbapenemases blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like were analyzed by PCR using primers as described previously [5-8]. The location of blaOXA-23-like and blaOXA-51-like with an insert element (ISAba1) was screened by PCR [11].\nIntegron analysis was conducted by using PCR with the class 1 integrase (int1 gene) as the targeting gene, as previously described [12]. Gene cassettes in class 1 integrase-positive isolates were performed by amplifying the sequence from the 5'-CS to the 3'-CS region [12]. The PCR products were purified and sequenced, either directly by using the PCR product, or by cloning into a pGEM®-T vector (Promega, Madison, WI, USA). All sequencing reactions were performed using an ABI prism sequencer 3730 (Applied Biosystems, Foster City, CA, USA), and the sequence data were analyzed by using BLAST at the NCBI website (http://www.ncbi.nlm.nih.gov).",
"MLST was performed on A. baumannii as described by Bartual et al. [20]. Briefly, 7 housekeeping genes, i.e., gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, were amplified, followed by sequencing on the ABI prism sequencer 3730 (Applied Biosystems). The sequences of these 7 housekeeping genes were analyzed using the Pubmlst database (http://pubmlst.org/abaumannii/). The sequence type (ST) was designated according to the allelic profiles in the database. The eBURST algorithm (version 3; http://eburst.mlst.net/) was used to assign STs to CCs and to assess the genetic relationship with definition of the groups sharing alleles at ≥6 of 7 loci [21]. The CC comprises a founding ST as a common ancestor and several other closely related STs descending from the predicted founding genotype.",
"Resistance rates for each antibiotic were compared between CNSAb and carbapenem-susceptible A. baumannii (CSAb), ST92-CNSAb and ST92-CSAb, and the int1-positive group and int1-negative group, using Fisher's exact test. A P value <0.05 was considered as significant difference.",
"In this study, 33 non-duplicate A. baumannii isolates were isolated from patients hospitalized in our medical ICU. All of these isolates were confirmed by 16S-23S rRNA internal spacer analysis as previously described [17]. Among these isolates (26 from lower respiratory tract, 4 from urinary tract, and 2 from blood, and 1 from pus), 54.5% (18/33) isolates were resistant to either imipenem, meropenem, or both. The resistance rate of colistin was 3% (1/33), which coincides with the observation that colistin is the most effective antibiotic to treat A. baumannii infections in our area. From a nationwide surveillance conducted during 2007, the resistance rates of imipenem and meropenem in China were calculated to be 40% and 35%, respectively [22]. However, from a new surveillance report generated in 2010, the prevalence of CNSAb isolates was dramatically increased to 80% [23]. Therefore, the CNSAb isolate is becoming a major concern as a nosocomial infection in China.",
"According to the MLST, 33 A. baumannii isolates can be grouped into 5 existing STs (ST92, 1-3-3-2-2-7-3; ST75, 1-3-3-2-2-11-3; ST112, 1-12-56-36-4-61-26; ST145, 21-35-2-28-1-52-4; ST345, 46-12-110-1-16-141-50) and 1 new ST, which has a G-A mutation at nt268 on the rpoD40 loci of ST251(32-48-54-35-1-11-40) (Table 1). All CNSAb isolates comprised of ST92 and ST75 only, accounting for 77.8% (14/18) and 22.2% (4/18), respectively.\nClonal relation analysis showed that both ST92 and ST75 belong to the CC92, which is a globally disseminated CC for A. baumannii (Fig. 1). This result suggests that CC92 is the major clone that spreads in the ICU of our hospital. eBURST analysis [21] showed that ST92 is the founder of CC92. Previous studies [14, 15, 24] showed that several STs exist, e.g., ST75, ST88, ST90, ST136, ST137, and ST138, which are SLVs of the founder ST92. However, we still found other types, e.g., ST381 and ST375, which are double locus variants (DLVs) of ST92.",
"No blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-24-like, and blaOXA-58-like genes were observed among these isolates, and no MBLs producing these isolates were detected by the EDTA double-disk synergy test. Most (94.4%, 17/18) ST92-CNSAb isolates harbor the blaOXA-23 gene, as shown in Table 2. Interestingly, there was 1 ST92-CSAb isolate that could be detected as being positive for the blaOXA-23 gene. However, ISAba1\nblaOXA-23 gene mapping showed that no ISAba1 gene could be detected in this isolate. Therefore, this result confirmed the importance of the ISAba1 gene in A. baumannii isolates, which exhibit the carbapenem resistance phenotype. ISAba1 was detected in most (88.2%, 15/17) blaOXA-23-positive isolates, which are located upstream of blaOXA-23. The blaOXA-51-like gene was detected in all isolates in this study, which is consistent with the blaOXA-51-like gene as an intrinsic carbapenemase. However, there were no ISAba1 genes located with the blaOXA-51-like gene as determined by PCR mapping in CSAb isolates. All blaOXA-51-like genes were confirmed as blaOXA-66 genes by sequencing of CNSAb isolates.\nIntegron analysis revealed that all CNSAb isolates have a class 1 integrase (Table 2). When comparing the antibiotic resistance profiles (Table 1), most antibiotics showed resistance rates that were much higher in the int1-positive isolates than in the negative ones. Integron gene cassette analysis showed that there were 3 distinct antibiotic gene arrays, i.e., aacA4-catB8-aadA1 (77.3%, 17/22; Genebank accession no. EU340419), aacA4 (22.7%, 5/22; Genebank accession no. EU523056), and aacC1-orfX-orfX'-aadA1 (4.5%, 1/22; Genebank accession no. AY307113). Functional analysis showed that these genes were only associated with aminoglycoside and chloramphenicol resistance, which are not related to carbapenem resistance. It seems that carbapenem-resistant genes might not be transmitted via integron structures in our area."
] |
[
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"METHODS",
"1. Bacterial strains and antimicrobial susceptibility test",
"2. Carbapenemase genes and class 1 integron analysis",
"3. MLST",
"4. Statistical analysis",
"RESULTS",
"1. Characteristics of A. baumannii isolates",
"2. MLST and clonal relationship of A. baumannii isolates",
"3. Genetic determinants in CNSAb isolates",
"DISCUSSION"
] |
[
"Acinetobacter baumannii is an opportunistic human pathogen associated with an increasing incidence of nosocomial infections, including bacteremia, urinary tract infections, and ventilator-associated pneumonia [1-3]. Most A. baumannii isolates have a multidrug resistant phenotype (MDRAb) with an increasing prevalence in the intensive care unit (ICU) [2, 4]. Although carbapenem is a common clinical choice to treat MDRAb infections, the resistance rate has increased dramatically over the past decade. Carbapenem resistance is caused by the production of carbapenemases, including class D β-lactamases (OXA-type carbapenemases) and class B metallo-β-lactamases (MBLs) as previously described [5-8]. The OXA-type carbapenemases are comprised of 4 kinds of genes: blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and an intrinsic blaOXA-51-like type as previously reported [9, 10]. In addition, ISAba1, an insertion sequence (IS), was found in A. baumannii isolates, which can enhance the expression of OXA-type carbapenemase genes and mobilize these genes among the strains [11].\nIntegron is a widely studied vehicle for gene capture, which is highly related to antibiotic resistant gene dissemination [12, 13]. Among MDRAb isolates, the class 1 integron is commonly detected, which infers a high association with carbapenem resistance. The integron structure contains an integrase, followed by an attI site for the integration of cassettes and the recognition of integrase, and a promoter to drive its expression. It can appear on a plasmid or chromosome, and can be classified into different types (class 1, class 2, and class 3 integron) on the basis of their integrases. The most classical integron, class 1 integron, has 2 conserved terminal sequences. Therefore, the primer set (5'-conserved sequence [CS] and 3'-CS), which targets 2 terminal sequences in the integron, is commonly used for integron cassette analysis.\nMulti locus sequence typing (MLST) is an unambiguous typing method for identifying accurate and portable nucleotide sequences of internal fragments of multiple housekeeping genes. Recently, some studies [14-16] revealed that the clonal complex (CC) 92 in the carbapenem non-susceptible A. baumannii (CNSAb) isolate has a worldwide dissemination. The ST92 A. baumannii isolate is the largest clone and is predicted to be the founder of this clonal complex. Molecular epidemiological surveillance not only provides a better understanding of nosocomial infections in certain hospitals, but also provides a chance to understand the route of global dissemination. However, the genetic diversity and molecular characteristics of A. baumannii in our region remain unclear. Therefore, in order to better understand the epidemiological characteristics of A. baumannii in the ICU and the relationship of carbapenem resistance with integrons, in this study, we performed MLST typing and a molecular investigation of antibiotic resistance determinants in A. baumannii isolates collected in our medical ICU.",
" 1. Bacterial strains and antimicrobial susceptibility test In total, 33 non-duplicated clinical A. baumannii isolates were collected by standard isolation methods between January and December 2009 in our medical ICU. All isolates were identified by conventional methods and further identified by 16S-23S rRNA gene spacer region analysis as described previously [17]. All of these isolates were stored at -80℃ in brain-heart infusion broth containing 50% (v/v) glycerol before inclusion in this study.\nAntimicrobial susceptibility of isolates to cefepime, ceftazidime, piperacillin, amikacin, gentamicin, ciprofloxacin, ampicillin/sulbactam, and piperacillin/tazobactam were determined by using the disc diffusion method. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by using the agar dilution method, and that of colistin was determined by using the E-test (AB bioMérieux, Marcy-l'Etoile, France). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for the antimicrobial susceptibility test. The results were interpreted according to the manufacturer's instructions and CLSI guidelines [18].\nIn total, 33 non-duplicated clinical A. baumannii isolates were collected by standard isolation methods between January and December 2009 in our medical ICU. All isolates were identified by conventional methods and further identified by 16S-23S rRNA gene spacer region analysis as described previously [17]. All of these isolates were stored at -80℃ in brain-heart infusion broth containing 50% (v/v) glycerol before inclusion in this study.\nAntimicrobial susceptibility of isolates to cefepime, ceftazidime, piperacillin, amikacin, gentamicin, ciprofloxacin, ampicillin/sulbactam, and piperacillin/tazobactam were determined by using the disc diffusion method. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by using the agar dilution method, and that of colistin was determined by using the E-test (AB bioMérieux, Marcy-l'Etoile, France). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for the antimicrobial susceptibility test. The results were interpreted according to the manufacturer's instructions and CLSI guidelines [18].\n 2. Carbapenemase genes and class 1 integron analysis Genomic DNA from A. baumannii was extracted using a genomic DNA extraction Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. MBL-producing isolates were screened using the imipenem-EDTA double-disk synergy test [19]. Metallo-β-lactamase (IPM4)-producing A. baumannii was used as a positive control. The genes encoding carbapenemases blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like were analyzed by PCR using primers as described previously [5-8]. The location of blaOXA-23-like and blaOXA-51-like with an insert element (ISAba1) was screened by PCR [11].\nIntegron analysis was conducted by using PCR with the class 1 integrase (int1 gene) as the targeting gene, as previously described [12]. Gene cassettes in class 1 integrase-positive isolates were performed by amplifying the sequence from the 5'-CS to the 3'-CS region [12]. The PCR products were purified and sequenced, either directly by using the PCR product, or by cloning into a pGEM®-T vector (Promega, Madison, WI, USA). All sequencing reactions were performed using an ABI prism sequencer 3730 (Applied Biosystems, Foster City, CA, USA), and the sequence data were analyzed by using BLAST at the NCBI website (http://www.ncbi.nlm.nih.gov).\nGenomic DNA from A. baumannii was extracted using a genomic DNA extraction Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. MBL-producing isolates were screened using the imipenem-EDTA double-disk synergy test [19]. Metallo-β-lactamase (IPM4)-producing A. baumannii was used as a positive control. The genes encoding carbapenemases blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like were analyzed by PCR using primers as described previously [5-8]. The location of blaOXA-23-like and blaOXA-51-like with an insert element (ISAba1) was screened by PCR [11].\nIntegron analysis was conducted by using PCR with the class 1 integrase (int1 gene) as the targeting gene, as previously described [12]. Gene cassettes in class 1 integrase-positive isolates were performed by amplifying the sequence from the 5'-CS to the 3'-CS region [12]. The PCR products were purified and sequenced, either directly by using the PCR product, or by cloning into a pGEM®-T vector (Promega, Madison, WI, USA). All sequencing reactions were performed using an ABI prism sequencer 3730 (Applied Biosystems, Foster City, CA, USA), and the sequence data were analyzed by using BLAST at the NCBI website (http://www.ncbi.nlm.nih.gov).\n 3. MLST MLST was performed on A. baumannii as described by Bartual et al. [20]. Briefly, 7 housekeeping genes, i.e., gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, were amplified, followed by sequencing on the ABI prism sequencer 3730 (Applied Biosystems). The sequences of these 7 housekeeping genes were analyzed using the Pubmlst database (http://pubmlst.org/abaumannii/). The sequence type (ST) was designated according to the allelic profiles in the database. The eBURST algorithm (version 3; http://eburst.mlst.net/) was used to assign STs to CCs and to assess the genetic relationship with definition of the groups sharing alleles at ≥6 of 7 loci [21]. The CC comprises a founding ST as a common ancestor and several other closely related STs descending from the predicted founding genotype.\nMLST was performed on A. baumannii as described by Bartual et al. [20]. Briefly, 7 housekeeping genes, i.e., gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, were amplified, followed by sequencing on the ABI prism sequencer 3730 (Applied Biosystems). The sequences of these 7 housekeeping genes were analyzed using the Pubmlst database (http://pubmlst.org/abaumannii/). The sequence type (ST) was designated according to the allelic profiles in the database. The eBURST algorithm (version 3; http://eburst.mlst.net/) was used to assign STs to CCs and to assess the genetic relationship with definition of the groups sharing alleles at ≥6 of 7 loci [21]. The CC comprises a founding ST as a common ancestor and several other closely related STs descending from the predicted founding genotype.\n 4. Statistical analysis Resistance rates for each antibiotic were compared between CNSAb and carbapenem-susceptible A. baumannii (CSAb), ST92-CNSAb and ST92-CSAb, and the int1-positive group and int1-negative group, using Fisher's exact test. A P value <0.05 was considered as significant difference.\nResistance rates for each antibiotic were compared between CNSAb and carbapenem-susceptible A. baumannii (CSAb), ST92-CNSAb and ST92-CSAb, and the int1-positive group and int1-negative group, using Fisher's exact test. A P value <0.05 was considered as significant difference.",
"In total, 33 non-duplicated clinical A. baumannii isolates were collected by standard isolation methods between January and December 2009 in our medical ICU. All isolates were identified by conventional methods and further identified by 16S-23S rRNA gene spacer region analysis as described previously [17]. All of these isolates were stored at -80℃ in brain-heart infusion broth containing 50% (v/v) glycerol before inclusion in this study.\nAntimicrobial susceptibility of isolates to cefepime, ceftazidime, piperacillin, amikacin, gentamicin, ciprofloxacin, ampicillin/sulbactam, and piperacillin/tazobactam were determined by using the disc diffusion method. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by using the agar dilution method, and that of colistin was determined by using the E-test (AB bioMérieux, Marcy-l'Etoile, France). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for the antimicrobial susceptibility test. The results were interpreted according to the manufacturer's instructions and CLSI guidelines [18].",
"Genomic DNA from A. baumannii was extracted using a genomic DNA extraction Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. MBL-producing isolates were screened using the imipenem-EDTA double-disk synergy test [19]. Metallo-β-lactamase (IPM4)-producing A. baumannii was used as a positive control. The genes encoding carbapenemases blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like were analyzed by PCR using primers as described previously [5-8]. The location of blaOXA-23-like and blaOXA-51-like with an insert element (ISAba1) was screened by PCR [11].\nIntegron analysis was conducted by using PCR with the class 1 integrase (int1 gene) as the targeting gene, as previously described [12]. Gene cassettes in class 1 integrase-positive isolates were performed by amplifying the sequence from the 5'-CS to the 3'-CS region [12]. The PCR products were purified and sequenced, either directly by using the PCR product, or by cloning into a pGEM®-T vector (Promega, Madison, WI, USA). All sequencing reactions were performed using an ABI prism sequencer 3730 (Applied Biosystems, Foster City, CA, USA), and the sequence data were analyzed by using BLAST at the NCBI website (http://www.ncbi.nlm.nih.gov).",
"MLST was performed on A. baumannii as described by Bartual et al. [20]. Briefly, 7 housekeeping genes, i.e., gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, were amplified, followed by sequencing on the ABI prism sequencer 3730 (Applied Biosystems). The sequences of these 7 housekeeping genes were analyzed using the Pubmlst database (http://pubmlst.org/abaumannii/). The sequence type (ST) was designated according to the allelic profiles in the database. The eBURST algorithm (version 3; http://eburst.mlst.net/) was used to assign STs to CCs and to assess the genetic relationship with definition of the groups sharing alleles at ≥6 of 7 loci [21]. The CC comprises a founding ST as a common ancestor and several other closely related STs descending from the predicted founding genotype.",
"Resistance rates for each antibiotic were compared between CNSAb and carbapenem-susceptible A. baumannii (CSAb), ST92-CNSAb and ST92-CSAb, and the int1-positive group and int1-negative group, using Fisher's exact test. A P value <0.05 was considered as significant difference.",
" 1. Characteristics of A. baumannii isolates In this study, 33 non-duplicate A. baumannii isolates were isolated from patients hospitalized in our medical ICU. All of these isolates were confirmed by 16S-23S rRNA internal spacer analysis as previously described [17]. Among these isolates (26 from lower respiratory tract, 4 from urinary tract, and 2 from blood, and 1 from pus), 54.5% (18/33) isolates were resistant to either imipenem, meropenem, or both. The resistance rate of colistin was 3% (1/33), which coincides with the observation that colistin is the most effective antibiotic to treat A. baumannii infections in our area. From a nationwide surveillance conducted during 2007, the resistance rates of imipenem and meropenem in China were calculated to be 40% and 35%, respectively [22]. However, from a new surveillance report generated in 2010, the prevalence of CNSAb isolates was dramatically increased to 80% [23]. Therefore, the CNSAb isolate is becoming a major concern as a nosocomial infection in China.\nIn this study, 33 non-duplicate A. baumannii isolates were isolated from patients hospitalized in our medical ICU. All of these isolates were confirmed by 16S-23S rRNA internal spacer analysis as previously described [17]. Among these isolates (26 from lower respiratory tract, 4 from urinary tract, and 2 from blood, and 1 from pus), 54.5% (18/33) isolates were resistant to either imipenem, meropenem, or both. The resistance rate of colistin was 3% (1/33), which coincides with the observation that colistin is the most effective antibiotic to treat A. baumannii infections in our area. From a nationwide surveillance conducted during 2007, the resistance rates of imipenem and meropenem in China were calculated to be 40% and 35%, respectively [22]. However, from a new surveillance report generated in 2010, the prevalence of CNSAb isolates was dramatically increased to 80% [23]. Therefore, the CNSAb isolate is becoming a major concern as a nosocomial infection in China.\n 2. MLST and clonal relationship of A. baumannii isolates According to the MLST, 33 A. baumannii isolates can be grouped into 5 existing STs (ST92, 1-3-3-2-2-7-3; ST75, 1-3-3-2-2-11-3; ST112, 1-12-56-36-4-61-26; ST145, 21-35-2-28-1-52-4; ST345, 46-12-110-1-16-141-50) and 1 new ST, which has a G-A mutation at nt268 on the rpoD40 loci of ST251(32-48-54-35-1-11-40) (Table 1). All CNSAb isolates comprised of ST92 and ST75 only, accounting for 77.8% (14/18) and 22.2% (4/18), respectively.\nClonal relation analysis showed that both ST92 and ST75 belong to the CC92, which is a globally disseminated CC for A. baumannii (Fig. 1). This result suggests that CC92 is the major clone that spreads in the ICU of our hospital. eBURST analysis [21] showed that ST92 is the founder of CC92. Previous studies [14, 15, 24] showed that several STs exist, e.g., ST75, ST88, ST90, ST136, ST137, and ST138, which are SLVs of the founder ST92. However, we still found other types, e.g., ST381 and ST375, which are double locus variants (DLVs) of ST92.\nAccording to the MLST, 33 A. baumannii isolates can be grouped into 5 existing STs (ST92, 1-3-3-2-2-7-3; ST75, 1-3-3-2-2-11-3; ST112, 1-12-56-36-4-61-26; ST145, 21-35-2-28-1-52-4; ST345, 46-12-110-1-16-141-50) and 1 new ST, which has a G-A mutation at nt268 on the rpoD40 loci of ST251(32-48-54-35-1-11-40) (Table 1). All CNSAb isolates comprised of ST92 and ST75 only, accounting for 77.8% (14/18) and 22.2% (4/18), respectively.\nClonal relation analysis showed that both ST92 and ST75 belong to the CC92, which is a globally disseminated CC for A. baumannii (Fig. 1). This result suggests that CC92 is the major clone that spreads in the ICU of our hospital. eBURST analysis [21] showed that ST92 is the founder of CC92. Previous studies [14, 15, 24] showed that several STs exist, e.g., ST75, ST88, ST90, ST136, ST137, and ST138, which are SLVs of the founder ST92. However, we still found other types, e.g., ST381 and ST375, which are double locus variants (DLVs) of ST92.\n 3. Genetic determinants in CNSAb isolates No blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-24-like, and blaOXA-58-like genes were observed among these isolates, and no MBLs producing these isolates were detected by the EDTA double-disk synergy test. Most (94.4%, 17/18) ST92-CNSAb isolates harbor the blaOXA-23 gene, as shown in Table 2. Interestingly, there was 1 ST92-CSAb isolate that could be detected as being positive for the blaOXA-23 gene. However, ISAba1\nblaOXA-23 gene mapping showed that no ISAba1 gene could be detected in this isolate. Therefore, this result confirmed the importance of the ISAba1 gene in A. baumannii isolates, which exhibit the carbapenem resistance phenotype. ISAba1 was detected in most (88.2%, 15/17) blaOXA-23-positive isolates, which are located upstream of blaOXA-23. The blaOXA-51-like gene was detected in all isolates in this study, which is consistent with the blaOXA-51-like gene as an intrinsic carbapenemase. However, there were no ISAba1 genes located with the blaOXA-51-like gene as determined by PCR mapping in CSAb isolates. All blaOXA-51-like genes were confirmed as blaOXA-66 genes by sequencing of CNSAb isolates.\nIntegron analysis revealed that all CNSAb isolates have a class 1 integrase (Table 2). When comparing the antibiotic resistance profiles (Table 1), most antibiotics showed resistance rates that were much higher in the int1-positive isolates than in the negative ones. Integron gene cassette analysis showed that there were 3 distinct antibiotic gene arrays, i.e., aacA4-catB8-aadA1 (77.3%, 17/22; Genebank accession no. EU340419), aacA4 (22.7%, 5/22; Genebank accession no. EU523056), and aacC1-orfX-orfX'-aadA1 (4.5%, 1/22; Genebank accession no. AY307113). Functional analysis showed that these genes were only associated with aminoglycoside and chloramphenicol resistance, which are not related to carbapenem resistance. It seems that carbapenem-resistant genes might not be transmitted via integron structures in our area.\nNo blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-24-like, and blaOXA-58-like genes were observed among these isolates, and no MBLs producing these isolates were detected by the EDTA double-disk synergy test. Most (94.4%, 17/18) ST92-CNSAb isolates harbor the blaOXA-23 gene, as shown in Table 2. Interestingly, there was 1 ST92-CSAb isolate that could be detected as being positive for the blaOXA-23 gene. However, ISAba1\nblaOXA-23 gene mapping showed that no ISAba1 gene could be detected in this isolate. Therefore, this result confirmed the importance of the ISAba1 gene in A. baumannii isolates, which exhibit the carbapenem resistance phenotype. ISAba1 was detected in most (88.2%, 15/17) blaOXA-23-positive isolates, which are located upstream of blaOXA-23. The blaOXA-51-like gene was detected in all isolates in this study, which is consistent with the blaOXA-51-like gene as an intrinsic carbapenemase. However, there were no ISAba1 genes located with the blaOXA-51-like gene as determined by PCR mapping in CSAb isolates. All blaOXA-51-like genes were confirmed as blaOXA-66 genes by sequencing of CNSAb isolates.\nIntegron analysis revealed that all CNSAb isolates have a class 1 integrase (Table 2). When comparing the antibiotic resistance profiles (Table 1), most antibiotics showed resistance rates that were much higher in the int1-positive isolates than in the negative ones. Integron gene cassette analysis showed that there were 3 distinct antibiotic gene arrays, i.e., aacA4-catB8-aadA1 (77.3%, 17/22; Genebank accession no. EU340419), aacA4 (22.7%, 5/22; Genebank accession no. EU523056), and aacC1-orfX-orfX'-aadA1 (4.5%, 1/22; Genebank accession no. AY307113). Functional analysis showed that these genes were only associated with aminoglycoside and chloramphenicol resistance, which are not related to carbapenem resistance. It seems that carbapenem-resistant genes might not be transmitted via integron structures in our area.",
"In this study, 33 non-duplicate A. baumannii isolates were isolated from patients hospitalized in our medical ICU. All of these isolates were confirmed by 16S-23S rRNA internal spacer analysis as previously described [17]. Among these isolates (26 from lower respiratory tract, 4 from urinary tract, and 2 from blood, and 1 from pus), 54.5% (18/33) isolates were resistant to either imipenem, meropenem, or both. The resistance rate of colistin was 3% (1/33), which coincides with the observation that colistin is the most effective antibiotic to treat A. baumannii infections in our area. From a nationwide surveillance conducted during 2007, the resistance rates of imipenem and meropenem in China were calculated to be 40% and 35%, respectively [22]. However, from a new surveillance report generated in 2010, the prevalence of CNSAb isolates was dramatically increased to 80% [23]. Therefore, the CNSAb isolate is becoming a major concern as a nosocomial infection in China.",
"According to the MLST, 33 A. baumannii isolates can be grouped into 5 existing STs (ST92, 1-3-3-2-2-7-3; ST75, 1-3-3-2-2-11-3; ST112, 1-12-56-36-4-61-26; ST145, 21-35-2-28-1-52-4; ST345, 46-12-110-1-16-141-50) and 1 new ST, which has a G-A mutation at nt268 on the rpoD40 loci of ST251(32-48-54-35-1-11-40) (Table 1). All CNSAb isolates comprised of ST92 and ST75 only, accounting for 77.8% (14/18) and 22.2% (4/18), respectively.\nClonal relation analysis showed that both ST92 and ST75 belong to the CC92, which is a globally disseminated CC for A. baumannii (Fig. 1). This result suggests that CC92 is the major clone that spreads in the ICU of our hospital. eBURST analysis [21] showed that ST92 is the founder of CC92. Previous studies [14, 15, 24] showed that several STs exist, e.g., ST75, ST88, ST90, ST136, ST137, and ST138, which are SLVs of the founder ST92. However, we still found other types, e.g., ST381 and ST375, which are double locus variants (DLVs) of ST92.",
"No blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-24-like, and blaOXA-58-like genes were observed among these isolates, and no MBLs producing these isolates were detected by the EDTA double-disk synergy test. Most (94.4%, 17/18) ST92-CNSAb isolates harbor the blaOXA-23 gene, as shown in Table 2. Interestingly, there was 1 ST92-CSAb isolate that could be detected as being positive for the blaOXA-23 gene. However, ISAba1\nblaOXA-23 gene mapping showed that no ISAba1 gene could be detected in this isolate. Therefore, this result confirmed the importance of the ISAba1 gene in A. baumannii isolates, which exhibit the carbapenem resistance phenotype. ISAba1 was detected in most (88.2%, 15/17) blaOXA-23-positive isolates, which are located upstream of blaOXA-23. The blaOXA-51-like gene was detected in all isolates in this study, which is consistent with the blaOXA-51-like gene as an intrinsic carbapenemase. However, there were no ISAba1 genes located with the blaOXA-51-like gene as determined by PCR mapping in CSAb isolates. All blaOXA-51-like genes were confirmed as blaOXA-66 genes by sequencing of CNSAb isolates.\nIntegron analysis revealed that all CNSAb isolates have a class 1 integrase (Table 2). When comparing the antibiotic resistance profiles (Table 1), most antibiotics showed resistance rates that were much higher in the int1-positive isolates than in the negative ones. Integron gene cassette analysis showed that there were 3 distinct antibiotic gene arrays, i.e., aacA4-catB8-aadA1 (77.3%, 17/22; Genebank accession no. EU340419), aacA4 (22.7%, 5/22; Genebank accession no. EU523056), and aacC1-orfX-orfX'-aadA1 (4.5%, 1/22; Genebank accession no. AY307113). Functional analysis showed that these genes were only associated with aminoglycoside and chloramphenicol resistance, which are not related to carbapenem resistance. It seems that carbapenem-resistant genes might not be transmitted via integron structures in our area.",
"In this study, we analyzed the molecular typing and characteristics of A. baumannii isolates in our ICU. From the MLST result, the predominant clone is ST92 in CNSAb isolates in our hospital, and this observation is also consistent with previous studies [14-16, 25], which revealed that ST92 has spread worldwide. However, limited diversity was observed in CNSAb isolates in our ICU, while there was a higher diversity in CSAb isolates. Interestingly, there were still 8 CSAb isolates being assigned as ST92, but none of the CSAb isolates belonged to ST75. This characteristic indicates that these 2 types might exhibit differences in clinical outcomes or have antibiotic resistance patterns. Although there were no significant differences in antibiotic resistance rates for the ST92 and ST75 CNSAb isolates, they still exhibited slight differences on their MIC ranges. As shown in Table 2, all ST92 CNSAb isolates were resistant to imipenem, with MICs ranging from 32-128 mg/L; most of them (71.4%, 10/14) were also resistant to meropenem with the same MIC range. However, the MIC of the imipenem-resistant ST75 isolates ranged from 64 to 128 mg/L, which is a more narrow resistance profile than that of ST92 (Table 2). However, there was no difference in the MIC range of meropenem for these 2 types. These results indicate that ST75 might have a more severe imipenem resistance range than ST92.\nGenetic analysis showed that ST75 is the single locus variant (SLV) of ST92, which differs in its gpi loci. While ST92 isolates show moderate carbapenem resistance rates, all ST75 types are resistant to carbapenem with a higher MIC range. Therefore, we hypothesized that mutations of the gpi loci in ST92, e.g., ST75, might benefit its survival when encountering carbapenem selection. In contrast, in our collection, there were some ST92-type CSAb isolates, but none was ST75, which also inferred that ST75 CNSAb isolates might represent a severe epidemic marker in nosocomial infections in our area. Moreover, a study by He et al. [15] showed that more ST138-CNSAb isolates are associated with ST92 in western China, which could give rise to the interesting assumption that the composition of CNSAb STs could exhibit geographic differences. These differences might be caused by different antibiotic usage habits, which may influence the evolution direction of ST92. Therefore, ST75 accompanied by ST92 might be the epidemical feature in eastern China.\nIn this study, we observed more diversity in CSAb isolates (5 ST types) than in CNSAb isolates (only 2 ST types). Interestingly, we observed some of CSAb isolates is ST92-CSAb type, and this type strain is the majority component in the epidemic CNSAb clonal CC92. While the other 4 STs, including ST112, ST145, ST345, and STn are singleton in the A. baumannii population, which shows no relation with epidemic strain CC92. As is known, natural selection is the major force that shapes the population structure of microbial communities in certain environments. A higher diversity in CSAb isolates might be due to fewer encounters with natural selection forces, e.g., antibiotics. Antibiotic resistance profiles showed that most antibiotics caused more resistance in the ST92-CNSAb group than in the ST92-CSAb group (Table 1). However, ST92-CSAb strains could potentially be a source of CNSAb strains, having acquired carbapenem resistance. Therefore, ST92-CSAb strains should be considered as important along with CNSAb isolates for future nosocomial infection control.\nGenetic determinants analysis shows MBLs are not the major resistant determinants in CNSAb isolates in our hosptial, which is consistent with previous study [26]. However, in some distinct areas of China, the MBL plays a more important role than the OXA-type carbapenemase in CNSAb [27]. This implies that a geographical characteristic plays an important role in CNSAb isolates formation, and it could be due to different antibiotic usage in distinct areas. Integron analysis shows a high prevalence of this gene capture machinery. Although in this study, we only recovered 3 type of class 1 integron cassette, there still shows integron is highly associated with antibiotic resistance. However, there are some limitations in this integron cassette analysis method, e.g., some gene cassettes failed to be recovered due to the lack of a conserved terminal [28]. However, there are several studies [29, 30] showing that there are multiple gene cassettes that contain the OXA gene in A. baumannii isolates. This might indicate that transmission of the carbapenemase resistant gene might be mediated by a very complex mechanism. Notably, whole genome sequencing of MDRAbs [31-34] revealed a correlation between carbapenem resistance and the integron component. Most MDRAb strains harbor an antibiotic resistance island (AbaR) in their genome, which provides great potential for antibiotic resistance. Moreover, in the present study, we observed that these gene cassettes are shared among these strains and other species from the integron database [29], which provides a clue for the transmission of integrons among inner- and interspecies. Interestingly, several environmental isolates in our ICU possessed a class 1 integrase from a previous collection, while 1 isolate was detected with an empty gene cassette (data not shown). Similarly, there is a recent report that shows 1 environmental isolate of P. aeruginosa with an empty cassette [35]. This empty cassette potentially represents a reservoir with an increased capacity to adapt to the environment.\nIn conclusion, we found that there are 2 predominant distinct clones (ST92 and ST75) of CNSAb isolates, which clustered to CC92 in our medical ICU. ST92-A. baumannii is considered to be the clone with a worldwide dissemination, while ST75 could be another major clone accompanying the CNSAb isolates in our region. Moreover, the ST75 clone might act as a severe epidemic marker in the carbapenem-resistant A. baumannii isolates in our area. Therefore, CC92-CNSAb plays an important role in causing nosocomial infections in our medical ICU and thus, should be investigated as part of hospital infection control in the future."
] |
[
null,
"methods",
null,
null,
null,
null,
"results",
null,
null,
null,
"discussion"
] |
[
"MLST",
"Molecular epidemiology",
"\nAcinetobacter baumannii\n"
] |
INTRODUCTION:
Acinetobacter baumannii is an opportunistic human pathogen associated with an increasing incidence of nosocomial infections, including bacteremia, urinary tract infections, and ventilator-associated pneumonia [1-3]. Most A. baumannii isolates have a multidrug resistant phenotype (MDRAb) with an increasing prevalence in the intensive care unit (ICU) [2, 4]. Although carbapenem is a common clinical choice to treat MDRAb infections, the resistance rate has increased dramatically over the past decade. Carbapenem resistance is caused by the production of carbapenemases, including class D β-lactamases (OXA-type carbapenemases) and class B metallo-β-lactamases (MBLs) as previously described [5-8]. The OXA-type carbapenemases are comprised of 4 kinds of genes: blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and an intrinsic blaOXA-51-like type as previously reported [9, 10]. In addition, ISAba1, an insertion sequence (IS), was found in A. baumannii isolates, which can enhance the expression of OXA-type carbapenemase genes and mobilize these genes among the strains [11].
Integron is a widely studied vehicle for gene capture, which is highly related to antibiotic resistant gene dissemination [12, 13]. Among MDRAb isolates, the class 1 integron is commonly detected, which infers a high association with carbapenem resistance. The integron structure contains an integrase, followed by an attI site for the integration of cassettes and the recognition of integrase, and a promoter to drive its expression. It can appear on a plasmid or chromosome, and can be classified into different types (class 1, class 2, and class 3 integron) on the basis of their integrases. The most classical integron, class 1 integron, has 2 conserved terminal sequences. Therefore, the primer set (5'-conserved sequence [CS] and 3'-CS), which targets 2 terminal sequences in the integron, is commonly used for integron cassette analysis.
Multi locus sequence typing (MLST) is an unambiguous typing method for identifying accurate and portable nucleotide sequences of internal fragments of multiple housekeeping genes. Recently, some studies [14-16] revealed that the clonal complex (CC) 92 in the carbapenem non-susceptible A. baumannii (CNSAb) isolate has a worldwide dissemination. The ST92 A. baumannii isolate is the largest clone and is predicted to be the founder of this clonal complex. Molecular epidemiological surveillance not only provides a better understanding of nosocomial infections in certain hospitals, but also provides a chance to understand the route of global dissemination. However, the genetic diversity and molecular characteristics of A. baumannii in our region remain unclear. Therefore, in order to better understand the epidemiological characteristics of A. baumannii in the ICU and the relationship of carbapenem resistance with integrons, in this study, we performed MLST typing and a molecular investigation of antibiotic resistance determinants in A. baumannii isolates collected in our medical ICU.
METHODS:
1. Bacterial strains and antimicrobial susceptibility test In total, 33 non-duplicated clinical A. baumannii isolates were collected by standard isolation methods between January and December 2009 in our medical ICU. All isolates were identified by conventional methods and further identified by 16S-23S rRNA gene spacer region analysis as described previously [17]. All of these isolates were stored at -80℃ in brain-heart infusion broth containing 50% (v/v) glycerol before inclusion in this study.
Antimicrobial susceptibility of isolates to cefepime, ceftazidime, piperacillin, amikacin, gentamicin, ciprofloxacin, ampicillin/sulbactam, and piperacillin/tazobactam were determined by using the disc diffusion method. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by using the agar dilution method, and that of colistin was determined by using the E-test (AB bioMérieux, Marcy-l'Etoile, France). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for the antimicrobial susceptibility test. The results were interpreted according to the manufacturer's instructions and CLSI guidelines [18].
In total, 33 non-duplicated clinical A. baumannii isolates were collected by standard isolation methods between January and December 2009 in our medical ICU. All isolates were identified by conventional methods and further identified by 16S-23S rRNA gene spacer region analysis as described previously [17]. All of these isolates were stored at -80℃ in brain-heart infusion broth containing 50% (v/v) glycerol before inclusion in this study.
Antimicrobial susceptibility of isolates to cefepime, ceftazidime, piperacillin, amikacin, gentamicin, ciprofloxacin, ampicillin/sulbactam, and piperacillin/tazobactam were determined by using the disc diffusion method. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by using the agar dilution method, and that of colistin was determined by using the E-test (AB bioMérieux, Marcy-l'Etoile, France). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for the antimicrobial susceptibility test. The results were interpreted according to the manufacturer's instructions and CLSI guidelines [18].
2. Carbapenemase genes and class 1 integron analysis Genomic DNA from A. baumannii was extracted using a genomic DNA extraction Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. MBL-producing isolates were screened using the imipenem-EDTA double-disk synergy test [19]. Metallo-β-lactamase (IPM4)-producing A. baumannii was used as a positive control. The genes encoding carbapenemases blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like were analyzed by PCR using primers as described previously [5-8]. The location of blaOXA-23-like and blaOXA-51-like with an insert element (ISAba1) was screened by PCR [11].
Integron analysis was conducted by using PCR with the class 1 integrase (int1 gene) as the targeting gene, as previously described [12]. Gene cassettes in class 1 integrase-positive isolates were performed by amplifying the sequence from the 5'-CS to the 3'-CS region [12]. The PCR products were purified and sequenced, either directly by using the PCR product, or by cloning into a pGEM®-T vector (Promega, Madison, WI, USA). All sequencing reactions were performed using an ABI prism sequencer 3730 (Applied Biosystems, Foster City, CA, USA), and the sequence data were analyzed by using BLAST at the NCBI website (http://www.ncbi.nlm.nih.gov).
Genomic DNA from A. baumannii was extracted using a genomic DNA extraction Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. MBL-producing isolates were screened using the imipenem-EDTA double-disk synergy test [19]. Metallo-β-lactamase (IPM4)-producing A. baumannii was used as a positive control. The genes encoding carbapenemases blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like were analyzed by PCR using primers as described previously [5-8]. The location of blaOXA-23-like and blaOXA-51-like with an insert element (ISAba1) was screened by PCR [11].
Integron analysis was conducted by using PCR with the class 1 integrase (int1 gene) as the targeting gene, as previously described [12]. Gene cassettes in class 1 integrase-positive isolates were performed by amplifying the sequence from the 5'-CS to the 3'-CS region [12]. The PCR products were purified and sequenced, either directly by using the PCR product, or by cloning into a pGEM®-T vector (Promega, Madison, WI, USA). All sequencing reactions were performed using an ABI prism sequencer 3730 (Applied Biosystems, Foster City, CA, USA), and the sequence data were analyzed by using BLAST at the NCBI website (http://www.ncbi.nlm.nih.gov).
3. MLST MLST was performed on A. baumannii as described by Bartual et al. [20]. Briefly, 7 housekeeping genes, i.e., gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, were amplified, followed by sequencing on the ABI prism sequencer 3730 (Applied Biosystems). The sequences of these 7 housekeeping genes were analyzed using the Pubmlst database (http://pubmlst.org/abaumannii/). The sequence type (ST) was designated according to the allelic profiles in the database. The eBURST algorithm (version 3; http://eburst.mlst.net/) was used to assign STs to CCs and to assess the genetic relationship with definition of the groups sharing alleles at ≥6 of 7 loci [21]. The CC comprises a founding ST as a common ancestor and several other closely related STs descending from the predicted founding genotype.
MLST was performed on A. baumannii as described by Bartual et al. [20]. Briefly, 7 housekeeping genes, i.e., gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, were amplified, followed by sequencing on the ABI prism sequencer 3730 (Applied Biosystems). The sequences of these 7 housekeeping genes were analyzed using the Pubmlst database (http://pubmlst.org/abaumannii/). The sequence type (ST) was designated according to the allelic profiles in the database. The eBURST algorithm (version 3; http://eburst.mlst.net/) was used to assign STs to CCs and to assess the genetic relationship with definition of the groups sharing alleles at ≥6 of 7 loci [21]. The CC comprises a founding ST as a common ancestor and several other closely related STs descending from the predicted founding genotype.
4. Statistical analysis Resistance rates for each antibiotic were compared between CNSAb and carbapenem-susceptible A. baumannii (CSAb), ST92-CNSAb and ST92-CSAb, and the int1-positive group and int1-negative group, using Fisher's exact test. A P value <0.05 was considered as significant difference.
Resistance rates for each antibiotic were compared between CNSAb and carbapenem-susceptible A. baumannii (CSAb), ST92-CNSAb and ST92-CSAb, and the int1-positive group and int1-negative group, using Fisher's exact test. A P value <0.05 was considered as significant difference.
1. Bacterial strains and antimicrobial susceptibility test:
In total, 33 non-duplicated clinical A. baumannii isolates were collected by standard isolation methods between January and December 2009 in our medical ICU. All isolates were identified by conventional methods and further identified by 16S-23S rRNA gene spacer region analysis as described previously [17]. All of these isolates were stored at -80℃ in brain-heart infusion broth containing 50% (v/v) glycerol before inclusion in this study.
Antimicrobial susceptibility of isolates to cefepime, ceftazidime, piperacillin, amikacin, gentamicin, ciprofloxacin, ampicillin/sulbactam, and piperacillin/tazobactam were determined by using the disc diffusion method. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by using the agar dilution method, and that of colistin was determined by using the E-test (AB bioMérieux, Marcy-l'Etoile, France). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for the antimicrobial susceptibility test. The results were interpreted according to the manufacturer's instructions and CLSI guidelines [18].
2. Carbapenemase genes and class 1 integron analysis:
Genomic DNA from A. baumannii was extracted using a genomic DNA extraction Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. MBL-producing isolates were screened using the imipenem-EDTA double-disk synergy test [19]. Metallo-β-lactamase (IPM4)-producing A. baumannii was used as a positive control. The genes encoding carbapenemases blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like were analyzed by PCR using primers as described previously [5-8]. The location of blaOXA-23-like and blaOXA-51-like with an insert element (ISAba1) was screened by PCR [11].
Integron analysis was conducted by using PCR with the class 1 integrase (int1 gene) as the targeting gene, as previously described [12]. Gene cassettes in class 1 integrase-positive isolates were performed by amplifying the sequence from the 5'-CS to the 3'-CS region [12]. The PCR products were purified and sequenced, either directly by using the PCR product, or by cloning into a pGEM®-T vector (Promega, Madison, WI, USA). All sequencing reactions were performed using an ABI prism sequencer 3730 (Applied Biosystems, Foster City, CA, USA), and the sequence data were analyzed by using BLAST at the NCBI website (http://www.ncbi.nlm.nih.gov).
3. MLST:
MLST was performed on A. baumannii as described by Bartual et al. [20]. Briefly, 7 housekeeping genes, i.e., gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, were amplified, followed by sequencing on the ABI prism sequencer 3730 (Applied Biosystems). The sequences of these 7 housekeeping genes were analyzed using the Pubmlst database (http://pubmlst.org/abaumannii/). The sequence type (ST) was designated according to the allelic profiles in the database. The eBURST algorithm (version 3; http://eburst.mlst.net/) was used to assign STs to CCs and to assess the genetic relationship with definition of the groups sharing alleles at ≥6 of 7 loci [21]. The CC comprises a founding ST as a common ancestor and several other closely related STs descending from the predicted founding genotype.
4. Statistical analysis:
Resistance rates for each antibiotic were compared between CNSAb and carbapenem-susceptible A. baumannii (CSAb), ST92-CNSAb and ST92-CSAb, and the int1-positive group and int1-negative group, using Fisher's exact test. A P value <0.05 was considered as significant difference.
RESULTS:
1. Characteristics of A. baumannii isolates In this study, 33 non-duplicate A. baumannii isolates were isolated from patients hospitalized in our medical ICU. All of these isolates were confirmed by 16S-23S rRNA internal spacer analysis as previously described [17]. Among these isolates (26 from lower respiratory tract, 4 from urinary tract, and 2 from blood, and 1 from pus), 54.5% (18/33) isolates were resistant to either imipenem, meropenem, or both. The resistance rate of colistin was 3% (1/33), which coincides with the observation that colistin is the most effective antibiotic to treat A. baumannii infections in our area. From a nationwide surveillance conducted during 2007, the resistance rates of imipenem and meropenem in China were calculated to be 40% and 35%, respectively [22]. However, from a new surveillance report generated in 2010, the prevalence of CNSAb isolates was dramatically increased to 80% [23]. Therefore, the CNSAb isolate is becoming a major concern as a nosocomial infection in China.
In this study, 33 non-duplicate A. baumannii isolates were isolated from patients hospitalized in our medical ICU. All of these isolates were confirmed by 16S-23S rRNA internal spacer analysis as previously described [17]. Among these isolates (26 from lower respiratory tract, 4 from urinary tract, and 2 from blood, and 1 from pus), 54.5% (18/33) isolates were resistant to either imipenem, meropenem, or both. The resistance rate of colistin was 3% (1/33), which coincides with the observation that colistin is the most effective antibiotic to treat A. baumannii infections in our area. From a nationwide surveillance conducted during 2007, the resistance rates of imipenem and meropenem in China were calculated to be 40% and 35%, respectively [22]. However, from a new surveillance report generated in 2010, the prevalence of CNSAb isolates was dramatically increased to 80% [23]. Therefore, the CNSAb isolate is becoming a major concern as a nosocomial infection in China.
2. MLST and clonal relationship of A. baumannii isolates According to the MLST, 33 A. baumannii isolates can be grouped into 5 existing STs (ST92, 1-3-3-2-2-7-3; ST75, 1-3-3-2-2-11-3; ST112, 1-12-56-36-4-61-26; ST145, 21-35-2-28-1-52-4; ST345, 46-12-110-1-16-141-50) and 1 new ST, which has a G-A mutation at nt268 on the rpoD40 loci of ST251(32-48-54-35-1-11-40) (Table 1). All CNSAb isolates comprised of ST92 and ST75 only, accounting for 77.8% (14/18) and 22.2% (4/18), respectively.
Clonal relation analysis showed that both ST92 and ST75 belong to the CC92, which is a globally disseminated CC for A. baumannii (Fig. 1). This result suggests that CC92 is the major clone that spreads in the ICU of our hospital. eBURST analysis [21] showed that ST92 is the founder of CC92. Previous studies [14, 15, 24] showed that several STs exist, e.g., ST75, ST88, ST90, ST136, ST137, and ST138, which are SLVs of the founder ST92. However, we still found other types, e.g., ST381 and ST375, which are double locus variants (DLVs) of ST92.
According to the MLST, 33 A. baumannii isolates can be grouped into 5 existing STs (ST92, 1-3-3-2-2-7-3; ST75, 1-3-3-2-2-11-3; ST112, 1-12-56-36-4-61-26; ST145, 21-35-2-28-1-52-4; ST345, 46-12-110-1-16-141-50) and 1 new ST, which has a G-A mutation at nt268 on the rpoD40 loci of ST251(32-48-54-35-1-11-40) (Table 1). All CNSAb isolates comprised of ST92 and ST75 only, accounting for 77.8% (14/18) and 22.2% (4/18), respectively.
Clonal relation analysis showed that both ST92 and ST75 belong to the CC92, which is a globally disseminated CC for A. baumannii (Fig. 1). This result suggests that CC92 is the major clone that spreads in the ICU of our hospital. eBURST analysis [21] showed that ST92 is the founder of CC92. Previous studies [14, 15, 24] showed that several STs exist, e.g., ST75, ST88, ST90, ST136, ST137, and ST138, which are SLVs of the founder ST92. However, we still found other types, e.g., ST381 and ST375, which are double locus variants (DLVs) of ST92.
3. Genetic determinants in CNSAb isolates No blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-24-like, and blaOXA-58-like genes were observed among these isolates, and no MBLs producing these isolates were detected by the EDTA double-disk synergy test. Most (94.4%, 17/18) ST92-CNSAb isolates harbor the blaOXA-23 gene, as shown in Table 2. Interestingly, there was 1 ST92-CSAb isolate that could be detected as being positive for the blaOXA-23 gene. However, ISAba1
blaOXA-23 gene mapping showed that no ISAba1 gene could be detected in this isolate. Therefore, this result confirmed the importance of the ISAba1 gene in A. baumannii isolates, which exhibit the carbapenem resistance phenotype. ISAba1 was detected in most (88.2%, 15/17) blaOXA-23-positive isolates, which are located upstream of blaOXA-23. The blaOXA-51-like gene was detected in all isolates in this study, which is consistent with the blaOXA-51-like gene as an intrinsic carbapenemase. However, there were no ISAba1 genes located with the blaOXA-51-like gene as determined by PCR mapping in CSAb isolates. All blaOXA-51-like genes were confirmed as blaOXA-66 genes by sequencing of CNSAb isolates.
Integron analysis revealed that all CNSAb isolates have a class 1 integrase (Table 2). When comparing the antibiotic resistance profiles (Table 1), most antibiotics showed resistance rates that were much higher in the int1-positive isolates than in the negative ones. Integron gene cassette analysis showed that there were 3 distinct antibiotic gene arrays, i.e., aacA4-catB8-aadA1 (77.3%, 17/22; Genebank accession no. EU340419), aacA4 (22.7%, 5/22; Genebank accession no. EU523056), and aacC1-orfX-orfX'-aadA1 (4.5%, 1/22; Genebank accession no. AY307113). Functional analysis showed that these genes were only associated with aminoglycoside and chloramphenicol resistance, which are not related to carbapenem resistance. It seems that carbapenem-resistant genes might not be transmitted via integron structures in our area.
No blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-24-like, and blaOXA-58-like genes were observed among these isolates, and no MBLs producing these isolates were detected by the EDTA double-disk synergy test. Most (94.4%, 17/18) ST92-CNSAb isolates harbor the blaOXA-23 gene, as shown in Table 2. Interestingly, there was 1 ST92-CSAb isolate that could be detected as being positive for the blaOXA-23 gene. However, ISAba1
blaOXA-23 gene mapping showed that no ISAba1 gene could be detected in this isolate. Therefore, this result confirmed the importance of the ISAba1 gene in A. baumannii isolates, which exhibit the carbapenem resistance phenotype. ISAba1 was detected in most (88.2%, 15/17) blaOXA-23-positive isolates, which are located upstream of blaOXA-23. The blaOXA-51-like gene was detected in all isolates in this study, which is consistent with the blaOXA-51-like gene as an intrinsic carbapenemase. However, there were no ISAba1 genes located with the blaOXA-51-like gene as determined by PCR mapping in CSAb isolates. All blaOXA-51-like genes were confirmed as blaOXA-66 genes by sequencing of CNSAb isolates.
Integron analysis revealed that all CNSAb isolates have a class 1 integrase (Table 2). When comparing the antibiotic resistance profiles (Table 1), most antibiotics showed resistance rates that were much higher in the int1-positive isolates than in the negative ones. Integron gene cassette analysis showed that there were 3 distinct antibiotic gene arrays, i.e., aacA4-catB8-aadA1 (77.3%, 17/22; Genebank accession no. EU340419), aacA4 (22.7%, 5/22; Genebank accession no. EU523056), and aacC1-orfX-orfX'-aadA1 (4.5%, 1/22; Genebank accession no. AY307113). Functional analysis showed that these genes were only associated with aminoglycoside and chloramphenicol resistance, which are not related to carbapenem resistance. It seems that carbapenem-resistant genes might not be transmitted via integron structures in our area.
1. Characteristics of A. baumannii isolates:
In this study, 33 non-duplicate A. baumannii isolates were isolated from patients hospitalized in our medical ICU. All of these isolates were confirmed by 16S-23S rRNA internal spacer analysis as previously described [17]. Among these isolates (26 from lower respiratory tract, 4 from urinary tract, and 2 from blood, and 1 from pus), 54.5% (18/33) isolates were resistant to either imipenem, meropenem, or both. The resistance rate of colistin was 3% (1/33), which coincides with the observation that colistin is the most effective antibiotic to treat A. baumannii infections in our area. From a nationwide surveillance conducted during 2007, the resistance rates of imipenem and meropenem in China were calculated to be 40% and 35%, respectively [22]. However, from a new surveillance report generated in 2010, the prevalence of CNSAb isolates was dramatically increased to 80% [23]. Therefore, the CNSAb isolate is becoming a major concern as a nosocomial infection in China.
2. MLST and clonal relationship of A. baumannii isolates:
According to the MLST, 33 A. baumannii isolates can be grouped into 5 existing STs (ST92, 1-3-3-2-2-7-3; ST75, 1-3-3-2-2-11-3; ST112, 1-12-56-36-4-61-26; ST145, 21-35-2-28-1-52-4; ST345, 46-12-110-1-16-141-50) and 1 new ST, which has a G-A mutation at nt268 on the rpoD40 loci of ST251(32-48-54-35-1-11-40) (Table 1). All CNSAb isolates comprised of ST92 and ST75 only, accounting for 77.8% (14/18) and 22.2% (4/18), respectively.
Clonal relation analysis showed that both ST92 and ST75 belong to the CC92, which is a globally disseminated CC for A. baumannii (Fig. 1). This result suggests that CC92 is the major clone that spreads in the ICU of our hospital. eBURST analysis [21] showed that ST92 is the founder of CC92. Previous studies [14, 15, 24] showed that several STs exist, e.g., ST75, ST88, ST90, ST136, ST137, and ST138, which are SLVs of the founder ST92. However, we still found other types, e.g., ST381 and ST375, which are double locus variants (DLVs) of ST92.
3. Genetic determinants in CNSAb isolates:
No blaIMP-type, blaVIM-type, blaSIM-1, blaOXA-24-like, and blaOXA-58-like genes were observed among these isolates, and no MBLs producing these isolates were detected by the EDTA double-disk synergy test. Most (94.4%, 17/18) ST92-CNSAb isolates harbor the blaOXA-23 gene, as shown in Table 2. Interestingly, there was 1 ST92-CSAb isolate that could be detected as being positive for the blaOXA-23 gene. However, ISAba1
blaOXA-23 gene mapping showed that no ISAba1 gene could be detected in this isolate. Therefore, this result confirmed the importance of the ISAba1 gene in A. baumannii isolates, which exhibit the carbapenem resistance phenotype. ISAba1 was detected in most (88.2%, 15/17) blaOXA-23-positive isolates, which are located upstream of blaOXA-23. The blaOXA-51-like gene was detected in all isolates in this study, which is consistent with the blaOXA-51-like gene as an intrinsic carbapenemase. However, there were no ISAba1 genes located with the blaOXA-51-like gene as determined by PCR mapping in CSAb isolates. All blaOXA-51-like genes were confirmed as blaOXA-66 genes by sequencing of CNSAb isolates.
Integron analysis revealed that all CNSAb isolates have a class 1 integrase (Table 2). When comparing the antibiotic resistance profiles (Table 1), most antibiotics showed resistance rates that were much higher in the int1-positive isolates than in the negative ones. Integron gene cassette analysis showed that there were 3 distinct antibiotic gene arrays, i.e., aacA4-catB8-aadA1 (77.3%, 17/22; Genebank accession no. EU340419), aacA4 (22.7%, 5/22; Genebank accession no. EU523056), and aacC1-orfX-orfX'-aadA1 (4.5%, 1/22; Genebank accession no. AY307113). Functional analysis showed that these genes were only associated with aminoglycoside and chloramphenicol resistance, which are not related to carbapenem resistance. It seems that carbapenem-resistant genes might not be transmitted via integron structures in our area.
DISCUSSION:
In this study, we analyzed the molecular typing and characteristics of A. baumannii isolates in our ICU. From the MLST result, the predominant clone is ST92 in CNSAb isolates in our hospital, and this observation is also consistent with previous studies [14-16, 25], which revealed that ST92 has spread worldwide. However, limited diversity was observed in CNSAb isolates in our ICU, while there was a higher diversity in CSAb isolates. Interestingly, there were still 8 CSAb isolates being assigned as ST92, but none of the CSAb isolates belonged to ST75. This characteristic indicates that these 2 types might exhibit differences in clinical outcomes or have antibiotic resistance patterns. Although there were no significant differences in antibiotic resistance rates for the ST92 and ST75 CNSAb isolates, they still exhibited slight differences on their MIC ranges. As shown in Table 2, all ST92 CNSAb isolates were resistant to imipenem, with MICs ranging from 32-128 mg/L; most of them (71.4%, 10/14) were also resistant to meropenem with the same MIC range. However, the MIC of the imipenem-resistant ST75 isolates ranged from 64 to 128 mg/L, which is a more narrow resistance profile than that of ST92 (Table 2). However, there was no difference in the MIC range of meropenem for these 2 types. These results indicate that ST75 might have a more severe imipenem resistance range than ST92.
Genetic analysis showed that ST75 is the single locus variant (SLV) of ST92, which differs in its gpi loci. While ST92 isolates show moderate carbapenem resistance rates, all ST75 types are resistant to carbapenem with a higher MIC range. Therefore, we hypothesized that mutations of the gpi loci in ST92, e.g., ST75, might benefit its survival when encountering carbapenem selection. In contrast, in our collection, there were some ST92-type CSAb isolates, but none was ST75, which also inferred that ST75 CNSAb isolates might represent a severe epidemic marker in nosocomial infections in our area. Moreover, a study by He et al. [15] showed that more ST138-CNSAb isolates are associated with ST92 in western China, which could give rise to the interesting assumption that the composition of CNSAb STs could exhibit geographic differences. These differences might be caused by different antibiotic usage habits, which may influence the evolution direction of ST92. Therefore, ST75 accompanied by ST92 might be the epidemical feature in eastern China.
In this study, we observed more diversity in CSAb isolates (5 ST types) than in CNSAb isolates (only 2 ST types). Interestingly, we observed some of CSAb isolates is ST92-CSAb type, and this type strain is the majority component in the epidemic CNSAb clonal CC92. While the other 4 STs, including ST112, ST145, ST345, and STn are singleton in the A. baumannii population, which shows no relation with epidemic strain CC92. As is known, natural selection is the major force that shapes the population structure of microbial communities in certain environments. A higher diversity in CSAb isolates might be due to fewer encounters with natural selection forces, e.g., antibiotics. Antibiotic resistance profiles showed that most antibiotics caused more resistance in the ST92-CNSAb group than in the ST92-CSAb group (Table 1). However, ST92-CSAb strains could potentially be a source of CNSAb strains, having acquired carbapenem resistance. Therefore, ST92-CSAb strains should be considered as important along with CNSAb isolates for future nosocomial infection control.
Genetic determinants analysis shows MBLs are not the major resistant determinants in CNSAb isolates in our hosptial, which is consistent with previous study [26]. However, in some distinct areas of China, the MBL plays a more important role than the OXA-type carbapenemase in CNSAb [27]. This implies that a geographical characteristic plays an important role in CNSAb isolates formation, and it could be due to different antibiotic usage in distinct areas. Integron analysis shows a high prevalence of this gene capture machinery. Although in this study, we only recovered 3 type of class 1 integron cassette, there still shows integron is highly associated with antibiotic resistance. However, there are some limitations in this integron cassette analysis method, e.g., some gene cassettes failed to be recovered due to the lack of a conserved terminal [28]. However, there are several studies [29, 30] showing that there are multiple gene cassettes that contain the OXA gene in A. baumannii isolates. This might indicate that transmission of the carbapenemase resistant gene might be mediated by a very complex mechanism. Notably, whole genome sequencing of MDRAbs [31-34] revealed a correlation between carbapenem resistance and the integron component. Most MDRAb strains harbor an antibiotic resistance island (AbaR) in their genome, which provides great potential for antibiotic resistance. Moreover, in the present study, we observed that these gene cassettes are shared among these strains and other species from the integron database [29], which provides a clue for the transmission of integrons among inner- and interspecies. Interestingly, several environmental isolates in our ICU possessed a class 1 integrase from a previous collection, while 1 isolate was detected with an empty gene cassette (data not shown). Similarly, there is a recent report that shows 1 environmental isolate of P. aeruginosa with an empty cassette [35]. This empty cassette potentially represents a reservoir with an increased capacity to adapt to the environment.
In conclusion, we found that there are 2 predominant distinct clones (ST92 and ST75) of CNSAb isolates, which clustered to CC92 in our medical ICU. ST92-A. baumannii is considered to be the clone with a worldwide dissemination, while ST75 could be another major clone accompanying the CNSAb isolates in our region. Moreover, the ST75 clone might act as a severe epidemic marker in the carbapenem-resistant A. baumannii isolates in our area. Therefore, CC92-CNSAb plays an important role in causing nosocomial infections in our medical ICU and thus, should be investigated as part of hospital infection control in the future.
|
Background:
This study was aimed to investigate the genetic diversity and antibiotic resistance profile of the nosocomial infection agent Acinetobacter baumannii from a medical intensive care unit (ICU) in a teaching hospital in Suzhou, China.
Methods:
The genetic relationship among A. baumannii isolates in an ICU was investigated using multilocus sequence typing (MLST). The antibiotic resistance pattern was determined by performing an antibiotic susceptible test, which included an agar dilution method and an E-test method. Resistant determinants, e.g., carbapenemase genes, metallo-β-lactamases, and class 1 integron, were analyzed by specific PCR and DNA sequencing.
Results:
In the present study, 33 non-duplicate isolates were identified as 5 existing sequence types (STs) (ST92, ST75, ST112, ST145, and ST345) and 1 new sequence type STn, which has a G-A mutation at nt268 on ropD40 of ST251. These results reveal limited diversity in carbapenem non-susceptible A. baumannii (CNSAb) isolates in our ICU, which are comprised of only 2 distinct STs, with ST92 and ST75 clustering into a clonal complex (CC) 92. Most CNSAb isolates (94.4%, 17/18) harbored the OXA-23 gene, while no carbapenem-susceptible A. baumannii (CSAb) isolates harbored it. In addition, 66.7% (22/33) isolates were positive for class 1 integrase, and gene cassette analysis showed there are 3 gene arrays among them, i.e., aacA4-catB8-aadA1 (77.3%, 17/22), aacA4 (22.7%, 5/22), and aacC1-orfX-orfX'-aadA1 (4.5%, 1/22).
Conclusions:
When all these data are combined, the antibiotic resistance and wide distribution of CNSAb isolates in our ICU are probably caused by expansion of the CC92 clone.
| null | null | 6,417 | 344 | 11 |
[
"isolates",
"blaoxa",
"st92",
"gene",
"baumannii",
"cnsab",
"resistance",
"like",
"analysis",
"genes"
] |
[
"test",
"test"
] | null | null |
[CONTENT] MLST | Molecular epidemiology |
Acinetobacter baumannii
[SUMMARY]
|
[CONTENT] MLST | Molecular epidemiology |
Acinetobacter baumannii
[SUMMARY]
|
[CONTENT] MLST | Molecular epidemiology |
Acinetobacter baumannii
[SUMMARY]
| null |
[CONTENT] MLST | Molecular epidemiology |
Acinetobacter baumannii
[SUMMARY]
| null |
[CONTENT] Acinetobacter baumannii | Anti-Bacterial Agents | Bacterial Proteins | Carbapenems | China | Drug Resistance, Bacterial | Hospitals, Teaching | Humans | Integrases | Intensive Care Units | Microbial Sensitivity Tests | Mutation | Polymerase Chain Reaction | Sequence Analysis, DNA | beta-Lactamases [SUMMARY]
|
[CONTENT] Acinetobacter baumannii | Anti-Bacterial Agents | Bacterial Proteins | Carbapenems | China | Drug Resistance, Bacterial | Hospitals, Teaching | Humans | Integrases | Intensive Care Units | Microbial Sensitivity Tests | Mutation | Polymerase Chain Reaction | Sequence Analysis, DNA | beta-Lactamases [SUMMARY]
|
[CONTENT] Acinetobacter baumannii | Anti-Bacterial Agents | Bacterial Proteins | Carbapenems | China | Drug Resistance, Bacterial | Hospitals, Teaching | Humans | Integrases | Intensive Care Units | Microbial Sensitivity Tests | Mutation | Polymerase Chain Reaction | Sequence Analysis, DNA | beta-Lactamases [SUMMARY]
| null |
[CONTENT] Acinetobacter baumannii | Anti-Bacterial Agents | Bacterial Proteins | Carbapenems | China | Drug Resistance, Bacterial | Hospitals, Teaching | Humans | Integrases | Intensive Care Units | Microbial Sensitivity Tests | Mutation | Polymerase Chain Reaction | Sequence Analysis, DNA | beta-Lactamases [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] isolates | blaoxa | st92 | gene | baumannii | cnsab | resistance | like | analysis | genes [SUMMARY]
|
[CONTENT] isolates | blaoxa | st92 | gene | baumannii | cnsab | resistance | like | analysis | genes [SUMMARY]
|
[CONTENT] isolates | blaoxa | st92 | gene | baumannii | cnsab | resistance | like | analysis | genes [SUMMARY]
| null |
[CONTENT] isolates | blaoxa | st92 | gene | baumannii | cnsab | resistance | like | analysis | genes [SUMMARY]
| null |
[CONTENT] integron | class | baumannii | carbapenem | infections | dissemination | typing | oxa type | oxa | molecular [SUMMARY]
|
[CONTENT] blaoxa | like | pcr | usa | isolates | test | like blaoxa | http | antimicrobial susceptibility | susceptibility [SUMMARY]
|
[CONTENT] isolates | blaoxa | gene | showed | 22 | st92 | like | resistance | detected | cnsab isolates [SUMMARY]
| null |
[CONTENT] isolates | blaoxa | st92 | like | gene | cnsab | resistance | baumannii | genes | st75 [SUMMARY]
| null |
[CONTENT] Acinetobacter baumannii | ICU | Suzhou | China [SUMMARY]
|
[CONTENT] ICU ||| ||| 1 | PCR [SUMMARY]
|
[CONTENT] 33 | 5 | ST112 | ST145 | ST345 | 1 | nt268 | ST251 ||| ICU | only 2 | CC | 92 ||| 94.4% | 17/18 ||| 66.7% | 22/33 | 1 | 3 | 77.3% | 17/22 | 22.7% | 5/22 | 4.5% | 1/22 [SUMMARY]
| null |
[CONTENT] Acinetobacter baumannii | ICU | Suzhou | China ||| ICU ||| ||| 1 | PCR ||| ||| 33 | 5 | ST112 | ST145 | ST345 | 1 | nt268 | ST251 ||| ICU | only 2 | CC | 92 ||| 94.4% | 17/18 ||| 66.7% | 22/33 | 1 | 3 | 77.3% | 17/22 | 22.7% | 5/22 | 4.5% | 1/22 ||| ICU [SUMMARY]
| null |
||||
Factors associated with adolescent school girl's pregnancy in Kumbo East Health District North West region Cameroon.
|
31037198
|
Teenage pregnancy is a social problem in Cameroon in general and in Kumbo East in particular. This results in physical, psychological and socio-economic consequences on the teenage mother, family and the society as a whole. In spite of studies and interventions that have been and are being implemented, the prevalence of unplanned teenage pregnancy in Kumbo East Health District is still high, suggesting that more efforts are required to achieve effective preventive measures. The aim of this study was to determine factors associated with adolescent school girl's pregnancy in Kumbo East health district.
|
INTRODUCTION
|
A cross-sectional descriptive study design was used and a simple random sampling technique was used to select 293 respondents aged 15 to 19year. The district hospital antenatal clinics and the Health Centres were selected. Data was obtained from 292 participants under the age of 20 years who were willing using a questionnaire administered through face-to-face interviews.
|
METHODS
|
The study show a high prevalence (60.75%) of teenage pregnancy in the sampled antenatal clinics of Kumbo East Health District attributable to inadequate considerations given to factors associated with school girl's pregnancy. This study has indicated that the age of teenager at first pregnancy, low contraceptive use, socio-economic status and physical violence are factors that are greatly associated with teenage pregnancy. Among the reasons contributing to the low use of contraceptives are: sexually activity, lack of knowledge, fear of side effects, including sterility, condoms disappearing in the womb and inequality of power with sexual partners. This study shows that teenagers obtain information mainly from school (53%) and relatives (20%).
|
RESULTS
|
The use of contraceptive alone may not reduce teenage pregnancy, however double method is very effective but addressing the impact of poverty on teenagers, empowering them on their rights and information in order to make right choices is very important.
|
CONCLUSION
|
[
"Adolescent",
"Cameroon",
"Contraception",
"Contraception Behavior",
"Cross-Sectional Studies",
"Female",
"Health Knowledge, Attitudes, Practice",
"Humans",
"Poverty",
"Pregnancy",
"Pregnancy in Adolescence",
"Prevalence",
"Sexual Behavior",
"Socioeconomic Factors",
"Surveys and Questionnaires",
"Young Adult"
] |
6462492
|
Introduction
|
Teenage pregnancy is considered as one occurring in a young woman who has not reached her 20th birthday. This definition is applicable irrespective of the legal status of the marriage of the woman or legal age to consider an individual as adult [1]. 16 million girls aged 15-19 years give birth each year, most prevalent in low and middle-income countries located in sub-Saharan Africa [1]. In the developing world, one-third to one-half of women become mothers before the age of 20 and pregnancy related complications have become the leading causes of death among them [1, 2]. Teenage pregnancies are a global phenomenon. The pregnancy rate among teenagers in USA was 6.78% of pregnancies per 1,000 women aged 15-19 in 2008. [3]. Among the countries in the Western Europe, the United Kingdom [UK] has the highest teenage conception and abortion rates [2, 4]. The report presents an update on the current situation of pregnancies among girls less than 18 years of age and adolescents 15-19 years of age; trends during the last 10 years; variations across geographic, cultural and economic settings; interventions available to minimize pregnancy among adolescents; evidence for these programmatic approaches; and challenges that nations will have to deal with in the next 20 years given current population momentum [5]. The concentration of adolescent girls aged 10 to 17 will also change significantly, with the largest increase occurring in sub-Saharan Africa, where adolescent pregnancy is most common, and the rate of contraceptive use the lowest in the world [5]. A study conducted in Malawi showed that 57% of teenage girls opt to risk pregnancy rather than asking a partner to use a condom [2]. In Malawi, there is a high prevalence of casual sex among teenagers who shun condoms although they engage in multiple relationships. Scholars in the field argue that, because of the risk associated with high prevalence of early sexual behaviour, low contraceptive use, and many early pregnancies, adolescents in Cameroon are an important target group for sexual and reproductive health programs [6, 7]. In order to prevent early age pregnancies, it is important to make sure that adolescents have the means to make informed and healthy choices concerning their sexual and reproductive health. Yet, as it stands, reproductive health and family planning services in Cameroon mainly target older married women, and adolescents often remain largely overlooked [5].
Adolescent school girl's pregnancies vary from country to country and in Cameroon, reproductive health remains a major public health challenge with elevated maternal mortality rate. The maternal mortality rate is estimated at 782 deaths per 100,000 live births [8]. This high mortality rate remains a dilemma because it involves the young mothers at the moment where they are giving birth. In addition, adolescent contribute 28% of maternal mortality in Cameroon [8]. According to DHS 2011, 25.6% of adolescents 15 to 19 have started sexual intercourse and 21% of then have had a child and 4% are pregnant for their first pregnancy. Still from the same source fertility rate of this age group is 127‰ which increase rapidly and attained a maximum of 250‰ within the age group 25-29yrs, suggesting that teenage pregnancy may be on the increase. The proportion of adolescents who have started fertility grow rapidly with age, 5% for 15yrs to 48% for 19yrs and the rate stood at 18% in the north west region [8]. Therefore, reducing teenage pregnancy, chiefly by promoting the use of contraceptives, will be necessary in order to prevent consequences that are associated with teenage pregnancy. Nevertheless, lessons from Zimbabwe and South Africa suggest that promoting the use of contraceptives alone does not necessarily reduce teenage pregnancy in developing as in developed countries. Therefore, other factors may be playing a major role as mentioned earlier. The general objective was to determine factors associated with adolescent school girl's pregnancy in Kumbo East Health District.
|
Methods
|
A crossectional descriptive study was selected because it incorporates a descriptive component that enables the calculation of the prevalence of teenage pregnancy in antenatal clinics of KEHD. The socio-economic status where measured by asking respondents questions concerning their househood items they possessed; bicycle, car , family income were collected and the knowledge was assessed by structured questions to evaluate the knowledge of respondents to factors associated with teenage pregnancy. The questionnaire was adopted from WHO standard guide and from the research of other researchers. The questionnaire was then pre-tested and adjusted to fit the context. Four midwives' students who were in their final year and had some experience in data collection assisted in the study. They were given orientation on the process for data collection and management prior to the commencement of field studies. They were trained for a period of three days in the administering of the questionnaire and research ethics, with an emphasis on informed consent. Each research assistant was given a code that was used for identification in case of any queries. Data was then collected entered into the computer and stored in external hard drive and USB keys. Data was then assembling and the analysis started. Our research period was for 10 months with the study population consisting of all school girl's pregnant aged [15-19yrs] from Kumbo East Health District. The sample size were then pregnant teens (15 to 19yrs) attending antenatal clinics in selected health centres in Kumbo East Health District at the time of data collection. Antenatal and infant welfare clinics were used in order to access pregnant adolescents.
Inclusion criteria: All school teens age 15 to 19 yrs who are pregnant who attend: ANC consultation irrespective of their trimester at SEGH and selected health centre, infant welfare clinics, and who give their consent.
Exclusion criteria: Pregnant teens who came to the clinic and didn't give their consent were excluded. Although teenage pregnancy concerns both sexes, male teenagers were excluded as they rarely attend antenatal services with their partners.
Sample method and sample type: The sampling method used was a simple random technique; the names of the health centres within was written on a piece of paper and placed in a container, four names were picked at random from the container. Jakiri integrated health centre, sub-divisional hospital Jakiri, St. Jude clinic and Shisong General Hospital. Our sample type was a convenience sampling. The Kumbo East District Hospital [Shisong] represents urban teenage mothers who may have experienced different factors from the rural setting. To obtain an adequate sample, all pregnant women visiting the antenatal clinics and infant welfare clinics over a three-month period at the selected sites under the age of 20 were interviewed.
Sample size in a cross sectional study: To calculate our sample size, we are going to use the Lorenz formula which stipulates, P= Prevalence of adolescent 15 to 19 (p = 0.25), t = confidence level [95%= 1.96], e = error margin [0.05], N = Sample size.
Statistical application of this formula, resulted to a sample size of 293, implying that the study will recruit a total of 293 participants from the selected hospitals.
Data collection technique: Data was collected over a period of 3 months from August to October 2017, because of the Anglophone crisis with numerous “ghost towns” that reduce Antennal consultation by pregnant women difficult and hence leading to a long period of time to collect data. Using a standardized questionnaire administered through face-to-face interviews. The Questionnaire consisted mostly of closed questions. Each questionnaire took 20-40 minutes to administer. There were no refusals - all participants willingly consented to participate in the study. The arrangements for confidentiality and privacy ensured that respondents accepted to be interviewed and spoke more freely on the subject. In addition, it was also possible that respondents felt that there was somebody to listen to their problems. The face-to-face interviews facilitated responses and the quality of information and this method was convenient since most of the participants had low literacy levels.
Statistical consideration: Data collected were entered into the CDC-Epi-Info version 7.2.2.2, transferred to MS-Excel and then exported to SPSS version 21.0 software. Data cleaning was performed with MS-Excel to check for inconsistencies in data entry and responses, prior to analyses. Associations between planed [Yes/ No] pregnancy and various variables were evaluated using Pearson and Yate's chi square [φ2] tests. Measures of association; OR and Pearson's chi square [φ2] tests were calculated by use of CDC-Epi-info version 7.2.2.2 and SPSS version 21.0 for the establishment of associations or differences (un) planed pregnancy and various variables.
Ethical considerations: in order to protect the rights of the interviewees and meet requirements for research involving people, clearances were obtained from authorities and informed consent from the participants. The authorization was also obtained from the North West Regional Delegation of Health. Ethical clearances were obtained from the Committee for Research on Human Subjects of the Catholic University of Central Africa (UCAC). Clearances to use health facilities were also obtained from the Kumbo East District Health Office. The following points were clarified: participation in the study was voluntary; participants were free to withdraw at any time without coercion and there were neither direct benefits nor known risks at any time. To ensure anonymity and privacy, numbers were used instead of names on the questionnaires. Interviews were conducted in private rooms which were provided at the clinics for this purpose. The completed questionnaires that were not entered to the computer were locked in a cupboard accessible to the researcher only to ensure confidentiality.
|
Results
|
Socio-demographic characteristics: A total of 293 participants of average age 18.88yrs were sampled from 13 shortlisted localities of KEHD; Jakiri had the highest proportion 65 [22.18%] of participants while Noi and Tan had the lowest 11 [3.75%] each. Out of the 293 participants, 154 [52.56%] admitted currently schooling. Out of the 293 ladies sampled, 153 (52.22%) were single. Out of 293 sampled in the study, 175 [59.73%] who had not delivered, while the remainder had delivered at least a child, either in marriage or out of marriage. Socio-demographic data was collected for variables such age, marital status, alcohol and drug/substance attitudes, as well as information concerning partner (Table 1).
Demographics; age, religion, school attendance and parity
ATR = African Traditional Religion
Sources of information on sexual and reproductive health: Despite the diversity of the sources of information on a variety of issues, 20.48% and 2.05% claimed ignorance of information on sex and sexuality as well as the use of contraceptives. However, a majority of the participants; 79.52% and 97.95% had at least one of the variety of sources of information on sex and sexuality and use of contraceptives. A majority of the participants had learned of sex and sexuality as well as use of contraceptives from school; 38.20% and 52.90%, respectively. Only 13.31%, of mothers educated their daughters on sex and sexuality. Books, Counsellors, local radio and peers respectively served as 6.83%, 19.80%, 13.65% and 20.48%, sources of information on sex and sexuality to adolescents. Out of the 293 respondents, 11.60%, 13.31%, and 20.14% had learned of contraceptives from friends, hospital, and relatives respectively (Table 2).
Sources of information on SRH and contraceptive use
Dichotomy group tabulated at value 2
Knowledge of information on sexual and reproductive health (SRH): Considering the respondents answers to question ask on whether their menstrual and reproductive characteristics , their knowledge of information on sexual and reproductive health (SRH) was assessed, a majority of the respondents; 66.2%, 66.6%, 59.7% and 93.2% out rightly admitted that it is true that; amenorrhoea leads to accumulation of dirt and sickness, disappearance of a condom into a woman during sex, most pregnancies occur in the middle of the menstrual cycle as well as, one can become pregnant during the first sexual intercourse. Another majority; 93.2%, 53.2%, 65.9% and 53.6% actually admitted as outright false the fact that; frequent sex prevents pregnancy, sterility arises from the use of contraceptive use, washing of genitals after sex prevents pregnancy and the consumption of sachet whisky prevents conception (Table 3).
Knowledge of information on sexual and reproductive health (SRH)
Factors associated with unplanned teenage pregnancy in KEHD: A list of factors were provided in the questionnaire for the respondent to tick and out of the 293 respondents, 61.09% claimed never to have used the condom, and 52.56% said their partner was not the father of their child. 46.42% of the 293 participants said they do not use contraceptives, 13.65% failed to disclose the method of contraceptive used, by simply saying “none of the above”, when asked, when they started using contraceptive, 58.02%, said they have never used contraceptives (Table 4).
Factors associated with adolescent school girl’s pregnancy
Lack of knowledge on sexual reproductive health, circumstances of first sex, physical, sexual and substance abuse: From the answers provided by the respondent concerning knowledge assessment, 20.48% of the respondents had information on sex and reproductive health from their peers and, another 20.8% could not really identify their source of information on sex and reproductive health. 11.60% and 2.05% of the respondents learned of how to use contraceptives from friends and no defined sources (Table 5).
Circumstances of first sex, first sexual experience, relationship control and duration of relation
Characteristics of sexual partner and gender power relations in partnership: The sexual partners of respondents were of the age range 16-32 years (mean age of 22.815). At their age, a majority 67.24% of the respondents claimed their partners do not use drugs, while 6.49%, 15.02% and 3.75% respectively claimed their partners; do any injectable drugs, any other drugs and marijuana. On alcohol consumption frequency; 49.83% claimed not to know the drinking rate of their spouses, 7.85%, 20.82% and 21.50% said their partners drink at the rate of; only at weekend, less than once a month and a few times a month respectively (Table 6).
Alcohol/ drug attitude of partner
|
Conclusion
|
The study factors associated with adolescent school girl's pregnancy in KEHD was aimed at examining the factors associated with teens pregnancy. A descriptive study was used and the teens 15 to 19yrs were selected. Teenage pregnancy whether planned or unplanned is detrimental to the health and socio economic status of the teenagers. This study shows that there is a high prevalence (60.75%) of teenage pregnancy in the sampled antenatal clinics of Kumbo East Health District attributable to the low contraceptive use and socio-economic status, lack of reproductive and sexual knowledge, circumstances at first sex, including force and rape, physical and sexual violence, early marriage, age of sexual partner and alcohol abuse. Among reasons contributing to the low use of contraceptives are; lack of knowledge, fear of side effects, including sterility, condoms disappearing in the womb and inequality of power with sexual partners. Teenagers obtain information mainly from school (53%) and relatives (20%). The high prevalence of unplanned teenage pregnancy, low contraceptive use, myths and misconceptions surrounding the use of contraceptives indicate that teenagers are receiving inaccurate information about reproductive and sexual health, a problem which may be compounded by low educational levels. Therefore, the lack of knowledge about reproductive health is a factor for teenage pregnancy. The lack of basic necessities is one of the factors that force teenagers to engage in transactional sex. Based on the study it is clear that teenagers have no control over sexual and reproductive health since their sexual partners are the sore decision makers making physical and sexual violence a factor for teenage pregnancy. However, the influence of cultural practices and religion, substance abuse, relationship control, and fear of parents and intimidating attitude of health service providers have been supported as factors. The associations suggested by the study point towards a need for greater emphasis in reproduction and sexual health promotion interventions. In addition, strengthening awareness and giving information to dispel fears, misconceptions and rumours about contraceptive use will prepare teenagers for the physical changes they might experience when adopting contraceptive methods. Nevertheless, contraceptive use alone may not reduce teenage pregnancy, but addressing the impact of poverty on teenagers, empowering them on their rights and information in order to make right choices is very important.
Recommendations: The study recognizes efforts being made by government and non-governmental organizations to solve the problem of teenage pregnancy in north west region and Kumbo East in particular. These efforts include promoting the use of contraceptives, education of girls and poverty alleviation. However, the low use of contraceptives and socio-economic status, continued sexual and physical violence, the feeling of inferiority due to age differences with sexual partners and early marriages indicate that factors for teenage pregnancy still persist in the study area. Therefore, there is a need to develop programmes to address factors identified in this study. Solutions to the problem require multidisciplinary implementing teams, including parents, schools, communities, NGOs and government sectors. The following recommendations are suggested.
What is known about this topic Knowledge of this geographic distribution can provide useful information for updating and strengthening adolescent reproductive health strategies in Cameroon;
In Cameroon, the prevalence of adolescent pregnancy is between 40% to 50%;
Adolescent age 15 to 19 contribute to 28% of maternal mortality in Cameroon.
Knowledge of this geographic distribution can provide useful information for updating and strengthening adolescent reproductive health strategies in Cameroon;
In Cameroon, the prevalence of adolescent pregnancy is between 40% to 50%;
Adolescent age 15 to 19 contribute to 28% of maternal mortality in Cameroon.
What this study adds Study will help provide few answers to this problem and may be prick health leaders to react towards the amelioration of some of their health strategies concerning reproductive health especially adolescent health in rural areas of Cameroon;
Moreover, it will also go a long way to convince national and international partners to continue to largely invest in the reproductive health section;
This will stand as a pillar to help adolescent to adopt responsible behaviours in regard to adolescent pregnancy especially at individual, community and health systems levels.
Study will help provide few answers to this problem and may be prick health leaders to react towards the amelioration of some of their health strategies concerning reproductive health especially adolescent health in rural areas of Cameroon;
Moreover, it will also go a long way to convince national and international partners to continue to largely invest in the reproductive health section;
This will stand as a pillar to help adolescent to adopt responsible behaviours in regard to adolescent pregnancy especially at individual, community and health systems levels.
|
[
"General patterns of teenage pregnancy in Kumbo East Health District",
"Knowledge and sources of information on reproductive and sexual health",
"Age of the teenagers",
"Knowledge contraceptive use",
"Levels of education, cultural practices and economic factor",
"Gender power relations in partnerships",
"Lack of knowledge on sexual reproductive health, circumstances of first sex, physical, sexual and substance abuse",
"What is known about this topic",
"What this study adds"
] |
[
"The prevalence of teenage pregnancy in Cameroon is 28% and in Kumbo East Health District the results on this study shows that the prevalence of unplanned teenage pregnancy is 60.75% which very high this could be attributed to the fact that schools were not operational at the time of this study due to political unrest in the country at the time of data collection. Out of the 293 respondents, 61.09% claimed never to have used the condom, and 52.56% said their partner was not the father of their child. 46.42% of the 293 participants said they do not use contraceptives, 13.65% failed to disclose the method of contraceptive used, by simply saying “none of the above”, when asked, when they started using contraceptive, 58.02% said they have never used contraceptives. This implies that there is still a need for more efforts. Possible factors contributing to the high prevalence in this study area include age at first pregnancy, low contraceptive use, educational levels and socio-economic status, circumstances at first sex, lack of knowledge on reproductive and sexual health and physical and sexual violence.",
"Considering the respondents answers to question ask on whether their menstrual and reproductive characteristics , their knowledge of information on sexual and reproductive health [SRH], a majority of the respondents; 66.2%, 66.6%, 59.7% and 93.2% out rightly admitted that it is true that; amenorrhoea leads to accumulation of dirt and sickness, disappearance of a condom into a woman during sex, most pregnancies occur in the middle of the menstrual cycle as well as, one can become pregnant during the first sexual intercourse. Another majority, 93.2%, 53.2%, 65.9% and 53.6% actually admitted as outright false the fact that; frequent sex prevents pregnancy, sterility arises from the use of contraceptive use, washing of genitals after sex prevents pregnancy and the consumption of sachet whisky prevents conception (Table 4).",
"The ages of participants ranged from 15-27 years, with mean age 18.88 years and modal age group of 15 -< 20 years with 227 [< 77.41%]. A large majority of participants 59.73% had no child, while 32.42% had one child, 0.68% had two children, 6.14% had three children and 1.02% had four children. In Cameroon, Kenya, South Africa and Canada, the average age difference between teenagers and their sexual partners is 15, 7, 5 and 2.6 years, respectively. In addition, teenagers who marry older men often have less power in decision making around sexual intercourse, childbearing and the use of contraceptive [1].",
"On the usage of contraceptives, 15.02% used the condom, 5.12% claim to use implants, 8.19% claim to use IUCD, while 11.60% use pills. On when they began using contraceptives; 2.05% began this year, 24.23% began last year, 7.51% began in the year 2015, 2.73% began in 2014 and 2013 as well as 2012 (Table 4). Respondents also advanced possible fears and reasons for not using contraceptive; 6.83, were scared of nurses, 20.14% were scared of their parents, 6.83% claimed it was against their religion and 12.97% admitted their partners were not in favour of it (Table 1). The fears, beliefs and perceptions of sexual inactivity associated with contraceptive use may be attributed to the lack of knowledge and low levels of awareness. This is conformity with a similar study Contraceptive prevalence in Cameroon is low among married women and shows uneven distribution varying from 2.6% in North Cameroon to 43.9% in Yaoundé [6, 7]. The low prevalence use among married couples suggests that once a woman is married she is exposed to pregnancy irrespective of her age. In Cameroon, unmet needs for family planning are 22%. And the use of modern contraception in Cameroon is about 16%. Reasons for non-contraceptive use include, religious and cultural beliefs, poor quality of services, including the negative attitude of service providers, fear of exposure of their bodies, having adults at the same services and inability to negotiate contraceptive use with sexual partners. Furthermore, misconceptions, fear of side effects and stigma associated with the use of contraceptives as adolescents may be labelled as being promiscuous can also be considered as contributing factors for non-contraceptive use [1]. Similarly, in South Africa, a study revealed that teenage pregnancy is attributed to the low utilization of contraceptives, especially on their first intercourse, due to the lack of access to medical information on reproductive system, inaccessible family planning services, gender inequality and decision on the social life, fears about contraception on fertility and menstruation and condoms could be left inside the vagina or womb [4]. It was therefore envisaged that the use of contraceptives among teenagers in the study area could be low, making it a factor for pregnancy.",
"The ages of the partners of participants ranged from 16-32 years; Half, 50.17% of partners of adolescent school girls were in the age group 20 - < 25 years. 17.41% were in the age group 16 -< 20 years, 25.26% in the group 25 -< 30 years and 7.17% in the age group 30 - 32 years. With 51.88% of the 293 participant's partners had secondary school education, followed by those with tertiary education 40.27%, those with no formal education were 6.83% and primary education were 1.02%. Of the 293 participants, the partners of 44.37%, 40.96% and 14.68% were respectively studying, unemployed and working (Table 2). The study agrees that Teenage girls may indulge in sexual activity in exchange for goods, money and experiences such as taking meals in hotels [1]. A study in Malawi, found that 66% of adolescents had accepted money or gifts in exchange for sex and in some cases, parents may encourage their daughters into relationships with men for consumer goods or a girl may go out with men because her parents cannot give her the basic needs. Teenagers with unplanned pregnancy are more likely to come from low socio-economic status than with planned pregnancy [1]. The level of education of parents, especially the mother, may have an influence on the adolescent towards teenage pregnancy as she acts as a role model [9] which may be a preventive factor of teenage pregnancy. In Kenya it was reported that women with no education had first sexual intercourse three years earlier than their counterparts with at least a secondary school education [1]. The low literacy levels may lead to low paying jobs, causing early marriage and influencing non-contraceptive use, thereby increasing the prevalence of teenage pregnancy. Therefore, it was expected in the study area that the low literacy levels may have an impact on unplanned teenage pregnancy. Culturally, amongst factors associated with unplanned teenage pregnancy were, age at onset of menstruation, age at first sex, who they live with as well as the marital status of their parents. It was difficult to prove that culture is a factor. Economically, these were age, religious and delivery status of participants (Table 1), marital status of participants and parents, partner's educational and occupational status.",
"There were a variety of circumstances that could lead to sex; partner violence, reasons for involvement in a relationship and first sexual experience. For partner violence; 19.80%, had their funds managed by their partners, 33.11% were forced by their partners into sex, 20.14% would be hit by their partners and 40.27% had their funds managed by their partners. 6.48%, 13.65% and 12.97% of respondents respectively got involved in a relationship to be provided with clothes, to be married and to have a good time. For the first sexual experience; 46.42% were persuaded either by a relation or boyfriend, 6.48% were raped, 21.84% were raped, 7.51% were willing and collaborated or 17.75% refused to disclose their first sexual experience (Table 5). This means that male partner is the one who decides what happen in a relationship.",
"20.48% of the respondents had information on sex and reproductive health from their peers and, another 20.8% could not really identify their source of information on sex and reproductive health. 11.60% and 2.05% of the respondents learned of how to use contraceptives from friends and no defined sources (Table 3). Most studies conducted in developing countries report that adolescent girls often lack basic knowledge about reproductive and sexual health [1]. This study contrast with that of findings of Nanze. C.C (2006 on risk factors of unwanted/uplanned pregnancy in zomba district, Malawi) indicate that communication about reproductive and sexual matters within families is limited, forcing girls to get information chiefly from peers, boyfriends and teachers. On drug and substance use, a wide majority (80.55%, said they do not take drugs 12.97% said they took any drug, while 6.48% admitted smoking “banga” (Table 7). Similar findings have been reported in South Africa [9]. Unlike wise in Cameroon, it is not common for teenage girls to use drug substances due to cultural values. However, there may have been underreporting since drug use is illegal, making it a sensitive topic.\nCircumstances of first sex, first sexual experience, relationship control and duration of relation",
"Knowledge of this geographic distribution can provide useful information for updating and strengthening adolescent reproductive health strategies in Cameroon;\nIn Cameroon, the prevalence of adolescent pregnancy is between 40% to 50%;\nAdolescent age 15 to 19 contribute to 28% of maternal mortality in Cameroon.",
"Study will help provide few answers to this problem and may be prick health leaders to react towards the amelioration of some of their health strategies concerning reproductive health especially adolescent health in rural areas of Cameroon;\nMoreover, it will also go a long way to convince national and international partners to continue to largely invest in the reproductive health section;\nThis will stand as a pillar to help adolescent to adopt responsible behaviours in regard to adolescent pregnancy especially at individual, community and health systems levels."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Introduction",
"Methods",
"Results",
"Discussion",
"General patterns of teenage pregnancy in Kumbo East Health District",
"Knowledge and sources of information on reproductive and sexual health",
"Age of the teenagers",
"Knowledge contraceptive use",
"Levels of education, cultural practices and economic factor",
"Gender power relations in partnerships",
"Lack of knowledge on sexual reproductive health, circumstances of first sex, physical, sexual and substance abuse",
"Conclusion",
"What is known about this topic",
"What this study adds",
"Competing interests"
] |
[
"Teenage pregnancy is considered as one occurring in a young woman who has not reached her 20th birthday. This definition is applicable irrespective of the legal status of the marriage of the woman or legal age to consider an individual as adult [1]. 16 million girls aged 15-19 years give birth each year, most prevalent in low and middle-income countries located in sub-Saharan Africa [1]. In the developing world, one-third to one-half of women become mothers before the age of 20 and pregnancy related complications have become the leading causes of death among them [1, 2]. Teenage pregnancies are a global phenomenon. The pregnancy rate among teenagers in USA was 6.78% of pregnancies per 1,000 women aged 15-19 in 2008. [3]. Among the countries in the Western Europe, the United Kingdom [UK] has the highest teenage conception and abortion rates [2, 4]. The report presents an update on the current situation of pregnancies among girls less than 18 years of age and adolescents 15-19 years of age; trends during the last 10 years; variations across geographic, cultural and economic settings; interventions available to minimize pregnancy among adolescents; evidence for these programmatic approaches; and challenges that nations will have to deal with in the next 20 years given current population momentum [5]. The concentration of adolescent girls aged 10 to 17 will also change significantly, with the largest increase occurring in sub-Saharan Africa, where adolescent pregnancy is most common, and the rate of contraceptive use the lowest in the world [5]. A study conducted in Malawi showed that 57% of teenage girls opt to risk pregnancy rather than asking a partner to use a condom [2]. In Malawi, there is a high prevalence of casual sex among teenagers who shun condoms although they engage in multiple relationships. Scholars in the field argue that, because of the risk associated with high prevalence of early sexual behaviour, low contraceptive use, and many early pregnancies, adolescents in Cameroon are an important target group for sexual and reproductive health programs [6, 7]. In order to prevent early age pregnancies, it is important to make sure that adolescents have the means to make informed and healthy choices concerning their sexual and reproductive health. Yet, as it stands, reproductive health and family planning services in Cameroon mainly target older married women, and adolescents often remain largely overlooked [5].\nAdolescent school girl's pregnancies vary from country to country and in Cameroon, reproductive health remains a major public health challenge with elevated maternal mortality rate. The maternal mortality rate is estimated at 782 deaths per 100,000 live births [8]. This high mortality rate remains a dilemma because it involves the young mothers at the moment where they are giving birth. In addition, adolescent contribute 28% of maternal mortality in Cameroon [8]. According to DHS 2011, 25.6% of adolescents 15 to 19 have started sexual intercourse and 21% of then have had a child and 4% are pregnant for their first pregnancy. Still from the same source fertility rate of this age group is 127‰ which increase rapidly and attained a maximum of 250‰ within the age group 25-29yrs, suggesting that teenage pregnancy may be on the increase. The proportion of adolescents who have started fertility grow rapidly with age, 5% for 15yrs to 48% for 19yrs and the rate stood at 18% in the north west region [8]. Therefore, reducing teenage pregnancy, chiefly by promoting the use of contraceptives, will be necessary in order to prevent consequences that are associated with teenage pregnancy. Nevertheless, lessons from Zimbabwe and South Africa suggest that promoting the use of contraceptives alone does not necessarily reduce teenage pregnancy in developing as in developed countries. Therefore, other factors may be playing a major role as mentioned earlier. The general objective was to determine factors associated with adolescent school girl's pregnancy in Kumbo East Health District.",
"A crossectional descriptive study was selected because it incorporates a descriptive component that enables the calculation of the prevalence of teenage pregnancy in antenatal clinics of KEHD. The socio-economic status where measured by asking respondents questions concerning their househood items they possessed; bicycle, car , family income were collected and the knowledge was assessed by structured questions to evaluate the knowledge of respondents to factors associated with teenage pregnancy. The questionnaire was adopted from WHO standard guide and from the research of other researchers. The questionnaire was then pre-tested and adjusted to fit the context. Four midwives' students who were in their final year and had some experience in data collection assisted in the study. They were given orientation on the process for data collection and management prior to the commencement of field studies. They were trained for a period of three days in the administering of the questionnaire and research ethics, with an emphasis on informed consent. Each research assistant was given a code that was used for identification in case of any queries. Data was then collected entered into the computer and stored in external hard drive and USB keys. Data was then assembling and the analysis started. Our research period was for 10 months with the study population consisting of all school girl's pregnant aged [15-19yrs] from Kumbo East Health District. The sample size were then pregnant teens (15 to 19yrs) attending antenatal clinics in selected health centres in Kumbo East Health District at the time of data collection. Antenatal and infant welfare clinics were used in order to access pregnant adolescents.\nInclusion criteria: All school teens age 15 to 19 yrs who are pregnant who attend: ANC consultation irrespective of their trimester at SEGH and selected health centre, infant welfare clinics, and who give their consent.\nExclusion criteria: Pregnant teens who came to the clinic and didn't give their consent were excluded. Although teenage pregnancy concerns both sexes, male teenagers were excluded as they rarely attend antenatal services with their partners.\nSample method and sample type: The sampling method used was a simple random technique; the names of the health centres within was written on a piece of paper and placed in a container, four names were picked at random from the container. Jakiri integrated health centre, sub-divisional hospital Jakiri, St. Jude clinic and Shisong General Hospital. Our sample type was a convenience sampling. The Kumbo East District Hospital [Shisong] represents urban teenage mothers who may have experienced different factors from the rural setting. To obtain an adequate sample, all pregnant women visiting the antenatal clinics and infant welfare clinics over a three-month period at the selected sites under the age of 20 were interviewed.\nSample size in a cross sectional study: To calculate our sample size, we are going to use the Lorenz formula which stipulates, P= Prevalence of adolescent 15 to 19 (p = 0.25), t = confidence level [95%= 1.96], e = error margin [0.05], N = Sample size.\nStatistical application of this formula, resulted to a sample size of 293, implying that the study will recruit a total of 293 participants from the selected hospitals.\nData collection technique: Data was collected over a period of 3 months from August to October 2017, because of the Anglophone crisis with numerous “ghost towns” that reduce Antennal consultation by pregnant women difficult and hence leading to a long period of time to collect data. Using a standardized questionnaire administered through face-to-face interviews. The Questionnaire consisted mostly of closed questions. Each questionnaire took 20-40 minutes to administer. There were no refusals - all participants willingly consented to participate in the study. The arrangements for confidentiality and privacy ensured that respondents accepted to be interviewed and spoke more freely on the subject. In addition, it was also possible that respondents felt that there was somebody to listen to their problems. The face-to-face interviews facilitated responses and the quality of information and this method was convenient since most of the participants had low literacy levels.\nStatistical consideration: Data collected were entered into the CDC-Epi-Info version 7.2.2.2, transferred to MS-Excel and then exported to SPSS version 21.0 software. Data cleaning was performed with MS-Excel to check for inconsistencies in data entry and responses, prior to analyses. Associations between planed [Yes/ No] pregnancy and various variables were evaluated using Pearson and Yate's chi square [φ2] tests. Measures of association; OR and Pearson's chi square [φ2] tests were calculated by use of CDC-Epi-info version 7.2.2.2 and SPSS version 21.0 for the establishment of associations or differences (un) planed pregnancy and various variables.\nEthical considerations: in order to protect the rights of the interviewees and meet requirements for research involving people, clearances were obtained from authorities and informed consent from the participants. The authorization was also obtained from the North West Regional Delegation of Health. Ethical clearances were obtained from the Committee for Research on Human Subjects of the Catholic University of Central Africa (UCAC). Clearances to use health facilities were also obtained from the Kumbo East District Health Office. The following points were clarified: participation in the study was voluntary; participants were free to withdraw at any time without coercion and there were neither direct benefits nor known risks at any time. To ensure anonymity and privacy, numbers were used instead of names on the questionnaires. Interviews were conducted in private rooms which were provided at the clinics for this purpose. The completed questionnaires that were not entered to the computer were locked in a cupboard accessible to the researcher only to ensure confidentiality.",
"Socio-demographic characteristics: A total of 293 participants of average age 18.88yrs were sampled from 13 shortlisted localities of KEHD; Jakiri had the highest proportion 65 [22.18%] of participants while Noi and Tan had the lowest 11 [3.75%] each. Out of the 293 participants, 154 [52.56%] admitted currently schooling. Out of the 293 ladies sampled, 153 (52.22%) were single. Out of 293 sampled in the study, 175 [59.73%] who had not delivered, while the remainder had delivered at least a child, either in marriage or out of marriage. Socio-demographic data was collected for variables such age, marital status, alcohol and drug/substance attitudes, as well as information concerning partner (Table 1).\nDemographics; age, religion, school attendance and parity\nATR = African Traditional Religion\nSources of information on sexual and reproductive health: Despite the diversity of the sources of information on a variety of issues, 20.48% and 2.05% claimed ignorance of information on sex and sexuality as well as the use of contraceptives. However, a majority of the participants; 79.52% and 97.95% had at least one of the variety of sources of information on sex and sexuality and use of contraceptives. A majority of the participants had learned of sex and sexuality as well as use of contraceptives from school; 38.20% and 52.90%, respectively. Only 13.31%, of mothers educated their daughters on sex and sexuality. Books, Counsellors, local radio and peers respectively served as 6.83%, 19.80%, 13.65% and 20.48%, sources of information on sex and sexuality to adolescents. Out of the 293 respondents, 11.60%, 13.31%, and 20.14% had learned of contraceptives from friends, hospital, and relatives respectively (Table 2).\nSources of information on SRH and contraceptive use\nDichotomy group tabulated at value 2\nKnowledge of information on sexual and reproductive health (SRH): Considering the respondents answers to question ask on whether their menstrual and reproductive characteristics , their knowledge of information on sexual and reproductive health (SRH) was assessed, a majority of the respondents; 66.2%, 66.6%, 59.7% and 93.2% out rightly admitted that it is true that; amenorrhoea leads to accumulation of dirt and sickness, disappearance of a condom into a woman during sex, most pregnancies occur in the middle of the menstrual cycle as well as, one can become pregnant during the first sexual intercourse. Another majority; 93.2%, 53.2%, 65.9% and 53.6% actually admitted as outright false the fact that; frequent sex prevents pregnancy, sterility arises from the use of contraceptive use, washing of genitals after sex prevents pregnancy and the consumption of sachet whisky prevents conception (Table 3).\nKnowledge of information on sexual and reproductive health (SRH)\nFactors associated with unplanned teenage pregnancy in KEHD: A list of factors were provided in the questionnaire for the respondent to tick and out of the 293 respondents, 61.09% claimed never to have used the condom, and 52.56% said their partner was not the father of their child. 46.42% of the 293 participants said they do not use contraceptives, 13.65% failed to disclose the method of contraceptive used, by simply saying “none of the above”, when asked, when they started using contraceptive, 58.02%, said they have never used contraceptives (Table 4).\nFactors associated with adolescent school girl’s pregnancy\nLack of knowledge on sexual reproductive health, circumstances of first sex, physical, sexual and substance abuse: From the answers provided by the respondent concerning knowledge assessment, 20.48% of the respondents had information on sex and reproductive health from their peers and, another 20.8% could not really identify their source of information on sex and reproductive health. 11.60% and 2.05% of the respondents learned of how to use contraceptives from friends and no defined sources (Table 5).\nCircumstances of first sex, first sexual experience, relationship control and duration of relation\nCharacteristics of sexual partner and gender power relations in partnership: The sexual partners of respondents were of the age range 16-32 years (mean age of 22.815). At their age, a majority 67.24% of the respondents claimed their partners do not use drugs, while 6.49%, 15.02% and 3.75% respectively claimed their partners; do any injectable drugs, any other drugs and marijuana. On alcohol consumption frequency; 49.83% claimed not to know the drinking rate of their spouses, 7.85%, 20.82% and 21.50% said their partners drink at the rate of; only at weekend, less than once a month and a few times a month respectively (Table 6).\nAlcohol/ drug attitude of partner",
" General patterns of teenage pregnancy in Kumbo East Health District The prevalence of teenage pregnancy in Cameroon is 28% and in Kumbo East Health District the results on this study shows that the prevalence of unplanned teenage pregnancy is 60.75% which very high this could be attributed to the fact that schools were not operational at the time of this study due to political unrest in the country at the time of data collection. Out of the 293 respondents, 61.09% claimed never to have used the condom, and 52.56% said their partner was not the father of their child. 46.42% of the 293 participants said they do not use contraceptives, 13.65% failed to disclose the method of contraceptive used, by simply saying “none of the above”, when asked, when they started using contraceptive, 58.02% said they have never used contraceptives. This implies that there is still a need for more efforts. Possible factors contributing to the high prevalence in this study area include age at first pregnancy, low contraceptive use, educational levels and socio-economic status, circumstances at first sex, lack of knowledge on reproductive and sexual health and physical and sexual violence.\nThe prevalence of teenage pregnancy in Cameroon is 28% and in Kumbo East Health District the results on this study shows that the prevalence of unplanned teenage pregnancy is 60.75% which very high this could be attributed to the fact that schools were not operational at the time of this study due to political unrest in the country at the time of data collection. Out of the 293 respondents, 61.09% claimed never to have used the condom, and 52.56% said their partner was not the father of their child. 46.42% of the 293 participants said they do not use contraceptives, 13.65% failed to disclose the method of contraceptive used, by simply saying “none of the above”, when asked, when they started using contraceptive, 58.02% said they have never used contraceptives. This implies that there is still a need for more efforts. Possible factors contributing to the high prevalence in this study area include age at first pregnancy, low contraceptive use, educational levels and socio-economic status, circumstances at first sex, lack of knowledge on reproductive and sexual health and physical and sexual violence.\n Knowledge and sources of information on reproductive and sexual health Considering the respondents answers to question ask on whether their menstrual and reproductive characteristics , their knowledge of information on sexual and reproductive health [SRH], a majority of the respondents; 66.2%, 66.6%, 59.7% and 93.2% out rightly admitted that it is true that; amenorrhoea leads to accumulation of dirt and sickness, disappearance of a condom into a woman during sex, most pregnancies occur in the middle of the menstrual cycle as well as, one can become pregnant during the first sexual intercourse. Another majority, 93.2%, 53.2%, 65.9% and 53.6% actually admitted as outright false the fact that; frequent sex prevents pregnancy, sterility arises from the use of contraceptive use, washing of genitals after sex prevents pregnancy and the consumption of sachet whisky prevents conception (Table 4).\nConsidering the respondents answers to question ask on whether their menstrual and reproductive characteristics , their knowledge of information on sexual and reproductive health [SRH], a majority of the respondents; 66.2%, 66.6%, 59.7% and 93.2% out rightly admitted that it is true that; amenorrhoea leads to accumulation of dirt and sickness, disappearance of a condom into a woman during sex, most pregnancies occur in the middle of the menstrual cycle as well as, one can become pregnant during the first sexual intercourse. Another majority, 93.2%, 53.2%, 65.9% and 53.6% actually admitted as outright false the fact that; frequent sex prevents pregnancy, sterility arises from the use of contraceptive use, washing of genitals after sex prevents pregnancy and the consumption of sachet whisky prevents conception (Table 4).\n Age of the teenagers The ages of participants ranged from 15-27 years, with mean age 18.88 years and modal age group of 15 -< 20 years with 227 [< 77.41%]. A large majority of participants 59.73% had no child, while 32.42% had one child, 0.68% had two children, 6.14% had three children and 1.02% had four children. In Cameroon, Kenya, South Africa and Canada, the average age difference between teenagers and their sexual partners is 15, 7, 5 and 2.6 years, respectively. In addition, teenagers who marry older men often have less power in decision making around sexual intercourse, childbearing and the use of contraceptive [1].\nThe ages of participants ranged from 15-27 years, with mean age 18.88 years and modal age group of 15 -< 20 years with 227 [< 77.41%]. A large majority of participants 59.73% had no child, while 32.42% had one child, 0.68% had two children, 6.14% had three children and 1.02% had four children. In Cameroon, Kenya, South Africa and Canada, the average age difference between teenagers and their sexual partners is 15, 7, 5 and 2.6 years, respectively. In addition, teenagers who marry older men often have less power in decision making around sexual intercourse, childbearing and the use of contraceptive [1].\n Knowledge contraceptive use On the usage of contraceptives, 15.02% used the condom, 5.12% claim to use implants, 8.19% claim to use IUCD, while 11.60% use pills. On when they began using contraceptives; 2.05% began this year, 24.23% began last year, 7.51% began in the year 2015, 2.73% began in 2014 and 2013 as well as 2012 (Table 4). Respondents also advanced possible fears and reasons for not using contraceptive; 6.83, were scared of nurses, 20.14% were scared of their parents, 6.83% claimed it was against their religion and 12.97% admitted their partners were not in favour of it (Table 1). The fears, beliefs and perceptions of sexual inactivity associated with contraceptive use may be attributed to the lack of knowledge and low levels of awareness. This is conformity with a similar study Contraceptive prevalence in Cameroon is low among married women and shows uneven distribution varying from 2.6% in North Cameroon to 43.9% in Yaoundé [6, 7]. The low prevalence use among married couples suggests that once a woman is married she is exposed to pregnancy irrespective of her age. In Cameroon, unmet needs for family planning are 22%. And the use of modern contraception in Cameroon is about 16%. Reasons for non-contraceptive use include, religious and cultural beliefs, poor quality of services, including the negative attitude of service providers, fear of exposure of their bodies, having adults at the same services and inability to negotiate contraceptive use with sexual partners. Furthermore, misconceptions, fear of side effects and stigma associated with the use of contraceptives as adolescents may be labelled as being promiscuous can also be considered as contributing factors for non-contraceptive use [1]. Similarly, in South Africa, a study revealed that teenage pregnancy is attributed to the low utilization of contraceptives, especially on their first intercourse, due to the lack of access to medical information on reproductive system, inaccessible family planning services, gender inequality and decision on the social life, fears about contraception on fertility and menstruation and condoms could be left inside the vagina or womb [4]. It was therefore envisaged that the use of contraceptives among teenagers in the study area could be low, making it a factor for pregnancy.\nOn the usage of contraceptives, 15.02% used the condom, 5.12% claim to use implants, 8.19% claim to use IUCD, while 11.60% use pills. On when they began using contraceptives; 2.05% began this year, 24.23% began last year, 7.51% began in the year 2015, 2.73% began in 2014 and 2013 as well as 2012 (Table 4). Respondents also advanced possible fears and reasons for not using contraceptive; 6.83, were scared of nurses, 20.14% were scared of their parents, 6.83% claimed it was against their religion and 12.97% admitted their partners were not in favour of it (Table 1). The fears, beliefs and perceptions of sexual inactivity associated with contraceptive use may be attributed to the lack of knowledge and low levels of awareness. This is conformity with a similar study Contraceptive prevalence in Cameroon is low among married women and shows uneven distribution varying from 2.6% in North Cameroon to 43.9% in Yaoundé [6, 7]. The low prevalence use among married couples suggests that once a woman is married she is exposed to pregnancy irrespective of her age. In Cameroon, unmet needs for family planning are 22%. And the use of modern contraception in Cameroon is about 16%. Reasons for non-contraceptive use include, religious and cultural beliefs, poor quality of services, including the negative attitude of service providers, fear of exposure of their bodies, having adults at the same services and inability to negotiate contraceptive use with sexual partners. Furthermore, misconceptions, fear of side effects and stigma associated with the use of contraceptives as adolescents may be labelled as being promiscuous can also be considered as contributing factors for non-contraceptive use [1]. Similarly, in South Africa, a study revealed that teenage pregnancy is attributed to the low utilization of contraceptives, especially on their first intercourse, due to the lack of access to medical information on reproductive system, inaccessible family planning services, gender inequality and decision on the social life, fears about contraception on fertility and menstruation and condoms could be left inside the vagina or womb [4]. It was therefore envisaged that the use of contraceptives among teenagers in the study area could be low, making it a factor for pregnancy.\n Levels of education, cultural practices and economic factor The ages of the partners of participants ranged from 16-32 years; Half, 50.17% of partners of adolescent school girls were in the age group 20 - < 25 years. 17.41% were in the age group 16 -< 20 years, 25.26% in the group 25 -< 30 years and 7.17% in the age group 30 - 32 years. With 51.88% of the 293 participant's partners had secondary school education, followed by those with tertiary education 40.27%, those with no formal education were 6.83% and primary education were 1.02%. Of the 293 participants, the partners of 44.37%, 40.96% and 14.68% were respectively studying, unemployed and working (Table 2). The study agrees that Teenage girls may indulge in sexual activity in exchange for goods, money and experiences such as taking meals in hotels [1]. A study in Malawi, found that 66% of adolescents had accepted money or gifts in exchange for sex and in some cases, parents may encourage their daughters into relationships with men for consumer goods or a girl may go out with men because her parents cannot give her the basic needs. Teenagers with unplanned pregnancy are more likely to come from low socio-economic status than with planned pregnancy [1]. The level of education of parents, especially the mother, may have an influence on the adolescent towards teenage pregnancy as she acts as a role model [9] which may be a preventive factor of teenage pregnancy. In Kenya it was reported that women with no education had first sexual intercourse three years earlier than their counterparts with at least a secondary school education [1]. The low literacy levels may lead to low paying jobs, causing early marriage and influencing non-contraceptive use, thereby increasing the prevalence of teenage pregnancy. Therefore, it was expected in the study area that the low literacy levels may have an impact on unplanned teenage pregnancy. Culturally, amongst factors associated with unplanned teenage pregnancy were, age at onset of menstruation, age at first sex, who they live with as well as the marital status of their parents. It was difficult to prove that culture is a factor. Economically, these were age, religious and delivery status of participants (Table 1), marital status of participants and parents, partner's educational and occupational status.\nThe ages of the partners of participants ranged from 16-32 years; Half, 50.17% of partners of adolescent school girls were in the age group 20 - < 25 years. 17.41% were in the age group 16 -< 20 years, 25.26% in the group 25 -< 30 years and 7.17% in the age group 30 - 32 years. With 51.88% of the 293 participant's partners had secondary school education, followed by those with tertiary education 40.27%, those with no formal education were 6.83% and primary education were 1.02%. Of the 293 participants, the partners of 44.37%, 40.96% and 14.68% were respectively studying, unemployed and working (Table 2). The study agrees that Teenage girls may indulge in sexual activity in exchange for goods, money and experiences such as taking meals in hotels [1]. A study in Malawi, found that 66% of adolescents had accepted money or gifts in exchange for sex and in some cases, parents may encourage their daughters into relationships with men for consumer goods or a girl may go out with men because her parents cannot give her the basic needs. Teenagers with unplanned pregnancy are more likely to come from low socio-economic status than with planned pregnancy [1]. The level of education of parents, especially the mother, may have an influence on the adolescent towards teenage pregnancy as she acts as a role model [9] which may be a preventive factor of teenage pregnancy. In Kenya it was reported that women with no education had first sexual intercourse three years earlier than their counterparts with at least a secondary school education [1]. The low literacy levels may lead to low paying jobs, causing early marriage and influencing non-contraceptive use, thereby increasing the prevalence of teenage pregnancy. Therefore, it was expected in the study area that the low literacy levels may have an impact on unplanned teenage pregnancy. Culturally, amongst factors associated with unplanned teenage pregnancy were, age at onset of menstruation, age at first sex, who they live with as well as the marital status of their parents. It was difficult to prove that culture is a factor. Economically, these were age, religious and delivery status of participants (Table 1), marital status of participants and parents, partner's educational and occupational status.\n Gender power relations in partnerships There were a variety of circumstances that could lead to sex; partner violence, reasons for involvement in a relationship and first sexual experience. For partner violence; 19.80%, had their funds managed by their partners, 33.11% were forced by their partners into sex, 20.14% would be hit by their partners and 40.27% had their funds managed by their partners. 6.48%, 13.65% and 12.97% of respondents respectively got involved in a relationship to be provided with clothes, to be married and to have a good time. For the first sexual experience; 46.42% were persuaded either by a relation or boyfriend, 6.48% were raped, 21.84% were raped, 7.51% were willing and collaborated or 17.75% refused to disclose their first sexual experience (Table 5). This means that male partner is the one who decides what happen in a relationship.\nThere were a variety of circumstances that could lead to sex; partner violence, reasons for involvement in a relationship and first sexual experience. For partner violence; 19.80%, had their funds managed by their partners, 33.11% were forced by their partners into sex, 20.14% would be hit by their partners and 40.27% had their funds managed by their partners. 6.48%, 13.65% and 12.97% of respondents respectively got involved in a relationship to be provided with clothes, to be married and to have a good time. For the first sexual experience; 46.42% were persuaded either by a relation or boyfriend, 6.48% were raped, 21.84% were raped, 7.51% were willing and collaborated or 17.75% refused to disclose their first sexual experience (Table 5). This means that male partner is the one who decides what happen in a relationship.\n Lack of knowledge on sexual reproductive health, circumstances of first sex, physical, sexual and substance abuse 20.48% of the respondents had information on sex and reproductive health from their peers and, another 20.8% could not really identify their source of information on sex and reproductive health. 11.60% and 2.05% of the respondents learned of how to use contraceptives from friends and no defined sources (Table 3). Most studies conducted in developing countries report that adolescent girls often lack basic knowledge about reproductive and sexual health [1]. This study contrast with that of findings of Nanze. C.C (2006 on risk factors of unwanted/uplanned pregnancy in zomba district, Malawi) indicate that communication about reproductive and sexual matters within families is limited, forcing girls to get information chiefly from peers, boyfriends and teachers. On drug and substance use, a wide majority (80.55%, said they do not take drugs 12.97% said they took any drug, while 6.48% admitted smoking “banga” (Table 7). Similar findings have been reported in South Africa [9]. Unlike wise in Cameroon, it is not common for teenage girls to use drug substances due to cultural values. However, there may have been underreporting since drug use is illegal, making it a sensitive topic.\nCircumstances of first sex, first sexual experience, relationship control and duration of relation\n20.48% of the respondents had information on sex and reproductive health from their peers and, another 20.8% could not really identify their source of information on sex and reproductive health. 11.60% and 2.05% of the respondents learned of how to use contraceptives from friends and no defined sources (Table 3). Most studies conducted in developing countries report that adolescent girls often lack basic knowledge about reproductive and sexual health [1]. This study contrast with that of findings of Nanze. C.C (2006 on risk factors of unwanted/uplanned pregnancy in zomba district, Malawi) indicate that communication about reproductive and sexual matters within families is limited, forcing girls to get information chiefly from peers, boyfriends and teachers. On drug and substance use, a wide majority (80.55%, said they do not take drugs 12.97% said they took any drug, while 6.48% admitted smoking “banga” (Table 7). Similar findings have been reported in South Africa [9]. Unlike wise in Cameroon, it is not common for teenage girls to use drug substances due to cultural values. However, there may have been underreporting since drug use is illegal, making it a sensitive topic.\nCircumstances of first sex, first sexual experience, relationship control and duration of relation",
"The prevalence of teenage pregnancy in Cameroon is 28% and in Kumbo East Health District the results on this study shows that the prevalence of unplanned teenage pregnancy is 60.75% which very high this could be attributed to the fact that schools were not operational at the time of this study due to political unrest in the country at the time of data collection. Out of the 293 respondents, 61.09% claimed never to have used the condom, and 52.56% said their partner was not the father of their child. 46.42% of the 293 participants said they do not use contraceptives, 13.65% failed to disclose the method of contraceptive used, by simply saying “none of the above”, when asked, when they started using contraceptive, 58.02% said they have never used contraceptives. This implies that there is still a need for more efforts. Possible factors contributing to the high prevalence in this study area include age at first pregnancy, low contraceptive use, educational levels and socio-economic status, circumstances at first sex, lack of knowledge on reproductive and sexual health and physical and sexual violence.",
"Considering the respondents answers to question ask on whether their menstrual and reproductive characteristics , their knowledge of information on sexual and reproductive health [SRH], a majority of the respondents; 66.2%, 66.6%, 59.7% and 93.2% out rightly admitted that it is true that; amenorrhoea leads to accumulation of dirt and sickness, disappearance of a condom into a woman during sex, most pregnancies occur in the middle of the menstrual cycle as well as, one can become pregnant during the first sexual intercourse. Another majority, 93.2%, 53.2%, 65.9% and 53.6% actually admitted as outright false the fact that; frequent sex prevents pregnancy, sterility arises from the use of contraceptive use, washing of genitals after sex prevents pregnancy and the consumption of sachet whisky prevents conception (Table 4).",
"The ages of participants ranged from 15-27 years, with mean age 18.88 years and modal age group of 15 -< 20 years with 227 [< 77.41%]. A large majority of participants 59.73% had no child, while 32.42% had one child, 0.68% had two children, 6.14% had three children and 1.02% had four children. In Cameroon, Kenya, South Africa and Canada, the average age difference between teenagers and their sexual partners is 15, 7, 5 and 2.6 years, respectively. In addition, teenagers who marry older men often have less power in decision making around sexual intercourse, childbearing and the use of contraceptive [1].",
"On the usage of contraceptives, 15.02% used the condom, 5.12% claim to use implants, 8.19% claim to use IUCD, while 11.60% use pills. On when they began using contraceptives; 2.05% began this year, 24.23% began last year, 7.51% began in the year 2015, 2.73% began in 2014 and 2013 as well as 2012 (Table 4). Respondents also advanced possible fears and reasons for not using contraceptive; 6.83, were scared of nurses, 20.14% were scared of their parents, 6.83% claimed it was against their religion and 12.97% admitted their partners were not in favour of it (Table 1). The fears, beliefs and perceptions of sexual inactivity associated with contraceptive use may be attributed to the lack of knowledge and low levels of awareness. This is conformity with a similar study Contraceptive prevalence in Cameroon is low among married women and shows uneven distribution varying from 2.6% in North Cameroon to 43.9% in Yaoundé [6, 7]. The low prevalence use among married couples suggests that once a woman is married she is exposed to pregnancy irrespective of her age. In Cameroon, unmet needs for family planning are 22%. And the use of modern contraception in Cameroon is about 16%. Reasons for non-contraceptive use include, religious and cultural beliefs, poor quality of services, including the negative attitude of service providers, fear of exposure of their bodies, having adults at the same services and inability to negotiate contraceptive use with sexual partners. Furthermore, misconceptions, fear of side effects and stigma associated with the use of contraceptives as adolescents may be labelled as being promiscuous can also be considered as contributing factors for non-contraceptive use [1]. Similarly, in South Africa, a study revealed that teenage pregnancy is attributed to the low utilization of contraceptives, especially on their first intercourse, due to the lack of access to medical information on reproductive system, inaccessible family planning services, gender inequality and decision on the social life, fears about contraception on fertility and menstruation and condoms could be left inside the vagina or womb [4]. It was therefore envisaged that the use of contraceptives among teenagers in the study area could be low, making it a factor for pregnancy.",
"The ages of the partners of participants ranged from 16-32 years; Half, 50.17% of partners of adolescent school girls were in the age group 20 - < 25 years. 17.41% were in the age group 16 -< 20 years, 25.26% in the group 25 -< 30 years and 7.17% in the age group 30 - 32 years. With 51.88% of the 293 participant's partners had secondary school education, followed by those with tertiary education 40.27%, those with no formal education were 6.83% and primary education were 1.02%. Of the 293 participants, the partners of 44.37%, 40.96% and 14.68% were respectively studying, unemployed and working (Table 2). The study agrees that Teenage girls may indulge in sexual activity in exchange for goods, money and experiences such as taking meals in hotels [1]. A study in Malawi, found that 66% of adolescents had accepted money or gifts in exchange for sex and in some cases, parents may encourage their daughters into relationships with men for consumer goods or a girl may go out with men because her parents cannot give her the basic needs. Teenagers with unplanned pregnancy are more likely to come from low socio-economic status than with planned pregnancy [1]. The level of education of parents, especially the mother, may have an influence on the adolescent towards teenage pregnancy as she acts as a role model [9] which may be a preventive factor of teenage pregnancy. In Kenya it was reported that women with no education had first sexual intercourse three years earlier than their counterparts with at least a secondary school education [1]. The low literacy levels may lead to low paying jobs, causing early marriage and influencing non-contraceptive use, thereby increasing the prevalence of teenage pregnancy. Therefore, it was expected in the study area that the low literacy levels may have an impact on unplanned teenage pregnancy. Culturally, amongst factors associated with unplanned teenage pregnancy were, age at onset of menstruation, age at first sex, who they live with as well as the marital status of their parents. It was difficult to prove that culture is a factor. Economically, these were age, religious and delivery status of participants (Table 1), marital status of participants and parents, partner's educational and occupational status.",
"There were a variety of circumstances that could lead to sex; partner violence, reasons for involvement in a relationship and first sexual experience. For partner violence; 19.80%, had their funds managed by their partners, 33.11% were forced by their partners into sex, 20.14% would be hit by their partners and 40.27% had their funds managed by their partners. 6.48%, 13.65% and 12.97% of respondents respectively got involved in a relationship to be provided with clothes, to be married and to have a good time. For the first sexual experience; 46.42% were persuaded either by a relation or boyfriend, 6.48% were raped, 21.84% were raped, 7.51% were willing and collaborated or 17.75% refused to disclose their first sexual experience (Table 5). This means that male partner is the one who decides what happen in a relationship.",
"20.48% of the respondents had information on sex and reproductive health from their peers and, another 20.8% could not really identify their source of information on sex and reproductive health. 11.60% and 2.05% of the respondents learned of how to use contraceptives from friends and no defined sources (Table 3). Most studies conducted in developing countries report that adolescent girls often lack basic knowledge about reproductive and sexual health [1]. This study contrast with that of findings of Nanze. C.C (2006 on risk factors of unwanted/uplanned pregnancy in zomba district, Malawi) indicate that communication about reproductive and sexual matters within families is limited, forcing girls to get information chiefly from peers, boyfriends and teachers. On drug and substance use, a wide majority (80.55%, said they do not take drugs 12.97% said they took any drug, while 6.48% admitted smoking “banga” (Table 7). Similar findings have been reported in South Africa [9]. Unlike wise in Cameroon, it is not common for teenage girls to use drug substances due to cultural values. However, there may have been underreporting since drug use is illegal, making it a sensitive topic.\nCircumstances of first sex, first sexual experience, relationship control and duration of relation",
"The study factors associated with adolescent school girl's pregnancy in KEHD was aimed at examining the factors associated with teens pregnancy. A descriptive study was used and the teens 15 to 19yrs were selected. Teenage pregnancy whether planned or unplanned is detrimental to the health and socio economic status of the teenagers. This study shows that there is a high prevalence (60.75%) of teenage pregnancy in the sampled antenatal clinics of Kumbo East Health District attributable to the low contraceptive use and socio-economic status, lack of reproductive and sexual knowledge, circumstances at first sex, including force and rape, physical and sexual violence, early marriage, age of sexual partner and alcohol abuse. Among reasons contributing to the low use of contraceptives are; lack of knowledge, fear of side effects, including sterility, condoms disappearing in the womb and inequality of power with sexual partners. Teenagers obtain information mainly from school (53%) and relatives (20%). The high prevalence of unplanned teenage pregnancy, low contraceptive use, myths and misconceptions surrounding the use of contraceptives indicate that teenagers are receiving inaccurate information about reproductive and sexual health, a problem which may be compounded by low educational levels. Therefore, the lack of knowledge about reproductive health is a factor for teenage pregnancy. The lack of basic necessities is one of the factors that force teenagers to engage in transactional sex. Based on the study it is clear that teenagers have no control over sexual and reproductive health since their sexual partners are the sore decision makers making physical and sexual violence a factor for teenage pregnancy. However, the influence of cultural practices and religion, substance abuse, relationship control, and fear of parents and intimidating attitude of health service providers have been supported as factors. The associations suggested by the study point towards a need for greater emphasis in reproduction and sexual health promotion interventions. In addition, strengthening awareness and giving information to dispel fears, misconceptions and rumours about contraceptive use will prepare teenagers for the physical changes they might experience when adopting contraceptive methods. Nevertheless, contraceptive use alone may not reduce teenage pregnancy, but addressing the impact of poverty on teenagers, empowering them on their rights and information in order to make right choices is very important.\nRecommendations: The study recognizes efforts being made by government and non-governmental organizations to solve the problem of teenage pregnancy in north west region and Kumbo East in particular. These efforts include promoting the use of contraceptives, education of girls and poverty alleviation. However, the low use of contraceptives and socio-economic status, continued sexual and physical violence, the feeling of inferiority due to age differences with sexual partners and early marriages indicate that factors for teenage pregnancy still persist in the study area. Therefore, there is a need to develop programmes to address factors identified in this study. Solutions to the problem require multidisciplinary implementing teams, including parents, schools, communities, NGOs and government sectors. The following recommendations are suggested.\n What is known about this topic Knowledge of this geographic distribution can provide useful information for updating and strengthening adolescent reproductive health strategies in Cameroon;\nIn Cameroon, the prevalence of adolescent pregnancy is between 40% to 50%;\nAdolescent age 15 to 19 contribute to 28% of maternal mortality in Cameroon.\nKnowledge of this geographic distribution can provide useful information for updating and strengthening adolescent reproductive health strategies in Cameroon;\nIn Cameroon, the prevalence of adolescent pregnancy is between 40% to 50%;\nAdolescent age 15 to 19 contribute to 28% of maternal mortality in Cameroon.\n What this study adds Study will help provide few answers to this problem and may be prick health leaders to react towards the amelioration of some of their health strategies concerning reproductive health especially adolescent health in rural areas of Cameroon;\nMoreover, it will also go a long way to convince national and international partners to continue to largely invest in the reproductive health section;\nThis will stand as a pillar to help adolescent to adopt responsible behaviours in regard to adolescent pregnancy especially at individual, community and health systems levels.\nStudy will help provide few answers to this problem and may be prick health leaders to react towards the amelioration of some of their health strategies concerning reproductive health especially adolescent health in rural areas of Cameroon;\nMoreover, it will also go a long way to convince national and international partners to continue to largely invest in the reproductive health section;\nThis will stand as a pillar to help adolescent to adopt responsible behaviours in regard to adolescent pregnancy especially at individual, community and health systems levels.",
"Knowledge of this geographic distribution can provide useful information for updating and strengthening adolescent reproductive health strategies in Cameroon;\nIn Cameroon, the prevalence of adolescent pregnancy is between 40% to 50%;\nAdolescent age 15 to 19 contribute to 28% of maternal mortality in Cameroon.",
"Study will help provide few answers to this problem and may be prick health leaders to react towards the amelioration of some of their health strategies concerning reproductive health especially adolescent health in rural areas of Cameroon;\nMoreover, it will also go a long way to convince national and international partners to continue to largely invest in the reproductive health section;\nThis will stand as a pillar to help adolescent to adopt responsible behaviours in regard to adolescent pregnancy especially at individual, community and health systems levels.",
"Authors declare no competing interests."
] |
[
"intro",
"methods",
"results",
"discussion",
null,
null,
null,
null,
null,
null,
null,
"conclusion",
null,
null,
"COI-statement"
] |
[
"Adolescents",
"factors",
"pregnancy",
"health district"
] |
Introduction:
Teenage pregnancy is considered as one occurring in a young woman who has not reached her 20th birthday. This definition is applicable irrespective of the legal status of the marriage of the woman or legal age to consider an individual as adult [1]. 16 million girls aged 15-19 years give birth each year, most prevalent in low and middle-income countries located in sub-Saharan Africa [1]. In the developing world, one-third to one-half of women become mothers before the age of 20 and pregnancy related complications have become the leading causes of death among them [1, 2]. Teenage pregnancies are a global phenomenon. The pregnancy rate among teenagers in USA was 6.78% of pregnancies per 1,000 women aged 15-19 in 2008. [3]. Among the countries in the Western Europe, the United Kingdom [UK] has the highest teenage conception and abortion rates [2, 4]. The report presents an update on the current situation of pregnancies among girls less than 18 years of age and adolescents 15-19 years of age; trends during the last 10 years; variations across geographic, cultural and economic settings; interventions available to minimize pregnancy among adolescents; evidence for these programmatic approaches; and challenges that nations will have to deal with in the next 20 years given current population momentum [5]. The concentration of adolescent girls aged 10 to 17 will also change significantly, with the largest increase occurring in sub-Saharan Africa, where adolescent pregnancy is most common, and the rate of contraceptive use the lowest in the world [5]. A study conducted in Malawi showed that 57% of teenage girls opt to risk pregnancy rather than asking a partner to use a condom [2]. In Malawi, there is a high prevalence of casual sex among teenagers who shun condoms although they engage in multiple relationships. Scholars in the field argue that, because of the risk associated with high prevalence of early sexual behaviour, low contraceptive use, and many early pregnancies, adolescents in Cameroon are an important target group for sexual and reproductive health programs [6, 7]. In order to prevent early age pregnancies, it is important to make sure that adolescents have the means to make informed and healthy choices concerning their sexual and reproductive health. Yet, as it stands, reproductive health and family planning services in Cameroon mainly target older married women, and adolescents often remain largely overlooked [5].
Adolescent school girl's pregnancies vary from country to country and in Cameroon, reproductive health remains a major public health challenge with elevated maternal mortality rate. The maternal mortality rate is estimated at 782 deaths per 100,000 live births [8]. This high mortality rate remains a dilemma because it involves the young mothers at the moment where they are giving birth. In addition, adolescent contribute 28% of maternal mortality in Cameroon [8]. According to DHS 2011, 25.6% of adolescents 15 to 19 have started sexual intercourse and 21% of then have had a child and 4% are pregnant for their first pregnancy. Still from the same source fertility rate of this age group is 127‰ which increase rapidly and attained a maximum of 250‰ within the age group 25-29yrs, suggesting that teenage pregnancy may be on the increase. The proportion of adolescents who have started fertility grow rapidly with age, 5% for 15yrs to 48% for 19yrs and the rate stood at 18% in the north west region [8]. Therefore, reducing teenage pregnancy, chiefly by promoting the use of contraceptives, will be necessary in order to prevent consequences that are associated with teenage pregnancy. Nevertheless, lessons from Zimbabwe and South Africa suggest that promoting the use of contraceptives alone does not necessarily reduce teenage pregnancy in developing as in developed countries. Therefore, other factors may be playing a major role as mentioned earlier. The general objective was to determine factors associated with adolescent school girl's pregnancy in Kumbo East Health District.
Methods:
A crossectional descriptive study was selected because it incorporates a descriptive component that enables the calculation of the prevalence of teenage pregnancy in antenatal clinics of KEHD. The socio-economic status where measured by asking respondents questions concerning their househood items they possessed; bicycle, car , family income were collected and the knowledge was assessed by structured questions to evaluate the knowledge of respondents to factors associated with teenage pregnancy. The questionnaire was adopted from WHO standard guide and from the research of other researchers. The questionnaire was then pre-tested and adjusted to fit the context. Four midwives' students who were in their final year and had some experience in data collection assisted in the study. They were given orientation on the process for data collection and management prior to the commencement of field studies. They were trained for a period of three days in the administering of the questionnaire and research ethics, with an emphasis on informed consent. Each research assistant was given a code that was used for identification in case of any queries. Data was then collected entered into the computer and stored in external hard drive and USB keys. Data was then assembling and the analysis started. Our research period was for 10 months with the study population consisting of all school girl's pregnant aged [15-19yrs] from Kumbo East Health District. The sample size were then pregnant teens (15 to 19yrs) attending antenatal clinics in selected health centres in Kumbo East Health District at the time of data collection. Antenatal and infant welfare clinics were used in order to access pregnant adolescents.
Inclusion criteria: All school teens age 15 to 19 yrs who are pregnant who attend: ANC consultation irrespective of their trimester at SEGH and selected health centre, infant welfare clinics, and who give their consent.
Exclusion criteria: Pregnant teens who came to the clinic and didn't give their consent were excluded. Although teenage pregnancy concerns both sexes, male teenagers were excluded as they rarely attend antenatal services with their partners.
Sample method and sample type: The sampling method used was a simple random technique; the names of the health centres within was written on a piece of paper and placed in a container, four names were picked at random from the container. Jakiri integrated health centre, sub-divisional hospital Jakiri, St. Jude clinic and Shisong General Hospital. Our sample type was a convenience sampling. The Kumbo East District Hospital [Shisong] represents urban teenage mothers who may have experienced different factors from the rural setting. To obtain an adequate sample, all pregnant women visiting the antenatal clinics and infant welfare clinics over a three-month period at the selected sites under the age of 20 were interviewed.
Sample size in a cross sectional study: To calculate our sample size, we are going to use the Lorenz formula which stipulates, P= Prevalence of adolescent 15 to 19 (p = 0.25), t = confidence level [95%= 1.96], e = error margin [0.05], N = Sample size.
Statistical application of this formula, resulted to a sample size of 293, implying that the study will recruit a total of 293 participants from the selected hospitals.
Data collection technique: Data was collected over a period of 3 months from August to October 2017, because of the Anglophone crisis with numerous “ghost towns” that reduce Antennal consultation by pregnant women difficult and hence leading to a long period of time to collect data. Using a standardized questionnaire administered through face-to-face interviews. The Questionnaire consisted mostly of closed questions. Each questionnaire took 20-40 minutes to administer. There were no refusals - all participants willingly consented to participate in the study. The arrangements for confidentiality and privacy ensured that respondents accepted to be interviewed and spoke more freely on the subject. In addition, it was also possible that respondents felt that there was somebody to listen to their problems. The face-to-face interviews facilitated responses and the quality of information and this method was convenient since most of the participants had low literacy levels.
Statistical consideration: Data collected were entered into the CDC-Epi-Info version 7.2.2.2, transferred to MS-Excel and then exported to SPSS version 21.0 software. Data cleaning was performed with MS-Excel to check for inconsistencies in data entry and responses, prior to analyses. Associations between planed [Yes/ No] pregnancy and various variables were evaluated using Pearson and Yate's chi square [φ2] tests. Measures of association; OR and Pearson's chi square [φ2] tests were calculated by use of CDC-Epi-info version 7.2.2.2 and SPSS version 21.0 for the establishment of associations or differences (un) planed pregnancy and various variables.
Ethical considerations: in order to protect the rights of the interviewees and meet requirements for research involving people, clearances were obtained from authorities and informed consent from the participants. The authorization was also obtained from the North West Regional Delegation of Health. Ethical clearances were obtained from the Committee for Research on Human Subjects of the Catholic University of Central Africa (UCAC). Clearances to use health facilities were also obtained from the Kumbo East District Health Office. The following points were clarified: participation in the study was voluntary; participants were free to withdraw at any time without coercion and there were neither direct benefits nor known risks at any time. To ensure anonymity and privacy, numbers were used instead of names on the questionnaires. Interviews were conducted in private rooms which were provided at the clinics for this purpose. The completed questionnaires that were not entered to the computer were locked in a cupboard accessible to the researcher only to ensure confidentiality.
Results:
Socio-demographic characteristics: A total of 293 participants of average age 18.88yrs were sampled from 13 shortlisted localities of KEHD; Jakiri had the highest proportion 65 [22.18%] of participants while Noi and Tan had the lowest 11 [3.75%] each. Out of the 293 participants, 154 [52.56%] admitted currently schooling. Out of the 293 ladies sampled, 153 (52.22%) were single. Out of 293 sampled in the study, 175 [59.73%] who had not delivered, while the remainder had delivered at least a child, either in marriage or out of marriage. Socio-demographic data was collected for variables such age, marital status, alcohol and drug/substance attitudes, as well as information concerning partner (Table 1).
Demographics; age, religion, school attendance and parity
ATR = African Traditional Religion
Sources of information on sexual and reproductive health: Despite the diversity of the sources of information on a variety of issues, 20.48% and 2.05% claimed ignorance of information on sex and sexuality as well as the use of contraceptives. However, a majority of the participants; 79.52% and 97.95% had at least one of the variety of sources of information on sex and sexuality and use of contraceptives. A majority of the participants had learned of sex and sexuality as well as use of contraceptives from school; 38.20% and 52.90%, respectively. Only 13.31%, of mothers educated their daughters on sex and sexuality. Books, Counsellors, local radio and peers respectively served as 6.83%, 19.80%, 13.65% and 20.48%, sources of information on sex and sexuality to adolescents. Out of the 293 respondents, 11.60%, 13.31%, and 20.14% had learned of contraceptives from friends, hospital, and relatives respectively (Table 2).
Sources of information on SRH and contraceptive use
Dichotomy group tabulated at value 2
Knowledge of information on sexual and reproductive health (SRH): Considering the respondents answers to question ask on whether their menstrual and reproductive characteristics , their knowledge of information on sexual and reproductive health (SRH) was assessed, a majority of the respondents; 66.2%, 66.6%, 59.7% and 93.2% out rightly admitted that it is true that; amenorrhoea leads to accumulation of dirt and sickness, disappearance of a condom into a woman during sex, most pregnancies occur in the middle of the menstrual cycle as well as, one can become pregnant during the first sexual intercourse. Another majority; 93.2%, 53.2%, 65.9% and 53.6% actually admitted as outright false the fact that; frequent sex prevents pregnancy, sterility arises from the use of contraceptive use, washing of genitals after sex prevents pregnancy and the consumption of sachet whisky prevents conception (Table 3).
Knowledge of information on sexual and reproductive health (SRH)
Factors associated with unplanned teenage pregnancy in KEHD: A list of factors were provided in the questionnaire for the respondent to tick and out of the 293 respondents, 61.09% claimed never to have used the condom, and 52.56% said their partner was not the father of their child. 46.42% of the 293 participants said they do not use contraceptives, 13.65% failed to disclose the method of contraceptive used, by simply saying “none of the above”, when asked, when they started using contraceptive, 58.02%, said they have never used contraceptives (Table 4).
Factors associated with adolescent school girl’s pregnancy
Lack of knowledge on sexual reproductive health, circumstances of first sex, physical, sexual and substance abuse: From the answers provided by the respondent concerning knowledge assessment, 20.48% of the respondents had information on sex and reproductive health from their peers and, another 20.8% could not really identify their source of information on sex and reproductive health. 11.60% and 2.05% of the respondents learned of how to use contraceptives from friends and no defined sources (Table 5).
Circumstances of first sex, first sexual experience, relationship control and duration of relation
Characteristics of sexual partner and gender power relations in partnership: The sexual partners of respondents were of the age range 16-32 years (mean age of 22.815). At their age, a majority 67.24% of the respondents claimed their partners do not use drugs, while 6.49%, 15.02% and 3.75% respectively claimed their partners; do any injectable drugs, any other drugs and marijuana. On alcohol consumption frequency; 49.83% claimed not to know the drinking rate of their spouses, 7.85%, 20.82% and 21.50% said their partners drink at the rate of; only at weekend, less than once a month and a few times a month respectively (Table 6).
Alcohol/ drug attitude of partner
Discussion:
General patterns of teenage pregnancy in Kumbo East Health District The prevalence of teenage pregnancy in Cameroon is 28% and in Kumbo East Health District the results on this study shows that the prevalence of unplanned teenage pregnancy is 60.75% which very high this could be attributed to the fact that schools were not operational at the time of this study due to political unrest in the country at the time of data collection. Out of the 293 respondents, 61.09% claimed never to have used the condom, and 52.56% said their partner was not the father of their child. 46.42% of the 293 participants said they do not use contraceptives, 13.65% failed to disclose the method of contraceptive used, by simply saying “none of the above”, when asked, when they started using contraceptive, 58.02% said they have never used contraceptives. This implies that there is still a need for more efforts. Possible factors contributing to the high prevalence in this study area include age at first pregnancy, low contraceptive use, educational levels and socio-economic status, circumstances at first sex, lack of knowledge on reproductive and sexual health and physical and sexual violence.
The prevalence of teenage pregnancy in Cameroon is 28% and in Kumbo East Health District the results on this study shows that the prevalence of unplanned teenage pregnancy is 60.75% which very high this could be attributed to the fact that schools were not operational at the time of this study due to political unrest in the country at the time of data collection. Out of the 293 respondents, 61.09% claimed never to have used the condom, and 52.56% said their partner was not the father of their child. 46.42% of the 293 participants said they do not use contraceptives, 13.65% failed to disclose the method of contraceptive used, by simply saying “none of the above”, when asked, when they started using contraceptive, 58.02% said they have never used contraceptives. This implies that there is still a need for more efforts. Possible factors contributing to the high prevalence in this study area include age at first pregnancy, low contraceptive use, educational levels and socio-economic status, circumstances at first sex, lack of knowledge on reproductive and sexual health and physical and sexual violence.
Knowledge and sources of information on reproductive and sexual health Considering the respondents answers to question ask on whether their menstrual and reproductive characteristics , their knowledge of information on sexual and reproductive health [SRH], a majority of the respondents; 66.2%, 66.6%, 59.7% and 93.2% out rightly admitted that it is true that; amenorrhoea leads to accumulation of dirt and sickness, disappearance of a condom into a woman during sex, most pregnancies occur in the middle of the menstrual cycle as well as, one can become pregnant during the first sexual intercourse. Another majority, 93.2%, 53.2%, 65.9% and 53.6% actually admitted as outright false the fact that; frequent sex prevents pregnancy, sterility arises from the use of contraceptive use, washing of genitals after sex prevents pregnancy and the consumption of sachet whisky prevents conception (Table 4).
Considering the respondents answers to question ask on whether their menstrual and reproductive characteristics , their knowledge of information on sexual and reproductive health [SRH], a majority of the respondents; 66.2%, 66.6%, 59.7% and 93.2% out rightly admitted that it is true that; amenorrhoea leads to accumulation of dirt and sickness, disappearance of a condom into a woman during sex, most pregnancies occur in the middle of the menstrual cycle as well as, one can become pregnant during the first sexual intercourse. Another majority, 93.2%, 53.2%, 65.9% and 53.6% actually admitted as outright false the fact that; frequent sex prevents pregnancy, sterility arises from the use of contraceptive use, washing of genitals after sex prevents pregnancy and the consumption of sachet whisky prevents conception (Table 4).
Age of the teenagers The ages of participants ranged from 15-27 years, with mean age 18.88 years and modal age group of 15 -< 20 years with 227 [< 77.41%]. A large majority of participants 59.73% had no child, while 32.42% had one child, 0.68% had two children, 6.14% had three children and 1.02% had four children. In Cameroon, Kenya, South Africa and Canada, the average age difference between teenagers and their sexual partners is 15, 7, 5 and 2.6 years, respectively. In addition, teenagers who marry older men often have less power in decision making around sexual intercourse, childbearing and the use of contraceptive [1].
The ages of participants ranged from 15-27 years, with mean age 18.88 years and modal age group of 15 -< 20 years with 227 [< 77.41%]. A large majority of participants 59.73% had no child, while 32.42% had one child, 0.68% had two children, 6.14% had three children and 1.02% had four children. In Cameroon, Kenya, South Africa and Canada, the average age difference between teenagers and their sexual partners is 15, 7, 5 and 2.6 years, respectively. In addition, teenagers who marry older men often have less power in decision making around sexual intercourse, childbearing and the use of contraceptive [1].
Knowledge contraceptive use On the usage of contraceptives, 15.02% used the condom, 5.12% claim to use implants, 8.19% claim to use IUCD, while 11.60% use pills. On when they began using contraceptives; 2.05% began this year, 24.23% began last year, 7.51% began in the year 2015, 2.73% began in 2014 and 2013 as well as 2012 (Table 4). Respondents also advanced possible fears and reasons for not using contraceptive; 6.83, were scared of nurses, 20.14% were scared of their parents, 6.83% claimed it was against their religion and 12.97% admitted their partners were not in favour of it (Table 1). The fears, beliefs and perceptions of sexual inactivity associated with contraceptive use may be attributed to the lack of knowledge and low levels of awareness. This is conformity with a similar study Contraceptive prevalence in Cameroon is low among married women and shows uneven distribution varying from 2.6% in North Cameroon to 43.9% in Yaoundé [6, 7]. The low prevalence use among married couples suggests that once a woman is married she is exposed to pregnancy irrespective of her age. In Cameroon, unmet needs for family planning are 22%. And the use of modern contraception in Cameroon is about 16%. Reasons for non-contraceptive use include, religious and cultural beliefs, poor quality of services, including the negative attitude of service providers, fear of exposure of their bodies, having adults at the same services and inability to negotiate contraceptive use with sexual partners. Furthermore, misconceptions, fear of side effects and stigma associated with the use of contraceptives as adolescents may be labelled as being promiscuous can also be considered as contributing factors for non-contraceptive use [1]. Similarly, in South Africa, a study revealed that teenage pregnancy is attributed to the low utilization of contraceptives, especially on their first intercourse, due to the lack of access to medical information on reproductive system, inaccessible family planning services, gender inequality and decision on the social life, fears about contraception on fertility and menstruation and condoms could be left inside the vagina or womb [4]. It was therefore envisaged that the use of contraceptives among teenagers in the study area could be low, making it a factor for pregnancy.
On the usage of contraceptives, 15.02% used the condom, 5.12% claim to use implants, 8.19% claim to use IUCD, while 11.60% use pills. On when they began using contraceptives; 2.05% began this year, 24.23% began last year, 7.51% began in the year 2015, 2.73% began in 2014 and 2013 as well as 2012 (Table 4). Respondents also advanced possible fears and reasons for not using contraceptive; 6.83, were scared of nurses, 20.14% were scared of their parents, 6.83% claimed it was against their religion and 12.97% admitted their partners were not in favour of it (Table 1). The fears, beliefs and perceptions of sexual inactivity associated with contraceptive use may be attributed to the lack of knowledge and low levels of awareness. This is conformity with a similar study Contraceptive prevalence in Cameroon is low among married women and shows uneven distribution varying from 2.6% in North Cameroon to 43.9% in Yaoundé [6, 7]. The low prevalence use among married couples suggests that once a woman is married she is exposed to pregnancy irrespective of her age. In Cameroon, unmet needs for family planning are 22%. And the use of modern contraception in Cameroon is about 16%. Reasons for non-contraceptive use include, religious and cultural beliefs, poor quality of services, including the negative attitude of service providers, fear of exposure of their bodies, having adults at the same services and inability to negotiate contraceptive use with sexual partners. Furthermore, misconceptions, fear of side effects and stigma associated with the use of contraceptives as adolescents may be labelled as being promiscuous can also be considered as contributing factors for non-contraceptive use [1]. Similarly, in South Africa, a study revealed that teenage pregnancy is attributed to the low utilization of contraceptives, especially on their first intercourse, due to the lack of access to medical information on reproductive system, inaccessible family planning services, gender inequality and decision on the social life, fears about contraception on fertility and menstruation and condoms could be left inside the vagina or womb [4]. It was therefore envisaged that the use of contraceptives among teenagers in the study area could be low, making it a factor for pregnancy.
Levels of education, cultural practices and economic factor The ages of the partners of participants ranged from 16-32 years; Half, 50.17% of partners of adolescent school girls were in the age group 20 - < 25 years. 17.41% were in the age group 16 -< 20 years, 25.26% in the group 25 -< 30 years and 7.17% in the age group 30 - 32 years. With 51.88% of the 293 participant's partners had secondary school education, followed by those with tertiary education 40.27%, those with no formal education were 6.83% and primary education were 1.02%. Of the 293 participants, the partners of 44.37%, 40.96% and 14.68% were respectively studying, unemployed and working (Table 2). The study agrees that Teenage girls may indulge in sexual activity in exchange for goods, money and experiences such as taking meals in hotels [1]. A study in Malawi, found that 66% of adolescents had accepted money or gifts in exchange for sex and in some cases, parents may encourage their daughters into relationships with men for consumer goods or a girl may go out with men because her parents cannot give her the basic needs. Teenagers with unplanned pregnancy are more likely to come from low socio-economic status than with planned pregnancy [1]. The level of education of parents, especially the mother, may have an influence on the adolescent towards teenage pregnancy as she acts as a role model [9] which may be a preventive factor of teenage pregnancy. In Kenya it was reported that women with no education had first sexual intercourse three years earlier than their counterparts with at least a secondary school education [1]. The low literacy levels may lead to low paying jobs, causing early marriage and influencing non-contraceptive use, thereby increasing the prevalence of teenage pregnancy. Therefore, it was expected in the study area that the low literacy levels may have an impact on unplanned teenage pregnancy. Culturally, amongst factors associated with unplanned teenage pregnancy were, age at onset of menstruation, age at first sex, who they live with as well as the marital status of their parents. It was difficult to prove that culture is a factor. Economically, these were age, religious and delivery status of participants (Table 1), marital status of participants and parents, partner's educational and occupational status.
The ages of the partners of participants ranged from 16-32 years; Half, 50.17% of partners of adolescent school girls were in the age group 20 - < 25 years. 17.41% were in the age group 16 -< 20 years, 25.26% in the group 25 -< 30 years and 7.17% in the age group 30 - 32 years. With 51.88% of the 293 participant's partners had secondary school education, followed by those with tertiary education 40.27%, those with no formal education were 6.83% and primary education were 1.02%. Of the 293 participants, the partners of 44.37%, 40.96% and 14.68% were respectively studying, unemployed and working (Table 2). The study agrees that Teenage girls may indulge in sexual activity in exchange for goods, money and experiences such as taking meals in hotels [1]. A study in Malawi, found that 66% of adolescents had accepted money or gifts in exchange for sex and in some cases, parents may encourage their daughters into relationships with men for consumer goods or a girl may go out with men because her parents cannot give her the basic needs. Teenagers with unplanned pregnancy are more likely to come from low socio-economic status than with planned pregnancy [1]. The level of education of parents, especially the mother, may have an influence on the adolescent towards teenage pregnancy as she acts as a role model [9] which may be a preventive factor of teenage pregnancy. In Kenya it was reported that women with no education had first sexual intercourse three years earlier than their counterparts with at least a secondary school education [1]. The low literacy levels may lead to low paying jobs, causing early marriage and influencing non-contraceptive use, thereby increasing the prevalence of teenage pregnancy. Therefore, it was expected in the study area that the low literacy levels may have an impact on unplanned teenage pregnancy. Culturally, amongst factors associated with unplanned teenage pregnancy were, age at onset of menstruation, age at first sex, who they live with as well as the marital status of their parents. It was difficult to prove that culture is a factor. Economically, these were age, religious and delivery status of participants (Table 1), marital status of participants and parents, partner's educational and occupational status.
Gender power relations in partnerships There were a variety of circumstances that could lead to sex; partner violence, reasons for involvement in a relationship and first sexual experience. For partner violence; 19.80%, had their funds managed by their partners, 33.11% were forced by their partners into sex, 20.14% would be hit by their partners and 40.27% had their funds managed by their partners. 6.48%, 13.65% and 12.97% of respondents respectively got involved in a relationship to be provided with clothes, to be married and to have a good time. For the first sexual experience; 46.42% were persuaded either by a relation or boyfriend, 6.48% were raped, 21.84% were raped, 7.51% were willing and collaborated or 17.75% refused to disclose their first sexual experience (Table 5). This means that male partner is the one who decides what happen in a relationship.
There were a variety of circumstances that could lead to sex; partner violence, reasons for involvement in a relationship and first sexual experience. For partner violence; 19.80%, had their funds managed by their partners, 33.11% were forced by their partners into sex, 20.14% would be hit by their partners and 40.27% had their funds managed by their partners. 6.48%, 13.65% and 12.97% of respondents respectively got involved in a relationship to be provided with clothes, to be married and to have a good time. For the first sexual experience; 46.42% were persuaded either by a relation or boyfriend, 6.48% were raped, 21.84% were raped, 7.51% were willing and collaborated or 17.75% refused to disclose their first sexual experience (Table 5). This means that male partner is the one who decides what happen in a relationship.
Lack of knowledge on sexual reproductive health, circumstances of first sex, physical, sexual and substance abuse 20.48% of the respondents had information on sex and reproductive health from their peers and, another 20.8% could not really identify their source of information on sex and reproductive health. 11.60% and 2.05% of the respondents learned of how to use contraceptives from friends and no defined sources (Table 3). Most studies conducted in developing countries report that adolescent girls often lack basic knowledge about reproductive and sexual health [1]. This study contrast with that of findings of Nanze. C.C (2006 on risk factors of unwanted/uplanned pregnancy in zomba district, Malawi) indicate that communication about reproductive and sexual matters within families is limited, forcing girls to get information chiefly from peers, boyfriends and teachers. On drug and substance use, a wide majority (80.55%, said they do not take drugs 12.97% said they took any drug, while 6.48% admitted smoking “banga” (Table 7). Similar findings have been reported in South Africa [9]. Unlike wise in Cameroon, it is not common for teenage girls to use drug substances due to cultural values. However, there may have been underreporting since drug use is illegal, making it a sensitive topic.
Circumstances of first sex, first sexual experience, relationship control and duration of relation
20.48% of the respondents had information on sex and reproductive health from their peers and, another 20.8% could not really identify their source of information on sex and reproductive health. 11.60% and 2.05% of the respondents learned of how to use contraceptives from friends and no defined sources (Table 3). Most studies conducted in developing countries report that adolescent girls often lack basic knowledge about reproductive and sexual health [1]. This study contrast with that of findings of Nanze. C.C (2006 on risk factors of unwanted/uplanned pregnancy in zomba district, Malawi) indicate that communication about reproductive and sexual matters within families is limited, forcing girls to get information chiefly from peers, boyfriends and teachers. On drug and substance use, a wide majority (80.55%, said they do not take drugs 12.97% said they took any drug, while 6.48% admitted smoking “banga” (Table 7). Similar findings have been reported in South Africa [9]. Unlike wise in Cameroon, it is not common for teenage girls to use drug substances due to cultural values. However, there may have been underreporting since drug use is illegal, making it a sensitive topic.
Circumstances of first sex, first sexual experience, relationship control and duration of relation
General patterns of teenage pregnancy in Kumbo East Health District:
The prevalence of teenage pregnancy in Cameroon is 28% and in Kumbo East Health District the results on this study shows that the prevalence of unplanned teenage pregnancy is 60.75% which very high this could be attributed to the fact that schools were not operational at the time of this study due to political unrest in the country at the time of data collection. Out of the 293 respondents, 61.09% claimed never to have used the condom, and 52.56% said their partner was not the father of their child. 46.42% of the 293 participants said they do not use contraceptives, 13.65% failed to disclose the method of contraceptive used, by simply saying “none of the above”, when asked, when they started using contraceptive, 58.02% said they have never used contraceptives. This implies that there is still a need for more efforts. Possible factors contributing to the high prevalence in this study area include age at first pregnancy, low contraceptive use, educational levels and socio-economic status, circumstances at first sex, lack of knowledge on reproductive and sexual health and physical and sexual violence.
Knowledge and sources of information on reproductive and sexual health:
Considering the respondents answers to question ask on whether their menstrual and reproductive characteristics , their knowledge of information on sexual and reproductive health [SRH], a majority of the respondents; 66.2%, 66.6%, 59.7% and 93.2% out rightly admitted that it is true that; amenorrhoea leads to accumulation of dirt and sickness, disappearance of a condom into a woman during sex, most pregnancies occur in the middle of the menstrual cycle as well as, one can become pregnant during the first sexual intercourse. Another majority, 93.2%, 53.2%, 65.9% and 53.6% actually admitted as outright false the fact that; frequent sex prevents pregnancy, sterility arises from the use of contraceptive use, washing of genitals after sex prevents pregnancy and the consumption of sachet whisky prevents conception (Table 4).
Age of the teenagers:
The ages of participants ranged from 15-27 years, with mean age 18.88 years and modal age group of 15 -< 20 years with 227 [< 77.41%]. A large majority of participants 59.73% had no child, while 32.42% had one child, 0.68% had two children, 6.14% had three children and 1.02% had four children. In Cameroon, Kenya, South Africa and Canada, the average age difference between teenagers and their sexual partners is 15, 7, 5 and 2.6 years, respectively. In addition, teenagers who marry older men often have less power in decision making around sexual intercourse, childbearing and the use of contraceptive [1].
Knowledge contraceptive use:
On the usage of contraceptives, 15.02% used the condom, 5.12% claim to use implants, 8.19% claim to use IUCD, while 11.60% use pills. On when they began using contraceptives; 2.05% began this year, 24.23% began last year, 7.51% began in the year 2015, 2.73% began in 2014 and 2013 as well as 2012 (Table 4). Respondents also advanced possible fears and reasons for not using contraceptive; 6.83, were scared of nurses, 20.14% were scared of their parents, 6.83% claimed it was against their religion and 12.97% admitted their partners were not in favour of it (Table 1). The fears, beliefs and perceptions of sexual inactivity associated with contraceptive use may be attributed to the lack of knowledge and low levels of awareness. This is conformity with a similar study Contraceptive prevalence in Cameroon is low among married women and shows uneven distribution varying from 2.6% in North Cameroon to 43.9% in Yaoundé [6, 7]. The low prevalence use among married couples suggests that once a woman is married she is exposed to pregnancy irrespective of her age. In Cameroon, unmet needs for family planning are 22%. And the use of modern contraception in Cameroon is about 16%. Reasons for non-contraceptive use include, religious and cultural beliefs, poor quality of services, including the negative attitude of service providers, fear of exposure of their bodies, having adults at the same services and inability to negotiate contraceptive use with sexual partners. Furthermore, misconceptions, fear of side effects and stigma associated with the use of contraceptives as adolescents may be labelled as being promiscuous can also be considered as contributing factors for non-contraceptive use [1]. Similarly, in South Africa, a study revealed that teenage pregnancy is attributed to the low utilization of contraceptives, especially on their first intercourse, due to the lack of access to medical information on reproductive system, inaccessible family planning services, gender inequality and decision on the social life, fears about contraception on fertility and menstruation and condoms could be left inside the vagina or womb [4]. It was therefore envisaged that the use of contraceptives among teenagers in the study area could be low, making it a factor for pregnancy.
Levels of education, cultural practices and economic factor:
The ages of the partners of participants ranged from 16-32 years; Half, 50.17% of partners of adolescent school girls were in the age group 20 - < 25 years. 17.41% were in the age group 16 -< 20 years, 25.26% in the group 25 -< 30 years and 7.17% in the age group 30 - 32 years. With 51.88% of the 293 participant's partners had secondary school education, followed by those with tertiary education 40.27%, those with no formal education were 6.83% and primary education were 1.02%. Of the 293 participants, the partners of 44.37%, 40.96% and 14.68% were respectively studying, unemployed and working (Table 2). The study agrees that Teenage girls may indulge in sexual activity in exchange for goods, money and experiences such as taking meals in hotels [1]. A study in Malawi, found that 66% of adolescents had accepted money or gifts in exchange for sex and in some cases, parents may encourage their daughters into relationships with men for consumer goods or a girl may go out with men because her parents cannot give her the basic needs. Teenagers with unplanned pregnancy are more likely to come from low socio-economic status than with planned pregnancy [1]. The level of education of parents, especially the mother, may have an influence on the adolescent towards teenage pregnancy as she acts as a role model [9] which may be a preventive factor of teenage pregnancy. In Kenya it was reported that women with no education had first sexual intercourse three years earlier than their counterparts with at least a secondary school education [1]. The low literacy levels may lead to low paying jobs, causing early marriage and influencing non-contraceptive use, thereby increasing the prevalence of teenage pregnancy. Therefore, it was expected in the study area that the low literacy levels may have an impact on unplanned teenage pregnancy. Culturally, amongst factors associated with unplanned teenage pregnancy were, age at onset of menstruation, age at first sex, who they live with as well as the marital status of their parents. It was difficult to prove that culture is a factor. Economically, these were age, religious and delivery status of participants (Table 1), marital status of participants and parents, partner's educational and occupational status.
Gender power relations in partnerships:
There were a variety of circumstances that could lead to sex; partner violence, reasons for involvement in a relationship and first sexual experience. For partner violence; 19.80%, had their funds managed by their partners, 33.11% were forced by their partners into sex, 20.14% would be hit by their partners and 40.27% had their funds managed by their partners. 6.48%, 13.65% and 12.97% of respondents respectively got involved in a relationship to be provided with clothes, to be married and to have a good time. For the first sexual experience; 46.42% were persuaded either by a relation or boyfriend, 6.48% were raped, 21.84% were raped, 7.51% were willing and collaborated or 17.75% refused to disclose their first sexual experience (Table 5). This means that male partner is the one who decides what happen in a relationship.
Lack of knowledge on sexual reproductive health, circumstances of first sex, physical, sexual and substance abuse:
20.48% of the respondents had information on sex and reproductive health from their peers and, another 20.8% could not really identify their source of information on sex and reproductive health. 11.60% and 2.05% of the respondents learned of how to use contraceptives from friends and no defined sources (Table 3). Most studies conducted in developing countries report that adolescent girls often lack basic knowledge about reproductive and sexual health [1]. This study contrast with that of findings of Nanze. C.C (2006 on risk factors of unwanted/uplanned pregnancy in zomba district, Malawi) indicate that communication about reproductive and sexual matters within families is limited, forcing girls to get information chiefly from peers, boyfriends and teachers. On drug and substance use, a wide majority (80.55%, said they do not take drugs 12.97% said they took any drug, while 6.48% admitted smoking “banga” (Table 7). Similar findings have been reported in South Africa [9]. Unlike wise in Cameroon, it is not common for teenage girls to use drug substances due to cultural values. However, there may have been underreporting since drug use is illegal, making it a sensitive topic.
Circumstances of first sex, first sexual experience, relationship control and duration of relation
Conclusion:
The study factors associated with adolescent school girl's pregnancy in KEHD was aimed at examining the factors associated with teens pregnancy. A descriptive study was used and the teens 15 to 19yrs were selected. Teenage pregnancy whether planned or unplanned is detrimental to the health and socio economic status of the teenagers. This study shows that there is a high prevalence (60.75%) of teenage pregnancy in the sampled antenatal clinics of Kumbo East Health District attributable to the low contraceptive use and socio-economic status, lack of reproductive and sexual knowledge, circumstances at first sex, including force and rape, physical and sexual violence, early marriage, age of sexual partner and alcohol abuse. Among reasons contributing to the low use of contraceptives are; lack of knowledge, fear of side effects, including sterility, condoms disappearing in the womb and inequality of power with sexual partners. Teenagers obtain information mainly from school (53%) and relatives (20%). The high prevalence of unplanned teenage pregnancy, low contraceptive use, myths and misconceptions surrounding the use of contraceptives indicate that teenagers are receiving inaccurate information about reproductive and sexual health, a problem which may be compounded by low educational levels. Therefore, the lack of knowledge about reproductive health is a factor for teenage pregnancy. The lack of basic necessities is one of the factors that force teenagers to engage in transactional sex. Based on the study it is clear that teenagers have no control over sexual and reproductive health since their sexual partners are the sore decision makers making physical and sexual violence a factor for teenage pregnancy. However, the influence of cultural practices and religion, substance abuse, relationship control, and fear of parents and intimidating attitude of health service providers have been supported as factors. The associations suggested by the study point towards a need for greater emphasis in reproduction and sexual health promotion interventions. In addition, strengthening awareness and giving information to dispel fears, misconceptions and rumours about contraceptive use will prepare teenagers for the physical changes they might experience when adopting contraceptive methods. Nevertheless, contraceptive use alone may not reduce teenage pregnancy, but addressing the impact of poverty on teenagers, empowering them on their rights and information in order to make right choices is very important.
Recommendations: The study recognizes efforts being made by government and non-governmental organizations to solve the problem of teenage pregnancy in north west region and Kumbo East in particular. These efforts include promoting the use of contraceptives, education of girls and poverty alleviation. However, the low use of contraceptives and socio-economic status, continued sexual and physical violence, the feeling of inferiority due to age differences with sexual partners and early marriages indicate that factors for teenage pregnancy still persist in the study area. Therefore, there is a need to develop programmes to address factors identified in this study. Solutions to the problem require multidisciplinary implementing teams, including parents, schools, communities, NGOs and government sectors. The following recommendations are suggested.
What is known about this topic Knowledge of this geographic distribution can provide useful information for updating and strengthening adolescent reproductive health strategies in Cameroon;
In Cameroon, the prevalence of adolescent pregnancy is between 40% to 50%;
Adolescent age 15 to 19 contribute to 28% of maternal mortality in Cameroon.
Knowledge of this geographic distribution can provide useful information for updating and strengthening adolescent reproductive health strategies in Cameroon;
In Cameroon, the prevalence of adolescent pregnancy is between 40% to 50%;
Adolescent age 15 to 19 contribute to 28% of maternal mortality in Cameroon.
What this study adds Study will help provide few answers to this problem and may be prick health leaders to react towards the amelioration of some of their health strategies concerning reproductive health especially adolescent health in rural areas of Cameroon;
Moreover, it will also go a long way to convince national and international partners to continue to largely invest in the reproductive health section;
This will stand as a pillar to help adolescent to adopt responsible behaviours in regard to adolescent pregnancy especially at individual, community and health systems levels.
Study will help provide few answers to this problem and may be prick health leaders to react towards the amelioration of some of their health strategies concerning reproductive health especially adolescent health in rural areas of Cameroon;
Moreover, it will also go a long way to convince national and international partners to continue to largely invest in the reproductive health section;
This will stand as a pillar to help adolescent to adopt responsible behaviours in regard to adolescent pregnancy especially at individual, community and health systems levels.
What is known about this topic:
Knowledge of this geographic distribution can provide useful information for updating and strengthening adolescent reproductive health strategies in Cameroon;
In Cameroon, the prevalence of adolescent pregnancy is between 40% to 50%;
Adolescent age 15 to 19 contribute to 28% of maternal mortality in Cameroon.
What this study adds:
Study will help provide few answers to this problem and may be prick health leaders to react towards the amelioration of some of their health strategies concerning reproductive health especially adolescent health in rural areas of Cameroon;
Moreover, it will also go a long way to convince national and international partners to continue to largely invest in the reproductive health section;
This will stand as a pillar to help adolescent to adopt responsible behaviours in regard to adolescent pregnancy especially at individual, community and health systems levels.
Competing interests:
Authors declare no competing interests.
|
Background:
Teenage pregnancy is a social problem in Cameroon in general and in Kumbo East in particular. This results in physical, psychological and socio-economic consequences on the teenage mother, family and the society as a whole. In spite of studies and interventions that have been and are being implemented, the prevalence of unplanned teenage pregnancy in Kumbo East Health District is still high, suggesting that more efforts are required to achieve effective preventive measures. The aim of this study was to determine factors associated with adolescent school girl's pregnancy in Kumbo East health district.
Methods:
A cross-sectional descriptive study design was used and a simple random sampling technique was used to select 293 respondents aged 15 to 19year. The district hospital antenatal clinics and the Health Centres were selected. Data was obtained from 292 participants under the age of 20 years who were willing using a questionnaire administered through face-to-face interviews.
Results:
The study show a high prevalence (60.75%) of teenage pregnancy in the sampled antenatal clinics of Kumbo East Health District attributable to inadequate considerations given to factors associated with school girl's pregnancy. This study has indicated that the age of teenager at first pregnancy, low contraceptive use, socio-economic status and physical violence are factors that are greatly associated with teenage pregnancy. Among the reasons contributing to the low use of contraceptives are: sexually activity, lack of knowledge, fear of side effects, including sterility, condoms disappearing in the womb and inequality of power with sexual partners. This study shows that teenagers obtain information mainly from school (53%) and relatives (20%).
Conclusions:
The use of contraceptive alone may not reduce teenage pregnancy, however double method is very effective but addressing the impact of poverty on teenagers, empowering them on their rights and information in order to make right choices is very important.
|
Introduction:
Teenage pregnancy is considered as one occurring in a young woman who has not reached her 20th birthday. This definition is applicable irrespective of the legal status of the marriage of the woman or legal age to consider an individual as adult [1]. 16 million girls aged 15-19 years give birth each year, most prevalent in low and middle-income countries located in sub-Saharan Africa [1]. In the developing world, one-third to one-half of women become mothers before the age of 20 and pregnancy related complications have become the leading causes of death among them [1, 2]. Teenage pregnancies are a global phenomenon. The pregnancy rate among teenagers in USA was 6.78% of pregnancies per 1,000 women aged 15-19 in 2008. [3]. Among the countries in the Western Europe, the United Kingdom [UK] has the highest teenage conception and abortion rates [2, 4]. The report presents an update on the current situation of pregnancies among girls less than 18 years of age and adolescents 15-19 years of age; trends during the last 10 years; variations across geographic, cultural and economic settings; interventions available to minimize pregnancy among adolescents; evidence for these programmatic approaches; and challenges that nations will have to deal with in the next 20 years given current population momentum [5]. The concentration of adolescent girls aged 10 to 17 will also change significantly, with the largest increase occurring in sub-Saharan Africa, where adolescent pregnancy is most common, and the rate of contraceptive use the lowest in the world [5]. A study conducted in Malawi showed that 57% of teenage girls opt to risk pregnancy rather than asking a partner to use a condom [2]. In Malawi, there is a high prevalence of casual sex among teenagers who shun condoms although they engage in multiple relationships. Scholars in the field argue that, because of the risk associated with high prevalence of early sexual behaviour, low contraceptive use, and many early pregnancies, adolescents in Cameroon are an important target group for sexual and reproductive health programs [6, 7]. In order to prevent early age pregnancies, it is important to make sure that adolescents have the means to make informed and healthy choices concerning their sexual and reproductive health. Yet, as it stands, reproductive health and family planning services in Cameroon mainly target older married women, and adolescents often remain largely overlooked [5].
Adolescent school girl's pregnancies vary from country to country and in Cameroon, reproductive health remains a major public health challenge with elevated maternal mortality rate. The maternal mortality rate is estimated at 782 deaths per 100,000 live births [8]. This high mortality rate remains a dilemma because it involves the young mothers at the moment where they are giving birth. In addition, adolescent contribute 28% of maternal mortality in Cameroon [8]. According to DHS 2011, 25.6% of adolescents 15 to 19 have started sexual intercourse and 21% of then have had a child and 4% are pregnant for their first pregnancy. Still from the same source fertility rate of this age group is 127‰ which increase rapidly and attained a maximum of 250‰ within the age group 25-29yrs, suggesting that teenage pregnancy may be on the increase. The proportion of adolescents who have started fertility grow rapidly with age, 5% for 15yrs to 48% for 19yrs and the rate stood at 18% in the north west region [8]. Therefore, reducing teenage pregnancy, chiefly by promoting the use of contraceptives, will be necessary in order to prevent consequences that are associated with teenage pregnancy. Nevertheless, lessons from Zimbabwe and South Africa suggest that promoting the use of contraceptives alone does not necessarily reduce teenage pregnancy in developing as in developed countries. Therefore, other factors may be playing a major role as mentioned earlier. The general objective was to determine factors associated with adolescent school girl's pregnancy in Kumbo East Health District.
Conclusion:
The study factors associated with adolescent school girl's pregnancy in KEHD was aimed at examining the factors associated with teens pregnancy. A descriptive study was used and the teens 15 to 19yrs were selected. Teenage pregnancy whether planned or unplanned is detrimental to the health and socio economic status of the teenagers. This study shows that there is a high prevalence (60.75%) of teenage pregnancy in the sampled antenatal clinics of Kumbo East Health District attributable to the low contraceptive use and socio-economic status, lack of reproductive and sexual knowledge, circumstances at first sex, including force and rape, physical and sexual violence, early marriage, age of sexual partner and alcohol abuse. Among reasons contributing to the low use of contraceptives are; lack of knowledge, fear of side effects, including sterility, condoms disappearing in the womb and inequality of power with sexual partners. Teenagers obtain information mainly from school (53%) and relatives (20%). The high prevalence of unplanned teenage pregnancy, low contraceptive use, myths and misconceptions surrounding the use of contraceptives indicate that teenagers are receiving inaccurate information about reproductive and sexual health, a problem which may be compounded by low educational levels. Therefore, the lack of knowledge about reproductive health is a factor for teenage pregnancy. The lack of basic necessities is one of the factors that force teenagers to engage in transactional sex. Based on the study it is clear that teenagers have no control over sexual and reproductive health since their sexual partners are the sore decision makers making physical and sexual violence a factor for teenage pregnancy. However, the influence of cultural practices and religion, substance abuse, relationship control, and fear of parents and intimidating attitude of health service providers have been supported as factors. The associations suggested by the study point towards a need for greater emphasis in reproduction and sexual health promotion interventions. In addition, strengthening awareness and giving information to dispel fears, misconceptions and rumours about contraceptive use will prepare teenagers for the physical changes they might experience when adopting contraceptive methods. Nevertheless, contraceptive use alone may not reduce teenage pregnancy, but addressing the impact of poverty on teenagers, empowering them on their rights and information in order to make right choices is very important.
Recommendations: The study recognizes efforts being made by government and non-governmental organizations to solve the problem of teenage pregnancy in north west region and Kumbo East in particular. These efforts include promoting the use of contraceptives, education of girls and poverty alleviation. However, the low use of contraceptives and socio-economic status, continued sexual and physical violence, the feeling of inferiority due to age differences with sexual partners and early marriages indicate that factors for teenage pregnancy still persist in the study area. Therefore, there is a need to develop programmes to address factors identified in this study. Solutions to the problem require multidisciplinary implementing teams, including parents, schools, communities, NGOs and government sectors. The following recommendations are suggested.
What is known about this topic Knowledge of this geographic distribution can provide useful information for updating and strengthening adolescent reproductive health strategies in Cameroon;
In Cameroon, the prevalence of adolescent pregnancy is between 40% to 50%;
Adolescent age 15 to 19 contribute to 28% of maternal mortality in Cameroon.
Knowledge of this geographic distribution can provide useful information for updating and strengthening adolescent reproductive health strategies in Cameroon;
In Cameroon, the prevalence of adolescent pregnancy is between 40% to 50%;
Adolescent age 15 to 19 contribute to 28% of maternal mortality in Cameroon.
What this study adds Study will help provide few answers to this problem and may be prick health leaders to react towards the amelioration of some of their health strategies concerning reproductive health especially adolescent health in rural areas of Cameroon;
Moreover, it will also go a long way to convince national and international partners to continue to largely invest in the reproductive health section;
This will stand as a pillar to help adolescent to adopt responsible behaviours in regard to adolescent pregnancy especially at individual, community and health systems levels.
Study will help provide few answers to this problem and may be prick health leaders to react towards the amelioration of some of their health strategies concerning reproductive health especially adolescent health in rural areas of Cameroon;
Moreover, it will also go a long way to convince national and international partners to continue to largely invest in the reproductive health section;
This will stand as a pillar to help adolescent to adopt responsible behaviours in regard to adolescent pregnancy especially at individual, community and health systems levels.
|
Background:
Teenage pregnancy is a social problem in Cameroon in general and in Kumbo East in particular. This results in physical, psychological and socio-economic consequences on the teenage mother, family and the society as a whole. In spite of studies and interventions that have been and are being implemented, the prevalence of unplanned teenage pregnancy in Kumbo East Health District is still high, suggesting that more efforts are required to achieve effective preventive measures. The aim of this study was to determine factors associated with adolescent school girl's pregnancy in Kumbo East health district.
Methods:
A cross-sectional descriptive study design was used and a simple random sampling technique was used to select 293 respondents aged 15 to 19year. The district hospital antenatal clinics and the Health Centres were selected. Data was obtained from 292 participants under the age of 20 years who were willing using a questionnaire administered through face-to-face interviews.
Results:
The study show a high prevalence (60.75%) of teenage pregnancy in the sampled antenatal clinics of Kumbo East Health District attributable to inadequate considerations given to factors associated with school girl's pregnancy. This study has indicated that the age of teenager at first pregnancy, low contraceptive use, socio-economic status and physical violence are factors that are greatly associated with teenage pregnancy. Among the reasons contributing to the low use of contraceptives are: sexually activity, lack of knowledge, fear of side effects, including sterility, condoms disappearing in the womb and inequality of power with sexual partners. This study shows that teenagers obtain information mainly from school (53%) and relatives (20%).
Conclusions:
The use of contraceptive alone may not reduce teenage pregnancy, however double method is very effective but addressing the impact of poverty on teenagers, empowering them on their rights and information in order to make right choices is very important.
| 9,305 | 359 | 15 |
[
"use",
"pregnancy",
"sexual",
"health",
"age",
"teenage",
"reproductive",
"study",
"sex",
"contraceptive"
] |
[
"test",
"test"
] |
[CONTENT] Adolescents | factors | pregnancy | health district [SUMMARY]
|
[CONTENT] Adolescents | factors | pregnancy | health district [SUMMARY]
|
[CONTENT] Adolescents | factors | pregnancy | health district [SUMMARY]
|
[CONTENT] Adolescents | factors | pregnancy | health district [SUMMARY]
|
[CONTENT] Adolescents | factors | pregnancy | health district [SUMMARY]
|
[CONTENT] Adolescents | factors | pregnancy | health district [SUMMARY]
|
[CONTENT] Adolescent | Cameroon | Contraception | Contraception Behavior | Cross-Sectional Studies | Female | Health Knowledge, Attitudes, Practice | Humans | Poverty | Pregnancy | Pregnancy in Adolescence | Prevalence | Sexual Behavior | Socioeconomic Factors | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Cameroon | Contraception | Contraception Behavior | Cross-Sectional Studies | Female | Health Knowledge, Attitudes, Practice | Humans | Poverty | Pregnancy | Pregnancy in Adolescence | Prevalence | Sexual Behavior | Socioeconomic Factors | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Cameroon | Contraception | Contraception Behavior | Cross-Sectional Studies | Female | Health Knowledge, Attitudes, Practice | Humans | Poverty | Pregnancy | Pregnancy in Adolescence | Prevalence | Sexual Behavior | Socioeconomic Factors | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Cameroon | Contraception | Contraception Behavior | Cross-Sectional Studies | Female | Health Knowledge, Attitudes, Practice | Humans | Poverty | Pregnancy | Pregnancy in Adolescence | Prevalence | Sexual Behavior | Socioeconomic Factors | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Cameroon | Contraception | Contraception Behavior | Cross-Sectional Studies | Female | Health Knowledge, Attitudes, Practice | Humans | Poverty | Pregnancy | Pregnancy in Adolescence | Prevalence | Sexual Behavior | Socioeconomic Factors | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Cameroon | Contraception | Contraception Behavior | Cross-Sectional Studies | Female | Health Knowledge, Attitudes, Practice | Humans | Poverty | Pregnancy | Pregnancy in Adolescence | Prevalence | Sexual Behavior | Socioeconomic Factors | Surveys and Questionnaires | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] use | pregnancy | sexual | health | age | teenage | reproductive | study | sex | contraceptive [SUMMARY]
|
[CONTENT] use | pregnancy | sexual | health | age | teenage | reproductive | study | sex | contraceptive [SUMMARY]
|
[CONTENT] use | pregnancy | sexual | health | age | teenage | reproductive | study | sex | contraceptive [SUMMARY]
|
[CONTENT] use | pregnancy | sexual | health | age | teenage | reproductive | study | sex | contraceptive [SUMMARY]
|
[CONTENT] use | pregnancy | sexual | health | age | teenage | reproductive | study | sex | contraceptive [SUMMARY]
|
[CONTENT] use | pregnancy | sexual | health | age | teenage | reproductive | study | sex | contraceptive [SUMMARY]
|
[CONTENT] rate | pregnancy | pregnancies | adolescents | teenage | age | years | mortality | mortality rate | increase [SUMMARY]
|
[CONTENT] sample | data | clinics | research | questionnaire | sample size | period | size | pregnant | antenatal [SUMMARY]
|
[CONTENT] information | sex | sexuality | sex sexuality | sources | 293 | sources information | sexual | respondents | 52 [SUMMARY]
|
[CONTENT] health | adolescent | pregnancy | study | sexual | reproductive | problem | reproductive health | teenage pregnancy | teenagers [SUMMARY]
|
[CONTENT] health | use | pregnancy | sexual | reproductive | age | adolescent | sex | cameroon | years [SUMMARY]
|
[CONTENT] health | use | pregnancy | sexual | reproductive | age | adolescent | sex | cameroon | years [SUMMARY]
|
[CONTENT] Cameroon | Kumbo East ||| ||| Kumbo East Health District ||| Kumbo East [SUMMARY]
|
[CONTENT] 293 | 15 to 19year ||| the Health Centres ||| 292 | the age of 20 years [SUMMARY]
|
[CONTENT] 60.75% | Kumbo East Health District ||| first ||| ||| 53% | 20% [SUMMARY]
|
[CONTENT] [SUMMARY]
|
[CONTENT] Cameroon | Kumbo East ||| ||| Kumbo East Health District ||| Kumbo East ||| 293 | 15 to 19year ||| the Health Centres ||| 292 | the age of 20 years ||| ||| 60.75% | Kumbo East Health District ||| first ||| ||| 53% | 20% ||| [SUMMARY]
|
[CONTENT] Cameroon | Kumbo East ||| ||| Kumbo East Health District ||| Kumbo East ||| 293 | 15 to 19year ||| the Health Centres ||| 292 | the age of 20 years ||| ||| 60.75% | Kumbo East Health District ||| first ||| ||| 53% | 20% ||| [SUMMARY]
|
||||||
Physical Activity and Low Glycemic Index Mediterranean Diet: Main and Modification Effects on NAFLD Score. Results from a Randomized Clinical Trial.
|
33379253
|
Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common chronic liver disease worldwide, and lifestyle modification is the current standard treatment. The aim of the study was to estimate the effect of two different physical activity (PA) programs, a Low Glycemic Index Mediterranean Diet (LGIMD), and their combined effect on the NAFLD score as measured by FibroScan®.
|
BACKGROUND
|
Moderate or severe NAFLD subjects (n = 144) were randomly assigned to six intervention arms during three months. Interventions arms were a control diet, LGIMD, aerobic activity program (PA1), combined activity program (PA2), and LGIMD plus PA1 or LGIMD plus PA2. The data were compared at baseline, at 45 days, and at 90 days. Analysis of variance was performed under the intention-to-treat principle.
|
METHODS
|
There was a statistically significant reduction in the NAFLD score after 45 days of treatment in every working arm except for Arm 1 (control diet). After 90 days, the best results were shown by the intervention arms in which LGIMD was associated with PA: LGIMD plus PA1 (-61.56 95% CI -89.61, -33.50) and LGIMD plus PA2 (-38.15 95% CI -64.53, -11.77).
|
RESULTS
|
All treatments were effective to reduce NAFLD scores, but LGIMD plus PA1 was the most efficient.
|
CONCLUSION
|
[
"Adult",
"Behavior Therapy",
"Diet, Mediterranean",
"Exercise",
"Female",
"Glycemic Index",
"Humans",
"Life Style",
"Male",
"Middle Aged",
"Non-alcoholic Fatty Liver Disease"
] |
7823843
|
1. Introduction
|
Non-Alcoholic Fatty Liver Disease (NAFLD) is a broad spectrum of liver diseases. It is characterized by fat accumulation in the liver in the absence of excessive alcohol consumption or any other specific causes of hepatic steatosis [1]. NAFLD is a major public health challenge as it is associated with the rising prevalence of obesity and Diabetes Mellitus type 2 worldwide and is a risk factor for Cardiovascular Diseases. Moreover, NAFLD is the most common cause of chronic liver disease and increases the 5-year direct and indirect health care costs by an estimated 26% [2]. The global prevalence of NAFLD is currently estimated to be 24% and is continually rising [3]. Our studies, conducted in the population of Castellana Grotte and Putignano (district of Bari, Apulia, Italy), evidenced an NAFLD prevalence of about 30%, with a higher prevalence in male and older patients [4,5]. Therefore, accurate diagnosis of NAFLD is important in patient management [6]. Until now, the gold standard for hepatic steatosis diagnosis has been liver biopsy, but it is an invasive procedure that has several disadvantages such as low acceptance for patients (especially for volunteers), complications, and even a small risk of death (0.01%) [7,8]. Today, many imaging methods are available for the non-invasive assessment of NAFLD [9], but they have limitations such as high costs or operator-dependency of Ultrasound Scan (US) [10,11]. FibroScan® is a non-invasive examination that appears to have good repeatability, which can be used as indices of continuous observation [12]. Data suggest that the controlled attenuation parameter (CAP), measured with FibroScan®, can be compared to liver biopsy for the detection and quantification of steatosis [13], and recent studies have evaluated its accuracy in NAFLD [14]. Furthermore, Lee et al. 2016 demonstrated that CAP and liver stiffness can be used as non-invasive and predictive markers of steatosis and fibrosis in patients with NAFLD [15].
Lifestyle modification is the standard treatment for NAFLD [16]. This approach encompasses dietary modifications, exercise, reduced alcohol intake, and weight loss, as they offer a range of health benefits. The Mediterranean Diet (MD) has long been associated with favorable health outcomes [17,18], and good adherence to MD has been reported to have a beneficial effect on the severity of NAFLD [19] as well as of Cardiovascular Diseases [20]. In particular, the Low Carbohydrate Diet, similar to the Low Glycemic Index Mediterranean Diet, is characterized by a reduction in the intake of several ultra-processed foods, refined grains starches, and foods rich in simple or added sugars, and it leads to a number of benefits such as weight loss and weight loss maintenance, reduction of Diastolic Blood Pressure (DBP), reduction of low-density lipoprotein cholesterol (LDL-C) and Triglycerides levels, increase of high-density lipoprotein cholesterol (HDL-C) levels, improvements in insulin resistance, and reduction of glycated hemoglobin (HbA1c) levels [21].
In addition, different forms of exercise have been shown to have similar effects on liver fat [22,23] if physical activity (PA) guidelines recommendations are followed [24]. Minimal physical activity (PA) decreases by 6% the risk for NAFLD compared to sedentary behavior [25]. The European Association for the Study of the Liver (EASL), the European Association for the Study of Diabetes (EASD) and the European Association for the Study of Obesity (EASO) guidelines recommend 150 to 200 min/week of moderate-intensity aerobic physical activity in three to five sessions [26]. Aerobic activity and resistance training seem to have similar improvement in NAFLD but with different mechanisms [27]. Nevertheless, there are still no universal recommendations on the optimal intensity, dose, and type of physical activity in NAFLD treatment. Two studies conducted in this area have confirmed the efficacy of Low Glycemic Index Mediterranean Diet (LGIMD) [28] and the efficiency of aerobic PA on NAFLD [29]. In this context, and as studies about the effect of diet and PA on NAFLD are scarce, we hypothesized that different PA programs and LGIMD or their combination could have different degrees of efficacy on NAFLD severity. We built a local version of the Mediterranean diet based on the work of Trichopoulos et al. and Elia, which contains no more than 10% of total daily calories deriving from saturated fats and a low glycemic index with an extensive use of olive oil [30,31]. Furthermore, the PA programs were built following European guidelines, which comprise aerobic PA as well as resistance training with precision indication about frequency, duration and intensity [26]. This work was aimed estimate the effect of healthy diet (LGIMD), two different PA programs, and their combination on the score of NAFLD as measured in a continuous scale by FibroScan®. Moreover, we aimed to explore biochemical, anthropometric, and body composition changes by different degrees of severity of NAFLD in geographical area where the Mediterranean diet is the most prevalent way of eating.
| null | null |
3. Results
|
One hundred and sixty-six subjects were assessed for eligibility: 17 subjects were excluded because they did not satisfy the inclusion criteria and 5 were excluded for various other reasons. The study design is shown in Figure 1. The following subjects were lost to follow-up: 5, 3, 7, 6, 9, and 4 from Arms 1, 2, 3, 4, 5 and 6, respectively.
Finally, 144 subjects were included and randomized to the intervention arms. Characteristics of the participants are shown in Table 1 and Table 2 and in Appendix A, Table A1.
As expected, the age–sex distribution of the population under study reflected the age–sex distribution of the NAFLD condition in the population [4]. About 62% of the sample were men, and the mean age was 49.9 years (10.08), but few subjects were under 40 years old (12.5%). As expected, all subjects were overweight or obese with increased waist circumference. At baseline, almost every participant had low levels of HDL-C and a high HOMA-IR. Most subjects were married and had attended secondary school or higher (Table 2). All parameters considered were equally distributed among groups at baseline with the exception of NAFLD severity. More subjects had severe than moderate NAFLD, and the CAP value was over 323 dB/m in all groups with a successive decreasing in intermediate and final observation times.
The behavior of anthropometric, body composition, and biochemical profiles by time and intervention arm are shown in Appendix A, Table A1. Overall, there were improvements in all markers, especially at 45 days. These improvements were particularly evident for intervention arms PA1 + LGIMD and PA2 + LGIMD.
Field Fitness Test results by gender and time are shown in Table 3.
Overall, there was a significant improvement of the fitness index in both men and women. The compliance to physical activity programs is shown in Table 4.
Overall, adherence was above 80% to both PA intervention program in both genders and in all weeks.
Compliance to LGIMD, as measured by MAI, by age class, gender, and week is shown in Table 5.
Compliance with the control diet showed a median MAI of 1.29 (0.77, 2.63). MAI was lower amongst males (1.07) than females (2.14), whereas compliance with LGMID evidenced a median MAI of 11.2 (6.4, 22.8). Adherence was lower among women (10.0) than men (11.3).
Table 6 shows the changes in the CAP value by intervention arms and time (at 45 days and at 90 days).
Apart from the CD, there were statistically significant principal effects of all the other intervention arms, Arms 3 and 5 showing the strongest effects (PA1: −166.35, 95%CI −242.01; −90.68 and LGIMD plus PA1: −94.10, 95% CI −170.87; −19.12). After the analysis, no statistical significance principal effect of time in reducing CAP was observed. All comparisons between different time points and Intervention Arms, except for Intervention Arm 1, showed a statistically significant decreased value for CAP. The strongest effect was observed for LGIMD plus PA1 at 45 days (−52.96, 95% CI −79.03; −26.88) and 90 days (−61.56, 95% CI −89.61; −33.50).
Results from separate analysis by NAFLD severity are shown in Appendix A, Table A2 and Table A3. The relative decrease of CAP values was more relevant among severe NAFLD subjects. HOMA-IR decreased in all intervention arms with the exception of LGIMD in moderate NAFLD subjects. In both moderate and severe NAFLD subjects, there was a modest effect of only-diet intervention arms on CAP. Only-PA intervention arms effect on CAP values evidenced a decrease among severe NAFLD subjects but not among moderate ones. The most intense effect in decreasing CAP values was observed among severe NAFLD subjects who underwent PA1+LGIMD. An improvement in all anthropometric, body composition, and biochemical parameters was observed (most evident at day 45). Noteworthy, the improvement in HDL-C was accompanied by an improvement in FFM only in moderate NAFLD subjects. Table 7 shows the changes in the CAP value by NAFLD severity, intervention arms, and time (at 45 days and at 90 days).
There was an effect that was statistically significant only in subjects with moderate NAFLD allocated to Arm 6 after 90 days, whereas among severe NAFLD subjects, there were statistically significant effects in all times and arms with the exception of CD and Arm 6 at 90 days. The strongest estimated effect was observed in the PA1 + LGIMD intervention arm.
The results of predictive margins from analysis of variance are graphically displayed in Figure 2, which highlights the strong effect of LGIMD plus PA1. Hypothesis testing about the equality of measurements at baseline showed no statistically significant differences in CAP among intervention arms (p > 0.05).
|
5. Conclusions
|
It is clear that PA single (aerobic or resistance exercise) or combined (aerobic plus resistance exercise) should be integrated into NAFLD management [78]. Moreover, aerobic exercise programs are simple to implement and the cost-efficiency is higher. These should be included in programs for the primary prevention of metabolic and cardiovascular diseases.
In conclusion, a multidisciplinary team including dietitians, a psychologist, and physical activity supervisors is needed to ensure the best management of NAFLD patients [78].
|
[
"2. Materials and Methods",
"2.2. Study Design",
"2.3. Sample Size",
"2.4. Data Collection",
"2.5. Randomization and Masking",
"2.6. Dietary Interventions",
"2.7. Physical Activity Interventions",
"2.7.1. Aerobic Activity Program",
"2.7.2. Combination of Aerobic Exercise and Resistance Training",
"2.8. Outcome Assessment",
"2.9. Statistical Analysis"
] |
[
" 2.1. Participants Details about the study have been published elsewhere [32]. Briefly, 166 subjects, referred to the Laboratory of Epidemiology and Biostatistics of the National Institute of Digestive Diseases, IRCCS “S. de Bellis”, Castellana Grotte, Italy, by their General Practitioners or identified during the NutriEp [5] or MICOL [33] enrollment or follow-up process, were assessed for eligibility. The trial was conducted from March 2015 to December 2016. Patients were consecutively enrolled, and the trial was registered at www.https://clinicaltrials.gov (registration number CT02347696). All subjects provided informed consent and the study was conducted in accordance with the Helsinki Declaration and approved by the Ethical Committee (Prot. n. 10/CE/De Bellis, 3 February 2015).\nDetails about the study have been published elsewhere [32]. Briefly, 166 subjects, referred to the Laboratory of Epidemiology and Biostatistics of the National Institute of Digestive Diseases, IRCCS “S. de Bellis”, Castellana Grotte, Italy, by their General Practitioners or identified during the NutriEp [5] or MICOL [33] enrollment or follow-up process, were assessed for eligibility. The trial was conducted from March 2015 to December 2016. Patients were consecutively enrolled, and the trial was registered at www.https://clinicaltrials.gov (registration number CT02347696). All subjects provided informed consent and the study was conducted in accordance with the Helsinki Declaration and approved by the Ethical Committee (Prot. n. 10/CE/De Bellis, 3 February 2015).\n 2.2. Study Design This study was a parallel group randomized controlled clinical trial. Inclusion criteria were Body Mass Index (BMI) ≥25, expressed as weight in kilograms divided by square height in meters (kg/m2); age >30 years old and <60 years old; moderate or severe NAFLD as assessed by the controlled attenuation parameter (CAP). Exclusion criteria included (1) overt cardiovascular disease and revascularization procedures; (2) stroke; (3) clinical peripheral artery disease; (4) T2DM (current treatment with insulin or oral hypoglycemic drugs, fasting glucose >126 mg/dL, or casual glucose >200 mg/dL; (5) more than 20 gr/daily of alcohol intake; (6) any severe medical condition that could prevent the subject’s participation in a nutritional intervention study; (7) subjects following a special diet or involved in a program for weight loss, or who had experienced recent weight loss, and (8) inability to follow a LGIMD for religious or other reasons [32].\nThis study was a parallel group randomized controlled clinical trial. Inclusion criteria were Body Mass Index (BMI) ≥25, expressed as weight in kilograms divided by square height in meters (kg/m2); age >30 years old and <60 years old; moderate or severe NAFLD as assessed by the controlled attenuation parameter (CAP). Exclusion criteria included (1) overt cardiovascular disease and revascularization procedures; (2) stroke; (3) clinical peripheral artery disease; (4) T2DM (current treatment with insulin or oral hypoglycemic drugs, fasting glucose >126 mg/dL, or casual glucose >200 mg/dL; (5) more than 20 gr/daily of alcohol intake; (6) any severe medical condition that could prevent the subject’s participation in a nutritional intervention study; (7) subjects following a special diet or involved in a program for weight loss, or who had experienced recent weight loss, and (8) inability to follow a LGIMD for religious or other reasons [32].\n 2.3. Sample Size Sample size was estimated considering the repeated measurements nature of the outcome. From a previous study [34], the mean ± Standard Deviation (SD) score of NAFLD was estimated to be 4.5 (1) and 4.0 (0.5) for the treatment and control groups, respectively. We hypothesized a decrease by 20 units of the CAP score after three months of intervention. Probabilistic type I error was fixed at 0.05 (one sided) and probabilistic error type II was fixed at 0.10; statistical power was fixed at 0.9. The correlation between baseline/follow-up measurements of the outcome was set at 0.4. A sample size of n1 = n2 = n3 = n4 = n5 = n6 = 20 was estimated.\nSample size was estimated considering the repeated measurements nature of the outcome. From a previous study [34], the mean ± Standard Deviation (SD) score of NAFLD was estimated to be 4.5 (1) and 4.0 (0.5) for the treatment and control groups, respectively. We hypothesized a decrease by 20 units of the CAP score after three months of intervention. Probabilistic type I error was fixed at 0.05 (one sided) and probabilistic error type II was fixed at 0.10; statistical power was fixed at 0.9. The correlation between baseline/follow-up measurements of the outcome was set at 0.4. A sample size of n1 = n2 = n3 = n4 = n5 = n6 = 20 was estimated.\n 2.4. Data Collection During enrollment, patients were interviewed by trained nutritionists to collect information on socio-demographic aspects, medical history, and lifestyle. Information about physical activity was collected using the validated questionnaire International Physical Activity Questionnaire Long Form (IPAQ-LF) [35]. The European Prospective Investigation into Cancer and Nutrition Food Frequency Questionnaire (EPIC FFQ) was used to probe alcohol intake and eating behavior [36]. Anthropometric measurements (weight, height, waist circumference) were taken in a standard manner. Weight and height measurements were taken using SECA instruments (Model 700; and Model 206; 220 cm; SECA, Hamburg, Germany). Blood samples, taken by venous puncture, were drawn in the morning after overnight fasting and were collected in tubes containing ethylenediamine tetra acetic acid (K-EDTA) anticoagulant. Biochemical measurements were performed using standard methods.\nDuring enrollment, patients were interviewed by trained nutritionists to collect information on socio-demographic aspects, medical history, and lifestyle. Information about physical activity was collected using the validated questionnaire International Physical Activity Questionnaire Long Form (IPAQ-LF) [35]. The European Prospective Investigation into Cancer and Nutrition Food Frequency Questionnaire (EPIC FFQ) was used to probe alcohol intake and eating behavior [36]. Anthropometric measurements (weight, height, waist circumference) were taken in a standard manner. Weight and height measurements were taken using SECA instruments (Model 700; and Model 206; 220 cm; SECA, Hamburg, Germany). Blood samples, taken by venous puncture, were drawn in the morning after overnight fasting and were collected in tubes containing ethylenediamine tetra acetic acid (K-EDTA) anticoagulant. Biochemical measurements were performed using standard methods.\n 2.5. Randomization and Masking According to a computerized random numbers sequence, subjects were randomized to six arms as follows: (1) Control Diet (CD) based on CREA-AN (Research Center for Food and Nutrition, Council for Agricultural Research and Economics, Rome, Italy) guidelines [37]; (2) Low Glycemic Index Mediterranean Diet (LGIMD); (3) Physical Activity aerobic program (PA1); (4) Physical Activity combined program (aerobic activity and resistance training) (PA2); (5) LGIMD plus PA1 and (6) LGIMD plus PA2. The trial flowchart is shown in Figure 1.\nAccording to a computerized random numbers sequence, subjects were randomized to six arms as follows: (1) Control Diet (CD) based on CREA-AN (Research Center for Food and Nutrition, Council for Agricultural Research and Economics, Rome, Italy) guidelines [37]; (2) Low Glycemic Index Mediterranean Diet (LGIMD); (3) Physical Activity aerobic program (PA1); (4) Physical Activity combined program (aerobic activity and resistance training) (PA2); (5) LGIMD plus PA1 and (6) LGIMD plus PA2. The trial flowchart is shown in Figure 1.\n 2.6. Dietary Interventions Two types of diets were prescribed: a Control Diet (CD) based on CREA-AN guidelines [37] and a Low Glycemic Index Mediterranean Diet (LGIMD) [28]. No indications were given regarding the total calories to be consumed. Foods in LGIMD have all a low Glycemic Index (GI) and no more than 10% of total daily calories coming from saturated fats. The LGIMD was high in monounsaturated fatty acids (MUFA) from olive oil and contained also omega-3 polyunsaturated fatty acids (ω3PUFA), from both plant and marine sources. The prescribed diets were provided in brochure format, with graphic explanations. All participants were asked to record what they ate on a daily diary. The Mediterranean Adequacy Index (MAI) was chosen as a relevant measure to evaluate the adherence to both the intervention diet and the control diet [38]. Detailed information relating to the LGIMD intervention and control (recommended by the WHO and followed by INRAN [39]) is shown in Figure S1 and Figure S2.\nTwo types of diets were prescribed: a Control Diet (CD) based on CREA-AN guidelines [37] and a Low Glycemic Index Mediterranean Diet (LGIMD) [28]. No indications were given regarding the total calories to be consumed. Foods in LGIMD have all a low Glycemic Index (GI) and no more than 10% of total daily calories coming from saturated fats. The LGIMD was high in monounsaturated fatty acids (MUFA) from olive oil and contained also omega-3 polyunsaturated fatty acids (ω3PUFA), from both plant and marine sources. The prescribed diets were provided in brochure format, with graphic explanations. All participants were asked to record what they ate on a daily diary. The Mediterranean Adequacy Index (MAI) was chosen as a relevant measure to evaluate the adherence to both the intervention diet and the control diet [38]. Detailed information relating to the LGIMD intervention and control (recommended by the WHO and followed by INRAN [39]) is shown in Figure S1 and Figure S2.\n 2.7. Physical Activity Interventions To evaluate the initial physical condition, the right training program, and to compare the initial with the FU assessment of physical condition, three field tests were carried out: cardiorespiratory [40], strength [41], and flexibility fitness [42]. Cardio-respiratory fitness was assessed by means of the 2 km walking test, which is suitable for adults [40], whereas strength and flexibility fitness were evaluated by means of the push-up test (also called press-up test) and the Sit and Reach test, respectively. Subjects randomized to an intervention arm that included physical activity underwent the tests at baseline, after 45, and after 90 days, whereas subjects randomized to the diet arms underwent the field fitness tests at baseline and after 90 days. Physical activity interventions included two different types of exercise programs: Physical Activity 1 (PA1), based on the aerobic activity program, and Physical Activity 2 (PA2), based on the combination of aerobic activity and resistance training. The primary methods used for determining the intensity of prescribed exercise are maximum heart rate (Max HR), through heart-rate monitors [43], and metabolic equivalent (MET) [44,45]. To establish the age-predicted maximal heart rate, we used the formulas of Tanaka [46]. All subjects were randomized for one of the two physical activity programs based on the results of the tests performed. A progressive change of the monthly target has been planned based on the results obtained.\n 2.7.1. Aerobic Activity Program Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\nThree non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\n 2.7.2. Combination of Aerobic Exercise and Resistance Training Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.\nThree non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.\nTo evaluate the initial physical condition, the right training program, and to compare the initial with the FU assessment of physical condition, three field tests were carried out: cardiorespiratory [40], strength [41], and flexibility fitness [42]. Cardio-respiratory fitness was assessed by means of the 2 km walking test, which is suitable for adults [40], whereas strength and flexibility fitness were evaluated by means of the push-up test (also called press-up test) and the Sit and Reach test, respectively. Subjects randomized to an intervention arm that included physical activity underwent the tests at baseline, after 45, and after 90 days, whereas subjects randomized to the diet arms underwent the field fitness tests at baseline and after 90 days. Physical activity interventions included two different types of exercise programs: Physical Activity 1 (PA1), based on the aerobic activity program, and Physical Activity 2 (PA2), based on the combination of aerobic activity and resistance training. The primary methods used for determining the intensity of prescribed exercise are maximum heart rate (Max HR), through heart-rate monitors [43], and metabolic equivalent (MET) [44,45]. To establish the age-predicted maximal heart rate, we used the formulas of Tanaka [46]. All subjects were randomized for one of the two physical activity programs based on the results of the tests performed. A progressive change of the monthly target has been planned based on the results obtained.\n 2.7.1. Aerobic Activity Program Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\nThree non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\n 2.7.2. Combination of Aerobic Exercise and Resistance Training Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.\nThree non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.\n 2.8. Outcome Assessment To quantify and detect hepatic steatosis, the controlled attenuation parameter (CAP) score was used. CAP measures the degree of ultrasound attenuation due to hepatic fat at the standardized frequency of 3.5 MHz through a vibration-controlled elastography (VCTE) implemented on FibroScan® (Echosens, Paris, France). Values <248, 248–267, 268–279 and ≥280 dB/m indicate absence, mild, moderate, or severe NAFLD, respectively [47].\nTo quantify and detect hepatic steatosis, the controlled attenuation parameter (CAP) score was used. CAP measures the degree of ultrasound attenuation due to hepatic fat at the standardized frequency of 3.5 MHz through a vibration-controlled elastography (VCTE) implemented on FibroScan® (Echosens, Paris, France). Values <248, 248–267, 268–279 and ≥280 dB/m indicate absence, mild, moderate, or severe NAFLD, respectively [47].\n 2.9. Statistical Analysis The intention-to-treat principle was applied in all analysis. A description of the data was performed by Means (±SD), Median (IQR), and Frequencies (%) as appropriate. Data were also described separately by NAFLD severity. Biochemical markers were included only for descriptive purposes. Adherence to PA programs was evaluated as the proportion of effective frequency/time/intensity spent on gym or field sessions divided by the expected weekly frequency/time/intensity per session for the aerobic exercise group, plus expected load for the combined exercise group. The adherence was estimated by age group, gender, and month, and it was expressed as percentage. The third to tenth weeks of intervention were considered. The first two weeks were excluded to give subjects the opportunity to adapt to the programs training, and the last two weeks were excluded because the compliance could be decreased. The adherence to the LGIMD was measured with MAI [38]. Random week and weekend days were chosen from the second to 9th weeks of intervention. The first two weeks were excluded to allow participants to learn about the new diet and weeks successive to ninth were excluded because the compliance could decrease. To clearly describe the adherence and compliance to the LGIMD, MAI was estimated by gender, age, and week. Compliance was defined as previously described [38]. A median value of 7.5 was expected, as established by the reference Italian Mediterranean Diet [38].\nTo analyze differences among groups (Diet by Time), an Analysis of Variance for repeated measures was applied to the data. This statistical technique was chosen because it is relatively robust against violations of its assumptions, allows for multiple comparisons, and can be applied to a variety of experimental designs. Analysis of variance for repeated measurements was also performed by NAFLD severity. CAP was normally distributed, so no transformation was needed. Post estimation procedures were performed to compare estimated means with the reference category (control diet at baseline). To display graphically prediction from the fitted model, margins post estimation commands were performed. Margins statistics were calculated from predictions of a previously fitted Analysis of Variance model and displayed in graph form. Results are all expressed in the natural scale of CAP measurements as long as 95% Confidence Interval (95% CI).\nStata statistical (version 16.1) package was used to perform statistical analysis (StataCorp, 4905 Lakeway Drive, College Station, TX, USA); the Stata code is available upon request from the email: [email protected]).\nThe intention-to-treat principle was applied in all analysis. A description of the data was performed by Means (±SD), Median (IQR), and Frequencies (%) as appropriate. Data were also described separately by NAFLD severity. Biochemical markers were included only for descriptive purposes. Adherence to PA programs was evaluated as the proportion of effective frequency/time/intensity spent on gym or field sessions divided by the expected weekly frequency/time/intensity per session for the aerobic exercise group, plus expected load for the combined exercise group. The adherence was estimated by age group, gender, and month, and it was expressed as percentage. The third to tenth weeks of intervention were considered. The first two weeks were excluded to give subjects the opportunity to adapt to the programs training, and the last two weeks were excluded because the compliance could be decreased. The adherence to the LGIMD was measured with MAI [38]. Random week and weekend days were chosen from the second to 9th weeks of intervention. The first two weeks were excluded to allow participants to learn about the new diet and weeks successive to ninth were excluded because the compliance could decrease. To clearly describe the adherence and compliance to the LGIMD, MAI was estimated by gender, age, and week. Compliance was defined as previously described [38]. A median value of 7.5 was expected, as established by the reference Italian Mediterranean Diet [38].\nTo analyze differences among groups (Diet by Time), an Analysis of Variance for repeated measures was applied to the data. This statistical technique was chosen because it is relatively robust against violations of its assumptions, allows for multiple comparisons, and can be applied to a variety of experimental designs. Analysis of variance for repeated measurements was also performed by NAFLD severity. CAP was normally distributed, so no transformation was needed. Post estimation procedures were performed to compare estimated means with the reference category (control diet at baseline). To display graphically prediction from the fitted model, margins post estimation commands were performed. Margins statistics were calculated from predictions of a previously fitted Analysis of Variance model and displayed in graph form. Results are all expressed in the natural scale of CAP measurements as long as 95% Confidence Interval (95% CI).\nStata statistical (version 16.1) package was used to perform statistical analysis (StataCorp, 4905 Lakeway Drive, College Station, TX, USA); the Stata code is available upon request from the email: [email protected]).",
"This study was a parallel group randomized controlled clinical trial. Inclusion criteria were Body Mass Index (BMI) ≥25, expressed as weight in kilograms divided by square height in meters (kg/m2); age >30 years old and <60 years old; moderate or severe NAFLD as assessed by the controlled attenuation parameter (CAP). Exclusion criteria included (1) overt cardiovascular disease and revascularization procedures; (2) stroke; (3) clinical peripheral artery disease; (4) T2DM (current treatment with insulin or oral hypoglycemic drugs, fasting glucose >126 mg/dL, or casual glucose >200 mg/dL; (5) more than 20 gr/daily of alcohol intake; (6) any severe medical condition that could prevent the subject’s participation in a nutritional intervention study; (7) subjects following a special diet or involved in a program for weight loss, or who had experienced recent weight loss, and (8) inability to follow a LGIMD for religious or other reasons [32].",
"Sample size was estimated considering the repeated measurements nature of the outcome. From a previous study [34], the mean ± Standard Deviation (SD) score of NAFLD was estimated to be 4.5 (1) and 4.0 (0.5) for the treatment and control groups, respectively. We hypothesized a decrease by 20 units of the CAP score after three months of intervention. Probabilistic type I error was fixed at 0.05 (one sided) and probabilistic error type II was fixed at 0.10; statistical power was fixed at 0.9. The correlation between baseline/follow-up measurements of the outcome was set at 0.4. A sample size of n1 = n2 = n3 = n4 = n5 = n6 = 20 was estimated.",
"During enrollment, patients were interviewed by trained nutritionists to collect information on socio-demographic aspects, medical history, and lifestyle. Information about physical activity was collected using the validated questionnaire International Physical Activity Questionnaire Long Form (IPAQ-LF) [35]. The European Prospective Investigation into Cancer and Nutrition Food Frequency Questionnaire (EPIC FFQ) was used to probe alcohol intake and eating behavior [36]. Anthropometric measurements (weight, height, waist circumference) were taken in a standard manner. Weight and height measurements were taken using SECA instruments (Model 700; and Model 206; 220 cm; SECA, Hamburg, Germany). Blood samples, taken by venous puncture, were drawn in the morning after overnight fasting and were collected in tubes containing ethylenediamine tetra acetic acid (K-EDTA) anticoagulant. Biochemical measurements were performed using standard methods.",
"According to a computerized random numbers sequence, subjects were randomized to six arms as follows: (1) Control Diet (CD) based on CREA-AN (Research Center for Food and Nutrition, Council for Agricultural Research and Economics, Rome, Italy) guidelines [37]; (2) Low Glycemic Index Mediterranean Diet (LGIMD); (3) Physical Activity aerobic program (PA1); (4) Physical Activity combined program (aerobic activity and resistance training) (PA2); (5) LGIMD plus PA1 and (6) LGIMD plus PA2. The trial flowchart is shown in Figure 1.",
"Two types of diets were prescribed: a Control Diet (CD) based on CREA-AN guidelines [37] and a Low Glycemic Index Mediterranean Diet (LGIMD) [28]. No indications were given regarding the total calories to be consumed. Foods in LGIMD have all a low Glycemic Index (GI) and no more than 10% of total daily calories coming from saturated fats. The LGIMD was high in monounsaturated fatty acids (MUFA) from olive oil and contained also omega-3 polyunsaturated fatty acids (ω3PUFA), from both plant and marine sources. The prescribed diets were provided in brochure format, with graphic explanations. All participants were asked to record what they ate on a daily diary. The Mediterranean Adequacy Index (MAI) was chosen as a relevant measure to evaluate the adherence to both the intervention diet and the control diet [38]. Detailed information relating to the LGIMD intervention and control (recommended by the WHO and followed by INRAN [39]) is shown in Figure S1 and Figure S2.",
"To evaluate the initial physical condition, the right training program, and to compare the initial with the FU assessment of physical condition, three field tests were carried out: cardiorespiratory [40], strength [41], and flexibility fitness [42]. Cardio-respiratory fitness was assessed by means of the 2 km walking test, which is suitable for adults [40], whereas strength and flexibility fitness were evaluated by means of the push-up test (also called press-up test) and the Sit and Reach test, respectively. Subjects randomized to an intervention arm that included physical activity underwent the tests at baseline, after 45, and after 90 days, whereas subjects randomized to the diet arms underwent the field fitness tests at baseline and after 90 days. Physical activity interventions included two different types of exercise programs: Physical Activity 1 (PA1), based on the aerobic activity program, and Physical Activity 2 (PA2), based on the combination of aerobic activity and resistance training. The primary methods used for determining the intensity of prescribed exercise are maximum heart rate (Max HR), through heart-rate monitors [43], and metabolic equivalent (MET) [44,45]. To establish the age-predicted maximal heart rate, we used the formulas of Tanaka [46]. All subjects were randomized for one of the two physical activity programs based on the results of the tests performed. A progressive change of the monthly target has been planned based on the results obtained.\n 2.7.1. Aerobic Activity Program Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\nThree non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\n 2.7.2. Combination of Aerobic Exercise and Resistance Training Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.\nThree non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.",
"Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.",
"Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.",
"To quantify and detect hepatic steatosis, the controlled attenuation parameter (CAP) score was used. CAP measures the degree of ultrasound attenuation due to hepatic fat at the standardized frequency of 3.5 MHz through a vibration-controlled elastography (VCTE) implemented on FibroScan® (Echosens, Paris, France). Values <248, 248–267, 268–279 and ≥280 dB/m indicate absence, mild, moderate, or severe NAFLD, respectively [47].",
"The intention-to-treat principle was applied in all analysis. A description of the data was performed by Means (±SD), Median (IQR), and Frequencies (%) as appropriate. Data were also described separately by NAFLD severity. Biochemical markers were included only for descriptive purposes. Adherence to PA programs was evaluated as the proportion of effective frequency/time/intensity spent on gym or field sessions divided by the expected weekly frequency/time/intensity per session for the aerobic exercise group, plus expected load for the combined exercise group. The adherence was estimated by age group, gender, and month, and it was expressed as percentage. The third to tenth weeks of intervention were considered. The first two weeks were excluded to give subjects the opportunity to adapt to the programs training, and the last two weeks were excluded because the compliance could be decreased. The adherence to the LGIMD was measured with MAI [38]. Random week and weekend days were chosen from the second to 9th weeks of intervention. The first two weeks were excluded to allow participants to learn about the new diet and weeks successive to ninth were excluded because the compliance could decrease. To clearly describe the adherence and compliance to the LGIMD, MAI was estimated by gender, age, and week. Compliance was defined as previously described [38]. A median value of 7.5 was expected, as established by the reference Italian Mediterranean Diet [38].\nTo analyze differences among groups (Diet by Time), an Analysis of Variance for repeated measures was applied to the data. This statistical technique was chosen because it is relatively robust against violations of its assumptions, allows for multiple comparisons, and can be applied to a variety of experimental designs. Analysis of variance for repeated measurements was also performed by NAFLD severity. CAP was normally distributed, so no transformation was needed. Post estimation procedures were performed to compare estimated means with the reference category (control diet at baseline). To display graphically prediction from the fitted model, margins post estimation commands were performed. Margins statistics were calculated from predictions of a previously fitted Analysis of Variance model and displayed in graph form. Results are all expressed in the natural scale of CAP measurements as long as 95% Confidence Interval (95% CI).\nStata statistical (version 16.1) package was used to perform statistical analysis (StataCorp, 4905 Lakeway Drive, College Station, TX, USA); the Stata code is available upon request from the email: [email protected])."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"1. Introduction",
"2. Materials and Methods",
"2.1. Participants",
"2.2. Study Design",
"2.3. Sample Size",
"2.4. Data Collection",
"2.5. Randomization and Masking",
"2.6. Dietary Interventions",
"2.7. Physical Activity Interventions",
"2.7.1. Aerobic Activity Program",
"2.7.2. Combination of Aerobic Exercise and Resistance Training",
"2.8. Outcome Assessment",
"2.9. Statistical Analysis",
"3. Results",
"4. Discussion",
"5. Conclusions"
] |
[
"Non-Alcoholic Fatty Liver Disease (NAFLD) is a broad spectrum of liver diseases. It is characterized by fat accumulation in the liver in the absence of excessive alcohol consumption or any other specific causes of hepatic steatosis [1]. NAFLD is a major public health challenge as it is associated with the rising prevalence of obesity and Diabetes Mellitus type 2 worldwide and is a risk factor for Cardiovascular Diseases. Moreover, NAFLD is the most common cause of chronic liver disease and increases the 5-year direct and indirect health care costs by an estimated 26% [2]. The global prevalence of NAFLD is currently estimated to be 24% and is continually rising [3]. Our studies, conducted in the population of Castellana Grotte and Putignano (district of Bari, Apulia, Italy), evidenced an NAFLD prevalence of about 30%, with a higher prevalence in male and older patients [4,5]. Therefore, accurate diagnosis of NAFLD is important in patient management [6]. Until now, the gold standard for hepatic steatosis diagnosis has been liver biopsy, but it is an invasive procedure that has several disadvantages such as low acceptance for patients (especially for volunteers), complications, and even a small risk of death (0.01%) [7,8]. Today, many imaging methods are available for the non-invasive assessment of NAFLD [9], but they have limitations such as high costs or operator-dependency of Ultrasound Scan (US) [10,11]. FibroScan® is a non-invasive examination that appears to have good repeatability, which can be used as indices of continuous observation [12]. Data suggest that the controlled attenuation parameter (CAP), measured with FibroScan®, can be compared to liver biopsy for the detection and quantification of steatosis [13], and recent studies have evaluated its accuracy in NAFLD [14]. Furthermore, Lee et al. 2016 demonstrated that CAP and liver stiffness can be used as non-invasive and predictive markers of steatosis and fibrosis in patients with NAFLD [15].\nLifestyle modification is the standard treatment for NAFLD [16]. This approach encompasses dietary modifications, exercise, reduced alcohol intake, and weight loss, as they offer a range of health benefits. The Mediterranean Diet (MD) has long been associated with favorable health outcomes [17,18], and good adherence to MD has been reported to have a beneficial effect on the severity of NAFLD [19] as well as of Cardiovascular Diseases [20]. In particular, the Low Carbohydrate Diet, similar to the Low Glycemic Index Mediterranean Diet, is characterized by a reduction in the intake of several ultra-processed foods, refined grains starches, and foods rich in simple or added sugars, and it leads to a number of benefits such as weight loss and weight loss maintenance, reduction of Diastolic Blood Pressure (DBP), reduction of low-density lipoprotein cholesterol (LDL-C) and Triglycerides levels, increase of high-density lipoprotein cholesterol (HDL-C) levels, improvements in insulin resistance, and reduction of glycated hemoglobin (HbA1c) levels [21].\nIn addition, different forms of exercise have been shown to have similar effects on liver fat [22,23] if physical activity (PA) guidelines recommendations are followed [24]. Minimal physical activity (PA) decreases by 6% the risk for NAFLD compared to sedentary behavior [25]. The European Association for the Study of the Liver (EASL), the European Association for the Study of Diabetes (EASD) and the European Association for the Study of Obesity (EASO) guidelines recommend 150 to 200 min/week of moderate-intensity aerobic physical activity in three to five sessions [26]. Aerobic activity and resistance training seem to have similar improvement in NAFLD but with different mechanisms [27]. Nevertheless, there are still no universal recommendations on the optimal intensity, dose, and type of physical activity in NAFLD treatment. Two studies conducted in this area have confirmed the efficacy of Low Glycemic Index Mediterranean Diet (LGIMD) [28] and the efficiency of aerobic PA on NAFLD [29]. In this context, and as studies about the effect of diet and PA on NAFLD are scarce, we hypothesized that different PA programs and LGIMD or their combination could have different degrees of efficacy on NAFLD severity. We built a local version of the Mediterranean diet based on the work of Trichopoulos et al. and Elia, which contains no more than 10% of total daily calories deriving from saturated fats and a low glycemic index with an extensive use of olive oil [30,31]. Furthermore, the PA programs were built following European guidelines, which comprise aerobic PA as well as resistance training with precision indication about frequency, duration and intensity [26]. This work was aimed estimate the effect of healthy diet (LGIMD), two different PA programs, and their combination on the score of NAFLD as measured in a continuous scale by FibroScan®. Moreover, we aimed to explore biochemical, anthropometric, and body composition changes by different degrees of severity of NAFLD in geographical area where the Mediterranean diet is the most prevalent way of eating.",
" 2.1. Participants Details about the study have been published elsewhere [32]. Briefly, 166 subjects, referred to the Laboratory of Epidemiology and Biostatistics of the National Institute of Digestive Diseases, IRCCS “S. de Bellis”, Castellana Grotte, Italy, by their General Practitioners or identified during the NutriEp [5] or MICOL [33] enrollment or follow-up process, were assessed for eligibility. The trial was conducted from March 2015 to December 2016. Patients were consecutively enrolled, and the trial was registered at www.https://clinicaltrials.gov (registration number CT02347696). All subjects provided informed consent and the study was conducted in accordance with the Helsinki Declaration and approved by the Ethical Committee (Prot. n. 10/CE/De Bellis, 3 February 2015).\nDetails about the study have been published elsewhere [32]. Briefly, 166 subjects, referred to the Laboratory of Epidemiology and Biostatistics of the National Institute of Digestive Diseases, IRCCS “S. de Bellis”, Castellana Grotte, Italy, by their General Practitioners or identified during the NutriEp [5] or MICOL [33] enrollment or follow-up process, were assessed for eligibility. The trial was conducted from March 2015 to December 2016. Patients were consecutively enrolled, and the trial was registered at www.https://clinicaltrials.gov (registration number CT02347696). All subjects provided informed consent and the study was conducted in accordance with the Helsinki Declaration and approved by the Ethical Committee (Prot. n. 10/CE/De Bellis, 3 February 2015).\n 2.2. Study Design This study was a parallel group randomized controlled clinical trial. Inclusion criteria were Body Mass Index (BMI) ≥25, expressed as weight in kilograms divided by square height in meters (kg/m2); age >30 years old and <60 years old; moderate or severe NAFLD as assessed by the controlled attenuation parameter (CAP). Exclusion criteria included (1) overt cardiovascular disease and revascularization procedures; (2) stroke; (3) clinical peripheral artery disease; (4) T2DM (current treatment with insulin or oral hypoglycemic drugs, fasting glucose >126 mg/dL, or casual glucose >200 mg/dL; (5) more than 20 gr/daily of alcohol intake; (6) any severe medical condition that could prevent the subject’s participation in a nutritional intervention study; (7) subjects following a special diet or involved in a program for weight loss, or who had experienced recent weight loss, and (8) inability to follow a LGIMD for religious or other reasons [32].\nThis study was a parallel group randomized controlled clinical trial. Inclusion criteria were Body Mass Index (BMI) ≥25, expressed as weight in kilograms divided by square height in meters (kg/m2); age >30 years old and <60 years old; moderate or severe NAFLD as assessed by the controlled attenuation parameter (CAP). Exclusion criteria included (1) overt cardiovascular disease and revascularization procedures; (2) stroke; (3) clinical peripheral artery disease; (4) T2DM (current treatment with insulin or oral hypoglycemic drugs, fasting glucose >126 mg/dL, or casual glucose >200 mg/dL; (5) more than 20 gr/daily of alcohol intake; (6) any severe medical condition that could prevent the subject’s participation in a nutritional intervention study; (7) subjects following a special diet or involved in a program for weight loss, or who had experienced recent weight loss, and (8) inability to follow a LGIMD for religious or other reasons [32].\n 2.3. Sample Size Sample size was estimated considering the repeated measurements nature of the outcome. From a previous study [34], the mean ± Standard Deviation (SD) score of NAFLD was estimated to be 4.5 (1) and 4.0 (0.5) for the treatment and control groups, respectively. We hypothesized a decrease by 20 units of the CAP score after three months of intervention. Probabilistic type I error was fixed at 0.05 (one sided) and probabilistic error type II was fixed at 0.10; statistical power was fixed at 0.9. The correlation between baseline/follow-up measurements of the outcome was set at 0.4. A sample size of n1 = n2 = n3 = n4 = n5 = n6 = 20 was estimated.\nSample size was estimated considering the repeated measurements nature of the outcome. From a previous study [34], the mean ± Standard Deviation (SD) score of NAFLD was estimated to be 4.5 (1) and 4.0 (0.5) for the treatment and control groups, respectively. We hypothesized a decrease by 20 units of the CAP score after three months of intervention. Probabilistic type I error was fixed at 0.05 (one sided) and probabilistic error type II was fixed at 0.10; statistical power was fixed at 0.9. The correlation between baseline/follow-up measurements of the outcome was set at 0.4. A sample size of n1 = n2 = n3 = n4 = n5 = n6 = 20 was estimated.\n 2.4. Data Collection During enrollment, patients were interviewed by trained nutritionists to collect information on socio-demographic aspects, medical history, and lifestyle. Information about physical activity was collected using the validated questionnaire International Physical Activity Questionnaire Long Form (IPAQ-LF) [35]. The European Prospective Investigation into Cancer and Nutrition Food Frequency Questionnaire (EPIC FFQ) was used to probe alcohol intake and eating behavior [36]. Anthropometric measurements (weight, height, waist circumference) were taken in a standard manner. Weight and height measurements were taken using SECA instruments (Model 700; and Model 206; 220 cm; SECA, Hamburg, Germany). Blood samples, taken by venous puncture, were drawn in the morning after overnight fasting and were collected in tubes containing ethylenediamine tetra acetic acid (K-EDTA) anticoagulant. Biochemical measurements were performed using standard methods.\nDuring enrollment, patients were interviewed by trained nutritionists to collect information on socio-demographic aspects, medical history, and lifestyle. Information about physical activity was collected using the validated questionnaire International Physical Activity Questionnaire Long Form (IPAQ-LF) [35]. The European Prospective Investigation into Cancer and Nutrition Food Frequency Questionnaire (EPIC FFQ) was used to probe alcohol intake and eating behavior [36]. Anthropometric measurements (weight, height, waist circumference) were taken in a standard manner. Weight and height measurements were taken using SECA instruments (Model 700; and Model 206; 220 cm; SECA, Hamburg, Germany). Blood samples, taken by venous puncture, were drawn in the morning after overnight fasting and were collected in tubes containing ethylenediamine tetra acetic acid (K-EDTA) anticoagulant. Biochemical measurements were performed using standard methods.\n 2.5. Randomization and Masking According to a computerized random numbers sequence, subjects were randomized to six arms as follows: (1) Control Diet (CD) based on CREA-AN (Research Center for Food and Nutrition, Council for Agricultural Research and Economics, Rome, Italy) guidelines [37]; (2) Low Glycemic Index Mediterranean Diet (LGIMD); (3) Physical Activity aerobic program (PA1); (4) Physical Activity combined program (aerobic activity and resistance training) (PA2); (5) LGIMD plus PA1 and (6) LGIMD plus PA2. The trial flowchart is shown in Figure 1.\nAccording to a computerized random numbers sequence, subjects were randomized to six arms as follows: (1) Control Diet (CD) based on CREA-AN (Research Center for Food and Nutrition, Council for Agricultural Research and Economics, Rome, Italy) guidelines [37]; (2) Low Glycemic Index Mediterranean Diet (LGIMD); (3) Physical Activity aerobic program (PA1); (4) Physical Activity combined program (aerobic activity and resistance training) (PA2); (5) LGIMD plus PA1 and (6) LGIMD plus PA2. The trial flowchart is shown in Figure 1.\n 2.6. Dietary Interventions Two types of diets were prescribed: a Control Diet (CD) based on CREA-AN guidelines [37] and a Low Glycemic Index Mediterranean Diet (LGIMD) [28]. No indications were given regarding the total calories to be consumed. Foods in LGIMD have all a low Glycemic Index (GI) and no more than 10% of total daily calories coming from saturated fats. The LGIMD was high in monounsaturated fatty acids (MUFA) from olive oil and contained also omega-3 polyunsaturated fatty acids (ω3PUFA), from both plant and marine sources. The prescribed diets were provided in brochure format, with graphic explanations. All participants were asked to record what they ate on a daily diary. The Mediterranean Adequacy Index (MAI) was chosen as a relevant measure to evaluate the adherence to both the intervention diet and the control diet [38]. Detailed information relating to the LGIMD intervention and control (recommended by the WHO and followed by INRAN [39]) is shown in Figure S1 and Figure S2.\nTwo types of diets were prescribed: a Control Diet (CD) based on CREA-AN guidelines [37] and a Low Glycemic Index Mediterranean Diet (LGIMD) [28]. No indications were given regarding the total calories to be consumed. Foods in LGIMD have all a low Glycemic Index (GI) and no more than 10% of total daily calories coming from saturated fats. The LGIMD was high in monounsaturated fatty acids (MUFA) from olive oil and contained also omega-3 polyunsaturated fatty acids (ω3PUFA), from both plant and marine sources. The prescribed diets were provided in brochure format, with graphic explanations. All participants were asked to record what they ate on a daily diary. The Mediterranean Adequacy Index (MAI) was chosen as a relevant measure to evaluate the adherence to both the intervention diet and the control diet [38]. Detailed information relating to the LGIMD intervention and control (recommended by the WHO and followed by INRAN [39]) is shown in Figure S1 and Figure S2.\n 2.7. Physical Activity Interventions To evaluate the initial physical condition, the right training program, and to compare the initial with the FU assessment of physical condition, three field tests were carried out: cardiorespiratory [40], strength [41], and flexibility fitness [42]. Cardio-respiratory fitness was assessed by means of the 2 km walking test, which is suitable for adults [40], whereas strength and flexibility fitness were evaluated by means of the push-up test (also called press-up test) and the Sit and Reach test, respectively. Subjects randomized to an intervention arm that included physical activity underwent the tests at baseline, after 45, and after 90 days, whereas subjects randomized to the diet arms underwent the field fitness tests at baseline and after 90 days. Physical activity interventions included two different types of exercise programs: Physical Activity 1 (PA1), based on the aerobic activity program, and Physical Activity 2 (PA2), based on the combination of aerobic activity and resistance training. The primary methods used for determining the intensity of prescribed exercise are maximum heart rate (Max HR), through heart-rate monitors [43], and metabolic equivalent (MET) [44,45]. To establish the age-predicted maximal heart rate, we used the formulas of Tanaka [46]. All subjects were randomized for one of the two physical activity programs based on the results of the tests performed. A progressive change of the monthly target has been planned based on the results obtained.\n 2.7.1. Aerobic Activity Program Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\nThree non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\n 2.7.2. Combination of Aerobic Exercise and Resistance Training Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.\nThree non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.\nTo evaluate the initial physical condition, the right training program, and to compare the initial with the FU assessment of physical condition, three field tests were carried out: cardiorespiratory [40], strength [41], and flexibility fitness [42]. Cardio-respiratory fitness was assessed by means of the 2 km walking test, which is suitable for adults [40], whereas strength and flexibility fitness were evaluated by means of the push-up test (also called press-up test) and the Sit and Reach test, respectively. Subjects randomized to an intervention arm that included physical activity underwent the tests at baseline, after 45, and after 90 days, whereas subjects randomized to the diet arms underwent the field fitness tests at baseline and after 90 days. Physical activity interventions included two different types of exercise programs: Physical Activity 1 (PA1), based on the aerobic activity program, and Physical Activity 2 (PA2), based on the combination of aerobic activity and resistance training. The primary methods used for determining the intensity of prescribed exercise are maximum heart rate (Max HR), through heart-rate monitors [43], and metabolic equivalent (MET) [44,45]. To establish the age-predicted maximal heart rate, we used the formulas of Tanaka [46]. All subjects were randomized for one of the two physical activity programs based on the results of the tests performed. A progressive change of the monthly target has been planned based on the results obtained.\n 2.7.1. Aerobic Activity Program Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\nThree non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\n 2.7.2. Combination of Aerobic Exercise and Resistance Training Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.\nThree non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.\n 2.8. Outcome Assessment To quantify and detect hepatic steatosis, the controlled attenuation parameter (CAP) score was used. CAP measures the degree of ultrasound attenuation due to hepatic fat at the standardized frequency of 3.5 MHz through a vibration-controlled elastography (VCTE) implemented on FibroScan® (Echosens, Paris, France). Values <248, 248–267, 268–279 and ≥280 dB/m indicate absence, mild, moderate, or severe NAFLD, respectively [47].\nTo quantify and detect hepatic steatosis, the controlled attenuation parameter (CAP) score was used. CAP measures the degree of ultrasound attenuation due to hepatic fat at the standardized frequency of 3.5 MHz through a vibration-controlled elastography (VCTE) implemented on FibroScan® (Echosens, Paris, France). Values <248, 248–267, 268–279 and ≥280 dB/m indicate absence, mild, moderate, or severe NAFLD, respectively [47].\n 2.9. Statistical Analysis The intention-to-treat principle was applied in all analysis. A description of the data was performed by Means (±SD), Median (IQR), and Frequencies (%) as appropriate. Data were also described separately by NAFLD severity. Biochemical markers were included only for descriptive purposes. Adherence to PA programs was evaluated as the proportion of effective frequency/time/intensity spent on gym or field sessions divided by the expected weekly frequency/time/intensity per session for the aerobic exercise group, plus expected load for the combined exercise group. The adherence was estimated by age group, gender, and month, and it was expressed as percentage. The third to tenth weeks of intervention were considered. The first two weeks were excluded to give subjects the opportunity to adapt to the programs training, and the last two weeks were excluded because the compliance could be decreased. The adherence to the LGIMD was measured with MAI [38]. Random week and weekend days were chosen from the second to 9th weeks of intervention. The first two weeks were excluded to allow participants to learn about the new diet and weeks successive to ninth were excluded because the compliance could decrease. To clearly describe the adherence and compliance to the LGIMD, MAI was estimated by gender, age, and week. Compliance was defined as previously described [38]. A median value of 7.5 was expected, as established by the reference Italian Mediterranean Diet [38].\nTo analyze differences among groups (Diet by Time), an Analysis of Variance for repeated measures was applied to the data. This statistical technique was chosen because it is relatively robust against violations of its assumptions, allows for multiple comparisons, and can be applied to a variety of experimental designs. Analysis of variance for repeated measurements was also performed by NAFLD severity. CAP was normally distributed, so no transformation was needed. Post estimation procedures were performed to compare estimated means with the reference category (control diet at baseline). To display graphically prediction from the fitted model, margins post estimation commands were performed. Margins statistics were calculated from predictions of a previously fitted Analysis of Variance model and displayed in graph form. Results are all expressed in the natural scale of CAP measurements as long as 95% Confidence Interval (95% CI).\nStata statistical (version 16.1) package was used to perform statistical analysis (StataCorp, 4905 Lakeway Drive, College Station, TX, USA); the Stata code is available upon request from the email: [email protected]).\nThe intention-to-treat principle was applied in all analysis. A description of the data was performed by Means (±SD), Median (IQR), and Frequencies (%) as appropriate. Data were also described separately by NAFLD severity. Biochemical markers were included only for descriptive purposes. Adherence to PA programs was evaluated as the proportion of effective frequency/time/intensity spent on gym or field sessions divided by the expected weekly frequency/time/intensity per session for the aerobic exercise group, plus expected load for the combined exercise group. The adherence was estimated by age group, gender, and month, and it was expressed as percentage. The third to tenth weeks of intervention were considered. The first two weeks were excluded to give subjects the opportunity to adapt to the programs training, and the last two weeks were excluded because the compliance could be decreased. The adherence to the LGIMD was measured with MAI [38]. Random week and weekend days were chosen from the second to 9th weeks of intervention. The first two weeks were excluded to allow participants to learn about the new diet and weeks successive to ninth were excluded because the compliance could decrease. To clearly describe the adherence and compliance to the LGIMD, MAI was estimated by gender, age, and week. Compliance was defined as previously described [38]. A median value of 7.5 was expected, as established by the reference Italian Mediterranean Diet [38].\nTo analyze differences among groups (Diet by Time), an Analysis of Variance for repeated measures was applied to the data. This statistical technique was chosen because it is relatively robust against violations of its assumptions, allows for multiple comparisons, and can be applied to a variety of experimental designs. Analysis of variance for repeated measurements was also performed by NAFLD severity. CAP was normally distributed, so no transformation was needed. Post estimation procedures were performed to compare estimated means with the reference category (control diet at baseline). To display graphically prediction from the fitted model, margins post estimation commands were performed. Margins statistics were calculated from predictions of a previously fitted Analysis of Variance model and displayed in graph form. Results are all expressed in the natural scale of CAP measurements as long as 95% Confidence Interval (95% CI).\nStata statistical (version 16.1) package was used to perform statistical analysis (StataCorp, 4905 Lakeway Drive, College Station, TX, USA); the Stata code is available upon request from the email: [email protected]).",
"Details about the study have been published elsewhere [32]. Briefly, 166 subjects, referred to the Laboratory of Epidemiology and Biostatistics of the National Institute of Digestive Diseases, IRCCS “S. de Bellis”, Castellana Grotte, Italy, by their General Practitioners or identified during the NutriEp [5] or MICOL [33] enrollment or follow-up process, were assessed for eligibility. The trial was conducted from March 2015 to December 2016. Patients were consecutively enrolled, and the trial was registered at www.https://clinicaltrials.gov (registration number CT02347696). All subjects provided informed consent and the study was conducted in accordance with the Helsinki Declaration and approved by the Ethical Committee (Prot. n. 10/CE/De Bellis, 3 February 2015).",
"This study was a parallel group randomized controlled clinical trial. Inclusion criteria were Body Mass Index (BMI) ≥25, expressed as weight in kilograms divided by square height in meters (kg/m2); age >30 years old and <60 years old; moderate or severe NAFLD as assessed by the controlled attenuation parameter (CAP). Exclusion criteria included (1) overt cardiovascular disease and revascularization procedures; (2) stroke; (3) clinical peripheral artery disease; (4) T2DM (current treatment with insulin or oral hypoglycemic drugs, fasting glucose >126 mg/dL, or casual glucose >200 mg/dL; (5) more than 20 gr/daily of alcohol intake; (6) any severe medical condition that could prevent the subject’s participation in a nutritional intervention study; (7) subjects following a special diet or involved in a program for weight loss, or who had experienced recent weight loss, and (8) inability to follow a LGIMD for religious or other reasons [32].",
"Sample size was estimated considering the repeated measurements nature of the outcome. From a previous study [34], the mean ± Standard Deviation (SD) score of NAFLD was estimated to be 4.5 (1) and 4.0 (0.5) for the treatment and control groups, respectively. We hypothesized a decrease by 20 units of the CAP score after three months of intervention. Probabilistic type I error was fixed at 0.05 (one sided) and probabilistic error type II was fixed at 0.10; statistical power was fixed at 0.9. The correlation between baseline/follow-up measurements of the outcome was set at 0.4. A sample size of n1 = n2 = n3 = n4 = n5 = n6 = 20 was estimated.",
"During enrollment, patients were interviewed by trained nutritionists to collect information on socio-demographic aspects, medical history, and lifestyle. Information about physical activity was collected using the validated questionnaire International Physical Activity Questionnaire Long Form (IPAQ-LF) [35]. The European Prospective Investigation into Cancer and Nutrition Food Frequency Questionnaire (EPIC FFQ) was used to probe alcohol intake and eating behavior [36]. Anthropometric measurements (weight, height, waist circumference) were taken in a standard manner. Weight and height measurements were taken using SECA instruments (Model 700; and Model 206; 220 cm; SECA, Hamburg, Germany). Blood samples, taken by venous puncture, were drawn in the morning after overnight fasting and were collected in tubes containing ethylenediamine tetra acetic acid (K-EDTA) anticoagulant. Biochemical measurements were performed using standard methods.",
"According to a computerized random numbers sequence, subjects were randomized to six arms as follows: (1) Control Diet (CD) based on CREA-AN (Research Center for Food and Nutrition, Council for Agricultural Research and Economics, Rome, Italy) guidelines [37]; (2) Low Glycemic Index Mediterranean Diet (LGIMD); (3) Physical Activity aerobic program (PA1); (4) Physical Activity combined program (aerobic activity and resistance training) (PA2); (5) LGIMD plus PA1 and (6) LGIMD plus PA2. The trial flowchart is shown in Figure 1.",
"Two types of diets were prescribed: a Control Diet (CD) based on CREA-AN guidelines [37] and a Low Glycemic Index Mediterranean Diet (LGIMD) [28]. No indications were given regarding the total calories to be consumed. Foods in LGIMD have all a low Glycemic Index (GI) and no more than 10% of total daily calories coming from saturated fats. The LGIMD was high in monounsaturated fatty acids (MUFA) from olive oil and contained also omega-3 polyunsaturated fatty acids (ω3PUFA), from both plant and marine sources. The prescribed diets were provided in brochure format, with graphic explanations. All participants were asked to record what they ate on a daily diary. The Mediterranean Adequacy Index (MAI) was chosen as a relevant measure to evaluate the adherence to both the intervention diet and the control diet [38]. Detailed information relating to the LGIMD intervention and control (recommended by the WHO and followed by INRAN [39]) is shown in Figure S1 and Figure S2.",
"To evaluate the initial physical condition, the right training program, and to compare the initial with the FU assessment of physical condition, three field tests were carried out: cardiorespiratory [40], strength [41], and flexibility fitness [42]. Cardio-respiratory fitness was assessed by means of the 2 km walking test, which is suitable for adults [40], whereas strength and flexibility fitness were evaluated by means of the push-up test (also called press-up test) and the Sit and Reach test, respectively. Subjects randomized to an intervention arm that included physical activity underwent the tests at baseline, after 45, and after 90 days, whereas subjects randomized to the diet arms underwent the field fitness tests at baseline and after 90 days. Physical activity interventions included two different types of exercise programs: Physical Activity 1 (PA1), based on the aerobic activity program, and Physical Activity 2 (PA2), based on the combination of aerobic activity and resistance training. The primary methods used for determining the intensity of prescribed exercise are maximum heart rate (Max HR), through heart-rate monitors [43], and metabolic equivalent (MET) [44,45]. To establish the age-predicted maximal heart rate, we used the formulas of Tanaka [46]. All subjects were randomized for one of the two physical activity programs based on the results of the tests performed. A progressive change of the monthly target has been planned based on the results obtained.\n 2.7.1. Aerobic Activity Program Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\nThree non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.\n 2.7.2. Combination of Aerobic Exercise and Resistance Training Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.\nThree non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.",
"Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.",
"Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.",
"To quantify and detect hepatic steatosis, the controlled attenuation parameter (CAP) score was used. CAP measures the degree of ultrasound attenuation due to hepatic fat at the standardized frequency of 3.5 MHz through a vibration-controlled elastography (VCTE) implemented on FibroScan® (Echosens, Paris, France). Values <248, 248–267, 268–279 and ≥280 dB/m indicate absence, mild, moderate, or severe NAFLD, respectively [47].",
"The intention-to-treat principle was applied in all analysis. A description of the data was performed by Means (±SD), Median (IQR), and Frequencies (%) as appropriate. Data were also described separately by NAFLD severity. Biochemical markers were included only for descriptive purposes. Adherence to PA programs was evaluated as the proportion of effective frequency/time/intensity spent on gym or field sessions divided by the expected weekly frequency/time/intensity per session for the aerobic exercise group, plus expected load for the combined exercise group. The adherence was estimated by age group, gender, and month, and it was expressed as percentage. The third to tenth weeks of intervention were considered. The first two weeks were excluded to give subjects the opportunity to adapt to the programs training, and the last two weeks were excluded because the compliance could be decreased. The adherence to the LGIMD was measured with MAI [38]. Random week and weekend days were chosen from the second to 9th weeks of intervention. The first two weeks were excluded to allow participants to learn about the new diet and weeks successive to ninth were excluded because the compliance could decrease. To clearly describe the adherence and compliance to the LGIMD, MAI was estimated by gender, age, and week. Compliance was defined as previously described [38]. A median value of 7.5 was expected, as established by the reference Italian Mediterranean Diet [38].\nTo analyze differences among groups (Diet by Time), an Analysis of Variance for repeated measures was applied to the data. This statistical technique was chosen because it is relatively robust against violations of its assumptions, allows for multiple comparisons, and can be applied to a variety of experimental designs. Analysis of variance for repeated measurements was also performed by NAFLD severity. CAP was normally distributed, so no transformation was needed. Post estimation procedures were performed to compare estimated means with the reference category (control diet at baseline). To display graphically prediction from the fitted model, margins post estimation commands were performed. Margins statistics were calculated from predictions of a previously fitted Analysis of Variance model and displayed in graph form. Results are all expressed in the natural scale of CAP measurements as long as 95% Confidence Interval (95% CI).\nStata statistical (version 16.1) package was used to perform statistical analysis (StataCorp, 4905 Lakeway Drive, College Station, TX, USA); the Stata code is available upon request from the email: [email protected]).",
"One hundred and sixty-six subjects were assessed for eligibility: 17 subjects were excluded because they did not satisfy the inclusion criteria and 5 were excluded for various other reasons. The study design is shown in Figure 1. The following subjects were lost to follow-up: 5, 3, 7, 6, 9, and 4 from Arms 1, 2, 3, 4, 5 and 6, respectively.\nFinally, 144 subjects were included and randomized to the intervention arms. Characteristics of the participants are shown in Table 1 and Table 2 and in Appendix A, Table A1.\nAs expected, the age–sex distribution of the population under study reflected the age–sex distribution of the NAFLD condition in the population [4]. About 62% of the sample were men, and the mean age was 49.9 years (10.08), but few subjects were under 40 years old (12.5%). As expected, all subjects were overweight or obese with increased waist circumference. At baseline, almost every participant had low levels of HDL-C and a high HOMA-IR. Most subjects were married and had attended secondary school or higher (Table 2). All parameters considered were equally distributed among groups at baseline with the exception of NAFLD severity. More subjects had severe than moderate NAFLD, and the CAP value was over 323 dB/m in all groups with a successive decreasing in intermediate and final observation times.\nThe behavior of anthropometric, body composition, and biochemical profiles by time and intervention arm are shown in Appendix A, Table A1. Overall, there were improvements in all markers, especially at 45 days. These improvements were particularly evident for intervention arms PA1 + LGIMD and PA2 + LGIMD.\nField Fitness Test results by gender and time are shown in Table 3.\nOverall, there was a significant improvement of the fitness index in both men and women. The compliance to physical activity programs is shown in Table 4.\nOverall, adherence was above 80% to both PA intervention program in both genders and in all weeks.\nCompliance to LGIMD, as measured by MAI, by age class, gender, and week is shown in Table 5.\nCompliance with the control diet showed a median MAI of 1.29 (0.77, 2.63). MAI was lower amongst males (1.07) than females (2.14), whereas compliance with LGMID evidenced a median MAI of 11.2 (6.4, 22.8). Adherence was lower among women (10.0) than men (11.3).\nTable 6 shows the changes in the CAP value by intervention arms and time (at 45 days and at 90 days).\nApart from the CD, there were statistically significant principal effects of all the other intervention arms, Arms 3 and 5 showing the strongest effects (PA1: −166.35, 95%CI −242.01; −90.68 and LGIMD plus PA1: −94.10, 95% CI −170.87; −19.12). After the analysis, no statistical significance principal effect of time in reducing CAP was observed. All comparisons between different time points and Intervention Arms, except for Intervention Arm 1, showed a statistically significant decreased value for CAP. The strongest effect was observed for LGIMD plus PA1 at 45 days (−52.96, 95% CI −79.03; −26.88) and 90 days (−61.56, 95% CI −89.61; −33.50).\nResults from separate analysis by NAFLD severity are shown in Appendix A, Table A2 and Table A3. The relative decrease of CAP values was more relevant among severe NAFLD subjects. HOMA-IR decreased in all intervention arms with the exception of LGIMD in moderate NAFLD subjects. In both moderate and severe NAFLD subjects, there was a modest effect of only-diet intervention arms on CAP. Only-PA intervention arms effect on CAP values evidenced a decrease among severe NAFLD subjects but not among moderate ones. The most intense effect in decreasing CAP values was observed among severe NAFLD subjects who underwent PA1+LGIMD. An improvement in all anthropometric, body composition, and biochemical parameters was observed (most evident at day 45). Noteworthy, the improvement in HDL-C was accompanied by an improvement in FFM only in moderate NAFLD subjects. Table 7 shows the changes in the CAP value by NAFLD severity, intervention arms, and time (at 45 days and at 90 days).\nThere was an effect that was statistically significant only in subjects with moderate NAFLD allocated to Arm 6 after 90 days, whereas among severe NAFLD subjects, there were statistically significant effects in all times and arms with the exception of CD and Arm 6 at 90 days. The strongest estimated effect was observed in the PA1 + LGIMD intervention arm.\nThe results of predictive margins from analysis of variance are graphically displayed in Figure 2, which highlights the strong effect of LGIMD plus PA1. Hypothesis testing about the equality of measurements at baseline showed no statistically significant differences in CAP among intervention arms (p > 0.05).",
"In this RCT, the combination of the aerobic activity program and an LGIMD was associated with the strongest reduction of the CAP, as measured by FibroScan®, compared to the other intervention arms. Overall, this effect was greater until 45 days of intervention. However, the effect of Aerobic PA plus LGIMD was stronger during the complete intervention time in severe than moderate NAFLD subjects. The effect on CAP was accompanied by an overall improvement in all anthropometric, body composition and biochemical parameters.\nCurrently, a lifestyle change focused on diet and physical activity seems to be the best therapy in the treatment of NAFLD, combined with pharmacologic therapy when necessary [48]. Regular physical activity is now considered a therapy to prevent the onset and progression of several chronic diseases, including NAFLD [49]. Aerobic, resistance and combination exercise programs may enhance systemic indicators of hepatic functions and intrahepatic fats in NAFLD patients in both mild and advanced cases [50].\nThe adoption of either aerobic or strength PA programs showed to influence hepatic metabolism with a resulting decrease in hepatic fat accumulation, an increased insulin sensitivity, and fat oxidation. Moreover, these effects have been observed even without weight loss [51].\nAlthough the risk of developing NAFLD or more advanced grades of liver diseases seems to be exercise dose-related [52], no differences among different intensity aerobic exercise regimens were recently found: all of them reduced liver fat [53].\nAt the community level, a reduced infiltration of liver fat has been documented after a combined exercise program [54]. This effect was proportional to weight loss rate and event without it.\nLGIMD was locally created by integrating the works of Trichopolou et al. [30] and Elia [31]. Moreover, we have adapted it to our population, and it is similar to the diet suggested by other authors for the metabolic syndrome [55]. LGIMD, which contains no more than 10% of total daily calories deriving from saturated fats and a low glycemic index, was associated with a more intense reduction of the NAFLD score [28]. The LGIMD is rich in monounsaturated fatty acids (MUFA) and also contains omega-3 polyunsaturated fatty acids (ω3PUFA).\nIn this RCT, in working Arm 2, where the subjects only followed a LGIMD, we observed a statistically significant improvement of the score of NAFLD, reducing the CAP value, especially at 45 days. However, after 45 days, a slight increase in the CAP value was observed. Several studies have shown that lifestyle changes are difficult to implement and maintain for a long time, although individuals know the importance of having a healthier lifestyle [56,57]. In our study, adherence was almost homogenously distributed during the intervention time but, as it has been shown, participants could have given answers that are socially desirable [58].\nWhen comparing the effectiveness of the two different physical activity programs, it is clear that both the aerobic and the combined exercise program significantly reduced the CAP value. Our data are confirmed by previous studies that have demonstrated the effectiveness of aerobic activity on liver function [59,60]. Moreover, recently, resistance training has also been found to be effective in NAFLD treatment, regardless of weight loss [61,62,63].\nBoth aerobic exercise and combined exercise significantly reduced the CAP value from 45 days onward until the end of the project. The literature exhaustively showed the efficacy of both combined and resistance training in NAFLD [64]. Furthermore, aerobic activity and resistance training have been recently shown, although with different mechanisms, to improve NAFLD score [23,65]. At least three mechanisms have been proposed for aerobic PA: activation of lipolysis in different tissues, upregulation of the uncoupling protein-1 and peroxisome proliferator-activated receptor γ pathways, and alteration of adipokine levels [23]; on the other hand, resistance training stimulates the hypertrophy of type II muscle fibers, alters myokine levels, and activates glucose transporter 4, AMP-activated protein kinase, and caveolins [23]. Moreover, resistance training appeared to improve NAFLD with less energy consumption. A recent study conducted in mice with diethyl nitrosamine-induced hepatocellular carcinoma has shown that in early stages, voluntary exercise decreases the proliferation rate of dysplastic hepatocytes [66].\nTherefore, PA could be hypothesized as a preventive measure for liver cancer development, as NAFLD is a stage of a broad spectrum of conditions that lead to hepatocellular carcinoma. In this sense, after an energy restriction dietary intervention and/or regular PA, several facts have been observed such as histological improvements, resolution of liver fat, as well as necroinflammation and fibrosis. These changes have been observed even after 7% to 10% of weight loss [67,68].\nHealthy dietary habits and healthy lifestyle, similar to any regular physical activity, can improve NAFLD more than weight loss alone [69].\nWeight loss achieved by dietary treatment must be done taking into account quantitative and qualitative characteristics, as energy restriction per se is not sufficient to improve NAFLD [22]. Moreover, a dietary composition modulating both macro and micronutrients is crucial [70]. Therefore, balanced nutrition such as LGIMD, moderate weight loss, and a physical activity program can be considered as the best therapeutic approach in NAFLD.\nThis seems to be confirmed in our study where the effect of intervention was more evident in the severe NAFLD group, highlighting that the combination of diet and physical activity can be an effective therapeutic tool in the prevention not only of the complications of NAFLD but also of the associated cardiovascular risk. The observed effect in the severe NAFLD group could be due to the particular distribution of NAFLD severity, as 75% of participants (data not shown) had severe NAFLD at baseline. Moreover, the use of FibroScan® as the NAFLD assessment method could have enhanced the frequency of severe NAFLD diagnosis. It is known that FibroScan® has a greater sensibility [71] than Liver Ultrasound for fat infiltration >25–30% of hepatocytes [72].\nThe diminution of intrahepatic fat content is not the only marker of interest in this study. The metabolic pattern resulting from the interventions was explored, and the results are almost always in line with the literature. The negative association between adherence to Mediterranean diet and NAFLD severity has been confirmed in this study as well as HOMA-IR decreasing, which has been more strong in severe NAFLD subjects [19]. As noted above, we built a local version of the Mediterranean diet following the European Association for the study of the Liver, the European Association for the study of Diabetes, and the European Association for the study of Obesity guidelines [26]. After the intervention period, our results are similar to those of Ryan et al., who found an improvement in fatty liver content and HOMA-IR [73]. Moreover, a recent systematic review and meta-analysis has documented beneficial changes of the Mediterranean diet on metabolic health, including anthropometric and biochemical markers [74].\nAs regards PA, a network meta-analysis has shown that aerobic exercise plus diet and aerobic exercise alone exert an improvement on BMI and HOMA-IR but progressive resistance training has an effect only on HOMA-IR but not on BMI [75]. While diet seems to be more effective at improving intrahepatic markers of liver damage, PA plus diet improves insulin resistance and BMI [76]. However, in our study, there was a modest effect of only the LGIMD intervention arm. In the geographical area where the study was conducted, the Mediterranean diet is the most prevalent dietary pattern with a high adherence at population level, which may be the cause of the modest observed effect [76].\nThis study has several strengths and limitations. Strengths include the study design, the measured compliance to both dietary and PA interventions as well as their controlled application, and an adequate sample size. Moreover, a well-validated assessment of the outcome such as FibroScan® has been implemented. The applied intention-to-treat analytical strategy prevents the design from introducing bias relating to non-adherence to the protocol to the prognosis; therefore, this RCT provides an unbiased assessment of treatment efficacy [77]. It is worthy to note that in this area, the most prevalent dietary pattern is the local version of Mediterranean Diet; then, a dilution bias could be present. Another limitation may be the duration of the intervention, which prevents a wide application in the clinical field, However, the objective of the study was to estimate the effect of the intervention in order to establish its efficacy.",
"It is clear that PA single (aerobic or resistance exercise) or combined (aerobic plus resistance exercise) should be integrated into NAFLD management [78]. Moreover, aerobic exercise programs are simple to implement and the cost-efficiency is higher. These should be included in programs for the primary prevention of metabolic and cardiovascular diseases.\nIn conclusion, a multidisciplinary team including dietitians, a psychologist, and physical activity supervisors is needed to ensure the best management of NAFLD patients [78]."
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[
"physical activity",
"non-alcoholic fatty liver disease",
"diet",
"controlled attenuation parameter (CAP)",
"aerobic exercise",
"combined exercise"
] |
1. Introduction:
Non-Alcoholic Fatty Liver Disease (NAFLD) is a broad spectrum of liver diseases. It is characterized by fat accumulation in the liver in the absence of excessive alcohol consumption or any other specific causes of hepatic steatosis [1]. NAFLD is a major public health challenge as it is associated with the rising prevalence of obesity and Diabetes Mellitus type 2 worldwide and is a risk factor for Cardiovascular Diseases. Moreover, NAFLD is the most common cause of chronic liver disease and increases the 5-year direct and indirect health care costs by an estimated 26% [2]. The global prevalence of NAFLD is currently estimated to be 24% and is continually rising [3]. Our studies, conducted in the population of Castellana Grotte and Putignano (district of Bari, Apulia, Italy), evidenced an NAFLD prevalence of about 30%, with a higher prevalence in male and older patients [4,5]. Therefore, accurate diagnosis of NAFLD is important in patient management [6]. Until now, the gold standard for hepatic steatosis diagnosis has been liver biopsy, but it is an invasive procedure that has several disadvantages such as low acceptance for patients (especially for volunteers), complications, and even a small risk of death (0.01%) [7,8]. Today, many imaging methods are available for the non-invasive assessment of NAFLD [9], but they have limitations such as high costs or operator-dependency of Ultrasound Scan (US) [10,11]. FibroScan® is a non-invasive examination that appears to have good repeatability, which can be used as indices of continuous observation [12]. Data suggest that the controlled attenuation parameter (CAP), measured with FibroScan®, can be compared to liver biopsy for the detection and quantification of steatosis [13], and recent studies have evaluated its accuracy in NAFLD [14]. Furthermore, Lee et al. 2016 demonstrated that CAP and liver stiffness can be used as non-invasive and predictive markers of steatosis and fibrosis in patients with NAFLD [15].
Lifestyle modification is the standard treatment for NAFLD [16]. This approach encompasses dietary modifications, exercise, reduced alcohol intake, and weight loss, as they offer a range of health benefits. The Mediterranean Diet (MD) has long been associated with favorable health outcomes [17,18], and good adherence to MD has been reported to have a beneficial effect on the severity of NAFLD [19] as well as of Cardiovascular Diseases [20]. In particular, the Low Carbohydrate Diet, similar to the Low Glycemic Index Mediterranean Diet, is characterized by a reduction in the intake of several ultra-processed foods, refined grains starches, and foods rich in simple or added sugars, and it leads to a number of benefits such as weight loss and weight loss maintenance, reduction of Diastolic Blood Pressure (DBP), reduction of low-density lipoprotein cholesterol (LDL-C) and Triglycerides levels, increase of high-density lipoprotein cholesterol (HDL-C) levels, improvements in insulin resistance, and reduction of glycated hemoglobin (HbA1c) levels [21].
In addition, different forms of exercise have been shown to have similar effects on liver fat [22,23] if physical activity (PA) guidelines recommendations are followed [24]. Minimal physical activity (PA) decreases by 6% the risk for NAFLD compared to sedentary behavior [25]. The European Association for the Study of the Liver (EASL), the European Association for the Study of Diabetes (EASD) and the European Association for the Study of Obesity (EASO) guidelines recommend 150 to 200 min/week of moderate-intensity aerobic physical activity in three to five sessions [26]. Aerobic activity and resistance training seem to have similar improvement in NAFLD but with different mechanisms [27]. Nevertheless, there are still no universal recommendations on the optimal intensity, dose, and type of physical activity in NAFLD treatment. Two studies conducted in this area have confirmed the efficacy of Low Glycemic Index Mediterranean Diet (LGIMD) [28] and the efficiency of aerobic PA on NAFLD [29]. In this context, and as studies about the effect of diet and PA on NAFLD are scarce, we hypothesized that different PA programs and LGIMD or their combination could have different degrees of efficacy on NAFLD severity. We built a local version of the Mediterranean diet based on the work of Trichopoulos et al. and Elia, which contains no more than 10% of total daily calories deriving from saturated fats and a low glycemic index with an extensive use of olive oil [30,31]. Furthermore, the PA programs were built following European guidelines, which comprise aerobic PA as well as resistance training with precision indication about frequency, duration and intensity [26]. This work was aimed estimate the effect of healthy diet (LGIMD), two different PA programs, and their combination on the score of NAFLD as measured in a continuous scale by FibroScan®. Moreover, we aimed to explore biochemical, anthropometric, and body composition changes by different degrees of severity of NAFLD in geographical area where the Mediterranean diet is the most prevalent way of eating.
2. Materials and Methods:
2.1. Participants Details about the study have been published elsewhere [32]. Briefly, 166 subjects, referred to the Laboratory of Epidemiology and Biostatistics of the National Institute of Digestive Diseases, IRCCS “S. de Bellis”, Castellana Grotte, Italy, by their General Practitioners or identified during the NutriEp [5] or MICOL [33] enrollment or follow-up process, were assessed for eligibility. The trial was conducted from March 2015 to December 2016. Patients were consecutively enrolled, and the trial was registered at www.https://clinicaltrials.gov (registration number CT02347696). All subjects provided informed consent and the study was conducted in accordance with the Helsinki Declaration and approved by the Ethical Committee (Prot. n. 10/CE/De Bellis, 3 February 2015).
Details about the study have been published elsewhere [32]. Briefly, 166 subjects, referred to the Laboratory of Epidemiology and Biostatistics of the National Institute of Digestive Diseases, IRCCS “S. de Bellis”, Castellana Grotte, Italy, by their General Practitioners or identified during the NutriEp [5] or MICOL [33] enrollment or follow-up process, were assessed for eligibility. The trial was conducted from March 2015 to December 2016. Patients were consecutively enrolled, and the trial was registered at www.https://clinicaltrials.gov (registration number CT02347696). All subjects provided informed consent and the study was conducted in accordance with the Helsinki Declaration and approved by the Ethical Committee (Prot. n. 10/CE/De Bellis, 3 February 2015).
2.2. Study Design This study was a parallel group randomized controlled clinical trial. Inclusion criteria were Body Mass Index (BMI) ≥25, expressed as weight in kilograms divided by square height in meters (kg/m2); age >30 years old and <60 years old; moderate or severe NAFLD as assessed by the controlled attenuation parameter (CAP). Exclusion criteria included (1) overt cardiovascular disease and revascularization procedures; (2) stroke; (3) clinical peripheral artery disease; (4) T2DM (current treatment with insulin or oral hypoglycemic drugs, fasting glucose >126 mg/dL, or casual glucose >200 mg/dL; (5) more than 20 gr/daily of alcohol intake; (6) any severe medical condition that could prevent the subject’s participation in a nutritional intervention study; (7) subjects following a special diet or involved in a program for weight loss, or who had experienced recent weight loss, and (8) inability to follow a LGIMD for religious or other reasons [32].
This study was a parallel group randomized controlled clinical trial. Inclusion criteria were Body Mass Index (BMI) ≥25, expressed as weight in kilograms divided by square height in meters (kg/m2); age >30 years old and <60 years old; moderate or severe NAFLD as assessed by the controlled attenuation parameter (CAP). Exclusion criteria included (1) overt cardiovascular disease and revascularization procedures; (2) stroke; (3) clinical peripheral artery disease; (4) T2DM (current treatment with insulin or oral hypoglycemic drugs, fasting glucose >126 mg/dL, or casual glucose >200 mg/dL; (5) more than 20 gr/daily of alcohol intake; (6) any severe medical condition that could prevent the subject’s participation in a nutritional intervention study; (7) subjects following a special diet or involved in a program for weight loss, or who had experienced recent weight loss, and (8) inability to follow a LGIMD for religious or other reasons [32].
2.3. Sample Size Sample size was estimated considering the repeated measurements nature of the outcome. From a previous study [34], the mean ± Standard Deviation (SD) score of NAFLD was estimated to be 4.5 (1) and 4.0 (0.5) for the treatment and control groups, respectively. We hypothesized a decrease by 20 units of the CAP score after three months of intervention. Probabilistic type I error was fixed at 0.05 (one sided) and probabilistic error type II was fixed at 0.10; statistical power was fixed at 0.9. The correlation between baseline/follow-up measurements of the outcome was set at 0.4. A sample size of n1 = n2 = n3 = n4 = n5 = n6 = 20 was estimated.
Sample size was estimated considering the repeated measurements nature of the outcome. From a previous study [34], the mean ± Standard Deviation (SD) score of NAFLD was estimated to be 4.5 (1) and 4.0 (0.5) for the treatment and control groups, respectively. We hypothesized a decrease by 20 units of the CAP score after three months of intervention. Probabilistic type I error was fixed at 0.05 (one sided) and probabilistic error type II was fixed at 0.10; statistical power was fixed at 0.9. The correlation between baseline/follow-up measurements of the outcome was set at 0.4. A sample size of n1 = n2 = n3 = n4 = n5 = n6 = 20 was estimated.
2.4. Data Collection During enrollment, patients were interviewed by trained nutritionists to collect information on socio-demographic aspects, medical history, and lifestyle. Information about physical activity was collected using the validated questionnaire International Physical Activity Questionnaire Long Form (IPAQ-LF) [35]. The European Prospective Investigation into Cancer and Nutrition Food Frequency Questionnaire (EPIC FFQ) was used to probe alcohol intake and eating behavior [36]. Anthropometric measurements (weight, height, waist circumference) were taken in a standard manner. Weight and height measurements were taken using SECA instruments (Model 700; and Model 206; 220 cm; SECA, Hamburg, Germany). Blood samples, taken by venous puncture, were drawn in the morning after overnight fasting and were collected in tubes containing ethylenediamine tetra acetic acid (K-EDTA) anticoagulant. Biochemical measurements were performed using standard methods.
During enrollment, patients were interviewed by trained nutritionists to collect information on socio-demographic aspects, medical history, and lifestyle. Information about physical activity was collected using the validated questionnaire International Physical Activity Questionnaire Long Form (IPAQ-LF) [35]. The European Prospective Investigation into Cancer and Nutrition Food Frequency Questionnaire (EPIC FFQ) was used to probe alcohol intake and eating behavior [36]. Anthropometric measurements (weight, height, waist circumference) were taken in a standard manner. Weight and height measurements were taken using SECA instruments (Model 700; and Model 206; 220 cm; SECA, Hamburg, Germany). Blood samples, taken by venous puncture, were drawn in the morning after overnight fasting and were collected in tubes containing ethylenediamine tetra acetic acid (K-EDTA) anticoagulant. Biochemical measurements were performed using standard methods.
2.5. Randomization and Masking According to a computerized random numbers sequence, subjects were randomized to six arms as follows: (1) Control Diet (CD) based on CREA-AN (Research Center for Food and Nutrition, Council for Agricultural Research and Economics, Rome, Italy) guidelines [37]; (2) Low Glycemic Index Mediterranean Diet (LGIMD); (3) Physical Activity aerobic program (PA1); (4) Physical Activity combined program (aerobic activity and resistance training) (PA2); (5) LGIMD plus PA1 and (6) LGIMD plus PA2. The trial flowchart is shown in Figure 1.
According to a computerized random numbers sequence, subjects were randomized to six arms as follows: (1) Control Diet (CD) based on CREA-AN (Research Center for Food and Nutrition, Council for Agricultural Research and Economics, Rome, Italy) guidelines [37]; (2) Low Glycemic Index Mediterranean Diet (LGIMD); (3) Physical Activity aerobic program (PA1); (4) Physical Activity combined program (aerobic activity and resistance training) (PA2); (5) LGIMD plus PA1 and (6) LGIMD plus PA2. The trial flowchart is shown in Figure 1.
2.6. Dietary Interventions Two types of diets were prescribed: a Control Diet (CD) based on CREA-AN guidelines [37] and a Low Glycemic Index Mediterranean Diet (LGIMD) [28]. No indications were given regarding the total calories to be consumed. Foods in LGIMD have all a low Glycemic Index (GI) and no more than 10% of total daily calories coming from saturated fats. The LGIMD was high in monounsaturated fatty acids (MUFA) from olive oil and contained also omega-3 polyunsaturated fatty acids (ω3PUFA), from both plant and marine sources. The prescribed diets were provided in brochure format, with graphic explanations. All participants were asked to record what they ate on a daily diary. The Mediterranean Adequacy Index (MAI) was chosen as a relevant measure to evaluate the adherence to both the intervention diet and the control diet [38]. Detailed information relating to the LGIMD intervention and control (recommended by the WHO and followed by INRAN [39]) is shown in Figure S1 and Figure S2.
Two types of diets were prescribed: a Control Diet (CD) based on CREA-AN guidelines [37] and a Low Glycemic Index Mediterranean Diet (LGIMD) [28]. No indications were given regarding the total calories to be consumed. Foods in LGIMD have all a low Glycemic Index (GI) and no more than 10% of total daily calories coming from saturated fats. The LGIMD was high in monounsaturated fatty acids (MUFA) from olive oil and contained also omega-3 polyunsaturated fatty acids (ω3PUFA), from both plant and marine sources. The prescribed diets were provided in brochure format, with graphic explanations. All participants were asked to record what they ate on a daily diary. The Mediterranean Adequacy Index (MAI) was chosen as a relevant measure to evaluate the adherence to both the intervention diet and the control diet [38]. Detailed information relating to the LGIMD intervention and control (recommended by the WHO and followed by INRAN [39]) is shown in Figure S1 and Figure S2.
2.7. Physical Activity Interventions To evaluate the initial physical condition, the right training program, and to compare the initial with the FU assessment of physical condition, three field tests were carried out: cardiorespiratory [40], strength [41], and flexibility fitness [42]. Cardio-respiratory fitness was assessed by means of the 2 km walking test, which is suitable for adults [40], whereas strength and flexibility fitness were evaluated by means of the push-up test (also called press-up test) and the Sit and Reach test, respectively. Subjects randomized to an intervention arm that included physical activity underwent the tests at baseline, after 45, and after 90 days, whereas subjects randomized to the diet arms underwent the field fitness tests at baseline and after 90 days. Physical activity interventions included two different types of exercise programs: Physical Activity 1 (PA1), based on the aerobic activity program, and Physical Activity 2 (PA2), based on the combination of aerobic activity and resistance training. The primary methods used for determining the intensity of prescribed exercise are maximum heart rate (Max HR), through heart-rate monitors [43], and metabolic equivalent (MET) [44,45]. To establish the age-predicted maximal heart rate, we used the formulas of Tanaka [46]. All subjects were randomized for one of the two physical activity programs based on the results of the tests performed. A progressive change of the monthly target has been planned based on the results obtained.
2.7.1. Aerobic Activity Program Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.
Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.
2.7.2. Combination of Aerobic Exercise and Resistance Training Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.
Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.
To evaluate the initial physical condition, the right training program, and to compare the initial with the FU assessment of physical condition, three field tests were carried out: cardiorespiratory [40], strength [41], and flexibility fitness [42]. Cardio-respiratory fitness was assessed by means of the 2 km walking test, which is suitable for adults [40], whereas strength and flexibility fitness were evaluated by means of the push-up test (also called press-up test) and the Sit and Reach test, respectively. Subjects randomized to an intervention arm that included physical activity underwent the tests at baseline, after 45, and after 90 days, whereas subjects randomized to the diet arms underwent the field fitness tests at baseline and after 90 days. Physical activity interventions included two different types of exercise programs: Physical Activity 1 (PA1), based on the aerobic activity program, and Physical Activity 2 (PA2), based on the combination of aerobic activity and resistance training. The primary methods used for determining the intensity of prescribed exercise are maximum heart rate (Max HR), through heart-rate monitors [43], and metabolic equivalent (MET) [44,45]. To establish the age-predicted maximal heart rate, we used the formulas of Tanaka [46]. All subjects were randomized for one of the two physical activity programs based on the results of the tests performed. A progressive change of the monthly target has been planned based on the results obtained.
2.7.1. Aerobic Activity Program Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.
Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.
2.7.2. Combination of Aerobic Exercise and Resistance Training Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.
Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.
2.8. Outcome Assessment To quantify and detect hepatic steatosis, the controlled attenuation parameter (CAP) score was used. CAP measures the degree of ultrasound attenuation due to hepatic fat at the standardized frequency of 3.5 MHz through a vibration-controlled elastography (VCTE) implemented on FibroScan® (Echosens, Paris, France). Values <248, 248–267, 268–279 and ≥280 dB/m indicate absence, mild, moderate, or severe NAFLD, respectively [47].
To quantify and detect hepatic steatosis, the controlled attenuation parameter (CAP) score was used. CAP measures the degree of ultrasound attenuation due to hepatic fat at the standardized frequency of 3.5 MHz through a vibration-controlled elastography (VCTE) implemented on FibroScan® (Echosens, Paris, France). Values <248, 248–267, 268–279 and ≥280 dB/m indicate absence, mild, moderate, or severe NAFLD, respectively [47].
2.9. Statistical Analysis The intention-to-treat principle was applied in all analysis. A description of the data was performed by Means (±SD), Median (IQR), and Frequencies (%) as appropriate. Data were also described separately by NAFLD severity. Biochemical markers were included only for descriptive purposes. Adherence to PA programs was evaluated as the proportion of effective frequency/time/intensity spent on gym or field sessions divided by the expected weekly frequency/time/intensity per session for the aerobic exercise group, plus expected load for the combined exercise group. The adherence was estimated by age group, gender, and month, and it was expressed as percentage. The third to tenth weeks of intervention were considered. The first two weeks were excluded to give subjects the opportunity to adapt to the programs training, and the last two weeks were excluded because the compliance could be decreased. The adherence to the LGIMD was measured with MAI [38]. Random week and weekend days were chosen from the second to 9th weeks of intervention. The first two weeks were excluded to allow participants to learn about the new diet and weeks successive to ninth were excluded because the compliance could decrease. To clearly describe the adherence and compliance to the LGIMD, MAI was estimated by gender, age, and week. Compliance was defined as previously described [38]. A median value of 7.5 was expected, as established by the reference Italian Mediterranean Diet [38].
To analyze differences among groups (Diet by Time), an Analysis of Variance for repeated measures was applied to the data. This statistical technique was chosen because it is relatively robust against violations of its assumptions, allows for multiple comparisons, and can be applied to a variety of experimental designs. Analysis of variance for repeated measurements was also performed by NAFLD severity. CAP was normally distributed, so no transformation was needed. Post estimation procedures were performed to compare estimated means with the reference category (control diet at baseline). To display graphically prediction from the fitted model, margins post estimation commands were performed. Margins statistics were calculated from predictions of a previously fitted Analysis of Variance model and displayed in graph form. Results are all expressed in the natural scale of CAP measurements as long as 95% Confidence Interval (95% CI).
Stata statistical (version 16.1) package was used to perform statistical analysis (StataCorp, 4905 Lakeway Drive, College Station, TX, USA); the Stata code is available upon request from the email: [email protected]).
The intention-to-treat principle was applied in all analysis. A description of the data was performed by Means (±SD), Median (IQR), and Frequencies (%) as appropriate. Data were also described separately by NAFLD severity. Biochemical markers were included only for descriptive purposes. Adherence to PA programs was evaluated as the proportion of effective frequency/time/intensity spent on gym or field sessions divided by the expected weekly frequency/time/intensity per session for the aerobic exercise group, plus expected load for the combined exercise group. The adherence was estimated by age group, gender, and month, and it was expressed as percentage. The third to tenth weeks of intervention were considered. The first two weeks were excluded to give subjects the opportunity to adapt to the programs training, and the last two weeks were excluded because the compliance could be decreased. The adherence to the LGIMD was measured with MAI [38]. Random week and weekend days were chosen from the second to 9th weeks of intervention. The first two weeks were excluded to allow participants to learn about the new diet and weeks successive to ninth were excluded because the compliance could decrease. To clearly describe the adherence and compliance to the LGIMD, MAI was estimated by gender, age, and week. Compliance was defined as previously described [38]. A median value of 7.5 was expected, as established by the reference Italian Mediterranean Diet [38].
To analyze differences among groups (Diet by Time), an Analysis of Variance for repeated measures was applied to the data. This statistical technique was chosen because it is relatively robust against violations of its assumptions, allows for multiple comparisons, and can be applied to a variety of experimental designs. Analysis of variance for repeated measurements was also performed by NAFLD severity. CAP was normally distributed, so no transformation was needed. Post estimation procedures were performed to compare estimated means with the reference category (control diet at baseline). To display graphically prediction from the fitted model, margins post estimation commands were performed. Margins statistics were calculated from predictions of a previously fitted Analysis of Variance model and displayed in graph form. Results are all expressed in the natural scale of CAP measurements as long as 95% Confidence Interval (95% CI).
Stata statistical (version 16.1) package was used to perform statistical analysis (StataCorp, 4905 Lakeway Drive, College Station, TX, USA); the Stata code is available upon request from the email: [email protected]).
2.1. Participants:
Details about the study have been published elsewhere [32]. Briefly, 166 subjects, referred to the Laboratory of Epidemiology and Biostatistics of the National Institute of Digestive Diseases, IRCCS “S. de Bellis”, Castellana Grotte, Italy, by their General Practitioners or identified during the NutriEp [5] or MICOL [33] enrollment or follow-up process, were assessed for eligibility. The trial was conducted from March 2015 to December 2016. Patients were consecutively enrolled, and the trial was registered at www.https://clinicaltrials.gov (registration number CT02347696). All subjects provided informed consent and the study was conducted in accordance with the Helsinki Declaration and approved by the Ethical Committee (Prot. n. 10/CE/De Bellis, 3 February 2015).
2.2. Study Design:
This study was a parallel group randomized controlled clinical trial. Inclusion criteria were Body Mass Index (BMI) ≥25, expressed as weight in kilograms divided by square height in meters (kg/m2); age >30 years old and <60 years old; moderate or severe NAFLD as assessed by the controlled attenuation parameter (CAP). Exclusion criteria included (1) overt cardiovascular disease and revascularization procedures; (2) stroke; (3) clinical peripheral artery disease; (4) T2DM (current treatment with insulin or oral hypoglycemic drugs, fasting glucose >126 mg/dL, or casual glucose >200 mg/dL; (5) more than 20 gr/daily of alcohol intake; (6) any severe medical condition that could prevent the subject’s participation in a nutritional intervention study; (7) subjects following a special diet or involved in a program for weight loss, or who had experienced recent weight loss, and (8) inability to follow a LGIMD for religious or other reasons [32].
2.3. Sample Size:
Sample size was estimated considering the repeated measurements nature of the outcome. From a previous study [34], the mean ± Standard Deviation (SD) score of NAFLD was estimated to be 4.5 (1) and 4.0 (0.5) for the treatment and control groups, respectively. We hypothesized a decrease by 20 units of the CAP score after three months of intervention. Probabilistic type I error was fixed at 0.05 (one sided) and probabilistic error type II was fixed at 0.10; statistical power was fixed at 0.9. The correlation between baseline/follow-up measurements of the outcome was set at 0.4. A sample size of n1 = n2 = n3 = n4 = n5 = n6 = 20 was estimated.
2.4. Data Collection:
During enrollment, patients were interviewed by trained nutritionists to collect information on socio-demographic aspects, medical history, and lifestyle. Information about physical activity was collected using the validated questionnaire International Physical Activity Questionnaire Long Form (IPAQ-LF) [35]. The European Prospective Investigation into Cancer and Nutrition Food Frequency Questionnaire (EPIC FFQ) was used to probe alcohol intake and eating behavior [36]. Anthropometric measurements (weight, height, waist circumference) were taken in a standard manner. Weight and height measurements were taken using SECA instruments (Model 700; and Model 206; 220 cm; SECA, Hamburg, Germany). Blood samples, taken by venous puncture, were drawn in the morning after overnight fasting and were collected in tubes containing ethylenediamine tetra acetic acid (K-EDTA) anticoagulant. Biochemical measurements were performed using standard methods.
2.5. Randomization and Masking:
According to a computerized random numbers sequence, subjects were randomized to six arms as follows: (1) Control Diet (CD) based on CREA-AN (Research Center for Food and Nutrition, Council for Agricultural Research and Economics, Rome, Italy) guidelines [37]; (2) Low Glycemic Index Mediterranean Diet (LGIMD); (3) Physical Activity aerobic program (PA1); (4) Physical Activity combined program (aerobic activity and resistance training) (PA2); (5) LGIMD plus PA1 and (6) LGIMD plus PA2. The trial flowchart is shown in Figure 1.
2.6. Dietary Interventions:
Two types of diets were prescribed: a Control Diet (CD) based on CREA-AN guidelines [37] and a Low Glycemic Index Mediterranean Diet (LGIMD) [28]. No indications were given regarding the total calories to be consumed. Foods in LGIMD have all a low Glycemic Index (GI) and no more than 10% of total daily calories coming from saturated fats. The LGIMD was high in monounsaturated fatty acids (MUFA) from olive oil and contained also omega-3 polyunsaturated fatty acids (ω3PUFA), from both plant and marine sources. The prescribed diets were provided in brochure format, with graphic explanations. All participants were asked to record what they ate on a daily diary. The Mediterranean Adequacy Index (MAI) was chosen as a relevant measure to evaluate the adherence to both the intervention diet and the control diet [38]. Detailed information relating to the LGIMD intervention and control (recommended by the WHO and followed by INRAN [39]) is shown in Figure S1 and Figure S2.
2.7. Physical Activity Interventions:
To evaluate the initial physical condition, the right training program, and to compare the initial with the FU assessment of physical condition, three field tests were carried out: cardiorespiratory [40], strength [41], and flexibility fitness [42]. Cardio-respiratory fitness was assessed by means of the 2 km walking test, which is suitable for adults [40], whereas strength and flexibility fitness were evaluated by means of the push-up test (also called press-up test) and the Sit and Reach test, respectively. Subjects randomized to an intervention arm that included physical activity underwent the tests at baseline, after 45, and after 90 days, whereas subjects randomized to the diet arms underwent the field fitness tests at baseline and after 90 days. Physical activity interventions included two different types of exercise programs: Physical Activity 1 (PA1), based on the aerobic activity program, and Physical Activity 2 (PA2), based on the combination of aerobic activity and resistance training. The primary methods used for determining the intensity of prescribed exercise are maximum heart rate (Max HR), through heart-rate monitors [43], and metabolic equivalent (MET) [44,45]. To establish the age-predicted maximal heart rate, we used the formulas of Tanaka [46]. All subjects were randomized for one of the two physical activity programs based on the results of the tests performed. A progressive change of the monthly target has been planned based on the results obtained.
2.7.1. Aerobic Activity Program Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.
Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.
2.7.2. Combination of Aerobic Exercise and Resistance Training Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.
Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.
2.7.1. Aerobic Activity Program:
Three non-consecutive sessions per week of moderate intensity aerobic activity (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1). The approximate duration of each session was between 50 and 60 min. Aerobic exercise intensity was monitored every 5 min using an automated heart rate monitor. Total weekly exercise duration was 150/180 min. Exercise modalities included treadmill walking, cycling, cross-training, and rowing.
2.7.2. Combination of Aerobic Exercise and Resistance Training:
Three non-consecutive sessions including (1) 45 min of moderate intensity aerobic exercise on treadmill, cycling, rowing, and cross-trainer (60–75% max HR, 3.0–5.9 METs distributed as follows: week 1–4: 14.2 kcal ∗ kg−1 ∗ week−1; week 5–8: 18.9 kcal ∗ kg−1 ∗ week−1; week 9–12: 23.6 kcal ∗ kg−1 ∗ week−1); (2) 3 sets of 12 exercises, each to volitional fatigue like leg press, adductor/abductor machine, gluteus machine, bicep curls, triceps extension, three different abdominal exercises, leg machine, low row, shoulder flexion. The weight lifting was increased by 1–2.5 kg ∗ week−1 when 10 repetitions were completed in good form. Total weekly exercise duration was 180/240 min. All physical activity programs were carried out in a local gym, and all subjects participated in the physical training program, deciding on the times and days for the training, and interacting with the specialist at any time.
2.8. Outcome Assessment:
To quantify and detect hepatic steatosis, the controlled attenuation parameter (CAP) score was used. CAP measures the degree of ultrasound attenuation due to hepatic fat at the standardized frequency of 3.5 MHz through a vibration-controlled elastography (VCTE) implemented on FibroScan® (Echosens, Paris, France). Values <248, 248–267, 268–279 and ≥280 dB/m indicate absence, mild, moderate, or severe NAFLD, respectively [47].
2.9. Statistical Analysis:
The intention-to-treat principle was applied in all analysis. A description of the data was performed by Means (±SD), Median (IQR), and Frequencies (%) as appropriate. Data were also described separately by NAFLD severity. Biochemical markers were included only for descriptive purposes. Adherence to PA programs was evaluated as the proportion of effective frequency/time/intensity spent on gym or field sessions divided by the expected weekly frequency/time/intensity per session for the aerobic exercise group, plus expected load for the combined exercise group. The adherence was estimated by age group, gender, and month, and it was expressed as percentage. The third to tenth weeks of intervention were considered. The first two weeks were excluded to give subjects the opportunity to adapt to the programs training, and the last two weeks were excluded because the compliance could be decreased. The adherence to the LGIMD was measured with MAI [38]. Random week and weekend days were chosen from the second to 9th weeks of intervention. The first two weeks were excluded to allow participants to learn about the new diet and weeks successive to ninth were excluded because the compliance could decrease. To clearly describe the adherence and compliance to the LGIMD, MAI was estimated by gender, age, and week. Compliance was defined as previously described [38]. A median value of 7.5 was expected, as established by the reference Italian Mediterranean Diet [38].
To analyze differences among groups (Diet by Time), an Analysis of Variance for repeated measures was applied to the data. This statistical technique was chosen because it is relatively robust against violations of its assumptions, allows for multiple comparisons, and can be applied to a variety of experimental designs. Analysis of variance for repeated measurements was also performed by NAFLD severity. CAP was normally distributed, so no transformation was needed. Post estimation procedures were performed to compare estimated means with the reference category (control diet at baseline). To display graphically prediction from the fitted model, margins post estimation commands were performed. Margins statistics were calculated from predictions of a previously fitted Analysis of Variance model and displayed in graph form. Results are all expressed in the natural scale of CAP measurements as long as 95% Confidence Interval (95% CI).
Stata statistical (version 16.1) package was used to perform statistical analysis (StataCorp, 4905 Lakeway Drive, College Station, TX, USA); the Stata code is available upon request from the email: [email protected]).
3. Results:
One hundred and sixty-six subjects were assessed for eligibility: 17 subjects were excluded because they did not satisfy the inclusion criteria and 5 were excluded for various other reasons. The study design is shown in Figure 1. The following subjects were lost to follow-up: 5, 3, 7, 6, 9, and 4 from Arms 1, 2, 3, 4, 5 and 6, respectively.
Finally, 144 subjects were included and randomized to the intervention arms. Characteristics of the participants are shown in Table 1 and Table 2 and in Appendix A, Table A1.
As expected, the age–sex distribution of the population under study reflected the age–sex distribution of the NAFLD condition in the population [4]. About 62% of the sample were men, and the mean age was 49.9 years (10.08), but few subjects were under 40 years old (12.5%). As expected, all subjects were overweight or obese with increased waist circumference. At baseline, almost every participant had low levels of HDL-C and a high HOMA-IR. Most subjects were married and had attended secondary school or higher (Table 2). All parameters considered were equally distributed among groups at baseline with the exception of NAFLD severity. More subjects had severe than moderate NAFLD, and the CAP value was over 323 dB/m in all groups with a successive decreasing in intermediate and final observation times.
The behavior of anthropometric, body composition, and biochemical profiles by time and intervention arm are shown in Appendix A, Table A1. Overall, there were improvements in all markers, especially at 45 days. These improvements were particularly evident for intervention arms PA1 + LGIMD and PA2 + LGIMD.
Field Fitness Test results by gender and time are shown in Table 3.
Overall, there was a significant improvement of the fitness index in both men and women. The compliance to physical activity programs is shown in Table 4.
Overall, adherence was above 80% to both PA intervention program in both genders and in all weeks.
Compliance to LGIMD, as measured by MAI, by age class, gender, and week is shown in Table 5.
Compliance with the control diet showed a median MAI of 1.29 (0.77, 2.63). MAI was lower amongst males (1.07) than females (2.14), whereas compliance with LGMID evidenced a median MAI of 11.2 (6.4, 22.8). Adherence was lower among women (10.0) than men (11.3).
Table 6 shows the changes in the CAP value by intervention arms and time (at 45 days and at 90 days).
Apart from the CD, there were statistically significant principal effects of all the other intervention arms, Arms 3 and 5 showing the strongest effects (PA1: −166.35, 95%CI −242.01; −90.68 and LGIMD plus PA1: −94.10, 95% CI −170.87; −19.12). After the analysis, no statistical significance principal effect of time in reducing CAP was observed. All comparisons between different time points and Intervention Arms, except for Intervention Arm 1, showed a statistically significant decreased value for CAP. The strongest effect was observed for LGIMD plus PA1 at 45 days (−52.96, 95% CI −79.03; −26.88) and 90 days (−61.56, 95% CI −89.61; −33.50).
Results from separate analysis by NAFLD severity are shown in Appendix A, Table A2 and Table A3. The relative decrease of CAP values was more relevant among severe NAFLD subjects. HOMA-IR decreased in all intervention arms with the exception of LGIMD in moderate NAFLD subjects. In both moderate and severe NAFLD subjects, there was a modest effect of only-diet intervention arms on CAP. Only-PA intervention arms effect on CAP values evidenced a decrease among severe NAFLD subjects but not among moderate ones. The most intense effect in decreasing CAP values was observed among severe NAFLD subjects who underwent PA1+LGIMD. An improvement in all anthropometric, body composition, and biochemical parameters was observed (most evident at day 45). Noteworthy, the improvement in HDL-C was accompanied by an improvement in FFM only in moderate NAFLD subjects. Table 7 shows the changes in the CAP value by NAFLD severity, intervention arms, and time (at 45 days and at 90 days).
There was an effect that was statistically significant only in subjects with moderate NAFLD allocated to Arm 6 after 90 days, whereas among severe NAFLD subjects, there were statistically significant effects in all times and arms with the exception of CD and Arm 6 at 90 days. The strongest estimated effect was observed in the PA1 + LGIMD intervention arm.
The results of predictive margins from analysis of variance are graphically displayed in Figure 2, which highlights the strong effect of LGIMD plus PA1. Hypothesis testing about the equality of measurements at baseline showed no statistically significant differences in CAP among intervention arms (p > 0.05).
4. Discussion:
In this RCT, the combination of the aerobic activity program and an LGIMD was associated with the strongest reduction of the CAP, as measured by FibroScan®, compared to the other intervention arms. Overall, this effect was greater until 45 days of intervention. However, the effect of Aerobic PA plus LGIMD was stronger during the complete intervention time in severe than moderate NAFLD subjects. The effect on CAP was accompanied by an overall improvement in all anthropometric, body composition and biochemical parameters.
Currently, a lifestyle change focused on diet and physical activity seems to be the best therapy in the treatment of NAFLD, combined with pharmacologic therapy when necessary [48]. Regular physical activity is now considered a therapy to prevent the onset and progression of several chronic diseases, including NAFLD [49]. Aerobic, resistance and combination exercise programs may enhance systemic indicators of hepatic functions and intrahepatic fats in NAFLD patients in both mild and advanced cases [50].
The adoption of either aerobic or strength PA programs showed to influence hepatic metabolism with a resulting decrease in hepatic fat accumulation, an increased insulin sensitivity, and fat oxidation. Moreover, these effects have been observed even without weight loss [51].
Although the risk of developing NAFLD or more advanced grades of liver diseases seems to be exercise dose-related [52], no differences among different intensity aerobic exercise regimens were recently found: all of them reduced liver fat [53].
At the community level, a reduced infiltration of liver fat has been documented after a combined exercise program [54]. This effect was proportional to weight loss rate and event without it.
LGIMD was locally created by integrating the works of Trichopolou et al. [30] and Elia [31]. Moreover, we have adapted it to our population, and it is similar to the diet suggested by other authors for the metabolic syndrome [55]. LGIMD, which contains no more than 10% of total daily calories deriving from saturated fats and a low glycemic index, was associated with a more intense reduction of the NAFLD score [28]. The LGIMD is rich in monounsaturated fatty acids (MUFA) and also contains omega-3 polyunsaturated fatty acids (ω3PUFA).
In this RCT, in working Arm 2, where the subjects only followed a LGIMD, we observed a statistically significant improvement of the score of NAFLD, reducing the CAP value, especially at 45 days. However, after 45 days, a slight increase in the CAP value was observed. Several studies have shown that lifestyle changes are difficult to implement and maintain for a long time, although individuals know the importance of having a healthier lifestyle [56,57]. In our study, adherence was almost homogenously distributed during the intervention time but, as it has been shown, participants could have given answers that are socially desirable [58].
When comparing the effectiveness of the two different physical activity programs, it is clear that both the aerobic and the combined exercise program significantly reduced the CAP value. Our data are confirmed by previous studies that have demonstrated the effectiveness of aerobic activity on liver function [59,60]. Moreover, recently, resistance training has also been found to be effective in NAFLD treatment, regardless of weight loss [61,62,63].
Both aerobic exercise and combined exercise significantly reduced the CAP value from 45 days onward until the end of the project. The literature exhaustively showed the efficacy of both combined and resistance training in NAFLD [64]. Furthermore, aerobic activity and resistance training have been recently shown, although with different mechanisms, to improve NAFLD score [23,65]. At least three mechanisms have been proposed for aerobic PA: activation of lipolysis in different tissues, upregulation of the uncoupling protein-1 and peroxisome proliferator-activated receptor γ pathways, and alteration of adipokine levels [23]; on the other hand, resistance training stimulates the hypertrophy of type II muscle fibers, alters myokine levels, and activates glucose transporter 4, AMP-activated protein kinase, and caveolins [23]. Moreover, resistance training appeared to improve NAFLD with less energy consumption. A recent study conducted in mice with diethyl nitrosamine-induced hepatocellular carcinoma has shown that in early stages, voluntary exercise decreases the proliferation rate of dysplastic hepatocytes [66].
Therefore, PA could be hypothesized as a preventive measure for liver cancer development, as NAFLD is a stage of a broad spectrum of conditions that lead to hepatocellular carcinoma. In this sense, after an energy restriction dietary intervention and/or regular PA, several facts have been observed such as histological improvements, resolution of liver fat, as well as necroinflammation and fibrosis. These changes have been observed even after 7% to 10% of weight loss [67,68].
Healthy dietary habits and healthy lifestyle, similar to any regular physical activity, can improve NAFLD more than weight loss alone [69].
Weight loss achieved by dietary treatment must be done taking into account quantitative and qualitative characteristics, as energy restriction per se is not sufficient to improve NAFLD [22]. Moreover, a dietary composition modulating both macro and micronutrients is crucial [70]. Therefore, balanced nutrition such as LGIMD, moderate weight loss, and a physical activity program can be considered as the best therapeutic approach in NAFLD.
This seems to be confirmed in our study where the effect of intervention was more evident in the severe NAFLD group, highlighting that the combination of diet and physical activity can be an effective therapeutic tool in the prevention not only of the complications of NAFLD but also of the associated cardiovascular risk. The observed effect in the severe NAFLD group could be due to the particular distribution of NAFLD severity, as 75% of participants (data not shown) had severe NAFLD at baseline. Moreover, the use of FibroScan® as the NAFLD assessment method could have enhanced the frequency of severe NAFLD diagnosis. It is known that FibroScan® has a greater sensibility [71] than Liver Ultrasound for fat infiltration >25–30% of hepatocytes [72].
The diminution of intrahepatic fat content is not the only marker of interest in this study. The metabolic pattern resulting from the interventions was explored, and the results are almost always in line with the literature. The negative association between adherence to Mediterranean diet and NAFLD severity has been confirmed in this study as well as HOMA-IR decreasing, which has been more strong in severe NAFLD subjects [19]. As noted above, we built a local version of the Mediterranean diet following the European Association for the study of the Liver, the European Association for the study of Diabetes, and the European Association for the study of Obesity guidelines [26]. After the intervention period, our results are similar to those of Ryan et al., who found an improvement in fatty liver content and HOMA-IR [73]. Moreover, a recent systematic review and meta-analysis has documented beneficial changes of the Mediterranean diet on metabolic health, including anthropometric and biochemical markers [74].
As regards PA, a network meta-analysis has shown that aerobic exercise plus diet and aerobic exercise alone exert an improvement on BMI and HOMA-IR but progressive resistance training has an effect only on HOMA-IR but not on BMI [75]. While diet seems to be more effective at improving intrahepatic markers of liver damage, PA plus diet improves insulin resistance and BMI [76]. However, in our study, there was a modest effect of only the LGIMD intervention arm. In the geographical area where the study was conducted, the Mediterranean diet is the most prevalent dietary pattern with a high adherence at population level, which may be the cause of the modest observed effect [76].
This study has several strengths and limitations. Strengths include the study design, the measured compliance to both dietary and PA interventions as well as their controlled application, and an adequate sample size. Moreover, a well-validated assessment of the outcome such as FibroScan® has been implemented. The applied intention-to-treat analytical strategy prevents the design from introducing bias relating to non-adherence to the protocol to the prognosis; therefore, this RCT provides an unbiased assessment of treatment efficacy [77]. It is worthy to note that in this area, the most prevalent dietary pattern is the local version of Mediterranean Diet; then, a dilution bias could be present. Another limitation may be the duration of the intervention, which prevents a wide application in the clinical field, However, the objective of the study was to estimate the effect of the intervention in order to establish its efficacy.
5. Conclusions:
It is clear that PA single (aerobic or resistance exercise) or combined (aerobic plus resistance exercise) should be integrated into NAFLD management [78]. Moreover, aerobic exercise programs are simple to implement and the cost-efficiency is higher. These should be included in programs for the primary prevention of metabolic and cardiovascular diseases.
In conclusion, a multidisciplinary team including dietitians, a psychologist, and physical activity supervisors is needed to ensure the best management of NAFLD patients [78].
|
Background:
Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common chronic liver disease worldwide, and lifestyle modification is the current standard treatment. The aim of the study was to estimate the effect of two different physical activity (PA) programs, a Low Glycemic Index Mediterranean Diet (LGIMD), and their combined effect on the NAFLD score as measured by FibroScan®.
Methods:
Moderate or severe NAFLD subjects (n = 144) were randomly assigned to six intervention arms during three months. Interventions arms were a control diet, LGIMD, aerobic activity program (PA1), combined activity program (PA2), and LGIMD plus PA1 or LGIMD plus PA2. The data were compared at baseline, at 45 days, and at 90 days. Analysis of variance was performed under the intention-to-treat principle.
Results:
There was a statistically significant reduction in the NAFLD score after 45 days of treatment in every working arm except for Arm 1 (control diet). After 90 days, the best results were shown by the intervention arms in which LGIMD was associated with PA: LGIMD plus PA1 (-61.56 95% CI -89.61, -33.50) and LGIMD plus PA2 (-38.15 95% CI -64.53, -11.77).
Conclusions:
All treatments were effective to reduce NAFLD scores, but LGIMD plus PA1 was the most efficient.
|
1. Introduction:
Non-Alcoholic Fatty Liver Disease (NAFLD) is a broad spectrum of liver diseases. It is characterized by fat accumulation in the liver in the absence of excessive alcohol consumption or any other specific causes of hepatic steatosis [1]. NAFLD is a major public health challenge as it is associated with the rising prevalence of obesity and Diabetes Mellitus type 2 worldwide and is a risk factor for Cardiovascular Diseases. Moreover, NAFLD is the most common cause of chronic liver disease and increases the 5-year direct and indirect health care costs by an estimated 26% [2]. The global prevalence of NAFLD is currently estimated to be 24% and is continually rising [3]. Our studies, conducted in the population of Castellana Grotte and Putignano (district of Bari, Apulia, Italy), evidenced an NAFLD prevalence of about 30%, with a higher prevalence in male and older patients [4,5]. Therefore, accurate diagnosis of NAFLD is important in patient management [6]. Until now, the gold standard for hepatic steatosis diagnosis has been liver biopsy, but it is an invasive procedure that has several disadvantages such as low acceptance for patients (especially for volunteers), complications, and even a small risk of death (0.01%) [7,8]. Today, many imaging methods are available for the non-invasive assessment of NAFLD [9], but they have limitations such as high costs or operator-dependency of Ultrasound Scan (US) [10,11]. FibroScan® is a non-invasive examination that appears to have good repeatability, which can be used as indices of continuous observation [12]. Data suggest that the controlled attenuation parameter (CAP), measured with FibroScan®, can be compared to liver biopsy for the detection and quantification of steatosis [13], and recent studies have evaluated its accuracy in NAFLD [14]. Furthermore, Lee et al. 2016 demonstrated that CAP and liver stiffness can be used as non-invasive and predictive markers of steatosis and fibrosis in patients with NAFLD [15].
Lifestyle modification is the standard treatment for NAFLD [16]. This approach encompasses dietary modifications, exercise, reduced alcohol intake, and weight loss, as they offer a range of health benefits. The Mediterranean Diet (MD) has long been associated with favorable health outcomes [17,18], and good adherence to MD has been reported to have a beneficial effect on the severity of NAFLD [19] as well as of Cardiovascular Diseases [20]. In particular, the Low Carbohydrate Diet, similar to the Low Glycemic Index Mediterranean Diet, is characterized by a reduction in the intake of several ultra-processed foods, refined grains starches, and foods rich in simple or added sugars, and it leads to a number of benefits such as weight loss and weight loss maintenance, reduction of Diastolic Blood Pressure (DBP), reduction of low-density lipoprotein cholesterol (LDL-C) and Triglycerides levels, increase of high-density lipoprotein cholesterol (HDL-C) levels, improvements in insulin resistance, and reduction of glycated hemoglobin (HbA1c) levels [21].
In addition, different forms of exercise have been shown to have similar effects on liver fat [22,23] if physical activity (PA) guidelines recommendations are followed [24]. Minimal physical activity (PA) decreases by 6% the risk for NAFLD compared to sedentary behavior [25]. The European Association for the Study of the Liver (EASL), the European Association for the Study of Diabetes (EASD) and the European Association for the Study of Obesity (EASO) guidelines recommend 150 to 200 min/week of moderate-intensity aerobic physical activity in three to five sessions [26]. Aerobic activity and resistance training seem to have similar improvement in NAFLD but with different mechanisms [27]. Nevertheless, there are still no universal recommendations on the optimal intensity, dose, and type of physical activity in NAFLD treatment. Two studies conducted in this area have confirmed the efficacy of Low Glycemic Index Mediterranean Diet (LGIMD) [28] and the efficiency of aerobic PA on NAFLD [29]. In this context, and as studies about the effect of diet and PA on NAFLD are scarce, we hypothesized that different PA programs and LGIMD or their combination could have different degrees of efficacy on NAFLD severity. We built a local version of the Mediterranean diet based on the work of Trichopoulos et al. and Elia, which contains no more than 10% of total daily calories deriving from saturated fats and a low glycemic index with an extensive use of olive oil [30,31]. Furthermore, the PA programs were built following European guidelines, which comprise aerobic PA as well as resistance training with precision indication about frequency, duration and intensity [26]. This work was aimed estimate the effect of healthy diet (LGIMD), two different PA programs, and their combination on the score of NAFLD as measured in a continuous scale by FibroScan®. Moreover, we aimed to explore biochemical, anthropometric, and body composition changes by different degrees of severity of NAFLD in geographical area where the Mediterranean diet is the most prevalent way of eating.
5. Conclusions:
It is clear that PA single (aerobic or resistance exercise) or combined (aerobic plus resistance exercise) should be integrated into NAFLD management [78]. Moreover, aerobic exercise programs are simple to implement and the cost-efficiency is higher. These should be included in programs for the primary prevention of metabolic and cardiovascular diseases.
In conclusion, a multidisciplinary team including dietitians, a psychologist, and physical activity supervisors is needed to ensure the best management of NAFLD patients [78].
|
Background:
Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common chronic liver disease worldwide, and lifestyle modification is the current standard treatment. The aim of the study was to estimate the effect of two different physical activity (PA) programs, a Low Glycemic Index Mediterranean Diet (LGIMD), and their combined effect on the NAFLD score as measured by FibroScan®.
Methods:
Moderate or severe NAFLD subjects (n = 144) were randomly assigned to six intervention arms during three months. Interventions arms were a control diet, LGIMD, aerobic activity program (PA1), combined activity program (PA2), and LGIMD plus PA1 or LGIMD plus PA2. The data were compared at baseline, at 45 days, and at 90 days. Analysis of variance was performed under the intention-to-treat principle.
Results:
There was a statistically significant reduction in the NAFLD score after 45 days of treatment in every working arm except for Arm 1 (control diet). After 90 days, the best results were shown by the intervention arms in which LGIMD was associated with PA: LGIMD plus PA1 (-61.56 95% CI -89.61, -33.50) and LGIMD plus PA2 (-38.15 95% CI -64.53, -11.77).
Conclusions:
All treatments were effective to reduce NAFLD scores, but LGIMD plus PA1 was the most efficient.
| 11,346 | 262 | 16 |
[
"week",
"nafld",
"activity",
"exercise",
"aerobic",
"physical",
"diet",
"kg",
"subjects",
"lgimd"
] |
[
"test",
"test"
] | null |
[CONTENT] physical activity | non-alcoholic fatty liver disease | diet | controlled attenuation parameter (CAP) | aerobic exercise | combined exercise [SUMMARY]
| null |
[CONTENT] physical activity | non-alcoholic fatty liver disease | diet | controlled attenuation parameter (CAP) | aerobic exercise | combined exercise [SUMMARY]
|
[CONTENT] physical activity | non-alcoholic fatty liver disease | diet | controlled attenuation parameter (CAP) | aerobic exercise | combined exercise [SUMMARY]
|
[CONTENT] physical activity | non-alcoholic fatty liver disease | diet | controlled attenuation parameter (CAP) | aerobic exercise | combined exercise [SUMMARY]
|
[CONTENT] physical activity | non-alcoholic fatty liver disease | diet | controlled attenuation parameter (CAP) | aerobic exercise | combined exercise [SUMMARY]
|
[CONTENT] Adult | Behavior Therapy | Diet, Mediterranean | Exercise | Female | Glycemic Index | Humans | Life Style | Male | Middle Aged | Non-alcoholic Fatty Liver Disease [SUMMARY]
| null |
[CONTENT] Adult | Behavior Therapy | Diet, Mediterranean | Exercise | Female | Glycemic Index | Humans | Life Style | Male | Middle Aged | Non-alcoholic Fatty Liver Disease [SUMMARY]
|
[CONTENT] Adult | Behavior Therapy | Diet, Mediterranean | Exercise | Female | Glycemic Index | Humans | Life Style | Male | Middle Aged | Non-alcoholic Fatty Liver Disease [SUMMARY]
|
[CONTENT] Adult | Behavior Therapy | Diet, Mediterranean | Exercise | Female | Glycemic Index | Humans | Life Style | Male | Middle Aged | Non-alcoholic Fatty Liver Disease [SUMMARY]
|
[CONTENT] Adult | Behavior Therapy | Diet, Mediterranean | Exercise | Female | Glycemic Index | Humans | Life Style | Male | Middle Aged | Non-alcoholic Fatty Liver Disease [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] week | nafld | activity | exercise | aerobic | physical | diet | kg | subjects | lgimd [SUMMARY]
| null |
[CONTENT] week | nafld | activity | exercise | aerobic | physical | diet | kg | subjects | lgimd [SUMMARY]
|
[CONTENT] week | nafld | activity | exercise | aerobic | physical | diet | kg | subjects | lgimd [SUMMARY]
|
[CONTENT] week | nafld | activity | exercise | aerobic | physical | diet | kg | subjects | lgimd [SUMMARY]
|
[CONTENT] week | nafld | activity | exercise | aerobic | physical | diet | kg | subjects | lgimd [SUMMARY]
|
[CONTENT] nafld | liver | pa | prevalence | invasive | diet | different | reduction | studies | health [SUMMARY]
| null |
[CONTENT] table | intervention arms | arms | subjects | intervention | nafld | effect | nafld subjects | days | significant [SUMMARY]
|
[CONTENT] 78 | resistance exercise | management | exercise | aerobic | resistance | programs | pa single | supervisors needed ensure best | supervisors needed ensure [SUMMARY]
|
[CONTENT] week | nafld | exercise | kg week | kg | aerobic | activity | lgimd | diet | kcal kg [SUMMARY]
|
[CONTENT] week | nafld | exercise | kg week | kg | aerobic | activity | lgimd | diet | kcal kg [SUMMARY]
|
[CONTENT] Non-Alcoholic Fatty Liver Disease | NAFLD ||| two | LGIMD | NAFLD [SUMMARY]
| null |
[CONTENT] NAFLD | 45 days | 1 ||| 90 days | LGIMD | LGIMD | 95% | CI | LGIMD | 95% | CI [SUMMARY]
|
[CONTENT] LGIMD [SUMMARY]
|
[CONTENT] NAFLD ||| two | LGIMD | NAFLD ||| NAFLD | 144 | six | three months ||| LGIMD | LGIMD | LGIMD ||| 45 days | 90 days ||| ||| NAFLD | 45 days | 1 ||| 90 days | LGIMD | LGIMD | 95% | CI | LGIMD | 95% | CI ||| LGIMD [SUMMARY]
|
[CONTENT] NAFLD ||| two | LGIMD | NAFLD ||| NAFLD | 144 | six | three months ||| LGIMD | LGIMD | LGIMD ||| 45 days | 90 days ||| ||| NAFLD | 45 days | 1 ||| 90 days | LGIMD | LGIMD | 95% | CI | LGIMD | 95% | CI ||| LGIMD [SUMMARY]
|
|||||
Factors leading to dyspepsia in renal transplant recipients.
|
29515738
|
Renal transplantation is the definitive treatment for end stage renal disease. Patients subjected to transplantation require lifelong immunosuppression and are prone to several gastrointestinal disorders. Dyspepsia is a common disorder in these patients. The objective of this study was to determine factors leading to dyspepsia in renal (kidney) transplant recipients.
|
INTRODUCTION
|
It was a cross sectional study conducted at department of hepatogastroenterology and transplant sciences, SIUT Karachi, from 1-6-15 to 1-12-15 for six months. All renal transplanted patients having dyspeptic symptoms for more than 6 weeks. EGD was performed, biopsy specimens obtained from antrum and duodenum, these were sent for histopathological examination. Frequency and percentages were obtained for categorical variables, mean ± SD was calculated for continuous variables. Chi square test was used for categorical variable and student t-test for continuous variables.
|
METHODS
|
Ninety patients were included in the study out of which 64 (71.1%) were males, mean age was 35.82 ± 10.04 years (range: 18-65 years). Gastritis (non H.pylori associated) in 78 (78.6%), duodenitis in 35 (38.9%) and H. pylori infection in 29 (32.2%), renal transplant recipients. Most of the patients belonged to Sindhi ethnicity, 27 (30%), followed by Punjabi. Hypertension was the most common co-morbid condition in our patients found in 29 (32.2%), while most of them don't have any co morbid condition. Duodenitis was found to be associated with tacrolimus use (p = 0.037).
|
RESULTS
|
Gastritis is the most common factor accountable for this symptoms, followed by duodenitis and H. Pylori. Patients taking tacrolimus as immunosuppressant are more prone to develop duodenitis.
|
CONCLUSION
|
[
"Adolescent",
"Adult",
"Aged",
"Cross-Sectional Studies",
"Duodenitis",
"Dyspepsia",
"Female",
"Gastritis",
"Helicobacter Infections",
"Helicobacter pylori",
"Humans",
"Immunosuppressive Agents",
"Kidney Failure, Chronic",
"Kidney Transplantation",
"Male",
"Middle Aged",
"Pakistan",
"Risk Factors",
"Tacrolimus",
"Young Adult"
] |
5837158
|
Introduction
|
Dyspepsia and gastroesophageal reflux disease (GERD) are the most common diseases encountered by the gastroenterologist, its prevalence in general population is about 10-20% in the Western world and lower in Asia. Six percent of the patients have heartburn; around 38% had dyspepsia while regurgitation was reported in 16% [1, 2]. GERD develops when increased acidic gastric secretion refluxes into the esophagus [3, 4]. Gastrointestinal (GI) symptoms are commonly experienced in solid organ transplant recipients and can effects any part of the GI tract [5]. After three years of renal transplant, about 23% of the patients have GERD, reflux esophagitis (RE) and dyspepsia in 20%, 5% and 6%, respectively [6]. Renal transplantation, chronic renal disease, immunosuppressive therapy, H. pylori infection, female gender, pre transplant upper gastrointestinal disease, older age, obesity, Caucasian and African-American race, are considered as potential risk factors for the development of GERD and dyspepsia [6–9]. Neri et al concluded that female gender (39.9%), hypertension (22.5%) and renal failure secondary to type I DM (12.1%) have increased risk of dyspepsia [6]. Raised creatinine is one of the risk factor of dyspepsia in post renal transplant era [7, 8]. In normal population increased age and smoking are risk factors of dyspepsia [9]. Darji et al found that 50% of patients had considerable improvement in dyspeptic symptoms after substituting mycophenolate mofetil (MMF) with its enteric coated formulation [10]. Besides Webster et al concluded that in comparison to cyclosporine, the use of tacrolimus was associated with more risk of dyspepsia, post-transplant diabetes mellitus (PTDM), diarrhea, tremor and headache [11] Ozgur et al inducted 54 renal transplanted patients with dyspepsia out of which H. pylori infection was seen in 70%, gastritis in 65%, duodenitis in 13% and peptic ulcer in 4% [8]. Khidmat et al found duodenal ulcer, erosive gastritis and H. pylori infection in 8%, 20.5% and 40% respectively in post renal transplant [12]. Neri et al proposed that renal transplanted patients having GERD, dyspepsia or reflux esophagitis are at increased risk of graft failure and death [6]. Kim et al reported severe complications in around 10% resulting in graft loss and death [5]. The rationale of this study is to determine factors leading to dyspepsia in renal transplant recipients, as no local study have been done so far for evaluation of dyspepsia in these patients. Evaluation of factors leading to dyspepsia, its early diagnosis and prompt treatment is crucial in these patients as it can lead to graft failure and increased morbidity and mortality.
|
Methods
|
The study was descriptive cross sectional study, conducted in the department of Hepatogastroenterology, Sindh Institute of Urology and Transplantation (SIUT), Karachi from 1st June 2015 to 30th November 2015. Those patients who had dyspepsia3 for at least 6 weeks after 6 months of renal transplantation of both genders and aged 18 and above were included in the study. Following patients were excluded: those who had taken proton pump inhibitors at least two weeks before EGD, had congestive heart failure (i.e: ejection fraction is less than 40% on echocardiography), bleeding disorder: thrombocytopenia or coagulopathy (Platelets < 50000/μL or International normalized ratio (INR) > 1.5, hemodynamically unstable (B.P < 70/40 mmHg). Eight factors which lead to dyspepsia were taken into consideration in this study, factors were gender, hypertension, diabetes mellitus, obesity, immunosuppressive drugs, H.pylori gastritis, non H.pylori gastritis and duodenitis. Sample size was calculated by using open epi calculator prevalence of duodenitis with dyspeptic symptoms was found to be 13% [8] in renal transplant recipients, d = 7%, confidence interval = 95%, estimated sample size was at least n = 89. All the cases were subjected to endoscopic evaluation. Venous blood sample collected for laboratory parameters; urea, creatinine, complete blood count and prothrombin time. Esophagogastroduodenoscopy (EGD) was done in all enrolled patients as per indication, performed by researcher under supervision of supervisor using video endoscope (Olympus GIF-XP180). Two biopsies were taken from antrum and duodenum for histopathological examination and were sent to the histopathology department at SIUT, to rule out gastritis, duodenitis or H. pylori infection. Data was analyzed by using statistical software SPSS-20.0. Mean and standard deviation (SD) was calculated for continuous variables (age, BMI, duration of symptoms). Frequencies and percentages were evaluated for categorical variables (Gender, co-morbids ie DM and HTN, addictions, H.pylorigastritis, duodenitis, gastritis, immunosuppressants which includes steroids, azathioprine, everolimus, tacrolimus, cyclosporine, mycophenolate mofetil and obesity). Stratification with respect to age, addiction, ethnicity, duration since renal transplant and duration of symptoms was done. Post stratification Chi square test was applied. A p-value ≤ 0.05 was taken as significant.
|
Results
|
Total 90 renal transplant recipients were enrolled in this study. Most of the patients were male i.e 64 (71.1%) (Table 1). Mean age was 35.82 ± 10.04 years (Range: 18-65 years) (Table 2). Most of the patients belonged to Sindhi ethnicity, 27(30%) followed by Punjabi 21 (23.3%) (Table 3). Almost half of the patients had no known co- morbid 47 (52.2%). Hypertension, DM and hepatitis B was observed in 29 (32.2%), 4 (4.4%) and 4 (4.4%) respectively (Table 4). Upper GI symptoms before transplant were also uncommon in these patients, only around one-fourth of subjects had history of dyspeptic symptoms prior to transplant i.e: 23 (25.6%). Sixty nine patients (76.7%) had no history of substance abuse while tobacco smoking was observed in 12 (13.3%) subjects followed by alcoholism in 6 (6.7%) (Table 1). In the study subjects, urea was ranged from 17-230 mg/dL with mean of 58.34 ± 39.30 mg/dl, while creatinine ranged from 0.72-6.08 mg/dL, with mean of 2.11 ± 1.11 mg/dL (Table 2). Renal transplant patients were receiving multiple immunosuppressant, all of them were on steroids (prednisolone) along with other agents. Azathioprine was used in 54 (60%), followed by cyclosporine in 35 (38.9%), tacrolimus in 27 (30%), MMF in 26 (28.9%) and everolimus in 8 (8.9%) patients. Gastritis (non H. pylori infection) seen in 78 (86.7%), duodenitis in 35 (38.9%) and H. pylori infection in 29 (32.2%) recipients as cause of dyspepsia (Table 1). No endoscopy related complication was observed in any patient. Analysis of factors leading to dyspepsia, revealed statistically significant association in patients having duodenitis on tacrolimus (p = 0.037) (Table 5), however, one tailed p-value was significant in patients having gastritis and BMI of more than 18.92 (p = 0.053). No significant association was found with other immunosuppressive agents in patients having H. Pylori, non H. Pylori gastritis, duodenitis or female gender.
Demographic and clinical characteristics of study population (n=90)
Descriptive statistics of continuous variables (n=90)
Ethnicity of study population (n=90)
Co morbid of study Population (n=90)
Analysis of factors leading to dyspepsia: duodenitis (n=90)
|
Conclusion
|
The common cause of dyspepsia in our renal transplanted patients was gastritis (non H. pylori associated) followed by duodenitis and H. pylori infection. Use of immunosuppression tacrolimus was associated with duodenitis.
What is known about this topic Dyspepsia is common in uremic patients;
Renal transplant recipients are prone to have dyspepsia because of immunosuppressive agents.
Dyspepsia is common in uremic patients;
Renal transplant recipients are prone to have dyspepsia because of immunosuppressive agents.
What this study adds Most common cause of dyspepsia is non H.pylori gastritis;
Duodenitis is associated with the use of tacrolimus as immunosuppressant.
Most common cause of dyspepsia is non H.pylori gastritis;
Duodenitis is associated with the use of tacrolimus as immunosuppressant.
|
[
"What is known about this topic",
"What this study adds",
"Competing interests"
] |
[
"Dyspepsia is common in uremic patients;\nRenal transplant recipients are prone to have dyspepsia because of immunosuppressive agents.",
"Most common cause of dyspepsia is non H.pylori gastritis;\nDuodenitis is associated with the use of tacrolimus as immunosuppressant.",
"The authors declare no competing interests."
] |
[
null,
null,
null
] |
[
"Introduction",
"Methods",
"Results",
"Discussion",
"Conclusion",
"What is known about this topic",
"What this study adds",
"Competing interests"
] |
[
"Dyspepsia and gastroesophageal reflux disease (GERD) are the most common diseases encountered by the gastroenterologist, its prevalence in general population is about 10-20% in the Western world and lower in Asia. Six percent of the patients have heartburn; around 38% had dyspepsia while regurgitation was reported in 16% [1, 2]. GERD develops when increased acidic gastric secretion refluxes into the esophagus [3, 4]. Gastrointestinal (GI) symptoms are commonly experienced in solid organ transplant recipients and can effects any part of the GI tract [5]. After three years of renal transplant, about 23% of the patients have GERD, reflux esophagitis (RE) and dyspepsia in 20%, 5% and 6%, respectively [6]. Renal transplantation, chronic renal disease, immunosuppressive therapy, H. pylori infection, female gender, pre transplant upper gastrointestinal disease, older age, obesity, Caucasian and African-American race, are considered as potential risk factors for the development of GERD and dyspepsia [6–9]. Neri et al concluded that female gender (39.9%), hypertension (22.5%) and renal failure secondary to type I DM (12.1%) have increased risk of dyspepsia [6]. Raised creatinine is one of the risk factor of dyspepsia in post renal transplant era [7, 8]. In normal population increased age and smoking are risk factors of dyspepsia [9]. Darji et al found that 50% of patients had considerable improvement in dyspeptic symptoms after substituting mycophenolate mofetil (MMF) with its enteric coated formulation [10]. Besides Webster et al concluded that in comparison to cyclosporine, the use of tacrolimus was associated with more risk of dyspepsia, post-transplant diabetes mellitus (PTDM), diarrhea, tremor and headache [11] Ozgur et al inducted 54 renal transplanted patients with dyspepsia out of which H. pylori infection was seen in 70%, gastritis in 65%, duodenitis in 13% and peptic ulcer in 4% [8]. Khidmat et al found duodenal ulcer, erosive gastritis and H. pylori infection in 8%, 20.5% and 40% respectively in post renal transplant [12]. Neri et al proposed that renal transplanted patients having GERD, dyspepsia or reflux esophagitis are at increased risk of graft failure and death [6]. Kim et al reported severe complications in around 10% resulting in graft loss and death [5]. The rationale of this study is to determine factors leading to dyspepsia in renal transplant recipients, as no local study have been done so far for evaluation of dyspepsia in these patients. Evaluation of factors leading to dyspepsia, its early diagnosis and prompt treatment is crucial in these patients as it can lead to graft failure and increased morbidity and mortality.",
"The study was descriptive cross sectional study, conducted in the department of Hepatogastroenterology, Sindh Institute of Urology and Transplantation (SIUT), Karachi from 1st June 2015 to 30th November 2015. Those patients who had dyspepsia3 for at least 6 weeks after 6 months of renal transplantation of both genders and aged 18 and above were included in the study. Following patients were excluded: those who had taken proton pump inhibitors at least two weeks before EGD, had congestive heart failure (i.e: ejection fraction is less than 40% on echocardiography), bleeding disorder: thrombocytopenia or coagulopathy (Platelets < 50000/μL or International normalized ratio (INR) > 1.5, hemodynamically unstable (B.P < 70/40 mmHg). Eight factors which lead to dyspepsia were taken into consideration in this study, factors were gender, hypertension, diabetes mellitus, obesity, immunosuppressive drugs, H.pylori gastritis, non H.pylori gastritis and duodenitis. Sample size was calculated by using open epi calculator prevalence of duodenitis with dyspeptic symptoms was found to be 13% [8] in renal transplant recipients, d = 7%, confidence interval = 95%, estimated sample size was at least n = 89. All the cases were subjected to endoscopic evaluation. Venous blood sample collected for laboratory parameters; urea, creatinine, complete blood count and prothrombin time. Esophagogastroduodenoscopy (EGD) was done in all enrolled patients as per indication, performed by researcher under supervision of supervisor using video endoscope (Olympus GIF-XP180). Two biopsies were taken from antrum and duodenum for histopathological examination and were sent to the histopathology department at SIUT, to rule out gastritis, duodenitis or H. pylori infection. Data was analyzed by using statistical software SPSS-20.0. Mean and standard deviation (SD) was calculated for continuous variables (age, BMI, duration of symptoms). Frequencies and percentages were evaluated for categorical variables (Gender, co-morbids ie DM and HTN, addictions, H.pylorigastritis, duodenitis, gastritis, immunosuppressants which includes steroids, azathioprine, everolimus, tacrolimus, cyclosporine, mycophenolate mofetil and obesity). Stratification with respect to age, addiction, ethnicity, duration since renal transplant and duration of symptoms was done. Post stratification Chi square test was applied. A p-value ≤ 0.05 was taken as significant.",
"Total 90 renal transplant recipients were enrolled in this study. Most of the patients were male i.e 64 (71.1%) (Table 1). Mean age was 35.82 ± 10.04 years (Range: 18-65 years) (Table 2). Most of the patients belonged to Sindhi ethnicity, 27(30%) followed by Punjabi 21 (23.3%) (Table 3). Almost half of the patients had no known co- morbid 47 (52.2%). Hypertension, DM and hepatitis B was observed in 29 (32.2%), 4 (4.4%) and 4 (4.4%) respectively (Table 4). Upper GI symptoms before transplant were also uncommon in these patients, only around one-fourth of subjects had history of dyspeptic symptoms prior to transplant i.e: 23 (25.6%). Sixty nine patients (76.7%) had no history of substance abuse while tobacco smoking was observed in 12 (13.3%) subjects followed by alcoholism in 6 (6.7%) (Table 1). In the study subjects, urea was ranged from 17-230 mg/dL with mean of 58.34 ± 39.30 mg/dl, while creatinine ranged from 0.72-6.08 mg/dL, with mean of 2.11 ± 1.11 mg/dL (Table 2). Renal transplant patients were receiving multiple immunosuppressant, all of them were on steroids (prednisolone) along with other agents. Azathioprine was used in 54 (60%), followed by cyclosporine in 35 (38.9%), tacrolimus in 27 (30%), MMF in 26 (28.9%) and everolimus in 8 (8.9%) patients. Gastritis (non H. pylori infection) seen in 78 (86.7%), duodenitis in 35 (38.9%) and H. pylori infection in 29 (32.2%) recipients as cause of dyspepsia (Table 1). No endoscopy related complication was observed in any patient. Analysis of factors leading to dyspepsia, revealed statistically significant association in patients having duodenitis on tacrolimus (p = 0.037) (Table 5), however, one tailed p-value was significant in patients having gastritis and BMI of more than 18.92 (p = 0.053). No significant association was found with other immunosuppressive agents in patients having H. Pylori, non H. Pylori gastritis, duodenitis or female gender.\nDemographic and clinical characteristics of study population (n=90)\nDescriptive statistics of continuous variables (n=90)\nEthnicity of study population (n=90)\nCo morbid of study Population (n=90)\nAnalysis of factors leading to dyspepsia: duodenitis (n=90)",
"Renal transplantation is the definitive management for patients suffering from of end stage renal disease (ESRD) [13]. Gastrointestinal disorders in this population are more common as compared to general population, which includes oral ulcers, esophagitis, peptic ulcer disease, diarrhea and dyspepsia [14]. Post renal transplant dyspepsia not only worsens the quality of life, but is also associated with increased risk of graft failure, resulting in high morbidity and mortality [6]. Among the demographic characteristics, the gender distribution in our study was similar in comparison with other studies. Our study reported that around 70% of the patients were male, consistent with multiple other studies in which around 60-70% of the patients were male [6–8, 15–17]. There is no statistical significance seen in our study as well as in previous studies. Mean age of our study group was 35.82 ± 10.04 years, which was lower than that seen in other regions of world. Around 35% of the patients, who underwent renal transplant in USA belonged to the age group of 45-60 years [6], similar characteristics were seen by Ozgur et al [8] and Savas [15]. Our contrasting results could possibly be explained by the fact that we belong to a developing country in the stone belt and are unable to identify early detection of renal diseases and implement strategies to delay the progression of renal failure. These factors probably play an important role in the development of dyspepsia in renal transplanted patients and ESRD at younger age in our country [13]. Most of the patients in our study had normal BMI with mean of 19.67 ± 3.65, which is in contrast to the study done at USA on renal transplanted subjects, [6] they had mean BMI of 44 ± 12.5 kg/m2. This huge difference of BMI is likely because our patients belonged to a third world country with limited resources. Our transplanted patients follow stringent criteria of weight maintenance, as advised by nutritionist and most of them are compliant.\nIn our study, no statistically significant association of dyspepsia was found with ethnicity and obesity, which was different from the results of Neri et al [6] that found ethnicity as important factor and concluded that whites, Hispanic and African-Americans who are obese and have greater BMI were more prone to develop GERD and dyspepsia. Most of our patients in our study underwent renal transplant were non-diabetics, hypertension was the most common co morbid condition, this is probably because we live in the region of stone belt and likely this would be the common cause of ESRD in our transplanted patients [13]. Neri et al also have same results, found HTN to be the most common cause of renal failure, followed by DM [6]. In our region most of the diabetic patients are unable to avail this treatment option because of multiple co morbidities and complications related with DM. In this study about one-fourth of subjects that is, 23 (25.6%), had dyspeptic symptoms before transplant; however no significance of this observation can be elicited because there was no control group. It was seen by Neri et al that those patients who had upper GI symptoms, prior to transplant, were more prone to experience it post-transplant as well [6]. Mean urea and creatinine in our study was 58.34 ± 39.3 mg/dL and 2.11 ± 1.1 mg/dL respectively, this was higher than that of Saudi Arabia's population, have mean creatinine of 1.6 ± 1.1 mg/dL, but this had no association with factors leading to dyspepsia [7]. Deranged urea and creatinine, has been considered as one of the known cause of dyspepsia in these patients [6].\nThe most common factor leading to dyspepsia in this study was gastritis seen in 78 (86.7%), followed by duodenitis and H. Pylori infection. A study done at Istanbul also finds gastritis as the most common finding on endoscopy, seen in 55 (85.9%) patients [15]. Abdur Rehman and Al Qurain's observation was contrasting and found that GERD was more common in renal transplanted patients in comparison with gastritis [7]. In our study frequency of H. Pylori was less as compare to the other studies. Thirty two percent of our patients were found to have H. pylori infection in comparison to Ozgur et al and Hurby's, who reported it this infection in 70 and 62% respectively [8, 17]. Another study done at the same institute as ours (SIUT) reported H. pylori infection in 11 patients out of 119 examined gastric biopsies, showing decrease prevalence of this infection in our renal transplanted population [18]. A possible explanation of the differences of these results is yet to be known. This study has highlighted the statistical significance of tacrolimus with duodenitis. A case report has been published by Suzuki et al he found severe duodenitis along with graft dysfunction following renal transplantation, the patient had various immunosuppressants to avoid rejection including tacrolimus [19]. To the best of our knowledge, previously no such association has been reported in a study. Although this study has tried to identify various factors leading to dyspepsia, there is still need for further research work in this regard. It was a single center study, with small sample size. Therefore, further studies are needed to assess other factors leading to dyspepsia in renal transplant recipients.",
"The common cause of dyspepsia in our renal transplanted patients was gastritis (non H. pylori associated) followed by duodenitis and H. pylori infection. Use of immunosuppression tacrolimus was associated with duodenitis.\n What is known about this topic Dyspepsia is common in uremic patients;\nRenal transplant recipients are prone to have dyspepsia because of immunosuppressive agents.\nDyspepsia is common in uremic patients;\nRenal transplant recipients are prone to have dyspepsia because of immunosuppressive agents.\n What this study adds Most common cause of dyspepsia is non H.pylori gastritis;\nDuodenitis is associated with the use of tacrolimus as immunosuppressant.\nMost common cause of dyspepsia is non H.pylori gastritis;\nDuodenitis is associated with the use of tacrolimus as immunosuppressant.",
"Dyspepsia is common in uremic patients;\nRenal transplant recipients are prone to have dyspepsia because of immunosuppressive agents.",
"Most common cause of dyspepsia is non H.pylori gastritis;\nDuodenitis is associated with the use of tacrolimus as immunosuppressant.",
"The authors declare no competing interests."
] |
[
"intro",
"methods",
"results",
"discussion",
"conclusions",
null,
null,
null
] |
[
"Renal transplantation",
"dyspepsia",
"tacrolimus"
] |
Introduction:
Dyspepsia and gastroesophageal reflux disease (GERD) are the most common diseases encountered by the gastroenterologist, its prevalence in general population is about 10-20% in the Western world and lower in Asia. Six percent of the patients have heartburn; around 38% had dyspepsia while regurgitation was reported in 16% [1, 2]. GERD develops when increased acidic gastric secretion refluxes into the esophagus [3, 4]. Gastrointestinal (GI) symptoms are commonly experienced in solid organ transplant recipients and can effects any part of the GI tract [5]. After three years of renal transplant, about 23% of the patients have GERD, reflux esophagitis (RE) and dyspepsia in 20%, 5% and 6%, respectively [6]. Renal transplantation, chronic renal disease, immunosuppressive therapy, H. pylori infection, female gender, pre transplant upper gastrointestinal disease, older age, obesity, Caucasian and African-American race, are considered as potential risk factors for the development of GERD and dyspepsia [6–9]. Neri et al concluded that female gender (39.9%), hypertension (22.5%) and renal failure secondary to type I DM (12.1%) have increased risk of dyspepsia [6]. Raised creatinine is one of the risk factor of dyspepsia in post renal transplant era [7, 8]. In normal population increased age and smoking are risk factors of dyspepsia [9]. Darji et al found that 50% of patients had considerable improvement in dyspeptic symptoms after substituting mycophenolate mofetil (MMF) with its enteric coated formulation [10]. Besides Webster et al concluded that in comparison to cyclosporine, the use of tacrolimus was associated with more risk of dyspepsia, post-transplant diabetes mellitus (PTDM), diarrhea, tremor and headache [11] Ozgur et al inducted 54 renal transplanted patients with dyspepsia out of which H. pylori infection was seen in 70%, gastritis in 65%, duodenitis in 13% and peptic ulcer in 4% [8]. Khidmat et al found duodenal ulcer, erosive gastritis and H. pylori infection in 8%, 20.5% and 40% respectively in post renal transplant [12]. Neri et al proposed that renal transplanted patients having GERD, dyspepsia or reflux esophagitis are at increased risk of graft failure and death [6]. Kim et al reported severe complications in around 10% resulting in graft loss and death [5]. The rationale of this study is to determine factors leading to dyspepsia in renal transplant recipients, as no local study have been done so far for evaluation of dyspepsia in these patients. Evaluation of factors leading to dyspepsia, its early diagnosis and prompt treatment is crucial in these patients as it can lead to graft failure and increased morbidity and mortality.
Methods:
The study was descriptive cross sectional study, conducted in the department of Hepatogastroenterology, Sindh Institute of Urology and Transplantation (SIUT), Karachi from 1st June 2015 to 30th November 2015. Those patients who had dyspepsia3 for at least 6 weeks after 6 months of renal transplantation of both genders and aged 18 and above were included in the study. Following patients were excluded: those who had taken proton pump inhibitors at least two weeks before EGD, had congestive heart failure (i.e: ejection fraction is less than 40% on echocardiography), bleeding disorder: thrombocytopenia or coagulopathy (Platelets < 50000/μL or International normalized ratio (INR) > 1.5, hemodynamically unstable (B.P < 70/40 mmHg). Eight factors which lead to dyspepsia were taken into consideration in this study, factors were gender, hypertension, diabetes mellitus, obesity, immunosuppressive drugs, H.pylori gastritis, non H.pylori gastritis and duodenitis. Sample size was calculated by using open epi calculator prevalence of duodenitis with dyspeptic symptoms was found to be 13% [8] in renal transplant recipients, d = 7%, confidence interval = 95%, estimated sample size was at least n = 89. All the cases were subjected to endoscopic evaluation. Venous blood sample collected for laboratory parameters; urea, creatinine, complete blood count and prothrombin time. Esophagogastroduodenoscopy (EGD) was done in all enrolled patients as per indication, performed by researcher under supervision of supervisor using video endoscope (Olympus GIF-XP180). Two biopsies were taken from antrum and duodenum for histopathological examination and were sent to the histopathology department at SIUT, to rule out gastritis, duodenitis or H. pylori infection. Data was analyzed by using statistical software SPSS-20.0. Mean and standard deviation (SD) was calculated for continuous variables (age, BMI, duration of symptoms). Frequencies and percentages were evaluated for categorical variables (Gender, co-morbids ie DM and HTN, addictions, H.pylorigastritis, duodenitis, gastritis, immunosuppressants which includes steroids, azathioprine, everolimus, tacrolimus, cyclosporine, mycophenolate mofetil and obesity). Stratification with respect to age, addiction, ethnicity, duration since renal transplant and duration of symptoms was done. Post stratification Chi square test was applied. A p-value ≤ 0.05 was taken as significant.
Results:
Total 90 renal transplant recipients were enrolled in this study. Most of the patients were male i.e 64 (71.1%) (Table 1). Mean age was 35.82 ± 10.04 years (Range: 18-65 years) (Table 2). Most of the patients belonged to Sindhi ethnicity, 27(30%) followed by Punjabi 21 (23.3%) (Table 3). Almost half of the patients had no known co- morbid 47 (52.2%). Hypertension, DM and hepatitis B was observed in 29 (32.2%), 4 (4.4%) and 4 (4.4%) respectively (Table 4). Upper GI symptoms before transplant were also uncommon in these patients, only around one-fourth of subjects had history of dyspeptic symptoms prior to transplant i.e: 23 (25.6%). Sixty nine patients (76.7%) had no history of substance abuse while tobacco smoking was observed in 12 (13.3%) subjects followed by alcoholism in 6 (6.7%) (Table 1). In the study subjects, urea was ranged from 17-230 mg/dL with mean of 58.34 ± 39.30 mg/dl, while creatinine ranged from 0.72-6.08 mg/dL, with mean of 2.11 ± 1.11 mg/dL (Table 2). Renal transplant patients were receiving multiple immunosuppressant, all of them were on steroids (prednisolone) along with other agents. Azathioprine was used in 54 (60%), followed by cyclosporine in 35 (38.9%), tacrolimus in 27 (30%), MMF in 26 (28.9%) and everolimus in 8 (8.9%) patients. Gastritis (non H. pylori infection) seen in 78 (86.7%), duodenitis in 35 (38.9%) and H. pylori infection in 29 (32.2%) recipients as cause of dyspepsia (Table 1). No endoscopy related complication was observed in any patient. Analysis of factors leading to dyspepsia, revealed statistically significant association in patients having duodenitis on tacrolimus (p = 0.037) (Table 5), however, one tailed p-value was significant in patients having gastritis and BMI of more than 18.92 (p = 0.053). No significant association was found with other immunosuppressive agents in patients having H. Pylori, non H. Pylori gastritis, duodenitis or female gender.
Demographic and clinical characteristics of study population (n=90)
Descriptive statistics of continuous variables (n=90)
Ethnicity of study population (n=90)
Co morbid of study Population (n=90)
Analysis of factors leading to dyspepsia: duodenitis (n=90)
Discussion:
Renal transplantation is the definitive management for patients suffering from of end stage renal disease (ESRD) [13]. Gastrointestinal disorders in this population are more common as compared to general population, which includes oral ulcers, esophagitis, peptic ulcer disease, diarrhea and dyspepsia [14]. Post renal transplant dyspepsia not only worsens the quality of life, but is also associated with increased risk of graft failure, resulting in high morbidity and mortality [6]. Among the demographic characteristics, the gender distribution in our study was similar in comparison with other studies. Our study reported that around 70% of the patients were male, consistent with multiple other studies in which around 60-70% of the patients were male [6–8, 15–17]. There is no statistical significance seen in our study as well as in previous studies. Mean age of our study group was 35.82 ± 10.04 years, which was lower than that seen in other regions of world. Around 35% of the patients, who underwent renal transplant in USA belonged to the age group of 45-60 years [6], similar characteristics were seen by Ozgur et al [8] and Savas [15]. Our contrasting results could possibly be explained by the fact that we belong to a developing country in the stone belt and are unable to identify early detection of renal diseases and implement strategies to delay the progression of renal failure. These factors probably play an important role in the development of dyspepsia in renal transplanted patients and ESRD at younger age in our country [13]. Most of the patients in our study had normal BMI with mean of 19.67 ± 3.65, which is in contrast to the study done at USA on renal transplanted subjects, [6] they had mean BMI of 44 ± 12.5 kg/m2. This huge difference of BMI is likely because our patients belonged to a third world country with limited resources. Our transplanted patients follow stringent criteria of weight maintenance, as advised by nutritionist and most of them are compliant.
In our study, no statistically significant association of dyspepsia was found with ethnicity and obesity, which was different from the results of Neri et al [6] that found ethnicity as important factor and concluded that whites, Hispanic and African-Americans who are obese and have greater BMI were more prone to develop GERD and dyspepsia. Most of our patients in our study underwent renal transplant were non-diabetics, hypertension was the most common co morbid condition, this is probably because we live in the region of stone belt and likely this would be the common cause of ESRD in our transplanted patients [13]. Neri et al also have same results, found HTN to be the most common cause of renal failure, followed by DM [6]. In our region most of the diabetic patients are unable to avail this treatment option because of multiple co morbidities and complications related with DM. In this study about one-fourth of subjects that is, 23 (25.6%), had dyspeptic symptoms before transplant; however no significance of this observation can be elicited because there was no control group. It was seen by Neri et al that those patients who had upper GI symptoms, prior to transplant, were more prone to experience it post-transplant as well [6]. Mean urea and creatinine in our study was 58.34 ± 39.3 mg/dL and 2.11 ± 1.1 mg/dL respectively, this was higher than that of Saudi Arabia's population, have mean creatinine of 1.6 ± 1.1 mg/dL, but this had no association with factors leading to dyspepsia [7]. Deranged urea and creatinine, has been considered as one of the known cause of dyspepsia in these patients [6].
The most common factor leading to dyspepsia in this study was gastritis seen in 78 (86.7%), followed by duodenitis and H. Pylori infection. A study done at Istanbul also finds gastritis as the most common finding on endoscopy, seen in 55 (85.9%) patients [15]. Abdur Rehman and Al Qurain's observation was contrasting and found that GERD was more common in renal transplanted patients in comparison with gastritis [7]. In our study frequency of H. Pylori was less as compare to the other studies. Thirty two percent of our patients were found to have H. pylori infection in comparison to Ozgur et al and Hurby's, who reported it this infection in 70 and 62% respectively [8, 17]. Another study done at the same institute as ours (SIUT) reported H. pylori infection in 11 patients out of 119 examined gastric biopsies, showing decrease prevalence of this infection in our renal transplanted population [18]. A possible explanation of the differences of these results is yet to be known. This study has highlighted the statistical significance of tacrolimus with duodenitis. A case report has been published by Suzuki et al he found severe duodenitis along with graft dysfunction following renal transplantation, the patient had various immunosuppressants to avoid rejection including tacrolimus [19]. To the best of our knowledge, previously no such association has been reported in a study. Although this study has tried to identify various factors leading to dyspepsia, there is still need for further research work in this regard. It was a single center study, with small sample size. Therefore, further studies are needed to assess other factors leading to dyspepsia in renal transplant recipients.
Conclusion:
The common cause of dyspepsia in our renal transplanted patients was gastritis (non H. pylori associated) followed by duodenitis and H. pylori infection. Use of immunosuppression tacrolimus was associated with duodenitis.
What is known about this topic Dyspepsia is common in uremic patients;
Renal transplant recipients are prone to have dyspepsia because of immunosuppressive agents.
Dyspepsia is common in uremic patients;
Renal transplant recipients are prone to have dyspepsia because of immunosuppressive agents.
What this study adds Most common cause of dyspepsia is non H.pylori gastritis;
Duodenitis is associated with the use of tacrolimus as immunosuppressant.
Most common cause of dyspepsia is non H.pylori gastritis;
Duodenitis is associated with the use of tacrolimus as immunosuppressant.
What is known about this topic:
Dyspepsia is common in uremic patients;
Renal transplant recipients are prone to have dyspepsia because of immunosuppressive agents.
What this study adds:
Most common cause of dyspepsia is non H.pylori gastritis;
Duodenitis is associated with the use of tacrolimus as immunosuppressant.
Competing interests:
The authors declare no competing interests.
|
Background:
Renal transplantation is the definitive treatment for end stage renal disease. Patients subjected to transplantation require lifelong immunosuppression and are prone to several gastrointestinal disorders. Dyspepsia is a common disorder in these patients. The objective of this study was to determine factors leading to dyspepsia in renal (kidney) transplant recipients.
Methods:
It was a cross sectional study conducted at department of hepatogastroenterology and transplant sciences, SIUT Karachi, from 1-6-15 to 1-12-15 for six months. All renal transplanted patients having dyspeptic symptoms for more than 6 weeks. EGD was performed, biopsy specimens obtained from antrum and duodenum, these were sent for histopathological examination. Frequency and percentages were obtained for categorical variables, mean ± SD was calculated for continuous variables. Chi square test was used for categorical variable and student t-test for continuous variables.
Results:
Ninety patients were included in the study out of which 64 (71.1%) were males, mean age was 35.82 ± 10.04 years (range: 18-65 years). Gastritis (non H.pylori associated) in 78 (78.6%), duodenitis in 35 (38.9%) and H. pylori infection in 29 (32.2%), renal transplant recipients. Most of the patients belonged to Sindhi ethnicity, 27 (30%), followed by Punjabi. Hypertension was the most common co-morbid condition in our patients found in 29 (32.2%), while most of them don't have any co morbid condition. Duodenitis was found to be associated with tacrolimus use (p = 0.037).
Conclusions:
Gastritis is the most common factor accountable for this symptoms, followed by duodenitis and H. Pylori. Patients taking tacrolimus as immunosuppressant are more prone to develop duodenitis.
|
Introduction:
Dyspepsia and gastroesophageal reflux disease (GERD) are the most common diseases encountered by the gastroenterologist, its prevalence in general population is about 10-20% in the Western world and lower in Asia. Six percent of the patients have heartburn; around 38% had dyspepsia while regurgitation was reported in 16% [1, 2]. GERD develops when increased acidic gastric secretion refluxes into the esophagus [3, 4]. Gastrointestinal (GI) symptoms are commonly experienced in solid organ transplant recipients and can effects any part of the GI tract [5]. After three years of renal transplant, about 23% of the patients have GERD, reflux esophagitis (RE) and dyspepsia in 20%, 5% and 6%, respectively [6]. Renal transplantation, chronic renal disease, immunosuppressive therapy, H. pylori infection, female gender, pre transplant upper gastrointestinal disease, older age, obesity, Caucasian and African-American race, are considered as potential risk factors for the development of GERD and dyspepsia [6–9]. Neri et al concluded that female gender (39.9%), hypertension (22.5%) and renal failure secondary to type I DM (12.1%) have increased risk of dyspepsia [6]. Raised creatinine is one of the risk factor of dyspepsia in post renal transplant era [7, 8]. In normal population increased age and smoking are risk factors of dyspepsia [9]. Darji et al found that 50% of patients had considerable improvement in dyspeptic symptoms after substituting mycophenolate mofetil (MMF) with its enteric coated formulation [10]. Besides Webster et al concluded that in comparison to cyclosporine, the use of tacrolimus was associated with more risk of dyspepsia, post-transplant diabetes mellitus (PTDM), diarrhea, tremor and headache [11] Ozgur et al inducted 54 renal transplanted patients with dyspepsia out of which H. pylori infection was seen in 70%, gastritis in 65%, duodenitis in 13% and peptic ulcer in 4% [8]. Khidmat et al found duodenal ulcer, erosive gastritis and H. pylori infection in 8%, 20.5% and 40% respectively in post renal transplant [12]. Neri et al proposed that renal transplanted patients having GERD, dyspepsia or reflux esophagitis are at increased risk of graft failure and death [6]. Kim et al reported severe complications in around 10% resulting in graft loss and death [5]. The rationale of this study is to determine factors leading to dyspepsia in renal transplant recipients, as no local study have been done so far for evaluation of dyspepsia in these patients. Evaluation of factors leading to dyspepsia, its early diagnosis and prompt treatment is crucial in these patients as it can lead to graft failure and increased morbidity and mortality.
Conclusion:
The common cause of dyspepsia in our renal transplanted patients was gastritis (non H. pylori associated) followed by duodenitis and H. pylori infection. Use of immunosuppression tacrolimus was associated with duodenitis.
What is known about this topic Dyspepsia is common in uremic patients;
Renal transplant recipients are prone to have dyspepsia because of immunosuppressive agents.
Dyspepsia is common in uremic patients;
Renal transplant recipients are prone to have dyspepsia because of immunosuppressive agents.
What this study adds Most common cause of dyspepsia is non H.pylori gastritis;
Duodenitis is associated with the use of tacrolimus as immunosuppressant.
Most common cause of dyspepsia is non H.pylori gastritis;
Duodenitis is associated with the use of tacrolimus as immunosuppressant.
|
Background:
Renal transplantation is the definitive treatment for end stage renal disease. Patients subjected to transplantation require lifelong immunosuppression and are prone to several gastrointestinal disorders. Dyspepsia is a common disorder in these patients. The objective of this study was to determine factors leading to dyspepsia in renal (kidney) transplant recipients.
Methods:
It was a cross sectional study conducted at department of hepatogastroenterology and transplant sciences, SIUT Karachi, from 1-6-15 to 1-12-15 for six months. All renal transplanted patients having dyspeptic symptoms for more than 6 weeks. EGD was performed, biopsy specimens obtained from antrum and duodenum, these were sent for histopathological examination. Frequency and percentages were obtained for categorical variables, mean ± SD was calculated for continuous variables. Chi square test was used for categorical variable and student t-test for continuous variables.
Results:
Ninety patients were included in the study out of which 64 (71.1%) were males, mean age was 35.82 ± 10.04 years (range: 18-65 years). Gastritis (non H.pylori associated) in 78 (78.6%), duodenitis in 35 (38.9%) and H. pylori infection in 29 (32.2%), renal transplant recipients. Most of the patients belonged to Sindhi ethnicity, 27 (30%), followed by Punjabi. Hypertension was the most common co-morbid condition in our patients found in 29 (32.2%), while most of them don't have any co morbid condition. Duodenitis was found to be associated with tacrolimus use (p = 0.037).
Conclusions:
Gastritis is the most common factor accountable for this symptoms, followed by duodenitis and H. Pylori. Patients taking tacrolimus as immunosuppressant are more prone to develop duodenitis.
| 2,703 | 337 | 8 |
[
"patients",
"dyspepsia",
"renal",
"study",
"transplant",
"pylori",
"duodenitis",
"gastritis",
"renal transplant",
"common"
] |
[
"test",
"test"
] |
[CONTENT] Renal transplantation | dyspepsia | tacrolimus [SUMMARY]
|
[CONTENT] Renal transplantation | dyspepsia | tacrolimus [SUMMARY]
|
[CONTENT] Renal transplantation | dyspepsia | tacrolimus [SUMMARY]
|
[CONTENT] Renal transplantation | dyspepsia | tacrolimus [SUMMARY]
|
[CONTENT] Renal transplantation | dyspepsia | tacrolimus [SUMMARY]
|
[CONTENT] Renal transplantation | dyspepsia | tacrolimus [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Cross-Sectional Studies | Duodenitis | Dyspepsia | Female | Gastritis | Helicobacter Infections | Helicobacter pylori | Humans | Immunosuppressive Agents | Kidney Failure, Chronic | Kidney Transplantation | Male | Middle Aged | Pakistan | Risk Factors | Tacrolimus | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Cross-Sectional Studies | Duodenitis | Dyspepsia | Female | Gastritis | Helicobacter Infections | Helicobacter pylori | Humans | Immunosuppressive Agents | Kidney Failure, Chronic | Kidney Transplantation | Male | Middle Aged | Pakistan | Risk Factors | Tacrolimus | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Cross-Sectional Studies | Duodenitis | Dyspepsia | Female | Gastritis | Helicobacter Infections | Helicobacter pylori | Humans | Immunosuppressive Agents | Kidney Failure, Chronic | Kidney Transplantation | Male | Middle Aged | Pakistan | Risk Factors | Tacrolimus | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Cross-Sectional Studies | Duodenitis | Dyspepsia | Female | Gastritis | Helicobacter Infections | Helicobacter pylori | Humans | Immunosuppressive Agents | Kidney Failure, Chronic | Kidney Transplantation | Male | Middle Aged | Pakistan | Risk Factors | Tacrolimus | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Cross-Sectional Studies | Duodenitis | Dyspepsia | Female | Gastritis | Helicobacter Infections | Helicobacter pylori | Humans | Immunosuppressive Agents | Kidney Failure, Chronic | Kidney Transplantation | Male | Middle Aged | Pakistan | Risk Factors | Tacrolimus | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Cross-Sectional Studies | Duodenitis | Dyspepsia | Female | Gastritis | Helicobacter Infections | Helicobacter pylori | Humans | Immunosuppressive Agents | Kidney Failure, Chronic | Kidney Transplantation | Male | Middle Aged | Pakistan | Risk Factors | Tacrolimus | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] patients | dyspepsia | renal | study | transplant | pylori | duodenitis | gastritis | renal transplant | common [SUMMARY]
|
[CONTENT] patients | dyspepsia | renal | study | transplant | pylori | duodenitis | gastritis | renal transplant | common [SUMMARY]
|
[CONTENT] patients | dyspepsia | renal | study | transplant | pylori | duodenitis | gastritis | renal transplant | common [SUMMARY]
|
[CONTENT] patients | dyspepsia | renal | study | transplant | pylori | duodenitis | gastritis | renal transplant | common [SUMMARY]
|
[CONTENT] patients | dyspepsia | renal | study | transplant | pylori | duodenitis | gastritis | renal transplant | common [SUMMARY]
|
[CONTENT] patients | dyspepsia | renal | study | transplant | pylori | duodenitis | gastritis | renal transplant | common [SUMMARY]
|
[CONTENT] dyspepsia | risk | renal | gerd | increased | patients | transplant | reflux | factors | 20 [SUMMARY]
|
[CONTENT] taken | duration | sample | study | stratification | blood | egd | duration symptoms | calculated | department [SUMMARY]
|
[CONTENT] table | 90 | patients | mg | dl | mg dl | population 90 | study population | observed | study population 90 [SUMMARY]
|
[CONTENT] dyspepsia | common | associated | common cause dyspepsia | common cause | use | duodenitis | pylori | cause dyspepsia | non pylori [SUMMARY]
|
[CONTENT] dyspepsia | patients | renal | common | study | transplant | pylori | duodenitis | gastritis | renal transplant [SUMMARY]
|
[CONTENT] dyspepsia | patients | renal | common | study | transplant | pylori | duodenitis | gastritis | renal transplant [SUMMARY]
|
[CONTENT] ||| ||| Dyspepsia ||| [SUMMARY]
|
[CONTENT] SIUT Karachi | 1-6-15 | six months ||| more than 6 weeks ||| EGD ||| ||| [SUMMARY]
|
[CONTENT] Ninety | 64 | 71.1% | 35.82 | 10.04 years | 18-65 years ||| 78 | 78.6% | 35 | 38.9% | 29 | 32.2% ||| Sindhi | 27 | 30% ||| 29 | 32.2% ||| 0.037 [SUMMARY]
|
[CONTENT] Gastritis | H. Pylori ||| [SUMMARY]
|
[CONTENT] ||| ||| Dyspepsia ||| ||| SIUT Karachi | 1-6-15 | six months ||| more than 6 weeks ||| EGD ||| ||| ||| Ninety | 64 | 71.1% | 35.82 | 10.04 years | 18-65 years ||| 78 | 78.6% | 35 | 38.9% | 29 | 32.2% ||| Sindhi | 27 | 30% ||| 29 | 32.2% ||| 0.037 ||| Gastritis | H. Pylori ||| [SUMMARY]
|
[CONTENT] ||| ||| Dyspepsia ||| ||| SIUT Karachi | 1-6-15 | six months ||| more than 6 weeks ||| EGD ||| ||| ||| Ninety | 64 | 71.1% | 35.82 | 10.04 years | 18-65 years ||| 78 | 78.6% | 35 | 38.9% | 29 | 32.2% ||| Sindhi | 27 | 30% ||| 29 | 32.2% ||| 0.037 ||| Gastritis | H. Pylori ||| [SUMMARY]
|
||||||
Repellent and insecticidal efficacy of a new combination of fipronil and permethrin against the main vector of canine leishmaniosis in Europe (Phlebotomus perniciosus).
|
25622922
|
Two successive laboratory experiments (A and B) were conducted to confirm the efficacy of a new fipronil and permethrin combination to repel and kill Phlebotomus perniciosus sandflies when applied once topically on dogs.
|
BACKGROUND
|
Due to the difficulty to get enough available dogs and sandflies in one run, the study was divided into 2 experiments which had exactly the same design, and were conducted at the same place, with the same technicians. They compared dogs treated with a combination containing 67.6 mg/mL fipronil + 504.8 mg/mL permethrin (Frontect/Frontline Tri-Act, Merial) to untreated dogs. The treatments were applied topically once on Day 0. Sandfly exposures were performed on Days 1, 7, 14, 21 and 29 with 80 P. perniciosus female sandflies. After 60 min, sandflies were assessed for vitality and engorgement status. Live sandflies were kept in an insectary and observed for mortality counts 4 h after the exposure period ended.
|
METHODS
|
Percent sandfly repellency on treated dogs was 98.2, 98.5, 99.2, 90.9 and 90.3%, for Days 1, 7, 14, 21, and 29, respectively. There was a significant difference (p ≤ 0.05) between the treated and control groups in both experiments and for the pooled data on every assessment day. Insecticidal efficacy on treated dogs at 4 h post-exposure on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9%, respectively. There was a significant difference between the treated and control groups for live sandflies observed at 4 h post-exposure for all assessment days (p < 0.05).
|
RESULTS
|
A single topical administration of a new combination of fipronil and permethrin demonstrated a significant repellent effect (i.e., > 80%) against P. perniciosus which lasted for 29 days after application. The repellent effect was accompanied by a significant insecticidal effect on sandflies. The results suggest that in endemic areas, the application of the fipronil-permethrin combination could be integrated into canine leishmaniosis prevention program.
|
CONCLUSIONS
|
[
"Administration, Topical",
"Animals",
"Dog Diseases",
"Dogs",
"Female",
"Insect Control",
"Insect Repellents",
"Insect Vectors",
"Insecticides",
"Leishmania",
"Leishmaniasis",
"Male",
"Permethrin",
"Phlebotomus",
"Pyrazoles"
] |
4316642
|
Background
|
Leishmaniosis is a serious parasitic disease caused by flagellated protozoa of the genus Leishmania. The protozoa are transmitted to animals and humans by haematophagous female sandflies of the genus Phlebotomus in the Old World and Lutzomyia in the New World. Although certain wild mammals may be involved in the transmission of leishmaniosis, domestic dogs appear to be the principal reservoir of Leishmania infantum throughout the world [1]. In Europe, there is a tendency for leishmaniosis but also other canine vector-borne diseases to have an increased distribution [2]. This is related to several factors, including climate and social changes [3].
The prevention of canine leishmaniosis in dogs is based on several measures, including anti-Leishmania vaccines and methods for protecting healthy dogs against sandfly bites [4,5]. The studies reported here were conducted to assess the repellent and insecticidal efficacies of a new spot on topical combination of fipronil and permethrin (Frontect®/Frontline Tri-Act®, Merial) against the main vector of canine leishmaniosis in Europe (Phlebotomus perniciosus). Such a combination is intended to provide both repellent and insecticidal-acaricidal effects against several ectoparasites of dogs [6,7].
| null | null |
Results
|
No health abnormalities related to treatment were observed throughout the studies, including during hourly observations conducted for 4 h immediately after treatment.
In Exp. B, Dog 6277 in the control group vomited on Day 0 and then was normal through Day 7. The dog did not eat well from Days 8 to 14 and was not considered to be suitable for anesthesia and subsequent exposure to sandflies on Day 14. An intussusception was observed at ultrasonography on Day 15 and the dog was removed from the study, therefore the control group of Exp. B moved from 8 to 7 dogs on Days 14, 21 and 29.
Untreated control dogs had high numbers of engorged sandflies at the end of the exposure period at all time-points with means between 54.6 and 68.2 out of 80 (Table 1). With at least 68% of feeding behaviour on control dogs, it showed a robust sandfly strain population. The survival rate was also very good with at least 73.1% sandflies surviving until 4 h after the end of the exposure times in the control group (Table 2).Table 1
Percent repellency of
Phlebotomus perniciosus
in dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on geometric means
Number of engorged sandflies
Exposure day experiment (A/B)
Dogs (n)
Untreated dogs
Dogs (n)
Treated dogs
Repellency (%)
1 (A)560.053.494.4*
98.2%
*
1 (B)864.480.499.4*7 (A)560.553.294.7*
98.5%
*
7 (B)861.380.299.7*14 (A)559.350.499.3*
99.2%
*
14 (B)763.180.699.1*21 (A)554.857.386.6*
90.9%
*
21 (B)764.784.692.9*29 (A)554.651.597.3*
90.3%
*
29 (B)768.2812.681.5**Significant difference between the population means of the treated and control groups (p < 0.05).Right column gives the % repellency based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of repellency calculated for each experiment.Table 2
Percent insecticidal efficacy against
Phlebotomus perniciosus
observed at 4 h in dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on Geometric means
Number of live sandflies
Exposure day experiment (A/B)
Dogs (n)
Untreated dogs
Dogs (n)
Treated dogs
Efficacy (%)
1 (A)574.350.399.6*
98.7%
*
1 (B)873.581.697.9*7 (A)575.250100*
99.7%
*
7 (B)872.180.499.5*14 (A)574.555.792.3*
96.8%
*
14 (B)772.181.298.4*21 (A)575.2533.954.9*
93.4%
*
21 (B)774.081.098.7*29 (A)574.257.090.6*
78.9%
*
29 (B)775.1825.765.7**Significant difference between the population means of the treated and control groups (p < 0.05).Right column gives the % insecticidal activity based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of insecticidal activity calculated for each experiment.
Percent repellency of
Phlebotomus perniciosus
in dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on geometric means
*Significant difference between the population means of the treated and control groups (p < 0.05).
Right column gives the % repellency based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of repellency calculated for each experiment.
Percent insecticidal efficacy against
Phlebotomus perniciosus
observed at 4 h in dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on Geometric means
*Significant difference between the population means of the treated and control groups (p < 0.05).
Right column gives the % insecticidal activity based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of insecticidal activity calculated for each experiment.
Repellency Treated dogs had significantly fewer fed sandflies at the end of the exposure period than untreated control dogs for all study days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008). The percent sandfly repellency after 60 min exposure was 98.2, 98.5, 99.2, 90.9 and 90.3% (Table 1), for Days 1, 7, 14, 21, and 29, respectively.
Treated dogs had significantly fewer fed sandflies at the end of the exposure period than untreated control dogs for all study days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008). The percent sandfly repellency after 60 min exposure was 98.2, 98.5, 99.2, 90.9 and 90.3% (Table 1), for Days 1, 7, 14, 21, and 29, respectively.
Insecticidal efficacy Treated dogs had significantly more dead sandflies at 4 h post-exposure than untreated control dogs for all challenge days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008).
Percent insecticidal efficacy of the treated group at 4 h compared to the control group on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9% (Table 2), respectively. In Exp. A, the insecticidal efficacy was 54.9% on Day 21 and got back to 90.6% on Day 29.
Treated dogs had significantly more dead sandflies at 4 h post-exposure than untreated control dogs for all challenge days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008).
Percent insecticidal efficacy of the treated group at 4 h compared to the control group on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9% (Table 2), respectively. In Exp. A, the insecticidal efficacy was 54.9% on Day 21 and got back to 90.6% on Day 29.
|
Conclusions
|
The new combination of fipronil and permethrin demonstrated a significant repellent effect against P. perniciosus bites as soon as it was applied on the dogs, and its repellent efficacy lasted 4 weeks. The results suggest that in endemic areas, the regular application of the new combination could be integrated in a canine leishmaniosis prevention program.
|
[
"Animals",
"Sandfly exposures",
"Allocation and treatment",
"Data analysis",
"Percent sandfly repellency",
"Percent insecticidal efficacy",
"Repellency",
"Insecticidal efficacy"
] |
[
"Adult Beagle dogs were used in the experiements and had not been exposed to ectoparasiticides having a monthly efficacy or shorter 3 months prior to treatment, and were never exposed to long lasting ectoparasiticides (Experiment A (Exp. A): 6 males and 4 females, 10.7 to 11.4 months of age, weighing 6.9–9.2 kg; Experiment B (Exp. B): 8 males and 8 females, 14.1 to 14.9 months of age, weighing 8.1–10.4 kg). The dogs were housed individually in stainless steel cages with an exercise area, under controlled environmental conditions, fed with a commercial dry dog food ration, with water available ad libitum. No concurrent medication was given during the study. They were managed similarly and with due regard for their well-being. Animals were handled in compliance with Merial Animal Care and Use Guidelines and in compliance with the French regulatory requirements and ethical committee of Toulouse Veterinary School. The dogs were acclimated to the study conditions for at least 11 days prior to treatment and were observed for general health conditions throughout the study.",
"Sandfly exposures were performed one week prior to treatment for allocation purposes, and after treatment on Days 1, 7, 14, 21 and 29. Prior to each exposure, animals were anesthetized with intra-muscular injections of 0.02 to 0.03 mg/kg dexmedetomidine (Dextomidor®, Pfizer), 3.8 to 6.8 mg/kg ketamine (Imalgene® 1000, Merial) and 0.46 to 0.65 mg/kg of Diazepam (Valium®, Roche); placed individually into a sandfly proof exposure cage; and exposed to 80 P. perniciosus unfed female sandflies (strain originated from Lisbon, Portugal and maintained under laboratory conditions for 9 years). After approximately 60 min, live sandflies were removed from the exposure cage, counted and categorized as engorged (fed) or non-engorged (unfed). Dead sandflies remaining in the exposure cage or on the dog were counted and categorized as engorged or non-engorged. The dogs were then returned to their normal housing. Live sandflies recovered from each exposure (except pre-treatment) were maintained at appropriate environmental conditions for approximately 4 h after the exposure period ended, and dead phlebotomes were recovered and counted from each container.",
"To allocate the dogs, blocks were formed, based on descending pre-treatment counts of fed sandflies and 26 dogs were randomly allocated to the two treatment groups (10 for Exp. A and 16 for Exp. B). Dogs in Group 1 served as untreated control dogs. Dogs in Group 2 were treated on Day 0 with the topical combination of permethrin and fipronil. In Exp. A, 5 dogs were treated at the minimum recommended dose (0.1 mL/kg based on Day 0 body weight, corresponding to a dose of 6.73 mg/kg fipronil and 50.16 mg/kg permethrin) whereas in Exp. B, 8 dogs were treated at the recommended commercial dose (1.0 mL for dogs <10.0 kg, and 2.0 mL for dogs >10.0 to 20 kg, based on Day 0 body weight, delivering a minimum dose of 6.76 mg/kg fipronil and 50.48 mg/kg permethrin).\nThe dose was applied by parting the hair and applying the formulation directly onto the skin on the dorsal midline of the neck. The total volume was divided into two approximately equal portions. One fraction was applied between the base of the skull and the shoulder blades and the other fraction was applied at the front of the shoulder blades. The dogs were observed prior to treatment and hourly for 4 h following treatment administration.",
" Percent sandfly repellency The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.\nThe total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.\n Percent insecticidal efficacy All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.\nThe treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.\nAll sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.\nThe treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.",
"The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.",
"All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.\nThe treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.",
"Treated dogs had significantly fewer fed sandflies at the end of the exposure period than untreated control dogs for all study days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008). The percent sandfly repellency after 60 min exposure was 98.2, 98.5, 99.2, 90.9 and 90.3% (Table 1), for Days 1, 7, 14, 21, and 29, respectively.",
"Treated dogs had significantly more dead sandflies at 4 h post-exposure than untreated control dogs for all challenge days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008).\nPercent insecticidal efficacy of the treated group at 4 h compared to the control group on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9% (Table 2), respectively. In Exp. A, the insecticidal efficacy was 54.9% on Day 21 and got back to 90.6% on Day 29."
] |
[
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Animals",
"Sandfly exposures",
"Allocation and treatment",
"Data analysis",
"Percent sandfly repellency",
"Percent insecticidal efficacy",
"Results",
"Repellency",
"Insecticidal efficacy",
"Discussion",
"Conclusions"
] |
[
"Leishmaniosis is a serious parasitic disease caused by flagellated protozoa of the genus Leishmania. The protozoa are transmitted to animals and humans by haematophagous female sandflies of the genus Phlebotomus in the Old World and Lutzomyia in the New World. Although certain wild mammals may be involved in the transmission of leishmaniosis, domestic dogs appear to be the principal reservoir of Leishmania infantum throughout the world [1]. In Europe, there is a tendency for leishmaniosis but also other canine vector-borne diseases to have an increased distribution [2]. This is related to several factors, including climate and social changes [3].\nThe prevention of canine leishmaniosis in dogs is based on several measures, including anti-Leishmania vaccines and methods for protecting healthy dogs against sandfly bites [4,5]. The studies reported here were conducted to assess the repellent and insecticidal efficacies of a new spot on topical combination of fipronil and permethrin (Frontect®/Frontline Tri-Act®, Merial) against the main vector of canine leishmaniosis in Europe (Phlebotomus perniciosus). Such a combination is intended to provide both repellent and insecticidal-acaricidal effects against several ectoparasites of dogs [6,7].",
"Two successive experiments were conducted at the Ecole Nationale Vétérinaire de Toulouse. The 2 experiments were necessary due to the difficulty to include many dogs and to produce enough sandfly. Experiment A was also considered as a preliminary exploratory study whereas Experiment B was conducted according to Good Clinical Practices (GCP) as described in the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guideline 9 [8].\n Animals Adult Beagle dogs were used in the experiements and had not been exposed to ectoparasiticides having a monthly efficacy or shorter 3 months prior to treatment, and were never exposed to long lasting ectoparasiticides (Experiment A (Exp. A): 6 males and 4 females, 10.7 to 11.4 months of age, weighing 6.9–9.2 kg; Experiment B (Exp. B): 8 males and 8 females, 14.1 to 14.9 months of age, weighing 8.1–10.4 kg). The dogs were housed individually in stainless steel cages with an exercise area, under controlled environmental conditions, fed with a commercial dry dog food ration, with water available ad libitum. No concurrent medication was given during the study. They were managed similarly and with due regard for their well-being. Animals were handled in compliance with Merial Animal Care and Use Guidelines and in compliance with the French regulatory requirements and ethical committee of Toulouse Veterinary School. The dogs were acclimated to the study conditions for at least 11 days prior to treatment and were observed for general health conditions throughout the study.\nAdult Beagle dogs were used in the experiements and had not been exposed to ectoparasiticides having a monthly efficacy or shorter 3 months prior to treatment, and were never exposed to long lasting ectoparasiticides (Experiment A (Exp. A): 6 males and 4 females, 10.7 to 11.4 months of age, weighing 6.9–9.2 kg; Experiment B (Exp. B): 8 males and 8 females, 14.1 to 14.9 months of age, weighing 8.1–10.4 kg). The dogs were housed individually in stainless steel cages with an exercise area, under controlled environmental conditions, fed with a commercial dry dog food ration, with water available ad libitum. No concurrent medication was given during the study. They were managed similarly and with due regard for their well-being. Animals were handled in compliance with Merial Animal Care and Use Guidelines and in compliance with the French regulatory requirements and ethical committee of Toulouse Veterinary School. The dogs were acclimated to the study conditions for at least 11 days prior to treatment and were observed for general health conditions throughout the study.\n Sandfly exposures Sandfly exposures were performed one week prior to treatment for allocation purposes, and after treatment on Days 1, 7, 14, 21 and 29. Prior to each exposure, animals were anesthetized with intra-muscular injections of 0.02 to 0.03 mg/kg dexmedetomidine (Dextomidor®, Pfizer), 3.8 to 6.8 mg/kg ketamine (Imalgene® 1000, Merial) and 0.46 to 0.65 mg/kg of Diazepam (Valium®, Roche); placed individually into a sandfly proof exposure cage; and exposed to 80 P. perniciosus unfed female sandflies (strain originated from Lisbon, Portugal and maintained under laboratory conditions for 9 years). After approximately 60 min, live sandflies were removed from the exposure cage, counted and categorized as engorged (fed) or non-engorged (unfed). Dead sandflies remaining in the exposure cage or on the dog were counted and categorized as engorged or non-engorged. The dogs were then returned to their normal housing. Live sandflies recovered from each exposure (except pre-treatment) were maintained at appropriate environmental conditions for approximately 4 h after the exposure period ended, and dead phlebotomes were recovered and counted from each container.\nSandfly exposures were performed one week prior to treatment for allocation purposes, and after treatment on Days 1, 7, 14, 21 and 29. Prior to each exposure, animals were anesthetized with intra-muscular injections of 0.02 to 0.03 mg/kg dexmedetomidine (Dextomidor®, Pfizer), 3.8 to 6.8 mg/kg ketamine (Imalgene® 1000, Merial) and 0.46 to 0.65 mg/kg of Diazepam (Valium®, Roche); placed individually into a sandfly proof exposure cage; and exposed to 80 P. perniciosus unfed female sandflies (strain originated from Lisbon, Portugal and maintained under laboratory conditions for 9 years). After approximately 60 min, live sandflies were removed from the exposure cage, counted and categorized as engorged (fed) or non-engorged (unfed). Dead sandflies remaining in the exposure cage or on the dog were counted and categorized as engorged or non-engorged. The dogs were then returned to their normal housing. Live sandflies recovered from each exposure (except pre-treatment) were maintained at appropriate environmental conditions for approximately 4 h after the exposure period ended, and dead phlebotomes were recovered and counted from each container.\n Allocation and treatment To allocate the dogs, blocks were formed, based on descending pre-treatment counts of fed sandflies and 26 dogs were randomly allocated to the two treatment groups (10 for Exp. A and 16 for Exp. B). Dogs in Group 1 served as untreated control dogs. Dogs in Group 2 were treated on Day 0 with the topical combination of permethrin and fipronil. In Exp. A, 5 dogs were treated at the minimum recommended dose (0.1 mL/kg based on Day 0 body weight, corresponding to a dose of 6.73 mg/kg fipronil and 50.16 mg/kg permethrin) whereas in Exp. B, 8 dogs were treated at the recommended commercial dose (1.0 mL for dogs <10.0 kg, and 2.0 mL for dogs >10.0 to 20 kg, based on Day 0 body weight, delivering a minimum dose of 6.76 mg/kg fipronil and 50.48 mg/kg permethrin).\nThe dose was applied by parting the hair and applying the formulation directly onto the skin on the dorsal midline of the neck. The total volume was divided into two approximately equal portions. One fraction was applied between the base of the skull and the shoulder blades and the other fraction was applied at the front of the shoulder blades. The dogs were observed prior to treatment and hourly for 4 h following treatment administration.\nTo allocate the dogs, blocks were formed, based on descending pre-treatment counts of fed sandflies and 26 dogs were randomly allocated to the two treatment groups (10 for Exp. A and 16 for Exp. B). Dogs in Group 1 served as untreated control dogs. Dogs in Group 2 were treated on Day 0 with the topical combination of permethrin and fipronil. In Exp. A, 5 dogs were treated at the minimum recommended dose (0.1 mL/kg based on Day 0 body weight, corresponding to a dose of 6.73 mg/kg fipronil and 50.16 mg/kg permethrin) whereas in Exp. B, 8 dogs were treated at the recommended commercial dose (1.0 mL for dogs <10.0 kg, and 2.0 mL for dogs >10.0 to 20 kg, based on Day 0 body weight, delivering a minimum dose of 6.76 mg/kg fipronil and 50.48 mg/kg permethrin).\nThe dose was applied by parting the hair and applying the formulation directly onto the skin on the dorsal midline of the neck. The total volume was divided into two approximately equal portions. One fraction was applied between the base of the skull and the shoulder blades and the other fraction was applied at the front of the shoulder blades. The dogs were observed prior to treatment and hourly for 4 h following treatment administration.\n Data analysis Percent sandfly repellency The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.\nThe total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.\n Percent insecticidal efficacy All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.\nThe treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.\nAll sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.\nThe treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.\n Percent sandfly repellency The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.\nThe total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.\n Percent insecticidal efficacy All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.\nThe treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.\nAll sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.\nThe treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.",
"Adult Beagle dogs were used in the experiements and had not been exposed to ectoparasiticides having a monthly efficacy or shorter 3 months prior to treatment, and were never exposed to long lasting ectoparasiticides (Experiment A (Exp. A): 6 males and 4 females, 10.7 to 11.4 months of age, weighing 6.9–9.2 kg; Experiment B (Exp. B): 8 males and 8 females, 14.1 to 14.9 months of age, weighing 8.1–10.4 kg). The dogs were housed individually in stainless steel cages with an exercise area, under controlled environmental conditions, fed with a commercial dry dog food ration, with water available ad libitum. No concurrent medication was given during the study. They were managed similarly and with due regard for their well-being. Animals were handled in compliance with Merial Animal Care and Use Guidelines and in compliance with the French regulatory requirements and ethical committee of Toulouse Veterinary School. The dogs were acclimated to the study conditions for at least 11 days prior to treatment and were observed for general health conditions throughout the study.",
"Sandfly exposures were performed one week prior to treatment for allocation purposes, and after treatment on Days 1, 7, 14, 21 and 29. Prior to each exposure, animals were anesthetized with intra-muscular injections of 0.02 to 0.03 mg/kg dexmedetomidine (Dextomidor®, Pfizer), 3.8 to 6.8 mg/kg ketamine (Imalgene® 1000, Merial) and 0.46 to 0.65 mg/kg of Diazepam (Valium®, Roche); placed individually into a sandfly proof exposure cage; and exposed to 80 P. perniciosus unfed female sandflies (strain originated from Lisbon, Portugal and maintained under laboratory conditions for 9 years). After approximately 60 min, live sandflies were removed from the exposure cage, counted and categorized as engorged (fed) or non-engorged (unfed). Dead sandflies remaining in the exposure cage or on the dog were counted and categorized as engorged or non-engorged. The dogs were then returned to their normal housing. Live sandflies recovered from each exposure (except pre-treatment) were maintained at appropriate environmental conditions for approximately 4 h after the exposure period ended, and dead phlebotomes were recovered and counted from each container.",
"To allocate the dogs, blocks were formed, based on descending pre-treatment counts of fed sandflies and 26 dogs were randomly allocated to the two treatment groups (10 for Exp. A and 16 for Exp. B). Dogs in Group 1 served as untreated control dogs. Dogs in Group 2 were treated on Day 0 with the topical combination of permethrin and fipronil. In Exp. A, 5 dogs were treated at the minimum recommended dose (0.1 mL/kg based on Day 0 body weight, corresponding to a dose of 6.73 mg/kg fipronil and 50.16 mg/kg permethrin) whereas in Exp. B, 8 dogs were treated at the recommended commercial dose (1.0 mL for dogs <10.0 kg, and 2.0 mL for dogs >10.0 to 20 kg, based on Day 0 body weight, delivering a minimum dose of 6.76 mg/kg fipronil and 50.48 mg/kg permethrin).\nThe dose was applied by parting the hair and applying the formulation directly onto the skin on the dorsal midline of the neck. The total volume was divided into two approximately equal portions. One fraction was applied between the base of the skull and the shoulder blades and the other fraction was applied at the front of the shoulder blades. The dogs were observed prior to treatment and hourly for 4 h following treatment administration.",
" Percent sandfly repellency The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.\nThe total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.\n Percent insecticidal efficacy All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.\nThe treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.\nAll sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.\nThe treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.",
"The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.",
"All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.\nThe treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.",
"No health abnormalities related to treatment were observed throughout the studies, including during hourly observations conducted for 4 h immediately after treatment.\nIn Exp. B, Dog 6277 in the control group vomited on Day 0 and then was normal through Day 7. The dog did not eat well from Days 8 to 14 and was not considered to be suitable for anesthesia and subsequent exposure to sandflies on Day 14. An intussusception was observed at ultrasonography on Day 15 and the dog was removed from the study, therefore the control group of Exp. B moved from 8 to 7 dogs on Days 14, 21 and 29.\nUntreated control dogs had high numbers of engorged sandflies at the end of the exposure period at all time-points with means between 54.6 and 68.2 out of 80 (Table 1). With at least 68% of feeding behaviour on control dogs, it showed a robust sandfly strain population. The survival rate was also very good with at least 73.1% sandflies surviving until 4 h after the end of the exposure times in the control group (Table 2).Table 1\nPercent repellency of\nPhlebotomus perniciosus\nin dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on geometric means\n\nNumber of engorged sandflies\n\nExposure day experiment (A/B)\n\nDogs (n)\n\nUntreated dogs\n\nDogs (n)\n\nTreated dogs\n\nRepellency (%)\n1 (A)560.053.494.4*\n98.2%\n*\n1 (B)864.480.499.4*7 (A)560.553.294.7*\n98.5%\n*\n7 (B)861.380.299.7*14 (A)559.350.499.3*\n99.2%\n*\n14 (B)763.180.699.1*21 (A)554.857.386.6*\n90.9%\n*\n21 (B)764.784.692.9*29 (A)554.651.597.3*\n90.3%\n*\n29 (B)768.2812.681.5**Significant difference between the population means of the treated and control groups (p < 0.05).Right column gives the % repellency based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of repellency calculated for each experiment.Table 2\nPercent insecticidal efficacy against\nPhlebotomus perniciosus\nobserved at 4 h in dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on Geometric means\n\nNumber of live sandflies\n\nExposure day experiment (A/B)\n\nDogs (n)\n\nUntreated dogs\n\nDogs (n)\n\nTreated dogs\n\nEfficacy (%)\n1 (A)574.350.399.6*\n98.7%\n*\n1 (B)873.581.697.9*7 (A)575.250100*\n99.7%\n*\n7 (B)872.180.499.5*14 (A)574.555.792.3*\n96.8%\n*\n14 (B)772.181.298.4*21 (A)575.2533.954.9*\n93.4%\n*\n21 (B)774.081.098.7*29 (A)574.257.090.6*\n78.9%\n*\n29 (B)775.1825.765.7**Significant difference between the population means of the treated and control groups (p < 0.05).Right column gives the % insecticidal activity based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of insecticidal activity calculated for each experiment.\n\nPercent repellency of\nPhlebotomus perniciosus\nin dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on geometric means\n\n*Significant difference between the population means of the treated and control groups (p < 0.05).\nRight column gives the % repellency based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of repellency calculated for each experiment.\n\nPercent insecticidal efficacy against\nPhlebotomus perniciosus\nobserved at 4 h in dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on Geometric means\n\n*Significant difference between the population means of the treated and control groups (p < 0.05).\nRight column gives the % insecticidal activity based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of insecticidal activity calculated for each experiment.\n Repellency Treated dogs had significantly fewer fed sandflies at the end of the exposure period than untreated control dogs for all study days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008). The percent sandfly repellency after 60 min exposure was 98.2, 98.5, 99.2, 90.9 and 90.3% (Table 1), for Days 1, 7, 14, 21, and 29, respectively.\nTreated dogs had significantly fewer fed sandflies at the end of the exposure period than untreated control dogs for all study days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008). The percent sandfly repellency after 60 min exposure was 98.2, 98.5, 99.2, 90.9 and 90.3% (Table 1), for Days 1, 7, 14, 21, and 29, respectively.\n Insecticidal efficacy Treated dogs had significantly more dead sandflies at 4 h post-exposure than untreated control dogs for all challenge days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008).\nPercent insecticidal efficacy of the treated group at 4 h compared to the control group on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9% (Table 2), respectively. In Exp. A, the insecticidal efficacy was 54.9% on Day 21 and got back to 90.6% on Day 29.\nTreated dogs had significantly more dead sandflies at 4 h post-exposure than untreated control dogs for all challenge days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008).\nPercent insecticidal efficacy of the treated group at 4 h compared to the control group on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9% (Table 2), respectively. In Exp. A, the insecticidal efficacy was 54.9% on Day 21 and got back to 90.6% on Day 29.",
"Treated dogs had significantly fewer fed sandflies at the end of the exposure period than untreated control dogs for all study days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008). The percent sandfly repellency after 60 min exposure was 98.2, 98.5, 99.2, 90.9 and 90.3% (Table 1), for Days 1, 7, 14, 21, and 29, respectively.",
"Treated dogs had significantly more dead sandflies at 4 h post-exposure than untreated control dogs for all challenge days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008).\nPercent insecticidal efficacy of the treated group at 4 h compared to the control group on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9% (Table 2), respectively. In Exp. A, the insecticidal efficacy was 54.9% on Day 21 and got back to 90.6% on Day 29.",
"The number of female sandflies used for each exposure was limited to 80 instead of a more classical number of 100 females [8,9] because of the large number of dogs in each study (10 dogs in Exp. A and 16 dogs in Exp. B) and the difficulty of rearing large numbers of female sandflies under laboratory conditions. There was no impact on the outcome of the studies due to the high rates of feeding and viability in the control groups.\nThe feeding behaviour of the sandflies on control dogs showed a robust population on all exposures days with at least 68% having fed after 60 min in the control group. Sandfly survival was also very good with at least 73.1% surviving until 4 h after the end of the exposure times in the control group.\nThe potential for a topical product to provide protection against sandfly-transmitted diseases depends on its ability to prevent the sandflies from taking a blood meal. Hence, the prevention of sandfly feeding was the major focus of this study. The new combination product showed a high repellency rate over one month with a plateau lasting until 21 days post-treatment (≥99.2%) and a progressive decrease to 90.3% on Day 29. A repellency over 80% is considered as a minimum threshold by the registration agencies and other authors [8,9]. This level of efficacy was also observed in other similar studies testing topical combinations including permethrin. Miro et al. [10] evaluated the activity of imidacloprid + permethrin (Advantix®) against P. perniciosus and reported ≥96.45% until Day 14, 92.73% on Day 21 and 74.7% on Day 28. Lienard et al. [11] assessed the repellent efficacy of another topical combination of dinotefuran + permethrin + pyriproxyfen (Vectra 3D), under the same experimental conditions, in the same location, and with the same Phlebotomus strain as in the present investigation. The repellency was 96.9, 99.7, 98.7, 83.5 and 87.0% on days 1, 7, 14, 21 and 28, respectively. The mortality effect was 97.8, 99.8, 73.7, 27.5 and 39.6% on days 1, 7, 14, 21 and 28, respectively. Against P. papatasi, Mencke et al. [12] found Advantix® efficacy to be ≥93.3% until Day 8, then 80.0, 72.8 and 55.9% on Days 15, 22 and 29, respectively. Molina et al. [13] tested a topical combination of permethrin alone (Exspot®) against P. perniciosus and reported efficacy ≥93.4% until Day 8 then 86.8, 67.6 and 61.0% on Days 15, 22 and 29, respectively. The observed results for the new topical combination of fipronil and permethrin are clearly close to what has been already published with other spot on formulation containing permethrin.\nInsecticidal efficacy, as a secondary parameter, was high until Day 21 (93.4%), decreasing to 78.9% on Day 29. The insecticidal effect can be attributed to the action of both fipronil and permethrin, whereas the repellent activity of the product is likely due to the permethrin. Even if not a direct comparison, the mortality effect looks higher with the fipronil-permethrin combination than with the dinotefuran-permethrin-pyriproxyfen [7,11], under the same experimental conditions in the same laboratory and with the same sandfly strain.\nIn Exp. A, the insecticidal activity dropped on Day 21 to 54.9% and was 90.6% on Day 29, whereas it was 98.7% on Day 21 in Exp. B and then 65.7% on Day 29. The regular decrease was as expected in Exp. B. The lower insecticidal activity observed in treated dogs in Exp. A on Day 21 is difficult to explain as no particular variation was observed for the repellent effect, and no variation in the controls. It shows how it is important to include enough dogs in a study: 5 dogs is too limited and the 8 dogs included in Exp. B were important to compensate these observations. We could argue that the number of dogs is probably not sufficient and that a repetition of studies, using the same design, could confirm observations. This is certainly why the registration agencies request at least 2 efficacy studies (called dose confirmations) to confirm an indication [8,9].",
"The new combination of fipronil and permethrin demonstrated a significant repellent effect against P. perniciosus bites as soon as it was applied on the dogs, and its repellent efficacy lasted 4 weeks. The results suggest that in endemic areas, the regular application of the new combination could be integrated in a canine leishmaniosis prevention program."
] |
[
"introduction",
"materials|methods",
null,
null,
null,
null,
null,
null,
"results",
null,
null,
"discussion",
"conclusion"
] |
[
"Repellent",
"\nPhlebotomus perniciosus\n",
"Sandflies",
"Fipronil",
"Permethrin"
] |
Background:
Leishmaniosis is a serious parasitic disease caused by flagellated protozoa of the genus Leishmania. The protozoa are transmitted to animals and humans by haematophagous female sandflies of the genus Phlebotomus in the Old World and Lutzomyia in the New World. Although certain wild mammals may be involved in the transmission of leishmaniosis, domestic dogs appear to be the principal reservoir of Leishmania infantum throughout the world [1]. In Europe, there is a tendency for leishmaniosis but also other canine vector-borne diseases to have an increased distribution [2]. This is related to several factors, including climate and social changes [3].
The prevention of canine leishmaniosis in dogs is based on several measures, including anti-Leishmania vaccines and methods for protecting healthy dogs against sandfly bites [4,5]. The studies reported here were conducted to assess the repellent and insecticidal efficacies of a new spot on topical combination of fipronil and permethrin (Frontect®/Frontline Tri-Act®, Merial) against the main vector of canine leishmaniosis in Europe (Phlebotomus perniciosus). Such a combination is intended to provide both repellent and insecticidal-acaricidal effects against several ectoparasites of dogs [6,7].
Methods:
Two successive experiments were conducted at the Ecole Nationale Vétérinaire de Toulouse. The 2 experiments were necessary due to the difficulty to include many dogs and to produce enough sandfly. Experiment A was also considered as a preliminary exploratory study whereas Experiment B was conducted according to Good Clinical Practices (GCP) as described in the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guideline 9 [8].
Animals Adult Beagle dogs were used in the experiements and had not been exposed to ectoparasiticides having a monthly efficacy or shorter 3 months prior to treatment, and were never exposed to long lasting ectoparasiticides (Experiment A (Exp. A): 6 males and 4 females, 10.7 to 11.4 months of age, weighing 6.9–9.2 kg; Experiment B (Exp. B): 8 males and 8 females, 14.1 to 14.9 months of age, weighing 8.1–10.4 kg). The dogs were housed individually in stainless steel cages with an exercise area, under controlled environmental conditions, fed with a commercial dry dog food ration, with water available ad libitum. No concurrent medication was given during the study. They were managed similarly and with due regard for their well-being. Animals were handled in compliance with Merial Animal Care and Use Guidelines and in compliance with the French regulatory requirements and ethical committee of Toulouse Veterinary School. The dogs were acclimated to the study conditions for at least 11 days prior to treatment and were observed for general health conditions throughout the study.
Adult Beagle dogs were used in the experiements and had not been exposed to ectoparasiticides having a monthly efficacy or shorter 3 months prior to treatment, and were never exposed to long lasting ectoparasiticides (Experiment A (Exp. A): 6 males and 4 females, 10.7 to 11.4 months of age, weighing 6.9–9.2 kg; Experiment B (Exp. B): 8 males and 8 females, 14.1 to 14.9 months of age, weighing 8.1–10.4 kg). The dogs were housed individually in stainless steel cages with an exercise area, under controlled environmental conditions, fed with a commercial dry dog food ration, with water available ad libitum. No concurrent medication was given during the study. They were managed similarly and with due regard for their well-being. Animals were handled in compliance with Merial Animal Care and Use Guidelines and in compliance with the French regulatory requirements and ethical committee of Toulouse Veterinary School. The dogs were acclimated to the study conditions for at least 11 days prior to treatment and were observed for general health conditions throughout the study.
Sandfly exposures Sandfly exposures were performed one week prior to treatment for allocation purposes, and after treatment on Days 1, 7, 14, 21 and 29. Prior to each exposure, animals were anesthetized with intra-muscular injections of 0.02 to 0.03 mg/kg dexmedetomidine (Dextomidor®, Pfizer), 3.8 to 6.8 mg/kg ketamine (Imalgene® 1000, Merial) and 0.46 to 0.65 mg/kg of Diazepam (Valium®, Roche); placed individually into a sandfly proof exposure cage; and exposed to 80 P. perniciosus unfed female sandflies (strain originated from Lisbon, Portugal and maintained under laboratory conditions for 9 years). After approximately 60 min, live sandflies were removed from the exposure cage, counted and categorized as engorged (fed) or non-engorged (unfed). Dead sandflies remaining in the exposure cage or on the dog were counted and categorized as engorged or non-engorged. The dogs were then returned to their normal housing. Live sandflies recovered from each exposure (except pre-treatment) were maintained at appropriate environmental conditions for approximately 4 h after the exposure period ended, and dead phlebotomes were recovered and counted from each container.
Sandfly exposures were performed one week prior to treatment for allocation purposes, and after treatment on Days 1, 7, 14, 21 and 29. Prior to each exposure, animals were anesthetized with intra-muscular injections of 0.02 to 0.03 mg/kg dexmedetomidine (Dextomidor®, Pfizer), 3.8 to 6.8 mg/kg ketamine (Imalgene® 1000, Merial) and 0.46 to 0.65 mg/kg of Diazepam (Valium®, Roche); placed individually into a sandfly proof exposure cage; and exposed to 80 P. perniciosus unfed female sandflies (strain originated from Lisbon, Portugal and maintained under laboratory conditions for 9 years). After approximately 60 min, live sandflies were removed from the exposure cage, counted and categorized as engorged (fed) or non-engorged (unfed). Dead sandflies remaining in the exposure cage or on the dog were counted and categorized as engorged or non-engorged. The dogs were then returned to their normal housing. Live sandflies recovered from each exposure (except pre-treatment) were maintained at appropriate environmental conditions for approximately 4 h after the exposure period ended, and dead phlebotomes were recovered and counted from each container.
Allocation and treatment To allocate the dogs, blocks were formed, based on descending pre-treatment counts of fed sandflies and 26 dogs were randomly allocated to the two treatment groups (10 for Exp. A and 16 for Exp. B). Dogs in Group 1 served as untreated control dogs. Dogs in Group 2 were treated on Day 0 with the topical combination of permethrin and fipronil. In Exp. A, 5 dogs were treated at the minimum recommended dose (0.1 mL/kg based on Day 0 body weight, corresponding to a dose of 6.73 mg/kg fipronil and 50.16 mg/kg permethrin) whereas in Exp. B, 8 dogs were treated at the recommended commercial dose (1.0 mL for dogs <10.0 kg, and 2.0 mL for dogs >10.0 to 20 kg, based on Day 0 body weight, delivering a minimum dose of 6.76 mg/kg fipronil and 50.48 mg/kg permethrin).
The dose was applied by parting the hair and applying the formulation directly onto the skin on the dorsal midline of the neck. The total volume was divided into two approximately equal portions. One fraction was applied between the base of the skull and the shoulder blades and the other fraction was applied at the front of the shoulder blades. The dogs were observed prior to treatment and hourly for 4 h following treatment administration.
To allocate the dogs, blocks were formed, based on descending pre-treatment counts of fed sandflies and 26 dogs were randomly allocated to the two treatment groups (10 for Exp. A and 16 for Exp. B). Dogs in Group 1 served as untreated control dogs. Dogs in Group 2 were treated on Day 0 with the topical combination of permethrin and fipronil. In Exp. A, 5 dogs were treated at the minimum recommended dose (0.1 mL/kg based on Day 0 body weight, corresponding to a dose of 6.73 mg/kg fipronil and 50.16 mg/kg permethrin) whereas in Exp. B, 8 dogs were treated at the recommended commercial dose (1.0 mL for dogs <10.0 kg, and 2.0 mL for dogs >10.0 to 20 kg, based on Day 0 body weight, delivering a minimum dose of 6.76 mg/kg fipronil and 50.48 mg/kg permethrin).
The dose was applied by parting the hair and applying the formulation directly onto the skin on the dorsal midline of the neck. The total volume was divided into two approximately equal portions. One fraction was applied between the base of the skull and the shoulder blades and the other fraction was applied at the front of the shoulder blades. The dogs were observed prior to treatment and hourly for 4 h following treatment administration.
Data analysis Percent sandfly repellency The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.
The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.
Percent insecticidal efficacy All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.
The treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.
All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.
The treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.
Percent sandfly repellency The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.
The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.
Percent insecticidal efficacy All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.
The treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.
All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.
The treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.
Animals:
Adult Beagle dogs were used in the experiements and had not been exposed to ectoparasiticides having a monthly efficacy or shorter 3 months prior to treatment, and were never exposed to long lasting ectoparasiticides (Experiment A (Exp. A): 6 males and 4 females, 10.7 to 11.4 months of age, weighing 6.9–9.2 kg; Experiment B (Exp. B): 8 males and 8 females, 14.1 to 14.9 months of age, weighing 8.1–10.4 kg). The dogs were housed individually in stainless steel cages with an exercise area, under controlled environmental conditions, fed with a commercial dry dog food ration, with water available ad libitum. No concurrent medication was given during the study. They were managed similarly and with due regard for their well-being. Animals were handled in compliance with Merial Animal Care and Use Guidelines and in compliance with the French regulatory requirements and ethical committee of Toulouse Veterinary School. The dogs were acclimated to the study conditions for at least 11 days prior to treatment and were observed for general health conditions throughout the study.
Sandfly exposures:
Sandfly exposures were performed one week prior to treatment for allocation purposes, and after treatment on Days 1, 7, 14, 21 and 29. Prior to each exposure, animals were anesthetized with intra-muscular injections of 0.02 to 0.03 mg/kg dexmedetomidine (Dextomidor®, Pfizer), 3.8 to 6.8 mg/kg ketamine (Imalgene® 1000, Merial) and 0.46 to 0.65 mg/kg of Diazepam (Valium®, Roche); placed individually into a sandfly proof exposure cage; and exposed to 80 P. perniciosus unfed female sandflies (strain originated from Lisbon, Portugal and maintained under laboratory conditions for 9 years). After approximately 60 min, live sandflies were removed from the exposure cage, counted and categorized as engorged (fed) or non-engorged (unfed). Dead sandflies remaining in the exposure cage or on the dog were counted and categorized as engorged or non-engorged. The dogs were then returned to their normal housing. Live sandflies recovered from each exposure (except pre-treatment) were maintained at appropriate environmental conditions for approximately 4 h after the exposure period ended, and dead phlebotomes were recovered and counted from each container.
Allocation and treatment:
To allocate the dogs, blocks were formed, based on descending pre-treatment counts of fed sandflies and 26 dogs were randomly allocated to the two treatment groups (10 for Exp. A and 16 for Exp. B). Dogs in Group 1 served as untreated control dogs. Dogs in Group 2 were treated on Day 0 with the topical combination of permethrin and fipronil. In Exp. A, 5 dogs were treated at the minimum recommended dose (0.1 mL/kg based on Day 0 body weight, corresponding to a dose of 6.73 mg/kg fipronil and 50.16 mg/kg permethrin) whereas in Exp. B, 8 dogs were treated at the recommended commercial dose (1.0 mL for dogs <10.0 kg, and 2.0 mL for dogs >10.0 to 20 kg, based on Day 0 body weight, delivering a minimum dose of 6.76 mg/kg fipronil and 50.48 mg/kg permethrin).
The dose was applied by parting the hair and applying the formulation directly onto the skin on the dorsal midline of the neck. The total volume was divided into two approximately equal portions. One fraction was applied between the base of the skull and the shoulder blades and the other fraction was applied at the front of the shoulder blades. The dogs were observed prior to treatment and hourly for 4 h following treatment administration.
Data analysis:
Percent sandfly repellency The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.
The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.
Percent insecticidal efficacy All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.
The treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.
All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.
The treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.
Percent sandfly repellency:
The total number of engorged (alive + dead) sandflies at the end of each post-treatment exposure period was transformed to the natural logarithm of (count + 1) for calculation of geometric means (GM) by treatment group. Percent repellency was expressed as the percent reduction in fed sandflies of the treated group compared to the control group at each post-treatment exposure day: 100 × [(C-T)/C], where C is the GM of the control group and T is the GM of the treated group.
Percent insecticidal efficacy:
All sandflies were collected at 60 min after exposure and classified as dead or alive. The live sandflies were put in containers and observed at 4 h post-exposure. The number of live sandflies after each post-treatment exposure was calculated by subtracting the number of dead sandflies at 4 h and the number of dead sandflies observed at the end of the 60 min exposure. The number of live sandflies was transformed to the natural logarithm of (count + 1) for calculation of geometric means. Percent insecticidal efficacy at 4 h post-exposure was calculated as 100 × [(C-T)/C], where C is the mean of live sandflies in the control group and T is the mean in the treated group.
The treated group was compared to the control group using Friedman’s rank test with blocks defined as the allocation blocks. The testing was two-sided and used a significance level of 5%.
Results:
No health abnormalities related to treatment were observed throughout the studies, including during hourly observations conducted for 4 h immediately after treatment.
In Exp. B, Dog 6277 in the control group vomited on Day 0 and then was normal through Day 7. The dog did not eat well from Days 8 to 14 and was not considered to be suitable for anesthesia and subsequent exposure to sandflies on Day 14. An intussusception was observed at ultrasonography on Day 15 and the dog was removed from the study, therefore the control group of Exp. B moved from 8 to 7 dogs on Days 14, 21 and 29.
Untreated control dogs had high numbers of engorged sandflies at the end of the exposure period at all time-points with means between 54.6 and 68.2 out of 80 (Table 1). With at least 68% of feeding behaviour on control dogs, it showed a robust sandfly strain population. The survival rate was also very good with at least 73.1% sandflies surviving until 4 h after the end of the exposure times in the control group (Table 2).Table 1
Percent repellency of
Phlebotomus perniciosus
in dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on geometric means
Number of engorged sandflies
Exposure day experiment (A/B)
Dogs (n)
Untreated dogs
Dogs (n)
Treated dogs
Repellency (%)
1 (A)560.053.494.4*
98.2%
*
1 (B)864.480.499.4*7 (A)560.553.294.7*
98.5%
*
7 (B)861.380.299.7*14 (A)559.350.499.3*
99.2%
*
14 (B)763.180.699.1*21 (A)554.857.386.6*
90.9%
*
21 (B)764.784.692.9*29 (A)554.651.597.3*
90.3%
*
29 (B)768.2812.681.5**Significant difference between the population means of the treated and control groups (p < 0.05).Right column gives the % repellency based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of repellency calculated for each experiment.Table 2
Percent insecticidal efficacy against
Phlebotomus perniciosus
observed at 4 h in dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on Geometric means
Number of live sandflies
Exposure day experiment (A/B)
Dogs (n)
Untreated dogs
Dogs (n)
Treated dogs
Efficacy (%)
1 (A)574.350.399.6*
98.7%
*
1 (B)873.581.697.9*7 (A)575.250100*
99.7%
*
7 (B)872.180.499.5*14 (A)574.555.792.3*
96.8%
*
14 (B)772.181.298.4*21 (A)575.2533.954.9*
93.4%
*
21 (B)774.081.098.7*29 (A)574.257.090.6*
78.9%
*
29 (B)775.1825.765.7**Significant difference between the population means of the treated and control groups (p < 0.05).Right column gives the % insecticidal activity based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of insecticidal activity calculated for each experiment.
Percent repellency of
Phlebotomus perniciosus
in dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on geometric means
*Significant difference between the population means of the treated and control groups (p < 0.05).
Right column gives the % repellency based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of repellency calculated for each experiment.
Percent insecticidal efficacy against
Phlebotomus perniciosus
observed at 4 h in dogs treated with the combination of fipronil and permethrin (Experiments A & B) based on Geometric means
*Significant difference between the population means of the treated and control groups (p < 0.05).
Right column gives the % insecticidal activity based on geometric means obtained for each dog, pooling the two experiments, it is not the simple mean of the two % of insecticidal activity calculated for each experiment.
Repellency Treated dogs had significantly fewer fed sandflies at the end of the exposure period than untreated control dogs for all study days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008). The percent sandfly repellency after 60 min exposure was 98.2, 98.5, 99.2, 90.9 and 90.3% (Table 1), for Days 1, 7, 14, 21, and 29, respectively.
Treated dogs had significantly fewer fed sandflies at the end of the exposure period than untreated control dogs for all study days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008). The percent sandfly repellency after 60 min exposure was 98.2, 98.5, 99.2, 90.9 and 90.3% (Table 1), for Days 1, 7, 14, 21, and 29, respectively.
Insecticidal efficacy Treated dogs had significantly more dead sandflies at 4 h post-exposure than untreated control dogs for all challenge days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008).
Percent insecticidal efficacy of the treated group at 4 h compared to the control group on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9% (Table 2), respectively. In Exp. A, the insecticidal efficacy was 54.9% on Day 21 and got back to 90.6% on Day 29.
Treated dogs had significantly more dead sandflies at 4 h post-exposure than untreated control dogs for all challenge days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008).
Percent insecticidal efficacy of the treated group at 4 h compared to the control group on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9% (Table 2), respectively. In Exp. A, the insecticidal efficacy was 54.9% on Day 21 and got back to 90.6% on Day 29.
Repellency:
Treated dogs had significantly fewer fed sandflies at the end of the exposure period than untreated control dogs for all study days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008). The percent sandfly repellency after 60 min exposure was 98.2, 98.5, 99.2, 90.9 and 90.3% (Table 1), for Days 1, 7, 14, 21, and 29, respectively.
Insecticidal efficacy:
Treated dogs had significantly more dead sandflies at 4 h post-exposure than untreated control dogs for all challenge days (Exp. A, p = 0.025, Exp. B, p ≤ 0.008).
Percent insecticidal efficacy of the treated group at 4 h compared to the control group on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9% (Table 2), respectively. In Exp. A, the insecticidal efficacy was 54.9% on Day 21 and got back to 90.6% on Day 29.
Discussion:
The number of female sandflies used for each exposure was limited to 80 instead of a more classical number of 100 females [8,9] because of the large number of dogs in each study (10 dogs in Exp. A and 16 dogs in Exp. B) and the difficulty of rearing large numbers of female sandflies under laboratory conditions. There was no impact on the outcome of the studies due to the high rates of feeding and viability in the control groups.
The feeding behaviour of the sandflies on control dogs showed a robust population on all exposures days with at least 68% having fed after 60 min in the control group. Sandfly survival was also very good with at least 73.1% surviving until 4 h after the end of the exposure times in the control group.
The potential for a topical product to provide protection against sandfly-transmitted diseases depends on its ability to prevent the sandflies from taking a blood meal. Hence, the prevention of sandfly feeding was the major focus of this study. The new combination product showed a high repellency rate over one month with a plateau lasting until 21 days post-treatment (≥99.2%) and a progressive decrease to 90.3% on Day 29. A repellency over 80% is considered as a minimum threshold by the registration agencies and other authors [8,9]. This level of efficacy was also observed in other similar studies testing topical combinations including permethrin. Miro et al. [10] evaluated the activity of imidacloprid + permethrin (Advantix®) against P. perniciosus and reported ≥96.45% until Day 14, 92.73% on Day 21 and 74.7% on Day 28. Lienard et al. [11] assessed the repellent efficacy of another topical combination of dinotefuran + permethrin + pyriproxyfen (Vectra 3D), under the same experimental conditions, in the same location, and with the same Phlebotomus strain as in the present investigation. The repellency was 96.9, 99.7, 98.7, 83.5 and 87.0% on days 1, 7, 14, 21 and 28, respectively. The mortality effect was 97.8, 99.8, 73.7, 27.5 and 39.6% on days 1, 7, 14, 21 and 28, respectively. Against P. papatasi, Mencke et al. [12] found Advantix® efficacy to be ≥93.3% until Day 8, then 80.0, 72.8 and 55.9% on Days 15, 22 and 29, respectively. Molina et al. [13] tested a topical combination of permethrin alone (Exspot®) against P. perniciosus and reported efficacy ≥93.4% until Day 8 then 86.8, 67.6 and 61.0% on Days 15, 22 and 29, respectively. The observed results for the new topical combination of fipronil and permethrin are clearly close to what has been already published with other spot on formulation containing permethrin.
Insecticidal efficacy, as a secondary parameter, was high until Day 21 (93.4%), decreasing to 78.9% on Day 29. The insecticidal effect can be attributed to the action of both fipronil and permethrin, whereas the repellent activity of the product is likely due to the permethrin. Even if not a direct comparison, the mortality effect looks higher with the fipronil-permethrin combination than with the dinotefuran-permethrin-pyriproxyfen [7,11], under the same experimental conditions in the same laboratory and with the same sandfly strain.
In Exp. A, the insecticidal activity dropped on Day 21 to 54.9% and was 90.6% on Day 29, whereas it was 98.7% on Day 21 in Exp. B and then 65.7% on Day 29. The regular decrease was as expected in Exp. B. The lower insecticidal activity observed in treated dogs in Exp. A on Day 21 is difficult to explain as no particular variation was observed for the repellent effect, and no variation in the controls. It shows how it is important to include enough dogs in a study: 5 dogs is too limited and the 8 dogs included in Exp. B were important to compensate these observations. We could argue that the number of dogs is probably not sufficient and that a repetition of studies, using the same design, could confirm observations. This is certainly why the registration agencies request at least 2 efficacy studies (called dose confirmations) to confirm an indication [8,9].
Conclusions:
The new combination of fipronil and permethrin demonstrated a significant repellent effect against P. perniciosus bites as soon as it was applied on the dogs, and its repellent efficacy lasted 4 weeks. The results suggest that in endemic areas, the regular application of the new combination could be integrated in a canine leishmaniosis prevention program.
|
Background:
Two successive laboratory experiments (A and B) were conducted to confirm the efficacy of a new fipronil and permethrin combination to repel and kill Phlebotomus perniciosus sandflies when applied once topically on dogs.
Methods:
Due to the difficulty to get enough available dogs and sandflies in one run, the study was divided into 2 experiments which had exactly the same design, and were conducted at the same place, with the same technicians. They compared dogs treated with a combination containing 67.6 mg/mL fipronil + 504.8 mg/mL permethrin (Frontect/Frontline Tri-Act, Merial) to untreated dogs. The treatments were applied topically once on Day 0. Sandfly exposures were performed on Days 1, 7, 14, 21 and 29 with 80 P. perniciosus female sandflies. After 60 min, sandflies were assessed for vitality and engorgement status. Live sandflies were kept in an insectary and observed for mortality counts 4 h after the exposure period ended.
Results:
Percent sandfly repellency on treated dogs was 98.2, 98.5, 99.2, 90.9 and 90.3%, for Days 1, 7, 14, 21, and 29, respectively. There was a significant difference (p ≤ 0.05) between the treated and control groups in both experiments and for the pooled data on every assessment day. Insecticidal efficacy on treated dogs at 4 h post-exposure on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9%, respectively. There was a significant difference between the treated and control groups for live sandflies observed at 4 h post-exposure for all assessment days (p < 0.05).
Conclusions:
A single topical administration of a new combination of fipronil and permethrin demonstrated a significant repellent effect (i.e., > 80%) against P. perniciosus which lasted for 29 days after application. The repellent effect was accompanied by a significant insecticidal effect on sandflies. The results suggest that in endemic areas, the application of the fipronil-permethrin combination could be integrated into canine leishmaniosis prevention program.
|
Background:
Leishmaniosis is a serious parasitic disease caused by flagellated protozoa of the genus Leishmania. The protozoa are transmitted to animals and humans by haematophagous female sandflies of the genus Phlebotomus in the Old World and Lutzomyia in the New World. Although certain wild mammals may be involved in the transmission of leishmaniosis, domestic dogs appear to be the principal reservoir of Leishmania infantum throughout the world [1]. In Europe, there is a tendency for leishmaniosis but also other canine vector-borne diseases to have an increased distribution [2]. This is related to several factors, including climate and social changes [3].
The prevention of canine leishmaniosis in dogs is based on several measures, including anti-Leishmania vaccines and methods for protecting healthy dogs against sandfly bites [4,5]. The studies reported here were conducted to assess the repellent and insecticidal efficacies of a new spot on topical combination of fipronil and permethrin (Frontect®/Frontline Tri-Act®, Merial) against the main vector of canine leishmaniosis in Europe (Phlebotomus perniciosus). Such a combination is intended to provide both repellent and insecticidal-acaricidal effects against several ectoparasites of dogs [6,7].
Conclusions:
The new combination of fipronil and permethrin demonstrated a significant repellent effect against P. perniciosus bites as soon as it was applied on the dogs, and its repellent efficacy lasted 4 weeks. The results suggest that in endemic areas, the regular application of the new combination could be integrated in a canine leishmaniosis prevention program.
|
Background:
Two successive laboratory experiments (A and B) were conducted to confirm the efficacy of a new fipronil and permethrin combination to repel and kill Phlebotomus perniciosus sandflies when applied once topically on dogs.
Methods:
Due to the difficulty to get enough available dogs and sandflies in one run, the study was divided into 2 experiments which had exactly the same design, and were conducted at the same place, with the same technicians. They compared dogs treated with a combination containing 67.6 mg/mL fipronil + 504.8 mg/mL permethrin (Frontect/Frontline Tri-Act, Merial) to untreated dogs. The treatments were applied topically once on Day 0. Sandfly exposures were performed on Days 1, 7, 14, 21 and 29 with 80 P. perniciosus female sandflies. After 60 min, sandflies were assessed for vitality and engorgement status. Live sandflies were kept in an insectary and observed for mortality counts 4 h after the exposure period ended.
Results:
Percent sandfly repellency on treated dogs was 98.2, 98.5, 99.2, 90.9 and 90.3%, for Days 1, 7, 14, 21, and 29, respectively. There was a significant difference (p ≤ 0.05) between the treated and control groups in both experiments and for the pooled data on every assessment day. Insecticidal efficacy on treated dogs at 4 h post-exposure on Days 1, 7, 14, 21 and 29 was 98.7, 99.7, 96.8, 93.4, and 78.9%, respectively. There was a significant difference between the treated and control groups for live sandflies observed at 4 h post-exposure for all assessment days (p < 0.05).
Conclusions:
A single topical administration of a new combination of fipronil and permethrin demonstrated a significant repellent effect (i.e., > 80%) against P. perniciosus which lasted for 29 days after application. The repellent effect was accompanied by a significant insecticidal effect on sandflies. The results suggest that in endemic areas, the application of the fipronil-permethrin combination could be integrated into canine leishmaniosis prevention program.
| 6,777 | 400 | 13 |
[
"sandflies",
"dogs",
"exposure",
"group",
"treatment",
"treated",
"control",
"exp",
"day",
"number"
] |
[
"test",
"test"
] | null |
[CONTENT] Repellent |
Phlebotomus perniciosus
| Sandflies | Fipronil | Permethrin [SUMMARY]
| null |
[CONTENT] Repellent |
Phlebotomus perniciosus
| Sandflies | Fipronil | Permethrin [SUMMARY]
|
[CONTENT] Repellent |
Phlebotomus perniciosus
| Sandflies | Fipronil | Permethrin [SUMMARY]
|
[CONTENT] Repellent |
Phlebotomus perniciosus
| Sandflies | Fipronil | Permethrin [SUMMARY]
|
[CONTENT] Repellent |
Phlebotomus perniciosus
| Sandflies | Fipronil | Permethrin [SUMMARY]
|
[CONTENT] Administration, Topical | Animals | Dog Diseases | Dogs | Female | Insect Control | Insect Repellents | Insect Vectors | Insecticides | Leishmania | Leishmaniasis | Male | Permethrin | Phlebotomus | Pyrazoles [SUMMARY]
| null |
[CONTENT] Administration, Topical | Animals | Dog Diseases | Dogs | Female | Insect Control | Insect Repellents | Insect Vectors | Insecticides | Leishmania | Leishmaniasis | Male | Permethrin | Phlebotomus | Pyrazoles [SUMMARY]
|
[CONTENT] Administration, Topical | Animals | Dog Diseases | Dogs | Female | Insect Control | Insect Repellents | Insect Vectors | Insecticides | Leishmania | Leishmaniasis | Male | Permethrin | Phlebotomus | Pyrazoles [SUMMARY]
|
[CONTENT] Administration, Topical | Animals | Dog Diseases | Dogs | Female | Insect Control | Insect Repellents | Insect Vectors | Insecticides | Leishmania | Leishmaniasis | Male | Permethrin | Phlebotomus | Pyrazoles [SUMMARY]
|
[CONTENT] Administration, Topical | Animals | Dog Diseases | Dogs | Female | Insect Control | Insect Repellents | Insect Vectors | Insecticides | Leishmania | Leishmaniasis | Male | Permethrin | Phlebotomus | Pyrazoles [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] sandflies | dogs | exposure | group | treatment | treated | control | exp | day | number [SUMMARY]
| null |
[CONTENT] sandflies | dogs | exposure | group | treatment | treated | control | exp | day | number [SUMMARY]
|
[CONTENT] sandflies | dogs | exposure | group | treatment | treated | control | exp | day | number [SUMMARY]
|
[CONTENT] sandflies | dogs | exposure | group | treatment | treated | control | exp | day | number [SUMMARY]
|
[CONTENT] sandflies | dogs | exposure | group | treatment | treated | control | exp | day | number [SUMMARY]
|
[CONTENT] leishmaniosis | world | leishmania | canine | europe | protozoa | vector | repellent insecticidal | genus | canine leishmaniosis [SUMMARY]
| null |
[CONTENT] dogs | means | based geometric means | based geometric | treated | experiments | control | exp | 21 | 29 [SUMMARY]
|
[CONTENT] new combination | new | repellent | combination | areas regular application | dogs repellent efficacy lasted | combination integrated canine | soon | soon applied | combination integrated canine leishmaniosis [SUMMARY]
|
[CONTENT] group | exposure | dogs | sandflies | exp | treated | treatment | control | kg | day [SUMMARY]
|
[CONTENT] group | exposure | dogs | sandflies | exp | treated | treatment | control | kg | day [SUMMARY]
|
[CONTENT] Two [SUMMARY]
| null |
[CONTENT] 98.2 | 98.5 | 99.2 | 90.9 | 90.3% | Days 1, 7 | 14, 21 | 29 ||| ||| 4 | Days 1, 7 | 14 | 21 | 29 | 98.7 | 99.7 | 96.8 | 93.4 | 78.9% ||| 4 | 0.05 [SUMMARY]
|
[CONTENT] 80% | P. | 29 days ||| ||| [SUMMARY]
|
[CONTENT] Two ||| one | 2 ||| 67.6 | Frontect/Frontline Tri-Act ||| Day 0 ||| Days 1, 7 | 14 | 21 | 29 | 80 ||| 60 ||| 4 ||| 98.2 | 98.5 | 99.2 | 90.9 | 90.3% | Days 1, 7 | 14, 21 | 29 ||| ||| 4 | Days 1, 7 | 14 | 21 | 29 | 98.7 | 99.7 | 96.8 | 93.4 | 78.9% ||| 4 | 0.05 ||| 80% | P. | 29 days ||| ||| [SUMMARY]
|
[CONTENT] Two ||| one | 2 ||| 67.6 | Frontect/Frontline Tri-Act ||| Day 0 ||| Days 1, 7 | 14 | 21 | 29 | 80 ||| 60 ||| 4 ||| 98.2 | 98.5 | 99.2 | 90.9 | 90.3% | Days 1, 7 | 14, 21 | 29 ||| ||| 4 | Days 1, 7 | 14 | 21 | 29 | 98.7 | 99.7 | 96.8 | 93.4 | 78.9% ||| 4 | 0.05 ||| 80% | P. | 29 days ||| ||| [SUMMARY]
|
|||||
Serum lipid concentrations in patients with cholesterol and pigment gallstones.
|
25135323
|
Gallstones (GS) are formed as a result of impaired metabolic regulation of the human body. Abnormal lipid metabolism is partly responsible for the pathogenesis of GS mainly rich in cholesterol. Thus abnormalities of serum lipids would reflect the possibilities of the formation of cholesterol GS. This study aims to identify the significance of serum lipids on the development of GS disease.
|
BACKGROUND
|
Serum lipid profiles were estimated in 73 patients with symptomatic GS, admitted to the Teaching Hospital, Peradeniya, Sri Lanka for GS removal surgeries from May 2011 to December 2012. Patients with normal serum bilirubin level and not being on lipid lowering drugs were recruited for the study. Serum lipid profile of each patient was analyzed by enzymatic kit assays and the chemical composition of GS was analyzed by Fourier Transform Infrared spectroscopy.
|
METHODS
|
Of the 73 patients, 37 (51%) had cholesterol GS while 36 (49%) had pigment GS. Serum lipid parameters of a majority of patients were within the normal range. Body mass index values of the patients with two types of GS were not significantly different (Two sample t test, p = 0.335). Out of the lipid parameters tested, only serum triglyceride concentration was significantly high in patients with cholesterol GS than that of pigment GS (Two sample t test, p = 0.038). None of the lipid parameters were significantly different between males and females (Two sample t test, p > 0.05). Compared to the patients with pigment GS who were aged below 50 years, mean total cholesterol and triglyceride concentrations were higher in the same age category patients with cholesterol GS.
|
RESULTS
|
Abnormal serum lipid profiles doesn't seem to be an essential feature in patients with cholesterol GS. However when the two groups of patients with cholesterol and pigment GS with no significant difference of body mass indexes were compared, patients with cholesterol GS are more likely to have serum lipid parameters towards the undesirable cutoff levels of their respective normal ranges. However an effect of serum lipid concentrations on high incidence of GS among females has not been identified.
|
CONCLUSION
|
[
"Adult",
"Cholesterol",
"Female",
"Gallstones",
"Humans",
"Lipids",
"Male",
"Middle Aged",
"Pigments, Biological"
] |
4143546
|
Background
|
Gallstones (GS) are formed in the gall bladder and biliary tract and are of two types: namely cholesterol and pigment stones [1]. GS is one of the main causes for number of upper gastrointestinal surgical casualties [2]. Despite the wider exploration into the aetiopathogenesis of GS, knowledge on exact pathogenesis of GS is yet incomplete. Complex interaction of multiple environmental risk factors with the genetic risk factor is the most possible explanation given for this incomplete understanding. Biliary cholesterol supersaturation is identified as a main prerequisite for the formation of cholesterol GS [3] and elevated unconjugated bilirubin in bile is considered as the primary cause for the pigment GS [4]. Thus GS are formed as a result of impaired metabolic regulation of human body. Biliary cholesterol hypersecretion is the main cause for biliary cholesterol supersaturation and bile stasis also plays an additional role [5]. Impaired lipid homeostasis can give rise to cholesterol hypersecretion from biliary canaliculi. Therefore high incidence of cholesterol GS compared to pigment GS can be expected in patients with impaired lipid homeostasis.
Even though a positive correlation between serum triglycerides (TG) and nucleation time of cholesterol in bile is identified, a relationship between serum lipids and biliary cholesterol saturation index has not been identified [6]. Moreover, controversial results have been obtained for the serum lipid profiles of patients with GS. In some case control studies, serum hypertriglyceridemia and low HDL-cholesterol (HDL-C) have shown a significant association with GS disease [7–9]. However it has not been a common observation [10].
If impaired lipid homeostasis is the main cause for cholesterol supersaturation and crystallization, a significant difference between serum lipid profiles of patients with cholesterol and pigment GS should be observed, as cholesterol hypersecretion is not a recognized cause for the pathogenesis of pigment stones. Therefore comparison of lipid profiles in patients with two types of GS would be important in detecting the significance of different lipid parameters on the development of compositionally different GS. Moreover serum lipids might have an effect on the high incidence of GS disease among females [11] and on the age related changes in the chemical composition of GS [12]. The objective of this study was to identify the relationship between serum lipids and the pathogenesis of different types of GS.
|
Methods
|
The study was carried out during May 2011 to December 2012 in a cohort of patients with symptomatic GS, admitted to Teaching hospital, Peradeniya, Sri Lanka for surgical removal of GS. Inclusion criteria for the study were, age more than 18 years, residing in Kandy district, Sri Lanka for more than five years, normal total bilirubin concentration [13] and not being on lipid lowering drugs. Informed written consent was taken from the patients at the time of recruitment. An interviewer administered formatted data sheet which included age, gender, comorbidities and drug history of the patient was used in data collection. Weight and height of each patient was measured using standard techniques [14] to calculate the body mass index (BMI). According to BMI values, patients were classified into two groups as normal and overweight or obese based on the WHO BMI cutoff values [15]. A sample of 2 ml venous blood was taken from each patient after a fasting period of 12 hours [16]. Serum samples were stored at -20°C and the lipid profiles were analyzed within 4 weeks of sample collection. Total Cholesterol (TC), TG and HDL- C in serum were analyzed by enzymatic methods using enzyme assay kits purchased from Aggape Diagnostics, Germany. All the tests were done in duplicates and mean value was taken as the end result. Measurements of the standard samples were taken at the beginning, middle and end of the test samples to improve the accuracy of the readings. LDL- cholesterol (LDL-C) was calculated using Friedewald equation [17].
Gallstones removed at the time of surgery were preserved appropriately and the chemical composition was identified by the physical characteristics and Fourier Transform Infrared spectroscopy (FTIR, Shimadzu IR prestige 21). Previously described methods were used in the analysis of chemical composition of the GS by FTIR [18, 19]. GS were divided in to two groups as, cholesterol and pigment GS, based on the chemical composition [1] and the lipid profile results of the two groups were compared in the final analysis. In addition, serum lipid profiles of patients with the two types of GS were compared after categorizing them in to two age groups each as 50 years or less and above 50 years. This was done based on the median age of menopause of Sinhalese females [20].
Data obtained were analyzed by Minitab 14 software in which two sample t test was used to analyze the continuous data, while chi square and Fisher’s exact tests were used in the analysis of categorical data.
The cutoff values used to determine the desirable levels of serum lipids were; serum TC below 200 mg/dl, TG below 150 mg/dl, HDL-C above 40 mg/dl and LDL-C below 100 mg/dl [21]. Ethical clearance for the study was obtained from the Ethical Review Committee, Faculty of Medicine, University of Peradeniya, Sri Lanka.
|
Results
|
A total of 73 patients were recruited to the study, based on the inclusion criteria. Mean age of the total study group was 44.6 ± 10.4 years and majority was females. (n = 60, 82%). Mean ages of females and males were 44.0 ± 10.4 and 47.1 ± 10.0 years respectively. A greater proportion (n = 52, 71%) of patients in the study group were aged 50 years or less.
Of the total, 37 (51%) had cholesterol GS while 36 (49%) had pigment GS. Age and gender distribution of patients with two types of GS are shown in Table 1. Mean age of the patients with cholesterol GS was 41.1 ± 9.2 years and those with pigment GS was 48.5 ± 10.3 years, denoting a significant difference (Two sample t test, p = 0.002).Table 1
Age and gender distribution of patients with two types of GS
Physical parameterCholesterol GSPigment GSP valueAge (mean ± SD) years41.1 ± 9.248.5 ± 10.30.002*Gender (n,%)Females34 (57%)26 (43%)0.035$
Males03 (23%)10 (77%)*-Two sample t test.
$-Fisher’s exact test.
Age and gender distribution of patients with two types of GS
*-Two sample t test.
$-Fisher’s exact test.
Among the 60 females, majority (n = 34, 57%) had cholesterol GS, while majority of males (n = 10, 77%) had pigment GS. Compared to males the cholesterol GS were common among females (Fisher’s exact test, p = 0.035).
In the group of patients aged ≤ 50 years, majority (n = 31, 60%) had cholesterol stones whereas majority (n = 15, 71%) of patients aged > 50 years had pigment stones. Frequency of detecting cholesterol GS among the patients aged ≤50 years was significantly common compared to that of patients aged > 50 years (Chi square test, p = 0.010). Mean BMI of the patients with cholesterol GS (26.06 ± 3.15 kg/m2) was not statistically significant (Two sample t test, p = 0.335) from that of the patients with pigment GS (25.28 ± 3.28 kg/m2). Mean concentrations of serum total cholesterol, TG, HDL-C and LDL-C in the total study group were 169.9 ± 27.6, 149.1 ± 24.7, 52.9 ± 9.6 and 87.2 ± 30.7 mg/dl respectively.
Even though none of the patients in this study cohort had a history of dyslipidemia few had serum lipid parameters in undesirable range (Table 2). Serum lipid concentrations in patients with cholesterol and pigment GS are shown in Table 3. In the comparison of serum lipids among patients with two types of GS, serum TG, TC and HDL-C were higher in patients with cholesterol GS than pigment GS. However a statistical significance was seen only for serum TG (Two sample t test, p = 0.038).Table 2
Undesirable lipid parameters in patients with cholesterol and pigment GS
Serum lipid parameterNo of patients in undesirable levelCholesterol GS n(%)Pigment GS n(%)TC07 (19)03 (08)TG05 (14)03 (08)HDL-C00 (00)03 (08)LDL-C07 (19)12 (33)Table 3
Comparison of serum lipids in patients with cholesterol and pigment GS
Serum lipid parameter (mg/dl)Cholesterol GS (n = 37) Mean ± SDPigment GS (n = 36) Mean ± SDP value*TC172.0 ± 30.1167.8 ± 25.10.515TG155. 0 ± 20.9143.00 ± 27.10.038HDL-C54.05 ± 9.1951.5 ± 10.00.261LDL-C86.9 ± 33.088.5 ± 29.00.826*Two sample t test.
Undesirable lipid parameters in patients with cholesterol and pigment GS
Comparison of serum lipids in patients with cholesterol and pigment GS
*Two sample t test.
Serum lipids among male and female patients are shown in Table 4. None of the serum lipid parameter was significantly different between females and males (Two sample t test, P > 0.05).Table 4
Comparison of serum lipids in females and males in the total study group
Serum lipid parameter (mg/dl)Females (n=60) Mean ± SDMales (n=13) Mean ± SDP value*TC171.1 ± 28.8164.7 ± 21.60.374TG149.4 ± 23.8147.9 ± 29.60.866HDL-C53.82 ± 8.3948.11 ± 9.640.065LDL-C87.8 ± 32.087.0 ± 26.00.924*Two sample t test.
Comparison of serum lipids in females and males in the total study group
*Two sample t test.
Serum lipid profiles in patients with two types of gallstones were further analyzed after categorizing them into two age groups as age ≤ 50 years and > 50 years and are summarized in Tables 5 and 6. Irrespective of the age category high serum TC and TG were seen among the patients with cholesterol GS. However none of these parameters were significantly different (Two sample t test, p > 0.05) in both age categories.Table 5
Comparison of serum lipids in patients aged ≤ 50 years with two types of GS
Serum lipid parameter (mg/dl)Cholesterol GS (n = 31) Mean ± SDPigment GS (n = 21) Mean ± SDP value*TC171.10 ± 32.0165.1 ± 22.40.431TG153.50 ± 20.2141.00 ± 30.70.108HDL-C54.86 ± 9.5056.08 ± 9.340.650LDL-C85.50 ± 34.580.80 ± 26.20.581*Two sample t test.Table 6
Comparison of serum lipids in patients aged > 50 years with two types of GS
Serum lipid parameter (mg/dl)Cholesterol GS (n = 6) Mean ± SDPigment GS (n = 15) Mean ± SDP value*TC176.94 ± 17.94171.57 ± 28.800.615TG162.70 ± 24.9145.97 ± 21.760.188HDL-C51.29 ± 7.1645.13 ± 6.650.107LDL-C93.12 ± 21.1297.25 ± 30.980.731*Two sample t test.
Comparison of serum lipids in patients aged ≤ 50 years with two types of GS
*Two sample t test.
Comparison of serum lipids in patients aged > 50 years with two types of GS
*Two sample t test.
|
Conclusion
|
Abnormal serum lipid profiles doesn’t seem to be an essential feature in patients with cholesterol GS. However when the two groups of patients with cholesterol and pigment GS with no significant difference in body mass indexes were compared, patients with cholesterol GS are more likely to have serum lipid parameters towards the undesirable cutoff levels of their respective normal ranges. However an effect of serum lipid concentrations on high incidence of GS among females has not been identified.
|
[
"Background"
] |
[
"Gallstones (GS) are formed in the gall bladder and biliary tract and are of two types: namely cholesterol and pigment stones [1]. GS is one of the main causes for number of upper gastrointestinal surgical casualties [2]. Despite the wider exploration into the aetiopathogenesis of GS, knowledge on exact pathogenesis of GS is yet incomplete. Complex interaction of multiple environmental risk factors with the genetic risk factor is the most possible explanation given for this incomplete understanding. Biliary cholesterol supersaturation is identified as a main prerequisite for the formation of cholesterol GS [3] and elevated unconjugated bilirubin in bile is considered as the primary cause for the pigment GS [4]. Thus GS are formed as a result of impaired metabolic regulation of human body. Biliary cholesterol hypersecretion is the main cause for biliary cholesterol supersaturation and bile stasis also plays an additional role [5]. Impaired lipid homeostasis can give rise to cholesterol hypersecretion from biliary canaliculi. Therefore high incidence of cholesterol GS compared to pigment GS can be expected in patients with impaired lipid homeostasis.\nEven though a positive correlation between serum triglycerides (TG) and nucleation time of cholesterol in bile is identified, a relationship between serum lipids and biliary cholesterol saturation index has not been identified [6]. Moreover, controversial results have been obtained for the serum lipid profiles of patients with GS. In some case control studies, serum hypertriglyceridemia and low HDL-cholesterol (HDL-C) have shown a significant association with GS disease [7–9]. However it has not been a common observation [10].\nIf impaired lipid homeostasis is the main cause for cholesterol supersaturation and crystallization, a significant difference between serum lipid profiles of patients with cholesterol and pigment GS should be observed, as cholesterol hypersecretion is not a recognized cause for the pathogenesis of pigment stones. Therefore comparison of lipid profiles in patients with two types of GS would be important in detecting the significance of different lipid parameters on the development of compositionally different GS. Moreover serum lipids might have an effect on the high incidence of GS disease among females [11] and on the age related changes in the chemical composition of GS [12]. The objective of this study was to identify the relationship between serum lipids and the pathogenesis of different types of GS."
] |
[
null
] |
[
"Background",
"Methods",
"Results",
"Discussion",
"Conclusion"
] |
[
"Gallstones (GS) are formed in the gall bladder and biliary tract and are of two types: namely cholesterol and pigment stones [1]. GS is one of the main causes for number of upper gastrointestinal surgical casualties [2]. Despite the wider exploration into the aetiopathogenesis of GS, knowledge on exact pathogenesis of GS is yet incomplete. Complex interaction of multiple environmental risk factors with the genetic risk factor is the most possible explanation given for this incomplete understanding. Biliary cholesterol supersaturation is identified as a main prerequisite for the formation of cholesterol GS [3] and elevated unconjugated bilirubin in bile is considered as the primary cause for the pigment GS [4]. Thus GS are formed as a result of impaired metabolic regulation of human body. Biliary cholesterol hypersecretion is the main cause for biliary cholesterol supersaturation and bile stasis also plays an additional role [5]. Impaired lipid homeostasis can give rise to cholesterol hypersecretion from biliary canaliculi. Therefore high incidence of cholesterol GS compared to pigment GS can be expected in patients with impaired lipid homeostasis.\nEven though a positive correlation between serum triglycerides (TG) and nucleation time of cholesterol in bile is identified, a relationship between serum lipids and biliary cholesterol saturation index has not been identified [6]. Moreover, controversial results have been obtained for the serum lipid profiles of patients with GS. In some case control studies, serum hypertriglyceridemia and low HDL-cholesterol (HDL-C) have shown a significant association with GS disease [7–9]. However it has not been a common observation [10].\nIf impaired lipid homeostasis is the main cause for cholesterol supersaturation and crystallization, a significant difference between serum lipid profiles of patients with cholesterol and pigment GS should be observed, as cholesterol hypersecretion is not a recognized cause for the pathogenesis of pigment stones. Therefore comparison of lipid profiles in patients with two types of GS would be important in detecting the significance of different lipid parameters on the development of compositionally different GS. Moreover serum lipids might have an effect on the high incidence of GS disease among females [11] and on the age related changes in the chemical composition of GS [12]. The objective of this study was to identify the relationship between serum lipids and the pathogenesis of different types of GS.",
"The study was carried out during May 2011 to December 2012 in a cohort of patients with symptomatic GS, admitted to Teaching hospital, Peradeniya, Sri Lanka for surgical removal of GS. Inclusion criteria for the study were, age more than 18 years, residing in Kandy district, Sri Lanka for more than five years, normal total bilirubin concentration [13] and not being on lipid lowering drugs. Informed written consent was taken from the patients at the time of recruitment. An interviewer administered formatted data sheet which included age, gender, comorbidities and drug history of the patient was used in data collection. Weight and height of each patient was measured using standard techniques [14] to calculate the body mass index (BMI). According to BMI values, patients were classified into two groups as normal and overweight or obese based on the WHO BMI cutoff values [15]. A sample of 2 ml venous blood was taken from each patient after a fasting period of 12 hours [16]. Serum samples were stored at -20°C and the lipid profiles were analyzed within 4 weeks of sample collection. Total Cholesterol (TC), TG and HDL- C in serum were analyzed by enzymatic methods using enzyme assay kits purchased from Aggape Diagnostics, Germany. All the tests were done in duplicates and mean value was taken as the end result. Measurements of the standard samples were taken at the beginning, middle and end of the test samples to improve the accuracy of the readings. LDL- cholesterol (LDL-C) was calculated using Friedewald equation [17].\nGallstones removed at the time of surgery were preserved appropriately and the chemical composition was identified by the physical characteristics and Fourier Transform Infrared spectroscopy (FTIR, Shimadzu IR prestige 21). Previously described methods were used in the analysis of chemical composition of the GS by FTIR [18, 19]. GS were divided in to two groups as, cholesterol and pigment GS, based on the chemical composition [1] and the lipid profile results of the two groups were compared in the final analysis. In addition, serum lipid profiles of patients with the two types of GS were compared after categorizing them in to two age groups each as 50 years or less and above 50 years. This was done based on the median age of menopause of Sinhalese females [20].\nData obtained were analyzed by Minitab 14 software in which two sample t test was used to analyze the continuous data, while chi square and Fisher’s exact tests were used in the analysis of categorical data.\nThe cutoff values used to determine the desirable levels of serum lipids were; serum TC below 200 mg/dl, TG below 150 mg/dl, HDL-C above 40 mg/dl and LDL-C below 100 mg/dl [21]. Ethical clearance for the study was obtained from the Ethical Review Committee, Faculty of Medicine, University of Peradeniya, Sri Lanka.",
"A total of 73 patients were recruited to the study, based on the inclusion criteria. Mean age of the total study group was 44.6 ± 10.4 years and majority was females. (n = 60, 82%). Mean ages of females and males were 44.0 ± 10.4 and 47.1 ± 10.0 years respectively. A greater proportion (n = 52, 71%) of patients in the study group were aged 50 years or less.\nOf the total, 37 (51%) had cholesterol GS while 36 (49%) had pigment GS. Age and gender distribution of patients with two types of GS are shown in Table 1. Mean age of the patients with cholesterol GS was 41.1 ± 9.2 years and those with pigment GS was 48.5 ± 10.3 years, denoting a significant difference (Two sample t test, p = 0.002).Table 1\nAge and gender distribution of patients with two types of GS\nPhysical parameterCholesterol GSPigment GSP valueAge (mean ± SD) years41.1 ± 9.248.5 ± 10.30.002*Gender (n,%)Females34 (57%)26 (43%)0.035$\nMales03 (23%)10 (77%)*-Two sample t test.\n$-Fisher’s exact test.\n\nAge and gender distribution of patients with two types of GS\n\n*-Two sample t test.\n\n$-Fisher’s exact test.\nAmong the 60 females, majority (n = 34, 57%) had cholesterol GS, while majority of males (n = 10, 77%) had pigment GS. Compared to males the cholesterol GS were common among females (Fisher’s exact test, p = 0.035).\nIn the group of patients aged ≤ 50 years, majority (n = 31, 60%) had cholesterol stones whereas majority (n = 15, 71%) of patients aged > 50 years had pigment stones. Frequency of detecting cholesterol GS among the patients aged ≤50 years was significantly common compared to that of patients aged > 50 years (Chi square test, p = 0.010). Mean BMI of the patients with cholesterol GS (26.06 ± 3.15 kg/m2) was not statistically significant (Two sample t test, p = 0.335) from that of the patients with pigment GS (25.28 ± 3.28 kg/m2). Mean concentrations of serum total cholesterol, TG, HDL-C and LDL-C in the total study group were 169.9 ± 27.6, 149.1 ± 24.7, 52.9 ± 9.6 and 87.2 ± 30.7 mg/dl respectively.\nEven though none of the patients in this study cohort had a history of dyslipidemia few had serum lipid parameters in undesirable range (Table 2). Serum lipid concentrations in patients with cholesterol and pigment GS are shown in Table 3. In the comparison of serum lipids among patients with two types of GS, serum TG, TC and HDL-C were higher in patients with cholesterol GS than pigment GS. However a statistical significance was seen only for serum TG (Two sample t test, p = 0.038).Table 2\nUndesirable lipid parameters in patients with cholesterol and pigment GS\nSerum lipid parameterNo of patients in undesirable levelCholesterol GS n(%)Pigment GS n(%)TC07 (19)03 (08)TG05 (14)03 (08)HDL-C00 (00)03 (08)LDL-C07 (19)12 (33)Table 3\nComparison of serum lipids in patients with cholesterol and pigment GS\nSerum lipid parameter (mg/dl)Cholesterol GS (n = 37) Mean ± SDPigment GS (n = 36) Mean ± SDP value*TC172.0 ± 30.1167.8 ± 25.10.515TG155. 0 ± 20.9143.00 ± 27.10.038HDL-C54.05 ± 9.1951.5 ± 10.00.261LDL-C86.9 ± 33.088.5 ± 29.00.826*Two sample t test.\n\nUndesirable lipid parameters in patients with cholesterol and pigment GS\n\n\nComparison of serum lipids in patients with cholesterol and pigment GS\n\n*Two sample t test.\nSerum lipids among male and female patients are shown in Table 4. None of the serum lipid parameter was significantly different between females and males (Two sample t test, P > 0.05).Table 4\nComparison of serum lipids in females and males in the total study group\nSerum lipid parameter (mg/dl)Females (n=60) Mean ± SDMales (n=13) Mean ± SDP value*TC171.1 ± 28.8164.7 ± 21.60.374TG149.4 ± 23.8147.9 ± 29.60.866HDL-C53.82 ± 8.3948.11 ± 9.640.065LDL-C87.8 ± 32.087.0 ± 26.00.924*Two sample t test.\n\nComparison of serum lipids in females and males in the total study group\n\n*Two sample t test.\nSerum lipid profiles in patients with two types of gallstones were further analyzed after categorizing them into two age groups as age ≤ 50 years and > 50 years and are summarized in Tables 5 and 6. Irrespective of the age category high serum TC and TG were seen among the patients with cholesterol GS. However none of these parameters were significantly different (Two sample t test, p > 0.05) in both age categories.Table 5\nComparison of serum lipids in patients aged ≤ 50 years with two types of GS\nSerum lipid parameter (mg/dl)Cholesterol GS (n = 31) Mean ± SDPigment GS (n = 21) Mean ± SDP value*TC171.10 ± 32.0165.1 ± 22.40.431TG153.50 ± 20.2141.00 ± 30.70.108HDL-C54.86 ± 9.5056.08 ± 9.340.650LDL-C85.50 ± 34.580.80 ± 26.20.581*Two sample t test.Table 6\nComparison of serum lipids in patients aged > 50 years with two types of GS\nSerum lipid parameter (mg/dl)Cholesterol GS (n = 6) Mean ± SDPigment GS (n = 15) Mean ± SDP value*TC176.94 ± 17.94171.57 ± 28.800.615TG162.70 ± 24.9145.97 ± 21.760.188HDL-C51.29 ± 7.1645.13 ± 6.650.107LDL-C93.12 ± 21.1297.25 ± 30.980.731*Two sample t test.\n\nComparison of serum lipids in patients aged ≤ 50 years with two types of GS\n\n*Two sample t test.\n\nComparison of serum lipids in patients aged > 50 years with two types of GS\n\n*Two sample t test.",
"This study describes the pattern of serum lipid concentrations in a cohort of patients with cholesterol and pigment GS. Further in this study cohort, BMI values of the patients with two types of GS were not significantly different. Despite that serum TG and TC were high among the patients with cholesterol GS compared to that of pigment GS. Of these two parameters, serum TG showed a significant difference as well. Further serum TG and TC concentrations were high even in young patients with cholesterol GS compared to that of pigment GS. This signifies the possible involvement of serum TG and TC on the development of cholesterol GS even in young patients. Moreover, none of the lipid parameters were significantly different between females and males. As the study was carried out among the patients with GS disease with no prior diagnosis of dyslipidaemia, the lipid parameters were normal in majority of patients. Findings similar to the current study have been observed among Indian patients with GS disease [22] and this signifies the possibility of developing GS even in people with normal lipid parameters. Detection of significantly high serum TG in patients with cholesterol GS is a supportive evidence to indicate hypertriglyceridemia as a risk factor of cholesterol GS [23]. High serum TG in patients with GS is revealed in case control studies carried out in countries with high prevalence of GS disease as well [7–9]. The current finding further illustrates the significant role of serum TG in the determination of chemical composition of GS among the stone formers. Hypertriglyceridemia is identified as a cause for gall bladder (GB) hypomotility as it reduces the GB sensitivity to cholecystokinin [24], a paracrine hormone that regulates the GB contraction. GB hypomotility is one of the main causes for cholesterol crystallization [25]. Therefore the negative effect of serum TG on GB motility can be considered as a cause to develop cholesterol GS than pigment GS in people with high risk of GS disease.\nThe prevalence of GS depends on the gender where females have a higher incidence [11]. Multiple reasons have been evaluated in the identification of the cause for this gender difference. However a possible effect of serum lipids causing high incidence of GS among females was not identified in this study. Interestingly female patients in the study group had high serum HDL-C than that of male patients. The favorable effects of female reproductive hormones on serum lipids can be considered as a possible reason for such observation, as mean age of females in the study group was below the menopausal age which is considered as 51 years for Sinhalese [20]. However, a high risk of GS among patients with low HDL-C has been observed [8].\nIn this study cohort, patients with pigment GS were significantly older than those with cholesterol GS. In spite of this, the serum lipid abnormalities were more common among patients with cholesterol GS showing the possibilities of developing cholesterol GS even in young patients with impaired serum lipids. Importantly high serum TG concentrations even at younger ages can be considered as a risk factor to have cholesterol GS. Moreover, aging is associated with significant changes in lipid profile and as well as in the chemical composition of GS. High incidence of pigment GS is observed in elderly population as cholesterol content in GS gradually reduces with aging [12]. Similar changes in the GS composition were seen in patients in this study cohort as well. Though cholesterol GS was significantly high among patients aged ≤ 50 years, none of the lipid parameters were significantly different between young (age ≤ 50 years) and elderly patients (age > 50 years) in the two types of GS. Though it was not statistically significant, the results obtained for serum lipids for cholesterol GS patients aged ≤ 50 years indicate a higher tendency to have abnormal lipid profiles in their future lives than that of pigment GS patients. Importantly the lipid profile pattern seen among these patients should be evaluated with caution as impaired lipid homeostasis is a main risk factor for the cardiovascular disease as well [21]. Thus the development of GS disease can be considered as a warning sign to have high risk of cardiovascular disease for the patients with cholesterol GS. Increased mortality due to cardiovascular disease among patients with GS in USA has been observed [26].\nIn the current study patients with cholesterol GS aged > 50 years had high serum HDL-C compared to that of patients pigment GS and such finding was not observed among the patients aged ≤ 50 years. However, the presence of high serum TC and TG among cholesterol GS patients in both age categories signifies the effect of serum TC and TG on the formation of cholesterol GS over pigment GS irrespective of the age.",
"Abnormal serum lipid profiles doesn’t seem to be an essential feature in patients with cholesterol GS. However when the two groups of patients with cholesterol and pigment GS with no significant difference in body mass indexes were compared, patients with cholesterol GS are more likely to have serum lipid parameters towards the undesirable cutoff levels of their respective normal ranges. However an effect of serum lipid concentrations on high incidence of GS among females has not been identified."
] |
[
null,
"methods",
"results",
"discussion",
"conclusions"
] |
[
"Cholesterol gallstones",
"Pigment gallstones",
"Lipid profile"
] |
Background:
Gallstones (GS) are formed in the gall bladder and biliary tract and are of two types: namely cholesterol and pigment stones [1]. GS is one of the main causes for number of upper gastrointestinal surgical casualties [2]. Despite the wider exploration into the aetiopathogenesis of GS, knowledge on exact pathogenesis of GS is yet incomplete. Complex interaction of multiple environmental risk factors with the genetic risk factor is the most possible explanation given for this incomplete understanding. Biliary cholesterol supersaturation is identified as a main prerequisite for the formation of cholesterol GS [3] and elevated unconjugated bilirubin in bile is considered as the primary cause for the pigment GS [4]. Thus GS are formed as a result of impaired metabolic regulation of human body. Biliary cholesterol hypersecretion is the main cause for biliary cholesterol supersaturation and bile stasis also plays an additional role [5]. Impaired lipid homeostasis can give rise to cholesterol hypersecretion from biliary canaliculi. Therefore high incidence of cholesterol GS compared to pigment GS can be expected in patients with impaired lipid homeostasis.
Even though a positive correlation between serum triglycerides (TG) and nucleation time of cholesterol in bile is identified, a relationship between serum lipids and biliary cholesterol saturation index has not been identified [6]. Moreover, controversial results have been obtained for the serum lipid profiles of patients with GS. In some case control studies, serum hypertriglyceridemia and low HDL-cholesterol (HDL-C) have shown a significant association with GS disease [7–9]. However it has not been a common observation [10].
If impaired lipid homeostasis is the main cause for cholesterol supersaturation and crystallization, a significant difference between serum lipid profiles of patients with cholesterol and pigment GS should be observed, as cholesterol hypersecretion is not a recognized cause for the pathogenesis of pigment stones. Therefore comparison of lipid profiles in patients with two types of GS would be important in detecting the significance of different lipid parameters on the development of compositionally different GS. Moreover serum lipids might have an effect on the high incidence of GS disease among females [11] and on the age related changes in the chemical composition of GS [12]. The objective of this study was to identify the relationship between serum lipids and the pathogenesis of different types of GS.
Methods:
The study was carried out during May 2011 to December 2012 in a cohort of patients with symptomatic GS, admitted to Teaching hospital, Peradeniya, Sri Lanka for surgical removal of GS. Inclusion criteria for the study were, age more than 18 years, residing in Kandy district, Sri Lanka for more than five years, normal total bilirubin concentration [13] and not being on lipid lowering drugs. Informed written consent was taken from the patients at the time of recruitment. An interviewer administered formatted data sheet which included age, gender, comorbidities and drug history of the patient was used in data collection. Weight and height of each patient was measured using standard techniques [14] to calculate the body mass index (BMI). According to BMI values, patients were classified into two groups as normal and overweight or obese based on the WHO BMI cutoff values [15]. A sample of 2 ml venous blood was taken from each patient after a fasting period of 12 hours [16]. Serum samples were stored at -20°C and the lipid profiles were analyzed within 4 weeks of sample collection. Total Cholesterol (TC), TG and HDL- C in serum were analyzed by enzymatic methods using enzyme assay kits purchased from Aggape Diagnostics, Germany. All the tests were done in duplicates and mean value was taken as the end result. Measurements of the standard samples were taken at the beginning, middle and end of the test samples to improve the accuracy of the readings. LDL- cholesterol (LDL-C) was calculated using Friedewald equation [17].
Gallstones removed at the time of surgery were preserved appropriately and the chemical composition was identified by the physical characteristics and Fourier Transform Infrared spectroscopy (FTIR, Shimadzu IR prestige 21). Previously described methods were used in the analysis of chemical composition of the GS by FTIR [18, 19]. GS were divided in to two groups as, cholesterol and pigment GS, based on the chemical composition [1] and the lipid profile results of the two groups were compared in the final analysis. In addition, serum lipid profiles of patients with the two types of GS were compared after categorizing them in to two age groups each as 50 years or less and above 50 years. This was done based on the median age of menopause of Sinhalese females [20].
Data obtained were analyzed by Minitab 14 software in which two sample t test was used to analyze the continuous data, while chi square and Fisher’s exact tests were used in the analysis of categorical data.
The cutoff values used to determine the desirable levels of serum lipids were; serum TC below 200 mg/dl, TG below 150 mg/dl, HDL-C above 40 mg/dl and LDL-C below 100 mg/dl [21]. Ethical clearance for the study was obtained from the Ethical Review Committee, Faculty of Medicine, University of Peradeniya, Sri Lanka.
Results:
A total of 73 patients were recruited to the study, based on the inclusion criteria. Mean age of the total study group was 44.6 ± 10.4 years and majority was females. (n = 60, 82%). Mean ages of females and males were 44.0 ± 10.4 and 47.1 ± 10.0 years respectively. A greater proportion (n = 52, 71%) of patients in the study group were aged 50 years or less.
Of the total, 37 (51%) had cholesterol GS while 36 (49%) had pigment GS. Age and gender distribution of patients with two types of GS are shown in Table 1. Mean age of the patients with cholesterol GS was 41.1 ± 9.2 years and those with pigment GS was 48.5 ± 10.3 years, denoting a significant difference (Two sample t test, p = 0.002).Table 1
Age and gender distribution of patients with two types of GS
Physical parameterCholesterol GSPigment GSP valueAge (mean ± SD) years41.1 ± 9.248.5 ± 10.30.002*Gender (n,%)Females34 (57%)26 (43%)0.035$
Males03 (23%)10 (77%)*-Two sample t test.
$-Fisher’s exact test.
Age and gender distribution of patients with two types of GS
*-Two sample t test.
$-Fisher’s exact test.
Among the 60 females, majority (n = 34, 57%) had cholesterol GS, while majority of males (n = 10, 77%) had pigment GS. Compared to males the cholesterol GS were common among females (Fisher’s exact test, p = 0.035).
In the group of patients aged ≤ 50 years, majority (n = 31, 60%) had cholesterol stones whereas majority (n = 15, 71%) of patients aged > 50 years had pigment stones. Frequency of detecting cholesterol GS among the patients aged ≤50 years was significantly common compared to that of patients aged > 50 years (Chi square test, p = 0.010). Mean BMI of the patients with cholesterol GS (26.06 ± 3.15 kg/m2) was not statistically significant (Two sample t test, p = 0.335) from that of the patients with pigment GS (25.28 ± 3.28 kg/m2). Mean concentrations of serum total cholesterol, TG, HDL-C and LDL-C in the total study group were 169.9 ± 27.6, 149.1 ± 24.7, 52.9 ± 9.6 and 87.2 ± 30.7 mg/dl respectively.
Even though none of the patients in this study cohort had a history of dyslipidemia few had serum lipid parameters in undesirable range (Table 2). Serum lipid concentrations in patients with cholesterol and pigment GS are shown in Table 3. In the comparison of serum lipids among patients with two types of GS, serum TG, TC and HDL-C were higher in patients with cholesterol GS than pigment GS. However a statistical significance was seen only for serum TG (Two sample t test, p = 0.038).Table 2
Undesirable lipid parameters in patients with cholesterol and pigment GS
Serum lipid parameterNo of patients in undesirable levelCholesterol GS n(%)Pigment GS n(%)TC07 (19)03 (08)TG05 (14)03 (08)HDL-C00 (00)03 (08)LDL-C07 (19)12 (33)Table 3
Comparison of serum lipids in patients with cholesterol and pigment GS
Serum lipid parameter (mg/dl)Cholesterol GS (n = 37) Mean ± SDPigment GS (n = 36) Mean ± SDP value*TC172.0 ± 30.1167.8 ± 25.10.515TG155. 0 ± 20.9143.00 ± 27.10.038HDL-C54.05 ± 9.1951.5 ± 10.00.261LDL-C86.9 ± 33.088.5 ± 29.00.826*Two sample t test.
Undesirable lipid parameters in patients with cholesterol and pigment GS
Comparison of serum lipids in patients with cholesterol and pigment GS
*Two sample t test.
Serum lipids among male and female patients are shown in Table 4. None of the serum lipid parameter was significantly different between females and males (Two sample t test, P > 0.05).Table 4
Comparison of serum lipids in females and males in the total study group
Serum lipid parameter (mg/dl)Females (n=60) Mean ± SDMales (n=13) Mean ± SDP value*TC171.1 ± 28.8164.7 ± 21.60.374TG149.4 ± 23.8147.9 ± 29.60.866HDL-C53.82 ± 8.3948.11 ± 9.640.065LDL-C87.8 ± 32.087.0 ± 26.00.924*Two sample t test.
Comparison of serum lipids in females and males in the total study group
*Two sample t test.
Serum lipid profiles in patients with two types of gallstones were further analyzed after categorizing them into two age groups as age ≤ 50 years and > 50 years and are summarized in Tables 5 and 6. Irrespective of the age category high serum TC and TG were seen among the patients with cholesterol GS. However none of these parameters were significantly different (Two sample t test, p > 0.05) in both age categories.Table 5
Comparison of serum lipids in patients aged ≤ 50 years with two types of GS
Serum lipid parameter (mg/dl)Cholesterol GS (n = 31) Mean ± SDPigment GS (n = 21) Mean ± SDP value*TC171.10 ± 32.0165.1 ± 22.40.431TG153.50 ± 20.2141.00 ± 30.70.108HDL-C54.86 ± 9.5056.08 ± 9.340.650LDL-C85.50 ± 34.580.80 ± 26.20.581*Two sample t test.Table 6
Comparison of serum lipids in patients aged > 50 years with two types of GS
Serum lipid parameter (mg/dl)Cholesterol GS (n = 6) Mean ± SDPigment GS (n = 15) Mean ± SDP value*TC176.94 ± 17.94171.57 ± 28.800.615TG162.70 ± 24.9145.97 ± 21.760.188HDL-C51.29 ± 7.1645.13 ± 6.650.107LDL-C93.12 ± 21.1297.25 ± 30.980.731*Two sample t test.
Comparison of serum lipids in patients aged ≤ 50 years with two types of GS
*Two sample t test.
Comparison of serum lipids in patients aged > 50 years with two types of GS
*Two sample t test.
Discussion:
This study describes the pattern of serum lipid concentrations in a cohort of patients with cholesterol and pigment GS. Further in this study cohort, BMI values of the patients with two types of GS were not significantly different. Despite that serum TG and TC were high among the patients with cholesterol GS compared to that of pigment GS. Of these two parameters, serum TG showed a significant difference as well. Further serum TG and TC concentrations were high even in young patients with cholesterol GS compared to that of pigment GS. This signifies the possible involvement of serum TG and TC on the development of cholesterol GS even in young patients. Moreover, none of the lipid parameters were significantly different between females and males. As the study was carried out among the patients with GS disease with no prior diagnosis of dyslipidaemia, the lipid parameters were normal in majority of patients. Findings similar to the current study have been observed among Indian patients with GS disease [22] and this signifies the possibility of developing GS even in people with normal lipid parameters. Detection of significantly high serum TG in patients with cholesterol GS is a supportive evidence to indicate hypertriglyceridemia as a risk factor of cholesterol GS [23]. High serum TG in patients with GS is revealed in case control studies carried out in countries with high prevalence of GS disease as well [7–9]. The current finding further illustrates the significant role of serum TG in the determination of chemical composition of GS among the stone formers. Hypertriglyceridemia is identified as a cause for gall bladder (GB) hypomotility as it reduces the GB sensitivity to cholecystokinin [24], a paracrine hormone that regulates the GB contraction. GB hypomotility is one of the main causes for cholesterol crystallization [25]. Therefore the negative effect of serum TG on GB motility can be considered as a cause to develop cholesterol GS than pigment GS in people with high risk of GS disease.
The prevalence of GS depends on the gender where females have a higher incidence [11]. Multiple reasons have been evaluated in the identification of the cause for this gender difference. However a possible effect of serum lipids causing high incidence of GS among females was not identified in this study. Interestingly female patients in the study group had high serum HDL-C than that of male patients. The favorable effects of female reproductive hormones on serum lipids can be considered as a possible reason for such observation, as mean age of females in the study group was below the menopausal age which is considered as 51 years for Sinhalese [20]. However, a high risk of GS among patients with low HDL-C has been observed [8].
In this study cohort, patients with pigment GS were significantly older than those with cholesterol GS. In spite of this, the serum lipid abnormalities were more common among patients with cholesterol GS showing the possibilities of developing cholesterol GS even in young patients with impaired serum lipids. Importantly high serum TG concentrations even at younger ages can be considered as a risk factor to have cholesterol GS. Moreover, aging is associated with significant changes in lipid profile and as well as in the chemical composition of GS. High incidence of pigment GS is observed in elderly population as cholesterol content in GS gradually reduces with aging [12]. Similar changes in the GS composition were seen in patients in this study cohort as well. Though cholesterol GS was significantly high among patients aged ≤ 50 years, none of the lipid parameters were significantly different between young (age ≤ 50 years) and elderly patients (age > 50 years) in the two types of GS. Though it was not statistically significant, the results obtained for serum lipids for cholesterol GS patients aged ≤ 50 years indicate a higher tendency to have abnormal lipid profiles in their future lives than that of pigment GS patients. Importantly the lipid profile pattern seen among these patients should be evaluated with caution as impaired lipid homeostasis is a main risk factor for the cardiovascular disease as well [21]. Thus the development of GS disease can be considered as a warning sign to have high risk of cardiovascular disease for the patients with cholesterol GS. Increased mortality due to cardiovascular disease among patients with GS in USA has been observed [26].
In the current study patients with cholesterol GS aged > 50 years had high serum HDL-C compared to that of patients pigment GS and such finding was not observed among the patients aged ≤ 50 years. However, the presence of high serum TC and TG among cholesterol GS patients in both age categories signifies the effect of serum TC and TG on the formation of cholesterol GS over pigment GS irrespective of the age.
Conclusion:
Abnormal serum lipid profiles doesn’t seem to be an essential feature in patients with cholesterol GS. However when the two groups of patients with cholesterol and pigment GS with no significant difference in body mass indexes were compared, patients with cholesterol GS are more likely to have serum lipid parameters towards the undesirable cutoff levels of their respective normal ranges. However an effect of serum lipid concentrations on high incidence of GS among females has not been identified.
|
Background:
Gallstones (GS) are formed as a result of impaired metabolic regulation of the human body. Abnormal lipid metabolism is partly responsible for the pathogenesis of GS mainly rich in cholesterol. Thus abnormalities of serum lipids would reflect the possibilities of the formation of cholesterol GS. This study aims to identify the significance of serum lipids on the development of GS disease.
Methods:
Serum lipid profiles were estimated in 73 patients with symptomatic GS, admitted to the Teaching Hospital, Peradeniya, Sri Lanka for GS removal surgeries from May 2011 to December 2012. Patients with normal serum bilirubin level and not being on lipid lowering drugs were recruited for the study. Serum lipid profile of each patient was analyzed by enzymatic kit assays and the chemical composition of GS was analyzed by Fourier Transform Infrared spectroscopy.
Results:
Of the 73 patients, 37 (51%) had cholesterol GS while 36 (49%) had pigment GS. Serum lipid parameters of a majority of patients were within the normal range. Body mass index values of the patients with two types of GS were not significantly different (Two sample t test, p = 0.335). Out of the lipid parameters tested, only serum triglyceride concentration was significantly high in patients with cholesterol GS than that of pigment GS (Two sample t test, p = 0.038). None of the lipid parameters were significantly different between males and females (Two sample t test, p > 0.05). Compared to the patients with pigment GS who were aged below 50 years, mean total cholesterol and triglyceride concentrations were higher in the same age category patients with cholesterol GS.
Conclusions:
Abnormal serum lipid profiles doesn't seem to be an essential feature in patients with cholesterol GS. However when the two groups of patients with cholesterol and pigment GS with no significant difference of body mass indexes were compared, patients with cholesterol GS are more likely to have serum lipid parameters towards the undesirable cutoff levels of their respective normal ranges. However an effect of serum lipid concentrations on high incidence of GS among females has not been identified.
|
Background:
Gallstones (GS) are formed in the gall bladder and biliary tract and are of two types: namely cholesterol and pigment stones [1]. GS is one of the main causes for number of upper gastrointestinal surgical casualties [2]. Despite the wider exploration into the aetiopathogenesis of GS, knowledge on exact pathogenesis of GS is yet incomplete. Complex interaction of multiple environmental risk factors with the genetic risk factor is the most possible explanation given for this incomplete understanding. Biliary cholesterol supersaturation is identified as a main prerequisite for the formation of cholesterol GS [3] and elevated unconjugated bilirubin in bile is considered as the primary cause for the pigment GS [4]. Thus GS are formed as a result of impaired metabolic regulation of human body. Biliary cholesterol hypersecretion is the main cause for biliary cholesterol supersaturation and bile stasis also plays an additional role [5]. Impaired lipid homeostasis can give rise to cholesterol hypersecretion from biliary canaliculi. Therefore high incidence of cholesterol GS compared to pigment GS can be expected in patients with impaired lipid homeostasis.
Even though a positive correlation between serum triglycerides (TG) and nucleation time of cholesterol in bile is identified, a relationship between serum lipids and biliary cholesterol saturation index has not been identified [6]. Moreover, controversial results have been obtained for the serum lipid profiles of patients with GS. In some case control studies, serum hypertriglyceridemia and low HDL-cholesterol (HDL-C) have shown a significant association with GS disease [7–9]. However it has not been a common observation [10].
If impaired lipid homeostasis is the main cause for cholesterol supersaturation and crystallization, a significant difference between serum lipid profiles of patients with cholesterol and pigment GS should be observed, as cholesterol hypersecretion is not a recognized cause for the pathogenesis of pigment stones. Therefore comparison of lipid profiles in patients with two types of GS would be important in detecting the significance of different lipid parameters on the development of compositionally different GS. Moreover serum lipids might have an effect on the high incidence of GS disease among females [11] and on the age related changes in the chemical composition of GS [12]. The objective of this study was to identify the relationship between serum lipids and the pathogenesis of different types of GS.
Conclusion:
Abnormal serum lipid profiles doesn’t seem to be an essential feature in patients with cholesterol GS. However when the two groups of patients with cholesterol and pigment GS with no significant difference in body mass indexes were compared, patients with cholesterol GS are more likely to have serum lipid parameters towards the undesirable cutoff levels of their respective normal ranges. However an effect of serum lipid concentrations on high incidence of GS among females has not been identified.
|
Background:
Gallstones (GS) are formed as a result of impaired metabolic regulation of the human body. Abnormal lipid metabolism is partly responsible for the pathogenesis of GS mainly rich in cholesterol. Thus abnormalities of serum lipids would reflect the possibilities of the formation of cholesterol GS. This study aims to identify the significance of serum lipids on the development of GS disease.
Methods:
Serum lipid profiles were estimated in 73 patients with symptomatic GS, admitted to the Teaching Hospital, Peradeniya, Sri Lanka for GS removal surgeries from May 2011 to December 2012. Patients with normal serum bilirubin level and not being on lipid lowering drugs were recruited for the study. Serum lipid profile of each patient was analyzed by enzymatic kit assays and the chemical composition of GS was analyzed by Fourier Transform Infrared spectroscopy.
Results:
Of the 73 patients, 37 (51%) had cholesterol GS while 36 (49%) had pigment GS. Serum lipid parameters of a majority of patients were within the normal range. Body mass index values of the patients with two types of GS were not significantly different (Two sample t test, p = 0.335). Out of the lipid parameters tested, only serum triglyceride concentration was significantly high in patients with cholesterol GS than that of pigment GS (Two sample t test, p = 0.038). None of the lipid parameters were significantly different between males and females (Two sample t test, p > 0.05). Compared to the patients with pigment GS who were aged below 50 years, mean total cholesterol and triglyceride concentrations were higher in the same age category patients with cholesterol GS.
Conclusions:
Abnormal serum lipid profiles doesn't seem to be an essential feature in patients with cholesterol GS. However when the two groups of patients with cholesterol and pigment GS with no significant difference of body mass indexes were compared, patients with cholesterol GS are more likely to have serum lipid parameters towards the undesirable cutoff levels of their respective normal ranges. However an effect of serum lipid concentrations on high incidence of GS among females has not been identified.
| 3,276 | 403 | 5 |
[
"gs",
"patients",
"serum",
"cholesterol",
"lipid",
"cholesterol gs",
"pigment",
"years",
"pigment gs",
"study"
] |
[
"test",
"test"
] |
[CONTENT] Cholesterol gallstones | Pigment gallstones | Lipid profile [SUMMARY]
|
[CONTENT] Cholesterol gallstones | Pigment gallstones | Lipid profile [SUMMARY]
|
[CONTENT] Cholesterol gallstones | Pigment gallstones | Lipid profile [SUMMARY]
|
[CONTENT] Cholesterol gallstones | Pigment gallstones | Lipid profile [SUMMARY]
|
[CONTENT] Cholesterol gallstones | Pigment gallstones | Lipid profile [SUMMARY]
|
[CONTENT] Cholesterol gallstones | Pigment gallstones | Lipid profile [SUMMARY]
|
[CONTENT] Adult | Cholesterol | Female | Gallstones | Humans | Lipids | Male | Middle Aged | Pigments, Biological [SUMMARY]
|
[CONTENT] Adult | Cholesterol | Female | Gallstones | Humans | Lipids | Male | Middle Aged | Pigments, Biological [SUMMARY]
|
[CONTENT] Adult | Cholesterol | Female | Gallstones | Humans | Lipids | Male | Middle Aged | Pigments, Biological [SUMMARY]
|
[CONTENT] Adult | Cholesterol | Female | Gallstones | Humans | Lipids | Male | Middle Aged | Pigments, Biological [SUMMARY]
|
[CONTENT] Adult | Cholesterol | Female | Gallstones | Humans | Lipids | Male | Middle Aged | Pigments, Biological [SUMMARY]
|
[CONTENT] Adult | Cholesterol | Female | Gallstones | Humans | Lipids | Male | Middle Aged | Pigments, Biological [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] gs | patients | serum | cholesterol | lipid | cholesterol gs | pigment | years | pigment gs | study [SUMMARY]
|
[CONTENT] gs | patients | serum | cholesterol | lipid | cholesterol gs | pigment | years | pigment gs | study [SUMMARY]
|
[CONTENT] gs | patients | serum | cholesterol | lipid | cholesterol gs | pigment | years | pigment gs | study [SUMMARY]
|
[CONTENT] gs | patients | serum | cholesterol | lipid | cholesterol gs | pigment | years | pigment gs | study [SUMMARY]
|
[CONTENT] gs | patients | serum | cholesterol | lipid | cholesterol gs | pigment | years | pigment gs | study [SUMMARY]
|
[CONTENT] gs | patients | serum | cholesterol | lipid | cholesterol gs | pigment | years | pigment gs | study [SUMMARY]
|
[CONTENT] gs | cholesterol | biliary | biliary cholesterol | lipid | serum | cause | main | impaired | bile [SUMMARY]
|
[CONTENT] data | taken | dl | mg | mg dl | sri | analysis | samples | patient | lanka [SUMMARY]
|
[CONTENT] gs | test | patients | sample | sample test | serum | years | table | mean | comparison serum [SUMMARY]
|
[CONTENT] gs | patients cholesterol | serum | cholesterol | serum lipid | patients | lipid | patients cholesterol gs | cholesterol gs | levels respective normal [SUMMARY]
|
[CONTENT] gs | patients | cholesterol | serum | lipid | cholesterol gs | patients cholesterol | years | pigment | test [SUMMARY]
|
[CONTENT] gs | patients | cholesterol | serum | lipid | cholesterol gs | patients cholesterol | years | pigment | test [SUMMARY]
|
[CONTENT] ||| ||| GS ||| [SUMMARY]
|
[CONTENT] 73 | the Teaching Hospital | Peradeniya | Sri Lanka | May 2011 to December 2012 ||| ||| GS | Fourier Transform Infrared [SUMMARY]
|
[CONTENT] 73 | 37 | 51% | GS | 36 | 49% | GS ||| ||| two | Two | 0.335 ||| GS | Two | 0.038 ||| Two | 0.05 ||| below 50 years | GS [SUMMARY]
|
[CONTENT] GS ||| two ||| [SUMMARY]
|
[CONTENT] ||| ||| GS ||| ||| 73 | the Teaching Hospital | Peradeniya | Sri Lanka | May 2011 to December 2012 ||| ||| GS | Fourier Transform Infrared ||| 73 | 37 | 51% | GS | 36 | 49% | GS ||| ||| two | Two | 0.335 ||| GS | Two | 0.038 ||| Two | 0.05 ||| below 50 years ||| GS ||| two ||| [SUMMARY]
|
[CONTENT] ||| ||| GS ||| ||| 73 | the Teaching Hospital | Peradeniya | Sri Lanka | May 2011 to December 2012 ||| ||| GS | Fourier Transform Infrared ||| 73 | 37 | 51% | GS | 36 | 49% | GS ||| ||| two | Two | 0.335 ||| GS | Two | 0.038 ||| Two | 0.05 ||| below 50 years ||| GS ||| two ||| [SUMMARY]
|
||||||
Energy Expenditure, Carbohydrate Oxidation and Appetitive Responses to Sucrose or Sucralose in Humans: A Pilot Study.
|
31374985
|
In light of obesity, replacing sugar with non-nutritive sweeteners is commonly used to reduce sugar content of food products. This study aimed to compare human energy expenditure (EE), carbohydrate oxidation and food intake after the ingestion of test foods sweetened with sucrose or a non-nutritive sweetener.
|
BACKGROUND
|
This was an acute crossover feeding study that entailed consumption of three test foods: jelly sweetened with 50 g sucrose (SUCROSE), with 120 mg of sucralose only (NNS), or 120 mg sucralose but matched in carbohydrate with 50 g maltodextrin (MALT). On test days, participants arrived at the research facility after an overnight fast. Resting energy expenditure (indirect calorimeter) was measured for 30 min followed by jelly consumption. Participants' EE and substrate oxidation were measured for 90 min subsequently. After EE assessment, participants completed a meal challenge before leaving the research facility, and recorded food intake for the remaining day. Subjective appetite ratings were assessed before and after test foods and meal challenge.
|
METHODS
|
Eleven participants completed the study. EE was higher in SUCROSE and MALT than NNS, but not statistically significant. Carbohydrate oxidation was SUCROSE > MALT > NNS (p < 0.001). Earlier and bigger rise in carbohydrate oxidation was observed in SUCROSE than MALT, although both were carbohydrate-matched. NNS did not promote energy expenditure, carbohydrate oxidation or stimulate appetite.
|
RESULTS
|
Foods sweetened with sucrose or non-nutritive sweeteners but matched in carbohydrate content have different effects on human EE and carbohydrate oxidation. Sucralose alone did not affect EE, but lower energy in the test food from sugar replacement was eventually fully compensated. Findings from this pilot study should be verified with bigger clinical studies in the future to establish clinical relevance.
|
CONCLUSIONS
|
[
"Adolescent",
"Adult",
"Aged",
"Appetite Regulation",
"Cross-Over Studies",
"Energy Metabolism",
"Female",
"Humans",
"Male",
"Middle Aged",
"Non-Nutritive Sweeteners",
"Oxidation-Reduction",
"Pilot Projects",
"Sucrose",
"Young Adult"
] |
6723924
|
1. Introduction
|
The global prevalence of overweight and obesity is increasing and it increases the risk of several chronic diseases such as type 2 diabetes and cardiovascular disease [1,2]. Together with its co-morbidities, overweight and obesity increases mortality rates and adds burden to the healthcare system.
An increase in energy intake is one of several factors responsible for the rising prevalence of overweight and obesity [3]. In recent years, epidemiological studies have linked obesity with dietary sugar intake [4,5], and concluded that reducing sugar intake reduces prevalence of overweight and obesity [6]. The World Health Organization recommends a reduction in free sugar intake to no more than 10% of total daily intake [7]. A number of countries such as Mexico, Brazil and France have successfully reduced the demand for sugar-sweetened beverages through the introduction of sugar tax, and this may drive the reduction of added sugar during food processing by the food industry [8].
The intake of natural sugars from major food groups such as fruits, vegetables and dairy foods should not be limited as they are good sources of nutrients such as vitamin A, vitamin C, vitamin D, potassium, calcium, etc. Therefore, a common strategy to reduce total sugar intake is to avoid consumption of high-sugary discretionary foods, or choose healthier alternatives where sugars are replaced with non-nutritive sweeteners (NNS).
Sugar alcohols (e.g., sorbitol, xylitol, maltitol, etc.) are a group of NNS that provide sweetness that is comparable to table sugars [9]. Although sugar alcohols contain energy, these sweeteners are not well-digested and absorbed by the body and; therefore, provide minimal energy when consumed [10]. Another category of NNS possesses high sweetness potency [9] and some examples from this category include acesulfame potassium, alitame, rebaudioside A, aspartame, cyclamate, neotame, saccharin and sucralose. Because of the high sweetness potency, a very small amount of NNS is required to match the sweetness levels provided by sugars (often in milligrams). Hence, these sweeteners provide little or no energy to the body. All NNS have different chemical structures and their metabolism and health implications have been extensively discussed in a previous review [11]. Of all NNS, sucralose has a chemical structure that has the closest resemblance to sucrose (C12H22O11), where three hydrogen atoms in sucrose are replaced with chlorine in sucralose (C12H19Cl3O8). Sucralose is pH and heat-stable, and it is widely used in food products that requires heat application [12,13].
Currently, eight NNS (i.e., advantame, aspartame, acesulfame-K, neotame, saccharin, sucralose, stevia and monk fruit extracts) are considered to be generally recognized as safe (GRAS) by the US Food and Drug Authority and available to consumers [14]. Unlike sugars, NNS do not increase blood glucose [15]. However, there are also concerns that NNS may stimulate appetite and food intake, and subsequently weight gain [16,17]. These concerns appear to be supported by findings from a number of epidemiological studies, where the consumption of NNS foods or beverages was associated with higher body weight [18,19]. However, scientists also pointed out that such relationship may be reverse-causation, where people with higher body weight tend to include diet products to assist in weight management. Therefore, a better understanding of how NNS may influence acute energy balance, namely energy expenditure and energy intake, is needed.
The primary aim of this study was to compare energy expenditure and carbohydrate oxidation patterns after the consumption of test foods sweetened with table sugar (SUCROSE), sucralose (NNS), or sucralose but matched SUCROSE in energy and carbohydrate content with the addition of bland carbohydrate maltodextrin (MALT). The secondary aim was to compare appetitive responses and total daily intake following these test foods.
| null | null |
3. Results
|
Fifteen participants were recruited and 11 completed the study. Four withdrew from the study prior to the commencement of study due to difficulty in scheduling study visits. Participants who completed all test visits were eight males three females, aged = 24.9 ± 6.1 years, baseline body weight = 72.9 ± 19.0 kg (males = 81.0 ± 19.8 kg; females = 54.8 ± 8.4 kg), BMI = 25.0 ± 4.7 kgm−2 (males = 26.4 ± 5.5 kgm−2; females = 21.5 ± 3.1 kgm−2), and body fat = 20.3% ± 4.7% (males = 20.3% ± 8.7%; females = 22.3% ± 7.9%). Throughout all test visits, participants’ body weight did not change significantly from baseline: visit 1 = 0.0 ± 0.9 kg, visit 2 = −0.2 ± 0.8 kg, and visit 3 = 0.0 ± 0.7 kg. The jellies were rated as equally sweet by participants after consumption: SUCROSE = 62 ± 17 mm, NNS = 61 ± 13 mm, MALT = 64 ± 21 mm (repeated measures ANOVA, p = 0.911).
Table 1 summarises the unadjusted and adjusted (for body weight) values for total EE, carbohydrate and fat oxidation over the 90 min measurement period using an indirect calorimeter following test food was ingestion. The unadjusted variables were significantly different between all test foods (p < 0.05); the 90 min EE was almost statistically significant after adjustment for body weight (p = 0.050). The unadjusted EE were significantly higher in both SUCROSE and MALT than NNS. Carbohydrate oxidation rates were SUCROSE > MALT > NNS, and fat oxidation was significantly lower in SUCROSE than MALT and NNS.
As resting EE and carbohydrate oxidation rates were not significantly different between all study visits, data on these outcomes were also presented as patterns of changes from resting values after the ingestion of test foods in Figure 2. There were significant time effects on EE changes (p = 0.001) but they did not differ between test foods (F (8.3, 125.0) = 0.811, p = 0.599) (Figure 2A). However, significant interaction effects were found for carbohydrate oxidation (F (10.2, 153.5) = 6.117, p < 0.001) during the 90 min postprandial assessment (Figure 2B).
Appetite ratings pre- and post-test food ingestion, as well as pre- and post-meal challenge are presented in Table 2. No significant interaction effects of test foods on appetite ratings were found. Food intakes at meal challenge (bread with jam, p = 0.863; bread with ham, p = 0.874), in a free-living environment (p = 0.585), and total daily intake including test foods (p = 0.838) did not differ between test foods (Figure 3).
|
5. Conclusions
|
This pilot study provides early findings that the replacement of sugars with NNS during food product formulation, with or without accompanying carbohydrate from maltodextrin, may have implications on human energy balance, especially human energy expenditure and carbohydrate oxidation patterns. Acutely, humans appear to compensate for energy shortfall from NNS but did not promote appetite or overeating beyond that. Findings from this pilot study should be verified and confirmed with bigger clinical trials in the future.
|
[
"2. Materials and Methods",
"2.1. Study Design",
"2.2. Experimental Protocol",
"2.4. Study Test Foods",
"2.5. Measures",
"2.6. Statistical Analysis"
] |
[
" 2.1. Study Design This was an acute randomised, crossover study that included one baseline visit and three test visits.\nThis was an acute randomised, crossover study that included one baseline visit and three test visits.\n 2.2. Experimental Protocol At baseline, participants provided their written consent and had their height, body weight and eating behaviours assessed. Prior to each test day, the participants were reminded to avoid alcohol and strenuous exercise, consume their habitual dinner, and fast overnight for 10 h. On the test days, participants arrived between 08:00 and 09:00 am, voided urine, and had their body weight measured in a standing position with minimal clothing on. Following that, the participants rested for 30 min before their resting energy expenditure (REE) was assessed for 30 min. After REE, the participants consumed a test food within 10 min, followed by the measurements of postprandial gaseous exchanges (oxygen consumption and carbon dioxide production) for 90 min using the same indirect calorimeter, with a 10 min break every 30 min indirect calorimeter measurement, where the participants remained in a supine position without the hood. After the total 90 min calorimeter measurements, participants completed a meal challenge, where they were provided with 300 ml of plain water and bread with jam (sweet) and ham (savoury) toppings. Participants were asked to eat until they were comfortably full, which was defined as a fullness level where participants will not be eating other foods in the subsequent 3 to 4 h [20,21]. Appetite sensations were assessed pre- and post-consumption of test foods, as well as pre- and post-meal challenge, and sweetness levels of test foods were assessed immediately after ingestion of test foods. After the meal challenge, participants recorded food intake to determine their free-living energy intake for the rest of the day. The experimental protocol on test day is depicted in Figure 1. Test visits were repeated at least three days apart to normalize habitual food intake for males, and four weeks for females to ensure that experiment was performed at the same phase of the menstrual cycle, until all test foods were consumed. Participants were also asked to maintain their habitual dietary intake and physical activity level during the washout period. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Human Research Ethics Committee (approval number #0000033058). Written informed consent was obtained from all participants, and they were able to withdraw from the study at any time without consequences. This trial has also been registered with the Australian and New Zealand Clinical Trial Registry (ACTRN12614000879662).\nAt baseline, participants provided their written consent and had their height, body weight and eating behaviours assessed. Prior to each test day, the participants were reminded to avoid alcohol and strenuous exercise, consume their habitual dinner, and fast overnight for 10 h. On the test days, participants arrived between 08:00 and 09:00 am, voided urine, and had their body weight measured in a standing position with minimal clothing on. Following that, the participants rested for 30 min before their resting energy expenditure (REE) was assessed for 30 min. After REE, the participants consumed a test food within 10 min, followed by the measurements of postprandial gaseous exchanges (oxygen consumption and carbon dioxide production) for 90 min using the same indirect calorimeter, with a 10 min break every 30 min indirect calorimeter measurement, where the participants remained in a supine position without the hood. After the total 90 min calorimeter measurements, participants completed a meal challenge, where they were provided with 300 ml of plain water and bread with jam (sweet) and ham (savoury) toppings. Participants were asked to eat until they were comfortably full, which was defined as a fullness level where participants will not be eating other foods in the subsequent 3 to 4 h [20,21]. Appetite sensations were assessed pre- and post-consumption of test foods, as well as pre- and post-meal challenge, and sweetness levels of test foods were assessed immediately after ingestion of test foods. After the meal challenge, participants recorded food intake to determine their free-living energy intake for the rest of the day. The experimental protocol on test day is depicted in Figure 1. Test visits were repeated at least three days apart to normalize habitual food intake for males, and four weeks for females to ensure that experiment was performed at the same phase of the menstrual cycle, until all test foods were consumed. Participants were also asked to maintain their habitual dietary intake and physical activity level during the washout period. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Human Research Ethics Committee (approval number #0000033058). Written informed consent was obtained from all participants, and they were able to withdraw from the study at any time without consequences. This trial has also been registered with the Australian and New Zealand Clinical Trial Registry (ACTRN12614000879662).\n 2.3. Study Participants Participants were recruited from both the University and the local community. The inclusion criteria of this study were: (1) adults males (18–65 years old) or females (18–45 years old, pre-menopausal), (2) non-smokers, (3) normal or overweight (BMI 18.5–30.0 kgm−2), (4) weight changes of <5 kg in the past 3 months, (5) no diseases or not taking any medications that affect metabolism and appetite, and (6) no food allergies and have consumed diet food products containing NNS before.\nParticipants were recruited from both the University and the local community. The inclusion criteria of this study were: (1) adults males (18–65 years old) or females (18–45 years old, pre-menopausal), (2) non-smokers, (3) normal or overweight (BMI 18.5–30.0 kgm−2), (4) weight changes of <5 kg in the past 3 months, (5) no diseases or not taking any medications that affect metabolism and appetite, and (6) no food allergies and have consumed diet food products containing NNS before.\n 2.4. Study Test Foods This study included three types of jelly made with 100 g low-fat milk (274 kJ, 5.8 g carbohydrate, 7.7 g protein, 1.2 g fat) and 4.7 g gelling powder (carrageenan, brand: Jel-it-in, Queen Fine Foods Pty. Ltd., Alderley, QLD, Australia), sweetened with: (1) SUCROSE: 50 g sucrose (sweet carbohydrate), (2) NNS: 120 mg of sucralose only (sweet, no carbohydrate), and (3) MALT: 120 mg sucralose and matched 50 g carbohydrate as bland maltodextrin (Poly-Joule, Nutricia) (sweet and carbohydrate). The mixture was heated to dissolve all ingredients. The amount of sucralose used in MALT and NNS provided equal level of sweetness compared to SUCROSE, as determined through a sensory evaluation prior to this experiment.\nBoth SUCROSE and NNS test foods were equally sweet, but they differed in energy and carbohydrate content and may; therefore, have differential effects on human energy expenditure and carbohydrate oxidation in this study [22]. For this reason, a third test food, MALT, was included and its sweet taste came from sucralose but it has same energy and carbohydrate content (from maltodextrin) as the SUCROSE test food. Maltodextrin is a common ingredient used in food productions, is bland and hence did not alter the sweetness levels when added to the NNS test food containing 120 mg of sucralose. Therefore, SUCROSE and MALT were matched in sweetness levels, energy, and carbohydrate content to allow fair comparisons of metabolic and appetitive responses between types of sweeteners. NNS test food was chosen to examine the effects of non-nutritive sweetener alone. The order of test food consumption was randomised using an online research randomizer (www.randomizer.org) and participants were blinded to the type of test food they received at each test visit.\nThis study included three types of jelly made with 100 g low-fat milk (274 kJ, 5.8 g carbohydrate, 7.7 g protein, 1.2 g fat) and 4.7 g gelling powder (carrageenan, brand: Jel-it-in, Queen Fine Foods Pty. Ltd., Alderley, QLD, Australia), sweetened with: (1) SUCROSE: 50 g sucrose (sweet carbohydrate), (2) NNS: 120 mg of sucralose only (sweet, no carbohydrate), and (3) MALT: 120 mg sucralose and matched 50 g carbohydrate as bland maltodextrin (Poly-Joule, Nutricia) (sweet and carbohydrate). The mixture was heated to dissolve all ingredients. The amount of sucralose used in MALT and NNS provided equal level of sweetness compared to SUCROSE, as determined through a sensory evaluation prior to this experiment.\nBoth SUCROSE and NNS test foods were equally sweet, but they differed in energy and carbohydrate content and may; therefore, have differential effects on human energy expenditure and carbohydrate oxidation in this study [22]. For this reason, a third test food, MALT, was included and its sweet taste came from sucralose but it has same energy and carbohydrate content (from maltodextrin) as the SUCROSE test food. Maltodextrin is a common ingredient used in food productions, is bland and hence did not alter the sweetness levels when added to the NNS test food containing 120 mg of sucralose. Therefore, SUCROSE and MALT were matched in sweetness levels, energy, and carbohydrate content to allow fair comparisons of metabolic and appetitive responses between types of sweeteners. NNS test food was chosen to examine the effects of non-nutritive sweetener alone. The order of test food consumption was randomised using an online research randomizer (www.randomizer.org) and participants were blinded to the type of test food they received at each test visit.\n 2.5. Measures Anthropometry: Height was measured with a Seca stadiometer (to the nearest 1 mm) and body weight with a Tanita scale (Tanita BF-680, Tanita Inc., Japan, to the nearest 0.1 kg). Height and weight measurements were taken in duplicates and averaged. A third measurement was taken when height and weight measurements differed by more than 5 mm or 0.5 kg, respectively, and the average of the two closest measurements was taken. Eating behaviour was assessed at baseline using the validated three-factor eating questionnaire [23] to eliminate volunteers who may have unusual eating behaviour that could affect appetite ratings and food intake during meal challenge.\nEnergy expenditure and substrate oxidation (primary outcomes): Energy expenditure and substrate oxidation was assessed using a ventilated-hood indirect calorimeter (TrueMax 2400, Parvomedic Inc., Sandy, UT, USA). The machine was calibrated prior to each test visit using a standard 3.0 L gas syringe and standard gas (16.00% O2, 0.986% CO2 and balance nitrogen) to ensure accuracy of readings. Measurements were converted to standard temperature, pressure, and dry. Continuous gaseous exchanges were calculated as 10 min averages. For resting energy expenditure measurement, the final 20 min (stable state) of the 30 min measurement was used. Oxygen consumption and carbon dioxide production were used to calculate energy expenditure [24] and non-protein substrate oxidation rates [25] using previously published equations. \nAppetite and food intake (secondary outcomes): The appetite ratings of participants were assessed with validated 100-mm visual analogue scales (VAS) [26]. Sweetness level of test foods was also assessed using a horizontal 100-mm VAS anchored at “not sweet at all” on the left and “extremely sweet” on the right. A food diary was used to record free-living food intake for the remaining day. An explanatory page with instructions on how to document food intake accurately was provided with the diary, and food diaries were checked by researchers for completeness when they were returned. Dietary analysis was performed using the AUSNUT 2013 database in FoodWorks Professional (Xyris Software Pty. Ltd., Spring Hill, QLD, Australia).\nAnthropometry: Height was measured with a Seca stadiometer (to the nearest 1 mm) and body weight with a Tanita scale (Tanita BF-680, Tanita Inc., Japan, to the nearest 0.1 kg). Height and weight measurements were taken in duplicates and averaged. A third measurement was taken when height and weight measurements differed by more than 5 mm or 0.5 kg, respectively, and the average of the two closest measurements was taken. Eating behaviour was assessed at baseline using the validated three-factor eating questionnaire [23] to eliminate volunteers who may have unusual eating behaviour that could affect appetite ratings and food intake during meal challenge.\nEnergy expenditure and substrate oxidation (primary outcomes): Energy expenditure and substrate oxidation was assessed using a ventilated-hood indirect calorimeter (TrueMax 2400, Parvomedic Inc., Sandy, UT, USA). The machine was calibrated prior to each test visit using a standard 3.0 L gas syringe and standard gas (16.00% O2, 0.986% CO2 and balance nitrogen) to ensure accuracy of readings. Measurements were converted to standard temperature, pressure, and dry. Continuous gaseous exchanges were calculated as 10 min averages. For resting energy expenditure measurement, the final 20 min (stable state) of the 30 min measurement was used. Oxygen consumption and carbon dioxide production were used to calculate energy expenditure [24] and non-protein substrate oxidation rates [25] using previously published equations. \nAppetite and food intake (secondary outcomes): The appetite ratings of participants were assessed with validated 100-mm visual analogue scales (VAS) [26]. Sweetness level of test foods was also assessed using a horizontal 100-mm VAS anchored at “not sweet at all” on the left and “extremely sweet” on the right. A food diary was used to record free-living food intake for the remaining day. An explanatory page with instructions on how to document food intake accurately was provided with the diary, and food diaries were checked by researchers for completeness when they were returned. Dietary analysis was performed using the AUSNUT 2013 database in FoodWorks Professional (Xyris Software Pty. Ltd., Spring Hill, QLD, Australia).\n 2.6. Statistical Analysis Descriptive statistics are presented as mean ± standard deviation. Postprandial EE and carbohydrate oxidation were calculated and presented as changes from baseline, as well as total over 90 min study period as area under the curve (AUC) using trapezoidal rules. The 90 min EE and carbohydrate oxidation was also adjusted for body weight taken on the test days, using regression residuals from a regression equation as following:Y = a0 + a1 × Body Weight (kg),\nwhere a0 is a constant and a1 is a coefficient.\nThe comparisons AUC and changes in EE, carbohydrate, appetite and sweetness ratings, and dietary intake following the consumption of three test foods were compared using general linear model for repeated measures ANOVA with Bonferroni corrections. All statistical analyses were performed with statistical package SPSS (version 23.0.0, IBM Corp, Armonk, NY, USA). As there was no previous study comparing the postprandial substrate oxidation effects induced by different sweeteners, sample size was guided by a study we previously conducted [27], where significant differences in substrate oxidation were detected with a study sample of 12 participants. Fifteen participants were targeted to account for attrition of 20%.\nDescriptive statistics are presented as mean ± standard deviation. Postprandial EE and carbohydrate oxidation were calculated and presented as changes from baseline, as well as total over 90 min study period as area under the curve (AUC) using trapezoidal rules. The 90 min EE and carbohydrate oxidation was also adjusted for body weight taken on the test days, using regression residuals from a regression equation as following:Y = a0 + a1 × Body Weight (kg),\nwhere a0 is a constant and a1 is a coefficient.\nThe comparisons AUC and changes in EE, carbohydrate, appetite and sweetness ratings, and dietary intake following the consumption of three test foods were compared using general linear model for repeated measures ANOVA with Bonferroni corrections. All statistical analyses were performed with statistical package SPSS (version 23.0.0, IBM Corp, Armonk, NY, USA). As there was no previous study comparing the postprandial substrate oxidation effects induced by different sweeteners, sample size was guided by a study we previously conducted [27], where significant differences in substrate oxidation were detected with a study sample of 12 participants. Fifteen participants were targeted to account for attrition of 20%.",
"This was an acute randomised, crossover study that included one baseline visit and three test visits.",
"At baseline, participants provided their written consent and had their height, body weight and eating behaviours assessed. Prior to each test day, the participants were reminded to avoid alcohol and strenuous exercise, consume their habitual dinner, and fast overnight for 10 h. On the test days, participants arrived between 08:00 and 09:00 am, voided urine, and had their body weight measured in a standing position with minimal clothing on. Following that, the participants rested for 30 min before their resting energy expenditure (REE) was assessed for 30 min. After REE, the participants consumed a test food within 10 min, followed by the measurements of postprandial gaseous exchanges (oxygen consumption and carbon dioxide production) for 90 min using the same indirect calorimeter, with a 10 min break every 30 min indirect calorimeter measurement, where the participants remained in a supine position without the hood. After the total 90 min calorimeter measurements, participants completed a meal challenge, where they were provided with 300 ml of plain water and bread with jam (sweet) and ham (savoury) toppings. Participants were asked to eat until they were comfortably full, which was defined as a fullness level where participants will not be eating other foods in the subsequent 3 to 4 h [20,21]. Appetite sensations were assessed pre- and post-consumption of test foods, as well as pre- and post-meal challenge, and sweetness levels of test foods were assessed immediately after ingestion of test foods. After the meal challenge, participants recorded food intake to determine their free-living energy intake for the rest of the day. The experimental protocol on test day is depicted in Figure 1. Test visits were repeated at least three days apart to normalize habitual food intake for males, and four weeks for females to ensure that experiment was performed at the same phase of the menstrual cycle, until all test foods were consumed. Participants were also asked to maintain their habitual dietary intake and physical activity level during the washout period. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Human Research Ethics Committee (approval number #0000033058). Written informed consent was obtained from all participants, and they were able to withdraw from the study at any time without consequences. This trial has also been registered with the Australian and New Zealand Clinical Trial Registry (ACTRN12614000879662).",
"This study included three types of jelly made with 100 g low-fat milk (274 kJ, 5.8 g carbohydrate, 7.7 g protein, 1.2 g fat) and 4.7 g gelling powder (carrageenan, brand: Jel-it-in, Queen Fine Foods Pty. Ltd., Alderley, QLD, Australia), sweetened with: (1) SUCROSE: 50 g sucrose (sweet carbohydrate), (2) NNS: 120 mg of sucralose only (sweet, no carbohydrate), and (3) MALT: 120 mg sucralose and matched 50 g carbohydrate as bland maltodextrin (Poly-Joule, Nutricia) (sweet and carbohydrate). The mixture was heated to dissolve all ingredients. The amount of sucralose used in MALT and NNS provided equal level of sweetness compared to SUCROSE, as determined through a sensory evaluation prior to this experiment.\nBoth SUCROSE and NNS test foods were equally sweet, but they differed in energy and carbohydrate content and may; therefore, have differential effects on human energy expenditure and carbohydrate oxidation in this study [22]. For this reason, a third test food, MALT, was included and its sweet taste came from sucralose but it has same energy and carbohydrate content (from maltodextrin) as the SUCROSE test food. Maltodextrin is a common ingredient used in food productions, is bland and hence did not alter the sweetness levels when added to the NNS test food containing 120 mg of sucralose. Therefore, SUCROSE and MALT were matched in sweetness levels, energy, and carbohydrate content to allow fair comparisons of metabolic and appetitive responses between types of sweeteners. NNS test food was chosen to examine the effects of non-nutritive sweetener alone. The order of test food consumption was randomised using an online research randomizer (www.randomizer.org) and participants were blinded to the type of test food they received at each test visit.",
"Anthropometry: Height was measured with a Seca stadiometer (to the nearest 1 mm) and body weight with a Tanita scale (Tanita BF-680, Tanita Inc., Japan, to the nearest 0.1 kg). Height and weight measurements were taken in duplicates and averaged. A third measurement was taken when height and weight measurements differed by more than 5 mm or 0.5 kg, respectively, and the average of the two closest measurements was taken. Eating behaviour was assessed at baseline using the validated three-factor eating questionnaire [23] to eliminate volunteers who may have unusual eating behaviour that could affect appetite ratings and food intake during meal challenge.\nEnergy expenditure and substrate oxidation (primary outcomes): Energy expenditure and substrate oxidation was assessed using a ventilated-hood indirect calorimeter (TrueMax 2400, Parvomedic Inc., Sandy, UT, USA). The machine was calibrated prior to each test visit using a standard 3.0 L gas syringe and standard gas (16.00% O2, 0.986% CO2 and balance nitrogen) to ensure accuracy of readings. Measurements were converted to standard temperature, pressure, and dry. Continuous gaseous exchanges were calculated as 10 min averages. For resting energy expenditure measurement, the final 20 min (stable state) of the 30 min measurement was used. Oxygen consumption and carbon dioxide production were used to calculate energy expenditure [24] and non-protein substrate oxidation rates [25] using previously published equations. \nAppetite and food intake (secondary outcomes): The appetite ratings of participants were assessed with validated 100-mm visual analogue scales (VAS) [26]. Sweetness level of test foods was also assessed using a horizontal 100-mm VAS anchored at “not sweet at all” on the left and “extremely sweet” on the right. A food diary was used to record free-living food intake for the remaining day. An explanatory page with instructions on how to document food intake accurately was provided with the diary, and food diaries were checked by researchers for completeness when they were returned. Dietary analysis was performed using the AUSNUT 2013 database in FoodWorks Professional (Xyris Software Pty. Ltd., Spring Hill, QLD, Australia).",
"Descriptive statistics are presented as mean ± standard deviation. Postprandial EE and carbohydrate oxidation were calculated and presented as changes from baseline, as well as total over 90 min study period as area under the curve (AUC) using trapezoidal rules. The 90 min EE and carbohydrate oxidation was also adjusted for body weight taken on the test days, using regression residuals from a regression equation as following:Y = a0 + a1 × Body Weight (kg),\nwhere a0 is a constant and a1 is a coefficient.\nThe comparisons AUC and changes in EE, carbohydrate, appetite and sweetness ratings, and dietary intake following the consumption of three test foods were compared using general linear model for repeated measures ANOVA with Bonferroni corrections. All statistical analyses were performed with statistical package SPSS (version 23.0.0, IBM Corp, Armonk, NY, USA). As there was no previous study comparing the postprandial substrate oxidation effects induced by different sweeteners, sample size was guided by a study we previously conducted [27], where significant differences in substrate oxidation were detected with a study sample of 12 participants. Fifteen participants were targeted to account for attrition of 20%."
] |
[
null,
null,
null,
null,
null,
null
] |
[
"1. Introduction",
"2. Materials and Methods",
"2.1. Study Design",
"2.2. Experimental Protocol",
"2.3. Study Participants",
"2.4. Study Test Foods",
"2.5. Measures",
"2.6. Statistical Analysis",
"3. Results",
"4. Discussion",
"5. Conclusions"
] |
[
"The global prevalence of overweight and obesity is increasing and it increases the risk of several chronic diseases such as type 2 diabetes and cardiovascular disease [1,2]. Together with its co-morbidities, overweight and obesity increases mortality rates and adds burden to the healthcare system.\nAn increase in energy intake is one of several factors responsible for the rising prevalence of overweight and obesity [3]. In recent years, epidemiological studies have linked obesity with dietary sugar intake [4,5], and concluded that reducing sugar intake reduces prevalence of overweight and obesity [6]. The World Health Organization recommends a reduction in free sugar intake to no more than 10% of total daily intake [7]. A number of countries such as Mexico, Brazil and France have successfully reduced the demand for sugar-sweetened beverages through the introduction of sugar tax, and this may drive the reduction of added sugar during food processing by the food industry [8].\nThe intake of natural sugars from major food groups such as fruits, vegetables and dairy foods should not be limited as they are good sources of nutrients such as vitamin A, vitamin C, vitamin D, potassium, calcium, etc. Therefore, a common strategy to reduce total sugar intake is to avoid consumption of high-sugary discretionary foods, or choose healthier alternatives where sugars are replaced with non-nutritive sweeteners (NNS).\nSugar alcohols (e.g., sorbitol, xylitol, maltitol, etc.) are a group of NNS that provide sweetness that is comparable to table sugars [9]. Although sugar alcohols contain energy, these sweeteners are not well-digested and absorbed by the body and; therefore, provide minimal energy when consumed [10]. Another category of NNS possesses high sweetness potency [9] and some examples from this category include acesulfame potassium, alitame, rebaudioside A, aspartame, cyclamate, neotame, saccharin and sucralose. Because of the high sweetness potency, a very small amount of NNS is required to match the sweetness levels provided by sugars (often in milligrams). Hence, these sweeteners provide little or no energy to the body. All NNS have different chemical structures and their metabolism and health implications have been extensively discussed in a previous review [11]. Of all NNS, sucralose has a chemical structure that has the closest resemblance to sucrose (C12H22O11), where three hydrogen atoms in sucrose are replaced with chlorine in sucralose (C12H19Cl3O8). Sucralose is pH and heat-stable, and it is widely used in food products that requires heat application [12,13].\nCurrently, eight NNS (i.e., advantame, aspartame, acesulfame-K, neotame, saccharin, sucralose, stevia and monk fruit extracts) are considered to be generally recognized as safe (GRAS) by the US Food and Drug Authority and available to consumers [14]. Unlike sugars, NNS do not increase blood glucose [15]. However, there are also concerns that NNS may stimulate appetite and food intake, and subsequently weight gain [16,17]. These concerns appear to be supported by findings from a number of epidemiological studies, where the consumption of NNS foods or beverages was associated with higher body weight [18,19]. However, scientists also pointed out that such relationship may be reverse-causation, where people with higher body weight tend to include diet products to assist in weight management. Therefore, a better understanding of how NNS may influence acute energy balance, namely energy expenditure and energy intake, is needed.\nThe primary aim of this study was to compare energy expenditure and carbohydrate oxidation patterns after the consumption of test foods sweetened with table sugar (SUCROSE), sucralose (NNS), or sucralose but matched SUCROSE in energy and carbohydrate content with the addition of bland carbohydrate maltodextrin (MALT). The secondary aim was to compare appetitive responses and total daily intake following these test foods.",
" 2.1. Study Design This was an acute randomised, crossover study that included one baseline visit and three test visits.\nThis was an acute randomised, crossover study that included one baseline visit and three test visits.\n 2.2. Experimental Protocol At baseline, participants provided their written consent and had their height, body weight and eating behaviours assessed. Prior to each test day, the participants were reminded to avoid alcohol and strenuous exercise, consume their habitual dinner, and fast overnight for 10 h. On the test days, participants arrived between 08:00 and 09:00 am, voided urine, and had their body weight measured in a standing position with minimal clothing on. Following that, the participants rested for 30 min before their resting energy expenditure (REE) was assessed for 30 min. After REE, the participants consumed a test food within 10 min, followed by the measurements of postprandial gaseous exchanges (oxygen consumption and carbon dioxide production) for 90 min using the same indirect calorimeter, with a 10 min break every 30 min indirect calorimeter measurement, where the participants remained in a supine position without the hood. After the total 90 min calorimeter measurements, participants completed a meal challenge, where they were provided with 300 ml of plain water and bread with jam (sweet) and ham (savoury) toppings. Participants were asked to eat until they were comfortably full, which was defined as a fullness level where participants will not be eating other foods in the subsequent 3 to 4 h [20,21]. Appetite sensations were assessed pre- and post-consumption of test foods, as well as pre- and post-meal challenge, and sweetness levels of test foods were assessed immediately after ingestion of test foods. After the meal challenge, participants recorded food intake to determine their free-living energy intake for the rest of the day. The experimental protocol on test day is depicted in Figure 1. Test visits were repeated at least three days apart to normalize habitual food intake for males, and four weeks for females to ensure that experiment was performed at the same phase of the menstrual cycle, until all test foods were consumed. Participants were also asked to maintain their habitual dietary intake and physical activity level during the washout period. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Human Research Ethics Committee (approval number #0000033058). Written informed consent was obtained from all participants, and they were able to withdraw from the study at any time without consequences. This trial has also been registered with the Australian and New Zealand Clinical Trial Registry (ACTRN12614000879662).\nAt baseline, participants provided their written consent and had their height, body weight and eating behaviours assessed. Prior to each test day, the participants were reminded to avoid alcohol and strenuous exercise, consume their habitual dinner, and fast overnight for 10 h. On the test days, participants arrived between 08:00 and 09:00 am, voided urine, and had their body weight measured in a standing position with minimal clothing on. Following that, the participants rested for 30 min before their resting energy expenditure (REE) was assessed for 30 min. After REE, the participants consumed a test food within 10 min, followed by the measurements of postprandial gaseous exchanges (oxygen consumption and carbon dioxide production) for 90 min using the same indirect calorimeter, with a 10 min break every 30 min indirect calorimeter measurement, where the participants remained in a supine position without the hood. After the total 90 min calorimeter measurements, participants completed a meal challenge, where they were provided with 300 ml of plain water and bread with jam (sweet) and ham (savoury) toppings. Participants were asked to eat until they were comfortably full, which was defined as a fullness level where participants will not be eating other foods in the subsequent 3 to 4 h [20,21]. Appetite sensations were assessed pre- and post-consumption of test foods, as well as pre- and post-meal challenge, and sweetness levels of test foods were assessed immediately after ingestion of test foods. After the meal challenge, participants recorded food intake to determine their free-living energy intake for the rest of the day. The experimental protocol on test day is depicted in Figure 1. Test visits were repeated at least three days apart to normalize habitual food intake for males, and four weeks for females to ensure that experiment was performed at the same phase of the menstrual cycle, until all test foods were consumed. Participants were also asked to maintain their habitual dietary intake and physical activity level during the washout period. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Human Research Ethics Committee (approval number #0000033058). Written informed consent was obtained from all participants, and they were able to withdraw from the study at any time without consequences. This trial has also been registered with the Australian and New Zealand Clinical Trial Registry (ACTRN12614000879662).\n 2.3. Study Participants Participants were recruited from both the University and the local community. The inclusion criteria of this study were: (1) adults males (18–65 years old) or females (18–45 years old, pre-menopausal), (2) non-smokers, (3) normal or overweight (BMI 18.5–30.0 kgm−2), (4) weight changes of <5 kg in the past 3 months, (5) no diseases or not taking any medications that affect metabolism and appetite, and (6) no food allergies and have consumed diet food products containing NNS before.\nParticipants were recruited from both the University and the local community. The inclusion criteria of this study were: (1) adults males (18–65 years old) or females (18–45 years old, pre-menopausal), (2) non-smokers, (3) normal or overweight (BMI 18.5–30.0 kgm−2), (4) weight changes of <5 kg in the past 3 months, (5) no diseases or not taking any medications that affect metabolism and appetite, and (6) no food allergies and have consumed diet food products containing NNS before.\n 2.4. Study Test Foods This study included three types of jelly made with 100 g low-fat milk (274 kJ, 5.8 g carbohydrate, 7.7 g protein, 1.2 g fat) and 4.7 g gelling powder (carrageenan, brand: Jel-it-in, Queen Fine Foods Pty. Ltd., Alderley, QLD, Australia), sweetened with: (1) SUCROSE: 50 g sucrose (sweet carbohydrate), (2) NNS: 120 mg of sucralose only (sweet, no carbohydrate), and (3) MALT: 120 mg sucralose and matched 50 g carbohydrate as bland maltodextrin (Poly-Joule, Nutricia) (sweet and carbohydrate). The mixture was heated to dissolve all ingredients. The amount of sucralose used in MALT and NNS provided equal level of sweetness compared to SUCROSE, as determined through a sensory evaluation prior to this experiment.\nBoth SUCROSE and NNS test foods were equally sweet, but they differed in energy and carbohydrate content and may; therefore, have differential effects on human energy expenditure and carbohydrate oxidation in this study [22]. For this reason, a third test food, MALT, was included and its sweet taste came from sucralose but it has same energy and carbohydrate content (from maltodextrin) as the SUCROSE test food. Maltodextrin is a common ingredient used in food productions, is bland and hence did not alter the sweetness levels when added to the NNS test food containing 120 mg of sucralose. Therefore, SUCROSE and MALT were matched in sweetness levels, energy, and carbohydrate content to allow fair comparisons of metabolic and appetitive responses between types of sweeteners. NNS test food was chosen to examine the effects of non-nutritive sweetener alone. The order of test food consumption was randomised using an online research randomizer (www.randomizer.org) and participants were blinded to the type of test food they received at each test visit.\nThis study included three types of jelly made with 100 g low-fat milk (274 kJ, 5.8 g carbohydrate, 7.7 g protein, 1.2 g fat) and 4.7 g gelling powder (carrageenan, brand: Jel-it-in, Queen Fine Foods Pty. Ltd., Alderley, QLD, Australia), sweetened with: (1) SUCROSE: 50 g sucrose (sweet carbohydrate), (2) NNS: 120 mg of sucralose only (sweet, no carbohydrate), and (3) MALT: 120 mg sucralose and matched 50 g carbohydrate as bland maltodextrin (Poly-Joule, Nutricia) (sweet and carbohydrate). The mixture was heated to dissolve all ingredients. The amount of sucralose used in MALT and NNS provided equal level of sweetness compared to SUCROSE, as determined through a sensory evaluation prior to this experiment.\nBoth SUCROSE and NNS test foods were equally sweet, but they differed in energy and carbohydrate content and may; therefore, have differential effects on human energy expenditure and carbohydrate oxidation in this study [22]. For this reason, a third test food, MALT, was included and its sweet taste came from sucralose but it has same energy and carbohydrate content (from maltodextrin) as the SUCROSE test food. Maltodextrin is a common ingredient used in food productions, is bland and hence did not alter the sweetness levels when added to the NNS test food containing 120 mg of sucralose. Therefore, SUCROSE and MALT were matched in sweetness levels, energy, and carbohydrate content to allow fair comparisons of metabolic and appetitive responses between types of sweeteners. NNS test food was chosen to examine the effects of non-nutritive sweetener alone. The order of test food consumption was randomised using an online research randomizer (www.randomizer.org) and participants were blinded to the type of test food they received at each test visit.\n 2.5. Measures Anthropometry: Height was measured with a Seca stadiometer (to the nearest 1 mm) and body weight with a Tanita scale (Tanita BF-680, Tanita Inc., Japan, to the nearest 0.1 kg). Height and weight measurements were taken in duplicates and averaged. A third measurement was taken when height and weight measurements differed by more than 5 mm or 0.5 kg, respectively, and the average of the two closest measurements was taken. Eating behaviour was assessed at baseline using the validated three-factor eating questionnaire [23] to eliminate volunteers who may have unusual eating behaviour that could affect appetite ratings and food intake during meal challenge.\nEnergy expenditure and substrate oxidation (primary outcomes): Energy expenditure and substrate oxidation was assessed using a ventilated-hood indirect calorimeter (TrueMax 2400, Parvomedic Inc., Sandy, UT, USA). The machine was calibrated prior to each test visit using a standard 3.0 L gas syringe and standard gas (16.00% O2, 0.986% CO2 and balance nitrogen) to ensure accuracy of readings. Measurements were converted to standard temperature, pressure, and dry. Continuous gaseous exchanges were calculated as 10 min averages. For resting energy expenditure measurement, the final 20 min (stable state) of the 30 min measurement was used. Oxygen consumption and carbon dioxide production were used to calculate energy expenditure [24] and non-protein substrate oxidation rates [25] using previously published equations. \nAppetite and food intake (secondary outcomes): The appetite ratings of participants were assessed with validated 100-mm visual analogue scales (VAS) [26]. Sweetness level of test foods was also assessed using a horizontal 100-mm VAS anchored at “not sweet at all” on the left and “extremely sweet” on the right. A food diary was used to record free-living food intake for the remaining day. An explanatory page with instructions on how to document food intake accurately was provided with the diary, and food diaries were checked by researchers for completeness when they were returned. Dietary analysis was performed using the AUSNUT 2013 database in FoodWorks Professional (Xyris Software Pty. Ltd., Spring Hill, QLD, Australia).\nAnthropometry: Height was measured with a Seca stadiometer (to the nearest 1 mm) and body weight with a Tanita scale (Tanita BF-680, Tanita Inc., Japan, to the nearest 0.1 kg). Height and weight measurements were taken in duplicates and averaged. A third measurement was taken when height and weight measurements differed by more than 5 mm or 0.5 kg, respectively, and the average of the two closest measurements was taken. Eating behaviour was assessed at baseline using the validated three-factor eating questionnaire [23] to eliminate volunteers who may have unusual eating behaviour that could affect appetite ratings and food intake during meal challenge.\nEnergy expenditure and substrate oxidation (primary outcomes): Energy expenditure and substrate oxidation was assessed using a ventilated-hood indirect calorimeter (TrueMax 2400, Parvomedic Inc., Sandy, UT, USA). The machine was calibrated prior to each test visit using a standard 3.0 L gas syringe and standard gas (16.00% O2, 0.986% CO2 and balance nitrogen) to ensure accuracy of readings. Measurements were converted to standard temperature, pressure, and dry. Continuous gaseous exchanges were calculated as 10 min averages. For resting energy expenditure measurement, the final 20 min (stable state) of the 30 min measurement was used. Oxygen consumption and carbon dioxide production were used to calculate energy expenditure [24] and non-protein substrate oxidation rates [25] using previously published equations. \nAppetite and food intake (secondary outcomes): The appetite ratings of participants were assessed with validated 100-mm visual analogue scales (VAS) [26]. Sweetness level of test foods was also assessed using a horizontal 100-mm VAS anchored at “not sweet at all” on the left and “extremely sweet” on the right. A food diary was used to record free-living food intake for the remaining day. An explanatory page with instructions on how to document food intake accurately was provided with the diary, and food diaries were checked by researchers for completeness when they were returned. Dietary analysis was performed using the AUSNUT 2013 database in FoodWorks Professional (Xyris Software Pty. Ltd., Spring Hill, QLD, Australia).\n 2.6. Statistical Analysis Descriptive statistics are presented as mean ± standard deviation. Postprandial EE and carbohydrate oxidation were calculated and presented as changes from baseline, as well as total over 90 min study period as area under the curve (AUC) using trapezoidal rules. The 90 min EE and carbohydrate oxidation was also adjusted for body weight taken on the test days, using regression residuals from a regression equation as following:Y = a0 + a1 × Body Weight (kg),\nwhere a0 is a constant and a1 is a coefficient.\nThe comparisons AUC and changes in EE, carbohydrate, appetite and sweetness ratings, and dietary intake following the consumption of three test foods were compared using general linear model for repeated measures ANOVA with Bonferroni corrections. All statistical analyses were performed with statistical package SPSS (version 23.0.0, IBM Corp, Armonk, NY, USA). As there was no previous study comparing the postprandial substrate oxidation effects induced by different sweeteners, sample size was guided by a study we previously conducted [27], where significant differences in substrate oxidation were detected with a study sample of 12 participants. Fifteen participants were targeted to account for attrition of 20%.\nDescriptive statistics are presented as mean ± standard deviation. Postprandial EE and carbohydrate oxidation were calculated and presented as changes from baseline, as well as total over 90 min study period as area under the curve (AUC) using trapezoidal rules. The 90 min EE and carbohydrate oxidation was also adjusted for body weight taken on the test days, using regression residuals from a regression equation as following:Y = a0 + a1 × Body Weight (kg),\nwhere a0 is a constant and a1 is a coefficient.\nThe comparisons AUC and changes in EE, carbohydrate, appetite and sweetness ratings, and dietary intake following the consumption of three test foods were compared using general linear model for repeated measures ANOVA with Bonferroni corrections. All statistical analyses were performed with statistical package SPSS (version 23.0.0, IBM Corp, Armonk, NY, USA). As there was no previous study comparing the postprandial substrate oxidation effects induced by different sweeteners, sample size was guided by a study we previously conducted [27], where significant differences in substrate oxidation were detected with a study sample of 12 participants. Fifteen participants were targeted to account for attrition of 20%.",
"This was an acute randomised, crossover study that included one baseline visit and three test visits.",
"At baseline, participants provided their written consent and had their height, body weight and eating behaviours assessed. Prior to each test day, the participants were reminded to avoid alcohol and strenuous exercise, consume their habitual dinner, and fast overnight for 10 h. On the test days, participants arrived between 08:00 and 09:00 am, voided urine, and had their body weight measured in a standing position with minimal clothing on. Following that, the participants rested for 30 min before their resting energy expenditure (REE) was assessed for 30 min. After REE, the participants consumed a test food within 10 min, followed by the measurements of postprandial gaseous exchanges (oxygen consumption and carbon dioxide production) for 90 min using the same indirect calorimeter, with a 10 min break every 30 min indirect calorimeter measurement, where the participants remained in a supine position without the hood. After the total 90 min calorimeter measurements, participants completed a meal challenge, where they were provided with 300 ml of plain water and bread with jam (sweet) and ham (savoury) toppings. Participants were asked to eat until they were comfortably full, which was defined as a fullness level where participants will not be eating other foods in the subsequent 3 to 4 h [20,21]. Appetite sensations were assessed pre- and post-consumption of test foods, as well as pre- and post-meal challenge, and sweetness levels of test foods were assessed immediately after ingestion of test foods. After the meal challenge, participants recorded food intake to determine their free-living energy intake for the rest of the day. The experimental protocol on test day is depicted in Figure 1. Test visits were repeated at least three days apart to normalize habitual food intake for males, and four weeks for females to ensure that experiment was performed at the same phase of the menstrual cycle, until all test foods were consumed. Participants were also asked to maintain their habitual dietary intake and physical activity level during the washout period. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Human Research Ethics Committee (approval number #0000033058). Written informed consent was obtained from all participants, and they were able to withdraw from the study at any time without consequences. This trial has also been registered with the Australian and New Zealand Clinical Trial Registry (ACTRN12614000879662).",
"Participants were recruited from both the University and the local community. The inclusion criteria of this study were: (1) adults males (18–65 years old) or females (18–45 years old, pre-menopausal), (2) non-smokers, (3) normal or overweight (BMI 18.5–30.0 kgm−2), (4) weight changes of <5 kg in the past 3 months, (5) no diseases or not taking any medications that affect metabolism and appetite, and (6) no food allergies and have consumed diet food products containing NNS before.",
"This study included three types of jelly made with 100 g low-fat milk (274 kJ, 5.8 g carbohydrate, 7.7 g protein, 1.2 g fat) and 4.7 g gelling powder (carrageenan, brand: Jel-it-in, Queen Fine Foods Pty. Ltd., Alderley, QLD, Australia), sweetened with: (1) SUCROSE: 50 g sucrose (sweet carbohydrate), (2) NNS: 120 mg of sucralose only (sweet, no carbohydrate), and (3) MALT: 120 mg sucralose and matched 50 g carbohydrate as bland maltodextrin (Poly-Joule, Nutricia) (sweet and carbohydrate). The mixture was heated to dissolve all ingredients. The amount of sucralose used in MALT and NNS provided equal level of sweetness compared to SUCROSE, as determined through a sensory evaluation prior to this experiment.\nBoth SUCROSE and NNS test foods were equally sweet, but they differed in energy and carbohydrate content and may; therefore, have differential effects on human energy expenditure and carbohydrate oxidation in this study [22]. For this reason, a third test food, MALT, was included and its sweet taste came from sucralose but it has same energy and carbohydrate content (from maltodextrin) as the SUCROSE test food. Maltodextrin is a common ingredient used in food productions, is bland and hence did not alter the sweetness levels when added to the NNS test food containing 120 mg of sucralose. Therefore, SUCROSE and MALT were matched in sweetness levels, energy, and carbohydrate content to allow fair comparisons of metabolic and appetitive responses between types of sweeteners. NNS test food was chosen to examine the effects of non-nutritive sweetener alone. The order of test food consumption was randomised using an online research randomizer (www.randomizer.org) and participants were blinded to the type of test food they received at each test visit.",
"Anthropometry: Height was measured with a Seca stadiometer (to the nearest 1 mm) and body weight with a Tanita scale (Tanita BF-680, Tanita Inc., Japan, to the nearest 0.1 kg). Height and weight measurements were taken in duplicates and averaged. A third measurement was taken when height and weight measurements differed by more than 5 mm or 0.5 kg, respectively, and the average of the two closest measurements was taken. Eating behaviour was assessed at baseline using the validated three-factor eating questionnaire [23] to eliminate volunteers who may have unusual eating behaviour that could affect appetite ratings and food intake during meal challenge.\nEnergy expenditure and substrate oxidation (primary outcomes): Energy expenditure and substrate oxidation was assessed using a ventilated-hood indirect calorimeter (TrueMax 2400, Parvomedic Inc., Sandy, UT, USA). The machine was calibrated prior to each test visit using a standard 3.0 L gas syringe and standard gas (16.00% O2, 0.986% CO2 and balance nitrogen) to ensure accuracy of readings. Measurements were converted to standard temperature, pressure, and dry. Continuous gaseous exchanges were calculated as 10 min averages. For resting energy expenditure measurement, the final 20 min (stable state) of the 30 min measurement was used. Oxygen consumption and carbon dioxide production were used to calculate energy expenditure [24] and non-protein substrate oxidation rates [25] using previously published equations. \nAppetite and food intake (secondary outcomes): The appetite ratings of participants were assessed with validated 100-mm visual analogue scales (VAS) [26]. Sweetness level of test foods was also assessed using a horizontal 100-mm VAS anchored at “not sweet at all” on the left and “extremely sweet” on the right. A food diary was used to record free-living food intake for the remaining day. An explanatory page with instructions on how to document food intake accurately was provided with the diary, and food diaries were checked by researchers for completeness when they were returned. Dietary analysis was performed using the AUSNUT 2013 database in FoodWorks Professional (Xyris Software Pty. Ltd., Spring Hill, QLD, Australia).",
"Descriptive statistics are presented as mean ± standard deviation. Postprandial EE and carbohydrate oxidation were calculated and presented as changes from baseline, as well as total over 90 min study period as area under the curve (AUC) using trapezoidal rules. The 90 min EE and carbohydrate oxidation was also adjusted for body weight taken on the test days, using regression residuals from a regression equation as following:Y = a0 + a1 × Body Weight (kg),\nwhere a0 is a constant and a1 is a coefficient.\nThe comparisons AUC and changes in EE, carbohydrate, appetite and sweetness ratings, and dietary intake following the consumption of three test foods were compared using general linear model for repeated measures ANOVA with Bonferroni corrections. All statistical analyses were performed with statistical package SPSS (version 23.0.0, IBM Corp, Armonk, NY, USA). As there was no previous study comparing the postprandial substrate oxidation effects induced by different sweeteners, sample size was guided by a study we previously conducted [27], where significant differences in substrate oxidation were detected with a study sample of 12 participants. Fifteen participants were targeted to account for attrition of 20%.",
"Fifteen participants were recruited and 11 completed the study. Four withdrew from the study prior to the commencement of study due to difficulty in scheduling study visits. Participants who completed all test visits were eight males three females, aged = 24.9 ± 6.1 years, baseline body weight = 72.9 ± 19.0 kg (males = 81.0 ± 19.8 kg; females = 54.8 ± 8.4 kg), BMI = 25.0 ± 4.7 kgm−2 (males = 26.4 ± 5.5 kgm−2; females = 21.5 ± 3.1 kgm−2), and body fat = 20.3% ± 4.7% (males = 20.3% ± 8.7%; females = 22.3% ± 7.9%). Throughout all test visits, participants’ body weight did not change significantly from baseline: visit 1 = 0.0 ± 0.9 kg, visit 2 = −0.2 ± 0.8 kg, and visit 3 = 0.0 ± 0.7 kg. The jellies were rated as equally sweet by participants after consumption: SUCROSE = 62 ± 17 mm, NNS = 61 ± 13 mm, MALT = 64 ± 21 mm (repeated measures ANOVA, p = 0.911).\nTable 1 summarises the unadjusted and adjusted (for body weight) values for total EE, carbohydrate and fat oxidation over the 90 min measurement period using an indirect calorimeter following test food was ingestion. The unadjusted variables were significantly different between all test foods (p < 0.05); the 90 min EE was almost statistically significant after adjustment for body weight (p = 0.050). The unadjusted EE were significantly higher in both SUCROSE and MALT than NNS. Carbohydrate oxidation rates were SUCROSE > MALT > NNS, and fat oxidation was significantly lower in SUCROSE than MALT and NNS.\nAs resting EE and carbohydrate oxidation rates were not significantly different between all study visits, data on these outcomes were also presented as patterns of changes from resting values after the ingestion of test foods in Figure 2. There were significant time effects on EE changes (p = 0.001) but they did not differ between test foods (F (8.3, 125.0) = 0.811, p = 0.599) (Figure 2A). However, significant interaction effects were found for carbohydrate oxidation (F (10.2, 153.5) = 6.117, p < 0.001) during the 90 min postprandial assessment (Figure 2B).\nAppetite ratings pre- and post-test food ingestion, as well as pre- and post-meal challenge are presented in Table 2. No significant interaction effects of test foods on appetite ratings were found. Food intakes at meal challenge (bread with jam, p = 0.863; bread with ham, p = 0.874), in a free-living environment (p = 0.585), and total daily intake including test foods (p = 0.838) did not differ between test foods (Figure 3).",
"This study was designed to examine how the re-formulation of a high-sugar food product with sucralose to reduce added sugar content may affect energy expenditure, carbohydrate oxidation, appetite and food intake. During the 90 min postprandial period, the EE was comparable between the SUCROSE and MALT test foods but both were higher than in NNS (Table 1 and Figure 2A). This observation was consistent with the evidence that postprandial EE is predominantly determined by the energy content of a meal [28].\nPostprandial carbohydrate oxidation rate was significantly lower in NNS than SUCROSE and MALT (Table 1), which is likely to be explained by the higher carbohydrate content in SUCROSE and MALT. The temporal carbohydrate oxidation patterns of all jellies differ significantly (Figure 2B) although NNS did not alter carbohydrate oxidation. Interestingly, SUCROSE and MALT jellies produced different carbohydrate oxidation patterns although they have matched carbohydrate content. A higher and earlier carbohydrate oxidation peak was observed in SUCROSE, which subsequently converged with the MALT carbohydrate oxidation rates from 70 min onwards. As a result, the carbohydrate oxidation rate of SUCROSE was also significantly higher than MALT over 90 min.\nThere are two potential explanations for our novel finding on the differential carbohydrate oxidation patterns between SUCROSE and MALT. In the first scenario, maltodextrin is a polysaccharide and may be digested and absorbed slower than sugars (disaccharide) in SUCROSE test food, hence producing a lower and delayed carbohydrate oxidation. A potential counterargument is that maltodextrin is considered as a short-chain carbohydrates that is rapidly digested and release glucose for absorption [29]. In the second scenario, the differential carbohydrate oxidation patterns may be explained by sweet taste stimulation from two different types of sweeteners. Sweet taste perception stimulated by sugars has been shown to initiate carbohydrate-specific physiological responses such as salivary alpha-amylase content and hormones that are involved in blood glucose regulation such as insulin and GLP-1 [30,31,32]. Such effects were specific to sugars and were not seen when sweet taste was stimulated by NNS [15,30,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47], which may be due to two distinctive pathways [48] and part of brain [49] of sweet taste activation. Previously, sensory stimulation has also been shown to alter energy expenditure [50,51]. Therefore, it is likely that sweet taste from sugars explained the earlier and greater increase in carbohydrate oxidation after SUCROSE test food. Carefully designed studies are needed to confirm this speculation.\nThere were indications from animal studies that NNS may stimulate appetite and food intake [10]. However, our study did not observe significant acute heightened appetite from the consumption of the NNS test food. However, it should be highlighted that energy gaps between NNS and SUCROSE and MALT were fully and slightly over-compensated for the remaining day. Our observations were consistent with a systematic review of observational and short-term interventional trials, where NNS use did not increase appetite, food intake or weight gain in humans [52]. In addition, preference for sweet foods was also not observed after jellies made with sucralose (MALT and NNS), as demonstrated by the comparable amount of bread with jam consumed during the meal challenge. \nThis study has a number of strengths. The crossover design and carefully matched sweetness level of test foods allowed the participants to be tested in a blinded manner, hence minimise the cognitive compensation in food intake after test sessions. This study was also an early study to investigate how product formulation may have implications on energy balance via energy expenditure and energy intake. However, there were also limitations that restricted the interpretation of our data. First, this was a pilot study with small sample size to compare how sweetener types affect energy expenditure and carbohydrate oxidation in humans, which has not been studied before. Second, the digestion rates between sucrose and maltodextrin were not measured in this study and should; therefore, be considered in future studies. Future studies could measure blood glucose concurrently over 90 min to confirm if the digestion of SUCROSE and MALT and absorption of glucose may explain differential carbohydrate oxidation patterns observed in this study. Third, bread with jam is not a traditional lunch meal, and the cultural influence or expectation may have diluted the preference for sweet foods after sweet test foods. Fourth, the length of the study was not long enough to allow carbohydrate oxidation to return to baseline in order to examine if the total carbohydrate oxidized following SUCROSE and MALT are comparable. Fifth, although our study has sufficient statistical power to detect the differential carbohydrate oxidation rates between test foods, the relatively small sample size and young population in this exploratory study may not be representative of the general population. Sixth, this study assessed only one type of non-nutritive sweetener and it is unknown if findings can be extended to other non-nutritive sweeteners as they may have different chemical structures and sweet taste potency [9]. A recent randomized, controlled study showed that different non-nutritive sweeteners affected energy intake and have different effects on weight change over 12 weeks [53]. Therefore, potential differences in energy expenditure and carbohydrate after different non-nutritive sweeteners (e.g., sugar alcohols, saccharin, aspartame, acesulfame K, etc.) ingestion should be investigated in the future.\nObservations in our study on appetite ratings and food intake for 24 h, albeit interesting, were not able to draw a conclusion due to the lack of statistical power (this study was powered based on primary outcomes). Our study was not powered to detect differences between dietary intakes after test foods originally due to the absence of other similar human studies. When calculated retrospectively using our own results, at least 117 (Glass’ delta, effect size = 0. 26) participants were needed to detect statistically significant differences in energy intake at α = 0.05 at 80% power, which was beyond the exploratory nature of this study.",
"This pilot study provides early findings that the replacement of sugars with NNS during food product formulation, with or without accompanying carbohydrate from maltodextrin, may have implications on human energy balance, especially human energy expenditure and carbohydrate oxidation patterns. Acutely, humans appear to compensate for energy shortfall from NNS but did not promote appetite or overeating beyond that. Findings from this pilot study should be verified and confirmed with bigger clinical trials in the future."
] |
[
"intro",
null,
null,
null,
"subjects",
null,
null,
null,
"results",
"discussion",
"conclusions"
] |
[
"non-nutritive sweeteners",
"carbohydrate",
"oxidation",
"appetite",
"food intake"
] |
1. Introduction:
The global prevalence of overweight and obesity is increasing and it increases the risk of several chronic diseases such as type 2 diabetes and cardiovascular disease [1,2]. Together with its co-morbidities, overweight and obesity increases mortality rates and adds burden to the healthcare system.
An increase in energy intake is one of several factors responsible for the rising prevalence of overweight and obesity [3]. In recent years, epidemiological studies have linked obesity with dietary sugar intake [4,5], and concluded that reducing sugar intake reduces prevalence of overweight and obesity [6]. The World Health Organization recommends a reduction in free sugar intake to no more than 10% of total daily intake [7]. A number of countries such as Mexico, Brazil and France have successfully reduced the demand for sugar-sweetened beverages through the introduction of sugar tax, and this may drive the reduction of added sugar during food processing by the food industry [8].
The intake of natural sugars from major food groups such as fruits, vegetables and dairy foods should not be limited as they are good sources of nutrients such as vitamin A, vitamin C, vitamin D, potassium, calcium, etc. Therefore, a common strategy to reduce total sugar intake is to avoid consumption of high-sugary discretionary foods, or choose healthier alternatives where sugars are replaced with non-nutritive sweeteners (NNS).
Sugar alcohols (e.g., sorbitol, xylitol, maltitol, etc.) are a group of NNS that provide sweetness that is comparable to table sugars [9]. Although sugar alcohols contain energy, these sweeteners are not well-digested and absorbed by the body and; therefore, provide minimal energy when consumed [10]. Another category of NNS possesses high sweetness potency [9] and some examples from this category include acesulfame potassium, alitame, rebaudioside A, aspartame, cyclamate, neotame, saccharin and sucralose. Because of the high sweetness potency, a very small amount of NNS is required to match the sweetness levels provided by sugars (often in milligrams). Hence, these sweeteners provide little or no energy to the body. All NNS have different chemical structures and their metabolism and health implications have been extensively discussed in a previous review [11]. Of all NNS, sucralose has a chemical structure that has the closest resemblance to sucrose (C12H22O11), where three hydrogen atoms in sucrose are replaced with chlorine in sucralose (C12H19Cl3O8). Sucralose is pH and heat-stable, and it is widely used in food products that requires heat application [12,13].
Currently, eight NNS (i.e., advantame, aspartame, acesulfame-K, neotame, saccharin, sucralose, stevia and monk fruit extracts) are considered to be generally recognized as safe (GRAS) by the US Food and Drug Authority and available to consumers [14]. Unlike sugars, NNS do not increase blood glucose [15]. However, there are also concerns that NNS may stimulate appetite and food intake, and subsequently weight gain [16,17]. These concerns appear to be supported by findings from a number of epidemiological studies, where the consumption of NNS foods or beverages was associated with higher body weight [18,19]. However, scientists also pointed out that such relationship may be reverse-causation, where people with higher body weight tend to include diet products to assist in weight management. Therefore, a better understanding of how NNS may influence acute energy balance, namely energy expenditure and energy intake, is needed.
The primary aim of this study was to compare energy expenditure and carbohydrate oxidation patterns after the consumption of test foods sweetened with table sugar (SUCROSE), sucralose (NNS), or sucralose but matched SUCROSE in energy and carbohydrate content with the addition of bland carbohydrate maltodextrin (MALT). The secondary aim was to compare appetitive responses and total daily intake following these test foods.
2. Materials and Methods:
2.1. Study Design This was an acute randomised, crossover study that included one baseline visit and three test visits.
This was an acute randomised, crossover study that included one baseline visit and three test visits.
2.2. Experimental Protocol At baseline, participants provided their written consent and had their height, body weight and eating behaviours assessed. Prior to each test day, the participants were reminded to avoid alcohol and strenuous exercise, consume their habitual dinner, and fast overnight for 10 h. On the test days, participants arrived between 08:00 and 09:00 am, voided urine, and had their body weight measured in a standing position with minimal clothing on. Following that, the participants rested for 30 min before their resting energy expenditure (REE) was assessed for 30 min. After REE, the participants consumed a test food within 10 min, followed by the measurements of postprandial gaseous exchanges (oxygen consumption and carbon dioxide production) for 90 min using the same indirect calorimeter, with a 10 min break every 30 min indirect calorimeter measurement, where the participants remained in a supine position without the hood. After the total 90 min calorimeter measurements, participants completed a meal challenge, where they were provided with 300 ml of plain water and bread with jam (sweet) and ham (savoury) toppings. Participants were asked to eat until they were comfortably full, which was defined as a fullness level where participants will not be eating other foods in the subsequent 3 to 4 h [20,21]. Appetite sensations were assessed pre- and post-consumption of test foods, as well as pre- and post-meal challenge, and sweetness levels of test foods were assessed immediately after ingestion of test foods. After the meal challenge, participants recorded food intake to determine their free-living energy intake for the rest of the day. The experimental protocol on test day is depicted in Figure 1. Test visits were repeated at least three days apart to normalize habitual food intake for males, and four weeks for females to ensure that experiment was performed at the same phase of the menstrual cycle, until all test foods were consumed. Participants were also asked to maintain their habitual dietary intake and physical activity level during the washout period. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Human Research Ethics Committee (approval number #0000033058). Written informed consent was obtained from all participants, and they were able to withdraw from the study at any time without consequences. This trial has also been registered with the Australian and New Zealand Clinical Trial Registry (ACTRN12614000879662).
At baseline, participants provided their written consent and had their height, body weight and eating behaviours assessed. Prior to each test day, the participants were reminded to avoid alcohol and strenuous exercise, consume their habitual dinner, and fast overnight for 10 h. On the test days, participants arrived between 08:00 and 09:00 am, voided urine, and had their body weight measured in a standing position with minimal clothing on. Following that, the participants rested for 30 min before their resting energy expenditure (REE) was assessed for 30 min. After REE, the participants consumed a test food within 10 min, followed by the measurements of postprandial gaseous exchanges (oxygen consumption and carbon dioxide production) for 90 min using the same indirect calorimeter, with a 10 min break every 30 min indirect calorimeter measurement, where the participants remained in a supine position without the hood. After the total 90 min calorimeter measurements, participants completed a meal challenge, where they were provided with 300 ml of plain water and bread with jam (sweet) and ham (savoury) toppings. Participants were asked to eat until they were comfortably full, which was defined as a fullness level where participants will not be eating other foods in the subsequent 3 to 4 h [20,21]. Appetite sensations were assessed pre- and post-consumption of test foods, as well as pre- and post-meal challenge, and sweetness levels of test foods were assessed immediately after ingestion of test foods. After the meal challenge, participants recorded food intake to determine their free-living energy intake for the rest of the day. The experimental protocol on test day is depicted in Figure 1. Test visits were repeated at least three days apart to normalize habitual food intake for males, and four weeks for females to ensure that experiment was performed at the same phase of the menstrual cycle, until all test foods were consumed. Participants were also asked to maintain their habitual dietary intake and physical activity level during the washout period. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Human Research Ethics Committee (approval number #0000033058). Written informed consent was obtained from all participants, and they were able to withdraw from the study at any time without consequences. This trial has also been registered with the Australian and New Zealand Clinical Trial Registry (ACTRN12614000879662).
2.3. Study Participants Participants were recruited from both the University and the local community. The inclusion criteria of this study were: (1) adults males (18–65 years old) or females (18–45 years old, pre-menopausal), (2) non-smokers, (3) normal or overweight (BMI 18.5–30.0 kgm−2), (4) weight changes of <5 kg in the past 3 months, (5) no diseases or not taking any medications that affect metabolism and appetite, and (6) no food allergies and have consumed diet food products containing NNS before.
Participants were recruited from both the University and the local community. The inclusion criteria of this study were: (1) adults males (18–65 years old) or females (18–45 years old, pre-menopausal), (2) non-smokers, (3) normal or overweight (BMI 18.5–30.0 kgm−2), (4) weight changes of <5 kg in the past 3 months, (5) no diseases or not taking any medications that affect metabolism and appetite, and (6) no food allergies and have consumed diet food products containing NNS before.
2.4. Study Test Foods This study included three types of jelly made with 100 g low-fat milk (274 kJ, 5.8 g carbohydrate, 7.7 g protein, 1.2 g fat) and 4.7 g gelling powder (carrageenan, brand: Jel-it-in, Queen Fine Foods Pty. Ltd., Alderley, QLD, Australia), sweetened with: (1) SUCROSE: 50 g sucrose (sweet carbohydrate), (2) NNS: 120 mg of sucralose only (sweet, no carbohydrate), and (3) MALT: 120 mg sucralose and matched 50 g carbohydrate as bland maltodextrin (Poly-Joule, Nutricia) (sweet and carbohydrate). The mixture was heated to dissolve all ingredients. The amount of sucralose used in MALT and NNS provided equal level of sweetness compared to SUCROSE, as determined through a sensory evaluation prior to this experiment.
Both SUCROSE and NNS test foods were equally sweet, but they differed in energy and carbohydrate content and may; therefore, have differential effects on human energy expenditure and carbohydrate oxidation in this study [22]. For this reason, a third test food, MALT, was included and its sweet taste came from sucralose but it has same energy and carbohydrate content (from maltodextrin) as the SUCROSE test food. Maltodextrin is a common ingredient used in food productions, is bland and hence did not alter the sweetness levels when added to the NNS test food containing 120 mg of sucralose. Therefore, SUCROSE and MALT were matched in sweetness levels, energy, and carbohydrate content to allow fair comparisons of metabolic and appetitive responses between types of sweeteners. NNS test food was chosen to examine the effects of non-nutritive sweetener alone. The order of test food consumption was randomised using an online research randomizer (www.randomizer.org) and participants were blinded to the type of test food they received at each test visit.
This study included three types of jelly made with 100 g low-fat milk (274 kJ, 5.8 g carbohydrate, 7.7 g protein, 1.2 g fat) and 4.7 g gelling powder (carrageenan, brand: Jel-it-in, Queen Fine Foods Pty. Ltd., Alderley, QLD, Australia), sweetened with: (1) SUCROSE: 50 g sucrose (sweet carbohydrate), (2) NNS: 120 mg of sucralose only (sweet, no carbohydrate), and (3) MALT: 120 mg sucralose and matched 50 g carbohydrate as bland maltodextrin (Poly-Joule, Nutricia) (sweet and carbohydrate). The mixture was heated to dissolve all ingredients. The amount of sucralose used in MALT and NNS provided equal level of sweetness compared to SUCROSE, as determined through a sensory evaluation prior to this experiment.
Both SUCROSE and NNS test foods were equally sweet, but they differed in energy and carbohydrate content and may; therefore, have differential effects on human energy expenditure and carbohydrate oxidation in this study [22]. For this reason, a third test food, MALT, was included and its sweet taste came from sucralose but it has same energy and carbohydrate content (from maltodextrin) as the SUCROSE test food. Maltodextrin is a common ingredient used in food productions, is bland and hence did not alter the sweetness levels when added to the NNS test food containing 120 mg of sucralose. Therefore, SUCROSE and MALT were matched in sweetness levels, energy, and carbohydrate content to allow fair comparisons of metabolic and appetitive responses between types of sweeteners. NNS test food was chosen to examine the effects of non-nutritive sweetener alone. The order of test food consumption was randomised using an online research randomizer (www.randomizer.org) and participants were blinded to the type of test food they received at each test visit.
2.5. Measures Anthropometry: Height was measured with a Seca stadiometer (to the nearest 1 mm) and body weight with a Tanita scale (Tanita BF-680, Tanita Inc., Japan, to the nearest 0.1 kg). Height and weight measurements were taken in duplicates and averaged. A third measurement was taken when height and weight measurements differed by more than 5 mm or 0.5 kg, respectively, and the average of the two closest measurements was taken. Eating behaviour was assessed at baseline using the validated three-factor eating questionnaire [23] to eliminate volunteers who may have unusual eating behaviour that could affect appetite ratings and food intake during meal challenge.
Energy expenditure and substrate oxidation (primary outcomes): Energy expenditure and substrate oxidation was assessed using a ventilated-hood indirect calorimeter (TrueMax 2400, Parvomedic Inc., Sandy, UT, USA). The machine was calibrated prior to each test visit using a standard 3.0 L gas syringe and standard gas (16.00% O2, 0.986% CO2 and balance nitrogen) to ensure accuracy of readings. Measurements were converted to standard temperature, pressure, and dry. Continuous gaseous exchanges were calculated as 10 min averages. For resting energy expenditure measurement, the final 20 min (stable state) of the 30 min measurement was used. Oxygen consumption and carbon dioxide production were used to calculate energy expenditure [24] and non-protein substrate oxidation rates [25] using previously published equations.
Appetite and food intake (secondary outcomes): The appetite ratings of participants were assessed with validated 100-mm visual analogue scales (VAS) [26]. Sweetness level of test foods was also assessed using a horizontal 100-mm VAS anchored at “not sweet at all” on the left and “extremely sweet” on the right. A food diary was used to record free-living food intake for the remaining day. An explanatory page with instructions on how to document food intake accurately was provided with the diary, and food diaries were checked by researchers for completeness when they were returned. Dietary analysis was performed using the AUSNUT 2013 database in FoodWorks Professional (Xyris Software Pty. Ltd., Spring Hill, QLD, Australia).
Anthropometry: Height was measured with a Seca stadiometer (to the nearest 1 mm) and body weight with a Tanita scale (Tanita BF-680, Tanita Inc., Japan, to the nearest 0.1 kg). Height and weight measurements were taken in duplicates and averaged. A third measurement was taken when height and weight measurements differed by more than 5 mm or 0.5 kg, respectively, and the average of the two closest measurements was taken. Eating behaviour was assessed at baseline using the validated three-factor eating questionnaire [23] to eliminate volunteers who may have unusual eating behaviour that could affect appetite ratings and food intake during meal challenge.
Energy expenditure and substrate oxidation (primary outcomes): Energy expenditure and substrate oxidation was assessed using a ventilated-hood indirect calorimeter (TrueMax 2400, Parvomedic Inc., Sandy, UT, USA). The machine was calibrated prior to each test visit using a standard 3.0 L gas syringe and standard gas (16.00% O2, 0.986% CO2 and balance nitrogen) to ensure accuracy of readings. Measurements were converted to standard temperature, pressure, and dry. Continuous gaseous exchanges were calculated as 10 min averages. For resting energy expenditure measurement, the final 20 min (stable state) of the 30 min measurement was used. Oxygen consumption and carbon dioxide production were used to calculate energy expenditure [24] and non-protein substrate oxidation rates [25] using previously published equations.
Appetite and food intake (secondary outcomes): The appetite ratings of participants were assessed with validated 100-mm visual analogue scales (VAS) [26]. Sweetness level of test foods was also assessed using a horizontal 100-mm VAS anchored at “not sweet at all” on the left and “extremely sweet” on the right. A food diary was used to record free-living food intake for the remaining day. An explanatory page with instructions on how to document food intake accurately was provided with the diary, and food diaries were checked by researchers for completeness when they were returned. Dietary analysis was performed using the AUSNUT 2013 database in FoodWorks Professional (Xyris Software Pty. Ltd., Spring Hill, QLD, Australia).
2.6. Statistical Analysis Descriptive statistics are presented as mean ± standard deviation. Postprandial EE and carbohydrate oxidation were calculated and presented as changes from baseline, as well as total over 90 min study period as area under the curve (AUC) using trapezoidal rules. The 90 min EE and carbohydrate oxidation was also adjusted for body weight taken on the test days, using regression residuals from a regression equation as following:Y = a0 + a1 × Body Weight (kg),
where a0 is a constant and a1 is a coefficient.
The comparisons AUC and changes in EE, carbohydrate, appetite and sweetness ratings, and dietary intake following the consumption of three test foods were compared using general linear model for repeated measures ANOVA with Bonferroni corrections. All statistical analyses were performed with statistical package SPSS (version 23.0.0, IBM Corp, Armonk, NY, USA). As there was no previous study comparing the postprandial substrate oxidation effects induced by different sweeteners, sample size was guided by a study we previously conducted [27], where significant differences in substrate oxidation were detected with a study sample of 12 participants. Fifteen participants were targeted to account for attrition of 20%.
Descriptive statistics are presented as mean ± standard deviation. Postprandial EE and carbohydrate oxidation were calculated and presented as changes from baseline, as well as total over 90 min study period as area under the curve (AUC) using trapezoidal rules. The 90 min EE and carbohydrate oxidation was also adjusted for body weight taken on the test days, using regression residuals from a regression equation as following:Y = a0 + a1 × Body Weight (kg),
where a0 is a constant and a1 is a coefficient.
The comparisons AUC and changes in EE, carbohydrate, appetite and sweetness ratings, and dietary intake following the consumption of three test foods were compared using general linear model for repeated measures ANOVA with Bonferroni corrections. All statistical analyses were performed with statistical package SPSS (version 23.0.0, IBM Corp, Armonk, NY, USA). As there was no previous study comparing the postprandial substrate oxidation effects induced by different sweeteners, sample size was guided by a study we previously conducted [27], where significant differences in substrate oxidation were detected with a study sample of 12 participants. Fifteen participants were targeted to account for attrition of 20%.
2.1. Study Design:
This was an acute randomised, crossover study that included one baseline visit and three test visits.
2.2. Experimental Protocol:
At baseline, participants provided their written consent and had their height, body weight and eating behaviours assessed. Prior to each test day, the participants were reminded to avoid alcohol and strenuous exercise, consume their habitual dinner, and fast overnight for 10 h. On the test days, participants arrived between 08:00 and 09:00 am, voided urine, and had their body weight measured in a standing position with minimal clothing on. Following that, the participants rested for 30 min before their resting energy expenditure (REE) was assessed for 30 min. After REE, the participants consumed a test food within 10 min, followed by the measurements of postprandial gaseous exchanges (oxygen consumption and carbon dioxide production) for 90 min using the same indirect calorimeter, with a 10 min break every 30 min indirect calorimeter measurement, where the participants remained in a supine position without the hood. After the total 90 min calorimeter measurements, participants completed a meal challenge, where they were provided with 300 ml of plain water and bread with jam (sweet) and ham (savoury) toppings. Participants were asked to eat until they were comfortably full, which was defined as a fullness level where participants will not be eating other foods in the subsequent 3 to 4 h [20,21]. Appetite sensations were assessed pre- and post-consumption of test foods, as well as pre- and post-meal challenge, and sweetness levels of test foods were assessed immediately after ingestion of test foods. After the meal challenge, participants recorded food intake to determine their free-living energy intake for the rest of the day. The experimental protocol on test day is depicted in Figure 1. Test visits were repeated at least three days apart to normalize habitual food intake for males, and four weeks for females to ensure that experiment was performed at the same phase of the menstrual cycle, until all test foods were consumed. Participants were also asked to maintain their habitual dietary intake and physical activity level during the washout period. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Human Research Ethics Committee (approval number #0000033058). Written informed consent was obtained from all participants, and they were able to withdraw from the study at any time without consequences. This trial has also been registered with the Australian and New Zealand Clinical Trial Registry (ACTRN12614000879662).
2.3. Study Participants:
Participants were recruited from both the University and the local community. The inclusion criteria of this study were: (1) adults males (18–65 years old) or females (18–45 years old, pre-menopausal), (2) non-smokers, (3) normal or overweight (BMI 18.5–30.0 kgm−2), (4) weight changes of <5 kg in the past 3 months, (5) no diseases or not taking any medications that affect metabolism and appetite, and (6) no food allergies and have consumed diet food products containing NNS before.
2.4. Study Test Foods:
This study included three types of jelly made with 100 g low-fat milk (274 kJ, 5.8 g carbohydrate, 7.7 g protein, 1.2 g fat) and 4.7 g gelling powder (carrageenan, brand: Jel-it-in, Queen Fine Foods Pty. Ltd., Alderley, QLD, Australia), sweetened with: (1) SUCROSE: 50 g sucrose (sweet carbohydrate), (2) NNS: 120 mg of sucralose only (sweet, no carbohydrate), and (3) MALT: 120 mg sucralose and matched 50 g carbohydrate as bland maltodextrin (Poly-Joule, Nutricia) (sweet and carbohydrate). The mixture was heated to dissolve all ingredients. The amount of sucralose used in MALT and NNS provided equal level of sweetness compared to SUCROSE, as determined through a sensory evaluation prior to this experiment.
Both SUCROSE and NNS test foods were equally sweet, but they differed in energy and carbohydrate content and may; therefore, have differential effects on human energy expenditure and carbohydrate oxidation in this study [22]. For this reason, a third test food, MALT, was included and its sweet taste came from sucralose but it has same energy and carbohydrate content (from maltodextrin) as the SUCROSE test food. Maltodextrin is a common ingredient used in food productions, is bland and hence did not alter the sweetness levels when added to the NNS test food containing 120 mg of sucralose. Therefore, SUCROSE and MALT were matched in sweetness levels, energy, and carbohydrate content to allow fair comparisons of metabolic and appetitive responses between types of sweeteners. NNS test food was chosen to examine the effects of non-nutritive sweetener alone. The order of test food consumption was randomised using an online research randomizer (www.randomizer.org) and participants were blinded to the type of test food they received at each test visit.
2.5. Measures:
Anthropometry: Height was measured with a Seca stadiometer (to the nearest 1 mm) and body weight with a Tanita scale (Tanita BF-680, Tanita Inc., Japan, to the nearest 0.1 kg). Height and weight measurements were taken in duplicates and averaged. A third measurement was taken when height and weight measurements differed by more than 5 mm or 0.5 kg, respectively, and the average of the two closest measurements was taken. Eating behaviour was assessed at baseline using the validated three-factor eating questionnaire [23] to eliminate volunteers who may have unusual eating behaviour that could affect appetite ratings and food intake during meal challenge.
Energy expenditure and substrate oxidation (primary outcomes): Energy expenditure and substrate oxidation was assessed using a ventilated-hood indirect calorimeter (TrueMax 2400, Parvomedic Inc., Sandy, UT, USA). The machine was calibrated prior to each test visit using a standard 3.0 L gas syringe and standard gas (16.00% O2, 0.986% CO2 and balance nitrogen) to ensure accuracy of readings. Measurements were converted to standard temperature, pressure, and dry. Continuous gaseous exchanges were calculated as 10 min averages. For resting energy expenditure measurement, the final 20 min (stable state) of the 30 min measurement was used. Oxygen consumption and carbon dioxide production were used to calculate energy expenditure [24] and non-protein substrate oxidation rates [25] using previously published equations.
Appetite and food intake (secondary outcomes): The appetite ratings of participants were assessed with validated 100-mm visual analogue scales (VAS) [26]. Sweetness level of test foods was also assessed using a horizontal 100-mm VAS anchored at “not sweet at all” on the left and “extremely sweet” on the right. A food diary was used to record free-living food intake for the remaining day. An explanatory page with instructions on how to document food intake accurately was provided with the diary, and food diaries were checked by researchers for completeness when they were returned. Dietary analysis was performed using the AUSNUT 2013 database in FoodWorks Professional (Xyris Software Pty. Ltd., Spring Hill, QLD, Australia).
2.6. Statistical Analysis:
Descriptive statistics are presented as mean ± standard deviation. Postprandial EE and carbohydrate oxidation were calculated and presented as changes from baseline, as well as total over 90 min study period as area under the curve (AUC) using trapezoidal rules. The 90 min EE and carbohydrate oxidation was also adjusted for body weight taken on the test days, using regression residuals from a regression equation as following:Y = a0 + a1 × Body Weight (kg),
where a0 is a constant and a1 is a coefficient.
The comparisons AUC and changes in EE, carbohydrate, appetite and sweetness ratings, and dietary intake following the consumption of three test foods were compared using general linear model for repeated measures ANOVA with Bonferroni corrections. All statistical analyses were performed with statistical package SPSS (version 23.0.0, IBM Corp, Armonk, NY, USA). As there was no previous study comparing the postprandial substrate oxidation effects induced by different sweeteners, sample size was guided by a study we previously conducted [27], where significant differences in substrate oxidation were detected with a study sample of 12 participants. Fifteen participants were targeted to account for attrition of 20%.
3. Results:
Fifteen participants were recruited and 11 completed the study. Four withdrew from the study prior to the commencement of study due to difficulty in scheduling study visits. Participants who completed all test visits were eight males three females, aged = 24.9 ± 6.1 years, baseline body weight = 72.9 ± 19.0 kg (males = 81.0 ± 19.8 kg; females = 54.8 ± 8.4 kg), BMI = 25.0 ± 4.7 kgm−2 (males = 26.4 ± 5.5 kgm−2; females = 21.5 ± 3.1 kgm−2), and body fat = 20.3% ± 4.7% (males = 20.3% ± 8.7%; females = 22.3% ± 7.9%). Throughout all test visits, participants’ body weight did not change significantly from baseline: visit 1 = 0.0 ± 0.9 kg, visit 2 = −0.2 ± 0.8 kg, and visit 3 = 0.0 ± 0.7 kg. The jellies were rated as equally sweet by participants after consumption: SUCROSE = 62 ± 17 mm, NNS = 61 ± 13 mm, MALT = 64 ± 21 mm (repeated measures ANOVA, p = 0.911).
Table 1 summarises the unadjusted and adjusted (for body weight) values for total EE, carbohydrate and fat oxidation over the 90 min measurement period using an indirect calorimeter following test food was ingestion. The unadjusted variables were significantly different between all test foods (p < 0.05); the 90 min EE was almost statistically significant after adjustment for body weight (p = 0.050). The unadjusted EE were significantly higher in both SUCROSE and MALT than NNS. Carbohydrate oxidation rates were SUCROSE > MALT > NNS, and fat oxidation was significantly lower in SUCROSE than MALT and NNS.
As resting EE and carbohydrate oxidation rates were not significantly different between all study visits, data on these outcomes were also presented as patterns of changes from resting values after the ingestion of test foods in Figure 2. There were significant time effects on EE changes (p = 0.001) but they did not differ between test foods (F (8.3, 125.0) = 0.811, p = 0.599) (Figure 2A). However, significant interaction effects were found for carbohydrate oxidation (F (10.2, 153.5) = 6.117, p < 0.001) during the 90 min postprandial assessment (Figure 2B).
Appetite ratings pre- and post-test food ingestion, as well as pre- and post-meal challenge are presented in Table 2. No significant interaction effects of test foods on appetite ratings were found. Food intakes at meal challenge (bread with jam, p = 0.863; bread with ham, p = 0.874), in a free-living environment (p = 0.585), and total daily intake including test foods (p = 0.838) did not differ between test foods (Figure 3).
4. Discussion:
This study was designed to examine how the re-formulation of a high-sugar food product with sucralose to reduce added sugar content may affect energy expenditure, carbohydrate oxidation, appetite and food intake. During the 90 min postprandial period, the EE was comparable between the SUCROSE and MALT test foods but both were higher than in NNS (Table 1 and Figure 2A). This observation was consistent with the evidence that postprandial EE is predominantly determined by the energy content of a meal [28].
Postprandial carbohydrate oxidation rate was significantly lower in NNS than SUCROSE and MALT (Table 1), which is likely to be explained by the higher carbohydrate content in SUCROSE and MALT. The temporal carbohydrate oxidation patterns of all jellies differ significantly (Figure 2B) although NNS did not alter carbohydrate oxidation. Interestingly, SUCROSE and MALT jellies produced different carbohydrate oxidation patterns although they have matched carbohydrate content. A higher and earlier carbohydrate oxidation peak was observed in SUCROSE, which subsequently converged with the MALT carbohydrate oxidation rates from 70 min onwards. As a result, the carbohydrate oxidation rate of SUCROSE was also significantly higher than MALT over 90 min.
There are two potential explanations for our novel finding on the differential carbohydrate oxidation patterns between SUCROSE and MALT. In the first scenario, maltodextrin is a polysaccharide and may be digested and absorbed slower than sugars (disaccharide) in SUCROSE test food, hence producing a lower and delayed carbohydrate oxidation. A potential counterargument is that maltodextrin is considered as a short-chain carbohydrates that is rapidly digested and release glucose for absorption [29]. In the second scenario, the differential carbohydrate oxidation patterns may be explained by sweet taste stimulation from two different types of sweeteners. Sweet taste perception stimulated by sugars has been shown to initiate carbohydrate-specific physiological responses such as salivary alpha-amylase content and hormones that are involved in blood glucose regulation such as insulin and GLP-1 [30,31,32]. Such effects were specific to sugars and were not seen when sweet taste was stimulated by NNS [15,30,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47], which may be due to two distinctive pathways [48] and part of brain [49] of sweet taste activation. Previously, sensory stimulation has also been shown to alter energy expenditure [50,51]. Therefore, it is likely that sweet taste from sugars explained the earlier and greater increase in carbohydrate oxidation after SUCROSE test food. Carefully designed studies are needed to confirm this speculation.
There were indications from animal studies that NNS may stimulate appetite and food intake [10]. However, our study did not observe significant acute heightened appetite from the consumption of the NNS test food. However, it should be highlighted that energy gaps between NNS and SUCROSE and MALT were fully and slightly over-compensated for the remaining day. Our observations were consistent with a systematic review of observational and short-term interventional trials, where NNS use did not increase appetite, food intake or weight gain in humans [52]. In addition, preference for sweet foods was also not observed after jellies made with sucralose (MALT and NNS), as demonstrated by the comparable amount of bread with jam consumed during the meal challenge.
This study has a number of strengths. The crossover design and carefully matched sweetness level of test foods allowed the participants to be tested in a blinded manner, hence minimise the cognitive compensation in food intake after test sessions. This study was also an early study to investigate how product formulation may have implications on energy balance via energy expenditure and energy intake. However, there were also limitations that restricted the interpretation of our data. First, this was a pilot study with small sample size to compare how sweetener types affect energy expenditure and carbohydrate oxidation in humans, which has not been studied before. Second, the digestion rates between sucrose and maltodextrin were not measured in this study and should; therefore, be considered in future studies. Future studies could measure blood glucose concurrently over 90 min to confirm if the digestion of SUCROSE and MALT and absorption of glucose may explain differential carbohydrate oxidation patterns observed in this study. Third, bread with jam is not a traditional lunch meal, and the cultural influence or expectation may have diluted the preference for sweet foods after sweet test foods. Fourth, the length of the study was not long enough to allow carbohydrate oxidation to return to baseline in order to examine if the total carbohydrate oxidized following SUCROSE and MALT are comparable. Fifth, although our study has sufficient statistical power to detect the differential carbohydrate oxidation rates between test foods, the relatively small sample size and young population in this exploratory study may not be representative of the general population. Sixth, this study assessed only one type of non-nutritive sweetener and it is unknown if findings can be extended to other non-nutritive sweeteners as they may have different chemical structures and sweet taste potency [9]. A recent randomized, controlled study showed that different non-nutritive sweeteners affected energy intake and have different effects on weight change over 12 weeks [53]. Therefore, potential differences in energy expenditure and carbohydrate after different non-nutritive sweeteners (e.g., sugar alcohols, saccharin, aspartame, acesulfame K, etc.) ingestion should be investigated in the future.
Observations in our study on appetite ratings and food intake for 24 h, albeit interesting, were not able to draw a conclusion due to the lack of statistical power (this study was powered based on primary outcomes). Our study was not powered to detect differences between dietary intakes after test foods originally due to the absence of other similar human studies. When calculated retrospectively using our own results, at least 117 (Glass’ delta, effect size = 0. 26) participants were needed to detect statistically significant differences in energy intake at α = 0.05 at 80% power, which was beyond the exploratory nature of this study.
5. Conclusions:
This pilot study provides early findings that the replacement of sugars with NNS during food product formulation, with or without accompanying carbohydrate from maltodextrin, may have implications on human energy balance, especially human energy expenditure and carbohydrate oxidation patterns. Acutely, humans appear to compensate for energy shortfall from NNS but did not promote appetite or overeating beyond that. Findings from this pilot study should be verified and confirmed with bigger clinical trials in the future.
|
Background:
In light of obesity, replacing sugar with non-nutritive sweeteners is commonly used to reduce sugar content of food products. This study aimed to compare human energy expenditure (EE), carbohydrate oxidation and food intake after the ingestion of test foods sweetened with sucrose or a non-nutritive sweetener.
Methods:
This was an acute crossover feeding study that entailed consumption of three test foods: jelly sweetened with 50 g sucrose (SUCROSE), with 120 mg of sucralose only (NNS), or 120 mg sucralose but matched in carbohydrate with 50 g maltodextrin (MALT). On test days, participants arrived at the research facility after an overnight fast. Resting energy expenditure (indirect calorimeter) was measured for 30 min followed by jelly consumption. Participants' EE and substrate oxidation were measured for 90 min subsequently. After EE assessment, participants completed a meal challenge before leaving the research facility, and recorded food intake for the remaining day. Subjective appetite ratings were assessed before and after test foods and meal challenge.
Results:
Eleven participants completed the study. EE was higher in SUCROSE and MALT than NNS, but not statistically significant. Carbohydrate oxidation was SUCROSE > MALT > NNS (p < 0.001). Earlier and bigger rise in carbohydrate oxidation was observed in SUCROSE than MALT, although both were carbohydrate-matched. NNS did not promote energy expenditure, carbohydrate oxidation or stimulate appetite.
Conclusions:
Foods sweetened with sucrose or non-nutritive sweeteners but matched in carbohydrate content have different effects on human EE and carbohydrate oxidation. Sucralose alone did not affect EE, but lower energy in the test food from sugar replacement was eventually fully compensated. Findings from this pilot study should be verified with bigger clinical studies in the future to establish clinical relevance.
|
1. Introduction:
The global prevalence of overweight and obesity is increasing and it increases the risk of several chronic diseases such as type 2 diabetes and cardiovascular disease [1,2]. Together with its co-morbidities, overweight and obesity increases mortality rates and adds burden to the healthcare system.
An increase in energy intake is one of several factors responsible for the rising prevalence of overweight and obesity [3]. In recent years, epidemiological studies have linked obesity with dietary sugar intake [4,5], and concluded that reducing sugar intake reduces prevalence of overweight and obesity [6]. The World Health Organization recommends a reduction in free sugar intake to no more than 10% of total daily intake [7]. A number of countries such as Mexico, Brazil and France have successfully reduced the demand for sugar-sweetened beverages through the introduction of sugar tax, and this may drive the reduction of added sugar during food processing by the food industry [8].
The intake of natural sugars from major food groups such as fruits, vegetables and dairy foods should not be limited as they are good sources of nutrients such as vitamin A, vitamin C, vitamin D, potassium, calcium, etc. Therefore, a common strategy to reduce total sugar intake is to avoid consumption of high-sugary discretionary foods, or choose healthier alternatives where sugars are replaced with non-nutritive sweeteners (NNS).
Sugar alcohols (e.g., sorbitol, xylitol, maltitol, etc.) are a group of NNS that provide sweetness that is comparable to table sugars [9]. Although sugar alcohols contain energy, these sweeteners are not well-digested and absorbed by the body and; therefore, provide minimal energy when consumed [10]. Another category of NNS possesses high sweetness potency [9] and some examples from this category include acesulfame potassium, alitame, rebaudioside A, aspartame, cyclamate, neotame, saccharin and sucralose. Because of the high sweetness potency, a very small amount of NNS is required to match the sweetness levels provided by sugars (often in milligrams). Hence, these sweeteners provide little or no energy to the body. All NNS have different chemical structures and their metabolism and health implications have been extensively discussed in a previous review [11]. Of all NNS, sucralose has a chemical structure that has the closest resemblance to sucrose (C12H22O11), where three hydrogen atoms in sucrose are replaced with chlorine in sucralose (C12H19Cl3O8). Sucralose is pH and heat-stable, and it is widely used in food products that requires heat application [12,13].
Currently, eight NNS (i.e., advantame, aspartame, acesulfame-K, neotame, saccharin, sucralose, stevia and monk fruit extracts) are considered to be generally recognized as safe (GRAS) by the US Food and Drug Authority and available to consumers [14]. Unlike sugars, NNS do not increase blood glucose [15]. However, there are also concerns that NNS may stimulate appetite and food intake, and subsequently weight gain [16,17]. These concerns appear to be supported by findings from a number of epidemiological studies, where the consumption of NNS foods or beverages was associated with higher body weight [18,19]. However, scientists also pointed out that such relationship may be reverse-causation, where people with higher body weight tend to include diet products to assist in weight management. Therefore, a better understanding of how NNS may influence acute energy balance, namely energy expenditure and energy intake, is needed.
The primary aim of this study was to compare energy expenditure and carbohydrate oxidation patterns after the consumption of test foods sweetened with table sugar (SUCROSE), sucralose (NNS), or sucralose but matched SUCROSE in energy and carbohydrate content with the addition of bland carbohydrate maltodextrin (MALT). The secondary aim was to compare appetitive responses and total daily intake following these test foods.
5. Conclusions:
This pilot study provides early findings that the replacement of sugars with NNS during food product formulation, with or without accompanying carbohydrate from maltodextrin, may have implications on human energy balance, especially human energy expenditure and carbohydrate oxidation patterns. Acutely, humans appear to compensate for energy shortfall from NNS but did not promote appetite or overeating beyond that. Findings from this pilot study should be verified and confirmed with bigger clinical trials in the future.
|
Background:
In light of obesity, replacing sugar with non-nutritive sweeteners is commonly used to reduce sugar content of food products. This study aimed to compare human energy expenditure (EE), carbohydrate oxidation and food intake after the ingestion of test foods sweetened with sucrose or a non-nutritive sweetener.
Methods:
This was an acute crossover feeding study that entailed consumption of three test foods: jelly sweetened with 50 g sucrose (SUCROSE), with 120 mg of sucralose only (NNS), or 120 mg sucralose but matched in carbohydrate with 50 g maltodextrin (MALT). On test days, participants arrived at the research facility after an overnight fast. Resting energy expenditure (indirect calorimeter) was measured for 30 min followed by jelly consumption. Participants' EE and substrate oxidation were measured for 90 min subsequently. After EE assessment, participants completed a meal challenge before leaving the research facility, and recorded food intake for the remaining day. Subjective appetite ratings were assessed before and after test foods and meal challenge.
Results:
Eleven participants completed the study. EE was higher in SUCROSE and MALT than NNS, but not statistically significant. Carbohydrate oxidation was SUCROSE > MALT > NNS (p < 0.001). Earlier and bigger rise in carbohydrate oxidation was observed in SUCROSE than MALT, although both were carbohydrate-matched. NNS did not promote energy expenditure, carbohydrate oxidation or stimulate appetite.
Conclusions:
Foods sweetened with sucrose or non-nutritive sweeteners but matched in carbohydrate content have different effects on human EE and carbohydrate oxidation. Sucralose alone did not affect EE, but lower energy in the test food from sugar replacement was eventually fully compensated. Findings from this pilot study should be verified with bigger clinical studies in the future to establish clinical relevance.
| 7,280 | 340 | 11 |
[
"test",
"food",
"carbohydrate",
"study",
"participants",
"energy",
"foods",
"oxidation",
"intake",
"nns"
] |
[
"test",
"test"
] | null |
[CONTENT] non-nutritive sweeteners | carbohydrate | oxidation | appetite | food intake [SUMMARY]
| null |
[CONTENT] non-nutritive sweeteners | carbohydrate | oxidation | appetite | food intake [SUMMARY]
|
[CONTENT] non-nutritive sweeteners | carbohydrate | oxidation | appetite | food intake [SUMMARY]
|
[CONTENT] non-nutritive sweeteners | carbohydrate | oxidation | appetite | food intake [SUMMARY]
|
[CONTENT] non-nutritive sweeteners | carbohydrate | oxidation | appetite | food intake [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Appetite Regulation | Cross-Over Studies | Energy Metabolism | Female | Humans | Male | Middle Aged | Non-Nutritive Sweeteners | Oxidation-Reduction | Pilot Projects | Sucrose | Young Adult [SUMMARY]
| null |
[CONTENT] Adolescent | Adult | Aged | Appetite Regulation | Cross-Over Studies | Energy Metabolism | Female | Humans | Male | Middle Aged | Non-Nutritive Sweeteners | Oxidation-Reduction | Pilot Projects | Sucrose | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Appetite Regulation | Cross-Over Studies | Energy Metabolism | Female | Humans | Male | Middle Aged | Non-Nutritive Sweeteners | Oxidation-Reduction | Pilot Projects | Sucrose | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Appetite Regulation | Cross-Over Studies | Energy Metabolism | Female | Humans | Male | Middle Aged | Non-Nutritive Sweeteners | Oxidation-Reduction | Pilot Projects | Sucrose | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Appetite Regulation | Cross-Over Studies | Energy Metabolism | Female | Humans | Male | Middle Aged | Non-Nutritive Sweeteners | Oxidation-Reduction | Pilot Projects | Sucrose | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | food | carbohydrate | study | participants | energy | foods | oxidation | intake | nns [SUMMARY]
| null |
[CONTENT] test | food | carbohydrate | study | participants | energy | foods | oxidation | intake | nns [SUMMARY]
|
[CONTENT] test | food | carbohydrate | study | participants | energy | foods | oxidation | intake | nns [SUMMARY]
|
[CONTENT] test | food | carbohydrate | study | participants | energy | foods | oxidation | intake | nns [SUMMARY]
|
[CONTENT] test | food | carbohydrate | study | participants | energy | foods | oxidation | intake | nns [SUMMARY]
|
[CONTENT] sugar | nns | intake | obesity | sucralose | energy | sugar intake | overweight obesity | sugars | overweight [SUMMARY]
| null |
[CONTENT] significantly | test | kg | ee | visit kg | unadjusted | sucrose malt nns | test foods | foods | body [SUMMARY]
|
[CONTENT] pilot study | pilot | energy | human energy | findings | human | carbohydrate | nns | especially human energy | product formulation accompanying carbohydrate [SUMMARY]
|
[CONTENT] test | carbohydrate | food | energy | study | nns | participants | sucrose | oxidation | min [SUMMARY]
|
[CONTENT] test | carbohydrate | food | energy | study | nns | participants | sucrose | oxidation | min [SUMMARY]
|
[CONTENT] ||| [SUMMARY]
| null |
[CONTENT] Eleven ||| SUCROSE | MALT | NNS ||| SUCROSE ||| MALT ||| NNS ||| SUCROSE | MALT ||| NNS [SUMMARY]
|
[CONTENT] ||| ||| [SUMMARY]
|
[CONTENT] ||| ||| three | 50 | SUCROSE | 120 | NNS | 120 | 50 g maltodextrin | MALT ||| test days | overnight ||| 30 ||| 90 ||| the remaining day ||| ||| ||| Eleven ||| SUCROSE | MALT | NNS ||| SUCROSE ||| MALT ||| NNS ||| SUCROSE | MALT ||| NNS ||| ||| ||| [SUMMARY]
|
[CONTENT] ||| ||| three | 50 | SUCROSE | 120 | NNS | 120 | 50 g maltodextrin | MALT ||| test days | overnight ||| 30 ||| 90 ||| the remaining day ||| ||| ||| Eleven ||| SUCROSE | MALT | NNS ||| SUCROSE ||| MALT ||| NNS ||| SUCROSE | MALT ||| NNS ||| ||| ||| [SUMMARY]
|
|||||
Identification of Myocardial Insulin Resistance by Using Liver Tests: A Simple Approach for Clinical Practice.
|
35955920
|
We report that myocardial insulin resistance (mIR) occurs in around 60% of patients with type 2 diabetes (T2D) and was associated with higher cardiovascular risk in comparison with patients with insulin-sensitive myocardium (mIS). These two phenotypes (mIR vs. mIS) can only be assessed using time-consuming and expensive methods. The aim of the present study is to search a simple and reliable surrogate to identify both phenotypes.
|
BACKGROUND
|
Forty-seven patients with T2D underwent myocardial [18F]FDG PET/CT at baseline and after a hyperinsulinemic-euglycemic clamp (HEC) to determine mIR were prospectively recruited. Biochemical assessments were performed before and after the HEC. Baseline hepatic steatosis index and index of hepatic fibrosis (FIB-4) were calculated. Furthermore, liver stiffness measurement was performed using transient elastography.
|
METHODS
|
The best model to predict the presence of mIR was the combination of transaminases, protein levels, FIB-4 score and HOMA (AUC = 0.95; sensibility: 0.81; specificity: 0.95). We observed significantly higher levels of fibrosis in patients with mIR than in those with mIS (p = 0.034). In addition, we found that patients with mIR presented a reduced glucose uptake by the liver in comparison with patients with mIS.
|
RESULTS
|
The combination of HOMA, protein, transaminases and FIB-4 is a simple and reliable tool for identifying mIR in patients with T2D. This information will be useful to improve the stratification of cardiovascular risk in T2D.
|
CONCLUSIONS
|
[
"Diabetes Mellitus, Type 2",
"Fibrosis",
"Humans",
"Insulin Resistance",
"Liver",
"Myocardium",
"Non-alcoholic Fatty Liver Disease",
"Positron Emission Tomography Computed Tomography",
"Transaminases"
] |
9369008
|
1. Introduction
|
Type 2 diabetes (T2D) is a highly prevalent multifactorial disease caused by a combination of genetic and lifestyle factors which represents a huge societal problem associated with a significant economic burden for all healthcare systems [1,2]. T2D is characterized by high levels of plasma glucose caused mainly by pancreatic β-cell dysfunction and inefficient action of insulin, known as insulin resistance (IR), which occurs in cells of insulin-sensitive key organs and tissues such as the heart or skeletal muscle [1,3]. These high levels of plasma glucose cause mainly micro- and macrovascular complications [4,5]. Likewise, IR causes cells to not respond to the action of insulin to incorporate glucose and, thus, obtain energy by maintaining a normal metabolism [1,6]. The deficit of glucose in cells causes metabolic alterations and changes in oxidizable substrates to obtain energy, triggering severe pathological complications [7,8]. Therefore, both chronic hyperglycemia and IR have been related to the appearance of cardiovascular diseases (CVD) such as coronary artery diseases (CAD), stroke, and cardiomyopathies in T2D [6,8,9,10].
Myocardium is a striated muscle that constitutes the main tissue of the walls of the heart, and plays crucial roles for contraction, the basic function of the heart [11]. The main cells of the muscle are cardiomyocytes that are insulin-sensitive cells and, consequently, can be affected by the IR related to T2D. Many clinical trials have shown that systemic IR is an independent risk factor for heart failure and cardiovascular death [12]. In addition, increasing evidence points to IR as the primary etiologic factor in the development of non-ischemic heart failure [13,14].
A number of preclinical studies have shown that myocardial IR (mIR), which is characterized by inefficient energy metabolism, co-occurs with systemic insulin resistance and contributes to post-ischemic heart failure [15]. The presence of mIR should be put in the context of the general IR, in which the main contributors are the liver, muscles other than myocardium, and the adipose tissue. Some researchers have suggested not only that the heart is a target organ of systemic insulin resistance, but also that local mIR is a distinct risk factor for heart failure [16]. The myocardium normally responds to injury by altering substrate metabolism to increase energy efficiency. Insulin resistance prevents this adaptive response and can lead to further injury by contributing to lipotoxicity, sympathetic up-regulation, inflammation, oxidative stress, and fibrosis [13].
We have recently reported the presence of mIR in around 60% of patients with T2D patients without previous CVD [17]. These patients present higher myocardial radiodensity and a significantly higher coronary artery calcium score (CACs), a reliable biomarker of cardiovascular risk, when compared with those patients with insulin-sensitive myocardium (mIS). In addition, we observed that systemic IR is mainly accounted by patients with mIR, thus underlying the important role of the myocardium in glucose metabolism. This study was performed using 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET/CT) before and after a hyperinsulinemic–euglycemic clamp (HEC). This is an expensive, radioactive, time-consuming and cumbersome examination that cannot be proposed for daily clinical practice. Therefore, a more accessible, cheap and widely applicable surrogate to identify patients with mIR is needed.
In the present study, we examined a myriad of plasma biochemical parameters aimed at obtaining a reliable surrogate that permits us to differentiate the two phenotypes of T2D patients: mIR and mIS.
| null | null |
2. Results
|
The general results of PET/CT before and after HEC were previously reported [17]. In summary, the first (baseline) PET/CT showed in all patients a very low left ventricular [18]FDG uptake. After HEC, we were able to observe two clear behaviors: (1) A pronounced enhancement in [18]FDG uptake in 21 cases (44.7%), thus revealing an insulin-sensitive myocardium (mIS). (2) A marginal increase in [18]FDG uptake in the remaining 26 (55.30%) patients, thus indicating the presence of an insulin-resistant myocardium (mIR).
2.1. General Characteristics of T2D Patients The main clinical and biochemical variables of T2D patients included in the study, taking into account the myocardial insulin resistance (mIS vs. mIR), are shown in Table 1. The antidiabetic treatment is displayed in Table 2.
The main clinical and biochemical variables of T2D patients included in the study, taking into account the myocardial insulin resistance (mIS vs. mIR), are shown in Table 1. The antidiabetic treatment is displayed in Table 2.
2.2. Biomarkers Discriminating between mIR and mIS Baseline and post-HEC biochemical variables were divided by mIS and mIR subgroups and analyzed. The variables significantly different between both groups at baseline are shown in Table 1. As can be seen, HOMA-IR, ALT, AST, GGT and protein were significantly higher in the mIR group than in the mIS group. However, after HEC, we found that only ALT and FFA were significantly increased in the mIR group: 29 IU/L [16:42] vs. 18 IU/L [16:21] (p = 0.0180), and 0.32 mmol/L [0.18:0.38] vs. 0.20 mmol/L p = 0.0149, respectively. We did not find any difference between males and females when comparing mIR vs. mIS and neither of the biochemical variables were useful for discriminating both phenotypes both at baseline and after HEC.
Since liver enzymes were the most closely associated with mIR, we wanted to explore whether the hepatic steatosis index (HSI), a simple screening tool reflecting non-alcoholic fatty liver disease (NAFLD), was different between mIR and mIS patients [18]. We found a higher HIS in patients with mIR in comparison with those patients with mIS but the differences did not reach statistical significance (2.35 [1.06:5.62] vs. 1.16 [0.53:1.80]; p = 0.055) (Figure 1A). In addition, we examined whether the non-alcoholic fatty liver disease–liver fat score (NAFLD-LFS) [19], an index that integrates IR and hepatic damage, could be able to discriminate patients with and without mIR. We found that patients with mIR presented higher values than patients with mIS (2.58 [1.08:8.31] vs. 1.05 [0.47:1.76]; p = 0.0154) (Figure 1B). Finally, the usefulness of an index of hepatic fibrosis (FIB-4) was assessed [20]. We found a significantly higher FIB-4 score in patients with mIR in comparison with patients with mIS (1.65 [1.07:2.73] vs. 1.18 [0.82:1.49]; p = 0.025) (Figure 1C).
Since the baseline (fasting state) is the current state for performing biochemical analysis, thresholds and AUC for optimal discrimination at baseline between both groups (mIR and mIS) were obtained for the variables significantly different between groups (Figure 2).
In addition, different models combining the variables displayed in Figure 2 were analyzed to dichotomize both phenotypes (mIR vs. mIS). The results are shown in Figure 3.
The best model was the combination of Protein, AST, ALT, GGT, FIB and HOMA (AUC = 0.95; sensibility: 0.81; and specificity: 0.95). Since HOMA is not always available in the daily clinical practice and it cannot be used in those patients receiving treatment with insulin, we wanted to examine the result obtained by leaving HOMA out of the model, and the result was AUC = 0.87; sensibility: 0.82; and specificity: 0.96.
Baseline and post-HEC biochemical variables were divided by mIS and mIR subgroups and analyzed. The variables significantly different between both groups at baseline are shown in Table 1. As can be seen, HOMA-IR, ALT, AST, GGT and protein were significantly higher in the mIR group than in the mIS group. However, after HEC, we found that only ALT and FFA were significantly increased in the mIR group: 29 IU/L [16:42] vs. 18 IU/L [16:21] (p = 0.0180), and 0.32 mmol/L [0.18:0.38] vs. 0.20 mmol/L p = 0.0149, respectively. We did not find any difference between males and females when comparing mIR vs. mIS and neither of the biochemical variables were useful for discriminating both phenotypes both at baseline and after HEC.
Since liver enzymes were the most closely associated with mIR, we wanted to explore whether the hepatic steatosis index (HSI), a simple screening tool reflecting non-alcoholic fatty liver disease (NAFLD), was different between mIR and mIS patients [18]. We found a higher HIS in patients with mIR in comparison with those patients with mIS but the differences did not reach statistical significance (2.35 [1.06:5.62] vs. 1.16 [0.53:1.80]; p = 0.055) (Figure 1A). In addition, we examined whether the non-alcoholic fatty liver disease–liver fat score (NAFLD-LFS) [19], an index that integrates IR and hepatic damage, could be able to discriminate patients with and without mIR. We found that patients with mIR presented higher values than patients with mIS (2.58 [1.08:8.31] vs. 1.05 [0.47:1.76]; p = 0.0154) (Figure 1B). Finally, the usefulness of an index of hepatic fibrosis (FIB-4) was assessed [20]. We found a significantly higher FIB-4 score in patients with mIR in comparison with patients with mIS (1.65 [1.07:2.73] vs. 1.18 [0.82:1.49]; p = 0.025) (Figure 1C).
Since the baseline (fasting state) is the current state for performing biochemical analysis, thresholds and AUC for optimal discrimination at baseline between both groups (mIR and mIS) were obtained for the variables significantly different between groups (Figure 2).
In addition, different models combining the variables displayed in Figure 2 were analyzed to dichotomize both phenotypes (mIR vs. mIS). The results are shown in Figure 3.
The best model was the combination of Protein, AST, ALT, GGT, FIB and HOMA (AUC = 0.95; sensibility: 0.81; and specificity: 0.95). Since HOMA is not always available in the daily clinical practice and it cannot be used in those patients receiving treatment with insulin, we wanted to examine the result obtained by leaving HOMA out of the model, and the result was AUC = 0.87; sensibility: 0.82; and specificity: 0.96.
2.3. Hepatic Fibrosis Measured by Fibroscan Since FIB-4 is a reliable index of hepatic fibrosis, we wanted to examine whether the degree of hepatic fibrosis measured by fibroscan was related to the presence of mIR. We observed significantly higher levels of fibrosis in patients with mIR than in those with mIS (9.10 [5.43:16.08] vs. 5.35 [3.70:6.38]; p = 0.034) Figure 4. ROC analysis of fibroscan data showed an AUC = 0.73 with a sensibility of 0.80 and specificity of 0.71.
In addition, patients with mIR presented a reduced glucose uptake by the liver in comparison with patients with mIS (Figure 5).
Since FIB-4 is a reliable index of hepatic fibrosis, we wanted to examine whether the degree of hepatic fibrosis measured by fibroscan was related to the presence of mIR. We observed significantly higher levels of fibrosis in patients with mIR than in those with mIS (9.10 [5.43:16.08] vs. 5.35 [3.70:6.38]; p = 0.034) Figure 4. ROC analysis of fibroscan data showed an AUC = 0.73 with a sensibility of 0.80 and specificity of 0.71.
In addition, patients with mIR presented a reduced glucose uptake by the liver in comparison with patients with mIS (Figure 5).
| null | null |
[
"2.2. Biomarkers Discriminating between mIR and mIS",
"2.3. Hepatic Fibrosis Measured by Fibroscan",
"4. Material and Methods",
"4.1. Subjects",
"4.2. Study Design",
"4.3. Biochemical Analysis",
"4.4. Fibroscan",
"4.5. Statistical Analysis"
] |
[
"Baseline and post-HEC biochemical variables were divided by mIS and mIR subgroups and analyzed. The variables significantly different between both groups at baseline are shown in Table 1. As can be seen, HOMA-IR, ALT, AST, GGT and protein were significantly higher in the mIR group than in the mIS group. However, after HEC, we found that only ALT and FFA were significantly increased in the mIR group: 29 IU/L [16:42] vs. 18 IU/L [16:21] (p = 0.0180), and 0.32 mmol/L [0.18:0.38] vs. 0.20 mmol/L p = 0.0149, respectively. We did not find any difference between males and females when comparing mIR vs. mIS and neither of the biochemical variables were useful for discriminating both phenotypes both at baseline and after HEC.\nSince liver enzymes were the most closely associated with mIR, we wanted to explore whether the hepatic steatosis index (HSI), a simple screening tool reflecting non-alcoholic fatty liver disease (NAFLD), was different between mIR and mIS patients [18]. We found a higher HIS in patients with mIR in comparison with those patients with mIS but the differences did not reach statistical significance (2.35 [1.06:5.62] vs. 1.16 [0.53:1.80]; p = 0.055) (Figure 1A). In addition, we examined whether the non-alcoholic fatty liver disease–liver fat score (NAFLD-LFS) [19], an index that integrates IR and hepatic damage, could be able to discriminate patients with and without mIR. We found that patients with mIR presented higher values than patients with mIS (2.58 [1.08:8.31] vs. 1.05 [0.47:1.76]; p = 0.0154) (Figure 1B). Finally, the usefulness of an index of hepatic fibrosis (FIB-4) was assessed [20]. We found a significantly higher FIB-4 score in patients with mIR in comparison with patients with mIS (1.65 [1.07:2.73] vs. 1.18 [0.82:1.49]; p = 0.025) (Figure 1C).\nSince the baseline (fasting state) is the current state for performing biochemical analysis, thresholds and AUC for optimal discrimination at baseline between both groups (mIR and mIS) were obtained for the variables significantly different between groups (Figure 2).\nIn addition, different models combining the variables displayed in Figure 2 were analyzed to dichotomize both phenotypes (mIR vs. mIS). The results are shown in Figure 3.\nThe best model was the combination of Protein, AST, ALT, GGT, FIB and HOMA (AUC = 0.95; sensibility: 0.81; and specificity: 0.95). Since HOMA is not always available in the daily clinical practice and it cannot be used in those patients receiving treatment with insulin, we wanted to examine the result obtained by leaving HOMA out of the model, and the result was AUC = 0.87; sensibility: 0.82; and specificity: 0.96.",
"Since FIB-4 is a reliable index of hepatic fibrosis, we wanted to examine whether the degree of hepatic fibrosis measured by fibroscan was related to the presence of mIR. We observed significantly higher levels of fibrosis in patients with mIR than in those with mIS (9.10 [5.43:16.08] vs. 5.35 [3.70:6.38]; p = 0.034) Figure 4. ROC analysis of fibroscan data showed an AUC = 0.73 with a sensibility of 0.80 and specificity of 0.71.\nIn addition, patients with mIR presented a reduced glucose uptake by the liver in comparison with patients with mIS (Figure 5).",
" 4.1. Subjects This proof-of-concept observational study comprised forty-seven T2D patients and was conducted according to the tenets of the Helsinki Declaration after approval by the Ethic Committee of the Vall d’Hebron University Hospital (ClinicalTrials.gov: NCT02248311). Written informed consent of all participants was obtained.\nThe inclusion criteria were: T2D patients from 50 to 79 years old, with at least 5 years from the diagnosis. The exclusion criteria were: (1) type 1 diabetes, (2) any previous CVD event, (3) patients with known cardiac pathology, (4) any contraindication or claustrophobia for the PET/CT, (5) any pathology related to a short life expectancy, (6) daily alcohol drinker, (7) smokers who did not stop smoking at least 1 year before (non-smoker and ex-smoker were recorded), (8) treatment with any cardiotoxic medication.\nSubjects were recruited at the Endocrinology Department of Vall d’Hebron University Hospital. Among a total of 250 clinical records of T2D patients reviewed, 72 met the inclusion criteria, but only 51 agreed to participate in the clinical trial. Four patients dropped out of the study before its completion.\nThis proof-of-concept observational study comprised forty-seven T2D patients and was conducted according to the tenets of the Helsinki Declaration after approval by the Ethic Committee of the Vall d’Hebron University Hospital (ClinicalTrials.gov: NCT02248311). Written informed consent of all participants was obtained.\nThe inclusion criteria were: T2D patients from 50 to 79 years old, with at least 5 years from the diagnosis. The exclusion criteria were: (1) type 1 diabetes, (2) any previous CVD event, (3) patients with known cardiac pathology, (4) any contraindication or claustrophobia for the PET/CT, (5) any pathology related to a short life expectancy, (6) daily alcohol drinker, (7) smokers who did not stop smoking at least 1 year before (non-smoker and ex-smoker were recorded), (8) treatment with any cardiotoxic medication.\nSubjects were recruited at the Endocrinology Department of Vall d’Hebron University Hospital. Among a total of 250 clinical records of T2D patients reviewed, 72 met the inclusion criteria, but only 51 agreed to participate in the clinical trial. Four patients dropped out of the study before its completion.\n 4.2. Study Design Two [18F]FDG PET/CT studies per patient were implemented: one at baseline and another after performing a HEC. The HEC procedure was performed as previously reported with minor modifications [48,49]. The two [18F]FDG PET/CT scans were performed for each patient in a random order within two days under at least 8 h of fasting conditions and after the withdrawal of any medication the day before. A dose of 1.9 MBq/Kg of [18]FDG was IV-administered to patients before each scan session. PET/CT acquisitions protocols and data processing and analysis were described by our group previously [17]. Several anthropometric measurements and blood samples for biochemical analysis were obtained during the procedure following the flow chart shown in Figure 6.\nTwo [18F]FDG PET/CT studies per patient were implemented: one at baseline and another after performing a HEC. The HEC procedure was performed as previously reported with minor modifications [48,49]. The two [18F]FDG PET/CT scans were performed for each patient in a random order within two days under at least 8 h of fasting conditions and after the withdrawal of any medication the day before. A dose of 1.9 MBq/Kg of [18]FDG was IV-administered to patients before each scan session. PET/CT acquisitions protocols and data processing and analysis were described by our group previously [17]. Several anthropometric measurements and blood samples for biochemical analysis were obtained during the procedure following the flow chart shown in Figure 6.\n 4.3. Biochemical Analysis Peripheral blood samples were collected under fasting conditions. Plasma samples for 1H-RMN analysis were stored at −80 °C at Vall d’Hebron Biobank. Biochemical analysis was performed at the Biochemistry Core Facilities of Vall d’Hebron University Hospital using standardized and validated routine methodologies. The following parameters were determined: blood count, glucose, HbA1c, fructosamine (FA), urea, creatinine (Cr), glomerular filtration (GF), sodium (Na), potassium (K), phosphate (PH), calcium (Ca), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamiltransferase (GGT), alkaline phosphatase (ALP), total cholesterol (C), high-density lipoprotein (HDL) cholesterol (C-HDL), low-density lipoprotein cholesterol (C-LDL), triglyceride (TG), total free fatty acids (FFA), protein (P), albumin, C-peptide, troponin (TN), chloride (Cl), total bilirubin (BR) and esterified bilirubin (E-BR). Non-routine laboratory parameters such as insulin, leptin and IL4 were also determined. Insulin was assessed by chemiluminescence immunoassay (Atellica IM, Siemens Healthineers, Erlangen, Germany), leptin analysis was performed by ELISA (Diagnostics Biochem Canada Inc., London, ON, Canada), and IL6 by electrochemiluminescence immunoassay (Cobas e2801, Roche Diagnostics, Basel, Switzerland) following strictly the instructions provided by the manufacturer.\nThe fibrosis index based on four factors (FIB-4) was determined by the standard equations using age, ALT, AST and platelet counting. 1H-RMN analysis was performed following the previous methodology described by our group to determine VLDL (CH) and VLDL (aliphatic chain) [50].\nPeripheral blood samples were collected under fasting conditions. Plasma samples for 1H-RMN analysis were stored at −80 °C at Vall d’Hebron Biobank. Biochemical analysis was performed at the Biochemistry Core Facilities of Vall d’Hebron University Hospital using standardized and validated routine methodologies. The following parameters were determined: blood count, glucose, HbA1c, fructosamine (FA), urea, creatinine (Cr), glomerular filtration (GF), sodium (Na), potassium (K), phosphate (PH), calcium (Ca), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamiltransferase (GGT), alkaline phosphatase (ALP), total cholesterol (C), high-density lipoprotein (HDL) cholesterol (C-HDL), low-density lipoprotein cholesterol (C-LDL), triglyceride (TG), total free fatty acids (FFA), protein (P), albumin, C-peptide, troponin (TN), chloride (Cl), total bilirubin (BR) and esterified bilirubin (E-BR). Non-routine laboratory parameters such as insulin, leptin and IL4 were also determined. Insulin was assessed by chemiluminescence immunoassay (Atellica IM, Siemens Healthineers, Erlangen, Germany), leptin analysis was performed by ELISA (Diagnostics Biochem Canada Inc., London, ON, Canada), and IL6 by electrochemiluminescence immunoassay (Cobas e2801, Roche Diagnostics, Basel, Switzerland) following strictly the instructions provided by the manufacturer.\nThe fibrosis index based on four factors (FIB-4) was determined by the standard equations using age, ALT, AST and platelet counting. 1H-RMN analysis was performed following the previous methodology described by our group to determine VLDL (CH) and VLDL (aliphatic chain) [50].\n 4.4. Fibroscan Liver stiffness measurement (LSM) was performed using transient elastography (Fibroscan 502 Touch, Echosens, Paris, France). According to the usual standard procedure, measurements were performed under fasting conditions with at least 10 valid measurements and an interquartile to median ratio ≤ 30% [51]. Data are expressed in kilopascals (kPa). Normal LSM values varied between 4 and 6 kPa and values ≥ 6 kPa were considered abnormal and suggestive of liver mild fibrosis/disease.\nLiver stiffness measurement (LSM) was performed using transient elastography (Fibroscan 502 Touch, Echosens, Paris, France). According to the usual standard procedure, measurements were performed under fasting conditions with at least 10 valid measurements and an interquartile to median ratio ≤ 30% [51]. Data are expressed in kilopascals (kPa). Normal LSM values varied between 4 and 6 kPa and values ≥ 6 kPa were considered abnormal and suggestive of liver mild fibrosis/disease.\n 4.5. Statistical Analysis Data are expressed as median and interquartile range (IQR). Data normality was assessed with the Shapiro–Wilk test. The Kolmogorov–Smirnov test was used for group comparisons, as appropriate. Spearman’s correlation analysis and ROC curves were performed for variable associations. An ROC analysis was also performed to determine the utility of variables to dichotomize groups and obtain thresholds for fingerprints. With the extracted altered parameters for T2D phenotype segregation, new multi-variable functions were computed using the generalized linear model approach with specifications for binomial distribution and logistic-type canonical link function. This was carried out to compute alternative variable definitions for patient classification based on a combination of the mentioned parameters. ROC curves using the newly defined variables were obtained to assess the best combination according to the highest AUC value. Thresholds for each of the included parameters as well as for the combination of them were obtained by means of the same procedure as in the independent-variable assessment. All data were analyzed in GraphPad Prism (Version 6.01, San Diego, CA, USA) or R commander (Version 2.3-1, Hamilton, ON, Canada). p values below 0.05 were considered statistically significant.\nData are expressed as median and interquartile range (IQR). Data normality was assessed with the Shapiro–Wilk test. The Kolmogorov–Smirnov test was used for group comparisons, as appropriate. Spearman’s correlation analysis and ROC curves were performed for variable associations. An ROC analysis was also performed to determine the utility of variables to dichotomize groups and obtain thresholds for fingerprints. With the extracted altered parameters for T2D phenotype segregation, new multi-variable functions were computed using the generalized linear model approach with specifications for binomial distribution and logistic-type canonical link function. This was carried out to compute alternative variable definitions for patient classification based on a combination of the mentioned parameters. ROC curves using the newly defined variables were obtained to assess the best combination according to the highest AUC value. Thresholds for each of the included parameters as well as for the combination of them were obtained by means of the same procedure as in the independent-variable assessment. All data were analyzed in GraphPad Prism (Version 6.01, San Diego, CA, USA) or R commander (Version 2.3-1, Hamilton, ON, Canada). p values below 0.05 were considered statistically significant.",
"This proof-of-concept observational study comprised forty-seven T2D patients and was conducted according to the tenets of the Helsinki Declaration after approval by the Ethic Committee of the Vall d’Hebron University Hospital (ClinicalTrials.gov: NCT02248311). Written informed consent of all participants was obtained.\nThe inclusion criteria were: T2D patients from 50 to 79 years old, with at least 5 years from the diagnosis. The exclusion criteria were: (1) type 1 diabetes, (2) any previous CVD event, (3) patients with known cardiac pathology, (4) any contraindication or claustrophobia for the PET/CT, (5) any pathology related to a short life expectancy, (6) daily alcohol drinker, (7) smokers who did not stop smoking at least 1 year before (non-smoker and ex-smoker were recorded), (8) treatment with any cardiotoxic medication.\nSubjects were recruited at the Endocrinology Department of Vall d’Hebron University Hospital. Among a total of 250 clinical records of T2D patients reviewed, 72 met the inclusion criteria, but only 51 agreed to participate in the clinical trial. Four patients dropped out of the study before its completion.",
"Two [18F]FDG PET/CT studies per patient were implemented: one at baseline and another after performing a HEC. The HEC procedure was performed as previously reported with minor modifications [48,49]. The two [18F]FDG PET/CT scans were performed for each patient in a random order within two days under at least 8 h of fasting conditions and after the withdrawal of any medication the day before. A dose of 1.9 MBq/Kg of [18]FDG was IV-administered to patients before each scan session. PET/CT acquisitions protocols and data processing and analysis were described by our group previously [17]. Several anthropometric measurements and blood samples for biochemical analysis were obtained during the procedure following the flow chart shown in Figure 6.",
"Peripheral blood samples were collected under fasting conditions. Plasma samples for 1H-RMN analysis were stored at −80 °C at Vall d’Hebron Biobank. Biochemical analysis was performed at the Biochemistry Core Facilities of Vall d’Hebron University Hospital using standardized and validated routine methodologies. The following parameters were determined: blood count, glucose, HbA1c, fructosamine (FA), urea, creatinine (Cr), glomerular filtration (GF), sodium (Na), potassium (K), phosphate (PH), calcium (Ca), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamiltransferase (GGT), alkaline phosphatase (ALP), total cholesterol (C), high-density lipoprotein (HDL) cholesterol (C-HDL), low-density lipoprotein cholesterol (C-LDL), triglyceride (TG), total free fatty acids (FFA), protein (P), albumin, C-peptide, troponin (TN), chloride (Cl), total bilirubin (BR) and esterified bilirubin (E-BR). Non-routine laboratory parameters such as insulin, leptin and IL4 were also determined. Insulin was assessed by chemiluminescence immunoassay (Atellica IM, Siemens Healthineers, Erlangen, Germany), leptin analysis was performed by ELISA (Diagnostics Biochem Canada Inc., London, ON, Canada), and IL6 by electrochemiluminescence immunoassay (Cobas e2801, Roche Diagnostics, Basel, Switzerland) following strictly the instructions provided by the manufacturer.\nThe fibrosis index based on four factors (FIB-4) was determined by the standard equations using age, ALT, AST and platelet counting. 1H-RMN analysis was performed following the previous methodology described by our group to determine VLDL (CH) and VLDL (aliphatic chain) [50].",
"Liver stiffness measurement (LSM) was performed using transient elastography (Fibroscan 502 Touch, Echosens, Paris, France). According to the usual standard procedure, measurements were performed under fasting conditions with at least 10 valid measurements and an interquartile to median ratio ≤ 30% [51]. Data are expressed in kilopascals (kPa). Normal LSM values varied between 4 and 6 kPa and values ≥ 6 kPa were considered abnormal and suggestive of liver mild fibrosis/disease.",
"Data are expressed as median and interquartile range (IQR). Data normality was assessed with the Shapiro–Wilk test. The Kolmogorov–Smirnov test was used for group comparisons, as appropriate. Spearman’s correlation analysis and ROC curves were performed for variable associations. An ROC analysis was also performed to determine the utility of variables to dichotomize groups and obtain thresholds for fingerprints. With the extracted altered parameters for T2D phenotype segregation, new multi-variable functions were computed using the generalized linear model approach with specifications for binomial distribution and logistic-type canonical link function. This was carried out to compute alternative variable definitions for patient classification based on a combination of the mentioned parameters. ROC curves using the newly defined variables were obtained to assess the best combination according to the highest AUC value. Thresholds for each of the included parameters as well as for the combination of them were obtained by means of the same procedure as in the independent-variable assessment. All data were analyzed in GraphPad Prism (Version 6.01, San Diego, CA, USA) or R commander (Version 2.3-1, Hamilton, ON, Canada). p values below 0.05 were considered statistically significant."
] |
[
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"1. Introduction",
"2. Results",
"2.1. General Characteristics of T2D Patients",
"2.2. Biomarkers Discriminating between mIR and mIS",
"2.3. Hepatic Fibrosis Measured by Fibroscan",
"3. Discussion",
"4. Material and Methods",
"4.1. Subjects",
"4.2. Study Design",
"4.3. Biochemical Analysis",
"4.4. Fibroscan",
"4.5. Statistical Analysis"
] |
[
"Type 2 diabetes (T2D) is a highly prevalent multifactorial disease caused by a combination of genetic and lifestyle factors which represents a huge societal problem associated with a significant economic burden for all healthcare systems [1,2]. T2D is characterized by high levels of plasma glucose caused mainly by pancreatic β-cell dysfunction and inefficient action of insulin, known as insulin resistance (IR), which occurs in cells of insulin-sensitive key organs and tissues such as the heart or skeletal muscle [1,3]. These high levels of plasma glucose cause mainly micro- and macrovascular complications [4,5]. Likewise, IR causes cells to not respond to the action of insulin to incorporate glucose and, thus, obtain energy by maintaining a normal metabolism [1,6]. The deficit of glucose in cells causes metabolic alterations and changes in oxidizable substrates to obtain energy, triggering severe pathological complications [7,8]. Therefore, both chronic hyperglycemia and IR have been related to the appearance of cardiovascular diseases (CVD) such as coronary artery diseases (CAD), stroke, and cardiomyopathies in T2D [6,8,9,10].\nMyocardium is a striated muscle that constitutes the main tissue of the walls of the heart, and plays crucial roles for contraction, the basic function of the heart [11]. The main cells of the muscle are cardiomyocytes that are insulin-sensitive cells and, consequently, can be affected by the IR related to T2D. Many clinical trials have shown that systemic IR is an independent risk factor for heart failure and cardiovascular death [12]. In addition, increasing evidence points to IR as the primary etiologic factor in the development of non-ischemic heart failure [13,14].\nA number of preclinical studies have shown that myocardial IR (mIR), which is characterized by inefficient energy metabolism, co-occurs with systemic insulin resistance and contributes to post-ischemic heart failure [15]. The presence of mIR should be put in the context of the general IR, in which the main contributors are the liver, muscles other than myocardium, and the adipose tissue. Some researchers have suggested not only that the heart is a target organ of systemic insulin resistance, but also that local mIR is a distinct risk factor for heart failure [16]. The myocardium normally responds to injury by altering substrate metabolism to increase energy efficiency. Insulin resistance prevents this adaptive response and can lead to further injury by contributing to lipotoxicity, sympathetic up-regulation, inflammation, oxidative stress, and fibrosis [13].\nWe have recently reported the presence of mIR in around 60% of patients with T2D patients without previous CVD [17]. These patients present higher myocardial radiodensity and a significantly higher coronary artery calcium score (CACs), a reliable biomarker of cardiovascular risk, when compared with those patients with insulin-sensitive myocardium (mIS). In addition, we observed that systemic IR is mainly accounted by patients with mIR, thus underlying the important role of the myocardium in glucose metabolism. This study was performed using 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET/CT) before and after a hyperinsulinemic–euglycemic clamp (HEC). This is an expensive, radioactive, time-consuming and cumbersome examination that cannot be proposed for daily clinical practice. Therefore, a more accessible, cheap and widely applicable surrogate to identify patients with mIR is needed.\nIn the present study, we examined a myriad of plasma biochemical parameters aimed at obtaining a reliable surrogate that permits us to differentiate the two phenotypes of T2D patients: mIR and mIS.",
"The general results of PET/CT before and after HEC were previously reported [17]. In summary, the first (baseline) PET/CT showed in all patients a very low left ventricular [18]FDG uptake. After HEC, we were able to observe two clear behaviors: (1) A pronounced enhancement in [18]FDG uptake in 21 cases (44.7%), thus revealing an insulin-sensitive myocardium (mIS). (2) A marginal increase in [18]FDG uptake in the remaining 26 (55.30%) patients, thus indicating the presence of an insulin-resistant myocardium (mIR).\n 2.1. General Characteristics of T2D Patients The main clinical and biochemical variables of T2D patients included in the study, taking into account the myocardial insulin resistance (mIS vs. mIR), are shown in Table 1. The antidiabetic treatment is displayed in Table 2.\nThe main clinical and biochemical variables of T2D patients included in the study, taking into account the myocardial insulin resistance (mIS vs. mIR), are shown in Table 1. The antidiabetic treatment is displayed in Table 2.\n 2.2. Biomarkers Discriminating between mIR and mIS Baseline and post-HEC biochemical variables were divided by mIS and mIR subgroups and analyzed. The variables significantly different between both groups at baseline are shown in Table 1. As can be seen, HOMA-IR, ALT, AST, GGT and protein were significantly higher in the mIR group than in the mIS group. However, after HEC, we found that only ALT and FFA were significantly increased in the mIR group: 29 IU/L [16:42] vs. 18 IU/L [16:21] (p = 0.0180), and 0.32 mmol/L [0.18:0.38] vs. 0.20 mmol/L p = 0.0149, respectively. We did not find any difference between males and females when comparing mIR vs. mIS and neither of the biochemical variables were useful for discriminating both phenotypes both at baseline and after HEC.\nSince liver enzymes were the most closely associated with mIR, we wanted to explore whether the hepatic steatosis index (HSI), a simple screening tool reflecting non-alcoholic fatty liver disease (NAFLD), was different between mIR and mIS patients [18]. We found a higher HIS in patients with mIR in comparison with those patients with mIS but the differences did not reach statistical significance (2.35 [1.06:5.62] vs. 1.16 [0.53:1.80]; p = 0.055) (Figure 1A). In addition, we examined whether the non-alcoholic fatty liver disease–liver fat score (NAFLD-LFS) [19], an index that integrates IR and hepatic damage, could be able to discriminate patients with and without mIR. We found that patients with mIR presented higher values than patients with mIS (2.58 [1.08:8.31] vs. 1.05 [0.47:1.76]; p = 0.0154) (Figure 1B). Finally, the usefulness of an index of hepatic fibrosis (FIB-4) was assessed [20]. We found a significantly higher FIB-4 score in patients with mIR in comparison with patients with mIS (1.65 [1.07:2.73] vs. 1.18 [0.82:1.49]; p = 0.025) (Figure 1C).\nSince the baseline (fasting state) is the current state for performing biochemical analysis, thresholds and AUC for optimal discrimination at baseline between both groups (mIR and mIS) were obtained for the variables significantly different between groups (Figure 2).\nIn addition, different models combining the variables displayed in Figure 2 were analyzed to dichotomize both phenotypes (mIR vs. mIS). The results are shown in Figure 3.\nThe best model was the combination of Protein, AST, ALT, GGT, FIB and HOMA (AUC = 0.95; sensibility: 0.81; and specificity: 0.95). Since HOMA is not always available in the daily clinical practice and it cannot be used in those patients receiving treatment with insulin, we wanted to examine the result obtained by leaving HOMA out of the model, and the result was AUC = 0.87; sensibility: 0.82; and specificity: 0.96.\nBaseline and post-HEC biochemical variables were divided by mIS and mIR subgroups and analyzed. The variables significantly different between both groups at baseline are shown in Table 1. As can be seen, HOMA-IR, ALT, AST, GGT and protein were significantly higher in the mIR group than in the mIS group. However, after HEC, we found that only ALT and FFA were significantly increased in the mIR group: 29 IU/L [16:42] vs. 18 IU/L [16:21] (p = 0.0180), and 0.32 mmol/L [0.18:0.38] vs. 0.20 mmol/L p = 0.0149, respectively. We did not find any difference between males and females when comparing mIR vs. mIS and neither of the biochemical variables were useful for discriminating both phenotypes both at baseline and after HEC.\nSince liver enzymes were the most closely associated with mIR, we wanted to explore whether the hepatic steatosis index (HSI), a simple screening tool reflecting non-alcoholic fatty liver disease (NAFLD), was different between mIR and mIS patients [18]. We found a higher HIS in patients with mIR in comparison with those patients with mIS but the differences did not reach statistical significance (2.35 [1.06:5.62] vs. 1.16 [0.53:1.80]; p = 0.055) (Figure 1A). In addition, we examined whether the non-alcoholic fatty liver disease–liver fat score (NAFLD-LFS) [19], an index that integrates IR and hepatic damage, could be able to discriminate patients with and without mIR. We found that patients with mIR presented higher values than patients with mIS (2.58 [1.08:8.31] vs. 1.05 [0.47:1.76]; p = 0.0154) (Figure 1B). Finally, the usefulness of an index of hepatic fibrosis (FIB-4) was assessed [20]. We found a significantly higher FIB-4 score in patients with mIR in comparison with patients with mIS (1.65 [1.07:2.73] vs. 1.18 [0.82:1.49]; p = 0.025) (Figure 1C).\nSince the baseline (fasting state) is the current state for performing biochemical analysis, thresholds and AUC for optimal discrimination at baseline between both groups (mIR and mIS) were obtained for the variables significantly different between groups (Figure 2).\nIn addition, different models combining the variables displayed in Figure 2 were analyzed to dichotomize both phenotypes (mIR vs. mIS). The results are shown in Figure 3.\nThe best model was the combination of Protein, AST, ALT, GGT, FIB and HOMA (AUC = 0.95; sensibility: 0.81; and specificity: 0.95). Since HOMA is not always available in the daily clinical practice and it cannot be used in those patients receiving treatment with insulin, we wanted to examine the result obtained by leaving HOMA out of the model, and the result was AUC = 0.87; sensibility: 0.82; and specificity: 0.96.\n 2.3. Hepatic Fibrosis Measured by Fibroscan Since FIB-4 is a reliable index of hepatic fibrosis, we wanted to examine whether the degree of hepatic fibrosis measured by fibroscan was related to the presence of mIR. We observed significantly higher levels of fibrosis in patients with mIR than in those with mIS (9.10 [5.43:16.08] vs. 5.35 [3.70:6.38]; p = 0.034) Figure 4. ROC analysis of fibroscan data showed an AUC = 0.73 with a sensibility of 0.80 and specificity of 0.71.\nIn addition, patients with mIR presented a reduced glucose uptake by the liver in comparison with patients with mIS (Figure 5).\nSince FIB-4 is a reliable index of hepatic fibrosis, we wanted to examine whether the degree of hepatic fibrosis measured by fibroscan was related to the presence of mIR. We observed significantly higher levels of fibrosis in patients with mIR than in those with mIS (9.10 [5.43:16.08] vs. 5.35 [3.70:6.38]; p = 0.034) Figure 4. ROC analysis of fibroscan data showed an AUC = 0.73 with a sensibility of 0.80 and specificity of 0.71.\nIn addition, patients with mIR presented a reduced glucose uptake by the liver in comparison with patients with mIS (Figure 5).",
"The main clinical and biochemical variables of T2D patients included in the study, taking into account the myocardial insulin resistance (mIS vs. mIR), are shown in Table 1. The antidiabetic treatment is displayed in Table 2.",
"Baseline and post-HEC biochemical variables were divided by mIS and mIR subgroups and analyzed. The variables significantly different between both groups at baseline are shown in Table 1. As can be seen, HOMA-IR, ALT, AST, GGT and protein were significantly higher in the mIR group than in the mIS group. However, after HEC, we found that only ALT and FFA were significantly increased in the mIR group: 29 IU/L [16:42] vs. 18 IU/L [16:21] (p = 0.0180), and 0.32 mmol/L [0.18:0.38] vs. 0.20 mmol/L p = 0.0149, respectively. We did not find any difference between males and females when comparing mIR vs. mIS and neither of the biochemical variables were useful for discriminating both phenotypes both at baseline and after HEC.\nSince liver enzymes were the most closely associated with mIR, we wanted to explore whether the hepatic steatosis index (HSI), a simple screening tool reflecting non-alcoholic fatty liver disease (NAFLD), was different between mIR and mIS patients [18]. We found a higher HIS in patients with mIR in comparison with those patients with mIS but the differences did not reach statistical significance (2.35 [1.06:5.62] vs. 1.16 [0.53:1.80]; p = 0.055) (Figure 1A). In addition, we examined whether the non-alcoholic fatty liver disease–liver fat score (NAFLD-LFS) [19], an index that integrates IR and hepatic damage, could be able to discriminate patients with and without mIR. We found that patients with mIR presented higher values than patients with mIS (2.58 [1.08:8.31] vs. 1.05 [0.47:1.76]; p = 0.0154) (Figure 1B). Finally, the usefulness of an index of hepatic fibrosis (FIB-4) was assessed [20]. We found a significantly higher FIB-4 score in patients with mIR in comparison with patients with mIS (1.65 [1.07:2.73] vs. 1.18 [0.82:1.49]; p = 0.025) (Figure 1C).\nSince the baseline (fasting state) is the current state for performing biochemical analysis, thresholds and AUC for optimal discrimination at baseline between both groups (mIR and mIS) were obtained for the variables significantly different between groups (Figure 2).\nIn addition, different models combining the variables displayed in Figure 2 were analyzed to dichotomize both phenotypes (mIR vs. mIS). The results are shown in Figure 3.\nThe best model was the combination of Protein, AST, ALT, GGT, FIB and HOMA (AUC = 0.95; sensibility: 0.81; and specificity: 0.95). Since HOMA is not always available in the daily clinical practice and it cannot be used in those patients receiving treatment with insulin, we wanted to examine the result obtained by leaving HOMA out of the model, and the result was AUC = 0.87; sensibility: 0.82; and specificity: 0.96.",
"Since FIB-4 is a reliable index of hepatic fibrosis, we wanted to examine whether the degree of hepatic fibrosis measured by fibroscan was related to the presence of mIR. We observed significantly higher levels of fibrosis in patients with mIR than in those with mIS (9.10 [5.43:16.08] vs. 5.35 [3.70:6.38]; p = 0.034) Figure 4. ROC analysis of fibroscan data showed an AUC = 0.73 with a sensibility of 0.80 and specificity of 0.71.\nIn addition, patients with mIR presented a reduced glucose uptake by the liver in comparison with patients with mIS (Figure 5).",
"Myocardial insulin resistance (mIR) has been hypothesized as a risk factor of CVD and cardiomyopathies in T2D patients [17,21]. Approaches based on dynamic PET/CT under HEC conditions have linked the myocardial [18F]FDG utilization rate to mIR [18,19]. Our group clearly identified T2D patients with mIR for the first time in a proof of concept using two [18F]FDG PET/CTs per patient (baseline and post-HEC) [17]. Since this methodology can hardly be proposed for current clinical practice, cost-effective and reliable surrogates are needed. In the present study, we provide evidence that transaminases and FIB-4 (an index of hepatic fibrosis) are the best predictors of mIR in patients with T2D without previous history of cardiovascular events.\nWe found that the best model to predict the presence of mIR was the combination of protein, AST, ALT, GGT, FIB-4 and HOMA-IR at baseline (AUC = 0.95; sensibility: 0.81; specificity: 0.95). Since HOMA-IR is not always available in the daily clinical practice and it cannot be used in those patients receiving treatment with insulin, we examined the result obtained by leaving HOMA-IR out of the model, and the result was AUC = 0.87 with a sensitivity of 0.82 and a specificity of 0.96. This means that general practitioners could have a useful index to estimate the presence of mIR in patients with T2D.\nRegarding the variables altered after the HEC procedure, the higher serum levels of FFA and ALT were found in the IRm subgroup. The increase in FFA in those patients with mIR could be expected because it is well-established that FFAs decrease myocardial [18F]FDG uptake [22]. It is well-established that the primary fuel for the myocardium is represented by FFAs in the fasting state and glucose in the fed state [23,24]. Myocardial glucose uptake is inversely associated with serum FFAs in both healthy and insulin-resistant conditions, and such a relationship explains the bulk of the individual variation in myocardial metabolism [24,25]. However, the fact that levels of FFA were significantly different between mIR and mIS patients only after HEC but not at the baseline/fasting state is intriguing and merits further investigation. The higher plasma levels of ALT after HEC in diabetic patients with mIR in comparison with patients with mIS have not previously reported and speak in favor of the robustness of ALT in dichotomizing both groups (mIR and mIS). Nevertheless, for the practical point of view, the fasting/baseline results are the only useful ones for clinical purposes.\nOverall, our results confirm previous findings suggesting the existence of a liver–heart axis [26,27,28,29]. To the best of our knowledge, this is the first report linking hepatic liver disease and mIR for T2D without previous CVD. In addition, we demonstrated that patients with mIR presented a significantly lower glucose uptake by the liver than patients with mIS. This result reveals the existence of an association between the myocardium and liver IR, which is essential to understand our findings. The liver is a central hub for lipid metabolism and endogenous glucose production; therefore, the liver is crucial for systemic glucose and lipid homeostasis. Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease, affecting approximately one-quarter of adults worldwide. NAFLD encompasses a pathological condition characterized by the ectopic deposition of adipose tissue in the liver, in the absence of secondary causes for hepatic fat accumulation, such as excessive alcohol intake, steatogenic drugs, and hereditary disorders [30]. Beyond liver-related morbidity and mortality, there is increasing evidence that patients with NAFLD are also at high risk of cardiovascular diseases (CVDs) [31]. Notably, 25% to 40% of patients with NAFLD also have CVD, and CVD accounts for a higher proportion of mortality than liver-related death in patients with NAFLD [32]. Recently, experts reached a consensus that NAFLD does not reflect the current knowledge, and metabolic-dysfunction-associated fatty liver disease (MAFLD) was suggested as a more appropriate term [33]. Although NAFLD or MAFLD is among the major forces driving CVD, the mechanistic relationship with CVD remains to be elucidated. Possible mechanisms include, but are not limited to, insulin resistance, low-grade systemic inflammation, oxidative stress and neuroendocrine activation [29]. Besides these common mechanisms, changes in protein secretion from the fatty liver also contribute to the pathogenesis of CVDs. In fact, the liver has recently been recognized as an endocrine organ that secretes hepatokines, which are proteins secreted by hepatocytes that can influence metabolic processes through autocrine, paracrine, and endocrine [34,35,36]. In the setting of MAFLD, several hepatokines can be downregulated such as sex hormone-binding globulin (SHBG), angiopoietin-like protein 4 (ANGPTL4) and adropin, whereas others such as fetuin-A, fetuin-B, hepassocin, leukocyte cell-derived chemotaxin 2 (LECT2), retinol-binding protein 4 (RBP4), selenoprotein P (SeP) and fibroblast growth factor 21 are upregulated [29,35]. The specific contribution of hepatokine imbalance on the uptake of glucose by the myocardium is beyond the scope of this paper but will be a piece of cutting-edge research in the coming years.\nLautamäki et al. demonstrated for the first time that liver fat content is an independent, significant mediator of myocardial glucose uptake in T2D patients with CAD [37]. In addition, they suggested a direct relationship between NAFLD and coronary atherosclerosis by showing impaired coronary flow reserve and an increment in markers of low-grade inflammation in patients with NAFLD. Our findings reinforce and extend this concept to patients with T2D without CVD. In addition, our findings suggest that, rather than steatosis, it is the presence of liver fibrosis which is the most important factor contributing to mIR. In fact, we found significantly higher levels of fibrosis in patients with mIR than in those with mIS. This result suggests that pro-fibrotic molecules might be essential players in linking MAFLD and mIR. Liver fibrosis has been suggested to be an independent predictor of the incidence of CVD and CV events in patients with NAFLD [38,39,40] and has a crucial role in the development of CVD through coronary microvascular dysfunction [41]. Recently, Liu et al. showed that liver fibrosis score metrics were significantly associated with the occurrence of recurrent cardiovascular events in patients with coronary artery disease and prior cardiovascular events [42]. It should be noted that hepatic lipid accumulation may not be toxic, and can also function as a protective mechanism to buffer the lipotoxic effects of FFA to hepatocytes. The progression of pure hepatic steatosis to liver fibrosis remains silent and underdiagnosed until its development into cirrhosis, characterized by severe liver fibrosis. In our cohort, we found a significantly higher proportion of fibrosis (≥6 Kpa) in patients with mIR than mIS (70% vs. 18.7%). Hepatic steatosis and incipient liver fibrosis may be reversible at early stages, and, in this regard, the accurate quantification of these hepatic phenotypes becomes central to the design of preventive strategies involving lifestyle modifications and therapeutic interventions [43,44]. It should be underlined that the level of transaminases in those patients with mIR was in the normal range. Furthermore, a high percentage of patients with T2D with liver fibrosis also presented transaminases in the normal range, thus making difficult the clinical suspicion. These findings are in agreement with previous reports [45,46,47] and suggest being proactive in identifying T2D at risk of having mIR by using the mentioned algorithms.\nFinally, the observed relationship between glucose uptake in the myocardium and the liver point mIR is a key factor in accounting for the increased CV risk in patients with MAFLD. The pathophysiological interactions between the liver and heart can be classified into three groups: (1) liver disease resulting from heart disease; (2) heart disease resulting from liver disease; (3) systemic conditions affecting the heart and the liver at the same time. Our findings suggest that T2D could be one of these systemic diseases altering, simultaneously, glucose uptake in both the liver and the heart. Nevertheless, as mentioned above, a primary pathogenic role of hepatokine mediator/s should be underlined.\nThere are some limitations in this study. First, we did not include a healthy control group, and the number of patients with T2D who were enrolled was low. The main reason is because PET/CT combined with HEC is cumbersome and time-consuming. In addition, using two PET/CTs on the same patient is limited due to ethical issues. Second, we did not confirm the presence and degree of hepatic fibrosis with additional histological assessments. Third, the withdrawal of the antidiabetic drugs one day before PET/CT could not be sufficient to assure a correct wash out. However, the insulin-mediated glucose uptake by using HEC overrides any eventual marginal action of insulin sensitizers or GLP-1 in terms of increasing the myocardial glucose uptake. In this regard, it should be noted that we observed a very low left-ventricular [18]FDG uptake in all patients in the first (baseline) PET/CT, and we did not observe any influence on [18]FDG uptake from antidiabetic drugs at both baseline and post [18F]FDG PET/CT. Fourth, although BMI was similar in mIR and mIR patients, a specific study on body composition was not performed and, therefore, a potential link between adipose tissue and mIR cannot be ruled out. Finally, studies on the long-term clinical consequences of mIR are needed.\nIn summary, our results add valuable information to understanding the “crosstalk” between the liver and the cardiovascular system. We provide evidence that T2D individuals with mIR can be identified by using a simple algorithm based on the plasma levels of proteins and transaminases. This information will be useful to improve the stratification of cardiovascular risk in patients with T2D, and will aid the development of new therapeutic strategies aimed at abrogating the underlying vicious circuits between the liver and the heart.",
" 4.1. Subjects This proof-of-concept observational study comprised forty-seven T2D patients and was conducted according to the tenets of the Helsinki Declaration after approval by the Ethic Committee of the Vall d’Hebron University Hospital (ClinicalTrials.gov: NCT02248311). Written informed consent of all participants was obtained.\nThe inclusion criteria were: T2D patients from 50 to 79 years old, with at least 5 years from the diagnosis. The exclusion criteria were: (1) type 1 diabetes, (2) any previous CVD event, (3) patients with known cardiac pathology, (4) any contraindication or claustrophobia for the PET/CT, (5) any pathology related to a short life expectancy, (6) daily alcohol drinker, (7) smokers who did not stop smoking at least 1 year before (non-smoker and ex-smoker were recorded), (8) treatment with any cardiotoxic medication.\nSubjects were recruited at the Endocrinology Department of Vall d’Hebron University Hospital. Among a total of 250 clinical records of T2D patients reviewed, 72 met the inclusion criteria, but only 51 agreed to participate in the clinical trial. Four patients dropped out of the study before its completion.\nThis proof-of-concept observational study comprised forty-seven T2D patients and was conducted according to the tenets of the Helsinki Declaration after approval by the Ethic Committee of the Vall d’Hebron University Hospital (ClinicalTrials.gov: NCT02248311). Written informed consent of all participants was obtained.\nThe inclusion criteria were: T2D patients from 50 to 79 years old, with at least 5 years from the diagnosis. The exclusion criteria were: (1) type 1 diabetes, (2) any previous CVD event, (3) patients with known cardiac pathology, (4) any contraindication or claustrophobia for the PET/CT, (5) any pathology related to a short life expectancy, (6) daily alcohol drinker, (7) smokers who did not stop smoking at least 1 year before (non-smoker and ex-smoker were recorded), (8) treatment with any cardiotoxic medication.\nSubjects were recruited at the Endocrinology Department of Vall d’Hebron University Hospital. Among a total of 250 clinical records of T2D patients reviewed, 72 met the inclusion criteria, but only 51 agreed to participate in the clinical trial. Four patients dropped out of the study before its completion.\n 4.2. Study Design Two [18F]FDG PET/CT studies per patient were implemented: one at baseline and another after performing a HEC. The HEC procedure was performed as previously reported with minor modifications [48,49]. The two [18F]FDG PET/CT scans were performed for each patient in a random order within two days under at least 8 h of fasting conditions and after the withdrawal of any medication the day before. A dose of 1.9 MBq/Kg of [18]FDG was IV-administered to patients before each scan session. PET/CT acquisitions protocols and data processing and analysis were described by our group previously [17]. Several anthropometric measurements and blood samples for biochemical analysis were obtained during the procedure following the flow chart shown in Figure 6.\nTwo [18F]FDG PET/CT studies per patient were implemented: one at baseline and another after performing a HEC. The HEC procedure was performed as previously reported with minor modifications [48,49]. The two [18F]FDG PET/CT scans were performed for each patient in a random order within two days under at least 8 h of fasting conditions and after the withdrawal of any medication the day before. A dose of 1.9 MBq/Kg of [18]FDG was IV-administered to patients before each scan session. PET/CT acquisitions protocols and data processing and analysis were described by our group previously [17]. Several anthropometric measurements and blood samples for biochemical analysis were obtained during the procedure following the flow chart shown in Figure 6.\n 4.3. Biochemical Analysis Peripheral blood samples were collected under fasting conditions. Plasma samples for 1H-RMN analysis were stored at −80 °C at Vall d’Hebron Biobank. Biochemical analysis was performed at the Biochemistry Core Facilities of Vall d’Hebron University Hospital using standardized and validated routine methodologies. The following parameters were determined: blood count, glucose, HbA1c, fructosamine (FA), urea, creatinine (Cr), glomerular filtration (GF), sodium (Na), potassium (K), phosphate (PH), calcium (Ca), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamiltransferase (GGT), alkaline phosphatase (ALP), total cholesterol (C), high-density lipoprotein (HDL) cholesterol (C-HDL), low-density lipoprotein cholesterol (C-LDL), triglyceride (TG), total free fatty acids (FFA), protein (P), albumin, C-peptide, troponin (TN), chloride (Cl), total bilirubin (BR) and esterified bilirubin (E-BR). Non-routine laboratory parameters such as insulin, leptin and IL4 were also determined. Insulin was assessed by chemiluminescence immunoassay (Atellica IM, Siemens Healthineers, Erlangen, Germany), leptin analysis was performed by ELISA (Diagnostics Biochem Canada Inc., London, ON, Canada), and IL6 by electrochemiluminescence immunoassay (Cobas e2801, Roche Diagnostics, Basel, Switzerland) following strictly the instructions provided by the manufacturer.\nThe fibrosis index based on four factors (FIB-4) was determined by the standard equations using age, ALT, AST and platelet counting. 1H-RMN analysis was performed following the previous methodology described by our group to determine VLDL (CH) and VLDL (aliphatic chain) [50].\nPeripheral blood samples were collected under fasting conditions. Plasma samples for 1H-RMN analysis were stored at −80 °C at Vall d’Hebron Biobank. Biochemical analysis was performed at the Biochemistry Core Facilities of Vall d’Hebron University Hospital using standardized and validated routine methodologies. The following parameters were determined: blood count, glucose, HbA1c, fructosamine (FA), urea, creatinine (Cr), glomerular filtration (GF), sodium (Na), potassium (K), phosphate (PH), calcium (Ca), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamiltransferase (GGT), alkaline phosphatase (ALP), total cholesterol (C), high-density lipoprotein (HDL) cholesterol (C-HDL), low-density lipoprotein cholesterol (C-LDL), triglyceride (TG), total free fatty acids (FFA), protein (P), albumin, C-peptide, troponin (TN), chloride (Cl), total bilirubin (BR) and esterified bilirubin (E-BR). Non-routine laboratory parameters such as insulin, leptin and IL4 were also determined. Insulin was assessed by chemiluminescence immunoassay (Atellica IM, Siemens Healthineers, Erlangen, Germany), leptin analysis was performed by ELISA (Diagnostics Biochem Canada Inc., London, ON, Canada), and IL6 by electrochemiluminescence immunoassay (Cobas e2801, Roche Diagnostics, Basel, Switzerland) following strictly the instructions provided by the manufacturer.\nThe fibrosis index based on four factors (FIB-4) was determined by the standard equations using age, ALT, AST and platelet counting. 1H-RMN analysis was performed following the previous methodology described by our group to determine VLDL (CH) and VLDL (aliphatic chain) [50].\n 4.4. Fibroscan Liver stiffness measurement (LSM) was performed using transient elastography (Fibroscan 502 Touch, Echosens, Paris, France). According to the usual standard procedure, measurements were performed under fasting conditions with at least 10 valid measurements and an interquartile to median ratio ≤ 30% [51]. Data are expressed in kilopascals (kPa). Normal LSM values varied between 4 and 6 kPa and values ≥ 6 kPa were considered abnormal and suggestive of liver mild fibrosis/disease.\nLiver stiffness measurement (LSM) was performed using transient elastography (Fibroscan 502 Touch, Echosens, Paris, France). According to the usual standard procedure, measurements were performed under fasting conditions with at least 10 valid measurements and an interquartile to median ratio ≤ 30% [51]. Data are expressed in kilopascals (kPa). Normal LSM values varied between 4 and 6 kPa and values ≥ 6 kPa were considered abnormal and suggestive of liver mild fibrosis/disease.\n 4.5. Statistical Analysis Data are expressed as median and interquartile range (IQR). Data normality was assessed with the Shapiro–Wilk test. The Kolmogorov–Smirnov test was used for group comparisons, as appropriate. Spearman’s correlation analysis and ROC curves were performed for variable associations. An ROC analysis was also performed to determine the utility of variables to dichotomize groups and obtain thresholds for fingerprints. With the extracted altered parameters for T2D phenotype segregation, new multi-variable functions were computed using the generalized linear model approach with specifications for binomial distribution and logistic-type canonical link function. This was carried out to compute alternative variable definitions for patient classification based on a combination of the mentioned parameters. ROC curves using the newly defined variables were obtained to assess the best combination according to the highest AUC value. Thresholds for each of the included parameters as well as for the combination of them were obtained by means of the same procedure as in the independent-variable assessment. All data were analyzed in GraphPad Prism (Version 6.01, San Diego, CA, USA) or R commander (Version 2.3-1, Hamilton, ON, Canada). p values below 0.05 were considered statistically significant.\nData are expressed as median and interquartile range (IQR). Data normality was assessed with the Shapiro–Wilk test. The Kolmogorov–Smirnov test was used for group comparisons, as appropriate. Spearman’s correlation analysis and ROC curves were performed for variable associations. An ROC analysis was also performed to determine the utility of variables to dichotomize groups and obtain thresholds for fingerprints. With the extracted altered parameters for T2D phenotype segregation, new multi-variable functions were computed using the generalized linear model approach with specifications for binomial distribution and logistic-type canonical link function. This was carried out to compute alternative variable definitions for patient classification based on a combination of the mentioned parameters. ROC curves using the newly defined variables were obtained to assess the best combination according to the highest AUC value. Thresholds for each of the included parameters as well as for the combination of them were obtained by means of the same procedure as in the independent-variable assessment. All data were analyzed in GraphPad Prism (Version 6.01, San Diego, CA, USA) or R commander (Version 2.3-1, Hamilton, ON, Canada). p values below 0.05 were considered statistically significant.",
"This proof-of-concept observational study comprised forty-seven T2D patients and was conducted according to the tenets of the Helsinki Declaration after approval by the Ethic Committee of the Vall d’Hebron University Hospital (ClinicalTrials.gov: NCT02248311). Written informed consent of all participants was obtained.\nThe inclusion criteria were: T2D patients from 50 to 79 years old, with at least 5 years from the diagnosis. The exclusion criteria were: (1) type 1 diabetes, (2) any previous CVD event, (3) patients with known cardiac pathology, (4) any contraindication or claustrophobia for the PET/CT, (5) any pathology related to a short life expectancy, (6) daily alcohol drinker, (7) smokers who did not stop smoking at least 1 year before (non-smoker and ex-smoker were recorded), (8) treatment with any cardiotoxic medication.\nSubjects were recruited at the Endocrinology Department of Vall d’Hebron University Hospital. Among a total of 250 clinical records of T2D patients reviewed, 72 met the inclusion criteria, but only 51 agreed to participate in the clinical trial. Four patients dropped out of the study before its completion.",
"Two [18F]FDG PET/CT studies per patient were implemented: one at baseline and another after performing a HEC. The HEC procedure was performed as previously reported with minor modifications [48,49]. The two [18F]FDG PET/CT scans were performed for each patient in a random order within two days under at least 8 h of fasting conditions and after the withdrawal of any medication the day before. A dose of 1.9 MBq/Kg of [18]FDG was IV-administered to patients before each scan session. PET/CT acquisitions protocols and data processing and analysis were described by our group previously [17]. Several anthropometric measurements and blood samples for biochemical analysis were obtained during the procedure following the flow chart shown in Figure 6.",
"Peripheral blood samples were collected under fasting conditions. Plasma samples for 1H-RMN analysis were stored at −80 °C at Vall d’Hebron Biobank. Biochemical analysis was performed at the Biochemistry Core Facilities of Vall d’Hebron University Hospital using standardized and validated routine methodologies. The following parameters were determined: blood count, glucose, HbA1c, fructosamine (FA), urea, creatinine (Cr), glomerular filtration (GF), sodium (Na), potassium (K), phosphate (PH), calcium (Ca), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamiltransferase (GGT), alkaline phosphatase (ALP), total cholesterol (C), high-density lipoprotein (HDL) cholesterol (C-HDL), low-density lipoprotein cholesterol (C-LDL), triglyceride (TG), total free fatty acids (FFA), protein (P), albumin, C-peptide, troponin (TN), chloride (Cl), total bilirubin (BR) and esterified bilirubin (E-BR). Non-routine laboratory parameters such as insulin, leptin and IL4 were also determined. Insulin was assessed by chemiluminescence immunoassay (Atellica IM, Siemens Healthineers, Erlangen, Germany), leptin analysis was performed by ELISA (Diagnostics Biochem Canada Inc., London, ON, Canada), and IL6 by electrochemiluminescence immunoassay (Cobas e2801, Roche Diagnostics, Basel, Switzerland) following strictly the instructions provided by the manufacturer.\nThe fibrosis index based on four factors (FIB-4) was determined by the standard equations using age, ALT, AST and platelet counting. 1H-RMN analysis was performed following the previous methodology described by our group to determine VLDL (CH) and VLDL (aliphatic chain) [50].",
"Liver stiffness measurement (LSM) was performed using transient elastography (Fibroscan 502 Touch, Echosens, Paris, France). According to the usual standard procedure, measurements were performed under fasting conditions with at least 10 valid measurements and an interquartile to median ratio ≤ 30% [51]. Data are expressed in kilopascals (kPa). Normal LSM values varied between 4 and 6 kPa and values ≥ 6 kPa were considered abnormal and suggestive of liver mild fibrosis/disease.",
"Data are expressed as median and interquartile range (IQR). Data normality was assessed with the Shapiro–Wilk test. The Kolmogorov–Smirnov test was used for group comparisons, as appropriate. Spearman’s correlation analysis and ROC curves were performed for variable associations. An ROC analysis was also performed to determine the utility of variables to dichotomize groups and obtain thresholds for fingerprints. With the extracted altered parameters for T2D phenotype segregation, new multi-variable functions were computed using the generalized linear model approach with specifications for binomial distribution and logistic-type canonical link function. This was carried out to compute alternative variable definitions for patient classification based on a combination of the mentioned parameters. ROC curves using the newly defined variables were obtained to assess the best combination according to the highest AUC value. Thresholds for each of the included parameters as well as for the combination of them were obtained by means of the same procedure as in the independent-variable assessment. All data were analyzed in GraphPad Prism (Version 6.01, San Diego, CA, USA) or R commander (Version 2.3-1, Hamilton, ON, Canada). p values below 0.05 were considered statistically significant."
] |
[
"intro",
"results",
"subjects",
null,
null,
"discussion",
null,
null,
null,
null,
null,
null
] |
[
"type 2 diabetes",
"myocardial insulin resistance",
"non-alcoholic fatty liver disease",
"cardiovascular risk"
] |
1. Introduction:
Type 2 diabetes (T2D) is a highly prevalent multifactorial disease caused by a combination of genetic and lifestyle factors which represents a huge societal problem associated with a significant economic burden for all healthcare systems [1,2]. T2D is characterized by high levels of plasma glucose caused mainly by pancreatic β-cell dysfunction and inefficient action of insulin, known as insulin resistance (IR), which occurs in cells of insulin-sensitive key organs and tissues such as the heart or skeletal muscle [1,3]. These high levels of plasma glucose cause mainly micro- and macrovascular complications [4,5]. Likewise, IR causes cells to not respond to the action of insulin to incorporate glucose and, thus, obtain energy by maintaining a normal metabolism [1,6]. The deficit of glucose in cells causes metabolic alterations and changes in oxidizable substrates to obtain energy, triggering severe pathological complications [7,8]. Therefore, both chronic hyperglycemia and IR have been related to the appearance of cardiovascular diseases (CVD) such as coronary artery diseases (CAD), stroke, and cardiomyopathies in T2D [6,8,9,10].
Myocardium is a striated muscle that constitutes the main tissue of the walls of the heart, and plays crucial roles for contraction, the basic function of the heart [11]. The main cells of the muscle are cardiomyocytes that are insulin-sensitive cells and, consequently, can be affected by the IR related to T2D. Many clinical trials have shown that systemic IR is an independent risk factor for heart failure and cardiovascular death [12]. In addition, increasing evidence points to IR as the primary etiologic factor in the development of non-ischemic heart failure [13,14].
A number of preclinical studies have shown that myocardial IR (mIR), which is characterized by inefficient energy metabolism, co-occurs with systemic insulin resistance and contributes to post-ischemic heart failure [15]. The presence of mIR should be put in the context of the general IR, in which the main contributors are the liver, muscles other than myocardium, and the adipose tissue. Some researchers have suggested not only that the heart is a target organ of systemic insulin resistance, but also that local mIR is a distinct risk factor for heart failure [16]. The myocardium normally responds to injury by altering substrate metabolism to increase energy efficiency. Insulin resistance prevents this adaptive response and can lead to further injury by contributing to lipotoxicity, sympathetic up-regulation, inflammation, oxidative stress, and fibrosis [13].
We have recently reported the presence of mIR in around 60% of patients with T2D patients without previous CVD [17]. These patients present higher myocardial radiodensity and a significantly higher coronary artery calcium score (CACs), a reliable biomarker of cardiovascular risk, when compared with those patients with insulin-sensitive myocardium (mIS). In addition, we observed that systemic IR is mainly accounted by patients with mIR, thus underlying the important role of the myocardium in glucose metabolism. This study was performed using 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET/CT) before and after a hyperinsulinemic–euglycemic clamp (HEC). This is an expensive, radioactive, time-consuming and cumbersome examination that cannot be proposed for daily clinical practice. Therefore, a more accessible, cheap and widely applicable surrogate to identify patients with mIR is needed.
In the present study, we examined a myriad of plasma biochemical parameters aimed at obtaining a reliable surrogate that permits us to differentiate the two phenotypes of T2D patients: mIR and mIS.
2. Results:
The general results of PET/CT before and after HEC were previously reported [17]. In summary, the first (baseline) PET/CT showed in all patients a very low left ventricular [18]FDG uptake. After HEC, we were able to observe two clear behaviors: (1) A pronounced enhancement in [18]FDG uptake in 21 cases (44.7%), thus revealing an insulin-sensitive myocardium (mIS). (2) A marginal increase in [18]FDG uptake in the remaining 26 (55.30%) patients, thus indicating the presence of an insulin-resistant myocardium (mIR).
2.1. General Characteristics of T2D Patients The main clinical and biochemical variables of T2D patients included in the study, taking into account the myocardial insulin resistance (mIS vs. mIR), are shown in Table 1. The antidiabetic treatment is displayed in Table 2.
The main clinical and biochemical variables of T2D patients included in the study, taking into account the myocardial insulin resistance (mIS vs. mIR), are shown in Table 1. The antidiabetic treatment is displayed in Table 2.
2.2. Biomarkers Discriminating between mIR and mIS Baseline and post-HEC biochemical variables were divided by mIS and mIR subgroups and analyzed. The variables significantly different between both groups at baseline are shown in Table 1. As can be seen, HOMA-IR, ALT, AST, GGT and protein were significantly higher in the mIR group than in the mIS group. However, after HEC, we found that only ALT and FFA were significantly increased in the mIR group: 29 IU/L [16:42] vs. 18 IU/L [16:21] (p = 0.0180), and 0.32 mmol/L [0.18:0.38] vs. 0.20 mmol/L p = 0.0149, respectively. We did not find any difference between males and females when comparing mIR vs. mIS and neither of the biochemical variables were useful for discriminating both phenotypes both at baseline and after HEC.
Since liver enzymes were the most closely associated with mIR, we wanted to explore whether the hepatic steatosis index (HSI), a simple screening tool reflecting non-alcoholic fatty liver disease (NAFLD), was different between mIR and mIS patients [18]. We found a higher HIS in patients with mIR in comparison with those patients with mIS but the differences did not reach statistical significance (2.35 [1.06:5.62] vs. 1.16 [0.53:1.80]; p = 0.055) (Figure 1A). In addition, we examined whether the non-alcoholic fatty liver disease–liver fat score (NAFLD-LFS) [19], an index that integrates IR and hepatic damage, could be able to discriminate patients with and without mIR. We found that patients with mIR presented higher values than patients with mIS (2.58 [1.08:8.31] vs. 1.05 [0.47:1.76]; p = 0.0154) (Figure 1B). Finally, the usefulness of an index of hepatic fibrosis (FIB-4) was assessed [20]. We found a significantly higher FIB-4 score in patients with mIR in comparison with patients with mIS (1.65 [1.07:2.73] vs. 1.18 [0.82:1.49]; p = 0.025) (Figure 1C).
Since the baseline (fasting state) is the current state for performing biochemical analysis, thresholds and AUC for optimal discrimination at baseline between both groups (mIR and mIS) were obtained for the variables significantly different between groups (Figure 2).
In addition, different models combining the variables displayed in Figure 2 were analyzed to dichotomize both phenotypes (mIR vs. mIS). The results are shown in Figure 3.
The best model was the combination of Protein, AST, ALT, GGT, FIB and HOMA (AUC = 0.95; sensibility: 0.81; and specificity: 0.95). Since HOMA is not always available in the daily clinical practice and it cannot be used in those patients receiving treatment with insulin, we wanted to examine the result obtained by leaving HOMA out of the model, and the result was AUC = 0.87; sensibility: 0.82; and specificity: 0.96.
Baseline and post-HEC biochemical variables were divided by mIS and mIR subgroups and analyzed. The variables significantly different between both groups at baseline are shown in Table 1. As can be seen, HOMA-IR, ALT, AST, GGT and protein were significantly higher in the mIR group than in the mIS group. However, after HEC, we found that only ALT and FFA were significantly increased in the mIR group: 29 IU/L [16:42] vs. 18 IU/L [16:21] (p = 0.0180), and 0.32 mmol/L [0.18:0.38] vs. 0.20 mmol/L p = 0.0149, respectively. We did not find any difference between males and females when comparing mIR vs. mIS and neither of the biochemical variables were useful for discriminating both phenotypes both at baseline and after HEC.
Since liver enzymes were the most closely associated with mIR, we wanted to explore whether the hepatic steatosis index (HSI), a simple screening tool reflecting non-alcoholic fatty liver disease (NAFLD), was different between mIR and mIS patients [18]. We found a higher HIS in patients with mIR in comparison with those patients with mIS but the differences did not reach statistical significance (2.35 [1.06:5.62] vs. 1.16 [0.53:1.80]; p = 0.055) (Figure 1A). In addition, we examined whether the non-alcoholic fatty liver disease–liver fat score (NAFLD-LFS) [19], an index that integrates IR and hepatic damage, could be able to discriminate patients with and without mIR. We found that patients with mIR presented higher values than patients with mIS (2.58 [1.08:8.31] vs. 1.05 [0.47:1.76]; p = 0.0154) (Figure 1B). Finally, the usefulness of an index of hepatic fibrosis (FIB-4) was assessed [20]. We found a significantly higher FIB-4 score in patients with mIR in comparison with patients with mIS (1.65 [1.07:2.73] vs. 1.18 [0.82:1.49]; p = 0.025) (Figure 1C).
Since the baseline (fasting state) is the current state for performing biochemical analysis, thresholds and AUC for optimal discrimination at baseline between both groups (mIR and mIS) were obtained for the variables significantly different between groups (Figure 2).
In addition, different models combining the variables displayed in Figure 2 were analyzed to dichotomize both phenotypes (mIR vs. mIS). The results are shown in Figure 3.
The best model was the combination of Protein, AST, ALT, GGT, FIB and HOMA (AUC = 0.95; sensibility: 0.81; and specificity: 0.95). Since HOMA is not always available in the daily clinical practice and it cannot be used in those patients receiving treatment with insulin, we wanted to examine the result obtained by leaving HOMA out of the model, and the result was AUC = 0.87; sensibility: 0.82; and specificity: 0.96.
2.3. Hepatic Fibrosis Measured by Fibroscan Since FIB-4 is a reliable index of hepatic fibrosis, we wanted to examine whether the degree of hepatic fibrosis measured by fibroscan was related to the presence of mIR. We observed significantly higher levels of fibrosis in patients with mIR than in those with mIS (9.10 [5.43:16.08] vs. 5.35 [3.70:6.38]; p = 0.034) Figure 4. ROC analysis of fibroscan data showed an AUC = 0.73 with a sensibility of 0.80 and specificity of 0.71.
In addition, patients with mIR presented a reduced glucose uptake by the liver in comparison with patients with mIS (Figure 5).
Since FIB-4 is a reliable index of hepatic fibrosis, we wanted to examine whether the degree of hepatic fibrosis measured by fibroscan was related to the presence of mIR. We observed significantly higher levels of fibrosis in patients with mIR than in those with mIS (9.10 [5.43:16.08] vs. 5.35 [3.70:6.38]; p = 0.034) Figure 4. ROC analysis of fibroscan data showed an AUC = 0.73 with a sensibility of 0.80 and specificity of 0.71.
In addition, patients with mIR presented a reduced glucose uptake by the liver in comparison with patients with mIS (Figure 5).
2.1. General Characteristics of T2D Patients:
The main clinical and biochemical variables of T2D patients included in the study, taking into account the myocardial insulin resistance (mIS vs. mIR), are shown in Table 1. The antidiabetic treatment is displayed in Table 2.
2.2. Biomarkers Discriminating between mIR and mIS:
Baseline and post-HEC biochemical variables were divided by mIS and mIR subgroups and analyzed. The variables significantly different between both groups at baseline are shown in Table 1. As can be seen, HOMA-IR, ALT, AST, GGT and protein were significantly higher in the mIR group than in the mIS group. However, after HEC, we found that only ALT and FFA were significantly increased in the mIR group: 29 IU/L [16:42] vs. 18 IU/L [16:21] (p = 0.0180), and 0.32 mmol/L [0.18:0.38] vs. 0.20 mmol/L p = 0.0149, respectively. We did not find any difference between males and females when comparing mIR vs. mIS and neither of the biochemical variables were useful for discriminating both phenotypes both at baseline and after HEC.
Since liver enzymes were the most closely associated with mIR, we wanted to explore whether the hepatic steatosis index (HSI), a simple screening tool reflecting non-alcoholic fatty liver disease (NAFLD), was different between mIR and mIS patients [18]. We found a higher HIS in patients with mIR in comparison with those patients with mIS but the differences did not reach statistical significance (2.35 [1.06:5.62] vs. 1.16 [0.53:1.80]; p = 0.055) (Figure 1A). In addition, we examined whether the non-alcoholic fatty liver disease–liver fat score (NAFLD-LFS) [19], an index that integrates IR and hepatic damage, could be able to discriminate patients with and without mIR. We found that patients with mIR presented higher values than patients with mIS (2.58 [1.08:8.31] vs. 1.05 [0.47:1.76]; p = 0.0154) (Figure 1B). Finally, the usefulness of an index of hepatic fibrosis (FIB-4) was assessed [20]. We found a significantly higher FIB-4 score in patients with mIR in comparison with patients with mIS (1.65 [1.07:2.73] vs. 1.18 [0.82:1.49]; p = 0.025) (Figure 1C).
Since the baseline (fasting state) is the current state for performing biochemical analysis, thresholds and AUC for optimal discrimination at baseline between both groups (mIR and mIS) were obtained for the variables significantly different between groups (Figure 2).
In addition, different models combining the variables displayed in Figure 2 were analyzed to dichotomize both phenotypes (mIR vs. mIS). The results are shown in Figure 3.
The best model was the combination of Protein, AST, ALT, GGT, FIB and HOMA (AUC = 0.95; sensibility: 0.81; and specificity: 0.95). Since HOMA is not always available in the daily clinical practice and it cannot be used in those patients receiving treatment with insulin, we wanted to examine the result obtained by leaving HOMA out of the model, and the result was AUC = 0.87; sensibility: 0.82; and specificity: 0.96.
2.3. Hepatic Fibrosis Measured by Fibroscan:
Since FIB-4 is a reliable index of hepatic fibrosis, we wanted to examine whether the degree of hepatic fibrosis measured by fibroscan was related to the presence of mIR. We observed significantly higher levels of fibrosis in patients with mIR than in those with mIS (9.10 [5.43:16.08] vs. 5.35 [3.70:6.38]; p = 0.034) Figure 4. ROC analysis of fibroscan data showed an AUC = 0.73 with a sensibility of 0.80 and specificity of 0.71.
In addition, patients with mIR presented a reduced glucose uptake by the liver in comparison with patients with mIS (Figure 5).
3. Discussion:
Myocardial insulin resistance (mIR) has been hypothesized as a risk factor of CVD and cardiomyopathies in T2D patients [17,21]. Approaches based on dynamic PET/CT under HEC conditions have linked the myocardial [18F]FDG utilization rate to mIR [18,19]. Our group clearly identified T2D patients with mIR for the first time in a proof of concept using two [18F]FDG PET/CTs per patient (baseline and post-HEC) [17]. Since this methodology can hardly be proposed for current clinical practice, cost-effective and reliable surrogates are needed. In the present study, we provide evidence that transaminases and FIB-4 (an index of hepatic fibrosis) are the best predictors of mIR in patients with T2D without previous history of cardiovascular events.
We found that the best model to predict the presence of mIR was the combination of protein, AST, ALT, GGT, FIB-4 and HOMA-IR at baseline (AUC = 0.95; sensibility: 0.81; specificity: 0.95). Since HOMA-IR is not always available in the daily clinical practice and it cannot be used in those patients receiving treatment with insulin, we examined the result obtained by leaving HOMA-IR out of the model, and the result was AUC = 0.87 with a sensitivity of 0.82 and a specificity of 0.96. This means that general practitioners could have a useful index to estimate the presence of mIR in patients with T2D.
Regarding the variables altered after the HEC procedure, the higher serum levels of FFA and ALT were found in the IRm subgroup. The increase in FFA in those patients with mIR could be expected because it is well-established that FFAs decrease myocardial [18F]FDG uptake [22]. It is well-established that the primary fuel for the myocardium is represented by FFAs in the fasting state and glucose in the fed state [23,24]. Myocardial glucose uptake is inversely associated with serum FFAs in both healthy and insulin-resistant conditions, and such a relationship explains the bulk of the individual variation in myocardial metabolism [24,25]. However, the fact that levels of FFA were significantly different between mIR and mIS patients only after HEC but not at the baseline/fasting state is intriguing and merits further investigation. The higher plasma levels of ALT after HEC in diabetic patients with mIR in comparison with patients with mIS have not previously reported and speak in favor of the robustness of ALT in dichotomizing both groups (mIR and mIS). Nevertheless, for the practical point of view, the fasting/baseline results are the only useful ones for clinical purposes.
Overall, our results confirm previous findings suggesting the existence of a liver–heart axis [26,27,28,29]. To the best of our knowledge, this is the first report linking hepatic liver disease and mIR for T2D without previous CVD. In addition, we demonstrated that patients with mIR presented a significantly lower glucose uptake by the liver than patients with mIS. This result reveals the existence of an association between the myocardium and liver IR, which is essential to understand our findings. The liver is a central hub for lipid metabolism and endogenous glucose production; therefore, the liver is crucial for systemic glucose and lipid homeostasis. Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease, affecting approximately one-quarter of adults worldwide. NAFLD encompasses a pathological condition characterized by the ectopic deposition of adipose tissue in the liver, in the absence of secondary causes for hepatic fat accumulation, such as excessive alcohol intake, steatogenic drugs, and hereditary disorders [30]. Beyond liver-related morbidity and mortality, there is increasing evidence that patients with NAFLD are also at high risk of cardiovascular diseases (CVDs) [31]. Notably, 25% to 40% of patients with NAFLD also have CVD, and CVD accounts for a higher proportion of mortality than liver-related death in patients with NAFLD [32]. Recently, experts reached a consensus that NAFLD does not reflect the current knowledge, and metabolic-dysfunction-associated fatty liver disease (MAFLD) was suggested as a more appropriate term [33]. Although NAFLD or MAFLD is among the major forces driving CVD, the mechanistic relationship with CVD remains to be elucidated. Possible mechanisms include, but are not limited to, insulin resistance, low-grade systemic inflammation, oxidative stress and neuroendocrine activation [29]. Besides these common mechanisms, changes in protein secretion from the fatty liver also contribute to the pathogenesis of CVDs. In fact, the liver has recently been recognized as an endocrine organ that secretes hepatokines, which are proteins secreted by hepatocytes that can influence metabolic processes through autocrine, paracrine, and endocrine [34,35,36]. In the setting of MAFLD, several hepatokines can be downregulated such as sex hormone-binding globulin (SHBG), angiopoietin-like protein 4 (ANGPTL4) and adropin, whereas others such as fetuin-A, fetuin-B, hepassocin, leukocyte cell-derived chemotaxin 2 (LECT2), retinol-binding protein 4 (RBP4), selenoprotein P (SeP) and fibroblast growth factor 21 are upregulated [29,35]. The specific contribution of hepatokine imbalance on the uptake of glucose by the myocardium is beyond the scope of this paper but will be a piece of cutting-edge research in the coming years.
Lautamäki et al. demonstrated for the first time that liver fat content is an independent, significant mediator of myocardial glucose uptake in T2D patients with CAD [37]. In addition, they suggested a direct relationship between NAFLD and coronary atherosclerosis by showing impaired coronary flow reserve and an increment in markers of low-grade inflammation in patients with NAFLD. Our findings reinforce and extend this concept to patients with T2D without CVD. In addition, our findings suggest that, rather than steatosis, it is the presence of liver fibrosis which is the most important factor contributing to mIR. In fact, we found significantly higher levels of fibrosis in patients with mIR than in those with mIS. This result suggests that pro-fibrotic molecules might be essential players in linking MAFLD and mIR. Liver fibrosis has been suggested to be an independent predictor of the incidence of CVD and CV events in patients with NAFLD [38,39,40] and has a crucial role in the development of CVD through coronary microvascular dysfunction [41]. Recently, Liu et al. showed that liver fibrosis score metrics were significantly associated with the occurrence of recurrent cardiovascular events in patients with coronary artery disease and prior cardiovascular events [42]. It should be noted that hepatic lipid accumulation may not be toxic, and can also function as a protective mechanism to buffer the lipotoxic effects of FFA to hepatocytes. The progression of pure hepatic steatosis to liver fibrosis remains silent and underdiagnosed until its development into cirrhosis, characterized by severe liver fibrosis. In our cohort, we found a significantly higher proportion of fibrosis (≥6 Kpa) in patients with mIR than mIS (70% vs. 18.7%). Hepatic steatosis and incipient liver fibrosis may be reversible at early stages, and, in this regard, the accurate quantification of these hepatic phenotypes becomes central to the design of preventive strategies involving lifestyle modifications and therapeutic interventions [43,44]. It should be underlined that the level of transaminases in those patients with mIR was in the normal range. Furthermore, a high percentage of patients with T2D with liver fibrosis also presented transaminases in the normal range, thus making difficult the clinical suspicion. These findings are in agreement with previous reports [45,46,47] and suggest being proactive in identifying T2D at risk of having mIR by using the mentioned algorithms.
Finally, the observed relationship between glucose uptake in the myocardium and the liver point mIR is a key factor in accounting for the increased CV risk in patients with MAFLD. The pathophysiological interactions between the liver and heart can be classified into three groups: (1) liver disease resulting from heart disease; (2) heart disease resulting from liver disease; (3) systemic conditions affecting the heart and the liver at the same time. Our findings suggest that T2D could be one of these systemic diseases altering, simultaneously, glucose uptake in both the liver and the heart. Nevertheless, as mentioned above, a primary pathogenic role of hepatokine mediator/s should be underlined.
There are some limitations in this study. First, we did not include a healthy control group, and the number of patients with T2D who were enrolled was low. The main reason is because PET/CT combined with HEC is cumbersome and time-consuming. In addition, using two PET/CTs on the same patient is limited due to ethical issues. Second, we did not confirm the presence and degree of hepatic fibrosis with additional histological assessments. Third, the withdrawal of the antidiabetic drugs one day before PET/CT could not be sufficient to assure a correct wash out. However, the insulin-mediated glucose uptake by using HEC overrides any eventual marginal action of insulin sensitizers or GLP-1 in terms of increasing the myocardial glucose uptake. In this regard, it should be noted that we observed a very low left-ventricular [18]FDG uptake in all patients in the first (baseline) PET/CT, and we did not observe any influence on [18]FDG uptake from antidiabetic drugs at both baseline and post [18F]FDG PET/CT. Fourth, although BMI was similar in mIR and mIR patients, a specific study on body composition was not performed and, therefore, a potential link between adipose tissue and mIR cannot be ruled out. Finally, studies on the long-term clinical consequences of mIR are needed.
In summary, our results add valuable information to understanding the “crosstalk” between the liver and the cardiovascular system. We provide evidence that T2D individuals with mIR can be identified by using a simple algorithm based on the plasma levels of proteins and transaminases. This information will be useful to improve the stratification of cardiovascular risk in patients with T2D, and will aid the development of new therapeutic strategies aimed at abrogating the underlying vicious circuits between the liver and the heart.
4. Material and Methods:
4.1. Subjects This proof-of-concept observational study comprised forty-seven T2D patients and was conducted according to the tenets of the Helsinki Declaration after approval by the Ethic Committee of the Vall d’Hebron University Hospital (ClinicalTrials.gov: NCT02248311). Written informed consent of all participants was obtained.
The inclusion criteria were: T2D patients from 50 to 79 years old, with at least 5 years from the diagnosis. The exclusion criteria were: (1) type 1 diabetes, (2) any previous CVD event, (3) patients with known cardiac pathology, (4) any contraindication or claustrophobia for the PET/CT, (5) any pathology related to a short life expectancy, (6) daily alcohol drinker, (7) smokers who did not stop smoking at least 1 year before (non-smoker and ex-smoker were recorded), (8) treatment with any cardiotoxic medication.
Subjects were recruited at the Endocrinology Department of Vall d’Hebron University Hospital. Among a total of 250 clinical records of T2D patients reviewed, 72 met the inclusion criteria, but only 51 agreed to participate in the clinical trial. Four patients dropped out of the study before its completion.
This proof-of-concept observational study comprised forty-seven T2D patients and was conducted according to the tenets of the Helsinki Declaration after approval by the Ethic Committee of the Vall d’Hebron University Hospital (ClinicalTrials.gov: NCT02248311). Written informed consent of all participants was obtained.
The inclusion criteria were: T2D patients from 50 to 79 years old, with at least 5 years from the diagnosis. The exclusion criteria were: (1) type 1 diabetes, (2) any previous CVD event, (3) patients with known cardiac pathology, (4) any contraindication or claustrophobia for the PET/CT, (5) any pathology related to a short life expectancy, (6) daily alcohol drinker, (7) smokers who did not stop smoking at least 1 year before (non-smoker and ex-smoker were recorded), (8) treatment with any cardiotoxic medication.
Subjects were recruited at the Endocrinology Department of Vall d’Hebron University Hospital. Among a total of 250 clinical records of T2D patients reviewed, 72 met the inclusion criteria, but only 51 agreed to participate in the clinical trial. Four patients dropped out of the study before its completion.
4.2. Study Design Two [18F]FDG PET/CT studies per patient were implemented: one at baseline and another after performing a HEC. The HEC procedure was performed as previously reported with minor modifications [48,49]. The two [18F]FDG PET/CT scans were performed for each patient in a random order within two days under at least 8 h of fasting conditions and after the withdrawal of any medication the day before. A dose of 1.9 MBq/Kg of [18]FDG was IV-administered to patients before each scan session. PET/CT acquisitions protocols and data processing and analysis were described by our group previously [17]. Several anthropometric measurements and blood samples for biochemical analysis were obtained during the procedure following the flow chart shown in Figure 6.
Two [18F]FDG PET/CT studies per patient were implemented: one at baseline and another after performing a HEC. The HEC procedure was performed as previously reported with minor modifications [48,49]. The two [18F]FDG PET/CT scans were performed for each patient in a random order within two days under at least 8 h of fasting conditions and after the withdrawal of any medication the day before. A dose of 1.9 MBq/Kg of [18]FDG was IV-administered to patients before each scan session. PET/CT acquisitions protocols and data processing and analysis were described by our group previously [17]. Several anthropometric measurements and blood samples for biochemical analysis were obtained during the procedure following the flow chart shown in Figure 6.
4.3. Biochemical Analysis Peripheral blood samples were collected under fasting conditions. Plasma samples for 1H-RMN analysis were stored at −80 °C at Vall d’Hebron Biobank. Biochemical analysis was performed at the Biochemistry Core Facilities of Vall d’Hebron University Hospital using standardized and validated routine methodologies. The following parameters were determined: blood count, glucose, HbA1c, fructosamine (FA), urea, creatinine (Cr), glomerular filtration (GF), sodium (Na), potassium (K), phosphate (PH), calcium (Ca), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamiltransferase (GGT), alkaline phosphatase (ALP), total cholesterol (C), high-density lipoprotein (HDL) cholesterol (C-HDL), low-density lipoprotein cholesterol (C-LDL), triglyceride (TG), total free fatty acids (FFA), protein (P), albumin, C-peptide, troponin (TN), chloride (Cl), total bilirubin (BR) and esterified bilirubin (E-BR). Non-routine laboratory parameters such as insulin, leptin and IL4 were also determined. Insulin was assessed by chemiluminescence immunoassay (Atellica IM, Siemens Healthineers, Erlangen, Germany), leptin analysis was performed by ELISA (Diagnostics Biochem Canada Inc., London, ON, Canada), and IL6 by electrochemiluminescence immunoassay (Cobas e2801, Roche Diagnostics, Basel, Switzerland) following strictly the instructions provided by the manufacturer.
The fibrosis index based on four factors (FIB-4) was determined by the standard equations using age, ALT, AST and platelet counting. 1H-RMN analysis was performed following the previous methodology described by our group to determine VLDL (CH) and VLDL (aliphatic chain) [50].
Peripheral blood samples were collected under fasting conditions. Plasma samples for 1H-RMN analysis were stored at −80 °C at Vall d’Hebron Biobank. Biochemical analysis was performed at the Biochemistry Core Facilities of Vall d’Hebron University Hospital using standardized and validated routine methodologies. The following parameters were determined: blood count, glucose, HbA1c, fructosamine (FA), urea, creatinine (Cr), glomerular filtration (GF), sodium (Na), potassium (K), phosphate (PH), calcium (Ca), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamiltransferase (GGT), alkaline phosphatase (ALP), total cholesterol (C), high-density lipoprotein (HDL) cholesterol (C-HDL), low-density lipoprotein cholesterol (C-LDL), triglyceride (TG), total free fatty acids (FFA), protein (P), albumin, C-peptide, troponin (TN), chloride (Cl), total bilirubin (BR) and esterified bilirubin (E-BR). Non-routine laboratory parameters such as insulin, leptin and IL4 were also determined. Insulin was assessed by chemiluminescence immunoassay (Atellica IM, Siemens Healthineers, Erlangen, Germany), leptin analysis was performed by ELISA (Diagnostics Biochem Canada Inc., London, ON, Canada), and IL6 by electrochemiluminescence immunoassay (Cobas e2801, Roche Diagnostics, Basel, Switzerland) following strictly the instructions provided by the manufacturer.
The fibrosis index based on four factors (FIB-4) was determined by the standard equations using age, ALT, AST and platelet counting. 1H-RMN analysis was performed following the previous methodology described by our group to determine VLDL (CH) and VLDL (aliphatic chain) [50].
4.4. Fibroscan Liver stiffness measurement (LSM) was performed using transient elastography (Fibroscan 502 Touch, Echosens, Paris, France). According to the usual standard procedure, measurements were performed under fasting conditions with at least 10 valid measurements and an interquartile to median ratio ≤ 30% [51]. Data are expressed in kilopascals (kPa). Normal LSM values varied between 4 and 6 kPa and values ≥ 6 kPa were considered abnormal and suggestive of liver mild fibrosis/disease.
Liver stiffness measurement (LSM) was performed using transient elastography (Fibroscan 502 Touch, Echosens, Paris, France). According to the usual standard procedure, measurements were performed under fasting conditions with at least 10 valid measurements and an interquartile to median ratio ≤ 30% [51]. Data are expressed in kilopascals (kPa). Normal LSM values varied between 4 and 6 kPa and values ≥ 6 kPa were considered abnormal and suggestive of liver mild fibrosis/disease.
4.5. Statistical Analysis Data are expressed as median and interquartile range (IQR). Data normality was assessed with the Shapiro–Wilk test. The Kolmogorov–Smirnov test was used for group comparisons, as appropriate. Spearman’s correlation analysis and ROC curves were performed for variable associations. An ROC analysis was also performed to determine the utility of variables to dichotomize groups and obtain thresholds for fingerprints. With the extracted altered parameters for T2D phenotype segregation, new multi-variable functions were computed using the generalized linear model approach with specifications for binomial distribution and logistic-type canonical link function. This was carried out to compute alternative variable definitions for patient classification based on a combination of the mentioned parameters. ROC curves using the newly defined variables were obtained to assess the best combination according to the highest AUC value. Thresholds for each of the included parameters as well as for the combination of them were obtained by means of the same procedure as in the independent-variable assessment. All data were analyzed in GraphPad Prism (Version 6.01, San Diego, CA, USA) or R commander (Version 2.3-1, Hamilton, ON, Canada). p values below 0.05 were considered statistically significant.
Data are expressed as median and interquartile range (IQR). Data normality was assessed with the Shapiro–Wilk test. The Kolmogorov–Smirnov test was used for group comparisons, as appropriate. Spearman’s correlation analysis and ROC curves were performed for variable associations. An ROC analysis was also performed to determine the utility of variables to dichotomize groups and obtain thresholds for fingerprints. With the extracted altered parameters for T2D phenotype segregation, new multi-variable functions were computed using the generalized linear model approach with specifications for binomial distribution and logistic-type canonical link function. This was carried out to compute alternative variable definitions for patient classification based on a combination of the mentioned parameters. ROC curves using the newly defined variables were obtained to assess the best combination according to the highest AUC value. Thresholds for each of the included parameters as well as for the combination of them were obtained by means of the same procedure as in the independent-variable assessment. All data were analyzed in GraphPad Prism (Version 6.01, San Diego, CA, USA) or R commander (Version 2.3-1, Hamilton, ON, Canada). p values below 0.05 were considered statistically significant.
4.1. Subjects:
This proof-of-concept observational study comprised forty-seven T2D patients and was conducted according to the tenets of the Helsinki Declaration after approval by the Ethic Committee of the Vall d’Hebron University Hospital (ClinicalTrials.gov: NCT02248311). Written informed consent of all participants was obtained.
The inclusion criteria were: T2D patients from 50 to 79 years old, with at least 5 years from the diagnosis. The exclusion criteria were: (1) type 1 diabetes, (2) any previous CVD event, (3) patients with known cardiac pathology, (4) any contraindication or claustrophobia for the PET/CT, (5) any pathology related to a short life expectancy, (6) daily alcohol drinker, (7) smokers who did not stop smoking at least 1 year before (non-smoker and ex-smoker were recorded), (8) treatment with any cardiotoxic medication.
Subjects were recruited at the Endocrinology Department of Vall d’Hebron University Hospital. Among a total of 250 clinical records of T2D patients reviewed, 72 met the inclusion criteria, but only 51 agreed to participate in the clinical trial. Four patients dropped out of the study before its completion.
4.2. Study Design:
Two [18F]FDG PET/CT studies per patient were implemented: one at baseline and another after performing a HEC. The HEC procedure was performed as previously reported with minor modifications [48,49]. The two [18F]FDG PET/CT scans were performed for each patient in a random order within two days under at least 8 h of fasting conditions and after the withdrawal of any medication the day before. A dose of 1.9 MBq/Kg of [18]FDG was IV-administered to patients before each scan session. PET/CT acquisitions protocols and data processing and analysis were described by our group previously [17]. Several anthropometric measurements and blood samples for biochemical analysis were obtained during the procedure following the flow chart shown in Figure 6.
4.3. Biochemical Analysis:
Peripheral blood samples were collected under fasting conditions. Plasma samples for 1H-RMN analysis were stored at −80 °C at Vall d’Hebron Biobank. Biochemical analysis was performed at the Biochemistry Core Facilities of Vall d’Hebron University Hospital using standardized and validated routine methodologies. The following parameters were determined: blood count, glucose, HbA1c, fructosamine (FA), urea, creatinine (Cr), glomerular filtration (GF), sodium (Na), potassium (K), phosphate (PH), calcium (Ca), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamiltransferase (GGT), alkaline phosphatase (ALP), total cholesterol (C), high-density lipoprotein (HDL) cholesterol (C-HDL), low-density lipoprotein cholesterol (C-LDL), triglyceride (TG), total free fatty acids (FFA), protein (P), albumin, C-peptide, troponin (TN), chloride (Cl), total bilirubin (BR) and esterified bilirubin (E-BR). Non-routine laboratory parameters such as insulin, leptin and IL4 were also determined. Insulin was assessed by chemiluminescence immunoassay (Atellica IM, Siemens Healthineers, Erlangen, Germany), leptin analysis was performed by ELISA (Diagnostics Biochem Canada Inc., London, ON, Canada), and IL6 by electrochemiluminescence immunoassay (Cobas e2801, Roche Diagnostics, Basel, Switzerland) following strictly the instructions provided by the manufacturer.
The fibrosis index based on four factors (FIB-4) was determined by the standard equations using age, ALT, AST and platelet counting. 1H-RMN analysis was performed following the previous methodology described by our group to determine VLDL (CH) and VLDL (aliphatic chain) [50].
4.4. Fibroscan:
Liver stiffness measurement (LSM) was performed using transient elastography (Fibroscan 502 Touch, Echosens, Paris, France). According to the usual standard procedure, measurements were performed under fasting conditions with at least 10 valid measurements and an interquartile to median ratio ≤ 30% [51]. Data are expressed in kilopascals (kPa). Normal LSM values varied between 4 and 6 kPa and values ≥ 6 kPa were considered abnormal and suggestive of liver mild fibrosis/disease.
4.5. Statistical Analysis:
Data are expressed as median and interquartile range (IQR). Data normality was assessed with the Shapiro–Wilk test. The Kolmogorov–Smirnov test was used for group comparisons, as appropriate. Spearman’s correlation analysis and ROC curves were performed for variable associations. An ROC analysis was also performed to determine the utility of variables to dichotomize groups and obtain thresholds for fingerprints. With the extracted altered parameters for T2D phenotype segregation, new multi-variable functions were computed using the generalized linear model approach with specifications for binomial distribution and logistic-type canonical link function. This was carried out to compute alternative variable definitions for patient classification based on a combination of the mentioned parameters. ROC curves using the newly defined variables were obtained to assess the best combination according to the highest AUC value. Thresholds for each of the included parameters as well as for the combination of them were obtained by means of the same procedure as in the independent-variable assessment. All data were analyzed in GraphPad Prism (Version 6.01, San Diego, CA, USA) or R commander (Version 2.3-1, Hamilton, ON, Canada). p values below 0.05 were considered statistically significant.
|
Background:
We report that myocardial insulin resistance (mIR) occurs in around 60% of patients with type 2 diabetes (T2D) and was associated with higher cardiovascular risk in comparison with patients with insulin-sensitive myocardium (mIS). These two phenotypes (mIR vs. mIS) can only be assessed using time-consuming and expensive methods. The aim of the present study is to search a simple and reliable surrogate to identify both phenotypes.
Methods:
Forty-seven patients with T2D underwent myocardial [18F]FDG PET/CT at baseline and after a hyperinsulinemic-euglycemic clamp (HEC) to determine mIR were prospectively recruited. Biochemical assessments were performed before and after the HEC. Baseline hepatic steatosis index and index of hepatic fibrosis (FIB-4) were calculated. Furthermore, liver stiffness measurement was performed using transient elastography.
Results:
The best model to predict the presence of mIR was the combination of transaminases, protein levels, FIB-4 score and HOMA (AUC = 0.95; sensibility: 0.81; specificity: 0.95). We observed significantly higher levels of fibrosis in patients with mIR than in those with mIS (p = 0.034). In addition, we found that patients with mIR presented a reduced glucose uptake by the liver in comparison with patients with mIS.
Conclusions:
The combination of HOMA, protein, transaminases and FIB-4 is a simple and reliable tool for identifying mIR in patients with T2D. This information will be useful to improve the stratification of cardiovascular risk in T2D.
| null | null | 8,040 | 284 | 12 |
[
"patients",
"mir",
"liver",
"mis",
"t2d",
"analysis",
"fibrosis",
"insulin",
"performed",
"patients mir"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] type 2 diabetes | myocardial insulin resistance | non-alcoholic fatty liver disease | cardiovascular risk [SUMMARY]
| null |
[CONTENT] type 2 diabetes | myocardial insulin resistance | non-alcoholic fatty liver disease | cardiovascular risk [SUMMARY]
| null |
[CONTENT] type 2 diabetes | myocardial insulin resistance | non-alcoholic fatty liver disease | cardiovascular risk [SUMMARY]
| null |
[CONTENT] Diabetes Mellitus, Type 2 | Fibrosis | Humans | Insulin Resistance | Liver | Myocardium | Non-alcoholic Fatty Liver Disease | Positron Emission Tomography Computed Tomography | Transaminases [SUMMARY]
| null |
[CONTENT] Diabetes Mellitus, Type 2 | Fibrosis | Humans | Insulin Resistance | Liver | Myocardium | Non-alcoholic Fatty Liver Disease | Positron Emission Tomography Computed Tomography | Transaminases [SUMMARY]
| null |
[CONTENT] Diabetes Mellitus, Type 2 | Fibrosis | Humans | Insulin Resistance | Liver | Myocardium | Non-alcoholic Fatty Liver Disease | Positron Emission Tomography Computed Tomography | Transaminases [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] patients | mir | liver | mis | t2d | analysis | fibrosis | insulin | performed | patients mir [SUMMARY]
| null |
[CONTENT] patients | mir | liver | mis | t2d | analysis | fibrosis | insulin | performed | patients mir [SUMMARY]
| null |
[CONTENT] patients | mir | liver | mis | t2d | analysis | fibrosis | insulin | performed | patients mir [SUMMARY]
| null |
[CONTENT] heart | ir | cells | insulin | failure | energy | heart failure | mir | myocardium | metabolism [SUMMARY]
| null |
[CONTENT] mir | mis | patients | vs | figure | hepatic | significantly | patients mir | baseline | 18 [SUMMARY]
| null |
[CONTENT] mir | patients | mis | liver | vs | t2d | performed | analysis | figure | patients mir [SUMMARY]
| null |
[CONTENT] around 60% | 2 ||| two ||| [SUMMARY]
| null |
[CONTENT] FIB-4 | 0.95 | 0.81 | 0.95 ||| mIS (p | 0.034 ||| [SUMMARY]
| null |
[CONTENT] around 60% | 2 ||| two ||| ||| Forty-seven ||| HEC ||| HEC ||| FIB-4 ||| ||| ||| FIB-4 | 0.95 | 0.81 | 0.95 ||| mIS (p | 0.034 ||| ||| HOMA | FIB-4 | T2D. This | T2D. [SUMMARY]
| null |
|||
Clinical, ultrasound and histopathological correlation of clinically N0 neck nodes in patients with cancers of the pharynx and larynx.
|
33544794
|
The presence of metastatic cervical adenopathy is essential for treatment planning and prognosis assessment. Treatment of patients with head and neck cancer with clinically negative cervical lymphadenopathy (N0) remains controversial. Neck palpation, as the method used in tumor, node, metastasis (TNM) staging, has limitations and can provide false negative results in some cases. Lymph node metastases are associated with a reduced survival rate but at the same time, neck dissection for the patient with N0 neck is not without risks or complications.
|
BACKGROUND
|
Forty-six patients with cancers of the pharynx and larynx that presented with a N0 neck were prospectively analyzed. They were divided in two groups: 23 patients operated with an external approach including the control of the lymph node areas, and a second group of 23 patients operated using endoscopy and carbon dioxide (CO2) laser, no neck dissection - "watchful waiting policy". All patients have had a flexible endoscopy of the pharynx and larynx, US of the neck and all received surgical treatment for their primary tumor. Imaging was performed in selected cases. All the removed lymph nodes were sent for histopathology. US was also used as a follow-up method. The US features of the examined lymph nodes were: diameters [longitudinal (L) and transverse (T)]; the ratio of the two diameters (L∕T); shape; lymph node area; central hypodensity; regular∕irregular margins; aspect (homogeneous or not).
|
PATIENTS, MATERIALS AND METHODS
|
US has detected 25 lymph nodes in the open surgery group and intraoperatively, we excised 31 (sensitivity of 80.6%). Ten lymph nodes showed metastases, with 100% accuracy of US, which have been confirmed both pathologically and immunohistochemically. US in the second group - patients treated with CO2 laser - detected at four patients 10 cervical lymph nodes that did not presented any malignant features. At recurrence alone, the US confirmed 100% presence of nodes metastases.
|
RESULTS
|
US was superior to palpation and this method can be recommended as a diagnostic tool in preoperative assessment of patients without palpable metastasis (N0).
|
CONCLUSIONS
|
[
"Female",
"Head and Neck Neoplasms",
"Humans",
"Larynx",
"Lymph Nodes",
"Male",
"Neoplasm Metastasis",
"Pharynx",
"Prospective Studies",
"Ultrasonography"
] |
7864314
|
⧉ Introduction
|
Malignant tumors are considered the first and most important problem of public healthcare, as their incidence has epidemic characteristics, they cause an immense burden on healthcare systems, they require large amounts of economic resources, they cause major social problems and seriously affect the individual and the family [1,2]. The present epidemiological data show a tendency of increase in the incidence, prevalence, and mortality for the next 40 years [1, 3].
Head and neck cancers represent the sixth most frequent form of cancer world widely, with approximately 630 000 new patients diagnosed every year and over 350 000 deaths every year [4,5]. Cancers located in the pharynx and larynx are the most frequent head and neck malignancies [6,7,8]. Smoking and alcohol intake represent major etiological factors for the pharynx and larynx cancer [9].
The incidence of laryngeal cancer varies a lot from one country to another, from 2.5 to 17.2 up to 100 000 per year and it represents approximately 3% of the new cases of malignant tumors diagnosed every year all over the world [10].
The hypopharynx cancer has a 3–5% morbidity of head and neck cancers, with a poor prognosis, as the tumor has an infiltrative progression alongside the pharynx mucosa, it has a low number of symptoms and gives early metastases, therefore when it is diagnosed, the cancer already presents metastases in the cervix ganglia [11,12].
The therapy of pharyngeal and laryngeal cancer is decided according to the results of the clinical examination, endoscopy, and imaging. According to the location of the primary tumor and the tumor, node, metastasis (TNM) staging, either surgical or non-surgical treatment are considered. The decision is influenced by the presence or absence of the cervical lymph node metastasis. For laryngeal cancers, if the decision is to perform open surgery, the control of the lymph node areas is usually associated. If the decision is to perform endoscopic surgery – usually with lasers – than a “watchful waiting policy” is employed if no lymph nodes are palpable (N0) neck. Palpation of the neck is not a very reliable method and it depends on many factors – anatomy of the neck (short, thick neck), size and location of the lymph nodes, etc. Ultrasonography (US) is a well-established method for diagnosis of the lymph nodes in the head and neck area and can provide details regarding the characteristics of the lymph nodes (benign or malignant) [13,14,15]. This noninvasive method can be used for postoperative screening of the involved lymph node areas for both surgical and non-surgical patients, as it is cheap and well tolerated.
Aim
In the present study, we proposed to evaluate the role of US within the diagnosis of cervix ganglion metastases in the pharynx and larynx cancer, in the patients where the clinical examination did not show the presence of adenopathy (N0).
| null | null |
⧉ Results
|
In our study, most patients were male, respectively 41, representing 89.1%. From the studied group, the women were in a small number, respectively five, representing 10.9%. The main symptoms were: dysphonia, which was present in 42 (91.3%) patients; dysphagia was found in eight (17.4%) patients; pain on swallowing was present in three (6.5%) patients, and significant weight loss was mentioned only in two (4.3%) patients. Palpation of the neck was carefully performed in all patients and only the N0 patients were included in the study. In our study, 45 (97.8%) patients had laryngeal tumors. One case presented a tumor located in the oropharynx, in the left tonsil, extending to the base of the tongue. The laryngeal tumors were located as follows: epiglottis – five (11.1%); extension beyond the epiglottis – two (4.4%); vocal cord – 14 (31.1%); vocal cord extended to anterior commissure – nine (20.1%); vocal cord extended to subglottic region – four (8.9%); vocal cord extended to the supraglottic region – 11 (24.4%). Staging was performed according to TNM Classification [American Joint Committee on Cancer (AJCC) and Union for International Cancer Control (UICC)] and is presented in Table 1. It can be seen that there are malignant laryngeal tumors that do not show lymphadenopathy even in advanced stages of the disease, respectively III and IV stages.
N0 neck patients according to T staging
Tumor (T) staging
No. of cases (%)
T I
18 (39.13%)
T II
8 (17.39%)
T III
6 (13.04%)
T IV
14 (30.43%)
Total
46 (100%)
Tumor (T) stage was the main factor in deciding which therapy was used. The open surgery was performed on a number of 23 (50%) patients, especially those in advanced (III and IV) stages of the disease (Table 2), while endoscopic surgery mainly in early (I and II) stages in 23 (50%) patients (Table 3).
N0 neck patients treated with open surgery according to T staging
Tumor (T) staging
No. of cases (%)
T I
1 (2.17%)
T II
2 (4.34%)
T III
6 (13.04%)
T IV
14 (30.45%)
Total
23 (50%)
N0 neck patients treated with endoscopic CO2 laser surgery according to T staging
Tumor (T) staging
No. of cases (%)
T I
17 (36.96%)
T II
6 (13.04%)
T III
0
T IV
0
Total
23 (50%)
US was performed in all patients both preoperatively – to help planning, and postoperatively – for follow-up. US has detected 25 lymph nodes in the open surgery group and intraoperatively we excised 31 (sensitivity of 80.6%).
The US features of the examined lymph nodes were: diameters [longitudinal (L) and transverse (T)]; the ratio of the two diameters (L/T); shape; lymph node area; central hypodensity; regular/irregular margins; aspect (homogeneous or not) (Table 4). US features for metastatic lymph nodes were as follows: size larger than 1.2 cm; L/T ratio less than 2; round shape; clustering; central hypodensity; irregular margins; non-homogeneous aspect. Not all of these features were present in every patient we examined. The only feature with 100% appearance was the round shape, followed by central hypodensity (75%) and L/T ratio (66%). Irregular shape and clustering were inconclusive (25%). While size and L/T ratio produced an over evaluation of the number of cases all other features produced an under evaluation. Clustering, L/T ratio and shape of the lymph nodes were features with 100% accuracy, while central hypodensity (Figure 1) had an accuracy of only 75% (Table 5). Irregular margins had no significance on diagnosis. This can be explained by the small size of the lymph nodes.
US in the second group (patients treated with CO2 laser) detected in four patients, 10 cervical lymph nodes that did not present any malignant features (Figures 2 and 3), and the attitude against them was a closed follow-up “watchful waiting policy” which revealed no change of the characteristics over time.
US features of the neck lymph nodes
US features
No. of lymph nodes (%)
Minimal and maximal diameter [cm]
▪ 0.8–1.2
25 (100%)
▪ >0.8–1.2
0
L/T ratio
▪ >1.8
19 (76%)
▪ <1.8
6 (24%)
Shape
▪ round
4 (16%)
▪ oval
21 (84%)
Lymph node clustering
▪ one lymph node
6 (24%)
▪ two lymph nodes
18 (72%)
▪ three lymph nodes
1 (4%)
Central hypodensity
▪ present
3 (12%)
▪ absent
22 (88%)
Margins
▪ regular
24 (96%)
▪ irregular
1 (4%)
Structure
▪ homogeneous
22 (88%)
▪ non-homogeneous
3 (12%)
L/T: Longitudinal/transverse; US: Ultrasonography
Lymph node metastasis, non-homogeneous (areas of central and peripheral necrosis) size 50/40 mm, regular margins, adjacent to the external jugular vein
US features of the lymph node metastasis in patients with N0 neck
Patient No.
Lymph node group
L/T ratio
Shape
Structure
Margins
Central hypodensity
1.
Two lymph nodes
1.12
Round
Hypodensity
Non-homogenous
Regular
Present
2.
Three lymph nodes
1.14
Round
Hypodensity
Non-homogenous
Regular
Present
3.
Two lymph nodes
1
Round
Hypodensity
Homogeneous
Regular
Absent
4.
Three lymph nodes
1.11
Round
Hypodensity
Non-homogenous
Regular
Present
L/T: Longitudinal/transverse; US: Ultrasonography
Ultrasound of the neck: reactive lymph nodes
Lymph node with hyperplastic secondary lymphatic follicles (HE staining, ×200).
Only one patient in this group developed local recurrence and it was treated with open surgery with neck dissection.
On neck dissection, we excised two lymph nodes – previously detected on US with malignant features, confirmed with metastases on HP examinations.
HP and IHC examination of the primary tumor was performed in all cases and for all removed lymph nodes. All tumors were squamous cell carcinomas with varying degrees of differentiation; most cases were moderately differentiated squamous cell carcinomas (Figures 4 and 5).
IHC study showed that both primary tumors and lymph node metastases were positive, more or less intense, of anti-Ki67 antibody, depending on the degree of tumor differentiation, weakly or moderately differentiated tumors showing a more intense reaction compared to well-differentiated tumors (Figures 6 and 7).
Regarding the reaction to the anti-AE1/AE3 antibody, an intense reaction was noted, both in the primary tumor and in the lymph node metastases (Figures 8 and 9). Also, laryngeal tumors that responded positively to p53, also showed positive lymph node metastases to the same antibody (Figures 10 and 11).
Over three-year follow-up there was only one case (4.3%) with local recurrence so the “wait and see” policy was justified. The benefits of this approach are obvious: shorter operating time, good exposure with the operating microscope, no external scars, no wound related complications, less postoperative pain, shorter hospitalization, and lower costs.
Islands of moderately differentiated squamous cell carcinoma, with intense inflammatory reaction around, in a case of laryngeal carcinoma (HE staining, ×200).
Moderately differentiated squamous cell carcinoma, with tumor cells arranged in coordinates, separated by a desmoplastic stroma (GS trichrome staining, ×200).
Image of well-differentiated squamous cell carcinoma with moderately positive reaction to anti-Ki67 antibody (Immunostaining with anti-Ki67 antibody, ×200).
Poorly differentiated laryngeal squamous cell carcinoma, with marked cellular and nuclear atypia, with intense reaction to anti-Ki67 antibody (Immunostaining with anti-Ki67 antibody, ×200).
Primitive laryngeal tumor, moderately differentiated, with intense reaction to anti-AE1/AE3 antibody (Immunostaining with anti-AE1/AE3 antibody, ×200)
Image of lymph node metastasis with intense reaction to anti-AE1/AE3 antibody (Immunostaining with anti-AE1/AE3 antibody, ×200).
Primitive laryngeal tumor: poorly differentiated squamous cell carcinoma, with intense reaction to anti-p53 antibody (Immunostaining with anti-p53 antibody, ×100).
Laterocervical ganglion metastasis consisting of cell islands or isolated cells, resulting from a moderately differentiated squamous laryngeal carcinoma (Immunostaining with anti-p53 antibody, ×100)
|
⧉ Conclusions
|
Selective neck dissection in N0 pharyngo-laryngeal cancers is still controversial. Palpation is an inaccurate method for detection of lymph node involvement in head and neck patients and depends highly on surgeon’s experience. US of the neck is a cheap, reliable, and very well tolerated method of investigation for the lymph node involvement in the cervical area both for diagnostic and follow-up purposes. HP and IHC examinations represent the most important methods of positive and differential diagnosis, but also for assessing the prognosis.
|
[] |
[] |
[] |
[
"⧉ Introduction",
"⧉ Patients, Materials and Methods",
"⧉ Results",
"⧉ Discussions",
"⧉ Conclusions",
"Conflict of interest"
] |
[
"Malignant tumors are considered the first and most important problem of public healthcare, as their incidence has epidemic characteristics, they cause an immense burden on healthcare systems, they require large amounts of economic resources, they cause major social problems and seriously affect the individual and the family [1,2]. The present epidemiological data show a tendency of increase in the incidence, prevalence, and mortality for the next 40 years [1, 3].\nHead and neck cancers represent the sixth most frequent form of cancer world widely, with approximately 630 000 new patients diagnosed every year and over 350 000 deaths every year [4,5]. Cancers located in the pharynx and larynx are the most frequent head and neck malignancies [6,7,8]. Smoking and alcohol intake represent major etiological factors for the pharynx and larynx cancer [9].\nThe incidence of laryngeal cancer varies a lot from one country to another, from 2.5 to 17.2 up to 100 000 per year and it represents approximately 3% of the new cases of malignant tumors diagnosed every year all over the world [10].\nThe hypopharynx cancer has a 3–5% morbidity of head and neck cancers, with a poor prognosis, as the tumor has an infiltrative progression alongside the pharynx mucosa, it has a low number of symptoms and gives early metastases, therefore when it is diagnosed, the cancer already presents metastases in the cervix ganglia [11,12].\nThe therapy of pharyngeal and laryngeal cancer is decided according to the results of the clinical examination, endoscopy, and imaging. According to the location of the primary tumor and the tumor, node, metastasis (TNM) staging, either surgical or non-surgical treatment are considered. The decision is influenced by the presence or absence of the cervical lymph node metastasis. For laryngeal cancers, if the decision is to perform open surgery, the control of the lymph node areas is usually associated. If the decision is to perform endoscopic surgery – usually with lasers – than a “watchful waiting policy” is employed if no lymph nodes are palpable (N0) neck. Palpation of the neck is not a very reliable method and it depends on many factors – anatomy of the neck (short, thick neck), size and location of the lymph nodes, etc. Ultrasonography (US) is a well-established method for diagnosis of the lymph nodes in the head and neck area and can provide details regarding the characteristics of the lymph nodes (benign or malignant) [13,14,15]. This noninvasive method can be used for postoperative screening of the involved lymph node areas for both surgical and non-surgical patients, as it is cheap and well tolerated.\n\nAim\n\nIn the present study, we proposed to evaluate the role of US within the diagnosis of cervix ganglion metastases in the pharynx and larynx cancer, in the patients where the clinical examination did not show the presence of adenopathy (N0).",
"We included in the study 46 patients with cancers of the pharynx and larynx that presented with a N0 neck in Ear, Nose and Throat (ENT) Department of Timişoara, Romania, for three years, having a three-year period follow-up. They were divided in two groups: 23 patients operated with an external approach including the control of the lymph node areas, and a second group of 23 patients operated using endoscopy and carbon dioxide (CO2) laser, no neck dissection – a “watchful waiting policy”. All patients have had a flexible endoscopy of the pharynx and larynx, US of the neck and all received surgical treatment for their primary tumor. Imaging was performed in all selected cases. US was also used as a follow-up method. US of the neck areas was performed using a wide band transducer (frequency of 6–13 MHz) at a frequency of 7.5 MHz. The patient is in a supine position with the neck in extension and the head rotated towards the opposite side. We searched for the following features of the lymph nodes: size, shape, margins, location, structure, and relation to the nearby structures.\nAll tumors and all excised lymph nodes were sent to the Laboratory of Pathological Anatomy for histopathological (HP) and immunohistochemical (IHC) studies.\nThe biological material was fixed in 10% neutral buffered formalin and included in paraffin, according to the usual HP protocol. After the microtome sectioning, there was performed Hematoxylin–Eosin (HE) and green light trichrome, the Goldner–Szekely (GS) technique; for the IHC study, there were used the following antibodies: anti-Ki67 (monoclonal mouse anti-human Ki67, clone MIB-1, 1/50 dilution, Dako); anti-AE1/AE3 (monoclonal mouse anti-human cytokeratin, clone AE1/AE3, 1/100 dilution, Dako); anti-p53 (monoclonal mouse anti-human p53 protein, clone DO-7, 1/100 dilution, Dako).",
"In our study, most patients were male, respectively 41, representing 89.1%. From the studied group, the women were in a small number, respectively five, representing 10.9%. The main symptoms were: dysphonia, which was present in 42 (91.3%) patients; dysphagia was found in eight (17.4%) patients; pain on swallowing was present in three (6.5%) patients, and significant weight loss was mentioned only in two (4.3%) patients. Palpation of the neck was carefully performed in all patients and only the N0 patients were included in the study. In our study, 45 (97.8%) patients had laryngeal tumors. One case presented a tumor located in the oropharynx, in the left tonsil, extending to the base of the tongue. The laryngeal tumors were located as follows: epiglottis – five (11.1%); extension beyond the epiglottis – two (4.4%); vocal cord – 14 (31.1%); vocal cord extended to anterior commissure – nine (20.1%); vocal cord extended to subglottic region – four (8.9%); vocal cord extended to the supraglottic region – 11 (24.4%). Staging was performed according to TNM Classification [American Joint Committee on Cancer (AJCC) and Union for International Cancer Control (UICC)] and is presented in Table 1. It can be seen that there are malignant laryngeal tumors that do not show lymphadenopathy even in advanced stages of the disease, respectively III and IV stages.\nN0 neck patients according to T staging\nTumor (T) staging\nNo. of cases (%)\nT I\n18 (39.13%)\nT II\n8 (17.39%)\nT III\n6 (13.04%)\nT IV\n14 (30.43%)\nTotal\n46 (100%)\nTumor (T) stage was the main factor in deciding which therapy was used. The open surgery was performed on a number of 23 (50%) patients, especially those in advanced (III and IV) stages of the disease (Table 2), while endoscopic surgery mainly in early (I and II) stages in 23 (50%) patients (Table 3).\nN0 neck patients treated with open surgery according to T staging\nTumor (T) staging\nNo. of cases (%)\nT I\n1 (2.17%)\nT II\n2 (4.34%)\nT III\n6 (13.04%)\nT IV\n14 (30.45%)\nTotal\n23 (50%)\nN0 neck patients treated with endoscopic CO2 laser surgery according to T staging\nTumor (T) staging\nNo. of cases (%)\nT I\n17 (36.96%)\nT II\n6 (13.04%)\nT III\n0\nT IV\n0\nTotal\n23 (50%)\nUS was performed in all patients both preoperatively – to help planning, and postoperatively – for follow-up. US has detected 25 lymph nodes in the open surgery group and intraoperatively we excised 31 (sensitivity of 80.6%).\nThe US features of the examined lymph nodes were: diameters [longitudinal (L) and transverse (T)]; the ratio of the two diameters (L/T); shape; lymph node area; central hypodensity; regular/irregular margins; aspect (homogeneous or not) (Table 4). US features for metastatic lymph nodes were as follows: size larger than 1.2 cm; L/T ratio less than 2; round shape; clustering; central hypodensity; irregular margins; non-homogeneous aspect. Not all of these features were present in every patient we examined. The only feature with 100% appearance was the round shape, followed by central hypodensity (75%) and L/T ratio (66%). Irregular shape and clustering were inconclusive (25%). While size and L/T ratio produced an over evaluation of the number of cases all other features produced an under evaluation. Clustering, L/T ratio and shape of the lymph nodes were features with 100% accuracy, while central hypodensity (Figure 1) had an accuracy of only 75% (Table 5). Irregular margins had no significance on diagnosis. This can be explained by the small size of the lymph nodes.\nUS in the second group (patients treated with CO2 laser) detected in four patients, 10 cervical lymph nodes that did not present any malignant features (Figures 2 and 3), and the attitude against them was a closed follow-up “watchful waiting policy” which revealed no change of the characteristics over time.\nUS features of the neck lymph nodes\nUS features\nNo. of lymph nodes (%)\nMinimal and maximal diameter [cm]\n \n▪ 0.8–1.2\n25 (100%)\n▪ >0.8–1.2\n0\nL/T ratio\n \n▪ >1.8\n19 (76%)\n▪ <1.8\n6 (24%)\nShape\n \n▪ round\n4 (16%)\n▪ oval\n21 (84%)\nLymph node clustering\n \n▪ one lymph node\n6 (24%)\n▪ two lymph nodes\n18 (72%)\n▪ three lymph nodes\n1 (4%)\nCentral hypodensity\n \n▪ present\n3 (12%)\n▪ absent\n22 (88%)\nMargins\n \n▪ regular\n24 (96%)\n▪ irregular\n1 (4%)\nStructure\n \n▪ homogeneous\n22 (88%)\n▪ non-homogeneous\n3 (12%)\n L/T: Longitudinal/transverse; US: Ultrasonography\nLymph node metastasis, non-homogeneous (areas of central and peripheral necrosis) size 50/40 mm, regular margins, adjacent to the external jugular vein\nUS features of the lymph node metastasis in patients with N0 neck\nPatient No.\nLymph node group\nL/T ratio\nShape\nStructure\nMargins\nCentral hypodensity\n1.\nTwo lymph nodes\n1.12\nRound\nHypodensity\nNon-homogenous\nRegular\nPresent\n2.\nThree lymph nodes\n1.14\nRound\nHypodensity\nNon-homogenous\nRegular\nPresent\n3.\nTwo lymph nodes\n1\nRound\nHypodensity\nHomogeneous\nRegular\nAbsent\n4.\nThree lymph nodes\n1.11\nRound\nHypodensity\nNon-homogenous\nRegular\nPresent\nL/T: Longitudinal/transverse; US: Ultrasonography \nUltrasound of the neck: reactive lymph nodes\nLymph node with hyperplastic secondary lymphatic follicles (HE staining, ×200).\nOnly one patient in this group developed local recurrence and it was treated with open surgery with neck dissection.\nOn neck dissection, we excised two lymph nodes – previously detected on US with malignant features, confirmed with metastases on HP examinations.\nHP and IHC examination of the primary tumor was performed in all cases and for all removed lymph nodes. All tumors were squamous cell carcinomas with varying degrees of differentiation; most cases were moderately differentiated squamous cell carcinomas (Figures 4 and 5).\nIHC study showed that both primary tumors and lymph node metastases were positive, more or less intense, of anti-Ki67 antibody, depending on the degree of tumor differentiation, weakly or moderately differentiated tumors showing a more intense reaction compared to well-differentiated tumors (Figures 6 and 7).\nRegarding the reaction to the anti-AE1/AE3 antibody, an intense reaction was noted, both in the primary tumor and in the lymph node metastases (Figures 8 and 9). Also, laryngeal tumors that responded positively to p53, also showed positive lymph node metastases to the same antibody (Figures 10 and 11).\nOver three-year follow-up there was only one case (4.3%) with local recurrence so the “wait and see” policy was justified. The benefits of this approach are obvious: shorter operating time, good exposure with the operating microscope, no external scars, no wound related complications, less postoperative pain, shorter hospitalization, and lower costs.\nIslands of moderately differentiated squamous cell carcinoma, with intense inflammatory reaction around, in a case of laryngeal carcinoma (HE staining, ×200).\nModerately differentiated squamous cell carcinoma, with tumor cells arranged in coordinates, separated by a desmoplastic stroma (GS trichrome staining, ×200).\nImage of well-differentiated squamous cell carcinoma with moderately positive reaction to anti-Ki67 antibody (Immunostaining with anti-Ki67 antibody, ×200).\nPoorly differentiated laryngeal squamous cell carcinoma, with marked cellular and nuclear atypia, with intense reaction to anti-Ki67 antibody (Immunostaining with anti-Ki67 antibody, ×200).\nPrimitive laryngeal tumor, moderately differentiated, with intense reaction to anti-AE1/AE3 antibody (Immunostaining with anti-AE1/AE3 antibody, ×200)\nImage of lymph node metastasis with intense reaction to anti-AE1/AE3 antibody (Immunostaining with anti-AE1/AE3 antibody, ×200).\nPrimitive laryngeal tumor: poorly differentiated squamous cell carcinoma, with intense reaction to anti-p53 antibody (Immunostaining with anti-p53 antibody, ×100).\nLaterocervical ganglion metastasis consisting of cell islands or isolated cells, resulting from a moderately differentiated squamous laryngeal carcinoma (Immunostaining with anti-p53 antibody, ×100)",
"Over 50% of the head and neck malignancies have cervical lymph node involvement at presentation [16], and lymph node metastases are associated with a reduced survival rate [16,17]. Palpation has its obvious limitations in detecting cervical metastasis. Sako et al. (1964) reported a 28% incidence of lymph node metastasis in 123 patients with head and neck tumors and N0 neck in which selective neck dissection was performed [18]. Szmeja et al. (1999) demonstrated the superiority of US over palpation in 810 patients with laryngeal cancer treated with either surgery or radiotherapy over a five-year period [19]. The same authors extended their study over seven years with 1120 patients with laryngeal cancer receiving surgical treatment. In the N0 group (505 cases), US examination detected 261 patients with lymph node involvement. Sixty-three (24.1%) patients had histology confirmation of metastatic lymph nodes. They found a correlation US–surgery of 95% and US–histology of 90% [20]. All the patients had US of the neck as a part of their follow-up. One hundred thirty-six patients developed lymph node recurrences and 117 presented initially with N0 neck (105 histologically confirmed). The authors recommend US as a very useful diagnostic tool and for the follow-up period. Schipper et al. (1999) [21] presented a study of 101 early (T1 and T2, N0) stage head and neck patients receiving US examination as part of their diagnostic procedure and 18–36 months follow-up period. US detected lymph node involvement in 30 cases and they received neck dissection as part of the treatment. Eight patients out of 30 presented with metastatic lymph nodes. The conclusion of the study was that the “watchful waiting policy” does not alter the prognosis and they recommend US for the follow-up in head and neck patients. Yoshida et al. (1998) perform histology in 117 lymph nodes detected by US in head and neck patients and find 26 (22.2%) metastases, so the sensitivity is 83% and sensibility is 97% with an accuracy of 95% [22]. In other study, an L/T ratio ≤2, non-hilar vascular pattern, parenchymal granular echoes, necrosis, and the presence of groups of three or more otherwise normal nodes in a high-risk area were considered good indicators of metastatic disease [23]. Wang et al. (2019), examining 89 patients with hypopharyngeal cancer showed that US plays an important role in the preoperative detection of tumor metastases in the cervical lymph nodes [24]. US diagnostic [13] criteria in the US differentiation of benign and malignant nodes of the neck are very useful (Table 6) as US is an important tool in the follow-up protocol in head and neck patients.\nUS diagnostic criteria in the US differentiation of benign and malignant nodes of the neck\nType\nBenign\nMalignant\nForm\nOval\nRound\nBoundary\nSharp\nBlurred\nHilar sign\nHyperechoic hilum\nNo hilum\nVessels\nHilar vascularization\nTangential vascularization\nDynamic behavior\nStationary\nIncrease of size\nIntranodal necrosis\nWithout\nAnechoic central region in heterogeneous lymph node texture\nUS: Ultrasonography \nUS of the neck was also successfully used as a follow-up method in oral cancers with only one patient out of 32 presenting a lymph node metastasis six months post-operatively [25]. US can be used for specifically monitoring of a certain lymph node area – the risk ones – jugulo-digastric (JDG) and jugulo-omohyoid (JO). 31.6% were not elliptical, more of the JO than the JDG nodes were round; there was a significant difference in roundness index between them (p<0.05) and significantly more of the lymph nodes with roundness index <2 lacked a visible echogenic hilum [26,27]. The follow-up for patients with N0 neck – especially in pharyngeal and laryngeal cancers – is of paramount importance as selective neck dissection is still controversial. Reports are still controversial: Birkeland et al. (2016), in 35 patients found 17% histology positive lymph nodes [28]. However, Deganello et al. (2014) [29] and Pezier et al. (2014) [30] found almost no lymph node involvement and recommend a conservative approach over the lymph nodes. When considering recurrence in laryngeal cancers neck dissection is mandatory in all cases of squamous cell carcinomas recurrence with N+, and routine elective neck dissection is recommended for recurrent supraglottic and transglottic cancers [31]. US can be also used intraoperatively in head and neck tumors. Stieve et al. (2012) in a study on 115 cases concluded that US was helpful providing information about the size of the tumor and involvement of the adjacent areas [32].",
"Selective neck dissection in N0 pharyngo-laryngeal cancers is still controversial. Palpation is an inaccurate method for detection of lymph node involvement in head and neck patients and depends highly on surgeon’s experience. US of the neck is a cheap, reliable, and very well tolerated method of investigation for the lymph node involvement in the cervical area both for diagnostic and follow-up purposes. HP and IHC examinations represent the most important methods of positive and differential diagnosis, but also for assessing the prognosis.",
"The authors declare that they have no conflict of interests."
] |
[
"intro",
"materials|methods",
"results",
"discussion",
"conclusions",
"COI-statement"
] |
[
"lymph nodes",
"palpation",
"ultrasonography",
"neck node metastasis"
] |
⧉ Introduction:
Malignant tumors are considered the first and most important problem of public healthcare, as their incidence has epidemic characteristics, they cause an immense burden on healthcare systems, they require large amounts of economic resources, they cause major social problems and seriously affect the individual and the family [1,2]. The present epidemiological data show a tendency of increase in the incidence, prevalence, and mortality for the next 40 years [1, 3].
Head and neck cancers represent the sixth most frequent form of cancer world widely, with approximately 630 000 new patients diagnosed every year and over 350 000 deaths every year [4,5]. Cancers located in the pharynx and larynx are the most frequent head and neck malignancies [6,7,8]. Smoking and alcohol intake represent major etiological factors for the pharynx and larynx cancer [9].
The incidence of laryngeal cancer varies a lot from one country to another, from 2.5 to 17.2 up to 100 000 per year and it represents approximately 3% of the new cases of malignant tumors diagnosed every year all over the world [10].
The hypopharynx cancer has a 3–5% morbidity of head and neck cancers, with a poor prognosis, as the tumor has an infiltrative progression alongside the pharynx mucosa, it has a low number of symptoms and gives early metastases, therefore when it is diagnosed, the cancer already presents metastases in the cervix ganglia [11,12].
The therapy of pharyngeal and laryngeal cancer is decided according to the results of the clinical examination, endoscopy, and imaging. According to the location of the primary tumor and the tumor, node, metastasis (TNM) staging, either surgical or non-surgical treatment are considered. The decision is influenced by the presence or absence of the cervical lymph node metastasis. For laryngeal cancers, if the decision is to perform open surgery, the control of the lymph node areas is usually associated. If the decision is to perform endoscopic surgery – usually with lasers – than a “watchful waiting policy” is employed if no lymph nodes are palpable (N0) neck. Palpation of the neck is not a very reliable method and it depends on many factors – anatomy of the neck (short, thick neck), size and location of the lymph nodes, etc. Ultrasonography (US) is a well-established method for diagnosis of the lymph nodes in the head and neck area and can provide details regarding the characteristics of the lymph nodes (benign or malignant) [13,14,15]. This noninvasive method can be used for postoperative screening of the involved lymph node areas for both surgical and non-surgical patients, as it is cheap and well tolerated.
Aim
In the present study, we proposed to evaluate the role of US within the diagnosis of cervix ganglion metastases in the pharynx and larynx cancer, in the patients where the clinical examination did not show the presence of adenopathy (N0).
⧉ Patients, Materials and Methods:
We included in the study 46 patients with cancers of the pharynx and larynx that presented with a N0 neck in Ear, Nose and Throat (ENT) Department of Timişoara, Romania, for three years, having a three-year period follow-up. They were divided in two groups: 23 patients operated with an external approach including the control of the lymph node areas, and a second group of 23 patients operated using endoscopy and carbon dioxide (CO2) laser, no neck dissection – a “watchful waiting policy”. All patients have had a flexible endoscopy of the pharynx and larynx, US of the neck and all received surgical treatment for their primary tumor. Imaging was performed in all selected cases. US was also used as a follow-up method. US of the neck areas was performed using a wide band transducer (frequency of 6–13 MHz) at a frequency of 7.5 MHz. The patient is in a supine position with the neck in extension and the head rotated towards the opposite side. We searched for the following features of the lymph nodes: size, shape, margins, location, structure, and relation to the nearby structures.
All tumors and all excised lymph nodes were sent to the Laboratory of Pathological Anatomy for histopathological (HP) and immunohistochemical (IHC) studies.
The biological material was fixed in 10% neutral buffered formalin and included in paraffin, according to the usual HP protocol. After the microtome sectioning, there was performed Hematoxylin–Eosin (HE) and green light trichrome, the Goldner–Szekely (GS) technique; for the IHC study, there were used the following antibodies: anti-Ki67 (monoclonal mouse anti-human Ki67, clone MIB-1, 1/50 dilution, Dako); anti-AE1/AE3 (monoclonal mouse anti-human cytokeratin, clone AE1/AE3, 1/100 dilution, Dako); anti-p53 (monoclonal mouse anti-human p53 protein, clone DO-7, 1/100 dilution, Dako).
⧉ Results:
In our study, most patients were male, respectively 41, representing 89.1%. From the studied group, the women were in a small number, respectively five, representing 10.9%. The main symptoms were: dysphonia, which was present in 42 (91.3%) patients; dysphagia was found in eight (17.4%) patients; pain on swallowing was present in three (6.5%) patients, and significant weight loss was mentioned only in two (4.3%) patients. Palpation of the neck was carefully performed in all patients and only the N0 patients were included in the study. In our study, 45 (97.8%) patients had laryngeal tumors. One case presented a tumor located in the oropharynx, in the left tonsil, extending to the base of the tongue. The laryngeal tumors were located as follows: epiglottis – five (11.1%); extension beyond the epiglottis – two (4.4%); vocal cord – 14 (31.1%); vocal cord extended to anterior commissure – nine (20.1%); vocal cord extended to subglottic region – four (8.9%); vocal cord extended to the supraglottic region – 11 (24.4%). Staging was performed according to TNM Classification [American Joint Committee on Cancer (AJCC) and Union for International Cancer Control (UICC)] and is presented in Table 1. It can be seen that there are malignant laryngeal tumors that do not show lymphadenopathy even in advanced stages of the disease, respectively III and IV stages.
N0 neck patients according to T staging
Tumor (T) staging
No. of cases (%)
T I
18 (39.13%)
T II
8 (17.39%)
T III
6 (13.04%)
T IV
14 (30.43%)
Total
46 (100%)
Tumor (T) stage was the main factor in deciding which therapy was used. The open surgery was performed on a number of 23 (50%) patients, especially those in advanced (III and IV) stages of the disease (Table 2), while endoscopic surgery mainly in early (I and II) stages in 23 (50%) patients (Table 3).
N0 neck patients treated with open surgery according to T staging
Tumor (T) staging
No. of cases (%)
T I
1 (2.17%)
T II
2 (4.34%)
T III
6 (13.04%)
T IV
14 (30.45%)
Total
23 (50%)
N0 neck patients treated with endoscopic CO2 laser surgery according to T staging
Tumor (T) staging
No. of cases (%)
T I
17 (36.96%)
T II
6 (13.04%)
T III
0
T IV
0
Total
23 (50%)
US was performed in all patients both preoperatively – to help planning, and postoperatively – for follow-up. US has detected 25 lymph nodes in the open surgery group and intraoperatively we excised 31 (sensitivity of 80.6%).
The US features of the examined lymph nodes were: diameters [longitudinal (L) and transverse (T)]; the ratio of the two diameters (L/T); shape; lymph node area; central hypodensity; regular/irregular margins; aspect (homogeneous or not) (Table 4). US features for metastatic lymph nodes were as follows: size larger than 1.2 cm; L/T ratio less than 2; round shape; clustering; central hypodensity; irregular margins; non-homogeneous aspect. Not all of these features were present in every patient we examined. The only feature with 100% appearance was the round shape, followed by central hypodensity (75%) and L/T ratio (66%). Irregular shape and clustering were inconclusive (25%). While size and L/T ratio produced an over evaluation of the number of cases all other features produced an under evaluation. Clustering, L/T ratio and shape of the lymph nodes were features with 100% accuracy, while central hypodensity (Figure 1) had an accuracy of only 75% (Table 5). Irregular margins had no significance on diagnosis. This can be explained by the small size of the lymph nodes.
US in the second group (patients treated with CO2 laser) detected in four patients, 10 cervical lymph nodes that did not present any malignant features (Figures 2 and 3), and the attitude against them was a closed follow-up “watchful waiting policy” which revealed no change of the characteristics over time.
US features of the neck lymph nodes
US features
No. of lymph nodes (%)
Minimal and maximal diameter [cm]
▪ 0.8–1.2
25 (100%)
▪ >0.8–1.2
0
L/T ratio
▪ >1.8
19 (76%)
▪ <1.8
6 (24%)
Shape
▪ round
4 (16%)
▪ oval
21 (84%)
Lymph node clustering
▪ one lymph node
6 (24%)
▪ two lymph nodes
18 (72%)
▪ three lymph nodes
1 (4%)
Central hypodensity
▪ present
3 (12%)
▪ absent
22 (88%)
Margins
▪ regular
24 (96%)
▪ irregular
1 (4%)
Structure
▪ homogeneous
22 (88%)
▪ non-homogeneous
3 (12%)
L/T: Longitudinal/transverse; US: Ultrasonography
Lymph node metastasis, non-homogeneous (areas of central and peripheral necrosis) size 50/40 mm, regular margins, adjacent to the external jugular vein
US features of the lymph node metastasis in patients with N0 neck
Patient No.
Lymph node group
L/T ratio
Shape
Structure
Margins
Central hypodensity
1.
Two lymph nodes
1.12
Round
Hypodensity
Non-homogenous
Regular
Present
2.
Three lymph nodes
1.14
Round
Hypodensity
Non-homogenous
Regular
Present
3.
Two lymph nodes
1
Round
Hypodensity
Homogeneous
Regular
Absent
4.
Three lymph nodes
1.11
Round
Hypodensity
Non-homogenous
Regular
Present
L/T: Longitudinal/transverse; US: Ultrasonography
Ultrasound of the neck: reactive lymph nodes
Lymph node with hyperplastic secondary lymphatic follicles (HE staining, ×200).
Only one patient in this group developed local recurrence and it was treated with open surgery with neck dissection.
On neck dissection, we excised two lymph nodes – previously detected on US with malignant features, confirmed with metastases on HP examinations.
HP and IHC examination of the primary tumor was performed in all cases and for all removed lymph nodes. All tumors were squamous cell carcinomas with varying degrees of differentiation; most cases were moderately differentiated squamous cell carcinomas (Figures 4 and 5).
IHC study showed that both primary tumors and lymph node metastases were positive, more or less intense, of anti-Ki67 antibody, depending on the degree of tumor differentiation, weakly or moderately differentiated tumors showing a more intense reaction compared to well-differentiated tumors (Figures 6 and 7).
Regarding the reaction to the anti-AE1/AE3 antibody, an intense reaction was noted, both in the primary tumor and in the lymph node metastases (Figures 8 and 9). Also, laryngeal tumors that responded positively to p53, also showed positive lymph node metastases to the same antibody (Figures 10 and 11).
Over three-year follow-up there was only one case (4.3%) with local recurrence so the “wait and see” policy was justified. The benefits of this approach are obvious: shorter operating time, good exposure with the operating microscope, no external scars, no wound related complications, less postoperative pain, shorter hospitalization, and lower costs.
Islands of moderately differentiated squamous cell carcinoma, with intense inflammatory reaction around, in a case of laryngeal carcinoma (HE staining, ×200).
Moderately differentiated squamous cell carcinoma, with tumor cells arranged in coordinates, separated by a desmoplastic stroma (GS trichrome staining, ×200).
Image of well-differentiated squamous cell carcinoma with moderately positive reaction to anti-Ki67 antibody (Immunostaining with anti-Ki67 antibody, ×200).
Poorly differentiated laryngeal squamous cell carcinoma, with marked cellular and nuclear atypia, with intense reaction to anti-Ki67 antibody (Immunostaining with anti-Ki67 antibody, ×200).
Primitive laryngeal tumor, moderately differentiated, with intense reaction to anti-AE1/AE3 antibody (Immunostaining with anti-AE1/AE3 antibody, ×200)
Image of lymph node metastasis with intense reaction to anti-AE1/AE3 antibody (Immunostaining with anti-AE1/AE3 antibody, ×200).
Primitive laryngeal tumor: poorly differentiated squamous cell carcinoma, with intense reaction to anti-p53 antibody (Immunostaining with anti-p53 antibody, ×100).
Laterocervical ganglion metastasis consisting of cell islands or isolated cells, resulting from a moderately differentiated squamous laryngeal carcinoma (Immunostaining with anti-p53 antibody, ×100)
⧉ Discussions:
Over 50% of the head and neck malignancies have cervical lymph node involvement at presentation [16], and lymph node metastases are associated with a reduced survival rate [16,17]. Palpation has its obvious limitations in detecting cervical metastasis. Sako et al. (1964) reported a 28% incidence of lymph node metastasis in 123 patients with head and neck tumors and N0 neck in which selective neck dissection was performed [18]. Szmeja et al. (1999) demonstrated the superiority of US over palpation in 810 patients with laryngeal cancer treated with either surgery or radiotherapy over a five-year period [19]. The same authors extended their study over seven years with 1120 patients with laryngeal cancer receiving surgical treatment. In the N0 group (505 cases), US examination detected 261 patients with lymph node involvement. Sixty-three (24.1%) patients had histology confirmation of metastatic lymph nodes. They found a correlation US–surgery of 95% and US–histology of 90% [20]. All the patients had US of the neck as a part of their follow-up. One hundred thirty-six patients developed lymph node recurrences and 117 presented initially with N0 neck (105 histologically confirmed). The authors recommend US as a very useful diagnostic tool and for the follow-up period. Schipper et al. (1999) [21] presented a study of 101 early (T1 and T2, N0) stage head and neck patients receiving US examination as part of their diagnostic procedure and 18–36 months follow-up period. US detected lymph node involvement in 30 cases and they received neck dissection as part of the treatment. Eight patients out of 30 presented with metastatic lymph nodes. The conclusion of the study was that the “watchful waiting policy” does not alter the prognosis and they recommend US for the follow-up in head and neck patients. Yoshida et al. (1998) perform histology in 117 lymph nodes detected by US in head and neck patients and find 26 (22.2%) metastases, so the sensitivity is 83% and sensibility is 97% with an accuracy of 95% [22]. In other study, an L/T ratio ≤2, non-hilar vascular pattern, parenchymal granular echoes, necrosis, and the presence of groups of three or more otherwise normal nodes in a high-risk area were considered good indicators of metastatic disease [23]. Wang et al. (2019), examining 89 patients with hypopharyngeal cancer showed that US plays an important role in the preoperative detection of tumor metastases in the cervical lymph nodes [24]. US diagnostic [13] criteria in the US differentiation of benign and malignant nodes of the neck are very useful (Table 6) as US is an important tool in the follow-up protocol in head and neck patients.
US diagnostic criteria in the US differentiation of benign and malignant nodes of the neck
Type
Benign
Malignant
Form
Oval
Round
Boundary
Sharp
Blurred
Hilar sign
Hyperechoic hilum
No hilum
Vessels
Hilar vascularization
Tangential vascularization
Dynamic behavior
Stationary
Increase of size
Intranodal necrosis
Without
Anechoic central region in heterogeneous lymph node texture
US: Ultrasonography
US of the neck was also successfully used as a follow-up method in oral cancers with only one patient out of 32 presenting a lymph node metastasis six months post-operatively [25]. US can be used for specifically monitoring of a certain lymph node area – the risk ones – jugulo-digastric (JDG) and jugulo-omohyoid (JO). 31.6% were not elliptical, more of the JO than the JDG nodes were round; there was a significant difference in roundness index between them (p<0.05) and significantly more of the lymph nodes with roundness index <2 lacked a visible echogenic hilum [26,27]. The follow-up for patients with N0 neck – especially in pharyngeal and laryngeal cancers – is of paramount importance as selective neck dissection is still controversial. Reports are still controversial: Birkeland et al. (2016), in 35 patients found 17% histology positive lymph nodes [28]. However, Deganello et al. (2014) [29] and Pezier et al. (2014) [30] found almost no lymph node involvement and recommend a conservative approach over the lymph nodes. When considering recurrence in laryngeal cancers neck dissection is mandatory in all cases of squamous cell carcinomas recurrence with N+, and routine elective neck dissection is recommended for recurrent supraglottic and transglottic cancers [31]. US can be also used intraoperatively in head and neck tumors. Stieve et al. (2012) in a study on 115 cases concluded that US was helpful providing information about the size of the tumor and involvement of the adjacent areas [32].
⧉ Conclusions:
Selective neck dissection in N0 pharyngo-laryngeal cancers is still controversial. Palpation is an inaccurate method for detection of lymph node involvement in head and neck patients and depends highly on surgeon’s experience. US of the neck is a cheap, reliable, and very well tolerated method of investigation for the lymph node involvement in the cervical area both for diagnostic and follow-up purposes. HP and IHC examinations represent the most important methods of positive and differential diagnosis, but also for assessing the prognosis.
Conflict of interest:
The authors declare that they have no conflict of interests.
|
Background:
The presence of metastatic cervical adenopathy is essential for treatment planning and prognosis assessment. Treatment of patients with head and neck cancer with clinically negative cervical lymphadenopathy (N0) remains controversial. Neck palpation, as the method used in tumor, node, metastasis (TNM) staging, has limitations and can provide false negative results in some cases. Lymph node metastases are associated with a reduced survival rate but at the same time, neck dissection for the patient with N0 neck is not without risks or complications.
Methods:
Forty-six patients with cancers of the pharynx and larynx that presented with a N0 neck were prospectively analyzed. They were divided in two groups: 23 patients operated with an external approach including the control of the lymph node areas, and a second group of 23 patients operated using endoscopy and carbon dioxide (CO2) laser, no neck dissection - "watchful waiting policy". All patients have had a flexible endoscopy of the pharynx and larynx, US of the neck and all received surgical treatment for their primary tumor. Imaging was performed in selected cases. All the removed lymph nodes were sent for histopathology. US was also used as a follow-up method. The US features of the examined lymph nodes were: diameters [longitudinal (L) and transverse (T)]; the ratio of the two diameters (L∕T); shape; lymph node area; central hypodensity; regular∕irregular margins; aspect (homogeneous or not).
Results:
US has detected 25 lymph nodes in the open surgery group and intraoperatively, we excised 31 (sensitivity of 80.6%). Ten lymph nodes showed metastases, with 100% accuracy of US, which have been confirmed both pathologically and immunohistochemically. US in the second group - patients treated with CO2 laser - detected at four patients 10 cervical lymph nodes that did not presented any malignant features. At recurrence alone, the US confirmed 100% presence of nodes metastases.
Conclusions:
US was superior to palpation and this method can be recommended as a diagnostic tool in preoperative assessment of patients without palpable metastasis (N0).
|
⧉ Introduction:
Malignant tumors are considered the first and most important problem of public healthcare, as their incidence has epidemic characteristics, they cause an immense burden on healthcare systems, they require large amounts of economic resources, they cause major social problems and seriously affect the individual and the family [1,2]. The present epidemiological data show a tendency of increase in the incidence, prevalence, and mortality for the next 40 years [1, 3].
Head and neck cancers represent the sixth most frequent form of cancer world widely, with approximately 630 000 new patients diagnosed every year and over 350 000 deaths every year [4,5]. Cancers located in the pharynx and larynx are the most frequent head and neck malignancies [6,7,8]. Smoking and alcohol intake represent major etiological factors for the pharynx and larynx cancer [9].
The incidence of laryngeal cancer varies a lot from one country to another, from 2.5 to 17.2 up to 100 000 per year and it represents approximately 3% of the new cases of malignant tumors diagnosed every year all over the world [10].
The hypopharynx cancer has a 3–5% morbidity of head and neck cancers, with a poor prognosis, as the tumor has an infiltrative progression alongside the pharynx mucosa, it has a low number of symptoms and gives early metastases, therefore when it is diagnosed, the cancer already presents metastases in the cervix ganglia [11,12].
The therapy of pharyngeal and laryngeal cancer is decided according to the results of the clinical examination, endoscopy, and imaging. According to the location of the primary tumor and the tumor, node, metastasis (TNM) staging, either surgical or non-surgical treatment are considered. The decision is influenced by the presence or absence of the cervical lymph node metastasis. For laryngeal cancers, if the decision is to perform open surgery, the control of the lymph node areas is usually associated. If the decision is to perform endoscopic surgery – usually with lasers – than a “watchful waiting policy” is employed if no lymph nodes are palpable (N0) neck. Palpation of the neck is not a very reliable method and it depends on many factors – anatomy of the neck (short, thick neck), size and location of the lymph nodes, etc. Ultrasonography (US) is a well-established method for diagnosis of the lymph nodes in the head and neck area and can provide details regarding the characteristics of the lymph nodes (benign or malignant) [13,14,15]. This noninvasive method can be used for postoperative screening of the involved lymph node areas for both surgical and non-surgical patients, as it is cheap and well tolerated.
Aim
In the present study, we proposed to evaluate the role of US within the diagnosis of cervix ganglion metastases in the pharynx and larynx cancer, in the patients where the clinical examination did not show the presence of adenopathy (N0).
⧉ Conclusions:
Selective neck dissection in N0 pharyngo-laryngeal cancers is still controversial. Palpation is an inaccurate method for detection of lymph node involvement in head and neck patients and depends highly on surgeon’s experience. US of the neck is a cheap, reliable, and very well tolerated method of investigation for the lymph node involvement in the cervical area both for diagnostic and follow-up purposes. HP and IHC examinations represent the most important methods of positive and differential diagnosis, but also for assessing the prognosis.
|
Background:
The presence of metastatic cervical adenopathy is essential for treatment planning and prognosis assessment. Treatment of patients with head and neck cancer with clinically negative cervical lymphadenopathy (N0) remains controversial. Neck palpation, as the method used in tumor, node, metastasis (TNM) staging, has limitations and can provide false negative results in some cases. Lymph node metastases are associated with a reduced survival rate but at the same time, neck dissection for the patient with N0 neck is not without risks or complications.
Methods:
Forty-six patients with cancers of the pharynx and larynx that presented with a N0 neck were prospectively analyzed. They were divided in two groups: 23 patients operated with an external approach including the control of the lymph node areas, and a second group of 23 patients operated using endoscopy and carbon dioxide (CO2) laser, no neck dissection - "watchful waiting policy". All patients have had a flexible endoscopy of the pharynx and larynx, US of the neck and all received surgical treatment for their primary tumor. Imaging was performed in selected cases. All the removed lymph nodes were sent for histopathology. US was also used as a follow-up method. The US features of the examined lymph nodes were: diameters [longitudinal (L) and transverse (T)]; the ratio of the two diameters (L∕T); shape; lymph node area; central hypodensity; regular∕irregular margins; aspect (homogeneous or not).
Results:
US has detected 25 lymph nodes in the open surgery group and intraoperatively, we excised 31 (sensitivity of 80.6%). Ten lymph nodes showed metastases, with 100% accuracy of US, which have been confirmed both pathologically and immunohistochemically. US in the second group - patients treated with CO2 laser - detected at four patients 10 cervical lymph nodes that did not presented any malignant features. At recurrence alone, the US confirmed 100% presence of nodes metastases.
Conclusions:
US was superior to palpation and this method can be recommended as a diagnostic tool in preoperative assessment of patients without palpable metastasis (N0).
| 3,811 | 404 | 6 |
[
"lymph",
"neck",
"patients",
"nodes",
"lymph nodes",
"node",
"lymph node",
"anti",
"tumor",
"laryngeal"
] |
[
"test",
"test"
] | null |
[CONTENT] lymph nodes | palpation | ultrasonography | neck node metastasis [SUMMARY]
| null |
[CONTENT] lymph nodes | palpation | ultrasonography | neck node metastasis [SUMMARY]
|
[CONTENT] lymph nodes | palpation | ultrasonography | neck node metastasis [SUMMARY]
|
[CONTENT] lymph nodes | palpation | ultrasonography | neck node metastasis [SUMMARY]
|
[CONTENT] lymph nodes | palpation | ultrasonography | neck node metastasis [SUMMARY]
|
[CONTENT] Female | Head and Neck Neoplasms | Humans | Larynx | Lymph Nodes | Male | Neoplasm Metastasis | Pharynx | Prospective Studies | Ultrasonography [SUMMARY]
| null |
[CONTENT] Female | Head and Neck Neoplasms | Humans | Larynx | Lymph Nodes | Male | Neoplasm Metastasis | Pharynx | Prospective Studies | Ultrasonography [SUMMARY]
|
[CONTENT] Female | Head and Neck Neoplasms | Humans | Larynx | Lymph Nodes | Male | Neoplasm Metastasis | Pharynx | Prospective Studies | Ultrasonography [SUMMARY]
|
[CONTENT] Female | Head and Neck Neoplasms | Humans | Larynx | Lymph Nodes | Male | Neoplasm Metastasis | Pharynx | Prospective Studies | Ultrasonography [SUMMARY]
|
[CONTENT] Female | Head and Neck Neoplasms | Humans | Larynx | Lymph Nodes | Male | Neoplasm Metastasis | Pharynx | Prospective Studies | Ultrasonography [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] lymph | neck | patients | nodes | lymph nodes | node | lymph node | anti | tumor | laryngeal [SUMMARY]
| null |
[CONTENT] lymph | neck | patients | nodes | lymph nodes | node | lymph node | anti | tumor | laryngeal [SUMMARY]
|
[CONTENT] lymph | neck | patients | nodes | lymph nodes | node | lymph node | anti | tumor | laryngeal [SUMMARY]
|
[CONTENT] lymph | neck | patients | nodes | lymph nodes | node | lymph node | anti | tumor | laryngeal [SUMMARY]
|
[CONTENT] lymph | neck | patients | nodes | lymph nodes | node | lymph node | anti | tumor | laryngeal [SUMMARY]
|
[CONTENT] cancer | neck | lymph | pharynx | decision | diagnosed | 000 | surgical | head neck | pharynx larynx [SUMMARY]
| null |
[CONTENT] lymph | antibody | anti | lymph nodes | nodes | hypodensity | differentiated | reaction | patients | features [SUMMARY]
|
[CONTENT] involvement | lymph node involvement | node involvement | neck | method | node | lymph | lymph node | n0 pharyngo laryngeal | palpation inaccurate method detection [SUMMARY]
|
[CONTENT] lymph | neck | patients | nodes | node | lymph nodes | lymph node | declare conflict interests | declare | interests [SUMMARY]
|
[CONTENT] lymph | neck | patients | nodes | node | lymph nodes | lymph node | declare conflict interests | declare | interests [SUMMARY]
|
[CONTENT] ||| ||| TNM ||| N0 [SUMMARY]
| null |
[CONTENT] US | 25 | 80.6% ||| Ten | 100% | US ||| US | second | CO2 | four | 10 ||| US | 100% [SUMMARY]
|
[CONTENT] US [SUMMARY]
|
[CONTENT] ||| ||| TNM ||| N0 ||| Forty-six | N0 ||| two | 23 | second | 23 ||| larynx | US ||| ||| ||| US ||| US ||| two | node ||| US | 25 | 80.6% ||| Ten | 100% | US ||| US | second | CO2 | four | 10 ||| US | 100% ||| US [SUMMARY]
|
[CONTENT] ||| ||| TNM ||| N0 ||| Forty-six | N0 ||| two | 23 | second | 23 ||| larynx | US ||| ||| ||| US ||| US ||| two | node ||| US | 25 | 80.6% ||| Ten | 100% | US ||| US | second | CO2 | four | 10 ||| US | 100% ||| US [SUMMARY]
|
|||||
Factors associated with severe occupational injuries at mining company in Zimbabwe, 2010: a cross-sectional study.
|
23504270
|
Injury rate among mining workers in Zimbabwe was 789/1000 workers in 2008. The proportion of severe occupational injuries increased from 18% in 2008 to 37% in 2009. We investigated factors associated with severe injuries at the mine.
|
INTRODUCTION
|
An unmatched 1:1 case-control study was carried out at the mine, a case was any worker who suffered severe occupational injury at the mine and was treated at the mine or district hospital from January 2008 to April 2010, a control was any worker who did not suffer occupational injury during same period. We randomly selected 156 cases and 156 controls and used interviewer administered questionnaires to collect data from participants.
|
METHODS
|
Majority of cases, 155(99.4%) and of controls 142(91%) were male, 127(81.4%) of cases and 48(30.8%) of controls worked underground. Majority (73.1%) of severe occupational injuries occurred during night shift. Underground temperatures reached 500C. Factors independently associated with getting severe occupational injuries included working underground (AOR=10.55; CI 5.97-18.65), having targets per shift (AOR=12.60; CI 3.46-45.84), inadequate PPE (AOR=3.65 CI 1.34-9.89) and working more than 8 hours per shift (AOR=8.65 CI 2.99-25.02).
|
RESULTS
|
Having targets exerts pressure to perform on workers. Prolonged working periods decrease workers' attention and concentration resulting in increased risk to severe injuries as workers become exhausted, lose focus and alertness. Underground work environment had environmental hazards so managers to install adequate ventilation and provide adequate PPE. Management agreed to standardize shifts to eight hours and workers in some departments have been supplied with adequate PPE.
|
CONCLUSION
|
[
"Adult",
"Case-Control Studies",
"Cross-Sectional Studies",
"Fatigue",
"Female",
"Goals",
"Humans",
"Job Satisfaction",
"Male",
"Middle Aged",
"Mining",
"Morbidity",
"Occupational Diseases",
"Occupational Injuries",
"Protective Devices",
"Risk Factors",
"Sampling Studies",
"Surveys and Questionnaires",
"Temperature",
"Ventilation",
"Work Schedule Tolerance",
"Workplace",
"Young Adult",
"Zimbabwe"
] |
3597909
|
Introduction
|
Occupational injuries present a major public health problem resulting in serious social and economic consequences that could be prevented if appropriate measures are taken. Majority of world′s workforce does not have access to occupational health services [1]. Estimated economic loss caused by work-related injuries and disease is equivalent to 4% of the world′s gross national product [2]. The impact is 10 to 20 times higher in developing countries [3]. According to Leigh, 100 million occupational injuries occur throughout the world each year [4].
National Institute of Occupational Safety and Health (NIOSH) USA estimates that at least 10,000,000 persons suffer injuries on job each year. About 30% of these injuries are severe. In Zimbabwe occupational injuries are among the top ten health priorities [5]. The highest numbers of occupational injuries in Zimbabwe occur in the construction, mining, and manufacturing industries. The injury rate among mining workers in Zimbabwe was 131 per 1000 exposed workers per year as of 1998 [6]. This figure rose to 789/1000 workers in 2008 [7]. A survey of 1585 informal/small scale workers in rural and urban Zimbabwe found occupational injury and mortality rates similar to those found in large scale/formal sector [8].
The mining company under study is a multiple mineral extracting company which was established in 1906. Since then it has transformed into one of the country′s largest mining companies with more than 3 000 employees in one of their divisions who are exposed to continuous potential risk of occupational injuries. The mine operates underground and open cast mines.
The proportion of severe occupational injuries at the mine increased from 18% in 2008 to 37% in 2009. The proportion is very high as compared to the maximum reported by NIOSH of 30% and the 21.9% recommended by ILO [9]. Factors contributing to severe occupational injuries were not clear to mine management; therefore we investigated factors associated with severe occupational injuries at the mine. Specifically we assessed personal, administrative, engineering factors associated with occupational injuries and the availability and use of personal protective equipment (PPE) at the mine form 2008 to 2010.
|
Methods
|
A 1:1 unmatched case-control study was carried out at the mine, with all mine workers as our study population. A case was any worker who suffered a severe occupational injury at the mine and was treated at the mine hospital during the period January 2008-April 2010. A control was any worker who did not suffer an occupational injury during the same period. We included any mine worker who suffered severe occupational injury at the mine and was treated at the mine hospital or the district hospital from January 2008 to April 2010 but excluded any mine worker who suffered an occupational injury at the mine but was not treated at the mine hospital or the district hospital from January 2008 to April 2010.
Using StatCalc function of EPI Info Version 3.5.1 of 2008 and calculating the sample size at 95% confidence level, 80% power and expected occupational injury rate of 25% among mine workers and using the variable of not having received pre-employment training, OR = 2.04; CI = 1.50-2.77, [10] a minimum sample size of 156 cases and 156 controls was calculated. A sampling frame was prepared from cases from the district hospital outpatients register and occupational injuries register at the mine. From the sampling frame, cases were selected through the process of simple random selection. Random numbers were generated using random number function of a scientific calculator and cases with corresponding numbers were selected into the study. Controls were randomly selected from the workers of the mine through the same procedure as for cases and using the employee register as the sampling frame.
A conceptual Framework PRECEDE and PROCEED Model was used to design the questionnaire to assess factors associated with severe occupational injuries at the mine. The questionnaire was developed using constructs in the framework which include: Predisposing, Enabling and Reinforcing factors. Two focus group discussions one with twenty eight people and the other with thirty three people were held to identify thematic areas which helped in designing the questionnaire for study participants. A pre-tested interviewer administered questionnaire was used to collect information from study participants. The questionnaire was pre tested at a different division of the mining company for validity. Medical records were reviewed to assess case classification and management.
A check list was used to assess PPE availability and use, availability of occupational safety policies and regulations, safety warning signs and maintenance of equipment. Mine management and occupational health officer were interviewed as key informants for information on administrative and engineering factors.
Actual measurement of temperatures, illumination and noise levels was performed to assess environmental stresses. Statistical analysis of quantitative data was done using Epi info 3.5.1 Permission to carry out research was obtained from Mine Management, Provincial Medical Director Midlands Province, and Health Studies Office. Informed written consent was obtained from participants and confidentiality was maintained throughout the study. Ethical clearance was obtained from Medical Research Council of Zimbabwe.
|
Results
|
We enrolled 156 cases and 156 controls for the study. The majority of study participants 155(99.4%) of cases and 142(91%) of controls were male, 127(81.4%) of cases and 48(30.8%) of controls worked underground and 127(81.4%) of cases and 17(11%) of controls had a maximum of primary school education. The median years in service was 13 (Q1 = 5 Q3 = 21) for cases and19 (Q1 = 11 Q3 = 26.5) for controls and median age was 37 (Q1 = 28 Q3 = 47) for cases and 41 (Q1 = 35 Q3 = 49.5) for controls (Table 1).
Demographic Characteristics of Cases and Controls at a Mining Company, 2010
The majority of cases 111 (71.1%) had severe injuries occurring on the arms, followed by leg injuries at 26.3% (41 workers), injuries on the head accounted for 9% (14 workers) and injuries on the trunk only contributed 0.6% (1worker) of all severe injuries. The majority, 114 (73.1%) of severe occupational injuries occurred while workers were on night shift.
On analysis of personal risk factors, we found that male miners were 15.3 times more likely to be severely injured than female miners; mine workers who had sleep problems were 9 times more likely to get severely injured than those who did not have and those who reported dissatisfaction with their work were 20 times more likely to get severely injured than those who were satisfied with their jobs.
Workers who worked more than eight hours per shift were 14.5 times more likely to get severely injured than those who worked eight or less hours per shift, those who had targets to meet had a 43.4 times more risk of getting severely injured than those who did not have targets set for them. Workers who were not trained in the use of the equipment they were using were 5.2 times more likely to get severely injured than those who had prior training. Those who worked underground were 9.8 times more likely to get severely injured than those who worked on the surface. On engineering factors, we found that mine workers who were using manual equipment were 10.2 times more likely to get severe injuries than those who used automatic equipment; those who reported that their equipment did not suit their needs were 18.6 times more likely to get severe occupational injuries than those who fitted well with their machines/equipment. On personal protective equipment (PPE) factors, we found that workers who had not been trained in correct use of their PPE were 2.8 times more likely to get severe occupational injuries than those who were trained, those who did not use PPE correctly were 3.8 times more likely to get severely injured than those who used it correctly and those who did not consistently use PPE while at work were 7.5 times more likely to be severely injured than those who consistently used PPE.
On multivariate analysis using stepwise logistic regression, working underground (AOR = 10.55; CI 5.97-18.65), having targets to achieve per work shift (AOR = 12.60; CI 3.46-45.84), inadequate PPE (AOR= 3.65; CI 1.34-9.89), working more than 8 hours per shift (AOR = 8.65; CI 2.99-25.02), using automatic equipment (AOR = 0.33; CI 0.13-0.81) and having at least secondary education (AOR = 0.08; CI 0.03-0.18) remained independently associated with experiencing severe occupational injuries at the mine (Table 2).
Factors Associated with Severe Occupational Injuries at a Mining Company in Zimbabwe, 2010
In all surface departments, Occupational Health and Safety policies were available and displayed in many places where workers could easily see and read. The same applied to danger warning signs. But steep slopes had no side rails and there were no guards on cutting edges and other sharp equipment except for the plant department. Ambient temperatures ranged from 29 to 29.8 degrees Celsius. The normal temperature for the day was 290C, noise levels were above recommended amounts of 85 dBA, ranging from 95 to 108 decibels, and illumination ranged from150 to 460 lux.
Underground, occupational health and safety policies were available at three workstations out of the six visited stations. There were no danger warning signs, no side rails to protect steep and slippery walkways and sharp edges were not guarded along all tunnels. Illumination was only provided by miners′ torches. Noise levels were from 150 up to 400 decibels during blasting and temperature was above 50°C in work areas where most of the activity was going on. The normal atmospheric temperature for the day was 28.6°C. Table 3 shows the environmental stressors and measurements observed against the threshold limit values (TLV).
Threshold Limit Values for Environmental Stressors and Measurements Observed at the Mine, 2010
|
Conclusion
|
Factors independently associated with getting severely injured were working underground, having inadequate PPE, having targets to achieve per work shift and working more than 8 hours per shift. Underground work environment was not conducive to safe work performance due to high temperatures, unguarded steep slopes and unavailability of danger warning signs.
We recommended that management should set achievable targets for workers and offer performance awards to motivate production. Management should rotate workers through departments to lessen the period a workers are exposed to the risky underground workstation, provide adequate PPE and install adequate fans to ventilate underground work areas. The administrative controls to prevent occupational injuries should be strengthened so that workers adhere to the OSH policy and appropriate PPE be given as a last resort. In the long term, management should invest in engineering controls. Management should institute research to evaluate economic losses due to occupational injuries and Public Health Officers should investigate the injury investigation procedure at the mine to find ways of making it worker friendly and workers’ representative and mine managers to encourage workers to report accidents. Public Health Actions taken so far include: the management has agreed to standardize work shifts to 8 hours, to supply every worker with adequate PPE and workers in the loading bay and the plant had already been given their stipulated PPE in adequate amounts.
|
[
"Introduction"
] |
[
"Occupational injuries present a major public health problem resulting in serious social and economic consequences that could be prevented if appropriate measures are taken. Majority of world′s workforce does not have access to occupational health services [1]. Estimated economic loss caused by work-related injuries and disease is equivalent to 4% of the world′s gross national product [2]. The impact is 10 to 20 times higher in developing countries [3]. According to Leigh, 100 million occupational injuries occur throughout the world each year [4].\nNational Institute of Occupational Safety and Health (NIOSH) USA estimates that at least 10,000,000 persons suffer injuries on job each year. About 30% of these injuries are severe. In Zimbabwe occupational injuries are among the top ten health priorities [5]. The highest numbers of occupational injuries in Zimbabwe occur in the construction, mining, and manufacturing industries. The injury rate among mining workers in Zimbabwe was 131 per 1000 exposed workers per year as of 1998 [6]. This figure rose to 789/1000 workers in 2008 [7]. A survey of 1585 informal/small scale workers in rural and urban Zimbabwe found occupational injury and mortality rates similar to those found in large scale/formal sector [8].\nThe mining company under study is a multiple mineral extracting company which was established in 1906. Since then it has transformed into one of the country′s largest mining companies with more than 3 000 employees in one of their divisions who are exposed to continuous potential risk of occupational injuries. The mine operates underground and open cast mines.\nThe proportion of severe occupational injuries at the mine increased from 18% in 2008 to 37% in 2009. The proportion is very high as compared to the maximum reported by NIOSH of 30% and the 21.9% recommended by ILO [9]. Factors contributing to severe occupational injuries were not clear to mine management; therefore we investigated factors associated with severe occupational injuries at the mine. Specifically we assessed personal, administrative, engineering factors associated with occupational injuries and the availability and use of personal protective equipment (PPE) at the mine form 2008 to 2010."
] |
[
null
] |
[
"Introduction",
"Methods",
"Results",
"Discussion",
"Conclusion"
] |
[
"Occupational injuries present a major public health problem resulting in serious social and economic consequences that could be prevented if appropriate measures are taken. Majority of world′s workforce does not have access to occupational health services [1]. Estimated economic loss caused by work-related injuries and disease is equivalent to 4% of the world′s gross national product [2]. The impact is 10 to 20 times higher in developing countries [3]. According to Leigh, 100 million occupational injuries occur throughout the world each year [4].\nNational Institute of Occupational Safety and Health (NIOSH) USA estimates that at least 10,000,000 persons suffer injuries on job each year. About 30% of these injuries are severe. In Zimbabwe occupational injuries are among the top ten health priorities [5]. The highest numbers of occupational injuries in Zimbabwe occur in the construction, mining, and manufacturing industries. The injury rate among mining workers in Zimbabwe was 131 per 1000 exposed workers per year as of 1998 [6]. This figure rose to 789/1000 workers in 2008 [7]. A survey of 1585 informal/small scale workers in rural and urban Zimbabwe found occupational injury and mortality rates similar to those found in large scale/formal sector [8].\nThe mining company under study is a multiple mineral extracting company which was established in 1906. Since then it has transformed into one of the country′s largest mining companies with more than 3 000 employees in one of their divisions who are exposed to continuous potential risk of occupational injuries. The mine operates underground and open cast mines.\nThe proportion of severe occupational injuries at the mine increased from 18% in 2008 to 37% in 2009. The proportion is very high as compared to the maximum reported by NIOSH of 30% and the 21.9% recommended by ILO [9]. Factors contributing to severe occupational injuries were not clear to mine management; therefore we investigated factors associated with severe occupational injuries at the mine. Specifically we assessed personal, administrative, engineering factors associated with occupational injuries and the availability and use of personal protective equipment (PPE) at the mine form 2008 to 2010.",
"A 1:1 unmatched case-control study was carried out at the mine, with all mine workers as our study population. A case was any worker who suffered a severe occupational injury at the mine and was treated at the mine hospital during the period January 2008-April 2010. A control was any worker who did not suffer an occupational injury during the same period. We included any mine worker who suffered severe occupational injury at the mine and was treated at the mine hospital or the district hospital from January 2008 to April 2010 but excluded any mine worker who suffered an occupational injury at the mine but was not treated at the mine hospital or the district hospital from January 2008 to April 2010.\nUsing StatCalc function of EPI Info Version 3.5.1 of 2008 and calculating the sample size at 95% confidence level, 80% power and expected occupational injury rate of 25% among mine workers and using the variable of not having received pre-employment training, OR = 2.04; CI = 1.50-2.77, [10] a minimum sample size of 156 cases and 156 controls was calculated. A sampling frame was prepared from cases from the district hospital outpatients register and occupational injuries register at the mine. From the sampling frame, cases were selected through the process of simple random selection. Random numbers were generated using random number function of a scientific calculator and cases with corresponding numbers were selected into the study. Controls were randomly selected from the workers of the mine through the same procedure as for cases and using the employee register as the sampling frame.\nA conceptual Framework PRECEDE and PROCEED Model was used to design the questionnaire to assess factors associated with severe occupational injuries at the mine. The questionnaire was developed using constructs in the framework which include: Predisposing, Enabling and Reinforcing factors. Two focus group discussions one with twenty eight people and the other with thirty three people were held to identify thematic areas which helped in designing the questionnaire for study participants. A pre-tested interviewer administered questionnaire was used to collect information from study participants. The questionnaire was pre tested at a different division of the mining company for validity. Medical records were reviewed to assess case classification and management.\nA check list was used to assess PPE availability and use, availability of occupational safety policies and regulations, safety warning signs and maintenance of equipment. Mine management and occupational health officer were interviewed as key informants for information on administrative and engineering factors.\nActual measurement of temperatures, illumination and noise levels was performed to assess environmental stresses. Statistical analysis of quantitative data was done using Epi info 3.5.1 Permission to carry out research was obtained from Mine Management, Provincial Medical Director Midlands Province, and Health Studies Office. Informed written consent was obtained from participants and confidentiality was maintained throughout the study. Ethical clearance was obtained from Medical Research Council of Zimbabwe.",
"We enrolled 156 cases and 156 controls for the study. The majority of study participants 155(99.4%) of cases and 142(91%) of controls were male, 127(81.4%) of cases and 48(30.8%) of controls worked underground and 127(81.4%) of cases and 17(11%) of controls had a maximum of primary school education. The median years in service was 13 (Q1 = 5 Q3 = 21) for cases and19 (Q1 = 11 Q3 = 26.5) for controls and median age was 37 (Q1 = 28 Q3 = 47) for cases and 41 (Q1 = 35 Q3 = 49.5) for controls (Table 1).\n\nDemographic Characteristics of Cases and Controls at a Mining Company, 2010\nThe majority of cases 111 (71.1%) had severe injuries occurring on the arms, followed by leg injuries at 26.3% (41 workers), injuries on the head accounted for 9% (14 workers) and injuries on the trunk only contributed 0.6% (1worker) of all severe injuries. The majority, 114 (73.1%) of severe occupational injuries occurred while workers were on night shift.\nOn analysis of personal risk factors, we found that male miners were 15.3 times more likely to be severely injured than female miners; mine workers who had sleep problems were 9 times more likely to get severely injured than those who did not have and those who reported dissatisfaction with their work were 20 times more likely to get severely injured than those who were satisfied with their jobs.\nWorkers who worked more than eight hours per shift were 14.5 times more likely to get severely injured than those who worked eight or less hours per shift, those who had targets to meet had a 43.4 times more risk of getting severely injured than those who did not have targets set for them. Workers who were not trained in the use of the equipment they were using were 5.2 times more likely to get severely injured than those who had prior training. Those who worked underground were 9.8 times more likely to get severely injured than those who worked on the surface. On engineering factors, we found that mine workers who were using manual equipment were 10.2 times more likely to get severe injuries than those who used automatic equipment; those who reported that their equipment did not suit their needs were 18.6 times more likely to get severe occupational injuries than those who fitted well with their machines/equipment. On personal protective equipment (PPE) factors, we found that workers who had not been trained in correct use of their PPE were 2.8 times more likely to get severe occupational injuries than those who were trained, those who did not use PPE correctly were 3.8 times more likely to get severely injured than those who used it correctly and those who did not consistently use PPE while at work were 7.5 times more likely to be severely injured than those who consistently used PPE.\nOn multivariate analysis using stepwise logistic regression, working underground (AOR = 10.55; CI 5.97-18.65), having targets to achieve per work shift (AOR = 12.60; CI 3.46-45.84), inadequate PPE (AOR= 3.65; CI 1.34-9.89), working more than 8 hours per shift (AOR = 8.65; CI 2.99-25.02), using automatic equipment (AOR = 0.33; CI 0.13-0.81) and having at least secondary education (AOR = 0.08; CI 0.03-0.18) remained independently associated with experiencing severe occupational injuries at the mine (Table 2).\n\nFactors Associated with Severe Occupational Injuries at a Mining Company in Zimbabwe, 2010\nIn all surface departments, Occupational Health and Safety policies were available and displayed in many places where workers could easily see and read. The same applied to danger warning signs. But steep slopes had no side rails and there were no guards on cutting edges and other sharp equipment except for the plant department. Ambient temperatures ranged from 29 to 29.8 degrees Celsius. The normal temperature for the day was 290C, noise levels were above recommended amounts of 85 dBA, ranging from 95 to 108 decibels, and illumination ranged from150 to 460 lux.\nUnderground, occupational health and safety policies were available at three workstations out of the six visited stations. There were no danger warning signs, no side rails to protect steep and slippery walkways and sharp edges were not guarded along all tunnels. Illumination was only provided by miners′ torches. Noise levels were from 150 up to 400 decibels during blasting and temperature was above 50°C in work areas where most of the activity was going on. The normal atmospheric temperature for the day was 28.6°C. Table 3 shows the environmental stressors and measurements observed against the threshold limit values (TLV).\n\nThreshold Limit Values for Environmental Stressors and Measurements Observed at the Mine, 2010",
"Having targets to achieve per shift was found to be associated with getting severely injured at the mine under study. Having targets set for a work shift exerts pressure to perform on workers. Targets make workers to focus on production and meeting the targets so much that they may disregard precautionary measures and put themselves at risk of being injured in the process. As production corresponds to remuneration, every effort would be directed towards achieving the targets so that workers get more money and short cuts may be taken, endangering workers. Findings from a study of transient risk factors for acute traumatic hand injury in China are consistent with our findings. They found that workers who were rushed to achieve targets were 15.5 times more likely to get injured than those who were not [11].\nWorking underground was significantly associated with getting severe occupational injuries. High temperatures and inadequate illumination negatively affects workers′ alertness leading to reduced concentration levels thereby increasing workers′ risk to severe occupational injuries. Lack of danger warning signs which keep reminding workers of areas which need extra precautions, steep slopes and slippery surfaces which had no side rails to support workers and prevent falls and sharp edges with no guards to prevent cuts, all contributed to making the underground work station a risky work area. Results of a study done in USA showed that occupational injury rate in underground mines was twice that of all other mining departments put together [12]. But contrary to our findings, a study by Hull et al showed that there was no difference in risk of occupational injuries by mine department [13].\nProlonged continuous working periods decrease workers′ attention and concentration levels resulting in increased risk to severe injuries.\nWorkers who work longer hours become very exhausted and lose focus and alertness due to tiredness. Results of a study done in USA on the impact of overtime and long working hours on occupational injuries and illnesses were consistent with our findings [14]. It was shown that working for more than 8 hours per shift resulted in a 61% increased risk for occupational injuries and that a strong dose response effect was observed where the longer the work shift resulted in increase in the risk of injuries [14].\nInadequate personal protective equipment and clothing was also significantly associated with getting severely injured at the mine. PPE puts a barrier between the worker and the hazard thereby protecting the worker from injury. Mining work is potentially risky and for a worker to work without adequate protective gear, is taking unnecessary risk. Our finding is consistent with findings from a study done in USA where results showed that 79% of the workers who got silicosis were putting on inadequate PPE in form of respirators [15].\nOur study was not without limitations, fatalities were excluded from the study yet they could have been valuable information which could have brought in the healthy worker effect in our results. Privacy was limited as workers were interviewed at their work places and we did not have adequate time with workers as they wanted to achieve their targets.",
"Factors independently associated with getting severely injured were working underground, having inadequate PPE, having targets to achieve per work shift and working more than 8 hours per shift. Underground work environment was not conducive to safe work performance due to high temperatures, unguarded steep slopes and unavailability of danger warning signs.\nWe recommended that management should set achievable targets for workers and offer performance awards to motivate production. Management should rotate workers through departments to lessen the period a workers are exposed to the risky underground workstation, provide adequate PPE and install adequate fans to ventilate underground work areas. The administrative controls to prevent occupational injuries should be strengthened so that workers adhere to the OSH policy and appropriate PPE be given as a last resort. In the long term, management should invest in engineering controls. Management should institute research to evaluate economic losses due to occupational injuries and Public Health Officers should investigate the injury investigation procedure at the mine to find ways of making it worker friendly and workers’ representative and mine managers to encourage workers to report accidents. Public Health Actions taken so far include: the management has agreed to standardize work shifts to 8 hours, to supply every worker with adequate PPE and workers in the loading bay and the plant had already been given their stipulated PPE in adequate amounts."
] |
[
null,
"methods",
"results",
"discussion",
"conclusion"
] |
[
"Severe occupational injuries",
"mine workers",
"Zimbabwe"
] |
Introduction:
Occupational injuries present a major public health problem resulting in serious social and economic consequences that could be prevented if appropriate measures are taken. Majority of world′s workforce does not have access to occupational health services [1]. Estimated economic loss caused by work-related injuries and disease is equivalent to 4% of the world′s gross national product [2]. The impact is 10 to 20 times higher in developing countries [3]. According to Leigh, 100 million occupational injuries occur throughout the world each year [4].
National Institute of Occupational Safety and Health (NIOSH) USA estimates that at least 10,000,000 persons suffer injuries on job each year. About 30% of these injuries are severe. In Zimbabwe occupational injuries are among the top ten health priorities [5]. The highest numbers of occupational injuries in Zimbabwe occur in the construction, mining, and manufacturing industries. The injury rate among mining workers in Zimbabwe was 131 per 1000 exposed workers per year as of 1998 [6]. This figure rose to 789/1000 workers in 2008 [7]. A survey of 1585 informal/small scale workers in rural and urban Zimbabwe found occupational injury and mortality rates similar to those found in large scale/formal sector [8].
The mining company under study is a multiple mineral extracting company which was established in 1906. Since then it has transformed into one of the country′s largest mining companies with more than 3 000 employees in one of their divisions who are exposed to continuous potential risk of occupational injuries. The mine operates underground and open cast mines.
The proportion of severe occupational injuries at the mine increased from 18% in 2008 to 37% in 2009. The proportion is very high as compared to the maximum reported by NIOSH of 30% and the 21.9% recommended by ILO [9]. Factors contributing to severe occupational injuries were not clear to mine management; therefore we investigated factors associated with severe occupational injuries at the mine. Specifically we assessed personal, administrative, engineering factors associated with occupational injuries and the availability and use of personal protective equipment (PPE) at the mine form 2008 to 2010.
Methods:
A 1:1 unmatched case-control study was carried out at the mine, with all mine workers as our study population. A case was any worker who suffered a severe occupational injury at the mine and was treated at the mine hospital during the period January 2008-April 2010. A control was any worker who did not suffer an occupational injury during the same period. We included any mine worker who suffered severe occupational injury at the mine and was treated at the mine hospital or the district hospital from January 2008 to April 2010 but excluded any mine worker who suffered an occupational injury at the mine but was not treated at the mine hospital or the district hospital from January 2008 to April 2010.
Using StatCalc function of EPI Info Version 3.5.1 of 2008 and calculating the sample size at 95% confidence level, 80% power and expected occupational injury rate of 25% among mine workers and using the variable of not having received pre-employment training, OR = 2.04; CI = 1.50-2.77, [10] a minimum sample size of 156 cases and 156 controls was calculated. A sampling frame was prepared from cases from the district hospital outpatients register and occupational injuries register at the mine. From the sampling frame, cases were selected through the process of simple random selection. Random numbers were generated using random number function of a scientific calculator and cases with corresponding numbers were selected into the study. Controls were randomly selected from the workers of the mine through the same procedure as for cases and using the employee register as the sampling frame.
A conceptual Framework PRECEDE and PROCEED Model was used to design the questionnaire to assess factors associated with severe occupational injuries at the mine. The questionnaire was developed using constructs in the framework which include: Predisposing, Enabling and Reinforcing factors. Two focus group discussions one with twenty eight people and the other with thirty three people were held to identify thematic areas which helped in designing the questionnaire for study participants. A pre-tested interviewer administered questionnaire was used to collect information from study participants. The questionnaire was pre tested at a different division of the mining company for validity. Medical records were reviewed to assess case classification and management.
A check list was used to assess PPE availability and use, availability of occupational safety policies and regulations, safety warning signs and maintenance of equipment. Mine management and occupational health officer were interviewed as key informants for information on administrative and engineering factors.
Actual measurement of temperatures, illumination and noise levels was performed to assess environmental stresses. Statistical analysis of quantitative data was done using Epi info 3.5.1 Permission to carry out research was obtained from Mine Management, Provincial Medical Director Midlands Province, and Health Studies Office. Informed written consent was obtained from participants and confidentiality was maintained throughout the study. Ethical clearance was obtained from Medical Research Council of Zimbabwe.
Results:
We enrolled 156 cases and 156 controls for the study. The majority of study participants 155(99.4%) of cases and 142(91%) of controls were male, 127(81.4%) of cases and 48(30.8%) of controls worked underground and 127(81.4%) of cases and 17(11%) of controls had a maximum of primary school education. The median years in service was 13 (Q1 = 5 Q3 = 21) for cases and19 (Q1 = 11 Q3 = 26.5) for controls and median age was 37 (Q1 = 28 Q3 = 47) for cases and 41 (Q1 = 35 Q3 = 49.5) for controls (Table 1).
Demographic Characteristics of Cases and Controls at a Mining Company, 2010
The majority of cases 111 (71.1%) had severe injuries occurring on the arms, followed by leg injuries at 26.3% (41 workers), injuries on the head accounted for 9% (14 workers) and injuries on the trunk only contributed 0.6% (1worker) of all severe injuries. The majority, 114 (73.1%) of severe occupational injuries occurred while workers were on night shift.
On analysis of personal risk factors, we found that male miners were 15.3 times more likely to be severely injured than female miners; mine workers who had sleep problems were 9 times more likely to get severely injured than those who did not have and those who reported dissatisfaction with their work were 20 times more likely to get severely injured than those who were satisfied with their jobs.
Workers who worked more than eight hours per shift were 14.5 times more likely to get severely injured than those who worked eight or less hours per shift, those who had targets to meet had a 43.4 times more risk of getting severely injured than those who did not have targets set for them. Workers who were not trained in the use of the equipment they were using were 5.2 times more likely to get severely injured than those who had prior training. Those who worked underground were 9.8 times more likely to get severely injured than those who worked on the surface. On engineering factors, we found that mine workers who were using manual equipment were 10.2 times more likely to get severe injuries than those who used automatic equipment; those who reported that their equipment did not suit their needs were 18.6 times more likely to get severe occupational injuries than those who fitted well with their machines/equipment. On personal protective equipment (PPE) factors, we found that workers who had not been trained in correct use of their PPE were 2.8 times more likely to get severe occupational injuries than those who were trained, those who did not use PPE correctly were 3.8 times more likely to get severely injured than those who used it correctly and those who did not consistently use PPE while at work were 7.5 times more likely to be severely injured than those who consistently used PPE.
On multivariate analysis using stepwise logistic regression, working underground (AOR = 10.55; CI 5.97-18.65), having targets to achieve per work shift (AOR = 12.60; CI 3.46-45.84), inadequate PPE (AOR= 3.65; CI 1.34-9.89), working more than 8 hours per shift (AOR = 8.65; CI 2.99-25.02), using automatic equipment (AOR = 0.33; CI 0.13-0.81) and having at least secondary education (AOR = 0.08; CI 0.03-0.18) remained independently associated with experiencing severe occupational injuries at the mine (Table 2).
Factors Associated with Severe Occupational Injuries at a Mining Company in Zimbabwe, 2010
In all surface departments, Occupational Health and Safety policies were available and displayed in many places where workers could easily see and read. The same applied to danger warning signs. But steep slopes had no side rails and there were no guards on cutting edges and other sharp equipment except for the plant department. Ambient temperatures ranged from 29 to 29.8 degrees Celsius. The normal temperature for the day was 290C, noise levels were above recommended amounts of 85 dBA, ranging from 95 to 108 decibels, and illumination ranged from150 to 460 lux.
Underground, occupational health and safety policies were available at three workstations out of the six visited stations. There were no danger warning signs, no side rails to protect steep and slippery walkways and sharp edges were not guarded along all tunnels. Illumination was only provided by miners′ torches. Noise levels were from 150 up to 400 decibels during blasting and temperature was above 50°C in work areas where most of the activity was going on. The normal atmospheric temperature for the day was 28.6°C. Table 3 shows the environmental stressors and measurements observed against the threshold limit values (TLV).
Threshold Limit Values for Environmental Stressors and Measurements Observed at the Mine, 2010
Discussion:
Having targets to achieve per shift was found to be associated with getting severely injured at the mine under study. Having targets set for a work shift exerts pressure to perform on workers. Targets make workers to focus on production and meeting the targets so much that they may disregard precautionary measures and put themselves at risk of being injured in the process. As production corresponds to remuneration, every effort would be directed towards achieving the targets so that workers get more money and short cuts may be taken, endangering workers. Findings from a study of transient risk factors for acute traumatic hand injury in China are consistent with our findings. They found that workers who were rushed to achieve targets were 15.5 times more likely to get injured than those who were not [11].
Working underground was significantly associated with getting severe occupational injuries. High temperatures and inadequate illumination negatively affects workers′ alertness leading to reduced concentration levels thereby increasing workers′ risk to severe occupational injuries. Lack of danger warning signs which keep reminding workers of areas which need extra precautions, steep slopes and slippery surfaces which had no side rails to support workers and prevent falls and sharp edges with no guards to prevent cuts, all contributed to making the underground work station a risky work area. Results of a study done in USA showed that occupational injury rate in underground mines was twice that of all other mining departments put together [12]. But contrary to our findings, a study by Hull et al showed that there was no difference in risk of occupational injuries by mine department [13].
Prolonged continuous working periods decrease workers′ attention and concentration levels resulting in increased risk to severe injuries.
Workers who work longer hours become very exhausted and lose focus and alertness due to tiredness. Results of a study done in USA on the impact of overtime and long working hours on occupational injuries and illnesses were consistent with our findings [14]. It was shown that working for more than 8 hours per shift resulted in a 61% increased risk for occupational injuries and that a strong dose response effect was observed where the longer the work shift resulted in increase in the risk of injuries [14].
Inadequate personal protective equipment and clothing was also significantly associated with getting severely injured at the mine. PPE puts a barrier between the worker and the hazard thereby protecting the worker from injury. Mining work is potentially risky and for a worker to work without adequate protective gear, is taking unnecessary risk. Our finding is consistent with findings from a study done in USA where results showed that 79% of the workers who got silicosis were putting on inadequate PPE in form of respirators [15].
Our study was not without limitations, fatalities were excluded from the study yet they could have been valuable information which could have brought in the healthy worker effect in our results. Privacy was limited as workers were interviewed at their work places and we did not have adequate time with workers as they wanted to achieve their targets.
Conclusion:
Factors independently associated with getting severely injured were working underground, having inadequate PPE, having targets to achieve per work shift and working more than 8 hours per shift. Underground work environment was not conducive to safe work performance due to high temperatures, unguarded steep slopes and unavailability of danger warning signs.
We recommended that management should set achievable targets for workers and offer performance awards to motivate production. Management should rotate workers through departments to lessen the period a workers are exposed to the risky underground workstation, provide adequate PPE and install adequate fans to ventilate underground work areas. The administrative controls to prevent occupational injuries should be strengthened so that workers adhere to the OSH policy and appropriate PPE be given as a last resort. In the long term, management should invest in engineering controls. Management should institute research to evaluate economic losses due to occupational injuries and Public Health Officers should investigate the injury investigation procedure at the mine to find ways of making it worker friendly and workers’ representative and mine managers to encourage workers to report accidents. Public Health Actions taken so far include: the management has agreed to standardize work shifts to 8 hours, to supply every worker with adequate PPE and workers in the loading bay and the plant had already been given their stipulated PPE in adequate amounts.
|
Background:
Injury rate among mining workers in Zimbabwe was 789/1000 workers in 2008. The proportion of severe occupational injuries increased from 18% in 2008 to 37% in 2009. We investigated factors associated with severe injuries at the mine.
Methods:
An unmatched 1:1 case-control study was carried out at the mine, a case was any worker who suffered severe occupational injury at the mine and was treated at the mine or district hospital from January 2008 to April 2010, a control was any worker who did not suffer occupational injury during same period. We randomly selected 156 cases and 156 controls and used interviewer administered questionnaires to collect data from participants.
Results:
Majority of cases, 155(99.4%) and of controls 142(91%) were male, 127(81.4%) of cases and 48(30.8%) of controls worked underground. Majority (73.1%) of severe occupational injuries occurred during night shift. Underground temperatures reached 500C. Factors independently associated with getting severe occupational injuries included working underground (AOR=10.55; CI 5.97-18.65), having targets per shift (AOR=12.60; CI 3.46-45.84), inadequate PPE (AOR=3.65 CI 1.34-9.89) and working more than 8 hours per shift (AOR=8.65 CI 2.99-25.02).
Conclusions:
Having targets exerts pressure to perform on workers. Prolonged working periods decrease workers' attention and concentration resulting in increased risk to severe injuries as workers become exhausted, lose focus and alertness. Underground work environment had environmental hazards so managers to install adequate ventilation and provide adequate PPE. Management agreed to standardize shifts to eight hours and workers in some departments have been supplied with adequate PPE.
|
Introduction:
Occupational injuries present a major public health problem resulting in serious social and economic consequences that could be prevented if appropriate measures are taken. Majority of world′s workforce does not have access to occupational health services [1]. Estimated economic loss caused by work-related injuries and disease is equivalent to 4% of the world′s gross national product [2]. The impact is 10 to 20 times higher in developing countries [3]. According to Leigh, 100 million occupational injuries occur throughout the world each year [4].
National Institute of Occupational Safety and Health (NIOSH) USA estimates that at least 10,000,000 persons suffer injuries on job each year. About 30% of these injuries are severe. In Zimbabwe occupational injuries are among the top ten health priorities [5]. The highest numbers of occupational injuries in Zimbabwe occur in the construction, mining, and manufacturing industries. The injury rate among mining workers in Zimbabwe was 131 per 1000 exposed workers per year as of 1998 [6]. This figure rose to 789/1000 workers in 2008 [7]. A survey of 1585 informal/small scale workers in rural and urban Zimbabwe found occupational injury and mortality rates similar to those found in large scale/formal sector [8].
The mining company under study is a multiple mineral extracting company which was established in 1906. Since then it has transformed into one of the country′s largest mining companies with more than 3 000 employees in one of their divisions who are exposed to continuous potential risk of occupational injuries. The mine operates underground and open cast mines.
The proportion of severe occupational injuries at the mine increased from 18% in 2008 to 37% in 2009. The proportion is very high as compared to the maximum reported by NIOSH of 30% and the 21.9% recommended by ILO [9]. Factors contributing to severe occupational injuries were not clear to mine management; therefore we investigated factors associated with severe occupational injuries at the mine. Specifically we assessed personal, administrative, engineering factors associated with occupational injuries and the availability and use of personal protective equipment (PPE) at the mine form 2008 to 2010.
Conclusion:
Factors independently associated with getting severely injured were working underground, having inadequate PPE, having targets to achieve per work shift and working more than 8 hours per shift. Underground work environment was not conducive to safe work performance due to high temperatures, unguarded steep slopes and unavailability of danger warning signs.
We recommended that management should set achievable targets for workers and offer performance awards to motivate production. Management should rotate workers through departments to lessen the period a workers are exposed to the risky underground workstation, provide adequate PPE and install adequate fans to ventilate underground work areas. The administrative controls to prevent occupational injuries should be strengthened so that workers adhere to the OSH policy and appropriate PPE be given as a last resort. In the long term, management should invest in engineering controls. Management should institute research to evaluate economic losses due to occupational injuries and Public Health Officers should investigate the injury investigation procedure at the mine to find ways of making it worker friendly and workers’ representative and mine managers to encourage workers to report accidents. Public Health Actions taken so far include: the management has agreed to standardize work shifts to 8 hours, to supply every worker with adequate PPE and workers in the loading bay and the plant had already been given their stipulated PPE in adequate amounts.
|
Background:
Injury rate among mining workers in Zimbabwe was 789/1000 workers in 2008. The proportion of severe occupational injuries increased from 18% in 2008 to 37% in 2009. We investigated factors associated with severe injuries at the mine.
Methods:
An unmatched 1:1 case-control study was carried out at the mine, a case was any worker who suffered severe occupational injury at the mine and was treated at the mine or district hospital from January 2008 to April 2010, a control was any worker who did not suffer occupational injury during same period. We randomly selected 156 cases and 156 controls and used interviewer administered questionnaires to collect data from participants.
Results:
Majority of cases, 155(99.4%) and of controls 142(91%) were male, 127(81.4%) of cases and 48(30.8%) of controls worked underground. Majority (73.1%) of severe occupational injuries occurred during night shift. Underground temperatures reached 500C. Factors independently associated with getting severe occupational injuries included working underground (AOR=10.55; CI 5.97-18.65), having targets per shift (AOR=12.60; CI 3.46-45.84), inadequate PPE (AOR=3.65 CI 1.34-9.89) and working more than 8 hours per shift (AOR=8.65 CI 2.99-25.02).
Conclusions:
Having targets exerts pressure to perform on workers. Prolonged working periods decrease workers' attention and concentration resulting in increased risk to severe injuries as workers become exhausted, lose focus and alertness. Underground work environment had environmental hazards so managers to install adequate ventilation and provide adequate PPE. Management agreed to standardize shifts to eight hours and workers in some departments have been supplied with adequate PPE.
| 2,682 | 312 | 5 |
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"workers",
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[
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[CONTENT] Severe occupational injuries | mine workers | Zimbabwe [SUMMARY]
|
[CONTENT] Severe occupational injuries | mine workers | Zimbabwe [SUMMARY]
|
[CONTENT] Severe occupational injuries | mine workers | Zimbabwe [SUMMARY]
|
[CONTENT] Severe occupational injuries | mine workers | Zimbabwe [SUMMARY]
|
[CONTENT] Severe occupational injuries | mine workers | Zimbabwe [SUMMARY]
|
[CONTENT] Severe occupational injuries | mine workers | Zimbabwe [SUMMARY]
|
[CONTENT] Adult | Case-Control Studies | Cross-Sectional Studies | Fatigue | Female | Goals | Humans | Job Satisfaction | Male | Middle Aged | Mining | Morbidity | Occupational Diseases | Occupational Injuries | Protective Devices | Risk Factors | Sampling Studies | Surveys and Questionnaires | Temperature | Ventilation | Work Schedule Tolerance | Workplace | Young Adult | Zimbabwe [SUMMARY]
|
[CONTENT] Adult | Case-Control Studies | Cross-Sectional Studies | Fatigue | Female | Goals | Humans | Job Satisfaction | Male | Middle Aged | Mining | Morbidity | Occupational Diseases | Occupational Injuries | Protective Devices | Risk Factors | Sampling Studies | Surveys and Questionnaires | Temperature | Ventilation | Work Schedule Tolerance | Workplace | Young Adult | Zimbabwe [SUMMARY]
|
[CONTENT] Adult | Case-Control Studies | Cross-Sectional Studies | Fatigue | Female | Goals | Humans | Job Satisfaction | Male | Middle Aged | Mining | Morbidity | Occupational Diseases | Occupational Injuries | Protective Devices | Risk Factors | Sampling Studies | Surveys and Questionnaires | Temperature | Ventilation | Work Schedule Tolerance | Workplace | Young Adult | Zimbabwe [SUMMARY]
|
[CONTENT] Adult | Case-Control Studies | Cross-Sectional Studies | Fatigue | Female | Goals | Humans | Job Satisfaction | Male | Middle Aged | Mining | Morbidity | Occupational Diseases | Occupational Injuries | Protective Devices | Risk Factors | Sampling Studies | Surveys and Questionnaires | Temperature | Ventilation | Work Schedule Tolerance | Workplace | Young Adult | Zimbabwe [SUMMARY]
|
[CONTENT] Adult | Case-Control Studies | Cross-Sectional Studies | Fatigue | Female | Goals | Humans | Job Satisfaction | Male | Middle Aged | Mining | Morbidity | Occupational Diseases | Occupational Injuries | Protective Devices | Risk Factors | Sampling Studies | Surveys and Questionnaires | Temperature | Ventilation | Work Schedule Tolerance | Workplace | Young Adult | Zimbabwe [SUMMARY]
|
[CONTENT] Adult | Case-Control Studies | Cross-Sectional Studies | Fatigue | Female | Goals | Humans | Job Satisfaction | Male | Middle Aged | Mining | Morbidity | Occupational Diseases | Occupational Injuries | Protective Devices | Risk Factors | Sampling Studies | Surveys and Questionnaires | Temperature | Ventilation | Work Schedule Tolerance | Workplace | Young Adult | Zimbabwe [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] workers | occupational | injuries | occupational injuries | work | severe | study | ppe | injured | times [SUMMARY]
|
[CONTENT] workers | occupational | injuries | occupational injuries | work | severe | study | ppe | injured | times [SUMMARY]
|
[CONTENT] workers | occupational | injuries | occupational injuries | work | severe | study | ppe | injured | times [SUMMARY]
|
[CONTENT] workers | occupational | injuries | occupational injuries | work | severe | study | ppe | injured | times [SUMMARY]
|
[CONTENT] workers | occupational | injuries | occupational injuries | work | severe | study | ppe | injured | times [SUMMARY]
|
[CONTENT] workers | occupational | injuries | occupational injuries | work | severe | study | ppe | injured | times [SUMMARY]
|
[CONTENT] injuries | occupational | occupational injuries | 000 | year | world | zimbabwe | 2008 | health | severe [SUMMARY]
|
[CONTENT] hospital | questionnaire | occupational | cases | assess | study | occupational injury | 2008 | case | suffered [SUMMARY]
|
[CONTENT] likely | times likely | times | times likely severely injured | times likely severely | likely severely | likely severely injured | cases | severely | injured [SUMMARY]
|
[CONTENT] management | workers | adequate | work | ppe | underground | given | performance | adequate ppe | public [SUMMARY]
|
[CONTENT] workers | occupational | injuries | occupational injuries | work | severe | study | cases | targets | injured [SUMMARY]
|
[CONTENT] workers | occupational | injuries | occupational injuries | work | severe | study | cases | targets | injured [SUMMARY]
|
[CONTENT] Zimbabwe | 2008 ||| 18% | 2008 | 37% | 2009 ||| [SUMMARY]
|
[CONTENT] 1:1 | January 2008 to April 2010 ||| 156 | 156 [SUMMARY]
|
[CONTENT] 155(99.4% | 142(91% | 127(81.4% | 48(30.8% ||| 73.1% ||| 500C. | CI | 5.97-18.65 | CI 3.46-45.84 | PPE | CI | 1.34-9.89 | more than 8 hours | CI | 2.99 [SUMMARY]
|
[CONTENT] ||| ||| PPE ||| eight hours | PPE [SUMMARY]
|
[CONTENT] Zimbabwe | 2008 ||| 18% | 2008 | 37% | 2009 ||| ||| 1:1 | January 2008 to April 2010 ||| 156 | 156 ||| 155(99.4% | 142(91% | 127(81.4% | 48(30.8% ||| 73.1% ||| 500C. | CI | 5.97-18.65 | CI 3.46-45.84 | PPE | CI | 1.34-9.89 | more than 8 hours | CI | 2.99 ||| ||| ||| PPE ||| eight hours | PPE [SUMMARY]
|
[CONTENT] Zimbabwe | 2008 ||| 18% | 2008 | 37% | 2009 ||| ||| 1:1 | January 2008 to April 2010 ||| 156 | 156 ||| 155(99.4% | 142(91% | 127(81.4% | 48(30.8% ||| 73.1% ||| 500C. | CI | 5.97-18.65 | CI 3.46-45.84 | PPE | CI | 1.34-9.89 | more than 8 hours | CI | 2.99 ||| ||| ||| PPE ||| eight hours | PPE [SUMMARY]
|
||||||
Clinical manifestations, treatment options, and comorbidities in COVID-19 relapse patients: A systematic review.
|
35396748
|
Interest revolving around coronavirus disease 2019 (COVID-19) reinfection is escalating rapidly. By definition, reinfection denotes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), PCR redetection, and COVID-19 recurrence within three months of the initial symptoms. The main aim of the current systematic review was to evaluate the features of COVID-19 relapse patients.
|
INTRODUCTION
|
For this study, we used a string of terms developed by a skilled librarian and through a systematical search in PubMed, Web of Science, and Embase for eligible studies. Clinical surveys of any type were included from January 2019 to March 2021. Eligible studies consisted of two positive assessments separated by a negative result via RT-PCR.
|
MATERIALS AND METHODS
|
Fifty-four studies included 207 cases of COVID-19 reinfection. Children were less likely to have COVID-19 relapse. However, the most patients were in the age group of 20-40 years. Asthenia (66.6%), headache (66.6%), and cough (54.7%) were prevalent symptoms in the first SARS-CoV-2 infection. Asthenia (62.9%), myalgia (62.9%), and headache (61.1%) were most frequent in the second one. The most common treatment options used in first COVID-19 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second infection, mostly antibiotics (100%), dexamethasone (100%), and remdesivir (80%) were used. In addition, obesity (32.5%), kidney failure (30.7%), and hypertension (30.1%) were the most common comorbidities. Unfortunately, approximately 4.5% of patients died.
|
RESULTS
|
We found the potency of COVID-19 recurrence as an outstanding issue. This feature should be regarded in the COVID-19 management. Furthermore, the first and second COVID-19 are similar in clinical features. For clinically practical comparison of the symptoms severity between two epochs of infection, uniform data of both are required. We suggest that future studies undertake a homogenous approach to establish the clinical patterns of the reinfection phenomena.
|
CONCLUSION
|
[
"Adult",
"Asthenia",
"COVID-19",
"Child",
"Headache",
"Humans",
"Reinfection",
"SARS-CoV-2",
"Young Adult"
] |
9102618
|
INTRODUCTION
|
This is not the first time that coronavirus has caused problems in the world. Viruses such as severe acute respiratory syndrome coronavirus (SARS‐CoV) and Middle East respiratory syndrome coronavirus (MERS‐CoV) have also been prevalent in recent years.
1
The mortality rates of SARS‐CoV and MERS‐CoV epidemics have been estimated to be 10% and 35%, respectively.
2
The main problem of today's global health communities is conflict over the novel coronavirus (2019‐nCoV). The new virus was first identified in China, but is now found in many countries around the world.
1
There are currently several variants of coronavirus circulating among people,
3
,
4
and the virus is mostly transmitted through the respiratory tract. Various symptoms have been described for patients with COVID‐19, ranging from asymptomatic to severe. Kidney damage has also been reported in some cases.
5
According to the World Health Organization (WHO), COVID‐19‐infected patients can leave home quarantine after the improvement of their infectious symptoms and also the confirmation of two negative RT‐PCR tests (within 24 h).
6
Reinfection, relapse, recurrence, and reactivation are terms used for people infected with coronavirus and have become positive again after a period of negativity.
7
Based on a report from Guangdong Province in China, about 14% of patients who recover from COVID‐19 become reinfected with the virus.
8
In addition, there have been reports of reinfection in Korea and Japan.
9
Duration of immunization against coronavirus reinfection in recovering individuals is six months.
10
People who become infected with coronavirus for the second time often have milder symptoms and recover more quickly than those infected for the first time.
11
However, there are concerns about reinfection in people recovering from the coronavirus. The objective of this systematic review was to evaluate the prevalence and frequency of reinfection in people recovering from COVID‐19 and their clinical signs, as well as to assess the treatment methods.
| null | null |
RESULTS
|
Study characteristics The search strategy yielded 1807 studies from three databases. Following the removal of duplicates, the title and abstract of 998 studies were examined. Among these studies, 152 were selected for full‐text assessment, and other 846 studies were eliminated due to irrelevancy. In all the selected studies, the relapse of coronavirus infection after a negative RT‐PCR test was reported. Among the 152 full‐text studies examined, only 54 studies were found to be eligible for data extraction (Figure 1). The included studies were original articles (5.5%, N = 3), case reports (72.2%, N = 39), and case series (22.2%, N = 12). Likewise, in the included studies, RT‐PCR tests were performed to detect both the first and the second infections. Thirteen studies were originated from Europe, 11 from the USA, 9 from China, and 6 from Brazil. These articles reported a total number of 207 patients who developed the second infection of coronavirus after a recovery, which was confirmed by a negative RT‐PCR test. Forty‐six studies reported the clinical features in the first infection; however, seven articles declared no symptoms. Only one study unrecorded the clinical features in the first infection. In addition, 42 investigations implied the medication and intervention.
Flow diagram detailing review process and study selection
In the second phase of infection, 43 articles reported the clinical manifestations, seven articles stated no sign, and four articles did not list any symptoms. Also, 37 studies reported specifically the treatment of the secondary infection. To compare the severity of symptoms between two phases of infection, sufficient information of both is required. Only 41 investigations presented features for both periods of infection. From these 41 studies, information of 63 cases was identified as qualified for the comparison of manifestations. Moreover, the approximate interval between negative and second positive RT‐PCR was obtained from 49 studies. The length of this interval reflects the characteristics of COVID‐19 relapse.
During diagnosis, evaluation, and treatment, attention to comorbidities is necessary. Among included studies, 44 articles specified comorbidities. Of these observations, 11 studies did not found any notable underlying conditions or disorders. In this survey, we examined the outcome of the COVID‐19 relapse, which was categorized into discharge or death. The outcome was reported for a total number of 199 patients from 49 studies. The detailed information of is summarized in Table 1.
The search strategy yielded 1807 studies from three databases. Following the removal of duplicates, the title and abstract of 998 studies were examined. Among these studies, 152 were selected for full‐text assessment, and other 846 studies were eliminated due to irrelevancy. In all the selected studies, the relapse of coronavirus infection after a negative RT‐PCR test was reported. Among the 152 full‐text studies examined, only 54 studies were found to be eligible for data extraction (Figure 1). The included studies were original articles (5.5%, N = 3), case reports (72.2%, N = 39), and case series (22.2%, N = 12). Likewise, in the included studies, RT‐PCR tests were performed to detect both the first and the second infections. Thirteen studies were originated from Europe, 11 from the USA, 9 from China, and 6 from Brazil. These articles reported a total number of 207 patients who developed the second infection of coronavirus after a recovery, which was confirmed by a negative RT‐PCR test. Forty‐six studies reported the clinical features in the first infection; however, seven articles declared no symptoms. Only one study unrecorded the clinical features in the first infection. In addition, 42 investigations implied the medication and intervention.
Flow diagram detailing review process and study selection
In the second phase of infection, 43 articles reported the clinical manifestations, seven articles stated no sign, and four articles did not list any symptoms. Also, 37 studies reported specifically the treatment of the secondary infection. To compare the severity of symptoms between two phases of infection, sufficient information of both is required. Only 41 investigations presented features for both periods of infection. From these 41 studies, information of 63 cases was identified as qualified for the comparison of manifestations. Moreover, the approximate interval between negative and second positive RT‐PCR was obtained from 49 studies. The length of this interval reflects the characteristics of COVID‐19 relapse.
During diagnosis, evaluation, and treatment, attention to comorbidities is necessary. Among included studies, 44 articles specified comorbidities. Of these observations, 11 studies did not found any notable underlying conditions or disorders. In this survey, we examined the outcome of the COVID‐19 relapse, which was categorized into discharge or death. The outcome was reported for a total number of 199 patients from 49 studies. The detailed information of is summarized in Table 1.
Demographic and general information Considering the studies included, we reviewed 207 patients presented with secondary infection of COVID‐19 after a period of recovery. A negative RT‐PCR confirmed the recovery from the first phase of disease. Among the included articles, there were only three observational studies that reported 132 cases. Of all 207 cases, 122 (58.9%) patients were female, and 85 (41.1%) patients were male. As shown in Table 2, children were less likely to have a recurrence of COVID‐19. However, the most patients were in the age group of 20–40 years. The studies reported a wide range of 2–240 days between two coronavirus infections. We classified this duration into three groups: n ≤ 30, 30 < n < 90, and n ≥ 90. Thirty‐eight (77.5%) studies reported n ≤ 30 or 30 < n < 90 for the recovery duration, and only 11 (22.5%) investigations implied more than 90 days (Table 2).
Summary of the findings
Abbreviations: HIV, human immunodeficiency virus; n, number of patients with any variables; N, the total number of patients with COVID‐19; ND, no difference; No, number; RT‐PCR, reverse transcription‐polymerase chain reaction.
To compare the severity of the first infection with secondary, the reported features and symptoms were reviewed and extracted from the articles. Of 63 patients, 29 presented more severe manifestations, while 25 cases showed an ameliorated status, and 9 other cases indicated similar symptoms in both phases of the infection. Forty‐nine studies recorded the outcome of 199 patients, among whom 190 cases were discharged with an improved status, but 9 cases succumbed in hospital. Indeed, the survival rate is required to be taken into account when determining the potency of coronavirus reactivation.
Considering the studies included, we reviewed 207 patients presented with secondary infection of COVID‐19 after a period of recovery. A negative RT‐PCR confirmed the recovery from the first phase of disease. Among the included articles, there were only three observational studies that reported 132 cases. Of all 207 cases, 122 (58.9%) patients were female, and 85 (41.1%) patients were male. As shown in Table 2, children were less likely to have a recurrence of COVID‐19. However, the most patients were in the age group of 20–40 years. The studies reported a wide range of 2–240 days between two coronavirus infections. We classified this duration into three groups: n ≤ 30, 30 < n < 90, and n ≥ 90. Thirty‐eight (77.5%) studies reported n ≤ 30 or 30 < n < 90 for the recovery duration, and only 11 (22.5%) investigations implied more than 90 days (Table 2).
Summary of the findings
Abbreviations: HIV, human immunodeficiency virus; n, number of patients with any variables; N, the total number of patients with COVID‐19; ND, no difference; No, number; RT‐PCR, reverse transcription‐polymerase chain reaction.
To compare the severity of the first infection with secondary, the reported features and symptoms were reviewed and extracted from the articles. Of 63 patients, 29 presented more severe manifestations, while 25 cases showed an ameliorated status, and 9 other cases indicated similar symptoms in both phases of the infection. Forty‐nine studies recorded the outcome of 199 patients, among whom 190 cases were discharged with an improved status, but 9 cases succumbed in hospital. Indeed, the survival rate is required to be taken into account when determining the potency of coronavirus reactivation.
Clinical manifestations Some clinical signs were most frequent between the two infections, but prevalence was different. Moreover, the most prevalent clinical signs in the first infection were asthenia (66.6%), headache (66.6%), cough (54.7%), fever (52.8%), sore throat (50.0%), and respiratory distress (48.1%). However, in the second infection, asthenia (62.9%), myalgia (62.6%), headache (61.11%), sore throat (52.54%), and dyspnea (48.53%) were the common. Respiratory alkalosis (21.43%), drowsiness (11.11%), and night sweat (11.11%) occurred only in the first infection. The most frequent symptoms observed only in the second infection were as follows: asthenia (62.9%), myalgia (62.9%), and headache (61.1%). COVID‐19 recurrence was manifested with more mild signs in 39.6% of patients, while 46.0% presented with more severe signs, and other 14.3% did not show any prominent change between the two infections.
Some clinical signs were most frequent between the two infections, but prevalence was different. Moreover, the most prevalent clinical signs in the first infection were asthenia (66.6%), headache (66.6%), cough (54.7%), fever (52.8%), sore throat (50.0%), and respiratory distress (48.1%). However, in the second infection, asthenia (62.9%), myalgia (62.6%), headache (61.11%), sore throat (52.54%), and dyspnea (48.53%) were the common. Respiratory alkalosis (21.43%), drowsiness (11.11%), and night sweat (11.11%) occurred only in the first infection. The most frequent symptoms observed only in the second infection were as follows: asthenia (62.9%), myalgia (62.9%), and headache (61.1%). COVID‐19 recurrence was manifested with more mild signs in 39.6% of patients, while 46.0% presented with more severe signs, and other 14.3% did not show any prominent change between the two infections.
Treatment Medications and treatments for the first COVID‐19 infection were reported in 40 studies. Among these treatments, hydroxychloroquine, azithromycin, and oxygen support were reported by 15, 11, and 11 articles, respectively. A total of 37 articles stated treatment for the second COVID‐19 infection. So that, oxygen support (17 studies), azithromycin (9 studies), dexamethasone (8 studies), and remdesivir (8 studies) were mostly reported. The most common treatment options used in first SARS‐CoV‐2 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second SARS‐CoV‐2 infection, mostly antibiotics (100%), dexamethasone (100%), and Remdesivir (80%) were used (Table 2).
Medications and treatments for the first COVID‐19 infection were reported in 40 studies. Among these treatments, hydroxychloroquine, azithromycin, and oxygen support were reported by 15, 11, and 11 articles, respectively. A total of 37 articles stated treatment for the second COVID‐19 infection. So that, oxygen support (17 studies), azithromycin (9 studies), dexamethasone (8 studies), and remdesivir (8 studies) were mostly reported. The most common treatment options used in first SARS‐CoV‐2 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second SARS‐CoV‐2 infection, mostly antibiotics (100%), dexamethasone (100%), and Remdesivir (80%) were used (Table 2).
Comorbidities The evaluating comorbidities and underlying conditions can enlighten some aspects of COVID‐19. Based on the extracted data, a number of underlying diseases and conditions had a notable frequency. Obesity was highlighted as a condition in 32.5% of patients by 15 articles, whereas 22 studies stated diabetes with an overall prevalence of 15.15%. Hypertension and heart failure were reported to be 30.16% and 26.09% in 24 and 17 articles, respectively. Neurodegenerative disorders such as Alzheimer's (9.52%) and Parkinson's (9.52%) diseases were found as comorbidities (Table 2). However, evidence established an association between these types of comorbidities and COVID‐19; further investigations could clarify the detailed mechanisms of these relations.
The evaluating comorbidities and underlying conditions can enlighten some aspects of COVID‐19. Based on the extracted data, a number of underlying diseases and conditions had a notable frequency. Obesity was highlighted as a condition in 32.5% of patients by 15 articles, whereas 22 studies stated diabetes with an overall prevalence of 15.15%. Hypertension and heart failure were reported to be 30.16% and 26.09% in 24 and 17 articles, respectively. Neurodegenerative disorders such as Alzheimer's (9.52%) and Parkinson's (9.52%) diseases were found as comorbidities (Table 2). However, evidence established an association between these types of comorbidities and COVID‐19; further investigations could clarify the detailed mechanisms of these relations.
|
CONCLUSION
|
The present study represents a large number of COVID‐19 reactivation over different countries. Overall, the recurrence of COVID‐19 in recovered patients may arises from various factors, including a false negative or positive in PCR, differences in tests, incorrect diagnosis by physicians to discharge a patient with COVID‐19, illness for reasons other than COVID‐19, the presence of various strains of SARS‐CoV‐2, and dysfunction of immune systems. Our results highlighted the potency of COVID‐19 recurrence as an outstanding issue. This feature needs to be regarded in the management of COVID‐19. The first and second COVID‐19 are the same in terms of clinical manifestations, but they are not distinguishable. So far, no acceptable marker has been found to predict the risk of reinfection. In addition, there is no validated test of whether a particular drug or treatment is associated with reinfection or reactivation. A careful follow‐up of discharged patients and accuracy in their discharge and removal from quarantine is of paramount importance to inhibit reinfection. Given the data discussed in this work, the first coronavirus infection can lead to the recurrence of COVID‐19. Regarding COVID‐19 infection, there are two hypothesis: (a) COVID‐19 infection reactivates following a period of dormancy, and (b) COVID‐19 increases the susceptibility to the second coronavirus invasion. Future experimental and clinical researches could examine these hypotheses and finally provide a clear view of COVID‐19 relapse.
|
[
"INTRODUCTION",
"Search strategy",
"Inclusion and exclusion criteria",
"Data extraction",
"Quality assessment",
"Study characteristics",
"Demographic and general information",
"Clinical manifestations",
"Treatment",
"Comorbidities",
"AUTHORS’ CONTRIBUTION",
"INFORMED CONSENT",
"CONSENT FOR PUBLICATION"
] |
[
"This is not the first time that coronavirus has caused problems in the world. Viruses such as severe acute respiratory syndrome coronavirus (SARS‐CoV) and Middle East respiratory syndrome coronavirus (MERS‐CoV) have also been prevalent in recent years.\n1\n The mortality rates of SARS‐CoV and MERS‐CoV epidemics have been estimated to be 10% and 35%, respectively.\n2\n The main problem of today's global health communities is conflict over the novel coronavirus (2019‐nCoV). The new virus was first identified in China, but is now found in many countries around the world.\n1\n There are currently several variants of coronavirus circulating among people,\n3\n, \n4\n and the virus is mostly transmitted through the respiratory tract. Various symptoms have been described for patients with COVID‐19, ranging from asymptomatic to severe. Kidney damage has also been reported in some cases.\n5\n According to the World Health Organization (WHO), COVID‐19‐infected patients can leave home quarantine after the improvement of their infectious symptoms and also the confirmation of two negative RT‐PCR tests (within 24 h).\n6\n\n\nReinfection, relapse, recurrence, and reactivation are terms used for people infected with coronavirus and have become positive again after a period of negativity.\n7\n Based on a report from Guangdong Province in China, about 14% of patients who recover from COVID‐19 become reinfected with the virus.\n8\n In addition, there have been reports of reinfection in Korea and Japan.\n9\n Duration of immunization against coronavirus reinfection in recovering individuals is six months.\n10\n People who become infected with coronavirus for the second time often have milder symptoms and recover more quickly than those infected for the first time.\n11\n However, there are concerns about reinfection in people recovering from the coronavirus. The objective of this systematic review was to evaluate the prevalence and frequency of reinfection in people recovering from COVID‐19 and their clinical signs, as well as to assess the treatment methods.",
"We used PubMed/Medline, Web of Science, and Embase for a systematic search from January 1, 2019, to March 7, 2021. The search was based on the following string of terms: (“recurrence” OR “relapse” OR “reinfection” OR “reactivation”) AND (“COVID‐19” OR “severe acute respiratory syndrome coronavirus 2” OR “novel coronavirus” OR “SARS‐CoV‐2” OR “nCoV disease” OR “SARS2” OR “2019‐nCoV” OR “coronavirus disease‐19” OR “coronavirus disease 2019” OR “2019 novel coronavirus” OR “Wuhan coronavirus” OR “Wuhan seafood market pneumonia virus” OR “Wuhan pneumonia”). There was no limitation on language, location, and type of studies.",
"All the studies reported the reactivation or second infection of COVID‐19 were considered in the search. Total records were retrieved and entered into EndNote X9 software (Thomson Reuters). Following duplicate exclusion, a three‐stage screening was carried out to exploit the eligible studies based on title, abstract, and full text. The whole eligible studies reported the patients who were recovered from primary infection, but then developed a secondary COVID‐19 infection. RT‐PCR test was necessary inclusion criteria. Patients with a positive RT‐PCR for the first phase of COVID‐19, a negative RT‐PCR for recovery, and a second positive RT‐PCR for COVID‐19 recurrence were examined in the study. We excluded articles that reported only a serologic diagnosis test, without a nasopharyngeal swab RT‐PCR test, as well as duplicate publication of same studies, congress abstracts, reviews, systematic reviews and meta‐analysis, cellular and molecular studies, and animal studies. All types of manifestations and treatments were regarded without any restriction, and there was no limitation on comorbidities and underlying disorders.",
"The following data were acquired from each article: first author's name, location, publication time, type of study, number of relapsed patients, age, gender, interval between two infections, clinical manifestations, treatment, relative status, comorbidities, and outcome. Two investigators independently extracted the data from full text of 54 included studies. Inconsistencies between reviewers were resolved by consensus. The retrieved data are represented in Table 1.\nCharacteristics of the included studies\nM 2\nF 2\nM 1\nF 1\nM 1\nF 1\nWorse 2\nBetter 1\nM 7\nF 26\nDied 1\nDischarged 32\nM 1\nF 1\nM 1\nF 3\nM 5\nF 2\nM 36\nF 57\nM 1\nF 2\nM 1\nF 1\nM 2\nF 4\nND 1\nBetter 3\nNR 2\nAbbreviations: ALB, albuterol; APAP, acetaminophen; ARB, arbidol; AZ, azithromycin; COPD, chronic obstructive pulmonary disease; CQ, chloroquine; CRO, ceftriaxone; CTM, Chinese traditional medicine; DEX, dexamethasone; HCQ, hydroxychloroquine; IFN, interferon; Ig, immunoglobulin; IVIG, intravenous immunoglobulin; IVR, ivermectin; LPV, lopinavir; LPV/r, lopinavir/ritonavir; MM, multiple myeloma; mPDRL, methylprednisolone; ND, no difference; NR, not reported; OTV, oseltamivir; PDRL, prednisolone; PRED, prednisone; RAN, ranitidine; RDV, remdesivir; RPV, ritonavir; TZP, piperacillin/tazobactam; UTI, urinary tract infection; VAN, vancomycin.",
"The critical appraisal checklist provided by the Joanna Briggs Institute (JBI) was used to perform a quality assessment of the studies.\n13\n\n",
"The search strategy yielded 1807 studies from three databases. Following the removal of duplicates, the title and abstract of 998 studies were examined. Among these studies, 152 were selected for full‐text assessment, and other 846 studies were eliminated due to irrelevancy. In all the selected studies, the relapse of coronavirus infection after a negative RT‐PCR test was reported. Among the 152 full‐text studies examined, only 54 studies were found to be eligible for data extraction (Figure 1). The included studies were original articles (5.5%, N = 3), case reports (72.2%, N = 39), and case series (22.2%, N = 12). Likewise, in the included studies, RT‐PCR tests were performed to detect both the first and the second infections. Thirteen studies were originated from Europe, 11 from the USA, 9 from China, and 6 from Brazil. These articles reported a total number of 207 patients who developed the second infection of coronavirus after a recovery, which was confirmed by a negative RT‐PCR test. Forty‐six studies reported the clinical features in the first infection; however, seven articles declared no symptoms. Only one study unrecorded the clinical features in the first infection. In addition, 42 investigations implied the medication and intervention.\nFlow diagram detailing review process and study selection\nIn the second phase of infection, 43 articles reported the clinical manifestations, seven articles stated no sign, and four articles did not list any symptoms. Also, 37 studies reported specifically the treatment of the secondary infection. To compare the severity of symptoms between two phases of infection, sufficient information of both is required. Only 41 investigations presented features for both periods of infection. From these 41 studies, information of 63 cases was identified as qualified for the comparison of manifestations. Moreover, the approximate interval between negative and second positive RT‐PCR was obtained from 49 studies. The length of this interval reflects the characteristics of COVID‐19 relapse.\nDuring diagnosis, evaluation, and treatment, attention to comorbidities is necessary. Among included studies, 44 articles specified comorbidities. Of these observations, 11 studies did not found any notable underlying conditions or disorders. In this survey, we examined the outcome of the COVID‐19 relapse, which was categorized into discharge or death. The outcome was reported for a total number of 199 patients from 49 studies. The detailed information of is summarized in Table 1.",
"Considering the studies included, we reviewed 207 patients presented with secondary infection of COVID‐19 after a period of recovery. A negative RT‐PCR confirmed the recovery from the first phase of disease. Among the included articles, there were only three observational studies that reported 132 cases. Of all 207 cases, 122 (58.9%) patients were female, and 85 (41.1%) patients were male. As shown in Table 2, children were less likely to have a recurrence of COVID‐19. However, the most patients were in the age group of 20–40 years. The studies reported a wide range of 2–240 days between two coronavirus infections. We classified this duration into three groups: n ≤ 30, 30 < n < 90, and n ≥ 90. Thirty‐eight (77.5%) studies reported n ≤ 30 or 30 < n < 90 for the recovery duration, and only 11 (22.5%) investigations implied more than 90 days (Table 2).\nSummary of the findings\nAbbreviations: HIV, human immunodeficiency virus; n, number of patients with any variables; N, the total number of patients with COVID‐19; ND, no difference; No, number; RT‐PCR, reverse transcription‐polymerase chain reaction.\nTo compare the severity of the first infection with secondary, the reported features and symptoms were reviewed and extracted from the articles. Of 63 patients, 29 presented more severe manifestations, while 25 cases showed an ameliorated status, and 9 other cases indicated similar symptoms in both phases of the infection. Forty‐nine studies recorded the outcome of 199 patients, among whom 190 cases were discharged with an improved status, but 9 cases succumbed in hospital. Indeed, the survival rate is required to be taken into account when determining the potency of coronavirus reactivation.",
"Some clinical signs were most frequent between the two infections, but prevalence was different. Moreover, the most prevalent clinical signs in the first infection were asthenia (66.6%), headache (66.6%), cough (54.7%), fever (52.8%), sore throat (50.0%), and respiratory distress (48.1%). However, in the second infection, asthenia (62.9%), myalgia (62.6%), headache (61.11%), sore throat (52.54%), and dyspnea (48.53%) were the common. Respiratory alkalosis (21.43%), drowsiness (11.11%), and night sweat (11.11%) occurred only in the first infection. The most frequent symptoms observed only in the second infection were as follows: asthenia (62.9%), myalgia (62.9%), and headache (61.1%). COVID‐19 recurrence was manifested with more mild signs in 39.6% of patients, while 46.0% presented with more severe signs, and other 14.3% did not show any prominent change between the two infections.",
"Medications and treatments for the first COVID‐19 infection were reported in 40 studies. Among these treatments, hydroxychloroquine, azithromycin, and oxygen support were reported by 15, 11, and 11 articles, respectively. A total of 37 articles stated treatment for the second COVID‐19 infection. So that, oxygen support (17 studies), azithromycin (9 studies), dexamethasone (8 studies), and remdesivir (8 studies) were mostly reported. The most common treatment options used in first SARS‐CoV‐2 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second SARS‐CoV‐2 infection, mostly antibiotics (100%), dexamethasone (100%), and Remdesivir (80%) were used (Table 2).",
"The evaluating comorbidities and underlying conditions can enlighten some aspects of COVID‐19. Based on the extracted data, a number of underlying diseases and conditions had a notable frequency. Obesity was highlighted as a condition in 32.5% of patients by 15 articles, whereas 22 studies stated diabetes with an overall prevalence of 15.15%. Hypertension and heart failure were reported to be 30.16% and 26.09% in 24 and 17 articles, respectively. Neurodegenerative disorders such as Alzheimer's (9.52%) and Parkinson's (9.52%) diseases were found as comorbidities (Table 2). However, evidence established an association between these types of comorbidities and COVID‐19; further investigations could clarify the detailed mechanisms of these relations.",
"Maryam Koupaei, Mohamad Hosein Mohamadi, Ilya Yashmi, Amir Hossein Shahabi, Amir Hosein Shabani, Mohsen Heidary, and Saeed Khoshnood contributed in revising and final approval of the version to be published. All authors agreed and confirmed the study for publication.",
"Not applicable.",
"Not applicable."
] |
[
null,
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[
"INTRODUCTION",
"MATERIALS AND METHODS",
"Search strategy",
"Inclusion and exclusion criteria",
"Data extraction",
"Quality assessment",
"RESULTS",
"Study characteristics",
"Demographic and general information",
"Clinical manifestations",
"Treatment",
"Comorbidities",
"DISCUSSION",
"CONCLUSION",
"CONFLICT OF INTEREST",
"AUTHORS’ CONTRIBUTION",
"INFORMED CONSENT",
"CONSENT FOR PUBLICATION"
] |
[
"This is not the first time that coronavirus has caused problems in the world. Viruses such as severe acute respiratory syndrome coronavirus (SARS‐CoV) and Middle East respiratory syndrome coronavirus (MERS‐CoV) have also been prevalent in recent years.\n1\n The mortality rates of SARS‐CoV and MERS‐CoV epidemics have been estimated to be 10% and 35%, respectively.\n2\n The main problem of today's global health communities is conflict over the novel coronavirus (2019‐nCoV). The new virus was first identified in China, but is now found in many countries around the world.\n1\n There are currently several variants of coronavirus circulating among people,\n3\n, \n4\n and the virus is mostly transmitted through the respiratory tract. Various symptoms have been described for patients with COVID‐19, ranging from asymptomatic to severe. Kidney damage has also been reported in some cases.\n5\n According to the World Health Organization (WHO), COVID‐19‐infected patients can leave home quarantine after the improvement of their infectious symptoms and also the confirmation of two negative RT‐PCR tests (within 24 h).\n6\n\n\nReinfection, relapse, recurrence, and reactivation are terms used for people infected with coronavirus and have become positive again after a period of negativity.\n7\n Based on a report from Guangdong Province in China, about 14% of patients who recover from COVID‐19 become reinfected with the virus.\n8\n In addition, there have been reports of reinfection in Korea and Japan.\n9\n Duration of immunization against coronavirus reinfection in recovering individuals is six months.\n10\n People who become infected with coronavirus for the second time often have milder symptoms and recover more quickly than those infected for the first time.\n11\n However, there are concerns about reinfection in people recovering from the coronavirus. The objective of this systematic review was to evaluate the prevalence and frequency of reinfection in people recovering from COVID‐19 and their clinical signs, as well as to assess the treatment methods.",
"The present systematic review was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) statements.\n12\n\n\n Search strategy We used PubMed/Medline, Web of Science, and Embase for a systematic search from January 1, 2019, to March 7, 2021. The search was based on the following string of terms: (“recurrence” OR “relapse” OR “reinfection” OR “reactivation”) AND (“COVID‐19” OR “severe acute respiratory syndrome coronavirus 2” OR “novel coronavirus” OR “SARS‐CoV‐2” OR “nCoV disease” OR “SARS2” OR “2019‐nCoV” OR “coronavirus disease‐19” OR “coronavirus disease 2019” OR “2019 novel coronavirus” OR “Wuhan coronavirus” OR “Wuhan seafood market pneumonia virus” OR “Wuhan pneumonia”). There was no limitation on language, location, and type of studies.\nWe used PubMed/Medline, Web of Science, and Embase for a systematic search from January 1, 2019, to March 7, 2021. The search was based on the following string of terms: (“recurrence” OR “relapse” OR “reinfection” OR “reactivation”) AND (“COVID‐19” OR “severe acute respiratory syndrome coronavirus 2” OR “novel coronavirus” OR “SARS‐CoV‐2” OR “nCoV disease” OR “SARS2” OR “2019‐nCoV” OR “coronavirus disease‐19” OR “coronavirus disease 2019” OR “2019 novel coronavirus” OR “Wuhan coronavirus” OR “Wuhan seafood market pneumonia virus” OR “Wuhan pneumonia”). There was no limitation on language, location, and type of studies.\n Inclusion and exclusion criteria All the studies reported the reactivation or second infection of COVID‐19 were considered in the search. Total records were retrieved and entered into EndNote X9 software (Thomson Reuters). Following duplicate exclusion, a three‐stage screening was carried out to exploit the eligible studies based on title, abstract, and full text. The whole eligible studies reported the patients who were recovered from primary infection, but then developed a secondary COVID‐19 infection. RT‐PCR test was necessary inclusion criteria. Patients with a positive RT‐PCR for the first phase of COVID‐19, a negative RT‐PCR for recovery, and a second positive RT‐PCR for COVID‐19 recurrence were examined in the study. We excluded articles that reported only a serologic diagnosis test, without a nasopharyngeal swab RT‐PCR test, as well as duplicate publication of same studies, congress abstracts, reviews, systematic reviews and meta‐analysis, cellular and molecular studies, and animal studies. All types of manifestations and treatments were regarded without any restriction, and there was no limitation on comorbidities and underlying disorders.\nAll the studies reported the reactivation or second infection of COVID‐19 were considered in the search. Total records were retrieved and entered into EndNote X9 software (Thomson Reuters). Following duplicate exclusion, a three‐stage screening was carried out to exploit the eligible studies based on title, abstract, and full text. The whole eligible studies reported the patients who were recovered from primary infection, but then developed a secondary COVID‐19 infection. RT‐PCR test was necessary inclusion criteria. Patients with a positive RT‐PCR for the first phase of COVID‐19, a negative RT‐PCR for recovery, and a second positive RT‐PCR for COVID‐19 recurrence were examined in the study. We excluded articles that reported only a serologic diagnosis test, without a nasopharyngeal swab RT‐PCR test, as well as duplicate publication of same studies, congress abstracts, reviews, systematic reviews and meta‐analysis, cellular and molecular studies, and animal studies. All types of manifestations and treatments were regarded without any restriction, and there was no limitation on comorbidities and underlying disorders.\n Data extraction The following data were acquired from each article: first author's name, location, publication time, type of study, number of relapsed patients, age, gender, interval between two infections, clinical manifestations, treatment, relative status, comorbidities, and outcome. Two investigators independently extracted the data from full text of 54 included studies. Inconsistencies between reviewers were resolved by consensus. The retrieved data are represented in Table 1.\nCharacteristics of the included studies\nM 2\nF 2\nM 1\nF 1\nM 1\nF 1\nWorse 2\nBetter 1\nM 7\nF 26\nDied 1\nDischarged 32\nM 1\nF 1\nM 1\nF 3\nM 5\nF 2\nM 36\nF 57\nM 1\nF 2\nM 1\nF 1\nM 2\nF 4\nND 1\nBetter 3\nNR 2\nAbbreviations: ALB, albuterol; APAP, acetaminophen; ARB, arbidol; AZ, azithromycin; COPD, chronic obstructive pulmonary disease; CQ, chloroquine; CRO, ceftriaxone; CTM, Chinese traditional medicine; DEX, dexamethasone; HCQ, hydroxychloroquine; IFN, interferon; Ig, immunoglobulin; IVIG, intravenous immunoglobulin; IVR, ivermectin; LPV, lopinavir; LPV/r, lopinavir/ritonavir; MM, multiple myeloma; mPDRL, methylprednisolone; ND, no difference; NR, not reported; OTV, oseltamivir; PDRL, prednisolone; PRED, prednisone; RAN, ranitidine; RDV, remdesivir; RPV, ritonavir; TZP, piperacillin/tazobactam; UTI, urinary tract infection; VAN, vancomycin.\nThe following data were acquired from each article: first author's name, location, publication time, type of study, number of relapsed patients, age, gender, interval between two infections, clinical manifestations, treatment, relative status, comorbidities, and outcome. Two investigators independently extracted the data from full text of 54 included studies. Inconsistencies between reviewers were resolved by consensus. The retrieved data are represented in Table 1.\nCharacteristics of the included studies\nM 2\nF 2\nM 1\nF 1\nM 1\nF 1\nWorse 2\nBetter 1\nM 7\nF 26\nDied 1\nDischarged 32\nM 1\nF 1\nM 1\nF 3\nM 5\nF 2\nM 36\nF 57\nM 1\nF 2\nM 1\nF 1\nM 2\nF 4\nND 1\nBetter 3\nNR 2\nAbbreviations: ALB, albuterol; APAP, acetaminophen; ARB, arbidol; AZ, azithromycin; COPD, chronic obstructive pulmonary disease; CQ, chloroquine; CRO, ceftriaxone; CTM, Chinese traditional medicine; DEX, dexamethasone; HCQ, hydroxychloroquine; IFN, interferon; Ig, immunoglobulin; IVIG, intravenous immunoglobulin; IVR, ivermectin; LPV, lopinavir; LPV/r, lopinavir/ritonavir; MM, multiple myeloma; mPDRL, methylprednisolone; ND, no difference; NR, not reported; OTV, oseltamivir; PDRL, prednisolone; PRED, prednisone; RAN, ranitidine; RDV, remdesivir; RPV, ritonavir; TZP, piperacillin/tazobactam; UTI, urinary tract infection; VAN, vancomycin.\n Quality assessment The critical appraisal checklist provided by the Joanna Briggs Institute (JBI) was used to perform a quality assessment of the studies.\n13\n\n\nThe critical appraisal checklist provided by the Joanna Briggs Institute (JBI) was used to perform a quality assessment of the studies.\n13\n\n",
"We used PubMed/Medline, Web of Science, and Embase for a systematic search from January 1, 2019, to March 7, 2021. The search was based on the following string of terms: (“recurrence” OR “relapse” OR “reinfection” OR “reactivation”) AND (“COVID‐19” OR “severe acute respiratory syndrome coronavirus 2” OR “novel coronavirus” OR “SARS‐CoV‐2” OR “nCoV disease” OR “SARS2” OR “2019‐nCoV” OR “coronavirus disease‐19” OR “coronavirus disease 2019” OR “2019 novel coronavirus” OR “Wuhan coronavirus” OR “Wuhan seafood market pneumonia virus” OR “Wuhan pneumonia”). There was no limitation on language, location, and type of studies.",
"All the studies reported the reactivation or second infection of COVID‐19 were considered in the search. Total records were retrieved and entered into EndNote X9 software (Thomson Reuters). Following duplicate exclusion, a three‐stage screening was carried out to exploit the eligible studies based on title, abstract, and full text. The whole eligible studies reported the patients who were recovered from primary infection, but then developed a secondary COVID‐19 infection. RT‐PCR test was necessary inclusion criteria. Patients with a positive RT‐PCR for the first phase of COVID‐19, a negative RT‐PCR for recovery, and a second positive RT‐PCR for COVID‐19 recurrence were examined in the study. We excluded articles that reported only a serologic diagnosis test, without a nasopharyngeal swab RT‐PCR test, as well as duplicate publication of same studies, congress abstracts, reviews, systematic reviews and meta‐analysis, cellular and molecular studies, and animal studies. All types of manifestations and treatments were regarded without any restriction, and there was no limitation on comorbidities and underlying disorders.",
"The following data were acquired from each article: first author's name, location, publication time, type of study, number of relapsed patients, age, gender, interval between two infections, clinical manifestations, treatment, relative status, comorbidities, and outcome. Two investigators independently extracted the data from full text of 54 included studies. Inconsistencies between reviewers were resolved by consensus. The retrieved data are represented in Table 1.\nCharacteristics of the included studies\nM 2\nF 2\nM 1\nF 1\nM 1\nF 1\nWorse 2\nBetter 1\nM 7\nF 26\nDied 1\nDischarged 32\nM 1\nF 1\nM 1\nF 3\nM 5\nF 2\nM 36\nF 57\nM 1\nF 2\nM 1\nF 1\nM 2\nF 4\nND 1\nBetter 3\nNR 2\nAbbreviations: ALB, albuterol; APAP, acetaminophen; ARB, arbidol; AZ, azithromycin; COPD, chronic obstructive pulmonary disease; CQ, chloroquine; CRO, ceftriaxone; CTM, Chinese traditional medicine; DEX, dexamethasone; HCQ, hydroxychloroquine; IFN, interferon; Ig, immunoglobulin; IVIG, intravenous immunoglobulin; IVR, ivermectin; LPV, lopinavir; LPV/r, lopinavir/ritonavir; MM, multiple myeloma; mPDRL, methylprednisolone; ND, no difference; NR, not reported; OTV, oseltamivir; PDRL, prednisolone; PRED, prednisone; RAN, ranitidine; RDV, remdesivir; RPV, ritonavir; TZP, piperacillin/tazobactam; UTI, urinary tract infection; VAN, vancomycin.",
"The critical appraisal checklist provided by the Joanna Briggs Institute (JBI) was used to perform a quality assessment of the studies.\n13\n\n",
" Study characteristics The search strategy yielded 1807 studies from three databases. Following the removal of duplicates, the title and abstract of 998 studies were examined. Among these studies, 152 were selected for full‐text assessment, and other 846 studies were eliminated due to irrelevancy. In all the selected studies, the relapse of coronavirus infection after a negative RT‐PCR test was reported. Among the 152 full‐text studies examined, only 54 studies were found to be eligible for data extraction (Figure 1). The included studies were original articles (5.5%, N = 3), case reports (72.2%, N = 39), and case series (22.2%, N = 12). Likewise, in the included studies, RT‐PCR tests were performed to detect both the first and the second infections. Thirteen studies were originated from Europe, 11 from the USA, 9 from China, and 6 from Brazil. These articles reported a total number of 207 patients who developed the second infection of coronavirus after a recovery, which was confirmed by a negative RT‐PCR test. Forty‐six studies reported the clinical features in the first infection; however, seven articles declared no symptoms. Only one study unrecorded the clinical features in the first infection. In addition, 42 investigations implied the medication and intervention.\nFlow diagram detailing review process and study selection\nIn the second phase of infection, 43 articles reported the clinical manifestations, seven articles stated no sign, and four articles did not list any symptoms. Also, 37 studies reported specifically the treatment of the secondary infection. To compare the severity of symptoms between two phases of infection, sufficient information of both is required. Only 41 investigations presented features for both periods of infection. From these 41 studies, information of 63 cases was identified as qualified for the comparison of manifestations. Moreover, the approximate interval between negative and second positive RT‐PCR was obtained from 49 studies. The length of this interval reflects the characteristics of COVID‐19 relapse.\nDuring diagnosis, evaluation, and treatment, attention to comorbidities is necessary. Among included studies, 44 articles specified comorbidities. Of these observations, 11 studies did not found any notable underlying conditions or disorders. In this survey, we examined the outcome of the COVID‐19 relapse, which was categorized into discharge or death. The outcome was reported for a total number of 199 patients from 49 studies. The detailed information of is summarized in Table 1.\nThe search strategy yielded 1807 studies from three databases. Following the removal of duplicates, the title and abstract of 998 studies were examined. Among these studies, 152 were selected for full‐text assessment, and other 846 studies were eliminated due to irrelevancy. In all the selected studies, the relapse of coronavirus infection after a negative RT‐PCR test was reported. Among the 152 full‐text studies examined, only 54 studies were found to be eligible for data extraction (Figure 1). The included studies were original articles (5.5%, N = 3), case reports (72.2%, N = 39), and case series (22.2%, N = 12). Likewise, in the included studies, RT‐PCR tests were performed to detect both the first and the second infections. Thirteen studies were originated from Europe, 11 from the USA, 9 from China, and 6 from Brazil. These articles reported a total number of 207 patients who developed the second infection of coronavirus after a recovery, which was confirmed by a negative RT‐PCR test. Forty‐six studies reported the clinical features in the first infection; however, seven articles declared no symptoms. Only one study unrecorded the clinical features in the first infection. In addition, 42 investigations implied the medication and intervention.\nFlow diagram detailing review process and study selection\nIn the second phase of infection, 43 articles reported the clinical manifestations, seven articles stated no sign, and four articles did not list any symptoms. Also, 37 studies reported specifically the treatment of the secondary infection. To compare the severity of symptoms between two phases of infection, sufficient information of both is required. Only 41 investigations presented features for both periods of infection. From these 41 studies, information of 63 cases was identified as qualified for the comparison of manifestations. Moreover, the approximate interval between negative and second positive RT‐PCR was obtained from 49 studies. The length of this interval reflects the characteristics of COVID‐19 relapse.\nDuring diagnosis, evaluation, and treatment, attention to comorbidities is necessary. Among included studies, 44 articles specified comorbidities. Of these observations, 11 studies did not found any notable underlying conditions or disorders. In this survey, we examined the outcome of the COVID‐19 relapse, which was categorized into discharge or death. The outcome was reported for a total number of 199 patients from 49 studies. The detailed information of is summarized in Table 1.\n Demographic and general information Considering the studies included, we reviewed 207 patients presented with secondary infection of COVID‐19 after a period of recovery. A negative RT‐PCR confirmed the recovery from the first phase of disease. Among the included articles, there were only three observational studies that reported 132 cases. Of all 207 cases, 122 (58.9%) patients were female, and 85 (41.1%) patients were male. As shown in Table 2, children were less likely to have a recurrence of COVID‐19. However, the most patients were in the age group of 20–40 years. The studies reported a wide range of 2–240 days between two coronavirus infections. We classified this duration into three groups: n ≤ 30, 30 < n < 90, and n ≥ 90. Thirty‐eight (77.5%) studies reported n ≤ 30 or 30 < n < 90 for the recovery duration, and only 11 (22.5%) investigations implied more than 90 days (Table 2).\nSummary of the findings\nAbbreviations: HIV, human immunodeficiency virus; n, number of patients with any variables; N, the total number of patients with COVID‐19; ND, no difference; No, number; RT‐PCR, reverse transcription‐polymerase chain reaction.\nTo compare the severity of the first infection with secondary, the reported features and symptoms were reviewed and extracted from the articles. Of 63 patients, 29 presented more severe manifestations, while 25 cases showed an ameliorated status, and 9 other cases indicated similar symptoms in both phases of the infection. Forty‐nine studies recorded the outcome of 199 patients, among whom 190 cases were discharged with an improved status, but 9 cases succumbed in hospital. Indeed, the survival rate is required to be taken into account when determining the potency of coronavirus reactivation.\nConsidering the studies included, we reviewed 207 patients presented with secondary infection of COVID‐19 after a period of recovery. A negative RT‐PCR confirmed the recovery from the first phase of disease. Among the included articles, there were only three observational studies that reported 132 cases. Of all 207 cases, 122 (58.9%) patients were female, and 85 (41.1%) patients were male. As shown in Table 2, children were less likely to have a recurrence of COVID‐19. However, the most patients were in the age group of 20–40 years. The studies reported a wide range of 2–240 days between two coronavirus infections. We classified this duration into three groups: n ≤ 30, 30 < n < 90, and n ≥ 90. Thirty‐eight (77.5%) studies reported n ≤ 30 or 30 < n < 90 for the recovery duration, and only 11 (22.5%) investigations implied more than 90 days (Table 2).\nSummary of the findings\nAbbreviations: HIV, human immunodeficiency virus; n, number of patients with any variables; N, the total number of patients with COVID‐19; ND, no difference; No, number; RT‐PCR, reverse transcription‐polymerase chain reaction.\nTo compare the severity of the first infection with secondary, the reported features and symptoms were reviewed and extracted from the articles. Of 63 patients, 29 presented more severe manifestations, while 25 cases showed an ameliorated status, and 9 other cases indicated similar symptoms in both phases of the infection. Forty‐nine studies recorded the outcome of 199 patients, among whom 190 cases were discharged with an improved status, but 9 cases succumbed in hospital. Indeed, the survival rate is required to be taken into account when determining the potency of coronavirus reactivation.\n Clinical manifestations Some clinical signs were most frequent between the two infections, but prevalence was different. Moreover, the most prevalent clinical signs in the first infection were asthenia (66.6%), headache (66.6%), cough (54.7%), fever (52.8%), sore throat (50.0%), and respiratory distress (48.1%). However, in the second infection, asthenia (62.9%), myalgia (62.6%), headache (61.11%), sore throat (52.54%), and dyspnea (48.53%) were the common. Respiratory alkalosis (21.43%), drowsiness (11.11%), and night sweat (11.11%) occurred only in the first infection. The most frequent symptoms observed only in the second infection were as follows: asthenia (62.9%), myalgia (62.9%), and headache (61.1%). COVID‐19 recurrence was manifested with more mild signs in 39.6% of patients, while 46.0% presented with more severe signs, and other 14.3% did not show any prominent change between the two infections.\nSome clinical signs were most frequent between the two infections, but prevalence was different. Moreover, the most prevalent clinical signs in the first infection were asthenia (66.6%), headache (66.6%), cough (54.7%), fever (52.8%), sore throat (50.0%), and respiratory distress (48.1%). However, in the second infection, asthenia (62.9%), myalgia (62.6%), headache (61.11%), sore throat (52.54%), and dyspnea (48.53%) were the common. Respiratory alkalosis (21.43%), drowsiness (11.11%), and night sweat (11.11%) occurred only in the first infection. The most frequent symptoms observed only in the second infection were as follows: asthenia (62.9%), myalgia (62.9%), and headache (61.1%). COVID‐19 recurrence was manifested with more mild signs in 39.6% of patients, while 46.0% presented with more severe signs, and other 14.3% did not show any prominent change between the two infections.\n Treatment Medications and treatments for the first COVID‐19 infection were reported in 40 studies. Among these treatments, hydroxychloroquine, azithromycin, and oxygen support were reported by 15, 11, and 11 articles, respectively. A total of 37 articles stated treatment for the second COVID‐19 infection. So that, oxygen support (17 studies), azithromycin (9 studies), dexamethasone (8 studies), and remdesivir (8 studies) were mostly reported. The most common treatment options used in first SARS‐CoV‐2 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second SARS‐CoV‐2 infection, mostly antibiotics (100%), dexamethasone (100%), and Remdesivir (80%) were used (Table 2).\nMedications and treatments for the first COVID‐19 infection were reported in 40 studies. Among these treatments, hydroxychloroquine, azithromycin, and oxygen support were reported by 15, 11, and 11 articles, respectively. A total of 37 articles stated treatment for the second COVID‐19 infection. So that, oxygen support (17 studies), azithromycin (9 studies), dexamethasone (8 studies), and remdesivir (8 studies) were mostly reported. The most common treatment options used in first SARS‐CoV‐2 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second SARS‐CoV‐2 infection, mostly antibiotics (100%), dexamethasone (100%), and Remdesivir (80%) were used (Table 2).\n Comorbidities The evaluating comorbidities and underlying conditions can enlighten some aspects of COVID‐19. Based on the extracted data, a number of underlying diseases and conditions had a notable frequency. Obesity was highlighted as a condition in 32.5% of patients by 15 articles, whereas 22 studies stated diabetes with an overall prevalence of 15.15%. Hypertension and heart failure were reported to be 30.16% and 26.09% in 24 and 17 articles, respectively. Neurodegenerative disorders such as Alzheimer's (9.52%) and Parkinson's (9.52%) diseases were found as comorbidities (Table 2). However, evidence established an association between these types of comorbidities and COVID‐19; further investigations could clarify the detailed mechanisms of these relations.\nThe evaluating comorbidities and underlying conditions can enlighten some aspects of COVID‐19. Based on the extracted data, a number of underlying diseases and conditions had a notable frequency. Obesity was highlighted as a condition in 32.5% of patients by 15 articles, whereas 22 studies stated diabetes with an overall prevalence of 15.15%. Hypertension and heart failure were reported to be 30.16% and 26.09% in 24 and 17 articles, respectively. Neurodegenerative disorders such as Alzheimer's (9.52%) and Parkinson's (9.52%) diseases were found as comorbidities (Table 2). However, evidence established an association between these types of comorbidities and COVID‐19; further investigations could clarify the detailed mechanisms of these relations.",
"The search strategy yielded 1807 studies from three databases. Following the removal of duplicates, the title and abstract of 998 studies were examined. Among these studies, 152 were selected for full‐text assessment, and other 846 studies were eliminated due to irrelevancy. In all the selected studies, the relapse of coronavirus infection after a negative RT‐PCR test was reported. Among the 152 full‐text studies examined, only 54 studies were found to be eligible for data extraction (Figure 1). The included studies were original articles (5.5%, N = 3), case reports (72.2%, N = 39), and case series (22.2%, N = 12). Likewise, in the included studies, RT‐PCR tests were performed to detect both the first and the second infections. Thirteen studies were originated from Europe, 11 from the USA, 9 from China, and 6 from Brazil. These articles reported a total number of 207 patients who developed the second infection of coronavirus after a recovery, which was confirmed by a negative RT‐PCR test. Forty‐six studies reported the clinical features in the first infection; however, seven articles declared no symptoms. Only one study unrecorded the clinical features in the first infection. In addition, 42 investigations implied the medication and intervention.\nFlow diagram detailing review process and study selection\nIn the second phase of infection, 43 articles reported the clinical manifestations, seven articles stated no sign, and four articles did not list any symptoms. Also, 37 studies reported specifically the treatment of the secondary infection. To compare the severity of symptoms between two phases of infection, sufficient information of both is required. Only 41 investigations presented features for both periods of infection. From these 41 studies, information of 63 cases was identified as qualified for the comparison of manifestations. Moreover, the approximate interval between negative and second positive RT‐PCR was obtained from 49 studies. The length of this interval reflects the characteristics of COVID‐19 relapse.\nDuring diagnosis, evaluation, and treatment, attention to comorbidities is necessary. Among included studies, 44 articles specified comorbidities. Of these observations, 11 studies did not found any notable underlying conditions or disorders. In this survey, we examined the outcome of the COVID‐19 relapse, which was categorized into discharge or death. The outcome was reported for a total number of 199 patients from 49 studies. The detailed information of is summarized in Table 1.",
"Considering the studies included, we reviewed 207 patients presented with secondary infection of COVID‐19 after a period of recovery. A negative RT‐PCR confirmed the recovery from the first phase of disease. Among the included articles, there were only three observational studies that reported 132 cases. Of all 207 cases, 122 (58.9%) patients were female, and 85 (41.1%) patients were male. As shown in Table 2, children were less likely to have a recurrence of COVID‐19. However, the most patients were in the age group of 20–40 years. The studies reported a wide range of 2–240 days between two coronavirus infections. We classified this duration into three groups: n ≤ 30, 30 < n < 90, and n ≥ 90. Thirty‐eight (77.5%) studies reported n ≤ 30 or 30 < n < 90 for the recovery duration, and only 11 (22.5%) investigations implied more than 90 days (Table 2).\nSummary of the findings\nAbbreviations: HIV, human immunodeficiency virus; n, number of patients with any variables; N, the total number of patients with COVID‐19; ND, no difference; No, number; RT‐PCR, reverse transcription‐polymerase chain reaction.\nTo compare the severity of the first infection with secondary, the reported features and symptoms were reviewed and extracted from the articles. Of 63 patients, 29 presented more severe manifestations, while 25 cases showed an ameliorated status, and 9 other cases indicated similar symptoms in both phases of the infection. Forty‐nine studies recorded the outcome of 199 patients, among whom 190 cases were discharged with an improved status, but 9 cases succumbed in hospital. Indeed, the survival rate is required to be taken into account when determining the potency of coronavirus reactivation.",
"Some clinical signs were most frequent between the two infections, but prevalence was different. Moreover, the most prevalent clinical signs in the first infection were asthenia (66.6%), headache (66.6%), cough (54.7%), fever (52.8%), sore throat (50.0%), and respiratory distress (48.1%). However, in the second infection, asthenia (62.9%), myalgia (62.6%), headache (61.11%), sore throat (52.54%), and dyspnea (48.53%) were the common. Respiratory alkalosis (21.43%), drowsiness (11.11%), and night sweat (11.11%) occurred only in the first infection. The most frequent symptoms observed only in the second infection were as follows: asthenia (62.9%), myalgia (62.9%), and headache (61.1%). COVID‐19 recurrence was manifested with more mild signs in 39.6% of patients, while 46.0% presented with more severe signs, and other 14.3% did not show any prominent change between the two infections.",
"Medications and treatments for the first COVID‐19 infection were reported in 40 studies. Among these treatments, hydroxychloroquine, azithromycin, and oxygen support were reported by 15, 11, and 11 articles, respectively. A total of 37 articles stated treatment for the second COVID‐19 infection. So that, oxygen support (17 studies), azithromycin (9 studies), dexamethasone (8 studies), and remdesivir (8 studies) were mostly reported. The most common treatment options used in first SARS‐CoV‐2 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second SARS‐CoV‐2 infection, mostly antibiotics (100%), dexamethasone (100%), and Remdesivir (80%) were used (Table 2).",
"The evaluating comorbidities and underlying conditions can enlighten some aspects of COVID‐19. Based on the extracted data, a number of underlying diseases and conditions had a notable frequency. Obesity was highlighted as a condition in 32.5% of patients by 15 articles, whereas 22 studies stated diabetes with an overall prevalence of 15.15%. Hypertension and heart failure were reported to be 30.16% and 26.09% in 24 and 17 articles, respectively. Neurodegenerative disorders such as Alzheimer's (9.52%) and Parkinson's (9.52%) diseases were found as comorbidities (Table 2). However, evidence established an association between these types of comorbidities and COVID‐19; further investigations could clarify the detailed mechanisms of these relations.",
"To prevent reinfection or reactivation, four criteria can be considered for patients’ discharge. First, the patient should not have a fever for at least three days. Second, the patient's respiratory symptoms should considerably be ameliorated. Third, the radiological abnormalities shown in the CT scan and X‐ray images should substantially be improved. Four, as per WHO recommendation, patients should have two consecutive negative RT‐PCR results with a 24‐h interval.\n14\n\n\nImproved or discharged patients are connected to the members of the community, and they, therefore, are presented as a latent source of infection.\n14\n In this study, we assessed the prevalence and frequency of recurrence or reinfection in patients with COVID‐19 and performed investigations from various aspects, including factors related to host, virus, and environment. The emergence of new virus mutations is the main hypothesis on mechanisms of the COVID‐19 reinfection. The new variants of the SARS‐CoV‐2 can bind to human cells, and the produced antibodies in the first infection could not efficiently opsonize them. Actually, these variants can lead to evading the immune response.\n15\n\n\nSeveral factors influencing reinfection, including the initial load of the virus and the type of genome, are virus‐dependent.\n16\n The average duration of SARS‐CoV‐2 shedding is 20 days, which in some cases is 37 days.\n17\n A survey has suggested an average viral shedding of 53 days, with a maximum of 83 days. Patients in whom clinical symptoms had started earlier tented to have a longer duration of viral shedding and more severe disease.\n18\n\n\nThe study by Elrashdy et al. found that the average time period between the previous discharge and the next positive test was 4–17 days.\n14\n In the present study, the highest incidence of reinfection was related to a period of less than 30 days (46.94%). In the periods of 30–90 days, the incidence of reinfection was 30.61%, and the lowest incidence (22.45%) was observed in the period of more than 90 days. Given the studies reviewed above, there is a discrepancy between the duration of subsequent coronavirus infection and the antibody‐induced immunity. Therefore, there is certainly other factors, such as the level of the individual's immune system or the accuracy of the tests, that affect this time period. Perhaps, the reason for the recurrence of the disease 7–14 days after discharge from the hospital is that the virus is still hidden in exosomes or extracellular vesicles and resumes activity after a period of \"silences\".\n14\n\n\nIn this study, RT‐PCR was a necessary inclusion criterion. Thus, patients with only a serologic diagnosis test, without a nasopharyngeal swab RT‐PCR were excluded. RT‐PCR is the gold standard for diagnosing SARS‐CoV‐2; however, this test has low sensitivity due to test error or insufficient sample size.\n19\n The accuracy of RT‐PCR is 97%,\n20\n and the occurrence of false negatives in PCR of SARS‐CoV‐2 has been reported to be 30%,\n21\n which in some cases increases due to sampling error.\n20\n One of the reasons for the error in RT‐PCR is the prolonged conversion of nucleic acid, which causes recurrence or \"turn positive\".\n22\n In the early stages of infection, the SARS‐CoV‐2 is readily detected in the upper respiratory tract. As the disease progresses, the virus appears in the lower respiratory tract and other organs such as the intestines and blood.\n23\n Therefore, it is impossible to identify SARS‐CoV‐2 in the throat, and some patients may have positive CT scan, despite the negative RT‐PCR.\n24\n\n\nIncorrect sampling is another reason for recurrence in improved individuals,\n14\n although it is unlikely to happen due to the use of devices such as gloves, masks, and caps.\n25\n\n\nAs the laboratory detection of virus nucleic acid can have false‐negative results, serological tests for specific IgG and IgM can be alternated.\n19\n Therefore, PCR alone is not adequate for discharging patients from hospital. Supplementary tests such as serological ones, together with the criteria recommended by the WHO and other specific health organizations, are needed to be performed in every country. There is a period of time between the apparent recovery in the clinic and the complete recovery from the SARS‐CoV‐2. Viral carriers with low symptoms pose a greater challenge to epidemic management and control.\n26\n Conducting two negative PCR at 24‐hour intervals is insufficient for detecting the virus; thus, repeating the test for a longer period of 48 h is recommended. In addition, immunological tests such as d‐dimer and absolute lymphocyte counts and even antibody testing should be performed. RT‐PCR results are negative on average 2.73 days after hospitalization.\n26\n Wolfel et al. in their study evaluated the hospitalized patients with COVID‐19. They demonstrated that after eight days of infection, the live virus is undetectable.\n27\n\n\nGender, old age, and the type of disease are host‐dependent factors influencing the occurrence of reinfection and require immune system suppression.\n16\n In the present study, women became more infected than men (58.94% vs. 41.06). Children were less likely to have COVID‐19 relapse. However, the most patients were in the age group of 20–40 years. In addition, obesity (32.5%), kidney failure (30.7%), and hypertension (30.1%) were the most frequent underlying comorbidities observed among COVID‐19 relapse patients. Although studies have shown that underlying conditions cause the severity of COVID‐19 disease, but how each of these factors contribute to reinfection should be examined by designing new studies determining these effects separately or in combination.\n28\n, \n29\n\n\nThe clinical features of patients with reinfection are similar to those of primary infection. The presence of asymptomatic patients among reactivated patients caused the recurrence of the asymptomatic contamination or infection with few symptoms.\n14\n, \n16\n\n\nIn an earlier study, rhesus macaques became reinfected after recovery, without showing any symptoms. This finding highlights the need for strict protection from SARS‐CoV‐2 and its control, to hider the development of this severe disease.\n30\n The second time of infection severity is varied; some cases show mild, and some others indicate more severe symptoms.\n10\n In a former study, 46.03% of patients had a worse condition, and 39.68% had a better condition in the second than the first infection. One of the reasons for the deterioration condition of patients in the second infection, compared with first one, is the occurrence of an antibody‐dependent enhancement (ADE) that increases the infectivity of virus in the secondary infection.\n31\n However, a patient with strong immunity and more immune memory cells and T‐cell mediation could decrease the severity of the second infection.\n10\n\n\nNormally, people with a primary infection with mild symptoms and those with a suppressed immune system are more likely to get COVID‐19 for the second time because they do not produce an adequate immune response. It has also been demonstrated that 95.48% of the patients reinfected with the SARS‐CoV‐2 were discharged from hospital and 4.52% were died. The rate of death would have possibly been reduced if patients had received more care during their first‐time hospitalization.\n10\n\n\nSARS‐CoV‐2 reactivation may occur when using any antiviral drug.\n16\n In this survey, the most recurrence of the disease occurred after taking lopinavir/ritonavir, oseltamivir, interferon, and Chinese traditional medicine. These drugs may not have been able to fully eradicate viruses from the body, and some of them may remain in the body, causing reinfection. However, further investigation can evaluate the effectiveness of different drugs in complete eradication of the virus to eliminate the possibility of reinfection.\nIn a study performed by Okhuese, the proportion of infected population will continue to grow in the world if unvaccinated. At the same time, the rate of recovery will continue slowly. In other words, in this situation, the mortality rate can be determined based on the ratio of infection to recovery rate. The rate of reinfection with clinical clearance of the virus from the improved population decreases to zero over time.\n32\n Contrary to the results achieved in Okhuese's study,\n33\n despite the high prevalence of SARS‐CoV‐2, the rate of reinfection is still high. Therefore, more experimental and laboratory studies are needed to determine the cause of reinfection and its frequency. Of note, reinfection differs from reactivation. Reinfection is caused by different variants of SARS‐CoV‐2 virus, but reinfection occurs with the same strain. The only way to discriminate the reinfection and reactivation is by sequencing and molecular techniques.\n34\n Regrettably, the first two actions happen only in 5%–10%.\n10\n Designing studies to sequence the virus genome in the first and second infections is highly recommended. In this way, the cause of COVID‐19 recurrence is clarified, and its prevalence in the community is determined.\nA number of limitations can be considered in this study. The first is the small number of original articles and short communication. The second is related to case series and case reports studies, which lack sufficient and accurate information on patients and are often reported descriptively. Therefore, accurate meta‐analysis calculations were impossible in this study.",
"The present study represents a large number of COVID‐19 reactivation over different countries. Overall, the recurrence of COVID‐19 in recovered patients may arises from various factors, including a false negative or positive in PCR, differences in tests, incorrect diagnosis by physicians to discharge a patient with COVID‐19, illness for reasons other than COVID‐19, the presence of various strains of SARS‐CoV‐2, and dysfunction of immune systems. Our results highlighted the potency of COVID‐19 recurrence as an outstanding issue. This feature needs to be regarded in the management of COVID‐19. The first and second COVID‐19 are the same in terms of clinical manifestations, but they are not distinguishable. So far, no acceptable marker has been found to predict the risk of reinfection. In addition, there is no validated test of whether a particular drug or treatment is associated with reinfection or reactivation. A careful follow‐up of discharged patients and accuracy in their discharge and removal from quarantine is of paramount importance to inhibit reinfection. Given the data discussed in this work, the first coronavirus infection can lead to the recurrence of COVID‐19. Regarding COVID‐19 infection, there are two hypothesis: (a) COVID‐19 infection reactivates following a period of dormancy, and (b) COVID‐19 increases the susceptibility to the second coronavirus invasion. Future experimental and clinical researches could examine these hypotheses and finally provide a clear view of COVID‐19 relapse.",
"The authors declare that they have no competing interests.",
"Maryam Koupaei, Mohamad Hosein Mohamadi, Ilya Yashmi, Amir Hossein Shahabi, Amir Hosein Shabani, Mohsen Heidary, and Saeed Khoshnood contributed in revising and final approval of the version to be published. All authors agreed and confirmed the study for publication.",
"Not applicable.",
"Not applicable."
] |
[
null,
"materials-and-methods",
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
"discussion",
"conclusions",
"COI-statement",
null,
null,
null
] |
[
"COVID‐19",
"recurrence",
"reinfection",
"relapse",
"SARS‐CoV2"
] |
INTRODUCTION:
This is not the first time that coronavirus has caused problems in the world. Viruses such as severe acute respiratory syndrome coronavirus (SARS‐CoV) and Middle East respiratory syndrome coronavirus (MERS‐CoV) have also been prevalent in recent years.
1
The mortality rates of SARS‐CoV and MERS‐CoV epidemics have been estimated to be 10% and 35%, respectively.
2
The main problem of today's global health communities is conflict over the novel coronavirus (2019‐nCoV). The new virus was first identified in China, but is now found in many countries around the world.
1
There are currently several variants of coronavirus circulating among people,
3
,
4
and the virus is mostly transmitted through the respiratory tract. Various symptoms have been described for patients with COVID‐19, ranging from asymptomatic to severe. Kidney damage has also been reported in some cases.
5
According to the World Health Organization (WHO), COVID‐19‐infected patients can leave home quarantine after the improvement of their infectious symptoms and also the confirmation of two negative RT‐PCR tests (within 24 h).
6
Reinfection, relapse, recurrence, and reactivation are terms used for people infected with coronavirus and have become positive again after a period of negativity.
7
Based on a report from Guangdong Province in China, about 14% of patients who recover from COVID‐19 become reinfected with the virus.
8
In addition, there have been reports of reinfection in Korea and Japan.
9
Duration of immunization against coronavirus reinfection in recovering individuals is six months.
10
People who become infected with coronavirus for the second time often have milder symptoms and recover more quickly than those infected for the first time.
11
However, there are concerns about reinfection in people recovering from the coronavirus. The objective of this systematic review was to evaluate the prevalence and frequency of reinfection in people recovering from COVID‐19 and their clinical signs, as well as to assess the treatment methods.
MATERIALS AND METHODS:
The present systematic review was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) statements.
12
Search strategy We used PubMed/Medline, Web of Science, and Embase for a systematic search from January 1, 2019, to March 7, 2021. The search was based on the following string of terms: (“recurrence” OR “relapse” OR “reinfection” OR “reactivation”) AND (“COVID‐19” OR “severe acute respiratory syndrome coronavirus 2” OR “novel coronavirus” OR “SARS‐CoV‐2” OR “nCoV disease” OR “SARS2” OR “2019‐nCoV” OR “coronavirus disease‐19” OR “coronavirus disease 2019” OR “2019 novel coronavirus” OR “Wuhan coronavirus” OR “Wuhan seafood market pneumonia virus” OR “Wuhan pneumonia”). There was no limitation on language, location, and type of studies.
We used PubMed/Medline, Web of Science, and Embase for a systematic search from January 1, 2019, to March 7, 2021. The search was based on the following string of terms: (“recurrence” OR “relapse” OR “reinfection” OR “reactivation”) AND (“COVID‐19” OR “severe acute respiratory syndrome coronavirus 2” OR “novel coronavirus” OR “SARS‐CoV‐2” OR “nCoV disease” OR “SARS2” OR “2019‐nCoV” OR “coronavirus disease‐19” OR “coronavirus disease 2019” OR “2019 novel coronavirus” OR “Wuhan coronavirus” OR “Wuhan seafood market pneumonia virus” OR “Wuhan pneumonia”). There was no limitation on language, location, and type of studies.
Inclusion and exclusion criteria All the studies reported the reactivation or second infection of COVID‐19 were considered in the search. Total records were retrieved and entered into EndNote X9 software (Thomson Reuters). Following duplicate exclusion, a three‐stage screening was carried out to exploit the eligible studies based on title, abstract, and full text. The whole eligible studies reported the patients who were recovered from primary infection, but then developed a secondary COVID‐19 infection. RT‐PCR test was necessary inclusion criteria. Patients with a positive RT‐PCR for the first phase of COVID‐19, a negative RT‐PCR for recovery, and a second positive RT‐PCR for COVID‐19 recurrence were examined in the study. We excluded articles that reported only a serologic diagnosis test, without a nasopharyngeal swab RT‐PCR test, as well as duplicate publication of same studies, congress abstracts, reviews, systematic reviews and meta‐analysis, cellular and molecular studies, and animal studies. All types of manifestations and treatments were regarded without any restriction, and there was no limitation on comorbidities and underlying disorders.
All the studies reported the reactivation or second infection of COVID‐19 were considered in the search. Total records were retrieved and entered into EndNote X9 software (Thomson Reuters). Following duplicate exclusion, a three‐stage screening was carried out to exploit the eligible studies based on title, abstract, and full text. The whole eligible studies reported the patients who were recovered from primary infection, but then developed a secondary COVID‐19 infection. RT‐PCR test was necessary inclusion criteria. Patients with a positive RT‐PCR for the first phase of COVID‐19, a negative RT‐PCR for recovery, and a second positive RT‐PCR for COVID‐19 recurrence were examined in the study. We excluded articles that reported only a serologic diagnosis test, without a nasopharyngeal swab RT‐PCR test, as well as duplicate publication of same studies, congress abstracts, reviews, systematic reviews and meta‐analysis, cellular and molecular studies, and animal studies. All types of manifestations and treatments were regarded without any restriction, and there was no limitation on comorbidities and underlying disorders.
Data extraction The following data were acquired from each article: first author's name, location, publication time, type of study, number of relapsed patients, age, gender, interval between two infections, clinical manifestations, treatment, relative status, comorbidities, and outcome. Two investigators independently extracted the data from full text of 54 included studies. Inconsistencies between reviewers were resolved by consensus. The retrieved data are represented in Table 1.
Characteristics of the included studies
M 2
F 2
M 1
F 1
M 1
F 1
Worse 2
Better 1
M 7
F 26
Died 1
Discharged 32
M 1
F 1
M 1
F 3
M 5
F 2
M 36
F 57
M 1
F 2
M 1
F 1
M 2
F 4
ND 1
Better 3
NR 2
Abbreviations: ALB, albuterol; APAP, acetaminophen; ARB, arbidol; AZ, azithromycin; COPD, chronic obstructive pulmonary disease; CQ, chloroquine; CRO, ceftriaxone; CTM, Chinese traditional medicine; DEX, dexamethasone; HCQ, hydroxychloroquine; IFN, interferon; Ig, immunoglobulin; IVIG, intravenous immunoglobulin; IVR, ivermectin; LPV, lopinavir; LPV/r, lopinavir/ritonavir; MM, multiple myeloma; mPDRL, methylprednisolone; ND, no difference; NR, not reported; OTV, oseltamivir; PDRL, prednisolone; PRED, prednisone; RAN, ranitidine; RDV, remdesivir; RPV, ritonavir; TZP, piperacillin/tazobactam; UTI, urinary tract infection; VAN, vancomycin.
The following data were acquired from each article: first author's name, location, publication time, type of study, number of relapsed patients, age, gender, interval between two infections, clinical manifestations, treatment, relative status, comorbidities, and outcome. Two investigators independently extracted the data from full text of 54 included studies. Inconsistencies between reviewers were resolved by consensus. The retrieved data are represented in Table 1.
Characteristics of the included studies
M 2
F 2
M 1
F 1
M 1
F 1
Worse 2
Better 1
M 7
F 26
Died 1
Discharged 32
M 1
F 1
M 1
F 3
M 5
F 2
M 36
F 57
M 1
F 2
M 1
F 1
M 2
F 4
ND 1
Better 3
NR 2
Abbreviations: ALB, albuterol; APAP, acetaminophen; ARB, arbidol; AZ, azithromycin; COPD, chronic obstructive pulmonary disease; CQ, chloroquine; CRO, ceftriaxone; CTM, Chinese traditional medicine; DEX, dexamethasone; HCQ, hydroxychloroquine; IFN, interferon; Ig, immunoglobulin; IVIG, intravenous immunoglobulin; IVR, ivermectin; LPV, lopinavir; LPV/r, lopinavir/ritonavir; MM, multiple myeloma; mPDRL, methylprednisolone; ND, no difference; NR, not reported; OTV, oseltamivir; PDRL, prednisolone; PRED, prednisone; RAN, ranitidine; RDV, remdesivir; RPV, ritonavir; TZP, piperacillin/tazobactam; UTI, urinary tract infection; VAN, vancomycin.
Quality assessment The critical appraisal checklist provided by the Joanna Briggs Institute (JBI) was used to perform a quality assessment of the studies.
13
The critical appraisal checklist provided by the Joanna Briggs Institute (JBI) was used to perform a quality assessment of the studies.
13
Search strategy:
We used PubMed/Medline, Web of Science, and Embase for a systematic search from January 1, 2019, to March 7, 2021. The search was based on the following string of terms: (“recurrence” OR “relapse” OR “reinfection” OR “reactivation”) AND (“COVID‐19” OR “severe acute respiratory syndrome coronavirus 2” OR “novel coronavirus” OR “SARS‐CoV‐2” OR “nCoV disease” OR “SARS2” OR “2019‐nCoV” OR “coronavirus disease‐19” OR “coronavirus disease 2019” OR “2019 novel coronavirus” OR “Wuhan coronavirus” OR “Wuhan seafood market pneumonia virus” OR “Wuhan pneumonia”). There was no limitation on language, location, and type of studies.
Inclusion and exclusion criteria:
All the studies reported the reactivation or second infection of COVID‐19 were considered in the search. Total records were retrieved and entered into EndNote X9 software (Thomson Reuters). Following duplicate exclusion, a three‐stage screening was carried out to exploit the eligible studies based on title, abstract, and full text. The whole eligible studies reported the patients who were recovered from primary infection, but then developed a secondary COVID‐19 infection. RT‐PCR test was necessary inclusion criteria. Patients with a positive RT‐PCR for the first phase of COVID‐19, a negative RT‐PCR for recovery, and a second positive RT‐PCR for COVID‐19 recurrence were examined in the study. We excluded articles that reported only a serologic diagnosis test, without a nasopharyngeal swab RT‐PCR test, as well as duplicate publication of same studies, congress abstracts, reviews, systematic reviews and meta‐analysis, cellular and molecular studies, and animal studies. All types of manifestations and treatments were regarded without any restriction, and there was no limitation on comorbidities and underlying disorders.
Data extraction:
The following data were acquired from each article: first author's name, location, publication time, type of study, number of relapsed patients, age, gender, interval between two infections, clinical manifestations, treatment, relative status, comorbidities, and outcome. Two investigators independently extracted the data from full text of 54 included studies. Inconsistencies between reviewers were resolved by consensus. The retrieved data are represented in Table 1.
Characteristics of the included studies
M 2
F 2
M 1
F 1
M 1
F 1
Worse 2
Better 1
M 7
F 26
Died 1
Discharged 32
M 1
F 1
M 1
F 3
M 5
F 2
M 36
F 57
M 1
F 2
M 1
F 1
M 2
F 4
ND 1
Better 3
NR 2
Abbreviations: ALB, albuterol; APAP, acetaminophen; ARB, arbidol; AZ, azithromycin; COPD, chronic obstructive pulmonary disease; CQ, chloroquine; CRO, ceftriaxone; CTM, Chinese traditional medicine; DEX, dexamethasone; HCQ, hydroxychloroquine; IFN, interferon; Ig, immunoglobulin; IVIG, intravenous immunoglobulin; IVR, ivermectin; LPV, lopinavir; LPV/r, lopinavir/ritonavir; MM, multiple myeloma; mPDRL, methylprednisolone; ND, no difference; NR, not reported; OTV, oseltamivir; PDRL, prednisolone; PRED, prednisone; RAN, ranitidine; RDV, remdesivir; RPV, ritonavir; TZP, piperacillin/tazobactam; UTI, urinary tract infection; VAN, vancomycin.
Quality assessment:
The critical appraisal checklist provided by the Joanna Briggs Institute (JBI) was used to perform a quality assessment of the studies.
13
RESULTS:
Study characteristics The search strategy yielded 1807 studies from three databases. Following the removal of duplicates, the title and abstract of 998 studies were examined. Among these studies, 152 were selected for full‐text assessment, and other 846 studies were eliminated due to irrelevancy. In all the selected studies, the relapse of coronavirus infection after a negative RT‐PCR test was reported. Among the 152 full‐text studies examined, only 54 studies were found to be eligible for data extraction (Figure 1). The included studies were original articles (5.5%, N = 3), case reports (72.2%, N = 39), and case series (22.2%, N = 12). Likewise, in the included studies, RT‐PCR tests were performed to detect both the first and the second infections. Thirteen studies were originated from Europe, 11 from the USA, 9 from China, and 6 from Brazil. These articles reported a total number of 207 patients who developed the second infection of coronavirus after a recovery, which was confirmed by a negative RT‐PCR test. Forty‐six studies reported the clinical features in the first infection; however, seven articles declared no symptoms. Only one study unrecorded the clinical features in the first infection. In addition, 42 investigations implied the medication and intervention.
Flow diagram detailing review process and study selection
In the second phase of infection, 43 articles reported the clinical manifestations, seven articles stated no sign, and four articles did not list any symptoms. Also, 37 studies reported specifically the treatment of the secondary infection. To compare the severity of symptoms between two phases of infection, sufficient information of both is required. Only 41 investigations presented features for both periods of infection. From these 41 studies, information of 63 cases was identified as qualified for the comparison of manifestations. Moreover, the approximate interval between negative and second positive RT‐PCR was obtained from 49 studies. The length of this interval reflects the characteristics of COVID‐19 relapse.
During diagnosis, evaluation, and treatment, attention to comorbidities is necessary. Among included studies, 44 articles specified comorbidities. Of these observations, 11 studies did not found any notable underlying conditions or disorders. In this survey, we examined the outcome of the COVID‐19 relapse, which was categorized into discharge or death. The outcome was reported for a total number of 199 patients from 49 studies. The detailed information of is summarized in Table 1.
The search strategy yielded 1807 studies from three databases. Following the removal of duplicates, the title and abstract of 998 studies were examined. Among these studies, 152 were selected for full‐text assessment, and other 846 studies were eliminated due to irrelevancy. In all the selected studies, the relapse of coronavirus infection after a negative RT‐PCR test was reported. Among the 152 full‐text studies examined, only 54 studies were found to be eligible for data extraction (Figure 1). The included studies were original articles (5.5%, N = 3), case reports (72.2%, N = 39), and case series (22.2%, N = 12). Likewise, in the included studies, RT‐PCR tests were performed to detect both the first and the second infections. Thirteen studies were originated from Europe, 11 from the USA, 9 from China, and 6 from Brazil. These articles reported a total number of 207 patients who developed the second infection of coronavirus after a recovery, which was confirmed by a negative RT‐PCR test. Forty‐six studies reported the clinical features in the first infection; however, seven articles declared no symptoms. Only one study unrecorded the clinical features in the first infection. In addition, 42 investigations implied the medication and intervention.
Flow diagram detailing review process and study selection
In the second phase of infection, 43 articles reported the clinical manifestations, seven articles stated no sign, and four articles did not list any symptoms. Also, 37 studies reported specifically the treatment of the secondary infection. To compare the severity of symptoms between two phases of infection, sufficient information of both is required. Only 41 investigations presented features for both periods of infection. From these 41 studies, information of 63 cases was identified as qualified for the comparison of manifestations. Moreover, the approximate interval between negative and second positive RT‐PCR was obtained from 49 studies. The length of this interval reflects the characteristics of COVID‐19 relapse.
During diagnosis, evaluation, and treatment, attention to comorbidities is necessary. Among included studies, 44 articles specified comorbidities. Of these observations, 11 studies did not found any notable underlying conditions or disorders. In this survey, we examined the outcome of the COVID‐19 relapse, which was categorized into discharge or death. The outcome was reported for a total number of 199 patients from 49 studies. The detailed information of is summarized in Table 1.
Demographic and general information Considering the studies included, we reviewed 207 patients presented with secondary infection of COVID‐19 after a period of recovery. A negative RT‐PCR confirmed the recovery from the first phase of disease. Among the included articles, there were only three observational studies that reported 132 cases. Of all 207 cases, 122 (58.9%) patients were female, and 85 (41.1%) patients were male. As shown in Table 2, children were less likely to have a recurrence of COVID‐19. However, the most patients were in the age group of 20–40 years. The studies reported a wide range of 2–240 days between two coronavirus infections. We classified this duration into three groups: n ≤ 30, 30 < n < 90, and n ≥ 90. Thirty‐eight (77.5%) studies reported n ≤ 30 or 30 < n < 90 for the recovery duration, and only 11 (22.5%) investigations implied more than 90 days (Table 2).
Summary of the findings
Abbreviations: HIV, human immunodeficiency virus; n, number of patients with any variables; N, the total number of patients with COVID‐19; ND, no difference; No, number; RT‐PCR, reverse transcription‐polymerase chain reaction.
To compare the severity of the first infection with secondary, the reported features and symptoms were reviewed and extracted from the articles. Of 63 patients, 29 presented more severe manifestations, while 25 cases showed an ameliorated status, and 9 other cases indicated similar symptoms in both phases of the infection. Forty‐nine studies recorded the outcome of 199 patients, among whom 190 cases were discharged with an improved status, but 9 cases succumbed in hospital. Indeed, the survival rate is required to be taken into account when determining the potency of coronavirus reactivation.
Considering the studies included, we reviewed 207 patients presented with secondary infection of COVID‐19 after a period of recovery. A negative RT‐PCR confirmed the recovery from the first phase of disease. Among the included articles, there were only three observational studies that reported 132 cases. Of all 207 cases, 122 (58.9%) patients were female, and 85 (41.1%) patients were male. As shown in Table 2, children were less likely to have a recurrence of COVID‐19. However, the most patients were in the age group of 20–40 years. The studies reported a wide range of 2–240 days between two coronavirus infections. We classified this duration into three groups: n ≤ 30, 30 < n < 90, and n ≥ 90. Thirty‐eight (77.5%) studies reported n ≤ 30 or 30 < n < 90 for the recovery duration, and only 11 (22.5%) investigations implied more than 90 days (Table 2).
Summary of the findings
Abbreviations: HIV, human immunodeficiency virus; n, number of patients with any variables; N, the total number of patients with COVID‐19; ND, no difference; No, number; RT‐PCR, reverse transcription‐polymerase chain reaction.
To compare the severity of the first infection with secondary, the reported features and symptoms were reviewed and extracted from the articles. Of 63 patients, 29 presented more severe manifestations, while 25 cases showed an ameliorated status, and 9 other cases indicated similar symptoms in both phases of the infection. Forty‐nine studies recorded the outcome of 199 patients, among whom 190 cases were discharged with an improved status, but 9 cases succumbed in hospital. Indeed, the survival rate is required to be taken into account when determining the potency of coronavirus reactivation.
Clinical manifestations Some clinical signs were most frequent between the two infections, but prevalence was different. Moreover, the most prevalent clinical signs in the first infection were asthenia (66.6%), headache (66.6%), cough (54.7%), fever (52.8%), sore throat (50.0%), and respiratory distress (48.1%). However, in the second infection, asthenia (62.9%), myalgia (62.6%), headache (61.11%), sore throat (52.54%), and dyspnea (48.53%) were the common. Respiratory alkalosis (21.43%), drowsiness (11.11%), and night sweat (11.11%) occurred only in the first infection. The most frequent symptoms observed only in the second infection were as follows: asthenia (62.9%), myalgia (62.9%), and headache (61.1%). COVID‐19 recurrence was manifested with more mild signs in 39.6% of patients, while 46.0% presented with more severe signs, and other 14.3% did not show any prominent change between the two infections.
Some clinical signs were most frequent between the two infections, but prevalence was different. Moreover, the most prevalent clinical signs in the first infection were asthenia (66.6%), headache (66.6%), cough (54.7%), fever (52.8%), sore throat (50.0%), and respiratory distress (48.1%). However, in the second infection, asthenia (62.9%), myalgia (62.6%), headache (61.11%), sore throat (52.54%), and dyspnea (48.53%) were the common. Respiratory alkalosis (21.43%), drowsiness (11.11%), and night sweat (11.11%) occurred only in the first infection. The most frequent symptoms observed only in the second infection were as follows: asthenia (62.9%), myalgia (62.9%), and headache (61.1%). COVID‐19 recurrence was manifested with more mild signs in 39.6% of patients, while 46.0% presented with more severe signs, and other 14.3% did not show any prominent change between the two infections.
Treatment Medications and treatments for the first COVID‐19 infection were reported in 40 studies. Among these treatments, hydroxychloroquine, azithromycin, and oxygen support were reported by 15, 11, and 11 articles, respectively. A total of 37 articles stated treatment for the second COVID‐19 infection. So that, oxygen support (17 studies), azithromycin (9 studies), dexamethasone (8 studies), and remdesivir (8 studies) were mostly reported. The most common treatment options used in first SARS‐CoV‐2 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second SARS‐CoV‐2 infection, mostly antibiotics (100%), dexamethasone (100%), and Remdesivir (80%) were used (Table 2).
Medications and treatments for the first COVID‐19 infection were reported in 40 studies. Among these treatments, hydroxychloroquine, azithromycin, and oxygen support were reported by 15, 11, and 11 articles, respectively. A total of 37 articles stated treatment for the second COVID‐19 infection. So that, oxygen support (17 studies), azithromycin (9 studies), dexamethasone (8 studies), and remdesivir (8 studies) were mostly reported. The most common treatment options used in first SARS‐CoV‐2 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second SARS‐CoV‐2 infection, mostly antibiotics (100%), dexamethasone (100%), and Remdesivir (80%) were used (Table 2).
Comorbidities The evaluating comorbidities and underlying conditions can enlighten some aspects of COVID‐19. Based on the extracted data, a number of underlying diseases and conditions had a notable frequency. Obesity was highlighted as a condition in 32.5% of patients by 15 articles, whereas 22 studies stated diabetes with an overall prevalence of 15.15%. Hypertension and heart failure were reported to be 30.16% and 26.09% in 24 and 17 articles, respectively. Neurodegenerative disorders such as Alzheimer's (9.52%) and Parkinson's (9.52%) diseases were found as comorbidities (Table 2). However, evidence established an association between these types of comorbidities and COVID‐19; further investigations could clarify the detailed mechanisms of these relations.
The evaluating comorbidities and underlying conditions can enlighten some aspects of COVID‐19. Based on the extracted data, a number of underlying diseases and conditions had a notable frequency. Obesity was highlighted as a condition in 32.5% of patients by 15 articles, whereas 22 studies stated diabetes with an overall prevalence of 15.15%. Hypertension and heart failure were reported to be 30.16% and 26.09% in 24 and 17 articles, respectively. Neurodegenerative disorders such as Alzheimer's (9.52%) and Parkinson's (9.52%) diseases were found as comorbidities (Table 2). However, evidence established an association between these types of comorbidities and COVID‐19; further investigations could clarify the detailed mechanisms of these relations.
Study characteristics:
The search strategy yielded 1807 studies from three databases. Following the removal of duplicates, the title and abstract of 998 studies were examined. Among these studies, 152 were selected for full‐text assessment, and other 846 studies were eliminated due to irrelevancy. In all the selected studies, the relapse of coronavirus infection after a negative RT‐PCR test was reported. Among the 152 full‐text studies examined, only 54 studies were found to be eligible for data extraction (Figure 1). The included studies were original articles (5.5%, N = 3), case reports (72.2%, N = 39), and case series (22.2%, N = 12). Likewise, in the included studies, RT‐PCR tests were performed to detect both the first and the second infections. Thirteen studies were originated from Europe, 11 from the USA, 9 from China, and 6 from Brazil. These articles reported a total number of 207 patients who developed the second infection of coronavirus after a recovery, which was confirmed by a negative RT‐PCR test. Forty‐six studies reported the clinical features in the first infection; however, seven articles declared no symptoms. Only one study unrecorded the clinical features in the first infection. In addition, 42 investigations implied the medication and intervention.
Flow diagram detailing review process and study selection
In the second phase of infection, 43 articles reported the clinical manifestations, seven articles stated no sign, and four articles did not list any symptoms. Also, 37 studies reported specifically the treatment of the secondary infection. To compare the severity of symptoms between two phases of infection, sufficient information of both is required. Only 41 investigations presented features for both periods of infection. From these 41 studies, information of 63 cases was identified as qualified for the comparison of manifestations. Moreover, the approximate interval between negative and second positive RT‐PCR was obtained from 49 studies. The length of this interval reflects the characteristics of COVID‐19 relapse.
During diagnosis, evaluation, and treatment, attention to comorbidities is necessary. Among included studies, 44 articles specified comorbidities. Of these observations, 11 studies did not found any notable underlying conditions or disorders. In this survey, we examined the outcome of the COVID‐19 relapse, which was categorized into discharge or death. The outcome was reported for a total number of 199 patients from 49 studies. The detailed information of is summarized in Table 1.
Demographic and general information:
Considering the studies included, we reviewed 207 patients presented with secondary infection of COVID‐19 after a period of recovery. A negative RT‐PCR confirmed the recovery from the first phase of disease. Among the included articles, there were only three observational studies that reported 132 cases. Of all 207 cases, 122 (58.9%) patients were female, and 85 (41.1%) patients were male. As shown in Table 2, children were less likely to have a recurrence of COVID‐19. However, the most patients were in the age group of 20–40 years. The studies reported a wide range of 2–240 days between two coronavirus infections. We classified this duration into three groups: n ≤ 30, 30 < n < 90, and n ≥ 90. Thirty‐eight (77.5%) studies reported n ≤ 30 or 30 < n < 90 for the recovery duration, and only 11 (22.5%) investigations implied more than 90 days (Table 2).
Summary of the findings
Abbreviations: HIV, human immunodeficiency virus; n, number of patients with any variables; N, the total number of patients with COVID‐19; ND, no difference; No, number; RT‐PCR, reverse transcription‐polymerase chain reaction.
To compare the severity of the first infection with secondary, the reported features and symptoms were reviewed and extracted from the articles. Of 63 patients, 29 presented more severe manifestations, while 25 cases showed an ameliorated status, and 9 other cases indicated similar symptoms in both phases of the infection. Forty‐nine studies recorded the outcome of 199 patients, among whom 190 cases were discharged with an improved status, but 9 cases succumbed in hospital. Indeed, the survival rate is required to be taken into account when determining the potency of coronavirus reactivation.
Clinical manifestations:
Some clinical signs were most frequent between the two infections, but prevalence was different. Moreover, the most prevalent clinical signs in the first infection were asthenia (66.6%), headache (66.6%), cough (54.7%), fever (52.8%), sore throat (50.0%), and respiratory distress (48.1%). However, in the second infection, asthenia (62.9%), myalgia (62.6%), headache (61.11%), sore throat (52.54%), and dyspnea (48.53%) were the common. Respiratory alkalosis (21.43%), drowsiness (11.11%), and night sweat (11.11%) occurred only in the first infection. The most frequent symptoms observed only in the second infection were as follows: asthenia (62.9%), myalgia (62.9%), and headache (61.1%). COVID‐19 recurrence was manifested with more mild signs in 39.6% of patients, while 46.0% presented with more severe signs, and other 14.3% did not show any prominent change between the two infections.
Treatment:
Medications and treatments for the first COVID‐19 infection were reported in 40 studies. Among these treatments, hydroxychloroquine, azithromycin, and oxygen support were reported by 15, 11, and 11 articles, respectively. A total of 37 articles stated treatment for the second COVID‐19 infection. So that, oxygen support (17 studies), azithromycin (9 studies), dexamethasone (8 studies), and remdesivir (8 studies) were mostly reported. The most common treatment options used in first SARS‐CoV‐2 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second SARS‐CoV‐2 infection, mostly antibiotics (100%), dexamethasone (100%), and Remdesivir (80%) were used (Table 2).
Comorbidities:
The evaluating comorbidities and underlying conditions can enlighten some aspects of COVID‐19. Based on the extracted data, a number of underlying diseases and conditions had a notable frequency. Obesity was highlighted as a condition in 32.5% of patients by 15 articles, whereas 22 studies stated diabetes with an overall prevalence of 15.15%. Hypertension and heart failure were reported to be 30.16% and 26.09% in 24 and 17 articles, respectively. Neurodegenerative disorders such as Alzheimer's (9.52%) and Parkinson's (9.52%) diseases were found as comorbidities (Table 2). However, evidence established an association between these types of comorbidities and COVID‐19; further investigations could clarify the detailed mechanisms of these relations.
DISCUSSION:
To prevent reinfection or reactivation, four criteria can be considered for patients’ discharge. First, the patient should not have a fever for at least three days. Second, the patient's respiratory symptoms should considerably be ameliorated. Third, the radiological abnormalities shown in the CT scan and X‐ray images should substantially be improved. Four, as per WHO recommendation, patients should have two consecutive negative RT‐PCR results with a 24‐h interval.
14
Improved or discharged patients are connected to the members of the community, and they, therefore, are presented as a latent source of infection.
14
In this study, we assessed the prevalence and frequency of recurrence or reinfection in patients with COVID‐19 and performed investigations from various aspects, including factors related to host, virus, and environment. The emergence of new virus mutations is the main hypothesis on mechanisms of the COVID‐19 reinfection. The new variants of the SARS‐CoV‐2 can bind to human cells, and the produced antibodies in the first infection could not efficiently opsonize them. Actually, these variants can lead to evading the immune response.
15
Several factors influencing reinfection, including the initial load of the virus and the type of genome, are virus‐dependent.
16
The average duration of SARS‐CoV‐2 shedding is 20 days, which in some cases is 37 days.
17
A survey has suggested an average viral shedding of 53 days, with a maximum of 83 days. Patients in whom clinical symptoms had started earlier tented to have a longer duration of viral shedding and more severe disease.
18
The study by Elrashdy et al. found that the average time period between the previous discharge and the next positive test was 4–17 days.
14
In the present study, the highest incidence of reinfection was related to a period of less than 30 days (46.94%). In the periods of 30–90 days, the incidence of reinfection was 30.61%, and the lowest incidence (22.45%) was observed in the period of more than 90 days. Given the studies reviewed above, there is a discrepancy between the duration of subsequent coronavirus infection and the antibody‐induced immunity. Therefore, there is certainly other factors, such as the level of the individual's immune system or the accuracy of the tests, that affect this time period. Perhaps, the reason for the recurrence of the disease 7–14 days after discharge from the hospital is that the virus is still hidden in exosomes or extracellular vesicles and resumes activity after a period of "silences".
14
In this study, RT‐PCR was a necessary inclusion criterion. Thus, patients with only a serologic diagnosis test, without a nasopharyngeal swab RT‐PCR were excluded. RT‐PCR is the gold standard for diagnosing SARS‐CoV‐2; however, this test has low sensitivity due to test error or insufficient sample size.
19
The accuracy of RT‐PCR is 97%,
20
and the occurrence of false negatives in PCR of SARS‐CoV‐2 has been reported to be 30%,
21
which in some cases increases due to sampling error.
20
One of the reasons for the error in RT‐PCR is the prolonged conversion of nucleic acid, which causes recurrence or "turn positive".
22
In the early stages of infection, the SARS‐CoV‐2 is readily detected in the upper respiratory tract. As the disease progresses, the virus appears in the lower respiratory tract and other organs such as the intestines and blood.
23
Therefore, it is impossible to identify SARS‐CoV‐2 in the throat, and some patients may have positive CT scan, despite the negative RT‐PCR.
24
Incorrect sampling is another reason for recurrence in improved individuals,
14
although it is unlikely to happen due to the use of devices such as gloves, masks, and caps.
25
As the laboratory detection of virus nucleic acid can have false‐negative results, serological tests for specific IgG and IgM can be alternated.
19
Therefore, PCR alone is not adequate for discharging patients from hospital. Supplementary tests such as serological ones, together with the criteria recommended by the WHO and other specific health organizations, are needed to be performed in every country. There is a period of time between the apparent recovery in the clinic and the complete recovery from the SARS‐CoV‐2. Viral carriers with low symptoms pose a greater challenge to epidemic management and control.
26
Conducting two negative PCR at 24‐hour intervals is insufficient for detecting the virus; thus, repeating the test for a longer period of 48 h is recommended. In addition, immunological tests such as d‐dimer and absolute lymphocyte counts and even antibody testing should be performed. RT‐PCR results are negative on average 2.73 days after hospitalization.
26
Wolfel et al. in their study evaluated the hospitalized patients with COVID‐19. They demonstrated that after eight days of infection, the live virus is undetectable.
27
Gender, old age, and the type of disease are host‐dependent factors influencing the occurrence of reinfection and require immune system suppression.
16
In the present study, women became more infected than men (58.94% vs. 41.06). Children were less likely to have COVID‐19 relapse. However, the most patients were in the age group of 20–40 years. In addition, obesity (32.5%), kidney failure (30.7%), and hypertension (30.1%) were the most frequent underlying comorbidities observed among COVID‐19 relapse patients. Although studies have shown that underlying conditions cause the severity of COVID‐19 disease, but how each of these factors contribute to reinfection should be examined by designing new studies determining these effects separately or in combination.
28
,
29
The clinical features of patients with reinfection are similar to those of primary infection. The presence of asymptomatic patients among reactivated patients caused the recurrence of the asymptomatic contamination or infection with few symptoms.
14
,
16
In an earlier study, rhesus macaques became reinfected after recovery, without showing any symptoms. This finding highlights the need for strict protection from SARS‐CoV‐2 and its control, to hider the development of this severe disease.
30
The second time of infection severity is varied; some cases show mild, and some others indicate more severe symptoms.
10
In a former study, 46.03% of patients had a worse condition, and 39.68% had a better condition in the second than the first infection. One of the reasons for the deterioration condition of patients in the second infection, compared with first one, is the occurrence of an antibody‐dependent enhancement (ADE) that increases the infectivity of virus in the secondary infection.
31
However, a patient with strong immunity and more immune memory cells and T‐cell mediation could decrease the severity of the second infection.
10
Normally, people with a primary infection with mild symptoms and those with a suppressed immune system are more likely to get COVID‐19 for the second time because they do not produce an adequate immune response. It has also been demonstrated that 95.48% of the patients reinfected with the SARS‐CoV‐2 were discharged from hospital and 4.52% were died. The rate of death would have possibly been reduced if patients had received more care during their first‐time hospitalization.
10
SARS‐CoV‐2 reactivation may occur when using any antiviral drug.
16
In this survey, the most recurrence of the disease occurred after taking lopinavir/ritonavir, oseltamivir, interferon, and Chinese traditional medicine. These drugs may not have been able to fully eradicate viruses from the body, and some of them may remain in the body, causing reinfection. However, further investigation can evaluate the effectiveness of different drugs in complete eradication of the virus to eliminate the possibility of reinfection.
In a study performed by Okhuese, the proportion of infected population will continue to grow in the world if unvaccinated. At the same time, the rate of recovery will continue slowly. In other words, in this situation, the mortality rate can be determined based on the ratio of infection to recovery rate. The rate of reinfection with clinical clearance of the virus from the improved population decreases to zero over time.
32
Contrary to the results achieved in Okhuese's study,
33
despite the high prevalence of SARS‐CoV‐2, the rate of reinfection is still high. Therefore, more experimental and laboratory studies are needed to determine the cause of reinfection and its frequency. Of note, reinfection differs from reactivation. Reinfection is caused by different variants of SARS‐CoV‐2 virus, but reinfection occurs with the same strain. The only way to discriminate the reinfection and reactivation is by sequencing and molecular techniques.
34
Regrettably, the first two actions happen only in 5%–10%.
10
Designing studies to sequence the virus genome in the first and second infections is highly recommended. In this way, the cause of COVID‐19 recurrence is clarified, and its prevalence in the community is determined.
A number of limitations can be considered in this study. The first is the small number of original articles and short communication. The second is related to case series and case reports studies, which lack sufficient and accurate information on patients and are often reported descriptively. Therefore, accurate meta‐analysis calculations were impossible in this study.
CONCLUSION:
The present study represents a large number of COVID‐19 reactivation over different countries. Overall, the recurrence of COVID‐19 in recovered patients may arises from various factors, including a false negative or positive in PCR, differences in tests, incorrect diagnosis by physicians to discharge a patient with COVID‐19, illness for reasons other than COVID‐19, the presence of various strains of SARS‐CoV‐2, and dysfunction of immune systems. Our results highlighted the potency of COVID‐19 recurrence as an outstanding issue. This feature needs to be regarded in the management of COVID‐19. The first and second COVID‐19 are the same in terms of clinical manifestations, but they are not distinguishable. So far, no acceptable marker has been found to predict the risk of reinfection. In addition, there is no validated test of whether a particular drug or treatment is associated with reinfection or reactivation. A careful follow‐up of discharged patients and accuracy in their discharge and removal from quarantine is of paramount importance to inhibit reinfection. Given the data discussed in this work, the first coronavirus infection can lead to the recurrence of COVID‐19. Regarding COVID‐19 infection, there are two hypothesis: (a) COVID‐19 infection reactivates following a period of dormancy, and (b) COVID‐19 increases the susceptibility to the second coronavirus invasion. Future experimental and clinical researches could examine these hypotheses and finally provide a clear view of COVID‐19 relapse.
CONFLICT OF INTEREST:
The authors declare that they have no competing interests.
AUTHORS’ CONTRIBUTION:
Maryam Koupaei, Mohamad Hosein Mohamadi, Ilya Yashmi, Amir Hossein Shahabi, Amir Hosein Shabani, Mohsen Heidary, and Saeed Khoshnood contributed in revising and final approval of the version to be published. All authors agreed and confirmed the study for publication.
INFORMED CONSENT:
Not applicable.
CONSENT FOR PUBLICATION:
Not applicable.
|
Background:
Interest revolving around coronavirus disease 2019 (COVID-19) reinfection is escalating rapidly. By definition, reinfection denotes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), PCR redetection, and COVID-19 recurrence within three months of the initial symptoms. The main aim of the current systematic review was to evaluate the features of COVID-19 relapse patients.
Methods:
For this study, we used a string of terms developed by a skilled librarian and through a systematical search in PubMed, Web of Science, and Embase for eligible studies. Clinical surveys of any type were included from January 2019 to March 2021. Eligible studies consisted of two positive assessments separated by a negative result via RT-PCR.
Results:
Fifty-four studies included 207 cases of COVID-19 reinfection. Children were less likely to have COVID-19 relapse. However, the most patients were in the age group of 20-40 years. Asthenia (66.6%), headache (66.6%), and cough (54.7%) were prevalent symptoms in the first SARS-CoV-2 infection. Asthenia (62.9%), myalgia (62.9%), and headache (61.1%) were most frequent in the second one. The most common treatment options used in first COVID-19 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second infection, mostly antibiotics (100%), dexamethasone (100%), and remdesivir (80%) were used. In addition, obesity (32.5%), kidney failure (30.7%), and hypertension (30.1%) were the most common comorbidities. Unfortunately, approximately 4.5% of patients died.
Conclusions:
We found the potency of COVID-19 recurrence as an outstanding issue. This feature should be regarded in the COVID-19 management. Furthermore, the first and second COVID-19 are similar in clinical features. For clinically practical comparison of the symptoms severity between two epochs of infection, uniform data of both are required. We suggest that future studies undertake a homogenous approach to establish the clinical patterns of the reinfection phenomena.
|
INTRODUCTION:
This is not the first time that coronavirus has caused problems in the world. Viruses such as severe acute respiratory syndrome coronavirus (SARS‐CoV) and Middle East respiratory syndrome coronavirus (MERS‐CoV) have also been prevalent in recent years.
1
The mortality rates of SARS‐CoV and MERS‐CoV epidemics have been estimated to be 10% and 35%, respectively.
2
The main problem of today's global health communities is conflict over the novel coronavirus (2019‐nCoV). The new virus was first identified in China, but is now found in many countries around the world.
1
There are currently several variants of coronavirus circulating among people,
3
,
4
and the virus is mostly transmitted through the respiratory tract. Various symptoms have been described for patients with COVID‐19, ranging from asymptomatic to severe. Kidney damage has also been reported in some cases.
5
According to the World Health Organization (WHO), COVID‐19‐infected patients can leave home quarantine after the improvement of their infectious symptoms and also the confirmation of two negative RT‐PCR tests (within 24 h).
6
Reinfection, relapse, recurrence, and reactivation are terms used for people infected with coronavirus and have become positive again after a period of negativity.
7
Based on a report from Guangdong Province in China, about 14% of patients who recover from COVID‐19 become reinfected with the virus.
8
In addition, there have been reports of reinfection in Korea and Japan.
9
Duration of immunization against coronavirus reinfection in recovering individuals is six months.
10
People who become infected with coronavirus for the second time often have milder symptoms and recover more quickly than those infected for the first time.
11
However, there are concerns about reinfection in people recovering from the coronavirus. The objective of this systematic review was to evaluate the prevalence and frequency of reinfection in people recovering from COVID‐19 and their clinical signs, as well as to assess the treatment methods.
CONCLUSION:
The present study represents a large number of COVID‐19 reactivation over different countries. Overall, the recurrence of COVID‐19 in recovered patients may arises from various factors, including a false negative or positive in PCR, differences in tests, incorrect diagnosis by physicians to discharge a patient with COVID‐19, illness for reasons other than COVID‐19, the presence of various strains of SARS‐CoV‐2, and dysfunction of immune systems. Our results highlighted the potency of COVID‐19 recurrence as an outstanding issue. This feature needs to be regarded in the management of COVID‐19. The first and second COVID‐19 are the same in terms of clinical manifestations, but they are not distinguishable. So far, no acceptable marker has been found to predict the risk of reinfection. In addition, there is no validated test of whether a particular drug or treatment is associated with reinfection or reactivation. A careful follow‐up of discharged patients and accuracy in their discharge and removal from quarantine is of paramount importance to inhibit reinfection. Given the data discussed in this work, the first coronavirus infection can lead to the recurrence of COVID‐19. Regarding COVID‐19 infection, there are two hypothesis: (a) COVID‐19 infection reactivates following a period of dormancy, and (b) COVID‐19 increases the susceptibility to the second coronavirus invasion. Future experimental and clinical researches could examine these hypotheses and finally provide a clear view of COVID‐19 relapse.
|
Background:
Interest revolving around coronavirus disease 2019 (COVID-19) reinfection is escalating rapidly. By definition, reinfection denotes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), PCR redetection, and COVID-19 recurrence within three months of the initial symptoms. The main aim of the current systematic review was to evaluate the features of COVID-19 relapse patients.
Methods:
For this study, we used a string of terms developed by a skilled librarian and through a systematical search in PubMed, Web of Science, and Embase for eligible studies. Clinical surveys of any type were included from January 2019 to March 2021. Eligible studies consisted of two positive assessments separated by a negative result via RT-PCR.
Results:
Fifty-four studies included 207 cases of COVID-19 reinfection. Children were less likely to have COVID-19 relapse. However, the most patients were in the age group of 20-40 years. Asthenia (66.6%), headache (66.6%), and cough (54.7%) were prevalent symptoms in the first SARS-CoV-2 infection. Asthenia (62.9%), myalgia (62.9%), and headache (61.1%) were most frequent in the second one. The most common treatment options used in first COVID-19 infection were lopinavir/ritonavir (80%), oxygen support (69.2%), and oseltamivir (66.6). However, for the treatment of second infection, mostly antibiotics (100%), dexamethasone (100%), and remdesivir (80%) were used. In addition, obesity (32.5%), kidney failure (30.7%), and hypertension (30.1%) were the most common comorbidities. Unfortunately, approximately 4.5% of patients died.
Conclusions:
We found the potency of COVID-19 recurrence as an outstanding issue. This feature should be regarded in the COVID-19 management. Furthermore, the first and second COVID-19 are similar in clinical features. For clinically practical comparison of the symptoms severity between two epochs of infection, uniform data of both are required. We suggest that future studies undertake a homogenous approach to establish the clinical patterns of the reinfection phenomena.
| 8,600 | 410 | 18 |
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[CONTENT] COVID‐19 | recurrence | reinfection | relapse | SARS‐CoV2 [SUMMARY]
| null |
[CONTENT] COVID‐19 | recurrence | reinfection | relapse | SARS‐CoV2 [SUMMARY]
|
[CONTENT] COVID‐19 | recurrence | reinfection | relapse | SARS‐CoV2 [SUMMARY]
|
[CONTENT] COVID‐19 | recurrence | reinfection | relapse | SARS‐CoV2 [SUMMARY]
|
[CONTENT] COVID‐19 | recurrence | reinfection | relapse | SARS‐CoV2 [SUMMARY]
|
[CONTENT] Adult | Asthenia | COVID-19 | Child | Headache | Humans | Reinfection | SARS-CoV-2 | Young Adult [SUMMARY]
| null |
[CONTENT] Adult | Asthenia | COVID-19 | Child | Headache | Humans | Reinfection | SARS-CoV-2 | Young Adult [SUMMARY]
|
[CONTENT] Adult | Asthenia | COVID-19 | Child | Headache | Humans | Reinfection | SARS-CoV-2 | Young Adult [SUMMARY]
|
[CONTENT] Adult | Asthenia | COVID-19 | Child | Headache | Humans | Reinfection | SARS-CoV-2 | Young Adult [SUMMARY]
|
[CONTENT] Adult | Asthenia | COVID-19 | Child | Headache | Humans | Reinfection | SARS-CoV-2 | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] studies | infection | 19 | covid 19 | covid | patients | reported | pcr | articles | rt [SUMMARY]
| null |
[CONTENT] studies | infection | 19 | covid 19 | covid | patients | reported | pcr | articles | rt [SUMMARY]
|
[CONTENT] studies | infection | 19 | covid 19 | covid | patients | reported | pcr | articles | rt [SUMMARY]
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[CONTENT] studies | infection | 19 | covid 19 | covid | patients | reported | pcr | articles | rt [SUMMARY]
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[CONTENT] studies | infection | 19 | covid 19 | covid | patients | reported | pcr | articles | rt [SUMMARY]
|
[CONTENT] coronavirus | people | infected | reinfection | recovering | world | cov | time | reinfection people recovering | mers cov [SUMMARY]
| null |
[CONTENT] studies | infection | articles | reported | 11 | patients | cases | covid | covid 19 | 19 [SUMMARY]
|
[CONTENT] covid 19 | covid | 19 | reinfection | recurrence covid | recurrence covid 19 | recurrence | discharge | infection | 19 infection [SUMMARY]
|
[CONTENT] applicable | studies | infection | 19 | covid | covid 19 | coronavirus | patients | reported | pcr [SUMMARY]
|
[CONTENT] applicable | studies | infection | 19 | covid | covid 19 | coronavirus | patients | reported | pcr [SUMMARY]
|
[CONTENT] 2019 | COVID-19 ||| 2 | PCR | COVID-19 | three months ||| COVID-19 [SUMMARY]
| null |
[CONTENT] Fifty-four | 207 | COVID-19 ||| COVID-19 ||| 20-40 years ||| Asthenia | 66.6% | 66.6% | 54.7% | first ||| Asthenia | 62.9% | myalgia | 62.9% | 61.1% | second ||| first | COVID-19 | 80% | 69.2% | 66.6 ||| second | 100% | 100% | 80% ||| 32.5% | 30.7% | 30.1% ||| approximately 4.5% [SUMMARY]
|
[CONTENT] COVID-19 ||| COVID-19 ||| first | second | COVID-19 ||| between two ||| [SUMMARY]
|
[CONTENT] ||| 2019 | COVID-19 ||| 2 | PCR | COVID-19 | three months ||| COVID-19 ||| PubMed | Embase ||| January 2019 to March 2021 ||| two | RT-PCR ||| ||| Fifty-four | 207 | COVID-19 ||| COVID-19 ||| 20-40 years ||| Asthenia | 66.6% | 66.6% | 54.7% | first ||| Asthenia | 62.9% | myalgia | 62.9% | 61.1% | second ||| first | COVID-19 | 80% | 69.2% | 66.6 ||| second | 100% | 100% | 80% ||| 32.5% | 30.7% | 30.1% ||| approximately 4.5% ||| COVID-19 ||| COVID-19 ||| first | second | COVID-19 ||| between two ||| [SUMMARY]
|
[CONTENT] ||| 2019 | COVID-19 ||| 2 | PCR | COVID-19 | three months ||| COVID-19 ||| PubMed | Embase ||| January 2019 to March 2021 ||| two | RT-PCR ||| ||| Fifty-four | 207 | COVID-19 ||| COVID-19 ||| 20-40 years ||| Asthenia | 66.6% | 66.6% | 54.7% | first ||| Asthenia | 62.9% | myalgia | 62.9% | 61.1% | second ||| first | COVID-19 | 80% | 69.2% | 66.6 ||| second | 100% | 100% | 80% ||| 32.5% | 30.7% | 30.1% ||| approximately 4.5% ||| COVID-19 ||| COVID-19 ||| first | second | COVID-19 ||| between two ||| [SUMMARY]
|
|||||
A long non-coding RNA OLBC15 promotes triple-negative breast cancer progression via enhancing ZNF326 degradation.
|
32329931
|
The long non-coding RNAs (lncRNAs) have been involved in various processes, including cancer. However, the function of many lncRNAs is still elusive in triple-negative breast cancer (TNBC).
|
BACKGROUND
|
LncRNA profiling was used to screen for novel lncRNAs related to TNBC. OLBC15 expression was measured via qRT-PCR. In vitro migration and viability assays were conducted to determine the oncogenic role of OLBC15. Xenograft and metastatic models were performed to further investigate effects in vivo. RNA immunoprecipitation (RIP), mass spectrometry (MS), and fluorescence in situ hybridization (FISH) strategies were designed to identify the interaction between ZNF326 and OLBC15.
|
METHODS
|
In the current study, we have identified a novel oncogenic lncRNA termed OLBC15 via lncRNA profiling. OLBC15 is highly expressed especially in triple-negative breast cancer. OLBC15 promoted viability and migration in breast cancer cells. Moreover, OLBC15 could accelerate metastasis and xenograft tumor growth. Mechanistic study suggested that OLBC15 could bind a well-characterized tumor suppressor ZNF326 and OLBC15-ZNF326 interaction resulted in ZNF326 destabilization. OLBC15 induced proteasomal ZNF326 degradation through enhanced ubiquitination. OLBC15 and ZNF326 protein expression is also negatively correlated in clinical specimens.
|
RESULTS
|
Collectively, OLBC15 may serve as an oncogenic lncRNA to facilitate TNBC progression and a putative target for therapeutic anti-breast cancer intervention.
|
CONCLUSIONS
|
[
"Breast",
"Carrier Proteins",
"Cell Line, Tumor",
"Disease Progression",
"Female",
"Humans",
"RNA, Long Noncoding",
"Triple Negative Breast Neoplasms"
] |
7439339
|
INTRODUCTION
|
The breast cancer (BC) has been reported to be a primary cause of malignancy and mortality in women, and the incidence rate worldwide has been on the rise.
1
The breast cancer possesses a heterogeneous nature and is classified into several subtypes.
2
Anti‐estrogen or combined anti‐HER2 therapeutic strategies are major treatments for luminal A, luminal B, and HER2‐positive (HER2+) breast cancer subtypes.
3
However, the highly aggressive and metastatic triple‐negative breast cancer (TNBC, negative in estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) is associated with high relapse even after chemotherapy or radiotherapy.
4
,
5
Therefore, unraveling novel and effective targets for TNBC intervention are of great significance.
Growing evidence has suggested that the non‐coding RNAs with no or minimal coding abilities contribute largely to tumor development.
6
A major constituent of non‐coding transcriptome is long non‐coding RNAs (lncRNAs).
6
The long non‐coding RNAs are RNA transcripts with more than 200 nucleotides in length and involved in numerous biological processes.
7
The lncRNAs can serve as an oncogene or tumor suppressor with an emerging effect on metastasis.
8
For instance, Tang et al identified that lncRNA PVT1 can stabilize KLF5 via BAP1 and increase its stability to promote TNBC progression.
9
Alipoor et al recently found that lncRNA MIAT increases miR‐302/miR‐150 expression, whereas miR‐29c expression was decreased.
10
Furthermore, MIAT also abolishes the expression of p16Ink4A and Cox2 to inhibit senescence commitment.
10
A TNBC‐specific lncRNA LINC00511 is markedly upregulated through gene amplification and significantly correlates with TNBC progression.
11
Sas‐Chen et al showed that lncRNA LIMT is a highly conserved tumor suppressor and its expression is suppressed by epidermal growth factor (EGF) signaling pathway.
12
Other reported lncRNAs (eg, RMST and BORG) are substantially associated with breast cancer development via diverse molecular mechanisms.
13
,
14
However, the function of lncRNAs especially in TNBC is still obscure.
In the current work, by lncRNA profiling, we have identified a novel lncRNA ENSG00000259457 as an oncogenic lncRNA in TNBC. It was termed as oncogenic lncRNA in breast cancer on chromosome 15 (OLBC15). We observed that OLBC15 is significantly upregulated in TNBC. OLBC15 acted as an oncogenic factor to promote breast cancer progression, and this effect was verified both in vivo and in vitro. Furthermore, by RNA immunoprecipitation we noted that zinc‐finger protein‐326 (ZNF326) might be an OLBC15 binding protein. The interaction of OLBC15 and ZNF326 decreased the stability of ZNF326 via enhanced ubiquitination and proteasomal degradation. OLBC15 also showed negative correlation with ZNF326 protein expression in clinical samples. These data collectively implied an oncogenic role of OLBC15 and may provide a novel anti‐TNBC target for therapeutic intervention.
| null | null |
RESULTS
|
Identifying OLBC15 in breast cancer via lncRNA profiling To screen for breast cancer (BC)‐related lncRNAs, we used lncRNA profiling assays in paired BC tissues and normal adjacent tissues (NATs) (Figure 1A). Overall, 73 significantly upregulated lncRNAs were detected (Figure 1A). These lncRNAs were candidate ones with oncogenic potential. Notably, two novel lncRNAs, RP11‐597D13.9 and OLBC15, were identified by lncRNA profiling (Table S2). Since lncRNA OLBC15 showed higher fold expression, we chose it for further analysis. OLBC15 was markedly upregulated in BC tissues compared with normal tissues (Figure 1B). OLBC15 is located on chromosome 15 with one annotated transcript (www.ensembl.org). The whole sequence for OLBC15 was shown (Figure S1A). Full‐length OLBC15 showed neglectable coding potential as evaluated by Coding Potential Assessment Tool (CPAT, coding probability ~ .032, Figure S1B). Moreover, OLBC15 was substantially enriched in triple‐negative breast cancer (TNBC) tissues compared with the other subtypes (Figure 1C). OLBC15 transcripts were highly increased in BC cell lines compared with MCF10A cells (Figure 1D). Patient characteristics demonstrated that OLBC15 expression significantly correlated with TNM stage, tumor size, and metastasis but was not significantly associated with age and histology (Table S3). Subcellular fractionation assay clarified that OLBC15 was primarily located in nucleus (Figure 1E). These data suggested that OLBC15 was upregulated in BC with nuclear distribution.
Identifying OLBC15 in breast cancer (BC). A, Heatmap representation of lncRNA profiling in normal adjacent tissues (NATs) and breast cancer tissues. Significantly upregulated and downregulated lncRNAs in BC tissues were 73 and 126, respectively. B, Relative OLBC15 expression in NAT and BC tissues. C, The relative OLBC15 levels in different breast cancer subtypes. The case numbers for luminal A (LA), luminal B (LB), HER2+ (HER2), and triple‐negative breast cancer (TNBC) were 40, 25, 26, and 51, respectively. D, Relative OLBC15 expression in transformed MCF10A and cancerous TNBC cell lines. E, Subcellular distribution of OLBC15. **: P < .01
To screen for breast cancer (BC)‐related lncRNAs, we used lncRNA profiling assays in paired BC tissues and normal adjacent tissues (NATs) (Figure 1A). Overall, 73 significantly upregulated lncRNAs were detected (Figure 1A). These lncRNAs were candidate ones with oncogenic potential. Notably, two novel lncRNAs, RP11‐597D13.9 and OLBC15, were identified by lncRNA profiling (Table S2). Since lncRNA OLBC15 showed higher fold expression, we chose it for further analysis. OLBC15 was markedly upregulated in BC tissues compared with normal tissues (Figure 1B). OLBC15 is located on chromosome 15 with one annotated transcript (www.ensembl.org). The whole sequence for OLBC15 was shown (Figure S1A). Full‐length OLBC15 showed neglectable coding potential as evaluated by Coding Potential Assessment Tool (CPAT, coding probability ~ .032, Figure S1B). Moreover, OLBC15 was substantially enriched in triple‐negative breast cancer (TNBC) tissues compared with the other subtypes (Figure 1C). OLBC15 transcripts were highly increased in BC cell lines compared with MCF10A cells (Figure 1D). Patient characteristics demonstrated that OLBC15 expression significantly correlated with TNM stage, tumor size, and metastasis but was not significantly associated with age and histology (Table S3). Subcellular fractionation assay clarified that OLBC15 was primarily located in nucleus (Figure 1E). These data suggested that OLBC15 was upregulated in BC with nuclear distribution.
Identifying OLBC15 in breast cancer (BC). A, Heatmap representation of lncRNA profiling in normal adjacent tissues (NATs) and breast cancer tissues. Significantly upregulated and downregulated lncRNAs in BC tissues were 73 and 126, respectively. B, Relative OLBC15 expression in NAT and BC tissues. C, The relative OLBC15 levels in different breast cancer subtypes. The case numbers for luminal A (LA), luminal B (LB), HER2+ (HER2), and triple‐negative breast cancer (TNBC) were 40, 25, 26, and 51, respectively. D, Relative OLBC15 expression in transformed MCF10A and cancerous TNBC cell lines. E, Subcellular distribution of OLBC15. **: P < .01
OLBC15 facilitates breast cancer progression in vitro We next checked whether OLBC15 had an effect in vitro. We first stably overexpressed or silenced OLBC15 expression in MDA‐MB‐231 and BT‐549 cells (Figure 2A,B). The knockdown and overexpression efficiency were verified (Figure 2A,B). As expected, OLBC15 overexpression significantly promoted the viability of BT‐549 and MDA‐MB‐231 cells (Figure 2C,D). Consistently, OLBC15 depletion (shOLBC15) strongly inhibited the viability of breast cancer cells (Figure 2C,D). Migration assay further suggested that OLBC15 overexpression advanced migration in BT‐549 and MDA‐MB‐231 cells (Figure 2E,F; P < .01). OLBC15 silence instead attenuated migratory capacity in breast cancer cells (Figure 2E,F). These results implied that OLBC15 might play an oncogenic role in vitro.
OLBC15 promotes TNBC in vitro. A, Overexpression efficiency by lentiviral OLBC15 transfection. B, Knockdown efficiency using shRNA targeting OLBC15. ShOLBC15 displayed higher efficiency and was selected as ShOLBC15. C‐D, Effect of OLBC15 on viability of BT‐549 (C) and MDA‐MB‐231 (D) cells transfected with shRNA scrambled control (ShCtrl), ShOLBC15, lentiviral control (OE‐control), or the lentiviral vector carrying OLBC15 (OE‐OLBC15). E, Effect of OLBC15 on migratory capacity of MDA‐MB‐231 (top) and BT‐549 (bottom) cells. F, Quantification of results in (E). **: P < .01
We next checked whether OLBC15 had an effect in vitro. We first stably overexpressed or silenced OLBC15 expression in MDA‐MB‐231 and BT‐549 cells (Figure 2A,B). The knockdown and overexpression efficiency were verified (Figure 2A,B). As expected, OLBC15 overexpression significantly promoted the viability of BT‐549 and MDA‐MB‐231 cells (Figure 2C,D). Consistently, OLBC15 depletion (shOLBC15) strongly inhibited the viability of breast cancer cells (Figure 2C,D). Migration assay further suggested that OLBC15 overexpression advanced migration in BT‐549 and MDA‐MB‐231 cells (Figure 2E,F; P < .01). OLBC15 silence instead attenuated migratory capacity in breast cancer cells (Figure 2E,F). These results implied that OLBC15 might play an oncogenic role in vitro.
OLBC15 promotes TNBC in vitro. A, Overexpression efficiency by lentiviral OLBC15 transfection. B, Knockdown efficiency using shRNA targeting OLBC15. ShOLBC15 displayed higher efficiency and was selected as ShOLBC15. C‐D, Effect of OLBC15 on viability of BT‐549 (C) and MDA‐MB‐231 (D) cells transfected with shRNA scrambled control (ShCtrl), ShOLBC15, lentiviral control (OE‐control), or the lentiviral vector carrying OLBC15 (OE‐OLBC15). E, Effect of OLBC15 on migratory capacity of MDA‐MB‐231 (top) and BT‐549 (bottom) cells. F, Quantification of results in (E). **: P < .01
OLBC15 promotes xenograft tumor growth and metastasis We then performed in vivo experiments to further investigate the function of OLBC15. Stably transfected MDA‐MB‐231 cells were used. OLBC15 knockdown inhibited tumor growth, whereas overexpressing OLBC15 promoted the tumor volume expansion (Figure 3A). A higher number of metastatic nodules were detected in OLBC15‐overexpressing group compared with the control (Figure 3B,C; P < .01). As expected, OLBC15 silence profoundly inhibited the occurrence of metastatic nodules (Figure 3B,C; P < .01). The xenograft tumor growth was evidently attenuated by OLBC15 depletion, whereas OLBC15 overexpression markedly accelerated the growth of xenograft tumors (Figure 3E,F; P < .01). These results further suggested that OLBC15 could exert an oncogenic effect in vivo.
OLBC15 advances TNBC in vivo. A, In vivo tumor volume growth of xenograft tumors with OLBC15 depletion or overexpression. B, Representative lung metastatic nodules with OLBC15 silence or overexpression. The description representing each numbered case was shown at the bottom. C, Quantification of metastatic nodules in (B). D, Xenograft tumors for MDA‐MB‐231 transplantation model with OLBC15 silence or overexpression. Numbered case description was shown at the bottom. E, Quantification data for (D). **: P < .01
We then performed in vivo experiments to further investigate the function of OLBC15. Stably transfected MDA‐MB‐231 cells were used. OLBC15 knockdown inhibited tumor growth, whereas overexpressing OLBC15 promoted the tumor volume expansion (Figure 3A). A higher number of metastatic nodules were detected in OLBC15‐overexpressing group compared with the control (Figure 3B,C; P < .01). As expected, OLBC15 silence profoundly inhibited the occurrence of metastatic nodules (Figure 3B,C; P < .01). The xenograft tumor growth was evidently attenuated by OLBC15 depletion, whereas OLBC15 overexpression markedly accelerated the growth of xenograft tumors (Figure 3E,F; P < .01). These results further suggested that OLBC15 could exert an oncogenic effect in vivo.
OLBC15 advances TNBC in vivo. A, In vivo tumor volume growth of xenograft tumors with OLBC15 depletion or overexpression. B, Representative lung metastatic nodules with OLBC15 silence or overexpression. The description representing each numbered case was shown at the bottom. C, Quantification of metastatic nodules in (B). D, Xenograft tumors for MDA‐MB‐231 transplantation model with OLBC15 silence or overexpression. Numbered case description was shown at the bottom. E, Quantification data for (D). **: P < .01
OLBC15 interacts with ZNF326 protein We further used RNA pulldown assays to unravel the potential mechanism of OLBC15‐induced oncogenic effect. One specific band was detected (Figure 4A; indicated by arrow). The enriched proteins in OLBC15 pulldowns were then analyzed using mass spectrometry, and the results showed five putative hits (Table S4). Immunoblot confirmed that ZNF326 may be binding factor (Figure 4B). OLBC15 was significantly enriched in fractions with antibody against ZNF326 as determined by measuring co‐precipitated RNA (Figure 4C). Fluorescence in situ hybridization (FISH) also revealed colocalization for OLBC15 and ZNF326 (Figure 4D). Full‐length and various ZNF326 truncated mutants were constructed (Figure 4E), and we found that depleting the C‐terminal region of ZNF326 markedly abolished its interaction with OLBC15 (Figure 4F). Various OLBC15 truncated forms were also created according to the predicted secondary structure (Figure 4G,H). The results showed that the N‐terminal domain of OLBC15 was responsible for ZNF326 binding (Figure 4I). These results showed that OLBC15 may interact with ZNF326 protein.
ZNF326 interacts with OLBC15. A, RNA pulldown for lncRNA OLBC15. The indicates band was trimmed and subject to mass spectrometry (MS) analyses. B, Proteins obtained from RNA pulldowns were analyzed by antibody for ZNF326. C, RNA immunoprecipitation (RIP) results for OLBC15. The lncRNA LINC00501 was used as a negative control. D, Fluorescence in situ hybridization (FISH) to show the colocalization of OLBC15 and ZNF326. E, Full‐length and depletion constructs of ZNF326 were used to locate the binding region for OLBC15. F, RNA immunoprecipitation (RIP) followed by qRT‐PCR in DMA‐MB‐231 cells transfected with ectopic expression for full‐length (FL) Flag‐tagged ZNF326 or various mutant constructs. G, The secondary structure of OLBC15 predicted by an online tool termed RNAfold (http://rna.tbi.univie.ac.at//cgi‐bin/RNAWebSuite/RNAfold.cgi). The black box depicts the binding region for ZNF326. H, Construction of full‐length (FL) or various OLBC15 truncates. I, RIP followed by qRT‐PCR to show the putative interacting domain for OLBC15 with ZNF326. Fold enrichment data were rescaled by comparing each input with Flag‐Vec
We further used RNA pulldown assays to unravel the potential mechanism of OLBC15‐induced oncogenic effect. One specific band was detected (Figure 4A; indicated by arrow). The enriched proteins in OLBC15 pulldowns were then analyzed using mass spectrometry, and the results showed five putative hits (Table S4). Immunoblot confirmed that ZNF326 may be binding factor (Figure 4B). OLBC15 was significantly enriched in fractions with antibody against ZNF326 as determined by measuring co‐precipitated RNA (Figure 4C). Fluorescence in situ hybridization (FISH) also revealed colocalization for OLBC15 and ZNF326 (Figure 4D). Full‐length and various ZNF326 truncated mutants were constructed (Figure 4E), and we found that depleting the C‐terminal region of ZNF326 markedly abolished its interaction with OLBC15 (Figure 4F). Various OLBC15 truncated forms were also created according to the predicted secondary structure (Figure 4G,H). The results showed that the N‐terminal domain of OLBC15 was responsible for ZNF326 binding (Figure 4I). These results showed that OLBC15 may interact with ZNF326 protein.
ZNF326 interacts with OLBC15. A, RNA pulldown for lncRNA OLBC15. The indicates band was trimmed and subject to mass spectrometry (MS) analyses. B, Proteins obtained from RNA pulldowns were analyzed by antibody for ZNF326. C, RNA immunoprecipitation (RIP) results for OLBC15. The lncRNA LINC00501 was used as a negative control. D, Fluorescence in situ hybridization (FISH) to show the colocalization of OLBC15 and ZNF326. E, Full‐length and depletion constructs of ZNF326 were used to locate the binding region for OLBC15. F, RNA immunoprecipitation (RIP) followed by qRT‐PCR in DMA‐MB‐231 cells transfected with ectopic expression for full‐length (FL) Flag‐tagged ZNF326 or various mutant constructs. G, The secondary structure of OLBC15 predicted by an online tool termed RNAfold (http://rna.tbi.univie.ac.at//cgi‐bin/RNAWebSuite/RNAfold.cgi). The black box depicts the binding region for ZNF326. H, Construction of full‐length (FL) or various OLBC15 truncates. I, RIP followed by qRT‐PCR to show the putative interacting domain for OLBC15 with ZNF326. Fold enrichment data were rescaled by comparing each input with Flag‐Vec
OLBC15 facilitates ZNF326 degradation To confirm the underlying mechanism of OLBC15‐ZNF326 binding, we knocked down OLBC15 expression in MDA‐MB‐231 cells and we found that ZNF326 abundance was increased (Figure 5A). Co‐treatment with MG132, a proteasomal inhibitor, however, could elevate ZNF326 levels irrespective of whether OLBC15 was silenced (Figure 5B). OLBC15 knockdown substantially inhibited ZNF326 ubiquitination (Figure 5C). However, OLBC15 overexpression or depletion did not affect ZNF326 mRNA levels (Figure 5D). Migration assays revealed that knocking down OLBC15 could significantly inhibit breast cancer cell migration (Figure 5E,F). However, this effect could be counteracted by combinatorial ZNF326 silence (Figure 5E,F). The efficiency of ZNF326 silence was also verified (Figure 5G). Previous study has shown that ZNF326 can bind the promoter region of Krüppel‐like factor 17 (KLF17) gene and induce KLF17 expression.
16
OLBC15 knockdown indeed dramatically upregulated KLF17 expression, whereas KLF17 expression was inhibited after ZNF326 was silenced (Figure S2A). Notably, KLF17 level was partially rescued with simultaneous ZNF326/OLBC15 silence (Figure S2A). The occupancies of ZNF326 to KLF17 promoter were also increased when OLBC15 was depleted (Figure S2B). These data suggested that OLBC15 could promote ZNF326 degradation via ubiquitination pathway.
OLBC15 destabilizes ZNF326 by increasing ubiquitination. A, ZNF326 expression in MDA‐MB‐231 cells transfected with ShCtrl or two shOLBC15 constructs. B, ZNF326 expression in MDA‐MB‐231 cells with or without OLBC15 silence treated with DMSO (MG132‐) or MG132 (MG132+). C, Ubiquitin ligation of ZNF326 in MDA‐MB‐231 cells with or without OLBC15 depletion expressing full‐length Flag‐tagged ZNF326. D, Relative expression of ZNF326 transcripts with either OLBC15 knockdown or overexpression. E, Migration assays for MDA‐MB‐231 cells with OLBC15 knockdown and/or ZNF326 shRNA. F, Quantification data for cellular migration in (E). G, Efficiency of ZNF326 silence on ZNF326 expression. ShZNF326‐2 showed higher efficiency and was selected as ShZNF326. **: P < .01
To confirm the underlying mechanism of OLBC15‐ZNF326 binding, we knocked down OLBC15 expression in MDA‐MB‐231 cells and we found that ZNF326 abundance was increased (Figure 5A). Co‐treatment with MG132, a proteasomal inhibitor, however, could elevate ZNF326 levels irrespective of whether OLBC15 was silenced (Figure 5B). OLBC15 knockdown substantially inhibited ZNF326 ubiquitination (Figure 5C). However, OLBC15 overexpression or depletion did not affect ZNF326 mRNA levels (Figure 5D). Migration assays revealed that knocking down OLBC15 could significantly inhibit breast cancer cell migration (Figure 5E,F). However, this effect could be counteracted by combinatorial ZNF326 silence (Figure 5E,F). The efficiency of ZNF326 silence was also verified (Figure 5G). Previous study has shown that ZNF326 can bind the promoter region of Krüppel‐like factor 17 (KLF17) gene and induce KLF17 expression.
16
OLBC15 knockdown indeed dramatically upregulated KLF17 expression, whereas KLF17 expression was inhibited after ZNF326 was silenced (Figure S2A). Notably, KLF17 level was partially rescued with simultaneous ZNF326/OLBC15 silence (Figure S2A). The occupancies of ZNF326 to KLF17 promoter were also increased when OLBC15 was depleted (Figure S2B). These data suggested that OLBC15 could promote ZNF326 degradation via ubiquitination pathway.
OLBC15 destabilizes ZNF326 by increasing ubiquitination. A, ZNF326 expression in MDA‐MB‐231 cells transfected with ShCtrl or two shOLBC15 constructs. B, ZNF326 expression in MDA‐MB‐231 cells with or without OLBC15 silence treated with DMSO (MG132‐) or MG132 (MG132+). C, Ubiquitin ligation of ZNF326 in MDA‐MB‐231 cells with or without OLBC15 depletion expressing full‐length Flag‐tagged ZNF326. D, Relative expression of ZNF326 transcripts with either OLBC15 knockdown or overexpression. E, Migration assays for MDA‐MB‐231 cells with OLBC15 knockdown and/or ZNF326 shRNA. F, Quantification data for cellular migration in (E). G, Efficiency of ZNF326 silence on ZNF326 expression. ShZNF326‐2 showed higher efficiency and was selected as ShZNF326. **: P < .01
Correlation between OLBC15 and ZNF326 in clinical samples We further investigated the clinical relevance of OLBC15 and ZNF326 in human samples. Immunohistochemistry (IHC) analyses showed various degrees of ZNF326 expression with different intensity scores which represented ZNF326 expression status (Figure 6A). Consistently, we found that OLBC15 level was not significantly associated with ZNF326 transcripts (Figure 6B). However, a significantly negative correlation was evident between ZNF326 protein expression and OLBC15 (Figure 6C). These results suggested that OLBC15 could inhibit ZNF326 expression in clinical specimens.
Correlation between OLBC15 and ZNF326 in clinical samples. A, Immunohistochemical (IHC) staining to identify ZNF326 in human specimens. Representative low (0), weak (1+), intermediate (2+), and strong staining (3+) cases using H‐score method were shown. Scale bar: 100 μm. B, Pearson correlation coefficient (R) between OLBC15 and ZNF326 mRNA abundance. The P value was shown. C, Correlation between OLBC15 levels and ZNF326 IHC scores (ie, protein abundance) in clinical samples
We further investigated the clinical relevance of OLBC15 and ZNF326 in human samples. Immunohistochemistry (IHC) analyses showed various degrees of ZNF326 expression with different intensity scores which represented ZNF326 expression status (Figure 6A). Consistently, we found that OLBC15 level was not significantly associated with ZNF326 transcripts (Figure 6B). However, a significantly negative correlation was evident between ZNF326 protein expression and OLBC15 (Figure 6C). These results suggested that OLBC15 could inhibit ZNF326 expression in clinical specimens.
Correlation between OLBC15 and ZNF326 in clinical samples. A, Immunohistochemical (IHC) staining to identify ZNF326 in human specimens. Representative low (0), weak (1+), intermediate (2+), and strong staining (3+) cases using H‐score method were shown. Scale bar: 100 μm. B, Pearson correlation coefficient (R) between OLBC15 and ZNF326 mRNA abundance. The P value was shown. C, Correlation between OLBC15 levels and ZNF326 IHC scores (ie, protein abundance) in clinical samples
| null | null |
[
"INTRODUCTION",
"Cell lines, lentiviral vectors, and reagents",
"Human samples",
"Migration assay",
"Viability assay",
"Statistical analysis",
"Identifying OLBC15 in breast cancer via lncRNA profiling",
"OLBC15 facilitates breast cancer progression in vitro",
"OLBC15 promotes xenograft tumor growth and metastasis",
"OLBC15 interacts with ZNF326 protein",
"OLBC15 facilitates ZNF326 degradation",
"Correlation between OLBC15 and ZNF326 in clinical samples"
] |
[
"The breast cancer (BC) has been reported to be a primary cause of malignancy and mortality in women, and the incidence rate worldwide has been on the rise.\n1\n The breast cancer possesses a heterogeneous nature and is classified into several subtypes.\n2\n Anti‐estrogen or combined anti‐HER2 therapeutic strategies are major treatments for luminal A, luminal B, and HER2‐positive (HER2+) breast cancer subtypes.\n3\n However, the highly aggressive and metastatic triple‐negative breast cancer (TNBC, negative in estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) is associated with high relapse even after chemotherapy or radiotherapy.\n4\n, \n5\n Therefore, unraveling novel and effective targets for TNBC intervention are of great significance.\nGrowing evidence has suggested that the non‐coding RNAs with no or minimal coding abilities contribute largely to tumor development.\n6\n A major constituent of non‐coding transcriptome is long non‐coding RNAs (lncRNAs).\n6\n The long non‐coding RNAs are RNA transcripts with more than 200 nucleotides in length and involved in numerous biological processes.\n7\n The lncRNAs can serve as an oncogene or tumor suppressor with an emerging effect on metastasis.\n8\n For instance, Tang et al identified that lncRNA PVT1 can stabilize KLF5 via BAP1 and increase its stability to promote TNBC progression.\n9\n Alipoor et al recently found that lncRNA MIAT increases miR‐302/miR‐150 expression, whereas miR‐29c expression was decreased.\n10\n Furthermore, MIAT also abolishes the expression of p16Ink4A and Cox2 to inhibit senescence commitment.\n10\n A TNBC‐specific lncRNA LINC00511 is markedly upregulated through gene amplification and significantly correlates with TNBC progression.\n11\n Sas‐Chen et al showed that lncRNA LIMT is a highly conserved tumor suppressor and its expression is suppressed by epidermal growth factor (EGF) signaling pathway.\n12\n Other reported lncRNAs (eg, RMST and BORG) are substantially associated with breast cancer development via diverse molecular mechanisms.\n13\n, \n14\n However, the function of lncRNAs especially in TNBC is still obscure.\nIn the current work, by lncRNA profiling, we have identified a novel lncRNA ENSG00000259457 as an oncogenic lncRNA in TNBC. It was termed as oncogenic lncRNA in breast cancer on chromosome 15 (OLBC15). We observed that OLBC15 is significantly upregulated in TNBC. OLBC15 acted as an oncogenic factor to promote breast cancer progression, and this effect was verified both in vivo and in vitro. Furthermore, by RNA immunoprecipitation we noted that zinc‐finger protein‐326 (ZNF326) might be an OLBC15 binding protein. The interaction of OLBC15 and ZNF326 decreased the stability of ZNF326 via enhanced ubiquitination and proteasomal degradation. OLBC15 also showed negative correlation with ZNF326 protein expression in clinical samples. These data collectively implied an oncogenic role of OLBC15 and may provide a novel anti‐TNBC target for therapeutic intervention.",
"The transformed MCF‐10A and breast cancer MDA‐MB‐231, BT‐549, HCC1806, Hs578t, and MDA‐MB‐468 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 7% FBS and 150 μg/mL streptomycin. The cell lines were all obtained from Cell Resource Center of Shanghai Institute of Biological Sciences (CAS). Puromycin was used for 2 days. OLBC15 was first cloned and was then inserted into a pWPXL vector (Biovector) to generate pWPXL‐OLBC15 vector (OE‐OLBC15). Lentiviral selection was then conducted using puromycin (No.A1113802, Thermo Fisher Scientific), and generated supernatants were treated with a syringe filter. An empty pWPXL vector serves as the control (ie, OE‐control). The short hairpin RNA (shRNA) for OLBC15 (ShOLBC15) together with a non‐targeting scrambled control (ShCtrl) was obtained from Biovector. Transfection was conducted using Lipofectamine 3000. Primers were listed in Table S1.",
"The breast cancer tissues were collected from patients following a protocol described previously\n15\n from January 2017 to November 2018. The samples were stored at −80℃ immediately after resection before use. Experiments associated with human specimens were approved by Human Research Ethics Committee (HREC) at Chongqing Three Gorges Central Hospital in accordance with the 1975 Declaration of Helsinki.",
"~5 × 105 cells were located on the top chamber of a 24‐well plate (BD Biosciences). The DMEM with 7% serum was loaded at the bottom. After an incubation for 24 hours, cells moving into the bottom chambers were fixed with 4% paraformaldehyde (No.16005, Sigma) and subject to staining by 0.4% crystal violet (No.C0775, Sigma).",
"We used Cell Counting Kit‐8 (CCK‐8, Dojindo, Japan) to measure the viability of breast cancer cells following the manufacturer's protocols. BT549 and MDA‐MB‐231 cells were resuspended and moved into a 12‐well plate (~2 × 105 cells/well). 30 µL CCK‐8 solutions were then added into the system. Optical density at 450 nm (OD 450 nm) was measured by a Spectramax M5 microplate monitor (Molecular Devices).",
"Statistical analyses were performed with SPSS (v16, SPSS, Inc). Data were represented as mean ± SD. The nonparametric Mann‐Whitney test was used to identify the statistical significance between two groups, and ANOVA was used for comparing multiple groups followed by the LSD post hoc test. P < .05 was considered statistically significant.",
"To screen for breast cancer (BC)‐related lncRNAs, we used lncRNA profiling assays in paired BC tissues and normal adjacent tissues (NATs) (Figure 1A). Overall, 73 significantly upregulated lncRNAs were detected (Figure 1A). These lncRNAs were candidate ones with oncogenic potential. Notably, two novel lncRNAs, RP11‐597D13.9 and OLBC15, were identified by lncRNA profiling (Table S2). Since lncRNA OLBC15 showed higher fold expression, we chose it for further analysis. OLBC15 was markedly upregulated in BC tissues compared with normal tissues (Figure 1B). OLBC15 is located on chromosome 15 with one annotated transcript (www.ensembl.org). The whole sequence for OLBC15 was shown (Figure S1A). Full‐length OLBC15 showed neglectable coding potential as evaluated by Coding Potential Assessment Tool (CPAT, coding probability ~ .032, Figure S1B). Moreover, OLBC15 was substantially enriched in triple‐negative breast cancer (TNBC) tissues compared with the other subtypes (Figure 1C). OLBC15 transcripts were highly increased in BC cell lines compared with MCF10A cells (Figure 1D). Patient characteristics demonstrated that OLBC15 expression significantly correlated with TNM stage, tumor size, and metastasis but was not significantly associated with age and histology (Table S3). Subcellular fractionation assay clarified that OLBC15 was primarily located in nucleus (Figure 1E). These data suggested that OLBC15 was upregulated in BC with nuclear distribution.\nIdentifying OLBC15 in breast cancer (BC). A, Heatmap representation of lncRNA profiling in normal adjacent tissues (NATs) and breast cancer tissues. Significantly upregulated and downregulated lncRNAs in BC tissues were 73 and 126, respectively. B, Relative OLBC15 expression in NAT and BC tissues. C, The relative OLBC15 levels in different breast cancer subtypes. The case numbers for luminal A (LA), luminal B (LB), HER2+ (HER2), and triple‐negative breast cancer (TNBC) were 40, 25, 26, and 51, respectively. D, Relative OLBC15 expression in transformed MCF10A and cancerous TNBC cell lines. E, Subcellular distribution of OLBC15. **: P < .01",
"We next checked whether OLBC15 had an effect in vitro. We first stably overexpressed or silenced OLBC15 expression in MDA‐MB‐231 and BT‐549 cells (Figure 2A,B). The knockdown and overexpression efficiency were verified (Figure 2A,B). As expected, OLBC15 overexpression significantly promoted the viability of BT‐549 and MDA‐MB‐231 cells (Figure 2C,D). Consistently, OLBC15 depletion (shOLBC15) strongly inhibited the viability of breast cancer cells (Figure 2C,D). Migration assay further suggested that OLBC15 overexpression advanced migration in BT‐549 and MDA‐MB‐231 cells (Figure 2E,F; P < .01). OLBC15 silence instead attenuated migratory capacity in breast cancer cells (Figure 2E,F). These results implied that OLBC15 might play an oncogenic role in vitro.\nOLBC15 promotes TNBC in vitro. A, Overexpression efficiency by lentiviral OLBC15 transfection. B, Knockdown efficiency using shRNA targeting OLBC15. ShOLBC15 displayed higher efficiency and was selected as ShOLBC15. C‐D, Effect of OLBC15 on viability of BT‐549 (C) and MDA‐MB‐231 (D) cells transfected with shRNA scrambled control (ShCtrl), ShOLBC15, lentiviral control (OE‐control), or the lentiviral vector carrying OLBC15 (OE‐OLBC15). E, Effect of OLBC15 on migratory capacity of MDA‐MB‐231 (top) and BT‐549 (bottom) cells. F, Quantification of results in (E). **: P < .01",
"We then performed in vivo experiments to further investigate the function of OLBC15. Stably transfected MDA‐MB‐231 cells were used. OLBC15 knockdown inhibited tumor growth, whereas overexpressing OLBC15 promoted the tumor volume expansion (Figure 3A). A higher number of metastatic nodules were detected in OLBC15‐overexpressing group compared with the control (Figure 3B,C; P < .01). As expected, OLBC15 silence profoundly inhibited the occurrence of metastatic nodules (Figure 3B,C; P < .01). The xenograft tumor growth was evidently attenuated by OLBC15 depletion, whereas OLBC15 overexpression markedly accelerated the growth of xenograft tumors (Figure 3E,F; P < .01). These results further suggested that OLBC15 could exert an oncogenic effect in vivo.\nOLBC15 advances TNBC in vivo. A, In vivo tumor volume growth of xenograft tumors with OLBC15 depletion or overexpression. B, Representative lung metastatic nodules with OLBC15 silence or overexpression. The description representing each numbered case was shown at the bottom. C, Quantification of metastatic nodules in (B). D, Xenograft tumors for MDA‐MB‐231 transplantation model with OLBC15 silence or overexpression. Numbered case description was shown at the bottom. E, Quantification data for (D). **: P < .01",
"We further used RNA pulldown assays to unravel the potential mechanism of OLBC15‐induced oncogenic effect. One specific band was detected (Figure 4A; indicated by arrow). The enriched proteins in OLBC15 pulldowns were then analyzed using mass spectrometry, and the results showed five putative hits (Table S4). Immunoblot confirmed that ZNF326 may be binding factor (Figure 4B). OLBC15 was significantly enriched in fractions with antibody against ZNF326 as determined by measuring co‐precipitated RNA (Figure 4C). Fluorescence in situ hybridization (FISH) also revealed colocalization for OLBC15 and ZNF326 (Figure 4D). Full‐length and various ZNF326 truncated mutants were constructed (Figure 4E), and we found that depleting the C‐terminal region of ZNF326 markedly abolished its interaction with OLBC15 (Figure 4F). Various OLBC15 truncated forms were also created according to the predicted secondary structure (Figure 4G,H). The results showed that the N‐terminal domain of OLBC15 was responsible for ZNF326 binding (Figure 4I). These results showed that OLBC15 may interact with ZNF326 protein.\nZNF326 interacts with OLBC15. A, RNA pulldown for lncRNA OLBC15. The indicates band was trimmed and subject to mass spectrometry (MS) analyses. B, Proteins obtained from RNA pulldowns were analyzed by antibody for ZNF326. C, RNA immunoprecipitation (RIP) results for OLBC15. The lncRNA LINC00501 was used as a negative control. D, Fluorescence in situ hybridization (FISH) to show the colocalization of OLBC15 and ZNF326. E, Full‐length and depletion constructs of ZNF326 were used to locate the binding region for OLBC15. F, RNA immunoprecipitation (RIP) followed by qRT‐PCR in DMA‐MB‐231 cells transfected with ectopic expression for full‐length (FL) Flag‐tagged ZNF326 or various mutant constructs. G, The secondary structure of OLBC15 predicted by an online tool termed RNAfold (http://rna.tbi.univie.ac.at//cgi‐bin/RNAWebSuite/RNAfold.cgi). The black box depicts the binding region for ZNF326. H, Construction of full‐length (FL) or various OLBC15 truncates. I, RIP followed by qRT‐PCR to show the putative interacting domain for OLBC15 with ZNF326. Fold enrichment data were rescaled by comparing each input with Flag‐Vec",
"To confirm the underlying mechanism of OLBC15‐ZNF326 binding, we knocked down OLBC15 expression in MDA‐MB‐231 cells and we found that ZNF326 abundance was increased (Figure 5A). Co‐treatment with MG132, a proteasomal inhibitor, however, could elevate ZNF326 levels irrespective of whether OLBC15 was silenced (Figure 5B). OLBC15 knockdown substantially inhibited ZNF326 ubiquitination (Figure 5C). However, OLBC15 overexpression or depletion did not affect ZNF326 mRNA levels (Figure 5D). Migration assays revealed that knocking down OLBC15 could significantly inhibit breast cancer cell migration (Figure 5E,F). However, this effect could be counteracted by combinatorial ZNF326 silence (Figure 5E,F). The efficiency of ZNF326 silence was also verified (Figure 5G). Previous study has shown that ZNF326 can bind the promoter region of Krüppel‐like factor 17 (KLF17) gene and induce KLF17 expression.\n16\n OLBC15 knockdown indeed dramatically upregulated KLF17 expression, whereas KLF17 expression was inhibited after ZNF326 was silenced (Figure S2A). Notably, KLF17 level was partially rescued with simultaneous ZNF326/OLBC15 silence (Figure S2A). The occupancies of ZNF326 to KLF17 promoter were also increased when OLBC15 was depleted (Figure S2B). These data suggested that OLBC15 could promote ZNF326 degradation via ubiquitination pathway.\nOLBC15 destabilizes ZNF326 by increasing ubiquitination. A, ZNF326 expression in MDA‐MB‐231 cells transfected with ShCtrl or two shOLBC15 constructs. B, ZNF326 expression in MDA‐MB‐231 cells with or without OLBC15 silence treated with DMSO (MG132‐) or MG132 (MG132+). C, Ubiquitin ligation of ZNF326 in MDA‐MB‐231 cells with or without OLBC15 depletion expressing full‐length Flag‐tagged ZNF326. D, Relative expression of ZNF326 transcripts with either OLBC15 knockdown or overexpression. E, Migration assays for MDA‐MB‐231 cells with OLBC15 knockdown and/or ZNF326 shRNA. F, Quantification data for cellular migration in (E). G, Efficiency of ZNF326 silence on ZNF326 expression. ShZNF326‐2 showed higher efficiency and was selected as ShZNF326. **: P < .01",
"We further investigated the clinical relevance of OLBC15 and ZNF326 in human samples. Immunohistochemistry (IHC) analyses showed various degrees of ZNF326 expression with different intensity scores which represented ZNF326 expression status (Figure 6A). Consistently, we found that OLBC15 level was not significantly associated with ZNF326 transcripts (Figure 6B). However, a significantly negative correlation was evident between ZNF326 protein expression and OLBC15 (Figure 6C). These results suggested that OLBC15 could inhibit ZNF326 expression in clinical specimens.\nCorrelation between OLBC15 and ZNF326 in clinical samples. A, Immunohistochemical (IHC) staining to identify ZNF326 in human specimens. Representative low (0), weak (1+), intermediate (2+), and strong staining (3+) cases using H‐score method were shown. Scale bar: 100 μm. B, Pearson correlation coefficient (R) between OLBC15 and ZNF326 mRNA abundance. The P value was shown. C, Correlation between OLBC15 levels and ZNF326 IHC scores (ie, protein abundance) in clinical samples"
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[
"INTRODUCTION",
"MATERIALS AND METHODS",
"Cell lines, lentiviral vectors, and reagents",
"Human samples",
"Migration assay",
"Viability assay",
"Statistical analysis",
"RESULTS",
"Identifying OLBC15 in breast cancer via lncRNA profiling",
"OLBC15 facilitates breast cancer progression in vitro",
"OLBC15 promotes xenograft tumor growth and metastasis",
"OLBC15 interacts with ZNF326 protein",
"OLBC15 facilitates ZNF326 degradation",
"Correlation between OLBC15 and ZNF326 in clinical samples",
"DISCUSSION",
"Supporting information"
] |
[
"The breast cancer (BC) has been reported to be a primary cause of malignancy and mortality in women, and the incidence rate worldwide has been on the rise.\n1\n The breast cancer possesses a heterogeneous nature and is classified into several subtypes.\n2\n Anti‐estrogen or combined anti‐HER2 therapeutic strategies are major treatments for luminal A, luminal B, and HER2‐positive (HER2+) breast cancer subtypes.\n3\n However, the highly aggressive and metastatic triple‐negative breast cancer (TNBC, negative in estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) is associated with high relapse even after chemotherapy or radiotherapy.\n4\n, \n5\n Therefore, unraveling novel and effective targets for TNBC intervention are of great significance.\nGrowing evidence has suggested that the non‐coding RNAs with no or minimal coding abilities contribute largely to tumor development.\n6\n A major constituent of non‐coding transcriptome is long non‐coding RNAs (lncRNAs).\n6\n The long non‐coding RNAs are RNA transcripts with more than 200 nucleotides in length and involved in numerous biological processes.\n7\n The lncRNAs can serve as an oncogene or tumor suppressor with an emerging effect on metastasis.\n8\n For instance, Tang et al identified that lncRNA PVT1 can stabilize KLF5 via BAP1 and increase its stability to promote TNBC progression.\n9\n Alipoor et al recently found that lncRNA MIAT increases miR‐302/miR‐150 expression, whereas miR‐29c expression was decreased.\n10\n Furthermore, MIAT also abolishes the expression of p16Ink4A and Cox2 to inhibit senescence commitment.\n10\n A TNBC‐specific lncRNA LINC00511 is markedly upregulated through gene amplification and significantly correlates with TNBC progression.\n11\n Sas‐Chen et al showed that lncRNA LIMT is a highly conserved tumor suppressor and its expression is suppressed by epidermal growth factor (EGF) signaling pathway.\n12\n Other reported lncRNAs (eg, RMST and BORG) are substantially associated with breast cancer development via diverse molecular mechanisms.\n13\n, \n14\n However, the function of lncRNAs especially in TNBC is still obscure.\nIn the current work, by lncRNA profiling, we have identified a novel lncRNA ENSG00000259457 as an oncogenic lncRNA in TNBC. It was termed as oncogenic lncRNA in breast cancer on chromosome 15 (OLBC15). We observed that OLBC15 is significantly upregulated in TNBC. OLBC15 acted as an oncogenic factor to promote breast cancer progression, and this effect was verified both in vivo and in vitro. Furthermore, by RNA immunoprecipitation we noted that zinc‐finger protein‐326 (ZNF326) might be an OLBC15 binding protein. The interaction of OLBC15 and ZNF326 decreased the stability of ZNF326 via enhanced ubiquitination and proteasomal degradation. OLBC15 also showed negative correlation with ZNF326 protein expression in clinical samples. These data collectively implied an oncogenic role of OLBC15 and may provide a novel anti‐TNBC target for therapeutic intervention.",
" Cell lines, lentiviral vectors, and reagents The transformed MCF‐10A and breast cancer MDA‐MB‐231, BT‐549, HCC1806, Hs578t, and MDA‐MB‐468 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 7% FBS and 150 μg/mL streptomycin. The cell lines were all obtained from Cell Resource Center of Shanghai Institute of Biological Sciences (CAS). Puromycin was used for 2 days. OLBC15 was first cloned and was then inserted into a pWPXL vector (Biovector) to generate pWPXL‐OLBC15 vector (OE‐OLBC15). Lentiviral selection was then conducted using puromycin (No.A1113802, Thermo Fisher Scientific), and generated supernatants were treated with a syringe filter. An empty pWPXL vector serves as the control (ie, OE‐control). The short hairpin RNA (shRNA) for OLBC15 (ShOLBC15) together with a non‐targeting scrambled control (ShCtrl) was obtained from Biovector. Transfection was conducted using Lipofectamine 3000. Primers were listed in Table S1.\nThe transformed MCF‐10A and breast cancer MDA‐MB‐231, BT‐549, HCC1806, Hs578t, and MDA‐MB‐468 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 7% FBS and 150 μg/mL streptomycin. The cell lines were all obtained from Cell Resource Center of Shanghai Institute of Biological Sciences (CAS). Puromycin was used for 2 days. OLBC15 was first cloned and was then inserted into a pWPXL vector (Biovector) to generate pWPXL‐OLBC15 vector (OE‐OLBC15). Lentiviral selection was then conducted using puromycin (No.A1113802, Thermo Fisher Scientific), and generated supernatants were treated with a syringe filter. An empty pWPXL vector serves as the control (ie, OE‐control). The short hairpin RNA (shRNA) for OLBC15 (ShOLBC15) together with a non‐targeting scrambled control (ShCtrl) was obtained from Biovector. Transfection was conducted using Lipofectamine 3000. Primers were listed in Table S1.\n Human samples The breast cancer tissues were collected from patients following a protocol described previously\n15\n from January 2017 to November 2018. The samples were stored at −80℃ immediately after resection before use. Experiments associated with human specimens were approved by Human Research Ethics Committee (HREC) at Chongqing Three Gorges Central Hospital in accordance with the 1975 Declaration of Helsinki.\nThe breast cancer tissues were collected from patients following a protocol described previously\n15\n from January 2017 to November 2018. The samples were stored at −80℃ immediately after resection before use. Experiments associated with human specimens were approved by Human Research Ethics Committee (HREC) at Chongqing Three Gorges Central Hospital in accordance with the 1975 Declaration of Helsinki.\n Migration assay ~5 × 105 cells were located on the top chamber of a 24‐well plate (BD Biosciences). The DMEM with 7% serum was loaded at the bottom. After an incubation for 24 hours, cells moving into the bottom chambers were fixed with 4% paraformaldehyde (No.16005, Sigma) and subject to staining by 0.4% crystal violet (No.C0775, Sigma).\n~5 × 105 cells were located on the top chamber of a 24‐well plate (BD Biosciences). The DMEM with 7% serum was loaded at the bottom. After an incubation for 24 hours, cells moving into the bottom chambers were fixed with 4% paraformaldehyde (No.16005, Sigma) and subject to staining by 0.4% crystal violet (No.C0775, Sigma).\n Viability assay We used Cell Counting Kit‐8 (CCK‐8, Dojindo, Japan) to measure the viability of breast cancer cells following the manufacturer's protocols. BT549 and MDA‐MB‐231 cells were resuspended and moved into a 12‐well plate (~2 × 105 cells/well). 30 µL CCK‐8 solutions were then added into the system. Optical density at 450 nm (OD 450 nm) was measured by a Spectramax M5 microplate monitor (Molecular Devices).\nWe used Cell Counting Kit‐8 (CCK‐8, Dojindo, Japan) to measure the viability of breast cancer cells following the manufacturer's protocols. BT549 and MDA‐MB‐231 cells were resuspended and moved into a 12‐well plate (~2 × 105 cells/well). 30 µL CCK‐8 solutions were then added into the system. Optical density at 450 nm (OD 450 nm) was measured by a Spectramax M5 microplate monitor (Molecular Devices).\n Statistical analysis Statistical analyses were performed with SPSS (v16, SPSS, Inc). Data were represented as mean ± SD. The nonparametric Mann‐Whitney test was used to identify the statistical significance between two groups, and ANOVA was used for comparing multiple groups followed by the LSD post hoc test. P < .05 was considered statistically significant.\nStatistical analyses were performed with SPSS (v16, SPSS, Inc). Data were represented as mean ± SD. The nonparametric Mann‐Whitney test was used to identify the statistical significance between two groups, and ANOVA was used for comparing multiple groups followed by the LSD post hoc test. P < .05 was considered statistically significant.",
"The transformed MCF‐10A and breast cancer MDA‐MB‐231, BT‐549, HCC1806, Hs578t, and MDA‐MB‐468 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 7% FBS and 150 μg/mL streptomycin. The cell lines were all obtained from Cell Resource Center of Shanghai Institute of Biological Sciences (CAS). Puromycin was used for 2 days. OLBC15 was first cloned and was then inserted into a pWPXL vector (Biovector) to generate pWPXL‐OLBC15 vector (OE‐OLBC15). Lentiviral selection was then conducted using puromycin (No.A1113802, Thermo Fisher Scientific), and generated supernatants were treated with a syringe filter. An empty pWPXL vector serves as the control (ie, OE‐control). The short hairpin RNA (shRNA) for OLBC15 (ShOLBC15) together with a non‐targeting scrambled control (ShCtrl) was obtained from Biovector. Transfection was conducted using Lipofectamine 3000. Primers were listed in Table S1.",
"The breast cancer tissues were collected from patients following a protocol described previously\n15\n from January 2017 to November 2018. The samples were stored at −80℃ immediately after resection before use. Experiments associated with human specimens were approved by Human Research Ethics Committee (HREC) at Chongqing Three Gorges Central Hospital in accordance with the 1975 Declaration of Helsinki.",
"~5 × 105 cells were located on the top chamber of a 24‐well plate (BD Biosciences). The DMEM with 7% serum was loaded at the bottom. After an incubation for 24 hours, cells moving into the bottom chambers were fixed with 4% paraformaldehyde (No.16005, Sigma) and subject to staining by 0.4% crystal violet (No.C0775, Sigma).",
"We used Cell Counting Kit‐8 (CCK‐8, Dojindo, Japan) to measure the viability of breast cancer cells following the manufacturer's protocols. BT549 and MDA‐MB‐231 cells were resuspended and moved into a 12‐well plate (~2 × 105 cells/well). 30 µL CCK‐8 solutions were then added into the system. Optical density at 450 nm (OD 450 nm) was measured by a Spectramax M5 microplate monitor (Molecular Devices).",
"Statistical analyses were performed with SPSS (v16, SPSS, Inc). Data were represented as mean ± SD. The nonparametric Mann‐Whitney test was used to identify the statistical significance between two groups, and ANOVA was used for comparing multiple groups followed by the LSD post hoc test. P < .05 was considered statistically significant.",
" Identifying OLBC15 in breast cancer via lncRNA profiling To screen for breast cancer (BC)‐related lncRNAs, we used lncRNA profiling assays in paired BC tissues and normal adjacent tissues (NATs) (Figure 1A). Overall, 73 significantly upregulated lncRNAs were detected (Figure 1A). These lncRNAs were candidate ones with oncogenic potential. Notably, two novel lncRNAs, RP11‐597D13.9 and OLBC15, were identified by lncRNA profiling (Table S2). Since lncRNA OLBC15 showed higher fold expression, we chose it for further analysis. OLBC15 was markedly upregulated in BC tissues compared with normal tissues (Figure 1B). OLBC15 is located on chromosome 15 with one annotated transcript (www.ensembl.org). The whole sequence for OLBC15 was shown (Figure S1A). Full‐length OLBC15 showed neglectable coding potential as evaluated by Coding Potential Assessment Tool (CPAT, coding probability ~ .032, Figure S1B). Moreover, OLBC15 was substantially enriched in triple‐negative breast cancer (TNBC) tissues compared with the other subtypes (Figure 1C). OLBC15 transcripts were highly increased in BC cell lines compared with MCF10A cells (Figure 1D). Patient characteristics demonstrated that OLBC15 expression significantly correlated with TNM stage, tumor size, and metastasis but was not significantly associated with age and histology (Table S3). Subcellular fractionation assay clarified that OLBC15 was primarily located in nucleus (Figure 1E). These data suggested that OLBC15 was upregulated in BC with nuclear distribution.\nIdentifying OLBC15 in breast cancer (BC). A, Heatmap representation of lncRNA profiling in normal adjacent tissues (NATs) and breast cancer tissues. Significantly upregulated and downregulated lncRNAs in BC tissues were 73 and 126, respectively. B, Relative OLBC15 expression in NAT and BC tissues. C, The relative OLBC15 levels in different breast cancer subtypes. The case numbers for luminal A (LA), luminal B (LB), HER2+ (HER2), and triple‐negative breast cancer (TNBC) were 40, 25, 26, and 51, respectively. D, Relative OLBC15 expression in transformed MCF10A and cancerous TNBC cell lines. E, Subcellular distribution of OLBC15. **: P < .01\nTo screen for breast cancer (BC)‐related lncRNAs, we used lncRNA profiling assays in paired BC tissues and normal adjacent tissues (NATs) (Figure 1A). Overall, 73 significantly upregulated lncRNAs were detected (Figure 1A). These lncRNAs were candidate ones with oncogenic potential. Notably, two novel lncRNAs, RP11‐597D13.9 and OLBC15, were identified by lncRNA profiling (Table S2). Since lncRNA OLBC15 showed higher fold expression, we chose it for further analysis. OLBC15 was markedly upregulated in BC tissues compared with normal tissues (Figure 1B). OLBC15 is located on chromosome 15 with one annotated transcript (www.ensembl.org). The whole sequence for OLBC15 was shown (Figure S1A). Full‐length OLBC15 showed neglectable coding potential as evaluated by Coding Potential Assessment Tool (CPAT, coding probability ~ .032, Figure S1B). Moreover, OLBC15 was substantially enriched in triple‐negative breast cancer (TNBC) tissues compared with the other subtypes (Figure 1C). OLBC15 transcripts were highly increased in BC cell lines compared with MCF10A cells (Figure 1D). Patient characteristics demonstrated that OLBC15 expression significantly correlated with TNM stage, tumor size, and metastasis but was not significantly associated with age and histology (Table S3). Subcellular fractionation assay clarified that OLBC15 was primarily located in nucleus (Figure 1E). These data suggested that OLBC15 was upregulated in BC with nuclear distribution.\nIdentifying OLBC15 in breast cancer (BC). A, Heatmap representation of lncRNA profiling in normal adjacent tissues (NATs) and breast cancer tissues. Significantly upregulated and downregulated lncRNAs in BC tissues were 73 and 126, respectively. B, Relative OLBC15 expression in NAT and BC tissues. C, The relative OLBC15 levels in different breast cancer subtypes. The case numbers for luminal A (LA), luminal B (LB), HER2+ (HER2), and triple‐negative breast cancer (TNBC) were 40, 25, 26, and 51, respectively. D, Relative OLBC15 expression in transformed MCF10A and cancerous TNBC cell lines. E, Subcellular distribution of OLBC15. **: P < .01\n OLBC15 facilitates breast cancer progression in vitro We next checked whether OLBC15 had an effect in vitro. We first stably overexpressed or silenced OLBC15 expression in MDA‐MB‐231 and BT‐549 cells (Figure 2A,B). The knockdown and overexpression efficiency were verified (Figure 2A,B). As expected, OLBC15 overexpression significantly promoted the viability of BT‐549 and MDA‐MB‐231 cells (Figure 2C,D). Consistently, OLBC15 depletion (shOLBC15) strongly inhibited the viability of breast cancer cells (Figure 2C,D). Migration assay further suggested that OLBC15 overexpression advanced migration in BT‐549 and MDA‐MB‐231 cells (Figure 2E,F; P < .01). OLBC15 silence instead attenuated migratory capacity in breast cancer cells (Figure 2E,F). These results implied that OLBC15 might play an oncogenic role in vitro.\nOLBC15 promotes TNBC in vitro. A, Overexpression efficiency by lentiviral OLBC15 transfection. B, Knockdown efficiency using shRNA targeting OLBC15. ShOLBC15 displayed higher efficiency and was selected as ShOLBC15. C‐D, Effect of OLBC15 on viability of BT‐549 (C) and MDA‐MB‐231 (D) cells transfected with shRNA scrambled control (ShCtrl), ShOLBC15, lentiviral control (OE‐control), or the lentiviral vector carrying OLBC15 (OE‐OLBC15). E, Effect of OLBC15 on migratory capacity of MDA‐MB‐231 (top) and BT‐549 (bottom) cells. F, Quantification of results in (E). **: P < .01\nWe next checked whether OLBC15 had an effect in vitro. We first stably overexpressed or silenced OLBC15 expression in MDA‐MB‐231 and BT‐549 cells (Figure 2A,B). The knockdown and overexpression efficiency were verified (Figure 2A,B). As expected, OLBC15 overexpression significantly promoted the viability of BT‐549 and MDA‐MB‐231 cells (Figure 2C,D). Consistently, OLBC15 depletion (shOLBC15) strongly inhibited the viability of breast cancer cells (Figure 2C,D). Migration assay further suggested that OLBC15 overexpression advanced migration in BT‐549 and MDA‐MB‐231 cells (Figure 2E,F; P < .01). OLBC15 silence instead attenuated migratory capacity in breast cancer cells (Figure 2E,F). These results implied that OLBC15 might play an oncogenic role in vitro.\nOLBC15 promotes TNBC in vitro. A, Overexpression efficiency by lentiviral OLBC15 transfection. B, Knockdown efficiency using shRNA targeting OLBC15. ShOLBC15 displayed higher efficiency and was selected as ShOLBC15. C‐D, Effect of OLBC15 on viability of BT‐549 (C) and MDA‐MB‐231 (D) cells transfected with shRNA scrambled control (ShCtrl), ShOLBC15, lentiviral control (OE‐control), or the lentiviral vector carrying OLBC15 (OE‐OLBC15). E, Effect of OLBC15 on migratory capacity of MDA‐MB‐231 (top) and BT‐549 (bottom) cells. F, Quantification of results in (E). **: P < .01\n OLBC15 promotes xenograft tumor growth and metastasis We then performed in vivo experiments to further investigate the function of OLBC15. Stably transfected MDA‐MB‐231 cells were used. OLBC15 knockdown inhibited tumor growth, whereas overexpressing OLBC15 promoted the tumor volume expansion (Figure 3A). A higher number of metastatic nodules were detected in OLBC15‐overexpressing group compared with the control (Figure 3B,C; P < .01). As expected, OLBC15 silence profoundly inhibited the occurrence of metastatic nodules (Figure 3B,C; P < .01). The xenograft tumor growth was evidently attenuated by OLBC15 depletion, whereas OLBC15 overexpression markedly accelerated the growth of xenograft tumors (Figure 3E,F; P < .01). These results further suggested that OLBC15 could exert an oncogenic effect in vivo.\nOLBC15 advances TNBC in vivo. A, In vivo tumor volume growth of xenograft tumors with OLBC15 depletion or overexpression. B, Representative lung metastatic nodules with OLBC15 silence or overexpression. The description representing each numbered case was shown at the bottom. C, Quantification of metastatic nodules in (B). D, Xenograft tumors for MDA‐MB‐231 transplantation model with OLBC15 silence or overexpression. Numbered case description was shown at the bottom. E, Quantification data for (D). **: P < .01\nWe then performed in vivo experiments to further investigate the function of OLBC15. Stably transfected MDA‐MB‐231 cells were used. OLBC15 knockdown inhibited tumor growth, whereas overexpressing OLBC15 promoted the tumor volume expansion (Figure 3A). A higher number of metastatic nodules were detected in OLBC15‐overexpressing group compared with the control (Figure 3B,C; P < .01). As expected, OLBC15 silence profoundly inhibited the occurrence of metastatic nodules (Figure 3B,C; P < .01). The xenograft tumor growth was evidently attenuated by OLBC15 depletion, whereas OLBC15 overexpression markedly accelerated the growth of xenograft tumors (Figure 3E,F; P < .01). These results further suggested that OLBC15 could exert an oncogenic effect in vivo.\nOLBC15 advances TNBC in vivo. A, In vivo tumor volume growth of xenograft tumors with OLBC15 depletion or overexpression. B, Representative lung metastatic nodules with OLBC15 silence or overexpression. The description representing each numbered case was shown at the bottom. C, Quantification of metastatic nodules in (B). D, Xenograft tumors for MDA‐MB‐231 transplantation model with OLBC15 silence or overexpression. Numbered case description was shown at the bottom. E, Quantification data for (D). **: P < .01\n OLBC15 interacts with ZNF326 protein We further used RNA pulldown assays to unravel the potential mechanism of OLBC15‐induced oncogenic effect. One specific band was detected (Figure 4A; indicated by arrow). The enriched proteins in OLBC15 pulldowns were then analyzed using mass spectrometry, and the results showed five putative hits (Table S4). Immunoblot confirmed that ZNF326 may be binding factor (Figure 4B). OLBC15 was significantly enriched in fractions with antibody against ZNF326 as determined by measuring co‐precipitated RNA (Figure 4C). Fluorescence in situ hybridization (FISH) also revealed colocalization for OLBC15 and ZNF326 (Figure 4D). Full‐length and various ZNF326 truncated mutants were constructed (Figure 4E), and we found that depleting the C‐terminal region of ZNF326 markedly abolished its interaction with OLBC15 (Figure 4F). Various OLBC15 truncated forms were also created according to the predicted secondary structure (Figure 4G,H). The results showed that the N‐terminal domain of OLBC15 was responsible for ZNF326 binding (Figure 4I). These results showed that OLBC15 may interact with ZNF326 protein.\nZNF326 interacts with OLBC15. A, RNA pulldown for lncRNA OLBC15. The indicates band was trimmed and subject to mass spectrometry (MS) analyses. B, Proteins obtained from RNA pulldowns were analyzed by antibody for ZNF326. C, RNA immunoprecipitation (RIP) results for OLBC15. The lncRNA LINC00501 was used as a negative control. D, Fluorescence in situ hybridization (FISH) to show the colocalization of OLBC15 and ZNF326. E, Full‐length and depletion constructs of ZNF326 were used to locate the binding region for OLBC15. F, RNA immunoprecipitation (RIP) followed by qRT‐PCR in DMA‐MB‐231 cells transfected with ectopic expression for full‐length (FL) Flag‐tagged ZNF326 or various mutant constructs. G, The secondary structure of OLBC15 predicted by an online tool termed RNAfold (http://rna.tbi.univie.ac.at//cgi‐bin/RNAWebSuite/RNAfold.cgi). The black box depicts the binding region for ZNF326. H, Construction of full‐length (FL) or various OLBC15 truncates. I, RIP followed by qRT‐PCR to show the putative interacting domain for OLBC15 with ZNF326. Fold enrichment data were rescaled by comparing each input with Flag‐Vec\nWe further used RNA pulldown assays to unravel the potential mechanism of OLBC15‐induced oncogenic effect. One specific band was detected (Figure 4A; indicated by arrow). The enriched proteins in OLBC15 pulldowns were then analyzed using mass spectrometry, and the results showed five putative hits (Table S4). Immunoblot confirmed that ZNF326 may be binding factor (Figure 4B). OLBC15 was significantly enriched in fractions with antibody against ZNF326 as determined by measuring co‐precipitated RNA (Figure 4C). Fluorescence in situ hybridization (FISH) also revealed colocalization for OLBC15 and ZNF326 (Figure 4D). Full‐length and various ZNF326 truncated mutants were constructed (Figure 4E), and we found that depleting the C‐terminal region of ZNF326 markedly abolished its interaction with OLBC15 (Figure 4F). Various OLBC15 truncated forms were also created according to the predicted secondary structure (Figure 4G,H). The results showed that the N‐terminal domain of OLBC15 was responsible for ZNF326 binding (Figure 4I). These results showed that OLBC15 may interact with ZNF326 protein.\nZNF326 interacts with OLBC15. A, RNA pulldown for lncRNA OLBC15. The indicates band was trimmed and subject to mass spectrometry (MS) analyses. B, Proteins obtained from RNA pulldowns were analyzed by antibody for ZNF326. C, RNA immunoprecipitation (RIP) results for OLBC15. The lncRNA LINC00501 was used as a negative control. D, Fluorescence in situ hybridization (FISH) to show the colocalization of OLBC15 and ZNF326. E, Full‐length and depletion constructs of ZNF326 were used to locate the binding region for OLBC15. F, RNA immunoprecipitation (RIP) followed by qRT‐PCR in DMA‐MB‐231 cells transfected with ectopic expression for full‐length (FL) Flag‐tagged ZNF326 or various mutant constructs. G, The secondary structure of OLBC15 predicted by an online tool termed RNAfold (http://rna.tbi.univie.ac.at//cgi‐bin/RNAWebSuite/RNAfold.cgi). The black box depicts the binding region for ZNF326. H, Construction of full‐length (FL) or various OLBC15 truncates. I, RIP followed by qRT‐PCR to show the putative interacting domain for OLBC15 with ZNF326. Fold enrichment data were rescaled by comparing each input with Flag‐Vec\n OLBC15 facilitates ZNF326 degradation To confirm the underlying mechanism of OLBC15‐ZNF326 binding, we knocked down OLBC15 expression in MDA‐MB‐231 cells and we found that ZNF326 abundance was increased (Figure 5A). Co‐treatment with MG132, a proteasomal inhibitor, however, could elevate ZNF326 levels irrespective of whether OLBC15 was silenced (Figure 5B). OLBC15 knockdown substantially inhibited ZNF326 ubiquitination (Figure 5C). However, OLBC15 overexpression or depletion did not affect ZNF326 mRNA levels (Figure 5D). Migration assays revealed that knocking down OLBC15 could significantly inhibit breast cancer cell migration (Figure 5E,F). However, this effect could be counteracted by combinatorial ZNF326 silence (Figure 5E,F). The efficiency of ZNF326 silence was also verified (Figure 5G). Previous study has shown that ZNF326 can bind the promoter region of Krüppel‐like factor 17 (KLF17) gene and induce KLF17 expression.\n16\n OLBC15 knockdown indeed dramatically upregulated KLF17 expression, whereas KLF17 expression was inhibited after ZNF326 was silenced (Figure S2A). Notably, KLF17 level was partially rescued with simultaneous ZNF326/OLBC15 silence (Figure S2A). The occupancies of ZNF326 to KLF17 promoter were also increased when OLBC15 was depleted (Figure S2B). These data suggested that OLBC15 could promote ZNF326 degradation via ubiquitination pathway.\nOLBC15 destabilizes ZNF326 by increasing ubiquitination. A, ZNF326 expression in MDA‐MB‐231 cells transfected with ShCtrl or two shOLBC15 constructs. B, ZNF326 expression in MDA‐MB‐231 cells with or without OLBC15 silence treated with DMSO (MG132‐) or MG132 (MG132+). C, Ubiquitin ligation of ZNF326 in MDA‐MB‐231 cells with or without OLBC15 depletion expressing full‐length Flag‐tagged ZNF326. D, Relative expression of ZNF326 transcripts with either OLBC15 knockdown or overexpression. E, Migration assays for MDA‐MB‐231 cells with OLBC15 knockdown and/or ZNF326 shRNA. F, Quantification data for cellular migration in (E). G, Efficiency of ZNF326 silence on ZNF326 expression. ShZNF326‐2 showed higher efficiency and was selected as ShZNF326. **: P < .01\nTo confirm the underlying mechanism of OLBC15‐ZNF326 binding, we knocked down OLBC15 expression in MDA‐MB‐231 cells and we found that ZNF326 abundance was increased (Figure 5A). Co‐treatment with MG132, a proteasomal inhibitor, however, could elevate ZNF326 levels irrespective of whether OLBC15 was silenced (Figure 5B). OLBC15 knockdown substantially inhibited ZNF326 ubiquitination (Figure 5C). However, OLBC15 overexpression or depletion did not affect ZNF326 mRNA levels (Figure 5D). Migration assays revealed that knocking down OLBC15 could significantly inhibit breast cancer cell migration (Figure 5E,F). However, this effect could be counteracted by combinatorial ZNF326 silence (Figure 5E,F). The efficiency of ZNF326 silence was also verified (Figure 5G). Previous study has shown that ZNF326 can bind the promoter region of Krüppel‐like factor 17 (KLF17) gene and induce KLF17 expression.\n16\n OLBC15 knockdown indeed dramatically upregulated KLF17 expression, whereas KLF17 expression was inhibited after ZNF326 was silenced (Figure S2A). Notably, KLF17 level was partially rescued with simultaneous ZNF326/OLBC15 silence (Figure S2A). The occupancies of ZNF326 to KLF17 promoter were also increased when OLBC15 was depleted (Figure S2B). These data suggested that OLBC15 could promote ZNF326 degradation via ubiquitination pathway.\nOLBC15 destabilizes ZNF326 by increasing ubiquitination. A, ZNF326 expression in MDA‐MB‐231 cells transfected with ShCtrl or two shOLBC15 constructs. B, ZNF326 expression in MDA‐MB‐231 cells with or without OLBC15 silence treated with DMSO (MG132‐) or MG132 (MG132+). C, Ubiquitin ligation of ZNF326 in MDA‐MB‐231 cells with or without OLBC15 depletion expressing full‐length Flag‐tagged ZNF326. D, Relative expression of ZNF326 transcripts with either OLBC15 knockdown or overexpression. E, Migration assays for MDA‐MB‐231 cells with OLBC15 knockdown and/or ZNF326 shRNA. F, Quantification data for cellular migration in (E). G, Efficiency of ZNF326 silence on ZNF326 expression. ShZNF326‐2 showed higher efficiency and was selected as ShZNF326. **: P < .01\n Correlation between OLBC15 and ZNF326 in clinical samples We further investigated the clinical relevance of OLBC15 and ZNF326 in human samples. Immunohistochemistry (IHC) analyses showed various degrees of ZNF326 expression with different intensity scores which represented ZNF326 expression status (Figure 6A). Consistently, we found that OLBC15 level was not significantly associated with ZNF326 transcripts (Figure 6B). However, a significantly negative correlation was evident between ZNF326 protein expression and OLBC15 (Figure 6C). These results suggested that OLBC15 could inhibit ZNF326 expression in clinical specimens.\nCorrelation between OLBC15 and ZNF326 in clinical samples. A, Immunohistochemical (IHC) staining to identify ZNF326 in human specimens. Representative low (0), weak (1+), intermediate (2+), and strong staining (3+) cases using H‐score method were shown. Scale bar: 100 μm. B, Pearson correlation coefficient (R) between OLBC15 and ZNF326 mRNA abundance. The P value was shown. C, Correlation between OLBC15 levels and ZNF326 IHC scores (ie, protein abundance) in clinical samples\nWe further investigated the clinical relevance of OLBC15 and ZNF326 in human samples. Immunohistochemistry (IHC) analyses showed various degrees of ZNF326 expression with different intensity scores which represented ZNF326 expression status (Figure 6A). Consistently, we found that OLBC15 level was not significantly associated with ZNF326 transcripts (Figure 6B). However, a significantly negative correlation was evident between ZNF326 protein expression and OLBC15 (Figure 6C). These results suggested that OLBC15 could inhibit ZNF326 expression in clinical specimens.\nCorrelation between OLBC15 and ZNF326 in clinical samples. A, Immunohistochemical (IHC) staining to identify ZNF326 in human specimens. Representative low (0), weak (1+), intermediate (2+), and strong staining (3+) cases using H‐score method were shown. Scale bar: 100 μm. B, Pearson correlation coefficient (R) between OLBC15 and ZNF326 mRNA abundance. The P value was shown. C, Correlation between OLBC15 levels and ZNF326 IHC scores (ie, protein abundance) in clinical samples",
"To screen for breast cancer (BC)‐related lncRNAs, we used lncRNA profiling assays in paired BC tissues and normal adjacent tissues (NATs) (Figure 1A). Overall, 73 significantly upregulated lncRNAs were detected (Figure 1A). These lncRNAs were candidate ones with oncogenic potential. Notably, two novel lncRNAs, RP11‐597D13.9 and OLBC15, were identified by lncRNA profiling (Table S2). Since lncRNA OLBC15 showed higher fold expression, we chose it for further analysis. OLBC15 was markedly upregulated in BC tissues compared with normal tissues (Figure 1B). OLBC15 is located on chromosome 15 with one annotated transcript (www.ensembl.org). The whole sequence for OLBC15 was shown (Figure S1A). Full‐length OLBC15 showed neglectable coding potential as evaluated by Coding Potential Assessment Tool (CPAT, coding probability ~ .032, Figure S1B). Moreover, OLBC15 was substantially enriched in triple‐negative breast cancer (TNBC) tissues compared with the other subtypes (Figure 1C). OLBC15 transcripts were highly increased in BC cell lines compared with MCF10A cells (Figure 1D). Patient characteristics demonstrated that OLBC15 expression significantly correlated with TNM stage, tumor size, and metastasis but was not significantly associated with age and histology (Table S3). Subcellular fractionation assay clarified that OLBC15 was primarily located in nucleus (Figure 1E). These data suggested that OLBC15 was upregulated in BC with nuclear distribution.\nIdentifying OLBC15 in breast cancer (BC). A, Heatmap representation of lncRNA profiling in normal adjacent tissues (NATs) and breast cancer tissues. Significantly upregulated and downregulated lncRNAs in BC tissues were 73 and 126, respectively. B, Relative OLBC15 expression in NAT and BC tissues. C, The relative OLBC15 levels in different breast cancer subtypes. The case numbers for luminal A (LA), luminal B (LB), HER2+ (HER2), and triple‐negative breast cancer (TNBC) were 40, 25, 26, and 51, respectively. D, Relative OLBC15 expression in transformed MCF10A and cancerous TNBC cell lines. E, Subcellular distribution of OLBC15. **: P < .01",
"We next checked whether OLBC15 had an effect in vitro. We first stably overexpressed or silenced OLBC15 expression in MDA‐MB‐231 and BT‐549 cells (Figure 2A,B). The knockdown and overexpression efficiency were verified (Figure 2A,B). As expected, OLBC15 overexpression significantly promoted the viability of BT‐549 and MDA‐MB‐231 cells (Figure 2C,D). Consistently, OLBC15 depletion (shOLBC15) strongly inhibited the viability of breast cancer cells (Figure 2C,D). Migration assay further suggested that OLBC15 overexpression advanced migration in BT‐549 and MDA‐MB‐231 cells (Figure 2E,F; P < .01). OLBC15 silence instead attenuated migratory capacity in breast cancer cells (Figure 2E,F). These results implied that OLBC15 might play an oncogenic role in vitro.\nOLBC15 promotes TNBC in vitro. A, Overexpression efficiency by lentiviral OLBC15 transfection. B, Knockdown efficiency using shRNA targeting OLBC15. ShOLBC15 displayed higher efficiency and was selected as ShOLBC15. C‐D, Effect of OLBC15 on viability of BT‐549 (C) and MDA‐MB‐231 (D) cells transfected with shRNA scrambled control (ShCtrl), ShOLBC15, lentiviral control (OE‐control), or the lentiviral vector carrying OLBC15 (OE‐OLBC15). E, Effect of OLBC15 on migratory capacity of MDA‐MB‐231 (top) and BT‐549 (bottom) cells. F, Quantification of results in (E). **: P < .01",
"We then performed in vivo experiments to further investigate the function of OLBC15. Stably transfected MDA‐MB‐231 cells were used. OLBC15 knockdown inhibited tumor growth, whereas overexpressing OLBC15 promoted the tumor volume expansion (Figure 3A). A higher number of metastatic nodules were detected in OLBC15‐overexpressing group compared with the control (Figure 3B,C; P < .01). As expected, OLBC15 silence profoundly inhibited the occurrence of metastatic nodules (Figure 3B,C; P < .01). The xenograft tumor growth was evidently attenuated by OLBC15 depletion, whereas OLBC15 overexpression markedly accelerated the growth of xenograft tumors (Figure 3E,F; P < .01). These results further suggested that OLBC15 could exert an oncogenic effect in vivo.\nOLBC15 advances TNBC in vivo. A, In vivo tumor volume growth of xenograft tumors with OLBC15 depletion or overexpression. B, Representative lung metastatic nodules with OLBC15 silence or overexpression. The description representing each numbered case was shown at the bottom. C, Quantification of metastatic nodules in (B). D, Xenograft tumors for MDA‐MB‐231 transplantation model with OLBC15 silence or overexpression. Numbered case description was shown at the bottom. E, Quantification data for (D). **: P < .01",
"We further used RNA pulldown assays to unravel the potential mechanism of OLBC15‐induced oncogenic effect. One specific band was detected (Figure 4A; indicated by arrow). The enriched proteins in OLBC15 pulldowns were then analyzed using mass spectrometry, and the results showed five putative hits (Table S4). Immunoblot confirmed that ZNF326 may be binding factor (Figure 4B). OLBC15 was significantly enriched in fractions with antibody against ZNF326 as determined by measuring co‐precipitated RNA (Figure 4C). Fluorescence in situ hybridization (FISH) also revealed colocalization for OLBC15 and ZNF326 (Figure 4D). Full‐length and various ZNF326 truncated mutants were constructed (Figure 4E), and we found that depleting the C‐terminal region of ZNF326 markedly abolished its interaction with OLBC15 (Figure 4F). Various OLBC15 truncated forms were also created according to the predicted secondary structure (Figure 4G,H). The results showed that the N‐terminal domain of OLBC15 was responsible for ZNF326 binding (Figure 4I). These results showed that OLBC15 may interact with ZNF326 protein.\nZNF326 interacts with OLBC15. A, RNA pulldown for lncRNA OLBC15. The indicates band was trimmed and subject to mass spectrometry (MS) analyses. B, Proteins obtained from RNA pulldowns were analyzed by antibody for ZNF326. C, RNA immunoprecipitation (RIP) results for OLBC15. The lncRNA LINC00501 was used as a negative control. D, Fluorescence in situ hybridization (FISH) to show the colocalization of OLBC15 and ZNF326. E, Full‐length and depletion constructs of ZNF326 were used to locate the binding region for OLBC15. F, RNA immunoprecipitation (RIP) followed by qRT‐PCR in DMA‐MB‐231 cells transfected with ectopic expression for full‐length (FL) Flag‐tagged ZNF326 or various mutant constructs. G, The secondary structure of OLBC15 predicted by an online tool termed RNAfold (http://rna.tbi.univie.ac.at//cgi‐bin/RNAWebSuite/RNAfold.cgi). The black box depicts the binding region for ZNF326. H, Construction of full‐length (FL) or various OLBC15 truncates. I, RIP followed by qRT‐PCR to show the putative interacting domain for OLBC15 with ZNF326. Fold enrichment data were rescaled by comparing each input with Flag‐Vec",
"To confirm the underlying mechanism of OLBC15‐ZNF326 binding, we knocked down OLBC15 expression in MDA‐MB‐231 cells and we found that ZNF326 abundance was increased (Figure 5A). Co‐treatment with MG132, a proteasomal inhibitor, however, could elevate ZNF326 levels irrespective of whether OLBC15 was silenced (Figure 5B). OLBC15 knockdown substantially inhibited ZNF326 ubiquitination (Figure 5C). However, OLBC15 overexpression or depletion did not affect ZNF326 mRNA levels (Figure 5D). Migration assays revealed that knocking down OLBC15 could significantly inhibit breast cancer cell migration (Figure 5E,F). However, this effect could be counteracted by combinatorial ZNF326 silence (Figure 5E,F). The efficiency of ZNF326 silence was also verified (Figure 5G). Previous study has shown that ZNF326 can bind the promoter region of Krüppel‐like factor 17 (KLF17) gene and induce KLF17 expression.\n16\n OLBC15 knockdown indeed dramatically upregulated KLF17 expression, whereas KLF17 expression was inhibited after ZNF326 was silenced (Figure S2A). Notably, KLF17 level was partially rescued with simultaneous ZNF326/OLBC15 silence (Figure S2A). The occupancies of ZNF326 to KLF17 promoter were also increased when OLBC15 was depleted (Figure S2B). These data suggested that OLBC15 could promote ZNF326 degradation via ubiquitination pathway.\nOLBC15 destabilizes ZNF326 by increasing ubiquitination. A, ZNF326 expression in MDA‐MB‐231 cells transfected with ShCtrl or two shOLBC15 constructs. B, ZNF326 expression in MDA‐MB‐231 cells with or without OLBC15 silence treated with DMSO (MG132‐) or MG132 (MG132+). C, Ubiquitin ligation of ZNF326 in MDA‐MB‐231 cells with or without OLBC15 depletion expressing full‐length Flag‐tagged ZNF326. D, Relative expression of ZNF326 transcripts with either OLBC15 knockdown or overexpression. E, Migration assays for MDA‐MB‐231 cells with OLBC15 knockdown and/or ZNF326 shRNA. F, Quantification data for cellular migration in (E). G, Efficiency of ZNF326 silence on ZNF326 expression. ShZNF326‐2 showed higher efficiency and was selected as ShZNF326. **: P < .01",
"We further investigated the clinical relevance of OLBC15 and ZNF326 in human samples. Immunohistochemistry (IHC) analyses showed various degrees of ZNF326 expression with different intensity scores which represented ZNF326 expression status (Figure 6A). Consistently, we found that OLBC15 level was not significantly associated with ZNF326 transcripts (Figure 6B). However, a significantly negative correlation was evident between ZNF326 protein expression and OLBC15 (Figure 6C). These results suggested that OLBC15 could inhibit ZNF326 expression in clinical specimens.\nCorrelation between OLBC15 and ZNF326 in clinical samples. A, Immunohistochemical (IHC) staining to identify ZNF326 in human specimens. Representative low (0), weak (1+), intermediate (2+), and strong staining (3+) cases using H‐score method were shown. Scale bar: 100 μm. B, Pearson correlation coefficient (R) between OLBC15 and ZNF326 mRNA abundance. The P value was shown. C, Correlation between OLBC15 levels and ZNF326 IHC scores (ie, protein abundance) in clinical samples",
"In the current study, we have identified a novel oncogenic lncRNA OLBC15. We found that OLBC15 depletion may exert profound inhibition to breast cancer progression implying that OLBC15 may act as a putative target for intervention. OLBC15 expression was dramatically increased in breast cancer tissues especially TNBC. Furthermore, we also observed an oncogenic effect of OLBC15 via both in vitro and in vivo experiments. Mechanistic study showed that OLBC15 could interact with ZNF326, which is a novel tumor suppressor in TNBC.\n16\n OLBC15 facilitates ZNF326 degradation via ubiquitination pathway. These data collectively suggested that OLBC15 fulfills its oncogenic function via destabilizing the tumor suppressor ZNF326. Notably, the ubiquitin ligase responsible for ZNF326 is still elusive and whether OLBC15 enhances the interaction between ZNF326 and its ubiquitin ligase remains to be determined. Moreover, the possibility of OLBC15 as a competitive endogenous RNA (ceRNA) during breast cancer progression remains to be evaluated.\n17\n\n\nWe found that OLBC15 interacts with ZNF326 to destabilize ZNF326 protein. The ZNF326 protein is initially identified in NIH3T3 cell line and modulates migration and growth.\n18\n Meanwhile, ZNF326 is also a transcriptional factor which activates neuronal differentiation.\n19\n ZNF326 can bind deleted in breast cancer 1 (DBC1) leading to the formation of DBIRD (DBC1/ZIRD) complex and result in its association with RNAPII.\n20\n Therefore, ZNF326 can actively participate in the process of alternative splicing in A/T‐rich regions of DNA.\n16\n Depleting ZNF326 can increase the expression of multiple genes involved in epithelial‐mesenchymal transition (EMT) in HEK293 cells and TNBC cell lines.\n16\n, \n20\n ZNF326 silence also leads to enhanced migratory and invasive capacity together with increased mammosphere formation in TNBC cells.\n16\n Consistently, ZNF326 knockdown also promotes orthologous transplant tumor formation, whereas overexpressing ZNF326 decreased xenograft tumor formation.\n16\n We have verified that OLBC15 silence can stabilize ZNF326 and as a result markedly augment the expression of KLF17, which is a well‐characterized tumor suppressor gene and negatively regulates EMT and metastasis in breast cancer.\n21\n Furthermore, increased expression of PRMT5/WDR77 complex in breast cancer cells results in ZNF326 methylation and is essential for Pol II elongation across A/T‐rich regions.\n22\n ZNF326 also represses breast cancer progression via diverse mechanisms.\n16\n These data support a tumor‐suppressive role of ZNF326 in TNBC. We should note that ZNF326 might play different roles in other types of cancer. For example, recent reports have demonstrated that cells with ZNF326 overexpression favor malignant phenotypes in glioma via increasing LDAC7 levels and activating Wnt signaling.\n18\n Wu et al have also identified that the C2H2 structure of ZNF326 can bind to the ERCC1 promoter and elevate ERCC1 expression in non–small‐cell lung cancer (NSCLC).\n23\n Therefore, ZNF326 can be a tumor suppressor and lncRNA OLBC15 promotes TNBC oncogenesis via destabilizing ZNF326 at least in TNBC. These data argue that OLBC15 may possibly be a tumor‐specific lncRNA in TNBC and the exact role of OLBC15 in other types of cancers remains to be investigated in future studies.\nNotably, patients with TNBC suffer greatly from poor outcomes and effective target therapy is still lacking in TNBC.\n5\n, \n24\n The programmed cell death protein 1 (PD‐1)/PD ligand 1 (PD‐L1) as well as androgen receptors (AR) might be possible targets for therapeutic intervention to repress breast cancer progression although the efficiency and the frequent off‐target effects have posed serious concerns.\n5\n Current data have supported OLBC15 as a potential oncogenic lncRNA especially in TNBC. Therefore, the efficacy of OLBC15 targeting deserves further investigation. RNA interference might be a possible strategy; however, the lowered stability and cellular uptake especially in nucleus remain an obstacle to OLBC15 depletion.\n25\n The antisense oligonucleotides (ASOs) may provide a promising way to annihilate OLBC15 in breast cancer cells, and ASOs specifically targeting EZH2 and AR are highly effective in recent work.\n26\n A clinical trial has demonstrated that an ASO termed IONIS‐APO(a)Rx can be of favorable efficiency.\n27\n Therefore, we argue that using ASOs targeting OLBC15 may present an alternative strategy to abolish OLBC15 expression and inhibit TNBC development. There is also a possibility to target multiple TNBC‐associated oncogenic lncRNAs via ASOs, and relevant investigations should be performed in future.\nOur current data have supported a novel transcript related to TNBC progression. OLBC15 directly interacts with ZNF326 tumor suppressor and interferes its post‐translational modification. OLBC15 has potentially clinical relevance and may aid in targeted design of anti‐cancer therapeutics especially in TNBC.",
"\nSupplementary Material\n\nClick here for additional data file."
] |
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"materials-and-methods",
null,
null,
null,
null,
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[
"breast cancer",
"OLBC15",
"oncogenesis",
"ZNF326"
] |
INTRODUCTION:
The breast cancer (BC) has been reported to be a primary cause of malignancy and mortality in women, and the incidence rate worldwide has been on the rise.
1
The breast cancer possesses a heterogeneous nature and is classified into several subtypes.
2
Anti‐estrogen or combined anti‐HER2 therapeutic strategies are major treatments for luminal A, luminal B, and HER2‐positive (HER2+) breast cancer subtypes.
3
However, the highly aggressive and metastatic triple‐negative breast cancer (TNBC, negative in estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) is associated with high relapse even after chemotherapy or radiotherapy.
4
,
5
Therefore, unraveling novel and effective targets for TNBC intervention are of great significance.
Growing evidence has suggested that the non‐coding RNAs with no or minimal coding abilities contribute largely to tumor development.
6
A major constituent of non‐coding transcriptome is long non‐coding RNAs (lncRNAs).
6
The long non‐coding RNAs are RNA transcripts with more than 200 nucleotides in length and involved in numerous biological processes.
7
The lncRNAs can serve as an oncogene or tumor suppressor with an emerging effect on metastasis.
8
For instance, Tang et al identified that lncRNA PVT1 can stabilize KLF5 via BAP1 and increase its stability to promote TNBC progression.
9
Alipoor et al recently found that lncRNA MIAT increases miR‐302/miR‐150 expression, whereas miR‐29c expression was decreased.
10
Furthermore, MIAT also abolishes the expression of p16Ink4A and Cox2 to inhibit senescence commitment.
10
A TNBC‐specific lncRNA LINC00511 is markedly upregulated through gene amplification and significantly correlates with TNBC progression.
11
Sas‐Chen et al showed that lncRNA LIMT is a highly conserved tumor suppressor and its expression is suppressed by epidermal growth factor (EGF) signaling pathway.
12
Other reported lncRNAs (eg, RMST and BORG) are substantially associated with breast cancer development via diverse molecular mechanisms.
13
,
14
However, the function of lncRNAs especially in TNBC is still obscure.
In the current work, by lncRNA profiling, we have identified a novel lncRNA ENSG00000259457 as an oncogenic lncRNA in TNBC. It was termed as oncogenic lncRNA in breast cancer on chromosome 15 (OLBC15). We observed that OLBC15 is significantly upregulated in TNBC. OLBC15 acted as an oncogenic factor to promote breast cancer progression, and this effect was verified both in vivo and in vitro. Furthermore, by RNA immunoprecipitation we noted that zinc‐finger protein‐326 (ZNF326) might be an OLBC15 binding protein. The interaction of OLBC15 and ZNF326 decreased the stability of ZNF326 via enhanced ubiquitination and proteasomal degradation. OLBC15 also showed negative correlation with ZNF326 protein expression in clinical samples. These data collectively implied an oncogenic role of OLBC15 and may provide a novel anti‐TNBC target for therapeutic intervention.
MATERIALS AND METHODS:
Cell lines, lentiviral vectors, and reagents The transformed MCF‐10A and breast cancer MDA‐MB‐231, BT‐549, HCC1806, Hs578t, and MDA‐MB‐468 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 7% FBS and 150 μg/mL streptomycin. The cell lines were all obtained from Cell Resource Center of Shanghai Institute of Biological Sciences (CAS). Puromycin was used for 2 days. OLBC15 was first cloned and was then inserted into a pWPXL vector (Biovector) to generate pWPXL‐OLBC15 vector (OE‐OLBC15). Lentiviral selection was then conducted using puromycin (No.A1113802, Thermo Fisher Scientific), and generated supernatants were treated with a syringe filter. An empty pWPXL vector serves as the control (ie, OE‐control). The short hairpin RNA (shRNA) for OLBC15 (ShOLBC15) together with a non‐targeting scrambled control (ShCtrl) was obtained from Biovector. Transfection was conducted using Lipofectamine 3000. Primers were listed in Table S1.
The transformed MCF‐10A and breast cancer MDA‐MB‐231, BT‐549, HCC1806, Hs578t, and MDA‐MB‐468 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 7% FBS and 150 μg/mL streptomycin. The cell lines were all obtained from Cell Resource Center of Shanghai Institute of Biological Sciences (CAS). Puromycin was used for 2 days. OLBC15 was first cloned and was then inserted into a pWPXL vector (Biovector) to generate pWPXL‐OLBC15 vector (OE‐OLBC15). Lentiviral selection was then conducted using puromycin (No.A1113802, Thermo Fisher Scientific), and generated supernatants were treated with a syringe filter. An empty pWPXL vector serves as the control (ie, OE‐control). The short hairpin RNA (shRNA) for OLBC15 (ShOLBC15) together with a non‐targeting scrambled control (ShCtrl) was obtained from Biovector. Transfection was conducted using Lipofectamine 3000. Primers were listed in Table S1.
Human samples The breast cancer tissues were collected from patients following a protocol described previously
15
from January 2017 to November 2018. The samples were stored at −80℃ immediately after resection before use. Experiments associated with human specimens were approved by Human Research Ethics Committee (HREC) at Chongqing Three Gorges Central Hospital in accordance with the 1975 Declaration of Helsinki.
The breast cancer tissues were collected from patients following a protocol described previously
15
from January 2017 to November 2018. The samples were stored at −80℃ immediately after resection before use. Experiments associated with human specimens were approved by Human Research Ethics Committee (HREC) at Chongqing Three Gorges Central Hospital in accordance with the 1975 Declaration of Helsinki.
Migration assay ~5 × 105 cells were located on the top chamber of a 24‐well plate (BD Biosciences). The DMEM with 7% serum was loaded at the bottom. After an incubation for 24 hours, cells moving into the bottom chambers were fixed with 4% paraformaldehyde (No.16005, Sigma) and subject to staining by 0.4% crystal violet (No.C0775, Sigma).
~5 × 105 cells were located on the top chamber of a 24‐well plate (BD Biosciences). The DMEM with 7% serum was loaded at the bottom. After an incubation for 24 hours, cells moving into the bottom chambers were fixed with 4% paraformaldehyde (No.16005, Sigma) and subject to staining by 0.4% crystal violet (No.C0775, Sigma).
Viability assay We used Cell Counting Kit‐8 (CCK‐8, Dojindo, Japan) to measure the viability of breast cancer cells following the manufacturer's protocols. BT549 and MDA‐MB‐231 cells were resuspended and moved into a 12‐well plate (~2 × 105 cells/well). 30 µL CCK‐8 solutions were then added into the system. Optical density at 450 nm (OD 450 nm) was measured by a Spectramax M5 microplate monitor (Molecular Devices).
We used Cell Counting Kit‐8 (CCK‐8, Dojindo, Japan) to measure the viability of breast cancer cells following the manufacturer's protocols. BT549 and MDA‐MB‐231 cells were resuspended and moved into a 12‐well plate (~2 × 105 cells/well). 30 µL CCK‐8 solutions were then added into the system. Optical density at 450 nm (OD 450 nm) was measured by a Spectramax M5 microplate monitor (Molecular Devices).
Statistical analysis Statistical analyses were performed with SPSS (v16, SPSS, Inc). Data were represented as mean ± SD. The nonparametric Mann‐Whitney test was used to identify the statistical significance between two groups, and ANOVA was used for comparing multiple groups followed by the LSD post hoc test. P < .05 was considered statistically significant.
Statistical analyses were performed with SPSS (v16, SPSS, Inc). Data were represented as mean ± SD. The nonparametric Mann‐Whitney test was used to identify the statistical significance between two groups, and ANOVA was used for comparing multiple groups followed by the LSD post hoc test. P < .05 was considered statistically significant.
Cell lines, lentiviral vectors, and reagents:
The transformed MCF‐10A and breast cancer MDA‐MB‐231, BT‐549, HCC1806, Hs578t, and MDA‐MB‐468 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 7% FBS and 150 μg/mL streptomycin. The cell lines were all obtained from Cell Resource Center of Shanghai Institute of Biological Sciences (CAS). Puromycin was used for 2 days. OLBC15 was first cloned and was then inserted into a pWPXL vector (Biovector) to generate pWPXL‐OLBC15 vector (OE‐OLBC15). Lentiviral selection was then conducted using puromycin (No.A1113802, Thermo Fisher Scientific), and generated supernatants were treated with a syringe filter. An empty pWPXL vector serves as the control (ie, OE‐control). The short hairpin RNA (shRNA) for OLBC15 (ShOLBC15) together with a non‐targeting scrambled control (ShCtrl) was obtained from Biovector. Transfection was conducted using Lipofectamine 3000. Primers were listed in Table S1.
Human samples:
The breast cancer tissues were collected from patients following a protocol described previously
15
from January 2017 to November 2018. The samples were stored at −80℃ immediately after resection before use. Experiments associated with human specimens were approved by Human Research Ethics Committee (HREC) at Chongqing Three Gorges Central Hospital in accordance with the 1975 Declaration of Helsinki.
Migration assay:
~5 × 105 cells were located on the top chamber of a 24‐well plate (BD Biosciences). The DMEM with 7% serum was loaded at the bottom. After an incubation for 24 hours, cells moving into the bottom chambers were fixed with 4% paraformaldehyde (No.16005, Sigma) and subject to staining by 0.4% crystal violet (No.C0775, Sigma).
Viability assay:
We used Cell Counting Kit‐8 (CCK‐8, Dojindo, Japan) to measure the viability of breast cancer cells following the manufacturer's protocols. BT549 and MDA‐MB‐231 cells were resuspended and moved into a 12‐well plate (~2 × 105 cells/well). 30 µL CCK‐8 solutions were then added into the system. Optical density at 450 nm (OD 450 nm) was measured by a Spectramax M5 microplate monitor (Molecular Devices).
Statistical analysis:
Statistical analyses were performed with SPSS (v16, SPSS, Inc). Data were represented as mean ± SD. The nonparametric Mann‐Whitney test was used to identify the statistical significance between two groups, and ANOVA was used for comparing multiple groups followed by the LSD post hoc test. P < .05 was considered statistically significant.
RESULTS:
Identifying OLBC15 in breast cancer via lncRNA profiling To screen for breast cancer (BC)‐related lncRNAs, we used lncRNA profiling assays in paired BC tissues and normal adjacent tissues (NATs) (Figure 1A). Overall, 73 significantly upregulated lncRNAs were detected (Figure 1A). These lncRNAs were candidate ones with oncogenic potential. Notably, two novel lncRNAs, RP11‐597D13.9 and OLBC15, were identified by lncRNA profiling (Table S2). Since lncRNA OLBC15 showed higher fold expression, we chose it for further analysis. OLBC15 was markedly upregulated in BC tissues compared with normal tissues (Figure 1B). OLBC15 is located on chromosome 15 with one annotated transcript (www.ensembl.org). The whole sequence for OLBC15 was shown (Figure S1A). Full‐length OLBC15 showed neglectable coding potential as evaluated by Coding Potential Assessment Tool (CPAT, coding probability ~ .032, Figure S1B). Moreover, OLBC15 was substantially enriched in triple‐negative breast cancer (TNBC) tissues compared with the other subtypes (Figure 1C). OLBC15 transcripts were highly increased in BC cell lines compared with MCF10A cells (Figure 1D). Patient characteristics demonstrated that OLBC15 expression significantly correlated with TNM stage, tumor size, and metastasis but was not significantly associated with age and histology (Table S3). Subcellular fractionation assay clarified that OLBC15 was primarily located in nucleus (Figure 1E). These data suggested that OLBC15 was upregulated in BC with nuclear distribution.
Identifying OLBC15 in breast cancer (BC). A, Heatmap representation of lncRNA profiling in normal adjacent tissues (NATs) and breast cancer tissues. Significantly upregulated and downregulated lncRNAs in BC tissues were 73 and 126, respectively. B, Relative OLBC15 expression in NAT and BC tissues. C, The relative OLBC15 levels in different breast cancer subtypes. The case numbers for luminal A (LA), luminal B (LB), HER2+ (HER2), and triple‐negative breast cancer (TNBC) were 40, 25, 26, and 51, respectively. D, Relative OLBC15 expression in transformed MCF10A and cancerous TNBC cell lines. E, Subcellular distribution of OLBC15. **: P < .01
To screen for breast cancer (BC)‐related lncRNAs, we used lncRNA profiling assays in paired BC tissues and normal adjacent tissues (NATs) (Figure 1A). Overall, 73 significantly upregulated lncRNAs were detected (Figure 1A). These lncRNAs were candidate ones with oncogenic potential. Notably, two novel lncRNAs, RP11‐597D13.9 and OLBC15, were identified by lncRNA profiling (Table S2). Since lncRNA OLBC15 showed higher fold expression, we chose it for further analysis. OLBC15 was markedly upregulated in BC tissues compared with normal tissues (Figure 1B). OLBC15 is located on chromosome 15 with one annotated transcript (www.ensembl.org). The whole sequence for OLBC15 was shown (Figure S1A). Full‐length OLBC15 showed neglectable coding potential as evaluated by Coding Potential Assessment Tool (CPAT, coding probability ~ .032, Figure S1B). Moreover, OLBC15 was substantially enriched in triple‐negative breast cancer (TNBC) tissues compared with the other subtypes (Figure 1C). OLBC15 transcripts were highly increased in BC cell lines compared with MCF10A cells (Figure 1D). Patient characteristics demonstrated that OLBC15 expression significantly correlated with TNM stage, tumor size, and metastasis but was not significantly associated with age and histology (Table S3). Subcellular fractionation assay clarified that OLBC15 was primarily located in nucleus (Figure 1E). These data suggested that OLBC15 was upregulated in BC with nuclear distribution.
Identifying OLBC15 in breast cancer (BC). A, Heatmap representation of lncRNA profiling in normal adjacent tissues (NATs) and breast cancer tissues. Significantly upregulated and downregulated lncRNAs in BC tissues were 73 and 126, respectively. B, Relative OLBC15 expression in NAT and BC tissues. C, The relative OLBC15 levels in different breast cancer subtypes. The case numbers for luminal A (LA), luminal B (LB), HER2+ (HER2), and triple‐negative breast cancer (TNBC) were 40, 25, 26, and 51, respectively. D, Relative OLBC15 expression in transformed MCF10A and cancerous TNBC cell lines. E, Subcellular distribution of OLBC15. **: P < .01
OLBC15 facilitates breast cancer progression in vitro We next checked whether OLBC15 had an effect in vitro. We first stably overexpressed or silenced OLBC15 expression in MDA‐MB‐231 and BT‐549 cells (Figure 2A,B). The knockdown and overexpression efficiency were verified (Figure 2A,B). As expected, OLBC15 overexpression significantly promoted the viability of BT‐549 and MDA‐MB‐231 cells (Figure 2C,D). Consistently, OLBC15 depletion (shOLBC15) strongly inhibited the viability of breast cancer cells (Figure 2C,D). Migration assay further suggested that OLBC15 overexpression advanced migration in BT‐549 and MDA‐MB‐231 cells (Figure 2E,F; P < .01). OLBC15 silence instead attenuated migratory capacity in breast cancer cells (Figure 2E,F). These results implied that OLBC15 might play an oncogenic role in vitro.
OLBC15 promotes TNBC in vitro. A, Overexpression efficiency by lentiviral OLBC15 transfection. B, Knockdown efficiency using shRNA targeting OLBC15. ShOLBC15 displayed higher efficiency and was selected as ShOLBC15. C‐D, Effect of OLBC15 on viability of BT‐549 (C) and MDA‐MB‐231 (D) cells transfected with shRNA scrambled control (ShCtrl), ShOLBC15, lentiviral control (OE‐control), or the lentiviral vector carrying OLBC15 (OE‐OLBC15). E, Effect of OLBC15 on migratory capacity of MDA‐MB‐231 (top) and BT‐549 (bottom) cells. F, Quantification of results in (E). **: P < .01
We next checked whether OLBC15 had an effect in vitro. We first stably overexpressed or silenced OLBC15 expression in MDA‐MB‐231 and BT‐549 cells (Figure 2A,B). The knockdown and overexpression efficiency were verified (Figure 2A,B). As expected, OLBC15 overexpression significantly promoted the viability of BT‐549 and MDA‐MB‐231 cells (Figure 2C,D). Consistently, OLBC15 depletion (shOLBC15) strongly inhibited the viability of breast cancer cells (Figure 2C,D). Migration assay further suggested that OLBC15 overexpression advanced migration in BT‐549 and MDA‐MB‐231 cells (Figure 2E,F; P < .01). OLBC15 silence instead attenuated migratory capacity in breast cancer cells (Figure 2E,F). These results implied that OLBC15 might play an oncogenic role in vitro.
OLBC15 promotes TNBC in vitro. A, Overexpression efficiency by lentiviral OLBC15 transfection. B, Knockdown efficiency using shRNA targeting OLBC15. ShOLBC15 displayed higher efficiency and was selected as ShOLBC15. C‐D, Effect of OLBC15 on viability of BT‐549 (C) and MDA‐MB‐231 (D) cells transfected with shRNA scrambled control (ShCtrl), ShOLBC15, lentiviral control (OE‐control), or the lentiviral vector carrying OLBC15 (OE‐OLBC15). E, Effect of OLBC15 on migratory capacity of MDA‐MB‐231 (top) and BT‐549 (bottom) cells. F, Quantification of results in (E). **: P < .01
OLBC15 promotes xenograft tumor growth and metastasis We then performed in vivo experiments to further investigate the function of OLBC15. Stably transfected MDA‐MB‐231 cells were used. OLBC15 knockdown inhibited tumor growth, whereas overexpressing OLBC15 promoted the tumor volume expansion (Figure 3A). A higher number of metastatic nodules were detected in OLBC15‐overexpressing group compared with the control (Figure 3B,C; P < .01). As expected, OLBC15 silence profoundly inhibited the occurrence of metastatic nodules (Figure 3B,C; P < .01). The xenograft tumor growth was evidently attenuated by OLBC15 depletion, whereas OLBC15 overexpression markedly accelerated the growth of xenograft tumors (Figure 3E,F; P < .01). These results further suggested that OLBC15 could exert an oncogenic effect in vivo.
OLBC15 advances TNBC in vivo. A, In vivo tumor volume growth of xenograft tumors with OLBC15 depletion or overexpression. B, Representative lung metastatic nodules with OLBC15 silence or overexpression. The description representing each numbered case was shown at the bottom. C, Quantification of metastatic nodules in (B). D, Xenograft tumors for MDA‐MB‐231 transplantation model with OLBC15 silence or overexpression. Numbered case description was shown at the bottom. E, Quantification data for (D). **: P < .01
We then performed in vivo experiments to further investigate the function of OLBC15. Stably transfected MDA‐MB‐231 cells were used. OLBC15 knockdown inhibited tumor growth, whereas overexpressing OLBC15 promoted the tumor volume expansion (Figure 3A). A higher number of metastatic nodules were detected in OLBC15‐overexpressing group compared with the control (Figure 3B,C; P < .01). As expected, OLBC15 silence profoundly inhibited the occurrence of metastatic nodules (Figure 3B,C; P < .01). The xenograft tumor growth was evidently attenuated by OLBC15 depletion, whereas OLBC15 overexpression markedly accelerated the growth of xenograft tumors (Figure 3E,F; P < .01). These results further suggested that OLBC15 could exert an oncogenic effect in vivo.
OLBC15 advances TNBC in vivo. A, In vivo tumor volume growth of xenograft tumors with OLBC15 depletion or overexpression. B, Representative lung metastatic nodules with OLBC15 silence or overexpression. The description representing each numbered case was shown at the bottom. C, Quantification of metastatic nodules in (B). D, Xenograft tumors for MDA‐MB‐231 transplantation model with OLBC15 silence or overexpression. Numbered case description was shown at the bottom. E, Quantification data for (D). **: P < .01
OLBC15 interacts with ZNF326 protein We further used RNA pulldown assays to unravel the potential mechanism of OLBC15‐induced oncogenic effect. One specific band was detected (Figure 4A; indicated by arrow). The enriched proteins in OLBC15 pulldowns were then analyzed using mass spectrometry, and the results showed five putative hits (Table S4). Immunoblot confirmed that ZNF326 may be binding factor (Figure 4B). OLBC15 was significantly enriched in fractions with antibody against ZNF326 as determined by measuring co‐precipitated RNA (Figure 4C). Fluorescence in situ hybridization (FISH) also revealed colocalization for OLBC15 and ZNF326 (Figure 4D). Full‐length and various ZNF326 truncated mutants were constructed (Figure 4E), and we found that depleting the C‐terminal region of ZNF326 markedly abolished its interaction with OLBC15 (Figure 4F). Various OLBC15 truncated forms were also created according to the predicted secondary structure (Figure 4G,H). The results showed that the N‐terminal domain of OLBC15 was responsible for ZNF326 binding (Figure 4I). These results showed that OLBC15 may interact with ZNF326 protein.
ZNF326 interacts with OLBC15. A, RNA pulldown for lncRNA OLBC15. The indicates band was trimmed and subject to mass spectrometry (MS) analyses. B, Proteins obtained from RNA pulldowns were analyzed by antibody for ZNF326. C, RNA immunoprecipitation (RIP) results for OLBC15. The lncRNA LINC00501 was used as a negative control. D, Fluorescence in situ hybridization (FISH) to show the colocalization of OLBC15 and ZNF326. E, Full‐length and depletion constructs of ZNF326 were used to locate the binding region for OLBC15. F, RNA immunoprecipitation (RIP) followed by qRT‐PCR in DMA‐MB‐231 cells transfected with ectopic expression for full‐length (FL) Flag‐tagged ZNF326 or various mutant constructs. G, The secondary structure of OLBC15 predicted by an online tool termed RNAfold (http://rna.tbi.univie.ac.at//cgi‐bin/RNAWebSuite/RNAfold.cgi). The black box depicts the binding region for ZNF326. H, Construction of full‐length (FL) or various OLBC15 truncates. I, RIP followed by qRT‐PCR to show the putative interacting domain for OLBC15 with ZNF326. Fold enrichment data were rescaled by comparing each input with Flag‐Vec
We further used RNA pulldown assays to unravel the potential mechanism of OLBC15‐induced oncogenic effect. One specific band was detected (Figure 4A; indicated by arrow). The enriched proteins in OLBC15 pulldowns were then analyzed using mass spectrometry, and the results showed five putative hits (Table S4). Immunoblot confirmed that ZNF326 may be binding factor (Figure 4B). OLBC15 was significantly enriched in fractions with antibody against ZNF326 as determined by measuring co‐precipitated RNA (Figure 4C). Fluorescence in situ hybridization (FISH) also revealed colocalization for OLBC15 and ZNF326 (Figure 4D). Full‐length and various ZNF326 truncated mutants were constructed (Figure 4E), and we found that depleting the C‐terminal region of ZNF326 markedly abolished its interaction with OLBC15 (Figure 4F). Various OLBC15 truncated forms were also created according to the predicted secondary structure (Figure 4G,H). The results showed that the N‐terminal domain of OLBC15 was responsible for ZNF326 binding (Figure 4I). These results showed that OLBC15 may interact with ZNF326 protein.
ZNF326 interacts with OLBC15. A, RNA pulldown for lncRNA OLBC15. The indicates band was trimmed and subject to mass spectrometry (MS) analyses. B, Proteins obtained from RNA pulldowns were analyzed by antibody for ZNF326. C, RNA immunoprecipitation (RIP) results for OLBC15. The lncRNA LINC00501 was used as a negative control. D, Fluorescence in situ hybridization (FISH) to show the colocalization of OLBC15 and ZNF326. E, Full‐length and depletion constructs of ZNF326 were used to locate the binding region for OLBC15. F, RNA immunoprecipitation (RIP) followed by qRT‐PCR in DMA‐MB‐231 cells transfected with ectopic expression for full‐length (FL) Flag‐tagged ZNF326 or various mutant constructs. G, The secondary structure of OLBC15 predicted by an online tool termed RNAfold (http://rna.tbi.univie.ac.at//cgi‐bin/RNAWebSuite/RNAfold.cgi). The black box depicts the binding region for ZNF326. H, Construction of full‐length (FL) or various OLBC15 truncates. I, RIP followed by qRT‐PCR to show the putative interacting domain for OLBC15 with ZNF326. Fold enrichment data were rescaled by comparing each input with Flag‐Vec
OLBC15 facilitates ZNF326 degradation To confirm the underlying mechanism of OLBC15‐ZNF326 binding, we knocked down OLBC15 expression in MDA‐MB‐231 cells and we found that ZNF326 abundance was increased (Figure 5A). Co‐treatment with MG132, a proteasomal inhibitor, however, could elevate ZNF326 levels irrespective of whether OLBC15 was silenced (Figure 5B). OLBC15 knockdown substantially inhibited ZNF326 ubiquitination (Figure 5C). However, OLBC15 overexpression or depletion did not affect ZNF326 mRNA levels (Figure 5D). Migration assays revealed that knocking down OLBC15 could significantly inhibit breast cancer cell migration (Figure 5E,F). However, this effect could be counteracted by combinatorial ZNF326 silence (Figure 5E,F). The efficiency of ZNF326 silence was also verified (Figure 5G). Previous study has shown that ZNF326 can bind the promoter region of Krüppel‐like factor 17 (KLF17) gene and induce KLF17 expression.
16
OLBC15 knockdown indeed dramatically upregulated KLF17 expression, whereas KLF17 expression was inhibited after ZNF326 was silenced (Figure S2A). Notably, KLF17 level was partially rescued with simultaneous ZNF326/OLBC15 silence (Figure S2A). The occupancies of ZNF326 to KLF17 promoter were also increased when OLBC15 was depleted (Figure S2B). These data suggested that OLBC15 could promote ZNF326 degradation via ubiquitination pathway.
OLBC15 destabilizes ZNF326 by increasing ubiquitination. A, ZNF326 expression in MDA‐MB‐231 cells transfected with ShCtrl or two shOLBC15 constructs. B, ZNF326 expression in MDA‐MB‐231 cells with or without OLBC15 silence treated with DMSO (MG132‐) or MG132 (MG132+). C, Ubiquitin ligation of ZNF326 in MDA‐MB‐231 cells with or without OLBC15 depletion expressing full‐length Flag‐tagged ZNF326. D, Relative expression of ZNF326 transcripts with either OLBC15 knockdown or overexpression. E, Migration assays for MDA‐MB‐231 cells with OLBC15 knockdown and/or ZNF326 shRNA. F, Quantification data for cellular migration in (E). G, Efficiency of ZNF326 silence on ZNF326 expression. ShZNF326‐2 showed higher efficiency and was selected as ShZNF326. **: P < .01
To confirm the underlying mechanism of OLBC15‐ZNF326 binding, we knocked down OLBC15 expression in MDA‐MB‐231 cells and we found that ZNF326 abundance was increased (Figure 5A). Co‐treatment with MG132, a proteasomal inhibitor, however, could elevate ZNF326 levels irrespective of whether OLBC15 was silenced (Figure 5B). OLBC15 knockdown substantially inhibited ZNF326 ubiquitination (Figure 5C). However, OLBC15 overexpression or depletion did not affect ZNF326 mRNA levels (Figure 5D). Migration assays revealed that knocking down OLBC15 could significantly inhibit breast cancer cell migration (Figure 5E,F). However, this effect could be counteracted by combinatorial ZNF326 silence (Figure 5E,F). The efficiency of ZNF326 silence was also verified (Figure 5G). Previous study has shown that ZNF326 can bind the promoter region of Krüppel‐like factor 17 (KLF17) gene and induce KLF17 expression.
16
OLBC15 knockdown indeed dramatically upregulated KLF17 expression, whereas KLF17 expression was inhibited after ZNF326 was silenced (Figure S2A). Notably, KLF17 level was partially rescued with simultaneous ZNF326/OLBC15 silence (Figure S2A). The occupancies of ZNF326 to KLF17 promoter were also increased when OLBC15 was depleted (Figure S2B). These data suggested that OLBC15 could promote ZNF326 degradation via ubiquitination pathway.
OLBC15 destabilizes ZNF326 by increasing ubiquitination. A, ZNF326 expression in MDA‐MB‐231 cells transfected with ShCtrl or two shOLBC15 constructs. B, ZNF326 expression in MDA‐MB‐231 cells with or without OLBC15 silence treated with DMSO (MG132‐) or MG132 (MG132+). C, Ubiquitin ligation of ZNF326 in MDA‐MB‐231 cells with or without OLBC15 depletion expressing full‐length Flag‐tagged ZNF326. D, Relative expression of ZNF326 transcripts with either OLBC15 knockdown or overexpression. E, Migration assays for MDA‐MB‐231 cells with OLBC15 knockdown and/or ZNF326 shRNA. F, Quantification data for cellular migration in (E). G, Efficiency of ZNF326 silence on ZNF326 expression. ShZNF326‐2 showed higher efficiency and was selected as ShZNF326. **: P < .01
Correlation between OLBC15 and ZNF326 in clinical samples We further investigated the clinical relevance of OLBC15 and ZNF326 in human samples. Immunohistochemistry (IHC) analyses showed various degrees of ZNF326 expression with different intensity scores which represented ZNF326 expression status (Figure 6A). Consistently, we found that OLBC15 level was not significantly associated with ZNF326 transcripts (Figure 6B). However, a significantly negative correlation was evident between ZNF326 protein expression and OLBC15 (Figure 6C). These results suggested that OLBC15 could inhibit ZNF326 expression in clinical specimens.
Correlation between OLBC15 and ZNF326 in clinical samples. A, Immunohistochemical (IHC) staining to identify ZNF326 in human specimens. Representative low (0), weak (1+), intermediate (2+), and strong staining (3+) cases using H‐score method were shown. Scale bar: 100 μm. B, Pearson correlation coefficient (R) between OLBC15 and ZNF326 mRNA abundance. The P value was shown. C, Correlation between OLBC15 levels and ZNF326 IHC scores (ie, protein abundance) in clinical samples
We further investigated the clinical relevance of OLBC15 and ZNF326 in human samples. Immunohistochemistry (IHC) analyses showed various degrees of ZNF326 expression with different intensity scores which represented ZNF326 expression status (Figure 6A). Consistently, we found that OLBC15 level was not significantly associated with ZNF326 transcripts (Figure 6B). However, a significantly negative correlation was evident between ZNF326 protein expression and OLBC15 (Figure 6C). These results suggested that OLBC15 could inhibit ZNF326 expression in clinical specimens.
Correlation between OLBC15 and ZNF326 in clinical samples. A, Immunohistochemical (IHC) staining to identify ZNF326 in human specimens. Representative low (0), weak (1+), intermediate (2+), and strong staining (3+) cases using H‐score method were shown. Scale bar: 100 μm. B, Pearson correlation coefficient (R) between OLBC15 and ZNF326 mRNA abundance. The P value was shown. C, Correlation between OLBC15 levels and ZNF326 IHC scores (ie, protein abundance) in clinical samples
Identifying OLBC15 in breast cancer via lncRNA profiling:
To screen for breast cancer (BC)‐related lncRNAs, we used lncRNA profiling assays in paired BC tissues and normal adjacent tissues (NATs) (Figure 1A). Overall, 73 significantly upregulated lncRNAs were detected (Figure 1A). These lncRNAs were candidate ones with oncogenic potential. Notably, two novel lncRNAs, RP11‐597D13.9 and OLBC15, were identified by lncRNA profiling (Table S2). Since lncRNA OLBC15 showed higher fold expression, we chose it for further analysis. OLBC15 was markedly upregulated in BC tissues compared with normal tissues (Figure 1B). OLBC15 is located on chromosome 15 with one annotated transcript (www.ensembl.org). The whole sequence for OLBC15 was shown (Figure S1A). Full‐length OLBC15 showed neglectable coding potential as evaluated by Coding Potential Assessment Tool (CPAT, coding probability ~ .032, Figure S1B). Moreover, OLBC15 was substantially enriched in triple‐negative breast cancer (TNBC) tissues compared with the other subtypes (Figure 1C). OLBC15 transcripts were highly increased in BC cell lines compared with MCF10A cells (Figure 1D). Patient characteristics demonstrated that OLBC15 expression significantly correlated with TNM stage, tumor size, and metastasis but was not significantly associated with age and histology (Table S3). Subcellular fractionation assay clarified that OLBC15 was primarily located in nucleus (Figure 1E). These data suggested that OLBC15 was upregulated in BC with nuclear distribution.
Identifying OLBC15 in breast cancer (BC). A, Heatmap representation of lncRNA profiling in normal adjacent tissues (NATs) and breast cancer tissues. Significantly upregulated and downregulated lncRNAs in BC tissues were 73 and 126, respectively. B, Relative OLBC15 expression in NAT and BC tissues. C, The relative OLBC15 levels in different breast cancer subtypes. The case numbers for luminal A (LA), luminal B (LB), HER2+ (HER2), and triple‐negative breast cancer (TNBC) were 40, 25, 26, and 51, respectively. D, Relative OLBC15 expression in transformed MCF10A and cancerous TNBC cell lines. E, Subcellular distribution of OLBC15. **: P < .01
OLBC15 facilitates breast cancer progression in vitro:
We next checked whether OLBC15 had an effect in vitro. We first stably overexpressed or silenced OLBC15 expression in MDA‐MB‐231 and BT‐549 cells (Figure 2A,B). The knockdown and overexpression efficiency were verified (Figure 2A,B). As expected, OLBC15 overexpression significantly promoted the viability of BT‐549 and MDA‐MB‐231 cells (Figure 2C,D). Consistently, OLBC15 depletion (shOLBC15) strongly inhibited the viability of breast cancer cells (Figure 2C,D). Migration assay further suggested that OLBC15 overexpression advanced migration in BT‐549 and MDA‐MB‐231 cells (Figure 2E,F; P < .01). OLBC15 silence instead attenuated migratory capacity in breast cancer cells (Figure 2E,F). These results implied that OLBC15 might play an oncogenic role in vitro.
OLBC15 promotes TNBC in vitro. A, Overexpression efficiency by lentiviral OLBC15 transfection. B, Knockdown efficiency using shRNA targeting OLBC15. ShOLBC15 displayed higher efficiency and was selected as ShOLBC15. C‐D, Effect of OLBC15 on viability of BT‐549 (C) and MDA‐MB‐231 (D) cells transfected with shRNA scrambled control (ShCtrl), ShOLBC15, lentiviral control (OE‐control), or the lentiviral vector carrying OLBC15 (OE‐OLBC15). E, Effect of OLBC15 on migratory capacity of MDA‐MB‐231 (top) and BT‐549 (bottom) cells. F, Quantification of results in (E). **: P < .01
OLBC15 promotes xenograft tumor growth and metastasis:
We then performed in vivo experiments to further investigate the function of OLBC15. Stably transfected MDA‐MB‐231 cells were used. OLBC15 knockdown inhibited tumor growth, whereas overexpressing OLBC15 promoted the tumor volume expansion (Figure 3A). A higher number of metastatic nodules were detected in OLBC15‐overexpressing group compared with the control (Figure 3B,C; P < .01). As expected, OLBC15 silence profoundly inhibited the occurrence of metastatic nodules (Figure 3B,C; P < .01). The xenograft tumor growth was evidently attenuated by OLBC15 depletion, whereas OLBC15 overexpression markedly accelerated the growth of xenograft tumors (Figure 3E,F; P < .01). These results further suggested that OLBC15 could exert an oncogenic effect in vivo.
OLBC15 advances TNBC in vivo. A, In vivo tumor volume growth of xenograft tumors with OLBC15 depletion or overexpression. B, Representative lung metastatic nodules with OLBC15 silence or overexpression. The description representing each numbered case was shown at the bottom. C, Quantification of metastatic nodules in (B). D, Xenograft tumors for MDA‐MB‐231 transplantation model with OLBC15 silence or overexpression. Numbered case description was shown at the bottom. E, Quantification data for (D). **: P < .01
OLBC15 interacts with ZNF326 protein:
We further used RNA pulldown assays to unravel the potential mechanism of OLBC15‐induced oncogenic effect. One specific band was detected (Figure 4A; indicated by arrow). The enriched proteins in OLBC15 pulldowns were then analyzed using mass spectrometry, and the results showed five putative hits (Table S4). Immunoblot confirmed that ZNF326 may be binding factor (Figure 4B). OLBC15 was significantly enriched in fractions with antibody against ZNF326 as determined by measuring co‐precipitated RNA (Figure 4C). Fluorescence in situ hybridization (FISH) also revealed colocalization for OLBC15 and ZNF326 (Figure 4D). Full‐length and various ZNF326 truncated mutants were constructed (Figure 4E), and we found that depleting the C‐terminal region of ZNF326 markedly abolished its interaction with OLBC15 (Figure 4F). Various OLBC15 truncated forms were also created according to the predicted secondary structure (Figure 4G,H). The results showed that the N‐terminal domain of OLBC15 was responsible for ZNF326 binding (Figure 4I). These results showed that OLBC15 may interact with ZNF326 protein.
ZNF326 interacts with OLBC15. A, RNA pulldown for lncRNA OLBC15. The indicates band was trimmed and subject to mass spectrometry (MS) analyses. B, Proteins obtained from RNA pulldowns were analyzed by antibody for ZNF326. C, RNA immunoprecipitation (RIP) results for OLBC15. The lncRNA LINC00501 was used as a negative control. D, Fluorescence in situ hybridization (FISH) to show the colocalization of OLBC15 and ZNF326. E, Full‐length and depletion constructs of ZNF326 were used to locate the binding region for OLBC15. F, RNA immunoprecipitation (RIP) followed by qRT‐PCR in DMA‐MB‐231 cells transfected with ectopic expression for full‐length (FL) Flag‐tagged ZNF326 or various mutant constructs. G, The secondary structure of OLBC15 predicted by an online tool termed RNAfold (http://rna.tbi.univie.ac.at//cgi‐bin/RNAWebSuite/RNAfold.cgi). The black box depicts the binding region for ZNF326. H, Construction of full‐length (FL) or various OLBC15 truncates. I, RIP followed by qRT‐PCR to show the putative interacting domain for OLBC15 with ZNF326. Fold enrichment data were rescaled by comparing each input with Flag‐Vec
OLBC15 facilitates ZNF326 degradation:
To confirm the underlying mechanism of OLBC15‐ZNF326 binding, we knocked down OLBC15 expression in MDA‐MB‐231 cells and we found that ZNF326 abundance was increased (Figure 5A). Co‐treatment with MG132, a proteasomal inhibitor, however, could elevate ZNF326 levels irrespective of whether OLBC15 was silenced (Figure 5B). OLBC15 knockdown substantially inhibited ZNF326 ubiquitination (Figure 5C). However, OLBC15 overexpression or depletion did not affect ZNF326 mRNA levels (Figure 5D). Migration assays revealed that knocking down OLBC15 could significantly inhibit breast cancer cell migration (Figure 5E,F). However, this effect could be counteracted by combinatorial ZNF326 silence (Figure 5E,F). The efficiency of ZNF326 silence was also verified (Figure 5G). Previous study has shown that ZNF326 can bind the promoter region of Krüppel‐like factor 17 (KLF17) gene and induce KLF17 expression.
16
OLBC15 knockdown indeed dramatically upregulated KLF17 expression, whereas KLF17 expression was inhibited after ZNF326 was silenced (Figure S2A). Notably, KLF17 level was partially rescued with simultaneous ZNF326/OLBC15 silence (Figure S2A). The occupancies of ZNF326 to KLF17 promoter were also increased when OLBC15 was depleted (Figure S2B). These data suggested that OLBC15 could promote ZNF326 degradation via ubiquitination pathway.
OLBC15 destabilizes ZNF326 by increasing ubiquitination. A, ZNF326 expression in MDA‐MB‐231 cells transfected with ShCtrl or two shOLBC15 constructs. B, ZNF326 expression in MDA‐MB‐231 cells with or without OLBC15 silence treated with DMSO (MG132‐) or MG132 (MG132+). C, Ubiquitin ligation of ZNF326 in MDA‐MB‐231 cells with or without OLBC15 depletion expressing full‐length Flag‐tagged ZNF326. D, Relative expression of ZNF326 transcripts with either OLBC15 knockdown or overexpression. E, Migration assays for MDA‐MB‐231 cells with OLBC15 knockdown and/or ZNF326 shRNA. F, Quantification data for cellular migration in (E). G, Efficiency of ZNF326 silence on ZNF326 expression. ShZNF326‐2 showed higher efficiency and was selected as ShZNF326. **: P < .01
Correlation between OLBC15 and ZNF326 in clinical samples:
We further investigated the clinical relevance of OLBC15 and ZNF326 in human samples. Immunohistochemistry (IHC) analyses showed various degrees of ZNF326 expression with different intensity scores which represented ZNF326 expression status (Figure 6A). Consistently, we found that OLBC15 level was not significantly associated with ZNF326 transcripts (Figure 6B). However, a significantly negative correlation was evident between ZNF326 protein expression and OLBC15 (Figure 6C). These results suggested that OLBC15 could inhibit ZNF326 expression in clinical specimens.
Correlation between OLBC15 and ZNF326 in clinical samples. A, Immunohistochemical (IHC) staining to identify ZNF326 in human specimens. Representative low (0), weak (1+), intermediate (2+), and strong staining (3+) cases using H‐score method were shown. Scale bar: 100 μm. B, Pearson correlation coefficient (R) between OLBC15 and ZNF326 mRNA abundance. The P value was shown. C, Correlation between OLBC15 levels and ZNF326 IHC scores (ie, protein abundance) in clinical samples
DISCUSSION:
In the current study, we have identified a novel oncogenic lncRNA OLBC15. We found that OLBC15 depletion may exert profound inhibition to breast cancer progression implying that OLBC15 may act as a putative target for intervention. OLBC15 expression was dramatically increased in breast cancer tissues especially TNBC. Furthermore, we also observed an oncogenic effect of OLBC15 via both in vitro and in vivo experiments. Mechanistic study showed that OLBC15 could interact with ZNF326, which is a novel tumor suppressor in TNBC.
16
OLBC15 facilitates ZNF326 degradation via ubiquitination pathway. These data collectively suggested that OLBC15 fulfills its oncogenic function via destabilizing the tumor suppressor ZNF326. Notably, the ubiquitin ligase responsible for ZNF326 is still elusive and whether OLBC15 enhances the interaction between ZNF326 and its ubiquitin ligase remains to be determined. Moreover, the possibility of OLBC15 as a competitive endogenous RNA (ceRNA) during breast cancer progression remains to be evaluated.
17
We found that OLBC15 interacts with ZNF326 to destabilize ZNF326 protein. The ZNF326 protein is initially identified in NIH3T3 cell line and modulates migration and growth.
18
Meanwhile, ZNF326 is also a transcriptional factor which activates neuronal differentiation.
19
ZNF326 can bind deleted in breast cancer 1 (DBC1) leading to the formation of DBIRD (DBC1/ZIRD) complex and result in its association with RNAPII.
20
Therefore, ZNF326 can actively participate in the process of alternative splicing in A/T‐rich regions of DNA.
16
Depleting ZNF326 can increase the expression of multiple genes involved in epithelial‐mesenchymal transition (EMT) in HEK293 cells and TNBC cell lines.
16
,
20
ZNF326 silence also leads to enhanced migratory and invasive capacity together with increased mammosphere formation in TNBC cells.
16
Consistently, ZNF326 knockdown also promotes orthologous transplant tumor formation, whereas overexpressing ZNF326 decreased xenograft tumor formation.
16
We have verified that OLBC15 silence can stabilize ZNF326 and as a result markedly augment the expression of KLF17, which is a well‐characterized tumor suppressor gene and negatively regulates EMT and metastasis in breast cancer.
21
Furthermore, increased expression of PRMT5/WDR77 complex in breast cancer cells results in ZNF326 methylation and is essential for Pol II elongation across A/T‐rich regions.
22
ZNF326 also represses breast cancer progression via diverse mechanisms.
16
These data support a tumor‐suppressive role of ZNF326 in TNBC. We should note that ZNF326 might play different roles in other types of cancer. For example, recent reports have demonstrated that cells with ZNF326 overexpression favor malignant phenotypes in glioma via increasing LDAC7 levels and activating Wnt signaling.
18
Wu et al have also identified that the C2H2 structure of ZNF326 can bind to the ERCC1 promoter and elevate ERCC1 expression in non–small‐cell lung cancer (NSCLC).
23
Therefore, ZNF326 can be a tumor suppressor and lncRNA OLBC15 promotes TNBC oncogenesis via destabilizing ZNF326 at least in TNBC. These data argue that OLBC15 may possibly be a tumor‐specific lncRNA in TNBC and the exact role of OLBC15 in other types of cancers remains to be investigated in future studies.
Notably, patients with TNBC suffer greatly from poor outcomes and effective target therapy is still lacking in TNBC.
5
,
24
The programmed cell death protein 1 (PD‐1)/PD ligand 1 (PD‐L1) as well as androgen receptors (AR) might be possible targets for therapeutic intervention to repress breast cancer progression although the efficiency and the frequent off‐target effects have posed serious concerns.
5
Current data have supported OLBC15 as a potential oncogenic lncRNA especially in TNBC. Therefore, the efficacy of OLBC15 targeting deserves further investigation. RNA interference might be a possible strategy; however, the lowered stability and cellular uptake especially in nucleus remain an obstacle to OLBC15 depletion.
25
The antisense oligonucleotides (ASOs) may provide a promising way to annihilate OLBC15 in breast cancer cells, and ASOs specifically targeting EZH2 and AR are highly effective in recent work.
26
A clinical trial has demonstrated that an ASO termed IONIS‐APO(a)Rx can be of favorable efficiency.
27
Therefore, we argue that using ASOs targeting OLBC15 may present an alternative strategy to abolish OLBC15 expression and inhibit TNBC development. There is also a possibility to target multiple TNBC‐associated oncogenic lncRNAs via ASOs, and relevant investigations should be performed in future.
Our current data have supported a novel transcript related to TNBC progression. OLBC15 directly interacts with ZNF326 tumor suppressor and interferes its post‐translational modification. OLBC15 has potentially clinical relevance and may aid in targeted design of anti‐cancer therapeutics especially in TNBC.
Supporting information:
Supplementary Material
Click here for additional data file.
|
Background:
The long non-coding RNAs (lncRNAs) have been involved in various processes, including cancer. However, the function of many lncRNAs is still elusive in triple-negative breast cancer (TNBC).
Methods:
LncRNA profiling was used to screen for novel lncRNAs related to TNBC. OLBC15 expression was measured via qRT-PCR. In vitro migration and viability assays were conducted to determine the oncogenic role of OLBC15. Xenograft and metastatic models were performed to further investigate effects in vivo. RNA immunoprecipitation (RIP), mass spectrometry (MS), and fluorescence in situ hybridization (FISH) strategies were designed to identify the interaction between ZNF326 and OLBC15.
Results:
In the current study, we have identified a novel oncogenic lncRNA termed OLBC15 via lncRNA profiling. OLBC15 is highly expressed especially in triple-negative breast cancer. OLBC15 promoted viability and migration in breast cancer cells. Moreover, OLBC15 could accelerate metastasis and xenograft tumor growth. Mechanistic study suggested that OLBC15 could bind a well-characterized tumor suppressor ZNF326 and OLBC15-ZNF326 interaction resulted in ZNF326 destabilization. OLBC15 induced proteasomal ZNF326 degradation through enhanced ubiquitination. OLBC15 and ZNF326 protein expression is also negatively correlated in clinical specimens.
Conclusions:
Collectively, OLBC15 may serve as an oncogenic lncRNA to facilitate TNBC progression and a putative target for therapeutic anti-breast cancer intervention.
| null | null | 8,682 | 259 | 16 |
[
"olbc15",
"znf326",
"figure",
"cells",
"expression",
"cancer",
"breast",
"breast cancer",
"mb",
"231"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] breast cancer | OLBC15 | oncogenesis | ZNF326 [SUMMARY]
| null |
[CONTENT] breast cancer | OLBC15 | oncogenesis | ZNF326 [SUMMARY]
| null |
[CONTENT] breast cancer | OLBC15 | oncogenesis | ZNF326 [SUMMARY]
| null |
[CONTENT] Breast | Carrier Proteins | Cell Line, Tumor | Disease Progression | Female | Humans | RNA, Long Noncoding | Triple Negative Breast Neoplasms [SUMMARY]
| null |
[CONTENT] Breast | Carrier Proteins | Cell Line, Tumor | Disease Progression | Female | Humans | RNA, Long Noncoding | Triple Negative Breast Neoplasms [SUMMARY]
| null |
[CONTENT] Breast | Carrier Proteins | Cell Line, Tumor | Disease Progression | Female | Humans | RNA, Long Noncoding | Triple Negative Breast Neoplasms [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] olbc15 | znf326 | figure | cells | expression | cancer | breast | breast cancer | mb | 231 [SUMMARY]
| null |
[CONTENT] olbc15 | znf326 | figure | cells | expression | cancer | breast | breast cancer | mb | 231 [SUMMARY]
| null |
[CONTENT] olbc15 | znf326 | figure | cells | expression | cancer | breast | breast cancer | mb | 231 [SUMMARY]
| null |
[CONTENT] tnbc | lncrna | non coding | coding | cancer | breast | breast cancer | olbc15 | rnas | coding rnas [SUMMARY]
| null |
[CONTENT] olbc15 | znf326 | figure | expression | 231 | mb | mb 231 | mda mb | mda | mda mb 231 [SUMMARY]
| null |
[CONTENT] olbc15 | znf326 | figure | cells | expression | cancer | breast cancer | breast | mda | mda mb [SUMMARY]
| null |
[CONTENT] ||| [SUMMARY]
| null |
[CONTENT] ||| ||| ||| ||| OLBC15 | ZNF326 | OLBC15-ZNF326 | ZNF326 ||| ||| ZNF326 [SUMMARY]
| null |
[CONTENT] ||| ||| TNBC ||| ||| ||| Xenograft ||| RNA | RIP | ZNF326 ||| ||| ||| ||| ||| ||| OLBC15 | ZNF326 | OLBC15-ZNF326 | ZNF326 ||| ||| ZNF326 ||| [SUMMARY]
| null |
|||
Effects of secukinumab and adalimumab on serum uric acid level in patients with plaque psoriasis.
|
35838407
|
Psoriasis is a chronic systemic inflammatory disease, and hyperuricemia is a common comorbidity in patients with psoriasis. However, there are limited reports on the relationship between serum uric acid levels and biological treatment efficacy. The purposes of this study were to compare the differences in serum uric acid levels between patients with psoriasis and healthy controls and analyze the risk of hyperuricemia.
|
BACKGROUND
|
A total of 196 patients with psoriasis and 191 age- and sex-matched healthy controls were enrolled in this retrospective cohort study. One hundred and twenty-seven patients with severe psoriasis were treated with biologics. Sixty-eight patients received adalimumab, and 59 patients received secukinumab. Serum uric acid levels were measured at baseline, week 24, and week 48 of treatment.
|
METHODS
|
Patients with psoriasis had higher serum uric acid levels than healthy controls (6.4 ± 1.7 mg/dL vs. 5.7 ± 1.5 mg/dL, P < 0.001). Hyperuricemia was found in 33.7% (66/196) of patients with psoriasis, which was significantly higher than that in healthy controls (13.1% [25/191], P < 0.001). Serum uric acid levels and hyperuricemia were not related to the severity of psoriasis ( P > 0.05). No significant changes in serum uric acid levels and hyperuricemia were observed following adalimumab treatment ( P > 0.05). The serum uric acid level in patients treated with secukinumab was 6.7 ± 1.6 mg/dL at week 24, which was not statistically different from that at baseline (6.6 ± 1.4 mg/dL, P = 0.885). Serum uric acid levels were significantly decreased at week 48 (6.3 ± 1.5 mg/dL vs. 6.6 ± 1.4 mg/dL, P = 0.007) in patients treated with secukinumab. Secukinumab had no significant effect on hyperuricemia either ( P > 0.05).
|
RESULTS
|
The serum uric acid levels and prevalence of hyperuricemia in patients with psoriasis were significantly higher than those in healthy controls. Secukinumab treatment for 48 weeks successfully decreased serum uric acid levels in patients with psoriasis, whereas adalimumab had no significant effect on serum uric acid levels.
|
CONCLUSIONS
|
[
"Adalimumab",
"Antibodies, Monoclonal, Humanized",
"Biological Products",
"Chronic Disease",
"Humans",
"Hyperuricemia",
"Psoriasis",
"Retrospective Studies",
"Treatment Outcome",
"Uric Acid"
] |
9481430
|
Introduction
|
Psoriasis is a chronic inflammatory disease.[1] In addition, patients with psoriasis have various comorbidities, such as arthritis, cardiovascular disease, metabolic syndrome, obesity, and diabetes mellitus.[2–5] In particular, psoriasis is associated with an increased risk of developing hyperuricemia.[6]
Uric acid is the end product of purine metabolism in humans, and it scavenges oxygen free radicals and prevents red blood cell membrane lipid oxidation. Urate deposits in joints, tendons, and other tissues in patients with hyperuricemia and induces gout. Hyperuricemia was found in 13.0% to 40.7% of patients with psoriasis,[7] and psoriasis was identified as an independent risk factor for gouty arthritis.[8] However, it was also reported that hyperuricemia and gout were not significantly associated with psoriasis.[9] Moreover, there have been few studies on the relationship between uric acid and psoriasis in Chinese patients.
The clinical symptoms of patients with psoriatic arthritis often appear 5 to 12 years after the initial skin manifestations.[10] The quality of life of patients with joint deformities is significantly decreased.[11] The use of biologics in patients with psoriasis can reduce the risk of developing psoriatic arthritis,[12] and multinational guidelines recommend biologics as first-line treatment options for psoriasis and psoriatic arthritis.[13,14] However, whether long-term treatment with biological agents affects serum uric acid levels in patients with psoriasis, especially Chinese psoriasis patients, remains unreported.
In summary, the purposes of our study were to compare the differences in serum uric acid levels between patients with psoriasis and healthy controls and analyze the risk of hyperuricemia. Furthermore, a 48-week follow-up was conducted to analyze the effects of adalimumab or secukinumab on uric acid levels in patients with psoriasis.
|
Methods
|
Ethical approval The study protocol was approved by the Ethics Committee of Peking University People's Hospital (No. 2020PHB303-01). Given the retrospective nature of the study, the requirement of written informed consent was waived.
The study protocol was approved by the Ethics Committee of Peking University People's Hospital (No. 2020PHB303-01). Given the retrospective nature of the study, the requirement of written informed consent was waived.
Study population The study population consisted of adult patients with plaque psoriasis in the Peking University People's Hospital from January 2019 to December 2020. All patients were diagnosed with plaque psoriasis clinically or histopathologically. The control group consisted of non-psoriatic healthy individuals who underwent regular health check-ups in our hospital during the same period. The severity of psoriasis was stratified according to body surface area (BSA) as follows: ≥10% indicated severe psoriasis, and BSA <10% indicated mild to moderate psoriasis.[15] Patients with severe plaque psoriasis who failed to respond, had a contraindication or were intolerant to other systemic therapies were divided into two groups and received adalimumab or secukinumab.
Excessive drinkers, subjects with advanced chronic kidney disease, or those taking drugs that affect serum uric acid levels (including allopurinol, diuretics, salicylate, ketoconazole, theophylline, pyrazinamide, and ethambutol) were excluded from the study. Other exclusion criteria for patients with biological treatment were previous exposure to active inflammatory or infectious diseases, hematologic or solid cancers, systemic treatments (including methotrexate, acitretin, and cyclosporine A) in the previous 1 month, and biological agents in the previous 3 months.
The sample size was calculated using PASS software (version 15.0.5, NCSS, Kaysville, UT, USA). Based on data from previous studies, we used the random meta-analysis model to estimate the mean difference in serum uric acid levels between patients with psoriasis and controls.[16–18] According to the results, the estimated mean difference in serum uric acid levels was 0.6 mg/dL. We assumed that the standard deviations of serum uric acid levels in patients with psoriasis and controls were 1.6 and 1.4 mg/dL, respectively, which were the largest values reported by previous studies.[16–18] With a two-sided significance level (alpha) of 0.05, a 1:1 group allocation ratio, and an estimated 20% non-response rate, we calculated that a total of 326 participants (163 for patients and 163 for controls) would be needed to obtain a 90% power to reject the null hypothesis using a two-sample unequal-variance t test.
The study population consisted of adult patients with plaque psoriasis in the Peking University People's Hospital from January 2019 to December 2020. All patients were diagnosed with plaque psoriasis clinically or histopathologically. The control group consisted of non-psoriatic healthy individuals who underwent regular health check-ups in our hospital during the same period. The severity of psoriasis was stratified according to body surface area (BSA) as follows: ≥10% indicated severe psoriasis, and BSA <10% indicated mild to moderate psoriasis.[15] Patients with severe plaque psoriasis who failed to respond, had a contraindication or were intolerant to other systemic therapies were divided into two groups and received adalimumab or secukinumab.
Excessive drinkers, subjects with advanced chronic kidney disease, or those taking drugs that affect serum uric acid levels (including allopurinol, diuretics, salicylate, ketoconazole, theophylline, pyrazinamide, and ethambutol) were excluded from the study. Other exclusion criteria for patients with biological treatment were previous exposure to active inflammatory or infectious diseases, hematologic or solid cancers, systemic treatments (including methotrexate, acitretin, and cyclosporine A) in the previous 1 month, and biological agents in the previous 3 months.
The sample size was calculated using PASS software (version 15.0.5, NCSS, Kaysville, UT, USA). Based on data from previous studies, we used the random meta-analysis model to estimate the mean difference in serum uric acid levels between patients with psoriasis and controls.[16–18] According to the results, the estimated mean difference in serum uric acid levels was 0.6 mg/dL. We assumed that the standard deviations of serum uric acid levels in patients with psoriasis and controls were 1.6 and 1.4 mg/dL, respectively, which were the largest values reported by previous studies.[16–18] With a two-sided significance level (alpha) of 0.05, a 1:1 group allocation ratio, and an estimated 20% non-response rate, we calculated that a total of 326 participants (163 for patients and 163 for controls) would be needed to obtain a 90% power to reject the null hypothesis using a two-sample unequal-variance t test.
Study design This was a retrospective cohort study. The baseline demographic and disease characteristics were collected. Physical examination and blood biochemistry analysis were performed at the first visit. Patients treated with biologics were followed up for 48 weeks after the first injection. Serum uric acid levels were measured at baseline, week 24, and week 48.
This was a retrospective cohort study. The baseline demographic and disease characteristics were collected. Physical examination and blood biochemistry analysis were performed at the first visit. Patients treated with biologics were followed up for 48 weeks after the first injection. Serum uric acid levels were measured at baseline, week 24, and week 48.
Primary outcomes and related parameters The primary outcomes of this study were the differences in mean serum uric acid levels between patients with psoriasis and healthy controls, the relationship between psoriasis and hyperuricemia, and the effects of biologics on serum uric levels. A patient was defined as developing hyperuricemia if their diagnosis was confirmed by a physician and/or fulfilled hyperuricemia criteria. Generally, hyperuricemia was defined as an increased serum uric acid level >7 mg/dL in men and >6 mg/dL in women at two different visits.[19,20]
The primary outcomes of this study were the differences in mean serum uric acid levels between patients with psoriasis and healthy controls, the relationship between psoriasis and hyperuricemia, and the effects of biologics on serum uric levels. A patient was defined as developing hyperuricemia if their diagnosis was confirmed by a physician and/or fulfilled hyperuricemia criteria. Generally, hyperuricemia was defined as an increased serum uric acid level >7 mg/dL in men and >6 mg/dL in women at two different visits.[19,20]
Statistical analysis All statistical analyses were performed with SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). Continuous variables were expressed as the mean ± standard deviation or median (interquartile range). Categorical data were expressed as a frequency (percentage). Quantitative indicators were compared using the Student t test or Mann–Whitney U test according to the data distribution. Categorical indicators were compared by the chi-square test and Fisher exact test. A generalized linear model was built to determine the correlative factors for hyperuricemia. The covariates included in the generalized linear model were sex, age, and body mass index (BMI). The serum uric acid levels and the prevalence of hyperuricemia in patients using biologics at baseline, week 24, and week 48 were analyzed by the paired t test and McNemar test. The missing values in this study were filled using the last observation carried forward (LOCF) method. Sensitivity analyses of the LOCF method were performed to further analyze the effects of biologics on serum uric acid levels and hyperuricemia. Statistical significance was defined as a P value < 0.05.
All statistical analyses were performed with SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). Continuous variables were expressed as the mean ± standard deviation or median (interquartile range). Categorical data were expressed as a frequency (percentage). Quantitative indicators were compared using the Student t test or Mann–Whitney U test according to the data distribution. Categorical indicators were compared by the chi-square test and Fisher exact test. A generalized linear model was built to determine the correlative factors for hyperuricemia. The covariates included in the generalized linear model were sex, age, and body mass index (BMI). The serum uric acid levels and the prevalence of hyperuricemia in patients using biologics at baseline, week 24, and week 48 were analyzed by the paired t test and McNemar test. The missing values in this study were filled using the last observation carried forward (LOCF) method. Sensitivity analyses of the LOCF method were performed to further analyze the effects of biologics on serum uric acid levels and hyperuricemia. Statistical significance was defined as a P value < 0.05.
|
Results
|
Study population characteristics The patients’ demographic characteristics at baseline are shown in [Table 1]. Based on the enrolment procedure, 196 patients (134 males, 62 females) with plaque psoriasis were included in this study. The average age was 40.4 ± 12.0 years, and the average BMI was 25.9 ± 4.4 kg/m2. The control group consisted of 191 adults (128 males, 63 females), and the average BMI was 24.4 ± 3.5 kg/m2.
Demographic and clinical characteristics of the study population at baseline.
Overweight and obesity were defined as BMI ≥25.0 and 30.0 kg/m2, respectively.[42] Data are presented as the mean ± standard deviation or n (%). Statistical significance was defined as a P value <0.05. BMI: Body mass index; BSA: Body surface area.
The patients’ demographic characteristics at baseline are shown in [Table 1]. Based on the enrolment procedure, 196 patients (134 males, 62 females) with plaque psoriasis were included in this study. The average age was 40.4 ± 12.0 years, and the average BMI was 25.9 ± 4.4 kg/m2. The control group consisted of 191 adults (128 males, 63 females), and the average BMI was 24.4 ± 3.5 kg/m2.
Demographic and clinical characteristics of the study population at baseline.
Overweight and obesity were defined as BMI ≥25.0 and 30.0 kg/m2, respectively.[42] Data are presented as the mean ± standard deviation or n (%). Statistical significance was defined as a P value <0.05. BMI: Body mass index; BSA: Body surface area.
Serum uric acid levels in patients with psoriasis and healthy controls Patients with psoriasis had higher serum uric acid levels compared with healthy controls (6.4 ± 1.7 mg/dL vs. 5.7 ± 1.5 mg/dL, P < 0.001). Hyperuricemia was found in 33.7% (66/196) of patients with psoriasis, which was significantly higher than that in healthy controls (13.1% [25/191], P < 0.001). No significant difference was found between patients with mild to moderate psoriasis and severe psoriasis (P > 0.05). Of note, the above results showed statistical significance after the calibration of BMI [Table 1].
Patients with psoriasis had higher serum uric acid levels compared with healthy controls (6.4 ± 1.7 mg/dL vs. 5.7 ± 1.5 mg/dL, P < 0.001). Hyperuricemia was found in 33.7% (66/196) of patients with psoriasis, which was significantly higher than that in healthy controls (13.1% [25/191], P < 0.001). No significant difference was found between patients with mild to moderate psoriasis and severe psoriasis (P > 0.05). Of note, the above results showed statistical significance after the calibration of BMI [Table 1].
Risk factors of hyperuricemia Male sex was associated with an increased risk of hyperuricemia Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).
Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).
Hyperuricemia was related to higher BMI Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).
Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).
Male sex was associated with an increased risk of hyperuricemia Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).
Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).
Hyperuricemia was related to higher BMI Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).
Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).
Effects of biologics on serum uric acid levels Effects of adalimumab At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].
Effects of adalimumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].
Effects of adalimumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Effects of secukinumab Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].
Effects of secukinumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].
Effects of secukinumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Effects of adalimumab At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].
Effects of adalimumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].
Effects of adalimumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Effects of secukinumab Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].
Effects of secukinumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].
Effects of secukinumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
| null | null |
[
"Ethical approval",
"Study population",
"Study design",
"Primary outcomes and related parameters",
"Statistical analysis",
"Study population characteristics",
"Risk factors of hyperuricemia",
"Male sex was associated with an increased risk of hyperuricemia",
"Hyperuricemia was related to higher BMI",
"Effects of biologics on serum uric acid levels",
"Effects of adalimumab",
"Effects of secukinumab",
"Acknowledgements"
] |
[
"The study protocol was approved by the Ethics Committee of Peking University People's Hospital (No. 2020PHB303-01). Given the retrospective nature of the study, the requirement of written informed consent was waived.",
"The study population consisted of adult patients with plaque psoriasis in the Peking University People's Hospital from January 2019 to December 2020. All patients were diagnosed with plaque psoriasis clinically or histopathologically. The control group consisted of non-psoriatic healthy individuals who underwent regular health check-ups in our hospital during the same period. The severity of psoriasis was stratified according to body surface area (BSA) as follows: ≥10% indicated severe psoriasis, and BSA <10% indicated mild to moderate psoriasis.[15] Patients with severe plaque psoriasis who failed to respond, had a contraindication or were intolerant to other systemic therapies were divided into two groups and received adalimumab or secukinumab.\nExcessive drinkers, subjects with advanced chronic kidney disease, or those taking drugs that affect serum uric acid levels (including allopurinol, diuretics, salicylate, ketoconazole, theophylline, pyrazinamide, and ethambutol) were excluded from the study. Other exclusion criteria for patients with biological treatment were previous exposure to active inflammatory or infectious diseases, hematologic or solid cancers, systemic treatments (including methotrexate, acitretin, and cyclosporine A) in the previous 1 month, and biological agents in the previous 3 months.\nThe sample size was calculated using PASS software (version 15.0.5, NCSS, Kaysville, UT, USA). Based on data from previous studies, we used the random meta-analysis model to estimate the mean difference in serum uric acid levels between patients with psoriasis and controls.[16–18] According to the results, the estimated mean difference in serum uric acid levels was 0.6 mg/dL. We assumed that the standard deviations of serum uric acid levels in patients with psoriasis and controls were 1.6 and 1.4 mg/dL, respectively, which were the largest values reported by previous studies.[16–18] With a two-sided significance level (alpha) of 0.05, a 1:1 group allocation ratio, and an estimated 20% non-response rate, we calculated that a total of 326 participants (163 for patients and 163 for controls) would be needed to obtain a 90% power to reject the null hypothesis using a two-sample unequal-variance t test.",
"This was a retrospective cohort study. The baseline demographic and disease characteristics were collected. Physical examination and blood biochemistry analysis were performed at the first visit. Patients treated with biologics were followed up for 48 weeks after the first injection. Serum uric acid levels were measured at baseline, week 24, and week 48.",
"The primary outcomes of this study were the differences in mean serum uric acid levels between patients with psoriasis and healthy controls, the relationship between psoriasis and hyperuricemia, and the effects of biologics on serum uric levels. A patient was defined as developing hyperuricemia if their diagnosis was confirmed by a physician and/or fulfilled hyperuricemia criteria. Generally, hyperuricemia was defined as an increased serum uric acid level >7 mg/dL in men and >6 mg/dL in women at two different visits.[19,20]",
"All statistical analyses were performed with SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). Continuous variables were expressed as the mean ± standard deviation or median (interquartile range). Categorical data were expressed as a frequency (percentage). Quantitative indicators were compared using the Student t test or Mann–Whitney U test according to the data distribution. Categorical indicators were compared by the chi-square test and Fisher exact test. A generalized linear model was built to determine the correlative factors for hyperuricemia. The covariates included in the generalized linear model were sex, age, and body mass index (BMI). The serum uric acid levels and the prevalence of hyperuricemia in patients using biologics at baseline, week 24, and week 48 were analyzed by the paired t test and McNemar test. The missing values in this study were filled using the last observation carried forward (LOCF) method. Sensitivity analyses of the LOCF method were performed to further analyze the effects of biologics on serum uric acid levels and hyperuricemia. Statistical significance was defined as a P value < 0.05.",
"The patients’ demographic characteristics at baseline are shown in [Table 1]. Based on the enrolment procedure, 196 patients (134 males, 62 females) with plaque psoriasis were included in this study. The average age was 40.4 ± 12.0 years, and the average BMI was 25.9 ± 4.4 kg/m2. The control group consisted of 191 adults (128 males, 63 females), and the average BMI was 24.4 ± 3.5 kg/m2.\nDemographic and clinical characteristics of the study population at baseline.\nOverweight and obesity were defined as BMI ≥25.0 and 30.0 kg/m2, respectively.[42] Data are presented as the mean ± standard deviation or n (%). Statistical significance was defined as a P value <0.05. BMI: Body mass index; BSA: Body surface area.",
" Male sex was associated with an increased risk of hyperuricemia Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).\nHyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).\n Hyperuricemia was related to higher BMI Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).\nHyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).",
"Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).",
"Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).",
" Effects of adalimumab At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].\nEffects of adalimumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\nAt baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].\nEffects of adalimumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\n Effects of secukinumab Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].\nEffects of secukinumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\nFifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].\nEffects of secukinumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.",
"At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].\nEffects of adalimumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.",
"Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].\nEffects of secukinumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.",
"We gratefully thank all doctors who support our registry database."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Introduction",
"Methods",
"Ethical approval",
"Study population",
"Study design",
"Primary outcomes and related parameters",
"Statistical analysis",
"Results",
"Study population characteristics",
"Serum uric acid levels in patients with psoriasis and healthy controls",
"Risk factors of hyperuricemia",
"Male sex was associated with an increased risk of hyperuricemia",
"Hyperuricemia was related to higher BMI",
"Effects of biologics on serum uric acid levels",
"Effects of adalimumab",
"Effects of secukinumab",
"Discussion",
"Acknowledgements",
"Conflicts of interest",
"Supplementary Material"
] |
[
"Psoriasis is a chronic inflammatory disease.[1] In addition, patients with psoriasis have various comorbidities, such as arthritis, cardiovascular disease, metabolic syndrome, obesity, and diabetes mellitus.[2–5] In particular, psoriasis is associated with an increased risk of developing hyperuricemia.[6]\nUric acid is the end product of purine metabolism in humans, and it scavenges oxygen free radicals and prevents red blood cell membrane lipid oxidation. Urate deposits in joints, tendons, and other tissues in patients with hyperuricemia and induces gout. Hyperuricemia was found in 13.0% to 40.7% of patients with psoriasis,[7] and psoriasis was identified as an independent risk factor for gouty arthritis.[8] However, it was also reported that hyperuricemia and gout were not significantly associated with psoriasis.[9] Moreover, there have been few studies on the relationship between uric acid and psoriasis in Chinese patients.\nThe clinical symptoms of patients with psoriatic arthritis often appear 5 to 12 years after the initial skin manifestations.[10] The quality of life of patients with joint deformities is significantly decreased.[11] The use of biologics in patients with psoriasis can reduce the risk of developing psoriatic arthritis,[12] and multinational guidelines recommend biologics as first-line treatment options for psoriasis and psoriatic arthritis.[13,14] However, whether long-term treatment with biological agents affects serum uric acid levels in patients with psoriasis, especially Chinese psoriasis patients, remains unreported.\nIn summary, the purposes of our study were to compare the differences in serum uric acid levels between patients with psoriasis and healthy controls and analyze the risk of hyperuricemia. Furthermore, a 48-week follow-up was conducted to analyze the effects of adalimumab or secukinumab on uric acid levels in patients with psoriasis.",
" Ethical approval The study protocol was approved by the Ethics Committee of Peking University People's Hospital (No. 2020PHB303-01). Given the retrospective nature of the study, the requirement of written informed consent was waived.\nThe study protocol was approved by the Ethics Committee of Peking University People's Hospital (No. 2020PHB303-01). Given the retrospective nature of the study, the requirement of written informed consent was waived.\n Study population The study population consisted of adult patients with plaque psoriasis in the Peking University People's Hospital from January 2019 to December 2020. All patients were diagnosed with plaque psoriasis clinically or histopathologically. The control group consisted of non-psoriatic healthy individuals who underwent regular health check-ups in our hospital during the same period. The severity of psoriasis was stratified according to body surface area (BSA) as follows: ≥10% indicated severe psoriasis, and BSA <10% indicated mild to moderate psoriasis.[15] Patients with severe plaque psoriasis who failed to respond, had a contraindication or were intolerant to other systemic therapies were divided into two groups and received adalimumab or secukinumab.\nExcessive drinkers, subjects with advanced chronic kidney disease, or those taking drugs that affect serum uric acid levels (including allopurinol, diuretics, salicylate, ketoconazole, theophylline, pyrazinamide, and ethambutol) were excluded from the study. Other exclusion criteria for patients with biological treatment were previous exposure to active inflammatory or infectious diseases, hematologic or solid cancers, systemic treatments (including methotrexate, acitretin, and cyclosporine A) in the previous 1 month, and biological agents in the previous 3 months.\nThe sample size was calculated using PASS software (version 15.0.5, NCSS, Kaysville, UT, USA). Based on data from previous studies, we used the random meta-analysis model to estimate the mean difference in serum uric acid levels between patients with psoriasis and controls.[16–18] According to the results, the estimated mean difference in serum uric acid levels was 0.6 mg/dL. We assumed that the standard deviations of serum uric acid levels in patients with psoriasis and controls were 1.6 and 1.4 mg/dL, respectively, which were the largest values reported by previous studies.[16–18] With a two-sided significance level (alpha) of 0.05, a 1:1 group allocation ratio, and an estimated 20% non-response rate, we calculated that a total of 326 participants (163 for patients and 163 for controls) would be needed to obtain a 90% power to reject the null hypothesis using a two-sample unequal-variance t test.\nThe study population consisted of adult patients with plaque psoriasis in the Peking University People's Hospital from January 2019 to December 2020. All patients were diagnosed with plaque psoriasis clinically or histopathologically. The control group consisted of non-psoriatic healthy individuals who underwent regular health check-ups in our hospital during the same period. The severity of psoriasis was stratified according to body surface area (BSA) as follows: ≥10% indicated severe psoriasis, and BSA <10% indicated mild to moderate psoriasis.[15] Patients with severe plaque psoriasis who failed to respond, had a contraindication or were intolerant to other systemic therapies were divided into two groups and received adalimumab or secukinumab.\nExcessive drinkers, subjects with advanced chronic kidney disease, or those taking drugs that affect serum uric acid levels (including allopurinol, diuretics, salicylate, ketoconazole, theophylline, pyrazinamide, and ethambutol) were excluded from the study. Other exclusion criteria for patients with biological treatment were previous exposure to active inflammatory or infectious diseases, hematologic or solid cancers, systemic treatments (including methotrexate, acitretin, and cyclosporine A) in the previous 1 month, and biological agents in the previous 3 months.\nThe sample size was calculated using PASS software (version 15.0.5, NCSS, Kaysville, UT, USA). Based on data from previous studies, we used the random meta-analysis model to estimate the mean difference in serum uric acid levels between patients with psoriasis and controls.[16–18] According to the results, the estimated mean difference in serum uric acid levels was 0.6 mg/dL. We assumed that the standard deviations of serum uric acid levels in patients with psoriasis and controls were 1.6 and 1.4 mg/dL, respectively, which were the largest values reported by previous studies.[16–18] With a two-sided significance level (alpha) of 0.05, a 1:1 group allocation ratio, and an estimated 20% non-response rate, we calculated that a total of 326 participants (163 for patients and 163 for controls) would be needed to obtain a 90% power to reject the null hypothesis using a two-sample unequal-variance t test.\n Study design This was a retrospective cohort study. The baseline demographic and disease characteristics were collected. Physical examination and blood biochemistry analysis were performed at the first visit. Patients treated with biologics were followed up for 48 weeks after the first injection. Serum uric acid levels were measured at baseline, week 24, and week 48.\nThis was a retrospective cohort study. The baseline demographic and disease characteristics were collected. Physical examination and blood biochemistry analysis were performed at the first visit. Patients treated with biologics were followed up for 48 weeks after the first injection. Serum uric acid levels were measured at baseline, week 24, and week 48.\n Primary outcomes and related parameters The primary outcomes of this study were the differences in mean serum uric acid levels between patients with psoriasis and healthy controls, the relationship between psoriasis and hyperuricemia, and the effects of biologics on serum uric levels. A patient was defined as developing hyperuricemia if their diagnosis was confirmed by a physician and/or fulfilled hyperuricemia criteria. Generally, hyperuricemia was defined as an increased serum uric acid level >7 mg/dL in men and >6 mg/dL in women at two different visits.[19,20]\nThe primary outcomes of this study were the differences in mean serum uric acid levels between patients with psoriasis and healthy controls, the relationship between psoriasis and hyperuricemia, and the effects of biologics on serum uric levels. A patient was defined as developing hyperuricemia if their diagnosis was confirmed by a physician and/or fulfilled hyperuricemia criteria. Generally, hyperuricemia was defined as an increased serum uric acid level >7 mg/dL in men and >6 mg/dL in women at two different visits.[19,20]\n Statistical analysis All statistical analyses were performed with SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). Continuous variables were expressed as the mean ± standard deviation or median (interquartile range). Categorical data were expressed as a frequency (percentage). Quantitative indicators were compared using the Student t test or Mann–Whitney U test according to the data distribution. Categorical indicators were compared by the chi-square test and Fisher exact test. A generalized linear model was built to determine the correlative factors for hyperuricemia. The covariates included in the generalized linear model were sex, age, and body mass index (BMI). The serum uric acid levels and the prevalence of hyperuricemia in patients using biologics at baseline, week 24, and week 48 were analyzed by the paired t test and McNemar test. The missing values in this study were filled using the last observation carried forward (LOCF) method. Sensitivity analyses of the LOCF method were performed to further analyze the effects of biologics on serum uric acid levels and hyperuricemia. Statistical significance was defined as a P value < 0.05.\nAll statistical analyses were performed with SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). Continuous variables were expressed as the mean ± standard deviation or median (interquartile range). Categorical data were expressed as a frequency (percentage). Quantitative indicators were compared using the Student t test or Mann–Whitney U test according to the data distribution. Categorical indicators were compared by the chi-square test and Fisher exact test. A generalized linear model was built to determine the correlative factors for hyperuricemia. The covariates included in the generalized linear model were sex, age, and body mass index (BMI). The serum uric acid levels and the prevalence of hyperuricemia in patients using biologics at baseline, week 24, and week 48 were analyzed by the paired t test and McNemar test. The missing values in this study were filled using the last observation carried forward (LOCF) method. Sensitivity analyses of the LOCF method were performed to further analyze the effects of biologics on serum uric acid levels and hyperuricemia. Statistical significance was defined as a P value < 0.05.",
"The study protocol was approved by the Ethics Committee of Peking University People's Hospital (No. 2020PHB303-01). Given the retrospective nature of the study, the requirement of written informed consent was waived.",
"The study population consisted of adult patients with plaque psoriasis in the Peking University People's Hospital from January 2019 to December 2020. All patients were diagnosed with plaque psoriasis clinically or histopathologically. The control group consisted of non-psoriatic healthy individuals who underwent regular health check-ups in our hospital during the same period. The severity of psoriasis was stratified according to body surface area (BSA) as follows: ≥10% indicated severe psoriasis, and BSA <10% indicated mild to moderate psoriasis.[15] Patients with severe plaque psoriasis who failed to respond, had a contraindication or were intolerant to other systemic therapies were divided into two groups and received adalimumab or secukinumab.\nExcessive drinkers, subjects with advanced chronic kidney disease, or those taking drugs that affect serum uric acid levels (including allopurinol, diuretics, salicylate, ketoconazole, theophylline, pyrazinamide, and ethambutol) were excluded from the study. Other exclusion criteria for patients with biological treatment were previous exposure to active inflammatory or infectious diseases, hematologic or solid cancers, systemic treatments (including methotrexate, acitretin, and cyclosporine A) in the previous 1 month, and biological agents in the previous 3 months.\nThe sample size was calculated using PASS software (version 15.0.5, NCSS, Kaysville, UT, USA). Based on data from previous studies, we used the random meta-analysis model to estimate the mean difference in serum uric acid levels between patients with psoriasis and controls.[16–18] According to the results, the estimated mean difference in serum uric acid levels was 0.6 mg/dL. We assumed that the standard deviations of serum uric acid levels in patients with psoriasis and controls were 1.6 and 1.4 mg/dL, respectively, which were the largest values reported by previous studies.[16–18] With a two-sided significance level (alpha) of 0.05, a 1:1 group allocation ratio, and an estimated 20% non-response rate, we calculated that a total of 326 participants (163 for patients and 163 for controls) would be needed to obtain a 90% power to reject the null hypothesis using a two-sample unequal-variance t test.",
"This was a retrospective cohort study. The baseline demographic and disease characteristics were collected. Physical examination and blood biochemistry analysis were performed at the first visit. Patients treated with biologics were followed up for 48 weeks after the first injection. Serum uric acid levels were measured at baseline, week 24, and week 48.",
"The primary outcomes of this study were the differences in mean serum uric acid levels between patients with psoriasis and healthy controls, the relationship between psoriasis and hyperuricemia, and the effects of biologics on serum uric levels. A patient was defined as developing hyperuricemia if their diagnosis was confirmed by a physician and/or fulfilled hyperuricemia criteria. Generally, hyperuricemia was defined as an increased serum uric acid level >7 mg/dL in men and >6 mg/dL in women at two different visits.[19,20]",
"All statistical analyses were performed with SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). Continuous variables were expressed as the mean ± standard deviation or median (interquartile range). Categorical data were expressed as a frequency (percentage). Quantitative indicators were compared using the Student t test or Mann–Whitney U test according to the data distribution. Categorical indicators were compared by the chi-square test and Fisher exact test. A generalized linear model was built to determine the correlative factors for hyperuricemia. The covariates included in the generalized linear model were sex, age, and body mass index (BMI). The serum uric acid levels and the prevalence of hyperuricemia in patients using biologics at baseline, week 24, and week 48 were analyzed by the paired t test and McNemar test. The missing values in this study were filled using the last observation carried forward (LOCF) method. Sensitivity analyses of the LOCF method were performed to further analyze the effects of biologics on serum uric acid levels and hyperuricemia. Statistical significance was defined as a P value < 0.05.",
" Study population characteristics The patients’ demographic characteristics at baseline are shown in [Table 1]. Based on the enrolment procedure, 196 patients (134 males, 62 females) with plaque psoriasis were included in this study. The average age was 40.4 ± 12.0 years, and the average BMI was 25.9 ± 4.4 kg/m2. The control group consisted of 191 adults (128 males, 63 females), and the average BMI was 24.4 ± 3.5 kg/m2.\nDemographic and clinical characteristics of the study population at baseline.\nOverweight and obesity were defined as BMI ≥25.0 and 30.0 kg/m2, respectively.[42] Data are presented as the mean ± standard deviation or n (%). Statistical significance was defined as a P value <0.05. BMI: Body mass index; BSA: Body surface area.\nThe patients’ demographic characteristics at baseline are shown in [Table 1]. Based on the enrolment procedure, 196 patients (134 males, 62 females) with plaque psoriasis were included in this study. The average age was 40.4 ± 12.0 years, and the average BMI was 25.9 ± 4.4 kg/m2. The control group consisted of 191 adults (128 males, 63 females), and the average BMI was 24.4 ± 3.5 kg/m2.\nDemographic and clinical characteristics of the study population at baseline.\nOverweight and obesity were defined as BMI ≥25.0 and 30.0 kg/m2, respectively.[42] Data are presented as the mean ± standard deviation or n (%). Statistical significance was defined as a P value <0.05. BMI: Body mass index; BSA: Body surface area.\n Serum uric acid levels in patients with psoriasis and healthy controls Patients with psoriasis had higher serum uric acid levels compared with healthy controls (6.4 ± 1.7 mg/dL vs. 5.7 ± 1.5 mg/dL, P < 0.001). Hyperuricemia was found in 33.7% (66/196) of patients with psoriasis, which was significantly higher than that in healthy controls (13.1% [25/191], P < 0.001). No significant difference was found between patients with mild to moderate psoriasis and severe psoriasis (P > 0.05). Of note, the above results showed statistical significance after the calibration of BMI [Table 1].\nPatients with psoriasis had higher serum uric acid levels compared with healthy controls (6.4 ± 1.7 mg/dL vs. 5.7 ± 1.5 mg/dL, P < 0.001). Hyperuricemia was found in 33.7% (66/196) of patients with psoriasis, which was significantly higher than that in healthy controls (13.1% [25/191], P < 0.001). No significant difference was found between patients with mild to moderate psoriasis and severe psoriasis (P > 0.05). Of note, the above results showed statistical significance after the calibration of BMI [Table 1].\n Risk factors of hyperuricemia Male sex was associated with an increased risk of hyperuricemia Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).\nHyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).\n Hyperuricemia was related to higher BMI Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).\nHyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).\n Male sex was associated with an increased risk of hyperuricemia Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).\nHyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).\n Hyperuricemia was related to higher BMI Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).\nHyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).\n Effects of biologics on serum uric acid levels Effects of adalimumab At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].\nEffects of adalimumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\nAt baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].\nEffects of adalimumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\n Effects of secukinumab Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].\nEffects of secukinumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\nFifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].\nEffects of secukinumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\n Effects of adalimumab At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].\nEffects of adalimumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\nAt baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].\nEffects of adalimumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\n Effects of secukinumab Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].\nEffects of secukinumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\nFifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].\nEffects of secukinumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.",
"The patients’ demographic characteristics at baseline are shown in [Table 1]. Based on the enrolment procedure, 196 patients (134 males, 62 females) with plaque psoriasis were included in this study. The average age was 40.4 ± 12.0 years, and the average BMI was 25.9 ± 4.4 kg/m2. The control group consisted of 191 adults (128 males, 63 females), and the average BMI was 24.4 ± 3.5 kg/m2.\nDemographic and clinical characteristics of the study population at baseline.\nOverweight and obesity were defined as BMI ≥25.0 and 30.0 kg/m2, respectively.[42] Data are presented as the mean ± standard deviation or n (%). Statistical significance was defined as a P value <0.05. BMI: Body mass index; BSA: Body surface area.",
"Patients with psoriasis had higher serum uric acid levels compared with healthy controls (6.4 ± 1.7 mg/dL vs. 5.7 ± 1.5 mg/dL, P < 0.001). Hyperuricemia was found in 33.7% (66/196) of patients with psoriasis, which was significantly higher than that in healthy controls (13.1% [25/191], P < 0.001). No significant difference was found between patients with mild to moderate psoriasis and severe psoriasis (P > 0.05). Of note, the above results showed statistical significance after the calibration of BMI [Table 1].",
" Male sex was associated with an increased risk of hyperuricemia Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).\nHyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).\n Hyperuricemia was related to higher BMI Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).\nHyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).",
"Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).",
"Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).",
" Effects of adalimumab At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].\nEffects of adalimumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\nAt baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].\nEffects of adalimumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\n Effects of secukinumab Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].\nEffects of secukinumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.\nFifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].\nEffects of secukinumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.",
"At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].\nEffects of adalimumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.",
"Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].\nEffects of secukinumab on serum uric acid levels in patients with psoriasis.\nData are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.",
"Psoriasis was previously reported to be associated with elevated serum uric acid levels.[6,8] Goldman[21] proposed that serum uric acid was involved in the pathogenesis of psoriasis. Tsuruta et al[22] also suggested that uric acid mediates inflammation.\nHyperuricemia is related to multiple factors, including gender, age, race, lifestyle, and environmental conditions.[23] The United States Health and Nutrition Examination Survey showed that the prevalence of hyperuricemia in men and women was 20.2% and 20.0%, respectively.[24] In addition, a Chinese study reported that the prevalence of hyperuricemia was 18.5% in males and 8.0% in females.[25] According to a meta-analysis, the prevalence of hyperuricemia in Mainland China was 13.3%.[26] In this study, we found that the prevalence of hyperuricemia was 17.2% in males and 4.8% in females, consistent with previous reports.\nThe relationship between psoriasis and hyperuricemia remains controversial. Gisondi et al[16] reported that hyperuricemia was frequently observed in patients with psoriasis. However, Kwon et al[27] reported that there was no significant difference in serum uric acid levels between patients with psoriasis and healthy controls. In this study, we found that the prevalence of hyperuricemia was significantly higher in patients with psoriasis than that in healthy controls.\nSimilarly, whether serum uric acid is related to the severity of psoriasis is controversial. Gisondi et al[16] found that serum uric acid levels in patients with Psoriasis Area and Severity Index (PASI) scores ≥10 were significantly higher than those in participants with PASI scores <10. Yilmaz et al[17] also found that the PASI score was positively correlated with serum uric acid levels. However, Gui et al[18] reported no significant correlation between serum uric acid levels and PASI scores in patients with psoriasis. Our study showed that the serum uric acid levels and prevalence of hyperuricemia were unrelated to the severity of psoriasis. Therefore, serum uric acid levels should be carefully considered, regardless of the severity of skin lesions.\nThe mechanism by which patients with psoriasis are prone to hyperuricemia remains unclear. Goldman[21] proposed that the replacement rate of keratinocytes in psoriasis patients was accelerated, and the increase in purine decomposition caused excessive uric acid production, subsequently increasing serum uric acid levels. Furthermore, urate crystals, a type of alarmin, activate innate immunity through NALP inflammasomes and increase cytokine production, including interleukin (IL)-1 and IL-8.[28] Uratsuji et al[29] confirmed that epidermal keratinocytes cultured in the presence of uric acid crystals formed NALP inflammasomes and produced large amounts of tumor necrosis factor (TNF)-α, IL-1a, IL-8/chemokine (CXC motif) ligand 8, and IL-6. These cytokines play an important role in the pathogenesis of psoriasis. In summary, psoriasis increases serum uric acid levels, and urate crystals may further promote the development of psoriasis.\nOther factors that may affect serum uric acid levels and hyperuricemia in patients with psoriasis include age, gender, and BMI. Gui et al[18] found that serum uric acid levels were significantly positively correlated with BMI (P < 0.001). Similarly, Lai et al[30] proposed that serum uric acid levels were positively correlated with BMI (P < 0.001), and overweight status showed the strongest correlation with hyperuricemia in patients with psoriatic arthritis (P < 0.001). Kwon et al[27] found that serum uric acid levels in patients with psoriasis had no significant correlation with age, age at psoriasis onset, or family history of psoriasis. Here, we also analyzed the correlation between serum uric acid levels and BMI. The results showed that serum uric acid levels in overweight and obese patients with psoriasis were significantly higher than those in normal-weight patients. Therefore, to reduce the risk of hyperuricemia, patients with psoriasis should be instructed to strictly control their diet and weight, especially male patients.\nLong-term dietary exposure to alcohol, red meat, seafood, and sugar-sweetened beverages can increase the risk of hyperuricemia.[31,32] Alcohol and fructose increase the production of urate, and alcohol consumption inhibits renal urate excretion. Conversely, the intake of dairy products and coffee reduces the risk of hyperuricemia.[33,34] Dietary management has been reported to lower serum uric acid levels by approximately 1.0 mg/dL.[35] Therefore, health education regarding a low-purine diet in patients with psoriasis is necessary.\nTNF-α is a key cytokine in the pathogenesis of psoriasis and associated comorbidities. TNF-α inhibitors are commonly used in the treatment of moderate to severe plaque psoriasis with good effectiveness and safety.[36] Montaudié et al[37] found no significant change in serum uric acid levels at the 3rd and 6th month of follow-up in patients with psoriasis treated with biological agents (etanercept, infliximab, adalimumab, and ustekinumab). However, Hasikova et al[38] reported increased serum uric acid levels in 128 patients with systemic autoimmune rheumatic diseases treated with TNF-α inhibitors for 3 months. Our study revealed no significant change in the serum uric acid levels or prevalence of hyperuricemia in patients with psoriasis after adalimumab treatment for 48 weeks.\nSecukinumab, a fully human monoclonal anti-IL-17a antibody, has shown good efficacy and safety in Chinese patients with moderate to severe plaque psoriasis.[39] Gerdes et al[40] reported that uric acid levels tended to decrease over 52 weeks of treatment with secukinumab in plaque psoriasis patients. However, Karataş et al[41] found that serum uric acid levels were not significantly different from baseline in 30 patients with ankylosing spondylitis and six patients with psoriatic arthritis treated with secukinumab for 6 months. Our study revealed slightly lower serum uric acid levels in patients treated with secukinumab for 48 weeks compared with baseline values, but the prevalence of hyperuricemia was not significantly different. The mechanism underlying the decrease in serum uric acid levels associated with secukinumab treatment remains elusive.\nIn conclusion, as a comorbid condition related to metabolism in psoriasis, hyperuricemia should attract our attention. Our study revealed that the serum uric acid levels and prevalence of hyperuricemia in patients with psoriasis were higher than those in healthy controls. Moreover, the serum uric acid levels and prevalence of hyperuricemia were related to obesity and gender rather than the severity of psoriasis. The above results suggested that patients with psoriasis should limit their diet and maintain a healthy weight regardless of disease severity, especially male patients. Compared with baseline values, adalimumab and secukinumab had no significant effect on the prevalence of hyperuricemia in patients with psoriasis after 48 weeks of treatment. Both biologics can be applied to psoriasis patients with hyperuricemia.\nThe limitations of our study include its retrospective nature, single-center design, and short-term observation period. In our hospital, there are more patients with severe psoriasis than patients with mild to moderate psoriasis. Therefore, selection bias cannot be avoided. Because this was a retrospective cohort study, the effects of biologics on patients lost to follow-up could not be determined. Therefore, a large sample, multi-center, long-term prospective study should be performed to demonstrate the correlation between psoriasis and serum uric acid levels. Although our study excluded patients with excessive alcohol consumption, the effects of dietary habits on serum uric acid should be considered. However, this study provides a theoretical basis for the prevention of hyperuricemia in patients with psoriasis and the development of biological treatment strategies.",
"We gratefully thank all doctors who support our registry database.",
"Lin Cai has participated in advisory boards as an investigator and/or speaker and received grants and/or honoraria from Novartis, AbbVie, Sanofi, and Eli Lilly Inc. Jianzhong Zhang has participated in advisory boards as an investigator and/or speaker and received grants and/or honoraria from LEO Pharma China, Novartis, Sanofi, AbbVie, Bayer, Janssen-Cilag, Henlius, Kyowa Kirin, and Pfizer Inc.",
""
] |
[
"intro",
"methods",
null,
null,
null,
null,
null,
"results",
null,
"subjects",
null,
null,
null,
null,
null,
null,
"discussion",
null,
"COI-statement",
"supplementary-material"
] |
[
"Psoriasis",
"Serum uric acid",
"Adalimumab",
"Secukinumab",
"Hyperuricemia"
] |
Introduction:
Psoriasis is a chronic inflammatory disease.[1] In addition, patients with psoriasis have various comorbidities, such as arthritis, cardiovascular disease, metabolic syndrome, obesity, and diabetes mellitus.[2–5] In particular, psoriasis is associated with an increased risk of developing hyperuricemia.[6]
Uric acid is the end product of purine metabolism in humans, and it scavenges oxygen free radicals and prevents red blood cell membrane lipid oxidation. Urate deposits in joints, tendons, and other tissues in patients with hyperuricemia and induces gout. Hyperuricemia was found in 13.0% to 40.7% of patients with psoriasis,[7] and psoriasis was identified as an independent risk factor for gouty arthritis.[8] However, it was also reported that hyperuricemia and gout were not significantly associated with psoriasis.[9] Moreover, there have been few studies on the relationship between uric acid and psoriasis in Chinese patients.
The clinical symptoms of patients with psoriatic arthritis often appear 5 to 12 years after the initial skin manifestations.[10] The quality of life of patients with joint deformities is significantly decreased.[11] The use of biologics in patients with psoriasis can reduce the risk of developing psoriatic arthritis,[12] and multinational guidelines recommend biologics as first-line treatment options for psoriasis and psoriatic arthritis.[13,14] However, whether long-term treatment with biological agents affects serum uric acid levels in patients with psoriasis, especially Chinese psoriasis patients, remains unreported.
In summary, the purposes of our study were to compare the differences in serum uric acid levels between patients with psoriasis and healthy controls and analyze the risk of hyperuricemia. Furthermore, a 48-week follow-up was conducted to analyze the effects of adalimumab or secukinumab on uric acid levels in patients with psoriasis.
Methods:
Ethical approval The study protocol was approved by the Ethics Committee of Peking University People's Hospital (No. 2020PHB303-01). Given the retrospective nature of the study, the requirement of written informed consent was waived.
The study protocol was approved by the Ethics Committee of Peking University People's Hospital (No. 2020PHB303-01). Given the retrospective nature of the study, the requirement of written informed consent was waived.
Study population The study population consisted of adult patients with plaque psoriasis in the Peking University People's Hospital from January 2019 to December 2020. All patients were diagnosed with plaque psoriasis clinically or histopathologically. The control group consisted of non-psoriatic healthy individuals who underwent regular health check-ups in our hospital during the same period. The severity of psoriasis was stratified according to body surface area (BSA) as follows: ≥10% indicated severe psoriasis, and BSA <10% indicated mild to moderate psoriasis.[15] Patients with severe plaque psoriasis who failed to respond, had a contraindication or were intolerant to other systemic therapies were divided into two groups and received adalimumab or secukinumab.
Excessive drinkers, subjects with advanced chronic kidney disease, or those taking drugs that affect serum uric acid levels (including allopurinol, diuretics, salicylate, ketoconazole, theophylline, pyrazinamide, and ethambutol) were excluded from the study. Other exclusion criteria for patients with biological treatment were previous exposure to active inflammatory or infectious diseases, hematologic or solid cancers, systemic treatments (including methotrexate, acitretin, and cyclosporine A) in the previous 1 month, and biological agents in the previous 3 months.
The sample size was calculated using PASS software (version 15.0.5, NCSS, Kaysville, UT, USA). Based on data from previous studies, we used the random meta-analysis model to estimate the mean difference in serum uric acid levels between patients with psoriasis and controls.[16–18] According to the results, the estimated mean difference in serum uric acid levels was 0.6 mg/dL. We assumed that the standard deviations of serum uric acid levels in patients with psoriasis and controls were 1.6 and 1.4 mg/dL, respectively, which were the largest values reported by previous studies.[16–18] With a two-sided significance level (alpha) of 0.05, a 1:1 group allocation ratio, and an estimated 20% non-response rate, we calculated that a total of 326 participants (163 for patients and 163 for controls) would be needed to obtain a 90% power to reject the null hypothesis using a two-sample unequal-variance t test.
The study population consisted of adult patients with plaque psoriasis in the Peking University People's Hospital from January 2019 to December 2020. All patients were diagnosed with plaque psoriasis clinically or histopathologically. The control group consisted of non-psoriatic healthy individuals who underwent regular health check-ups in our hospital during the same period. The severity of psoriasis was stratified according to body surface area (BSA) as follows: ≥10% indicated severe psoriasis, and BSA <10% indicated mild to moderate psoriasis.[15] Patients with severe plaque psoriasis who failed to respond, had a contraindication or were intolerant to other systemic therapies were divided into two groups and received adalimumab or secukinumab.
Excessive drinkers, subjects with advanced chronic kidney disease, or those taking drugs that affect serum uric acid levels (including allopurinol, diuretics, salicylate, ketoconazole, theophylline, pyrazinamide, and ethambutol) were excluded from the study. Other exclusion criteria for patients with biological treatment were previous exposure to active inflammatory or infectious diseases, hematologic or solid cancers, systemic treatments (including methotrexate, acitretin, and cyclosporine A) in the previous 1 month, and biological agents in the previous 3 months.
The sample size was calculated using PASS software (version 15.0.5, NCSS, Kaysville, UT, USA). Based on data from previous studies, we used the random meta-analysis model to estimate the mean difference in serum uric acid levels between patients with psoriasis and controls.[16–18] According to the results, the estimated mean difference in serum uric acid levels was 0.6 mg/dL. We assumed that the standard deviations of serum uric acid levels in patients with psoriasis and controls were 1.6 and 1.4 mg/dL, respectively, which were the largest values reported by previous studies.[16–18] With a two-sided significance level (alpha) of 0.05, a 1:1 group allocation ratio, and an estimated 20% non-response rate, we calculated that a total of 326 participants (163 for patients and 163 for controls) would be needed to obtain a 90% power to reject the null hypothesis using a two-sample unequal-variance t test.
Study design This was a retrospective cohort study. The baseline demographic and disease characteristics were collected. Physical examination and blood biochemistry analysis were performed at the first visit. Patients treated with biologics were followed up for 48 weeks after the first injection. Serum uric acid levels were measured at baseline, week 24, and week 48.
This was a retrospective cohort study. The baseline demographic and disease characteristics were collected. Physical examination and blood biochemistry analysis were performed at the first visit. Patients treated with biologics were followed up for 48 weeks after the first injection. Serum uric acid levels were measured at baseline, week 24, and week 48.
Primary outcomes and related parameters The primary outcomes of this study were the differences in mean serum uric acid levels between patients with psoriasis and healthy controls, the relationship between psoriasis and hyperuricemia, and the effects of biologics on serum uric levels. A patient was defined as developing hyperuricemia if their diagnosis was confirmed by a physician and/or fulfilled hyperuricemia criteria. Generally, hyperuricemia was defined as an increased serum uric acid level >7 mg/dL in men and >6 mg/dL in women at two different visits.[19,20]
The primary outcomes of this study were the differences in mean serum uric acid levels between patients with psoriasis and healthy controls, the relationship between psoriasis and hyperuricemia, and the effects of biologics on serum uric levels. A patient was defined as developing hyperuricemia if their diagnosis was confirmed by a physician and/or fulfilled hyperuricemia criteria. Generally, hyperuricemia was defined as an increased serum uric acid level >7 mg/dL in men and >6 mg/dL in women at two different visits.[19,20]
Statistical analysis All statistical analyses were performed with SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). Continuous variables were expressed as the mean ± standard deviation or median (interquartile range). Categorical data were expressed as a frequency (percentage). Quantitative indicators were compared using the Student t test or Mann–Whitney U test according to the data distribution. Categorical indicators were compared by the chi-square test and Fisher exact test. A generalized linear model was built to determine the correlative factors for hyperuricemia. The covariates included in the generalized linear model were sex, age, and body mass index (BMI). The serum uric acid levels and the prevalence of hyperuricemia in patients using biologics at baseline, week 24, and week 48 were analyzed by the paired t test and McNemar test. The missing values in this study were filled using the last observation carried forward (LOCF) method. Sensitivity analyses of the LOCF method were performed to further analyze the effects of biologics on serum uric acid levels and hyperuricemia. Statistical significance was defined as a P value < 0.05.
All statistical analyses were performed with SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). Continuous variables were expressed as the mean ± standard deviation or median (interquartile range). Categorical data were expressed as a frequency (percentage). Quantitative indicators were compared using the Student t test or Mann–Whitney U test according to the data distribution. Categorical indicators were compared by the chi-square test and Fisher exact test. A generalized linear model was built to determine the correlative factors for hyperuricemia. The covariates included in the generalized linear model were sex, age, and body mass index (BMI). The serum uric acid levels and the prevalence of hyperuricemia in patients using biologics at baseline, week 24, and week 48 were analyzed by the paired t test and McNemar test. The missing values in this study were filled using the last observation carried forward (LOCF) method. Sensitivity analyses of the LOCF method were performed to further analyze the effects of biologics on serum uric acid levels and hyperuricemia. Statistical significance was defined as a P value < 0.05.
Ethical approval:
The study protocol was approved by the Ethics Committee of Peking University People's Hospital (No. 2020PHB303-01). Given the retrospective nature of the study, the requirement of written informed consent was waived.
Study population:
The study population consisted of adult patients with plaque psoriasis in the Peking University People's Hospital from January 2019 to December 2020. All patients were diagnosed with plaque psoriasis clinically or histopathologically. The control group consisted of non-psoriatic healthy individuals who underwent regular health check-ups in our hospital during the same period. The severity of psoriasis was stratified according to body surface area (BSA) as follows: ≥10% indicated severe psoriasis, and BSA <10% indicated mild to moderate psoriasis.[15] Patients with severe plaque psoriasis who failed to respond, had a contraindication or were intolerant to other systemic therapies were divided into two groups and received adalimumab or secukinumab.
Excessive drinkers, subjects with advanced chronic kidney disease, or those taking drugs that affect serum uric acid levels (including allopurinol, diuretics, salicylate, ketoconazole, theophylline, pyrazinamide, and ethambutol) were excluded from the study. Other exclusion criteria for patients with biological treatment were previous exposure to active inflammatory or infectious diseases, hematologic or solid cancers, systemic treatments (including methotrexate, acitretin, and cyclosporine A) in the previous 1 month, and biological agents in the previous 3 months.
The sample size was calculated using PASS software (version 15.0.5, NCSS, Kaysville, UT, USA). Based on data from previous studies, we used the random meta-analysis model to estimate the mean difference in serum uric acid levels between patients with psoriasis and controls.[16–18] According to the results, the estimated mean difference in serum uric acid levels was 0.6 mg/dL. We assumed that the standard deviations of serum uric acid levels in patients with psoriasis and controls were 1.6 and 1.4 mg/dL, respectively, which were the largest values reported by previous studies.[16–18] With a two-sided significance level (alpha) of 0.05, a 1:1 group allocation ratio, and an estimated 20% non-response rate, we calculated that a total of 326 participants (163 for patients and 163 for controls) would be needed to obtain a 90% power to reject the null hypothesis using a two-sample unequal-variance t test.
Study design:
This was a retrospective cohort study. The baseline demographic and disease characteristics were collected. Physical examination and blood biochemistry analysis were performed at the first visit. Patients treated with biologics were followed up for 48 weeks after the first injection. Serum uric acid levels were measured at baseline, week 24, and week 48.
Primary outcomes and related parameters:
The primary outcomes of this study were the differences in mean serum uric acid levels between patients with psoriasis and healthy controls, the relationship between psoriasis and hyperuricemia, and the effects of biologics on serum uric levels. A patient was defined as developing hyperuricemia if their diagnosis was confirmed by a physician and/or fulfilled hyperuricemia criteria. Generally, hyperuricemia was defined as an increased serum uric acid level >7 mg/dL in men and >6 mg/dL in women at two different visits.[19,20]
Statistical analysis:
All statistical analyses were performed with SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). Continuous variables were expressed as the mean ± standard deviation or median (interquartile range). Categorical data were expressed as a frequency (percentage). Quantitative indicators were compared using the Student t test or Mann–Whitney U test according to the data distribution. Categorical indicators were compared by the chi-square test and Fisher exact test. A generalized linear model was built to determine the correlative factors for hyperuricemia. The covariates included in the generalized linear model were sex, age, and body mass index (BMI). The serum uric acid levels and the prevalence of hyperuricemia in patients using biologics at baseline, week 24, and week 48 were analyzed by the paired t test and McNemar test. The missing values in this study were filled using the last observation carried forward (LOCF) method. Sensitivity analyses of the LOCF method were performed to further analyze the effects of biologics on serum uric acid levels and hyperuricemia. Statistical significance was defined as a P value < 0.05.
Results:
Study population characteristics The patients’ demographic characteristics at baseline are shown in [Table 1]. Based on the enrolment procedure, 196 patients (134 males, 62 females) with plaque psoriasis were included in this study. The average age was 40.4 ± 12.0 years, and the average BMI was 25.9 ± 4.4 kg/m2. The control group consisted of 191 adults (128 males, 63 females), and the average BMI was 24.4 ± 3.5 kg/m2.
Demographic and clinical characteristics of the study population at baseline.
Overweight and obesity were defined as BMI ≥25.0 and 30.0 kg/m2, respectively.[42] Data are presented as the mean ± standard deviation or n (%). Statistical significance was defined as a P value <0.05. BMI: Body mass index; BSA: Body surface area.
The patients’ demographic characteristics at baseline are shown in [Table 1]. Based on the enrolment procedure, 196 patients (134 males, 62 females) with plaque psoriasis were included in this study. The average age was 40.4 ± 12.0 years, and the average BMI was 25.9 ± 4.4 kg/m2. The control group consisted of 191 adults (128 males, 63 females), and the average BMI was 24.4 ± 3.5 kg/m2.
Demographic and clinical characteristics of the study population at baseline.
Overweight and obesity were defined as BMI ≥25.0 and 30.0 kg/m2, respectively.[42] Data are presented as the mean ± standard deviation or n (%). Statistical significance was defined as a P value <0.05. BMI: Body mass index; BSA: Body surface area.
Serum uric acid levels in patients with psoriasis and healthy controls Patients with psoriasis had higher serum uric acid levels compared with healthy controls (6.4 ± 1.7 mg/dL vs. 5.7 ± 1.5 mg/dL, P < 0.001). Hyperuricemia was found in 33.7% (66/196) of patients with psoriasis, which was significantly higher than that in healthy controls (13.1% [25/191], P < 0.001). No significant difference was found between patients with mild to moderate psoriasis and severe psoriasis (P > 0.05). Of note, the above results showed statistical significance after the calibration of BMI [Table 1].
Patients with psoriasis had higher serum uric acid levels compared with healthy controls (6.4 ± 1.7 mg/dL vs. 5.7 ± 1.5 mg/dL, P < 0.001). Hyperuricemia was found in 33.7% (66/196) of patients with psoriasis, which was significantly higher than that in healthy controls (13.1% [25/191], P < 0.001). No significant difference was found between patients with mild to moderate psoriasis and severe psoriasis (P > 0.05). Of note, the above results showed statistical significance after the calibration of BMI [Table 1].
Risk factors of hyperuricemia Male sex was associated with an increased risk of hyperuricemia Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).
Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).
Hyperuricemia was related to higher BMI Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).
Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).
Male sex was associated with an increased risk of hyperuricemia Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).
Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).
Hyperuricemia was related to higher BMI Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).
Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).
Effects of biologics on serum uric acid levels Effects of adalimumab At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].
Effects of adalimumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].
Effects of adalimumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Effects of secukinumab Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].
Effects of secukinumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].
Effects of secukinumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Effects of adalimumab At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].
Effects of adalimumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].
Effects of adalimumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Effects of secukinumab Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].
Effects of secukinumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].
Effects of secukinumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Study population characteristics:
The patients’ demographic characteristics at baseline are shown in [Table 1]. Based on the enrolment procedure, 196 patients (134 males, 62 females) with plaque psoriasis were included in this study. The average age was 40.4 ± 12.0 years, and the average BMI was 25.9 ± 4.4 kg/m2. The control group consisted of 191 adults (128 males, 63 females), and the average BMI was 24.4 ± 3.5 kg/m2.
Demographic and clinical characteristics of the study population at baseline.
Overweight and obesity were defined as BMI ≥25.0 and 30.0 kg/m2, respectively.[42] Data are presented as the mean ± standard deviation or n (%). Statistical significance was defined as a P value <0.05. BMI: Body mass index; BSA: Body surface area.
Serum uric acid levels in patients with psoriasis and healthy controls:
Patients with psoriasis had higher serum uric acid levels compared with healthy controls (6.4 ± 1.7 mg/dL vs. 5.7 ± 1.5 mg/dL, P < 0.001). Hyperuricemia was found in 33.7% (66/196) of patients with psoriasis, which was significantly higher than that in healthy controls (13.1% [25/191], P < 0.001). No significant difference was found between patients with mild to moderate psoriasis and severe psoriasis (P > 0.05). Of note, the above results showed statistical significance after the calibration of BMI [Table 1].
Risk factors of hyperuricemia:
Male sex was associated with an increased risk of hyperuricemia Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).
Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).
Hyperuricemia was related to higher BMI Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).
Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).
Male sex was associated with an increased risk of hyperuricemia:
Hyperuricemia was found in 42.5% of male patients, which was significantly higher than that in female patients (14.5%, P = 0.001). Hyperuricemia was more frequent in male controls than in female controls (17.2% vs. 4.8%, P = 0.017).
Hyperuricemia was related to higher BMI:
Hyperuricemia was found in 44.1% of overweight and obese patients with psoriasis and 22.3% of normal-weight patients. Similarly, the prevalence of hyperuricemia in overweight and obese patients with psoriasis was higher than that in normal-weight patients (P = 0.002). In healthy controls, hyperuricemia was more frequent in overweight and obese subjects than in normal-weight subjects (22.1% vs. 7.0%, P = 0.004).
Effects of biologics on serum uric acid levels:
Effects of adalimumab At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].
Effects of adalimumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].
Effects of adalimumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Effects of secukinumab Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].
Effects of secukinumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].
Effects of secukinumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Effects of adalimumab:
At baseline, 68 patients were treated with adalimumab. During follow-up, 12 patients (17.6%) and 26 patients (38.2%) were lost to follow-up at week 24 and week 48, respectively, because of the epidemic, poor responses, and infectious diseases. The serum uric acid levels in patients with psoriasis were 6.2 ± 1.9, 6.2 ± 2.0, and 6.1 ± 1.9 mg/dL at baseline, week 24, and week 48, respectively. No significant difference was found in serum uric acid levels before and after adalimumab treatment (P = 0.762 and P = 0.235, respectively). The prevalence of hyperuricemia was 32.4%, 29.4%, and 27.9% at baseline, week 24, and week 48, respectively. There was no statistical difference at week 24 and week 48 compared with the baseline (P = 0.480 and P = 0.257, respectively) [Table 2]. Through the sensitivity analysis, the results after interpolation were consistent with those before interpolation [Supplementary Table 1].
Effects of adalimumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Effects of secukinumab:
Fifty-nine patients were treated with secukinumab at baseline. The proportion of loss to follow-up was 5.0% (3 patients) and 6.8% (4 patients) at week 24 and week 48, respectively. These patients dropped out because of the epidemic and poor responses. The mean serum uric acid levels were 6.6 ± 1.4 mg/dL at baseline and 6.7 ± 1.6 mg/dL at week 24 (P = 0.885). The serum uric acid level was 6.3 ± 1.5 mg/dL at week 48, which was significantly lower than that at baseline (P = 0.007). Treatment with secukinumab for 48 weeks significantly decreased serum uric acid levels. However, secukinumab had no effects on the prevalence of hyperuricemia [Table 3]. The results after interpolation were consistent with those before interpolation [Supplementary Table 2].
Effects of secukinumab on serum uric acid levels in patients with psoriasis.
Data are presented as the mean ± standard deviation or n (%). The missing values in this study were filled using the LOCF method. P1: the P value of week 24 compared with the baseline. P2: the P value of week 48 compared with the baseline. LOCF: Last observation carried forward.
Discussion:
Psoriasis was previously reported to be associated with elevated serum uric acid levels.[6,8] Goldman[21] proposed that serum uric acid was involved in the pathogenesis of psoriasis. Tsuruta et al[22] also suggested that uric acid mediates inflammation.
Hyperuricemia is related to multiple factors, including gender, age, race, lifestyle, and environmental conditions.[23] The United States Health and Nutrition Examination Survey showed that the prevalence of hyperuricemia in men and women was 20.2% and 20.0%, respectively.[24] In addition, a Chinese study reported that the prevalence of hyperuricemia was 18.5% in males and 8.0% in females.[25] According to a meta-analysis, the prevalence of hyperuricemia in Mainland China was 13.3%.[26] In this study, we found that the prevalence of hyperuricemia was 17.2% in males and 4.8% in females, consistent with previous reports.
The relationship between psoriasis and hyperuricemia remains controversial. Gisondi et al[16] reported that hyperuricemia was frequently observed in patients with psoriasis. However, Kwon et al[27] reported that there was no significant difference in serum uric acid levels between patients with psoriasis and healthy controls. In this study, we found that the prevalence of hyperuricemia was significantly higher in patients with psoriasis than that in healthy controls.
Similarly, whether serum uric acid is related to the severity of psoriasis is controversial. Gisondi et al[16] found that serum uric acid levels in patients with Psoriasis Area and Severity Index (PASI) scores ≥10 were significantly higher than those in participants with PASI scores <10. Yilmaz et al[17] also found that the PASI score was positively correlated with serum uric acid levels. However, Gui et al[18] reported no significant correlation between serum uric acid levels and PASI scores in patients with psoriasis. Our study showed that the serum uric acid levels and prevalence of hyperuricemia were unrelated to the severity of psoriasis. Therefore, serum uric acid levels should be carefully considered, regardless of the severity of skin lesions.
The mechanism by which patients with psoriasis are prone to hyperuricemia remains unclear. Goldman[21] proposed that the replacement rate of keratinocytes in psoriasis patients was accelerated, and the increase in purine decomposition caused excessive uric acid production, subsequently increasing serum uric acid levels. Furthermore, urate crystals, a type of alarmin, activate innate immunity through NALP inflammasomes and increase cytokine production, including interleukin (IL)-1 and IL-8.[28] Uratsuji et al[29] confirmed that epidermal keratinocytes cultured in the presence of uric acid crystals formed NALP inflammasomes and produced large amounts of tumor necrosis factor (TNF)-α, IL-1a, IL-8/chemokine (CXC motif) ligand 8, and IL-6. These cytokines play an important role in the pathogenesis of psoriasis. In summary, psoriasis increases serum uric acid levels, and urate crystals may further promote the development of psoriasis.
Other factors that may affect serum uric acid levels and hyperuricemia in patients with psoriasis include age, gender, and BMI. Gui et al[18] found that serum uric acid levels were significantly positively correlated with BMI (P < 0.001). Similarly, Lai et al[30] proposed that serum uric acid levels were positively correlated with BMI (P < 0.001), and overweight status showed the strongest correlation with hyperuricemia in patients with psoriatic arthritis (P < 0.001). Kwon et al[27] found that serum uric acid levels in patients with psoriasis had no significant correlation with age, age at psoriasis onset, or family history of psoriasis. Here, we also analyzed the correlation between serum uric acid levels and BMI. The results showed that serum uric acid levels in overweight and obese patients with psoriasis were significantly higher than those in normal-weight patients. Therefore, to reduce the risk of hyperuricemia, patients with psoriasis should be instructed to strictly control their diet and weight, especially male patients.
Long-term dietary exposure to alcohol, red meat, seafood, and sugar-sweetened beverages can increase the risk of hyperuricemia.[31,32] Alcohol and fructose increase the production of urate, and alcohol consumption inhibits renal urate excretion. Conversely, the intake of dairy products and coffee reduces the risk of hyperuricemia.[33,34] Dietary management has been reported to lower serum uric acid levels by approximately 1.0 mg/dL.[35] Therefore, health education regarding a low-purine diet in patients with psoriasis is necessary.
TNF-α is a key cytokine in the pathogenesis of psoriasis and associated comorbidities. TNF-α inhibitors are commonly used in the treatment of moderate to severe plaque psoriasis with good effectiveness and safety.[36] Montaudié et al[37] found no significant change in serum uric acid levels at the 3rd and 6th month of follow-up in patients with psoriasis treated with biological agents (etanercept, infliximab, adalimumab, and ustekinumab). However, Hasikova et al[38] reported increased serum uric acid levels in 128 patients with systemic autoimmune rheumatic diseases treated with TNF-α inhibitors for 3 months. Our study revealed no significant change in the serum uric acid levels or prevalence of hyperuricemia in patients with psoriasis after adalimumab treatment for 48 weeks.
Secukinumab, a fully human monoclonal anti-IL-17a antibody, has shown good efficacy and safety in Chinese patients with moderate to severe plaque psoriasis.[39] Gerdes et al[40] reported that uric acid levels tended to decrease over 52 weeks of treatment with secukinumab in plaque psoriasis patients. However, Karataş et al[41] found that serum uric acid levels were not significantly different from baseline in 30 patients with ankylosing spondylitis and six patients with psoriatic arthritis treated with secukinumab for 6 months. Our study revealed slightly lower serum uric acid levels in patients treated with secukinumab for 48 weeks compared with baseline values, but the prevalence of hyperuricemia was not significantly different. The mechanism underlying the decrease in serum uric acid levels associated with secukinumab treatment remains elusive.
In conclusion, as a comorbid condition related to metabolism in psoriasis, hyperuricemia should attract our attention. Our study revealed that the serum uric acid levels and prevalence of hyperuricemia in patients with psoriasis were higher than those in healthy controls. Moreover, the serum uric acid levels and prevalence of hyperuricemia were related to obesity and gender rather than the severity of psoriasis. The above results suggested that patients with psoriasis should limit their diet and maintain a healthy weight regardless of disease severity, especially male patients. Compared with baseline values, adalimumab and secukinumab had no significant effect on the prevalence of hyperuricemia in patients with psoriasis after 48 weeks of treatment. Both biologics can be applied to psoriasis patients with hyperuricemia.
The limitations of our study include its retrospective nature, single-center design, and short-term observation period. In our hospital, there are more patients with severe psoriasis than patients with mild to moderate psoriasis. Therefore, selection bias cannot be avoided. Because this was a retrospective cohort study, the effects of biologics on patients lost to follow-up could not be determined. Therefore, a large sample, multi-center, long-term prospective study should be performed to demonstrate the correlation between psoriasis and serum uric acid levels. Although our study excluded patients with excessive alcohol consumption, the effects of dietary habits on serum uric acid should be considered. However, this study provides a theoretical basis for the prevention of hyperuricemia in patients with psoriasis and the development of biological treatment strategies.
Acknowledgements:
We gratefully thank all doctors who support our registry database.
Conflicts of interest:
Lin Cai has participated in advisory boards as an investigator and/or speaker and received grants and/or honoraria from Novartis, AbbVie, Sanofi, and Eli Lilly Inc. Jianzhong Zhang has participated in advisory boards as an investigator and/or speaker and received grants and/or honoraria from LEO Pharma China, Novartis, Sanofi, AbbVie, Bayer, Janssen-Cilag, Henlius, Kyowa Kirin, and Pfizer Inc.
Supplementary Material:
|
Background:
Psoriasis is a chronic systemic inflammatory disease, and hyperuricemia is a common comorbidity in patients with psoriasis. However, there are limited reports on the relationship between serum uric acid levels and biological treatment efficacy. The purposes of this study were to compare the differences in serum uric acid levels between patients with psoriasis and healthy controls and analyze the risk of hyperuricemia.
Methods:
A total of 196 patients with psoriasis and 191 age- and sex-matched healthy controls were enrolled in this retrospective cohort study. One hundred and twenty-seven patients with severe psoriasis were treated with biologics. Sixty-eight patients received adalimumab, and 59 patients received secukinumab. Serum uric acid levels were measured at baseline, week 24, and week 48 of treatment.
Results:
Patients with psoriasis had higher serum uric acid levels than healthy controls (6.4 ± 1.7 mg/dL vs. 5.7 ± 1.5 mg/dL, P < 0.001). Hyperuricemia was found in 33.7% (66/196) of patients with psoriasis, which was significantly higher than that in healthy controls (13.1% [25/191], P < 0.001). Serum uric acid levels and hyperuricemia were not related to the severity of psoriasis ( P > 0.05). No significant changes in serum uric acid levels and hyperuricemia were observed following adalimumab treatment ( P > 0.05). The serum uric acid level in patients treated with secukinumab was 6.7 ± 1.6 mg/dL at week 24, which was not statistically different from that at baseline (6.6 ± 1.4 mg/dL, P = 0.885). Serum uric acid levels were significantly decreased at week 48 (6.3 ± 1.5 mg/dL vs. 6.6 ± 1.4 mg/dL, P = 0.007) in patients treated with secukinumab. Secukinumab had no significant effect on hyperuricemia either ( P > 0.05).
Conclusions:
The serum uric acid levels and prevalence of hyperuricemia in patients with psoriasis were significantly higher than those in healthy controls. Secukinumab treatment for 48 weeks successfully decreased serum uric acid levels in patients with psoriasis, whereas adalimumab had no significant effect on serum uric acid levels.
| null | null | 10,043 | 428 | 20 |
[
"patients",
"psoriasis",
"week",
"uric",
"uric acid",
"acid",
"serum",
"serum uric",
"serum uric acid",
"hyperuricemia"
] |
[
"test",
"test"
] | null | null |
[CONTENT] Psoriasis | Serum uric acid | Adalimumab | Secukinumab | Hyperuricemia [SUMMARY]
|
[CONTENT] Psoriasis | Serum uric acid | Adalimumab | Secukinumab | Hyperuricemia [SUMMARY]
|
[CONTENT] Psoriasis | Serum uric acid | Adalimumab | Secukinumab | Hyperuricemia [SUMMARY]
| null |
[CONTENT] Psoriasis | Serum uric acid | Adalimumab | Secukinumab | Hyperuricemia [SUMMARY]
| null |
[CONTENT] Adalimumab | Antibodies, Monoclonal, Humanized | Biological Products | Chronic Disease | Humans | Hyperuricemia | Psoriasis | Retrospective Studies | Treatment Outcome | Uric Acid [SUMMARY]
|
[CONTENT] Adalimumab | Antibodies, Monoclonal, Humanized | Biological Products | Chronic Disease | Humans | Hyperuricemia | Psoriasis | Retrospective Studies | Treatment Outcome | Uric Acid [SUMMARY]
|
[CONTENT] Adalimumab | Antibodies, Monoclonal, Humanized | Biological Products | Chronic Disease | Humans | Hyperuricemia | Psoriasis | Retrospective Studies | Treatment Outcome | Uric Acid [SUMMARY]
| null |
[CONTENT] Adalimumab | Antibodies, Monoclonal, Humanized | Biological Products | Chronic Disease | Humans | Hyperuricemia | Psoriasis | Retrospective Studies | Treatment Outcome | Uric Acid [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] patients | psoriasis | week | uric | uric acid | acid | serum | serum uric | serum uric acid | hyperuricemia [SUMMARY]
|
[CONTENT] patients | psoriasis | week | uric | uric acid | acid | serum | serum uric | serum uric acid | hyperuricemia [SUMMARY]
|
[CONTENT] patients | psoriasis | week | uric | uric acid | acid | serum | serum uric | serum uric acid | hyperuricemia [SUMMARY]
| null |
[CONTENT] patients | psoriasis | week | uric | uric acid | acid | serum | serum uric | serum uric acid | hyperuricemia [SUMMARY]
| null |
[CONTENT] psoriasis | arthritis | patients | risk | patients psoriasis | psoriatic arthritis | uric | uric acid | acid | psoriatic [SUMMARY]
|
[CONTENT] test | uric | serum | serum uric | psoriasis | acid | uric acid | serum uric acid | levels | previous [SUMMARY]
|
[CONTENT] week | patients | baseline | week 24 | week 48 | 48 | 24 | hyperuricemia | respectively | serum uric [SUMMARY]
| null |
[CONTENT] patients | week | psoriasis | hyperuricemia | uric | acid | uric acid | serum uric | serum | serum uric acid [SUMMARY]
| null |
[CONTENT] ||| ||| [SUMMARY]
|
[CONTENT] 196 | 191 ||| One hundred and twenty-seven ||| Sixty-eight | 59 ||| week 24, and week 48 [SUMMARY]
|
[CONTENT] 6.4 ± | 1.7 mg/dL | 5.7 ± | 1.5 mg/dL | P < 0.001 ||| 33.7% | 66/196 | 13.1% ||| 25/191 | P < 0.001 ||| 0.05 ||| 0.05 ||| 6.7 ± | week 24 | 6.6 ± | 1.4 | P | 0.885 ||| week 48 | 6.3 ± | 1.5 mg/dL | 6.6 ± | 1.4 | P | 0.007 ||| 0.05 [SUMMARY]
| null |
[CONTENT] ||| ||| ||| 196 | 191 ||| One hundred and twenty-seven ||| Sixty-eight | 59 ||| week 24, and week 48 ||| 6.4 ± | 1.7 mg/dL | 5.7 ± | 1.5 mg/dL | P < 0.001 ||| 33.7% | 66/196 | 13.1% ||| 25/191 | P < 0.001 ||| 0.05 ||| 0.05 ||| 6.7 ± | week 24 | 6.6 ± | 1.4 | P | 0.885 ||| week 48 | 6.3 ± | 1.5 mg/dL | 6.6 ± | 1.4 | P | 0.007 ||| 0.05 ||| ||| Secukinumab | 48 weeks [SUMMARY]
| null |
||||
Determinants and consequences of short birth interval in rural Bangladesh: a cross-sectional study.
|
25539669
|
Short birth intervals are known to have negative effects on pregnancy outcomes. We analysed data from a large population surveillance system in rural Bangladesh to identify predictors of short birth interval and determine consequences of short intervals on pregnancy outcomes.
|
BACKGROUND
|
The study was conducted in three districts of Bangladesh - Bogra, Moulavibazar and Faridpur (population 282,643, 54,668 women of reproductive age). We used data between January 2010 and June 2011 from a key informant surveillance system that recorded all births, deaths and stillbirths. Short birth interval was defined as an interval between consecutive births of less than 33 months. Initially, risk factors of a short birth interval were determined using a multivariate mixed effects logistic regression model. Independent risk factors were selected using a priori knowledge from literature review. An adjusted mixed effects logistic regression model was then used to determine the effect of up to 21-, 21-32-, 33-44- and 45-month and higher birth-to-birth intervals on pregnancy outcomes controlling for confounders selected through a directed acyclic graph.
|
METHODS
|
We analysed 5,571 second or higher order deliveries. Average birth interval was 55 months and 1368/5571 women (24.6%) had a short birth interval (<33 months). Younger women (AOR 1.11 95% CI 1.08-1.15 per year increase in age), women who started their reproductive life later (AOR 0.95, 0.92-0.98 per year) and those who achieve higher order parities were less likely to experience short birth intervals (AOR 0.28, 0.19-0.41 parity 4 compared to 1). Women who were socioeconomically disadvantaged were more likely to experience a short birth interval (AOR 1.42, 1.22-1.65) and a previous adverse outcome was an important determinant of interval (AOR 2.10, 1.83-2.40). Very short birth intervals of less than 21 months were associated with increased stillbirth rate (AOR 2.13, 95% CI 1.28-3.53) and neonatal mortality (AOR 2.28 95% CI 1.28-4.05).
|
RESULTS
|
Birth spacing remains a reproductive health problem in Bangladesh. Disadvantaged women are more likely to experience short birth intervals and to have increased perinatal deaths. Research into causal pathways and strategies to improve spacing between pregnancies should be intensified.
|
CONCLUSIONS
|
[
"Adolescent",
"Adult",
"Age Factors",
"Bangladesh",
"Birth Intervals",
"Cross-Sectional Studies",
"Educational Status",
"Female",
"Humans",
"Infant",
"Infant, Newborn",
"Parity",
"Perinatal Mortality",
"Population Surveillance",
"Pregnancy",
"Religion",
"Reproductive Behavior",
"Residence Characteristics",
"Rural Population",
"Stillbirth",
"Young Adult"
] |
4314752
|
Background
|
Short birth intervals, defined as the time between two births, are known to have negative effects on maternal, perinatal and neonatal outcomes as well as on child health, though the precise mechanisms are poorly understood [1]. Short intervals are associated with increased preterm birth, low birth weight, and small for gestational age births, as well as perinatal death [2,3]. Neonatal and infant mortality is also higher after a negative outcome of the previous delivery, which is mediated primarily through short birth interval [4,5].
Neonatal and infant mortality is highest for birth to pregnancy intervals of 18 months and is slightly increased for birth to pregnancy intervals of 18–27 months [6]. The most recent WHO recommendation for a healthy pregnancy interval is at least two years (24 months), which corresponds to a birth-to-birth interval of 33 months under the assumption of nine months gestation [7]. The Government of Bangladesh recommends an interval of three years between the last live birth and the next attempt to get pregnant, which corresponds to a birth-to-birth interval of 45 months under the assumption of nine months gestation [8].
Corresponding with increasing levels of contraceptive use over the past 30 years (from 7 per cent in 1975 to 54 per cent by 2000) [9], Bangladesh has witnessed large reductions in fertility [9] and marked increases in median birth interval, increasing from 35 months in 1993–1994 to 47 months in 2011 [10]. However, more than two-thirds of women in Bangladesh are married by the age of 18 and the median age at first birth is approximately 18 years [10]. These young women have birth intervals of just 27 months and large differences are seen between socio-economic groups, with poorer, less educated women having shorter intervals [10-13]. The length of the birth interval is closely associated with the survival of the previous sibling [4,6]. The Bangladesh Demographic and Health Survey (BDHS) 2011 identifies a median birth interval 15 months shorter for children whose previous sibling died than for children whose previous sibling is alive (26 and 41 months, respectively) [10].
Mother’s age at first birth, parity, previous birth interval, mother’s working status, gender composition of the living children, and mass media are also described as determinants of birth intervals [11-13].
National strategies focused on family planning, reproductive health and safe motherhood have been well described [8] and are likely to represent a dynamic reproductive health context in Bangladesh that warrants contemporary description. Our study seeks to do this by not only describing birth spacing and its consequences on pregnancy outcomes, but also exploring the socioeconomic, demographic and reproductive factors that are associated with short birth intervals using prospective surveillance data from three districts in rural Bangladesh. Our findings are discussed in terms of opportunities for intervention strategies to prevent short birth intervals and the risks associated with them.
| null | null |
Results
|
In the period January 2010-June 2011, 9,797 deliveries were registered with 5,911 second or higher order deliveries. The average birth interval was 4 years and 7 months (55 months). The average age of first pregnancy was 18.4 years and the average age of delivery 26.4 years (Table 1).Table 1
Crude and adjusted model of determinants of short birth interval defined as birth-to-birth interval <33 months
Recommended birth interval (at least 33 months)
Short birth interval < 33 months
Crude OR
95% CI
OR
†
95% CI
Age at onset of birth interval
22.0 year$
22.8 year$
1.041.031.061.111.081.15
Parity at onset of birth interval
12,81150.5%70851.8%
reference
reference
21,47026.4%32924.0%0.860.740.990.530.440.63370812.7%16812.3%0.920.761.120.380.290.514+58210.4%16311.9%1.160.951.410.280.190.41
Age at first pregnancy
18.3 year$
18.6 year$
1.041.021.070.950.920.98
Adverse outcome of any previous pregnancy
Yes2,02136.3%65147.6%2.281.952.672.101.832.40No3,55063.7%71752.4%
reference
reference
Religion
Muslim (%)4,60582.6%1,15284.2%
reference
reference
Other (%)96617.3%21615.8%0.860.731.020.680.530.87
Education
None or primary education only3,40861.2%81059.2%
reference
reference
Secondary or above2,16338.8%55840.8%1.110.981.261.261.091.45
Household assets
0-34,04372.7%104076.0%1.221.061.411.421.221.654+1,52027.3%32824.0%
reference
reference
Tea garden resident
Yes66311.9%16912.4%1.060.881.281.411.071.87No480988.1%1.19987.6%
reference
reference
†Adjustment for all other variables. $Average.
Crude and adjusted model of determinants of short birth interval defined as birth-to-birth interval <33 months
†Adjustment for all other variables. $Average.
We checked for data completeness and 340 observations (5.8%) had missing data. 330 observations had a missing birth interval and 10 observations missed other covariates (parity, education). Neonatal mortality was higher among those cases with missing data and younger age, lower parity, previous adverse outcomes, Christian or Hindu religion and higher education groups were overrepresented [see Additional file 2]. A complete case analysis was performed, using 5,571 pregnancies without any missing data.
Determinants of birth interval <33 months 1,368 women (24.6%) had a birth interval shorter than 33 months. The determinants of short birth intervals are described in detail in Table 1. Results from the mixed effects multivariate analysis indicate that the risk of having a short interval increases with age. The likelihood of a short birth interval increased by 11% with each additional year of age (adjusted odds ratio (AOR) 1.11 95% confidence interval (CI) 1.08-1.15). Women who delivered a third or higher order baby were less likely to xperience a short birth interval compared to women who delivered their second baby. Parity of four or more was associated with 72% decrease in the odds of a short birth interval compared to a parity of one at the start of the birth interval (AOR 0.28, 0.19-0.41). The likelihood of a short birth interval decreased 5% for every year that a woman’s first pregnancy was delayed (AOR 0.95, 0.92-0.98). Women who experienced a previous adverse outcome of pregnancy were twice as likely to have a birth interval of less than 33 months compared to those women whose babies survived the neonatal period (AOR 2.10, 1.83-2.40). Christian or Hindu women were 32% less likely to experience a short birth interval than Muslim women (AOR 0.68, 0.53-0.87). Women with secondary education or higher had a 26% increased likelihood of a short birth interval (AOR 1.26, 1.09-1.45) whilst women from a household with up to 3 assets were 42% more likely to have a short interval compared to those with four or more assets (AOR 1.42, 1.22-1.65). Tea-garden residence was associated with a 41% increase in the odds of a short birth interval (AOR 1.41, 1.07-1.87). The ROC showed that the model was a reasonable fit (0.63).
1,368 women (24.6%) had a birth interval shorter than 33 months. The determinants of short birth intervals are described in detail in Table 1. Results from the mixed effects multivariate analysis indicate that the risk of having a short interval increases with age. The likelihood of a short birth interval increased by 11% with each additional year of age (adjusted odds ratio (AOR) 1.11 95% confidence interval (CI) 1.08-1.15). Women who delivered a third or higher order baby were less likely to xperience a short birth interval compared to women who delivered their second baby. Parity of four or more was associated with 72% decrease in the odds of a short birth interval compared to a parity of one at the start of the birth interval (AOR 0.28, 0.19-0.41). The likelihood of a short birth interval decreased 5% for every year that a woman’s first pregnancy was delayed (AOR 0.95, 0.92-0.98). Women who experienced a previous adverse outcome of pregnancy were twice as likely to have a birth interval of less than 33 months compared to those women whose babies survived the neonatal period (AOR 2.10, 1.83-2.40). Christian or Hindu women were 32% less likely to experience a short birth interval than Muslim women (AOR 0.68, 0.53-0.87). Women with secondary education or higher had a 26% increased likelihood of a short birth interval (AOR 1.26, 1.09-1.45) whilst women from a household with up to 3 assets were 42% more likely to have a short interval compared to those with four or more assets (AOR 1.42, 1.22-1.65). Tea-garden residence was associated with a 41% increase in the odds of a short birth interval (AOR 1.41, 1.07-1.87). The ROC showed that the model was a reasonable fit (0.63).
Short birth interval and adverse outcome of pregnancy Compared to birth intervals of 45 months or longer, birth intervals of less than 21 months were associated with a greater than two-fold increased risk of adverse pregnancy outcome (AOR 2.23 95% CI 1.51-3.29), as well as increased risk of perinatal mortality (AOR 2.33, 95% CI 1.55-3.50), stillbirth (AOR 2.13, 95% CI 1.28-3.53), neonatal mortality (AOR 2.28 95% CI 1.28-4.05) (Table 2). There was no evidence of increased risk of adverse pregnancy outcomes associated with birth intervals of 21–32 months and 33–44 compared to longer intervals.Table 2
Exposure models of adverse outcomes of pregnancy by birth interval
Birth interval (months)
N
Events
Crude OR*
95% CI
Adjusted OR
†
95% CI
N
/1,000
Adverse outcome of pregnancy
>453,17314345.1
reference
reference
33-441,0305452.41.190.861.651.210.871.7021-328794247.81.070.751.521.130.781.65<214894490.0
2.12
1.493.03
2.23
1.513.29
Perinatal mortality
>453,17312439.1
reference
reference
33-441,0305048.51.290.921.821.310.921.8721-328793742.11.090.751.591.150.771.71<214894183.8
2.31
1.60
3.35
2.33
1.553.50
Still birth rate
>453,1737924.9
reference
reference
33-441,0302827.21.090.701.691.100.701.7321-328791921.60.850.511.420.920.541.56<214892551.12.101.323.332.131.283.53
Neonatal mortality
>4530946420.7
reference
reference
33-4410022625.91.310.822.091.350.832.2021-328602326.71.320.822.161.380.832.32<214641940.92.101.243.552.281.284.05
Early neonatal mortality
>4530944514.5
reference
reference
33-4410022222.01.660.982.811.680.651.8421-328601820.91.510.872.651.540.552.23<214641634.52.631.464.742.590.532.73
*The crude analyses were adjusted for clustering.
†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.
Exposure models of adverse outcomes of pregnancy by birth interval
*The crude analyses were adjusted for clustering.
†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.
Compared to birth intervals of 45 months or longer, birth intervals of less than 21 months were associated with a greater than two-fold increased risk of adverse pregnancy outcome (AOR 2.23 95% CI 1.51-3.29), as well as increased risk of perinatal mortality (AOR 2.33, 95% CI 1.55-3.50), stillbirth (AOR 2.13, 95% CI 1.28-3.53), neonatal mortality (AOR 2.28 95% CI 1.28-4.05) (Table 2). There was no evidence of increased risk of adverse pregnancy outcomes associated with birth intervals of 21–32 months and 33–44 compared to longer intervals.Table 2
Exposure models of adverse outcomes of pregnancy by birth interval
Birth interval (months)
N
Events
Crude OR*
95% CI
Adjusted OR
†
95% CI
N
/1,000
Adverse outcome of pregnancy
>453,17314345.1
reference
reference
33-441,0305452.41.190.861.651.210.871.7021-328794247.81.070.751.521.130.781.65<214894490.0
2.12
1.493.03
2.23
1.513.29
Perinatal mortality
>453,17312439.1
reference
reference
33-441,0305048.51.290.921.821.310.921.8721-328793742.11.090.751.591.150.771.71<214894183.8
2.31
1.60
3.35
2.33
1.553.50
Still birth rate
>453,1737924.9
reference
reference
33-441,0302827.21.090.701.691.100.701.7321-328791921.60.850.511.420.920.541.56<214892551.12.101.323.332.131.283.53
Neonatal mortality
>4530946420.7
reference
reference
33-4410022625.91.310.822.091.350.832.2021-328602326.71.320.822.161.380.832.32<214641940.92.101.243.552.281.284.05
Early neonatal mortality
>4530944514.5
reference
reference
33-4410022222.01.660.982.811.680.651.8421-328601820.91.510.872.651.540.552.23<214641634.52.631.464.742.590.532.73
*The crude analyses were adjusted for clustering.
†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.
Exposure models of adverse outcomes of pregnancy by birth interval
*The crude analyses were adjusted for clustering.
†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.
|
Conclusion
|
Birth spacing remains an issue that deserves urgent attention in Bangladesh. Disadvantaged women are more likely to experience short birth intervals and suffer their consequences. Further research should look into causal pathways as well as strategies to improve spacing between pregnancies whilst efforts to prevent adverse pregnancy outcomes, an important determinant of birth spacing, should be intensified.
|
[
"Study population",
"Data collection",
"Definitions",
"Statistical methods",
"Ethics",
"Determinants of birth interval <33 months",
"Short birth interval and adverse outcome of pregnancy"
] |
[
"The study was conducted in nine unions (the lowest administrative level in Bangladesh) in three districts of Bangladesh – Bogra, Moulavibazar and Faridpur. The districts and unions represent part of a cluster randomised controlled trial of a community intervention to improve maternal and neonatal health described in detail elsewhere [14-16]. Only the control unions were included in the current descriptive study given that the aforementioned intervention could have influenced birth spacing or its consequences in intervention areas. The total population size of the nine unions was 282,643, representing 54,668 women of reproductive age residing in approximately 63,000 households. The study areas include tea-garden estates, which differ from much of non-tea garden areas in Bangladesh in terms of socioeconomics, access to services and history and are generally more disadvantaged than other areas [17,18].",
"Since 2004, a key informant surveillance system has been used in the study areas to record all births, neonatal deaths, and deaths of women of reproductive age [19]. 500 traditional birth attendants act as key informants, each monitoring a population of approximately 200 households or 1000 individuals. Most births in the study area are attended by traditional birth attendants, thus these women are aware of most if not all births in their community and are well placed to record pregnancy outcomes. Key informants are visited fortnightly by a monitor who collates data on births, either live or stillbirth, and neonatal and pregnancy-related deaths. 6–52 weeks after a registered event (birth or death), paid monitors visit women in their home to conduct a structured interview to gather data on socio-economic characteristics of the mother and her household, reproductive history, including birth interval, and history of previous pregnancy outcomes, as well as pregnancy, birth and postpartum experiences. Precise gestation cannot be reliably measured through these survey methods and so stillbirths are classified as such on the basis of reports of the birth of a child with no movement or other signs of life during the structured interview; it is unlikely that women’s reports or our survey tool would misclassify foetal deaths before 28 weeks gestation as stillbirths. All data are checked at the district office for quality and referred back to the field if incomplete or inaccurate. A random 10-20% sample of the respondents are revisited by supervisors to verify data quality. Data are sent to Dhaka for entry in an MS Access database, where further quality checks take place.\nThe current analysis was restricted to births taking place between January 2010 and June 2011. Observations were included if data on birth interval, pregnancy outcome and other predictors were complete.",
"We defined adverse pregnancy outcomes as the total number of stillbirths and neonatal deaths per 1,000 births. Perinatal mortality is comprised of the sum of stillbirths and deaths in the first week of life per 1,000 births. The neonatal mortality rate is presented as the number of newborn infants dying before reaching 28 days of age, per 1,000 live births and the early neonatal mortality rate as the number dying before reaching 7 days of age, per 1,000 live births.\nAge was defined as the age of the mother at the time of the previous birth. Parity was defined as the number of times that a woman has given birth, regardless of whether the child was born alive or not. Maternal religion was categorised as either Muslim or other religion which included Hinduism and Christianity. The number of household assets was counted using a standard list of household items that included electricity, radio/tape recorder, fan, TV, fridge, phone, generator and bicycle and then dichotomised as ‘up to 3’ or ‘4 or more’ assets. Educational attainment was classified as either none/ primary only or one or more years in secondary or higher education.\nOur data did not include the outcome of the immediately preceding pregnancy but did include the total number of pregnancies, miscarriages or abortions, stillbirths and live births for every mother as reported at the time of interview. These data were used to identify previous adverse pregnancy outcomes defined as a miscarriage or abortion, stillbirth or neonatal death resulting from any previous pregnancy.",
"Potential determinants of a short birth interval were chosen for inclusion in the statistical model and were arrived at a priori through a review of the literature. These included maternal age, parity, age at first pregnancy, maternal religion, education, household assets, tea garden residence and a history of a previous adverse pregnancy outcome. In examining the determinants of short birth interval we defined the outcome as an interval between consecutive births of any outcome (live or stillborn) of less than 33 months, corresponding to World Health Organisation recommendations. Initially, a univariate analysis was performed to determine the associations between individual predictors and short birth interval. A multivariate mixed effects logistic regression model, including all potential determinants of a short birth interval was then used to arrive at individually adjusted estimates. We assessed multicollinearity by calculating variance inflation factors (VIF). The VIF for parity was 3.60 and for age was 3.89, which indicates some, but acceptable collinearity. Given that we were interested in their joint effect, we kept both in the model. A likelihood ratio statistic was used to determine whether to include age and parity as a continuous or a categorical variable. Results indicate it was more appropriate to treat age as a discrete variable and parity as a categorical variable. The Receiver Operating Characteristic (ROC) was used to ascertain the predictive quality of the multivariate model.\nNext, we evaluated short birth intervals as a determinant of an adverse pregnancy outcome. For this, birth interval was categorized as an interval between consecutive births of any outcome (live or stillborn) of <21 months, 21–32 months, 33–44 months and 45 months or longer, corresponding to birth to pregnancy intervals of less than one year, 1–2 years, 2–3 years and 3 or more years under the assumption of 9 months gestation.\nWe identified potential confounders based on a review of the literature. A Directed Acyclic Graph (DAG) was then used to assist in the selection of appropriate confounders by modelling the relationships between potential confounders, short birth interval and stillbirth or neonatal death [see Additional file 1]. The DAG was created using a pre-determined selection criteria in order to minimize biases that have recently been shown to be present when using traditional methods of confounder selection [20]. The final confounders used to model the effects of birth interval on birth outcome were maternal age, parity, education, religion, household assets, tea garden residence and previous adverse pregnancy outcome. A mixed effects logistic regression model was used to evaluate the association between birth interval categories and adverse outcomes of pregnancy adjusting for confounders and the clustered design of the study.\nThe DAG was drawn in Dagitty, an online tool [21]. All analysis were conducted using Stata version 12.1 (StataCorp).",
"This study uses data gathered as part of a larger cluster randomised trial of a community intervention. Community consent was acquired for intervention implementation and all monitoring activities. Individual informed consent was obtained before each interview. The current analyses were not subject to specific ethical review as they fall within the scope of the trial and monitoring activities that were approved by the University College London research ethics committee (identification number 1488/001) and the ethical review committee of the Diabetic Association of Bangladesh.",
"1,368 women (24.6%) had a birth interval shorter than 33 months. The determinants of short birth intervals are described in detail in Table 1. Results from the mixed effects multivariate analysis indicate that the risk of having a short interval increases with age. The likelihood of a short birth interval increased by 11% with each additional year of age (adjusted odds ratio (AOR) 1.11 95% confidence interval (CI) 1.08-1.15). Women who delivered a third or higher order baby were less likely to xperience a short birth interval compared to women who delivered their second baby. Parity of four or more was associated with 72% decrease in the odds of a short birth interval compared to a parity of one at the start of the birth interval (AOR 0.28, 0.19-0.41). The likelihood of a short birth interval decreased 5% for every year that a woman’s first pregnancy was delayed (AOR 0.95, 0.92-0.98). Women who experienced a previous adverse outcome of pregnancy were twice as likely to have a birth interval of less than 33 months compared to those women whose babies survived the neonatal period (AOR 2.10, 1.83-2.40). Christian or Hindu women were 32% less likely to experience a short birth interval than Muslim women (AOR 0.68, 0.53-0.87). Women with secondary education or higher had a 26% increased likelihood of a short birth interval (AOR 1.26, 1.09-1.45) whilst women from a household with up to 3 assets were 42% more likely to have a short interval compared to those with four or more assets (AOR 1.42, 1.22-1.65). Tea-garden residence was associated with a 41% increase in the odds of a short birth interval (AOR 1.41, 1.07-1.87). The ROC showed that the model was a reasonable fit (0.63).",
"Compared to birth intervals of 45 months or longer, birth intervals of less than 21 months were associated with a greater than two-fold increased risk of adverse pregnancy outcome (AOR 2.23 95% CI 1.51-3.29), as well as increased risk of perinatal mortality (AOR 2.33, 95% CI 1.55-3.50), stillbirth (AOR 2.13, 95% CI 1.28-3.53), neonatal mortality (AOR 2.28 95% CI 1.28-4.05) (Table 2). There was no evidence of increased risk of adverse pregnancy outcomes associated with birth intervals of 21–32 months and 33–44 compared to longer intervals.Table 2\nExposure models of adverse outcomes of pregnancy by birth interval\n\nBirth interval (months)\n\nN\n\nEvents\n\nCrude OR*\n\n95% CI\n\nAdjusted OR\n†\n\n95% CI\n\nN\n\n/1,000\n\nAdverse outcome of pregnancy\n>453,17314345.1\nreference\n\nreference\n33-441,0305452.41.190.861.651.210.871.7021-328794247.81.070.751.521.130.781.65<214894490.0\n2.12\n1.493.03\n2.23\n1.513.29\nPerinatal mortality\n>453,17312439.1\nreference\n\nreference\n33-441,0305048.51.290.921.821.310.921.8721-328793742.11.090.751.591.150.771.71<214894183.8\n2.31\n\n1.60\n\n3.35\n\n2.33\n1.553.50\nStill birth rate\n>453,1737924.9\nreference\n\nreference\n33-441,0302827.21.090.701.691.100.701.7321-328791921.60.850.511.420.920.541.56<214892551.12.101.323.332.131.283.53\nNeonatal mortality\n>4530946420.7\nreference\n\nreference\n33-4410022625.91.310.822.091.350.832.2021-328602326.71.320.822.161.380.832.32<214641940.92.101.243.552.281.284.05\nEarly neonatal mortality\n>4530944514.5\nreference\n\nreference\n33-4410022222.01.660.982.811.680.651.8421-328601820.91.510.872.651.540.552.23<214641634.52.631.464.742.590.532.73\n*The crude analyses were adjusted for clustering.\n†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.\n\nExposure models of adverse outcomes of pregnancy by birth interval\n\n\n*The crude analyses were adjusted for clustering.\n\n†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering."
] |
[
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Study population",
"Data collection",
"Definitions",
"Statistical methods",
"Ethics",
"Results",
"Determinants of birth interval <33 months",
"Short birth interval and adverse outcome of pregnancy",
"Discussion",
"Conclusion"
] |
[
"Short birth intervals, defined as the time between two births, are known to have negative effects on maternal, perinatal and neonatal outcomes as well as on child health, though the precise mechanisms are poorly understood [1]. Short intervals are associated with increased preterm birth, low birth weight, and small for gestational age births, as well as perinatal death [2,3]. Neonatal and infant mortality is also higher after a negative outcome of the previous delivery, which is mediated primarily through short birth interval [4,5].\nNeonatal and infant mortality is highest for birth to pregnancy intervals of 18 months and is slightly increased for birth to pregnancy intervals of 18–27 months [6]. The most recent WHO recommendation for a healthy pregnancy interval is at least two years (24 months), which corresponds to a birth-to-birth interval of 33 months under the assumption of nine months gestation [7]. The Government of Bangladesh recommends an interval of three years between the last live birth and the next attempt to get pregnant, which corresponds to a birth-to-birth interval of 45 months under the assumption of nine months gestation [8].\nCorresponding with increasing levels of contraceptive use over the past 30 years (from 7 per cent in 1975 to 54 per cent by 2000) [9], Bangladesh has witnessed large reductions in fertility [9] and marked increases in median birth interval, increasing from 35 months in 1993–1994 to 47 months in 2011 [10]. However, more than two-thirds of women in Bangladesh are married by the age of 18 and the median age at first birth is approximately 18 years [10]. These young women have birth intervals of just 27 months and large differences are seen between socio-economic groups, with poorer, less educated women having shorter intervals [10-13]. The length of the birth interval is closely associated with the survival of the previous sibling [4,6]. The Bangladesh Demographic and Health Survey (BDHS) 2011 identifies a median birth interval 15 months shorter for children whose previous sibling died than for children whose previous sibling is alive (26 and 41 months, respectively) [10].\nMother’s age at first birth, parity, previous birth interval, mother’s working status, gender composition of the living children, and mass media are also described as determinants of birth intervals [11-13].\nNational strategies focused on family planning, reproductive health and safe motherhood have been well described [8] and are likely to represent a dynamic reproductive health context in Bangladesh that warrants contemporary description. Our study seeks to do this by not only describing birth spacing and its consequences on pregnancy outcomes, but also exploring the socioeconomic, demographic and reproductive factors that are associated with short birth intervals using prospective surveillance data from three districts in rural Bangladesh. Our findings are discussed in terms of opportunities for intervention strategies to prevent short birth intervals and the risks associated with them.",
" Study population The study was conducted in nine unions (the lowest administrative level in Bangladesh) in three districts of Bangladesh – Bogra, Moulavibazar and Faridpur. The districts and unions represent part of a cluster randomised controlled trial of a community intervention to improve maternal and neonatal health described in detail elsewhere [14-16]. Only the control unions were included in the current descriptive study given that the aforementioned intervention could have influenced birth spacing or its consequences in intervention areas. The total population size of the nine unions was 282,643, representing 54,668 women of reproductive age residing in approximately 63,000 households. The study areas include tea-garden estates, which differ from much of non-tea garden areas in Bangladesh in terms of socioeconomics, access to services and history and are generally more disadvantaged than other areas [17,18].\nThe study was conducted in nine unions (the lowest administrative level in Bangladesh) in three districts of Bangladesh – Bogra, Moulavibazar and Faridpur. The districts and unions represent part of a cluster randomised controlled trial of a community intervention to improve maternal and neonatal health described in detail elsewhere [14-16]. Only the control unions were included in the current descriptive study given that the aforementioned intervention could have influenced birth spacing or its consequences in intervention areas. The total population size of the nine unions was 282,643, representing 54,668 women of reproductive age residing in approximately 63,000 households. The study areas include tea-garden estates, which differ from much of non-tea garden areas in Bangladesh in terms of socioeconomics, access to services and history and are generally more disadvantaged than other areas [17,18].\n Data collection Since 2004, a key informant surveillance system has been used in the study areas to record all births, neonatal deaths, and deaths of women of reproductive age [19]. 500 traditional birth attendants act as key informants, each monitoring a population of approximately 200 households or 1000 individuals. Most births in the study area are attended by traditional birth attendants, thus these women are aware of most if not all births in their community and are well placed to record pregnancy outcomes. Key informants are visited fortnightly by a monitor who collates data on births, either live or stillbirth, and neonatal and pregnancy-related deaths. 6–52 weeks after a registered event (birth or death), paid monitors visit women in their home to conduct a structured interview to gather data on socio-economic characteristics of the mother and her household, reproductive history, including birth interval, and history of previous pregnancy outcomes, as well as pregnancy, birth and postpartum experiences. Precise gestation cannot be reliably measured through these survey methods and so stillbirths are classified as such on the basis of reports of the birth of a child with no movement or other signs of life during the structured interview; it is unlikely that women’s reports or our survey tool would misclassify foetal deaths before 28 weeks gestation as stillbirths. All data are checked at the district office for quality and referred back to the field if incomplete or inaccurate. A random 10-20% sample of the respondents are revisited by supervisors to verify data quality. Data are sent to Dhaka for entry in an MS Access database, where further quality checks take place.\nThe current analysis was restricted to births taking place between January 2010 and June 2011. Observations were included if data on birth interval, pregnancy outcome and other predictors were complete.\nSince 2004, a key informant surveillance system has been used in the study areas to record all births, neonatal deaths, and deaths of women of reproductive age [19]. 500 traditional birth attendants act as key informants, each monitoring a population of approximately 200 households or 1000 individuals. Most births in the study area are attended by traditional birth attendants, thus these women are aware of most if not all births in their community and are well placed to record pregnancy outcomes. Key informants are visited fortnightly by a monitor who collates data on births, either live or stillbirth, and neonatal and pregnancy-related deaths. 6–52 weeks after a registered event (birth or death), paid monitors visit women in their home to conduct a structured interview to gather data on socio-economic characteristics of the mother and her household, reproductive history, including birth interval, and history of previous pregnancy outcomes, as well as pregnancy, birth and postpartum experiences. Precise gestation cannot be reliably measured through these survey methods and so stillbirths are classified as such on the basis of reports of the birth of a child with no movement or other signs of life during the structured interview; it is unlikely that women’s reports or our survey tool would misclassify foetal deaths before 28 weeks gestation as stillbirths. All data are checked at the district office for quality and referred back to the field if incomplete or inaccurate. A random 10-20% sample of the respondents are revisited by supervisors to verify data quality. Data are sent to Dhaka for entry in an MS Access database, where further quality checks take place.\nThe current analysis was restricted to births taking place between January 2010 and June 2011. Observations were included if data on birth interval, pregnancy outcome and other predictors were complete.\n Definitions We defined adverse pregnancy outcomes as the total number of stillbirths and neonatal deaths per 1,000 births. Perinatal mortality is comprised of the sum of stillbirths and deaths in the first week of life per 1,000 births. The neonatal mortality rate is presented as the number of newborn infants dying before reaching 28 days of age, per 1,000 live births and the early neonatal mortality rate as the number dying before reaching 7 days of age, per 1,000 live births.\nAge was defined as the age of the mother at the time of the previous birth. Parity was defined as the number of times that a woman has given birth, regardless of whether the child was born alive or not. Maternal religion was categorised as either Muslim or other religion which included Hinduism and Christianity. The number of household assets was counted using a standard list of household items that included electricity, radio/tape recorder, fan, TV, fridge, phone, generator and bicycle and then dichotomised as ‘up to 3’ or ‘4 or more’ assets. Educational attainment was classified as either none/ primary only or one or more years in secondary or higher education.\nOur data did not include the outcome of the immediately preceding pregnancy but did include the total number of pregnancies, miscarriages or abortions, stillbirths and live births for every mother as reported at the time of interview. These data were used to identify previous adverse pregnancy outcomes defined as a miscarriage or abortion, stillbirth or neonatal death resulting from any previous pregnancy.\nWe defined adverse pregnancy outcomes as the total number of stillbirths and neonatal deaths per 1,000 births. Perinatal mortality is comprised of the sum of stillbirths and deaths in the first week of life per 1,000 births. The neonatal mortality rate is presented as the number of newborn infants dying before reaching 28 days of age, per 1,000 live births and the early neonatal mortality rate as the number dying before reaching 7 days of age, per 1,000 live births.\nAge was defined as the age of the mother at the time of the previous birth. Parity was defined as the number of times that a woman has given birth, regardless of whether the child was born alive or not. Maternal religion was categorised as either Muslim or other religion which included Hinduism and Christianity. The number of household assets was counted using a standard list of household items that included electricity, radio/tape recorder, fan, TV, fridge, phone, generator and bicycle and then dichotomised as ‘up to 3’ or ‘4 or more’ assets. Educational attainment was classified as either none/ primary only or one or more years in secondary or higher education.\nOur data did not include the outcome of the immediately preceding pregnancy but did include the total number of pregnancies, miscarriages or abortions, stillbirths and live births for every mother as reported at the time of interview. These data were used to identify previous adverse pregnancy outcomes defined as a miscarriage or abortion, stillbirth or neonatal death resulting from any previous pregnancy.\n Statistical methods Potential determinants of a short birth interval were chosen for inclusion in the statistical model and were arrived at a priori through a review of the literature. These included maternal age, parity, age at first pregnancy, maternal religion, education, household assets, tea garden residence and a history of a previous adverse pregnancy outcome. In examining the determinants of short birth interval we defined the outcome as an interval between consecutive births of any outcome (live or stillborn) of less than 33 months, corresponding to World Health Organisation recommendations. Initially, a univariate analysis was performed to determine the associations between individual predictors and short birth interval. A multivariate mixed effects logistic regression model, including all potential determinants of a short birth interval was then used to arrive at individually adjusted estimates. We assessed multicollinearity by calculating variance inflation factors (VIF). The VIF for parity was 3.60 and for age was 3.89, which indicates some, but acceptable collinearity. Given that we were interested in their joint effect, we kept both in the model. A likelihood ratio statistic was used to determine whether to include age and parity as a continuous or a categorical variable. Results indicate it was more appropriate to treat age as a discrete variable and parity as a categorical variable. The Receiver Operating Characteristic (ROC) was used to ascertain the predictive quality of the multivariate model.\nNext, we evaluated short birth intervals as a determinant of an adverse pregnancy outcome. For this, birth interval was categorized as an interval between consecutive births of any outcome (live or stillborn) of <21 months, 21–32 months, 33–44 months and 45 months or longer, corresponding to birth to pregnancy intervals of less than one year, 1–2 years, 2–3 years and 3 or more years under the assumption of 9 months gestation.\nWe identified potential confounders based on a review of the literature. A Directed Acyclic Graph (DAG) was then used to assist in the selection of appropriate confounders by modelling the relationships between potential confounders, short birth interval and stillbirth or neonatal death [see Additional file 1]. The DAG was created using a pre-determined selection criteria in order to minimize biases that have recently been shown to be present when using traditional methods of confounder selection [20]. The final confounders used to model the effects of birth interval on birth outcome were maternal age, parity, education, religion, household assets, tea garden residence and previous adverse pregnancy outcome. A mixed effects logistic regression model was used to evaluate the association between birth interval categories and adverse outcomes of pregnancy adjusting for confounders and the clustered design of the study.\nThe DAG was drawn in Dagitty, an online tool [21]. All analysis were conducted using Stata version 12.1 (StataCorp).\nPotential determinants of a short birth interval were chosen for inclusion in the statistical model and were arrived at a priori through a review of the literature. These included maternal age, parity, age at first pregnancy, maternal religion, education, household assets, tea garden residence and a history of a previous adverse pregnancy outcome. In examining the determinants of short birth interval we defined the outcome as an interval between consecutive births of any outcome (live or stillborn) of less than 33 months, corresponding to World Health Organisation recommendations. Initially, a univariate analysis was performed to determine the associations between individual predictors and short birth interval. A multivariate mixed effects logistic regression model, including all potential determinants of a short birth interval was then used to arrive at individually adjusted estimates. We assessed multicollinearity by calculating variance inflation factors (VIF). The VIF for parity was 3.60 and for age was 3.89, which indicates some, but acceptable collinearity. Given that we were interested in their joint effect, we kept both in the model. A likelihood ratio statistic was used to determine whether to include age and parity as a continuous or a categorical variable. Results indicate it was more appropriate to treat age as a discrete variable and parity as a categorical variable. The Receiver Operating Characteristic (ROC) was used to ascertain the predictive quality of the multivariate model.\nNext, we evaluated short birth intervals as a determinant of an adverse pregnancy outcome. For this, birth interval was categorized as an interval between consecutive births of any outcome (live or stillborn) of <21 months, 21–32 months, 33–44 months and 45 months or longer, corresponding to birth to pregnancy intervals of less than one year, 1–2 years, 2–3 years and 3 or more years under the assumption of 9 months gestation.\nWe identified potential confounders based on a review of the literature. A Directed Acyclic Graph (DAG) was then used to assist in the selection of appropriate confounders by modelling the relationships between potential confounders, short birth interval and stillbirth or neonatal death [see Additional file 1]. The DAG was created using a pre-determined selection criteria in order to minimize biases that have recently been shown to be present when using traditional methods of confounder selection [20]. The final confounders used to model the effects of birth interval on birth outcome were maternal age, parity, education, religion, household assets, tea garden residence and previous adverse pregnancy outcome. A mixed effects logistic regression model was used to evaluate the association between birth interval categories and adverse outcomes of pregnancy adjusting for confounders and the clustered design of the study.\nThe DAG was drawn in Dagitty, an online tool [21]. All analysis were conducted using Stata version 12.1 (StataCorp).\n Ethics This study uses data gathered as part of a larger cluster randomised trial of a community intervention. Community consent was acquired for intervention implementation and all monitoring activities. Individual informed consent was obtained before each interview. The current analyses were not subject to specific ethical review as they fall within the scope of the trial and monitoring activities that were approved by the University College London research ethics committee (identification number 1488/001) and the ethical review committee of the Diabetic Association of Bangladesh.\nThis study uses data gathered as part of a larger cluster randomised trial of a community intervention. Community consent was acquired for intervention implementation and all monitoring activities. Individual informed consent was obtained before each interview. The current analyses were not subject to specific ethical review as they fall within the scope of the trial and monitoring activities that were approved by the University College London research ethics committee (identification number 1488/001) and the ethical review committee of the Diabetic Association of Bangladesh.",
"The study was conducted in nine unions (the lowest administrative level in Bangladesh) in three districts of Bangladesh – Bogra, Moulavibazar and Faridpur. The districts and unions represent part of a cluster randomised controlled trial of a community intervention to improve maternal and neonatal health described in detail elsewhere [14-16]. Only the control unions were included in the current descriptive study given that the aforementioned intervention could have influenced birth spacing or its consequences in intervention areas. The total population size of the nine unions was 282,643, representing 54,668 women of reproductive age residing in approximately 63,000 households. The study areas include tea-garden estates, which differ from much of non-tea garden areas in Bangladesh in terms of socioeconomics, access to services and history and are generally more disadvantaged than other areas [17,18].",
"Since 2004, a key informant surveillance system has been used in the study areas to record all births, neonatal deaths, and deaths of women of reproductive age [19]. 500 traditional birth attendants act as key informants, each monitoring a population of approximately 200 households or 1000 individuals. Most births in the study area are attended by traditional birth attendants, thus these women are aware of most if not all births in their community and are well placed to record pregnancy outcomes. Key informants are visited fortnightly by a monitor who collates data on births, either live or stillbirth, and neonatal and pregnancy-related deaths. 6–52 weeks after a registered event (birth or death), paid monitors visit women in their home to conduct a structured interview to gather data on socio-economic characteristics of the mother and her household, reproductive history, including birth interval, and history of previous pregnancy outcomes, as well as pregnancy, birth and postpartum experiences. Precise gestation cannot be reliably measured through these survey methods and so stillbirths are classified as such on the basis of reports of the birth of a child with no movement or other signs of life during the structured interview; it is unlikely that women’s reports or our survey tool would misclassify foetal deaths before 28 weeks gestation as stillbirths. All data are checked at the district office for quality and referred back to the field if incomplete or inaccurate. A random 10-20% sample of the respondents are revisited by supervisors to verify data quality. Data are sent to Dhaka for entry in an MS Access database, where further quality checks take place.\nThe current analysis was restricted to births taking place between January 2010 and June 2011. Observations were included if data on birth interval, pregnancy outcome and other predictors were complete.",
"We defined adverse pregnancy outcomes as the total number of stillbirths and neonatal deaths per 1,000 births. Perinatal mortality is comprised of the sum of stillbirths and deaths in the first week of life per 1,000 births. The neonatal mortality rate is presented as the number of newborn infants dying before reaching 28 days of age, per 1,000 live births and the early neonatal mortality rate as the number dying before reaching 7 days of age, per 1,000 live births.\nAge was defined as the age of the mother at the time of the previous birth. Parity was defined as the number of times that a woman has given birth, regardless of whether the child was born alive or not. Maternal religion was categorised as either Muslim or other religion which included Hinduism and Christianity. The number of household assets was counted using a standard list of household items that included electricity, radio/tape recorder, fan, TV, fridge, phone, generator and bicycle and then dichotomised as ‘up to 3’ or ‘4 or more’ assets. Educational attainment was classified as either none/ primary only or one or more years in secondary or higher education.\nOur data did not include the outcome of the immediately preceding pregnancy but did include the total number of pregnancies, miscarriages or abortions, stillbirths and live births for every mother as reported at the time of interview. These data were used to identify previous adverse pregnancy outcomes defined as a miscarriage or abortion, stillbirth or neonatal death resulting from any previous pregnancy.",
"Potential determinants of a short birth interval were chosen for inclusion in the statistical model and were arrived at a priori through a review of the literature. These included maternal age, parity, age at first pregnancy, maternal religion, education, household assets, tea garden residence and a history of a previous adverse pregnancy outcome. In examining the determinants of short birth interval we defined the outcome as an interval between consecutive births of any outcome (live or stillborn) of less than 33 months, corresponding to World Health Organisation recommendations. Initially, a univariate analysis was performed to determine the associations between individual predictors and short birth interval. A multivariate mixed effects logistic regression model, including all potential determinants of a short birth interval was then used to arrive at individually adjusted estimates. We assessed multicollinearity by calculating variance inflation factors (VIF). The VIF for parity was 3.60 and for age was 3.89, which indicates some, but acceptable collinearity. Given that we were interested in their joint effect, we kept both in the model. A likelihood ratio statistic was used to determine whether to include age and parity as a continuous or a categorical variable. Results indicate it was more appropriate to treat age as a discrete variable and parity as a categorical variable. The Receiver Operating Characteristic (ROC) was used to ascertain the predictive quality of the multivariate model.\nNext, we evaluated short birth intervals as a determinant of an adverse pregnancy outcome. For this, birth interval was categorized as an interval between consecutive births of any outcome (live or stillborn) of <21 months, 21–32 months, 33–44 months and 45 months or longer, corresponding to birth to pregnancy intervals of less than one year, 1–2 years, 2–3 years and 3 or more years under the assumption of 9 months gestation.\nWe identified potential confounders based on a review of the literature. A Directed Acyclic Graph (DAG) was then used to assist in the selection of appropriate confounders by modelling the relationships between potential confounders, short birth interval and stillbirth or neonatal death [see Additional file 1]. The DAG was created using a pre-determined selection criteria in order to minimize biases that have recently been shown to be present when using traditional methods of confounder selection [20]. The final confounders used to model the effects of birth interval on birth outcome were maternal age, parity, education, religion, household assets, tea garden residence and previous adverse pregnancy outcome. A mixed effects logistic regression model was used to evaluate the association between birth interval categories and adverse outcomes of pregnancy adjusting for confounders and the clustered design of the study.\nThe DAG was drawn in Dagitty, an online tool [21]. All analysis were conducted using Stata version 12.1 (StataCorp).",
"This study uses data gathered as part of a larger cluster randomised trial of a community intervention. Community consent was acquired for intervention implementation and all monitoring activities. Individual informed consent was obtained before each interview. The current analyses were not subject to specific ethical review as they fall within the scope of the trial and monitoring activities that were approved by the University College London research ethics committee (identification number 1488/001) and the ethical review committee of the Diabetic Association of Bangladesh.",
"In the period January 2010-June 2011, 9,797 deliveries were registered with 5,911 second or higher order deliveries. The average birth interval was 4 years and 7 months (55 months). The average age of first pregnancy was 18.4 years and the average age of delivery 26.4 years (Table 1).Table 1\nCrude and adjusted model of determinants of short birth interval defined as birth-to-birth interval <33 months\n\nRecommended birth interval (at least 33 months)\n\nShort birth interval < 33 months\n\nCrude OR\n\n95% CI\n\nOR\n†\n\n95% CI\n\nAge at onset of birth interval\n22.0 year$\n22.8 year$\n1.041.031.061.111.081.15\nParity at onset of birth interval\n12,81150.5%70851.8%\nreference\n\nreference\n21,47026.4%32924.0%0.860.740.990.530.440.63370812.7%16812.3%0.920.761.120.380.290.514+58210.4%16311.9%1.160.951.410.280.190.41\nAge at first pregnancy\n18.3 year$\n18.6 year$\n1.041.021.070.950.920.98\nAdverse outcome of any previous pregnancy\nYes2,02136.3%65147.6%2.281.952.672.101.832.40No3,55063.7%71752.4%\nreference\n\nreference\n\nReligion\nMuslim (%)4,60582.6%1,15284.2%\nreference\n\nreference\nOther (%)96617.3%21615.8%0.860.731.020.680.530.87\nEducation\nNone or primary education only3,40861.2%81059.2%\nreference\n\nreference\nSecondary or above2,16338.8%55840.8%1.110.981.261.261.091.45\nHousehold assets\n0-34,04372.7%104076.0%1.221.061.411.421.221.654+1,52027.3%32824.0%\nreference\n\nreference\n\nTea garden resident\nYes66311.9%16912.4%1.060.881.281.411.071.87No480988.1%1.19987.6%\nreference\n\nreference\n\n†Adjustment for all other variables. $Average.\n\nCrude and adjusted model of determinants of short birth interval defined as birth-to-birth interval <33 months\n\n\n†Adjustment for all other variables. $Average.\nWe checked for data completeness and 340 observations (5.8%) had missing data. 330 observations had a missing birth interval and 10 observations missed other covariates (parity, education). Neonatal mortality was higher among those cases with missing data and younger age, lower parity, previous adverse outcomes, Christian or Hindu religion and higher education groups were overrepresented [see Additional file 2]. A complete case analysis was performed, using 5,571 pregnancies without any missing data.\n Determinants of birth interval <33 months 1,368 women (24.6%) had a birth interval shorter than 33 months. The determinants of short birth intervals are described in detail in Table 1. Results from the mixed effects multivariate analysis indicate that the risk of having a short interval increases with age. The likelihood of a short birth interval increased by 11% with each additional year of age (adjusted odds ratio (AOR) 1.11 95% confidence interval (CI) 1.08-1.15). Women who delivered a third or higher order baby were less likely to xperience a short birth interval compared to women who delivered their second baby. Parity of four or more was associated with 72% decrease in the odds of a short birth interval compared to a parity of one at the start of the birth interval (AOR 0.28, 0.19-0.41). The likelihood of a short birth interval decreased 5% for every year that a woman’s first pregnancy was delayed (AOR 0.95, 0.92-0.98). Women who experienced a previous adverse outcome of pregnancy were twice as likely to have a birth interval of less than 33 months compared to those women whose babies survived the neonatal period (AOR 2.10, 1.83-2.40). Christian or Hindu women were 32% less likely to experience a short birth interval than Muslim women (AOR 0.68, 0.53-0.87). Women with secondary education or higher had a 26% increased likelihood of a short birth interval (AOR 1.26, 1.09-1.45) whilst women from a household with up to 3 assets were 42% more likely to have a short interval compared to those with four or more assets (AOR 1.42, 1.22-1.65). Tea-garden residence was associated with a 41% increase in the odds of a short birth interval (AOR 1.41, 1.07-1.87). The ROC showed that the model was a reasonable fit (0.63).\n1,368 women (24.6%) had a birth interval shorter than 33 months. The determinants of short birth intervals are described in detail in Table 1. Results from the mixed effects multivariate analysis indicate that the risk of having a short interval increases with age. The likelihood of a short birth interval increased by 11% with each additional year of age (adjusted odds ratio (AOR) 1.11 95% confidence interval (CI) 1.08-1.15). Women who delivered a third or higher order baby were less likely to xperience a short birth interval compared to women who delivered their second baby. Parity of four or more was associated with 72% decrease in the odds of a short birth interval compared to a parity of one at the start of the birth interval (AOR 0.28, 0.19-0.41). The likelihood of a short birth interval decreased 5% for every year that a woman’s first pregnancy was delayed (AOR 0.95, 0.92-0.98). Women who experienced a previous adverse outcome of pregnancy were twice as likely to have a birth interval of less than 33 months compared to those women whose babies survived the neonatal period (AOR 2.10, 1.83-2.40). Christian or Hindu women were 32% less likely to experience a short birth interval than Muslim women (AOR 0.68, 0.53-0.87). Women with secondary education or higher had a 26% increased likelihood of a short birth interval (AOR 1.26, 1.09-1.45) whilst women from a household with up to 3 assets were 42% more likely to have a short interval compared to those with four or more assets (AOR 1.42, 1.22-1.65). Tea-garden residence was associated with a 41% increase in the odds of a short birth interval (AOR 1.41, 1.07-1.87). The ROC showed that the model was a reasonable fit (0.63).\n Short birth interval and adverse outcome of pregnancy Compared to birth intervals of 45 months or longer, birth intervals of less than 21 months were associated with a greater than two-fold increased risk of adverse pregnancy outcome (AOR 2.23 95% CI 1.51-3.29), as well as increased risk of perinatal mortality (AOR 2.33, 95% CI 1.55-3.50), stillbirth (AOR 2.13, 95% CI 1.28-3.53), neonatal mortality (AOR 2.28 95% CI 1.28-4.05) (Table 2). There was no evidence of increased risk of adverse pregnancy outcomes associated with birth intervals of 21–32 months and 33–44 compared to longer intervals.Table 2\nExposure models of adverse outcomes of pregnancy by birth interval\n\nBirth interval (months)\n\nN\n\nEvents\n\nCrude OR*\n\n95% CI\n\nAdjusted OR\n†\n\n95% CI\n\nN\n\n/1,000\n\nAdverse outcome of pregnancy\n>453,17314345.1\nreference\n\nreference\n33-441,0305452.41.190.861.651.210.871.7021-328794247.81.070.751.521.130.781.65<214894490.0\n2.12\n1.493.03\n2.23\n1.513.29\nPerinatal mortality\n>453,17312439.1\nreference\n\nreference\n33-441,0305048.51.290.921.821.310.921.8721-328793742.11.090.751.591.150.771.71<214894183.8\n2.31\n\n1.60\n\n3.35\n\n2.33\n1.553.50\nStill birth rate\n>453,1737924.9\nreference\n\nreference\n33-441,0302827.21.090.701.691.100.701.7321-328791921.60.850.511.420.920.541.56<214892551.12.101.323.332.131.283.53\nNeonatal mortality\n>4530946420.7\nreference\n\nreference\n33-4410022625.91.310.822.091.350.832.2021-328602326.71.320.822.161.380.832.32<214641940.92.101.243.552.281.284.05\nEarly neonatal mortality\n>4530944514.5\nreference\n\nreference\n33-4410022222.01.660.982.811.680.651.8421-328601820.91.510.872.651.540.552.23<214641634.52.631.464.742.590.532.73\n*The crude analyses were adjusted for clustering.\n†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.\n\nExposure models of adverse outcomes of pregnancy by birth interval\n\n\n*The crude analyses were adjusted for clustering.\n\n†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.\nCompared to birth intervals of 45 months or longer, birth intervals of less than 21 months were associated with a greater than two-fold increased risk of adverse pregnancy outcome (AOR 2.23 95% CI 1.51-3.29), as well as increased risk of perinatal mortality (AOR 2.33, 95% CI 1.55-3.50), stillbirth (AOR 2.13, 95% CI 1.28-3.53), neonatal mortality (AOR 2.28 95% CI 1.28-4.05) (Table 2). There was no evidence of increased risk of adverse pregnancy outcomes associated with birth intervals of 21–32 months and 33–44 compared to longer intervals.Table 2\nExposure models of adverse outcomes of pregnancy by birth interval\n\nBirth interval (months)\n\nN\n\nEvents\n\nCrude OR*\n\n95% CI\n\nAdjusted OR\n†\n\n95% CI\n\nN\n\n/1,000\n\nAdverse outcome of pregnancy\n>453,17314345.1\nreference\n\nreference\n33-441,0305452.41.190.861.651.210.871.7021-328794247.81.070.751.521.130.781.65<214894490.0\n2.12\n1.493.03\n2.23\n1.513.29\nPerinatal mortality\n>453,17312439.1\nreference\n\nreference\n33-441,0305048.51.290.921.821.310.921.8721-328793742.11.090.751.591.150.771.71<214894183.8\n2.31\n\n1.60\n\n3.35\n\n2.33\n1.553.50\nStill birth rate\n>453,1737924.9\nreference\n\nreference\n33-441,0302827.21.090.701.691.100.701.7321-328791921.60.850.511.420.920.541.56<214892551.12.101.323.332.131.283.53\nNeonatal mortality\n>4530946420.7\nreference\n\nreference\n33-4410022625.91.310.822.091.350.832.2021-328602326.71.320.822.161.380.832.32<214641940.92.101.243.552.281.284.05\nEarly neonatal mortality\n>4530944514.5\nreference\n\nreference\n33-4410022222.01.660.982.811.680.651.8421-328601820.91.510.872.651.540.552.23<214641634.52.631.464.742.590.532.73\n*The crude analyses were adjusted for clustering.\n†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.\n\nExposure models of adverse outcomes of pregnancy by birth interval\n\n\n*The crude analyses were adjusted for clustering.\n\n†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.",
"1,368 women (24.6%) had a birth interval shorter than 33 months. The determinants of short birth intervals are described in detail in Table 1. Results from the mixed effects multivariate analysis indicate that the risk of having a short interval increases with age. The likelihood of a short birth interval increased by 11% with each additional year of age (adjusted odds ratio (AOR) 1.11 95% confidence interval (CI) 1.08-1.15). Women who delivered a third or higher order baby were less likely to xperience a short birth interval compared to women who delivered their second baby. Parity of four or more was associated with 72% decrease in the odds of a short birth interval compared to a parity of one at the start of the birth interval (AOR 0.28, 0.19-0.41). The likelihood of a short birth interval decreased 5% for every year that a woman’s first pregnancy was delayed (AOR 0.95, 0.92-0.98). Women who experienced a previous adverse outcome of pregnancy were twice as likely to have a birth interval of less than 33 months compared to those women whose babies survived the neonatal period (AOR 2.10, 1.83-2.40). Christian or Hindu women were 32% less likely to experience a short birth interval than Muslim women (AOR 0.68, 0.53-0.87). Women with secondary education or higher had a 26% increased likelihood of a short birth interval (AOR 1.26, 1.09-1.45) whilst women from a household with up to 3 assets were 42% more likely to have a short interval compared to those with four or more assets (AOR 1.42, 1.22-1.65). Tea-garden residence was associated with a 41% increase in the odds of a short birth interval (AOR 1.41, 1.07-1.87). The ROC showed that the model was a reasonable fit (0.63).",
"Compared to birth intervals of 45 months or longer, birth intervals of less than 21 months were associated with a greater than two-fold increased risk of adverse pregnancy outcome (AOR 2.23 95% CI 1.51-3.29), as well as increased risk of perinatal mortality (AOR 2.33, 95% CI 1.55-3.50), stillbirth (AOR 2.13, 95% CI 1.28-3.53), neonatal mortality (AOR 2.28 95% CI 1.28-4.05) (Table 2). There was no evidence of increased risk of adverse pregnancy outcomes associated with birth intervals of 21–32 months and 33–44 compared to longer intervals.Table 2\nExposure models of adverse outcomes of pregnancy by birth interval\n\nBirth interval (months)\n\nN\n\nEvents\n\nCrude OR*\n\n95% CI\n\nAdjusted OR\n†\n\n95% CI\n\nN\n\n/1,000\n\nAdverse outcome of pregnancy\n>453,17314345.1\nreference\n\nreference\n33-441,0305452.41.190.861.651.210.871.7021-328794247.81.070.751.521.130.781.65<214894490.0\n2.12\n1.493.03\n2.23\n1.513.29\nPerinatal mortality\n>453,17312439.1\nreference\n\nreference\n33-441,0305048.51.290.921.821.310.921.8721-328793742.11.090.751.591.150.771.71<214894183.8\n2.31\n\n1.60\n\n3.35\n\n2.33\n1.553.50\nStill birth rate\n>453,1737924.9\nreference\n\nreference\n33-441,0302827.21.090.701.691.100.701.7321-328791921.60.850.511.420.920.541.56<214892551.12.101.323.332.131.283.53\nNeonatal mortality\n>4530946420.7\nreference\n\nreference\n33-4410022625.91.310.822.091.350.832.2021-328602326.71.320.822.161.380.832.32<214641940.92.101.243.552.281.284.05\nEarly neonatal mortality\n>4530944514.5\nreference\n\nreference\n33-4410022222.01.660.982.811.680.651.8421-328601820.91.510.872.651.540.552.23<214641634.52.631.464.742.590.532.73\n*The crude analyses were adjusted for clustering.\n†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.\n\nExposure models of adverse outcomes of pregnancy by birth interval\n\n\n*The crude analyses were adjusted for clustering.\n\n†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.",
"Using data from a large population based surveillance system from three districts in rural Bangladesh we identified demographic and reproductive predictors of short birth interval and identified important consequences of short intervals on pregnancy outcomes. Younger women, women who start their reproductive life later in life and those who achieve higher order parities are less likely to experience short birth intervals. A previous adverse outcome is an important determinant of short birth interval, which in turn increases the likelihood of subsequent adverse outcomes including stillbirth, perinatal and neonatal death, potentially creating a detrimental cycle of increased risk to reproductive, maternal and newbron health, particularly amongst the most socioeconomically disadvantaged groups.\nBetween the early 1950s and the early 2000s, the total fertility rate in Asia dropped from 5.7 to 2.4 births per woman [22]. This resulted in reduced family size and increases in average birth intervals and may in part be explained by the increase in contraceptive use – from 7 per cent in 1975 to 54 per cent by 2000 in Bangladesh [9]. Although the reduction in fertility rate has slowed, there is still a downward trend. Data limitations in the current analysis prevented us from examining the role of family planning, breastfeeding and fertility trends in our study setting, but our finding that young women are less likely than older women to experience short birth intervals suggests an overall shift in reproductive choices that may reflect these wider changes in family planning and fertility observed across the continent.\nThe current total fertility rate in Bangladesh is estimated as 2.3 children and average ideal family size is 2.2 children [10]. The observation that women who have achieved a parity of two or more and pursue a subsequent pregnancy are less likely to have a short birth interval than women with parity of one may not be surprising therefore. These women may have achieved their desired family size and may feel less pressure or be in less of a hurry to get pregnant again. However, it is difficult in our analysis to disentangle the effects of age, parity and age at first pregnancy, which will all correlate with average past birth intervals and are associated with the interval of interest. Nevertheless, including each of these parameters in our adjusted model allows some estimation of the independent effect of each.\nWe observe that women with a higher educational attainment have an increased risk of short birth intervals, which is surprising. Data from the DHS from Bangladesh as well as other countries suggest better educated women have longer birth intervals [10,23]. A hypothesis could be that more educated women in our study may have married later in life and subsequently hurried to establish a family. However, the relatively small difference in age at first pregnancy between education groups observed in our data is relatively small (18.27 compared to 18.63 years, p < 0.01) and does not support this. A further hypothesis is that better educated women may wish to compress childbearing into fewer years and participate in non-childbearing activities, but further quantitative and qualitative research is required to investigate this.\nWe have shown that a previous adverse outcome is a determinant of short birth interval and, in a separate analysis, that a short birth interval is a risk factor for an adverse pregnancy outcome. A limitation of our study is that we are unable to disentangle the possible “scarring” effect of a previous birth interval and unobserved family characteristics that cause both clustering of perinatal deaths and short birth intervals (family heterogeneity). A previous adverse outcome may have a “scarring” effect, because it causes women to rush into a next pregnancy (replacement) without properly recovering from the previous pregnancy. Women who experience a short birth interval may be subject to other factors that could result in clustering of adverse pregnancy outcomes, such as reduced access to health facilities which could reduce both access to family planning as well as delivery and perinatal care. A study by Saha et al., using advanced panel data analysis techniques and longitudinal data from Matlab, Bangladesh, demonstrates that a short birth interval is the most important pathway to explain neonatal death of the consequent child after a previous adverse neonatal death adjusted for unobserved family heterogeneity [4].\nOur observations confirm that the risk of a short birth interval is highest for those least well-off, both in terms of household assets and tea garden residence. Analysis of BDHS data also indicates that birth intervals are shortest and the difference between desired spacing and actual birth interval is biggest for the least well-off [23]. Differences in the use of family planning methods could explain the differences in birth intervals between socioeconomic groups. Rahman et al. demonstrated that replacement level fertility has almost been achieved in women who are better educated and socioeconomically better off, while replacement level fertility has not been achieved yet for the less well-off [9].\nVery short birth-to-pregnancy intervals of 18 months or shorter are associated with elevated risk of infant, neonatal and perinatal mortality, low birth weight, small size for gestational age, and pre-term delivery [2-6]. A birth-to-pregnancy interval of 18–27 months may pose some “residual” risk [2]. We observe that a very short birth interval less than 21 months (birth-to-pregnancy of less than 12 months when pregnancy is carried to term) is associated with an increased risk of adverse pregnancy outcomes, but intervals of 24–32 months (birth-to-pregnancy interval of 12–23 months when pregnancy is carried to term) and 33–44 months (birth-to-pregnancy interval of 24–35 months) do not appear to be. Based on these findings, the World Health Organisation’s recommendation to wait two years after a live birth before attempting a next pregnancy, and the Government of Bangladesh’s recommended birth-to-pregnancy interval of 3 years, may be overly cautious as far as perinatal outcomes are concerned.\nThe purpose of observational studies is to inform intervention, policy and practice. Our findings suggest that women from poor socioeconomic backgrounds and women who experienced an adverse previous pregnancy should receive particular attention from family planning programs in rural Bangladesh. Post-partum counselling including family planning counselling for bereaved mothers who experienced an adverse outcome is likely to be important in addressing short birth intervals and their consequences. An approach that may have potential in targeting women from poor socioeconomic backgrounds is community mobilisation through women’s groups on reproductive and women’s health. The approach has been shown to be effective at improving maternal and neonatal health, especially for the most marginalised women [15,24,25].",
"Birth spacing remains an issue that deserves urgent attention in Bangladesh. Disadvantaged women are more likely to experience short birth intervals and suffer their consequences. Further research should look into causal pathways as well as strategies to improve spacing between pregnancies whilst efforts to prevent adverse pregnancy outcomes, an important determinant of birth spacing, should be intensified."
] |
[
"introduction",
"materials|methods",
null,
null,
null,
null,
null,
"results",
null,
null,
"discussion",
"conclusion"
] |
[
"Birth intervals",
"Bangladesh",
"Perinatal mortality",
"Socioeconomic factors",
"Population surveillance"
] |
Background:
Short birth intervals, defined as the time between two births, are known to have negative effects on maternal, perinatal and neonatal outcomes as well as on child health, though the precise mechanisms are poorly understood [1]. Short intervals are associated with increased preterm birth, low birth weight, and small for gestational age births, as well as perinatal death [2,3]. Neonatal and infant mortality is also higher after a negative outcome of the previous delivery, which is mediated primarily through short birth interval [4,5].
Neonatal and infant mortality is highest for birth to pregnancy intervals of 18 months and is slightly increased for birth to pregnancy intervals of 18–27 months [6]. The most recent WHO recommendation for a healthy pregnancy interval is at least two years (24 months), which corresponds to a birth-to-birth interval of 33 months under the assumption of nine months gestation [7]. The Government of Bangladesh recommends an interval of three years between the last live birth and the next attempt to get pregnant, which corresponds to a birth-to-birth interval of 45 months under the assumption of nine months gestation [8].
Corresponding with increasing levels of contraceptive use over the past 30 years (from 7 per cent in 1975 to 54 per cent by 2000) [9], Bangladesh has witnessed large reductions in fertility [9] and marked increases in median birth interval, increasing from 35 months in 1993–1994 to 47 months in 2011 [10]. However, more than two-thirds of women in Bangladesh are married by the age of 18 and the median age at first birth is approximately 18 years [10]. These young women have birth intervals of just 27 months and large differences are seen between socio-economic groups, with poorer, less educated women having shorter intervals [10-13]. The length of the birth interval is closely associated with the survival of the previous sibling [4,6]. The Bangladesh Demographic and Health Survey (BDHS) 2011 identifies a median birth interval 15 months shorter for children whose previous sibling died than for children whose previous sibling is alive (26 and 41 months, respectively) [10].
Mother’s age at first birth, parity, previous birth interval, mother’s working status, gender composition of the living children, and mass media are also described as determinants of birth intervals [11-13].
National strategies focused on family planning, reproductive health and safe motherhood have been well described [8] and are likely to represent a dynamic reproductive health context in Bangladesh that warrants contemporary description. Our study seeks to do this by not only describing birth spacing and its consequences on pregnancy outcomes, but also exploring the socioeconomic, demographic and reproductive factors that are associated with short birth intervals using prospective surveillance data from three districts in rural Bangladesh. Our findings are discussed in terms of opportunities for intervention strategies to prevent short birth intervals and the risks associated with them.
Methods:
Study population The study was conducted in nine unions (the lowest administrative level in Bangladesh) in three districts of Bangladesh – Bogra, Moulavibazar and Faridpur. The districts and unions represent part of a cluster randomised controlled trial of a community intervention to improve maternal and neonatal health described in detail elsewhere [14-16]. Only the control unions were included in the current descriptive study given that the aforementioned intervention could have influenced birth spacing or its consequences in intervention areas. The total population size of the nine unions was 282,643, representing 54,668 women of reproductive age residing in approximately 63,000 households. The study areas include tea-garden estates, which differ from much of non-tea garden areas in Bangladesh in terms of socioeconomics, access to services and history and are generally more disadvantaged than other areas [17,18].
The study was conducted in nine unions (the lowest administrative level in Bangladesh) in three districts of Bangladesh – Bogra, Moulavibazar and Faridpur. The districts and unions represent part of a cluster randomised controlled trial of a community intervention to improve maternal and neonatal health described in detail elsewhere [14-16]. Only the control unions were included in the current descriptive study given that the aforementioned intervention could have influenced birth spacing or its consequences in intervention areas. The total population size of the nine unions was 282,643, representing 54,668 women of reproductive age residing in approximately 63,000 households. The study areas include tea-garden estates, which differ from much of non-tea garden areas in Bangladesh in terms of socioeconomics, access to services and history and are generally more disadvantaged than other areas [17,18].
Data collection Since 2004, a key informant surveillance system has been used in the study areas to record all births, neonatal deaths, and deaths of women of reproductive age [19]. 500 traditional birth attendants act as key informants, each monitoring a population of approximately 200 households or 1000 individuals. Most births in the study area are attended by traditional birth attendants, thus these women are aware of most if not all births in their community and are well placed to record pregnancy outcomes. Key informants are visited fortnightly by a monitor who collates data on births, either live or stillbirth, and neonatal and pregnancy-related deaths. 6–52 weeks after a registered event (birth or death), paid monitors visit women in their home to conduct a structured interview to gather data on socio-economic characteristics of the mother and her household, reproductive history, including birth interval, and history of previous pregnancy outcomes, as well as pregnancy, birth and postpartum experiences. Precise gestation cannot be reliably measured through these survey methods and so stillbirths are classified as such on the basis of reports of the birth of a child with no movement or other signs of life during the structured interview; it is unlikely that women’s reports or our survey tool would misclassify foetal deaths before 28 weeks gestation as stillbirths. All data are checked at the district office for quality and referred back to the field if incomplete or inaccurate. A random 10-20% sample of the respondents are revisited by supervisors to verify data quality. Data are sent to Dhaka for entry in an MS Access database, where further quality checks take place.
The current analysis was restricted to births taking place between January 2010 and June 2011. Observations were included if data on birth interval, pregnancy outcome and other predictors were complete.
Since 2004, a key informant surveillance system has been used in the study areas to record all births, neonatal deaths, and deaths of women of reproductive age [19]. 500 traditional birth attendants act as key informants, each monitoring a population of approximately 200 households or 1000 individuals. Most births in the study area are attended by traditional birth attendants, thus these women are aware of most if not all births in their community and are well placed to record pregnancy outcomes. Key informants are visited fortnightly by a monitor who collates data on births, either live or stillbirth, and neonatal and pregnancy-related deaths. 6–52 weeks after a registered event (birth or death), paid monitors visit women in their home to conduct a structured interview to gather data on socio-economic characteristics of the mother and her household, reproductive history, including birth interval, and history of previous pregnancy outcomes, as well as pregnancy, birth and postpartum experiences. Precise gestation cannot be reliably measured through these survey methods and so stillbirths are classified as such on the basis of reports of the birth of a child with no movement or other signs of life during the structured interview; it is unlikely that women’s reports or our survey tool would misclassify foetal deaths before 28 weeks gestation as stillbirths. All data are checked at the district office for quality and referred back to the field if incomplete or inaccurate. A random 10-20% sample of the respondents are revisited by supervisors to verify data quality. Data are sent to Dhaka for entry in an MS Access database, where further quality checks take place.
The current analysis was restricted to births taking place between January 2010 and June 2011. Observations were included if data on birth interval, pregnancy outcome and other predictors were complete.
Definitions We defined adverse pregnancy outcomes as the total number of stillbirths and neonatal deaths per 1,000 births. Perinatal mortality is comprised of the sum of stillbirths and deaths in the first week of life per 1,000 births. The neonatal mortality rate is presented as the number of newborn infants dying before reaching 28 days of age, per 1,000 live births and the early neonatal mortality rate as the number dying before reaching 7 days of age, per 1,000 live births.
Age was defined as the age of the mother at the time of the previous birth. Parity was defined as the number of times that a woman has given birth, regardless of whether the child was born alive or not. Maternal religion was categorised as either Muslim or other religion which included Hinduism and Christianity. The number of household assets was counted using a standard list of household items that included electricity, radio/tape recorder, fan, TV, fridge, phone, generator and bicycle and then dichotomised as ‘up to 3’ or ‘4 or more’ assets. Educational attainment was classified as either none/ primary only or one or more years in secondary or higher education.
Our data did not include the outcome of the immediately preceding pregnancy but did include the total number of pregnancies, miscarriages or abortions, stillbirths and live births for every mother as reported at the time of interview. These data were used to identify previous adverse pregnancy outcomes defined as a miscarriage or abortion, stillbirth or neonatal death resulting from any previous pregnancy.
We defined adverse pregnancy outcomes as the total number of stillbirths and neonatal deaths per 1,000 births. Perinatal mortality is comprised of the sum of stillbirths and deaths in the first week of life per 1,000 births. The neonatal mortality rate is presented as the number of newborn infants dying before reaching 28 days of age, per 1,000 live births and the early neonatal mortality rate as the number dying before reaching 7 days of age, per 1,000 live births.
Age was defined as the age of the mother at the time of the previous birth. Parity was defined as the number of times that a woman has given birth, regardless of whether the child was born alive or not. Maternal religion was categorised as either Muslim or other religion which included Hinduism and Christianity. The number of household assets was counted using a standard list of household items that included electricity, radio/tape recorder, fan, TV, fridge, phone, generator and bicycle and then dichotomised as ‘up to 3’ or ‘4 or more’ assets. Educational attainment was classified as either none/ primary only or one or more years in secondary or higher education.
Our data did not include the outcome of the immediately preceding pregnancy but did include the total number of pregnancies, miscarriages or abortions, stillbirths and live births for every mother as reported at the time of interview. These data were used to identify previous adverse pregnancy outcomes defined as a miscarriage or abortion, stillbirth or neonatal death resulting from any previous pregnancy.
Statistical methods Potential determinants of a short birth interval were chosen for inclusion in the statistical model and were arrived at a priori through a review of the literature. These included maternal age, parity, age at first pregnancy, maternal religion, education, household assets, tea garden residence and a history of a previous adverse pregnancy outcome. In examining the determinants of short birth interval we defined the outcome as an interval between consecutive births of any outcome (live or stillborn) of less than 33 months, corresponding to World Health Organisation recommendations. Initially, a univariate analysis was performed to determine the associations between individual predictors and short birth interval. A multivariate mixed effects logistic regression model, including all potential determinants of a short birth interval was then used to arrive at individually adjusted estimates. We assessed multicollinearity by calculating variance inflation factors (VIF). The VIF for parity was 3.60 and for age was 3.89, which indicates some, but acceptable collinearity. Given that we were interested in their joint effect, we kept both in the model. A likelihood ratio statistic was used to determine whether to include age and parity as a continuous or a categorical variable. Results indicate it was more appropriate to treat age as a discrete variable and parity as a categorical variable. The Receiver Operating Characteristic (ROC) was used to ascertain the predictive quality of the multivariate model.
Next, we evaluated short birth intervals as a determinant of an adverse pregnancy outcome. For this, birth interval was categorized as an interval between consecutive births of any outcome (live or stillborn) of <21 months, 21–32 months, 33–44 months and 45 months or longer, corresponding to birth to pregnancy intervals of less than one year, 1–2 years, 2–3 years and 3 or more years under the assumption of 9 months gestation.
We identified potential confounders based on a review of the literature. A Directed Acyclic Graph (DAG) was then used to assist in the selection of appropriate confounders by modelling the relationships between potential confounders, short birth interval and stillbirth or neonatal death [see Additional file 1]. The DAG was created using a pre-determined selection criteria in order to minimize biases that have recently been shown to be present when using traditional methods of confounder selection [20]. The final confounders used to model the effects of birth interval on birth outcome were maternal age, parity, education, religion, household assets, tea garden residence and previous adverse pregnancy outcome. A mixed effects logistic regression model was used to evaluate the association between birth interval categories and adverse outcomes of pregnancy adjusting for confounders and the clustered design of the study.
The DAG was drawn in Dagitty, an online tool [21]. All analysis were conducted using Stata version 12.1 (StataCorp).
Potential determinants of a short birth interval were chosen for inclusion in the statistical model and were arrived at a priori through a review of the literature. These included maternal age, parity, age at first pregnancy, maternal religion, education, household assets, tea garden residence and a history of a previous adverse pregnancy outcome. In examining the determinants of short birth interval we defined the outcome as an interval between consecutive births of any outcome (live or stillborn) of less than 33 months, corresponding to World Health Organisation recommendations. Initially, a univariate analysis was performed to determine the associations between individual predictors and short birth interval. A multivariate mixed effects logistic regression model, including all potential determinants of a short birth interval was then used to arrive at individually adjusted estimates. We assessed multicollinearity by calculating variance inflation factors (VIF). The VIF for parity was 3.60 and for age was 3.89, which indicates some, but acceptable collinearity. Given that we were interested in their joint effect, we kept both in the model. A likelihood ratio statistic was used to determine whether to include age and parity as a continuous or a categorical variable. Results indicate it was more appropriate to treat age as a discrete variable and parity as a categorical variable. The Receiver Operating Characteristic (ROC) was used to ascertain the predictive quality of the multivariate model.
Next, we evaluated short birth intervals as a determinant of an adverse pregnancy outcome. For this, birth interval was categorized as an interval between consecutive births of any outcome (live or stillborn) of <21 months, 21–32 months, 33–44 months and 45 months or longer, corresponding to birth to pregnancy intervals of less than one year, 1–2 years, 2–3 years and 3 or more years under the assumption of 9 months gestation.
We identified potential confounders based on a review of the literature. A Directed Acyclic Graph (DAG) was then used to assist in the selection of appropriate confounders by modelling the relationships between potential confounders, short birth interval and stillbirth or neonatal death [see Additional file 1]. The DAG was created using a pre-determined selection criteria in order to minimize biases that have recently been shown to be present when using traditional methods of confounder selection [20]. The final confounders used to model the effects of birth interval on birth outcome were maternal age, parity, education, religion, household assets, tea garden residence and previous adverse pregnancy outcome. A mixed effects logistic regression model was used to evaluate the association between birth interval categories and adverse outcomes of pregnancy adjusting for confounders and the clustered design of the study.
The DAG was drawn in Dagitty, an online tool [21]. All analysis were conducted using Stata version 12.1 (StataCorp).
Ethics This study uses data gathered as part of a larger cluster randomised trial of a community intervention. Community consent was acquired for intervention implementation and all monitoring activities. Individual informed consent was obtained before each interview. The current analyses were not subject to specific ethical review as they fall within the scope of the trial and monitoring activities that were approved by the University College London research ethics committee (identification number 1488/001) and the ethical review committee of the Diabetic Association of Bangladesh.
This study uses data gathered as part of a larger cluster randomised trial of a community intervention. Community consent was acquired for intervention implementation and all monitoring activities. Individual informed consent was obtained before each interview. The current analyses were not subject to specific ethical review as they fall within the scope of the trial and monitoring activities that were approved by the University College London research ethics committee (identification number 1488/001) and the ethical review committee of the Diabetic Association of Bangladesh.
Study population:
The study was conducted in nine unions (the lowest administrative level in Bangladesh) in three districts of Bangladesh – Bogra, Moulavibazar and Faridpur. The districts and unions represent part of a cluster randomised controlled trial of a community intervention to improve maternal and neonatal health described in detail elsewhere [14-16]. Only the control unions were included in the current descriptive study given that the aforementioned intervention could have influenced birth spacing or its consequences in intervention areas. The total population size of the nine unions was 282,643, representing 54,668 women of reproductive age residing in approximately 63,000 households. The study areas include tea-garden estates, which differ from much of non-tea garden areas in Bangladesh in terms of socioeconomics, access to services and history and are generally more disadvantaged than other areas [17,18].
Data collection:
Since 2004, a key informant surveillance system has been used in the study areas to record all births, neonatal deaths, and deaths of women of reproductive age [19]. 500 traditional birth attendants act as key informants, each monitoring a population of approximately 200 households or 1000 individuals. Most births in the study area are attended by traditional birth attendants, thus these women are aware of most if not all births in their community and are well placed to record pregnancy outcomes. Key informants are visited fortnightly by a monitor who collates data on births, either live or stillbirth, and neonatal and pregnancy-related deaths. 6–52 weeks after a registered event (birth or death), paid monitors visit women in their home to conduct a structured interview to gather data on socio-economic characteristics of the mother and her household, reproductive history, including birth interval, and history of previous pregnancy outcomes, as well as pregnancy, birth and postpartum experiences. Precise gestation cannot be reliably measured through these survey methods and so stillbirths are classified as such on the basis of reports of the birth of a child with no movement or other signs of life during the structured interview; it is unlikely that women’s reports or our survey tool would misclassify foetal deaths before 28 weeks gestation as stillbirths. All data are checked at the district office for quality and referred back to the field if incomplete or inaccurate. A random 10-20% sample of the respondents are revisited by supervisors to verify data quality. Data are sent to Dhaka for entry in an MS Access database, where further quality checks take place.
The current analysis was restricted to births taking place between January 2010 and June 2011. Observations were included if data on birth interval, pregnancy outcome and other predictors were complete.
Definitions:
We defined adverse pregnancy outcomes as the total number of stillbirths and neonatal deaths per 1,000 births. Perinatal mortality is comprised of the sum of stillbirths and deaths in the first week of life per 1,000 births. The neonatal mortality rate is presented as the number of newborn infants dying before reaching 28 days of age, per 1,000 live births and the early neonatal mortality rate as the number dying before reaching 7 days of age, per 1,000 live births.
Age was defined as the age of the mother at the time of the previous birth. Parity was defined as the number of times that a woman has given birth, regardless of whether the child was born alive or not. Maternal religion was categorised as either Muslim or other religion which included Hinduism and Christianity. The number of household assets was counted using a standard list of household items that included electricity, radio/tape recorder, fan, TV, fridge, phone, generator and bicycle and then dichotomised as ‘up to 3’ or ‘4 or more’ assets. Educational attainment was classified as either none/ primary only or one or more years in secondary or higher education.
Our data did not include the outcome of the immediately preceding pregnancy but did include the total number of pregnancies, miscarriages or abortions, stillbirths and live births for every mother as reported at the time of interview. These data were used to identify previous adverse pregnancy outcomes defined as a miscarriage or abortion, stillbirth or neonatal death resulting from any previous pregnancy.
Statistical methods:
Potential determinants of a short birth interval were chosen for inclusion in the statistical model and were arrived at a priori through a review of the literature. These included maternal age, parity, age at first pregnancy, maternal religion, education, household assets, tea garden residence and a history of a previous adverse pregnancy outcome. In examining the determinants of short birth interval we defined the outcome as an interval between consecutive births of any outcome (live or stillborn) of less than 33 months, corresponding to World Health Organisation recommendations. Initially, a univariate analysis was performed to determine the associations between individual predictors and short birth interval. A multivariate mixed effects logistic regression model, including all potential determinants of a short birth interval was then used to arrive at individually adjusted estimates. We assessed multicollinearity by calculating variance inflation factors (VIF). The VIF for parity was 3.60 and for age was 3.89, which indicates some, but acceptable collinearity. Given that we were interested in their joint effect, we kept both in the model. A likelihood ratio statistic was used to determine whether to include age and parity as a continuous or a categorical variable. Results indicate it was more appropriate to treat age as a discrete variable and parity as a categorical variable. The Receiver Operating Characteristic (ROC) was used to ascertain the predictive quality of the multivariate model.
Next, we evaluated short birth intervals as a determinant of an adverse pregnancy outcome. For this, birth interval was categorized as an interval between consecutive births of any outcome (live or stillborn) of <21 months, 21–32 months, 33–44 months and 45 months or longer, corresponding to birth to pregnancy intervals of less than one year, 1–2 years, 2–3 years and 3 or more years under the assumption of 9 months gestation.
We identified potential confounders based on a review of the literature. A Directed Acyclic Graph (DAG) was then used to assist in the selection of appropriate confounders by modelling the relationships between potential confounders, short birth interval and stillbirth or neonatal death [see Additional file 1]. The DAG was created using a pre-determined selection criteria in order to minimize biases that have recently been shown to be present when using traditional methods of confounder selection [20]. The final confounders used to model the effects of birth interval on birth outcome were maternal age, parity, education, religion, household assets, tea garden residence and previous adverse pregnancy outcome. A mixed effects logistic regression model was used to evaluate the association between birth interval categories and adverse outcomes of pregnancy adjusting for confounders and the clustered design of the study.
The DAG was drawn in Dagitty, an online tool [21]. All analysis were conducted using Stata version 12.1 (StataCorp).
Ethics:
This study uses data gathered as part of a larger cluster randomised trial of a community intervention. Community consent was acquired for intervention implementation and all monitoring activities. Individual informed consent was obtained before each interview. The current analyses were not subject to specific ethical review as they fall within the scope of the trial and monitoring activities that were approved by the University College London research ethics committee (identification number 1488/001) and the ethical review committee of the Diabetic Association of Bangladesh.
Results:
In the period January 2010-June 2011, 9,797 deliveries were registered with 5,911 second or higher order deliveries. The average birth interval was 4 years and 7 months (55 months). The average age of first pregnancy was 18.4 years and the average age of delivery 26.4 years (Table 1).Table 1
Crude and adjusted model of determinants of short birth interval defined as birth-to-birth interval <33 months
Recommended birth interval (at least 33 months)
Short birth interval < 33 months
Crude OR
95% CI
OR
†
95% CI
Age at onset of birth interval
22.0 year$
22.8 year$
1.041.031.061.111.081.15
Parity at onset of birth interval
12,81150.5%70851.8%
reference
reference
21,47026.4%32924.0%0.860.740.990.530.440.63370812.7%16812.3%0.920.761.120.380.290.514+58210.4%16311.9%1.160.951.410.280.190.41
Age at first pregnancy
18.3 year$
18.6 year$
1.041.021.070.950.920.98
Adverse outcome of any previous pregnancy
Yes2,02136.3%65147.6%2.281.952.672.101.832.40No3,55063.7%71752.4%
reference
reference
Religion
Muslim (%)4,60582.6%1,15284.2%
reference
reference
Other (%)96617.3%21615.8%0.860.731.020.680.530.87
Education
None or primary education only3,40861.2%81059.2%
reference
reference
Secondary or above2,16338.8%55840.8%1.110.981.261.261.091.45
Household assets
0-34,04372.7%104076.0%1.221.061.411.421.221.654+1,52027.3%32824.0%
reference
reference
Tea garden resident
Yes66311.9%16912.4%1.060.881.281.411.071.87No480988.1%1.19987.6%
reference
reference
†Adjustment for all other variables. $Average.
Crude and adjusted model of determinants of short birth interval defined as birth-to-birth interval <33 months
†Adjustment for all other variables. $Average.
We checked for data completeness and 340 observations (5.8%) had missing data. 330 observations had a missing birth interval and 10 observations missed other covariates (parity, education). Neonatal mortality was higher among those cases with missing data and younger age, lower parity, previous adverse outcomes, Christian or Hindu religion and higher education groups were overrepresented [see Additional file 2]. A complete case analysis was performed, using 5,571 pregnancies without any missing data.
Determinants of birth interval <33 months 1,368 women (24.6%) had a birth interval shorter than 33 months. The determinants of short birth intervals are described in detail in Table 1. Results from the mixed effects multivariate analysis indicate that the risk of having a short interval increases with age. The likelihood of a short birth interval increased by 11% with each additional year of age (adjusted odds ratio (AOR) 1.11 95% confidence interval (CI) 1.08-1.15). Women who delivered a third or higher order baby were less likely to xperience a short birth interval compared to women who delivered their second baby. Parity of four or more was associated with 72% decrease in the odds of a short birth interval compared to a parity of one at the start of the birth interval (AOR 0.28, 0.19-0.41). The likelihood of a short birth interval decreased 5% for every year that a woman’s first pregnancy was delayed (AOR 0.95, 0.92-0.98). Women who experienced a previous adverse outcome of pregnancy were twice as likely to have a birth interval of less than 33 months compared to those women whose babies survived the neonatal period (AOR 2.10, 1.83-2.40). Christian or Hindu women were 32% less likely to experience a short birth interval than Muslim women (AOR 0.68, 0.53-0.87). Women with secondary education or higher had a 26% increased likelihood of a short birth interval (AOR 1.26, 1.09-1.45) whilst women from a household with up to 3 assets were 42% more likely to have a short interval compared to those with four or more assets (AOR 1.42, 1.22-1.65). Tea-garden residence was associated with a 41% increase in the odds of a short birth interval (AOR 1.41, 1.07-1.87). The ROC showed that the model was a reasonable fit (0.63).
1,368 women (24.6%) had a birth interval shorter than 33 months. The determinants of short birth intervals are described in detail in Table 1. Results from the mixed effects multivariate analysis indicate that the risk of having a short interval increases with age. The likelihood of a short birth interval increased by 11% with each additional year of age (adjusted odds ratio (AOR) 1.11 95% confidence interval (CI) 1.08-1.15). Women who delivered a third or higher order baby were less likely to xperience a short birth interval compared to women who delivered their second baby. Parity of four or more was associated with 72% decrease in the odds of a short birth interval compared to a parity of one at the start of the birth interval (AOR 0.28, 0.19-0.41). The likelihood of a short birth interval decreased 5% for every year that a woman’s first pregnancy was delayed (AOR 0.95, 0.92-0.98). Women who experienced a previous adverse outcome of pregnancy were twice as likely to have a birth interval of less than 33 months compared to those women whose babies survived the neonatal period (AOR 2.10, 1.83-2.40). Christian or Hindu women were 32% less likely to experience a short birth interval than Muslim women (AOR 0.68, 0.53-0.87). Women with secondary education or higher had a 26% increased likelihood of a short birth interval (AOR 1.26, 1.09-1.45) whilst women from a household with up to 3 assets were 42% more likely to have a short interval compared to those with four or more assets (AOR 1.42, 1.22-1.65). Tea-garden residence was associated with a 41% increase in the odds of a short birth interval (AOR 1.41, 1.07-1.87). The ROC showed that the model was a reasonable fit (0.63).
Short birth interval and adverse outcome of pregnancy Compared to birth intervals of 45 months or longer, birth intervals of less than 21 months were associated with a greater than two-fold increased risk of adverse pregnancy outcome (AOR 2.23 95% CI 1.51-3.29), as well as increased risk of perinatal mortality (AOR 2.33, 95% CI 1.55-3.50), stillbirth (AOR 2.13, 95% CI 1.28-3.53), neonatal mortality (AOR 2.28 95% CI 1.28-4.05) (Table 2). There was no evidence of increased risk of adverse pregnancy outcomes associated with birth intervals of 21–32 months and 33–44 compared to longer intervals.Table 2
Exposure models of adverse outcomes of pregnancy by birth interval
Birth interval (months)
N
Events
Crude OR*
95% CI
Adjusted OR
†
95% CI
N
/1,000
Adverse outcome of pregnancy
>453,17314345.1
reference
reference
33-441,0305452.41.190.861.651.210.871.7021-328794247.81.070.751.521.130.781.65<214894490.0
2.12
1.493.03
2.23
1.513.29
Perinatal mortality
>453,17312439.1
reference
reference
33-441,0305048.51.290.921.821.310.921.8721-328793742.11.090.751.591.150.771.71<214894183.8
2.31
1.60
3.35
2.33
1.553.50
Still birth rate
>453,1737924.9
reference
reference
33-441,0302827.21.090.701.691.100.701.7321-328791921.60.850.511.420.920.541.56<214892551.12.101.323.332.131.283.53
Neonatal mortality
>4530946420.7
reference
reference
33-4410022625.91.310.822.091.350.832.2021-328602326.71.320.822.161.380.832.32<214641940.92.101.243.552.281.284.05
Early neonatal mortality
>4530944514.5
reference
reference
33-4410022222.01.660.982.811.680.651.8421-328601820.91.510.872.651.540.552.23<214641634.52.631.464.742.590.532.73
*The crude analyses were adjusted for clustering.
†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.
Exposure models of adverse outcomes of pregnancy by birth interval
*The crude analyses were adjusted for clustering.
†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.
Compared to birth intervals of 45 months or longer, birth intervals of less than 21 months were associated with a greater than two-fold increased risk of adverse pregnancy outcome (AOR 2.23 95% CI 1.51-3.29), as well as increased risk of perinatal mortality (AOR 2.33, 95% CI 1.55-3.50), stillbirth (AOR 2.13, 95% CI 1.28-3.53), neonatal mortality (AOR 2.28 95% CI 1.28-4.05) (Table 2). There was no evidence of increased risk of adverse pregnancy outcomes associated with birth intervals of 21–32 months and 33–44 compared to longer intervals.Table 2
Exposure models of adverse outcomes of pregnancy by birth interval
Birth interval (months)
N
Events
Crude OR*
95% CI
Adjusted OR
†
95% CI
N
/1,000
Adverse outcome of pregnancy
>453,17314345.1
reference
reference
33-441,0305452.41.190.861.651.210.871.7021-328794247.81.070.751.521.130.781.65<214894490.0
2.12
1.493.03
2.23
1.513.29
Perinatal mortality
>453,17312439.1
reference
reference
33-441,0305048.51.290.921.821.310.921.8721-328793742.11.090.751.591.150.771.71<214894183.8
2.31
1.60
3.35
2.33
1.553.50
Still birth rate
>453,1737924.9
reference
reference
33-441,0302827.21.090.701.691.100.701.7321-328791921.60.850.511.420.920.541.56<214892551.12.101.323.332.131.283.53
Neonatal mortality
>4530946420.7
reference
reference
33-4410022625.91.310.822.091.350.832.2021-328602326.71.320.822.161.380.832.32<214641940.92.101.243.552.281.284.05
Early neonatal mortality
>4530944514.5
reference
reference
33-4410022222.01.660.982.811.680.651.8421-328601820.91.510.872.651.540.552.23<214641634.52.631.464.742.590.532.73
*The crude analyses were adjusted for clustering.
†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.
Exposure models of adverse outcomes of pregnancy by birth interval
*The crude analyses were adjusted for clustering.
†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.
Determinants of birth interval <33 months:
1,368 women (24.6%) had a birth interval shorter than 33 months. The determinants of short birth intervals are described in detail in Table 1. Results from the mixed effects multivariate analysis indicate that the risk of having a short interval increases with age. The likelihood of a short birth interval increased by 11% with each additional year of age (adjusted odds ratio (AOR) 1.11 95% confidence interval (CI) 1.08-1.15). Women who delivered a third or higher order baby were less likely to xperience a short birth interval compared to women who delivered their second baby. Parity of four or more was associated with 72% decrease in the odds of a short birth interval compared to a parity of one at the start of the birth interval (AOR 0.28, 0.19-0.41). The likelihood of a short birth interval decreased 5% for every year that a woman’s first pregnancy was delayed (AOR 0.95, 0.92-0.98). Women who experienced a previous adverse outcome of pregnancy were twice as likely to have a birth interval of less than 33 months compared to those women whose babies survived the neonatal period (AOR 2.10, 1.83-2.40). Christian or Hindu women were 32% less likely to experience a short birth interval than Muslim women (AOR 0.68, 0.53-0.87). Women with secondary education or higher had a 26% increased likelihood of a short birth interval (AOR 1.26, 1.09-1.45) whilst women from a household with up to 3 assets were 42% more likely to have a short interval compared to those with four or more assets (AOR 1.42, 1.22-1.65). Tea-garden residence was associated with a 41% increase in the odds of a short birth interval (AOR 1.41, 1.07-1.87). The ROC showed that the model was a reasonable fit (0.63).
Short birth interval and adverse outcome of pregnancy:
Compared to birth intervals of 45 months or longer, birth intervals of less than 21 months were associated with a greater than two-fold increased risk of adverse pregnancy outcome (AOR 2.23 95% CI 1.51-3.29), as well as increased risk of perinatal mortality (AOR 2.33, 95% CI 1.55-3.50), stillbirth (AOR 2.13, 95% CI 1.28-3.53), neonatal mortality (AOR 2.28 95% CI 1.28-4.05) (Table 2). There was no evidence of increased risk of adverse pregnancy outcomes associated with birth intervals of 21–32 months and 33–44 compared to longer intervals.Table 2
Exposure models of adverse outcomes of pregnancy by birth interval
Birth interval (months)
N
Events
Crude OR*
95% CI
Adjusted OR
†
95% CI
N
/1,000
Adverse outcome of pregnancy
>453,17314345.1
reference
reference
33-441,0305452.41.190.861.651.210.871.7021-328794247.81.070.751.521.130.781.65<214894490.0
2.12
1.493.03
2.23
1.513.29
Perinatal mortality
>453,17312439.1
reference
reference
33-441,0305048.51.290.921.821.310.921.8721-328793742.11.090.751.591.150.771.71<214894183.8
2.31
1.60
3.35
2.33
1.553.50
Still birth rate
>453,1737924.9
reference
reference
33-441,0302827.21.090.701.691.100.701.7321-328791921.60.850.511.420.920.541.56<214892551.12.101.323.332.131.283.53
Neonatal mortality
>4530946420.7
reference
reference
33-4410022625.91.310.822.091.350.832.2021-328602326.71.320.822.161.380.832.32<214641940.92.101.243.552.281.284.05
Early neonatal mortality
>4530944514.5
reference
reference
33-4410022222.01.660.982.811.680.651.8421-328601820.91.510.872.651.540.552.23<214641634.52.631.464.742.590.532.73
*The crude analyses were adjusted for clustering.
†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.
Exposure models of adverse outcomes of pregnancy by birth interval
*The crude analyses were adjusted for clustering.
†The OR’s were adjusted for maternal age, parity, previous birth outcome, maternal religion, education and household assets, tea garden residence and clustering.
Discussion:
Using data from a large population based surveillance system from three districts in rural Bangladesh we identified demographic and reproductive predictors of short birth interval and identified important consequences of short intervals on pregnancy outcomes. Younger women, women who start their reproductive life later in life and those who achieve higher order parities are less likely to experience short birth intervals. A previous adverse outcome is an important determinant of short birth interval, which in turn increases the likelihood of subsequent adverse outcomes including stillbirth, perinatal and neonatal death, potentially creating a detrimental cycle of increased risk to reproductive, maternal and newbron health, particularly amongst the most socioeconomically disadvantaged groups.
Between the early 1950s and the early 2000s, the total fertility rate in Asia dropped from 5.7 to 2.4 births per woman [22]. This resulted in reduced family size and increases in average birth intervals and may in part be explained by the increase in contraceptive use – from 7 per cent in 1975 to 54 per cent by 2000 in Bangladesh [9]. Although the reduction in fertility rate has slowed, there is still a downward trend. Data limitations in the current analysis prevented us from examining the role of family planning, breastfeeding and fertility trends in our study setting, but our finding that young women are less likely than older women to experience short birth intervals suggests an overall shift in reproductive choices that may reflect these wider changes in family planning and fertility observed across the continent.
The current total fertility rate in Bangladesh is estimated as 2.3 children and average ideal family size is 2.2 children [10]. The observation that women who have achieved a parity of two or more and pursue a subsequent pregnancy are less likely to have a short birth interval than women with parity of one may not be surprising therefore. These women may have achieved their desired family size and may feel less pressure or be in less of a hurry to get pregnant again. However, it is difficult in our analysis to disentangle the effects of age, parity and age at first pregnancy, which will all correlate with average past birth intervals and are associated with the interval of interest. Nevertheless, including each of these parameters in our adjusted model allows some estimation of the independent effect of each.
We observe that women with a higher educational attainment have an increased risk of short birth intervals, which is surprising. Data from the DHS from Bangladesh as well as other countries suggest better educated women have longer birth intervals [10,23]. A hypothesis could be that more educated women in our study may have married later in life and subsequently hurried to establish a family. However, the relatively small difference in age at first pregnancy between education groups observed in our data is relatively small (18.27 compared to 18.63 years, p < 0.01) and does not support this. A further hypothesis is that better educated women may wish to compress childbearing into fewer years and participate in non-childbearing activities, but further quantitative and qualitative research is required to investigate this.
We have shown that a previous adverse outcome is a determinant of short birth interval and, in a separate analysis, that a short birth interval is a risk factor for an adverse pregnancy outcome. A limitation of our study is that we are unable to disentangle the possible “scarring” effect of a previous birth interval and unobserved family characteristics that cause both clustering of perinatal deaths and short birth intervals (family heterogeneity). A previous adverse outcome may have a “scarring” effect, because it causes women to rush into a next pregnancy (replacement) without properly recovering from the previous pregnancy. Women who experience a short birth interval may be subject to other factors that could result in clustering of adverse pregnancy outcomes, such as reduced access to health facilities which could reduce both access to family planning as well as delivery and perinatal care. A study by Saha et al., using advanced panel data analysis techniques and longitudinal data from Matlab, Bangladesh, demonstrates that a short birth interval is the most important pathway to explain neonatal death of the consequent child after a previous adverse neonatal death adjusted for unobserved family heterogeneity [4].
Our observations confirm that the risk of a short birth interval is highest for those least well-off, both in terms of household assets and tea garden residence. Analysis of BDHS data also indicates that birth intervals are shortest and the difference between desired spacing and actual birth interval is biggest for the least well-off [23]. Differences in the use of family planning methods could explain the differences in birth intervals between socioeconomic groups. Rahman et al. demonstrated that replacement level fertility has almost been achieved in women who are better educated and socioeconomically better off, while replacement level fertility has not been achieved yet for the less well-off [9].
Very short birth-to-pregnancy intervals of 18 months or shorter are associated with elevated risk of infant, neonatal and perinatal mortality, low birth weight, small size for gestational age, and pre-term delivery [2-6]. A birth-to-pregnancy interval of 18–27 months may pose some “residual” risk [2]. We observe that a very short birth interval less than 21 months (birth-to-pregnancy of less than 12 months when pregnancy is carried to term) is associated with an increased risk of adverse pregnancy outcomes, but intervals of 24–32 months (birth-to-pregnancy interval of 12–23 months when pregnancy is carried to term) and 33–44 months (birth-to-pregnancy interval of 24–35 months) do not appear to be. Based on these findings, the World Health Organisation’s recommendation to wait two years after a live birth before attempting a next pregnancy, and the Government of Bangladesh’s recommended birth-to-pregnancy interval of 3 years, may be overly cautious as far as perinatal outcomes are concerned.
The purpose of observational studies is to inform intervention, policy and practice. Our findings suggest that women from poor socioeconomic backgrounds and women who experienced an adverse previous pregnancy should receive particular attention from family planning programs in rural Bangladesh. Post-partum counselling including family planning counselling for bereaved mothers who experienced an adverse outcome is likely to be important in addressing short birth intervals and their consequences. An approach that may have potential in targeting women from poor socioeconomic backgrounds is community mobilisation through women’s groups on reproductive and women’s health. The approach has been shown to be effective at improving maternal and neonatal health, especially for the most marginalised women [15,24,25].
Conclusion:
Birth spacing remains an issue that deserves urgent attention in Bangladesh. Disadvantaged women are more likely to experience short birth intervals and suffer their consequences. Further research should look into causal pathways as well as strategies to improve spacing between pregnancies whilst efforts to prevent adverse pregnancy outcomes, an important determinant of birth spacing, should be intensified.
|
Background:
Short birth intervals are known to have negative effects on pregnancy outcomes. We analysed data from a large population surveillance system in rural Bangladesh to identify predictors of short birth interval and determine consequences of short intervals on pregnancy outcomes.
Methods:
The study was conducted in three districts of Bangladesh - Bogra, Moulavibazar and Faridpur (population 282,643, 54,668 women of reproductive age). We used data between January 2010 and June 2011 from a key informant surveillance system that recorded all births, deaths and stillbirths. Short birth interval was defined as an interval between consecutive births of less than 33 months. Initially, risk factors of a short birth interval were determined using a multivariate mixed effects logistic regression model. Independent risk factors were selected using a priori knowledge from literature review. An adjusted mixed effects logistic regression model was then used to determine the effect of up to 21-, 21-32-, 33-44- and 45-month and higher birth-to-birth intervals on pregnancy outcomes controlling for confounders selected through a directed acyclic graph.
Results:
We analysed 5,571 second or higher order deliveries. Average birth interval was 55 months and 1368/5571 women (24.6%) had a short birth interval (<33 months). Younger women (AOR 1.11 95% CI 1.08-1.15 per year increase in age), women who started their reproductive life later (AOR 0.95, 0.92-0.98 per year) and those who achieve higher order parities were less likely to experience short birth intervals (AOR 0.28, 0.19-0.41 parity 4 compared to 1). Women who were socioeconomically disadvantaged were more likely to experience a short birth interval (AOR 1.42, 1.22-1.65) and a previous adverse outcome was an important determinant of interval (AOR 2.10, 1.83-2.40). Very short birth intervals of less than 21 months were associated with increased stillbirth rate (AOR 2.13, 95% CI 1.28-3.53) and neonatal mortality (AOR 2.28 95% CI 1.28-4.05).
Conclusions:
Birth spacing remains a reproductive health problem in Bangladesh. Disadvantaged women are more likely to experience short birth intervals and to have increased perinatal deaths. Research into causal pathways and strategies to improve spacing between pregnancies should be intensified.
|
Background:
Short birth intervals, defined as the time between two births, are known to have negative effects on maternal, perinatal and neonatal outcomes as well as on child health, though the precise mechanisms are poorly understood [1]. Short intervals are associated with increased preterm birth, low birth weight, and small for gestational age births, as well as perinatal death [2,3]. Neonatal and infant mortality is also higher after a negative outcome of the previous delivery, which is mediated primarily through short birth interval [4,5].
Neonatal and infant mortality is highest for birth to pregnancy intervals of 18 months and is slightly increased for birth to pregnancy intervals of 18–27 months [6]. The most recent WHO recommendation for a healthy pregnancy interval is at least two years (24 months), which corresponds to a birth-to-birth interval of 33 months under the assumption of nine months gestation [7]. The Government of Bangladesh recommends an interval of three years between the last live birth and the next attempt to get pregnant, which corresponds to a birth-to-birth interval of 45 months under the assumption of nine months gestation [8].
Corresponding with increasing levels of contraceptive use over the past 30 years (from 7 per cent in 1975 to 54 per cent by 2000) [9], Bangladesh has witnessed large reductions in fertility [9] and marked increases in median birth interval, increasing from 35 months in 1993–1994 to 47 months in 2011 [10]. However, more than two-thirds of women in Bangladesh are married by the age of 18 and the median age at first birth is approximately 18 years [10]. These young women have birth intervals of just 27 months and large differences are seen between socio-economic groups, with poorer, less educated women having shorter intervals [10-13]. The length of the birth interval is closely associated with the survival of the previous sibling [4,6]. The Bangladesh Demographic and Health Survey (BDHS) 2011 identifies a median birth interval 15 months shorter for children whose previous sibling died than for children whose previous sibling is alive (26 and 41 months, respectively) [10].
Mother’s age at first birth, parity, previous birth interval, mother’s working status, gender composition of the living children, and mass media are also described as determinants of birth intervals [11-13].
National strategies focused on family planning, reproductive health and safe motherhood have been well described [8] and are likely to represent a dynamic reproductive health context in Bangladesh that warrants contemporary description. Our study seeks to do this by not only describing birth spacing and its consequences on pregnancy outcomes, but also exploring the socioeconomic, demographic and reproductive factors that are associated with short birth intervals using prospective surveillance data from three districts in rural Bangladesh. Our findings are discussed in terms of opportunities for intervention strategies to prevent short birth intervals and the risks associated with them.
Conclusion:
Birth spacing remains an issue that deserves urgent attention in Bangladesh. Disadvantaged women are more likely to experience short birth intervals and suffer their consequences. Further research should look into causal pathways as well as strategies to improve spacing between pregnancies whilst efforts to prevent adverse pregnancy outcomes, an important determinant of birth spacing, should be intensified.
|
Background:
Short birth intervals are known to have negative effects on pregnancy outcomes. We analysed data from a large population surveillance system in rural Bangladesh to identify predictors of short birth interval and determine consequences of short intervals on pregnancy outcomes.
Methods:
The study was conducted in three districts of Bangladesh - Bogra, Moulavibazar and Faridpur (population 282,643, 54,668 women of reproductive age). We used data between January 2010 and June 2011 from a key informant surveillance system that recorded all births, deaths and stillbirths. Short birth interval was defined as an interval between consecutive births of less than 33 months. Initially, risk factors of a short birth interval were determined using a multivariate mixed effects logistic regression model. Independent risk factors were selected using a priori knowledge from literature review. An adjusted mixed effects logistic regression model was then used to determine the effect of up to 21-, 21-32-, 33-44- and 45-month and higher birth-to-birth intervals on pregnancy outcomes controlling for confounders selected through a directed acyclic graph.
Results:
We analysed 5,571 second or higher order deliveries. Average birth interval was 55 months and 1368/5571 women (24.6%) had a short birth interval (<33 months). Younger women (AOR 1.11 95% CI 1.08-1.15 per year increase in age), women who started their reproductive life later (AOR 0.95, 0.92-0.98 per year) and those who achieve higher order parities were less likely to experience short birth intervals (AOR 0.28, 0.19-0.41 parity 4 compared to 1). Women who were socioeconomically disadvantaged were more likely to experience a short birth interval (AOR 1.42, 1.22-1.65) and a previous adverse outcome was an important determinant of interval (AOR 2.10, 1.83-2.40). Very short birth intervals of less than 21 months were associated with increased stillbirth rate (AOR 2.13, 95% CI 1.28-3.53) and neonatal mortality (AOR 2.28 95% CI 1.28-4.05).
Conclusions:
Birth spacing remains a reproductive health problem in Bangladesh. Disadvantaged women are more likely to experience short birth intervals and to have increased perinatal deaths. Research into causal pathways and strategies to improve spacing between pregnancies should be intensified.
| 8,718 | 433 | 12 |
[
"birth",
"interval",
"birth interval",
"pregnancy",
"short",
"short birth",
"women",
"months",
"age",
"adverse"
] |
[
"test",
"test"
] | null |
[CONTENT] Birth intervals | Bangladesh | Perinatal mortality | Socioeconomic factors | Population surveillance [SUMMARY]
| null |
[CONTENT] Birth intervals | Bangladesh | Perinatal mortality | Socioeconomic factors | Population surveillance [SUMMARY]
|
[CONTENT] Birth intervals | Bangladesh | Perinatal mortality | Socioeconomic factors | Population surveillance [SUMMARY]
|
[CONTENT] Birth intervals | Bangladesh | Perinatal mortality | Socioeconomic factors | Population surveillance [SUMMARY]
|
[CONTENT] Birth intervals | Bangladesh | Perinatal mortality | Socioeconomic factors | Population surveillance [SUMMARY]
|
[CONTENT] Adolescent | Adult | Age Factors | Bangladesh | Birth Intervals | Cross-Sectional Studies | Educational Status | Female | Humans | Infant | Infant, Newborn | Parity | Perinatal Mortality | Population Surveillance | Pregnancy | Religion | Reproductive Behavior | Residence Characteristics | Rural Population | Stillbirth | Young Adult [SUMMARY]
| null |
[CONTENT] Adolescent | Adult | Age Factors | Bangladesh | Birth Intervals | Cross-Sectional Studies | Educational Status | Female | Humans | Infant | Infant, Newborn | Parity | Perinatal Mortality | Population Surveillance | Pregnancy | Religion | Reproductive Behavior | Residence Characteristics | Rural Population | Stillbirth | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Age Factors | Bangladesh | Birth Intervals | Cross-Sectional Studies | Educational Status | Female | Humans | Infant | Infant, Newborn | Parity | Perinatal Mortality | Population Surveillance | Pregnancy | Religion | Reproductive Behavior | Residence Characteristics | Rural Population | Stillbirth | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Age Factors | Bangladesh | Birth Intervals | Cross-Sectional Studies | Educational Status | Female | Humans | Infant | Infant, Newborn | Parity | Perinatal Mortality | Population Surveillance | Pregnancy | Religion | Reproductive Behavior | Residence Characteristics | Rural Population | Stillbirth | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Age Factors | Bangladesh | Birth Intervals | Cross-Sectional Studies | Educational Status | Female | Humans | Infant | Infant, Newborn | Parity | Perinatal Mortality | Population Surveillance | Pregnancy | Religion | Reproductive Behavior | Residence Characteristics | Rural Population | Stillbirth | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] birth | interval | birth interval | pregnancy | short | short birth | women | months | age | adverse [SUMMARY]
| null |
[CONTENT] birth | interval | birth interval | pregnancy | short | short birth | women | months | age | adverse [SUMMARY]
|
[CONTENT] birth | interval | birth interval | pregnancy | short | short birth | women | months | age | adverse [SUMMARY]
|
[CONTENT] birth | interval | birth interval | pregnancy | short | short birth | women | months | age | adverse [SUMMARY]
|
[CONTENT] birth | interval | birth interval | pregnancy | short | short birth | women | months | age | adverse [SUMMARY]
|
[CONTENT] birth | months | intervals | interval | bangladesh | birth interval | previous sibling | median | sibling | short [SUMMARY]
| null |
[CONTENT] reference | interval | birth | aor | birth interval | reference reference | 95 | 33 | short | ci [SUMMARY]
|
[CONTENT] spacing | birth spacing | birth | urgent attention bangladesh | birth intervals suffer | birth intervals suffer consequences | pregnancies whilst | pregnancies whilst efforts | pregnancies whilst efforts prevent | strategies improve spacing pregnancies [SUMMARY]
|
[CONTENT] birth | interval | birth interval | pregnancy | short | short birth | women | months | reference | short birth interval [SUMMARY]
|
[CONTENT] birth | interval | birth interval | pregnancy | short | short birth | women | months | reference | short birth interval [SUMMARY]
|
[CONTENT] ||| Bangladesh [SUMMARY]
| null |
[CONTENT] 5,571 second ||| 55 months | 1368/5571 | 24.6% | months ||| 1.11 | 95% | CI | 1.08 | 0.92 | 0.19 | 4 | 1 ||| 1.42 | 1.22-1.65 | 2.10 | 1.83 ||| 2.13 | 95% | CI | 1.28-3.53 | 2.28 95% | CI | 1.28-4.05 [SUMMARY]
|
[CONTENT] Bangladesh ||| ||| [SUMMARY]
|
[CONTENT] ||| Bangladesh ||| three | Bangladesh - Bogra | Moulavibazar | Faridpur | 282,643 | 54,668 ||| January 2010 and June 2011 ||| less than 33 months ||| ||| ||| up to 21- | 21 | 33-44- | 45-month ||| ||| 5,571 second ||| 55 months | 1368/5571 | 24.6% | months ||| 1.11 | 95% | CI | 1.08 | 0.92 | 0.19 | 4 | 1 ||| 1.42 | 1.22-1.65 | 2.10 | 1.83 ||| 2.13 | 95% | CI | 1.28-3.53 | 2.28 95% | CI | 1.28-4.05 ||| Bangladesh ||| ||| [SUMMARY]
|
[CONTENT] ||| Bangladesh ||| three | Bangladesh - Bogra | Moulavibazar | Faridpur | 282,643 | 54,668 ||| January 2010 and June 2011 ||| less than 33 months ||| ||| ||| up to 21- | 21 | 33-44- | 45-month ||| ||| 5,571 second ||| 55 months | 1368/5571 | 24.6% | months ||| 1.11 | 95% | CI | 1.08 | 0.92 | 0.19 | 4 | 1 ||| 1.42 | 1.22-1.65 | 2.10 | 1.83 ||| 2.13 | 95% | CI | 1.28-3.53 | 2.28 95% | CI | 1.28-4.05 ||| Bangladesh ||| ||| [SUMMARY]
|
|||||
Predictive Factors for Efficacy of AST-120 Treatment in Diabetic Nephropathy: a Prospective Single-Arm, Open-Label, Multi-Center Study.
|
31001934
|
Removal of uremic toxins such as indoxyl sulfate by AST-120 is known to improve renal function and delay the initiation of dialysis in patients with advanced chronic kidney disease. However, it is unclear whether the addition of AST-120 to conventional treatments is effective in delaying the progression of renal dysfunction in patients with diabetic nephropathy.
|
BACKGROUND
|
A total of 100 patients with type 2 diabetes and renal dysfunction (serum creatinine levels ranging from 1.5 to 3.0 mg/dL) were recruited from eight centers in Korea and treated with AST-120 (6 g/day) for 24 weeks. The primary endpoint was improvement in renal function measured as the gradient of the reciprocal serum creatinine level (1/sCr) over time (i.e., the ratio of 1/sCr time slope for post- to pre-AST-120 therapy). A response was defined as a ratio change of the regression coefficient of 1/sCr ≤ 0.90.
|
METHODS
|
Renal function improved in 80.3% of patients (61/76) after 24 weeks of AST-120 treatment. There were no differences between responder and non-responder groups in baseline characteristics except for diastolic blood pressure (73.5 ± 9.5 mmHg in the responder group vs. 79.3 ± 11.1 mmHg in the non-responder group; P = 0.046). Serum lipid peroxidation level decreased significantly in the responder group (from 2.25 ± 0.56 μol/L to 1.91 ± 0.72 μol/L; P = 0.002) but not in the non-responder group.
|
RESULTS
|
The addition of AST-120 to conventional treatments may delay the progression of renal dysfunction in diabetic nephropathy. The antioxidant effect of AST-120 might contribute to improvement in renal function.
|
CONCLUSION
|
[
"Aged",
"Blood Pressure",
"Carbon",
"Creatinine",
"Diabetes Mellitus, Type 2",
"Diabetic Nephropathies",
"Dose-Response Relationship, Drug",
"Drug Administration Schedule",
"Female",
"Humans",
"Lipid Peroxidation",
"Male",
"Middle Aged",
"Oxides",
"Prospective Studies",
"Protective Agents",
"Severity of Illness Index",
"Treatment Outcome"
] |
6473095
|
INTRODUCTION
|
Diabetic nephropathy is one of the most common causes of end-stage renal disease (ESRD) worldwide.123 As the prevalence of diabetes has continued to increase, a parallel increase has been seen in the prevalence of dialysis patients and associated costs. This increase in the number of patients with chronic kidney disease (CKD) has led to social and economic problems in many countries.1 Moreover, the clinical course of diabetic nephropathy is usually characterized by relatively rapid progression compared with CKD due to other causes.4 In addition, as diabetic ESRD patients are more prone to cardiovascular morbidity and mortality than other ESRD patients, early identification of diabetic nephropathy and prompt renoprotective treatment are critical for the prevention of end organ damage.5 Previous studies have shown that intensive control of blood glucose and blood pressure are not sufficient to prevent or delay the development of diabetic nephropathy.56 However, effective therapy is not available yet, and integrated treatment strategies are therefore needed to improve the management of diabetic nephropathy.56
The so-called ‘uremic toxins,’ such as indoxyl sulfate, are biologically active substances involved in the progression of renal failure.7 Both clinical and experimental studies have suggested that indoxyl sulfate plays an important role in the progressive loss of intact nephrons through induction of an inflammatory reaction and enhanced expression of profibrotic cytokines.7 AST-120 (Kremezin®), a spherical adsorptive carbon preparation, absorbs and removes uremic toxins such as indoxyl sulfate in the digestive tract.8 To date, the renoprotective effects of AST-120 have been determined mostly in patients with advanced CKD with inconsistent results.91011 Furthermore, little is known about the efficacy of AST-120 and which clinical and biochemical variables predict response to AST-120 treatment in diabetic nephropathy.
We therefore aimed to determine whether the addition of AST-120 to conventional treatments is effective to attenuate the progression of renal dysfunction in patients with type 2 diabetes and CKD. We also examined which clinical variables and biochemical tests, particularly oxidative stress markers, could predict beneficial effects of AST-120 in diabetic nephropathy.
|
METHODS
|
Study subjects This study was a prospective, single-arm, open-label, multi-center study performed in subjects with overt diabetic nephropathy. Outpatients with type 2 diabetes and renal dysfunction who had not previously undergone dialysis from eight hospitals in Korea were prospectively enrolled in this study from 2008 to 2009. Study subjects had serum creatinine (sCr) levels ranging from 1.5 to 3.0 mg/dL measured at least three time points over a period of 12 months prior to treatment. We excluded any patients with active liver disease, acute infection, and those who had recent (< 6 months) acute coronary syndromes and cerebrovascular disease. After confirming a negative slope of the reciprocal serum creatinine level (1/sCr) time plot, oral administration of AST-120 6 grams per day was started and continued for 24 weeks.
This study was a prospective, single-arm, open-label, multi-center study performed in subjects with overt diabetic nephropathy. Outpatients with type 2 diabetes and renal dysfunction who had not previously undergone dialysis from eight hospitals in Korea were prospectively enrolled in this study from 2008 to 2009. Study subjects had serum creatinine (sCr) levels ranging from 1.5 to 3.0 mg/dL measured at least three time points over a period of 12 months prior to treatment. We excluded any patients with active liver disease, acute infection, and those who had recent (< 6 months) acute coronary syndromes and cerebrovascular disease. After confirming a negative slope of the reciprocal serum creatinine level (1/sCr) time plot, oral administration of AST-120 6 grams per day was started and continued for 24 weeks.
Clinical and laboratory examination Blood pressure was measured with the participant in a seated position after 5 minutes of quiet rest. Blood samples were collected after overnight fasting. Blood urea nitrogen, creatinine, glucose, and lipid profiles were measured. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance: [fasting insulin (μIU/mL) × fasting glucose (mmol/L)]/22.5.12 sCr was measured by the standard enzymatic method. Creatinine clearance (CrCl) was estimated by the Cockcroft-Gault equation; CrCl = [(140 − age) × weight (kg)]/sCr × 72 (× 0.85, if women).13 Glomerular filtration rate (GFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation.14 Diabetic retinopathy was diagnosed by an experienced ophthalmologist or diabetologist based on retinal images.15 Superoxide dismutase (SOD) activity in serum was determined with the Superoxide Dismutase Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), which utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Serum lipid peroxidation (LPO) was determined by a colorimetric assay using the Bioxytech LPO-586 kit (OxisResearch, Portland, OR, USA). Urinary excretion levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured with an enzyme-linked immunosorbent assay (8-Hydroxydeoxyguanosine Check; Japan Institute for the Control of Aging, Shizuoka, Japan).
The rate of progression of renal dysfunction was assessed as the gradient of the reciprocal serum creatinine level (1/sCr), as described by Mitch et al.,16 before and after the initiation of treatment. The 1/sCr was plotted against time, and the regression coefficient was determined by least-square linear regression analysis as previously described.17 In our study, the primary endpoint was improvement in renal function as the ratio of the 1/sCr-time slope for post- to pre- AST-120 therapy. Based on the ratio changes of the regression coefficients of 1/sCr before and after treatment, efficacy of this drug was defined as follows: 1) definitely improved: ≤ 0.30; 2) moderately improved: 0.31–0.60; 3) slightly improved: 0.61–0.90; 4) unchanged: 0.91–1.09; and 5) deteriorated: ≥ 1.10. Subjects in the definitely, moderately, or slightly improved groups (ratio ≤ 0.9) were classified as responders and those in the unchanged and deteriorated groups were considered non-responders. To determine whether baseline sCr levels affect the efficacy of AST-120 treatment, patients were divided into three groups according to the baseline sCr tertile values. The secondary endpoints were changes in 1) CrCl; 2) 1/sCr-time slope pre- and post- AST-120 treatment; and 3) oxidative stress markers. Changes in sCr, serum urea nitrogen, hematocrit, glucose, lipid profile, and systolic and diastolic blood pressure after AST-120 therapy were also compared.
Blood pressure was measured with the participant in a seated position after 5 minutes of quiet rest. Blood samples were collected after overnight fasting. Blood urea nitrogen, creatinine, glucose, and lipid profiles were measured. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance: [fasting insulin (μIU/mL) × fasting glucose (mmol/L)]/22.5.12 sCr was measured by the standard enzymatic method. Creatinine clearance (CrCl) was estimated by the Cockcroft-Gault equation; CrCl = [(140 − age) × weight (kg)]/sCr × 72 (× 0.85, if women).13 Glomerular filtration rate (GFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation.14 Diabetic retinopathy was diagnosed by an experienced ophthalmologist or diabetologist based on retinal images.15 Superoxide dismutase (SOD) activity in serum was determined with the Superoxide Dismutase Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), which utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Serum lipid peroxidation (LPO) was determined by a colorimetric assay using the Bioxytech LPO-586 kit (OxisResearch, Portland, OR, USA). Urinary excretion levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured with an enzyme-linked immunosorbent assay (8-Hydroxydeoxyguanosine Check; Japan Institute for the Control of Aging, Shizuoka, Japan).
The rate of progression of renal dysfunction was assessed as the gradient of the reciprocal serum creatinine level (1/sCr), as described by Mitch et al.,16 before and after the initiation of treatment. The 1/sCr was plotted against time, and the regression coefficient was determined by least-square linear regression analysis as previously described.17 In our study, the primary endpoint was improvement in renal function as the ratio of the 1/sCr-time slope for post- to pre- AST-120 therapy. Based on the ratio changes of the regression coefficients of 1/sCr before and after treatment, efficacy of this drug was defined as follows: 1) definitely improved: ≤ 0.30; 2) moderately improved: 0.31–0.60; 3) slightly improved: 0.61–0.90; 4) unchanged: 0.91–1.09; and 5) deteriorated: ≥ 1.10. Subjects in the definitely, moderately, or slightly improved groups (ratio ≤ 0.9) were classified as responders and those in the unchanged and deteriorated groups were considered non-responders. To determine whether baseline sCr levels affect the efficacy of AST-120 treatment, patients were divided into three groups according to the baseline sCr tertile values. The secondary endpoints were changes in 1) CrCl; 2) 1/sCr-time slope pre- and post- AST-120 treatment; and 3) oxidative stress markers. Changes in sCr, serum urea nitrogen, hematocrit, glucose, lipid profile, and systolic and diastolic blood pressure after AST-120 therapy were also compared.
Statistical analysis Data are expressed as means ± standard deviations. The χ2 test, Fisher's exact test, and Mann-Whitney test were used to compare the characteristics of study subjects. Parameters before and 24 weeks after AST-120 were compared by paired t-test. The clinical per-protocol (PP) population was defined as all patients taking the study medication for 24 weeks with a compliance rate of ≥ 70%, and 76 patients were enrolled for PP analysis. The primary end point was analyzed based on the PP population to better report additional efficacy.18 Other secondary endpoints were analyzed based on the intention-to-treat population, which consisted of all patients taking at least one dose of study medication. Missing data were input using the last-observation-carried-forward procedure. To assess the utility of the predictive factor for efficacy of AST-120, we conducted receiver operating characteristics (ROC) curves and calculated the areas under the curve (AUC). All statistical analyses were performed using PASW version 18.0 (SPSS, Chicago, IL, USA). A P value < 0.05 was considered significant.
Data are expressed as means ± standard deviations. The χ2 test, Fisher's exact test, and Mann-Whitney test were used to compare the characteristics of study subjects. Parameters before and 24 weeks after AST-120 were compared by paired t-test. The clinical per-protocol (PP) population was defined as all patients taking the study medication for 24 weeks with a compliance rate of ≥ 70%, and 76 patients were enrolled for PP analysis. The primary end point was analyzed based on the PP population to better report additional efficacy.18 Other secondary endpoints were analyzed based on the intention-to-treat population, which consisted of all patients taking at least one dose of study medication. Missing data were input using the last-observation-carried-forward procedure. To assess the utility of the predictive factor for efficacy of AST-120, we conducted receiver operating characteristics (ROC) curves and calculated the areas under the curve (AUC). All statistical analyses were performed using PASW version 18.0 (SPSS, Chicago, IL, USA). A P value < 0.05 was considered significant.
Ethics statement The investigators explained the purpose of the study to the patients at the time of study entry and written informed consent was obtained from all patients. The study was approved by the Institutional Review Board (IRB) of each participating center and was conducted in accordance with the Declaration of Helsinki (Samsung Medical Center IRB No. 2008-02-015).
The investigators explained the purpose of the study to the patients at the time of study entry and written informed consent was obtained from all patients. The study was approved by the Institutional Review Board (IRB) of each participating center and was conducted in accordance with the Declaration of Helsinki (Samsung Medical Center IRB No. 2008-02-015).
|
RESULTS
|
Table 1 shows the baseline characteristics of the patients. The mean age was 62.6 years and 77% of study participants were men. Eighty-nine percent of patients were taking an angiotensin-I-converting enzyme (ACE) inhibitor and/or angiotensin II receptor blocker, and 83% were taking a statin. The mean estimated GFR was 34.7 mL/min/1.73 m2 and 17, 51, 32 subjects were CKD stage 3a, 3b, and 4, respectively. We analyzed 76 subjects for the primary endpoint and 100 subjects for the secondary endpoints.
Data are expressed as means ± standard deviation or percentages.
ACEi = angiotensin-I-converting enzyme inhibitor, ARB = angiotensin-II-receptor blocker, HbA1c = hemoglobin A1c, HOMA-IR = homeostasis model assessment of insulin resistance, HDL = high-density lipoprotein, LDL = low-density lipoprotein, CrCl = creatinine clearance, GFR = glomerular filtration rate.
Of 76 patients (PP population) analyzed for the primary endpoint, 50 were definitely improved, 2 were moderately improved, and 9 were slightly improved responders after 24 weeks of treatment with AST-120. Among non-responders, 4 were unchanged and 11 were deteriorated based on the criteria for changes in sCr levels described above. As a result, 61 patients (80.3%) were categorized as responders and 15 patients (19.7%) were non-responders to the AST-120 therapy (Table 2).
aRatio change of regression coefficients of 1/sCr before and after AST-120 treatment.
Next, we determined baseline characteristics of the responder and non-responder groups. There were no differences in age, gender, previous treatment modalities, baseline sCr or CrCl, lipid profile, and blood glucose levels between the two groups; however, diastolic blood pressure was significantly different (Table 3). The non-responder group had a higher diastolic blood pressure than the responder group (79.3 ± 11.1 mmHg vs. 73.5 ± 9.5 mmHg, respectively; P = 0.046). We also analyzed the efficacy of 24-week AST-120 treatment according to baseline sCr tertile. All three groups demonstrated a significant improvement in renal function determined by the ratio changes of regression coefficients of 1/sCr after AST-120 treatment. In particular, individuals with lower baseline sCr (lower and middle tertiles) were more likely to show improved renal function after AST-120 treatment compared to individuals with higher baseline sCr (upper tertile) (P = 0.024) (Table 4).
Data are expressed as means ± standard deviation or number (percentages).
ACEi = angiotensin-I-converting enzyme inhibitor, ARB = angiotensin-II-receptor blocker, HbA1c = hemoglobin A1c, CrCl = creatinine clearance, GFR = glomerular filtration rate, NS = not significant.
P value for the comparison between responders and non-responders across the tertiles.
sCr = serum creatinine.
aRatio changes of regression coefficients of 1/sCr before and after AST-120 treatment.
Table 5 shows clinical and laboratory findings before and after AST-120 treatment in the responder and non-responder groups. Administration of AST-120 significantly increased serum SOD level in both the responder and non-responder groups. In addition, serum LPO level was significantly decreased in the responder group (from 2.25 ± 0.56 μmol/L to 1.91 ± 0.72; P = 0.002), but not in the non-responder group. Urine 8-OHdG levels showed no significant changes in either the responder or non-responder groups. AST-120 treatment had no significant effect on the other clinical or biochemical parameters tested including blood urea nitrogen, hematocrit, glucose, and lipid profile in either the responder or non-responder groups. The renal function in all patients was maintained and no patients started dialysis therapy.
Data are expressed as means ± standard deviation.
CrCl = creatinine clearance, HbA1c = hemoglobin A1c, LDL = low-density lipoprotein, HDL = high-density lipoprotein, SOD = superoxide dismutase, LPO = lipid peroxidation, 8-OHdG = 8-hydroxydeoxyguanosine, NS = not significant.
| null | null |
[
"Study subjects",
"Clinical and laboratory examination",
"Statistical analysis",
"Ethics statement"
] |
[
"This study was a prospective, single-arm, open-label, multi-center study performed in subjects with overt diabetic nephropathy. Outpatients with type 2 diabetes and renal dysfunction who had not previously undergone dialysis from eight hospitals in Korea were prospectively enrolled in this study from 2008 to 2009. Study subjects had serum creatinine (sCr) levels ranging from 1.5 to 3.0 mg/dL measured at least three time points over a period of 12 months prior to treatment. We excluded any patients with active liver disease, acute infection, and those who had recent (< 6 months) acute coronary syndromes and cerebrovascular disease. After confirming a negative slope of the reciprocal serum creatinine level (1/sCr) time plot, oral administration of AST-120 6 grams per day was started and continued for 24 weeks.",
"Blood pressure was measured with the participant in a seated position after 5 minutes of quiet rest. Blood samples were collected after overnight fasting. Blood urea nitrogen, creatinine, glucose, and lipid profiles were measured. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance: [fasting insulin (μIU/mL) × fasting glucose (mmol/L)]/22.5.12 sCr was measured by the standard enzymatic method. Creatinine clearance (CrCl) was estimated by the Cockcroft-Gault equation; CrCl = [(140 − age) × weight (kg)]/sCr × 72 (× 0.85, if women).13 Glomerular filtration rate (GFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation.14 Diabetic retinopathy was diagnosed by an experienced ophthalmologist or diabetologist based on retinal images.15 Superoxide dismutase (SOD) activity in serum was determined with the Superoxide Dismutase Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), which utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Serum lipid peroxidation (LPO) was determined by a colorimetric assay using the Bioxytech LPO-586 kit (OxisResearch, Portland, OR, USA). Urinary excretion levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured with an enzyme-linked immunosorbent assay (8-Hydroxydeoxyguanosine Check; Japan Institute for the Control of Aging, Shizuoka, Japan).\nThe rate of progression of renal dysfunction was assessed as the gradient of the reciprocal serum creatinine level (1/sCr), as described by Mitch et al.,16 before and after the initiation of treatment. The 1/sCr was plotted against time, and the regression coefficient was determined by least-square linear regression analysis as previously described.17 In our study, the primary endpoint was improvement in renal function as the ratio of the 1/sCr-time slope for post- to pre- AST-120 therapy. Based on the ratio changes of the regression coefficients of 1/sCr before and after treatment, efficacy of this drug was defined as follows: 1) definitely improved: ≤ 0.30; 2) moderately improved: 0.31–0.60; 3) slightly improved: 0.61–0.90; 4) unchanged: 0.91–1.09; and 5) deteriorated: ≥ 1.10. Subjects in the definitely, moderately, or slightly improved groups (ratio ≤ 0.9) were classified as responders and those in the unchanged and deteriorated groups were considered non-responders. To determine whether baseline sCr levels affect the efficacy of AST-120 treatment, patients were divided into three groups according to the baseline sCr tertile values. The secondary endpoints were changes in 1) CrCl; 2) 1/sCr-time slope pre- and post- AST-120 treatment; and 3) oxidative stress markers. Changes in sCr, serum urea nitrogen, hematocrit, glucose, lipid profile, and systolic and diastolic blood pressure after AST-120 therapy were also compared.",
"Data are expressed as means ± standard deviations. The χ2 test, Fisher's exact test, and Mann-Whitney test were used to compare the characteristics of study subjects. Parameters before and 24 weeks after AST-120 were compared by paired t-test. The clinical per-protocol (PP) population was defined as all patients taking the study medication for 24 weeks with a compliance rate of ≥ 70%, and 76 patients were enrolled for PP analysis. The primary end point was analyzed based on the PP population to better report additional efficacy.18 Other secondary endpoints were analyzed based on the intention-to-treat population, which consisted of all patients taking at least one dose of study medication. Missing data were input using the last-observation-carried-forward procedure. To assess the utility of the predictive factor for efficacy of AST-120, we conducted receiver operating characteristics (ROC) curves and calculated the areas under the curve (AUC). All statistical analyses were performed using PASW version 18.0 (SPSS, Chicago, IL, USA). A P value < 0.05 was considered significant.",
"The investigators explained the purpose of the study to the patients at the time of study entry and written informed consent was obtained from all patients. The study was approved by the Institutional Review Board (IRB) of each participating center and was conducted in accordance with the Declaration of Helsinki (Samsung Medical Center IRB No. 2008-02-015)."
] |
[
null,
null,
null,
null
] |
[
"INTRODUCTION",
"METHODS",
"Study subjects",
"Clinical and laboratory examination",
"Statistical analysis",
"Ethics statement",
"RESULTS",
"DISCUSSION"
] |
[
"Diabetic nephropathy is one of the most common causes of end-stage renal disease (ESRD) worldwide.123 As the prevalence of diabetes has continued to increase, a parallel increase has been seen in the prevalence of dialysis patients and associated costs. This increase in the number of patients with chronic kidney disease (CKD) has led to social and economic problems in many countries.1 Moreover, the clinical course of diabetic nephropathy is usually characterized by relatively rapid progression compared with CKD due to other causes.4 In addition, as diabetic ESRD patients are more prone to cardiovascular morbidity and mortality than other ESRD patients, early identification of diabetic nephropathy and prompt renoprotective treatment are critical for the prevention of end organ damage.5 Previous studies have shown that intensive control of blood glucose and blood pressure are not sufficient to prevent or delay the development of diabetic nephropathy.56 However, effective therapy is not available yet, and integrated treatment strategies are therefore needed to improve the management of diabetic nephropathy.56\nThe so-called ‘uremic toxins,’ such as indoxyl sulfate, are biologically active substances involved in the progression of renal failure.7 Both clinical and experimental studies have suggested that indoxyl sulfate plays an important role in the progressive loss of intact nephrons through induction of an inflammatory reaction and enhanced expression of profibrotic cytokines.7 AST-120 (Kremezin®), a spherical adsorptive carbon preparation, absorbs and removes uremic toxins such as indoxyl sulfate in the digestive tract.8 To date, the renoprotective effects of AST-120 have been determined mostly in patients with advanced CKD with inconsistent results.91011 Furthermore, little is known about the efficacy of AST-120 and which clinical and biochemical variables predict response to AST-120 treatment in diabetic nephropathy.\nWe therefore aimed to determine whether the addition of AST-120 to conventional treatments is effective to attenuate the progression of renal dysfunction in patients with type 2 diabetes and CKD. We also examined which clinical variables and biochemical tests, particularly oxidative stress markers, could predict beneficial effects of AST-120 in diabetic nephropathy.",
" Study subjects This study was a prospective, single-arm, open-label, multi-center study performed in subjects with overt diabetic nephropathy. Outpatients with type 2 diabetes and renal dysfunction who had not previously undergone dialysis from eight hospitals in Korea were prospectively enrolled in this study from 2008 to 2009. Study subjects had serum creatinine (sCr) levels ranging from 1.5 to 3.0 mg/dL measured at least three time points over a period of 12 months prior to treatment. We excluded any patients with active liver disease, acute infection, and those who had recent (< 6 months) acute coronary syndromes and cerebrovascular disease. After confirming a negative slope of the reciprocal serum creatinine level (1/sCr) time plot, oral administration of AST-120 6 grams per day was started and continued for 24 weeks.\nThis study was a prospective, single-arm, open-label, multi-center study performed in subjects with overt diabetic nephropathy. Outpatients with type 2 diabetes and renal dysfunction who had not previously undergone dialysis from eight hospitals in Korea were prospectively enrolled in this study from 2008 to 2009. Study subjects had serum creatinine (sCr) levels ranging from 1.5 to 3.0 mg/dL measured at least three time points over a period of 12 months prior to treatment. We excluded any patients with active liver disease, acute infection, and those who had recent (< 6 months) acute coronary syndromes and cerebrovascular disease. After confirming a negative slope of the reciprocal serum creatinine level (1/sCr) time plot, oral administration of AST-120 6 grams per day was started and continued for 24 weeks.\n Clinical and laboratory examination Blood pressure was measured with the participant in a seated position after 5 minutes of quiet rest. Blood samples were collected after overnight fasting. Blood urea nitrogen, creatinine, glucose, and lipid profiles were measured. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance: [fasting insulin (μIU/mL) × fasting glucose (mmol/L)]/22.5.12 sCr was measured by the standard enzymatic method. Creatinine clearance (CrCl) was estimated by the Cockcroft-Gault equation; CrCl = [(140 − age) × weight (kg)]/sCr × 72 (× 0.85, if women).13 Glomerular filtration rate (GFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation.14 Diabetic retinopathy was diagnosed by an experienced ophthalmologist or diabetologist based on retinal images.15 Superoxide dismutase (SOD) activity in serum was determined with the Superoxide Dismutase Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), which utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Serum lipid peroxidation (LPO) was determined by a colorimetric assay using the Bioxytech LPO-586 kit (OxisResearch, Portland, OR, USA). Urinary excretion levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured with an enzyme-linked immunosorbent assay (8-Hydroxydeoxyguanosine Check; Japan Institute for the Control of Aging, Shizuoka, Japan).\nThe rate of progression of renal dysfunction was assessed as the gradient of the reciprocal serum creatinine level (1/sCr), as described by Mitch et al.,16 before and after the initiation of treatment. The 1/sCr was plotted against time, and the regression coefficient was determined by least-square linear regression analysis as previously described.17 In our study, the primary endpoint was improvement in renal function as the ratio of the 1/sCr-time slope for post- to pre- AST-120 therapy. Based on the ratio changes of the regression coefficients of 1/sCr before and after treatment, efficacy of this drug was defined as follows: 1) definitely improved: ≤ 0.30; 2) moderately improved: 0.31–0.60; 3) slightly improved: 0.61–0.90; 4) unchanged: 0.91–1.09; and 5) deteriorated: ≥ 1.10. Subjects in the definitely, moderately, or slightly improved groups (ratio ≤ 0.9) were classified as responders and those in the unchanged and deteriorated groups were considered non-responders. To determine whether baseline sCr levels affect the efficacy of AST-120 treatment, patients were divided into three groups according to the baseline sCr tertile values. The secondary endpoints were changes in 1) CrCl; 2) 1/sCr-time slope pre- and post- AST-120 treatment; and 3) oxidative stress markers. Changes in sCr, serum urea nitrogen, hematocrit, glucose, lipid profile, and systolic and diastolic blood pressure after AST-120 therapy were also compared.\nBlood pressure was measured with the participant in a seated position after 5 minutes of quiet rest. Blood samples were collected after overnight fasting. Blood urea nitrogen, creatinine, glucose, and lipid profiles were measured. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance: [fasting insulin (μIU/mL) × fasting glucose (mmol/L)]/22.5.12 sCr was measured by the standard enzymatic method. Creatinine clearance (CrCl) was estimated by the Cockcroft-Gault equation; CrCl = [(140 − age) × weight (kg)]/sCr × 72 (× 0.85, if women).13 Glomerular filtration rate (GFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation.14 Diabetic retinopathy was diagnosed by an experienced ophthalmologist or diabetologist based on retinal images.15 Superoxide dismutase (SOD) activity in serum was determined with the Superoxide Dismutase Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), which utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Serum lipid peroxidation (LPO) was determined by a colorimetric assay using the Bioxytech LPO-586 kit (OxisResearch, Portland, OR, USA). Urinary excretion levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured with an enzyme-linked immunosorbent assay (8-Hydroxydeoxyguanosine Check; Japan Institute for the Control of Aging, Shizuoka, Japan).\nThe rate of progression of renal dysfunction was assessed as the gradient of the reciprocal serum creatinine level (1/sCr), as described by Mitch et al.,16 before and after the initiation of treatment. The 1/sCr was plotted against time, and the regression coefficient was determined by least-square linear regression analysis as previously described.17 In our study, the primary endpoint was improvement in renal function as the ratio of the 1/sCr-time slope for post- to pre- AST-120 therapy. Based on the ratio changes of the regression coefficients of 1/sCr before and after treatment, efficacy of this drug was defined as follows: 1) definitely improved: ≤ 0.30; 2) moderately improved: 0.31–0.60; 3) slightly improved: 0.61–0.90; 4) unchanged: 0.91–1.09; and 5) deteriorated: ≥ 1.10. Subjects in the definitely, moderately, or slightly improved groups (ratio ≤ 0.9) were classified as responders and those in the unchanged and deteriorated groups were considered non-responders. To determine whether baseline sCr levels affect the efficacy of AST-120 treatment, patients were divided into three groups according to the baseline sCr tertile values. The secondary endpoints were changes in 1) CrCl; 2) 1/sCr-time slope pre- and post- AST-120 treatment; and 3) oxidative stress markers. Changes in sCr, serum urea nitrogen, hematocrit, glucose, lipid profile, and systolic and diastolic blood pressure after AST-120 therapy were also compared.\n Statistical analysis Data are expressed as means ± standard deviations. The χ2 test, Fisher's exact test, and Mann-Whitney test were used to compare the characteristics of study subjects. Parameters before and 24 weeks after AST-120 were compared by paired t-test. The clinical per-protocol (PP) population was defined as all patients taking the study medication for 24 weeks with a compliance rate of ≥ 70%, and 76 patients were enrolled for PP analysis. The primary end point was analyzed based on the PP population to better report additional efficacy.18 Other secondary endpoints were analyzed based on the intention-to-treat population, which consisted of all patients taking at least one dose of study medication. Missing data were input using the last-observation-carried-forward procedure. To assess the utility of the predictive factor for efficacy of AST-120, we conducted receiver operating characteristics (ROC) curves and calculated the areas under the curve (AUC). All statistical analyses were performed using PASW version 18.0 (SPSS, Chicago, IL, USA). A P value < 0.05 was considered significant.\nData are expressed as means ± standard deviations. The χ2 test, Fisher's exact test, and Mann-Whitney test were used to compare the characteristics of study subjects. Parameters before and 24 weeks after AST-120 were compared by paired t-test. The clinical per-protocol (PP) population was defined as all patients taking the study medication for 24 weeks with a compliance rate of ≥ 70%, and 76 patients were enrolled for PP analysis. The primary end point was analyzed based on the PP population to better report additional efficacy.18 Other secondary endpoints were analyzed based on the intention-to-treat population, which consisted of all patients taking at least one dose of study medication. Missing data were input using the last-observation-carried-forward procedure. To assess the utility of the predictive factor for efficacy of AST-120, we conducted receiver operating characteristics (ROC) curves and calculated the areas under the curve (AUC). All statistical analyses were performed using PASW version 18.0 (SPSS, Chicago, IL, USA). A P value < 0.05 was considered significant.\n Ethics statement The investigators explained the purpose of the study to the patients at the time of study entry and written informed consent was obtained from all patients. The study was approved by the Institutional Review Board (IRB) of each participating center and was conducted in accordance with the Declaration of Helsinki (Samsung Medical Center IRB No. 2008-02-015).\nThe investigators explained the purpose of the study to the patients at the time of study entry and written informed consent was obtained from all patients. The study was approved by the Institutional Review Board (IRB) of each participating center and was conducted in accordance with the Declaration of Helsinki (Samsung Medical Center IRB No. 2008-02-015).",
"This study was a prospective, single-arm, open-label, multi-center study performed in subjects with overt diabetic nephropathy. Outpatients with type 2 diabetes and renal dysfunction who had not previously undergone dialysis from eight hospitals in Korea were prospectively enrolled in this study from 2008 to 2009. Study subjects had serum creatinine (sCr) levels ranging from 1.5 to 3.0 mg/dL measured at least three time points over a period of 12 months prior to treatment. We excluded any patients with active liver disease, acute infection, and those who had recent (< 6 months) acute coronary syndromes and cerebrovascular disease. After confirming a negative slope of the reciprocal serum creatinine level (1/sCr) time plot, oral administration of AST-120 6 grams per day was started and continued for 24 weeks.",
"Blood pressure was measured with the participant in a seated position after 5 minutes of quiet rest. Blood samples were collected after overnight fasting. Blood urea nitrogen, creatinine, glucose, and lipid profiles were measured. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance: [fasting insulin (μIU/mL) × fasting glucose (mmol/L)]/22.5.12 sCr was measured by the standard enzymatic method. Creatinine clearance (CrCl) was estimated by the Cockcroft-Gault equation; CrCl = [(140 − age) × weight (kg)]/sCr × 72 (× 0.85, if women).13 Glomerular filtration rate (GFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation.14 Diabetic retinopathy was diagnosed by an experienced ophthalmologist or diabetologist based on retinal images.15 Superoxide dismutase (SOD) activity in serum was determined with the Superoxide Dismutase Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), which utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Serum lipid peroxidation (LPO) was determined by a colorimetric assay using the Bioxytech LPO-586 kit (OxisResearch, Portland, OR, USA). Urinary excretion levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured with an enzyme-linked immunosorbent assay (8-Hydroxydeoxyguanosine Check; Japan Institute for the Control of Aging, Shizuoka, Japan).\nThe rate of progression of renal dysfunction was assessed as the gradient of the reciprocal serum creatinine level (1/sCr), as described by Mitch et al.,16 before and after the initiation of treatment. The 1/sCr was plotted against time, and the regression coefficient was determined by least-square linear regression analysis as previously described.17 In our study, the primary endpoint was improvement in renal function as the ratio of the 1/sCr-time slope for post- to pre- AST-120 therapy. Based on the ratio changes of the regression coefficients of 1/sCr before and after treatment, efficacy of this drug was defined as follows: 1) definitely improved: ≤ 0.30; 2) moderately improved: 0.31–0.60; 3) slightly improved: 0.61–0.90; 4) unchanged: 0.91–1.09; and 5) deteriorated: ≥ 1.10. Subjects in the definitely, moderately, or slightly improved groups (ratio ≤ 0.9) were classified as responders and those in the unchanged and deteriorated groups were considered non-responders. To determine whether baseline sCr levels affect the efficacy of AST-120 treatment, patients were divided into three groups according to the baseline sCr tertile values. The secondary endpoints were changes in 1) CrCl; 2) 1/sCr-time slope pre- and post- AST-120 treatment; and 3) oxidative stress markers. Changes in sCr, serum urea nitrogen, hematocrit, glucose, lipid profile, and systolic and diastolic blood pressure after AST-120 therapy were also compared.",
"Data are expressed as means ± standard deviations. The χ2 test, Fisher's exact test, and Mann-Whitney test were used to compare the characteristics of study subjects. Parameters before and 24 weeks after AST-120 were compared by paired t-test. The clinical per-protocol (PP) population was defined as all patients taking the study medication for 24 weeks with a compliance rate of ≥ 70%, and 76 patients were enrolled for PP analysis. The primary end point was analyzed based on the PP population to better report additional efficacy.18 Other secondary endpoints were analyzed based on the intention-to-treat population, which consisted of all patients taking at least one dose of study medication. Missing data were input using the last-observation-carried-forward procedure. To assess the utility of the predictive factor for efficacy of AST-120, we conducted receiver operating characteristics (ROC) curves and calculated the areas under the curve (AUC). All statistical analyses were performed using PASW version 18.0 (SPSS, Chicago, IL, USA). A P value < 0.05 was considered significant.",
"The investigators explained the purpose of the study to the patients at the time of study entry and written informed consent was obtained from all patients. The study was approved by the Institutional Review Board (IRB) of each participating center and was conducted in accordance with the Declaration of Helsinki (Samsung Medical Center IRB No. 2008-02-015).",
"Table 1 shows the baseline characteristics of the patients. The mean age was 62.6 years and 77% of study participants were men. Eighty-nine percent of patients were taking an angiotensin-I-converting enzyme (ACE) inhibitor and/or angiotensin II receptor blocker, and 83% were taking a statin. The mean estimated GFR was 34.7 mL/min/1.73 m2 and 17, 51, 32 subjects were CKD stage 3a, 3b, and 4, respectively. We analyzed 76 subjects for the primary endpoint and 100 subjects for the secondary endpoints.\nData are expressed as means ± standard deviation or percentages.\nACEi = angiotensin-I-converting enzyme inhibitor, ARB = angiotensin-II-receptor blocker, HbA1c = hemoglobin A1c, HOMA-IR = homeostasis model assessment of insulin resistance, HDL = high-density lipoprotein, LDL = low-density lipoprotein, CrCl = creatinine clearance, GFR = glomerular filtration rate.\nOf 76 patients (PP population) analyzed for the primary endpoint, 50 were definitely improved, 2 were moderately improved, and 9 were slightly improved responders after 24 weeks of treatment with AST-120. Among non-responders, 4 were unchanged and 11 were deteriorated based on the criteria for changes in sCr levels described above. As a result, 61 patients (80.3%) were categorized as responders and 15 patients (19.7%) were non-responders to the AST-120 therapy (Table 2).\naRatio change of regression coefficients of 1/sCr before and after AST-120 treatment.\nNext, we determined baseline characteristics of the responder and non-responder groups. There were no differences in age, gender, previous treatment modalities, baseline sCr or CrCl, lipid profile, and blood glucose levels between the two groups; however, diastolic blood pressure was significantly different (Table 3). The non-responder group had a higher diastolic blood pressure than the responder group (79.3 ± 11.1 mmHg vs. 73.5 ± 9.5 mmHg, respectively; P = 0.046). We also analyzed the efficacy of 24-week AST-120 treatment according to baseline sCr tertile. All three groups demonstrated a significant improvement in renal function determined by the ratio changes of regression coefficients of 1/sCr after AST-120 treatment. In particular, individuals with lower baseline sCr (lower and middle tertiles) were more likely to show improved renal function after AST-120 treatment compared to individuals with higher baseline sCr (upper tertile) (P = 0.024) (Table 4).\nData are expressed as means ± standard deviation or number (percentages).\nACEi = angiotensin-I-converting enzyme inhibitor, ARB = angiotensin-II-receptor blocker, HbA1c = hemoglobin A1c, CrCl = creatinine clearance, GFR = glomerular filtration rate, NS = not significant.\nP value for the comparison between responders and non-responders across the tertiles.\nsCr = serum creatinine.\naRatio changes of regression coefficients of 1/sCr before and after AST-120 treatment.\nTable 5 shows clinical and laboratory findings before and after AST-120 treatment in the responder and non-responder groups. Administration of AST-120 significantly increased serum SOD level in both the responder and non-responder groups. In addition, serum LPO level was significantly decreased in the responder group (from 2.25 ± 0.56 μmol/L to 1.91 ± 0.72; P = 0.002), but not in the non-responder group. Urine 8-OHdG levels showed no significant changes in either the responder or non-responder groups. AST-120 treatment had no significant effect on the other clinical or biochemical parameters tested including blood urea nitrogen, hematocrit, glucose, and lipid profile in either the responder or non-responder groups. The renal function in all patients was maintained and no patients started dialysis therapy.\nData are expressed as means ± standard deviation.\nCrCl = creatinine clearance, HbA1c = hemoglobin A1c, LDL = low-density lipoprotein, HDL = high-density lipoprotein, SOD = superoxide dismutase, LPO = lipid peroxidation, 8-OHdG = 8-hydroxydeoxyguanosine, NS = not significant.",
"The present study involved 100 undialyzed patients with diabetic nephropathy from eight tertiary centers in Korea. These patients were confirmed to have progressive renal dysfunction under conventional treatments based on negative 1/sCr gradients observed over a period of one year prior to treatment. Rates of decline in 1/sCr were compared before and after AST-120 treatment. Treatment with AST-120 for 24 weeks significantly attenuated the progression of renal dysfunction in 80.3% of patients with diabetic nephropathy. These results suggest that co-administration of AST-120 with conventional treatments may delay the progression of renal dysfunction in diabetic nephropathy. In addition, improvement in the 1/sCr slope was independent of sCr levels before AST-120 treatment. With the exception of diastolic blood pressure, there were no significant differences in other background factors including age, gender, previous treatment modalities, renal function, lipid profile, and fasting plasma glucose level between the responder and non-responder groups. These findings imply that the therapeutic effect of AST-120 is basically independent of previous treatment modalities, hyperlipidemia, anemia, and hyperglycemia, and indicate that AST-120 could be an effective treatment modality for diabetic nephropathy in patients with diverse baseline clinical features. However, lower baseline diastolic blood pressure may predict a better response to AST-120 treatment. When we performed the ROC analysis, the optimal discriminating cutoff value of diastolic blood pressure for detecting subjects with non-response was 79.5 mmHg, with a sensitivity of 60.0% and specificity of 68.9% (AUC = 0.67; 95% confidence interval, 0.51–0.83; P = 0.047; data not shown).\nOur findings are consistent with previous observations in patients with diabetic nephropathy.192021 In a study performed in 276 patients with advanced diabetic nephropathy and sCr levels ranging from 3.4 to 4.5 mg/dL, treatment with AST-120 for six months decreased the sCr level in 30% of patients. In addition, the beneficial effect of AST-120 was more evident in patients with normal blood pressure and a hematocrit of 30% or above.20 In another study performed in 16 patients with CKD and type 2 diabetes (sCr < 1.5 mg/dL and urinary protein > 0.5 g/day), administration of AST-120 initiated in overt diabetic nephropathy delayed the progression of renal dysfunction.19\nThe optimal time to initiate AST-120 treatment in patients with CKD remains unclear. It was suggested that CKD patients with sCr levels ≥ 3.0 mg/dL have increased serum indoxyl sulfate levels.22 Shoji et al.23 showed that AST-120 treatment in CKD patients tended to be more effective in delaying GFR decline in the high GFR (≥ 31.4 mL/min) group than in the low GFR (< 31.4 mL/min) group. In addition, other studies showed a renoprotective effect of AST-120 even in early-stage CKD. A Japanese study reported that AST-120 had renoprotective effects in patients with CKD whose creatinine concentrations were between 1.2 and 3.0 mg/dL.24 Furthermore, AST-120 treatment delayed an increase in sCr level even in patients with early diabetic nephropathy, in which mean baseline estimated GFR ranged from 60.0 to 84.2 mL/min/1.73 m2.19 Consistent with these observations, the baseline mean sCr level in our study was approximately 2.0 mg/dL and individuals with lower baseline sCr were more likely to show improved renal function after AST-120 treatment compared to individuals with higher baseline sCr. Although we are unsure of the underlying mechanism, it is possible that AST-120 treatment is more effective in delaying the deterioration of renal function in early stage CKD in which more severe metabolic abnormalities and irreversible structural changes in the renal parenchyma have not yet occurred. In addition, a recent post hoc analysis of the EPPIC study population demonstrated that treatment with AST-120 delayed time to dialysis and prevented depression in renal function in patients with higher baseline proteinuria and positive hematuria.25 Although we did not measure proteinuria or hematuria in the current study, it would be interesting to determine whether proteinuria or hematuria is a marker of response to AST-120 in patients with diabetic kidney disease. Disruption of antioxidant systems is one of the crucial factors that promote renal dysfunction.26 Superoxide anion radicals and their metabolites are well-known harmful reactive oxygen species. Oxidative stress produces oxidized low-density lipoproteins and degenerate lipoproteins. Products of LPO play important roles in vascular sclerosis including nephrosclerosis. Peroxidation of lipids is known to be one of the aggravating factors in the progression of CKD in humans and animal models.2728 In addition, SOD is a principal active component against oxidative stress and is disturbed in uremic state.29 In this study, we investigated the renoprotective mechanism of AST-120 focusing on changes in oxidative stress markers. As the lipid profile was not changed after AST-120 treatment in either the responders or non-responders, the renoprotective effect of AST-120 cannot be attributed to a reduction in serum cholesterol. However, AST-120 treatment significantly increased serum SOD levels in both the responder and non-responder groups. In particular, after AST-120 treatment, serum lipid peroxide levels significantly decreased only in the responder group. These results indicate that the antioxidant activity of AST-120 may play a critical role in delaying the progression of renal dysfunction in patients with diabetic nephropathy.\nThis study has some limitations. First, since all enrolled subjects were treated with 6 g/day AST-120 (Kremezin®) for 24 weeks, there was no control group. Second, we were not able to measure indoxyl sulfate or proteinuria during the experimental period. Third, due to the short duration of intervention, the development of ESRD events could not be assessed. Fourth, although not statistically significant, there were numerical differences in the percentage between responders and non-responders who took medications that may affect renal function including ACE inhibitor, angiotensin II receptor blocker and lipid-lowering drugs. Despite these limitations, our study demonstrates the renoprotective effect of AST-120 with a relatively large number of patients with diabetic nephropathy and suggests a potential renoprotective mechanism of AST-120.\nIn conclusion, our data suggest that co-administration of AST-120 with conventional treatments may delay the progression of renal dysfunction in diabetic nephropathy, and lower diastolic blood pressure could be a predictor for better response to AST-120 treatment. We also found that a decrease in oxidative stress might be responsible for the efficacy of AST-120 treatment in diabetic nephropathy. Given the increasing incidence of diabetic nephropathy as the underlying cause of CKD, our findings suggest that AST-120 may prevent deterioration in renal function in CKD. Further large-scale studies are warranted to determine the long-term cardiovascular benefits of AST-120 treatment."
] |
[
"intro",
"methods",
null,
null,
null,
null,
"results",
"discussion"
] |
[
"Diabetic Nephropathy",
"Indoxyl Sulfate",
"Creatinine",
"Oxidative Stress",
"Antioxidants"
] |
INTRODUCTION:
Diabetic nephropathy is one of the most common causes of end-stage renal disease (ESRD) worldwide.123 As the prevalence of diabetes has continued to increase, a parallel increase has been seen in the prevalence of dialysis patients and associated costs. This increase in the number of patients with chronic kidney disease (CKD) has led to social and economic problems in many countries.1 Moreover, the clinical course of diabetic nephropathy is usually characterized by relatively rapid progression compared with CKD due to other causes.4 In addition, as diabetic ESRD patients are more prone to cardiovascular morbidity and mortality than other ESRD patients, early identification of diabetic nephropathy and prompt renoprotective treatment are critical for the prevention of end organ damage.5 Previous studies have shown that intensive control of blood glucose and blood pressure are not sufficient to prevent or delay the development of diabetic nephropathy.56 However, effective therapy is not available yet, and integrated treatment strategies are therefore needed to improve the management of diabetic nephropathy.56
The so-called ‘uremic toxins,’ such as indoxyl sulfate, are biologically active substances involved in the progression of renal failure.7 Both clinical and experimental studies have suggested that indoxyl sulfate plays an important role in the progressive loss of intact nephrons through induction of an inflammatory reaction and enhanced expression of profibrotic cytokines.7 AST-120 (Kremezin®), a spherical adsorptive carbon preparation, absorbs and removes uremic toxins such as indoxyl sulfate in the digestive tract.8 To date, the renoprotective effects of AST-120 have been determined mostly in patients with advanced CKD with inconsistent results.91011 Furthermore, little is known about the efficacy of AST-120 and which clinical and biochemical variables predict response to AST-120 treatment in diabetic nephropathy.
We therefore aimed to determine whether the addition of AST-120 to conventional treatments is effective to attenuate the progression of renal dysfunction in patients with type 2 diabetes and CKD. We also examined which clinical variables and biochemical tests, particularly oxidative stress markers, could predict beneficial effects of AST-120 in diabetic nephropathy.
METHODS:
Study subjects This study was a prospective, single-arm, open-label, multi-center study performed in subjects with overt diabetic nephropathy. Outpatients with type 2 diabetes and renal dysfunction who had not previously undergone dialysis from eight hospitals in Korea were prospectively enrolled in this study from 2008 to 2009. Study subjects had serum creatinine (sCr) levels ranging from 1.5 to 3.0 mg/dL measured at least three time points over a period of 12 months prior to treatment. We excluded any patients with active liver disease, acute infection, and those who had recent (< 6 months) acute coronary syndromes and cerebrovascular disease. After confirming a negative slope of the reciprocal serum creatinine level (1/sCr) time plot, oral administration of AST-120 6 grams per day was started and continued for 24 weeks.
This study was a prospective, single-arm, open-label, multi-center study performed in subjects with overt diabetic nephropathy. Outpatients with type 2 diabetes and renal dysfunction who had not previously undergone dialysis from eight hospitals in Korea were prospectively enrolled in this study from 2008 to 2009. Study subjects had serum creatinine (sCr) levels ranging from 1.5 to 3.0 mg/dL measured at least three time points over a period of 12 months prior to treatment. We excluded any patients with active liver disease, acute infection, and those who had recent (< 6 months) acute coronary syndromes and cerebrovascular disease. After confirming a negative slope of the reciprocal serum creatinine level (1/sCr) time plot, oral administration of AST-120 6 grams per day was started and continued for 24 weeks.
Clinical and laboratory examination Blood pressure was measured with the participant in a seated position after 5 minutes of quiet rest. Blood samples were collected after overnight fasting. Blood urea nitrogen, creatinine, glucose, and lipid profiles were measured. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance: [fasting insulin (μIU/mL) × fasting glucose (mmol/L)]/22.5.12 sCr was measured by the standard enzymatic method. Creatinine clearance (CrCl) was estimated by the Cockcroft-Gault equation; CrCl = [(140 − age) × weight (kg)]/sCr × 72 (× 0.85, if women).13 Glomerular filtration rate (GFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation.14 Diabetic retinopathy was diagnosed by an experienced ophthalmologist or diabetologist based on retinal images.15 Superoxide dismutase (SOD) activity in serum was determined with the Superoxide Dismutase Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), which utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Serum lipid peroxidation (LPO) was determined by a colorimetric assay using the Bioxytech LPO-586 kit (OxisResearch, Portland, OR, USA). Urinary excretion levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured with an enzyme-linked immunosorbent assay (8-Hydroxydeoxyguanosine Check; Japan Institute for the Control of Aging, Shizuoka, Japan).
The rate of progression of renal dysfunction was assessed as the gradient of the reciprocal serum creatinine level (1/sCr), as described by Mitch et al.,16 before and after the initiation of treatment. The 1/sCr was plotted against time, and the regression coefficient was determined by least-square linear regression analysis as previously described.17 In our study, the primary endpoint was improvement in renal function as the ratio of the 1/sCr-time slope for post- to pre- AST-120 therapy. Based on the ratio changes of the regression coefficients of 1/sCr before and after treatment, efficacy of this drug was defined as follows: 1) definitely improved: ≤ 0.30; 2) moderately improved: 0.31–0.60; 3) slightly improved: 0.61–0.90; 4) unchanged: 0.91–1.09; and 5) deteriorated: ≥ 1.10. Subjects in the definitely, moderately, or slightly improved groups (ratio ≤ 0.9) were classified as responders and those in the unchanged and deteriorated groups were considered non-responders. To determine whether baseline sCr levels affect the efficacy of AST-120 treatment, patients were divided into three groups according to the baseline sCr tertile values. The secondary endpoints were changes in 1) CrCl; 2) 1/sCr-time slope pre- and post- AST-120 treatment; and 3) oxidative stress markers. Changes in sCr, serum urea nitrogen, hematocrit, glucose, lipid profile, and systolic and diastolic blood pressure after AST-120 therapy were also compared.
Blood pressure was measured with the participant in a seated position after 5 minutes of quiet rest. Blood samples were collected after overnight fasting. Blood urea nitrogen, creatinine, glucose, and lipid profiles were measured. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance: [fasting insulin (μIU/mL) × fasting glucose (mmol/L)]/22.5.12 sCr was measured by the standard enzymatic method. Creatinine clearance (CrCl) was estimated by the Cockcroft-Gault equation; CrCl = [(140 − age) × weight (kg)]/sCr × 72 (× 0.85, if women).13 Glomerular filtration rate (GFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation.14 Diabetic retinopathy was diagnosed by an experienced ophthalmologist or diabetologist based on retinal images.15 Superoxide dismutase (SOD) activity in serum was determined with the Superoxide Dismutase Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), which utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Serum lipid peroxidation (LPO) was determined by a colorimetric assay using the Bioxytech LPO-586 kit (OxisResearch, Portland, OR, USA). Urinary excretion levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured with an enzyme-linked immunosorbent assay (8-Hydroxydeoxyguanosine Check; Japan Institute for the Control of Aging, Shizuoka, Japan).
The rate of progression of renal dysfunction was assessed as the gradient of the reciprocal serum creatinine level (1/sCr), as described by Mitch et al.,16 before and after the initiation of treatment. The 1/sCr was plotted against time, and the regression coefficient was determined by least-square linear regression analysis as previously described.17 In our study, the primary endpoint was improvement in renal function as the ratio of the 1/sCr-time slope for post- to pre- AST-120 therapy. Based on the ratio changes of the regression coefficients of 1/sCr before and after treatment, efficacy of this drug was defined as follows: 1) definitely improved: ≤ 0.30; 2) moderately improved: 0.31–0.60; 3) slightly improved: 0.61–0.90; 4) unchanged: 0.91–1.09; and 5) deteriorated: ≥ 1.10. Subjects in the definitely, moderately, or slightly improved groups (ratio ≤ 0.9) were classified as responders and those in the unchanged and deteriorated groups were considered non-responders. To determine whether baseline sCr levels affect the efficacy of AST-120 treatment, patients were divided into three groups according to the baseline sCr tertile values. The secondary endpoints were changes in 1) CrCl; 2) 1/sCr-time slope pre- and post- AST-120 treatment; and 3) oxidative stress markers. Changes in sCr, serum urea nitrogen, hematocrit, glucose, lipid profile, and systolic and diastolic blood pressure after AST-120 therapy were also compared.
Statistical analysis Data are expressed as means ± standard deviations. The χ2 test, Fisher's exact test, and Mann-Whitney test were used to compare the characteristics of study subjects. Parameters before and 24 weeks after AST-120 were compared by paired t-test. The clinical per-protocol (PP) population was defined as all patients taking the study medication for 24 weeks with a compliance rate of ≥ 70%, and 76 patients were enrolled for PP analysis. The primary end point was analyzed based on the PP population to better report additional efficacy.18 Other secondary endpoints were analyzed based on the intention-to-treat population, which consisted of all patients taking at least one dose of study medication. Missing data were input using the last-observation-carried-forward procedure. To assess the utility of the predictive factor for efficacy of AST-120, we conducted receiver operating characteristics (ROC) curves and calculated the areas under the curve (AUC). All statistical analyses were performed using PASW version 18.0 (SPSS, Chicago, IL, USA). A P value < 0.05 was considered significant.
Data are expressed as means ± standard deviations. The χ2 test, Fisher's exact test, and Mann-Whitney test were used to compare the characteristics of study subjects. Parameters before and 24 weeks after AST-120 were compared by paired t-test. The clinical per-protocol (PP) population was defined as all patients taking the study medication for 24 weeks with a compliance rate of ≥ 70%, and 76 patients were enrolled for PP analysis. The primary end point was analyzed based on the PP population to better report additional efficacy.18 Other secondary endpoints were analyzed based on the intention-to-treat population, which consisted of all patients taking at least one dose of study medication. Missing data were input using the last-observation-carried-forward procedure. To assess the utility of the predictive factor for efficacy of AST-120, we conducted receiver operating characteristics (ROC) curves and calculated the areas under the curve (AUC). All statistical analyses were performed using PASW version 18.0 (SPSS, Chicago, IL, USA). A P value < 0.05 was considered significant.
Ethics statement The investigators explained the purpose of the study to the patients at the time of study entry and written informed consent was obtained from all patients. The study was approved by the Institutional Review Board (IRB) of each participating center and was conducted in accordance with the Declaration of Helsinki (Samsung Medical Center IRB No. 2008-02-015).
The investigators explained the purpose of the study to the patients at the time of study entry and written informed consent was obtained from all patients. The study was approved by the Institutional Review Board (IRB) of each participating center and was conducted in accordance with the Declaration of Helsinki (Samsung Medical Center IRB No. 2008-02-015).
Study subjects:
This study was a prospective, single-arm, open-label, multi-center study performed in subjects with overt diabetic nephropathy. Outpatients with type 2 diabetes and renal dysfunction who had not previously undergone dialysis from eight hospitals in Korea were prospectively enrolled in this study from 2008 to 2009. Study subjects had serum creatinine (sCr) levels ranging from 1.5 to 3.0 mg/dL measured at least three time points over a period of 12 months prior to treatment. We excluded any patients with active liver disease, acute infection, and those who had recent (< 6 months) acute coronary syndromes and cerebrovascular disease. After confirming a negative slope of the reciprocal serum creatinine level (1/sCr) time plot, oral administration of AST-120 6 grams per day was started and continued for 24 weeks.
Clinical and laboratory examination:
Blood pressure was measured with the participant in a seated position after 5 minutes of quiet rest. Blood samples were collected after overnight fasting. Blood urea nitrogen, creatinine, glucose, and lipid profiles were measured. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance: [fasting insulin (μIU/mL) × fasting glucose (mmol/L)]/22.5.12 sCr was measured by the standard enzymatic method. Creatinine clearance (CrCl) was estimated by the Cockcroft-Gault equation; CrCl = [(140 − age) × weight (kg)]/sCr × 72 (× 0.85, if women).13 Glomerular filtration rate (GFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation.14 Diabetic retinopathy was diagnosed by an experienced ophthalmologist or diabetologist based on retinal images.15 Superoxide dismutase (SOD) activity in serum was determined with the Superoxide Dismutase Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), which utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Serum lipid peroxidation (LPO) was determined by a colorimetric assay using the Bioxytech LPO-586 kit (OxisResearch, Portland, OR, USA). Urinary excretion levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured with an enzyme-linked immunosorbent assay (8-Hydroxydeoxyguanosine Check; Japan Institute for the Control of Aging, Shizuoka, Japan).
The rate of progression of renal dysfunction was assessed as the gradient of the reciprocal serum creatinine level (1/sCr), as described by Mitch et al.,16 before and after the initiation of treatment. The 1/sCr was plotted against time, and the regression coefficient was determined by least-square linear regression analysis as previously described.17 In our study, the primary endpoint was improvement in renal function as the ratio of the 1/sCr-time slope for post- to pre- AST-120 therapy. Based on the ratio changes of the regression coefficients of 1/sCr before and after treatment, efficacy of this drug was defined as follows: 1) definitely improved: ≤ 0.30; 2) moderately improved: 0.31–0.60; 3) slightly improved: 0.61–0.90; 4) unchanged: 0.91–1.09; and 5) deteriorated: ≥ 1.10. Subjects in the definitely, moderately, or slightly improved groups (ratio ≤ 0.9) were classified as responders and those in the unchanged and deteriorated groups were considered non-responders. To determine whether baseline sCr levels affect the efficacy of AST-120 treatment, patients were divided into three groups according to the baseline sCr tertile values. The secondary endpoints were changes in 1) CrCl; 2) 1/sCr-time slope pre- and post- AST-120 treatment; and 3) oxidative stress markers. Changes in sCr, serum urea nitrogen, hematocrit, glucose, lipid profile, and systolic and diastolic blood pressure after AST-120 therapy were also compared.
Statistical analysis:
Data are expressed as means ± standard deviations. The χ2 test, Fisher's exact test, and Mann-Whitney test were used to compare the characteristics of study subjects. Parameters before and 24 weeks after AST-120 were compared by paired t-test. The clinical per-protocol (PP) population was defined as all patients taking the study medication for 24 weeks with a compliance rate of ≥ 70%, and 76 patients were enrolled for PP analysis. The primary end point was analyzed based on the PP population to better report additional efficacy.18 Other secondary endpoints were analyzed based on the intention-to-treat population, which consisted of all patients taking at least one dose of study medication. Missing data were input using the last-observation-carried-forward procedure. To assess the utility of the predictive factor for efficacy of AST-120, we conducted receiver operating characteristics (ROC) curves and calculated the areas under the curve (AUC). All statistical analyses were performed using PASW version 18.0 (SPSS, Chicago, IL, USA). A P value < 0.05 was considered significant.
Ethics statement:
The investigators explained the purpose of the study to the patients at the time of study entry and written informed consent was obtained from all patients. The study was approved by the Institutional Review Board (IRB) of each participating center and was conducted in accordance with the Declaration of Helsinki (Samsung Medical Center IRB No. 2008-02-015).
RESULTS:
Table 1 shows the baseline characteristics of the patients. The mean age was 62.6 years and 77% of study participants were men. Eighty-nine percent of patients were taking an angiotensin-I-converting enzyme (ACE) inhibitor and/or angiotensin II receptor blocker, and 83% were taking a statin. The mean estimated GFR was 34.7 mL/min/1.73 m2 and 17, 51, 32 subjects were CKD stage 3a, 3b, and 4, respectively. We analyzed 76 subjects for the primary endpoint and 100 subjects for the secondary endpoints.
Data are expressed as means ± standard deviation or percentages.
ACEi = angiotensin-I-converting enzyme inhibitor, ARB = angiotensin-II-receptor blocker, HbA1c = hemoglobin A1c, HOMA-IR = homeostasis model assessment of insulin resistance, HDL = high-density lipoprotein, LDL = low-density lipoprotein, CrCl = creatinine clearance, GFR = glomerular filtration rate.
Of 76 patients (PP population) analyzed for the primary endpoint, 50 were definitely improved, 2 were moderately improved, and 9 were slightly improved responders after 24 weeks of treatment with AST-120. Among non-responders, 4 were unchanged and 11 were deteriorated based on the criteria for changes in sCr levels described above. As a result, 61 patients (80.3%) were categorized as responders and 15 patients (19.7%) were non-responders to the AST-120 therapy (Table 2).
aRatio change of regression coefficients of 1/sCr before and after AST-120 treatment.
Next, we determined baseline characteristics of the responder and non-responder groups. There were no differences in age, gender, previous treatment modalities, baseline sCr or CrCl, lipid profile, and blood glucose levels between the two groups; however, diastolic blood pressure was significantly different (Table 3). The non-responder group had a higher diastolic blood pressure than the responder group (79.3 ± 11.1 mmHg vs. 73.5 ± 9.5 mmHg, respectively; P = 0.046). We also analyzed the efficacy of 24-week AST-120 treatment according to baseline sCr tertile. All three groups demonstrated a significant improvement in renal function determined by the ratio changes of regression coefficients of 1/sCr after AST-120 treatment. In particular, individuals with lower baseline sCr (lower and middle tertiles) were more likely to show improved renal function after AST-120 treatment compared to individuals with higher baseline sCr (upper tertile) (P = 0.024) (Table 4).
Data are expressed as means ± standard deviation or number (percentages).
ACEi = angiotensin-I-converting enzyme inhibitor, ARB = angiotensin-II-receptor blocker, HbA1c = hemoglobin A1c, CrCl = creatinine clearance, GFR = glomerular filtration rate, NS = not significant.
P value for the comparison between responders and non-responders across the tertiles.
sCr = serum creatinine.
aRatio changes of regression coefficients of 1/sCr before and after AST-120 treatment.
Table 5 shows clinical and laboratory findings before and after AST-120 treatment in the responder and non-responder groups. Administration of AST-120 significantly increased serum SOD level in both the responder and non-responder groups. In addition, serum LPO level was significantly decreased in the responder group (from 2.25 ± 0.56 μmol/L to 1.91 ± 0.72; P = 0.002), but not in the non-responder group. Urine 8-OHdG levels showed no significant changes in either the responder or non-responder groups. AST-120 treatment had no significant effect on the other clinical or biochemical parameters tested including blood urea nitrogen, hematocrit, glucose, and lipid profile in either the responder or non-responder groups. The renal function in all patients was maintained and no patients started dialysis therapy.
Data are expressed as means ± standard deviation.
CrCl = creatinine clearance, HbA1c = hemoglobin A1c, LDL = low-density lipoprotein, HDL = high-density lipoprotein, SOD = superoxide dismutase, LPO = lipid peroxidation, 8-OHdG = 8-hydroxydeoxyguanosine, NS = not significant.
DISCUSSION:
The present study involved 100 undialyzed patients with diabetic nephropathy from eight tertiary centers in Korea. These patients were confirmed to have progressive renal dysfunction under conventional treatments based on negative 1/sCr gradients observed over a period of one year prior to treatment. Rates of decline in 1/sCr were compared before and after AST-120 treatment. Treatment with AST-120 for 24 weeks significantly attenuated the progression of renal dysfunction in 80.3% of patients with diabetic nephropathy. These results suggest that co-administration of AST-120 with conventional treatments may delay the progression of renal dysfunction in diabetic nephropathy. In addition, improvement in the 1/sCr slope was independent of sCr levels before AST-120 treatment. With the exception of diastolic blood pressure, there were no significant differences in other background factors including age, gender, previous treatment modalities, renal function, lipid profile, and fasting plasma glucose level between the responder and non-responder groups. These findings imply that the therapeutic effect of AST-120 is basically independent of previous treatment modalities, hyperlipidemia, anemia, and hyperglycemia, and indicate that AST-120 could be an effective treatment modality for diabetic nephropathy in patients with diverse baseline clinical features. However, lower baseline diastolic blood pressure may predict a better response to AST-120 treatment. When we performed the ROC analysis, the optimal discriminating cutoff value of diastolic blood pressure for detecting subjects with non-response was 79.5 mmHg, with a sensitivity of 60.0% and specificity of 68.9% (AUC = 0.67; 95% confidence interval, 0.51–0.83; P = 0.047; data not shown).
Our findings are consistent with previous observations in patients with diabetic nephropathy.192021 In a study performed in 276 patients with advanced diabetic nephropathy and sCr levels ranging from 3.4 to 4.5 mg/dL, treatment with AST-120 for six months decreased the sCr level in 30% of patients. In addition, the beneficial effect of AST-120 was more evident in patients with normal blood pressure and a hematocrit of 30% or above.20 In another study performed in 16 patients with CKD and type 2 diabetes (sCr < 1.5 mg/dL and urinary protein > 0.5 g/day), administration of AST-120 initiated in overt diabetic nephropathy delayed the progression of renal dysfunction.19
The optimal time to initiate AST-120 treatment in patients with CKD remains unclear. It was suggested that CKD patients with sCr levels ≥ 3.0 mg/dL have increased serum indoxyl sulfate levels.22 Shoji et al.23 showed that AST-120 treatment in CKD patients tended to be more effective in delaying GFR decline in the high GFR (≥ 31.4 mL/min) group than in the low GFR (< 31.4 mL/min) group. In addition, other studies showed a renoprotective effect of AST-120 even in early-stage CKD. A Japanese study reported that AST-120 had renoprotective effects in patients with CKD whose creatinine concentrations were between 1.2 and 3.0 mg/dL.24 Furthermore, AST-120 treatment delayed an increase in sCr level even in patients with early diabetic nephropathy, in which mean baseline estimated GFR ranged from 60.0 to 84.2 mL/min/1.73 m2.19 Consistent with these observations, the baseline mean sCr level in our study was approximately 2.0 mg/dL and individuals with lower baseline sCr were more likely to show improved renal function after AST-120 treatment compared to individuals with higher baseline sCr. Although we are unsure of the underlying mechanism, it is possible that AST-120 treatment is more effective in delaying the deterioration of renal function in early stage CKD in which more severe metabolic abnormalities and irreversible structural changes in the renal parenchyma have not yet occurred. In addition, a recent post hoc analysis of the EPPIC study population demonstrated that treatment with AST-120 delayed time to dialysis and prevented depression in renal function in patients with higher baseline proteinuria and positive hematuria.25 Although we did not measure proteinuria or hematuria in the current study, it would be interesting to determine whether proteinuria or hematuria is a marker of response to AST-120 in patients with diabetic kidney disease. Disruption of antioxidant systems is one of the crucial factors that promote renal dysfunction.26 Superoxide anion radicals and their metabolites are well-known harmful reactive oxygen species. Oxidative stress produces oxidized low-density lipoproteins and degenerate lipoproteins. Products of LPO play important roles in vascular sclerosis including nephrosclerosis. Peroxidation of lipids is known to be one of the aggravating factors in the progression of CKD in humans and animal models.2728 In addition, SOD is a principal active component against oxidative stress and is disturbed in uremic state.29 In this study, we investigated the renoprotective mechanism of AST-120 focusing on changes in oxidative stress markers. As the lipid profile was not changed after AST-120 treatment in either the responders or non-responders, the renoprotective effect of AST-120 cannot be attributed to a reduction in serum cholesterol. However, AST-120 treatment significantly increased serum SOD levels in both the responder and non-responder groups. In particular, after AST-120 treatment, serum lipid peroxide levels significantly decreased only in the responder group. These results indicate that the antioxidant activity of AST-120 may play a critical role in delaying the progression of renal dysfunction in patients with diabetic nephropathy.
This study has some limitations. First, since all enrolled subjects were treated with 6 g/day AST-120 (Kremezin®) for 24 weeks, there was no control group. Second, we were not able to measure indoxyl sulfate or proteinuria during the experimental period. Third, due to the short duration of intervention, the development of ESRD events could not be assessed. Fourth, although not statistically significant, there were numerical differences in the percentage between responders and non-responders who took medications that may affect renal function including ACE inhibitor, angiotensin II receptor blocker and lipid-lowering drugs. Despite these limitations, our study demonstrates the renoprotective effect of AST-120 with a relatively large number of patients with diabetic nephropathy and suggests a potential renoprotective mechanism of AST-120.
In conclusion, our data suggest that co-administration of AST-120 with conventional treatments may delay the progression of renal dysfunction in diabetic nephropathy, and lower diastolic blood pressure could be a predictor for better response to AST-120 treatment. We also found that a decrease in oxidative stress might be responsible for the efficacy of AST-120 treatment in diabetic nephropathy. Given the increasing incidence of diabetic nephropathy as the underlying cause of CKD, our findings suggest that AST-120 may prevent deterioration in renal function in CKD. Further large-scale studies are warranted to determine the long-term cardiovascular benefits of AST-120 treatment.
|
Background:
Removal of uremic toxins such as indoxyl sulfate by AST-120 is known to improve renal function and delay the initiation of dialysis in patients with advanced chronic kidney disease. However, it is unclear whether the addition of AST-120 to conventional treatments is effective in delaying the progression of renal dysfunction in patients with diabetic nephropathy.
Methods:
A total of 100 patients with type 2 diabetes and renal dysfunction (serum creatinine levels ranging from 1.5 to 3.0 mg/dL) were recruited from eight centers in Korea and treated with AST-120 (6 g/day) for 24 weeks. The primary endpoint was improvement in renal function measured as the gradient of the reciprocal serum creatinine level (1/sCr) over time (i.e., the ratio of 1/sCr time slope for post- to pre-AST-120 therapy). A response was defined as a ratio change of the regression coefficient of 1/sCr ≤ 0.90.
Results:
Renal function improved in 80.3% of patients (61/76) after 24 weeks of AST-120 treatment. There were no differences between responder and non-responder groups in baseline characteristics except for diastolic blood pressure (73.5 ± 9.5 mmHg in the responder group vs. 79.3 ± 11.1 mmHg in the non-responder group; P = 0.046). Serum lipid peroxidation level decreased significantly in the responder group (from 2.25 ± 0.56 μol/L to 1.91 ± 0.72 μol/L; P = 0.002) but not in the non-responder group.
Conclusions:
The addition of AST-120 to conventional treatments may delay the progression of renal dysfunction in diabetic nephropathy. The antioxidant effect of AST-120 might contribute to improvement in renal function.
| null | null | 5,345 | 316 | 8 |
[
"ast",
"ast 120",
"120",
"scr",
"patients",
"treatment",
"study",
"renal",
"diabetic",
"120 treatment"
] |
[
"test",
"test"
] | null | null |
[CONTENT] Diabetic Nephropathy | Indoxyl Sulfate | Creatinine | Oxidative Stress | Antioxidants [SUMMARY]
|
[CONTENT] Diabetic Nephropathy | Indoxyl Sulfate | Creatinine | Oxidative Stress | Antioxidants [SUMMARY]
|
[CONTENT] Diabetic Nephropathy | Indoxyl Sulfate | Creatinine | Oxidative Stress | Antioxidants [SUMMARY]
| null |
[CONTENT] Diabetic Nephropathy | Indoxyl Sulfate | Creatinine | Oxidative Stress | Antioxidants [SUMMARY]
| null |
[CONTENT] Aged | Blood Pressure | Carbon | Creatinine | Diabetes Mellitus, Type 2 | Diabetic Nephropathies | Dose-Response Relationship, Drug | Drug Administration Schedule | Female | Humans | Lipid Peroxidation | Male | Middle Aged | Oxides | Prospective Studies | Protective Agents | Severity of Illness Index | Treatment Outcome [SUMMARY]
|
[CONTENT] Aged | Blood Pressure | Carbon | Creatinine | Diabetes Mellitus, Type 2 | Diabetic Nephropathies | Dose-Response Relationship, Drug | Drug Administration Schedule | Female | Humans | Lipid Peroxidation | Male | Middle Aged | Oxides | Prospective Studies | Protective Agents | Severity of Illness Index | Treatment Outcome [SUMMARY]
|
[CONTENT] Aged | Blood Pressure | Carbon | Creatinine | Diabetes Mellitus, Type 2 | Diabetic Nephropathies | Dose-Response Relationship, Drug | Drug Administration Schedule | Female | Humans | Lipid Peroxidation | Male | Middle Aged | Oxides | Prospective Studies | Protective Agents | Severity of Illness Index | Treatment Outcome [SUMMARY]
| null |
[CONTENT] Aged | Blood Pressure | Carbon | Creatinine | Diabetes Mellitus, Type 2 | Diabetic Nephropathies | Dose-Response Relationship, Drug | Drug Administration Schedule | Female | Humans | Lipid Peroxidation | Male | Middle Aged | Oxides | Prospective Studies | Protective Agents | Severity of Illness Index | Treatment Outcome [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] ast | ast 120 | 120 | scr | patients | treatment | study | renal | diabetic | 120 treatment [SUMMARY]
|
[CONTENT] ast | ast 120 | 120 | scr | patients | treatment | study | renal | diabetic | 120 treatment [SUMMARY]
|
[CONTENT] ast | ast 120 | 120 | scr | patients | treatment | study | renal | diabetic | 120 treatment [SUMMARY]
| null |
[CONTENT] ast | ast 120 | 120 | scr | patients | treatment | study | renal | diabetic | 120 treatment [SUMMARY]
| null |
[CONTENT] diabetic | diabetic nephropathy | nephropathy | ckd | ast 120 | 120 | ast | increase | indoxyl sulfate | esrd [SUMMARY]
|
[CONTENT] scr | study | measured | serum | time | test | ast | ast 120 | 120 | creatinine [SUMMARY]
|
[CONTENT] responder | non | non responder | scr | angiotensin | table | treatment | ast 120 | ast | 120 [SUMMARY]
| null |
[CONTENT] scr | ast 120 | ast | 120 | study | patients | treatment | diabetic | diabetic nephropathy | nephropathy [SUMMARY]
| null |
[CONTENT] indoxyl ||| [SUMMARY]
|
[CONTENT] 100 | 2 | 1.5 | 3.0 mg/dL | eight | Korea | 6 g/day | 24 weeks ||| 1 | 1 ||| 1 [SUMMARY]
|
[CONTENT] 80.3% | 61/76 | 24 weeks ||| 73.5 | 9.5 | 79.3 | 11.1 | P | 0.046 ||| 2.25 | 0.56 | 0.72 | 0.002 [SUMMARY]
| null |
[CONTENT] indoxyl ||| ||| 100 | 2 | 1.5 | 3.0 mg/dL | eight | Korea | 6 g/day | 24 weeks ||| 1 | 1 ||| 1 ||| ||| 80.3% | 61/76 | 24 weeks ||| 73.5 | 9.5 | 79.3 | 11.1 | P | 0.046 ||| 2.25 | 0.56 | 0.72 | 0.002 ||| ||| [SUMMARY]
| null |
||||
Host genetic variants of ABCB1 and IL15 influence treatment outcome in paediatric acute lymphoblastic leukaemia.
|
24434428
|
Host germline variations and their potential prognostic importance is an emerging area of interest in paediatric ALL.
|
BACKGROUND
|
We investigated the associations between 20 germline variations and various clinical end points in 463 children with ALL.
|
METHODS
|
After adjusting for known prognostic factors, variants in two genes were found to be independently associated with poorer EFS: ABCB1 T/T at either 2677 (rs2032582) or 3435 (rs1045642) position (P=0.003) and IL15 67276493G/G (rs17015014; P=0.022). These variants showed a strong additive effect affecting outcome (P<0.001), whereby patients with both risk genotypes had the worst EFS (P=0.001), even after adjusting for MRD levels at the end of remission induction. The adverse effect of ABCB1 T/T genotypes was most pronounced in patients with favourable cytogenetics (P=0.011) while the IL15 67276493G/G genotype mainly affected patients without common chromosomal abnormalities (P=0.022). In both cytogenetic subgroups, increasing number of such risk genotypes still predicted worsening outcome (P<0.001 and=0.009, respectively).
|
RESULTS
|
These results point to the prognostic importance of host genetic variants, although the specific mechanisms remain unclarified. Inclusion of ABCB1 and IL15 variants may help improve risk assignment strategies in paediatric ALL.
|
CONCLUSION
|
[
"ATP Binding Cassette Transporter, Subfamily B",
"ATP Binding Cassette Transporter, Subfamily B, Member 1",
"Child",
"Child, Preschool",
"Female",
"Genotype",
"Humans",
"Infant",
"Interleukin-15",
"Linkage Disequilibrium",
"Male",
"Polymorphism, Single Nucleotide",
"Precursor Cell Lymphoblastic Leukemia-Lymphoma",
"Prognosis",
"Treatment Outcome"
] |
3960629
| null | null | null | null |
Results
|
Identification of polymorphisms of ABCB1 and IL15 as predictors of EFS The clinical and cytogenetic features at presentation and the prevalence of alleles studied are summarised in Table 1, Supplementary Tables S3a and S3b, respectively. All bi-allelic loci were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci). Of the presenting features examined, unfavourable cytogenetics was associated with a significantly poorer EFS (PCOX<0.001; Table 2), while NCI high-risk status had borderline significance (PCOX=0.091; Table 2); self-reported ethnicity, gender, and cell type were not significantly related to EFS. The complete genotype data as well as patient characteristics are available for research purposes upon request.
Among the 20 polymorphisms examined individually in preliminary test, polymorphisms in ABCB1 (rs2032582 and rs1045642) and IL15 (rs17015014) were found eligible for further analyses (adjusted P=0.038 and 0.048, respectively; Supplementary Table S4). They were subsequently analysed together in a Cox regression model adjusting for presenting clinical and cytogenetic features (Table 2). The homozygosity of either ABCB1 2677T/T or 3435T/T was still significantly related with a poorer EFS compared with other genotypes at these two loci (PKM=0.001; PCOX=0.003, HR=2.11, 95% CI=1.29–3.45; Figure 1A). There was moderate linkage disequilibrium (LD) between 2677T and 3435T (details shown in later section). IL15 67276493G/G was also associated with significantly poorer EFS compared with 67276493G/C and C/C (PKM=0.050; PCOX=0.022, HR=1.84, 95% CI=1.09–3.08; Figure 1B). Adjusting for MRD at the end of remission induction (Supplementary Figures S2 A–B) or excluding the cases of resistance from EFS analyses (Supplementary Table S5 and Supplementary Figures S3A–B) did not alter the significance of these polymorphisms identified.
The risk genotypes, that is, ABCB1 T/T at 2677 or 3435 or both as well as IL15 67276493G/G, exhibited an additive effect (PKM<0.001; PCOX<0.001, HR=1.97, 95% CI=1.38–2.82) demonstrating progressively poorer EFS in patients with an increasing number of risk genotypes (Table 2 and Figure 1C). Compared with the patients without any risk genotype, the hazard of adverse events raised 2.2 folds in patients with one risk genotype (PKM=0.005; PCOX=0.003, HR=2.20, 95% CI=1.30–3.70) and 3.6 folds in those with two risk genotypes (PKM=0.002; PCOX=0.001, HR=3.64, 95% CI=1.68–7.92). After adjusting for MRD at the end of remission induction (Supplementary Table S6 and Supplementary Figure S2C) or excluding the cases of resistance (Supplementary Table S7 and Supplementary Figure S3C), this additive effect remained significant. There was no significant EFS difference between patients with only ABCB1 risk genotype and those with only IL15 risk genotype (data not shown).
To further investigate the impact of ABCB1 2677G>T/A/3435C>T and IL15 67276493G>C genotypes on a specific event, we examined their influence on the cumulative incidence of relapse (including resistance), failure to achieve complete remission as well as treatment-related mortality. Homozygosity of either ABCB1 2677T/T or 3435T/T significantly increased the risk of relapse in both univariate (PG=0.001; Supplementary Figure S4A) and multivariate models after adjusting for clinical and cytogenetic features (PFG=0.011, HR=2.17, 95% CI=1.19–3.94; Supplementary Table S8). The risk of relapse in patients with at least one T/T was 20.4% compared with 9.0% in those without any T/T. Taking the end-of-induction MRD level into account did not alter the significance (PFG=0.027, HR=2.01, 95% CI=1.08–3.73). The risk of relapse also became higher with increasing number of risk genotypes (PG<0.001; Supplementary Figure S4B; PFG=0.011, HR=1.84, 95% CI=1.15–2.93; Supplementary Table S8). The trend remained significant after adjusting for the end-of-induction MRD level (PFG=0.026, HR=1.69, 95% CI=1.07–2.68).
IL15 67276493G/G was associated with higher risk of failure to achieve complete remission when compared with G/C and C/C genotypes (P=0.009, OR=3.14, 95% CI=1.34–7.36; Supplementary Table S9). G/G homozygosity also correlated with poorer overall survival (PKM=0.050; PCOX=0.005, HR=2.74, 95% CI=1.35–5.58; Supplementary Table S10 and Supplementary Figure S5), primarily due to more than 3-fold higher risk of treatment-related mortality in patients with IL15 67276493G/G (P=0.015, OR=3.40, 95% CI=1.27–9.11; Supplementary Table S11).
We also examined the associations of identified risk genotypes with known risk factors. Neither T/T homozygosity at ABCB1 2677 or 3435 nor IL15 67276493G/G was found to correlate with cytogenetics and MRD level after remission induction (Supplementary Table S12).
The clinical and cytogenetic features at presentation and the prevalence of alleles studied are summarised in Table 1, Supplementary Tables S3a and S3b, respectively. All bi-allelic loci were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci). Of the presenting features examined, unfavourable cytogenetics was associated with a significantly poorer EFS (PCOX<0.001; Table 2), while NCI high-risk status had borderline significance (PCOX=0.091; Table 2); self-reported ethnicity, gender, and cell type were not significantly related to EFS. The complete genotype data as well as patient characteristics are available for research purposes upon request.
Among the 20 polymorphisms examined individually in preliminary test, polymorphisms in ABCB1 (rs2032582 and rs1045642) and IL15 (rs17015014) were found eligible for further analyses (adjusted P=0.038 and 0.048, respectively; Supplementary Table S4). They were subsequently analysed together in a Cox regression model adjusting for presenting clinical and cytogenetic features (Table 2). The homozygosity of either ABCB1 2677T/T or 3435T/T was still significantly related with a poorer EFS compared with other genotypes at these two loci (PKM=0.001; PCOX=0.003, HR=2.11, 95% CI=1.29–3.45; Figure 1A). There was moderate linkage disequilibrium (LD) between 2677T and 3435T (details shown in later section). IL15 67276493G/G was also associated with significantly poorer EFS compared with 67276493G/C and C/C (PKM=0.050; PCOX=0.022, HR=1.84, 95% CI=1.09–3.08; Figure 1B). Adjusting for MRD at the end of remission induction (Supplementary Figures S2 A–B) or excluding the cases of resistance from EFS analyses (Supplementary Table S5 and Supplementary Figures S3A–B) did not alter the significance of these polymorphisms identified.
The risk genotypes, that is, ABCB1 T/T at 2677 or 3435 or both as well as IL15 67276493G/G, exhibited an additive effect (PKM<0.001; PCOX<0.001, HR=1.97, 95% CI=1.38–2.82) demonstrating progressively poorer EFS in patients with an increasing number of risk genotypes (Table 2 and Figure 1C). Compared with the patients without any risk genotype, the hazard of adverse events raised 2.2 folds in patients with one risk genotype (PKM=0.005; PCOX=0.003, HR=2.20, 95% CI=1.30–3.70) and 3.6 folds in those with two risk genotypes (PKM=0.002; PCOX=0.001, HR=3.64, 95% CI=1.68–7.92). After adjusting for MRD at the end of remission induction (Supplementary Table S6 and Supplementary Figure S2C) or excluding the cases of resistance (Supplementary Table S7 and Supplementary Figure S3C), this additive effect remained significant. There was no significant EFS difference between patients with only ABCB1 risk genotype and those with only IL15 risk genotype (data not shown).
To further investigate the impact of ABCB1 2677G>T/A/3435C>T and IL15 67276493G>C genotypes on a specific event, we examined their influence on the cumulative incidence of relapse (including resistance), failure to achieve complete remission as well as treatment-related mortality. Homozygosity of either ABCB1 2677T/T or 3435T/T significantly increased the risk of relapse in both univariate (PG=0.001; Supplementary Figure S4A) and multivariate models after adjusting for clinical and cytogenetic features (PFG=0.011, HR=2.17, 95% CI=1.19–3.94; Supplementary Table S8). The risk of relapse in patients with at least one T/T was 20.4% compared with 9.0% in those without any T/T. Taking the end-of-induction MRD level into account did not alter the significance (PFG=0.027, HR=2.01, 95% CI=1.08–3.73). The risk of relapse also became higher with increasing number of risk genotypes (PG<0.001; Supplementary Figure S4B; PFG=0.011, HR=1.84, 95% CI=1.15–2.93; Supplementary Table S8). The trend remained significant after adjusting for the end-of-induction MRD level (PFG=0.026, HR=1.69, 95% CI=1.07–2.68).
IL15 67276493G/G was associated with higher risk of failure to achieve complete remission when compared with G/C and C/C genotypes (P=0.009, OR=3.14, 95% CI=1.34–7.36; Supplementary Table S9). G/G homozygosity also correlated with poorer overall survival (PKM=0.050; PCOX=0.005, HR=2.74, 95% CI=1.35–5.58; Supplementary Table S10 and Supplementary Figure S5), primarily due to more than 3-fold higher risk of treatment-related mortality in patients with IL15 67276493G/G (P=0.015, OR=3.40, 95% CI=1.27–9.11; Supplementary Table S11).
We also examined the associations of identified risk genotypes with known risk factors. Neither T/T homozygosity at ABCB1 2677 or 3435 nor IL15 67276493G/G was found to correlate with cytogenetics and MRD level after remission induction (Supplementary Table S12).
Prognostic significance of ABCB1 and IL15 in patient subgroups In patients with favourable cytogenetics, T/T homozygosity at either ABCB1 2677 or 3435 position was associated with a significantly poorer EFS (PKM=0.011; PCOX=0.011, HR=4.02, 95% CI=1.38–11.75; Figure 2A). The EFS in patients carrying ABCB1 2677T/T or 3435T/T was 78.2%±7.6%, compared with 94.4%±1.9% in those carrying other genotypes. Such an association became even stronger after adjusting for MRD level (PCOX<0.001, HR=19.26, 95% CI=4.43–83.72; 80.6%±8.6% vs 98.9%±0.8%). In contrast, the impact of IL15 67276493 genotype was more pronounced in patients who do not carry chromosomal abnormalities of known prognosis, showing a significantly worse EFS in G/G carriers than the others (PKM=0.038; PCOX=0.022, HR=2.20, 95% CI=1.12–4.30; Figure 2C), with EFS of 73.9%±5.6% for G/G carriers and 86.7%±2.8% for G/C and C/C carriers, respectively. Adjustment for MRD level did not moderate the significance (PCOX=0.022, HR=2.30, 95% CI=1.13–4.70; 78.6%±5.4% vs 90.2%±2.7%).
In both cytogenetic subgroups, the additive effect of risk genotypes was found (PKM<0.001 and PCOX<0.001, Figure 2B; and PKM=0.013 and PCOX=0.009, Figure 2D) and remained significant after adjusting for MRD level (PCOX<0.001 and=0.008 respectively). In addition, we also examined the influence of risk genotypes in the two major ethnic groups, the Chinese and Malays, respectively. In both races, the additive effect of risk genotypes was also found (PKM<0.001 and PCOX=0.001 in the Chinese, Supplementary Figure S6A; PKM=0.075 and PCOX=0.036 in the Malays, Supplementary Figure S6B) and remained significant after adjusting for MRD level (PCOX=0.003 and=0.006, respectively).
In patients with favourable cytogenetics, T/T homozygosity at either ABCB1 2677 or 3435 position was associated with a significantly poorer EFS (PKM=0.011; PCOX=0.011, HR=4.02, 95% CI=1.38–11.75; Figure 2A). The EFS in patients carrying ABCB1 2677T/T or 3435T/T was 78.2%±7.6%, compared with 94.4%±1.9% in those carrying other genotypes. Such an association became even stronger after adjusting for MRD level (PCOX<0.001, HR=19.26, 95% CI=4.43–83.72; 80.6%±8.6% vs 98.9%±0.8%). In contrast, the impact of IL15 67276493 genotype was more pronounced in patients who do not carry chromosomal abnormalities of known prognosis, showing a significantly worse EFS in G/G carriers than the others (PKM=0.038; PCOX=0.022, HR=2.20, 95% CI=1.12–4.30; Figure 2C), with EFS of 73.9%±5.6% for G/G carriers and 86.7%±2.8% for G/C and C/C carriers, respectively. Adjustment for MRD level did not moderate the significance (PCOX=0.022, HR=2.30, 95% CI=1.13–4.70; 78.6%±5.4% vs 90.2%±2.7%).
In both cytogenetic subgroups, the additive effect of risk genotypes was found (PKM<0.001 and PCOX<0.001, Figure 2B; and PKM=0.013 and PCOX=0.009, Figure 2D) and remained significant after adjusting for MRD level (PCOX<0.001 and=0.008 respectively). In addition, we also examined the influence of risk genotypes in the two major ethnic groups, the Chinese and Malays, respectively. In both races, the additive effect of risk genotypes was also found (PKM<0.001 and PCOX=0.001 in the Chinese, Supplementary Figure S6A; PKM=0.075 and PCOX=0.036 in the Malays, Supplementary Figure S6B) and remained significant after adjusting for MRD level (PCOX=0.003 and=0.006, respectively).
Validation tests Patients were split into two halves based on the time of enrolment at each participating centre. In both cohorts, the additive effect of T/T homozygosity at ABCB1 2677 or 3435 and IL15 67276493G/G still remained significant (Supplementary Figure S7A, C), even after adjusting for MRD levels at the end of induction (Supplementary Figure S7B, D). In addition, we generated 100 random test sets by re-sampling. Of these 100 test scenarios, the significance was achieved for 86 test sets (Supplementary Figure S8A), and all results consistently showed that increasing number of risk genotypes corresponded to a poorer EFS (i.e., HR >1; Supplementary Figure S8B).
Patients were split into two halves based on the time of enrolment at each participating centre. In both cohorts, the additive effect of T/T homozygosity at ABCB1 2677 or 3435 and IL15 67276493G/G still remained significant (Supplementary Figure S7A, C), even after adjusting for MRD levels at the end of induction (Supplementary Figure S7B, D). In addition, we generated 100 random test sets by re-sampling. Of these 100 test scenarios, the significance was achieved for 86 test sets (Supplementary Figure S8A), and all results consistently showed that increasing number of risk genotypes corresponded to a poorer EFS (i.e., HR >1; Supplementary Figure S8B).
Linkage disequilibrium As multiple loci in ABCB1 and IL15 were genotyped, we calculated the linkage disequilibrium (LD) among different alleles. Two loci, ABCB1 2677 (rs2032582) and ABCB1 3435 (rs1045642), were in a moderate LD block. The LD was slightly stronger in the Chinese (r2=0.58 for entire patient cohort and r2=0.74 for the Chinese; Supplementary Figure S9), with two major haplotypes, 2677G-3435C (52.3%) and 2677T-3435T (36.2%). IL15 67276493G>C (rs17015014) was not in LD with any other studied loci of the IL15 gene (Supplementary Figure S10).
As multiple loci in ABCB1 and IL15 were genotyped, we calculated the linkage disequilibrium (LD) among different alleles. Two loci, ABCB1 2677 (rs2032582) and ABCB1 3435 (rs1045642), were in a moderate LD block. The LD was slightly stronger in the Chinese (r2=0.58 for entire patient cohort and r2=0.74 for the Chinese; Supplementary Figure S9), with two major haplotypes, 2677G-3435C (52.3%) and 2677T-3435T (36.2%). IL15 67276493G>C (rs17015014) was not in LD with any other studied loci of the IL15 gene (Supplementary Figure S10).
| null | null |
[
"Patients and samples",
"Cytogenetic subgroup, MRD, and genotype",
"Statistical analyses",
"Identification of polymorphisms of ABCB1 and IL15 as predictors of EFS",
"Prognostic significance of ABCB1 and IL15 in patient subgroups",
"Validation tests",
"Linkage disequilibrium"
] |
[
"We studied a total of 463 consecutive patients who were enrolled from July 2002 to August 2009 (median follow-up of 5.7 years upon data analyses) in the Malaysia-Singapore (Ma-Spore) ALL 2003 study from four participating centers in Singapore and Malaysia (Yeoh et al, 2012). This smaller cohort has similar EFS (Supplementary Figure S1) as well as demographic composition (data not shown) compared with the entire Ma-Spore ALL patient group. Genotyping assays were carried out in patients who had sufficient DNA. The patients are primarily comprised of Chinese, Malays and Indians, according to self-reported ethnicity. The treatment protocol was a modification of the ALL IC-BFM 2002 backbone (Fronkova et al, 2008) with additional CCG-augmented BFM regimen for high-risk patients (Nachman et al, 1997). Risk assignment was based on clinical- and cytogenetic-presenting features as well as early response to therapy as determined by prednisolone response and molecular MRD at the end of remission induction and consolidation (Supplementary Table S2).\nMononuclear cells were separated and collected from bone marrow aspirates or peripheral blood at diagnosis and after remission, using a Ficoll-Paque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. DNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendation.\nThe study was approved by the local institutional review boards at all participating institutions and informed consent was obtained from patients or their parents in accordance with the Declaration of Helsinki.",
"Morphological examination and immunophenotyping were performed in all patients. Karyotyping, DNA index, and oncogene fusion screening by reverse transcription PCR were performed in National University Hospital or KK Women's and Children's Hospital in Singapore. For the present study, t(9;22)/BCR-ABL1, t(11q23)/MLL rearrangements or hypodiploid ALL (modal chromosomes <45 or DNA index <1.0) were classified as unfavourable cytogenetic subgroup, while t(12;21)/ETV6-RUNX1, t(1:19)/TCF-PBX1 or hyperdiploid ALL (modal chromosomes >50 or DNA index ⩾1.16) were classified as a favourable subgroup.\nFor MRD monitoring, patient-specific, leukaemia-associated markers targeting the IgH, TCRδ, and IgK-Kde rearrangements were quantitated using BIOMED-II primers and protocols (McClure et al, 2006; Fronkova et al, 2008), and results were interpreted according to the criteria of the EuroMRD Study Group (Pongers-Willemse et al, 1999; van der Velden et al, 2006).\nGSTM1/T1 present/null and TYMS 28-bp enhancer repeat (rs34743033) were visualised on agarose gel directly after conventional PCR. Genotyping assays for other loci were performed using in-house allele-specific primer extension by real-time PCR. A qualified genotyping result must satisfy the following criteria: (a) at least one allele signal appears before the cycle threshold (CT) of 25; (b) for a homozygous call, CT difference between two allele signals exceeds four; and (c) for a heterozygous call, CT difference should be less than one. All bi-allelic loci studied were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci).",
"Homozygosity of the major allele in the single-nucleotide polymorphism (SNP) was used as the reference genotype unless otherwise specified. For each SNP, genotype was pooled as a dichotomous variable if applicable, and tested under both dominant and recessive models (1 degree of freedom). The grouping that yielded the smaller P-values was retained. To control for multiple testing while maintaining sufficient statistical power, we applied Benjamini–Hochberg Step-up FDR-controlling procedure for the results of preliminary test on individual SNP. Only the candidates with adjusted P-values of ⩽0.05 in multivariate tests were selected for further analyses. Subsequent investigation on the associations between shortlisted SNPs and various clinical end points was exploratory, where nominal P-values of ⩽0.05 were considered significant.\nPatients who abandoned treatment for more than six continuous weeks were excluded from all analyses provided that no event occurred before the abandonment. The EFS and OS were estimated by both Kaplan–Meier (PKM by log rank test) and Cox regression (PCOX by proportional hazards model) using PASW Statistics 18.0 (IBM, New York, NY, USA). The time-to-event was calculated from diagnosis until the occurrence of resistance (morphological blast count ⩾5% at the end of remission induction), relapse (at any site), or death (from any cause), whichever occurred first, in EFS analysis (N=74; the time-to-event for resistance and induction death was registered as 1 day), or until death in OS analysis (N=39). For patients in continuous complete remission (CCR), survival times were censored at the date of last follow-up or until 31 December 2012. Cumulative incidence of relapse (including resistance; N=51) was estimated in both univariate (PG by Gray's test) and multivariate analyses (PFG by Fine and Gray hazards model) using R (version 3.0.1) packages according to published guidelines (Scrucca et al, 2007, 2010), where death was considered as a competing event. Clinical-presenting features, that is, the race, sex, lineage, NCI risk group (the risk for infantile cases was set to ‘high' in statistical analyses), and cytogenetic subgroup, were used as co-variables in all multivariate analyses. The genotypes were examined together with clinical features in a full model (that is, non-stepwise mode). The particular genotypes that were associated with poorer EFS (termed as risk genotypes) were also tested for their additive effect on EFS, with an increasing number of such risk genotypes. When appropriate, Day 33 (end-of-induction) MRD was further added in the regression model to test the robustness of significant results discovered.\nTo investigate potential sampling bias, patients enrolled were split into two halves according to the time of enrolment at each participating center, and the additive effect of risk genotypes was re-examined. In addition, as a validation we performed re-sampling to generate 100 test sets each comprising 50% of events and 50% of continuous complete remission randomly selected from the original cohort and evaluated the additive effect of risk genotypes in each set. In both scenarios, clinical co-variables were included in analyses.\nBinary logistic regression was used to identify the genotypes associated with failure to achieve complete remission (that is, resistance or induction death; N=30) and treatment-related mortality (defined as the death during remission induction or in complete remission without prior leukemic relapse; N=23) after adjusting for clinical and cytogenetic features. The genotype frequencies in different categorical groups were compared by using the Pearson Chi-square test.\nHaplotype frequencies of ABCB1 and IL15 genes were calculated using Haploview (version 4.2) (Barrett et al, 2005).",
"The clinical and cytogenetic features at presentation and the prevalence of alleles studied are summarised in Table 1, Supplementary Tables S3a and S3b, respectively. All bi-allelic loci were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci). Of the presenting features examined, unfavourable cytogenetics was associated with a significantly poorer EFS (PCOX<0.001; Table 2), while NCI high-risk status had borderline significance (PCOX=0.091; Table 2); self-reported ethnicity, gender, and cell type were not significantly related to EFS. The complete genotype data as well as patient characteristics are available for research purposes upon request.\nAmong the 20 polymorphisms examined individually in preliminary test, polymorphisms in ABCB1 (rs2032582 and rs1045642) and IL15 (rs17015014) were found eligible for further analyses (adjusted P=0.038 and 0.048, respectively; Supplementary Table S4). They were subsequently analysed together in a Cox regression model adjusting for presenting clinical and cytogenetic features (Table 2). The homozygosity of either ABCB1 2677T/T or 3435T/T was still significantly related with a poorer EFS compared with other genotypes at these two loci (PKM=0.001; PCOX=0.003, HR=2.11, 95% CI=1.29–3.45; Figure 1A). There was moderate linkage disequilibrium (LD) between 2677T and 3435T (details shown in later section). IL15 67276493G/G was also associated with significantly poorer EFS compared with 67276493G/C and C/C (PKM=0.050; PCOX=0.022, HR=1.84, 95% CI=1.09–3.08; Figure 1B). Adjusting for MRD at the end of remission induction (Supplementary Figures S2 A–B) or excluding the cases of resistance from EFS analyses (Supplementary Table S5 and Supplementary Figures S3A–B) did not alter the significance of these polymorphisms identified.\nThe risk genotypes, that is, ABCB1 T/T at 2677 or 3435 or both as well as IL15 67276493G/G, exhibited an additive effect (PKM<0.001; PCOX<0.001, HR=1.97, 95% CI=1.38–2.82) demonstrating progressively poorer EFS in patients with an increasing number of risk genotypes (Table 2 and Figure 1C). Compared with the patients without any risk genotype, the hazard of adverse events raised 2.2 folds in patients with one risk genotype (PKM=0.005; PCOX=0.003, HR=2.20, 95% CI=1.30–3.70) and 3.6 folds in those with two risk genotypes (PKM=0.002; PCOX=0.001, HR=3.64, 95% CI=1.68–7.92). After adjusting for MRD at the end of remission induction (Supplementary Table S6 and Supplementary Figure S2C) or excluding the cases of resistance (Supplementary Table S7 and Supplementary Figure S3C), this additive effect remained significant. There was no significant EFS difference between patients with only ABCB1 risk genotype and those with only IL15 risk genotype (data not shown).\nTo further investigate the impact of ABCB1 2677G>T/A/3435C>T and IL15 67276493G>C genotypes on a specific event, we examined their influence on the cumulative incidence of relapse (including resistance), failure to achieve complete remission as well as treatment-related mortality. Homozygosity of either ABCB1 2677T/T or 3435T/T significantly increased the risk of relapse in both univariate (PG=0.001; Supplementary Figure S4A) and multivariate models after adjusting for clinical and cytogenetic features (PFG=0.011, HR=2.17, 95% CI=1.19–3.94; Supplementary Table S8). The risk of relapse in patients with at least one T/T was 20.4% compared with 9.0% in those without any T/T. Taking the end-of-induction MRD level into account did not alter the significance (PFG=0.027, HR=2.01, 95% CI=1.08–3.73). The risk of relapse also became higher with increasing number of risk genotypes (PG<0.001; Supplementary Figure S4B; PFG=0.011, HR=1.84, 95% CI=1.15–2.93; Supplementary Table S8). The trend remained significant after adjusting for the end-of-induction MRD level (PFG=0.026, HR=1.69, 95% CI=1.07–2.68).\nIL15 67276493G/G was associated with higher risk of failure to achieve complete remission when compared with G/C and C/C genotypes (P=0.009, OR=3.14, 95% CI=1.34–7.36; Supplementary Table S9). G/G homozygosity also correlated with poorer overall survival (PKM=0.050; PCOX=0.005, HR=2.74, 95% CI=1.35–5.58; Supplementary Table S10 and Supplementary Figure S5), primarily due to more than 3-fold higher risk of treatment-related mortality in patients with IL15 67276493G/G (P=0.015, OR=3.40, 95% CI=1.27–9.11; Supplementary Table S11).\nWe also examined the associations of identified risk genotypes with known risk factors. Neither T/T homozygosity at ABCB1 2677 or 3435 nor IL15 67276493G/G was found to correlate with cytogenetics and MRD level after remission induction (Supplementary Table S12).",
"In patients with favourable cytogenetics, T/T homozygosity at either ABCB1 2677 or 3435 position was associated with a significantly poorer EFS (PKM=0.011; PCOX=0.011, HR=4.02, 95% CI=1.38–11.75; Figure 2A). The EFS in patients carrying ABCB1 2677T/T or 3435T/T was 78.2%±7.6%, compared with 94.4%±1.9% in those carrying other genotypes. Such an association became even stronger after adjusting for MRD level (PCOX<0.001, HR=19.26, 95% CI=4.43–83.72; 80.6%±8.6% vs 98.9%±0.8%). In contrast, the impact of IL15 67276493 genotype was more pronounced in patients who do not carry chromosomal abnormalities of known prognosis, showing a significantly worse EFS in G/G carriers than the others (PKM=0.038; PCOX=0.022, HR=2.20, 95% CI=1.12–4.30; Figure 2C), with EFS of 73.9%±5.6% for G/G carriers and 86.7%±2.8% for G/C and C/C carriers, respectively. Adjustment for MRD level did not moderate the significance (PCOX=0.022, HR=2.30, 95% CI=1.13–4.70; 78.6%±5.4% vs 90.2%±2.7%).\nIn both cytogenetic subgroups, the additive effect of risk genotypes was found (PKM<0.001 and PCOX<0.001, Figure 2B; and PKM=0.013 and PCOX=0.009, Figure 2D) and remained significant after adjusting for MRD level (PCOX<0.001 and=0.008 respectively). In addition, we also examined the influence of risk genotypes in the two major ethnic groups, the Chinese and Malays, respectively. In both races, the additive effect of risk genotypes was also found (PKM<0.001 and PCOX=0.001 in the Chinese, Supplementary Figure S6A; PKM=0.075 and PCOX=0.036 in the Malays, Supplementary Figure S6B) and remained significant after adjusting for MRD level (PCOX=0.003 and=0.006, respectively).",
"Patients were split into two halves based on the time of enrolment at each participating centre. In both cohorts, the additive effect of T/T homozygosity at ABCB1 2677 or 3435 and IL15 67276493G/G still remained significant (Supplementary Figure S7A, C), even after adjusting for MRD levels at the end of induction (Supplementary Figure S7B, D). In addition, we generated 100 random test sets by re-sampling. Of these 100 test scenarios, the significance was achieved for 86 test sets (Supplementary Figure S8A), and all results consistently showed that increasing number of risk genotypes corresponded to a poorer EFS (i.e., HR >1; Supplementary Figure S8B).",
"As multiple loci in ABCB1 and IL15 were genotyped, we calculated the linkage disequilibrium (LD) among different alleles. Two loci, ABCB1 2677 (rs2032582) and ABCB1 3435 (rs1045642), were in a moderate LD block. The LD was slightly stronger in the Chinese (r2=0.58 for entire patient cohort and r2=0.74 for the Chinese; Supplementary Figure S9), with two major haplotypes, 2677G-3435C (52.3%) and 2677T-3435T (36.2%). IL15 67276493G>C (rs17015014) was not in LD with any other studied loci of the IL15 gene (Supplementary Figure S10)."
] |
[
null,
null,
null,
null,
null,
null,
null
] |
[
"Materials and methods",
"Patients and samples",
"Cytogenetic subgroup, MRD, and genotype",
"Statistical analyses",
"Results",
"Identification of polymorphisms of ABCB1 and IL15 as predictors of EFS",
"Prognostic significance of ABCB1 and IL15 in patient subgroups",
"Validation tests",
"Linkage disequilibrium",
"Discussion"
] |
[
" Patients and samples We studied a total of 463 consecutive patients who were enrolled from July 2002 to August 2009 (median follow-up of 5.7 years upon data analyses) in the Malaysia-Singapore (Ma-Spore) ALL 2003 study from four participating centers in Singapore and Malaysia (Yeoh et al, 2012). This smaller cohort has similar EFS (Supplementary Figure S1) as well as demographic composition (data not shown) compared with the entire Ma-Spore ALL patient group. Genotyping assays were carried out in patients who had sufficient DNA. The patients are primarily comprised of Chinese, Malays and Indians, according to self-reported ethnicity. The treatment protocol was a modification of the ALL IC-BFM 2002 backbone (Fronkova et al, 2008) with additional CCG-augmented BFM regimen for high-risk patients (Nachman et al, 1997). Risk assignment was based on clinical- and cytogenetic-presenting features as well as early response to therapy as determined by prednisolone response and molecular MRD at the end of remission induction and consolidation (Supplementary Table S2).\nMononuclear cells were separated and collected from bone marrow aspirates or peripheral blood at diagnosis and after remission, using a Ficoll-Paque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. DNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendation.\nThe study was approved by the local institutional review boards at all participating institutions and informed consent was obtained from patients or their parents in accordance with the Declaration of Helsinki.\nWe studied a total of 463 consecutive patients who were enrolled from July 2002 to August 2009 (median follow-up of 5.7 years upon data analyses) in the Malaysia-Singapore (Ma-Spore) ALL 2003 study from four participating centers in Singapore and Malaysia (Yeoh et al, 2012). This smaller cohort has similar EFS (Supplementary Figure S1) as well as demographic composition (data not shown) compared with the entire Ma-Spore ALL patient group. Genotyping assays were carried out in patients who had sufficient DNA. The patients are primarily comprised of Chinese, Malays and Indians, according to self-reported ethnicity. The treatment protocol was a modification of the ALL IC-BFM 2002 backbone (Fronkova et al, 2008) with additional CCG-augmented BFM regimen for high-risk patients (Nachman et al, 1997). Risk assignment was based on clinical- and cytogenetic-presenting features as well as early response to therapy as determined by prednisolone response and molecular MRD at the end of remission induction and consolidation (Supplementary Table S2).\nMononuclear cells were separated and collected from bone marrow aspirates or peripheral blood at diagnosis and after remission, using a Ficoll-Paque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. DNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendation.\nThe study was approved by the local institutional review boards at all participating institutions and informed consent was obtained from patients or their parents in accordance with the Declaration of Helsinki.\n Cytogenetic subgroup, MRD, and genotype Morphological examination and immunophenotyping were performed in all patients. Karyotyping, DNA index, and oncogene fusion screening by reverse transcription PCR were performed in National University Hospital or KK Women's and Children's Hospital in Singapore. For the present study, t(9;22)/BCR-ABL1, t(11q23)/MLL rearrangements or hypodiploid ALL (modal chromosomes <45 or DNA index <1.0) were classified as unfavourable cytogenetic subgroup, while t(12;21)/ETV6-RUNX1, t(1:19)/TCF-PBX1 or hyperdiploid ALL (modal chromosomes >50 or DNA index ⩾1.16) were classified as a favourable subgroup.\nFor MRD monitoring, patient-specific, leukaemia-associated markers targeting the IgH, TCRδ, and IgK-Kde rearrangements were quantitated using BIOMED-II primers and protocols (McClure et al, 2006; Fronkova et al, 2008), and results were interpreted according to the criteria of the EuroMRD Study Group (Pongers-Willemse et al, 1999; van der Velden et al, 2006).\nGSTM1/T1 present/null and TYMS 28-bp enhancer repeat (rs34743033) were visualised on agarose gel directly after conventional PCR. Genotyping assays for other loci were performed using in-house allele-specific primer extension by real-time PCR. A qualified genotyping result must satisfy the following criteria: (a) at least one allele signal appears before the cycle threshold (CT) of 25; (b) for a homozygous call, CT difference between two allele signals exceeds four; and (c) for a heterozygous call, CT difference should be less than one. All bi-allelic loci studied were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci).\nMorphological examination and immunophenotyping were performed in all patients. Karyotyping, DNA index, and oncogene fusion screening by reverse transcription PCR were performed in National University Hospital or KK Women's and Children's Hospital in Singapore. For the present study, t(9;22)/BCR-ABL1, t(11q23)/MLL rearrangements or hypodiploid ALL (modal chromosomes <45 or DNA index <1.0) were classified as unfavourable cytogenetic subgroup, while t(12;21)/ETV6-RUNX1, t(1:19)/TCF-PBX1 or hyperdiploid ALL (modal chromosomes >50 or DNA index ⩾1.16) were classified as a favourable subgroup.\nFor MRD monitoring, patient-specific, leukaemia-associated markers targeting the IgH, TCRδ, and IgK-Kde rearrangements were quantitated using BIOMED-II primers and protocols (McClure et al, 2006; Fronkova et al, 2008), and results were interpreted according to the criteria of the EuroMRD Study Group (Pongers-Willemse et al, 1999; van der Velden et al, 2006).\nGSTM1/T1 present/null and TYMS 28-bp enhancer repeat (rs34743033) were visualised on agarose gel directly after conventional PCR. Genotyping assays for other loci were performed using in-house allele-specific primer extension by real-time PCR. A qualified genotyping result must satisfy the following criteria: (a) at least one allele signal appears before the cycle threshold (CT) of 25; (b) for a homozygous call, CT difference between two allele signals exceeds four; and (c) for a heterozygous call, CT difference should be less than one. All bi-allelic loci studied were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci).\n Statistical analyses Homozygosity of the major allele in the single-nucleotide polymorphism (SNP) was used as the reference genotype unless otherwise specified. For each SNP, genotype was pooled as a dichotomous variable if applicable, and tested under both dominant and recessive models (1 degree of freedom). The grouping that yielded the smaller P-values was retained. To control for multiple testing while maintaining sufficient statistical power, we applied Benjamini–Hochberg Step-up FDR-controlling procedure for the results of preliminary test on individual SNP. Only the candidates with adjusted P-values of ⩽0.05 in multivariate tests were selected for further analyses. Subsequent investigation on the associations between shortlisted SNPs and various clinical end points was exploratory, where nominal P-values of ⩽0.05 were considered significant.\nPatients who abandoned treatment for more than six continuous weeks were excluded from all analyses provided that no event occurred before the abandonment. The EFS and OS were estimated by both Kaplan–Meier (PKM by log rank test) and Cox regression (PCOX by proportional hazards model) using PASW Statistics 18.0 (IBM, New York, NY, USA). The time-to-event was calculated from diagnosis until the occurrence of resistance (morphological blast count ⩾5% at the end of remission induction), relapse (at any site), or death (from any cause), whichever occurred first, in EFS analysis (N=74; the time-to-event for resistance and induction death was registered as 1 day), or until death in OS analysis (N=39). For patients in continuous complete remission (CCR), survival times were censored at the date of last follow-up or until 31 December 2012. Cumulative incidence of relapse (including resistance; N=51) was estimated in both univariate (PG by Gray's test) and multivariate analyses (PFG by Fine and Gray hazards model) using R (version 3.0.1) packages according to published guidelines (Scrucca et al, 2007, 2010), where death was considered as a competing event. Clinical-presenting features, that is, the race, sex, lineage, NCI risk group (the risk for infantile cases was set to ‘high' in statistical analyses), and cytogenetic subgroup, were used as co-variables in all multivariate analyses. The genotypes were examined together with clinical features in a full model (that is, non-stepwise mode). The particular genotypes that were associated with poorer EFS (termed as risk genotypes) were also tested for their additive effect on EFS, with an increasing number of such risk genotypes. When appropriate, Day 33 (end-of-induction) MRD was further added in the regression model to test the robustness of significant results discovered.\nTo investigate potential sampling bias, patients enrolled were split into two halves according to the time of enrolment at each participating center, and the additive effect of risk genotypes was re-examined. In addition, as a validation we performed re-sampling to generate 100 test sets each comprising 50% of events and 50% of continuous complete remission randomly selected from the original cohort and evaluated the additive effect of risk genotypes in each set. In both scenarios, clinical co-variables were included in analyses.\nBinary logistic regression was used to identify the genotypes associated with failure to achieve complete remission (that is, resistance or induction death; N=30) and treatment-related mortality (defined as the death during remission induction or in complete remission without prior leukemic relapse; N=23) after adjusting for clinical and cytogenetic features. The genotype frequencies in different categorical groups were compared by using the Pearson Chi-square test.\nHaplotype frequencies of ABCB1 and IL15 genes were calculated using Haploview (version 4.2) (Barrett et al, 2005).\nHomozygosity of the major allele in the single-nucleotide polymorphism (SNP) was used as the reference genotype unless otherwise specified. For each SNP, genotype was pooled as a dichotomous variable if applicable, and tested under both dominant and recessive models (1 degree of freedom). The grouping that yielded the smaller P-values was retained. To control for multiple testing while maintaining sufficient statistical power, we applied Benjamini–Hochberg Step-up FDR-controlling procedure for the results of preliminary test on individual SNP. Only the candidates with adjusted P-values of ⩽0.05 in multivariate tests were selected for further analyses. Subsequent investigation on the associations between shortlisted SNPs and various clinical end points was exploratory, where nominal P-values of ⩽0.05 were considered significant.\nPatients who abandoned treatment for more than six continuous weeks were excluded from all analyses provided that no event occurred before the abandonment. The EFS and OS were estimated by both Kaplan–Meier (PKM by log rank test) and Cox regression (PCOX by proportional hazards model) using PASW Statistics 18.0 (IBM, New York, NY, USA). The time-to-event was calculated from diagnosis until the occurrence of resistance (morphological blast count ⩾5% at the end of remission induction), relapse (at any site), or death (from any cause), whichever occurred first, in EFS analysis (N=74; the time-to-event for resistance and induction death was registered as 1 day), or until death in OS analysis (N=39). For patients in continuous complete remission (CCR), survival times were censored at the date of last follow-up or until 31 December 2012. Cumulative incidence of relapse (including resistance; N=51) was estimated in both univariate (PG by Gray's test) and multivariate analyses (PFG by Fine and Gray hazards model) using R (version 3.0.1) packages according to published guidelines (Scrucca et al, 2007, 2010), where death was considered as a competing event. Clinical-presenting features, that is, the race, sex, lineage, NCI risk group (the risk for infantile cases was set to ‘high' in statistical analyses), and cytogenetic subgroup, were used as co-variables in all multivariate analyses. The genotypes were examined together with clinical features in a full model (that is, non-stepwise mode). The particular genotypes that were associated with poorer EFS (termed as risk genotypes) were also tested for their additive effect on EFS, with an increasing number of such risk genotypes. When appropriate, Day 33 (end-of-induction) MRD was further added in the regression model to test the robustness of significant results discovered.\nTo investigate potential sampling bias, patients enrolled were split into two halves according to the time of enrolment at each participating center, and the additive effect of risk genotypes was re-examined. In addition, as a validation we performed re-sampling to generate 100 test sets each comprising 50% of events and 50% of continuous complete remission randomly selected from the original cohort and evaluated the additive effect of risk genotypes in each set. In both scenarios, clinical co-variables were included in analyses.\nBinary logistic regression was used to identify the genotypes associated with failure to achieve complete remission (that is, resistance or induction death; N=30) and treatment-related mortality (defined as the death during remission induction or in complete remission without prior leukemic relapse; N=23) after adjusting for clinical and cytogenetic features. The genotype frequencies in different categorical groups were compared by using the Pearson Chi-square test.\nHaplotype frequencies of ABCB1 and IL15 genes were calculated using Haploview (version 4.2) (Barrett et al, 2005).",
"We studied a total of 463 consecutive patients who were enrolled from July 2002 to August 2009 (median follow-up of 5.7 years upon data analyses) in the Malaysia-Singapore (Ma-Spore) ALL 2003 study from four participating centers in Singapore and Malaysia (Yeoh et al, 2012). This smaller cohort has similar EFS (Supplementary Figure S1) as well as demographic composition (data not shown) compared with the entire Ma-Spore ALL patient group. Genotyping assays were carried out in patients who had sufficient DNA. The patients are primarily comprised of Chinese, Malays and Indians, according to self-reported ethnicity. The treatment protocol was a modification of the ALL IC-BFM 2002 backbone (Fronkova et al, 2008) with additional CCG-augmented BFM regimen for high-risk patients (Nachman et al, 1997). Risk assignment was based on clinical- and cytogenetic-presenting features as well as early response to therapy as determined by prednisolone response and molecular MRD at the end of remission induction and consolidation (Supplementary Table S2).\nMononuclear cells were separated and collected from bone marrow aspirates or peripheral blood at diagnosis and after remission, using a Ficoll-Paque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. DNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendation.\nThe study was approved by the local institutional review boards at all participating institutions and informed consent was obtained from patients or their parents in accordance with the Declaration of Helsinki.",
"Morphological examination and immunophenotyping were performed in all patients. Karyotyping, DNA index, and oncogene fusion screening by reverse transcription PCR were performed in National University Hospital or KK Women's and Children's Hospital in Singapore. For the present study, t(9;22)/BCR-ABL1, t(11q23)/MLL rearrangements or hypodiploid ALL (modal chromosomes <45 or DNA index <1.0) were classified as unfavourable cytogenetic subgroup, while t(12;21)/ETV6-RUNX1, t(1:19)/TCF-PBX1 or hyperdiploid ALL (modal chromosomes >50 or DNA index ⩾1.16) were classified as a favourable subgroup.\nFor MRD monitoring, patient-specific, leukaemia-associated markers targeting the IgH, TCRδ, and IgK-Kde rearrangements were quantitated using BIOMED-II primers and protocols (McClure et al, 2006; Fronkova et al, 2008), and results were interpreted according to the criteria of the EuroMRD Study Group (Pongers-Willemse et al, 1999; van der Velden et al, 2006).\nGSTM1/T1 present/null and TYMS 28-bp enhancer repeat (rs34743033) were visualised on agarose gel directly after conventional PCR. Genotyping assays for other loci were performed using in-house allele-specific primer extension by real-time PCR. A qualified genotyping result must satisfy the following criteria: (a) at least one allele signal appears before the cycle threshold (CT) of 25; (b) for a homozygous call, CT difference between two allele signals exceeds four; and (c) for a heterozygous call, CT difference should be less than one. All bi-allelic loci studied were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci).",
"Homozygosity of the major allele in the single-nucleotide polymorphism (SNP) was used as the reference genotype unless otherwise specified. For each SNP, genotype was pooled as a dichotomous variable if applicable, and tested under both dominant and recessive models (1 degree of freedom). The grouping that yielded the smaller P-values was retained. To control for multiple testing while maintaining sufficient statistical power, we applied Benjamini–Hochberg Step-up FDR-controlling procedure for the results of preliminary test on individual SNP. Only the candidates with adjusted P-values of ⩽0.05 in multivariate tests were selected for further analyses. Subsequent investigation on the associations between shortlisted SNPs and various clinical end points was exploratory, where nominal P-values of ⩽0.05 were considered significant.\nPatients who abandoned treatment for more than six continuous weeks were excluded from all analyses provided that no event occurred before the abandonment. The EFS and OS were estimated by both Kaplan–Meier (PKM by log rank test) and Cox regression (PCOX by proportional hazards model) using PASW Statistics 18.0 (IBM, New York, NY, USA). The time-to-event was calculated from diagnosis until the occurrence of resistance (morphological blast count ⩾5% at the end of remission induction), relapse (at any site), or death (from any cause), whichever occurred first, in EFS analysis (N=74; the time-to-event for resistance and induction death was registered as 1 day), or until death in OS analysis (N=39). For patients in continuous complete remission (CCR), survival times were censored at the date of last follow-up or until 31 December 2012. Cumulative incidence of relapse (including resistance; N=51) was estimated in both univariate (PG by Gray's test) and multivariate analyses (PFG by Fine and Gray hazards model) using R (version 3.0.1) packages according to published guidelines (Scrucca et al, 2007, 2010), where death was considered as a competing event. Clinical-presenting features, that is, the race, sex, lineage, NCI risk group (the risk for infantile cases was set to ‘high' in statistical analyses), and cytogenetic subgroup, were used as co-variables in all multivariate analyses. The genotypes were examined together with clinical features in a full model (that is, non-stepwise mode). The particular genotypes that were associated with poorer EFS (termed as risk genotypes) were also tested for their additive effect on EFS, with an increasing number of such risk genotypes. When appropriate, Day 33 (end-of-induction) MRD was further added in the regression model to test the robustness of significant results discovered.\nTo investigate potential sampling bias, patients enrolled were split into two halves according to the time of enrolment at each participating center, and the additive effect of risk genotypes was re-examined. In addition, as a validation we performed re-sampling to generate 100 test sets each comprising 50% of events and 50% of continuous complete remission randomly selected from the original cohort and evaluated the additive effect of risk genotypes in each set. In both scenarios, clinical co-variables were included in analyses.\nBinary logistic regression was used to identify the genotypes associated with failure to achieve complete remission (that is, resistance or induction death; N=30) and treatment-related mortality (defined as the death during remission induction or in complete remission without prior leukemic relapse; N=23) after adjusting for clinical and cytogenetic features. The genotype frequencies in different categorical groups were compared by using the Pearson Chi-square test.\nHaplotype frequencies of ABCB1 and IL15 genes were calculated using Haploview (version 4.2) (Barrett et al, 2005).",
" Identification of polymorphisms of ABCB1 and IL15 as predictors of EFS The clinical and cytogenetic features at presentation and the prevalence of alleles studied are summarised in Table 1, Supplementary Tables S3a and S3b, respectively. All bi-allelic loci were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci). Of the presenting features examined, unfavourable cytogenetics was associated with a significantly poorer EFS (PCOX<0.001; Table 2), while NCI high-risk status had borderline significance (PCOX=0.091; Table 2); self-reported ethnicity, gender, and cell type were not significantly related to EFS. The complete genotype data as well as patient characteristics are available for research purposes upon request.\nAmong the 20 polymorphisms examined individually in preliminary test, polymorphisms in ABCB1 (rs2032582 and rs1045642) and IL15 (rs17015014) were found eligible for further analyses (adjusted P=0.038 and 0.048, respectively; Supplementary Table S4). They were subsequently analysed together in a Cox regression model adjusting for presenting clinical and cytogenetic features (Table 2). The homozygosity of either ABCB1 2677T/T or 3435T/T was still significantly related with a poorer EFS compared with other genotypes at these two loci (PKM=0.001; PCOX=0.003, HR=2.11, 95% CI=1.29–3.45; Figure 1A). There was moderate linkage disequilibrium (LD) between 2677T and 3435T (details shown in later section). IL15 67276493G/G was also associated with significantly poorer EFS compared with 67276493G/C and C/C (PKM=0.050; PCOX=0.022, HR=1.84, 95% CI=1.09–3.08; Figure 1B). Adjusting for MRD at the end of remission induction (Supplementary Figures S2 A–B) or excluding the cases of resistance from EFS analyses (Supplementary Table S5 and Supplementary Figures S3A–B) did not alter the significance of these polymorphisms identified.\nThe risk genotypes, that is, ABCB1 T/T at 2677 or 3435 or both as well as IL15 67276493G/G, exhibited an additive effect (PKM<0.001; PCOX<0.001, HR=1.97, 95% CI=1.38–2.82) demonstrating progressively poorer EFS in patients with an increasing number of risk genotypes (Table 2 and Figure 1C). Compared with the patients without any risk genotype, the hazard of adverse events raised 2.2 folds in patients with one risk genotype (PKM=0.005; PCOX=0.003, HR=2.20, 95% CI=1.30–3.70) and 3.6 folds in those with two risk genotypes (PKM=0.002; PCOX=0.001, HR=3.64, 95% CI=1.68–7.92). After adjusting for MRD at the end of remission induction (Supplementary Table S6 and Supplementary Figure S2C) or excluding the cases of resistance (Supplementary Table S7 and Supplementary Figure S3C), this additive effect remained significant. There was no significant EFS difference between patients with only ABCB1 risk genotype and those with only IL15 risk genotype (data not shown).\nTo further investigate the impact of ABCB1 2677G>T/A/3435C>T and IL15 67276493G>C genotypes on a specific event, we examined their influence on the cumulative incidence of relapse (including resistance), failure to achieve complete remission as well as treatment-related mortality. Homozygosity of either ABCB1 2677T/T or 3435T/T significantly increased the risk of relapse in both univariate (PG=0.001; Supplementary Figure S4A) and multivariate models after adjusting for clinical and cytogenetic features (PFG=0.011, HR=2.17, 95% CI=1.19–3.94; Supplementary Table S8). The risk of relapse in patients with at least one T/T was 20.4% compared with 9.0% in those without any T/T. Taking the end-of-induction MRD level into account did not alter the significance (PFG=0.027, HR=2.01, 95% CI=1.08–3.73). The risk of relapse also became higher with increasing number of risk genotypes (PG<0.001; Supplementary Figure S4B; PFG=0.011, HR=1.84, 95% CI=1.15–2.93; Supplementary Table S8). The trend remained significant after adjusting for the end-of-induction MRD level (PFG=0.026, HR=1.69, 95% CI=1.07–2.68).\nIL15 67276493G/G was associated with higher risk of failure to achieve complete remission when compared with G/C and C/C genotypes (P=0.009, OR=3.14, 95% CI=1.34–7.36; Supplementary Table S9). G/G homozygosity also correlated with poorer overall survival (PKM=0.050; PCOX=0.005, HR=2.74, 95% CI=1.35–5.58; Supplementary Table S10 and Supplementary Figure S5), primarily due to more than 3-fold higher risk of treatment-related mortality in patients with IL15 67276493G/G (P=0.015, OR=3.40, 95% CI=1.27–9.11; Supplementary Table S11).\nWe also examined the associations of identified risk genotypes with known risk factors. Neither T/T homozygosity at ABCB1 2677 or 3435 nor IL15 67276493G/G was found to correlate with cytogenetics and MRD level after remission induction (Supplementary Table S12).\nThe clinical and cytogenetic features at presentation and the prevalence of alleles studied are summarised in Table 1, Supplementary Tables S3a and S3b, respectively. All bi-allelic loci were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci). Of the presenting features examined, unfavourable cytogenetics was associated with a significantly poorer EFS (PCOX<0.001; Table 2), while NCI high-risk status had borderline significance (PCOX=0.091; Table 2); self-reported ethnicity, gender, and cell type were not significantly related to EFS. The complete genotype data as well as patient characteristics are available for research purposes upon request.\nAmong the 20 polymorphisms examined individually in preliminary test, polymorphisms in ABCB1 (rs2032582 and rs1045642) and IL15 (rs17015014) were found eligible for further analyses (adjusted P=0.038 and 0.048, respectively; Supplementary Table S4). They were subsequently analysed together in a Cox regression model adjusting for presenting clinical and cytogenetic features (Table 2). The homozygosity of either ABCB1 2677T/T or 3435T/T was still significantly related with a poorer EFS compared with other genotypes at these two loci (PKM=0.001; PCOX=0.003, HR=2.11, 95% CI=1.29–3.45; Figure 1A). There was moderate linkage disequilibrium (LD) between 2677T and 3435T (details shown in later section). IL15 67276493G/G was also associated with significantly poorer EFS compared with 67276493G/C and C/C (PKM=0.050; PCOX=0.022, HR=1.84, 95% CI=1.09–3.08; Figure 1B). Adjusting for MRD at the end of remission induction (Supplementary Figures S2 A–B) or excluding the cases of resistance from EFS analyses (Supplementary Table S5 and Supplementary Figures S3A–B) did not alter the significance of these polymorphisms identified.\nThe risk genotypes, that is, ABCB1 T/T at 2677 or 3435 or both as well as IL15 67276493G/G, exhibited an additive effect (PKM<0.001; PCOX<0.001, HR=1.97, 95% CI=1.38–2.82) demonstrating progressively poorer EFS in patients with an increasing number of risk genotypes (Table 2 and Figure 1C). Compared with the patients without any risk genotype, the hazard of adverse events raised 2.2 folds in patients with one risk genotype (PKM=0.005; PCOX=0.003, HR=2.20, 95% CI=1.30–3.70) and 3.6 folds in those with two risk genotypes (PKM=0.002; PCOX=0.001, HR=3.64, 95% CI=1.68–7.92). After adjusting for MRD at the end of remission induction (Supplementary Table S6 and Supplementary Figure S2C) or excluding the cases of resistance (Supplementary Table S7 and Supplementary Figure S3C), this additive effect remained significant. There was no significant EFS difference between patients with only ABCB1 risk genotype and those with only IL15 risk genotype (data not shown).\nTo further investigate the impact of ABCB1 2677G>T/A/3435C>T and IL15 67276493G>C genotypes on a specific event, we examined their influence on the cumulative incidence of relapse (including resistance), failure to achieve complete remission as well as treatment-related mortality. Homozygosity of either ABCB1 2677T/T or 3435T/T significantly increased the risk of relapse in both univariate (PG=0.001; Supplementary Figure S4A) and multivariate models after adjusting for clinical and cytogenetic features (PFG=0.011, HR=2.17, 95% CI=1.19–3.94; Supplementary Table S8). The risk of relapse in patients with at least one T/T was 20.4% compared with 9.0% in those without any T/T. Taking the end-of-induction MRD level into account did not alter the significance (PFG=0.027, HR=2.01, 95% CI=1.08–3.73). The risk of relapse also became higher with increasing number of risk genotypes (PG<0.001; Supplementary Figure S4B; PFG=0.011, HR=1.84, 95% CI=1.15–2.93; Supplementary Table S8). The trend remained significant after adjusting for the end-of-induction MRD level (PFG=0.026, HR=1.69, 95% CI=1.07–2.68).\nIL15 67276493G/G was associated with higher risk of failure to achieve complete remission when compared with G/C and C/C genotypes (P=0.009, OR=3.14, 95% CI=1.34–7.36; Supplementary Table S9). G/G homozygosity also correlated with poorer overall survival (PKM=0.050; PCOX=0.005, HR=2.74, 95% CI=1.35–5.58; Supplementary Table S10 and Supplementary Figure S5), primarily due to more than 3-fold higher risk of treatment-related mortality in patients with IL15 67276493G/G (P=0.015, OR=3.40, 95% CI=1.27–9.11; Supplementary Table S11).\nWe also examined the associations of identified risk genotypes with known risk factors. Neither T/T homozygosity at ABCB1 2677 or 3435 nor IL15 67276493G/G was found to correlate with cytogenetics and MRD level after remission induction (Supplementary Table S12).\n Prognostic significance of ABCB1 and IL15 in patient subgroups In patients with favourable cytogenetics, T/T homozygosity at either ABCB1 2677 or 3435 position was associated with a significantly poorer EFS (PKM=0.011; PCOX=0.011, HR=4.02, 95% CI=1.38–11.75; Figure 2A). The EFS in patients carrying ABCB1 2677T/T or 3435T/T was 78.2%±7.6%, compared with 94.4%±1.9% in those carrying other genotypes. Such an association became even stronger after adjusting for MRD level (PCOX<0.001, HR=19.26, 95% CI=4.43–83.72; 80.6%±8.6% vs 98.9%±0.8%). In contrast, the impact of IL15 67276493 genotype was more pronounced in patients who do not carry chromosomal abnormalities of known prognosis, showing a significantly worse EFS in G/G carriers than the others (PKM=0.038; PCOX=0.022, HR=2.20, 95% CI=1.12–4.30; Figure 2C), with EFS of 73.9%±5.6% for G/G carriers and 86.7%±2.8% for G/C and C/C carriers, respectively. Adjustment for MRD level did not moderate the significance (PCOX=0.022, HR=2.30, 95% CI=1.13–4.70; 78.6%±5.4% vs 90.2%±2.7%).\nIn both cytogenetic subgroups, the additive effect of risk genotypes was found (PKM<0.001 and PCOX<0.001, Figure 2B; and PKM=0.013 and PCOX=0.009, Figure 2D) and remained significant after adjusting for MRD level (PCOX<0.001 and=0.008 respectively). In addition, we also examined the influence of risk genotypes in the two major ethnic groups, the Chinese and Malays, respectively. In both races, the additive effect of risk genotypes was also found (PKM<0.001 and PCOX=0.001 in the Chinese, Supplementary Figure S6A; PKM=0.075 and PCOX=0.036 in the Malays, Supplementary Figure S6B) and remained significant after adjusting for MRD level (PCOX=0.003 and=0.006, respectively).\nIn patients with favourable cytogenetics, T/T homozygosity at either ABCB1 2677 or 3435 position was associated with a significantly poorer EFS (PKM=0.011; PCOX=0.011, HR=4.02, 95% CI=1.38–11.75; Figure 2A). The EFS in patients carrying ABCB1 2677T/T or 3435T/T was 78.2%±7.6%, compared with 94.4%±1.9% in those carrying other genotypes. Such an association became even stronger after adjusting for MRD level (PCOX<0.001, HR=19.26, 95% CI=4.43–83.72; 80.6%±8.6% vs 98.9%±0.8%). In contrast, the impact of IL15 67276493 genotype was more pronounced in patients who do not carry chromosomal abnormalities of known prognosis, showing a significantly worse EFS in G/G carriers than the others (PKM=0.038; PCOX=0.022, HR=2.20, 95% CI=1.12–4.30; Figure 2C), with EFS of 73.9%±5.6% for G/G carriers and 86.7%±2.8% for G/C and C/C carriers, respectively. Adjustment for MRD level did not moderate the significance (PCOX=0.022, HR=2.30, 95% CI=1.13–4.70; 78.6%±5.4% vs 90.2%±2.7%).\nIn both cytogenetic subgroups, the additive effect of risk genotypes was found (PKM<0.001 and PCOX<0.001, Figure 2B; and PKM=0.013 and PCOX=0.009, Figure 2D) and remained significant after adjusting for MRD level (PCOX<0.001 and=0.008 respectively). In addition, we also examined the influence of risk genotypes in the two major ethnic groups, the Chinese and Malays, respectively. In both races, the additive effect of risk genotypes was also found (PKM<0.001 and PCOX=0.001 in the Chinese, Supplementary Figure S6A; PKM=0.075 and PCOX=0.036 in the Malays, Supplementary Figure S6B) and remained significant after adjusting for MRD level (PCOX=0.003 and=0.006, respectively).\n Validation tests Patients were split into two halves based on the time of enrolment at each participating centre. In both cohorts, the additive effect of T/T homozygosity at ABCB1 2677 or 3435 and IL15 67276493G/G still remained significant (Supplementary Figure S7A, C), even after adjusting for MRD levels at the end of induction (Supplementary Figure S7B, D). In addition, we generated 100 random test sets by re-sampling. Of these 100 test scenarios, the significance was achieved for 86 test sets (Supplementary Figure S8A), and all results consistently showed that increasing number of risk genotypes corresponded to a poorer EFS (i.e., HR >1; Supplementary Figure S8B).\nPatients were split into two halves based on the time of enrolment at each participating centre. In both cohorts, the additive effect of T/T homozygosity at ABCB1 2677 or 3435 and IL15 67276493G/G still remained significant (Supplementary Figure S7A, C), even after adjusting for MRD levels at the end of induction (Supplementary Figure S7B, D). In addition, we generated 100 random test sets by re-sampling. Of these 100 test scenarios, the significance was achieved for 86 test sets (Supplementary Figure S8A), and all results consistently showed that increasing number of risk genotypes corresponded to a poorer EFS (i.e., HR >1; Supplementary Figure S8B).\n Linkage disequilibrium As multiple loci in ABCB1 and IL15 were genotyped, we calculated the linkage disequilibrium (LD) among different alleles. Two loci, ABCB1 2677 (rs2032582) and ABCB1 3435 (rs1045642), were in a moderate LD block. The LD was slightly stronger in the Chinese (r2=0.58 for entire patient cohort and r2=0.74 for the Chinese; Supplementary Figure S9), with two major haplotypes, 2677G-3435C (52.3%) and 2677T-3435T (36.2%). IL15 67276493G>C (rs17015014) was not in LD with any other studied loci of the IL15 gene (Supplementary Figure S10).\nAs multiple loci in ABCB1 and IL15 were genotyped, we calculated the linkage disequilibrium (LD) among different alleles. Two loci, ABCB1 2677 (rs2032582) and ABCB1 3435 (rs1045642), were in a moderate LD block. The LD was slightly stronger in the Chinese (r2=0.58 for entire patient cohort and r2=0.74 for the Chinese; Supplementary Figure S9), with two major haplotypes, 2677G-3435C (52.3%) and 2677T-3435T (36.2%). IL15 67276493G>C (rs17015014) was not in LD with any other studied loci of the IL15 gene (Supplementary Figure S10).",
"The clinical and cytogenetic features at presentation and the prevalence of alleles studied are summarised in Table 1, Supplementary Tables S3a and S3b, respectively. All bi-allelic loci were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci). Of the presenting features examined, unfavourable cytogenetics was associated with a significantly poorer EFS (PCOX<0.001; Table 2), while NCI high-risk status had borderline significance (PCOX=0.091; Table 2); self-reported ethnicity, gender, and cell type were not significantly related to EFS. The complete genotype data as well as patient characteristics are available for research purposes upon request.\nAmong the 20 polymorphisms examined individually in preliminary test, polymorphisms in ABCB1 (rs2032582 and rs1045642) and IL15 (rs17015014) were found eligible for further analyses (adjusted P=0.038 and 0.048, respectively; Supplementary Table S4). They were subsequently analysed together in a Cox regression model adjusting for presenting clinical and cytogenetic features (Table 2). The homozygosity of either ABCB1 2677T/T or 3435T/T was still significantly related with a poorer EFS compared with other genotypes at these two loci (PKM=0.001; PCOX=0.003, HR=2.11, 95% CI=1.29–3.45; Figure 1A). There was moderate linkage disequilibrium (LD) between 2677T and 3435T (details shown in later section). IL15 67276493G/G was also associated with significantly poorer EFS compared with 67276493G/C and C/C (PKM=0.050; PCOX=0.022, HR=1.84, 95% CI=1.09–3.08; Figure 1B). Adjusting for MRD at the end of remission induction (Supplementary Figures S2 A–B) or excluding the cases of resistance from EFS analyses (Supplementary Table S5 and Supplementary Figures S3A–B) did not alter the significance of these polymorphisms identified.\nThe risk genotypes, that is, ABCB1 T/T at 2677 or 3435 or both as well as IL15 67276493G/G, exhibited an additive effect (PKM<0.001; PCOX<0.001, HR=1.97, 95% CI=1.38–2.82) demonstrating progressively poorer EFS in patients with an increasing number of risk genotypes (Table 2 and Figure 1C). Compared with the patients without any risk genotype, the hazard of adverse events raised 2.2 folds in patients with one risk genotype (PKM=0.005; PCOX=0.003, HR=2.20, 95% CI=1.30–3.70) and 3.6 folds in those with two risk genotypes (PKM=0.002; PCOX=0.001, HR=3.64, 95% CI=1.68–7.92). After adjusting for MRD at the end of remission induction (Supplementary Table S6 and Supplementary Figure S2C) or excluding the cases of resistance (Supplementary Table S7 and Supplementary Figure S3C), this additive effect remained significant. There was no significant EFS difference between patients with only ABCB1 risk genotype and those with only IL15 risk genotype (data not shown).\nTo further investigate the impact of ABCB1 2677G>T/A/3435C>T and IL15 67276493G>C genotypes on a specific event, we examined their influence on the cumulative incidence of relapse (including resistance), failure to achieve complete remission as well as treatment-related mortality. Homozygosity of either ABCB1 2677T/T or 3435T/T significantly increased the risk of relapse in both univariate (PG=0.001; Supplementary Figure S4A) and multivariate models after adjusting for clinical and cytogenetic features (PFG=0.011, HR=2.17, 95% CI=1.19–3.94; Supplementary Table S8). The risk of relapse in patients with at least one T/T was 20.4% compared with 9.0% in those without any T/T. Taking the end-of-induction MRD level into account did not alter the significance (PFG=0.027, HR=2.01, 95% CI=1.08–3.73). The risk of relapse also became higher with increasing number of risk genotypes (PG<0.001; Supplementary Figure S4B; PFG=0.011, HR=1.84, 95% CI=1.15–2.93; Supplementary Table S8). The trend remained significant after adjusting for the end-of-induction MRD level (PFG=0.026, HR=1.69, 95% CI=1.07–2.68).\nIL15 67276493G/G was associated with higher risk of failure to achieve complete remission when compared with G/C and C/C genotypes (P=0.009, OR=3.14, 95% CI=1.34–7.36; Supplementary Table S9). G/G homozygosity also correlated with poorer overall survival (PKM=0.050; PCOX=0.005, HR=2.74, 95% CI=1.35–5.58; Supplementary Table S10 and Supplementary Figure S5), primarily due to more than 3-fold higher risk of treatment-related mortality in patients with IL15 67276493G/G (P=0.015, OR=3.40, 95% CI=1.27–9.11; Supplementary Table S11).\nWe also examined the associations of identified risk genotypes with known risk factors. Neither T/T homozygosity at ABCB1 2677 or 3435 nor IL15 67276493G/G was found to correlate with cytogenetics and MRD level after remission induction (Supplementary Table S12).",
"In patients with favourable cytogenetics, T/T homozygosity at either ABCB1 2677 or 3435 position was associated with a significantly poorer EFS (PKM=0.011; PCOX=0.011, HR=4.02, 95% CI=1.38–11.75; Figure 2A). The EFS in patients carrying ABCB1 2677T/T or 3435T/T was 78.2%±7.6%, compared with 94.4%±1.9% in those carrying other genotypes. Such an association became even stronger after adjusting for MRD level (PCOX<0.001, HR=19.26, 95% CI=4.43–83.72; 80.6%±8.6% vs 98.9%±0.8%). In contrast, the impact of IL15 67276493 genotype was more pronounced in patients who do not carry chromosomal abnormalities of known prognosis, showing a significantly worse EFS in G/G carriers than the others (PKM=0.038; PCOX=0.022, HR=2.20, 95% CI=1.12–4.30; Figure 2C), with EFS of 73.9%±5.6% for G/G carriers and 86.7%±2.8% for G/C and C/C carriers, respectively. Adjustment for MRD level did not moderate the significance (PCOX=0.022, HR=2.30, 95% CI=1.13–4.70; 78.6%±5.4% vs 90.2%±2.7%).\nIn both cytogenetic subgroups, the additive effect of risk genotypes was found (PKM<0.001 and PCOX<0.001, Figure 2B; and PKM=0.013 and PCOX=0.009, Figure 2D) and remained significant after adjusting for MRD level (PCOX<0.001 and=0.008 respectively). In addition, we also examined the influence of risk genotypes in the two major ethnic groups, the Chinese and Malays, respectively. In both races, the additive effect of risk genotypes was also found (PKM<0.001 and PCOX=0.001 in the Chinese, Supplementary Figure S6A; PKM=0.075 and PCOX=0.036 in the Malays, Supplementary Figure S6B) and remained significant after adjusting for MRD level (PCOX=0.003 and=0.006, respectively).",
"Patients were split into two halves based on the time of enrolment at each participating centre. In both cohorts, the additive effect of T/T homozygosity at ABCB1 2677 or 3435 and IL15 67276493G/G still remained significant (Supplementary Figure S7A, C), even after adjusting for MRD levels at the end of induction (Supplementary Figure S7B, D). In addition, we generated 100 random test sets by re-sampling. Of these 100 test scenarios, the significance was achieved for 86 test sets (Supplementary Figure S8A), and all results consistently showed that increasing number of risk genotypes corresponded to a poorer EFS (i.e., HR >1; Supplementary Figure S8B).",
"As multiple loci in ABCB1 and IL15 were genotyped, we calculated the linkage disequilibrium (LD) among different alleles. Two loci, ABCB1 2677 (rs2032582) and ABCB1 3435 (rs1045642), were in a moderate LD block. The LD was slightly stronger in the Chinese (r2=0.58 for entire patient cohort and r2=0.74 for the Chinese; Supplementary Figure S9), with two major haplotypes, 2677G-3435C (52.3%) and 2677T-3435T (36.2%). IL15 67276493G>C (rs17015014) was not in LD with any other studied loci of the IL15 gene (Supplementary Figure S10).",
"In childhood ALL, there is significant inter-individual variation in response to chemotherapy, which is not fully predicted by presenting features such as age and leukaemic cytogenetics. Newly described markers such as IKZF1 and CRLF2 have demonstrated prognostic impact in many studies (Mullighan et al, 2009; Buitenkamp et al, 2012; Chen et al, 2012; Feng and Tang, 2013), but their values still remain inconclusive in the Malaysia-Singapore ALL 2003 study according to our pilot investigations (data not shown). Here we showed that host germline variants of ABCB1 and IL15 genes are significant predictors of treatment outcome, suggesting that the characterisation of these genes' variants at diagnosis could contribute to risk-stratification strategies. In our study, the ABCB1 2677T/T or 3435T/T genotypes were associated with an increased risk of relapse/resistance. ABCB1 2677G>T/A introduces an Ala-to-Ser/Thr change in exon 21 while 3435C>T is a synonymous polymorphism in exon 26, which affects transport by altering substrate specificity (Kimchi-Sarfaty et al, 2007). Various studies have shown that not all synonymous SNPs are considered silent; they can cause changes in protein expression, conformation or function and are increasingly implicated in human disease risk and other complex traits (Sauna and Kimchi-Sarfaty, 2011).\nLarge studies in patients with acute myocardial infarction on oral anti-coagulant clopidogrel, a substrate of P-glycoprotein (P-gp), reported lower drug levels and poorer cardiovascular outcomes in patients with ABCB1 3435T/T homozygotes (Simon et al, 2009; Mega et al, 2010). Oral dexamethasone, used extensively in paediatric ALL treatment including our Ma-Spore ALL 2003 protocol, is also a known substrate for P-gp. Patients with ABCB1 2677T/T or 3435T/T may have lower systemic dexamethasone exposure due to higher ABCB1 expression on the apical villi of enterocytes (Nakamura et al, 2002), causing enhanced extrusion of drugs into the intestinal lumen. Supporting this hypothesis, Yang et al (2012) recently reported two ABCB1 variants (rs10264856 and rs4728709) that affected oral dexamethasone clearance and were associated with increased risk of relapse in the St Jude and COG cohorts, although we did not observe any LD between rs10264856/rs4728709 and rs2032582/rs1045642 in our study as well as the data from 1000 Genomes Project (the maximum r2 is only 0.14). Taken together with our findings on ABCB1 2677 and 3435, these strongly suggest the importance of ABCB1 variants in treatment of childhood ALL.\nHowever, Jamroziak et al (2004) and Stanulla et al (2005b) previously reported lower EFS (N=111) and higher CNS relapse (N=34), respectively, in ABCB1 3435C/C carriers treated on the BFM-ALL 86/90/95 protocols. Differences in the type of steroid used during induction therapy (prednisolone in the BFM-ALL 86/90/95 compared with dexamethasone in Ma-Spore ALL 2003) may account for this observed difference. In addition, the lower frequency of ABCB1 2677T and 3435T alleles among African Americans (10.0% and 20.2%, respectively) (Kroetz et al, 2003) compared with our studied population (43.6% and 40.5%, respectively) may mask the effect of ABCB1 variants on treatment outcome in the previously reported St Jude Total XIIIB protocol (Rocha et al, 2005).\nThe manner that ABCB1 variants affect the P-gp expression on the apical villi of enterocytes remains unresolved (Lin and Yamazaki, 2003; Leschziner et al, 2007; Hodges et al, 2011; Wolf et al, 2011). Nakamura et al (2002) reported that healthy Japanese subjects with 3435T/T had significantly higher duodenal ABCB1 mRNA expression and lower digoxin plasma level compared with C/C or C/T genotype carriers, while Hoffmeyer et al (2000) and Efferth et al (2003) disagreed with such a correlation. The effect of ABCB1 polymorphisms on P-gp activity is likely dependent on the substrates as well as the tissues (or cell types) studied, thus becoming a confounding factor when studying P-gp genotype–phenotype relationship. Further studies in correlating dexamethasone clearance and ABCB1 polymorphisms are required.\nConsidering the drug efflux capacity of P-gp, it is rational to speculate that the impact of ABCB1 genotype on the risk of relapse/resistance originates from its effect on MRD level. Although not statistically significant in our patients, Day 33 MRD high risk (i.e., ⩾10−2) tended to be enriched in the ABCB1 T/T group compared with the others (14.9% vs 8.8%, P=0.088, Supplementary Table S12). Published data on the relationship between ABCB1 gene and MRD are controversial. For instance, an earlier report showed that the median ABCB1 expression at diagnosis of childhood ALL were roughly comparable between the MRD-positive group and MRD-negative group at the end of induction therapy (Fedasenka et al, 2008). This finding was challenged by a very recent study reporting that mRNA expression of ABCB1 higher than a certain cutoff would raise the risk of MRD positivity by 12-fold after 1-year treatment in children with ALL (Rahgozar et al, 2014). Such discrepancies are likely due to (but not limited to) the diverse grouping criteria, the quantitation methods applied, the time points investigated, and sampled populations in different studies. As the determinants on early treatment response are known to be multifactorial, a clear correlation between ABCB1 genotype and MRD level may not be expected.\nThe IL15 67276493G/G genotype was associated with a poorer EFS and OS in our study, and carried a higher probability of treatment-related mortality. IL15 regulates T- and natural killer cell activation and proliferation, having central roles in cell-mediated immunity against microbes (Yoshikai and Nishimura, 2000). Administration of exogenous IL15 in animal models causes severe but reversible neutropenia due to redistribution of neutrophils out of the circulation (Berger et al, 2009; Waldmann et al, 2011). This SNP resides in the 3′UTR region of the gene, which exerts a negative regulatory effect on the expression of IL15; polymorphism at the 3′UTR may reduce this negative regulation, resulting in an enhanced expression. It is noteworthy that genetic variants in IL15, albeit different IL15 SNPs, have been previously inferred to significantly correlate with the MRD level at the end of induction (Yang et al, 2009), a strong surrogate marker to predict treatment outcome.\nABCB1 3435T/T or 2677T/T and IL15 67276493G/G showed a surprising additive effect in predicting a poorer EFS probability, even after adjusting for MRD levels at the end of remission induction. P-glycoprotein mediates in transmembrane transport of cytokines. It has been reported that ABCB1 2677T/T–3435T/T are associated with lower secretion of cytokines in PHA-stimulated lymphocytes (Pawlik et al, 2005). Conversely, in HIV-infected individuals, P-glycoprotein expression and function are decreased and restored only after IL15 stimulation (Chang et al, 2000). Therefore, we speculate that ABCB1 2677T/T or 3435T/T may alter secretion of IL15, which affects cellular immune responses and treatment outcome consequently.\nIn summary, host genetic variations in ABCB1 and IL15 genes influence treatment outcomes in paediatric ALL in this predominantly Asian population treated on the Ma-Spore ALL 2003. Inclusion of host variants in ABCB1 and IL15 genes may help future risk assignment strategies and deserves further study in other treatment cohorts."
] |
[
"materials|methods",
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion"
] |
[
"paediatric ALL",
"prognosis",
"single-nucleotide polymorphism",
"\nABCB1\n",
"\nIL15\n"
] |
Materials and methods:
Patients and samples We studied a total of 463 consecutive patients who were enrolled from July 2002 to August 2009 (median follow-up of 5.7 years upon data analyses) in the Malaysia-Singapore (Ma-Spore) ALL 2003 study from four participating centers in Singapore and Malaysia (Yeoh et al, 2012). This smaller cohort has similar EFS (Supplementary Figure S1) as well as demographic composition (data not shown) compared with the entire Ma-Spore ALL patient group. Genotyping assays were carried out in patients who had sufficient DNA. The patients are primarily comprised of Chinese, Malays and Indians, according to self-reported ethnicity. The treatment protocol was a modification of the ALL IC-BFM 2002 backbone (Fronkova et al, 2008) with additional CCG-augmented BFM regimen for high-risk patients (Nachman et al, 1997). Risk assignment was based on clinical- and cytogenetic-presenting features as well as early response to therapy as determined by prednisolone response and molecular MRD at the end of remission induction and consolidation (Supplementary Table S2).
Mononuclear cells were separated and collected from bone marrow aspirates or peripheral blood at diagnosis and after remission, using a Ficoll-Paque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. DNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendation.
The study was approved by the local institutional review boards at all participating institutions and informed consent was obtained from patients or their parents in accordance with the Declaration of Helsinki.
We studied a total of 463 consecutive patients who were enrolled from July 2002 to August 2009 (median follow-up of 5.7 years upon data analyses) in the Malaysia-Singapore (Ma-Spore) ALL 2003 study from four participating centers in Singapore and Malaysia (Yeoh et al, 2012). This smaller cohort has similar EFS (Supplementary Figure S1) as well as demographic composition (data not shown) compared with the entire Ma-Spore ALL patient group. Genotyping assays were carried out in patients who had sufficient DNA. The patients are primarily comprised of Chinese, Malays and Indians, according to self-reported ethnicity. The treatment protocol was a modification of the ALL IC-BFM 2002 backbone (Fronkova et al, 2008) with additional CCG-augmented BFM regimen for high-risk patients (Nachman et al, 1997). Risk assignment was based on clinical- and cytogenetic-presenting features as well as early response to therapy as determined by prednisolone response and molecular MRD at the end of remission induction and consolidation (Supplementary Table S2).
Mononuclear cells were separated and collected from bone marrow aspirates or peripheral blood at diagnosis and after remission, using a Ficoll-Paque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. DNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendation.
The study was approved by the local institutional review boards at all participating institutions and informed consent was obtained from patients or their parents in accordance with the Declaration of Helsinki.
Cytogenetic subgroup, MRD, and genotype Morphological examination and immunophenotyping were performed in all patients. Karyotyping, DNA index, and oncogene fusion screening by reverse transcription PCR were performed in National University Hospital or KK Women's and Children's Hospital in Singapore. For the present study, t(9;22)/BCR-ABL1, t(11q23)/MLL rearrangements or hypodiploid ALL (modal chromosomes <45 or DNA index <1.0) were classified as unfavourable cytogenetic subgroup, while t(12;21)/ETV6-RUNX1, t(1:19)/TCF-PBX1 or hyperdiploid ALL (modal chromosomes >50 or DNA index ⩾1.16) were classified as a favourable subgroup.
For MRD monitoring, patient-specific, leukaemia-associated markers targeting the IgH, TCRδ, and IgK-Kde rearrangements were quantitated using BIOMED-II primers and protocols (McClure et al, 2006; Fronkova et al, 2008), and results were interpreted according to the criteria of the EuroMRD Study Group (Pongers-Willemse et al, 1999; van der Velden et al, 2006).
GSTM1/T1 present/null and TYMS 28-bp enhancer repeat (rs34743033) were visualised on agarose gel directly after conventional PCR. Genotyping assays for other loci were performed using in-house allele-specific primer extension by real-time PCR. A qualified genotyping result must satisfy the following criteria: (a) at least one allele signal appears before the cycle threshold (CT) of 25; (b) for a homozygous call, CT difference between two allele signals exceeds four; and (c) for a heterozygous call, CT difference should be less than one. All bi-allelic loci studied were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci).
Morphological examination and immunophenotyping were performed in all patients. Karyotyping, DNA index, and oncogene fusion screening by reverse transcription PCR were performed in National University Hospital or KK Women's and Children's Hospital in Singapore. For the present study, t(9;22)/BCR-ABL1, t(11q23)/MLL rearrangements or hypodiploid ALL (modal chromosomes <45 or DNA index <1.0) were classified as unfavourable cytogenetic subgroup, while t(12;21)/ETV6-RUNX1, t(1:19)/TCF-PBX1 or hyperdiploid ALL (modal chromosomes >50 or DNA index ⩾1.16) were classified as a favourable subgroup.
For MRD monitoring, patient-specific, leukaemia-associated markers targeting the IgH, TCRδ, and IgK-Kde rearrangements were quantitated using BIOMED-II primers and protocols (McClure et al, 2006; Fronkova et al, 2008), and results were interpreted according to the criteria of the EuroMRD Study Group (Pongers-Willemse et al, 1999; van der Velden et al, 2006).
GSTM1/T1 present/null and TYMS 28-bp enhancer repeat (rs34743033) were visualised on agarose gel directly after conventional PCR. Genotyping assays for other loci were performed using in-house allele-specific primer extension by real-time PCR. A qualified genotyping result must satisfy the following criteria: (a) at least one allele signal appears before the cycle threshold (CT) of 25; (b) for a homozygous call, CT difference between two allele signals exceeds four; and (c) for a heterozygous call, CT difference should be less than one. All bi-allelic loci studied were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci).
Statistical analyses Homozygosity of the major allele in the single-nucleotide polymorphism (SNP) was used as the reference genotype unless otherwise specified. For each SNP, genotype was pooled as a dichotomous variable if applicable, and tested under both dominant and recessive models (1 degree of freedom). The grouping that yielded the smaller P-values was retained. To control for multiple testing while maintaining sufficient statistical power, we applied Benjamini–Hochberg Step-up FDR-controlling procedure for the results of preliminary test on individual SNP. Only the candidates with adjusted P-values of ⩽0.05 in multivariate tests were selected for further analyses. Subsequent investigation on the associations between shortlisted SNPs and various clinical end points was exploratory, where nominal P-values of ⩽0.05 were considered significant.
Patients who abandoned treatment for more than six continuous weeks were excluded from all analyses provided that no event occurred before the abandonment. The EFS and OS were estimated by both Kaplan–Meier (PKM by log rank test) and Cox regression (PCOX by proportional hazards model) using PASW Statistics 18.0 (IBM, New York, NY, USA). The time-to-event was calculated from diagnosis until the occurrence of resistance (morphological blast count ⩾5% at the end of remission induction), relapse (at any site), or death (from any cause), whichever occurred first, in EFS analysis (N=74; the time-to-event for resistance and induction death was registered as 1 day), or until death in OS analysis (N=39). For patients in continuous complete remission (CCR), survival times were censored at the date of last follow-up or until 31 December 2012. Cumulative incidence of relapse (including resistance; N=51) was estimated in both univariate (PG by Gray's test) and multivariate analyses (PFG by Fine and Gray hazards model) using R (version 3.0.1) packages according to published guidelines (Scrucca et al, 2007, 2010), where death was considered as a competing event. Clinical-presenting features, that is, the race, sex, lineage, NCI risk group (the risk for infantile cases was set to ‘high' in statistical analyses), and cytogenetic subgroup, were used as co-variables in all multivariate analyses. The genotypes were examined together with clinical features in a full model (that is, non-stepwise mode). The particular genotypes that were associated with poorer EFS (termed as risk genotypes) were also tested for their additive effect on EFS, with an increasing number of such risk genotypes. When appropriate, Day 33 (end-of-induction) MRD was further added in the regression model to test the robustness of significant results discovered.
To investigate potential sampling bias, patients enrolled were split into two halves according to the time of enrolment at each participating center, and the additive effect of risk genotypes was re-examined. In addition, as a validation we performed re-sampling to generate 100 test sets each comprising 50% of events and 50% of continuous complete remission randomly selected from the original cohort and evaluated the additive effect of risk genotypes in each set. In both scenarios, clinical co-variables were included in analyses.
Binary logistic regression was used to identify the genotypes associated with failure to achieve complete remission (that is, resistance or induction death; N=30) and treatment-related mortality (defined as the death during remission induction or in complete remission without prior leukemic relapse; N=23) after adjusting for clinical and cytogenetic features. The genotype frequencies in different categorical groups were compared by using the Pearson Chi-square test.
Haplotype frequencies of ABCB1 and IL15 genes were calculated using Haploview (version 4.2) (Barrett et al, 2005).
Homozygosity of the major allele in the single-nucleotide polymorphism (SNP) was used as the reference genotype unless otherwise specified. For each SNP, genotype was pooled as a dichotomous variable if applicable, and tested under both dominant and recessive models (1 degree of freedom). The grouping that yielded the smaller P-values was retained. To control for multiple testing while maintaining sufficient statistical power, we applied Benjamini–Hochberg Step-up FDR-controlling procedure for the results of preliminary test on individual SNP. Only the candidates with adjusted P-values of ⩽0.05 in multivariate tests were selected for further analyses. Subsequent investigation on the associations between shortlisted SNPs and various clinical end points was exploratory, where nominal P-values of ⩽0.05 were considered significant.
Patients who abandoned treatment for more than six continuous weeks were excluded from all analyses provided that no event occurred before the abandonment. The EFS and OS were estimated by both Kaplan–Meier (PKM by log rank test) and Cox regression (PCOX by proportional hazards model) using PASW Statistics 18.0 (IBM, New York, NY, USA). The time-to-event was calculated from diagnosis until the occurrence of resistance (morphological blast count ⩾5% at the end of remission induction), relapse (at any site), or death (from any cause), whichever occurred first, in EFS analysis (N=74; the time-to-event for resistance and induction death was registered as 1 day), or until death in OS analysis (N=39). For patients in continuous complete remission (CCR), survival times were censored at the date of last follow-up or until 31 December 2012. Cumulative incidence of relapse (including resistance; N=51) was estimated in both univariate (PG by Gray's test) and multivariate analyses (PFG by Fine and Gray hazards model) using R (version 3.0.1) packages according to published guidelines (Scrucca et al, 2007, 2010), where death was considered as a competing event. Clinical-presenting features, that is, the race, sex, lineage, NCI risk group (the risk for infantile cases was set to ‘high' in statistical analyses), and cytogenetic subgroup, were used as co-variables in all multivariate analyses. The genotypes were examined together with clinical features in a full model (that is, non-stepwise mode). The particular genotypes that were associated with poorer EFS (termed as risk genotypes) were also tested for their additive effect on EFS, with an increasing number of such risk genotypes. When appropriate, Day 33 (end-of-induction) MRD was further added in the regression model to test the robustness of significant results discovered.
To investigate potential sampling bias, patients enrolled were split into two halves according to the time of enrolment at each participating center, and the additive effect of risk genotypes was re-examined. In addition, as a validation we performed re-sampling to generate 100 test sets each comprising 50% of events and 50% of continuous complete remission randomly selected from the original cohort and evaluated the additive effect of risk genotypes in each set. In both scenarios, clinical co-variables were included in analyses.
Binary logistic regression was used to identify the genotypes associated with failure to achieve complete remission (that is, resistance or induction death; N=30) and treatment-related mortality (defined as the death during remission induction or in complete remission without prior leukemic relapse; N=23) after adjusting for clinical and cytogenetic features. The genotype frequencies in different categorical groups were compared by using the Pearson Chi-square test.
Haplotype frequencies of ABCB1 and IL15 genes were calculated using Haploview (version 4.2) (Barrett et al, 2005).
Patients and samples:
We studied a total of 463 consecutive patients who were enrolled from July 2002 to August 2009 (median follow-up of 5.7 years upon data analyses) in the Malaysia-Singapore (Ma-Spore) ALL 2003 study from four participating centers in Singapore and Malaysia (Yeoh et al, 2012). This smaller cohort has similar EFS (Supplementary Figure S1) as well as demographic composition (data not shown) compared with the entire Ma-Spore ALL patient group. Genotyping assays were carried out in patients who had sufficient DNA. The patients are primarily comprised of Chinese, Malays and Indians, according to self-reported ethnicity. The treatment protocol was a modification of the ALL IC-BFM 2002 backbone (Fronkova et al, 2008) with additional CCG-augmented BFM regimen for high-risk patients (Nachman et al, 1997). Risk assignment was based on clinical- and cytogenetic-presenting features as well as early response to therapy as determined by prednisolone response and molecular MRD at the end of remission induction and consolidation (Supplementary Table S2).
Mononuclear cells were separated and collected from bone marrow aspirates or peripheral blood at diagnosis and after remission, using a Ficoll-Paque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. DNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendation.
The study was approved by the local institutional review boards at all participating institutions and informed consent was obtained from patients or their parents in accordance with the Declaration of Helsinki.
Cytogenetic subgroup, MRD, and genotype:
Morphological examination and immunophenotyping were performed in all patients. Karyotyping, DNA index, and oncogene fusion screening by reverse transcription PCR were performed in National University Hospital or KK Women's and Children's Hospital in Singapore. For the present study, t(9;22)/BCR-ABL1, t(11q23)/MLL rearrangements or hypodiploid ALL (modal chromosomes <45 or DNA index <1.0) were classified as unfavourable cytogenetic subgroup, while t(12;21)/ETV6-RUNX1, t(1:19)/TCF-PBX1 or hyperdiploid ALL (modal chromosomes >50 or DNA index ⩾1.16) were classified as a favourable subgroup.
For MRD monitoring, patient-specific, leukaemia-associated markers targeting the IgH, TCRδ, and IgK-Kde rearrangements were quantitated using BIOMED-II primers and protocols (McClure et al, 2006; Fronkova et al, 2008), and results were interpreted according to the criteria of the EuroMRD Study Group (Pongers-Willemse et al, 1999; van der Velden et al, 2006).
GSTM1/T1 present/null and TYMS 28-bp enhancer repeat (rs34743033) were visualised on agarose gel directly after conventional PCR. Genotyping assays for other loci were performed using in-house allele-specific primer extension by real-time PCR. A qualified genotyping result must satisfy the following criteria: (a) at least one allele signal appears before the cycle threshold (CT) of 25; (b) for a homozygous call, CT difference between two allele signals exceeds four; and (c) for a heterozygous call, CT difference should be less than one. All bi-allelic loci studied were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci).
Statistical analyses:
Homozygosity of the major allele in the single-nucleotide polymorphism (SNP) was used as the reference genotype unless otherwise specified. For each SNP, genotype was pooled as a dichotomous variable if applicable, and tested under both dominant and recessive models (1 degree of freedom). The grouping that yielded the smaller P-values was retained. To control for multiple testing while maintaining sufficient statistical power, we applied Benjamini–Hochberg Step-up FDR-controlling procedure for the results of preliminary test on individual SNP. Only the candidates with adjusted P-values of ⩽0.05 in multivariate tests were selected for further analyses. Subsequent investigation on the associations between shortlisted SNPs and various clinical end points was exploratory, where nominal P-values of ⩽0.05 were considered significant.
Patients who abandoned treatment for more than six continuous weeks were excluded from all analyses provided that no event occurred before the abandonment. The EFS and OS were estimated by both Kaplan–Meier (PKM by log rank test) and Cox regression (PCOX by proportional hazards model) using PASW Statistics 18.0 (IBM, New York, NY, USA). The time-to-event was calculated from diagnosis until the occurrence of resistance (morphological blast count ⩾5% at the end of remission induction), relapse (at any site), or death (from any cause), whichever occurred first, in EFS analysis (N=74; the time-to-event for resistance and induction death was registered as 1 day), or until death in OS analysis (N=39). For patients in continuous complete remission (CCR), survival times were censored at the date of last follow-up or until 31 December 2012. Cumulative incidence of relapse (including resistance; N=51) was estimated in both univariate (PG by Gray's test) and multivariate analyses (PFG by Fine and Gray hazards model) using R (version 3.0.1) packages according to published guidelines (Scrucca et al, 2007, 2010), where death was considered as a competing event. Clinical-presenting features, that is, the race, sex, lineage, NCI risk group (the risk for infantile cases was set to ‘high' in statistical analyses), and cytogenetic subgroup, were used as co-variables in all multivariate analyses. The genotypes were examined together with clinical features in a full model (that is, non-stepwise mode). The particular genotypes that were associated with poorer EFS (termed as risk genotypes) were also tested for their additive effect on EFS, with an increasing number of such risk genotypes. When appropriate, Day 33 (end-of-induction) MRD was further added in the regression model to test the robustness of significant results discovered.
To investigate potential sampling bias, patients enrolled were split into two halves according to the time of enrolment at each participating center, and the additive effect of risk genotypes was re-examined. In addition, as a validation we performed re-sampling to generate 100 test sets each comprising 50% of events and 50% of continuous complete remission randomly selected from the original cohort and evaluated the additive effect of risk genotypes in each set. In both scenarios, clinical co-variables were included in analyses.
Binary logistic regression was used to identify the genotypes associated with failure to achieve complete remission (that is, resistance or induction death; N=30) and treatment-related mortality (defined as the death during remission induction or in complete remission without prior leukemic relapse; N=23) after adjusting for clinical and cytogenetic features. The genotype frequencies in different categorical groups were compared by using the Pearson Chi-square test.
Haplotype frequencies of ABCB1 and IL15 genes were calculated using Haploview (version 4.2) (Barrett et al, 2005).
Results:
Identification of polymorphisms of ABCB1 and IL15 as predictors of EFS The clinical and cytogenetic features at presentation and the prevalence of alleles studied are summarised in Table 1, Supplementary Tables S3a and S3b, respectively. All bi-allelic loci were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci). Of the presenting features examined, unfavourable cytogenetics was associated with a significantly poorer EFS (PCOX<0.001; Table 2), while NCI high-risk status had borderline significance (PCOX=0.091; Table 2); self-reported ethnicity, gender, and cell type were not significantly related to EFS. The complete genotype data as well as patient characteristics are available for research purposes upon request.
Among the 20 polymorphisms examined individually in preliminary test, polymorphisms in ABCB1 (rs2032582 and rs1045642) and IL15 (rs17015014) were found eligible for further analyses (adjusted P=0.038 and 0.048, respectively; Supplementary Table S4). They were subsequently analysed together in a Cox regression model adjusting for presenting clinical and cytogenetic features (Table 2). The homozygosity of either ABCB1 2677T/T or 3435T/T was still significantly related with a poorer EFS compared with other genotypes at these two loci (PKM=0.001; PCOX=0.003, HR=2.11, 95% CI=1.29–3.45; Figure 1A). There was moderate linkage disequilibrium (LD) between 2677T and 3435T (details shown in later section). IL15 67276493G/G was also associated with significantly poorer EFS compared with 67276493G/C and C/C (PKM=0.050; PCOX=0.022, HR=1.84, 95% CI=1.09–3.08; Figure 1B). Adjusting for MRD at the end of remission induction (Supplementary Figures S2 A–B) or excluding the cases of resistance from EFS analyses (Supplementary Table S5 and Supplementary Figures S3A–B) did not alter the significance of these polymorphisms identified.
The risk genotypes, that is, ABCB1 T/T at 2677 or 3435 or both as well as IL15 67276493G/G, exhibited an additive effect (PKM<0.001; PCOX<0.001, HR=1.97, 95% CI=1.38–2.82) demonstrating progressively poorer EFS in patients with an increasing number of risk genotypes (Table 2 and Figure 1C). Compared with the patients without any risk genotype, the hazard of adverse events raised 2.2 folds in patients with one risk genotype (PKM=0.005; PCOX=0.003, HR=2.20, 95% CI=1.30–3.70) and 3.6 folds in those with two risk genotypes (PKM=0.002; PCOX=0.001, HR=3.64, 95% CI=1.68–7.92). After adjusting for MRD at the end of remission induction (Supplementary Table S6 and Supplementary Figure S2C) or excluding the cases of resistance (Supplementary Table S7 and Supplementary Figure S3C), this additive effect remained significant. There was no significant EFS difference between patients with only ABCB1 risk genotype and those with only IL15 risk genotype (data not shown).
To further investigate the impact of ABCB1 2677G>T/A/3435C>T and IL15 67276493G>C genotypes on a specific event, we examined their influence on the cumulative incidence of relapse (including resistance), failure to achieve complete remission as well as treatment-related mortality. Homozygosity of either ABCB1 2677T/T or 3435T/T significantly increased the risk of relapse in both univariate (PG=0.001; Supplementary Figure S4A) and multivariate models after adjusting for clinical and cytogenetic features (PFG=0.011, HR=2.17, 95% CI=1.19–3.94; Supplementary Table S8). The risk of relapse in patients with at least one T/T was 20.4% compared with 9.0% in those without any T/T. Taking the end-of-induction MRD level into account did not alter the significance (PFG=0.027, HR=2.01, 95% CI=1.08–3.73). The risk of relapse also became higher with increasing number of risk genotypes (PG<0.001; Supplementary Figure S4B; PFG=0.011, HR=1.84, 95% CI=1.15–2.93; Supplementary Table S8). The trend remained significant after adjusting for the end-of-induction MRD level (PFG=0.026, HR=1.69, 95% CI=1.07–2.68).
IL15 67276493G/G was associated with higher risk of failure to achieve complete remission when compared with G/C and C/C genotypes (P=0.009, OR=3.14, 95% CI=1.34–7.36; Supplementary Table S9). G/G homozygosity also correlated with poorer overall survival (PKM=0.050; PCOX=0.005, HR=2.74, 95% CI=1.35–5.58; Supplementary Table S10 and Supplementary Figure S5), primarily due to more than 3-fold higher risk of treatment-related mortality in patients with IL15 67276493G/G (P=0.015, OR=3.40, 95% CI=1.27–9.11; Supplementary Table S11).
We also examined the associations of identified risk genotypes with known risk factors. Neither T/T homozygosity at ABCB1 2677 or 3435 nor IL15 67276493G/G was found to correlate with cytogenetics and MRD level after remission induction (Supplementary Table S12).
The clinical and cytogenetic features at presentation and the prevalence of alleles studied are summarised in Table 1, Supplementary Tables S3a and S3b, respectively. All bi-allelic loci were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci). Of the presenting features examined, unfavourable cytogenetics was associated with a significantly poorer EFS (PCOX<0.001; Table 2), while NCI high-risk status had borderline significance (PCOX=0.091; Table 2); self-reported ethnicity, gender, and cell type were not significantly related to EFS. The complete genotype data as well as patient characteristics are available for research purposes upon request.
Among the 20 polymorphisms examined individually in preliminary test, polymorphisms in ABCB1 (rs2032582 and rs1045642) and IL15 (rs17015014) were found eligible for further analyses (adjusted P=0.038 and 0.048, respectively; Supplementary Table S4). They were subsequently analysed together in a Cox regression model adjusting for presenting clinical and cytogenetic features (Table 2). The homozygosity of either ABCB1 2677T/T or 3435T/T was still significantly related with a poorer EFS compared with other genotypes at these two loci (PKM=0.001; PCOX=0.003, HR=2.11, 95% CI=1.29–3.45; Figure 1A). There was moderate linkage disequilibrium (LD) between 2677T and 3435T (details shown in later section). IL15 67276493G/G was also associated with significantly poorer EFS compared with 67276493G/C and C/C (PKM=0.050; PCOX=0.022, HR=1.84, 95% CI=1.09–3.08; Figure 1B). Adjusting for MRD at the end of remission induction (Supplementary Figures S2 A–B) or excluding the cases of resistance from EFS analyses (Supplementary Table S5 and Supplementary Figures S3A–B) did not alter the significance of these polymorphisms identified.
The risk genotypes, that is, ABCB1 T/T at 2677 or 3435 or both as well as IL15 67276493G/G, exhibited an additive effect (PKM<0.001; PCOX<0.001, HR=1.97, 95% CI=1.38–2.82) demonstrating progressively poorer EFS in patients with an increasing number of risk genotypes (Table 2 and Figure 1C). Compared with the patients without any risk genotype, the hazard of adverse events raised 2.2 folds in patients with one risk genotype (PKM=0.005; PCOX=0.003, HR=2.20, 95% CI=1.30–3.70) and 3.6 folds in those with two risk genotypes (PKM=0.002; PCOX=0.001, HR=3.64, 95% CI=1.68–7.92). After adjusting for MRD at the end of remission induction (Supplementary Table S6 and Supplementary Figure S2C) or excluding the cases of resistance (Supplementary Table S7 and Supplementary Figure S3C), this additive effect remained significant. There was no significant EFS difference between patients with only ABCB1 risk genotype and those with only IL15 risk genotype (data not shown).
To further investigate the impact of ABCB1 2677G>T/A/3435C>T and IL15 67276493G>C genotypes on a specific event, we examined their influence on the cumulative incidence of relapse (including resistance), failure to achieve complete remission as well as treatment-related mortality. Homozygosity of either ABCB1 2677T/T or 3435T/T significantly increased the risk of relapse in both univariate (PG=0.001; Supplementary Figure S4A) and multivariate models after adjusting for clinical and cytogenetic features (PFG=0.011, HR=2.17, 95% CI=1.19–3.94; Supplementary Table S8). The risk of relapse in patients with at least one T/T was 20.4% compared with 9.0% in those without any T/T. Taking the end-of-induction MRD level into account did not alter the significance (PFG=0.027, HR=2.01, 95% CI=1.08–3.73). The risk of relapse also became higher with increasing number of risk genotypes (PG<0.001; Supplementary Figure S4B; PFG=0.011, HR=1.84, 95% CI=1.15–2.93; Supplementary Table S8). The trend remained significant after adjusting for the end-of-induction MRD level (PFG=0.026, HR=1.69, 95% CI=1.07–2.68).
IL15 67276493G/G was associated with higher risk of failure to achieve complete remission when compared with G/C and C/C genotypes (P=0.009, OR=3.14, 95% CI=1.34–7.36; Supplementary Table S9). G/G homozygosity also correlated with poorer overall survival (PKM=0.050; PCOX=0.005, HR=2.74, 95% CI=1.35–5.58; Supplementary Table S10 and Supplementary Figure S5), primarily due to more than 3-fold higher risk of treatment-related mortality in patients with IL15 67276493G/G (P=0.015, OR=3.40, 95% CI=1.27–9.11; Supplementary Table S11).
We also examined the associations of identified risk genotypes with known risk factors. Neither T/T homozygosity at ABCB1 2677 or 3435 nor IL15 67276493G/G was found to correlate with cytogenetics and MRD level after remission induction (Supplementary Table S12).
Prognostic significance of ABCB1 and IL15 in patient subgroups In patients with favourable cytogenetics, T/T homozygosity at either ABCB1 2677 or 3435 position was associated with a significantly poorer EFS (PKM=0.011; PCOX=0.011, HR=4.02, 95% CI=1.38–11.75; Figure 2A). The EFS in patients carrying ABCB1 2677T/T or 3435T/T was 78.2%±7.6%, compared with 94.4%±1.9% in those carrying other genotypes. Such an association became even stronger after adjusting for MRD level (PCOX<0.001, HR=19.26, 95% CI=4.43–83.72; 80.6%±8.6% vs 98.9%±0.8%). In contrast, the impact of IL15 67276493 genotype was more pronounced in patients who do not carry chromosomal abnormalities of known prognosis, showing a significantly worse EFS in G/G carriers than the others (PKM=0.038; PCOX=0.022, HR=2.20, 95% CI=1.12–4.30; Figure 2C), with EFS of 73.9%±5.6% for G/G carriers and 86.7%±2.8% for G/C and C/C carriers, respectively. Adjustment for MRD level did not moderate the significance (PCOX=0.022, HR=2.30, 95% CI=1.13–4.70; 78.6%±5.4% vs 90.2%±2.7%).
In both cytogenetic subgroups, the additive effect of risk genotypes was found (PKM<0.001 and PCOX<0.001, Figure 2B; and PKM=0.013 and PCOX=0.009, Figure 2D) and remained significant after adjusting for MRD level (PCOX<0.001 and=0.008 respectively). In addition, we also examined the influence of risk genotypes in the two major ethnic groups, the Chinese and Malays, respectively. In both races, the additive effect of risk genotypes was also found (PKM<0.001 and PCOX=0.001 in the Chinese, Supplementary Figure S6A; PKM=0.075 and PCOX=0.036 in the Malays, Supplementary Figure S6B) and remained significant after adjusting for MRD level (PCOX=0.003 and=0.006, respectively).
In patients with favourable cytogenetics, T/T homozygosity at either ABCB1 2677 or 3435 position was associated with a significantly poorer EFS (PKM=0.011; PCOX=0.011, HR=4.02, 95% CI=1.38–11.75; Figure 2A). The EFS in patients carrying ABCB1 2677T/T or 3435T/T was 78.2%±7.6%, compared with 94.4%±1.9% in those carrying other genotypes. Such an association became even stronger after adjusting for MRD level (PCOX<0.001, HR=19.26, 95% CI=4.43–83.72; 80.6%±8.6% vs 98.9%±0.8%). In contrast, the impact of IL15 67276493 genotype was more pronounced in patients who do not carry chromosomal abnormalities of known prognosis, showing a significantly worse EFS in G/G carriers than the others (PKM=0.038; PCOX=0.022, HR=2.20, 95% CI=1.12–4.30; Figure 2C), with EFS of 73.9%±5.6% for G/G carriers and 86.7%±2.8% for G/C and C/C carriers, respectively. Adjustment for MRD level did not moderate the significance (PCOX=0.022, HR=2.30, 95% CI=1.13–4.70; 78.6%±5.4% vs 90.2%±2.7%).
In both cytogenetic subgroups, the additive effect of risk genotypes was found (PKM<0.001 and PCOX<0.001, Figure 2B; and PKM=0.013 and PCOX=0.009, Figure 2D) and remained significant after adjusting for MRD level (PCOX<0.001 and=0.008 respectively). In addition, we also examined the influence of risk genotypes in the two major ethnic groups, the Chinese and Malays, respectively. In both races, the additive effect of risk genotypes was also found (PKM<0.001 and PCOX=0.001 in the Chinese, Supplementary Figure S6A; PKM=0.075 and PCOX=0.036 in the Malays, Supplementary Figure S6B) and remained significant after adjusting for MRD level (PCOX=0.003 and=0.006, respectively).
Validation tests Patients were split into two halves based on the time of enrolment at each participating centre. In both cohorts, the additive effect of T/T homozygosity at ABCB1 2677 or 3435 and IL15 67276493G/G still remained significant (Supplementary Figure S7A, C), even after adjusting for MRD levels at the end of induction (Supplementary Figure S7B, D). In addition, we generated 100 random test sets by re-sampling. Of these 100 test scenarios, the significance was achieved for 86 test sets (Supplementary Figure S8A), and all results consistently showed that increasing number of risk genotypes corresponded to a poorer EFS (i.e., HR >1; Supplementary Figure S8B).
Patients were split into two halves based on the time of enrolment at each participating centre. In both cohorts, the additive effect of T/T homozygosity at ABCB1 2677 or 3435 and IL15 67276493G/G still remained significant (Supplementary Figure S7A, C), even after adjusting for MRD levels at the end of induction (Supplementary Figure S7B, D). In addition, we generated 100 random test sets by re-sampling. Of these 100 test scenarios, the significance was achieved for 86 test sets (Supplementary Figure S8A), and all results consistently showed that increasing number of risk genotypes corresponded to a poorer EFS (i.e., HR >1; Supplementary Figure S8B).
Linkage disequilibrium As multiple loci in ABCB1 and IL15 were genotyped, we calculated the linkage disequilibrium (LD) among different alleles. Two loci, ABCB1 2677 (rs2032582) and ABCB1 3435 (rs1045642), were in a moderate LD block. The LD was slightly stronger in the Chinese (r2=0.58 for entire patient cohort and r2=0.74 for the Chinese; Supplementary Figure S9), with two major haplotypes, 2677G-3435C (52.3%) and 2677T-3435T (36.2%). IL15 67276493G>C (rs17015014) was not in LD with any other studied loci of the IL15 gene (Supplementary Figure S10).
As multiple loci in ABCB1 and IL15 were genotyped, we calculated the linkage disequilibrium (LD) among different alleles. Two loci, ABCB1 2677 (rs2032582) and ABCB1 3435 (rs1045642), were in a moderate LD block. The LD was slightly stronger in the Chinese (r2=0.58 for entire patient cohort and r2=0.74 for the Chinese; Supplementary Figure S9), with two major haplotypes, 2677G-3435C (52.3%) and 2677T-3435T (36.2%). IL15 67276493G>C (rs17015014) was not in LD with any other studied loci of the IL15 gene (Supplementary Figure S10).
Identification of polymorphisms of ABCB1 and IL15 as predictors of EFS:
The clinical and cytogenetic features at presentation and the prevalence of alleles studied are summarised in Table 1, Supplementary Tables S3a and S3b, respectively. All bi-allelic loci were in Hardy–Weinberg equilibrium in each ethnic group (not applicable for GSTM1/T1 loci). Of the presenting features examined, unfavourable cytogenetics was associated with a significantly poorer EFS (PCOX<0.001; Table 2), while NCI high-risk status had borderline significance (PCOX=0.091; Table 2); self-reported ethnicity, gender, and cell type were not significantly related to EFS. The complete genotype data as well as patient characteristics are available for research purposes upon request.
Among the 20 polymorphisms examined individually in preliminary test, polymorphisms in ABCB1 (rs2032582 and rs1045642) and IL15 (rs17015014) were found eligible for further analyses (adjusted P=0.038 and 0.048, respectively; Supplementary Table S4). They were subsequently analysed together in a Cox regression model adjusting for presenting clinical and cytogenetic features (Table 2). The homozygosity of either ABCB1 2677T/T or 3435T/T was still significantly related with a poorer EFS compared with other genotypes at these two loci (PKM=0.001; PCOX=0.003, HR=2.11, 95% CI=1.29–3.45; Figure 1A). There was moderate linkage disequilibrium (LD) between 2677T and 3435T (details shown in later section). IL15 67276493G/G was also associated with significantly poorer EFS compared with 67276493G/C and C/C (PKM=0.050; PCOX=0.022, HR=1.84, 95% CI=1.09–3.08; Figure 1B). Adjusting for MRD at the end of remission induction (Supplementary Figures S2 A–B) or excluding the cases of resistance from EFS analyses (Supplementary Table S5 and Supplementary Figures S3A–B) did not alter the significance of these polymorphisms identified.
The risk genotypes, that is, ABCB1 T/T at 2677 or 3435 or both as well as IL15 67276493G/G, exhibited an additive effect (PKM<0.001; PCOX<0.001, HR=1.97, 95% CI=1.38–2.82) demonstrating progressively poorer EFS in patients with an increasing number of risk genotypes (Table 2 and Figure 1C). Compared with the patients without any risk genotype, the hazard of adverse events raised 2.2 folds in patients with one risk genotype (PKM=0.005; PCOX=0.003, HR=2.20, 95% CI=1.30–3.70) and 3.6 folds in those with two risk genotypes (PKM=0.002; PCOX=0.001, HR=3.64, 95% CI=1.68–7.92). After adjusting for MRD at the end of remission induction (Supplementary Table S6 and Supplementary Figure S2C) or excluding the cases of resistance (Supplementary Table S7 and Supplementary Figure S3C), this additive effect remained significant. There was no significant EFS difference between patients with only ABCB1 risk genotype and those with only IL15 risk genotype (data not shown).
To further investigate the impact of ABCB1 2677G>T/A/3435C>T and IL15 67276493G>C genotypes on a specific event, we examined their influence on the cumulative incidence of relapse (including resistance), failure to achieve complete remission as well as treatment-related mortality. Homozygosity of either ABCB1 2677T/T or 3435T/T significantly increased the risk of relapse in both univariate (PG=0.001; Supplementary Figure S4A) and multivariate models after adjusting for clinical and cytogenetic features (PFG=0.011, HR=2.17, 95% CI=1.19–3.94; Supplementary Table S8). The risk of relapse in patients with at least one T/T was 20.4% compared with 9.0% in those without any T/T. Taking the end-of-induction MRD level into account did not alter the significance (PFG=0.027, HR=2.01, 95% CI=1.08–3.73). The risk of relapse also became higher with increasing number of risk genotypes (PG<0.001; Supplementary Figure S4B; PFG=0.011, HR=1.84, 95% CI=1.15–2.93; Supplementary Table S8). The trend remained significant after adjusting for the end-of-induction MRD level (PFG=0.026, HR=1.69, 95% CI=1.07–2.68).
IL15 67276493G/G was associated with higher risk of failure to achieve complete remission when compared with G/C and C/C genotypes (P=0.009, OR=3.14, 95% CI=1.34–7.36; Supplementary Table S9). G/G homozygosity also correlated with poorer overall survival (PKM=0.050; PCOX=0.005, HR=2.74, 95% CI=1.35–5.58; Supplementary Table S10 and Supplementary Figure S5), primarily due to more than 3-fold higher risk of treatment-related mortality in patients with IL15 67276493G/G (P=0.015, OR=3.40, 95% CI=1.27–9.11; Supplementary Table S11).
We also examined the associations of identified risk genotypes with known risk factors. Neither T/T homozygosity at ABCB1 2677 or 3435 nor IL15 67276493G/G was found to correlate with cytogenetics and MRD level after remission induction (Supplementary Table S12).
Prognostic significance of ABCB1 and IL15 in patient subgroups:
In patients with favourable cytogenetics, T/T homozygosity at either ABCB1 2677 or 3435 position was associated with a significantly poorer EFS (PKM=0.011; PCOX=0.011, HR=4.02, 95% CI=1.38–11.75; Figure 2A). The EFS in patients carrying ABCB1 2677T/T or 3435T/T was 78.2%±7.6%, compared with 94.4%±1.9% in those carrying other genotypes. Such an association became even stronger after adjusting for MRD level (PCOX<0.001, HR=19.26, 95% CI=4.43–83.72; 80.6%±8.6% vs 98.9%±0.8%). In contrast, the impact of IL15 67276493 genotype was more pronounced in patients who do not carry chromosomal abnormalities of known prognosis, showing a significantly worse EFS in G/G carriers than the others (PKM=0.038; PCOX=0.022, HR=2.20, 95% CI=1.12–4.30; Figure 2C), with EFS of 73.9%±5.6% for G/G carriers and 86.7%±2.8% for G/C and C/C carriers, respectively. Adjustment for MRD level did not moderate the significance (PCOX=0.022, HR=2.30, 95% CI=1.13–4.70; 78.6%±5.4% vs 90.2%±2.7%).
In both cytogenetic subgroups, the additive effect of risk genotypes was found (PKM<0.001 and PCOX<0.001, Figure 2B; and PKM=0.013 and PCOX=0.009, Figure 2D) and remained significant after adjusting for MRD level (PCOX<0.001 and=0.008 respectively). In addition, we also examined the influence of risk genotypes in the two major ethnic groups, the Chinese and Malays, respectively. In both races, the additive effect of risk genotypes was also found (PKM<0.001 and PCOX=0.001 in the Chinese, Supplementary Figure S6A; PKM=0.075 and PCOX=0.036 in the Malays, Supplementary Figure S6B) and remained significant after adjusting for MRD level (PCOX=0.003 and=0.006, respectively).
Validation tests:
Patients were split into two halves based on the time of enrolment at each participating centre. In both cohorts, the additive effect of T/T homozygosity at ABCB1 2677 or 3435 and IL15 67276493G/G still remained significant (Supplementary Figure S7A, C), even after adjusting for MRD levels at the end of induction (Supplementary Figure S7B, D). In addition, we generated 100 random test sets by re-sampling. Of these 100 test scenarios, the significance was achieved for 86 test sets (Supplementary Figure S8A), and all results consistently showed that increasing number of risk genotypes corresponded to a poorer EFS (i.e., HR >1; Supplementary Figure S8B).
Linkage disequilibrium:
As multiple loci in ABCB1 and IL15 were genotyped, we calculated the linkage disequilibrium (LD) among different alleles. Two loci, ABCB1 2677 (rs2032582) and ABCB1 3435 (rs1045642), were in a moderate LD block. The LD was slightly stronger in the Chinese (r2=0.58 for entire patient cohort and r2=0.74 for the Chinese; Supplementary Figure S9), with two major haplotypes, 2677G-3435C (52.3%) and 2677T-3435T (36.2%). IL15 67276493G>C (rs17015014) was not in LD with any other studied loci of the IL15 gene (Supplementary Figure S10).
Discussion:
In childhood ALL, there is significant inter-individual variation in response to chemotherapy, which is not fully predicted by presenting features such as age and leukaemic cytogenetics. Newly described markers such as IKZF1 and CRLF2 have demonstrated prognostic impact in many studies (Mullighan et al, 2009; Buitenkamp et al, 2012; Chen et al, 2012; Feng and Tang, 2013), but their values still remain inconclusive in the Malaysia-Singapore ALL 2003 study according to our pilot investigations (data not shown). Here we showed that host germline variants of ABCB1 and IL15 genes are significant predictors of treatment outcome, suggesting that the characterisation of these genes' variants at diagnosis could contribute to risk-stratification strategies. In our study, the ABCB1 2677T/T or 3435T/T genotypes were associated with an increased risk of relapse/resistance. ABCB1 2677G>T/A introduces an Ala-to-Ser/Thr change in exon 21 while 3435C>T is a synonymous polymorphism in exon 26, which affects transport by altering substrate specificity (Kimchi-Sarfaty et al, 2007). Various studies have shown that not all synonymous SNPs are considered silent; they can cause changes in protein expression, conformation or function and are increasingly implicated in human disease risk and other complex traits (Sauna and Kimchi-Sarfaty, 2011).
Large studies in patients with acute myocardial infarction on oral anti-coagulant clopidogrel, a substrate of P-glycoprotein (P-gp), reported lower drug levels and poorer cardiovascular outcomes in patients with ABCB1 3435T/T homozygotes (Simon et al, 2009; Mega et al, 2010). Oral dexamethasone, used extensively in paediatric ALL treatment including our Ma-Spore ALL 2003 protocol, is also a known substrate for P-gp. Patients with ABCB1 2677T/T or 3435T/T may have lower systemic dexamethasone exposure due to higher ABCB1 expression on the apical villi of enterocytes (Nakamura et al, 2002), causing enhanced extrusion of drugs into the intestinal lumen. Supporting this hypothesis, Yang et al (2012) recently reported two ABCB1 variants (rs10264856 and rs4728709) that affected oral dexamethasone clearance and were associated with increased risk of relapse in the St Jude and COG cohorts, although we did not observe any LD between rs10264856/rs4728709 and rs2032582/rs1045642 in our study as well as the data from 1000 Genomes Project (the maximum r2 is only 0.14). Taken together with our findings on ABCB1 2677 and 3435, these strongly suggest the importance of ABCB1 variants in treatment of childhood ALL.
However, Jamroziak et al (2004) and Stanulla et al (2005b) previously reported lower EFS (N=111) and higher CNS relapse (N=34), respectively, in ABCB1 3435C/C carriers treated on the BFM-ALL 86/90/95 protocols. Differences in the type of steroid used during induction therapy (prednisolone in the BFM-ALL 86/90/95 compared with dexamethasone in Ma-Spore ALL 2003) may account for this observed difference. In addition, the lower frequency of ABCB1 2677T and 3435T alleles among African Americans (10.0% and 20.2%, respectively) (Kroetz et al, 2003) compared with our studied population (43.6% and 40.5%, respectively) may mask the effect of ABCB1 variants on treatment outcome in the previously reported St Jude Total XIIIB protocol (Rocha et al, 2005).
The manner that ABCB1 variants affect the P-gp expression on the apical villi of enterocytes remains unresolved (Lin and Yamazaki, 2003; Leschziner et al, 2007; Hodges et al, 2011; Wolf et al, 2011). Nakamura et al (2002) reported that healthy Japanese subjects with 3435T/T had significantly higher duodenal ABCB1 mRNA expression and lower digoxin plasma level compared with C/C or C/T genotype carriers, while Hoffmeyer et al (2000) and Efferth et al (2003) disagreed with such a correlation. The effect of ABCB1 polymorphisms on P-gp activity is likely dependent on the substrates as well as the tissues (or cell types) studied, thus becoming a confounding factor when studying P-gp genotype–phenotype relationship. Further studies in correlating dexamethasone clearance and ABCB1 polymorphisms are required.
Considering the drug efflux capacity of P-gp, it is rational to speculate that the impact of ABCB1 genotype on the risk of relapse/resistance originates from its effect on MRD level. Although not statistically significant in our patients, Day 33 MRD high risk (i.e., ⩾10−2) tended to be enriched in the ABCB1 T/T group compared with the others (14.9% vs 8.8%, P=0.088, Supplementary Table S12). Published data on the relationship between ABCB1 gene and MRD are controversial. For instance, an earlier report showed that the median ABCB1 expression at diagnosis of childhood ALL were roughly comparable between the MRD-positive group and MRD-negative group at the end of induction therapy (Fedasenka et al, 2008). This finding was challenged by a very recent study reporting that mRNA expression of ABCB1 higher than a certain cutoff would raise the risk of MRD positivity by 12-fold after 1-year treatment in children with ALL (Rahgozar et al, 2014). Such discrepancies are likely due to (but not limited to) the diverse grouping criteria, the quantitation methods applied, the time points investigated, and sampled populations in different studies. As the determinants on early treatment response are known to be multifactorial, a clear correlation between ABCB1 genotype and MRD level may not be expected.
The IL15 67276493G/G genotype was associated with a poorer EFS and OS in our study, and carried a higher probability of treatment-related mortality. IL15 regulates T- and natural killer cell activation and proliferation, having central roles in cell-mediated immunity against microbes (Yoshikai and Nishimura, 2000). Administration of exogenous IL15 in animal models causes severe but reversible neutropenia due to redistribution of neutrophils out of the circulation (Berger et al, 2009; Waldmann et al, 2011). This SNP resides in the 3′UTR region of the gene, which exerts a negative regulatory effect on the expression of IL15; polymorphism at the 3′UTR may reduce this negative regulation, resulting in an enhanced expression. It is noteworthy that genetic variants in IL15, albeit different IL15 SNPs, have been previously inferred to significantly correlate with the MRD level at the end of induction (Yang et al, 2009), a strong surrogate marker to predict treatment outcome.
ABCB1 3435T/T or 2677T/T and IL15 67276493G/G showed a surprising additive effect in predicting a poorer EFS probability, even after adjusting for MRD levels at the end of remission induction. P-glycoprotein mediates in transmembrane transport of cytokines. It has been reported that ABCB1 2677T/T–3435T/T are associated with lower secretion of cytokines in PHA-stimulated lymphocytes (Pawlik et al, 2005). Conversely, in HIV-infected individuals, P-glycoprotein expression and function are decreased and restored only after IL15 stimulation (Chang et al, 2000). Therefore, we speculate that ABCB1 2677T/T or 3435T/T may alter secretion of IL15, which affects cellular immune responses and treatment outcome consequently.
In summary, host genetic variations in ABCB1 and IL15 genes influence treatment outcomes in paediatric ALL in this predominantly Asian population treated on the Ma-Spore ALL 2003. Inclusion of host variants in ABCB1 and IL15 genes may help future risk assignment strategies and deserves further study in other treatment cohorts.
|
Background:
Host germline variations and their potential prognostic importance is an emerging area of interest in paediatric ALL.
Methods:
We investigated the associations between 20 germline variations and various clinical end points in 463 children with ALL.
Results:
After adjusting for known prognostic factors, variants in two genes were found to be independently associated with poorer EFS: ABCB1 T/T at either 2677 (rs2032582) or 3435 (rs1045642) position (P=0.003) and IL15 67276493G/G (rs17015014; P=0.022). These variants showed a strong additive effect affecting outcome (P<0.001), whereby patients with both risk genotypes had the worst EFS (P=0.001), even after adjusting for MRD levels at the end of remission induction. The adverse effect of ABCB1 T/T genotypes was most pronounced in patients with favourable cytogenetics (P=0.011) while the IL15 67276493G/G genotype mainly affected patients without common chromosomal abnormalities (P=0.022). In both cytogenetic subgroups, increasing number of such risk genotypes still predicted worsening outcome (P<0.001 and=0.009, respectively).
Conclusions:
These results point to the prognostic importance of host genetic variants, although the specific mechanisms remain unclarified. Inclusion of ABCB1 and IL15 variants may help improve risk assignment strategies in paediatric ALL.
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[CONTENT] paediatric ALL | prognosis | single-nucleotide polymorphism |
ABCB1
|
IL15
[SUMMARY]
| null |
[CONTENT] paediatric ALL | prognosis | single-nucleotide polymorphism |
ABCB1
|
IL15
[SUMMARY]
| null | null | null |
[CONTENT] ATP Binding Cassette Transporter, Subfamily B | ATP Binding Cassette Transporter, Subfamily B, Member 1 | Child | Child, Preschool | Female | Genotype | Humans | Infant | Interleukin-15 | Linkage Disequilibrium | Male | Polymorphism, Single Nucleotide | Precursor Cell Lymphoblastic Leukemia-Lymphoma | Prognosis | Treatment Outcome [SUMMARY]
| null |
[CONTENT] ATP Binding Cassette Transporter, Subfamily B | ATP Binding Cassette Transporter, Subfamily B, Member 1 | Child | Child, Preschool | Female | Genotype | Humans | Infant | Interleukin-15 | Linkage Disequilibrium | Male | Polymorphism, Single Nucleotide | Precursor Cell Lymphoblastic Leukemia-Lymphoma | Prognosis | Treatment Outcome [SUMMARY]
| null | null | null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null | null | null |
[CONTENT] risk | supplementary | abcb1 | patients | figure | genotypes | il15 | pcox | efs | mrd [SUMMARY]
| null |
[CONTENT] risk | supplementary | abcb1 | patients | figure | genotypes | il15 | pcox | efs | mrd [SUMMARY]
| null | null | null |
[CONTENT] 95 ci | ci | supplementary | pcox | 95 | hr | figure | 001 | table | risk [SUMMARY]
| null |
[CONTENT] supplementary | risk | figure | abcb1 | pcox | ci | 95 ci | genotypes | 95 | patients [SUMMARY]
| null | null | null |
[CONTENT] two | 2677 | rs2032582 | 3435 | 67276493G ||| MRD ||| ABCB1 | 67276493G ||| [SUMMARY]
| null |
[CONTENT] ||| between 20 | 463 ||| two | 2677 | rs2032582 | 3435 | 67276493G ||| MRD ||| ABCB1 | 67276493G ||| ||| ||| ALL [SUMMARY]
| null |
||
Pattern of diarrheal diseases in Atwima Nwabiagya District-Ghana, 2009- 2013.
|
28149440
|
Diarrheal diseases remain one of the most important public health challenges worldwide. In 2011, Ghana recorded average annual diarrheal cases of 2,218 per 100,000 populations for children under-five with Ashanti region recording the third highest. In the Atwima Nwabiagya District, summary statistics are done without detailed analysis. We analyzed diarrheal surveillance data to determine its pattern and to develop threshold levels for the disease in Atwima Nwabiagya District in the Ashanti Region of Ghana.
|
INTRODUCTION
|
District level diarrheal morbidity data from January 2009 to December 2013 was extracted from District Health Information Management System II, cleaned and analyzed. Descriptive analysis was done and expressed as frequencies and relative frequencies. Description of the data was done in time, place and person. We calculated diarrhea threshold using the C2 method.
|
METHODS
|
Overall, 51,131 cases were reported with 55.2% being females over the five year period. The highest episode of diarrhea by age-group occurred in children under-five during the study period. Changes in disease occurrence did not conform to a seasonal pattern. District analysis showed one outbreak whilst sub-district analysis revealed more than one outbreak.
|
RESULTS
|
Diarrheal disease pattern did not show a seasonal trend. Only one outbreak was observed at district level but each sub-district, showed more than one outbreak. The highest number of episodes of diarrhea per year occurred in Children under- five. Data analysis should be done at lower levels to inform interventions. Interventions should be targeted towards children under-five years.
|
CONCLUSION
|
[
"Adolescent",
"Adult",
"Age Distribution",
"Aged",
"Child",
"Child, Preschool",
"Diarrhea",
"Disease Outbreaks",
"Female",
"Ghana",
"Humans",
"Male",
"Middle Aged",
"Population Surveillance",
"Public Health",
"Sex Distribution",
"Young Adult"
] |
5257013
|
Introduction
|
Diarrheal diseases remain one of the most important public health challenges worldwide, particularly in developing countries where they cause high morbidity and mortality. Globally an estimated 1.7 billion diarrheal diseases are reported every year with majority occurring in Africa [1, 2]. Diarrhea is the second leading cause of death in children under five years and is also responsible for killing about 760 000 children every year globally[1]. Most deaths from diarrhea occur among children less than two years of age living in Southern Asia and sub-Saharan Africa [2]. In developing countries, children under three years old experience on average three episodes of diarrhea every year. Each episode deprives the child of the nutrition necessary for growth leading to malnutrition [1]. In 2011, Ghana recorded an average annual diarrheal cases of 2,218 per 100,000 population for children under-five with Ashanti region recording the third highest of 2,646 per 100,000 population [3]. Among children under-fives the highest prevalence of 33% was recorded in 12months-23months age group [4]. Diarrhea has been prioritized by many nations and attracts a lot of global attention. The integrated Global Action Plan for the Prevention and Control of Pneumonia and Diarrhea (GAPPD) aims at ending preventable childhood deaths due to pneumonia and diarrhea by 2025 [5]. Analysis of health-related data has proven useful for planning targeted interventions [6–8] to meet the GAPPD target. In Ghana, data on diarrheal diseases is collected routinely through the District Health Information Management System II (DHIMS II) but in-depth analysis is not done particularly at the sub-district and district level. In the Atwima Nwabiagya District, summary statistics are done without detailed analysis and action threshold levels are also not set to help guide interventions. As a result, public health officials are denied useful information for monitoring, controlling and prevention of diarrheal diseases. We therefore analyzed diarrheal surveillance data to determine its pattern and threshold levels in Atwima Nwabiagya District in the Ashanti Region of Ghana.
| null | null |
Results
|
A total of 51,131 cases of diarrhea were reported during the period, of which 28,233(55.2%) were females. Children under five years had the highest number of cases of diarrhea 19818(38.8%) and age group 70years or more had the least 2554(5.0%)(Table 1).
Characteristics of diarrheal cases in Atwima Nwabiagya district, 2009-2013
The highest episodes of diarrhea per year occurred in children under-five years for all the years under review, whiles 10 -14years age group had the least number of diarrhea episodes for four years (2009, 2011, 2012, and 2013). In 2010, children 5 years - 9 years had the least number of episodes per age group per year. For the under-fives, those under a year old had the highest episode for 2011-2013 and one to four years age group recorded the highest for 2009-2010. The episodes of diarrheal diseases per thousand populations increased steeply from 2009 to 2010 then gradually to 2012 and then decreases in 2013. From January 2009, pattern was irregular with different peaking months per year. In 2009, Diarrheal diseases peaked in September (984) and had the lowest number of cases in April (476) with 2010 recording the highest in September (1073) and the lowest in December (617). In 2011 the highest number of cases were recorded in March (1068) and the lowest in January (768) whiles 2012 peaked in November (1391) and the lowest in March (418). Finally 2013 had it highest number of cases in April (1247) and in December (443) had the least number of cases. Data over the five years shows that, changes in disease occurrence do not conform to a regular seasonal pattern (Figure 1). Asuofua had the highest episodes of diarrhea per sub-district population in two different years (2009 and 2013), Barekese in 2010 and Nkawie (2011 and 2012). Abuakwa recorded the lowest in 2009 and 2010 whiles Barekese recorded the lowest in 2011 and 2013. In 2012, Asuofua recorded the lowest episode of diarrhea per sub-district. Using the C2 method, July 2012 recorded higher than the expected number of cases. This is the only month which recorded observed cases being more than the threshold (recorded 1182, expected-1171). For the sub district analysis, each sub district recorded more than the expected number. District level analysis for children under-five showed that January 2010, February-March 2011 had more reported cases than the expected (Figure 2) whiles sub-district for children under-five years revealed different multiple possible outbreak at different months and years. A typical example is shown in Figure 3.
Trends of diarrheal cases with C2 threshold in AtwimaNwabiagya district from 2009-2013.
Trends of diarrheal cases and C2 threshold for children under-five years in Atwima Nwabiagya District from 2009-2013
Trends of diarrheal cases and C2 threshold for children under-five years in Abuakwa sub-district from 2009-2013
|
Conclusion
|
Overall, 51131 cases reported with 55.22% being females. The highest episode of diarrhea per age-group per year was recorded by children under-five. Changes in disease occurrence do not conform to a seasonal pattern. District analysis showed one outbreak whiles Sub district analysis revealed more than one outbreak.
|
[
"Methods"
] |
[
"Study design: all cause diarrhea surveillance data reported by health facilities in the district from 2009 to 2013 was extracted DHIMS 11 and analysis of this secondary data was done. DHIMS II is a database of priority diseases reported by all health facilities in the districts. Variables on diarrheal diseases in the DHIMS II were age, sex and sub district.\nStudy area: the Atwima Nwabiagya District is one of the 27 districts in Ashanti Region of Ghana. The District has five sub districts namely; Abuakwa, Akropong, Asuofua, Barekese and Nkawie. There are 17 health facilities which include hospitals, clinics, and health centers. It is peri-urban town with Barekese and Owabi dams all in Barekese Sub district which supply portable water to Kumasi and its environs including AtwimaNwabiagya District. The major rainfall season is from March to July and minor season is between August and mid-November. From 2010 population and housing census, the estimated 2013 population in the district is 161425 with 13.4% being children under-five.\nStudy period: the study was undertaken from 6th June to 11th July 2014.\nData collection, processing and analysis: diarrheal surveillance data from January 2009 to December 2013 for Atwima Nwabiagya District was extracted from DHIMS II database and exported into Microsoft Excel 2010. Data was cleaned and descriptive analysis was done and expressed as frequencies and relative frequencies. Description of the data was done in time, place and person. The thresholds for diarrhea were calculated using the C2 threshold method [9, 10]. The C2 method is defined as the sum of the mean and three standard deviations for seven preceding monthly diarrhea cases, skipping two most recent months.\nEthical issues: permission was obtained from the Regional and District Health Directorate of the Ghana Health Service. Data was not linked to patient identification; therefore one is unable to identify who has reported with an episode of diarrheal disease."
] |
[
null
] |
[
"Introduction",
"Methods",
"Results",
"Discussion",
"Conclusion"
] |
[
"Diarrheal diseases remain one of the most important public health challenges worldwide, particularly in developing countries where they cause high morbidity and mortality. Globally an estimated 1.7 billion diarrheal diseases are reported every year with majority occurring in Africa [1, 2]. Diarrhea is the second leading cause of death in children under five years and is also responsible for killing about 760 000 children every year globally[1]. Most deaths from diarrhea occur among children less than two years of age living in Southern Asia and sub-Saharan Africa [2]. In developing countries, children under three years old experience on average three episodes of diarrhea every year. Each episode deprives the child of the nutrition necessary for growth leading to malnutrition [1]. In 2011, Ghana recorded an average annual diarrheal cases of 2,218 per 100,000 population for children under-five with Ashanti region recording the third highest of 2,646 per 100,000 population [3]. Among children under-fives the highest prevalence of 33% was recorded in 12months-23months age group [4]. Diarrhea has been prioritized by many nations and attracts a lot of global attention. The integrated Global Action Plan for the Prevention and Control of Pneumonia and Diarrhea (GAPPD) aims at ending preventable childhood deaths due to pneumonia and diarrhea by 2025 [5]. Analysis of health-related data has proven useful for planning targeted interventions [6–8] to meet the GAPPD target. In Ghana, data on diarrheal diseases is collected routinely through the District Health Information Management System II (DHIMS II) but in-depth analysis is not done particularly at the sub-district and district level. In the Atwima Nwabiagya District, summary statistics are done without detailed analysis and action threshold levels are also not set to help guide interventions. As a result, public health officials are denied useful information for monitoring, controlling and prevention of diarrheal diseases. We therefore analyzed diarrheal surveillance data to determine its pattern and threshold levels in Atwima Nwabiagya District in the Ashanti Region of Ghana.",
"Study design: all cause diarrhea surveillance data reported by health facilities in the district from 2009 to 2013 was extracted DHIMS 11 and analysis of this secondary data was done. DHIMS II is a database of priority diseases reported by all health facilities in the districts. Variables on diarrheal diseases in the DHIMS II were age, sex and sub district.\nStudy area: the Atwima Nwabiagya District is one of the 27 districts in Ashanti Region of Ghana. The District has five sub districts namely; Abuakwa, Akropong, Asuofua, Barekese and Nkawie. There are 17 health facilities which include hospitals, clinics, and health centers. It is peri-urban town with Barekese and Owabi dams all in Barekese Sub district which supply portable water to Kumasi and its environs including AtwimaNwabiagya District. The major rainfall season is from March to July and minor season is between August and mid-November. From 2010 population and housing census, the estimated 2013 population in the district is 161425 with 13.4% being children under-five.\nStudy period: the study was undertaken from 6th June to 11th July 2014.\nData collection, processing and analysis: diarrheal surveillance data from January 2009 to December 2013 for Atwima Nwabiagya District was extracted from DHIMS II database and exported into Microsoft Excel 2010. Data was cleaned and descriptive analysis was done and expressed as frequencies and relative frequencies. Description of the data was done in time, place and person. The thresholds for diarrhea were calculated using the C2 threshold method [9, 10]. The C2 method is defined as the sum of the mean and three standard deviations for seven preceding monthly diarrhea cases, skipping two most recent months.\nEthical issues: permission was obtained from the Regional and District Health Directorate of the Ghana Health Service. Data was not linked to patient identification; therefore one is unable to identify who has reported with an episode of diarrheal disease.",
"A total of 51,131 cases of diarrhea were reported during the period, of which 28,233(55.2%) were females. Children under five years had the highest number of cases of diarrhea 19818(38.8%) and age group 70years or more had the least 2554(5.0%)(Table 1).\nCharacteristics of diarrheal cases in Atwima Nwabiagya district, 2009-2013\nThe highest episodes of diarrhea per year occurred in children under-five years for all the years under review, whiles 10 -14years age group had the least number of diarrhea episodes for four years (2009, 2011, 2012, and 2013). In 2010, children 5 years - 9 years had the least number of episodes per age group per year. For the under-fives, those under a year old had the highest episode for 2011-2013 and one to four years age group recorded the highest for 2009-2010. The episodes of diarrheal diseases per thousand populations increased steeply from 2009 to 2010 then gradually to 2012 and then decreases in 2013. From January 2009, pattern was irregular with different peaking months per year. In 2009, Diarrheal diseases peaked in September (984) and had the lowest number of cases in April (476) with 2010 recording the highest in September (1073) and the lowest in December (617). In 2011 the highest number of cases were recorded in March (1068) and the lowest in January (768) whiles 2012 peaked in November (1391) and the lowest in March (418). Finally 2013 had it highest number of cases in April (1247) and in December (443) had the least number of cases. Data over the five years shows that, changes in disease occurrence do not conform to a regular seasonal pattern (Figure 1). Asuofua had the highest episodes of diarrhea per sub-district population in two different years (2009 and 2013), Barekese in 2010 and Nkawie (2011 and 2012). Abuakwa recorded the lowest in 2009 and 2010 whiles Barekese recorded the lowest in 2011 and 2013. In 2012, Asuofua recorded the lowest episode of diarrhea per sub-district. Using the C2 method, July 2012 recorded higher than the expected number of cases. This is the only month which recorded observed cases being more than the threshold (recorded 1182, expected-1171). For the sub district analysis, each sub district recorded more than the expected number. District level analysis for children under-five showed that January 2010, February-March 2011 had more reported cases than the expected (Figure 2) whiles sub-district for children under-five years revealed different multiple possible outbreak at different months and years. A typical example is shown in Figure 3.\nTrends of diarrheal cases with C2 threshold in AtwimaNwabiagya district from 2009-2013.\nTrends of diarrheal cases and C2 threshold for children under-five years in Atwima Nwabiagya District from 2009-2013\nTrends of diarrheal cases and C2 threshold for children under-five years in Abuakwa sub-district from 2009-2013",
"This diarrheal surveillance data analysis was done to determine the pattern and to develop action threshold levels for the district. Few studies have used similar hospital data on diarrhea to define disease burden and trends in African countries [9–11]. Children under five years recorded the highest number of diarrhea episodes per year compared to the other age groups. This agrees with a research in Kenya that also revealed greater burden on children under five years than the other age groups [10] This may be because this age group include children who are crawling and put contaminated materials and fingers in their mouth. This may lead to a greater risk of having diarrhea and possible the reason why this age group had the highest in all the years. The episodes of diarrheal diseases per thousand populations increased steeply from 2009 to 2010 then gradually increased in 2012 and then finally decreased in 2013. The irregular increase and decrease agree by research [10] whiles others have recorded decrease over the years [12]. The increase may be as a result of the increase in health facilities over the period and the associated competition among health facilities which has improved quality of service by the various health facilities and therefore possibly attracting clients from adjacent districts. The decrease in 2013 may be as results of interventions put in place in 2012 by the district health management team, when the district had an alarming outbreak of cholera. The intervention included acceptable increase in chlorination of water by Ghana water and sewage, organizing and maintaining cleaning exercise. Sanitation task force increase surveillance and enforcement of sanitation laws This may have contributed to the decrease in the number of cases. Data over the five years shows that, changes in disease occurrence did not conform to a seasonal pattern which is the most reported form [10, 13]. In the district and sub-district analysis, the major peaks fell within and sometimes towards the end of the local rainy seasons of March to July and August to Mid-N November and this agrees with work done elsewhere [14, 15]. Sub-district having the highest rate of reported diarrhea varied from year to year and this findings agree with research [10]. Prevailing issues at a point in time may have influenced a particular sub district recording the highest prevalence of diarrheal diseases. Interventions to decrease diarrheal disease in some district may be better than others, so prevalence may vary among sub-district.\nDistrict analysis revealed only one month in which recorded cases were more than expected whiles sub-district analysis showed more than one over the five years. Sub-district analysis of children under-five revealed more months in which recorded cases were more than the expected and these occurred at different months and years compared to the district analysis. This means epidemiological patterns of diarrhea disease in the sub district may be different from the district picture and therefore the need to narrow down analysis to the lower level. Based on the findings we recommend to the District Health Management Team to do detailed sub district analysis to help them get clearer picture of what is happening at the lower level. This is important since aggregated data (district) may give a different picture compared to disaggregated data (sub-district). Interventions should be targeted at those under five and research should be conducted appreciate the reason for the trends seen.",
"Overall, 51131 cases reported with 55.22% being females. The highest episode of diarrhea per age-group per year was recorded by children under-five. Changes in disease occurrence do not conform to a seasonal pattern. District analysis showed one outbreak whiles Sub district analysis revealed more than one outbreak."
] |
[
"intro",
null,
"results",
"discussion",
"conclusion"
] |
[
"Health Information Management System II",
"DHIMS",
"Atwima Nwabiagya",
"diarrhea",
"C2-Method",
"Ghana"
] |
Introduction:
Diarrheal diseases remain one of the most important public health challenges worldwide, particularly in developing countries where they cause high morbidity and mortality. Globally an estimated 1.7 billion diarrheal diseases are reported every year with majority occurring in Africa [1, 2]. Diarrhea is the second leading cause of death in children under five years and is also responsible for killing about 760 000 children every year globally[1]. Most deaths from diarrhea occur among children less than two years of age living in Southern Asia and sub-Saharan Africa [2]. In developing countries, children under three years old experience on average three episodes of diarrhea every year. Each episode deprives the child of the nutrition necessary for growth leading to malnutrition [1]. In 2011, Ghana recorded an average annual diarrheal cases of 2,218 per 100,000 population for children under-five with Ashanti region recording the third highest of 2,646 per 100,000 population [3]. Among children under-fives the highest prevalence of 33% was recorded in 12months-23months age group [4]. Diarrhea has been prioritized by many nations and attracts a lot of global attention. The integrated Global Action Plan for the Prevention and Control of Pneumonia and Diarrhea (GAPPD) aims at ending preventable childhood deaths due to pneumonia and diarrhea by 2025 [5]. Analysis of health-related data has proven useful for planning targeted interventions [6–8] to meet the GAPPD target. In Ghana, data on diarrheal diseases is collected routinely through the District Health Information Management System II (DHIMS II) but in-depth analysis is not done particularly at the sub-district and district level. In the Atwima Nwabiagya District, summary statistics are done without detailed analysis and action threshold levels are also not set to help guide interventions. As a result, public health officials are denied useful information for monitoring, controlling and prevention of diarrheal diseases. We therefore analyzed diarrheal surveillance data to determine its pattern and threshold levels in Atwima Nwabiagya District in the Ashanti Region of Ghana.
Methods:
Study design: all cause diarrhea surveillance data reported by health facilities in the district from 2009 to 2013 was extracted DHIMS 11 and analysis of this secondary data was done. DHIMS II is a database of priority diseases reported by all health facilities in the districts. Variables on diarrheal diseases in the DHIMS II were age, sex and sub district.
Study area: the Atwima Nwabiagya District is one of the 27 districts in Ashanti Region of Ghana. The District has five sub districts namely; Abuakwa, Akropong, Asuofua, Barekese and Nkawie. There are 17 health facilities which include hospitals, clinics, and health centers. It is peri-urban town with Barekese and Owabi dams all in Barekese Sub district which supply portable water to Kumasi and its environs including AtwimaNwabiagya District. The major rainfall season is from March to July and minor season is between August and mid-November. From 2010 population and housing census, the estimated 2013 population in the district is 161425 with 13.4% being children under-five.
Study period: the study was undertaken from 6th June to 11th July 2014.
Data collection, processing and analysis: diarrheal surveillance data from January 2009 to December 2013 for Atwima Nwabiagya District was extracted from DHIMS II database and exported into Microsoft Excel 2010. Data was cleaned and descriptive analysis was done and expressed as frequencies and relative frequencies. Description of the data was done in time, place and person. The thresholds for diarrhea were calculated using the C2 threshold method [9, 10]. The C2 method is defined as the sum of the mean and three standard deviations for seven preceding monthly diarrhea cases, skipping two most recent months.
Ethical issues: permission was obtained from the Regional and District Health Directorate of the Ghana Health Service. Data was not linked to patient identification; therefore one is unable to identify who has reported with an episode of diarrheal disease.
Results:
A total of 51,131 cases of diarrhea were reported during the period, of which 28,233(55.2%) were females. Children under five years had the highest number of cases of diarrhea 19818(38.8%) and age group 70years or more had the least 2554(5.0%)(Table 1).
Characteristics of diarrheal cases in Atwima Nwabiagya district, 2009-2013
The highest episodes of diarrhea per year occurred in children under-five years for all the years under review, whiles 10 -14years age group had the least number of diarrhea episodes for four years (2009, 2011, 2012, and 2013). In 2010, children 5 years - 9 years had the least number of episodes per age group per year. For the under-fives, those under a year old had the highest episode for 2011-2013 and one to four years age group recorded the highest for 2009-2010. The episodes of diarrheal diseases per thousand populations increased steeply from 2009 to 2010 then gradually to 2012 and then decreases in 2013. From January 2009, pattern was irregular with different peaking months per year. In 2009, Diarrheal diseases peaked in September (984) and had the lowest number of cases in April (476) with 2010 recording the highest in September (1073) and the lowest in December (617). In 2011 the highest number of cases were recorded in March (1068) and the lowest in January (768) whiles 2012 peaked in November (1391) and the lowest in March (418). Finally 2013 had it highest number of cases in April (1247) and in December (443) had the least number of cases. Data over the five years shows that, changes in disease occurrence do not conform to a regular seasonal pattern (Figure 1). Asuofua had the highest episodes of diarrhea per sub-district population in two different years (2009 and 2013), Barekese in 2010 and Nkawie (2011 and 2012). Abuakwa recorded the lowest in 2009 and 2010 whiles Barekese recorded the lowest in 2011 and 2013. In 2012, Asuofua recorded the lowest episode of diarrhea per sub-district. Using the C2 method, July 2012 recorded higher than the expected number of cases. This is the only month which recorded observed cases being more than the threshold (recorded 1182, expected-1171). For the sub district analysis, each sub district recorded more than the expected number. District level analysis for children under-five showed that January 2010, February-March 2011 had more reported cases than the expected (Figure 2) whiles sub-district for children under-five years revealed different multiple possible outbreak at different months and years. A typical example is shown in Figure 3.
Trends of diarrheal cases with C2 threshold in AtwimaNwabiagya district from 2009-2013.
Trends of diarrheal cases and C2 threshold for children under-five years in Atwima Nwabiagya District from 2009-2013
Trends of diarrheal cases and C2 threshold for children under-five years in Abuakwa sub-district from 2009-2013
Discussion:
This diarrheal surveillance data analysis was done to determine the pattern and to develop action threshold levels for the district. Few studies have used similar hospital data on diarrhea to define disease burden and trends in African countries [9–11]. Children under five years recorded the highest number of diarrhea episodes per year compared to the other age groups. This agrees with a research in Kenya that also revealed greater burden on children under five years than the other age groups [10] This may be because this age group include children who are crawling and put contaminated materials and fingers in their mouth. This may lead to a greater risk of having diarrhea and possible the reason why this age group had the highest in all the years. The episodes of diarrheal diseases per thousand populations increased steeply from 2009 to 2010 then gradually increased in 2012 and then finally decreased in 2013. The irregular increase and decrease agree by research [10] whiles others have recorded decrease over the years [12]. The increase may be as a result of the increase in health facilities over the period and the associated competition among health facilities which has improved quality of service by the various health facilities and therefore possibly attracting clients from adjacent districts. The decrease in 2013 may be as results of interventions put in place in 2012 by the district health management team, when the district had an alarming outbreak of cholera. The intervention included acceptable increase in chlorination of water by Ghana water and sewage, organizing and maintaining cleaning exercise. Sanitation task force increase surveillance and enforcement of sanitation laws This may have contributed to the decrease in the number of cases. Data over the five years shows that, changes in disease occurrence did not conform to a seasonal pattern which is the most reported form [10, 13]. In the district and sub-district analysis, the major peaks fell within and sometimes towards the end of the local rainy seasons of March to July and August to Mid-N November and this agrees with work done elsewhere [14, 15]. Sub-district having the highest rate of reported diarrhea varied from year to year and this findings agree with research [10]. Prevailing issues at a point in time may have influenced a particular sub district recording the highest prevalence of diarrheal diseases. Interventions to decrease diarrheal disease in some district may be better than others, so prevalence may vary among sub-district.
District analysis revealed only one month in which recorded cases were more than expected whiles sub-district analysis showed more than one over the five years. Sub-district analysis of children under-five revealed more months in which recorded cases were more than the expected and these occurred at different months and years compared to the district analysis. This means epidemiological patterns of diarrhea disease in the sub district may be different from the district picture and therefore the need to narrow down analysis to the lower level. Based on the findings we recommend to the District Health Management Team to do detailed sub district analysis to help them get clearer picture of what is happening at the lower level. This is important since aggregated data (district) may give a different picture compared to disaggregated data (sub-district). Interventions should be targeted at those under five and research should be conducted appreciate the reason for the trends seen.
Conclusion:
Overall, 51131 cases reported with 55.22% being females. The highest episode of diarrhea per age-group per year was recorded by children under-five. Changes in disease occurrence do not conform to a seasonal pattern. District analysis showed one outbreak whiles Sub district analysis revealed more than one outbreak.
|
Background:
Diarrheal diseases remain one of the most important public health challenges worldwide. In 2011, Ghana recorded average annual diarrheal cases of 2,218 per 100,000 populations for children under-five with Ashanti region recording the third highest. In the Atwima Nwabiagya District, summary statistics are done without detailed analysis. We analyzed diarrheal surveillance data to determine its pattern and to develop threshold levels for the disease in Atwima Nwabiagya District in the Ashanti Region of Ghana.
Methods:
District level diarrheal morbidity data from January 2009 to December 2013 was extracted from District Health Information Management System II, cleaned and analyzed. Descriptive analysis was done and expressed as frequencies and relative frequencies. Description of the data was done in time, place and person. We calculated diarrhea threshold using the C2 method.
Results:
Overall, 51,131 cases were reported with 55.2% being females over the five year period. The highest episode of diarrhea by age-group occurred in children under-five during the study period. Changes in disease occurrence did not conform to a seasonal pattern. District analysis showed one outbreak whilst sub-district analysis revealed more than one outbreak.
Conclusions:
Diarrheal disease pattern did not show a seasonal trend. Only one outbreak was observed at district level but each sub-district, showed more than one outbreak. The highest number of episodes of diarrhea per year occurred in Children under- five. Data analysis should be done at lower levels to inform interventions. Interventions should be targeted towards children under-five years.
|
Introduction:
Diarrheal diseases remain one of the most important public health challenges worldwide, particularly in developing countries where they cause high morbidity and mortality. Globally an estimated 1.7 billion diarrheal diseases are reported every year with majority occurring in Africa [1, 2]. Diarrhea is the second leading cause of death in children under five years and is also responsible for killing about 760 000 children every year globally[1]. Most deaths from diarrhea occur among children less than two years of age living in Southern Asia and sub-Saharan Africa [2]. In developing countries, children under three years old experience on average three episodes of diarrhea every year. Each episode deprives the child of the nutrition necessary for growth leading to malnutrition [1]. In 2011, Ghana recorded an average annual diarrheal cases of 2,218 per 100,000 population for children under-five with Ashanti region recording the third highest of 2,646 per 100,000 population [3]. Among children under-fives the highest prevalence of 33% was recorded in 12months-23months age group [4]. Diarrhea has been prioritized by many nations and attracts a lot of global attention. The integrated Global Action Plan for the Prevention and Control of Pneumonia and Diarrhea (GAPPD) aims at ending preventable childhood deaths due to pneumonia and diarrhea by 2025 [5]. Analysis of health-related data has proven useful for planning targeted interventions [6–8] to meet the GAPPD target. In Ghana, data on diarrheal diseases is collected routinely through the District Health Information Management System II (DHIMS II) but in-depth analysis is not done particularly at the sub-district and district level. In the Atwima Nwabiagya District, summary statistics are done without detailed analysis and action threshold levels are also not set to help guide interventions. As a result, public health officials are denied useful information for monitoring, controlling and prevention of diarrheal diseases. We therefore analyzed diarrheal surveillance data to determine its pattern and threshold levels in Atwima Nwabiagya District in the Ashanti Region of Ghana.
Conclusion:
Overall, 51131 cases reported with 55.22% being females. The highest episode of diarrhea per age-group per year was recorded by children under-five. Changes in disease occurrence do not conform to a seasonal pattern. District analysis showed one outbreak whiles Sub district analysis revealed more than one outbreak.
|
Background:
Diarrheal diseases remain one of the most important public health challenges worldwide. In 2011, Ghana recorded average annual diarrheal cases of 2,218 per 100,000 populations for children under-five with Ashanti region recording the third highest. In the Atwima Nwabiagya District, summary statistics are done without detailed analysis. We analyzed diarrheal surveillance data to determine its pattern and to develop threshold levels for the disease in Atwima Nwabiagya District in the Ashanti Region of Ghana.
Methods:
District level diarrheal morbidity data from January 2009 to December 2013 was extracted from District Health Information Management System II, cleaned and analyzed. Descriptive analysis was done and expressed as frequencies and relative frequencies. Description of the data was done in time, place and person. We calculated diarrhea threshold using the C2 method.
Results:
Overall, 51,131 cases were reported with 55.2% being females over the five year period. The highest episode of diarrhea by age-group occurred in children under-five during the study period. Changes in disease occurrence did not conform to a seasonal pattern. District analysis showed one outbreak whilst sub-district analysis revealed more than one outbreak.
Conclusions:
Diarrheal disease pattern did not show a seasonal trend. Only one outbreak was observed at district level but each sub-district, showed more than one outbreak. The highest number of episodes of diarrhea per year occurred in Children under- five. Data analysis should be done at lower levels to inform interventions. Interventions should be targeted towards children under-five years.
| 2,013 | 289 | 5 |
[
"district",
"years",
"diarrhea",
"sub",
"children",
"sub district",
"diarrheal",
"cases",
"analysis",
"data"
] |
[
"test",
"test"
] | null |
[CONTENT] Health Information Management System II | DHIMS | Atwima Nwabiagya | diarrhea | C2-Method | Ghana [SUMMARY]
| null |
[CONTENT] Health Information Management System II | DHIMS | Atwima Nwabiagya | diarrhea | C2-Method | Ghana [SUMMARY]
|
[CONTENT] Health Information Management System II | DHIMS | Atwima Nwabiagya | diarrhea | C2-Method | Ghana [SUMMARY]
|
[CONTENT] Health Information Management System II | DHIMS | Atwima Nwabiagya | diarrhea | C2-Method | Ghana [SUMMARY]
|
[CONTENT] Health Information Management System II | DHIMS | Atwima Nwabiagya | diarrhea | C2-Method | Ghana [SUMMARY]
|
[CONTENT] Adolescent | Adult | Age Distribution | Aged | Child | Child, Preschool | Diarrhea | Disease Outbreaks | Female | Ghana | Humans | Male | Middle Aged | Population Surveillance | Public Health | Sex Distribution | Young Adult [SUMMARY]
| null |
[CONTENT] Adolescent | Adult | Age Distribution | Aged | Child | Child, Preschool | Diarrhea | Disease Outbreaks | Female | Ghana | Humans | Male | Middle Aged | Population Surveillance | Public Health | Sex Distribution | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Age Distribution | Aged | Child | Child, Preschool | Diarrhea | Disease Outbreaks | Female | Ghana | Humans | Male | Middle Aged | Population Surveillance | Public Health | Sex Distribution | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Age Distribution | Aged | Child | Child, Preschool | Diarrhea | Disease Outbreaks | Female | Ghana | Humans | Male | Middle Aged | Population Surveillance | Public Health | Sex Distribution | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Age Distribution | Aged | Child | Child, Preschool | Diarrhea | Disease Outbreaks | Female | Ghana | Humans | Male | Middle Aged | Population Surveillance | Public Health | Sex Distribution | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] district | years | diarrhea | sub | children | sub district | diarrheal | cases | analysis | data [SUMMARY]
| null |
[CONTENT] district | years | diarrhea | sub | children | sub district | diarrheal | cases | analysis | data [SUMMARY]
|
[CONTENT] district | years | diarrhea | sub | children | sub district | diarrheal | cases | analysis | data [SUMMARY]
|
[CONTENT] district | years | diarrhea | sub | children | sub district | diarrheal | cases | analysis | data [SUMMARY]
|
[CONTENT] district | years | diarrhea | sub | children | sub district | diarrheal | cases | analysis | data [SUMMARY]
|
[CONTENT] diarrheal | 000 | diarrhea | children | health | district | diseases | diarrheal diseases | children years | ghana [SUMMARY]
| null |
[CONTENT] years | 2009 | number | lowest | 2013 | cases | recorded | number cases | 2011 | 2012 [SUMMARY]
|
[CONTENT] district analysis | outbreak | cases reported | analysis showed outbreak | analysis revealed outbreak | diarrhea age | diarrhea age group | diarrhea age group year | recorded children | cases reported 55 [SUMMARY]
|
[CONTENT] district | years | diarrhea | analysis | health | diarrheal | sub | children | data | sub district [SUMMARY]
|
[CONTENT] district | years | diarrhea | analysis | health | diarrheal | sub | children | data | sub district [SUMMARY]
|
[CONTENT] Diarrheal | one ||| 2011 | Ghana | annual | 2,218 | 100,000 | under-five | Ashanti | third ||| the Atwima Nwabiagya District ||| Atwima Nwabiagya District | Ghana [SUMMARY]
| null |
[CONTENT] 51,131 | 55.2% | the five year ||| under-five ||| ||| one | more than one [SUMMARY]
|
[CONTENT] ||| Only one | more than one ||| diarrhea per year | Children ||| five ||| ||| under-five years [SUMMARY]
|
[CONTENT] Diarrheal | one ||| 2011 | Ghana | annual | 2,218 | 100,000 | under-five | Ashanti | third ||| the Atwima Nwabiagya District ||| Atwima Nwabiagya District | Ghana ||| January 2009 to December 2013 | District Health Information Management System II ||| ||| ||| C2 ||| 51,131 | 55.2% | the five year ||| under-five ||| ||| one | more than one ||| ||| Only one | more than one ||| diarrhea per year | Children ||| five ||| ||| under-five years [SUMMARY]
|
[CONTENT] Diarrheal | one ||| 2011 | Ghana | annual | 2,218 | 100,000 | under-five | Ashanti | third ||| the Atwima Nwabiagya District ||| Atwima Nwabiagya District | Ghana ||| January 2009 to December 2013 | District Health Information Management System II ||| ||| ||| C2 ||| 51,131 | 55.2% | the five year ||| under-five ||| ||| one | more than one ||| ||| Only one | more than one ||| diarrhea per year | Children ||| five ||| ||| under-five years [SUMMARY]
|
|||||
Cohort profile: the ages 2003 cohort study in Aichi, Japan.
|
21325730
|
The longevity of Japanese is thought to be associated with psychosocial factors such as sense of coherence, social support, and social capital. However, the actual factors responsible and the extent of their contribution to individual health status are not known.
|
BACKGROUND
|
The Aichi Gerontological Evaluation Study (AGES) 2003 Cohort Study is a prospective cohort study of community-dwelling, activities of daily living-independent people aged 65 or older living in 6 municipalities in Chita peninsula, Aichi Prefecture, Japan. Information on psychosocial factors and other individual- and community-level factors was collected in the second half of 2003 using a baseline questionnaire. Vital status and physical and cognitive decline have been followed using data derived from long-term care insurance certification. Geographical information on the study participants was also obtained.
|
METHODS
|
A total of 13 310 (6508 men; 6802 women) study participants were registered in the study. For an interim report, we followed the cohort for 48 months, yielding 24 753 person-years of observation among men and 26 456 person-years among women.
|
RESULTS
|
The AGES 2003 Cohort Study provides useful evidence for research in social epidemiology, gerontology, and health services.
|
CONCLUSIONS
|
[
"Aged",
"Aged, 80 and over",
"Cities",
"Female",
"Follow-Up Studies",
"Health Status",
"Humans",
"Japan",
"Longevity",
"Male",
"Prospective Studies",
"Residence Characteristics",
"Social Support",
"Surveys and Questionnaires"
] |
3899507
|
INTRODUCTION
|
“Why are Japanese living longer?”1 The question of Japanese longevity is fascinating, and the global community wants to know more about this phenomenon. In 2008, life expectancy was 79.19 years in Japanese men and 85.99 years in Japanese women, which outrank those of nearly all developed and developing countries.2,3 While some believe that genetic differences between Japanese and Western populations are a factor, research suggests that this is an unlikely explanation.4,5
Resources in society can improve the general health of individuals.6,7 Many people agree that the health of individuals is affected by several aspects of society, such as cohesion, lifestyle, customs, family structure, culture, and religious beliefs, all of which are generated through each country’s or community’s complexity and context over a period of many years.6,8 Large-scale cohort studies are being conducted in Japan to reveal the reasons for Japanese longevity, but these studies are limited in their ability to make causal inferences because of the complexity of the underlying mechanisms.9–20
In addition to the work of scholars, the national and local governments are also addressing the issue of aging in Japan. For instance, the nationwide universal long-term care insurance system was started in 2000,21,22 and an elder abuse prevention and caregiver support law was enacted in 2006.23 Nevertheless, a new type of health inequality may result from imbalances and lack of harmonization in the community’s supply and demand of health and social care.24,25
Within the natural course of aging (eg, from independence in activities of daily living [ADL], with only minor comorbidities, to ADL-dependence due to stroke sequelae and/or other medical conditions), what do older people and their families want from society? Which health services are efficient and fair? To answer these questions, we urgently need evidence-based approaches to living arrangements, social support, and social capital that are based on social epidemiology, gerontology, and health services research.
The Aichi Gerontological Evaluation Study (AGES) project was launched for this purpose in 1999. At the Center for Well-being and Society of Nihon Fukushi University in Aichi prefecture, Japan, one of the authors (KK) and colleagues are responsible for managing the project and thus take full responsibility for it. The organizations that provided funding for this research had no role in the conduct of the study or the presentation of its results.
|
METHODS
|
Study design, setting, and participants After a pilot cohort was generated and evaluated in 1999, we organized the AGES 2003 Cohort Study as a part of the AGES project. In this prospective study, the source population was community-dwelling individuals aged 65 years or older who lived in 6 targeted municipalities and were ADL-independent as of the second half of 2003 (the exact date varied by municipality). The targeted municipalities covered the entire southern part of the Chita peninsula in Aichi Prefecture (ie, Agui town, Handa city, Tokoname city, Taketoyo town, Mihama town, and Minami-Chita town). The 6 municipalities consist of 18 kyuuson (the second smallest administrative unit in Japan, based on the municipalities in 1950), which comprise urban, semi-urban, and rural settings.9 Older people who were not ADL-independent were excluded if they were eligible to receive benefits from public long-term care insurance (LTCI) services and or if they indicated that they were ADL-dependent in the baseline questionnaire.
The survey was conducted using a random sampling method in the 2 larger municipalities (Handa and Tokoname) and a complete census (complete enumeration) of the 4 smaller municipalities (Agui, Mihama, Minami-Chita, and Taketoyo) by municipal officers of the public LTCI system. A total of 49 707 residents aged 65 or older as of 1 October 2003 were targeted (Figure). The self-administered baseline questionnaire was mailed to 29 374 individuals selected by the sampling process as stated above. Then, 13 310 individuals (6508 men; 6802 women) were introduced to the AGES 2003 Cohort. There are no monetary or other incentives to encourage participation in the cohort. For data analysis, the municipality-level sample weight was calculated for selection probability, nonresponse, and other adjustments, to reflect the population proportion of those aged 65 or older in each municipality.
After a pilot cohort was generated and evaluated in 1999, we organized the AGES 2003 Cohort Study as a part of the AGES project. In this prospective study, the source population was community-dwelling individuals aged 65 years or older who lived in 6 targeted municipalities and were ADL-independent as of the second half of 2003 (the exact date varied by municipality). The targeted municipalities covered the entire southern part of the Chita peninsula in Aichi Prefecture (ie, Agui town, Handa city, Tokoname city, Taketoyo town, Mihama town, and Minami-Chita town). The 6 municipalities consist of 18 kyuuson (the second smallest administrative unit in Japan, based on the municipalities in 1950), which comprise urban, semi-urban, and rural settings.9 Older people who were not ADL-independent were excluded if they were eligible to receive benefits from public long-term care insurance (LTCI) services and or if they indicated that they were ADL-dependent in the baseline questionnaire.
The survey was conducted using a random sampling method in the 2 larger municipalities (Handa and Tokoname) and a complete census (complete enumeration) of the 4 smaller municipalities (Agui, Mihama, Minami-Chita, and Taketoyo) by municipal officers of the public LTCI system. A total of 49 707 residents aged 65 or older as of 1 October 2003 were targeted (Figure). The self-administered baseline questionnaire was mailed to 29 374 individuals selected by the sampling process as stated above. Then, 13 310 individuals (6508 men; 6802 women) were introduced to the AGES 2003 Cohort. There are no monetary or other incentives to encourage participation in the cohort. For data analysis, the municipality-level sample weight was calculated for selection probability, nonresponse, and other adjustments, to reflect the population proportion of those aged 65 or older in each municipality.
Baseline measures Table 1 summarizes the main measures that were collected in 2003. Other variables were also experimentally measured (some of which are not shown in Table 1). A summary of the measures is available at our website (http://square.umin.ac.jp/ages/index.html). The details of the questionnaire (including the exact wording of every question in Japanese and the translated version in English) have been published elsewhere.26–28
The baseline questionnaire included the following individual-level information: demographic factors (date of birth and sex), living arrangements (cohabitation status, marital status, and caregiving status), and socioeconomic status (education, household income, and current occupation status). Details of individual-level social support (receiving and providing emotional support and instrumental support), social participation, and sense of coherence29,30 were also obtained. Other factors included abuse of older people, negative life events (retirement, death of spouse, death of close relatives/friends, serious disease, a change in living arrangements such as moving, economic distress, or the beginning of informal family caregiving in the recent year), health-related behaviors (smoking, alcohol consumption, diet habits, physical activity [walking]), frequency of medical check-ups, and any hobbies. We regarded social participation as individual-level social capital and divided it into vertical and horizontal social capital. Vertical social capital was defined as participating in groups that encouraged hierarchical relations, and horizontal social capital was defined as participating in groups of social equals.18
Community-level measures were also obtained. Social capital (trust, reciprocity, and crime) was aggregated by individual-level variables.31 Other community-level variables of social factors (eg, the Gini coefficient) were aggregated by individual responses. For environmental exposures, information on airborne particle concentration (sulfur dioxide [SO2], nitrous monoxide [NO], carbon monoxide [CO], ozone [O3], and particulate matter with an aerodynamic diameter <10 µm [PM10])32 in the 6 municipalities (7 measuring points) was available from the Aichi prefecture website.33 We geocoded study participants’ home locations in each municipality, which enabled us to introduce municipality- or kyuuson-level multilevel analysis using Geographic Information System (GIS)-based methods.
Subjective general and mental health information included self-rated health,34 self-reported medical conditions (cancer, heart disease, stroke, hypertension, diabetes mellitus, hyperlipidemia, osteoporosis, joint/neurological disease, trauma/fracture, respiratory disease, gastrointestinal disease, liver disease, visual impairment, hearing impairment, urinary disorder, sleep disorder, and others), medication, sleeping, activities of daily living (Tokyo Metropolitan Institute of Gerontology Index of Competence [TMIG-IC]),35 Instrumental ADL,36 depression (Geriatric Depression Scale [GDS-15]),37,38 height, weight, body mass index (BMI), and dental status (number of remaining teeth).
Table 1 summarizes the main measures that were collected in 2003. Other variables were also experimentally measured (some of which are not shown in Table 1). A summary of the measures is available at our website (http://square.umin.ac.jp/ages/index.html). The details of the questionnaire (including the exact wording of every question in Japanese and the translated version in English) have been published elsewhere.26–28
The baseline questionnaire included the following individual-level information: demographic factors (date of birth and sex), living arrangements (cohabitation status, marital status, and caregiving status), and socioeconomic status (education, household income, and current occupation status). Details of individual-level social support (receiving and providing emotional support and instrumental support), social participation, and sense of coherence29,30 were also obtained. Other factors included abuse of older people, negative life events (retirement, death of spouse, death of close relatives/friends, serious disease, a change in living arrangements such as moving, economic distress, or the beginning of informal family caregiving in the recent year), health-related behaviors (smoking, alcohol consumption, diet habits, physical activity [walking]), frequency of medical check-ups, and any hobbies. We regarded social participation as individual-level social capital and divided it into vertical and horizontal social capital. Vertical social capital was defined as participating in groups that encouraged hierarchical relations, and horizontal social capital was defined as participating in groups of social equals.18
Community-level measures were also obtained. Social capital (trust, reciprocity, and crime) was aggregated by individual-level variables.31 Other community-level variables of social factors (eg, the Gini coefficient) were aggregated by individual responses. For environmental exposures, information on airborne particle concentration (sulfur dioxide [SO2], nitrous monoxide [NO], carbon monoxide [CO], ozone [O3], and particulate matter with an aerodynamic diameter <10 µm [PM10])32 in the 6 municipalities (7 measuring points) was available from the Aichi prefecture website.33 We geocoded study participants’ home locations in each municipality, which enabled us to introduce municipality- or kyuuson-level multilevel analysis using Geographic Information System (GIS)-based methods.
Subjective general and mental health information included self-rated health,34 self-reported medical conditions (cancer, heart disease, stroke, hypertension, diabetes mellitus, hyperlipidemia, osteoporosis, joint/neurological disease, trauma/fracture, respiratory disease, gastrointestinal disease, liver disease, visual impairment, hearing impairment, urinary disorder, sleep disorder, and others), medication, sleeping, activities of daily living (Tokyo Metropolitan Institute of Gerontology Index of Competence [TMIG-IC]),35 Instrumental ADL,36 depression (Geriatric Depression Scale [GDS-15]),37,38 height, weight, body mass index (BMI), and dental status (number of remaining teeth).
Follow-up and outcome measures Survival time was monitored since the second half of 2003 (the time when self-administered questionnaires were distributed varied by municipality). When individuals died or moved during follow-up, the date they died or left the study area was recorded. We obtained information on physical and cognitive disability by using the LTCI database maintained by the municipalities. The qualification was based on a standardized multistep assessment with several levels of care need, ranging from support levels 1 and 2 to care levels 1 through 5.21
Survival time was monitored since the second half of 2003 (the time when self-administered questionnaires were distributed varied by municipality). When individuals died or moved during follow-up, the date they died or left the study area was recorded. We obtained information on physical and cognitive disability by using the LTCI database maintained by the municipalities. The qualification was based on a standardized multistep assessment with several levels of care need, ranging from support levels 1 and 2 to care levels 1 through 5.21
Ethical issues Our study protocol and informed consent procedure were approved by the Ethics Committee on the Research of Human Subjects at Nihon Fukushi University.
Our study protocol and informed consent procedure were approved by the Ethics Committee on the Research of Human Subjects at Nihon Fukushi University.
Statistical analysis All analyses were performed using the computer software STATA/IC 11.0 (StataCorp LP, College Station, TX, USA).
All analyses were performed using the computer software STATA/IC 11.0 (StataCorp LP, College Station, TX, USA).
|
RESULTS
|
The baseline characteristics of the participants are shown in Table 2. Living alone, being divorced/widowed, lower education status, no current occupation, no smoking, and no alcohol consumption were more frequent in women than in men. As age increased, the proportions of those with a tertiary education decreased, as did current smokers, drinkers, and positive horizontal social capital.
aUnweighted values are reported only for cells indicating numbers of individuals.
During a 48-month follow-up period, which is the current maximum, men were observed for 24 753 person-years and women for 26 456 person-years. In total, 0.8% of the study participants were lost to follow-up because they left the study area during the 4-year period (n = 105). The cumulative survival rate for the study population was 96.8% (men, 95.6%; women, 98.0%) at 24 months and 92.1% (men, 89.4%; women, 94.7%) at 48 months. Sex and age-group differences among individual-level core variables are shown in Table 2. Overall and sex-stratified cumulative survival rates at 24 and 48 months for individual-level core variables are shown in Table 3.
| null | null |
[
"INTRODUCTION",
"Study design, setting, and participants",
"Baseline measures",
"Follow-up and outcome measures",
"Ethical issues",
"Statistical analysis"
] |
[
"“Why are Japanese living longer?”1 The question of Japanese longevity is fascinating, and the global community wants to know more about this phenomenon. In 2008, life expectancy was 79.19 years in Japanese men and 85.99 years in Japanese women, which outrank those of nearly all developed and developing countries.2,3 While some believe that genetic differences between Japanese and Western populations are a factor, research suggests that this is an unlikely explanation.4,5\nResources in society can improve the general health of individuals.6,7 Many people agree that the health of individuals is affected by several aspects of society, such as cohesion, lifestyle, customs, family structure, culture, and religious beliefs, all of which are generated through each country’s or community’s complexity and context over a period of many years.6,8 Large-scale cohort studies are being conducted in Japan to reveal the reasons for Japanese longevity, but these studies are limited in their ability to make causal inferences because of the complexity of the underlying mechanisms.9–20\nIn addition to the work of scholars, the national and local governments are also addressing the issue of aging in Japan. For instance, the nationwide universal long-term care insurance system was started in 2000,21,22 and an elder abuse prevention and caregiver support law was enacted in 2006.23 Nevertheless, a new type of health inequality may result from imbalances and lack of harmonization in the community’s supply and demand of health and social care.24,25\nWithin the natural course of aging (eg, from independence in activities of daily living [ADL], with only minor comorbidities, to ADL-dependence due to stroke sequelae and/or other medical conditions), what do older people and their families want from society? Which health services are efficient and fair? To answer these questions, we urgently need evidence-based approaches to living arrangements, social support, and social capital that are based on social epidemiology, gerontology, and health services research.\nThe Aichi Gerontological Evaluation Study (AGES) project was launched for this purpose in 1999. At the Center for Well-being and Society of Nihon Fukushi University in Aichi prefecture, Japan, one of the authors (KK) and colleagues are responsible for managing the project and thus take full responsibility for it. The organizations that provided funding for this research had no role in the conduct of the study or the presentation of its results.",
"After a pilot cohort was generated and evaluated in 1999, we organized the AGES 2003 Cohort Study as a part of the AGES project. In this prospective study, the source population was community-dwelling individuals aged 65 years or older who lived in 6 targeted municipalities and were ADL-independent as of the second half of 2003 (the exact date varied by municipality). The targeted municipalities covered the entire southern part of the Chita peninsula in Aichi Prefecture (ie, Agui town, Handa city, Tokoname city, Taketoyo town, Mihama town, and Minami-Chita town). The 6 municipalities consist of 18 kyuuson (the second smallest administrative unit in Japan, based on the municipalities in 1950), which comprise urban, semi-urban, and rural settings.9 Older people who were not ADL-independent were excluded if they were eligible to receive benefits from public long-term care insurance (LTCI) services and or if they indicated that they were ADL-dependent in the baseline questionnaire.\nThe survey was conducted using a random sampling method in the 2 larger municipalities (Handa and Tokoname) and a complete census (complete enumeration) of the 4 smaller municipalities (Agui, Mihama, Minami-Chita, and Taketoyo) by municipal officers of the public LTCI system. A total of 49 707 residents aged 65 or older as of 1 October 2003 were targeted (Figure). The self-administered baseline questionnaire was mailed to 29 374 individuals selected by the sampling process as stated above. Then, 13 310 individuals (6508 men; 6802 women) were introduced to the AGES 2003 Cohort. There are no monetary or other incentives to encourage participation in the cohort. For data analysis, the municipality-level sample weight was calculated for selection probability, nonresponse, and other adjustments, to reflect the population proportion of those aged 65 or older in each municipality.",
"Table 1 summarizes the main measures that were collected in 2003. Other variables were also experimentally measured (some of which are not shown in Table 1). A summary of the measures is available at our website (http://square.umin.ac.jp/ages/index.html). The details of the questionnaire (including the exact wording of every question in Japanese and the translated version in English) have been published elsewhere.26–28\nThe baseline questionnaire included the following individual-level information: demographic factors (date of birth and sex), living arrangements (cohabitation status, marital status, and caregiving status), and socioeconomic status (education, household income, and current occupation status). Details of individual-level social support (receiving and providing emotional support and instrumental support), social participation, and sense of coherence29,30 were also obtained. Other factors included abuse of older people, negative life events (retirement, death of spouse, death of close relatives/friends, serious disease, a change in living arrangements such as moving, economic distress, or the beginning of informal family caregiving in the recent year), health-related behaviors (smoking, alcohol consumption, diet habits, physical activity [walking]), frequency of medical check-ups, and any hobbies. We regarded social participation as individual-level social capital and divided it into vertical and horizontal social capital. Vertical social capital was defined as participating in groups that encouraged hierarchical relations, and horizontal social capital was defined as participating in groups of social equals.18\nCommunity-level measures were also obtained. Social capital (trust, reciprocity, and crime) was aggregated by individual-level variables.31 Other community-level variables of social factors (eg, the Gini coefficient) were aggregated by individual responses. For environmental exposures, information on airborne particle concentration (sulfur dioxide [SO2], nitrous monoxide [NO], carbon monoxide [CO], ozone [O3], and particulate matter with an aerodynamic diameter <10 µm [PM10])32 in the 6 municipalities (7 measuring points) was available from the Aichi prefecture website.33 We geocoded study participants’ home locations in each municipality, which enabled us to introduce municipality- or kyuuson-level multilevel analysis using Geographic Information System (GIS)-based methods.\nSubjective general and mental health information included self-rated health,34 self-reported medical conditions (cancer, heart disease, stroke, hypertension, diabetes mellitus, hyperlipidemia, osteoporosis, joint/neurological disease, trauma/fracture, respiratory disease, gastrointestinal disease, liver disease, visual impairment, hearing impairment, urinary disorder, sleep disorder, and others), medication, sleeping, activities of daily living (Tokyo Metropolitan Institute of Gerontology Index of Competence [TMIG-IC]),35 Instrumental ADL,36 depression (Geriatric Depression Scale [GDS-15]),37,38 height, weight, body mass index (BMI), and dental status (number of remaining teeth).",
"Survival time was monitored since the second half of 2003 (the time when self-administered questionnaires were distributed varied by municipality). When individuals died or moved during follow-up, the date they died or left the study area was recorded. We obtained information on physical and cognitive disability by using the LTCI database maintained by the municipalities. The qualification was based on a standardized multistep assessment with several levels of care need, ranging from support levels 1 and 2 to care levels 1 through 5.21",
"Our study protocol and informed consent procedure were approved by the Ethics Committee on the Research of Human Subjects at Nihon Fukushi University.",
"All analyses were performed using the computer software STATA/IC 11.0 (StataCorp LP, College Station, TX, USA)."
] |
[
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"METHODS",
"Study design, setting, and participants",
"Baseline measures",
"Follow-up and outcome measures",
"Ethical issues",
"Statistical analysis",
"RESULTS",
"DISCUSSION"
] |
[
"“Why are Japanese living longer?”1 The question of Japanese longevity is fascinating, and the global community wants to know more about this phenomenon. In 2008, life expectancy was 79.19 years in Japanese men and 85.99 years in Japanese women, which outrank those of nearly all developed and developing countries.2,3 While some believe that genetic differences between Japanese and Western populations are a factor, research suggests that this is an unlikely explanation.4,5\nResources in society can improve the general health of individuals.6,7 Many people agree that the health of individuals is affected by several aspects of society, such as cohesion, lifestyle, customs, family structure, culture, and religious beliefs, all of which are generated through each country’s or community’s complexity and context over a period of many years.6,8 Large-scale cohort studies are being conducted in Japan to reveal the reasons for Japanese longevity, but these studies are limited in their ability to make causal inferences because of the complexity of the underlying mechanisms.9–20\nIn addition to the work of scholars, the national and local governments are also addressing the issue of aging in Japan. For instance, the nationwide universal long-term care insurance system was started in 2000,21,22 and an elder abuse prevention and caregiver support law was enacted in 2006.23 Nevertheless, a new type of health inequality may result from imbalances and lack of harmonization in the community’s supply and demand of health and social care.24,25\nWithin the natural course of aging (eg, from independence in activities of daily living [ADL], with only minor comorbidities, to ADL-dependence due to stroke sequelae and/or other medical conditions), what do older people and their families want from society? Which health services are efficient and fair? To answer these questions, we urgently need evidence-based approaches to living arrangements, social support, and social capital that are based on social epidemiology, gerontology, and health services research.\nThe Aichi Gerontological Evaluation Study (AGES) project was launched for this purpose in 1999. At the Center for Well-being and Society of Nihon Fukushi University in Aichi prefecture, Japan, one of the authors (KK) and colleagues are responsible for managing the project and thus take full responsibility for it. The organizations that provided funding for this research had no role in the conduct of the study or the presentation of its results.",
" Study design, setting, and participants After a pilot cohort was generated and evaluated in 1999, we organized the AGES 2003 Cohort Study as a part of the AGES project. In this prospective study, the source population was community-dwelling individuals aged 65 years or older who lived in 6 targeted municipalities and were ADL-independent as of the second half of 2003 (the exact date varied by municipality). The targeted municipalities covered the entire southern part of the Chita peninsula in Aichi Prefecture (ie, Agui town, Handa city, Tokoname city, Taketoyo town, Mihama town, and Minami-Chita town). The 6 municipalities consist of 18 kyuuson (the second smallest administrative unit in Japan, based on the municipalities in 1950), which comprise urban, semi-urban, and rural settings.9 Older people who were not ADL-independent were excluded if they were eligible to receive benefits from public long-term care insurance (LTCI) services and or if they indicated that they were ADL-dependent in the baseline questionnaire.\nThe survey was conducted using a random sampling method in the 2 larger municipalities (Handa and Tokoname) and a complete census (complete enumeration) of the 4 smaller municipalities (Agui, Mihama, Minami-Chita, and Taketoyo) by municipal officers of the public LTCI system. A total of 49 707 residents aged 65 or older as of 1 October 2003 were targeted (Figure). The self-administered baseline questionnaire was mailed to 29 374 individuals selected by the sampling process as stated above. Then, 13 310 individuals (6508 men; 6802 women) were introduced to the AGES 2003 Cohort. There are no monetary or other incentives to encourage participation in the cohort. For data analysis, the municipality-level sample weight was calculated for selection probability, nonresponse, and other adjustments, to reflect the population proportion of those aged 65 or older in each municipality.\nAfter a pilot cohort was generated and evaluated in 1999, we organized the AGES 2003 Cohort Study as a part of the AGES project. In this prospective study, the source population was community-dwelling individuals aged 65 years or older who lived in 6 targeted municipalities and were ADL-independent as of the second half of 2003 (the exact date varied by municipality). The targeted municipalities covered the entire southern part of the Chita peninsula in Aichi Prefecture (ie, Agui town, Handa city, Tokoname city, Taketoyo town, Mihama town, and Minami-Chita town). The 6 municipalities consist of 18 kyuuson (the second smallest administrative unit in Japan, based on the municipalities in 1950), which comprise urban, semi-urban, and rural settings.9 Older people who were not ADL-independent were excluded if they were eligible to receive benefits from public long-term care insurance (LTCI) services and or if they indicated that they were ADL-dependent in the baseline questionnaire.\nThe survey was conducted using a random sampling method in the 2 larger municipalities (Handa and Tokoname) and a complete census (complete enumeration) of the 4 smaller municipalities (Agui, Mihama, Minami-Chita, and Taketoyo) by municipal officers of the public LTCI system. A total of 49 707 residents aged 65 or older as of 1 October 2003 were targeted (Figure). The self-administered baseline questionnaire was mailed to 29 374 individuals selected by the sampling process as stated above. Then, 13 310 individuals (6508 men; 6802 women) were introduced to the AGES 2003 Cohort. There are no monetary or other incentives to encourage participation in the cohort. For data analysis, the municipality-level sample weight was calculated for selection probability, nonresponse, and other adjustments, to reflect the population proportion of those aged 65 or older in each municipality.\n Baseline measures Table 1 summarizes the main measures that were collected in 2003. Other variables were also experimentally measured (some of which are not shown in Table 1). A summary of the measures is available at our website (http://square.umin.ac.jp/ages/index.html). The details of the questionnaire (including the exact wording of every question in Japanese and the translated version in English) have been published elsewhere.26–28\nThe baseline questionnaire included the following individual-level information: demographic factors (date of birth and sex), living arrangements (cohabitation status, marital status, and caregiving status), and socioeconomic status (education, household income, and current occupation status). Details of individual-level social support (receiving and providing emotional support and instrumental support), social participation, and sense of coherence29,30 were also obtained. Other factors included abuse of older people, negative life events (retirement, death of spouse, death of close relatives/friends, serious disease, a change in living arrangements such as moving, economic distress, or the beginning of informal family caregiving in the recent year), health-related behaviors (smoking, alcohol consumption, diet habits, physical activity [walking]), frequency of medical check-ups, and any hobbies. We regarded social participation as individual-level social capital and divided it into vertical and horizontal social capital. Vertical social capital was defined as participating in groups that encouraged hierarchical relations, and horizontal social capital was defined as participating in groups of social equals.18\nCommunity-level measures were also obtained. Social capital (trust, reciprocity, and crime) was aggregated by individual-level variables.31 Other community-level variables of social factors (eg, the Gini coefficient) were aggregated by individual responses. For environmental exposures, information on airborne particle concentration (sulfur dioxide [SO2], nitrous monoxide [NO], carbon monoxide [CO], ozone [O3], and particulate matter with an aerodynamic diameter <10 µm [PM10])32 in the 6 municipalities (7 measuring points) was available from the Aichi prefecture website.33 We geocoded study participants’ home locations in each municipality, which enabled us to introduce municipality- or kyuuson-level multilevel analysis using Geographic Information System (GIS)-based methods.\nSubjective general and mental health information included self-rated health,34 self-reported medical conditions (cancer, heart disease, stroke, hypertension, diabetes mellitus, hyperlipidemia, osteoporosis, joint/neurological disease, trauma/fracture, respiratory disease, gastrointestinal disease, liver disease, visual impairment, hearing impairment, urinary disorder, sleep disorder, and others), medication, sleeping, activities of daily living (Tokyo Metropolitan Institute of Gerontology Index of Competence [TMIG-IC]),35 Instrumental ADL,36 depression (Geriatric Depression Scale [GDS-15]),37,38 height, weight, body mass index (BMI), and dental status (number of remaining teeth).\nTable 1 summarizes the main measures that were collected in 2003. Other variables were also experimentally measured (some of which are not shown in Table 1). A summary of the measures is available at our website (http://square.umin.ac.jp/ages/index.html). The details of the questionnaire (including the exact wording of every question in Japanese and the translated version in English) have been published elsewhere.26–28\nThe baseline questionnaire included the following individual-level information: demographic factors (date of birth and sex), living arrangements (cohabitation status, marital status, and caregiving status), and socioeconomic status (education, household income, and current occupation status). Details of individual-level social support (receiving and providing emotional support and instrumental support), social participation, and sense of coherence29,30 were also obtained. Other factors included abuse of older people, negative life events (retirement, death of spouse, death of close relatives/friends, serious disease, a change in living arrangements such as moving, economic distress, or the beginning of informal family caregiving in the recent year), health-related behaviors (smoking, alcohol consumption, diet habits, physical activity [walking]), frequency of medical check-ups, and any hobbies. We regarded social participation as individual-level social capital and divided it into vertical and horizontal social capital. Vertical social capital was defined as participating in groups that encouraged hierarchical relations, and horizontal social capital was defined as participating in groups of social equals.18\nCommunity-level measures were also obtained. Social capital (trust, reciprocity, and crime) was aggregated by individual-level variables.31 Other community-level variables of social factors (eg, the Gini coefficient) were aggregated by individual responses. For environmental exposures, information on airborne particle concentration (sulfur dioxide [SO2], nitrous monoxide [NO], carbon monoxide [CO], ozone [O3], and particulate matter with an aerodynamic diameter <10 µm [PM10])32 in the 6 municipalities (7 measuring points) was available from the Aichi prefecture website.33 We geocoded study participants’ home locations in each municipality, which enabled us to introduce municipality- or kyuuson-level multilevel analysis using Geographic Information System (GIS)-based methods.\nSubjective general and mental health information included self-rated health,34 self-reported medical conditions (cancer, heart disease, stroke, hypertension, diabetes mellitus, hyperlipidemia, osteoporosis, joint/neurological disease, trauma/fracture, respiratory disease, gastrointestinal disease, liver disease, visual impairment, hearing impairment, urinary disorder, sleep disorder, and others), medication, sleeping, activities of daily living (Tokyo Metropolitan Institute of Gerontology Index of Competence [TMIG-IC]),35 Instrumental ADL,36 depression (Geriatric Depression Scale [GDS-15]),37,38 height, weight, body mass index (BMI), and dental status (number of remaining teeth).\n Follow-up and outcome measures Survival time was monitored since the second half of 2003 (the time when self-administered questionnaires were distributed varied by municipality). When individuals died or moved during follow-up, the date they died or left the study area was recorded. We obtained information on physical and cognitive disability by using the LTCI database maintained by the municipalities. The qualification was based on a standardized multistep assessment with several levels of care need, ranging from support levels 1 and 2 to care levels 1 through 5.21\nSurvival time was monitored since the second half of 2003 (the time when self-administered questionnaires were distributed varied by municipality). When individuals died or moved during follow-up, the date they died or left the study area was recorded. We obtained information on physical and cognitive disability by using the LTCI database maintained by the municipalities. The qualification was based on a standardized multistep assessment with several levels of care need, ranging from support levels 1 and 2 to care levels 1 through 5.21\n Ethical issues Our study protocol and informed consent procedure were approved by the Ethics Committee on the Research of Human Subjects at Nihon Fukushi University.\nOur study protocol and informed consent procedure were approved by the Ethics Committee on the Research of Human Subjects at Nihon Fukushi University.\n Statistical analysis All analyses were performed using the computer software STATA/IC 11.0 (StataCorp LP, College Station, TX, USA).\nAll analyses were performed using the computer software STATA/IC 11.0 (StataCorp LP, College Station, TX, USA).",
"After a pilot cohort was generated and evaluated in 1999, we organized the AGES 2003 Cohort Study as a part of the AGES project. In this prospective study, the source population was community-dwelling individuals aged 65 years or older who lived in 6 targeted municipalities and were ADL-independent as of the second half of 2003 (the exact date varied by municipality). The targeted municipalities covered the entire southern part of the Chita peninsula in Aichi Prefecture (ie, Agui town, Handa city, Tokoname city, Taketoyo town, Mihama town, and Minami-Chita town). The 6 municipalities consist of 18 kyuuson (the second smallest administrative unit in Japan, based on the municipalities in 1950), which comprise urban, semi-urban, and rural settings.9 Older people who were not ADL-independent were excluded if they were eligible to receive benefits from public long-term care insurance (LTCI) services and or if they indicated that they were ADL-dependent in the baseline questionnaire.\nThe survey was conducted using a random sampling method in the 2 larger municipalities (Handa and Tokoname) and a complete census (complete enumeration) of the 4 smaller municipalities (Agui, Mihama, Minami-Chita, and Taketoyo) by municipal officers of the public LTCI system. A total of 49 707 residents aged 65 or older as of 1 October 2003 were targeted (Figure). The self-administered baseline questionnaire was mailed to 29 374 individuals selected by the sampling process as stated above. Then, 13 310 individuals (6508 men; 6802 women) were introduced to the AGES 2003 Cohort. There are no monetary or other incentives to encourage participation in the cohort. For data analysis, the municipality-level sample weight was calculated for selection probability, nonresponse, and other adjustments, to reflect the population proportion of those aged 65 or older in each municipality.",
"Table 1 summarizes the main measures that were collected in 2003. Other variables were also experimentally measured (some of which are not shown in Table 1). A summary of the measures is available at our website (http://square.umin.ac.jp/ages/index.html). The details of the questionnaire (including the exact wording of every question in Japanese and the translated version in English) have been published elsewhere.26–28\nThe baseline questionnaire included the following individual-level information: demographic factors (date of birth and sex), living arrangements (cohabitation status, marital status, and caregiving status), and socioeconomic status (education, household income, and current occupation status). Details of individual-level social support (receiving and providing emotional support and instrumental support), social participation, and sense of coherence29,30 were also obtained. Other factors included abuse of older people, negative life events (retirement, death of spouse, death of close relatives/friends, serious disease, a change in living arrangements such as moving, economic distress, or the beginning of informal family caregiving in the recent year), health-related behaviors (smoking, alcohol consumption, diet habits, physical activity [walking]), frequency of medical check-ups, and any hobbies. We regarded social participation as individual-level social capital and divided it into vertical and horizontal social capital. Vertical social capital was defined as participating in groups that encouraged hierarchical relations, and horizontal social capital was defined as participating in groups of social equals.18\nCommunity-level measures were also obtained. Social capital (trust, reciprocity, and crime) was aggregated by individual-level variables.31 Other community-level variables of social factors (eg, the Gini coefficient) were aggregated by individual responses. For environmental exposures, information on airborne particle concentration (sulfur dioxide [SO2], nitrous monoxide [NO], carbon monoxide [CO], ozone [O3], and particulate matter with an aerodynamic diameter <10 µm [PM10])32 in the 6 municipalities (7 measuring points) was available from the Aichi prefecture website.33 We geocoded study participants’ home locations in each municipality, which enabled us to introduce municipality- or kyuuson-level multilevel analysis using Geographic Information System (GIS)-based methods.\nSubjective general and mental health information included self-rated health,34 self-reported medical conditions (cancer, heart disease, stroke, hypertension, diabetes mellitus, hyperlipidemia, osteoporosis, joint/neurological disease, trauma/fracture, respiratory disease, gastrointestinal disease, liver disease, visual impairment, hearing impairment, urinary disorder, sleep disorder, and others), medication, sleeping, activities of daily living (Tokyo Metropolitan Institute of Gerontology Index of Competence [TMIG-IC]),35 Instrumental ADL,36 depression (Geriatric Depression Scale [GDS-15]),37,38 height, weight, body mass index (BMI), and dental status (number of remaining teeth).",
"Survival time was monitored since the second half of 2003 (the time when self-administered questionnaires were distributed varied by municipality). When individuals died or moved during follow-up, the date they died or left the study area was recorded. We obtained information on physical and cognitive disability by using the LTCI database maintained by the municipalities. The qualification was based on a standardized multistep assessment with several levels of care need, ranging from support levels 1 and 2 to care levels 1 through 5.21",
"Our study protocol and informed consent procedure were approved by the Ethics Committee on the Research of Human Subjects at Nihon Fukushi University.",
"All analyses were performed using the computer software STATA/IC 11.0 (StataCorp LP, College Station, TX, USA).",
"The baseline characteristics of the participants are shown in Table 2. Living alone, being divorced/widowed, lower education status, no current occupation, no smoking, and no alcohol consumption were more frequent in women than in men. As age increased, the proportions of those with a tertiary education decreased, as did current smokers, drinkers, and positive horizontal social capital.\naUnweighted values are reported only for cells indicating numbers of individuals.\nDuring a 48-month follow-up period, which is the current maximum, men were observed for 24 753 person-years and women for 26 456 person-years. In total, 0.8% of the study participants were lost to follow-up because they left the study area during the 4-year period (n = 105). The cumulative survival rate for the study population was 96.8% (men, 95.6%; women, 98.0%) at 24 months and 92.1% (men, 89.4%; women, 94.7%) at 48 months. Sex and age-group differences among individual-level core variables are shown in Table 2. Overall and sex-stratified cumulative survival rates at 24 and 48 months for individual-level core variables are shown in Table 3.",
"The main strengths of the AGES 2003 Cohort Study are: (1) the location of the study—Japan leads the world in the pace of population aging, (2) the linkage with geographical data via the Geographic Information System, (3) the quality and length of follow-up—there is no administrative loss during follow-up, (4) the large variability of health determinants (health-related behavior, socioeconomic status, social support, and social capital at the individual and community level) minimizes omitted variable bias, and (5) the availability of multilevel analysis.\nThe main weaknesses of the study are the moderate response rate and the limited generalizability of the findings, as the data are not obtained from a national representative sample. Nevertheless, urban, semi-urban, and rural municipalities were included. Regarding the moderate response rate of the AGES 2003 Cohort Study, differences between respondents and nonrespondents at baseline were examined with respect to some demographic characteristics.26,28 Although the available information did not encompass all 6 municipalities, individuals who were younger than 80 years and those with a household income higher than the median were more likely to answer the baseline questionnaire (P < 0.10 and P < 0.001, respectively). There was no difference between men and women.\nOur study group is currently conducting quantitative research using data on all-cause mortality from the AGES 2003 Cohort Study. However, several peer-reviewed articles and a book (and its English translation) have already been published on the AGES project.9,18–20,26,28 Using cross-sectional data from 2003, with a baseline that extended across 15 municipalities in 3 prefectures, Kondo and colleagues described the relationship between socioeconomic status and self-reported health status.26,28\nMurata and colleagues reported that lower SES and residential area were significantly associated with depression.20 A study by Ichida and colleagues was the first in Japan to use multilevel analysis to support the relative income hypothesis.9 Aida and colleagues also used multilevel analysis to reveal that horizontal social capital but not vertical social capital had beneficial effects on the number of remaining teeth in older Japanese adults.18 Another study identified the risk factors for eligible care level under the national long-term care insurance from 2003 to 2006–2007. Kondo and colleagues reported that relative deprivation may be a mechanism underlying the relationship between income inequality and disability during old age, at least among men.\nIn conclusion, the AGES 2003 Cohort Study has provided useful and observable quantitative findings for use in social epidemiology, gerontology, and health services research. We reported the baseline profile and the progress of the AGES 2003 Cohort Study under the AGES project, which is an open, general-purpose epidemiological/gerontological laboratory. An additional survey was implemented in 2006–2007 and is planned for 2010. We are certain to obtain additional useful data in the 2010 survey. The data in the AGES project is available to academic investigators on an approval basis. The first step is to contact Katsunori Kondo. Please refer to the AGES project website (http://square.umin.ac.jp/ages/index.html) for details.27"
] |
[
null,
"methods",
null,
null,
null,
null,
null,
"results",
"discussion"
] |
[
"Japanese longevity",
"social support",
"social capital",
"cohort profile",
"the AGES project"
] |
INTRODUCTION:
“Why are Japanese living longer?”1 The question of Japanese longevity is fascinating, and the global community wants to know more about this phenomenon. In 2008, life expectancy was 79.19 years in Japanese men and 85.99 years in Japanese women, which outrank those of nearly all developed and developing countries.2,3 While some believe that genetic differences between Japanese and Western populations are a factor, research suggests that this is an unlikely explanation.4,5
Resources in society can improve the general health of individuals.6,7 Many people agree that the health of individuals is affected by several aspects of society, such as cohesion, lifestyle, customs, family structure, culture, and religious beliefs, all of which are generated through each country’s or community’s complexity and context over a period of many years.6,8 Large-scale cohort studies are being conducted in Japan to reveal the reasons for Japanese longevity, but these studies are limited in their ability to make causal inferences because of the complexity of the underlying mechanisms.9–20
In addition to the work of scholars, the national and local governments are also addressing the issue of aging in Japan. For instance, the nationwide universal long-term care insurance system was started in 2000,21,22 and an elder abuse prevention and caregiver support law was enacted in 2006.23 Nevertheless, a new type of health inequality may result from imbalances and lack of harmonization in the community’s supply and demand of health and social care.24,25
Within the natural course of aging (eg, from independence in activities of daily living [ADL], with only minor comorbidities, to ADL-dependence due to stroke sequelae and/or other medical conditions), what do older people and their families want from society? Which health services are efficient and fair? To answer these questions, we urgently need evidence-based approaches to living arrangements, social support, and social capital that are based on social epidemiology, gerontology, and health services research.
The Aichi Gerontological Evaluation Study (AGES) project was launched for this purpose in 1999. At the Center for Well-being and Society of Nihon Fukushi University in Aichi prefecture, Japan, one of the authors (KK) and colleagues are responsible for managing the project and thus take full responsibility for it. The organizations that provided funding for this research had no role in the conduct of the study or the presentation of its results.
METHODS:
Study design, setting, and participants After a pilot cohort was generated and evaluated in 1999, we organized the AGES 2003 Cohort Study as a part of the AGES project. In this prospective study, the source population was community-dwelling individuals aged 65 years or older who lived in 6 targeted municipalities and were ADL-independent as of the second half of 2003 (the exact date varied by municipality). The targeted municipalities covered the entire southern part of the Chita peninsula in Aichi Prefecture (ie, Agui town, Handa city, Tokoname city, Taketoyo town, Mihama town, and Minami-Chita town). The 6 municipalities consist of 18 kyuuson (the second smallest administrative unit in Japan, based on the municipalities in 1950), which comprise urban, semi-urban, and rural settings.9 Older people who were not ADL-independent were excluded if they were eligible to receive benefits from public long-term care insurance (LTCI) services and or if they indicated that they were ADL-dependent in the baseline questionnaire.
The survey was conducted using a random sampling method in the 2 larger municipalities (Handa and Tokoname) and a complete census (complete enumeration) of the 4 smaller municipalities (Agui, Mihama, Minami-Chita, and Taketoyo) by municipal officers of the public LTCI system. A total of 49 707 residents aged 65 or older as of 1 October 2003 were targeted (Figure). The self-administered baseline questionnaire was mailed to 29 374 individuals selected by the sampling process as stated above. Then, 13 310 individuals (6508 men; 6802 women) were introduced to the AGES 2003 Cohort. There are no monetary or other incentives to encourage participation in the cohort. For data analysis, the municipality-level sample weight was calculated for selection probability, nonresponse, and other adjustments, to reflect the population proportion of those aged 65 or older in each municipality.
After a pilot cohort was generated and evaluated in 1999, we organized the AGES 2003 Cohort Study as a part of the AGES project. In this prospective study, the source population was community-dwelling individuals aged 65 years or older who lived in 6 targeted municipalities and were ADL-independent as of the second half of 2003 (the exact date varied by municipality). The targeted municipalities covered the entire southern part of the Chita peninsula in Aichi Prefecture (ie, Agui town, Handa city, Tokoname city, Taketoyo town, Mihama town, and Minami-Chita town). The 6 municipalities consist of 18 kyuuson (the second smallest administrative unit in Japan, based on the municipalities in 1950), which comprise urban, semi-urban, and rural settings.9 Older people who were not ADL-independent were excluded if they were eligible to receive benefits from public long-term care insurance (LTCI) services and or if they indicated that they were ADL-dependent in the baseline questionnaire.
The survey was conducted using a random sampling method in the 2 larger municipalities (Handa and Tokoname) and a complete census (complete enumeration) of the 4 smaller municipalities (Agui, Mihama, Minami-Chita, and Taketoyo) by municipal officers of the public LTCI system. A total of 49 707 residents aged 65 or older as of 1 October 2003 were targeted (Figure). The self-administered baseline questionnaire was mailed to 29 374 individuals selected by the sampling process as stated above. Then, 13 310 individuals (6508 men; 6802 women) were introduced to the AGES 2003 Cohort. There are no monetary or other incentives to encourage participation in the cohort. For data analysis, the municipality-level sample weight was calculated for selection probability, nonresponse, and other adjustments, to reflect the population proportion of those aged 65 or older in each municipality.
Baseline measures Table 1 summarizes the main measures that were collected in 2003. Other variables were also experimentally measured (some of which are not shown in Table 1). A summary of the measures is available at our website (http://square.umin.ac.jp/ages/index.html). The details of the questionnaire (including the exact wording of every question in Japanese and the translated version in English) have been published elsewhere.26–28
The baseline questionnaire included the following individual-level information: demographic factors (date of birth and sex), living arrangements (cohabitation status, marital status, and caregiving status), and socioeconomic status (education, household income, and current occupation status). Details of individual-level social support (receiving and providing emotional support and instrumental support), social participation, and sense of coherence29,30 were also obtained. Other factors included abuse of older people, negative life events (retirement, death of spouse, death of close relatives/friends, serious disease, a change in living arrangements such as moving, economic distress, or the beginning of informal family caregiving in the recent year), health-related behaviors (smoking, alcohol consumption, diet habits, physical activity [walking]), frequency of medical check-ups, and any hobbies. We regarded social participation as individual-level social capital and divided it into vertical and horizontal social capital. Vertical social capital was defined as participating in groups that encouraged hierarchical relations, and horizontal social capital was defined as participating in groups of social equals.18
Community-level measures were also obtained. Social capital (trust, reciprocity, and crime) was aggregated by individual-level variables.31 Other community-level variables of social factors (eg, the Gini coefficient) were aggregated by individual responses. For environmental exposures, information on airborne particle concentration (sulfur dioxide [SO2], nitrous monoxide [NO], carbon monoxide [CO], ozone [O3], and particulate matter with an aerodynamic diameter <10 µm [PM10])32 in the 6 municipalities (7 measuring points) was available from the Aichi prefecture website.33 We geocoded study participants’ home locations in each municipality, which enabled us to introduce municipality- or kyuuson-level multilevel analysis using Geographic Information System (GIS)-based methods.
Subjective general and mental health information included self-rated health,34 self-reported medical conditions (cancer, heart disease, stroke, hypertension, diabetes mellitus, hyperlipidemia, osteoporosis, joint/neurological disease, trauma/fracture, respiratory disease, gastrointestinal disease, liver disease, visual impairment, hearing impairment, urinary disorder, sleep disorder, and others), medication, sleeping, activities of daily living (Tokyo Metropolitan Institute of Gerontology Index of Competence [TMIG-IC]),35 Instrumental ADL,36 depression (Geriatric Depression Scale [GDS-15]),37,38 height, weight, body mass index (BMI), and dental status (number of remaining teeth).
Table 1 summarizes the main measures that were collected in 2003. Other variables were also experimentally measured (some of which are not shown in Table 1). A summary of the measures is available at our website (http://square.umin.ac.jp/ages/index.html). The details of the questionnaire (including the exact wording of every question in Japanese and the translated version in English) have been published elsewhere.26–28
The baseline questionnaire included the following individual-level information: demographic factors (date of birth and sex), living arrangements (cohabitation status, marital status, and caregiving status), and socioeconomic status (education, household income, and current occupation status). Details of individual-level social support (receiving and providing emotional support and instrumental support), social participation, and sense of coherence29,30 were also obtained. Other factors included abuse of older people, negative life events (retirement, death of spouse, death of close relatives/friends, serious disease, a change in living arrangements such as moving, economic distress, or the beginning of informal family caregiving in the recent year), health-related behaviors (smoking, alcohol consumption, diet habits, physical activity [walking]), frequency of medical check-ups, and any hobbies. We regarded social participation as individual-level social capital and divided it into vertical and horizontal social capital. Vertical social capital was defined as participating in groups that encouraged hierarchical relations, and horizontal social capital was defined as participating in groups of social equals.18
Community-level measures were also obtained. Social capital (trust, reciprocity, and crime) was aggregated by individual-level variables.31 Other community-level variables of social factors (eg, the Gini coefficient) were aggregated by individual responses. For environmental exposures, information on airborne particle concentration (sulfur dioxide [SO2], nitrous monoxide [NO], carbon monoxide [CO], ozone [O3], and particulate matter with an aerodynamic diameter <10 µm [PM10])32 in the 6 municipalities (7 measuring points) was available from the Aichi prefecture website.33 We geocoded study participants’ home locations in each municipality, which enabled us to introduce municipality- or kyuuson-level multilevel analysis using Geographic Information System (GIS)-based methods.
Subjective general and mental health information included self-rated health,34 self-reported medical conditions (cancer, heart disease, stroke, hypertension, diabetes mellitus, hyperlipidemia, osteoporosis, joint/neurological disease, trauma/fracture, respiratory disease, gastrointestinal disease, liver disease, visual impairment, hearing impairment, urinary disorder, sleep disorder, and others), medication, sleeping, activities of daily living (Tokyo Metropolitan Institute of Gerontology Index of Competence [TMIG-IC]),35 Instrumental ADL,36 depression (Geriatric Depression Scale [GDS-15]),37,38 height, weight, body mass index (BMI), and dental status (number of remaining teeth).
Follow-up and outcome measures Survival time was monitored since the second half of 2003 (the time when self-administered questionnaires were distributed varied by municipality). When individuals died or moved during follow-up, the date they died or left the study area was recorded. We obtained information on physical and cognitive disability by using the LTCI database maintained by the municipalities. The qualification was based on a standardized multistep assessment with several levels of care need, ranging from support levels 1 and 2 to care levels 1 through 5.21
Survival time was monitored since the second half of 2003 (the time when self-administered questionnaires were distributed varied by municipality). When individuals died or moved during follow-up, the date they died or left the study area was recorded. We obtained information on physical and cognitive disability by using the LTCI database maintained by the municipalities. The qualification was based on a standardized multistep assessment with several levels of care need, ranging from support levels 1 and 2 to care levels 1 through 5.21
Ethical issues Our study protocol and informed consent procedure were approved by the Ethics Committee on the Research of Human Subjects at Nihon Fukushi University.
Our study protocol and informed consent procedure were approved by the Ethics Committee on the Research of Human Subjects at Nihon Fukushi University.
Statistical analysis All analyses were performed using the computer software STATA/IC 11.0 (StataCorp LP, College Station, TX, USA).
All analyses were performed using the computer software STATA/IC 11.0 (StataCorp LP, College Station, TX, USA).
Study design, setting, and participants:
After a pilot cohort was generated and evaluated in 1999, we organized the AGES 2003 Cohort Study as a part of the AGES project. In this prospective study, the source population was community-dwelling individuals aged 65 years or older who lived in 6 targeted municipalities and were ADL-independent as of the second half of 2003 (the exact date varied by municipality). The targeted municipalities covered the entire southern part of the Chita peninsula in Aichi Prefecture (ie, Agui town, Handa city, Tokoname city, Taketoyo town, Mihama town, and Minami-Chita town). The 6 municipalities consist of 18 kyuuson (the second smallest administrative unit in Japan, based on the municipalities in 1950), which comprise urban, semi-urban, and rural settings.9 Older people who were not ADL-independent were excluded if they were eligible to receive benefits from public long-term care insurance (LTCI) services and or if they indicated that they were ADL-dependent in the baseline questionnaire.
The survey was conducted using a random sampling method in the 2 larger municipalities (Handa and Tokoname) and a complete census (complete enumeration) of the 4 smaller municipalities (Agui, Mihama, Minami-Chita, and Taketoyo) by municipal officers of the public LTCI system. A total of 49 707 residents aged 65 or older as of 1 October 2003 were targeted (Figure). The self-administered baseline questionnaire was mailed to 29 374 individuals selected by the sampling process as stated above. Then, 13 310 individuals (6508 men; 6802 women) were introduced to the AGES 2003 Cohort. There are no monetary or other incentives to encourage participation in the cohort. For data analysis, the municipality-level sample weight was calculated for selection probability, nonresponse, and other adjustments, to reflect the population proportion of those aged 65 or older in each municipality.
Baseline measures:
Table 1 summarizes the main measures that were collected in 2003. Other variables were also experimentally measured (some of which are not shown in Table 1). A summary of the measures is available at our website (http://square.umin.ac.jp/ages/index.html). The details of the questionnaire (including the exact wording of every question in Japanese and the translated version in English) have been published elsewhere.26–28
The baseline questionnaire included the following individual-level information: demographic factors (date of birth and sex), living arrangements (cohabitation status, marital status, and caregiving status), and socioeconomic status (education, household income, and current occupation status). Details of individual-level social support (receiving and providing emotional support and instrumental support), social participation, and sense of coherence29,30 were also obtained. Other factors included abuse of older people, negative life events (retirement, death of spouse, death of close relatives/friends, serious disease, a change in living arrangements such as moving, economic distress, or the beginning of informal family caregiving in the recent year), health-related behaviors (smoking, alcohol consumption, diet habits, physical activity [walking]), frequency of medical check-ups, and any hobbies. We regarded social participation as individual-level social capital and divided it into vertical and horizontal social capital. Vertical social capital was defined as participating in groups that encouraged hierarchical relations, and horizontal social capital was defined as participating in groups of social equals.18
Community-level measures were also obtained. Social capital (trust, reciprocity, and crime) was aggregated by individual-level variables.31 Other community-level variables of social factors (eg, the Gini coefficient) were aggregated by individual responses. For environmental exposures, information on airborne particle concentration (sulfur dioxide [SO2], nitrous monoxide [NO], carbon monoxide [CO], ozone [O3], and particulate matter with an aerodynamic diameter <10 µm [PM10])32 in the 6 municipalities (7 measuring points) was available from the Aichi prefecture website.33 We geocoded study participants’ home locations in each municipality, which enabled us to introduce municipality- or kyuuson-level multilevel analysis using Geographic Information System (GIS)-based methods.
Subjective general and mental health information included self-rated health,34 self-reported medical conditions (cancer, heart disease, stroke, hypertension, diabetes mellitus, hyperlipidemia, osteoporosis, joint/neurological disease, trauma/fracture, respiratory disease, gastrointestinal disease, liver disease, visual impairment, hearing impairment, urinary disorder, sleep disorder, and others), medication, sleeping, activities of daily living (Tokyo Metropolitan Institute of Gerontology Index of Competence [TMIG-IC]),35 Instrumental ADL,36 depression (Geriatric Depression Scale [GDS-15]),37,38 height, weight, body mass index (BMI), and dental status (number of remaining teeth).
Follow-up and outcome measures:
Survival time was monitored since the second half of 2003 (the time when self-administered questionnaires were distributed varied by municipality). When individuals died or moved during follow-up, the date they died or left the study area was recorded. We obtained information on physical and cognitive disability by using the LTCI database maintained by the municipalities. The qualification was based on a standardized multistep assessment with several levels of care need, ranging from support levels 1 and 2 to care levels 1 through 5.21
Ethical issues:
Our study protocol and informed consent procedure were approved by the Ethics Committee on the Research of Human Subjects at Nihon Fukushi University.
Statistical analysis:
All analyses were performed using the computer software STATA/IC 11.0 (StataCorp LP, College Station, TX, USA).
RESULTS:
The baseline characteristics of the participants are shown in Table 2. Living alone, being divorced/widowed, lower education status, no current occupation, no smoking, and no alcohol consumption were more frequent in women than in men. As age increased, the proportions of those with a tertiary education decreased, as did current smokers, drinkers, and positive horizontal social capital.
aUnweighted values are reported only for cells indicating numbers of individuals.
During a 48-month follow-up period, which is the current maximum, men were observed for 24 753 person-years and women for 26 456 person-years. In total, 0.8% of the study participants were lost to follow-up because they left the study area during the 4-year period (n = 105). The cumulative survival rate for the study population was 96.8% (men, 95.6%; women, 98.0%) at 24 months and 92.1% (men, 89.4%; women, 94.7%) at 48 months. Sex and age-group differences among individual-level core variables are shown in Table 2. Overall and sex-stratified cumulative survival rates at 24 and 48 months for individual-level core variables are shown in Table 3.
DISCUSSION:
The main strengths of the AGES 2003 Cohort Study are: (1) the location of the study—Japan leads the world in the pace of population aging, (2) the linkage with geographical data via the Geographic Information System, (3) the quality and length of follow-up—there is no administrative loss during follow-up, (4) the large variability of health determinants (health-related behavior, socioeconomic status, social support, and social capital at the individual and community level) minimizes omitted variable bias, and (5) the availability of multilevel analysis.
The main weaknesses of the study are the moderate response rate and the limited generalizability of the findings, as the data are not obtained from a national representative sample. Nevertheless, urban, semi-urban, and rural municipalities were included. Regarding the moderate response rate of the AGES 2003 Cohort Study, differences between respondents and nonrespondents at baseline were examined with respect to some demographic characteristics.26,28 Although the available information did not encompass all 6 municipalities, individuals who were younger than 80 years and those with a household income higher than the median were more likely to answer the baseline questionnaire (P < 0.10 and P < 0.001, respectively). There was no difference between men and women.
Our study group is currently conducting quantitative research using data on all-cause mortality from the AGES 2003 Cohort Study. However, several peer-reviewed articles and a book (and its English translation) have already been published on the AGES project.9,18–20,26,28 Using cross-sectional data from 2003, with a baseline that extended across 15 municipalities in 3 prefectures, Kondo and colleagues described the relationship between socioeconomic status and self-reported health status.26,28
Murata and colleagues reported that lower SES and residential area were significantly associated with depression.20 A study by Ichida and colleagues was the first in Japan to use multilevel analysis to support the relative income hypothesis.9 Aida and colleagues also used multilevel analysis to reveal that horizontal social capital but not vertical social capital had beneficial effects on the number of remaining teeth in older Japanese adults.18 Another study identified the risk factors for eligible care level under the national long-term care insurance from 2003 to 2006–2007. Kondo and colleagues reported that relative deprivation may be a mechanism underlying the relationship between income inequality and disability during old age, at least among men.
In conclusion, the AGES 2003 Cohort Study has provided useful and observable quantitative findings for use in social epidemiology, gerontology, and health services research. We reported the baseline profile and the progress of the AGES 2003 Cohort Study under the AGES project, which is an open, general-purpose epidemiological/gerontological laboratory. An additional survey was implemented in 2006–2007 and is planned for 2010. We are certain to obtain additional useful data in the 2010 survey. The data in the AGES project is available to academic investigators on an approval basis. The first step is to contact Katsunori Kondo. Please refer to the AGES project website (http://square.umin.ac.jp/ages/index.html) for details.27
|
Background:
The longevity of Japanese is thought to be associated with psychosocial factors such as sense of coherence, social support, and social capital. However, the actual factors responsible and the extent of their contribution to individual health status are not known.
Methods:
The Aichi Gerontological Evaluation Study (AGES) 2003 Cohort Study is a prospective cohort study of community-dwelling, activities of daily living-independent people aged 65 or older living in 6 municipalities in Chita peninsula, Aichi Prefecture, Japan. Information on psychosocial factors and other individual- and community-level factors was collected in the second half of 2003 using a baseline questionnaire. Vital status and physical and cognitive decline have been followed using data derived from long-term care insurance certification. Geographical information on the study participants was also obtained.
Results:
A total of 13 310 (6508 men; 6802 women) study participants were registered in the study. For an interim report, we followed the cohort for 48 months, yielding 24 753 person-years of observation among men and 26 456 person-years among women.
Conclusions:
The AGES 2003 Cohort Study provides useful evidence for research in social epidemiology, gerontology, and health services.
| null | null | 4,443 | 233 | 9 |
[
"social",
"study",
"level",
"municipalities",
"2003",
"ages",
"status",
"social capital",
"capital",
"health"
] |
[
"test",
"test"
] | null | null |
[CONTENT] Japanese longevity | social support | social capital | cohort profile | the AGES project [SUMMARY]
|
[CONTENT] Japanese longevity | social support | social capital | cohort profile | the AGES project [SUMMARY]
|
[CONTENT] Japanese longevity | social support | social capital | cohort profile | the AGES project [SUMMARY]
| null |
[CONTENT] Japanese longevity | social support | social capital | cohort profile | the AGES project [SUMMARY]
| null |
[CONTENT] Aged | Aged, 80 and over | Cities | Female | Follow-Up Studies | Health Status | Humans | Japan | Longevity | Male | Prospective Studies | Residence Characteristics | Social Support | Surveys and Questionnaires [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Cities | Female | Follow-Up Studies | Health Status | Humans | Japan | Longevity | Male | Prospective Studies | Residence Characteristics | Social Support | Surveys and Questionnaires [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Cities | Female | Follow-Up Studies | Health Status | Humans | Japan | Longevity | Male | Prospective Studies | Residence Characteristics | Social Support | Surveys and Questionnaires [SUMMARY]
| null |
[CONTENT] Aged | Aged, 80 and over | Cities | Female | Follow-Up Studies | Health Status | Humans | Japan | Longevity | Male | Prospective Studies | Residence Characteristics | Social Support | Surveys and Questionnaires [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] social | study | level | municipalities | 2003 | ages | status | social capital | capital | health [SUMMARY]
|
[CONTENT] social | study | level | municipalities | 2003 | ages | status | social capital | capital | health [SUMMARY]
|
[CONTENT] social | study | level | municipalities | 2003 | ages | status | social capital | capital | health [SUMMARY]
| null |
[CONTENT] social | study | level | municipalities | 2003 | ages | status | social capital | capital | health [SUMMARY]
| null |
[CONTENT] society | japanese | health | social | japanese longevity | longevity | health individuals | complexity | studies | years japanese [SUMMARY]
|
[CONTENT] social | disease | level | municipalities | municipality | status | 2003 | town | measures | information [SUMMARY]
|
[CONTENT] months | 48 | 24 | women | men | current | shown | shown table | table | cumulative [SUMMARY]
| null |
[CONTENT] social | study | municipalities | 2003 | level | ages | health | status | levels | cohort [SUMMARY]
| null |
[CONTENT] Japanese ||| [SUMMARY]
|
[CONTENT] The Aichi Gerontological Evaluation Study | 2003 | daily | 65 or | 6 | Chita | Aichi Prefecture | Japan ||| the second half of 2003 ||| ||| [SUMMARY]
|
[CONTENT] 13 310 | 6508 | 6802 ||| 48 months | 24 753 | 26 456 | years [SUMMARY]
| null |
[CONTENT] Japanese ||| ||| The Aichi Gerontological Evaluation Study | 2003 | daily | 65 or | 6 | Chita | Aichi Prefecture | Japan ||| the second half of 2003 ||| ||| ||| 13 310 | 6508 | 6802 ||| 48 months | 24 753 | 26 456 | years ||| [SUMMARY]
| null |
||||
In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii.
|
23414290
|
The aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Černjavski & Soška (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC).
|
BACKGROUND
|
The aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors.
|
METHODS
|
The investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways.
|
RESULTS
|
Extracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells.
|
CONCLUSION
|
[
"Antineoplastic Agents, Phytogenic",
"Apoptosis",
"Cell Proliferation",
"Dose-Response Relationship, Drug",
"Female",
"HeLa Cells",
"Helichrysum",
"Humans",
"Leukemia, Myeloid",
"Leukocytes, Mononuclear",
"Melanoma",
"Neoplasms",
"Phytotherapy",
"Plant Extracts",
"Signal Transduction",
"Uterine Cervical Neoplasms"
] |
3585823
|
Background
|
Bioactive constituents of medicinal plants are in the center of attention of modern anticancer research due to their prospective roles in suppressing the different stages of malignant transformation. The antitumor potential of plant extracts and compounds could be attributed to their ability to induce changes in the regulation of target molecules in oncogenic signal transduction pathways implicated in cell growth, replication, apoptosis, as well as in angiogenesis, invasion and metastasis of cancer cells
[1-4]. To evaluate the anticancer properties of novel chemotherapeutic agents, the selectivity of their actions against malignant cells in comparison to healthy non-transformed cells, especially immunocompetent cells involved in the immune control of tumor suppression, needs to be carefully examined.
Helichrysum zivojinii Černjavski & Soška is an endemic plant species that grows in the National Park "Galičica" in Macedonia. Some of the plant species from the large genus Helichrysum are used in different regions of the world in traditional medicine for treating wounds, respiratory tract infections and gastro-intestinal disorders
[5-8]. This plant genus is a valuable source of several different secondary metabolites/phytochemicals, such as flavonoids, acetophenones, phloroglucinols, pyrones, diterpenes and sesquiterpenes
[5]. Different morphological groups of Helichrysum species often display unique qualitative and quantitative chemical compositions
[5]. It has been reported that extracts and individual constituents of these plants possess significant biological and pharmacological properties, including antibacterial, antiviral, antifungal, antioxidant, anti-inflammatory and antidiabetic activities
[9-16]. A search through the literature suggests that plants from the genus Helichrysum could be a significant source of compounds with potential anticancer activities
[17-20].
The main goal of this research was to investigate the cytotoxic activities of five extracts isolated as fractions from the endemic plant Helichrysum zivojinii towards selected human malignant cell lines. To assess the sensitivity of normal immunocompetent cells included in the antitumor immune response, the cytotoxicity of these extracts was also tested against human peripheral blood mononuclear cells (PBMC) – both unstimulated and stimulated to proliferate by the mitogen phytohemagglutinin (PHA). To elucidate the molecular mechanisms of the cytotoxic effects of the tested extracts, the distribution of target HeLa cells at specific phases of the cell cycle after the actions of these agents was also analyzed. The mode of HeLa cell death induced by the extracts was also investigated. Elucidation of the signaling pathways implicated in the induction of apoptosis by the tested extracts was conducted by identification of target caspases.
|
Methods
|
Plant extracts The plant material was collected at Tomoros (ca. 1700 altitude), mountain Galičica (Macedonia) during the flowering (17 July 2010) and identified by Vlado Matevski, Institute of Biology, Faculty of Natural Sciences and Mathematics,
http://Ss. Cyril and Methodius University of Skopje, where the voucher specimen is deposited at Macedonian National Herbarium (MKNH) under the number MKNH121335.
Air-dried and powdered aerial parts of Helichrysum zivojinii (330 g) were extracted twice with n-hexane in an ultrasonic bath for 45 min. The combined extracts were concentrated in a vacuum to obtain a hexane extract (4.2 g). The plant material was successively extracted in the same manner with solvents of rising polarity to obtain a dichloromethane extract (1.4 g), an ethyl-acetate extract (0.7 g), a n-butanol extract (5.4 g) and finally a methanol extract (12.4 g).
Stock solutions of the investigated extracts were made in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml.
The plant material was collected at Tomoros (ca. 1700 altitude), mountain Galičica (Macedonia) during the flowering (17 July 2010) and identified by Vlado Matevski, Institute of Biology, Faculty of Natural Sciences and Mathematics,
http://Ss. Cyril and Methodius University of Skopje, where the voucher specimen is deposited at Macedonian National Herbarium (MKNH) under the number MKNH121335.
Air-dried and powdered aerial parts of Helichrysum zivojinii (330 g) were extracted twice with n-hexane in an ultrasonic bath for 45 min. The combined extracts were concentrated in a vacuum to obtain a hexane extract (4.2 g). The plant material was successively extracted in the same manner with solvents of rising polarity to obtain a dichloromethane extract (1.4 g), an ethyl-acetate extract (0.7 g), a n-butanol extract (5.4 g) and finally a methanol extract (12.4 g).
Stock solutions of the investigated extracts were made in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml.
Instrumentation and chromatographic conditions 1HNMR spectra were recorded with Varian Gemini 200 in CDCl3 and DMSO-d6 with TMS as an internal standard. HPLC-MS analysis was performed with an Agilent 1100 Series chromatography system equipped with a binary pump, degasser, autosampler, column Li Chrospher 100 RP 18 (250 × 4,0 mm i.d. 5 μm), and DAD detector in combination with 6210 Time of Flight MS (Agilent Technologies). The mobile phase consisted of 0.2% formic acid in water (solvent A) and 100% acetonitrile (solvent B) with the following gradient elution: 0–5 min 10–20% B, 5–10 min 20% B, 10–20 min 20–30% B, 20–30 min 30–70% B, 30–35 min 70–100% B, 35–40 min 70% B, 40–41 min 100–10% B, 41–45 min 10% B, at a flow rate of 1 ml/min. The injection volume was 10 μL, the column temperature was 25°C. The effluent was monitored with DAD (190–550 nm) and a mass detector (ESI) which operated in negative mode at atmospheric pressure; the mass range was from m/z 100–2500, with the following ESI parameters: capillary voltage: 4000 V; gas temperature: 350°C; nebulizer pressure: 45 psig; fragmentor voltage: 140 V. Mass Hunter Workstation software was used for data analysis.
1HNMR spectra were recorded with Varian Gemini 200 in CDCl3 and DMSO-d6 with TMS as an internal standard. HPLC-MS analysis was performed with an Agilent 1100 Series chromatography system equipped with a binary pump, degasser, autosampler, column Li Chrospher 100 RP 18 (250 × 4,0 mm i.d. 5 μm), and DAD detector in combination with 6210 Time of Flight MS (Agilent Technologies). The mobile phase consisted of 0.2% formic acid in water (solvent A) and 100% acetonitrile (solvent B) with the following gradient elution: 0–5 min 10–20% B, 5–10 min 20% B, 10–20 min 20–30% B, 20–30 min 30–70% B, 30–35 min 70–100% B, 35–40 min 70% B, 40–41 min 100–10% B, 41–45 min 10% B, at a flow rate of 1 ml/min. The injection volume was 10 μL, the column temperature was 25°C. The effluent was monitored with DAD (190–550 nm) and a mass detector (ESI) which operated in negative mode at atmospheric pressure; the mass range was from m/z 100–2500, with the following ESI parameters: capillary voltage: 4000 V; gas temperature: 350°C; nebulizer pressure: 45 psig; fragmentor voltage: 140 V. Mass Hunter Workstation software was used for data analysis.
Cell culture Human cervix adenocarcinoma HeLa, human melanoma Fem-x and human breast adenocarcinoma MDA-MB-361 cells were cultured as monolayers. Human chronic myelogenous leukemia K562 cells were grown in a suspension in nutrient medium. Cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The complete nutrient medium was RPMI 1640 supplemented with 3 mM L-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, 10% heat-inactivated (56°C) fetal bovine serum and 25 mM Hepes adjusted to pH 7.2 with a bicarbonate solution. The cells were grown at 37°C in an atmosphere of 5% CO2 and humidified air. RPMI 1640, L-glutamine and Hepes were obtained from PAA (Pasching, Austria).
Human cervix adenocarcinoma HeLa, human melanoma Fem-x and human breast adenocarcinoma MDA-MB-361 cells were cultured as monolayers. Human chronic myelogenous leukemia K562 cells were grown in a suspension in nutrient medium. Cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The complete nutrient medium was RPMI 1640 supplemented with 3 mM L-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, 10% heat-inactivated (56°C) fetal bovine serum and 25 mM Hepes adjusted to pH 7.2 with a bicarbonate solution. The cells were grown at 37°C in an atmosphere of 5% CO2 and humidified air. RPMI 1640, L-glutamine and Hepes were obtained from PAA (Pasching, Austria).
Preparation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of two healthy volunteers by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl- and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in nutrient medium. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia. Written informed consent was obtained from each healthy donor.
Peripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of two healthy volunteers by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl- and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in nutrient medium. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia. Written informed consent was obtained from each healthy donor.
Treatment of cancer cell lines HeLa (2,000 cells per well), Fem-x (2,000 cells per well), MDA-MB-361 (10,000 cells per well) were seeded into 96-well microtiter plates and 20 h later, after cell adherence, five different concentrations of the tested extracts were added to the wells. Nutrient medium was only added to the cells in the control wells. K562 cells (5,000 cells per well) were seeded 2 h before addition of the extracts. Stock solutions of plant extracts were diluted with complete nutrient medium and applied to target cells at different final concentrations that ranged from 6.25 μg/ml to 100 μg/ml for extracts 1–4, and from 12.5 μg/ml to 150 μg/ml or 200 μg/ml for extract 5. All experiments were done in triplicate. Cisplatin was used as a positive control.
HeLa (2,000 cells per well), Fem-x (2,000 cells per well), MDA-MB-361 (10,000 cells per well) were seeded into 96-well microtiter plates and 20 h later, after cell adherence, five different concentrations of the tested extracts were added to the wells. Nutrient medium was only added to the cells in the control wells. K562 cells (5,000 cells per well) were seeded 2 h before addition of the extracts. Stock solutions of plant extracts were diluted with complete nutrient medium and applied to target cells at different final concentrations that ranged from 6.25 μg/ml to 100 μg/ml for extracts 1–4, and from 12.5 μg/ml to 150 μg/ml or 200 μg/ml for extract 5. All experiments were done in triplicate. Cisplatin was used as a positive control.
Treatment of PBMC PBMC (150,000 cells per well) were seeded into nutrient medium or in nutrient medium enriched with (5 μg/ml) (PHA) in 96-well microtiter plates. After 2 h, five different concentrations of the plant extracts were added to the individual wells, in triplicate, except to the control wells where a nutrient medium only was added to the cells. The final concentrations of the tested extracts ranged from 12.5 μg/ml to 200 μg/ml. PHA was obtained from INEP (Belgrade, Serbia). Cisplatin was used as a positive control.
PBMC (150,000 cells per well) were seeded into nutrient medium or in nutrient medium enriched with (5 μg/ml) (PHA) in 96-well microtiter plates. After 2 h, five different concentrations of the plant extracts were added to the individual wells, in triplicate, except to the control wells where a nutrient medium only was added to the cells. The final concentrations of the tested extracts ranged from 12.5 μg/ml to 200 μg/ml. PHA was obtained from INEP (Belgrade, Serbia). Cisplatin was used as a positive control.
Determination of target cell survival Cell survival was determined by the MTT test according to the method of Mosmann
[21] and modified by Ohno and Abe
[22]. Briefly, after the treatment with plant extracts for 72 h, 10 μl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 μl of 10% SDS. Absorbance at 570 nm was measured the next day.
To quantify cell survival (S%), the absorbance of a sample with cells grown in the presence of different concentrations of the investigated agents was divided by the absorbance of the control cells grown only in the nutrient medium, and multiplied by 100. It is implied that the absorbance of the blank was always subtracted from the absorbance of the corresponding sample with target cells. The IC50 was defined as the concentration of the agent that inhibited cell survival by 50%, compared to the vehicle-treated control.
Cell survival was determined by the MTT test according to the method of Mosmann
[21] and modified by Ohno and Abe
[22]. Briefly, after the treatment with plant extracts for 72 h, 10 μl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 μl of 10% SDS. Absorbance at 570 nm was measured the next day.
To quantify cell survival (S%), the absorbance of a sample with cells grown in the presence of different concentrations of the investigated agents was divided by the absorbance of the control cells grown only in the nutrient medium, and multiplied by 100. It is implied that the absorbance of the blank was always subtracted from the absorbance of the corresponding sample with target cells. The IC50 was defined as the concentration of the agent that inhibited cell survival by 50%, compared to the vehicle-treated control.
Morphological evaluation of HeLa cell death To evaluate whether the extracts from the endemic plant Helichrysum zivojinii induce apoptosis in HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide-stained target cells was performed. HeLa cells were seeded overnight on coverslips (100,000 cells) in 2 ml of complete medium. The next day, cells were treated with plant extracts for 24 h at concentrations corresponding to IC90 values that were obtained after treatments that lasted 72 h. After this period, the target cells were stained with 18 μl of a mixture of the DNA dyes acridine orange and ethidium bromide (3 μg/ml AO and 10 μg/ml EB in PBS), and visualized under a fluorescence microscope using a fluorescein isothiocyanate (FITC) filter set.
To evaluate whether the extracts from the endemic plant Helichrysum zivojinii induce apoptosis in HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide-stained target cells was performed. HeLa cells were seeded overnight on coverslips (100,000 cells) in 2 ml of complete medium. The next day, cells were treated with plant extracts for 24 h at concentrations corresponding to IC90 values that were obtained after treatments that lasted 72 h. After this period, the target cells were stained with 18 μl of a mixture of the DNA dyes acridine orange and ethidium bromide (3 μg/ml AO and 10 μg/ml EB in PBS), and visualized under a fluorescence microscope using a fluorescein isothiocyanate (FITC) filter set.
Cell cycle analysis HeLa cells were incubated in the presence of two different concentrations (corresponding to the IC50 and IC90 values determined after 72 h) of the examined Helichrysum zivojinii extracts for 24, 48 and 72 h. After these incubation times, the target cells were collected, washed and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before staining. HeLa cells were washed in PBS, resuspended in 500 μl of staining solution (PBS containing RNAse A at a final concentration of 200 μg/ml, and propidium iodide (PI) at a final concentration of 20 μg/ml), and incubated for 30 min at 37°C.
Cell cycle phase distribution was determined using a FACSCalibur Flow Cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data (10,000 events collected for each sample) were analyzed using CELLQuest software (BD Biosciences).
HeLa cells were incubated in the presence of two different concentrations (corresponding to the IC50 and IC90 values determined after 72 h) of the examined Helichrysum zivojinii extracts for 24, 48 and 72 h. After these incubation times, the target cells were collected, washed and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before staining. HeLa cells were washed in PBS, resuspended in 500 μl of staining solution (PBS containing RNAse A at a final concentration of 200 μg/ml, and propidium iodide (PI) at a final concentration of 20 μg/ml), and incubated for 30 min at 37°C.
Cell cycle phase distribution was determined using a FACSCalibur Flow Cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data (10,000 events collected for each sample) were analyzed using CELLQuest software (BD Biosciences).
Determination of target caspases To identify the caspases involved in the apoptotic cell death pathway induced by the investigated extracts, the percentages of HeLa cells pretreated with caspase inhibitors in the subG1 phase were determined. HeLa cells were preincubated for 2 h with specific caspase inhibitors (at a final concentration of 40 μM). These were: Z-DEVD-FMK, a caspase-3 inhibitor, Z-IETD-FMK, a caspase-8 inhibitor and Z-LEHD-FMK, a caspase-9 inhibitor. Caspase inhibitors were purchased from R&D Systems (Minneapolis, USA). Five tested extracts were applied to the HeLa cells at concentrations that corresponded to the IC90 values obtained after 72 h. For each extract, one sample of HeLa cells was not treated with an inhibitor and served as a reference sample. After 24 h of incubation, cells were harvested and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before PI staining. Changes in the percentages of cells in the subG1 phase were determined using a FACSCalibur Flow Cytometer and analyzed using CELLQuest software.
To identify the caspases involved in the apoptotic cell death pathway induced by the investigated extracts, the percentages of HeLa cells pretreated with caspase inhibitors in the subG1 phase were determined. HeLa cells were preincubated for 2 h with specific caspase inhibitors (at a final concentration of 40 μM). These were: Z-DEVD-FMK, a caspase-3 inhibitor, Z-IETD-FMK, a caspase-8 inhibitor and Z-LEHD-FMK, a caspase-9 inhibitor. Caspase inhibitors were purchased from R&D Systems (Minneapolis, USA). Five tested extracts were applied to the HeLa cells at concentrations that corresponded to the IC90 values obtained after 72 h. For each extract, one sample of HeLa cells was not treated with an inhibitor and served as a reference sample. After 24 h of incubation, cells were harvested and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before PI staining. Changes in the percentages of cells in the subG1 phase were determined using a FACSCalibur Flow Cytometer and analyzed using CELLQuest software.
|
Results
|
Chemical analysis of plant extracts The hexane (1) and dichloromethane extracts (2) presented similar 1H NMR spectra (recorded in CDCl3). The signals in the 1H NMR spectra of both extracts pointed to a complex mixture with prevailing relatively non-polar substances (region δ 0.8–2.2). LC/DAD analysis revealed the presence of a flavonoid with the molecular formula C18H16O7 (344) in the hexane extract, while the flavanone naringenin and flavone apigenin were confirmed in the dichloromethane extract, also by LC/DAD analysis. Three compounds found in both extracts are presented in Table
1, with their presumed molecular formulae and measured m/z values corresponding to identified ions obtained by ESI ToF mass spectrometry. According to LC/DAD analysis, these compounds unfortunately did not absorb in the UV spectrum, suggesting the presence of structures without a chromophore. The ethyl-acetate extract (3) and n-butanol extracts (4) presented 1H NMR spectra (recorded in DMSO-d6) with approximately identical groups of signals in the regions δ 12.5–13.9 (hydroxy protons), δ 5.9-8.1 (protons on the aromatic ring that is substituted with one or more hydroxy groups), and δ 4.7–5.5 (group of protons corresponding to a sugar unit). The 1H NMR spectrum of the most abundant methanol extract (5), also recorded in DMSO-d6, resembles the spectra of the ethyl-acetate and n-butanol extracts by the presence of signals in the region δ 5.9–8.1 (aromatic protons), as well as by the presence of a signal at δ 13.9 (hydroxy proton); it differs from the last two spectra by possessing a more pronounced region at δ 4.7–5.5, which is responsible for the protons of sugar units. LC/DAD analysis and ESI ToF mass spectrometry of these three more polar extracts (3, 4 and 5) revealed quite similar constituents in each of them. Apart from chlorogenic acid, two groups of flavonoid compounds were detected: flavonoid O-glycosides, and flavonoid aglycons. Among the O-glycosides, the glucoside (or possibly galactoside) of quercetin, the glucoside of apigenin, the glucoside of kaempferol (or possibly luteolin), and another glucoside of kaempferol (possibly luteolin or 6-hydroxyapigenin), were present. Flavonoid aglycones, such as apigenin and naringenin, were detected. Phthalic acid was found in extracts 2–5.
Components of five
Helichrysum zivojinii
extracts
aMore abundant on the expense of other fraction constituents.
The hexane (1) and dichloromethane extracts (2) presented similar 1H NMR spectra (recorded in CDCl3). The signals in the 1H NMR spectra of both extracts pointed to a complex mixture with prevailing relatively non-polar substances (region δ 0.8–2.2). LC/DAD analysis revealed the presence of a flavonoid with the molecular formula C18H16O7 (344) in the hexane extract, while the flavanone naringenin and flavone apigenin were confirmed in the dichloromethane extract, also by LC/DAD analysis. Three compounds found in both extracts are presented in Table
1, with their presumed molecular formulae and measured m/z values corresponding to identified ions obtained by ESI ToF mass spectrometry. According to LC/DAD analysis, these compounds unfortunately did not absorb in the UV spectrum, suggesting the presence of structures without a chromophore. The ethyl-acetate extract (3) and n-butanol extracts (4) presented 1H NMR spectra (recorded in DMSO-d6) with approximately identical groups of signals in the regions δ 12.5–13.9 (hydroxy protons), δ 5.9-8.1 (protons on the aromatic ring that is substituted with one or more hydroxy groups), and δ 4.7–5.5 (group of protons corresponding to a sugar unit). The 1H NMR spectrum of the most abundant methanol extract (5), also recorded in DMSO-d6, resembles the spectra of the ethyl-acetate and n-butanol extracts by the presence of signals in the region δ 5.9–8.1 (aromatic protons), as well as by the presence of a signal at δ 13.9 (hydroxy proton); it differs from the last two spectra by possessing a more pronounced region at δ 4.7–5.5, which is responsible for the protons of sugar units. LC/DAD analysis and ESI ToF mass spectrometry of these three more polar extracts (3, 4 and 5) revealed quite similar constituents in each of them. Apart from chlorogenic acid, two groups of flavonoid compounds were detected: flavonoid O-glycosides, and flavonoid aglycons. Among the O-glycosides, the glucoside (or possibly galactoside) of quercetin, the glucoside of apigenin, the glucoside of kaempferol (or possibly luteolin), and another glucoside of kaempferol (possibly luteolin or 6-hydroxyapigenin), were present. Flavonoid aglycones, such as apigenin and naringenin, were detected. Phthalic acid was found in extracts 2–5.
Components of five
Helichrysum zivojinii
extracts
aMore abundant on the expense of other fraction constituents.
In vitro cytotoxic activity The cytotoxicity of the five isolated extracts was tested against selected cancer cell lines: human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562 and human breast adenocarcinoma MDA-MB-361 cells. All investigated extracts exerted selective dose-dependent cytotoxic actions on malignant cells. The decrease in survival of target cancer cells induced by the five Helichrysum zivojinii extracts is shown in Figure
1 and Table
2.
Survival of HeLa (A), Fem-x (B), K562 (C) and MDA-MB-361 cells (D) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
Concentrations of five
Helichrysum zivojinii
extracts, which induced 50% decrease in the target cancer cell survival, determined by MTT test
Time of continuous agent’s action was 72 h.
In general, extracts 1 and 2 (as well as cisplatin, which served as a positive control) exhibited the highest cytotoxic actions against target malignant cell lines; extracts 3 and 4 displayed less pronounced cytotoxicity; extract 5 had the lowest cytotoxic action.
With regard to the specific sensitivities of the different cells to the cytotoxic activities of the extracts, it is important to note that K562 cells were the most sensitive to the cytotoxic actions of extracts 1 and 3. HeLa and Fem-x cells exhibited a lower sensitivity, while the sensitivity of breast cancer MDA-MB-361 cells to the toxic actions of the tested extracts was the lowest (being several times lower than that of the other cell lines to extracts 1, 2 and 3 especially).
Considering the possible effects of applied antitumor drugs on normal healthy immunocompetent cells, components of the antitumor immune response, their viability is significant for tumor control. For that reason, the activities of the investigated Helichrysum zivojinii extracts were evaluated against healthy unstimulated and PHA-stimulated PBMC (Figure
2 and Table
3). It should be noted that these extracts overall exhibited weaker cytotoxic effects against unstimulated PBMC than against stimulated PBMC. Moreover, extracts 2 and 3 exerted a more pronounced cytotoxicity against unstimulated and PHA-stimulated PBMC than extracts 1, 4 and 5.
Survival of resting PBMC (A) and PHA-stimulated PBMC (B) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
Concentrations of five
Helichrysum zivojinii
extracts, which induced 50% decrease in target PBMC survival, determined by MTT test
Time of continuous agent’s action was 72 h.
In order to further evaluate the anticancer potential of the extracts, the selectivity in the antitumor action against specific malignant cell line in comparison to healthy PBMC was determined as well. These data are presented in Table
4 from which it can be observed that extract 1 exhibited highly selective antitumor action, especially against K562 cells. Extract 2 also displayed good selectivity in its antitumor action.
Selectivity in the antitumor action of five
Helichrysum zivojinii
extracts
The cytotoxicity of the five isolated extracts was tested against selected cancer cell lines: human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562 and human breast adenocarcinoma MDA-MB-361 cells. All investigated extracts exerted selective dose-dependent cytotoxic actions on malignant cells. The decrease in survival of target cancer cells induced by the five Helichrysum zivojinii extracts is shown in Figure
1 and Table
2.
Survival of HeLa (A), Fem-x (B), K562 (C) and MDA-MB-361 cells (D) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
Concentrations of five
Helichrysum zivojinii
extracts, which induced 50% decrease in the target cancer cell survival, determined by MTT test
Time of continuous agent’s action was 72 h.
In general, extracts 1 and 2 (as well as cisplatin, which served as a positive control) exhibited the highest cytotoxic actions against target malignant cell lines; extracts 3 and 4 displayed less pronounced cytotoxicity; extract 5 had the lowest cytotoxic action.
With regard to the specific sensitivities of the different cells to the cytotoxic activities of the extracts, it is important to note that K562 cells were the most sensitive to the cytotoxic actions of extracts 1 and 3. HeLa and Fem-x cells exhibited a lower sensitivity, while the sensitivity of breast cancer MDA-MB-361 cells to the toxic actions of the tested extracts was the lowest (being several times lower than that of the other cell lines to extracts 1, 2 and 3 especially).
Considering the possible effects of applied antitumor drugs on normal healthy immunocompetent cells, components of the antitumor immune response, their viability is significant for tumor control. For that reason, the activities of the investigated Helichrysum zivojinii extracts were evaluated against healthy unstimulated and PHA-stimulated PBMC (Figure
2 and Table
3). It should be noted that these extracts overall exhibited weaker cytotoxic effects against unstimulated PBMC than against stimulated PBMC. Moreover, extracts 2 and 3 exerted a more pronounced cytotoxicity against unstimulated and PHA-stimulated PBMC than extracts 1, 4 and 5.
Survival of resting PBMC (A) and PHA-stimulated PBMC (B) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
Concentrations of five
Helichrysum zivojinii
extracts, which induced 50% decrease in target PBMC survival, determined by MTT test
Time of continuous agent’s action was 72 h.
In order to further evaluate the anticancer potential of the extracts, the selectivity in the antitumor action against specific malignant cell line in comparison to healthy PBMC was determined as well. These data are presented in Table
4 from which it can be observed that extract 1 exhibited highly selective antitumor action, especially against K562 cells. Extract 2 also displayed good selectivity in its antitumor action.
Selectivity in the antitumor action of five
Helichrysum zivojinii
extracts
Morphological analysis of HeLa cell death mode In order to determine whether the investigated plant extracts have pro-apoptotic activities, we performed morphological analysis by fluorescent microscopy of acridine orange/ethidium bromide-stained HeLa cells, exposed to the extracts. Microscopic examination revealed that all five extracts applied at IC90 concentrations induced apoptosis in target HeLa cells after 24 h treatment (Figure
3). The morphological characteristics of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange-red stained cells at late stages of apoptosis or secondary necrosis (the latter was observed in extract 1-treated HeLa cells) and apoptotic bodies. These analyses confirmed that the cytotoxicity of the Helichrysum zivojinii extracts is based on their prominent pro-apoptotic effects.
Photomicrographs of acridine orange/ethidium bromide-stained control HeLa cells (A), and HeLa cells treated with different
Helichrysum zivojinii
extracts for 24 h (extracts 1–5, photomicrographs B-F, consecutively).
In order to determine whether the investigated plant extracts have pro-apoptotic activities, we performed morphological analysis by fluorescent microscopy of acridine orange/ethidium bromide-stained HeLa cells, exposed to the extracts. Microscopic examination revealed that all five extracts applied at IC90 concentrations induced apoptosis in target HeLa cells after 24 h treatment (Figure
3). The morphological characteristics of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange-red stained cells at late stages of apoptosis or secondary necrosis (the latter was observed in extract 1-treated HeLa cells) and apoptotic bodies. These analyses confirmed that the cytotoxicity of the Helichrysum zivojinii extracts is based on their prominent pro-apoptotic effects.
Photomicrographs of acridine orange/ethidium bromide-stained control HeLa cells (A), and HeLa cells treated with different
Helichrysum zivojinii
extracts for 24 h (extracts 1–5, photomicrographs B-F, consecutively).
Analysis of changes in cell cycle phase distribution Examination of changes in the cell cycle phase distribution of HeLa cells treated with these extracts for 24, 48 and 72 h was done to elucidate the mechanisms of the observed cytotoxic actions (Figure
4). Results from this analysis showed a time - dependent increase in the percentages of HeLa cells in the subG1 phase after exposure to an IC50 concentration of all of the tested extracts. Additionally, exposure to extracts at IC90 concentrations induced significant increases in the percentages of cells in the subG1 phase 24 h after exposure. It should be mentioned that the investigated extracts induced a slight accumulation of HeLa cells in the S phase after 72 h. Examination of the cell cycle changes that were induced after exposure for 72 h to IC90 for each extract was not performed because at this time point and at this extract concentration low numbers of mostly dead or dying cells were present in the sample.
Changes in the cell cycle phase distribution of HeLa cells induced by the Helichrysum zivojinii extracts after 24 (A,B), 48 (C,D) and 72 h (E) treatment (applied concentrations of tested extracts corresponded to IC50 and IC90 values determined for 72 h). (C- control HeLa cells, 1 – 5 – corresponding extracts are numbered consecutively). Representative graphs are shown.
Since the Helichrysum zivojinii extracts exhibited the pro-apoptotic activities against cervix adenocarcinoma HeLa cells, the identification of target caspases involved in the apoptotic pathway was performed. The presence of the specific caspase inhibitors (caspase-3 inhibitor, caspase-8 inhibitor or caspase-9 inhibitor) significantly reduced the percentages of apoptotic subG1 HeLa cells treated with each of the five plant extracts, as shown in Figure
5. The effect of the caspase-3 inhibitor on HeLa cells treated with extracts is shown in Figure
6. It can be seen that there is an increase in rounded, but attached and live HeLa cells treated with the caspase-3 inhibitor before the addition of the extracts in relation to target HeLa cells only exposed to the tested extracts.
Effects of specific caspase inhibitors on the percentages of apoptotic subG1 HeLa cells treated with Helichrysum zivojinii extracts for 24 h (A - extract 1, B - extract 2, C - extract 3, D - extract 4, E - extract 5). (Z-DEVD-FMK - caspase-3 inhibitor; Z-IETD-FMK -caspase-8 inhibitor; Z-LEHD-FMK - caspase-9 inhibitor).
Effects of pretreatment of HeLa cells with caspase-3 inhibitor (Z-DEVD-FMK), exposed to Helichrysum zivojinii extracts (applied concentrations of tested extracts corresponded to IC90 values determined for 72 h). A – control; B – Extract 1, C – Extract 1 + Z-DEVD-FMK; D – Extract 2, E – Extract 2 + Z-DEVD-FMK; F – Extract 3, G – Extract 3 + Z-DEVD-FMK; H – Extract 4, I – Extract 4 + Z-DEVD-FMK; J – Extract 5, K – Extract 5 + Z-DEVD-FMK.
Examination of changes in the cell cycle phase distribution of HeLa cells treated with these extracts for 24, 48 and 72 h was done to elucidate the mechanisms of the observed cytotoxic actions (Figure
4). Results from this analysis showed a time - dependent increase in the percentages of HeLa cells in the subG1 phase after exposure to an IC50 concentration of all of the tested extracts. Additionally, exposure to extracts at IC90 concentrations induced significant increases in the percentages of cells in the subG1 phase 24 h after exposure. It should be mentioned that the investigated extracts induced a slight accumulation of HeLa cells in the S phase after 72 h. Examination of the cell cycle changes that were induced after exposure for 72 h to IC90 for each extract was not performed because at this time point and at this extract concentration low numbers of mostly dead or dying cells were present in the sample.
Changes in the cell cycle phase distribution of HeLa cells induced by the Helichrysum zivojinii extracts after 24 (A,B), 48 (C,D) and 72 h (E) treatment (applied concentrations of tested extracts corresponded to IC50 and IC90 values determined for 72 h). (C- control HeLa cells, 1 – 5 – corresponding extracts are numbered consecutively). Representative graphs are shown.
Since the Helichrysum zivojinii extracts exhibited the pro-apoptotic activities against cervix adenocarcinoma HeLa cells, the identification of target caspases involved in the apoptotic pathway was performed. The presence of the specific caspase inhibitors (caspase-3 inhibitor, caspase-8 inhibitor or caspase-9 inhibitor) significantly reduced the percentages of apoptotic subG1 HeLa cells treated with each of the five plant extracts, as shown in Figure
5. The effect of the caspase-3 inhibitor on HeLa cells treated with extracts is shown in Figure
6. It can be seen that there is an increase in rounded, but attached and live HeLa cells treated with the caspase-3 inhibitor before the addition of the extracts in relation to target HeLa cells only exposed to the tested extracts.
Effects of specific caspase inhibitors on the percentages of apoptotic subG1 HeLa cells treated with Helichrysum zivojinii extracts for 24 h (A - extract 1, B - extract 2, C - extract 3, D - extract 4, E - extract 5). (Z-DEVD-FMK - caspase-3 inhibitor; Z-IETD-FMK -caspase-8 inhibitor; Z-LEHD-FMK - caspase-9 inhibitor).
Effects of pretreatment of HeLa cells with caspase-3 inhibitor (Z-DEVD-FMK), exposed to Helichrysum zivojinii extracts (applied concentrations of tested extracts corresponded to IC90 values determined for 72 h). A – control; B – Extract 1, C – Extract 1 + Z-DEVD-FMK; D – Extract 2, E – Extract 2 + Z-DEVD-FMK; F – Extract 3, G – Extract 3 + Z-DEVD-FMK; H – Extract 4, I – Extract 4 + Z-DEVD-FMK; J – Extract 5, K – Extract 5 + Z-DEVD-FMK.
|
Conclusions
|
Data from this in vitro study clearly demonstrate the prominent antitumor potential of five extracts prepared from the endemic plant species Helichrysum zivojinii, which can be attributed to their selective and pronounced antiproliferative and pro-apoptotic actions towards specific malignant cells in comparison to healthy PBMC. Prospective cancer-suppressive effects of the tested extracts should be further evaluated in in vivo experiments.
|
[
"Background",
"Plant extracts",
"Instrumentation and chromatographic conditions",
"Cell culture",
"Preparation of peripheral blood mononuclear cells",
"Treatment of cancer cell lines",
"Treatment of PBMC",
"Determination of target cell survival",
"Morphological evaluation of HeLa cell death",
"Cell cycle analysis",
"Determination of target caspases",
"Chemical analysis of plant extracts",
"In vitro cytotoxic activity",
"Morphological analysis of HeLa cell death mode",
"Analysis of changes in cell cycle phase distribution",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Bioactive constituents of medicinal plants are in the center of attention of modern anticancer research due to their prospective roles in suppressing the different stages of malignant transformation. The antitumor potential of plant extracts and compounds could be attributed to their ability to induce changes in the regulation of target molecules in oncogenic signal transduction pathways implicated in cell growth, replication, apoptosis, as well as in angiogenesis, invasion and metastasis of cancer cells\n[1-4]. To evaluate the anticancer properties of novel chemotherapeutic agents, the selectivity of their actions against malignant cells in comparison to healthy non-transformed cells, especially immunocompetent cells involved in the immune control of tumor suppression, needs to be carefully examined.\nHelichrysum zivojinii Černjavski & Soška is an endemic plant species that grows in the National Park \"Galičica\" in Macedonia. Some of the plant species from the large genus Helichrysum are used in different regions of the world in traditional medicine for treating wounds, respiratory tract infections and gastro-intestinal disorders\n[5-8]. This plant genus is a valuable source of several different secondary metabolites/phytochemicals, such as flavonoids, acetophenones, phloroglucinols, pyrones, diterpenes and sesquiterpenes\n[5]. Different morphological groups of Helichrysum species often display unique qualitative and quantitative chemical compositions\n[5]. It has been reported that extracts and individual constituents of these plants possess significant biological and pharmacological properties, including antibacterial, antiviral, antifungal, antioxidant, anti-inflammatory and antidiabetic activities\n[9-16]. A search through the literature suggests that plants from the genus Helichrysum could be a significant source of compounds with potential anticancer activities\n[17-20].\nThe main goal of this research was to investigate the cytotoxic activities of five extracts isolated as fractions from the endemic plant Helichrysum zivojinii towards selected human malignant cell lines. To assess the sensitivity of normal immunocompetent cells included in the antitumor immune response, the cytotoxicity of these extracts was also tested against human peripheral blood mononuclear cells (PBMC) – both unstimulated and stimulated to proliferate by the mitogen phytohemagglutinin (PHA). To elucidate the molecular mechanisms of the cytotoxic effects of the tested extracts, the distribution of target HeLa cells at specific phases of the cell cycle after the actions of these agents was also analyzed. The mode of HeLa cell death induced by the extracts was also investigated. Elucidation of the signaling pathways implicated in the induction of apoptosis by the tested extracts was conducted by identification of target caspases.",
"The plant material was collected at Tomoros (ca. 1700 altitude), mountain Galičica (Macedonia) during the flowering (17 July 2010) and identified by Vlado Matevski, Institute of Biology, Faculty of Natural Sciences and Mathematics,\nhttp://Ss. Cyril and Methodius University of Skopje, where the voucher specimen is deposited at Macedonian National Herbarium (MKNH) under the number MKNH121335.\nAir-dried and powdered aerial parts of Helichrysum zivojinii (330 g) were extracted twice with n-hexane in an ultrasonic bath for 45 min. The combined extracts were concentrated in a vacuum to obtain a hexane extract (4.2 g). The plant material was successively extracted in the same manner with solvents of rising polarity to obtain a dichloromethane extract (1.4 g), an ethyl-acetate extract (0.7 g), a n-butanol extract (5.4 g) and finally a methanol extract (12.4 g).\nStock solutions of the investigated extracts were made in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml.",
"1HNMR spectra were recorded with Varian Gemini 200 in CDCl3 and DMSO-d6 with TMS as an internal standard. HPLC-MS analysis was performed with an Agilent 1100 Series chromatography system equipped with a binary pump, degasser, autosampler, column Li Chrospher 100 RP 18 (250 × 4,0 mm i.d. 5 μm), and DAD detector in combination with 6210 Time of Flight MS (Agilent Technologies). The mobile phase consisted of 0.2% formic acid in water (solvent A) and 100% acetonitrile (solvent B) with the following gradient elution: 0–5 min 10–20% B, 5–10 min 20% B, 10–20 min 20–30% B, 20–30 min 30–70% B, 30–35 min 70–100% B, 35–40 min 70% B, 40–41 min 100–10% B, 41–45 min 10% B, at a flow rate of 1 ml/min. The injection volume was 10 μL, the column temperature was 25°C. The effluent was monitored with DAD (190–550 nm) and a mass detector (ESI) which operated in negative mode at atmospheric pressure; the mass range was from m/z 100–2500, with the following ESI parameters: capillary voltage: 4000 V; gas temperature: 350°C; nebulizer pressure: 45 psig; fragmentor voltage: 140 V. Mass Hunter Workstation software was used for data analysis.",
"Human cervix adenocarcinoma HeLa, human melanoma Fem-x and human breast adenocarcinoma MDA-MB-361 cells were cultured as monolayers. Human chronic myelogenous leukemia K562 cells were grown in a suspension in nutrient medium. Cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The complete nutrient medium was RPMI 1640 supplemented with 3 mM L-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, 10% heat-inactivated (56°C) fetal bovine serum and 25 mM Hepes adjusted to pH 7.2 with a bicarbonate solution. The cells were grown at 37°C in an atmosphere of 5% CO2 and humidified air. RPMI 1640, L-glutamine and Hepes were obtained from PAA (Pasching, Austria).",
"Peripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of two healthy volunteers by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl- and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in nutrient medium. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia. Written informed consent was obtained from each healthy donor.",
"HeLa (2,000 cells per well), Fem-x (2,000 cells per well), MDA-MB-361 (10,000 cells per well) were seeded into 96-well microtiter plates and 20 h later, after cell adherence, five different concentrations of the tested extracts were added to the wells. Nutrient medium was only added to the cells in the control wells. K562 cells (5,000 cells per well) were seeded 2 h before addition of the extracts. Stock solutions of plant extracts were diluted with complete nutrient medium and applied to target cells at different final concentrations that ranged from 6.25 μg/ml to 100 μg/ml for extracts 1–4, and from 12.5 μg/ml to 150 μg/ml or 200 μg/ml for extract 5. All experiments were done in triplicate. Cisplatin was used as a positive control.",
"PBMC (150,000 cells per well) were seeded into nutrient medium or in nutrient medium enriched with (5 μg/ml) (PHA) in 96-well microtiter plates. After 2 h, five different concentrations of the plant extracts were added to the individual wells, in triplicate, except to the control wells where a nutrient medium only was added to the cells. The final concentrations of the tested extracts ranged from 12.5 μg/ml to 200 μg/ml. PHA was obtained from INEP (Belgrade, Serbia). Cisplatin was used as a positive control.",
"Cell survival was determined by the MTT test according to the method of Mosmann\n[21] and modified by Ohno and Abe\n[22]. Briefly, after the treatment with plant extracts for 72 h, 10 μl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 μl of 10% SDS. Absorbance at 570 nm was measured the next day.\nTo quantify cell survival (S%), the absorbance of a sample with cells grown in the presence of different concentrations of the investigated agents was divided by the absorbance of the control cells grown only in the nutrient medium, and multiplied by 100. It is implied that the absorbance of the blank was always subtracted from the absorbance of the corresponding sample with target cells. The IC50 was defined as the concentration of the agent that inhibited cell survival by 50%, compared to the vehicle-treated control.",
"To evaluate whether the extracts from the endemic plant Helichrysum zivojinii induce apoptosis in HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide-stained target cells was performed. HeLa cells were seeded overnight on coverslips (100,000 cells) in 2 ml of complete medium. The next day, cells were treated with plant extracts for 24 h at concentrations corresponding to IC90 values that were obtained after treatments that lasted 72 h. After this period, the target cells were stained with 18 μl of a mixture of the DNA dyes acridine orange and ethidium bromide (3 μg/ml AO and 10 μg/ml EB in PBS), and visualized under a fluorescence microscope using a fluorescein isothiocyanate (FITC) filter set.",
"HeLa cells were incubated in the presence of two different concentrations (corresponding to the IC50 and IC90 values determined after 72 h) of the examined Helichrysum zivojinii extracts for 24, 48 and 72 h. After these incubation times, the target cells were collected, washed and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before staining. HeLa cells were washed in PBS, resuspended in 500 μl of staining solution (PBS containing RNAse A at a final concentration of 200 μg/ml, and propidium iodide (PI) at a final concentration of 20 μg/ml), and incubated for 30 min at 37°C.\nCell cycle phase distribution was determined using a FACSCalibur Flow Cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data (10,000 events collected for each sample) were analyzed using CELLQuest software (BD Biosciences).",
"To identify the caspases involved in the apoptotic cell death pathway induced by the investigated extracts, the percentages of HeLa cells pretreated with caspase inhibitors in the subG1 phase were determined. HeLa cells were preincubated for 2 h with specific caspase inhibitors (at a final concentration of 40 μM). These were: Z-DEVD-FMK, a caspase-3 inhibitor, Z-IETD-FMK, a caspase-8 inhibitor and Z-LEHD-FMK, a caspase-9 inhibitor. Caspase inhibitors were purchased from R&D Systems (Minneapolis, USA). Five tested extracts were applied to the HeLa cells at concentrations that corresponded to the IC90 values obtained after 72 h. For each extract, one sample of HeLa cells was not treated with an inhibitor and served as a reference sample. After 24 h of incubation, cells were harvested and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before PI staining. Changes in the percentages of cells in the subG1 phase were determined using a FACSCalibur Flow Cytometer and analyzed using CELLQuest software.",
"The hexane (1) and dichloromethane extracts (2) presented similar 1H NMR spectra (recorded in CDCl3). The signals in the 1H NMR spectra of both extracts pointed to a complex mixture with prevailing relatively non-polar substances (region δ 0.8–2.2). LC/DAD analysis revealed the presence of a flavonoid with the molecular formula C18H16O7 (344) in the hexane extract, while the flavanone naringenin and flavone apigenin were confirmed in the dichloromethane extract, also by LC/DAD analysis. Three compounds found in both extracts are presented in Table \n1, with their presumed molecular formulae and measured m/z values corresponding to identified ions obtained by ESI ToF mass spectrometry. According to LC/DAD analysis, these compounds unfortunately did not absorb in the UV spectrum, suggesting the presence of structures without a chromophore. The ethyl-acetate extract (3) and n-butanol extracts (4) presented 1H NMR spectra (recorded in DMSO-d6) with approximately identical groups of signals in the regions δ 12.5–13.9 (hydroxy protons), δ 5.9-8.1 (protons on the aromatic ring that is substituted with one or more hydroxy groups), and δ 4.7–5.5 (group of protons corresponding to a sugar unit). The 1H NMR spectrum of the most abundant methanol extract (5), also recorded in DMSO-d6, resembles the spectra of the ethyl-acetate and n-butanol extracts by the presence of signals in the region δ 5.9–8.1 (aromatic protons), as well as by the presence of a signal at δ 13.9 (hydroxy proton); it differs from the last two spectra by possessing a more pronounced region at δ 4.7–5.5, which is responsible for the protons of sugar units. LC/DAD analysis and ESI ToF mass spectrometry of these three more polar extracts (3, 4 and 5) revealed quite similar constituents in each of them. Apart from chlorogenic acid, two groups of flavonoid compounds were detected: flavonoid O-glycosides, and flavonoid aglycons. Among the O-glycosides, the glucoside (or possibly galactoside) of quercetin, the glucoside of apigenin, the glucoside of kaempferol (or possibly luteolin), and another glucoside of kaempferol (possibly luteolin or 6-hydroxyapigenin), were present. Flavonoid aglycones, such as apigenin and naringenin, were detected. Phthalic acid was found in extracts 2–5.\n\nComponents of five \n\nHelichrysum zivojinii \n\nextracts\n\naMore abundant on the expense of other fraction constituents.",
"The cytotoxicity of the five isolated extracts was tested against selected cancer cell lines: human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562 and human breast adenocarcinoma MDA-MB-361 cells. All investigated extracts exerted selective dose-dependent cytotoxic actions on malignant cells. The decrease in survival of target cancer cells induced by the five Helichrysum zivojinii extracts is shown in Figure \n1 and Table \n2.\nSurvival of HeLa (A), Fem-x (B), K562 (C) and MDA-MB-361 cells (D) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.\n\nConcentrations of five \n\nHelichrysum zivojinii \n\nextracts, which induced 50% decrease in the target cancer cell survival, determined by MTT test\n\nTime of continuous agent’s action was 72 h.\nIn general, extracts 1 and 2 (as well as cisplatin, which served as a positive control) exhibited the highest cytotoxic actions against target malignant cell lines; extracts 3 and 4 displayed less pronounced cytotoxicity; extract 5 had the lowest cytotoxic action.\nWith regard to the specific sensitivities of the different cells to the cytotoxic activities of the extracts, it is important to note that K562 cells were the most sensitive to the cytotoxic actions of extracts 1 and 3. HeLa and Fem-x cells exhibited a lower sensitivity, while the sensitivity of breast cancer MDA-MB-361 cells to the toxic actions of the tested extracts was the lowest (being several times lower than that of the other cell lines to extracts 1, 2 and 3 especially).\nConsidering the possible effects of applied antitumor drugs on normal healthy immunocompetent cells, components of the antitumor immune response, their viability is significant for tumor control. For that reason, the activities of the investigated Helichrysum zivojinii extracts were evaluated against healthy unstimulated and PHA-stimulated PBMC (Figure \n2 and Table \n3). It should be noted that these extracts overall exhibited weaker cytotoxic effects against unstimulated PBMC than against stimulated PBMC. Moreover, extracts 2 and 3 exerted a more pronounced cytotoxicity against unstimulated and PHA-stimulated PBMC than extracts 1, 4 and 5.\nSurvival of resting PBMC (A) and PHA-stimulated PBMC (B) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.\n\nConcentrations of five \n\nHelichrysum zivojinii \n\nextracts, which induced 50% decrease in target PBMC survival, determined by MTT test\n\nTime of continuous agent’s action was 72 h.\nIn order to further evaluate the anticancer potential of the extracts, the selectivity in the antitumor action against specific malignant cell line in comparison to healthy PBMC was determined as well. These data are presented in Table \n4 from which it can be observed that extract 1 exhibited highly selective antitumor action, especially against K562 cells. Extract 2 also displayed good selectivity in its antitumor action.\n\nSelectivity in the antitumor action of five \n\nHelichrysum zivojinii \n\nextracts\n",
"In order to determine whether the investigated plant extracts have pro-apoptotic activities, we performed morphological analysis by fluorescent microscopy of acridine orange/ethidium bromide-stained HeLa cells, exposed to the extracts. Microscopic examination revealed that all five extracts applied at IC90 concentrations induced apoptosis in target HeLa cells after 24 h treatment (Figure \n3). The morphological characteristics of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange-red stained cells at late stages of apoptosis or secondary necrosis (the latter was observed in extract 1-treated HeLa cells) and apoptotic bodies. These analyses confirmed that the cytotoxicity of the Helichrysum zivojinii extracts is based on their prominent pro-apoptotic effects.\n\nPhotomicrographs of acridine orange/ethidium bromide-stained control HeLa cells (A), and HeLa cells treated with different \n\nHelichrysum zivojinii \n\nextracts for 24 h (extracts 1–5, photomicrographs B-F, consecutively).\n",
"Examination of changes in the cell cycle phase distribution of HeLa cells treated with these extracts for 24, 48 and 72 h was done to elucidate the mechanisms of the observed cytotoxic actions (Figure \n4). Results from this analysis showed a time - dependent increase in the percentages of HeLa cells in the subG1 phase after exposure to an IC50 concentration of all of the tested extracts. Additionally, exposure to extracts at IC90 concentrations induced significant increases in the percentages of cells in the subG1 phase 24 h after exposure. It should be mentioned that the investigated extracts induced a slight accumulation of HeLa cells in the S phase after 72 h. Examination of the cell cycle changes that were induced after exposure for 72 h to IC90 for each extract was not performed because at this time point and at this extract concentration low numbers of mostly dead or dying cells were present in the sample.\nChanges in the cell cycle phase distribution of HeLa cells induced by the Helichrysum zivojinii extracts after 24 (A,B), 48 (C,D) and 72 h (E) treatment (applied concentrations of tested extracts corresponded to IC50 and IC90 values determined for 72 h). (C- control HeLa cells, 1 – 5 – corresponding extracts are numbered consecutively). Representative graphs are shown.\nSince the Helichrysum zivojinii extracts exhibited the pro-apoptotic activities against cervix adenocarcinoma HeLa cells, the identification of target caspases involved in the apoptotic pathway was performed. The presence of the specific caspase inhibitors (caspase-3 inhibitor, caspase-8 inhibitor or caspase-9 inhibitor) significantly reduced the percentages of apoptotic subG1 HeLa cells treated with each of the five plant extracts, as shown in Figure \n5. The effect of the caspase-3 inhibitor on HeLa cells treated with extracts is shown in Figure \n6. It can be seen that there is an increase in rounded, but attached and live HeLa cells treated with the caspase-3 inhibitor before the addition of the extracts in relation to target HeLa cells only exposed to the tested extracts.\nEffects of specific caspase inhibitors on the percentages of apoptotic subG1 HeLa cells treated with Helichrysum zivojinii extracts for 24 h (A - extract 1, B - extract 2, C - extract 3, D - extract 4, E - extract 5). (Z-DEVD-FMK - caspase-3 inhibitor; Z-IETD-FMK -caspase-8 inhibitor; Z-LEHD-FMK - caspase-9 inhibitor).\nEffects of pretreatment of HeLa cells with caspase-3 inhibitor (Z-DEVD-FMK), exposed to Helichrysum zivojinii extracts (applied concentrations of tested extracts corresponded to IC90 values determined for 72 h). A – control; B – Extract 1, C – Extract 1 + Z-DEVD-FMK; D – Extract 2, E – Extract 2 + Z-DEVD-FMK; F – Extract 3, G – Extract 3 + Z-DEVD-FMK; H – Extract 4, I – Extract 4 + Z-DEVD-FMK; J – Extract 5, K – Extract 5 + Z-DEVD-FMK.",
"The authors declare that they have no competing interests.",
"IM performed all analyses of the anticancer properties of investigated extracts, interpreted obtained data and wrote the first and last version of the manuscript. IA participated in design of the study, prepared extracts, performed chemical characterization, interpreted data and wrote the part of the manuscript. ŽŽ participated in acquisition and analysis of data. MJ carried out chemical analyses of the extracts. VV and SM participated in design of the study and interpreted obtained data. ZJ designed the research on anticancer properties of tested extracts, interpreted obtained data, participated in writing the manuscript and critically revised the manuscript. All authors have read and approved the final version of the manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/13/36/prepub\n"
] |
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[
"Background",
"Methods",
"Plant extracts",
"Instrumentation and chromatographic conditions",
"Cell culture",
"Preparation of peripheral blood mononuclear cells",
"Treatment of cancer cell lines",
"Treatment of PBMC",
"Determination of target cell survival",
"Morphological evaluation of HeLa cell death",
"Cell cycle analysis",
"Determination of target caspases",
"Results",
"Chemical analysis of plant extracts",
"In vitro cytotoxic activity",
"Morphological analysis of HeLa cell death mode",
"Analysis of changes in cell cycle phase distribution",
"Discussion",
"Conclusions",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Bioactive constituents of medicinal plants are in the center of attention of modern anticancer research due to their prospective roles in suppressing the different stages of malignant transformation. The antitumor potential of plant extracts and compounds could be attributed to their ability to induce changes in the regulation of target molecules in oncogenic signal transduction pathways implicated in cell growth, replication, apoptosis, as well as in angiogenesis, invasion and metastasis of cancer cells\n[1-4]. To evaluate the anticancer properties of novel chemotherapeutic agents, the selectivity of their actions against malignant cells in comparison to healthy non-transformed cells, especially immunocompetent cells involved in the immune control of tumor suppression, needs to be carefully examined.\nHelichrysum zivojinii Černjavski & Soška is an endemic plant species that grows in the National Park \"Galičica\" in Macedonia. Some of the plant species from the large genus Helichrysum are used in different regions of the world in traditional medicine for treating wounds, respiratory tract infections and gastro-intestinal disorders\n[5-8]. This plant genus is a valuable source of several different secondary metabolites/phytochemicals, such as flavonoids, acetophenones, phloroglucinols, pyrones, diterpenes and sesquiterpenes\n[5]. Different morphological groups of Helichrysum species often display unique qualitative and quantitative chemical compositions\n[5]. It has been reported that extracts and individual constituents of these plants possess significant biological and pharmacological properties, including antibacterial, antiviral, antifungal, antioxidant, anti-inflammatory and antidiabetic activities\n[9-16]. A search through the literature suggests that plants from the genus Helichrysum could be a significant source of compounds with potential anticancer activities\n[17-20].\nThe main goal of this research was to investigate the cytotoxic activities of five extracts isolated as fractions from the endemic plant Helichrysum zivojinii towards selected human malignant cell lines. To assess the sensitivity of normal immunocompetent cells included in the antitumor immune response, the cytotoxicity of these extracts was also tested against human peripheral blood mononuclear cells (PBMC) – both unstimulated and stimulated to proliferate by the mitogen phytohemagglutinin (PHA). To elucidate the molecular mechanisms of the cytotoxic effects of the tested extracts, the distribution of target HeLa cells at specific phases of the cell cycle after the actions of these agents was also analyzed. The mode of HeLa cell death induced by the extracts was also investigated. Elucidation of the signaling pathways implicated in the induction of apoptosis by the tested extracts was conducted by identification of target caspases.",
" Plant extracts The plant material was collected at Tomoros (ca. 1700 altitude), mountain Galičica (Macedonia) during the flowering (17 July 2010) and identified by Vlado Matevski, Institute of Biology, Faculty of Natural Sciences and Mathematics,\nhttp://Ss. Cyril and Methodius University of Skopje, where the voucher specimen is deposited at Macedonian National Herbarium (MKNH) under the number MKNH121335.\nAir-dried and powdered aerial parts of Helichrysum zivojinii (330 g) were extracted twice with n-hexane in an ultrasonic bath for 45 min. The combined extracts were concentrated in a vacuum to obtain a hexane extract (4.2 g). The plant material was successively extracted in the same manner with solvents of rising polarity to obtain a dichloromethane extract (1.4 g), an ethyl-acetate extract (0.7 g), a n-butanol extract (5.4 g) and finally a methanol extract (12.4 g).\nStock solutions of the investigated extracts were made in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml.\nThe plant material was collected at Tomoros (ca. 1700 altitude), mountain Galičica (Macedonia) during the flowering (17 July 2010) and identified by Vlado Matevski, Institute of Biology, Faculty of Natural Sciences and Mathematics,\nhttp://Ss. Cyril and Methodius University of Skopje, where the voucher specimen is deposited at Macedonian National Herbarium (MKNH) under the number MKNH121335.\nAir-dried and powdered aerial parts of Helichrysum zivojinii (330 g) were extracted twice with n-hexane in an ultrasonic bath for 45 min. The combined extracts were concentrated in a vacuum to obtain a hexane extract (4.2 g). The plant material was successively extracted in the same manner with solvents of rising polarity to obtain a dichloromethane extract (1.4 g), an ethyl-acetate extract (0.7 g), a n-butanol extract (5.4 g) and finally a methanol extract (12.4 g).\nStock solutions of the investigated extracts were made in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml.\n Instrumentation and chromatographic conditions 1HNMR spectra were recorded with Varian Gemini 200 in CDCl3 and DMSO-d6 with TMS as an internal standard. HPLC-MS analysis was performed with an Agilent 1100 Series chromatography system equipped with a binary pump, degasser, autosampler, column Li Chrospher 100 RP 18 (250 × 4,0 mm i.d. 5 μm), and DAD detector in combination with 6210 Time of Flight MS (Agilent Technologies). The mobile phase consisted of 0.2% formic acid in water (solvent A) and 100% acetonitrile (solvent B) with the following gradient elution: 0–5 min 10–20% B, 5–10 min 20% B, 10–20 min 20–30% B, 20–30 min 30–70% B, 30–35 min 70–100% B, 35–40 min 70% B, 40–41 min 100–10% B, 41–45 min 10% B, at a flow rate of 1 ml/min. The injection volume was 10 μL, the column temperature was 25°C. The effluent was monitored with DAD (190–550 nm) and a mass detector (ESI) which operated in negative mode at atmospheric pressure; the mass range was from m/z 100–2500, with the following ESI parameters: capillary voltage: 4000 V; gas temperature: 350°C; nebulizer pressure: 45 psig; fragmentor voltage: 140 V. Mass Hunter Workstation software was used for data analysis.\n1HNMR spectra were recorded with Varian Gemini 200 in CDCl3 and DMSO-d6 with TMS as an internal standard. HPLC-MS analysis was performed with an Agilent 1100 Series chromatography system equipped with a binary pump, degasser, autosampler, column Li Chrospher 100 RP 18 (250 × 4,0 mm i.d. 5 μm), and DAD detector in combination with 6210 Time of Flight MS (Agilent Technologies). The mobile phase consisted of 0.2% formic acid in water (solvent A) and 100% acetonitrile (solvent B) with the following gradient elution: 0–5 min 10–20% B, 5–10 min 20% B, 10–20 min 20–30% B, 20–30 min 30–70% B, 30–35 min 70–100% B, 35–40 min 70% B, 40–41 min 100–10% B, 41–45 min 10% B, at a flow rate of 1 ml/min. The injection volume was 10 μL, the column temperature was 25°C. The effluent was monitored with DAD (190–550 nm) and a mass detector (ESI) which operated in negative mode at atmospheric pressure; the mass range was from m/z 100–2500, with the following ESI parameters: capillary voltage: 4000 V; gas temperature: 350°C; nebulizer pressure: 45 psig; fragmentor voltage: 140 V. Mass Hunter Workstation software was used for data analysis.\n Cell culture Human cervix adenocarcinoma HeLa, human melanoma Fem-x and human breast adenocarcinoma MDA-MB-361 cells were cultured as monolayers. Human chronic myelogenous leukemia K562 cells were grown in a suspension in nutrient medium. Cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The complete nutrient medium was RPMI 1640 supplemented with 3 mM L-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, 10% heat-inactivated (56°C) fetal bovine serum and 25 mM Hepes adjusted to pH 7.2 with a bicarbonate solution. The cells were grown at 37°C in an atmosphere of 5% CO2 and humidified air. RPMI 1640, L-glutamine and Hepes were obtained from PAA (Pasching, Austria).\nHuman cervix adenocarcinoma HeLa, human melanoma Fem-x and human breast adenocarcinoma MDA-MB-361 cells were cultured as monolayers. Human chronic myelogenous leukemia K562 cells were grown in a suspension in nutrient medium. Cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The complete nutrient medium was RPMI 1640 supplemented with 3 mM L-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, 10% heat-inactivated (56°C) fetal bovine serum and 25 mM Hepes adjusted to pH 7.2 with a bicarbonate solution. The cells were grown at 37°C in an atmosphere of 5% CO2 and humidified air. RPMI 1640, L-glutamine and Hepes were obtained from PAA (Pasching, Austria).\n Preparation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of two healthy volunteers by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl- and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in nutrient medium. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia. Written informed consent was obtained from each healthy donor.\nPeripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of two healthy volunteers by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl- and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in nutrient medium. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia. Written informed consent was obtained from each healthy donor.\n Treatment of cancer cell lines HeLa (2,000 cells per well), Fem-x (2,000 cells per well), MDA-MB-361 (10,000 cells per well) were seeded into 96-well microtiter plates and 20 h later, after cell adherence, five different concentrations of the tested extracts were added to the wells. Nutrient medium was only added to the cells in the control wells. K562 cells (5,000 cells per well) were seeded 2 h before addition of the extracts. Stock solutions of plant extracts were diluted with complete nutrient medium and applied to target cells at different final concentrations that ranged from 6.25 μg/ml to 100 μg/ml for extracts 1–4, and from 12.5 μg/ml to 150 μg/ml or 200 μg/ml for extract 5. All experiments were done in triplicate. Cisplatin was used as a positive control.\nHeLa (2,000 cells per well), Fem-x (2,000 cells per well), MDA-MB-361 (10,000 cells per well) were seeded into 96-well microtiter plates and 20 h later, after cell adherence, five different concentrations of the tested extracts were added to the wells. Nutrient medium was only added to the cells in the control wells. K562 cells (5,000 cells per well) were seeded 2 h before addition of the extracts. Stock solutions of plant extracts were diluted with complete nutrient medium and applied to target cells at different final concentrations that ranged from 6.25 μg/ml to 100 μg/ml for extracts 1–4, and from 12.5 μg/ml to 150 μg/ml or 200 μg/ml for extract 5. All experiments were done in triplicate. Cisplatin was used as a positive control.\n Treatment of PBMC PBMC (150,000 cells per well) were seeded into nutrient medium or in nutrient medium enriched with (5 μg/ml) (PHA) in 96-well microtiter plates. After 2 h, five different concentrations of the plant extracts were added to the individual wells, in triplicate, except to the control wells where a nutrient medium only was added to the cells. The final concentrations of the tested extracts ranged from 12.5 μg/ml to 200 μg/ml. PHA was obtained from INEP (Belgrade, Serbia). Cisplatin was used as a positive control.\nPBMC (150,000 cells per well) were seeded into nutrient medium or in nutrient medium enriched with (5 μg/ml) (PHA) in 96-well microtiter plates. After 2 h, five different concentrations of the plant extracts were added to the individual wells, in triplicate, except to the control wells where a nutrient medium only was added to the cells. The final concentrations of the tested extracts ranged from 12.5 μg/ml to 200 μg/ml. PHA was obtained from INEP (Belgrade, Serbia). Cisplatin was used as a positive control.\n Determination of target cell survival Cell survival was determined by the MTT test according to the method of Mosmann\n[21] and modified by Ohno and Abe\n[22]. Briefly, after the treatment with plant extracts for 72 h, 10 μl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 μl of 10% SDS. Absorbance at 570 nm was measured the next day.\nTo quantify cell survival (S%), the absorbance of a sample with cells grown in the presence of different concentrations of the investigated agents was divided by the absorbance of the control cells grown only in the nutrient medium, and multiplied by 100. It is implied that the absorbance of the blank was always subtracted from the absorbance of the corresponding sample with target cells. The IC50 was defined as the concentration of the agent that inhibited cell survival by 50%, compared to the vehicle-treated control.\nCell survival was determined by the MTT test according to the method of Mosmann\n[21] and modified by Ohno and Abe\n[22]. Briefly, after the treatment with plant extracts for 72 h, 10 μl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 μl of 10% SDS. Absorbance at 570 nm was measured the next day.\nTo quantify cell survival (S%), the absorbance of a sample with cells grown in the presence of different concentrations of the investigated agents was divided by the absorbance of the control cells grown only in the nutrient medium, and multiplied by 100. It is implied that the absorbance of the blank was always subtracted from the absorbance of the corresponding sample with target cells. The IC50 was defined as the concentration of the agent that inhibited cell survival by 50%, compared to the vehicle-treated control.\n Morphological evaluation of HeLa cell death To evaluate whether the extracts from the endemic plant Helichrysum zivojinii induce apoptosis in HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide-stained target cells was performed. HeLa cells were seeded overnight on coverslips (100,000 cells) in 2 ml of complete medium. The next day, cells were treated with plant extracts for 24 h at concentrations corresponding to IC90 values that were obtained after treatments that lasted 72 h. After this period, the target cells were stained with 18 μl of a mixture of the DNA dyes acridine orange and ethidium bromide (3 μg/ml AO and 10 μg/ml EB in PBS), and visualized under a fluorescence microscope using a fluorescein isothiocyanate (FITC) filter set.\nTo evaluate whether the extracts from the endemic plant Helichrysum zivojinii induce apoptosis in HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide-stained target cells was performed. HeLa cells were seeded overnight on coverslips (100,000 cells) in 2 ml of complete medium. The next day, cells were treated with plant extracts for 24 h at concentrations corresponding to IC90 values that were obtained after treatments that lasted 72 h. After this period, the target cells were stained with 18 μl of a mixture of the DNA dyes acridine orange and ethidium bromide (3 μg/ml AO and 10 μg/ml EB in PBS), and visualized under a fluorescence microscope using a fluorescein isothiocyanate (FITC) filter set.\n Cell cycle analysis HeLa cells were incubated in the presence of two different concentrations (corresponding to the IC50 and IC90 values determined after 72 h) of the examined Helichrysum zivojinii extracts for 24, 48 and 72 h. After these incubation times, the target cells were collected, washed and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before staining. HeLa cells were washed in PBS, resuspended in 500 μl of staining solution (PBS containing RNAse A at a final concentration of 200 μg/ml, and propidium iodide (PI) at a final concentration of 20 μg/ml), and incubated for 30 min at 37°C.\nCell cycle phase distribution was determined using a FACSCalibur Flow Cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data (10,000 events collected for each sample) were analyzed using CELLQuest software (BD Biosciences).\nHeLa cells were incubated in the presence of two different concentrations (corresponding to the IC50 and IC90 values determined after 72 h) of the examined Helichrysum zivojinii extracts for 24, 48 and 72 h. After these incubation times, the target cells were collected, washed and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before staining. HeLa cells were washed in PBS, resuspended in 500 μl of staining solution (PBS containing RNAse A at a final concentration of 200 μg/ml, and propidium iodide (PI) at a final concentration of 20 μg/ml), and incubated for 30 min at 37°C.\nCell cycle phase distribution was determined using a FACSCalibur Flow Cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data (10,000 events collected for each sample) were analyzed using CELLQuest software (BD Biosciences).\n Determination of target caspases To identify the caspases involved in the apoptotic cell death pathway induced by the investigated extracts, the percentages of HeLa cells pretreated with caspase inhibitors in the subG1 phase were determined. HeLa cells were preincubated for 2 h with specific caspase inhibitors (at a final concentration of 40 μM). These were: Z-DEVD-FMK, a caspase-3 inhibitor, Z-IETD-FMK, a caspase-8 inhibitor and Z-LEHD-FMK, a caspase-9 inhibitor. Caspase inhibitors were purchased from R&D Systems (Minneapolis, USA). Five tested extracts were applied to the HeLa cells at concentrations that corresponded to the IC90 values obtained after 72 h. For each extract, one sample of HeLa cells was not treated with an inhibitor and served as a reference sample. After 24 h of incubation, cells were harvested and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before PI staining. Changes in the percentages of cells in the subG1 phase were determined using a FACSCalibur Flow Cytometer and analyzed using CELLQuest software.\nTo identify the caspases involved in the apoptotic cell death pathway induced by the investigated extracts, the percentages of HeLa cells pretreated with caspase inhibitors in the subG1 phase were determined. HeLa cells were preincubated for 2 h with specific caspase inhibitors (at a final concentration of 40 μM). These were: Z-DEVD-FMK, a caspase-3 inhibitor, Z-IETD-FMK, a caspase-8 inhibitor and Z-LEHD-FMK, a caspase-9 inhibitor. Caspase inhibitors were purchased from R&D Systems (Minneapolis, USA). Five tested extracts were applied to the HeLa cells at concentrations that corresponded to the IC90 values obtained after 72 h. For each extract, one sample of HeLa cells was not treated with an inhibitor and served as a reference sample. After 24 h of incubation, cells were harvested and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before PI staining. Changes in the percentages of cells in the subG1 phase were determined using a FACSCalibur Flow Cytometer and analyzed using CELLQuest software.",
"The plant material was collected at Tomoros (ca. 1700 altitude), mountain Galičica (Macedonia) during the flowering (17 July 2010) and identified by Vlado Matevski, Institute of Biology, Faculty of Natural Sciences and Mathematics,\nhttp://Ss. Cyril and Methodius University of Skopje, where the voucher specimen is deposited at Macedonian National Herbarium (MKNH) under the number MKNH121335.\nAir-dried and powdered aerial parts of Helichrysum zivojinii (330 g) were extracted twice with n-hexane in an ultrasonic bath for 45 min. The combined extracts were concentrated in a vacuum to obtain a hexane extract (4.2 g). The plant material was successively extracted in the same manner with solvents of rising polarity to obtain a dichloromethane extract (1.4 g), an ethyl-acetate extract (0.7 g), a n-butanol extract (5.4 g) and finally a methanol extract (12.4 g).\nStock solutions of the investigated extracts were made in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml.",
"1HNMR spectra were recorded with Varian Gemini 200 in CDCl3 and DMSO-d6 with TMS as an internal standard. HPLC-MS analysis was performed with an Agilent 1100 Series chromatography system equipped with a binary pump, degasser, autosampler, column Li Chrospher 100 RP 18 (250 × 4,0 mm i.d. 5 μm), and DAD detector in combination with 6210 Time of Flight MS (Agilent Technologies). The mobile phase consisted of 0.2% formic acid in water (solvent A) and 100% acetonitrile (solvent B) with the following gradient elution: 0–5 min 10–20% B, 5–10 min 20% B, 10–20 min 20–30% B, 20–30 min 30–70% B, 30–35 min 70–100% B, 35–40 min 70% B, 40–41 min 100–10% B, 41–45 min 10% B, at a flow rate of 1 ml/min. The injection volume was 10 μL, the column temperature was 25°C. The effluent was monitored with DAD (190–550 nm) and a mass detector (ESI) which operated in negative mode at atmospheric pressure; the mass range was from m/z 100–2500, with the following ESI parameters: capillary voltage: 4000 V; gas temperature: 350°C; nebulizer pressure: 45 psig; fragmentor voltage: 140 V. Mass Hunter Workstation software was used for data analysis.",
"Human cervix adenocarcinoma HeLa, human melanoma Fem-x and human breast adenocarcinoma MDA-MB-361 cells were cultured as monolayers. Human chronic myelogenous leukemia K562 cells were grown in a suspension in nutrient medium. Cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The complete nutrient medium was RPMI 1640 supplemented with 3 mM L-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, 10% heat-inactivated (56°C) fetal bovine serum and 25 mM Hepes adjusted to pH 7.2 with a bicarbonate solution. The cells were grown at 37°C in an atmosphere of 5% CO2 and humidified air. RPMI 1640, L-glutamine and Hepes were obtained from PAA (Pasching, Austria).",
"Peripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of two healthy volunteers by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl- and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in nutrient medium. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia. Written informed consent was obtained from each healthy donor.",
"HeLa (2,000 cells per well), Fem-x (2,000 cells per well), MDA-MB-361 (10,000 cells per well) were seeded into 96-well microtiter plates and 20 h later, after cell adherence, five different concentrations of the tested extracts were added to the wells. Nutrient medium was only added to the cells in the control wells. K562 cells (5,000 cells per well) were seeded 2 h before addition of the extracts. Stock solutions of plant extracts were diluted with complete nutrient medium and applied to target cells at different final concentrations that ranged from 6.25 μg/ml to 100 μg/ml for extracts 1–4, and from 12.5 μg/ml to 150 μg/ml or 200 μg/ml for extract 5. All experiments were done in triplicate. Cisplatin was used as a positive control.",
"PBMC (150,000 cells per well) were seeded into nutrient medium or in nutrient medium enriched with (5 μg/ml) (PHA) in 96-well microtiter plates. After 2 h, five different concentrations of the plant extracts were added to the individual wells, in triplicate, except to the control wells where a nutrient medium only was added to the cells. The final concentrations of the tested extracts ranged from 12.5 μg/ml to 200 μg/ml. PHA was obtained from INEP (Belgrade, Serbia). Cisplatin was used as a positive control.",
"Cell survival was determined by the MTT test according to the method of Mosmann\n[21] and modified by Ohno and Abe\n[22]. Briefly, after the treatment with plant extracts for 72 h, 10 μl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 μl of 10% SDS. Absorbance at 570 nm was measured the next day.\nTo quantify cell survival (S%), the absorbance of a sample with cells grown in the presence of different concentrations of the investigated agents was divided by the absorbance of the control cells grown only in the nutrient medium, and multiplied by 100. It is implied that the absorbance of the blank was always subtracted from the absorbance of the corresponding sample with target cells. The IC50 was defined as the concentration of the agent that inhibited cell survival by 50%, compared to the vehicle-treated control.",
"To evaluate whether the extracts from the endemic plant Helichrysum zivojinii induce apoptosis in HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide-stained target cells was performed. HeLa cells were seeded overnight on coverslips (100,000 cells) in 2 ml of complete medium. The next day, cells were treated with plant extracts for 24 h at concentrations corresponding to IC90 values that were obtained after treatments that lasted 72 h. After this period, the target cells were stained with 18 μl of a mixture of the DNA dyes acridine orange and ethidium bromide (3 μg/ml AO and 10 μg/ml EB in PBS), and visualized under a fluorescence microscope using a fluorescein isothiocyanate (FITC) filter set.",
"HeLa cells were incubated in the presence of two different concentrations (corresponding to the IC50 and IC90 values determined after 72 h) of the examined Helichrysum zivojinii extracts for 24, 48 and 72 h. After these incubation times, the target cells were collected, washed and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before staining. HeLa cells were washed in PBS, resuspended in 500 μl of staining solution (PBS containing RNAse A at a final concentration of 200 μg/ml, and propidium iodide (PI) at a final concentration of 20 μg/ml), and incubated for 30 min at 37°C.\nCell cycle phase distribution was determined using a FACSCalibur Flow Cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data (10,000 events collected for each sample) were analyzed using CELLQuest software (BD Biosciences).",
"To identify the caspases involved in the apoptotic cell death pathway induced by the investigated extracts, the percentages of HeLa cells pretreated with caspase inhibitors in the subG1 phase were determined. HeLa cells were preincubated for 2 h with specific caspase inhibitors (at a final concentration of 40 μM). These were: Z-DEVD-FMK, a caspase-3 inhibitor, Z-IETD-FMK, a caspase-8 inhibitor and Z-LEHD-FMK, a caspase-9 inhibitor. Caspase inhibitors were purchased from R&D Systems (Minneapolis, USA). Five tested extracts were applied to the HeLa cells at concentrations that corresponded to the IC90 values obtained after 72 h. For each extract, one sample of HeLa cells was not treated with an inhibitor and served as a reference sample. After 24 h of incubation, cells were harvested and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before PI staining. Changes in the percentages of cells in the subG1 phase were determined using a FACSCalibur Flow Cytometer and analyzed using CELLQuest software.",
" Chemical analysis of plant extracts The hexane (1) and dichloromethane extracts (2) presented similar 1H NMR spectra (recorded in CDCl3). The signals in the 1H NMR spectra of both extracts pointed to a complex mixture with prevailing relatively non-polar substances (region δ 0.8–2.2). LC/DAD analysis revealed the presence of a flavonoid with the molecular formula C18H16O7 (344) in the hexane extract, while the flavanone naringenin and flavone apigenin were confirmed in the dichloromethane extract, also by LC/DAD analysis. Three compounds found in both extracts are presented in Table \n1, with their presumed molecular formulae and measured m/z values corresponding to identified ions obtained by ESI ToF mass spectrometry. According to LC/DAD analysis, these compounds unfortunately did not absorb in the UV spectrum, suggesting the presence of structures without a chromophore. The ethyl-acetate extract (3) and n-butanol extracts (4) presented 1H NMR spectra (recorded in DMSO-d6) with approximately identical groups of signals in the regions δ 12.5–13.9 (hydroxy protons), δ 5.9-8.1 (protons on the aromatic ring that is substituted with one or more hydroxy groups), and δ 4.7–5.5 (group of protons corresponding to a sugar unit). The 1H NMR spectrum of the most abundant methanol extract (5), also recorded in DMSO-d6, resembles the spectra of the ethyl-acetate and n-butanol extracts by the presence of signals in the region δ 5.9–8.1 (aromatic protons), as well as by the presence of a signal at δ 13.9 (hydroxy proton); it differs from the last two spectra by possessing a more pronounced region at δ 4.7–5.5, which is responsible for the protons of sugar units. LC/DAD analysis and ESI ToF mass spectrometry of these three more polar extracts (3, 4 and 5) revealed quite similar constituents in each of them. Apart from chlorogenic acid, two groups of flavonoid compounds were detected: flavonoid O-glycosides, and flavonoid aglycons. Among the O-glycosides, the glucoside (or possibly galactoside) of quercetin, the glucoside of apigenin, the glucoside of kaempferol (or possibly luteolin), and another glucoside of kaempferol (possibly luteolin or 6-hydroxyapigenin), were present. Flavonoid aglycones, such as apigenin and naringenin, were detected. Phthalic acid was found in extracts 2–5.\n\nComponents of five \n\nHelichrysum zivojinii \n\nextracts\n\naMore abundant on the expense of other fraction constituents.\nThe hexane (1) and dichloromethane extracts (2) presented similar 1H NMR spectra (recorded in CDCl3). The signals in the 1H NMR spectra of both extracts pointed to a complex mixture with prevailing relatively non-polar substances (region δ 0.8–2.2). LC/DAD analysis revealed the presence of a flavonoid with the molecular formula C18H16O7 (344) in the hexane extract, while the flavanone naringenin and flavone apigenin were confirmed in the dichloromethane extract, also by LC/DAD analysis. Three compounds found in both extracts are presented in Table \n1, with their presumed molecular formulae and measured m/z values corresponding to identified ions obtained by ESI ToF mass spectrometry. According to LC/DAD analysis, these compounds unfortunately did not absorb in the UV spectrum, suggesting the presence of structures without a chromophore. The ethyl-acetate extract (3) and n-butanol extracts (4) presented 1H NMR spectra (recorded in DMSO-d6) with approximately identical groups of signals in the regions δ 12.5–13.9 (hydroxy protons), δ 5.9-8.1 (protons on the aromatic ring that is substituted with one or more hydroxy groups), and δ 4.7–5.5 (group of protons corresponding to a sugar unit). The 1H NMR spectrum of the most abundant methanol extract (5), also recorded in DMSO-d6, resembles the spectra of the ethyl-acetate and n-butanol extracts by the presence of signals in the region δ 5.9–8.1 (aromatic protons), as well as by the presence of a signal at δ 13.9 (hydroxy proton); it differs from the last two spectra by possessing a more pronounced region at δ 4.7–5.5, which is responsible for the protons of sugar units. LC/DAD analysis and ESI ToF mass spectrometry of these three more polar extracts (3, 4 and 5) revealed quite similar constituents in each of them. Apart from chlorogenic acid, two groups of flavonoid compounds were detected: flavonoid O-glycosides, and flavonoid aglycons. Among the O-glycosides, the glucoside (or possibly galactoside) of quercetin, the glucoside of apigenin, the glucoside of kaempferol (or possibly luteolin), and another glucoside of kaempferol (possibly luteolin or 6-hydroxyapigenin), were present. Flavonoid aglycones, such as apigenin and naringenin, were detected. Phthalic acid was found in extracts 2–5.\n\nComponents of five \n\nHelichrysum zivojinii \n\nextracts\n\naMore abundant on the expense of other fraction constituents.\n In vitro cytotoxic activity The cytotoxicity of the five isolated extracts was tested against selected cancer cell lines: human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562 and human breast adenocarcinoma MDA-MB-361 cells. All investigated extracts exerted selective dose-dependent cytotoxic actions on malignant cells. The decrease in survival of target cancer cells induced by the five Helichrysum zivojinii extracts is shown in Figure \n1 and Table \n2.\nSurvival of HeLa (A), Fem-x (B), K562 (C) and MDA-MB-361 cells (D) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.\n\nConcentrations of five \n\nHelichrysum zivojinii \n\nextracts, which induced 50% decrease in the target cancer cell survival, determined by MTT test\n\nTime of continuous agent’s action was 72 h.\nIn general, extracts 1 and 2 (as well as cisplatin, which served as a positive control) exhibited the highest cytotoxic actions against target malignant cell lines; extracts 3 and 4 displayed less pronounced cytotoxicity; extract 5 had the lowest cytotoxic action.\nWith regard to the specific sensitivities of the different cells to the cytotoxic activities of the extracts, it is important to note that K562 cells were the most sensitive to the cytotoxic actions of extracts 1 and 3. HeLa and Fem-x cells exhibited a lower sensitivity, while the sensitivity of breast cancer MDA-MB-361 cells to the toxic actions of the tested extracts was the lowest (being several times lower than that of the other cell lines to extracts 1, 2 and 3 especially).\nConsidering the possible effects of applied antitumor drugs on normal healthy immunocompetent cells, components of the antitumor immune response, their viability is significant for tumor control. For that reason, the activities of the investigated Helichrysum zivojinii extracts were evaluated against healthy unstimulated and PHA-stimulated PBMC (Figure \n2 and Table \n3). It should be noted that these extracts overall exhibited weaker cytotoxic effects against unstimulated PBMC than against stimulated PBMC. Moreover, extracts 2 and 3 exerted a more pronounced cytotoxicity against unstimulated and PHA-stimulated PBMC than extracts 1, 4 and 5.\nSurvival of resting PBMC (A) and PHA-stimulated PBMC (B) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.\n\nConcentrations of five \n\nHelichrysum zivojinii \n\nextracts, which induced 50% decrease in target PBMC survival, determined by MTT test\n\nTime of continuous agent’s action was 72 h.\nIn order to further evaluate the anticancer potential of the extracts, the selectivity in the antitumor action against specific malignant cell line in comparison to healthy PBMC was determined as well. These data are presented in Table \n4 from which it can be observed that extract 1 exhibited highly selective antitumor action, especially against K562 cells. Extract 2 also displayed good selectivity in its antitumor action.\n\nSelectivity in the antitumor action of five \n\nHelichrysum zivojinii \n\nextracts\n\nThe cytotoxicity of the five isolated extracts was tested against selected cancer cell lines: human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562 and human breast adenocarcinoma MDA-MB-361 cells. All investigated extracts exerted selective dose-dependent cytotoxic actions on malignant cells. The decrease in survival of target cancer cells induced by the five Helichrysum zivojinii extracts is shown in Figure \n1 and Table \n2.\nSurvival of HeLa (A), Fem-x (B), K562 (C) and MDA-MB-361 cells (D) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.\n\nConcentrations of five \n\nHelichrysum zivojinii \n\nextracts, which induced 50% decrease in the target cancer cell survival, determined by MTT test\n\nTime of continuous agent’s action was 72 h.\nIn general, extracts 1 and 2 (as well as cisplatin, which served as a positive control) exhibited the highest cytotoxic actions against target malignant cell lines; extracts 3 and 4 displayed less pronounced cytotoxicity; extract 5 had the lowest cytotoxic action.\nWith regard to the specific sensitivities of the different cells to the cytotoxic activities of the extracts, it is important to note that K562 cells were the most sensitive to the cytotoxic actions of extracts 1 and 3. HeLa and Fem-x cells exhibited a lower sensitivity, while the sensitivity of breast cancer MDA-MB-361 cells to the toxic actions of the tested extracts was the lowest (being several times lower than that of the other cell lines to extracts 1, 2 and 3 especially).\nConsidering the possible effects of applied antitumor drugs on normal healthy immunocompetent cells, components of the antitumor immune response, their viability is significant for tumor control. For that reason, the activities of the investigated Helichrysum zivojinii extracts were evaluated against healthy unstimulated and PHA-stimulated PBMC (Figure \n2 and Table \n3). It should be noted that these extracts overall exhibited weaker cytotoxic effects against unstimulated PBMC than against stimulated PBMC. Moreover, extracts 2 and 3 exerted a more pronounced cytotoxicity against unstimulated and PHA-stimulated PBMC than extracts 1, 4 and 5.\nSurvival of resting PBMC (A) and PHA-stimulated PBMC (B) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.\n\nConcentrations of five \n\nHelichrysum zivojinii \n\nextracts, which induced 50% decrease in target PBMC survival, determined by MTT test\n\nTime of continuous agent’s action was 72 h.\nIn order to further evaluate the anticancer potential of the extracts, the selectivity in the antitumor action against specific malignant cell line in comparison to healthy PBMC was determined as well. These data are presented in Table \n4 from which it can be observed that extract 1 exhibited highly selective antitumor action, especially against K562 cells. Extract 2 also displayed good selectivity in its antitumor action.\n\nSelectivity in the antitumor action of five \n\nHelichrysum zivojinii \n\nextracts\n\n Morphological analysis of HeLa cell death mode In order to determine whether the investigated plant extracts have pro-apoptotic activities, we performed morphological analysis by fluorescent microscopy of acridine orange/ethidium bromide-stained HeLa cells, exposed to the extracts. Microscopic examination revealed that all five extracts applied at IC90 concentrations induced apoptosis in target HeLa cells after 24 h treatment (Figure \n3). The morphological characteristics of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange-red stained cells at late stages of apoptosis or secondary necrosis (the latter was observed in extract 1-treated HeLa cells) and apoptotic bodies. These analyses confirmed that the cytotoxicity of the Helichrysum zivojinii extracts is based on their prominent pro-apoptotic effects.\n\nPhotomicrographs of acridine orange/ethidium bromide-stained control HeLa cells (A), and HeLa cells treated with different \n\nHelichrysum zivojinii \n\nextracts for 24 h (extracts 1–5, photomicrographs B-F, consecutively).\n\nIn order to determine whether the investigated plant extracts have pro-apoptotic activities, we performed morphological analysis by fluorescent microscopy of acridine orange/ethidium bromide-stained HeLa cells, exposed to the extracts. Microscopic examination revealed that all five extracts applied at IC90 concentrations induced apoptosis in target HeLa cells after 24 h treatment (Figure \n3). The morphological characteristics of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange-red stained cells at late stages of apoptosis or secondary necrosis (the latter was observed in extract 1-treated HeLa cells) and apoptotic bodies. These analyses confirmed that the cytotoxicity of the Helichrysum zivojinii extracts is based on their prominent pro-apoptotic effects.\n\nPhotomicrographs of acridine orange/ethidium bromide-stained control HeLa cells (A), and HeLa cells treated with different \n\nHelichrysum zivojinii \n\nextracts for 24 h (extracts 1–5, photomicrographs B-F, consecutively).\n\n Analysis of changes in cell cycle phase distribution Examination of changes in the cell cycle phase distribution of HeLa cells treated with these extracts for 24, 48 and 72 h was done to elucidate the mechanisms of the observed cytotoxic actions (Figure \n4). Results from this analysis showed a time - dependent increase in the percentages of HeLa cells in the subG1 phase after exposure to an IC50 concentration of all of the tested extracts. Additionally, exposure to extracts at IC90 concentrations induced significant increases in the percentages of cells in the subG1 phase 24 h after exposure. It should be mentioned that the investigated extracts induced a slight accumulation of HeLa cells in the S phase after 72 h. Examination of the cell cycle changes that were induced after exposure for 72 h to IC90 for each extract was not performed because at this time point and at this extract concentration low numbers of mostly dead or dying cells were present in the sample.\nChanges in the cell cycle phase distribution of HeLa cells induced by the Helichrysum zivojinii extracts after 24 (A,B), 48 (C,D) and 72 h (E) treatment (applied concentrations of tested extracts corresponded to IC50 and IC90 values determined for 72 h). (C- control HeLa cells, 1 – 5 – corresponding extracts are numbered consecutively). Representative graphs are shown.\nSince the Helichrysum zivojinii extracts exhibited the pro-apoptotic activities against cervix adenocarcinoma HeLa cells, the identification of target caspases involved in the apoptotic pathway was performed. The presence of the specific caspase inhibitors (caspase-3 inhibitor, caspase-8 inhibitor or caspase-9 inhibitor) significantly reduced the percentages of apoptotic subG1 HeLa cells treated with each of the five plant extracts, as shown in Figure \n5. The effect of the caspase-3 inhibitor on HeLa cells treated with extracts is shown in Figure \n6. It can be seen that there is an increase in rounded, but attached and live HeLa cells treated with the caspase-3 inhibitor before the addition of the extracts in relation to target HeLa cells only exposed to the tested extracts.\nEffects of specific caspase inhibitors on the percentages of apoptotic subG1 HeLa cells treated with Helichrysum zivojinii extracts for 24 h (A - extract 1, B - extract 2, C - extract 3, D - extract 4, E - extract 5). (Z-DEVD-FMK - caspase-3 inhibitor; Z-IETD-FMK -caspase-8 inhibitor; Z-LEHD-FMK - caspase-9 inhibitor).\nEffects of pretreatment of HeLa cells with caspase-3 inhibitor (Z-DEVD-FMK), exposed to Helichrysum zivojinii extracts (applied concentrations of tested extracts corresponded to IC90 values determined for 72 h). A – control; B – Extract 1, C – Extract 1 + Z-DEVD-FMK; D – Extract 2, E – Extract 2 + Z-DEVD-FMK; F – Extract 3, G – Extract 3 + Z-DEVD-FMK; H – Extract 4, I – Extract 4 + Z-DEVD-FMK; J – Extract 5, K – Extract 5 + Z-DEVD-FMK.\nExamination of changes in the cell cycle phase distribution of HeLa cells treated with these extracts for 24, 48 and 72 h was done to elucidate the mechanisms of the observed cytotoxic actions (Figure \n4). Results from this analysis showed a time - dependent increase in the percentages of HeLa cells in the subG1 phase after exposure to an IC50 concentration of all of the tested extracts. Additionally, exposure to extracts at IC90 concentrations induced significant increases in the percentages of cells in the subG1 phase 24 h after exposure. It should be mentioned that the investigated extracts induced a slight accumulation of HeLa cells in the S phase after 72 h. Examination of the cell cycle changes that were induced after exposure for 72 h to IC90 for each extract was not performed because at this time point and at this extract concentration low numbers of mostly dead or dying cells were present in the sample.\nChanges in the cell cycle phase distribution of HeLa cells induced by the Helichrysum zivojinii extracts after 24 (A,B), 48 (C,D) and 72 h (E) treatment (applied concentrations of tested extracts corresponded to IC50 and IC90 values determined for 72 h). (C- control HeLa cells, 1 – 5 – corresponding extracts are numbered consecutively). Representative graphs are shown.\nSince the Helichrysum zivojinii extracts exhibited the pro-apoptotic activities against cervix adenocarcinoma HeLa cells, the identification of target caspases involved in the apoptotic pathway was performed. The presence of the specific caspase inhibitors (caspase-3 inhibitor, caspase-8 inhibitor or caspase-9 inhibitor) significantly reduced the percentages of apoptotic subG1 HeLa cells treated with each of the five plant extracts, as shown in Figure \n5. The effect of the caspase-3 inhibitor on HeLa cells treated with extracts is shown in Figure \n6. It can be seen that there is an increase in rounded, but attached and live HeLa cells treated with the caspase-3 inhibitor before the addition of the extracts in relation to target HeLa cells only exposed to the tested extracts.\nEffects of specific caspase inhibitors on the percentages of apoptotic subG1 HeLa cells treated with Helichrysum zivojinii extracts for 24 h (A - extract 1, B - extract 2, C - extract 3, D - extract 4, E - extract 5). (Z-DEVD-FMK - caspase-3 inhibitor; Z-IETD-FMK -caspase-8 inhibitor; Z-LEHD-FMK - caspase-9 inhibitor).\nEffects of pretreatment of HeLa cells with caspase-3 inhibitor (Z-DEVD-FMK), exposed to Helichrysum zivojinii extracts (applied concentrations of tested extracts corresponded to IC90 values determined for 72 h). A – control; B – Extract 1, C – Extract 1 + Z-DEVD-FMK; D – Extract 2, E – Extract 2 + Z-DEVD-FMK; F – Extract 3, G – Extract 3 + Z-DEVD-FMK; H – Extract 4, I – Extract 4 + Z-DEVD-FMK; J – Extract 5, K – Extract 5 + Z-DEVD-FMK.",
"The hexane (1) and dichloromethane extracts (2) presented similar 1H NMR spectra (recorded in CDCl3). The signals in the 1H NMR spectra of both extracts pointed to a complex mixture with prevailing relatively non-polar substances (region δ 0.8–2.2). LC/DAD analysis revealed the presence of a flavonoid with the molecular formula C18H16O7 (344) in the hexane extract, while the flavanone naringenin and flavone apigenin were confirmed in the dichloromethane extract, also by LC/DAD analysis. Three compounds found in both extracts are presented in Table \n1, with their presumed molecular formulae and measured m/z values corresponding to identified ions obtained by ESI ToF mass spectrometry. According to LC/DAD analysis, these compounds unfortunately did not absorb in the UV spectrum, suggesting the presence of structures without a chromophore. The ethyl-acetate extract (3) and n-butanol extracts (4) presented 1H NMR spectra (recorded in DMSO-d6) with approximately identical groups of signals in the regions δ 12.5–13.9 (hydroxy protons), δ 5.9-8.1 (protons on the aromatic ring that is substituted with one or more hydroxy groups), and δ 4.7–5.5 (group of protons corresponding to a sugar unit). The 1H NMR spectrum of the most abundant methanol extract (5), also recorded in DMSO-d6, resembles the spectra of the ethyl-acetate and n-butanol extracts by the presence of signals in the region δ 5.9–8.1 (aromatic protons), as well as by the presence of a signal at δ 13.9 (hydroxy proton); it differs from the last two spectra by possessing a more pronounced region at δ 4.7–5.5, which is responsible for the protons of sugar units. LC/DAD analysis and ESI ToF mass spectrometry of these three more polar extracts (3, 4 and 5) revealed quite similar constituents in each of them. Apart from chlorogenic acid, two groups of flavonoid compounds were detected: flavonoid O-glycosides, and flavonoid aglycons. Among the O-glycosides, the glucoside (or possibly galactoside) of quercetin, the glucoside of apigenin, the glucoside of kaempferol (or possibly luteolin), and another glucoside of kaempferol (possibly luteolin or 6-hydroxyapigenin), were present. Flavonoid aglycones, such as apigenin and naringenin, were detected. Phthalic acid was found in extracts 2–5.\n\nComponents of five \n\nHelichrysum zivojinii \n\nextracts\n\naMore abundant on the expense of other fraction constituents.",
"The cytotoxicity of the five isolated extracts was tested against selected cancer cell lines: human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562 and human breast adenocarcinoma MDA-MB-361 cells. All investigated extracts exerted selective dose-dependent cytotoxic actions on malignant cells. The decrease in survival of target cancer cells induced by the five Helichrysum zivojinii extracts is shown in Figure \n1 and Table \n2.\nSurvival of HeLa (A), Fem-x (B), K562 (C) and MDA-MB-361 cells (D) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.\n\nConcentrations of five \n\nHelichrysum zivojinii \n\nextracts, which induced 50% decrease in the target cancer cell survival, determined by MTT test\n\nTime of continuous agent’s action was 72 h.\nIn general, extracts 1 and 2 (as well as cisplatin, which served as a positive control) exhibited the highest cytotoxic actions against target malignant cell lines; extracts 3 and 4 displayed less pronounced cytotoxicity; extract 5 had the lowest cytotoxic action.\nWith regard to the specific sensitivities of the different cells to the cytotoxic activities of the extracts, it is important to note that K562 cells were the most sensitive to the cytotoxic actions of extracts 1 and 3. HeLa and Fem-x cells exhibited a lower sensitivity, while the sensitivity of breast cancer MDA-MB-361 cells to the toxic actions of the tested extracts was the lowest (being several times lower than that of the other cell lines to extracts 1, 2 and 3 especially).\nConsidering the possible effects of applied antitumor drugs on normal healthy immunocompetent cells, components of the antitumor immune response, their viability is significant for tumor control. For that reason, the activities of the investigated Helichrysum zivojinii extracts were evaluated against healthy unstimulated and PHA-stimulated PBMC (Figure \n2 and Table \n3). It should be noted that these extracts overall exhibited weaker cytotoxic effects against unstimulated PBMC than against stimulated PBMC. Moreover, extracts 2 and 3 exerted a more pronounced cytotoxicity against unstimulated and PHA-stimulated PBMC than extracts 1, 4 and 5.\nSurvival of resting PBMC (A) and PHA-stimulated PBMC (B) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.\n\nConcentrations of five \n\nHelichrysum zivojinii \n\nextracts, which induced 50% decrease in target PBMC survival, determined by MTT test\n\nTime of continuous agent’s action was 72 h.\nIn order to further evaluate the anticancer potential of the extracts, the selectivity in the antitumor action against specific malignant cell line in comparison to healthy PBMC was determined as well. These data are presented in Table \n4 from which it can be observed that extract 1 exhibited highly selective antitumor action, especially against K562 cells. Extract 2 also displayed good selectivity in its antitumor action.\n\nSelectivity in the antitumor action of five \n\nHelichrysum zivojinii \n\nextracts\n",
"In order to determine whether the investigated plant extracts have pro-apoptotic activities, we performed morphological analysis by fluorescent microscopy of acridine orange/ethidium bromide-stained HeLa cells, exposed to the extracts. Microscopic examination revealed that all five extracts applied at IC90 concentrations induced apoptosis in target HeLa cells after 24 h treatment (Figure \n3). The morphological characteristics of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange-red stained cells at late stages of apoptosis or secondary necrosis (the latter was observed in extract 1-treated HeLa cells) and apoptotic bodies. These analyses confirmed that the cytotoxicity of the Helichrysum zivojinii extracts is based on their prominent pro-apoptotic effects.\n\nPhotomicrographs of acridine orange/ethidium bromide-stained control HeLa cells (A), and HeLa cells treated with different \n\nHelichrysum zivojinii \n\nextracts for 24 h (extracts 1–5, photomicrographs B-F, consecutively).\n",
"Examination of changes in the cell cycle phase distribution of HeLa cells treated with these extracts for 24, 48 and 72 h was done to elucidate the mechanisms of the observed cytotoxic actions (Figure \n4). Results from this analysis showed a time - dependent increase in the percentages of HeLa cells in the subG1 phase after exposure to an IC50 concentration of all of the tested extracts. Additionally, exposure to extracts at IC90 concentrations induced significant increases in the percentages of cells in the subG1 phase 24 h after exposure. It should be mentioned that the investigated extracts induced a slight accumulation of HeLa cells in the S phase after 72 h. Examination of the cell cycle changes that were induced after exposure for 72 h to IC90 for each extract was not performed because at this time point and at this extract concentration low numbers of mostly dead or dying cells were present in the sample.\nChanges in the cell cycle phase distribution of HeLa cells induced by the Helichrysum zivojinii extracts after 24 (A,B), 48 (C,D) and 72 h (E) treatment (applied concentrations of tested extracts corresponded to IC50 and IC90 values determined for 72 h). (C- control HeLa cells, 1 – 5 – corresponding extracts are numbered consecutively). Representative graphs are shown.\nSince the Helichrysum zivojinii extracts exhibited the pro-apoptotic activities against cervix adenocarcinoma HeLa cells, the identification of target caspases involved in the apoptotic pathway was performed. The presence of the specific caspase inhibitors (caspase-3 inhibitor, caspase-8 inhibitor or caspase-9 inhibitor) significantly reduced the percentages of apoptotic subG1 HeLa cells treated with each of the five plant extracts, as shown in Figure \n5. The effect of the caspase-3 inhibitor on HeLa cells treated with extracts is shown in Figure \n6. It can be seen that there is an increase in rounded, but attached and live HeLa cells treated with the caspase-3 inhibitor before the addition of the extracts in relation to target HeLa cells only exposed to the tested extracts.\nEffects of specific caspase inhibitors on the percentages of apoptotic subG1 HeLa cells treated with Helichrysum zivojinii extracts for 24 h (A - extract 1, B - extract 2, C - extract 3, D - extract 4, E - extract 5). (Z-DEVD-FMK - caspase-3 inhibitor; Z-IETD-FMK -caspase-8 inhibitor; Z-LEHD-FMK - caspase-9 inhibitor).\nEffects of pretreatment of HeLa cells with caspase-3 inhibitor (Z-DEVD-FMK), exposed to Helichrysum zivojinii extracts (applied concentrations of tested extracts corresponded to IC90 values determined for 72 h). A – control; B – Extract 1, C – Extract 1 + Z-DEVD-FMK; D – Extract 2, E – Extract 2 + Z-DEVD-FMK; F – Extract 3, G – Extract 3 + Z-DEVD-FMK; H – Extract 4, I – Extract 4 + Z-DEVD-FMK; J – Extract 5, K – Extract 5 + Z-DEVD-FMK.",
"The plant kingdom provides a rich source of compounds with promising cancer chemopreventive and cancer therapeutic potential. The main drugs currently used in clinical practice in the treatment of malignant diseases originate from plants: vinca alkaloids, taxanes, camptothecins and epipodophyllotoxins\n[23]. Over the past years, the focus of modern anticancer drug discovery has been on a wide variety of natural compounds, especially on phenolic compounds. Phytochemicals have been reported to affect different intracellular signaling pathways implicated in the initiation, promotion and progression of cancer. The antitumor effects of plant constituents have been associated with the induction of carcinogen detoxifying enzymes, the scavenging of free radicals, anti-inflammatory activity, cell cycle arrest, the triggering of apoptosis, inhibition of tumor angiogenesis and invasiveness\n[1-4].\nThe antioxidant and anti-inflammatory activities of extracts and isolated compounds from plants belonging to the large genus Helichrysum have been well documented\n[9,11,15,18]. Arzanol, a phloroglucinyl α-pyrone, is a constituent of Helichrysum italicum that has been reported to inhibit the NF-κB transcription factor, cyclooxygenase and lipooxygenase, as well as the release of proinflammatory cytokines\n[11,18]. Regarding the link between inflammation and cancer, chemicals with anti-inflammatory properties targeting the molecules of signaling cascades implicated in inflammation and carcinogenesis may be useful as cancer chemopreventive drugs.\nOn the other hand, data about the potential anticancer activity of extracts and phytochemicals of plants from the genus Helichrysum are scarce. The antiproliferative effect of the ethanol extract from Helichrysum maracandicum towards SENCAR mouse skin transformed cells has been demonstrated\n[20]. This extract suppressed the expression of p38 MAP kinase. An examination of the arzanol properties showed that this compound, which was isolated from Helichrysum italicum, did not exert cytotoxic action against monkey VERO cells at concentrations up to 40 μM\n[24]. In contrast, another study showed that arzanol at a concentration of 50 μM significantly suppressed the survival of human lung carcinoma A549 cells\n[18]. It should be mentioned that methanolic extracts prepared from different Helichrysum species were found to inhibit DNA topoisomerase I\n[19]. Moreover, the cytotoxicity of Helichrysum gymnocephalum essential oil towards human breast adenocarcinoma MCF-7 cells has been documented\n[17].\nThe results presented herein demonstrate the selective dose-dependent cytotoxic actions of the five extracts isolated from the endemic plant species Helichrysum zivojinii against target cancer cell lines and against healthy immunocompetent PBMC that have been stimulated to proliferate, while their cytotoxic actions were not as pronounced against unstimulated PBMC. The observed selectivity in the antitumor effects of the extracts against specific malignant cell types could be attributed to the actions of different Helichrysum zivojinii constituents on target molecules of the signal transduction pathways that regulate cell proliferation and apoptosis. Furthermore, each of the investigated extracts exhibited considerably stronger cytotoxicity to HeLa, Fem-x and K562 cells when compared to PBMC, both resting and PHA-stimulated, which points to the cancer specificity of their actions. It is noteworthy that when these extracts were applied at concentrations that were highly cytotoxic to malignant cells, they demonstrated very low toxicity towards healthy immunocompetent PBMC, the key players in immune defenses against tumors. The good selectivity of their antitumor actions highlights the significant anticancer potential of Helichrysum zivojinii extracts. The prominent antitumor properties of these extracts need to be examined further in in vivo studies.\nIt should be stressed that all of these extracts exhibited weaker cytotoxic effects against unstimulated PBMC in comparison to stimulated PBMC. This finding indicates that the extracts possess the ability to inhibit the proliferation of PHA-stimulated PBMC. Thus, these agents may even suppress certain immune functions, particularly non-specific antigen stimulation. Additionally, the observed lower activities against resting PBMC than against mitogen-stimulated PBMC point to components in pathways regulating cell proliferation as the possible molecular targets of the Helichrysum zivojinii extracts. However, it is very important to note that when extracts 1, 2 and 3 were applied at lower concentrations, they stimulated the proliferation of resting PBMC. This growth stimulation effect of lower concentrations of extracts is in accordance with the well-known effect of very small doses of X rays on enhanced proliferation of irradiated cells\n[25]. The observed effects of low concentrations of these extracts on one of the main components of the immune response point to the possibility of their use to enhance immunity. It would be interesting to investigate their action towards different PBMC subpopulations and elucidate the potential mechanisms through which they stimulate proliferation. The possible immunostimulatory effects of extracts at lower concentrations might be explained by a modulation in lymphocyte cytokine production, including IL-2, IFN-γ, as well as IL-4 and IL-6. The immunoregulatory actions of phenolic compounds, such as quercetin, kaempferol and apigenin, have been reported\n[26-28]. Considering the presented results, the effects of the extracts on PBMC might be mediated through NF-κB.\nExamination of in vitro cytotoxicity revealed that extracts 1 and 2 might be a significant source of novel promising anticancer compounds in view of their pronounced cytotoxic activities against HeLa, Fem-x and especially against K562 cells, as well as their high selectivity in the antitumor actions against cancer cells in comparison to healthy PBMC. Chemical analyses of the Helichrysum zivojinii extracts showed the presence of phenolic compounds whose antitumor potential has already been documented. The bioactive flavone apigenin that was found in extracts 2–5 has been reported to exhibit anticancer activities against different types of malignant cells including breast, cervical, ovarian, prostate, colon, gastric, liver and lung cancers, as well as skin and thyroid cancer, diverse hematological malignancies and neuroblastoma\n[28] and references cited therein]. The cytotoxic activities of extracts 2–5 may be at least in part due to the flavonoid naringenin. This flavonoid has been shown to exert cytotoxicity towards various malignant cell lines, such as breast cancer cell lines (MCF-7, MDA-MB-231), cervix adenocarcinoma (HeLa), liver cancer (HepG2, Hep3B, Huh7), pancreas cancer (PK-1), colon cancer (Caco-2), stomach cancer (KATOIII, MKN-7) and leukemia cells (Jurkat, HL-60, U937, NALM-6, THP-1)\n[29-31]. Additionally, the cancer-preventive and cancer-suppressive properties of quercetin, whose O-glycosides were identified in extracts 3, 4 and 5, have been documented as well\n[32]. The antiproliferative and pro-apoptotic effects of quercetin were shown against the HeLa cell line\n[33].\nMorphological analysis of the mode of HeLa cell death, together with the cell cycle analysis, showed that the treatment of HeLa cells with higher concentrations of the examined extracts induced apoptotic cell death. To confirm the pro-apoptotic action of the tested Helichrysum zivojinii extracts and to identify the caspases implicated in the employed apoptotic pathways, specific caspase inhibitors were used (Z-DEVD-FMK, Z-IETD-FMK, Z-LEHD-FMK). A prominent decrease in the percentages of subG1 apoptotic HeLa cells after treatments with each of the tested extracts in combination with specific caspase inhibitors compared to the percentages of subG1 cells after treatments with only the corresponding extracts, indicates that each of the five extracts induced apoptosis through the activation of caspase-3, the main effector caspase, as well as through the activation of caspase-8 and caspase-9. We conclude that the constituents of the Helichrysum zivojinii extracts triggered apoptosis in HeLa cells through the intrinsic pathway mediated by caspase-9, and the extrinsic pathway mediated by caspase-8. In addition, the crosstalk between these two apoptotic pathways should also be considered. Due to their ability to promote apoptotic cell death in cancer cells, the investigated extracts and their constituents may have significant anticancer potential. It is worth noting that antitumor drugs that induce apoptosis and thereby suppress the further growth of tumors play important roles in the clinical treatment of malignancies. In addition to the pronounced inhibition of proliferation and survival of target malignant cells, the lower cytotoxicity of Helichrysum zivojinii extracts against healthy PBMC is a promising lead for future studies.",
"Data from this in vitro study clearly demonstrate the prominent antitumor potential of five extracts prepared from the endemic plant species Helichrysum zivojinii, which can be attributed to their selective and pronounced antiproliferative and pro-apoptotic actions towards specific malignant cells in comparison to healthy PBMC. Prospective cancer-suppressive effects of the tested extracts should be further evaluated in in vivo experiments.",
"The authors declare that they have no competing interests.",
"IM performed all analyses of the anticancer properties of investigated extracts, interpreted obtained data and wrote the first and last version of the manuscript. IA participated in design of the study, prepared extracts, performed chemical characterization, interpreted data and wrote the part of the manuscript. ŽŽ participated in acquisition and analysis of data. MJ carried out chemical analyses of the extracts. VV and SM participated in design of the study and interpreted obtained data. ZJ designed the research on anticancer properties of tested extracts, interpreted obtained data, participated in writing the manuscript and critically revised the manuscript. All authors have read and approved the final version of the manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/13/36/prepub\n"
] |
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"methods",
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"results",
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"discussion",
"conclusions",
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[
"\nHelichrysum zivojinii\n",
"Cytotoxicity",
"Cancer cells",
"Peripheral blood mononuclear cells",
"Apoptosis"
] |
Background:
Bioactive constituents of medicinal plants are in the center of attention of modern anticancer research due to their prospective roles in suppressing the different stages of malignant transformation. The antitumor potential of plant extracts and compounds could be attributed to their ability to induce changes in the regulation of target molecules in oncogenic signal transduction pathways implicated in cell growth, replication, apoptosis, as well as in angiogenesis, invasion and metastasis of cancer cells
[1-4]. To evaluate the anticancer properties of novel chemotherapeutic agents, the selectivity of their actions against malignant cells in comparison to healthy non-transformed cells, especially immunocompetent cells involved in the immune control of tumor suppression, needs to be carefully examined.
Helichrysum zivojinii Černjavski & Soška is an endemic plant species that grows in the National Park "Galičica" in Macedonia. Some of the plant species from the large genus Helichrysum are used in different regions of the world in traditional medicine for treating wounds, respiratory tract infections and gastro-intestinal disorders
[5-8]. This plant genus is a valuable source of several different secondary metabolites/phytochemicals, such as flavonoids, acetophenones, phloroglucinols, pyrones, diterpenes and sesquiterpenes
[5]. Different morphological groups of Helichrysum species often display unique qualitative and quantitative chemical compositions
[5]. It has been reported that extracts and individual constituents of these plants possess significant biological and pharmacological properties, including antibacterial, antiviral, antifungal, antioxidant, anti-inflammatory and antidiabetic activities
[9-16]. A search through the literature suggests that plants from the genus Helichrysum could be a significant source of compounds with potential anticancer activities
[17-20].
The main goal of this research was to investigate the cytotoxic activities of five extracts isolated as fractions from the endemic plant Helichrysum zivojinii towards selected human malignant cell lines. To assess the sensitivity of normal immunocompetent cells included in the antitumor immune response, the cytotoxicity of these extracts was also tested against human peripheral blood mononuclear cells (PBMC) – both unstimulated and stimulated to proliferate by the mitogen phytohemagglutinin (PHA). To elucidate the molecular mechanisms of the cytotoxic effects of the tested extracts, the distribution of target HeLa cells at specific phases of the cell cycle after the actions of these agents was also analyzed. The mode of HeLa cell death induced by the extracts was also investigated. Elucidation of the signaling pathways implicated in the induction of apoptosis by the tested extracts was conducted by identification of target caspases.
Methods:
Plant extracts The plant material was collected at Tomoros (ca. 1700 altitude), mountain Galičica (Macedonia) during the flowering (17 July 2010) and identified by Vlado Matevski, Institute of Biology, Faculty of Natural Sciences and Mathematics,
http://Ss. Cyril and Methodius University of Skopje, where the voucher specimen is deposited at Macedonian National Herbarium (MKNH) under the number MKNH121335.
Air-dried and powdered aerial parts of Helichrysum zivojinii (330 g) were extracted twice with n-hexane in an ultrasonic bath for 45 min. The combined extracts were concentrated in a vacuum to obtain a hexane extract (4.2 g). The plant material was successively extracted in the same manner with solvents of rising polarity to obtain a dichloromethane extract (1.4 g), an ethyl-acetate extract (0.7 g), a n-butanol extract (5.4 g) and finally a methanol extract (12.4 g).
Stock solutions of the investigated extracts were made in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml.
The plant material was collected at Tomoros (ca. 1700 altitude), mountain Galičica (Macedonia) during the flowering (17 July 2010) and identified by Vlado Matevski, Institute of Biology, Faculty of Natural Sciences and Mathematics,
http://Ss. Cyril and Methodius University of Skopje, where the voucher specimen is deposited at Macedonian National Herbarium (MKNH) under the number MKNH121335.
Air-dried and powdered aerial parts of Helichrysum zivojinii (330 g) were extracted twice with n-hexane in an ultrasonic bath for 45 min. The combined extracts were concentrated in a vacuum to obtain a hexane extract (4.2 g). The plant material was successively extracted in the same manner with solvents of rising polarity to obtain a dichloromethane extract (1.4 g), an ethyl-acetate extract (0.7 g), a n-butanol extract (5.4 g) and finally a methanol extract (12.4 g).
Stock solutions of the investigated extracts were made in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml.
Instrumentation and chromatographic conditions 1HNMR spectra were recorded with Varian Gemini 200 in CDCl3 and DMSO-d6 with TMS as an internal standard. HPLC-MS analysis was performed with an Agilent 1100 Series chromatography system equipped with a binary pump, degasser, autosampler, column Li Chrospher 100 RP 18 (250 × 4,0 mm i.d. 5 μm), and DAD detector in combination with 6210 Time of Flight MS (Agilent Technologies). The mobile phase consisted of 0.2% formic acid in water (solvent A) and 100% acetonitrile (solvent B) with the following gradient elution: 0–5 min 10–20% B, 5–10 min 20% B, 10–20 min 20–30% B, 20–30 min 30–70% B, 30–35 min 70–100% B, 35–40 min 70% B, 40–41 min 100–10% B, 41–45 min 10% B, at a flow rate of 1 ml/min. The injection volume was 10 μL, the column temperature was 25°C. The effluent was monitored with DAD (190–550 nm) and a mass detector (ESI) which operated in negative mode at atmospheric pressure; the mass range was from m/z 100–2500, with the following ESI parameters: capillary voltage: 4000 V; gas temperature: 350°C; nebulizer pressure: 45 psig; fragmentor voltage: 140 V. Mass Hunter Workstation software was used for data analysis.
1HNMR spectra were recorded with Varian Gemini 200 in CDCl3 and DMSO-d6 with TMS as an internal standard. HPLC-MS analysis was performed with an Agilent 1100 Series chromatography system equipped with a binary pump, degasser, autosampler, column Li Chrospher 100 RP 18 (250 × 4,0 mm i.d. 5 μm), and DAD detector in combination with 6210 Time of Flight MS (Agilent Technologies). The mobile phase consisted of 0.2% formic acid in water (solvent A) and 100% acetonitrile (solvent B) with the following gradient elution: 0–5 min 10–20% B, 5–10 min 20% B, 10–20 min 20–30% B, 20–30 min 30–70% B, 30–35 min 70–100% B, 35–40 min 70% B, 40–41 min 100–10% B, 41–45 min 10% B, at a flow rate of 1 ml/min. The injection volume was 10 μL, the column temperature was 25°C. The effluent was monitored with DAD (190–550 nm) and a mass detector (ESI) which operated in negative mode at atmospheric pressure; the mass range was from m/z 100–2500, with the following ESI parameters: capillary voltage: 4000 V; gas temperature: 350°C; nebulizer pressure: 45 psig; fragmentor voltage: 140 V. Mass Hunter Workstation software was used for data analysis.
Cell culture Human cervix adenocarcinoma HeLa, human melanoma Fem-x and human breast adenocarcinoma MDA-MB-361 cells were cultured as monolayers. Human chronic myelogenous leukemia K562 cells were grown in a suspension in nutrient medium. Cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The complete nutrient medium was RPMI 1640 supplemented with 3 mM L-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, 10% heat-inactivated (56°C) fetal bovine serum and 25 mM Hepes adjusted to pH 7.2 with a bicarbonate solution. The cells were grown at 37°C in an atmosphere of 5% CO2 and humidified air. RPMI 1640, L-glutamine and Hepes were obtained from PAA (Pasching, Austria).
Human cervix adenocarcinoma HeLa, human melanoma Fem-x and human breast adenocarcinoma MDA-MB-361 cells were cultured as monolayers. Human chronic myelogenous leukemia K562 cells were grown in a suspension in nutrient medium. Cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The complete nutrient medium was RPMI 1640 supplemented with 3 mM L-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, 10% heat-inactivated (56°C) fetal bovine serum and 25 mM Hepes adjusted to pH 7.2 with a bicarbonate solution. The cells were grown at 37°C in an atmosphere of 5% CO2 and humidified air. RPMI 1640, L-glutamine and Hepes were obtained from PAA (Pasching, Austria).
Preparation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of two healthy volunteers by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl- and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in nutrient medium. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia. Written informed consent was obtained from each healthy donor.
Peripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of two healthy volunteers by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl- and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in nutrient medium. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia. Written informed consent was obtained from each healthy donor.
Treatment of cancer cell lines HeLa (2,000 cells per well), Fem-x (2,000 cells per well), MDA-MB-361 (10,000 cells per well) were seeded into 96-well microtiter plates and 20 h later, after cell adherence, five different concentrations of the tested extracts were added to the wells. Nutrient medium was only added to the cells in the control wells. K562 cells (5,000 cells per well) were seeded 2 h before addition of the extracts. Stock solutions of plant extracts were diluted with complete nutrient medium and applied to target cells at different final concentrations that ranged from 6.25 μg/ml to 100 μg/ml for extracts 1–4, and from 12.5 μg/ml to 150 μg/ml or 200 μg/ml for extract 5. All experiments were done in triplicate. Cisplatin was used as a positive control.
HeLa (2,000 cells per well), Fem-x (2,000 cells per well), MDA-MB-361 (10,000 cells per well) were seeded into 96-well microtiter plates and 20 h later, after cell adherence, five different concentrations of the tested extracts were added to the wells. Nutrient medium was only added to the cells in the control wells. K562 cells (5,000 cells per well) were seeded 2 h before addition of the extracts. Stock solutions of plant extracts were diluted with complete nutrient medium and applied to target cells at different final concentrations that ranged from 6.25 μg/ml to 100 μg/ml for extracts 1–4, and from 12.5 μg/ml to 150 μg/ml or 200 μg/ml for extract 5. All experiments were done in triplicate. Cisplatin was used as a positive control.
Treatment of PBMC PBMC (150,000 cells per well) were seeded into nutrient medium or in nutrient medium enriched with (5 μg/ml) (PHA) in 96-well microtiter plates. After 2 h, five different concentrations of the plant extracts were added to the individual wells, in triplicate, except to the control wells where a nutrient medium only was added to the cells. The final concentrations of the tested extracts ranged from 12.5 μg/ml to 200 μg/ml. PHA was obtained from INEP (Belgrade, Serbia). Cisplatin was used as a positive control.
PBMC (150,000 cells per well) were seeded into nutrient medium or in nutrient medium enriched with (5 μg/ml) (PHA) in 96-well microtiter plates. After 2 h, five different concentrations of the plant extracts were added to the individual wells, in triplicate, except to the control wells where a nutrient medium only was added to the cells. The final concentrations of the tested extracts ranged from 12.5 μg/ml to 200 μg/ml. PHA was obtained from INEP (Belgrade, Serbia). Cisplatin was used as a positive control.
Determination of target cell survival Cell survival was determined by the MTT test according to the method of Mosmann
[21] and modified by Ohno and Abe
[22]. Briefly, after the treatment with plant extracts for 72 h, 10 μl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 μl of 10% SDS. Absorbance at 570 nm was measured the next day.
To quantify cell survival (S%), the absorbance of a sample with cells grown in the presence of different concentrations of the investigated agents was divided by the absorbance of the control cells grown only in the nutrient medium, and multiplied by 100. It is implied that the absorbance of the blank was always subtracted from the absorbance of the corresponding sample with target cells. The IC50 was defined as the concentration of the agent that inhibited cell survival by 50%, compared to the vehicle-treated control.
Cell survival was determined by the MTT test according to the method of Mosmann
[21] and modified by Ohno and Abe
[22]. Briefly, after the treatment with plant extracts for 72 h, 10 μl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 μl of 10% SDS. Absorbance at 570 nm was measured the next day.
To quantify cell survival (S%), the absorbance of a sample with cells grown in the presence of different concentrations of the investigated agents was divided by the absorbance of the control cells grown only in the nutrient medium, and multiplied by 100. It is implied that the absorbance of the blank was always subtracted from the absorbance of the corresponding sample with target cells. The IC50 was defined as the concentration of the agent that inhibited cell survival by 50%, compared to the vehicle-treated control.
Morphological evaluation of HeLa cell death To evaluate whether the extracts from the endemic plant Helichrysum zivojinii induce apoptosis in HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide-stained target cells was performed. HeLa cells were seeded overnight on coverslips (100,000 cells) in 2 ml of complete medium. The next day, cells were treated with plant extracts for 24 h at concentrations corresponding to IC90 values that were obtained after treatments that lasted 72 h. After this period, the target cells were stained with 18 μl of a mixture of the DNA dyes acridine orange and ethidium bromide (3 μg/ml AO and 10 μg/ml EB in PBS), and visualized under a fluorescence microscope using a fluorescein isothiocyanate (FITC) filter set.
To evaluate whether the extracts from the endemic plant Helichrysum zivojinii induce apoptosis in HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide-stained target cells was performed. HeLa cells were seeded overnight on coverslips (100,000 cells) in 2 ml of complete medium. The next day, cells were treated with plant extracts for 24 h at concentrations corresponding to IC90 values that were obtained after treatments that lasted 72 h. After this period, the target cells were stained with 18 μl of a mixture of the DNA dyes acridine orange and ethidium bromide (3 μg/ml AO and 10 μg/ml EB in PBS), and visualized under a fluorescence microscope using a fluorescein isothiocyanate (FITC) filter set.
Cell cycle analysis HeLa cells were incubated in the presence of two different concentrations (corresponding to the IC50 and IC90 values determined after 72 h) of the examined Helichrysum zivojinii extracts for 24, 48 and 72 h. After these incubation times, the target cells were collected, washed and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before staining. HeLa cells were washed in PBS, resuspended in 500 μl of staining solution (PBS containing RNAse A at a final concentration of 200 μg/ml, and propidium iodide (PI) at a final concentration of 20 μg/ml), and incubated for 30 min at 37°C.
Cell cycle phase distribution was determined using a FACSCalibur Flow Cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data (10,000 events collected for each sample) were analyzed using CELLQuest software (BD Biosciences).
HeLa cells were incubated in the presence of two different concentrations (corresponding to the IC50 and IC90 values determined after 72 h) of the examined Helichrysum zivojinii extracts for 24, 48 and 72 h. After these incubation times, the target cells were collected, washed and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before staining. HeLa cells were washed in PBS, resuspended in 500 μl of staining solution (PBS containing RNAse A at a final concentration of 200 μg/ml, and propidium iodide (PI) at a final concentration of 20 μg/ml), and incubated for 30 min at 37°C.
Cell cycle phase distribution was determined using a FACSCalibur Flow Cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data (10,000 events collected for each sample) were analyzed using CELLQuest software (BD Biosciences).
Determination of target caspases To identify the caspases involved in the apoptotic cell death pathway induced by the investigated extracts, the percentages of HeLa cells pretreated with caspase inhibitors in the subG1 phase were determined. HeLa cells were preincubated for 2 h with specific caspase inhibitors (at a final concentration of 40 μM). These were: Z-DEVD-FMK, a caspase-3 inhibitor, Z-IETD-FMK, a caspase-8 inhibitor and Z-LEHD-FMK, a caspase-9 inhibitor. Caspase inhibitors were purchased from R&D Systems (Minneapolis, USA). Five tested extracts were applied to the HeLa cells at concentrations that corresponded to the IC90 values obtained after 72 h. For each extract, one sample of HeLa cells was not treated with an inhibitor and served as a reference sample. After 24 h of incubation, cells were harvested and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before PI staining. Changes in the percentages of cells in the subG1 phase were determined using a FACSCalibur Flow Cytometer and analyzed using CELLQuest software.
To identify the caspases involved in the apoptotic cell death pathway induced by the investigated extracts, the percentages of HeLa cells pretreated with caspase inhibitors in the subG1 phase were determined. HeLa cells were preincubated for 2 h with specific caspase inhibitors (at a final concentration of 40 μM). These were: Z-DEVD-FMK, a caspase-3 inhibitor, Z-IETD-FMK, a caspase-8 inhibitor and Z-LEHD-FMK, a caspase-9 inhibitor. Caspase inhibitors were purchased from R&D Systems (Minneapolis, USA). Five tested extracts were applied to the HeLa cells at concentrations that corresponded to the IC90 values obtained after 72 h. For each extract, one sample of HeLa cells was not treated with an inhibitor and served as a reference sample. After 24 h of incubation, cells were harvested and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before PI staining. Changes in the percentages of cells in the subG1 phase were determined using a FACSCalibur Flow Cytometer and analyzed using CELLQuest software.
Plant extracts:
The plant material was collected at Tomoros (ca. 1700 altitude), mountain Galičica (Macedonia) during the flowering (17 July 2010) and identified by Vlado Matevski, Institute of Biology, Faculty of Natural Sciences and Mathematics,
http://Ss. Cyril and Methodius University of Skopje, where the voucher specimen is deposited at Macedonian National Herbarium (MKNH) under the number MKNH121335.
Air-dried and powdered aerial parts of Helichrysum zivojinii (330 g) were extracted twice with n-hexane in an ultrasonic bath for 45 min. The combined extracts were concentrated in a vacuum to obtain a hexane extract (4.2 g). The plant material was successively extracted in the same manner with solvents of rising polarity to obtain a dichloromethane extract (1.4 g), an ethyl-acetate extract (0.7 g), a n-butanol extract (5.4 g) and finally a methanol extract (12.4 g).
Stock solutions of the investigated extracts were made in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml.
Instrumentation and chromatographic conditions:
1HNMR spectra were recorded with Varian Gemini 200 in CDCl3 and DMSO-d6 with TMS as an internal standard. HPLC-MS analysis was performed with an Agilent 1100 Series chromatography system equipped with a binary pump, degasser, autosampler, column Li Chrospher 100 RP 18 (250 × 4,0 mm i.d. 5 μm), and DAD detector in combination with 6210 Time of Flight MS (Agilent Technologies). The mobile phase consisted of 0.2% formic acid in water (solvent A) and 100% acetonitrile (solvent B) with the following gradient elution: 0–5 min 10–20% B, 5–10 min 20% B, 10–20 min 20–30% B, 20–30 min 30–70% B, 30–35 min 70–100% B, 35–40 min 70% B, 40–41 min 100–10% B, 41–45 min 10% B, at a flow rate of 1 ml/min. The injection volume was 10 μL, the column temperature was 25°C. The effluent was monitored with DAD (190–550 nm) and a mass detector (ESI) which operated in negative mode at atmospheric pressure; the mass range was from m/z 100–2500, with the following ESI parameters: capillary voltage: 4000 V; gas temperature: 350°C; nebulizer pressure: 45 psig; fragmentor voltage: 140 V. Mass Hunter Workstation software was used for data analysis.
Cell culture:
Human cervix adenocarcinoma HeLa, human melanoma Fem-x and human breast adenocarcinoma MDA-MB-361 cells were cultured as monolayers. Human chronic myelogenous leukemia K562 cells were grown in a suspension in nutrient medium. Cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The complete nutrient medium was RPMI 1640 supplemented with 3 mM L-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, 10% heat-inactivated (56°C) fetal bovine serum and 25 mM Hepes adjusted to pH 7.2 with a bicarbonate solution. The cells were grown at 37°C in an atmosphere of 5% CO2 and humidified air. RPMI 1640, L-glutamine and Hepes were obtained from PAA (Pasching, Austria).
Preparation of peripheral blood mononuclear cells:
Peripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of two healthy volunteers by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl- and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in nutrient medium. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia. Written informed consent was obtained from each healthy donor.
Treatment of cancer cell lines:
HeLa (2,000 cells per well), Fem-x (2,000 cells per well), MDA-MB-361 (10,000 cells per well) were seeded into 96-well microtiter plates and 20 h later, after cell adherence, five different concentrations of the tested extracts were added to the wells. Nutrient medium was only added to the cells in the control wells. K562 cells (5,000 cells per well) were seeded 2 h before addition of the extracts. Stock solutions of plant extracts were diluted with complete nutrient medium and applied to target cells at different final concentrations that ranged from 6.25 μg/ml to 100 μg/ml for extracts 1–4, and from 12.5 μg/ml to 150 μg/ml or 200 μg/ml for extract 5. All experiments were done in triplicate. Cisplatin was used as a positive control.
Treatment of PBMC:
PBMC (150,000 cells per well) were seeded into nutrient medium or in nutrient medium enriched with (5 μg/ml) (PHA) in 96-well microtiter plates. After 2 h, five different concentrations of the plant extracts were added to the individual wells, in triplicate, except to the control wells where a nutrient medium only was added to the cells. The final concentrations of the tested extracts ranged from 12.5 μg/ml to 200 μg/ml. PHA was obtained from INEP (Belgrade, Serbia). Cisplatin was used as a positive control.
Determination of target cell survival:
Cell survival was determined by the MTT test according to the method of Mosmann
[21] and modified by Ohno and Abe
[22]. Briefly, after the treatment with plant extracts for 72 h, 10 μl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 μl of 10% SDS. Absorbance at 570 nm was measured the next day.
To quantify cell survival (S%), the absorbance of a sample with cells grown in the presence of different concentrations of the investigated agents was divided by the absorbance of the control cells grown only in the nutrient medium, and multiplied by 100. It is implied that the absorbance of the blank was always subtracted from the absorbance of the corresponding sample with target cells. The IC50 was defined as the concentration of the agent that inhibited cell survival by 50%, compared to the vehicle-treated control.
Morphological evaluation of HeLa cell death:
To evaluate whether the extracts from the endemic plant Helichrysum zivojinii induce apoptosis in HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide-stained target cells was performed. HeLa cells were seeded overnight on coverslips (100,000 cells) in 2 ml of complete medium. The next day, cells were treated with plant extracts for 24 h at concentrations corresponding to IC90 values that were obtained after treatments that lasted 72 h. After this period, the target cells were stained with 18 μl of a mixture of the DNA dyes acridine orange and ethidium bromide (3 μg/ml AO and 10 μg/ml EB in PBS), and visualized under a fluorescence microscope using a fluorescein isothiocyanate (FITC) filter set.
Cell cycle analysis:
HeLa cells were incubated in the presence of two different concentrations (corresponding to the IC50 and IC90 values determined after 72 h) of the examined Helichrysum zivojinii extracts for 24, 48 and 72 h. After these incubation times, the target cells were collected, washed and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before staining. HeLa cells were washed in PBS, resuspended in 500 μl of staining solution (PBS containing RNAse A at a final concentration of 200 μg/ml, and propidium iodide (PI) at a final concentration of 20 μg/ml), and incubated for 30 min at 37°C.
Cell cycle phase distribution was determined using a FACSCalibur Flow Cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data (10,000 events collected for each sample) were analyzed using CELLQuest software (BD Biosciences).
Determination of target caspases:
To identify the caspases involved in the apoptotic cell death pathway induced by the investigated extracts, the percentages of HeLa cells pretreated with caspase inhibitors in the subG1 phase were determined. HeLa cells were preincubated for 2 h with specific caspase inhibitors (at a final concentration of 40 μM). These were: Z-DEVD-FMK, a caspase-3 inhibitor, Z-IETD-FMK, a caspase-8 inhibitor and Z-LEHD-FMK, a caspase-9 inhibitor. Caspase inhibitors were purchased from R&D Systems (Minneapolis, USA). Five tested extracts were applied to the HeLa cells at concentrations that corresponded to the IC90 values obtained after 72 h. For each extract, one sample of HeLa cells was not treated with an inhibitor and served as a reference sample. After 24 h of incubation, cells were harvested and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before PI staining. Changes in the percentages of cells in the subG1 phase were determined using a FACSCalibur Flow Cytometer and analyzed using CELLQuest software.
Results:
Chemical analysis of plant extracts The hexane (1) and dichloromethane extracts (2) presented similar 1H NMR spectra (recorded in CDCl3). The signals in the 1H NMR spectra of both extracts pointed to a complex mixture with prevailing relatively non-polar substances (region δ 0.8–2.2). LC/DAD analysis revealed the presence of a flavonoid with the molecular formula C18H16O7 (344) in the hexane extract, while the flavanone naringenin and flavone apigenin were confirmed in the dichloromethane extract, also by LC/DAD analysis. Three compounds found in both extracts are presented in Table
1, with their presumed molecular formulae and measured m/z values corresponding to identified ions obtained by ESI ToF mass spectrometry. According to LC/DAD analysis, these compounds unfortunately did not absorb in the UV spectrum, suggesting the presence of structures without a chromophore. The ethyl-acetate extract (3) and n-butanol extracts (4) presented 1H NMR spectra (recorded in DMSO-d6) with approximately identical groups of signals in the regions δ 12.5–13.9 (hydroxy protons), δ 5.9-8.1 (protons on the aromatic ring that is substituted with one or more hydroxy groups), and δ 4.7–5.5 (group of protons corresponding to a sugar unit). The 1H NMR spectrum of the most abundant methanol extract (5), also recorded in DMSO-d6, resembles the spectra of the ethyl-acetate and n-butanol extracts by the presence of signals in the region δ 5.9–8.1 (aromatic protons), as well as by the presence of a signal at δ 13.9 (hydroxy proton); it differs from the last two spectra by possessing a more pronounced region at δ 4.7–5.5, which is responsible for the protons of sugar units. LC/DAD analysis and ESI ToF mass spectrometry of these three more polar extracts (3, 4 and 5) revealed quite similar constituents in each of them. Apart from chlorogenic acid, two groups of flavonoid compounds were detected: flavonoid O-glycosides, and flavonoid aglycons. Among the O-glycosides, the glucoside (or possibly galactoside) of quercetin, the glucoside of apigenin, the glucoside of kaempferol (or possibly luteolin), and another glucoside of kaempferol (possibly luteolin or 6-hydroxyapigenin), were present. Flavonoid aglycones, such as apigenin and naringenin, were detected. Phthalic acid was found in extracts 2–5.
Components of five
Helichrysum zivojinii
extracts
aMore abundant on the expense of other fraction constituents.
The hexane (1) and dichloromethane extracts (2) presented similar 1H NMR spectra (recorded in CDCl3). The signals in the 1H NMR spectra of both extracts pointed to a complex mixture with prevailing relatively non-polar substances (region δ 0.8–2.2). LC/DAD analysis revealed the presence of a flavonoid with the molecular formula C18H16O7 (344) in the hexane extract, while the flavanone naringenin and flavone apigenin were confirmed in the dichloromethane extract, also by LC/DAD analysis. Three compounds found in both extracts are presented in Table
1, with their presumed molecular formulae and measured m/z values corresponding to identified ions obtained by ESI ToF mass spectrometry. According to LC/DAD analysis, these compounds unfortunately did not absorb in the UV spectrum, suggesting the presence of structures without a chromophore. The ethyl-acetate extract (3) and n-butanol extracts (4) presented 1H NMR spectra (recorded in DMSO-d6) with approximately identical groups of signals in the regions δ 12.5–13.9 (hydroxy protons), δ 5.9-8.1 (protons on the aromatic ring that is substituted with one or more hydroxy groups), and δ 4.7–5.5 (group of protons corresponding to a sugar unit). The 1H NMR spectrum of the most abundant methanol extract (5), also recorded in DMSO-d6, resembles the spectra of the ethyl-acetate and n-butanol extracts by the presence of signals in the region δ 5.9–8.1 (aromatic protons), as well as by the presence of a signal at δ 13.9 (hydroxy proton); it differs from the last two spectra by possessing a more pronounced region at δ 4.7–5.5, which is responsible for the protons of sugar units. LC/DAD analysis and ESI ToF mass spectrometry of these three more polar extracts (3, 4 and 5) revealed quite similar constituents in each of them. Apart from chlorogenic acid, two groups of flavonoid compounds were detected: flavonoid O-glycosides, and flavonoid aglycons. Among the O-glycosides, the glucoside (or possibly galactoside) of quercetin, the glucoside of apigenin, the glucoside of kaempferol (or possibly luteolin), and another glucoside of kaempferol (possibly luteolin or 6-hydroxyapigenin), were present. Flavonoid aglycones, such as apigenin and naringenin, were detected. Phthalic acid was found in extracts 2–5.
Components of five
Helichrysum zivojinii
extracts
aMore abundant on the expense of other fraction constituents.
In vitro cytotoxic activity The cytotoxicity of the five isolated extracts was tested against selected cancer cell lines: human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562 and human breast adenocarcinoma MDA-MB-361 cells. All investigated extracts exerted selective dose-dependent cytotoxic actions on malignant cells. The decrease in survival of target cancer cells induced by the five Helichrysum zivojinii extracts is shown in Figure
1 and Table
2.
Survival of HeLa (A), Fem-x (B), K562 (C) and MDA-MB-361 cells (D) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
Concentrations of five
Helichrysum zivojinii
extracts, which induced 50% decrease in the target cancer cell survival, determined by MTT test
Time of continuous agent’s action was 72 h.
In general, extracts 1 and 2 (as well as cisplatin, which served as a positive control) exhibited the highest cytotoxic actions against target malignant cell lines; extracts 3 and 4 displayed less pronounced cytotoxicity; extract 5 had the lowest cytotoxic action.
With regard to the specific sensitivities of the different cells to the cytotoxic activities of the extracts, it is important to note that K562 cells were the most sensitive to the cytotoxic actions of extracts 1 and 3. HeLa and Fem-x cells exhibited a lower sensitivity, while the sensitivity of breast cancer MDA-MB-361 cells to the toxic actions of the tested extracts was the lowest (being several times lower than that of the other cell lines to extracts 1, 2 and 3 especially).
Considering the possible effects of applied antitumor drugs on normal healthy immunocompetent cells, components of the antitumor immune response, their viability is significant for tumor control. For that reason, the activities of the investigated Helichrysum zivojinii extracts were evaluated against healthy unstimulated and PHA-stimulated PBMC (Figure
2 and Table
3). It should be noted that these extracts overall exhibited weaker cytotoxic effects against unstimulated PBMC than against stimulated PBMC. Moreover, extracts 2 and 3 exerted a more pronounced cytotoxicity against unstimulated and PHA-stimulated PBMC than extracts 1, 4 and 5.
Survival of resting PBMC (A) and PHA-stimulated PBMC (B) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
Concentrations of five
Helichrysum zivojinii
extracts, which induced 50% decrease in target PBMC survival, determined by MTT test
Time of continuous agent’s action was 72 h.
In order to further evaluate the anticancer potential of the extracts, the selectivity in the antitumor action against specific malignant cell line in comparison to healthy PBMC was determined as well. These data are presented in Table
4 from which it can be observed that extract 1 exhibited highly selective antitumor action, especially against K562 cells. Extract 2 also displayed good selectivity in its antitumor action.
Selectivity in the antitumor action of five
Helichrysum zivojinii
extracts
The cytotoxicity of the five isolated extracts was tested against selected cancer cell lines: human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562 and human breast adenocarcinoma MDA-MB-361 cells. All investigated extracts exerted selective dose-dependent cytotoxic actions on malignant cells. The decrease in survival of target cancer cells induced by the five Helichrysum zivojinii extracts is shown in Figure
1 and Table
2.
Survival of HeLa (A), Fem-x (B), K562 (C) and MDA-MB-361 cells (D) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
Concentrations of five
Helichrysum zivojinii
extracts, which induced 50% decrease in the target cancer cell survival, determined by MTT test
Time of continuous agent’s action was 72 h.
In general, extracts 1 and 2 (as well as cisplatin, which served as a positive control) exhibited the highest cytotoxic actions against target malignant cell lines; extracts 3 and 4 displayed less pronounced cytotoxicity; extract 5 had the lowest cytotoxic action.
With regard to the specific sensitivities of the different cells to the cytotoxic activities of the extracts, it is important to note that K562 cells were the most sensitive to the cytotoxic actions of extracts 1 and 3. HeLa and Fem-x cells exhibited a lower sensitivity, while the sensitivity of breast cancer MDA-MB-361 cells to the toxic actions of the tested extracts was the lowest (being several times lower than that of the other cell lines to extracts 1, 2 and 3 especially).
Considering the possible effects of applied antitumor drugs on normal healthy immunocompetent cells, components of the antitumor immune response, their viability is significant for tumor control. For that reason, the activities of the investigated Helichrysum zivojinii extracts were evaluated against healthy unstimulated and PHA-stimulated PBMC (Figure
2 and Table
3). It should be noted that these extracts overall exhibited weaker cytotoxic effects against unstimulated PBMC than against stimulated PBMC. Moreover, extracts 2 and 3 exerted a more pronounced cytotoxicity against unstimulated and PHA-stimulated PBMC than extracts 1, 4 and 5.
Survival of resting PBMC (A) and PHA-stimulated PBMC (B) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
Concentrations of five
Helichrysum zivojinii
extracts, which induced 50% decrease in target PBMC survival, determined by MTT test
Time of continuous agent’s action was 72 h.
In order to further evaluate the anticancer potential of the extracts, the selectivity in the antitumor action against specific malignant cell line in comparison to healthy PBMC was determined as well. These data are presented in Table
4 from which it can be observed that extract 1 exhibited highly selective antitumor action, especially against K562 cells. Extract 2 also displayed good selectivity in its antitumor action.
Selectivity in the antitumor action of five
Helichrysum zivojinii
extracts
Morphological analysis of HeLa cell death mode In order to determine whether the investigated plant extracts have pro-apoptotic activities, we performed morphological analysis by fluorescent microscopy of acridine orange/ethidium bromide-stained HeLa cells, exposed to the extracts. Microscopic examination revealed that all five extracts applied at IC90 concentrations induced apoptosis in target HeLa cells after 24 h treatment (Figure
3). The morphological characteristics of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange-red stained cells at late stages of apoptosis or secondary necrosis (the latter was observed in extract 1-treated HeLa cells) and apoptotic bodies. These analyses confirmed that the cytotoxicity of the Helichrysum zivojinii extracts is based on their prominent pro-apoptotic effects.
Photomicrographs of acridine orange/ethidium bromide-stained control HeLa cells (A), and HeLa cells treated with different
Helichrysum zivojinii
extracts for 24 h (extracts 1–5, photomicrographs B-F, consecutively).
In order to determine whether the investigated plant extracts have pro-apoptotic activities, we performed morphological analysis by fluorescent microscopy of acridine orange/ethidium bromide-stained HeLa cells, exposed to the extracts. Microscopic examination revealed that all five extracts applied at IC90 concentrations induced apoptosis in target HeLa cells after 24 h treatment (Figure
3). The morphological characteristics of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange-red stained cells at late stages of apoptosis or secondary necrosis (the latter was observed in extract 1-treated HeLa cells) and apoptotic bodies. These analyses confirmed that the cytotoxicity of the Helichrysum zivojinii extracts is based on their prominent pro-apoptotic effects.
Photomicrographs of acridine orange/ethidium bromide-stained control HeLa cells (A), and HeLa cells treated with different
Helichrysum zivojinii
extracts for 24 h (extracts 1–5, photomicrographs B-F, consecutively).
Analysis of changes in cell cycle phase distribution Examination of changes in the cell cycle phase distribution of HeLa cells treated with these extracts for 24, 48 and 72 h was done to elucidate the mechanisms of the observed cytotoxic actions (Figure
4). Results from this analysis showed a time - dependent increase in the percentages of HeLa cells in the subG1 phase after exposure to an IC50 concentration of all of the tested extracts. Additionally, exposure to extracts at IC90 concentrations induced significant increases in the percentages of cells in the subG1 phase 24 h after exposure. It should be mentioned that the investigated extracts induced a slight accumulation of HeLa cells in the S phase after 72 h. Examination of the cell cycle changes that were induced after exposure for 72 h to IC90 for each extract was not performed because at this time point and at this extract concentration low numbers of mostly dead or dying cells were present in the sample.
Changes in the cell cycle phase distribution of HeLa cells induced by the Helichrysum zivojinii extracts after 24 (A,B), 48 (C,D) and 72 h (E) treatment (applied concentrations of tested extracts corresponded to IC50 and IC90 values determined for 72 h). (C- control HeLa cells, 1 – 5 – corresponding extracts are numbered consecutively). Representative graphs are shown.
Since the Helichrysum zivojinii extracts exhibited the pro-apoptotic activities against cervix adenocarcinoma HeLa cells, the identification of target caspases involved in the apoptotic pathway was performed. The presence of the specific caspase inhibitors (caspase-3 inhibitor, caspase-8 inhibitor or caspase-9 inhibitor) significantly reduced the percentages of apoptotic subG1 HeLa cells treated with each of the five plant extracts, as shown in Figure
5. The effect of the caspase-3 inhibitor on HeLa cells treated with extracts is shown in Figure
6. It can be seen that there is an increase in rounded, but attached and live HeLa cells treated with the caspase-3 inhibitor before the addition of the extracts in relation to target HeLa cells only exposed to the tested extracts.
Effects of specific caspase inhibitors on the percentages of apoptotic subG1 HeLa cells treated with Helichrysum zivojinii extracts for 24 h (A - extract 1, B - extract 2, C - extract 3, D - extract 4, E - extract 5). (Z-DEVD-FMK - caspase-3 inhibitor; Z-IETD-FMK -caspase-8 inhibitor; Z-LEHD-FMK - caspase-9 inhibitor).
Effects of pretreatment of HeLa cells with caspase-3 inhibitor (Z-DEVD-FMK), exposed to Helichrysum zivojinii extracts (applied concentrations of tested extracts corresponded to IC90 values determined for 72 h). A – control; B – Extract 1, C – Extract 1 + Z-DEVD-FMK; D – Extract 2, E – Extract 2 + Z-DEVD-FMK; F – Extract 3, G – Extract 3 + Z-DEVD-FMK; H – Extract 4, I – Extract 4 + Z-DEVD-FMK; J – Extract 5, K – Extract 5 + Z-DEVD-FMK.
Examination of changes in the cell cycle phase distribution of HeLa cells treated with these extracts for 24, 48 and 72 h was done to elucidate the mechanisms of the observed cytotoxic actions (Figure
4). Results from this analysis showed a time - dependent increase in the percentages of HeLa cells in the subG1 phase after exposure to an IC50 concentration of all of the tested extracts. Additionally, exposure to extracts at IC90 concentrations induced significant increases in the percentages of cells in the subG1 phase 24 h after exposure. It should be mentioned that the investigated extracts induced a slight accumulation of HeLa cells in the S phase after 72 h. Examination of the cell cycle changes that were induced after exposure for 72 h to IC90 for each extract was not performed because at this time point and at this extract concentration low numbers of mostly dead or dying cells were present in the sample.
Changes in the cell cycle phase distribution of HeLa cells induced by the Helichrysum zivojinii extracts after 24 (A,B), 48 (C,D) and 72 h (E) treatment (applied concentrations of tested extracts corresponded to IC50 and IC90 values determined for 72 h). (C- control HeLa cells, 1 – 5 – corresponding extracts are numbered consecutively). Representative graphs are shown.
Since the Helichrysum zivojinii extracts exhibited the pro-apoptotic activities against cervix adenocarcinoma HeLa cells, the identification of target caspases involved in the apoptotic pathway was performed. The presence of the specific caspase inhibitors (caspase-3 inhibitor, caspase-8 inhibitor or caspase-9 inhibitor) significantly reduced the percentages of apoptotic subG1 HeLa cells treated with each of the five plant extracts, as shown in Figure
5. The effect of the caspase-3 inhibitor on HeLa cells treated with extracts is shown in Figure
6. It can be seen that there is an increase in rounded, but attached and live HeLa cells treated with the caspase-3 inhibitor before the addition of the extracts in relation to target HeLa cells only exposed to the tested extracts.
Effects of specific caspase inhibitors on the percentages of apoptotic subG1 HeLa cells treated with Helichrysum zivojinii extracts for 24 h (A - extract 1, B - extract 2, C - extract 3, D - extract 4, E - extract 5). (Z-DEVD-FMK - caspase-3 inhibitor; Z-IETD-FMK -caspase-8 inhibitor; Z-LEHD-FMK - caspase-9 inhibitor).
Effects of pretreatment of HeLa cells with caspase-3 inhibitor (Z-DEVD-FMK), exposed to Helichrysum zivojinii extracts (applied concentrations of tested extracts corresponded to IC90 values determined for 72 h). A – control; B – Extract 1, C – Extract 1 + Z-DEVD-FMK; D – Extract 2, E – Extract 2 + Z-DEVD-FMK; F – Extract 3, G – Extract 3 + Z-DEVD-FMK; H – Extract 4, I – Extract 4 + Z-DEVD-FMK; J – Extract 5, K – Extract 5 + Z-DEVD-FMK.
Chemical analysis of plant extracts:
The hexane (1) and dichloromethane extracts (2) presented similar 1H NMR spectra (recorded in CDCl3). The signals in the 1H NMR spectra of both extracts pointed to a complex mixture with prevailing relatively non-polar substances (region δ 0.8–2.2). LC/DAD analysis revealed the presence of a flavonoid with the molecular formula C18H16O7 (344) in the hexane extract, while the flavanone naringenin and flavone apigenin were confirmed in the dichloromethane extract, also by LC/DAD analysis. Three compounds found in both extracts are presented in Table
1, with their presumed molecular formulae and measured m/z values corresponding to identified ions obtained by ESI ToF mass spectrometry. According to LC/DAD analysis, these compounds unfortunately did not absorb in the UV spectrum, suggesting the presence of structures without a chromophore. The ethyl-acetate extract (3) and n-butanol extracts (4) presented 1H NMR spectra (recorded in DMSO-d6) with approximately identical groups of signals in the regions δ 12.5–13.9 (hydroxy protons), δ 5.9-8.1 (protons on the aromatic ring that is substituted with one or more hydroxy groups), and δ 4.7–5.5 (group of protons corresponding to a sugar unit). The 1H NMR spectrum of the most abundant methanol extract (5), also recorded in DMSO-d6, resembles the spectra of the ethyl-acetate and n-butanol extracts by the presence of signals in the region δ 5.9–8.1 (aromatic protons), as well as by the presence of a signal at δ 13.9 (hydroxy proton); it differs from the last two spectra by possessing a more pronounced region at δ 4.7–5.5, which is responsible for the protons of sugar units. LC/DAD analysis and ESI ToF mass spectrometry of these three more polar extracts (3, 4 and 5) revealed quite similar constituents in each of them. Apart from chlorogenic acid, two groups of flavonoid compounds were detected: flavonoid O-glycosides, and flavonoid aglycons. Among the O-glycosides, the glucoside (or possibly galactoside) of quercetin, the glucoside of apigenin, the glucoside of kaempferol (or possibly luteolin), and another glucoside of kaempferol (possibly luteolin or 6-hydroxyapigenin), were present. Flavonoid aglycones, such as apigenin and naringenin, were detected. Phthalic acid was found in extracts 2–5.
Components of five
Helichrysum zivojinii
extracts
aMore abundant on the expense of other fraction constituents.
In vitro cytotoxic activity:
The cytotoxicity of the five isolated extracts was tested against selected cancer cell lines: human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562 and human breast adenocarcinoma MDA-MB-361 cells. All investigated extracts exerted selective dose-dependent cytotoxic actions on malignant cells. The decrease in survival of target cancer cells induced by the five Helichrysum zivojinii extracts is shown in Figure
1 and Table
2.
Survival of HeLa (A), Fem-x (B), K562 (C) and MDA-MB-361 cells (D) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
Concentrations of five
Helichrysum zivojinii
extracts, which induced 50% decrease in the target cancer cell survival, determined by MTT test
Time of continuous agent’s action was 72 h.
In general, extracts 1 and 2 (as well as cisplatin, which served as a positive control) exhibited the highest cytotoxic actions against target malignant cell lines; extracts 3 and 4 displayed less pronounced cytotoxicity; extract 5 had the lowest cytotoxic action.
With regard to the specific sensitivities of the different cells to the cytotoxic activities of the extracts, it is important to note that K562 cells were the most sensitive to the cytotoxic actions of extracts 1 and 3. HeLa and Fem-x cells exhibited a lower sensitivity, while the sensitivity of breast cancer MDA-MB-361 cells to the toxic actions of the tested extracts was the lowest (being several times lower than that of the other cell lines to extracts 1, 2 and 3 especially).
Considering the possible effects of applied antitumor drugs on normal healthy immunocompetent cells, components of the antitumor immune response, their viability is significant for tumor control. For that reason, the activities of the investigated Helichrysum zivojinii extracts were evaluated against healthy unstimulated and PHA-stimulated PBMC (Figure
2 and Table
3). It should be noted that these extracts overall exhibited weaker cytotoxic effects against unstimulated PBMC than against stimulated PBMC. Moreover, extracts 2 and 3 exerted a more pronounced cytotoxicity against unstimulated and PHA-stimulated PBMC than extracts 1, 4 and 5.
Survival of resting PBMC (A) and PHA-stimulated PBMC (B) grown for 72 h in the presence of increasing concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
Concentrations of five
Helichrysum zivojinii
extracts, which induced 50% decrease in target PBMC survival, determined by MTT test
Time of continuous agent’s action was 72 h.
In order to further evaluate the anticancer potential of the extracts, the selectivity in the antitumor action against specific malignant cell line in comparison to healthy PBMC was determined as well. These data are presented in Table
4 from which it can be observed that extract 1 exhibited highly selective antitumor action, especially against K562 cells. Extract 2 also displayed good selectivity in its antitumor action.
Selectivity in the antitumor action of five
Helichrysum zivojinii
extracts
Morphological analysis of HeLa cell death mode:
In order to determine whether the investigated plant extracts have pro-apoptotic activities, we performed morphological analysis by fluorescent microscopy of acridine orange/ethidium bromide-stained HeLa cells, exposed to the extracts. Microscopic examination revealed that all five extracts applied at IC90 concentrations induced apoptosis in target HeLa cells after 24 h treatment (Figure
3). The morphological characteristics of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange-red stained cells at late stages of apoptosis or secondary necrosis (the latter was observed in extract 1-treated HeLa cells) and apoptotic bodies. These analyses confirmed that the cytotoxicity of the Helichrysum zivojinii extracts is based on their prominent pro-apoptotic effects.
Photomicrographs of acridine orange/ethidium bromide-stained control HeLa cells (A), and HeLa cells treated with different
Helichrysum zivojinii
extracts for 24 h (extracts 1–5, photomicrographs B-F, consecutively).
Analysis of changes in cell cycle phase distribution:
Examination of changes in the cell cycle phase distribution of HeLa cells treated with these extracts for 24, 48 and 72 h was done to elucidate the mechanisms of the observed cytotoxic actions (Figure
4). Results from this analysis showed a time - dependent increase in the percentages of HeLa cells in the subG1 phase after exposure to an IC50 concentration of all of the tested extracts. Additionally, exposure to extracts at IC90 concentrations induced significant increases in the percentages of cells in the subG1 phase 24 h after exposure. It should be mentioned that the investigated extracts induced a slight accumulation of HeLa cells in the S phase after 72 h. Examination of the cell cycle changes that were induced after exposure for 72 h to IC90 for each extract was not performed because at this time point and at this extract concentration low numbers of mostly dead or dying cells were present in the sample.
Changes in the cell cycle phase distribution of HeLa cells induced by the Helichrysum zivojinii extracts after 24 (A,B), 48 (C,D) and 72 h (E) treatment (applied concentrations of tested extracts corresponded to IC50 and IC90 values determined for 72 h). (C- control HeLa cells, 1 – 5 – corresponding extracts are numbered consecutively). Representative graphs are shown.
Since the Helichrysum zivojinii extracts exhibited the pro-apoptotic activities against cervix adenocarcinoma HeLa cells, the identification of target caspases involved in the apoptotic pathway was performed. The presence of the specific caspase inhibitors (caspase-3 inhibitor, caspase-8 inhibitor or caspase-9 inhibitor) significantly reduced the percentages of apoptotic subG1 HeLa cells treated with each of the five plant extracts, as shown in Figure
5. The effect of the caspase-3 inhibitor on HeLa cells treated with extracts is shown in Figure
6. It can be seen that there is an increase in rounded, but attached and live HeLa cells treated with the caspase-3 inhibitor before the addition of the extracts in relation to target HeLa cells only exposed to the tested extracts.
Effects of specific caspase inhibitors on the percentages of apoptotic subG1 HeLa cells treated with Helichrysum zivojinii extracts for 24 h (A - extract 1, B - extract 2, C - extract 3, D - extract 4, E - extract 5). (Z-DEVD-FMK - caspase-3 inhibitor; Z-IETD-FMK -caspase-8 inhibitor; Z-LEHD-FMK - caspase-9 inhibitor).
Effects of pretreatment of HeLa cells with caspase-3 inhibitor (Z-DEVD-FMK), exposed to Helichrysum zivojinii extracts (applied concentrations of tested extracts corresponded to IC90 values determined for 72 h). A – control; B – Extract 1, C – Extract 1 + Z-DEVD-FMK; D – Extract 2, E – Extract 2 + Z-DEVD-FMK; F – Extract 3, G – Extract 3 + Z-DEVD-FMK; H – Extract 4, I – Extract 4 + Z-DEVD-FMK; J – Extract 5, K – Extract 5 + Z-DEVD-FMK.
Discussion:
The plant kingdom provides a rich source of compounds with promising cancer chemopreventive and cancer therapeutic potential. The main drugs currently used in clinical practice in the treatment of malignant diseases originate from plants: vinca alkaloids, taxanes, camptothecins and epipodophyllotoxins
[23]. Over the past years, the focus of modern anticancer drug discovery has been on a wide variety of natural compounds, especially on phenolic compounds. Phytochemicals have been reported to affect different intracellular signaling pathways implicated in the initiation, promotion and progression of cancer. The antitumor effects of plant constituents have been associated with the induction of carcinogen detoxifying enzymes, the scavenging of free radicals, anti-inflammatory activity, cell cycle arrest, the triggering of apoptosis, inhibition of tumor angiogenesis and invasiveness
[1-4].
The antioxidant and anti-inflammatory activities of extracts and isolated compounds from plants belonging to the large genus Helichrysum have been well documented
[9,11,15,18]. Arzanol, a phloroglucinyl α-pyrone, is a constituent of Helichrysum italicum that has been reported to inhibit the NF-κB transcription factor, cyclooxygenase and lipooxygenase, as well as the release of proinflammatory cytokines
[11,18]. Regarding the link between inflammation and cancer, chemicals with anti-inflammatory properties targeting the molecules of signaling cascades implicated in inflammation and carcinogenesis may be useful as cancer chemopreventive drugs.
On the other hand, data about the potential anticancer activity of extracts and phytochemicals of plants from the genus Helichrysum are scarce. The antiproliferative effect of the ethanol extract from Helichrysum maracandicum towards SENCAR mouse skin transformed cells has been demonstrated
[20]. This extract suppressed the expression of p38 MAP kinase. An examination of the arzanol properties showed that this compound, which was isolated from Helichrysum italicum, did not exert cytotoxic action against monkey VERO cells at concentrations up to 40 μM
[24]. In contrast, another study showed that arzanol at a concentration of 50 μM significantly suppressed the survival of human lung carcinoma A549 cells
[18]. It should be mentioned that methanolic extracts prepared from different Helichrysum species were found to inhibit DNA topoisomerase I
[19]. Moreover, the cytotoxicity of Helichrysum gymnocephalum essential oil towards human breast adenocarcinoma MCF-7 cells has been documented
[17].
The results presented herein demonstrate the selective dose-dependent cytotoxic actions of the five extracts isolated from the endemic plant species Helichrysum zivojinii against target cancer cell lines and against healthy immunocompetent PBMC that have been stimulated to proliferate, while their cytotoxic actions were not as pronounced against unstimulated PBMC. The observed selectivity in the antitumor effects of the extracts against specific malignant cell types could be attributed to the actions of different Helichrysum zivojinii constituents on target molecules of the signal transduction pathways that regulate cell proliferation and apoptosis. Furthermore, each of the investigated extracts exhibited considerably stronger cytotoxicity to HeLa, Fem-x and K562 cells when compared to PBMC, both resting and PHA-stimulated, which points to the cancer specificity of their actions. It is noteworthy that when these extracts were applied at concentrations that were highly cytotoxic to malignant cells, they demonstrated very low toxicity towards healthy immunocompetent PBMC, the key players in immune defenses against tumors. The good selectivity of their antitumor actions highlights the significant anticancer potential of Helichrysum zivojinii extracts. The prominent antitumor properties of these extracts need to be examined further in in vivo studies.
It should be stressed that all of these extracts exhibited weaker cytotoxic effects against unstimulated PBMC in comparison to stimulated PBMC. This finding indicates that the extracts possess the ability to inhibit the proliferation of PHA-stimulated PBMC. Thus, these agents may even suppress certain immune functions, particularly non-specific antigen stimulation. Additionally, the observed lower activities against resting PBMC than against mitogen-stimulated PBMC point to components in pathways regulating cell proliferation as the possible molecular targets of the Helichrysum zivojinii extracts. However, it is very important to note that when extracts 1, 2 and 3 were applied at lower concentrations, they stimulated the proliferation of resting PBMC. This growth stimulation effect of lower concentrations of extracts is in accordance with the well-known effect of very small doses of X rays on enhanced proliferation of irradiated cells
[25]. The observed effects of low concentrations of these extracts on one of the main components of the immune response point to the possibility of their use to enhance immunity. It would be interesting to investigate their action towards different PBMC subpopulations and elucidate the potential mechanisms through which they stimulate proliferation. The possible immunostimulatory effects of extracts at lower concentrations might be explained by a modulation in lymphocyte cytokine production, including IL-2, IFN-γ, as well as IL-4 and IL-6. The immunoregulatory actions of phenolic compounds, such as quercetin, kaempferol and apigenin, have been reported
[26-28]. Considering the presented results, the effects of the extracts on PBMC might be mediated through NF-κB.
Examination of in vitro cytotoxicity revealed that extracts 1 and 2 might be a significant source of novel promising anticancer compounds in view of their pronounced cytotoxic activities against HeLa, Fem-x and especially against K562 cells, as well as their high selectivity in the antitumor actions against cancer cells in comparison to healthy PBMC. Chemical analyses of the Helichrysum zivojinii extracts showed the presence of phenolic compounds whose antitumor potential has already been documented. The bioactive flavone apigenin that was found in extracts 2–5 has been reported to exhibit anticancer activities against different types of malignant cells including breast, cervical, ovarian, prostate, colon, gastric, liver and lung cancers, as well as skin and thyroid cancer, diverse hematological malignancies and neuroblastoma
[28] and references cited therein]. The cytotoxic activities of extracts 2–5 may be at least in part due to the flavonoid naringenin. This flavonoid has been shown to exert cytotoxicity towards various malignant cell lines, such as breast cancer cell lines (MCF-7, MDA-MB-231), cervix adenocarcinoma (HeLa), liver cancer (HepG2, Hep3B, Huh7), pancreas cancer (PK-1), colon cancer (Caco-2), stomach cancer (KATOIII, MKN-7) and leukemia cells (Jurkat, HL-60, U937, NALM-6, THP-1)
[29-31]. Additionally, the cancer-preventive and cancer-suppressive properties of quercetin, whose O-glycosides were identified in extracts 3, 4 and 5, have been documented as well
[32]. The antiproliferative and pro-apoptotic effects of quercetin were shown against the HeLa cell line
[33].
Morphological analysis of the mode of HeLa cell death, together with the cell cycle analysis, showed that the treatment of HeLa cells with higher concentrations of the examined extracts induced apoptotic cell death. To confirm the pro-apoptotic action of the tested Helichrysum zivojinii extracts and to identify the caspases implicated in the employed apoptotic pathways, specific caspase inhibitors were used (Z-DEVD-FMK, Z-IETD-FMK, Z-LEHD-FMK). A prominent decrease in the percentages of subG1 apoptotic HeLa cells after treatments with each of the tested extracts in combination with specific caspase inhibitors compared to the percentages of subG1 cells after treatments with only the corresponding extracts, indicates that each of the five extracts induced apoptosis through the activation of caspase-3, the main effector caspase, as well as through the activation of caspase-8 and caspase-9. We conclude that the constituents of the Helichrysum zivojinii extracts triggered apoptosis in HeLa cells through the intrinsic pathway mediated by caspase-9, and the extrinsic pathway mediated by caspase-8. In addition, the crosstalk between these two apoptotic pathways should also be considered. Due to their ability to promote apoptotic cell death in cancer cells, the investigated extracts and their constituents may have significant anticancer potential. It is worth noting that antitumor drugs that induce apoptosis and thereby suppress the further growth of tumors play important roles in the clinical treatment of malignancies. In addition to the pronounced inhibition of proliferation and survival of target malignant cells, the lower cytotoxicity of Helichrysum zivojinii extracts against healthy PBMC is a promising lead for future studies.
Conclusions:
Data from this in vitro study clearly demonstrate the prominent antitumor potential of five extracts prepared from the endemic plant species Helichrysum zivojinii, which can be attributed to their selective and pronounced antiproliferative and pro-apoptotic actions towards specific malignant cells in comparison to healthy PBMC. Prospective cancer-suppressive effects of the tested extracts should be further evaluated in in vivo experiments.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
IM performed all analyses of the anticancer properties of investigated extracts, interpreted obtained data and wrote the first and last version of the manuscript. IA participated in design of the study, prepared extracts, performed chemical characterization, interpreted data and wrote the part of the manuscript. ŽŽ participated in acquisition and analysis of data. MJ carried out chemical analyses of the extracts. VV and SM participated in design of the study and interpreted obtained data. ZJ designed the research on anticancer properties of tested extracts, interpreted obtained data, participated in writing the manuscript and critically revised the manuscript. All authors have read and approved the final version of the manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1472-6882/13/36/prepub
|
Background:
The aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Černjavski & Soška (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC).
Methods:
The aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors.
Results:
The investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways.
Conclusions:
Extracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells.
|
Background:
Bioactive constituents of medicinal plants are in the center of attention of modern anticancer research due to their prospective roles in suppressing the different stages of malignant transformation. The antitumor potential of plant extracts and compounds could be attributed to their ability to induce changes in the regulation of target molecules in oncogenic signal transduction pathways implicated in cell growth, replication, apoptosis, as well as in angiogenesis, invasion and metastasis of cancer cells
[1-4]. To evaluate the anticancer properties of novel chemotherapeutic agents, the selectivity of their actions against malignant cells in comparison to healthy non-transformed cells, especially immunocompetent cells involved in the immune control of tumor suppression, needs to be carefully examined.
Helichrysum zivojinii Černjavski & Soška is an endemic plant species that grows in the National Park "Galičica" in Macedonia. Some of the plant species from the large genus Helichrysum are used in different regions of the world in traditional medicine for treating wounds, respiratory tract infections and gastro-intestinal disorders
[5-8]. This plant genus is a valuable source of several different secondary metabolites/phytochemicals, such as flavonoids, acetophenones, phloroglucinols, pyrones, diterpenes and sesquiterpenes
[5]. Different morphological groups of Helichrysum species often display unique qualitative and quantitative chemical compositions
[5]. It has been reported that extracts and individual constituents of these plants possess significant biological and pharmacological properties, including antibacterial, antiviral, antifungal, antioxidant, anti-inflammatory and antidiabetic activities
[9-16]. A search through the literature suggests that plants from the genus Helichrysum could be a significant source of compounds with potential anticancer activities
[17-20].
The main goal of this research was to investigate the cytotoxic activities of five extracts isolated as fractions from the endemic plant Helichrysum zivojinii towards selected human malignant cell lines. To assess the sensitivity of normal immunocompetent cells included in the antitumor immune response, the cytotoxicity of these extracts was also tested against human peripheral blood mononuclear cells (PBMC) – both unstimulated and stimulated to proliferate by the mitogen phytohemagglutinin (PHA). To elucidate the molecular mechanisms of the cytotoxic effects of the tested extracts, the distribution of target HeLa cells at specific phases of the cell cycle after the actions of these agents was also analyzed. The mode of HeLa cell death induced by the extracts was also investigated. Elucidation of the signaling pathways implicated in the induction of apoptosis by the tested extracts was conducted by identification of target caspases.
Conclusions:
Data from this in vitro study clearly demonstrate the prominent antitumor potential of five extracts prepared from the endemic plant species Helichrysum zivojinii, which can be attributed to their selective and pronounced antiproliferative and pro-apoptotic actions towards specific malignant cells in comparison to healthy PBMC. Prospective cancer-suppressive effects of the tested extracts should be further evaluated in in vivo experiments.
|
Background:
The aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Černjavski & Soška (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC).
Methods:
The aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors.
Results:
The investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways.
Conclusions:
Extracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells.
| 13,169 | 348 | 22 |
[
"extracts",
"cells",
"hela",
"extract",
"cell",
"hela cells",
"helichrysum",
"zivojinii",
"helichrysum zivojinii",
"caspase"
] |
[
"test",
"test"
] |
[CONTENT]
Helichrysum zivojinii
| Cytotoxicity | Cancer cells | Peripheral blood mononuclear cells | Apoptosis [SUMMARY]
|
[CONTENT]
Helichrysum zivojinii
| Cytotoxicity | Cancer cells | Peripheral blood mononuclear cells | Apoptosis [SUMMARY]
|
[CONTENT]
Helichrysum zivojinii
| Cytotoxicity | Cancer cells | Peripheral blood mononuclear cells | Apoptosis [SUMMARY]
|
[CONTENT]
Helichrysum zivojinii
| Cytotoxicity | Cancer cells | Peripheral blood mononuclear cells | Apoptosis [SUMMARY]
|
[CONTENT]
Helichrysum zivojinii
| Cytotoxicity | Cancer cells | Peripheral blood mononuclear cells | Apoptosis [SUMMARY]
|
[CONTENT]
Helichrysum zivojinii
| Cytotoxicity | Cancer cells | Peripheral blood mononuclear cells | Apoptosis [SUMMARY]
|
[CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Proliferation | Dose-Response Relationship, Drug | Female | HeLa Cells | Helichrysum | Humans | Leukemia, Myeloid | Leukocytes, Mononuclear | Melanoma | Neoplasms | Phytotherapy | Plant Extracts | Signal Transduction | Uterine Cervical Neoplasms [SUMMARY]
|
[CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Proliferation | Dose-Response Relationship, Drug | Female | HeLa Cells | Helichrysum | Humans | Leukemia, Myeloid | Leukocytes, Mononuclear | Melanoma | Neoplasms | Phytotherapy | Plant Extracts | Signal Transduction | Uterine Cervical Neoplasms [SUMMARY]
|
[CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Proliferation | Dose-Response Relationship, Drug | Female | HeLa Cells | Helichrysum | Humans | Leukemia, Myeloid | Leukocytes, Mononuclear | Melanoma | Neoplasms | Phytotherapy | Plant Extracts | Signal Transduction | Uterine Cervical Neoplasms [SUMMARY]
|
[CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Proliferation | Dose-Response Relationship, Drug | Female | HeLa Cells | Helichrysum | Humans | Leukemia, Myeloid | Leukocytes, Mononuclear | Melanoma | Neoplasms | Phytotherapy | Plant Extracts | Signal Transduction | Uterine Cervical Neoplasms [SUMMARY]
|
[CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Proliferation | Dose-Response Relationship, Drug | Female | HeLa Cells | Helichrysum | Humans | Leukemia, Myeloid | Leukocytes, Mononuclear | Melanoma | Neoplasms | Phytotherapy | Plant Extracts | Signal Transduction | Uterine Cervical Neoplasms [SUMMARY]
|
[CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Proliferation | Dose-Response Relationship, Drug | Female | HeLa Cells | Helichrysum | Humans | Leukemia, Myeloid | Leukocytes, Mononuclear | Melanoma | Neoplasms | Phytotherapy | Plant Extracts | Signal Transduction | Uterine Cervical Neoplasms [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] extracts | cells | hela | extract | cell | hela cells | helichrysum | zivojinii | helichrysum zivojinii | caspase [SUMMARY]
|
[CONTENT] extracts | cells | hela | extract | cell | hela cells | helichrysum | zivojinii | helichrysum zivojinii | caspase [SUMMARY]
|
[CONTENT] extracts | cells | hela | extract | cell | hela cells | helichrysum | zivojinii | helichrysum zivojinii | caspase [SUMMARY]
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[CONTENT] extracts | cells | hela | extract | cell | hela cells | helichrysum | zivojinii | helichrysum zivojinii | caspase [SUMMARY]
|
[CONTENT] extracts | cells | hela | extract | cell | hela cells | helichrysum | zivojinii | helichrysum zivojinii | caspase [SUMMARY]
|
[CONTENT] extracts | cells | hela | extract | cell | hela cells | helichrysum | zivojinii | helichrysum zivojinii | caspase [SUMMARY]
|
[CONTENT] cells | plants | genus | extracts | species | helichrysum | plant | anticancer | malignant | different [SUMMARY]
|
[CONTENT] cells | ml | μg | μg ml | min | 10 | 100 | medium | nutrient | nutrient medium [SUMMARY]
|
[CONTENT] extracts | extract | cells | hela | hela cells | extract extract | helichrysum zivojinii extracts | zivojinii extracts | caspase | caspase inhibitor [SUMMARY]
|
[CONTENT] data vitro study | healthy pbmc prospective | pronounced antiproliferative pro apoptotic | pronounced antiproliferative pro | pronounced antiproliferative | zivojinii attributed | zivojinii attributed selective | zivojinii attributed selective pronounced | effects tested extracts evaluated | clearly [SUMMARY]
|
[CONTENT] cells | extracts | hela | extract | hela cells | ml | μg ml | μg | caspase | cell [SUMMARY]
|
[CONTENT] cells | extracts | hela | extract | hela cells | ml | μg ml | μg | caspase | cell [SUMMARY]
|
[CONTENT] five | Helichrysum | Černjavski & Soška | Asteraceae ||| [SUMMARY]
|
[CONTENT] hexane ||| HeLa | K562 | MTT ||| HeLa ||| HeLa ||| [SUMMARY]
|
[CONTENT] Helichrysum ||| HeLa | K562 ||| ||| five | HeLa [SUMMARY]
|
[CONTENT] Helichrysum zivojinii [SUMMARY]
|
[CONTENT] five | Helichrysum | Černjavski & Soška | Asteraceae ||| ||| hexane ||| HeLa | K562 | MTT ||| HeLa ||| HeLa ||| ||| Helichrysum ||| HeLa | K562 ||| ||| five | HeLa ||| Helichrysum zivojinii [SUMMARY]
|
[CONTENT] five | Helichrysum | Černjavski & Soška | Asteraceae ||| ||| hexane ||| HeLa | K562 | MTT ||| HeLa ||| HeLa ||| ||| Helichrysum ||| HeLa | K562 ||| ||| five | HeLa ||| Helichrysum zivojinii [SUMMARY]
|
||||||
A systematic assessment of the current capacity to act in nutrition in West Africa: cross-country similarities and differences.
|
25034256
|
Although it is widely accepted that lack of capacity is one of the barriers to scaling up nutrition in West Africa, there is a paucity of information about what capacities exist and the capacities that need to be developed to accelerate progress toward improved nutrition outcomes in the region.
|
BACKGROUND
|
Data were collected from 13 West African countries through interviews with government officials, key development partners, tertiary-level training institutions, and health professional schools. The assessment was based on a conceptual framework of four interdependent levels (tools; skills; staff and infrastructure; and structures, systems and roles). In each of the surveyed countries, we assessed capacity assets and gaps at individual, organizational, and systemic levels.
|
DESIGN
|
Important similarities and differences in capacity assets and gaps emerged across all the surveyed countries. There was strong momentum to improve nutrition in nearly all the surveyed countries. Most of the countries had a set of policies on nutrition in place and had set up multisectoral, multi-stakeholder platforms to coordinate nutrition activities, although much remained to be done to improve the effectiveness of these platforms. Many initiatives aimed to reduce undernutrition were ongoing in the region, but there did not seem to be clear coordination between them. Insufficient financial resources to implement nutrition activities were a major problem in all countries. The bulk of financial allocations for nutrition was provided by development partners, even though some countries, such as Niger, Nigeria, and Senegal, had a national budget line for nutrition. Sporadic stock-outs of nutrition supplies were reported in most of the countries as a result of a weak logistic and supply chain system. They also had a critical shortage of skilled nutrition professionals. There was limited supervision of nutrition activities, especially at lower levels. Nigeria and Ghana emerged as the countries with the greatest capacities to support the expansion of a nutrition workforce, although a significant proportion of their trained nutritionists were not employed in the nutrition sector. None of the countries had in place a unified nutrition information system that could guide decision-making processes across the different sectors.
|
RESULTS
|
There is an urgent need for a shift toward wider reforms for nutrition capacity development in the West Africa region. Addressing these unmet needs is a critical first step toward improved capacity for action in nutrition in the region.
|
CONCLUSIONS
|
[
"Africa, Western",
"Capacity Building",
"Humans",
"Interviews as Topic",
"Malnutrition",
"Nutritional Sciences",
"Nutritional Status",
"Qualitative Research"
] |
4102835
| null | null |
Methods
|
This qualitative study is part of a larger capacity needs assessment conducted in the West Africa region. The assessment was conducted within the framework of the WANCDI implemented under the auspices of WAHO.
The West Africa region was defined according to the United Nations country classifications (11). It has a total 16 countries comprising Mauritania and the 15 member states of the ECOWAS. The region has a total population of about 340 million people and covers an area of approximately 6.1 million square km (12). The region can be categorized into three major language groups:Anglophone countries (Ghana, Liberia, Nigeria, Sierra Leone, and The Gambia)Francophone countries (Benin, Burkina Faso, Cote d'Ivoire, Guinea, Mali, Mauritania, Niger, Senegal, and Togo)Lusophone countries (Cape Verde and Guinea-Bissau).
Anglophone countries (Ghana, Liberia, Nigeria, Sierra Leone, and The Gambia)
Francophone countries (Benin, Burkina Faso, Cote d'Ivoire, Guinea, Mali, Mauritania, Niger, Senegal, and Togo)
Lusophone countries (Cape Verde and Guinea-Bissau).
Data collection Data were collected in 13 of the 16 West African countries between March and August 2013. We did not conduct field visits in three countries (Cape Verde, Guinea-Bissau, and Gambia) because our preliminary contacts with in-country key informants revealed that tertiary-level nutrition training activities were either non-existent or were not well-developed. Data were collected by three experienced interviewers through self-administered questionnaires or face-to-face interviews during country visits. Data were completed (either manually or electronically) by the interviewers in the presence of respondents during in-person meetings. Completed self-administered questionnaires were emailed to the research team. Interviews were not audio or videotaped, so data transcription was not performed.
Data for this assessment were derived from five main sources:Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.
Interviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).
Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.
Interviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.
Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).
Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.
Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).
Data were collected in 13 of the 16 West African countries between March and August 2013. We did not conduct field visits in three countries (Cape Verde, Guinea-Bissau, and Gambia) because our preliminary contacts with in-country key informants revealed that tertiary-level nutrition training activities were either non-existent or were not well-developed. Data were collected by three experienced interviewers through self-administered questionnaires or face-to-face interviews during country visits. Data were completed (either manually or electronically) by the interviewers in the presence of respondents during in-person meetings. Completed self-administered questionnaires were emailed to the research team. Interviews were not audio or videotaped, so data transcription was not performed.
Data for this assessment were derived from five main sources:Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.
Interviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).
Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.
Interviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.
Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).
Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.
Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).
Data analyses Data were entered into Microsoft Excel 2010. They were analyzed and interpreted using content analysis. We used the analytical framework developed by Potter and Brough (7) to systematically assess the current capacity to act in nutrition at the individual, organizational, and systemic levels (Fig. 1). The framework considers capacity development as a hierarchy of needs, portrayed as a pyramid of four interdependent levels, starting from the ‘easier to achieve’ at the apex of the pyramid (tools, skills, staff, and infrastructure) to the ‘much harder’ to implement at its bottom (systems, structures, and roles). The different levels of the pyramid are further divided into nine interdependent sub-components. The topics covered in each of the surveyed countries, for each sub-component, are highlighted in Table 1.
Capacity building pyramid-Potter and Brough (7).
Capacity development issues covered in each of the surveyed countries
Derived from Potter and Brough (7).
Data were entered into Microsoft Excel 2010. They were analyzed and interpreted using content analysis. We used the analytical framework developed by Potter and Brough (7) to systematically assess the current capacity to act in nutrition at the individual, organizational, and systemic levels (Fig. 1). The framework considers capacity development as a hierarchy of needs, portrayed as a pyramid of four interdependent levels, starting from the ‘easier to achieve’ at the apex of the pyramid (tools, skills, staff, and infrastructure) to the ‘much harder’ to implement at its bottom (systems, structures, and roles). The different levels of the pyramid are further divided into nine interdependent sub-components. The topics covered in each of the surveyed countries, for each sub-component, are highlighted in Table 1.
Capacity building pyramid-Potter and Brough (7).
Capacity development issues covered in each of the surveyed countries
Derived from Potter and Brough (7).
Ethical considerations Administrative authorization was sought by sending a formal correspondence to the head of all of the targeted institutions prior to data collection. We did not seek ethical approval for the study as there was no data collection on human subjects. All respondents were fully informed about the purposes of the study. They gave their full verbal consent before participating in the study.
Administrative authorization was sought by sending a formal correspondence to the head of all of the targeted institutions prior to data collection. We did not seek ethical approval for the study as there was no data collection on human subjects. All respondents were fully informed about the purposes of the study. They gave their full verbal consent before participating in the study.
|
Results
|
In the 13 surveyed countries, semi-structured interviews were conducted with 44 government officials and 51 stakeholders, namely representatives of non-governmental organizations, UN agencies, and donors (Table 2). We identified 305 nutrition degree programs organized by 113 tertiary-level institutions and 127 training programs organized by 52 health professional schools.
Data collection methods and participating institutions
Capacity at individual level (tools and skills) We report the findings relating to logistics, information management, funding, and the quality of nutrition training. In all the 13 surveyed countries, nutrition service providers, in general, had the needed equipment (weighing scales, measuring boards, mid-upper arm circumference tapes, behavior change materials, etc.) and supplies to perform nutrition-related tasks, although they were not available everywhere or at all service delivery points. Equipment and nutrition supplies (especially ready-to-use therapeutic foods) were mainly provided by development partners. Concerted efforts had been made to ensure the availability of these nutrition products at all service delivery points. Sporadic stock-outs of nutrition supplies were reported in most of the countries as a result of a weak logistic and supply chain system, poor forecasting of nutrition supplies at lower levels, and inadequate transportation systems. As of the time of our study, development partners were playing a strong role in the procurement and distribution of these nutrition supplies.
A common challenge for all 13 countries was the scarcity of financial resources for nutrition activities (Table 3). Although some countries, such as Ghana, Mauritania, Nigeria, and Senegal, experienced an increasing trend in the budget allocated to nutrition activities over the past years, there still remained some important financial gaps. At the time of our study, only a few countries in the region (Niger, Nigeria, and Senegal) had succeeded in securing a budgetary line for nutrition activities in their national budgets. Other countries, such as Benin, Burkina Faso, Mauritania, and Ghana, were in the process of doing so. As a result, the bulk of resources allocated for nutrition activities in the region was provided by donors and development partners. In Burkina-Faso, some nutrition activities were financed through a pooled fund portfolio, which contained mostly funds from donors and development partners.
Another common observation was the difficulty in getting accurate information about nutrition financing. The fact that resources come from various sources and cut across many sectors made it difficult to track investments and expenditures on nutrition. In Ghana for example, each government unit carrying out nutrition activities had its own budget that was often embedded in a higher level budgetary category. Efforts were underway in most of the countries to improve clarity in overall nutrition budgeting. In Ghana, a sub-committee on resource mobilization had been established, within the Cross-Sectoral Planning Group (CSPG) of the national SUN movement secretariat, to track resource allocation for nutrition in the country.
Inadequate human resources for nutrition were a major barrier to the implementation of nutrition activities (Table 3). In all the 13 countries, there was a critical shortage of skilled nutrition professionals, especially at lower levels. Nutrition graduates did not have practical experience and were not adequately prepared to tackle real-life nutrition issues. There were indeed important gaps in tertiary-level nutrition training in all the 13 countries. Nutrition training curricula were not aligned to regional nutrition priorities. In addition, there did not seem to be a coordinated approach toward harmonization of nutrition training curricula. Existing nutrition degree programs did not provide a full coverage of all essential aspects of human nutrition. Details about the current capacity for nutrition training in the surveyed countries have been reported elsewhere (13).
In the absence of a critical mass of skilled nutrition professionals, the bulk of nutrition work was done by health workers who did not have a good understanding of the application of nutrition principles to public health or clinical practice. As a result, they did not have broad enough skills to provide quality nutrition services. We noted that some countries were taking steps to enhance the performance of health workers through in-service training and mentoring in nutrition. However, these efforts were mainly oriented toward the management of acute malnutrition. In addition, continuing training efforts were mainly concentrated on staff at national and regional levels, leaving staff at lower levels with inadequate capacities in nutrition.
Similarities and differences in capacity assets and gaps across all the surveyed countries
We report the findings relating to logistics, information management, funding, and the quality of nutrition training. In all the 13 surveyed countries, nutrition service providers, in general, had the needed equipment (weighing scales, measuring boards, mid-upper arm circumference tapes, behavior change materials, etc.) and supplies to perform nutrition-related tasks, although they were not available everywhere or at all service delivery points. Equipment and nutrition supplies (especially ready-to-use therapeutic foods) were mainly provided by development partners. Concerted efforts had been made to ensure the availability of these nutrition products at all service delivery points. Sporadic stock-outs of nutrition supplies were reported in most of the countries as a result of a weak logistic and supply chain system, poor forecasting of nutrition supplies at lower levels, and inadequate transportation systems. As of the time of our study, development partners were playing a strong role in the procurement and distribution of these nutrition supplies.
A common challenge for all 13 countries was the scarcity of financial resources for nutrition activities (Table 3). Although some countries, such as Ghana, Mauritania, Nigeria, and Senegal, experienced an increasing trend in the budget allocated to nutrition activities over the past years, there still remained some important financial gaps. At the time of our study, only a few countries in the region (Niger, Nigeria, and Senegal) had succeeded in securing a budgetary line for nutrition activities in their national budgets. Other countries, such as Benin, Burkina Faso, Mauritania, and Ghana, were in the process of doing so. As a result, the bulk of resources allocated for nutrition activities in the region was provided by donors and development partners. In Burkina-Faso, some nutrition activities were financed through a pooled fund portfolio, which contained mostly funds from donors and development partners.
Another common observation was the difficulty in getting accurate information about nutrition financing. The fact that resources come from various sources and cut across many sectors made it difficult to track investments and expenditures on nutrition. In Ghana for example, each government unit carrying out nutrition activities had its own budget that was often embedded in a higher level budgetary category. Efforts were underway in most of the countries to improve clarity in overall nutrition budgeting. In Ghana, a sub-committee on resource mobilization had been established, within the Cross-Sectoral Planning Group (CSPG) of the national SUN movement secretariat, to track resource allocation for nutrition in the country.
Inadequate human resources for nutrition were a major barrier to the implementation of nutrition activities (Table 3). In all the 13 countries, there was a critical shortage of skilled nutrition professionals, especially at lower levels. Nutrition graduates did not have practical experience and were not adequately prepared to tackle real-life nutrition issues. There were indeed important gaps in tertiary-level nutrition training in all the 13 countries. Nutrition training curricula were not aligned to regional nutrition priorities. In addition, there did not seem to be a coordinated approach toward harmonization of nutrition training curricula. Existing nutrition degree programs did not provide a full coverage of all essential aspects of human nutrition. Details about the current capacity for nutrition training in the surveyed countries have been reported elsewhere (13).
In the absence of a critical mass of skilled nutrition professionals, the bulk of nutrition work was done by health workers who did not have a good understanding of the application of nutrition principles to public health or clinical practice. As a result, they did not have broad enough skills to provide quality nutrition services. We noted that some countries were taking steps to enhance the performance of health workers through in-service training and mentoring in nutrition. However, these efforts were mainly oriented toward the management of acute malnutrition. In addition, continuing training efforts were mainly concentrated on staff at national and regional levels, leaving staff at lower levels with inadequate capacities in nutrition.
Similarities and differences in capacity assets and gaps across all the surveyed countries
Capacity at organizational level (staff and infrastructure) The results on capacity at the organizational level relate to the production of human resources, staff placement, and supervision. A common characteristic for all the surveyed countries was a critical shortage of nutritionists (Table 3). Although at least 2,000 nutritionists are needed every year in the region, the current output was just at 700. The surveyed countries differed, however, in their capacity to support the expansion of nutrition workforce. Although the capacity to produce nutrition graduates was weak in most of the countries, there seemed to be a reasonable number of trained nutritionists in Nigeria, Ghana, and Sierra Leone. In 2013, there were about 1,000 undergraduate and 200 graduates enrolled in Nigeria, whereas Ghana had about 275 undergraduates and 50 graduates enrolled in nutrition. In Sierra Leone, the current output (45) outweighed the annual need of the country. However, the distribution of the nutrition workforce was inadequate in these three countries. In Nigeria, for example, the vast majority of trained nutritionists worked in urban areas. In addition, a significant proportion of them worked outside the nutrition sector. In Ghana, there was a critical shortage of nutritionists at the district level. This situation put considerable demand on health workers who had to perform nutrition-related tasks in the absence of skilled nutrition professionals. Because these health workers were overwhelmed by other equally important public health tasks, they tended to pay insufficient attention to nutrition. As a result, there was a lack of commitment to nutrition activities in all the surveyed countries.
In all the surveyed countries, training institutions supporting the development of nutrition workforce faced many challenges with teaching resources. They were poorly staffed, financially constrained, and had weak research capacity. They lacked teaching materials and equipment, infrastructure, libraries, and access to advanced technology resources. In most of the countries, there did not seem to be a coordinated effort toward interactions between academia, policy makers, and program managers. As a result, nutrition training institutions operated in silos and rarely participated in policy development or service delivery.
The countries also differed significantly in their approach with regard to staff placement (Table 3). Although some countries, such as Ghana, Liberia, Niger, Nigeria, and Sierra Leone, had a formal cadre of placement of nutritionists through the civil service commission, the other surveyed countries did not yet have a formal cadre of recognized nutritionists. A common pattern for all 13 countries was the lack of regulation of the nutrition profession. None of the countries had a set of minimum standards to qualify as a nutritionist in place. As a result, professionals from other sectors or people with little background in nutrition could be recruited and given the title of nutritionist.
Most of the countries faced challenges in supervising nutrition activities at lower levels. We noted that supportive supervision and mentoring on the job were not systematically or widely carried out. There was limited monitoring because of inadequate logistics and funding. All countries agreed that the provision of additional means of transport would enhance supervision and thereby strengthen program implementation.
The results on capacity at the organizational level relate to the production of human resources, staff placement, and supervision. A common characteristic for all the surveyed countries was a critical shortage of nutritionists (Table 3). Although at least 2,000 nutritionists are needed every year in the region, the current output was just at 700. The surveyed countries differed, however, in their capacity to support the expansion of nutrition workforce. Although the capacity to produce nutrition graduates was weak in most of the countries, there seemed to be a reasonable number of trained nutritionists in Nigeria, Ghana, and Sierra Leone. In 2013, there were about 1,000 undergraduate and 200 graduates enrolled in Nigeria, whereas Ghana had about 275 undergraduates and 50 graduates enrolled in nutrition. In Sierra Leone, the current output (45) outweighed the annual need of the country. However, the distribution of the nutrition workforce was inadequate in these three countries. In Nigeria, for example, the vast majority of trained nutritionists worked in urban areas. In addition, a significant proportion of them worked outside the nutrition sector. In Ghana, there was a critical shortage of nutritionists at the district level. This situation put considerable demand on health workers who had to perform nutrition-related tasks in the absence of skilled nutrition professionals. Because these health workers were overwhelmed by other equally important public health tasks, they tended to pay insufficient attention to nutrition. As a result, there was a lack of commitment to nutrition activities in all the surveyed countries.
In all the surveyed countries, training institutions supporting the development of nutrition workforce faced many challenges with teaching resources. They were poorly staffed, financially constrained, and had weak research capacity. They lacked teaching materials and equipment, infrastructure, libraries, and access to advanced technology resources. In most of the countries, there did not seem to be a coordinated effort toward interactions between academia, policy makers, and program managers. As a result, nutrition training institutions operated in silos and rarely participated in policy development or service delivery.
The countries also differed significantly in their approach with regard to staff placement (Table 3). Although some countries, such as Ghana, Liberia, Niger, Nigeria, and Sierra Leone, had a formal cadre of placement of nutritionists through the civil service commission, the other surveyed countries did not yet have a formal cadre of recognized nutritionists. A common pattern for all 13 countries was the lack of regulation of the nutrition profession. None of the countries had a set of minimum standards to qualify as a nutritionist in place. As a result, professionals from other sectors or people with little background in nutrition could be recruited and given the title of nutritionist.
Most of the countries faced challenges in supervising nutrition activities at lower levels. We noted that supportive supervision and mentoring on the job were not systematically or widely carried out. There was limited monitoring because of inadequate logistics and funding. All countries agreed that the provision of additional means of transport would enhance supervision and thereby strengthen program implementation.
Capacity at systemic level (structures, systems, and roles) The results on capacity at systemic level relate to governance, coordination mechanisms, and information management. There was strong momentum to improve nutrition in most of the countries. This was illustrated by the existence of a set of policies and plans on nutrition, which are used to prioritize actions and track progress toward the achievement of stated objectives (Table 3). However, much remained to be done to align the efforts of all stakeholders to these national nutrition policies and plans. The fact that all the surveyed countries had signed up to the SUN movement was a positive move toward improved nutrition governance in the West Africa region.
Most of the countries had also set up national multisectoral, multi-stakeholder platforms that brought together all the major stakeholders in the nutrition area. These platforms offered the opportunity to share lessons and best practices, as well as to reduce duplication of efforts. Their location differed in the surveyed countries. In Benin for example, the coordinating body was placed directly under the Office of the President, whereas it was within the Prime Minister's office in Niger and Senegal. Ghana and Mauritania had set up a separate government body that oversaw nutrition activities at national level. The Ministry of Health continued to coordinate nutrition activities in Burkina Faso, Cote d'Ivoire, Liberia, Mali, and Nigeria. Cote d'Ivoire and Liberia were, however, taking steps to establish, within the framework of their involvement in the SUN movement, a multisectoral, multi-stakeholder coordination mechanism for nutrition activities. Other new initiatives aimed at improving nutrition security were also ongoing in the surveyed countries. The Renewed Efforts Against Child Hunger and undernutrition (REACH) had also gained momentum in five countries in West Africa (Ghana, Mali, Mauritania, Niger, and Sierra Leone). The European Union and other development partners had also launched the Global Alliance for Resilience Initiative (AGIR).
A common weakness in all countries was the lack of or inadequate coordination mechanisms at lower levels. The cross-disciplinary nature of nutrition made it difficult to coordinate nutrition activities, leading to a high degree of fragmentation of nutrition activities across several ministries, departments, and agencies. In Ghana, coordination at the national, regional, and district levels was being strengthened, institutionalized, and empowered to enhance their effectiveness. Countries such as Benin, Burkina Faso, and Nigeria were taking steps to strengthen the coordination of nutrition activities at their policy and operational levels.
The vast majority of the surveyed countries had made considerable efforts to strengthen the nutrition data collection system. Nutrition surveys, using the SMART methodology, were now being conducted on a regular basis in most of the countries and the survey results were being used for decision-making. Nutrition data were also generated through Demographic and Health Surveys (DHS) or Multiple Indicator Cluster Surveys (MICS). Despite these advancements, there were still some unmet needs. None of the countries had as yet succeeded in putting in place a single, multisectoral information system for nutrition that will guide decision-making processes across the different sectors (Table 3). In addition, much remained to be done to improve the accuracy of routine nutrition data collected at lower levels.
The results on capacity at systemic level relate to governance, coordination mechanisms, and information management. There was strong momentum to improve nutrition in most of the countries. This was illustrated by the existence of a set of policies and plans on nutrition, which are used to prioritize actions and track progress toward the achievement of stated objectives (Table 3). However, much remained to be done to align the efforts of all stakeholders to these national nutrition policies and plans. The fact that all the surveyed countries had signed up to the SUN movement was a positive move toward improved nutrition governance in the West Africa region.
Most of the countries had also set up national multisectoral, multi-stakeholder platforms that brought together all the major stakeholders in the nutrition area. These platforms offered the opportunity to share lessons and best practices, as well as to reduce duplication of efforts. Their location differed in the surveyed countries. In Benin for example, the coordinating body was placed directly under the Office of the President, whereas it was within the Prime Minister's office in Niger and Senegal. Ghana and Mauritania had set up a separate government body that oversaw nutrition activities at national level. The Ministry of Health continued to coordinate nutrition activities in Burkina Faso, Cote d'Ivoire, Liberia, Mali, and Nigeria. Cote d'Ivoire and Liberia were, however, taking steps to establish, within the framework of their involvement in the SUN movement, a multisectoral, multi-stakeholder coordination mechanism for nutrition activities. Other new initiatives aimed at improving nutrition security were also ongoing in the surveyed countries. The Renewed Efforts Against Child Hunger and undernutrition (REACH) had also gained momentum in five countries in West Africa (Ghana, Mali, Mauritania, Niger, and Sierra Leone). The European Union and other development partners had also launched the Global Alliance for Resilience Initiative (AGIR).
A common weakness in all countries was the lack of or inadequate coordination mechanisms at lower levels. The cross-disciplinary nature of nutrition made it difficult to coordinate nutrition activities, leading to a high degree of fragmentation of nutrition activities across several ministries, departments, and agencies. In Ghana, coordination at the national, regional, and district levels was being strengthened, institutionalized, and empowered to enhance their effectiveness. Countries such as Benin, Burkina Faso, and Nigeria were taking steps to strengthen the coordination of nutrition activities at their policy and operational levels.
The vast majority of the surveyed countries had made considerable efforts to strengthen the nutrition data collection system. Nutrition surveys, using the SMART methodology, were now being conducted on a regular basis in most of the countries and the survey results were being used for decision-making. Nutrition data were also generated through Demographic and Health Surveys (DHS) or Multiple Indicator Cluster Surveys (MICS). Despite these advancements, there were still some unmet needs. None of the countries had as yet succeeded in putting in place a single, multisectoral information system for nutrition that will guide decision-making processes across the different sectors (Table 3). In addition, much remained to be done to improve the accuracy of routine nutrition data collected at lower levels.
|
Conclusions
|
Nutrition programs in West Africa are characterized by a critical shortage of skilled human resources, limited funding, high dependency on donor resources, weak logistic and infrastructure systems, and lack of supervision as well as coordination of nutrition activities at lower levels. These factors hamper the ability to act in nutrition in the West Africa region. Addressing these unmet needs is critical in moving toward improved nutrition outcomes in the region. There is an urgent need for a shift toward wider reforms for nutrition capacity development in the West Africa region.
|
[
"Data collection",
"Data analyses",
"Ethical considerations",
"Capacity at individual level (tools and skills)",
"Capacity at organizational level (staff and infrastructure)",
"Capacity at systemic level (structures, systems, and roles)"
] |
[
"Data were collected in 13 of the 16 West African countries between March and August 2013. We did not conduct field visits in three countries (Cape Verde, Guinea-Bissau, and Gambia) because our preliminary contacts with in-country key informants revealed that tertiary-level nutrition training activities were either non-existent or were not well-developed. Data were collected by three experienced interviewers through self-administered questionnaires or face-to-face interviews during country visits. Data were completed (either manually or electronically) by the interviewers in the presence of respondents during in-person meetings. Completed self-administered questionnaires were emailed to the research team. Interviews were not audio or videotaped, so data transcription was not performed.\nData for this assessment were derived from five main sources:Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.\nInterviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).\n\nInterviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.\n\nInterviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.\nInterviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).\nCountry presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.\nLiterature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).",
"Data were entered into Microsoft Excel 2010. They were analyzed and interpreted using content analysis. We used the analytical framework developed by Potter and Brough (7) to systematically assess the current capacity to act in nutrition at the individual, organizational, and systemic levels (Fig. 1). The framework considers capacity development as a hierarchy of needs, portrayed as a pyramid of four interdependent levels, starting from the ‘easier to achieve’ at the apex of the pyramid (tools, skills, staff, and infrastructure) to the ‘much harder’ to implement at its bottom (systems, structures, and roles). The different levels of the pyramid are further divided into nine interdependent sub-components. The topics covered in each of the surveyed countries, for each sub-component, are highlighted in Table 1.\nCapacity building pyramid-Potter and Brough (7).\nCapacity development issues covered in each of the surveyed countries\nDerived from Potter and Brough (7).",
"Administrative authorization was sought by sending a formal correspondence to the head of all of the targeted institutions prior to data collection. We did not seek ethical approval for the study as there was no data collection on human subjects. All respondents were fully informed about the purposes of the study. They gave their full verbal consent before participating in the study.",
"We report the findings relating to logistics, information management, funding, and the quality of nutrition training. In all the 13 surveyed countries, nutrition service providers, in general, had the needed equipment (weighing scales, measuring boards, mid-upper arm circumference tapes, behavior change materials, etc.) and supplies to perform nutrition-related tasks, although they were not available everywhere or at all service delivery points. Equipment and nutrition supplies (especially ready-to-use therapeutic foods) were mainly provided by development partners. Concerted efforts had been made to ensure the availability of these nutrition products at all service delivery points. Sporadic stock-outs of nutrition supplies were reported in most of the countries as a result of a weak logistic and supply chain system, poor forecasting of nutrition supplies at lower levels, and inadequate transportation systems. As of the time of our study, development partners were playing a strong role in the procurement and distribution of these nutrition supplies.\nA common challenge for all 13 countries was the scarcity of financial resources for nutrition activities (Table 3). Although some countries, such as Ghana, Mauritania, Nigeria, and Senegal, experienced an increasing trend in the budget allocated to nutrition activities over the past years, there still remained some important financial gaps. At the time of our study, only a few countries in the region (Niger, Nigeria, and Senegal) had succeeded in securing a budgetary line for nutrition activities in their national budgets. Other countries, such as Benin, Burkina Faso, Mauritania, and Ghana, were in the process of doing so. As a result, the bulk of resources allocated for nutrition activities in the region was provided by donors and development partners. In Burkina-Faso, some nutrition activities were financed through a pooled fund portfolio, which contained mostly funds from donors and development partners.\nAnother common observation was the difficulty in getting accurate information about nutrition financing. The fact that resources come from various sources and cut across many sectors made it difficult to track investments and expenditures on nutrition. In Ghana for example, each government unit carrying out nutrition activities had its own budget that was often embedded in a higher level budgetary category. Efforts were underway in most of the countries to improve clarity in overall nutrition budgeting. In Ghana, a sub-committee on resource mobilization had been established, within the Cross-Sectoral Planning Group (CSPG) of the national SUN movement secretariat, to track resource allocation for nutrition in the country.\nInadequate human resources for nutrition were a major barrier to the implementation of nutrition activities (Table 3). In all the 13 countries, there was a critical shortage of skilled nutrition professionals, especially at lower levels. Nutrition graduates did not have practical experience and were not adequately prepared to tackle real-life nutrition issues. There were indeed important gaps in tertiary-level nutrition training in all the 13 countries. Nutrition training curricula were not aligned to regional nutrition priorities. In addition, there did not seem to be a coordinated approach toward harmonization of nutrition training curricula. Existing nutrition degree programs did not provide a full coverage of all essential aspects of human nutrition. Details about the current capacity for nutrition training in the surveyed countries have been reported elsewhere (13).\nIn the absence of a critical mass of skilled nutrition professionals, the bulk of nutrition work was done by health workers who did not have a good understanding of the application of nutrition principles to public health or clinical practice. As a result, they did not have broad enough skills to provide quality nutrition services. We noted that some countries were taking steps to enhance the performance of health workers through in-service training and mentoring in nutrition. However, these efforts were mainly oriented toward the management of acute malnutrition. In addition, continuing training efforts were mainly concentrated on staff at national and regional levels, leaving staff at lower levels with inadequate capacities in nutrition.\nSimilarities and differences in capacity assets and gaps across all the surveyed countries",
"The results on capacity at the organizational level relate to the production of human resources, staff placement, and supervision. A common characteristic for all the surveyed countries was a critical shortage of nutritionists (Table 3). Although at least 2,000 nutritionists are needed every year in the region, the current output was just at 700. The surveyed countries differed, however, in their capacity to support the expansion of nutrition workforce. Although the capacity to produce nutrition graduates was weak in most of the countries, there seemed to be a reasonable number of trained nutritionists in Nigeria, Ghana, and Sierra Leone. In 2013, there were about 1,000 undergraduate and 200 graduates enrolled in Nigeria, whereas Ghana had about 275 undergraduates and 50 graduates enrolled in nutrition. In Sierra Leone, the current output (45) outweighed the annual need of the country. However, the distribution of the nutrition workforce was inadequate in these three countries. In Nigeria, for example, the vast majority of trained nutritionists worked in urban areas. In addition, a significant proportion of them worked outside the nutrition sector. In Ghana, there was a critical shortage of nutritionists at the district level. This situation put considerable demand on health workers who had to perform nutrition-related tasks in the absence of skilled nutrition professionals. Because these health workers were overwhelmed by other equally important public health tasks, they tended to pay insufficient attention to nutrition. As a result, there was a lack of commitment to nutrition activities in all the surveyed countries.\nIn all the surveyed countries, training institutions supporting the development of nutrition workforce faced many challenges with teaching resources. They were poorly staffed, financially constrained, and had weak research capacity. They lacked teaching materials and equipment, infrastructure, libraries, and access to advanced technology resources. In most of the countries, there did not seem to be a coordinated effort toward interactions between academia, policy makers, and program managers. As a result, nutrition training institutions operated in silos and rarely participated in policy development or service delivery.\nThe countries also differed significantly in their approach with regard to staff placement (Table 3). Although some countries, such as Ghana, Liberia, Niger, Nigeria, and Sierra Leone, had a formal cadre of placement of nutritionists through the civil service commission, the other surveyed countries did not yet have a formal cadre of recognized nutritionists. A common pattern for all 13 countries was the lack of regulation of the nutrition profession. None of the countries had a set of minimum standards to qualify as a nutritionist in place. As a result, professionals from other sectors or people with little background in nutrition could be recruited and given the title of nutritionist.\nMost of the countries faced challenges in supervising nutrition activities at lower levels. We noted that supportive supervision and mentoring on the job were not systematically or widely carried out. There was limited monitoring because of inadequate logistics and funding. All countries agreed that the provision of additional means of transport would enhance supervision and thereby strengthen program implementation.",
"The results on capacity at systemic level relate to governance, coordination mechanisms, and information management. There was strong momentum to improve nutrition in most of the countries. This was illustrated by the existence of a set of policies and plans on nutrition, which are used to prioritize actions and track progress toward the achievement of stated objectives (Table 3). However, much remained to be done to align the efforts of all stakeholders to these national nutrition policies and plans. The fact that all the surveyed countries had signed up to the SUN movement was a positive move toward improved nutrition governance in the West Africa region.\nMost of the countries had also set up national multisectoral, multi-stakeholder platforms that brought together all the major stakeholders in the nutrition area. These platforms offered the opportunity to share lessons and best practices, as well as to reduce duplication of efforts. Their location differed in the surveyed countries. In Benin for example, the coordinating body was placed directly under the Office of the President, whereas it was within the Prime Minister's office in Niger and Senegal. Ghana and Mauritania had set up a separate government body that oversaw nutrition activities at national level. The Ministry of Health continued to coordinate nutrition activities in Burkina Faso, Cote d'Ivoire, Liberia, Mali, and Nigeria. Cote d'Ivoire and Liberia were, however, taking steps to establish, within the framework of their involvement in the SUN movement, a multisectoral, multi-stakeholder coordination mechanism for nutrition activities. Other new initiatives aimed at improving nutrition security were also ongoing in the surveyed countries. The Renewed Efforts Against Child Hunger and undernutrition (REACH) had also gained momentum in five countries in West Africa (Ghana, Mali, Mauritania, Niger, and Sierra Leone). The European Union and other development partners had also launched the Global Alliance for Resilience Initiative (AGIR).\nA common weakness in all countries was the lack of or inadequate coordination mechanisms at lower levels. The cross-disciplinary nature of nutrition made it difficult to coordinate nutrition activities, leading to a high degree of fragmentation of nutrition activities across several ministries, departments, and agencies. In Ghana, coordination at the national, regional, and district levels was being strengthened, institutionalized, and empowered to enhance their effectiveness. Countries such as Benin, Burkina Faso, and Nigeria were taking steps to strengthen the coordination of nutrition activities at their policy and operational levels.\nThe vast majority of the surveyed countries had made considerable efforts to strengthen the nutrition data collection system. Nutrition surveys, using the SMART methodology, were now being conducted on a regular basis in most of the countries and the survey results were being used for decision-making. Nutrition data were also generated through Demographic and Health Surveys (DHS) or Multiple Indicator Cluster Surveys (MICS). Despite these advancements, there were still some unmet needs. None of the countries had as yet succeeded in putting in place a single, multisectoral information system for nutrition that will guide decision-making processes across the different sectors (Table 3). In addition, much remained to be done to improve the accuracy of routine nutrition data collected at lower levels."
] |
[
null,
null,
null,
null,
null,
null
] |
[
"Methods",
"Data collection",
"Data analyses",
"Ethical considerations",
"Results",
"Capacity at individual level (tools and skills)",
"Capacity at organizational level (staff and infrastructure)",
"Capacity at systemic level (structures, systems, and roles)",
"Discussion",
"Conclusions"
] |
[
"This qualitative study is part of a larger capacity needs assessment conducted in the West Africa region. The assessment was conducted within the framework of the WANCDI implemented under the auspices of WAHO.\nThe West Africa region was defined according to the United Nations country classifications (11). It has a total 16 countries comprising Mauritania and the 15 member states of the ECOWAS. The region has a total population of about 340 million people and covers an area of approximately 6.1 million square km (12). The region can be categorized into three major language groups:Anglophone countries (Ghana, Liberia, Nigeria, Sierra Leone, and The Gambia)Francophone countries (Benin, Burkina Faso, Cote d'Ivoire, Guinea, Mali, Mauritania, Niger, Senegal, and Togo)Lusophone countries (Cape Verde and Guinea-Bissau).\n\nAnglophone countries (Ghana, Liberia, Nigeria, Sierra Leone, and The Gambia)\nFrancophone countries (Benin, Burkina Faso, Cote d'Ivoire, Guinea, Mali, Mauritania, Niger, Senegal, and Togo)\nLusophone countries (Cape Verde and Guinea-Bissau).\n Data collection Data were collected in 13 of the 16 West African countries between March and August 2013. We did not conduct field visits in three countries (Cape Verde, Guinea-Bissau, and Gambia) because our preliminary contacts with in-country key informants revealed that tertiary-level nutrition training activities were either non-existent or were not well-developed. Data were collected by three experienced interviewers through self-administered questionnaires or face-to-face interviews during country visits. Data were completed (either manually or electronically) by the interviewers in the presence of respondents during in-person meetings. Completed self-administered questionnaires were emailed to the research team. Interviews were not audio or videotaped, so data transcription was not performed.\nData for this assessment were derived from five main sources:Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.\nInterviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).\n\nInterviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.\n\nInterviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.\nInterviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).\nCountry presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.\nLiterature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).\nData were collected in 13 of the 16 West African countries between March and August 2013. We did not conduct field visits in three countries (Cape Verde, Guinea-Bissau, and Gambia) because our preliminary contacts with in-country key informants revealed that tertiary-level nutrition training activities were either non-existent or were not well-developed. Data were collected by three experienced interviewers through self-administered questionnaires or face-to-face interviews during country visits. Data were completed (either manually or electronically) by the interviewers in the presence of respondents during in-person meetings. Completed self-administered questionnaires were emailed to the research team. Interviews were not audio or videotaped, so data transcription was not performed.\nData for this assessment were derived from five main sources:Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.\nInterviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).\n\nInterviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.\n\nInterviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.\nInterviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).\nCountry presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.\nLiterature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).\n Data analyses Data were entered into Microsoft Excel 2010. They were analyzed and interpreted using content analysis. We used the analytical framework developed by Potter and Brough (7) to systematically assess the current capacity to act in nutrition at the individual, organizational, and systemic levels (Fig. 1). The framework considers capacity development as a hierarchy of needs, portrayed as a pyramid of four interdependent levels, starting from the ‘easier to achieve’ at the apex of the pyramid (tools, skills, staff, and infrastructure) to the ‘much harder’ to implement at its bottom (systems, structures, and roles). The different levels of the pyramid are further divided into nine interdependent sub-components. The topics covered in each of the surveyed countries, for each sub-component, are highlighted in Table 1.\nCapacity building pyramid-Potter and Brough (7).\nCapacity development issues covered in each of the surveyed countries\nDerived from Potter and Brough (7).\nData were entered into Microsoft Excel 2010. They were analyzed and interpreted using content analysis. We used the analytical framework developed by Potter and Brough (7) to systematically assess the current capacity to act in nutrition at the individual, organizational, and systemic levels (Fig. 1). The framework considers capacity development as a hierarchy of needs, portrayed as a pyramid of four interdependent levels, starting from the ‘easier to achieve’ at the apex of the pyramid (tools, skills, staff, and infrastructure) to the ‘much harder’ to implement at its bottom (systems, structures, and roles). The different levels of the pyramid are further divided into nine interdependent sub-components. The topics covered in each of the surveyed countries, for each sub-component, are highlighted in Table 1.\nCapacity building pyramid-Potter and Brough (7).\nCapacity development issues covered in each of the surveyed countries\nDerived from Potter and Brough (7).\n Ethical considerations Administrative authorization was sought by sending a formal correspondence to the head of all of the targeted institutions prior to data collection. We did not seek ethical approval for the study as there was no data collection on human subjects. All respondents were fully informed about the purposes of the study. They gave their full verbal consent before participating in the study.\nAdministrative authorization was sought by sending a formal correspondence to the head of all of the targeted institutions prior to data collection. We did not seek ethical approval for the study as there was no data collection on human subjects. All respondents were fully informed about the purposes of the study. They gave their full verbal consent before participating in the study.",
"Data were collected in 13 of the 16 West African countries between March and August 2013. We did not conduct field visits in three countries (Cape Verde, Guinea-Bissau, and Gambia) because our preliminary contacts with in-country key informants revealed that tertiary-level nutrition training activities were either non-existent or were not well-developed. Data were collected by three experienced interviewers through self-administered questionnaires or face-to-face interviews during country visits. Data were completed (either manually or electronically) by the interviewers in the presence of respondents during in-person meetings. Completed self-administered questionnaires were emailed to the research team. Interviews were not audio or videotaped, so data transcription was not performed.\nData for this assessment were derived from five main sources:Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.\nInterviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).\n\nInterviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.\n\nInterviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.\nInterviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).\nCountry presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.\nLiterature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).",
"Data were entered into Microsoft Excel 2010. They were analyzed and interpreted using content analysis. We used the analytical framework developed by Potter and Brough (7) to systematically assess the current capacity to act in nutrition at the individual, organizational, and systemic levels (Fig. 1). The framework considers capacity development as a hierarchy of needs, portrayed as a pyramid of four interdependent levels, starting from the ‘easier to achieve’ at the apex of the pyramid (tools, skills, staff, and infrastructure) to the ‘much harder’ to implement at its bottom (systems, structures, and roles). The different levels of the pyramid are further divided into nine interdependent sub-components. The topics covered in each of the surveyed countries, for each sub-component, are highlighted in Table 1.\nCapacity building pyramid-Potter and Brough (7).\nCapacity development issues covered in each of the surveyed countries\nDerived from Potter and Brough (7).",
"Administrative authorization was sought by sending a formal correspondence to the head of all of the targeted institutions prior to data collection. We did not seek ethical approval for the study as there was no data collection on human subjects. All respondents were fully informed about the purposes of the study. They gave their full verbal consent before participating in the study.",
"In the 13 surveyed countries, semi-structured interviews were conducted with 44 government officials and 51 stakeholders, namely representatives of non-governmental organizations, UN agencies, and donors (Table 2). We identified 305 nutrition degree programs organized by 113 tertiary-level institutions and 127 training programs organized by 52 health professional schools.\nData collection methods and participating institutions\n Capacity at individual level (tools and skills) We report the findings relating to logistics, information management, funding, and the quality of nutrition training. In all the 13 surveyed countries, nutrition service providers, in general, had the needed equipment (weighing scales, measuring boards, mid-upper arm circumference tapes, behavior change materials, etc.) and supplies to perform nutrition-related tasks, although they were not available everywhere or at all service delivery points. Equipment and nutrition supplies (especially ready-to-use therapeutic foods) were mainly provided by development partners. Concerted efforts had been made to ensure the availability of these nutrition products at all service delivery points. Sporadic stock-outs of nutrition supplies were reported in most of the countries as a result of a weak logistic and supply chain system, poor forecasting of nutrition supplies at lower levels, and inadequate transportation systems. As of the time of our study, development partners were playing a strong role in the procurement and distribution of these nutrition supplies.\nA common challenge for all 13 countries was the scarcity of financial resources for nutrition activities (Table 3). Although some countries, such as Ghana, Mauritania, Nigeria, and Senegal, experienced an increasing trend in the budget allocated to nutrition activities over the past years, there still remained some important financial gaps. At the time of our study, only a few countries in the region (Niger, Nigeria, and Senegal) had succeeded in securing a budgetary line for nutrition activities in their national budgets. Other countries, such as Benin, Burkina Faso, Mauritania, and Ghana, were in the process of doing so. As a result, the bulk of resources allocated for nutrition activities in the region was provided by donors and development partners. In Burkina-Faso, some nutrition activities were financed through a pooled fund portfolio, which contained mostly funds from donors and development partners.\nAnother common observation was the difficulty in getting accurate information about nutrition financing. The fact that resources come from various sources and cut across many sectors made it difficult to track investments and expenditures on nutrition. In Ghana for example, each government unit carrying out nutrition activities had its own budget that was often embedded in a higher level budgetary category. Efforts were underway in most of the countries to improve clarity in overall nutrition budgeting. In Ghana, a sub-committee on resource mobilization had been established, within the Cross-Sectoral Planning Group (CSPG) of the national SUN movement secretariat, to track resource allocation for nutrition in the country.\nInadequate human resources for nutrition were a major barrier to the implementation of nutrition activities (Table 3). In all the 13 countries, there was a critical shortage of skilled nutrition professionals, especially at lower levels. Nutrition graduates did not have practical experience and were not adequately prepared to tackle real-life nutrition issues. There were indeed important gaps in tertiary-level nutrition training in all the 13 countries. Nutrition training curricula were not aligned to regional nutrition priorities. In addition, there did not seem to be a coordinated approach toward harmonization of nutrition training curricula. Existing nutrition degree programs did not provide a full coverage of all essential aspects of human nutrition. Details about the current capacity for nutrition training in the surveyed countries have been reported elsewhere (13).\nIn the absence of a critical mass of skilled nutrition professionals, the bulk of nutrition work was done by health workers who did not have a good understanding of the application of nutrition principles to public health or clinical practice. As a result, they did not have broad enough skills to provide quality nutrition services. We noted that some countries were taking steps to enhance the performance of health workers through in-service training and mentoring in nutrition. However, these efforts were mainly oriented toward the management of acute malnutrition. In addition, continuing training efforts were mainly concentrated on staff at national and regional levels, leaving staff at lower levels with inadequate capacities in nutrition.\nSimilarities and differences in capacity assets and gaps across all the surveyed countries\nWe report the findings relating to logistics, information management, funding, and the quality of nutrition training. In all the 13 surveyed countries, nutrition service providers, in general, had the needed equipment (weighing scales, measuring boards, mid-upper arm circumference tapes, behavior change materials, etc.) and supplies to perform nutrition-related tasks, although they were not available everywhere or at all service delivery points. Equipment and nutrition supplies (especially ready-to-use therapeutic foods) were mainly provided by development partners. Concerted efforts had been made to ensure the availability of these nutrition products at all service delivery points. Sporadic stock-outs of nutrition supplies were reported in most of the countries as a result of a weak logistic and supply chain system, poor forecasting of nutrition supplies at lower levels, and inadequate transportation systems. As of the time of our study, development partners were playing a strong role in the procurement and distribution of these nutrition supplies.\nA common challenge for all 13 countries was the scarcity of financial resources for nutrition activities (Table 3). Although some countries, such as Ghana, Mauritania, Nigeria, and Senegal, experienced an increasing trend in the budget allocated to nutrition activities over the past years, there still remained some important financial gaps. At the time of our study, only a few countries in the region (Niger, Nigeria, and Senegal) had succeeded in securing a budgetary line for nutrition activities in their national budgets. Other countries, such as Benin, Burkina Faso, Mauritania, and Ghana, were in the process of doing so. As a result, the bulk of resources allocated for nutrition activities in the region was provided by donors and development partners. In Burkina-Faso, some nutrition activities were financed through a pooled fund portfolio, which contained mostly funds from donors and development partners.\nAnother common observation was the difficulty in getting accurate information about nutrition financing. The fact that resources come from various sources and cut across many sectors made it difficult to track investments and expenditures on nutrition. In Ghana for example, each government unit carrying out nutrition activities had its own budget that was often embedded in a higher level budgetary category. Efforts were underway in most of the countries to improve clarity in overall nutrition budgeting. In Ghana, a sub-committee on resource mobilization had been established, within the Cross-Sectoral Planning Group (CSPG) of the national SUN movement secretariat, to track resource allocation for nutrition in the country.\nInadequate human resources for nutrition were a major barrier to the implementation of nutrition activities (Table 3). In all the 13 countries, there was a critical shortage of skilled nutrition professionals, especially at lower levels. Nutrition graduates did not have practical experience and were not adequately prepared to tackle real-life nutrition issues. There were indeed important gaps in tertiary-level nutrition training in all the 13 countries. Nutrition training curricula were not aligned to regional nutrition priorities. In addition, there did not seem to be a coordinated approach toward harmonization of nutrition training curricula. Existing nutrition degree programs did not provide a full coverage of all essential aspects of human nutrition. Details about the current capacity for nutrition training in the surveyed countries have been reported elsewhere (13).\nIn the absence of a critical mass of skilled nutrition professionals, the bulk of nutrition work was done by health workers who did not have a good understanding of the application of nutrition principles to public health or clinical practice. As a result, they did not have broad enough skills to provide quality nutrition services. We noted that some countries were taking steps to enhance the performance of health workers through in-service training and mentoring in nutrition. However, these efforts were mainly oriented toward the management of acute malnutrition. In addition, continuing training efforts were mainly concentrated on staff at national and regional levels, leaving staff at lower levels with inadequate capacities in nutrition.\nSimilarities and differences in capacity assets and gaps across all the surveyed countries\n Capacity at organizational level (staff and infrastructure) The results on capacity at the organizational level relate to the production of human resources, staff placement, and supervision. A common characteristic for all the surveyed countries was a critical shortage of nutritionists (Table 3). Although at least 2,000 nutritionists are needed every year in the region, the current output was just at 700. The surveyed countries differed, however, in their capacity to support the expansion of nutrition workforce. Although the capacity to produce nutrition graduates was weak in most of the countries, there seemed to be a reasonable number of trained nutritionists in Nigeria, Ghana, and Sierra Leone. In 2013, there were about 1,000 undergraduate and 200 graduates enrolled in Nigeria, whereas Ghana had about 275 undergraduates and 50 graduates enrolled in nutrition. In Sierra Leone, the current output (45) outweighed the annual need of the country. However, the distribution of the nutrition workforce was inadequate in these three countries. In Nigeria, for example, the vast majority of trained nutritionists worked in urban areas. In addition, a significant proportion of them worked outside the nutrition sector. In Ghana, there was a critical shortage of nutritionists at the district level. This situation put considerable demand on health workers who had to perform nutrition-related tasks in the absence of skilled nutrition professionals. Because these health workers were overwhelmed by other equally important public health tasks, they tended to pay insufficient attention to nutrition. As a result, there was a lack of commitment to nutrition activities in all the surveyed countries.\nIn all the surveyed countries, training institutions supporting the development of nutrition workforce faced many challenges with teaching resources. They were poorly staffed, financially constrained, and had weak research capacity. They lacked teaching materials and equipment, infrastructure, libraries, and access to advanced technology resources. In most of the countries, there did not seem to be a coordinated effort toward interactions between academia, policy makers, and program managers. As a result, nutrition training institutions operated in silos and rarely participated in policy development or service delivery.\nThe countries also differed significantly in their approach with regard to staff placement (Table 3). Although some countries, such as Ghana, Liberia, Niger, Nigeria, and Sierra Leone, had a formal cadre of placement of nutritionists through the civil service commission, the other surveyed countries did not yet have a formal cadre of recognized nutritionists. A common pattern for all 13 countries was the lack of regulation of the nutrition profession. None of the countries had a set of minimum standards to qualify as a nutritionist in place. As a result, professionals from other sectors or people with little background in nutrition could be recruited and given the title of nutritionist.\nMost of the countries faced challenges in supervising nutrition activities at lower levels. We noted that supportive supervision and mentoring on the job were not systematically or widely carried out. There was limited monitoring because of inadequate logistics and funding. All countries agreed that the provision of additional means of transport would enhance supervision and thereby strengthen program implementation.\nThe results on capacity at the organizational level relate to the production of human resources, staff placement, and supervision. A common characteristic for all the surveyed countries was a critical shortage of nutritionists (Table 3). Although at least 2,000 nutritionists are needed every year in the region, the current output was just at 700. The surveyed countries differed, however, in their capacity to support the expansion of nutrition workforce. Although the capacity to produce nutrition graduates was weak in most of the countries, there seemed to be a reasonable number of trained nutritionists in Nigeria, Ghana, and Sierra Leone. In 2013, there were about 1,000 undergraduate and 200 graduates enrolled in Nigeria, whereas Ghana had about 275 undergraduates and 50 graduates enrolled in nutrition. In Sierra Leone, the current output (45) outweighed the annual need of the country. However, the distribution of the nutrition workforce was inadequate in these three countries. In Nigeria, for example, the vast majority of trained nutritionists worked in urban areas. In addition, a significant proportion of them worked outside the nutrition sector. In Ghana, there was a critical shortage of nutritionists at the district level. This situation put considerable demand on health workers who had to perform nutrition-related tasks in the absence of skilled nutrition professionals. Because these health workers were overwhelmed by other equally important public health tasks, they tended to pay insufficient attention to nutrition. As a result, there was a lack of commitment to nutrition activities in all the surveyed countries.\nIn all the surveyed countries, training institutions supporting the development of nutrition workforce faced many challenges with teaching resources. They were poorly staffed, financially constrained, and had weak research capacity. They lacked teaching materials and equipment, infrastructure, libraries, and access to advanced technology resources. In most of the countries, there did not seem to be a coordinated effort toward interactions between academia, policy makers, and program managers. As a result, nutrition training institutions operated in silos and rarely participated in policy development or service delivery.\nThe countries also differed significantly in their approach with regard to staff placement (Table 3). Although some countries, such as Ghana, Liberia, Niger, Nigeria, and Sierra Leone, had a formal cadre of placement of nutritionists through the civil service commission, the other surveyed countries did not yet have a formal cadre of recognized nutritionists. A common pattern for all 13 countries was the lack of regulation of the nutrition profession. None of the countries had a set of minimum standards to qualify as a nutritionist in place. As a result, professionals from other sectors or people with little background in nutrition could be recruited and given the title of nutritionist.\nMost of the countries faced challenges in supervising nutrition activities at lower levels. We noted that supportive supervision and mentoring on the job were not systematically or widely carried out. There was limited monitoring because of inadequate logistics and funding. All countries agreed that the provision of additional means of transport would enhance supervision and thereby strengthen program implementation.\n Capacity at systemic level (structures, systems, and roles) The results on capacity at systemic level relate to governance, coordination mechanisms, and information management. There was strong momentum to improve nutrition in most of the countries. This was illustrated by the existence of a set of policies and plans on nutrition, which are used to prioritize actions and track progress toward the achievement of stated objectives (Table 3). However, much remained to be done to align the efforts of all stakeholders to these national nutrition policies and plans. The fact that all the surveyed countries had signed up to the SUN movement was a positive move toward improved nutrition governance in the West Africa region.\nMost of the countries had also set up national multisectoral, multi-stakeholder platforms that brought together all the major stakeholders in the nutrition area. These platforms offered the opportunity to share lessons and best practices, as well as to reduce duplication of efforts. Their location differed in the surveyed countries. In Benin for example, the coordinating body was placed directly under the Office of the President, whereas it was within the Prime Minister's office in Niger and Senegal. Ghana and Mauritania had set up a separate government body that oversaw nutrition activities at national level. The Ministry of Health continued to coordinate nutrition activities in Burkina Faso, Cote d'Ivoire, Liberia, Mali, and Nigeria. Cote d'Ivoire and Liberia were, however, taking steps to establish, within the framework of their involvement in the SUN movement, a multisectoral, multi-stakeholder coordination mechanism for nutrition activities. Other new initiatives aimed at improving nutrition security were also ongoing in the surveyed countries. The Renewed Efforts Against Child Hunger and undernutrition (REACH) had also gained momentum in five countries in West Africa (Ghana, Mali, Mauritania, Niger, and Sierra Leone). The European Union and other development partners had also launched the Global Alliance for Resilience Initiative (AGIR).\nA common weakness in all countries was the lack of or inadequate coordination mechanisms at lower levels. The cross-disciplinary nature of nutrition made it difficult to coordinate nutrition activities, leading to a high degree of fragmentation of nutrition activities across several ministries, departments, and agencies. In Ghana, coordination at the national, regional, and district levels was being strengthened, institutionalized, and empowered to enhance their effectiveness. Countries such as Benin, Burkina Faso, and Nigeria were taking steps to strengthen the coordination of nutrition activities at their policy and operational levels.\nThe vast majority of the surveyed countries had made considerable efforts to strengthen the nutrition data collection system. Nutrition surveys, using the SMART methodology, were now being conducted on a regular basis in most of the countries and the survey results were being used for decision-making. Nutrition data were also generated through Demographic and Health Surveys (DHS) or Multiple Indicator Cluster Surveys (MICS). Despite these advancements, there were still some unmet needs. None of the countries had as yet succeeded in putting in place a single, multisectoral information system for nutrition that will guide decision-making processes across the different sectors (Table 3). In addition, much remained to be done to improve the accuracy of routine nutrition data collected at lower levels.\nThe results on capacity at systemic level relate to governance, coordination mechanisms, and information management. There was strong momentum to improve nutrition in most of the countries. This was illustrated by the existence of a set of policies and plans on nutrition, which are used to prioritize actions and track progress toward the achievement of stated objectives (Table 3). However, much remained to be done to align the efforts of all stakeholders to these national nutrition policies and plans. The fact that all the surveyed countries had signed up to the SUN movement was a positive move toward improved nutrition governance in the West Africa region.\nMost of the countries had also set up national multisectoral, multi-stakeholder platforms that brought together all the major stakeholders in the nutrition area. These platforms offered the opportunity to share lessons and best practices, as well as to reduce duplication of efforts. Their location differed in the surveyed countries. In Benin for example, the coordinating body was placed directly under the Office of the President, whereas it was within the Prime Minister's office in Niger and Senegal. Ghana and Mauritania had set up a separate government body that oversaw nutrition activities at national level. The Ministry of Health continued to coordinate nutrition activities in Burkina Faso, Cote d'Ivoire, Liberia, Mali, and Nigeria. Cote d'Ivoire and Liberia were, however, taking steps to establish, within the framework of their involvement in the SUN movement, a multisectoral, multi-stakeholder coordination mechanism for nutrition activities. Other new initiatives aimed at improving nutrition security were also ongoing in the surveyed countries. The Renewed Efforts Against Child Hunger and undernutrition (REACH) had also gained momentum in five countries in West Africa (Ghana, Mali, Mauritania, Niger, and Sierra Leone). The European Union and other development partners had also launched the Global Alliance for Resilience Initiative (AGIR).\nA common weakness in all countries was the lack of or inadequate coordination mechanisms at lower levels. The cross-disciplinary nature of nutrition made it difficult to coordinate nutrition activities, leading to a high degree of fragmentation of nutrition activities across several ministries, departments, and agencies. In Ghana, coordination at the national, regional, and district levels was being strengthened, institutionalized, and empowered to enhance their effectiveness. Countries such as Benin, Burkina Faso, and Nigeria were taking steps to strengthen the coordination of nutrition activities at their policy and operational levels.\nThe vast majority of the surveyed countries had made considerable efforts to strengthen the nutrition data collection system. Nutrition surveys, using the SMART methodology, were now being conducted on a regular basis in most of the countries and the survey results were being used for decision-making. Nutrition data were also generated through Demographic and Health Surveys (DHS) or Multiple Indicator Cluster Surveys (MICS). Despite these advancements, there were still some unmet needs. None of the countries had as yet succeeded in putting in place a single, multisectoral information system for nutrition that will guide decision-making processes across the different sectors (Table 3). In addition, much remained to be done to improve the accuracy of routine nutrition data collected at lower levels.",
"We report the findings relating to logistics, information management, funding, and the quality of nutrition training. In all the 13 surveyed countries, nutrition service providers, in general, had the needed equipment (weighing scales, measuring boards, mid-upper arm circumference tapes, behavior change materials, etc.) and supplies to perform nutrition-related tasks, although they were not available everywhere or at all service delivery points. Equipment and nutrition supplies (especially ready-to-use therapeutic foods) were mainly provided by development partners. Concerted efforts had been made to ensure the availability of these nutrition products at all service delivery points. Sporadic stock-outs of nutrition supplies were reported in most of the countries as a result of a weak logistic and supply chain system, poor forecasting of nutrition supplies at lower levels, and inadequate transportation systems. As of the time of our study, development partners were playing a strong role in the procurement and distribution of these nutrition supplies.\nA common challenge for all 13 countries was the scarcity of financial resources for nutrition activities (Table 3). Although some countries, such as Ghana, Mauritania, Nigeria, and Senegal, experienced an increasing trend in the budget allocated to nutrition activities over the past years, there still remained some important financial gaps. At the time of our study, only a few countries in the region (Niger, Nigeria, and Senegal) had succeeded in securing a budgetary line for nutrition activities in their national budgets. Other countries, such as Benin, Burkina Faso, Mauritania, and Ghana, were in the process of doing so. As a result, the bulk of resources allocated for nutrition activities in the region was provided by donors and development partners. In Burkina-Faso, some nutrition activities were financed through a pooled fund portfolio, which contained mostly funds from donors and development partners.\nAnother common observation was the difficulty in getting accurate information about nutrition financing. The fact that resources come from various sources and cut across many sectors made it difficult to track investments and expenditures on nutrition. In Ghana for example, each government unit carrying out nutrition activities had its own budget that was often embedded in a higher level budgetary category. Efforts were underway in most of the countries to improve clarity in overall nutrition budgeting. In Ghana, a sub-committee on resource mobilization had been established, within the Cross-Sectoral Planning Group (CSPG) of the national SUN movement secretariat, to track resource allocation for nutrition in the country.\nInadequate human resources for nutrition were a major barrier to the implementation of nutrition activities (Table 3). In all the 13 countries, there was a critical shortage of skilled nutrition professionals, especially at lower levels. Nutrition graduates did not have practical experience and were not adequately prepared to tackle real-life nutrition issues. There were indeed important gaps in tertiary-level nutrition training in all the 13 countries. Nutrition training curricula were not aligned to regional nutrition priorities. In addition, there did not seem to be a coordinated approach toward harmonization of nutrition training curricula. Existing nutrition degree programs did not provide a full coverage of all essential aspects of human nutrition. Details about the current capacity for nutrition training in the surveyed countries have been reported elsewhere (13).\nIn the absence of a critical mass of skilled nutrition professionals, the bulk of nutrition work was done by health workers who did not have a good understanding of the application of nutrition principles to public health or clinical practice. As a result, they did not have broad enough skills to provide quality nutrition services. We noted that some countries were taking steps to enhance the performance of health workers through in-service training and mentoring in nutrition. However, these efforts were mainly oriented toward the management of acute malnutrition. In addition, continuing training efforts were mainly concentrated on staff at national and regional levels, leaving staff at lower levels with inadequate capacities in nutrition.\nSimilarities and differences in capacity assets and gaps across all the surveyed countries",
"The results on capacity at the organizational level relate to the production of human resources, staff placement, and supervision. A common characteristic for all the surveyed countries was a critical shortage of nutritionists (Table 3). Although at least 2,000 nutritionists are needed every year in the region, the current output was just at 700. The surveyed countries differed, however, in their capacity to support the expansion of nutrition workforce. Although the capacity to produce nutrition graduates was weak in most of the countries, there seemed to be a reasonable number of trained nutritionists in Nigeria, Ghana, and Sierra Leone. In 2013, there were about 1,000 undergraduate and 200 graduates enrolled in Nigeria, whereas Ghana had about 275 undergraduates and 50 graduates enrolled in nutrition. In Sierra Leone, the current output (45) outweighed the annual need of the country. However, the distribution of the nutrition workforce was inadequate in these three countries. In Nigeria, for example, the vast majority of trained nutritionists worked in urban areas. In addition, a significant proportion of them worked outside the nutrition sector. In Ghana, there was a critical shortage of nutritionists at the district level. This situation put considerable demand on health workers who had to perform nutrition-related tasks in the absence of skilled nutrition professionals. Because these health workers were overwhelmed by other equally important public health tasks, they tended to pay insufficient attention to nutrition. As a result, there was a lack of commitment to nutrition activities in all the surveyed countries.\nIn all the surveyed countries, training institutions supporting the development of nutrition workforce faced many challenges with teaching resources. They were poorly staffed, financially constrained, and had weak research capacity. They lacked teaching materials and equipment, infrastructure, libraries, and access to advanced technology resources. In most of the countries, there did not seem to be a coordinated effort toward interactions between academia, policy makers, and program managers. As a result, nutrition training institutions operated in silos and rarely participated in policy development or service delivery.\nThe countries also differed significantly in their approach with regard to staff placement (Table 3). Although some countries, such as Ghana, Liberia, Niger, Nigeria, and Sierra Leone, had a formal cadre of placement of nutritionists through the civil service commission, the other surveyed countries did not yet have a formal cadre of recognized nutritionists. A common pattern for all 13 countries was the lack of regulation of the nutrition profession. None of the countries had a set of minimum standards to qualify as a nutritionist in place. As a result, professionals from other sectors or people with little background in nutrition could be recruited and given the title of nutritionist.\nMost of the countries faced challenges in supervising nutrition activities at lower levels. We noted that supportive supervision and mentoring on the job were not systematically or widely carried out. There was limited monitoring because of inadequate logistics and funding. All countries agreed that the provision of additional means of transport would enhance supervision and thereby strengthen program implementation.",
"The results on capacity at systemic level relate to governance, coordination mechanisms, and information management. There was strong momentum to improve nutrition in most of the countries. This was illustrated by the existence of a set of policies and plans on nutrition, which are used to prioritize actions and track progress toward the achievement of stated objectives (Table 3). However, much remained to be done to align the efforts of all stakeholders to these national nutrition policies and plans. The fact that all the surveyed countries had signed up to the SUN movement was a positive move toward improved nutrition governance in the West Africa region.\nMost of the countries had also set up national multisectoral, multi-stakeholder platforms that brought together all the major stakeholders in the nutrition area. These platforms offered the opportunity to share lessons and best practices, as well as to reduce duplication of efforts. Their location differed in the surveyed countries. In Benin for example, the coordinating body was placed directly under the Office of the President, whereas it was within the Prime Minister's office in Niger and Senegal. Ghana and Mauritania had set up a separate government body that oversaw nutrition activities at national level. The Ministry of Health continued to coordinate nutrition activities in Burkina Faso, Cote d'Ivoire, Liberia, Mali, and Nigeria. Cote d'Ivoire and Liberia were, however, taking steps to establish, within the framework of their involvement in the SUN movement, a multisectoral, multi-stakeholder coordination mechanism for nutrition activities. Other new initiatives aimed at improving nutrition security were also ongoing in the surveyed countries. The Renewed Efforts Against Child Hunger and undernutrition (REACH) had also gained momentum in five countries in West Africa (Ghana, Mali, Mauritania, Niger, and Sierra Leone). The European Union and other development partners had also launched the Global Alliance for Resilience Initiative (AGIR).\nA common weakness in all countries was the lack of or inadequate coordination mechanisms at lower levels. The cross-disciplinary nature of nutrition made it difficult to coordinate nutrition activities, leading to a high degree of fragmentation of nutrition activities across several ministries, departments, and agencies. In Ghana, coordination at the national, regional, and district levels was being strengthened, institutionalized, and empowered to enhance their effectiveness. Countries such as Benin, Burkina Faso, and Nigeria were taking steps to strengthen the coordination of nutrition activities at their policy and operational levels.\nThe vast majority of the surveyed countries had made considerable efforts to strengthen the nutrition data collection system. Nutrition surveys, using the SMART methodology, were now being conducted on a regular basis in most of the countries and the survey results were being used for decision-making. Nutrition data were also generated through Demographic and Health Surveys (DHS) or Multiple Indicator Cluster Surveys (MICS). Despite these advancements, there were still some unmet needs. None of the countries had as yet succeeded in putting in place a single, multisectoral information system for nutrition that will guide decision-making processes across the different sectors (Table 3). In addition, much remained to be done to improve the accuracy of routine nutrition data collected at lower levels.",
"In our region-wide assessment of the current capacity to act in nutrition in West Africa, we analyzed key capacity assets and gaps as well as cross-country similarities and differences. Our findings reveal that important efforts have been made over the past years to raise the profile of nutrition to a high priority in the surveyed countries. The nutrition landscape has been evolving in most of the countries in West Africa since 2011. In addition, advocacy efforts are underway to sustain this momentum and keep governments involved. This is a positive move toward the achievement of nutrition-related MDGs in the region. Donors and development partners have also played an important role in this process. The different nutrition initiatives supported by development partners (SUN, REACH, AGIR, etc.) have greatly contributed to the advancement of nutrition in the region.\n\nDespite these positive developments, there are still some challenges and unmet needs. There is a need for clear divisions of tasks between ongoing nutrition initiatives. For example, five of the 13 surveyed countries (Ghana, Mali, Mauritania, Niger, and Sierra Leone) are both SUN and REACH countries. It is critical to ensure good coordination between these two important initiatives as both aim to support country-led processes to improve nutrition governance and enable multisectoral, multi-stakeholders approach to addressing undernutrition (15, 16). This could help prevent potential misunderstandings that may arise. The confusion in multiple nutrition initiatives has been reported in Niger (17).\nOur findings also indicate that lack of funding is a major barrier for the implementation of nutrition activities. There is indeed a clear disconnect between national nutrition goals and the resources available for the implementation of nutrition activities. This situation slows the translation of governments’ commitments for nutrition into tangible actions on the ground. We found a high dependency on donor resources for nutrition activities as a result of low government investments. It is critical that governments in the West Africa region take on greater responsibility for nutrition financing.\nAnother major challenge that was common was the inability to track investments and expenditures on nutrition activities. The importance of having accurate information on nutrition financing cannot be overemphasized. These data are not only crucial for planning purposes (to make projections for nutrition financing), but are also vital in identifying financial gaps and ensuring efficiency and accountability. There is an urgent need to improve the systems for tracking allocations and expenditures for nutrition activities in the surveyed countries. The workshop on costing and tracking investments in nutrition, co-organized by UNICEF and the SUN movement secretariat in Nairobi, Kenya, in November 2013 (18), was a preliminary step in moving toward that direction. Much remains to be done to improve transparency and clarity over nutrition budgeting in the West Africa region. It is gratifying that the SUN movement secretariat continues to work with countries to ensure that nutrition programs are costed and that resources allocated to nutrition activities are tracked (14).\nThe results of our study indicate that shortage of skilled human resources to deliver nutrition interventions is a major problem in all countries. There is a clear need to support the expansion of the nutrition workforce in the region and provide incentives for nutrition service providers to stay in service. Training institutions should be empowered to support the expansion of the nutrition workforce. In addition, attention should be paid to the pre-service and in-service training in nutrition of health professionals. Our findings also indicate that there is a lack of regulation of the nutrition profession. The need to set a minimum standard to qualify as a nutritionist in the region has been previously stressed (19, 20). It would also be important to create a proper cadre of placement for nutrition service providers and clearly specify their roles and responsibilities.\nOverall, our data are consistent with findings in previous studies in some of the West African countries (9, 10). They underscore the urgent need to strengthen capacity for nutrition at individual, organizational, and systemic levels. Our results are also in line with findings in previous studies in other sub-Saharan African countries such as Malawi (21), Namibia (22), and Angola (23). Some common elements are the limited financial resources for nutrition activities, the lack of human resources for nutrition, the lack of pre-service nutrition training programs, the limited capacity to support the expansion of a nutrition workforce, and the lack of cross-sectoral coordination. Clearly, support is urgently needed to build the needed capacity to accelerate progress for nutrition in Africa.\nIt is not our intention in this paper to make specific recommendations on how to develop capacity for nutrition in the West Africa region. We believe that this should be done in a participatory manner, with the involvement of all stakeholders. For instance, we are planning to convene a regional workshop to discuss the findings from this study and build consensus on the way forward. The ultimate goal of this workshop would be to create a unified discourse between stakeholders on how to strengthen the capacity to act in nutrition in the West Africa region. This will ultimately lead to the development of a consensual regional capacity development strategy that will help shape future nutrition actions.\nOur study has several limitations. It was difficult to obtain quantitative data on financial investments in nutrition as they were not readily available in the surveyed countries. In addition, we did not have the opportunity to assess the adequacy of infrastructure for nutrition services through direct observations. Given the rapidly evolving nutrition environment in West Africa, it is possible that some of the information reported in this paper may not fully reflect the current situation on the ground. However, we believe that our study provides a comprehensive view on the current capacity to act in nutrition in the West Africa region.",
"Nutrition programs in West Africa are characterized by a critical shortage of skilled human resources, limited funding, high dependency on donor resources, weak logistic and infrastructure systems, and lack of supervision as well as coordination of nutrition activities at lower levels. These factors hamper the ability to act in nutrition in the West Africa region. Addressing these unmet needs is critical in moving toward improved nutrition outcomes in the region. There is an urgent need for a shift toward wider reforms for nutrition capacity development in the West Africa region."
] |
[
"methods",
null,
null,
null,
"results",
null,
null,
null,
"discussion",
"conclusions"
] |
[
"capacity development",
"nutrition",
"undernutrition",
"nutrition workforce",
"West Africa"
] |
Methods:
This qualitative study is part of a larger capacity needs assessment conducted in the West Africa region. The assessment was conducted within the framework of the WANCDI implemented under the auspices of WAHO.
The West Africa region was defined according to the United Nations country classifications (11). It has a total 16 countries comprising Mauritania and the 15 member states of the ECOWAS. The region has a total population of about 340 million people and covers an area of approximately 6.1 million square km (12). The region can be categorized into three major language groups:Anglophone countries (Ghana, Liberia, Nigeria, Sierra Leone, and The Gambia)Francophone countries (Benin, Burkina Faso, Cote d'Ivoire, Guinea, Mali, Mauritania, Niger, Senegal, and Togo)Lusophone countries (Cape Verde and Guinea-Bissau).
Anglophone countries (Ghana, Liberia, Nigeria, Sierra Leone, and The Gambia)
Francophone countries (Benin, Burkina Faso, Cote d'Ivoire, Guinea, Mali, Mauritania, Niger, Senegal, and Togo)
Lusophone countries (Cape Verde and Guinea-Bissau).
Data collection Data were collected in 13 of the 16 West African countries between March and August 2013. We did not conduct field visits in three countries (Cape Verde, Guinea-Bissau, and Gambia) because our preliminary contacts with in-country key informants revealed that tertiary-level nutrition training activities were either non-existent or were not well-developed. Data were collected by three experienced interviewers through self-administered questionnaires or face-to-face interviews during country visits. Data were completed (either manually or electronically) by the interviewers in the presence of respondents during in-person meetings. Completed self-administered questionnaires were emailed to the research team. Interviews were not audio or videotaped, so data transcription was not performed.
Data for this assessment were derived from five main sources:Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.
Interviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).
Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.
Interviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.
Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).
Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.
Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).
Data were collected in 13 of the 16 West African countries between March and August 2013. We did not conduct field visits in three countries (Cape Verde, Guinea-Bissau, and Gambia) because our preliminary contacts with in-country key informants revealed that tertiary-level nutrition training activities were either non-existent or were not well-developed. Data were collected by three experienced interviewers through self-administered questionnaires or face-to-face interviews during country visits. Data were completed (either manually or electronically) by the interviewers in the presence of respondents during in-person meetings. Completed self-administered questionnaires were emailed to the research team. Interviews were not audio or videotaped, so data transcription was not performed.
Data for this assessment were derived from five main sources:Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.
Interviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).
Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.
Interviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.
Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).
Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.
Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).
Data analyses Data were entered into Microsoft Excel 2010. They were analyzed and interpreted using content analysis. We used the analytical framework developed by Potter and Brough (7) to systematically assess the current capacity to act in nutrition at the individual, organizational, and systemic levels (Fig. 1). The framework considers capacity development as a hierarchy of needs, portrayed as a pyramid of four interdependent levels, starting from the ‘easier to achieve’ at the apex of the pyramid (tools, skills, staff, and infrastructure) to the ‘much harder’ to implement at its bottom (systems, structures, and roles). The different levels of the pyramid are further divided into nine interdependent sub-components. The topics covered in each of the surveyed countries, for each sub-component, are highlighted in Table 1.
Capacity building pyramid-Potter and Brough (7).
Capacity development issues covered in each of the surveyed countries
Derived from Potter and Brough (7).
Data were entered into Microsoft Excel 2010. They were analyzed and interpreted using content analysis. We used the analytical framework developed by Potter and Brough (7) to systematically assess the current capacity to act in nutrition at the individual, organizational, and systemic levels (Fig. 1). The framework considers capacity development as a hierarchy of needs, portrayed as a pyramid of four interdependent levels, starting from the ‘easier to achieve’ at the apex of the pyramid (tools, skills, staff, and infrastructure) to the ‘much harder’ to implement at its bottom (systems, structures, and roles). The different levels of the pyramid are further divided into nine interdependent sub-components. The topics covered in each of the surveyed countries, for each sub-component, are highlighted in Table 1.
Capacity building pyramid-Potter and Brough (7).
Capacity development issues covered in each of the surveyed countries
Derived from Potter and Brough (7).
Ethical considerations Administrative authorization was sought by sending a formal correspondence to the head of all of the targeted institutions prior to data collection. We did not seek ethical approval for the study as there was no data collection on human subjects. All respondents were fully informed about the purposes of the study. They gave their full verbal consent before participating in the study.
Administrative authorization was sought by sending a formal correspondence to the head of all of the targeted institutions prior to data collection. We did not seek ethical approval for the study as there was no data collection on human subjects. All respondents were fully informed about the purposes of the study. They gave their full verbal consent before participating in the study.
Data collection:
Data were collected in 13 of the 16 West African countries between March and August 2013. We did not conduct field visits in three countries (Cape Verde, Guinea-Bissau, and Gambia) because our preliminary contacts with in-country key informants revealed that tertiary-level nutrition training activities were either non-existent or were not well-developed. Data were collected by three experienced interviewers through self-administered questionnaires or face-to-face interviews during country visits. Data were completed (either manually or electronically) by the interviewers in the presence of respondents during in-person meetings. Completed self-administered questionnaires were emailed to the research team. Interviews were not audio or videotaped, so data transcription was not performed.
Data for this assessment were derived from five main sources:Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.
Interviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).
Interviews with government officials in charge of nutrition: A semi-structured questionnaire (available upon request) was administered to the nutrition focal person in each of the surveyed countries to explore the three interdependent levels (individual, organizational, and systemic) of nutrition capacity development. The questionnaire was structured around four key elements: workforce planning and leadership, management of workforce, work environment, and human resources.
Interviews with key stakeholders involved in nutrition: Data were also collected from other sources to support the findings from discussions with government officials. Interviews were conducted in each of the surveyed countries with key stakeholders, namely non-governmental organizations, United Nations (UN) agencies, and donors. A strengths, weaknesses, opportunities, and threats (SWOT) analysis was conducted with all stakeholders.
Interviews with nutrition training institutions and health professional schools: We conducted a detailed inventory of the current capacity for nutrition training in each of the surveyed countries. Data were collected using a semi-structured questionnaire that was either administrated during face-to-face discussion or self-administrated by tertiary-level training institutions offering nutrition degree programs. Health professional schools offering nutrition courses as part of their training curricula were also interviewed. Information was collected on the content and status of nutrition courses; profile of faculty members; students and graduates; funding; facilities; prevailing teaching methods; school ownership; institutional affiliation; main funding source; and institutional collaborations. Details about this inventory have been described elsewhere (13).
Country presentations from the ECOWAS nutrition meeting: Data were also collected during the ECOWAS nutrition forum mid-term review meeting held in Monrovia, Liberia, 26–27 November 2013. The workshop was organized by WAHO and was attended by the nutrition focal points of all the surveyed countries. Data were also gathered from country presentations made by the nutrition focal points during the meeting.
Literature review and internet searches: Besides the interviews, we did a comprehensive literature review that looked beyond individual training needs and focused on previous reports addressing nutrition capacity development in West Africa. We also checked several web portals, including the SUN movement website (14).
Data analyses:
Data were entered into Microsoft Excel 2010. They were analyzed and interpreted using content analysis. We used the analytical framework developed by Potter and Brough (7) to systematically assess the current capacity to act in nutrition at the individual, organizational, and systemic levels (Fig. 1). The framework considers capacity development as a hierarchy of needs, portrayed as a pyramid of four interdependent levels, starting from the ‘easier to achieve’ at the apex of the pyramid (tools, skills, staff, and infrastructure) to the ‘much harder’ to implement at its bottom (systems, structures, and roles). The different levels of the pyramid are further divided into nine interdependent sub-components. The topics covered in each of the surveyed countries, for each sub-component, are highlighted in Table 1.
Capacity building pyramid-Potter and Brough (7).
Capacity development issues covered in each of the surveyed countries
Derived from Potter and Brough (7).
Ethical considerations:
Administrative authorization was sought by sending a formal correspondence to the head of all of the targeted institutions prior to data collection. We did not seek ethical approval for the study as there was no data collection on human subjects. All respondents were fully informed about the purposes of the study. They gave their full verbal consent before participating in the study.
Results:
In the 13 surveyed countries, semi-structured interviews were conducted with 44 government officials and 51 stakeholders, namely representatives of non-governmental organizations, UN agencies, and donors (Table 2). We identified 305 nutrition degree programs organized by 113 tertiary-level institutions and 127 training programs organized by 52 health professional schools.
Data collection methods and participating institutions
Capacity at individual level (tools and skills) We report the findings relating to logistics, information management, funding, and the quality of nutrition training. In all the 13 surveyed countries, nutrition service providers, in general, had the needed equipment (weighing scales, measuring boards, mid-upper arm circumference tapes, behavior change materials, etc.) and supplies to perform nutrition-related tasks, although they were not available everywhere or at all service delivery points. Equipment and nutrition supplies (especially ready-to-use therapeutic foods) were mainly provided by development partners. Concerted efforts had been made to ensure the availability of these nutrition products at all service delivery points. Sporadic stock-outs of nutrition supplies were reported in most of the countries as a result of a weak logistic and supply chain system, poor forecasting of nutrition supplies at lower levels, and inadequate transportation systems. As of the time of our study, development partners were playing a strong role in the procurement and distribution of these nutrition supplies.
A common challenge for all 13 countries was the scarcity of financial resources for nutrition activities (Table 3). Although some countries, such as Ghana, Mauritania, Nigeria, and Senegal, experienced an increasing trend in the budget allocated to nutrition activities over the past years, there still remained some important financial gaps. At the time of our study, only a few countries in the region (Niger, Nigeria, and Senegal) had succeeded in securing a budgetary line for nutrition activities in their national budgets. Other countries, such as Benin, Burkina Faso, Mauritania, and Ghana, were in the process of doing so. As a result, the bulk of resources allocated for nutrition activities in the region was provided by donors and development partners. In Burkina-Faso, some nutrition activities were financed through a pooled fund portfolio, which contained mostly funds from donors and development partners.
Another common observation was the difficulty in getting accurate information about nutrition financing. The fact that resources come from various sources and cut across many sectors made it difficult to track investments and expenditures on nutrition. In Ghana for example, each government unit carrying out nutrition activities had its own budget that was often embedded in a higher level budgetary category. Efforts were underway in most of the countries to improve clarity in overall nutrition budgeting. In Ghana, a sub-committee on resource mobilization had been established, within the Cross-Sectoral Planning Group (CSPG) of the national SUN movement secretariat, to track resource allocation for nutrition in the country.
Inadequate human resources for nutrition were a major barrier to the implementation of nutrition activities (Table 3). In all the 13 countries, there was a critical shortage of skilled nutrition professionals, especially at lower levels. Nutrition graduates did not have practical experience and were not adequately prepared to tackle real-life nutrition issues. There were indeed important gaps in tertiary-level nutrition training in all the 13 countries. Nutrition training curricula were not aligned to regional nutrition priorities. In addition, there did not seem to be a coordinated approach toward harmonization of nutrition training curricula. Existing nutrition degree programs did not provide a full coverage of all essential aspects of human nutrition. Details about the current capacity for nutrition training in the surveyed countries have been reported elsewhere (13).
In the absence of a critical mass of skilled nutrition professionals, the bulk of nutrition work was done by health workers who did not have a good understanding of the application of nutrition principles to public health or clinical practice. As a result, they did not have broad enough skills to provide quality nutrition services. We noted that some countries were taking steps to enhance the performance of health workers through in-service training and mentoring in nutrition. However, these efforts were mainly oriented toward the management of acute malnutrition. In addition, continuing training efforts were mainly concentrated on staff at national and regional levels, leaving staff at lower levels with inadequate capacities in nutrition.
Similarities and differences in capacity assets and gaps across all the surveyed countries
We report the findings relating to logistics, information management, funding, and the quality of nutrition training. In all the 13 surveyed countries, nutrition service providers, in general, had the needed equipment (weighing scales, measuring boards, mid-upper arm circumference tapes, behavior change materials, etc.) and supplies to perform nutrition-related tasks, although they were not available everywhere or at all service delivery points. Equipment and nutrition supplies (especially ready-to-use therapeutic foods) were mainly provided by development partners. Concerted efforts had been made to ensure the availability of these nutrition products at all service delivery points. Sporadic stock-outs of nutrition supplies were reported in most of the countries as a result of a weak logistic and supply chain system, poor forecasting of nutrition supplies at lower levels, and inadequate transportation systems. As of the time of our study, development partners were playing a strong role in the procurement and distribution of these nutrition supplies.
A common challenge for all 13 countries was the scarcity of financial resources for nutrition activities (Table 3). Although some countries, such as Ghana, Mauritania, Nigeria, and Senegal, experienced an increasing trend in the budget allocated to nutrition activities over the past years, there still remained some important financial gaps. At the time of our study, only a few countries in the region (Niger, Nigeria, and Senegal) had succeeded in securing a budgetary line for nutrition activities in their national budgets. Other countries, such as Benin, Burkina Faso, Mauritania, and Ghana, were in the process of doing so. As a result, the bulk of resources allocated for nutrition activities in the region was provided by donors and development partners. In Burkina-Faso, some nutrition activities were financed through a pooled fund portfolio, which contained mostly funds from donors and development partners.
Another common observation was the difficulty in getting accurate information about nutrition financing. The fact that resources come from various sources and cut across many sectors made it difficult to track investments and expenditures on nutrition. In Ghana for example, each government unit carrying out nutrition activities had its own budget that was often embedded in a higher level budgetary category. Efforts were underway in most of the countries to improve clarity in overall nutrition budgeting. In Ghana, a sub-committee on resource mobilization had been established, within the Cross-Sectoral Planning Group (CSPG) of the national SUN movement secretariat, to track resource allocation for nutrition in the country.
Inadequate human resources for nutrition were a major barrier to the implementation of nutrition activities (Table 3). In all the 13 countries, there was a critical shortage of skilled nutrition professionals, especially at lower levels. Nutrition graduates did not have practical experience and were not adequately prepared to tackle real-life nutrition issues. There were indeed important gaps in tertiary-level nutrition training in all the 13 countries. Nutrition training curricula were not aligned to regional nutrition priorities. In addition, there did not seem to be a coordinated approach toward harmonization of nutrition training curricula. Existing nutrition degree programs did not provide a full coverage of all essential aspects of human nutrition. Details about the current capacity for nutrition training in the surveyed countries have been reported elsewhere (13).
In the absence of a critical mass of skilled nutrition professionals, the bulk of nutrition work was done by health workers who did not have a good understanding of the application of nutrition principles to public health or clinical practice. As a result, they did not have broad enough skills to provide quality nutrition services. We noted that some countries were taking steps to enhance the performance of health workers through in-service training and mentoring in nutrition. However, these efforts were mainly oriented toward the management of acute malnutrition. In addition, continuing training efforts were mainly concentrated on staff at national and regional levels, leaving staff at lower levels with inadequate capacities in nutrition.
Similarities and differences in capacity assets and gaps across all the surveyed countries
Capacity at organizational level (staff and infrastructure) The results on capacity at the organizational level relate to the production of human resources, staff placement, and supervision. A common characteristic for all the surveyed countries was a critical shortage of nutritionists (Table 3). Although at least 2,000 nutritionists are needed every year in the region, the current output was just at 700. The surveyed countries differed, however, in their capacity to support the expansion of nutrition workforce. Although the capacity to produce nutrition graduates was weak in most of the countries, there seemed to be a reasonable number of trained nutritionists in Nigeria, Ghana, and Sierra Leone. In 2013, there were about 1,000 undergraduate and 200 graduates enrolled in Nigeria, whereas Ghana had about 275 undergraduates and 50 graduates enrolled in nutrition. In Sierra Leone, the current output (45) outweighed the annual need of the country. However, the distribution of the nutrition workforce was inadequate in these three countries. In Nigeria, for example, the vast majority of trained nutritionists worked in urban areas. In addition, a significant proportion of them worked outside the nutrition sector. In Ghana, there was a critical shortage of nutritionists at the district level. This situation put considerable demand on health workers who had to perform nutrition-related tasks in the absence of skilled nutrition professionals. Because these health workers were overwhelmed by other equally important public health tasks, they tended to pay insufficient attention to nutrition. As a result, there was a lack of commitment to nutrition activities in all the surveyed countries.
In all the surveyed countries, training institutions supporting the development of nutrition workforce faced many challenges with teaching resources. They were poorly staffed, financially constrained, and had weak research capacity. They lacked teaching materials and equipment, infrastructure, libraries, and access to advanced technology resources. In most of the countries, there did not seem to be a coordinated effort toward interactions between academia, policy makers, and program managers. As a result, nutrition training institutions operated in silos and rarely participated in policy development or service delivery.
The countries also differed significantly in their approach with regard to staff placement (Table 3). Although some countries, such as Ghana, Liberia, Niger, Nigeria, and Sierra Leone, had a formal cadre of placement of nutritionists through the civil service commission, the other surveyed countries did not yet have a formal cadre of recognized nutritionists. A common pattern for all 13 countries was the lack of regulation of the nutrition profession. None of the countries had a set of minimum standards to qualify as a nutritionist in place. As a result, professionals from other sectors or people with little background in nutrition could be recruited and given the title of nutritionist.
Most of the countries faced challenges in supervising nutrition activities at lower levels. We noted that supportive supervision and mentoring on the job were not systematically or widely carried out. There was limited monitoring because of inadequate logistics and funding. All countries agreed that the provision of additional means of transport would enhance supervision and thereby strengthen program implementation.
The results on capacity at the organizational level relate to the production of human resources, staff placement, and supervision. A common characteristic for all the surveyed countries was a critical shortage of nutritionists (Table 3). Although at least 2,000 nutritionists are needed every year in the region, the current output was just at 700. The surveyed countries differed, however, in their capacity to support the expansion of nutrition workforce. Although the capacity to produce nutrition graduates was weak in most of the countries, there seemed to be a reasonable number of trained nutritionists in Nigeria, Ghana, and Sierra Leone. In 2013, there were about 1,000 undergraduate and 200 graduates enrolled in Nigeria, whereas Ghana had about 275 undergraduates and 50 graduates enrolled in nutrition. In Sierra Leone, the current output (45) outweighed the annual need of the country. However, the distribution of the nutrition workforce was inadequate in these three countries. In Nigeria, for example, the vast majority of trained nutritionists worked in urban areas. In addition, a significant proportion of them worked outside the nutrition sector. In Ghana, there was a critical shortage of nutritionists at the district level. This situation put considerable demand on health workers who had to perform nutrition-related tasks in the absence of skilled nutrition professionals. Because these health workers were overwhelmed by other equally important public health tasks, they tended to pay insufficient attention to nutrition. As a result, there was a lack of commitment to nutrition activities in all the surveyed countries.
In all the surveyed countries, training institutions supporting the development of nutrition workforce faced many challenges with teaching resources. They were poorly staffed, financially constrained, and had weak research capacity. They lacked teaching materials and equipment, infrastructure, libraries, and access to advanced technology resources. In most of the countries, there did not seem to be a coordinated effort toward interactions between academia, policy makers, and program managers. As a result, nutrition training institutions operated in silos and rarely participated in policy development or service delivery.
The countries also differed significantly in their approach with regard to staff placement (Table 3). Although some countries, such as Ghana, Liberia, Niger, Nigeria, and Sierra Leone, had a formal cadre of placement of nutritionists through the civil service commission, the other surveyed countries did not yet have a formal cadre of recognized nutritionists. A common pattern for all 13 countries was the lack of regulation of the nutrition profession. None of the countries had a set of minimum standards to qualify as a nutritionist in place. As a result, professionals from other sectors or people with little background in nutrition could be recruited and given the title of nutritionist.
Most of the countries faced challenges in supervising nutrition activities at lower levels. We noted that supportive supervision and mentoring on the job were not systematically or widely carried out. There was limited monitoring because of inadequate logistics and funding. All countries agreed that the provision of additional means of transport would enhance supervision and thereby strengthen program implementation.
Capacity at systemic level (structures, systems, and roles) The results on capacity at systemic level relate to governance, coordination mechanisms, and information management. There was strong momentum to improve nutrition in most of the countries. This was illustrated by the existence of a set of policies and plans on nutrition, which are used to prioritize actions and track progress toward the achievement of stated objectives (Table 3). However, much remained to be done to align the efforts of all stakeholders to these national nutrition policies and plans. The fact that all the surveyed countries had signed up to the SUN movement was a positive move toward improved nutrition governance in the West Africa region.
Most of the countries had also set up national multisectoral, multi-stakeholder platforms that brought together all the major stakeholders in the nutrition area. These platforms offered the opportunity to share lessons and best practices, as well as to reduce duplication of efforts. Their location differed in the surveyed countries. In Benin for example, the coordinating body was placed directly under the Office of the President, whereas it was within the Prime Minister's office in Niger and Senegal. Ghana and Mauritania had set up a separate government body that oversaw nutrition activities at national level. The Ministry of Health continued to coordinate nutrition activities in Burkina Faso, Cote d'Ivoire, Liberia, Mali, and Nigeria. Cote d'Ivoire and Liberia were, however, taking steps to establish, within the framework of their involvement in the SUN movement, a multisectoral, multi-stakeholder coordination mechanism for nutrition activities. Other new initiatives aimed at improving nutrition security were also ongoing in the surveyed countries. The Renewed Efforts Against Child Hunger and undernutrition (REACH) had also gained momentum in five countries in West Africa (Ghana, Mali, Mauritania, Niger, and Sierra Leone). The European Union and other development partners had also launched the Global Alliance for Resilience Initiative (AGIR).
A common weakness in all countries was the lack of or inadequate coordination mechanisms at lower levels. The cross-disciplinary nature of nutrition made it difficult to coordinate nutrition activities, leading to a high degree of fragmentation of nutrition activities across several ministries, departments, and agencies. In Ghana, coordination at the national, regional, and district levels was being strengthened, institutionalized, and empowered to enhance their effectiveness. Countries such as Benin, Burkina Faso, and Nigeria were taking steps to strengthen the coordination of nutrition activities at their policy and operational levels.
The vast majority of the surveyed countries had made considerable efforts to strengthen the nutrition data collection system. Nutrition surveys, using the SMART methodology, were now being conducted on a regular basis in most of the countries and the survey results were being used for decision-making. Nutrition data were also generated through Demographic and Health Surveys (DHS) or Multiple Indicator Cluster Surveys (MICS). Despite these advancements, there were still some unmet needs. None of the countries had as yet succeeded in putting in place a single, multisectoral information system for nutrition that will guide decision-making processes across the different sectors (Table 3). In addition, much remained to be done to improve the accuracy of routine nutrition data collected at lower levels.
The results on capacity at systemic level relate to governance, coordination mechanisms, and information management. There was strong momentum to improve nutrition in most of the countries. This was illustrated by the existence of a set of policies and plans on nutrition, which are used to prioritize actions and track progress toward the achievement of stated objectives (Table 3). However, much remained to be done to align the efforts of all stakeholders to these national nutrition policies and plans. The fact that all the surveyed countries had signed up to the SUN movement was a positive move toward improved nutrition governance in the West Africa region.
Most of the countries had also set up national multisectoral, multi-stakeholder platforms that brought together all the major stakeholders in the nutrition area. These platforms offered the opportunity to share lessons and best practices, as well as to reduce duplication of efforts. Their location differed in the surveyed countries. In Benin for example, the coordinating body was placed directly under the Office of the President, whereas it was within the Prime Minister's office in Niger and Senegal. Ghana and Mauritania had set up a separate government body that oversaw nutrition activities at national level. The Ministry of Health continued to coordinate nutrition activities in Burkina Faso, Cote d'Ivoire, Liberia, Mali, and Nigeria. Cote d'Ivoire and Liberia were, however, taking steps to establish, within the framework of their involvement in the SUN movement, a multisectoral, multi-stakeholder coordination mechanism for nutrition activities. Other new initiatives aimed at improving nutrition security were also ongoing in the surveyed countries. The Renewed Efforts Against Child Hunger and undernutrition (REACH) had also gained momentum in five countries in West Africa (Ghana, Mali, Mauritania, Niger, and Sierra Leone). The European Union and other development partners had also launched the Global Alliance for Resilience Initiative (AGIR).
A common weakness in all countries was the lack of or inadequate coordination mechanisms at lower levels. The cross-disciplinary nature of nutrition made it difficult to coordinate nutrition activities, leading to a high degree of fragmentation of nutrition activities across several ministries, departments, and agencies. In Ghana, coordination at the national, regional, and district levels was being strengthened, institutionalized, and empowered to enhance their effectiveness. Countries such as Benin, Burkina Faso, and Nigeria were taking steps to strengthen the coordination of nutrition activities at their policy and operational levels.
The vast majority of the surveyed countries had made considerable efforts to strengthen the nutrition data collection system. Nutrition surveys, using the SMART methodology, were now being conducted on a regular basis in most of the countries and the survey results were being used for decision-making. Nutrition data were also generated through Demographic and Health Surveys (DHS) or Multiple Indicator Cluster Surveys (MICS). Despite these advancements, there were still some unmet needs. None of the countries had as yet succeeded in putting in place a single, multisectoral information system for nutrition that will guide decision-making processes across the different sectors (Table 3). In addition, much remained to be done to improve the accuracy of routine nutrition data collected at lower levels.
Capacity at individual level (tools and skills):
We report the findings relating to logistics, information management, funding, and the quality of nutrition training. In all the 13 surveyed countries, nutrition service providers, in general, had the needed equipment (weighing scales, measuring boards, mid-upper arm circumference tapes, behavior change materials, etc.) and supplies to perform nutrition-related tasks, although they were not available everywhere or at all service delivery points. Equipment and nutrition supplies (especially ready-to-use therapeutic foods) were mainly provided by development partners. Concerted efforts had been made to ensure the availability of these nutrition products at all service delivery points. Sporadic stock-outs of nutrition supplies were reported in most of the countries as a result of a weak logistic and supply chain system, poor forecasting of nutrition supplies at lower levels, and inadequate transportation systems. As of the time of our study, development partners were playing a strong role in the procurement and distribution of these nutrition supplies.
A common challenge for all 13 countries was the scarcity of financial resources for nutrition activities (Table 3). Although some countries, such as Ghana, Mauritania, Nigeria, and Senegal, experienced an increasing trend in the budget allocated to nutrition activities over the past years, there still remained some important financial gaps. At the time of our study, only a few countries in the region (Niger, Nigeria, and Senegal) had succeeded in securing a budgetary line for nutrition activities in their national budgets. Other countries, such as Benin, Burkina Faso, Mauritania, and Ghana, were in the process of doing so. As a result, the bulk of resources allocated for nutrition activities in the region was provided by donors and development partners. In Burkina-Faso, some nutrition activities were financed through a pooled fund portfolio, which contained mostly funds from donors and development partners.
Another common observation was the difficulty in getting accurate information about nutrition financing. The fact that resources come from various sources and cut across many sectors made it difficult to track investments and expenditures on nutrition. In Ghana for example, each government unit carrying out nutrition activities had its own budget that was often embedded in a higher level budgetary category. Efforts were underway in most of the countries to improve clarity in overall nutrition budgeting. In Ghana, a sub-committee on resource mobilization had been established, within the Cross-Sectoral Planning Group (CSPG) of the national SUN movement secretariat, to track resource allocation for nutrition in the country.
Inadequate human resources for nutrition were a major barrier to the implementation of nutrition activities (Table 3). In all the 13 countries, there was a critical shortage of skilled nutrition professionals, especially at lower levels. Nutrition graduates did not have practical experience and were not adequately prepared to tackle real-life nutrition issues. There were indeed important gaps in tertiary-level nutrition training in all the 13 countries. Nutrition training curricula were not aligned to regional nutrition priorities. In addition, there did not seem to be a coordinated approach toward harmonization of nutrition training curricula. Existing nutrition degree programs did not provide a full coverage of all essential aspects of human nutrition. Details about the current capacity for nutrition training in the surveyed countries have been reported elsewhere (13).
In the absence of a critical mass of skilled nutrition professionals, the bulk of nutrition work was done by health workers who did not have a good understanding of the application of nutrition principles to public health or clinical practice. As a result, they did not have broad enough skills to provide quality nutrition services. We noted that some countries were taking steps to enhance the performance of health workers through in-service training and mentoring in nutrition. However, these efforts were mainly oriented toward the management of acute malnutrition. In addition, continuing training efforts were mainly concentrated on staff at national and regional levels, leaving staff at lower levels with inadequate capacities in nutrition.
Similarities and differences in capacity assets and gaps across all the surveyed countries
Capacity at organizational level (staff and infrastructure):
The results on capacity at the organizational level relate to the production of human resources, staff placement, and supervision. A common characteristic for all the surveyed countries was a critical shortage of nutritionists (Table 3). Although at least 2,000 nutritionists are needed every year in the region, the current output was just at 700. The surveyed countries differed, however, in their capacity to support the expansion of nutrition workforce. Although the capacity to produce nutrition graduates was weak in most of the countries, there seemed to be a reasonable number of trained nutritionists in Nigeria, Ghana, and Sierra Leone. In 2013, there were about 1,000 undergraduate and 200 graduates enrolled in Nigeria, whereas Ghana had about 275 undergraduates and 50 graduates enrolled in nutrition. In Sierra Leone, the current output (45) outweighed the annual need of the country. However, the distribution of the nutrition workforce was inadequate in these three countries. In Nigeria, for example, the vast majority of trained nutritionists worked in urban areas. In addition, a significant proportion of them worked outside the nutrition sector. In Ghana, there was a critical shortage of nutritionists at the district level. This situation put considerable demand on health workers who had to perform nutrition-related tasks in the absence of skilled nutrition professionals. Because these health workers were overwhelmed by other equally important public health tasks, they tended to pay insufficient attention to nutrition. As a result, there was a lack of commitment to nutrition activities in all the surveyed countries.
In all the surveyed countries, training institutions supporting the development of nutrition workforce faced many challenges with teaching resources. They were poorly staffed, financially constrained, and had weak research capacity. They lacked teaching materials and equipment, infrastructure, libraries, and access to advanced technology resources. In most of the countries, there did not seem to be a coordinated effort toward interactions between academia, policy makers, and program managers. As a result, nutrition training institutions operated in silos and rarely participated in policy development or service delivery.
The countries also differed significantly in their approach with regard to staff placement (Table 3). Although some countries, such as Ghana, Liberia, Niger, Nigeria, and Sierra Leone, had a formal cadre of placement of nutritionists through the civil service commission, the other surveyed countries did not yet have a formal cadre of recognized nutritionists. A common pattern for all 13 countries was the lack of regulation of the nutrition profession. None of the countries had a set of minimum standards to qualify as a nutritionist in place. As a result, professionals from other sectors or people with little background in nutrition could be recruited and given the title of nutritionist.
Most of the countries faced challenges in supervising nutrition activities at lower levels. We noted that supportive supervision and mentoring on the job were not systematically or widely carried out. There was limited monitoring because of inadequate logistics and funding. All countries agreed that the provision of additional means of transport would enhance supervision and thereby strengthen program implementation.
Capacity at systemic level (structures, systems, and roles):
The results on capacity at systemic level relate to governance, coordination mechanisms, and information management. There was strong momentum to improve nutrition in most of the countries. This was illustrated by the existence of a set of policies and plans on nutrition, which are used to prioritize actions and track progress toward the achievement of stated objectives (Table 3). However, much remained to be done to align the efforts of all stakeholders to these national nutrition policies and plans. The fact that all the surveyed countries had signed up to the SUN movement was a positive move toward improved nutrition governance in the West Africa region.
Most of the countries had also set up national multisectoral, multi-stakeholder platforms that brought together all the major stakeholders in the nutrition area. These platforms offered the opportunity to share lessons and best practices, as well as to reduce duplication of efforts. Their location differed in the surveyed countries. In Benin for example, the coordinating body was placed directly under the Office of the President, whereas it was within the Prime Minister's office in Niger and Senegal. Ghana and Mauritania had set up a separate government body that oversaw nutrition activities at national level. The Ministry of Health continued to coordinate nutrition activities in Burkina Faso, Cote d'Ivoire, Liberia, Mali, and Nigeria. Cote d'Ivoire and Liberia were, however, taking steps to establish, within the framework of their involvement in the SUN movement, a multisectoral, multi-stakeholder coordination mechanism for nutrition activities. Other new initiatives aimed at improving nutrition security were also ongoing in the surveyed countries. The Renewed Efforts Against Child Hunger and undernutrition (REACH) had also gained momentum in five countries in West Africa (Ghana, Mali, Mauritania, Niger, and Sierra Leone). The European Union and other development partners had also launched the Global Alliance for Resilience Initiative (AGIR).
A common weakness in all countries was the lack of or inadequate coordination mechanisms at lower levels. The cross-disciplinary nature of nutrition made it difficult to coordinate nutrition activities, leading to a high degree of fragmentation of nutrition activities across several ministries, departments, and agencies. In Ghana, coordination at the national, regional, and district levels was being strengthened, institutionalized, and empowered to enhance their effectiveness. Countries such as Benin, Burkina Faso, and Nigeria were taking steps to strengthen the coordination of nutrition activities at their policy and operational levels.
The vast majority of the surveyed countries had made considerable efforts to strengthen the nutrition data collection system. Nutrition surveys, using the SMART methodology, were now being conducted on a regular basis in most of the countries and the survey results were being used for decision-making. Nutrition data were also generated through Demographic and Health Surveys (DHS) or Multiple Indicator Cluster Surveys (MICS). Despite these advancements, there were still some unmet needs. None of the countries had as yet succeeded in putting in place a single, multisectoral information system for nutrition that will guide decision-making processes across the different sectors (Table 3). In addition, much remained to be done to improve the accuracy of routine nutrition data collected at lower levels.
Discussion:
In our region-wide assessment of the current capacity to act in nutrition in West Africa, we analyzed key capacity assets and gaps as well as cross-country similarities and differences. Our findings reveal that important efforts have been made over the past years to raise the profile of nutrition to a high priority in the surveyed countries. The nutrition landscape has been evolving in most of the countries in West Africa since 2011. In addition, advocacy efforts are underway to sustain this momentum and keep governments involved. This is a positive move toward the achievement of nutrition-related MDGs in the region. Donors and development partners have also played an important role in this process. The different nutrition initiatives supported by development partners (SUN, REACH, AGIR, etc.) have greatly contributed to the advancement of nutrition in the region.
Despite these positive developments, there are still some challenges and unmet needs. There is a need for clear divisions of tasks between ongoing nutrition initiatives. For example, five of the 13 surveyed countries (Ghana, Mali, Mauritania, Niger, and Sierra Leone) are both SUN and REACH countries. It is critical to ensure good coordination between these two important initiatives as both aim to support country-led processes to improve nutrition governance and enable multisectoral, multi-stakeholders approach to addressing undernutrition (15, 16). This could help prevent potential misunderstandings that may arise. The confusion in multiple nutrition initiatives has been reported in Niger (17).
Our findings also indicate that lack of funding is a major barrier for the implementation of nutrition activities. There is indeed a clear disconnect between national nutrition goals and the resources available for the implementation of nutrition activities. This situation slows the translation of governments’ commitments for nutrition into tangible actions on the ground. We found a high dependency on donor resources for nutrition activities as a result of low government investments. It is critical that governments in the West Africa region take on greater responsibility for nutrition financing.
Another major challenge that was common was the inability to track investments and expenditures on nutrition activities. The importance of having accurate information on nutrition financing cannot be overemphasized. These data are not only crucial for planning purposes (to make projections for nutrition financing), but are also vital in identifying financial gaps and ensuring efficiency and accountability. There is an urgent need to improve the systems for tracking allocations and expenditures for nutrition activities in the surveyed countries. The workshop on costing and tracking investments in nutrition, co-organized by UNICEF and the SUN movement secretariat in Nairobi, Kenya, in November 2013 (18), was a preliminary step in moving toward that direction. Much remains to be done to improve transparency and clarity over nutrition budgeting in the West Africa region. It is gratifying that the SUN movement secretariat continues to work with countries to ensure that nutrition programs are costed and that resources allocated to nutrition activities are tracked (14).
The results of our study indicate that shortage of skilled human resources to deliver nutrition interventions is a major problem in all countries. There is a clear need to support the expansion of the nutrition workforce in the region and provide incentives for nutrition service providers to stay in service. Training institutions should be empowered to support the expansion of the nutrition workforce. In addition, attention should be paid to the pre-service and in-service training in nutrition of health professionals. Our findings also indicate that there is a lack of regulation of the nutrition profession. The need to set a minimum standard to qualify as a nutritionist in the region has been previously stressed (19, 20). It would also be important to create a proper cadre of placement for nutrition service providers and clearly specify their roles and responsibilities.
Overall, our data are consistent with findings in previous studies in some of the West African countries (9, 10). They underscore the urgent need to strengthen capacity for nutrition at individual, organizational, and systemic levels. Our results are also in line with findings in previous studies in other sub-Saharan African countries such as Malawi (21), Namibia (22), and Angola (23). Some common elements are the limited financial resources for nutrition activities, the lack of human resources for nutrition, the lack of pre-service nutrition training programs, the limited capacity to support the expansion of a nutrition workforce, and the lack of cross-sectoral coordination. Clearly, support is urgently needed to build the needed capacity to accelerate progress for nutrition in Africa.
It is not our intention in this paper to make specific recommendations on how to develop capacity for nutrition in the West Africa region. We believe that this should be done in a participatory manner, with the involvement of all stakeholders. For instance, we are planning to convene a regional workshop to discuss the findings from this study and build consensus on the way forward. The ultimate goal of this workshop would be to create a unified discourse between stakeholders on how to strengthen the capacity to act in nutrition in the West Africa region. This will ultimately lead to the development of a consensual regional capacity development strategy that will help shape future nutrition actions.
Our study has several limitations. It was difficult to obtain quantitative data on financial investments in nutrition as they were not readily available in the surveyed countries. In addition, we did not have the opportunity to assess the adequacy of infrastructure for nutrition services through direct observations. Given the rapidly evolving nutrition environment in West Africa, it is possible that some of the information reported in this paper may not fully reflect the current situation on the ground. However, we believe that our study provides a comprehensive view on the current capacity to act in nutrition in the West Africa region.
Conclusions:
Nutrition programs in West Africa are characterized by a critical shortage of skilled human resources, limited funding, high dependency on donor resources, weak logistic and infrastructure systems, and lack of supervision as well as coordination of nutrition activities at lower levels. These factors hamper the ability to act in nutrition in the West Africa region. Addressing these unmet needs is critical in moving toward improved nutrition outcomes in the region. There is an urgent need for a shift toward wider reforms for nutrition capacity development in the West Africa region.
|
Background:
Although it is widely accepted that lack of capacity is one of the barriers to scaling up nutrition in West Africa, there is a paucity of information about what capacities exist and the capacities that need to be developed to accelerate progress toward improved nutrition outcomes in the region.
Methods:
Data were collected from 13 West African countries through interviews with government officials, key development partners, tertiary-level training institutions, and health professional schools. The assessment was based on a conceptual framework of four interdependent levels (tools; skills; staff and infrastructure; and structures, systems and roles). In each of the surveyed countries, we assessed capacity assets and gaps at individual, organizational, and systemic levels.
Results:
Important similarities and differences in capacity assets and gaps emerged across all the surveyed countries. There was strong momentum to improve nutrition in nearly all the surveyed countries. Most of the countries had a set of policies on nutrition in place and had set up multisectoral, multi-stakeholder platforms to coordinate nutrition activities, although much remained to be done to improve the effectiveness of these platforms. Many initiatives aimed to reduce undernutrition were ongoing in the region, but there did not seem to be clear coordination between them. Insufficient financial resources to implement nutrition activities were a major problem in all countries. The bulk of financial allocations for nutrition was provided by development partners, even though some countries, such as Niger, Nigeria, and Senegal, had a national budget line for nutrition. Sporadic stock-outs of nutrition supplies were reported in most of the countries as a result of a weak logistic and supply chain system. They also had a critical shortage of skilled nutrition professionals. There was limited supervision of nutrition activities, especially at lower levels. Nigeria and Ghana emerged as the countries with the greatest capacities to support the expansion of a nutrition workforce, although a significant proportion of their trained nutritionists were not employed in the nutrition sector. None of the countries had in place a unified nutrition information system that could guide decision-making processes across the different sectors.
Conclusions:
There is an urgent need for a shift toward wider reforms for nutrition capacity development in the West Africa region. Addressing these unmet needs is a critical first step toward improved capacity for action in nutrition in the region.
| null | null | 11,091 | 443 | 10 |
[
"nutrition",
"countries",
"surveyed countries",
"surveyed",
"capacity",
"data",
"training",
"activities",
"nutrition activities",
"development"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] capacity development | nutrition | undernutrition | nutrition workforce | West Africa [SUMMARY]
|
[CONTENT] capacity development | nutrition | undernutrition | nutrition workforce | West Africa [SUMMARY]
|
[CONTENT] capacity development | nutrition | undernutrition | nutrition workforce | West Africa [SUMMARY]
|
[CONTENT] capacity development | nutrition | undernutrition | nutrition workforce | West Africa [SUMMARY]
| null | null |
[CONTENT] Africa, Western | Capacity Building | Humans | Interviews as Topic | Malnutrition | Nutritional Sciences | Nutritional Status | Qualitative Research [SUMMARY]
|
[CONTENT] Africa, Western | Capacity Building | Humans | Interviews as Topic | Malnutrition | Nutritional Sciences | Nutritional Status | Qualitative Research [SUMMARY]
|
[CONTENT] Africa, Western | Capacity Building | Humans | Interviews as Topic | Malnutrition | Nutritional Sciences | Nutritional Status | Qualitative Research [SUMMARY]
|
[CONTENT] Africa, Western | Capacity Building | Humans | Interviews as Topic | Malnutrition | Nutritional Sciences | Nutritional Status | Qualitative Research [SUMMARY]
| null | null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null | null |
[CONTENT] nutrition | countries | surveyed countries | surveyed | capacity | data | training | activities | nutrition activities | development [SUMMARY]
|
[CONTENT] nutrition | countries | surveyed countries | surveyed | capacity | data | training | activities | nutrition activities | development [SUMMARY]
|
[CONTENT] nutrition | countries | surveyed countries | surveyed | capacity | data | training | activities | nutrition activities | development [SUMMARY]
|
[CONTENT] nutrition | countries | surveyed countries | surveyed | capacity | data | training | activities | nutrition activities | development [SUMMARY]
| null | null |
[CONTENT] nutrition | interviews | data | countries | collected | training | data collected | key | face | nutrition focal [SUMMARY]
|
[CONTENT] nutrition | countries | nutrition activities | activities | nutritionists | ghana | surveyed countries | surveyed | efforts | training [SUMMARY]
|
[CONTENT] nutrition | west | africa | west africa | region | africa region | critical | west africa region | weak logistic infrastructure systems | weak logistic infrastructure [SUMMARY]
|
[CONTENT] nutrition | countries | data | surveyed | surveyed countries | nutrition activities | capacity | training | activities | west [SUMMARY]
| null | null |
[CONTENT] 13 | West African | tertiary ||| four ||| [SUMMARY]
|
[CONTENT] ||| ||| ||| ||| ||| Niger, Nigeria | Senegal ||| ||| ||| ||| Nigeria | Ghana ||| [SUMMARY]
|
[CONTENT] West Africa ||| first [SUMMARY]
|
[CONTENT] ||| West Africa ||| 13 | West African | tertiary ||| four ||| ||| ||| ||| ||| ||| ||| Niger, Nigeria | Senegal ||| ||| ||| ||| Nigeria | Ghana ||| ||| West Africa ||| first [SUMMARY]
| null |
||||
Neuropilin-2 induced by transforming growth factor-β augments migration of hepatocellular carcinoma cells.
|
26573807
|
Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third most lethal cancer worldwide. The epithelial to mesenchymal transition (EMT) describes the transformation of well-differentiated epithelial cells to a de-differentiated phenotype and plays a central role in the invasion and intrahepatic metastasis of HCC cells. Modulation of the transforming growth factor-β (TGF-β) signaling is known to induce various tumor-promoting and EMT-inducing pathways in HCC. The meta-analysis of a panel of EMT gene expression studies revealed that neuropilin 2 (NRP2) is significantly upregulated in cells that have undergone EMT induced by TGF-β. In this study we assessed the functional role of NRP2 in epithelial and mesenchymal-like HCC cells and focused on the molecular interplay between NRP2 and TGF-β/Smad signaling.
|
BACKGROUND
|
NRP2 expression was analyzed in human HCC cell lines and tissue arrays comprising 133 HCC samples. Cell migration was examined by wound healing and Transwell assays in the presence and absence of siRNA against NRP2. NRP2 and TGF-β signaling were analyzed by Western blotting and confocal immunofluorescence microscopy.
|
METHODS
|
We show that NRP2 is particularly expressed in HCC cell lines with a dedifferentiated, mesenchymal-like phenotype. NRP2 expression is upregulated by the canonical TGF-β/Smad signaling while NRP2 expression has no impact on TGF-β signaling in HCC cells. Reduced expression of NRP2 by knock-down or inhibition of TGF-β signaling resulted in diminished cell migration independently of each other, suggesting that NRP2 fails to collaborate with TGF-β signaling in cell movement. In accordance with these data, elevated levels of NRP2 correlated with a higher tumor grade and less differentiation in a large collection of human HCC specimens.
|
RESULTS
|
These data suggest that NRP2 associates with a less differentiated, mesenchymal-like HCC phenotype and that NRP2 plays an important role in tumor cell migration upon TGF-β-dependent HCC progression.
|
CONCLUSIONS
|
[
"Blotting, Western",
"Carcinoma, Hepatocellular",
"Cell Movement",
"Humans",
"Liver Neoplasms",
"Neuropilin-2",
"Phenotype",
"Signal Transduction",
"Sulfhydryl Reagents",
"Tissue Array Analysis",
"Transforming Growth Factor beta",
"Tumor Cells, Cultured"
] |
4647494
|
Background
|
Liver cancer is the sixth most common cancer in the world and ranks second in the list of most deadly cancers [1]. The vast majority of liver cancers are hepatocellular carcinomas (HCC) representing up to 90 % of all liver malignancies [2, 3]. The main risk factors for HCC are chronic infections with either hepatitis B virus (HBV) or hepatitis C virus (HCV), making up approximately 75–85 % of all cases, as well as excessive alcohol consumption, which is responsible for about 40 % of HCC development in Western countries [2, 4–7]. Chronic inflammation and tissue damage by these agents leads to cirrhosis which is the underlying condition for the majority of HCC cases [8].
The dissemination of primary tumor cells into the body drastically worsens the prognosis of cancer patients [9]. Metastasis of HCC cells most frequently occurs intrahepatically rather than extrahepatically to distal sites such as the lung [10]. For spreading of HCC cells, individual cell movement by an epithelial to mesenchymal transition (EMT) has been considered to be essentially involved [11]. Upon EMT and progression in malignancy, highly differentiated epithelial cells such as hepatocytes de-differentiate into a mesenchymal-like phenotype that exhibits strong migratory abilities. Various signaling cascades are known to induce EMT, such as the Wnt/β-catenin, PI3K/AKT/mTOR, Hedgehog, Ras/Raf/MEK/ERK, Notch and NFκB pathways, as well as transforming growth factor (TGF)-β [12–15]. These signals mostly converge on EMT-transcription factors (EMT-TFs) such as Snail, ZEB1 or Twist1/2 which transcriptionally repress E-cadherin and other epithelial junctional proteins as well as activate a mesenchymal gene expression signature. In HCC, TGF-β signaling has been shown to activate EMT-TFs and to repress their negative feedback loops by the downregulation of miRNAs that antagonize EMT-TFs [16, 17].
A recently performed meta-analysis compared 24 published EMT gene expression data sets and generated a core list of genes that are most frequently altered during EMT [18]. One of the genes found upregulated in several studies of TGF-β-induced EMT coded for the protein neuropilin-2 (NRP2). There are two homologs of the NRP family, NRP1 and NRP2, which are 130 kDa single-pass transmembrane glycoproteins that act as non-tyrosine kinase co-receptors [19]. They contain 4 distinct domains including a CUB domain, a FV/FVIII domain, a MAM domain and a domain that contains the transmembrane and short cytoplasmic region [20]. Both homologues can homo- and heteromultimerize [21] and can bind different members of the semaphorin family, as well as members of the vascular endothelial growth factor (VEGF) family [22]. In addition, NRPs are receptors of hepatocyte growth factor, platelet-derived growth factor BB, fibroblast growth factor, epidermal growth factor, placenta growth factor, and importantly of TGF-β1 [23–25]. NRPs are therefore critical regulators of angiogenesis, lymphangiogenesis and tumor progression. NRP2 expression is correlated with lymph node metastasis in breast cancer and blocking of NRP2 leads to decreased metastasis formation [26–28]. Clinical data show that high NRP levels, in particular NRP2, correlate with poor prognosis and survival in various cancer types [29, 30]. NRP2 was suggested to play a direct role in EMT and a cross-talk between NRP2 and TGF-β1 signaling promotes colorectal cancer progression [31]. In the context of HCC, the role of NRP2 is so far unknown [32].
In this study, we show that NRP2 expression strongly correlates with a mesenchymal phenotype in HCC cell lines and that reduced levels of NRP2 dramatically impair the migratory abilities of HCC cells. We further provide evidence that NRP2 expression is controlled by canonical TGF-β signaling, while no direct impact of NRP2 on TGF-β signaling could be observed. Translation of these data into the HCC patient situation revealed a correlation of NRP2 levels with a poorly differentiated HCC, suggesting a role of NRP2 in TGF-β regulated HCC progression.
| null | null |
Results
|
NRP2 is upregulated in high-grade HCC and highly expressed in mesenchymal-like HCC cell lines We employed a tissue array containing 133 HCC cases for the analysis of NRP2 expression and found a significant correlation of NRP2 levels with higher tumor grading. While well-differentiated HCC (grade 1) showed NRP2 expression in 32 % of cases, less-differentiated HCC samples displayed a nearly twice as much higher frequency of NRP2 presence. In particular, 59 % of HCC with grade 2 and 56 % of HCC with grade 3 tumors showed NRP2 expression (Fig. 1a). Generally, NRP2 was found distributed in a patchy fashion (Fig. 1b) and interestingly, NRP2 was exclusively expressed in those patient samples that showed TGF-β1 expression (Additional file 1: Figure S1). Noteworthy, normal liver did not display NRP2 expression (Fig. 1b) which confirmed recent observations that NRP2 is not expressed in hepatocytes under physiological conditions [35].Fig. 1NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01
NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01
To correlate NRP2 expression with differentiation of hepatoma cells more closely, we analyzed various human HCC cell lines with differentiated and epithelial traits versus those exhibiting a de-differentiated and mesenchymal-like phenotype. Western blot (Fig. 1c) and qPCR analyses (Fig. 1d) revealed that NRP2 expression strongly correlated with a mesenchymal-like HCC phenotype in 3sp, SNU-398, SNU-423, SNU-449, SNU-475 and FLC-4 cells. In contrast, NRP2 was expressed in only one out of five epithelial HCC cell lines, i.e., HuH-6 cells, and showed undetectable levels of NRP2 in the other epithelial 3p, Hep3B, HepG2 and PLC cells. Together, these data suggest that NRP2 expression associates with less-differentiated high-grade tumors and TGF-β1 expression in HCC patients. In agreement, NRP2 expression was found in de-differentiated mesenchymal-like HCC cells in vitro which supports the idea that NRP2 expression correlates with a de-differentiated phenotype.
We employed a tissue array containing 133 HCC cases for the analysis of NRP2 expression and found a significant correlation of NRP2 levels with higher tumor grading. While well-differentiated HCC (grade 1) showed NRP2 expression in 32 % of cases, less-differentiated HCC samples displayed a nearly twice as much higher frequency of NRP2 presence. In particular, 59 % of HCC with grade 2 and 56 % of HCC with grade 3 tumors showed NRP2 expression (Fig. 1a). Generally, NRP2 was found distributed in a patchy fashion (Fig. 1b) and interestingly, NRP2 was exclusively expressed in those patient samples that showed TGF-β1 expression (Additional file 1: Figure S1). Noteworthy, normal liver did not display NRP2 expression (Fig. 1b) which confirmed recent observations that NRP2 is not expressed in hepatocytes under physiological conditions [35].Fig. 1NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01
NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01
To correlate NRP2 expression with differentiation of hepatoma cells more closely, we analyzed various human HCC cell lines with differentiated and epithelial traits versus those exhibiting a de-differentiated and mesenchymal-like phenotype. Western blot (Fig. 1c) and qPCR analyses (Fig. 1d) revealed that NRP2 expression strongly correlated with a mesenchymal-like HCC phenotype in 3sp, SNU-398, SNU-423, SNU-449, SNU-475 and FLC-4 cells. In contrast, NRP2 was expressed in only one out of five epithelial HCC cell lines, i.e., HuH-6 cells, and showed undetectable levels of NRP2 in the other epithelial 3p, Hep3B, HepG2 and PLC cells. Together, these data suggest that NRP2 expression associates with less-differentiated high-grade tumors and TGF-β1 expression in HCC patients. In agreement, NRP2 expression was found in de-differentiated mesenchymal-like HCC cells in vitro which supports the idea that NRP2 expression correlates with a de-differentiated phenotype.
NRP2 regulates HCC cell migration and invasion Next we analyzed the functional impact of NRP2 expression on cell proliferation and migration. Neither knock-down nor exogenous overexpression of NRP2 significantly affected proliferation kinetics of mesenchymal-like HCC cells (data not shown). However, Transwell migration assays employing the mesenchymal-like cell lines 3sp and SNU-449 revealed that reduced expression of NRP2 results in impaired migratory abilities (Fig. 2a). Comparable results were obtained in Transwell invasion and transendothelial migration assays (Fig. 2b, c). In addition, assessment of cell migration by wound healing assays of SNU-449 cells confirmed elevated migratory capabilities dependent on NRP2 (Fig. 2d). Interestingly, overexpression of NRP2 could not enhance migration or invasion in any of the employed cell lines (3p, PLC, 3sp, SNU-449; data not shown). Thus, these results suggest that NRP2 is crucially involved in upregulating cell movement of de-differentiated HCC cells.Fig. 2Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001
Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001
Next we analyzed the functional impact of NRP2 expression on cell proliferation and migration. Neither knock-down nor exogenous overexpression of NRP2 significantly affected proliferation kinetics of mesenchymal-like HCC cells (data not shown). However, Transwell migration assays employing the mesenchymal-like cell lines 3sp and SNU-449 revealed that reduced expression of NRP2 results in impaired migratory abilities (Fig. 2a). Comparable results were obtained in Transwell invasion and transendothelial migration assays (Fig. 2b, c). In addition, assessment of cell migration by wound healing assays of SNU-449 cells confirmed elevated migratory capabilities dependent on NRP2 (Fig. 2d). Interestingly, overexpression of NRP2 could not enhance migration or invasion in any of the employed cell lines (3p, PLC, 3sp, SNU-449; data not shown). Thus, these results suggest that NRP2 is crucially involved in upregulating cell movement of de-differentiated HCC cells.Fig. 2Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001
Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001
TGF-β induces NRP2 in a Smad-dependent fashion Since NRP2 expression correlated with TGF-β1 expression (Additional file 1: Figure S1) and TGF-β signaling is a common trigger of EMT in HCC, we asked whether it directly influences NRP2 expression in HCC cells. Remarkably, TGF-β treatment of epithelial 3p, Hep3B and PLC cells, as well as mesenchymal-like 3sp, SNU-423 and SNU-449 cells resulted in increased NRP2 protein expression (Fig. 3a). Upregulation of NRP2 was essentially confirmed at transcript levels by qPCR analysis of epithelial (3p, Hep3B, PLC) and mesenchymal-like HCC cells (3sp, SNU-423, SNU-449) (Fig. 3b). Interestingly, blocking of TGF-β signaling by the TGF-β receptor inhibitor LY2109761 resulted in downregulation of NRP2 mRNA and protein to levels even lower than those of untreated cells (Fig. 3a–c), suggesting autoregulatory TGF-β loops in respective HCC cells. Importantly, cells with interference of canonical TGF-β/Smad signaling by knock-down of Smad4 were insensitive to TGF-β-induced upregulation of NRP2 (Fig. 3c). These data indicate that augmented NRP2 expression is caused by canonical TGF-β/Smad signaling.Fig. 3NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates
NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates
Since NRP2 expression correlated with TGF-β1 expression (Additional file 1: Figure S1) and TGF-β signaling is a common trigger of EMT in HCC, we asked whether it directly influences NRP2 expression in HCC cells. Remarkably, TGF-β treatment of epithelial 3p, Hep3B and PLC cells, as well as mesenchymal-like 3sp, SNU-423 and SNU-449 cells resulted in increased NRP2 protein expression (Fig. 3a). Upregulation of NRP2 was essentially confirmed at transcript levels by qPCR analysis of epithelial (3p, Hep3B, PLC) and mesenchymal-like HCC cells (3sp, SNU-423, SNU-449) (Fig. 3b). Interestingly, blocking of TGF-β signaling by the TGF-β receptor inhibitor LY2109761 resulted in downregulation of NRP2 mRNA and protein to levels even lower than those of untreated cells (Fig. 3a–c), suggesting autoregulatory TGF-β loops in respective HCC cells. Importantly, cells with interference of canonical TGF-β/Smad signaling by knock-down of Smad4 were insensitive to TGF-β-induced upregulation of NRP2 (Fig. 3c). These data indicate that augmented NRP2 expression is caused by canonical TGF-β/Smad signaling.Fig. 3NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates
NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates
TGF-β-independent activity of NRP2 We next examined whether NRP2 affects canonical TGF-β signaling in a feedback loop. First, we analyzed the level of phosphorylated Smad2 protein that displays TGF-β signaling in SNU-449 cells with and without a NRP2 knock-down after treatment with TGF-β. No difference in pSmad2 levels were observed, independently whether or not NRP2 was expressed (Fig. 4a). To confirm these data, we performed confocal immunofluorescence analysis of TGF-β-treated NRP2 knock-down cells and analyzed nuclear Smad2/3 staining (Fig. 4b). In accordance, no differences in nuclear Smad2/3 localization between control and NRP2 knock-down cells were detected, suggesting that NRP2 does not influence the canonical TGF-β/Smad signaling.Fig. 4Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001
Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001
Interference with TGF-β signaling also leads to a reduced migration of mesenchymal-like HCC cells. Hence, we further analyzed whether the effect of reduced NRP2 on cell migration is dependent on TGF-β signaling or not by employing NRP2 knock-down and control cells after treatment with TGF-β inhibitor LY2109761 (Fig. 4c). Cells treated with LY2109761 reached approximately 64 % migration efficiency compared to control siNT cells. NRP2 knock-down cells without inhibitor treatment were even slower migrating, reaching about 33 % of cell motility. Importantly, the cells treated with both siRNA against NRP2 and LY2109761 migrated even significantly slower than those treated with LY2109761 only, reaching about 10 % compared to reference. In conclusion, these findings indicate that NRP2 has a role in cell migration that is independent of TGF-β signaling.
We next examined whether NRP2 affects canonical TGF-β signaling in a feedback loop. First, we analyzed the level of phosphorylated Smad2 protein that displays TGF-β signaling in SNU-449 cells with and without a NRP2 knock-down after treatment with TGF-β. No difference in pSmad2 levels were observed, independently whether or not NRP2 was expressed (Fig. 4a). To confirm these data, we performed confocal immunofluorescence analysis of TGF-β-treated NRP2 knock-down cells and analyzed nuclear Smad2/3 staining (Fig. 4b). In accordance, no differences in nuclear Smad2/3 localization between control and NRP2 knock-down cells were detected, suggesting that NRP2 does not influence the canonical TGF-β/Smad signaling.Fig. 4Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001
Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001
Interference with TGF-β signaling also leads to a reduced migration of mesenchymal-like HCC cells. Hence, we further analyzed whether the effect of reduced NRP2 on cell migration is dependent on TGF-β signaling or not by employing NRP2 knock-down and control cells after treatment with TGF-β inhibitor LY2109761 (Fig. 4c). Cells treated with LY2109761 reached approximately 64 % migration efficiency compared to control siNT cells. NRP2 knock-down cells without inhibitor treatment were even slower migrating, reaching about 33 % of cell motility. Importantly, the cells treated with both siRNA against NRP2 and LY2109761 migrated even significantly slower than those treated with LY2109761 only, reaching about 10 % compared to reference. In conclusion, these findings indicate that NRP2 has a role in cell migration that is independent of TGF-β signaling.
|
Conclusions
|
TGF-β shows anti-proliferative and tumor-suppressing functions in healthy liver tissue but upon HCC development, aberrant TGF-β signaling is a major driver of HCC progression. We found that NRP2 is induced by canonical TGF-β/Smad signaling in HCC cells and that NRP2 is a potent regulator of HCC cell migration and invasion. NRP2 associates with a mesenchymal-like phenotype in vitro and accordingly, NRP2 correlates with a higher tumor grade in vivo indicating less differentiation. NRP2 is therefore thought to play an important role in TGF-β-dependent HCC progression.
|
[
"Tissue array analysis",
"Cell culture",
"siRNA knock-down",
"Western blot analysis",
"Transwell migration and invasion",
"Wound healing assay",
"Quantitative real-time polymerase chain reaction (qPCR)",
"Confocal immunofluorescence microscopy",
"Statistical analysis",
"NRP2 is upregulated in high-grade HCC and highly expressed in mesenchymal-like HCC cell lines",
"NRP2 regulates HCC cell migration and invasion",
"TGF-β induces NRP2 in a Smad-dependent fashion",
"TGF-β-independent activity of NRP2"
] |
[
"Tissue arrays contained paraffin-embedded specimens of tumors and adjacent normal tissue collected from 133 female and male HCC patients. All patients have undergone orthotopic liver transplantation for HCC at the Department of Transplantation Surgery, Medical University of Vienna, between 1982 and 2002, as described [33]. All specimens were reviewed for histological type and grade by 2 individual board certified pathologists. 4 μm thick sections of core biopsies were arrayed in triplicate and stained with anti-NRP2 antibody (R&D Systems, Minneapolis, USA) or anti-TGF-β1 antibody (Santa Cruz, Dallas, USA) at a dilution of 1:100. After incubation with secondary antibodies, visualization was performed using the vectastain ABC system (Vector Laboratories, Burlingame, USA). Triplicates of stained tissues were evaluated by two independent researchers (P.W. and M.G.) who were blinded regarding patient details. Immunostaining for NRP2 was scored by arbitrary scaling of no and low-to-high staining. Immunohistochemical analysis of paraffin-embedded tissues and retrospective analysis of patient data were approved by the ethics committee of the Medical University of Vienna.",
"The human hepatoma cell lines 3p, 3sp, SNU-398, SNU-423, SNU-449 and SNU-475 were grown in RPMI-1640 medium plus 10 % fetal calf serum (FCS) and antibiotics. Hep3B, HepG2 and FLC-4 cells were cultivated in Eagle’s Minimum Essential Medium (EMEM) plus 10 % FCS and antibiotics. PLC and HuH-6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10 % FCS and antibiotics. Human hepatic sinusoidal endothelial cells (HSECs) received endothelial cell medium (Lonza, Basel, Switzerland). All cells were kept at 37 °C and 5 % CO2. TGF-β signaling was stimulated by supplementing the medium with 2.5 ng/mL TGF-β1 (R&D Systems, Minneapolis, USA) for 24 h. For inhibition of TGF-β signaling, LY2109761 (Santa Cruz, Dallas, USA) antagonizing both TGF-β receptors I/II was used at a concentration of 10 μM for 24 h. All HCC cell lines were validated by short tandem repeat analysis.",
"Cells were seeded on 6-well plates and either transfected with 80 nM of non-target small interfering (si)RNA or with 80 nM of siRNA against human NRP2 (Dharmacon, Town, UK). Cells were processed for further analysis after 48 h of siRNA transfection.",
"Immunoblotting was performed as described [34]. Dilutions of primary antibodies were as follows: NRP2 (R&D Systems, Minneapolis, USA), 1:1000; β-actin (Sigma, St. Louis, USA), 1:2500; Smad2/3 (BD Biosciences, NJ, USA), 1:1000; pSmad2 (Upstate, NY, USA), 1:500; Smad4 (Cell Signaling Technology, Danvers, USA), 1:1000. Secondary antibodies conjugated to horseradish peroxidase were used for detection.",
"2.5 × 104 hepatoma cells were resuspended in 100 μL medium containing 1 % FCS and transferred onto 24-well cell culture inserts with a pore size of 8 μm (Corning, Tewksbury, USA). 600 μL medium containing 10 % FCS was added to lower chambers to generate a gradient for cell migration. Medium was removed and migrated cells on membranes were fixed with 4 % paraformaldehyde after 16 h. Cell nuclei of migrated cells at the bottom side of the membrane were stained with Hoechst 33342 (Life Technologies, Green Island, USA) and counted under the fluorescence microscope (Nikon, Tokyo, Japan). To analyze cell invasion, 24-well cell culture inserts were coated with rat-tail collagen (BD Biosciences, NJ, USA) prior to seeding of hepatoma cells. To examine transendothelial invasion, 2 × 105 HSECs were plated in 100 μL endothelial cell medium (Lonza, Basel, Switzerland) onto coated 24-well cell culture inserts and allowed to form a monolayer for 48 h. Subsequently, endothelial cell medium was aspirated and hepatoma cells were seeded in 100 μL medium containing 1 % FCS onto 24-well inserts. Transmigrated cells were visualized after 16 h and quantified as described for cell migration.",
"For studying cell migration in a scratch wound assay, cells were seeded in 6-well plates and artificial wounds were inflicted to the cell layer by scratching with sterile pipette tips. For each condition, three scratches were inflicted in three independent wells of a 6-well plate. From each of these scratches eight images were taken for a total of 24 images per condition and time point. Images were performed by phase contrast microscopy (Nikon, Tokyo, Japan) immediately after wounding and after 24 h. The migrated area of cells into the wound was quantified with ImageJ software.",
"RNA isolation was performed using an RNeasy purification kit according to the manufacturer’s instructions (Quiagen, Hilden, Germany). For reverse transcription of RNA into DNA, the QuantiTect Reverse Transcription kit was employed (Quiagen, Hilden, Germany). For quantitative RT-PCR (qPCR), aliquots of cDNA were employed for Fast SYBR green qPCR (Applied Biosystems, Foster City, USA) and quantified with the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, USA). RPL41 as HCC housekeeping gene and the average ΔCT value of the HCC cell line 3sp were used to calculate the ΔΔCT values for all cell lines. The ΔΔCT values were used to graph the fold-change (RQ) via the formula RQ = 2^(−ΔΔCT). Error bars show SE of ΔΔCT, calculated with the following formula: SE(ΔΔCT) = √((SE(ΔCT Control)^2) ± (SE(ΔCT Target)^2)). The primer sequences are: NRP2 (forward), 5’-CTGTGGGTCATCCGTGAGGAC-3’ and NRP2 (reverse) 5’-ATGGGTTCCATGCAGTTCTCCAG-3’; RPL41 (forward), 5’-CAAGTGGAGGAAGAAGCGA-3’ and RPL41 (reverse), 5’-TTACTTGGACCTCTGCCTC-3’.",
"Cells were seeded on glass cover slips coated with rat-tail collagen (BD Biosciences, NJ, USA) and fixed with 4 % formaldehyde. After permeabilization with 0.25 % Triton-X 100 and blocking with 5 % horse serum, cells were stained with primary antibody against Smad2/3 (BD Biosciences, NJ, USA) at a concentration of 1:100 and further incubated with secondary antibody (1:200), phalloidin (1:750) and DAPI (1:1000). Images were obtained by confocal immunofluorescence microscopy (Zeiss, Oberkochen, Germany).",
"The statistical significance of differences was evaluated using two-sided Student’s t-test. Data are expressed as means ± standard deviation (SD) or means ± standard error of the mean (SEM) for tissue array data.",
"We employed a tissue array containing 133 HCC cases for the analysis of NRP2 expression and found a significant correlation of NRP2 levels with higher tumor grading. While well-differentiated HCC (grade 1) showed NRP2 expression in 32 % of cases, less-differentiated HCC samples displayed a nearly twice as much higher frequency of NRP2 presence. In particular, 59 % of HCC with grade 2 and 56 % of HCC with grade 3 tumors showed NRP2 expression (Fig. 1a). Generally, NRP2 was found distributed in a patchy fashion (Fig. 1b) and interestingly, NRP2 was exclusively expressed in those patient samples that showed TGF-β1 expression (Additional file 1: Figure S1). Noteworthy, normal liver did not display NRP2 expression (Fig. 1b) which confirmed recent observations that NRP2 is not expressed in hepatocytes under physiological conditions [35].Fig. 1NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01\nNRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01\nTo correlate NRP2 expression with differentiation of hepatoma cells more closely, we analyzed various human HCC cell lines with differentiated and epithelial traits versus those exhibiting a de-differentiated and mesenchymal-like phenotype. Western blot (Fig. 1c) and qPCR analyses (Fig. 1d) revealed that NRP2 expression strongly correlated with a mesenchymal-like HCC phenotype in 3sp, SNU-398, SNU-423, SNU-449, SNU-475 and FLC-4 cells. In contrast, NRP2 was expressed in only one out of five epithelial HCC cell lines, i.e., HuH-6 cells, and showed undetectable levels of NRP2 in the other epithelial 3p, Hep3B, HepG2 and PLC cells. Together, these data suggest that NRP2 expression associates with less-differentiated high-grade tumors and TGF-β1 expression in HCC patients. In agreement, NRP2 expression was found in de-differentiated mesenchymal-like HCC cells in vitro which supports the idea that NRP2 expression correlates with a de-differentiated phenotype.",
"Next we analyzed the functional impact of NRP2 expression on cell proliferation and migration. Neither knock-down nor exogenous overexpression of NRP2 significantly affected proliferation kinetics of mesenchymal-like HCC cells (data not shown). However, Transwell migration assays employing the mesenchymal-like cell lines 3sp and SNU-449 revealed that reduced expression of NRP2 results in impaired migratory abilities (Fig. 2a). Comparable results were obtained in Transwell invasion and transendothelial migration assays (Fig. 2b, c). In addition, assessment of cell migration by wound healing assays of SNU-449 cells confirmed elevated migratory capabilities dependent on NRP2 (Fig. 2d). Interestingly, overexpression of NRP2 could not enhance migration or invasion in any of the employed cell lines (3p, PLC, 3sp, SNU-449; data not shown). Thus, these results suggest that NRP2 is crucially involved in upregulating cell movement of de-differentiated HCC cells.Fig. 2Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001\nLoss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001",
"Since NRP2 expression correlated with TGF-β1 expression (Additional file 1: Figure S1) and TGF-β signaling is a common trigger of EMT in HCC, we asked whether it directly influences NRP2 expression in HCC cells. Remarkably, TGF-β treatment of epithelial 3p, Hep3B and PLC cells, as well as mesenchymal-like 3sp, SNU-423 and SNU-449 cells resulted in increased NRP2 protein expression (Fig. 3a). Upregulation of NRP2 was essentially confirmed at transcript levels by qPCR analysis of epithelial (3p, Hep3B, PLC) and mesenchymal-like HCC cells (3sp, SNU-423, SNU-449) (Fig. 3b). Interestingly, blocking of TGF-β signaling by the TGF-β receptor inhibitor LY2109761 resulted in downregulation of NRP2 mRNA and protein to levels even lower than those of untreated cells (Fig. 3a–c), suggesting autoregulatory TGF-β loops in respective HCC cells. Importantly, cells with interference of canonical TGF-β/Smad signaling by knock-down of Smad4 were insensitive to TGF-β-induced upregulation of NRP2 (Fig. 3c). These data indicate that augmented NRP2 expression is caused by canonical TGF-β/Smad signaling.Fig. 3NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates\nNRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates",
"We next examined whether NRP2 affects canonical TGF-β signaling in a feedback loop. First, we analyzed the level of phosphorylated Smad2 protein that displays TGF-β signaling in SNU-449 cells with and without a NRP2 knock-down after treatment with TGF-β. No difference in pSmad2 levels were observed, independently whether or not NRP2 was expressed (Fig. 4a). To confirm these data, we performed confocal immunofluorescence analysis of TGF-β-treated NRP2 knock-down cells and analyzed nuclear Smad2/3 staining (Fig. 4b). In accordance, no differences in nuclear Smad2/3 localization between control and NRP2 knock-down cells were detected, suggesting that NRP2 does not influence the canonical TGF-β/Smad signaling.Fig. 4Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001\nEffects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001\nInterference with TGF-β signaling also leads to a reduced migration of mesenchymal-like HCC cells. Hence, we further analyzed whether the effect of reduced NRP2 on cell migration is dependent on TGF-β signaling or not by employing NRP2 knock-down and control cells after treatment with TGF-β inhibitor LY2109761 (Fig. 4c). Cells treated with LY2109761 reached approximately 64 % migration efficiency compared to control siNT cells. NRP2 knock-down cells without inhibitor treatment were even slower migrating, reaching about 33 % of cell motility. Importantly, the cells treated with both siRNA against NRP2 and LY2109761 migrated even significantly slower than those treated with LY2109761 only, reaching about 10 % compared to reference. In conclusion, these findings indicate that NRP2 has a role in cell migration that is independent of TGF-β signaling."
] |
[
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null
] |
[
"Background",
"Methods",
"Tissue array analysis",
"Cell culture",
"siRNA knock-down",
"Western blot analysis",
"Transwell migration and invasion",
"Wound healing assay",
"Quantitative real-time polymerase chain reaction (qPCR)",
"Confocal immunofluorescence microscopy",
"Statistical analysis",
"Results",
"NRP2 is upregulated in high-grade HCC and highly expressed in mesenchymal-like HCC cell lines",
"NRP2 regulates HCC cell migration and invasion",
"TGF-β induces NRP2 in a Smad-dependent fashion",
"TGF-β-independent activity of NRP2",
"Discussion",
"Conclusions"
] |
[
"Liver cancer is the sixth most common cancer in the world and ranks second in the list of most deadly cancers [1]. The vast majority of liver cancers are hepatocellular carcinomas (HCC) representing up to 90 % of all liver malignancies [2, 3]. The main risk factors for HCC are chronic infections with either hepatitis B virus (HBV) or hepatitis C virus (HCV), making up approximately 75–85 % of all cases, as well as excessive alcohol consumption, which is responsible for about 40 % of HCC development in Western countries [2, 4–7]. Chronic inflammation and tissue damage by these agents leads to cirrhosis which is the underlying condition for the majority of HCC cases [8].\nThe dissemination of primary tumor cells into the body drastically worsens the prognosis of cancer patients [9]. Metastasis of HCC cells most frequently occurs intrahepatically rather than extrahepatically to distal sites such as the lung [10]. For spreading of HCC cells, individual cell movement by an epithelial to mesenchymal transition (EMT) has been considered to be essentially involved [11]. Upon EMT and progression in malignancy, highly differentiated epithelial cells such as hepatocytes de-differentiate into a mesenchymal-like phenotype that exhibits strong migratory abilities. Various signaling cascades are known to induce EMT, such as the Wnt/β-catenin, PI3K/AKT/mTOR, Hedgehog, Ras/Raf/MEK/ERK, Notch and NFκB pathways, as well as transforming growth factor (TGF)-β [12–15]. These signals mostly converge on EMT-transcription factors (EMT-TFs) such as Snail, ZEB1 or Twist1/2 which transcriptionally repress E-cadherin and other epithelial junctional proteins as well as activate a mesenchymal gene expression signature. In HCC, TGF-β signaling has been shown to activate EMT-TFs and to repress their negative feedback loops by the downregulation of miRNAs that antagonize EMT-TFs [16, 17].\nA recently performed meta-analysis compared 24 published EMT gene expression data sets and generated a core list of genes that are most frequently altered during EMT [18]. One of the genes found upregulated in several studies of TGF-β-induced EMT coded for the protein neuropilin-2 (NRP2). There are two homologs of the NRP family, NRP1 and NRP2, which are 130 kDa single-pass transmembrane glycoproteins that act as non-tyrosine kinase co-receptors [19]. They contain 4 distinct domains including a CUB domain, a FV/FVIII domain, a MAM domain and a domain that contains the transmembrane and short cytoplasmic region [20]. Both homologues can homo- and heteromultimerize [21] and can bind different members of the semaphorin family, as well as members of the vascular endothelial growth factor (VEGF) family [22]. In addition, NRPs are receptors of hepatocyte growth factor, platelet-derived growth factor BB, fibroblast growth factor, epidermal growth factor, placenta growth factor, and importantly of TGF-β1 [23–25]. NRPs are therefore critical regulators of angiogenesis, lymphangiogenesis and tumor progression. NRP2 expression is correlated with lymph node metastasis in breast cancer and blocking of NRP2 leads to decreased metastasis formation [26–28]. Clinical data show that high NRP levels, in particular NRP2, correlate with poor prognosis and survival in various cancer types [29, 30]. NRP2 was suggested to play a direct role in EMT and a cross-talk between NRP2 and TGF-β1 signaling promotes colorectal cancer progression [31]. In the context of HCC, the role of NRP2 is so far unknown [32].\nIn this study, we show that NRP2 expression strongly correlates with a mesenchymal phenotype in HCC cell lines and that reduced levels of NRP2 dramatically impair the migratory abilities of HCC cells. We further provide evidence that NRP2 expression is controlled by canonical TGF-β signaling, while no direct impact of NRP2 on TGF-β signaling could be observed. Translation of these data into the HCC patient situation revealed a correlation of NRP2 levels with a poorly differentiated HCC, suggesting a role of NRP2 in TGF-β regulated HCC progression.",
" Tissue array analysis Tissue arrays contained paraffin-embedded specimens of tumors and adjacent normal tissue collected from 133 female and male HCC patients. All patients have undergone orthotopic liver transplantation for HCC at the Department of Transplantation Surgery, Medical University of Vienna, between 1982 and 2002, as described [33]. All specimens were reviewed for histological type and grade by 2 individual board certified pathologists. 4 μm thick sections of core biopsies were arrayed in triplicate and stained with anti-NRP2 antibody (R&D Systems, Minneapolis, USA) or anti-TGF-β1 antibody (Santa Cruz, Dallas, USA) at a dilution of 1:100. After incubation with secondary antibodies, visualization was performed using the vectastain ABC system (Vector Laboratories, Burlingame, USA). Triplicates of stained tissues were evaluated by two independent researchers (P.W. and M.G.) who were blinded regarding patient details. Immunostaining for NRP2 was scored by arbitrary scaling of no and low-to-high staining. Immunohistochemical analysis of paraffin-embedded tissues and retrospective analysis of patient data were approved by the ethics committee of the Medical University of Vienna.\nTissue arrays contained paraffin-embedded specimens of tumors and adjacent normal tissue collected from 133 female and male HCC patients. All patients have undergone orthotopic liver transplantation for HCC at the Department of Transplantation Surgery, Medical University of Vienna, between 1982 and 2002, as described [33]. All specimens were reviewed for histological type and grade by 2 individual board certified pathologists. 4 μm thick sections of core biopsies were arrayed in triplicate and stained with anti-NRP2 antibody (R&D Systems, Minneapolis, USA) or anti-TGF-β1 antibody (Santa Cruz, Dallas, USA) at a dilution of 1:100. After incubation with secondary antibodies, visualization was performed using the vectastain ABC system (Vector Laboratories, Burlingame, USA). Triplicates of stained tissues were evaluated by two independent researchers (P.W. and M.G.) who were blinded regarding patient details. Immunostaining for NRP2 was scored by arbitrary scaling of no and low-to-high staining. Immunohistochemical analysis of paraffin-embedded tissues and retrospective analysis of patient data were approved by the ethics committee of the Medical University of Vienna.\n Cell culture The human hepatoma cell lines 3p, 3sp, SNU-398, SNU-423, SNU-449 and SNU-475 were grown in RPMI-1640 medium plus 10 % fetal calf serum (FCS) and antibiotics. Hep3B, HepG2 and FLC-4 cells were cultivated in Eagle’s Minimum Essential Medium (EMEM) plus 10 % FCS and antibiotics. PLC and HuH-6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10 % FCS and antibiotics. Human hepatic sinusoidal endothelial cells (HSECs) received endothelial cell medium (Lonza, Basel, Switzerland). All cells were kept at 37 °C and 5 % CO2. TGF-β signaling was stimulated by supplementing the medium with 2.5 ng/mL TGF-β1 (R&D Systems, Minneapolis, USA) for 24 h. For inhibition of TGF-β signaling, LY2109761 (Santa Cruz, Dallas, USA) antagonizing both TGF-β receptors I/II was used at a concentration of 10 μM for 24 h. All HCC cell lines were validated by short tandem repeat analysis.\nThe human hepatoma cell lines 3p, 3sp, SNU-398, SNU-423, SNU-449 and SNU-475 were grown in RPMI-1640 medium plus 10 % fetal calf serum (FCS) and antibiotics. Hep3B, HepG2 and FLC-4 cells were cultivated in Eagle’s Minimum Essential Medium (EMEM) plus 10 % FCS and antibiotics. PLC and HuH-6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10 % FCS and antibiotics. Human hepatic sinusoidal endothelial cells (HSECs) received endothelial cell medium (Lonza, Basel, Switzerland). All cells were kept at 37 °C and 5 % CO2. TGF-β signaling was stimulated by supplementing the medium with 2.5 ng/mL TGF-β1 (R&D Systems, Minneapolis, USA) for 24 h. For inhibition of TGF-β signaling, LY2109761 (Santa Cruz, Dallas, USA) antagonizing both TGF-β receptors I/II was used at a concentration of 10 μM for 24 h. All HCC cell lines were validated by short tandem repeat analysis.\n siRNA knock-down Cells were seeded on 6-well plates and either transfected with 80 nM of non-target small interfering (si)RNA or with 80 nM of siRNA against human NRP2 (Dharmacon, Town, UK). Cells were processed for further analysis after 48 h of siRNA transfection.\nCells were seeded on 6-well plates and either transfected with 80 nM of non-target small interfering (si)RNA or with 80 nM of siRNA against human NRP2 (Dharmacon, Town, UK). Cells were processed for further analysis after 48 h of siRNA transfection.\n Western blot analysis Immunoblotting was performed as described [34]. Dilutions of primary antibodies were as follows: NRP2 (R&D Systems, Minneapolis, USA), 1:1000; β-actin (Sigma, St. Louis, USA), 1:2500; Smad2/3 (BD Biosciences, NJ, USA), 1:1000; pSmad2 (Upstate, NY, USA), 1:500; Smad4 (Cell Signaling Technology, Danvers, USA), 1:1000. Secondary antibodies conjugated to horseradish peroxidase were used for detection.\nImmunoblotting was performed as described [34]. Dilutions of primary antibodies were as follows: NRP2 (R&D Systems, Minneapolis, USA), 1:1000; β-actin (Sigma, St. Louis, USA), 1:2500; Smad2/3 (BD Biosciences, NJ, USA), 1:1000; pSmad2 (Upstate, NY, USA), 1:500; Smad4 (Cell Signaling Technology, Danvers, USA), 1:1000. Secondary antibodies conjugated to horseradish peroxidase were used for detection.\n Transwell migration and invasion 2.5 × 104 hepatoma cells were resuspended in 100 μL medium containing 1 % FCS and transferred onto 24-well cell culture inserts with a pore size of 8 μm (Corning, Tewksbury, USA). 600 μL medium containing 10 % FCS was added to lower chambers to generate a gradient for cell migration. Medium was removed and migrated cells on membranes were fixed with 4 % paraformaldehyde after 16 h. Cell nuclei of migrated cells at the bottom side of the membrane were stained with Hoechst 33342 (Life Technologies, Green Island, USA) and counted under the fluorescence microscope (Nikon, Tokyo, Japan). To analyze cell invasion, 24-well cell culture inserts were coated with rat-tail collagen (BD Biosciences, NJ, USA) prior to seeding of hepatoma cells. To examine transendothelial invasion, 2 × 105 HSECs were plated in 100 μL endothelial cell medium (Lonza, Basel, Switzerland) onto coated 24-well cell culture inserts and allowed to form a monolayer for 48 h. Subsequently, endothelial cell medium was aspirated and hepatoma cells were seeded in 100 μL medium containing 1 % FCS onto 24-well inserts. Transmigrated cells were visualized after 16 h and quantified as described for cell migration.\n2.5 × 104 hepatoma cells were resuspended in 100 μL medium containing 1 % FCS and transferred onto 24-well cell culture inserts with a pore size of 8 μm (Corning, Tewksbury, USA). 600 μL medium containing 10 % FCS was added to lower chambers to generate a gradient for cell migration. Medium was removed and migrated cells on membranes were fixed with 4 % paraformaldehyde after 16 h. Cell nuclei of migrated cells at the bottom side of the membrane were stained with Hoechst 33342 (Life Technologies, Green Island, USA) and counted under the fluorescence microscope (Nikon, Tokyo, Japan). To analyze cell invasion, 24-well cell culture inserts were coated with rat-tail collagen (BD Biosciences, NJ, USA) prior to seeding of hepatoma cells. To examine transendothelial invasion, 2 × 105 HSECs were plated in 100 μL endothelial cell medium (Lonza, Basel, Switzerland) onto coated 24-well cell culture inserts and allowed to form a monolayer for 48 h. Subsequently, endothelial cell medium was aspirated and hepatoma cells were seeded in 100 μL medium containing 1 % FCS onto 24-well inserts. Transmigrated cells were visualized after 16 h and quantified as described for cell migration.\n Wound healing assay For studying cell migration in a scratch wound assay, cells were seeded in 6-well plates and artificial wounds were inflicted to the cell layer by scratching with sterile pipette tips. For each condition, three scratches were inflicted in three independent wells of a 6-well plate. From each of these scratches eight images were taken for a total of 24 images per condition and time point. Images were performed by phase contrast microscopy (Nikon, Tokyo, Japan) immediately after wounding and after 24 h. The migrated area of cells into the wound was quantified with ImageJ software.\nFor studying cell migration in a scratch wound assay, cells were seeded in 6-well plates and artificial wounds were inflicted to the cell layer by scratching with sterile pipette tips. For each condition, three scratches were inflicted in three independent wells of a 6-well plate. From each of these scratches eight images were taken for a total of 24 images per condition and time point. Images were performed by phase contrast microscopy (Nikon, Tokyo, Japan) immediately after wounding and after 24 h. The migrated area of cells into the wound was quantified with ImageJ software.\n Quantitative real-time polymerase chain reaction (qPCR) RNA isolation was performed using an RNeasy purification kit according to the manufacturer’s instructions (Quiagen, Hilden, Germany). For reverse transcription of RNA into DNA, the QuantiTect Reverse Transcription kit was employed (Quiagen, Hilden, Germany). For quantitative RT-PCR (qPCR), aliquots of cDNA were employed for Fast SYBR green qPCR (Applied Biosystems, Foster City, USA) and quantified with the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, USA). RPL41 as HCC housekeeping gene and the average ΔCT value of the HCC cell line 3sp were used to calculate the ΔΔCT values for all cell lines. The ΔΔCT values were used to graph the fold-change (RQ) via the formula RQ = 2^(−ΔΔCT). Error bars show SE of ΔΔCT, calculated with the following formula: SE(ΔΔCT) = √((SE(ΔCT Control)^2) ± (SE(ΔCT Target)^2)). The primer sequences are: NRP2 (forward), 5’-CTGTGGGTCATCCGTGAGGAC-3’ and NRP2 (reverse) 5’-ATGGGTTCCATGCAGTTCTCCAG-3’; RPL41 (forward), 5’-CAAGTGGAGGAAGAAGCGA-3’ and RPL41 (reverse), 5’-TTACTTGGACCTCTGCCTC-3’.\nRNA isolation was performed using an RNeasy purification kit according to the manufacturer’s instructions (Quiagen, Hilden, Germany). For reverse transcription of RNA into DNA, the QuantiTect Reverse Transcription kit was employed (Quiagen, Hilden, Germany). For quantitative RT-PCR (qPCR), aliquots of cDNA were employed for Fast SYBR green qPCR (Applied Biosystems, Foster City, USA) and quantified with the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, USA). RPL41 as HCC housekeeping gene and the average ΔCT value of the HCC cell line 3sp were used to calculate the ΔΔCT values for all cell lines. The ΔΔCT values were used to graph the fold-change (RQ) via the formula RQ = 2^(−ΔΔCT). Error bars show SE of ΔΔCT, calculated with the following formula: SE(ΔΔCT) = √((SE(ΔCT Control)^2) ± (SE(ΔCT Target)^2)). The primer sequences are: NRP2 (forward), 5’-CTGTGGGTCATCCGTGAGGAC-3’ and NRP2 (reverse) 5’-ATGGGTTCCATGCAGTTCTCCAG-3’; RPL41 (forward), 5’-CAAGTGGAGGAAGAAGCGA-3’ and RPL41 (reverse), 5’-TTACTTGGACCTCTGCCTC-3’.\n Confocal immunofluorescence microscopy Cells were seeded on glass cover slips coated with rat-tail collagen (BD Biosciences, NJ, USA) and fixed with 4 % formaldehyde. After permeabilization with 0.25 % Triton-X 100 and blocking with 5 % horse serum, cells were stained with primary antibody against Smad2/3 (BD Biosciences, NJ, USA) at a concentration of 1:100 and further incubated with secondary antibody (1:200), phalloidin (1:750) and DAPI (1:1000). Images were obtained by confocal immunofluorescence microscopy (Zeiss, Oberkochen, Germany).\nCells were seeded on glass cover slips coated with rat-tail collagen (BD Biosciences, NJ, USA) and fixed with 4 % formaldehyde. After permeabilization with 0.25 % Triton-X 100 and blocking with 5 % horse serum, cells were stained with primary antibody against Smad2/3 (BD Biosciences, NJ, USA) at a concentration of 1:100 and further incubated with secondary antibody (1:200), phalloidin (1:750) and DAPI (1:1000). Images were obtained by confocal immunofluorescence microscopy (Zeiss, Oberkochen, Germany).\n Statistical analysis The statistical significance of differences was evaluated using two-sided Student’s t-test. Data are expressed as means ± standard deviation (SD) or means ± standard error of the mean (SEM) for tissue array data.\nThe statistical significance of differences was evaluated using two-sided Student’s t-test. Data are expressed as means ± standard deviation (SD) or means ± standard error of the mean (SEM) for tissue array data.",
"Tissue arrays contained paraffin-embedded specimens of tumors and adjacent normal tissue collected from 133 female and male HCC patients. All patients have undergone orthotopic liver transplantation for HCC at the Department of Transplantation Surgery, Medical University of Vienna, between 1982 and 2002, as described [33]. All specimens were reviewed for histological type and grade by 2 individual board certified pathologists. 4 μm thick sections of core biopsies were arrayed in triplicate and stained with anti-NRP2 antibody (R&D Systems, Minneapolis, USA) or anti-TGF-β1 antibody (Santa Cruz, Dallas, USA) at a dilution of 1:100. After incubation with secondary antibodies, visualization was performed using the vectastain ABC system (Vector Laboratories, Burlingame, USA). Triplicates of stained tissues were evaluated by two independent researchers (P.W. and M.G.) who were blinded regarding patient details. Immunostaining for NRP2 was scored by arbitrary scaling of no and low-to-high staining. Immunohistochemical analysis of paraffin-embedded tissues and retrospective analysis of patient data were approved by the ethics committee of the Medical University of Vienna.",
"The human hepatoma cell lines 3p, 3sp, SNU-398, SNU-423, SNU-449 and SNU-475 were grown in RPMI-1640 medium plus 10 % fetal calf serum (FCS) and antibiotics. Hep3B, HepG2 and FLC-4 cells were cultivated in Eagle’s Minimum Essential Medium (EMEM) plus 10 % FCS and antibiotics. PLC and HuH-6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10 % FCS and antibiotics. Human hepatic sinusoidal endothelial cells (HSECs) received endothelial cell medium (Lonza, Basel, Switzerland). All cells were kept at 37 °C and 5 % CO2. TGF-β signaling was stimulated by supplementing the medium with 2.5 ng/mL TGF-β1 (R&D Systems, Minneapolis, USA) for 24 h. For inhibition of TGF-β signaling, LY2109761 (Santa Cruz, Dallas, USA) antagonizing both TGF-β receptors I/II was used at a concentration of 10 μM for 24 h. All HCC cell lines were validated by short tandem repeat analysis.",
"Cells were seeded on 6-well plates and either transfected with 80 nM of non-target small interfering (si)RNA or with 80 nM of siRNA against human NRP2 (Dharmacon, Town, UK). Cells were processed for further analysis after 48 h of siRNA transfection.",
"Immunoblotting was performed as described [34]. Dilutions of primary antibodies were as follows: NRP2 (R&D Systems, Minneapolis, USA), 1:1000; β-actin (Sigma, St. Louis, USA), 1:2500; Smad2/3 (BD Biosciences, NJ, USA), 1:1000; pSmad2 (Upstate, NY, USA), 1:500; Smad4 (Cell Signaling Technology, Danvers, USA), 1:1000. Secondary antibodies conjugated to horseradish peroxidase were used for detection.",
"2.5 × 104 hepatoma cells were resuspended in 100 μL medium containing 1 % FCS and transferred onto 24-well cell culture inserts with a pore size of 8 μm (Corning, Tewksbury, USA). 600 μL medium containing 10 % FCS was added to lower chambers to generate a gradient for cell migration. Medium was removed and migrated cells on membranes were fixed with 4 % paraformaldehyde after 16 h. Cell nuclei of migrated cells at the bottom side of the membrane were stained with Hoechst 33342 (Life Technologies, Green Island, USA) and counted under the fluorescence microscope (Nikon, Tokyo, Japan). To analyze cell invasion, 24-well cell culture inserts were coated with rat-tail collagen (BD Biosciences, NJ, USA) prior to seeding of hepatoma cells. To examine transendothelial invasion, 2 × 105 HSECs were plated in 100 μL endothelial cell medium (Lonza, Basel, Switzerland) onto coated 24-well cell culture inserts and allowed to form a monolayer for 48 h. Subsequently, endothelial cell medium was aspirated and hepatoma cells were seeded in 100 μL medium containing 1 % FCS onto 24-well inserts. Transmigrated cells were visualized after 16 h and quantified as described for cell migration.",
"For studying cell migration in a scratch wound assay, cells were seeded in 6-well plates and artificial wounds were inflicted to the cell layer by scratching with sterile pipette tips. For each condition, three scratches were inflicted in three independent wells of a 6-well plate. From each of these scratches eight images were taken for a total of 24 images per condition and time point. Images were performed by phase contrast microscopy (Nikon, Tokyo, Japan) immediately after wounding and after 24 h. The migrated area of cells into the wound was quantified with ImageJ software.",
"RNA isolation was performed using an RNeasy purification kit according to the manufacturer’s instructions (Quiagen, Hilden, Germany). For reverse transcription of RNA into DNA, the QuantiTect Reverse Transcription kit was employed (Quiagen, Hilden, Germany). For quantitative RT-PCR (qPCR), aliquots of cDNA were employed for Fast SYBR green qPCR (Applied Biosystems, Foster City, USA) and quantified with the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, USA). RPL41 as HCC housekeeping gene and the average ΔCT value of the HCC cell line 3sp were used to calculate the ΔΔCT values for all cell lines. The ΔΔCT values were used to graph the fold-change (RQ) via the formula RQ = 2^(−ΔΔCT). Error bars show SE of ΔΔCT, calculated with the following formula: SE(ΔΔCT) = √((SE(ΔCT Control)^2) ± (SE(ΔCT Target)^2)). The primer sequences are: NRP2 (forward), 5’-CTGTGGGTCATCCGTGAGGAC-3’ and NRP2 (reverse) 5’-ATGGGTTCCATGCAGTTCTCCAG-3’; RPL41 (forward), 5’-CAAGTGGAGGAAGAAGCGA-3’ and RPL41 (reverse), 5’-TTACTTGGACCTCTGCCTC-3’.",
"Cells were seeded on glass cover slips coated with rat-tail collagen (BD Biosciences, NJ, USA) and fixed with 4 % formaldehyde. After permeabilization with 0.25 % Triton-X 100 and blocking with 5 % horse serum, cells were stained with primary antibody against Smad2/3 (BD Biosciences, NJ, USA) at a concentration of 1:100 and further incubated with secondary antibody (1:200), phalloidin (1:750) and DAPI (1:1000). Images were obtained by confocal immunofluorescence microscopy (Zeiss, Oberkochen, Germany).",
"The statistical significance of differences was evaluated using two-sided Student’s t-test. Data are expressed as means ± standard deviation (SD) or means ± standard error of the mean (SEM) for tissue array data.",
" NRP2 is upregulated in high-grade HCC and highly expressed in mesenchymal-like HCC cell lines We employed a tissue array containing 133 HCC cases for the analysis of NRP2 expression and found a significant correlation of NRP2 levels with higher tumor grading. While well-differentiated HCC (grade 1) showed NRP2 expression in 32 % of cases, less-differentiated HCC samples displayed a nearly twice as much higher frequency of NRP2 presence. In particular, 59 % of HCC with grade 2 and 56 % of HCC with grade 3 tumors showed NRP2 expression (Fig. 1a). Generally, NRP2 was found distributed in a patchy fashion (Fig. 1b) and interestingly, NRP2 was exclusively expressed in those patient samples that showed TGF-β1 expression (Additional file 1: Figure S1). Noteworthy, normal liver did not display NRP2 expression (Fig. 1b) which confirmed recent observations that NRP2 is not expressed in hepatocytes under physiological conditions [35].Fig. 1NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01\nNRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01\nTo correlate NRP2 expression with differentiation of hepatoma cells more closely, we analyzed various human HCC cell lines with differentiated and epithelial traits versus those exhibiting a de-differentiated and mesenchymal-like phenotype. Western blot (Fig. 1c) and qPCR analyses (Fig. 1d) revealed that NRP2 expression strongly correlated with a mesenchymal-like HCC phenotype in 3sp, SNU-398, SNU-423, SNU-449, SNU-475 and FLC-4 cells. In contrast, NRP2 was expressed in only one out of five epithelial HCC cell lines, i.e., HuH-6 cells, and showed undetectable levels of NRP2 in the other epithelial 3p, Hep3B, HepG2 and PLC cells. Together, these data suggest that NRP2 expression associates with less-differentiated high-grade tumors and TGF-β1 expression in HCC patients. In agreement, NRP2 expression was found in de-differentiated mesenchymal-like HCC cells in vitro which supports the idea that NRP2 expression correlates with a de-differentiated phenotype.\nWe employed a tissue array containing 133 HCC cases for the analysis of NRP2 expression and found a significant correlation of NRP2 levels with higher tumor grading. While well-differentiated HCC (grade 1) showed NRP2 expression in 32 % of cases, less-differentiated HCC samples displayed a nearly twice as much higher frequency of NRP2 presence. In particular, 59 % of HCC with grade 2 and 56 % of HCC with grade 3 tumors showed NRP2 expression (Fig. 1a). Generally, NRP2 was found distributed in a patchy fashion (Fig. 1b) and interestingly, NRP2 was exclusively expressed in those patient samples that showed TGF-β1 expression (Additional file 1: Figure S1). Noteworthy, normal liver did not display NRP2 expression (Fig. 1b) which confirmed recent observations that NRP2 is not expressed in hepatocytes under physiological conditions [35].Fig. 1NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01\nNRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01\nTo correlate NRP2 expression with differentiation of hepatoma cells more closely, we analyzed various human HCC cell lines with differentiated and epithelial traits versus those exhibiting a de-differentiated and mesenchymal-like phenotype. Western blot (Fig. 1c) and qPCR analyses (Fig. 1d) revealed that NRP2 expression strongly correlated with a mesenchymal-like HCC phenotype in 3sp, SNU-398, SNU-423, SNU-449, SNU-475 and FLC-4 cells. In contrast, NRP2 was expressed in only one out of five epithelial HCC cell lines, i.e., HuH-6 cells, and showed undetectable levels of NRP2 in the other epithelial 3p, Hep3B, HepG2 and PLC cells. Together, these data suggest that NRP2 expression associates with less-differentiated high-grade tumors and TGF-β1 expression in HCC patients. In agreement, NRP2 expression was found in de-differentiated mesenchymal-like HCC cells in vitro which supports the idea that NRP2 expression correlates with a de-differentiated phenotype.\n NRP2 regulates HCC cell migration and invasion Next we analyzed the functional impact of NRP2 expression on cell proliferation and migration. Neither knock-down nor exogenous overexpression of NRP2 significantly affected proliferation kinetics of mesenchymal-like HCC cells (data not shown). However, Transwell migration assays employing the mesenchymal-like cell lines 3sp and SNU-449 revealed that reduced expression of NRP2 results in impaired migratory abilities (Fig. 2a). Comparable results were obtained in Transwell invasion and transendothelial migration assays (Fig. 2b, c). In addition, assessment of cell migration by wound healing assays of SNU-449 cells confirmed elevated migratory capabilities dependent on NRP2 (Fig. 2d). Interestingly, overexpression of NRP2 could not enhance migration or invasion in any of the employed cell lines (3p, PLC, 3sp, SNU-449; data not shown). Thus, these results suggest that NRP2 is crucially involved in upregulating cell movement of de-differentiated HCC cells.Fig. 2Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001\nLoss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001\nNext we analyzed the functional impact of NRP2 expression on cell proliferation and migration. Neither knock-down nor exogenous overexpression of NRP2 significantly affected proliferation kinetics of mesenchymal-like HCC cells (data not shown). However, Transwell migration assays employing the mesenchymal-like cell lines 3sp and SNU-449 revealed that reduced expression of NRP2 results in impaired migratory abilities (Fig. 2a). Comparable results were obtained in Transwell invasion and transendothelial migration assays (Fig. 2b, c). In addition, assessment of cell migration by wound healing assays of SNU-449 cells confirmed elevated migratory capabilities dependent on NRP2 (Fig. 2d). Interestingly, overexpression of NRP2 could not enhance migration or invasion in any of the employed cell lines (3p, PLC, 3sp, SNU-449; data not shown). Thus, these results suggest that NRP2 is crucially involved in upregulating cell movement of de-differentiated HCC cells.Fig. 2Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001\nLoss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001\n TGF-β induces NRP2 in a Smad-dependent fashion Since NRP2 expression correlated with TGF-β1 expression (Additional file 1: Figure S1) and TGF-β signaling is a common trigger of EMT in HCC, we asked whether it directly influences NRP2 expression in HCC cells. Remarkably, TGF-β treatment of epithelial 3p, Hep3B and PLC cells, as well as mesenchymal-like 3sp, SNU-423 and SNU-449 cells resulted in increased NRP2 protein expression (Fig. 3a). Upregulation of NRP2 was essentially confirmed at transcript levels by qPCR analysis of epithelial (3p, Hep3B, PLC) and mesenchymal-like HCC cells (3sp, SNU-423, SNU-449) (Fig. 3b). Interestingly, blocking of TGF-β signaling by the TGF-β receptor inhibitor LY2109761 resulted in downregulation of NRP2 mRNA and protein to levels even lower than those of untreated cells (Fig. 3a–c), suggesting autoregulatory TGF-β loops in respective HCC cells. Importantly, cells with interference of canonical TGF-β/Smad signaling by knock-down of Smad4 were insensitive to TGF-β-induced upregulation of NRP2 (Fig. 3c). These data indicate that augmented NRP2 expression is caused by canonical TGF-β/Smad signaling.Fig. 3NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates\nNRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates\nSince NRP2 expression correlated with TGF-β1 expression (Additional file 1: Figure S1) and TGF-β signaling is a common trigger of EMT in HCC, we asked whether it directly influences NRP2 expression in HCC cells. Remarkably, TGF-β treatment of epithelial 3p, Hep3B and PLC cells, as well as mesenchymal-like 3sp, SNU-423 and SNU-449 cells resulted in increased NRP2 protein expression (Fig. 3a). Upregulation of NRP2 was essentially confirmed at transcript levels by qPCR analysis of epithelial (3p, Hep3B, PLC) and mesenchymal-like HCC cells (3sp, SNU-423, SNU-449) (Fig. 3b). Interestingly, blocking of TGF-β signaling by the TGF-β receptor inhibitor LY2109761 resulted in downregulation of NRP2 mRNA and protein to levels even lower than those of untreated cells (Fig. 3a–c), suggesting autoregulatory TGF-β loops in respective HCC cells. Importantly, cells with interference of canonical TGF-β/Smad signaling by knock-down of Smad4 were insensitive to TGF-β-induced upregulation of NRP2 (Fig. 3c). These data indicate that augmented NRP2 expression is caused by canonical TGF-β/Smad signaling.Fig. 3NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates\nNRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates\n TGF-β-independent activity of NRP2 We next examined whether NRP2 affects canonical TGF-β signaling in a feedback loop. First, we analyzed the level of phosphorylated Smad2 protein that displays TGF-β signaling in SNU-449 cells with and without a NRP2 knock-down after treatment with TGF-β. No difference in pSmad2 levels were observed, independently whether or not NRP2 was expressed (Fig. 4a). To confirm these data, we performed confocal immunofluorescence analysis of TGF-β-treated NRP2 knock-down cells and analyzed nuclear Smad2/3 staining (Fig. 4b). In accordance, no differences in nuclear Smad2/3 localization between control and NRP2 knock-down cells were detected, suggesting that NRP2 does not influence the canonical TGF-β/Smad signaling.Fig. 4Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001\nEffects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001\nInterference with TGF-β signaling also leads to a reduced migration of mesenchymal-like HCC cells. Hence, we further analyzed whether the effect of reduced NRP2 on cell migration is dependent on TGF-β signaling or not by employing NRP2 knock-down and control cells after treatment with TGF-β inhibitor LY2109761 (Fig. 4c). Cells treated with LY2109761 reached approximately 64 % migration efficiency compared to control siNT cells. NRP2 knock-down cells without inhibitor treatment were even slower migrating, reaching about 33 % of cell motility. Importantly, the cells treated with both siRNA against NRP2 and LY2109761 migrated even significantly slower than those treated with LY2109761 only, reaching about 10 % compared to reference. In conclusion, these findings indicate that NRP2 has a role in cell migration that is independent of TGF-β signaling.\nWe next examined whether NRP2 affects canonical TGF-β signaling in a feedback loop. First, we analyzed the level of phosphorylated Smad2 protein that displays TGF-β signaling in SNU-449 cells with and without a NRP2 knock-down after treatment with TGF-β. No difference in pSmad2 levels were observed, independently whether or not NRP2 was expressed (Fig. 4a). To confirm these data, we performed confocal immunofluorescence analysis of TGF-β-treated NRP2 knock-down cells and analyzed nuclear Smad2/3 staining (Fig. 4b). In accordance, no differences in nuclear Smad2/3 localization between control and NRP2 knock-down cells were detected, suggesting that NRP2 does not influence the canonical TGF-β/Smad signaling.Fig. 4Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001\nEffects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001\nInterference with TGF-β signaling also leads to a reduced migration of mesenchymal-like HCC cells. Hence, we further analyzed whether the effect of reduced NRP2 on cell migration is dependent on TGF-β signaling or not by employing NRP2 knock-down and control cells after treatment with TGF-β inhibitor LY2109761 (Fig. 4c). Cells treated with LY2109761 reached approximately 64 % migration efficiency compared to control siNT cells. NRP2 knock-down cells without inhibitor treatment were even slower migrating, reaching about 33 % of cell motility. Importantly, the cells treated with both siRNA against NRP2 and LY2109761 migrated even significantly slower than those treated with LY2109761 only, reaching about 10 % compared to reference. In conclusion, these findings indicate that NRP2 has a role in cell migration that is independent of TGF-β signaling.",
"We employed a tissue array containing 133 HCC cases for the analysis of NRP2 expression and found a significant correlation of NRP2 levels with higher tumor grading. While well-differentiated HCC (grade 1) showed NRP2 expression in 32 % of cases, less-differentiated HCC samples displayed a nearly twice as much higher frequency of NRP2 presence. In particular, 59 % of HCC with grade 2 and 56 % of HCC with grade 3 tumors showed NRP2 expression (Fig. 1a). Generally, NRP2 was found distributed in a patchy fashion (Fig. 1b) and interestingly, NRP2 was exclusively expressed in those patient samples that showed TGF-β1 expression (Additional file 1: Figure S1). Noteworthy, normal liver did not display NRP2 expression (Fig. 1b) which confirmed recent observations that NRP2 is not expressed in hepatocytes under physiological conditions [35].Fig. 1NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01\nNRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01\nTo correlate NRP2 expression with differentiation of hepatoma cells more closely, we analyzed various human HCC cell lines with differentiated and epithelial traits versus those exhibiting a de-differentiated and mesenchymal-like phenotype. Western blot (Fig. 1c) and qPCR analyses (Fig. 1d) revealed that NRP2 expression strongly correlated with a mesenchymal-like HCC phenotype in 3sp, SNU-398, SNU-423, SNU-449, SNU-475 and FLC-4 cells. In contrast, NRP2 was expressed in only one out of five epithelial HCC cell lines, i.e., HuH-6 cells, and showed undetectable levels of NRP2 in the other epithelial 3p, Hep3B, HepG2 and PLC cells. Together, these data suggest that NRP2 expression associates with less-differentiated high-grade tumors and TGF-β1 expression in HCC patients. In agreement, NRP2 expression was found in de-differentiated mesenchymal-like HCC cells in vitro which supports the idea that NRP2 expression correlates with a de-differentiated phenotype.",
"Next we analyzed the functional impact of NRP2 expression on cell proliferation and migration. Neither knock-down nor exogenous overexpression of NRP2 significantly affected proliferation kinetics of mesenchymal-like HCC cells (data not shown). However, Transwell migration assays employing the mesenchymal-like cell lines 3sp and SNU-449 revealed that reduced expression of NRP2 results in impaired migratory abilities (Fig. 2a). Comparable results were obtained in Transwell invasion and transendothelial migration assays (Fig. 2b, c). In addition, assessment of cell migration by wound healing assays of SNU-449 cells confirmed elevated migratory capabilities dependent on NRP2 (Fig. 2d). Interestingly, overexpression of NRP2 could not enhance migration or invasion in any of the employed cell lines (3p, PLC, 3sp, SNU-449; data not shown). Thus, these results suggest that NRP2 is crucially involved in upregulating cell movement of de-differentiated HCC cells.Fig. 2Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001\nLoss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001",
"Since NRP2 expression correlated with TGF-β1 expression (Additional file 1: Figure S1) and TGF-β signaling is a common trigger of EMT in HCC, we asked whether it directly influences NRP2 expression in HCC cells. Remarkably, TGF-β treatment of epithelial 3p, Hep3B and PLC cells, as well as mesenchymal-like 3sp, SNU-423 and SNU-449 cells resulted in increased NRP2 protein expression (Fig. 3a). Upregulation of NRP2 was essentially confirmed at transcript levels by qPCR analysis of epithelial (3p, Hep3B, PLC) and mesenchymal-like HCC cells (3sp, SNU-423, SNU-449) (Fig. 3b). Interestingly, blocking of TGF-β signaling by the TGF-β receptor inhibitor LY2109761 resulted in downregulation of NRP2 mRNA and protein to levels even lower than those of untreated cells (Fig. 3a–c), suggesting autoregulatory TGF-β loops in respective HCC cells. Importantly, cells with interference of canonical TGF-β/Smad signaling by knock-down of Smad4 were insensitive to TGF-β-induced upregulation of NRP2 (Fig. 3c). These data indicate that augmented NRP2 expression is caused by canonical TGF-β/Smad signaling.Fig. 3NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates\nNRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates",
"We next examined whether NRP2 affects canonical TGF-β signaling in a feedback loop. First, we analyzed the level of phosphorylated Smad2 protein that displays TGF-β signaling in SNU-449 cells with and without a NRP2 knock-down after treatment with TGF-β. No difference in pSmad2 levels were observed, independently whether or not NRP2 was expressed (Fig. 4a). To confirm these data, we performed confocal immunofluorescence analysis of TGF-β-treated NRP2 knock-down cells and analyzed nuclear Smad2/3 staining (Fig. 4b). In accordance, no differences in nuclear Smad2/3 localization between control and NRP2 knock-down cells were detected, suggesting that NRP2 does not influence the canonical TGF-β/Smad signaling.Fig. 4Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001\nEffects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001\nInterference with TGF-β signaling also leads to a reduced migration of mesenchymal-like HCC cells. Hence, we further analyzed whether the effect of reduced NRP2 on cell migration is dependent on TGF-β signaling or not by employing NRP2 knock-down and control cells after treatment with TGF-β inhibitor LY2109761 (Fig. 4c). Cells treated with LY2109761 reached approximately 64 % migration efficiency compared to control siNT cells. NRP2 knock-down cells without inhibitor treatment were even slower migrating, reaching about 33 % of cell motility. Importantly, the cells treated with both siRNA against NRP2 and LY2109761 migrated even significantly slower than those treated with LY2109761 only, reaching about 10 % compared to reference. In conclusion, these findings indicate that NRP2 has a role in cell migration that is independent of TGF-β signaling.",
"Of the two neuropilin isoforms, NRP1 is the far more examined protein. Its role in various types of cancer is well documented and often linked to angiogenesis via VEGF. In contrast, few studies were dedicated to NRP2 and its role in tumorigenesis. To our knowledge no attempts were made to elucidate its role in HCC.\nIn this study, we showed that NRP2 expression correlates with less differentiation in HCC patients as well as with a de-differentiated, mesenchymal-like phenotype of HCC cell lines (Fig. 1). Functionally, NRP2 expression severely enhances migration of mesenchymal-like HCC cells and is induced by the canonical TGF-β/Smad signaling. In this setting, NRP2 can be considered as a biomarker of both the activation of TGF-β/Smad signaling and loss of differentiation by EMT. Thus, NRP2 represents a novel marker for EMT-transformed cells that can be used apart from the classical ones such as increased vimentin and N-cadherin or loss of ZO-1 and E-cadherin expression [36].\nDiminished migration of HCC cells was similar either after reducing NRP2 levels or after blocking TGF-β signaling (Fig. 4c). TGF-β inhibition on its own reduced NRP2 expression but could not completely block it (Fig. 3a, b), as the remaining NRP2 levels might allow cells to better migrate as compared to those cells with strongly reduced NRP2 levels after RNA interference. Yet, if HCC cells were blocked for both NRP2 and TGF-β signaling, a further decrease in HCC cell migration was observed (Fig. 4c). This additive operation of NRP2 and TGF-β signaling suggests that TGF-β additionally affects cell migration that is independent of NRP2. Moreover, neither overexpression of exogenous NRP2 nor treatment with TGF-β1 had any detectable positive effect on migration (data not shown), suggesting that neither NRP2 expression nor TGF-β signaling are rate limiting in migration of mesenchymal-like HCC cell lines. In addition, the consequences of NRP2 expression on migration and invasion as determined by the passing of cells through unchanged or collagen-coated Transwell membranes was stronger than in follow-up experiments in which cells were allowed to transmigrate through a layer of endothelial cells. This suggests that NRP2 mainly affects the migratory potential of HCC cells rather than their ability of breaking through endothelial barriers.\nWe could show for the first time that NRP2 expression is tightly controlled by the canonical Smad2/3-Smad4 signaling cascade in HCC cells (Fig. 3c). NRP2 is therefore considered as an effector of active TGF-β signaling. Interference with TGF-β signaling decreases NRP2 expression below levels of untreated cells (Fig. 3a), indicating autocrine regulatory TGF-β signaling in mesenchymal-like HCC cells [37]. In this line, NRP2 expression could be used as marker of persistent TGF-β activity in HCC patients which is indicative of HCC progression via the tumor promoting arm of TGF-β [38, 39]. This could explain why NRP2 expression is associated with higher grading in HCC specimens (Fig. 1a). Interestingly, no other significant correlations between NRP2 expression and e.g., tumor staging or vessel invasion could be found which might be due to limitations in the immunohistochemical analysis of tissue arrays.\nTGF-β is molecularly linked to NRP2 expression as TGF-β induces NRP2 levels (Fig. 3a, b). However, we found no impact of NRP2 on canonical TGF-β signaling (Fig. 4a, b) despite the fact that NRP2 has been described to act as TGF-β co-receptor [31]. Interestingly, a role of NRP2 in canonical TGF-β signaling could also not be confirmed in lung cancer cells [40], however, this study showed that NRP2 affected ERK signaling supposedly via non-canonical TGF-β signaling activity. In HCC cells, we could also not detect NRP2-driven non-canonical TGF-β signaling (data not shown). The question how NRP2 exerts its influence on cell migration remains open. NRP2 could act on other signaling pathways controlling migration or NRP2 could directly act as a receptor that conducts signals via its intracellular domain. These aspects are the basis for further investigations and allow assessing whether NRP2 represents a valuable target for HCC intervention.",
"TGF-β shows anti-proliferative and tumor-suppressing functions in healthy liver tissue but upon HCC development, aberrant TGF-β signaling is a major driver of HCC progression. We found that NRP2 is induced by canonical TGF-β/Smad signaling in HCC cells and that NRP2 is a potent regulator of HCC cell migration and invasion. NRP2 associates with a mesenchymal-like phenotype in vitro and accordingly, NRP2 correlates with a higher tumor grade in vivo indicating less differentiation. NRP2 is therefore thought to play an important role in TGF-β-dependent HCC progression."
] |
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"results",
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] |
[
"Neuropilin-2",
"Transforming growth factor-β",
"Epithelial to mesenchymal transition",
"Hepatocellular carcinoma"
] |
Background:
Liver cancer is the sixth most common cancer in the world and ranks second in the list of most deadly cancers [1]. The vast majority of liver cancers are hepatocellular carcinomas (HCC) representing up to 90 % of all liver malignancies [2, 3]. The main risk factors for HCC are chronic infections with either hepatitis B virus (HBV) or hepatitis C virus (HCV), making up approximately 75–85 % of all cases, as well as excessive alcohol consumption, which is responsible for about 40 % of HCC development in Western countries [2, 4–7]. Chronic inflammation and tissue damage by these agents leads to cirrhosis which is the underlying condition for the majority of HCC cases [8].
The dissemination of primary tumor cells into the body drastically worsens the prognosis of cancer patients [9]. Metastasis of HCC cells most frequently occurs intrahepatically rather than extrahepatically to distal sites such as the lung [10]. For spreading of HCC cells, individual cell movement by an epithelial to mesenchymal transition (EMT) has been considered to be essentially involved [11]. Upon EMT and progression in malignancy, highly differentiated epithelial cells such as hepatocytes de-differentiate into a mesenchymal-like phenotype that exhibits strong migratory abilities. Various signaling cascades are known to induce EMT, such as the Wnt/β-catenin, PI3K/AKT/mTOR, Hedgehog, Ras/Raf/MEK/ERK, Notch and NFκB pathways, as well as transforming growth factor (TGF)-β [12–15]. These signals mostly converge on EMT-transcription factors (EMT-TFs) such as Snail, ZEB1 or Twist1/2 which transcriptionally repress E-cadherin and other epithelial junctional proteins as well as activate a mesenchymal gene expression signature. In HCC, TGF-β signaling has been shown to activate EMT-TFs and to repress their negative feedback loops by the downregulation of miRNAs that antagonize EMT-TFs [16, 17].
A recently performed meta-analysis compared 24 published EMT gene expression data sets and generated a core list of genes that are most frequently altered during EMT [18]. One of the genes found upregulated in several studies of TGF-β-induced EMT coded for the protein neuropilin-2 (NRP2). There are two homologs of the NRP family, NRP1 and NRP2, which are 130 kDa single-pass transmembrane glycoproteins that act as non-tyrosine kinase co-receptors [19]. They contain 4 distinct domains including a CUB domain, a FV/FVIII domain, a MAM domain and a domain that contains the transmembrane and short cytoplasmic region [20]. Both homologues can homo- and heteromultimerize [21] and can bind different members of the semaphorin family, as well as members of the vascular endothelial growth factor (VEGF) family [22]. In addition, NRPs are receptors of hepatocyte growth factor, platelet-derived growth factor BB, fibroblast growth factor, epidermal growth factor, placenta growth factor, and importantly of TGF-β1 [23–25]. NRPs are therefore critical regulators of angiogenesis, lymphangiogenesis and tumor progression. NRP2 expression is correlated with lymph node metastasis in breast cancer and blocking of NRP2 leads to decreased metastasis formation [26–28]. Clinical data show that high NRP levels, in particular NRP2, correlate with poor prognosis and survival in various cancer types [29, 30]. NRP2 was suggested to play a direct role in EMT and a cross-talk between NRP2 and TGF-β1 signaling promotes colorectal cancer progression [31]. In the context of HCC, the role of NRP2 is so far unknown [32].
In this study, we show that NRP2 expression strongly correlates with a mesenchymal phenotype in HCC cell lines and that reduced levels of NRP2 dramatically impair the migratory abilities of HCC cells. We further provide evidence that NRP2 expression is controlled by canonical TGF-β signaling, while no direct impact of NRP2 on TGF-β signaling could be observed. Translation of these data into the HCC patient situation revealed a correlation of NRP2 levels with a poorly differentiated HCC, suggesting a role of NRP2 in TGF-β regulated HCC progression.
Methods:
Tissue array analysis Tissue arrays contained paraffin-embedded specimens of tumors and adjacent normal tissue collected from 133 female and male HCC patients. All patients have undergone orthotopic liver transplantation for HCC at the Department of Transplantation Surgery, Medical University of Vienna, between 1982 and 2002, as described [33]. All specimens were reviewed for histological type and grade by 2 individual board certified pathologists. 4 μm thick sections of core biopsies were arrayed in triplicate and stained with anti-NRP2 antibody (R&D Systems, Minneapolis, USA) or anti-TGF-β1 antibody (Santa Cruz, Dallas, USA) at a dilution of 1:100. After incubation with secondary antibodies, visualization was performed using the vectastain ABC system (Vector Laboratories, Burlingame, USA). Triplicates of stained tissues were evaluated by two independent researchers (P.W. and M.G.) who were blinded regarding patient details. Immunostaining for NRP2 was scored by arbitrary scaling of no and low-to-high staining. Immunohistochemical analysis of paraffin-embedded tissues and retrospective analysis of patient data were approved by the ethics committee of the Medical University of Vienna.
Tissue arrays contained paraffin-embedded specimens of tumors and adjacent normal tissue collected from 133 female and male HCC patients. All patients have undergone orthotopic liver transplantation for HCC at the Department of Transplantation Surgery, Medical University of Vienna, between 1982 and 2002, as described [33]. All specimens were reviewed for histological type and grade by 2 individual board certified pathologists. 4 μm thick sections of core biopsies were arrayed in triplicate and stained with anti-NRP2 antibody (R&D Systems, Minneapolis, USA) or anti-TGF-β1 antibody (Santa Cruz, Dallas, USA) at a dilution of 1:100. After incubation with secondary antibodies, visualization was performed using the vectastain ABC system (Vector Laboratories, Burlingame, USA). Triplicates of stained tissues were evaluated by two independent researchers (P.W. and M.G.) who were blinded regarding patient details. Immunostaining for NRP2 was scored by arbitrary scaling of no and low-to-high staining. Immunohistochemical analysis of paraffin-embedded tissues and retrospective analysis of patient data were approved by the ethics committee of the Medical University of Vienna.
Cell culture The human hepatoma cell lines 3p, 3sp, SNU-398, SNU-423, SNU-449 and SNU-475 were grown in RPMI-1640 medium plus 10 % fetal calf serum (FCS) and antibiotics. Hep3B, HepG2 and FLC-4 cells were cultivated in Eagle’s Minimum Essential Medium (EMEM) plus 10 % FCS and antibiotics. PLC and HuH-6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10 % FCS and antibiotics. Human hepatic sinusoidal endothelial cells (HSECs) received endothelial cell medium (Lonza, Basel, Switzerland). All cells were kept at 37 °C and 5 % CO2. TGF-β signaling was stimulated by supplementing the medium with 2.5 ng/mL TGF-β1 (R&D Systems, Minneapolis, USA) for 24 h. For inhibition of TGF-β signaling, LY2109761 (Santa Cruz, Dallas, USA) antagonizing both TGF-β receptors I/II was used at a concentration of 10 μM for 24 h. All HCC cell lines were validated by short tandem repeat analysis.
The human hepatoma cell lines 3p, 3sp, SNU-398, SNU-423, SNU-449 and SNU-475 were grown in RPMI-1640 medium plus 10 % fetal calf serum (FCS) and antibiotics. Hep3B, HepG2 and FLC-4 cells were cultivated in Eagle’s Minimum Essential Medium (EMEM) plus 10 % FCS and antibiotics. PLC and HuH-6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10 % FCS and antibiotics. Human hepatic sinusoidal endothelial cells (HSECs) received endothelial cell medium (Lonza, Basel, Switzerland). All cells were kept at 37 °C and 5 % CO2. TGF-β signaling was stimulated by supplementing the medium with 2.5 ng/mL TGF-β1 (R&D Systems, Minneapolis, USA) for 24 h. For inhibition of TGF-β signaling, LY2109761 (Santa Cruz, Dallas, USA) antagonizing both TGF-β receptors I/II was used at a concentration of 10 μM for 24 h. All HCC cell lines were validated by short tandem repeat analysis.
siRNA knock-down Cells were seeded on 6-well plates and either transfected with 80 nM of non-target small interfering (si)RNA or with 80 nM of siRNA against human NRP2 (Dharmacon, Town, UK). Cells were processed for further analysis after 48 h of siRNA transfection.
Cells were seeded on 6-well plates and either transfected with 80 nM of non-target small interfering (si)RNA or with 80 nM of siRNA against human NRP2 (Dharmacon, Town, UK). Cells were processed for further analysis after 48 h of siRNA transfection.
Western blot analysis Immunoblotting was performed as described [34]. Dilutions of primary antibodies were as follows: NRP2 (R&D Systems, Minneapolis, USA), 1:1000; β-actin (Sigma, St. Louis, USA), 1:2500; Smad2/3 (BD Biosciences, NJ, USA), 1:1000; pSmad2 (Upstate, NY, USA), 1:500; Smad4 (Cell Signaling Technology, Danvers, USA), 1:1000. Secondary antibodies conjugated to horseradish peroxidase were used for detection.
Immunoblotting was performed as described [34]. Dilutions of primary antibodies were as follows: NRP2 (R&D Systems, Minneapolis, USA), 1:1000; β-actin (Sigma, St. Louis, USA), 1:2500; Smad2/3 (BD Biosciences, NJ, USA), 1:1000; pSmad2 (Upstate, NY, USA), 1:500; Smad4 (Cell Signaling Technology, Danvers, USA), 1:1000. Secondary antibodies conjugated to horseradish peroxidase were used for detection.
Transwell migration and invasion 2.5 × 104 hepatoma cells were resuspended in 100 μL medium containing 1 % FCS and transferred onto 24-well cell culture inserts with a pore size of 8 μm (Corning, Tewksbury, USA). 600 μL medium containing 10 % FCS was added to lower chambers to generate a gradient for cell migration. Medium was removed and migrated cells on membranes were fixed with 4 % paraformaldehyde after 16 h. Cell nuclei of migrated cells at the bottom side of the membrane were stained with Hoechst 33342 (Life Technologies, Green Island, USA) and counted under the fluorescence microscope (Nikon, Tokyo, Japan). To analyze cell invasion, 24-well cell culture inserts were coated with rat-tail collagen (BD Biosciences, NJ, USA) prior to seeding of hepatoma cells. To examine transendothelial invasion, 2 × 105 HSECs were plated in 100 μL endothelial cell medium (Lonza, Basel, Switzerland) onto coated 24-well cell culture inserts and allowed to form a monolayer for 48 h. Subsequently, endothelial cell medium was aspirated and hepatoma cells were seeded in 100 μL medium containing 1 % FCS onto 24-well inserts. Transmigrated cells were visualized after 16 h and quantified as described for cell migration.
2.5 × 104 hepatoma cells were resuspended in 100 μL medium containing 1 % FCS and transferred onto 24-well cell culture inserts with a pore size of 8 μm (Corning, Tewksbury, USA). 600 μL medium containing 10 % FCS was added to lower chambers to generate a gradient for cell migration. Medium was removed and migrated cells on membranes were fixed with 4 % paraformaldehyde after 16 h. Cell nuclei of migrated cells at the bottom side of the membrane were stained with Hoechst 33342 (Life Technologies, Green Island, USA) and counted under the fluorescence microscope (Nikon, Tokyo, Japan). To analyze cell invasion, 24-well cell culture inserts were coated with rat-tail collagen (BD Biosciences, NJ, USA) prior to seeding of hepatoma cells. To examine transendothelial invasion, 2 × 105 HSECs were plated in 100 μL endothelial cell medium (Lonza, Basel, Switzerland) onto coated 24-well cell culture inserts and allowed to form a monolayer for 48 h. Subsequently, endothelial cell medium was aspirated and hepatoma cells were seeded in 100 μL medium containing 1 % FCS onto 24-well inserts. Transmigrated cells were visualized after 16 h and quantified as described for cell migration.
Wound healing assay For studying cell migration in a scratch wound assay, cells were seeded in 6-well plates and artificial wounds were inflicted to the cell layer by scratching with sterile pipette tips. For each condition, three scratches were inflicted in three independent wells of a 6-well plate. From each of these scratches eight images were taken for a total of 24 images per condition and time point. Images were performed by phase contrast microscopy (Nikon, Tokyo, Japan) immediately after wounding and after 24 h. The migrated area of cells into the wound was quantified with ImageJ software.
For studying cell migration in a scratch wound assay, cells were seeded in 6-well plates and artificial wounds were inflicted to the cell layer by scratching with sterile pipette tips. For each condition, three scratches were inflicted in three independent wells of a 6-well plate. From each of these scratches eight images were taken for a total of 24 images per condition and time point. Images were performed by phase contrast microscopy (Nikon, Tokyo, Japan) immediately after wounding and after 24 h. The migrated area of cells into the wound was quantified with ImageJ software.
Quantitative real-time polymerase chain reaction (qPCR) RNA isolation was performed using an RNeasy purification kit according to the manufacturer’s instructions (Quiagen, Hilden, Germany). For reverse transcription of RNA into DNA, the QuantiTect Reverse Transcription kit was employed (Quiagen, Hilden, Germany). For quantitative RT-PCR (qPCR), aliquots of cDNA were employed for Fast SYBR green qPCR (Applied Biosystems, Foster City, USA) and quantified with the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, USA). RPL41 as HCC housekeeping gene and the average ΔCT value of the HCC cell line 3sp were used to calculate the ΔΔCT values for all cell lines. The ΔΔCT values were used to graph the fold-change (RQ) via the formula RQ = 2^(−ΔΔCT). Error bars show SE of ΔΔCT, calculated with the following formula: SE(ΔΔCT) = √((SE(ΔCT Control)^2) ± (SE(ΔCT Target)^2)). The primer sequences are: NRP2 (forward), 5’-CTGTGGGTCATCCGTGAGGAC-3’ and NRP2 (reverse) 5’-ATGGGTTCCATGCAGTTCTCCAG-3’; RPL41 (forward), 5’-CAAGTGGAGGAAGAAGCGA-3’ and RPL41 (reverse), 5’-TTACTTGGACCTCTGCCTC-3’.
RNA isolation was performed using an RNeasy purification kit according to the manufacturer’s instructions (Quiagen, Hilden, Germany). For reverse transcription of RNA into DNA, the QuantiTect Reverse Transcription kit was employed (Quiagen, Hilden, Germany). For quantitative RT-PCR (qPCR), aliquots of cDNA were employed for Fast SYBR green qPCR (Applied Biosystems, Foster City, USA) and quantified with the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, USA). RPL41 as HCC housekeeping gene and the average ΔCT value of the HCC cell line 3sp were used to calculate the ΔΔCT values for all cell lines. The ΔΔCT values were used to graph the fold-change (RQ) via the formula RQ = 2^(−ΔΔCT). Error bars show SE of ΔΔCT, calculated with the following formula: SE(ΔΔCT) = √((SE(ΔCT Control)^2) ± (SE(ΔCT Target)^2)). The primer sequences are: NRP2 (forward), 5’-CTGTGGGTCATCCGTGAGGAC-3’ and NRP2 (reverse) 5’-ATGGGTTCCATGCAGTTCTCCAG-3’; RPL41 (forward), 5’-CAAGTGGAGGAAGAAGCGA-3’ and RPL41 (reverse), 5’-TTACTTGGACCTCTGCCTC-3’.
Confocal immunofluorescence microscopy Cells were seeded on glass cover slips coated with rat-tail collagen (BD Biosciences, NJ, USA) and fixed with 4 % formaldehyde. After permeabilization with 0.25 % Triton-X 100 and blocking with 5 % horse serum, cells were stained with primary antibody against Smad2/3 (BD Biosciences, NJ, USA) at a concentration of 1:100 and further incubated with secondary antibody (1:200), phalloidin (1:750) and DAPI (1:1000). Images were obtained by confocal immunofluorescence microscopy (Zeiss, Oberkochen, Germany).
Cells were seeded on glass cover slips coated with rat-tail collagen (BD Biosciences, NJ, USA) and fixed with 4 % formaldehyde. After permeabilization with 0.25 % Triton-X 100 and blocking with 5 % horse serum, cells were stained with primary antibody against Smad2/3 (BD Biosciences, NJ, USA) at a concentration of 1:100 and further incubated with secondary antibody (1:200), phalloidin (1:750) and DAPI (1:1000). Images were obtained by confocal immunofluorescence microscopy (Zeiss, Oberkochen, Germany).
Statistical analysis The statistical significance of differences was evaluated using two-sided Student’s t-test. Data are expressed as means ± standard deviation (SD) or means ± standard error of the mean (SEM) for tissue array data.
The statistical significance of differences was evaluated using two-sided Student’s t-test. Data are expressed as means ± standard deviation (SD) or means ± standard error of the mean (SEM) for tissue array data.
Tissue array analysis:
Tissue arrays contained paraffin-embedded specimens of tumors and adjacent normal tissue collected from 133 female and male HCC patients. All patients have undergone orthotopic liver transplantation for HCC at the Department of Transplantation Surgery, Medical University of Vienna, between 1982 and 2002, as described [33]. All specimens were reviewed for histological type and grade by 2 individual board certified pathologists. 4 μm thick sections of core biopsies were arrayed in triplicate and stained with anti-NRP2 antibody (R&D Systems, Minneapolis, USA) or anti-TGF-β1 antibody (Santa Cruz, Dallas, USA) at a dilution of 1:100. After incubation with secondary antibodies, visualization was performed using the vectastain ABC system (Vector Laboratories, Burlingame, USA). Triplicates of stained tissues were evaluated by two independent researchers (P.W. and M.G.) who were blinded regarding patient details. Immunostaining for NRP2 was scored by arbitrary scaling of no and low-to-high staining. Immunohistochemical analysis of paraffin-embedded tissues and retrospective analysis of patient data were approved by the ethics committee of the Medical University of Vienna.
Cell culture:
The human hepatoma cell lines 3p, 3sp, SNU-398, SNU-423, SNU-449 and SNU-475 were grown in RPMI-1640 medium plus 10 % fetal calf serum (FCS) and antibiotics. Hep3B, HepG2 and FLC-4 cells were cultivated in Eagle’s Minimum Essential Medium (EMEM) plus 10 % FCS and antibiotics. PLC and HuH-6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10 % FCS and antibiotics. Human hepatic sinusoidal endothelial cells (HSECs) received endothelial cell medium (Lonza, Basel, Switzerland). All cells were kept at 37 °C and 5 % CO2. TGF-β signaling was stimulated by supplementing the medium with 2.5 ng/mL TGF-β1 (R&D Systems, Minneapolis, USA) for 24 h. For inhibition of TGF-β signaling, LY2109761 (Santa Cruz, Dallas, USA) antagonizing both TGF-β receptors I/II was used at a concentration of 10 μM for 24 h. All HCC cell lines were validated by short tandem repeat analysis.
siRNA knock-down:
Cells were seeded on 6-well plates and either transfected with 80 nM of non-target small interfering (si)RNA or with 80 nM of siRNA against human NRP2 (Dharmacon, Town, UK). Cells were processed for further analysis after 48 h of siRNA transfection.
Western blot analysis:
Immunoblotting was performed as described [34]. Dilutions of primary antibodies were as follows: NRP2 (R&D Systems, Minneapolis, USA), 1:1000; β-actin (Sigma, St. Louis, USA), 1:2500; Smad2/3 (BD Biosciences, NJ, USA), 1:1000; pSmad2 (Upstate, NY, USA), 1:500; Smad4 (Cell Signaling Technology, Danvers, USA), 1:1000. Secondary antibodies conjugated to horseradish peroxidase were used for detection.
Transwell migration and invasion:
2.5 × 104 hepatoma cells were resuspended in 100 μL medium containing 1 % FCS and transferred onto 24-well cell culture inserts with a pore size of 8 μm (Corning, Tewksbury, USA). 600 μL medium containing 10 % FCS was added to lower chambers to generate a gradient for cell migration. Medium was removed and migrated cells on membranes were fixed with 4 % paraformaldehyde after 16 h. Cell nuclei of migrated cells at the bottom side of the membrane were stained with Hoechst 33342 (Life Technologies, Green Island, USA) and counted under the fluorescence microscope (Nikon, Tokyo, Japan). To analyze cell invasion, 24-well cell culture inserts were coated with rat-tail collagen (BD Biosciences, NJ, USA) prior to seeding of hepatoma cells. To examine transendothelial invasion, 2 × 105 HSECs were plated in 100 μL endothelial cell medium (Lonza, Basel, Switzerland) onto coated 24-well cell culture inserts and allowed to form a monolayer for 48 h. Subsequently, endothelial cell medium was aspirated and hepatoma cells were seeded in 100 μL medium containing 1 % FCS onto 24-well inserts. Transmigrated cells were visualized after 16 h and quantified as described for cell migration.
Wound healing assay:
For studying cell migration in a scratch wound assay, cells were seeded in 6-well plates and artificial wounds were inflicted to the cell layer by scratching with sterile pipette tips. For each condition, three scratches were inflicted in three independent wells of a 6-well plate. From each of these scratches eight images were taken for a total of 24 images per condition and time point. Images were performed by phase contrast microscopy (Nikon, Tokyo, Japan) immediately after wounding and after 24 h. The migrated area of cells into the wound was quantified with ImageJ software.
Quantitative real-time polymerase chain reaction (qPCR):
RNA isolation was performed using an RNeasy purification kit according to the manufacturer’s instructions (Quiagen, Hilden, Germany). For reverse transcription of RNA into DNA, the QuantiTect Reverse Transcription kit was employed (Quiagen, Hilden, Germany). For quantitative RT-PCR (qPCR), aliquots of cDNA were employed for Fast SYBR green qPCR (Applied Biosystems, Foster City, USA) and quantified with the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, USA). RPL41 as HCC housekeeping gene and the average ΔCT value of the HCC cell line 3sp were used to calculate the ΔΔCT values for all cell lines. The ΔΔCT values were used to graph the fold-change (RQ) via the formula RQ = 2^(−ΔΔCT). Error bars show SE of ΔΔCT, calculated with the following formula: SE(ΔΔCT) = √((SE(ΔCT Control)^2) ± (SE(ΔCT Target)^2)). The primer sequences are: NRP2 (forward), 5’-CTGTGGGTCATCCGTGAGGAC-3’ and NRP2 (reverse) 5’-ATGGGTTCCATGCAGTTCTCCAG-3’; RPL41 (forward), 5’-CAAGTGGAGGAAGAAGCGA-3’ and RPL41 (reverse), 5’-TTACTTGGACCTCTGCCTC-3’.
Confocal immunofluorescence microscopy:
Cells were seeded on glass cover slips coated with rat-tail collagen (BD Biosciences, NJ, USA) and fixed with 4 % formaldehyde. After permeabilization with 0.25 % Triton-X 100 and blocking with 5 % horse serum, cells were stained with primary antibody against Smad2/3 (BD Biosciences, NJ, USA) at a concentration of 1:100 and further incubated with secondary antibody (1:200), phalloidin (1:750) and DAPI (1:1000). Images were obtained by confocal immunofluorescence microscopy (Zeiss, Oberkochen, Germany).
Statistical analysis:
The statistical significance of differences was evaluated using two-sided Student’s t-test. Data are expressed as means ± standard deviation (SD) or means ± standard error of the mean (SEM) for tissue array data.
Results:
NRP2 is upregulated in high-grade HCC and highly expressed in mesenchymal-like HCC cell lines We employed a tissue array containing 133 HCC cases for the analysis of NRP2 expression and found a significant correlation of NRP2 levels with higher tumor grading. While well-differentiated HCC (grade 1) showed NRP2 expression in 32 % of cases, less-differentiated HCC samples displayed a nearly twice as much higher frequency of NRP2 presence. In particular, 59 % of HCC with grade 2 and 56 % of HCC with grade 3 tumors showed NRP2 expression (Fig. 1a). Generally, NRP2 was found distributed in a patchy fashion (Fig. 1b) and interestingly, NRP2 was exclusively expressed in those patient samples that showed TGF-β1 expression (Additional file 1: Figure S1). Noteworthy, normal liver did not display NRP2 expression (Fig. 1b) which confirmed recent observations that NRP2 is not expressed in hepatocytes under physiological conditions [35].Fig. 1NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01
NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01
To correlate NRP2 expression with differentiation of hepatoma cells more closely, we analyzed various human HCC cell lines with differentiated and epithelial traits versus those exhibiting a de-differentiated and mesenchymal-like phenotype. Western blot (Fig. 1c) and qPCR analyses (Fig. 1d) revealed that NRP2 expression strongly correlated with a mesenchymal-like HCC phenotype in 3sp, SNU-398, SNU-423, SNU-449, SNU-475 and FLC-4 cells. In contrast, NRP2 was expressed in only one out of five epithelial HCC cell lines, i.e., HuH-6 cells, and showed undetectable levels of NRP2 in the other epithelial 3p, Hep3B, HepG2 and PLC cells. Together, these data suggest that NRP2 expression associates with less-differentiated high-grade tumors and TGF-β1 expression in HCC patients. In agreement, NRP2 expression was found in de-differentiated mesenchymal-like HCC cells in vitro which supports the idea that NRP2 expression correlates with a de-differentiated phenotype.
We employed a tissue array containing 133 HCC cases for the analysis of NRP2 expression and found a significant correlation of NRP2 levels with higher tumor grading. While well-differentiated HCC (grade 1) showed NRP2 expression in 32 % of cases, less-differentiated HCC samples displayed a nearly twice as much higher frequency of NRP2 presence. In particular, 59 % of HCC with grade 2 and 56 % of HCC with grade 3 tumors showed NRP2 expression (Fig. 1a). Generally, NRP2 was found distributed in a patchy fashion (Fig. 1b) and interestingly, NRP2 was exclusively expressed in those patient samples that showed TGF-β1 expression (Additional file 1: Figure S1). Noteworthy, normal liver did not display NRP2 expression (Fig. 1b) which confirmed recent observations that NRP2 is not expressed in hepatocytes under physiological conditions [35].Fig. 1NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01
NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01
To correlate NRP2 expression with differentiation of hepatoma cells more closely, we analyzed various human HCC cell lines with differentiated and epithelial traits versus those exhibiting a de-differentiated and mesenchymal-like phenotype. Western blot (Fig. 1c) and qPCR analyses (Fig. 1d) revealed that NRP2 expression strongly correlated with a mesenchymal-like HCC phenotype in 3sp, SNU-398, SNU-423, SNU-449, SNU-475 and FLC-4 cells. In contrast, NRP2 was expressed in only one out of five epithelial HCC cell lines, i.e., HuH-6 cells, and showed undetectable levels of NRP2 in the other epithelial 3p, Hep3B, HepG2 and PLC cells. Together, these data suggest that NRP2 expression associates with less-differentiated high-grade tumors and TGF-β1 expression in HCC patients. In agreement, NRP2 expression was found in de-differentiated mesenchymal-like HCC cells in vitro which supports the idea that NRP2 expression correlates with a de-differentiated phenotype.
NRP2 regulates HCC cell migration and invasion Next we analyzed the functional impact of NRP2 expression on cell proliferation and migration. Neither knock-down nor exogenous overexpression of NRP2 significantly affected proliferation kinetics of mesenchymal-like HCC cells (data not shown). However, Transwell migration assays employing the mesenchymal-like cell lines 3sp and SNU-449 revealed that reduced expression of NRP2 results in impaired migratory abilities (Fig. 2a). Comparable results were obtained in Transwell invasion and transendothelial migration assays (Fig. 2b, c). In addition, assessment of cell migration by wound healing assays of SNU-449 cells confirmed elevated migratory capabilities dependent on NRP2 (Fig. 2d). Interestingly, overexpression of NRP2 could not enhance migration or invasion in any of the employed cell lines (3p, PLC, 3sp, SNU-449; data not shown). Thus, these results suggest that NRP2 is crucially involved in upregulating cell movement of de-differentiated HCC cells.Fig. 2Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001
Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001
Next we analyzed the functional impact of NRP2 expression on cell proliferation and migration. Neither knock-down nor exogenous overexpression of NRP2 significantly affected proliferation kinetics of mesenchymal-like HCC cells (data not shown). However, Transwell migration assays employing the mesenchymal-like cell lines 3sp and SNU-449 revealed that reduced expression of NRP2 results in impaired migratory abilities (Fig. 2a). Comparable results were obtained in Transwell invasion and transendothelial migration assays (Fig. 2b, c). In addition, assessment of cell migration by wound healing assays of SNU-449 cells confirmed elevated migratory capabilities dependent on NRP2 (Fig. 2d). Interestingly, overexpression of NRP2 could not enhance migration or invasion in any of the employed cell lines (3p, PLC, 3sp, SNU-449; data not shown). Thus, these results suggest that NRP2 is crucially involved in upregulating cell movement of de-differentiated HCC cells.Fig. 2Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001
Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001
TGF-β induces NRP2 in a Smad-dependent fashion Since NRP2 expression correlated with TGF-β1 expression (Additional file 1: Figure S1) and TGF-β signaling is a common trigger of EMT in HCC, we asked whether it directly influences NRP2 expression in HCC cells. Remarkably, TGF-β treatment of epithelial 3p, Hep3B and PLC cells, as well as mesenchymal-like 3sp, SNU-423 and SNU-449 cells resulted in increased NRP2 protein expression (Fig. 3a). Upregulation of NRP2 was essentially confirmed at transcript levels by qPCR analysis of epithelial (3p, Hep3B, PLC) and mesenchymal-like HCC cells (3sp, SNU-423, SNU-449) (Fig. 3b). Interestingly, blocking of TGF-β signaling by the TGF-β receptor inhibitor LY2109761 resulted in downregulation of NRP2 mRNA and protein to levels even lower than those of untreated cells (Fig. 3a–c), suggesting autoregulatory TGF-β loops in respective HCC cells. Importantly, cells with interference of canonical TGF-β/Smad signaling by knock-down of Smad4 were insensitive to TGF-β-induced upregulation of NRP2 (Fig. 3c). These data indicate that augmented NRP2 expression is caused by canonical TGF-β/Smad signaling.Fig. 3NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates
NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates
Since NRP2 expression correlated with TGF-β1 expression (Additional file 1: Figure S1) and TGF-β signaling is a common trigger of EMT in HCC, we asked whether it directly influences NRP2 expression in HCC cells. Remarkably, TGF-β treatment of epithelial 3p, Hep3B and PLC cells, as well as mesenchymal-like 3sp, SNU-423 and SNU-449 cells resulted in increased NRP2 protein expression (Fig. 3a). Upregulation of NRP2 was essentially confirmed at transcript levels by qPCR analysis of epithelial (3p, Hep3B, PLC) and mesenchymal-like HCC cells (3sp, SNU-423, SNU-449) (Fig. 3b). Interestingly, blocking of TGF-β signaling by the TGF-β receptor inhibitor LY2109761 resulted in downregulation of NRP2 mRNA and protein to levels even lower than those of untreated cells (Fig. 3a–c), suggesting autoregulatory TGF-β loops in respective HCC cells. Importantly, cells with interference of canonical TGF-β/Smad signaling by knock-down of Smad4 were insensitive to TGF-β-induced upregulation of NRP2 (Fig. 3c). These data indicate that augmented NRP2 expression is caused by canonical TGF-β/Smad signaling.Fig. 3NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates
NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates
TGF-β-independent activity of NRP2 We next examined whether NRP2 affects canonical TGF-β signaling in a feedback loop. First, we analyzed the level of phosphorylated Smad2 protein that displays TGF-β signaling in SNU-449 cells with and without a NRP2 knock-down after treatment with TGF-β. No difference in pSmad2 levels were observed, independently whether or not NRP2 was expressed (Fig. 4a). To confirm these data, we performed confocal immunofluorescence analysis of TGF-β-treated NRP2 knock-down cells and analyzed nuclear Smad2/3 staining (Fig. 4b). In accordance, no differences in nuclear Smad2/3 localization between control and NRP2 knock-down cells were detected, suggesting that NRP2 does not influence the canonical TGF-β/Smad signaling.Fig. 4Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001
Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001
Interference with TGF-β signaling also leads to a reduced migration of mesenchymal-like HCC cells. Hence, we further analyzed whether the effect of reduced NRP2 on cell migration is dependent on TGF-β signaling or not by employing NRP2 knock-down and control cells after treatment with TGF-β inhibitor LY2109761 (Fig. 4c). Cells treated with LY2109761 reached approximately 64 % migration efficiency compared to control siNT cells. NRP2 knock-down cells without inhibitor treatment were even slower migrating, reaching about 33 % of cell motility. Importantly, the cells treated with both siRNA against NRP2 and LY2109761 migrated even significantly slower than those treated with LY2109761 only, reaching about 10 % compared to reference. In conclusion, these findings indicate that NRP2 has a role in cell migration that is independent of TGF-β signaling.
We next examined whether NRP2 affects canonical TGF-β signaling in a feedback loop. First, we analyzed the level of phosphorylated Smad2 protein that displays TGF-β signaling in SNU-449 cells with and without a NRP2 knock-down after treatment with TGF-β. No difference in pSmad2 levels were observed, independently whether or not NRP2 was expressed (Fig. 4a). To confirm these data, we performed confocal immunofluorescence analysis of TGF-β-treated NRP2 knock-down cells and analyzed nuclear Smad2/3 staining (Fig. 4b). In accordance, no differences in nuclear Smad2/3 localization between control and NRP2 knock-down cells were detected, suggesting that NRP2 does not influence the canonical TGF-β/Smad signaling.Fig. 4Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001
Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001
Interference with TGF-β signaling also leads to a reduced migration of mesenchymal-like HCC cells. Hence, we further analyzed whether the effect of reduced NRP2 on cell migration is dependent on TGF-β signaling or not by employing NRP2 knock-down and control cells after treatment with TGF-β inhibitor LY2109761 (Fig. 4c). Cells treated with LY2109761 reached approximately 64 % migration efficiency compared to control siNT cells. NRP2 knock-down cells without inhibitor treatment were even slower migrating, reaching about 33 % of cell motility. Importantly, the cells treated with both siRNA against NRP2 and LY2109761 migrated even significantly slower than those treated with LY2109761 only, reaching about 10 % compared to reference. In conclusion, these findings indicate that NRP2 has a role in cell migration that is independent of TGF-β signaling.
NRP2 is upregulated in high-grade HCC and highly expressed in mesenchymal-like HCC cell lines:
We employed a tissue array containing 133 HCC cases for the analysis of NRP2 expression and found a significant correlation of NRP2 levels with higher tumor grading. While well-differentiated HCC (grade 1) showed NRP2 expression in 32 % of cases, less-differentiated HCC samples displayed a nearly twice as much higher frequency of NRP2 presence. In particular, 59 % of HCC with grade 2 and 56 % of HCC with grade 3 tumors showed NRP2 expression (Fig. 1a). Generally, NRP2 was found distributed in a patchy fashion (Fig. 1b) and interestingly, NRP2 was exclusively expressed in those patient samples that showed TGF-β1 expression (Additional file 1: Figure S1). Noteworthy, normal liver did not display NRP2 expression (Fig. 1b) which confirmed recent observations that NRP2 is not expressed in hepatocytes under physiological conditions [35].Fig. 1NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01
NRP2 expression in primary HCC tissue and HCC cell lines. a NRP2 expression correlated with less differentiated HCC of grade 2 and 3. b Representative images of no (left) and low-to-high NRP2 expression (right). c, d Western blot (c) and qPCR analysis (d) of epithelial and mesenchymal-like HCC cells. Expression of actin is shown as loading control. NRP2 expression in 3sp cells was set to a value of 1 to allow comparison of NRP2 levels in the various cell lines. Error bars depict SD from 3 independent experiments that were performed in triplicates.*, p < 0.05; **, p < 0.01
To correlate NRP2 expression with differentiation of hepatoma cells more closely, we analyzed various human HCC cell lines with differentiated and epithelial traits versus those exhibiting a de-differentiated and mesenchymal-like phenotype. Western blot (Fig. 1c) and qPCR analyses (Fig. 1d) revealed that NRP2 expression strongly correlated with a mesenchymal-like HCC phenotype in 3sp, SNU-398, SNU-423, SNU-449, SNU-475 and FLC-4 cells. In contrast, NRP2 was expressed in only one out of five epithelial HCC cell lines, i.e., HuH-6 cells, and showed undetectable levels of NRP2 in the other epithelial 3p, Hep3B, HepG2 and PLC cells. Together, these data suggest that NRP2 expression associates with less-differentiated high-grade tumors and TGF-β1 expression in HCC patients. In agreement, NRP2 expression was found in de-differentiated mesenchymal-like HCC cells in vitro which supports the idea that NRP2 expression correlates with a de-differentiated phenotype.
NRP2 regulates HCC cell migration and invasion:
Next we analyzed the functional impact of NRP2 expression on cell proliferation and migration. Neither knock-down nor exogenous overexpression of NRP2 significantly affected proliferation kinetics of mesenchymal-like HCC cells (data not shown). However, Transwell migration assays employing the mesenchymal-like cell lines 3sp and SNU-449 revealed that reduced expression of NRP2 results in impaired migratory abilities (Fig. 2a). Comparable results were obtained in Transwell invasion and transendothelial migration assays (Fig. 2b, c). In addition, assessment of cell migration by wound healing assays of SNU-449 cells confirmed elevated migratory capabilities dependent on NRP2 (Fig. 2d). Interestingly, overexpression of NRP2 could not enhance migration or invasion in any of the employed cell lines (3p, PLC, 3sp, SNU-449; data not shown). Thus, these results suggest that NRP2 is crucially involved in upregulating cell movement of de-differentiated HCC cells.Fig. 2Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001
Loss of NRP2 impairs migration of HCC cells. a Transwell migration of 3sp and SNU-449 cells either untreated or treated with non-target siRNA (siNT) or siRNA against NRP2 (siNRP2). b, cTranswell invasion (b) and transendothelial invasion (c). d Migration of SNU-449 analyzed by wound healing assays. Images show migration of cells after 24 h (left panel). Quantification of migrated cells (right panel). The migration of untreated parental SNU-449 cells was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. **, p < 0.01; ***, p < 0.001
TGF-β induces NRP2 in a Smad-dependent fashion:
Since NRP2 expression correlated with TGF-β1 expression (Additional file 1: Figure S1) and TGF-β signaling is a common trigger of EMT in HCC, we asked whether it directly influences NRP2 expression in HCC cells. Remarkably, TGF-β treatment of epithelial 3p, Hep3B and PLC cells, as well as mesenchymal-like 3sp, SNU-423 and SNU-449 cells resulted in increased NRP2 protein expression (Fig. 3a). Upregulation of NRP2 was essentially confirmed at transcript levels by qPCR analysis of epithelial (3p, Hep3B, PLC) and mesenchymal-like HCC cells (3sp, SNU-423, SNU-449) (Fig. 3b). Interestingly, blocking of TGF-β signaling by the TGF-β receptor inhibitor LY2109761 resulted in downregulation of NRP2 mRNA and protein to levels even lower than those of untreated cells (Fig. 3a–c), suggesting autoregulatory TGF-β loops in respective HCC cells. Importantly, cells with interference of canonical TGF-β/Smad signaling by knock-down of Smad4 were insensitive to TGF-β-induced upregulation of NRP2 (Fig. 3c). These data indicate that augmented NRP2 expression is caused by canonical TGF-β/Smad signaling.Fig. 3NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates
NRP2 expression depends on TGF-β/Smad signaling. a NRP2 protein expression after stimulation with 2.5 ng/mL TGF-β1 for 24 h as determined by Western blotting. Actin is shown as loading control. b qPCR analysis showing NRP2 mRNA levels in epithelial and mesenchymal-like HCC cell lines treated with either 2.5 ng/mL TGF-β1 or with 10 μM TGF-β inhibitor LY2109761 (LY) for 24 h. c SNU-449 hepatoma cells treated with control siRNA (siNT) and either administrated with 2.5 ng/mL TGF-β or 10 μM LY2109761 (LY) alone or in combination and analyzed for NRP2 and Smad4 expression by Western blotting. Smad4 knock-down (siSmad4) of SNU-449 cells treated with 2.5 ng/mL TGF-β1 for 24 h did not show modulation of NRP2 levels. Error bars depict SD from 3 independent experiments that were performed in triplicates
TGF-β-independent activity of NRP2:
We next examined whether NRP2 affects canonical TGF-β signaling in a feedback loop. First, we analyzed the level of phosphorylated Smad2 protein that displays TGF-β signaling in SNU-449 cells with and without a NRP2 knock-down after treatment with TGF-β. No difference in pSmad2 levels were observed, independently whether or not NRP2 was expressed (Fig. 4a). To confirm these data, we performed confocal immunofluorescence analysis of TGF-β-treated NRP2 knock-down cells and analyzed nuclear Smad2/3 staining (Fig. 4b). In accordance, no differences in nuclear Smad2/3 localization between control and NRP2 knock-down cells were detected, suggesting that NRP2 does not influence the canonical TGF-β/Smad signaling.Fig. 4Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001
Effects of NRP2 on TGF-β Signaling and synergistic effects on migration. a Knock-down of NRP2 does not affect phosphorylation of Smad2 (pSmad2) in SNU-449 cells after treatment with 2.5 ng/mL TGF-β1 for 6 h. Actin was used as loading control. b Translocation of Smad2/3 into the nucleus of SNU-449 cells treated with control siRNA (siNT) or siRNA against NRP2 (siNRP2) after treatment with 2.5 ng/mL TGF-β1 for 1 h. c Transwell assays of SNU-449 cells assessing the migratory impact of reduced TGF-β signaling by LY2109761 (LY) and diminished NRP2 expression by knock-down (siNRP2), either alone or in combination. The treatment of cells with non-target siRNA (siNT) was set to 100 %. Error bars depict SD from 3 independent experiments that were carried out in triplicates. ***, p < 0.001
Interference with TGF-β signaling also leads to a reduced migration of mesenchymal-like HCC cells. Hence, we further analyzed whether the effect of reduced NRP2 on cell migration is dependent on TGF-β signaling or not by employing NRP2 knock-down and control cells after treatment with TGF-β inhibitor LY2109761 (Fig. 4c). Cells treated with LY2109761 reached approximately 64 % migration efficiency compared to control siNT cells. NRP2 knock-down cells without inhibitor treatment were even slower migrating, reaching about 33 % of cell motility. Importantly, the cells treated with both siRNA against NRP2 and LY2109761 migrated even significantly slower than those treated with LY2109761 only, reaching about 10 % compared to reference. In conclusion, these findings indicate that NRP2 has a role in cell migration that is independent of TGF-β signaling.
Discussion:
Of the two neuropilin isoforms, NRP1 is the far more examined protein. Its role in various types of cancer is well documented and often linked to angiogenesis via VEGF. In contrast, few studies were dedicated to NRP2 and its role in tumorigenesis. To our knowledge no attempts were made to elucidate its role in HCC.
In this study, we showed that NRP2 expression correlates with less differentiation in HCC patients as well as with a de-differentiated, mesenchymal-like phenotype of HCC cell lines (Fig. 1). Functionally, NRP2 expression severely enhances migration of mesenchymal-like HCC cells and is induced by the canonical TGF-β/Smad signaling. In this setting, NRP2 can be considered as a biomarker of both the activation of TGF-β/Smad signaling and loss of differentiation by EMT. Thus, NRP2 represents a novel marker for EMT-transformed cells that can be used apart from the classical ones such as increased vimentin and N-cadherin or loss of ZO-1 and E-cadherin expression [36].
Diminished migration of HCC cells was similar either after reducing NRP2 levels or after blocking TGF-β signaling (Fig. 4c). TGF-β inhibition on its own reduced NRP2 expression but could not completely block it (Fig. 3a, b), as the remaining NRP2 levels might allow cells to better migrate as compared to those cells with strongly reduced NRP2 levels after RNA interference. Yet, if HCC cells were blocked for both NRP2 and TGF-β signaling, a further decrease in HCC cell migration was observed (Fig. 4c). This additive operation of NRP2 and TGF-β signaling suggests that TGF-β additionally affects cell migration that is independent of NRP2. Moreover, neither overexpression of exogenous NRP2 nor treatment with TGF-β1 had any detectable positive effect on migration (data not shown), suggesting that neither NRP2 expression nor TGF-β signaling are rate limiting in migration of mesenchymal-like HCC cell lines. In addition, the consequences of NRP2 expression on migration and invasion as determined by the passing of cells through unchanged or collagen-coated Transwell membranes was stronger than in follow-up experiments in which cells were allowed to transmigrate through a layer of endothelial cells. This suggests that NRP2 mainly affects the migratory potential of HCC cells rather than their ability of breaking through endothelial barriers.
We could show for the first time that NRP2 expression is tightly controlled by the canonical Smad2/3-Smad4 signaling cascade in HCC cells (Fig. 3c). NRP2 is therefore considered as an effector of active TGF-β signaling. Interference with TGF-β signaling decreases NRP2 expression below levels of untreated cells (Fig. 3a), indicating autocrine regulatory TGF-β signaling in mesenchymal-like HCC cells [37]. In this line, NRP2 expression could be used as marker of persistent TGF-β activity in HCC patients which is indicative of HCC progression via the tumor promoting arm of TGF-β [38, 39]. This could explain why NRP2 expression is associated with higher grading in HCC specimens (Fig. 1a). Interestingly, no other significant correlations between NRP2 expression and e.g., tumor staging or vessel invasion could be found which might be due to limitations in the immunohistochemical analysis of tissue arrays.
TGF-β is molecularly linked to NRP2 expression as TGF-β induces NRP2 levels (Fig. 3a, b). However, we found no impact of NRP2 on canonical TGF-β signaling (Fig. 4a, b) despite the fact that NRP2 has been described to act as TGF-β co-receptor [31]. Interestingly, a role of NRP2 in canonical TGF-β signaling could also not be confirmed in lung cancer cells [40], however, this study showed that NRP2 affected ERK signaling supposedly via non-canonical TGF-β signaling activity. In HCC cells, we could also not detect NRP2-driven non-canonical TGF-β signaling (data not shown). The question how NRP2 exerts its influence on cell migration remains open. NRP2 could act on other signaling pathways controlling migration or NRP2 could directly act as a receptor that conducts signals via its intracellular domain. These aspects are the basis for further investigations and allow assessing whether NRP2 represents a valuable target for HCC intervention.
Conclusions:
TGF-β shows anti-proliferative and tumor-suppressing functions in healthy liver tissue but upon HCC development, aberrant TGF-β signaling is a major driver of HCC progression. We found that NRP2 is induced by canonical TGF-β/Smad signaling in HCC cells and that NRP2 is a potent regulator of HCC cell migration and invasion. NRP2 associates with a mesenchymal-like phenotype in vitro and accordingly, NRP2 correlates with a higher tumor grade in vivo indicating less differentiation. NRP2 is therefore thought to play an important role in TGF-β-dependent HCC progression.
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Background:
Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third most lethal cancer worldwide. The epithelial to mesenchymal transition (EMT) describes the transformation of well-differentiated epithelial cells to a de-differentiated phenotype and plays a central role in the invasion and intrahepatic metastasis of HCC cells. Modulation of the transforming growth factor-β (TGF-β) signaling is known to induce various tumor-promoting and EMT-inducing pathways in HCC. The meta-analysis of a panel of EMT gene expression studies revealed that neuropilin 2 (NRP2) is significantly upregulated in cells that have undergone EMT induced by TGF-β. In this study we assessed the functional role of NRP2 in epithelial and mesenchymal-like HCC cells and focused on the molecular interplay between NRP2 and TGF-β/Smad signaling.
Methods:
NRP2 expression was analyzed in human HCC cell lines and tissue arrays comprising 133 HCC samples. Cell migration was examined by wound healing and Transwell assays in the presence and absence of siRNA against NRP2. NRP2 and TGF-β signaling were analyzed by Western blotting and confocal immunofluorescence microscopy.
Results:
We show that NRP2 is particularly expressed in HCC cell lines with a dedifferentiated, mesenchymal-like phenotype. NRP2 expression is upregulated by the canonical TGF-β/Smad signaling while NRP2 expression has no impact on TGF-β signaling in HCC cells. Reduced expression of NRP2 by knock-down or inhibition of TGF-β signaling resulted in diminished cell migration independently of each other, suggesting that NRP2 fails to collaborate with TGF-β signaling in cell movement. In accordance with these data, elevated levels of NRP2 correlated with a higher tumor grade and less differentiation in a large collection of human HCC specimens.
Conclusions:
These data suggest that NRP2 associates with a less differentiated, mesenchymal-like HCC phenotype and that NRP2 plays an important role in tumor cell migration upon TGF-β-dependent HCC progression.
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Background:
Liver cancer is the sixth most common cancer in the world and ranks second in the list of most deadly cancers [1]. The vast majority of liver cancers are hepatocellular carcinomas (HCC) representing up to 90 % of all liver malignancies [2, 3]. The main risk factors for HCC are chronic infections with either hepatitis B virus (HBV) or hepatitis C virus (HCV), making up approximately 75–85 % of all cases, as well as excessive alcohol consumption, which is responsible for about 40 % of HCC development in Western countries [2, 4–7]. Chronic inflammation and tissue damage by these agents leads to cirrhosis which is the underlying condition for the majority of HCC cases [8].
The dissemination of primary tumor cells into the body drastically worsens the prognosis of cancer patients [9]. Metastasis of HCC cells most frequently occurs intrahepatically rather than extrahepatically to distal sites such as the lung [10]. For spreading of HCC cells, individual cell movement by an epithelial to mesenchymal transition (EMT) has been considered to be essentially involved [11]. Upon EMT and progression in malignancy, highly differentiated epithelial cells such as hepatocytes de-differentiate into a mesenchymal-like phenotype that exhibits strong migratory abilities. Various signaling cascades are known to induce EMT, such as the Wnt/β-catenin, PI3K/AKT/mTOR, Hedgehog, Ras/Raf/MEK/ERK, Notch and NFκB pathways, as well as transforming growth factor (TGF)-β [12–15]. These signals mostly converge on EMT-transcription factors (EMT-TFs) such as Snail, ZEB1 or Twist1/2 which transcriptionally repress E-cadherin and other epithelial junctional proteins as well as activate a mesenchymal gene expression signature. In HCC, TGF-β signaling has been shown to activate EMT-TFs and to repress their negative feedback loops by the downregulation of miRNAs that antagonize EMT-TFs [16, 17].
A recently performed meta-analysis compared 24 published EMT gene expression data sets and generated a core list of genes that are most frequently altered during EMT [18]. One of the genes found upregulated in several studies of TGF-β-induced EMT coded for the protein neuropilin-2 (NRP2). There are two homologs of the NRP family, NRP1 and NRP2, which are 130 kDa single-pass transmembrane glycoproteins that act as non-tyrosine kinase co-receptors [19]. They contain 4 distinct domains including a CUB domain, a FV/FVIII domain, a MAM domain and a domain that contains the transmembrane and short cytoplasmic region [20]. Both homologues can homo- and heteromultimerize [21] and can bind different members of the semaphorin family, as well as members of the vascular endothelial growth factor (VEGF) family [22]. In addition, NRPs are receptors of hepatocyte growth factor, platelet-derived growth factor BB, fibroblast growth factor, epidermal growth factor, placenta growth factor, and importantly of TGF-β1 [23–25]. NRPs are therefore critical regulators of angiogenesis, lymphangiogenesis and tumor progression. NRP2 expression is correlated with lymph node metastasis in breast cancer and blocking of NRP2 leads to decreased metastasis formation [26–28]. Clinical data show that high NRP levels, in particular NRP2, correlate with poor prognosis and survival in various cancer types [29, 30]. NRP2 was suggested to play a direct role in EMT and a cross-talk between NRP2 and TGF-β1 signaling promotes colorectal cancer progression [31]. In the context of HCC, the role of NRP2 is so far unknown [32].
In this study, we show that NRP2 expression strongly correlates with a mesenchymal phenotype in HCC cell lines and that reduced levels of NRP2 dramatically impair the migratory abilities of HCC cells. We further provide evidence that NRP2 expression is controlled by canonical TGF-β signaling, while no direct impact of NRP2 on TGF-β signaling could be observed. Translation of these data into the HCC patient situation revealed a correlation of NRP2 levels with a poorly differentiated HCC, suggesting a role of NRP2 in TGF-β regulated HCC progression.
Conclusions:
TGF-β shows anti-proliferative and tumor-suppressing functions in healthy liver tissue but upon HCC development, aberrant TGF-β signaling is a major driver of HCC progression. We found that NRP2 is induced by canonical TGF-β/Smad signaling in HCC cells and that NRP2 is a potent regulator of HCC cell migration and invasion. NRP2 associates with a mesenchymal-like phenotype in vitro and accordingly, NRP2 correlates with a higher tumor grade in vivo indicating less differentiation. NRP2 is therefore thought to play an important role in TGF-β-dependent HCC progression.
|
Background:
Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third most lethal cancer worldwide. The epithelial to mesenchymal transition (EMT) describes the transformation of well-differentiated epithelial cells to a de-differentiated phenotype and plays a central role in the invasion and intrahepatic metastasis of HCC cells. Modulation of the transforming growth factor-β (TGF-β) signaling is known to induce various tumor-promoting and EMT-inducing pathways in HCC. The meta-analysis of a panel of EMT gene expression studies revealed that neuropilin 2 (NRP2) is significantly upregulated in cells that have undergone EMT induced by TGF-β. In this study we assessed the functional role of NRP2 in epithelial and mesenchymal-like HCC cells and focused on the molecular interplay between NRP2 and TGF-β/Smad signaling.
Methods:
NRP2 expression was analyzed in human HCC cell lines and tissue arrays comprising 133 HCC samples. Cell migration was examined by wound healing and Transwell assays in the presence and absence of siRNA against NRP2. NRP2 and TGF-β signaling were analyzed by Western blotting and confocal immunofluorescence microscopy.
Results:
We show that NRP2 is particularly expressed in HCC cell lines with a dedifferentiated, mesenchymal-like phenotype. NRP2 expression is upregulated by the canonical TGF-β/Smad signaling while NRP2 expression has no impact on TGF-β signaling in HCC cells. Reduced expression of NRP2 by knock-down or inhibition of TGF-β signaling resulted in diminished cell migration independently of each other, suggesting that NRP2 fails to collaborate with TGF-β signaling in cell movement. In accordance with these data, elevated levels of NRP2 correlated with a higher tumor grade and less differentiation in a large collection of human HCC specimens.
Conclusions:
These data suggest that NRP2 associates with a less differentiated, mesenchymal-like HCC phenotype and that NRP2 plays an important role in tumor cell migration upon TGF-β-dependent HCC progression.
| 12,691 | 377 | 18 |
[
"nrp2",
"cells",
"tgf",
"hcc",
"expression",
"cell",
"snu",
"migration",
"nrp2 expression",
"signaling"
] |
[
"test",
"test"
] | null |
[CONTENT] Neuropilin-2 | Transforming growth factor-β | Epithelial to mesenchymal transition | Hepatocellular carcinoma [SUMMARY]
| null |
[CONTENT] Neuropilin-2 | Transforming growth factor-β | Epithelial to mesenchymal transition | Hepatocellular carcinoma [SUMMARY]
|
[CONTENT] Neuropilin-2 | Transforming growth factor-β | Epithelial to mesenchymal transition | Hepatocellular carcinoma [SUMMARY]
|
[CONTENT] Neuropilin-2 | Transforming growth factor-β | Epithelial to mesenchymal transition | Hepatocellular carcinoma [SUMMARY]
|
[CONTENT] Neuropilin-2 | Transforming growth factor-β | Epithelial to mesenchymal transition | Hepatocellular carcinoma [SUMMARY]
|
[CONTENT] Blotting, Western | Carcinoma, Hepatocellular | Cell Movement | Humans | Liver Neoplasms | Neuropilin-2 | Phenotype | Signal Transduction | Sulfhydryl Reagents | Tissue Array Analysis | Transforming Growth Factor beta | Tumor Cells, Cultured [SUMMARY]
| null |
[CONTENT] Blotting, Western | Carcinoma, Hepatocellular | Cell Movement | Humans | Liver Neoplasms | Neuropilin-2 | Phenotype | Signal Transduction | Sulfhydryl Reagents | Tissue Array Analysis | Transforming Growth Factor beta | Tumor Cells, Cultured [SUMMARY]
|
[CONTENT] Blotting, Western | Carcinoma, Hepatocellular | Cell Movement | Humans | Liver Neoplasms | Neuropilin-2 | Phenotype | Signal Transduction | Sulfhydryl Reagents | Tissue Array Analysis | Transforming Growth Factor beta | Tumor Cells, Cultured [SUMMARY]
|
[CONTENT] Blotting, Western | Carcinoma, Hepatocellular | Cell Movement | Humans | Liver Neoplasms | Neuropilin-2 | Phenotype | Signal Transduction | Sulfhydryl Reagents | Tissue Array Analysis | Transforming Growth Factor beta | Tumor Cells, Cultured [SUMMARY]
|
[CONTENT] Blotting, Western | Carcinoma, Hepatocellular | Cell Movement | Humans | Liver Neoplasms | Neuropilin-2 | Phenotype | Signal Transduction | Sulfhydryl Reagents | Tissue Array Analysis | Transforming Growth Factor beta | Tumor Cells, Cultured [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] nrp2 | cells | tgf | hcc | expression | cell | snu | migration | nrp2 expression | signaling [SUMMARY]
| null |
[CONTENT] nrp2 | cells | tgf | hcc | expression | cell | snu | migration | nrp2 expression | signaling [SUMMARY]
|
[CONTENT] nrp2 | cells | tgf | hcc | expression | cell | snu | migration | nrp2 expression | signaling [SUMMARY]
|
[CONTENT] nrp2 | cells | tgf | hcc | expression | cell | snu | migration | nrp2 expression | signaling [SUMMARY]
|
[CONTENT] nrp2 | cells | tgf | hcc | expression | cell | snu | migration | nrp2 expression | signaling [SUMMARY]
|
[CONTENT] emt | growth factor | growth | factor | nrp2 | hcc | cancer | tgf | domain | progression [SUMMARY]
| null |
[CONTENT] nrp2 | expression | tgf | cells | snu | snu 449 | 449 | nrp2 expression | hcc | fig [SUMMARY]
|
[CONTENT] hcc | nrp2 | tgf | hcc progression | progression | tumor | signaling | liver tissue hcc development | nrp2 thought play important | found nrp2 induced [SUMMARY]
|
[CONTENT] nrp2 | cells | tgf | hcc | expression | usa | cell | snu | signaling | migration [SUMMARY]
|
[CONTENT] nrp2 | cells | tgf | hcc | expression | usa | cell | snu | signaling | migration [SUMMARY]
|
[CONTENT] third ||| EMT | HCC ||| EMT | HCC ||| EMT | neuropilin | 2 | EMT ||| NRP2 | HCC | NRP2 [SUMMARY]
| null |
[CONTENT] HCC ||| HCC ||| ||| [SUMMARY]
|
[CONTENT] [SUMMARY]
|
[CONTENT] third ||| EMT | HCC ||| EMT | HCC ||| EMT | neuropilin | 2 | EMT ||| NRP2 | HCC | NRP2 ||| 133 | HCC ||| Transwell | NRP2 ||| ||| ||| HCC ||| HCC ||| ||| ||| [SUMMARY]
|
[CONTENT] third ||| EMT | HCC ||| EMT | HCC ||| EMT | neuropilin | 2 | EMT ||| NRP2 | HCC | NRP2 ||| 133 | HCC ||| Transwell | NRP2 ||| ||| ||| HCC ||| HCC ||| ||| ||| [SUMMARY]
|
|||||
Barriers to Cancer Screening Uptake in Women: A Qualitative Study from Tamil Nadu, India.
|
32334474
|
The uptake for cancer screening has been consistently poor in India despite the efforts of nation-wide screening programs. Understanding the barriers and enablers among community women would aid in increasing the proportion of cancer screening uptake.
|
BACKGROUND
|
Nineteen key informants including community women, service providers and a cancer survivor were interviewed using a semi-structured interview guide. Interviews were recorded and transcribed by the interviewers. Manual descriptive thematic analysis was conducted using deductive approach. Codes were given and extracted into categories which were later grouped to form themes.
|
METHODS
|
The mean age of participants was 38 years. Among the participants, 38.9% and 16.7% underwent breast and cervical cancer screening respectively. The psychosocial factors were the major barriers for screening uptake such as fear of screening procedure and fear of being diagnosed with cancer. The other factors include lack of awareness, cultural beliefs, in addition to financial difficulties and health care system-related factors. Change in government policies to conduct mandatory screening programs, incentivization and creating awareness were reported as enablers for increasing the screening uptake among women.
|
RESULTS
|
Psychosocial factors, the major barriers for screening uptake in women have remained unchanged over the years. Increasing awareness campaigns, usage of decision-making aids and changes in government policies are crucial for improving the rate of uptake and successful implementation of national screening programs.
|
CONCLUSION
|
[
"Adult",
"Early Detection of Cancer",
"Female",
"Follow-Up Studies",
"Genital Neoplasms, Female",
"Health Knowledge, Attitudes, Practice",
"Health Personnel",
"Humans",
"India",
"Male",
"Middle Aged",
"Patient Acceptance of Health Care",
"Prognosis",
"Qualitative Research"
] |
7445965
|
Introduction
|
In India, breast and cervix-uterus are the first and third most common sites of cancers contributing to about 144,937 cases (Globocan, 2018; Patil et al., 2019). Reports from the Tamil Nadu Cancer Registry Project (2017) observed that 56% of women were affected by cancer, out of which gynecological cancers (breast, cervix and ovary) comprised of 50%. The survival rates of breast and cervical cancers can be improved by early diagnosis (National Programme for Prevention and Control of Cancer, Diabetes, CVD and Stroke, 2017). Yet, the universal availability and accessibility of screening are debatable (Jacob, 2012), particularly in developing countries like India (Gakidou et al., 2008, Van Dyne et al., 2019, Gupta et al., 2019). While coverage is a concern, the low screening uptake by targeted women has been reported as the major challenge in cancer screening. Significant information asymmetry, economic, cultural and psychosocial factors have been identified as barriers for the low cancer screening uptake among women (Nyblade et al., 2017).
Although the nationwide screening program (National Programme for Prevention and Control of Cancer, Diabetes, CVD and Stroke, 2017) is being implemented in India in a phased manner, not understanding and addressing the barriers will hinder the success of the program. This qualitative study attempted to explore the current barriers and enablers to breast and cervical cancer screening uptake among women in Tamil Nadu.
| null | null |
Results
|
Socio-demographic characteristics of participants
There were no refusals of consent or dropouts during participation, with a total of 19 KI, of which one KI was a male service provider (Medical Oncologist). Of the remaining 18, nine were community women, eight were service providers from health care and cancer screening setting and one was a cancer survivor. The mean age of KI was 38 years, ranging from 32 to 58 years. Only 38.9% and 16.7% of the KI underwent breast and cervical cancer screening respectively. Table 1 shows the characteristics of participants.
Barriers and Enablers
Table 2 shows the barriers and enablers for cancer screening reported by KI in the context of themes, categories and codes. Three broad themes for barriers and two broad themes for enablers are described below.
Barriers
Theme 1: Psychosocial and Individual Factors
Women were found to have psychological barriers like fear, anxiety, embarrassment, shyness, negligence which were influenced by social convictions and lack of awareness.
i. Nihilistic attitude towards cancer
Many participants reported that fear of being diagnosed with cancer was a major barrier to screening uptake. It was also associated with fear of discrimination by family and society, stigma related to cancer (that cancer is equivalent to death) and the lack of understanding about cancer (that cancer is contagious). A few stated,
“I am scared, what will I do, if they say, I have cancer” (CW1, CW3,CW8,SP1)
“[I’m] Scared, the family members may not mingle with us casually. If the society comes to know that is all, [I] fear [that], they will isolate us” (CW6)
“If they say, we have the disease after the screening, our life is gone after that, the life is over, that kind of negative thoughts [prevent going]”(CW5)
“This is like a contagious disease, if one is diagnosed of cancer, we should not allow children near them” (CW1)
The same was reiterated by SPs as well, “Scared that the life will be over with that, and they will die” (SP1)
Few women expressed concerns about the prolonged treatment and multiple visits and associated financial implications.
“If we have to go for screening, the situation is that we need to go to hospital eight to ten days” (CW6, CW1)
“One day, they should spend” (SP1)
“Time will be waste, if we go it will take one day” (CW2)
“The treatment expenses [of cancer] are high” (CW5, CW6)
ii. Lack of awareness about cancer screening
All the participants perceived that lack of awareness about cancer screening was a barrier for not participating in the screening program.
“There is no adequate amount of awareness [about cancer screening] among people” (CW4, CS1).
Cancer screening was frequently described as a painful and uncomfortable process.
“We cannot bear the pain” (CW1)
“First of all, that [mammogram] will be very painful, that is also one reason for my fear” (CW2)
“The fear related to the pain during the screening is also one of the important reasons” (CW8)
Most of the women reported that being asymptomatic, feeling fit or healthy does not necessitate screening and the cause for not approaching the doctors for screening. Procrastination was also observed due to the lack of understanding of the rationale of screening.
“Thinking that we can do little later, maybe after 15 days” (CW1)
“If there are any symptoms, we can go, otherwise why should we go. The attitude is that after 40 or 45 years, the system will start functioning slow, then we can do, what is the urgency”(CW2)
“I don’t have any symptoms, why should I check” (SP6)
“I am fine only” (SP3, SP8).
Lack of awareness also found among the family members and elders was stated as,
“Even before, we go to the screening, they will stop us saying, you will not get such diseases, why are you imagining yourself” (CW1)
“A few relatives will ask, why are you going to the hospital when you don’t have any problem” (SP3)
iii. Negligence
An attitude of carelessness regarding their health was seen in women as mentioned by a CW.
“We are fine only if anything comes let us see at that time” (CW9)
iv. Embarrassment or shyness
The embarrassment of revealing their body parts was a commonly perceived notion that caused hesitation in women. It was difficult for women to break their conventional mindset imbibed with cultural norms for the purpose of screening.
“Shyness, they need to expose the private parts to doctors (Breast and cervix)” (SP2, SP3)
“People are shy, that is why refusing to go” (CW3)
v. Superstitious beliefs
A common social stigma was identified among CW that related to the occurrence of cancer with their misdeed. They believed that going to church could cure them of their sins as well as the disease.
“I did not commit any sin” (CW3, CS1)
“This disease will come only to people who commit sin” (SP8)
“If we go to church, it will be alright” (CS1)
Theme 2: Cultural and financial factors
Cultural hindrances were found to be interlaced with familial barriers like lack of family support, household responsibilities. Financial difficulties were also reported as a major concern by the women.
i. Family-related barriers
Women reported that they needed a companion as some were not habituated to going out alone. There was no support from the family to go for screening in some cases.
“More than 50% of the spouses do not co-operate” (CS1)
“Sometimes, the husband will say not to go for screening” (CW6)
Women with household and childrearing responsibilities were not willing to give up on their duties in order to attend the screening.
“Family ladies have so much at home, for example, taking care of children” (CW8)
“If they have adolescent girls, they will say they cannot leave them at home alone and come” (SP7)
ii. Economic barriers
In many families women were still the bread-winners, working daily-wage jobs, and would lose a day’s wage if they attended the screening. Women felt the screening cost to be high and an additional expense.
“[If we go to screening], we lose our daily wages” (CS1)
“Firstly, if we go for screening, it will cost about Rs.4000 for breast and cervical screening. Many people may think [hesitate], because they need to spend more money”(CW1)
Theme 3: Health care system-related factors
i. Lack of trust in doctors and hospitals
Pervasive distrust on the health care facilities, both private and Government, was observed in the community. The KI mentioned that doctors from government hospitals were unavailable or unreceptive based on their previous experiences.
“Don’t believe corporate hospitals, they will prescribe investigations unnecessarily” (CW2)
“At the Government hospitals, there is no regulation on the doctor’s visiting time to the hospital” (CW6)
“... [At government hospital], he [the doctor] said, we cannot do anything hereafter, that is all, they behaved very harsh, …” (CS1)
ii. Poor accessibility due to geographic location
Women reported that screening was inaccessible to those in villages due to inadequate facilities.
“In villages, there are no facilities at the primary health centres to do the cancer screening” (CW8)
iii. Male health service providers
Screening conducted by male physician or technicians was found to be an obstacle in cancer screening. Women felt diffident and reluctant to undergo cancer screening if male health care providers conducted the screening procedures.
“Men are conducting the screening test [mammogram], it will be ok if women take [mammogram]. If we go to scan centrs, more than ladies, in many places men are only there, that is why women refuse to go”(CW3)
“We hesitate to show to male doctors” (CW4)
“If the doctor is sometimes male, people definitely feel shy and hesitate to show their breast to them” (CW8, CS1)
Enablers
Theme: Intensification of culture-specific IEC activities - Awareness generation
The participants mentioned that creating awareness and educating the public about the need for screening could help reach to the community. Media was considered an essential tool to spread knowledge among people.
“Need to conduct awareness programs, can insert handbills through newspapers, through Television…” (CW4)
“Can show someone who is cured of cancer as a role model” (CS1)
It was also suggested that regular camps should be conducted extensively.
“Can conduct screening camps” (CW2)
Theme 5: Policy changes – screen and treat, financial support
Policy changes to increase the availability and accessibility to screening facilities, appointing female staff incentivization were suggested by the KIs.
i. Government policies
Participants recommended that the government should make it compulsory for women to undergo cancer screening and that women have increased access to these services.
“The Government should bring a policy to screen all the women compulsorily” (SP1)
“The screening facilities should be made available in all the hospitals” (CW7, CW8)
“The camp should be near their place” (CW2)
“The facilities should be made available near their villages” (SP2)
ii. Financial support
As for the financial burden, it was suggested that the screening should be done at low cost or incentives could be provided to attend cancer screening.
“We should advertise that the screening will be done at low cost” (CW4)
“Incentives can be provided. Then the people from below poverty line will come forward to do cancer screening” (SP1)
iii. Appointing female service providers
Appointing more women doctors and technicians for the screening programs could help women overcome fear and embarrassment leading to a higher proportion of women attending the cancer screening. Appointing doctors from the same community will improve the level of comfort and trust in the system.
“We need to take them to familiar doctors, then without fear, they can do the screening” (CW5)
“If women do the screening/mammogram it will be good” (CW3)
Thematic Questions Used during in-Depth Interviews
Participant Details
SD, Standard Deviation
Perspectives of Community Women, Survivor and Health Care Providers on Barriers and Enablers for Cancer Screening
| null | null |
[] |
[] |
[] |
[
"Introduction",
"Materials and Methods",
"Results",
"Discussion"
] |
[
"In India, breast and cervix-uterus are the first and third most common sites of cancers contributing to about 144,937 cases (Globocan, 2018; Patil et al., 2019). Reports from the Tamil Nadu Cancer Registry Project (2017) observed that 56% of women were affected by cancer, out of which gynecological cancers (breast, cervix and ovary) comprised of 50%. The survival rates of breast and cervical cancers can be improved by early diagnosis (National Programme for Prevention and Control of Cancer, Diabetes, CVD and Stroke, 2017). Yet, the universal availability and accessibility of screening are debatable (Jacob, 2012), particularly in developing countries like India (Gakidou et al., 2008, Van Dyne et al., 2019, Gupta et al., 2019). While coverage is a concern, the low screening uptake by targeted women has been reported as the major challenge in cancer screening. Significant information asymmetry, economic, cultural and psychosocial factors have been identified as barriers for the low cancer screening uptake among women (Nyblade et al., 2017). \nAlthough the nationwide screening program (National Programme for Prevention and Control of Cancer, Diabetes, CVD and Stroke, 2017) is being implemented in India in a phased manner, not understanding and addressing the barriers will hinder the success of the program. This qualitative study attempted to explore the current barriers and enablers to breast and cervical cancer screening uptake among women in Tamil Nadu. ",
"\nStudy design\n\nDescriptive qualitative study design was conducted using in-depth interviews among the selected key informants (KI). Experiences and perceptions of cancer screening were explored with particular focus on barriers to screening uptake and possible solutions.\n\nStudy participants\n\nAll participants in the study were purposively selected to ensure representation of various sects from the population. To obtain a triangular perspective, cancer survivor (CS), community women (CW) with and without symptoms, community service providers (SP) and professionals from oncology and community medicine were included in the study. \n\nData collection\n\nInterviews were conducted using a semi-structured interview guide with open-ended questions between January and March 2019 (Box 1) by a qualified psycho-oncologist (M.Phil) trained in qualitative research. Written informed consent was obtained from all participants and briefing about the cancer screening. The socio-demographic characteristics of KI was collected using a structured pro forma. The KI were inquired about barriers to cancer screening in general followed by their own experiences in screening. Among SP, their experiences about cancer screening in their routine clinical setting were explored. All the interviews were conducted face-to-face at their residence or workplace in the regional language and audio-recorded. The questions proceeded from general to specific topics to reduce interviewer and participant bias. Probing questions were asked wherever appropriate. The verbatim was transcribed on the same day by the interviewers in the same language (Tamil). The duration of interviews ranged from 20 to 30 minutes. Data collection was carried out until saturation was achieved and redundancy of information was observed. \n\nData analysis\n\nTwo trained researchers read the transcripts to become familiar with the data and conducted the manual descriptive thematic analysis using a deductive approach to reduce researcher bias and improve interpretive credibility. The decision on coding rules and theme generation was done using standard procedures (Saldana, 2010). The contents of participants’ verbatim quotes were shortened and coded with names. The codes that covered similar ideas were merged into specific categories. Finally, the categories with similar context were grouped into themes (Creswell and Clark, 2007). Any differences between the researchers were resolved by discussion. To ensure that the results were a reflection of data, codes were related to the original data (Lincoln and Guba, 1985). Final stage of the analysis was carried out by the two researchers. The naming of categories and themes were discussed until agreement was reached. In this manuscript, the verbatim is reported in double quotations and italicized, the author explanations within quotes in square brackets and the respondents’ identities are given in round brackets and italicized. The findings were reported using ‘Consolidated Criteria for Reporting Qualitative Research (COREQ) guidelines (Tong et al., 2007).\n\nEthical considerations\n\nThe study was approved by the doctoral committee appointed by Rajiv Gandhi National Institute of Youth Development for the research degree of the first author.",
"\nSocio-demographic characteristics of participants\n\nThere were no refusals of consent or dropouts during participation, with a total of 19 KI, of which one KI was a male service provider (Medical Oncologist). Of the remaining 18, nine were community women, eight were service providers from health care and cancer screening setting and one was a cancer survivor. The mean age of KI was 38 years, ranging from 32 to 58 years. Only 38.9% and 16.7% of the KI underwent breast and cervical cancer screening respectively. Table 1 shows the characteristics of participants.\n\nBarriers and Enablers\n\n\nTable 2 shows the barriers and enablers for cancer screening reported by KI in the context of themes, categories and codes. Three broad themes for barriers and two broad themes for enablers are described below. \n\nBarriers\n\n\nTheme 1: Psychosocial and Individual Factors\n\nWomen were found to have psychological barriers like fear, anxiety, embarrassment, shyness, negligence which were influenced by social convictions and lack of awareness.\n\ni. Nihilistic attitude towards cancer\n\nMany participants reported that fear of being diagnosed with cancer was a major barrier to screening uptake. It was also associated with fear of discrimination by family and society, stigma related to cancer (that cancer is equivalent to death) and the lack of understanding about cancer (that cancer is contagious). A few stated, \n\n“I am scared, what will I do, if they say, I have cancer” (CW1, CW3,CW8,SP1)\n\n\n“[I’m] Scared, the family members may not mingle with us casually. If the society comes to know that is all, [I] fear [that], they will isolate us” (CW6)\n\n\n“If they say, we have the disease after the screening, our life is gone after that, the life is over, that kind of negative thoughts [prevent going]”(CW5)\n\n\n“This is like a contagious disease, if one is diagnosed of cancer, we should not allow children near them” (CW1)\n\nThe same was reiterated by SPs as well, “Scared that the life will be over with that, and they will die” (SP1)\nFew women expressed concerns about the prolonged treatment and multiple visits and associated financial implications.\n\n“If we have to go for screening, the situation is that we need to go to hospital eight to ten days” (CW6, CW1) \n\n\n“One day, they should spend” (SP1)\n\n\n“Time will be waste, if we go it will take one day” (CW2)\n\n\n “The treatment expenses [of cancer] are high” (CW5, CW6) \n\n\nii. Lack of awareness about cancer screening\n\nAll the participants perceived that lack of awareness about cancer screening was a barrier for not participating in the screening program. \n\n“There is no adequate amount of awareness [about cancer screening] among people” (CW4, CS1).\n\nCancer screening was frequently described as a painful and uncomfortable process.\n\n“We cannot bear the pain” (CW1)\n\n\n“First of all, that [mammogram] will be very painful, that is also one reason for my fear” (CW2)\n\n\n“The fear related to the pain during the screening is also one of the important reasons” (CW8)\n\nMost of the women reported that being asymptomatic, feeling fit or healthy does not necessitate screening and the cause for not approaching the doctors for screening. Procrastination was also observed due to the lack of understanding of the rationale of screening. \n\n“Thinking that we can do little later, maybe after 15 days” (CW1)\n\n\n“If there are any symptoms, we can go, otherwise why should we go. The attitude is that after 40 or 45 years, the system will start functioning slow, then we can do, what is the urgency”(CW2)\n\n\n“I don’t have any symptoms, why should I check” (SP6)\n\n\n“I am fine only” (SP3, SP8).\n\nLack of awareness also found among the family members and elders was stated as,\n\n“Even before, we go to the screening, they will stop us saying, you will not get such diseases, why are you imagining yourself” (CW1)\n\n\n“A few relatives will ask, why are you going to the hospital when you don’t have any problem” (SP3)\n\n\niii. Negligence\n\nAn attitude of carelessness regarding their health was seen in women as mentioned by a CW.\n\n“We are fine only if anything comes let us see at that time” (CW9)\n\n\niv. Embarrassment or shyness\n\nThe embarrassment of revealing their body parts was a commonly perceived notion that caused hesitation in women. It was difficult for women to break their conventional mindset imbibed with cultural norms for the purpose of screening. \n\n“Shyness, they need to expose the private parts to doctors (Breast and cervix)” (SP2, SP3) \n\n\n“People are shy, that is why refusing to go” (CW3)\n\nv. Superstitious beliefs \nA common social stigma was identified among CW that related to the occurrence of cancer with their misdeed. They believed that going to church could cure them of their sins as well as the disease.\n\n“I did not commit any sin” (CW3, CS1)\n\n\n“This disease will come only to people who commit sin” (SP8)\n\n\n“If we go to church, it will be alright” (CS1)\n\n\nTheme 2: Cultural and financial factors\n\nCultural hindrances were found to be interlaced with familial barriers like lack of family support, household responsibilities. Financial difficulties were also reported as a major concern by the women. \n\ni. Family-related barriers\n\nWomen reported that they needed a companion as some were not habituated to going out alone. There was no support from the family to go for screening in some cases. \n\n“More than 50% of the spouses do not co-operate” (CS1)\n\n\n“Sometimes, the husband will say not to go for screening” (CW6)\n\nWomen with household and childrearing responsibilities were not willing to give up on their duties in order to attend the screening.\n\n“Family ladies have so much at home, for example, taking care of children” (CW8)\n\n\n“If they have adolescent girls, they will say they cannot leave them at home alone and come” (SP7)\n\n\nii. Economic barriers\n\nIn many families women were still the bread-winners, working daily-wage jobs, and would lose a day’s wage if they attended the screening. Women felt the screening cost to be high and an additional expense.\n\n“[If we go to screening], we lose our daily wages” (CS1)\n\n\n“Firstly, if we go for screening, it will cost about Rs.4000 for breast and cervical screening. Many people may think [hesitate], because they need to spend more money”(CW1)\n\n\nTheme 3: Health care system-related factors\n\n\ni. Lack of trust in doctors and hospitals\n\nPervasive distrust on the health care facilities, both private and Government, was observed in the community. The KI mentioned that doctors from government hospitals were unavailable or unreceptive based on their previous experiences.\n\n“Don’t believe corporate hospitals, they will prescribe investigations unnecessarily” (CW2)\n\n\n“At the Government hospitals, there is no regulation on the doctor’s visiting time to the hospital” (CW6)\n\n\n“... [At government hospital], he [the doctor] said, we cannot do anything hereafter, that is all, they behaved very harsh, …” (CS1)\n\n\nii. Poor accessibility due to geographic location\n\nWomen reported that screening was inaccessible to those in villages due to inadequate facilities.\n\n“In villages, there are no facilities at the primary health centres to do the cancer screening” (CW8)\n\n\niii. Male health service providers\n\nScreening conducted by male physician or technicians was found to be an obstacle in cancer screening. Women felt diffident and reluctant to undergo cancer screening if male health care providers conducted the screening procedures. \n\n“Men are conducting the screening test [mammogram], it will be ok if women take [mammogram]. If we go to scan centrs, more than ladies, in many places men are only there, that is why women refuse to go”(CW3)\n\n\n“We hesitate to show to male doctors” (CW4)\n\n\n“If the doctor is sometimes male, people definitely feel shy and hesitate to show their breast to them” (CW8, CS1)\n\n\nEnablers\n\n\nTheme: Intensification of culture-specific IEC activities - Awareness generation \n\nThe participants mentioned that creating awareness and educating the public about the need for screening could help reach to the community. Media was considered an essential tool to spread knowledge among people.\n\n“Need to conduct awareness programs, can insert handbills through newspapers, through Television…” (CW4)\n\n\n“Can show someone who is cured of cancer as a role model” (CS1)\n\n\nIt was also suggested that regular camps should be conducted extensively.\n\n\n“Can conduct screening camps” (CW2)\n\n\nTheme 5: Policy changes – screen and treat, financial support\n\nPolicy changes to increase the availability and accessibility to screening facilities, appointing female staff incentivization were suggested by the KIs.\n\ni. Government policies\n\nParticipants recommended that the government should make it compulsory for women to undergo cancer screening and that women have increased access to these services.\n\n“The Government should bring a policy to screen all the women compulsorily” (SP1)\n\n\n“The screening facilities should be made available in all the hospitals” (CW7, CW8)\n\n\n“The camp should be near their place” (CW2)\n\n\n“The facilities should be made available near their villages” (SP2)\n\n\nii. Financial support\n\nAs for the financial burden, it was suggested that the screening should be done at low cost or incentives could be provided to attend cancer screening. \n\n“We should advertise that the screening will be done at low cost” (CW4)\n\n\n“Incentives can be provided. Then the people from below poverty line will come forward to do cancer screening” (SP1)\n\n\niii. Appointing female service providers\n\nAppointing more women doctors and technicians for the screening programs could help women overcome fear and embarrassment leading to a higher proportion of women attending the cancer screening. Appointing doctors from the same community will improve the level of comfort and trust in the system. \n\n“We need to take them to familiar doctors, then without fear, they can do the screening” (CW5)\n\n\n“If women do the screening/mammogram it will be good” (CW3)\n\nThematic Questions Used during in-Depth Interviews\nParticipant Details\nSD, Standard Deviation\nPerspectives of Community Women, Survivor and Health Care Providers on Barriers and Enablers for Cancer Screening",
"The study qualitatively explored the barriers and enablers for screening uptake among women. Psychosocial, economic, cultural and system related factors were identified. \nThe strength of this study was that, the population consisted of educated and uneducated male and female participants from rural and urban communities, professionals including oncologists, epidemiologist and other health care providers belonging to different socio-economic backgrounds representing variation in the study population. Hence the study could acquire different dimensions in its viewpoint. There were some limitations in our study. Firstly, the study population was small and there was not enough representation from each category. Secondly, as the study was conducted in a specific Indian region, generalizability is difficult.\nFear of being diagnosed or of the examination procedure were reported as barriers in previous studies (Agurto et al., 2004; Allahverdipour et al., 2011; Malhotra et al., 2016) as in the current study. Embarrassment to reveal their body parts, especially with male health service providers, not necessitating screening in asymptomatic conditions and lack of family support were also found in the current study confirming previous findings (Devarapalli et al., 2018; Marlow et al., 2015; Nyblade et al., 2017; Szalacha et al., 2017).\nIn previous literatures, cultural beliefs have been identified as a significant barrier (cancer as sin, the result of immorality) in women to undergo cancer screening as they prominently influence the level of understanding and knowledge about these cancers (de Cuevas et al., 2018; Gupta et al., 2015; Lee, 2015; Meana et al., 2001; Modibbo et al., 2016; Szalacha et al., 2017) and interventions addressing them have also produced results (Adunlin et al., 2019; Pratt et al., 2019). Financial concern was reported both in the current study and previous studies (Malhotra et al., 2016). Nevertheless, screening conducted at no cost for all eligible women by the current nationwide program in India might help in addressing this issue. The main enablers mentioned in our study were creating awareness, policy changes by the government including availability of facilities and incentivization. Government has been providing incentives for Tuberculosis patients to continue receiving treatment and to mothers undergoing institutional deliveries in an attempt to reduce infant and maternal mortality rate. Similar incentivization to women attending cancer screening could improve the rate of screening uptake. \nThe currently prevailing barriers and enablers in the society, mentioned in this study have been consistently reported as determinants of screening uptake over the years (de Cuevas et al., 2018; Devarapalli et al., 2018; Dinshaw et al., 2007; Gupta et al., 2015; Kulkarni et al., 2019). Despite repeated researches conducted with this focus, barriers influencing cancer screening uptake have not changed for the past two decades. This might be attributed to the society’s low sensitivity about screening of women related cancers. Awareness for women cancers were reported as poor (Bora et al., 2016; Khokhar, 2009) and lack of media attention towards cancer control in India is prevalent (Gupta et al., 2015). \nUsage of decision aids will help in increasing the rate of acceptance in contemplating stage, thus improving the screening uptake. Decision aids have been shown to reduce indecisiveness, improve consensus on values and choices as well as improve knowledge (Barratt et al., 2004). IEC and screening are the two main components in cancer prevention recommended by the World Health Organization (Bora et al., 2016). The interim results of a community-based cancer screening conducted in Mumbai showed a compliance rate of 85% and 70% for breast and cervical cancer screening where the women were sensitized about the reproductive organs, cancer symptoms and early detection (Mishra et al., 2015). IEC method and targeted intervention programs have been effective to improve participation in breast and cervical cancer screenings (Jacob, 2012; Rao et al., 2005). Although decision making aids are widely used for cancer treatment, its role in cancer screening is still unexploited. In India, massive mobilization of the community, effective micro-planning and compound communication approach have been the foundation to eradicate polio in the country (Thacker et al., 2016). Such galvanizing social movement regarding women cancer screening is crucial to reach out to maximum population. Empowering through awareness and use of decision making aids are the need of the hour for mass mobilization of the society and improving the uptake of women cancer screening.\nIn Conclusion, This study reiterates the psychosocial barriers and enablers that have been prevalent in the community women over the period of time. It was inferred that the factors hindering the cancer screening uptake have remained unchanged for about two decades. Addressing these barriers by creating awareness, using decision making aids and changes in government policy are the means to strengthen the current NPCDCS program. "
] |
[
"intro",
"materials|methods",
"results",
"discussion"
] |
[
"Cervical cancer",
"breast cancer",
"opportunistic screening",
"cancer prevention",
"psycho oncology"
] |
Introduction:
In India, breast and cervix-uterus are the first and third most common sites of cancers contributing to about 144,937 cases (Globocan, 2018; Patil et al., 2019). Reports from the Tamil Nadu Cancer Registry Project (2017) observed that 56% of women were affected by cancer, out of which gynecological cancers (breast, cervix and ovary) comprised of 50%. The survival rates of breast and cervical cancers can be improved by early diagnosis (National Programme for Prevention and Control of Cancer, Diabetes, CVD and Stroke, 2017). Yet, the universal availability and accessibility of screening are debatable (Jacob, 2012), particularly in developing countries like India (Gakidou et al., 2008, Van Dyne et al., 2019, Gupta et al., 2019). While coverage is a concern, the low screening uptake by targeted women has been reported as the major challenge in cancer screening. Significant information asymmetry, economic, cultural and psychosocial factors have been identified as barriers for the low cancer screening uptake among women (Nyblade et al., 2017).
Although the nationwide screening program (National Programme for Prevention and Control of Cancer, Diabetes, CVD and Stroke, 2017) is being implemented in India in a phased manner, not understanding and addressing the barriers will hinder the success of the program. This qualitative study attempted to explore the current barriers and enablers to breast and cervical cancer screening uptake among women in Tamil Nadu.
Materials and Methods:
Study design
Descriptive qualitative study design was conducted using in-depth interviews among the selected key informants (KI). Experiences and perceptions of cancer screening were explored with particular focus on barriers to screening uptake and possible solutions.
Study participants
All participants in the study were purposively selected to ensure representation of various sects from the population. To obtain a triangular perspective, cancer survivor (CS), community women (CW) with and without symptoms, community service providers (SP) and professionals from oncology and community medicine were included in the study.
Data collection
Interviews were conducted using a semi-structured interview guide with open-ended questions between January and March 2019 (Box 1) by a qualified psycho-oncologist (M.Phil) trained in qualitative research. Written informed consent was obtained from all participants and briefing about the cancer screening. The socio-demographic characteristics of KI was collected using a structured pro forma. The KI were inquired about barriers to cancer screening in general followed by their own experiences in screening. Among SP, their experiences about cancer screening in their routine clinical setting were explored. All the interviews were conducted face-to-face at their residence or workplace in the regional language and audio-recorded. The questions proceeded from general to specific topics to reduce interviewer and participant bias. Probing questions were asked wherever appropriate. The verbatim was transcribed on the same day by the interviewers in the same language (Tamil). The duration of interviews ranged from 20 to 30 minutes. Data collection was carried out until saturation was achieved and redundancy of information was observed.
Data analysis
Two trained researchers read the transcripts to become familiar with the data and conducted the manual descriptive thematic analysis using a deductive approach to reduce researcher bias and improve interpretive credibility. The decision on coding rules and theme generation was done using standard procedures (Saldana, 2010). The contents of participants’ verbatim quotes were shortened and coded with names. The codes that covered similar ideas were merged into specific categories. Finally, the categories with similar context were grouped into themes (Creswell and Clark, 2007). Any differences between the researchers were resolved by discussion. To ensure that the results were a reflection of data, codes were related to the original data (Lincoln and Guba, 1985). Final stage of the analysis was carried out by the two researchers. The naming of categories and themes were discussed until agreement was reached. In this manuscript, the verbatim is reported in double quotations and italicized, the author explanations within quotes in square brackets and the respondents’ identities are given in round brackets and italicized. The findings were reported using ‘Consolidated Criteria for Reporting Qualitative Research (COREQ) guidelines (Tong et al., 2007).
Ethical considerations
The study was approved by the doctoral committee appointed by Rajiv Gandhi National Institute of Youth Development for the research degree of the first author.
Results:
Socio-demographic characteristics of participants
There were no refusals of consent or dropouts during participation, with a total of 19 KI, of which one KI was a male service provider (Medical Oncologist). Of the remaining 18, nine were community women, eight were service providers from health care and cancer screening setting and one was a cancer survivor. The mean age of KI was 38 years, ranging from 32 to 58 years. Only 38.9% and 16.7% of the KI underwent breast and cervical cancer screening respectively. Table 1 shows the characteristics of participants.
Barriers and Enablers
Table 2 shows the barriers and enablers for cancer screening reported by KI in the context of themes, categories and codes. Three broad themes for barriers and two broad themes for enablers are described below.
Barriers
Theme 1: Psychosocial and Individual Factors
Women were found to have psychological barriers like fear, anxiety, embarrassment, shyness, negligence which were influenced by social convictions and lack of awareness.
i. Nihilistic attitude towards cancer
Many participants reported that fear of being diagnosed with cancer was a major barrier to screening uptake. It was also associated with fear of discrimination by family and society, stigma related to cancer (that cancer is equivalent to death) and the lack of understanding about cancer (that cancer is contagious). A few stated,
“I am scared, what will I do, if they say, I have cancer” (CW1, CW3,CW8,SP1)
“[I’m] Scared, the family members may not mingle with us casually. If the society comes to know that is all, [I] fear [that], they will isolate us” (CW6)
“If they say, we have the disease after the screening, our life is gone after that, the life is over, that kind of negative thoughts [prevent going]”(CW5)
“This is like a contagious disease, if one is diagnosed of cancer, we should not allow children near them” (CW1)
The same was reiterated by SPs as well, “Scared that the life will be over with that, and they will die” (SP1)
Few women expressed concerns about the prolonged treatment and multiple visits and associated financial implications.
“If we have to go for screening, the situation is that we need to go to hospital eight to ten days” (CW6, CW1)
“One day, they should spend” (SP1)
“Time will be waste, if we go it will take one day” (CW2)
“The treatment expenses [of cancer] are high” (CW5, CW6)
ii. Lack of awareness about cancer screening
All the participants perceived that lack of awareness about cancer screening was a barrier for not participating in the screening program.
“There is no adequate amount of awareness [about cancer screening] among people” (CW4, CS1).
Cancer screening was frequently described as a painful and uncomfortable process.
“We cannot bear the pain” (CW1)
“First of all, that [mammogram] will be very painful, that is also one reason for my fear” (CW2)
“The fear related to the pain during the screening is also one of the important reasons” (CW8)
Most of the women reported that being asymptomatic, feeling fit or healthy does not necessitate screening and the cause for not approaching the doctors for screening. Procrastination was also observed due to the lack of understanding of the rationale of screening.
“Thinking that we can do little later, maybe after 15 days” (CW1)
“If there are any symptoms, we can go, otherwise why should we go. The attitude is that after 40 or 45 years, the system will start functioning slow, then we can do, what is the urgency”(CW2)
“I don’t have any symptoms, why should I check” (SP6)
“I am fine only” (SP3, SP8).
Lack of awareness also found among the family members and elders was stated as,
“Even before, we go to the screening, they will stop us saying, you will not get such diseases, why are you imagining yourself” (CW1)
“A few relatives will ask, why are you going to the hospital when you don’t have any problem” (SP3)
iii. Negligence
An attitude of carelessness regarding their health was seen in women as mentioned by a CW.
“We are fine only if anything comes let us see at that time” (CW9)
iv. Embarrassment or shyness
The embarrassment of revealing their body parts was a commonly perceived notion that caused hesitation in women. It was difficult for women to break their conventional mindset imbibed with cultural norms for the purpose of screening.
“Shyness, they need to expose the private parts to doctors (Breast and cervix)” (SP2, SP3)
“People are shy, that is why refusing to go” (CW3)
v. Superstitious beliefs
A common social stigma was identified among CW that related to the occurrence of cancer with their misdeed. They believed that going to church could cure them of their sins as well as the disease.
“I did not commit any sin” (CW3, CS1)
“This disease will come only to people who commit sin” (SP8)
“If we go to church, it will be alright” (CS1)
Theme 2: Cultural and financial factors
Cultural hindrances were found to be interlaced with familial barriers like lack of family support, household responsibilities. Financial difficulties were also reported as a major concern by the women.
i. Family-related barriers
Women reported that they needed a companion as some were not habituated to going out alone. There was no support from the family to go for screening in some cases.
“More than 50% of the spouses do not co-operate” (CS1)
“Sometimes, the husband will say not to go for screening” (CW6)
Women with household and childrearing responsibilities were not willing to give up on their duties in order to attend the screening.
“Family ladies have so much at home, for example, taking care of children” (CW8)
“If they have adolescent girls, they will say they cannot leave them at home alone and come” (SP7)
ii. Economic barriers
In many families women were still the bread-winners, working daily-wage jobs, and would lose a day’s wage if they attended the screening. Women felt the screening cost to be high and an additional expense.
“[If we go to screening], we lose our daily wages” (CS1)
“Firstly, if we go for screening, it will cost about Rs.4000 for breast and cervical screening. Many people may think [hesitate], because they need to spend more money”(CW1)
Theme 3: Health care system-related factors
i. Lack of trust in doctors and hospitals
Pervasive distrust on the health care facilities, both private and Government, was observed in the community. The KI mentioned that doctors from government hospitals were unavailable or unreceptive based on their previous experiences.
“Don’t believe corporate hospitals, they will prescribe investigations unnecessarily” (CW2)
“At the Government hospitals, there is no regulation on the doctor’s visiting time to the hospital” (CW6)
“... [At government hospital], he [the doctor] said, we cannot do anything hereafter, that is all, they behaved very harsh, …” (CS1)
ii. Poor accessibility due to geographic location
Women reported that screening was inaccessible to those in villages due to inadequate facilities.
“In villages, there are no facilities at the primary health centres to do the cancer screening” (CW8)
iii. Male health service providers
Screening conducted by male physician or technicians was found to be an obstacle in cancer screening. Women felt diffident and reluctant to undergo cancer screening if male health care providers conducted the screening procedures.
“Men are conducting the screening test [mammogram], it will be ok if women take [mammogram]. If we go to scan centrs, more than ladies, in many places men are only there, that is why women refuse to go”(CW3)
“We hesitate to show to male doctors” (CW4)
“If the doctor is sometimes male, people definitely feel shy and hesitate to show their breast to them” (CW8, CS1)
Enablers
Theme: Intensification of culture-specific IEC activities - Awareness generation
The participants mentioned that creating awareness and educating the public about the need for screening could help reach to the community. Media was considered an essential tool to spread knowledge among people.
“Need to conduct awareness programs, can insert handbills through newspapers, through Television…” (CW4)
“Can show someone who is cured of cancer as a role model” (CS1)
It was also suggested that regular camps should be conducted extensively.
“Can conduct screening camps” (CW2)
Theme 5: Policy changes – screen and treat, financial support
Policy changes to increase the availability and accessibility to screening facilities, appointing female staff incentivization were suggested by the KIs.
i. Government policies
Participants recommended that the government should make it compulsory for women to undergo cancer screening and that women have increased access to these services.
“The Government should bring a policy to screen all the women compulsorily” (SP1)
“The screening facilities should be made available in all the hospitals” (CW7, CW8)
“The camp should be near their place” (CW2)
“The facilities should be made available near their villages” (SP2)
ii. Financial support
As for the financial burden, it was suggested that the screening should be done at low cost or incentives could be provided to attend cancer screening.
“We should advertise that the screening will be done at low cost” (CW4)
“Incentives can be provided. Then the people from below poverty line will come forward to do cancer screening” (SP1)
iii. Appointing female service providers
Appointing more women doctors and technicians for the screening programs could help women overcome fear and embarrassment leading to a higher proportion of women attending the cancer screening. Appointing doctors from the same community will improve the level of comfort and trust in the system.
“We need to take them to familiar doctors, then without fear, they can do the screening” (CW5)
“If women do the screening/mammogram it will be good” (CW3)
Thematic Questions Used during in-Depth Interviews
Participant Details
SD, Standard Deviation
Perspectives of Community Women, Survivor and Health Care Providers on Barriers and Enablers for Cancer Screening
Discussion:
The study qualitatively explored the barriers and enablers for screening uptake among women. Psychosocial, economic, cultural and system related factors were identified.
The strength of this study was that, the population consisted of educated and uneducated male and female participants from rural and urban communities, professionals including oncologists, epidemiologist and other health care providers belonging to different socio-economic backgrounds representing variation in the study population. Hence the study could acquire different dimensions in its viewpoint. There were some limitations in our study. Firstly, the study population was small and there was not enough representation from each category. Secondly, as the study was conducted in a specific Indian region, generalizability is difficult.
Fear of being diagnosed or of the examination procedure were reported as barriers in previous studies (Agurto et al., 2004; Allahverdipour et al., 2011; Malhotra et al., 2016) as in the current study. Embarrassment to reveal their body parts, especially with male health service providers, not necessitating screening in asymptomatic conditions and lack of family support were also found in the current study confirming previous findings (Devarapalli et al., 2018; Marlow et al., 2015; Nyblade et al., 2017; Szalacha et al., 2017).
In previous literatures, cultural beliefs have been identified as a significant barrier (cancer as sin, the result of immorality) in women to undergo cancer screening as they prominently influence the level of understanding and knowledge about these cancers (de Cuevas et al., 2018; Gupta et al., 2015; Lee, 2015; Meana et al., 2001; Modibbo et al., 2016; Szalacha et al., 2017) and interventions addressing them have also produced results (Adunlin et al., 2019; Pratt et al., 2019). Financial concern was reported both in the current study and previous studies (Malhotra et al., 2016). Nevertheless, screening conducted at no cost for all eligible women by the current nationwide program in India might help in addressing this issue. The main enablers mentioned in our study were creating awareness, policy changes by the government including availability of facilities and incentivization. Government has been providing incentives for Tuberculosis patients to continue receiving treatment and to mothers undergoing institutional deliveries in an attempt to reduce infant and maternal mortality rate. Similar incentivization to women attending cancer screening could improve the rate of screening uptake.
The currently prevailing barriers and enablers in the society, mentioned in this study have been consistently reported as determinants of screening uptake over the years (de Cuevas et al., 2018; Devarapalli et al., 2018; Dinshaw et al., 2007; Gupta et al., 2015; Kulkarni et al., 2019). Despite repeated researches conducted with this focus, barriers influencing cancer screening uptake have not changed for the past two decades. This might be attributed to the society’s low sensitivity about screening of women related cancers. Awareness for women cancers were reported as poor (Bora et al., 2016; Khokhar, 2009) and lack of media attention towards cancer control in India is prevalent (Gupta et al., 2015).
Usage of decision aids will help in increasing the rate of acceptance in contemplating stage, thus improving the screening uptake. Decision aids have been shown to reduce indecisiveness, improve consensus on values and choices as well as improve knowledge (Barratt et al., 2004). IEC and screening are the two main components in cancer prevention recommended by the World Health Organization (Bora et al., 2016). The interim results of a community-based cancer screening conducted in Mumbai showed a compliance rate of 85% and 70% for breast and cervical cancer screening where the women were sensitized about the reproductive organs, cancer symptoms and early detection (Mishra et al., 2015). IEC method and targeted intervention programs have been effective to improve participation in breast and cervical cancer screenings (Jacob, 2012; Rao et al., 2005). Although decision making aids are widely used for cancer treatment, its role in cancer screening is still unexploited. In India, massive mobilization of the community, effective micro-planning and compound communication approach have been the foundation to eradicate polio in the country (Thacker et al., 2016). Such galvanizing social movement regarding women cancer screening is crucial to reach out to maximum population. Empowering through awareness and use of decision making aids are the need of the hour for mass mobilization of the society and improving the uptake of women cancer screening.
In Conclusion, This study reiterates the psychosocial barriers and enablers that have been prevalent in the community women over the period of time. It was inferred that the factors hindering the cancer screening uptake have remained unchanged for about two decades. Addressing these barriers by creating awareness, using decision making aids and changes in government policy are the means to strengthen the current NPCDCS program.
|
Background:
The uptake for cancer screening has been consistently poor in India despite the efforts of nation-wide screening programs. Understanding the barriers and enablers among community women would aid in increasing the proportion of cancer screening uptake.
Methods:
Nineteen key informants including community women, service providers and a cancer survivor were interviewed using a semi-structured interview guide. Interviews were recorded and transcribed by the interviewers. Manual descriptive thematic analysis was conducted using deductive approach. Codes were given and extracted into categories which were later grouped to form themes.
Results:
The mean age of participants was 38 years. Among the participants, 38.9% and 16.7% underwent breast and cervical cancer screening respectively. The psychosocial factors were the major barriers for screening uptake such as fear of screening procedure and fear of being diagnosed with cancer. The other factors include lack of awareness, cultural beliefs, in addition to financial difficulties and health care system-related factors. Change in government policies to conduct mandatory screening programs, incentivization and creating awareness were reported as enablers for increasing the screening uptake among women.
Conclusions:
Psychosocial factors, the major barriers for screening uptake in women have remained unchanged over the years. Increasing awareness campaigns, usage of decision-making aids and changes in government policies are crucial for improving the rate of uptake and successful implementation of national screening programs.
| null | null | 3,924 | 261 | 4 |
[
"screening",
"cancer",
"women",
"cancer screening",
"barriers",
"study",
"reported",
"awareness",
"uptake",
"community"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] Cervical cancer | breast cancer | opportunistic screening | cancer prevention | psycho oncology [SUMMARY]
| null |
[CONTENT] Cervical cancer | breast cancer | opportunistic screening | cancer prevention | psycho oncology [SUMMARY]
| null |
[CONTENT] Cervical cancer | breast cancer | opportunistic screening | cancer prevention | psycho oncology [SUMMARY]
| null |
[CONTENT] Adult | Early Detection of Cancer | Female | Follow-Up Studies | Genital Neoplasms, Female | Health Knowledge, Attitudes, Practice | Health Personnel | Humans | India | Male | Middle Aged | Patient Acceptance of Health Care | Prognosis | Qualitative Research [SUMMARY]
| null |
[CONTENT] Adult | Early Detection of Cancer | Female | Follow-Up Studies | Genital Neoplasms, Female | Health Knowledge, Attitudes, Practice | Health Personnel | Humans | India | Male | Middle Aged | Patient Acceptance of Health Care | Prognosis | Qualitative Research [SUMMARY]
| null |
[CONTENT] Adult | Early Detection of Cancer | Female | Follow-Up Studies | Genital Neoplasms, Female | Health Knowledge, Attitudes, Practice | Health Personnel | Humans | India | Male | Middle Aged | Patient Acceptance of Health Care | Prognosis | Qualitative Research [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] screening | cancer | women | cancer screening | barriers | study | reported | awareness | uptake | community [SUMMARY]
| null |
[CONTENT] screening | cancer | women | cancer screening | barriers | study | reported | awareness | uptake | community [SUMMARY]
| null |
[CONTENT] screening | cancer | women | cancer screening | barriers | study | reported | awareness | uptake | community [SUMMARY]
| null |
[CONTENT] cancer | 2017 | screening | breast | india | cancers | women | cancer diabetes | prevention control cancer | national programme prevention control [SUMMARY]
| null |
[CONTENT] screening | cancer | women | cs1 | doctors | cancer screening | cw1 | people | health | awareness [SUMMARY]
| null |
[CONTENT] screening | cancer | women | cancer screening | study | barriers | 2017 | data | breast | uptake [SUMMARY]
| null |
[CONTENT] India ||| [SUMMARY]
| null |
[CONTENT] 38 years ||| 38.9% | 16.7% ||| ||| ||| [SUMMARY]
| null |
[CONTENT] India ||| ||| Nineteen ||| Interviews ||| ||| ||| ||| 38 years ||| 38.9% | 16.7% ||| ||| ||| ||| the years ||| [SUMMARY]
| null |
|||
A simple and safe minimally invasive technique for laparoscopic gastrostomy.
|
20529529
|
Percutaneous endoscopic gastrostomy (PEG) is the procedure of choice in the nutritional management of patients requiring gastrostomies. However, PEG tubes are not always feasible. The aim of the present study was to determine the feasibility, complications, and adequacy of feeding support of a novel laparoscopic gastrostomy technique in adults where PEG tubes were neither feasible nor safe.
|
INTRODUCTION
|
A retrospective chart review of patients who underwent a laparoscopic gastrostomy from August 2007 to July 2008 was performed. Demographic and outcome data were abstracted.
|
METHODS
|
Fourteen patients underwent laparoscopic gastrostomy. Nine had obstructing head/neck cancer, 2 had severe head trauma, and one was morbidly obese. Nine patients had previous abdominal surgery. The mean operative time was 29.8 minutes (+/-7.2). There were no conversions to open gastrostomy. Two ports (5mm and 10mm) were used in the majority of patients (78.5%). No major complications were observed. The mean follow-up was 3.1 months (range, 2 to 8).
|
RESULTS
|
This innovative 2-port laparoscopic technique for gastrostomy tube placement is safe and effective. It allows for the quick, accurate, and safe insertion of the feeding tube under direct visualization and avoids open techniques in patients where PEG tubes are not feasible.
|
CONCLUSION
|
[
"Adult",
"Aged",
"Aged, 80 and over",
"Enteral Nutrition",
"Feasibility Studies",
"Female",
"Gastrostomy",
"Head and Neck Neoplasms",
"Humans",
"Laparoscopy",
"Male",
"Middle Aged",
"Obesity, Morbid",
"Retrospective Studies",
"Suture Techniques"
] |
3021287
|
INTRODUCTION
|
Enteral access is the treatment of choice for malnourished patients with a normally functioning gastrointestinal tract. Percutaneous endoscopic gastrostomy (PEG) was developed in the early 1980s and has become the standard procedure for enteral nutrition in these patients.1 However, PEG tubes are not always feasible. The aim of this study was to evaluate the feasibility and safety of the laparoscopic gastrostomy (LG) performed in an adult population for morbid obesity, obstructing pharyngeal or esophageal cancers, severe head trauma, or history of multiple abdominal surgeries. In this article, we report our early experiences with this method.
|
Procedure
|
With the patient under general anesthesia, a supraumbilical or infraumbilical incision is used to establish pneumoperitoneum with either an open or closed technique. Pneumoperitoneum is created with a CO2 pressure between 5mm Hg to 10mm Hg. A 5-mm port is placed, and a 30° angled laparoscope is inserted. The table is then tilted in a reverse Trendelenburg position and by using digital palpation and laparoscopic screening; the site for the gastrostomy tube placement is chosen (Figure 1). Ideally, placement for the gastrostomy tube should be along the greater curvature. A 10- to 11-mm port is then introduced under direct vision over the designated site for the tube placement (Figure 2). This site should be at least 2cm caudal to the costal margin. The gastric wall is then grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site while simultaneously removing the trocar and decreasing the pneumoperitoneal pressure to 0mm Hg (Figure 2). Once exteriorized, the gastric wall is secured with 2 Babcock forceps. Double purse-string sutures (00) are then placed at the exposed stomach, which will be used later as anchoring sutures to the peritoneum. A gastrostomy is opened at the center of the loop by diathermy. A balloon gastrostomy tube is then inserted (Figure 3). A large-size Foley catheter, mushroom catheter, or Moss tube can be used. The balloon of the feeding tube should then be positioned behind the inner purse string. The incision is sometimes enlarged up to an additional 1cm for placement of the purse-string sutures. The stomach is then pushed back to the abdominal cavity and the anchoring sutures used to attach the stomach to the anterior abdominal wall. Pneumoperitoneum (10mm Hg) is recreated to check for hemostasis and any evidence for leakage around the gastrostomy insertion site. The feeding tube is then placed to gravity, and tubal feeding is started the next day.
Digital palpation at the site of planned gastrostomy under 5-mm camera direct visualization.
Gastric wall is grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site whilst the trocar is simultaneously removed and the pneumoperitoneal pressure is decreased to 0mm Hg
The stomach is opened at the center of the purse-string suture by diathermy and a balloon gastrostomy tube is then inserted and positioned behind the inner purse string.
|
RESULTS
|
Fourteen patients underwent LG during the study period. All the adults were selected for this procedure after a primary PEG procedure was considered not feasible or unsafe by the surgeon. Nine patients had obstructing head/neck cancer, 2 patients had severe head trauma, 1 was morbidly obese (Body Mass Index 56kg/m2), and 2 had multiple upper abdominal surgeries. The mean age was 59.1 years (range, 19 to 95); 3 patients were female. The mean operative time was 29.8 minutes (±7.2). There were no conversions to open. Two ports (5mm and 10/ 11mm, Ethicon Endosurgery, Inc., Newark, NJ) were used in the majority of patients (78.5%); however, up to 4 ports had to be placed (3 of 14 patients; 21.5%) when lysis of adhesions was required. Lysis of adhesions was needed in 3 of 9 patients who had previous abdominal surgery. No major intraoperative or postoperative complications were observed. Minor postoperative complications included 3 superficial wound infections and 2 premature dislodgments. These dislodgments were at 3 and 16 days postoperatively and were managed by placement of a new feeding tube under fluoroscopy. Superficial wound infections were managed conservatively with local wound care and oral antibiotics. Every patient had a successful tubal feeding within 24 hours from the operation. The mean follow-up was 3.1 months (range, 2 to 8).
|
CONCLUSION
|
We have shown that our 2-port placement of a laparoscopic gastrostomy is not only safe and effective but also has clear benefits over previously reported laparoscopic techniques. The characteristic feature of our technique is that it requires only 2 ports and no intracorporeal suturing. In addition, our technique does not require the stomach to be insufflated during the procedure. These in turn reduce operative time, expense, and difficulty. Therefore, our LG procedure should be considered an alternative to provide enteral access for malnourished patients when PEG is neither feasible nor safe.
|
[
"INTRODUCTION",
"METHODS"
] |
[
"Enteral access is the treatment of choice for malnourished patients with a normally functioning gastrointestinal tract. Percutaneous endoscopic gastrostomy (PEG) was developed in the early 1980s and has become the standard procedure for enteral nutrition in these patients.1 However, PEG tubes are not always feasible. The aim of this study was to evaluate the feasibility and safety of the laparoscopic gastrostomy (LG) performed in an adult population for morbid obesity, obstructing pharyngeal or esophageal cancers, severe head trauma, or history of multiple abdominal surgeries. In this article, we report our early experiences with this method.",
"A retrospective chart review was performed of all patients who underwent LG from August 1, 2007 to May 21, 2008 at Tulane University. Collected data included patient age, sex, indication for the procedure, number of previous abdominal surgeries, and both procedure-specific and nonspecific complications. Approval for the study was obtained from the Tulane School of Medicine Institutional Review Board. Data are presented as the mean ± standard errors.\n Procedure With the patient under general anesthesia, a supraumbilical or infraumbilical incision is used to establish pneumoperitoneum with either an open or closed technique. Pneumoperitoneum is created with a CO2 pressure between 5mm Hg to 10mm Hg. A 5-mm port is placed, and a 30° angled laparoscope is inserted. The table is then tilted in a reverse Trendelenburg position and by using digital palpation and laparoscopic screening; the site for the gastrostomy tube placement is chosen (Figure 1). Ideally, placement for the gastrostomy tube should be along the greater curvature. A 10- to 11-mm port is then introduced under direct vision over the designated site for the tube placement (Figure 2). This site should be at least 2cm caudal to the costal margin. The gastric wall is then grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site while simultaneously removing the trocar and decreasing the pneumoperitoneal pressure to 0mm Hg (Figure 2). Once exteriorized, the gastric wall is secured with 2 Babcock forceps. Double purse-string sutures (00) are then placed at the exposed stomach, which will be used later as anchoring sutures to the peritoneum. A gastrostomy is opened at the center of the loop by diathermy. A balloon gastrostomy tube is then inserted (Figure 3). A large-size Foley catheter, mushroom catheter, or Moss tube can be used. The balloon of the feeding tube should then be positioned behind the inner purse string. The incision is sometimes enlarged up to an additional 1cm for placement of the purse-string sutures. The stomach is then pushed back to the abdominal cavity and the anchoring sutures used to attach the stomach to the anterior abdominal wall. Pneumoperitoneum (10mm Hg) is recreated to check for hemostasis and any evidence for leakage around the gastrostomy insertion site. The feeding tube is then placed to gravity, and tubal feeding is started the next day.\nDigital palpation at the site of planned gastrostomy under 5-mm camera direct visualization.\nGastric wall is grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site whilst the trocar is simultaneously removed and the pneumoperitoneal pressure is decreased to 0mm Hg\nThe stomach is opened at the center of the purse-string suture by diathermy and a balloon gastrostomy tube is then inserted and positioned behind the inner purse string.\nWith the patient under general anesthesia, a supraumbilical or infraumbilical incision is used to establish pneumoperitoneum with either an open or closed technique. Pneumoperitoneum is created with a CO2 pressure between 5mm Hg to 10mm Hg. A 5-mm port is placed, and a 30° angled laparoscope is inserted. The table is then tilted in a reverse Trendelenburg position and by using digital palpation and laparoscopic screening; the site for the gastrostomy tube placement is chosen (Figure 1). Ideally, placement for the gastrostomy tube should be along the greater curvature. A 10- to 11-mm port is then introduced under direct vision over the designated site for the tube placement (Figure 2). This site should be at least 2cm caudal to the costal margin. The gastric wall is then grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site while simultaneously removing the trocar and decreasing the pneumoperitoneal pressure to 0mm Hg (Figure 2). Once exteriorized, the gastric wall is secured with 2 Babcock forceps. Double purse-string sutures (00) are then placed at the exposed stomach, which will be used later as anchoring sutures to the peritoneum. A gastrostomy is opened at the center of the loop by diathermy. A balloon gastrostomy tube is then inserted (Figure 3). A large-size Foley catheter, mushroom catheter, or Moss tube can be used. The balloon of the feeding tube should then be positioned behind the inner purse string. The incision is sometimes enlarged up to an additional 1cm for placement of the purse-string sutures. The stomach is then pushed back to the abdominal cavity and the anchoring sutures used to attach the stomach to the anterior abdominal wall. Pneumoperitoneum (10mm Hg) is recreated to check for hemostasis and any evidence for leakage around the gastrostomy insertion site. The feeding tube is then placed to gravity, and tubal feeding is started the next day.\nDigital palpation at the site of planned gastrostomy under 5-mm camera direct visualization.\nGastric wall is grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site whilst the trocar is simultaneously removed and the pneumoperitoneal pressure is decreased to 0mm Hg\nThe stomach is opened at the center of the purse-string suture by diathermy and a balloon gastrostomy tube is then inserted and positioned behind the inner purse string."
] |
[
null,
"methods"
] |
[
"INTRODUCTION",
"METHODS",
"Procedure",
"RESULTS",
"DISCUSSION",
"CONCLUSION"
] |
[
"Enteral access is the treatment of choice for malnourished patients with a normally functioning gastrointestinal tract. Percutaneous endoscopic gastrostomy (PEG) was developed in the early 1980s and has become the standard procedure for enteral nutrition in these patients.1 However, PEG tubes are not always feasible. The aim of this study was to evaluate the feasibility and safety of the laparoscopic gastrostomy (LG) performed in an adult population for morbid obesity, obstructing pharyngeal or esophageal cancers, severe head trauma, or history of multiple abdominal surgeries. In this article, we report our early experiences with this method.",
"A retrospective chart review was performed of all patients who underwent LG from August 1, 2007 to May 21, 2008 at Tulane University. Collected data included patient age, sex, indication for the procedure, number of previous abdominal surgeries, and both procedure-specific and nonspecific complications. Approval for the study was obtained from the Tulane School of Medicine Institutional Review Board. Data are presented as the mean ± standard errors.\n Procedure With the patient under general anesthesia, a supraumbilical or infraumbilical incision is used to establish pneumoperitoneum with either an open or closed technique. Pneumoperitoneum is created with a CO2 pressure between 5mm Hg to 10mm Hg. A 5-mm port is placed, and a 30° angled laparoscope is inserted. The table is then tilted in a reverse Trendelenburg position and by using digital palpation and laparoscopic screening; the site for the gastrostomy tube placement is chosen (Figure 1). Ideally, placement for the gastrostomy tube should be along the greater curvature. A 10- to 11-mm port is then introduced under direct vision over the designated site for the tube placement (Figure 2). This site should be at least 2cm caudal to the costal margin. The gastric wall is then grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site while simultaneously removing the trocar and decreasing the pneumoperitoneal pressure to 0mm Hg (Figure 2). Once exteriorized, the gastric wall is secured with 2 Babcock forceps. Double purse-string sutures (00) are then placed at the exposed stomach, which will be used later as anchoring sutures to the peritoneum. A gastrostomy is opened at the center of the loop by diathermy. A balloon gastrostomy tube is then inserted (Figure 3). A large-size Foley catheter, mushroom catheter, or Moss tube can be used. The balloon of the feeding tube should then be positioned behind the inner purse string. The incision is sometimes enlarged up to an additional 1cm for placement of the purse-string sutures. The stomach is then pushed back to the abdominal cavity and the anchoring sutures used to attach the stomach to the anterior abdominal wall. Pneumoperitoneum (10mm Hg) is recreated to check for hemostasis and any evidence for leakage around the gastrostomy insertion site. The feeding tube is then placed to gravity, and tubal feeding is started the next day.\nDigital palpation at the site of planned gastrostomy under 5-mm camera direct visualization.\nGastric wall is grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site whilst the trocar is simultaneously removed and the pneumoperitoneal pressure is decreased to 0mm Hg\nThe stomach is opened at the center of the purse-string suture by diathermy and a balloon gastrostomy tube is then inserted and positioned behind the inner purse string.\nWith the patient under general anesthesia, a supraumbilical or infraumbilical incision is used to establish pneumoperitoneum with either an open or closed technique. Pneumoperitoneum is created with a CO2 pressure between 5mm Hg to 10mm Hg. A 5-mm port is placed, and a 30° angled laparoscope is inserted. The table is then tilted in a reverse Trendelenburg position and by using digital palpation and laparoscopic screening; the site for the gastrostomy tube placement is chosen (Figure 1). Ideally, placement for the gastrostomy tube should be along the greater curvature. A 10- to 11-mm port is then introduced under direct vision over the designated site for the tube placement (Figure 2). This site should be at least 2cm caudal to the costal margin. The gastric wall is then grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site while simultaneously removing the trocar and decreasing the pneumoperitoneal pressure to 0mm Hg (Figure 2). Once exteriorized, the gastric wall is secured with 2 Babcock forceps. Double purse-string sutures (00) are then placed at the exposed stomach, which will be used later as anchoring sutures to the peritoneum. A gastrostomy is opened at the center of the loop by diathermy. A balloon gastrostomy tube is then inserted (Figure 3). A large-size Foley catheter, mushroom catheter, or Moss tube can be used. The balloon of the feeding tube should then be positioned behind the inner purse string. The incision is sometimes enlarged up to an additional 1cm for placement of the purse-string sutures. The stomach is then pushed back to the abdominal cavity and the anchoring sutures used to attach the stomach to the anterior abdominal wall. Pneumoperitoneum (10mm Hg) is recreated to check for hemostasis and any evidence for leakage around the gastrostomy insertion site. The feeding tube is then placed to gravity, and tubal feeding is started the next day.\nDigital palpation at the site of planned gastrostomy under 5-mm camera direct visualization.\nGastric wall is grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site whilst the trocar is simultaneously removed and the pneumoperitoneal pressure is decreased to 0mm Hg\nThe stomach is opened at the center of the purse-string suture by diathermy and a balloon gastrostomy tube is then inserted and positioned behind the inner purse string.",
"With the patient under general anesthesia, a supraumbilical or infraumbilical incision is used to establish pneumoperitoneum with either an open or closed technique. Pneumoperitoneum is created with a CO2 pressure between 5mm Hg to 10mm Hg. A 5-mm port is placed, and a 30° angled laparoscope is inserted. The table is then tilted in a reverse Trendelenburg position and by using digital palpation and laparoscopic screening; the site for the gastrostomy tube placement is chosen (Figure 1). Ideally, placement for the gastrostomy tube should be along the greater curvature. A 10- to 11-mm port is then introduced under direct vision over the designated site for the tube placement (Figure 2). This site should be at least 2cm caudal to the costal margin. The gastric wall is then grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site while simultaneously removing the trocar and decreasing the pneumoperitoneal pressure to 0mm Hg (Figure 2). Once exteriorized, the gastric wall is secured with 2 Babcock forceps. Double purse-string sutures (00) are then placed at the exposed stomach, which will be used later as anchoring sutures to the peritoneum. A gastrostomy is opened at the center of the loop by diathermy. A balloon gastrostomy tube is then inserted (Figure 3). A large-size Foley catheter, mushroom catheter, or Moss tube can be used. The balloon of the feeding tube should then be positioned behind the inner purse string. The incision is sometimes enlarged up to an additional 1cm for placement of the purse-string sutures. The stomach is then pushed back to the abdominal cavity and the anchoring sutures used to attach the stomach to the anterior abdominal wall. Pneumoperitoneum (10mm Hg) is recreated to check for hemostasis and any evidence for leakage around the gastrostomy insertion site. The feeding tube is then placed to gravity, and tubal feeding is started the next day.\nDigital palpation at the site of planned gastrostomy under 5-mm camera direct visualization.\nGastric wall is grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site whilst the trocar is simultaneously removed and the pneumoperitoneal pressure is decreased to 0mm Hg\nThe stomach is opened at the center of the purse-string suture by diathermy and a balloon gastrostomy tube is then inserted and positioned behind the inner purse string.",
"Fourteen patients underwent LG during the study period. All the adults were selected for this procedure after a primary PEG procedure was considered not feasible or unsafe by the surgeon. Nine patients had obstructing head/neck cancer, 2 patients had severe head trauma, 1 was morbidly obese (Body Mass Index 56kg/m2), and 2 had multiple upper abdominal surgeries. The mean age was 59.1 years (range, 19 to 95); 3 patients were female. The mean operative time was 29.8 minutes (±7.2). There were no conversions to open. Two ports (5mm and 10/ 11mm, Ethicon Endosurgery, Inc., Newark, NJ) were used in the majority of patients (78.5%); however, up to 4 ports had to be placed (3 of 14 patients; 21.5%) when lysis of adhesions was required. Lysis of adhesions was needed in 3 of 9 patients who had previous abdominal surgery. No major intraoperative or postoperative complications were observed. Minor postoperative complications included 3 superficial wound infections and 2 premature dislodgments. These dislodgments were at 3 and 16 days postoperatively and were managed by placement of a new feeding tube under fluoroscopy. Superficial wound infections were managed conservatively with local wound care and oral antibiotics. Every patient had a successful tubal feeding within 24 hours from the operation. The mean follow-up was 3.1 months (range, 2 to 8).",
"Since its first description in 1979 by Gauderer1 for use in the pediatric population, percutaneous endoscopic gastrostomy (PEG) tubes have become the gold standard for nutritional assistance in adult patients who are dysphagic secondary to neurologic deficits or secondary to treatment of obstruction from head and neck cancers. However, PEG tube placement requires that a light source be passed from the esophagus and into the stomach to act as a guide for the percutaneous procedure. This method is neither always feasible nor safe in cases of obstruction from head and neck cancer or head trauma. Additionally, there might be an inability to transilluminate due to morbid obesity, ascites, or colon or liver overlying the stomach. Consequently, several alternative methods for laparoscopic feeding tube insertion have been described. However, unlike our technique, these methods are sometimes expensive, complicated, and time consuming.2,3 Hsieh et al2 used a 3-port method in an adult population of 48 patients with obstructing head and neck cancers. Interestingly, not only was the insertion time longer in this published report compared with our technique (62.4±11 min vs. 29.8±7.2 min, respectively), but these authors also used an additional port for their LG method.\nOur innovative, simple, 2-port laparoscopic technique makes it possible to place the gastrostomy tube with ease. It allows direct viewing and manipulation of the stomach into a position where it can be safely punctured for placement of a gastrostomy tube, and because it is performed under direct vision it also minimizes the risk of inadvertent visceral injury. Consequently, our procedure carries a low rate of complications. In fact, in our series, the complication rate of 12% is lower than the incidence reported by others (23%).3\nBesides LG, laparoscopic-assisted percutaneous endoscopic gastrostomy (LAPEG) has also been used in instances where PEG placement has either failed or is contraindicated.4,5 This technique involves placing one laparoscopic optical trocar into the right midabdomen then utilizing direct visualization to identify the needle track for PEG placement.4 Unlike our 2-port method, however, LAPEG cannot be used in severely stenotic or obstructing upper digestive tracts due to the inherent need to pass a light source through the esophagus. In addition, our 2-port method allows the added advantage of doing a diagnostic laparoscopy and gastrostomy tube placement in one procedure versus converting to a 2-port method after LAPEG.",
"We have shown that our 2-port placement of a laparoscopic gastrostomy is not only safe and effective but also has clear benefits over previously reported laparoscopic techniques. The characteristic feature of our technique is that it requires only 2 ports and no intracorporeal suturing. In addition, our technique does not require the stomach to be insufflated during the procedure. These in turn reduce operative time, expense, and difficulty. Therefore, our LG procedure should be considered an alternative to provide enteral access for malnourished patients when PEG is neither feasible nor safe."
] |
[
null,
"methods",
"methods",
"results",
"discussion",
"conclusions"
] |
[
"Laparoscopy",
"Percutaneous endoscopic gastrostomy"
] |
INTRODUCTION:
Enteral access is the treatment of choice for malnourished patients with a normally functioning gastrointestinal tract. Percutaneous endoscopic gastrostomy (PEG) was developed in the early 1980s and has become the standard procedure for enteral nutrition in these patients.1 However, PEG tubes are not always feasible. The aim of this study was to evaluate the feasibility and safety of the laparoscopic gastrostomy (LG) performed in an adult population for morbid obesity, obstructing pharyngeal or esophageal cancers, severe head trauma, or history of multiple abdominal surgeries. In this article, we report our early experiences with this method.
METHODS:
A retrospective chart review was performed of all patients who underwent LG from August 1, 2007 to May 21, 2008 at Tulane University. Collected data included patient age, sex, indication for the procedure, number of previous abdominal surgeries, and both procedure-specific and nonspecific complications. Approval for the study was obtained from the Tulane School of Medicine Institutional Review Board. Data are presented as the mean ± standard errors.
Procedure With the patient under general anesthesia, a supraumbilical or infraumbilical incision is used to establish pneumoperitoneum with either an open or closed technique. Pneumoperitoneum is created with a CO2 pressure between 5mm Hg to 10mm Hg. A 5-mm port is placed, and a 30° angled laparoscope is inserted. The table is then tilted in a reverse Trendelenburg position and by using digital palpation and laparoscopic screening; the site for the gastrostomy tube placement is chosen (Figure 1). Ideally, placement for the gastrostomy tube should be along the greater curvature. A 10- to 11-mm port is then introduced under direct vision over the designated site for the tube placement (Figure 2). This site should be at least 2cm caudal to the costal margin. The gastric wall is then grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site while simultaneously removing the trocar and decreasing the pneumoperitoneal pressure to 0mm Hg (Figure 2). Once exteriorized, the gastric wall is secured with 2 Babcock forceps. Double purse-string sutures (00) are then placed at the exposed stomach, which will be used later as anchoring sutures to the peritoneum. A gastrostomy is opened at the center of the loop by diathermy. A balloon gastrostomy tube is then inserted (Figure 3). A large-size Foley catheter, mushroom catheter, or Moss tube can be used. The balloon of the feeding tube should then be positioned behind the inner purse string. The incision is sometimes enlarged up to an additional 1cm for placement of the purse-string sutures. The stomach is then pushed back to the abdominal cavity and the anchoring sutures used to attach the stomach to the anterior abdominal wall. Pneumoperitoneum (10mm Hg) is recreated to check for hemostasis and any evidence for leakage around the gastrostomy insertion site. The feeding tube is then placed to gravity, and tubal feeding is started the next day.
Digital palpation at the site of planned gastrostomy under 5-mm camera direct visualization.
Gastric wall is grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site whilst the trocar is simultaneously removed and the pneumoperitoneal pressure is decreased to 0mm Hg
The stomach is opened at the center of the purse-string suture by diathermy and a balloon gastrostomy tube is then inserted and positioned behind the inner purse string.
With the patient under general anesthesia, a supraumbilical or infraumbilical incision is used to establish pneumoperitoneum with either an open or closed technique. Pneumoperitoneum is created with a CO2 pressure between 5mm Hg to 10mm Hg. A 5-mm port is placed, and a 30° angled laparoscope is inserted. The table is then tilted in a reverse Trendelenburg position and by using digital palpation and laparoscopic screening; the site for the gastrostomy tube placement is chosen (Figure 1). Ideally, placement for the gastrostomy tube should be along the greater curvature. A 10- to 11-mm port is then introduced under direct vision over the designated site for the tube placement (Figure 2). This site should be at least 2cm caudal to the costal margin. The gastric wall is then grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site while simultaneously removing the trocar and decreasing the pneumoperitoneal pressure to 0mm Hg (Figure 2). Once exteriorized, the gastric wall is secured with 2 Babcock forceps. Double purse-string sutures (00) are then placed at the exposed stomach, which will be used later as anchoring sutures to the peritoneum. A gastrostomy is opened at the center of the loop by diathermy. A balloon gastrostomy tube is then inserted (Figure 3). A large-size Foley catheter, mushroom catheter, or Moss tube can be used. The balloon of the feeding tube should then be positioned behind the inner purse string. The incision is sometimes enlarged up to an additional 1cm for placement of the purse-string sutures. The stomach is then pushed back to the abdominal cavity and the anchoring sutures used to attach the stomach to the anterior abdominal wall. Pneumoperitoneum (10mm Hg) is recreated to check for hemostasis and any evidence for leakage around the gastrostomy insertion site. The feeding tube is then placed to gravity, and tubal feeding is started the next day.
Digital palpation at the site of planned gastrostomy under 5-mm camera direct visualization.
Gastric wall is grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site whilst the trocar is simultaneously removed and the pneumoperitoneal pressure is decreased to 0mm Hg
The stomach is opened at the center of the purse-string suture by diathermy and a balloon gastrostomy tube is then inserted and positioned behind the inner purse string.
Procedure:
With the patient under general anesthesia, a supraumbilical or infraumbilical incision is used to establish pneumoperitoneum with either an open or closed technique. Pneumoperitoneum is created with a CO2 pressure between 5mm Hg to 10mm Hg. A 5-mm port is placed, and a 30° angled laparoscope is inserted. The table is then tilted in a reverse Trendelenburg position and by using digital palpation and laparoscopic screening; the site for the gastrostomy tube placement is chosen (Figure 1). Ideally, placement for the gastrostomy tube should be along the greater curvature. A 10- to 11-mm port is then introduced under direct vision over the designated site for the tube placement (Figure 2). This site should be at least 2cm caudal to the costal margin. The gastric wall is then grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site while simultaneously removing the trocar and decreasing the pneumoperitoneal pressure to 0mm Hg (Figure 2). Once exteriorized, the gastric wall is secured with 2 Babcock forceps. Double purse-string sutures (00) are then placed at the exposed stomach, which will be used later as anchoring sutures to the peritoneum. A gastrostomy is opened at the center of the loop by diathermy. A balloon gastrostomy tube is then inserted (Figure 3). A large-size Foley catheter, mushroom catheter, or Moss tube can be used. The balloon of the feeding tube should then be positioned behind the inner purse string. The incision is sometimes enlarged up to an additional 1cm for placement of the purse-string sutures. The stomach is then pushed back to the abdominal cavity and the anchoring sutures used to attach the stomach to the anterior abdominal wall. Pneumoperitoneum (10mm Hg) is recreated to check for hemostasis and any evidence for leakage around the gastrostomy insertion site. The feeding tube is then placed to gravity, and tubal feeding is started the next day.
Digital palpation at the site of planned gastrostomy under 5-mm camera direct visualization.
Gastric wall is grasped with a 10-mm laparoscopic Babcock forceps and brought through the port site whilst the trocar is simultaneously removed and the pneumoperitoneal pressure is decreased to 0mm Hg
The stomach is opened at the center of the purse-string suture by diathermy and a balloon gastrostomy tube is then inserted and positioned behind the inner purse string.
RESULTS:
Fourteen patients underwent LG during the study period. All the adults were selected for this procedure after a primary PEG procedure was considered not feasible or unsafe by the surgeon. Nine patients had obstructing head/neck cancer, 2 patients had severe head trauma, 1 was morbidly obese (Body Mass Index 56kg/m2), and 2 had multiple upper abdominal surgeries. The mean age was 59.1 years (range, 19 to 95); 3 patients were female. The mean operative time was 29.8 minutes (±7.2). There were no conversions to open. Two ports (5mm and 10/ 11mm, Ethicon Endosurgery, Inc., Newark, NJ) were used in the majority of patients (78.5%); however, up to 4 ports had to be placed (3 of 14 patients; 21.5%) when lysis of adhesions was required. Lysis of adhesions was needed in 3 of 9 patients who had previous abdominal surgery. No major intraoperative or postoperative complications were observed. Minor postoperative complications included 3 superficial wound infections and 2 premature dislodgments. These dislodgments were at 3 and 16 days postoperatively and were managed by placement of a new feeding tube under fluoroscopy. Superficial wound infections were managed conservatively with local wound care and oral antibiotics. Every patient had a successful tubal feeding within 24 hours from the operation. The mean follow-up was 3.1 months (range, 2 to 8).
DISCUSSION:
Since its first description in 1979 by Gauderer1 for use in the pediatric population, percutaneous endoscopic gastrostomy (PEG) tubes have become the gold standard for nutritional assistance in adult patients who are dysphagic secondary to neurologic deficits or secondary to treatment of obstruction from head and neck cancers. However, PEG tube placement requires that a light source be passed from the esophagus and into the stomach to act as a guide for the percutaneous procedure. This method is neither always feasible nor safe in cases of obstruction from head and neck cancer or head trauma. Additionally, there might be an inability to transilluminate due to morbid obesity, ascites, or colon or liver overlying the stomach. Consequently, several alternative methods for laparoscopic feeding tube insertion have been described. However, unlike our technique, these methods are sometimes expensive, complicated, and time consuming.2,3 Hsieh et al2 used a 3-port method in an adult population of 48 patients with obstructing head and neck cancers. Interestingly, not only was the insertion time longer in this published report compared with our technique (62.4±11 min vs. 29.8±7.2 min, respectively), but these authors also used an additional port for their LG method.
Our innovative, simple, 2-port laparoscopic technique makes it possible to place the gastrostomy tube with ease. It allows direct viewing and manipulation of the stomach into a position where it can be safely punctured for placement of a gastrostomy tube, and because it is performed under direct vision it also minimizes the risk of inadvertent visceral injury. Consequently, our procedure carries a low rate of complications. In fact, in our series, the complication rate of 12% is lower than the incidence reported by others (23%).3
Besides LG, laparoscopic-assisted percutaneous endoscopic gastrostomy (LAPEG) has also been used in instances where PEG placement has either failed or is contraindicated.4,5 This technique involves placing one laparoscopic optical trocar into the right midabdomen then utilizing direct visualization to identify the needle track for PEG placement.4 Unlike our 2-port method, however, LAPEG cannot be used in severely stenotic or obstructing upper digestive tracts due to the inherent need to pass a light source through the esophagus. In addition, our 2-port method allows the added advantage of doing a diagnostic laparoscopy and gastrostomy tube placement in one procedure versus converting to a 2-port method after LAPEG.
CONCLUSION:
We have shown that our 2-port placement of a laparoscopic gastrostomy is not only safe and effective but also has clear benefits over previously reported laparoscopic techniques. The characteristic feature of our technique is that it requires only 2 ports and no intracorporeal suturing. In addition, our technique does not require the stomach to be insufflated during the procedure. These in turn reduce operative time, expense, and difficulty. Therefore, our LG procedure should be considered an alternative to provide enteral access for malnourished patients when PEG is neither feasible nor safe.
|
Background:
Percutaneous endoscopic gastrostomy (PEG) is the procedure of choice in the nutritional management of patients requiring gastrostomies. However, PEG tubes are not always feasible. The aim of the present study was to determine the feasibility, complications, and adequacy of feeding support of a novel laparoscopic gastrostomy technique in adults where PEG tubes were neither feasible nor safe.
Methods:
A retrospective chart review of patients who underwent a laparoscopic gastrostomy from August 2007 to July 2008 was performed. Demographic and outcome data were abstracted.
Results:
Fourteen patients underwent laparoscopic gastrostomy. Nine had obstructing head/neck cancer, 2 had severe head trauma, and one was morbidly obese. Nine patients had previous abdominal surgery. The mean operative time was 29.8 minutes (+/-7.2). There were no conversions to open gastrostomy. Two ports (5mm and 10mm) were used in the majority of patients (78.5%). No major complications were observed. The mean follow-up was 3.1 months (range, 2 to 8).
Conclusions:
This innovative 2-port laparoscopic technique for gastrostomy tube placement is safe and effective. It allows for the quick, accurate, and safe insertion of the feeding tube under direct visualization and avoids open techniques in patients where PEG tubes are not feasible.
|
INTRODUCTION:
Enteral access is the treatment of choice for malnourished patients with a normally functioning gastrointestinal tract. Percutaneous endoscopic gastrostomy (PEG) was developed in the early 1980s and has become the standard procedure for enteral nutrition in these patients.1 However, PEG tubes are not always feasible. The aim of this study was to evaluate the feasibility and safety of the laparoscopic gastrostomy (LG) performed in an adult population for morbid obesity, obstructing pharyngeal or esophageal cancers, severe head trauma, or history of multiple abdominal surgeries. In this article, we report our early experiences with this method.
CONCLUSION:
We have shown that our 2-port placement of a laparoscopic gastrostomy is not only safe and effective but also has clear benefits over previously reported laparoscopic techniques. The characteristic feature of our technique is that it requires only 2 ports and no intracorporeal suturing. In addition, our technique does not require the stomach to be insufflated during the procedure. These in turn reduce operative time, expense, and difficulty. Therefore, our LG procedure should be considered an alternative to provide enteral access for malnourished patients when PEG is neither feasible nor safe.
|
Background:
Percutaneous endoscopic gastrostomy (PEG) is the procedure of choice in the nutritional management of patients requiring gastrostomies. However, PEG tubes are not always feasible. The aim of the present study was to determine the feasibility, complications, and adequacy of feeding support of a novel laparoscopic gastrostomy technique in adults where PEG tubes were neither feasible nor safe.
Methods:
A retrospective chart review of patients who underwent a laparoscopic gastrostomy from August 2007 to July 2008 was performed. Demographic and outcome data were abstracted.
Results:
Fourteen patients underwent laparoscopic gastrostomy. Nine had obstructing head/neck cancer, 2 had severe head trauma, and one was morbidly obese. Nine patients had previous abdominal surgery. The mean operative time was 29.8 minutes (+/-7.2). There were no conversions to open gastrostomy. Two ports (5mm and 10mm) were used in the majority of patients (78.5%). No major complications were observed. The mean follow-up was 3.1 months (range, 2 to 8).
Conclusions:
This innovative 2-port laparoscopic technique for gastrostomy tube placement is safe and effective. It allows for the quick, accurate, and safe insertion of the feeding tube under direct visualization and avoids open techniques in patients where PEG tubes are not feasible.
| 2,385 | 249 | 6 |
[
"tube",
"gastrostomy",
"site",
"port",
"placement",
"laparoscopic",
"stomach",
"purse",
"mm",
"gastrostomy tube"
] |
[
"test",
"test"
] |
[CONTENT] Laparoscopy | Percutaneous endoscopic gastrostomy [SUMMARY]
|
[CONTENT] Laparoscopy | Percutaneous endoscopic gastrostomy [SUMMARY]
|
[CONTENT] Laparoscopy | Percutaneous endoscopic gastrostomy [SUMMARY]
|
[CONTENT] Laparoscopy | Percutaneous endoscopic gastrostomy [SUMMARY]
|
[CONTENT] Laparoscopy | Percutaneous endoscopic gastrostomy [SUMMARY]
|
[CONTENT] Laparoscopy | Percutaneous endoscopic gastrostomy [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Enteral Nutrition | Feasibility Studies | Female | Gastrostomy | Head and Neck Neoplasms | Humans | Laparoscopy | Male | Middle Aged | Obesity, Morbid | Retrospective Studies | Suture Techniques [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Enteral Nutrition | Feasibility Studies | Female | Gastrostomy | Head and Neck Neoplasms | Humans | Laparoscopy | Male | Middle Aged | Obesity, Morbid | Retrospective Studies | Suture Techniques [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Enteral Nutrition | Feasibility Studies | Female | Gastrostomy | Head and Neck Neoplasms | Humans | Laparoscopy | Male | Middle Aged | Obesity, Morbid | Retrospective Studies | Suture Techniques [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Enteral Nutrition | Feasibility Studies | Female | Gastrostomy | Head and Neck Neoplasms | Humans | Laparoscopy | Male | Middle Aged | Obesity, Morbid | Retrospective Studies | Suture Techniques [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Enteral Nutrition | Feasibility Studies | Female | Gastrostomy | Head and Neck Neoplasms | Humans | Laparoscopy | Male | Middle Aged | Obesity, Morbid | Retrospective Studies | Suture Techniques [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Enteral Nutrition | Feasibility Studies | Female | Gastrostomy | Head and Neck Neoplasms | Humans | Laparoscopy | Male | Middle Aged | Obesity, Morbid | Retrospective Studies | Suture Techniques [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] tube | gastrostomy | site | port | placement | laparoscopic | stomach | purse | mm | gastrostomy tube [SUMMARY]
|
[CONTENT] tube | gastrostomy | site | port | placement | laparoscopic | stomach | purse | mm | gastrostomy tube [SUMMARY]
|
[CONTENT] tube | gastrostomy | site | port | placement | laparoscopic | stomach | purse | mm | gastrostomy tube [SUMMARY]
|
[CONTENT] tube | gastrostomy | site | port | placement | laparoscopic | stomach | purse | mm | gastrostomy tube [SUMMARY]
|
[CONTENT] tube | gastrostomy | site | port | placement | laparoscopic | stomach | purse | mm | gastrostomy tube [SUMMARY]
|
[CONTENT] tube | gastrostomy | site | port | placement | laparoscopic | stomach | purse | mm | gastrostomy tube [SUMMARY]
|
[CONTENT] early | enteral | peg | gastrostomy | patients | endoscopic gastrostomy peg developed | gastrointestinal tract percutaneous endoscopic | feasible aim study evaluate | obstructing pharyngeal esophageal | obstructing pharyngeal [SUMMARY]
|
[CONTENT] site | tube | purse string | mm | purse | hg | string | gastrostomy | wall | figure [SUMMARY]
|
[CONTENT] patients | wound | mean | superficial wound infections | superficial wound | superficial | postoperative complications | postoperative | infections | managed [SUMMARY]
|
[CONTENT] safe | technique | procedure | laparoscopic | reported laparoscopic | insufflated procedure turn reduce | insufflated procedure turn | insufflated procedure | insufflated | shown port placement laparoscopic [SUMMARY]
|
[CONTENT] tube | gastrostomy | site | port | patients | laparoscopic | placement | string | hg | purse [SUMMARY]
|
[CONTENT] tube | gastrostomy | site | port | patients | laparoscopic | placement | string | hg | purse [SUMMARY]
|
[CONTENT] PEG ||| PEG ||| PEG [SUMMARY]
|
[CONTENT] August 2007 to July 2008 ||| [SUMMARY]
|
[CONTENT] Fourteen ||| 2 | obese ||| Nine ||| 29.8 minutes ||| ||| Two | 5mm and | 10mm | 78.5% ||| ||| 3.1 months | 2 [SUMMARY]
|
[CONTENT] 2 ||| PEG [SUMMARY]
|
[CONTENT] PEG ||| PEG ||| PEG ||| August 2007 to July 2008 ||| ||| ||| Fourteen ||| 2 | obese ||| Nine ||| 29.8 minutes ||| ||| Two | 5mm and | 10mm | 78.5% ||| ||| 3.1 months | 2 ||| 2 ||| PEG [SUMMARY]
|
[CONTENT] PEG ||| PEG ||| PEG ||| August 2007 to July 2008 ||| ||| ||| Fourteen ||| 2 | obese ||| Nine ||| 29.8 minutes ||| ||| Two | 5mm and | 10mm | 78.5% ||| ||| 3.1 months | 2 ||| 2 ||| PEG [SUMMARY]
|
||||||
TMPRSS6 rs855791 polymorphism influences the susceptibility to iron deficiency anemia in women at reproductive age.
|
24782651
|
Genome-wide-association studies have identified the TMPRSS6 polymorphism rs855791 has the strongest association with red blood cell indices or iron parameters in general population. Whether this genetic variant influences the susceptibility of iron deficiency anemia (IDA) in women with menstruation has not been well studied.
|
BACKGROUND
|
In this case-control study, we enrolled 67 women with IDA and 107 healthy volunteers, and analyzed their complete blood counts, rs855791 genotypes, and menstrual amounts. Menstrual blood loss was evaluated with a pictorial blood-loss assessment chart.
|
METHODS
|
There were significantly fewer rs855791 C homozygotes in the IDA group than in the healthy group (11.9% vs. 25.2%, p = 0.03). The odds ratio (OR) of C homozygotes having IDA versus non-CC subjects having IDA was 0.4 (95% CI, 0.17 - 0.95, p = 0.04). When the analysis was confined to study subjects with menorrhagia, this difference became more prominent (9.6% vs. 28.6%, p = 0.01; OR, 0.27, 95% CI, 0.09 - 0.77, p = 0.01). For women with non-CC genotypes, there was an inverse correlation between hemoglobin levels and menstrual loss (p < 0.001); however, this association was not found for those with genotypes CC (p = 0.15).
|
RESULTS
|
Our study suggests homozygosity for TMPRSS6 rs855791 C genotype has a protective role against IDA in women at reproductive age, especially in those with menorrhagia.
|
CONCLUSIONS
|
[
"Adult",
"Anemia, Iron-Deficiency",
"Case-Control Studies",
"Female",
"Genetic Association Studies",
"Genetic Predisposition to Disease",
"Genotype",
"Humans",
"Membrane Proteins",
"Menorrhagia",
"Menstruation",
"Middle Aged",
"Risk Factors",
"Serine Endopeptidases"
] |
4003547
|
Introduction
|
Iron deficiency is the most common nutritional disorder worldwide. As well as affecting a large number of children and women in non-industrialized countries, it is the only nutrient deficiency which is also significant prevalent in many industrialized nations 1. Menstruating women have been recognized to be among the most likely individuals to develop iron deficiency anemia (IDA) and its prevalence is 6-22% in developed countries 2-5. Even though menorrhagia serve as a major contributing factor of IDA in this population, it is not uncommon that women with heavy menstrual loss have normal hemoglobin (Hb) levels and adequate iron stores 2. Individual susceptibility to IDA varies. However, whether genetic variants play a role in IDA pathogenesis remains unclear.
Hepcidin is the core of iron metabolism and is tightly regulated by several mediators 6. Matriptase-2 is an important one and down regulates hepcidin expression through cleaving membrane-bound hemojuvelin, which can enhance hepcidin transcription 6. Complete loss of function mutation of matriptase-2 leads to a rare disease, iron-refractory iron deficiency anemia (IRIDA) 7, 8. Patients with IRIDA have high serum hepcidin levels and cannot absorb iron from intestine. It is of interest that whether genetic variants with incomplete loss of function of matriptase-2 will be associated with IDA in general population.
Recently, several genome-wide association studies identified several single nucleotide polymorphisms (SNPs) that influence blood cell phenotypes 9-13. Among these SNPs, rs855791 has the strongest association with red blood cell indices and iron parameters in general population 12, 13. This SNP is located in the functional part of TMPRSS6 and the rs855791 (2321 C>T) causes a nonsynonymous substitution that reduces the ability of the enzyme to inhibit hepcidin transcription 14. To investigate the role of this genetic variant rs855791 on the susceptibility of IDA in menstruating women, we conducted this case-control study.
| null | null |
Results
|
A total of 67 IDA subjects and 107 healthy women were eligible for analysis. Characteristics of both groups are shown in Table 1. IDA patients were significantly older than control subjects (39.5 ± 9.2 vs. 32.7 ± 6.2 years; p < 0.001). All red blood cell indices and platelet counts differed significantly between two groups (Table 1). PBAC scores were not available for 2 IDA subjects. The PBAC scores of the 65 IDA patients were significantly higher than those of the 107 healthy controls (362.5 ± 352.4 vs. 185.2 ± 193.6; p < 0.001). Menorrhagia (PBAC score > 100) was more common in IDA patients than in the control group (80.0% vs. 65.4%, p = 0.04).
The frequency distribution of the rs855791 TT, TC, and CC genotypes in both groups showed borderline difference (p = 0.06; Table 2). However, the proportion of the C homozygotes was significantly lower in IDA patients (11.9% vs. 25.2%, p = 0.03; Figure 2). The odds ratio (OR) of C homozygotes having IDA versus those with TX (TT and TC) genotypes having IDA was 0.4 (95% CI, 0.17 - 0.95, p = 0.04). The C allele frequencies in the IDA and control group did not differ (43.3% vs. 48.6%, p= 0.33).
We further performed subgroup analysis based on menorrhagia (PBAC score > 100). For menorrhagic subjects, the frequency distribution of the 3 genotypes of the rs855791 SNP differed significantly in both groups (p = 0.02; Table 3). The frequency of the CC genotype was much lower in the IDA group (9.6% vs. 28.6%, p = 0.01, Figure 3a). Menorrhagic women with homozygous C genotype have lower risk to have IDA than those with TX genotypes (OR = 0.27, 95% CI, 0.09-0.77, p = 0.01). However, for those without menorrhagia, no difference between IDA and healthy women was observed in the frequency distribution of TT, TC, and CC genotypes (p = 0.91; Table 3) or of the CC and TX genotypes (OR = 0.78, 95% CI, 0.14-4.34, p = 0.78; Figure 3b). The frequency of the C allele did not differ between the groups whether or not the study subjects had menorrhagia (for menorrhagic subjects, 42.3% vs. 50.7%, p = 0.19; for non-menorrhagic subjects, 46.2% vs. 44.6%, p = 0.89).
Then, we pooled data from both groups, and analyzed the correlation between Hb levels and menstrual blood loss (PBAC scores). For women with genotypes TX, there was a strong inverse correlation between Hb levels and menstrual amounts; the equation used was Hb = 11.95 - 0.003 × PBAC score (R2 = 0.135, p < 0.001). In contrast, the correlation was not statistically significant for those with C homozygotes (R2 = 0.062, p = 0.15). The results are also shown in Figure 4. Because subjects in the IDA group were older and had more menstrual blood loss than the control group, we therefore analyzed the correlation between age and PBAC scores in all subjects. We found that older women had more menstrual blood loss (p = 0.002, data not shown). This result explained the difference in the age distribution between two groups.
Finally, we investigated the influence of rs855791 on RBC parameters, ferritin levels, and log(ferritin) value based on the control group data. However, we did not find a difference between the genotypes in our study (data not shown).
| null | null |
[
"Study population",
"Assessment of menstrual bleeding",
"Red blood cell parameters and ferritin levels",
"Genotyping",
"Statistical analysis"
] |
[
"Female patients with microcytic anemia were screened at hematologic clinics for IDA. IDA was defined as a hemoglobin level < 12 g/dL and a ferritin level < 10 ng/mL 2, 15. The evaluation for IDA included a physical examination, history taking, renal function test, and stool occult blood test. For those without an adequate response to iron replacement (oral elemental iron 300 mg/day for 2-4 weeks), Hb electrophoresis and brilliant cresyl blue staining tests were used to exclude thalassemia. Healthy women with regular menstruation were enrolled as the control group; most of them were staff in our institute. Complete blood counts and ferritin levels were measured and only those with normal Hb and ferritin levels were included in the analysis. An in-person questionnaire was administered to every subject in this study, which included information on menstrual, medical and surgical history. The exclusion criteria for both groups included gastrointestinal bleeding, thalassemia, renal insufficiency, menopause, and history of gastrointestinal resection. This study was performed in accordance with the declaration of Helsinki, and approved by the Ethics Committee of Chang-Gung Memorial Hospital (99-0482B) and informed consent was obtained from all participants.",
"Menstrual blood loss was evaluated with the pictorial blood-loss assessment chart (PBAC) 16, a semi-quantitative method used to estimate menstrual amount and has been validated in many studies 17-19. Menorrhagia was defined as a PBAC score of more than 100 during one menstrual period, which corresponds to a blood loss of more than 80 mL 16.",
"Red blood cell numbers, Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width and platelet counts were determined by the XE-2100 system (Sysmex Co, Kobe, Japan). Serum ferritin levels were determined using a commercial EIA kit (Advia Centaur, Siemens, NY).",
"Genomic DNA was extracted from peripheral leukocytes using the Genomic DNA Purification Kit (Gentra Systems, Inc. Chicago, IL, USA). The TMPRSS6 rs855791 C>T polymorphism was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)(Figure 1). The primers used were 5'-tag aga aca ggg gct cca gg-3' (forward) and 5'-atg tgg gca gca tcc ttt c-3' (reverse). The reaction conditions for amplification were as follows: 95°C for 5 min; 30 cycles of 95°C for 45 s, 63°C for 45 s, and 72°C for 45 s; and a final extension at 72°C for 5 min. Following amplification, the PCR products (249 bp) were digested with the restriction endonuclease Stu I (New England Biolabs, Inc. Hitchin, Herts, UK). Genotype was determined by fragment size, and 10% of the samples were directly sequenced to confirm the genotyping results.",
"Continuous variables are presented as mean ± standard deviation; categorical variables are presented as numbers and percentages. Categorical variables were analyzed by Chi-square or Fisher's exact tests. Student's t-test was used to examine continuous variables with normal distributions. Linear correlation and regression was used to evaluate the correlations between age, Hb levels and menstrual amounts. Odds ratio (OR) and confidence interval (CI) were tested by binary logistic regression. A p-value of less than 0.05 was considered statistically significant."
] |
[
null,
null,
null,
null,
null
] |
[
"Introduction",
"Materials and Methods",
"Study population",
"Assessment of menstrual bleeding",
"Red blood cell parameters and ferritin levels",
"Genotyping",
"Statistical analysis",
"Results",
"Discussion"
] |
[
"Iron deficiency is the most common nutritional disorder worldwide. As well as affecting a large number of children and women in non-industrialized countries, it is the only nutrient deficiency which is also significant prevalent in many industrialized nations 1. Menstruating women have been recognized to be among the most likely individuals to develop iron deficiency anemia (IDA) and its prevalence is 6-22% in developed countries 2-5. Even though menorrhagia serve as a major contributing factor of IDA in this population, it is not uncommon that women with heavy menstrual loss have normal hemoglobin (Hb) levels and adequate iron stores 2. Individual susceptibility to IDA varies. However, whether genetic variants play a role in IDA pathogenesis remains unclear.\nHepcidin is the core of iron metabolism and is tightly regulated by several mediators 6. Matriptase-2 is an important one and down regulates hepcidin expression through cleaving membrane-bound hemojuvelin, which can enhance hepcidin transcription 6. Complete loss of function mutation of matriptase-2 leads to a rare disease, iron-refractory iron deficiency anemia (IRIDA) 7, 8. Patients with IRIDA have high serum hepcidin levels and cannot absorb iron from intestine. It is of interest that whether genetic variants with incomplete loss of function of matriptase-2 will be associated with IDA in general population.\nRecently, several genome-wide association studies identified several single nucleotide polymorphisms (SNPs) that influence blood cell phenotypes 9-13. Among these SNPs, rs855791 has the strongest association with red blood cell indices and iron parameters in general population 12, 13. This SNP is located in the functional part of TMPRSS6 and the rs855791 (2321 C>T) causes a nonsynonymous substitution that reduces the ability of the enzyme to inhibit hepcidin transcription 14. To investigate the role of this genetic variant rs855791 on the susceptibility of IDA in menstruating women, we conducted this case-control study.",
" Study population Female patients with microcytic anemia were screened at hematologic clinics for IDA. IDA was defined as a hemoglobin level < 12 g/dL and a ferritin level < 10 ng/mL 2, 15. The evaluation for IDA included a physical examination, history taking, renal function test, and stool occult blood test. For those without an adequate response to iron replacement (oral elemental iron 300 mg/day for 2-4 weeks), Hb electrophoresis and brilliant cresyl blue staining tests were used to exclude thalassemia. Healthy women with regular menstruation were enrolled as the control group; most of them were staff in our institute. Complete blood counts and ferritin levels were measured and only those with normal Hb and ferritin levels were included in the analysis. An in-person questionnaire was administered to every subject in this study, which included information on menstrual, medical and surgical history. The exclusion criteria for both groups included gastrointestinal bleeding, thalassemia, renal insufficiency, menopause, and history of gastrointestinal resection. This study was performed in accordance with the declaration of Helsinki, and approved by the Ethics Committee of Chang-Gung Memorial Hospital (99-0482B) and informed consent was obtained from all participants.\nFemale patients with microcytic anemia were screened at hematologic clinics for IDA. IDA was defined as a hemoglobin level < 12 g/dL and a ferritin level < 10 ng/mL 2, 15. The evaluation for IDA included a physical examination, history taking, renal function test, and stool occult blood test. For those without an adequate response to iron replacement (oral elemental iron 300 mg/day for 2-4 weeks), Hb electrophoresis and brilliant cresyl blue staining tests were used to exclude thalassemia. Healthy women with regular menstruation were enrolled as the control group; most of them were staff in our institute. Complete blood counts and ferritin levels were measured and only those with normal Hb and ferritin levels were included in the analysis. An in-person questionnaire was administered to every subject in this study, which included information on menstrual, medical and surgical history. The exclusion criteria for both groups included gastrointestinal bleeding, thalassemia, renal insufficiency, menopause, and history of gastrointestinal resection. This study was performed in accordance with the declaration of Helsinki, and approved by the Ethics Committee of Chang-Gung Memorial Hospital (99-0482B) and informed consent was obtained from all participants.\n Assessment of menstrual bleeding Menstrual blood loss was evaluated with the pictorial blood-loss assessment chart (PBAC) 16, a semi-quantitative method used to estimate menstrual amount and has been validated in many studies 17-19. Menorrhagia was defined as a PBAC score of more than 100 during one menstrual period, which corresponds to a blood loss of more than 80 mL 16.\nMenstrual blood loss was evaluated with the pictorial blood-loss assessment chart (PBAC) 16, a semi-quantitative method used to estimate menstrual amount and has been validated in many studies 17-19. Menorrhagia was defined as a PBAC score of more than 100 during one menstrual period, which corresponds to a blood loss of more than 80 mL 16.\n Red blood cell parameters and ferritin levels Red blood cell numbers, Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width and platelet counts were determined by the XE-2100 system (Sysmex Co, Kobe, Japan). Serum ferritin levels were determined using a commercial EIA kit (Advia Centaur, Siemens, NY).\nRed blood cell numbers, Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width and platelet counts were determined by the XE-2100 system (Sysmex Co, Kobe, Japan). Serum ferritin levels were determined using a commercial EIA kit (Advia Centaur, Siemens, NY).\n Genotyping Genomic DNA was extracted from peripheral leukocytes using the Genomic DNA Purification Kit (Gentra Systems, Inc. Chicago, IL, USA). The TMPRSS6 rs855791 C>T polymorphism was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)(Figure 1). The primers used were 5'-tag aga aca ggg gct cca gg-3' (forward) and 5'-atg tgg gca gca tcc ttt c-3' (reverse). The reaction conditions for amplification were as follows: 95°C for 5 min; 30 cycles of 95°C for 45 s, 63°C for 45 s, and 72°C for 45 s; and a final extension at 72°C for 5 min. Following amplification, the PCR products (249 bp) were digested with the restriction endonuclease Stu I (New England Biolabs, Inc. Hitchin, Herts, UK). Genotype was determined by fragment size, and 10% of the samples were directly sequenced to confirm the genotyping results.\nGenomic DNA was extracted from peripheral leukocytes using the Genomic DNA Purification Kit (Gentra Systems, Inc. Chicago, IL, USA). The TMPRSS6 rs855791 C>T polymorphism was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)(Figure 1). The primers used were 5'-tag aga aca ggg gct cca gg-3' (forward) and 5'-atg tgg gca gca tcc ttt c-3' (reverse). The reaction conditions for amplification were as follows: 95°C for 5 min; 30 cycles of 95°C for 45 s, 63°C for 45 s, and 72°C for 45 s; and a final extension at 72°C for 5 min. Following amplification, the PCR products (249 bp) were digested with the restriction endonuclease Stu I (New England Biolabs, Inc. Hitchin, Herts, UK). Genotype was determined by fragment size, and 10% of the samples were directly sequenced to confirm the genotyping results.\n Statistical analysis Continuous variables are presented as mean ± standard deviation; categorical variables are presented as numbers and percentages. Categorical variables were analyzed by Chi-square or Fisher's exact tests. Student's t-test was used to examine continuous variables with normal distributions. Linear correlation and regression was used to evaluate the correlations between age, Hb levels and menstrual amounts. Odds ratio (OR) and confidence interval (CI) were tested by binary logistic regression. A p-value of less than 0.05 was considered statistically significant.\nContinuous variables are presented as mean ± standard deviation; categorical variables are presented as numbers and percentages. Categorical variables were analyzed by Chi-square or Fisher's exact tests. Student's t-test was used to examine continuous variables with normal distributions. Linear correlation and regression was used to evaluate the correlations between age, Hb levels and menstrual amounts. Odds ratio (OR) and confidence interval (CI) were tested by binary logistic regression. A p-value of less than 0.05 was considered statistically significant.",
"Female patients with microcytic anemia were screened at hematologic clinics for IDA. IDA was defined as a hemoglobin level < 12 g/dL and a ferritin level < 10 ng/mL 2, 15. The evaluation for IDA included a physical examination, history taking, renal function test, and stool occult blood test. For those without an adequate response to iron replacement (oral elemental iron 300 mg/day for 2-4 weeks), Hb electrophoresis and brilliant cresyl blue staining tests were used to exclude thalassemia. Healthy women with regular menstruation were enrolled as the control group; most of them were staff in our institute. Complete blood counts and ferritin levels were measured and only those with normal Hb and ferritin levels were included in the analysis. An in-person questionnaire was administered to every subject in this study, which included information on menstrual, medical and surgical history. The exclusion criteria for both groups included gastrointestinal bleeding, thalassemia, renal insufficiency, menopause, and history of gastrointestinal resection. This study was performed in accordance with the declaration of Helsinki, and approved by the Ethics Committee of Chang-Gung Memorial Hospital (99-0482B) and informed consent was obtained from all participants.",
"Menstrual blood loss was evaluated with the pictorial blood-loss assessment chart (PBAC) 16, a semi-quantitative method used to estimate menstrual amount and has been validated in many studies 17-19. Menorrhagia was defined as a PBAC score of more than 100 during one menstrual period, which corresponds to a blood loss of more than 80 mL 16.",
"Red blood cell numbers, Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width and platelet counts were determined by the XE-2100 system (Sysmex Co, Kobe, Japan). Serum ferritin levels were determined using a commercial EIA kit (Advia Centaur, Siemens, NY).",
"Genomic DNA was extracted from peripheral leukocytes using the Genomic DNA Purification Kit (Gentra Systems, Inc. Chicago, IL, USA). The TMPRSS6 rs855791 C>T polymorphism was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)(Figure 1). The primers used were 5'-tag aga aca ggg gct cca gg-3' (forward) and 5'-atg tgg gca gca tcc ttt c-3' (reverse). The reaction conditions for amplification were as follows: 95°C for 5 min; 30 cycles of 95°C for 45 s, 63°C for 45 s, and 72°C for 45 s; and a final extension at 72°C for 5 min. Following amplification, the PCR products (249 bp) were digested with the restriction endonuclease Stu I (New England Biolabs, Inc. Hitchin, Herts, UK). Genotype was determined by fragment size, and 10% of the samples were directly sequenced to confirm the genotyping results.",
"Continuous variables are presented as mean ± standard deviation; categorical variables are presented as numbers and percentages. Categorical variables were analyzed by Chi-square or Fisher's exact tests. Student's t-test was used to examine continuous variables with normal distributions. Linear correlation and regression was used to evaluate the correlations between age, Hb levels and menstrual amounts. Odds ratio (OR) and confidence interval (CI) were tested by binary logistic regression. A p-value of less than 0.05 was considered statistically significant.",
"A total of 67 IDA subjects and 107 healthy women were eligible for analysis. Characteristics of both groups are shown in Table 1. IDA patients were significantly older than control subjects (39.5 ± 9.2 vs. 32.7 ± 6.2 years; p < 0.001). All red blood cell indices and platelet counts differed significantly between two groups (Table 1). PBAC scores were not available for 2 IDA subjects. The PBAC scores of the 65 IDA patients were significantly higher than those of the 107 healthy controls (362.5 ± 352.4 vs. 185.2 ± 193.6; p < 0.001). Menorrhagia (PBAC score > 100) was more common in IDA patients than in the control group (80.0% vs. 65.4%, p = 0.04).\nThe frequency distribution of the rs855791 TT, TC, and CC genotypes in both groups showed borderline difference (p = 0.06; Table 2). However, the proportion of the C homozygotes was significantly lower in IDA patients (11.9% vs. 25.2%, p = 0.03; Figure 2). The odds ratio (OR) of C homozygotes having IDA versus those with TX (TT and TC) genotypes having IDA was 0.4 (95% CI, 0.17 - 0.95, p = 0.04). The C allele frequencies in the IDA and control group did not differ (43.3% vs. 48.6%, p= 0.33).\nWe further performed subgroup analysis based on menorrhagia (PBAC score > 100). For menorrhagic subjects, the frequency distribution of the 3 genotypes of the rs855791 SNP differed significantly in both groups (p = 0.02; Table 3). The frequency of the CC genotype was much lower in the IDA group (9.6% vs. 28.6%, p = 0.01, Figure 3a). Menorrhagic women with homozygous C genotype have lower risk to have IDA than those with TX genotypes (OR = 0.27, 95% CI, 0.09-0.77, p = 0.01). However, for those without menorrhagia, no difference between IDA and healthy women was observed in the frequency distribution of TT, TC, and CC genotypes (p = 0.91; Table 3) or of the CC and TX genotypes (OR = 0.78, 95% CI, 0.14-4.34, p = 0.78; Figure 3b). The frequency of the C allele did not differ between the groups whether or not the study subjects had menorrhagia (for menorrhagic subjects, 42.3% vs. 50.7%, p = 0.19; for non-menorrhagic subjects, 46.2% vs. 44.6%, p = 0.89).\nThen, we pooled data from both groups, and analyzed the correlation between Hb levels and menstrual blood loss (PBAC scores). For women with genotypes TX, there was a strong inverse correlation between Hb levels and menstrual amounts; the equation used was Hb = 11.95 - 0.003 × PBAC score (R2 = 0.135, p < 0.001). In contrast, the correlation was not statistically significant for those with C homozygotes (R2 = 0.062, p = 0.15). The results are also shown in Figure 4. Because subjects in the IDA group were older and had more menstrual blood loss than the control group, we therefore analyzed the correlation between age and PBAC scores in all subjects. We found that older women had more menstrual blood loss (p = 0.002, data not shown). This result explained the difference in the age distribution between two groups.\nFinally, we investigated the influence of rs855791 on RBC parameters, ferritin levels, and log(ferritin) value based on the control group data. However, we did not find a difference between the genotypes in our study (data not shown).",
"In this study, we evaluated whether the TMPRSS6 rs855791 polymorphism influencing IDA susceptibility in menstruating women. There were fewer women with CC genotype in the IDA group and this difference became more obvious when the analysis was confined to those with menorrhagia. This result suggested that rs855791 C homozygotes have a lower risk to have IDA, especially those with menorrhagia. Then, we explored the influence of this SNP on menstrual amount effect to IDA. For women with genotypes TX, Hb levels were inversely correlated with the PBAC scores. This result was compatible with the general concept that more menstrual loss would result in greater iron loss and subsequent IDA. However, this correlation was not significant in the C homozygotes. This provides evidence that rs855791 C homozygotes serve a genetic protective factor against IDA. For the first time, we demonstrate that the higher ferritin levels of 736A homozygotes in the general population will transform into the lower susceptibility to IDA in women, especially for those with menorrhagia 13.\nIn our study, menorrhagia is still the major cause of IDA in reproductive-age women 2, 20. Menstrual amount increased with age and the mean age of IDA group was older than control group; these phenomena were in line with previous reports the prevalence of menorrhagia increases with age, as does the risk of IDA 21. However, the association between menstrual blood loss and IDA was no more significant for women with CC genotype. Addition to well-known risks contributing to IDA in this population, such as vegetarian and gastrointestinal bleeding, our result demonstrates that a genetic factor can also affect the susceptibility of menorrhagia-associated IDA.\nOur study also provided the first report of rs855791 allelic frequency in Taiwan, located in East Asia. The C allele frequency in our control group was 48.6% (104/214) which is similar to that in European epidemiologic studies (approximately 50%) and higher than that in reported Asian countries (39-47%) 22. However, the C allele frequency is reported to be 82 to 93% in African population; this high prevalence may confer an advantage of enhanced iron absorption during food shortages or provide some protection against parasites that invade RBCs, such as malaria 22.\nThe SNP rs855791 locates in the catalytic domain of matriptase-2. Nai et al. recently showed that the rs855791 C genotype inhibits hepcidin more efficiently than the T genotype in vitro and then confirmed that C homozygous individuals have lower serum hepcidin levels and higher transferrin saturation than those with T homozygotes in the general population 22. However, they excluded iron-deficient subjects, a population of great interest to us. Very recently, a population-based study showed the TMPRSS6 genetic variants, including rs855791, are associated with IDA susceptibility in elderly women 23. The OR of the protective allele was relative insignificant (OR, 0.56; 95% CI, 0.51 - 0.63) based on their large sample size (n = 2,139); and the reason may be that most of their study population was post-menopausal and at relatively low risk for IDA. In our study, we selected a population at high-risk for IDA, that is, menstruating women. With the enhancement of menstrual blood loss, the role of this SNP would be highlighted. Furthermore, most of the subjects in our study (>98%) have PBAC scores and these data allowed us to perform additional analysis according to menstrual amount. Then, we could clarify the protective role of this polymorphism in menorrhagic women.\nWe did not measure hepcidin levels in our subjects. Therefore, we could not analyze the correlation between rs855791 and hepcidin levels. According to Nai's study, C homozygotes have lower hepcidin levels than T homozygotes in the general population 22. We speculate that the protective role of C homozygous rs855791 in our study is due to decreased hepcidin levels. Besides, because mammals lack a regulated pathway for iron excretion 6, it is likely that women with the CC genotype increase the iron absorption from the intestine through decreased hepcidin production and then can offset the menstrual losses. From clinical aspect, iron replacement till 3 to 6 months after relieving anemia is recommended 15. Then, how to prevent relapse may be determined by this genetic variant. For those with CC genotypes, diet modification may be enough to keep iron balance; otherwise, long-term iron replacement till menopause may be considered.\nIn conclusion, our data showed that TMPRSS6 rs855791 CC genotype is less frequent in reproductive-age women with IDA than in healthy women. Menorrhagia is the major risk factor for IDA in menstruating women with TX genotypes, but not for those with CC genotype. Homozygosity for rs855791 C genotype plays a protective role against IDA, especially for those with menorrhagia."
] |
[
"intro",
"materials|methods",
null,
null,
null,
null,
null,
"results",
"discussion"
] |
[
"iron deficiency anemia",
"menorrhagia",
"single nucleotide polymorphism",
"rs855791",
"transmembrane protease serine 6 (TMPRSS6)"
] |
Introduction:
Iron deficiency is the most common nutritional disorder worldwide. As well as affecting a large number of children and women in non-industrialized countries, it is the only nutrient deficiency which is also significant prevalent in many industrialized nations 1. Menstruating women have been recognized to be among the most likely individuals to develop iron deficiency anemia (IDA) and its prevalence is 6-22% in developed countries 2-5. Even though menorrhagia serve as a major contributing factor of IDA in this population, it is not uncommon that women with heavy menstrual loss have normal hemoglobin (Hb) levels and adequate iron stores 2. Individual susceptibility to IDA varies. However, whether genetic variants play a role in IDA pathogenesis remains unclear.
Hepcidin is the core of iron metabolism and is tightly regulated by several mediators 6. Matriptase-2 is an important one and down regulates hepcidin expression through cleaving membrane-bound hemojuvelin, which can enhance hepcidin transcription 6. Complete loss of function mutation of matriptase-2 leads to a rare disease, iron-refractory iron deficiency anemia (IRIDA) 7, 8. Patients with IRIDA have high serum hepcidin levels and cannot absorb iron from intestine. It is of interest that whether genetic variants with incomplete loss of function of matriptase-2 will be associated with IDA in general population.
Recently, several genome-wide association studies identified several single nucleotide polymorphisms (SNPs) that influence blood cell phenotypes 9-13. Among these SNPs, rs855791 has the strongest association with red blood cell indices and iron parameters in general population 12, 13. This SNP is located in the functional part of TMPRSS6 and the rs855791 (2321 C>T) causes a nonsynonymous substitution that reduces the ability of the enzyme to inhibit hepcidin transcription 14. To investigate the role of this genetic variant rs855791 on the susceptibility of IDA in menstruating women, we conducted this case-control study.
Materials and Methods:
Study population Female patients with microcytic anemia were screened at hematologic clinics for IDA. IDA was defined as a hemoglobin level < 12 g/dL and a ferritin level < 10 ng/mL 2, 15. The evaluation for IDA included a physical examination, history taking, renal function test, and stool occult blood test. For those without an adequate response to iron replacement (oral elemental iron 300 mg/day for 2-4 weeks), Hb electrophoresis and brilliant cresyl blue staining tests were used to exclude thalassemia. Healthy women with regular menstruation were enrolled as the control group; most of them were staff in our institute. Complete blood counts and ferritin levels were measured and only those with normal Hb and ferritin levels were included in the analysis. An in-person questionnaire was administered to every subject in this study, which included information on menstrual, medical and surgical history. The exclusion criteria for both groups included gastrointestinal bleeding, thalassemia, renal insufficiency, menopause, and history of gastrointestinal resection. This study was performed in accordance with the declaration of Helsinki, and approved by the Ethics Committee of Chang-Gung Memorial Hospital (99-0482B) and informed consent was obtained from all participants.
Female patients with microcytic anemia were screened at hematologic clinics for IDA. IDA was defined as a hemoglobin level < 12 g/dL and a ferritin level < 10 ng/mL 2, 15. The evaluation for IDA included a physical examination, history taking, renal function test, and stool occult blood test. For those without an adequate response to iron replacement (oral elemental iron 300 mg/day for 2-4 weeks), Hb electrophoresis and brilliant cresyl blue staining tests were used to exclude thalassemia. Healthy women with regular menstruation were enrolled as the control group; most of them were staff in our institute. Complete blood counts and ferritin levels were measured and only those with normal Hb and ferritin levels were included in the analysis. An in-person questionnaire was administered to every subject in this study, which included information on menstrual, medical and surgical history. The exclusion criteria for both groups included gastrointestinal bleeding, thalassemia, renal insufficiency, menopause, and history of gastrointestinal resection. This study was performed in accordance with the declaration of Helsinki, and approved by the Ethics Committee of Chang-Gung Memorial Hospital (99-0482B) and informed consent was obtained from all participants.
Assessment of menstrual bleeding Menstrual blood loss was evaluated with the pictorial blood-loss assessment chart (PBAC) 16, a semi-quantitative method used to estimate menstrual amount and has been validated in many studies 17-19. Menorrhagia was defined as a PBAC score of more than 100 during one menstrual period, which corresponds to a blood loss of more than 80 mL 16.
Menstrual blood loss was evaluated with the pictorial blood-loss assessment chart (PBAC) 16, a semi-quantitative method used to estimate menstrual amount and has been validated in many studies 17-19. Menorrhagia was defined as a PBAC score of more than 100 during one menstrual period, which corresponds to a blood loss of more than 80 mL 16.
Red blood cell parameters and ferritin levels Red blood cell numbers, Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width and platelet counts were determined by the XE-2100 system (Sysmex Co, Kobe, Japan). Serum ferritin levels were determined using a commercial EIA kit (Advia Centaur, Siemens, NY).
Red blood cell numbers, Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width and platelet counts were determined by the XE-2100 system (Sysmex Co, Kobe, Japan). Serum ferritin levels were determined using a commercial EIA kit (Advia Centaur, Siemens, NY).
Genotyping Genomic DNA was extracted from peripheral leukocytes using the Genomic DNA Purification Kit (Gentra Systems, Inc. Chicago, IL, USA). The TMPRSS6 rs855791 C>T polymorphism was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)(Figure 1). The primers used were 5'-tag aga aca ggg gct cca gg-3' (forward) and 5'-atg tgg gca gca tcc ttt c-3' (reverse). The reaction conditions for amplification were as follows: 95°C for 5 min; 30 cycles of 95°C for 45 s, 63°C for 45 s, and 72°C for 45 s; and a final extension at 72°C for 5 min. Following amplification, the PCR products (249 bp) were digested with the restriction endonuclease Stu I (New England Biolabs, Inc. Hitchin, Herts, UK). Genotype was determined by fragment size, and 10% of the samples were directly sequenced to confirm the genotyping results.
Genomic DNA was extracted from peripheral leukocytes using the Genomic DNA Purification Kit (Gentra Systems, Inc. Chicago, IL, USA). The TMPRSS6 rs855791 C>T polymorphism was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)(Figure 1). The primers used were 5'-tag aga aca ggg gct cca gg-3' (forward) and 5'-atg tgg gca gca tcc ttt c-3' (reverse). The reaction conditions for amplification were as follows: 95°C for 5 min; 30 cycles of 95°C for 45 s, 63°C for 45 s, and 72°C for 45 s; and a final extension at 72°C for 5 min. Following amplification, the PCR products (249 bp) were digested with the restriction endonuclease Stu I (New England Biolabs, Inc. Hitchin, Herts, UK). Genotype was determined by fragment size, and 10% of the samples were directly sequenced to confirm the genotyping results.
Statistical analysis Continuous variables are presented as mean ± standard deviation; categorical variables are presented as numbers and percentages. Categorical variables were analyzed by Chi-square or Fisher's exact tests. Student's t-test was used to examine continuous variables with normal distributions. Linear correlation and regression was used to evaluate the correlations between age, Hb levels and menstrual amounts. Odds ratio (OR) and confidence interval (CI) were tested by binary logistic regression. A p-value of less than 0.05 was considered statistically significant.
Continuous variables are presented as mean ± standard deviation; categorical variables are presented as numbers and percentages. Categorical variables were analyzed by Chi-square or Fisher's exact tests. Student's t-test was used to examine continuous variables with normal distributions. Linear correlation and regression was used to evaluate the correlations between age, Hb levels and menstrual amounts. Odds ratio (OR) and confidence interval (CI) were tested by binary logistic regression. A p-value of less than 0.05 was considered statistically significant.
Study population:
Female patients with microcytic anemia were screened at hematologic clinics for IDA. IDA was defined as a hemoglobin level < 12 g/dL and a ferritin level < 10 ng/mL 2, 15. The evaluation for IDA included a physical examination, history taking, renal function test, and stool occult blood test. For those without an adequate response to iron replacement (oral elemental iron 300 mg/day for 2-4 weeks), Hb electrophoresis and brilliant cresyl blue staining tests were used to exclude thalassemia. Healthy women with regular menstruation were enrolled as the control group; most of them were staff in our institute. Complete blood counts and ferritin levels were measured and only those with normal Hb and ferritin levels were included in the analysis. An in-person questionnaire was administered to every subject in this study, which included information on menstrual, medical and surgical history. The exclusion criteria for both groups included gastrointestinal bleeding, thalassemia, renal insufficiency, menopause, and history of gastrointestinal resection. This study was performed in accordance with the declaration of Helsinki, and approved by the Ethics Committee of Chang-Gung Memorial Hospital (99-0482B) and informed consent was obtained from all participants.
Assessment of menstrual bleeding:
Menstrual blood loss was evaluated with the pictorial blood-loss assessment chart (PBAC) 16, a semi-quantitative method used to estimate menstrual amount and has been validated in many studies 17-19. Menorrhagia was defined as a PBAC score of more than 100 during one menstrual period, which corresponds to a blood loss of more than 80 mL 16.
Red blood cell parameters and ferritin levels:
Red blood cell numbers, Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width and platelet counts were determined by the XE-2100 system (Sysmex Co, Kobe, Japan). Serum ferritin levels were determined using a commercial EIA kit (Advia Centaur, Siemens, NY).
Genotyping:
Genomic DNA was extracted from peripheral leukocytes using the Genomic DNA Purification Kit (Gentra Systems, Inc. Chicago, IL, USA). The TMPRSS6 rs855791 C>T polymorphism was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)(Figure 1). The primers used were 5'-tag aga aca ggg gct cca gg-3' (forward) and 5'-atg tgg gca gca tcc ttt c-3' (reverse). The reaction conditions for amplification were as follows: 95°C for 5 min; 30 cycles of 95°C for 45 s, 63°C for 45 s, and 72°C for 45 s; and a final extension at 72°C for 5 min. Following amplification, the PCR products (249 bp) were digested with the restriction endonuclease Stu I (New England Biolabs, Inc. Hitchin, Herts, UK). Genotype was determined by fragment size, and 10% of the samples were directly sequenced to confirm the genotyping results.
Statistical analysis:
Continuous variables are presented as mean ± standard deviation; categorical variables are presented as numbers and percentages. Categorical variables were analyzed by Chi-square or Fisher's exact tests. Student's t-test was used to examine continuous variables with normal distributions. Linear correlation and regression was used to evaluate the correlations between age, Hb levels and menstrual amounts. Odds ratio (OR) and confidence interval (CI) were tested by binary logistic regression. A p-value of less than 0.05 was considered statistically significant.
Results:
A total of 67 IDA subjects and 107 healthy women were eligible for analysis. Characteristics of both groups are shown in Table 1. IDA patients were significantly older than control subjects (39.5 ± 9.2 vs. 32.7 ± 6.2 years; p < 0.001). All red blood cell indices and platelet counts differed significantly between two groups (Table 1). PBAC scores were not available for 2 IDA subjects. The PBAC scores of the 65 IDA patients were significantly higher than those of the 107 healthy controls (362.5 ± 352.4 vs. 185.2 ± 193.6; p < 0.001). Menorrhagia (PBAC score > 100) was more common in IDA patients than in the control group (80.0% vs. 65.4%, p = 0.04).
The frequency distribution of the rs855791 TT, TC, and CC genotypes in both groups showed borderline difference (p = 0.06; Table 2). However, the proportion of the C homozygotes was significantly lower in IDA patients (11.9% vs. 25.2%, p = 0.03; Figure 2). The odds ratio (OR) of C homozygotes having IDA versus those with TX (TT and TC) genotypes having IDA was 0.4 (95% CI, 0.17 - 0.95, p = 0.04). The C allele frequencies in the IDA and control group did not differ (43.3% vs. 48.6%, p= 0.33).
We further performed subgroup analysis based on menorrhagia (PBAC score > 100). For menorrhagic subjects, the frequency distribution of the 3 genotypes of the rs855791 SNP differed significantly in both groups (p = 0.02; Table 3). The frequency of the CC genotype was much lower in the IDA group (9.6% vs. 28.6%, p = 0.01, Figure 3a). Menorrhagic women with homozygous C genotype have lower risk to have IDA than those with TX genotypes (OR = 0.27, 95% CI, 0.09-0.77, p = 0.01). However, for those without menorrhagia, no difference between IDA and healthy women was observed in the frequency distribution of TT, TC, and CC genotypes (p = 0.91; Table 3) or of the CC and TX genotypes (OR = 0.78, 95% CI, 0.14-4.34, p = 0.78; Figure 3b). The frequency of the C allele did not differ between the groups whether or not the study subjects had menorrhagia (for menorrhagic subjects, 42.3% vs. 50.7%, p = 0.19; for non-menorrhagic subjects, 46.2% vs. 44.6%, p = 0.89).
Then, we pooled data from both groups, and analyzed the correlation between Hb levels and menstrual blood loss (PBAC scores). For women with genotypes TX, there was a strong inverse correlation between Hb levels and menstrual amounts; the equation used was Hb = 11.95 - 0.003 × PBAC score (R2 = 0.135, p < 0.001). In contrast, the correlation was not statistically significant for those with C homozygotes (R2 = 0.062, p = 0.15). The results are also shown in Figure 4. Because subjects in the IDA group were older and had more menstrual blood loss than the control group, we therefore analyzed the correlation between age and PBAC scores in all subjects. We found that older women had more menstrual blood loss (p = 0.002, data not shown). This result explained the difference in the age distribution between two groups.
Finally, we investigated the influence of rs855791 on RBC parameters, ferritin levels, and log(ferritin) value based on the control group data. However, we did not find a difference between the genotypes in our study (data not shown).
Discussion:
In this study, we evaluated whether the TMPRSS6 rs855791 polymorphism influencing IDA susceptibility in menstruating women. There were fewer women with CC genotype in the IDA group and this difference became more obvious when the analysis was confined to those with menorrhagia. This result suggested that rs855791 C homozygotes have a lower risk to have IDA, especially those with menorrhagia. Then, we explored the influence of this SNP on menstrual amount effect to IDA. For women with genotypes TX, Hb levels were inversely correlated with the PBAC scores. This result was compatible with the general concept that more menstrual loss would result in greater iron loss and subsequent IDA. However, this correlation was not significant in the C homozygotes. This provides evidence that rs855791 C homozygotes serve a genetic protective factor against IDA. For the first time, we demonstrate that the higher ferritin levels of 736A homozygotes in the general population will transform into the lower susceptibility to IDA in women, especially for those with menorrhagia 13.
In our study, menorrhagia is still the major cause of IDA in reproductive-age women 2, 20. Menstrual amount increased with age and the mean age of IDA group was older than control group; these phenomena were in line with previous reports the prevalence of menorrhagia increases with age, as does the risk of IDA 21. However, the association between menstrual blood loss and IDA was no more significant for women with CC genotype. Addition to well-known risks contributing to IDA in this population, such as vegetarian and gastrointestinal bleeding, our result demonstrates that a genetic factor can also affect the susceptibility of menorrhagia-associated IDA.
Our study also provided the first report of rs855791 allelic frequency in Taiwan, located in East Asia. The C allele frequency in our control group was 48.6% (104/214) which is similar to that in European epidemiologic studies (approximately 50%) and higher than that in reported Asian countries (39-47%) 22. However, the C allele frequency is reported to be 82 to 93% in African population; this high prevalence may confer an advantage of enhanced iron absorption during food shortages or provide some protection against parasites that invade RBCs, such as malaria 22.
The SNP rs855791 locates in the catalytic domain of matriptase-2. Nai et al. recently showed that the rs855791 C genotype inhibits hepcidin more efficiently than the T genotype in vitro and then confirmed that C homozygous individuals have lower serum hepcidin levels and higher transferrin saturation than those with T homozygotes in the general population 22. However, they excluded iron-deficient subjects, a population of great interest to us. Very recently, a population-based study showed the TMPRSS6 genetic variants, including rs855791, are associated with IDA susceptibility in elderly women 23. The OR of the protective allele was relative insignificant (OR, 0.56; 95% CI, 0.51 - 0.63) based on their large sample size (n = 2,139); and the reason may be that most of their study population was post-menopausal and at relatively low risk for IDA. In our study, we selected a population at high-risk for IDA, that is, menstruating women. With the enhancement of menstrual blood loss, the role of this SNP would be highlighted. Furthermore, most of the subjects in our study (>98%) have PBAC scores and these data allowed us to perform additional analysis according to menstrual amount. Then, we could clarify the protective role of this polymorphism in menorrhagic women.
We did not measure hepcidin levels in our subjects. Therefore, we could not analyze the correlation between rs855791 and hepcidin levels. According to Nai's study, C homozygotes have lower hepcidin levels than T homozygotes in the general population 22. We speculate that the protective role of C homozygous rs855791 in our study is due to decreased hepcidin levels. Besides, because mammals lack a regulated pathway for iron excretion 6, it is likely that women with the CC genotype increase the iron absorption from the intestine through decreased hepcidin production and then can offset the menstrual losses. From clinical aspect, iron replacement till 3 to 6 months after relieving anemia is recommended 15. Then, how to prevent relapse may be determined by this genetic variant. For those with CC genotypes, diet modification may be enough to keep iron balance; otherwise, long-term iron replacement till menopause may be considered.
In conclusion, our data showed that TMPRSS6 rs855791 CC genotype is less frequent in reproductive-age women with IDA than in healthy women. Menorrhagia is the major risk factor for IDA in menstruating women with TX genotypes, but not for those with CC genotype. Homozygosity for rs855791 C genotype plays a protective role against IDA, especially for those with menorrhagia.
|
Background:
Genome-wide-association studies have identified the TMPRSS6 polymorphism rs855791 has the strongest association with red blood cell indices or iron parameters in general population. Whether this genetic variant influences the susceptibility of iron deficiency anemia (IDA) in women with menstruation has not been well studied.
Methods:
In this case-control study, we enrolled 67 women with IDA and 107 healthy volunteers, and analyzed their complete blood counts, rs855791 genotypes, and menstrual amounts. Menstrual blood loss was evaluated with a pictorial blood-loss assessment chart.
Results:
There were significantly fewer rs855791 C homozygotes in the IDA group than in the healthy group (11.9% vs. 25.2%, p = 0.03). The odds ratio (OR) of C homozygotes having IDA versus non-CC subjects having IDA was 0.4 (95% CI, 0.17 - 0.95, p = 0.04). When the analysis was confined to study subjects with menorrhagia, this difference became more prominent (9.6% vs. 28.6%, p = 0.01; OR, 0.27, 95% CI, 0.09 - 0.77, p = 0.01). For women with non-CC genotypes, there was an inverse correlation between hemoglobin levels and menstrual loss (p < 0.001); however, this association was not found for those with genotypes CC (p = 0.15).
Conclusions:
Our study suggests homozygosity for TMPRSS6 rs855791 C genotype has a protective role against IDA in women at reproductive age, especially in those with menorrhagia.
| null | null | 3,954 | 289 | 9 |
[
"ida",
"menstrual",
"blood",
"levels",
"women",
"iron",
"study",
"rs855791",
"loss",
"hb"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] iron deficiency anemia | menorrhagia | single nucleotide polymorphism | rs855791 | transmembrane protease serine 6 (TMPRSS6) [SUMMARY]
| null |
[CONTENT] iron deficiency anemia | menorrhagia | single nucleotide polymorphism | rs855791 | transmembrane protease serine 6 (TMPRSS6) [SUMMARY]
| null |
[CONTENT] iron deficiency anemia | menorrhagia | single nucleotide polymorphism | rs855791 | transmembrane protease serine 6 (TMPRSS6) [SUMMARY]
| null |
[CONTENT] Adult | Anemia, Iron-Deficiency | Case-Control Studies | Female | Genetic Association Studies | Genetic Predisposition to Disease | Genotype | Humans | Membrane Proteins | Menorrhagia | Menstruation | Middle Aged | Risk Factors | Serine Endopeptidases [SUMMARY]
| null |
[CONTENT] Adult | Anemia, Iron-Deficiency | Case-Control Studies | Female | Genetic Association Studies | Genetic Predisposition to Disease | Genotype | Humans | Membrane Proteins | Menorrhagia | Menstruation | Middle Aged | Risk Factors | Serine Endopeptidases [SUMMARY]
| null |
[CONTENT] Adult | Anemia, Iron-Deficiency | Case-Control Studies | Female | Genetic Association Studies | Genetic Predisposition to Disease | Genotype | Humans | Membrane Proteins | Menorrhagia | Menstruation | Middle Aged | Risk Factors | Serine Endopeptidases [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] ida | menstrual | blood | levels | women | iron | study | rs855791 | loss | hb [SUMMARY]
| null |
[CONTENT] ida | menstrual | blood | levels | women | iron | study | rs855791 | loss | hb [SUMMARY]
| null |
[CONTENT] ida | menstrual | blood | levels | women | iron | study | rs855791 | loss | hb [SUMMARY]
| null |
[CONTENT] iron | hepcidin | deficiency | ida | iron deficiency | genetic | matriptase | women | population | snps [SUMMARY]
| null |
[CONTENT] vs | subjects | ida | genotypes | groups | table | significantly | pbac | frequency | ida patients [SUMMARY]
| null |
[CONTENT] ida | blood | menstrual | iron | loss | variables | women | blood loss | levels | mean [SUMMARY]
| null |
[CONTENT] TMPRSS6 ||| IDA [SUMMARY]
| null |
[CONTENT] rs855791 C | IDA | 11.9% | 25.2% | 0.03 ||| IDA | non-CC | IDA | 0.4 | 95% | CI | 0.17 | 0.04 ||| 9.6% | 28.6% | 0.01 | 0.27 | 95% | CI | 0.09 | 0.01 ||| CC | 0.15 [SUMMARY]
| null |
[CONTENT] TMPRSS6 ||| IDA ||| 67 | IDA | 107 ||| ||| rs855791 C | IDA | 11.9% | 25.2% | 0.03 ||| IDA | non-CC | IDA | 0.4 | 95% | CI | 0.17 | 0.04 ||| 9.6% | 28.6% | 0.01 | 0.27 | 95% | CI | 0.09 | 0.01 ||| CC | 0.15 ||| TMPRSS6 rs855791 C | IDA [SUMMARY]
| null |
|||
[Robot-assisted surgery as an elective-fascinating lesson(s)?]
|
35037971
|
Even though robot-assisted operations have evolved to a standard procedure in surgery, they are underrepresented in the curriculum of current medical students.
|
BACKGROUND
|
Ten undergraduates in their final years were taught the theoretical basics and practical skills in robot-assisted surgery within six lessons each lasting 2 h, including the opportunity to observe a live robot-assisted surgery. The increase of knowledge (ten multiple-choice questions) and skills (exercises Camera 0, Clutch, and Sea Spikes 1) on a robotic simulation device were quantified including an evaluation of the student's perspective.
|
MATERIALS AND METHODS
|
The 10 participants had a significant increase in knowledge and gave at a median of 3.5 additional correct answers in the final assessment (p = 0.011). For two out of three practical exercises, the overall score significantly increased (Camera 0 and Sea Spikes 1, for both p < 0.05), but for the exercise "Clutch", only economy of motion significantly improved (p = 0.028). The elective was evaluated (very) good and the willingness of the participants to become urologists significantly increased (p = 0.007).
|
RESULTS
|
There is a great interest of many undergraduate medical students in robot-assisted surgery. Offering an elective appears to be an excellent format to teach the theoretical background and practical skills in robotic (urologic) surgery. Moreover, such an elective could raise more attention to the field of urology and might attract future colleagues.
|
CONCLUSION
|
[
"Clinical Competence",
"Curriculum",
"Humans",
"Robotic Surgical Procedures",
"Robotics",
"Urology"
] |
9005389
| null | null | null | null | null | null |
Fazit für die Praxis
|
Vor dem Hintergrund der weiten Verbreitung roboterassistierter Operationen ist die robotische Chirurgie im Lehrplan heutiger Medizinstudierender unterrepräsentiert.Es besteht offensichtlich ein Bedarf von studentischer Seite, sich entsprechend zu bilden.Ein Wahlfach „Robotische Chirurgie“ erscheint in diesem Zusammenhang als geeignetes Format, theoretische Grundlagen und praktische Fertigkeiten in der robotischen (urologischen) Chirurgie zu vermitteln.Organisatorisch bietet sich die Aufteilung in einen Theorie-, mehrere Praxisteile und eine Hospitation an.Die Gruppen sollten nicht zu groß sein, insbesondere im Praxisteil maximal 5 Personen umfassen.Jenseits der edukativen Perspektive kann ein solches Wahlfach auf die faszinierende Vielfältigkeit der Urologie aufmerksam machen und möglicherweise auch neue Kolleginnen und Kollegen gewinnen.
Vor dem Hintergrund der weiten Verbreitung roboterassistierter Operationen ist die robotische Chirurgie im Lehrplan heutiger Medizinstudierender unterrepräsentiert.
Es besteht offensichtlich ein Bedarf von studentischer Seite, sich entsprechend zu bilden.
Ein Wahlfach „Robotische Chirurgie“ erscheint in diesem Zusammenhang als geeignetes Format, theoretische Grundlagen und praktische Fertigkeiten in der robotischen (urologischen) Chirurgie zu vermitteln.
Organisatorisch bietet sich die Aufteilung in einen Theorie-, mehrere Praxisteile und eine Hospitation an.
Die Gruppen sollten nicht zu groß sein, insbesondere im Praxisteil maximal 5 Personen umfassen.
Jenseits der edukativen Perspektive kann ein solches Wahlfach auf die faszinierende Vielfältigkeit der Urologie aufmerksam machen und möglicherweise auch neue Kolleginnen und Kollegen gewinnen.
|
[
"Einleitung",
"Methodik",
"Ergebnisse",
"Diskussion",
"Ausblick",
"Infobox 1 Mehr Informationen zum Thema",
""
] |
[
"Seit der Food and Drug (FDA)-Zulassung des DaVinci®-Operationssystems im Jahr 2000 hat sich die robotische Chirurgie als Standardverfahren in der Urologie und in vielen weiteren operativen Fächern etabliert. Auch wenn eine signifikante Zahl (mittel)großer Kliniken heute roboterassistierte Operationen anbietet, spielt die robotische Chirurgie im Lehrplan von Medizinstudenten nur eine untergeordnete Rolle.\nDemgegenüber existieren bereits diverse strukturierte Curricula und Trainingsprogramme für angehende robotische Operateure, zu denen u. a. das 6‑monatige Deutsche Robotercurriculum der Deutschen Gesellschaft für Robotische Urologie (DRGU) oder als europäisches Pendant das ERUS (European Association of Urology Robotic Urology Section) Robotic Curriculum zählen [3]. Diesen Programmen liegt die Erkenntnis zugrunde, dass ein strukturiertes Training insbesondere unter Einbeziehung von Simulatortraining in kürzerer Zeit einen größeren Lernerfolg mit besseren operativen Ergebnissen ermöglicht [14]. Trainingsprogramme für den in der Regel unerlässlichen Bedside-Assistenten sind schon deutlich seltener, auch wenn gezeigt werden konnte, dass dessen Erfahrung bei komplexen robotischen Operateuren ebenso einen signifikanten Einfluss auf das operative Ergebnis hat [2, 23]. Prinzipiell könnte es noch sinnvoller sein, mit einer strukturierten Ausbildung in der robotischen Chirurgie bereits im Medizinstudium zu beginnen. Einerseits sind robotische Operationen aus dem klinischen Alltag in vielen urologischen Kliniken nicht mehr wegzudenken und sollten deswegen Bestandteil der urologischen Lehre sein. Andererseits könnte ein solches Programm auch dazu dienen, aus berufspolitischer Sicht vermehrtes Interesse an der Urologie zu erzeugen und talentierte zukünftige Kolleginnen und Kollegen frühzeitig zu gewinnen [16, 21].\nUnsere Klinik verfügt über zwei DaVinci®-Operationssysteme vom Typ „X“ und einen DaVinci® SimNow ®-Simulator (Intuitive Surgical, Sunnyvale, CA, USA), der ein virtuelles Üben und Operieren an der Operationskonsole ermöglicht. Um Erfahrungen in der Implementierung der robotischen Chirurgie in die urologische Lehre zu sammeln, haben wir kürzlich erstmalig für eine urologische Universitätsklinik in Deutschland das Wahlfach „Robotische Chirurgie“ in einer Kleingruppe von 10 Studierenden aus den klinischen Semestern organisiert. In 6 Treffen à 2 h wurden theoretische Grundlagen und praktische Fertigkeiten in der robotischen Chirurgie vermittelt inklusive einer Hospitation während einer roboterassistierten Operation. Der Zuwachs an Wissen und Fertigkeiten wurde quantifiziert und die studentische Einschätzung evaluiert.",
"Nach Verzögerungen aufgrund der Coronapandemie konnte der erste Termin unter strenger Einhaltung der coronabedingten Vorsichtsmaßnahmen (Abstandsregeln, Tragen eines medizinischen Mund-Nase-Schutz, Nachweis einer abgeschlossenen Impfung, durchgemachten Infektion oder tagesaktueller negativer Schnelltest) auf Mitte Juni 2021 festgesetzt wurde. Alle Treffen fanden im leeren robotischen Operationssaal der Klinik für Urologie und Kinderurologie statt.\nDer Ablauf des Wahlfachs ist in Abb. 1 dargestellt. Während des Eingangstestats am ersten Treffen wurde das Vorwissen der Studierenden in einer theoretischen Prüfung (10 Multiple-choice-Fragen, s. Infobox 1) erhoben. Zudem erfolgte die Messung der praktischen Fähigkeiten mit drei verschiedenen Übungen an der Konsole eines DaVinci®-X-Systems mit angedocktem SimNow®-Simulator, bewusst mit nur sehr kurzer vorheriger Einführung (Clutch: Verwendung der Kupplung, Camera 0: Steuerung der Kamera, Sea Spikes 1: Bedienung der Greifarme). Im zweiten Treffen erfolgte online die Vermittlung der theoretischen Grundlagen zur Historie robotischer Chirurgie, eine ausführliche Einführung in das robotische Operationssystem inklusive verschiedener Docking-Strategien und ein kritischer Vergleich von robotischem, laparoskopischen und offenen Operieren. Zudem wurden Besonderheiten der Robotik, wie die Notwendigkeit einer guten Kommunikation zwischen Bedside-Assistent und Operateur besprochen und gemeinsam eine vorher aufgezeichnete robotische radikale Prostatektomie analysiert. An Treffen 3 und 4 lag der Schwerpunkt auf einem möglichst großen Praxisanteil mit Einweisung in den Umgang mit dem Patientenwagen, Feinheiten der Trokareinlage und weiteren Übungen am Modell sowie Simulator. Eine Hospitation als 5. Bestandteil des Moduls wurde individuell in Zweiergruppen vereinbart. Während des letzten 6.Treffens wurden zur Projekteffektemessung dieselbe theoretische und praktische Prüfung wiederholt und eine anonyme Evaluation durchgeführt. Eine individuelle Bewertung der Teilnehmenden entfiel, weil es sich um ein rein freiwilliges Wahlfach handelte, für das keine Benotung vorgesehen ist.\nDie statistische Auswertung erfolgte mit SPSS (IBM, Armonk, USA). Häufigkeiten wurden als Median (Range) angegeben, die verbundenen Stichproben mit dem Wilcoxon-Test verglichen, p < 0,05 galt als statistisch signifikant. Die Durchführung des Wahlfachs erfolgte im Rahmen des Teach-the-teacher-Kurses des Universitätsklinikums des Saarlandes und wurde durch den Studiendekan genehmigt.",
"Bei mehr als 30 Anfragen wurde das Wahlfach mit 6 Studentinnen und 4 Studenten aus dem 6. bis 10. Semester begonnen, die Teilnehmerauswahl erfolgte nach dem Prinzip „first come, first serve“. Die durchschnittliche Teilnahmequote betrug 83 %, wobei bisher noch nicht alle individuellen Hospitationen stattgefunden haben. Ein Teilnehmender hatte zuvor ein Pflegepraktikum in der Urologie absolviert und 2 weitere eine Famulatur in einer urologischen Klinik.\nDie praktischen Treffen bedurften einer guten Planung und Abstimmung innerhalb der Abteilung wie interdisziplinär mit der Anästhesie, weil sichergestellt werden musste, dass ein bestimmter OP-Saal von 17 bis 19 Uhr trotz etwaiger Notoperationen frei sein musste. Um die Wartezeiten zu verkürzen, bis die Teilnehmenden wieder am Simulator üben können, wurde die Gruppe ab dem 3. Treffen zweigeteilt und dann in noch kleineren Teams mit 5 Teilnehmenden je 1 h unterrichtet.\nIm theoretischen Ein- und Ausgangstestat ließ sich ein signifikanter Wissenszuwachs aller Teilnehmender feststellen. Bei anfänglich im Median 6 (Range 4–8) richtigen Antworten wurden im Ausgangstestat 9,5 (9–10) und damit signifikant mehr Fragen korrekt beantwortet (p = 0,011, s. Abb. 2). Im Praxisteil wurden in den Übungen „Camera 0“ und „Sea Spikes 1“ am Ende ebenfalls signifikant höhere Punktwerte erzielt (p = 0,036 und p = 0,018). Bei der Übung „Clutch“ ließ sich keine signifikante Veränderung in der Gesamtpunktzahl feststellen. Bereits im Eingangstestat wurden hier im Median 98 (Range 74–100) von 100 möglichen Punkten erreicht, am Ende des Wahlfachs 100 (83–100) Punkte. Trotzdem lag auch bei dieser Übung eine signifikant verbesserte Bewegungsökonomie vor, da die mediane Bewegungsstrecke von 214,8 cm (Range 166,1–379,5) auf 166,9 cm (160,6–212,5) abnahm (p = 0,028).\nAcht von 10 Studierenden nahmen an der Evaluation teil und bewerteten das Modul als (sehr) gut (s. Tab. 1). Im Freitext wurde als besonders positiv hervorgehoben, dass sich in einer Kleingruppe mit hohem Praxisanteil viel Zeit für Fragen genommen wurde. Es wurde vorgeschlagen, noch einen zweiten Teil für „Fortgeschrittene“ anzubieten und die einzelnen Treffen länger zu gestalten, damit noch mehr Übungszeit an der Konsole besteht.123456xMedWie bewerten Sie das Eingangstestat?7–––––1,0Wie bewerten Sie den Theorieteil?34––––1,5Wie bewerten Sie den dritten Praxisteil?7–––––1,0Wie bewerten Sie den vierten Praxisteil?61––––1,14Wie bewerten Sie die Hospitation?4–––––1,0Wie bewerten Sie das Ausgangstestat?7–––––1,0Wie bewerten Sie das Wahlfach insgesamt?8–––––1,0Wie bewerten Sie die Organisation des Wahlfachs?8–––––1,0Wie bewerten Sie den didaktischen Aufbau?8–––––1,0Wie bewerten Sie die Motivation des Dozenten?8–––––1,0\nZum Zeitpunkt des Wahlfachs befand sich noch kein Teilnehmender im praktischen Jahr (PJ), einer hatte schon vorher beabsichtigt, dies in der Urologie abzuleisten. Nach Abschluss des Wahlfachs haben 4 Studierende ihr PJ fest ohne Urologie geplant, wobei zumindest ein Teilnehmender dies möglicherweise noch ändern möchte. Bei 4 weiteren Studierenden zählt Urologie mit zur engeren Auswahl.\nAm Ende des Wahlfachs konnten sich die Studierenden deutlich besser vorstellen, später als Urologe/in tätig zu werden (p = 0,007, s. Abb. 3). Auch wenn die Bereitschaft, später als Chirurg/in zu arbeiten, ebenfalls zunahm, war dieser Unterschied nicht statistisch signifikant (p = 0,066).",
"Roboterassistierte Operationen sind heute nicht mehr aus dem Alltag vieler urologischer Kliniken wegzudenken. Im Jahr 2018 wurden in Deutschland von insgesamt 11.492 radikalen Prostatektomien 39,1 % robotisch und damit fast doppelt so viele wie offen (2253, entsprechend 19,6 %) durchgeführt. Der Anteil laparoskopischer radikaler Prostatektomien war mit 41,3 % nur knapp höher [7]. Bei den Nierenteilresektionen im Jahr 2019 in Deutschland wurden demgegenüber mehr als doppelt so viele Eingriffe robotisch wie laparoskopisch durchgeführt (28,8 % vs. 13 %), und dieser Anteil stieg im Jahr 2020 auf 36,9 % (3577 von 9700 Eingriffen; [24]). Demzufolge sollte die Robotik auch ein fester Bestandteil der (urologischen) Lehre im Medizinstudium sein. Das Medizinstudium befindet sich derzeit mit dem „Masterplan Medizinstudium 2020“ in einem Umbruch und der nationale kompetenzbasierte Lernzielkatalog (NKLM) wurde kürzlich überarbeitet [4, 5]. Dadurch ergeben sich einerseits Chancen für die Urologie, da ein stärkerer Fokus auf die Anwendungsebene im Medizinstudium gelegt wird und viele Prüfungen bspw. im OSCE-Format („objective structured clinical examination“) abgehalten werden sollen [18]. Andererseits wird auch die Digitalisierung in der Medizin einen weiteren Kernaspekt im Medizinstudium spielen, die roboterassistierte Chirurgie ist jedoch weiterhin unterrepräsentiert [22]. Um die urologische Lehre in unserer Klinik um Lehrinhalte der robotischen Chirurgie zu erweitern, haben wir kürzlich deutschlandweit erstmalig in einer urologischen Klinik das Wahlfach „Robotische Chirurgie“ angeboten. In einer Kleingruppe von 10 Studierenden der klinischen Semester wurden in 6 Treffen à 2 h theoretische Inhalte und praktische Fertigkeiten vermittelt. Die Teilnehmenden hatten nicht nur einen signifikanten Zuwachs an Wissen und Fertigkeiten, auch ihre Bereitschaft, später in der Urologie tätig zu werden, stieg messbar an.\nBislang existieren nur wenige strukturierte und evaluierte Lehrkonzepte der robotischen Chirurgie für Studierende. Das 2‑wöchige Curriculum von Mullens et al. ähnelt einem Intensivkurs mit sehr hohem Praxisanteil und einer Vielzahl an Übungen am Simulator, aber auch im Wet Lab sowie einer Assistenz während einer robotischen Operation [13]. Moglia et al. konzipierten demgegenüber ein strukturiertes 4‑stündiges Training sowohl am Laparoskopie- als auch am Robotiksimulator [12]. Auch hier geht den praktischen Anteilen ein theoretischer Abschnitt mit Prüfung voraus. Kuhn et al. legen in ihrem deutlich weiter gefassten Lehrkonzept zur „Medizin im digitalen Zeitalter“ nur in einem von fünf Modulen den Fokus auf die Robotik, neben Virtual und Augmented Reality [9]. Es sollen nicht nur Basisfertigkeiten am Simulator für robotische Chirurgie geübt werden, sondern auch immersive Simulationen laparoskopischer Cholezystektomien oder eine leberchirurgische OP-Planung mittels Augmented Reality durchgeführt werden. Auch wenn sich diese Programme teils deutlich von dem hier präsentierten Wahlfach unterscheiden, enthalten sie sowohl theoretische als auch möglichst viele praktische Anteile. Mullens et al. bieten ebenso eine Hospitation während einer robotischen Operation an.\nIn der theoretischen Eingangsprüfung unseres Wahlfachs (s. Supplement) konnten fast alle Teilnehmende ohne Vorbereitung bereits mehr als die Hälfte der Fragen beantworten, was auf ein gewisses Maß an Vorwissen hindeutet. Im Ausgangstestat wurden jedoch im Median 3,5 von 10 Fragen mehr richtig beantwortet. Folglich fand eine nachhaltige Vermittlung theoretischen Wissens statt, da zu diesem Zeitpunkt der Vortrag über die theoretischen Grundlagen bereits 4 Wochen zurücklag. An dieser Stelle sei explizit hervorgehoben, dass im Theorieteil nicht nur die Vor- sondern auch Nachteile roboterassistierten Operierens (insbesondere die im Vergleich zur Laparoskopie höheren Kosten) explizit diskutiert wurden, um den Studierenden ehrliche und vorurteilsfreie Einblicke zu geben.\nBei den praktischen Übungen am robotischen Simulationssystem direkt an der Operationskonsole war nur in 2 von 3 Fällen eine signifikante Verbesserung der Gesamtpunktzahl bei den Teilnehmenden messbar. Die Auswahl der Übungen zielte darauf ab, grundlegende Fähigkeiten im Umgang mit der robotischen Operationskonsole zu testen (Clutch: Verwendung der Kupplung, Camera 0: Steuerung der Kamera, Sea Spikes 1: Bewegung der Greifarme). Die Übung „Clutch“ ist aber offensichtlich so wenig anspruchsvoll, dass eine tatsächliche Verbesserung in der Bedienung anhand der Gesamtpunktzahl nicht abzubilden ist: Auch ohne vorherige Einweisung wurden bei der allerersten Durchführung im Median 98 von 100 möglichen Punkten erreicht. Die mit den Instrumenten zurückgelegte Bewegungsstrecke während der Übung verdeutlicht jedoch, dass sich die Bewegungsökonomie der Teilnehmenden verbesserte, da die zurückgelegte Strecke um 30 % abnahm. Dass sich die Teilnehmenden durch das Training in ihren praktischen Fähigkeiten verbesserten, ist nicht verwunderlich, war zu erwarten und konnte bereits bei anderen laparoskopischen Simulatortrainings gezeigt werden [6, 14]. Dabei muss hervorgehoben werden, dass es letztlich unerheblich zu sein scheint, ob komplett virtuell am Simulator oder im Wet Lab bspw. an echtem biologischen Gewebe geübt wird, denn der Lernzuwachs ist vergleichbar [1]. In zukünftigen Wahlfächern könnte insofern also nicht nur rein virtuell, sondern auch mit den eigens dafür konzipierten Übungsinstrumenten für das robotische Operationssystem an einem Hands-on-Phantommodell geübt werden.\nAls organisatorischer Flaschenhals im Praxisteil stellte sich der Zugang zum Simulatorsystem dar, weil zumindest in unserer Klinik die Robotersysteme regelhaft im Tagesprogramm ausgelastet sind. Der DaVinci® SimNow®-Simulator als Nachfolger des DaVinci® Skills®-Simulator wird ebenfalls auf der Rückseite der Operationskonsole befestigt und ermöglicht das Erlernen grundlegender Steuerungsfunktionen der Konsole bis hin zur Simulation komplexer Operationen (bspw. der radikalen Prostatektomie). Er kann allerdings nur dann verwendet werden, wenn die Konsole nicht gerade bei einer Operation eingesetzt wird. Daher wurde der Beginn des Wahlfachs auf den späten Nachmittag angesetzt und damit einen Zeitpunkt, zu dem das reguläre Operationsprogramm bereits beendet ist. Im Optimalfall kann natürlich auf ein System zurückgegriffen werden, das ausschließlich dem Trainieren dient, was aber im Falle des hier eingesetzten robotischen Simulationssystems mit hohen fünfstelligen Kosten verbunden ist, da eine eigene Operationskonsole benötigt wird. Als Alternativen existieren auch Standalone-Systeme von anderen Anbietern, wie der dV-Trainer® (Mimic Technologies, Seattle, WA, USA) oder RobotiX Mentor® (3D Systems, ehemals Simbionix, Beit Golan, Israel; [10, 11, 20]). Diese Systeme ermöglichen ebenfalls ein Erlernen der grundlegenden Funktionen der Robotersysteme mit entsprechenden Trainingsprogrammen, wobei diese durchaus unterschiedlich strukturiert sind und ebenfalls schnell mehr als 100.000 US$ kosten [2]. Da immer nur ein Teilnehmender alleine an der Konsole üben kann, haben wir die Gruppe von 10 Studierenden auf zwei Teams à 5 Personen an den Praxisterminen verkleinert, um die Wartezeiten zu reduzieren. Zusätzlich wurde die Operationskonsole an einen externen Monitor angeschlossen, damit die Wartenden dem Übenden zuschauen können und daraus einen zusätzlichen Lerneffekt erzielen. Es ist natürlich auch denkbar, mehrere Übungsstationen gleichzeitig anzubieten, zwischen denen rotiert wird (bspw. unter Einbeziehung studentischer Hilfskräfte). Diese studentischen Hilfskräfte könnten dann auch das ärztliche Personal bei der Durchführung des Wahlfachs entlasten, zumal sich der Zeitaufwand in der Vor- und Nachbereitung als nicht unerheblich erwies. Darüber hinaus könnte das Wahlfach gleichzeitig als Rekrutierungsweg für zukünftige Doktorandinnen und Doktoranden dienen, indem die Teilnehmer für eine Promotionsstelle angeworben werden, im Folgenden selber ein Wahlfach leiten und später andere Doktorandinnen und Doktoranden im Rahmen eines Mentoringprogramms betreuen. Schließlich erscheint aus der wissenschaftlichen Perspektive heraus ein solches Lehrkonzept auch gut für die Durchführung von Trainingsstudien an Robotik- oder Laparoskopietrainern geeignet, die aufgrund ihres zeitlichen Aufwands kaum alleine von Ärzten geleitet werden können [15, 17].\nDas Wahlfach sollte aber nicht nur theoretische und praktische Anteile enthalten, sondern auch eine Hospitation während einer robotischen Operation. Diese stellt eine sinnvolle Ergänzung dar, weil gewonnenes Wissen und erlernte Fertigkeiten direkt auf die intraoperative Situation übertragen werden können, was sehr positiv evaluiert wurde. Zum gegenwärtigen Zeitpunkt haben noch nicht alle Teilnehmenden während einer robotischen Operation hospitiert. Dies ist damit zu erklären, dass das Wahlfach nicht wie geplant zu Semesteranfang starten konnte und sich in den vergangenen Wochen fast alle Teilnehmenden auf Prüfungen am Semesterende vorbereiten mussten.\nEinen für Urologen/innen berufspolitisch äußerst bedeutsamen Aspekt ist der Umstand, dass die Bereitschaft, möglicherweise in der Urologie tätig zu werden, am Ende des Wahlfachs bei den Teilnehmenden signifikant höher ausfiel. Die Arbeitsgemeinschaft Junge Urologen hat im Rahmen der „Zukunftsoffensive 2025“ klar zur Thematik der Nachwuchsförderung Stellung genommen und eine verstärkte Studentenbindung unter dem Slogan „Urologen der Zukunft“ als eines der Hauptziele benannt [19]. Die Faszination an der robotischen Chirurgie stellt eine bedeutsame Facette unseres Fachbereichs dar, die zukünftig noch stärker dafür eingesetzt werden sollte, aktiv für und um den urologischen Nachwuchs zu werben. In diesem Zusammenhang ist es sicher kein Zufall, dass das auf dem 71. Jahreskongress der DGU in Hamburg im Jahr 2019 vorgestellte Image-Video für unseren Fachbereich mit dem Motto „Die Vielfalt wartet. Worauf wartest du?“ als Teil der „Zukunftsoffensive Urologie“ direkt mit einer robotischen Operation beginnt. Insgesamt stieß unser strukturiertes Lehrkonzept mit mehr als 30 Anfragen für 10 Plätze auf großes Interesse. Es besteht offensichtlich ein großer Bedarf von studentischer Seite, sich in der robotischen Chirurgie zu bilden, weswegen bisher eine eigene Vorlesung über urologische Robotik gehalten wurde. Auch andere Fachbereiche haben das Potenzial der Faszination um robotische Chirurgie bereits erkannt und möchten dieses zukünftig stärker nutzen [8].\nAls Limitation dieser Arbeit ist zu benennen, dass es sich hierbei um die allererste Durchführung mit einer limitierten Zahl an Teilnehmenden handelt, die Ergebnisse sind somit als präliminär zu bewerten. Es besteht die Möglichkeit, dass ein Selektionsbias in der Gruppenzusammensetzung vorliegt, weil bei den Teilnehmenden möglicherweise eine ohnehin schon vergleichsweise hohe Affinität zu chirurgischen Fächern bestand und deswegen die Effekte des Trainings überschätzt werden. Hierbei ist einschränkend anzumerken, dass vor dem Wahlfach zumindest bei den meisten Teilnehmern keine besondere Verbindung zur Urologie bestand. Eine unmittelbare Übertragbarkeit auf andere Kliniken ist aufgrund der hohen Kosten für ein robotisches Simulatorsystem nur bedingt gegeben, sofern dieses noch angeschafft werden müsste. Alternativ bieten sich hier natürlich auch Laparoskopietrainer an, die mit weniger Kosten verbunden sind – aber das robotische Add-on nicht bieten können. Nicht zuletzt wird sich erst in der Zukunft zeigen, ob die Teilnehmenden tatsächlich eine Famulatur oder ein Teil des praktischen Jahres in der Urologie ableisten – oder gar in einer urologischen Klinik anfangen werden, zu arbeiten. Dieser longitudinale Aspekt lässt sich nur im Verlauf beantworten und steht noch aus.",
"Diese ersten Erfahrungen mit dem Wahlfach „Robotische Chirurgie“ zeigen, dass offensichtlich ein Bedarf von studentischer Seite besteht, sich in der roboterassistierten Chirurgie zu bilden. Das Wahlfach ermöglicht einerseits, den Studierenden theoretische wie praktische Inhalte der Robotik zu vermitteln. Andererseits erscheint es auch als ein gutes berufspolitisches Instrument, auf unser Fachgebiet aufmerksam zu machen, Interesse zu wecken und die Faszination an der robotischen urologischen Chirurgie zu transportieren. Darüber hinaus erscheinen Wahlfächer wie diese als geeignetes Medium, wissenschaftliche Fragestellungen bspw. zu Lernkurvenanalysen zu beantworten. Aus diesem Grund wird es zumindest in unserer Klinik kein einmaliges Angebot bleiben, und zukünftige Wahlfächer „Robotische Chirurgie“ werden folgen.\n Infobox 1 Mehr Informationen zum Thema \nDRUS robotisches Curriculum: https://www.dgru.de/ERUS robotisches Curriculum: http://uroweb.org/section/erus/educationDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)\n\nDRUS robotisches Curriculum: https://www.dgru.de/\nERUS robotisches Curriculum: http://uroweb.org/section/erus/education\nDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)\n\nDRUS robotisches Curriculum: https://www.dgru.de/ERUS robotisches Curriculum: http://uroweb.org/section/erus/educationDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)\n\nDRUS robotisches Curriculum: https://www.dgru.de/\nERUS robotisches Curriculum: http://uroweb.org/section/erus/education\nDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)",
"\nDRUS robotisches Curriculum: https://www.dgru.de/ERUS robotisches Curriculum: http://uroweb.org/section/erus/educationDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)\n\nDRUS robotisches Curriculum: https://www.dgru.de/\nERUS robotisches Curriculum: http://uroweb.org/section/erus/education\nDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)",
"\n\n"
] |
[
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null,
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[
"Einleitung",
"Methodik",
"Ergebnisse",
"Diskussion",
"Ausblick",
"Infobox 1 Mehr Informationen zum Thema",
"Fazit für die Praxis",
"Supplementary Information",
""
] |
[
"Seit der Food and Drug (FDA)-Zulassung des DaVinci®-Operationssystems im Jahr 2000 hat sich die robotische Chirurgie als Standardverfahren in der Urologie und in vielen weiteren operativen Fächern etabliert. Auch wenn eine signifikante Zahl (mittel)großer Kliniken heute roboterassistierte Operationen anbietet, spielt die robotische Chirurgie im Lehrplan von Medizinstudenten nur eine untergeordnete Rolle.\nDemgegenüber existieren bereits diverse strukturierte Curricula und Trainingsprogramme für angehende robotische Operateure, zu denen u. a. das 6‑monatige Deutsche Robotercurriculum der Deutschen Gesellschaft für Robotische Urologie (DRGU) oder als europäisches Pendant das ERUS (European Association of Urology Robotic Urology Section) Robotic Curriculum zählen [3]. Diesen Programmen liegt die Erkenntnis zugrunde, dass ein strukturiertes Training insbesondere unter Einbeziehung von Simulatortraining in kürzerer Zeit einen größeren Lernerfolg mit besseren operativen Ergebnissen ermöglicht [14]. Trainingsprogramme für den in der Regel unerlässlichen Bedside-Assistenten sind schon deutlich seltener, auch wenn gezeigt werden konnte, dass dessen Erfahrung bei komplexen robotischen Operateuren ebenso einen signifikanten Einfluss auf das operative Ergebnis hat [2, 23]. Prinzipiell könnte es noch sinnvoller sein, mit einer strukturierten Ausbildung in der robotischen Chirurgie bereits im Medizinstudium zu beginnen. Einerseits sind robotische Operationen aus dem klinischen Alltag in vielen urologischen Kliniken nicht mehr wegzudenken und sollten deswegen Bestandteil der urologischen Lehre sein. Andererseits könnte ein solches Programm auch dazu dienen, aus berufspolitischer Sicht vermehrtes Interesse an der Urologie zu erzeugen und talentierte zukünftige Kolleginnen und Kollegen frühzeitig zu gewinnen [16, 21].\nUnsere Klinik verfügt über zwei DaVinci®-Operationssysteme vom Typ „X“ und einen DaVinci® SimNow ®-Simulator (Intuitive Surgical, Sunnyvale, CA, USA), der ein virtuelles Üben und Operieren an der Operationskonsole ermöglicht. Um Erfahrungen in der Implementierung der robotischen Chirurgie in die urologische Lehre zu sammeln, haben wir kürzlich erstmalig für eine urologische Universitätsklinik in Deutschland das Wahlfach „Robotische Chirurgie“ in einer Kleingruppe von 10 Studierenden aus den klinischen Semestern organisiert. In 6 Treffen à 2 h wurden theoretische Grundlagen und praktische Fertigkeiten in der robotischen Chirurgie vermittelt inklusive einer Hospitation während einer roboterassistierten Operation. Der Zuwachs an Wissen und Fertigkeiten wurde quantifiziert und die studentische Einschätzung evaluiert.",
"Nach Verzögerungen aufgrund der Coronapandemie konnte der erste Termin unter strenger Einhaltung der coronabedingten Vorsichtsmaßnahmen (Abstandsregeln, Tragen eines medizinischen Mund-Nase-Schutz, Nachweis einer abgeschlossenen Impfung, durchgemachten Infektion oder tagesaktueller negativer Schnelltest) auf Mitte Juni 2021 festgesetzt wurde. Alle Treffen fanden im leeren robotischen Operationssaal der Klinik für Urologie und Kinderurologie statt.\nDer Ablauf des Wahlfachs ist in Abb. 1 dargestellt. Während des Eingangstestats am ersten Treffen wurde das Vorwissen der Studierenden in einer theoretischen Prüfung (10 Multiple-choice-Fragen, s. Infobox 1) erhoben. Zudem erfolgte die Messung der praktischen Fähigkeiten mit drei verschiedenen Übungen an der Konsole eines DaVinci®-X-Systems mit angedocktem SimNow®-Simulator, bewusst mit nur sehr kurzer vorheriger Einführung (Clutch: Verwendung der Kupplung, Camera 0: Steuerung der Kamera, Sea Spikes 1: Bedienung der Greifarme). Im zweiten Treffen erfolgte online die Vermittlung der theoretischen Grundlagen zur Historie robotischer Chirurgie, eine ausführliche Einführung in das robotische Operationssystem inklusive verschiedener Docking-Strategien und ein kritischer Vergleich von robotischem, laparoskopischen und offenen Operieren. Zudem wurden Besonderheiten der Robotik, wie die Notwendigkeit einer guten Kommunikation zwischen Bedside-Assistent und Operateur besprochen und gemeinsam eine vorher aufgezeichnete robotische radikale Prostatektomie analysiert. An Treffen 3 und 4 lag der Schwerpunkt auf einem möglichst großen Praxisanteil mit Einweisung in den Umgang mit dem Patientenwagen, Feinheiten der Trokareinlage und weiteren Übungen am Modell sowie Simulator. Eine Hospitation als 5. Bestandteil des Moduls wurde individuell in Zweiergruppen vereinbart. Während des letzten 6.Treffens wurden zur Projekteffektemessung dieselbe theoretische und praktische Prüfung wiederholt und eine anonyme Evaluation durchgeführt. Eine individuelle Bewertung der Teilnehmenden entfiel, weil es sich um ein rein freiwilliges Wahlfach handelte, für das keine Benotung vorgesehen ist.\nDie statistische Auswertung erfolgte mit SPSS (IBM, Armonk, USA). Häufigkeiten wurden als Median (Range) angegeben, die verbundenen Stichproben mit dem Wilcoxon-Test verglichen, p < 0,05 galt als statistisch signifikant. Die Durchführung des Wahlfachs erfolgte im Rahmen des Teach-the-teacher-Kurses des Universitätsklinikums des Saarlandes und wurde durch den Studiendekan genehmigt.",
"Bei mehr als 30 Anfragen wurde das Wahlfach mit 6 Studentinnen und 4 Studenten aus dem 6. bis 10. Semester begonnen, die Teilnehmerauswahl erfolgte nach dem Prinzip „first come, first serve“. Die durchschnittliche Teilnahmequote betrug 83 %, wobei bisher noch nicht alle individuellen Hospitationen stattgefunden haben. Ein Teilnehmender hatte zuvor ein Pflegepraktikum in der Urologie absolviert und 2 weitere eine Famulatur in einer urologischen Klinik.\nDie praktischen Treffen bedurften einer guten Planung und Abstimmung innerhalb der Abteilung wie interdisziplinär mit der Anästhesie, weil sichergestellt werden musste, dass ein bestimmter OP-Saal von 17 bis 19 Uhr trotz etwaiger Notoperationen frei sein musste. Um die Wartezeiten zu verkürzen, bis die Teilnehmenden wieder am Simulator üben können, wurde die Gruppe ab dem 3. Treffen zweigeteilt und dann in noch kleineren Teams mit 5 Teilnehmenden je 1 h unterrichtet.\nIm theoretischen Ein- und Ausgangstestat ließ sich ein signifikanter Wissenszuwachs aller Teilnehmender feststellen. Bei anfänglich im Median 6 (Range 4–8) richtigen Antworten wurden im Ausgangstestat 9,5 (9–10) und damit signifikant mehr Fragen korrekt beantwortet (p = 0,011, s. Abb. 2). Im Praxisteil wurden in den Übungen „Camera 0“ und „Sea Spikes 1“ am Ende ebenfalls signifikant höhere Punktwerte erzielt (p = 0,036 und p = 0,018). Bei der Übung „Clutch“ ließ sich keine signifikante Veränderung in der Gesamtpunktzahl feststellen. Bereits im Eingangstestat wurden hier im Median 98 (Range 74–100) von 100 möglichen Punkten erreicht, am Ende des Wahlfachs 100 (83–100) Punkte. Trotzdem lag auch bei dieser Übung eine signifikant verbesserte Bewegungsökonomie vor, da die mediane Bewegungsstrecke von 214,8 cm (Range 166,1–379,5) auf 166,9 cm (160,6–212,5) abnahm (p = 0,028).\nAcht von 10 Studierenden nahmen an der Evaluation teil und bewerteten das Modul als (sehr) gut (s. Tab. 1). Im Freitext wurde als besonders positiv hervorgehoben, dass sich in einer Kleingruppe mit hohem Praxisanteil viel Zeit für Fragen genommen wurde. Es wurde vorgeschlagen, noch einen zweiten Teil für „Fortgeschrittene“ anzubieten und die einzelnen Treffen länger zu gestalten, damit noch mehr Übungszeit an der Konsole besteht.123456xMedWie bewerten Sie das Eingangstestat?7–––––1,0Wie bewerten Sie den Theorieteil?34––––1,5Wie bewerten Sie den dritten Praxisteil?7–––––1,0Wie bewerten Sie den vierten Praxisteil?61––––1,14Wie bewerten Sie die Hospitation?4–––––1,0Wie bewerten Sie das Ausgangstestat?7–––––1,0Wie bewerten Sie das Wahlfach insgesamt?8–––––1,0Wie bewerten Sie die Organisation des Wahlfachs?8–––––1,0Wie bewerten Sie den didaktischen Aufbau?8–––––1,0Wie bewerten Sie die Motivation des Dozenten?8–––––1,0\nZum Zeitpunkt des Wahlfachs befand sich noch kein Teilnehmender im praktischen Jahr (PJ), einer hatte schon vorher beabsichtigt, dies in der Urologie abzuleisten. Nach Abschluss des Wahlfachs haben 4 Studierende ihr PJ fest ohne Urologie geplant, wobei zumindest ein Teilnehmender dies möglicherweise noch ändern möchte. Bei 4 weiteren Studierenden zählt Urologie mit zur engeren Auswahl.\nAm Ende des Wahlfachs konnten sich die Studierenden deutlich besser vorstellen, später als Urologe/in tätig zu werden (p = 0,007, s. Abb. 3). Auch wenn die Bereitschaft, später als Chirurg/in zu arbeiten, ebenfalls zunahm, war dieser Unterschied nicht statistisch signifikant (p = 0,066).",
"Roboterassistierte Operationen sind heute nicht mehr aus dem Alltag vieler urologischer Kliniken wegzudenken. Im Jahr 2018 wurden in Deutschland von insgesamt 11.492 radikalen Prostatektomien 39,1 % robotisch und damit fast doppelt so viele wie offen (2253, entsprechend 19,6 %) durchgeführt. Der Anteil laparoskopischer radikaler Prostatektomien war mit 41,3 % nur knapp höher [7]. Bei den Nierenteilresektionen im Jahr 2019 in Deutschland wurden demgegenüber mehr als doppelt so viele Eingriffe robotisch wie laparoskopisch durchgeführt (28,8 % vs. 13 %), und dieser Anteil stieg im Jahr 2020 auf 36,9 % (3577 von 9700 Eingriffen; [24]). Demzufolge sollte die Robotik auch ein fester Bestandteil der (urologischen) Lehre im Medizinstudium sein. Das Medizinstudium befindet sich derzeit mit dem „Masterplan Medizinstudium 2020“ in einem Umbruch und der nationale kompetenzbasierte Lernzielkatalog (NKLM) wurde kürzlich überarbeitet [4, 5]. Dadurch ergeben sich einerseits Chancen für die Urologie, da ein stärkerer Fokus auf die Anwendungsebene im Medizinstudium gelegt wird und viele Prüfungen bspw. im OSCE-Format („objective structured clinical examination“) abgehalten werden sollen [18]. Andererseits wird auch die Digitalisierung in der Medizin einen weiteren Kernaspekt im Medizinstudium spielen, die roboterassistierte Chirurgie ist jedoch weiterhin unterrepräsentiert [22]. Um die urologische Lehre in unserer Klinik um Lehrinhalte der robotischen Chirurgie zu erweitern, haben wir kürzlich deutschlandweit erstmalig in einer urologischen Klinik das Wahlfach „Robotische Chirurgie“ angeboten. In einer Kleingruppe von 10 Studierenden der klinischen Semester wurden in 6 Treffen à 2 h theoretische Inhalte und praktische Fertigkeiten vermittelt. Die Teilnehmenden hatten nicht nur einen signifikanten Zuwachs an Wissen und Fertigkeiten, auch ihre Bereitschaft, später in der Urologie tätig zu werden, stieg messbar an.\nBislang existieren nur wenige strukturierte und evaluierte Lehrkonzepte der robotischen Chirurgie für Studierende. Das 2‑wöchige Curriculum von Mullens et al. ähnelt einem Intensivkurs mit sehr hohem Praxisanteil und einer Vielzahl an Übungen am Simulator, aber auch im Wet Lab sowie einer Assistenz während einer robotischen Operation [13]. Moglia et al. konzipierten demgegenüber ein strukturiertes 4‑stündiges Training sowohl am Laparoskopie- als auch am Robotiksimulator [12]. Auch hier geht den praktischen Anteilen ein theoretischer Abschnitt mit Prüfung voraus. Kuhn et al. legen in ihrem deutlich weiter gefassten Lehrkonzept zur „Medizin im digitalen Zeitalter“ nur in einem von fünf Modulen den Fokus auf die Robotik, neben Virtual und Augmented Reality [9]. Es sollen nicht nur Basisfertigkeiten am Simulator für robotische Chirurgie geübt werden, sondern auch immersive Simulationen laparoskopischer Cholezystektomien oder eine leberchirurgische OP-Planung mittels Augmented Reality durchgeführt werden. Auch wenn sich diese Programme teils deutlich von dem hier präsentierten Wahlfach unterscheiden, enthalten sie sowohl theoretische als auch möglichst viele praktische Anteile. Mullens et al. bieten ebenso eine Hospitation während einer robotischen Operation an.\nIn der theoretischen Eingangsprüfung unseres Wahlfachs (s. Supplement) konnten fast alle Teilnehmende ohne Vorbereitung bereits mehr als die Hälfte der Fragen beantworten, was auf ein gewisses Maß an Vorwissen hindeutet. Im Ausgangstestat wurden jedoch im Median 3,5 von 10 Fragen mehr richtig beantwortet. Folglich fand eine nachhaltige Vermittlung theoretischen Wissens statt, da zu diesem Zeitpunkt der Vortrag über die theoretischen Grundlagen bereits 4 Wochen zurücklag. An dieser Stelle sei explizit hervorgehoben, dass im Theorieteil nicht nur die Vor- sondern auch Nachteile roboterassistierten Operierens (insbesondere die im Vergleich zur Laparoskopie höheren Kosten) explizit diskutiert wurden, um den Studierenden ehrliche und vorurteilsfreie Einblicke zu geben.\nBei den praktischen Übungen am robotischen Simulationssystem direkt an der Operationskonsole war nur in 2 von 3 Fällen eine signifikante Verbesserung der Gesamtpunktzahl bei den Teilnehmenden messbar. Die Auswahl der Übungen zielte darauf ab, grundlegende Fähigkeiten im Umgang mit der robotischen Operationskonsole zu testen (Clutch: Verwendung der Kupplung, Camera 0: Steuerung der Kamera, Sea Spikes 1: Bewegung der Greifarme). Die Übung „Clutch“ ist aber offensichtlich so wenig anspruchsvoll, dass eine tatsächliche Verbesserung in der Bedienung anhand der Gesamtpunktzahl nicht abzubilden ist: Auch ohne vorherige Einweisung wurden bei der allerersten Durchführung im Median 98 von 100 möglichen Punkten erreicht. Die mit den Instrumenten zurückgelegte Bewegungsstrecke während der Übung verdeutlicht jedoch, dass sich die Bewegungsökonomie der Teilnehmenden verbesserte, da die zurückgelegte Strecke um 30 % abnahm. Dass sich die Teilnehmenden durch das Training in ihren praktischen Fähigkeiten verbesserten, ist nicht verwunderlich, war zu erwarten und konnte bereits bei anderen laparoskopischen Simulatortrainings gezeigt werden [6, 14]. Dabei muss hervorgehoben werden, dass es letztlich unerheblich zu sein scheint, ob komplett virtuell am Simulator oder im Wet Lab bspw. an echtem biologischen Gewebe geübt wird, denn der Lernzuwachs ist vergleichbar [1]. In zukünftigen Wahlfächern könnte insofern also nicht nur rein virtuell, sondern auch mit den eigens dafür konzipierten Übungsinstrumenten für das robotische Operationssystem an einem Hands-on-Phantommodell geübt werden.\nAls organisatorischer Flaschenhals im Praxisteil stellte sich der Zugang zum Simulatorsystem dar, weil zumindest in unserer Klinik die Robotersysteme regelhaft im Tagesprogramm ausgelastet sind. Der DaVinci® SimNow®-Simulator als Nachfolger des DaVinci® Skills®-Simulator wird ebenfalls auf der Rückseite der Operationskonsole befestigt und ermöglicht das Erlernen grundlegender Steuerungsfunktionen der Konsole bis hin zur Simulation komplexer Operationen (bspw. der radikalen Prostatektomie). Er kann allerdings nur dann verwendet werden, wenn die Konsole nicht gerade bei einer Operation eingesetzt wird. Daher wurde der Beginn des Wahlfachs auf den späten Nachmittag angesetzt und damit einen Zeitpunkt, zu dem das reguläre Operationsprogramm bereits beendet ist. Im Optimalfall kann natürlich auf ein System zurückgegriffen werden, das ausschließlich dem Trainieren dient, was aber im Falle des hier eingesetzten robotischen Simulationssystems mit hohen fünfstelligen Kosten verbunden ist, da eine eigene Operationskonsole benötigt wird. Als Alternativen existieren auch Standalone-Systeme von anderen Anbietern, wie der dV-Trainer® (Mimic Technologies, Seattle, WA, USA) oder RobotiX Mentor® (3D Systems, ehemals Simbionix, Beit Golan, Israel; [10, 11, 20]). Diese Systeme ermöglichen ebenfalls ein Erlernen der grundlegenden Funktionen der Robotersysteme mit entsprechenden Trainingsprogrammen, wobei diese durchaus unterschiedlich strukturiert sind und ebenfalls schnell mehr als 100.000 US$ kosten [2]. Da immer nur ein Teilnehmender alleine an der Konsole üben kann, haben wir die Gruppe von 10 Studierenden auf zwei Teams à 5 Personen an den Praxisterminen verkleinert, um die Wartezeiten zu reduzieren. Zusätzlich wurde die Operationskonsole an einen externen Monitor angeschlossen, damit die Wartenden dem Übenden zuschauen können und daraus einen zusätzlichen Lerneffekt erzielen. Es ist natürlich auch denkbar, mehrere Übungsstationen gleichzeitig anzubieten, zwischen denen rotiert wird (bspw. unter Einbeziehung studentischer Hilfskräfte). Diese studentischen Hilfskräfte könnten dann auch das ärztliche Personal bei der Durchführung des Wahlfachs entlasten, zumal sich der Zeitaufwand in der Vor- und Nachbereitung als nicht unerheblich erwies. Darüber hinaus könnte das Wahlfach gleichzeitig als Rekrutierungsweg für zukünftige Doktorandinnen und Doktoranden dienen, indem die Teilnehmer für eine Promotionsstelle angeworben werden, im Folgenden selber ein Wahlfach leiten und später andere Doktorandinnen und Doktoranden im Rahmen eines Mentoringprogramms betreuen. Schließlich erscheint aus der wissenschaftlichen Perspektive heraus ein solches Lehrkonzept auch gut für die Durchführung von Trainingsstudien an Robotik- oder Laparoskopietrainern geeignet, die aufgrund ihres zeitlichen Aufwands kaum alleine von Ärzten geleitet werden können [15, 17].\nDas Wahlfach sollte aber nicht nur theoretische und praktische Anteile enthalten, sondern auch eine Hospitation während einer robotischen Operation. Diese stellt eine sinnvolle Ergänzung dar, weil gewonnenes Wissen und erlernte Fertigkeiten direkt auf die intraoperative Situation übertragen werden können, was sehr positiv evaluiert wurde. Zum gegenwärtigen Zeitpunkt haben noch nicht alle Teilnehmenden während einer robotischen Operation hospitiert. Dies ist damit zu erklären, dass das Wahlfach nicht wie geplant zu Semesteranfang starten konnte und sich in den vergangenen Wochen fast alle Teilnehmenden auf Prüfungen am Semesterende vorbereiten mussten.\nEinen für Urologen/innen berufspolitisch äußerst bedeutsamen Aspekt ist der Umstand, dass die Bereitschaft, möglicherweise in der Urologie tätig zu werden, am Ende des Wahlfachs bei den Teilnehmenden signifikant höher ausfiel. Die Arbeitsgemeinschaft Junge Urologen hat im Rahmen der „Zukunftsoffensive 2025“ klar zur Thematik der Nachwuchsförderung Stellung genommen und eine verstärkte Studentenbindung unter dem Slogan „Urologen der Zukunft“ als eines der Hauptziele benannt [19]. Die Faszination an der robotischen Chirurgie stellt eine bedeutsame Facette unseres Fachbereichs dar, die zukünftig noch stärker dafür eingesetzt werden sollte, aktiv für und um den urologischen Nachwuchs zu werben. In diesem Zusammenhang ist es sicher kein Zufall, dass das auf dem 71. Jahreskongress der DGU in Hamburg im Jahr 2019 vorgestellte Image-Video für unseren Fachbereich mit dem Motto „Die Vielfalt wartet. Worauf wartest du?“ als Teil der „Zukunftsoffensive Urologie“ direkt mit einer robotischen Operation beginnt. Insgesamt stieß unser strukturiertes Lehrkonzept mit mehr als 30 Anfragen für 10 Plätze auf großes Interesse. Es besteht offensichtlich ein großer Bedarf von studentischer Seite, sich in der robotischen Chirurgie zu bilden, weswegen bisher eine eigene Vorlesung über urologische Robotik gehalten wurde. Auch andere Fachbereiche haben das Potenzial der Faszination um robotische Chirurgie bereits erkannt und möchten dieses zukünftig stärker nutzen [8].\nAls Limitation dieser Arbeit ist zu benennen, dass es sich hierbei um die allererste Durchführung mit einer limitierten Zahl an Teilnehmenden handelt, die Ergebnisse sind somit als präliminär zu bewerten. Es besteht die Möglichkeit, dass ein Selektionsbias in der Gruppenzusammensetzung vorliegt, weil bei den Teilnehmenden möglicherweise eine ohnehin schon vergleichsweise hohe Affinität zu chirurgischen Fächern bestand und deswegen die Effekte des Trainings überschätzt werden. Hierbei ist einschränkend anzumerken, dass vor dem Wahlfach zumindest bei den meisten Teilnehmern keine besondere Verbindung zur Urologie bestand. Eine unmittelbare Übertragbarkeit auf andere Kliniken ist aufgrund der hohen Kosten für ein robotisches Simulatorsystem nur bedingt gegeben, sofern dieses noch angeschafft werden müsste. Alternativ bieten sich hier natürlich auch Laparoskopietrainer an, die mit weniger Kosten verbunden sind – aber das robotische Add-on nicht bieten können. Nicht zuletzt wird sich erst in der Zukunft zeigen, ob die Teilnehmenden tatsächlich eine Famulatur oder ein Teil des praktischen Jahres in der Urologie ableisten – oder gar in einer urologischen Klinik anfangen werden, zu arbeiten. Dieser longitudinale Aspekt lässt sich nur im Verlauf beantworten und steht noch aus.",
"Diese ersten Erfahrungen mit dem Wahlfach „Robotische Chirurgie“ zeigen, dass offensichtlich ein Bedarf von studentischer Seite besteht, sich in der roboterassistierten Chirurgie zu bilden. Das Wahlfach ermöglicht einerseits, den Studierenden theoretische wie praktische Inhalte der Robotik zu vermitteln. Andererseits erscheint es auch als ein gutes berufspolitisches Instrument, auf unser Fachgebiet aufmerksam zu machen, Interesse zu wecken und die Faszination an der robotischen urologischen Chirurgie zu transportieren. Darüber hinaus erscheinen Wahlfächer wie diese als geeignetes Medium, wissenschaftliche Fragestellungen bspw. zu Lernkurvenanalysen zu beantworten. Aus diesem Grund wird es zumindest in unserer Klinik kein einmaliges Angebot bleiben, und zukünftige Wahlfächer „Robotische Chirurgie“ werden folgen.\n Infobox 1 Mehr Informationen zum Thema \nDRUS robotisches Curriculum: https://www.dgru.de/ERUS robotisches Curriculum: http://uroweb.org/section/erus/educationDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)\n\nDRUS robotisches Curriculum: https://www.dgru.de/\nERUS robotisches Curriculum: http://uroweb.org/section/erus/education\nDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)\n\nDRUS robotisches Curriculum: https://www.dgru.de/ERUS robotisches Curriculum: http://uroweb.org/section/erus/educationDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)\n\nDRUS robotisches Curriculum: https://www.dgru.de/\nERUS robotisches Curriculum: http://uroweb.org/section/erus/education\nDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)",
"\nDRUS robotisches Curriculum: https://www.dgru.de/ERUS robotisches Curriculum: http://uroweb.org/section/erus/educationDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)\n\nDRUS robotisches Curriculum: https://www.dgru.de/\nERUS robotisches Curriculum: http://uroweb.org/section/erus/education\nDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)",
"\nVor dem Hintergrund der weiten Verbreitung roboterassistierter Operationen ist die robotische Chirurgie im Lehrplan heutiger Medizinstudierender unterrepräsentiert.Es besteht offensichtlich ein Bedarf von studentischer Seite, sich entsprechend zu bilden.Ein Wahlfach „Robotische Chirurgie“ erscheint in diesem Zusammenhang als geeignetes Format, theoretische Grundlagen und praktische Fertigkeiten in der robotischen (urologischen) Chirurgie zu vermitteln.Organisatorisch bietet sich die Aufteilung in einen Theorie-, mehrere Praxisteile und eine Hospitation an.Die Gruppen sollten nicht zu groß sein, insbesondere im Praxisteil maximal 5 Personen umfassen.Jenseits der edukativen Perspektive kann ein solches Wahlfach auf die faszinierende Vielfältigkeit der Urologie aufmerksam machen und möglicherweise auch neue Kolleginnen und Kollegen gewinnen.\n\nVor dem Hintergrund der weiten Verbreitung roboterassistierter Operationen ist die robotische Chirurgie im Lehrplan heutiger Medizinstudierender unterrepräsentiert.\nEs besteht offensichtlich ein Bedarf von studentischer Seite, sich entsprechend zu bilden.\nEin Wahlfach „Robotische Chirurgie“ erscheint in diesem Zusammenhang als geeignetes Format, theoretische Grundlagen und praktische Fertigkeiten in der robotischen (urologischen) Chirurgie zu vermitteln.\nOrganisatorisch bietet sich die Aufteilung in einen Theorie-, mehrere Praxisteile und eine Hospitation an.\nDie Gruppen sollten nicht zu groß sein, insbesondere im Praxisteil maximal 5 Personen umfassen.\nJenseits der edukativen Perspektive kann ein solches Wahlfach auf die faszinierende Vielfältigkeit der Urologie aufmerksam machen und möglicherweise auch neue Kolleginnen und Kollegen gewinnen.",
" \n\n\n\n\n",
"\n\n"
] |
[
null,
null,
null,
null,
null,
null,
"conclusion",
"supplementary-material",
null
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[
"Robotische Chirurgie",
"Robotik",
"Simulationstraining",
"Medizinische Lehre",
"Medizinstudium",
"Robotic surgical procedures",
"Robotics",
"Simulation training",
"Medical education",
"Medical studies"
] |
Einleitung:
Seit der Food and Drug (FDA)-Zulassung des DaVinci®-Operationssystems im Jahr 2000 hat sich die robotische Chirurgie als Standardverfahren in der Urologie und in vielen weiteren operativen Fächern etabliert. Auch wenn eine signifikante Zahl (mittel)großer Kliniken heute roboterassistierte Operationen anbietet, spielt die robotische Chirurgie im Lehrplan von Medizinstudenten nur eine untergeordnete Rolle.
Demgegenüber existieren bereits diverse strukturierte Curricula und Trainingsprogramme für angehende robotische Operateure, zu denen u. a. das 6‑monatige Deutsche Robotercurriculum der Deutschen Gesellschaft für Robotische Urologie (DRGU) oder als europäisches Pendant das ERUS (European Association of Urology Robotic Urology Section) Robotic Curriculum zählen [3]. Diesen Programmen liegt die Erkenntnis zugrunde, dass ein strukturiertes Training insbesondere unter Einbeziehung von Simulatortraining in kürzerer Zeit einen größeren Lernerfolg mit besseren operativen Ergebnissen ermöglicht [14]. Trainingsprogramme für den in der Regel unerlässlichen Bedside-Assistenten sind schon deutlich seltener, auch wenn gezeigt werden konnte, dass dessen Erfahrung bei komplexen robotischen Operateuren ebenso einen signifikanten Einfluss auf das operative Ergebnis hat [2, 23]. Prinzipiell könnte es noch sinnvoller sein, mit einer strukturierten Ausbildung in der robotischen Chirurgie bereits im Medizinstudium zu beginnen. Einerseits sind robotische Operationen aus dem klinischen Alltag in vielen urologischen Kliniken nicht mehr wegzudenken und sollten deswegen Bestandteil der urologischen Lehre sein. Andererseits könnte ein solches Programm auch dazu dienen, aus berufspolitischer Sicht vermehrtes Interesse an der Urologie zu erzeugen und talentierte zukünftige Kolleginnen und Kollegen frühzeitig zu gewinnen [16, 21].
Unsere Klinik verfügt über zwei DaVinci®-Operationssysteme vom Typ „X“ und einen DaVinci® SimNow ®-Simulator (Intuitive Surgical, Sunnyvale, CA, USA), der ein virtuelles Üben und Operieren an der Operationskonsole ermöglicht. Um Erfahrungen in der Implementierung der robotischen Chirurgie in die urologische Lehre zu sammeln, haben wir kürzlich erstmalig für eine urologische Universitätsklinik in Deutschland das Wahlfach „Robotische Chirurgie“ in einer Kleingruppe von 10 Studierenden aus den klinischen Semestern organisiert. In 6 Treffen à 2 h wurden theoretische Grundlagen und praktische Fertigkeiten in der robotischen Chirurgie vermittelt inklusive einer Hospitation während einer roboterassistierten Operation. Der Zuwachs an Wissen und Fertigkeiten wurde quantifiziert und die studentische Einschätzung evaluiert.
Methodik:
Nach Verzögerungen aufgrund der Coronapandemie konnte der erste Termin unter strenger Einhaltung der coronabedingten Vorsichtsmaßnahmen (Abstandsregeln, Tragen eines medizinischen Mund-Nase-Schutz, Nachweis einer abgeschlossenen Impfung, durchgemachten Infektion oder tagesaktueller negativer Schnelltest) auf Mitte Juni 2021 festgesetzt wurde. Alle Treffen fanden im leeren robotischen Operationssaal der Klinik für Urologie und Kinderurologie statt.
Der Ablauf des Wahlfachs ist in Abb. 1 dargestellt. Während des Eingangstestats am ersten Treffen wurde das Vorwissen der Studierenden in einer theoretischen Prüfung (10 Multiple-choice-Fragen, s. Infobox 1) erhoben. Zudem erfolgte die Messung der praktischen Fähigkeiten mit drei verschiedenen Übungen an der Konsole eines DaVinci®-X-Systems mit angedocktem SimNow®-Simulator, bewusst mit nur sehr kurzer vorheriger Einführung (Clutch: Verwendung der Kupplung, Camera 0: Steuerung der Kamera, Sea Spikes 1: Bedienung der Greifarme). Im zweiten Treffen erfolgte online die Vermittlung der theoretischen Grundlagen zur Historie robotischer Chirurgie, eine ausführliche Einführung in das robotische Operationssystem inklusive verschiedener Docking-Strategien und ein kritischer Vergleich von robotischem, laparoskopischen und offenen Operieren. Zudem wurden Besonderheiten der Robotik, wie die Notwendigkeit einer guten Kommunikation zwischen Bedside-Assistent und Operateur besprochen und gemeinsam eine vorher aufgezeichnete robotische radikale Prostatektomie analysiert. An Treffen 3 und 4 lag der Schwerpunkt auf einem möglichst großen Praxisanteil mit Einweisung in den Umgang mit dem Patientenwagen, Feinheiten der Trokareinlage und weiteren Übungen am Modell sowie Simulator. Eine Hospitation als 5. Bestandteil des Moduls wurde individuell in Zweiergruppen vereinbart. Während des letzten 6.Treffens wurden zur Projekteffektemessung dieselbe theoretische und praktische Prüfung wiederholt und eine anonyme Evaluation durchgeführt. Eine individuelle Bewertung der Teilnehmenden entfiel, weil es sich um ein rein freiwilliges Wahlfach handelte, für das keine Benotung vorgesehen ist.
Die statistische Auswertung erfolgte mit SPSS (IBM, Armonk, USA). Häufigkeiten wurden als Median (Range) angegeben, die verbundenen Stichproben mit dem Wilcoxon-Test verglichen, p < 0,05 galt als statistisch signifikant. Die Durchführung des Wahlfachs erfolgte im Rahmen des Teach-the-teacher-Kurses des Universitätsklinikums des Saarlandes und wurde durch den Studiendekan genehmigt.
Ergebnisse:
Bei mehr als 30 Anfragen wurde das Wahlfach mit 6 Studentinnen und 4 Studenten aus dem 6. bis 10. Semester begonnen, die Teilnehmerauswahl erfolgte nach dem Prinzip „first come, first serve“. Die durchschnittliche Teilnahmequote betrug 83 %, wobei bisher noch nicht alle individuellen Hospitationen stattgefunden haben. Ein Teilnehmender hatte zuvor ein Pflegepraktikum in der Urologie absolviert und 2 weitere eine Famulatur in einer urologischen Klinik.
Die praktischen Treffen bedurften einer guten Planung und Abstimmung innerhalb der Abteilung wie interdisziplinär mit der Anästhesie, weil sichergestellt werden musste, dass ein bestimmter OP-Saal von 17 bis 19 Uhr trotz etwaiger Notoperationen frei sein musste. Um die Wartezeiten zu verkürzen, bis die Teilnehmenden wieder am Simulator üben können, wurde die Gruppe ab dem 3. Treffen zweigeteilt und dann in noch kleineren Teams mit 5 Teilnehmenden je 1 h unterrichtet.
Im theoretischen Ein- und Ausgangstestat ließ sich ein signifikanter Wissenszuwachs aller Teilnehmender feststellen. Bei anfänglich im Median 6 (Range 4–8) richtigen Antworten wurden im Ausgangstestat 9,5 (9–10) und damit signifikant mehr Fragen korrekt beantwortet (p = 0,011, s. Abb. 2). Im Praxisteil wurden in den Übungen „Camera 0“ und „Sea Spikes 1“ am Ende ebenfalls signifikant höhere Punktwerte erzielt (p = 0,036 und p = 0,018). Bei der Übung „Clutch“ ließ sich keine signifikante Veränderung in der Gesamtpunktzahl feststellen. Bereits im Eingangstestat wurden hier im Median 98 (Range 74–100) von 100 möglichen Punkten erreicht, am Ende des Wahlfachs 100 (83–100) Punkte. Trotzdem lag auch bei dieser Übung eine signifikant verbesserte Bewegungsökonomie vor, da die mediane Bewegungsstrecke von 214,8 cm (Range 166,1–379,5) auf 166,9 cm (160,6–212,5) abnahm (p = 0,028).
Acht von 10 Studierenden nahmen an der Evaluation teil und bewerteten das Modul als (sehr) gut (s. Tab. 1). Im Freitext wurde als besonders positiv hervorgehoben, dass sich in einer Kleingruppe mit hohem Praxisanteil viel Zeit für Fragen genommen wurde. Es wurde vorgeschlagen, noch einen zweiten Teil für „Fortgeschrittene“ anzubieten und die einzelnen Treffen länger zu gestalten, damit noch mehr Übungszeit an der Konsole besteht.123456xMedWie bewerten Sie das Eingangstestat?7–––––1,0Wie bewerten Sie den Theorieteil?34––––1,5Wie bewerten Sie den dritten Praxisteil?7–––––1,0Wie bewerten Sie den vierten Praxisteil?61––––1,14Wie bewerten Sie die Hospitation?4–––––1,0Wie bewerten Sie das Ausgangstestat?7–––––1,0Wie bewerten Sie das Wahlfach insgesamt?8–––––1,0Wie bewerten Sie die Organisation des Wahlfachs?8–––––1,0Wie bewerten Sie den didaktischen Aufbau?8–––––1,0Wie bewerten Sie die Motivation des Dozenten?8–––––1,0
Zum Zeitpunkt des Wahlfachs befand sich noch kein Teilnehmender im praktischen Jahr (PJ), einer hatte schon vorher beabsichtigt, dies in der Urologie abzuleisten. Nach Abschluss des Wahlfachs haben 4 Studierende ihr PJ fest ohne Urologie geplant, wobei zumindest ein Teilnehmender dies möglicherweise noch ändern möchte. Bei 4 weiteren Studierenden zählt Urologie mit zur engeren Auswahl.
Am Ende des Wahlfachs konnten sich die Studierenden deutlich besser vorstellen, später als Urologe/in tätig zu werden (p = 0,007, s. Abb. 3). Auch wenn die Bereitschaft, später als Chirurg/in zu arbeiten, ebenfalls zunahm, war dieser Unterschied nicht statistisch signifikant (p = 0,066).
Diskussion:
Roboterassistierte Operationen sind heute nicht mehr aus dem Alltag vieler urologischer Kliniken wegzudenken. Im Jahr 2018 wurden in Deutschland von insgesamt 11.492 radikalen Prostatektomien 39,1 % robotisch und damit fast doppelt so viele wie offen (2253, entsprechend 19,6 %) durchgeführt. Der Anteil laparoskopischer radikaler Prostatektomien war mit 41,3 % nur knapp höher [7]. Bei den Nierenteilresektionen im Jahr 2019 in Deutschland wurden demgegenüber mehr als doppelt so viele Eingriffe robotisch wie laparoskopisch durchgeführt (28,8 % vs. 13 %), und dieser Anteil stieg im Jahr 2020 auf 36,9 % (3577 von 9700 Eingriffen; [24]). Demzufolge sollte die Robotik auch ein fester Bestandteil der (urologischen) Lehre im Medizinstudium sein. Das Medizinstudium befindet sich derzeit mit dem „Masterplan Medizinstudium 2020“ in einem Umbruch und der nationale kompetenzbasierte Lernzielkatalog (NKLM) wurde kürzlich überarbeitet [4, 5]. Dadurch ergeben sich einerseits Chancen für die Urologie, da ein stärkerer Fokus auf die Anwendungsebene im Medizinstudium gelegt wird und viele Prüfungen bspw. im OSCE-Format („objective structured clinical examination“) abgehalten werden sollen [18]. Andererseits wird auch die Digitalisierung in der Medizin einen weiteren Kernaspekt im Medizinstudium spielen, die roboterassistierte Chirurgie ist jedoch weiterhin unterrepräsentiert [22]. Um die urologische Lehre in unserer Klinik um Lehrinhalte der robotischen Chirurgie zu erweitern, haben wir kürzlich deutschlandweit erstmalig in einer urologischen Klinik das Wahlfach „Robotische Chirurgie“ angeboten. In einer Kleingruppe von 10 Studierenden der klinischen Semester wurden in 6 Treffen à 2 h theoretische Inhalte und praktische Fertigkeiten vermittelt. Die Teilnehmenden hatten nicht nur einen signifikanten Zuwachs an Wissen und Fertigkeiten, auch ihre Bereitschaft, später in der Urologie tätig zu werden, stieg messbar an.
Bislang existieren nur wenige strukturierte und evaluierte Lehrkonzepte der robotischen Chirurgie für Studierende. Das 2‑wöchige Curriculum von Mullens et al. ähnelt einem Intensivkurs mit sehr hohem Praxisanteil und einer Vielzahl an Übungen am Simulator, aber auch im Wet Lab sowie einer Assistenz während einer robotischen Operation [13]. Moglia et al. konzipierten demgegenüber ein strukturiertes 4‑stündiges Training sowohl am Laparoskopie- als auch am Robotiksimulator [12]. Auch hier geht den praktischen Anteilen ein theoretischer Abschnitt mit Prüfung voraus. Kuhn et al. legen in ihrem deutlich weiter gefassten Lehrkonzept zur „Medizin im digitalen Zeitalter“ nur in einem von fünf Modulen den Fokus auf die Robotik, neben Virtual und Augmented Reality [9]. Es sollen nicht nur Basisfertigkeiten am Simulator für robotische Chirurgie geübt werden, sondern auch immersive Simulationen laparoskopischer Cholezystektomien oder eine leberchirurgische OP-Planung mittels Augmented Reality durchgeführt werden. Auch wenn sich diese Programme teils deutlich von dem hier präsentierten Wahlfach unterscheiden, enthalten sie sowohl theoretische als auch möglichst viele praktische Anteile. Mullens et al. bieten ebenso eine Hospitation während einer robotischen Operation an.
In der theoretischen Eingangsprüfung unseres Wahlfachs (s. Supplement) konnten fast alle Teilnehmende ohne Vorbereitung bereits mehr als die Hälfte der Fragen beantworten, was auf ein gewisses Maß an Vorwissen hindeutet. Im Ausgangstestat wurden jedoch im Median 3,5 von 10 Fragen mehr richtig beantwortet. Folglich fand eine nachhaltige Vermittlung theoretischen Wissens statt, da zu diesem Zeitpunkt der Vortrag über die theoretischen Grundlagen bereits 4 Wochen zurücklag. An dieser Stelle sei explizit hervorgehoben, dass im Theorieteil nicht nur die Vor- sondern auch Nachteile roboterassistierten Operierens (insbesondere die im Vergleich zur Laparoskopie höheren Kosten) explizit diskutiert wurden, um den Studierenden ehrliche und vorurteilsfreie Einblicke zu geben.
Bei den praktischen Übungen am robotischen Simulationssystem direkt an der Operationskonsole war nur in 2 von 3 Fällen eine signifikante Verbesserung der Gesamtpunktzahl bei den Teilnehmenden messbar. Die Auswahl der Übungen zielte darauf ab, grundlegende Fähigkeiten im Umgang mit der robotischen Operationskonsole zu testen (Clutch: Verwendung der Kupplung, Camera 0: Steuerung der Kamera, Sea Spikes 1: Bewegung der Greifarme). Die Übung „Clutch“ ist aber offensichtlich so wenig anspruchsvoll, dass eine tatsächliche Verbesserung in der Bedienung anhand der Gesamtpunktzahl nicht abzubilden ist: Auch ohne vorherige Einweisung wurden bei der allerersten Durchführung im Median 98 von 100 möglichen Punkten erreicht. Die mit den Instrumenten zurückgelegte Bewegungsstrecke während der Übung verdeutlicht jedoch, dass sich die Bewegungsökonomie der Teilnehmenden verbesserte, da die zurückgelegte Strecke um 30 % abnahm. Dass sich die Teilnehmenden durch das Training in ihren praktischen Fähigkeiten verbesserten, ist nicht verwunderlich, war zu erwarten und konnte bereits bei anderen laparoskopischen Simulatortrainings gezeigt werden [6, 14]. Dabei muss hervorgehoben werden, dass es letztlich unerheblich zu sein scheint, ob komplett virtuell am Simulator oder im Wet Lab bspw. an echtem biologischen Gewebe geübt wird, denn der Lernzuwachs ist vergleichbar [1]. In zukünftigen Wahlfächern könnte insofern also nicht nur rein virtuell, sondern auch mit den eigens dafür konzipierten Übungsinstrumenten für das robotische Operationssystem an einem Hands-on-Phantommodell geübt werden.
Als organisatorischer Flaschenhals im Praxisteil stellte sich der Zugang zum Simulatorsystem dar, weil zumindest in unserer Klinik die Robotersysteme regelhaft im Tagesprogramm ausgelastet sind. Der DaVinci® SimNow®-Simulator als Nachfolger des DaVinci® Skills®-Simulator wird ebenfalls auf der Rückseite der Operationskonsole befestigt und ermöglicht das Erlernen grundlegender Steuerungsfunktionen der Konsole bis hin zur Simulation komplexer Operationen (bspw. der radikalen Prostatektomie). Er kann allerdings nur dann verwendet werden, wenn die Konsole nicht gerade bei einer Operation eingesetzt wird. Daher wurde der Beginn des Wahlfachs auf den späten Nachmittag angesetzt und damit einen Zeitpunkt, zu dem das reguläre Operationsprogramm bereits beendet ist. Im Optimalfall kann natürlich auf ein System zurückgegriffen werden, das ausschließlich dem Trainieren dient, was aber im Falle des hier eingesetzten robotischen Simulationssystems mit hohen fünfstelligen Kosten verbunden ist, da eine eigene Operationskonsole benötigt wird. Als Alternativen existieren auch Standalone-Systeme von anderen Anbietern, wie der dV-Trainer® (Mimic Technologies, Seattle, WA, USA) oder RobotiX Mentor® (3D Systems, ehemals Simbionix, Beit Golan, Israel; [10, 11, 20]). Diese Systeme ermöglichen ebenfalls ein Erlernen der grundlegenden Funktionen der Robotersysteme mit entsprechenden Trainingsprogrammen, wobei diese durchaus unterschiedlich strukturiert sind und ebenfalls schnell mehr als 100.000 US$ kosten [2]. Da immer nur ein Teilnehmender alleine an der Konsole üben kann, haben wir die Gruppe von 10 Studierenden auf zwei Teams à 5 Personen an den Praxisterminen verkleinert, um die Wartezeiten zu reduzieren. Zusätzlich wurde die Operationskonsole an einen externen Monitor angeschlossen, damit die Wartenden dem Übenden zuschauen können und daraus einen zusätzlichen Lerneffekt erzielen. Es ist natürlich auch denkbar, mehrere Übungsstationen gleichzeitig anzubieten, zwischen denen rotiert wird (bspw. unter Einbeziehung studentischer Hilfskräfte). Diese studentischen Hilfskräfte könnten dann auch das ärztliche Personal bei der Durchführung des Wahlfachs entlasten, zumal sich der Zeitaufwand in der Vor- und Nachbereitung als nicht unerheblich erwies. Darüber hinaus könnte das Wahlfach gleichzeitig als Rekrutierungsweg für zukünftige Doktorandinnen und Doktoranden dienen, indem die Teilnehmer für eine Promotionsstelle angeworben werden, im Folgenden selber ein Wahlfach leiten und später andere Doktorandinnen und Doktoranden im Rahmen eines Mentoringprogramms betreuen. Schließlich erscheint aus der wissenschaftlichen Perspektive heraus ein solches Lehrkonzept auch gut für die Durchführung von Trainingsstudien an Robotik- oder Laparoskopietrainern geeignet, die aufgrund ihres zeitlichen Aufwands kaum alleine von Ärzten geleitet werden können [15, 17].
Das Wahlfach sollte aber nicht nur theoretische und praktische Anteile enthalten, sondern auch eine Hospitation während einer robotischen Operation. Diese stellt eine sinnvolle Ergänzung dar, weil gewonnenes Wissen und erlernte Fertigkeiten direkt auf die intraoperative Situation übertragen werden können, was sehr positiv evaluiert wurde. Zum gegenwärtigen Zeitpunkt haben noch nicht alle Teilnehmenden während einer robotischen Operation hospitiert. Dies ist damit zu erklären, dass das Wahlfach nicht wie geplant zu Semesteranfang starten konnte und sich in den vergangenen Wochen fast alle Teilnehmenden auf Prüfungen am Semesterende vorbereiten mussten.
Einen für Urologen/innen berufspolitisch äußerst bedeutsamen Aspekt ist der Umstand, dass die Bereitschaft, möglicherweise in der Urologie tätig zu werden, am Ende des Wahlfachs bei den Teilnehmenden signifikant höher ausfiel. Die Arbeitsgemeinschaft Junge Urologen hat im Rahmen der „Zukunftsoffensive 2025“ klar zur Thematik der Nachwuchsförderung Stellung genommen und eine verstärkte Studentenbindung unter dem Slogan „Urologen der Zukunft“ als eines der Hauptziele benannt [19]. Die Faszination an der robotischen Chirurgie stellt eine bedeutsame Facette unseres Fachbereichs dar, die zukünftig noch stärker dafür eingesetzt werden sollte, aktiv für und um den urologischen Nachwuchs zu werben. In diesem Zusammenhang ist es sicher kein Zufall, dass das auf dem 71. Jahreskongress der DGU in Hamburg im Jahr 2019 vorgestellte Image-Video für unseren Fachbereich mit dem Motto „Die Vielfalt wartet. Worauf wartest du?“ als Teil der „Zukunftsoffensive Urologie“ direkt mit einer robotischen Operation beginnt. Insgesamt stieß unser strukturiertes Lehrkonzept mit mehr als 30 Anfragen für 10 Plätze auf großes Interesse. Es besteht offensichtlich ein großer Bedarf von studentischer Seite, sich in der robotischen Chirurgie zu bilden, weswegen bisher eine eigene Vorlesung über urologische Robotik gehalten wurde. Auch andere Fachbereiche haben das Potenzial der Faszination um robotische Chirurgie bereits erkannt und möchten dieses zukünftig stärker nutzen [8].
Als Limitation dieser Arbeit ist zu benennen, dass es sich hierbei um die allererste Durchführung mit einer limitierten Zahl an Teilnehmenden handelt, die Ergebnisse sind somit als präliminär zu bewerten. Es besteht die Möglichkeit, dass ein Selektionsbias in der Gruppenzusammensetzung vorliegt, weil bei den Teilnehmenden möglicherweise eine ohnehin schon vergleichsweise hohe Affinität zu chirurgischen Fächern bestand und deswegen die Effekte des Trainings überschätzt werden. Hierbei ist einschränkend anzumerken, dass vor dem Wahlfach zumindest bei den meisten Teilnehmern keine besondere Verbindung zur Urologie bestand. Eine unmittelbare Übertragbarkeit auf andere Kliniken ist aufgrund der hohen Kosten für ein robotisches Simulatorsystem nur bedingt gegeben, sofern dieses noch angeschafft werden müsste. Alternativ bieten sich hier natürlich auch Laparoskopietrainer an, die mit weniger Kosten verbunden sind – aber das robotische Add-on nicht bieten können. Nicht zuletzt wird sich erst in der Zukunft zeigen, ob die Teilnehmenden tatsächlich eine Famulatur oder ein Teil des praktischen Jahres in der Urologie ableisten – oder gar in einer urologischen Klinik anfangen werden, zu arbeiten. Dieser longitudinale Aspekt lässt sich nur im Verlauf beantworten und steht noch aus.
Ausblick:
Diese ersten Erfahrungen mit dem Wahlfach „Robotische Chirurgie“ zeigen, dass offensichtlich ein Bedarf von studentischer Seite besteht, sich in der roboterassistierten Chirurgie zu bilden. Das Wahlfach ermöglicht einerseits, den Studierenden theoretische wie praktische Inhalte der Robotik zu vermitteln. Andererseits erscheint es auch als ein gutes berufspolitisches Instrument, auf unser Fachgebiet aufmerksam zu machen, Interesse zu wecken und die Faszination an der robotischen urologischen Chirurgie zu transportieren. Darüber hinaus erscheinen Wahlfächer wie diese als geeignetes Medium, wissenschaftliche Fragestellungen bspw. zu Lernkurvenanalysen zu beantworten. Aus diesem Grund wird es zumindest in unserer Klinik kein einmaliges Angebot bleiben, und zukünftige Wahlfächer „Robotische Chirurgie“ werden folgen.
Infobox 1 Mehr Informationen zum Thema
DRUS robotisches Curriculum: https://www.dgru.de/ERUS robotisches Curriculum: http://uroweb.org/section/erus/educationDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)
DRUS robotisches Curriculum: https://www.dgru.de/
ERUS robotisches Curriculum: http://uroweb.org/section/erus/education
DGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)
DRUS robotisches Curriculum: https://www.dgru.de/ERUS robotisches Curriculum: http://uroweb.org/section/erus/educationDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)
DRUS robotisches Curriculum: https://www.dgru.de/
ERUS robotisches Curriculum: http://uroweb.org/section/erus/education
DGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)
Infobox 1 Mehr Informationen zum Thema:
DRUS robotisches Curriculum: https://www.dgru.de/ERUS robotisches Curriculum: http://uroweb.org/section/erus/educationDGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)
DRUS robotisches Curriculum: https://www.dgru.de/
ERUS robotisches Curriculum: http://uroweb.org/section/erus/education
DGU-Imagefilm „Die Vielfalt wartet – worauf wartest du?“: https://www.youtube.com/watch?v=omEGZcgU754 (Alternativ unter urologenportal.de > Fachbesucher > DGU-Imagefilm)
Fazit für die Praxis:
Vor dem Hintergrund der weiten Verbreitung roboterassistierter Operationen ist die robotische Chirurgie im Lehrplan heutiger Medizinstudierender unterrepräsentiert.Es besteht offensichtlich ein Bedarf von studentischer Seite, sich entsprechend zu bilden.Ein Wahlfach „Robotische Chirurgie“ erscheint in diesem Zusammenhang als geeignetes Format, theoretische Grundlagen und praktische Fertigkeiten in der robotischen (urologischen) Chirurgie zu vermitteln.Organisatorisch bietet sich die Aufteilung in einen Theorie-, mehrere Praxisteile und eine Hospitation an.Die Gruppen sollten nicht zu groß sein, insbesondere im Praxisteil maximal 5 Personen umfassen.Jenseits der edukativen Perspektive kann ein solches Wahlfach auf die faszinierende Vielfältigkeit der Urologie aufmerksam machen und möglicherweise auch neue Kolleginnen und Kollegen gewinnen.
Vor dem Hintergrund der weiten Verbreitung roboterassistierter Operationen ist die robotische Chirurgie im Lehrplan heutiger Medizinstudierender unterrepräsentiert.
Es besteht offensichtlich ein Bedarf von studentischer Seite, sich entsprechend zu bilden.
Ein Wahlfach „Robotische Chirurgie“ erscheint in diesem Zusammenhang als geeignetes Format, theoretische Grundlagen und praktische Fertigkeiten in der robotischen (urologischen) Chirurgie zu vermitteln.
Organisatorisch bietet sich die Aufteilung in einen Theorie-, mehrere Praxisteile und eine Hospitation an.
Die Gruppen sollten nicht zu groß sein, insbesondere im Praxisteil maximal 5 Personen umfassen.
Jenseits der edukativen Perspektive kann ein solches Wahlfach auf die faszinierende Vielfältigkeit der Urologie aufmerksam machen und möglicherweise auch neue Kolleginnen und Kollegen gewinnen.
Supplementary Information:
:
|
Background:
Even though robot-assisted operations have evolved to a standard procedure in surgery, they are underrepresented in the curriculum of current medical students.
Methods:
Ten undergraduates in their final years were taught the theoretical basics and practical skills in robot-assisted surgery within six lessons each lasting 2 h, including the opportunity to observe a live robot-assisted surgery. The increase of knowledge (ten multiple-choice questions) and skills (exercises Camera 0, Clutch, and Sea Spikes 1) on a robotic simulation device were quantified including an evaluation of the student's perspective.
Results:
The 10 participants had a significant increase in knowledge and gave at a median of 3.5 additional correct answers in the final assessment (p = 0.011). For two out of three practical exercises, the overall score significantly increased (Camera 0 and Sea Spikes 1, for both p < 0.05), but for the exercise "Clutch", only economy of motion significantly improved (p = 0.028). The elective was evaluated (very) good and the willingness of the participants to become urologists significantly increased (p = 0.007).
Conclusions:
There is a great interest of many undergraduate medical students in robot-assisted surgery. Offering an elective appears to be an excellent format to teach the theoretical background and practical skills in robotic (urologic) surgery. Moreover, such an elective could raise more attention to the field of urology and might attract future colleagues.
| null | null | 3,962 | 303 | 9 |
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[CONTENT] Robotische Chirurgie | Robotik | Simulationstraining | Medizinische Lehre | Medizinstudium | Robotic surgical procedures | Robotics | Simulation training | Medical education | Medical studies [SUMMARY]
|
[CONTENT] Robotische Chirurgie | Robotik | Simulationstraining | Medizinische Lehre | Medizinstudium | Robotic surgical procedures | Robotics | Simulation training | Medical education | Medical studies [SUMMARY]
| null | null | null | null |
[CONTENT] Clinical Competence | Curriculum | Humans | Robotic Surgical Procedures | Robotics | Urology [SUMMARY]
|
[CONTENT] Clinical Competence | Curriculum | Humans | Robotic Surgical Procedures | Robotics | Urology [SUMMARY]
| null | null | null | null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null | null | null | null |
[CONTENT] der | die | und | im | zu | ein | als | mit | das | eine [SUMMARY]
|
[CONTENT] der | die | und | im | zu | ein | als | mit | das | eine [SUMMARY]
| null | null | null | null |
[CONTENT] der | und | die | chirurgie | zu | ein | robotische chirurgie | robotische | im | sich [SUMMARY]
|
[CONTENT] der | und | die | zu | im | ein | https www | robotisches curriculum | imagefilm | de [SUMMARY]
| null | null | null | null |
[CONTENT] ||| ||| [SUMMARY]
|
[CONTENT] ||| Ten | their final years | six lessons | 2 ||| ten | Camera | Clutch | Sea Spikes ||| ||| 10 | 3.5 | 0.011 ||| two | three | Camera 0 | Sea Spikes | 1 | 0.05 | 0.028 ||| 0.007 ||| ||| ||| [SUMMARY]
| null |
||
Evaluation of an Intervention to Promote Self-Management Regarding Cardiovascular Disease: The Social Engagement Framework for Addressing the Chronic-Disease-Challenge (SEFAC).
|
36293726
|
Cardiovascular diseases (CVD) are predominantly lifestyle related. Mental health issues also influence CVD progression and quality of life. Self-management of lifestyle behaviors and mental well-being may play a significant role in reducing the CVD burden. Previous studies have shown that mindfulness practices are associated with psychological well-being, but their effects on CVD self-management are mainly unknown.
|
BACKGROUND
|
The study had a before-after design and included adults over 50 years with CVD and/or one or more risk factors from three European countries. Follow-up was six months. The intervention was a 7-week mindfulness-based intervention (MBI) in a group setting focusing on chronic disease self-management. Outcomes were measured with validated self-report questionnaires at baseline and follow-up: self-efficacy, physical activity, nutrition, smoking, alcohol use, sleep and fatigue, social support, stress, depression, medication adherence, and self-rated health.
|
METHODS
|
Among 352 participants, 324 (92%) attended ≥4 of the 7 group sessions and completed follow-up. During follow-up, self-efficacy, stress, social support, depressive symptoms, and self-rated health significantly improved. No significant changes were detected for other outcomes.
|
RESULTS
|
A 7-week MBI focusing on chronic disease self-management was conducive to improved self-efficacy, emotional well-being, social support, and self-rated overall health during six months. These findings support the use of MBIs for improving self-management in cardiovascular care. ISRCTN registry-number ISRCTN11248135.
|
CONCLUSIONS
|
[
"Adult",
"Humans",
"Self-Management",
"Cardiovascular Diseases",
"Social Participation",
"Quality of Life",
"Chronic Disease"
] |
9603702
|
1. Introduction
|
Despite substantial improvements in outcomes in recent decades, cardiovascular diseases (CVD) are still the leading causes of morbidity and mortality globally, with an estimated 17 million deaths each year [1].
Much of the global burden of CVD is attributable to uncontrolled behavioral risk factors, including poor diet, physical inactivity, and smoking [2]. In addition, psychological distress, such as chronic stress, depression, anxiety, and post-traumatic stress disorder, contributes to poorer quality of life and increases the risk of cardiovascular events and all-cause mortality [3,4]. Conversely, favorable lifestyle behaviors and psychological well-being promote cardiovascular health [5]. For the primary and secondary prevention of atherosclerotic vascular disease, heart failure, atrial fibrillation, and other cardiovascular conditions, lifestyle behaviors and mental health are of great importance [6]. Since patients spend very little time with healthcare providers, individuals largely depend on self-care to promote physical and emotional health and manage chronic illness. Despite the importance and effectiveness of self-care in preventing and managing CVD, many individuals find it challenging to make enduring modifications in lifestyle behaviors, take care of their mental health, and deal with chronic conditions [7,8]. Programs to enhance self-management skills based on behavioral interventions are only modestly effective, and novel intervention targets are needed to improve their impact [9].
One such intervention target is mindfulness, commonly defined as moment-to-moment awareness of one’s experience without judgement [10]. Mindfulness is considered a metacognitive process that enhances the capacity for self-regulation, i.e., adaptively regulating one’s attention, emotions, cognition, and behavior to respond effectively to internal and external demands. Mindfulness is deeply rooted in ancient Eastern philosophies and has received considerable public interest in recent decades. This universal human capacity can be strengthened through meditation, mind–body practices, such as yoga, and the application of mindful attention in daily life. A growing body of evidence indicates that mindfulness-based interventions (MBIs) can help people cope across a broad range of medical and psychological conditions, including depression, stress, anxiety, and chronic pain [11,12]. MBIs are commonly offered as secular, manualized, group-based interventions, the most popular of which are mindfulness-based stress reduction (MBSR) and mindfulness-based cognitive therapy. Typically, a package of mindfulness practices is provided for eight weeks, including body scan, sitting meditation, walking meditation, and gentle yoga exercises. Although mindfulness practices also produce relaxation in the body, relaxation is not the primary objective. Instead, these practices teach participants to focus on present-moment experiences with an orientation of openness, kindness, curiosity, and acceptance instead of ruminating about the past or worrying about the future. By cultivating an even-minded, witnessing relationship with (distressing) physical sensations, emotions, and thoughts as passing events arising in the body-mind system, practitioners learn to undo automatic habitual responses, counter negative thought patterns, and increase cognitive flexibility. Through modulation of attention control, emotion regulation, self-awareness, motivation and learning, mindfulness is postulated to influence self-regulation and behaviors that affect physical or mental health and quality of life [13].
Although preliminary research suggests that mindfulness may influence blood pressure and glycemic control, the potential of mindfulness training in facilitating self-management of CVD has received limited attention [14]. Therefore, the Social Engagement Framework for Addressing the Chronic-disease-challenge (SEFAC) study aimed to examine the effects of the 7-week SEFAC intervention targeting health behaviors and psychosocial factors for improving self-management in adults over 50 with or at increased risk of CVD.
|
2. Methods
|
2.1. Study Design The SEFAC study had a before–after single-group design and was conducted between 28 November 2018 and 26 October 2020 in 4 European countries (Croatia, Italy, United Kingdom (UK), and the Netherlands). For full details of study design and protocol, see Zhang et al. [15] ISRCTN registry number is ISRCTN11248135, the date of registration is 30 August 2018 (retrospectively registered). Ethics approval was provided by the human research ethics boards of the study sites [15]. All participants provided written informed consent. This article reports on health outcomes as assessed at six months follow-up. We used the SPIRIT reporting guidelines [16].
The SEFAC study had a before–after single-group design and was conducted between 28 November 2018 and 26 October 2020 in 4 European countries (Croatia, Italy, United Kingdom (UK), and the Netherlands). For full details of study design and protocol, see Zhang et al. [15] ISRCTN registry number is ISRCTN11248135, the date of registration is 30 August 2018 (retrospectively registered). Ethics approval was provided by the human research ethics boards of the study sites [15]. All participants provided written informed consent. This article reports on health outcomes as assessed at six months follow-up. We used the SPIRIT reporting guidelines [16].
2.2. Recruitment and Eligibility Criteria Recruitment for participation in a study investigating a new intervention to improve the self-management of chronic cardiovascular diseases through mindfulness training, social engagement, and e-health support was conducted in the study sites (located in Rijeka, Croatia; Treviso, Italy; Camborne, United Kingdom; and Rotterdam, the Netherlands). Community-dwelling citizens were recruited using public events and announcements, patient and volunteer organizations, and social media. Main eligibility criteria were age over 50 years and established CVD (i.e., ischemic heart disease, cerebrovascular disease, peripheral artery disease, heart failure, or other cardiovascular conditions) and/or one or more risk factors for the development of CVD (including diabetes, smoking, hypertension, hypercholesterolemia, obesity, positive family history). In addition, citizens were not eligible to participate in the study when they were diagnosed with mild or severe cognitive impairment, were terminally ill or scheduled to enter secondary or tertiary care settings for a long period, not able to comprehend the information provided in the local language, or not able to make an informed decision regarding participation in the study.
Recruitment for participation in a study investigating a new intervention to improve the self-management of chronic cardiovascular diseases through mindfulness training, social engagement, and e-health support was conducted in the study sites (located in Rijeka, Croatia; Treviso, Italy; Camborne, United Kingdom; and Rotterdam, the Netherlands). Community-dwelling citizens were recruited using public events and announcements, patient and volunteer organizations, and social media. Main eligibility criteria were age over 50 years and established CVD (i.e., ischemic heart disease, cerebrovascular disease, peripheral artery disease, heart failure, or other cardiovascular conditions) and/or one or more risk factors for the development of CVD (including diabetes, smoking, hypertension, hypercholesterolemia, obesity, positive family history). In addition, citizens were not eligible to participate in the study when they were diagnosed with mild or severe cognitive impairment, were terminally ill or scheduled to enter secondary or tertiary care settings for a long period, not able to comprehend the information provided in the local language, or not able to make an informed decision regarding participation in the study.
2.3. Intervention The SEFAC intervention to promote health behaviors and psychological well-being had a duration of 7 weeks, and consisted of three elements, (i) weekly mindfulness-based sessions for seven weeks, (ii) support by social engagement, and (iii) e-health support by the SEFAC app [15].
Ad (i): The mindfulness-based sessions were offered in a series of seven weekly in-person group sessions (11–12 participants), lasting 2–2.5 h each for which a workbook was available. The sessions focused on mindset, habit change, relationships, and living with chronic conditions and included elements of positive psychology and health coaching in addition to mindfulness training. They were conducted by certified mindfulness professionals and/or health care professionals with training in mindfulness. Furthermore, local volunteers facilitated the intervention during and between the sessions in terms of logistics, raising awareness for self-management, motivating participants to commit to the intervention, and assisting them with e-health support. The intervention incorporated the core elements of the standard MBSR protocol: (a) mental and physical mindfulness exercises (body scan, sitting meditation, walking meditation, gentle yoga exercises; Supplement Table S1; (b) how to apply mindfulness to everyday situations and stress management; (c) sharing of experiences of mindfulness and insights into automatic patterns and habits among participants. During the sessions, participants were trained to foster greater awareness of present moment experience to promote mental well-being, build self-efficacy, enhance quality of life, adopt a healthy lifestyle, and connect to their community.
Ad (ii): Social engagement was used to support the mindfulness-based sessions [17]. Volunteers facilitated the organization and the sessions (see above). The group sessions provided an environment for peer-to-peer support allowing participants to share their experiences, offer encouragement, and share advice. Group factors such as normalizing one’s experience, a sense of shared group identity, and the empathic and anxiety-relieving nature of the group setting are considered helpful [18].
Ad (iii): In addition to the weekly sessions, participants were encouraged to engage in the practices (described in Supplement Table S1) and reflections in between the sessions using e-health support, consisting of the free SEFAC app developed for the Android operating system. The app included audio recordings of the mindfulness practices, tips and reflections on nutrition, stress reduction, emotional health, and physical activity. In addition, it allowed the user to set personal goals to support habit change. Participants were encouraged to download the app on their mobile phone or tablet at the start of the SEFAC intervention and use it as digital support during the seven weeks of mindfulness-based sessions; it should be noted that the app stayed available to be used without support and on a voluntary basis until six months after the start of the intervention.
The SEFAC intervention to promote health behaviors and psychological well-being had a duration of 7 weeks, and consisted of three elements, (i) weekly mindfulness-based sessions for seven weeks, (ii) support by social engagement, and (iii) e-health support by the SEFAC app [15].
Ad (i): The mindfulness-based sessions were offered in a series of seven weekly in-person group sessions (11–12 participants), lasting 2–2.5 h each for which a workbook was available. The sessions focused on mindset, habit change, relationships, and living with chronic conditions and included elements of positive psychology and health coaching in addition to mindfulness training. They were conducted by certified mindfulness professionals and/or health care professionals with training in mindfulness. Furthermore, local volunteers facilitated the intervention during and between the sessions in terms of logistics, raising awareness for self-management, motivating participants to commit to the intervention, and assisting them with e-health support. The intervention incorporated the core elements of the standard MBSR protocol: (a) mental and physical mindfulness exercises (body scan, sitting meditation, walking meditation, gentle yoga exercises; Supplement Table S1; (b) how to apply mindfulness to everyday situations and stress management; (c) sharing of experiences of mindfulness and insights into automatic patterns and habits among participants. During the sessions, participants were trained to foster greater awareness of present moment experience to promote mental well-being, build self-efficacy, enhance quality of life, adopt a healthy lifestyle, and connect to their community.
Ad (ii): Social engagement was used to support the mindfulness-based sessions [17]. Volunteers facilitated the organization and the sessions (see above). The group sessions provided an environment for peer-to-peer support allowing participants to share their experiences, offer encouragement, and share advice. Group factors such as normalizing one’s experience, a sense of shared group identity, and the empathic and anxiety-relieving nature of the group setting are considered helpful [18].
Ad (iii): In addition to the weekly sessions, participants were encouraged to engage in the practices (described in Supplement Table S1) and reflections in between the sessions using e-health support, consisting of the free SEFAC app developed for the Android operating system. The app included audio recordings of the mindfulness practices, tips and reflections on nutrition, stress reduction, emotional health, and physical activity. In addition, it allowed the user to set personal goals to support habit change. Participants were encouraged to download the app on their mobile phone or tablet at the start of the SEFAC intervention and use it as digital support during the seven weeks of mindfulness-based sessions; it should be noted that the app stayed available to be used without support and on a voluntary basis until six months after the start of the intervention.
2.4. Outcome Measures Outcomes were obtained through self-report questionnaires at the start of the first session (baseline) and at 6-month follow-up to assess the ‘mid-term’ effects (i.e., the sustainability of the effects four months after finishing the intervention), as suggested by Ory et al. and Barkan et al. [19,20]. The internal consistency was assessed for each multi-item scale by computing Cronbach’s alphas, which was considered sufficient when over 0.70, and preferably under 0.95 [21]. The degree of self-efficacy was determined with four instruments: Self-Efficacy for Managing Chronic Diseases 6-item scale (SEMCD-6), Cronbach’s alpha 0.88 General Self-efficacy Scale (GSES), Cronbach’s alpha 0.91; Physical Exercise Self-Efficacy Scale (PESES), Cronbach’s alpha 0.94; Nutrition Self-Efficacy Scale (NSES), Cronbach’s alpha 0.95, with higher values indicating more self-efficacy [22,23,24]. Health behaviors were assessed in six domains, in line with the American College of Lifestyle Medicine [25]: (1) Healthy eating: three items on the intake of fruit and vegetables as well as having breakfast; (2) Physical activity: six items on physical exercise [26]; one item on sedentary behavior: International Physical Activity Questionnaire (IPAQ) [27]; (3) Substance use: current smoking, yes/no; frequency of alcohol use, one item from the AUDIT-C [28]; (4) Stress management: Perceived Stress 10-item Scale (PSS-10), range 0–40, Cronbach’s alpha 0.86, with higher values indicating more stress, score <14 corresponds to low stress and score ≥14 to moderate or high perceived stress [29]; (5) Sleep and fatigue: visual analogue scales (VAS), range 0–10, with higher values indicating worse sleep/more fatigue); (6) Relationships: Oslo Social Support 3-item scale (OSSS-3), Cronbach’s alpha 0.54 [30]. Medication adherence was assessed with the Simplified Medication Adherence Questionnaire (SMAQ), Cronbach’s alpha 0.57 [31]. Depression severity was assessed with the Patient Health Questionnaire 8-item scale (PHQ-8), range 0–24, Cronbach’s alpha 0.82, with higher values indicating higher severity, score ≥10 corresponding to current depression [32]. Health-related quality of life (HR-QoL) was assessed with the Short-Form 12-item health survey (SF-12), range 0–100, Cronbach’s alpha 0.67; the EuroQol-5 Dimensions-5 levels (EQ-5D-5L), using the UK value sets, range 0–1, Cronbach’s alpha 0.73, with higher values indicating better health utility [33,34]; and EQ-VAS, range 0–100, with higher values indicating better HR-QoL.
Outcomes were obtained through self-report questionnaires at the start of the first session (baseline) and at 6-month follow-up to assess the ‘mid-term’ effects (i.e., the sustainability of the effects four months after finishing the intervention), as suggested by Ory et al. and Barkan et al. [19,20]. The internal consistency was assessed for each multi-item scale by computing Cronbach’s alphas, which was considered sufficient when over 0.70, and preferably under 0.95 [21]. The degree of self-efficacy was determined with four instruments: Self-Efficacy for Managing Chronic Diseases 6-item scale (SEMCD-6), Cronbach’s alpha 0.88 General Self-efficacy Scale (GSES), Cronbach’s alpha 0.91; Physical Exercise Self-Efficacy Scale (PESES), Cronbach’s alpha 0.94; Nutrition Self-Efficacy Scale (NSES), Cronbach’s alpha 0.95, with higher values indicating more self-efficacy [22,23,24]. Health behaviors were assessed in six domains, in line with the American College of Lifestyle Medicine [25]: (1) Healthy eating: three items on the intake of fruit and vegetables as well as having breakfast; (2) Physical activity: six items on physical exercise [26]; one item on sedentary behavior: International Physical Activity Questionnaire (IPAQ) [27]; (3) Substance use: current smoking, yes/no; frequency of alcohol use, one item from the AUDIT-C [28]; (4) Stress management: Perceived Stress 10-item Scale (PSS-10), range 0–40, Cronbach’s alpha 0.86, with higher values indicating more stress, score <14 corresponds to low stress and score ≥14 to moderate or high perceived stress [29]; (5) Sleep and fatigue: visual analogue scales (VAS), range 0–10, with higher values indicating worse sleep/more fatigue); (6) Relationships: Oslo Social Support 3-item scale (OSSS-3), Cronbach’s alpha 0.54 [30]. Medication adherence was assessed with the Simplified Medication Adherence Questionnaire (SMAQ), Cronbach’s alpha 0.57 [31]. Depression severity was assessed with the Patient Health Questionnaire 8-item scale (PHQ-8), range 0–24, Cronbach’s alpha 0.82, with higher values indicating higher severity, score ≥10 corresponding to current depression [32]. Health-related quality of life (HR-QoL) was assessed with the Short-Form 12-item health survey (SF-12), range 0–100, Cronbach’s alpha 0.67; the EuroQol-5 Dimensions-5 levels (EQ-5D-5L), using the UK value sets, range 0–1, Cronbach’s alpha 0.73, with higher values indicating better health utility [33,34]; and EQ-VAS, range 0–100, with higher values indicating better HR-QoL.
2.5. Other Measures Sociodemographic characteristics (age, sex, household composition, educational level, income, migration background), BMI (body mass index), and the presence of chronic conditions were assessed by self-report questionnaires. Good adherence to interventions was defined a priori as attending ≥4 of 7 group sessions [35]. The satisfaction with the SEFAC intervention was evaluated with a questionnaire consisting of eight items at 6-month follow-up. Seven items were rated on a 5-point scale, ranging from strongly agree to strongly disagree. One item was rated on a 1-to-10 rating scale, with higher values indicating higher satisfaction.
Sociodemographic characteristics (age, sex, household composition, educational level, income, migration background), BMI (body mass index), and the presence of chronic conditions were assessed by self-report questionnaires. Good adherence to interventions was defined a priori as attending ≥4 of 7 group sessions [35]. The satisfaction with the SEFAC intervention was evaluated with a questionnaire consisting of eight items at 6-month follow-up. Seven items were rated on a 5-point scale, ranging from strongly agree to strongly disagree. One item was rated on a 1-to-10 rating scale, with higher values indicating higher satisfaction.
2.6. Power Considerations The power considerations of the study were described previously [15]. It was planned to include 452 participants at baseline. By assuming a loss to follow-up of 20%, it was expected to have data of 360 participants with a baseline and a follow-up measure. Assuming equal standard deviations (SD) at baseline and follow-up, an alpha of 0.05 and power of 0.80, and by taking into account an average cluster size of 90 participants (360/4) and an intra-class correlation coefficient of 0.02, a difference of 0.24 SD between baseline and follow-up can be established for continuous outcome measures, such as the SF-12 [15].
The power considerations of the study were described previously [15]. It was planned to include 452 participants at baseline. By assuming a loss to follow-up of 20%, it was expected to have data of 360 participants with a baseline and a follow-up measure. Assuming equal standard deviations (SD) at baseline and follow-up, an alpha of 0.05 and power of 0.80, and by taking into account an average cluster size of 90 participants (360/4) and an intra-class correlation coefficient of 0.02, a difference of 0.24 SD between baseline and follow-up can be established for continuous outcome measures, such as the SF-12 [15].
2.7. Statistical Methods Participant characteristics were described using mean (SD) or number of participants (%) for the total study sample. The effects of the intervention were assessed in the participants who completed the baseline and follow-up questionnaires and attended ≥4 sessions. A paired samples t-test was performed to assess the effects of the intervention on continuous outcome measures. Cohen’s d within-group effect size was computed for all significant outcomes [36]. For dichotomous outcome measures, the paired McNemar test was used. We stratified the descriptive statistics (see Supplement Table S2) and effects of the intervention on the outcome measures for the subgroups ‘History of CVD’ and ‘At risk of CVD’ (see Supplement Table S3a,b). The mean change in outcome measures for both subgroups was compared using an independent samples t-test for continuous outcomes and a z-test for dichotomous outcomes (see Supplement Table S3c). We did not impute missing data as only <5% of the data per variable were missing. Analyses were conducted with SPSS version 25.0 (IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA). The 2-sided significance threshold, after Bonferroni correction for multiple testing, was set at p = 0.05/20 = 0.0025.
Participant characteristics were described using mean (SD) or number of participants (%) for the total study sample. The effects of the intervention were assessed in the participants who completed the baseline and follow-up questionnaires and attended ≥4 sessions. A paired samples t-test was performed to assess the effects of the intervention on continuous outcome measures. Cohen’s d within-group effect size was computed for all significant outcomes [36]. For dichotomous outcome measures, the paired McNemar test was used. We stratified the descriptive statistics (see Supplement Table S2) and effects of the intervention on the outcome measures for the subgroups ‘History of CVD’ and ‘At risk of CVD’ (see Supplement Table S3a,b). The mean change in outcome measures for both subgroups was compared using an independent samples t-test for continuous outcomes and a z-test for dichotomous outcomes (see Supplement Table S3c). We did not impute missing data as only <5% of the data per variable were missing. Analyses were conducted with SPSS version 25.0 (IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA). The 2-sided significance threshold, after Bonferroni correction for multiple testing, was set at p = 0.05/20 = 0.0025.
|
3. Results
|
3.1. Description of Participants Between 28 November 2018 and 20 February 2020, 352 participants who fulfilled the inclusion criteria provided informed consent and started the intervention; 77 (21.9%) with a history of CVD and 275 (78.1%) at increased risk of CVD due to the presence of one or more risk factors. Participants were included in 3 European study sites: Rijeka, Croatia (n = 147); Treviso, Italy (n = 97); Rotterdam, the Netherlands (n = 84). At the pilot site in the UK (n = 91), outcome measurements with the self-report questionnaires were not feasible because of a high prevalence of health literacy problems; hence, these participants were not included for further analyses. Table 1 shows the baseline characteristics of the study sample.
Between 28 November 2018 and 20 February 2020, 352 participants who fulfilled the inclusion criteria provided informed consent and started the intervention; 77 (21.9%) with a history of CVD and 275 (78.1%) at increased risk of CVD due to the presence of one or more risk factors. Participants were included in 3 European study sites: Rijeka, Croatia (n = 147); Treviso, Italy (n = 97); Rotterdam, the Netherlands (n = 84). At the pilot site in the UK (n = 91), outcome measurements with the self-report questionnaires were not feasible because of a high prevalence of health literacy problems; hence, these participants were not included for further analyses. Table 1 shows the baseline characteristics of the study sample.
3.2. Adherence to Interventions and Follow-Up Attrition Of all included participants (n = 352), 343 (97.4%) attended four or more of the seven group sessions. A median of 6 out of 7 group sessions (interquartile range [IQR], 6–7) were attended. The number of participants taking part in the 6-month follow-up assessment was 327 (92.9%). Overall, 324/352 participants (92.0%) completed the baseline and 6-month follow-up questionnaires, attended ≥4 sessions, and were included as the study sample for further analyses. Baseline characteristics of the 28 dropout participants were not significantly different from the included participants, except for the study site. The participant flow chart is shown in Figure 1.
Of all included participants (n = 352), 343 (97.4%) attended four or more of the seven group sessions. A median of 6 out of 7 group sessions (interquartile range [IQR], 6–7) were attended. The number of participants taking part in the 6-month follow-up assessment was 327 (92.9%). Overall, 324/352 participants (92.0%) completed the baseline and 6-month follow-up questionnaires, attended ≥4 sessions, and were included as the study sample for further analyses. Baseline characteristics of the 28 dropout participants were not significantly different from the included participants, except for the study site. The participant flow chart is shown in Figure 1.
3.3. Outcomes Table 2 presents the outcome scores at baseline and follow-up of the 324 participants who completed both the baseline and 6-month follow-up questionnaires and who attended ≥4 sessions. Three of the four self-efficacy outcomes significantly improved, except for physical exercise self-efficacy. At the 6-month follow-up, there was a significant reduction in perceived stress and PHQ depression scores and significantly more social support as compared to baseline. Participants of the SEFAC intervention also experienced significant improvements in self-rated overall health and health utility (all p < 0.0025). Over time, no significant changes were observed for nutrition, physical activity, substance use, sleep, medication adherence, or mental and physical HR-QoL.
Table 2 presents the outcome scores at baseline and follow-up of the 324 participants who completed both the baseline and 6-month follow-up questionnaires and who attended ≥4 sessions. Three of the four self-efficacy outcomes significantly improved, except for physical exercise self-efficacy. At the 6-month follow-up, there was a significant reduction in perceived stress and PHQ depression scores and significantly more social support as compared to baseline. Participants of the SEFAC intervention also experienced significant improvements in self-rated overall health and health utility (all p < 0.0025). Over time, no significant changes were observed for nutrition, physical activity, substance use, sleep, medication adherence, or mental and physical HR-QoL.
3.4. Satisfaction and Adverse Events Of those completing the questions on satisfaction with the intervention at the 6-month follow-up, participants reported that the intervention improved self-awareness (84.8%) and stimulated them to work on a healthy lifestyle (85.4%). The average satisfaction score was 8.2 ± 1.6 on a scale from 1 (lowest) to 10 (highest). The majority of participants (75.0%) perceived the SEFAC app as supportive of a healthy lifestyle through mindfulness exercises. (See Supplement Table S4). One participant died during the 6-month follow-up; this event was judged as unrelated to the intervention.
Of those completing the questions on satisfaction with the intervention at the 6-month follow-up, participants reported that the intervention improved self-awareness (84.8%) and stimulated them to work on a healthy lifestyle (85.4%). The average satisfaction score was 8.2 ± 1.6 on a scale from 1 (lowest) to 10 (highest). The majority of participants (75.0%) perceived the SEFAC app as supportive of a healthy lifestyle through mindfulness exercises. (See Supplement Table S4). One participant died during the 6-month follow-up; this event was judged as unrelated to the intervention.
|
5. Conclusions
|
In conclusion, the present study shows promising effects of a mindfulness-based intervention, in combination with social engagement and e-health support. It enhances self-management as a complementary approach for modifying psychosocial factors relevant to cardiovascular care. Our findings deserve further study in a larger, long-term randomized controlled trial to explore the potential of mindfulness training in catalyzing chronic disease self-management. An intervention offering access to high-quality therapeutic lifestyle interventions focusing on nutrition, physical activity, and substance use, in conjunction with ongoing encouragement for mindfulness practice with digital and community resources, may be a sustainable approach for promoting cardiovascular health.
|
[
"2.1. Study Design",
"2.2. Recruitment and Eligibility Criteria",
"2.3. Intervention",
"2.4. Outcome Measures",
"2.5. Other Measures",
"2.6. Power Considerations",
"2.7. Statistical Methods",
"3.2. Adherence to Interventions and Follow-Up Attrition",
"3.3. Outcomes",
"3.4. Satisfaction and Adverse Events"
] |
[
"The SEFAC study had a before–after single-group design and was conducted between 28 November 2018 and 26 October 2020 in 4 European countries (Croatia, Italy, United Kingdom (UK), and the Netherlands). For full details of study design and protocol, see Zhang et al. [15] ISRCTN registry number is ISRCTN11248135, the date of registration is 30 August 2018 (retrospectively registered). Ethics approval was provided by the human research ethics boards of the study sites [15]. All participants provided written informed consent. This article reports on health outcomes as assessed at six months follow-up. We used the SPIRIT reporting guidelines [16].",
"Recruitment for participation in a study investigating a new intervention to improve the self-management of chronic cardiovascular diseases through mindfulness training, social engagement, and e-health support was conducted in the study sites (located in Rijeka, Croatia; Treviso, Italy; Camborne, United Kingdom; and Rotterdam, the Netherlands). Community-dwelling citizens were recruited using public events and announcements, patient and volunteer organizations, and social media. Main eligibility criteria were age over 50 years and established CVD (i.e., ischemic heart disease, cerebrovascular disease, peripheral artery disease, heart failure, or other cardiovascular conditions) and/or one or more risk factors for the development of CVD (including diabetes, smoking, hypertension, hypercholesterolemia, obesity, positive family history). In addition, citizens were not eligible to participate in the study when they were diagnosed with mild or severe cognitive impairment, were terminally ill or scheduled to enter secondary or tertiary care settings for a long period, not able to comprehend the information provided in the local language, or not able to make an informed decision regarding participation in the study. ",
"The SEFAC intervention to promote health behaviors and psychological well-being had a duration of 7 weeks, and consisted of three elements, (i) weekly mindfulness-based sessions for seven weeks, (ii) support by social engagement, and (iii) e-health support by the SEFAC app [15]. \nAd (i): The mindfulness-based sessions were offered in a series of seven weekly in-person group sessions (11–12 participants), lasting 2–2.5 h each for which a workbook was available. The sessions focused on mindset, habit change, relationships, and living with chronic conditions and included elements of positive psychology and health coaching in addition to mindfulness training. They were conducted by certified mindfulness professionals and/or health care professionals with training in mindfulness. Furthermore, local volunteers facilitated the intervention during and between the sessions in terms of logistics, raising awareness for self-management, motivating participants to commit to the intervention, and assisting them with e-health support. The intervention incorporated the core elements of the standard MBSR protocol: (a) mental and physical mindfulness exercises (body scan, sitting meditation, walking meditation, gentle yoga exercises; Supplement Table S1; (b) how to apply mindfulness to everyday situations and stress management; (c) sharing of experiences of mindfulness and insights into automatic patterns and habits among participants. During the sessions, participants were trained to foster greater awareness of present moment experience to promote mental well-being, build self-efficacy, enhance quality of life, adopt a healthy lifestyle, and connect to their community. \nAd (ii): Social engagement was used to support the mindfulness-based sessions [17]. Volunteers facilitated the organization and the sessions (see above). The group sessions provided an environment for peer-to-peer support allowing participants to share their experiences, offer encouragement, and share advice. Group factors such as normalizing one’s experience, a sense of shared group identity, and the empathic and anxiety-relieving nature of the group setting are considered helpful [18]. \nAd (iii): In addition to the weekly sessions, participants were encouraged to engage in the practices (described in Supplement Table S1) and reflections in between the sessions using e-health support, consisting of the free SEFAC app developed for the Android operating system. The app included audio recordings of the mindfulness practices, tips and reflections on nutrition, stress reduction, emotional health, and physical activity. In addition, it allowed the user to set personal goals to support habit change. Participants were encouraged to download the app on their mobile phone or tablet at the start of the SEFAC intervention and use it as digital support during the seven weeks of mindfulness-based sessions; it should be noted that the app stayed available to be used without support and on a voluntary basis until six months after the start of the intervention. ",
"Outcomes were obtained through self-report questionnaires at the start of the first session (baseline) and at 6-month follow-up to assess the ‘mid-term’ effects (i.e., the sustainability of the effects four months after finishing the intervention), as suggested by Ory et al. and Barkan et al. [19,20]. The internal consistency was assessed for each multi-item scale by computing Cronbach’s alphas, which was considered sufficient when over 0.70, and preferably under 0.95 [21]. The degree of self-efficacy was determined with four instruments: Self-Efficacy for Managing Chronic Diseases 6-item scale (SEMCD-6), Cronbach’s alpha 0.88 General Self-efficacy Scale (GSES), Cronbach’s alpha 0.91; Physical Exercise Self-Efficacy Scale (PESES), Cronbach’s alpha 0.94; Nutrition Self-Efficacy Scale (NSES), Cronbach’s alpha 0.95, with higher values indicating more self-efficacy [22,23,24]. Health behaviors were assessed in six domains, in line with the American College of Lifestyle Medicine [25]: (1) Healthy eating: three items on the intake of fruit and vegetables as well as having breakfast; (2) Physical activity: six items on physical exercise [26]; one item on sedentary behavior: International Physical Activity Questionnaire (IPAQ) [27]; (3) Substance use: current smoking, yes/no; frequency of alcohol use, one item from the AUDIT-C [28]; (4) Stress management: Perceived Stress 10-item Scale (PSS-10), range 0–40, Cronbach’s alpha 0.86, with higher values indicating more stress, score <14 corresponds to low stress and score ≥14 to moderate or high perceived stress [29]; (5) Sleep and fatigue: visual analogue scales (VAS), range 0–10, with higher values indicating worse sleep/more fatigue); (6) Relationships: Oslo Social Support 3-item scale (OSSS-3), Cronbach’s alpha 0.54 [30]. Medication adherence was assessed with the Simplified Medication Adherence Questionnaire (SMAQ), Cronbach’s alpha 0.57 [31]. Depression severity was assessed with the Patient Health Questionnaire 8-item scale (PHQ-8), range 0–24, Cronbach’s alpha 0.82, with higher values indicating higher severity, score ≥10 corresponding to current depression [32]. Health-related quality of life (HR-QoL) was assessed with the Short-Form 12-item health survey (SF-12), range 0–100, Cronbach’s alpha 0.67; the EuroQol-5 Dimensions-5 levels (EQ-5D-5L), using the UK value sets, range 0–1, Cronbach’s alpha 0.73, with higher values indicating better health utility [33,34]; and EQ-VAS, range 0–100, with higher values indicating better HR-QoL. ",
"Sociodemographic characteristics (age, sex, household composition, educational level, income, migration background), BMI (body mass index), and the presence of chronic conditions were assessed by self-report questionnaires. Good adherence to interventions was defined a priori as attending ≥4 of 7 group sessions [35]. The satisfaction with the SEFAC intervention was evaluated with a questionnaire consisting of eight items at 6-month follow-up. Seven items were rated on a 5-point scale, ranging from strongly agree to strongly disagree. One item was rated on a 1-to-10 rating scale, with higher values indicating higher satisfaction. ",
"The power considerations of the study were described previously [15]. It was planned to include 452 participants at baseline. By assuming a loss to follow-up of 20%, it was expected to have data of 360 participants with a baseline and a follow-up measure. Assuming equal standard deviations (SD) at baseline and follow-up, an alpha of 0.05 and power of 0.80, and by taking into account an average cluster size of 90 participants (360/4) and an intra-class correlation coefficient of 0.02, a difference of 0.24 SD between baseline and follow-up can be established for continuous outcome measures, such as the SF-12 [15].",
"Participant characteristics were described using mean (SD) or number of participants (%) for the total study sample. The effects of the intervention were assessed in the participants who completed the baseline and follow-up questionnaires and attended ≥4 sessions. A paired samples t-test was performed to assess the effects of the intervention on continuous outcome measures. Cohen’s d within-group effect size was computed for all significant outcomes [36]. For dichotomous outcome measures, the paired McNemar test was used. We stratified the descriptive statistics (see Supplement Table S2) and effects of the intervention on the outcome measures for the subgroups ‘History of CVD’ and ‘At risk of CVD’ (see Supplement Table S3a,b). The mean change in outcome measures for both subgroups was compared using an independent samples t-test for continuous outcomes and a z-test for dichotomous outcomes (see Supplement Table S3c). We did not impute missing data as only <5% of the data per variable were missing. Analyses were conducted with SPSS version 25.0 (IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA). The 2-sided significance threshold, after Bonferroni correction for multiple testing, was set at p = 0.05/20 = 0.0025. ",
"Of all included participants (n = 352), 343 (97.4%) attended four or more of the seven group sessions. A median of 6 out of 7 group sessions (interquartile range [IQR], 6–7) were attended. The number of participants taking part in the 6-month follow-up assessment was 327 (92.9%). Overall, 324/352 participants (92.0%) completed the baseline and 6-month follow-up questionnaires, attended ≥4 sessions, and were included as the study sample for further analyses. Baseline characteristics of the 28 dropout participants were not significantly different from the included participants, except for the study site. The participant flow chart is shown in Figure 1.",
"Table 2 presents the outcome scores at baseline and follow-up of the 324 participants who completed both the baseline and 6-month follow-up questionnaires and who attended ≥4 sessions. Three of the four self-efficacy outcomes significantly improved, except for physical exercise self-efficacy. At the 6-month follow-up, there was a significant reduction in perceived stress and PHQ depression scores and significantly more social support as compared to baseline. Participants of the SEFAC intervention also experienced significant improvements in self-rated overall health and health utility (all p < 0.0025). Over time, no significant changes were observed for nutrition, physical activity, substance use, sleep, medication adherence, or mental and physical HR-QoL. ",
"Of those completing the questions on satisfaction with the intervention at the 6-month follow-up, participants reported that the intervention improved self-awareness (84.8%) and stimulated them to work on a healthy lifestyle (85.4%). The average satisfaction score was 8.2 ± 1.6 on a scale from 1 (lowest) to 10 (highest). The majority of participants (75.0%) perceived the SEFAC app as supportive of a healthy lifestyle through mindfulness exercises. (See Supplement Table S4). One participant died during the 6-month follow-up; this event was judged as unrelated to the intervention."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"1. Introduction",
"2. Methods",
"2.1. Study Design",
"2.2. Recruitment and Eligibility Criteria",
"2.3. Intervention",
"2.4. Outcome Measures",
"2.5. Other Measures",
"2.6. Power Considerations",
"2.7. Statistical Methods",
"3. Results",
"3.1. Description of Participants",
"3.2. Adherence to Interventions and Follow-Up Attrition",
"3.3. Outcomes",
"3.4. Satisfaction and Adverse Events",
"4. Discussion",
"5. Conclusions"
] |
[
"Despite substantial improvements in outcomes in recent decades, cardiovascular diseases (CVD) are still the leading causes of morbidity and mortality globally, with an estimated 17 million deaths each year [1]. \nMuch of the global burden of CVD is attributable to uncontrolled behavioral risk factors, including poor diet, physical inactivity, and smoking [2]. In addition, psychological distress, such as chronic stress, depression, anxiety, and post-traumatic stress disorder, contributes to poorer quality of life and increases the risk of cardiovascular events and all-cause mortality [3,4]. Conversely, favorable lifestyle behaviors and psychological well-being promote cardiovascular health [5]. For the primary and secondary prevention of atherosclerotic vascular disease, heart failure, atrial fibrillation, and other cardiovascular conditions, lifestyle behaviors and mental health are of great importance [6]. Since patients spend very little time with healthcare providers, individuals largely depend on self-care to promote physical and emotional health and manage chronic illness. Despite the importance and effectiveness of self-care in preventing and managing CVD, many individuals find it challenging to make enduring modifications in lifestyle behaviors, take care of their mental health, and deal with chronic conditions [7,8]. Programs to enhance self-management skills based on behavioral interventions are only modestly effective, and novel intervention targets are needed to improve their impact [9]. \nOne such intervention target is mindfulness, commonly defined as moment-to-moment awareness of one’s experience without judgement [10]. Mindfulness is considered a metacognitive process that enhances the capacity for self-regulation, i.e., adaptively regulating one’s attention, emotions, cognition, and behavior to respond effectively to internal and external demands. Mindfulness is deeply rooted in ancient Eastern philosophies and has received considerable public interest in recent decades. This universal human capacity can be strengthened through meditation, mind–body practices, such as yoga, and the application of mindful attention in daily life. A growing body of evidence indicates that mindfulness-based interventions (MBIs) can help people cope across a broad range of medical and psychological conditions, including depression, stress, anxiety, and chronic pain [11,12]. MBIs are commonly offered as secular, manualized, group-based interventions, the most popular of which are mindfulness-based stress reduction (MBSR) and mindfulness-based cognitive therapy. Typically, a package of mindfulness practices is provided for eight weeks, including body scan, sitting meditation, walking meditation, and gentle yoga exercises. Although mindfulness practices also produce relaxation in the body, relaxation is not the primary objective. Instead, these practices teach participants to focus on present-moment experiences with an orientation of openness, kindness, curiosity, and acceptance instead of ruminating about the past or worrying about the future. By cultivating an even-minded, witnessing relationship with (distressing) physical sensations, emotions, and thoughts as passing events arising in the body-mind system, practitioners learn to undo automatic habitual responses, counter negative thought patterns, and increase cognitive flexibility. Through modulation of attention control, emotion regulation, self-awareness, motivation and learning, mindfulness is postulated to influence self-regulation and behaviors that affect physical or mental health and quality of life [13].\nAlthough preliminary research suggests that mindfulness may influence blood pressure and glycemic control, the potential of mindfulness training in facilitating self-management of CVD has received limited attention [14]. Therefore, the Social Engagement Framework for Addressing the Chronic-disease-challenge (SEFAC) study aimed to examine the effects of the 7-week SEFAC intervention targeting health behaviors and psychosocial factors for improving self-management in adults over 50 with or at increased risk of CVD.",
" 2.1. Study Design The SEFAC study had a before–after single-group design and was conducted between 28 November 2018 and 26 October 2020 in 4 European countries (Croatia, Italy, United Kingdom (UK), and the Netherlands). For full details of study design and protocol, see Zhang et al. [15] ISRCTN registry number is ISRCTN11248135, the date of registration is 30 August 2018 (retrospectively registered). Ethics approval was provided by the human research ethics boards of the study sites [15]. All participants provided written informed consent. This article reports on health outcomes as assessed at six months follow-up. We used the SPIRIT reporting guidelines [16].\nThe SEFAC study had a before–after single-group design and was conducted between 28 November 2018 and 26 October 2020 in 4 European countries (Croatia, Italy, United Kingdom (UK), and the Netherlands). For full details of study design and protocol, see Zhang et al. [15] ISRCTN registry number is ISRCTN11248135, the date of registration is 30 August 2018 (retrospectively registered). Ethics approval was provided by the human research ethics boards of the study sites [15]. All participants provided written informed consent. This article reports on health outcomes as assessed at six months follow-up. We used the SPIRIT reporting guidelines [16].\n 2.2. Recruitment and Eligibility Criteria Recruitment for participation in a study investigating a new intervention to improve the self-management of chronic cardiovascular diseases through mindfulness training, social engagement, and e-health support was conducted in the study sites (located in Rijeka, Croatia; Treviso, Italy; Camborne, United Kingdom; and Rotterdam, the Netherlands). Community-dwelling citizens were recruited using public events and announcements, patient and volunteer organizations, and social media. Main eligibility criteria were age over 50 years and established CVD (i.e., ischemic heart disease, cerebrovascular disease, peripheral artery disease, heart failure, or other cardiovascular conditions) and/or one or more risk factors for the development of CVD (including diabetes, smoking, hypertension, hypercholesterolemia, obesity, positive family history). In addition, citizens were not eligible to participate in the study when they were diagnosed with mild or severe cognitive impairment, were terminally ill or scheduled to enter secondary or tertiary care settings for a long period, not able to comprehend the information provided in the local language, or not able to make an informed decision regarding participation in the study. \nRecruitment for participation in a study investigating a new intervention to improve the self-management of chronic cardiovascular diseases through mindfulness training, social engagement, and e-health support was conducted in the study sites (located in Rijeka, Croatia; Treviso, Italy; Camborne, United Kingdom; and Rotterdam, the Netherlands). Community-dwelling citizens were recruited using public events and announcements, patient and volunteer organizations, and social media. Main eligibility criteria were age over 50 years and established CVD (i.e., ischemic heart disease, cerebrovascular disease, peripheral artery disease, heart failure, or other cardiovascular conditions) and/or one or more risk factors for the development of CVD (including diabetes, smoking, hypertension, hypercholesterolemia, obesity, positive family history). In addition, citizens were not eligible to participate in the study when they were diagnosed with mild or severe cognitive impairment, were terminally ill or scheduled to enter secondary or tertiary care settings for a long period, not able to comprehend the information provided in the local language, or not able to make an informed decision regarding participation in the study. \n 2.3. Intervention The SEFAC intervention to promote health behaviors and psychological well-being had a duration of 7 weeks, and consisted of three elements, (i) weekly mindfulness-based sessions for seven weeks, (ii) support by social engagement, and (iii) e-health support by the SEFAC app [15]. \nAd (i): The mindfulness-based sessions were offered in a series of seven weekly in-person group sessions (11–12 participants), lasting 2–2.5 h each for which a workbook was available. The sessions focused on mindset, habit change, relationships, and living with chronic conditions and included elements of positive psychology and health coaching in addition to mindfulness training. They were conducted by certified mindfulness professionals and/or health care professionals with training in mindfulness. Furthermore, local volunteers facilitated the intervention during and between the sessions in terms of logistics, raising awareness for self-management, motivating participants to commit to the intervention, and assisting them with e-health support. The intervention incorporated the core elements of the standard MBSR protocol: (a) mental and physical mindfulness exercises (body scan, sitting meditation, walking meditation, gentle yoga exercises; Supplement Table S1; (b) how to apply mindfulness to everyday situations and stress management; (c) sharing of experiences of mindfulness and insights into automatic patterns and habits among participants. During the sessions, participants were trained to foster greater awareness of present moment experience to promote mental well-being, build self-efficacy, enhance quality of life, adopt a healthy lifestyle, and connect to their community. \nAd (ii): Social engagement was used to support the mindfulness-based sessions [17]. Volunteers facilitated the organization and the sessions (see above). The group sessions provided an environment for peer-to-peer support allowing participants to share their experiences, offer encouragement, and share advice. Group factors such as normalizing one’s experience, a sense of shared group identity, and the empathic and anxiety-relieving nature of the group setting are considered helpful [18]. \nAd (iii): In addition to the weekly sessions, participants were encouraged to engage in the practices (described in Supplement Table S1) and reflections in between the sessions using e-health support, consisting of the free SEFAC app developed for the Android operating system. The app included audio recordings of the mindfulness practices, tips and reflections on nutrition, stress reduction, emotional health, and physical activity. In addition, it allowed the user to set personal goals to support habit change. Participants were encouraged to download the app on their mobile phone or tablet at the start of the SEFAC intervention and use it as digital support during the seven weeks of mindfulness-based sessions; it should be noted that the app stayed available to be used without support and on a voluntary basis until six months after the start of the intervention. \nThe SEFAC intervention to promote health behaviors and psychological well-being had a duration of 7 weeks, and consisted of three elements, (i) weekly mindfulness-based sessions for seven weeks, (ii) support by social engagement, and (iii) e-health support by the SEFAC app [15]. \nAd (i): The mindfulness-based sessions were offered in a series of seven weekly in-person group sessions (11–12 participants), lasting 2–2.5 h each for which a workbook was available. The sessions focused on mindset, habit change, relationships, and living with chronic conditions and included elements of positive psychology and health coaching in addition to mindfulness training. They were conducted by certified mindfulness professionals and/or health care professionals with training in mindfulness. Furthermore, local volunteers facilitated the intervention during and between the sessions in terms of logistics, raising awareness for self-management, motivating participants to commit to the intervention, and assisting them with e-health support. The intervention incorporated the core elements of the standard MBSR protocol: (a) mental and physical mindfulness exercises (body scan, sitting meditation, walking meditation, gentle yoga exercises; Supplement Table S1; (b) how to apply mindfulness to everyday situations and stress management; (c) sharing of experiences of mindfulness and insights into automatic patterns and habits among participants. During the sessions, participants were trained to foster greater awareness of present moment experience to promote mental well-being, build self-efficacy, enhance quality of life, adopt a healthy lifestyle, and connect to their community. \nAd (ii): Social engagement was used to support the mindfulness-based sessions [17]. Volunteers facilitated the organization and the sessions (see above). The group sessions provided an environment for peer-to-peer support allowing participants to share their experiences, offer encouragement, and share advice. Group factors such as normalizing one’s experience, a sense of shared group identity, and the empathic and anxiety-relieving nature of the group setting are considered helpful [18]. \nAd (iii): In addition to the weekly sessions, participants were encouraged to engage in the practices (described in Supplement Table S1) and reflections in between the sessions using e-health support, consisting of the free SEFAC app developed for the Android operating system. The app included audio recordings of the mindfulness practices, tips and reflections on nutrition, stress reduction, emotional health, and physical activity. In addition, it allowed the user to set personal goals to support habit change. Participants were encouraged to download the app on their mobile phone or tablet at the start of the SEFAC intervention and use it as digital support during the seven weeks of mindfulness-based sessions; it should be noted that the app stayed available to be used without support and on a voluntary basis until six months after the start of the intervention. \n 2.4. Outcome Measures Outcomes were obtained through self-report questionnaires at the start of the first session (baseline) and at 6-month follow-up to assess the ‘mid-term’ effects (i.e., the sustainability of the effects four months after finishing the intervention), as suggested by Ory et al. and Barkan et al. [19,20]. The internal consistency was assessed for each multi-item scale by computing Cronbach’s alphas, which was considered sufficient when over 0.70, and preferably under 0.95 [21]. The degree of self-efficacy was determined with four instruments: Self-Efficacy for Managing Chronic Diseases 6-item scale (SEMCD-6), Cronbach’s alpha 0.88 General Self-efficacy Scale (GSES), Cronbach’s alpha 0.91; Physical Exercise Self-Efficacy Scale (PESES), Cronbach’s alpha 0.94; Nutrition Self-Efficacy Scale (NSES), Cronbach’s alpha 0.95, with higher values indicating more self-efficacy [22,23,24]. Health behaviors were assessed in six domains, in line with the American College of Lifestyle Medicine [25]: (1) Healthy eating: three items on the intake of fruit and vegetables as well as having breakfast; (2) Physical activity: six items on physical exercise [26]; one item on sedentary behavior: International Physical Activity Questionnaire (IPAQ) [27]; (3) Substance use: current smoking, yes/no; frequency of alcohol use, one item from the AUDIT-C [28]; (4) Stress management: Perceived Stress 10-item Scale (PSS-10), range 0–40, Cronbach’s alpha 0.86, with higher values indicating more stress, score <14 corresponds to low stress and score ≥14 to moderate or high perceived stress [29]; (5) Sleep and fatigue: visual analogue scales (VAS), range 0–10, with higher values indicating worse sleep/more fatigue); (6) Relationships: Oslo Social Support 3-item scale (OSSS-3), Cronbach’s alpha 0.54 [30]. Medication adherence was assessed with the Simplified Medication Adherence Questionnaire (SMAQ), Cronbach’s alpha 0.57 [31]. Depression severity was assessed with the Patient Health Questionnaire 8-item scale (PHQ-8), range 0–24, Cronbach’s alpha 0.82, with higher values indicating higher severity, score ≥10 corresponding to current depression [32]. Health-related quality of life (HR-QoL) was assessed with the Short-Form 12-item health survey (SF-12), range 0–100, Cronbach’s alpha 0.67; the EuroQol-5 Dimensions-5 levels (EQ-5D-5L), using the UK value sets, range 0–1, Cronbach’s alpha 0.73, with higher values indicating better health utility [33,34]; and EQ-VAS, range 0–100, with higher values indicating better HR-QoL. \nOutcomes were obtained through self-report questionnaires at the start of the first session (baseline) and at 6-month follow-up to assess the ‘mid-term’ effects (i.e., the sustainability of the effects four months after finishing the intervention), as suggested by Ory et al. and Barkan et al. [19,20]. The internal consistency was assessed for each multi-item scale by computing Cronbach’s alphas, which was considered sufficient when over 0.70, and preferably under 0.95 [21]. The degree of self-efficacy was determined with four instruments: Self-Efficacy for Managing Chronic Diseases 6-item scale (SEMCD-6), Cronbach’s alpha 0.88 General Self-efficacy Scale (GSES), Cronbach’s alpha 0.91; Physical Exercise Self-Efficacy Scale (PESES), Cronbach’s alpha 0.94; Nutrition Self-Efficacy Scale (NSES), Cronbach’s alpha 0.95, with higher values indicating more self-efficacy [22,23,24]. Health behaviors were assessed in six domains, in line with the American College of Lifestyle Medicine [25]: (1) Healthy eating: three items on the intake of fruit and vegetables as well as having breakfast; (2) Physical activity: six items on physical exercise [26]; one item on sedentary behavior: International Physical Activity Questionnaire (IPAQ) [27]; (3) Substance use: current smoking, yes/no; frequency of alcohol use, one item from the AUDIT-C [28]; (4) Stress management: Perceived Stress 10-item Scale (PSS-10), range 0–40, Cronbach’s alpha 0.86, with higher values indicating more stress, score <14 corresponds to low stress and score ≥14 to moderate or high perceived stress [29]; (5) Sleep and fatigue: visual analogue scales (VAS), range 0–10, with higher values indicating worse sleep/more fatigue); (6) Relationships: Oslo Social Support 3-item scale (OSSS-3), Cronbach’s alpha 0.54 [30]. Medication adherence was assessed with the Simplified Medication Adherence Questionnaire (SMAQ), Cronbach’s alpha 0.57 [31]. Depression severity was assessed with the Patient Health Questionnaire 8-item scale (PHQ-8), range 0–24, Cronbach’s alpha 0.82, with higher values indicating higher severity, score ≥10 corresponding to current depression [32]. Health-related quality of life (HR-QoL) was assessed with the Short-Form 12-item health survey (SF-12), range 0–100, Cronbach’s alpha 0.67; the EuroQol-5 Dimensions-5 levels (EQ-5D-5L), using the UK value sets, range 0–1, Cronbach’s alpha 0.73, with higher values indicating better health utility [33,34]; and EQ-VAS, range 0–100, with higher values indicating better HR-QoL. \n 2.5. Other Measures Sociodemographic characteristics (age, sex, household composition, educational level, income, migration background), BMI (body mass index), and the presence of chronic conditions were assessed by self-report questionnaires. Good adherence to interventions was defined a priori as attending ≥4 of 7 group sessions [35]. The satisfaction with the SEFAC intervention was evaluated with a questionnaire consisting of eight items at 6-month follow-up. Seven items were rated on a 5-point scale, ranging from strongly agree to strongly disagree. One item was rated on a 1-to-10 rating scale, with higher values indicating higher satisfaction. \nSociodemographic characteristics (age, sex, household composition, educational level, income, migration background), BMI (body mass index), and the presence of chronic conditions were assessed by self-report questionnaires. Good adherence to interventions was defined a priori as attending ≥4 of 7 group sessions [35]. The satisfaction with the SEFAC intervention was evaluated with a questionnaire consisting of eight items at 6-month follow-up. Seven items were rated on a 5-point scale, ranging from strongly agree to strongly disagree. One item was rated on a 1-to-10 rating scale, with higher values indicating higher satisfaction. \n 2.6. Power Considerations The power considerations of the study were described previously [15]. It was planned to include 452 participants at baseline. By assuming a loss to follow-up of 20%, it was expected to have data of 360 participants with a baseline and a follow-up measure. Assuming equal standard deviations (SD) at baseline and follow-up, an alpha of 0.05 and power of 0.80, and by taking into account an average cluster size of 90 participants (360/4) and an intra-class correlation coefficient of 0.02, a difference of 0.24 SD between baseline and follow-up can be established for continuous outcome measures, such as the SF-12 [15].\nThe power considerations of the study were described previously [15]. It was planned to include 452 participants at baseline. By assuming a loss to follow-up of 20%, it was expected to have data of 360 participants with a baseline and a follow-up measure. Assuming equal standard deviations (SD) at baseline and follow-up, an alpha of 0.05 and power of 0.80, and by taking into account an average cluster size of 90 participants (360/4) and an intra-class correlation coefficient of 0.02, a difference of 0.24 SD between baseline and follow-up can be established for continuous outcome measures, such as the SF-12 [15].\n 2.7. Statistical Methods Participant characteristics were described using mean (SD) or number of participants (%) for the total study sample. The effects of the intervention were assessed in the participants who completed the baseline and follow-up questionnaires and attended ≥4 sessions. A paired samples t-test was performed to assess the effects of the intervention on continuous outcome measures. Cohen’s d within-group effect size was computed for all significant outcomes [36]. For dichotomous outcome measures, the paired McNemar test was used. We stratified the descriptive statistics (see Supplement Table S2) and effects of the intervention on the outcome measures for the subgroups ‘History of CVD’ and ‘At risk of CVD’ (see Supplement Table S3a,b). The mean change in outcome measures for both subgroups was compared using an independent samples t-test for continuous outcomes and a z-test for dichotomous outcomes (see Supplement Table S3c). We did not impute missing data as only <5% of the data per variable were missing. Analyses were conducted with SPSS version 25.0 (IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA). The 2-sided significance threshold, after Bonferroni correction for multiple testing, was set at p = 0.05/20 = 0.0025. \nParticipant characteristics were described using mean (SD) or number of participants (%) for the total study sample. The effects of the intervention were assessed in the participants who completed the baseline and follow-up questionnaires and attended ≥4 sessions. A paired samples t-test was performed to assess the effects of the intervention on continuous outcome measures. Cohen’s d within-group effect size was computed for all significant outcomes [36]. For dichotomous outcome measures, the paired McNemar test was used. We stratified the descriptive statistics (see Supplement Table S2) and effects of the intervention on the outcome measures for the subgroups ‘History of CVD’ and ‘At risk of CVD’ (see Supplement Table S3a,b). The mean change in outcome measures for both subgroups was compared using an independent samples t-test for continuous outcomes and a z-test for dichotomous outcomes (see Supplement Table S3c). We did not impute missing data as only <5% of the data per variable were missing. Analyses were conducted with SPSS version 25.0 (IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA). The 2-sided significance threshold, after Bonferroni correction for multiple testing, was set at p = 0.05/20 = 0.0025. ",
"The SEFAC study had a before–after single-group design and was conducted between 28 November 2018 and 26 October 2020 in 4 European countries (Croatia, Italy, United Kingdom (UK), and the Netherlands). For full details of study design and protocol, see Zhang et al. [15] ISRCTN registry number is ISRCTN11248135, the date of registration is 30 August 2018 (retrospectively registered). Ethics approval was provided by the human research ethics boards of the study sites [15]. All participants provided written informed consent. This article reports on health outcomes as assessed at six months follow-up. We used the SPIRIT reporting guidelines [16].",
"Recruitment for participation in a study investigating a new intervention to improve the self-management of chronic cardiovascular diseases through mindfulness training, social engagement, and e-health support was conducted in the study sites (located in Rijeka, Croatia; Treviso, Italy; Camborne, United Kingdom; and Rotterdam, the Netherlands). Community-dwelling citizens were recruited using public events and announcements, patient and volunteer organizations, and social media. Main eligibility criteria were age over 50 years and established CVD (i.e., ischemic heart disease, cerebrovascular disease, peripheral artery disease, heart failure, or other cardiovascular conditions) and/or one or more risk factors for the development of CVD (including diabetes, smoking, hypertension, hypercholesterolemia, obesity, positive family history). In addition, citizens were not eligible to participate in the study when they were diagnosed with mild or severe cognitive impairment, were terminally ill or scheduled to enter secondary or tertiary care settings for a long period, not able to comprehend the information provided in the local language, or not able to make an informed decision regarding participation in the study. ",
"The SEFAC intervention to promote health behaviors and psychological well-being had a duration of 7 weeks, and consisted of three elements, (i) weekly mindfulness-based sessions for seven weeks, (ii) support by social engagement, and (iii) e-health support by the SEFAC app [15]. \nAd (i): The mindfulness-based sessions were offered in a series of seven weekly in-person group sessions (11–12 participants), lasting 2–2.5 h each for which a workbook was available. The sessions focused on mindset, habit change, relationships, and living with chronic conditions and included elements of positive psychology and health coaching in addition to mindfulness training. They were conducted by certified mindfulness professionals and/or health care professionals with training in mindfulness. Furthermore, local volunteers facilitated the intervention during and between the sessions in terms of logistics, raising awareness for self-management, motivating participants to commit to the intervention, and assisting them with e-health support. The intervention incorporated the core elements of the standard MBSR protocol: (a) mental and physical mindfulness exercises (body scan, sitting meditation, walking meditation, gentle yoga exercises; Supplement Table S1; (b) how to apply mindfulness to everyday situations and stress management; (c) sharing of experiences of mindfulness and insights into automatic patterns and habits among participants. During the sessions, participants were trained to foster greater awareness of present moment experience to promote mental well-being, build self-efficacy, enhance quality of life, adopt a healthy lifestyle, and connect to their community. \nAd (ii): Social engagement was used to support the mindfulness-based sessions [17]. Volunteers facilitated the organization and the sessions (see above). The group sessions provided an environment for peer-to-peer support allowing participants to share their experiences, offer encouragement, and share advice. Group factors such as normalizing one’s experience, a sense of shared group identity, and the empathic and anxiety-relieving nature of the group setting are considered helpful [18]. \nAd (iii): In addition to the weekly sessions, participants were encouraged to engage in the practices (described in Supplement Table S1) and reflections in between the sessions using e-health support, consisting of the free SEFAC app developed for the Android operating system. The app included audio recordings of the mindfulness practices, tips and reflections on nutrition, stress reduction, emotional health, and physical activity. In addition, it allowed the user to set personal goals to support habit change. Participants were encouraged to download the app on their mobile phone or tablet at the start of the SEFAC intervention and use it as digital support during the seven weeks of mindfulness-based sessions; it should be noted that the app stayed available to be used without support and on a voluntary basis until six months after the start of the intervention. ",
"Outcomes were obtained through self-report questionnaires at the start of the first session (baseline) and at 6-month follow-up to assess the ‘mid-term’ effects (i.e., the sustainability of the effects four months after finishing the intervention), as suggested by Ory et al. and Barkan et al. [19,20]. The internal consistency was assessed for each multi-item scale by computing Cronbach’s alphas, which was considered sufficient when over 0.70, and preferably under 0.95 [21]. The degree of self-efficacy was determined with four instruments: Self-Efficacy for Managing Chronic Diseases 6-item scale (SEMCD-6), Cronbach’s alpha 0.88 General Self-efficacy Scale (GSES), Cronbach’s alpha 0.91; Physical Exercise Self-Efficacy Scale (PESES), Cronbach’s alpha 0.94; Nutrition Self-Efficacy Scale (NSES), Cronbach’s alpha 0.95, with higher values indicating more self-efficacy [22,23,24]. Health behaviors were assessed in six domains, in line with the American College of Lifestyle Medicine [25]: (1) Healthy eating: three items on the intake of fruit and vegetables as well as having breakfast; (2) Physical activity: six items on physical exercise [26]; one item on sedentary behavior: International Physical Activity Questionnaire (IPAQ) [27]; (3) Substance use: current smoking, yes/no; frequency of alcohol use, one item from the AUDIT-C [28]; (4) Stress management: Perceived Stress 10-item Scale (PSS-10), range 0–40, Cronbach’s alpha 0.86, with higher values indicating more stress, score <14 corresponds to low stress and score ≥14 to moderate or high perceived stress [29]; (5) Sleep and fatigue: visual analogue scales (VAS), range 0–10, with higher values indicating worse sleep/more fatigue); (6) Relationships: Oslo Social Support 3-item scale (OSSS-3), Cronbach’s alpha 0.54 [30]. Medication adherence was assessed with the Simplified Medication Adherence Questionnaire (SMAQ), Cronbach’s alpha 0.57 [31]. Depression severity was assessed with the Patient Health Questionnaire 8-item scale (PHQ-8), range 0–24, Cronbach’s alpha 0.82, with higher values indicating higher severity, score ≥10 corresponding to current depression [32]. Health-related quality of life (HR-QoL) was assessed with the Short-Form 12-item health survey (SF-12), range 0–100, Cronbach’s alpha 0.67; the EuroQol-5 Dimensions-5 levels (EQ-5D-5L), using the UK value sets, range 0–1, Cronbach’s alpha 0.73, with higher values indicating better health utility [33,34]; and EQ-VAS, range 0–100, with higher values indicating better HR-QoL. ",
"Sociodemographic characteristics (age, sex, household composition, educational level, income, migration background), BMI (body mass index), and the presence of chronic conditions were assessed by self-report questionnaires. Good adherence to interventions was defined a priori as attending ≥4 of 7 group sessions [35]. The satisfaction with the SEFAC intervention was evaluated with a questionnaire consisting of eight items at 6-month follow-up. Seven items were rated on a 5-point scale, ranging from strongly agree to strongly disagree. One item was rated on a 1-to-10 rating scale, with higher values indicating higher satisfaction. ",
"The power considerations of the study were described previously [15]. It was planned to include 452 participants at baseline. By assuming a loss to follow-up of 20%, it was expected to have data of 360 participants with a baseline and a follow-up measure. Assuming equal standard deviations (SD) at baseline and follow-up, an alpha of 0.05 and power of 0.80, and by taking into account an average cluster size of 90 participants (360/4) and an intra-class correlation coefficient of 0.02, a difference of 0.24 SD between baseline and follow-up can be established for continuous outcome measures, such as the SF-12 [15].",
"Participant characteristics were described using mean (SD) or number of participants (%) for the total study sample. The effects of the intervention were assessed in the participants who completed the baseline and follow-up questionnaires and attended ≥4 sessions. A paired samples t-test was performed to assess the effects of the intervention on continuous outcome measures. Cohen’s d within-group effect size was computed for all significant outcomes [36]. For dichotomous outcome measures, the paired McNemar test was used. We stratified the descriptive statistics (see Supplement Table S2) and effects of the intervention on the outcome measures for the subgroups ‘History of CVD’ and ‘At risk of CVD’ (see Supplement Table S3a,b). The mean change in outcome measures for both subgroups was compared using an independent samples t-test for continuous outcomes and a z-test for dichotomous outcomes (see Supplement Table S3c). We did not impute missing data as only <5% of the data per variable were missing. Analyses were conducted with SPSS version 25.0 (IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA). The 2-sided significance threshold, after Bonferroni correction for multiple testing, was set at p = 0.05/20 = 0.0025. ",
" 3.1. Description of Participants Between 28 November 2018 and 20 February 2020, 352 participants who fulfilled the inclusion criteria provided informed consent and started the intervention; 77 (21.9%) with a history of CVD and 275 (78.1%) at increased risk of CVD due to the presence of one or more risk factors. Participants were included in 3 European study sites: Rijeka, Croatia (n = 147); Treviso, Italy (n = 97); Rotterdam, the Netherlands (n = 84). At the pilot site in the UK (n = 91), outcome measurements with the self-report questionnaires were not feasible because of a high prevalence of health literacy problems; hence, these participants were not included for further analyses. Table 1 shows the baseline characteristics of the study sample. \nBetween 28 November 2018 and 20 February 2020, 352 participants who fulfilled the inclusion criteria provided informed consent and started the intervention; 77 (21.9%) with a history of CVD and 275 (78.1%) at increased risk of CVD due to the presence of one or more risk factors. Participants were included in 3 European study sites: Rijeka, Croatia (n = 147); Treviso, Italy (n = 97); Rotterdam, the Netherlands (n = 84). At the pilot site in the UK (n = 91), outcome measurements with the self-report questionnaires were not feasible because of a high prevalence of health literacy problems; hence, these participants were not included for further analyses. Table 1 shows the baseline characteristics of the study sample. \n 3.2. Adherence to Interventions and Follow-Up Attrition Of all included participants (n = 352), 343 (97.4%) attended four or more of the seven group sessions. A median of 6 out of 7 group sessions (interquartile range [IQR], 6–7) were attended. The number of participants taking part in the 6-month follow-up assessment was 327 (92.9%). Overall, 324/352 participants (92.0%) completed the baseline and 6-month follow-up questionnaires, attended ≥4 sessions, and were included as the study sample for further analyses. Baseline characteristics of the 28 dropout participants were not significantly different from the included participants, except for the study site. The participant flow chart is shown in Figure 1.\nOf all included participants (n = 352), 343 (97.4%) attended four or more of the seven group sessions. A median of 6 out of 7 group sessions (interquartile range [IQR], 6–7) were attended. The number of participants taking part in the 6-month follow-up assessment was 327 (92.9%). Overall, 324/352 participants (92.0%) completed the baseline and 6-month follow-up questionnaires, attended ≥4 sessions, and were included as the study sample for further analyses. Baseline characteristics of the 28 dropout participants were not significantly different from the included participants, except for the study site. The participant flow chart is shown in Figure 1.\n 3.3. Outcomes Table 2 presents the outcome scores at baseline and follow-up of the 324 participants who completed both the baseline and 6-month follow-up questionnaires and who attended ≥4 sessions. Three of the four self-efficacy outcomes significantly improved, except for physical exercise self-efficacy. At the 6-month follow-up, there was a significant reduction in perceived stress and PHQ depression scores and significantly more social support as compared to baseline. Participants of the SEFAC intervention also experienced significant improvements in self-rated overall health and health utility (all p < 0.0025). Over time, no significant changes were observed for nutrition, physical activity, substance use, sleep, medication adherence, or mental and physical HR-QoL. \nTable 2 presents the outcome scores at baseline and follow-up of the 324 participants who completed both the baseline and 6-month follow-up questionnaires and who attended ≥4 sessions. Three of the four self-efficacy outcomes significantly improved, except for physical exercise self-efficacy. At the 6-month follow-up, there was a significant reduction in perceived stress and PHQ depression scores and significantly more social support as compared to baseline. Participants of the SEFAC intervention also experienced significant improvements in self-rated overall health and health utility (all p < 0.0025). Over time, no significant changes were observed for nutrition, physical activity, substance use, sleep, medication adherence, or mental and physical HR-QoL. \n 3.4. Satisfaction and Adverse Events Of those completing the questions on satisfaction with the intervention at the 6-month follow-up, participants reported that the intervention improved self-awareness (84.8%) and stimulated them to work on a healthy lifestyle (85.4%). The average satisfaction score was 8.2 ± 1.6 on a scale from 1 (lowest) to 10 (highest). The majority of participants (75.0%) perceived the SEFAC app as supportive of a healthy lifestyle through mindfulness exercises. (See Supplement Table S4). One participant died during the 6-month follow-up; this event was judged as unrelated to the intervention.\nOf those completing the questions on satisfaction with the intervention at the 6-month follow-up, participants reported that the intervention improved self-awareness (84.8%) and stimulated them to work on a healthy lifestyle (85.4%). The average satisfaction score was 8.2 ± 1.6 on a scale from 1 (lowest) to 10 (highest). The majority of participants (75.0%) perceived the SEFAC app as supportive of a healthy lifestyle through mindfulness exercises. (See Supplement Table S4). One participant died during the 6-month follow-up; this event was judged as unrelated to the intervention.",
"Between 28 November 2018 and 20 February 2020, 352 participants who fulfilled the inclusion criteria provided informed consent and started the intervention; 77 (21.9%) with a history of CVD and 275 (78.1%) at increased risk of CVD due to the presence of one or more risk factors. Participants were included in 3 European study sites: Rijeka, Croatia (n = 147); Treviso, Italy (n = 97); Rotterdam, the Netherlands (n = 84). At the pilot site in the UK (n = 91), outcome measurements with the self-report questionnaires were not feasible because of a high prevalence of health literacy problems; hence, these participants were not included for further analyses. Table 1 shows the baseline characteristics of the study sample. ",
"Of all included participants (n = 352), 343 (97.4%) attended four or more of the seven group sessions. A median of 6 out of 7 group sessions (interquartile range [IQR], 6–7) were attended. The number of participants taking part in the 6-month follow-up assessment was 327 (92.9%). Overall, 324/352 participants (92.0%) completed the baseline and 6-month follow-up questionnaires, attended ≥4 sessions, and were included as the study sample for further analyses. Baseline characteristics of the 28 dropout participants were not significantly different from the included participants, except for the study site. The participant flow chart is shown in Figure 1.",
"Table 2 presents the outcome scores at baseline and follow-up of the 324 participants who completed both the baseline and 6-month follow-up questionnaires and who attended ≥4 sessions. Three of the four self-efficacy outcomes significantly improved, except for physical exercise self-efficacy. At the 6-month follow-up, there was a significant reduction in perceived stress and PHQ depression scores and significantly more social support as compared to baseline. Participants of the SEFAC intervention also experienced significant improvements in self-rated overall health and health utility (all p < 0.0025). Over time, no significant changes were observed for nutrition, physical activity, substance use, sleep, medication adherence, or mental and physical HR-QoL. ",
"Of those completing the questions on satisfaction with the intervention at the 6-month follow-up, participants reported that the intervention improved self-awareness (84.8%) and stimulated them to work on a healthy lifestyle (85.4%). The average satisfaction score was 8.2 ± 1.6 on a scale from 1 (lowest) to 10 (highest). The majority of participants (75.0%) perceived the SEFAC app as supportive of a healthy lifestyle through mindfulness exercises. (See Supplement Table S4). One participant died during the 6-month follow-up; this event was judged as unrelated to the intervention.",
"This multicenter study involving over 300 individuals with or at increased risk of CVD showed modest effects from a 7-week MBI on self-efficacy, stress, depressive symptoms, social support, and self-rated overall health. \nThe SEFAC MBI was primarily designed to enhance self-management as an approach to improving health behavior and well-being for individuals with or at increased risk of CVD. Self-efficacy, defined as the belief that one has the ability to accomplish a specific task or reach a goal [37], is considered a key modifiable mediator of self-management skills in chronic disease. Individuals with high self-efficacy are more likely to believe they can master challenging problems and recover quickly from setbacks and disappointments. Therefore, it is hypothesized that interventions that enhance self-efficacy can improve patient self-management of chronic conditions [26]. The participants’ self-efficacy in our intervention increased, except for physical exercise self-efficacy. The mean changes in self-efficacy were comparable to those found in other self-management interventions with a similar follow-up duration and have been shown to be associated with better health outcomes [23]. Our findings align with a recent study showing that an 8-week mindfulness training facilitated self-management skills among primary care patients diagnosed with mental health conditions [38]. Though self-efficacy is considered essential for initiating and maintaining health behavior change, the improvements in self-efficacy in our participants did not translate into healthier lifestyle behaviors relevant to cardiovascular outcomes, such as nutrition, physical activity, smoking cessation, and sleep. \nDespite widespread awareness that behavior change is key to the prevention and treatment of CVD, it remains challenging to change behavior [39]. Theoretically, training the mind to hold each moment in awareness may be paramount to change habit patterns [40]. Based on the limited number of studies, there is no definitive agreement yet in the literature concerning the effects of mindfulness interventions alone on health behaviors. Mindfulness has been shown to promote favorable changes in selected behaviors related to psychiatric conditions, such as several substance-use disorders and eating disorders [13]. However, our study’s lack of effect on nutrition, physical activity and other lifestyle factors does not support the benefits of a 7-week MBI for health behavior change related to CVD. This effect may, in part, be related to the way clinicians, patients, and the research community incorrectly conceptualize contemplative practices as expedient interventions for health problems. Training the mind in awareness and understanding the subtleties of one’s existence take time and consistent practice. A 7-week MBI is probably inadequate to achieve a level of expertise needed to transform behavior that depends on mastery of complex biological, mental and emotional processes. However, the results of this study do support the inclusion of mindfulness training in the arsenal of lifestyle interventions to combat CVD together with evidence-based nutrition and physical activity interventions.\nParticipation in the SEFAC MBI promoted psychological well-being, including reducing perceived stress, less depressive symptoms and better self-rated overall health. Previous meta-analyses demonstrated comparable small-to-moderate beneficial effects for MBIs on stress, depression, anxiety, and quality of life in individuals with a range of chronic conditions [11,12]. Psychological well-being has been identified as a positive cardiovascular health asset [4]. It is associated with improved CVD outcomes, whereas adverse psychological factors, such as stress and depression, are associated with an increased risk of cardiovascular events [41,42]. Psychological well-being, including positive thoughts and feelings, purpose in life, and optimism, is linked with cardiovascular health via neurobiological processes, behavioral pathways, and psychosocial resources that protect health and buffer stress effects [3,5]. Several guidelines and scientific statements on CVD prevention and management recommend screening for psychosocial risk factors and considering tailored interventions to enhance the quality of life as well as life expectancy and to reduce cardiovascular events [6,43]. In line with this, the observed improvement in psychological health six months after our 7-week MBI may benefit cardiovascular health. \nPrevious studies have indicated that social isolation is a risk factor for the poor prognosis of individuals with cardiovascular disease. Isolated and lonely persons have a 1.5-fold increased risk of myocardial infarction, stroke and death [44]. Most of this excess risk is attributable to conventional risk factors, such as obesity, smoking, low education and pre-existing chronic illness [44]. In our study, one-third of the participants lived alone, and social support significantly improved following the MBI. Mindfulness practices may effectively reduce social risk by addressing both the subjective perception of loneliness and objective social isolation. In addition to the social context of a group-based MBI, the development of mindfulness-specific attention monitoring and acceptance skills is associated with improved social functioning [45]. These findings support group-based MBIs to promote the prevention and treatment of CVD in individuals with poor social connections. \nOne of the strengths of our study is that it was conducted in multiple European study sites with heterogeneous populations of middle-aged and older adults with representative cardiovascular risk profiles. The results suggest that the SEFAC MBI applies to a broad range of individuals with or at increased risk of chronic CVD. The statistical power of the study was considered sufficient; instead of 360 participants, data of 352 and 324 participants were analyzed. Because of the limited sample sizes per study site, we could not generalize about differences in the effects of the MBI among the different study sites. Only about 10% of participants were lost to follow-up, which is a low dropout rate compared to similar behavioral interventions. Dropped out participants were not different from those with complete follow-up in terms of baseline characteristics, making attrition bias less likely.\nThis study has several limitations. First, due to the lack of a control group, it cannot be ruled out that the observed changes are due to nonspecific effects unrelated to the intervention, including attention, expectations for improvement, social support and sense of community deriving from participation in a group-based intervention. Furthermore, although our MBI was based on and included the mindfulness exercises of the well-tested MBSR curriculum, mindfulness itself as a psychological construct was not assessed in this study. All outcome measures were self-reported, which may be less accurate than objectively measured outcomes. In addition, we cannot exclude the presence of a selection or social desirability bias. Our mindfulness-based intervention lasted seven weeks, unlike other existing programs that last eight weeks. However, the SEFAC mindfulness-based intervention took place in relatively small groups, with a similar duration as the MBSR and MBCT; therefore, the intensity of the intervention was assumed to be relatively high. Nevertheless, we recommend that future studies consider possibilities to strengthen the intervention, including making it last eight weeks. In addition, for future studies with larger varied samples, we advise exploring the differences regarding the effects between subgroup participants with cardiovascular disease and subgroup with participants at risk of cardiovascular disease. We recommend future studies to apply a randomized controlled trial (RCT) with pre-defined primary and secondary outcomes. This study only evaluated the ‘mid-term’ effects of the intervention after six months; we recommend future studies to measure both ‘immediate’ effects after finishing the intervention (i.e., after seven weeks in the current intervention), and the ‘mid-term’ effects after six months. In our study, three scales showed a Cronbach’s Alpha <0.70; we recommend that future studies pay attention to the reliability of the measurements. Moreover, an intervention such as the current one that is applied only one time might have been too short since changing lifestyle patterns is complex and requires continuous attention, practice, and support. Finally, the predefined 6-month follow-up period may have been too short to assess the durability of the observed effects. ",
"In conclusion, the present study shows promising effects of a mindfulness-based intervention, in combination with social engagement and e-health support. It enhances self-management as a complementary approach for modifying psychosocial factors relevant to cardiovascular care. Our findings deserve further study in a larger, long-term randomized controlled trial to explore the potential of mindfulness training in catalyzing chronic disease self-management. An intervention offering access to high-quality therapeutic lifestyle interventions focusing on nutrition, physical activity, and substance use, in conjunction with ongoing encouragement for mindfulness practice with digital and community resources, may be a sustainable approach for promoting cardiovascular health. "
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"results",
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[
"mindfulness",
"cardiovascular disease",
"risk factors",
"self management",
"chronic disease management"
] |
1. Introduction:
Despite substantial improvements in outcomes in recent decades, cardiovascular diseases (CVD) are still the leading causes of morbidity and mortality globally, with an estimated 17 million deaths each year [1].
Much of the global burden of CVD is attributable to uncontrolled behavioral risk factors, including poor diet, physical inactivity, and smoking [2]. In addition, psychological distress, such as chronic stress, depression, anxiety, and post-traumatic stress disorder, contributes to poorer quality of life and increases the risk of cardiovascular events and all-cause mortality [3,4]. Conversely, favorable lifestyle behaviors and psychological well-being promote cardiovascular health [5]. For the primary and secondary prevention of atherosclerotic vascular disease, heart failure, atrial fibrillation, and other cardiovascular conditions, lifestyle behaviors and mental health are of great importance [6]. Since patients spend very little time with healthcare providers, individuals largely depend on self-care to promote physical and emotional health and manage chronic illness. Despite the importance and effectiveness of self-care in preventing and managing CVD, many individuals find it challenging to make enduring modifications in lifestyle behaviors, take care of their mental health, and deal with chronic conditions [7,8]. Programs to enhance self-management skills based on behavioral interventions are only modestly effective, and novel intervention targets are needed to improve their impact [9].
One such intervention target is mindfulness, commonly defined as moment-to-moment awareness of one’s experience without judgement [10]. Mindfulness is considered a metacognitive process that enhances the capacity for self-regulation, i.e., adaptively regulating one’s attention, emotions, cognition, and behavior to respond effectively to internal and external demands. Mindfulness is deeply rooted in ancient Eastern philosophies and has received considerable public interest in recent decades. This universal human capacity can be strengthened through meditation, mind–body practices, such as yoga, and the application of mindful attention in daily life. A growing body of evidence indicates that mindfulness-based interventions (MBIs) can help people cope across a broad range of medical and psychological conditions, including depression, stress, anxiety, and chronic pain [11,12]. MBIs are commonly offered as secular, manualized, group-based interventions, the most popular of which are mindfulness-based stress reduction (MBSR) and mindfulness-based cognitive therapy. Typically, a package of mindfulness practices is provided for eight weeks, including body scan, sitting meditation, walking meditation, and gentle yoga exercises. Although mindfulness practices also produce relaxation in the body, relaxation is not the primary objective. Instead, these practices teach participants to focus on present-moment experiences with an orientation of openness, kindness, curiosity, and acceptance instead of ruminating about the past or worrying about the future. By cultivating an even-minded, witnessing relationship with (distressing) physical sensations, emotions, and thoughts as passing events arising in the body-mind system, practitioners learn to undo automatic habitual responses, counter negative thought patterns, and increase cognitive flexibility. Through modulation of attention control, emotion regulation, self-awareness, motivation and learning, mindfulness is postulated to influence self-regulation and behaviors that affect physical or mental health and quality of life [13].
Although preliminary research suggests that mindfulness may influence blood pressure and glycemic control, the potential of mindfulness training in facilitating self-management of CVD has received limited attention [14]. Therefore, the Social Engagement Framework for Addressing the Chronic-disease-challenge (SEFAC) study aimed to examine the effects of the 7-week SEFAC intervention targeting health behaviors and psychosocial factors for improving self-management in adults over 50 with or at increased risk of CVD.
2. Methods:
2.1. Study Design The SEFAC study had a before–after single-group design and was conducted between 28 November 2018 and 26 October 2020 in 4 European countries (Croatia, Italy, United Kingdom (UK), and the Netherlands). For full details of study design and protocol, see Zhang et al. [15] ISRCTN registry number is ISRCTN11248135, the date of registration is 30 August 2018 (retrospectively registered). Ethics approval was provided by the human research ethics boards of the study sites [15]. All participants provided written informed consent. This article reports on health outcomes as assessed at six months follow-up. We used the SPIRIT reporting guidelines [16].
The SEFAC study had a before–after single-group design and was conducted between 28 November 2018 and 26 October 2020 in 4 European countries (Croatia, Italy, United Kingdom (UK), and the Netherlands). For full details of study design and protocol, see Zhang et al. [15] ISRCTN registry number is ISRCTN11248135, the date of registration is 30 August 2018 (retrospectively registered). Ethics approval was provided by the human research ethics boards of the study sites [15]. All participants provided written informed consent. This article reports on health outcomes as assessed at six months follow-up. We used the SPIRIT reporting guidelines [16].
2.2. Recruitment and Eligibility Criteria Recruitment for participation in a study investigating a new intervention to improve the self-management of chronic cardiovascular diseases through mindfulness training, social engagement, and e-health support was conducted in the study sites (located in Rijeka, Croatia; Treviso, Italy; Camborne, United Kingdom; and Rotterdam, the Netherlands). Community-dwelling citizens were recruited using public events and announcements, patient and volunteer organizations, and social media. Main eligibility criteria were age over 50 years and established CVD (i.e., ischemic heart disease, cerebrovascular disease, peripheral artery disease, heart failure, or other cardiovascular conditions) and/or one or more risk factors for the development of CVD (including diabetes, smoking, hypertension, hypercholesterolemia, obesity, positive family history). In addition, citizens were not eligible to participate in the study when they were diagnosed with mild or severe cognitive impairment, were terminally ill or scheduled to enter secondary or tertiary care settings for a long period, not able to comprehend the information provided in the local language, or not able to make an informed decision regarding participation in the study.
Recruitment for participation in a study investigating a new intervention to improve the self-management of chronic cardiovascular diseases through mindfulness training, social engagement, and e-health support was conducted in the study sites (located in Rijeka, Croatia; Treviso, Italy; Camborne, United Kingdom; and Rotterdam, the Netherlands). Community-dwelling citizens were recruited using public events and announcements, patient and volunteer organizations, and social media. Main eligibility criteria were age over 50 years and established CVD (i.e., ischemic heart disease, cerebrovascular disease, peripheral artery disease, heart failure, or other cardiovascular conditions) and/or one or more risk factors for the development of CVD (including diabetes, smoking, hypertension, hypercholesterolemia, obesity, positive family history). In addition, citizens were not eligible to participate in the study when they were diagnosed with mild or severe cognitive impairment, were terminally ill or scheduled to enter secondary or tertiary care settings for a long period, not able to comprehend the information provided in the local language, or not able to make an informed decision regarding participation in the study.
2.3. Intervention The SEFAC intervention to promote health behaviors and psychological well-being had a duration of 7 weeks, and consisted of three elements, (i) weekly mindfulness-based sessions for seven weeks, (ii) support by social engagement, and (iii) e-health support by the SEFAC app [15].
Ad (i): The mindfulness-based sessions were offered in a series of seven weekly in-person group sessions (11–12 participants), lasting 2–2.5 h each for which a workbook was available. The sessions focused on mindset, habit change, relationships, and living with chronic conditions and included elements of positive psychology and health coaching in addition to mindfulness training. They were conducted by certified mindfulness professionals and/or health care professionals with training in mindfulness. Furthermore, local volunteers facilitated the intervention during and between the sessions in terms of logistics, raising awareness for self-management, motivating participants to commit to the intervention, and assisting them with e-health support. The intervention incorporated the core elements of the standard MBSR protocol: (a) mental and physical mindfulness exercises (body scan, sitting meditation, walking meditation, gentle yoga exercises; Supplement Table S1; (b) how to apply mindfulness to everyday situations and stress management; (c) sharing of experiences of mindfulness and insights into automatic patterns and habits among participants. During the sessions, participants were trained to foster greater awareness of present moment experience to promote mental well-being, build self-efficacy, enhance quality of life, adopt a healthy lifestyle, and connect to their community.
Ad (ii): Social engagement was used to support the mindfulness-based sessions [17]. Volunteers facilitated the organization and the sessions (see above). The group sessions provided an environment for peer-to-peer support allowing participants to share their experiences, offer encouragement, and share advice. Group factors such as normalizing one’s experience, a sense of shared group identity, and the empathic and anxiety-relieving nature of the group setting are considered helpful [18].
Ad (iii): In addition to the weekly sessions, participants were encouraged to engage in the practices (described in Supplement Table S1) and reflections in between the sessions using e-health support, consisting of the free SEFAC app developed for the Android operating system. The app included audio recordings of the mindfulness practices, tips and reflections on nutrition, stress reduction, emotional health, and physical activity. In addition, it allowed the user to set personal goals to support habit change. Participants were encouraged to download the app on their mobile phone or tablet at the start of the SEFAC intervention and use it as digital support during the seven weeks of mindfulness-based sessions; it should be noted that the app stayed available to be used without support and on a voluntary basis until six months after the start of the intervention.
The SEFAC intervention to promote health behaviors and psychological well-being had a duration of 7 weeks, and consisted of three elements, (i) weekly mindfulness-based sessions for seven weeks, (ii) support by social engagement, and (iii) e-health support by the SEFAC app [15].
Ad (i): The mindfulness-based sessions were offered in a series of seven weekly in-person group sessions (11–12 participants), lasting 2–2.5 h each for which a workbook was available. The sessions focused on mindset, habit change, relationships, and living with chronic conditions and included elements of positive psychology and health coaching in addition to mindfulness training. They were conducted by certified mindfulness professionals and/or health care professionals with training in mindfulness. Furthermore, local volunteers facilitated the intervention during and between the sessions in terms of logistics, raising awareness for self-management, motivating participants to commit to the intervention, and assisting them with e-health support. The intervention incorporated the core elements of the standard MBSR protocol: (a) mental and physical mindfulness exercises (body scan, sitting meditation, walking meditation, gentle yoga exercises; Supplement Table S1; (b) how to apply mindfulness to everyday situations and stress management; (c) sharing of experiences of mindfulness and insights into automatic patterns and habits among participants. During the sessions, participants were trained to foster greater awareness of present moment experience to promote mental well-being, build self-efficacy, enhance quality of life, adopt a healthy lifestyle, and connect to their community.
Ad (ii): Social engagement was used to support the mindfulness-based sessions [17]. Volunteers facilitated the organization and the sessions (see above). The group sessions provided an environment for peer-to-peer support allowing participants to share their experiences, offer encouragement, and share advice. Group factors such as normalizing one’s experience, a sense of shared group identity, and the empathic and anxiety-relieving nature of the group setting are considered helpful [18].
Ad (iii): In addition to the weekly sessions, participants were encouraged to engage in the practices (described in Supplement Table S1) and reflections in between the sessions using e-health support, consisting of the free SEFAC app developed for the Android operating system. The app included audio recordings of the mindfulness practices, tips and reflections on nutrition, stress reduction, emotional health, and physical activity. In addition, it allowed the user to set personal goals to support habit change. Participants were encouraged to download the app on their mobile phone or tablet at the start of the SEFAC intervention and use it as digital support during the seven weeks of mindfulness-based sessions; it should be noted that the app stayed available to be used without support and on a voluntary basis until six months after the start of the intervention.
2.4. Outcome Measures Outcomes were obtained through self-report questionnaires at the start of the first session (baseline) and at 6-month follow-up to assess the ‘mid-term’ effects (i.e., the sustainability of the effects four months after finishing the intervention), as suggested by Ory et al. and Barkan et al. [19,20]. The internal consistency was assessed for each multi-item scale by computing Cronbach’s alphas, which was considered sufficient when over 0.70, and preferably under 0.95 [21]. The degree of self-efficacy was determined with four instruments: Self-Efficacy for Managing Chronic Diseases 6-item scale (SEMCD-6), Cronbach’s alpha 0.88 General Self-efficacy Scale (GSES), Cronbach’s alpha 0.91; Physical Exercise Self-Efficacy Scale (PESES), Cronbach’s alpha 0.94; Nutrition Self-Efficacy Scale (NSES), Cronbach’s alpha 0.95, with higher values indicating more self-efficacy [22,23,24]. Health behaviors were assessed in six domains, in line with the American College of Lifestyle Medicine [25]: (1) Healthy eating: three items on the intake of fruit and vegetables as well as having breakfast; (2) Physical activity: six items on physical exercise [26]; one item on sedentary behavior: International Physical Activity Questionnaire (IPAQ) [27]; (3) Substance use: current smoking, yes/no; frequency of alcohol use, one item from the AUDIT-C [28]; (4) Stress management: Perceived Stress 10-item Scale (PSS-10), range 0–40, Cronbach’s alpha 0.86, with higher values indicating more stress, score <14 corresponds to low stress and score ≥14 to moderate or high perceived stress [29]; (5) Sleep and fatigue: visual analogue scales (VAS), range 0–10, with higher values indicating worse sleep/more fatigue); (6) Relationships: Oslo Social Support 3-item scale (OSSS-3), Cronbach’s alpha 0.54 [30]. Medication adherence was assessed with the Simplified Medication Adherence Questionnaire (SMAQ), Cronbach’s alpha 0.57 [31]. Depression severity was assessed with the Patient Health Questionnaire 8-item scale (PHQ-8), range 0–24, Cronbach’s alpha 0.82, with higher values indicating higher severity, score ≥10 corresponding to current depression [32]. Health-related quality of life (HR-QoL) was assessed with the Short-Form 12-item health survey (SF-12), range 0–100, Cronbach’s alpha 0.67; the EuroQol-5 Dimensions-5 levels (EQ-5D-5L), using the UK value sets, range 0–1, Cronbach’s alpha 0.73, with higher values indicating better health utility [33,34]; and EQ-VAS, range 0–100, with higher values indicating better HR-QoL.
Outcomes were obtained through self-report questionnaires at the start of the first session (baseline) and at 6-month follow-up to assess the ‘mid-term’ effects (i.e., the sustainability of the effects four months after finishing the intervention), as suggested by Ory et al. and Barkan et al. [19,20]. The internal consistency was assessed for each multi-item scale by computing Cronbach’s alphas, which was considered sufficient when over 0.70, and preferably under 0.95 [21]. The degree of self-efficacy was determined with four instruments: Self-Efficacy for Managing Chronic Diseases 6-item scale (SEMCD-6), Cronbach’s alpha 0.88 General Self-efficacy Scale (GSES), Cronbach’s alpha 0.91; Physical Exercise Self-Efficacy Scale (PESES), Cronbach’s alpha 0.94; Nutrition Self-Efficacy Scale (NSES), Cronbach’s alpha 0.95, with higher values indicating more self-efficacy [22,23,24]. Health behaviors were assessed in six domains, in line with the American College of Lifestyle Medicine [25]: (1) Healthy eating: three items on the intake of fruit and vegetables as well as having breakfast; (2) Physical activity: six items on physical exercise [26]; one item on sedentary behavior: International Physical Activity Questionnaire (IPAQ) [27]; (3) Substance use: current smoking, yes/no; frequency of alcohol use, one item from the AUDIT-C [28]; (4) Stress management: Perceived Stress 10-item Scale (PSS-10), range 0–40, Cronbach’s alpha 0.86, with higher values indicating more stress, score <14 corresponds to low stress and score ≥14 to moderate or high perceived stress [29]; (5) Sleep and fatigue: visual analogue scales (VAS), range 0–10, with higher values indicating worse sleep/more fatigue); (6) Relationships: Oslo Social Support 3-item scale (OSSS-3), Cronbach’s alpha 0.54 [30]. Medication adherence was assessed with the Simplified Medication Adherence Questionnaire (SMAQ), Cronbach’s alpha 0.57 [31]. Depression severity was assessed with the Patient Health Questionnaire 8-item scale (PHQ-8), range 0–24, Cronbach’s alpha 0.82, with higher values indicating higher severity, score ≥10 corresponding to current depression [32]. Health-related quality of life (HR-QoL) was assessed with the Short-Form 12-item health survey (SF-12), range 0–100, Cronbach’s alpha 0.67; the EuroQol-5 Dimensions-5 levels (EQ-5D-5L), using the UK value sets, range 0–1, Cronbach’s alpha 0.73, with higher values indicating better health utility [33,34]; and EQ-VAS, range 0–100, with higher values indicating better HR-QoL.
2.5. Other Measures Sociodemographic characteristics (age, sex, household composition, educational level, income, migration background), BMI (body mass index), and the presence of chronic conditions were assessed by self-report questionnaires. Good adherence to interventions was defined a priori as attending ≥4 of 7 group sessions [35]. The satisfaction with the SEFAC intervention was evaluated with a questionnaire consisting of eight items at 6-month follow-up. Seven items were rated on a 5-point scale, ranging from strongly agree to strongly disagree. One item was rated on a 1-to-10 rating scale, with higher values indicating higher satisfaction.
Sociodemographic characteristics (age, sex, household composition, educational level, income, migration background), BMI (body mass index), and the presence of chronic conditions were assessed by self-report questionnaires. Good adherence to interventions was defined a priori as attending ≥4 of 7 group sessions [35]. The satisfaction with the SEFAC intervention was evaluated with a questionnaire consisting of eight items at 6-month follow-up. Seven items were rated on a 5-point scale, ranging from strongly agree to strongly disagree. One item was rated on a 1-to-10 rating scale, with higher values indicating higher satisfaction.
2.6. Power Considerations The power considerations of the study were described previously [15]. It was planned to include 452 participants at baseline. By assuming a loss to follow-up of 20%, it was expected to have data of 360 participants with a baseline and a follow-up measure. Assuming equal standard deviations (SD) at baseline and follow-up, an alpha of 0.05 and power of 0.80, and by taking into account an average cluster size of 90 participants (360/4) and an intra-class correlation coefficient of 0.02, a difference of 0.24 SD between baseline and follow-up can be established for continuous outcome measures, such as the SF-12 [15].
The power considerations of the study were described previously [15]. It was planned to include 452 participants at baseline. By assuming a loss to follow-up of 20%, it was expected to have data of 360 participants with a baseline and a follow-up measure. Assuming equal standard deviations (SD) at baseline and follow-up, an alpha of 0.05 and power of 0.80, and by taking into account an average cluster size of 90 participants (360/4) and an intra-class correlation coefficient of 0.02, a difference of 0.24 SD between baseline and follow-up can be established for continuous outcome measures, such as the SF-12 [15].
2.7. Statistical Methods Participant characteristics were described using mean (SD) or number of participants (%) for the total study sample. The effects of the intervention were assessed in the participants who completed the baseline and follow-up questionnaires and attended ≥4 sessions. A paired samples t-test was performed to assess the effects of the intervention on continuous outcome measures. Cohen’s d within-group effect size was computed for all significant outcomes [36]. For dichotomous outcome measures, the paired McNemar test was used. We stratified the descriptive statistics (see Supplement Table S2) and effects of the intervention on the outcome measures for the subgroups ‘History of CVD’ and ‘At risk of CVD’ (see Supplement Table S3a,b). The mean change in outcome measures for both subgroups was compared using an independent samples t-test for continuous outcomes and a z-test for dichotomous outcomes (see Supplement Table S3c). We did not impute missing data as only <5% of the data per variable were missing. Analyses were conducted with SPSS version 25.0 (IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA). The 2-sided significance threshold, after Bonferroni correction for multiple testing, was set at p = 0.05/20 = 0.0025.
Participant characteristics were described using mean (SD) or number of participants (%) for the total study sample. The effects of the intervention were assessed in the participants who completed the baseline and follow-up questionnaires and attended ≥4 sessions. A paired samples t-test was performed to assess the effects of the intervention on continuous outcome measures. Cohen’s d within-group effect size was computed for all significant outcomes [36]. For dichotomous outcome measures, the paired McNemar test was used. We stratified the descriptive statistics (see Supplement Table S2) and effects of the intervention on the outcome measures for the subgroups ‘History of CVD’ and ‘At risk of CVD’ (see Supplement Table S3a,b). The mean change in outcome measures for both subgroups was compared using an independent samples t-test for continuous outcomes and a z-test for dichotomous outcomes (see Supplement Table S3c). We did not impute missing data as only <5% of the data per variable were missing. Analyses were conducted with SPSS version 25.0 (IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA). The 2-sided significance threshold, after Bonferroni correction for multiple testing, was set at p = 0.05/20 = 0.0025.
2.1. Study Design:
The SEFAC study had a before–after single-group design and was conducted between 28 November 2018 and 26 October 2020 in 4 European countries (Croatia, Italy, United Kingdom (UK), and the Netherlands). For full details of study design and protocol, see Zhang et al. [15] ISRCTN registry number is ISRCTN11248135, the date of registration is 30 August 2018 (retrospectively registered). Ethics approval was provided by the human research ethics boards of the study sites [15]. All participants provided written informed consent. This article reports on health outcomes as assessed at six months follow-up. We used the SPIRIT reporting guidelines [16].
2.2. Recruitment and Eligibility Criteria:
Recruitment for participation in a study investigating a new intervention to improve the self-management of chronic cardiovascular diseases through mindfulness training, social engagement, and e-health support was conducted in the study sites (located in Rijeka, Croatia; Treviso, Italy; Camborne, United Kingdom; and Rotterdam, the Netherlands). Community-dwelling citizens were recruited using public events and announcements, patient and volunteer organizations, and social media. Main eligibility criteria were age over 50 years and established CVD (i.e., ischemic heart disease, cerebrovascular disease, peripheral artery disease, heart failure, or other cardiovascular conditions) and/or one or more risk factors for the development of CVD (including diabetes, smoking, hypertension, hypercholesterolemia, obesity, positive family history). In addition, citizens were not eligible to participate in the study when they were diagnosed with mild or severe cognitive impairment, were terminally ill or scheduled to enter secondary or tertiary care settings for a long period, not able to comprehend the information provided in the local language, or not able to make an informed decision regarding participation in the study.
2.3. Intervention:
The SEFAC intervention to promote health behaviors and psychological well-being had a duration of 7 weeks, and consisted of three elements, (i) weekly mindfulness-based sessions for seven weeks, (ii) support by social engagement, and (iii) e-health support by the SEFAC app [15].
Ad (i): The mindfulness-based sessions were offered in a series of seven weekly in-person group sessions (11–12 participants), lasting 2–2.5 h each for which a workbook was available. The sessions focused on mindset, habit change, relationships, and living with chronic conditions and included elements of positive psychology and health coaching in addition to mindfulness training. They were conducted by certified mindfulness professionals and/or health care professionals with training in mindfulness. Furthermore, local volunteers facilitated the intervention during and between the sessions in terms of logistics, raising awareness for self-management, motivating participants to commit to the intervention, and assisting them with e-health support. The intervention incorporated the core elements of the standard MBSR protocol: (a) mental and physical mindfulness exercises (body scan, sitting meditation, walking meditation, gentle yoga exercises; Supplement Table S1; (b) how to apply mindfulness to everyday situations and stress management; (c) sharing of experiences of mindfulness and insights into automatic patterns and habits among participants. During the sessions, participants were trained to foster greater awareness of present moment experience to promote mental well-being, build self-efficacy, enhance quality of life, adopt a healthy lifestyle, and connect to their community.
Ad (ii): Social engagement was used to support the mindfulness-based sessions [17]. Volunteers facilitated the organization and the sessions (see above). The group sessions provided an environment for peer-to-peer support allowing participants to share their experiences, offer encouragement, and share advice. Group factors such as normalizing one’s experience, a sense of shared group identity, and the empathic and anxiety-relieving nature of the group setting are considered helpful [18].
Ad (iii): In addition to the weekly sessions, participants were encouraged to engage in the practices (described in Supplement Table S1) and reflections in between the sessions using e-health support, consisting of the free SEFAC app developed for the Android operating system. The app included audio recordings of the mindfulness practices, tips and reflections on nutrition, stress reduction, emotional health, and physical activity. In addition, it allowed the user to set personal goals to support habit change. Participants were encouraged to download the app on their mobile phone or tablet at the start of the SEFAC intervention and use it as digital support during the seven weeks of mindfulness-based sessions; it should be noted that the app stayed available to be used without support and on a voluntary basis until six months after the start of the intervention.
2.4. Outcome Measures:
Outcomes were obtained through self-report questionnaires at the start of the first session (baseline) and at 6-month follow-up to assess the ‘mid-term’ effects (i.e., the sustainability of the effects four months after finishing the intervention), as suggested by Ory et al. and Barkan et al. [19,20]. The internal consistency was assessed for each multi-item scale by computing Cronbach’s alphas, which was considered sufficient when over 0.70, and preferably under 0.95 [21]. The degree of self-efficacy was determined with four instruments: Self-Efficacy for Managing Chronic Diseases 6-item scale (SEMCD-6), Cronbach’s alpha 0.88 General Self-efficacy Scale (GSES), Cronbach’s alpha 0.91; Physical Exercise Self-Efficacy Scale (PESES), Cronbach’s alpha 0.94; Nutrition Self-Efficacy Scale (NSES), Cronbach’s alpha 0.95, with higher values indicating more self-efficacy [22,23,24]. Health behaviors were assessed in six domains, in line with the American College of Lifestyle Medicine [25]: (1) Healthy eating: three items on the intake of fruit and vegetables as well as having breakfast; (2) Physical activity: six items on physical exercise [26]; one item on sedentary behavior: International Physical Activity Questionnaire (IPAQ) [27]; (3) Substance use: current smoking, yes/no; frequency of alcohol use, one item from the AUDIT-C [28]; (4) Stress management: Perceived Stress 10-item Scale (PSS-10), range 0–40, Cronbach’s alpha 0.86, with higher values indicating more stress, score <14 corresponds to low stress and score ≥14 to moderate or high perceived stress [29]; (5) Sleep and fatigue: visual analogue scales (VAS), range 0–10, with higher values indicating worse sleep/more fatigue); (6) Relationships: Oslo Social Support 3-item scale (OSSS-3), Cronbach’s alpha 0.54 [30]. Medication adherence was assessed with the Simplified Medication Adherence Questionnaire (SMAQ), Cronbach’s alpha 0.57 [31]. Depression severity was assessed with the Patient Health Questionnaire 8-item scale (PHQ-8), range 0–24, Cronbach’s alpha 0.82, with higher values indicating higher severity, score ≥10 corresponding to current depression [32]. Health-related quality of life (HR-QoL) was assessed with the Short-Form 12-item health survey (SF-12), range 0–100, Cronbach’s alpha 0.67; the EuroQol-5 Dimensions-5 levels (EQ-5D-5L), using the UK value sets, range 0–1, Cronbach’s alpha 0.73, with higher values indicating better health utility [33,34]; and EQ-VAS, range 0–100, with higher values indicating better HR-QoL.
2.5. Other Measures:
Sociodemographic characteristics (age, sex, household composition, educational level, income, migration background), BMI (body mass index), and the presence of chronic conditions were assessed by self-report questionnaires. Good adherence to interventions was defined a priori as attending ≥4 of 7 group sessions [35]. The satisfaction with the SEFAC intervention was evaluated with a questionnaire consisting of eight items at 6-month follow-up. Seven items were rated on a 5-point scale, ranging from strongly agree to strongly disagree. One item was rated on a 1-to-10 rating scale, with higher values indicating higher satisfaction.
2.6. Power Considerations:
The power considerations of the study were described previously [15]. It was planned to include 452 participants at baseline. By assuming a loss to follow-up of 20%, it was expected to have data of 360 participants with a baseline and a follow-up measure. Assuming equal standard deviations (SD) at baseline and follow-up, an alpha of 0.05 and power of 0.80, and by taking into account an average cluster size of 90 participants (360/4) and an intra-class correlation coefficient of 0.02, a difference of 0.24 SD between baseline and follow-up can be established for continuous outcome measures, such as the SF-12 [15].
2.7. Statistical Methods:
Participant characteristics were described using mean (SD) or number of participants (%) for the total study sample. The effects of the intervention were assessed in the participants who completed the baseline and follow-up questionnaires and attended ≥4 sessions. A paired samples t-test was performed to assess the effects of the intervention on continuous outcome measures. Cohen’s d within-group effect size was computed for all significant outcomes [36]. For dichotomous outcome measures, the paired McNemar test was used. We stratified the descriptive statistics (see Supplement Table S2) and effects of the intervention on the outcome measures for the subgroups ‘History of CVD’ and ‘At risk of CVD’ (see Supplement Table S3a,b). The mean change in outcome measures for both subgroups was compared using an independent samples t-test for continuous outcomes and a z-test for dichotomous outcomes (see Supplement Table S3c). We did not impute missing data as only <5% of the data per variable were missing. Analyses were conducted with SPSS version 25.0 (IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA). The 2-sided significance threshold, after Bonferroni correction for multiple testing, was set at p = 0.05/20 = 0.0025.
3. Results:
3.1. Description of Participants Between 28 November 2018 and 20 February 2020, 352 participants who fulfilled the inclusion criteria provided informed consent and started the intervention; 77 (21.9%) with a history of CVD and 275 (78.1%) at increased risk of CVD due to the presence of one or more risk factors. Participants were included in 3 European study sites: Rijeka, Croatia (n = 147); Treviso, Italy (n = 97); Rotterdam, the Netherlands (n = 84). At the pilot site in the UK (n = 91), outcome measurements with the self-report questionnaires were not feasible because of a high prevalence of health literacy problems; hence, these participants were not included for further analyses. Table 1 shows the baseline characteristics of the study sample.
Between 28 November 2018 and 20 February 2020, 352 participants who fulfilled the inclusion criteria provided informed consent and started the intervention; 77 (21.9%) with a history of CVD and 275 (78.1%) at increased risk of CVD due to the presence of one or more risk factors. Participants were included in 3 European study sites: Rijeka, Croatia (n = 147); Treviso, Italy (n = 97); Rotterdam, the Netherlands (n = 84). At the pilot site in the UK (n = 91), outcome measurements with the self-report questionnaires were not feasible because of a high prevalence of health literacy problems; hence, these participants were not included for further analyses. Table 1 shows the baseline characteristics of the study sample.
3.2. Adherence to Interventions and Follow-Up Attrition Of all included participants (n = 352), 343 (97.4%) attended four or more of the seven group sessions. A median of 6 out of 7 group sessions (interquartile range [IQR], 6–7) were attended. The number of participants taking part in the 6-month follow-up assessment was 327 (92.9%). Overall, 324/352 participants (92.0%) completed the baseline and 6-month follow-up questionnaires, attended ≥4 sessions, and were included as the study sample for further analyses. Baseline characteristics of the 28 dropout participants were not significantly different from the included participants, except for the study site. The participant flow chart is shown in Figure 1.
Of all included participants (n = 352), 343 (97.4%) attended four or more of the seven group sessions. A median of 6 out of 7 group sessions (interquartile range [IQR], 6–7) were attended. The number of participants taking part in the 6-month follow-up assessment was 327 (92.9%). Overall, 324/352 participants (92.0%) completed the baseline and 6-month follow-up questionnaires, attended ≥4 sessions, and were included as the study sample for further analyses. Baseline characteristics of the 28 dropout participants were not significantly different from the included participants, except for the study site. The participant flow chart is shown in Figure 1.
3.3. Outcomes Table 2 presents the outcome scores at baseline and follow-up of the 324 participants who completed both the baseline and 6-month follow-up questionnaires and who attended ≥4 sessions. Three of the four self-efficacy outcomes significantly improved, except for physical exercise self-efficacy. At the 6-month follow-up, there was a significant reduction in perceived stress and PHQ depression scores and significantly more social support as compared to baseline. Participants of the SEFAC intervention also experienced significant improvements in self-rated overall health and health utility (all p < 0.0025). Over time, no significant changes were observed for nutrition, physical activity, substance use, sleep, medication adherence, or mental and physical HR-QoL.
Table 2 presents the outcome scores at baseline and follow-up of the 324 participants who completed both the baseline and 6-month follow-up questionnaires and who attended ≥4 sessions. Three of the four self-efficacy outcomes significantly improved, except for physical exercise self-efficacy. At the 6-month follow-up, there was a significant reduction in perceived stress and PHQ depression scores and significantly more social support as compared to baseline. Participants of the SEFAC intervention also experienced significant improvements in self-rated overall health and health utility (all p < 0.0025). Over time, no significant changes were observed for nutrition, physical activity, substance use, sleep, medication adherence, or mental and physical HR-QoL.
3.4. Satisfaction and Adverse Events Of those completing the questions on satisfaction with the intervention at the 6-month follow-up, participants reported that the intervention improved self-awareness (84.8%) and stimulated them to work on a healthy lifestyle (85.4%). The average satisfaction score was 8.2 ± 1.6 on a scale from 1 (lowest) to 10 (highest). The majority of participants (75.0%) perceived the SEFAC app as supportive of a healthy lifestyle through mindfulness exercises. (See Supplement Table S4). One participant died during the 6-month follow-up; this event was judged as unrelated to the intervention.
Of those completing the questions on satisfaction with the intervention at the 6-month follow-up, participants reported that the intervention improved self-awareness (84.8%) and stimulated them to work on a healthy lifestyle (85.4%). The average satisfaction score was 8.2 ± 1.6 on a scale from 1 (lowest) to 10 (highest). The majority of participants (75.0%) perceived the SEFAC app as supportive of a healthy lifestyle through mindfulness exercises. (See Supplement Table S4). One participant died during the 6-month follow-up; this event was judged as unrelated to the intervention.
3.1. Description of Participants:
Between 28 November 2018 and 20 February 2020, 352 participants who fulfilled the inclusion criteria provided informed consent and started the intervention; 77 (21.9%) with a history of CVD and 275 (78.1%) at increased risk of CVD due to the presence of one or more risk factors. Participants were included in 3 European study sites: Rijeka, Croatia (n = 147); Treviso, Italy (n = 97); Rotterdam, the Netherlands (n = 84). At the pilot site in the UK (n = 91), outcome measurements with the self-report questionnaires were not feasible because of a high prevalence of health literacy problems; hence, these participants were not included for further analyses. Table 1 shows the baseline characteristics of the study sample.
3.2. Adherence to Interventions and Follow-Up Attrition:
Of all included participants (n = 352), 343 (97.4%) attended four or more of the seven group sessions. A median of 6 out of 7 group sessions (interquartile range [IQR], 6–7) were attended. The number of participants taking part in the 6-month follow-up assessment was 327 (92.9%). Overall, 324/352 participants (92.0%) completed the baseline and 6-month follow-up questionnaires, attended ≥4 sessions, and were included as the study sample for further analyses. Baseline characteristics of the 28 dropout participants were not significantly different from the included participants, except for the study site. The participant flow chart is shown in Figure 1.
3.3. Outcomes:
Table 2 presents the outcome scores at baseline and follow-up of the 324 participants who completed both the baseline and 6-month follow-up questionnaires and who attended ≥4 sessions. Three of the four self-efficacy outcomes significantly improved, except for physical exercise self-efficacy. At the 6-month follow-up, there was a significant reduction in perceived stress and PHQ depression scores and significantly more social support as compared to baseline. Participants of the SEFAC intervention also experienced significant improvements in self-rated overall health and health utility (all p < 0.0025). Over time, no significant changes were observed for nutrition, physical activity, substance use, sleep, medication adherence, or mental and physical HR-QoL.
3.4. Satisfaction and Adverse Events:
Of those completing the questions on satisfaction with the intervention at the 6-month follow-up, participants reported that the intervention improved self-awareness (84.8%) and stimulated them to work on a healthy lifestyle (85.4%). The average satisfaction score was 8.2 ± 1.6 on a scale from 1 (lowest) to 10 (highest). The majority of participants (75.0%) perceived the SEFAC app as supportive of a healthy lifestyle through mindfulness exercises. (See Supplement Table S4). One participant died during the 6-month follow-up; this event was judged as unrelated to the intervention.
4. Discussion:
This multicenter study involving over 300 individuals with or at increased risk of CVD showed modest effects from a 7-week MBI on self-efficacy, stress, depressive symptoms, social support, and self-rated overall health.
The SEFAC MBI was primarily designed to enhance self-management as an approach to improving health behavior and well-being for individuals with or at increased risk of CVD. Self-efficacy, defined as the belief that one has the ability to accomplish a specific task or reach a goal [37], is considered a key modifiable mediator of self-management skills in chronic disease. Individuals with high self-efficacy are more likely to believe they can master challenging problems and recover quickly from setbacks and disappointments. Therefore, it is hypothesized that interventions that enhance self-efficacy can improve patient self-management of chronic conditions [26]. The participants’ self-efficacy in our intervention increased, except for physical exercise self-efficacy. The mean changes in self-efficacy were comparable to those found in other self-management interventions with a similar follow-up duration and have been shown to be associated with better health outcomes [23]. Our findings align with a recent study showing that an 8-week mindfulness training facilitated self-management skills among primary care patients diagnosed with mental health conditions [38]. Though self-efficacy is considered essential for initiating and maintaining health behavior change, the improvements in self-efficacy in our participants did not translate into healthier lifestyle behaviors relevant to cardiovascular outcomes, such as nutrition, physical activity, smoking cessation, and sleep.
Despite widespread awareness that behavior change is key to the prevention and treatment of CVD, it remains challenging to change behavior [39]. Theoretically, training the mind to hold each moment in awareness may be paramount to change habit patterns [40]. Based on the limited number of studies, there is no definitive agreement yet in the literature concerning the effects of mindfulness interventions alone on health behaviors. Mindfulness has been shown to promote favorable changes in selected behaviors related to psychiatric conditions, such as several substance-use disorders and eating disorders [13]. However, our study’s lack of effect on nutrition, physical activity and other lifestyle factors does not support the benefits of a 7-week MBI for health behavior change related to CVD. This effect may, in part, be related to the way clinicians, patients, and the research community incorrectly conceptualize contemplative practices as expedient interventions for health problems. Training the mind in awareness and understanding the subtleties of one’s existence take time and consistent practice. A 7-week MBI is probably inadequate to achieve a level of expertise needed to transform behavior that depends on mastery of complex biological, mental and emotional processes. However, the results of this study do support the inclusion of mindfulness training in the arsenal of lifestyle interventions to combat CVD together with evidence-based nutrition and physical activity interventions.
Participation in the SEFAC MBI promoted psychological well-being, including reducing perceived stress, less depressive symptoms and better self-rated overall health. Previous meta-analyses demonstrated comparable small-to-moderate beneficial effects for MBIs on stress, depression, anxiety, and quality of life in individuals with a range of chronic conditions [11,12]. Psychological well-being has been identified as a positive cardiovascular health asset [4]. It is associated with improved CVD outcomes, whereas adverse psychological factors, such as stress and depression, are associated with an increased risk of cardiovascular events [41,42]. Psychological well-being, including positive thoughts and feelings, purpose in life, and optimism, is linked with cardiovascular health via neurobiological processes, behavioral pathways, and psychosocial resources that protect health and buffer stress effects [3,5]. Several guidelines and scientific statements on CVD prevention and management recommend screening for psychosocial risk factors and considering tailored interventions to enhance the quality of life as well as life expectancy and to reduce cardiovascular events [6,43]. In line with this, the observed improvement in psychological health six months after our 7-week MBI may benefit cardiovascular health.
Previous studies have indicated that social isolation is a risk factor for the poor prognosis of individuals with cardiovascular disease. Isolated and lonely persons have a 1.5-fold increased risk of myocardial infarction, stroke and death [44]. Most of this excess risk is attributable to conventional risk factors, such as obesity, smoking, low education and pre-existing chronic illness [44]. In our study, one-third of the participants lived alone, and social support significantly improved following the MBI. Mindfulness practices may effectively reduce social risk by addressing both the subjective perception of loneliness and objective social isolation. In addition to the social context of a group-based MBI, the development of mindfulness-specific attention monitoring and acceptance skills is associated with improved social functioning [45]. These findings support group-based MBIs to promote the prevention and treatment of CVD in individuals with poor social connections.
One of the strengths of our study is that it was conducted in multiple European study sites with heterogeneous populations of middle-aged and older adults with representative cardiovascular risk profiles. The results suggest that the SEFAC MBI applies to a broad range of individuals with or at increased risk of chronic CVD. The statistical power of the study was considered sufficient; instead of 360 participants, data of 352 and 324 participants were analyzed. Because of the limited sample sizes per study site, we could not generalize about differences in the effects of the MBI among the different study sites. Only about 10% of participants were lost to follow-up, which is a low dropout rate compared to similar behavioral interventions. Dropped out participants were not different from those with complete follow-up in terms of baseline characteristics, making attrition bias less likely.
This study has several limitations. First, due to the lack of a control group, it cannot be ruled out that the observed changes are due to nonspecific effects unrelated to the intervention, including attention, expectations for improvement, social support and sense of community deriving from participation in a group-based intervention. Furthermore, although our MBI was based on and included the mindfulness exercises of the well-tested MBSR curriculum, mindfulness itself as a psychological construct was not assessed in this study. All outcome measures were self-reported, which may be less accurate than objectively measured outcomes. In addition, we cannot exclude the presence of a selection or social desirability bias. Our mindfulness-based intervention lasted seven weeks, unlike other existing programs that last eight weeks. However, the SEFAC mindfulness-based intervention took place in relatively small groups, with a similar duration as the MBSR and MBCT; therefore, the intensity of the intervention was assumed to be relatively high. Nevertheless, we recommend that future studies consider possibilities to strengthen the intervention, including making it last eight weeks. In addition, for future studies with larger varied samples, we advise exploring the differences regarding the effects between subgroup participants with cardiovascular disease and subgroup with participants at risk of cardiovascular disease. We recommend future studies to apply a randomized controlled trial (RCT) with pre-defined primary and secondary outcomes. This study only evaluated the ‘mid-term’ effects of the intervention after six months; we recommend future studies to measure both ‘immediate’ effects after finishing the intervention (i.e., after seven weeks in the current intervention), and the ‘mid-term’ effects after six months. In our study, three scales showed a Cronbach’s Alpha <0.70; we recommend that future studies pay attention to the reliability of the measurements. Moreover, an intervention such as the current one that is applied only one time might have been too short since changing lifestyle patterns is complex and requires continuous attention, practice, and support. Finally, the predefined 6-month follow-up period may have been too short to assess the durability of the observed effects.
5. Conclusions:
In conclusion, the present study shows promising effects of a mindfulness-based intervention, in combination with social engagement and e-health support. It enhances self-management as a complementary approach for modifying psychosocial factors relevant to cardiovascular care. Our findings deserve further study in a larger, long-term randomized controlled trial to explore the potential of mindfulness training in catalyzing chronic disease self-management. An intervention offering access to high-quality therapeutic lifestyle interventions focusing on nutrition, physical activity, and substance use, in conjunction with ongoing encouragement for mindfulness practice with digital and community resources, may be a sustainable approach for promoting cardiovascular health.
|
Background:
Cardiovascular diseases (CVD) are predominantly lifestyle related. Mental health issues also influence CVD progression and quality of life. Self-management of lifestyle behaviors and mental well-being may play a significant role in reducing the CVD burden. Previous studies have shown that mindfulness practices are associated with psychological well-being, but their effects on CVD self-management are mainly unknown.
Methods:
The study had a before-after design and included adults over 50 years with CVD and/or one or more risk factors from three European countries. Follow-up was six months. The intervention was a 7-week mindfulness-based intervention (MBI) in a group setting focusing on chronic disease self-management. Outcomes were measured with validated self-report questionnaires at baseline and follow-up: self-efficacy, physical activity, nutrition, smoking, alcohol use, sleep and fatigue, social support, stress, depression, medication adherence, and self-rated health.
Results:
Among 352 participants, 324 (92%) attended ≥4 of the 7 group sessions and completed follow-up. During follow-up, self-efficacy, stress, social support, depressive symptoms, and self-rated health significantly improved. No significant changes were detected for other outcomes.
Conclusions:
A 7-week MBI focusing on chronic disease self-management was conducive to improved self-efficacy, emotional well-being, social support, and self-rated overall health during six months. These findings support the use of MBIs for improving self-management in cardiovascular care. ISRCTN registry-number ISRCTN11248135.
|
1. Introduction:
Despite substantial improvements in outcomes in recent decades, cardiovascular diseases (CVD) are still the leading causes of morbidity and mortality globally, with an estimated 17 million deaths each year [1].
Much of the global burden of CVD is attributable to uncontrolled behavioral risk factors, including poor diet, physical inactivity, and smoking [2]. In addition, psychological distress, such as chronic stress, depression, anxiety, and post-traumatic stress disorder, contributes to poorer quality of life and increases the risk of cardiovascular events and all-cause mortality [3,4]. Conversely, favorable lifestyle behaviors and psychological well-being promote cardiovascular health [5]. For the primary and secondary prevention of atherosclerotic vascular disease, heart failure, atrial fibrillation, and other cardiovascular conditions, lifestyle behaviors and mental health are of great importance [6]. Since patients spend very little time with healthcare providers, individuals largely depend on self-care to promote physical and emotional health and manage chronic illness. Despite the importance and effectiveness of self-care in preventing and managing CVD, many individuals find it challenging to make enduring modifications in lifestyle behaviors, take care of their mental health, and deal with chronic conditions [7,8]. Programs to enhance self-management skills based on behavioral interventions are only modestly effective, and novel intervention targets are needed to improve their impact [9].
One such intervention target is mindfulness, commonly defined as moment-to-moment awareness of one’s experience without judgement [10]. Mindfulness is considered a metacognitive process that enhances the capacity for self-regulation, i.e., adaptively regulating one’s attention, emotions, cognition, and behavior to respond effectively to internal and external demands. Mindfulness is deeply rooted in ancient Eastern philosophies and has received considerable public interest in recent decades. This universal human capacity can be strengthened through meditation, mind–body practices, such as yoga, and the application of mindful attention in daily life. A growing body of evidence indicates that mindfulness-based interventions (MBIs) can help people cope across a broad range of medical and psychological conditions, including depression, stress, anxiety, and chronic pain [11,12]. MBIs are commonly offered as secular, manualized, group-based interventions, the most popular of which are mindfulness-based stress reduction (MBSR) and mindfulness-based cognitive therapy. Typically, a package of mindfulness practices is provided for eight weeks, including body scan, sitting meditation, walking meditation, and gentle yoga exercises. Although mindfulness practices also produce relaxation in the body, relaxation is not the primary objective. Instead, these practices teach participants to focus on present-moment experiences with an orientation of openness, kindness, curiosity, and acceptance instead of ruminating about the past or worrying about the future. By cultivating an even-minded, witnessing relationship with (distressing) physical sensations, emotions, and thoughts as passing events arising in the body-mind system, practitioners learn to undo automatic habitual responses, counter negative thought patterns, and increase cognitive flexibility. Through modulation of attention control, emotion regulation, self-awareness, motivation and learning, mindfulness is postulated to influence self-regulation and behaviors that affect physical or mental health and quality of life [13].
Although preliminary research suggests that mindfulness may influence blood pressure and glycemic control, the potential of mindfulness training in facilitating self-management of CVD has received limited attention [14]. Therefore, the Social Engagement Framework for Addressing the Chronic-disease-challenge (SEFAC) study aimed to examine the effects of the 7-week SEFAC intervention targeting health behaviors and psychosocial factors for improving self-management in adults over 50 with or at increased risk of CVD.
5. Conclusions:
In conclusion, the present study shows promising effects of a mindfulness-based intervention, in combination with social engagement and e-health support. It enhances self-management as a complementary approach for modifying psychosocial factors relevant to cardiovascular care. Our findings deserve further study in a larger, long-term randomized controlled trial to explore the potential of mindfulness training in catalyzing chronic disease self-management. An intervention offering access to high-quality therapeutic lifestyle interventions focusing on nutrition, physical activity, and substance use, in conjunction with ongoing encouragement for mindfulness practice with digital and community resources, may be a sustainable approach for promoting cardiovascular health.
|
Background:
Cardiovascular diseases (CVD) are predominantly lifestyle related. Mental health issues also influence CVD progression and quality of life. Self-management of lifestyle behaviors and mental well-being may play a significant role in reducing the CVD burden. Previous studies have shown that mindfulness practices are associated with psychological well-being, but their effects on CVD self-management are mainly unknown.
Methods:
The study had a before-after design and included adults over 50 years with CVD and/or one or more risk factors from three European countries. Follow-up was six months. The intervention was a 7-week mindfulness-based intervention (MBI) in a group setting focusing on chronic disease self-management. Outcomes were measured with validated self-report questionnaires at baseline and follow-up: self-efficacy, physical activity, nutrition, smoking, alcohol use, sleep and fatigue, social support, stress, depression, medication adherence, and self-rated health.
Results:
Among 352 participants, 324 (92%) attended ≥4 of the 7 group sessions and completed follow-up. During follow-up, self-efficacy, stress, social support, depressive symptoms, and self-rated health significantly improved. No significant changes were detected for other outcomes.
Conclusions:
A 7-week MBI focusing on chronic disease self-management was conducive to improved self-efficacy, emotional well-being, social support, and self-rated overall health during six months. These findings support the use of MBIs for improving self-management in cardiovascular care. ISRCTN registry-number ISRCTN11248135.
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[CONTENT] mindfulness | cardiovascular disease | risk factors | self management | chronic disease management [SUMMARY]
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[CONTENT] mindfulness | cardiovascular disease | risk factors | self management | chronic disease management [SUMMARY]
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[CONTENT] mindfulness | cardiovascular disease | risk factors | self management | chronic disease management [SUMMARY]
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[CONTENT] mindfulness | cardiovascular disease | risk factors | self management | chronic disease management [SUMMARY]
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[CONTENT] mindfulness | cardiovascular disease | risk factors | self management | chronic disease management [SUMMARY]
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[CONTENT] mindfulness | cardiovascular disease | risk factors | self management | chronic disease management [SUMMARY]
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[CONTENT] Adult | Humans | Self-Management | Cardiovascular Diseases | Social Participation | Quality of Life | Chronic Disease [SUMMARY]
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[CONTENT] Adult | Humans | Self-Management | Cardiovascular Diseases | Social Participation | Quality of Life | Chronic Disease [SUMMARY]
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[CONTENT] Adult | Humans | Self-Management | Cardiovascular Diseases | Social Participation | Quality of Life | Chronic Disease [SUMMARY]
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[CONTENT] Adult | Humans | Self-Management | Cardiovascular Diseases | Social Participation | Quality of Life | Chronic Disease [SUMMARY]
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[CONTENT] Adult | Humans | Self-Management | Cardiovascular Diseases | Social Participation | Quality of Life | Chronic Disease [SUMMARY]
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[CONTENT] Adult | Humans | Self-Management | Cardiovascular Diseases | Social Participation | Quality of Life | Chronic Disease [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] participants | self | health | intervention | mindfulness | study | sessions | follow | support | baseline [SUMMARY]
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[CONTENT] participants | self | health | intervention | mindfulness | study | sessions | follow | support | baseline [SUMMARY]
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[CONTENT] participants | self | health | intervention | mindfulness | study | sessions | follow | support | baseline [SUMMARY]
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[CONTENT] participants | self | health | intervention | mindfulness | study | sessions | follow | support | baseline [SUMMARY]
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[CONTENT] participants | self | health | intervention | mindfulness | study | sessions | follow | support | baseline [SUMMARY]
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[CONTENT] participants | self | health | intervention | mindfulness | study | sessions | follow | support | baseline [SUMMARY]
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[CONTENT] mindfulness | body | attention | behaviors | based | self | regulation | practices | cvd | lifestyle behaviors [SUMMARY]
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[CONTENT] cronbach | sessions | cronbach alpha | alpha | item | higher | scale | mindfulness | health | support [SUMMARY]
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[CONTENT] participants | follow | month follow | month | included | baseline | attended | sessions | significant | significantly [SUMMARY]
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[CONTENT] approach | mindfulness | cardiovascular | self management | management | high quality | long term randomized | complementary approach | complementary approach modifying | complementary approach modifying psychosocial [SUMMARY]
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[CONTENT] participants | mindfulness | self | study | health | follow | sessions | intervention | baseline | month [SUMMARY]
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[CONTENT] participants | mindfulness | self | study | health | follow | sessions | intervention | baseline | month [SUMMARY]
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[CONTENT] ||| ||| CVD ||| CVD [SUMMARY]
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[CONTENT] over 50 years | CVD | one | three | European ||| six months ||| 7-week ||| [SUMMARY]
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[CONTENT] 352 | 324 | 92% | ≥4 of the 7 group sessions ||| ||| [SUMMARY]
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[CONTENT] 7-week | six months ||| ||| [SUMMARY]
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[CONTENT] ||| ||| CVD ||| CVD ||| over 50 years | CVD | one | three | European ||| six months ||| 7-week ||| ||| ||| 352 | 324 | 92% | ≥4 of the 7 group sessions ||| ||| ||| 7-week | six months ||| ||| [SUMMARY]
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[CONTENT] ||| ||| CVD ||| CVD ||| over 50 years | CVD | one | three | European ||| six months ||| 7-week ||| ||| ||| 352 | 324 | 92% | ≥4 of the 7 group sessions ||| ||| ||| 7-week | six months ||| ||| [SUMMARY]
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||||||
Resistance to temephos and deltamethrin in Aedes aegypti from Brazil between 1985 and 2017.
|
31038548
|
Aedes aegypti populations in Brazil have been subjected to insecticide selection pressures with variable levels and sources since 1967. Therefore, the Brazilian Ministry of Health (MoH) coordinated the activities of an Ae. aegypti insecticide resistance monitoring network (MoReNAa) from 1999 to 2012.
|
BACKGROUND
|
Data were gathered from two sources: a bibliographic review of studies published from 1985 to 2017, and unpublished data produced by our team within the MoReNAa between 1998 and 2012. A total of 146 municipalities were included, many of which were evaluated several times, totalling 457 evaluations for temephos and 274 for deltamethrin. Insecticide resistance data from the five Brazilian regions were examined separately using annual records of both the MoH supply of insecticides to each state and the dengue incidence in each evaluated municipality.
|
METHODS
|
Ae. aegypti resistance to temephos and deltamethrin, the main larvicide and adulticide, respectively, employed against mosquitoes in Brazil for a long time, was found to be widespread in the country, although with some regional variations. Comparisons between metabolic and target-site resistance mechanisms showed that one or another of these was the main component of pesticide resistance in each studied population.
|
FINDINGS
|
(i) A robust dataset on the assessments of the insecticide resistance of Brazilian Ae. aegypti populations performed since 1985 was made available through our study. (ii) Our findings call into question the efficacy of chemical control as the sole methodology of vector control. (iii) It is necessary to ensure that sustainable insecticide resistance monitoring is maintained as a key component of integrated vector management. (iv) Consideration of additional parameters, beyond the supply of insecticides distributed by the MoH or the diverse local dynamics of dengue incidence, is necessary to find consistent correlations with heterogeneous vector resistance profiles.
|
MAIN CONCLUSIONS
|
[
"Aedes",
"Animals",
"Biological Assay",
"Brazil",
"Dengue",
"Incidence",
"Insecticide Resistance",
"Insecticides",
"Mosquito Vectors",
"Nitriles",
"Pyrethrins",
"Temefos"
] |
6489372
| null | null | null | null |
RESULTS
|
This study presented the results of evaluations of 146 Brazilian municipalities widely dispersed throughout the country, conducted between 1998 and 2012. Fig. 2 depicts the most current results of the 457 evaluations for temephos and 274 evaluations for deltamethrin, comprising the resistance status of Brazilian Ae. aegypti populations from 514 combinations of municipalities and years (‘municipalities/years’) and totalling 2,390 records. For each ‘municipality/year’, the total number of records was variable, and depended on both the insecticides evaluated and the tests performed (qualitative/quantitative). One ‘register’ was defined for each test: (1) in the case of qualitative assays, this was the mean mortality value; (2) for dose-response tests, this was each available LC (50, 80, 90, and 95) and the corresponding RR; and (3) for biochemical assays, this was the activity quantified for each enzyme class (ACE, MFO, GST, α-EST, β-EST, and ρNPA-EST). Allelic frequencies of kdr at the Na
V 1016 and 1534 positions, which are strongly associated with PY resistance,
55
,
56
were also reported whenever available, although these were not considered as main ‘registers’ herein.
Fig. 2:resistance status of larvae and adults of Brazilian Aedes aegypti populations against temephos and deltamethrin, respectively. For each municipality, the results of the most recent bioassay are shown. For deltamethrin, in addition to the results of quantitative (circles) bioassays, those of qualitative assays (triangles) are also depicted.
Data were presented separately for each geographical Region of Brazil, and organized sequentially by state and then municipality. For each municipality, the annual results were grouped chronologically. Supplementary data (Tables) are included that depict: the annual incidence of dengue in each municipality in the period evaluated [Supplementary data (Table II)]; the history of insecticide distribution by the MoH to each state for use against larvae [Supplementary data (Table III)] and adults [Supplementary data (Table V)]; results of bioassays of larvae exposed to temephos [Supplementary data (Table IV), quantitative assays]; and results of adult bioassays against deltamethrin [Supplementary data (Tables VI-VII) for qualitative and quantitative assays, respectively]. Supplementary data (Table VIII) exhibits the results obtained concerning the resistance status and mechanisms for all samples for which biochemical or molecular data were available.
In the text, in addition to sections dealing with each geographic region, two further sections were included: the first compared the results of CDC and WHO qualitative bioassays with adults, and the second analysed the relative participation of metabolic and target-site mechanisms in the resistance observed.
Our aim was to present, as completely as possible, the historical sequence of the development of resistance in Brazilian Ae. aegypti populations to insecticides used by public managers. It is noteworthy that 96.8% of these records were generated in laboratories belonging to the MoReNAa.
North Region
Dengue incidence - Most of the evaluated municipalities in this region were concentrated in Pará state, where dengue outbreaks occurred with a high, but sporadic, incidence [Supplementary data (Table II)]. However, some epidemics were reported in other states, even in consecutive years, and sometimes with incidences exceeding the ‘threshold’ of 0.3% by 5-30 times, including: Rio Branco, AC (2009-2011), Oiapoque, AP (2007-2011), and Boa Vista, RR (between 2001-2003 and 2008-2010). In Pacaraima, RR and Palmas, TO, high dengue incidence values were registered during most of the evaluated period.
Resistance against the larvicide temephos - During the evaluated period, temephos was almost continuously supplied throughout the North Region. In general, other products, such as Bti or IGRs, did not replace this larvicide, but were sometimes employed together with it [Supplementary data (Table III)]. Accordingly, resistance to temephos was found to be widespread in nearly all 20 evaluated municipalities in the North Region. The only exceptions were Porto Velho, RO in 2002
54
and Boa Vista in 2010 [Supplementary data (Table IV)].
For the most part, the temephos RR95 values of mosquitoes in the North Region ranged from 4-12 in the studied period. The main exception was Oiapoque in 2009, in which very high resistance levels, including a RR50 of 42.1 and RR95 of 102.5, were recorded.
57
This latter value was five times higher than the second largest RR95 in the region across the whole studied period: 21.4, in Dom Eliseu, PA in 2003.
20
More recent trials with samples from Amapá (collected between December 2014 and January 2015) showed a significant reduction in temephos resistance, including a RR50 of 21.8 for mosquitoes from Oiapoque and of 6.5 for those from Macapá.
58
It is worth remembering that temephos has been gradually replaced by IGRs since 2010, and its supplying to Amapá stopped in 2013 [Supplementary data (Table III)].
Oiapoque is located along the Brazilian border with French Guiana. The high temephos RR in Oiapoque in 2009 (RR95 = 102.5) may have been the consequence of the intense vector control actions applied in this border area by both countries. Dengue incidence was roughly 10 times higher than the threshold that defines epidemics in 2007-2011. This profile, however, does not seem to be general, as different patterns occurred in: (1) Palmas, TO, which presented RR values lower than 10 in two evaluations (2006 and 2009), with a high incidence of dengue in all years between sample collections; and (2) Rio Branco, which had a RR value below 10 in 2011, after four successive years of high incidences of dengue, with rates that reached 30 times the epidemic threshold. Therefore, it is not simply the epidemic per se that is associated with resistance, but the kind of response enacted to it, in particular the relative importance attached to chemical control of vectors.
Resistance to deltamethrin - In the North Region, deltamethrin was widely used in the control of adult mosquitoes over the period examined. Malathion was supplied to Pará in 2011, and later began to be employed in some other states [Supplementary data (Table V)]. Qualitative bioassays with deltamethrin using the WHO methodology classified 15 of the 18 evaluated mosquito populations as resistant, and none were considered susceptible.
43
On the other hand, trials with the CDC methodology resulted in higher mortalities, and of the 11 evaluated populations, only two were classified as resistant [Supplementary data (Table VI)]. A comparison of these two methodologies is presented below (section: ‘Qualitative bioassays with adults: CDC and WHO methodologies’).
Although performed with mosquitoes from municipalities distinct from the sources of those used in qualitative tests, the results obtained with quantitative assays corroborated the changes in deltamethrin susceptibility suggested by the results of the former trials [Supplementary data (Tables VI-VII)]. The deltamethrin RR95, a parameter evaluated since 2009, was always high, usually between 8.5 and 26.6. However, for mosquitoes from Pacaraima, RR and Marabá, PA evaluated in 2011, the deltamethrin RR95 was extremely high, 60.3 and 70.7, respectively. Pacaraima borders Santa Helena, Venezuela, where there is more intense chemical control of malaria vectors; this and the high dengue fever incidence there since 2007 [Supplementary data (Table II)] may explain the elevated deltamethrin RR95 in mosquitoes from Pacaraima. On the other hand, in Marabá, despite the low incidence of dengue [Supplementary data (Table II)], there was an eight-fold increase in the RR95 in a two-year interval, from 8.8 in 2009 to 70.7 in 2011 [Supplementary data (Table VII)]. This was the highest deltamethrin resistance ratio found in the region. Altogether, for the North Region it was not possible to make the general conclusion that high dengue incidence corresponds with increased PY resistance. The increase in the use of PYs in general observed during epidemics due to blocking actions undertaken by public health services, intensification of domestic use, and non-integrated use of PYs against malaria vectors
10
,
13
,
14
,
59
could explain the high level of resistance seen in mosquitoes from Pacaraima, but not in those from Marabá.
Resistance mechanisms - In the North Region, the activities of two enzymatic classes related to metabolic resistance were consistently altered: GSTs and, more intensely, ESTs, which in the latter case were evaluated with different substrates [Supplementary data (Table VIII)]. Few vector populations showed altered MFO activity. In only three of the 20 samples available, the total activity (AChE) of ACE, the target of OPs, appeared to be increased. However, in all cases ACE was considered sensitive to inhibition by insecticides according to WHO criteria (data not shown). Unfortunately, the metabolic resistance of larvae from a same locality in the North Region was never evaluated at two consecutive periods. However, there were simultaneous evaluations of larvae and adults on six occasions, and in all these cases the activity was more strongly altered in adults [Supplementary data (Table VIII)].
Northeast Region
Dengue incidence - Dengue dynamics between 2001 and 2012 in the Northeast Region presented four three-year periods with distinct disease incidences [Supplementary data (Table II)]; in the first and last of these, 2001-2003 and 2010-2012, dengue incidences were high and consistent with epidemic conditions in most of the evaluated locations in all states. In the second triennium (2004-2006), incidence values were the lowest observed, while in the third triennium (2007-2009) the dengue incidence was variable. It is noteworthy that this parameter was very high in Bahia during 2009. However, in the 2007-2009 triennium, the dengue incidence tended to be low in two periods and sections: in the beginning (2007), in the southernmost states of the Northeast Region (SE, BA); and in the last year (2009), in the remaining ones. In general, the number of ‘municipalities/years’ with a dengue incidence more than 0.3% corroborated this scenario: the first and fourth triennia registered higher numbers of 81 and 70 ‘municipalities/years’ that were above this cut-off point, respectively; whereas recorded values in the second triennium were lower (16), and in the third triennium they were intermediate (48).
Northeast Brazil was strongly affected by dengue over the evaluated period: in two states, CE and RN, at least 50% of ‘municipalities/years’ presented incidences indicating dengue epidemics. Even in MA, the least strongly affected state, more than 12% of the ‘municipalities/years’ presented high dengue incidences. The percentage of cases with incidences above 1,000 cases per 100,000 inhabitants, an arbitrary value equivalent to 1% of the population and more than three times the cut-off point defined by the Ministry of Health, was also evaluated. In this case, CE and RN were confirmed as the states that were the most strongly affected by dengue during the studied period. High incidences were also found for AL and BA.
Temephos resistance - Except for RN, temephos was continuously distributed to all states in the Northeast Region until 2011-2014 [Supplementary data (Table III)]. Bti was simultaneously applied in several states (MA, CE, PE, and AL) until 2009-2010; IGRs were also introduced during this period in most states in the Northeast Region. By 2014 almost all states exclusively employed IGRs.
In the Northeast Region, resistance to temephos was detected in all municipalities evaluated, with the only exception being Fernando de Noronha Island in 2009 [Supplementary data (Table IV)]. Although to date the highest temephos resistance levels in Brazil occur in the Northeast Region, the RR there varies significantly: a temephos RR95 above 100 was found in 20% of cases, but in almost 40% of cases the RR95 was below 10. The lowest dengue incidences and also the lowest temephos RR95 values (always below 10) were found in the state of MA. In contrast, extremely high temephos RR95 values above 100 (sometimes above 200) were concentrated in PE (six municipalities), southern CE (two), eastern BA (five), and the countryside of AL (one) [Supplementary data (Fig. 1)].
Much variation was noted in several aspects of insecticide resistance monitoring in the Northeast Region, including: (1) the number of municipalities evaluated in each state (ranging from only two in MA and PI, up to more than 10 in PE and BA); (2) the frequency of evaluation of the different municipalities, with each municipality evaluated only once in some states, such as MA and PB, while some municipalities in other states were evaluated five (Juazeiro do Norte, CE and Natal, RN), six (Fortaleza, CE and Maceió, AL), or even seven (Aracaju, SE) times over the evaluated period; and (3) the regularity of monitoring, as for example on the one hand five of the six evaluations were performed in 2003 for PB, while on the other hand in BA only three out of 23 evaluations were done before 2008.
For municipalities that were evaluated more than once, temephos resistance levels were compared with both their dengue incidence and supply of insecticides [Supplementary data (Tables II, III, IV). Although several different parameters influence dengue dynamics, some correlations were suggested, as outlined below:
― Crato, in the south of CE state, where the temephos RR95 in 2009 was higher than 190, presented a history of severe dengue epidemics between 2001 and 2007, which were five years with high values of dengue incidence, including two years when this was greater than 1%. This scenario may have contributed to the intensification of chemical control and the consequent increase in temephos resistance levels in this municipality. However, although dengue incidence remained high in this municipality until at least 2012, in 2013 resistance to temephos had declined more than three-fold (RR95 = 65), probably due to the interruption of its supply to the state in 2011.
― In Fortaleza, CE, the temephos RR95 increased from 8-10 in 2002 and 2006 to 43 in 2007. The annual dengue incidence was above the MoH cut-off point five times between 2001 and 2007, pointing to the intensification of chemical control and a consequent RR increase. Unfortunately, although there were several periods of high dengue incidence until 2012 in Fortaleza, no further temephos evaluations were reported there.
― With the exception of Santana do Ipanema, in the state of AL relatively stable temephos RR50-95 values, varying between four and 17, were found. These were consistent data, since these municipalities were assessed three to six times during the studied period. These results are also coherent with the supply of larvicides provided to AL state, since temephos was continuously but not exclusively provided between 2003 and 2013, being always concomitantly applied with Bti and/or an IGR.
― In contrast, in the state of RN, although temephos was provided only in 2004 [Supplementary data (Table II)], the RR95 of mosquitoes for this OP fluctuated over the same range as those in AL, and was around 12 (from 10 to 19) between 2004 and 2011. These are also consistent data, as of the five municipalities monitored, four were evaluated three to five times. This situation suggests that dengue incidence and records of insecticide supply by the MoH were not sufficient to explain the temephos resistance dynamics observed in RN state. Additional investigations could reveal other parameters that should be taken into account for the interpretation of these data.
― Accordingly, in Barra dos Coqueiros, SE, neither the low dengue incidence, nor the exclusive supplying of temephos as a larvicide over the evaluated period, were able to explain the increase in the temephos RR by seven times between 2000 (3.2) and 2004 (23.5).
In BA state, temephos was supplied continuously between 2003 and 2013, and this was the only larvicide used between 2004 and 2009. Among Bahia municipalities monitored more than once, two distinct scenarios should be mentioned: (1) records of high dengue incidence and stable levels of temephos resistance were observed in Barreiras, which was subjected to two years of very high dengue incidence between 2008 and 2012 (up to 1.6%), although the temephos RR95 there remained around 5.0 throughout the evaluated period; and (2) records of high dengue incidence and increasing temephos resistance levels were noted in such municipalities as Itabuna, Jequié, and Jacobina. In Itabuna, the temephos RR95 increased from 18.6 in 2004 to 55.8 in 2013, and records of dengue incidence in this municipality were also extremely high, in the range of 3-7%, in 2009 and 2012. Jequié exhibited low dengue incidences between 2004 and 2008, but high values between 2009 and 2012, and this last period was concomitant with a three-fold increase in the temephos RR95 value of mosquitoes from there. In Jacobina, temephos resistance levels increased 10-fold in one year, between 2008 and 2009, when the RR95 reached 104. Four years later, the temephos RR95 more than doubled, reaching almost 230. Dengue incidence in this municipality was high, or very high, during almost the entire period evaluated (2001-2012).
Resistance to deltamethrin - The whole Northeast Region received an uninterrupted supply of PY insecticides between 2003 and 2014 [Supplementary data (Table V)]. Malathion was delivered to CE in 2006, and to BA in 2011. This OP started to be continuously distributed in addition to deltamethrin in CE in 2008, and in some other states in the Northeast Region as of 2010.
Mosquitoes from all of the 39 ‘municipalities/years’ from the Northeast Region evaluated for deltamethrin resistance with qualitative tests using the WHO methodology were classified as resistant (26) or with incipient resistance (13). Among the 14 samples evaluated with the CDC methodology, 11 were considered resistant (two) or with incipient resistance (nine). In 13 cases, discussed in a section below, the two methodologies were applied simultaneously [Supplementary data (Table VI)].
Quantitative tests were performed in eight municipalities from four states [Supplementary data (Table VII)], and confirmed that resistance to PY was prevalent throughout the Northeast Region. The RR95 values were always above 7.0 and, except for Mossoró in 2011 and Crato in 2013, remained below 20.
In the Northeast Region the deltamethrin resistance status was evaluated twice in two localities, Mossoró, RN and Aracaju, SE. In both cases, the deltamethrin RR95 increased and, accordingly, the frequency of kdr alleles at the PY target site also increased [Supplementary data (Table VIII)].
Resistance mechanisms - The activities of two classes of detoxifying enzymes, GSTs and ESTs, were altered by pesticides [Supplementary data (Table VIII)]. Both α-EST and β-EST activity were affected in larvae, while α-EST was the most strongly altered class in adults. Total ACE activity presented increased levels in only two cases. However, according to WHO criteria, ACE was sensitive to insecticide inhibition throughout the Northeast Region (data not shown). MFO and ρNPA-EST activities were altered in some cases, mainly in adults. In eight instances, larvae and adults were simultaneously evaluated, with greater enzyme activity alterations always observed in adults [Supplementary data (Table VIII)]. The two municipalities evaluated at more than one point in time, Aracaju and Caicó, exhibited a general increase in the activity of detoxifying enzymes in both larvae and adults.
Southeast Region
Dengue incidence - In the Southeast Region, SP state stands out, as detailed below. SP presented the longest insecticide resistance monitoring history and the lowest dengue incidence of all evaluated states; it is an example of the positive effects of both entomological surveillance and resistance management on dengue prevention. During the evaluated period (2001-2012), the dengue incidence was above 0.3%, the MoH cut-off point, in less than 20% of ‘municipalities/years’, and only 10% of cases presented a dengue incidence of more than 1% [Supplementary data (Table II)]. In contrast, ES was the state in the Southeast Region that was the most strongly affected by dengue in the studied period, as in more than 50% of the ‘municipalities/years’ in ES the dengue incidence was above the MoH recommendation, and 20% of these had a dengue incidence of more than 1%. RJ and MG states presented intermediate values: 45-55% and 15-20% of the ‘municipalities/years’ in these states exhibited dengue incidences above 0.3 and 1%, respectively.
Temephos resistance - In the Southeast Region in general, temephos was distributed continuously throughout the period evaluated. Bti was supplied simultaneously until 2007-2010, and thereafter IGRs were used [Supplementary data (Table III)]. RJ was an exception, since temephos and IGRs were provided intermittently from 2004 onward in this state, while IGRs began to be supplied continuously after 2008, before they were supplied to the other states in the Southeast Region.
Temephos resistance was evaluated in 42 municipalities, totalling 250 ‘municipalities/years’, in this Region. SP accounted for more than 80% of the records, among which 17 municipalities out of 20 were assessed several times, some almost annually and, in many cases, from 14 to 18 times over the 1998-2015 period. In contrast, in other states in the Southeast Region, the accomplishment of temephos bioassays, which started from 1999 (RJ) and 2005 (MG and ES) onward, extended only until 2011. In these states, most municipalities were evaluated once or twice in the studied period, with a maximum of five evaluations in the case of Rio de Janeiro city [Supplementary data (Table IV)].
This differential resistance monitoring and management effort was reflected in the susceptibility profiles obtained: the average temephos RR95 of mosquitoes from SP (3.5) was at least three times lower than that of those in the other states in the Southeast Region (RJ, MG, and ES: RR95 = 10.1, 11.4, and 13.3, respectively). In SP, the temephos RR95 was above 3.0 in 60% of cases, in contrast with it being above this value for 100% of the municipalities in the other states, with the only exception to this trend being São José do Vale do Rio Preto, RJ (RR95 = 3.0). In SP, the temephos RR95 was above 10.0 in mosquitoes from only 1% of ‘municipalities/years’ (two out of a total of 194 records). In contrast, more than 50% of the records for the other states in the Southeast Region reached this value. Out of the 42 municipalities assessed in the Southeast Region, four were classified as having mosquitoes susceptible to temephos (RR95 below 3.0), three of which were located in SP. In particular, just one among the four municipalities was evaluated more than once; this was Botucatu, SP, the mosquitoes from which remained susceptible to temephos during all six monitoring rounds carried out between 2004 and 2014. This municipality did not exhibit a dengue incidence above the epidemic ‘threshold’ over the entire evaluated period [Supplementary data (Table II)].
In SP, a temephos RR95 above 10.0 was detected only in mosquitoes from São Sebastião in 2004 and from Santos in 2005. In both municipalities, resistance ratio values decreased later on, culminating at levels indicating susceptibility in 2015. In SP, the highest temephos RR95 levels were reported in Santos, São Sebastião, São Paulo, and Itapevi. The first two of these were among the five SP cities evaluated herein with the highest dengue incidences in the period of 2001-2012.
On the other hand, in the Southeast Region, the ES state presented the highest dengue incidence and also the highest temephos RR95 in the evaluated period. These were consistent data, which were always derived from more than one evaluation. In the case of RJ, although most of the locations were evaluated more than once, there were gaps in the RR95 records, and in MG only the capital, Belo Horizonte, was evaluated more than once.
Resistance to deltamethrin - Supplementary data (Table V) shows the supply of adulticides provided by the MoH for all vector control programs, not just those targeting Ae. aegypti (see ‘Discussion’). PYs were widely distributed throughout the Southeast Region: SP received malathion during the whole period, and other states received it from 2007 onward, or later. It should be mentioned that SP stopped using PYs against the dengue vector in 2000, almost 10 years before their replacement in the rest of the country.
60
Deltamethrin qualitative bioassays were performed for 115 and 75 ‘municipalities/years’ with the WHO and CDC methodologies, respectively [Supplementary data (Table VI)]. In all cases, established or incipient resistance was detected. Established resistance prevailed in 92% of the trials (n = 106) done with the WHO methodology and in 72% of the trials (n = 54) done with the CDC methodology. Simultaneous evaluations with the WHO and CDC methodologies were performed for 46 ‘municipalities/years’ (see section below in which both methodologies were compared). Accordingly, deltamethrin quantitative bioassays revealed high resistance levels in all cases [Supplementary data (Table VII)].
Resistance mechanisms - GSTs were the enzymes whose activities were the most strongly altered by pesticides in samples of larvae and adults from the Southeast Region [Supplementary data (Table VIII)]. In adults, the activity of EST enzymes, mainly a-EST and ρNPA-EST, were also altered. Changes in MFO activity were also identified in some samples. Increases in total ACE activity were detected in roughly 30% of the evaluated samples. However, ACE activity was never altered in both stages evaluated from the same ‘municipality/year’. In addition, the ACE activity in all samples was considered sensitive to insecticide inhibition (AChI) when the WHO criterion was used (data not shown). Whenever biochemical assays were available for both life stages (eight pairs of samples), metabolic resistance was higher in adults. Metabolic resistance was never evaluated twice in larvae from the same locality. However, for five municipalities, the metabolic resistance of adults was evaluated 2-3 times; despite this, there were no concurrent data on the application of quantitative bioassays or on Na
V allele frequencies, a situation that precludes the analysis of the main mechanisms involved in resistance in these cases.
South Region
Dengue incidence - Due in part to its milder climate, the South Region was the least strongly affected by dengue. Most municipalities in SC and RS states evaluated over the studied period did not register dengue epidemics. The exception was Ijuí, RS in 2010, where the dengue incidence reached 3.7%, more than 12 times the MoH cut-off point [Supplementary data (Table II)]. The state of PR was the most affected by dengue. In the South Region, a dengue outbreak first occurred in 2002 in Foz do Iguaçu, PR, which is located on the triple border of Brazil with Paraguay and Argentina. In 2003, there was an epidemic in three municipalities in northern central Paraná, Cambé, Ipiporã, and Londrina. From then on, new records of high dengue incidences only occurred in this state in 2007, and then again in 2010/2011. In the period 2007-2011, Foz do Iguaçu endured three years of extremely high dengue incidences 3-12 times the MoH threshold of 0.3%.
Temephos resistance - All states in the South Region received the larvicide temephos almost continuously between 2003 and 2014 [Supplementary data (Table III)]. RS also received Bti in 2006 and 2009. The supplying of IGR larvicides to PR occurred from 2010 onward. Both SC and RS received IGRs in 2014 for the first time.
The temephos resistance status was evaluated in 12 municipalities in the South Region, totalling 29 ‘municipalities/years’ [Supplementary data (Table IV)]. Foz do Iguaçu and Maringá, both in PR, were evaluated eight and six times, respectively, between 2001 and 2009. Among the remaining municipalities, six were evaluated only once, and four were evaluated 2-3 times. Although in 55% of the bioassays the RR95 was higher than 3.0, the MoH cut-off point, it was never higher than 7.0. Mosquitoes from Maringá in 2005 and Ibiporã in 2006 exhibited the highest resistance levels. In general, a slight increase in resistance to OP was noted in the South Region over time.
Resistance to deltamethrin - Pyrethroids were provided almost continuously and used nearly exclusively in the South Region [Supplementary data (Table V)]. PR also received the OP malathion from 2010 onward. Qualitative deltamethrin bioassays were performed for three PR locations. Eight ‘municipalities/years’ were evaluated by the WHO method, and four of these were also evaluated using the CDC methodology [Supplementary data (Table VI)], and susceptibility was not detected in any case. In the South Region, results of deltamethrin quantitative bioassays were only available for mosquitoes from Santa Rosa, RS, for which there was a RR95 of 33.3 in 2011 [Supplementary data (Table VII)].
Resistance mechanisms - In the South Region, resistance mechanisms were investigated only in mosquito larvae and adults from Santa Rosa, RS in 2011. The larval enzyme activity profile was unaltered, which was consistent with their susceptibility to temephos [Supplementary data (Table VIII)]. In adults that were resistant to deltamethrin, changes in EST activity were detected, particularly in the activities of α-EST and ρNPA-EST. The participation of Na
V alterations in resistance was also identified in this case. Despite showing an increase in their total ACE activity, mosquitoes from Santa Rosa were considered to be sensitive to insecticide inhibition according to the WHO criteria (data not shown).
Centre-West Region
Dengue incidence - All municipalities evaluated in this Region experienced at least one dengue epidemic year between 2001 and 2012 [Supplementary data (Table II)]. Brazil’s capital, Brasília, in the Federal District (DF), was the least strongly affected locality, with there being high dengue incidence only in 2010. In the MS municipalities evaluated, dengue incidences generally increased from 2006 onward and reached values up to 6.3%, more than 20 times the MoH threshold, as was seen in Campo Grande in 2007. In GO, the dengue incidence also reached very high values in several localities. In particular, high dengue incidences were observed in two neighbouring municipalities, Goiânia and Aparecida de Goiânia, throughout the period evaluated (2001-2012). In 2010, a strong dengue epidemic affected the entire Centre-West Region: in 16 of the 17 municipalities evaluated, the dengue incidence was above 0.3%, and in four this value was even above 3.0%.
Temephos resistance - Except in the DF, temephos was supplied continuously between 2003 and 2013 throughout the Region. In MS, Bti was used in addition to temephos between 2003 and 2009. IGRs were introduced from 2009-2010, becoming the sole larvicides provided by the MoH throughout the Centre-West Region in 2014 [Supplementary data (Table III)].
Mosquitoes from all municipalities in the Centre-West Region evaluated were resistant to temephos [Supplementary data (Table IV)], the only exception being those from Brasilia in 2008. Brasilia presented the highest number of evaluations, and also the lowest temephos resistance levels and the lowest dengue incidence rates, over the evaluated period. Only Cuiabá in 2005 was evaluated in MT state; its temephos RR95 was also among the lowest in the region, in line with the moderate dengue incidence rates recorded there up to this time.
The temephos RR95 in MS was always below 8.0 [Supplementary data (Table IV)], even in 2011, after a period of extremely high dengue incidence in 2006-2010 [Supplementary data (Table II)]. The simultaneous use of temephos and Bti [Supplementary data (Table III)] may have contributed to there being less selection pressure for OP resistance in this state.
In contrast, mosquitoes from GO showed the highest temephos resistance levels in the region, as 10 out of the 15 municipalities for which the RR95 was evaluated in this state had values that were above 10.0 [Supplementary data (Table IV)]. Notably, São Miguel do Araguaia, GO in 2012 presented the highest temephos resistance level in the Centre-West Region, even though 2012 corresponded to the second year of low dengue incidence after five consecutive years of high incidences in this locality. The contiguous municipalities of Goiânia and Aparecida de Goiânia are clear examples of how differences in chemical control management can have an impact on the development of resistance. Although dengue incidence was high in both municipalities during the entire evaluated period, temephos resistance levels were much lower in Goiânia than in Aparecida de Goiânia, suggesting there were distinct selection pressures in both localities. Indeed, in the routine insecticide resistance monitoring of Brazilian Ae. aegypti populations, some adjacent municipalities with different local dynamics of infestation control were purposely chosen for just this reason. This was done to emphasize to municipal public officials how much both surveillance and infestation control strategies can impact insecticide resistance and, consequently, influence the effectiveness of vector control.
Resistance to deltamethrin - Pyrethroid adulticides were distributed widely in the Centre-West region between 2003 and 2014 [Supplementary data (Table V)]. The introduction of malathion, in addition to deltamethrin, began in 2009.
All 20 sites submitted to qualitative trials using the WHO methodology [Supplementary data (Table VI)] were classified as having mosquitoes that were resistant (18) or with incipient resistance (two). Of these, 11 were also evaluated using the CDC methodology, and two were classified as susceptible, while the others were considered resistant (five) or with incipient resistance (four).
Results of deltamethrin quantitative bioassays were only available for municipalities in GO state since 2009, and the results mainly came from 2011 [Supplementary data (Table VII)]. In all cases, data on resistance levels were obtained one or two years after dengue outbreaks [Supplementary data (Tables II, VII). In particular, deltamethrin resistance levels in mosquitoes from Luziânia were so high that the RR95 could not be readily estimated from the adult specimens available, as their RR80 was already almost 170. It is remarkable that Luziânia was considered susceptible to this PY three years before this evaluation (2008) according to a qualitative bioassay done using the CDC methodology [Supplementary data (Table VI)]. Additionally, Luziânia was one of the municipalities in the Centre-West Region that was the least strongly affected by dengue epidemics in the studied period [Supplementary data (Table II)]. This high deltamethrin resistance level in Luziânia in 2011 can be explained if one takes into account that the samples used for these bioassays were collected during the periods with the highest dengue incidence in this locality, and precisely at the end of the epidemic period [Supplementary data (Fig. 2)]. Thus, the high resistance levels may have reflected the seasonality of infestations, as well as the intensification of the use of adulticides during epidemic periods. The participation of kdr mutations, which contribute to PY resistance, was also detected in mosquitoes from Luziânia; unfortunately, the absence of biochemical data precludes the conclusive evaluation of the main mechanisms involved in this resistance.
Resistance mechanisms - The activities of two enzyme classes, GSTs and ESTs, were greatly altered in both vector stages [Supplementary data (Table VIII)]. In some cases, there was also a high alteration of MFO activity, but mainly in adults. Although the total ACE activity was altered in mosquitoes from two populations, this enzyme was classified in all cases as being sensitive to insecticide inhibition according to the WHO criteria (data not shown). Larvae and adults from Aparecida de Goiânia, Goiânia and Rio Verde were evaluated more than once. In general, they showed increased alterations to the activities of their metabolic resistance enzymes over time. Metabolic resistance tended to be higher in adults in nine out of the 10 available pairs of simultaneous evaluations of both larvae and adults.
Qualitative bioassays with adults: CDC and WHO methodologies - In the routine evaluation of the resistance of adult Culicidae to neurotoxic insecticides, two qualitative bioassay methodologies are available and globally used, although there is only limited agreement between them.
45
Originally, the CDC and WHO methodologies were designed to evaluate different parameters, being knockdown and mortality, respectively. However, the assessment of mosquito mortality with the CDC methodology after recovery from insecticide exposure for 24 hours has already been employed in previous studies.
23
,
24
,
29
,
36
,
40
This approach is especially useful in evaluating resistance to pyrethroids, which have a strong knockdown effect. In practice, this evaluation procedure, when performed after 24 h of recovery, tends to increase the agreement between both methodologies, as stated elsewhere.
45
In the present study, mosquitoes from 82 ‘municipalities/years’ were submitted to bioassays using both procedures, and the mortality results obtained from these tests after 24 h of exposure to deltamethrin were then compared. The WHO methodology classified 87.8% of the populations as resistant, 12.2% as presenting incipient resistance, and none as susceptible [Supplementary data (Table VI)]. Meanwhile, mortality levels obtained with the CDC methodology tended to be higher, resulting in only 43.9% of the same populations being classified as resistant, 45.1% as having incipient resistance, and 11% as susceptible.
We identified a weak positive correlation between the results of both methodologies (n = 82, R2 = 0.014, p < 0.001). However, regional differences were observed: a weak negative correlation between results was found for samples from the Southeast and South Regions, but the correlation was positive for samples from the remaining Regions. In particular, a strong and highly significant correlation was found for samples from the North Region (n = 8, R2 = 0.751, p < 0.002).
The Cohen’s kappa coefficient (κ) values obtained showed that there was reasonable agreement between the two methodologies’ results for the 82 evaluated pairs of samples (κ = 0.39084) when the criterion proposed by David and Zahar
43
was employed. This criterion considers populations with mortality below 80% in these bioassays as being resistant. However, the agreement increased considerably (κ = 0.64995) when the most recent criterion proposed by the WHO,
46
which uses 90% mortality as the cut-off point for resistance, was used instead. It is worth noting that all of the results used herein came from laboratories belonging to the MoReNAa, in which both methodologies were adopted following standardised procedures in the monitoring routine of Ae. aegypti adults for the country of Brazil.
Several particular aspects of the two methodologies, discussed in detail elsewhere,
45
are thought to account for their differences in performance. Additionally, mortalities tend to be higher in the bottle assays used in the CDC methodology, wherein the mosquitoes are forced to make direct contact with the insecticide, since the entire inner surface of the bottle, including the cap, is impregnated with the product. In contrast, the WHO methodology includes non-insecticidal spots that can be used as refuges, specifically the screens at the ends of the cylindrical tubes. In principle, the CDC methodology is more versatile, since the user can impregnate their bottles relatively easily on their own, while the impregnation of papers depends on a more delicate process, or on their acquisition already impregnated with the product. However, there are limitations to the use of impregnated bottles, but not WHO tubes, in humid places, especially in the field, as there may be condensation under such conditions that may cause mosquitoes to adhere to the walls of the bottles.
Resistance to pyrethroids: metabolic and target-site mechanisms - Some studies have suggested that metabolic changes tend to result in lower levels of resistance to pyrethroids than changes in their target site, Na
V .
37
,
61
,
62
To test this possibility, deltamethrin resistance ratios were compared with both metabolic resistance and kdr frequencies. Direct comparisons were also made between the two resistance mechanisms, metabolic and target-site. As stated in the ‘Materials and methods’ section, for each population the mean level of the changes in activity found for all enzymatic classes related to metabolic resistance was used as a metabolic resistance index. The sum of the allelic frequencies of kdr mutations at positions 1016 and 1534 was used to evaluate changes in the PY target site.
When all available data from the entire country were combined, the deltamethrin RR was directly proportional to the frequency of kdr mutations (n = 18, R2 = 0.301, p < 0.001) [Supplementary data (Fig. 3A)]. Therefore, the higher the resistance level, the greater the participation of target-site alterations was in the resistance observed. Simultaneously, the deltamethrin RR and frequency of metabolic resistance tended to be inversely proportional; in other words, higher PY resistance levels tended to be correlated with less participation by detoxifying enzymes in resistance (n = 24, R2 = 0.004, p = 0.288) [Supplementary data (Fig. 3B)]. However, the correlation in the latter case was much weaker and non-significant, which makes sense when one considers that one of the variables evaluated in this case is a secondary parameter, derived from the activity of several enzymatic classes with variable specificity.
The direct comparison between both resistance mechanisms, metabolic and target-site, confirmed that one mechanism or the other tended to participate in the development of resistance; in other words, the frequency of metabolic resistance seemed to be higher when the kdr frequency was lower (n = 19, R2 = 0.012, p = < 0.001) [Supplementary data (Fig. 3C)].
| null | null |
[] |
[] |
[] |
[
"MATERIALS AND METHODS",
"RESULTS",
"DISCUSSION"
] |
[
"\nMosquitoes - Samples were collected in the field between 2005 and 2011. Ae. aegypti eggs were collected with ovitraps following MoReNAa recommendations.\n20\n\n,\n\n30\n\n,\n\n31\n In the laboratory, 1,000 larvae were raised per plastic basin (27 × 19 × 7 cm) containing 1L of partially dechlorinated water at 26 ± 1ºC. Every three days, 0.5 g of cat food (Friskies®, Purina, São Paulo, SP) was added to the basin as food for the larvae.\n13\n Pupae were transferred to cylindrical cardboard cages (18 × 17 cm), and the identification and sorting of the Ae. aegypti adults that emerged from them was done according to Consoli and Lourenço-de-Oliveira.\n32\n Guinea pigs, anesthetised with ketamine and xylazine,\n33\n were used as a blood meal source for the adults once a week for four weeks. Whenever possible, F1 or F2 mosquitoes were employed in the assays performed.\n\nInsecticides - Data on the temephos and deltamethrin sources employed in the assays performed are presented in Supplementary data (Table I).\n\nQuantitative bioassays - Temephos dose-response bioassays with larvae were performed following WHO\n34\n procedures, a protocol that was also described by Lima et al.\n30\n and Braga et al.\n31\n. For each municipality, at least three trials were carried out on different days, totalling at least 2,400 exposed larvae. Each trial consisted of 10 different temephos concentrations, four replicates per concentration, and 20 larvae per replicate. Mortality was recorded after 24 hours.\nStarting in 2009, adults of some vector populations were also submitted to deltamethrin dose-response bioassays, using an adapted version of the impregnated paper methodology.\n13\n\n,\n\n35\n\n,\n\n36\n\n,\n\n37\n Each replicate employed 15-20 females, which were one to five days old and not fed blood. Three replicates were used per concentration, and between eight and 10 concentrations were tested per assay. At least three trials were performed for each population on different days, totalling 1,080 to 1,800 females. Mosquitoes were exposed to the insecticide for one hour, and then transferred to recovery tubes, without insecticide, and any mortality was recorded 24 h later.\nIn all cases, the susceptibility reference lineage Rockefeller\n38\n was used to calibrate the assays and to calculate RR95 values.\n\nQualitative bioassays - In many cases, adult resistance to deltamethrin was investigated with diagnostic-dose (DD) assays using either insecticide-impregnated bottles or papers, based on the methodologies of the Centers for Disease Control and Prevention (CDC) and WHO, respectively.\n35\n\n,\n\n39\n In both bioassays, replicates of 15-20 females, one to five days old and not fed blood, were used. For each vector population, at least three trials were performed on different days.\nFor the CDC methodology, a control bottle impregnated with solvent alone (acetone) and three bottles impregnated with 5 μg of deltamethrin were used per test. According to this methodology, the DD is defined as the lowest deltamethrin concentration that kills 100% of Rockefeller mosquitoes in 30 min. The assay took 30-120 min, and knockdown records were taken every 15 min. Females were then transferred to recovery cardboard cages, and mortality counts were made 24 h later.\n23\n\n,\n\n39\n\n,\n\n40\n\n\nFor the WHO methodology,\n35\n each assay consisted of three replicates with papers impregnated with 3.65 mg deltamethrin/m2 (0.009%) and one control tube containing paper impregnated only with silicone oil.\n36\n In the WHO methodology, the DD is equivalent to twice the lethal concentration that kills 99% (LC99) of Rockefeller mosquitoes. After being in contact with the insecticide for 1 h, the mosquitoes were transferred to recovery tubes, and their percent mortality was determined 24 h later.\n\nBiochemical tests - Enzymes related to metabolic resistance were investigated in individual specimens following the protocols described by Valle et al.\n41\n and Montella et al.\n20\n for adults, and by Viana-Medeiros\n42\n for larvae. For each population, approximately 90 larvae (L3 final or L4 initial) or 90 adult females (24 h old and not fed blood) were used. The activities of MFO, EST, and GST enzymes were then quantified. For ESTs, three different substrates were used: alpha-naphthyl (α-EST), beta-naphthyl (β-EST), or p-nitrophenyl (ρNPA-EST) acetate. The activity of the OP target, ACE, was also measured; in this case, in addition to its inhibition by propoxur (AChI), the ACE total activity (AChE) was also quantified.\n\nData analyses - The following criteria proposed by David and Zahar\n43\n were adopted in the present study to interpret the results of qualitative bioassays: less than 80% mortality indicates resistance, mortality above 98% indicates susceptibility, and intermediate values point to incipient resistance.\nCohen’s kappa coefficient\n44\n\n,\n\n45\n was used to compare the results of CDC and WHO adult qualitative assays. This coefficient (κ) is a proxy for data agreement, which varies between zero and one. Two mortality cut-off points were used in these comparisons, 80%\n43\n and 90%\n46\n.\nIn the case of dose-response assays, lethal concentrations (LC) were calculated with Polo-PC software\n47\n via probit analysis\n48\n and used to calculate RR95 values through comparisons with Rockefeller data. For both temephos and deltamethrin, the functional criterion adopted by the MoReNAa in 2006 was used,\n11\n\n,\n\n20\n which states that a RR95 value above 3.0 is indicative of resistance.\nResults of biochemical assays were classified following Valle et al.\n41\n and Montella et al.,\n20\n using the Rockefeller 99th percentile obtained for each enzyme as a cut-off point. For each population and class of enzymes, the activity was then classified as unchanged, altered, or highly altered by the insecticide if the percentage of the samples above the Rockefeller 99th percentile was below 15%, between 15% and 50%, or above 50%, respectively. These percent activity change results for EST, MFO, and GST enzymes were also used to calculate the mean percent change in metabolic resistance (‘X_Metab_Resist’), a secondary measure corresponding to the mean of the changes in EST, MFO, and GST activities in each population. This latter measure was used to compare metabolic and target-site resistance mechanisms, as described in detail in the ‘Results’ section. AChE activity was evaluated as described above, through comparisons with the Rockefeller 99th percentile. Additionally, AChI assays were classified according to WHO criteria, which state that activity inhibition above 70% indicates unaltered ACE activity.\n49\n\n\n\nDelivery of insecticides used in public health - In Brazil, the MoH is responsible for distributing the insecticides used to control important disease vectors with relevance to public health to all states.\n13\n The annual delivery records of insecticides used against Ae. aegypti from 2003 to 2014 are presented herein. The larvicides employed were temephos, Bti, and IGRs (the chitin synthesis inhibitors novaluron and diflubenzuron, and the juvenile hormone analogue pyriproxifen). The adulticides employed were malathion (an OP) and several PY insecticides (deltamethrin, cypermethrin, etofenprox, and alpha-cypermethrin; see ‘Discussion’ section).\n\nDengue incidence - The history of dengue cases was scrutinized using the SINAN NET database (a Brazilian Health Information System).\n50\n According to the PNCD, all confirmed dengue cases have been recorded in this database. Human population growth over the period examined was estimated based on the results of the censuses of the years 2000 and 2010, which were obtained at the website of the Brazilian Institute of Geography and Statistics (IBGE).\n51\n These values were used to calculate the dengue incidence between 2000 and 2012. The dengue incidence was calculated as: (N/P) × 100,000 inhabitants, where N is the number of confirmed new dengue cases and P is the total resident population in a given time period.\n52\n According to the MoH, values of dengue incidence above 300 cases per 100,000 inhabitants (or 0.3%) are considered epidemic.\n3\n\n\n\nBibliographical research and criteria for data inclusion in the survey - An update on the temephos and deltamethrin resistance status of Brazilian Ae. aegypti populations from 146 municipalities is presented herein. The data included in the survey consisted of: (1) unpublished data generated by our team within the MoReNAa; (2) data from the recent Moyes et al.\n29\n review, which encompassed populations from around the world, and updated the work of Ranson et al.\n28\n; (3) a systematic bibliographic survey of data from 1 January 1985 to 31 July 2017 in the databases contained in the National Center for Biotechnology Information (NCBI) (PubMed and PubMed Central ® - PMC) and in Google Scholar. In this survey the following search terms were used: ‘Brazil’, ‘Aedes aegypti’, ‘insecticide resistance’, ‘organophosphate’, and ‘pyrethroid’. Study selection was initially based on the relevance of the title and compatibility of the abstract with the theme of this study. The chosen articles were then read in full and submitted to a new sorting stage, this time according to other criteria: (1) the sample representativeness of each municipality, and (2) the presence of data on temephos and deltamethrin susceptibility status. Only articles describing representative field collections were kept (in some cases, the origin of the samples was not even reported). Fig. 1 shows the information flow in this systematic bibliographic review, and the quantity of unpublished records presented in this study; results were assembled according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) methodology proposed by Moher et al.\n53\n\n\n\nFig. 1:preferred reporting items for systematic reviews and meta-analyses (PRISMA) diagram, showing the different phases of the bibliographic survey and consolidation of the data regarding the insecticide resistance of Brazilian Aedes aegypti populations between 1985 and 2017. The following criteria were adopted for the inclusion of articles in the systematic review: (1) articles with a title and abstract related to the theme of this study were included. (2) Consistency of susceptibility status data against temephos or deltamethrin: for qualitative trials, those studies reporting the diagnostic concentration(s) of pesticide tested and the percent mortality obtained were included; for quantitative trials, those reporting LCs (50, 90, and 95) and the corresponding RRs were included. (3) Field collection: articles were included that provided information on the field sample size collected, taking into account their representativeness in relation to the municipality area and the collection period. For samples collected in districts or in other municipality subdivisions, the average RR of the reported values was considered; in these cases, the entire range of LC confidence intervals was used. Papers that did not mention the year of sample collection were discarded, except for those described by Braga et al.(54), whose records are in our team’s possession. (4) The articles already covered by Moyes et al.(29) were excluded from the present analysis.\n\n\nEthics statement - The use of anesthetised guinea pig to blood feed mosquitoes was authorised by the Fiocruz Ethical Committee for Animal Use (CEUA/Fiocruz), under license numbers P-0147/02, L-011/09, and LW-20/14.",
"This study presented the results of evaluations of 146 Brazilian municipalities widely dispersed throughout the country, conducted between 1998 and 2012. Fig. 2 depicts the most current results of the 457 evaluations for temephos and 274 evaluations for deltamethrin, comprising the resistance status of Brazilian Ae. aegypti populations from 514 combinations of municipalities and years (‘municipalities/years’) and totalling 2,390 records. For each ‘municipality/year’, the total number of records was variable, and depended on both the insecticides evaluated and the tests performed (qualitative/quantitative). One ‘register’ was defined for each test: (1) in the case of qualitative assays, this was the mean mortality value; (2) for dose-response tests, this was each available LC (50, 80, 90, and 95) and the corresponding RR; and (3) for biochemical assays, this was the activity quantified for each enzyme class (ACE, MFO, GST, α-EST, β-EST, and ρNPA-EST). Allelic frequencies of kdr at the Na\nV 1016 and 1534 positions, which are strongly associated with PY resistance,\n55\n\n,\n\n56\n were also reported whenever available, although these were not considered as main ‘registers’ herein.\n\nFig. 2:resistance status of larvae and adults of Brazilian Aedes aegypti populations against temephos and deltamethrin, respectively. For each municipality, the results of the most recent bioassay are shown. For deltamethrin, in addition to the results of quantitative (circles) bioassays, those of qualitative assays (triangles) are also depicted.\n\nData were presented separately for each geographical Region of Brazil, and organized sequentially by state and then municipality. For each municipality, the annual results were grouped chronologically. Supplementary data (Tables) are included that depict: the annual incidence of dengue in each municipality in the period evaluated [Supplementary data (Table II)]; the history of insecticide distribution by the MoH to each state for use against larvae [Supplementary data (Table III)] and adults [Supplementary data (Table V)]; results of bioassays of larvae exposed to temephos [Supplementary data (Table IV), quantitative assays]; and results of adult bioassays against deltamethrin [Supplementary data (Tables VI-VII) for qualitative and quantitative assays, respectively]. Supplementary data (Table VIII) exhibits the results obtained concerning the resistance status and mechanisms for all samples for which biochemical or molecular data were available.\nIn the text, in addition to sections dealing with each geographic region, two further sections were included: the first compared the results of CDC and WHO qualitative bioassays with adults, and the second analysed the relative participation of metabolic and target-site mechanisms in the resistance observed.\nOur aim was to present, as completely as possible, the historical sequence of the development of resistance in Brazilian Ae. aegypti populations to insecticides used by public managers. It is noteworthy that 96.8% of these records were generated in laboratories belonging to the MoReNAa.\nNorth Region\n\nDengue incidence - Most of the evaluated municipalities in this region were concentrated in Pará state, where dengue outbreaks occurred with a high, but sporadic, incidence [Supplementary data (Table II)]. However, some epidemics were reported in other states, even in consecutive years, and sometimes with incidences exceeding the ‘threshold’ of 0.3% by 5-30 times, including: Rio Branco, AC (2009-2011), Oiapoque, AP (2007-2011), and Boa Vista, RR (between 2001-2003 and 2008-2010). In Pacaraima, RR and Palmas, TO, high dengue incidence values were registered during most of the evaluated period.\n\nResistance against the larvicide temephos - During the evaluated period, temephos was almost continuously supplied throughout the North Region. In general, other products, such as Bti or IGRs, did not replace this larvicide, but were sometimes employed together with it [Supplementary data (Table III)]. Accordingly, resistance to temephos was found to be widespread in nearly all 20 evaluated municipalities in the North Region. The only exceptions were Porto Velho, RO in 2002\n54\n and Boa Vista in 2010 [Supplementary data (Table IV)].\nFor the most part, the temephos RR95 values of mosquitoes in the North Region ranged from 4-12 in the studied period. The main exception was Oiapoque in 2009, in which very high resistance levels, including a RR50 of 42.1 and RR95 of 102.5, were recorded.\n57\n This latter value was five times higher than the second largest RR95 in the region across the whole studied period: 21.4, in Dom Eliseu, PA in 2003.\n20\n More recent trials with samples from Amapá (collected between December 2014 and January 2015) showed a significant reduction in temephos resistance, including a RR50 of 21.8 for mosquitoes from Oiapoque and of 6.5 for those from Macapá.\n58\n It is worth remembering that temephos has been gradually replaced by IGRs since 2010, and its supplying to Amapá stopped in 2013 [Supplementary data (Table III)].\nOiapoque is located along the Brazilian border with French Guiana. The high temephos RR in Oiapoque in 2009 (RR95 = 102.5) may have been the consequence of the intense vector control actions applied in this border area by both countries. Dengue incidence was roughly 10 times higher than the threshold that defines epidemics in 2007-2011. This profile, however, does not seem to be general, as different patterns occurred in: (1) Palmas, TO, which presented RR values lower than 10 in two evaluations (2006 and 2009), with a high incidence of dengue in all years between sample collections; and (2) Rio Branco, which had a RR value below 10 in 2011, after four successive years of high incidences of dengue, with rates that reached 30 times the epidemic threshold. Therefore, it is not simply the epidemic per se that is associated with resistance, but the kind of response enacted to it, in particular the relative importance attached to chemical control of vectors.\n\nResistance to deltamethrin - In the North Region, deltamethrin was widely used in the control of adult mosquitoes over the period examined. Malathion was supplied to Pará in 2011, and later began to be employed in some other states [Supplementary data (Table V)]. Qualitative bioassays with deltamethrin using the WHO methodology classified 15 of the 18 evaluated mosquito populations as resistant, and none were considered susceptible.\n43\n On the other hand, trials with the CDC methodology resulted in higher mortalities, and of the 11 evaluated populations, only two were classified as resistant [Supplementary data (Table VI)]. A comparison of these two methodologies is presented below (section: ‘Qualitative bioassays with adults: CDC and WHO methodologies’).\nAlthough performed with mosquitoes from municipalities distinct from the sources of those used in qualitative tests, the results obtained with quantitative assays corroborated the changes in deltamethrin susceptibility suggested by the results of the former trials [Supplementary data (Tables VI-VII)]. The deltamethrin RR95, a parameter evaluated since 2009, was always high, usually between 8.5 and 26.6. However, for mosquitoes from Pacaraima, RR and Marabá, PA evaluated in 2011, the deltamethrin RR95 was extremely high, 60.3 and 70.7, respectively. Pacaraima borders Santa Helena, Venezuela, where there is more intense chemical control of malaria vectors; this and the high dengue fever incidence there since 2007 [Supplementary data (Table II)] may explain the elevated deltamethrin RR95 in mosquitoes from Pacaraima. On the other hand, in Marabá, despite the low incidence of dengue [Supplementary data (Table II)], there was an eight-fold increase in the RR95 in a two-year interval, from 8.8 in 2009 to 70.7 in 2011 [Supplementary data (Table VII)]. This was the highest deltamethrin resistance ratio found in the region. Altogether, for the North Region it was not possible to make the general conclusion that high dengue incidence corresponds with increased PY resistance. The increase in the use of PYs in general observed during epidemics due to blocking actions undertaken by public health services, intensification of domestic use, and non-integrated use of PYs against malaria vectors\n10\n\n,\n\n13\n\n,\n\n14\n\n,\n\n59\n could explain the high level of resistance seen in mosquitoes from Pacaraima, but not in those from Marabá.\n\nResistance mechanisms - In the North Region, the activities of two enzymatic classes related to metabolic resistance were consistently altered: GSTs and, more intensely, ESTs, which in the latter case were evaluated with different substrates [Supplementary data (Table VIII)]. Few vector populations showed altered MFO activity. In only three of the 20 samples available, the total activity (AChE) of ACE, the target of OPs, appeared to be increased. However, in all cases ACE was considered sensitive to inhibition by insecticides according to WHO criteria (data not shown). Unfortunately, the metabolic resistance of larvae from a same locality in the North Region was never evaluated at two consecutive periods. However, there were simultaneous evaluations of larvae and adults on six occasions, and in all these cases the activity was more strongly altered in adults [Supplementary data (Table VIII)].\nNortheast Region\n\nDengue incidence - Dengue dynamics between 2001 and 2012 in the Northeast Region presented four three-year periods with distinct disease incidences [Supplementary data (Table II)]; in the first and last of these, 2001-2003 and 2010-2012, dengue incidences were high and consistent with epidemic conditions in most of the evaluated locations in all states. In the second triennium (2004-2006), incidence values were the lowest observed, while in the third triennium (2007-2009) the dengue incidence was variable. It is noteworthy that this parameter was very high in Bahia during 2009. However, in the 2007-2009 triennium, the dengue incidence tended to be low in two periods and sections: in the beginning (2007), in the southernmost states of the Northeast Region (SE, BA); and in the last year (2009), in the remaining ones. In general, the number of ‘municipalities/years’ with a dengue incidence more than 0.3% corroborated this scenario: the first and fourth triennia registered higher numbers of 81 and 70 ‘municipalities/years’ that were above this cut-off point, respectively; whereas recorded values in the second triennium were lower (16), and in the third triennium they were intermediate (48).\nNortheast Brazil was strongly affected by dengue over the evaluated period: in two states, CE and RN, at least 50% of ‘municipalities/years’ presented incidences indicating dengue epidemics. Even in MA, the least strongly affected state, more than 12% of the ‘municipalities/years’ presented high dengue incidences. The percentage of cases with incidences above 1,000 cases per 100,000 inhabitants, an arbitrary value equivalent to 1% of the population and more than three times the cut-off point defined by the Ministry of Health, was also evaluated. In this case, CE and RN were confirmed as the states that were the most strongly affected by dengue during the studied period. High incidences were also found for AL and BA.\n\nTemephos resistance - Except for RN, temephos was continuously distributed to all states in the Northeast Region until 2011-2014 [Supplementary data (Table III)]. Bti was simultaneously applied in several states (MA, CE, PE, and AL) until 2009-2010; IGRs were also introduced during this period in most states in the Northeast Region. By 2014 almost all states exclusively employed IGRs.\nIn the Northeast Region, resistance to temephos was detected in all municipalities evaluated, with the only exception being Fernando de Noronha Island in 2009 [Supplementary data (Table IV)]. Although to date the highest temephos resistance levels in Brazil occur in the Northeast Region, the RR there varies significantly: a temephos RR95 above 100 was found in 20% of cases, but in almost 40% of cases the RR95 was below 10. The lowest dengue incidences and also the lowest temephos RR95 values (always below 10) were found in the state of MA. In contrast, extremely high temephos RR95 values above 100 (sometimes above 200) were concentrated in PE (six municipalities), southern CE (two), eastern BA (five), and the countryside of AL (one) [Supplementary data (Fig. 1)].\nMuch variation was noted in several aspects of insecticide resistance monitoring in the Northeast Region, including: (1) the number of municipalities evaluated in each state (ranging from only two in MA and PI, up to more than 10 in PE and BA); (2) the frequency of evaluation of the different municipalities, with each municipality evaluated only once in some states, such as MA and PB, while some municipalities in other states were evaluated five (Juazeiro do Norte, CE and Natal, RN), six (Fortaleza, CE and Maceió, AL), or even seven (Aracaju, SE) times over the evaluated period; and (3) the regularity of monitoring, as for example on the one hand five of the six evaluations were performed in 2003 for PB, while on the other hand in BA only three out of 23 evaluations were done before 2008.\nFor municipalities that were evaluated more than once, temephos resistance levels were compared with both their dengue incidence and supply of insecticides [Supplementary data (Tables II, III, IV). Although several different parameters influence dengue dynamics, some correlations were suggested, as outlined below:\n― Crato, in the south of CE state, where the temephos RR95 in 2009 was higher than 190, presented a history of severe dengue epidemics between 2001 and 2007, which were five years with high values of dengue incidence, including two years when this was greater than 1%. This scenario may have contributed to the intensification of chemical control and the consequent increase in temephos resistance levels in this municipality. However, although dengue incidence remained high in this municipality until at least 2012, in 2013 resistance to temephos had declined more than three-fold (RR95 = 65), probably due to the interruption of its supply to the state in 2011.\n― In Fortaleza, CE, the temephos RR95 increased from 8-10 in 2002 and 2006 to 43 in 2007. The annual dengue incidence was above the MoH cut-off point five times between 2001 and 2007, pointing to the intensification of chemical control and a consequent RR increase. Unfortunately, although there were several periods of high dengue incidence until 2012 in Fortaleza, no further temephos evaluations were reported there.\n― With the exception of Santana do Ipanema, in the state of AL relatively stable temephos RR50-95 values, varying between four and 17, were found. These were consistent data, since these municipalities were assessed three to six times during the studied period. These results are also coherent with the supply of larvicides provided to AL state, since temephos was continuously but not exclusively provided between 2003 and 2013, being always concomitantly applied with Bti and/or an IGR.\n― In contrast, in the state of RN, although temephos was provided only in 2004 [Supplementary data (Table II)], the RR95 of mosquitoes for this OP fluctuated over the same range as those in AL, and was around 12 (from 10 to 19) between 2004 and 2011. These are also consistent data, as of the five municipalities monitored, four were evaluated three to five times. This situation suggests that dengue incidence and records of insecticide supply by the MoH were not sufficient to explain the temephos resistance dynamics observed in RN state. Additional investigations could reveal other parameters that should be taken into account for the interpretation of these data.\n― Accordingly, in Barra dos Coqueiros, SE, neither the low dengue incidence, nor the exclusive supplying of temephos as a larvicide over the evaluated period, were able to explain the increase in the temephos RR by seven times between 2000 (3.2) and 2004 (23.5).\nIn BA state, temephos was supplied continuously between 2003 and 2013, and this was the only larvicide used between 2004 and 2009. Among Bahia municipalities monitored more than once, two distinct scenarios should be mentioned: (1) records of high dengue incidence and stable levels of temephos resistance were observed in Barreiras, which was subjected to two years of very high dengue incidence between 2008 and 2012 (up to 1.6%), although the temephos RR95 there remained around 5.0 throughout the evaluated period; and (2) records of high dengue incidence and increasing temephos resistance levels were noted in such municipalities as Itabuna, Jequié, and Jacobina. In Itabuna, the temephos RR95 increased from 18.6 in 2004 to 55.8 in 2013, and records of dengue incidence in this municipality were also extremely high, in the range of 3-7%, in 2009 and 2012. Jequié exhibited low dengue incidences between 2004 and 2008, but high values between 2009 and 2012, and this last period was concomitant with a three-fold increase in the temephos RR95 value of mosquitoes from there. In Jacobina, temephos resistance levels increased 10-fold in one year, between 2008 and 2009, when the RR95 reached 104. Four years later, the temephos RR95 more than doubled, reaching almost 230. Dengue incidence in this municipality was high, or very high, during almost the entire period evaluated (2001-2012).\n\nResistance to deltamethrin - The whole Northeast Region received an uninterrupted supply of PY insecticides between 2003 and 2014 [Supplementary data (Table V)]. Malathion was delivered to CE in 2006, and to BA in 2011. This OP started to be continuously distributed in addition to deltamethrin in CE in 2008, and in some other states in the Northeast Region as of 2010.\nMosquitoes from all of the 39 ‘municipalities/years’ from the Northeast Region evaluated for deltamethrin resistance with qualitative tests using the WHO methodology were classified as resistant (26) or with incipient resistance (13). Among the 14 samples evaluated with the CDC methodology, 11 were considered resistant (two) or with incipient resistance (nine). In 13 cases, discussed in a section below, the two methodologies were applied simultaneously [Supplementary data (Table VI)].\nQuantitative tests were performed in eight municipalities from four states [Supplementary data (Table VII)], and confirmed that resistance to PY was prevalent throughout the Northeast Region. The RR95 values were always above 7.0 and, except for Mossoró in 2011 and Crato in 2013, remained below 20.\nIn the Northeast Region the deltamethrin resistance status was evaluated twice in two localities, Mossoró, RN and Aracaju, SE. In both cases, the deltamethrin RR95 increased and, accordingly, the frequency of kdr alleles at the PY target site also increased [Supplementary data (Table VIII)].\n\nResistance mechanisms - The activities of two classes of detoxifying enzymes, GSTs and ESTs, were altered by pesticides [Supplementary data (Table VIII)]. Both α-EST and β-EST activity were affected in larvae, while α-EST was the most strongly altered class in adults. Total ACE activity presented increased levels in only two cases. However, according to WHO criteria, ACE was sensitive to insecticide inhibition throughout the Northeast Region (data not shown). MFO and ρNPA-EST activities were altered in some cases, mainly in adults. In eight instances, larvae and adults were simultaneously evaluated, with greater enzyme activity alterations always observed in adults [Supplementary data (Table VIII)]. The two municipalities evaluated at more than one point in time, Aracaju and Caicó, exhibited a general increase in the activity of detoxifying enzymes in both larvae and adults.\nSoutheast Region\n\nDengue incidence - In the Southeast Region, SP state stands out, as detailed below. SP presented the longest insecticide resistance monitoring history and the lowest dengue incidence of all evaluated states; it is an example of the positive effects of both entomological surveillance and resistance management on dengue prevention. During the evaluated period (2001-2012), the dengue incidence was above 0.3%, the MoH cut-off point, in less than 20% of ‘municipalities/years’, and only 10% of cases presented a dengue incidence of more than 1% [Supplementary data (Table II)]. In contrast, ES was the state in the Southeast Region that was the most strongly affected by dengue in the studied period, as in more than 50% of the ‘municipalities/years’ in ES the dengue incidence was above the MoH recommendation, and 20% of these had a dengue incidence of more than 1%. RJ and MG states presented intermediate values: 45-55% and 15-20% of the ‘municipalities/years’ in these states exhibited dengue incidences above 0.3 and 1%, respectively.\n\nTemephos resistance - In the Southeast Region in general, temephos was distributed continuously throughout the period evaluated. Bti was supplied simultaneously until 2007-2010, and thereafter IGRs were used [Supplementary data (Table III)]. RJ was an exception, since temephos and IGRs were provided intermittently from 2004 onward in this state, while IGRs began to be supplied continuously after 2008, before they were supplied to the other states in the Southeast Region.\nTemephos resistance was evaluated in 42 municipalities, totalling 250 ‘municipalities/years’, in this Region. SP accounted for more than 80% of the records, among which 17 municipalities out of 20 were assessed several times, some almost annually and, in many cases, from 14 to 18 times over the 1998-2015 period. In contrast, in other states in the Southeast Region, the accomplishment of temephos bioassays, which started from 1999 (RJ) and 2005 (MG and ES) onward, extended only until 2011. In these states, most municipalities were evaluated once or twice in the studied period, with a maximum of five evaluations in the case of Rio de Janeiro city [Supplementary data (Table IV)].\nThis differential resistance monitoring and management effort was reflected in the susceptibility profiles obtained: the average temephos RR95 of mosquitoes from SP (3.5) was at least three times lower than that of those in the other states in the Southeast Region (RJ, MG, and ES: RR95 = 10.1, 11.4, and 13.3, respectively). In SP, the temephos RR95 was above 3.0 in 60% of cases, in contrast with it being above this value for 100% of the municipalities in the other states, with the only exception to this trend being São José do Vale do Rio Preto, RJ (RR95 = 3.0). In SP, the temephos RR95 was above 10.0 in mosquitoes from only 1% of ‘municipalities/years’ (two out of a total of 194 records). In contrast, more than 50% of the records for the other states in the Southeast Region reached this value. Out of the 42 municipalities assessed in the Southeast Region, four were classified as having mosquitoes susceptible to temephos (RR95 below 3.0), three of which were located in SP. In particular, just one among the four municipalities was evaluated more than once; this was Botucatu, SP, the mosquitoes from which remained susceptible to temephos during all six monitoring rounds carried out between 2004 and 2014. This municipality did not exhibit a dengue incidence above the epidemic ‘threshold’ over the entire evaluated period [Supplementary data (Table II)].\nIn SP, a temephos RR95 above 10.0 was detected only in mosquitoes from São Sebastião in 2004 and from Santos in 2005. In both municipalities, resistance ratio values decreased later on, culminating at levels indicating susceptibility in 2015. In SP, the highest temephos RR95 levels were reported in Santos, São Sebastião, São Paulo, and Itapevi. The first two of these were among the five SP cities evaluated herein with the highest dengue incidences in the period of 2001-2012.\nOn the other hand, in the Southeast Region, the ES state presented the highest dengue incidence and also the highest temephos RR95 in the evaluated period. These were consistent data, which were always derived from more than one evaluation. In the case of RJ, although most of the locations were evaluated more than once, there were gaps in the RR95 records, and in MG only the capital, Belo Horizonte, was evaluated more than once.\n\nResistance to deltamethrin - Supplementary data (Table V) shows the supply of adulticides provided by the MoH for all vector control programs, not just those targeting Ae. aegypti (see ‘Discussion’). PYs were widely distributed throughout the Southeast Region: SP received malathion during the whole period, and other states received it from 2007 onward, or later. It should be mentioned that SP stopped using PYs against the dengue vector in 2000, almost 10 years before their replacement in the rest of the country.\n60\n\n\nDeltamethrin qualitative bioassays were performed for 115 and 75 ‘municipalities/years’ with the WHO and CDC methodologies, respectively [Supplementary data (Table VI)]. In all cases, established or incipient resistance was detected. Established resistance prevailed in 92% of the trials (n = 106) done with the WHO methodology and in 72% of the trials (n = 54) done with the CDC methodology. Simultaneous evaluations with the WHO and CDC methodologies were performed for 46 ‘municipalities/years’ (see section below in which both methodologies were compared). Accordingly, deltamethrin quantitative bioassays revealed high resistance levels in all cases [Supplementary data (Table VII)].\n\nResistance mechanisms - GSTs were the enzymes whose activities were the most strongly altered by pesticides in samples of larvae and adults from the Southeast Region [Supplementary data (Table VIII)]. In adults, the activity of EST enzymes, mainly a-EST and ρNPA-EST, were also altered. Changes in MFO activity were also identified in some samples. Increases in total ACE activity were detected in roughly 30% of the evaluated samples. However, ACE activity was never altered in both stages evaluated from the same ‘municipality/year’. In addition, the ACE activity in all samples was considered sensitive to insecticide inhibition (AChI) when the WHO criterion was used (data not shown). Whenever biochemical assays were available for both life stages (eight pairs of samples), metabolic resistance was higher in adults. Metabolic resistance was never evaluated twice in larvae from the same locality. However, for five municipalities, the metabolic resistance of adults was evaluated 2-3 times; despite this, there were no concurrent data on the application of quantitative bioassays or on Na\nV allele frequencies, a situation that precludes the analysis of the main mechanisms involved in resistance in these cases.\nSouth Region\n\nDengue incidence - Due in part to its milder climate, the South Region was the least strongly affected by dengue. Most municipalities in SC and RS states evaluated over the studied period did not register dengue epidemics. The exception was Ijuí, RS in 2010, where the dengue incidence reached 3.7%, more than 12 times the MoH cut-off point [Supplementary data (Table II)]. The state of PR was the most affected by dengue. In the South Region, a dengue outbreak first occurred in 2002 in Foz do Iguaçu, PR, which is located on the triple border of Brazil with Paraguay and Argentina. In 2003, there was an epidemic in three municipalities in northern central Paraná, Cambé, Ipiporã, and Londrina. From then on, new records of high dengue incidences only occurred in this state in 2007, and then again in 2010/2011. In the period 2007-2011, Foz do Iguaçu endured three years of extremely high dengue incidences 3-12 times the MoH threshold of 0.3%.\n\nTemephos resistance - All states in the South Region received the larvicide temephos almost continuously between 2003 and 2014 [Supplementary data (Table III)]. RS also received Bti in 2006 and 2009. The supplying of IGR larvicides to PR occurred from 2010 onward. Both SC and RS received IGRs in 2014 for the first time.\nThe temephos resistance status was evaluated in 12 municipalities in the South Region, totalling 29 ‘municipalities/years’ [Supplementary data (Table IV)]. Foz do Iguaçu and Maringá, both in PR, were evaluated eight and six times, respectively, between 2001 and 2009. Among the remaining municipalities, six were evaluated only once, and four were evaluated 2-3 times. Although in 55% of the bioassays the RR95 was higher than 3.0, the MoH cut-off point, it was never higher than 7.0. Mosquitoes from Maringá in 2005 and Ibiporã in 2006 exhibited the highest resistance levels. In general, a slight increase in resistance to OP was noted in the South Region over time.\n\nResistance to deltamethrin - Pyrethroids were provided almost continuously and used nearly exclusively in the South Region [Supplementary data (Table V)]. PR also received the OP malathion from 2010 onward. Qualitative deltamethrin bioassays were performed for three PR locations. Eight ‘municipalities/years’ were evaluated by the WHO method, and four of these were also evaluated using the CDC methodology [Supplementary data (Table VI)], and susceptibility was not detected in any case. In the South Region, results of deltamethrin quantitative bioassays were only available for mosquitoes from Santa Rosa, RS, for which there was a RR95 of 33.3 in 2011 [Supplementary data (Table VII)].\n\nResistance mechanisms - In the South Region, resistance mechanisms were investigated only in mosquito larvae and adults from Santa Rosa, RS in 2011. The larval enzyme activity profile was unaltered, which was consistent with their susceptibility to temephos [Supplementary data (Table VIII)]. In adults that were resistant to deltamethrin, changes in EST activity were detected, particularly in the activities of α-EST and ρNPA-EST. The participation of Na\nV alterations in resistance was also identified in this case. Despite showing an increase in their total ACE activity, mosquitoes from Santa Rosa were considered to be sensitive to insecticide inhibition according to the WHO criteria (data not shown).\nCentre-West Region\n\nDengue incidence - All municipalities evaluated in this Region experienced at least one dengue epidemic year between 2001 and 2012 [Supplementary data (Table II)]. Brazil’s capital, Brasília, in the Federal District (DF), was the least strongly affected locality, with there being high dengue incidence only in 2010. In the MS municipalities evaluated, dengue incidences generally increased from 2006 onward and reached values up to 6.3%, more than 20 times the MoH threshold, as was seen in Campo Grande in 2007. In GO, the dengue incidence also reached very high values in several localities. In particular, high dengue incidences were observed in two neighbouring municipalities, Goiânia and Aparecida de Goiânia, throughout the period evaluated (2001-2012). In 2010, a strong dengue epidemic affected the entire Centre-West Region: in 16 of the 17 municipalities evaluated, the dengue incidence was above 0.3%, and in four this value was even above 3.0%.\n\nTemephos resistance - Except in the DF, temephos was supplied continuously between 2003 and 2013 throughout the Region. In MS, Bti was used in addition to temephos between 2003 and 2009. IGRs were introduced from 2009-2010, becoming the sole larvicides provided by the MoH throughout the Centre-West Region in 2014 [Supplementary data (Table III)].\nMosquitoes from all municipalities in the Centre-West Region evaluated were resistant to temephos [Supplementary data (Table IV)], the only exception being those from Brasilia in 2008. Brasilia presented the highest number of evaluations, and also the lowest temephos resistance levels and the lowest dengue incidence rates, over the evaluated period. Only Cuiabá in 2005 was evaluated in MT state; its temephos RR95 was also among the lowest in the region, in line with the moderate dengue incidence rates recorded there up to this time.\nThe temephos RR95 in MS was always below 8.0 [Supplementary data (Table IV)], even in 2011, after a period of extremely high dengue incidence in 2006-2010 [Supplementary data (Table II)]. The simultaneous use of temephos and Bti [Supplementary data (Table III)] may have contributed to there being less selection pressure for OP resistance in this state.\nIn contrast, mosquitoes from GO showed the highest temephos resistance levels in the region, as 10 out of the 15 municipalities for which the RR95 was evaluated in this state had values that were above 10.0 [Supplementary data (Table IV)]. Notably, São Miguel do Araguaia, GO in 2012 presented the highest temephos resistance level in the Centre-West Region, even though 2012 corresponded to the second year of low dengue incidence after five consecutive years of high incidences in this locality. The contiguous municipalities of Goiânia and Aparecida de Goiânia are clear examples of how differences in chemical control management can have an impact on the development of resistance. Although dengue incidence was high in both municipalities during the entire evaluated period, temephos resistance levels were much lower in Goiânia than in Aparecida de Goiânia, suggesting there were distinct selection pressures in both localities. Indeed, in the routine insecticide resistance monitoring of Brazilian Ae. aegypti populations, some adjacent municipalities with different local dynamics of infestation control were purposely chosen for just this reason. This was done to emphasize to municipal public officials how much both surveillance and infestation control strategies can impact insecticide resistance and, consequently, influence the effectiveness of vector control.\n\nResistance to deltamethrin - Pyrethroid adulticides were distributed widely in the Centre-West region between 2003 and 2014 [Supplementary data (Table V)]. The introduction of malathion, in addition to deltamethrin, began in 2009.\nAll 20 sites submitted to qualitative trials using the WHO methodology [Supplementary data (Table VI)] were classified as having mosquitoes that were resistant (18) or with incipient resistance (two). Of these, 11 were also evaluated using the CDC methodology, and two were classified as susceptible, while the others were considered resistant (five) or with incipient resistance (four).\nResults of deltamethrin quantitative bioassays were only available for municipalities in GO state since 2009, and the results mainly came from 2011 [Supplementary data (Table VII)]. In all cases, data on resistance levels were obtained one or two years after dengue outbreaks [Supplementary data (Tables II, VII). In particular, deltamethrin resistance levels in mosquitoes from Luziânia were so high that the RR95 could not be readily estimated from the adult specimens available, as their RR80 was already almost 170. It is remarkable that Luziânia was considered susceptible to this PY three years before this evaluation (2008) according to a qualitative bioassay done using the CDC methodology [Supplementary data (Table VI)]. Additionally, Luziânia was one of the municipalities in the Centre-West Region that was the least strongly affected by dengue epidemics in the studied period [Supplementary data (Table II)]. This high deltamethrin resistance level in Luziânia in 2011 can be explained if one takes into account that the samples used for these bioassays were collected during the periods with the highest dengue incidence in this locality, and precisely at the end of the epidemic period [Supplementary data (Fig. 2)]. Thus, the high resistance levels may have reflected the seasonality of infestations, as well as the intensification of the use of adulticides during epidemic periods. The participation of kdr mutations, which contribute to PY resistance, was also detected in mosquitoes from Luziânia; unfortunately, the absence of biochemical data precludes the conclusive evaluation of the main mechanisms involved in this resistance.\n\nResistance mechanisms - The activities of two enzyme classes, GSTs and ESTs, were greatly altered in both vector stages [Supplementary data (Table VIII)]. In some cases, there was also a high alteration of MFO activity, but mainly in adults. Although the total ACE activity was altered in mosquitoes from two populations, this enzyme was classified in all cases as being sensitive to insecticide inhibition according to the WHO criteria (data not shown). Larvae and adults from Aparecida de Goiânia, Goiânia and Rio Verde were evaluated more than once. In general, they showed increased alterations to the activities of their metabolic resistance enzymes over time. Metabolic resistance tended to be higher in adults in nine out of the 10 available pairs of simultaneous evaluations of both larvae and adults.\n\nQualitative bioassays with adults: CDC and WHO methodologies - In the routine evaluation of the resistance of adult Culicidae to neurotoxic insecticides, two qualitative bioassay methodologies are available and globally used, although there is only limited agreement between them.\n45\n Originally, the CDC and WHO methodologies were designed to evaluate different parameters, being knockdown and mortality, respectively. However, the assessment of mosquito mortality with the CDC methodology after recovery from insecticide exposure for 24 hours has already been employed in previous studies.\n23\n\n,\n\n24\n\n,\n\n29\n\n,\n\n36\n\n,\n\n40\n This approach is especially useful in evaluating resistance to pyrethroids, which have a strong knockdown effect. In practice, this evaluation procedure, when performed after 24 h of recovery, tends to increase the agreement between both methodologies, as stated elsewhere.\n45\n\n\nIn the present study, mosquitoes from 82 ‘municipalities/years’ were submitted to bioassays using both procedures, and the mortality results obtained from these tests after 24 h of exposure to deltamethrin were then compared. The WHO methodology classified 87.8% of the populations as resistant, 12.2% as presenting incipient resistance, and none as susceptible [Supplementary data (Table VI)]. Meanwhile, mortality levels obtained with the CDC methodology tended to be higher, resulting in only 43.9% of the same populations being classified as resistant, 45.1% as having incipient resistance, and 11% as susceptible.\nWe identified a weak positive correlation between the results of both methodologies (n = 82, R2 = 0.014, p < 0.001). However, regional differences were observed: a weak negative correlation between results was found for samples from the Southeast and South Regions, but the correlation was positive for samples from the remaining Regions. In particular, a strong and highly significant correlation was found for samples from the North Region (n = 8, R2 = 0.751, p < 0.002).\nThe Cohen’s kappa coefficient (κ) values obtained showed that there was reasonable agreement between the two methodologies’ results for the 82 evaluated pairs of samples (κ = 0.39084) when the criterion proposed by David and Zahar\n43\n was employed. This criterion considers populations with mortality below 80% in these bioassays as being resistant. However, the agreement increased considerably (κ = 0.64995) when the most recent criterion proposed by the WHO,\n46\n which uses 90% mortality as the cut-off point for resistance, was used instead. It is worth noting that all of the results used herein came from laboratories belonging to the MoReNAa, in which both methodologies were adopted following standardised procedures in the monitoring routine of Ae. aegypti adults for the country of Brazil.\nSeveral particular aspects of the two methodologies, discussed in detail elsewhere,\n45\n are thought to account for their differences in performance. Additionally, mortalities tend to be higher in the bottle assays used in the CDC methodology, wherein the mosquitoes are forced to make direct contact with the insecticide, since the entire inner surface of the bottle, including the cap, is impregnated with the product. In contrast, the WHO methodology includes non-insecticidal spots that can be used as refuges, specifically the screens at the ends of the cylindrical tubes. In principle, the CDC methodology is more versatile, since the user can impregnate their bottles relatively easily on their own, while the impregnation of papers depends on a more delicate process, or on their acquisition already impregnated with the product. However, there are limitations to the use of impregnated bottles, but not WHO tubes, in humid places, especially in the field, as there may be condensation under such conditions that may cause mosquitoes to adhere to the walls of the bottles.\n\nResistance to pyrethroids: metabolic and target-site mechanisms - Some studies have suggested that metabolic changes tend to result in lower levels of resistance to pyrethroids than changes in their target site, Na\nV .\n37\n\n,\n\n61\n\n,\n\n62\n To test this possibility, deltamethrin resistance ratios were compared with both metabolic resistance and kdr frequencies. Direct comparisons were also made between the two resistance mechanisms, metabolic and target-site. As stated in the ‘Materials and methods’ section, for each population the mean level of the changes in activity found for all enzymatic classes related to metabolic resistance was used as a metabolic resistance index. The sum of the allelic frequencies of kdr mutations at positions 1016 and 1534 was used to evaluate changes in the PY target site.\nWhen all available data from the entire country were combined, the deltamethrin RR was directly proportional to the frequency of kdr mutations (n = 18, R2 = 0.301, p < 0.001) [Supplementary data (Fig. 3A)]. Therefore, the higher the resistance level, the greater the participation of target-site alterations was in the resistance observed. Simultaneously, the deltamethrin RR and frequency of metabolic resistance tended to be inversely proportional; in other words, higher PY resistance levels tended to be correlated with less participation by detoxifying enzymes in resistance (n = 24, R2 = 0.004, p = 0.288) [Supplementary data (Fig. 3B)]. However, the correlation in the latter case was much weaker and non-significant, which makes sense when one considers that one of the variables evaluated in this case is a secondary parameter, derived from the activity of several enzymatic classes with variable specificity.\nThe direct comparison between both resistance mechanisms, metabolic and target-site, confirmed that one mechanism or the other tended to participate in the development of resistance; in other words, the frequency of metabolic resistance seemed to be higher when the kdr frequency was lower (n = 19, R2 = 0.012, p = < 0.001) [Supplementary data (Fig. 3C)].",
"Although in the ‘Results’ section the details of the data collected were presented separately for each Region, we also attempted to identify national patterns and trends in relation to each of the main topics addressed: (1) the dengue incidence in the period studied; (2) the supply of the main insecticides used in vector control by the MoH to each state; (3) temephos and deltamethrin resistance profiles; and (4) the main and potential resistance mechanisms involved.\n\nDengue incidence - Only the dengue incidence in localities monitored for insecticide resistance was presented herein. As mentioned above, 96.8% of these records were generated within the MoReNAa, which attempted to define ‘sentinel municipalities’ that were representative of the dengue epidemiology in their geographical areas. Hence, it was possible to sketch an approximate picture of the recent history of dengue outbreaks and vector control in different Brazilian Regions. Except for the South Region, which was affected by dengue epidemics later than the rest of the country, the dengue incidence in all other regions was higher than the MoH cut-off point of 0.3% in more than 30% of municipalities [Supplementary data (Table IX)]. In addition, except for the South Region, extremely high dengue incidences above 1% were observed in more than 10% of the cases examined. In particular, in the Centre-West Region almost 20% of the records were above 1% over the studied period.\n\nSupplementary data (Tables X, XI) depict the dengue incidence in each year in each Region. In general, the high incidence of dengue fever recorded in 2001-2002 and from 2008 onward\n63\n can be seen in these data. However, epidemics did not occur simultaneously in all regions of the country. In particular, it is worth noting (1) the late inclusion of the South Region in the area of epidemiological importance for dengue, and (2) the magnitude and persistence of dengue epidemics in the Centre-West Region, most notably between 2006 and 2010. Climate changes, reflected in changes in temperature and precipitation patterns over the evaluated period, together with distinct macro- and micro-scale social determinants, tended to favour the spread of the vector.\n64\n However, the non-simultaneous transmission of dengue in the country may be the consequence of heterogeneous variations in such climatic changes among the different geographical regions, which would have had unequal effects on the biology of the vector therein.\n\nProvision of insecticides by the Brazilian Ministry of Health - We used the annual MoH registry of the insecticides supplied to the different states aiming to control different arthropod vectors between 2003 and 2014. Although the available documents did not specify the target vectors for the different products, we sought to restrict the analysis to the insecticides known to be used against Ae. aegypti. However, in the case of adulticides, we opted to include all PY insecticides provided by the MoH, since there is partial overlap between Ae. aegypti occurrence and that of other vectors targeted by PYs that are relevant to public health. In addition, the common mode of action of PY insecticides, and consequently their potential to induce selection for the same resistance mechanisms, were considered.\nTemephos was the most heavily used larvicide in the period of 2003-2014, when it was supplied almost continuously to nearly all Brazilian Regions. In fact, for a long time temephos was the only larvicide approved by the WHO for use in potable water containers. Since at least 2003, Bti has also been used throughout the country, usually in addition to temephos. Subsequently, IGRs were introduced, and their use as larvicides was established in almost the entire country from 2009-2010, except for the South Region, where these larvicides were introduced later. It is worth mentioning that since 2012\n7\n Brazil has adopted a larvicide rotation approach to preserve the supply of the few available products in any given year. Chitin synthesis inhibitors, mainly diflubenzuron and novaluron, were the first IGRs to be employed in Brazil, and were later succeeded by juvenile hormone analogues, such as pyriproxyfen. In 2013, temephos ceased to be the first-choice larvicide for use in Ae. aegypti control, and since 2014 it has no longer been used, and has instead been replaced by IGR for use in larval control.\n65\n\n\nAccording to the WHO, there are only a few options to use as adulticides, all of which are PY products except for the OP malathion.\n27\n In the 2003-2014 period, deltamethrin was used practically throughout the whole country except in SP state, where due to resistance PYs have not been employed in the control of Ae. aegypti adults since 2000.\n14\n\n,\n\n60\n Despite this, SP continued to receive PYs to control other arthropod vectors. Malathion started to be distributed in 2006-2008 to some states, mainly in the Northeast (CE in 2006) and Southeast (RJ in 2007 and MG in 2008) Regions. Later, due to the dissemination of high levels of PY resistance in Ae. aegypti populations throughout the country, malathion was gradually introduced instead for adult control,\n25\n\n,\n\n26\n and its use was well-established in the country by around 2009-2011.\n\nResistance to the larvicide temephos - Since 2006, Brazil has considered Ae. aegypti populations with a RR95 to temephos of 3.0 or higher to be resistant to this larvicide. This change in classification, from 10.0 to 3.0, was based on functional and operational criteria. For functional validation, simulated field trials confirmed the reduction of the effectiveness of temephos in populations with RR95 values above 3.0.\n20\n The operational criteria were related to the logistics of the insecticide substitution procedure in the country, which depends on coordination among the three public management levels (federal, state, and municipal), meaning that, in practice, it can take up to two years for a recommendation at the central level to be implemented in the field.\nToday, temephos resistance is so widespread in Brazil that this OP is no longer considered as the first-choice larvicide for use against Ae. aegypti, and it has been replaced by other, non-neurotoxic products. Varied temephos resistance levels were observed in the country. In some cases, high temephos resistance levels occurred simultaneously with, or soon after, periods of elevated dengue incidence, suggesting the intensification of chemical control in response to outbreaks; in other cases, resistance levels were associated with the temephos supply, or its interruption. However, in many cases these indicators were not enough to explain the variety of situations observed. For instance, the South Region presented the lowest RR95 values throughout the period evaluated. This result is compatible with the late inclusion of the South in the ‘dengue scenario’. Low temephos resistance levels were also found in SP state, but in this case they were probably due to there being strong and coordinated monitoring and resistance management. The Northeast Region presented the greatest variation in temephos resistance levels, as well as the highest registered values, as the RR95 was above 100 in 20% of cases there, and in 6% of cases it was higher than 200 (five municipalities, three in PE and two in BA). According to Moyes et al.,\n29\n Brazil is the country with the highest levels of resistance by mosquitoes to temephos in the world. In Martinique, the country with the second highest resistance levels on this scale, the highest recorded RR50 was 35 in Gros Morne in 2009.\n66\n In general, these results revealed the influence of local public health actions on the establishment of temephos resistance and the fragility of general guidelines, which should contribute to the more rational use of chemical control and, ultimately, its preservation as a complementary alternative for vector control.\n\nResistance to deltamethrin - In Brazil, a few decades elapsed until the spread of temephos resistance reached levels that could compromise its use in Ae. aegypti control. However, in the case of PYs, which were introduced by the PNCD across the whole country in 2000, only a few years were enough to induce high resistance levels in populations of this vector to this class of pesticide.\n10\n\n,\n\n13\n\n,\n\n23\n\n,\n\n67\n\n\nCompared with larvae, the evaluation of adult resistance is more labour intensive. It depends on the use of devices and consumable materials that are not always easy to obtain. In addition, since adult females are used instead of larvae, tests become more time-consuming and dependent on the availability of specimens. For these reasons, routine tests are generally qualitative ones, in which specimens are exposed to only one insecticide dose and samples are classified as resistant, susceptible, or with incipient resistance. Two main methodologies, referred to herein as the CDC and WHO methodologies, are available for such tests and use bottles or papers, respectively, which are impregnated with insecticide.\n35\n\n,\n\n39\n The CDC methodology tends to result in higher mortality levels due to the nature of the assay itself. In Brazil, both methodologies have been employed to evaluate deltamethrin resistance. The WHO methodology did not identify any susceptible populations, and with the CDC approach only 8% of the samples were susceptible. Although both the CDC and WHO methodologies are effective to assess the susceptibility profile of adult mosquitoes, the variable correlation between the results of the two methods inhibits, and even precludes, the ability for comparisons to be made between their results. In this sense, we tend to agree with Owusu et al.,\n45\n who suggested that dose-response trials should be adopted to produce more consistent evaluations.\nIn this sense, within the scope of the MoReNAa we started to introduce the use of quantitative deltamethrin assays using impregnated papers directed at adult specimens in 2009.\n26\n\n,\n\n68\n To our knowledge this was the first quantitative evaluation of adults performed in the Ae. aegypti resistance monitoring routine. It was necessary to establish a series of conditions and parameters for this technique, as well as to define evaluation criteria. In the latter case, we adopted the cut-off established in Brazil in 2006 for temephos: that a RR95 above 3.0 is indicative of resistance.\n19\n\n\nQuantitative trials with deltamethrin for 41 ‘municipalities/years’ were identified herein. Most tests (30) were performed in 2011-2012, and confirmed the dissemination of resistance of Ae. aegypti to PYs in Brazil. Except for the South Region, very high RR95 values around 50-70 were detected in all Regions, and were more frequent in municipalities in the Centre-West Region. The Northeast Region tended to house the vector populations with the lowest PY resistance levels. There have been reports in the literature that Ae. aegypti resistance to PYs is more strongly influenced by vector seasonality, and in particular by the seasonality of dengue epidemics, than larvicides.\n10\n\n,\n\n13\n\n,\n\n67\n This is a clear manifestation of the conceptual confusion between ‘vector control’ and ‘chemical control of adult vectors’. Indeed, this is one of the hypotheses that explains the rapid spread of PY resistance throughout the world.\n13\n\n,\n\n14\n\n,\n\n55\n\n,\n\n67\n\n,\n\n69\n\n,\n\n70\n\n,\n\n71\n\n,\n\n72\n\n\nWhen the introduction of DENV-4 was detected in Brazil during the course of a dengue outbreak in Boa Vista, RR in the North Region, a quick and significant increase in PY resistance was detected throughout the city, including in areas that had not received ultra-low volume applications from the public health sector.\n10\n Additionally, in SP state, Ae. aegypti resistance to pyrethroid insecticides continued to persist in several localities, even 10 years after their discontinuation in the control of the dengue vector by SP state municipal health secretaries.\n14\n In relation to the data presented herein, in several cases high deltamethrin resistance levels were preceded by dengue epidemics, confirming that intense and indiscriminate use of pyrethroids occurred during outbreaks. The scenario found in the Centre-West Region is quite illustrative of this point: in practically all municipalities evaluated in 2011/2012, the RR95 was around 50. Since 2006, this region has been subjected to dengue outbreaks, culminating in 2010, which was precisely when the average dengue incidence in the municipalities evaluated was approximately 1.7%, almost six times the MoH cut-off point. The case of Luziânia, GO is also particularly informative: in 2011, the highest dengue rates so far registered (above 1.4%), and also extremely high levels of deltamethrin resistance (RR80 = 167), were found, and a closer look at the data revealed that the collection of field material for these bioassays was performed at the end of the epidemic season [Supplementary data (Fig. 2)], showing the influence of the intensification of adulticide application on the vector populations’ pyrethroid resistance status.\n\nResistance mechanisms - Metabolic resistance is the result of changes in the activities of enzymes involved in the metabolism, sequestration, and excretion of insecticides. The activities of three major enzyme classes, MFOs, ESTs (Phase I), and GSTs (Phase II), were evaluated in larval and adult female samples using the routine monitoring methodology applied in Brazil. In general, adults exhibited greater alterations to their enzymes’ activities than larvae.\nThe multifactorial nature of insecticide resistance was confirmed in this study, as has been reported for other vector populations worldwide.\n73\n\n,\n\n74\n\n,\n\n75\n The most strongly affected mechanisms of metabolic resistance throughout Brazil were the activities of the EST and GST enzyme superfamilies, which were closely related to OP and PY resistance in Brazilian samples.\n13\n\n,\n\n20\n\n,\n\n57\n\n,\n\n67\n\n,\n\n76\n Changes in MFO activity were less prominent, as only around 30% and less than 20% of adult and larval samples, respectively, exhibited changes in MFO activity. This metabolic profile contrasts with those reported by studies done elsewhere,\n56\n\n,\n\n70\n\n,\n\n77\n\n,\n\n78\n\n,\n\n79\n and this difference was probably due to the distinct nature of the assays examined herein. Specifically, these biochemical assays (which are adopted in routine monitoring programs) quantify levels of different enzyme classes, and thus may underestimate activity alterations; in contrast, the detection of MFO participation in metabolic resistance is generally derived from molecular assays that consist of more specific approaches to investigate gene expression changes. Efforts to understand the dynamics of metabolic resistance in Brazilian vector samples at the gene expression level, including those of MFOs, are currently underway.\nThe OP target is encoded by two genes, ace1 and ace2.\n80\n The relationship between some ace1 mutations in mosquitoes belonging to the genera Culex and Anopheles and their OP resistance has been well-documented.\n80\n\n,\n\n81\n The participation of two recently detected point mutations in ace1 in OP resistance in Ae. aegypti populations in India and Indonesia has also been suggested.\n82\n\n,\n\n83\n However, it is intriguing that for the mutations reported in Aedes mosquitoes, only a small difference in the inhibition profile of ACE has been suggested, which is different from the impacts of similar mutations observed in Culex and Anopheles species. The present study detected changes in total ACE activity (AChE) in 17% of the analysed samples, but in no case did the remaining ACE activity after insecticide inhibition (AChI) signal the alteration of this endpoint. However, it is important to note that the criteria applied to classify the ACE status in both assays, AChE and AChI, are quite different, which indicates the need for further, deeper analyses of this enzyme in Ae. aegypti field populations.\nIn relation to PY resistance, some research groups have argued that metabolic changes tend to result in lower resistance levels than alterations in the PY target site, Na\nV .\n37\n\n,\n\n61\n\n,\n\n62\n We presented evidence that the participation of metabolic mechanisms indeed seems to be limited in populations with high deltamethrin resistance levels. Accordingly, it has been shown elsewhere that there is less participation of the metabolic detoxification pathway in the resistance of mosquito populations presenting Na\nV\nkdr mutations.\n61\n In that same study, the authors suggested that different Ae. aegypti populations may respond to selection pressure in distinct ways, hindering or even preventing attempts to identify metabolic resistance components that could be diagnostic of PY resistance in general.\nThe history of temephos and deltamethrin resistance status examined herein provides evidence of the spread of resistance against the main insecticides used by the PNCD in the control of Brazilian Ae. aegypti populations. The high temephos resistance levels observed are the consequence of its use for more than 40 years in Brazil. Rapid dissemination of adult resistance to deltamethrin was correlated with the intensified chemical control of adults during dengue epidemic seasons. High dengue incidences are recurrent in Brazil, a hyperendemic country for this arbovirus. This dataset highlights the limitations of chemical vector control as the methodology of choice for the sustainable reduction of dengue incidence. In this sense, the rotation of insecticides has already been adopted in Brazil as a strategy to preserve these chemical pesticides as effective vector control tools. The multifactorial character of pesticide resistance was shown in this study. In view of the insecticide resistance of Ae. aegypti populations in Brazil, alternative approaches, such as community engagement and mechanical control, should be considered when planning interventions to reduce dengue transmission. It is worth emphasizing the importance of respecting the complementary character of insecticides, meaning that they should be reserved for the treatment of vector breeding places that cannot be eliminated and for specific places and situations, such as outbreak blockage or intervention at strategic points."
] |
[
"materials|methods",
"results",
"discussion"
] |
[
"Aedes aegypti",
"Brazil",
"insecticide resistance",
"temephos",
"deltamethrin",
"vector control"
] |
MATERIALS AND METHODS:
Mosquitoes - Samples were collected in the field between 2005 and 2011. Ae. aegypti eggs were collected with ovitraps following MoReNAa recommendations.
20
,
30
,
31
In the laboratory, 1,000 larvae were raised per plastic basin (27 × 19 × 7 cm) containing 1L of partially dechlorinated water at 26 ± 1ºC. Every three days, 0.5 g of cat food (Friskies®, Purina, São Paulo, SP) was added to the basin as food for the larvae.
13
Pupae were transferred to cylindrical cardboard cages (18 × 17 cm), and the identification and sorting of the Ae. aegypti adults that emerged from them was done according to Consoli and Lourenço-de-Oliveira.
32
Guinea pigs, anesthetised with ketamine and xylazine,
33
were used as a blood meal source for the adults once a week for four weeks. Whenever possible, F1 or F2 mosquitoes were employed in the assays performed.
Insecticides - Data on the temephos and deltamethrin sources employed in the assays performed are presented in Supplementary data (Table I).
Quantitative bioassays - Temephos dose-response bioassays with larvae were performed following WHO
34
procedures, a protocol that was also described by Lima et al.
30
and Braga et al.
31
. For each municipality, at least three trials were carried out on different days, totalling at least 2,400 exposed larvae. Each trial consisted of 10 different temephos concentrations, four replicates per concentration, and 20 larvae per replicate. Mortality was recorded after 24 hours.
Starting in 2009, adults of some vector populations were also submitted to deltamethrin dose-response bioassays, using an adapted version of the impregnated paper methodology.
13
,
35
,
36
,
37
Each replicate employed 15-20 females, which were one to five days old and not fed blood. Three replicates were used per concentration, and between eight and 10 concentrations were tested per assay. At least three trials were performed for each population on different days, totalling 1,080 to 1,800 females. Mosquitoes were exposed to the insecticide for one hour, and then transferred to recovery tubes, without insecticide, and any mortality was recorded 24 h later.
In all cases, the susceptibility reference lineage Rockefeller
38
was used to calibrate the assays and to calculate RR95 values.
Qualitative bioassays - In many cases, adult resistance to deltamethrin was investigated with diagnostic-dose (DD) assays using either insecticide-impregnated bottles or papers, based on the methodologies of the Centers for Disease Control and Prevention (CDC) and WHO, respectively.
35
,
39
In both bioassays, replicates of 15-20 females, one to five days old and not fed blood, were used. For each vector population, at least three trials were performed on different days.
For the CDC methodology, a control bottle impregnated with solvent alone (acetone) and three bottles impregnated with 5 μg of deltamethrin were used per test. According to this methodology, the DD is defined as the lowest deltamethrin concentration that kills 100% of Rockefeller mosquitoes in 30 min. The assay took 30-120 min, and knockdown records were taken every 15 min. Females were then transferred to recovery cardboard cages, and mortality counts were made 24 h later.
23
,
39
,
40
For the WHO methodology,
35
each assay consisted of three replicates with papers impregnated with 3.65 mg deltamethrin/m2 (0.009%) and one control tube containing paper impregnated only with silicone oil.
36
In the WHO methodology, the DD is equivalent to twice the lethal concentration that kills 99% (LC99) of Rockefeller mosquitoes. After being in contact with the insecticide for 1 h, the mosquitoes were transferred to recovery tubes, and their percent mortality was determined 24 h later.
Biochemical tests - Enzymes related to metabolic resistance were investigated in individual specimens following the protocols described by Valle et al.
41
and Montella et al.
20
for adults, and by Viana-Medeiros
42
for larvae. For each population, approximately 90 larvae (L3 final or L4 initial) or 90 adult females (24 h old and not fed blood) were used. The activities of MFO, EST, and GST enzymes were then quantified. For ESTs, three different substrates were used: alpha-naphthyl (α-EST), beta-naphthyl (β-EST), or p-nitrophenyl (ρNPA-EST) acetate. The activity of the OP target, ACE, was also measured; in this case, in addition to its inhibition by propoxur (AChI), the ACE total activity (AChE) was also quantified.
Data analyses - The following criteria proposed by David and Zahar
43
were adopted in the present study to interpret the results of qualitative bioassays: less than 80% mortality indicates resistance, mortality above 98% indicates susceptibility, and intermediate values point to incipient resistance.
Cohen’s kappa coefficient
44
,
45
was used to compare the results of CDC and WHO adult qualitative assays. This coefficient (κ) is a proxy for data agreement, which varies between zero and one. Two mortality cut-off points were used in these comparisons, 80%
43
and 90%
46
.
In the case of dose-response assays, lethal concentrations (LC) were calculated with Polo-PC software
47
via probit analysis
48
and used to calculate RR95 values through comparisons with Rockefeller data. For both temephos and deltamethrin, the functional criterion adopted by the MoReNAa in 2006 was used,
11
,
20
which states that a RR95 value above 3.0 is indicative of resistance.
Results of biochemical assays were classified following Valle et al.
41
and Montella et al.,
20
using the Rockefeller 99th percentile obtained for each enzyme as a cut-off point. For each population and class of enzymes, the activity was then classified as unchanged, altered, or highly altered by the insecticide if the percentage of the samples above the Rockefeller 99th percentile was below 15%, between 15% and 50%, or above 50%, respectively. These percent activity change results for EST, MFO, and GST enzymes were also used to calculate the mean percent change in metabolic resistance (‘X_Metab_Resist’), a secondary measure corresponding to the mean of the changes in EST, MFO, and GST activities in each population. This latter measure was used to compare metabolic and target-site resistance mechanisms, as described in detail in the ‘Results’ section. AChE activity was evaluated as described above, through comparisons with the Rockefeller 99th percentile. Additionally, AChI assays were classified according to WHO criteria, which state that activity inhibition above 70% indicates unaltered ACE activity.
49
Delivery of insecticides used in public health - In Brazil, the MoH is responsible for distributing the insecticides used to control important disease vectors with relevance to public health to all states.
13
The annual delivery records of insecticides used against Ae. aegypti from 2003 to 2014 are presented herein. The larvicides employed were temephos, Bti, and IGRs (the chitin synthesis inhibitors novaluron and diflubenzuron, and the juvenile hormone analogue pyriproxifen). The adulticides employed were malathion (an OP) and several PY insecticides (deltamethrin, cypermethrin, etofenprox, and alpha-cypermethrin; see ‘Discussion’ section).
Dengue incidence - The history of dengue cases was scrutinized using the SINAN NET database (a Brazilian Health Information System).
50
According to the PNCD, all confirmed dengue cases have been recorded in this database. Human population growth over the period examined was estimated based on the results of the censuses of the years 2000 and 2010, which were obtained at the website of the Brazilian Institute of Geography and Statistics (IBGE).
51
These values were used to calculate the dengue incidence between 2000 and 2012. The dengue incidence was calculated as: (N/P) × 100,000 inhabitants, where N is the number of confirmed new dengue cases and P is the total resident population in a given time period.
52
According to the MoH, values of dengue incidence above 300 cases per 100,000 inhabitants (or 0.3%) are considered epidemic.
3
Bibliographical research and criteria for data inclusion in the survey - An update on the temephos and deltamethrin resistance status of Brazilian Ae. aegypti populations from 146 municipalities is presented herein. The data included in the survey consisted of: (1) unpublished data generated by our team within the MoReNAa; (2) data from the recent Moyes et al.
29
review, which encompassed populations from around the world, and updated the work of Ranson et al.
28
; (3) a systematic bibliographic survey of data from 1 January 1985 to 31 July 2017 in the databases contained in the National Center for Biotechnology Information (NCBI) (PubMed and PubMed Central ® - PMC) and in Google Scholar. In this survey the following search terms were used: ‘Brazil’, ‘Aedes aegypti’, ‘insecticide resistance’, ‘organophosphate’, and ‘pyrethroid’. Study selection was initially based on the relevance of the title and compatibility of the abstract with the theme of this study. The chosen articles were then read in full and submitted to a new sorting stage, this time according to other criteria: (1) the sample representativeness of each municipality, and (2) the presence of data on temephos and deltamethrin susceptibility status. Only articles describing representative field collections were kept (in some cases, the origin of the samples was not even reported). Fig. 1 shows the information flow in this systematic bibliographic review, and the quantity of unpublished records presented in this study; results were assembled according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) methodology proposed by Moher et al.
53
Fig. 1:preferred reporting items for systematic reviews and meta-analyses (PRISMA) diagram, showing the different phases of the bibliographic survey and consolidation of the data regarding the insecticide resistance of Brazilian Aedes aegypti populations between 1985 and 2017. The following criteria were adopted for the inclusion of articles in the systematic review: (1) articles with a title and abstract related to the theme of this study were included. (2) Consistency of susceptibility status data against temephos or deltamethrin: for qualitative trials, those studies reporting the diagnostic concentration(s) of pesticide tested and the percent mortality obtained were included; for quantitative trials, those reporting LCs (50, 90, and 95) and the corresponding RRs were included. (3) Field collection: articles were included that provided information on the field sample size collected, taking into account their representativeness in relation to the municipality area and the collection period. For samples collected in districts or in other municipality subdivisions, the average RR of the reported values was considered; in these cases, the entire range of LC confidence intervals was used. Papers that did not mention the year of sample collection were discarded, except for those described by Braga et al.(54), whose records are in our team’s possession. (4) The articles already covered by Moyes et al.(29) were excluded from the present analysis.
Ethics statement - The use of anesthetised guinea pig to blood feed mosquitoes was authorised by the Fiocruz Ethical Committee for Animal Use (CEUA/Fiocruz), under license numbers P-0147/02, L-011/09, and LW-20/14.
RESULTS:
This study presented the results of evaluations of 146 Brazilian municipalities widely dispersed throughout the country, conducted between 1998 and 2012. Fig. 2 depicts the most current results of the 457 evaluations for temephos and 274 evaluations for deltamethrin, comprising the resistance status of Brazilian Ae. aegypti populations from 514 combinations of municipalities and years (‘municipalities/years’) and totalling 2,390 records. For each ‘municipality/year’, the total number of records was variable, and depended on both the insecticides evaluated and the tests performed (qualitative/quantitative). One ‘register’ was defined for each test: (1) in the case of qualitative assays, this was the mean mortality value; (2) for dose-response tests, this was each available LC (50, 80, 90, and 95) and the corresponding RR; and (3) for biochemical assays, this was the activity quantified for each enzyme class (ACE, MFO, GST, α-EST, β-EST, and ρNPA-EST). Allelic frequencies of kdr at the Na
V 1016 and 1534 positions, which are strongly associated with PY resistance,
55
,
56
were also reported whenever available, although these were not considered as main ‘registers’ herein.
Fig. 2:resistance status of larvae and adults of Brazilian Aedes aegypti populations against temephos and deltamethrin, respectively. For each municipality, the results of the most recent bioassay are shown. For deltamethrin, in addition to the results of quantitative (circles) bioassays, those of qualitative assays (triangles) are also depicted.
Data were presented separately for each geographical Region of Brazil, and organized sequentially by state and then municipality. For each municipality, the annual results were grouped chronologically. Supplementary data (Tables) are included that depict: the annual incidence of dengue in each municipality in the period evaluated [Supplementary data (Table II)]; the history of insecticide distribution by the MoH to each state for use against larvae [Supplementary data (Table III)] and adults [Supplementary data (Table V)]; results of bioassays of larvae exposed to temephos [Supplementary data (Table IV), quantitative assays]; and results of adult bioassays against deltamethrin [Supplementary data (Tables VI-VII) for qualitative and quantitative assays, respectively]. Supplementary data (Table VIII) exhibits the results obtained concerning the resistance status and mechanisms for all samples for which biochemical or molecular data were available.
In the text, in addition to sections dealing with each geographic region, two further sections were included: the first compared the results of CDC and WHO qualitative bioassays with adults, and the second analysed the relative participation of metabolic and target-site mechanisms in the resistance observed.
Our aim was to present, as completely as possible, the historical sequence of the development of resistance in Brazilian Ae. aegypti populations to insecticides used by public managers. It is noteworthy that 96.8% of these records were generated in laboratories belonging to the MoReNAa.
North Region
Dengue incidence - Most of the evaluated municipalities in this region were concentrated in Pará state, where dengue outbreaks occurred with a high, but sporadic, incidence [Supplementary data (Table II)]. However, some epidemics were reported in other states, even in consecutive years, and sometimes with incidences exceeding the ‘threshold’ of 0.3% by 5-30 times, including: Rio Branco, AC (2009-2011), Oiapoque, AP (2007-2011), and Boa Vista, RR (between 2001-2003 and 2008-2010). In Pacaraima, RR and Palmas, TO, high dengue incidence values were registered during most of the evaluated period.
Resistance against the larvicide temephos - During the evaluated period, temephos was almost continuously supplied throughout the North Region. In general, other products, such as Bti or IGRs, did not replace this larvicide, but were sometimes employed together with it [Supplementary data (Table III)]. Accordingly, resistance to temephos was found to be widespread in nearly all 20 evaluated municipalities in the North Region. The only exceptions were Porto Velho, RO in 2002
54
and Boa Vista in 2010 [Supplementary data (Table IV)].
For the most part, the temephos RR95 values of mosquitoes in the North Region ranged from 4-12 in the studied period. The main exception was Oiapoque in 2009, in which very high resistance levels, including a RR50 of 42.1 and RR95 of 102.5, were recorded.
57
This latter value was five times higher than the second largest RR95 in the region across the whole studied period: 21.4, in Dom Eliseu, PA in 2003.
20
More recent trials with samples from Amapá (collected between December 2014 and January 2015) showed a significant reduction in temephos resistance, including a RR50 of 21.8 for mosquitoes from Oiapoque and of 6.5 for those from Macapá.
58
It is worth remembering that temephos has been gradually replaced by IGRs since 2010, and its supplying to Amapá stopped in 2013 [Supplementary data (Table III)].
Oiapoque is located along the Brazilian border with French Guiana. The high temephos RR in Oiapoque in 2009 (RR95 = 102.5) may have been the consequence of the intense vector control actions applied in this border area by both countries. Dengue incidence was roughly 10 times higher than the threshold that defines epidemics in 2007-2011. This profile, however, does not seem to be general, as different patterns occurred in: (1) Palmas, TO, which presented RR values lower than 10 in two evaluations (2006 and 2009), with a high incidence of dengue in all years between sample collections; and (2) Rio Branco, which had a RR value below 10 in 2011, after four successive years of high incidences of dengue, with rates that reached 30 times the epidemic threshold. Therefore, it is not simply the epidemic per se that is associated with resistance, but the kind of response enacted to it, in particular the relative importance attached to chemical control of vectors.
Resistance to deltamethrin - In the North Region, deltamethrin was widely used in the control of adult mosquitoes over the period examined. Malathion was supplied to Pará in 2011, and later began to be employed in some other states [Supplementary data (Table V)]. Qualitative bioassays with deltamethrin using the WHO methodology classified 15 of the 18 evaluated mosquito populations as resistant, and none were considered susceptible.
43
On the other hand, trials with the CDC methodology resulted in higher mortalities, and of the 11 evaluated populations, only two were classified as resistant [Supplementary data (Table VI)]. A comparison of these two methodologies is presented below (section: ‘Qualitative bioassays with adults: CDC and WHO methodologies’).
Although performed with mosquitoes from municipalities distinct from the sources of those used in qualitative tests, the results obtained with quantitative assays corroborated the changes in deltamethrin susceptibility suggested by the results of the former trials [Supplementary data (Tables VI-VII)]. The deltamethrin RR95, a parameter evaluated since 2009, was always high, usually between 8.5 and 26.6. However, for mosquitoes from Pacaraima, RR and Marabá, PA evaluated in 2011, the deltamethrin RR95 was extremely high, 60.3 and 70.7, respectively. Pacaraima borders Santa Helena, Venezuela, where there is more intense chemical control of malaria vectors; this and the high dengue fever incidence there since 2007 [Supplementary data (Table II)] may explain the elevated deltamethrin RR95 in mosquitoes from Pacaraima. On the other hand, in Marabá, despite the low incidence of dengue [Supplementary data (Table II)], there was an eight-fold increase in the RR95 in a two-year interval, from 8.8 in 2009 to 70.7 in 2011 [Supplementary data (Table VII)]. This was the highest deltamethrin resistance ratio found in the region. Altogether, for the North Region it was not possible to make the general conclusion that high dengue incidence corresponds with increased PY resistance. The increase in the use of PYs in general observed during epidemics due to blocking actions undertaken by public health services, intensification of domestic use, and non-integrated use of PYs against malaria vectors
10
,
13
,
14
,
59
could explain the high level of resistance seen in mosquitoes from Pacaraima, but not in those from Marabá.
Resistance mechanisms - In the North Region, the activities of two enzymatic classes related to metabolic resistance were consistently altered: GSTs and, more intensely, ESTs, which in the latter case were evaluated with different substrates [Supplementary data (Table VIII)]. Few vector populations showed altered MFO activity. In only three of the 20 samples available, the total activity (AChE) of ACE, the target of OPs, appeared to be increased. However, in all cases ACE was considered sensitive to inhibition by insecticides according to WHO criteria (data not shown). Unfortunately, the metabolic resistance of larvae from a same locality in the North Region was never evaluated at two consecutive periods. However, there were simultaneous evaluations of larvae and adults on six occasions, and in all these cases the activity was more strongly altered in adults [Supplementary data (Table VIII)].
Northeast Region
Dengue incidence - Dengue dynamics between 2001 and 2012 in the Northeast Region presented four three-year periods with distinct disease incidences [Supplementary data (Table II)]; in the first and last of these, 2001-2003 and 2010-2012, dengue incidences were high and consistent with epidemic conditions in most of the evaluated locations in all states. In the second triennium (2004-2006), incidence values were the lowest observed, while in the third triennium (2007-2009) the dengue incidence was variable. It is noteworthy that this parameter was very high in Bahia during 2009. However, in the 2007-2009 triennium, the dengue incidence tended to be low in two periods and sections: in the beginning (2007), in the southernmost states of the Northeast Region (SE, BA); and in the last year (2009), in the remaining ones. In general, the number of ‘municipalities/years’ with a dengue incidence more than 0.3% corroborated this scenario: the first and fourth triennia registered higher numbers of 81 and 70 ‘municipalities/years’ that were above this cut-off point, respectively; whereas recorded values in the second triennium were lower (16), and in the third triennium they were intermediate (48).
Northeast Brazil was strongly affected by dengue over the evaluated period: in two states, CE and RN, at least 50% of ‘municipalities/years’ presented incidences indicating dengue epidemics. Even in MA, the least strongly affected state, more than 12% of the ‘municipalities/years’ presented high dengue incidences. The percentage of cases with incidences above 1,000 cases per 100,000 inhabitants, an arbitrary value equivalent to 1% of the population and more than three times the cut-off point defined by the Ministry of Health, was also evaluated. In this case, CE and RN were confirmed as the states that were the most strongly affected by dengue during the studied period. High incidences were also found for AL and BA.
Temephos resistance - Except for RN, temephos was continuously distributed to all states in the Northeast Region until 2011-2014 [Supplementary data (Table III)]. Bti was simultaneously applied in several states (MA, CE, PE, and AL) until 2009-2010; IGRs were also introduced during this period in most states in the Northeast Region. By 2014 almost all states exclusively employed IGRs.
In the Northeast Region, resistance to temephos was detected in all municipalities evaluated, with the only exception being Fernando de Noronha Island in 2009 [Supplementary data (Table IV)]. Although to date the highest temephos resistance levels in Brazil occur in the Northeast Region, the RR there varies significantly: a temephos RR95 above 100 was found in 20% of cases, but in almost 40% of cases the RR95 was below 10. The lowest dengue incidences and also the lowest temephos RR95 values (always below 10) were found in the state of MA. In contrast, extremely high temephos RR95 values above 100 (sometimes above 200) were concentrated in PE (six municipalities), southern CE (two), eastern BA (five), and the countryside of AL (one) [Supplementary data (Fig. 1)].
Much variation was noted in several aspects of insecticide resistance monitoring in the Northeast Region, including: (1) the number of municipalities evaluated in each state (ranging from only two in MA and PI, up to more than 10 in PE and BA); (2) the frequency of evaluation of the different municipalities, with each municipality evaluated only once in some states, such as MA and PB, while some municipalities in other states were evaluated five (Juazeiro do Norte, CE and Natal, RN), six (Fortaleza, CE and Maceió, AL), or even seven (Aracaju, SE) times over the evaluated period; and (3) the regularity of monitoring, as for example on the one hand five of the six evaluations were performed in 2003 for PB, while on the other hand in BA only three out of 23 evaluations were done before 2008.
For municipalities that were evaluated more than once, temephos resistance levels were compared with both their dengue incidence and supply of insecticides [Supplementary data (Tables II, III, IV). Although several different parameters influence dengue dynamics, some correlations were suggested, as outlined below:
― Crato, in the south of CE state, where the temephos RR95 in 2009 was higher than 190, presented a history of severe dengue epidemics between 2001 and 2007, which were five years with high values of dengue incidence, including two years when this was greater than 1%. This scenario may have contributed to the intensification of chemical control and the consequent increase in temephos resistance levels in this municipality. However, although dengue incidence remained high in this municipality until at least 2012, in 2013 resistance to temephos had declined more than three-fold (RR95 = 65), probably due to the interruption of its supply to the state in 2011.
― In Fortaleza, CE, the temephos RR95 increased from 8-10 in 2002 and 2006 to 43 in 2007. The annual dengue incidence was above the MoH cut-off point five times between 2001 and 2007, pointing to the intensification of chemical control and a consequent RR increase. Unfortunately, although there were several periods of high dengue incidence until 2012 in Fortaleza, no further temephos evaluations were reported there.
― With the exception of Santana do Ipanema, in the state of AL relatively stable temephos RR50-95 values, varying between four and 17, were found. These were consistent data, since these municipalities were assessed three to six times during the studied period. These results are also coherent with the supply of larvicides provided to AL state, since temephos was continuously but not exclusively provided between 2003 and 2013, being always concomitantly applied with Bti and/or an IGR.
― In contrast, in the state of RN, although temephos was provided only in 2004 [Supplementary data (Table II)], the RR95 of mosquitoes for this OP fluctuated over the same range as those in AL, and was around 12 (from 10 to 19) between 2004 and 2011. These are also consistent data, as of the five municipalities monitored, four were evaluated three to five times. This situation suggests that dengue incidence and records of insecticide supply by the MoH were not sufficient to explain the temephos resistance dynamics observed in RN state. Additional investigations could reveal other parameters that should be taken into account for the interpretation of these data.
― Accordingly, in Barra dos Coqueiros, SE, neither the low dengue incidence, nor the exclusive supplying of temephos as a larvicide over the evaluated period, were able to explain the increase in the temephos RR by seven times between 2000 (3.2) and 2004 (23.5).
In BA state, temephos was supplied continuously between 2003 and 2013, and this was the only larvicide used between 2004 and 2009. Among Bahia municipalities monitored more than once, two distinct scenarios should be mentioned: (1) records of high dengue incidence and stable levels of temephos resistance were observed in Barreiras, which was subjected to two years of very high dengue incidence between 2008 and 2012 (up to 1.6%), although the temephos RR95 there remained around 5.0 throughout the evaluated period; and (2) records of high dengue incidence and increasing temephos resistance levels were noted in such municipalities as Itabuna, Jequié, and Jacobina. In Itabuna, the temephos RR95 increased from 18.6 in 2004 to 55.8 in 2013, and records of dengue incidence in this municipality were also extremely high, in the range of 3-7%, in 2009 and 2012. Jequié exhibited low dengue incidences between 2004 and 2008, but high values between 2009 and 2012, and this last period was concomitant with a three-fold increase in the temephos RR95 value of mosquitoes from there. In Jacobina, temephos resistance levels increased 10-fold in one year, between 2008 and 2009, when the RR95 reached 104. Four years later, the temephos RR95 more than doubled, reaching almost 230. Dengue incidence in this municipality was high, or very high, during almost the entire period evaluated (2001-2012).
Resistance to deltamethrin - The whole Northeast Region received an uninterrupted supply of PY insecticides between 2003 and 2014 [Supplementary data (Table V)]. Malathion was delivered to CE in 2006, and to BA in 2011. This OP started to be continuously distributed in addition to deltamethrin in CE in 2008, and in some other states in the Northeast Region as of 2010.
Mosquitoes from all of the 39 ‘municipalities/years’ from the Northeast Region evaluated for deltamethrin resistance with qualitative tests using the WHO methodology were classified as resistant (26) or with incipient resistance (13). Among the 14 samples evaluated with the CDC methodology, 11 were considered resistant (two) or with incipient resistance (nine). In 13 cases, discussed in a section below, the two methodologies were applied simultaneously [Supplementary data (Table VI)].
Quantitative tests were performed in eight municipalities from four states [Supplementary data (Table VII)], and confirmed that resistance to PY was prevalent throughout the Northeast Region. The RR95 values were always above 7.0 and, except for Mossoró in 2011 and Crato in 2013, remained below 20.
In the Northeast Region the deltamethrin resistance status was evaluated twice in two localities, Mossoró, RN and Aracaju, SE. In both cases, the deltamethrin RR95 increased and, accordingly, the frequency of kdr alleles at the PY target site also increased [Supplementary data (Table VIII)].
Resistance mechanisms - The activities of two classes of detoxifying enzymes, GSTs and ESTs, were altered by pesticides [Supplementary data (Table VIII)]. Both α-EST and β-EST activity were affected in larvae, while α-EST was the most strongly altered class in adults. Total ACE activity presented increased levels in only two cases. However, according to WHO criteria, ACE was sensitive to insecticide inhibition throughout the Northeast Region (data not shown). MFO and ρNPA-EST activities were altered in some cases, mainly in adults. In eight instances, larvae and adults were simultaneously evaluated, with greater enzyme activity alterations always observed in adults [Supplementary data (Table VIII)]. The two municipalities evaluated at more than one point in time, Aracaju and Caicó, exhibited a general increase in the activity of detoxifying enzymes in both larvae and adults.
Southeast Region
Dengue incidence - In the Southeast Region, SP state stands out, as detailed below. SP presented the longest insecticide resistance monitoring history and the lowest dengue incidence of all evaluated states; it is an example of the positive effects of both entomological surveillance and resistance management on dengue prevention. During the evaluated period (2001-2012), the dengue incidence was above 0.3%, the MoH cut-off point, in less than 20% of ‘municipalities/years’, and only 10% of cases presented a dengue incidence of more than 1% [Supplementary data (Table II)]. In contrast, ES was the state in the Southeast Region that was the most strongly affected by dengue in the studied period, as in more than 50% of the ‘municipalities/years’ in ES the dengue incidence was above the MoH recommendation, and 20% of these had a dengue incidence of more than 1%. RJ and MG states presented intermediate values: 45-55% and 15-20% of the ‘municipalities/years’ in these states exhibited dengue incidences above 0.3 and 1%, respectively.
Temephos resistance - In the Southeast Region in general, temephos was distributed continuously throughout the period evaluated. Bti was supplied simultaneously until 2007-2010, and thereafter IGRs were used [Supplementary data (Table III)]. RJ was an exception, since temephos and IGRs were provided intermittently from 2004 onward in this state, while IGRs began to be supplied continuously after 2008, before they were supplied to the other states in the Southeast Region.
Temephos resistance was evaluated in 42 municipalities, totalling 250 ‘municipalities/years’, in this Region. SP accounted for more than 80% of the records, among which 17 municipalities out of 20 were assessed several times, some almost annually and, in many cases, from 14 to 18 times over the 1998-2015 period. In contrast, in other states in the Southeast Region, the accomplishment of temephos bioassays, which started from 1999 (RJ) and 2005 (MG and ES) onward, extended only until 2011. In these states, most municipalities were evaluated once or twice in the studied period, with a maximum of five evaluations in the case of Rio de Janeiro city [Supplementary data (Table IV)].
This differential resistance monitoring and management effort was reflected in the susceptibility profiles obtained: the average temephos RR95 of mosquitoes from SP (3.5) was at least three times lower than that of those in the other states in the Southeast Region (RJ, MG, and ES: RR95 = 10.1, 11.4, and 13.3, respectively). In SP, the temephos RR95 was above 3.0 in 60% of cases, in contrast with it being above this value for 100% of the municipalities in the other states, with the only exception to this trend being São José do Vale do Rio Preto, RJ (RR95 = 3.0). In SP, the temephos RR95 was above 10.0 in mosquitoes from only 1% of ‘municipalities/years’ (two out of a total of 194 records). In contrast, more than 50% of the records for the other states in the Southeast Region reached this value. Out of the 42 municipalities assessed in the Southeast Region, four were classified as having mosquitoes susceptible to temephos (RR95 below 3.0), three of which were located in SP. In particular, just one among the four municipalities was evaluated more than once; this was Botucatu, SP, the mosquitoes from which remained susceptible to temephos during all six monitoring rounds carried out between 2004 and 2014. This municipality did not exhibit a dengue incidence above the epidemic ‘threshold’ over the entire evaluated period [Supplementary data (Table II)].
In SP, a temephos RR95 above 10.0 was detected only in mosquitoes from São Sebastião in 2004 and from Santos in 2005. In both municipalities, resistance ratio values decreased later on, culminating at levels indicating susceptibility in 2015. In SP, the highest temephos RR95 levels were reported in Santos, São Sebastião, São Paulo, and Itapevi. The first two of these were among the five SP cities evaluated herein with the highest dengue incidences in the period of 2001-2012.
On the other hand, in the Southeast Region, the ES state presented the highest dengue incidence and also the highest temephos RR95 in the evaluated period. These were consistent data, which were always derived from more than one evaluation. In the case of RJ, although most of the locations were evaluated more than once, there were gaps in the RR95 records, and in MG only the capital, Belo Horizonte, was evaluated more than once.
Resistance to deltamethrin - Supplementary data (Table V) shows the supply of adulticides provided by the MoH for all vector control programs, not just those targeting Ae. aegypti (see ‘Discussion’). PYs were widely distributed throughout the Southeast Region: SP received malathion during the whole period, and other states received it from 2007 onward, or later. It should be mentioned that SP stopped using PYs against the dengue vector in 2000, almost 10 years before their replacement in the rest of the country.
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Deltamethrin qualitative bioassays were performed for 115 and 75 ‘municipalities/years’ with the WHO and CDC methodologies, respectively [Supplementary data (Table VI)]. In all cases, established or incipient resistance was detected. Established resistance prevailed in 92% of the trials (n = 106) done with the WHO methodology and in 72% of the trials (n = 54) done with the CDC methodology. Simultaneous evaluations with the WHO and CDC methodologies were performed for 46 ‘municipalities/years’ (see section below in which both methodologies were compared). Accordingly, deltamethrin quantitative bioassays revealed high resistance levels in all cases [Supplementary data (Table VII)].
Resistance mechanisms - GSTs were the enzymes whose activities were the most strongly altered by pesticides in samples of larvae and adults from the Southeast Region [Supplementary data (Table VIII)]. In adults, the activity of EST enzymes, mainly a-EST and ρNPA-EST, were also altered. Changes in MFO activity were also identified in some samples. Increases in total ACE activity were detected in roughly 30% of the evaluated samples. However, ACE activity was never altered in both stages evaluated from the same ‘municipality/year’. In addition, the ACE activity in all samples was considered sensitive to insecticide inhibition (AChI) when the WHO criterion was used (data not shown). Whenever biochemical assays were available for both life stages (eight pairs of samples), metabolic resistance was higher in adults. Metabolic resistance was never evaluated twice in larvae from the same locality. However, for five municipalities, the metabolic resistance of adults was evaluated 2-3 times; despite this, there were no concurrent data on the application of quantitative bioassays or on Na
V allele frequencies, a situation that precludes the analysis of the main mechanisms involved in resistance in these cases.
South Region
Dengue incidence - Due in part to its milder climate, the South Region was the least strongly affected by dengue. Most municipalities in SC and RS states evaluated over the studied period did not register dengue epidemics. The exception was Ijuí, RS in 2010, where the dengue incidence reached 3.7%, more than 12 times the MoH cut-off point [Supplementary data (Table II)]. The state of PR was the most affected by dengue. In the South Region, a dengue outbreak first occurred in 2002 in Foz do Iguaçu, PR, which is located on the triple border of Brazil with Paraguay and Argentina. In 2003, there was an epidemic in three municipalities in northern central Paraná, Cambé, Ipiporã, and Londrina. From then on, new records of high dengue incidences only occurred in this state in 2007, and then again in 2010/2011. In the period 2007-2011, Foz do Iguaçu endured three years of extremely high dengue incidences 3-12 times the MoH threshold of 0.3%.
Temephos resistance - All states in the South Region received the larvicide temephos almost continuously between 2003 and 2014 [Supplementary data (Table III)]. RS also received Bti in 2006 and 2009. The supplying of IGR larvicides to PR occurred from 2010 onward. Both SC and RS received IGRs in 2014 for the first time.
The temephos resistance status was evaluated in 12 municipalities in the South Region, totalling 29 ‘municipalities/years’ [Supplementary data (Table IV)]. Foz do Iguaçu and Maringá, both in PR, were evaluated eight and six times, respectively, between 2001 and 2009. Among the remaining municipalities, six were evaluated only once, and four were evaluated 2-3 times. Although in 55% of the bioassays the RR95 was higher than 3.0, the MoH cut-off point, it was never higher than 7.0. Mosquitoes from Maringá in 2005 and Ibiporã in 2006 exhibited the highest resistance levels. In general, a slight increase in resistance to OP was noted in the South Region over time.
Resistance to deltamethrin - Pyrethroids were provided almost continuously and used nearly exclusively in the South Region [Supplementary data (Table V)]. PR also received the OP malathion from 2010 onward. Qualitative deltamethrin bioassays were performed for three PR locations. Eight ‘municipalities/years’ were evaluated by the WHO method, and four of these were also evaluated using the CDC methodology [Supplementary data (Table VI)], and susceptibility was not detected in any case. In the South Region, results of deltamethrin quantitative bioassays were only available for mosquitoes from Santa Rosa, RS, for which there was a RR95 of 33.3 in 2011 [Supplementary data (Table VII)].
Resistance mechanisms - In the South Region, resistance mechanisms were investigated only in mosquito larvae and adults from Santa Rosa, RS in 2011. The larval enzyme activity profile was unaltered, which was consistent with their susceptibility to temephos [Supplementary data (Table VIII)]. In adults that were resistant to deltamethrin, changes in EST activity were detected, particularly in the activities of α-EST and ρNPA-EST. The participation of Na
V alterations in resistance was also identified in this case. Despite showing an increase in their total ACE activity, mosquitoes from Santa Rosa were considered to be sensitive to insecticide inhibition according to the WHO criteria (data not shown).
Centre-West Region
Dengue incidence - All municipalities evaluated in this Region experienced at least one dengue epidemic year between 2001 and 2012 [Supplementary data (Table II)]. Brazil’s capital, Brasília, in the Federal District (DF), was the least strongly affected locality, with there being high dengue incidence only in 2010. In the MS municipalities evaluated, dengue incidences generally increased from 2006 onward and reached values up to 6.3%, more than 20 times the MoH threshold, as was seen in Campo Grande in 2007. In GO, the dengue incidence also reached very high values in several localities. In particular, high dengue incidences were observed in two neighbouring municipalities, Goiânia and Aparecida de Goiânia, throughout the period evaluated (2001-2012). In 2010, a strong dengue epidemic affected the entire Centre-West Region: in 16 of the 17 municipalities evaluated, the dengue incidence was above 0.3%, and in four this value was even above 3.0%.
Temephos resistance - Except in the DF, temephos was supplied continuously between 2003 and 2013 throughout the Region. In MS, Bti was used in addition to temephos between 2003 and 2009. IGRs were introduced from 2009-2010, becoming the sole larvicides provided by the MoH throughout the Centre-West Region in 2014 [Supplementary data (Table III)].
Mosquitoes from all municipalities in the Centre-West Region evaluated were resistant to temephos [Supplementary data (Table IV)], the only exception being those from Brasilia in 2008. Brasilia presented the highest number of evaluations, and also the lowest temephos resistance levels and the lowest dengue incidence rates, over the evaluated period. Only Cuiabá in 2005 was evaluated in MT state; its temephos RR95 was also among the lowest in the region, in line with the moderate dengue incidence rates recorded there up to this time.
The temephos RR95 in MS was always below 8.0 [Supplementary data (Table IV)], even in 2011, after a period of extremely high dengue incidence in 2006-2010 [Supplementary data (Table II)]. The simultaneous use of temephos and Bti [Supplementary data (Table III)] may have contributed to there being less selection pressure for OP resistance in this state.
In contrast, mosquitoes from GO showed the highest temephos resistance levels in the region, as 10 out of the 15 municipalities for which the RR95 was evaluated in this state had values that were above 10.0 [Supplementary data (Table IV)]. Notably, São Miguel do Araguaia, GO in 2012 presented the highest temephos resistance level in the Centre-West Region, even though 2012 corresponded to the second year of low dengue incidence after five consecutive years of high incidences in this locality. The contiguous municipalities of Goiânia and Aparecida de Goiânia are clear examples of how differences in chemical control management can have an impact on the development of resistance. Although dengue incidence was high in both municipalities during the entire evaluated period, temephos resistance levels were much lower in Goiânia than in Aparecida de Goiânia, suggesting there were distinct selection pressures in both localities. Indeed, in the routine insecticide resistance monitoring of Brazilian Ae. aegypti populations, some adjacent municipalities with different local dynamics of infestation control were purposely chosen for just this reason. This was done to emphasize to municipal public officials how much both surveillance and infestation control strategies can impact insecticide resistance and, consequently, influence the effectiveness of vector control.
Resistance to deltamethrin - Pyrethroid adulticides were distributed widely in the Centre-West region between 2003 and 2014 [Supplementary data (Table V)]. The introduction of malathion, in addition to deltamethrin, began in 2009.
All 20 sites submitted to qualitative trials using the WHO methodology [Supplementary data (Table VI)] were classified as having mosquitoes that were resistant (18) or with incipient resistance (two). Of these, 11 were also evaluated using the CDC methodology, and two were classified as susceptible, while the others were considered resistant (five) or with incipient resistance (four).
Results of deltamethrin quantitative bioassays were only available for municipalities in GO state since 2009, and the results mainly came from 2011 [Supplementary data (Table VII)]. In all cases, data on resistance levels were obtained one or two years after dengue outbreaks [Supplementary data (Tables II, VII). In particular, deltamethrin resistance levels in mosquitoes from Luziânia were so high that the RR95 could not be readily estimated from the adult specimens available, as their RR80 was already almost 170. It is remarkable that Luziânia was considered susceptible to this PY three years before this evaluation (2008) according to a qualitative bioassay done using the CDC methodology [Supplementary data (Table VI)]. Additionally, Luziânia was one of the municipalities in the Centre-West Region that was the least strongly affected by dengue epidemics in the studied period [Supplementary data (Table II)]. This high deltamethrin resistance level in Luziânia in 2011 can be explained if one takes into account that the samples used for these bioassays were collected during the periods with the highest dengue incidence in this locality, and precisely at the end of the epidemic period [Supplementary data (Fig. 2)]. Thus, the high resistance levels may have reflected the seasonality of infestations, as well as the intensification of the use of adulticides during epidemic periods. The participation of kdr mutations, which contribute to PY resistance, was also detected in mosquitoes from Luziânia; unfortunately, the absence of biochemical data precludes the conclusive evaluation of the main mechanisms involved in this resistance.
Resistance mechanisms - The activities of two enzyme classes, GSTs and ESTs, were greatly altered in both vector stages [Supplementary data (Table VIII)]. In some cases, there was also a high alteration of MFO activity, but mainly in adults. Although the total ACE activity was altered in mosquitoes from two populations, this enzyme was classified in all cases as being sensitive to insecticide inhibition according to the WHO criteria (data not shown). Larvae and adults from Aparecida de Goiânia, Goiânia and Rio Verde were evaluated more than once. In general, they showed increased alterations to the activities of their metabolic resistance enzymes over time. Metabolic resistance tended to be higher in adults in nine out of the 10 available pairs of simultaneous evaluations of both larvae and adults.
Qualitative bioassays with adults: CDC and WHO methodologies - In the routine evaluation of the resistance of adult Culicidae to neurotoxic insecticides, two qualitative bioassay methodologies are available and globally used, although there is only limited agreement between them.
45
Originally, the CDC and WHO methodologies were designed to evaluate different parameters, being knockdown and mortality, respectively. However, the assessment of mosquito mortality with the CDC methodology after recovery from insecticide exposure for 24 hours has already been employed in previous studies.
23
,
24
,
29
,
36
,
40
This approach is especially useful in evaluating resistance to pyrethroids, which have a strong knockdown effect. In practice, this evaluation procedure, when performed after 24 h of recovery, tends to increase the agreement between both methodologies, as stated elsewhere.
45
In the present study, mosquitoes from 82 ‘municipalities/years’ were submitted to bioassays using both procedures, and the mortality results obtained from these tests after 24 h of exposure to deltamethrin were then compared. The WHO methodology classified 87.8% of the populations as resistant, 12.2% as presenting incipient resistance, and none as susceptible [Supplementary data (Table VI)]. Meanwhile, mortality levels obtained with the CDC methodology tended to be higher, resulting in only 43.9% of the same populations being classified as resistant, 45.1% as having incipient resistance, and 11% as susceptible.
We identified a weak positive correlation between the results of both methodologies (n = 82, R2 = 0.014, p < 0.001). However, regional differences were observed: a weak negative correlation between results was found for samples from the Southeast and South Regions, but the correlation was positive for samples from the remaining Regions. In particular, a strong and highly significant correlation was found for samples from the North Region (n = 8, R2 = 0.751, p < 0.002).
The Cohen’s kappa coefficient (κ) values obtained showed that there was reasonable agreement between the two methodologies’ results for the 82 evaluated pairs of samples (κ = 0.39084) when the criterion proposed by David and Zahar
43
was employed. This criterion considers populations with mortality below 80% in these bioassays as being resistant. However, the agreement increased considerably (κ = 0.64995) when the most recent criterion proposed by the WHO,
46
which uses 90% mortality as the cut-off point for resistance, was used instead. It is worth noting that all of the results used herein came from laboratories belonging to the MoReNAa, in which both methodologies were adopted following standardised procedures in the monitoring routine of Ae. aegypti adults for the country of Brazil.
Several particular aspects of the two methodologies, discussed in detail elsewhere,
45
are thought to account for their differences in performance. Additionally, mortalities tend to be higher in the bottle assays used in the CDC methodology, wherein the mosquitoes are forced to make direct contact with the insecticide, since the entire inner surface of the bottle, including the cap, is impregnated with the product. In contrast, the WHO methodology includes non-insecticidal spots that can be used as refuges, specifically the screens at the ends of the cylindrical tubes. In principle, the CDC methodology is more versatile, since the user can impregnate their bottles relatively easily on their own, while the impregnation of papers depends on a more delicate process, or on their acquisition already impregnated with the product. However, there are limitations to the use of impregnated bottles, but not WHO tubes, in humid places, especially in the field, as there may be condensation under such conditions that may cause mosquitoes to adhere to the walls of the bottles.
Resistance to pyrethroids: metabolic and target-site mechanisms - Some studies have suggested that metabolic changes tend to result in lower levels of resistance to pyrethroids than changes in their target site, Na
V .
37
,
61
,
62
To test this possibility, deltamethrin resistance ratios were compared with both metabolic resistance and kdr frequencies. Direct comparisons were also made between the two resistance mechanisms, metabolic and target-site. As stated in the ‘Materials and methods’ section, for each population the mean level of the changes in activity found for all enzymatic classes related to metabolic resistance was used as a metabolic resistance index. The sum of the allelic frequencies of kdr mutations at positions 1016 and 1534 was used to evaluate changes in the PY target site.
When all available data from the entire country were combined, the deltamethrin RR was directly proportional to the frequency of kdr mutations (n = 18, R2 = 0.301, p < 0.001) [Supplementary data (Fig. 3A)]. Therefore, the higher the resistance level, the greater the participation of target-site alterations was in the resistance observed. Simultaneously, the deltamethrin RR and frequency of metabolic resistance tended to be inversely proportional; in other words, higher PY resistance levels tended to be correlated with less participation by detoxifying enzymes in resistance (n = 24, R2 = 0.004, p = 0.288) [Supplementary data (Fig. 3B)]. However, the correlation in the latter case was much weaker and non-significant, which makes sense when one considers that one of the variables evaluated in this case is a secondary parameter, derived from the activity of several enzymatic classes with variable specificity.
The direct comparison between both resistance mechanisms, metabolic and target-site, confirmed that one mechanism or the other tended to participate in the development of resistance; in other words, the frequency of metabolic resistance seemed to be higher when the kdr frequency was lower (n = 19, R2 = 0.012, p = < 0.001) [Supplementary data (Fig. 3C)].
DISCUSSION:
Although in the ‘Results’ section the details of the data collected were presented separately for each Region, we also attempted to identify national patterns and trends in relation to each of the main topics addressed: (1) the dengue incidence in the period studied; (2) the supply of the main insecticides used in vector control by the MoH to each state; (3) temephos and deltamethrin resistance profiles; and (4) the main and potential resistance mechanisms involved.
Dengue incidence - Only the dengue incidence in localities monitored for insecticide resistance was presented herein. As mentioned above, 96.8% of these records were generated within the MoReNAa, which attempted to define ‘sentinel municipalities’ that were representative of the dengue epidemiology in their geographical areas. Hence, it was possible to sketch an approximate picture of the recent history of dengue outbreaks and vector control in different Brazilian Regions. Except for the South Region, which was affected by dengue epidemics later than the rest of the country, the dengue incidence in all other regions was higher than the MoH cut-off point of 0.3% in more than 30% of municipalities [Supplementary data (Table IX)]. In addition, except for the South Region, extremely high dengue incidences above 1% were observed in more than 10% of the cases examined. In particular, in the Centre-West Region almost 20% of the records were above 1% over the studied period.
Supplementary data (Tables X, XI) depict the dengue incidence in each year in each Region. In general, the high incidence of dengue fever recorded in 2001-2002 and from 2008 onward
63
can be seen in these data. However, epidemics did not occur simultaneously in all regions of the country. In particular, it is worth noting (1) the late inclusion of the South Region in the area of epidemiological importance for dengue, and (2) the magnitude and persistence of dengue epidemics in the Centre-West Region, most notably between 2006 and 2010. Climate changes, reflected in changes in temperature and precipitation patterns over the evaluated period, together with distinct macro- and micro-scale social determinants, tended to favour the spread of the vector.
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However, the non-simultaneous transmission of dengue in the country may be the consequence of heterogeneous variations in such climatic changes among the different geographical regions, which would have had unequal effects on the biology of the vector therein.
Provision of insecticides by the Brazilian Ministry of Health - We used the annual MoH registry of the insecticides supplied to the different states aiming to control different arthropod vectors between 2003 and 2014. Although the available documents did not specify the target vectors for the different products, we sought to restrict the analysis to the insecticides known to be used against Ae. aegypti. However, in the case of adulticides, we opted to include all PY insecticides provided by the MoH, since there is partial overlap between Ae. aegypti occurrence and that of other vectors targeted by PYs that are relevant to public health. In addition, the common mode of action of PY insecticides, and consequently their potential to induce selection for the same resistance mechanisms, were considered.
Temephos was the most heavily used larvicide in the period of 2003-2014, when it was supplied almost continuously to nearly all Brazilian Regions. In fact, for a long time temephos was the only larvicide approved by the WHO for use in potable water containers. Since at least 2003, Bti has also been used throughout the country, usually in addition to temephos. Subsequently, IGRs were introduced, and their use as larvicides was established in almost the entire country from 2009-2010, except for the South Region, where these larvicides were introduced later. It is worth mentioning that since 2012
7
Brazil has adopted a larvicide rotation approach to preserve the supply of the few available products in any given year. Chitin synthesis inhibitors, mainly diflubenzuron and novaluron, were the first IGRs to be employed in Brazil, and were later succeeded by juvenile hormone analogues, such as pyriproxyfen. In 2013, temephos ceased to be the first-choice larvicide for use in Ae. aegypti control, and since 2014 it has no longer been used, and has instead been replaced by IGR for use in larval control.
65
According to the WHO, there are only a few options to use as adulticides, all of which are PY products except for the OP malathion.
27
In the 2003-2014 period, deltamethrin was used practically throughout the whole country except in SP state, where due to resistance PYs have not been employed in the control of Ae. aegypti adults since 2000.
14
,
60
Despite this, SP continued to receive PYs to control other arthropod vectors. Malathion started to be distributed in 2006-2008 to some states, mainly in the Northeast (CE in 2006) and Southeast (RJ in 2007 and MG in 2008) Regions. Later, due to the dissemination of high levels of PY resistance in Ae. aegypti populations throughout the country, malathion was gradually introduced instead for adult control,
25
,
26
and its use was well-established in the country by around 2009-2011.
Resistance to the larvicide temephos - Since 2006, Brazil has considered Ae. aegypti populations with a RR95 to temephos of 3.0 or higher to be resistant to this larvicide. This change in classification, from 10.0 to 3.0, was based on functional and operational criteria. For functional validation, simulated field trials confirmed the reduction of the effectiveness of temephos in populations with RR95 values above 3.0.
20
The operational criteria were related to the logistics of the insecticide substitution procedure in the country, which depends on coordination among the three public management levels (federal, state, and municipal), meaning that, in practice, it can take up to two years for a recommendation at the central level to be implemented in the field.
Today, temephos resistance is so widespread in Brazil that this OP is no longer considered as the first-choice larvicide for use against Ae. aegypti, and it has been replaced by other, non-neurotoxic products. Varied temephos resistance levels were observed in the country. In some cases, high temephos resistance levels occurred simultaneously with, or soon after, periods of elevated dengue incidence, suggesting the intensification of chemical control in response to outbreaks; in other cases, resistance levels were associated with the temephos supply, or its interruption. However, in many cases these indicators were not enough to explain the variety of situations observed. For instance, the South Region presented the lowest RR95 values throughout the period evaluated. This result is compatible with the late inclusion of the South in the ‘dengue scenario’. Low temephos resistance levels were also found in SP state, but in this case they were probably due to there being strong and coordinated monitoring and resistance management. The Northeast Region presented the greatest variation in temephos resistance levels, as well as the highest registered values, as the RR95 was above 100 in 20% of cases there, and in 6% of cases it was higher than 200 (five municipalities, three in PE and two in BA). According to Moyes et al.,
29
Brazil is the country with the highest levels of resistance by mosquitoes to temephos in the world. In Martinique, the country with the second highest resistance levels on this scale, the highest recorded RR50 was 35 in Gros Morne in 2009.
66
In general, these results revealed the influence of local public health actions on the establishment of temephos resistance and the fragility of general guidelines, which should contribute to the more rational use of chemical control and, ultimately, its preservation as a complementary alternative for vector control.
Resistance to deltamethrin - In Brazil, a few decades elapsed until the spread of temephos resistance reached levels that could compromise its use in Ae. aegypti control. However, in the case of PYs, which were introduced by the PNCD across the whole country in 2000, only a few years were enough to induce high resistance levels in populations of this vector to this class of pesticide.
10
,
13
,
23
,
67
Compared with larvae, the evaluation of adult resistance is more labour intensive. It depends on the use of devices and consumable materials that are not always easy to obtain. In addition, since adult females are used instead of larvae, tests become more time-consuming and dependent on the availability of specimens. For these reasons, routine tests are generally qualitative ones, in which specimens are exposed to only one insecticide dose and samples are classified as resistant, susceptible, or with incipient resistance. Two main methodologies, referred to herein as the CDC and WHO methodologies, are available for such tests and use bottles or papers, respectively, which are impregnated with insecticide.
35
,
39
The CDC methodology tends to result in higher mortality levels due to the nature of the assay itself. In Brazil, both methodologies have been employed to evaluate deltamethrin resistance. The WHO methodology did not identify any susceptible populations, and with the CDC approach only 8% of the samples were susceptible. Although both the CDC and WHO methodologies are effective to assess the susceptibility profile of adult mosquitoes, the variable correlation between the results of the two methods inhibits, and even precludes, the ability for comparisons to be made between their results. In this sense, we tend to agree with Owusu et al.,
45
who suggested that dose-response trials should be adopted to produce more consistent evaluations.
In this sense, within the scope of the MoReNAa we started to introduce the use of quantitative deltamethrin assays using impregnated papers directed at adult specimens in 2009.
26
,
68
To our knowledge this was the first quantitative evaluation of adults performed in the Ae. aegypti resistance monitoring routine. It was necessary to establish a series of conditions and parameters for this technique, as well as to define evaluation criteria. In the latter case, we adopted the cut-off established in Brazil in 2006 for temephos: that a RR95 above 3.0 is indicative of resistance.
19
Quantitative trials with deltamethrin for 41 ‘municipalities/years’ were identified herein. Most tests (30) were performed in 2011-2012, and confirmed the dissemination of resistance of Ae. aegypti to PYs in Brazil. Except for the South Region, very high RR95 values around 50-70 were detected in all Regions, and were more frequent in municipalities in the Centre-West Region. The Northeast Region tended to house the vector populations with the lowest PY resistance levels. There have been reports in the literature that Ae. aegypti resistance to PYs is more strongly influenced by vector seasonality, and in particular by the seasonality of dengue epidemics, than larvicides.
10
,
13
,
67
This is a clear manifestation of the conceptual confusion between ‘vector control’ and ‘chemical control of adult vectors’. Indeed, this is one of the hypotheses that explains the rapid spread of PY resistance throughout the world.
13
,
14
,
55
,
67
,
69
,
70
,
71
,
72
When the introduction of DENV-4 was detected in Brazil during the course of a dengue outbreak in Boa Vista, RR in the North Region, a quick and significant increase in PY resistance was detected throughout the city, including in areas that had not received ultra-low volume applications from the public health sector.
10
Additionally, in SP state, Ae. aegypti resistance to pyrethroid insecticides continued to persist in several localities, even 10 years after their discontinuation in the control of the dengue vector by SP state municipal health secretaries.
14
In relation to the data presented herein, in several cases high deltamethrin resistance levels were preceded by dengue epidemics, confirming that intense and indiscriminate use of pyrethroids occurred during outbreaks. The scenario found in the Centre-West Region is quite illustrative of this point: in practically all municipalities evaluated in 2011/2012, the RR95 was around 50. Since 2006, this region has been subjected to dengue outbreaks, culminating in 2010, which was precisely when the average dengue incidence in the municipalities evaluated was approximately 1.7%, almost six times the MoH cut-off point. The case of Luziânia, GO is also particularly informative: in 2011, the highest dengue rates so far registered (above 1.4%), and also extremely high levels of deltamethrin resistance (RR80 = 167), were found, and a closer look at the data revealed that the collection of field material for these bioassays was performed at the end of the epidemic season [Supplementary data (Fig. 2)], showing the influence of the intensification of adulticide application on the vector populations’ pyrethroid resistance status.
Resistance mechanisms - Metabolic resistance is the result of changes in the activities of enzymes involved in the metabolism, sequestration, and excretion of insecticides. The activities of three major enzyme classes, MFOs, ESTs (Phase I), and GSTs (Phase II), were evaluated in larval and adult female samples using the routine monitoring methodology applied in Brazil. In general, adults exhibited greater alterations to their enzymes’ activities than larvae.
The multifactorial nature of insecticide resistance was confirmed in this study, as has been reported for other vector populations worldwide.
73
,
74
,
75
The most strongly affected mechanisms of metabolic resistance throughout Brazil were the activities of the EST and GST enzyme superfamilies, which were closely related to OP and PY resistance in Brazilian samples.
13
,
20
,
57
,
67
,
76
Changes in MFO activity were less prominent, as only around 30% and less than 20% of adult and larval samples, respectively, exhibited changes in MFO activity. This metabolic profile contrasts with those reported by studies done elsewhere,
56
,
70
,
77
,
78
,
79
and this difference was probably due to the distinct nature of the assays examined herein. Specifically, these biochemical assays (which are adopted in routine monitoring programs) quantify levels of different enzyme classes, and thus may underestimate activity alterations; in contrast, the detection of MFO participation in metabolic resistance is generally derived from molecular assays that consist of more specific approaches to investigate gene expression changes. Efforts to understand the dynamics of metabolic resistance in Brazilian vector samples at the gene expression level, including those of MFOs, are currently underway.
The OP target is encoded by two genes, ace1 and ace2.
80
The relationship between some ace1 mutations in mosquitoes belonging to the genera Culex and Anopheles and their OP resistance has been well-documented.
80
,
81
The participation of two recently detected point mutations in ace1 in OP resistance in Ae. aegypti populations in India and Indonesia has also been suggested.
82
,
83
However, it is intriguing that for the mutations reported in Aedes mosquitoes, only a small difference in the inhibition profile of ACE has been suggested, which is different from the impacts of similar mutations observed in Culex and Anopheles species. The present study detected changes in total ACE activity (AChE) in 17% of the analysed samples, but in no case did the remaining ACE activity after insecticide inhibition (AChI) signal the alteration of this endpoint. However, it is important to note that the criteria applied to classify the ACE status in both assays, AChE and AChI, are quite different, which indicates the need for further, deeper analyses of this enzyme in Ae. aegypti field populations.
In relation to PY resistance, some research groups have argued that metabolic changes tend to result in lower resistance levels than alterations in the PY target site, Na
V .
37
,
61
,
62
We presented evidence that the participation of metabolic mechanisms indeed seems to be limited in populations with high deltamethrin resistance levels. Accordingly, it has been shown elsewhere that there is less participation of the metabolic detoxification pathway in the resistance of mosquito populations presenting Na
V
kdr mutations.
61
In that same study, the authors suggested that different Ae. aegypti populations may respond to selection pressure in distinct ways, hindering or even preventing attempts to identify metabolic resistance components that could be diagnostic of PY resistance in general.
The history of temephos and deltamethrin resistance status examined herein provides evidence of the spread of resistance against the main insecticides used by the PNCD in the control of Brazilian Ae. aegypti populations. The high temephos resistance levels observed are the consequence of its use for more than 40 years in Brazil. Rapid dissemination of adult resistance to deltamethrin was correlated with the intensified chemical control of adults during dengue epidemic seasons. High dengue incidences are recurrent in Brazil, a hyperendemic country for this arbovirus. This dataset highlights the limitations of chemical vector control as the methodology of choice for the sustainable reduction of dengue incidence. In this sense, the rotation of insecticides has already been adopted in Brazil as a strategy to preserve these chemical pesticides as effective vector control tools. The multifactorial character of pesticide resistance was shown in this study. In view of the insecticide resistance of Ae. aegypti populations in Brazil, alternative approaches, such as community engagement and mechanical control, should be considered when planning interventions to reduce dengue transmission. It is worth emphasizing the importance of respecting the complementary character of insecticides, meaning that they should be reserved for the treatment of vector breeding places that cannot be eliminated and for specific places and situations, such as outbreak blockage or intervention at strategic points.
|
Background:
Aedes aegypti populations in Brazil have been subjected to insecticide selection pressures with variable levels and sources since 1967. Therefore, the Brazilian Ministry of Health (MoH) coordinated the activities of an Ae. aegypti insecticide resistance monitoring network (MoReNAa) from 1999 to 2012.
Methods:
Data were gathered from two sources: a bibliographic review of studies published from 1985 to 2017, and unpublished data produced by our team within the MoReNAa between 1998 and 2012. A total of 146 municipalities were included, many of which were evaluated several times, totalling 457 evaluations for temephos and 274 for deltamethrin. Insecticide resistance data from the five Brazilian regions were examined separately using annual records of both the MoH supply of insecticides to each state and the dengue incidence in each evaluated municipality.
Results:
Ae. aegypti resistance to temephos and deltamethrin, the main larvicide and adulticide, respectively, employed against mosquitoes in Brazil for a long time, was found to be widespread in the country, although with some regional variations. Comparisons between metabolic and target-site resistance mechanisms showed that one or another of these was the main component of pesticide resistance in each studied population.
Conclusions:
(i) A robust dataset on the assessments of the insecticide resistance of Brazilian Ae. aegypti populations performed since 1985 was made available through our study. (ii) Our findings call into question the efficacy of chemical control as the sole methodology of vector control. (iii) It is necessary to ensure that sustainable insecticide resistance monitoring is maintained as a key component of integrated vector management. (iv) Consideration of additional parameters, beyond the supply of insecticides distributed by the MoH or the diverse local dynamics of dengue incidence, is necessary to find consistent correlations with heterogeneous vector resistance profiles.
| null | null | 13,881 | 339 | 3 |
[
"resistance",
"dengue",
"data",
"temephos",
"region",
"evaluated",
"supplementary",
"supplementary data",
"municipalities",
"incidence"
] |
[
"test",
"test"
] | null | null | null | null | null | null |
[CONTENT] Aedes aegypti | Brazil | insecticide resistance | temephos | deltamethrin | vector control [SUMMARY]
| null |
[CONTENT] Aedes aegypti | Brazil | insecticide resistance | temephos | deltamethrin | vector control [SUMMARY]
| null | null | null |
[CONTENT] Aedes | Animals | Biological Assay | Brazil | Dengue | Incidence | Insecticide Resistance | Insecticides | Mosquito Vectors | Nitriles | Pyrethrins | Temefos [SUMMARY]
| null |
[CONTENT] Aedes | Animals | Biological Assay | Brazil | Dengue | Incidence | Insecticide Resistance | Insecticides | Mosquito Vectors | Nitriles | Pyrethrins | Temefos [SUMMARY]
| null | null | null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null | null | null |
[CONTENT] resistance | dengue | data | temephos | region | evaluated | supplementary | supplementary data | municipalities | incidence [SUMMARY]
| null |
[CONTENT] resistance | dengue | data | temephos | region | evaluated | supplementary | supplementary data | municipalities | incidence [SUMMARY]
| null | null | null |
[CONTENT] resistance | data | region | dengue | temephos | supplementary data | supplementary | evaluated | municipalities | data table [SUMMARY]
| null |
[CONTENT] resistance | dengue | data | temephos | region | deltamethrin | levels | high | evaluated | incidence [SUMMARY]
| null | null | null |
[CONTENT] Brazil ||| one [SUMMARY]
| null |
[CONTENT] Brazil | 1967 ||| the Brazilian Ministry of Health | MoH | 1999 | 2012 ||| two | 1985 to 2017 | between 1998 and 2012 ||| 146 | 457 | 274 ||| five | Brazilian | annual | MoH ||| Brazil ||| one ||| ||| Brazilian ||| 1985 ||| ||| ||| MoH [SUMMARY]
| null |
||
Isoflavones inhibit poly(I:C)-induced serum, brain, and skin inflammatory mediators - relevance to chronic fatigue syndrome.
|
25359293
|
Chronic Fatigue Syndrome (CFS) is a neuroimmunoendocrine disease affecting about 1% of the US population, mostly women. It is characterized by debilitating fatigue for six or more months in the absence of cancer or other systemic diseases. Many CFS patients also have fibromyalgia and skin hypersensitivity that worsen with stress. Corticotropin-releasing hormone (CRH) and neurotensin (NT), secreted under stress, activate mast cells (MC) necessary for allergic reactions to release inflammatory mediators that could contribute to CFS symptoms.
|
BACKGROUND
|
Female C57BL/6 mice were randomly divided into four groups: (a) control/no-swim, (b) control/swim, (c) polyinosinic:polycytidylic acid (poly(I:C))/no swim, and (d) polyinosinic:polycytidylic acid (poly(I:C))/swim. Mice were provided with chow low or high in isoflavones for 2 weeks prior to ip injection with 20 mg/kg poly(I:C) followed or not by swim stress for 15 minutes. Locomotor activity was monitored overnight and animals were sacrificed the following day. Brain and skin gene expression, as well as serum levels, of inflammatory mediators were measured. Data were analyzed using the non-parametric Mann-Whitney U-test.
|
METHODS
|
Poly(I:C)-treated mice had decreased locomotor activity over 24 hours, and increased serum levels of TNF-α, IL-6, KC (IL-8/CXCL8 murine homolog), CCL2,3,4,5, CXCL10, as well as brain and skin gene expression of TNF, IL-6, KC (Cxcl1, IL8 murine homolog), CCL2, CCL4, CCL5 and CXCL10. Histidine decarboxylase (HDC) and NT expression were also increased, but only in the skin, over the same period. High isoflavone diet reversed these effects.
|
RESULTS
|
Poly(I:C) treatment decreased mouse locomotor activity and increased serum levels and brain and skin gene expression of inflammatory mediators. These effects were inhibited by isoflavones that may prove useful in CFS.
|
CONCLUSION
|
[
"Animals",
"Biomarkers",
"Brain",
"Fatigue Syndrome, Chronic",
"Female",
"Inflammation Mediators",
"Isoflavones",
"Mice",
"Mice, Inbred C57BL",
"Motor Activity",
"Poly I-C",
"Skin",
"Swimming"
] |
4236420
|
Background
|
Chronic Fatigue Syndrome (CFS) is a complex disease, which has also been called ‘neurasthenia’, ‘post viral fatigue’, and ‘chronic mononucleosis’ [1-3]. Its prevalence may be as high as 1% in the US population [4] with a female to male ratio of 4 to 1 [5]. CFS involves the muscular, nervous, hormonal and immune systems. Patients complain of overwhelming fatigue, sleep disturbances, malaise, muscle aches, gastrointestinal symptoms, dizziness on standing, and cognitive problems [6]. Clinical and subclinical viral infections have been suspected, but never confirmed [7,8].
CFS is often comorbid with other disorders, including fibromyalgia, Pelvic Bladder Syndrome/Interstitial Cystitis (PBS/IC), irritable bowel syndrome (IBS), and migraines [9], all of which are characterized by central nervous system (CNS) dysfunction [10], and worsened by stress [11-15]. Many CFS patients demonstrate abnormal hypothalamic-pituitary-adrenal (HPA) axis activity [16,17]. Anxiety is common in CFS [18] and patients are particularly vulnerable to stress [14]. Many CFS symptoms could derive from the possible release of inflammatory mediators that could affect brain function [19,20]. As of today, there are no FDA approved drugs for the treatment of CFS [21]; psychological, physical, and pharmacological interventions used currently are not very effective [22].
An abnormal immune component may be involved in CFS [23-25], but the neuroimmune and neuroendocrine interactions involved are still unknown [26]. Mast cells (MC) and their mediators have been implicated in all diseases that are comorbid with CFS [9]. There is higher number of skin MC in patients with CFS [27,28], and such patients also show increased skin hypersensitivity [29]. Furthermore, CFS also occurs more often in patients with chronic urticaria, that also involves MC [30]. In fact, there is hyperresponsiveness in the bronchi of CFS patients, implying MC activation [31].
Activated MC release a number of chemokines and cytokines that could contribute to CFS symptoms [32,33]. MC are located perivascularly in close proximity to neurons [34], especially in the diencephalon [35,36], where functional MC-neuron interactions have been documented [36,37] in response to corticotropin-releasing hormone (CRH) [38]. In vivo activation of MC by CRH is augmented by neurotensin (NT) [39]. Moreover, NT is induced in the hypothalamus in response to bacterial lipopolysaccharide (LPS) and regulates the HPA axis [40].
Unfortunately, there are neither effective CFS treatments nor human MC inhibitors clinically available that may also be used in CFS. Flavonoids are natural compounds with strong antioxidant and anti-inflammatory activity [41]. Certain flavonoids also inhibit MC [42] and have neuroprotective effects [41,43].
Here we report that treatment of mice with poly(I:C) results in reduced locomotor activity and increased serum levels, as well as brain and gene expression, of inflammatory mediators, all of which are reversed by treatment with the isoflavones daidzein and genistein.
| null | null |
Results
|
Effect of poly(I:C) and isoflavones on locomotor activity Poly(I:C)/swim and poly(I:C)/no swim-treated mice on low isoflavones had reduced maximum (max) locomotor activity (over the 10-hour night period), denoted by the dark bar (P = 0.008 and P = 0.036) compared to the control/swim and control/no swim-treated mice, respectively (Figure 1A). High isoflavone chow reversed this decrease (Figure 1B), and the difference between the two diets was statistically significant (P = 0.032).Figure 1
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1
A) or high (Figure 1
B) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1
A) or high (Figure 1
B) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.
Poly(I:C)/swim and poly(I:C)/no swim-treated mice on low isoflavones had reduced maximum (max) locomotor activity (over the 10-hour night period), denoted by the dark bar (P = 0.008 and P = 0.036) compared to the control/swim and control/no swim-treated mice, respectively (Figure 1A). High isoflavone chow reversed this decrease (Figure 1B), and the difference between the two diets was statistically significant (P = 0.032).Figure 1
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1
A) or high (Figure 1
B) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1
A) or high (Figure 1
B) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.
Effect of poly(I:C) and isoflavones on serum inflammatory mediators Poly(I:C)-treated mice on low isoflavones had increased serum levels of TNF-α (Figure 2A), IL-6 (Figure 2B), KC (Figure 2C), and CCL4 (Figure 2D), as well as CCL2, CCL3, CCL5, and CXCL10 (Additional file 2: Table S2). High isoflavones reduced the poly(I:C)-increased serum levels of all the inflammatory markers. All IFNγ, IL-1β, IL-4, IL-9, IL-10, IL-12p70, IL-17 and VEGFα serum levels were below the detection limit, while IL-1α serum levels were similar between the different treatment groups. Swim stress augmented the poly(I:C) effect on TNF-α, CCL4, and CCL5 serum levels.Figure 2
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2
A), IL-6 (Figure 2
B), KC (Figure 2
C), and CCL4 (Figure 2
D) using the Milliplex microbead assay.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2
A), IL-6 (Figure 2
B), KC (Figure 2
C), and CCL4 (Figure 2
D) using the Milliplex microbead assay.
More specifically, TNF-α serum levels in poly(I:C)/swim-treated mice on low isoflavones were increased (55 ± 14 pg/ml) compared to control/swim-treated mice (32 ± 0 pg/ml, P = 0.0011) and poly(I:C)/no swim-treated mice (37 ± 10 pg/ml,P = 0.0221). High isoflavones decreased these high TNF-α serum levels (32 ± 0 pg/ml, P = 0.0042) (Figure 2A). IL-6 serum levels in poly(I:C)-swim and poly(I:C)/no swim-treated mice on low isoflavones were also increased (229 ± 110 pg/ml and 141 ± 11 pg/ml) compared to the corresponding control/swim and control/no swim mice on low isoflavones (32 ± 0 pg/ml, P = 0.0011 and 32 ± 0 pg/ml, P = 0.0114) (Figure 2B). High isoflavones reduced IL-6 serum levels of poly(I:C)/swim-treated mice (35 ± 5 pg/ml, P = 0.0054) and poly(I:C)/no swim-treated (32 ± 0 pg/ml, P = 0.0298) compared to the corresponding groups on low isoflavones (Figure 2B).
Similarly, KC serum levels in poly(I:C)-treated mice on low isoflavones were increased (735 ± 579 pg/ml and 626 ± 523 pg/ml) compared to the corresponding controls (173 ± 43 pg/ml, P = 0.0379 and 132 ± 52 pg/ml, P = 0.0006, respectively) (Figure 2C). Although KC serum levels in poly(I:C)/swim-treated mice on high isoflavones were also increased (451 ± 107 pg/ml) compared to control poly(I:C)/swim-treated mice (100 ± 45 pg/ml, P = 0.0079), high isoflavone diet decreased KC serum levels in poly(I:C)/no swim-treated mice (128 ± 43 pg/ml, P = 0.0025) compared to those on low isoflavones (Figure 2C).
Moreover, CCL4 serum levels were increased in poly(I:C)-treated mice on low isoflavones (1,256 ± 556 pg/ml and 395 ± 245 pg/ml) compared to the corresponding controls (160 ± 0 pg/ml, P = 0.0011 and 160 ± 0 pg/ml, P = 0.0037, respectively) (Figure 2D). Swim further increased CCL4 serum levels (1,256 ± 556 pg/ml, P = 0.0023). Although CCL4 serum levels in poly(I:C)-treated mice on high isoflavones were also increased (217 ± 55 pg/ml and 265 ± 111 pg/ml) compared to their controls (160 ± 0 pg/ml, P = 0.0075 and 160 ± 0 pg/ml, P = 0.0254, respectively)., high isoflavones decreased CCL4 serum levels in poly(I:C)/swim-treated mice (217 ± 55 pg/ml, P = 0.0025) (Figure 2D).
Poly(I:C) treatment also increased CCL2, CCL3, CCL5 and CXCL10 serum levels and high isoflavones reduced those increases (Additional file 3).
Poly(I:C)-treated mice on low isoflavones had increased serum levels of TNF-α (Figure 2A), IL-6 (Figure 2B), KC (Figure 2C), and CCL4 (Figure 2D), as well as CCL2, CCL3, CCL5, and CXCL10 (Additional file 2: Table S2). High isoflavones reduced the poly(I:C)-increased serum levels of all the inflammatory markers. All IFNγ, IL-1β, IL-4, IL-9, IL-10, IL-12p70, IL-17 and VEGFα serum levels were below the detection limit, while IL-1α serum levels were similar between the different treatment groups. Swim stress augmented the poly(I:C) effect on TNF-α, CCL4, and CCL5 serum levels.Figure 2
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2
A), IL-6 (Figure 2
B), KC (Figure 2
C), and CCL4 (Figure 2
D) using the Milliplex microbead assay.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2
A), IL-6 (Figure 2
B), KC (Figure 2
C), and CCL4 (Figure 2
D) using the Milliplex microbead assay.
More specifically, TNF-α serum levels in poly(I:C)/swim-treated mice on low isoflavones were increased (55 ± 14 pg/ml) compared to control/swim-treated mice (32 ± 0 pg/ml, P = 0.0011) and poly(I:C)/no swim-treated mice (37 ± 10 pg/ml,P = 0.0221). High isoflavones decreased these high TNF-α serum levels (32 ± 0 pg/ml, P = 0.0042) (Figure 2A). IL-6 serum levels in poly(I:C)-swim and poly(I:C)/no swim-treated mice on low isoflavones were also increased (229 ± 110 pg/ml and 141 ± 11 pg/ml) compared to the corresponding control/swim and control/no swim mice on low isoflavones (32 ± 0 pg/ml, P = 0.0011 and 32 ± 0 pg/ml, P = 0.0114) (Figure 2B). High isoflavones reduced IL-6 serum levels of poly(I:C)/swim-treated mice (35 ± 5 pg/ml, P = 0.0054) and poly(I:C)/no swim-treated (32 ± 0 pg/ml, P = 0.0298) compared to the corresponding groups on low isoflavones (Figure 2B).
Similarly, KC serum levels in poly(I:C)-treated mice on low isoflavones were increased (735 ± 579 pg/ml and 626 ± 523 pg/ml) compared to the corresponding controls (173 ± 43 pg/ml, P = 0.0379 and 132 ± 52 pg/ml, P = 0.0006, respectively) (Figure 2C). Although KC serum levels in poly(I:C)/swim-treated mice on high isoflavones were also increased (451 ± 107 pg/ml) compared to control poly(I:C)/swim-treated mice (100 ± 45 pg/ml, P = 0.0079), high isoflavone diet decreased KC serum levels in poly(I:C)/no swim-treated mice (128 ± 43 pg/ml, P = 0.0025) compared to those on low isoflavones (Figure 2C).
Moreover, CCL4 serum levels were increased in poly(I:C)-treated mice on low isoflavones (1,256 ± 556 pg/ml and 395 ± 245 pg/ml) compared to the corresponding controls (160 ± 0 pg/ml, P = 0.0011 and 160 ± 0 pg/ml, P = 0.0037, respectively) (Figure 2D). Swim further increased CCL4 serum levels (1,256 ± 556 pg/ml, P = 0.0023). Although CCL4 serum levels in poly(I:C)-treated mice on high isoflavones were also increased (217 ± 55 pg/ml and 265 ± 111 pg/ml) compared to their controls (160 ± 0 pg/ml, P = 0.0075 and 160 ± 0 pg/ml, P = 0.0254, respectively)., high isoflavones decreased CCL4 serum levels in poly(I:C)/swim-treated mice (217 ± 55 pg/ml, P = 0.0025) (Figure 2D).
Poly(I:C) treatment also increased CCL2, CCL3, CCL5 and CXCL10 serum levels and high isoflavones reduced those increases (Additional file 3).
Effect of poly(I:C) and isoflavones on brain gene expression of inflammatory mediators TNF-α (Figure 3A), IL-6 (Figure 3B), KC (Figure 3C), CCL4 (Figure 3D), as well as CCL2, CCL5 and CXCL10 brain gene expression were increased in the poly(I:C)-treated mice. High isoflavones reduced the increased TNF, IL6, KC, CCL4 and CCL2 brain gene expression. There was no difference in HDC and NT gene expression between the different treatment groups (Additional file 4: Table S3).Figure 3
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3
A), IL-6 (Figure 3
B), KC (Figure 3
C), and CCL4 (Figure 3
D) brain gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3
A), IL-6 (Figure 3
B), KC (Figure 3
C), and CCL4 (Figure 3
D) brain gene expression using qPCR.
More specifically TNF-α gene expression was increased in the brain of poly(I:C)-treated mice on low isoflavones (relative fold change of 22 ± 7 and 12 ± 9) compared to their controls (1.1 ± 0.9, P = 0.0006 and 1 ± 0.7, P = 0.0012, respectively) (Figure 3A). Although poly(I:C)-treated mice on high isoflavones had increased TNF-α brain gene expression (6 ± 1 and 9 ± 8) compared to their corresponding controls (1.2 ± 0.5, P = 0.0079 and 1 ± 0.3, P = 0.0079, respectively), high isoflavones decreased TNF-α gene expression in the brain of poly(I:C)/swim-treated mice (6 ± 1, P = 0.0025) compared to those on low isoflavones (Figure 3A). There was no effect of poly(I:C) treatment on IL-6 brain gene expression (Figure 3B), but high isoflavones still decreased IL-6 gene expression in the brain of poly(I:C)/swim-treated mice (0.5 ± 0.1, P = 0.0228) compared to those on low isoflavones (2.1 ± 2.4) (Figure 3B).
KC gene expression in the brain of poly(I:C)-treated mice on low isoflavones was increased (18 ± 13 and 40 ± 52) compared to their controls (0.9 ± 0.3, P = 0.0006 and 1 ± 0.3, P = 0.0183, respectively) (Figure 3C). Although poly(I:C)/swim-treated mice on high isoflavones still had increased KC gene expression in their brain (2 ± 0.9) compared to control/swim-treated mice (0.6 ± 0.1, P = 0.0159), high isoflavones reduced this increase in KC gene expression (2 ± 0.9, P = 0.0177) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3C).
Likewise, CCL4 gene expression in the brain of poly(I:C)-treated mice on low isoflavones was also increased (6.5 ± 4 and 2.9 ± 1.6) compared to their controls (1 ± 0.3, P = 0.0006 and 1 ± 0.4, P = 0.0350, respectively) (Figure 3D). Although, poly(I:C)/swim-treated mice on high isoflavones still had increased CCL4 gene expression (1.8 ± 0.4) compared to control/swim-treated mice (1 ± 0.4, P = 0.0317), high isoflavones reduced this increase in CCL4 gene expression (1.8 ± 0.4, P = 0.0101) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3D). Poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the brains of the mice. High isoflavones reduced this noted CCL2 brain gene expression increase (Additional file 3).
TNF-α (Figure 3A), IL-6 (Figure 3B), KC (Figure 3C), CCL4 (Figure 3D), as well as CCL2, CCL5 and CXCL10 brain gene expression were increased in the poly(I:C)-treated mice. High isoflavones reduced the increased TNF, IL6, KC, CCL4 and CCL2 brain gene expression. There was no difference in HDC and NT gene expression between the different treatment groups (Additional file 4: Table S3).Figure 3
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3
A), IL-6 (Figure 3
B), KC (Figure 3
C), and CCL4 (Figure 3
D) brain gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3
A), IL-6 (Figure 3
B), KC (Figure 3
C), and CCL4 (Figure 3
D) brain gene expression using qPCR.
More specifically TNF-α gene expression was increased in the brain of poly(I:C)-treated mice on low isoflavones (relative fold change of 22 ± 7 and 12 ± 9) compared to their controls (1.1 ± 0.9, P = 0.0006 and 1 ± 0.7, P = 0.0012, respectively) (Figure 3A). Although poly(I:C)-treated mice on high isoflavones had increased TNF-α brain gene expression (6 ± 1 and 9 ± 8) compared to their corresponding controls (1.2 ± 0.5, P = 0.0079 and 1 ± 0.3, P = 0.0079, respectively), high isoflavones decreased TNF-α gene expression in the brain of poly(I:C)/swim-treated mice (6 ± 1, P = 0.0025) compared to those on low isoflavones (Figure 3A). There was no effect of poly(I:C) treatment on IL-6 brain gene expression (Figure 3B), but high isoflavones still decreased IL-6 gene expression in the brain of poly(I:C)/swim-treated mice (0.5 ± 0.1, P = 0.0228) compared to those on low isoflavones (2.1 ± 2.4) (Figure 3B).
KC gene expression in the brain of poly(I:C)-treated mice on low isoflavones was increased (18 ± 13 and 40 ± 52) compared to their controls (0.9 ± 0.3, P = 0.0006 and 1 ± 0.3, P = 0.0183, respectively) (Figure 3C). Although poly(I:C)/swim-treated mice on high isoflavones still had increased KC gene expression in their brain (2 ± 0.9) compared to control/swim-treated mice (0.6 ± 0.1, P = 0.0159), high isoflavones reduced this increase in KC gene expression (2 ± 0.9, P = 0.0177) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3C).
Likewise, CCL4 gene expression in the brain of poly(I:C)-treated mice on low isoflavones was also increased (6.5 ± 4 and 2.9 ± 1.6) compared to their controls (1 ± 0.3, P = 0.0006 and 1 ± 0.4, P = 0.0350, respectively) (Figure 3D). Although, poly(I:C)/swim-treated mice on high isoflavones still had increased CCL4 gene expression (1.8 ± 0.4) compared to control/swim-treated mice (1 ± 0.4, P = 0.0317), high isoflavones reduced this increase in CCL4 gene expression (1.8 ± 0.4, P = 0.0101) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3D). Poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the brains of the mice. High isoflavones reduced this noted CCL2 brain gene expression increase (Additional file 3).
Effect of poly(I:C) and isoflavones on skin gene expression of inflammatory mediators TNF-α (Figure 4A), IL-6 (Figure 4B), KC (Figure 4C), CCL4 (Figure 4D), as well as CCL2, CCL5 and CXCL10 (Additional file 5: Table S4) skin gene expression were increased in the poly(I:C)-treated mice. Moreover, HDC and NT gene expression were also increased in the skin (Figure 5A and B). High isoflavone diet reduced the increased TNF-α, IL-6 and KC skin gene expression.Figure 4
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4
A), IL-6 (Figure 4
B), KC (Figure 4
C) and CCL4 (Figure 4
D) skin gene expression using qPCR.Figure 5
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5
A) and NT (Figure 5
B) skin gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4
A), IL-6 (Figure 4
B), KC (Figure 4
C) and CCL4 (Figure 4
D) skin gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5
A) and NT (Figure 5
B) skin gene expression using qPCR.
More specifically, TNF-α gene expression in the skin of poly(I:C)/swim-treated mice on low isoflavones, was increased (5 ± 0.9) compared to their controls (1.1 ± 0.4, P = 0.0006) (Figure 4A) and high isoflavones decreased this increase in TNF-α gene expression (2.3 ± 1.3, P = 0.0101) (Figure 4A). Likewise, IL-6 gene expression in the skin of poly(I:C)-treated mice on low isoflavones was increased (14 ± 4 and 22 ± 16) compared to their controls (0.9 ± 0.8, P = 0.0006 and 1 ± 0.4, P = 0.0012, respectively) (Figure 4B). Although poly(I:C)-treated mice on high isoflavones still had increased IL-6 gene expression (5.5 ± 3 and 3.4 ± 1.7) compared to their controls (1.4 ± 0.7, P = 0.0079 and 0.7 ± 0.4, P = 0.0159, respectively), high isoflavones reduced this increase in the IL-6 gene expression (5.5 ± 3 P = 0.0101) (Figure 4B).
Similarly, KC gene expression in the skin of poly(I:C)-treated mice on low isoflavones was also increased (53 ± 55 and 69 ± 83) compared to their controls (0.3 ± 0.2, P = 0.0021 and 1 ± 1.5, P = 0.0012, respectively) (Figure 4C). High isoflavones reduced this increase in the KC gene expression of poly(I:C)-treated mice (2.8 ± 3.5, P = 0.0057 and 3.4 ± 1.9, P = 0.0043) (Figure 4C).
CCL4 gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (24 ± 15 and 21 ± 19) compared to their controls (0.4 ± 0.3, P = 0.0021 and 1 ± 1.7, P = 0.0023, respectively) (Figure 4D). CCL4 gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (20 ± 24 and 27 ± 20) compared to their controls (0.8 ± 0.7, P = 0.0079 and 1 ± 1.3, P = 0.0079, respectively) and high isoflavones did have an effect on reducing this increase.
In the same way, HDC gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (4 ± 2 and 4 ± 2) compared to their controls (0.6 ± 0.4, P = 0.0003 and 1 ± 0.8, P = 0.0123, respectively) (Figure 5A). HDC gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (3 ± 1.1 and 3 ± 1) compared to their controls (0.9 ± 0.3, P = 0.0079 and 1 ± 0.2, P = 0.0317, respectively).
Similar to HDC gene expression, NT gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (8 ± 4 and 9 ± 10) compared to their controls (0.6 ± 0.4, P = 0.0025 and 1 ± 0.7, P = 0.0012, respectively) (Figure 5B). NT gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (8 ± 4, and 6 ± 6) compared to their controls (1.9 ± 0.8, P = 0.0317 and 1 ± 0.2, P = 0.0079, respectively).
Finally, poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the skin of the mice. High isoflavones did not have any statistical effect on CCL4, CCL2, CCL5, CXCL10, HDC and NT skin gene expression (Additional file 5: Table S4).
TNF-α (Figure 4A), IL-6 (Figure 4B), KC (Figure 4C), CCL4 (Figure 4D), as well as CCL2, CCL5 and CXCL10 (Additional file 5: Table S4) skin gene expression were increased in the poly(I:C)-treated mice. Moreover, HDC and NT gene expression were also increased in the skin (Figure 5A and B). High isoflavone diet reduced the increased TNF-α, IL-6 and KC skin gene expression.Figure 4
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4
A), IL-6 (Figure 4
B), KC (Figure 4
C) and CCL4 (Figure 4
D) skin gene expression using qPCR.Figure 5
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5
A) and NT (Figure 5
B) skin gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4
A), IL-6 (Figure 4
B), KC (Figure 4
C) and CCL4 (Figure 4
D) skin gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5
A) and NT (Figure 5
B) skin gene expression using qPCR.
More specifically, TNF-α gene expression in the skin of poly(I:C)/swim-treated mice on low isoflavones, was increased (5 ± 0.9) compared to their controls (1.1 ± 0.4, P = 0.0006) (Figure 4A) and high isoflavones decreased this increase in TNF-α gene expression (2.3 ± 1.3, P = 0.0101) (Figure 4A). Likewise, IL-6 gene expression in the skin of poly(I:C)-treated mice on low isoflavones was increased (14 ± 4 and 22 ± 16) compared to their controls (0.9 ± 0.8, P = 0.0006 and 1 ± 0.4, P = 0.0012, respectively) (Figure 4B). Although poly(I:C)-treated mice on high isoflavones still had increased IL-6 gene expression (5.5 ± 3 and 3.4 ± 1.7) compared to their controls (1.4 ± 0.7, P = 0.0079 and 0.7 ± 0.4, P = 0.0159, respectively), high isoflavones reduced this increase in the IL-6 gene expression (5.5 ± 3 P = 0.0101) (Figure 4B).
Similarly, KC gene expression in the skin of poly(I:C)-treated mice on low isoflavones was also increased (53 ± 55 and 69 ± 83) compared to their controls (0.3 ± 0.2, P = 0.0021 and 1 ± 1.5, P = 0.0012, respectively) (Figure 4C). High isoflavones reduced this increase in the KC gene expression of poly(I:C)-treated mice (2.8 ± 3.5, P = 0.0057 and 3.4 ± 1.9, P = 0.0043) (Figure 4C).
CCL4 gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (24 ± 15 and 21 ± 19) compared to their controls (0.4 ± 0.3, P = 0.0021 and 1 ± 1.7, P = 0.0023, respectively) (Figure 4D). CCL4 gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (20 ± 24 and 27 ± 20) compared to their controls (0.8 ± 0.7, P = 0.0079 and 1 ± 1.3, P = 0.0079, respectively) and high isoflavones did have an effect on reducing this increase.
In the same way, HDC gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (4 ± 2 and 4 ± 2) compared to their controls (0.6 ± 0.4, P = 0.0003 and 1 ± 0.8, P = 0.0123, respectively) (Figure 5A). HDC gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (3 ± 1.1 and 3 ± 1) compared to their controls (0.9 ± 0.3, P = 0.0079 and 1 ± 0.2, P = 0.0317, respectively).
Similar to HDC gene expression, NT gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (8 ± 4 and 9 ± 10) compared to their controls (0.6 ± 0.4, P = 0.0025 and 1 ± 0.7, P = 0.0012, respectively) (Figure 5B). NT gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (8 ± 4, and 6 ± 6) compared to their controls (1.9 ± 0.8, P = 0.0317 and 1 ± 0.2, P = 0.0079, respectively).
Finally, poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the skin of the mice. High isoflavones did not have any statistical effect on CCL4, CCL2, CCL5, CXCL10, HDC and NT skin gene expression (Additional file 5: Table S4).
|
Conclusion
|
Here we report that treatment of mice with poly(I:C) resulted in reduced locomotor activity and increased inflammatory markers in serum, brain and skin, all of which were reversed by treatment with isoflavones. Our findings also demonstrate significant variability among mouse ‘models’ as was recently reported [109]. There is need for the establishment of a more reliable animal model for CFS. Nevertheless, certain flavonoids appear promising for use in pilot clinical trials with CFS patients.
|
[
"Chemicals and reagents",
"Animals",
"Treatment conditions",
"Assessment of behavioral parameter-locomotor activity",
"Sample collection",
"Serum levels of inflammatory mediators",
"Quantitative PCR",
"Statistical analysis",
"Effect of poly(I:C) and isoflavones on locomotor activity",
"Effect of poly(I:C) and isoflavones on serum inflammatory mediators",
"Effect of poly(I:C) and isoflavones on brain gene expression of inflammatory mediators",
"Effect of poly(I:C) and isoflavones on skin gene expression of inflammatory mediators"
] |
[
"Polyinosinic-polycytidylic acid-TLR3-based adjuvant, poly(I:C), HMW VacciGrade, (catalog# vac-pic) was purchased from Invivogen (San Diego, CA, USA). Substance P (SP, catalog# S6883), neurotensin (NT, catalog# N6383) and corticotropin-releasing hormone (CRF, catalog# C3042) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aliquots of the above were prepared according to the manufacturer’s instructions. Teklad lab animal diets (catalog# 2918X, and 2920X) were purchased from Harlan (Indianapolis, IN, USA).",
"C57BL/6 female mice, nine to twelve weeks old, (Jackson Laboratories, Bar Harbor, ME, USA) were kept in virus-free sections of a modern animal facility and were allowed ad libitum access to food and water. They were maintained on a 14:10 hour light-dark cycle (the standard light-dark cycle used by the Department of Animal Health). Female mice were chosen because published reports indicate a female to male ratio of 4:1 [5], while the US Centers for Disease Control and Prevention (CDC) specify a female to male ratio of 4:1 [5]. Mice were kept in cages of five mice/cage until the day of the experiments. Both low and high isoflavone diets (2018X and 2020X) were sterile with similar ingredients other than isoflavone content. We monitored weight changes for 21 days prior to the beginning of the experiments. Only poly(I:C)/no swim-treated mice showed a slight decrease in weight change. Poly(I:C)/swim-treated mice, as well as their corresponding control mice, slightly increased their weight over the three-week observation period. However, by the end of this period, there was no statistical difference in weight change. The protocol was approved by Tufts Medical Center IACUC under number B 2011-88.",
"Mice were provided with chow containing either non-detectable to low (ND-20 mg/kg, Teklad 2920X) or high (150 to 250 mg/kg, Teklad 2918X) isoflavone (daidzein plus genistein) levels for two weeks. Conditions included four groups: (a) control (normal saline intraperitoneal (ip) injection)/no swim, (b) control/swim, (c) poly(I:C)/no swim, and (d) poly(I:C)/swim (n = 5 to 7/group). Mice were injected ip with 20 mg/kg of poly(I:C) or normal saline the first day. Subsequently, they were subjected to swim for 15 minutes, individually in a transparent plastic cylindrical jar (17 cm × 25 cm) containing 15 cm-deep water at room temperature (23 ± 1°C). This approach reflects both exercise and the stress of water immersion. Mice were then placed individually into specific cages and locomotor activity was monitored overnight.",
"After the experimental procedures, animals were placed individually into standard plastic housing cages with food and water available ad libitum and overnight locomotor activity (for a total of 16 hours) was monitored with the Neuroscience Behavior Core’s mouse SmartFrame® Cage Rack System (Kinder Scientific, Poway, CA, USA). This system consists of 20 PC-interfaced horizontal photobeam frames. The frame (containing 12 photocells; arranged on a 8 L × 4 W grid) surrounds one home cage environment and continuously tracks the animal’s movement. This fully automated system allows the user to quantify horizontal ambulation by counting breaks in infrared photocell beams using MotorMonitor® software (Hamilton-Kinder Scientific, Poway, CA, USA). Data were collected and subsequently analyzed in time bins (every hour) or as a total over the course of collection to the ‘Total Distance Travelled’ (in cm) parameter for each zone.",
"Mice were euthanized 24 hours post poly(I:C) ip injection using isoflurane overdose and thoracotomy. Blood was collected by cardiac puncture and was used to determine inflammatory mediator levels in the serum. Brain (diencephalon) and skin (back shaved with a electric shaver the day before) samples were collected and immersed into RNAlater (catalog# AM7021) purchased from Invitrogen (Grand Island, NY, USA). Samples were stored at -80°C.",
"TNF-α, VEGFα, IL-1α, IL-1β, IL-4, IL-6, KC (IL-8/(CXC motif) ligand (CXC)L8 murine homolog, IL-9, IL-10, IL-12p70, IL-17, (CC motif) ligand (CCL)2, CCL3, CCL4, CCL5, CXCL10 and IFNγ mouse serum levels were determined using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Assay (MCYTOMAG-70 K custom made panel for the specific analytes mentioned above). Measurements were performed blindly by Millipore (St. Charles, MI, USA). Millipore's minimum assay reporting range for TNF-α and IL-6 levels was 32 pg/ml, for CCL2 was 800 pg/ml, while for CCL3 and CCL4 it was 160 pg/ml. Therefore, we consider all values < × pg/ml as equal to x pg/ml.",
"Total RNA from mouse tissues was extracted using RNeasy Plus Mini kit (catalog# 74134) and RNeasy Fibrous Tissue Mini Kit (catalog# 74704), purchased from QIAGEN (Valencia, CA, USA). Reverse transcription was performed with 300 ng of total RNA using the iScript cDNA synthesis kit (catalog# 170-8891) purchased from Bio-Rad (Hercules, CA, USA). Real-time quantitative polymerase chain reaction (qPCR) was carried out in a 7300 Sequence Detector, according to TaqMan Gene Expression Assay instructions from Life Technologies, Applied Biosystems (Grand Island, NY, USA) using Taqman primer/probe sets (Additional file 1: Table S1). Samples were analyzed for Tnf (TNF-α), Il4, Il6 (IL-6), KC (Cxcl1, IL-8 homolog in the mouse), Ccl2 (CCL2), Ccl4 (CCL4), Ccl5 (CCL5), Cxcl10 (CXCL10), histidine decarboxylase-Hdc (HDC), and Nts (NT) gene expression using Gapdh as internal control.Thermal cycling proceeded at 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, for 40 cycles, and 60°C for 1 minute. Negative controls included samples with water instead of template. Assays were performed in triplicate for each data point. Results were normalized against the endogenous gene GAPDH and were expressed relative to the mean of the control for each gene (relative fold change).",
"Data were analyzed using the non-parametric Mann-Whitney U-test. Results are presented as mean ± SE. Statistical significance is defined as P < 0.05. We analyzed separately the different treatment subgroups within each dietary group, using Mann-Whitney U-test to compare poly(I:C)/no swim and poly(I:C)/swim-treated mice with the control/no swim and control/swim-treated groups, accordingly. We then analyzed the same treatment subgroups between the two dietary groups, using Mann-Whitney U-test to compare, for example, poly(I:C)/swim-treated mice with low isoflavone diet to poly(I:C)/swim-treated mice that were provided with high isoflavone diet. Due to space limitations, we submitted figures composed of all the experimental conditions from the two different diets and all the statistical significant results were marked in these figures.",
"Poly(I:C)/swim and poly(I:C)/no swim-treated mice on low isoflavones had reduced maximum (max) locomotor activity (over the 10-hour night period), denoted by the dark bar (P = 0.008 and P = 0.036) compared to the control/swim and control/no swim-treated mice, respectively (Figure 1A). High isoflavone chow reversed this decrease (Figure 1B), and the difference between the two diets was statistically significant (P = 0.032).Figure 1\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1\nA) or high (Figure 1\nB) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1\nA) or high (Figure 1\nB) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.",
"Poly(I:C)-treated mice on low isoflavones had increased serum levels of TNF-α (Figure 2A), IL-6 (Figure 2B), KC (Figure 2C), and CCL4 (Figure 2D), as well as CCL2, CCL3, CCL5, and CXCL10 (Additional file 2: Table S2). High isoflavones reduced the poly(I:C)-increased serum levels of all the inflammatory markers. All IFNγ, IL-1β, IL-4, IL-9, IL-10, IL-12p70, IL-17 and VEGFα serum levels were below the detection limit, while IL-1α serum levels were similar between the different treatment groups. Swim stress augmented the poly(I:C) effect on TNF-α, CCL4, and CCL5 serum levels.Figure 2\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2\nA), IL-6 (Figure 2\nB), KC (Figure 2\nC), and CCL4 (Figure 2\nD) using the Milliplex microbead assay.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2\nA), IL-6 (Figure 2\nB), KC (Figure 2\nC), and CCL4 (Figure 2\nD) using the Milliplex microbead assay.\nMore specifically, TNF-α serum levels in poly(I:C)/swim-treated mice on low isoflavones were increased (55 ± 14 pg/ml) compared to control/swim-treated mice (32 ± 0 pg/ml, P = 0.0011) and poly(I:C)/no swim-treated mice (37 ± 10 pg/ml,P = 0.0221). High isoflavones decreased these high TNF-α serum levels (32 ± 0 pg/ml, P = 0.0042) (Figure 2A). IL-6 serum levels in poly(I:C)-swim and poly(I:C)/no swim-treated mice on low isoflavones were also increased (229 ± 110 pg/ml and 141 ± 11 pg/ml) compared to the corresponding control/swim and control/no swim mice on low isoflavones (32 ± 0 pg/ml, P = 0.0011 and 32 ± 0 pg/ml, P = 0.0114) (Figure 2B). High isoflavones reduced IL-6 serum levels of poly(I:C)/swim-treated mice (35 ± 5 pg/ml, P = 0.0054) and poly(I:C)/no swim-treated (32 ± 0 pg/ml, P = 0.0298) compared to the corresponding groups on low isoflavones (Figure 2B).\nSimilarly, KC serum levels in poly(I:C)-treated mice on low isoflavones were increased (735 ± 579 pg/ml and 626 ± 523 pg/ml) compared to the corresponding controls (173 ± 43 pg/ml, P = 0.0379 and 132 ± 52 pg/ml, P = 0.0006, respectively) (Figure 2C). Although KC serum levels in poly(I:C)/swim-treated mice on high isoflavones were also increased (451 ± 107 pg/ml) compared to control poly(I:C)/swim-treated mice (100 ± 45 pg/ml, P = 0.0079), high isoflavone diet decreased KC serum levels in poly(I:C)/no swim-treated mice (128 ± 43 pg/ml, P = 0.0025) compared to those on low isoflavones (Figure 2C).\nMoreover, CCL4 serum levels were increased in poly(I:C)-treated mice on low isoflavones (1,256 ± 556 pg/ml and 395 ± 245 pg/ml) compared to the corresponding controls (160 ± 0 pg/ml, P = 0.0011 and 160 ± 0 pg/ml, P = 0.0037, respectively) (Figure 2D). Swim further increased CCL4 serum levels (1,256 ± 556 pg/ml, P = 0.0023). Although CCL4 serum levels in poly(I:C)-treated mice on high isoflavones were also increased (217 ± 55 pg/ml and 265 ± 111 pg/ml) compared to their controls (160 ± 0 pg/ml, P = 0.0075 and 160 ± 0 pg/ml, P = 0.0254, respectively)., high isoflavones decreased CCL4 serum levels in poly(I:C)/swim-treated mice (217 ± 55 pg/ml, P = 0.0025) (Figure 2D).\nPoly(I:C) treatment also increased CCL2, CCL3, CCL5 and CXCL10 serum levels and high isoflavones reduced those increases (Additional file 3).",
"TNF-α (Figure 3A), IL-6 (Figure 3B), KC (Figure 3C), CCL4 (Figure 3D), as well as CCL2, CCL5 and CXCL10 brain gene expression were increased in the poly(I:C)-treated mice. High isoflavones reduced the increased TNF, IL6, KC, CCL4 and CCL2 brain gene expression. There was no difference in HDC and NT gene expression between the different treatment groups (Additional file 4: Table S3).Figure 3\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3\nA), IL-6 (Figure 3\nB), KC (Figure 3\nC), and CCL4 (Figure 3\nD) brain gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3\nA), IL-6 (Figure 3\nB), KC (Figure 3\nC), and CCL4 (Figure 3\nD) brain gene expression using qPCR.\nMore specifically TNF-α gene expression was increased in the brain of poly(I:C)-treated mice on low isoflavones (relative fold change of 22 ± 7 and 12 ± 9) compared to their controls (1.1 ± 0.9, P = 0.0006 and 1 ± 0.7, P = 0.0012, respectively) (Figure 3A). Although poly(I:C)-treated mice on high isoflavones had increased TNF-α brain gene expression (6 ± 1 and 9 ± 8) compared to their corresponding controls (1.2 ± 0.5, P = 0.0079 and 1 ± 0.3, P = 0.0079, respectively), high isoflavones decreased TNF-α gene expression in the brain of poly(I:C)/swim-treated mice (6 ± 1, P = 0.0025) compared to those on low isoflavones (Figure 3A). There was no effect of poly(I:C) treatment on IL-6 brain gene expression (Figure 3B), but high isoflavones still decreased IL-6 gene expression in the brain of poly(I:C)/swim-treated mice (0.5 ± 0.1, P = 0.0228) compared to those on low isoflavones (2.1 ± 2.4) (Figure 3B).\nKC gene expression in the brain of poly(I:C)-treated mice on low isoflavones was increased (18 ± 13 and 40 ± 52) compared to their controls (0.9 ± 0.3, P = 0.0006 and 1 ± 0.3, P = 0.0183, respectively) (Figure 3C). Although poly(I:C)/swim-treated mice on high isoflavones still had increased KC gene expression in their brain (2 ± 0.9) compared to control/swim-treated mice (0.6 ± 0.1, P = 0.0159), high isoflavones reduced this increase in KC gene expression (2 ± 0.9, P = 0.0177) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3C).\nLikewise, CCL4 gene expression in the brain of poly(I:C)-treated mice on low isoflavones was also increased (6.5 ± 4 and 2.9 ± 1.6) compared to their controls (1 ± 0.3, P = 0.0006 and 1 ± 0.4, P = 0.0350, respectively) (Figure 3D). Although, poly(I:C)/swim-treated mice on high isoflavones still had increased CCL4 gene expression (1.8 ± 0.4) compared to control/swim-treated mice (1 ± 0.4, P = 0.0317), high isoflavones reduced this increase in CCL4 gene expression (1.8 ± 0.4, P = 0.0101) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3D). Poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the brains of the mice. High isoflavones reduced this noted CCL2 brain gene expression increase (Additional file 3).",
"TNF-α (Figure 4A), IL-6 (Figure 4B), KC (Figure 4C), CCL4 (Figure 4D), as well as CCL2, CCL5 and CXCL10 (Additional file 5: Table S4) skin gene expression were increased in the poly(I:C)-treated mice. Moreover, HDC and NT gene expression were also increased in the skin (Figure 5A and B). High isoflavone diet reduced the increased TNF-α, IL-6 and KC skin gene expression.Figure 4\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4\nA), IL-6 (Figure 4\nB), KC (Figure 4\nC) and CCL4 (Figure 4\nD) skin gene expression using qPCR.Figure 5\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5\nA) and NT (Figure 5\nB) skin gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4\nA), IL-6 (Figure 4\nB), KC (Figure 4\nC) and CCL4 (Figure 4\nD) skin gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5\nA) and NT (Figure 5\nB) skin gene expression using qPCR.\nMore specifically, TNF-α gene expression in the skin of poly(I:C)/swim-treated mice on low isoflavones, was increased (5 ± 0.9) compared to their controls (1.1 ± 0.4, P = 0.0006) (Figure 4A) and high isoflavones decreased this increase in TNF-α gene expression (2.3 ± 1.3, P = 0.0101) (Figure 4A). Likewise, IL-6 gene expression in the skin of poly(I:C)-treated mice on low isoflavones was increased (14 ± 4 and 22 ± 16) compared to their controls (0.9 ± 0.8, P = 0.0006 and 1 ± 0.4, P = 0.0012, respectively) (Figure 4B). Although poly(I:C)-treated mice on high isoflavones still had increased IL-6 gene expression (5.5 ± 3 and 3.4 ± 1.7) compared to their controls (1.4 ± 0.7, P = 0.0079 and 0.7 ± 0.4, P = 0.0159, respectively), high isoflavones reduced this increase in the IL-6 gene expression (5.5 ± 3 P = 0.0101) (Figure 4B).\nSimilarly, KC gene expression in the skin of poly(I:C)-treated mice on low isoflavones was also increased (53 ± 55 and 69 ± 83) compared to their controls (0.3 ± 0.2, P = 0.0021 and 1 ± 1.5, P = 0.0012, respectively) (Figure 4C). High isoflavones reduced this increase in the KC gene expression of poly(I:C)-treated mice (2.8 ± 3.5, P = 0.0057 and 3.4 ± 1.9, P = 0.0043) (Figure 4C).\nCCL4 gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (24 ± 15 and 21 ± 19) compared to their controls (0.4 ± 0.3, P = 0.0021 and 1 ± 1.7, P = 0.0023, respectively) (Figure 4D). CCL4 gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (20 ± 24 and 27 ± 20) compared to their controls (0.8 ± 0.7, P = 0.0079 and 1 ± 1.3, P = 0.0079, respectively) and high isoflavones did have an effect on reducing this increase.\nIn the same way, HDC gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (4 ± 2 and 4 ± 2) compared to their controls (0.6 ± 0.4, P = 0.0003 and 1 ± 0.8, P = 0.0123, respectively) (Figure 5A). HDC gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (3 ± 1.1 and 3 ± 1) compared to their controls (0.9 ± 0.3, P = 0.0079 and 1 ± 0.2, P = 0.0317, respectively).\nSimilar to HDC gene expression, NT gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (8 ± 4 and 9 ± 10) compared to their controls (0.6 ± 0.4, P = 0.0025 and 1 ± 0.7, P = 0.0012, respectively) (Figure 5B). NT gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (8 ± 4, and 6 ± 6) compared to their controls (1.9 ± 0.8, P = 0.0317 and 1 ± 0.2, P = 0.0079, respectively).\nFinally, poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the skin of the mice. High isoflavones did not have any statistical effect on CCL4, CCL2, CCL5, CXCL10, HDC and NT skin gene expression (Additional file 5: Table S4)."
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[
"Background",
"Methods",
"Chemicals and reagents",
"Animals",
"Treatment conditions",
"Assessment of behavioral parameter-locomotor activity",
"Sample collection",
"Serum levels of inflammatory mediators",
"Quantitative PCR",
"Statistical analysis",
"Results",
"Effect of poly(I:C) and isoflavones on locomotor activity",
"Effect of poly(I:C) and isoflavones on serum inflammatory mediators",
"Effect of poly(I:C) and isoflavones on brain gene expression of inflammatory mediators",
"Effect of poly(I:C) and isoflavones on skin gene expression of inflammatory mediators",
"Discussion",
"Conclusion"
] |
[
"Chronic Fatigue Syndrome (CFS) is a complex disease, which has also been called ‘neurasthenia’, ‘post viral fatigue’, and ‘chronic mononucleosis’ [1-3]. Its prevalence may be as high as 1% in the US population [4] with a female to male ratio of 4 to 1 [5]. CFS involves the muscular, nervous, hormonal and immune systems. Patients complain of overwhelming fatigue, sleep disturbances, malaise, muscle aches, gastrointestinal symptoms, dizziness on standing, and cognitive problems [6]. Clinical and subclinical viral infections have been suspected, but never confirmed [7,8].\nCFS is often comorbid with other disorders, including fibromyalgia, Pelvic Bladder Syndrome/Interstitial Cystitis (PBS/IC), irritable bowel syndrome (IBS), and migraines [9], all of which are characterized by central nervous system (CNS) dysfunction [10], and worsened by stress [11-15]. Many CFS patients demonstrate abnormal hypothalamic-pituitary-adrenal (HPA) axis activity [16,17]. Anxiety is common in CFS [18] and patients are particularly vulnerable to stress [14]. Many CFS symptoms could derive from the possible release of inflammatory mediators that could affect brain function [19,20]. As of today, there are no FDA approved drugs for the treatment of CFS [21]; psychological, physical, and pharmacological interventions used currently are not very effective [22].\nAn abnormal immune component may be involved in CFS [23-25], but the neuroimmune and neuroendocrine interactions involved are still unknown [26]. Mast cells (MC) and their mediators have been implicated in all diseases that are comorbid with CFS [9]. There is higher number of skin MC in patients with CFS [27,28], and such patients also show increased skin hypersensitivity [29]. Furthermore, CFS also occurs more often in patients with chronic urticaria, that also involves MC [30]. In fact, there is hyperresponsiveness in the bronchi of CFS patients, implying MC activation [31].\nActivated MC release a number of chemokines and cytokines that could contribute to CFS symptoms [32,33]. MC are located perivascularly in close proximity to neurons [34], especially in the diencephalon [35,36], where functional MC-neuron interactions have been documented [36,37] in response to corticotropin-releasing hormone (CRH) [38]. In vivo activation of MC by CRH is augmented by neurotensin (NT) [39]. Moreover, NT is induced in the hypothalamus in response to bacterial lipopolysaccharide (LPS) and regulates the HPA axis [40].\nUnfortunately, there are neither effective CFS treatments nor human MC inhibitors clinically available that may also be used in CFS. Flavonoids are natural compounds with strong antioxidant and anti-inflammatory activity [41]. Certain flavonoids also inhibit MC [42] and have neuroprotective effects [41,43].\nHere we report that treatment of mice with poly(I:C) results in reduced locomotor activity and increased serum levels, as well as brain and gene expression, of inflammatory mediators, all of which are reversed by treatment with the isoflavones daidzein and genistein.",
" Chemicals and reagents Polyinosinic-polycytidylic acid-TLR3-based adjuvant, poly(I:C), HMW VacciGrade, (catalog# vac-pic) was purchased from Invivogen (San Diego, CA, USA). Substance P (SP, catalog# S6883), neurotensin (NT, catalog# N6383) and corticotropin-releasing hormone (CRF, catalog# C3042) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aliquots of the above were prepared according to the manufacturer’s instructions. Teklad lab animal diets (catalog# 2918X, and 2920X) were purchased from Harlan (Indianapolis, IN, USA).\nPolyinosinic-polycytidylic acid-TLR3-based adjuvant, poly(I:C), HMW VacciGrade, (catalog# vac-pic) was purchased from Invivogen (San Diego, CA, USA). Substance P (SP, catalog# S6883), neurotensin (NT, catalog# N6383) and corticotropin-releasing hormone (CRF, catalog# C3042) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aliquots of the above were prepared according to the manufacturer’s instructions. Teklad lab animal diets (catalog# 2918X, and 2920X) were purchased from Harlan (Indianapolis, IN, USA).\n Animals C57BL/6 female mice, nine to twelve weeks old, (Jackson Laboratories, Bar Harbor, ME, USA) were kept in virus-free sections of a modern animal facility and were allowed ad libitum access to food and water. They were maintained on a 14:10 hour light-dark cycle (the standard light-dark cycle used by the Department of Animal Health). Female mice were chosen because published reports indicate a female to male ratio of 4:1 [5], while the US Centers for Disease Control and Prevention (CDC) specify a female to male ratio of 4:1 [5]. Mice were kept in cages of five mice/cage until the day of the experiments. Both low and high isoflavone diets (2018X and 2020X) were sterile with similar ingredients other than isoflavone content. We monitored weight changes for 21 days prior to the beginning of the experiments. Only poly(I:C)/no swim-treated mice showed a slight decrease in weight change. Poly(I:C)/swim-treated mice, as well as their corresponding control mice, slightly increased their weight over the three-week observation period. However, by the end of this period, there was no statistical difference in weight change. The protocol was approved by Tufts Medical Center IACUC under number B 2011-88.\nC57BL/6 female mice, nine to twelve weeks old, (Jackson Laboratories, Bar Harbor, ME, USA) were kept in virus-free sections of a modern animal facility and were allowed ad libitum access to food and water. They were maintained on a 14:10 hour light-dark cycle (the standard light-dark cycle used by the Department of Animal Health). Female mice were chosen because published reports indicate a female to male ratio of 4:1 [5], while the US Centers for Disease Control and Prevention (CDC) specify a female to male ratio of 4:1 [5]. Mice were kept in cages of five mice/cage until the day of the experiments. Both low and high isoflavone diets (2018X and 2020X) were sterile with similar ingredients other than isoflavone content. We monitored weight changes for 21 days prior to the beginning of the experiments. Only poly(I:C)/no swim-treated mice showed a slight decrease in weight change. Poly(I:C)/swim-treated mice, as well as their corresponding control mice, slightly increased their weight over the three-week observation period. However, by the end of this period, there was no statistical difference in weight change. The protocol was approved by Tufts Medical Center IACUC under number B 2011-88.\n Treatment conditions Mice were provided with chow containing either non-detectable to low (ND-20 mg/kg, Teklad 2920X) or high (150 to 250 mg/kg, Teklad 2918X) isoflavone (daidzein plus genistein) levels for two weeks. Conditions included four groups: (a) control (normal saline intraperitoneal (ip) injection)/no swim, (b) control/swim, (c) poly(I:C)/no swim, and (d) poly(I:C)/swim (n = 5 to 7/group). Mice were injected ip with 20 mg/kg of poly(I:C) or normal saline the first day. Subsequently, they were subjected to swim for 15 minutes, individually in a transparent plastic cylindrical jar (17 cm × 25 cm) containing 15 cm-deep water at room temperature (23 ± 1°C). This approach reflects both exercise and the stress of water immersion. Mice were then placed individually into specific cages and locomotor activity was monitored overnight.\nMice were provided with chow containing either non-detectable to low (ND-20 mg/kg, Teklad 2920X) or high (150 to 250 mg/kg, Teklad 2918X) isoflavone (daidzein plus genistein) levels for two weeks. Conditions included four groups: (a) control (normal saline intraperitoneal (ip) injection)/no swim, (b) control/swim, (c) poly(I:C)/no swim, and (d) poly(I:C)/swim (n = 5 to 7/group). Mice were injected ip with 20 mg/kg of poly(I:C) or normal saline the first day. Subsequently, they were subjected to swim for 15 minutes, individually in a transparent plastic cylindrical jar (17 cm × 25 cm) containing 15 cm-deep water at room temperature (23 ± 1°C). This approach reflects both exercise and the stress of water immersion. Mice were then placed individually into specific cages and locomotor activity was monitored overnight.\n Assessment of behavioral parameter-locomotor activity After the experimental procedures, animals were placed individually into standard plastic housing cages with food and water available ad libitum and overnight locomotor activity (for a total of 16 hours) was monitored with the Neuroscience Behavior Core’s mouse SmartFrame® Cage Rack System (Kinder Scientific, Poway, CA, USA). This system consists of 20 PC-interfaced horizontal photobeam frames. The frame (containing 12 photocells; arranged on a 8 L × 4 W grid) surrounds one home cage environment and continuously tracks the animal’s movement. This fully automated system allows the user to quantify horizontal ambulation by counting breaks in infrared photocell beams using MotorMonitor® software (Hamilton-Kinder Scientific, Poway, CA, USA). Data were collected and subsequently analyzed in time bins (every hour) or as a total over the course of collection to the ‘Total Distance Travelled’ (in cm) parameter for each zone.\nAfter the experimental procedures, animals were placed individually into standard plastic housing cages with food and water available ad libitum and overnight locomotor activity (for a total of 16 hours) was monitored with the Neuroscience Behavior Core’s mouse SmartFrame® Cage Rack System (Kinder Scientific, Poway, CA, USA). This system consists of 20 PC-interfaced horizontal photobeam frames. The frame (containing 12 photocells; arranged on a 8 L × 4 W grid) surrounds one home cage environment and continuously tracks the animal’s movement. This fully automated system allows the user to quantify horizontal ambulation by counting breaks in infrared photocell beams using MotorMonitor® software (Hamilton-Kinder Scientific, Poway, CA, USA). Data were collected and subsequently analyzed in time bins (every hour) or as a total over the course of collection to the ‘Total Distance Travelled’ (in cm) parameter for each zone.\n Sample collection Mice were euthanized 24 hours post poly(I:C) ip injection using isoflurane overdose and thoracotomy. Blood was collected by cardiac puncture and was used to determine inflammatory mediator levels in the serum. Brain (diencephalon) and skin (back shaved with a electric shaver the day before) samples were collected and immersed into RNAlater (catalog# AM7021) purchased from Invitrogen (Grand Island, NY, USA). Samples were stored at -80°C.\nMice were euthanized 24 hours post poly(I:C) ip injection using isoflurane overdose and thoracotomy. Blood was collected by cardiac puncture and was used to determine inflammatory mediator levels in the serum. Brain (diencephalon) and skin (back shaved with a electric shaver the day before) samples were collected and immersed into RNAlater (catalog# AM7021) purchased from Invitrogen (Grand Island, NY, USA). Samples were stored at -80°C.\n Serum levels of inflammatory mediators TNF-α, VEGFα, IL-1α, IL-1β, IL-4, IL-6, KC (IL-8/(CXC motif) ligand (CXC)L8 murine homolog, IL-9, IL-10, IL-12p70, IL-17, (CC motif) ligand (CCL)2, CCL3, CCL4, CCL5, CXCL10 and IFNγ mouse serum levels were determined using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Assay (MCYTOMAG-70 K custom made panel for the specific analytes mentioned above). Measurements were performed blindly by Millipore (St. Charles, MI, USA). Millipore's minimum assay reporting range for TNF-α and IL-6 levels was 32 pg/ml, for CCL2 was 800 pg/ml, while for CCL3 and CCL4 it was 160 pg/ml. Therefore, we consider all values < × pg/ml as equal to x pg/ml.\nTNF-α, VEGFα, IL-1α, IL-1β, IL-4, IL-6, KC (IL-8/(CXC motif) ligand (CXC)L8 murine homolog, IL-9, IL-10, IL-12p70, IL-17, (CC motif) ligand (CCL)2, CCL3, CCL4, CCL5, CXCL10 and IFNγ mouse serum levels were determined using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Assay (MCYTOMAG-70 K custom made panel for the specific analytes mentioned above). Measurements were performed blindly by Millipore (St. Charles, MI, USA). Millipore's minimum assay reporting range for TNF-α and IL-6 levels was 32 pg/ml, for CCL2 was 800 pg/ml, while for CCL3 and CCL4 it was 160 pg/ml. Therefore, we consider all values < × pg/ml as equal to x pg/ml.\n Quantitative PCR Total RNA from mouse tissues was extracted using RNeasy Plus Mini kit (catalog# 74134) and RNeasy Fibrous Tissue Mini Kit (catalog# 74704), purchased from QIAGEN (Valencia, CA, USA). Reverse transcription was performed with 300 ng of total RNA using the iScript cDNA synthesis kit (catalog# 170-8891) purchased from Bio-Rad (Hercules, CA, USA). Real-time quantitative polymerase chain reaction (qPCR) was carried out in a 7300 Sequence Detector, according to TaqMan Gene Expression Assay instructions from Life Technologies, Applied Biosystems (Grand Island, NY, USA) using Taqman primer/probe sets (Additional file 1: Table S1). Samples were analyzed for Tnf (TNF-α), Il4, Il6 (IL-6), KC (Cxcl1, IL-8 homolog in the mouse), Ccl2 (CCL2), Ccl4 (CCL4), Ccl5 (CCL5), Cxcl10 (CXCL10), histidine decarboxylase-Hdc (HDC), and Nts (NT) gene expression using Gapdh as internal control.Thermal cycling proceeded at 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, for 40 cycles, and 60°C for 1 minute. Negative controls included samples with water instead of template. Assays were performed in triplicate for each data point. Results were normalized against the endogenous gene GAPDH and were expressed relative to the mean of the control for each gene (relative fold change).\nTotal RNA from mouse tissues was extracted using RNeasy Plus Mini kit (catalog# 74134) and RNeasy Fibrous Tissue Mini Kit (catalog# 74704), purchased from QIAGEN (Valencia, CA, USA). Reverse transcription was performed with 300 ng of total RNA using the iScript cDNA synthesis kit (catalog# 170-8891) purchased from Bio-Rad (Hercules, CA, USA). Real-time quantitative polymerase chain reaction (qPCR) was carried out in a 7300 Sequence Detector, according to TaqMan Gene Expression Assay instructions from Life Technologies, Applied Biosystems (Grand Island, NY, USA) using Taqman primer/probe sets (Additional file 1: Table S1). Samples were analyzed for Tnf (TNF-α), Il4, Il6 (IL-6), KC (Cxcl1, IL-8 homolog in the mouse), Ccl2 (CCL2), Ccl4 (CCL4), Ccl5 (CCL5), Cxcl10 (CXCL10), histidine decarboxylase-Hdc (HDC), and Nts (NT) gene expression using Gapdh as internal control.Thermal cycling proceeded at 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, for 40 cycles, and 60°C for 1 minute. Negative controls included samples with water instead of template. Assays were performed in triplicate for each data point. Results were normalized against the endogenous gene GAPDH and were expressed relative to the mean of the control for each gene (relative fold change).\n Statistical analysis Data were analyzed using the non-parametric Mann-Whitney U-test. Results are presented as mean ± SE. Statistical significance is defined as P < 0.05. We analyzed separately the different treatment subgroups within each dietary group, using Mann-Whitney U-test to compare poly(I:C)/no swim and poly(I:C)/swim-treated mice with the control/no swim and control/swim-treated groups, accordingly. We then analyzed the same treatment subgroups between the two dietary groups, using Mann-Whitney U-test to compare, for example, poly(I:C)/swim-treated mice with low isoflavone diet to poly(I:C)/swim-treated mice that were provided with high isoflavone diet. Due to space limitations, we submitted figures composed of all the experimental conditions from the two different diets and all the statistical significant results were marked in these figures.\nData were analyzed using the non-parametric Mann-Whitney U-test. Results are presented as mean ± SE. Statistical significance is defined as P < 0.05. We analyzed separately the different treatment subgroups within each dietary group, using Mann-Whitney U-test to compare poly(I:C)/no swim and poly(I:C)/swim-treated mice with the control/no swim and control/swim-treated groups, accordingly. We then analyzed the same treatment subgroups between the two dietary groups, using Mann-Whitney U-test to compare, for example, poly(I:C)/swim-treated mice with low isoflavone diet to poly(I:C)/swim-treated mice that were provided with high isoflavone diet. Due to space limitations, we submitted figures composed of all the experimental conditions from the two different diets and all the statistical significant results were marked in these figures.",
"Polyinosinic-polycytidylic acid-TLR3-based adjuvant, poly(I:C), HMW VacciGrade, (catalog# vac-pic) was purchased from Invivogen (San Diego, CA, USA). Substance P (SP, catalog# S6883), neurotensin (NT, catalog# N6383) and corticotropin-releasing hormone (CRF, catalog# C3042) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aliquots of the above were prepared according to the manufacturer’s instructions. Teklad lab animal diets (catalog# 2918X, and 2920X) were purchased from Harlan (Indianapolis, IN, USA).",
"C57BL/6 female mice, nine to twelve weeks old, (Jackson Laboratories, Bar Harbor, ME, USA) were kept in virus-free sections of a modern animal facility and were allowed ad libitum access to food and water. They were maintained on a 14:10 hour light-dark cycle (the standard light-dark cycle used by the Department of Animal Health). Female mice were chosen because published reports indicate a female to male ratio of 4:1 [5], while the US Centers for Disease Control and Prevention (CDC) specify a female to male ratio of 4:1 [5]. Mice were kept in cages of five mice/cage until the day of the experiments. Both low and high isoflavone diets (2018X and 2020X) were sterile with similar ingredients other than isoflavone content. We monitored weight changes for 21 days prior to the beginning of the experiments. Only poly(I:C)/no swim-treated mice showed a slight decrease in weight change. Poly(I:C)/swim-treated mice, as well as their corresponding control mice, slightly increased their weight over the three-week observation period. However, by the end of this period, there was no statistical difference in weight change. The protocol was approved by Tufts Medical Center IACUC under number B 2011-88.",
"Mice were provided with chow containing either non-detectable to low (ND-20 mg/kg, Teklad 2920X) or high (150 to 250 mg/kg, Teklad 2918X) isoflavone (daidzein plus genistein) levels for two weeks. Conditions included four groups: (a) control (normal saline intraperitoneal (ip) injection)/no swim, (b) control/swim, (c) poly(I:C)/no swim, and (d) poly(I:C)/swim (n = 5 to 7/group). Mice were injected ip with 20 mg/kg of poly(I:C) or normal saline the first day. Subsequently, they were subjected to swim for 15 minutes, individually in a transparent plastic cylindrical jar (17 cm × 25 cm) containing 15 cm-deep water at room temperature (23 ± 1°C). This approach reflects both exercise and the stress of water immersion. Mice were then placed individually into specific cages and locomotor activity was monitored overnight.",
"After the experimental procedures, animals were placed individually into standard plastic housing cages with food and water available ad libitum and overnight locomotor activity (for a total of 16 hours) was monitored with the Neuroscience Behavior Core’s mouse SmartFrame® Cage Rack System (Kinder Scientific, Poway, CA, USA). This system consists of 20 PC-interfaced horizontal photobeam frames. The frame (containing 12 photocells; arranged on a 8 L × 4 W grid) surrounds one home cage environment and continuously tracks the animal’s movement. This fully automated system allows the user to quantify horizontal ambulation by counting breaks in infrared photocell beams using MotorMonitor® software (Hamilton-Kinder Scientific, Poway, CA, USA). Data were collected and subsequently analyzed in time bins (every hour) or as a total over the course of collection to the ‘Total Distance Travelled’ (in cm) parameter for each zone.",
"Mice were euthanized 24 hours post poly(I:C) ip injection using isoflurane overdose and thoracotomy. Blood was collected by cardiac puncture and was used to determine inflammatory mediator levels in the serum. Brain (diencephalon) and skin (back shaved with a electric shaver the day before) samples were collected and immersed into RNAlater (catalog# AM7021) purchased from Invitrogen (Grand Island, NY, USA). Samples were stored at -80°C.",
"TNF-α, VEGFα, IL-1α, IL-1β, IL-4, IL-6, KC (IL-8/(CXC motif) ligand (CXC)L8 murine homolog, IL-9, IL-10, IL-12p70, IL-17, (CC motif) ligand (CCL)2, CCL3, CCL4, CCL5, CXCL10 and IFNγ mouse serum levels were determined using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Assay (MCYTOMAG-70 K custom made panel for the specific analytes mentioned above). Measurements were performed blindly by Millipore (St. Charles, MI, USA). Millipore's minimum assay reporting range for TNF-α and IL-6 levels was 32 pg/ml, for CCL2 was 800 pg/ml, while for CCL3 and CCL4 it was 160 pg/ml. Therefore, we consider all values < × pg/ml as equal to x pg/ml.",
"Total RNA from mouse tissues was extracted using RNeasy Plus Mini kit (catalog# 74134) and RNeasy Fibrous Tissue Mini Kit (catalog# 74704), purchased from QIAGEN (Valencia, CA, USA). Reverse transcription was performed with 300 ng of total RNA using the iScript cDNA synthesis kit (catalog# 170-8891) purchased from Bio-Rad (Hercules, CA, USA). Real-time quantitative polymerase chain reaction (qPCR) was carried out in a 7300 Sequence Detector, according to TaqMan Gene Expression Assay instructions from Life Technologies, Applied Biosystems (Grand Island, NY, USA) using Taqman primer/probe sets (Additional file 1: Table S1). Samples were analyzed for Tnf (TNF-α), Il4, Il6 (IL-6), KC (Cxcl1, IL-8 homolog in the mouse), Ccl2 (CCL2), Ccl4 (CCL4), Ccl5 (CCL5), Cxcl10 (CXCL10), histidine decarboxylase-Hdc (HDC), and Nts (NT) gene expression using Gapdh as internal control.Thermal cycling proceeded at 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, for 40 cycles, and 60°C for 1 minute. Negative controls included samples with water instead of template. Assays were performed in triplicate for each data point. Results were normalized against the endogenous gene GAPDH and were expressed relative to the mean of the control for each gene (relative fold change).",
"Data were analyzed using the non-parametric Mann-Whitney U-test. Results are presented as mean ± SE. Statistical significance is defined as P < 0.05. We analyzed separately the different treatment subgroups within each dietary group, using Mann-Whitney U-test to compare poly(I:C)/no swim and poly(I:C)/swim-treated mice with the control/no swim and control/swim-treated groups, accordingly. We then analyzed the same treatment subgroups between the two dietary groups, using Mann-Whitney U-test to compare, for example, poly(I:C)/swim-treated mice with low isoflavone diet to poly(I:C)/swim-treated mice that were provided with high isoflavone diet. Due to space limitations, we submitted figures composed of all the experimental conditions from the two different diets and all the statistical significant results were marked in these figures.",
" Effect of poly(I:C) and isoflavones on locomotor activity Poly(I:C)/swim and poly(I:C)/no swim-treated mice on low isoflavones had reduced maximum (max) locomotor activity (over the 10-hour night period), denoted by the dark bar (P = 0.008 and P = 0.036) compared to the control/swim and control/no swim-treated mice, respectively (Figure 1A). High isoflavone chow reversed this decrease (Figure 1B), and the difference between the two diets was statistically significant (P = 0.032).Figure 1\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1\nA) or high (Figure 1\nB) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1\nA) or high (Figure 1\nB) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.\nPoly(I:C)/swim and poly(I:C)/no swim-treated mice on low isoflavones had reduced maximum (max) locomotor activity (over the 10-hour night period), denoted by the dark bar (P = 0.008 and P = 0.036) compared to the control/swim and control/no swim-treated mice, respectively (Figure 1A). High isoflavone chow reversed this decrease (Figure 1B), and the difference between the two diets was statistically significant (P = 0.032).Figure 1\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1\nA) or high (Figure 1\nB) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1\nA) or high (Figure 1\nB) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.\n Effect of poly(I:C) and isoflavones on serum inflammatory mediators Poly(I:C)-treated mice on low isoflavones had increased serum levels of TNF-α (Figure 2A), IL-6 (Figure 2B), KC (Figure 2C), and CCL4 (Figure 2D), as well as CCL2, CCL3, CCL5, and CXCL10 (Additional file 2: Table S2). High isoflavones reduced the poly(I:C)-increased serum levels of all the inflammatory markers. All IFNγ, IL-1β, IL-4, IL-9, IL-10, IL-12p70, IL-17 and VEGFα serum levels were below the detection limit, while IL-1α serum levels were similar between the different treatment groups. Swim stress augmented the poly(I:C) effect on TNF-α, CCL4, and CCL5 serum levels.Figure 2\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2\nA), IL-6 (Figure 2\nB), KC (Figure 2\nC), and CCL4 (Figure 2\nD) using the Milliplex microbead assay.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2\nA), IL-6 (Figure 2\nB), KC (Figure 2\nC), and CCL4 (Figure 2\nD) using the Milliplex microbead assay.\nMore specifically, TNF-α serum levels in poly(I:C)/swim-treated mice on low isoflavones were increased (55 ± 14 pg/ml) compared to control/swim-treated mice (32 ± 0 pg/ml, P = 0.0011) and poly(I:C)/no swim-treated mice (37 ± 10 pg/ml,P = 0.0221). High isoflavones decreased these high TNF-α serum levels (32 ± 0 pg/ml, P = 0.0042) (Figure 2A). IL-6 serum levels in poly(I:C)-swim and poly(I:C)/no swim-treated mice on low isoflavones were also increased (229 ± 110 pg/ml and 141 ± 11 pg/ml) compared to the corresponding control/swim and control/no swim mice on low isoflavones (32 ± 0 pg/ml, P = 0.0011 and 32 ± 0 pg/ml, P = 0.0114) (Figure 2B). High isoflavones reduced IL-6 serum levels of poly(I:C)/swim-treated mice (35 ± 5 pg/ml, P = 0.0054) and poly(I:C)/no swim-treated (32 ± 0 pg/ml, P = 0.0298) compared to the corresponding groups on low isoflavones (Figure 2B).\nSimilarly, KC serum levels in poly(I:C)-treated mice on low isoflavones were increased (735 ± 579 pg/ml and 626 ± 523 pg/ml) compared to the corresponding controls (173 ± 43 pg/ml, P = 0.0379 and 132 ± 52 pg/ml, P = 0.0006, respectively) (Figure 2C). Although KC serum levels in poly(I:C)/swim-treated mice on high isoflavones were also increased (451 ± 107 pg/ml) compared to control poly(I:C)/swim-treated mice (100 ± 45 pg/ml, P = 0.0079), high isoflavone diet decreased KC serum levels in poly(I:C)/no swim-treated mice (128 ± 43 pg/ml, P = 0.0025) compared to those on low isoflavones (Figure 2C).\nMoreover, CCL4 serum levels were increased in poly(I:C)-treated mice on low isoflavones (1,256 ± 556 pg/ml and 395 ± 245 pg/ml) compared to the corresponding controls (160 ± 0 pg/ml, P = 0.0011 and 160 ± 0 pg/ml, P = 0.0037, respectively) (Figure 2D). Swim further increased CCL4 serum levels (1,256 ± 556 pg/ml, P = 0.0023). Although CCL4 serum levels in poly(I:C)-treated mice on high isoflavones were also increased (217 ± 55 pg/ml and 265 ± 111 pg/ml) compared to their controls (160 ± 0 pg/ml, P = 0.0075 and 160 ± 0 pg/ml, P = 0.0254, respectively)., high isoflavones decreased CCL4 serum levels in poly(I:C)/swim-treated mice (217 ± 55 pg/ml, P = 0.0025) (Figure 2D).\nPoly(I:C) treatment also increased CCL2, CCL3, CCL5 and CXCL10 serum levels and high isoflavones reduced those increases (Additional file 3).\nPoly(I:C)-treated mice on low isoflavones had increased serum levels of TNF-α (Figure 2A), IL-6 (Figure 2B), KC (Figure 2C), and CCL4 (Figure 2D), as well as CCL2, CCL3, CCL5, and CXCL10 (Additional file 2: Table S2). High isoflavones reduced the poly(I:C)-increased serum levels of all the inflammatory markers. All IFNγ, IL-1β, IL-4, IL-9, IL-10, IL-12p70, IL-17 and VEGFα serum levels were below the detection limit, while IL-1α serum levels were similar between the different treatment groups. Swim stress augmented the poly(I:C) effect on TNF-α, CCL4, and CCL5 serum levels.Figure 2\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2\nA), IL-6 (Figure 2\nB), KC (Figure 2\nC), and CCL4 (Figure 2\nD) using the Milliplex microbead assay.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2\nA), IL-6 (Figure 2\nB), KC (Figure 2\nC), and CCL4 (Figure 2\nD) using the Milliplex microbead assay.\nMore specifically, TNF-α serum levels in poly(I:C)/swim-treated mice on low isoflavones were increased (55 ± 14 pg/ml) compared to control/swim-treated mice (32 ± 0 pg/ml, P = 0.0011) and poly(I:C)/no swim-treated mice (37 ± 10 pg/ml,P = 0.0221). High isoflavones decreased these high TNF-α serum levels (32 ± 0 pg/ml, P = 0.0042) (Figure 2A). IL-6 serum levels in poly(I:C)-swim and poly(I:C)/no swim-treated mice on low isoflavones were also increased (229 ± 110 pg/ml and 141 ± 11 pg/ml) compared to the corresponding control/swim and control/no swim mice on low isoflavones (32 ± 0 pg/ml, P = 0.0011 and 32 ± 0 pg/ml, P = 0.0114) (Figure 2B). High isoflavones reduced IL-6 serum levels of poly(I:C)/swim-treated mice (35 ± 5 pg/ml, P = 0.0054) and poly(I:C)/no swim-treated (32 ± 0 pg/ml, P = 0.0298) compared to the corresponding groups on low isoflavones (Figure 2B).\nSimilarly, KC serum levels in poly(I:C)-treated mice on low isoflavones were increased (735 ± 579 pg/ml and 626 ± 523 pg/ml) compared to the corresponding controls (173 ± 43 pg/ml, P = 0.0379 and 132 ± 52 pg/ml, P = 0.0006, respectively) (Figure 2C). Although KC serum levels in poly(I:C)/swim-treated mice on high isoflavones were also increased (451 ± 107 pg/ml) compared to control poly(I:C)/swim-treated mice (100 ± 45 pg/ml, P = 0.0079), high isoflavone diet decreased KC serum levels in poly(I:C)/no swim-treated mice (128 ± 43 pg/ml, P = 0.0025) compared to those on low isoflavones (Figure 2C).\nMoreover, CCL4 serum levels were increased in poly(I:C)-treated mice on low isoflavones (1,256 ± 556 pg/ml and 395 ± 245 pg/ml) compared to the corresponding controls (160 ± 0 pg/ml, P = 0.0011 and 160 ± 0 pg/ml, P = 0.0037, respectively) (Figure 2D). Swim further increased CCL4 serum levels (1,256 ± 556 pg/ml, P = 0.0023). Although CCL4 serum levels in poly(I:C)-treated mice on high isoflavones were also increased (217 ± 55 pg/ml and 265 ± 111 pg/ml) compared to their controls (160 ± 0 pg/ml, P = 0.0075 and 160 ± 0 pg/ml, P = 0.0254, respectively)., high isoflavones decreased CCL4 serum levels in poly(I:C)/swim-treated mice (217 ± 55 pg/ml, P = 0.0025) (Figure 2D).\nPoly(I:C) treatment also increased CCL2, CCL3, CCL5 and CXCL10 serum levels and high isoflavones reduced those increases (Additional file 3).\n Effect of poly(I:C) and isoflavones on brain gene expression of inflammatory mediators TNF-α (Figure 3A), IL-6 (Figure 3B), KC (Figure 3C), CCL4 (Figure 3D), as well as CCL2, CCL5 and CXCL10 brain gene expression were increased in the poly(I:C)-treated mice. High isoflavones reduced the increased TNF, IL6, KC, CCL4 and CCL2 brain gene expression. There was no difference in HDC and NT gene expression between the different treatment groups (Additional file 4: Table S3).Figure 3\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3\nA), IL-6 (Figure 3\nB), KC (Figure 3\nC), and CCL4 (Figure 3\nD) brain gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3\nA), IL-6 (Figure 3\nB), KC (Figure 3\nC), and CCL4 (Figure 3\nD) brain gene expression using qPCR.\nMore specifically TNF-α gene expression was increased in the brain of poly(I:C)-treated mice on low isoflavones (relative fold change of 22 ± 7 and 12 ± 9) compared to their controls (1.1 ± 0.9, P = 0.0006 and 1 ± 0.7, P = 0.0012, respectively) (Figure 3A). Although poly(I:C)-treated mice on high isoflavones had increased TNF-α brain gene expression (6 ± 1 and 9 ± 8) compared to their corresponding controls (1.2 ± 0.5, P = 0.0079 and 1 ± 0.3, P = 0.0079, respectively), high isoflavones decreased TNF-α gene expression in the brain of poly(I:C)/swim-treated mice (6 ± 1, P = 0.0025) compared to those on low isoflavones (Figure 3A). There was no effect of poly(I:C) treatment on IL-6 brain gene expression (Figure 3B), but high isoflavones still decreased IL-6 gene expression in the brain of poly(I:C)/swim-treated mice (0.5 ± 0.1, P = 0.0228) compared to those on low isoflavones (2.1 ± 2.4) (Figure 3B).\nKC gene expression in the brain of poly(I:C)-treated mice on low isoflavones was increased (18 ± 13 and 40 ± 52) compared to their controls (0.9 ± 0.3, P = 0.0006 and 1 ± 0.3, P = 0.0183, respectively) (Figure 3C). Although poly(I:C)/swim-treated mice on high isoflavones still had increased KC gene expression in their brain (2 ± 0.9) compared to control/swim-treated mice (0.6 ± 0.1, P = 0.0159), high isoflavones reduced this increase in KC gene expression (2 ± 0.9, P = 0.0177) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3C).\nLikewise, CCL4 gene expression in the brain of poly(I:C)-treated mice on low isoflavones was also increased (6.5 ± 4 and 2.9 ± 1.6) compared to their controls (1 ± 0.3, P = 0.0006 and 1 ± 0.4, P = 0.0350, respectively) (Figure 3D). Although, poly(I:C)/swim-treated mice on high isoflavones still had increased CCL4 gene expression (1.8 ± 0.4) compared to control/swim-treated mice (1 ± 0.4, P = 0.0317), high isoflavones reduced this increase in CCL4 gene expression (1.8 ± 0.4, P = 0.0101) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3D). Poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the brains of the mice. High isoflavones reduced this noted CCL2 brain gene expression increase (Additional file 3).\nTNF-α (Figure 3A), IL-6 (Figure 3B), KC (Figure 3C), CCL4 (Figure 3D), as well as CCL2, CCL5 and CXCL10 brain gene expression were increased in the poly(I:C)-treated mice. High isoflavones reduced the increased TNF, IL6, KC, CCL4 and CCL2 brain gene expression. There was no difference in HDC and NT gene expression between the different treatment groups (Additional file 4: Table S3).Figure 3\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3\nA), IL-6 (Figure 3\nB), KC (Figure 3\nC), and CCL4 (Figure 3\nD) brain gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3\nA), IL-6 (Figure 3\nB), KC (Figure 3\nC), and CCL4 (Figure 3\nD) brain gene expression using qPCR.\nMore specifically TNF-α gene expression was increased in the brain of poly(I:C)-treated mice on low isoflavones (relative fold change of 22 ± 7 and 12 ± 9) compared to their controls (1.1 ± 0.9, P = 0.0006 and 1 ± 0.7, P = 0.0012, respectively) (Figure 3A). Although poly(I:C)-treated mice on high isoflavones had increased TNF-α brain gene expression (6 ± 1 and 9 ± 8) compared to their corresponding controls (1.2 ± 0.5, P = 0.0079 and 1 ± 0.3, P = 0.0079, respectively), high isoflavones decreased TNF-α gene expression in the brain of poly(I:C)/swim-treated mice (6 ± 1, P = 0.0025) compared to those on low isoflavones (Figure 3A). There was no effect of poly(I:C) treatment on IL-6 brain gene expression (Figure 3B), but high isoflavones still decreased IL-6 gene expression in the brain of poly(I:C)/swim-treated mice (0.5 ± 0.1, P = 0.0228) compared to those on low isoflavones (2.1 ± 2.4) (Figure 3B).\nKC gene expression in the brain of poly(I:C)-treated mice on low isoflavones was increased (18 ± 13 and 40 ± 52) compared to their controls (0.9 ± 0.3, P = 0.0006 and 1 ± 0.3, P = 0.0183, respectively) (Figure 3C). Although poly(I:C)/swim-treated mice on high isoflavones still had increased KC gene expression in their brain (2 ± 0.9) compared to control/swim-treated mice (0.6 ± 0.1, P = 0.0159), high isoflavones reduced this increase in KC gene expression (2 ± 0.9, P = 0.0177) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3C).\nLikewise, CCL4 gene expression in the brain of poly(I:C)-treated mice on low isoflavones was also increased (6.5 ± 4 and 2.9 ± 1.6) compared to their controls (1 ± 0.3, P = 0.0006 and 1 ± 0.4, P = 0.0350, respectively) (Figure 3D). Although, poly(I:C)/swim-treated mice on high isoflavones still had increased CCL4 gene expression (1.8 ± 0.4) compared to control/swim-treated mice (1 ± 0.4, P = 0.0317), high isoflavones reduced this increase in CCL4 gene expression (1.8 ± 0.4, P = 0.0101) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3D). Poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the brains of the mice. High isoflavones reduced this noted CCL2 brain gene expression increase (Additional file 3).\n Effect of poly(I:C) and isoflavones on skin gene expression of inflammatory mediators TNF-α (Figure 4A), IL-6 (Figure 4B), KC (Figure 4C), CCL4 (Figure 4D), as well as CCL2, CCL5 and CXCL10 (Additional file 5: Table S4) skin gene expression were increased in the poly(I:C)-treated mice. Moreover, HDC and NT gene expression were also increased in the skin (Figure 5A and B). High isoflavone diet reduced the increased TNF-α, IL-6 and KC skin gene expression.Figure 4\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4\nA), IL-6 (Figure 4\nB), KC (Figure 4\nC) and CCL4 (Figure 4\nD) skin gene expression using qPCR.Figure 5\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5\nA) and NT (Figure 5\nB) skin gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4\nA), IL-6 (Figure 4\nB), KC (Figure 4\nC) and CCL4 (Figure 4\nD) skin gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5\nA) and NT (Figure 5\nB) skin gene expression using qPCR.\nMore specifically, TNF-α gene expression in the skin of poly(I:C)/swim-treated mice on low isoflavones, was increased (5 ± 0.9) compared to their controls (1.1 ± 0.4, P = 0.0006) (Figure 4A) and high isoflavones decreased this increase in TNF-α gene expression (2.3 ± 1.3, P = 0.0101) (Figure 4A). Likewise, IL-6 gene expression in the skin of poly(I:C)-treated mice on low isoflavones was increased (14 ± 4 and 22 ± 16) compared to their controls (0.9 ± 0.8, P = 0.0006 and 1 ± 0.4, P = 0.0012, respectively) (Figure 4B). Although poly(I:C)-treated mice on high isoflavones still had increased IL-6 gene expression (5.5 ± 3 and 3.4 ± 1.7) compared to their controls (1.4 ± 0.7, P = 0.0079 and 0.7 ± 0.4, P = 0.0159, respectively), high isoflavones reduced this increase in the IL-6 gene expression (5.5 ± 3 P = 0.0101) (Figure 4B).\nSimilarly, KC gene expression in the skin of poly(I:C)-treated mice on low isoflavones was also increased (53 ± 55 and 69 ± 83) compared to their controls (0.3 ± 0.2, P = 0.0021 and 1 ± 1.5, P = 0.0012, respectively) (Figure 4C). High isoflavones reduced this increase in the KC gene expression of poly(I:C)-treated mice (2.8 ± 3.5, P = 0.0057 and 3.4 ± 1.9, P = 0.0043) (Figure 4C).\nCCL4 gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (24 ± 15 and 21 ± 19) compared to their controls (0.4 ± 0.3, P = 0.0021 and 1 ± 1.7, P = 0.0023, respectively) (Figure 4D). CCL4 gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (20 ± 24 and 27 ± 20) compared to their controls (0.8 ± 0.7, P = 0.0079 and 1 ± 1.3, P = 0.0079, respectively) and high isoflavones did have an effect on reducing this increase.\nIn the same way, HDC gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (4 ± 2 and 4 ± 2) compared to their controls (0.6 ± 0.4, P = 0.0003 and 1 ± 0.8, P = 0.0123, respectively) (Figure 5A). HDC gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (3 ± 1.1 and 3 ± 1) compared to their controls (0.9 ± 0.3, P = 0.0079 and 1 ± 0.2, P = 0.0317, respectively).\nSimilar to HDC gene expression, NT gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (8 ± 4 and 9 ± 10) compared to their controls (0.6 ± 0.4, P = 0.0025 and 1 ± 0.7, P = 0.0012, respectively) (Figure 5B). NT gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (8 ± 4, and 6 ± 6) compared to their controls (1.9 ± 0.8, P = 0.0317 and 1 ± 0.2, P = 0.0079, respectively).\nFinally, poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the skin of the mice. High isoflavones did not have any statistical effect on CCL4, CCL2, CCL5, CXCL10, HDC and NT skin gene expression (Additional file 5: Table S4).\nTNF-α (Figure 4A), IL-6 (Figure 4B), KC (Figure 4C), CCL4 (Figure 4D), as well as CCL2, CCL5 and CXCL10 (Additional file 5: Table S4) skin gene expression were increased in the poly(I:C)-treated mice. Moreover, HDC and NT gene expression were also increased in the skin (Figure 5A and B). High isoflavone diet reduced the increased TNF-α, IL-6 and KC skin gene expression.Figure 4\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4\nA), IL-6 (Figure 4\nB), KC (Figure 4\nC) and CCL4 (Figure 4\nD) skin gene expression using qPCR.Figure 5\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5\nA) and NT (Figure 5\nB) skin gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4\nA), IL-6 (Figure 4\nB), KC (Figure 4\nC) and CCL4 (Figure 4\nD) skin gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5\nA) and NT (Figure 5\nB) skin gene expression using qPCR.\nMore specifically, TNF-α gene expression in the skin of poly(I:C)/swim-treated mice on low isoflavones, was increased (5 ± 0.9) compared to their controls (1.1 ± 0.4, P = 0.0006) (Figure 4A) and high isoflavones decreased this increase in TNF-α gene expression (2.3 ± 1.3, P = 0.0101) (Figure 4A). Likewise, IL-6 gene expression in the skin of poly(I:C)-treated mice on low isoflavones was increased (14 ± 4 and 22 ± 16) compared to their controls (0.9 ± 0.8, P = 0.0006 and 1 ± 0.4, P = 0.0012, respectively) (Figure 4B). Although poly(I:C)-treated mice on high isoflavones still had increased IL-6 gene expression (5.5 ± 3 and 3.4 ± 1.7) compared to their controls (1.4 ± 0.7, P = 0.0079 and 0.7 ± 0.4, P = 0.0159, respectively), high isoflavones reduced this increase in the IL-6 gene expression (5.5 ± 3 P = 0.0101) (Figure 4B).\nSimilarly, KC gene expression in the skin of poly(I:C)-treated mice on low isoflavones was also increased (53 ± 55 and 69 ± 83) compared to their controls (0.3 ± 0.2, P = 0.0021 and 1 ± 1.5, P = 0.0012, respectively) (Figure 4C). High isoflavones reduced this increase in the KC gene expression of poly(I:C)-treated mice (2.8 ± 3.5, P = 0.0057 and 3.4 ± 1.9, P = 0.0043) (Figure 4C).\nCCL4 gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (24 ± 15 and 21 ± 19) compared to their controls (0.4 ± 0.3, P = 0.0021 and 1 ± 1.7, P = 0.0023, respectively) (Figure 4D). CCL4 gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (20 ± 24 and 27 ± 20) compared to their controls (0.8 ± 0.7, P = 0.0079 and 1 ± 1.3, P = 0.0079, respectively) and high isoflavones did have an effect on reducing this increase.\nIn the same way, HDC gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (4 ± 2 and 4 ± 2) compared to their controls (0.6 ± 0.4, P = 0.0003 and 1 ± 0.8, P = 0.0123, respectively) (Figure 5A). HDC gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (3 ± 1.1 and 3 ± 1) compared to their controls (0.9 ± 0.3, P = 0.0079 and 1 ± 0.2, P = 0.0317, respectively).\nSimilar to HDC gene expression, NT gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (8 ± 4 and 9 ± 10) compared to their controls (0.6 ± 0.4, P = 0.0025 and 1 ± 0.7, P = 0.0012, respectively) (Figure 5B). NT gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (8 ± 4, and 6 ± 6) compared to their controls (1.9 ± 0.8, P = 0.0317 and 1 ± 0.2, P = 0.0079, respectively).\nFinally, poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the skin of the mice. High isoflavones did not have any statistical effect on CCL4, CCL2, CCL5, CXCL10, HDC and NT skin gene expression (Additional file 5: Table S4).",
"Poly(I:C)/swim and poly(I:C)/no swim-treated mice on low isoflavones had reduced maximum (max) locomotor activity (over the 10-hour night period), denoted by the dark bar (P = 0.008 and P = 0.036) compared to the control/swim and control/no swim-treated mice, respectively (Figure 1A). High isoflavone chow reversed this decrease (Figure 1B), and the difference between the two diets was statistically significant (P = 0.032).Figure 1\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1\nA) or high (Figure 1\nB) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1\nA) or high (Figure 1\nB) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.",
"Poly(I:C)-treated mice on low isoflavones had increased serum levels of TNF-α (Figure 2A), IL-6 (Figure 2B), KC (Figure 2C), and CCL4 (Figure 2D), as well as CCL2, CCL3, CCL5, and CXCL10 (Additional file 2: Table S2). High isoflavones reduced the poly(I:C)-increased serum levels of all the inflammatory markers. All IFNγ, IL-1β, IL-4, IL-9, IL-10, IL-12p70, IL-17 and VEGFα serum levels were below the detection limit, while IL-1α serum levels were similar between the different treatment groups. Swim stress augmented the poly(I:C) effect on TNF-α, CCL4, and CCL5 serum levels.Figure 2\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2\nA), IL-6 (Figure 2\nB), KC (Figure 2\nC), and CCL4 (Figure 2\nD) using the Milliplex microbead assay.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2\nA), IL-6 (Figure 2\nB), KC (Figure 2\nC), and CCL4 (Figure 2\nD) using the Milliplex microbead assay.\nMore specifically, TNF-α serum levels in poly(I:C)/swim-treated mice on low isoflavones were increased (55 ± 14 pg/ml) compared to control/swim-treated mice (32 ± 0 pg/ml, P = 0.0011) and poly(I:C)/no swim-treated mice (37 ± 10 pg/ml,P = 0.0221). High isoflavones decreased these high TNF-α serum levels (32 ± 0 pg/ml, P = 0.0042) (Figure 2A). IL-6 serum levels in poly(I:C)-swim and poly(I:C)/no swim-treated mice on low isoflavones were also increased (229 ± 110 pg/ml and 141 ± 11 pg/ml) compared to the corresponding control/swim and control/no swim mice on low isoflavones (32 ± 0 pg/ml, P = 0.0011 and 32 ± 0 pg/ml, P = 0.0114) (Figure 2B). High isoflavones reduced IL-6 serum levels of poly(I:C)/swim-treated mice (35 ± 5 pg/ml, P = 0.0054) and poly(I:C)/no swim-treated (32 ± 0 pg/ml, P = 0.0298) compared to the corresponding groups on low isoflavones (Figure 2B).\nSimilarly, KC serum levels in poly(I:C)-treated mice on low isoflavones were increased (735 ± 579 pg/ml and 626 ± 523 pg/ml) compared to the corresponding controls (173 ± 43 pg/ml, P = 0.0379 and 132 ± 52 pg/ml, P = 0.0006, respectively) (Figure 2C). Although KC serum levels in poly(I:C)/swim-treated mice on high isoflavones were also increased (451 ± 107 pg/ml) compared to control poly(I:C)/swim-treated mice (100 ± 45 pg/ml, P = 0.0079), high isoflavone diet decreased KC serum levels in poly(I:C)/no swim-treated mice (128 ± 43 pg/ml, P = 0.0025) compared to those on low isoflavones (Figure 2C).\nMoreover, CCL4 serum levels were increased in poly(I:C)-treated mice on low isoflavones (1,256 ± 556 pg/ml and 395 ± 245 pg/ml) compared to the corresponding controls (160 ± 0 pg/ml, P = 0.0011 and 160 ± 0 pg/ml, P = 0.0037, respectively) (Figure 2D). Swim further increased CCL4 serum levels (1,256 ± 556 pg/ml, P = 0.0023). Although CCL4 serum levels in poly(I:C)-treated mice on high isoflavones were also increased (217 ± 55 pg/ml and 265 ± 111 pg/ml) compared to their controls (160 ± 0 pg/ml, P = 0.0075 and 160 ± 0 pg/ml, P = 0.0254, respectively)., high isoflavones decreased CCL4 serum levels in poly(I:C)/swim-treated mice (217 ± 55 pg/ml, P = 0.0025) (Figure 2D).\nPoly(I:C) treatment also increased CCL2, CCL3, CCL5 and CXCL10 serum levels and high isoflavones reduced those increases (Additional file 3).",
"TNF-α (Figure 3A), IL-6 (Figure 3B), KC (Figure 3C), CCL4 (Figure 3D), as well as CCL2, CCL5 and CXCL10 brain gene expression were increased in the poly(I:C)-treated mice. High isoflavones reduced the increased TNF, IL6, KC, CCL4 and CCL2 brain gene expression. There was no difference in HDC and NT gene expression between the different treatment groups (Additional file 4: Table S3).Figure 3\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3\nA), IL-6 (Figure 3\nB), KC (Figure 3\nC), and CCL4 (Figure 3\nD) brain gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3\nA), IL-6 (Figure 3\nB), KC (Figure 3\nC), and CCL4 (Figure 3\nD) brain gene expression using qPCR.\nMore specifically TNF-α gene expression was increased in the brain of poly(I:C)-treated mice on low isoflavones (relative fold change of 22 ± 7 and 12 ± 9) compared to their controls (1.1 ± 0.9, P = 0.0006 and 1 ± 0.7, P = 0.0012, respectively) (Figure 3A). Although poly(I:C)-treated mice on high isoflavones had increased TNF-α brain gene expression (6 ± 1 and 9 ± 8) compared to their corresponding controls (1.2 ± 0.5, P = 0.0079 and 1 ± 0.3, P = 0.0079, respectively), high isoflavones decreased TNF-α gene expression in the brain of poly(I:C)/swim-treated mice (6 ± 1, P = 0.0025) compared to those on low isoflavones (Figure 3A). There was no effect of poly(I:C) treatment on IL-6 brain gene expression (Figure 3B), but high isoflavones still decreased IL-6 gene expression in the brain of poly(I:C)/swim-treated mice (0.5 ± 0.1, P = 0.0228) compared to those on low isoflavones (2.1 ± 2.4) (Figure 3B).\nKC gene expression in the brain of poly(I:C)-treated mice on low isoflavones was increased (18 ± 13 and 40 ± 52) compared to their controls (0.9 ± 0.3, P = 0.0006 and 1 ± 0.3, P = 0.0183, respectively) (Figure 3C). Although poly(I:C)/swim-treated mice on high isoflavones still had increased KC gene expression in their brain (2 ± 0.9) compared to control/swim-treated mice (0.6 ± 0.1, P = 0.0159), high isoflavones reduced this increase in KC gene expression (2 ± 0.9, P = 0.0177) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3C).\nLikewise, CCL4 gene expression in the brain of poly(I:C)-treated mice on low isoflavones was also increased (6.5 ± 4 and 2.9 ± 1.6) compared to their controls (1 ± 0.3, P = 0.0006 and 1 ± 0.4, P = 0.0350, respectively) (Figure 3D). Although, poly(I:C)/swim-treated mice on high isoflavones still had increased CCL4 gene expression (1.8 ± 0.4) compared to control/swim-treated mice (1 ± 0.4, P = 0.0317), high isoflavones reduced this increase in CCL4 gene expression (1.8 ± 0.4, P = 0.0101) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3D). Poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the brains of the mice. High isoflavones reduced this noted CCL2 brain gene expression increase (Additional file 3).",
"TNF-α (Figure 4A), IL-6 (Figure 4B), KC (Figure 4C), CCL4 (Figure 4D), as well as CCL2, CCL5 and CXCL10 (Additional file 5: Table S4) skin gene expression were increased in the poly(I:C)-treated mice. Moreover, HDC and NT gene expression were also increased in the skin (Figure 5A and B). High isoflavone diet reduced the increased TNF-α, IL-6 and KC skin gene expression.Figure 4\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4\nA), IL-6 (Figure 4\nB), KC (Figure 4\nC) and CCL4 (Figure 4\nD) skin gene expression using qPCR.Figure 5\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5\nA) and NT (Figure 5\nB) skin gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4\nA), IL-6 (Figure 4\nB), KC (Figure 4\nC) and CCL4 (Figure 4\nD) skin gene expression using qPCR.\n\nPolyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5\nA) and NT (Figure 5\nB) skin gene expression using qPCR.\nMore specifically, TNF-α gene expression in the skin of poly(I:C)/swim-treated mice on low isoflavones, was increased (5 ± 0.9) compared to their controls (1.1 ± 0.4, P = 0.0006) (Figure 4A) and high isoflavones decreased this increase in TNF-α gene expression (2.3 ± 1.3, P = 0.0101) (Figure 4A). Likewise, IL-6 gene expression in the skin of poly(I:C)-treated mice on low isoflavones was increased (14 ± 4 and 22 ± 16) compared to their controls (0.9 ± 0.8, P = 0.0006 and 1 ± 0.4, P = 0.0012, respectively) (Figure 4B). Although poly(I:C)-treated mice on high isoflavones still had increased IL-6 gene expression (5.5 ± 3 and 3.4 ± 1.7) compared to their controls (1.4 ± 0.7, P = 0.0079 and 0.7 ± 0.4, P = 0.0159, respectively), high isoflavones reduced this increase in the IL-6 gene expression (5.5 ± 3 P = 0.0101) (Figure 4B).\nSimilarly, KC gene expression in the skin of poly(I:C)-treated mice on low isoflavones was also increased (53 ± 55 and 69 ± 83) compared to their controls (0.3 ± 0.2, P = 0.0021 and 1 ± 1.5, P = 0.0012, respectively) (Figure 4C). High isoflavones reduced this increase in the KC gene expression of poly(I:C)-treated mice (2.8 ± 3.5, P = 0.0057 and 3.4 ± 1.9, P = 0.0043) (Figure 4C).\nCCL4 gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (24 ± 15 and 21 ± 19) compared to their controls (0.4 ± 0.3, P = 0.0021 and 1 ± 1.7, P = 0.0023, respectively) (Figure 4D). CCL4 gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (20 ± 24 and 27 ± 20) compared to their controls (0.8 ± 0.7, P = 0.0079 and 1 ± 1.3, P = 0.0079, respectively) and high isoflavones did have an effect on reducing this increase.\nIn the same way, HDC gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (4 ± 2 and 4 ± 2) compared to their controls (0.6 ± 0.4, P = 0.0003 and 1 ± 0.8, P = 0.0123, respectively) (Figure 5A). HDC gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (3 ± 1.1 and 3 ± 1) compared to their controls (0.9 ± 0.3, P = 0.0079 and 1 ± 0.2, P = 0.0317, respectively).\nSimilar to HDC gene expression, NT gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (8 ± 4 and 9 ± 10) compared to their controls (0.6 ± 0.4, P = 0.0025 and 1 ± 0.7, P = 0.0012, respectively) (Figure 5B). NT gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (8 ± 4, and 6 ± 6) compared to their controls (1.9 ± 0.8, P = 0.0317 and 1 ± 0.2, P = 0.0079, respectively).\nFinally, poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the skin of the mice. High isoflavones did not have any statistical effect on CCL4, CCL2, CCL5, CXCL10, HDC and NT skin gene expression (Additional file 5: Table S4).",
"Here we show that poly(I:C) significantly reduced locomotor activity over the first 24 hours, in comparison to control mice. Forced swim did not have any effect on its own, but augmented the effect of poly(I:C) on increasing TNF-α, CCL3 and CCL5 serum levels. We also used BALBc mice in an effort to investigate the possibility of strain differences, but there was no difference from our findings with C57BL/6 mice (results not shown).\nUse of poly(I:C) increased serum levels of molecules that have been associated with inflammation and fatigue including TNF-α, IL-6, KC, CCL2, CCL3, CCL4, CCL5, and CXCL10. Moreover, poly(I:C) also increased brain and skin gene expression of TNF-α, IL-6, KC, CCL2, CCL4, CCL5, CXCL10, while HDC and NT gene expression were only increased in the skin, suggesting that NT and histamine may explain the skin findings in CFS patients. Forced swim stress had no additional effect to that of poly(I:C), except for augmenting TNF-α serum levels. TNF-α and IL-6 serum levels were actually increased in CFS [20,44]. Such pro-inflammatory mediators can increase blood-brain barrier (BBB) permeability [45] and permit entry of circulating leukocytes leading to brain inflammation. TNF-α was shown to be released along with histamine from rat brain MC [46], and was involved in brain inflammation [47]. MC can also interact with T cells [48-50] and superactivate them through TNF-α [51].\nUnlike our present results, one group reported that daily forced swim stress for two to three weeks, with or without an immunological trigger administered on day one (lipopolysaccharide or Brucella abortus antigen), induced ‘chronic fatigue’, but only in Albino laca mice [52-55] and Wistar rats [56]. In these papers, behavioral parameters, such as immobility time, time to start grooming after swim stress, roda rod test, and elevated plus maze test were increased indicating post stress fatigue and anxiety. Also, biochemical measurements indicative of brain oxidative stress were increased. In another paper, BALBc mice were injected with Brucella abortus and developed decreased running activity that lasted one week [57]. Forced swim of Charles Foster albino rats for twenty-one days also increased immobility time, anxiety as assessed by elevated plus maze test, and brain oxidative stress [58]. These findings maybe due to the differences in the strains and triggers used.\nChemokines interact with their receptors and attract immune cells to inflammation sites [59-61]. CXCR1, CXCR2, CXCR3, CXCR4, CX3CR1, CCR1, CCR3, CCR4 and CCR5 are expressed by human MC of different origins [62]. Stimulated MC release CCL2, CCL3, CCL4 [63]. Murine fetal skin-derived cultured MC (FSMCs) release CCL3, CCL4, CCL5, IL-6 and TNF-α upon TLR3-poly(I:C) activation [64,65]. Peritoneal MC from C57BL/6 mice activated by poly(I:C)-TLR3 have increased CCL5 and CXCL10 expression [66,67].\nHere we also show that poly(I:C) with or without NT or CRH did not have any effect on TNF-α, CXCL8 and VEGFα release from human cultured LAD2 MC (results not shown), but increased only TNF-α gene expression when used together with CRH, NT, or SP. Poly(I:C) alone did not have any effect on human MC, but increased TNF-α gene expression at 24 hours when used together with CRH or NT.\nToll-like receptors’ (TLRs) [68,69] activation is important in the development of innate immunity to invading pathogens, leading to release of different cytokines [64,70,71]. Antigen-mediated MC reactivity is amplified through prolonged TLR-ligand treatment [72].\nHuman umbilical cord blood-derived MC express TLR-3, activation of which produced IFNα and IFNβ in response to double-stranded RNA [73]. A recent publication reported that MC respond to intracellular, but not extracellular, poly(I:C) by inducing mainly IFNα and TNF-α; moreover, infection of MC with live Sendai virus induces an anti-viral response similar to that of intracellular poly(I:C) [74].\nWe also used CRHR-1 knockout (KO) mice in order to investigate the possible involvement of CRHR in any stress effect. However, forced swim stress alone did not have an effect and there was no difference using these mice (results not shown). Nevertheless, rats exposed to water immersion stress had a four-fold increase in plasma histamine levels that was absent in W/Wv MC-deficient rats [75]. Acute stress also increased serum histamine and IL-6 levels, both of which were also absent in MC deficient W/Wv mice [76].\nHere we also show that the isoflavones genistein and daidzein reversed the effect of poly(I:C) on mice. Genistein has been reported to attenuate muscle fatigue [77], protect against endothelial barrier dysfunction [78] and suppress LPS-induced inflammatory response in macrophages [79]. Isoflavones also suppress MC expression of the high affinity IgE receptor (FcεRI) [80]. However, isoflavones have estrogenic activity and may not be desirable in certain clinical settings. The flavonoids epigallocatechin, naringin, and curcumin ameliorated ‘chronic fatigue’ [53-56]. Other papers reported similar effects for the Astragalus flavonoids [81] and for the olive extract [82].\nFlavonoids exert potent anti-inflammatory effects via various pathways [83-87]. A review of human randomized controlled trial studies summarized some significant benefits to cognitive function after isoflavone supplementation [88]: improvements in executive function, working memory and processing speed [88]. Specifically, two studies reported significant effects of 60 mg/day treatment with isoflavones in processing speed and psychomotor speed [89,90].\nQuercetin increases exercise tolerance in mice [91]. Oral administration of quercetin leads to accumulation in brain tissue and attenuates the increased oxidative stress in the hippocampus and striatum of rats exposed to chronic forced swimming [92,93]. Quercetin has potent anti-oxidant and anti-inflammatory activity [41,42], and inhibits MC degranulation [94,95], as well as TNF-α, IL-6, and IL-8 secretion [95,96]. Moreover, it reverses acute stress-induced behavioral changes and reduces brain glutathione levels in mice [97].\nRecent studies with antigen-stimulated MC show that epigallocatechin gallate (EGCG) inhibits MC degranulation, leukotriene C4 secretion, as well as the production of TNF-α, IL-6 and IL-8 [94,96]. The quercetin-related flavone luteolin inhibits MC activation [98] and MC-dependent stimulation of activated T cells [51]. Luteolin also inhibits IL-6 release from microglia cells [99] and from astrocytes [100]. Recent reviews have addressed the possible use of flavonoids for the treatment of CNS diseases [101,102].\nIncreasing evidence implicates CNS inflammation [103], as well as MC-microglia interactions, in neuropsychiatric diseases [104,105]. MC are important for allergic reactions, but also in immunity [106,107], and inflammatory diseases [15,108]. TLRs have also been implicated in CNS dysfunction through MC and glial activation [104].",
"Here we report that treatment of mice with poly(I:C) resulted in reduced locomotor activity and increased inflammatory markers in serum, brain and skin, all of which were reversed by treatment with isoflavones. Our findings also demonstrate significant variability among mouse ‘models’ as was recently reported [109]. There is need for the establishment of a more reliable animal model for CFS. Nevertheless, certain flavonoids appear promising for use in pilot clinical trials with CFS patients."
] |
[
"introduction",
"materials|methods",
null,
null,
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
"conclusion"
] |
[
"Brain",
"Fatigue",
"Inflammation",
"Isoflavones",
"Mast cells",
"Polyinosinic:polycytidylic acid",
"Skin",
"Stress",
"Swim"
] |
Background:
Chronic Fatigue Syndrome (CFS) is a complex disease, which has also been called ‘neurasthenia’, ‘post viral fatigue’, and ‘chronic mononucleosis’ [1-3]. Its prevalence may be as high as 1% in the US population [4] with a female to male ratio of 4 to 1 [5]. CFS involves the muscular, nervous, hormonal and immune systems. Patients complain of overwhelming fatigue, sleep disturbances, malaise, muscle aches, gastrointestinal symptoms, dizziness on standing, and cognitive problems [6]. Clinical and subclinical viral infections have been suspected, but never confirmed [7,8].
CFS is often comorbid with other disorders, including fibromyalgia, Pelvic Bladder Syndrome/Interstitial Cystitis (PBS/IC), irritable bowel syndrome (IBS), and migraines [9], all of which are characterized by central nervous system (CNS) dysfunction [10], and worsened by stress [11-15]. Many CFS patients demonstrate abnormal hypothalamic-pituitary-adrenal (HPA) axis activity [16,17]. Anxiety is common in CFS [18] and patients are particularly vulnerable to stress [14]. Many CFS symptoms could derive from the possible release of inflammatory mediators that could affect brain function [19,20]. As of today, there are no FDA approved drugs for the treatment of CFS [21]; psychological, physical, and pharmacological interventions used currently are not very effective [22].
An abnormal immune component may be involved in CFS [23-25], but the neuroimmune and neuroendocrine interactions involved are still unknown [26]. Mast cells (MC) and their mediators have been implicated in all diseases that are comorbid with CFS [9]. There is higher number of skin MC in patients with CFS [27,28], and such patients also show increased skin hypersensitivity [29]. Furthermore, CFS also occurs more often in patients with chronic urticaria, that also involves MC [30]. In fact, there is hyperresponsiveness in the bronchi of CFS patients, implying MC activation [31].
Activated MC release a number of chemokines and cytokines that could contribute to CFS symptoms [32,33]. MC are located perivascularly in close proximity to neurons [34], especially in the diencephalon [35,36], where functional MC-neuron interactions have been documented [36,37] in response to corticotropin-releasing hormone (CRH) [38]. In vivo activation of MC by CRH is augmented by neurotensin (NT) [39]. Moreover, NT is induced in the hypothalamus in response to bacterial lipopolysaccharide (LPS) and regulates the HPA axis [40].
Unfortunately, there are neither effective CFS treatments nor human MC inhibitors clinically available that may also be used in CFS. Flavonoids are natural compounds with strong antioxidant and anti-inflammatory activity [41]. Certain flavonoids also inhibit MC [42] and have neuroprotective effects [41,43].
Here we report that treatment of mice with poly(I:C) results in reduced locomotor activity and increased serum levels, as well as brain and gene expression, of inflammatory mediators, all of which are reversed by treatment with the isoflavones daidzein and genistein.
Methods:
Chemicals and reagents Polyinosinic-polycytidylic acid-TLR3-based adjuvant, poly(I:C), HMW VacciGrade, (catalog# vac-pic) was purchased from Invivogen (San Diego, CA, USA). Substance P (SP, catalog# S6883), neurotensin (NT, catalog# N6383) and corticotropin-releasing hormone (CRF, catalog# C3042) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aliquots of the above were prepared according to the manufacturer’s instructions. Teklad lab animal diets (catalog# 2918X, and 2920X) were purchased from Harlan (Indianapolis, IN, USA).
Polyinosinic-polycytidylic acid-TLR3-based adjuvant, poly(I:C), HMW VacciGrade, (catalog# vac-pic) was purchased from Invivogen (San Diego, CA, USA). Substance P (SP, catalog# S6883), neurotensin (NT, catalog# N6383) and corticotropin-releasing hormone (CRF, catalog# C3042) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aliquots of the above were prepared according to the manufacturer’s instructions. Teklad lab animal diets (catalog# 2918X, and 2920X) were purchased from Harlan (Indianapolis, IN, USA).
Animals C57BL/6 female mice, nine to twelve weeks old, (Jackson Laboratories, Bar Harbor, ME, USA) were kept in virus-free sections of a modern animal facility and were allowed ad libitum access to food and water. They were maintained on a 14:10 hour light-dark cycle (the standard light-dark cycle used by the Department of Animal Health). Female mice were chosen because published reports indicate a female to male ratio of 4:1 [5], while the US Centers for Disease Control and Prevention (CDC) specify a female to male ratio of 4:1 [5]. Mice were kept in cages of five mice/cage until the day of the experiments. Both low and high isoflavone diets (2018X and 2020X) were sterile with similar ingredients other than isoflavone content. We monitored weight changes for 21 days prior to the beginning of the experiments. Only poly(I:C)/no swim-treated mice showed a slight decrease in weight change. Poly(I:C)/swim-treated mice, as well as their corresponding control mice, slightly increased their weight over the three-week observation period. However, by the end of this period, there was no statistical difference in weight change. The protocol was approved by Tufts Medical Center IACUC under number B 2011-88.
C57BL/6 female mice, nine to twelve weeks old, (Jackson Laboratories, Bar Harbor, ME, USA) were kept in virus-free sections of a modern animal facility and were allowed ad libitum access to food and water. They were maintained on a 14:10 hour light-dark cycle (the standard light-dark cycle used by the Department of Animal Health). Female mice were chosen because published reports indicate a female to male ratio of 4:1 [5], while the US Centers for Disease Control and Prevention (CDC) specify a female to male ratio of 4:1 [5]. Mice were kept in cages of five mice/cage until the day of the experiments. Both low and high isoflavone diets (2018X and 2020X) were sterile with similar ingredients other than isoflavone content. We monitored weight changes for 21 days prior to the beginning of the experiments. Only poly(I:C)/no swim-treated mice showed a slight decrease in weight change. Poly(I:C)/swim-treated mice, as well as their corresponding control mice, slightly increased their weight over the three-week observation period. However, by the end of this period, there was no statistical difference in weight change. The protocol was approved by Tufts Medical Center IACUC under number B 2011-88.
Treatment conditions Mice were provided with chow containing either non-detectable to low (ND-20 mg/kg, Teklad 2920X) or high (150 to 250 mg/kg, Teklad 2918X) isoflavone (daidzein plus genistein) levels for two weeks. Conditions included four groups: (a) control (normal saline intraperitoneal (ip) injection)/no swim, (b) control/swim, (c) poly(I:C)/no swim, and (d) poly(I:C)/swim (n = 5 to 7/group). Mice were injected ip with 20 mg/kg of poly(I:C) or normal saline the first day. Subsequently, they were subjected to swim for 15 minutes, individually in a transparent plastic cylindrical jar (17 cm × 25 cm) containing 15 cm-deep water at room temperature (23 ± 1°C). This approach reflects both exercise and the stress of water immersion. Mice were then placed individually into specific cages and locomotor activity was monitored overnight.
Mice were provided with chow containing either non-detectable to low (ND-20 mg/kg, Teklad 2920X) or high (150 to 250 mg/kg, Teklad 2918X) isoflavone (daidzein plus genistein) levels for two weeks. Conditions included four groups: (a) control (normal saline intraperitoneal (ip) injection)/no swim, (b) control/swim, (c) poly(I:C)/no swim, and (d) poly(I:C)/swim (n = 5 to 7/group). Mice were injected ip with 20 mg/kg of poly(I:C) or normal saline the first day. Subsequently, they were subjected to swim for 15 minutes, individually in a transparent plastic cylindrical jar (17 cm × 25 cm) containing 15 cm-deep water at room temperature (23 ± 1°C). This approach reflects both exercise and the stress of water immersion. Mice were then placed individually into specific cages and locomotor activity was monitored overnight.
Assessment of behavioral parameter-locomotor activity After the experimental procedures, animals were placed individually into standard plastic housing cages with food and water available ad libitum and overnight locomotor activity (for a total of 16 hours) was monitored with the Neuroscience Behavior Core’s mouse SmartFrame® Cage Rack System (Kinder Scientific, Poway, CA, USA). This system consists of 20 PC-interfaced horizontal photobeam frames. The frame (containing 12 photocells; arranged on a 8 L × 4 W grid) surrounds one home cage environment and continuously tracks the animal’s movement. This fully automated system allows the user to quantify horizontal ambulation by counting breaks in infrared photocell beams using MotorMonitor® software (Hamilton-Kinder Scientific, Poway, CA, USA). Data were collected and subsequently analyzed in time bins (every hour) or as a total over the course of collection to the ‘Total Distance Travelled’ (in cm) parameter for each zone.
After the experimental procedures, animals were placed individually into standard plastic housing cages with food and water available ad libitum and overnight locomotor activity (for a total of 16 hours) was monitored with the Neuroscience Behavior Core’s mouse SmartFrame® Cage Rack System (Kinder Scientific, Poway, CA, USA). This system consists of 20 PC-interfaced horizontal photobeam frames. The frame (containing 12 photocells; arranged on a 8 L × 4 W grid) surrounds one home cage environment and continuously tracks the animal’s movement. This fully automated system allows the user to quantify horizontal ambulation by counting breaks in infrared photocell beams using MotorMonitor® software (Hamilton-Kinder Scientific, Poway, CA, USA). Data were collected and subsequently analyzed in time bins (every hour) or as a total over the course of collection to the ‘Total Distance Travelled’ (in cm) parameter for each zone.
Sample collection Mice were euthanized 24 hours post poly(I:C) ip injection using isoflurane overdose and thoracotomy. Blood was collected by cardiac puncture and was used to determine inflammatory mediator levels in the serum. Brain (diencephalon) and skin (back shaved with a electric shaver the day before) samples were collected and immersed into RNAlater (catalog# AM7021) purchased from Invitrogen (Grand Island, NY, USA). Samples were stored at -80°C.
Mice were euthanized 24 hours post poly(I:C) ip injection using isoflurane overdose and thoracotomy. Blood was collected by cardiac puncture and was used to determine inflammatory mediator levels in the serum. Brain (diencephalon) and skin (back shaved with a electric shaver the day before) samples were collected and immersed into RNAlater (catalog# AM7021) purchased from Invitrogen (Grand Island, NY, USA). Samples were stored at -80°C.
Serum levels of inflammatory mediators TNF-α, VEGFα, IL-1α, IL-1β, IL-4, IL-6, KC (IL-8/(CXC motif) ligand (CXC)L8 murine homolog, IL-9, IL-10, IL-12p70, IL-17, (CC motif) ligand (CCL)2, CCL3, CCL4, CCL5, CXCL10 and IFNγ mouse serum levels were determined using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Assay (MCYTOMAG-70 K custom made panel for the specific analytes mentioned above). Measurements were performed blindly by Millipore (St. Charles, MI, USA). Millipore's minimum assay reporting range for TNF-α and IL-6 levels was 32 pg/ml, for CCL2 was 800 pg/ml, while for CCL3 and CCL4 it was 160 pg/ml. Therefore, we consider all values < × pg/ml as equal to x pg/ml.
TNF-α, VEGFα, IL-1α, IL-1β, IL-4, IL-6, KC (IL-8/(CXC motif) ligand (CXC)L8 murine homolog, IL-9, IL-10, IL-12p70, IL-17, (CC motif) ligand (CCL)2, CCL3, CCL4, CCL5, CXCL10 and IFNγ mouse serum levels were determined using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Assay (MCYTOMAG-70 K custom made panel for the specific analytes mentioned above). Measurements were performed blindly by Millipore (St. Charles, MI, USA). Millipore's minimum assay reporting range for TNF-α and IL-6 levels was 32 pg/ml, for CCL2 was 800 pg/ml, while for CCL3 and CCL4 it was 160 pg/ml. Therefore, we consider all values < × pg/ml as equal to x pg/ml.
Quantitative PCR Total RNA from mouse tissues was extracted using RNeasy Plus Mini kit (catalog# 74134) and RNeasy Fibrous Tissue Mini Kit (catalog# 74704), purchased from QIAGEN (Valencia, CA, USA). Reverse transcription was performed with 300 ng of total RNA using the iScript cDNA synthesis kit (catalog# 170-8891) purchased from Bio-Rad (Hercules, CA, USA). Real-time quantitative polymerase chain reaction (qPCR) was carried out in a 7300 Sequence Detector, according to TaqMan Gene Expression Assay instructions from Life Technologies, Applied Biosystems (Grand Island, NY, USA) using Taqman primer/probe sets (Additional file 1: Table S1). Samples were analyzed for Tnf (TNF-α), Il4, Il6 (IL-6), KC (Cxcl1, IL-8 homolog in the mouse), Ccl2 (CCL2), Ccl4 (CCL4), Ccl5 (CCL5), Cxcl10 (CXCL10), histidine decarboxylase-Hdc (HDC), and Nts (NT) gene expression using Gapdh as internal control.Thermal cycling proceeded at 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, for 40 cycles, and 60°C for 1 minute. Negative controls included samples with water instead of template. Assays were performed in triplicate for each data point. Results were normalized against the endogenous gene GAPDH and were expressed relative to the mean of the control for each gene (relative fold change).
Total RNA from mouse tissues was extracted using RNeasy Plus Mini kit (catalog# 74134) and RNeasy Fibrous Tissue Mini Kit (catalog# 74704), purchased from QIAGEN (Valencia, CA, USA). Reverse transcription was performed with 300 ng of total RNA using the iScript cDNA synthesis kit (catalog# 170-8891) purchased from Bio-Rad (Hercules, CA, USA). Real-time quantitative polymerase chain reaction (qPCR) was carried out in a 7300 Sequence Detector, according to TaqMan Gene Expression Assay instructions from Life Technologies, Applied Biosystems (Grand Island, NY, USA) using Taqman primer/probe sets (Additional file 1: Table S1). Samples were analyzed for Tnf (TNF-α), Il4, Il6 (IL-6), KC (Cxcl1, IL-8 homolog in the mouse), Ccl2 (CCL2), Ccl4 (CCL4), Ccl5 (CCL5), Cxcl10 (CXCL10), histidine decarboxylase-Hdc (HDC), and Nts (NT) gene expression using Gapdh as internal control.Thermal cycling proceeded at 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, for 40 cycles, and 60°C for 1 minute. Negative controls included samples with water instead of template. Assays were performed in triplicate for each data point. Results were normalized against the endogenous gene GAPDH and were expressed relative to the mean of the control for each gene (relative fold change).
Statistical analysis Data were analyzed using the non-parametric Mann-Whitney U-test. Results are presented as mean ± SE. Statistical significance is defined as P < 0.05. We analyzed separately the different treatment subgroups within each dietary group, using Mann-Whitney U-test to compare poly(I:C)/no swim and poly(I:C)/swim-treated mice with the control/no swim and control/swim-treated groups, accordingly. We then analyzed the same treatment subgroups between the two dietary groups, using Mann-Whitney U-test to compare, for example, poly(I:C)/swim-treated mice with low isoflavone diet to poly(I:C)/swim-treated mice that were provided with high isoflavone diet. Due to space limitations, we submitted figures composed of all the experimental conditions from the two different diets and all the statistical significant results were marked in these figures.
Data were analyzed using the non-parametric Mann-Whitney U-test. Results are presented as mean ± SE. Statistical significance is defined as P < 0.05. We analyzed separately the different treatment subgroups within each dietary group, using Mann-Whitney U-test to compare poly(I:C)/no swim and poly(I:C)/swim-treated mice with the control/no swim and control/swim-treated groups, accordingly. We then analyzed the same treatment subgroups between the two dietary groups, using Mann-Whitney U-test to compare, for example, poly(I:C)/swim-treated mice with low isoflavone diet to poly(I:C)/swim-treated mice that were provided with high isoflavone diet. Due to space limitations, we submitted figures composed of all the experimental conditions from the two different diets and all the statistical significant results were marked in these figures.
Chemicals and reagents:
Polyinosinic-polycytidylic acid-TLR3-based adjuvant, poly(I:C), HMW VacciGrade, (catalog# vac-pic) was purchased from Invivogen (San Diego, CA, USA). Substance P (SP, catalog# S6883), neurotensin (NT, catalog# N6383) and corticotropin-releasing hormone (CRF, catalog# C3042) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aliquots of the above were prepared according to the manufacturer’s instructions. Teklad lab animal diets (catalog# 2918X, and 2920X) were purchased from Harlan (Indianapolis, IN, USA).
Animals:
C57BL/6 female mice, nine to twelve weeks old, (Jackson Laboratories, Bar Harbor, ME, USA) were kept in virus-free sections of a modern animal facility and were allowed ad libitum access to food and water. They were maintained on a 14:10 hour light-dark cycle (the standard light-dark cycle used by the Department of Animal Health). Female mice were chosen because published reports indicate a female to male ratio of 4:1 [5], while the US Centers for Disease Control and Prevention (CDC) specify a female to male ratio of 4:1 [5]. Mice were kept in cages of five mice/cage until the day of the experiments. Both low and high isoflavone diets (2018X and 2020X) were sterile with similar ingredients other than isoflavone content. We monitored weight changes for 21 days prior to the beginning of the experiments. Only poly(I:C)/no swim-treated mice showed a slight decrease in weight change. Poly(I:C)/swim-treated mice, as well as their corresponding control mice, slightly increased their weight over the three-week observation period. However, by the end of this period, there was no statistical difference in weight change. The protocol was approved by Tufts Medical Center IACUC under number B 2011-88.
Treatment conditions:
Mice were provided with chow containing either non-detectable to low (ND-20 mg/kg, Teklad 2920X) or high (150 to 250 mg/kg, Teklad 2918X) isoflavone (daidzein plus genistein) levels for two weeks. Conditions included four groups: (a) control (normal saline intraperitoneal (ip) injection)/no swim, (b) control/swim, (c) poly(I:C)/no swim, and (d) poly(I:C)/swim (n = 5 to 7/group). Mice were injected ip with 20 mg/kg of poly(I:C) or normal saline the first day. Subsequently, they were subjected to swim for 15 minutes, individually in a transparent plastic cylindrical jar (17 cm × 25 cm) containing 15 cm-deep water at room temperature (23 ± 1°C). This approach reflects both exercise and the stress of water immersion. Mice were then placed individually into specific cages and locomotor activity was monitored overnight.
Assessment of behavioral parameter-locomotor activity:
After the experimental procedures, animals were placed individually into standard plastic housing cages with food and water available ad libitum and overnight locomotor activity (for a total of 16 hours) was monitored with the Neuroscience Behavior Core’s mouse SmartFrame® Cage Rack System (Kinder Scientific, Poway, CA, USA). This system consists of 20 PC-interfaced horizontal photobeam frames. The frame (containing 12 photocells; arranged on a 8 L × 4 W grid) surrounds one home cage environment and continuously tracks the animal’s movement. This fully automated system allows the user to quantify horizontal ambulation by counting breaks in infrared photocell beams using MotorMonitor® software (Hamilton-Kinder Scientific, Poway, CA, USA). Data were collected and subsequently analyzed in time bins (every hour) or as a total over the course of collection to the ‘Total Distance Travelled’ (in cm) parameter for each zone.
Sample collection:
Mice were euthanized 24 hours post poly(I:C) ip injection using isoflurane overdose and thoracotomy. Blood was collected by cardiac puncture and was used to determine inflammatory mediator levels in the serum. Brain (diencephalon) and skin (back shaved with a electric shaver the day before) samples were collected and immersed into RNAlater (catalog# AM7021) purchased from Invitrogen (Grand Island, NY, USA). Samples were stored at -80°C.
Serum levels of inflammatory mediators:
TNF-α, VEGFα, IL-1α, IL-1β, IL-4, IL-6, KC (IL-8/(CXC motif) ligand (CXC)L8 murine homolog, IL-9, IL-10, IL-12p70, IL-17, (CC motif) ligand (CCL)2, CCL3, CCL4, CCL5, CXCL10 and IFNγ mouse serum levels were determined using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Assay (MCYTOMAG-70 K custom made panel for the specific analytes mentioned above). Measurements were performed blindly by Millipore (St. Charles, MI, USA). Millipore's minimum assay reporting range for TNF-α and IL-6 levels was 32 pg/ml, for CCL2 was 800 pg/ml, while for CCL3 and CCL4 it was 160 pg/ml. Therefore, we consider all values < × pg/ml as equal to x pg/ml.
Quantitative PCR:
Total RNA from mouse tissues was extracted using RNeasy Plus Mini kit (catalog# 74134) and RNeasy Fibrous Tissue Mini Kit (catalog# 74704), purchased from QIAGEN (Valencia, CA, USA). Reverse transcription was performed with 300 ng of total RNA using the iScript cDNA synthesis kit (catalog# 170-8891) purchased from Bio-Rad (Hercules, CA, USA). Real-time quantitative polymerase chain reaction (qPCR) was carried out in a 7300 Sequence Detector, according to TaqMan Gene Expression Assay instructions from Life Technologies, Applied Biosystems (Grand Island, NY, USA) using Taqman primer/probe sets (Additional file 1: Table S1). Samples were analyzed for Tnf (TNF-α), Il4, Il6 (IL-6), KC (Cxcl1, IL-8 homolog in the mouse), Ccl2 (CCL2), Ccl4 (CCL4), Ccl5 (CCL5), Cxcl10 (CXCL10), histidine decarboxylase-Hdc (HDC), and Nts (NT) gene expression using Gapdh as internal control.Thermal cycling proceeded at 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, for 40 cycles, and 60°C for 1 minute. Negative controls included samples with water instead of template. Assays were performed in triplicate for each data point. Results were normalized against the endogenous gene GAPDH and were expressed relative to the mean of the control for each gene (relative fold change).
Statistical analysis:
Data were analyzed using the non-parametric Mann-Whitney U-test. Results are presented as mean ± SE. Statistical significance is defined as P < 0.05. We analyzed separately the different treatment subgroups within each dietary group, using Mann-Whitney U-test to compare poly(I:C)/no swim and poly(I:C)/swim-treated mice with the control/no swim and control/swim-treated groups, accordingly. We then analyzed the same treatment subgroups between the two dietary groups, using Mann-Whitney U-test to compare, for example, poly(I:C)/swim-treated mice with low isoflavone diet to poly(I:C)/swim-treated mice that were provided with high isoflavone diet. Due to space limitations, we submitted figures composed of all the experimental conditions from the two different diets and all the statistical significant results were marked in these figures.
Results:
Effect of poly(I:C) and isoflavones on locomotor activity Poly(I:C)/swim and poly(I:C)/no swim-treated mice on low isoflavones had reduced maximum (max) locomotor activity (over the 10-hour night period), denoted by the dark bar (P = 0.008 and P = 0.036) compared to the control/swim and control/no swim-treated mice, respectively (Figure 1A). High isoflavone chow reversed this decrease (Figure 1B), and the difference between the two diets was statistically significant (P = 0.032).Figure 1
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1
A) or high (Figure 1
B) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1
A) or high (Figure 1
B) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.
Poly(I:C)/swim and poly(I:C)/no swim-treated mice on low isoflavones had reduced maximum (max) locomotor activity (over the 10-hour night period), denoted by the dark bar (P = 0.008 and P = 0.036) compared to the control/swim and control/no swim-treated mice, respectively (Figure 1A). High isoflavone chow reversed this decrease (Figure 1B), and the difference between the two diets was statistically significant (P = 0.032).Figure 1
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1
A) or high (Figure 1
B) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1
A) or high (Figure 1
B) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.
Effect of poly(I:C) and isoflavones on serum inflammatory mediators Poly(I:C)-treated mice on low isoflavones had increased serum levels of TNF-α (Figure 2A), IL-6 (Figure 2B), KC (Figure 2C), and CCL4 (Figure 2D), as well as CCL2, CCL3, CCL5, and CXCL10 (Additional file 2: Table S2). High isoflavones reduced the poly(I:C)-increased serum levels of all the inflammatory markers. All IFNγ, IL-1β, IL-4, IL-9, IL-10, IL-12p70, IL-17 and VEGFα serum levels were below the detection limit, while IL-1α serum levels were similar between the different treatment groups. Swim stress augmented the poly(I:C) effect on TNF-α, CCL4, and CCL5 serum levels.Figure 2
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2
A), IL-6 (Figure 2
B), KC (Figure 2
C), and CCL4 (Figure 2
D) using the Milliplex microbead assay.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2
A), IL-6 (Figure 2
B), KC (Figure 2
C), and CCL4 (Figure 2
D) using the Milliplex microbead assay.
More specifically, TNF-α serum levels in poly(I:C)/swim-treated mice on low isoflavones were increased (55 ± 14 pg/ml) compared to control/swim-treated mice (32 ± 0 pg/ml, P = 0.0011) and poly(I:C)/no swim-treated mice (37 ± 10 pg/ml,P = 0.0221). High isoflavones decreased these high TNF-α serum levels (32 ± 0 pg/ml, P = 0.0042) (Figure 2A). IL-6 serum levels in poly(I:C)-swim and poly(I:C)/no swim-treated mice on low isoflavones were also increased (229 ± 110 pg/ml and 141 ± 11 pg/ml) compared to the corresponding control/swim and control/no swim mice on low isoflavones (32 ± 0 pg/ml, P = 0.0011 and 32 ± 0 pg/ml, P = 0.0114) (Figure 2B). High isoflavones reduced IL-6 serum levels of poly(I:C)/swim-treated mice (35 ± 5 pg/ml, P = 0.0054) and poly(I:C)/no swim-treated (32 ± 0 pg/ml, P = 0.0298) compared to the corresponding groups on low isoflavones (Figure 2B).
Similarly, KC serum levels in poly(I:C)-treated mice on low isoflavones were increased (735 ± 579 pg/ml and 626 ± 523 pg/ml) compared to the corresponding controls (173 ± 43 pg/ml, P = 0.0379 and 132 ± 52 pg/ml, P = 0.0006, respectively) (Figure 2C). Although KC serum levels in poly(I:C)/swim-treated mice on high isoflavones were also increased (451 ± 107 pg/ml) compared to control poly(I:C)/swim-treated mice (100 ± 45 pg/ml, P = 0.0079), high isoflavone diet decreased KC serum levels in poly(I:C)/no swim-treated mice (128 ± 43 pg/ml, P = 0.0025) compared to those on low isoflavones (Figure 2C).
Moreover, CCL4 serum levels were increased in poly(I:C)-treated mice on low isoflavones (1,256 ± 556 pg/ml and 395 ± 245 pg/ml) compared to the corresponding controls (160 ± 0 pg/ml, P = 0.0011 and 160 ± 0 pg/ml, P = 0.0037, respectively) (Figure 2D). Swim further increased CCL4 serum levels (1,256 ± 556 pg/ml, P = 0.0023). Although CCL4 serum levels in poly(I:C)-treated mice on high isoflavones were also increased (217 ± 55 pg/ml and 265 ± 111 pg/ml) compared to their controls (160 ± 0 pg/ml, P = 0.0075 and 160 ± 0 pg/ml, P = 0.0254, respectively)., high isoflavones decreased CCL4 serum levels in poly(I:C)/swim-treated mice (217 ± 55 pg/ml, P = 0.0025) (Figure 2D).
Poly(I:C) treatment also increased CCL2, CCL3, CCL5 and CXCL10 serum levels and high isoflavones reduced those increases (Additional file 3).
Poly(I:C)-treated mice on low isoflavones had increased serum levels of TNF-α (Figure 2A), IL-6 (Figure 2B), KC (Figure 2C), and CCL4 (Figure 2D), as well as CCL2, CCL3, CCL5, and CXCL10 (Additional file 2: Table S2). High isoflavones reduced the poly(I:C)-increased serum levels of all the inflammatory markers. All IFNγ, IL-1β, IL-4, IL-9, IL-10, IL-12p70, IL-17 and VEGFα serum levels were below the detection limit, while IL-1α serum levels were similar between the different treatment groups. Swim stress augmented the poly(I:C) effect on TNF-α, CCL4, and CCL5 serum levels.Figure 2
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2
A), IL-6 (Figure 2
B), KC (Figure 2
C), and CCL4 (Figure 2
D) using the Milliplex microbead assay.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2
A), IL-6 (Figure 2
B), KC (Figure 2
C), and CCL4 (Figure 2
D) using the Milliplex microbead assay.
More specifically, TNF-α serum levels in poly(I:C)/swim-treated mice on low isoflavones were increased (55 ± 14 pg/ml) compared to control/swim-treated mice (32 ± 0 pg/ml, P = 0.0011) and poly(I:C)/no swim-treated mice (37 ± 10 pg/ml,P = 0.0221). High isoflavones decreased these high TNF-α serum levels (32 ± 0 pg/ml, P = 0.0042) (Figure 2A). IL-6 serum levels in poly(I:C)-swim and poly(I:C)/no swim-treated mice on low isoflavones were also increased (229 ± 110 pg/ml and 141 ± 11 pg/ml) compared to the corresponding control/swim and control/no swim mice on low isoflavones (32 ± 0 pg/ml, P = 0.0011 and 32 ± 0 pg/ml, P = 0.0114) (Figure 2B). High isoflavones reduced IL-6 serum levels of poly(I:C)/swim-treated mice (35 ± 5 pg/ml, P = 0.0054) and poly(I:C)/no swim-treated (32 ± 0 pg/ml, P = 0.0298) compared to the corresponding groups on low isoflavones (Figure 2B).
Similarly, KC serum levels in poly(I:C)-treated mice on low isoflavones were increased (735 ± 579 pg/ml and 626 ± 523 pg/ml) compared to the corresponding controls (173 ± 43 pg/ml, P = 0.0379 and 132 ± 52 pg/ml, P = 0.0006, respectively) (Figure 2C). Although KC serum levels in poly(I:C)/swim-treated mice on high isoflavones were also increased (451 ± 107 pg/ml) compared to control poly(I:C)/swim-treated mice (100 ± 45 pg/ml, P = 0.0079), high isoflavone diet decreased KC serum levels in poly(I:C)/no swim-treated mice (128 ± 43 pg/ml, P = 0.0025) compared to those on low isoflavones (Figure 2C).
Moreover, CCL4 serum levels were increased in poly(I:C)-treated mice on low isoflavones (1,256 ± 556 pg/ml and 395 ± 245 pg/ml) compared to the corresponding controls (160 ± 0 pg/ml, P = 0.0011 and 160 ± 0 pg/ml, P = 0.0037, respectively) (Figure 2D). Swim further increased CCL4 serum levels (1,256 ± 556 pg/ml, P = 0.0023). Although CCL4 serum levels in poly(I:C)-treated mice on high isoflavones were also increased (217 ± 55 pg/ml and 265 ± 111 pg/ml) compared to their controls (160 ± 0 pg/ml, P = 0.0075 and 160 ± 0 pg/ml, P = 0.0254, respectively)., high isoflavones decreased CCL4 serum levels in poly(I:C)/swim-treated mice (217 ± 55 pg/ml, P = 0.0025) (Figure 2D).
Poly(I:C) treatment also increased CCL2, CCL3, CCL5 and CXCL10 serum levels and high isoflavones reduced those increases (Additional file 3).
Effect of poly(I:C) and isoflavones on brain gene expression of inflammatory mediators TNF-α (Figure 3A), IL-6 (Figure 3B), KC (Figure 3C), CCL4 (Figure 3D), as well as CCL2, CCL5 and CXCL10 brain gene expression were increased in the poly(I:C)-treated mice. High isoflavones reduced the increased TNF, IL6, KC, CCL4 and CCL2 brain gene expression. There was no difference in HDC and NT gene expression between the different treatment groups (Additional file 4: Table S3).Figure 3
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3
A), IL-6 (Figure 3
B), KC (Figure 3
C), and CCL4 (Figure 3
D) brain gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3
A), IL-6 (Figure 3
B), KC (Figure 3
C), and CCL4 (Figure 3
D) brain gene expression using qPCR.
More specifically TNF-α gene expression was increased in the brain of poly(I:C)-treated mice on low isoflavones (relative fold change of 22 ± 7 and 12 ± 9) compared to their controls (1.1 ± 0.9, P = 0.0006 and 1 ± 0.7, P = 0.0012, respectively) (Figure 3A). Although poly(I:C)-treated mice on high isoflavones had increased TNF-α brain gene expression (6 ± 1 and 9 ± 8) compared to their corresponding controls (1.2 ± 0.5, P = 0.0079 and 1 ± 0.3, P = 0.0079, respectively), high isoflavones decreased TNF-α gene expression in the brain of poly(I:C)/swim-treated mice (6 ± 1, P = 0.0025) compared to those on low isoflavones (Figure 3A). There was no effect of poly(I:C) treatment on IL-6 brain gene expression (Figure 3B), but high isoflavones still decreased IL-6 gene expression in the brain of poly(I:C)/swim-treated mice (0.5 ± 0.1, P = 0.0228) compared to those on low isoflavones (2.1 ± 2.4) (Figure 3B).
KC gene expression in the brain of poly(I:C)-treated mice on low isoflavones was increased (18 ± 13 and 40 ± 52) compared to their controls (0.9 ± 0.3, P = 0.0006 and 1 ± 0.3, P = 0.0183, respectively) (Figure 3C). Although poly(I:C)/swim-treated mice on high isoflavones still had increased KC gene expression in their brain (2 ± 0.9) compared to control/swim-treated mice (0.6 ± 0.1, P = 0.0159), high isoflavones reduced this increase in KC gene expression (2 ± 0.9, P = 0.0177) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3C).
Likewise, CCL4 gene expression in the brain of poly(I:C)-treated mice on low isoflavones was also increased (6.5 ± 4 and 2.9 ± 1.6) compared to their controls (1 ± 0.3, P = 0.0006 and 1 ± 0.4, P = 0.0350, respectively) (Figure 3D). Although, poly(I:C)/swim-treated mice on high isoflavones still had increased CCL4 gene expression (1.8 ± 0.4) compared to control/swim-treated mice (1 ± 0.4, P = 0.0317), high isoflavones reduced this increase in CCL4 gene expression (1.8 ± 0.4, P = 0.0101) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3D). Poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the brains of the mice. High isoflavones reduced this noted CCL2 brain gene expression increase (Additional file 3).
TNF-α (Figure 3A), IL-6 (Figure 3B), KC (Figure 3C), CCL4 (Figure 3D), as well as CCL2, CCL5 and CXCL10 brain gene expression were increased in the poly(I:C)-treated mice. High isoflavones reduced the increased TNF, IL6, KC, CCL4 and CCL2 brain gene expression. There was no difference in HDC and NT gene expression between the different treatment groups (Additional file 4: Table S3).Figure 3
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3
A), IL-6 (Figure 3
B), KC (Figure 3
C), and CCL4 (Figure 3
D) brain gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3
A), IL-6 (Figure 3
B), KC (Figure 3
C), and CCL4 (Figure 3
D) brain gene expression using qPCR.
More specifically TNF-α gene expression was increased in the brain of poly(I:C)-treated mice on low isoflavones (relative fold change of 22 ± 7 and 12 ± 9) compared to their controls (1.1 ± 0.9, P = 0.0006 and 1 ± 0.7, P = 0.0012, respectively) (Figure 3A). Although poly(I:C)-treated mice on high isoflavones had increased TNF-α brain gene expression (6 ± 1 and 9 ± 8) compared to their corresponding controls (1.2 ± 0.5, P = 0.0079 and 1 ± 0.3, P = 0.0079, respectively), high isoflavones decreased TNF-α gene expression in the brain of poly(I:C)/swim-treated mice (6 ± 1, P = 0.0025) compared to those on low isoflavones (Figure 3A). There was no effect of poly(I:C) treatment on IL-6 brain gene expression (Figure 3B), but high isoflavones still decreased IL-6 gene expression in the brain of poly(I:C)/swim-treated mice (0.5 ± 0.1, P = 0.0228) compared to those on low isoflavones (2.1 ± 2.4) (Figure 3B).
KC gene expression in the brain of poly(I:C)-treated mice on low isoflavones was increased (18 ± 13 and 40 ± 52) compared to their controls (0.9 ± 0.3, P = 0.0006 and 1 ± 0.3, P = 0.0183, respectively) (Figure 3C). Although poly(I:C)/swim-treated mice on high isoflavones still had increased KC gene expression in their brain (2 ± 0.9) compared to control/swim-treated mice (0.6 ± 0.1, P = 0.0159), high isoflavones reduced this increase in KC gene expression (2 ± 0.9, P = 0.0177) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3C).
Likewise, CCL4 gene expression in the brain of poly(I:C)-treated mice on low isoflavones was also increased (6.5 ± 4 and 2.9 ± 1.6) compared to their controls (1 ± 0.3, P = 0.0006 and 1 ± 0.4, P = 0.0350, respectively) (Figure 3D). Although, poly(I:C)/swim-treated mice on high isoflavones still had increased CCL4 gene expression (1.8 ± 0.4) compared to control/swim-treated mice (1 ± 0.4, P = 0.0317), high isoflavones reduced this increase in CCL4 gene expression (1.8 ± 0.4, P = 0.0101) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3D). Poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the brains of the mice. High isoflavones reduced this noted CCL2 brain gene expression increase (Additional file 3).
Effect of poly(I:C) and isoflavones on skin gene expression of inflammatory mediators TNF-α (Figure 4A), IL-6 (Figure 4B), KC (Figure 4C), CCL4 (Figure 4D), as well as CCL2, CCL5 and CXCL10 (Additional file 5: Table S4) skin gene expression were increased in the poly(I:C)-treated mice. Moreover, HDC and NT gene expression were also increased in the skin (Figure 5A and B). High isoflavone diet reduced the increased TNF-α, IL-6 and KC skin gene expression.Figure 4
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4
A), IL-6 (Figure 4
B), KC (Figure 4
C) and CCL4 (Figure 4
D) skin gene expression using qPCR.Figure 5
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5
A) and NT (Figure 5
B) skin gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4
A), IL-6 (Figure 4
B), KC (Figure 4
C) and CCL4 (Figure 4
D) skin gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5
A) and NT (Figure 5
B) skin gene expression using qPCR.
More specifically, TNF-α gene expression in the skin of poly(I:C)/swim-treated mice on low isoflavones, was increased (5 ± 0.9) compared to their controls (1.1 ± 0.4, P = 0.0006) (Figure 4A) and high isoflavones decreased this increase in TNF-α gene expression (2.3 ± 1.3, P = 0.0101) (Figure 4A). Likewise, IL-6 gene expression in the skin of poly(I:C)-treated mice on low isoflavones was increased (14 ± 4 and 22 ± 16) compared to their controls (0.9 ± 0.8, P = 0.0006 and 1 ± 0.4, P = 0.0012, respectively) (Figure 4B). Although poly(I:C)-treated mice on high isoflavones still had increased IL-6 gene expression (5.5 ± 3 and 3.4 ± 1.7) compared to their controls (1.4 ± 0.7, P = 0.0079 and 0.7 ± 0.4, P = 0.0159, respectively), high isoflavones reduced this increase in the IL-6 gene expression (5.5 ± 3 P = 0.0101) (Figure 4B).
Similarly, KC gene expression in the skin of poly(I:C)-treated mice on low isoflavones was also increased (53 ± 55 and 69 ± 83) compared to their controls (0.3 ± 0.2, P = 0.0021 and 1 ± 1.5, P = 0.0012, respectively) (Figure 4C). High isoflavones reduced this increase in the KC gene expression of poly(I:C)-treated mice (2.8 ± 3.5, P = 0.0057 and 3.4 ± 1.9, P = 0.0043) (Figure 4C).
CCL4 gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (24 ± 15 and 21 ± 19) compared to their controls (0.4 ± 0.3, P = 0.0021 and 1 ± 1.7, P = 0.0023, respectively) (Figure 4D). CCL4 gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (20 ± 24 and 27 ± 20) compared to their controls (0.8 ± 0.7, P = 0.0079 and 1 ± 1.3, P = 0.0079, respectively) and high isoflavones did have an effect on reducing this increase.
In the same way, HDC gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (4 ± 2 and 4 ± 2) compared to their controls (0.6 ± 0.4, P = 0.0003 and 1 ± 0.8, P = 0.0123, respectively) (Figure 5A). HDC gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (3 ± 1.1 and 3 ± 1) compared to their controls (0.9 ± 0.3, P = 0.0079 and 1 ± 0.2, P = 0.0317, respectively).
Similar to HDC gene expression, NT gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (8 ± 4 and 9 ± 10) compared to their controls (0.6 ± 0.4, P = 0.0025 and 1 ± 0.7, P = 0.0012, respectively) (Figure 5B). NT gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (8 ± 4, and 6 ± 6) compared to their controls (1.9 ± 0.8, P = 0.0317 and 1 ± 0.2, P = 0.0079, respectively).
Finally, poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the skin of the mice. High isoflavones did not have any statistical effect on CCL4, CCL2, CCL5, CXCL10, HDC and NT skin gene expression (Additional file 5: Table S4).
TNF-α (Figure 4A), IL-6 (Figure 4B), KC (Figure 4C), CCL4 (Figure 4D), as well as CCL2, CCL5 and CXCL10 (Additional file 5: Table S4) skin gene expression were increased in the poly(I:C)-treated mice. Moreover, HDC and NT gene expression were also increased in the skin (Figure 5A and B). High isoflavone diet reduced the increased TNF-α, IL-6 and KC skin gene expression.Figure 4
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4
A), IL-6 (Figure 4
B), KC (Figure 4
C) and CCL4 (Figure 4
D) skin gene expression using qPCR.Figure 5
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5
A) and NT (Figure 5
B) skin gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4
A), IL-6 (Figure 4
B), KC (Figure 4
C) and CCL4 (Figure 4
D) skin gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5
A) and NT (Figure 5
B) skin gene expression using qPCR.
More specifically, TNF-α gene expression in the skin of poly(I:C)/swim-treated mice on low isoflavones, was increased (5 ± 0.9) compared to their controls (1.1 ± 0.4, P = 0.0006) (Figure 4A) and high isoflavones decreased this increase in TNF-α gene expression (2.3 ± 1.3, P = 0.0101) (Figure 4A). Likewise, IL-6 gene expression in the skin of poly(I:C)-treated mice on low isoflavones was increased (14 ± 4 and 22 ± 16) compared to their controls (0.9 ± 0.8, P = 0.0006 and 1 ± 0.4, P = 0.0012, respectively) (Figure 4B). Although poly(I:C)-treated mice on high isoflavones still had increased IL-6 gene expression (5.5 ± 3 and 3.4 ± 1.7) compared to their controls (1.4 ± 0.7, P = 0.0079 and 0.7 ± 0.4, P = 0.0159, respectively), high isoflavones reduced this increase in the IL-6 gene expression (5.5 ± 3 P = 0.0101) (Figure 4B).
Similarly, KC gene expression in the skin of poly(I:C)-treated mice on low isoflavones was also increased (53 ± 55 and 69 ± 83) compared to their controls (0.3 ± 0.2, P = 0.0021 and 1 ± 1.5, P = 0.0012, respectively) (Figure 4C). High isoflavones reduced this increase in the KC gene expression of poly(I:C)-treated mice (2.8 ± 3.5, P = 0.0057 and 3.4 ± 1.9, P = 0.0043) (Figure 4C).
CCL4 gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (24 ± 15 and 21 ± 19) compared to their controls (0.4 ± 0.3, P = 0.0021 and 1 ± 1.7, P = 0.0023, respectively) (Figure 4D). CCL4 gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (20 ± 24 and 27 ± 20) compared to their controls (0.8 ± 0.7, P = 0.0079 and 1 ± 1.3, P = 0.0079, respectively) and high isoflavones did have an effect on reducing this increase.
In the same way, HDC gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (4 ± 2 and 4 ± 2) compared to their controls (0.6 ± 0.4, P = 0.0003 and 1 ± 0.8, P = 0.0123, respectively) (Figure 5A). HDC gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (3 ± 1.1 and 3 ± 1) compared to their controls (0.9 ± 0.3, P = 0.0079 and 1 ± 0.2, P = 0.0317, respectively).
Similar to HDC gene expression, NT gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (8 ± 4 and 9 ± 10) compared to their controls (0.6 ± 0.4, P = 0.0025 and 1 ± 0.7, P = 0.0012, respectively) (Figure 5B). NT gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (8 ± 4, and 6 ± 6) compared to their controls (1.9 ± 0.8, P = 0.0317 and 1 ± 0.2, P = 0.0079, respectively).
Finally, poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the skin of the mice. High isoflavones did not have any statistical effect on CCL4, CCL2, CCL5, CXCL10, HDC and NT skin gene expression (Additional file 5: Table S4).
Effect of poly(I:C) and isoflavones on locomotor activity:
Poly(I:C)/swim and poly(I:C)/no swim-treated mice on low isoflavones had reduced maximum (max) locomotor activity (over the 10-hour night period), denoted by the dark bar (P = 0.008 and P = 0.036) compared to the control/swim and control/no swim-treated mice, respectively (Figure 1A). High isoflavone chow reversed this decrease (Figure 1B), and the difference between the two diets was statistically significant (P = 0.032).Figure 1
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1
A) or high (Figure 1
B) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on mouse locomotor activity. Mice were provided with either low (Figure 1
A) or high (Figure 1
B) isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight.
Effect of poly(I:C) and isoflavones on serum inflammatory mediators:
Poly(I:C)-treated mice on low isoflavones had increased serum levels of TNF-α (Figure 2A), IL-6 (Figure 2B), KC (Figure 2C), and CCL4 (Figure 2D), as well as CCL2, CCL3, CCL5, and CXCL10 (Additional file 2: Table S2). High isoflavones reduced the poly(I:C)-increased serum levels of all the inflammatory markers. All IFNγ, IL-1β, IL-4, IL-9, IL-10, IL-12p70, IL-17 and VEGFα serum levels were below the detection limit, while IL-1α serum levels were similar between the different treatment groups. Swim stress augmented the poly(I:C) effect on TNF-α, CCL4, and CCL5 serum levels.Figure 2
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2
A), IL-6 (Figure 2
B), KC (Figure 2
C), and CCL4 (Figure 2
D) using the Milliplex microbead assay.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on serum levels of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, serum samples were collected and analyzed for TNF-α (Figure 2
A), IL-6 (Figure 2
B), KC (Figure 2
C), and CCL4 (Figure 2
D) using the Milliplex microbead assay.
More specifically, TNF-α serum levels in poly(I:C)/swim-treated mice on low isoflavones were increased (55 ± 14 pg/ml) compared to control/swim-treated mice (32 ± 0 pg/ml, P = 0.0011) and poly(I:C)/no swim-treated mice (37 ± 10 pg/ml,P = 0.0221). High isoflavones decreased these high TNF-α serum levels (32 ± 0 pg/ml, P = 0.0042) (Figure 2A). IL-6 serum levels in poly(I:C)-swim and poly(I:C)/no swim-treated mice on low isoflavones were also increased (229 ± 110 pg/ml and 141 ± 11 pg/ml) compared to the corresponding control/swim and control/no swim mice on low isoflavones (32 ± 0 pg/ml, P = 0.0011 and 32 ± 0 pg/ml, P = 0.0114) (Figure 2B). High isoflavones reduced IL-6 serum levels of poly(I:C)/swim-treated mice (35 ± 5 pg/ml, P = 0.0054) and poly(I:C)/no swim-treated (32 ± 0 pg/ml, P = 0.0298) compared to the corresponding groups on low isoflavones (Figure 2B).
Similarly, KC serum levels in poly(I:C)-treated mice on low isoflavones were increased (735 ± 579 pg/ml and 626 ± 523 pg/ml) compared to the corresponding controls (173 ± 43 pg/ml, P = 0.0379 and 132 ± 52 pg/ml, P = 0.0006, respectively) (Figure 2C). Although KC serum levels in poly(I:C)/swim-treated mice on high isoflavones were also increased (451 ± 107 pg/ml) compared to control poly(I:C)/swim-treated mice (100 ± 45 pg/ml, P = 0.0079), high isoflavone diet decreased KC serum levels in poly(I:C)/no swim-treated mice (128 ± 43 pg/ml, P = 0.0025) compared to those on low isoflavones (Figure 2C).
Moreover, CCL4 serum levels were increased in poly(I:C)-treated mice on low isoflavones (1,256 ± 556 pg/ml and 395 ± 245 pg/ml) compared to the corresponding controls (160 ± 0 pg/ml, P = 0.0011 and 160 ± 0 pg/ml, P = 0.0037, respectively) (Figure 2D). Swim further increased CCL4 serum levels (1,256 ± 556 pg/ml, P = 0.0023). Although CCL4 serum levels in poly(I:C)-treated mice on high isoflavones were also increased (217 ± 55 pg/ml and 265 ± 111 pg/ml) compared to their controls (160 ± 0 pg/ml, P = 0.0075 and 160 ± 0 pg/ml, P = 0.0254, respectively)., high isoflavones decreased CCL4 serum levels in poly(I:C)/swim-treated mice (217 ± 55 pg/ml, P = 0.0025) (Figure 2D).
Poly(I:C) treatment also increased CCL2, CCL3, CCL5 and CXCL10 serum levels and high isoflavones reduced those increases (Additional file 3).
Effect of poly(I:C) and isoflavones on brain gene expression of inflammatory mediators:
TNF-α (Figure 3A), IL-6 (Figure 3B), KC (Figure 3C), CCL4 (Figure 3D), as well as CCL2, CCL5 and CXCL10 brain gene expression were increased in the poly(I:C)-treated mice. High isoflavones reduced the increased TNF, IL6, KC, CCL4 and CCL2 brain gene expression. There was no difference in HDC and NT gene expression between the different treatment groups (Additional file 4: Table S3).Figure 3
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3
A), IL-6 (Figure 3
B), KC (Figure 3
C), and CCL4 (Figure 3
D) brain gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on brain gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, brain samples were collected and analyzed for TNF-α (Figure 3
A), IL-6 (Figure 3
B), KC (Figure 3
C), and CCL4 (Figure 3
D) brain gene expression using qPCR.
More specifically TNF-α gene expression was increased in the brain of poly(I:C)-treated mice on low isoflavones (relative fold change of 22 ± 7 and 12 ± 9) compared to their controls (1.1 ± 0.9, P = 0.0006 and 1 ± 0.7, P = 0.0012, respectively) (Figure 3A). Although poly(I:C)-treated mice on high isoflavones had increased TNF-α brain gene expression (6 ± 1 and 9 ± 8) compared to their corresponding controls (1.2 ± 0.5, P = 0.0079 and 1 ± 0.3, P = 0.0079, respectively), high isoflavones decreased TNF-α gene expression in the brain of poly(I:C)/swim-treated mice (6 ± 1, P = 0.0025) compared to those on low isoflavones (Figure 3A). There was no effect of poly(I:C) treatment on IL-6 brain gene expression (Figure 3B), but high isoflavones still decreased IL-6 gene expression in the brain of poly(I:C)/swim-treated mice (0.5 ± 0.1, P = 0.0228) compared to those on low isoflavones (2.1 ± 2.4) (Figure 3B).
KC gene expression in the brain of poly(I:C)-treated mice on low isoflavones was increased (18 ± 13 and 40 ± 52) compared to their controls (0.9 ± 0.3, P = 0.0006 and 1 ± 0.3, P = 0.0183, respectively) (Figure 3C). Although poly(I:C)/swim-treated mice on high isoflavones still had increased KC gene expression in their brain (2 ± 0.9) compared to control/swim-treated mice (0.6 ± 0.1, P = 0.0159), high isoflavones reduced this increase in KC gene expression (2 ± 0.9, P = 0.0177) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3C).
Likewise, CCL4 gene expression in the brain of poly(I:C)-treated mice on low isoflavones was also increased (6.5 ± 4 and 2.9 ± 1.6) compared to their controls (1 ± 0.3, P = 0.0006 and 1 ± 0.4, P = 0.0350, respectively) (Figure 3D). Although, poly(I:C)/swim-treated mice on high isoflavones still had increased CCL4 gene expression (1.8 ± 0.4) compared to control/swim-treated mice (1 ± 0.4, P = 0.0317), high isoflavones reduced this increase in CCL4 gene expression (1.8 ± 0.4, P = 0.0101) compared to poly(I:C)/swim-treated mice on low isoflavones (Figure 3D). Poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the brains of the mice. High isoflavones reduced this noted CCL2 brain gene expression increase (Additional file 3).
Effect of poly(I:C) and isoflavones on skin gene expression of inflammatory mediators:
TNF-α (Figure 4A), IL-6 (Figure 4B), KC (Figure 4C), CCL4 (Figure 4D), as well as CCL2, CCL5 and CXCL10 (Additional file 5: Table S4) skin gene expression were increased in the poly(I:C)-treated mice. Moreover, HDC and NT gene expression were also increased in the skin (Figure 5A and B). High isoflavone diet reduced the increased TNF-α, IL-6 and KC skin gene expression.Figure 4
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4
A), IL-6 (Figure 4
B), KC (Figure 4
C) and CCL4 (Figure 4
D) skin gene expression using qPCR.Figure 5
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5
A) and NT (Figure 5
B) skin gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for TNF-α (Figure 4
A), IL-6 (Figure 4
B), KC (Figure 4
C) and CCL4 (Figure 4
D) skin gene expression using qPCR.
Polyinosinic:polycytidylic acid (poly(I:C)) and isoflavone effect on skin gene expression of inflammatory mediators. Mice were provided with either low or high isoflavone diet ad libitum. Mice were injected intraperitoneally (ip) with 20 mg/kg poly(I:C) and were forced to swim for 15 minutes. Following this, they were individually placed into specific cages and locomotor activity was monitored overnight. The next day, skin samples were collected and analyzed for HDC (Figure 5
A) and NT (Figure 5
B) skin gene expression using qPCR.
More specifically, TNF-α gene expression in the skin of poly(I:C)/swim-treated mice on low isoflavones, was increased (5 ± 0.9) compared to their controls (1.1 ± 0.4, P = 0.0006) (Figure 4A) and high isoflavones decreased this increase in TNF-α gene expression (2.3 ± 1.3, P = 0.0101) (Figure 4A). Likewise, IL-6 gene expression in the skin of poly(I:C)-treated mice on low isoflavones was increased (14 ± 4 and 22 ± 16) compared to their controls (0.9 ± 0.8, P = 0.0006 and 1 ± 0.4, P = 0.0012, respectively) (Figure 4B). Although poly(I:C)-treated mice on high isoflavones still had increased IL-6 gene expression (5.5 ± 3 and 3.4 ± 1.7) compared to their controls (1.4 ± 0.7, P = 0.0079 and 0.7 ± 0.4, P = 0.0159, respectively), high isoflavones reduced this increase in the IL-6 gene expression (5.5 ± 3 P = 0.0101) (Figure 4B).
Similarly, KC gene expression in the skin of poly(I:C)-treated mice on low isoflavones was also increased (53 ± 55 and 69 ± 83) compared to their controls (0.3 ± 0.2, P = 0.0021 and 1 ± 1.5, P = 0.0012, respectively) (Figure 4C). High isoflavones reduced this increase in the KC gene expression of poly(I:C)-treated mice (2.8 ± 3.5, P = 0.0057 and 3.4 ± 1.9, P = 0.0043) (Figure 4C).
CCL4 gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (24 ± 15 and 21 ± 19) compared to their controls (0.4 ± 0.3, P = 0.0021 and 1 ± 1.7, P = 0.0023, respectively) (Figure 4D). CCL4 gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (20 ± 24 and 27 ± 20) compared to their controls (0.8 ± 0.7, P = 0.0079 and 1 ± 1.3, P = 0.0079, respectively) and high isoflavones did have an effect on reducing this increase.
In the same way, HDC gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (4 ± 2 and 4 ± 2) compared to their controls (0.6 ± 0.4, P = 0.0003 and 1 ± 0.8, P = 0.0123, respectively) (Figure 5A). HDC gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (3 ± 1.1 and 3 ± 1) compared to their controls (0.9 ± 0.3, P = 0.0079 and 1 ± 0.2, P = 0.0317, respectively).
Similar to HDC gene expression, NT gene expression was increased in the skin of poly(I:C)-treated mice on low isoflavones (8 ± 4 and 9 ± 10) compared to their controls (0.6 ± 0.4, P = 0.0025 and 1 ± 0.7, P = 0.0012, respectively) (Figure 5B). NT gene expression was also increased in the skin of poly(I:C)-treated mice on high isoflavones (8 ± 4, and 6 ± 6) compared to their controls (1.9 ± 0.8, P = 0.0317 and 1 ± 0.2, P = 0.0079, respectively).
Finally, poly(I:C) treatment also increased CCL2, CCL5 and CXCL10 gene expression in the skin of the mice. High isoflavones did not have any statistical effect on CCL4, CCL2, CCL5, CXCL10, HDC and NT skin gene expression (Additional file 5: Table S4).
Discussion:
Here we show that poly(I:C) significantly reduced locomotor activity over the first 24 hours, in comparison to control mice. Forced swim did not have any effect on its own, but augmented the effect of poly(I:C) on increasing TNF-α, CCL3 and CCL5 serum levels. We also used BALBc mice in an effort to investigate the possibility of strain differences, but there was no difference from our findings with C57BL/6 mice (results not shown).
Use of poly(I:C) increased serum levels of molecules that have been associated with inflammation and fatigue including TNF-α, IL-6, KC, CCL2, CCL3, CCL4, CCL5, and CXCL10. Moreover, poly(I:C) also increased brain and skin gene expression of TNF-α, IL-6, KC, CCL2, CCL4, CCL5, CXCL10, while HDC and NT gene expression were only increased in the skin, suggesting that NT and histamine may explain the skin findings in CFS patients. Forced swim stress had no additional effect to that of poly(I:C), except for augmenting TNF-α serum levels. TNF-α and IL-6 serum levels were actually increased in CFS [20,44]. Such pro-inflammatory mediators can increase blood-brain barrier (BBB) permeability [45] and permit entry of circulating leukocytes leading to brain inflammation. TNF-α was shown to be released along with histamine from rat brain MC [46], and was involved in brain inflammation [47]. MC can also interact with T cells [48-50] and superactivate them through TNF-α [51].
Unlike our present results, one group reported that daily forced swim stress for two to three weeks, with or without an immunological trigger administered on day one (lipopolysaccharide or Brucella abortus antigen), induced ‘chronic fatigue’, but only in Albino laca mice [52-55] and Wistar rats [56]. In these papers, behavioral parameters, such as immobility time, time to start grooming after swim stress, roda rod test, and elevated plus maze test were increased indicating post stress fatigue and anxiety. Also, biochemical measurements indicative of brain oxidative stress were increased. In another paper, BALBc mice were injected with Brucella abortus and developed decreased running activity that lasted one week [57]. Forced swim of Charles Foster albino rats for twenty-one days also increased immobility time, anxiety as assessed by elevated plus maze test, and brain oxidative stress [58]. These findings maybe due to the differences in the strains and triggers used.
Chemokines interact with their receptors and attract immune cells to inflammation sites [59-61]. CXCR1, CXCR2, CXCR3, CXCR4, CX3CR1, CCR1, CCR3, CCR4 and CCR5 are expressed by human MC of different origins [62]. Stimulated MC release CCL2, CCL3, CCL4 [63]. Murine fetal skin-derived cultured MC (FSMCs) release CCL3, CCL4, CCL5, IL-6 and TNF-α upon TLR3-poly(I:C) activation [64,65]. Peritoneal MC from C57BL/6 mice activated by poly(I:C)-TLR3 have increased CCL5 and CXCL10 expression [66,67].
Here we also show that poly(I:C) with or without NT or CRH did not have any effect on TNF-α, CXCL8 and VEGFα release from human cultured LAD2 MC (results not shown), but increased only TNF-α gene expression when used together with CRH, NT, or SP. Poly(I:C) alone did not have any effect on human MC, but increased TNF-α gene expression at 24 hours when used together with CRH or NT.
Toll-like receptors’ (TLRs) [68,69] activation is important in the development of innate immunity to invading pathogens, leading to release of different cytokines [64,70,71]. Antigen-mediated MC reactivity is amplified through prolonged TLR-ligand treatment [72].
Human umbilical cord blood-derived MC express TLR-3, activation of which produced IFNα and IFNβ in response to double-stranded RNA [73]. A recent publication reported that MC respond to intracellular, but not extracellular, poly(I:C) by inducing mainly IFNα and TNF-α; moreover, infection of MC with live Sendai virus induces an anti-viral response similar to that of intracellular poly(I:C) [74].
We also used CRHR-1 knockout (KO) mice in order to investigate the possible involvement of CRHR in any stress effect. However, forced swim stress alone did not have an effect and there was no difference using these mice (results not shown). Nevertheless, rats exposed to water immersion stress had a four-fold increase in plasma histamine levels that was absent in W/Wv MC-deficient rats [75]. Acute stress also increased serum histamine and IL-6 levels, both of which were also absent in MC deficient W/Wv mice [76].
Here we also show that the isoflavones genistein and daidzein reversed the effect of poly(I:C) on mice. Genistein has been reported to attenuate muscle fatigue [77], protect against endothelial barrier dysfunction [78] and suppress LPS-induced inflammatory response in macrophages [79]. Isoflavones also suppress MC expression of the high affinity IgE receptor (FcεRI) [80]. However, isoflavones have estrogenic activity and may not be desirable in certain clinical settings. The flavonoids epigallocatechin, naringin, and curcumin ameliorated ‘chronic fatigue’ [53-56]. Other papers reported similar effects for the Astragalus flavonoids [81] and for the olive extract [82].
Flavonoids exert potent anti-inflammatory effects via various pathways [83-87]. A review of human randomized controlled trial studies summarized some significant benefits to cognitive function after isoflavone supplementation [88]: improvements in executive function, working memory and processing speed [88]. Specifically, two studies reported significant effects of 60 mg/day treatment with isoflavones in processing speed and psychomotor speed [89,90].
Quercetin increases exercise tolerance in mice [91]. Oral administration of quercetin leads to accumulation in brain tissue and attenuates the increased oxidative stress in the hippocampus and striatum of rats exposed to chronic forced swimming [92,93]. Quercetin has potent anti-oxidant and anti-inflammatory activity [41,42], and inhibits MC degranulation [94,95], as well as TNF-α, IL-6, and IL-8 secretion [95,96]. Moreover, it reverses acute stress-induced behavioral changes and reduces brain glutathione levels in mice [97].
Recent studies with antigen-stimulated MC show that epigallocatechin gallate (EGCG) inhibits MC degranulation, leukotriene C4 secretion, as well as the production of TNF-α, IL-6 and IL-8 [94,96]. The quercetin-related flavone luteolin inhibits MC activation [98] and MC-dependent stimulation of activated T cells [51]. Luteolin also inhibits IL-6 release from microglia cells [99] and from astrocytes [100]. Recent reviews have addressed the possible use of flavonoids for the treatment of CNS diseases [101,102].
Increasing evidence implicates CNS inflammation [103], as well as MC-microglia interactions, in neuropsychiatric diseases [104,105]. MC are important for allergic reactions, but also in immunity [106,107], and inflammatory diseases [15,108]. TLRs have also been implicated in CNS dysfunction through MC and glial activation [104].
Conclusion:
Here we report that treatment of mice with poly(I:C) resulted in reduced locomotor activity and increased inflammatory markers in serum, brain and skin, all of which were reversed by treatment with isoflavones. Our findings also demonstrate significant variability among mouse ‘models’ as was recently reported [109]. There is need for the establishment of a more reliable animal model for CFS. Nevertheless, certain flavonoids appear promising for use in pilot clinical trials with CFS patients.
|
Background:
Chronic Fatigue Syndrome (CFS) is a neuroimmunoendocrine disease affecting about 1% of the US population, mostly women. It is characterized by debilitating fatigue for six or more months in the absence of cancer or other systemic diseases. Many CFS patients also have fibromyalgia and skin hypersensitivity that worsen with stress. Corticotropin-releasing hormone (CRH) and neurotensin (NT), secreted under stress, activate mast cells (MC) necessary for allergic reactions to release inflammatory mediators that could contribute to CFS symptoms.
Methods:
Female C57BL/6 mice were randomly divided into four groups: (a) control/no-swim, (b) control/swim, (c) polyinosinic:polycytidylic acid (poly(I:C))/no swim, and (d) polyinosinic:polycytidylic acid (poly(I:C))/swim. Mice were provided with chow low or high in isoflavones for 2 weeks prior to ip injection with 20 mg/kg poly(I:C) followed or not by swim stress for 15 minutes. Locomotor activity was monitored overnight and animals were sacrificed the following day. Brain and skin gene expression, as well as serum levels, of inflammatory mediators were measured. Data were analyzed using the non-parametric Mann-Whitney U-test.
Results:
Poly(I:C)-treated mice had decreased locomotor activity over 24 hours, and increased serum levels of TNF-α, IL-6, KC (IL-8/CXCL8 murine homolog), CCL2,3,4,5, CXCL10, as well as brain and skin gene expression of TNF, IL-6, KC (Cxcl1, IL8 murine homolog), CCL2, CCL4, CCL5 and CXCL10. Histidine decarboxylase (HDC) and NT expression were also increased, but only in the skin, over the same period. High isoflavone diet reversed these effects.
Conclusions:
Poly(I:C) treatment decreased mouse locomotor activity and increased serum levels and brain and skin gene expression of inflammatory mediators. These effects were inhibited by isoflavones that may prove useful in CFS.
|
Background:
Chronic Fatigue Syndrome (CFS) is a complex disease, which has also been called ‘neurasthenia’, ‘post viral fatigue’, and ‘chronic mononucleosis’ [1-3]. Its prevalence may be as high as 1% in the US population [4] with a female to male ratio of 4 to 1 [5]. CFS involves the muscular, nervous, hormonal and immune systems. Patients complain of overwhelming fatigue, sleep disturbances, malaise, muscle aches, gastrointestinal symptoms, dizziness on standing, and cognitive problems [6]. Clinical and subclinical viral infections have been suspected, but never confirmed [7,8].
CFS is often comorbid with other disorders, including fibromyalgia, Pelvic Bladder Syndrome/Interstitial Cystitis (PBS/IC), irritable bowel syndrome (IBS), and migraines [9], all of which are characterized by central nervous system (CNS) dysfunction [10], and worsened by stress [11-15]. Many CFS patients demonstrate abnormal hypothalamic-pituitary-adrenal (HPA) axis activity [16,17]. Anxiety is common in CFS [18] and patients are particularly vulnerable to stress [14]. Many CFS symptoms could derive from the possible release of inflammatory mediators that could affect brain function [19,20]. As of today, there are no FDA approved drugs for the treatment of CFS [21]; psychological, physical, and pharmacological interventions used currently are not very effective [22].
An abnormal immune component may be involved in CFS [23-25], but the neuroimmune and neuroendocrine interactions involved are still unknown [26]. Mast cells (MC) and their mediators have been implicated in all diseases that are comorbid with CFS [9]. There is higher number of skin MC in patients with CFS [27,28], and such patients also show increased skin hypersensitivity [29]. Furthermore, CFS also occurs more often in patients with chronic urticaria, that also involves MC [30]. In fact, there is hyperresponsiveness in the bronchi of CFS patients, implying MC activation [31].
Activated MC release a number of chemokines and cytokines that could contribute to CFS symptoms [32,33]. MC are located perivascularly in close proximity to neurons [34], especially in the diencephalon [35,36], where functional MC-neuron interactions have been documented [36,37] in response to corticotropin-releasing hormone (CRH) [38]. In vivo activation of MC by CRH is augmented by neurotensin (NT) [39]. Moreover, NT is induced in the hypothalamus in response to bacterial lipopolysaccharide (LPS) and regulates the HPA axis [40].
Unfortunately, there are neither effective CFS treatments nor human MC inhibitors clinically available that may also be used in CFS. Flavonoids are natural compounds with strong antioxidant and anti-inflammatory activity [41]. Certain flavonoids also inhibit MC [42] and have neuroprotective effects [41,43].
Here we report that treatment of mice with poly(I:C) results in reduced locomotor activity and increased serum levels, as well as brain and gene expression, of inflammatory mediators, all of which are reversed by treatment with the isoflavones daidzein and genistein.
Conclusion:
Here we report that treatment of mice with poly(I:C) resulted in reduced locomotor activity and increased inflammatory markers in serum, brain and skin, all of which were reversed by treatment with isoflavones. Our findings also demonstrate significant variability among mouse ‘models’ as was recently reported [109]. There is need for the establishment of a more reliable animal model for CFS. Nevertheless, certain flavonoids appear promising for use in pilot clinical trials with CFS patients.
|
Background:
Chronic Fatigue Syndrome (CFS) is a neuroimmunoendocrine disease affecting about 1% of the US population, mostly women. It is characterized by debilitating fatigue for six or more months in the absence of cancer or other systemic diseases. Many CFS patients also have fibromyalgia and skin hypersensitivity that worsen with stress. Corticotropin-releasing hormone (CRH) and neurotensin (NT), secreted under stress, activate mast cells (MC) necessary for allergic reactions to release inflammatory mediators that could contribute to CFS symptoms.
Methods:
Female C57BL/6 mice were randomly divided into four groups: (a) control/no-swim, (b) control/swim, (c) polyinosinic:polycytidylic acid (poly(I:C))/no swim, and (d) polyinosinic:polycytidylic acid (poly(I:C))/swim. Mice were provided with chow low or high in isoflavones for 2 weeks prior to ip injection with 20 mg/kg poly(I:C) followed or not by swim stress for 15 minutes. Locomotor activity was monitored overnight and animals were sacrificed the following day. Brain and skin gene expression, as well as serum levels, of inflammatory mediators were measured. Data were analyzed using the non-parametric Mann-Whitney U-test.
Results:
Poly(I:C)-treated mice had decreased locomotor activity over 24 hours, and increased serum levels of TNF-α, IL-6, KC (IL-8/CXCL8 murine homolog), CCL2,3,4,5, CXCL10, as well as brain and skin gene expression of TNF, IL-6, KC (Cxcl1, IL8 murine homolog), CCL2, CCL4, CCL5 and CXCL10. Histidine decarboxylase (HDC) and NT expression were also increased, but only in the skin, over the same period. High isoflavone diet reversed these effects.
Conclusions:
Poly(I:C) treatment decreased mouse locomotor activity and increased serum levels and brain and skin gene expression of inflammatory mediators. These effects were inhibited by isoflavones that may prove useful in CFS.
| 17,988 | 380 | 17 |
[
"poly",
"mice",
"figure",
"gene",
"swim",
"expression",
"gene expression",
"isoflavones",
"treated",
"treated mice"
] |
[
"test",
"test"
] | null |
[CONTENT] Brain | Fatigue | Inflammation | Isoflavones | Mast cells | Polyinosinic:polycytidylic acid | Skin | Stress | Swim [SUMMARY]
| null |
[CONTENT] Brain | Fatigue | Inflammation | Isoflavones | Mast cells | Polyinosinic:polycytidylic acid | Skin | Stress | Swim [SUMMARY]
|
[CONTENT] Brain | Fatigue | Inflammation | Isoflavones | Mast cells | Polyinosinic:polycytidylic acid | Skin | Stress | Swim [SUMMARY]
|
[CONTENT] Brain | Fatigue | Inflammation | Isoflavones | Mast cells | Polyinosinic:polycytidylic acid | Skin | Stress | Swim [SUMMARY]
|
[CONTENT] Brain | Fatigue | Inflammation | Isoflavones | Mast cells | Polyinosinic:polycytidylic acid | Skin | Stress | Swim [SUMMARY]
|
[CONTENT] Animals | Biomarkers | Brain | Fatigue Syndrome, Chronic | Female | Inflammation Mediators | Isoflavones | Mice | Mice, Inbred C57BL | Motor Activity | Poly I-C | Skin | Swimming [SUMMARY]
| null |
[CONTENT] Animals | Biomarkers | Brain | Fatigue Syndrome, Chronic | Female | Inflammation Mediators | Isoflavones | Mice | Mice, Inbred C57BL | Motor Activity | Poly I-C | Skin | Swimming [SUMMARY]
|
[CONTENT] Animals | Biomarkers | Brain | Fatigue Syndrome, Chronic | Female | Inflammation Mediators | Isoflavones | Mice | Mice, Inbred C57BL | Motor Activity | Poly I-C | Skin | Swimming [SUMMARY]
|
[CONTENT] Animals | Biomarkers | Brain | Fatigue Syndrome, Chronic | Female | Inflammation Mediators | Isoflavones | Mice | Mice, Inbred C57BL | Motor Activity | Poly I-C | Skin | Swimming [SUMMARY]
|
[CONTENT] Animals | Biomarkers | Brain | Fatigue Syndrome, Chronic | Female | Inflammation Mediators | Isoflavones | Mice | Mice, Inbred C57BL | Motor Activity | Poly I-C | Skin | Swimming [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] poly | mice | figure | gene | swim | expression | gene expression | isoflavones | treated | treated mice [SUMMARY]
| null |
[CONTENT] poly | mice | figure | gene | swim | expression | gene expression | isoflavones | treated | treated mice [SUMMARY]
|
[CONTENT] poly | mice | figure | gene | swim | expression | gene expression | isoflavones | treated | treated mice [SUMMARY]
|
[CONTENT] poly | mice | figure | gene | swim | expression | gene expression | isoflavones | treated | treated mice [SUMMARY]
|
[CONTENT] poly | mice | figure | gene | swim | expression | gene expression | isoflavones | treated | treated mice [SUMMARY]
|
[CONTENT] cfs | mc | patients | symptoms | syndrome | chronic | fatigue | axis | hpa axis | hpa [SUMMARY]
| null |
[CONTENT] figure | gene expression | gene | expression | isoflavones | poly | mice | treated | treated mice | pg ml [SUMMARY]
|
[CONTENT] cfs | clinical trials cfs patients | variability | reported 109 | appear promising use pilot | appear promising use | appear promising | variability mouse models recently | variability mouse models | variability mouse [SUMMARY]
|
[CONTENT] figure | mice | poly | swim | il | gene | treated | expression | gene expression | treated mice [SUMMARY]
|
[CONTENT] figure | mice | poly | swim | il | gene | treated | expression | gene expression | treated mice [SUMMARY]
|
[CONTENT] Fatigue Syndrome | CFS | about 1% | US ||| six or more months ||| CFS ||| [SUMMARY]
| null |
[CONTENT] 24 hours | TNF | IL-6 | KC | TNF | IL-6 | KC | Cxcl1 | CCL2 | CCL4 | CCL5 ||| ||| [SUMMARY]
|
[CONTENT] ||| CFS [SUMMARY]
|
[CONTENT] CFS | about 1% | US ||| six or more months ||| CFS ||| ||| four ||| 2 weeks | 20 | 15 minutes ||| overnight ||| ||| Mann-Whitney U-test ||| 24 hours | TNF | IL-6 | KC | TNF | IL-6 | KC | Cxcl1 | CCL2 | CCL4 | CCL5 ||| ||| ||| ||| CFS [SUMMARY]
|
[CONTENT] CFS | about 1% | US ||| six or more months ||| CFS ||| ||| four ||| 2 weeks | 20 | 15 minutes ||| overnight ||| ||| Mann-Whitney U-test ||| 24 hours | TNF | IL-6 | KC | TNF | IL-6 | KC | Cxcl1 | CCL2 | CCL4 | CCL5 ||| ||| ||| ||| CFS [SUMMARY]
|
|||||
Laparoscopic completely extraperitoneal repair of inguinal hernia in children: a single-institute experience with 1,257 repairs compared with cut-down herniorrhaphy.
|
19343444
|
Conventional open herniorrhaphy in children has been reported to have 0.3-3.8% recurrence and 5.6-30% postoperative contralateral hernia rates. We developed a unique technique to achieve completely extraperitoneal ligation of PPV without any skip areas under laparoscopic control. This report introduces our technique and results compared with the cut-down herniorrhaphy.
|
BACKGROUND
|
A consecutive series of 1,585 children with inguinal hernia/hydrocele (1996-2006) was analyzed. In laparoscopic patent processus vaginalis (PPV) closure (LPC), an orifice of PPV was encircled with a 2-0 suture extraperitoneally by a specially devised Endoneedle and tied up from outside of the body achieving completely extraperitoneal ligation of the ring. The round ligament was included in the ligation, whereas the spermatic cord and testicular vessels were excluded by advancing the needle across them behind the peritoneum. Cut-down herniorrhaphy (CD), with or without diagnostic laparoscopy, or LPC was selected according to parental preference under informed consent.
|
METHODS
|
Parents gave more preference to LPC (LPC in 1,257 children, CD in 308, and miscellaneous in 20). Age ranges were equal for both groups. Sex distribution showed female preponderance in the LPC group (44.8% vs. 26.6%, p < 0.001) and umbilical hernia/cysts were predominantly included in the LPC group (11.9% vs. 2.9%, p < 0.001). Mean operation times were equal for both groups for unilateral repair (28.2 +/- 9.2 for LPC vs. 27.8 +/- 13.5 for CD) and were shorter for bilateral repair in the LPC group (35.8 +/- 11.6 vs. 46.7 +/- 17.7). The incidence of postoperative hernia recurrence and contralateral hernia in the LPC group was 0.2% and 0.8%. Two children in the CD group had injuries to their reproductive system during the operation (0.6%).
|
RESULTS
|
The advantages of our technique include following: technically simple, short operation time, inspection of bilateral IIRs with simultaneous closure of cPPV, reproductive systems remain intact, routine addition of umbilicoplasty if desired, and essentially indiscernible wounds.
|
CONCLUSIONS
|
[
"Adolescent",
"Child",
"Child, Preschool",
"Fallopian Tubes",
"Female",
"Follow-Up Studies",
"Hernia, Inguinal",
"Humans",
"Incidence",
"Infant",
"Infant, Newborn",
"Intraoperative Complications",
"Laparoscopy",
"Laparotomy",
"Ligation",
"Male",
"Parents",
"Recurrence",
"Retrospective Studies",
"Round Ligament of Uterus",
"Suture Techniques",
"Testicular Hydrocele",
"Vas Deferens",
"Young Adult"
] |
2710496
| null | null | null | null |
Results
|
Parental perspective and choice Parents showed a greater preference for LPC, accounting for 1,257 children compared with 308 who underwent CD with or without diagnostic laparoscopy (CDA, 62; CDL, 246). The reasons why the parents chose LPC were postoperative cosmetic superiority, inspection for cPPV, and simultaneous repair if it was present, the ability for a second look at the previous operation site in cases of recurrence or contralateral occurrence, availability of simultaneous umbilicoplasty for umbilical hernia, or an ugly umbilicus. Diagnostic laparoscopy was selected for inspection for cPPV and simultaneous repair if it was present. The cut-down procedure was chosen because of strong disagreement of family members, including grandparents and relations based on fear of laparoscopic procedures, experience regarding siblings or other family members who had previously undergone cut-down herniorrhaphy with good results or no problems, and the lack of long-term follow-up data with the innovative technique.
Parents showed a greater preference for LPC, accounting for 1,257 children compared with 308 who underwent CD with or without diagnostic laparoscopy (CDA, 62; CDL, 246). The reasons why the parents chose LPC were postoperative cosmetic superiority, inspection for cPPV, and simultaneous repair if it was present, the ability for a second look at the previous operation site in cases of recurrence or contralateral occurrence, availability of simultaneous umbilicoplasty for umbilical hernia, or an ugly umbilicus. Diagnostic laparoscopy was selected for inspection for cPPV and simultaneous repair if it was present. The cut-down procedure was chosen because of strong disagreement of family members, including grandparents and relations based on fear of laparoscopic procedures, experience regarding siblings or other family members who had previously undergone cut-down herniorrhaphy with good results or no problems, and the lack of long-term follow-up data with the innovative technique.
Characteristics of patients who underwent each chosen procedure The data are shown in Table 1. Age ranges were equal at approximately 3 years for both the LPC and CD groups. Sex distribution showed female predominance in the LPC group (44.8% in LPC vs. 26.6% in CD, p < 0.001). Differences in the hernia side were not significant between the groups. No statistically different distributions of associated morbidities necessitating combined operation were seen, except umbilical hernia/cyst. Umbilical morbidities, such as umbilical hernia, cystic degeneration of the umbilicus, or an ugly-looking umbilicus, were seen four times more in the LPC group (p < 0.001).Table 1Characteristics of patients who underwent chosen procedureLPCCDDifferenceNo. of patients1,257308 (246 CDL, 62 CDA)Age (range)1 month to 24 years1 month to 22 years Mean ± SD3.8 ± 2.9 years3.7 ± 3.2 yearsNSSex694 males, 563 females226 males, 82 females % of females44.826.6p < 0.001Side of hernia745 right, 456 left, 56 bilateral177 right, 117 left, 14 bilateral % of lateralityRight (58.4), left (35.8), bilateral (4.4)Right (57.5), left (38), bilateral (4.5)NSAssociated morbidities necessitate combined operation Maldescended testis41 (3.3%)11 (3.6)NS Umbilical hernia/cyst149 (11.9%)9 (2.9%)p < 0.001 Visceral sliding/incarceration Omentum15 (1.2%)1 (0.3%)NS Bowel loop, cecum, appendix14 (1.1%)2 (0.6%)NS Ovarium, ovarian duct42 (3.3%)10 (3.2%)NS After primary herniorrhaphy Recurrence7 (0.6%)2 (0.6%)NS Contralateral hernia26 (2.1%)4 (1.3%)NS Miscellaneous15 (1.2%)9 (2.9%)NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Characteristics of patients who underwent chosen procedure
LPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
The data are shown in Table 1. Age ranges were equal at approximately 3 years for both the LPC and CD groups. Sex distribution showed female predominance in the LPC group (44.8% in LPC vs. 26.6% in CD, p < 0.001). Differences in the hernia side were not significant between the groups. No statistically different distributions of associated morbidities necessitating combined operation were seen, except umbilical hernia/cyst. Umbilical morbidities, such as umbilical hernia, cystic degeneration of the umbilicus, or an ugly-looking umbilicus, were seen four times more in the LPC group (p < 0.001).Table 1Characteristics of patients who underwent chosen procedureLPCCDDifferenceNo. of patients1,257308 (246 CDL, 62 CDA)Age (range)1 month to 24 years1 month to 22 years Mean ± SD3.8 ± 2.9 years3.7 ± 3.2 yearsNSSex694 males, 563 females226 males, 82 females % of females44.826.6p < 0.001Side of hernia745 right, 456 left, 56 bilateral177 right, 117 left, 14 bilateral % of lateralityRight (58.4), left (35.8), bilateral (4.4)Right (57.5), left (38), bilateral (4.5)NSAssociated morbidities necessitate combined operation Maldescended testis41 (3.3%)11 (3.6)NS Umbilical hernia/cyst149 (11.9%)9 (2.9%)p < 0.001 Visceral sliding/incarceration Omentum15 (1.2%)1 (0.3%)NS Bowel loop, cecum, appendix14 (1.1%)2 (0.6%)NS Ovarium, ovarian duct42 (3.3%)10 (3.2%)NS After primary herniorrhaphy Recurrence7 (0.6%)2 (0.6%)NS Contralateral hernia26 (2.1%)4 (1.3%)NS Miscellaneous15 (1.2%)9 (2.9%)NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Characteristics of patients who underwent chosen procedure
LPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Operative findings Regarding laparoscopic inspection of the contralateral IIR, the presence of cPPVs was more dominant in the LPC group than in the CDL group (47% vs. 21.6%, p < 0.001). Mean operation times for unilateral repair were equal in both LPC and CD groups (28.2 ± 9.2 min for LPC vs. 27.8 ± 13.5 min for CD) and were shorter for LPC in bilateral repair (35.8 ± 11.6 vs. 46.7 ± 17.7 min, p < 0.001). When comparing the CDL with the CDA, CDL took an average of 6 min longer for a unilateral and 8.6 min longer for a bilateral closure than CDA (p < 0.05). The difference between males and females was significant in the LPC group (p < 0.001), accounting for a 4.4-min increase for unilateral and a 6.9-min increase for bilateral closure in males (Table 2). In the LPC group, incidental umbilical hernia, ugly umbilicus, and other abnormalities, such as intraumbilical epidermoid cysts, also were repaired simultaneously during closure of the laparoscopic wound, whereas in the CD group an umbilicoplasty was performed as another definitive surgery.Table 2Operative findingsLPCCDDifferenceTotalCDLCDALaterality of PPV Right42514311132Left211997821Bilateral62166579Total187837430371% of contralateral PPV4721.6p < 0.001Operation time (mean ± SD, min) Unilateraln = 591n = 216n = 171n = 4528.2 ± 9.227.8 ± 13.529.1 ± 12.323.1 ± 16.8 DifferenceNSp < 0.05 Bilateraln = 542n = 60n = 50n = 1035.8 ± 11.646.7 ± 17.748.1 ± 18.339.5 ± 15 Differencep < 0.001p < 0.05Male versus femaleMaleFemale Unilateral LPCn = 34530 ± 8n = 24625.6 ± 10p < 0.001 Bilateral LPCn = 25339.5 ± 10.6n = 28932.6 ± 11.5p < 0.001 Unilateral CDn = 15428 ± 12.1n = 6227.3 ± 16.6NS Bilateral CDn = 4348.6 ± 18.5n = 1741.8 ± 14.1NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Operative findings
LPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
One boy in the LPC group had a small stab injury on the anterior wall of the rectum during the placement of a port for the grasper through the abdominal wall, which was immediately repaired with the laparoscopic technique without sequelae. A boy and a girl in the CD group had respective accidental severance of the spermatic duct and ovarian duct, which was anastomosed under surgical microscopy during the same session. There was a statistically significant difference in the incidence of intraoperative injuries to the reproductive system between the LPC and CD groups (0% vs. 0.6%, p < 0.005).
Regarding laparoscopic inspection of the contralateral IIR, the presence of cPPVs was more dominant in the LPC group than in the CDL group (47% vs. 21.6%, p < 0.001). Mean operation times for unilateral repair were equal in both LPC and CD groups (28.2 ± 9.2 min for LPC vs. 27.8 ± 13.5 min for CD) and were shorter for LPC in bilateral repair (35.8 ± 11.6 vs. 46.7 ± 17.7 min, p < 0.001). When comparing the CDL with the CDA, CDL took an average of 6 min longer for a unilateral and 8.6 min longer for a bilateral closure than CDA (p < 0.05). The difference between males and females was significant in the LPC group (p < 0.001), accounting for a 4.4-min increase for unilateral and a 6.9-min increase for bilateral closure in males (Table 2). In the LPC group, incidental umbilical hernia, ugly umbilicus, and other abnormalities, such as intraumbilical epidermoid cysts, also were repaired simultaneously during closure of the laparoscopic wound, whereas in the CD group an umbilicoplasty was performed as another definitive surgery.Table 2Operative findingsLPCCDDifferenceTotalCDLCDALaterality of PPV Right42514311132Left211997821Bilateral62166579Total187837430371% of contralateral PPV4721.6p < 0.001Operation time (mean ± SD, min) Unilateraln = 591n = 216n = 171n = 4528.2 ± 9.227.8 ± 13.529.1 ± 12.323.1 ± 16.8 DifferenceNSp < 0.05 Bilateraln = 542n = 60n = 50n = 1035.8 ± 11.646.7 ± 17.748.1 ± 18.339.5 ± 15 Differencep < 0.001p < 0.05Male versus femaleMaleFemale Unilateral LPCn = 34530 ± 8n = 24625.6 ± 10p < 0.001 Bilateral LPCn = 25339.5 ± 10.6n = 28932.6 ± 11.5p < 0.001 Unilateral CDn = 15428 ± 12.1n = 6227.3 ± 16.6NS Bilateral CDn = 4348.6 ± 18.5n = 1741.8 ± 14.1NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Operative findings
LPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
One boy in the LPC group had a small stab injury on the anterior wall of the rectum during the placement of a port for the grasper through the abdominal wall, which was immediately repaired with the laparoscopic technique without sequelae. A boy and a girl in the CD group had respective accidental severance of the spermatic duct and ovarian duct, which was anastomosed under surgical microscopy during the same session. There was a statistically significant difference in the incidence of intraoperative injuries to the reproductive system between the LPC and CD groups (0% vs. 0.6%, p < 0.005).
Postoperative findings In the LPC group, the operation was performed satisfactorily in all patients. Preoperative and postoperative photographs of the IIR are shown in Fig. 4, and cosmetic results were excellent with almost invisible scars. In second-look operation for postoperative contralateral hernia, the primarily closed IIR was found to be completely covered with thick cicatricial tissue (Fig. 4C). Postoperative hernia recurrence was seen in 2 of 1,257 patients (0.16%) and 1,878 PPVs (0.11%) in the LPC group, and in 2 of 308 patients (0.65%) and 374 PPVs (0.53%) in the CD group. Contralateral hernia developed in five of the LPC group (0.79% of unilateral PPV closure) and in four of the CD group (1.67%; 3 CDL (1.59%) and 1 CDA (1.89%)). The incidence of the postoperative recurrence and contralateral hernia were lower in the LPC group, but with no statistically significant difference. Postoperative direct hernia occurred in one patient in the CD group. As for minor postoperative complications, fugitive stitch granuloma occurred at the umbilicus in seven patients of the LPC group and at the crease incision site in one patient of the CD group. Transient fluid accumulation in a sac with omental remnant occurred in 1 LPC patient with omental incarceration. Postoperative testicular atrophy was found in none of the both groups.Fig. 4G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places
G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places
In the LPC group, the operation was performed satisfactorily in all patients. Preoperative and postoperative photographs of the IIR are shown in Fig. 4, and cosmetic results were excellent with almost invisible scars. In second-look operation for postoperative contralateral hernia, the primarily closed IIR was found to be completely covered with thick cicatricial tissue (Fig. 4C). Postoperative hernia recurrence was seen in 2 of 1,257 patients (0.16%) and 1,878 PPVs (0.11%) in the LPC group, and in 2 of 308 patients (0.65%) and 374 PPVs (0.53%) in the CD group. Contralateral hernia developed in five of the LPC group (0.79% of unilateral PPV closure) and in four of the CD group (1.67%; 3 CDL (1.59%) and 1 CDA (1.89%)). The incidence of the postoperative recurrence and contralateral hernia were lower in the LPC group, but with no statistically significant difference. Postoperative direct hernia occurred in one patient in the CD group. As for minor postoperative complications, fugitive stitch granuloma occurred at the umbilicus in seven patients of the LPC group and at the crease incision site in one patient of the CD group. Transient fluid accumulation in a sac with omental remnant occurred in 1 LPC patient with omental incarceration. Postoperative testicular atrophy was found in none of the both groups.Fig. 4G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places
G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places
|
Conclusions
|
Although we must currently accept that laparoscopic hernia repair has not been around long enough to rate fully the risk of late complications, we believe that this procedure with the Endoneedle can be a routine procedure with results comparable or superior to those with open procedures. The advantages of our technique include the following: technically simple, short operation time, inspection of bilateral IIRs with simultaneous closure of cPPV, the reproductive system remains intact, routine addition of umbilicoplasty if desired by the parents of patients, and essentially indiscernible wounds.
|
[
"Study design",
"Statistical analysis",
"Operative procedures",
"Parental perspective and choice",
"Characteristics of patients who underwent each chosen procedure",
"Operative findings",
"Postoperative findings"
] |
[
"A consecutive series of 1,585 children with inguinal hernia or hydrocele, or both, experienced during 1996–2006, was analyzed. Regarding the operative procedures, the cut-down procedure (CD) and laparoscopic PPV closure (LPC) were proposed to the parents of the patients. CD was further divided into CD for the affected side only (CDA) and CD with diagnostic laparoscopy (CDL). CDA, CDL, or LPC was selected according to parental preference under informed consent. The medical records of these children were analyzed in terms of parental selection, distribution of sex, age, presence of contralateral patent processus vaginalis (cPPV), operation time, and complications among the groups.\nTwenty patients who had various procedures during the period of development of laparoscopic herniorrhaphy were excluded from the analysis, and patients who underwent combined procedures affecting definitive herniorrhaphy also were excluded from the analysis of the operation time. The patients were followed up regularly in our outpatient clinic until 7 months, and at the visit for any complaints or other morbidities after that time. The follow-up periods ranged from 1 to 11 years.",
"Continuous data were expressed as mean ± standard deviation (SD). Statistical significance was calculated with a two-tailed t test or the Mann–Whitney U test. For proportion data, the χ2 test was used.",
"In laparoscopic PPV closure, a 2–0 suture, placed in the lower half of the internal inguinal ring through a 16-gauge sheath needle advanced extraperitoneally across the cord and vessels, was retrieved through the upper half of the ring by a specially devised needle (Fig. 1) and was tied up extracorporeally, achieving completely extraperitoneal ligation of the ring. Technical details are illustrated in Fig. 2. The procedure for girls was as described previously [7]. For infants younger than aged 1 year 6 months, the IIR was closed with double ligation (Fig. 3). If a cPPV was identified, it was closed.Fig. 1Instrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the EndoneedleFig. 2Laparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedureFig. 3Double ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture\nInstrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the Endoneedle\nLaparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedure\nDouble ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture\nIn cut-down herniorrhaphy, the traditional procedure described in the text was performed through a crease incision at the affected side [8]. Diagnostic laparoscopy was performed with a 70-degree laparoscope via ipsilateral hernia sac. A 2-mm grasper was inserted through the paraumbilical port to manipulate the peritoneum at the contralateral IIR if needed. When a cPPV was noted, it was closed through a crease incision.",
"Parents showed a greater preference for LPC, accounting for 1,257 children compared with 308 who underwent CD with or without diagnostic laparoscopy (CDA, 62; CDL, 246). The reasons why the parents chose LPC were postoperative cosmetic superiority, inspection for cPPV, and simultaneous repair if it was present, the ability for a second look at the previous operation site in cases of recurrence or contralateral occurrence, availability of simultaneous umbilicoplasty for umbilical hernia, or an ugly umbilicus. Diagnostic laparoscopy was selected for inspection for cPPV and simultaneous repair if it was present. The cut-down procedure was chosen because of strong disagreement of family members, including grandparents and relations based on fear of laparoscopic procedures, experience regarding siblings or other family members who had previously undergone cut-down herniorrhaphy with good results or no problems, and the lack of long-term follow-up data with the innovative technique.",
"The data are shown in Table 1. Age ranges were equal at approximately 3 years for both the LPC and CD groups. Sex distribution showed female predominance in the LPC group (44.8% in LPC vs. 26.6% in CD, p < 0.001). Differences in the hernia side were not significant between the groups. No statistically different distributions of associated morbidities necessitating combined operation were seen, except umbilical hernia/cyst. Umbilical morbidities, such as umbilical hernia, cystic degeneration of the umbilicus, or an ugly-looking umbilicus, were seen four times more in the LPC group (p < 0.001).Table 1Characteristics of patients who underwent chosen procedureLPCCDDifferenceNo. of patients1,257308 (246 CDL, 62 CDA)Age (range)1 month to 24 years1 month to 22 years Mean ± SD3.8 ± 2.9 years3.7 ± 3.2 yearsNSSex694 males, 563 females226 males, 82 females % of females44.826.6p < 0.001Side of hernia745 right, 456 left, 56 bilateral177 right, 117 left, 14 bilateral % of lateralityRight (58.4), left (35.8), bilateral (4.4)Right (57.5), left (38), bilateral (4.5)NSAssociated morbidities necessitate combined operation Maldescended testis41 (3.3%)11 (3.6)NS Umbilical hernia/cyst149 (11.9%)9 (2.9%)p < 0.001 Visceral sliding/incarceration Omentum15 (1.2%)1 (0.3%)NS Bowel loop, cecum, appendix14 (1.1%)2 (0.6%)NS Ovarium, ovarian duct42 (3.3%)10 (3.2%)NS After primary herniorrhaphy Recurrence7 (0.6%)2 (0.6%)NS Contralateral hernia26 (2.1%)4 (1.3%)NS Miscellaneous15 (1.2%)9 (2.9%)NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nCharacteristics of patients who underwent chosen procedure\nLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant",
"Regarding laparoscopic inspection of the contralateral IIR, the presence of cPPVs was more dominant in the LPC group than in the CDL group (47% vs. 21.6%, p < 0.001). Mean operation times for unilateral repair were equal in both LPC and CD groups (28.2 ± 9.2 min for LPC vs. 27.8 ± 13.5 min for CD) and were shorter for LPC in bilateral repair (35.8 ± 11.6 vs. 46.7 ± 17.7 min, p < 0.001). When comparing the CDL with the CDA, CDL took an average of 6 min longer for a unilateral and 8.6 min longer for a bilateral closure than CDA (p < 0.05). The difference between males and females was significant in the LPC group (p < 0.001), accounting for a 4.4-min increase for unilateral and a 6.9-min increase for bilateral closure in males (Table 2). In the LPC group, incidental umbilical hernia, ugly umbilicus, and other abnormalities, such as intraumbilical epidermoid cysts, also were repaired simultaneously during closure of the laparoscopic wound, whereas in the CD group an umbilicoplasty was performed as another definitive surgery.Table 2Operative findingsLPCCDDifferenceTotalCDLCDALaterality of PPV Right42514311132Left211997821Bilateral62166579Total187837430371% of contralateral PPV4721.6p < 0.001Operation time (mean ± SD, min) Unilateraln = 591n = 216n = 171n = 4528.2 ± 9.227.8 ± 13.529.1 ± 12.323.1 ± 16.8 DifferenceNSp < 0.05 Bilateraln = 542n = 60n = 50n = 1035.8 ± 11.646.7 ± 17.748.1 ± 18.339.5 ± 15 Differencep < 0.001p < 0.05Male versus femaleMaleFemale Unilateral LPCn = 34530 ± 8n = 24625.6 ± 10p < 0.001 Bilateral LPCn = 25339.5 ± 10.6n = 28932.6 ± 11.5p < 0.001 Unilateral CDn = 15428 ± 12.1n = 6227.3 ± 16.6NS Bilateral CDn = 4348.6 ± 18.5n = 1741.8 ± 14.1NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nOperative findings\nLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nOne boy in the LPC group had a small stab injury on the anterior wall of the rectum during the placement of a port for the grasper through the abdominal wall, which was immediately repaired with the laparoscopic technique without sequelae. A boy and a girl in the CD group had respective accidental severance of the spermatic duct and ovarian duct, which was anastomosed under surgical microscopy during the same session. There was a statistically significant difference in the incidence of intraoperative injuries to the reproductive system between the LPC and CD groups (0% vs. 0.6%, p < 0.005).",
"In the LPC group, the operation was performed satisfactorily in all patients. Preoperative and postoperative photographs of the IIR are shown in Fig. 4, and cosmetic results were excellent with almost invisible scars. In second-look operation for postoperative contralateral hernia, the primarily closed IIR was found to be completely covered with thick cicatricial tissue (Fig. 4C). Postoperative hernia recurrence was seen in 2 of 1,257 patients (0.16%) and 1,878 PPVs (0.11%) in the LPC group, and in 2 of 308 patients (0.65%) and 374 PPVs (0.53%) in the CD group. Contralateral hernia developed in five of the LPC group (0.79% of unilateral PPV closure) and in four of the CD group (1.67%; 3 CDL (1.59%) and 1 CDA (1.89%)). The incidence of the postoperative recurrence and contralateral hernia were lower in the LPC group, but with no statistically significant difference. Postoperative direct hernia occurred in one patient in the CD group. As for minor postoperative complications, fugitive stitch granuloma occurred at the umbilicus in seven patients of the LPC group and at the crease incision site in one patient of the CD group. Transient fluid accumulation in a sac with omental remnant occurred in 1 LPC patient with omental incarceration. Postoperative testicular atrophy was found in none of the both groups.Fig. 4G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places\nG.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places"
] |
[
null,
null,
null,
null,
null,
null,
null
] |
[
"Materials and methods",
"Study design",
"Statistical analysis",
"Operative procedures",
"Results",
"Parental perspective and choice",
"Characteristics of patients who underwent each chosen procedure",
"Operative findings",
"Postoperative findings",
"Discussion",
"Conclusions"
] |
[
" Study design A consecutive series of 1,585 children with inguinal hernia or hydrocele, or both, experienced during 1996–2006, was analyzed. Regarding the operative procedures, the cut-down procedure (CD) and laparoscopic PPV closure (LPC) were proposed to the parents of the patients. CD was further divided into CD for the affected side only (CDA) and CD with diagnostic laparoscopy (CDL). CDA, CDL, or LPC was selected according to parental preference under informed consent. The medical records of these children were analyzed in terms of parental selection, distribution of sex, age, presence of contralateral patent processus vaginalis (cPPV), operation time, and complications among the groups.\nTwenty patients who had various procedures during the period of development of laparoscopic herniorrhaphy were excluded from the analysis, and patients who underwent combined procedures affecting definitive herniorrhaphy also were excluded from the analysis of the operation time. The patients were followed up regularly in our outpatient clinic until 7 months, and at the visit for any complaints or other morbidities after that time. The follow-up periods ranged from 1 to 11 years.\nA consecutive series of 1,585 children with inguinal hernia or hydrocele, or both, experienced during 1996–2006, was analyzed. Regarding the operative procedures, the cut-down procedure (CD) and laparoscopic PPV closure (LPC) were proposed to the parents of the patients. CD was further divided into CD for the affected side only (CDA) and CD with diagnostic laparoscopy (CDL). CDA, CDL, or LPC was selected according to parental preference under informed consent. The medical records of these children were analyzed in terms of parental selection, distribution of sex, age, presence of contralateral patent processus vaginalis (cPPV), operation time, and complications among the groups.\nTwenty patients who had various procedures during the period of development of laparoscopic herniorrhaphy were excluded from the analysis, and patients who underwent combined procedures affecting definitive herniorrhaphy also were excluded from the analysis of the operation time. The patients were followed up regularly in our outpatient clinic until 7 months, and at the visit for any complaints or other morbidities after that time. The follow-up periods ranged from 1 to 11 years.\n Statistical analysis Continuous data were expressed as mean ± standard deviation (SD). Statistical significance was calculated with a two-tailed t test or the Mann–Whitney U test. For proportion data, the χ2 test was used.\nContinuous data were expressed as mean ± standard deviation (SD). Statistical significance was calculated with a two-tailed t test or the Mann–Whitney U test. For proportion data, the χ2 test was used.\n Operative procedures In laparoscopic PPV closure, a 2–0 suture, placed in the lower half of the internal inguinal ring through a 16-gauge sheath needle advanced extraperitoneally across the cord and vessels, was retrieved through the upper half of the ring by a specially devised needle (Fig. 1) and was tied up extracorporeally, achieving completely extraperitoneal ligation of the ring. Technical details are illustrated in Fig. 2. The procedure for girls was as described previously [7]. For infants younger than aged 1 year 6 months, the IIR was closed with double ligation (Fig. 3). If a cPPV was identified, it was closed.Fig. 1Instrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the EndoneedleFig. 2Laparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedureFig. 3Double ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture\nInstrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the Endoneedle\nLaparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedure\nDouble ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture\nIn cut-down herniorrhaphy, the traditional procedure described in the text was performed through a crease incision at the affected side [8]. Diagnostic laparoscopy was performed with a 70-degree laparoscope via ipsilateral hernia sac. A 2-mm grasper was inserted through the paraumbilical port to manipulate the peritoneum at the contralateral IIR if needed. When a cPPV was noted, it was closed through a crease incision.\nIn laparoscopic PPV closure, a 2–0 suture, placed in the lower half of the internal inguinal ring through a 16-gauge sheath needle advanced extraperitoneally across the cord and vessels, was retrieved through the upper half of the ring by a specially devised needle (Fig. 1) and was tied up extracorporeally, achieving completely extraperitoneal ligation of the ring. Technical details are illustrated in Fig. 2. The procedure for girls was as described previously [7]. For infants younger than aged 1 year 6 months, the IIR was closed with double ligation (Fig. 3). If a cPPV was identified, it was closed.Fig. 1Instrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the EndoneedleFig. 2Laparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedureFig. 3Double ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture\nInstrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the Endoneedle\nLaparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedure\nDouble ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture\nIn cut-down herniorrhaphy, the traditional procedure described in the text was performed through a crease incision at the affected side [8]. Diagnostic laparoscopy was performed with a 70-degree laparoscope via ipsilateral hernia sac. A 2-mm grasper was inserted through the paraumbilical port to manipulate the peritoneum at the contralateral IIR if needed. When a cPPV was noted, it was closed through a crease incision.",
"A consecutive series of 1,585 children with inguinal hernia or hydrocele, or both, experienced during 1996–2006, was analyzed. Regarding the operative procedures, the cut-down procedure (CD) and laparoscopic PPV closure (LPC) were proposed to the parents of the patients. CD was further divided into CD for the affected side only (CDA) and CD with diagnostic laparoscopy (CDL). CDA, CDL, or LPC was selected according to parental preference under informed consent. The medical records of these children were analyzed in terms of parental selection, distribution of sex, age, presence of contralateral patent processus vaginalis (cPPV), operation time, and complications among the groups.\nTwenty patients who had various procedures during the period of development of laparoscopic herniorrhaphy were excluded from the analysis, and patients who underwent combined procedures affecting definitive herniorrhaphy also were excluded from the analysis of the operation time. The patients were followed up regularly in our outpatient clinic until 7 months, and at the visit for any complaints or other morbidities after that time. The follow-up periods ranged from 1 to 11 years.",
"Continuous data were expressed as mean ± standard deviation (SD). Statistical significance was calculated with a two-tailed t test or the Mann–Whitney U test. For proportion data, the χ2 test was used.",
"In laparoscopic PPV closure, a 2–0 suture, placed in the lower half of the internal inguinal ring through a 16-gauge sheath needle advanced extraperitoneally across the cord and vessels, was retrieved through the upper half of the ring by a specially devised needle (Fig. 1) and was tied up extracorporeally, achieving completely extraperitoneal ligation of the ring. Technical details are illustrated in Fig. 2. The procedure for girls was as described previously [7]. For infants younger than aged 1 year 6 months, the IIR was closed with double ligation (Fig. 3). If a cPPV was identified, it was closed.Fig. 1Instrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the EndoneedleFig. 2Laparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedureFig. 3Double ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture\nInstrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the Endoneedle\nLaparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedure\nDouble ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture\nIn cut-down herniorrhaphy, the traditional procedure described in the text was performed through a crease incision at the affected side [8]. Diagnostic laparoscopy was performed with a 70-degree laparoscope via ipsilateral hernia sac. A 2-mm grasper was inserted through the paraumbilical port to manipulate the peritoneum at the contralateral IIR if needed. When a cPPV was noted, it was closed through a crease incision.",
" Parental perspective and choice Parents showed a greater preference for LPC, accounting for 1,257 children compared with 308 who underwent CD with or without diagnostic laparoscopy (CDA, 62; CDL, 246). The reasons why the parents chose LPC were postoperative cosmetic superiority, inspection for cPPV, and simultaneous repair if it was present, the ability for a second look at the previous operation site in cases of recurrence or contralateral occurrence, availability of simultaneous umbilicoplasty for umbilical hernia, or an ugly umbilicus. Diagnostic laparoscopy was selected for inspection for cPPV and simultaneous repair if it was present. The cut-down procedure was chosen because of strong disagreement of family members, including grandparents and relations based on fear of laparoscopic procedures, experience regarding siblings or other family members who had previously undergone cut-down herniorrhaphy with good results or no problems, and the lack of long-term follow-up data with the innovative technique.\nParents showed a greater preference for LPC, accounting for 1,257 children compared with 308 who underwent CD with or without diagnostic laparoscopy (CDA, 62; CDL, 246). The reasons why the parents chose LPC were postoperative cosmetic superiority, inspection for cPPV, and simultaneous repair if it was present, the ability for a second look at the previous operation site in cases of recurrence or contralateral occurrence, availability of simultaneous umbilicoplasty for umbilical hernia, or an ugly umbilicus. Diagnostic laparoscopy was selected for inspection for cPPV and simultaneous repair if it was present. The cut-down procedure was chosen because of strong disagreement of family members, including grandparents and relations based on fear of laparoscopic procedures, experience regarding siblings or other family members who had previously undergone cut-down herniorrhaphy with good results or no problems, and the lack of long-term follow-up data with the innovative technique.\n Characteristics of patients who underwent each chosen procedure The data are shown in Table 1. Age ranges were equal at approximately 3 years for both the LPC and CD groups. Sex distribution showed female predominance in the LPC group (44.8% in LPC vs. 26.6% in CD, p < 0.001). Differences in the hernia side were not significant between the groups. No statistically different distributions of associated morbidities necessitating combined operation were seen, except umbilical hernia/cyst. Umbilical morbidities, such as umbilical hernia, cystic degeneration of the umbilicus, or an ugly-looking umbilicus, were seen four times more in the LPC group (p < 0.001).Table 1Characteristics of patients who underwent chosen procedureLPCCDDifferenceNo. of patients1,257308 (246 CDL, 62 CDA)Age (range)1 month to 24 years1 month to 22 years Mean ± SD3.8 ± 2.9 years3.7 ± 3.2 yearsNSSex694 males, 563 females226 males, 82 females % of females44.826.6p < 0.001Side of hernia745 right, 456 left, 56 bilateral177 right, 117 left, 14 bilateral % of lateralityRight (58.4), left (35.8), bilateral (4.4)Right (57.5), left (38), bilateral (4.5)NSAssociated morbidities necessitate combined operation Maldescended testis41 (3.3%)11 (3.6)NS Umbilical hernia/cyst149 (11.9%)9 (2.9%)p < 0.001 Visceral sliding/incarceration Omentum15 (1.2%)1 (0.3%)NS Bowel loop, cecum, appendix14 (1.1%)2 (0.6%)NS Ovarium, ovarian duct42 (3.3%)10 (3.2%)NS After primary herniorrhaphy Recurrence7 (0.6%)2 (0.6%)NS Contralateral hernia26 (2.1%)4 (1.3%)NS Miscellaneous15 (1.2%)9 (2.9%)NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nCharacteristics of patients who underwent chosen procedure\nLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nThe data are shown in Table 1. Age ranges were equal at approximately 3 years for both the LPC and CD groups. Sex distribution showed female predominance in the LPC group (44.8% in LPC vs. 26.6% in CD, p < 0.001). Differences in the hernia side were not significant between the groups. No statistically different distributions of associated morbidities necessitating combined operation were seen, except umbilical hernia/cyst. Umbilical morbidities, such as umbilical hernia, cystic degeneration of the umbilicus, or an ugly-looking umbilicus, were seen four times more in the LPC group (p < 0.001).Table 1Characteristics of patients who underwent chosen procedureLPCCDDifferenceNo. of patients1,257308 (246 CDL, 62 CDA)Age (range)1 month to 24 years1 month to 22 years Mean ± SD3.8 ± 2.9 years3.7 ± 3.2 yearsNSSex694 males, 563 females226 males, 82 females % of females44.826.6p < 0.001Side of hernia745 right, 456 left, 56 bilateral177 right, 117 left, 14 bilateral % of lateralityRight (58.4), left (35.8), bilateral (4.4)Right (57.5), left (38), bilateral (4.5)NSAssociated morbidities necessitate combined operation Maldescended testis41 (3.3%)11 (3.6)NS Umbilical hernia/cyst149 (11.9%)9 (2.9%)p < 0.001 Visceral sliding/incarceration Omentum15 (1.2%)1 (0.3%)NS Bowel loop, cecum, appendix14 (1.1%)2 (0.6%)NS Ovarium, ovarian duct42 (3.3%)10 (3.2%)NS After primary herniorrhaphy Recurrence7 (0.6%)2 (0.6%)NS Contralateral hernia26 (2.1%)4 (1.3%)NS Miscellaneous15 (1.2%)9 (2.9%)NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nCharacteristics of patients who underwent chosen procedure\nLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\n Operative findings Regarding laparoscopic inspection of the contralateral IIR, the presence of cPPVs was more dominant in the LPC group than in the CDL group (47% vs. 21.6%, p < 0.001). Mean operation times for unilateral repair were equal in both LPC and CD groups (28.2 ± 9.2 min for LPC vs. 27.8 ± 13.5 min for CD) and were shorter for LPC in bilateral repair (35.8 ± 11.6 vs. 46.7 ± 17.7 min, p < 0.001). When comparing the CDL with the CDA, CDL took an average of 6 min longer for a unilateral and 8.6 min longer for a bilateral closure than CDA (p < 0.05). The difference between males and females was significant in the LPC group (p < 0.001), accounting for a 4.4-min increase for unilateral and a 6.9-min increase for bilateral closure in males (Table 2). In the LPC group, incidental umbilical hernia, ugly umbilicus, and other abnormalities, such as intraumbilical epidermoid cysts, also were repaired simultaneously during closure of the laparoscopic wound, whereas in the CD group an umbilicoplasty was performed as another definitive surgery.Table 2Operative findingsLPCCDDifferenceTotalCDLCDALaterality of PPV Right42514311132Left211997821Bilateral62166579Total187837430371% of contralateral PPV4721.6p < 0.001Operation time (mean ± SD, min) Unilateraln = 591n = 216n = 171n = 4528.2 ± 9.227.8 ± 13.529.1 ± 12.323.1 ± 16.8 DifferenceNSp < 0.05 Bilateraln = 542n = 60n = 50n = 1035.8 ± 11.646.7 ± 17.748.1 ± 18.339.5 ± 15 Differencep < 0.001p < 0.05Male versus femaleMaleFemale Unilateral LPCn = 34530 ± 8n = 24625.6 ± 10p < 0.001 Bilateral LPCn = 25339.5 ± 10.6n = 28932.6 ± 11.5p < 0.001 Unilateral CDn = 15428 ± 12.1n = 6227.3 ± 16.6NS Bilateral CDn = 4348.6 ± 18.5n = 1741.8 ± 14.1NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nOperative findings\nLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nOne boy in the LPC group had a small stab injury on the anterior wall of the rectum during the placement of a port for the grasper through the abdominal wall, which was immediately repaired with the laparoscopic technique without sequelae. A boy and a girl in the CD group had respective accidental severance of the spermatic duct and ovarian duct, which was anastomosed under surgical microscopy during the same session. There was a statistically significant difference in the incidence of intraoperative injuries to the reproductive system between the LPC and CD groups (0% vs. 0.6%, p < 0.005).\nRegarding laparoscopic inspection of the contralateral IIR, the presence of cPPVs was more dominant in the LPC group than in the CDL group (47% vs. 21.6%, p < 0.001). Mean operation times for unilateral repair were equal in both LPC and CD groups (28.2 ± 9.2 min for LPC vs. 27.8 ± 13.5 min for CD) and were shorter for LPC in bilateral repair (35.8 ± 11.6 vs. 46.7 ± 17.7 min, p < 0.001). When comparing the CDL with the CDA, CDL took an average of 6 min longer for a unilateral and 8.6 min longer for a bilateral closure than CDA (p < 0.05). The difference between males and females was significant in the LPC group (p < 0.001), accounting for a 4.4-min increase for unilateral and a 6.9-min increase for bilateral closure in males (Table 2). In the LPC group, incidental umbilical hernia, ugly umbilicus, and other abnormalities, such as intraumbilical epidermoid cysts, also were repaired simultaneously during closure of the laparoscopic wound, whereas in the CD group an umbilicoplasty was performed as another definitive surgery.Table 2Operative findingsLPCCDDifferenceTotalCDLCDALaterality of PPV Right42514311132Left211997821Bilateral62166579Total187837430371% of contralateral PPV4721.6p < 0.001Operation time (mean ± SD, min) Unilateraln = 591n = 216n = 171n = 4528.2 ± 9.227.8 ± 13.529.1 ± 12.323.1 ± 16.8 DifferenceNSp < 0.05 Bilateraln = 542n = 60n = 50n = 1035.8 ± 11.646.7 ± 17.748.1 ± 18.339.5 ± 15 Differencep < 0.001p < 0.05Male versus femaleMaleFemale Unilateral LPCn = 34530 ± 8n = 24625.6 ± 10p < 0.001 Bilateral LPCn = 25339.5 ± 10.6n = 28932.6 ± 11.5p < 0.001 Unilateral CDn = 15428 ± 12.1n = 6227.3 ± 16.6NS Bilateral CDn = 4348.6 ± 18.5n = 1741.8 ± 14.1NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nOperative findings\nLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nOne boy in the LPC group had a small stab injury on the anterior wall of the rectum during the placement of a port for the grasper through the abdominal wall, which was immediately repaired with the laparoscopic technique without sequelae. A boy and a girl in the CD group had respective accidental severance of the spermatic duct and ovarian duct, which was anastomosed under surgical microscopy during the same session. There was a statistically significant difference in the incidence of intraoperative injuries to the reproductive system between the LPC and CD groups (0% vs. 0.6%, p < 0.005).\n Postoperative findings In the LPC group, the operation was performed satisfactorily in all patients. Preoperative and postoperative photographs of the IIR are shown in Fig. 4, and cosmetic results were excellent with almost invisible scars. In second-look operation for postoperative contralateral hernia, the primarily closed IIR was found to be completely covered with thick cicatricial tissue (Fig. 4C). Postoperative hernia recurrence was seen in 2 of 1,257 patients (0.16%) and 1,878 PPVs (0.11%) in the LPC group, and in 2 of 308 patients (0.65%) and 374 PPVs (0.53%) in the CD group. Contralateral hernia developed in five of the LPC group (0.79% of unilateral PPV closure) and in four of the CD group (1.67%; 3 CDL (1.59%) and 1 CDA (1.89%)). The incidence of the postoperative recurrence and contralateral hernia were lower in the LPC group, but with no statistically significant difference. Postoperative direct hernia occurred in one patient in the CD group. As for minor postoperative complications, fugitive stitch granuloma occurred at the umbilicus in seven patients of the LPC group and at the crease incision site in one patient of the CD group. Transient fluid accumulation in a sac with omental remnant occurred in 1 LPC patient with omental incarceration. Postoperative testicular atrophy was found in none of the both groups.Fig. 4G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places\nG.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places\nIn the LPC group, the operation was performed satisfactorily in all patients. Preoperative and postoperative photographs of the IIR are shown in Fig. 4, and cosmetic results were excellent with almost invisible scars. In second-look operation for postoperative contralateral hernia, the primarily closed IIR was found to be completely covered with thick cicatricial tissue (Fig. 4C). Postoperative hernia recurrence was seen in 2 of 1,257 patients (0.16%) and 1,878 PPVs (0.11%) in the LPC group, and in 2 of 308 patients (0.65%) and 374 PPVs (0.53%) in the CD group. Contralateral hernia developed in five of the LPC group (0.79% of unilateral PPV closure) and in four of the CD group (1.67%; 3 CDL (1.59%) and 1 CDA (1.89%)). The incidence of the postoperative recurrence and contralateral hernia were lower in the LPC group, but with no statistically significant difference. Postoperative direct hernia occurred in one patient in the CD group. As for minor postoperative complications, fugitive stitch granuloma occurred at the umbilicus in seven patients of the LPC group and at the crease incision site in one patient of the CD group. Transient fluid accumulation in a sac with omental remnant occurred in 1 LPC patient with omental incarceration. Postoperative testicular atrophy was found in none of the both groups.Fig. 4G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places\nG.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places",
"Parents showed a greater preference for LPC, accounting for 1,257 children compared with 308 who underwent CD with or without diagnostic laparoscopy (CDA, 62; CDL, 246). The reasons why the parents chose LPC were postoperative cosmetic superiority, inspection for cPPV, and simultaneous repair if it was present, the ability for a second look at the previous operation site in cases of recurrence or contralateral occurrence, availability of simultaneous umbilicoplasty for umbilical hernia, or an ugly umbilicus. Diagnostic laparoscopy was selected for inspection for cPPV and simultaneous repair if it was present. The cut-down procedure was chosen because of strong disagreement of family members, including grandparents and relations based on fear of laparoscopic procedures, experience regarding siblings or other family members who had previously undergone cut-down herniorrhaphy with good results or no problems, and the lack of long-term follow-up data with the innovative technique.",
"The data are shown in Table 1. Age ranges were equal at approximately 3 years for both the LPC and CD groups. Sex distribution showed female predominance in the LPC group (44.8% in LPC vs. 26.6% in CD, p < 0.001). Differences in the hernia side were not significant between the groups. No statistically different distributions of associated morbidities necessitating combined operation were seen, except umbilical hernia/cyst. Umbilical morbidities, such as umbilical hernia, cystic degeneration of the umbilicus, or an ugly-looking umbilicus, were seen four times more in the LPC group (p < 0.001).Table 1Characteristics of patients who underwent chosen procedureLPCCDDifferenceNo. of patients1,257308 (246 CDL, 62 CDA)Age (range)1 month to 24 years1 month to 22 years Mean ± SD3.8 ± 2.9 years3.7 ± 3.2 yearsNSSex694 males, 563 females226 males, 82 females % of females44.826.6p < 0.001Side of hernia745 right, 456 left, 56 bilateral177 right, 117 left, 14 bilateral % of lateralityRight (58.4), left (35.8), bilateral (4.4)Right (57.5), left (38), bilateral (4.5)NSAssociated morbidities necessitate combined operation Maldescended testis41 (3.3%)11 (3.6)NS Umbilical hernia/cyst149 (11.9%)9 (2.9%)p < 0.001 Visceral sliding/incarceration Omentum15 (1.2%)1 (0.3%)NS Bowel loop, cecum, appendix14 (1.1%)2 (0.6%)NS Ovarium, ovarian duct42 (3.3%)10 (3.2%)NS After primary herniorrhaphy Recurrence7 (0.6%)2 (0.6%)NS Contralateral hernia26 (2.1%)4 (1.3%)NS Miscellaneous15 (1.2%)9 (2.9%)NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nCharacteristics of patients who underwent chosen procedure\nLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant",
"Regarding laparoscopic inspection of the contralateral IIR, the presence of cPPVs was more dominant in the LPC group than in the CDL group (47% vs. 21.6%, p < 0.001). Mean operation times for unilateral repair were equal in both LPC and CD groups (28.2 ± 9.2 min for LPC vs. 27.8 ± 13.5 min for CD) and were shorter for LPC in bilateral repair (35.8 ± 11.6 vs. 46.7 ± 17.7 min, p < 0.001). When comparing the CDL with the CDA, CDL took an average of 6 min longer for a unilateral and 8.6 min longer for a bilateral closure than CDA (p < 0.05). The difference between males and females was significant in the LPC group (p < 0.001), accounting for a 4.4-min increase for unilateral and a 6.9-min increase for bilateral closure in males (Table 2). In the LPC group, incidental umbilical hernia, ugly umbilicus, and other abnormalities, such as intraumbilical epidermoid cysts, also were repaired simultaneously during closure of the laparoscopic wound, whereas in the CD group an umbilicoplasty was performed as another definitive surgery.Table 2Operative findingsLPCCDDifferenceTotalCDLCDALaterality of PPV Right42514311132Left211997821Bilateral62166579Total187837430371% of contralateral PPV4721.6p < 0.001Operation time (mean ± SD, min) Unilateraln = 591n = 216n = 171n = 4528.2 ± 9.227.8 ± 13.529.1 ± 12.323.1 ± 16.8 DifferenceNSp < 0.05 Bilateraln = 542n = 60n = 50n = 1035.8 ± 11.646.7 ± 17.748.1 ± 18.339.5 ± 15 Differencep < 0.001p < 0.05Male versus femaleMaleFemale Unilateral LPCn = 34530 ± 8n = 24625.6 ± 10p < 0.001 Bilateral LPCn = 25339.5 ± 10.6n = 28932.6 ± 11.5p < 0.001 Unilateral CDn = 15428 ± 12.1n = 6227.3 ± 16.6NS Bilateral CDn = 4348.6 ± 18.5n = 1741.8 ± 14.1NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nOperative findings\nLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant\nOne boy in the LPC group had a small stab injury on the anterior wall of the rectum during the placement of a port for the grasper through the abdominal wall, which was immediately repaired with the laparoscopic technique without sequelae. A boy and a girl in the CD group had respective accidental severance of the spermatic duct and ovarian duct, which was anastomosed under surgical microscopy during the same session. There was a statistically significant difference in the incidence of intraoperative injuries to the reproductive system between the LPC and CD groups (0% vs. 0.6%, p < 0.005).",
"In the LPC group, the operation was performed satisfactorily in all patients. Preoperative and postoperative photographs of the IIR are shown in Fig. 4, and cosmetic results were excellent with almost invisible scars. In second-look operation for postoperative contralateral hernia, the primarily closed IIR was found to be completely covered with thick cicatricial tissue (Fig. 4C). Postoperative hernia recurrence was seen in 2 of 1,257 patients (0.16%) and 1,878 PPVs (0.11%) in the LPC group, and in 2 of 308 patients (0.65%) and 374 PPVs (0.53%) in the CD group. Contralateral hernia developed in five of the LPC group (0.79% of unilateral PPV closure) and in four of the CD group (1.67%; 3 CDL (1.59%) and 1 CDA (1.89%)). The incidence of the postoperative recurrence and contralateral hernia were lower in the LPC group, but with no statistically significant difference. Postoperative direct hernia occurred in one patient in the CD group. As for minor postoperative complications, fugitive stitch granuloma occurred at the umbilicus in seven patients of the LPC group and at the crease incision site in one patient of the CD group. Transient fluid accumulation in a sac with omental remnant occurred in 1 LPC patient with omental incarceration. Postoperative testicular atrophy was found in none of the both groups.Fig. 4G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places\nG.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places",
"Conventional open herniorrhaphy has a problem of whether contralateral exploration is necessary in children with an indirect hernia [9]. Laparoscopic hernia repair resolves this question with opportunity to close both PPVs simultaneously, when a cPPV is found, without the addition of a crease incision on the opposite side. The second problem is postoperative hernia recurrence. The main factors affecting recurrence have been recognized as (1) failure to ligate the sac high enough at the internal ring; (2) injury to the floor of the inguinal canal due to operative trauma; (3) failure to close the internal ring in girls; and (4) postoperative wound infection and hematoma [10]. The laparoscopic technique has proven to be a method that can avoid all these possible causes of recurrence [11]. The third problem is injury to the reproductive system. Childhood inguinal herniorrhaphy has been said to be one of the most frequent causes of infertility [12].\nThe goal of our project was based on the principle of the traditional cut-down technique, which involves completely extraperitoneal high ligation of the PPV, minimizing the above-mentioned drawbacks with a simple technique. We devised the needles to accomplish circumferential ligation of the PPV via the extraperitoneal route more easily, safely, and completely. The needle goes beneath the ligamentum teres uteri distal to the U-turned ovarian duct in girls, involving the ligament inside of the ligature. To avoid damage to the spermatic cord and testicular vessels in boys, the needle is advanced between the peritoneum and cord and vessels.\nAt the preoperative guidance session, three methods were proposed to the patients’ parents: traditional cut-down repair, additional diagnostic laparoscopy for contralateral IIR inspection with simultaneous closure of a cPPV, and laparoscopic repair. The parents chose the laparoscopic repair more frequently based on the reasons described in the Results, which were occasioned by the predominance of girls and associated morbidities of the umbilicus in the LPC group. The parents were very satisfied with the wound cosmesis in LPC group patients. Hand-in-hand with experience-related advances in technique and patient feedback to family doctors, the incidence of LPC being chosen has markedly increased and the procedure garnered the position of the new standard of herniorrhaphy in our hospital.\nRegarding the intraoperative findings, incidences of cPPV have been reported to range from 20–40% [13–16]. The outstanding point in our series was the difference of cPPV rates between the CDL (21.6%) and LPS (47%) groups. This difference might be due to the technical differences of laparoscopic examination. In diagnostic laparoscopy during the cut-down procedure, an endoscope was inserted through the ipsilateral hernia sac with or without an assistant grasper. On the other hand, during an LPS procedure the laparoscope through the umbilicus is capable of visualizing both IIRs directly and always with the assistance of a grasper. Grossmann et al. described some difficulty in visualization of the contralateral side with an endoscope inserted from the ipsilateral hernia sac [17]. Furthermore, after experiences of contralateral development of clinical hernia in two girls, we have adopted more strict criteria for negative cPPV in LPC.\nThe average operation time for a unilateral hernia in the LPS group was comparable with the CD group, but was shorter for bilateral hernias. According to the difference in time between unilateral and bilateral closure, the time needed for closure of the IIR itself accounted for 8 min. The time for diagnostic laparoscopy via an ipsilateral hernia sac in cut-down herniorrhaphy was calculated at 6 min, which is compatible with a previous report [13]. Differences in time between boys and girls came from the care required around the spermatic cord. Because of tight contact between the spermatic cord and the peritoneum, separation of these structures in advance using electrocautery and a grasper is advisable, although in skilled hands, this step can be abridged, the result of which saves 4 min for a unilateral repair.\nAs for postoperative complications, contralateral hernia developed in eight children who underwent LPC or CDL. The patency had been overlooked in two of them because of a peritoneal veil covering the orifice completely. The remaining six children had had a pinhole orifice or shallow depression, development of a metachronous hernia from which was not thought to occur. The incidence of metachronous hernia of children who underwent laparoscopic inspection was 1.1%, which was far less compared with reported traditional unilateral open repair.\nIt is very difficult to eradicate postoperative recurrence as a fate of herniorrhaphy. Despite the fact that the laparoscopic approach theoretically provides high ligation of the PPV more proximally than open repair, higher rates of recurrence have been reported with this approach. Laparoscopic repairs involving closure of the hernia opening by suturing within the abdominal cavity in the pursestring or Z-type suture fashion where the suture material is tied off intracorporeally may have an intrinsic risk of reopening the vaginal process, leading to the recurrence of the hernia or development of a hydrocele. Reported recurrence rates were 3.1–4.4% [18–20]. In another procedure in which a circuit suture was placed extraperitoneally around the hernial orifice, crossing over the spermatic cord or the testicular vessels to leave them untouched and spare them from injury, small spaces are left above these structures. Reported recurrences in these techniques are 0.8–2.8%, which is lower than intraperitoneal closures [21–23]. Methods that allow complete encircling of the PPV, such as the intraperitoneal pursestring stitch passing between the peritoneum and the cord and vessel structures so as not to leave any skipped area, or a laparoscopic technique that produces every step of the open procedure involving complete division and stitching up of the PPV at the IIR, achieved the lowest recurrence rate from 0–1.3% [11, 24].\nOur technique has fundamentally the same concept as the latter-mentioned techniques, but with more simplicity. Hernia recurrence occurred in two boys in the LPC group (0.2%): one due to early rejection of the suture, and the other due to provable loosening of the suture knot, prompting a modification in technique. The modification was made by double-ligation of the proximal end of the sac for infants younger than 1 year 6 months in whom the external inguinal ring is located so close to the IIR that the shortened inguinal canal becomes uncovered by musculature and is vulnerable to increased intra-abdominal pressure. The doubly ligated IIR is expected to hold against pressure until wound healing is completely accomplished.\nDrawbacks associated with the reproductive system are a hidden but not negligible problem. One report suggests that vas deferens or epididymis was found in 0.53% of hernial sacs removed during herniorrhaphy [25]. The incidence of vasal injury during inguinal herniorrhaphy has been estimated at 0.5% [26]. Fallopian tube obstruction in a woman with a history of childhood bilateral inguinal herniorrhaphy was reported as the cause of infertility [27]. We had two episodes of injury to the reproductive system during cut-down repair. In addition, testicular atrophy, ascent of the testis, ovarian malposition, and bladder injury have been reported, none of which occurred in our laparoscopic series.\nDespite increasing reports regarding the laparoscopic approach, there has been only one comparative study between laparoscopic and open repair. Chan et al. emphasized the superiority of laparoscopic repair from the points of less pain, prompter recovery, and better cosmesis [28]. Operation times were longer for unilateral and equal for bilateral repair compared with open repair. In our series, the majority of parents preferred laparoscopic repair based on postoperative cosmetic superiority, bilateral IIR inspection, and simultaneous repair for an unpleasant-looking umbilicus. Laparoscopic operation times were equal for unilateral and shorter for bilateral repair compared with open repair. There was no injury to the reproductive system in contrast to the cut-down procedure. Postoperative recurrence and contralateral hernia were less in the laparoscopic group, although we could not achieve 0% incidence.",
"Although we must currently accept that laparoscopic hernia repair has not been around long enough to rate fully the risk of late complications, we believe that this procedure with the Endoneedle can be a routine procedure with results comparable or superior to those with open procedures. The advantages of our technique include the following: technically simple, short operation time, inspection of bilateral IIRs with simultaneous closure of cPPV, the reproductive system remains intact, routine addition of umbilicoplasty if desired by the parents of patients, and essentially indiscernible wounds."
] |
[
"materials|methods",
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
"conclusion"
] |
[
"Laparoscopic herniorrhaphy",
"Indirect inguinal hernia",
"Extraperitoneal repair",
"Cut-down herniorrhaphy",
"Laparoscopic repair"
] |
Materials and methods:
Study design A consecutive series of 1,585 children with inguinal hernia or hydrocele, or both, experienced during 1996–2006, was analyzed. Regarding the operative procedures, the cut-down procedure (CD) and laparoscopic PPV closure (LPC) were proposed to the parents of the patients. CD was further divided into CD for the affected side only (CDA) and CD with diagnostic laparoscopy (CDL). CDA, CDL, or LPC was selected according to parental preference under informed consent. The medical records of these children were analyzed in terms of parental selection, distribution of sex, age, presence of contralateral patent processus vaginalis (cPPV), operation time, and complications among the groups.
Twenty patients who had various procedures during the period of development of laparoscopic herniorrhaphy were excluded from the analysis, and patients who underwent combined procedures affecting definitive herniorrhaphy also were excluded from the analysis of the operation time. The patients were followed up regularly in our outpatient clinic until 7 months, and at the visit for any complaints or other morbidities after that time. The follow-up periods ranged from 1 to 11 years.
A consecutive series of 1,585 children with inguinal hernia or hydrocele, or both, experienced during 1996–2006, was analyzed. Regarding the operative procedures, the cut-down procedure (CD) and laparoscopic PPV closure (LPC) were proposed to the parents of the patients. CD was further divided into CD for the affected side only (CDA) and CD with diagnostic laparoscopy (CDL). CDA, CDL, or LPC was selected according to parental preference under informed consent. The medical records of these children were analyzed in terms of parental selection, distribution of sex, age, presence of contralateral patent processus vaginalis (cPPV), operation time, and complications among the groups.
Twenty patients who had various procedures during the period of development of laparoscopic herniorrhaphy were excluded from the analysis, and patients who underwent combined procedures affecting definitive herniorrhaphy also were excluded from the analysis of the operation time. The patients were followed up regularly in our outpatient clinic until 7 months, and at the visit for any complaints or other morbidities after that time. The follow-up periods ranged from 1 to 11 years.
Statistical analysis Continuous data were expressed as mean ± standard deviation (SD). Statistical significance was calculated with a two-tailed t test or the Mann–Whitney U test. For proportion data, the χ2 test was used.
Continuous data were expressed as mean ± standard deviation (SD). Statistical significance was calculated with a two-tailed t test or the Mann–Whitney U test. For proportion data, the χ2 test was used.
Operative procedures In laparoscopic PPV closure, a 2–0 suture, placed in the lower half of the internal inguinal ring through a 16-gauge sheath needle advanced extraperitoneally across the cord and vessels, was retrieved through the upper half of the ring by a specially devised needle (Fig. 1) and was tied up extracorporeally, achieving completely extraperitoneal ligation of the ring. Technical details are illustrated in Fig. 2. The procedure for girls was as described previously [7]. For infants younger than aged 1 year 6 months, the IIR was closed with double ligation (Fig. 3). If a cPPV was identified, it was closed.Fig. 1Instrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the EndoneedleFig. 2Laparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedureFig. 3Double ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture
Instrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the Endoneedle
Laparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedure
Double ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture
In cut-down herniorrhaphy, the traditional procedure described in the text was performed through a crease incision at the affected side [8]. Diagnostic laparoscopy was performed with a 70-degree laparoscope via ipsilateral hernia sac. A 2-mm grasper was inserted through the paraumbilical port to manipulate the peritoneum at the contralateral IIR if needed. When a cPPV was noted, it was closed through a crease incision.
In laparoscopic PPV closure, a 2–0 suture, placed in the lower half of the internal inguinal ring through a 16-gauge sheath needle advanced extraperitoneally across the cord and vessels, was retrieved through the upper half of the ring by a specially devised needle (Fig. 1) and was tied up extracorporeally, achieving completely extraperitoneal ligation of the ring. Technical details are illustrated in Fig. 2. The procedure for girls was as described previously [7]. For infants younger than aged 1 year 6 months, the IIR was closed with double ligation (Fig. 3). If a cPPV was identified, it was closed.Fig. 1Instrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the EndoneedleFig. 2Laparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedureFig. 3Double ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture
Instrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the Endoneedle
Laparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedure
Double ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture
In cut-down herniorrhaphy, the traditional procedure described in the text was performed through a crease incision at the affected side [8]. Diagnostic laparoscopy was performed with a 70-degree laparoscope via ipsilateral hernia sac. A 2-mm grasper was inserted through the paraumbilical port to manipulate the peritoneum at the contralateral IIR if needed. When a cPPV was noted, it was closed through a crease incision.
Study design:
A consecutive series of 1,585 children with inguinal hernia or hydrocele, or both, experienced during 1996–2006, was analyzed. Regarding the operative procedures, the cut-down procedure (CD) and laparoscopic PPV closure (LPC) were proposed to the parents of the patients. CD was further divided into CD for the affected side only (CDA) and CD with diagnostic laparoscopy (CDL). CDA, CDL, or LPC was selected according to parental preference under informed consent. The medical records of these children were analyzed in terms of parental selection, distribution of sex, age, presence of contralateral patent processus vaginalis (cPPV), operation time, and complications among the groups.
Twenty patients who had various procedures during the period of development of laparoscopic herniorrhaphy were excluded from the analysis, and patients who underwent combined procedures affecting definitive herniorrhaphy also were excluded from the analysis of the operation time. The patients were followed up regularly in our outpatient clinic until 7 months, and at the visit for any complaints or other morbidities after that time. The follow-up periods ranged from 1 to 11 years.
Statistical analysis:
Continuous data were expressed as mean ± standard deviation (SD). Statistical significance was calculated with a two-tailed t test or the Mann–Whitney U test. For proportion data, the χ2 test was used.
Operative procedures:
In laparoscopic PPV closure, a 2–0 suture, placed in the lower half of the internal inguinal ring through a 16-gauge sheath needle advanced extraperitoneally across the cord and vessels, was retrieved through the upper half of the ring by a specially devised needle (Fig. 1) and was tied up extracorporeally, achieving completely extraperitoneal ligation of the ring. Technical details are illustrated in Fig. 2. The procedure for girls was as described previously [7]. For infants younger than aged 1 year 6 months, the IIR was closed with double ligation (Fig. 3). If a cPPV was identified, it was closed.Fig. 1Instrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the EndoneedleFig. 2Laparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedureFig. 3Double ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture
Instrumentation consisting of 14-gauge sheath needle as a port for 15-gauge grasper with electrocautery, 16-gauge sheath needle for puncture, and 19-gauge Endoneedle for sending and retrieving a suture. A metal filament is used for setting a 2–0 nylon twine as a suture into the Endoneedle
Laparoscopic completely extraperitoneal closure of right-sided PPV. (1) Anatomy of male IIR. 1, umbilical plica; 2, inferior epigastric vessels; 3, external iliac vein; 4, transverse abdominal muscle; 5, orifice of PPV; 6, spermatic duct; 7, testicular vessels. (2) A small opening is made on the peritoneum between spermatic duct and testicular vessels using 15-gauge grasper with electrocautery. (3) The spermatic duct is separated from covering peritoneum by the grasper. (4) 16-gauge sheath needle goes along lower half of the IIR extraperitoneally crossing over the testicular vessels and spermatic duct beneath the peritoneum. (5) After the puncture needle penetrates the peritoneum at the opposite side, a 2–0 suture is send by Endoneedle. (6) Free end of the suture is bitten into the Endoneedle that has come along upper half of the orifice and drawn out together with the needle. (7) The orifice of PPV has been encircled without any skip areas. The suture is tied from outside. (8) End of the procedure
Double ligation for infant younger than aged 1 year 6 months. An internal pursestring suture is placed, skipping over the spermatic cord and testicular vessels, proximally to the previously placed encircling suture
In cut-down herniorrhaphy, the traditional procedure described in the text was performed through a crease incision at the affected side [8]. Diagnostic laparoscopy was performed with a 70-degree laparoscope via ipsilateral hernia sac. A 2-mm grasper was inserted through the paraumbilical port to manipulate the peritoneum at the contralateral IIR if needed. When a cPPV was noted, it was closed through a crease incision.
Results:
Parental perspective and choice Parents showed a greater preference for LPC, accounting for 1,257 children compared with 308 who underwent CD with or without diagnostic laparoscopy (CDA, 62; CDL, 246). The reasons why the parents chose LPC were postoperative cosmetic superiority, inspection for cPPV, and simultaneous repair if it was present, the ability for a second look at the previous operation site in cases of recurrence or contralateral occurrence, availability of simultaneous umbilicoplasty for umbilical hernia, or an ugly umbilicus. Diagnostic laparoscopy was selected for inspection for cPPV and simultaneous repair if it was present. The cut-down procedure was chosen because of strong disagreement of family members, including grandparents and relations based on fear of laparoscopic procedures, experience regarding siblings or other family members who had previously undergone cut-down herniorrhaphy with good results or no problems, and the lack of long-term follow-up data with the innovative technique.
Parents showed a greater preference for LPC, accounting for 1,257 children compared with 308 who underwent CD with or without diagnostic laparoscopy (CDA, 62; CDL, 246). The reasons why the parents chose LPC were postoperative cosmetic superiority, inspection for cPPV, and simultaneous repair if it was present, the ability for a second look at the previous operation site in cases of recurrence or contralateral occurrence, availability of simultaneous umbilicoplasty for umbilical hernia, or an ugly umbilicus. Diagnostic laparoscopy was selected for inspection for cPPV and simultaneous repair if it was present. The cut-down procedure was chosen because of strong disagreement of family members, including grandparents and relations based on fear of laparoscopic procedures, experience regarding siblings or other family members who had previously undergone cut-down herniorrhaphy with good results or no problems, and the lack of long-term follow-up data with the innovative technique.
Characteristics of patients who underwent each chosen procedure The data are shown in Table 1. Age ranges were equal at approximately 3 years for both the LPC and CD groups. Sex distribution showed female predominance in the LPC group (44.8% in LPC vs. 26.6% in CD, p < 0.001). Differences in the hernia side were not significant between the groups. No statistically different distributions of associated morbidities necessitating combined operation were seen, except umbilical hernia/cyst. Umbilical morbidities, such as umbilical hernia, cystic degeneration of the umbilicus, or an ugly-looking umbilicus, were seen four times more in the LPC group (p < 0.001).Table 1Characteristics of patients who underwent chosen procedureLPCCDDifferenceNo. of patients1,257308 (246 CDL, 62 CDA)Age (range)1 month to 24 years1 month to 22 years Mean ± SD3.8 ± 2.9 years3.7 ± 3.2 yearsNSSex694 males, 563 females226 males, 82 females % of females44.826.6p < 0.001Side of hernia745 right, 456 left, 56 bilateral177 right, 117 left, 14 bilateral % of lateralityRight (58.4), left (35.8), bilateral (4.4)Right (57.5), left (38), bilateral (4.5)NSAssociated morbidities necessitate combined operation Maldescended testis41 (3.3%)11 (3.6)NS Umbilical hernia/cyst149 (11.9%)9 (2.9%)p < 0.001 Visceral sliding/incarceration Omentum15 (1.2%)1 (0.3%)NS Bowel loop, cecum, appendix14 (1.1%)2 (0.6%)NS Ovarium, ovarian duct42 (3.3%)10 (3.2%)NS After primary herniorrhaphy Recurrence7 (0.6%)2 (0.6%)NS Contralateral hernia26 (2.1%)4 (1.3%)NS Miscellaneous15 (1.2%)9 (2.9%)NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Characteristics of patients who underwent chosen procedure
LPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
The data are shown in Table 1. Age ranges were equal at approximately 3 years for both the LPC and CD groups. Sex distribution showed female predominance in the LPC group (44.8% in LPC vs. 26.6% in CD, p < 0.001). Differences in the hernia side were not significant between the groups. No statistically different distributions of associated morbidities necessitating combined operation were seen, except umbilical hernia/cyst. Umbilical morbidities, such as umbilical hernia, cystic degeneration of the umbilicus, or an ugly-looking umbilicus, were seen four times more in the LPC group (p < 0.001).Table 1Characteristics of patients who underwent chosen procedureLPCCDDifferenceNo. of patients1,257308 (246 CDL, 62 CDA)Age (range)1 month to 24 years1 month to 22 years Mean ± SD3.8 ± 2.9 years3.7 ± 3.2 yearsNSSex694 males, 563 females226 males, 82 females % of females44.826.6p < 0.001Side of hernia745 right, 456 left, 56 bilateral177 right, 117 left, 14 bilateral % of lateralityRight (58.4), left (35.8), bilateral (4.4)Right (57.5), left (38), bilateral (4.5)NSAssociated morbidities necessitate combined operation Maldescended testis41 (3.3%)11 (3.6)NS Umbilical hernia/cyst149 (11.9%)9 (2.9%)p < 0.001 Visceral sliding/incarceration Omentum15 (1.2%)1 (0.3%)NS Bowel loop, cecum, appendix14 (1.1%)2 (0.6%)NS Ovarium, ovarian duct42 (3.3%)10 (3.2%)NS After primary herniorrhaphy Recurrence7 (0.6%)2 (0.6%)NS Contralateral hernia26 (2.1%)4 (1.3%)NS Miscellaneous15 (1.2%)9 (2.9%)NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Characteristics of patients who underwent chosen procedure
LPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Operative findings Regarding laparoscopic inspection of the contralateral IIR, the presence of cPPVs was more dominant in the LPC group than in the CDL group (47% vs. 21.6%, p < 0.001). Mean operation times for unilateral repair were equal in both LPC and CD groups (28.2 ± 9.2 min for LPC vs. 27.8 ± 13.5 min for CD) and were shorter for LPC in bilateral repair (35.8 ± 11.6 vs. 46.7 ± 17.7 min, p < 0.001). When comparing the CDL with the CDA, CDL took an average of 6 min longer for a unilateral and 8.6 min longer for a bilateral closure than CDA (p < 0.05). The difference between males and females was significant in the LPC group (p < 0.001), accounting for a 4.4-min increase for unilateral and a 6.9-min increase for bilateral closure in males (Table 2). In the LPC group, incidental umbilical hernia, ugly umbilicus, and other abnormalities, such as intraumbilical epidermoid cysts, also were repaired simultaneously during closure of the laparoscopic wound, whereas in the CD group an umbilicoplasty was performed as another definitive surgery.Table 2Operative findingsLPCCDDifferenceTotalCDLCDALaterality of PPV Right42514311132Left211997821Bilateral62166579Total187837430371% of contralateral PPV4721.6p < 0.001Operation time (mean ± SD, min) Unilateraln = 591n = 216n = 171n = 4528.2 ± 9.227.8 ± 13.529.1 ± 12.323.1 ± 16.8 DifferenceNSp < 0.05 Bilateraln = 542n = 60n = 50n = 1035.8 ± 11.646.7 ± 17.748.1 ± 18.339.5 ± 15 Differencep < 0.001p < 0.05Male versus femaleMaleFemale Unilateral LPCn = 34530 ± 8n = 24625.6 ± 10p < 0.001 Bilateral LPCn = 25339.5 ± 10.6n = 28932.6 ± 11.5p < 0.001 Unilateral CDn = 15428 ± 12.1n = 6227.3 ± 16.6NS Bilateral CDn = 4348.6 ± 18.5n = 1741.8 ± 14.1NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Operative findings
LPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
One boy in the LPC group had a small stab injury on the anterior wall of the rectum during the placement of a port for the grasper through the abdominal wall, which was immediately repaired with the laparoscopic technique without sequelae. A boy and a girl in the CD group had respective accidental severance of the spermatic duct and ovarian duct, which was anastomosed under surgical microscopy during the same session. There was a statistically significant difference in the incidence of intraoperative injuries to the reproductive system between the LPC and CD groups (0% vs. 0.6%, p < 0.005).
Regarding laparoscopic inspection of the contralateral IIR, the presence of cPPVs was more dominant in the LPC group than in the CDL group (47% vs. 21.6%, p < 0.001). Mean operation times for unilateral repair were equal in both LPC and CD groups (28.2 ± 9.2 min for LPC vs. 27.8 ± 13.5 min for CD) and were shorter for LPC in bilateral repair (35.8 ± 11.6 vs. 46.7 ± 17.7 min, p < 0.001). When comparing the CDL with the CDA, CDL took an average of 6 min longer for a unilateral and 8.6 min longer for a bilateral closure than CDA (p < 0.05). The difference between males and females was significant in the LPC group (p < 0.001), accounting for a 4.4-min increase for unilateral and a 6.9-min increase for bilateral closure in males (Table 2). In the LPC group, incidental umbilical hernia, ugly umbilicus, and other abnormalities, such as intraumbilical epidermoid cysts, also were repaired simultaneously during closure of the laparoscopic wound, whereas in the CD group an umbilicoplasty was performed as another definitive surgery.Table 2Operative findingsLPCCDDifferenceTotalCDLCDALaterality of PPV Right42514311132Left211997821Bilateral62166579Total187837430371% of contralateral PPV4721.6p < 0.001Operation time (mean ± SD, min) Unilateraln = 591n = 216n = 171n = 4528.2 ± 9.227.8 ± 13.529.1 ± 12.323.1 ± 16.8 DifferenceNSp < 0.05 Bilateraln = 542n = 60n = 50n = 1035.8 ± 11.646.7 ± 17.748.1 ± 18.339.5 ± 15 Differencep < 0.001p < 0.05Male versus femaleMaleFemale Unilateral LPCn = 34530 ± 8n = 24625.6 ± 10p < 0.001 Bilateral LPCn = 25339.5 ± 10.6n = 28932.6 ± 11.5p < 0.001 Unilateral CDn = 15428 ± 12.1n = 6227.3 ± 16.6NS Bilateral CDn = 4348.6 ± 18.5n = 1741.8 ± 14.1NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Operative findings
LPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
One boy in the LPC group had a small stab injury on the anterior wall of the rectum during the placement of a port for the grasper through the abdominal wall, which was immediately repaired with the laparoscopic technique without sequelae. A boy and a girl in the CD group had respective accidental severance of the spermatic duct and ovarian duct, which was anastomosed under surgical microscopy during the same session. There was a statistically significant difference in the incidence of intraoperative injuries to the reproductive system between the LPC and CD groups (0% vs. 0.6%, p < 0.005).
Postoperative findings In the LPC group, the operation was performed satisfactorily in all patients. Preoperative and postoperative photographs of the IIR are shown in Fig. 4, and cosmetic results were excellent with almost invisible scars. In second-look operation for postoperative contralateral hernia, the primarily closed IIR was found to be completely covered with thick cicatricial tissue (Fig. 4C). Postoperative hernia recurrence was seen in 2 of 1,257 patients (0.16%) and 1,878 PPVs (0.11%) in the LPC group, and in 2 of 308 patients (0.65%) and 374 PPVs (0.53%) in the CD group. Contralateral hernia developed in five of the LPC group (0.79% of unilateral PPV closure) and in four of the CD group (1.67%; 3 CDL (1.59%) and 1 CDA (1.89%)). The incidence of the postoperative recurrence and contralateral hernia were lower in the LPC group, but with no statistically significant difference. Postoperative direct hernia occurred in one patient in the CD group. As for minor postoperative complications, fugitive stitch granuloma occurred at the umbilicus in seven patients of the LPC group and at the crease incision site in one patient of the CD group. Transient fluid accumulation in a sac with omental remnant occurred in 1 LPC patient with omental incarceration. Postoperative testicular atrophy was found in none of the both groups.Fig. 4G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places
G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places
In the LPC group, the operation was performed satisfactorily in all patients. Preoperative and postoperative photographs of the IIR are shown in Fig. 4, and cosmetic results were excellent with almost invisible scars. In second-look operation for postoperative contralateral hernia, the primarily closed IIR was found to be completely covered with thick cicatricial tissue (Fig. 4C). Postoperative hernia recurrence was seen in 2 of 1,257 patients (0.16%) and 1,878 PPVs (0.11%) in the LPC group, and in 2 of 308 patients (0.65%) and 374 PPVs (0.53%) in the CD group. Contralateral hernia developed in five of the LPC group (0.79% of unilateral PPV closure) and in four of the CD group (1.67%; 3 CDL (1.59%) and 1 CDA (1.89%)). The incidence of the postoperative recurrence and contralateral hernia were lower in the LPC group, but with no statistically significant difference. Postoperative direct hernia occurred in one patient in the CD group. As for minor postoperative complications, fugitive stitch granuloma occurred at the umbilicus in seven patients of the LPC group and at the crease incision site in one patient of the CD group. Transient fluid accumulation in a sac with omental remnant occurred in 1 LPC patient with omental incarceration. Postoperative testicular atrophy was found in none of the both groups.Fig. 4G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places
G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places
Parental perspective and choice:
Parents showed a greater preference for LPC, accounting for 1,257 children compared with 308 who underwent CD with or without diagnostic laparoscopy (CDA, 62; CDL, 246). The reasons why the parents chose LPC were postoperative cosmetic superiority, inspection for cPPV, and simultaneous repair if it was present, the ability for a second look at the previous operation site in cases of recurrence or contralateral occurrence, availability of simultaneous umbilicoplasty for umbilical hernia, or an ugly umbilicus. Diagnostic laparoscopy was selected for inspection for cPPV and simultaneous repair if it was present. The cut-down procedure was chosen because of strong disagreement of family members, including grandparents and relations based on fear of laparoscopic procedures, experience regarding siblings or other family members who had previously undergone cut-down herniorrhaphy with good results or no problems, and the lack of long-term follow-up data with the innovative technique.
Characteristics of patients who underwent each chosen procedure:
The data are shown in Table 1. Age ranges were equal at approximately 3 years for both the LPC and CD groups. Sex distribution showed female predominance in the LPC group (44.8% in LPC vs. 26.6% in CD, p < 0.001). Differences in the hernia side were not significant between the groups. No statistically different distributions of associated morbidities necessitating combined operation were seen, except umbilical hernia/cyst. Umbilical morbidities, such as umbilical hernia, cystic degeneration of the umbilicus, or an ugly-looking umbilicus, were seen four times more in the LPC group (p < 0.001).Table 1Characteristics of patients who underwent chosen procedureLPCCDDifferenceNo. of patients1,257308 (246 CDL, 62 CDA)Age (range)1 month to 24 years1 month to 22 years Mean ± SD3.8 ± 2.9 years3.7 ± 3.2 yearsNSSex694 males, 563 females226 males, 82 females % of females44.826.6p < 0.001Side of hernia745 right, 456 left, 56 bilateral177 right, 117 left, 14 bilateral % of lateralityRight (58.4), left (35.8), bilateral (4.4)Right (57.5), left (38), bilateral (4.5)NSAssociated morbidities necessitate combined operation Maldescended testis41 (3.3%)11 (3.6)NS Umbilical hernia/cyst149 (11.9%)9 (2.9%)p < 0.001 Visceral sliding/incarceration Omentum15 (1.2%)1 (0.3%)NS Bowel loop, cecum, appendix14 (1.1%)2 (0.6%)NS Ovarium, ovarian duct42 (3.3%)10 (3.2%)NS After primary herniorrhaphy Recurrence7 (0.6%)2 (0.6%)NS Contralateral hernia26 (2.1%)4 (1.3%)NS Miscellaneous15 (1.2%)9 (2.9%)NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Characteristics of patients who underwent chosen procedure
LPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Operative findings:
Regarding laparoscopic inspection of the contralateral IIR, the presence of cPPVs was more dominant in the LPC group than in the CDL group (47% vs. 21.6%, p < 0.001). Mean operation times for unilateral repair were equal in both LPC and CD groups (28.2 ± 9.2 min for LPC vs. 27.8 ± 13.5 min for CD) and were shorter for LPC in bilateral repair (35.8 ± 11.6 vs. 46.7 ± 17.7 min, p < 0.001). When comparing the CDL with the CDA, CDL took an average of 6 min longer for a unilateral and 8.6 min longer for a bilateral closure than CDA (p < 0.05). The difference between males and females was significant in the LPC group (p < 0.001), accounting for a 4.4-min increase for unilateral and a 6.9-min increase for bilateral closure in males (Table 2). In the LPC group, incidental umbilical hernia, ugly umbilicus, and other abnormalities, such as intraumbilical epidermoid cysts, also were repaired simultaneously during closure of the laparoscopic wound, whereas in the CD group an umbilicoplasty was performed as another definitive surgery.Table 2Operative findingsLPCCDDifferenceTotalCDLCDALaterality of PPV Right42514311132Left211997821Bilateral62166579Total187837430371% of contralateral PPV4721.6p < 0.001Operation time (mean ± SD, min) Unilateraln = 591n = 216n = 171n = 4528.2 ± 9.227.8 ± 13.529.1 ± 12.323.1 ± 16.8 DifferenceNSp < 0.05 Bilateraln = 542n = 60n = 50n = 1035.8 ± 11.646.7 ± 17.748.1 ± 18.339.5 ± 15 Differencep < 0.001p < 0.05Male versus femaleMaleFemale Unilateral LPCn = 34530 ± 8n = 24625.6 ± 10p < 0.001 Bilateral LPCn = 25339.5 ± 10.6n = 28932.6 ± 11.5p < 0.001 Unilateral CDn = 15428 ± 12.1n = 6227.3 ± 16.6NS Bilateral CDn = 4348.6 ± 18.5n = 1741.8 ± 14.1NSLPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
Operative findings
LPC laparoscopic patent processus vaginalis closure, CD cut-down herniorrhaphy, NS not significant
One boy in the LPC group had a small stab injury on the anterior wall of the rectum during the placement of a port for the grasper through the abdominal wall, which was immediately repaired with the laparoscopic technique without sequelae. A boy and a girl in the CD group had respective accidental severance of the spermatic duct and ovarian duct, which was anastomosed under surgical microscopy during the same session. There was a statistically significant difference in the incidence of intraoperative injuries to the reproductive system between the LPC and CD groups (0% vs. 0.6%, p < 0.005).
Postoperative findings:
In the LPC group, the operation was performed satisfactorily in all patients. Preoperative and postoperative photographs of the IIR are shown in Fig. 4, and cosmetic results were excellent with almost invisible scars. In second-look operation for postoperative contralateral hernia, the primarily closed IIR was found to be completely covered with thick cicatricial tissue (Fig. 4C). Postoperative hernia recurrence was seen in 2 of 1,257 patients (0.16%) and 1,878 PPVs (0.11%) in the LPC group, and in 2 of 308 patients (0.65%) and 374 PPVs (0.53%) in the CD group. Contralateral hernia developed in five of the LPC group (0.79% of unilateral PPV closure) and in four of the CD group (1.67%; 3 CDL (1.59%) and 1 CDA (1.89%)). The incidence of the postoperative recurrence and contralateral hernia were lower in the LPC group, but with no statistically significant difference. Postoperative direct hernia occurred in one patient in the CD group. As for minor postoperative complications, fugitive stitch granuloma occurred at the umbilicus in seven patients of the LPC group and at the crease incision site in one patient of the CD group. Transient fluid accumulation in a sac with omental remnant occurred in 1 LPC patient with omental incarceration. Postoperative testicular atrophy was found in none of the both groups.Fig. 4G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places
G.K., 2-year-old boy right indirect inguinal hernia. A Preoperative findings; 1 spermatic cord, 2 testicular vessels, 3 inferior epigastric vessels. B Immediately after closure; 4 Umbilical plica. Umbilical plica has been drawn toward the center of ligation. The spermatic cord and testicular vessels run apart from the ligation. C Revisit for contralateral hernia developed after 3 months; 5 suture knot. The primarily closed IIR has been covered by thick cicatricial tissue resulting in super-high ligation. The spermatic cord and testicular vessels have returned to preoperative places
Discussion:
Conventional open herniorrhaphy has a problem of whether contralateral exploration is necessary in children with an indirect hernia [9]. Laparoscopic hernia repair resolves this question with opportunity to close both PPVs simultaneously, when a cPPV is found, without the addition of a crease incision on the opposite side. The second problem is postoperative hernia recurrence. The main factors affecting recurrence have been recognized as (1) failure to ligate the sac high enough at the internal ring; (2) injury to the floor of the inguinal canal due to operative trauma; (3) failure to close the internal ring in girls; and (4) postoperative wound infection and hematoma [10]. The laparoscopic technique has proven to be a method that can avoid all these possible causes of recurrence [11]. The third problem is injury to the reproductive system. Childhood inguinal herniorrhaphy has been said to be one of the most frequent causes of infertility [12].
The goal of our project was based on the principle of the traditional cut-down technique, which involves completely extraperitoneal high ligation of the PPV, minimizing the above-mentioned drawbacks with a simple technique. We devised the needles to accomplish circumferential ligation of the PPV via the extraperitoneal route more easily, safely, and completely. The needle goes beneath the ligamentum teres uteri distal to the U-turned ovarian duct in girls, involving the ligament inside of the ligature. To avoid damage to the spermatic cord and testicular vessels in boys, the needle is advanced between the peritoneum and cord and vessels.
At the preoperative guidance session, three methods were proposed to the patients’ parents: traditional cut-down repair, additional diagnostic laparoscopy for contralateral IIR inspection with simultaneous closure of a cPPV, and laparoscopic repair. The parents chose the laparoscopic repair more frequently based on the reasons described in the Results, which were occasioned by the predominance of girls and associated morbidities of the umbilicus in the LPC group. The parents were very satisfied with the wound cosmesis in LPC group patients. Hand-in-hand with experience-related advances in technique and patient feedback to family doctors, the incidence of LPC being chosen has markedly increased and the procedure garnered the position of the new standard of herniorrhaphy in our hospital.
Regarding the intraoperative findings, incidences of cPPV have been reported to range from 20–40% [13–16]. The outstanding point in our series was the difference of cPPV rates between the CDL (21.6%) and LPS (47%) groups. This difference might be due to the technical differences of laparoscopic examination. In diagnostic laparoscopy during the cut-down procedure, an endoscope was inserted through the ipsilateral hernia sac with or without an assistant grasper. On the other hand, during an LPS procedure the laparoscope through the umbilicus is capable of visualizing both IIRs directly and always with the assistance of a grasper. Grossmann et al. described some difficulty in visualization of the contralateral side with an endoscope inserted from the ipsilateral hernia sac [17]. Furthermore, after experiences of contralateral development of clinical hernia in two girls, we have adopted more strict criteria for negative cPPV in LPC.
The average operation time for a unilateral hernia in the LPS group was comparable with the CD group, but was shorter for bilateral hernias. According to the difference in time between unilateral and bilateral closure, the time needed for closure of the IIR itself accounted for 8 min. The time for diagnostic laparoscopy via an ipsilateral hernia sac in cut-down herniorrhaphy was calculated at 6 min, which is compatible with a previous report [13]. Differences in time between boys and girls came from the care required around the spermatic cord. Because of tight contact between the spermatic cord and the peritoneum, separation of these structures in advance using electrocautery and a grasper is advisable, although in skilled hands, this step can be abridged, the result of which saves 4 min for a unilateral repair.
As for postoperative complications, contralateral hernia developed in eight children who underwent LPC or CDL. The patency had been overlooked in two of them because of a peritoneal veil covering the orifice completely. The remaining six children had had a pinhole orifice or shallow depression, development of a metachronous hernia from which was not thought to occur. The incidence of metachronous hernia of children who underwent laparoscopic inspection was 1.1%, which was far less compared with reported traditional unilateral open repair.
It is very difficult to eradicate postoperative recurrence as a fate of herniorrhaphy. Despite the fact that the laparoscopic approach theoretically provides high ligation of the PPV more proximally than open repair, higher rates of recurrence have been reported with this approach. Laparoscopic repairs involving closure of the hernia opening by suturing within the abdominal cavity in the pursestring or Z-type suture fashion where the suture material is tied off intracorporeally may have an intrinsic risk of reopening the vaginal process, leading to the recurrence of the hernia or development of a hydrocele. Reported recurrence rates were 3.1–4.4% [18–20]. In another procedure in which a circuit suture was placed extraperitoneally around the hernial orifice, crossing over the spermatic cord or the testicular vessels to leave them untouched and spare them from injury, small spaces are left above these structures. Reported recurrences in these techniques are 0.8–2.8%, which is lower than intraperitoneal closures [21–23]. Methods that allow complete encircling of the PPV, such as the intraperitoneal pursestring stitch passing between the peritoneum and the cord and vessel structures so as not to leave any skipped area, or a laparoscopic technique that produces every step of the open procedure involving complete division and stitching up of the PPV at the IIR, achieved the lowest recurrence rate from 0–1.3% [11, 24].
Our technique has fundamentally the same concept as the latter-mentioned techniques, but with more simplicity. Hernia recurrence occurred in two boys in the LPC group (0.2%): one due to early rejection of the suture, and the other due to provable loosening of the suture knot, prompting a modification in technique. The modification was made by double-ligation of the proximal end of the sac for infants younger than 1 year 6 months in whom the external inguinal ring is located so close to the IIR that the shortened inguinal canal becomes uncovered by musculature and is vulnerable to increased intra-abdominal pressure. The doubly ligated IIR is expected to hold against pressure until wound healing is completely accomplished.
Drawbacks associated with the reproductive system are a hidden but not negligible problem. One report suggests that vas deferens or epididymis was found in 0.53% of hernial sacs removed during herniorrhaphy [25]. The incidence of vasal injury during inguinal herniorrhaphy has been estimated at 0.5% [26]. Fallopian tube obstruction in a woman with a history of childhood bilateral inguinal herniorrhaphy was reported as the cause of infertility [27]. We had two episodes of injury to the reproductive system during cut-down repair. In addition, testicular atrophy, ascent of the testis, ovarian malposition, and bladder injury have been reported, none of which occurred in our laparoscopic series.
Despite increasing reports regarding the laparoscopic approach, there has been only one comparative study between laparoscopic and open repair. Chan et al. emphasized the superiority of laparoscopic repair from the points of less pain, prompter recovery, and better cosmesis [28]. Operation times were longer for unilateral and equal for bilateral repair compared with open repair. In our series, the majority of parents preferred laparoscopic repair based on postoperative cosmetic superiority, bilateral IIR inspection, and simultaneous repair for an unpleasant-looking umbilicus. Laparoscopic operation times were equal for unilateral and shorter for bilateral repair compared with open repair. There was no injury to the reproductive system in contrast to the cut-down procedure. Postoperative recurrence and contralateral hernia were less in the laparoscopic group, although we could not achieve 0% incidence.
Conclusions:
Although we must currently accept that laparoscopic hernia repair has not been around long enough to rate fully the risk of late complications, we believe that this procedure with the Endoneedle can be a routine procedure with results comparable or superior to those with open procedures. The advantages of our technique include the following: technically simple, short operation time, inspection of bilateral IIRs with simultaneous closure of cPPV, the reproductive system remains intact, routine addition of umbilicoplasty if desired by the parents of patients, and essentially indiscernible wounds.
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Background:
Conventional open herniorrhaphy in children has been reported to have 0.3-3.8% recurrence and 5.6-30% postoperative contralateral hernia rates. We developed a unique technique to achieve completely extraperitoneal ligation of PPV without any skip areas under laparoscopic control. This report introduces our technique and results compared with the cut-down herniorrhaphy.
Methods:
A consecutive series of 1,585 children with inguinal hernia/hydrocele (1996-2006) was analyzed. In laparoscopic patent processus vaginalis (PPV) closure (LPC), an orifice of PPV was encircled with a 2-0 suture extraperitoneally by a specially devised Endoneedle and tied up from outside of the body achieving completely extraperitoneal ligation of the ring. The round ligament was included in the ligation, whereas the spermatic cord and testicular vessels were excluded by advancing the needle across them behind the peritoneum. Cut-down herniorrhaphy (CD), with or without diagnostic laparoscopy, or LPC was selected according to parental preference under informed consent.
Results:
Parents gave more preference to LPC (LPC in 1,257 children, CD in 308, and miscellaneous in 20). Age ranges were equal for both groups. Sex distribution showed female preponderance in the LPC group (44.8% vs. 26.6%, p < 0.001) and umbilical hernia/cysts were predominantly included in the LPC group (11.9% vs. 2.9%, p < 0.001). Mean operation times were equal for both groups for unilateral repair (28.2 +/- 9.2 for LPC vs. 27.8 +/- 13.5 for CD) and were shorter for bilateral repair in the LPC group (35.8 +/- 11.6 vs. 46.7 +/- 17.7). The incidence of postoperative hernia recurrence and contralateral hernia in the LPC group was 0.2% and 0.8%. Two children in the CD group had injuries to their reproductive system during the operation (0.6%).
Conclusions:
The advantages of our technique include following: technically simple, short operation time, inspection of bilateral IIRs with simultaneous closure of cPPV, reproductive systems remain intact, routine addition of umbilicoplasty if desired, and essentially indiscernible wounds.
| null | null | 9,605 | 399 | 11 |
[
"lpc",
"hernia",
"cd",
"vessels",
"group",
"suture",
"spermatic",
"laparoscopic",
"testicular",
"closure"
] |
[
"test",
"test"
] | null | null | null | null | null |
[CONTENT] Laparoscopic herniorrhaphy | Indirect inguinal hernia | Extraperitoneal repair | Cut-down herniorrhaphy | Laparoscopic repair [SUMMARY]
|
[CONTENT] Laparoscopic herniorrhaphy | Indirect inguinal hernia | Extraperitoneal repair | Cut-down herniorrhaphy | Laparoscopic repair [SUMMARY]
|
[CONTENT] Laparoscopic herniorrhaphy | Indirect inguinal hernia | Extraperitoneal repair | Cut-down herniorrhaphy | Laparoscopic repair [SUMMARY]
| null | null | null |
[CONTENT] Adolescent | Child | Child, Preschool | Fallopian Tubes | Female | Follow-Up Studies | Hernia, Inguinal | Humans | Incidence | Infant | Infant, Newborn | Intraoperative Complications | Laparoscopy | Laparotomy | Ligation | Male | Parents | Recurrence | Retrospective Studies | Round Ligament of Uterus | Suture Techniques | Testicular Hydrocele | Vas Deferens | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Child | Child, Preschool | Fallopian Tubes | Female | Follow-Up Studies | Hernia, Inguinal | Humans | Incidence | Infant | Infant, Newborn | Intraoperative Complications | Laparoscopy | Laparotomy | Ligation | Male | Parents | Recurrence | Retrospective Studies | Round Ligament of Uterus | Suture Techniques | Testicular Hydrocele | Vas Deferens | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Child | Child, Preschool | Fallopian Tubes | Female | Follow-Up Studies | Hernia, Inguinal | Humans | Incidence | Infant | Infant, Newborn | Intraoperative Complications | Laparoscopy | Laparotomy | Ligation | Male | Parents | Recurrence | Retrospective Studies | Round Ligament of Uterus | Suture Techniques | Testicular Hydrocele | Vas Deferens | Young Adult [SUMMARY]
| null | null | null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null | null | null |
[CONTENT] lpc | hernia | cd | vessels | group | suture | spermatic | laparoscopic | testicular | closure [SUMMARY]
|
[CONTENT] lpc | hernia | cd | vessels | group | suture | spermatic | laparoscopic | testicular | closure [SUMMARY]
|
[CONTENT] lpc | hernia | cd | vessels | group | suture | spermatic | laparoscopic | testicular | closure [SUMMARY]
| null | null | null |
[CONTENT] group | lpc | ns | cd | lpc group | min | 001 | hernia | postoperative | significant [SUMMARY]
|
[CONTENT] routine | iirs simultaneous | bilateral iirs simultaneous closure | simple short | comparable superior | comparable superior open | comparable superior open procedures | operation time inspection | bilateral iirs | bilateral iirs simultaneous [SUMMARY]
|
[CONTENT] lpc | group | cd | vessels | hernia | suture | laparoscopic | ns | spermatic | gauge [SUMMARY]
| null | null | null |
[CONTENT] LPC | 1,257 | 308 | 20 ||| ||| LPC | 44.8% | 26.6% | p < 0.001 | LPC | 11.9% | 2.9% | p < 0.001 ||| 28.2 + | 9.2 | LPC | 27.8 + | 13.5 | LPC | 35.8 | 11.6 | 46.7 | 17.7 ||| LPC | 0.2% | 0.8% ||| Two | 0.6% [SUMMARY]
|
[CONTENT] cPPV [SUMMARY]
|
[CONTENT] 0.3-3.8% | 5.6-30% | hernia ||| PPV ||| ||| 1,585 | hernia | 1996-2006 ||| PPV | PPV | 2 | Endoneedle ||| ||| LPC ||| LPC | 1,257 | 308 | 20 ||| ||| LPC | 44.8% | 26.6% | p < 0.001 | LPC | 11.9% | 2.9% | p < 0.001 ||| 28.2 + | 9.2 | LPC | 27.8 + | 13.5 | LPC | 35.8 | 11.6 | 46.7 | 17.7 ||| LPC | 0.2% | 0.8% ||| Two | 0.6% ||| cPPV [SUMMARY]
| null |
|||
Chronologically matched toenail-Hg to hair-Hg ratio: temporal analysis within the Japanese community (U.S.).
|
23113987
|
Toenail-Hg levels are being used as a marker of methylmercury (MeHg) exposure in efforts to associate exposure with effects such as cardiovascular disease. There is a need to correlate this marker with more established biomarkers that presently underlie existing dose-response relationships in order to compare these relationships across studies.
|
BACKGROUND
|
As part of the Arsenic Mercury Intake Biometric Study, toenail clippings were collected at three time points over a period of one year amongst females from within the population of Japanese living near Puget Sound in Washington State (US). Variability in temporal intra-individual toenail-Hg levels was examined and chronologically matched hair and toenail samples were compared to more accurately define the toxicokinetic variability of Hg levels observed between the two compartments.
|
METHODS
|
Mean toenail-Hg values (n=43) for the 1st, 2nd and 3rd visits were 0.60, 0.60 and 0.56 ng/mg. Correlations were as follows: r=0.92 between 1st and 2nd clinic visits, r=0.75 between 1st and 3rd visits and r=0.87 between 2nd and 3rd visits. With few exceptions, toenail-Hg values from any visit were within 50-150% of the individual's mean toenail-Hg level. Nearly all participants had less than a two-fold change in toenail-Hg levels across the study period. A regression model of the relationship between toenail-Hg and hair-Hg (n = 41) levels representing the same time period of exposure, gave a slope (Hg ng/mg) of 2.79 for hair relative to toenail (r=0.954).
|
RESULTS
|
A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that consumes fish regularly and in quantity. Intra-individual variation in toenail-Hg levels was less than two-fold and may represent dietary-based fluctuations in body burden for individuals consuming various fish species with different contaminant levels. The chronologically matched ratio will be useful for relating MeHg exposure and dose-response derived from toenail-Hg measurements to those derived from hair-Hg measurements in other studies, and may be useful in future investigations as an indicator of stable MeHg body burden within a population.
|
CONCLUSIONS
|
[
"Adolescent",
"Adult",
"Asian People",
"Environmental Monitoring",
"Environmental Pollutants",
"Female",
"Hair",
"Humans",
"Mercury",
"Middle Aged",
"Nails",
"Time Factors",
"Young Adult"
] |
3511224
|
Background
|
As most human exposures to methylmercury (MeHg) result from fish consumption, federal and state consumption advisories have been released as a public health protective measure [1,2]. Most state and federal advice to fish consumers reflects consideration of the US EPA reference dose (RfD) for MeHg [3]. The RfD, as well as the general recognition of the hazardous nature of MeHg exposure is in turn based upon extensive neurological research that has been repeatedly summarized e.g., [4-6]. In addition to concerns over neurophysiological deficits that can occur in children exposed in utero to MeHg, concern has been raised about the possible role of MeHg in cardiovascular disease in adults. There have been several major epidemiological studies conducted that investigated the relationship between MeHg exposure and the incidence of myocardial infarction [7-11]. The findings from four of these five studies along with additional data relating MeHg exposure with, for example, heart rate variability and oxidative stress were critical to a panel assembled under contract to the US EPA that recommended the development of a dose–response function for myocardial infarction and MeHg exposure in a regulatory benefits analyses addressing Hg emissions [12]. Roman et al. [12] did not address findings from the subsequently published fifth study which did not observe an association between MeHg exposure and coronary heart disease or stroke [11].
Hair-Hg, blood-Hg and toenail-Hg levels have been used as biomarkers of exposure for MeHg with each biomarker having been significantly correlated with fish consumption which is the primary source of MeHg exposure [4,6,13-15]. Analyses of hair-Hg and blood-Hg levels in fish consumers and non-fish consumers have been conducted allowing for these compartments to be used to characterize exposure to MeHg within a population [4]. Hair- and blood- Hg concentration have been the biomarkers of MeHg exposure used most frequently in studies investigating the relationship between MeHg exposure and neurodevelopmental outcomes as well as in several of the major studies investigating cardiovascular effects [7,10,16-18]. However, several important epidemiological studies investigating the relationship between exposure and cardiovascular outcomes have relied on toenail-Hg levels as the biomarker for MeHg exposure [8,9,11]. Data on the use of toenail-Hg levels as a biomarker of exposure is less robust compared to data from hair and blood compartments. The foundation of the toenail compartment as a biomarker is primarily based on observed correlations with hair-Hg levels and fish consumption with several studies having assessed MeHg exposures using toenails and a few having compared measurements across multiple markers [13-15,19-26]. Several studies within this group have provided for data allowing hair-Hg to toenail-Hg ratios to be established based on mean values and have developed regression models examining the relationship between hair-Hg and toenail-Hg levels [13,15,21,22,26]. However, despite these available data comparing Hg concentrations between two compartments, data are not available comparing hair and toenail biomarker results from exposure that occurred over the same time period.
As part of the Arsenic Mercury Intake Biometric Study (AMIBS), we compared Hg levels from hair segments and toenail clippings reflecting the same time period of exposure and examined longitudinal data for toenail-Hg levels obtained at three time points within the population of Japanese women living in the Puget Sound area of Washington State (US). Previous work has examined fish intake and MeHg body burden in this population and results indicated that this Japanese population consumes substantially more fish than the national average and that MeHg exposure within this group is among the highest in the US [27,28]. The goals of this work were two-fold: to more accurately define the toxicokinetic variability of Hg levels observed between the hair and toenail compartment, and to examine variability in temporal toenail-Hg levels that were obtained over a period of approximately one year. This information will permit an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure and may be useful in future studies examining the relationship between neurological effects and MeHg exposure. It will also facilitate the comparison between the results of studies employing toenail- and hair-Hg levels as biomarkers of exposure.
|
Methods
|
AMIBS As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.
Informed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.
As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.
Informed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.
Hair and toenail sampling To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.
Toenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.
The time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.
In general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:
1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and
2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.
Accordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.
The hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.
To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.
Toenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.
The time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.
In general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:
1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and
2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.
Accordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.
The hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.
Hair- and toenail-Hg analyses Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.
Toenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).
Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.
Toenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).
Statistical analyses A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).
A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).
|
Results
|
Toenail and hair samples were collected from 43 Japanese participants across three clinic visits. Mean toenail growth rate was 0.048 ±0.012 mm/day (22.4 ±6.5 days/mm) and mean hair growth rate was 0.42 ±0.07 mm/day (24.5 ±5.3 days/cm). No significant difference in toenail-Hg or in hair-Hg levels was observed between pregnant (n = 11) and non-pregnant participants (n = 32) (p > 0.05). The average number of days between visits was 135 (± 18) days with the shortest interval being 100 days and the longest, 189 days. The interquartile range was 126 to 141 days. The mean toenail growth length across the average number of days between visits was 6.48 mm (135 days x 0.048 mm/day). The mean time period spanned by the three visits was 270 days (range, 226 to 350). Interquartile Hg-concentration ranges for the three visits were 0.33-0.67, 0.34-0.66 and 0.34-0.67 ng/mg, respectively. Respective 95th percentile confidence intervals were ± 0.12, 0.13 and 0.10. Arithmetic mean toenail-Hg values with standard deviations and coefficients of variation in parentheses for the 1st, 2nd and 3rd visits were 0.60 ±0.42 (0.70), 0.60 ±0.43 (0.72) and 0.56 ±0.35 (0.63) ng/mg with box plots depicting visit distributional data provided in Figure 1. No significant difference in toenail-Hg levels was observed among the three sampling visits, but the relatively small number of samples limited the power to detect a small difference. Pearson’s product–moment correlation coefficients of Hg concentration between sampling intervals were as follows: r = 0.92 between 1st and 2nd clinic visits, r = 0.87 between 2nd and 3rd visits and r = 0.75 between 1st and 3rd visits.
Toenail-Hg levels for each visit (n = 43). Medians are middle lines within box. Top and bottom of box represents upper and lower quartile values, respectively. Upper and lower whiskers represent sample maximum and minimum values, respectively. Distribution mean values do not differ significantly (p <0.05).
The absolute change in toenail-Hg levels on an individual basis between successive samples across the three visits is depicted in Figure 2. With the exception of three toenail-Hg levels (out of 129), all values for separate clinic visits were within 50% to 150% of the individual’s mean toenail-Hg level. The extent of intra-individual change among participants depicted as the ratio of highest to lowest toenail-Hg levels from the visits for each individual is provided as a histogram in Figure 3. Nearly all participants (39 of 43) had less than a two-fold change in toenail-Hg levels across the one-year time period encompassed by the three samples with half (22 of 43) not exceeding a 1.5-fold change. Range of change was 1.08 - 3.44.
Intra-individual toenail-Hg variability across three successive clinic visits.
Ratio of intra-individual toenail-Hg levels (n = 43).
Chronologically matched sample evaluation was conducted by comparing toenail-Hg levels with hair-Hg values representing the same time period of exposure (n = 41). Chronological matching of samples was not possible for all individuals due to factors such as timing of sample collections and length of hair samples. A regression model of the relationship between chronologically matched toenail-Hg and hair-Hg samples reflecting the same period of exposure provided for a slope (Hg ng/mg) of 2.79 for hair relative to tonail (r=0.954) (Figure 4). For purposes of comparison with previously conducted studies, the regression line of hair-Hg on toenail-Hg using results from samples obtained at the same point in time (i.e. during a clinic visit) and thus not reflecting the same window of exposure (chronologically unmatched) was determined and yielded a slope of 2.39 (r = 0.871)(data not shown). A comparison of results on group mean biomarker-Hg levels identified from the literature are provided in Table 1. The table gives ratios of hair-Hg to toenail-Hg based on study mean values with the addition of slope values from regression models of hair-Hg on toenail-Hg provided from three of the studies [13,15, this study]. The ratio of hair- to toenail-Hg based on arithmetic mean values for this study was 3.08 and 2.77 based on chronologically matched and unmatched samples respectively. The unmatched sample ratio of 2.77 was similar to the ratio (2.56) obtained by Ohno and co-workers [15] within a similar population (non-occupationally exposed group of Japanese women) and also similar to ratios obtained from a control group (2.4) in a cohort study of dentists as well as from one of two cohorts (2.5) studied in Finland [13,26]. The slope from the regression model using chronologically unmatched sample results (2.39) was similar to the slope (2.44) obtained by Ohno and co-workers [15] and by Alfthan [13] in one of two cohorts (2.24). For comparative purposes, a ratio of 3.16 was derived and is presented in Table 1 using geometric mean values for hair-Hg and toenail-Hg from matched samples; 1.41 ng/mg and 0.45 ng/mg, respectively.
Relationship between toenail- and hair- Hg levels (ng/mg) among participants (n= 41).
Hair-Hg to toenail-Hg ratios determined from regression models and mean values obtained from literature
a. Study data provided for blood-Hg and toenail-Hg values along with regression model. Hair-Hg to blood-Hg ratio of 250:1 was used to obtain hair-Hg: toenail-Hg and slope of regression model of blood-Hg on toenail-Hg [4].
b. One individual had previous occupational exposure as a dental nurse.
c. Ohno and co-workers [15] and Alfthan [13] provided regression models of hair-Hg on toenail-Hg with resulting slope factors. Slope in this study from linear regression model based on chronologically matched and unmatched samples are provided.
d. For this study, geometric mean values for hair-Hg and toenail-Hg from matched samples were 1.410 ng/mg and 0.446 ng/mg, respectively.
|
Conclusions
|
The data presented in this study will allow for an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure. A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that has a MeHg body-burden level that is relatively stable over time. Intra-individual variation was less than two-fold, which may represent normal body burden fluctuation for individuals consuming elevated amounts of various fish species with different contaminant levels. The use of biomarker-based estimates reflecting body-burden levels that are chronologically distant from manifestation of deleterious outcome, or that are used as the sole indicator reflecting chronic exposure, need to be considered carefully when testing a hypothesis as individual body-burden levels may not remain constant across lengthy time periods such as a decade.
|
[
"Background",
"AMIBS",
"Hair and toenail sampling",
"Hair- and toenail-Hg analyses",
"Statistical analyses",
"Abbreviations",
"Competing interests",
"Authors’ contributions",
"Disclaimer"
] |
[
"As most human exposures to methylmercury (MeHg) result from fish consumption, federal and state consumption advisories have been released as a public health protective measure [1,2]. Most state and federal advice to fish consumers reflects consideration of the US EPA reference dose (RfD) for MeHg [3]. The RfD, as well as the general recognition of the hazardous nature of MeHg exposure is in turn based upon extensive neurological research that has been repeatedly summarized e.g., [4-6]. In addition to concerns over neurophysiological deficits that can occur in children exposed in utero to MeHg, concern has been raised about the possible role of MeHg in cardiovascular disease in adults. There have been several major epidemiological studies conducted that investigated the relationship between MeHg exposure and the incidence of myocardial infarction [7-11]. The findings from four of these five studies along with additional data relating MeHg exposure with, for example, heart rate variability and oxidative stress were critical to a panel assembled under contract to the US EPA that recommended the development of a dose–response function for myocardial infarction and MeHg exposure in a regulatory benefits analyses addressing Hg emissions [12]. Roman et al. [12] did not address findings from the subsequently published fifth study which did not observe an association between MeHg exposure and coronary heart disease or stroke [11].\nHair-Hg, blood-Hg and toenail-Hg levels have been used as biomarkers of exposure for MeHg with each biomarker having been significantly correlated with fish consumption which is the primary source of MeHg exposure [4,6,13-15]. Analyses of hair-Hg and blood-Hg levels in fish consumers and non-fish consumers have been conducted allowing for these compartments to be used to characterize exposure to MeHg within a population [4]. Hair- and blood- Hg concentration have been the biomarkers of MeHg exposure used most frequently in studies investigating the relationship between MeHg exposure and neurodevelopmental outcomes as well as in several of the major studies investigating cardiovascular effects [7,10,16-18]. However, several important epidemiological studies investigating the relationship between exposure and cardiovascular outcomes have relied on toenail-Hg levels as the biomarker for MeHg exposure [8,9,11]. Data on the use of toenail-Hg levels as a biomarker of exposure is less robust compared to data from hair and blood compartments. The foundation of the toenail compartment as a biomarker is primarily based on observed correlations with hair-Hg levels and fish consumption with several studies having assessed MeHg exposures using toenails and a few having compared measurements across multiple markers [13-15,19-26]. Several studies within this group have provided for data allowing hair-Hg to toenail-Hg ratios to be established based on mean values and have developed regression models examining the relationship between hair-Hg and toenail-Hg levels [13,15,21,22,26]. However, despite these available data comparing Hg concentrations between two compartments, data are not available comparing hair and toenail biomarker results from exposure that occurred over the same time period.\nAs part of the Arsenic Mercury Intake Biometric Study (AMIBS), we compared Hg levels from hair segments and toenail clippings reflecting the same time period of exposure and examined longitudinal data for toenail-Hg levels obtained at three time points within the population of Japanese women living in the Puget Sound area of Washington State (US). Previous work has examined fish intake and MeHg body burden in this population and results indicated that this Japanese population consumes substantially more fish than the national average and that MeHg exposure within this group is among the highest in the US [27,28]. The goals of this work were two-fold: to more accurately define the toxicokinetic variability of Hg levels observed between the hair and toenail compartment, and to examine variability in temporal toenail-Hg levels that were obtained over a period of approximately one year. This information will permit an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure and may be useful in future studies examining the relationship between neurological effects and MeHg exposure. It will also facilitate the comparison between the results of studies employing toenail- and hair-Hg levels as biomarkers of exposure.",
"As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.",
"To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.",
"Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).",
"A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).",
"AMIBS: Arsenic Mercury Intake Biometric Study; EPA: Environmental Protection Agency; GM: Geometric Mean; Hg: Mercury; MeHg: MethylMercury; RfD: Reference Dose; US: United States; WA: Washington State.",
"All authors declare they have no actual or potential competing financial or non-financial interests in this work.",
"TH conducted the hair-Hg and toenail-Hg analyses and provided the Supplemental Materials. AT was a significant contributor to almost all aspects of this project involving the Korean and Japanese populations. AMIBS would not have been as successful without her. AS aided in writing the manuscript. TMB contributed significantly to study design and data interpretation. EM aided in the writing of the manuscript and contributed to the acquisition of the data. All authors contributed substantially to the discussion of the data and their analyses, and provided editorial comments to the draft manuscript.",
"The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views or policies of the U.S. EPA, NIH/National Institute of Environmental Health, National Science Foundation, New Jersey Department of Environmental Protection, or Washington State Department of Health."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"AMIBS",
"Hair and toenail sampling",
"Hair- and toenail-Hg analyses",
"Statistical analyses",
"Results",
"Discussion",
"Conclusions",
"Abbreviations",
"Competing interests",
"Authors’ contributions",
"Disclaimer",
"Supplementary Material"
] |
[
"As most human exposures to methylmercury (MeHg) result from fish consumption, federal and state consumption advisories have been released as a public health protective measure [1,2]. Most state and federal advice to fish consumers reflects consideration of the US EPA reference dose (RfD) for MeHg [3]. The RfD, as well as the general recognition of the hazardous nature of MeHg exposure is in turn based upon extensive neurological research that has been repeatedly summarized e.g., [4-6]. In addition to concerns over neurophysiological deficits that can occur in children exposed in utero to MeHg, concern has been raised about the possible role of MeHg in cardiovascular disease in adults. There have been several major epidemiological studies conducted that investigated the relationship between MeHg exposure and the incidence of myocardial infarction [7-11]. The findings from four of these five studies along with additional data relating MeHg exposure with, for example, heart rate variability and oxidative stress were critical to a panel assembled under contract to the US EPA that recommended the development of a dose–response function for myocardial infarction and MeHg exposure in a regulatory benefits analyses addressing Hg emissions [12]. Roman et al. [12] did not address findings from the subsequently published fifth study which did not observe an association between MeHg exposure and coronary heart disease or stroke [11].\nHair-Hg, blood-Hg and toenail-Hg levels have been used as biomarkers of exposure for MeHg with each biomarker having been significantly correlated with fish consumption which is the primary source of MeHg exposure [4,6,13-15]. Analyses of hair-Hg and blood-Hg levels in fish consumers and non-fish consumers have been conducted allowing for these compartments to be used to characterize exposure to MeHg within a population [4]. Hair- and blood- Hg concentration have been the biomarkers of MeHg exposure used most frequently in studies investigating the relationship between MeHg exposure and neurodevelopmental outcomes as well as in several of the major studies investigating cardiovascular effects [7,10,16-18]. However, several important epidemiological studies investigating the relationship between exposure and cardiovascular outcomes have relied on toenail-Hg levels as the biomarker for MeHg exposure [8,9,11]. Data on the use of toenail-Hg levels as a biomarker of exposure is less robust compared to data from hair and blood compartments. The foundation of the toenail compartment as a biomarker is primarily based on observed correlations with hair-Hg levels and fish consumption with several studies having assessed MeHg exposures using toenails and a few having compared measurements across multiple markers [13-15,19-26]. Several studies within this group have provided for data allowing hair-Hg to toenail-Hg ratios to be established based on mean values and have developed regression models examining the relationship between hair-Hg and toenail-Hg levels [13,15,21,22,26]. However, despite these available data comparing Hg concentrations between two compartments, data are not available comparing hair and toenail biomarker results from exposure that occurred over the same time period.\nAs part of the Arsenic Mercury Intake Biometric Study (AMIBS), we compared Hg levels from hair segments and toenail clippings reflecting the same time period of exposure and examined longitudinal data for toenail-Hg levels obtained at three time points within the population of Japanese women living in the Puget Sound area of Washington State (US). Previous work has examined fish intake and MeHg body burden in this population and results indicated that this Japanese population consumes substantially more fish than the national average and that MeHg exposure within this group is among the highest in the US [27,28]. The goals of this work were two-fold: to more accurately define the toxicokinetic variability of Hg levels observed between the hair and toenail compartment, and to examine variability in temporal toenail-Hg levels that were obtained over a period of approximately one year. This information will permit an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure and may be useful in future studies examining the relationship between neurological effects and MeHg exposure. It will also facilitate the comparison between the results of studies employing toenail- and hair-Hg levels as biomarkers of exposure.",
" AMIBS As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.\nAs a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.\n Hair and toenail sampling To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.\nTo assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.\n Hair- and toenail-Hg analyses Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).\nSpecimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).\n Statistical analyses A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).\nA repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).",
"As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.\nInformed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.",
"To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.\nToenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.\nThe time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.\nIn general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:\n1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and\n2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.\nAccordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.\nThe hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.",
"Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.\nToenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).",
"A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).",
"Toenail and hair samples were collected from 43 Japanese participants across three clinic visits. Mean toenail growth rate was 0.048 ±0.012 mm/day (22.4 ±6.5 days/mm) and mean hair growth rate was 0.42 ±0.07 mm/day (24.5 ±5.3 days/cm). No significant difference in toenail-Hg or in hair-Hg levels was observed between pregnant (n = 11) and non-pregnant participants (n = 32) (p > 0.05). The average number of days between visits was 135 (± 18) days with the shortest interval being 100 days and the longest, 189 days. The interquartile range was 126 to 141 days. The mean toenail growth length across the average number of days between visits was 6.48 mm (135 days x 0.048 mm/day). The mean time period spanned by the three visits was 270 days (range, 226 to 350). Interquartile Hg-concentration ranges for the three visits were 0.33-0.67, 0.34-0.66 and 0.34-0.67 ng/mg, respectively. Respective 95th percentile confidence intervals were ± 0.12, 0.13 and 0.10. Arithmetic mean toenail-Hg values with standard deviations and coefficients of variation in parentheses for the 1st, 2nd and 3rd visits were 0.60 ±0.42 (0.70), 0.60 ±0.43 (0.72) and 0.56 ±0.35 (0.63) ng/mg with box plots depicting visit distributional data provided in Figure 1. No significant difference in toenail-Hg levels was observed among the three sampling visits, but the relatively small number of samples limited the power to detect a small difference. Pearson’s product–moment correlation coefficients of Hg concentration between sampling intervals were as follows: r = 0.92 between 1st and 2nd clinic visits, r = 0.87 between 2nd and 3rd visits and r = 0.75 between 1st and 3rd visits.\nToenail-Hg levels for each visit (n = 43). Medians are middle lines within box. Top and bottom of box represents upper and lower quartile values, respectively. Upper and lower whiskers represent sample maximum and minimum values, respectively. Distribution mean values do not differ significantly (p <0.05).\nThe absolute change in toenail-Hg levels on an individual basis between successive samples across the three visits is depicted in Figure 2. With the exception of three toenail-Hg levels (out of 129), all values for separate clinic visits were within 50% to 150% of the individual’s mean toenail-Hg level. The extent of intra-individual change among participants depicted as the ratio of highest to lowest toenail-Hg levels from the visits for each individual is provided as a histogram in Figure 3. Nearly all participants (39 of 43) had less than a two-fold change in toenail-Hg levels across the one-year time period encompassed by the three samples with half (22 of 43) not exceeding a 1.5-fold change. Range of change was 1.08 - 3.44.\nIntra-individual toenail-Hg variability across three successive clinic visits.\nRatio of intra-individual toenail-Hg levels (n = 43).\nChronologically matched sample evaluation was conducted by comparing toenail-Hg levels with hair-Hg values representing the same time period of exposure (n = 41). Chronological matching of samples was not possible for all individuals due to factors such as timing of sample collections and length of hair samples. A regression model of the relationship between chronologically matched toenail-Hg and hair-Hg samples reflecting the same period of exposure provided for a slope (Hg ng/mg) of 2.79 for hair relative to tonail (r=0.954) (Figure 4). For purposes of comparison with previously conducted studies, the regression line of hair-Hg on toenail-Hg using results from samples obtained at the same point in time (i.e. during a clinic visit) and thus not reflecting the same window of exposure (chronologically unmatched) was determined and yielded a slope of 2.39 (r = 0.871)(data not shown). A comparison of results on group mean biomarker-Hg levels identified from the literature are provided in Table 1. The table gives ratios of hair-Hg to toenail-Hg based on study mean values with the addition of slope values from regression models of hair-Hg on toenail-Hg provided from three of the studies [13,15, this study]. The ratio of hair- to toenail-Hg based on arithmetic mean values for this study was 3.08 and 2.77 based on chronologically matched and unmatched samples respectively. The unmatched sample ratio of 2.77 was similar to the ratio (2.56) obtained by Ohno and co-workers [15] within a similar population (non-occupationally exposed group of Japanese women) and also similar to ratios obtained from a control group (2.4) in a cohort study of dentists as well as from one of two cohorts (2.5) studied in Finland [13,26]. The slope from the regression model using chronologically unmatched sample results (2.39) was similar to the slope (2.44) obtained by Ohno and co-workers [15] and by Alfthan [13] in one of two cohorts (2.24). For comparative purposes, a ratio of 3.16 was derived and is presented in Table 1 using geometric mean values for hair-Hg and toenail-Hg from matched samples; 1.41 ng/mg and 0.45 ng/mg, respectively.\nRelationship between toenail- and hair- Hg levels (ng/mg) among participants (n= 41).\nHair-Hg to toenail-Hg ratios determined from regression models and mean values obtained from literature\na. Study data provided for blood-Hg and toenail-Hg values along with regression model. Hair-Hg to blood-Hg ratio of 250:1 was used to obtain hair-Hg: toenail-Hg and slope of regression model of blood-Hg on toenail-Hg [4].\nb. One individual had previous occupational exposure as a dental nurse.\nc. Ohno and co-workers [15] and Alfthan [13] provided regression models of hair-Hg on toenail-Hg with resulting slope factors. Slope in this study from linear regression model based on chronologically matched and unmatched samples are provided.\nd. For this study, geometric mean values for hair-Hg and toenail-Hg from matched samples were 1.410 ng/mg and 0.446 ng/mg, respectively.",
"Toenail clippings from females were examined over a period of approximately one year to investigate variability in temporal intra-individual toenail-Hg levels, and chronologically matched hair and toenail samples were compared to more accurately define the toxicokinetic variability of MeHg levels observed between the two compartments.\nHair-Hg levels from this population of females have previously been investigated with results showing no significant change in MeHg body burden levels across the study period suggesting that if MeHg population-based body burden levels were changing they were doing so slowly and across a time period larger than the one investigated [35]. This study corroborates that conclusion and further, indicates that while there was temporal stability of the three toenail-Hg levels for the group as a whole, individuals had variable MeHg body-burden levels but the levels did not fluctuate by more than two-fold across the one-year period investigated. The fluctuation in individual body burden levels is likely due to changes in MeHg intake across sample intervals while intra-individual toxicokinetic variability may also be a contributing factor. The intra-individual change in Hg levels observed across the study period which did not exceed 1.5-fold for half of the participants and two-fold for nearly the entire study group can be considered normal behavioral variability that is non-directional fluctuation within longer term temporal stability. For individuals with stable body burden levels, a single data point value, such as toenail-Hg levels obtained at a given time, could be used to reflect long term MeHg body burden; with the individual’s mean value being within the interval defined by one-third to twice the obtained toenail-Hg level. It must be noted however, that for identifying body burden levels across a short time period, even within those having temporal stability, biomarker based measurements from a single time point used to estimate exposure can miss short term elevated MeHg exposure periods consisting of single exposures to those lasting weeks [36-38].\nFor toenails to be used as a biomarker for long term exposures such as years, there is a need to either verify temporal stability within the study population by comparing biomarkers reflecting body-burden levels against an empirically derived ratio as defined herein, or repeated biomarker determinations would be needed so as to understand long term body burden characteristics. If a population has stable MeHg body burden levels, or if long term averages can be identified, biomarkers such as toenail could be used to identify causal association even though the biomarker may be chronologically removed from disease manifestation. These determinants are relevant to the relationship between ongoing exposures and endpoints resulting from chronic exposure (i.e. cardiovascular disease) but would not be as applicable to circumstances associated with the population consisting of women of child bearing age. Long term variability will not be as relevant for gestational exposure as present day understanding suggests a finite window of susceptibility for women during pregnancy may exist that can result in deleterious effects; possibly even from a single exposure resulting in a large bolus dose at a critical time period during fetal development.\nA limitation of this work is that the intra-individual temporal variability observed in toenail-Hg levels, even though only two fold, could be a factor leading to imprecision in exposure measurement. Estimates of imprecision for biomarker parameters such as hair and blood that can impact dose estimations in dose–response models have already been provided and the toenail compartment as a reservoir reflecting MeHg body burden may include similar imprecision [39]. When investigating dose–response relationships, unbiased imprecision can lead to an underestimation of the relationship between dose and response. In the present work, where two toxicokinetic compartments are being compared, the imprecision will not lead to bias but will provide for a random error effect.\nChronologically matching hair with toenails provided for high correlation and yielded a slope of 2.79 based on a regression model (Table 1). Data that were obtained from samples collected at the same point in time (i.e. during a clinic visit) and thus not chronologically matched with respect to exposure period were also shown to correlate and were similar to results found in other studies comparing data from biomarkers collected at the same time but not representing the same period of exposure (Table 1). Nonetheless, the chronologically matched ratio should be used to estimate Hg levels in the hair compartment based on toenail levels. The chronologically matched ratio would allow for estimating the corresponding Hg levels in hair that have been widely used as a biomarker for investigating multiple toxicological effects, including cardiovascular outcomes and, would allow for comparison across studies as the hair-Hg data set within the literature is robust.\nFor any given population, if the hair- and toenail-Hg ratios from chronologically matched and unmatched samples are in reasonable agreement, we may conclude that their body burdens are relatively stable over time. From the existing population data sets (Table 1), this reasoning would suggest that the non-occupationally exposed Japanese women [15], one group of non-occupationally exposed Finns [13] and possibly the control group from within the dentists study [26] may have stable body burden levels. However, even if the body burden in a population is found to be, on average, stable over a given time period, it cannot necessarily be assumed that such stability can be extrapolated to much longer time periods. Thus, attempts at identifying stability in body burden within a study population should be made if biomarkers chronologically removed from disease manifestation are to be used to investigate a dose response relationship.",
"The data presented in this study will allow for an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure. A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that has a MeHg body-burden level that is relatively stable over time. Intra-individual variation was less than two-fold, which may represent normal body burden fluctuation for individuals consuming elevated amounts of various fish species with different contaminant levels. The use of biomarker-based estimates reflecting body-burden levels that are chronologically distant from manifestation of deleterious outcome, or that are used as the sole indicator reflecting chronic exposure, need to be considered carefully when testing a hypothesis as individual body-burden levels may not remain constant across lengthy time periods such as a decade.",
"AMIBS: Arsenic Mercury Intake Biometric Study; EPA: Environmental Protection Agency; GM: Geometric Mean; Hg: Mercury; MeHg: MethylMercury; RfD: Reference Dose; US: United States; WA: Washington State.",
"All authors declare they have no actual or potential competing financial or non-financial interests in this work.",
"TH conducted the hair-Hg and toenail-Hg analyses and provided the Supplemental Materials. AT was a significant contributor to almost all aspects of this project involving the Korean and Japanese populations. AMIBS would not have been as successful without her. AS aided in writing the manuscript. TMB contributed significantly to study design and data interpretation. EM aided in the writing of the manuscript and contributed to the acquisition of the data. All authors contributed substantially to the discussion of the data and their analyses, and provided editorial comments to the draft manuscript.",
"The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views or policies of the U.S. EPA, NIH/National Institute of Environmental Health, National Science Foundation, New Jersey Department of Environmental Protection, or Washington State Department of Health.",
"Supplemental Material. Provides additional text to description found in Methods section on hair-Hg representativeness for each hair sample.\nClick here for file"
] |
[
null,
"methods",
null,
null,
null,
null,
"results",
"discussion",
"conclusions",
null,
null,
null,
null,
"supplementary-material"
] |
[
"Mercury",
"Methylmercury",
"Cardiovascular",
"Hair",
"Toenail",
"Chronological",
"Biomarker"
] |
Background:
As most human exposures to methylmercury (MeHg) result from fish consumption, federal and state consumption advisories have been released as a public health protective measure [1,2]. Most state and federal advice to fish consumers reflects consideration of the US EPA reference dose (RfD) for MeHg [3]. The RfD, as well as the general recognition of the hazardous nature of MeHg exposure is in turn based upon extensive neurological research that has been repeatedly summarized e.g., [4-6]. In addition to concerns over neurophysiological deficits that can occur in children exposed in utero to MeHg, concern has been raised about the possible role of MeHg in cardiovascular disease in adults. There have been several major epidemiological studies conducted that investigated the relationship between MeHg exposure and the incidence of myocardial infarction [7-11]. The findings from four of these five studies along with additional data relating MeHg exposure with, for example, heart rate variability and oxidative stress were critical to a panel assembled under contract to the US EPA that recommended the development of a dose–response function for myocardial infarction and MeHg exposure in a regulatory benefits analyses addressing Hg emissions [12]. Roman et al. [12] did not address findings from the subsequently published fifth study which did not observe an association between MeHg exposure and coronary heart disease or stroke [11].
Hair-Hg, blood-Hg and toenail-Hg levels have been used as biomarkers of exposure for MeHg with each biomarker having been significantly correlated with fish consumption which is the primary source of MeHg exposure [4,6,13-15]. Analyses of hair-Hg and blood-Hg levels in fish consumers and non-fish consumers have been conducted allowing for these compartments to be used to characterize exposure to MeHg within a population [4]. Hair- and blood- Hg concentration have been the biomarkers of MeHg exposure used most frequently in studies investigating the relationship between MeHg exposure and neurodevelopmental outcomes as well as in several of the major studies investigating cardiovascular effects [7,10,16-18]. However, several important epidemiological studies investigating the relationship between exposure and cardiovascular outcomes have relied on toenail-Hg levels as the biomarker for MeHg exposure [8,9,11]. Data on the use of toenail-Hg levels as a biomarker of exposure is less robust compared to data from hair and blood compartments. The foundation of the toenail compartment as a biomarker is primarily based on observed correlations with hair-Hg levels and fish consumption with several studies having assessed MeHg exposures using toenails and a few having compared measurements across multiple markers [13-15,19-26]. Several studies within this group have provided for data allowing hair-Hg to toenail-Hg ratios to be established based on mean values and have developed regression models examining the relationship between hair-Hg and toenail-Hg levels [13,15,21,22,26]. However, despite these available data comparing Hg concentrations between two compartments, data are not available comparing hair and toenail biomarker results from exposure that occurred over the same time period.
As part of the Arsenic Mercury Intake Biometric Study (AMIBS), we compared Hg levels from hair segments and toenail clippings reflecting the same time period of exposure and examined longitudinal data for toenail-Hg levels obtained at three time points within the population of Japanese women living in the Puget Sound area of Washington State (US). Previous work has examined fish intake and MeHg body burden in this population and results indicated that this Japanese population consumes substantially more fish than the national average and that MeHg exposure within this group is among the highest in the US [27,28]. The goals of this work were two-fold: to more accurately define the toxicokinetic variability of Hg levels observed between the hair and toenail compartment, and to examine variability in temporal toenail-Hg levels that were obtained over a period of approximately one year. This information will permit an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure and may be useful in future studies examining the relationship between neurological effects and MeHg exposure. It will also facilitate the comparison between the results of studies employing toenail- and hair-Hg levels as biomarkers of exposure.
Methods:
AMIBS As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.
Informed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.
As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.
Informed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.
Hair and toenail sampling To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.
Toenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.
The time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.
In general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:
1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and
2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.
Accordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.
The hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.
To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.
Toenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.
The time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.
In general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:
1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and
2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.
Accordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.
The hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.
Hair- and toenail-Hg analyses Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.
Toenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).
Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.
Toenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).
Statistical analyses A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).
A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).
AMIBS:
As a continuation of AMIBS, hair and toenail data were collected from a subset of the 106 Japanese women participants. The participants were sampled three times across 14 months and this manuscript describes the results from 43 women who provided toenail samples during all three clinic visits and 41 women who provided a toenail and hair sample whose length allowed chronological matching with a toenail sample. Chronologically matching biomarker samples allowed for the same exposure period to be compared. AMIBS has been previously described in detail [27,28]. Briefly, the study consisted of women of childbearing age (18 – 45 years of age), who identified themselves as Korean, Japanese or, of Japanese or Korean descent, and who had lived in the Puget Sound area of Washington State, US, for at least 6 months. The AMIBS group consisted of 214 women (108 Korean and 106 Japanese participants). Participation in the study required that the women provide a hair sample from the nape of the neck (0.5 cm in diameter) and complete a fish consumption survey. Participants also completed a self-administered food frequency questionnaire and could provide additional biological samples (urine, blood and/or toenails) at their discretion.
Informed consent was obtained from all study participants, and the study design and materials were approved by the State of Washington Department of Social and Health Services Human Research Review Board.
Hair and toenail sampling:
To assess growth rates for hair and toenail, the capillary-tube method and the etching method were applied, respectively [29,30]. Hair growth rate determinations using the method described by Saito et al. [29] are provided with some frequency in the literature, yet the method described by Dawber [30] of etching the nail for determining toenail growth rates is less often provided. Toenail growth rate was determined by mechanically etching a groove in the nail plate at the highest point of the lunula perpendicular to the direction of nail plate growth during the participant’s first clinic visit. After etching the nail plate, the distance from the top of the lunula to the eponychium was measured. Upon return visit, the distance between the marking and the top of the lunula as well as between the marking to the eponychium were measured. As both the size and shape of lunula and the eponychium can change slightly over time, the average of both measurements was used along with the time span between visits to determine growth rates.
Toenail growth rates for 35 of the 43 women participants and hair growth rates for 29 of the 43 women participants were obtained during the study period. Individual growth rates were used to determine the date at which the nail emerged from beneath the eponychium as well as when the hair initially protruded from the scalp. For example, by dividing the length of the nail from the eponychium to the distal end of the nail by nail growth rate, the number of days since the nail protruded from beneath the eponychium was obtained.
The time point of exposure identified by the distal end of the toenail clipping, which represents the oldest portion of the nail, was used to determine which hair segment (from hair strands obtained at an earlier clinic visit) was required for analysis. The hair segment was identified based on the date the hair strands were obtained (cut from the nape of the neck) and the individual’s hair growth rate value. With the same time point of exposure identified on the nail and hair segment, chronologically matching samples were acquired for analyses. For those 8 and 14 women, respectively, for whom toenail and hair growth rates were not obtained, the average growth rates from those individuals for whom data were available, were used in calculations to obtain chronologically equivalent samples.
In general, the axial lengths of toenail clippings were between approximately 1mm and 3mm. These toenail clippings reflect not just the point in time defined by the distal end of the clippings, but represent a few weeks of growth. Accordingly, the hair sample segment was specifically identified to encompass the exposure period of a toenail clipping as defined from the distal to the proximal end of the clipping (the axial length). The length of the hair segment sample analyzed represented the window of exposure on the toenail identified as being between:
1) The distal end of the toenail clipping to a time point 60 days prior (which on the toenail is equivalent to 60 days of growth in the direction of proximal end of clipping and exceeds the time period identified by the axial lengths of toenail samples), and
2) The distal end of the toenail clipping to a time point 30 days beyond the exposure time point identified by the distal end of the toenail clipping.
Accordingly, from the location on the hair strand considered to be chronologically equivalent to the time point identified by the distal end of the toenail sample, a hair segment was analyzed that included 30 days growth in the direction of the distal end of the hair segment as well as a period of growth representing 60 days towards the proximal end of the hair segment. In total, a period of approximately 90 days exposure was reflected by the hair segment so as to insure that the time period of exposure represented by the entire toenail clipping was included within the hair segment length analyzed.
The hair segment representing an exposure window of 90 days was chosen so as to encompass the period of exposure identified by the axial length of the toenail sample as well as to address areas of uncertainty associated with uptake of Hg into hair and toenail. Although we assume that the incorporation of Hg into the nail matrix and hair follicle is directly proportional to the level of Hg in blood, there were several factors identified that resulted in a hair segment encompassing an exposure window exceeding that produced by the toenail sample. First, there are no available data regarding the time required between MeHg intake and uptake into the nail root and nail matrix so as to become visible at the eponychium. Second, the time delay between uptake of Hg from blood and into the hair shaft that protrudes above the scalp has been estimated to be between 20 and 60 days [23,31,32]. Third, the axial thicknesses of the toenail samples varied between individuals and it varied within each individual’s sample (i.e., due to tapering of the toenail clippings at each end, the axial thickness of each clipping was not uniform). This lack of precision in toenail clipping samples as well as uncertainty in Hg uptake into the biological markers, did not make it feasible to determine the exact exposure period encompassed between the distal and proximal end of the toenail clipping nor did it allow for determination of the precise position along the hair strand reflecting the time point associated with the distal end of the toenail clipping. Accordingly, a segment of hair growth was analyzed considered to encompass the time period of exposure represented by the toenail clipping.
Hair- and toenail-Hg analyses:
Specimens of hair (0.5-cm diameter bundles from the nape of the neck) and of protruding nail (from both big toes) were collected at three visits. Each chronological hair segment matching the toenail sample was obtained from strands individually cut after being suspended between two self-closing bent forceps and viewed under 1.75x magnification. Accuracy of hair-strand cutting was ensured with the use of suture (stitch) scissors (where the wider notched blade, designed to lift the suture, allowed close positioning to the hair strand without premature cutting) in combination with resting the notched scissor blade in the desired-length groove of a stainless-steel ruler, which permitted the magnified viewing to be unceasingly focused on aligning the free-end of the fiber until the scissor blades were closed. Segments not cut within 0.02 cm of required length (as viewed under 4.25x magnification) were rejected to minimize segmenting as a variable. For each hair sample, chronological hair segments from several fibers were combined to provide at least 1.00 mg (see Additional file 1 for a discussion of hair-Hg representativeness). Chronological hair segments for nearly all the participants were obtained within the first 8 cm from the proximal end of the hair strand.
Toenail clippings were obtained from the big toes using stainless steel scissors. The big toe was selected because the nail beds differ in length among the toes; this results in the available nail segments on each toe having been formed at different times. Clippings were stored in labeled micro-centrifuge tubes, and transported in plastic bags. The toenail clippings were wiped with an alcohol pad (individually packaged) to remove any adhering dermal tissue and dried to constant weight at ambient conditions. Hg analyses for hair and toenails were conducted as described previously using a direct analyzer (Milestone, Inc., Shelton, CT) following EPA Method 7473 [28,33]. The instrument detection limit was near 0.01 ng (corresponding to 0.001 ng/mg for a 10 mg hair or toenail sample). Quality assurance included the measurement of reference materials before and after sample analyses and, participation in Health Canada Mercury-in-Hair Interlaboratory Comparison Program [34]. Because our QC plan specified that Hg results for reference materials that exceeded ±10% of the nominal Hg mean concentration (adjusted for the moisture content) would be used to normalize the data from samples obtained during the same measurement session, raw data for the reference materials (primarily Albacore Tuna, NBS RM 50) do not reflect the accuracy for the samples. Using this calibration-normalization procedure, our blind results for 21 specimens from the Health Canada Mercury-in-Hair Interlaboratory Comparison Program were 100.4 ± 2.9% (mean ± SD) of the consensus Hg means (range 0.92 to 10.05 μg/g, median 4.66 μg/g) from 19 to 25 laboratories. Our measurement precision was sufficient to distinguish (p <0.001) between two Health Canada specimens that differed in Hg by only 3% (2.35 vs 2.28 μg/g). Individual toenail-Hg concentrations from left and right foot were averaged for this study as the relative percent difference (RPD) between individual left and right toenail mercury levels was small (RPD = 6.5%, range 0.1 to 16%).
Statistical analyses:
A repeated-measures one-way ANOVA was used to compare toenail-Hg levels among sample intervals. Student’s t-test was applied to compare means of toenail-Hg levels as well as hair-Hg levels based on pregnancy status. Pearson product–moment correlation coefficients were determined to compare toenail-Hg levels with hair-Hg values. Statistical analyses were performed using a significance level of 5% (p < 0.05) using Stata (Stata Corporation, College Station, Texas), Excel (Microsoft Corporation, Redmond, Washington), VassarStats (http://faculty.vassar.edu/lowry/VassarStats.html) and SPSS (SPSS Inc., Chicago, Illinois).
Results:
Toenail and hair samples were collected from 43 Japanese participants across three clinic visits. Mean toenail growth rate was 0.048 ±0.012 mm/day (22.4 ±6.5 days/mm) and mean hair growth rate was 0.42 ±0.07 mm/day (24.5 ±5.3 days/cm). No significant difference in toenail-Hg or in hair-Hg levels was observed between pregnant (n = 11) and non-pregnant participants (n = 32) (p > 0.05). The average number of days between visits was 135 (± 18) days with the shortest interval being 100 days and the longest, 189 days. The interquartile range was 126 to 141 days. The mean toenail growth length across the average number of days between visits was 6.48 mm (135 days x 0.048 mm/day). The mean time period spanned by the three visits was 270 days (range, 226 to 350). Interquartile Hg-concentration ranges for the three visits were 0.33-0.67, 0.34-0.66 and 0.34-0.67 ng/mg, respectively. Respective 95th percentile confidence intervals were ± 0.12, 0.13 and 0.10. Arithmetic mean toenail-Hg values with standard deviations and coefficients of variation in parentheses for the 1st, 2nd and 3rd visits were 0.60 ±0.42 (0.70), 0.60 ±0.43 (0.72) and 0.56 ±0.35 (0.63) ng/mg with box plots depicting visit distributional data provided in Figure 1. No significant difference in toenail-Hg levels was observed among the three sampling visits, but the relatively small number of samples limited the power to detect a small difference. Pearson’s product–moment correlation coefficients of Hg concentration between sampling intervals were as follows: r = 0.92 between 1st and 2nd clinic visits, r = 0.87 between 2nd and 3rd visits and r = 0.75 between 1st and 3rd visits.
Toenail-Hg levels for each visit (n = 43). Medians are middle lines within box. Top and bottom of box represents upper and lower quartile values, respectively. Upper and lower whiskers represent sample maximum and minimum values, respectively. Distribution mean values do not differ significantly (p <0.05).
The absolute change in toenail-Hg levels on an individual basis between successive samples across the three visits is depicted in Figure 2. With the exception of three toenail-Hg levels (out of 129), all values for separate clinic visits were within 50% to 150% of the individual’s mean toenail-Hg level. The extent of intra-individual change among participants depicted as the ratio of highest to lowest toenail-Hg levels from the visits for each individual is provided as a histogram in Figure 3. Nearly all participants (39 of 43) had less than a two-fold change in toenail-Hg levels across the one-year time period encompassed by the three samples with half (22 of 43) not exceeding a 1.5-fold change. Range of change was 1.08 - 3.44.
Intra-individual toenail-Hg variability across three successive clinic visits.
Ratio of intra-individual toenail-Hg levels (n = 43).
Chronologically matched sample evaluation was conducted by comparing toenail-Hg levels with hair-Hg values representing the same time period of exposure (n = 41). Chronological matching of samples was not possible for all individuals due to factors such as timing of sample collections and length of hair samples. A regression model of the relationship between chronologically matched toenail-Hg and hair-Hg samples reflecting the same period of exposure provided for a slope (Hg ng/mg) of 2.79 for hair relative to tonail (r=0.954) (Figure 4). For purposes of comparison with previously conducted studies, the regression line of hair-Hg on toenail-Hg using results from samples obtained at the same point in time (i.e. during a clinic visit) and thus not reflecting the same window of exposure (chronologically unmatched) was determined and yielded a slope of 2.39 (r = 0.871)(data not shown). A comparison of results on group mean biomarker-Hg levels identified from the literature are provided in Table 1. The table gives ratios of hair-Hg to toenail-Hg based on study mean values with the addition of slope values from regression models of hair-Hg on toenail-Hg provided from three of the studies [13,15, this study]. The ratio of hair- to toenail-Hg based on arithmetic mean values for this study was 3.08 and 2.77 based on chronologically matched and unmatched samples respectively. The unmatched sample ratio of 2.77 was similar to the ratio (2.56) obtained by Ohno and co-workers [15] within a similar population (non-occupationally exposed group of Japanese women) and also similar to ratios obtained from a control group (2.4) in a cohort study of dentists as well as from one of two cohorts (2.5) studied in Finland [13,26]. The slope from the regression model using chronologically unmatched sample results (2.39) was similar to the slope (2.44) obtained by Ohno and co-workers [15] and by Alfthan [13] in one of two cohorts (2.24). For comparative purposes, a ratio of 3.16 was derived and is presented in Table 1 using geometric mean values for hair-Hg and toenail-Hg from matched samples; 1.41 ng/mg and 0.45 ng/mg, respectively.
Relationship between toenail- and hair- Hg levels (ng/mg) among participants (n= 41).
Hair-Hg to toenail-Hg ratios determined from regression models and mean values obtained from literature
a. Study data provided for blood-Hg and toenail-Hg values along with regression model. Hair-Hg to blood-Hg ratio of 250:1 was used to obtain hair-Hg: toenail-Hg and slope of regression model of blood-Hg on toenail-Hg [4].
b. One individual had previous occupational exposure as a dental nurse.
c. Ohno and co-workers [15] and Alfthan [13] provided regression models of hair-Hg on toenail-Hg with resulting slope factors. Slope in this study from linear regression model based on chronologically matched and unmatched samples are provided.
d. For this study, geometric mean values for hair-Hg and toenail-Hg from matched samples were 1.410 ng/mg and 0.446 ng/mg, respectively.
Discussion:
Toenail clippings from females were examined over a period of approximately one year to investigate variability in temporal intra-individual toenail-Hg levels, and chronologically matched hair and toenail samples were compared to more accurately define the toxicokinetic variability of MeHg levels observed between the two compartments.
Hair-Hg levels from this population of females have previously been investigated with results showing no significant change in MeHg body burden levels across the study period suggesting that if MeHg population-based body burden levels were changing they were doing so slowly and across a time period larger than the one investigated [35]. This study corroborates that conclusion and further, indicates that while there was temporal stability of the three toenail-Hg levels for the group as a whole, individuals had variable MeHg body-burden levels but the levels did not fluctuate by more than two-fold across the one-year period investigated. The fluctuation in individual body burden levels is likely due to changes in MeHg intake across sample intervals while intra-individual toxicokinetic variability may also be a contributing factor. The intra-individual change in Hg levels observed across the study period which did not exceed 1.5-fold for half of the participants and two-fold for nearly the entire study group can be considered normal behavioral variability that is non-directional fluctuation within longer term temporal stability. For individuals with stable body burden levels, a single data point value, such as toenail-Hg levels obtained at a given time, could be used to reflect long term MeHg body burden; with the individual’s mean value being within the interval defined by one-third to twice the obtained toenail-Hg level. It must be noted however, that for identifying body burden levels across a short time period, even within those having temporal stability, biomarker based measurements from a single time point used to estimate exposure can miss short term elevated MeHg exposure periods consisting of single exposures to those lasting weeks [36-38].
For toenails to be used as a biomarker for long term exposures such as years, there is a need to either verify temporal stability within the study population by comparing biomarkers reflecting body-burden levels against an empirically derived ratio as defined herein, or repeated biomarker determinations would be needed so as to understand long term body burden characteristics. If a population has stable MeHg body burden levels, or if long term averages can be identified, biomarkers such as toenail could be used to identify causal association even though the biomarker may be chronologically removed from disease manifestation. These determinants are relevant to the relationship between ongoing exposures and endpoints resulting from chronic exposure (i.e. cardiovascular disease) but would not be as applicable to circumstances associated with the population consisting of women of child bearing age. Long term variability will not be as relevant for gestational exposure as present day understanding suggests a finite window of susceptibility for women during pregnancy may exist that can result in deleterious effects; possibly even from a single exposure resulting in a large bolus dose at a critical time period during fetal development.
A limitation of this work is that the intra-individual temporal variability observed in toenail-Hg levels, even though only two fold, could be a factor leading to imprecision in exposure measurement. Estimates of imprecision for biomarker parameters such as hair and blood that can impact dose estimations in dose–response models have already been provided and the toenail compartment as a reservoir reflecting MeHg body burden may include similar imprecision [39]. When investigating dose–response relationships, unbiased imprecision can lead to an underestimation of the relationship between dose and response. In the present work, where two toxicokinetic compartments are being compared, the imprecision will not lead to bias but will provide for a random error effect.
Chronologically matching hair with toenails provided for high correlation and yielded a slope of 2.79 based on a regression model (Table 1). Data that were obtained from samples collected at the same point in time (i.e. during a clinic visit) and thus not chronologically matched with respect to exposure period were also shown to correlate and were similar to results found in other studies comparing data from biomarkers collected at the same time but not representing the same period of exposure (Table 1). Nonetheless, the chronologically matched ratio should be used to estimate Hg levels in the hair compartment based on toenail levels. The chronologically matched ratio would allow for estimating the corresponding Hg levels in hair that have been widely used as a biomarker for investigating multiple toxicological effects, including cardiovascular outcomes and, would allow for comparison across studies as the hair-Hg data set within the literature is robust.
For any given population, if the hair- and toenail-Hg ratios from chronologically matched and unmatched samples are in reasonable agreement, we may conclude that their body burdens are relatively stable over time. From the existing population data sets (Table 1), this reasoning would suggest that the non-occupationally exposed Japanese women [15], one group of non-occupationally exposed Finns [13] and possibly the control group from within the dentists study [26] may have stable body burden levels. However, even if the body burden in a population is found to be, on average, stable over a given time period, it cannot necessarily be assumed that such stability can be extrapolated to much longer time periods. Thus, attempts at identifying stability in body burden within a study population should be made if biomarkers chronologically removed from disease manifestation are to be used to investigate a dose response relationship.
Conclusions:
The data presented in this study will allow for an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure. A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that has a MeHg body-burden level that is relatively stable over time. Intra-individual variation was less than two-fold, which may represent normal body burden fluctuation for individuals consuming elevated amounts of various fish species with different contaminant levels. The use of biomarker-based estimates reflecting body-burden levels that are chronologically distant from manifestation of deleterious outcome, or that are used as the sole indicator reflecting chronic exposure, need to be considered carefully when testing a hypothesis as individual body-burden levels may not remain constant across lengthy time periods such as a decade.
Abbreviations:
AMIBS: Arsenic Mercury Intake Biometric Study; EPA: Environmental Protection Agency; GM: Geometric Mean; Hg: Mercury; MeHg: MethylMercury; RfD: Reference Dose; US: United States; WA: Washington State.
Competing interests:
All authors declare they have no actual or potential competing financial or non-financial interests in this work.
Authors’ contributions:
TH conducted the hair-Hg and toenail-Hg analyses and provided the Supplemental Materials. AT was a significant contributor to almost all aspects of this project involving the Korean and Japanese populations. AMIBS would not have been as successful without her. AS aided in writing the manuscript. TMB contributed significantly to study design and data interpretation. EM aided in the writing of the manuscript and contributed to the acquisition of the data. All authors contributed substantially to the discussion of the data and their analyses, and provided editorial comments to the draft manuscript.
Disclaimer:
The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views or policies of the U.S. EPA, NIH/National Institute of Environmental Health, National Science Foundation, New Jersey Department of Environmental Protection, or Washington State Department of Health.
Supplementary Material:
Supplemental Material. Provides additional text to description found in Methods section on hair-Hg representativeness for each hair sample.
Click here for file
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Background:
Toenail-Hg levels are being used as a marker of methylmercury (MeHg) exposure in efforts to associate exposure with effects such as cardiovascular disease. There is a need to correlate this marker with more established biomarkers that presently underlie existing dose-response relationships in order to compare these relationships across studies.
Methods:
As part of the Arsenic Mercury Intake Biometric Study, toenail clippings were collected at three time points over a period of one year amongst females from within the population of Japanese living near Puget Sound in Washington State (US). Variability in temporal intra-individual toenail-Hg levels was examined and chronologically matched hair and toenail samples were compared to more accurately define the toxicokinetic variability of Hg levels observed between the two compartments.
Results:
Mean toenail-Hg values (n=43) for the 1st, 2nd and 3rd visits were 0.60, 0.60 and 0.56 ng/mg. Correlations were as follows: r=0.92 between 1st and 2nd clinic visits, r=0.75 between 1st and 3rd visits and r=0.87 between 2nd and 3rd visits. With few exceptions, toenail-Hg values from any visit were within 50-150% of the individual's mean toenail-Hg level. Nearly all participants had less than a two-fold change in toenail-Hg levels across the study period. A regression model of the relationship between toenail-Hg and hair-Hg (n = 41) levels representing the same time period of exposure, gave a slope (Hg ng/mg) of 2.79 for hair relative to toenail (r=0.954).
Conclusions:
A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that consumes fish regularly and in quantity. Intra-individual variation in toenail-Hg levels was less than two-fold and may represent dietary-based fluctuations in body burden for individuals consuming various fish species with different contaminant levels. The chronologically matched ratio will be useful for relating MeHg exposure and dose-response derived from toenail-Hg measurements to those derived from hair-Hg measurements in other studies, and may be useful in future investigations as an indicator of stable MeHg body burden within a population.
|
Background:
As most human exposures to methylmercury (MeHg) result from fish consumption, federal and state consumption advisories have been released as a public health protective measure [1,2]. Most state and federal advice to fish consumers reflects consideration of the US EPA reference dose (RfD) for MeHg [3]. The RfD, as well as the general recognition of the hazardous nature of MeHg exposure is in turn based upon extensive neurological research that has been repeatedly summarized e.g., [4-6]. In addition to concerns over neurophysiological deficits that can occur in children exposed in utero to MeHg, concern has been raised about the possible role of MeHg in cardiovascular disease in adults. There have been several major epidemiological studies conducted that investigated the relationship between MeHg exposure and the incidence of myocardial infarction [7-11]. The findings from four of these five studies along with additional data relating MeHg exposure with, for example, heart rate variability and oxidative stress were critical to a panel assembled under contract to the US EPA that recommended the development of a dose–response function for myocardial infarction and MeHg exposure in a regulatory benefits analyses addressing Hg emissions [12]. Roman et al. [12] did not address findings from the subsequently published fifth study which did not observe an association between MeHg exposure and coronary heart disease or stroke [11].
Hair-Hg, blood-Hg and toenail-Hg levels have been used as biomarkers of exposure for MeHg with each biomarker having been significantly correlated with fish consumption which is the primary source of MeHg exposure [4,6,13-15]. Analyses of hair-Hg and blood-Hg levels in fish consumers and non-fish consumers have been conducted allowing for these compartments to be used to characterize exposure to MeHg within a population [4]. Hair- and blood- Hg concentration have been the biomarkers of MeHg exposure used most frequently in studies investigating the relationship between MeHg exposure and neurodevelopmental outcomes as well as in several of the major studies investigating cardiovascular effects [7,10,16-18]. However, several important epidemiological studies investigating the relationship between exposure and cardiovascular outcomes have relied on toenail-Hg levels as the biomarker for MeHg exposure [8,9,11]. Data on the use of toenail-Hg levels as a biomarker of exposure is less robust compared to data from hair and blood compartments. The foundation of the toenail compartment as a biomarker is primarily based on observed correlations with hair-Hg levels and fish consumption with several studies having assessed MeHg exposures using toenails and a few having compared measurements across multiple markers [13-15,19-26]. Several studies within this group have provided for data allowing hair-Hg to toenail-Hg ratios to be established based on mean values and have developed regression models examining the relationship between hair-Hg and toenail-Hg levels [13,15,21,22,26]. However, despite these available data comparing Hg concentrations between two compartments, data are not available comparing hair and toenail biomarker results from exposure that occurred over the same time period.
As part of the Arsenic Mercury Intake Biometric Study (AMIBS), we compared Hg levels from hair segments and toenail clippings reflecting the same time period of exposure and examined longitudinal data for toenail-Hg levels obtained at three time points within the population of Japanese women living in the Puget Sound area of Washington State (US). Previous work has examined fish intake and MeHg body burden in this population and results indicated that this Japanese population consumes substantially more fish than the national average and that MeHg exposure within this group is among the highest in the US [27,28]. The goals of this work were two-fold: to more accurately define the toxicokinetic variability of Hg levels observed between the hair and toenail compartment, and to examine variability in temporal toenail-Hg levels that were obtained over a period of approximately one year. This information will permit an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure and may be useful in future studies examining the relationship between neurological effects and MeHg exposure. It will also facilitate the comparison between the results of studies employing toenail- and hair-Hg levels as biomarkers of exposure.
Conclusions:
The data presented in this study will allow for an improved estimation of MeHg exposure based on the toenail compartment which is used as an exposure metric in studies examining the relationship between cardiovascular effects and MeHg exposure. A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that has a MeHg body-burden level that is relatively stable over time. Intra-individual variation was less than two-fold, which may represent normal body burden fluctuation for individuals consuming elevated amounts of various fish species with different contaminant levels. The use of biomarker-based estimates reflecting body-burden levels that are chronologically distant from manifestation of deleterious outcome, or that are used as the sole indicator reflecting chronic exposure, need to be considered carefully when testing a hypothesis as individual body-burden levels may not remain constant across lengthy time periods such as a decade.
|
Background:
Toenail-Hg levels are being used as a marker of methylmercury (MeHg) exposure in efforts to associate exposure with effects such as cardiovascular disease. There is a need to correlate this marker with more established biomarkers that presently underlie existing dose-response relationships in order to compare these relationships across studies.
Methods:
As part of the Arsenic Mercury Intake Biometric Study, toenail clippings were collected at three time points over a period of one year amongst females from within the population of Japanese living near Puget Sound in Washington State (US). Variability in temporal intra-individual toenail-Hg levels was examined and chronologically matched hair and toenail samples were compared to more accurately define the toxicokinetic variability of Hg levels observed between the two compartments.
Results:
Mean toenail-Hg values (n=43) for the 1st, 2nd and 3rd visits were 0.60, 0.60 and 0.56 ng/mg. Correlations were as follows: r=0.92 between 1st and 2nd clinic visits, r=0.75 between 1st and 3rd visits and r=0.87 between 2nd and 3rd visits. With few exceptions, toenail-Hg values from any visit were within 50-150% of the individual's mean toenail-Hg level. Nearly all participants had less than a two-fold change in toenail-Hg levels across the study period. A regression model of the relationship between toenail-Hg and hair-Hg (n = 41) levels representing the same time period of exposure, gave a slope (Hg ng/mg) of 2.79 for hair relative to toenail (r=0.954).
Conclusions:
A chronologically matched hair-Hg to toenail-Hg ratio has been identified within a population that consumes fish regularly and in quantity. Intra-individual variation in toenail-Hg levels was less than two-fold and may represent dietary-based fluctuations in body burden for individuals consuming various fish species with different contaminant levels. The chronologically matched ratio will be useful for relating MeHg exposure and dose-response derived from toenail-Hg measurements to those derived from hair-Hg measurements in other studies, and may be useful in future investigations as an indicator of stable MeHg body burden within a population.
| 9,675 | 414 | 14 |
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[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]
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[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]
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[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]
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[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]
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[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]
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[CONTENT] Mercury | Methylmercury | Cardiovascular | Hair | Toenail | Chronological | Biomarker [SUMMARY]
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[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]
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[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]
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[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]
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[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]
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[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]
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[CONTENT] Adolescent | Adult | Asian People | Environmental Monitoring | Environmental Pollutants | Female | Hair | Humans | Mercury | Middle Aged | Nails | Time Factors | Young Adult [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]
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[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]
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[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]
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[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]
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[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]
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[CONTENT] hair | toenail | hg | exposure | time | levels | growth | toenail hg | nail | sample [SUMMARY]
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[CONTENT] mehg | exposure | mehg exposure | hg | hg levels | studies | levels | toenail | fish | hair [SUMMARY]
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[CONTENT] hair | toenail | growth | nail | end | clipping | segment | distal | hair segment | time [SUMMARY]
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[CONTENT] hg | toenail | toenail hg | values | visits | days | mean | hair | samples | hg levels [SUMMARY]
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[CONTENT] body | body burden | burden | exposure | mehg | body burden levels | burden levels | levels | mehg exposure | time [SUMMARY]
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[CONTENT] toenail | hair | hg | levels | exposure | hg levels | mehg | toenail hg | time | growth [SUMMARY]
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[CONTENT] toenail | hair | hg | levels | exposure | hg levels | mehg | toenail hg | time | growth [SUMMARY]
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[CONTENT] Toenail-Hg | MeHg ||| [SUMMARY]
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[CONTENT] the Arsenic Mercury Intake Biometric Study | three | one year | Japanese | Washington State | US ||| two [SUMMARY]
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[CONTENT] the 1st, 2nd | 3rd | 0.60 | 0.60 | 0.56 ng ||| between 1st and 2nd | between 1st and | 3rd | r=0.87 | between 2nd and 3rd ||| 50-150% ||| two-fold ||| 41 | 2.79 [SUMMARY]
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[CONTENT] ||| Intra | less than two-fold ||| MeHg | MeHg [SUMMARY]
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[CONTENT] Toenail-Hg | MeHg ||| ||| the Arsenic Mercury Intake Biometric Study | three | one year | Japanese | Washington State | US ||| two ||| the 1st, 2nd | 3rd | 0.60 | 0.60 | 0.56 ng ||| between 1st and 2nd | between 1st and | 3rd | r=0.87 | between 2nd and 3rd ||| 50-150% ||| two-fold ||| 41 | 2.79 ||| ||| Intra | less than two-fold ||| MeHg | MeHg [SUMMARY]
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[CONTENT] Toenail-Hg | MeHg ||| ||| the Arsenic Mercury Intake Biometric Study | three | one year | Japanese | Washington State | US ||| two ||| the 1st, 2nd | 3rd | 0.60 | 0.60 | 0.56 ng ||| between 1st and 2nd | between 1st and | 3rd | r=0.87 | between 2nd and 3rd ||| 50-150% ||| two-fold ||| 41 | 2.79 ||| ||| Intra | less than two-fold ||| MeHg | MeHg [SUMMARY]
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Comparison of double antigen sandwich and indirect enzyme-linked immunosorbent assay for the diagnosis of hepatitis C virus antibodies.
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33245583
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The aim of this study is to compare double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA in the diagnosis of hepatitis C virus（HCV）infection.
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BACKGROUND
|
A total of 176 samples from the Tumor Hospital Affiliated to Xin Jiang Medical University were utilized to comparison. All serum samples were tested using double-antigen sandwich ELISA and indirect ELISA. Cohen's kappa statistics were used to assess the agreement between the two assays, and multivariate analysis was used to evaluate risk factors for the discordance between the double-antigen ELISA and indirect ELISA.
|
METHODS AND MATERIALS
|
The positivities of indirect ELISA (Beijing Wantai), double-antigen sandwich ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 74.43%, 68.75%, and 73.30%, respectively. The agreement between the indirect ELISA (Beijing Wantai) and double-antigen sandwich ELISA (Beijing Wantai) was high (κ = 0.829;P < .001), and the agreement between the double-antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847;P < .001). Variables associated with discordant results between the double-antigen sandwich and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05).
|
RESULTS
|
In detection of HCV, high agreement was found between the double-antigen sandwich ELISA and indirect ELISA. Female, younger age, and suffer from malignant tumor were significant risk factors for the discordance. Based on double-antigen sandwich ELISA has distinct methodological advantages over indirect ELISA. It is recommended for the diagnosis of HCV infection.
|
CONCLUSION
|
[
"Adolescent",
"Adult",
"Aged",
"Aged, 80 and over",
"Antigens, Viral",
"Enzyme-Linked Immunosorbent Assay",
"Female",
"Hepacivirus",
"Hepatitis C",
"Hepatitis C Antibodies",
"Humans",
"Immunologic Tests",
"Male",
"Middle Aged",
"Reproducibility of Results",
"Young Adult"
] |
7676215
|
INTRODUCTION
|
The virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.
1
,
2
The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.
3
,
4
Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.
5
For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.
6
WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.
7
People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.
8
Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.
9
,
10
Depending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.
11
In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection.
| null | null |
RESULTS
|
Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).
HCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)
Indirect ELISA
(Wantai), number(%)
S/CO
Double‐antigen Sandwich ELISA (Wantai), number(%)
S/CO
Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).
HCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)
Indirect ELISA
(Wantai), number(%)
S/CO
Double‐antigen Sandwich ELISA (Wantai), number(%)
S/CO
Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).
HCV antibody detection by double‐antigen sandwich
Indirect ELISA
(Jinhao), number(%)
S/CO
Double‐Antigen Sandwich ELISA (Wantai), number(%)
S/CO
ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).
Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).
HCV antibody detection by double‐antigen sandwich
Indirect ELISA
(Jinhao), number(%)
S/CO
Double‐Antigen Sandwich ELISA (Wantai), number(%)
S/CO
ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).
Discordance among the test results Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).
Univariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody
Abbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.
Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).
Univariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody
Abbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.
|
CONCLUSION
|
In detection of HCV infection, high agreement was found between the double‐antigen sandwich ELISA and indirect ELISA. Based on double‐antigen sandwich ELISA has distinct methodological advantages over indirect ELISA, considering double‐antigen sandwich ELISA has high clinical popularization value in the screening of HCV infection. Female, younger age, and malignancy were significant risk factors for the discordance. It is recommended that relevant laboratories should pay more attention to the existence of the above significant risk factors by using indirect ELISA and suggest to detect anti‐HCV by using double‐antigen sandwich ELISA if condition permits.
|
[
"INTRODUCTION",
"Subjects",
"Commercial assays",
"Statistical analysis",
"Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)",
"Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao)",
"Discordance among the test results"
] |
[
"The virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.\n1\n, \n2\n The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.\n3\n, \n4\n Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.\n5\n For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.\n6\n WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.\n7\n People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.\n8\n Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.\n9\n, \n10\n\n\nDepending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.\n11\n In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection.",
"A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.",
"Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.",
"Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.",
"Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO",
"Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).",
"Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin."
] |
[
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"MATERIAL AND METHOD",
"Subjects",
"Commercial assays",
"Statistical analysis",
"RESULTS",
"Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)",
"Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao)",
"Discordance among the test results",
"DISCUSSION",
"CONCLUSION"
] |
[
"The virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.\n1\n, \n2\n The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.\n3\n, \n4\n Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.\n5\n For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.\n6\n WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.\n7\n People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.\n8\n Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.\n9\n, \n10\n\n\nDepending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.\n11\n In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection.",
" Subjects A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.\nA total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.\n Commercial assays Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.\nDouble‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.\n Statistical analysis Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.\nStatistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.",
"A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.",
"Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.\nIndirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.\nIn addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.",
"Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.",
" Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO\n Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\nAmong the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).\n Discordance among the test results Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.\nVariables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.",
"Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).\nHCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)\nIndirect ELISA\n(Wantai), number(%)\nS/CO\nDouble‐antigen Sandwich ELISA (Wantai), number(%)\nS/CO",
"Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).\nHCV antibody detection by double‐antigen sandwich\nIndirect ELISA\n(Jinhao), number(%)\nS/CO\nDouble‐Antigen Sandwich ELISA (Wantai), number(%)\nS/CO\nELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).",
"Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).\nUnivariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody\nAbbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.",
"There are no symptoms or only mild symptoms such as right upper abdominal discomfort and oil aversion in the incubation period after HCV infection, and it is likely to take 20‐40 years to developed into serious clinical complication such as liver fibrosis or even hepatocellular carcinoma.\n12\n, \n13\n The exact time of HCV infection is difficult to determine the symptoms are mild or absent, easily transform into chronic, and higher chronic rate was 55% to 85%. Additionally, the understanding degree of the disease at a lower level, autonomous screening rate is low and public lacks attention with this disease. It caused tremendous burden on both China and the world economy. Along with a deeper understanding of the HCV life cycle, direct antiviral drugs (DAAs) have become widely used since its introduction, which is highly effective, less adverse reactions and a short course of treatment available to fight HCV.\n14\n, \n15\n DAAs are working directly on the non‐structural protein (NS) 3/4A, 5A, and 5B to achieve virus clearance, so the deadlock of clinical treatment of viral hepatitis C was broken even promising to get complete cure; thus, WHO sets targets to eliminate hepatitis C as a public health threat by 2030.\n16\n, \n17\n However, related research suggests that DAAs are still at a high price, at the same time, reinfection cannot be avoided, and the cured patients still have the risk of hepatocellular carcinoma.\n18\n, \n19\n, \n20\n Vaccine is the most cost‐effective and effective measure to prevent and combat the spread of disease. However, there is not any effective vaccine to prevent HCV infection.\n21\n The successful development and clinical application of HCV vaccine are unpredictable.\n22\n In summary, the focal problems for hepatitis C are shifting from the therapeutic strategies to screening program launched in high‐risk groups.\nTo date, early screening and diagnosis of HCV infection are based on three different methods, which may be utilized alone or in combination. These are (a) detection of HCV core antigen (HCV‐Ag); (b) nucleic acid testing (NAT) to detect HCV‐RNA by PCR; and (c) detection of an IgG antibody by ELISA (anti‐HCV).\n23\n Related literature reports that there is a good correlation between the contents of HCV‐Ag and the quantity of HCV‐RNA as viral load exceeds 3000 IU/mL in the serum samples of HCV patients, so HCV core antigen can be used as early diagnostic indicators of HCV infection.\n24\n Meanwhile, detection of HCV‐Ag has the advantages of relatively low cost, handiness, and simplicity and not need to add special instruments. However, the structure of the HCV‐cAg is very special and the preparation of antibody is more difficult, and detection sensitivity of HCV‐cAg is lower than that of anti‐HCV by using ELISA. It can be used as a supplement to anti‐HCV detection but cannot replace the determination of antibody. The samples were tested HCV‐RNA positive are the reliable index of viral replication activity, and the HCV‐RNA methodology can be subdivided into qualitative and quantitative methods.\n25\n, \n26\n Qualitative analysis has played a key role in blood screening of blood banks, it as a means of indicating the presence or absence of viremia but not measure the viral load. In recent years, the real‐time PCR (RT‐PCR) is gradually replacing qualitative analysis as a common method used in detecting HCV‐RNA, as it features high sensitivity and specificity. However, it cannot be used as a routine method because of its complicated operation and higher experimental conditions needed. In particular, most hospitals, especially primary healthcare facilities, do not have the capacity to detect HCV‐RNA and provide accurate results.\n27\n\n\nAccording to guidelines for the prevention and treatment of hepatitis C (2019 version) revised by the Chinese Medical Association, along with WS 213—2018 Diagnosis for hepatitis C (National Health and Family Planning Commission of the People's Republic of China). Compared with (HCV‐Ag) and nucleic acid testing (NAT), the anti‐HCV immunoassays are the most frequently used laboratory tests for detection of HCV infection. And now it has the following advantages in the detection of HCV antibodies with the rapid development of genetic engineering technology: high sensitivity and specificity, better stability, automation, low cost, and convenience. Indirect ELISA had already gone through three reformation, the first generation anti‐HCV assay using recombinant c100‐3 epitope from the NS4 protein, which had limited sensitivity and specificity. Second generation assay was developed using epitopes from the core, NS3, and NS4 proteins, a multi‐antigen format that can reduce the time required and increase the sensitivity. More recently, an highly conserved NS5 antigen have adopted in third generation assays. If the sample contains anti‐HCV, incubate for a certain period of time, solid‐phase antigen will fully bind to the anti‐HCV in the serum, and then react with the added IgG‐HRP. Tetramethylbenzidine (TMB) color development or not to indicate present or absent of anti‐HCV in human serum or plasma.\n28\n However, indirect ELISA can always result in false positives due to interfering factors, including high gamma globulin levels, nephritic syndrome, pregnancy, sample hemolysis, the splashing, and pollution of the blood sample.\n29\n\n\nDouble‐antigen sandwich ELISA is a developing technology with great potential.\n30\n At the beginning of development, it faces two major obstacles, the HCV antigen labeled with HRP directly is easy to cause the space steric hindrance and ratio of enzyme‐labeled antigens is difficult to control. Subsequently, preparation of growth arm biotinylated HCV antigen as a sandwich antigen using genetic engineering, with horseradish peroxidase mildew affinity tag chain element as enzymes, so anti‐HCV could perform twice specific binding in the sample and amplification of the signal was achieved using biotin–streptavidin system. Related research shows that compared to traditional indirect ELISA, double‐antigen sandwich ELISA displays higher specificity and better sensitivity, and it can utilize to detect IgM.\n31\n Double‐antigen sandwich ELISA could make up for the methodological deficiencies inherent in indirect ELISA and has clinical utility in both screening and diagnosis of HCV infection.\nObjective of our study is to investigate the consistency of three commercially available anti‐HCV assays, and analysis of influencing factors to disagreement between the double‐antigen sandwich and indirect ELISA. So as to provide pertinent evidence for the selection of methods of detecting anti‐HCV in clinical laboratories. In our study, among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai), indirect ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 68.75%, 74.43%, and 73.30%, respectively. The agreement between the double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) was high (κ = 0.829; P < .001). The agreement between the double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847; P < .001). But there are still inconsistent results in the analysis of anti‐HCV by using double‐antigen sandwich and indirect ELISA. It was found that among the fifty‐five specimens that were tested negative using by double‐antigen sandwich ELISA (Beijing Wantai), eleven were positive using by indirect ELISA (Beijing Wantai) and ten were positive using by indirect ELISA (Beijing Jinhao), and the possible cause of this problem perhaps is that nonspecific immunoglobulin, rheumatoid factor, hydrophilic antibodies, and other interfering substances in the specimen, which can be nonspecifically combined with the enzyme‐labeled secondary antibody, lead to false‐positive results. Among one hundred and twenty‐one samples tested positive using by double‐antigen sandwich ELISA (Beijing Wantai), one was negative using by indirect ELISA (Beijing Wantai) and two were negative by using indirect ELISA (Beijing Jinhao). The probable reason is that the patients are in the window period of infection and the body has not yet produced IgG. Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR: 1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05). Analysis of possible reasons were as follows: (a) More women predisposed to autoimmune diseases and autoantibody positivity was higher than men; (b) positive antinuclear antibodies in malignancies than benign tumor; and (c) pregnant woman in people younger than 35 years old was high and the presence of some interfering substances may be affect the results significantly. These discrepancies emphasize the need for further research, and we will follow up in subsequent studies.",
"In detection of HCV infection, high agreement was found between the double‐antigen sandwich ELISA and indirect ELISA. Based on double‐antigen sandwich ELISA has distinct methodological advantages over indirect ELISA, considering double‐antigen sandwich ELISA has high clinical popularization value in the screening of HCV infection. Female, younger age, and malignancy were significant risk factors for the discordance. It is recommended that relevant laboratories should pay more attention to the existence of the above significant risk factors by using indirect ELISA and suggest to detect anti‐HCV by using double‐antigen sandwich ELISA if condition permits."
] |
[
null,
"materials-and-methods",
null,
null,
null,
"results",
null,
null,
null,
"discussion",
"conclusions"
] |
[
"double‐antigen sandwich ELISA",
"hepatitis C antibodies",
"indirect ELISA"
] |
INTRODUCTION:
The virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.
1
,
2
The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.
3
,
4
Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.
5
For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.
6
WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.
7
People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.
8
Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.
9
,
10
Depending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.
11
In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection.
MATERIAL AND METHOD:
Subjects A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.
A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.
Commercial assays Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.
Indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.
In addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.
Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.
Indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.
In addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.
Statistical analysis Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.
Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.
Subjects:
A total of 176 samples from outpatient and inpatient were used for comparison, 80 males and 96 females (mean age of 55.41 years, SD = 13.52 years, and range = 18‐86 years old). We are collecting specimens from the Tumor Hospital Affiliated to Xin Jiang Medical University from January 2018 to October 2018. Venous blood samples were collected from studying subjects for serum separation. The same protocol of sample collection was used in both outpatients and inpatients. Study subjects’ whole blood, obtained by venipuncture, was collected in 4 mL BD Vacutainer Plus Plastic Serum Tubes. Samples were allowed to clot for 30 min at room temperature, followed by centrifugation at 1728g for 10 minutes, and then aliquots of 1 mL serum were transferred to 1.5mL sterile microcentrifuge tubes using a disposable transfer pipet. The serum samples of the patients were withdrawn from the storage refrigerator and mixed thoroughly. All serum samples were tested using double‐antigen sandwich ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd), indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) and indirect ELISA (Beijing Jinhao bio Pharmaceutical Co., Ltd), respectively. Quality control materials, and positive and negative controlled substance were used in parallel. All assays were performed according to the manufacturers’ instructions.
Commercial assays:
Double‐antigen sandwich ELISA kit (Beijing Wantai bio Pharmaceutical Co., Ltd) is a qualitative test intended for the detection of HCV infection in human serum or plasma samples. This kit using horseradish peroxidase (HRP) tagged biotinylated recombinant antigen and solid‐phase multi‐epitope gene recombination antigens (HCV core, NS3, NS4, NS5, et al) to specifically bind to anti‐HCV with twice. Meanwhile, this commercial assay kit used the biotin‐streptavidin binding system to amplify the detection signal and it can be used for detecting both IgM and IgG antibodies. The cutoff value was calculated according to kit instructions (Cutoff value = Positive control mean + 0.12). The anti‐HCV results of sample are negative when absorbance values are less than the cutoff value, and there is positive result when absorbance value is greater than or equal to cutoff value.
Indirect ELISA (Beijing Wantai bio Pharmaceutical Co., Ltd) also is qualitative test. Indirect ELISA kit was the most widely used method for early screening and auxiliary tool in the diagnosis of HCV infection. Both kits used HRP‐labeled nonspecific anti‐human antibody as secondary antibody, meanwhile, recombinant antigens binding to solid support. There are a lot of factors that can influence the absorbance measurement. To see whether the sample was positive or not, we need to calculate cutoff value according to the reagent kit instructions (Cutoff value = Negative control mean + 0.12, Beijing Wantai; Cutoff value = 0.13 × Positive control mean + Negative control mean, Beijing Jinhao). Samples giving an absorbance less than the cutoff were scored as negative, while those giving an absorbance equal to or greater than the cutoff were scored as positive.
In addition, a few parameters like total bilirubin (TBil), direct bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma‐glutamyl transpeptidase (GGT), and a‐L‐Fucosidase (AFU) related to serological indicators of liver function were recorded. And above‐mentioned biochemical indexes were tested by Roche modular fully automatic biochemical analyzer.
Statistical analysis:
Statistical analysis was performed using the SPSS statistical software package (SPSS version 18). Qualitative variables are presented as frequency (percentage), and quantitative statistics are presented as mean ± standard deviation (SD). We used the Cohen kappa statistics to evaluate concordance of test results by double‐antigen sandwich and indirect ELISA, and multiple factor analysis was assessed by multivariate logistic regression analysis to calculate the odds ratio (OR), and a P value of less than 0.05 was taken to indicate a statistically significant difference.
RESULTS:
Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).
HCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)
Indirect ELISA
(Wantai), number(%)
S/CO
Double‐antigen Sandwich ELISA (Wantai), number(%)
S/CO
Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).
HCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)
Indirect ELISA
(Wantai), number(%)
S/CO
Double‐antigen Sandwich ELISA (Wantai), number(%)
S/CO
Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).
HCV antibody detection by double‐antigen sandwich
Indirect ELISA
(Jinhao), number(%)
S/CO
Double‐Antigen Sandwich ELISA (Wantai), number(%)
S/CO
ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).
Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).
HCV antibody detection by double‐antigen sandwich
Indirect ELISA
(Jinhao), number(%)
S/CO
Double‐Antigen Sandwich ELISA (Wantai), number(%)
S/CO
ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).
Discordance among the test results Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).
Univariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody
Abbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.
Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).
Univariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody
Abbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.
Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai):
Among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) were 68.75% and 74.43%, respectively. The agreement between the two approaches was high (κ = 0.829;P < .001) (Table 1).
HCV antibody detection by double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai)
Indirect ELISA
(Wantai), number(%)
S/CO
Double‐antigen Sandwich ELISA (Wantai), number(%)
S/CO
Comparison of consistency between double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao):
Among the 176 subjects, the positivities of double‐antigen sandwich ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao) were 68.75% and 73.30%, respectively. The agreement between the two approaches was high (κ = 0.847; P < .001) (Table 2).
HCV antibody detection by double‐antigen sandwich
Indirect ELISA
(Jinhao), number(%)
S/CO
Double‐Antigen Sandwich ELISA (Wantai), number(%)
S/CO
ELISA(Beijing Wantai) and indirect ELISA (Beijing Jinhao).
Discordance among the test results:
Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05) (Table 3).
Univariate analysis of risk factors associated with discordance between the double‐antigen sandwich and indirect ELIS in detection of HCV antibody
Abbreviations: AFU, a‐L‐Fucosidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBil, direct bilirubin; r‐ GGT, gamma‐glutamyl transpeptidase; TBil, total bilirubin.
DISCUSSION:
There are no symptoms or only mild symptoms such as right upper abdominal discomfort and oil aversion in the incubation period after HCV infection, and it is likely to take 20‐40 years to developed into serious clinical complication such as liver fibrosis or even hepatocellular carcinoma.
12
,
13
The exact time of HCV infection is difficult to determine the symptoms are mild or absent, easily transform into chronic, and higher chronic rate was 55% to 85%. Additionally, the understanding degree of the disease at a lower level, autonomous screening rate is low and public lacks attention with this disease. It caused tremendous burden on both China and the world economy. Along with a deeper understanding of the HCV life cycle, direct antiviral drugs (DAAs) have become widely used since its introduction, which is highly effective, less adverse reactions and a short course of treatment available to fight HCV.
14
,
15
DAAs are working directly on the non‐structural protein (NS) 3/4A, 5A, and 5B to achieve virus clearance, so the deadlock of clinical treatment of viral hepatitis C was broken even promising to get complete cure; thus, WHO sets targets to eliminate hepatitis C as a public health threat by 2030.
16
,
17
However, related research suggests that DAAs are still at a high price, at the same time, reinfection cannot be avoided, and the cured patients still have the risk of hepatocellular carcinoma.
18
,
19
,
20
Vaccine is the most cost‐effective and effective measure to prevent and combat the spread of disease. However, there is not any effective vaccine to prevent HCV infection.
21
The successful development and clinical application of HCV vaccine are unpredictable.
22
In summary, the focal problems for hepatitis C are shifting from the therapeutic strategies to screening program launched in high‐risk groups.
To date, early screening and diagnosis of HCV infection are based on three different methods, which may be utilized alone or in combination. These are (a) detection of HCV core antigen (HCV‐Ag); (b) nucleic acid testing (NAT) to detect HCV‐RNA by PCR; and (c) detection of an IgG antibody by ELISA (anti‐HCV).
23
Related literature reports that there is a good correlation between the contents of HCV‐Ag and the quantity of HCV‐RNA as viral load exceeds 3000 IU/mL in the serum samples of HCV patients, so HCV core antigen can be used as early diagnostic indicators of HCV infection.
24
Meanwhile, detection of HCV‐Ag has the advantages of relatively low cost, handiness, and simplicity and not need to add special instruments. However, the structure of the HCV‐cAg is very special and the preparation of antibody is more difficult, and detection sensitivity of HCV‐cAg is lower than that of anti‐HCV by using ELISA. It can be used as a supplement to anti‐HCV detection but cannot replace the determination of antibody. The samples were tested HCV‐RNA positive are the reliable index of viral replication activity, and the HCV‐RNA methodology can be subdivided into qualitative and quantitative methods.
25
,
26
Qualitative analysis has played a key role in blood screening of blood banks, it as a means of indicating the presence or absence of viremia but not measure the viral load. In recent years, the real‐time PCR (RT‐PCR) is gradually replacing qualitative analysis as a common method used in detecting HCV‐RNA, as it features high sensitivity and specificity. However, it cannot be used as a routine method because of its complicated operation and higher experimental conditions needed. In particular, most hospitals, especially primary healthcare facilities, do not have the capacity to detect HCV‐RNA and provide accurate results.
27
According to guidelines for the prevention and treatment of hepatitis C (2019 version) revised by the Chinese Medical Association, along with WS 213—2018 Diagnosis for hepatitis C (National Health and Family Planning Commission of the People's Republic of China). Compared with (HCV‐Ag) and nucleic acid testing (NAT), the anti‐HCV immunoassays are the most frequently used laboratory tests for detection of HCV infection. And now it has the following advantages in the detection of HCV antibodies with the rapid development of genetic engineering technology: high sensitivity and specificity, better stability, automation, low cost, and convenience. Indirect ELISA had already gone through three reformation, the first generation anti‐HCV assay using recombinant c100‐3 epitope from the NS4 protein, which had limited sensitivity and specificity. Second generation assay was developed using epitopes from the core, NS3, and NS4 proteins, a multi‐antigen format that can reduce the time required and increase the sensitivity. More recently, an highly conserved NS5 antigen have adopted in third generation assays. If the sample contains anti‐HCV, incubate for a certain period of time, solid‐phase antigen will fully bind to the anti‐HCV in the serum, and then react with the added IgG‐HRP. Tetramethylbenzidine (TMB) color development or not to indicate present or absent of anti‐HCV in human serum or plasma.
28
However, indirect ELISA can always result in false positives due to interfering factors, including high gamma globulin levels, nephritic syndrome, pregnancy, sample hemolysis, the splashing, and pollution of the blood sample.
29
Double‐antigen sandwich ELISA is a developing technology with great potential.
30
At the beginning of development, it faces two major obstacles, the HCV antigen labeled with HRP directly is easy to cause the space steric hindrance and ratio of enzyme‐labeled antigens is difficult to control. Subsequently, preparation of growth arm biotinylated HCV antigen as a sandwich antigen using genetic engineering, with horseradish peroxidase mildew affinity tag chain element as enzymes, so anti‐HCV could perform twice specific binding in the sample and amplification of the signal was achieved using biotin–streptavidin system. Related research shows that compared to traditional indirect ELISA, double‐antigen sandwich ELISA displays higher specificity and better sensitivity, and it can utilize to detect IgM.
31
Double‐antigen sandwich ELISA could make up for the methodological deficiencies inherent in indirect ELISA and has clinical utility in both screening and diagnosis of HCV infection.
Objective of our study is to investigate the consistency of three commercially available anti‐HCV assays, and analysis of influencing factors to disagreement between the double‐antigen sandwich and indirect ELISA. So as to provide pertinent evidence for the selection of methods of detecting anti‐HCV in clinical laboratories. In our study, among the 176 subjects, the positivities of double‐antigen sandwich ELISA (Beijing Wantai), indirect ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 68.75%, 74.43%, and 73.30%, respectively. The agreement between the double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Wantai) was high (κ = 0.829; P < .001). The agreement between the double‐antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847; P < .001). But there are still inconsistent results in the analysis of anti‐HCV by using double‐antigen sandwich and indirect ELISA. It was found that among the fifty‐five specimens that were tested negative using by double‐antigen sandwich ELISA (Beijing Wantai), eleven were positive using by indirect ELISA (Beijing Wantai) and ten were positive using by indirect ELISA (Beijing Jinhao), and the possible cause of this problem perhaps is that nonspecific immunoglobulin, rheumatoid factor, hydrophilic antibodies, and other interfering substances in the specimen, which can be nonspecifically combined with the enzyme‐labeled secondary antibody, lead to false‐positive results. Among one hundred and twenty‐one samples tested positive using by double‐antigen sandwich ELISA (Beijing Wantai), one was negative using by indirect ELISA (Beijing Wantai) and two were negative by using indirect ELISA (Beijing Jinhao). The probable reason is that the patients are in the window period of infection and the body has not yet produced IgG. Variables associated with discordant results between the double‐antigen sandwich ELISA and indirect ELISA in multivariate analysis were as follows: female (OR: 1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05). Analysis of possible reasons were as follows: (a) More women predisposed to autoimmune diseases and autoantibody positivity was higher than men; (b) positive antinuclear antibodies in malignancies than benign tumor; and (c) pregnant woman in people younger than 35 years old was high and the presence of some interfering substances may be affect the results significantly. These discrepancies emphasize the need for further research, and we will follow up in subsequent studies.
CONCLUSION:
In detection of HCV infection, high agreement was found between the double‐antigen sandwich ELISA and indirect ELISA. Based on double‐antigen sandwich ELISA has distinct methodological advantages over indirect ELISA, considering double‐antigen sandwich ELISA has high clinical popularization value in the screening of HCV infection. Female, younger age, and malignancy were significant risk factors for the discordance. It is recommended that relevant laboratories should pay more attention to the existence of the above significant risk factors by using indirect ELISA and suggest to detect anti‐HCV by using double‐antigen sandwich ELISA if condition permits.
|
Background:
The aim of this study is to compare double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA in the diagnosis of hepatitis C virus（HCV）infection.
Methods:
A total of 176 samples from the Tumor Hospital Affiliated to Xin Jiang Medical University were utilized to comparison. All serum samples were tested using double-antigen sandwich ELISA and indirect ELISA. Cohen's kappa statistics were used to assess the agreement between the two assays, and multivariate analysis was used to evaluate risk factors for the discordance between the double-antigen ELISA and indirect ELISA.
Results:
The positivities of indirect ELISA (Beijing Wantai), double-antigen sandwich ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 74.43%, 68.75%, and 73.30%, respectively. The agreement between the indirect ELISA (Beijing Wantai) and double-antigen sandwich ELISA (Beijing Wantai) was high (κ = 0.829;P < .001), and the agreement between the double-antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847;P < .001). Variables associated with discordant results between the double-antigen sandwich and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05).
Conclusions:
In detection of HCV, high agreement was found between the double-antigen sandwich ELISA and indirect ELISA. Female, younger age, and suffer from malignant tumor were significant risk factors for the discordance. Based on double-antigen sandwich ELISA has distinct methodological advantages over indirect ELISA. It is recommended for the diagnosis of HCV infection.
|
INTRODUCTION:
The virus which is the most important etiologic agent of non‐A, non‐B hepatitis was officially named HCV, belongs to the family flaviviridae hepatitis virus genus and is a small, enveloped, positive‐sense single‐stranded RNA virus with a genome length of 9.4‐9.6 kb.
1
,
2
The genome sequence is substantial genetic diversity and can be divided into at least 7 genotypes and 67 subtypes, and there are general susceptibilities regardless of age, gender, and race.
3
,
4
Related studies have shown that some HCV genotypes have their specific distribution areas, and there were considerable differences in the natural course of disease after people infected with different genotype or subtypes.
5
For example, genotype la plays a vital role in North America, genotype lb is more common in Western Europe and Japan which is closely to invasive liver disease, and genotypes 1b and 2a predominate in China.
6
WHO estimates that HCV infects nearly 3% of the world population as a highly pathogenic virus.
7
People who infected with this virus tend to be mild in symptoms and more progressed to chronic hepatitis, and this character results approximately 400 000 people died in 2016.
8
Vaccination is the most effective method and cost‐effective manner in order to control the spread of contagious diseases, but there are still some problems inhibit development of an effective vaccine against HCV at this stage. The application of direct antiviral drugs (DAAs) has brought new hope to triumph over the HCV. The treatment of chronic HCV infection has entered the era of pan‐genotype with the intensive research and development of DAAs, but the high cost of DAAs threatens the affordability of patients with HCV infection.
9
,
10
Depending on the above, the principal contradiction of HCV by the treatment into screening. The diagnosis and monitoring of HCV infection are based on 2 types of methods: One is detecting HCV antigen‐specific antibodies and the other is detecting viral RNA or HCV core antigens. Making a clinical diagnosis was based on historical epidemiology, clinical manifestations, and HCV antibody screening results, and then making a definite diagnosis by viral nucleic acid testing. Thus, the screening of HCV antibody occupies an important position at this stage. Anti‐HCV was detected by indirect ELISA was always used as a screening tool.
11
In recent years, double‐antigen sandwich ELISA has received wide attention because of its key advantages in methodology. So the aim of this work was to compare three commercial ELISA kits in the diagnosis of HCV infection.
CONCLUSION:
In detection of HCV infection, high agreement was found between the double‐antigen sandwich ELISA and indirect ELISA. Based on double‐antigen sandwich ELISA has distinct methodological advantages over indirect ELISA, considering double‐antigen sandwich ELISA has high clinical popularization value in the screening of HCV infection. Female, younger age, and malignancy were significant risk factors for the discordance. It is recommended that relevant laboratories should pay more attention to the existence of the above significant risk factors by using indirect ELISA and suggest to detect anti‐HCV by using double‐antigen sandwich ELISA if condition permits.
|
Background:
The aim of this study is to compare double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA in the diagnosis of hepatitis C virus（HCV）infection.
Methods:
A total of 176 samples from the Tumor Hospital Affiliated to Xin Jiang Medical University were utilized to comparison. All serum samples were tested using double-antigen sandwich ELISA and indirect ELISA. Cohen's kappa statistics were used to assess the agreement between the two assays, and multivariate analysis was used to evaluate risk factors for the discordance between the double-antigen ELISA and indirect ELISA.
Results:
The positivities of indirect ELISA (Beijing Wantai), double-antigen sandwich ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 74.43%, 68.75%, and 73.30%, respectively. The agreement between the indirect ELISA (Beijing Wantai) and double-antigen sandwich ELISA (Beijing Wantai) was high (κ = 0.829;P < .001), and the agreement between the double-antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847;P < .001). Variables associated with discordant results between the double-antigen sandwich and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05).
Conclusions:
In detection of HCV, high agreement was found between the double-antigen sandwich ELISA and indirect ELISA. Female, younger age, and suffer from malignant tumor were significant risk factors for the discordance. Based on double-antigen sandwich ELISA has distinct methodological advantages over indirect ELISA. It is recommended for the diagnosis of HCV infection.
| 5,573 | 356 | 11 |
[
"elisa",
"hcv",
"beijing",
"antigen",
"indirect",
"indirect elisa",
"wantai",
"elisa beijing",
"sandwich",
"antigen sandwich"
] |
[
"test",
"test"
] | null |
[CONTENT] double‐antigen sandwich ELISA | hepatitis C antibodies | indirect ELISA [SUMMARY]
| null |
[CONTENT] double‐antigen sandwich ELISA | hepatitis C antibodies | indirect ELISA [SUMMARY]
|
[CONTENT] double‐antigen sandwich ELISA | hepatitis C antibodies | indirect ELISA [SUMMARY]
|
[CONTENT] double‐antigen sandwich ELISA | hepatitis C antibodies | indirect ELISA [SUMMARY]
|
[CONTENT] double‐antigen sandwich ELISA | hepatitis C antibodies | indirect ELISA [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Antigens, Viral | Enzyme-Linked Immunosorbent Assay | Female | Hepacivirus | Hepatitis C | Hepatitis C Antibodies | Humans | Immunologic Tests | Male | Middle Aged | Reproducibility of Results | Young Adult [SUMMARY]
| null |
[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Antigens, Viral | Enzyme-Linked Immunosorbent Assay | Female | Hepacivirus | Hepatitis C | Hepatitis C Antibodies | Humans | Immunologic Tests | Male | Middle Aged | Reproducibility of Results | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Antigens, Viral | Enzyme-Linked Immunosorbent Assay | Female | Hepacivirus | Hepatitis C | Hepatitis C Antibodies | Humans | Immunologic Tests | Male | Middle Aged | Reproducibility of Results | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Antigens, Viral | Enzyme-Linked Immunosorbent Assay | Female | Hepacivirus | Hepatitis C | Hepatitis C Antibodies | Humans | Immunologic Tests | Male | Middle Aged | Reproducibility of Results | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Antigens, Viral | Enzyme-Linked Immunosorbent Assay | Female | Hepacivirus | Hepatitis C | Hepatitis C Antibodies | Humans | Immunologic Tests | Male | Middle Aged | Reproducibility of Results | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] elisa | hcv | beijing | antigen | indirect | indirect elisa | wantai | elisa beijing | sandwich | antigen sandwich [SUMMARY]
| null |
[CONTENT] elisa | hcv | beijing | antigen | indirect | indirect elisa | wantai | elisa beijing | sandwich | antigen sandwich [SUMMARY]
|
[CONTENT] elisa | hcv | beijing | antigen | indirect | indirect elisa | wantai | elisa beijing | sandwich | antigen sandwich [SUMMARY]
|
[CONTENT] elisa | hcv | beijing | antigen | indirect | indirect elisa | wantai | elisa beijing | sandwich | antigen sandwich [SUMMARY]
|
[CONTENT] elisa | hcv | beijing | antigen | indirect | indirect elisa | wantai | elisa beijing | sandwich | antigen sandwich [SUMMARY]
|
[CONTENT] hcv | virus | genotype | genotypes | diagnosis | daas | people | effective | hepatitis | screening [SUMMARY]
| null |
[CONTENT] elisa | wantai | elisa beijing | beijing | beijing wantai indirect elisa | elisa beijing wantai indirect | wantai indirect | wantai indirect elisa | beijing wantai indirect | elisa beijing wantai [SUMMARY]
|
[CONTENT] elisa | significant risk | significant risk factors | double antigen sandwich elisa | sandwich elisa | antigen sandwich elisa | risk factors | significant | double | sandwich [SUMMARY]
|
[CONTENT] elisa | beijing | hcv | wantai | elisa beijing | antigen | indirect | beijing wantai | antigen sandwich | sandwich [SUMMARY]
|
[CONTENT] elisa | beijing | hcv | wantai | elisa beijing | antigen | indirect | beijing wantai | antigen sandwich | sandwich [SUMMARY]
|
[CONTENT] ELISA | ELISA [SUMMARY]
| null |
[CONTENT] ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao | 74.43% | 68.75% | 73.30% ||| ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao ||| ELISA | OR:1.462 | 35 years old | OR:3.621 [SUMMARY]
|
[CONTENT] ELISA | ELISA ||| ||| ELISA | ELISA ||| [SUMMARY]
|
[CONTENT] ELISA | ELISA ||| 176 | the Tumor Hospital Affiliated | Xin Jiang Medical University ||| ELISA | ELISA ||| Cohen | the two assays | ELISA | ELISA ||| ||| ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao | 74.43% | 68.75% | 73.30% ||| ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao ||| ELISA | OR:1.462 | 35 years old | OR:3.621 ||| ELISA | ELISA ||| ||| ELISA | ELISA ||| [SUMMARY]
|
[CONTENT] ELISA | ELISA ||| 176 | the Tumor Hospital Affiliated | Xin Jiang Medical University ||| ELISA | ELISA ||| Cohen | the two assays | ELISA | ELISA ||| ||| ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao | 74.43% | 68.75% | 73.30% ||| ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Wantai | ELISA | Beijing Jinhao ||| ELISA | OR:1.462 | 35 years old | OR:3.621 ||| ELISA | ELISA ||| ||| ELISA | ELISA ||| [SUMMARY]
|
|||||
Superior Effectiveness of Zidovudine Compared With Tenofovir When Combined With Nevirapine-based Antiretroviral Therapy in a Large Nigerian Cohort.
|
26561532
|
Despite sparse efficacy data, tenofovir-emtricitabine or tenofovir-lamivudine plus nevirapine is used in many resource-constrained settings.
|
BACKGROUND
|
This retrospective cohort study included patients initiating nevirapine-based antiretroviral therapy (ART) with either tenofovir-emtricitabine or lamivudine (tenofovir group) or zidovudine-lamivudine (zidovudine group). Clinical, virologic, and immunologic evaluations were performed at baseline and every 6 months. Virologic failure was defined as 2 consecutive human immunodeficiency virus (HIV)-RNA values >1000 copies/mL. Patients were included from ART initiation until time of failure, regimen switch, discontinuation, or last HIV-RNA measurement. Cox proportional hazards regression was used to model factors influencing time to failure. Bias due to dependent censoring was investigated via inverse probability weighted pooled logistic regression.
|
METHODS
|
A total of 5547 patients were evaluated; 1484 (26.8%) were in the tenofovir group and 4063 (73.2%) were in the zidovudine group. In the adjusted model, tenofovir regimen (hazard ratio [HR], 1.47; 95% confidence interval [CI], 1.21-1.79) and higher baseline log10 HIV-RNA (HR, 1.15; 95% CI, 1.03-1.28) were associated with virologic failure. Higher baseline log10 CD4+ cell count (HR, 0.50; 95% CI, .40-.63) and increasing age (HR, 0.98; 95% CI, .97-.99) decreased the risk of virologic failure. Inverse probability weighting results were consistent with the primary analysis.
|
RESULTS
|
Compared with zidovudine-lamivudine, the use of tenofovir-lamivudine or emtricitabine in combination with nevirapine was a strong predictor of virologic failure in our cohort, which was not explained by other risk factors or criteria for regimen selection.
|
CONCLUSIONS
|
[
"Adult",
"Anti-Retroviral Agents",
"Antiretroviral Therapy, Highly Active",
"CD4 Lymphocyte Count",
"Cohort Studies",
"Female",
"HIV Infections",
"Humans",
"Male",
"Nevirapine",
"Retrospective Studies",
"Tenofovir",
"Treatment Outcome",
"Viral Load",
"Young Adult",
"Zidovudine"
] |
4725384
| null | null |
METHODS
|
In this retrospective cohort study, prospectively collected data from a large clinical cohort in Nigeria were used. Between June 2004 and February 2012, the Harvard T. H. Chan School of Public Health (Harvard) collaborated with the AIDS Prevention Initiative in Nigeria Ltd./Gte. (APIN) to support HIV prevention, care, and treatment to over 160 000 patients, funded by the President's Emergency Plan for AIDS Relief (PEPFAR). Upon program entry, patients underwent a clinical examination and baseline laboratory testing including hematology and chemistry, CD4+ cell count (Cyflow, Partec), HIV-RNA (Cobas Amplicor Monitor Assay, Roche Diagnostics), hepatitis B virus (HBV) surface antigen (Monolisa HBsAg Ultra3, BioRad), and hepatitis C virus (HCV) antibody (DIA.PRO Diagnostic, Bioprobes srl) assessments. Patients eligible for ART, based on guidelines at the time of program entry, initiated standard first-line ART with 1 NNRTI and 2 NRTIs [11, 12]. Prescription refills were obtained monthly; clinical visits and laboratory tests were performed every 6 months, or earlier if necessary. All patient data were maintained in previously described electronic databases [13].
Ethical Considerations All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.
All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.
Patient Selection Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.
Patients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.
Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.
Patients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.
Study Definitions Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.
Study events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.
Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.
Study events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.
Statistical Analyses Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.
Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.
A Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.
Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.
A Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.
Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].
The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].
Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.
Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.
A Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.
Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.
A Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.
Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].
The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].
|
RESULTS
|
During the study period, 6575 patients were identified for inclusion and 5547 (84%) had data available for all variables in the final model—4063 (73.2%) in the zidovudine group and 1484 (26.8%) in the tenofovir group (Figure 1). The most common reason for exclusion was missing HBV or HCV status (11.0%). Baseline participant characteristics are summarized in Table 1. Consistent with clinical considerations for selecting an NRTI, HBV coinfection was more common among those in the tenofovir vs zidovudine group (27.2% vs 14.9%; P < .001), as was a lower median baseline hemoglobin (100 vs 110 g/L, P < .001). Proportionally, more women, patients with HCV coinfection, and those with more advanced HIV disease received tenofovir. Median (interquartile range [IQR]) duration of time to event or censor was slightly longer for the zidovudine group compared with the tenofovir group (392 [244] vs 376 [170] days, P < .001).
Table 1.Baseline Characteristics and Outcomes of Study ParticipantsTenofovir Group n = 1484Zidovudine Group n = 4063P ValueBaseline Characteristics Age (y)33 (11)32 (11).41 Female gender1255 (84.6)3085 (75.9)<.001 Hepatitis B virus coinfection404 (27.2)604 (14.9)<.001 Hepatitis C virus coinfection144 (9.7)294 (7.2).003 CD4 count (cells/mm3)122 (122)143 (115)<.001 Human immunodeficiency virus-RNA (log10 copies/mL)4.86 (1.1)4.70 (1.2)<.001 World Health Organization Stagen = 1335n = 3489<.001 1236 (17.7)923 (26.5) 2435 (32.6)1042 (29.9) 3494 (37.0)1045 (30.0) 4170 (12.7)479 (13.7) Tuberculosis coinfection294 (19.8)466 (11.5)<.001 Alanine transaminase (µkat/L)0.42 (0.37)0.40 (0.36).04 Hemoglobin (g/L)100 (24), n = 1462110 (20), n = 3948<.001 Creatinine (μmol/L)77 (31), n = 145877 (31), n = 3880.943Study Outcomes Virologic failure159 (10.7)298 (7.3)<.001 Antiretroviral therapy switch256 (17.3)622 (15.3).079 Discontinue308 (20.8)649 (16.0)<.001 No event761 (51.3)2494 (61.4)<.001Values shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.
Figure 1.Flow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.
Baseline Characteristics and Outcomes of Study Participants
Values shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.
Flow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.
Among patients in the tenofovir group, 1043 (70.2%) received emtricitabine as their second NRTI, 190 (12.8%) received lamivudine, and 252 (19.8%) switched between emtricitabine and lamivudine during the study period (median [IQR] time to switch was 172 [97] days). The number of virologic failure events did not differ significantly between patients receiving emtricitabine or lamivudine (P = .47); therefore, data were combined in the final analysis irrespective of emtricitabine or lamivudine use.
Overall, the median ART adherence rate was 95.3%, which was similar between the zidovudine and tenofovir groups (95.2% vs 95.8%). Among those who experienced virologic failure, more patients had adherence <95% compared with ≥95% (56.4% vs 43.6%; P < .001), but there was no difference in median adherence between those patients with failure in the zidovudine or tenofovir groups (91.9% vs 93.6%; P = .36).
Patients in the tenofovir group had a higher rate of virologic failure and discontinuation (Table 1). Figure 2 represents time to virologic failure between the groups. Of the 957 patients with a discontinuation event, 14 (1.5%) died, 849 (88.7%) were lost to follow-up, 92 (9.6%) transferred to another site, and 2 (0.2%) withdrew from the program. These distributions were similar between NRTI groups (P = .50).
Figure 2.Kaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.
Kaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.
Time to Virologic Failure After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).
Table 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.
Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy
All risk factors were measured at baseline.
Bold results indicate statistically significant results (P value <.05).
Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.
When the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).
After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).
Table 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.
Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy
All risk factors were measured at baseline.
Bold results indicate statistically significant results (P value <.05).
Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.
When the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).
Evaluation of Other Outcome Differences or Dependent Censoring Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.
Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.
| null | null |
[
"Ethical Considerations",
"Patient Selection",
"Study Definitions",
"Statistical Analyses",
"Time to Virologic Failure Regression Modeling",
"Evaluation of Dependent Censoring",
"Time to Virologic Failure",
"Evaluation of Other Outcome Differences or Dependent Censoring"
] |
[
"All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.",
"Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.",
"Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.",
"Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].",
"Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.",
"The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].",
"After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).",
"Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates."
] |
[
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"METHODS",
"Ethical Considerations",
"Patient Selection",
"Study Definitions",
"Statistical Analyses",
"Time to Virologic Failure Regression Modeling",
"Evaluation of Dependent Censoring",
"RESULTS",
"Time to Virologic Failure",
"Evaluation of Other Outcome Differences or Dependent Censoring",
"DISCUSSION",
"Supplementary Data"
] |
[
"In this retrospective cohort study, prospectively collected data from a large clinical cohort in Nigeria were used. Between June 2004 and February 2012, the Harvard T. H. Chan School of Public Health (Harvard) collaborated with the AIDS Prevention Initiative in Nigeria Ltd./Gte. (APIN) to support HIV prevention, care, and treatment to over 160 000 patients, funded by the President's Emergency Plan for AIDS Relief (PEPFAR). Upon program entry, patients underwent a clinical examination and baseline laboratory testing including hematology and chemistry, CD4+ cell count (Cyflow, Partec), HIV-RNA (Cobas Amplicor Monitor Assay, Roche Diagnostics), hepatitis B virus (HBV) surface antigen (Monolisa HBsAg Ultra3, BioRad), and hepatitis C virus (HCV) antibody (DIA.PRO Diagnostic, Bioprobes srl) assessments. Patients eligible for ART, based on guidelines at the time of program entry, initiated standard first-line ART with 1 NNRTI and 2 NRTIs [11, 12]. Prescription refills were obtained monthly; clinical visits and laboratory tests were performed every 6 months, or earlier if necessary. All patient data were maintained in previously described electronic databases [13].\n Ethical Considerations All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\nAll patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.\n Patient Selection Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\nAntiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.\n Study Definitions Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\nSex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.\n Statistical Analyses Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nSubject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].",
"All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.",
"Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.\nPatients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.",
"Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.\nStudy events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.",
"Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.\n Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\nBivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.\n Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].\nThe possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].",
"Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.\nA Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.",
"The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].",
"During the study period, 6575 patients were identified for inclusion and 5547 (84%) had data available for all variables in the final model—4063 (73.2%) in the zidovudine group and 1484 (26.8%) in the tenofovir group (Figure 1). The most common reason for exclusion was missing HBV or HCV status (11.0%). Baseline participant characteristics are summarized in Table 1. Consistent with clinical considerations for selecting an NRTI, HBV coinfection was more common among those in the tenofovir vs zidovudine group (27.2% vs 14.9%; P < .001), as was a lower median baseline hemoglobin (100 vs 110 g/L, P < .001). Proportionally, more women, patients with HCV coinfection, and those with more advanced HIV disease received tenofovir. Median (interquartile range [IQR]) duration of time to event or censor was slightly longer for the zidovudine group compared with the tenofovir group (392 [244] vs 376 [170] days, P < .001).\nTable 1.Baseline Characteristics and Outcomes of Study ParticipantsTenofovir Group n = 1484Zidovudine Group n = 4063P ValueBaseline Characteristics Age (y)33 (11)32 (11).41 Female gender1255 (84.6)3085 (75.9)<.001 Hepatitis B virus coinfection404 (27.2)604 (14.9)<.001 Hepatitis C virus coinfection144 (9.7)294 (7.2).003 CD4 count (cells/mm3)122 (122)143 (115)<.001 Human immunodeficiency virus-RNA (log10 copies/mL)4.86 (1.1)4.70 (1.2)<.001 World Health Organization Stagen = 1335n = 3489<.001 1236 (17.7)923 (26.5) 2435 (32.6)1042 (29.9) 3494 (37.0)1045 (30.0) 4170 (12.7)479 (13.7) Tuberculosis coinfection294 (19.8)466 (11.5)<.001 Alanine transaminase (µkat/L)0.42 (0.37)0.40 (0.36).04 Hemoglobin (g/L)100 (24), n = 1462110 (20), n = 3948<.001 Creatinine (μmol/L)77 (31), n = 145877 (31), n = 3880.943Study Outcomes Virologic failure159 (10.7)298 (7.3)<.001 Antiretroviral therapy switch256 (17.3)622 (15.3).079 Discontinue308 (20.8)649 (16.0)<.001 No event761 (51.3)2494 (61.4)<.001Values shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.\nFigure 1.Flow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.\nBaseline Characteristics and Outcomes of Study Participants\nValues shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.\nFlow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.\nAmong patients in the tenofovir group, 1043 (70.2%) received emtricitabine as their second NRTI, 190 (12.8%) received lamivudine, and 252 (19.8%) switched between emtricitabine and lamivudine during the study period (median [IQR] time to switch was 172 [97] days). The number of virologic failure events did not differ significantly between patients receiving emtricitabine or lamivudine (P = .47); therefore, data were combined in the final analysis irrespective of emtricitabine or lamivudine use.\nOverall, the median ART adherence rate was 95.3%, which was similar between the zidovudine and tenofovir groups (95.2% vs 95.8%). Among those who experienced virologic failure, more patients had adherence <95% compared with ≥95% (56.4% vs 43.6%; P < .001), but there was no difference in median adherence between those patients with failure in the zidovudine or tenofovir groups (91.9% vs 93.6%; P = .36).\nPatients in the tenofovir group had a higher rate of virologic failure and discontinuation (Table 1). Figure 2 represents time to virologic failure between the groups. Of the 957 patients with a discontinuation event, 14 (1.5%) died, 849 (88.7%) were lost to follow-up, 92 (9.6%) transferred to another site, and 2 (0.2%) withdrew from the program. These distributions were similar between NRTI groups (P = .50).\nFigure 2.Kaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.\nKaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.\n Time to Virologic Failure After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\nAfter adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).\n Evaluation of Other Outcome Differences or Dependent Censoring Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.\nAdditional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.",
"After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).\nTable 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nRisk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy\nAll risk factors were measured at baseline.\nBold results indicate statistically significant results (P value <.05).\nAbbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.\nWhen the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).",
"Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.",
"The Harvard–APIN program initially identified a high rate of confirmed virologic failure among patients on nevirapine plus tenofovir and emtricitabine or lamivudine compared with other WHO-recommended or alternative first-line regimens [7]. To evaluate the specific impact of the NRTI combination with nevirapine, we focused on patients initiating nevirapine-based ART combined with either zidovudine or tenofovir using Cox proportional hazards, time varying analysis, and IPW modeling. These results indicate that among patients initiating nevirapine, the use of tenofovir carried a 47% greater risk of virologic failure compared with the use of zidovudine; this difference persisted after adjusting for other variables that may influence regimen selection and treatment outcomes (Table 2). Tenofovir benefits were not observed in other outcomes; specifically, the tenofovir group had higher risk for regimen switch and similar risk for discontinuation. Notably, we did not observe significantly higher virologic failure in the tenofovir group after 12 months of ART, suggesting that patients already virologically suppressed on nevirapine plus tenofovir may not be at increased risk for virologic failure. These data have significant clinical implications for adults living in low- and middle-income countries who require an alternative to efavirenz for first-line ART [1].\nOur results are consistent with those from other evaluations of outcomes among patients who received ART containing nevirapine and tenofovir, thus supporting the belief that NNRTI effectiveness differs by NRTI selection. The DAUFIN study, which evaluated the effectiveness and tolerability of nevirapine given either with zidovudine–lamivudine or tenofovir–lamivudine in antiretroviral-naive patients, was terminated early due to high rates of early virologic failure in the tenofovir arm [6]; 9 of 36 patients receiving tenofovir experienced virologic failure, 8 during the first 12 weeks of the study, compared with only 1 of 35 patients receiving zidovudine. Lapadula et al also reported early virologic failure in patients receiving tenofovir–emtricitabine plus nevirapine compared with ritonavir-boosted atazanavir [5]. In another pilot study that described virologic outcomes among 23 patients receiving once-daily nevirapine plus tenofovir–lamivudine, only 10 patients achieved HIV-RNA <75 copies/mL by week 24 [3]. Turning to large clinical cohorts that evaluated this question, among 10 256 antiretroviral-naive patients initiating either zidovudine or tenofovir in combination with nevirapine in Zambia, 90-day mortality was higher among patients who received tenofovir (aHR, 1.45; 95% CI, 1.03–2.06); however, this difference did not remain after sensitivity analyses, including time-varying analysis [9]. In adjusted models from a large cohort study from southern Africa, patients on nevirapine plus tenofovir–emtricitabine or tenofovir–lamivudine experienced significantly higher rates of mortality but lower rates of loss to follow-up when compared with patients taking efavirenz plus similar NRTIs [21]. Consistent with these data, a metaanalysis of 33 studies comparing virologic efficacy among the WHO-recommended tenofovir-containing first-line ART regimens highlighted poorer outcomes with the nevirapine plus tenofovir-containing regimens [22].\nThe reason for the poorer performance of nevirapine–tenofovir ART is unclear. One proposed mechanism is an intracellular drug–drug interaction observed in vitro between tenofovir and nevirapine, resulting in lower intracellular concentrations of both antiretrovirals [23]. Differences in lamivudine vs emtricitabine pharmacokinetics have also been proposed. An observational Dutch cohort study identified a higher risk of virologic failure when lamivudine vs emtricitabine was combined with tenofovir and either nevirapine (aHR 2.01; 95% CI, 1.36–2.98) or efavirenz (aHR, 2.35; 95%CI, 1.61–3.42) [24]. Notably, our data did not identify a difference in outcomes among patients in the tenofovir group who received lamivudine or emtricitabine. Our results are consistent with those from a metaanalysis of 12 randomized controlled trials that compared 4913 patients randomized to lamivudine or emtricitabine in addition to various ART combinations and found that the 2 agents are clinically interchangeable [25].\nOur findings are limited by the study's retrospective cohort design. At the time of data collection, WHO guidelines recommended the use of either nevirapine or efavirenz in combination with zidovudine and lamivudine or with tenofovir and emtricitabine or lamivudine for first-line ART [11]. Regimen selection may have been influenced by factors that we were unable to measure or control for in our analysis, indicated by differences in baseline characteristics (Table 1). To account for some potential biases from dependent censoring, we generated an IPW pooled logistic regression model, and the close correspondence between these models testifies to the robustness of the Cox estimates. Additionally, there may be limitations to measuring adherence based on prescription refill data, particularly as it relates to pill burden. Although all patients received nevirapine twice daily, there was a difference between groups in the total number of pills per day. The zidovudine group received nevirapine plus a twice-daily FDC tablet of zidovudine–lamivudine (4 pills daily) or, beginning early 2007, a twice-daily full regimen FDC tablet (2 pills daily). The tenofovir group received nevirapine plus a once-daily FDC tablet of tenofovir–emtricitabine or tenofovir–lamivudine (3 pills daily). For twice-daily regimens, higher pill burden has previously been associated with lower rates of both adherence and virologic suppression (Spearman correlation, −0.67 and −0.75, respectively; both P < .01) [26]. However, a difference in adherence was not identified between our study groups and did not influence the associated risk of virologic failure related to regimen selection.\nIn conclusion, ART that includes tenofovir and nevirapine should be initiated with caution. The WHO recommends nevirapine as the alternative for patients who cannot take efavirenz, and nevirapine remains widely used for first-line ART in low- and middle-income countries [1, 27]. These results offer important information to guide selection of the NRTI combination when designing an HIV treatment regimen, indicating a preference for zidovudine over tenofovir when combined with nevirapine. These findings support the need for systematic evaluations of ART combinations before their inclusion in ART guidelines and subsequent widespread implementation.",
"Supplementary materials are available at http://cid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author."
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"zidovudine",
"tenofovir",
"nevirapine",
"virologic failure",
"antiretroviral therapy"
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METHODS:
In this retrospective cohort study, prospectively collected data from a large clinical cohort in Nigeria were used. Between June 2004 and February 2012, the Harvard T. H. Chan School of Public Health (Harvard) collaborated with the AIDS Prevention Initiative in Nigeria Ltd./Gte. (APIN) to support HIV prevention, care, and treatment to over 160 000 patients, funded by the President's Emergency Plan for AIDS Relief (PEPFAR). Upon program entry, patients underwent a clinical examination and baseline laboratory testing including hematology and chemistry, CD4+ cell count (Cyflow, Partec), HIV-RNA (Cobas Amplicor Monitor Assay, Roche Diagnostics), hepatitis B virus (HBV) surface antigen (Monolisa HBsAg Ultra3, BioRad), and hepatitis C virus (HCV) antibody (DIA.PRO Diagnostic, Bioprobes srl) assessments. Patients eligible for ART, based on guidelines at the time of program entry, initiated standard first-line ART with 1 NNRTI and 2 NRTIs [11, 12]. Prescription refills were obtained monthly; clinical visits and laboratory tests were performed every 6 months, or earlier if necessary. All patient data were maintained in previously described electronic databases [13].
Ethical Considerations All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.
All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.
Patient Selection Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.
Patients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.
Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.
Patients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.
Study Definitions Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.
Study events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.
Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.
Study events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.
Statistical Analyses Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.
Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.
A Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.
Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.
A Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.
Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].
The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].
Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.
Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.
A Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.
Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.
A Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.
Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].
The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].
Ethical Considerations:
All patients in the Harvard–APIN program provided written consent for care. Only those who consented to the use of their information for research were included in this analysis. Institutional review boards at Harvard and the individual clinics (Jos University Teaching Hospital, Nigerian Institute of Medical Research, 68 Military Hospital, and University of Maiduguri Teaching Hospital) approved the treatment protocol and data collection forms. Because we used de-identified data, the study was verified exempt from human subject review by institutional review boards.
Patient Selection:
Antiretroviral-naive, HIV-1–infected adults (aged ≥18 years) who initiated nevirapine-based ART with zidovudine–lamivudine (zidovudine group) or nevirapine-based ART with tenofovir–emtricitabine or tenofovir–lamivudine (tenofovir group) from 2006 to 2008 were included. Patients with prior antiretroviral experience, missing baseline HIV-RNA or CD4+ cell count data, no follow-up HIV-RNA after at least 6 months on treatment, no ART refills, or inconsistent ART regimens over 6 months (ie, regimen changed with each refill) were excluded. Patient data were included from time of ART initiation (baseline) through 18 months post-ART initiation.
Patients in the zidovudine group received zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg twice daily as a fixed dose combination (FDC) tablet (Aspen Pharmacare or Aurobindo Pharma Limited). Patients in the tenofovir group received nevirapine (Aurobindo) 200 mg twice daily with a once-daily FDC tablet of tenofovir–emtricitabine 300 mg–200 mg (Truvada, Aspen Pharmacare) or tenofovir–lamivudine 300 mg–300 mg (Matrix Laboratories Limited); use of the emtricitabine- or lamivudine-containing product was based on availability during the study period.
Study Definitions:
Sex, age, HBV or HCV coinfection, and WHO HIV clinical stage were evaluated at baseline. Tuberculosis coinfection and ART adherence were evaluated throughout the observation period. Adherence was estimated using ART refill data from baseline through the study-defined event or censoring [14, 15]. The average adherence rate was calculated as the proportion of time the patient had pills available (number of days supplied/number of days between refills) and was truncated at 100%. A prespecified adherence cutoff of 95% was used for categorical assessment of adherence.
Study events were confirmed virologic failure, ART switch, discontinuation, and unconfirmed virologic failure. Confirmed virologic failure was the primary outcome variable. In accordance with the clinical protocol, confirmed virologic failure was defined as 2 consecutive HIV-RNA >1000 copies/mL after 6 months on ART or switch to a second-line ART regimen, defined as protease inhibitor–based ART following an HIV-RNA >400 copies/mL. Time to virologic failure was the time from ART initiation to the first HIV-RNA >1000 copies/mL or the first protease inhibitor-based prescription. ART switch was defined as a change in ART for reasons other than virologic failure, with the exception of a switch between emtricitabine and lamivudine. The ART switch date was the date on which the new ART regimen was first dispensed. Discontinuation was defined as death, lost to follow-up (no ART refill for 6 months), or transferred or withdrew from the program. Time to discontinuation was calculated using the last recorded ART dispense date. Unconfirmed virologic failure was included to evaluate the potential influence of requiring 2 HIV-RNA results as our primary endpoint and was defined as HIV-RNA >1000 copies/mL without a consecutive confirmatory HIV-RNA measurement. Time to unconfirmed virologic failure was calculated using the date when the HIV-RNA measurement was >1000 copies/mL. Follow-up time was censored at the first event, and subsequent events were not included. For patients who did not experience a study event, follow-up was censored at last HIV-RNA within the study period.
Statistical Analyses:
Subject demographics were compared between groups using the Wilcoxon rank sum test for continuous variables or the χ2 test of independence for categorical variables.
Time to Virologic Failure Regression Modeling Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.
A Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.
Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.
A Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.
Evaluation of Dependent Censoring The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].
The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].
Time to Virologic Failure Regression Modeling:
Bivariate analyses were used to evaluate variables for inclusion in the Cox proportional hazards model; categorical variables were evaluated using the log-rank test, and continuous variables were evaluated using Cox proportional hazards regression [16]. Variables with a bivariate P < .25 and clinically relevant variables (adherence, age, serum creatinine) were considered candidates for inclusion in the initial Cox model. Kaplan–Meier graphs were generated for categorical variables to reveal violations of the Cox model's assumption of proportionality.
A Cox proportional hazards model was fitted by means of manual backward elimination of single covariates based on the likelihood ratio test and the comparison of hazard ratios between the model with and without the covariate in question. All statistically significant covariates, clinically relevant covariates (significant or not), effect modifiers, and confounders were retained in the final model. The scale of continuous covariates was evaluated using the design variable method. All interactions were examined using the likelihood ratio test. The proportionality assumption was assessed for covariates using Schoenfeld residuals, and the overall fit of the model was evaluated using Cox–Snell residuals.
Evaluation of Dependent Censoring:
The possibility of bias due to dependent censoring was evaluated by fitting a time-updated Cox regression model to assess the association between time-varying factors for each study-defined event [17]. Because clinical visits occur at 6-month intervals, laboratory values were carried forward up to 7 months to accommodate late visits. Percent adherence values were carried backward because adherence is calculated at the end of a prescription refill cycle. An inverse probability weighting (IPW) analysis was performed to assess potential bias due to dependent censoring by competing outcomes by fitting an IPW pooled logistic regression model using only uncensored person-time observations for the outcome of interest [18].
RESULTS:
During the study period, 6575 patients were identified for inclusion and 5547 (84%) had data available for all variables in the final model—4063 (73.2%) in the zidovudine group and 1484 (26.8%) in the tenofovir group (Figure 1). The most common reason for exclusion was missing HBV or HCV status (11.0%). Baseline participant characteristics are summarized in Table 1. Consistent with clinical considerations for selecting an NRTI, HBV coinfection was more common among those in the tenofovir vs zidovudine group (27.2% vs 14.9%; P < .001), as was a lower median baseline hemoglobin (100 vs 110 g/L, P < .001). Proportionally, more women, patients with HCV coinfection, and those with more advanced HIV disease received tenofovir. Median (interquartile range [IQR]) duration of time to event or censor was slightly longer for the zidovudine group compared with the tenofovir group (392 [244] vs 376 [170] days, P < .001).
Table 1.Baseline Characteristics and Outcomes of Study ParticipantsTenofovir Group n = 1484Zidovudine Group n = 4063P ValueBaseline Characteristics Age (y)33 (11)32 (11).41 Female gender1255 (84.6)3085 (75.9)<.001 Hepatitis B virus coinfection404 (27.2)604 (14.9)<.001 Hepatitis C virus coinfection144 (9.7)294 (7.2).003 CD4 count (cells/mm3)122 (122)143 (115)<.001 Human immunodeficiency virus-RNA (log10 copies/mL)4.86 (1.1)4.70 (1.2)<.001 World Health Organization Stagen = 1335n = 3489<.001 1236 (17.7)923 (26.5) 2435 (32.6)1042 (29.9) 3494 (37.0)1045 (30.0) 4170 (12.7)479 (13.7) Tuberculosis coinfection294 (19.8)466 (11.5)<.001 Alanine transaminase (µkat/L)0.42 (0.37)0.40 (0.36).04 Hemoglobin (g/L)100 (24), n = 1462110 (20), n = 3948<.001 Creatinine (μmol/L)77 (31), n = 145877 (31), n = 3880.943Study Outcomes Virologic failure159 (10.7)298 (7.3)<.001 Antiretroviral therapy switch256 (17.3)622 (15.3).079 Discontinue308 (20.8)649 (16.0)<.001 No event761 (51.3)2494 (61.4)<.001Values shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.
Figure 1.Flow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.
Baseline Characteristics and Outcomes of Study Participants
Values shown as either median (interquartile range) or n (%); percent is based on the total n (or category n) of the regimen group, as appropriate. The Wilcoxon rank sum test was used for continuous variables and the χ2 test of independence was used for categorical variables.
Flow diagram of eligible patients included in the cohort analysis in the final Cox proportional hazards model. Abbreviations: ART, antiretroviral therapy; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LTFU, lost to follow-up.
Among patients in the tenofovir group, 1043 (70.2%) received emtricitabine as their second NRTI, 190 (12.8%) received lamivudine, and 252 (19.8%) switched between emtricitabine and lamivudine during the study period (median [IQR] time to switch was 172 [97] days). The number of virologic failure events did not differ significantly between patients receiving emtricitabine or lamivudine (P = .47); therefore, data were combined in the final analysis irrespective of emtricitabine or lamivudine use.
Overall, the median ART adherence rate was 95.3%, which was similar between the zidovudine and tenofovir groups (95.2% vs 95.8%). Among those who experienced virologic failure, more patients had adherence <95% compared with ≥95% (56.4% vs 43.6%; P < .001), but there was no difference in median adherence between those patients with failure in the zidovudine or tenofovir groups (91.9% vs 93.6%; P = .36).
Patients in the tenofovir group had a higher rate of virologic failure and discontinuation (Table 1). Figure 2 represents time to virologic failure between the groups. Of the 957 patients with a discontinuation event, 14 (1.5%) died, 849 (88.7%) were lost to follow-up, 92 (9.6%) transferred to another site, and 2 (0.2%) withdrew from the program. These distributions were similar between NRTI groups (P = .50).
Figure 2.Kaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.
Kaplan–Meier graph of the time to confirmed virologic failure, defined as the time of the first human immunodeficiency virus-RNA >1000 copies/mL, for the tenofovir (TDF) and zidovudine (AZT) groups. Time is represented as days after initiating nevirapine (NVP)-containing first-line antiretroviral therapy. Abbreviations: 3TC, lamivudine; FTC, emtricitabine.
Time to Virologic Failure After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).
Table 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.
Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy
All risk factors were measured at baseline.
Bold results indicate statistically significant results (P value <.05).
Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.
When the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).
After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).
Table 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.
Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy
All risk factors were measured at baseline.
Bold results indicate statistically significant results (P value <.05).
Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.
When the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).
Evaluation of Other Outcome Differences or Dependent Censoring Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.
Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.
Time to Virologic Failure:
After adjusting for other baseline risk factors, patients in the tenofovir group had higher risk of virologic failure (adjusted hazard ratio [aHR], 1.47; 95% confidence interval [CI], 1.21–1.79; Table 2). Consistent with indicators of disease progression, the risk of virologic failure decreased with a higher baseline CD4+ cell count (aHR, 0.50; 95% CI, .40–.63), while a higher baseline HIV-RNA increased the risk of failure (aHR, 1.15; 95% CI, 1.03–1.28). Adherence was omitted from the final Cox model because it may have been on the causal pathway (ie, adherence is a potential mediator of the effect of regimen assignment) [19, 20]. Given its clinical importance, the Cox model that includes adherence is presented in Supplementary Table 1. While adherence <95% increased the risk of virologic failure (aHR, 1.28; 95% CI, 1.07–1.54), the inclusion of adherence in the Cox model had no notable effect on the HR or CI of the remaining covariates (tenofovir group aHR, 1.48; 95% CI, 1.21–1.80).
Table 2.Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral TherapyFactorCox Proportional Hazards Model (n = 5547 Patients)Inverse Probability Weighted Pooled Logistic Regression (5697 Patients, 81 642 Observations)HR95% CIP ValueOR95% CIP ValueTenofovir group1.471.21, 1.79.0011.571.28, 1.94.0001Age0.98.97, .99.0020.99.98, 1.00.122Female gender0.91.71, 1.17.4740.88.69, 1.13.309Hepatitis B virus coinfection0.96.75, 1.23.7170.98.78, 1.23.862Hepatitis C virus coinfection1.17.85, 1.61.3260.91.66, 1.26.554CD4 count (log10)0.50.40, .63<.0010.997.995, .998<.0001Human immunodeficiency virus-RNA (log10)1.151.03, 1.28.0111.201.01, 1.32.018Tuberculosis coinfection………0.72.50, 1.05.085Alanine transaminase (µkat/L)………1.111.00, 1.22.052Hemoglobin (g/L)………1.00.99, 1.00.415Creatinine (μmol/L)………0.997.993, .999.013All risk factors were measured at baseline.Bold results indicate statistically significant results (P value <.05).Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.
Risk Estimates and 95% Confidence Intervals for Virologic Failure During 18 Months of First Line Antiretroviral Therapy
All risk factors were measured at baseline.
Bold results indicate statistically significant results (P value <.05).
Abbreviations: CI, confidence interval; HR, hazard ratio; OR, odds ratio.
When the Cox analysis was restricted to virologic failures that occurred beyond 12 months, the regimen was no longer associated with virologic failure (aHR, 1.99; 95% CI, .94–4.21). However, the number of participants and the number of events that occurred during the 12–18 months were small (n = 3120 participants with 37 virologic failure events).
Evaluation of Other Outcome Differences or Dependent Censoring:
Additional outcome-specific, time-updated Cox models were constructed for each of our defined outcomes to evaluate the effect of regimen on events outside of virologic failure (Supplementary Table 2). Discontinuation and unconfirmed virologic failure outcomes were not associated with treatment regimen. ART switch was associated with treatment regimen, indicating that patients in the tenofovir group were 34% more likely to switch regimens (aHR, 1.34; 95% CI, 1.15–1.57). The effect of regimen on time to virologic failure was consistent between the primary and time-updated models and reflected a 47% increase in the risk of virologic failure for patients in the tenofovir group (aHR, 1.47; 95% CI, 1.24–1.75). While these models did not rule out dependent censoring, we found an overall agreement between IPW analysis results (Table 2) and the primary Cox proportional hazards results related to the impact of the study group on virologic failure, demonstrating that dependent censoring did not compromise the original nontime-updated Cox model estimates.
DISCUSSION:
The Harvard–APIN program initially identified a high rate of confirmed virologic failure among patients on nevirapine plus tenofovir and emtricitabine or lamivudine compared with other WHO-recommended or alternative first-line regimens [7]. To evaluate the specific impact of the NRTI combination with nevirapine, we focused on patients initiating nevirapine-based ART combined with either zidovudine or tenofovir using Cox proportional hazards, time varying analysis, and IPW modeling. These results indicate that among patients initiating nevirapine, the use of tenofovir carried a 47% greater risk of virologic failure compared with the use of zidovudine; this difference persisted after adjusting for other variables that may influence regimen selection and treatment outcomes (Table 2). Tenofovir benefits were not observed in other outcomes; specifically, the tenofovir group had higher risk for regimen switch and similar risk for discontinuation. Notably, we did not observe significantly higher virologic failure in the tenofovir group after 12 months of ART, suggesting that patients already virologically suppressed on nevirapine plus tenofovir may not be at increased risk for virologic failure. These data have significant clinical implications for adults living in low- and middle-income countries who require an alternative to efavirenz for first-line ART [1].
Our results are consistent with those from other evaluations of outcomes among patients who received ART containing nevirapine and tenofovir, thus supporting the belief that NNRTI effectiveness differs by NRTI selection. The DAUFIN study, which evaluated the effectiveness and tolerability of nevirapine given either with zidovudine–lamivudine or tenofovir–lamivudine in antiretroviral-naive patients, was terminated early due to high rates of early virologic failure in the tenofovir arm [6]; 9 of 36 patients receiving tenofovir experienced virologic failure, 8 during the first 12 weeks of the study, compared with only 1 of 35 patients receiving zidovudine. Lapadula et al also reported early virologic failure in patients receiving tenofovir–emtricitabine plus nevirapine compared with ritonavir-boosted atazanavir [5]. In another pilot study that described virologic outcomes among 23 patients receiving once-daily nevirapine plus tenofovir–lamivudine, only 10 patients achieved HIV-RNA <75 copies/mL by week 24 [3]. Turning to large clinical cohorts that evaluated this question, among 10 256 antiretroviral-naive patients initiating either zidovudine or tenofovir in combination with nevirapine in Zambia, 90-day mortality was higher among patients who received tenofovir (aHR, 1.45; 95% CI, 1.03–2.06); however, this difference did not remain after sensitivity analyses, including time-varying analysis [9]. In adjusted models from a large cohort study from southern Africa, patients on nevirapine plus tenofovir–emtricitabine or tenofovir–lamivudine experienced significantly higher rates of mortality but lower rates of loss to follow-up when compared with patients taking efavirenz plus similar NRTIs [21]. Consistent with these data, a metaanalysis of 33 studies comparing virologic efficacy among the WHO-recommended tenofovir-containing first-line ART regimens highlighted poorer outcomes with the nevirapine plus tenofovir-containing regimens [22].
The reason for the poorer performance of nevirapine–tenofovir ART is unclear. One proposed mechanism is an intracellular drug–drug interaction observed in vitro between tenofovir and nevirapine, resulting in lower intracellular concentrations of both antiretrovirals [23]. Differences in lamivudine vs emtricitabine pharmacokinetics have also been proposed. An observational Dutch cohort study identified a higher risk of virologic failure when lamivudine vs emtricitabine was combined with tenofovir and either nevirapine (aHR 2.01; 95% CI, 1.36–2.98) or efavirenz (aHR, 2.35; 95%CI, 1.61–3.42) [24]. Notably, our data did not identify a difference in outcomes among patients in the tenofovir group who received lamivudine or emtricitabine. Our results are consistent with those from a metaanalysis of 12 randomized controlled trials that compared 4913 patients randomized to lamivudine or emtricitabine in addition to various ART combinations and found that the 2 agents are clinically interchangeable [25].
Our findings are limited by the study's retrospective cohort design. At the time of data collection, WHO guidelines recommended the use of either nevirapine or efavirenz in combination with zidovudine and lamivudine or with tenofovir and emtricitabine or lamivudine for first-line ART [11]. Regimen selection may have been influenced by factors that we were unable to measure or control for in our analysis, indicated by differences in baseline characteristics (Table 1). To account for some potential biases from dependent censoring, we generated an IPW pooled logistic regression model, and the close correspondence between these models testifies to the robustness of the Cox estimates. Additionally, there may be limitations to measuring adherence based on prescription refill data, particularly as it relates to pill burden. Although all patients received nevirapine twice daily, there was a difference between groups in the total number of pills per day. The zidovudine group received nevirapine plus a twice-daily FDC tablet of zidovudine–lamivudine (4 pills daily) or, beginning early 2007, a twice-daily full regimen FDC tablet (2 pills daily). The tenofovir group received nevirapine plus a once-daily FDC tablet of tenofovir–emtricitabine or tenofovir–lamivudine (3 pills daily). For twice-daily regimens, higher pill burden has previously been associated with lower rates of both adherence and virologic suppression (Spearman correlation, −0.67 and −0.75, respectively; both P < .01) [26]. However, a difference in adherence was not identified between our study groups and did not influence the associated risk of virologic failure related to regimen selection.
In conclusion, ART that includes tenofovir and nevirapine should be initiated with caution. The WHO recommends nevirapine as the alternative for patients who cannot take efavirenz, and nevirapine remains widely used for first-line ART in low- and middle-income countries [1, 27]. These results offer important information to guide selection of the NRTI combination when designing an HIV treatment regimen, indicating a preference for zidovudine over tenofovir when combined with nevirapine. These findings support the need for systematic evaluations of ART combinations before their inclusion in ART guidelines and subsequent widespread implementation.
Supplementary Data:
Supplementary materials are available at http://cid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.
|
Background:
Despite sparse efficacy data, tenofovir-emtricitabine or tenofovir-lamivudine plus nevirapine is used in many resource-constrained settings.
Methods:
This retrospective cohort study included patients initiating nevirapine-based antiretroviral therapy (ART) with either tenofovir-emtricitabine or lamivudine (tenofovir group) or zidovudine-lamivudine (zidovudine group). Clinical, virologic, and immunologic evaluations were performed at baseline and every 6 months. Virologic failure was defined as 2 consecutive human immunodeficiency virus (HIV)-RNA values >1000 copies/mL. Patients were included from ART initiation until time of failure, regimen switch, discontinuation, or last HIV-RNA measurement. Cox proportional hazards regression was used to model factors influencing time to failure. Bias due to dependent censoring was investigated via inverse probability weighted pooled logistic regression.
Results:
A total of 5547 patients were evaluated; 1484 (26.8%) were in the tenofovir group and 4063 (73.2%) were in the zidovudine group. In the adjusted model, tenofovir regimen (hazard ratio [HR], 1.47; 95% confidence interval [CI], 1.21-1.79) and higher baseline log10 HIV-RNA (HR, 1.15; 95% CI, 1.03-1.28) were associated with virologic failure. Higher baseline log10 CD4+ cell count (HR, 0.50; 95% CI, .40-.63) and increasing age (HR, 0.98; 95% CI, .97-.99) decreased the risk of virologic failure. Inverse probability weighting results were consistent with the primary analysis.
Conclusions:
Compared with zidovudine-lamivudine, the use of tenofovir-lamivudine or emtricitabine in combination with nevirapine was a strong predictor of virologic failure in our cohort, which was not explained by other risk factors or criteria for regimen selection.
| null | null | 9,310 | 336 | 12 |
[
"virologic",
"failure",
"model",
"virologic failure",
"art",
"cox",
"time",
"tenofovir",
"patients",
"adherence"
] |
[
"test",
"test"
] | null | null | null | null |
[CONTENT] zidovudine | tenofovir | nevirapine | virologic failure | antiretroviral therapy [SUMMARY]
|
[CONTENT] zidovudine | tenofovir | nevirapine | virologic failure | antiretroviral therapy [SUMMARY]
| null |
[CONTENT] zidovudine | tenofovir | nevirapine | virologic failure | antiretroviral therapy [SUMMARY]
| null | null |
[CONTENT] Adult | Anti-Retroviral Agents | Antiretroviral Therapy, Highly Active | CD4 Lymphocyte Count | Cohort Studies | Female | HIV Infections | Humans | Male | Nevirapine | Retrospective Studies | Tenofovir | Treatment Outcome | Viral Load | Young Adult | Zidovudine [SUMMARY]
|
[CONTENT] Adult | Anti-Retroviral Agents | Antiretroviral Therapy, Highly Active | CD4 Lymphocyte Count | Cohort Studies | Female | HIV Infections | Humans | Male | Nevirapine | Retrospective Studies | Tenofovir | Treatment Outcome | Viral Load | Young Adult | Zidovudine [SUMMARY]
| null |
[CONTENT] Adult | Anti-Retroviral Agents | Antiretroviral Therapy, Highly Active | CD4 Lymphocyte Count | Cohort Studies | Female | HIV Infections | Humans | Male | Nevirapine | Retrospective Studies | Tenofovir | Treatment Outcome | Viral Load | Young Adult | Zidovudine [SUMMARY]
| null | null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null | null |
[CONTENT] virologic | failure | model | virologic failure | art | cox | time | tenofovir | patients | adherence [SUMMARY]
|
[CONTENT] virologic | failure | model | virologic failure | art | cox | time | tenofovir | patients | adherence [SUMMARY]
| null |
[CONTENT] virologic | failure | model | virologic failure | art | cox | time | tenofovir | patients | adherence [SUMMARY]
| null | null |
[CONTENT] art | variables | model | hiv | time | mg | evaluated | cox | covariates | hiv rna [SUMMARY]
|
[CONTENT] failure | virologic | virologic failure | 95 | ci | risk | 001 | virus | group | ahr [SUMMARY]
| null |
[CONTENT] virologic | art | model | failure | virologic failure | cox | tenofovir | time | variables | patients [SUMMARY]
| null | null |
[CONTENT] ||| every 6 months ||| Virologic | 2 | 1000 ||| ||| Bias [SUMMARY]
|
[CONTENT] 5547 | 1484 | 26.8% | 4063 | 73.2% ||| 1.47 | 95% | CI] | 1.21-1.79 | 1.15 | 95% | CI | 1.03-1.28 ||| 0.50 | 95% | CI | 0.98 | 95% | CI ||| [SUMMARY]
| null |
[CONTENT] ||| ||| every 6 months ||| Virologic | 2 | 1000 ||| ||| Bias ||| 5547 | 1484 | 26.8% | 4063 | 73.2% ||| 1.47 | 95% | CI] | 1.21-1.79 | 1.15 | 95% | CI | 1.03-1.28 ||| 0.50 | 95% | CI | 0.98 | 95% | CI ||| ||| [SUMMARY]
| null |
|||
Prognostic Value of Soluble ST2 During Hospitalization for ST-Segment Elevation Myocardial Infarction.
|
27139603
|
Studying the role of soluble ST2 (sST2) during hospitalization for myocardial infarction (MI) can be helpful for predicting the course of the hospitalization and development of complications.
|
BACKGROUND
|
We included 88 patients with MI (median age, 58 yr). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. On days 1 and 12 after MI, serum sST2 and N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured by ELISA.
|
METHODS
|
On day 1, the concentrations of sST2 and NT-proBNP increased 2.4- and 4.5-fold, compared with the controls. Measurements on day 12 showed a significant decrease in the sST2 level (P=0.001), whereas the NT-proBNP level did not change. On day 1, the sST2 level in the unfavorable outcome group was 2-fold higher than that in the favorable outcome group and 3.7-fold higher than in the controls. On day 12, the marker level decreased in both groups. On day 1, the NT-proBNP level in the unfavorable outcome group was 6.8-fold higher than in the controls and 1.8-fold higher than in the favorable outcome group. On day 12, the level of NT-proBNP remained elevated in both groups. Determining the levels of both sST2 and NT-proBNP increases their diagnostic significance (odds ratio [OR], 1.92; 95% confidence interval [CI], 1.7-3.2; areas under curve [AUC] 0.89; P=0.004).
|
RESULTS
|
The level of sST2 is a more sensitive indicator during MI hospitalization than NT-proBNP.
|
CONCLUSIONS
|
[
"Area Under Curve",
"Case-Control Studies",
"Electrocardiography",
"Enzyme-Linked Immunosorbent Assay",
"Female",
"Hospitalization",
"Humans",
"Logistic Models",
"Male",
"Middle Aged",
"Myocardial Infarction",
"Natriuretic Peptide, Brain",
"Odds Ratio",
"Peptide Fragments",
"Prognosis",
"Proportional Hazards Models",
"ROC Curve",
"Receptors, Somatostatin"
] |
4855050
|
INTRODUCTION
|
Myocardial infarction (MI) is accompanied by structural and geometric changes in the heart [1]. Early remodeling is characterized by the stretching and thinning of the myocardium and the dilation and spherification of the left ventricle. The acute stretching of the viable myocardium maintains the pumping function despite the decrease in its contracting function [1]. If more than 20% of the left ventricular mass is affected, the compensation is inadequate. A traditional indicator of the stretching of cardiomyocytes and the development of chronic heart failure is the level of the N-terminal pro-brain natriuretic peptide (NT-proBNP) [23]. However, the widespread application of this indicator is limited by its biological variation, as it varies according to sex, age, and body mass index. The levels of NT-proBNP may vary also in other pathologies, such as infections and kidney diseases [4].
ST2 is an early marker of myocardial remodeling, and this understudied growth-stimulating factor is expressed on macrovascular (aortic and coronary artery) and microvascular endothelial cells in the heart in humans [5] and on cardiomyocytes in rats and mice [3] when under biomechanical stress [6]; thus, it is a novel and promising marker. ST2 is a member of the family of interleukin (IL)-1 receptors. The main function of ST2, which potentiates IL-33, is to exert antihypertrophic and antifibrosing effects on cardiomyocytes that are under biomechanical stretching conditions [78]. However, an acute increase in the ST2 level has been observed when damage is accompanied by the inhibition of IL-33 and its favorable antihypertrophic effects. Studying the role of ST2 during hospitalization for MI can be helpful for predicting the course of the hospitalization and development of complications [391011]. The aim of this study was to determine the level of soluble ST2 (sST2) and its correlation with the level of NT-proBNP and with the clinical course of MI during hospitalization.
|
METHODS
|
1. Study population For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.
The control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.
The study protocol was approved by the local ethics committee of the Federal State Budgetary Institution "Research Institute for Complex Issues of Cardiovascular Disease" and was developed in accordance with the WMA Declaration of Helsinki "Ethical principles for medical research involving human subjects" (amended in 2000) and "Rules for clinical practice in the Russian Federation" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.
Among all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.
The complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).
Unless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.
For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.
The control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.
The study protocol was approved by the local ethics committee of the Federal State Budgetary Institution "Research Institute for Complex Issues of Cardiovascular Disease" and was developed in accordance with the WMA Declaration of Helsinki "Ethical principles for medical research involving human subjects" (amended in 2000) and "Rules for clinical practice in the Russian Federation" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.
Among all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.
The complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).
Unless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.
2. Assays Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.
Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.
3. Statistical analysis Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.
Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.
|
RESULTS
|
On day 1 of hospitalization for MI, the levels of sST2 and NT-proBNP increased by 2.4-fold and 4.5-fold, respectively, compared with the control group [44.75 (24.90; 93.56) ng/mL, P=0.002; 18.81 (15.12; 21.03) ng/mL] [36.84 (24.09; 89.26) fmol/mL, P=0.000; 8.23 (5.61; 11.12) fmol/mL)].
By day 12, the sST2 level significantly decreased by 2.5-fold [17.82 (15.30; 23.25 ng/mL, P=0.001], whereas the changes in the NT-proBNP level were not significant (Fig. 1).
The results of the correlation analyses indicated a moderate correlation between the levels of sST2 and NT-proBNP in both groups on days 1 (R=0.50, P=0.001) and 12 of MI hospitalization (R=0.55, P=0. 0002).
According to experimental data, sST2 in animals is expressed only in cardiomyocytes during myocardial injury; nevertheless, we were interested in the correlation analysis between troponin T (the classic marker of cardiomyocyte damage) and sST2 concentrations during the MI hospitalization period. The results of the correlation analyses indicated a direct correlation between the levels of sST2 and troponin T in both groups at the time of admission (R=0.65, P=0.002).
A comparative analysis of the levels of sST2 and NT-proBNP in both groups (favorable (n=58) and unfavorable (n=30) outcome) was performed, and the results are shown in Table 3. The level of sST2 in the unfavorable outcome group on day 1 was 2-fold higher than that in favorable outcome group. The sST2 levels increased by 1.9- and 3.7-fold in the favorable and unfavorable outcome groups, respectively, compared with those in the control group.
On day 12 after MI onset, the sST2 level was significantly lower (P=0.011) in subjects in both groups compared with the level in the control group.
In contrast to sST2, the level of NT-proBNP increased equally in both the favorable and unfavorable outcome groups up to day 12 of the study. The highest increase in the NT-proBNP level (6.8-fold) was observed in patients in the unfavorable outcome group on day 1.
A level of sST2 higher than 35 ng/mL is considered an indicator of adverse outcome development in cardiovascular pathology [1013]. Thus, in the next stage of our study, we analyzed the differences in the clinical and anamnestic characteristics of the subjects by categorizing the sST2 level as lower (Group 1) or higher (Group 2) than the critical level of 35 ng/mL (Table 2). In Group 2 (sST2 concentration above 35 ng/mL), according to the clinical and anamnestic characteristics, complications [MI (44.6%) and diabetes mellitus (23%)] during hospitalization were more common. In Group 1 (sST2 level lower than 35 ng/mL), the percentage of adverse events and complications during hospitalization was significantly lower (P=0.021).
Conversely, analysis of individual patient data indicated that five patients (15.6%) with a poor prognosis had an sST2 concentration lower than 35 ng/mL, whereas 31 (55.4%) patients with a favorable prognosis had an sST2 level higher than 35 ng/mL.
The results of the evaluation of the NT-proBNP level as a function of sST2 are shown in Table 4. On day 1, in patients with sST2 concentrations above 35 ng/mL, the concentration of NT-proBNP increased by 6.7-fold compared with the controls. At the same time, in patients with a moderate increase in the concentration of sST2 (less than 35 ng/mL), the concentration of NT-proBNP also increased, although to a lesser degree (4.6-fold compared with the controls). At day 12, the level of NT-proBNP remained elevated in both groups and did not differ significantly between the two (Table 4).
Logistic regression analysis showed that an increase in the concentration of sST2 increased the risk of complications during the hospitalization by 1.7-fold times (OR, 1.7; 95% CI, 1.6-2.8; areas under curve [AUC]=0.78; P=0.003), with sensitivity of 76.9% and a specificity of 69.4%. At the same time, the increase in the level of NT-proBNP was accompanied only by a 1.2-fold increase in adverse outcomes (OR, 1.2; 95% CI, 1.1-1.6; AUC=0.69; P=0.034), without affecting the high diagnostic sensitivity (69.6%) and specificity (65.3%).
Determining the level of sST2 in combination with NT-proBNP increases their diagnostic significance (OR, 1.92; 95% CI, 1.7-3.2). These indicators, when used together and measured at the early stages of MI, increased the quality of the model, with sensitivity of 81%, specificity of 72%, and AUC of 0.86.
| null | null |
[
"1. Study population",
"2. Assays",
"3. Statistical analysis"
] |
[
"For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.",
"Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.",
"Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant."
] |
[
null,
null,
null
] |
[
"INTRODUCTION",
"METHODS",
"1. Study population",
"2. Assays",
"3. Statistical analysis",
"RESULTS",
"DISCUSSION"
] |
[
"Myocardial infarction (MI) is accompanied by structural and geometric changes in the heart [1]. Early remodeling is characterized by the stretching and thinning of the myocardium and the dilation and spherification of the left ventricle. The acute stretching of the viable myocardium maintains the pumping function despite the decrease in its contracting function [1]. If more than 20% of the left ventricular mass is affected, the compensation is inadequate. A traditional indicator of the stretching of cardiomyocytes and the development of chronic heart failure is the level of the N-terminal pro-brain natriuretic peptide (NT-proBNP) [23]. However, the widespread application of this indicator is limited by its biological variation, as it varies according to sex, age, and body mass index. The levels of NT-proBNP may vary also in other pathologies, such as infections and kidney diseases [4].\nST2 is an early marker of myocardial remodeling, and this understudied growth-stimulating factor is expressed on macrovascular (aortic and coronary artery) and microvascular endothelial cells in the heart in humans [5] and on cardiomyocytes in rats and mice [3] when under biomechanical stress [6]; thus, it is a novel and promising marker. ST2 is a member of the family of interleukin (IL)-1 receptors. The main function of ST2, which potentiates IL-33, is to exert antihypertrophic and antifibrosing effects on cardiomyocytes that are under biomechanical stretching conditions [78]. However, an acute increase in the ST2 level has been observed when damage is accompanied by the inhibition of IL-33 and its favorable antihypertrophic effects. Studying the role of ST2 during hospitalization for MI can be helpful for predicting the course of the hospitalization and development of complications [391011]. The aim of this study was to determine the level of soluble ST2 (sST2) and its correlation with the level of NT-proBNP and with the clinical course of MI during hospitalization.",
" 1. Study population For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\nFor this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.\n 2. Assays Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\nSerum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.\n 3. Statistical analysis Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.\nStatistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.",
"For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.\nThe control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.\nThe study protocol was approved by the local ethics committee of the Federal State Budgetary Institution \"Research Institute for Complex Issues of Cardiovascular Disease\" and was developed in accordance with the WMA Declaration of Helsinki \"Ethical principles for medical research involving human subjects\" (amended in 2000) and \"Rules for clinical practice in the Russian Federation\" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.\nAmong all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.\nThe complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).\nUnless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.",
"Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.",
"Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.",
"On day 1 of hospitalization for MI, the levels of sST2 and NT-proBNP increased by 2.4-fold and 4.5-fold, respectively, compared with the control group [44.75 (24.90; 93.56) ng/mL, P=0.002; 18.81 (15.12; 21.03) ng/mL] [36.84 (24.09; 89.26) fmol/mL, P=0.000; 8.23 (5.61; 11.12) fmol/mL)].\nBy day 12, the sST2 level significantly decreased by 2.5-fold [17.82 (15.30; 23.25 ng/mL, P=0.001], whereas the changes in the NT-proBNP level were not significant (Fig. 1).\nThe results of the correlation analyses indicated a moderate correlation between the levels of sST2 and NT-proBNP in both groups on days 1 (R=0.50, P=0.001) and 12 of MI hospitalization (R=0.55, P=0. 0002).\nAccording to experimental data, sST2 in animals is expressed only in cardiomyocytes during myocardial injury; nevertheless, we were interested in the correlation analysis between troponin T (the classic marker of cardiomyocyte damage) and sST2 concentrations during the MI hospitalization period. The results of the correlation analyses indicated a direct correlation between the levels of sST2 and troponin T in both groups at the time of admission (R=0.65, P=0.002).\nA comparative analysis of the levels of sST2 and NT-proBNP in both groups (favorable (n=58) and unfavorable (n=30) outcome) was performed, and the results are shown in Table 3. The level of sST2 in the unfavorable outcome group on day 1 was 2-fold higher than that in favorable outcome group. The sST2 levels increased by 1.9- and 3.7-fold in the favorable and unfavorable outcome groups, respectively, compared with those in the control group.\nOn day 12 after MI onset, the sST2 level was significantly lower (P=0.011) in subjects in both groups compared with the level in the control group.\nIn contrast to sST2, the level of NT-proBNP increased equally in both the favorable and unfavorable outcome groups up to day 12 of the study. The highest increase in the NT-proBNP level (6.8-fold) was observed in patients in the unfavorable outcome group on day 1.\nA level of sST2 higher than 35 ng/mL is considered an indicator of adverse outcome development in cardiovascular pathology [1013]. Thus, in the next stage of our study, we analyzed the differences in the clinical and anamnestic characteristics of the subjects by categorizing the sST2 level as lower (Group 1) or higher (Group 2) than the critical level of 35 ng/mL (Table 2). In Group 2 (sST2 concentration above 35 ng/mL), according to the clinical and anamnestic characteristics, complications [MI (44.6%) and diabetes mellitus (23%)] during hospitalization were more common. In Group 1 (sST2 level lower than 35 ng/mL), the percentage of adverse events and complications during hospitalization was significantly lower (P=0.021).\nConversely, analysis of individual patient data indicated that five patients (15.6%) with a poor prognosis had an sST2 concentration lower than 35 ng/mL, whereas 31 (55.4%) patients with a favorable prognosis had an sST2 level higher than 35 ng/mL.\nThe results of the evaluation of the NT-proBNP level as a function of sST2 are shown in Table 4. On day 1, in patients with sST2 concentrations above 35 ng/mL, the concentration of NT-proBNP increased by 6.7-fold compared with the controls. At the same time, in patients with a moderate increase in the concentration of sST2 (less than 35 ng/mL), the concentration of NT-proBNP also increased, although to a lesser degree (4.6-fold compared with the controls). At day 12, the level of NT-proBNP remained elevated in both groups and did not differ significantly between the two (Table 4).\nLogistic regression analysis showed that an increase in the concentration of sST2 increased the risk of complications during the hospitalization by 1.7-fold times (OR, 1.7; 95% CI, 1.6-2.8; areas under curve [AUC]=0.78; P=0.003), with sensitivity of 76.9% and a specificity of 69.4%. At the same time, the increase in the level of NT-proBNP was accompanied only by a 1.2-fold increase in adverse outcomes (OR, 1.2; 95% CI, 1.1-1.6; AUC=0.69; P=0.034), without affecting the high diagnostic sensitivity (69.6%) and specificity (65.3%).\nDetermining the level of sST2 in combination with NT-proBNP increases their diagnostic significance (OR, 1.92; 95% CI, 1.7-3.2). These indicators, when used together and measured at the early stages of MI, increased the quality of the model, with sensitivity of 81%, specificity of 72%, and AUC of 0.86.",
"MI is accompanied by the mechanical deformation of cardiomyocytes, which may undergo adaptive and maladaptive changes, leading to chronic heart failure [1]. In response to increased wall tension of the heart ventricles, the intracardiac pressure increases, resulting in increased cardiomyocyte volume and the synthesis of factors such as NT-proBNP and sST2, which function to increase myocardial performance [3].\nOur study showed that on day 1 after MI, the NT-proBNP concentration increased 4.5-fold (Fig. 1) and remained elevated up to day 12 of the study. It was shown in BNP transgenic mice that following artificially induced MI, the infarcted area increased by about 5-fold during the 48 hr following MI onset and remained at an increased level over the next three to four weeks [1415]. When the disease course was unfavorable, the NT-proBNP concentration increased even more, to 6.8-fold compared with the control values. In addition to the mechanical stretching of the ventricles, there may be other mechanisms that stimulate the production of NT-proBNP, such as ischemia in various locations, arrhythmias, myocardial hypertrophy, and endothelial dysfunction [16]. However, in this study, the NT-proBNP level had a low diagnostic sensitivity and specificity as an indicator of an unfavorable course following MI, with its increase indicating only a 1.2-fold increase in the risk for complications.\nThe stimulating growth factor sST2 had a higher sensitivity for the development of a poor prognosis. With its increase on day 1, there was a 1.7-fold increase in the risk of an unfavorable outcome following MI. Compared with the controls, the sST2 level increased by 1.9-fold in cases with a favorable course of MI and increased 3.7-fold in cases of unfavorable outcomes (Table 2). Elevated levels of sST2 have been associated with an increase in the synthesis of sST2 in cardiac myocytes and fibroblasts due to biomechanical stress [31718].\nST2 is a member of the superfamily of IL-1 receptors. It exists in two forms: a transmembrane receptor (ST2L) and soluble receptor-trap (sST2). The ST2 ligand is IL-33, which contributes to the process reduction of the fibrosis and hypertrophy of tissues under mechanical loads [19]. sST2 acts as a decoy receptor by binding free IL-33 and preventing its signaling through ST2L [2021]. A moderate increase in the concentration of sST2 probably exerts a protective effect, which manifests itself in patients with a favorable course of MI [39]. The transmembrane form protects the myocardium from overcharging, whereas the soluble form of ST2 prevents this protective mechanism, binds to IL-33, and blocks its cardioprotective effect [320]. Perhaps the increase in the concentration of sST2 in cases of unfavorable outcomes is associated with an increased content of the soluble form of the marker following release by damaged cardiomyocytes.\nBoth sST2 and troponin levels have been measured in several other studies. In the study by Mueller et al. [22] in 2015, in contrast to our study, no correlation between sST2 and troponin levels was found. This difference can be explained by the fact that we measured biomarkers in patients in the acute phase of MI, whereas the patients in the study by Mueller et al. [22] had heart failure.\nIn the present study, by day 12 following MI, sST2 concentrations decreased to the level of control values, thus making it difficult to distinguish between the favorable and unfavorable outcome groups. The dynamics of the sST2 changes are somewhat similar to those of C-reactive protein (CRP). CRP levels increased acutely during acute MI; however, this marker tended to decrease by day 12 after MI onset [23]. This similarity indicates their common inflammatory nature. These results are consistent with those reported in the study by Weinberg et al. [24], which was performed on an experimental MI model (in vivo) by using C57/BL6J mice. After ligating the coronary artery, the maximal transcriptional induction of sST2 in cardiac myocytes occurred within 2 hr, was maintained for 9 hr, and decreased after 15 hr.\nStudies have shown that the sST2 threshold in patients with chronic heart failure is 35 ng/mL. Above this value, the risk of death increases dramatically within one year of the event [91018]. Kohli et al. [10] reported similar results, showing that high levels of sST2 (>35 ng/mL) in patients suffering from acute coronary syndrome predicted a 3-fold higher risk of cardiovascular death and heart failure within 30 days and one year. It is of particular interest to study such patterns in a cohort of patients with MI. In our study, the adverse outcomes during the early period of MI were not associated with sST2 levels above 35 ng/mL, as there were complications in patients with levels below (15.5%) and above (55.4%) the threshold. These results were probably affected not only by the level of sST2 but also by the presence of other factors, specifically the increase in NT-proBNP. Indeed, the use of these two markers together significantly increases their sensitivity and specificity of predicting the risk of unfavorable MI outcomes [25].\nOur findings are consistent with the study by Sabatine et al. [18], in which unfavorable outcomes were observed with high levels of both markers (risk of death or developing heart failure was 6.5-fold higher) during the 30-day observation period. Using the levels of NT-proBNP and sST2 to construct the receiver operating characteristics curve, the identification of combined cardiovascular mortality or heart failure significantly improved to 0.78 (95% CI, 0.74-0.83; P=0.0025).\nIn conclusion, the concentration of sST2 is a more sensitive indicator of the course of hospitalization for MI than the traditional indicator of the NT-proBNP concentration. Increased concentrations of sST2 on day 1 after MI were followed by an unfavorable hospitalization course, including progressive angina, arrhythmias, MI, and recurrent symptomatic AHF (Killip class II-IV)."
] |
[
"intro",
"methods",
null,
null,
null,
"results",
"discussion"
] |
[
"Myocardial infarction",
"NT-proBNP",
"sST2"
] |
INTRODUCTION:
Myocardial infarction (MI) is accompanied by structural and geometric changes in the heart [1]. Early remodeling is characterized by the stretching and thinning of the myocardium and the dilation and spherification of the left ventricle. The acute stretching of the viable myocardium maintains the pumping function despite the decrease in its contracting function [1]. If more than 20% of the left ventricular mass is affected, the compensation is inadequate. A traditional indicator of the stretching of cardiomyocytes and the development of chronic heart failure is the level of the N-terminal pro-brain natriuretic peptide (NT-proBNP) [23]. However, the widespread application of this indicator is limited by its biological variation, as it varies according to sex, age, and body mass index. The levels of NT-proBNP may vary also in other pathologies, such as infections and kidney diseases [4].
ST2 is an early marker of myocardial remodeling, and this understudied growth-stimulating factor is expressed on macrovascular (aortic and coronary artery) and microvascular endothelial cells in the heart in humans [5] and on cardiomyocytes in rats and mice [3] when under biomechanical stress [6]; thus, it is a novel and promising marker. ST2 is a member of the family of interleukin (IL)-1 receptors. The main function of ST2, which potentiates IL-33, is to exert antihypertrophic and antifibrosing effects on cardiomyocytes that are under biomechanical stretching conditions [78]. However, an acute increase in the ST2 level has been observed when damage is accompanied by the inhibition of IL-33 and its favorable antihypertrophic effects. Studying the role of ST2 during hospitalization for MI can be helpful for predicting the course of the hospitalization and development of complications [391011]. The aim of this study was to determine the level of soluble ST2 (sST2) and its correlation with the level of NT-proBNP and with the clinical course of MI during hospitalization.
METHODS:
1. Study population For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.
The control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.
The study protocol was approved by the local ethics committee of the Federal State Budgetary Institution "Research Institute for Complex Issues of Cardiovascular Disease" and was developed in accordance with the WMA Declaration of Helsinki "Ethical principles for medical research involving human subjects" (amended in 2000) and "Rules for clinical practice in the Russian Federation" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.
Among all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.
The complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).
Unless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.
For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.
The control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.
The study protocol was approved by the local ethics committee of the Federal State Budgetary Institution "Research Institute for Complex Issues of Cardiovascular Disease" and was developed in accordance with the WMA Declaration of Helsinki "Ethical principles for medical research involving human subjects" (amended in 2000) and "Rules for clinical practice in the Russian Federation" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.
Among all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.
The complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).
Unless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.
2. Assays Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.
Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.
3. Statistical analysis Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.
Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.
1. Study population:
For this study, 88 patients (64 men and 24 women with a median age of 58 [55;64)] yr) with MI between January 2011 and December 2013 were recruited and verified by using the All-Russian Scientific Society of Cardiology (2007) and ESC/ACCF/AHA/WHF [12] diagnostic criteria for the diagnosis of MI, namely, the presence of typical chest pain lasting longer than 20 min, ST-segment elevation of 0.1 mW in two or more contiguous leads, or the appearance of a complete left bundle branch block on an ECG, as well as laboratory findings (elevated CK [creatine phosphokinase], CK-MB, and troponin T levels [>0.1 ng/mL]). The exclusion criteria included previously or newly diagnosed type 2 diabetes at the time of the index event, diagnosis of severe diseases affecting prognosis (including anemia, renal and hepatic failure, cancer, acute infectious and inflammatory diseases), autoimmune diseases, long-term corticosteroid therapy, and death during the hospitalization. The demographic data of patients are presented in Table 1.
The control group included 30 participants without cardiovascular diseases or diabetes, and the members were comparable in age and sex ratio to the enrolled patients.
The study protocol was approved by the local ethics committee of the Federal State Budgetary Institution "Research Institute for Complex Issues of Cardiovascular Disease" and was developed in accordance with the WMA Declaration of Helsinki "Ethical principles for medical research involving human subjects" (amended in 2000) and "Rules for clinical practice in the Russian Federation" approved by the Ministry of Health of the Russian Federation on June 19, 2003. All patients provided written informed consent prior to their participation in the study.
Among all patients included in the study, 34 had a history of hypertension, and 13 had hypercholesterolemia. We identified 18 patients with angina of different functional classes and 4 with acute cerebrovascular accidents. Eleven patients had a family history of coronary artery disease. A total 26 patients were current smokers.
The complications observed during the hospitalization for MI were early postinfarction angina in eight patients (9.1%), rhythm disturbances in four (4.5%), recurrence of MI in six (6.8%), and clinical manifestations of acute heart failure (AHF) (Killip class II-IV) in 22 (25.1%). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. Depending on the concentration of sST2 35 ng/mL, all patients were divided into two groups below (Group 1) or above (Group 2) at this level (Table 2).
Unless contraindicated, all patients received combined coronary active, antithrombotic, and lipid-lowering therapy, including aspirin, clopidogrel, β-blockers, angiotensin-converting-enzyme (ACE) inhibitors, statins, and antianginal medications, during the hospitalization period in accordance with standard clinical practice.
2. Assays:
Serum was separated from venous blood by centrifugation at 3,000g for 20 min and stored at -70℃. sST2 levels were measured with the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA). This assay has a within-run CV < 6.5% and total CV < 9.1% at a mean concentration 16.9 ng/mL. We measured NT-proBNP with the Biomedica kit (Bratislava, Slovakia). The intra-assay CVs were 5 and 8% at a mean concentration of 13 fmol/mL. Troponin T levels were measured with Roche CARDIAC (Roche Diagnostics, Mannheim, Germany). All Roche assays were performed with the use of the Elecsys 2010 system (Roche Diagnostics): Troponin T (fourth generation) with a limit of detection of 0.01 ng/mL, a 99th-percentile cutoff point of less than 0.01 ng/mL, and a CV of less than 10% at 0.035 ng/mL.
3. Statistical analysis:
Statistical analysis was performed by using Statistica 6.1. (StatSoft, Tulsa, OK, USA) and SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Results are presented as the median (Me) and the Me 25 and 75% quartiles (Q1;Q3). Nonparametric tests were used to assess and analyze the data. The Mann-Whitney U test or the Kolmogorov-Smirnov method (more than 50 cases in each group) was used for quantitative comparisons of two independent groups. The Spearman rank correlation coefficient was used to investigate relationships between variables (P<0.05). The value of R (rank correlation coefficient) is 0.3 or less - low rates closeness of the connection; values greater than 0.4 but less than 0.7 - indicators of moderate closeness of the connection, and the values of 0.7 and more - high-performance connection tightness [13]. Stepwise logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CI) was used to determine the prognostic significance of parameters regarding long-term prognosis. Cox regression was used to evaluate the risk of unfavorable events; the impact of independent variables as predictors of risk was determined. A P<0.05 was considered statistically significant.
RESULTS:
On day 1 of hospitalization for MI, the levels of sST2 and NT-proBNP increased by 2.4-fold and 4.5-fold, respectively, compared with the control group [44.75 (24.90; 93.56) ng/mL, P=0.002; 18.81 (15.12; 21.03) ng/mL] [36.84 (24.09; 89.26) fmol/mL, P=0.000; 8.23 (5.61; 11.12) fmol/mL)].
By day 12, the sST2 level significantly decreased by 2.5-fold [17.82 (15.30; 23.25 ng/mL, P=0.001], whereas the changes in the NT-proBNP level were not significant (Fig. 1).
The results of the correlation analyses indicated a moderate correlation between the levels of sST2 and NT-proBNP in both groups on days 1 (R=0.50, P=0.001) and 12 of MI hospitalization (R=0.55, P=0. 0002).
According to experimental data, sST2 in animals is expressed only in cardiomyocytes during myocardial injury; nevertheless, we were interested in the correlation analysis between troponin T (the classic marker of cardiomyocyte damage) and sST2 concentrations during the MI hospitalization period. The results of the correlation analyses indicated a direct correlation between the levels of sST2 and troponin T in both groups at the time of admission (R=0.65, P=0.002).
A comparative analysis of the levels of sST2 and NT-proBNP in both groups (favorable (n=58) and unfavorable (n=30) outcome) was performed, and the results are shown in Table 3. The level of sST2 in the unfavorable outcome group on day 1 was 2-fold higher than that in favorable outcome group. The sST2 levels increased by 1.9- and 3.7-fold in the favorable and unfavorable outcome groups, respectively, compared with those in the control group.
On day 12 after MI onset, the sST2 level was significantly lower (P=0.011) in subjects in both groups compared with the level in the control group.
In contrast to sST2, the level of NT-proBNP increased equally in both the favorable and unfavorable outcome groups up to day 12 of the study. The highest increase in the NT-proBNP level (6.8-fold) was observed in patients in the unfavorable outcome group on day 1.
A level of sST2 higher than 35 ng/mL is considered an indicator of adverse outcome development in cardiovascular pathology [1013]. Thus, in the next stage of our study, we analyzed the differences in the clinical and anamnestic characteristics of the subjects by categorizing the sST2 level as lower (Group 1) or higher (Group 2) than the critical level of 35 ng/mL (Table 2). In Group 2 (sST2 concentration above 35 ng/mL), according to the clinical and anamnestic characteristics, complications [MI (44.6%) and diabetes mellitus (23%)] during hospitalization were more common. In Group 1 (sST2 level lower than 35 ng/mL), the percentage of adverse events and complications during hospitalization was significantly lower (P=0.021).
Conversely, analysis of individual patient data indicated that five patients (15.6%) with a poor prognosis had an sST2 concentration lower than 35 ng/mL, whereas 31 (55.4%) patients with a favorable prognosis had an sST2 level higher than 35 ng/mL.
The results of the evaluation of the NT-proBNP level as a function of sST2 are shown in Table 4. On day 1, in patients with sST2 concentrations above 35 ng/mL, the concentration of NT-proBNP increased by 6.7-fold compared with the controls. At the same time, in patients with a moderate increase in the concentration of sST2 (less than 35 ng/mL), the concentration of NT-proBNP also increased, although to a lesser degree (4.6-fold compared with the controls). At day 12, the level of NT-proBNP remained elevated in both groups and did not differ significantly between the two (Table 4).
Logistic regression analysis showed that an increase in the concentration of sST2 increased the risk of complications during the hospitalization by 1.7-fold times (OR, 1.7; 95% CI, 1.6-2.8; areas under curve [AUC]=0.78; P=0.003), with sensitivity of 76.9% and a specificity of 69.4%. At the same time, the increase in the level of NT-proBNP was accompanied only by a 1.2-fold increase in adverse outcomes (OR, 1.2; 95% CI, 1.1-1.6; AUC=0.69; P=0.034), without affecting the high diagnostic sensitivity (69.6%) and specificity (65.3%).
Determining the level of sST2 in combination with NT-proBNP increases their diagnostic significance (OR, 1.92; 95% CI, 1.7-3.2). These indicators, when used together and measured at the early stages of MI, increased the quality of the model, with sensitivity of 81%, specificity of 72%, and AUC of 0.86.
DISCUSSION:
MI is accompanied by the mechanical deformation of cardiomyocytes, which may undergo adaptive and maladaptive changes, leading to chronic heart failure [1]. In response to increased wall tension of the heart ventricles, the intracardiac pressure increases, resulting in increased cardiomyocyte volume and the synthesis of factors such as NT-proBNP and sST2, which function to increase myocardial performance [3].
Our study showed that on day 1 after MI, the NT-proBNP concentration increased 4.5-fold (Fig. 1) and remained elevated up to day 12 of the study. It was shown in BNP transgenic mice that following artificially induced MI, the infarcted area increased by about 5-fold during the 48 hr following MI onset and remained at an increased level over the next three to four weeks [1415]. When the disease course was unfavorable, the NT-proBNP concentration increased even more, to 6.8-fold compared with the control values. In addition to the mechanical stretching of the ventricles, there may be other mechanisms that stimulate the production of NT-proBNP, such as ischemia in various locations, arrhythmias, myocardial hypertrophy, and endothelial dysfunction [16]. However, in this study, the NT-proBNP level had a low diagnostic sensitivity and specificity as an indicator of an unfavorable course following MI, with its increase indicating only a 1.2-fold increase in the risk for complications.
The stimulating growth factor sST2 had a higher sensitivity for the development of a poor prognosis. With its increase on day 1, there was a 1.7-fold increase in the risk of an unfavorable outcome following MI. Compared with the controls, the sST2 level increased by 1.9-fold in cases with a favorable course of MI and increased 3.7-fold in cases of unfavorable outcomes (Table 2). Elevated levels of sST2 have been associated with an increase in the synthesis of sST2 in cardiac myocytes and fibroblasts due to biomechanical stress [31718].
ST2 is a member of the superfamily of IL-1 receptors. It exists in two forms: a transmembrane receptor (ST2L) and soluble receptor-trap (sST2). The ST2 ligand is IL-33, which contributes to the process reduction of the fibrosis and hypertrophy of tissues under mechanical loads [19]. sST2 acts as a decoy receptor by binding free IL-33 and preventing its signaling through ST2L [2021]. A moderate increase in the concentration of sST2 probably exerts a protective effect, which manifests itself in patients with a favorable course of MI [39]. The transmembrane form protects the myocardium from overcharging, whereas the soluble form of ST2 prevents this protective mechanism, binds to IL-33, and blocks its cardioprotective effect [320]. Perhaps the increase in the concentration of sST2 in cases of unfavorable outcomes is associated with an increased content of the soluble form of the marker following release by damaged cardiomyocytes.
Both sST2 and troponin levels have been measured in several other studies. In the study by Mueller et al. [22] in 2015, in contrast to our study, no correlation between sST2 and troponin levels was found. This difference can be explained by the fact that we measured biomarkers in patients in the acute phase of MI, whereas the patients in the study by Mueller et al. [22] had heart failure.
In the present study, by day 12 following MI, sST2 concentrations decreased to the level of control values, thus making it difficult to distinguish between the favorable and unfavorable outcome groups. The dynamics of the sST2 changes are somewhat similar to those of C-reactive protein (CRP). CRP levels increased acutely during acute MI; however, this marker tended to decrease by day 12 after MI onset [23]. This similarity indicates their common inflammatory nature. These results are consistent with those reported in the study by Weinberg et al. [24], which was performed on an experimental MI model (in vivo) by using C57/BL6J mice. After ligating the coronary artery, the maximal transcriptional induction of sST2 in cardiac myocytes occurred within 2 hr, was maintained for 9 hr, and decreased after 15 hr.
Studies have shown that the sST2 threshold in patients with chronic heart failure is 35 ng/mL. Above this value, the risk of death increases dramatically within one year of the event [91018]. Kohli et al. [10] reported similar results, showing that high levels of sST2 (>35 ng/mL) in patients suffering from acute coronary syndrome predicted a 3-fold higher risk of cardiovascular death and heart failure within 30 days and one year. It is of particular interest to study such patterns in a cohort of patients with MI. In our study, the adverse outcomes during the early period of MI were not associated with sST2 levels above 35 ng/mL, as there were complications in patients with levels below (15.5%) and above (55.4%) the threshold. These results were probably affected not only by the level of sST2 but also by the presence of other factors, specifically the increase in NT-proBNP. Indeed, the use of these two markers together significantly increases their sensitivity and specificity of predicting the risk of unfavorable MI outcomes [25].
Our findings are consistent with the study by Sabatine et al. [18], in which unfavorable outcomes were observed with high levels of both markers (risk of death or developing heart failure was 6.5-fold higher) during the 30-day observation period. Using the levels of NT-proBNP and sST2 to construct the receiver operating characteristics curve, the identification of combined cardiovascular mortality or heart failure significantly improved to 0.78 (95% CI, 0.74-0.83; P=0.0025).
In conclusion, the concentration of sST2 is a more sensitive indicator of the course of hospitalization for MI than the traditional indicator of the NT-proBNP concentration. Increased concentrations of sST2 on day 1 after MI were followed by an unfavorable hospitalization course, including progressive angina, arrhythmias, MI, and recurrent symptomatic AHF (Killip class II-IV).
|
Background:
Studying the role of soluble ST2 (sST2) during hospitalization for myocardial infarction (MI) can be helpful for predicting the course of the hospitalization and development of complications.
Methods:
We included 88 patients with MI (median age, 58 yr). Depending on the course of the hospitalization, the patients were divided into two groups: the favorable (n=58) and unfavorable (n=30) outcome groups. On days 1 and 12 after MI, serum sST2 and N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured by ELISA.
Results:
On day 1, the concentrations of sST2 and NT-proBNP increased 2.4- and 4.5-fold, compared with the controls. Measurements on day 12 showed a significant decrease in the sST2 level (P=0.001), whereas the NT-proBNP level did not change. On day 1, the sST2 level in the unfavorable outcome group was 2-fold higher than that in the favorable outcome group and 3.7-fold higher than in the controls. On day 12, the marker level decreased in both groups. On day 1, the NT-proBNP level in the unfavorable outcome group was 6.8-fold higher than in the controls and 1.8-fold higher than in the favorable outcome group. On day 12, the level of NT-proBNP remained elevated in both groups. Determining the levels of both sST2 and NT-proBNP increases their diagnostic significance (odds ratio [OR], 1.92; 95% confidence interval [CI], 1.7-3.2; areas under curve [AUC] 0.89; P=0.004).
Conclusions:
The level of sST2 is a more sensitive indicator during MI hospitalization than NT-proBNP.
| null | null | 5,484 | 329 | 7 |
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[CONTENT] Myocardial infarction | NT-proBNP | sST2 [SUMMARY]
|
[CONTENT] Myocardial infarction | NT-proBNP | sST2 [SUMMARY]
|
[CONTENT] Myocardial infarction | NT-proBNP | sST2 [SUMMARY]
| null |
[CONTENT] Myocardial infarction | NT-proBNP | sST2 [SUMMARY]
| null |
[CONTENT] Area Under Curve | Case-Control Studies | Electrocardiography | Enzyme-Linked Immunosorbent Assay | Female | Hospitalization | Humans | Logistic Models | Male | Middle Aged | Myocardial Infarction | Natriuretic Peptide, Brain | Odds Ratio | Peptide Fragments | Prognosis | Proportional Hazards Models | ROC Curve | Receptors, Somatostatin [SUMMARY]
|
[CONTENT] Area Under Curve | Case-Control Studies | Electrocardiography | Enzyme-Linked Immunosorbent Assay | Female | Hospitalization | Humans | Logistic Models | Male | Middle Aged | Myocardial Infarction | Natriuretic Peptide, Brain | Odds Ratio | Peptide Fragments | Prognosis | Proportional Hazards Models | ROC Curve | Receptors, Somatostatin [SUMMARY]
|
[CONTENT] Area Under Curve | Case-Control Studies | Electrocardiography | Enzyme-Linked Immunosorbent Assay | Female | Hospitalization | Humans | Logistic Models | Male | Middle Aged | Myocardial Infarction | Natriuretic Peptide, Brain | Odds Ratio | Peptide Fragments | Prognosis | Proportional Hazards Models | ROC Curve | Receptors, Somatostatin [SUMMARY]
| null |
[CONTENT] Area Under Curve | Case-Control Studies | Electrocardiography | Enzyme-Linked Immunosorbent Assay | Female | Hospitalization | Humans | Logistic Models | Male | Middle Aged | Myocardial Infarction | Natriuretic Peptide, Brain | Odds Ratio | Peptide Fragments | Prognosis | Proportional Hazards Models | ROC Curve | Receptors, Somatostatin [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] sst2 | patients | mi | ml | ng | ng ml | study | level | nt | nt probnp [SUMMARY]
|
[CONTENT] sst2 | patients | mi | ml | ng | ng ml | study | level | nt | nt probnp [SUMMARY]
|
[CONTENT] sst2 | patients | mi | ml | ng | ng ml | study | level | nt | nt probnp [SUMMARY]
| null |
[CONTENT] sst2 | patients | mi | ml | ng | ng ml | study | level | nt | nt probnp [SUMMARY]
| null |
[CONTENT] st2 | stretching | level | cardiomyocytes | function | effects | mass | remodeling | antihypertrophic | il [SUMMARY]
|
[CONTENT] patients | ml | roche | ng | ng ml | diseases | russian | connection | diagnostics | included [SUMMARY]
|
[CONTENT] sst2 | level | fold | ml | day | nt probnp | probnp | nt | group | ng ml [SUMMARY]
| null |
[CONTENT] patients | sst2 | ml | mi | ng ml | ng | level | nt probnp | nt | probnp [SUMMARY]
| null |
[CONTENT] [SUMMARY]
|
[CONTENT] 88 | 58 ||| two | n=58 ||| days 1 and 12 | ELISA [SUMMARY]
|
[CONTENT] day 1 | 2.4- | 4.5-fold ||| day 12 ||| day 1 | 2-fold | 3.7-fold ||| day 12 ||| day 1 | 6.8-fold | 1.8-fold ||| day 12 | NT-proBNP ||| ||| 1.92 | 95% ||| CI | 1.7 [SUMMARY]
| null |
[CONTENT] ||| 88 | 58 ||| two | n=58 ||| days 1 and 12 | ELISA ||| ||| day 1 | 2.4- | 4.5-fold ||| day 12 ||| day 1 | 2-fold | 3.7-fold ||| day 12 ||| day 1 | 6.8-fold | 1.8-fold ||| day 12 | NT-proBNP ||| ||| 1.92 | 95% ||| CI | 1.7 ||| [SUMMARY]
| null |
||||
Safety of combination antiretroviral prophylaxis in high-risk HIV-exposed newborns: a retrospective review of the Canadian experience.
|
26880241
|
The optimal management of infants born to HIV-positive mothers who are untreated or have detectable viral load prior to delivery remains controversial. Despite the increasing use of combination antiretroviral therapy (cART) for post-exposure prophylaxis (PEP) of neonates at high risk of HIV infection, there is little safety and pharmacokinetic data to support this approach. The objective of this study was to evaluate the safety and tolerability of cART for PEP in HIV-exposed neonates.
|
INTRODUCTION
|
Retrospective study on 148 cART and 145 Zidovudine (ZDV) monotherapy-exposed infants identified from four Canadian centres where cART for PEP has routinely been prescribed in high-risk situations. Physician-reported adverse events and clinical outcomes were extracted by chart review. Haematological and growth parameters at birth, one and six months of age were compared between cART and ZDV-exposed infants using multivariate mixed effects modelling.
|
METHODS
|
Non-specific signs and symptoms were reported in 10.2% of cART recipients versus none of the ZDV recipients. Treatment was discontinued prematurely in 9.5% of cART recipients versus 2.1% of ZDV recipients (p=0.01). In the multivariate model, cART recipients had lower mean haemoglobin (decrease of 2.07 g/L) over the 6-month period compared with ZDV recipients (p=0.04), but no effect was seen on absolute neutrophil count. cART recipients had lower weight and smaller head circumference at birth and one month of age compared with ZDV-exposed infants; these differences were no longer significant at six months of age.
|
RESULTS
|
cART administered at treatment doses for PEP in neonates was generally well tolerated, though a higher incidence of non-specific signs and symptoms and early treatment discontinuation occurred among cART recipients.
|
CONCLUSIONS
|
[
"Adult",
"Anti-HIV Agents",
"Canada",
"Drug Therapy, Combination",
"Female",
"HIV Infections",
"Humans",
"Infant, Newborn",
"Infectious Disease Transmission, Vertical",
"Male",
"Pregnancy",
"Retrospective Studies"
] |
4753845
|
Introduction
|
While the overall risk of perinatal HIV transmission in the developed world has decreased to less than 1%, transmission still occurs, most often due to failure to diagnose HIV in pregnant women in a timely manner, or because of poor maternal drug adherence to, and/or refusal of, antiretroviral therapy [1]. Despite the relatively high risk of transmission in these situations, the optimal management of infants born to HIV-positive mothers, who are untreated or have detectable viral load prior to delivery despite receiving antenatal therapy, remains controversial. Current US Department of Health and Human Services (DHHS) guidelines recommend a six-week course of Zidovudine (ZDV), along with three doses of Nevirapine (NVP) in the first week of life for infants born to mothers who have not received any treatment [2]. However, in practice, many paediatric HIV specialists prescribe various combinations of three or more drugs to such “high-risk” neonates. In a national web-based survey of US care providers between December 2009 and January 2010, 62% reported the use of combination antiretroviral therapy (cART) during the previous year [3]. In a recent study from the United Kingdom, cART was prescribed to 71% of infants born to untreated mothers [4].
Despite the increasing use of cART for prophylaxis in neonates at high risk of HIV infection, there is limited data on the safety of these different combination regimens, or on the appropriate dosing for the antiretroviral (ARV) medications used for this purpose. Transient haematological toxicity has been reported with the use of six-week post-natal ZDV [5], the recommended regimen in standard-risk situations, and a number of studies have suggested increased haematological toxicity associated with the addition of a second nucleoside analogue [6, 7]. Neither the safety nor the optimal dosing of additional drugs used for prophylaxis in neonates (non-nucleoside reverse transcriptase inhibitors or protease inhibitors (PIs)) has been well defined [8–13]. Furthermore, there is emerging concern about the long-term impact of increased ARV exposure on the growth of exposed infants [14, 15].
In four Canadian centres, triple cART at treatment doses has been routinely prescribed for the post-exposure prophylaxis (PEP) of neonates deemed at high risk of HIV infection, either because of detectable maternal viral load near delivery or in the absence of viral load results, clinical factors such as poor adherence or late diagnosis. In standard-risk situations, six weeks of ZDV monotherapy is prescribed. Given the limited data to support the use of cART, the primary objective of this study was to compare the safety and tolerability of cART versus ZDV monotherapy of PEP with respect to adverse events, haematological and growth parameters in exposed neonates.
|
Methods
|
HIV-1 exposed children were eligible for this study if they were started on treatment doses of cART within 72 hours of birth because of incomplete maternal virologic suppression at delivery or, in the absence of maternal viral load results, a maternal history of incomplete adherence or non-adherence to cART, or late pregnancy initiation of cART. Neonatal cART was defined as any regimen of three or more antiretroviral agents, prescribed at treatment doses, for a minimum of six weeks. Regimens included ZDV (2 mg/kg every six hours prior to 2011, and 4 mg/kg twice daily in 2011–2013) for six weeks, Lamivudine (3TC) (2 mg/kg twice daily) for six weeks, with the addition of either NVP (150 mg/m2 daily for 14 days, followed by 150 mg/m2 twice daily for 14 days) or Nelfinavir (NFV) 40–50 mg/kg twice daily for six weeks) or LPV/Ritonavir (LPV/r) (300 mg/m2 twice daily) for six weeks. Combination regimens containing single or three doses of NVP were excluded.
Subjects were identified for the period 1997–2013 from the clinical databases of four paediatric HIV-care institutions in Canada: The Hospital for Sick Children, Toronto; Children's Hospital of Eastern Ontario, Ottawa; Centre Hospitalier Universitaire (CHU) Sainte-Justine, Montreal; Stollery Children's Hospital, Edmonton. A control group consisting of all infants of HIV-positive mothers who were prescribed ZDV monotherapy during a three-year period (2010–2012) at the Hospital for Sick Children was selected (n=148). Approval for the study was obtained by the ethics review board of each participating institution; given the retrospective nature of the study, individual patient consent was not deemed necessary by the respective ethics review boards for the chart review.
Statistical analysis Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.
Multivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).
Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.
Multivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).
|
Results
|
One hundred and forty-eight infants received triple cART during the study period. NFV-based cART was the most common regimen (55%) followed by NVP-based cART (40%) and LPV/r-based cART (5%). The most common reason for prescribing cART was documented detectable maternal viral load (78.4%). Other overlapping factors included poor adherence (45%), late diagnosis (20%), no antenatal care (8%) and refusal of antenatal ARV therapy (8%). Fifteen of the cART-treated infants and five of the ZDV-treated infants had missing six-month data, either because they were lost to follow-up (n=8) or they did not attend the scheduled six-month visit (but were seen at 18 months of age or older) (n=12).
Baseline maternal and infant characteristics according to treatment group are described in Table 1. Mothers of cART-treated infants were younger, had higher viral loads and lower CD4 counts at the time of delivery, and were more likely to deliver vaginally and prematurely than mothers of ZDV monotherapy-treated infants.
Baseline demographic and clinical characteristics of mothers and infantsa
Reported means and standard deviation;
Chi Square test of proportions or Wilcoxon rank-sum.
Hematological and Growth Parameters by treatment group†
Reported means and standard deviation
Student T test, Wilcoxon rank sum, or Kruskal-Wallace test where appropriate.
n* observations recorded for each parameter out of a total of 148 cART recipients at birth, and 145 ZDV recipients.
Thirteen of the cART recipients were perinatally infected with HIV (8.8%). None of the ZDV recipients were infected. Five of the 13 (38.5%) had HIV-1 detected by PCR within 48 hours of birth suggesting in utero infection according to diagnostic standards. For the remaining eight, the timing of infection could not be ascertained as initial testing took place after 48 hours of life. Of the 13 infected children, four achieved long-term sustained virologic suppression, the details of which have been described elsewhere [16].
Non-specific signs and symptoms, including vomiting, diarrhoea, rash, jitteriness or irritability, potentially attributable to medication-related adverse effects, were reported in 10.2% of cART recipients and none of the ZDV recipients (p<0.001). ARV treatment was discontinued prematurely in 9.5% (n=14) of cART recipients (five NVP-treated and nine PI-treated infants) compared with 2.1% (n=3) ZDV recipients (p=0.01). Reasons given for premature treatment discontinuation among cART recipients included significant anaemia (n=3; haemoglobin at two, four and four weeks of 62, 76 and 92 g/L, respectively), neutropenia (n=1; neutrophil count 0.4 cells/mm3 at four weeks), non-specific symptoms including rash, vomiting, and diarrhoea (n=6), and parental decision (n=4). In the six cases of non-specific symptoms, treatment discontinuation was precautionary, as the treating physician indicated that the symptoms were most likely not medication related. In four cases of parental discontinuation, parents stopped the medication without evidence of toxicity or consultation with their healthcare provider. ZDV monotherapy was stopped early in three children, all because of significant anaemia (haemoglobin at four weeks of 76, 85 and 72 g/L).
The potential impact of cART on haematologic parameters was evaluated by comparing haemoglobin level and absolute neutrophil count for cART-treated and ZDV monotherapy-treated infants at one, two and six months of age (Table 2). In the unadjusted analysis, haemoglobin was lower in cART-treated infants compared with ZDV-treated infants at one month of age (mean 105±19 vs. 109±17 g/L, p=0.04), but not at two or six months of age (mean 104±12 vs. 105±9 g/L, p=0.31 and 118±11 vs. 121±8 g/L, p=0.15). In a subgroup analysis of specific treatment type, mean haemoglobin was lower among PI-treated infants compared with NVP-treated infants at one month of age (104±17 vs. 107±21, p=0.09), though only statistically significant at six months of age (mean 115±10 vs. 124±9 g/L, p<0.001) (Figure 1).
Haematological parameters according to treatment group.
Graphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.)
To study the overall effect of treatment type on haemoglobin over time, we constructed a multivariate model adjusting for chronological age and gestational age (Supplementary Table, not shown). Overall, the mean haemoglobin was 2.07 g/L lower among cART versus ZDV-treated infants (p=0.04) over the six-month period. In the subgroup analysis looking at specific treatment type, mean haemoglobin was 3.62 g/L lower in those receiving PI-based cART compared with those receiving ZDV monotherapy (p=0.002); no such effect was observed for NVP-based cART compared with ZDV monotherapy (p=0.82). There was no difference in absolute neutrophil count between NVP-based cART, PI-based cART and ZDV monotherapy-treated infants at one, two or six months of age in the unadjusted analysis; this finding was maintained in the multivariable model adjusting for visit number, ethnicity and gestational age.
Weight, length and HC were compared between all cART-treated and ZDV-treated infants, and between PI-based cART, NVP-based cART and ZDV at birth, one and six months of age (Table 2 and Figure 2). In the unadjusted analysis, cART-treated infants were smaller compared with ZDV-treated infants at birth (weight 2.91±0.60 vs. 3.09±0.56 kg, p=0.01; length 49.0±3.6 vs. 50.2±3.8 cm, p=0.03, head circumference 33.2±2.4 vs.33.6±2.3 cm, p=0.16). By six months of age, there was no difference in weight (8.03±1.12 kg vs. 8.25±1.12 kg, p=0.14) or head circumference (43.7±1.8 cm vs. 44.0±1.7, p=0.13), although the cART-treated group remained shorter than ZDV-treated infants (67.1±2.9 cm vs. 68.1±3.2 cm, p=0.01). In the subgroup analysis looking at specific treatment type, both NVP-and PI-treated infants were smaller than ZDV-treated infants across all growth parameters at birth; only the difference in length remained significant at six months of age.
Growth parameters according to treatment group.
Graphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.)
To study the overall effect of treatment type on growth parameters over time, we constructed a multivariate fixed effects model adjusting for birthweight, gestational age, chronological age and gender. Overall, there was a mean HC difference of −0.63 cm among cART compared with ZDV-treated infants over the six-month period (p<0.001) (Supplementary Table, not shown). A similar effect was seen with respect to weight and length, with a mean weight difference of −236 g and length difference of −0.92 cm over the six-month period (p<0.001 and p=0.002, respectively). In the subgroup multivariate analysis comparing PI-based cART versus ZDV and NVP-based cART versus ZDV, there was no discernable difference between the two cART treatment types and these growth parameters.
|
Conclusions
|
In summary, our study shows that cART at treatment doses administered as prophylaxis to “high-risk” HIV-exposed neonates is generally well tolerated and should be considered for newborn infants deemed at high risk of perinatally acquired HIV infection. However, close monitoring for anaemia, neutropenia and other side effects is important if such therapy is given. Until more robust pharmacokinetic data are available, the use of cART as prophylaxis in neonates may be limited to resource-rich settings where routine therapeutic drug monitoring is possible. However, further study is urgently needed in resource-limited settings where the vast majority of high-risk HIV-exposed infants are born. The potential benefits of cART, both to avert infection and, in case of transmission, preserve future therapeutic options and potentially limit the viral reservoir, may well outweigh the risks. Finally, if this strategy is used, counselling of parents and close follow-up is necessary to avoid premature treatment discontinuation and to ensure proper monitoring of cART-exposed infants.
|
[
"Statistical analysis"
] |
[
"Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA)."
] |
[
null
] |
[
"Introduction",
"Methods",
"Statistical analysis",
"Results",
"Discussion",
"Conclusions",
"Supplementary Material"
] |
[
"While the overall risk of perinatal HIV transmission in the developed world has decreased to less than 1%, transmission still occurs, most often due to failure to diagnose HIV in pregnant women in a timely manner, or because of poor maternal drug adherence to, and/or refusal of, antiretroviral therapy [1]. Despite the relatively high risk of transmission in these situations, the optimal management of infants born to HIV-positive mothers, who are untreated or have detectable viral load prior to delivery despite receiving antenatal therapy, remains controversial. Current US Department of Health and Human Services (DHHS) guidelines recommend a six-week course of Zidovudine (ZDV), along with three doses of Nevirapine (NVP) in the first week of life for infants born to mothers who have not received any treatment [2]. However, in practice, many paediatric HIV specialists prescribe various combinations of three or more drugs to such “high-risk” neonates. In a national web-based survey of US care providers between December 2009 and January 2010, 62% reported the use of combination antiretroviral therapy (cART) during the previous year [3]. In a recent study from the United Kingdom, cART was prescribed to 71% of infants born to untreated mothers [4].\nDespite the increasing use of cART for prophylaxis in neonates at high risk of HIV infection, there is limited data on the safety of these different combination regimens, or on the appropriate dosing for the antiretroviral (ARV) medications used for this purpose. Transient haematological toxicity has been reported with the use of six-week post-natal ZDV [5], the recommended regimen in standard-risk situations, and a number of studies have suggested increased haematological toxicity associated with the addition of a second nucleoside analogue [6, 7]. Neither the safety nor the optimal dosing of additional drugs used for prophylaxis in neonates (non-nucleoside reverse transcriptase inhibitors or protease inhibitors (PIs)) has been well defined [8–13]. Furthermore, there is emerging concern about the long-term impact of increased ARV exposure on the growth of exposed infants [14, 15].\nIn four Canadian centres, triple cART at treatment doses has been routinely prescribed for the post-exposure prophylaxis (PEP) of neonates deemed at high risk of HIV infection, either because of detectable maternal viral load near delivery or in the absence of viral load results, clinical factors such as poor adherence or late diagnosis. In standard-risk situations, six weeks of ZDV monotherapy is prescribed. Given the limited data to support the use of cART, the primary objective of this study was to compare the safety and tolerability of cART versus ZDV monotherapy of PEP with respect to adverse events, haematological and growth parameters in exposed neonates.",
"HIV-1 exposed children were eligible for this study if they were started on treatment doses of cART within 72 hours of birth because of incomplete maternal virologic suppression at delivery or, in the absence of maternal viral load results, a maternal history of incomplete adherence or non-adherence to cART, or late pregnancy initiation of cART. Neonatal cART was defined as any regimen of three or more antiretroviral agents, prescribed at treatment doses, for a minimum of six weeks. Regimens included ZDV (2 mg/kg every six hours prior to 2011, and 4 mg/kg twice daily in 2011–2013) for six weeks, Lamivudine (3TC) (2 mg/kg twice daily) for six weeks, with the addition of either NVP (150 mg/m2 daily for 14 days, followed by 150 mg/m2 twice daily for 14 days) or Nelfinavir (NFV) 40–50 mg/kg twice daily for six weeks) or LPV/Ritonavir (LPV/r) (300 mg/m2 twice daily) for six weeks. Combination regimens containing single or three doses of NVP were excluded.\nSubjects were identified for the period 1997–2013 from the clinical databases of four paediatric HIV-care institutions in Canada: The Hospital for Sick Children, Toronto; Children's Hospital of Eastern Ontario, Ottawa; Centre Hospitalier Universitaire (CHU) Sainte-Justine, Montreal; Stollery Children's Hospital, Edmonton. A control group consisting of all infants of HIV-positive mothers who were prescribed ZDV monotherapy during a three-year period (2010–2012) at the Hospital for Sick Children was selected (n=148). Approval for the study was obtained by the ethics review board of each participating institution; given the retrospective nature of the study, individual patient consent was not deemed necessary by the respective ethics review boards for the chart review.\n Statistical analysis Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).\nBaseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).",
"Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.\nMultivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).",
"One hundred and forty-eight infants received triple cART during the study period. NFV-based cART was the most common regimen (55%) followed by NVP-based cART (40%) and LPV/r-based cART (5%). The most common reason for prescribing cART was documented detectable maternal viral load (78.4%). Other overlapping factors included poor adherence (45%), late diagnosis (20%), no antenatal care (8%) and refusal of antenatal ARV therapy (8%). Fifteen of the cART-treated infants and five of the ZDV-treated infants had missing six-month data, either because they were lost to follow-up (n=8) or they did not attend the scheduled six-month visit (but were seen at 18 months of age or older) (n=12).\nBaseline maternal and infant characteristics according to treatment group are described in Table 1. Mothers of cART-treated infants were younger, had higher viral loads and lower CD4 counts at the time of delivery, and were more likely to deliver vaginally and prematurely than mothers of ZDV monotherapy-treated infants.\nBaseline demographic and clinical characteristics of mothers and infantsa\n\nReported means and standard deviation;\nChi Square test of proportions or Wilcoxon rank-sum.\nHematological and Growth Parameters by treatment group†\nReported means and standard deviation\nStudent T test, Wilcoxon rank sum, or Kruskal-Wallace test where appropriate.\nn* observations recorded for each parameter out of a total of 148 cART recipients at birth, and 145 ZDV recipients.\nThirteen of the cART recipients were perinatally infected with HIV (8.8%). None of the ZDV recipients were infected. Five of the 13 (38.5%) had HIV-1 detected by PCR within 48 hours of birth suggesting in utero infection according to diagnostic standards. For the remaining eight, the timing of infection could not be ascertained as initial testing took place after 48 hours of life. Of the 13 infected children, four achieved long-term sustained virologic suppression, the details of which have been described elsewhere [16].\nNon-specific signs and symptoms, including vomiting, diarrhoea, rash, jitteriness or irritability, potentially attributable to medication-related adverse effects, were reported in 10.2% of cART recipients and none of the ZDV recipients (p<0.001). ARV treatment was discontinued prematurely in 9.5% (n=14) of cART recipients (five NVP-treated and nine PI-treated infants) compared with 2.1% (n=3) ZDV recipients (p=0.01). Reasons given for premature treatment discontinuation among cART recipients included significant anaemia (n=3; haemoglobin at two, four and four weeks of 62, 76 and 92 g/L, respectively), neutropenia (n=1; neutrophil count 0.4 cells/mm3 at four weeks), non-specific symptoms including rash, vomiting, and diarrhoea (n=6), and parental decision (n=4). In the six cases of non-specific symptoms, treatment discontinuation was precautionary, as the treating physician indicated that the symptoms were most likely not medication related. In four cases of parental discontinuation, parents stopped the medication without evidence of toxicity or consultation with their healthcare provider. ZDV monotherapy was stopped early in three children, all because of significant anaemia (haemoglobin at four weeks of 76, 85 and 72 g/L).\nThe potential impact of cART on haematologic parameters was evaluated by comparing haemoglobin level and absolute neutrophil count for cART-treated and ZDV monotherapy-treated infants at one, two and six months of age (Table 2). In the unadjusted analysis, haemoglobin was lower in cART-treated infants compared with ZDV-treated infants at one month of age (mean 105±19 vs. 109±17 g/L, p=0.04), but not at two or six months of age (mean 104±12 vs. 105±9 g/L, p=0.31 and 118±11 vs. 121±8 g/L, p=0.15). In a subgroup analysis of specific treatment type, mean haemoglobin was lower among PI-treated infants compared with NVP-treated infants at one month of age (104±17 vs. 107±21, p=0.09), though only statistically significant at six months of age (mean 115±10 vs. 124±9 g/L, p<0.001) (Figure 1).\nHaematological parameters according to treatment group.\nGraphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.) \nTo study the overall effect of treatment type on haemoglobin over time, we constructed a multivariate model adjusting for chronological age and gestational age (Supplementary Table, not shown). Overall, the mean haemoglobin was 2.07 g/L lower among cART versus ZDV-treated infants (p=0.04) over the six-month period. In the subgroup analysis looking at specific treatment type, mean haemoglobin was 3.62 g/L lower in those receiving PI-based cART compared with those receiving ZDV monotherapy (p=0.002); no such effect was observed for NVP-based cART compared with ZDV monotherapy (p=0.82). There was no difference in absolute neutrophil count between NVP-based cART, PI-based cART and ZDV monotherapy-treated infants at one, two or six months of age in the unadjusted analysis; this finding was maintained in the multivariable model adjusting for visit number, ethnicity and gestational age.\nWeight, length and HC were compared between all cART-treated and ZDV-treated infants, and between PI-based cART, NVP-based cART and ZDV at birth, one and six months of age (Table 2 and Figure 2). In the unadjusted analysis, cART-treated infants were smaller compared with ZDV-treated infants at birth (weight 2.91±0.60 vs. 3.09±0.56 kg, p=0.01; length 49.0±3.6 vs. 50.2±3.8 cm, p=0.03, head circumference 33.2±2.4 vs.33.6±2.3 cm, p=0.16). By six months of age, there was no difference in weight (8.03±1.12 kg vs. 8.25±1.12 kg, p=0.14) or head circumference (43.7±1.8 cm vs. 44.0±1.7, p=0.13), although the cART-treated group remained shorter than ZDV-treated infants (67.1±2.9 cm vs. 68.1±3.2 cm, p=0.01). In the subgroup analysis looking at specific treatment type, both NVP-and PI-treated infants were smaller than ZDV-treated infants across all growth parameters at birth; only the difference in length remained significant at six months of age.\nGrowth parameters according to treatment group.\nGraphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.) \nTo study the overall effect of treatment type on growth parameters over time, we constructed a multivariate fixed effects model adjusting for birthweight, gestational age, chronological age and gender. Overall, there was a mean HC difference of −0.63 cm among cART compared with ZDV-treated infants over the six-month period (p<0.001) (Supplementary Table, not shown). A similar effect was seen with respect to weight and length, with a mean weight difference of −236 g and length difference of −0.92 cm over the six-month period (p<0.001 and p=0.002, respectively). In the subgroup multivariate analysis comparing PI-based cART versus ZDV and NVP-based cART versus ZDV, there was no discernable difference between the two cART treatment types and these growth parameters.",
"In this retrospective study, cART initiated empirically for newborns deemed at high risk for perinatal HIV infection was generally well-tolerated, though adverse effects and consequent premature discontinuation of therapy did occur more often in the cART versus ZDV-treated group (9.5% vs. 2.1%). Among the 14 cART-treated children in whom medications were prematurely discontinued, four had their medications stopped by the parents without consultation with the healthcare provider and in the absence of any side effects, and six for precautionary reasons by the clinician even though the symptoms were not thought to be medication related, highlighting a lower threshold for treatment discontinuation by both parents and physicians among cART-treated infants.\nWith respect to haematological toxicity, the frequency and degree of anaemia observed in our cART-treated neonates was higher than that observed in the ZDV-monotherapy neonates. In the subgroup analysis of cART-treated infants, haemoglobin was lower at one month of age among PI versus NVP-treated infants, suggesting perhaps less haematological toxicity with NVP-based cART. The lower mean haemoglobin observed in cART-treated subjects compared with ZDV-monotherapy-treated subjects at one month of age was no longer evident at two and six months of age after stopping treatment, confirming the reversible nature of the anaemia. Reassuringly, compared with ZDV monotherapy, the use of cART was not associated with any discernible change in neutrophil count.\nWhile in the multivariate mixed effects model cART-treated infants had lower mean growth measures (HC, weight and length) than ZDV-monotherapy-treated infants over time, there were no absolute differences in growth measures for head circumference or weight at six months of age, indicating good catch-up growth among the cART-treated infants. The observed lower mean growth over time in cART recipients was therefore likely due to factors associated with the indications for the use of cART. Specifically, the higher overall incidence of prematurity among cART-treated infants was likely a result of poorly controlled maternal HIV, an established risk factor for low birthweight and preterm delivery [17]. The higher viral load and lower CD4 counts of mothers of cART recipients in our study supports this hypothesis. While we adjusted for preterm delivery and gestational age in the multivariate models for growth, we were unable to adjust for in utero drug exposure, social situation, maternal health, nutritional status and education, which may be important predictors of infant growth and development.\nBecause of the selective use of cART for infants at higher risk of perinatal transmission in our study, it is not appropriate to compare the risk of transmission among cART versus ZDV-treated infants as the latter group would have received better antenatal preventive management. The 8.8% overall transmission rate observed among cART recipients in our cohort was similar to that in a previous trial in which neonates of untreated mothers were randomized to different cART regimens, evidence of the high risk of transmission despite prophylaxis in these situations [6]. Key potential benefits of cART at treatment doses initiated as prophylaxis for neonates later found to be infected is that such treatment will preserve health by controlling viral replication more rapidly, reduce the risk of generating antiretroviral medication resistance, as seen with single or multiple dose NVP in non-cART strategies, and limit the size of HIV viral reservoirs, which could have significant implications for achieving HIV remission in the future [18–20]. Given the limited number of therapeutic drug regimens available for children in resource-limited settings, preventing drug resistance to first-line therapy given to prevent perinatal transmission is a particularly important consideration.\nOur study has a number of limitations, including its retrospective nature, relatively small sample size, and potential differences in the patient population at different study sites which may have influenced biological and growth outcomes. All control group infants were selected from a single centre, and the choice of PI-based versus NVP-based cART was also site specific. Given that these were the comparison groups of interest, we could not adjust for site and treatment type in the final analysis, thereby limiting our conclusions on specific cART treatment type.",
"In summary, our study shows that cART at treatment doses administered as prophylaxis to “high-risk” HIV-exposed neonates is generally well tolerated and should be considered for newborn infants deemed at high risk of perinatally acquired HIV infection. However, close monitoring for anaemia, neutropenia and other side effects is important if such therapy is given. Until more robust pharmacokinetic data are available, the use of cART as prophylaxis in neonates may be limited to resource-rich settings where routine therapeutic drug monitoring is possible. However, further study is urgently needed in resource-limited settings where the vast majority of high-risk HIV-exposed infants are born. The potential benefits of cART, both to avert infection and, in case of transmission, preserve future therapeutic options and potentially limit the viral reservoir, may well outweigh the risks. Finally, if this strategy is used, counselling of parents and close follow-up is necessary to avoid premature treatment discontinuation and to ensure proper monitoring of cART-exposed infants.",
"Click here for additional data file."
] |
[
"intro",
"methods",
null,
"results",
"discussion",
"conclusions",
"supplementary-material"
] |
[
"Prevention of mother-to-child transmission",
"HIV",
"neonatal prophylaxis",
"safety",
"combination antiretroviral therapy"
] |
Introduction:
While the overall risk of perinatal HIV transmission in the developed world has decreased to less than 1%, transmission still occurs, most often due to failure to diagnose HIV in pregnant women in a timely manner, or because of poor maternal drug adherence to, and/or refusal of, antiretroviral therapy [1]. Despite the relatively high risk of transmission in these situations, the optimal management of infants born to HIV-positive mothers, who are untreated or have detectable viral load prior to delivery despite receiving antenatal therapy, remains controversial. Current US Department of Health and Human Services (DHHS) guidelines recommend a six-week course of Zidovudine (ZDV), along with three doses of Nevirapine (NVP) in the first week of life for infants born to mothers who have not received any treatment [2]. However, in practice, many paediatric HIV specialists prescribe various combinations of three or more drugs to such “high-risk” neonates. In a national web-based survey of US care providers between December 2009 and January 2010, 62% reported the use of combination antiretroviral therapy (cART) during the previous year [3]. In a recent study from the United Kingdom, cART was prescribed to 71% of infants born to untreated mothers [4].
Despite the increasing use of cART for prophylaxis in neonates at high risk of HIV infection, there is limited data on the safety of these different combination regimens, or on the appropriate dosing for the antiretroviral (ARV) medications used for this purpose. Transient haematological toxicity has been reported with the use of six-week post-natal ZDV [5], the recommended regimen in standard-risk situations, and a number of studies have suggested increased haematological toxicity associated with the addition of a second nucleoside analogue [6, 7]. Neither the safety nor the optimal dosing of additional drugs used for prophylaxis in neonates (non-nucleoside reverse transcriptase inhibitors or protease inhibitors (PIs)) has been well defined [8–13]. Furthermore, there is emerging concern about the long-term impact of increased ARV exposure on the growth of exposed infants [14, 15].
In four Canadian centres, triple cART at treatment doses has been routinely prescribed for the post-exposure prophylaxis (PEP) of neonates deemed at high risk of HIV infection, either because of detectable maternal viral load near delivery or in the absence of viral load results, clinical factors such as poor adherence or late diagnosis. In standard-risk situations, six weeks of ZDV monotherapy is prescribed. Given the limited data to support the use of cART, the primary objective of this study was to compare the safety and tolerability of cART versus ZDV monotherapy of PEP with respect to adverse events, haematological and growth parameters in exposed neonates.
Methods:
HIV-1 exposed children were eligible for this study if they were started on treatment doses of cART within 72 hours of birth because of incomplete maternal virologic suppression at delivery or, in the absence of maternal viral load results, a maternal history of incomplete adherence or non-adherence to cART, or late pregnancy initiation of cART. Neonatal cART was defined as any regimen of three or more antiretroviral agents, prescribed at treatment doses, for a minimum of six weeks. Regimens included ZDV (2 mg/kg every six hours prior to 2011, and 4 mg/kg twice daily in 2011–2013) for six weeks, Lamivudine (3TC) (2 mg/kg twice daily) for six weeks, with the addition of either NVP (150 mg/m2 daily for 14 days, followed by 150 mg/m2 twice daily for 14 days) or Nelfinavir (NFV) 40–50 mg/kg twice daily for six weeks) or LPV/Ritonavir (LPV/r) (300 mg/m2 twice daily) for six weeks. Combination regimens containing single or three doses of NVP were excluded.
Subjects were identified for the period 1997–2013 from the clinical databases of four paediatric HIV-care institutions in Canada: The Hospital for Sick Children, Toronto; Children's Hospital of Eastern Ontario, Ottawa; Centre Hospitalier Universitaire (CHU) Sainte-Justine, Montreal; Stollery Children's Hospital, Edmonton. A control group consisting of all infants of HIV-positive mothers who were prescribed ZDV monotherapy during a three-year period (2010–2012) at the Hospital for Sick Children was selected (n=148). Approval for the study was obtained by the ethics review board of each participating institution; given the retrospective nature of the study, individual patient consent was not deemed necessary by the respective ethics review boards for the chart review.
Statistical analysis Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.
Multivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).
Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.
Multivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).
Statistical analysis:
Baseline characteristics were reported as means with standard deviation (SD) for continuous variables and proportions for categorical variables. Comparisons between cART- and ZDV-exposed infants were performed using chi-square tests or Wilcoxon rank-sum. Haemoglobin, neutrophil count and growth parameters (head circumference (HC), weight and length) of cART- and ZDV-treated infants were compared at one, two and six months of age (haematological parameters) and at birth, one and six months of age (growth parameters) by Student's t-test or Wilcoxon rank-sum where appropriate. For the the three-way comparison between PI based-cART, NVP based cART and ZDV groups, the Kruskal-Wallace test was used.
Multivariate mixed models, allowing for repeated measures, were constructed to determine the overall effect of treatment category on haematological and growth parameters. For haematological parameters, effect of treatment category was adjusted for chronological age (variable for first, second or third visit corresponding to one, two and six months of age given the natural change in these parameters over time), gestational age and ethnicity (neutrophil count only). Due to a large number of missing values for haemoglobin and neutrophil count at baseline, these parameters were not included in the models. For growth parameters, effect of treatment was adjusted for chronological age, gestational age, gender and birthweight. All infants were included in the analysis of haematological toxicity and growth at one month; for all subsequent analyses infants confirmed to be HIV positive (n=13) were excluded from the analysis as all were maintained on ARV drugs. The analysis was performed using SAS statistical software, version 9.2 (SAS Institute, Cary, NC, USA).
Results:
One hundred and forty-eight infants received triple cART during the study period. NFV-based cART was the most common regimen (55%) followed by NVP-based cART (40%) and LPV/r-based cART (5%). The most common reason for prescribing cART was documented detectable maternal viral load (78.4%). Other overlapping factors included poor adherence (45%), late diagnosis (20%), no antenatal care (8%) and refusal of antenatal ARV therapy (8%). Fifteen of the cART-treated infants and five of the ZDV-treated infants had missing six-month data, either because they were lost to follow-up (n=8) or they did not attend the scheduled six-month visit (but were seen at 18 months of age or older) (n=12).
Baseline maternal and infant characteristics according to treatment group are described in Table 1. Mothers of cART-treated infants were younger, had higher viral loads and lower CD4 counts at the time of delivery, and were more likely to deliver vaginally and prematurely than mothers of ZDV monotherapy-treated infants.
Baseline demographic and clinical characteristics of mothers and infantsa
Reported means and standard deviation;
Chi Square test of proportions or Wilcoxon rank-sum.
Hematological and Growth Parameters by treatment group†
Reported means and standard deviation
Student T test, Wilcoxon rank sum, or Kruskal-Wallace test where appropriate.
n* observations recorded for each parameter out of a total of 148 cART recipients at birth, and 145 ZDV recipients.
Thirteen of the cART recipients were perinatally infected with HIV (8.8%). None of the ZDV recipients were infected. Five of the 13 (38.5%) had HIV-1 detected by PCR within 48 hours of birth suggesting in utero infection according to diagnostic standards. For the remaining eight, the timing of infection could not be ascertained as initial testing took place after 48 hours of life. Of the 13 infected children, four achieved long-term sustained virologic suppression, the details of which have been described elsewhere [16].
Non-specific signs and symptoms, including vomiting, diarrhoea, rash, jitteriness or irritability, potentially attributable to medication-related adverse effects, were reported in 10.2% of cART recipients and none of the ZDV recipients (p<0.001). ARV treatment was discontinued prematurely in 9.5% (n=14) of cART recipients (five NVP-treated and nine PI-treated infants) compared with 2.1% (n=3) ZDV recipients (p=0.01). Reasons given for premature treatment discontinuation among cART recipients included significant anaemia (n=3; haemoglobin at two, four and four weeks of 62, 76 and 92 g/L, respectively), neutropenia (n=1; neutrophil count 0.4 cells/mm3 at four weeks), non-specific symptoms including rash, vomiting, and diarrhoea (n=6), and parental decision (n=4). In the six cases of non-specific symptoms, treatment discontinuation was precautionary, as the treating physician indicated that the symptoms were most likely not medication related. In four cases of parental discontinuation, parents stopped the medication without evidence of toxicity or consultation with their healthcare provider. ZDV monotherapy was stopped early in three children, all because of significant anaemia (haemoglobin at four weeks of 76, 85 and 72 g/L).
The potential impact of cART on haematologic parameters was evaluated by comparing haemoglobin level and absolute neutrophil count for cART-treated and ZDV monotherapy-treated infants at one, two and six months of age (Table 2). In the unadjusted analysis, haemoglobin was lower in cART-treated infants compared with ZDV-treated infants at one month of age (mean 105±19 vs. 109±17 g/L, p=0.04), but not at two or six months of age (mean 104±12 vs. 105±9 g/L, p=0.31 and 118±11 vs. 121±8 g/L, p=0.15). In a subgroup analysis of specific treatment type, mean haemoglobin was lower among PI-treated infants compared with NVP-treated infants at one month of age (104±17 vs. 107±21, p=0.09), though only statistically significant at six months of age (mean 115±10 vs. 124±9 g/L, p<0.001) (Figure 1).
Haematological parameters according to treatment group.
Graphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.)
To study the overall effect of treatment type on haemoglobin over time, we constructed a multivariate model adjusting for chronological age and gestational age (Supplementary Table, not shown). Overall, the mean haemoglobin was 2.07 g/L lower among cART versus ZDV-treated infants (p=0.04) over the six-month period. In the subgroup analysis looking at specific treatment type, mean haemoglobin was 3.62 g/L lower in those receiving PI-based cART compared with those receiving ZDV monotherapy (p=0.002); no such effect was observed for NVP-based cART compared with ZDV monotherapy (p=0.82). There was no difference in absolute neutrophil count between NVP-based cART, PI-based cART and ZDV monotherapy-treated infants at one, two or six months of age in the unadjusted analysis; this finding was maintained in the multivariable model adjusting for visit number, ethnicity and gestational age.
Weight, length and HC were compared between all cART-treated and ZDV-treated infants, and between PI-based cART, NVP-based cART and ZDV at birth, one and six months of age (Table 2 and Figure 2). In the unadjusted analysis, cART-treated infants were smaller compared with ZDV-treated infants at birth (weight 2.91±0.60 vs. 3.09±0.56 kg, p=0.01; length 49.0±3.6 vs. 50.2±3.8 cm, p=0.03, head circumference 33.2±2.4 vs.33.6±2.3 cm, p=0.16). By six months of age, there was no difference in weight (8.03±1.12 kg vs. 8.25±1.12 kg, p=0.14) or head circumference (43.7±1.8 cm vs. 44.0±1.7, p=0.13), although the cART-treated group remained shorter than ZDV-treated infants (67.1±2.9 cm vs. 68.1±3.2 cm, p=0.01). In the subgroup analysis looking at specific treatment type, both NVP-and PI-treated infants were smaller than ZDV-treated infants across all growth parameters at birth; only the difference in length remained significant at six months of age.
Growth parameters according to treatment group.
Graphic representation of univariate analyses (Student's t-test, Wilcoxon rank-sum, or Kruskal-Wallace test where appropriate.)
To study the overall effect of treatment type on growth parameters over time, we constructed a multivariate fixed effects model adjusting for birthweight, gestational age, chronological age and gender. Overall, there was a mean HC difference of −0.63 cm among cART compared with ZDV-treated infants over the six-month period (p<0.001) (Supplementary Table, not shown). A similar effect was seen with respect to weight and length, with a mean weight difference of −236 g and length difference of −0.92 cm over the six-month period (p<0.001 and p=0.002, respectively). In the subgroup multivariate analysis comparing PI-based cART versus ZDV and NVP-based cART versus ZDV, there was no discernable difference between the two cART treatment types and these growth parameters.
Discussion:
In this retrospective study, cART initiated empirically for newborns deemed at high risk for perinatal HIV infection was generally well-tolerated, though adverse effects and consequent premature discontinuation of therapy did occur more often in the cART versus ZDV-treated group (9.5% vs. 2.1%). Among the 14 cART-treated children in whom medications were prematurely discontinued, four had their medications stopped by the parents without consultation with the healthcare provider and in the absence of any side effects, and six for precautionary reasons by the clinician even though the symptoms were not thought to be medication related, highlighting a lower threshold for treatment discontinuation by both parents and physicians among cART-treated infants.
With respect to haematological toxicity, the frequency and degree of anaemia observed in our cART-treated neonates was higher than that observed in the ZDV-monotherapy neonates. In the subgroup analysis of cART-treated infants, haemoglobin was lower at one month of age among PI versus NVP-treated infants, suggesting perhaps less haematological toxicity with NVP-based cART. The lower mean haemoglobin observed in cART-treated subjects compared with ZDV-monotherapy-treated subjects at one month of age was no longer evident at two and six months of age after stopping treatment, confirming the reversible nature of the anaemia. Reassuringly, compared with ZDV monotherapy, the use of cART was not associated with any discernible change in neutrophil count.
While in the multivariate mixed effects model cART-treated infants had lower mean growth measures (HC, weight and length) than ZDV-monotherapy-treated infants over time, there were no absolute differences in growth measures for head circumference or weight at six months of age, indicating good catch-up growth among the cART-treated infants. The observed lower mean growth over time in cART recipients was therefore likely due to factors associated with the indications for the use of cART. Specifically, the higher overall incidence of prematurity among cART-treated infants was likely a result of poorly controlled maternal HIV, an established risk factor for low birthweight and preterm delivery [17]. The higher viral load and lower CD4 counts of mothers of cART recipients in our study supports this hypothesis. While we adjusted for preterm delivery and gestational age in the multivariate models for growth, we were unable to adjust for in utero drug exposure, social situation, maternal health, nutritional status and education, which may be important predictors of infant growth and development.
Because of the selective use of cART for infants at higher risk of perinatal transmission in our study, it is not appropriate to compare the risk of transmission among cART versus ZDV-treated infants as the latter group would have received better antenatal preventive management. The 8.8% overall transmission rate observed among cART recipients in our cohort was similar to that in a previous trial in which neonates of untreated mothers were randomized to different cART regimens, evidence of the high risk of transmission despite prophylaxis in these situations [6]. Key potential benefits of cART at treatment doses initiated as prophylaxis for neonates later found to be infected is that such treatment will preserve health by controlling viral replication more rapidly, reduce the risk of generating antiretroviral medication resistance, as seen with single or multiple dose NVP in non-cART strategies, and limit the size of HIV viral reservoirs, which could have significant implications for achieving HIV remission in the future [18–20]. Given the limited number of therapeutic drug regimens available for children in resource-limited settings, preventing drug resistance to first-line therapy given to prevent perinatal transmission is a particularly important consideration.
Our study has a number of limitations, including its retrospective nature, relatively small sample size, and potential differences in the patient population at different study sites which may have influenced biological and growth outcomes. All control group infants were selected from a single centre, and the choice of PI-based versus NVP-based cART was also site specific. Given that these were the comparison groups of interest, we could not adjust for site and treatment type in the final analysis, thereby limiting our conclusions on specific cART treatment type.
Conclusions:
In summary, our study shows that cART at treatment doses administered as prophylaxis to “high-risk” HIV-exposed neonates is generally well tolerated and should be considered for newborn infants deemed at high risk of perinatally acquired HIV infection. However, close monitoring for anaemia, neutropenia and other side effects is important if such therapy is given. Until more robust pharmacokinetic data are available, the use of cART as prophylaxis in neonates may be limited to resource-rich settings where routine therapeutic drug monitoring is possible. However, further study is urgently needed in resource-limited settings where the vast majority of high-risk HIV-exposed infants are born. The potential benefits of cART, both to avert infection and, in case of transmission, preserve future therapeutic options and potentially limit the viral reservoir, may well outweigh the risks. Finally, if this strategy is used, counselling of parents and close follow-up is necessary to avoid premature treatment discontinuation and to ensure proper monitoring of cART-exposed infants.
Supplementary Material:
Click here for additional data file.
|
Background:
The optimal management of infants born to HIV-positive mothers who are untreated or have detectable viral load prior to delivery remains controversial. Despite the increasing use of combination antiretroviral therapy (cART) for post-exposure prophylaxis (PEP) of neonates at high risk of HIV infection, there is little safety and pharmacokinetic data to support this approach. The objective of this study was to evaluate the safety and tolerability of cART for PEP in HIV-exposed neonates.
Methods:
Retrospective study on 148 cART and 145 Zidovudine (ZDV) monotherapy-exposed infants identified from four Canadian centres where cART for PEP has routinely been prescribed in high-risk situations. Physician-reported adverse events and clinical outcomes were extracted by chart review. Haematological and growth parameters at birth, one and six months of age were compared between cART and ZDV-exposed infants using multivariate mixed effects modelling.
Results:
Non-specific signs and symptoms were reported in 10.2% of cART recipients versus none of the ZDV recipients. Treatment was discontinued prematurely in 9.5% of cART recipients versus 2.1% of ZDV recipients (p=0.01). In the multivariate model, cART recipients had lower mean haemoglobin (decrease of 2.07 g/L) over the 6-month period compared with ZDV recipients (p=0.04), but no effect was seen on absolute neutrophil count. cART recipients had lower weight and smaller head circumference at birth and one month of age compared with ZDV-exposed infants; these differences were no longer significant at six months of age.
Conclusions:
cART administered at treatment doses for PEP in neonates was generally well tolerated, though a higher incidence of non-specific signs and symptoms and early treatment discontinuation occurred among cART recipients.
|
Introduction:
While the overall risk of perinatal HIV transmission in the developed world has decreased to less than 1%, transmission still occurs, most often due to failure to diagnose HIV in pregnant women in a timely manner, or because of poor maternal drug adherence to, and/or refusal of, antiretroviral therapy [1]. Despite the relatively high risk of transmission in these situations, the optimal management of infants born to HIV-positive mothers, who are untreated or have detectable viral load prior to delivery despite receiving antenatal therapy, remains controversial. Current US Department of Health and Human Services (DHHS) guidelines recommend a six-week course of Zidovudine (ZDV), along with three doses of Nevirapine (NVP) in the first week of life for infants born to mothers who have not received any treatment [2]. However, in practice, many paediatric HIV specialists prescribe various combinations of three or more drugs to such “high-risk” neonates. In a national web-based survey of US care providers between December 2009 and January 2010, 62% reported the use of combination antiretroviral therapy (cART) during the previous year [3]. In a recent study from the United Kingdom, cART was prescribed to 71% of infants born to untreated mothers [4].
Despite the increasing use of cART for prophylaxis in neonates at high risk of HIV infection, there is limited data on the safety of these different combination regimens, or on the appropriate dosing for the antiretroviral (ARV) medications used for this purpose. Transient haematological toxicity has been reported with the use of six-week post-natal ZDV [5], the recommended regimen in standard-risk situations, and a number of studies have suggested increased haematological toxicity associated with the addition of a second nucleoside analogue [6, 7]. Neither the safety nor the optimal dosing of additional drugs used for prophylaxis in neonates (non-nucleoside reverse transcriptase inhibitors or protease inhibitors (PIs)) has been well defined [8–13]. Furthermore, there is emerging concern about the long-term impact of increased ARV exposure on the growth of exposed infants [14, 15].
In four Canadian centres, triple cART at treatment doses has been routinely prescribed for the post-exposure prophylaxis (PEP) of neonates deemed at high risk of HIV infection, either because of detectable maternal viral load near delivery or in the absence of viral load results, clinical factors such as poor adherence or late diagnosis. In standard-risk situations, six weeks of ZDV monotherapy is prescribed. Given the limited data to support the use of cART, the primary objective of this study was to compare the safety and tolerability of cART versus ZDV monotherapy of PEP with respect to adverse events, haematological and growth parameters in exposed neonates.
Conclusions:
In summary, our study shows that cART at treatment doses administered as prophylaxis to “high-risk” HIV-exposed neonates is generally well tolerated and should be considered for newborn infants deemed at high risk of perinatally acquired HIV infection. However, close monitoring for anaemia, neutropenia and other side effects is important if such therapy is given. Until more robust pharmacokinetic data are available, the use of cART as prophylaxis in neonates may be limited to resource-rich settings where routine therapeutic drug monitoring is possible. However, further study is urgently needed in resource-limited settings where the vast majority of high-risk HIV-exposed infants are born. The potential benefits of cART, both to avert infection and, in case of transmission, preserve future therapeutic options and potentially limit the viral reservoir, may well outweigh the risks. Finally, if this strategy is used, counselling of parents and close follow-up is necessary to avoid premature treatment discontinuation and to ensure proper monitoring of cART-exposed infants.
|
Background:
The optimal management of infants born to HIV-positive mothers who are untreated or have detectable viral load prior to delivery remains controversial. Despite the increasing use of combination antiretroviral therapy (cART) for post-exposure prophylaxis (PEP) of neonates at high risk of HIV infection, there is little safety and pharmacokinetic data to support this approach. The objective of this study was to evaluate the safety and tolerability of cART for PEP in HIV-exposed neonates.
Methods:
Retrospective study on 148 cART and 145 Zidovudine (ZDV) monotherapy-exposed infants identified from four Canadian centres where cART for PEP has routinely been prescribed in high-risk situations. Physician-reported adverse events and clinical outcomes were extracted by chart review. Haematological and growth parameters at birth, one and six months of age were compared between cART and ZDV-exposed infants using multivariate mixed effects modelling.
Results:
Non-specific signs and symptoms were reported in 10.2% of cART recipients versus none of the ZDV recipients. Treatment was discontinued prematurely in 9.5% of cART recipients versus 2.1% of ZDV recipients (p=0.01). In the multivariate model, cART recipients had lower mean haemoglobin (decrease of 2.07 g/L) over the 6-month period compared with ZDV recipients (p=0.04), but no effect was seen on absolute neutrophil count. cART recipients had lower weight and smaller head circumference at birth and one month of age compared with ZDV-exposed infants; these differences were no longer significant at six months of age.
Conclusions:
cART administered at treatment doses for PEP in neonates was generally well tolerated, though a higher incidence of non-specific signs and symptoms and early treatment discontinuation occurred among cART recipients.
| 4,271 | 331 | 7 |
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[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]
|
[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]
|
[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]
|
[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]
|
[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]
|
[CONTENT] Prevention of mother-to-child transmission | HIV | neonatal prophylaxis | safety | combination antiretroviral therapy [SUMMARY]
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[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]
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[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]
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[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]
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[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]
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[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]
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[CONTENT] Adult | Anti-HIV Agents | Canada | Drug Therapy, Combination | Female | HIV Infections | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Male | Pregnancy | Retrospective Studies [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]
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[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]
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[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]
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[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]
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[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]
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[CONTENT] cart | infants | zdv | age | treated | treatment | parameters | treated infants | growth | based [SUMMARY]
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[CONTENT] risk | neonates | week | safety | hiv | cart | high | high risk | use | born [SUMMARY]
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[CONTENT] parameters | age | mg | daily | cart | growth | twice | twice daily | growth parameters | zdv [SUMMARY]
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[CONTENT] treated | cart | treated infants | zdv | infants | age | vs | recipients | cm | difference [SUMMARY]
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[CONTENT] monitoring | risk | high risk | high | close | risk hiv exposed | high risk hiv exposed | cart | exposed | risk hiv [SUMMARY]
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[CONTENT] cart | age | infants | parameters | zdv | treated | growth | treatment | risk | treated infants [SUMMARY]
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[CONTENT] cart | age | infants | parameters | zdv | treated | growth | treatment | risk | treated infants [SUMMARY]
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[CONTENT] ||| ||| [SUMMARY]
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[CONTENT] 148 | 145 | ZDV | four | Canadian | PEP ||| ||| one and six months of age | ZDV [SUMMARY]
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[CONTENT] 10.2% | ZDV ||| 9.5% | 2.1% | ZDV ||| 2.07 | 6-month | ZDV | p=0.04 ||| birth and one month of age | ZDV | six months of age [SUMMARY]
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[CONTENT] [SUMMARY]
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[CONTENT] ||| ||| ||| 148 | 145 | ZDV | four | Canadian | PEP ||| ||| one and six months of age | ZDV ||| 10.2% | ZDV ||| 9.5% | 2.1% | ZDV ||| 2.07 | 6-month | ZDV | p=0.04 ||| birth and one month of age | ZDV | six months of age ||| [SUMMARY]
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[CONTENT] ||| ||| ||| 148 | 145 | ZDV | four | Canadian | PEP ||| ||| one and six months of age | ZDV ||| 10.2% | ZDV ||| 9.5% | 2.1% | ZDV ||| 2.07 | 6-month | ZDV | p=0.04 ||| birth and one month of age | ZDV | six months of age ||| [SUMMARY]
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Economic burden of stroke in a large county in Sweden.
|
23013284
|
Stroke remains to be a major burden of disease, often causing death or physical impairment or disability. This paper estimates the economic burden of stroke in a large county of 1.5 million inhabitants in western Sweden.
|
BACKGROUND
|
The economic burden of stroke was estimated from a societal perspective with an incidence approach. Data were collected from clinical registries and 3,074 patients were included. In the cost calculations, both direct and indirect costs were estimated and were based on costs for 12 months after a first-ever stroke.
|
METHODS
|
The total excess costs in the first 12 months after the first-ever stroke for a population of 1.5 million was 629 million SEK (€69 million). Men consumed more acute care in hospitals, whereas women consumed more rehabilitation and long-term care provided by the municipalities. Younger patients brought a significantly higher burden on society compared with older patients due to the loss of productivity and the increased use of resources in health care.
|
RESULTS
|
The results of this cost-of-illness study were based on an improved calculation process in a number of fields and are consistent with previous studies. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-time care and informal care and productivity loss explains 10% of total cost for the stroke disease. The result of this study can be used for further development of the methods for economic analyses as well as for analysis of improvements and investments in health care.
|
CONCLUSIONS
|
[
"Aged",
"Aged, 80 and over",
"Cost of Illness",
"Female",
"Health Care Costs",
"Health Services Research",
"Humans",
"Male",
"Middle Aged",
"Registries",
"Retrospective Studies",
"Stroke",
"Sweden"
] |
3506527
|
Background
|
Stroke continues to be a major burden of disease
[1,2] even when the risk of premature death has been reduced
[3]. Several assessments of the direct and indirect costs have been published
[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail
[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population
[11]. Thus, the financial burden of diseases such as stroke is important.
The Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure
1). All units of care regardless of the provider are financed by local taxes.
Providers in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.
A health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.
The aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke.
|
Methods
|
Epidemiology Stroke is a clinical syndrome with several different pathologies
[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)
[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden
[5]. The sex distribution in the whole group was equal (Table
1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men
[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table
1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table
2).
Demographic and clinical variables for patients with first-ever stroke in western Sweden during 2008
Total number of patients n = 3,074.
Data source: Local administrative register and The Stroke Register.
*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.
Age-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008
Total number of patients n = 3,074.
Data source: The Stroke register.
*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.
Stroke is a clinical syndrome with several different pathologies
[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)
[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden
[5]. The sex distribution in the whole group was equal (Table
1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men
[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table
1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table
2).
Demographic and clinical variables for patients with first-ever stroke in western Sweden during 2008
Total number of patients n = 3,074.
Data source: Local administrative register and The Stroke Register.
*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.
Age-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008
Total number of patients n = 3,074.
Data source: The Stroke register.
*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.
Data source To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure
2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden
[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland
[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.
Chain of health care for stroke survivors in Sweden.
To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure
2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden
[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland
[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.
Chain of health care for stroke survivors in Sweden.
Direct costs in health care Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table
3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table
3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.
Main unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008
Prices of year 2008 in SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
Data source: see methods.
Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table
3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table
3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.
Main unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008
Prices of year 2008 in SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
Data source: see methods.
Municipal cost As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.
As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.
Informal care volume Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.
Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.
Informal care costs The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner
[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden
[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%
[20] of the production loss value (Table
3).
The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner
[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden
[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%
[20] of the production loss value (Table
3).
Cost for loss of productivity Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county
[19] (Table
3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.
Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county
[19] (Table
3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.
Lifetime costs Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies
[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.
Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies
[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.
|
Results
|
Excess total costs This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table
4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.
Excess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008
Total number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table
4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.
Excess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008
Total number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
Excess cost per individual The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table
4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table
5).
Differences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males
Total number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table
4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table
5).
Differences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males
Total number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
Excess costs the first three years Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure
3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.
Cost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.
Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure
3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.
Cost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.
Generalizability and comparability Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study
[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.
Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study
[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.
|
Conclusions
|
The results of this cost of illness study are consistent with previous studies, although we have the cost calculation process in a number of fields. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-term care, informal care and productivity loss explain 10% of total cost for the stroke disease. The results of this study can be used as further development as well as for improvements and investments in health care as well as for development of econometric methods.
|
[
"Background",
"Epidemiology",
"Data source",
"Direct costs in health care",
"Municipal cost",
"Informal care volume",
"Informal care costs",
"Cost for loss of productivity",
"Lifetime costs",
"Excess total costs",
"Excess cost per individual",
"Excess costs the first three years",
"Generalizability and comparability",
"Methodological considerations",
"Analysis of result",
"Comparison with other studies",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Stroke continues to be a major burden of disease\n[1,2] even when the risk of premature death has been reduced\n[3]. Several assessments of the direct and indirect costs have been published\n[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail\n[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population\n[11]. Thus, the financial burden of diseases such as stroke is important.\nThe Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure\n1). All units of care regardless of the provider are financed by local taxes.\nProviders in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.\nA health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.\nThe aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke.",
"Stroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.",
"To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.",
"Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.",
"As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.",
"Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.",
"The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).",
"Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.",
"Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.",
"This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).",
"The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).",
"Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.",
"Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.",
"The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\n[11]. Thus, systematic data on informal care is an essential area for further research.",
"This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.",
"When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.",
"The study was performed upon request from the county council of Västra Götaland and the authors acknowledge financial support from the county council of Västra Götaland. However, none of the authors has received reimbursement, fees, funding, or salary from any other organisation. None of the authors hold any stock or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. None of the authors hold or are currently applying for any patents relating to the content of the manuscript or received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. None of the authors have any other financial or non-financial competing interests.",
"JP has written the manuscript and has made substantial contributions to analysis. JFN have made substantial contributions to analysis and revising the manuscript. IK have been involved in designing the study, drafting and revising the manuscript. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6963/12/341/prepub\n"
] |
[
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[
"Background",
"Methods",
"Epidemiology",
"Data source",
"Direct costs in health care",
"Municipal cost",
"Informal care volume",
"Informal care costs",
"Cost for loss of productivity",
"Lifetime costs",
"Results",
"Excess total costs",
"Excess cost per individual",
"Excess costs the first three years",
"Generalizability and comparability",
"Discussion",
"Methodological considerations",
"Analysis of result",
"Comparison with other studies",
"Conclusions",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Stroke continues to be a major burden of disease\n[1,2] even when the risk of premature death has been reduced\n[3]. Several assessments of the direct and indirect costs have been published\n[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail\n[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population\n[11]. Thus, the financial burden of diseases such as stroke is important.\nThe Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure\n1). All units of care regardless of the provider are financed by local taxes.\nProviders in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.\nA health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.\nThe aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke.",
" Epidemiology Stroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\nStroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.\n Data source To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.\nTo identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.\n Direct costs in health care Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.\nData were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.\n Municipal cost As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\nAs this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.\n Informal care volume Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\nEstimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.\n Informal care costs The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).\nThe opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).\n Cost for loss of productivity Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\nEstimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.\n Lifetime costs Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.\nEstimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.",
"Stroke is a clinical syndrome with several different pathologies\n[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)\n[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden\n[5]. The sex distribution in the whole group was equal (Table\n1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men\n[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table\n1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table\n2).\nDemographic and clinical variables for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: Local administrative register and The Stroke Register.\n*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.\nAge-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008\nTotal number of patients n = 3,074.\nData source: The Stroke register.\n*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.",
"To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure\n2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden\n[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland\n[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.\nChain of health care for stroke survivors in Sweden.",
"Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table\n3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table\n3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.\nMain unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008\nPrices of year 2008 in SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nData source: see methods.",
"As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.",
"Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.",
"The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner\n[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden\n[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%\n[20] of the production loss value (Table\n3).",
"Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county\n[19] (Table\n3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.",
"Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies\n[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.",
" Excess total costs This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nThis study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\n Excess cost per individual The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\nThe average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).\n Excess costs the first three years Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\nCosts after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.\n Generalizability and comparability Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.\nCosts for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.",
"This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table\n4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.\nExcess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).",
"The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table\n4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table\n5).\nDifferences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males\nTotal number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).\nBased on converting currency rate 1 June 2012 (1 SEK = €0,11).",
"Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure\n3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.\nCost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.",
"Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study\n[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.",
" Methodological considerations The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\n[11]. Thus, systematic data on informal care is an essential area for further research.\nThe estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\n[11]. Thus, systematic data on informal care is an essential area for further research.\n Analysis of result This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.\nThis study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.\n Comparison with other studies When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.\nWhen comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.",
"The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem\n[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses\n[11]. Thus, systematic data on informal care is an essential area for further research.",
"This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.",
"When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study\n[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).\nComparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen\n[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study\n[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days\n[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.",
"The results of this cost of illness study are consistent with previous studies, although we have the cost calculation process in a number of fields. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-term care, informal care and productivity loss explain 10% of total cost for the stroke disease. The results of this study can be used as further development as well as for improvements and investments in health care as well as for development of econometric methods.",
"The study was performed upon request from the county council of Västra Götaland and the authors acknowledge financial support from the county council of Västra Götaland. However, none of the authors has received reimbursement, fees, funding, or salary from any other organisation. None of the authors hold any stock or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. None of the authors hold or are currently applying for any patents relating to the content of the manuscript or received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. None of the authors have any other financial or non-financial competing interests.",
"JP has written the manuscript and has made substantial contributions to analysis. JFN have made substantial contributions to analysis and revising the manuscript. IK have been involved in designing the study, drafting and revising the manuscript. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6963/12/341/prepub\n"
] |
[
null,
"methods",
null,
null,
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
null,
null,
null,
"conclusions",
null,
null,
null
] |
[
"Burden",
"Cost of illness",
"Health economics",
"Incidence cost",
"Stroke",
"Sweden"
] |
Background:
Stroke continues to be a major burden of disease
[1,2] even when the risk of premature death has been reduced
[3]. Several assessments of the direct and indirect costs have been published
[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail
[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population
[11]. Thus, the financial burden of diseases such as stroke is important.
The Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure
1). All units of care regardless of the provider are financed by local taxes.
Providers in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.
A health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.
The aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke.
Methods:
Epidemiology Stroke is a clinical syndrome with several different pathologies
[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)
[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden
[5]. The sex distribution in the whole group was equal (Table
1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men
[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table
1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table
2).
Demographic and clinical variables for patients with first-ever stroke in western Sweden during 2008
Total number of patients n = 3,074.
Data source: Local administrative register and The Stroke Register.
*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.
Age-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008
Total number of patients n = 3,074.
Data source: The Stroke register.
*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.
Stroke is a clinical syndrome with several different pathologies
[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)
[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden
[5]. The sex distribution in the whole group was equal (Table
1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men
[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table
1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table
2).
Demographic and clinical variables for patients with first-ever stroke in western Sweden during 2008
Total number of patients n = 3,074.
Data source: Local administrative register and The Stroke Register.
*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.
Age-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008
Total number of patients n = 3,074.
Data source: The Stroke register.
*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.
Data source To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure
2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden
[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland
[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.
Chain of health care for stroke survivors in Sweden.
To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure
2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden
[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland
[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.
Chain of health care for stroke survivors in Sweden.
Direct costs in health care Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table
3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table
3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.
Main unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008
Prices of year 2008 in SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
Data source: see methods.
Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table
3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table
3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.
Main unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008
Prices of year 2008 in SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
Data source: see methods.
Municipal cost As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.
As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.
Informal care volume Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.
Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.
Informal care costs The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner
[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden
[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%
[20] of the production loss value (Table
3).
The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner
[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden
[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%
[20] of the production loss value (Table
3).
Cost for loss of productivity Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county
[19] (Table
3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.
Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county
[19] (Table
3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.
Lifetime costs Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies
[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.
Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies
[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.
Epidemiology:
Stroke is a clinical syndrome with several different pathologies
[12]. In this study we defined stroke as International Classification of Disease, 10th revision (ICD-10) codes I61 (intracerebral haemorrhage), I63 (cerebral infarction) and I64 (stroke, not specified as haemorrhage or infarction)
[13]. According to local data of health care consumption from the county council, 4,242 stroke events were reported in 2008, of which 3,074 cases (72%) were first-ever stroke. This proportion is in accordance with a previous study conducted in Sweden
[5]. The sex distribution in the whole group was equal (Table
1). However, in the age group below 55, the proportion of men was 67%, whereas in the age group above 85 the proportion of women was 66%. This could partly be explained by women living longer than men
[14]. Comorbidity such as atrial fibrillation and hypertension indicates risk factors for first-ever stroke (Table
1). Health-related quality of life decreased with age, i.e. patients over 85 had the lowest self-reported health-related quality of life. Women had lower self-reported health-related quality of life and impaired mobility compared with men (Table
2).
Demographic and clinical variables for patients with first-ever stroke in western Sweden during 2008
Total number of patients n = 3,074.
Data source: Local administrative register and The Stroke Register.
*Reference value for the age-matched Swedish population, according to The Swedish National Institute of Public Health.
Age-related differences in health-related quality of life and mobility three month after first-ever stroke onset for patients in western Sweden during 2008
Total number of patients n = 3,074.
Data source: The Stroke register.
*Reference value for the Swedish population. Source: Ferraz-Nunes J: Hälsa, ett värde utan pris (Health, a value without price), Holmberg & Weibull (red). Det nya samhället (The new society), SOM. Sweden: Gothenburg University; 2000:93–110.
Data source:
To identify, quantify and value events and contacts attributable to stroke, identifying the chain of healthcare is important. It is then possible to estimate all costs occurring in each step of the chain (Figure
2). Retrospective data were extracted from the local administrative register and a national clinical register, The Stroke Register, to calculate resources attributable to stroke. The clinical register contains information on stroke admissions in Sweden
[15], comorbidity, health-related quality of life, medication, admissions to municipal care and dependence on relatives. Collection of data into the national clinical registries is based on the law SFS 2008:355. Local data of health care consumption were extracted from the county council of Västra Götaland
[10], containing health care consumption for inpatients care as well as outpatient care. Data for inpatient care contained dates of admission and discharges, primary diagnosis (ICD-10) and type of ward, and data for outpatient care contained date visit, primary diagnosis (ICD-10), type of medical personnel and type of clinic/facility. To calculate the excess cost for stroke, health care consumption data were extracted one year before and one year after the stroke onset for each individual. Collection and utilisation of data for assessment is based on the law SFS 2008:355 and the Health Act HSL 36 §. To complement and verify the local administrative data, macro data were extracted from The Stroke Register. We did not use individual data from this source. We did not collect primary data and use individual data, and approval from the ethics committee was not relevant.
Chain of health care for stroke survivors in Sweden.
Direct costs in health care:
Data were collected, based in an incidence method, to estimate disease cost for first-ever stroke patients for a single year. Inpatient care costs were calculated according to age intervals, sex and diagnosis. The cost of emergency and inpatient care consists of the costs of all care episodes that began in the year 2008 for a period of 12 months after the stroke. Most patients had only one treatment episode, but some patients had more episodes during the 12-month period. Each episode includes basic, intensive care, nursing, diagnosis, blood transfusion and rehabilitation costs (Table
3). Costs for outpatient care varied to a larger extent than costs for inpatient care due to the differences between patients in age and disabilities caused by stroke. Cost-driving contacts after discharge were visits with doctors and nurses, rehabilitation with physiotherapists, occupational therapists, speech therapists and counsellors (Table
3). Data collection was based on number of visits over the 12-month period. Costs per contact and per hospitalisation day were based on prices in the county and includes overhead, equipment and personnel costs. The average cost per patient in each age group was calculated.
Main unit cost per resource use items in hospital care, municipal rehabilitation and aid service, average income and informal care for patients with first-ever stroke in western Sweden during 2008
Prices of year 2008 in SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
Data source: see methods.
Municipal cost:
As this is a study of excess costs, municipal costs were calculated based on the change in the use of rehabilitation as well as long-term care and home aid before and after the stroke, respectively based on information from The Stroke Register. Municipal excess costs were small for individuals who had already used a great deal of care before the stroke. Costs for patients who died during the first three months after the stroke were limited. The average per diem cost was based on information from municipal professional workers.
Informal care volume:
Estimates of the volume of hours spent on informal care were based on the information in The Stroke Register, which was verified by interviews with caregivers and employees within the municipalities. According to the register, 2,076 patients reported that they were entirely or partly dependent on their informal caregiver. It was estimated that those who were entirely dependent on their informal caregiver received four hours each day and those who were partly dependent on their informal caregiver received four hours each week. For patients above 85, this volume of informal care was halved because data from The Stroke Register on this age group indicated consumption of municipal care to a larger extent before the stroke.
Informal care costs:
The opportunity cost method was used to calculate the socioeconomic value of informal care, meaning the value of the informal caregiver’s best alternative use for this time, which may be the loss of income including social security contributions and/or loss of leisure time. According to previous studies, informal care is mainly given by the patient’s partner
[16-18]. Patients in the age group below 65 were estimated to have partners in working age, and were therefore estimated to have a loss of income of 200 SEK per hour based on the average income in western Sweden during 2008 calculated by from Statistics Sweden
[19]. This includes an estimation of 25% on sick leave or disability pension. Patients above 65 were estimated to have partners not in working age and therefore to have a loss of leisure time of 70 SEK per hour, which is 35%
[20] of the production loss value (Table
3).
Cost for loss of productivity:
Estimation of cost for loss of productivity was based on sick leave and early retirement due to the ICD-10 codes I61, I63 and I64 in the year 2008. This information was obtained from the social insurance authority, which registers all absence to work in Sweden. Another two weeks were added to the time the sick leave because this period is not included in the initial phase of sick leave covered by the insurance. The period was calculated in working days and then multiplied by average day-income including social security contributions based on the average monthly market income in the county
[19] (Table
3). Total potential productivity loss was recalculated with a factor that takes into account absence from work due other causes such as unemployment and other sicknesses. By this, the employment status of the patients before and after stroke was taken in consideration and a measure of lost social potential production was calculated. The period was calculated in working days and then multiplied by average income including social security contributions, which is a measure of output from a market perspective. The potential productivity loss was estimated based on the human capital approach.
Lifetime costs:
Estimation of lifetime costs was based on estimated excess total cost in the first three years after first-ever stroke. The calculation of the first year corresponds to the follow-up of patients at the individual level indicating the exact real consumption of health care services, community care and production loss. In the second year, costs were estimated based on information about the discharge of patients. This information includes mortality, survival rates, degree of handicap, change in the need of community service, health care and long sick leave. Future development of costs after three years were adjusted based on data from clinical registries and other studies
[5,21]. Future costs were discounted to present value using a discount rate of 3%. The structure of costs is substantially different in the first year after stroke compared with future costs. The weight of community costs increased after the first year.
Results:
Excess total costs This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table
4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.
Excess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008
Total number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table
4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.
Excess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008
Total number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
Excess cost per individual The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table
4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table
5).
Differences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males
Total number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table
4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table
5).
Differences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males
Total number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
Excess costs the first three years Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure
3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.
Cost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.
Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure
3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.
Cost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.
Generalizability and comparability Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study
[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.
Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study
[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.
Excess total costs:
This study estimates the total excess care costs in the first 12 months after first-ever stroke to be 629 million SEK (€69 million) per year for a population of 1.5 million in the Västra Götaland county, western Sweden (Table
4). The excess cost means that the consumption of non-stroke-related health care is ignored. Medical expenses relating to resources used in inpatient and outpatient care were estimated to be 49% of the total costs. Municipal care costs were estimated to be 34% of the total costs. Patient’s loss of productivity is equal to the value of lost production in society due to absence from work and was estimated to be 9% of the total costs. The socioeconomic value of the informal care costs for the first year after the stroke was estimated to be 6% of total costs.
Excess total costs for all patients, excess cost per patient and lifetime costs for patients with first-ever stroke in western Sweden during 2008
Total number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
Excess cost per individual:
The average excess cost per individual in the first year after a first-ever stroke was calculated to be 193,000 SEK (€21,200) (Table
4). However, this excess cost varied greatly between individuals depending on age, sex, and severity of the stroke. The average cost per individual below 55 was more than twice as high as the cost for individuals between 65 and 74. Of total costs, approximately 50% were related to health care and approximately 40% were related to municipal care. However, this ratio differs significantly between age groups. For individuals below 65, the proportion of health care costs was greater whereas for the older individuals the proportion of municipal care was greater. There were some differences when the costs per individual were grouped in the ICD-10 codes because a stroke caused by a cerebral haemorrhage (I61) incurs the largest cost per individual. Age-related differences in costs were similar among men and women; however, men used more resources in health care and women more municipal care (Table
5).
Differences in excess costs per patient with first-ever stroke in western Sweden during 2008 for females and males
Total number of patients n = 3,074. Prices of year 2008 in thousand SEK and (€).
Based on converting currency rate 1 June 2012 (1 SEK = €0,11).
Excess costs the first three years:
Costs after the first year after a first-ever stroke were significant but had another structure compared with the first year. During the first year, health care cost accounted for the largest share of the cost whereas the proportion of municipal service costs exceeded health care costs after the first year (Figure
3). Lifetime costs are extended through the remaining years of life and were estimated to be 768,000 SEK (€84,500) in present value (3% discount) per individual. However, the lifetime cost per individual varied greatly depending on age. For patients below 55, lifetime costs were almost six times higher than for patients above 84. This variation was mainly due to productivity loss for the working population and in addition the health care and municipal care for rehabilitation and aid that is needed for several years.
Cost per patient during three years after first-ever stroke in western Sweden during 2008. Year 2008 prices in thousand EURO.
Generalizability and comparability:
Costs for life time care are based on statistics for stroke and demography. Changing case-mix may have an influence on the assumptions. As indicated from our results, rising ages for patients with first stroke ever will lead to decreasing overall societal costs. Also, as a previous study
[22] indicates, the incidence of stroke increases among the younger population, especially among younger women. These are probable scenarios and might affect the burden of stroke in a different way than this study indicates.
Discussion:
Methodological considerations The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem
[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses
[11]. Thus, systematic data on informal care is an essential area for further research.
The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem
[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses
[11]. Thus, systematic data on informal care is an essential area for further research.
Analysis of result This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.
This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.
Comparison with other studies When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study
[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).
Comparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen
[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study
[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days
[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.
When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study
[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).
Comparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen
[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study
[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days
[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.
Methodological considerations:
The estimated excess cost in this study gives a value of the societal burden that could be saved if one stroke-incidence could be avoided. One of the main strengths of this study is the detailed information covering every part throughout the whole chain of health care in addition to societal costs. This study gives indication of the costs in the year 2008; however, there may be variations due to improvements the in health of the population as an effect of new treatments as well as preventions. By this, the result in this study can be used as a basis to evaluate new improvements. Also, this study could be used to initiate a discussion of cost and priorities. However, the calculations have some limitations. There is a lack of data at the individual level in municipal care because only the county health care registers data on individual level. Municipal care is for legal reasons not registered for single individuals, which makes it impossible to perform a regression analysis exploring causality. However, the calculation of average cost is not affected by this. Other published studies in the field had the same problem
[23]. Also, there are some uncertainties in calculating the informal care due lack of systematic evidence in the area, which may have a significant influence in health economic analyses
[11]. Thus, systematic data on informal care is an essential area for further research.
Analysis of result:
This study shows that the expenses for specialist health care in the first year causes most of the excess costs of stroke, but this cost varies between age groups. For individuals older than 75, most of the excess costs are within the municipal care, e.g. rehabilitation and long-time care. Within the health care, inpatient care costs dominated. The study also shows that the excess costs vary across age group, sex and diagnosis. Excess cost in the younger age group is significantly higher than in the older group due to more resources used in health and municipal care as well as productivity loss. Men consumed more resources within health care and had a higher level of productivity loss. Individuals who suffered a cerebral haemorrhage (I61) consumed more resources than other patients in all cost categories. Even though this kind of stroke affects only few individuals, the individuals who are affected are younger and require most resources, both within health care and municipal care. The estimated opportunity cost for informal care is an understatement due to lack of systematic data. However, this gives an indication of the family’s burden in monetary terms.
Comparison with other studies:
When comparing the result of this study with other studies on the topic, differences in calculation methods have to be considered. However, compared with previous studies in Sweden, our study showed similar results to other studies on an aggregate level. The most recent study
[5] in Sweden shows that the lifetime-cost per individual was 725,000 SEK (€80,000) converted to 2008 year prices and with a discount rate of 3%. This figure represents an average for Sweden. The life-time cost in this study is calculated to be 768,000 SEK (€84,500).
Comparisons with studies in other countries add even more uncertainties due to different health care systems. A European study by Porsdal and Boysen
[7] showed differences in the use of specific recourses within the health care chain. For example in Denmark and Finland, a large amount of rehabilitation resources are used. In our study, we only estimated the rehabilitation use to 10% compared with the 23% in Porsdal’s study
[7]. In this study, the length of stay was about 12 days, whereas the corresponding figure in Porsdal’s study was 33 days
[7]. This is an example of the different choices of resource allocation within the health care chain, which have an effect on the overall costs.
Conclusions:
The results of this cost of illness study are consistent with previous studies, although we have the cost calculation process in a number of fields. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-term care, informal care and productivity loss explain 10% of total cost for the stroke disease. The results of this study can be used as further development as well as for improvements and investments in health care as well as for development of econometric methods.
Competing interests:
The study was performed upon request from the county council of Västra Götaland and the authors acknowledge financial support from the county council of Västra Götaland. However, none of the authors has received reimbursement, fees, funding, or salary from any other organisation. None of the authors hold any stock or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. None of the authors hold or are currently applying for any patents relating to the content of the manuscript or received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. None of the authors have any other financial or non-financial competing interests.
Authors’ contributions:
JP has written the manuscript and has made substantial contributions to analysis. JFN have made substantial contributions to analysis and revising the manuscript. IK have been involved in designing the study, drafting and revising the manuscript. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1472-6963/12/341/prepub
|
Background:
Stroke remains to be a major burden of disease, often causing death or physical impairment or disability. This paper estimates the economic burden of stroke in a large county of 1.5 million inhabitants in western Sweden.
Methods:
The economic burden of stroke was estimated from a societal perspective with an incidence approach. Data were collected from clinical registries and 3,074 patients were included. In the cost calculations, both direct and indirect costs were estimated and were based on costs for 12 months after a first-ever stroke.
Results:
The total excess costs in the first 12 months after the first-ever stroke for a population of 1.5 million was 629 million SEK (€69 million). Men consumed more acute care in hospitals, whereas women consumed more rehabilitation and long-term care provided by the municipalities. Younger patients brought a significantly higher burden on society compared with older patients due to the loss of productivity and the increased use of resources in health care.
Conclusions:
The results of this cost-of-illness study were based on an improved calculation process in a number of fields and are consistent with previous studies. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-time care and informal care and productivity loss explains 10% of total cost for the stroke disease. The result of this study can be used for further development of the methods for economic analyses as well as for analysis of improvements and investments in health care.
|
Background:
Stroke continues to be a major burden of disease
[1,2] even when the risk of premature death has been reduced
[3]. Several assessments of the direct and indirect costs have been published
[4-9]. In Sweden, the comprehensive statistics from providers on the utilization of in-hospital, municipal and long-time care make it possible to evaluate the costs from a wide societal perspective in detail
[10]. Sweden, like many Organisation for Economic Co-operation and Development (OECD) countries, faces a problem of an increasing fraction of elderly persons, who will in the long run lead to a heavy burden on society and the working population
[11]. Thus, the financial burden of diseases such as stroke is important.
The Swedish health care model is based on the goal to provide good health and high-quality care on equal terms for the entire population. Residents in Sweden have equal rights of access to care based on the medical needs and in accordance with evidence-based medicine and proven experience. Those who are in greater need should take precedence, regardless of their ability to pay, place of residence, social status or other factors that are medically irrelevant. Health care should also be cost-effective, and the resources within the health care system should be used so that maximum gain is reached to meet the needs of the population. The Swedish health care system is highly decentralised with three levels, the state, the counties and the municipalities. The state sets the regulations but does not provide care. The counties provide all health care including hospital, acute care and primary care whereas the municipalities provide rehabilitation, long-time care and home aid (Figure
1). All units of care regardless of the provider are financed by local taxes.
Providers in Swedish health care system. Source: Integrated Care in Europe. Description and comparison of integrated care in six EU countries. Elsevier Gezondheidszorg, 2003.
A health economic analysis of a disease implies that there is information available regarding the use of resources at different levels in the chain of health care and society. By using the social security number, Sweden has a unique ability to follow one patient throughout the whole chain of health care within the Swedish administrative registries. In this paper, relevant data were selected to provide a complete picture of the burden of stroke in Västra Götaland, a county in western Sweden including health care consumption, municipal care, potential productivity loss and informal care by relatives.
The aim of this exploratory study was to present the societal costs of first-ever stroke during 2008 in Västra Götaland, a large county in western Sweden, which has 1.5 million inhabitants. The study identified, quantified and valued all costs for health care, rehabilitation, long-term care and home aid and potential productivity loss. Also, all costs for patients and families during the first 12 months after the first-ever stroke as well as lifetime costs, were estimated. This study analysed the costs arising due to the increase in resource consumption due to stroke.
Conclusions:
The results of this cost of illness study are consistent with previous studies, although we have the cost calculation process in a number of fields. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-term care, informal care and productivity loss explain 10% of total cost for the stroke disease. The results of this study can be used as further development as well as for improvements and investments in health care as well as for development of econometric methods.
|
Background:
Stroke remains to be a major burden of disease, often causing death or physical impairment or disability. This paper estimates the economic burden of stroke in a large county of 1.5 million inhabitants in western Sweden.
Methods:
The economic burden of stroke was estimated from a societal perspective with an incidence approach. Data were collected from clinical registries and 3,074 patients were included. In the cost calculations, both direct and indirect costs were estimated and were based on costs for 12 months after a first-ever stroke.
Results:
The total excess costs in the first 12 months after the first-ever stroke for a population of 1.5 million was 629 million SEK (€69 million). Men consumed more acute care in hospitals, whereas women consumed more rehabilitation and long-term care provided by the municipalities. Younger patients brought a significantly higher burden on society compared with older patients due to the loss of productivity and the increased use of resources in health care.
Conclusions:
The results of this cost-of-illness study were based on an improved calculation process in a number of fields and are consistent with previous studies. In essence, 50% of costs for stroke care fall on acute care hospital, 40% on rehabilitation and long-time care and informal care and productivity loss explains 10% of total cost for the stroke disease. The result of this study can be used for further development of the methods for economic analyses as well as for analysis of improvements and investments in health care.
| 10,986 | 293 | 23 |
[
"care",
"costs",
"stroke",
"health",
"cost",
"health care",
"patients",
"data",
"study",
"year"
] |
[
"test",
"test"
] |
[CONTENT] Burden | Cost of illness | Health economics | Incidence cost | Stroke | Sweden [SUMMARY]
|
[CONTENT] Burden | Cost of illness | Health economics | Incidence cost | Stroke | Sweden [SUMMARY]
|
[CONTENT] Burden | Cost of illness | Health economics | Incidence cost | Stroke | Sweden [SUMMARY]
|
[CONTENT] Burden | Cost of illness | Health economics | Incidence cost | Stroke | Sweden [SUMMARY]
|
[CONTENT] Burden | Cost of illness | Health economics | Incidence cost | Stroke | Sweden [SUMMARY]
|
[CONTENT] Burden | Cost of illness | Health economics | Incidence cost | Stroke | Sweden [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Cost of Illness | Female | Health Care Costs | Health Services Research | Humans | Male | Middle Aged | Registries | Retrospective Studies | Stroke | Sweden [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Cost of Illness | Female | Health Care Costs | Health Services Research | Humans | Male | Middle Aged | Registries | Retrospective Studies | Stroke | Sweden [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Cost of Illness | Female | Health Care Costs | Health Services Research | Humans | Male | Middle Aged | Registries | Retrospective Studies | Stroke | Sweden [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Cost of Illness | Female | Health Care Costs | Health Services Research | Humans | Male | Middle Aged | Registries | Retrospective Studies | Stroke | Sweden [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Cost of Illness | Female | Health Care Costs | Health Services Research | Humans | Male | Middle Aged | Registries | Retrospective Studies | Stroke | Sweden [SUMMARY]
|
[CONTENT] Aged | Aged, 80 and over | Cost of Illness | Female | Health Care Costs | Health Services Research | Humans | Male | Middle Aged | Registries | Retrospective Studies | Stroke | Sweden [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] care | costs | stroke | health | cost | health care | patients | data | study | year [SUMMARY]
|
[CONTENT] care | costs | stroke | health | cost | health care | patients | data | study | year [SUMMARY]
|
[CONTENT] care | costs | stroke | health | cost | health care | patients | data | study | year [SUMMARY]
|
[CONTENT] care | costs | stroke | health | cost | health care | patients | data | study | year [SUMMARY]
|
[CONTENT] care | costs | stroke | health | cost | health care | patients | data | study | year [SUMMARY]
|
[CONTENT] care | costs | stroke | health | cost | health care | patients | data | study | year [SUMMARY]
|
[CONTENT] care | health | provide | health care | swedish | system | health care system | swedish health care | swedish health | care system [SUMMARY]
|
[CONTENT] stroke | data | care | register | costs | based | patients | health | age | income [SUMMARY]
|
[CONTENT] costs | care | total | total costs | stroke | year | excess | cost | sek | individual [SUMMARY]
|
[CONTENT] care | development | results | cost | care development | process number fields | studies cost calculation | studies cost calculation process | process | process number [SUMMARY]
|
[CONTENT] care | costs | stroke | health | cost | study | health care | year | data | patients [SUMMARY]
|
[CONTENT] care | costs | stroke | health | cost | study | health care | year | data | patients [SUMMARY]
|
[CONTENT] ||| 1.5 million | Sweden [SUMMARY]
|
[CONTENT] ||| 3,074 ||| 12 months | first [SUMMARY]
|
[CONTENT] the first 12 months | first | 1.5 million | 629 million | SEK | €69 million ||| ||| [SUMMARY]
|
[CONTENT] ||| 50% | 40% | 10% ||| [SUMMARY]
|
[CONTENT] ||| 1.5 million | Sweden ||| ||| 3,074 ||| 12 months | first ||| ||| the first 12 months | first | 1.5 million | 629 million | SEK | €69 million ||| ||| ||| ||| 50% | 40% | 10% ||| [SUMMARY]
|
[CONTENT] ||| 1.5 million | Sweden ||| ||| 3,074 ||| 12 months | first ||| ||| the first 12 months | first | 1.5 million | 629 million | SEK | €69 million ||| ||| ||| ||| 50% | 40% | 10% ||| [SUMMARY]
|
||||||
Role of tight junction proteins in gastroesophageal reflux disease.
|
22994974
|
Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function that is regulated by cell-cell contacts. The aim of the study was to investigate the expression pattern of selected components involved in the formation of tight junctions in relation to GERD.
|
BACKGROUND
|
Eighty-four patients with GERD-related symptoms with endoscopic signs (erosive: n = 47) or without them (non-erosive: n = 37) as well as 26 patients lacking GERD-specific symptoms as controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and adapted Ismeil-Beigi criteria, respectively. Mucosal biopsies from distal esophagus were taken for analysis by histopathology, immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (RT-PCR) of five genes encoding tight junction components [Occludin, Claudin-1, -2, Zona occludens (ZO-1, -2)].
|
METHODS
|
Histopathology confirmed GERD-specific alterations as dilated intercellular spaces in the esophageal mucosa of patients with GERD compared to controls (P < 0.05). Claudin-1 and -2 were 2- to 6-fold upregulation on transcript (P < 0.01) and in part on protein level (P < 0.015) in GERD, while subgroup analysis of revealed this upregulation for ERD only. In both erosive and non-erosive reflux disease, expression levels of Occludin and ZO-1,-2 were not significantly affected. Notably, the induced expression of both claudins did not correlate with histopathological parameters (basal cell hyperplasia, dilated intercellular spaces) in patients with GERD.
|
RESULTS
|
Taken together, the missing correlation between the expression of tight junction-related components and histomorphological GERD-specific alterations does not support a major role of the five proteins studied in the pathogenesis of GERD.
|
CONCLUSIONS
|
[
"Adult",
"Aged",
"Esophagitis",
"Esophagoscopy",
"Female",
"Gastroesophageal Reflux",
"Gastroscopy",
"Gene Expression Regulation",
"Humans",
"Immunohistochemistry",
"Male",
"Middle Aged",
"Tight Junction Proteins",
"Young Adult"
] |
3503771
|
Background
|
Gastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world
[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)
[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification
[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD
[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD
[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium
[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located
[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD
[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD
[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function
[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others
[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma
[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models
[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4
[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4
[33].
Here, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD.
|
Methods
|
Study design and patients’ characteristics Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled
[34]. Patients with typical GERD-related symptoms based on Montreal classification
[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table
1A and Table
1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.
Patient groups analyzed by quantitative RT-PCR and immunohistochemistry
Functional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.
Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.
Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.
Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.
Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.
Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled
[34]. Patients with typical GERD-related symptoms based on Montreal classification
[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table
1A and Table
1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.
Patient groups analyzed by quantitative RT-PCR and immunohistochemistry
Functional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.
Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.
Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.
Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.
Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.
Endoscopy and histopathology The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”
[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.
In the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously
[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)
[34,36], molecules related to barrier functions
[37,38], desmosomal proteins
[39] and histopathological alterations
[34].
The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”
[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.
In the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously
[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)
[34,36], molecules related to barrier functions
[37,38], desmosomal proteins
[39] and histopathological alterations
[34].
Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously
[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table
2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method
[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.
Characteristics of primers, RT-PCR protocol and antibodies
mab: monoclonal antibody, fw: forward, rv: reverse.
Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously
[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table
2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method
[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.
Characteristics of primers, RT-PCR protocol and antibodies
mab: monoclonal antibody, fw: forward, rv: reverse.
Immunohistochemical analysis of tight junctional components Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously
[36]. Details for antigen retrieval and primary antibodies are illustrated in Table
2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied
[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.
Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously
[36]. Details for antigen retrieval and primary antibodies are illustrated in Table
2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied
[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.
Statistical analysis Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.
Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.
|
Results
|
Patients and GERD-specific histomorphological changes The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table
1). Histomorphological alterations are shown in Table
3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table
3).
Histopathological parameters
Parameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.
The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table
1). Histomorphological alterations are shown in Table
3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table
3).
Histopathological parameters
Parameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.
Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease As exemplarily demonstrated in figure
1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables
4A and
4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table
4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure
1).
Expression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables
4A, and B.
Expression of tight junction-related components in esophageal mucosa in patients with GERD
Transcript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.
In general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure
2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.
Immunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).
As exemplarily demonstrated in figure
1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables
4A and
4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table
4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure
1).
Expression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables
4A, and B.
Expression of tight junction-related components in esophageal mucosa in patients with GERD
Transcript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.
In general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure
2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.
Immunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).
Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure
3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table
5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table
5B).
Correlation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables
5A, and 5B.
Correlation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)
Panel A: The histopathological scores (Table
3) were correlated with gene expression levels (transcript levels, Table
4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.
In addition to the correlation based on transcript levels (Figure
3, Table
5), correlation analysis between protein expression levels (immunohistochemical scores, Table
4B) and histopathological alterations (Table
3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).
In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure
3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table
5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table
5B).
Correlation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables
5A, and 5B.
Correlation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)
Panel A: The histopathological scores (Table
3) were correlated with gene expression levels (transcript levels, Table
4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.
In addition to the correlation based on transcript levels (Figure
3, Table
5), correlation analysis between protein expression levels (immunohistochemical scores, Table
4B) and histopathological alterations (Table
3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).
|
Conclusions
|
In summary, this study demonstrates a partial upregulation of tight junction-related components, in particular Claudin-1, in relation to GERD. Since identified molecular changes do not correlate with histomorphological alterations in general, a major role of Claudin-1 as of the other four tight junction-related proteins in the pathogenesis of GERD can not be concluded from our study.
|
[
"Background",
"Study design and patients’ characteristics",
"Inclusion criteria",
"Exclusion criteria",
"Endoscopy and histopathology",
"Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes",
"Immunohistochemical analysis of tight junctional components",
"Statistical analysis",
"Patients and GERD-specific histomorphological changes",
"Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease",
"Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa",
"Abbreviations",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Gastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world\n[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)\n[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification\n[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD\n[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD\n[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium\n[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located\n[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD\n[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD\n[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function\n[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others\n[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma\n[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models\n[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4\n[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4\n[33].\nHere, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD.",
"Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.",
"Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.",
"Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.",
"The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].",
"Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.",
"Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.",
"Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.",
"The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.",
"As exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).",
"In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).",
"BE: Barrett’s esophagus; ERD: Erosive reflux disease; NERD: Nonerosive reflux disease; GEJ: Gastroesophageal junction; GERD: Gastroesophageal reflux disease; H2RA: Histamine-receptor antagonist; H. pylori: Helicobacter pylori; ICS: Intercellular spaces; INR: International normalized ratio; iNOS: Inducible nitro oxygen synthetase; JAM: Junctional adhesion molecule; NFκB: Nuclear factor kappa B; PAR-2: Protease activated receptor-2; PPI: Proton pump inhibitor; RDQ: Reflux disease questionnaire; ROS: Reactive oxygen species; RT-PCR: Reverse transcription polymerase chain reaction; ZO: Zona occludens.",
"The authors declare that they have no competing interest concerning the content of this article.",
"KM, PM and TW designed the study. KM, LCF enrolled the majority of patients. AK provided clinical data. TW coordinated and performed laboratory work. DK and AR provided histopathological and immunohistochemical data. TW and SK performed statistical analysis. The manuscript was drafted by TW, AK, DK, and reviewed for important intellectual content by KM and PM. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-230X/12/128/prepub\n"
] |
[
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] |
[
"Background",
"Methods",
"Study design and patients’ characteristics",
"Inclusion criteria",
"Exclusion criteria",
"Endoscopy and histopathology",
"Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes",
"Immunohistochemical analysis of tight junctional components",
"Statistical analysis",
"Results",
"Patients and GERD-specific histomorphological changes",
"Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease",
"Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa",
"Discussion",
"Conclusions",
"Abbreviations",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Gastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world\n[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)\n[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification\n[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD\n[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD\n[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium\n[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located\n[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD\n[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD\n[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function\n[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others\n[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma\n[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models\n[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4\n[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4\n[33].\nHere, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD.",
" Study design and patients’ characteristics Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nBetween 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\n Endoscopy and histopathology The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].\nThe patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].\n Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.\nExtraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.\n Immunohistochemical analysis of tight junctional components Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\nImmunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.\n Statistical analysis Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.\nData are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.",
"Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled\n[34]. Patients with typical GERD-related symptoms based on Montreal classification\n[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table\n1A and Table\n1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.\nPatient groups analyzed by quantitative RT-PCR and immunohistochemistry\nFunctional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.\n Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\nFemale or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.\n Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.\nUpper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.",
"Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.",
"Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.",
"The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”\n[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.\nIn the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously\n[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)\n[34,36], molecules related to barrier functions\n[37,38], desmosomal proteins\n[39] and histopathological alterations\n[34].",
"Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously\n[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table\n2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method\n[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.\nCharacteristics of primers, RT-PCR protocol and antibodies\nmab: monoclonal antibody, fw: forward, rv: reverse.",
"Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously\n[36]. Details for antigen retrieval and primary antibodies are illustrated in Table\n2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied\n[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.",
"Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.",
" Patients and GERD-specific histomorphological changes The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\nThe three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.\n Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease As exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\nAs exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).\n Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).\nIn order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).",
"The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table\n1). Histomorphological alterations are shown in Table\n3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table\n3).\nHistopathological parameters\nParameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.",
"As exemplarily demonstrated in figure\n1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables\n4A and\n4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table\n4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure\n1).\nExpression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables\n4A, and B.\nExpression of tight junction-related components in esophageal mucosa in patients with GERD\nTranscript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.\nIn general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure\n2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.\nImmunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).",
"In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure\n3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table\n5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table\n5B).\nCorrelation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables\n5A, and 5B.\nCorrelation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)\nPanel A: The histopathological scores (Table\n3) were correlated with gene expression levels (transcript levels, Table\n4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.\nIn addition to the correlation based on transcript levels (Figure\n3, Table\n5), correlation analysis between protein expression levels (immunohistochemical scores, Table\n4B) and histopathological alterations (Table\n3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).",
"In this study, we demonstrated (I) distinct expression patterns of five genes encoding for proteins involved in the formation of tight junctions in esophageal mucosa. In particular Claudin-1 in ERD and to lesser extent Claudin-2 was expressed at higher levels in patients with GERD. In contrast, ZO-1, ZO-2, and Occludin were not affected by the presence of GERD. (II) In general, altered gene expression of Claudin-1/-2 did not correlate with the degree of histomorphological changes in the esophageal mucosa of patients with GERD.\nTight junctions are composed of transmembrane proteins such as Occludin, 24 Claudins, several junctional adhesion molecules (JAMs) with different isoforms, E-Cadherin as well as cytosolic binding partners\n[43,44]. The selection of the five genes studied was based on functional aspects. Occludin is critical for the formation of tight junctions in most tissues\n[45]. Claudin-1 is one of the numerous Claudins that seals intercellular space leading to higher barrier function\n[46], while Claudin-2 is the only pore-forming member of this family resulting in increased permeability\n[47]. Zonula occludens (ZO)-1 and-2 are cytosolic partners of tight junctions in most epithelial surfaces\n[48,49]. The selected genes present important components of the tight junctional complex, and were considered to allow assessment about alterations of tight junctions in relation to GERD. A comprehensive analysis concerning the general expression pattern of other junctional proteins was not performed.\nRecently, several studies demonstrated characteristic histopathological alterations in esophageal mucosa of patients with GERD and a proinflammatory response including the activation of related pathways such as NFκB, PAR-2, ROS and iNOS\n[50,51]. Several in vitro and animal studies have provided evidence that incubation of esophageal mucosa or squamous cell lines either with acidified media with/without bile acids or proinflammatory cytokines can provoke changes in transepithelial electric resistance and increased transepithelial permeability\n[52-55]. Notably, several studies demonstrated a cytokine-mediated change of tight junction-related molecules in various cell models. For instance, IL-6 markedly induces Claudin-2 expression via MEK and PI3K signaling leading to increased tight junction permeability\n[56]. In a rabbit model of GERD, elevated IL-6 expression correlated with induction of several tight junction-related proteins (Claudin-1, Occludin, JAM-1, ZO-1)\n[57] and altered the motogenic activity of smooth muscle cells\n[58].\nAll together, there is sufficient data showing that the exposure of mixed gastric or gastroduodenal refluxate causes altered esophageal epithelial barrier function, inflammation and cellular damage, although the timely order of these processes is a matter of debate\n[20]. As today, it is well accepted that impaired epithelial barrier function of the distal esophagus presents a major pathophysiological process in GERD.\nThis study shows an upregulation of tight junction-related proteins in relation to ERD and NERD in mucosal samples. In particular, Claudin-1 and Claudin-2, though mediating opposite functionally effects, were induced, while cytoplasmic adapters and Occludin were rather unchanged in relation to controls. The higher expression of Claudin-1 (both on transcript and protein level) was the only significant difference identified between patients with ERD and NERD. The fact that all other identified changes were similar between NERD and ERD supports the concept of similar pathophysiological mechanisms between both diseases. Unexpectedly, these changes did not correlate with histomorphological alterations, in particular with dilated ICS in esophageal mucosa. This finding is in contrast to the recently identified correlation between histopathological alterations, in particular basal cell hyperplasia, and elevated gene expression of desmosomal proteins\n[39]. In this study, few borderline correlations were found for basal cell hyperplasia and some genes only, but notably these findings were mostly restricted to reflux-negative controls, whereas patients with GERD did not reveal significant correlations between histopathological alterations and transcript levels of the five genes. Since correlation analyses were performed in an explorative way (without adjustment for multiple comparison), the few significant correlations (with borderline significance) do not support a general role of these findings for the pathophysiology of GERD. Taken into consideration this limitation and the fact that that the overall majority of our comparisons (17 out of 20) revealed no correlations, we conclude that our data do not give evidence for an association between the gene expression of the five genes studied and the histopathological changes in our study groups. It is well known that extent of basal cell hyperplasia reflects proliferative status of esophageal mucosa\n[9]. Since the identified correlations between gene expression levels and basal cell hyperplasia were mostly restricted to controls, it is unlikely that elevated Claudin-1 levels in ERD reflect tissue repair in context to mucosal damage caused by refluxate in these patients. Since we and others demonstrated more severe histomorphological alterations in ERD than NERD, the overall consistent changes of the 5 genes and their corresponding proteins in both diseases seem to be of limited relevance to the mucosal integrity and function. Furthermore, it is notable that some of the stainings revealed not the typical membrane-restricted expression pattern as demonstrated for these tight junction-related molecules im most gastrointestinal tissues\n[59,60]. However, cytoplasmic or diffuse membranous expression patterns have been identified for Claudin-2\n[60] and ZO-1\n[61] in human gastrointestinal tissue and for Claudin-1 in esophageal mucosa of rat\n[62]. Occludin staining pattern or expression in esophageal mucosa differs frequently also from those identified in gastric or intestinal mucosa\n[60,63]. Overall, the subcellular distribution of the 5 tight junction-related proteins seems to differ partially from those identified in columnar-lined epithelium. However, the study was not aimed to analyze the subcellular distribution pattern of the molecules in esophageal mucosa on the subcellular level. The presence of appropriate negative and positive control stainings in other tissues, and the good concordance between expression data on transcript and protein level in general provide further indirect evidence for the specificity of immunohistochemical stainings.\nBased on the descriptive study design, it remains open whether the altered gene expression levels of Claudin-1 and −2 contribute to GERD pathophysiology or merely are markers for the existing disease. Furthermore it is notable that the majority of patients received GERD medications (PPI, H2RA) in the past before entering study. Even a stop of at least 2 weeks was mandatory to enter the study, we can not exclude that the effects of long-term therapy in the past or the changes induced by the 2-week stop of medication (e.g. acid rebound)\n[64] could have affect the expression of the five genes studied. Another limitation is the assessment of protein expression by an immunohistochemical score that can be done semiquantitatively at best. Besides this methodological aspect, posttranscriptional regulatory mechanisms can lead to different findings between gene expression analysis performed on transcript and protein levels. But as mentioned above, overall we observed a good concordance between both levels even not all significant findings were confirmed by both methodologies. Since we studied five selected components of tight junction complexes in GERD only, general conclusions can not be made. Assessment of other tight junction related molecules (e.g. Claudins, JAMs, Tricellulin)\n[44,46,65] in regard to GERD needs to be performed.",
"In summary, this study demonstrates a partial upregulation of tight junction-related components, in particular Claudin-1, in relation to GERD. Since identified molecular changes do not correlate with histomorphological alterations in general, a major role of Claudin-1 as of the other four tight junction-related proteins in the pathogenesis of GERD can not be concluded from our study.",
"BE: Barrett’s esophagus; ERD: Erosive reflux disease; NERD: Nonerosive reflux disease; GEJ: Gastroesophageal junction; GERD: Gastroesophageal reflux disease; H2RA: Histamine-receptor antagonist; H. pylori: Helicobacter pylori; ICS: Intercellular spaces; INR: International normalized ratio; iNOS: Inducible nitro oxygen synthetase; JAM: Junctional adhesion molecule; NFκB: Nuclear factor kappa B; PAR-2: Protease activated receptor-2; PPI: Proton pump inhibitor; RDQ: Reflux disease questionnaire; ROS: Reactive oxygen species; RT-PCR: Reverse transcription polymerase chain reaction; ZO: Zona occludens.",
"The authors declare that they have no competing interest concerning the content of this article.",
"KM, PM and TW designed the study. KM, LCF enrolled the majority of patients. AK provided clinical data. TW coordinated and performed laboratory work. DK and AR provided histopathological and immunohistochemical data. TW and SK performed statistical analysis. The manuscript was drafted by TW, AK, DK, and reviewed for important intellectual content by KM and PM. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-230X/12/128/prepub\n"
] |
[
null,
"methods",
null,
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
"discussion",
"conclusions",
null,
null,
null,
null
] |
[
"Gastroesophageal reflux disease",
"Tight junction",
"Claudins",
"Esophagitis",
"Inflammation"
] |
Background:
Gastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world
[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)
[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification
[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD
[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD
[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium
[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located
[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD
[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD
[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function
[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others
[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma
[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models
[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4
[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4
[33].
Here, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD.
Methods:
Study design and patients’ characteristics Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled
[34]. Patients with typical GERD-related symptoms based on Montreal classification
[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table
1A and Table
1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.
Patient groups analyzed by quantitative RT-PCR and immunohistochemistry
Functional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.
Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.
Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.
Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.
Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.
Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled
[34]. Patients with typical GERD-related symptoms based on Montreal classification
[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table
1A and Table
1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.
Patient groups analyzed by quantitative RT-PCR and immunohistochemistry
Functional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.
Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.
Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.
Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.
Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.
Endoscopy and histopathology The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”
[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.
In the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously
[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)
[34,36], molecules related to barrier functions
[37,38], desmosomal proteins
[39] and histopathological alterations
[34].
The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”
[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.
In the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously
[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)
[34,36], molecules related to barrier functions
[37,38], desmosomal proteins
[39] and histopathological alterations
[34].
Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously
[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table
2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method
[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.
Characteristics of primers, RT-PCR protocol and antibodies
mab: monoclonal antibody, fw: forward, rv: reverse.
Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously
[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table
2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method
[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.
Characteristics of primers, RT-PCR protocol and antibodies
mab: monoclonal antibody, fw: forward, rv: reverse.
Immunohistochemical analysis of tight junctional components Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously
[36]. Details for antigen retrieval and primary antibodies are illustrated in Table
2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied
[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.
Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously
[36]. Details for antigen retrieval and primary antibodies are illustrated in Table
2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied
[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.
Statistical analysis Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.
Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.
Study design and patients’ characteristics:
Between 2005 and 2007, a cohort of patients with GERD and individuals lacking any symptom or endoscopic sign of GERD as GERD-negative controls were enrolled
[34]. Patients with typical GERD-related symptoms based on Montreal classification
[4] and patients without any reflux-related clinical symptoms undergoing EGD for screening or non-reflux dyspepsia (GERD-negative controls with a reflux disease questionnaire, RDQ score of 0) were invited to participate. All the patients underwent a detailed history and physical examination. The demographic data and endoscopic findings of the study population are presented in Table
1A and Table
1B. Written informed consent was obtained from all patients before endoscopy, after the endoscopist had explained the procedure to the patient in detail and answered all questions. The study was approved by the ethical committee of our institution and conducted according to the ethical guidelines of the declaration of Helsinki as revised in 1989.
Patient groups analyzed by quantitative RT-PCR and immunohistochemistry
Functional investigations such as 24 hour-pH-metry or MII-pH analysis were performed in individual cases only, and could not be included as separate parameter. The assignment of NERD was additionally based on the responsiveness to PPI therapy that was subsequently assessed.
Inclusion criteria Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.
Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.
Exclusion criteria Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.
Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.
Inclusion criteria:
Female or male, age 18 to 80, able to provide written informed consent. Patients with typical reflux symptoms had to present symptoms at least three times a week. Typical reflux symptoms were defined as heartburn and regurgitation, as evaluated by the RDQ score. Patients with other types of reflux symptoms were not included in this study.
Exclusion criteria:
Upper gastrointestinal pathology (e.g. peptic ulcers, cancers, polyps, and Barrett’s mucosa), systemic inflammatory, neoplastic or malabsorptive diseases (e.g. Crohn’s disease, ulcerative colitis, vasculitis, celiac disease), and acute medical conditions such as pneumonia, stroke, coronary ischemia and acute renal failure. Patients with known abnormal coagulation parameters and thrombocytopenia at the time of the procedure (i.e. INR > 1.2, platelet count < 80,000) were also excluded. None of the patients had taken antibiotics, or bismuth compounds or any H2-blockers or proton-pump inhibitors (PPI) in the last 2 weeks before entering the study. It is notable that the majority of patients enrolled had various anti-secretory medications in their past, and does not present GERD-naïve patients. Each patient was assigned a coded number. Histopathological assessment was done by pathologist (DK) blinded to clinical data.
Endoscopy and histopathology:
The patients underwent the procedure after an overnight fast. The endoscopy was performed under conscious sedation with intravenous midazolam using a videogastroscope (Q160, Olympus, Hamburg). Endoscopic characterization of esophagitis was performed according to the “Los Angeles classification”
[35] describing the following endoscopic landmarks: gastroesophageal junction (GEJ), Z-line, beginning of the gastric folds and diaphragmatic pinch. The GEJ was defined as the beginning of the gastric folds, whereas the Z-line was defined as the squamocolumnar junction. The cardia was defined as the mucosa lying immediately below the GEJ.
In the distal esophagus, 3 biopsies were taken 2 cm above the squamous-columnar junction at the 3 o'clock position. In case of erosions, specimens were taken 2 cm above the tip of the erosion. One biopsy was snap-frozen in liquid nitrogen for molecular analysis. The two other biopsies were immediately fixed in 4 % neutral-buffered formalin and submitted for histopathological examinations using hematoxilin and eosin, modified Giemsa and PAS stain. In analogy to the Sydney classification for gastritis, the density of intraepithelial neutrophils/eosinophils and lymphocytes were scored to evaluation active and chronic inflammation. Furthermore, degree of basal cell hyperplasia, presence of papillary elongation and dilated intercellular spaces were semiquantitatively scored as either 0 (absent), 1 (mild), 2 (moderate), or 3 (severe) as described previously
[34]. Notably, several subgroups of the study cohort were published in regard to inflammatory mediators (e.g. cytokines, Protease-activated receptor 2)
[34,36], molecules related to barrier functions
[37,38], desmosomal proteins
[39] and histopathological alterations
[34].
Extraction of RNA and quantitative reverse transcription - polymerase chain reaction (RT-PCR) analysis of tight junction-related genes:
Extraction of total RNA and cDNA synthesis were performed by the “two-step” protocol as described previously
[40]. Transcript levels of Occludin, Claudin-1, -2, Zonula occludens-1, -2, and β-Actin were determined by quantitative real-time RT-PCR using an iCycler (BioRad, Munich, Germany) and the QuantiTect™ SYBR Green kit (Qiagen) using primers and standard conditions described in Table
2. Initial template mRNA amounts for all genes were calculated using iCycler software (Ct-values) and serial dilutions of plasmid DNA standard containing the corresponding PCR-fragments. Calculating template concentrations based on the Ct method and standard dilutions allowed an individual assessment of different efficiency for each PCR assay that were between 0.95 and 0.99. Gene-specific levels were normalized to the corresponding ß-actin level of the sample. Final results are expressed as arbitrary units (a.u.) and represent ratios between investigated gene and ß-Actin transcript amounts. All together, gene expression levels are identical to those calculated by the 2-∆∆ Ct-method
[41], but they are additionally adjusted to the assay-specific efficiency. Due to the primer design (usage of intron-spanning regions), amplification of genomic DNA was excluded. All amplification products were checked for their correct size by agarose gel electrophoresis. Therefore, gene expression levels (a.u.) illustrate the mRNA pool of the individual gene studied.
Characteristics of primers, RT-PCR protocol and antibodies
mab: monoclonal antibody, fw: forward, rv: reverse.
Immunohistochemical analysis of tight junctional components:
Immunohistochemistry was performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France) as described previously
[36]. Details for antigen retrieval and primary antibodies are illustrated in Table
2. Dilutions of primary antibodies were determined using appropriate positive and negative controls. For negative controls, primary antibody was replaced by irrelevant rabbit IgG that did not reveal specific signals (data not shown). Immunoreactivity was assessed in 5 representative high power fields (Zeiss Axioskop 50) of each sample by one blinded pathologist (DK). For semiquantitative assessment an adaptation of a score system originally described by Remmele et al. was applied
[42]. Briefly, staining intensity ([SI], 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells ([PPC], 1 = <10%, 2 = 10-50%, 3 = 51–80%, 4= > 80%) were scored semiquantitatively, resulting in an immunoreactive score [IRS = SI x PC] between 0 and 12. Furthermore, a score for membranous staining (0 = none, 1 = weak, 2 = moderate, 3 = strong/complete) was added resulting in a possible maximum of 15 points for each sample.
Statistical analysis:
Data are expressed as absolute number, relative proportion, median + range or mean ± standard deviation (SD) if not stated otherwise. Since the majority of data sets revealed skewed distribution, non-parametric Kruskal-Wallis test were applied for all comparisons made among the three groups (controls, NERD and ERD). If significant differences were identified (P < 0.05), post hoc analyses for pairwise comparisons between groups were performed using Mann–Whitney U test for gene expression analysis and immunohistochemistry. Age and histopathological parameters were analyzed by ANOVA and T test; frequencies by chi-square test. Non-parametric correlation analysis was performed by Spearman’s rank correlation test to investigate potential association between gene expression levels and histomorphological changes. Correlation analyses were performed in explorative manner only; adjustment for multiple comparisons was not performed. All tests were applied two-sided with a level of significance of P < 0.05.
Results:
Patients and GERD-specific histomorphological changes The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table
1). Histomorphological alterations are shown in Table
3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table
3).
Histopathological parameters
Parameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.
The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table
1). Histomorphological alterations are shown in Table
3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table
3).
Histopathological parameters
Parameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.
Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease As exemplarily demonstrated in figure
1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables
4A and
4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table
4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure
1).
Expression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables
4A, and B.
Expression of tight junction-related components in esophageal mucosa in patients with GERD
Transcript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.
In general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure
2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.
Immunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).
As exemplarily demonstrated in figure
1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables
4A and
4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table
4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure
1).
Expression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables
4A, and B.
Expression of tight junction-related components in esophageal mucosa in patients with GERD
Transcript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.
In general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure
2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.
Immunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).
Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure
3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table
5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table
5B).
Correlation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables
5A, and 5B.
Correlation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)
Panel A: The histopathological scores (Table
3) were correlated with gene expression levels (transcript levels, Table
4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.
In addition to the correlation based on transcript levels (Figure
3, Table
5), correlation analysis between protein expression levels (immunohistochemical scores, Table
4B) and histopathological alterations (Table
3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).
In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure
3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table
5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table
5B).
Correlation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables
5A, and 5B.
Correlation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)
Panel A: The histopathological scores (Table
3) were correlated with gene expression levels (transcript levels, Table
4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.
In addition to the correlation based on transcript levels (Figure
3, Table
5), correlation analysis between protein expression levels (immunohistochemical scores, Table
4B) and histopathological alterations (Table
3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).
Patients and GERD-specific histomorphological changes:
The three groups as well as the subgroups (randomly selected for immunohistochemistry) did not differ with respect to age and H. pylori status (Table
1). Histomorphological alterations are shown in Table
3. Activity and chronicity scores in esophageal mucosa were slightly higher in patients with NERD or ERD vs. controls without reaching significance. Basal cell hyperplasia, dilated intercellular spaces and elongation of papilla were significantly increased in both endoscopic entities (Table
3).
Histopathological parameters
Parameters were scored semiquantitatively as described in “Patients and Methods”. Data are presented as mean ± sd.
Upregulation of tight junction-related proteins in esophageal mucosa in context to the presence of gastroesophageal reflux disease:
As exemplarily demonstrated in figure
1, Claudin-1 transcript and protein levels in esophageal mucosa were significantly increased in patients with ERD, while a weaker increase was noted in NERD compared to controls. Corresponding data for the other four genes (Claudin-2, ZO-1, ZO-2; Occludin) including those of Claudin-1 are summarized in tables
4A and
4B. Claudin-2 had a similar expression pattern as Claudin-1, and both ZO-1 and ZO-2 showed a tendency to higher transcript levels in ERD and NERD (P-values <0.07, Table
4A). In addition to the upregulation in context to controls, both transcript levels and immunohistochemical scores of Claudin-1 were significantly higher in patients with ERD compared to those with NERD (Figure
1).
Expression of Claudin-1 in the esophageal mucosa of patients with NERD and ERD. The upper panel presents data of RT-PCR analysis; the lower panel illustrates immunohistochemical scores. Data are shown as boxplots illustrating 25, 75 percentile, median and 5–95 range. Note that due to skewed data distribution Claudin-1 (ERD) is presented as line only. Significant differences compared to controls are marked by a star (#); further details are presented in Tables
4A, and B.
Expression of tight junction-related components in esophageal mucosa in patients with GERD
Transcript levels are shown in relation to controls (Panel A). Immunohistochemical scores are illustrated similarly (Panel B). Statistical analyses for both datasets were done first by non-parametric Kruskal-Wallis (P-value italic style); if significant post-hoc analysis was done by Mann Whitney U test. Significant changes are demonstrated by bold letters. n.a.: not applicable.
In general, higher transcript levels were accompanied by higher immunohistochemical scores for most proteins. In addition to these quantitative changes in gene expression, different patterns of protein distribution within the cell compartment and within different mucosal layers were noted (Figure
2). In controls, the expression of tight junction-related proteins was mainly observed in the basal epithelial layers and in a cytoplasmatic pattern. In GERD, expansion of protein expression to the suprabasal und spinous epithelial layers was observed. Furthermore, expression of Claudin-1, Claudin-2 and ZO-1 was partly membrane-associated with a stronger intensity in GERD compared to controls.
Immunohistochemical stainings of tight junction-related proteins in esophageal mucosa. Occludin, Claudin-1, -2 and ZO-1,-2 are displayed by brown or red staining, respectively. Panels illustrate representative staining for controls and samples obtained from patients with NERD. Immunohistochemical staining was observed in the esophageal squamous epithelium mainly at the basal and suprabasal zone. Claudin-1/2 and ZO-1 showed partly a membranous staining. (Zeiss Axioskop 50; camera: Nikon coolpix 990).
Increased gene expression of tight junction-related molecules (transcript level) does not correlate with histomorphological changes in esophageal mucosa:
In order to study potential correlations between gene expression levels (transcript level) and the degree of histopathological alterations, all three groups were analyzed together in the first step. As exemplarily illustrated in figure
3, gene expression levels of Claudin-1 and Claudin-2 marginally correlated with the degree of basal cell hyperplasia, but not with dilated intercellular spaces and length of papilla (data not shown). Since most analyses were negative, these data are summarized in Table
5A for all five genes. Since basal cell hyperplasia revealed some even weak correlations in the complete study cohort, these correlation analyses were performed again for all three groups individually and for patients with GERD (NERD + ERD) combined. Only 3 out of 20 subanalyses revealed marginally significant correlations (without adjustment for multiple comparisons), two of those were identified in controls (data summarized in Table
5B).
Correlation of Claudin-1 and Claudin-2 with histomorphological changes in esophageal mucosa. Panels illustrate correlations between transcript levels and histomorphological alterations as indicated. Data are shown as open dot plots; medians are presented filled dot. Non-parametric correlation analysis was performed by Spearman’s rank correlation test; P values are presented in figure. Detailed data of other correlations are presented in Tables
5A, and 5B.
Correlation between GERD-specific histopathological alterations and gene expression level of tight junction-related genes (transcript level)
Panel A: The histopathological scores (Table
3) were correlated with gene expression levels (transcript levels, Table
4) for each gene individually in the combined study cohort (n = 110). Data (r-, and P-values) represent potential correlations between these parameters (n.s. = not significant). Panel B: Since basal cell hyperplasia demonstrated significant correlations in global analysis (Panel A), this parameter was further analyzed by correlating expression values for all five genes with basal cell hyperplasia within each group and for patients with GERD individually as identified in table.
In addition to the correlation based on transcript levels (Figure
3, Table
5), correlation analysis between protein expression levels (immunohistochemical scores, Table
4B) and histopathological alterations (Table
3) was performed. Here, only one significant correlation (between Claudin-1 and activity of inflammation, r = 0.51, P < 0.01) was identified (data not shown).
Discussion:
In this study, we demonstrated (I) distinct expression patterns of five genes encoding for proteins involved in the formation of tight junctions in esophageal mucosa. In particular Claudin-1 in ERD and to lesser extent Claudin-2 was expressed at higher levels in patients with GERD. In contrast, ZO-1, ZO-2, and Occludin were not affected by the presence of GERD. (II) In general, altered gene expression of Claudin-1/-2 did not correlate with the degree of histomorphological changes in the esophageal mucosa of patients with GERD.
Tight junctions are composed of transmembrane proteins such as Occludin, 24 Claudins, several junctional adhesion molecules (JAMs) with different isoforms, E-Cadherin as well as cytosolic binding partners
[43,44]. The selection of the five genes studied was based on functional aspects. Occludin is critical for the formation of tight junctions in most tissues
[45]. Claudin-1 is one of the numerous Claudins that seals intercellular space leading to higher barrier function
[46], while Claudin-2 is the only pore-forming member of this family resulting in increased permeability
[47]. Zonula occludens (ZO)-1 and-2 are cytosolic partners of tight junctions in most epithelial surfaces
[48,49]. The selected genes present important components of the tight junctional complex, and were considered to allow assessment about alterations of tight junctions in relation to GERD. A comprehensive analysis concerning the general expression pattern of other junctional proteins was not performed.
Recently, several studies demonstrated characteristic histopathological alterations in esophageal mucosa of patients with GERD and a proinflammatory response including the activation of related pathways such as NFκB, PAR-2, ROS and iNOS
[50,51]. Several in vitro and animal studies have provided evidence that incubation of esophageal mucosa or squamous cell lines either with acidified media with/without bile acids or proinflammatory cytokines can provoke changes in transepithelial electric resistance and increased transepithelial permeability
[52-55]. Notably, several studies demonstrated a cytokine-mediated change of tight junction-related molecules in various cell models. For instance, IL-6 markedly induces Claudin-2 expression via MEK and PI3K signaling leading to increased tight junction permeability
[56]. In a rabbit model of GERD, elevated IL-6 expression correlated with induction of several tight junction-related proteins (Claudin-1, Occludin, JAM-1, ZO-1)
[57] and altered the motogenic activity of smooth muscle cells
[58].
All together, there is sufficient data showing that the exposure of mixed gastric or gastroduodenal refluxate causes altered esophageal epithelial barrier function, inflammation and cellular damage, although the timely order of these processes is a matter of debate
[20]. As today, it is well accepted that impaired epithelial barrier function of the distal esophagus presents a major pathophysiological process in GERD.
This study shows an upregulation of tight junction-related proteins in relation to ERD and NERD in mucosal samples. In particular, Claudin-1 and Claudin-2, though mediating opposite functionally effects, were induced, while cytoplasmic adapters and Occludin were rather unchanged in relation to controls. The higher expression of Claudin-1 (both on transcript and protein level) was the only significant difference identified between patients with ERD and NERD. The fact that all other identified changes were similar between NERD and ERD supports the concept of similar pathophysiological mechanisms between both diseases. Unexpectedly, these changes did not correlate with histomorphological alterations, in particular with dilated ICS in esophageal mucosa. This finding is in contrast to the recently identified correlation between histopathological alterations, in particular basal cell hyperplasia, and elevated gene expression of desmosomal proteins
[39]. In this study, few borderline correlations were found for basal cell hyperplasia and some genes only, but notably these findings were mostly restricted to reflux-negative controls, whereas patients with GERD did not reveal significant correlations between histopathological alterations and transcript levels of the five genes. Since correlation analyses were performed in an explorative way (without adjustment for multiple comparison), the few significant correlations (with borderline significance) do not support a general role of these findings for the pathophysiology of GERD. Taken into consideration this limitation and the fact that that the overall majority of our comparisons (17 out of 20) revealed no correlations, we conclude that our data do not give evidence for an association between the gene expression of the five genes studied and the histopathological changes in our study groups. It is well known that extent of basal cell hyperplasia reflects proliferative status of esophageal mucosa
[9]. Since the identified correlations between gene expression levels and basal cell hyperplasia were mostly restricted to controls, it is unlikely that elevated Claudin-1 levels in ERD reflect tissue repair in context to mucosal damage caused by refluxate in these patients. Since we and others demonstrated more severe histomorphological alterations in ERD than NERD, the overall consistent changes of the 5 genes and their corresponding proteins in both diseases seem to be of limited relevance to the mucosal integrity and function. Furthermore, it is notable that some of the stainings revealed not the typical membrane-restricted expression pattern as demonstrated for these tight junction-related molecules im most gastrointestinal tissues
[59,60]. However, cytoplasmic or diffuse membranous expression patterns have been identified for Claudin-2
[60] and ZO-1
[61] in human gastrointestinal tissue and for Claudin-1 in esophageal mucosa of rat
[62]. Occludin staining pattern or expression in esophageal mucosa differs frequently also from those identified in gastric or intestinal mucosa
[60,63]. Overall, the subcellular distribution of the 5 tight junction-related proteins seems to differ partially from those identified in columnar-lined epithelium. However, the study was not aimed to analyze the subcellular distribution pattern of the molecules in esophageal mucosa on the subcellular level. The presence of appropriate negative and positive control stainings in other tissues, and the good concordance between expression data on transcript and protein level in general provide further indirect evidence for the specificity of immunohistochemical stainings.
Based on the descriptive study design, it remains open whether the altered gene expression levels of Claudin-1 and −2 contribute to GERD pathophysiology or merely are markers for the existing disease. Furthermore it is notable that the majority of patients received GERD medications (PPI, H2RA) in the past before entering study. Even a stop of at least 2 weeks was mandatory to enter the study, we can not exclude that the effects of long-term therapy in the past or the changes induced by the 2-week stop of medication (e.g. acid rebound)
[64] could have affect the expression of the five genes studied. Another limitation is the assessment of protein expression by an immunohistochemical score that can be done semiquantitatively at best. Besides this methodological aspect, posttranscriptional regulatory mechanisms can lead to different findings between gene expression analysis performed on transcript and protein levels. But as mentioned above, overall we observed a good concordance between both levels even not all significant findings were confirmed by both methodologies. Since we studied five selected components of tight junction complexes in GERD only, general conclusions can not be made. Assessment of other tight junction related molecules (e.g. Claudins, JAMs, Tricellulin)
[44,46,65] in regard to GERD needs to be performed.
Conclusions:
In summary, this study demonstrates a partial upregulation of tight junction-related components, in particular Claudin-1, in relation to GERD. Since identified molecular changes do not correlate with histomorphological alterations in general, a major role of Claudin-1 as of the other four tight junction-related proteins in the pathogenesis of GERD can not be concluded from our study.
Abbreviations:
BE: Barrett’s esophagus; ERD: Erosive reflux disease; NERD: Nonerosive reflux disease; GEJ: Gastroesophageal junction; GERD: Gastroesophageal reflux disease; H2RA: Histamine-receptor antagonist; H. pylori: Helicobacter pylori; ICS: Intercellular spaces; INR: International normalized ratio; iNOS: Inducible nitro oxygen synthetase; JAM: Junctional adhesion molecule; NFκB: Nuclear factor kappa B; PAR-2: Protease activated receptor-2; PPI: Proton pump inhibitor; RDQ: Reflux disease questionnaire; ROS: Reactive oxygen species; RT-PCR: Reverse transcription polymerase chain reaction; ZO: Zona occludens.
Competing interests:
The authors declare that they have no competing interest concerning the content of this article.
Authors’ contributions:
KM, PM and TW designed the study. KM, LCF enrolled the majority of patients. AK provided clinical data. TW coordinated and performed laboratory work. DK and AR provided histopathological and immunohistochemical data. TW and SK performed statistical analysis. The manuscript was drafted by TW, AK, DK, and reviewed for important intellectual content by KM and PM. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-230X/12/128/prepub
|
Background:
Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function that is regulated by cell-cell contacts. The aim of the study was to investigate the expression pattern of selected components involved in the formation of tight junctions in relation to GERD.
Methods:
Eighty-four patients with GERD-related symptoms with endoscopic signs (erosive: n = 47) or without them (non-erosive: n = 37) as well as 26 patients lacking GERD-specific symptoms as controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and adapted Ismeil-Beigi criteria, respectively. Mucosal biopsies from distal esophagus were taken for analysis by histopathology, immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (RT-PCR) of five genes encoding tight junction components [Occludin, Claudin-1, -2, Zona occludens (ZO-1, -2)].
Results:
Histopathology confirmed GERD-specific alterations as dilated intercellular spaces in the esophageal mucosa of patients with GERD compared to controls (P < 0.05). Claudin-1 and -2 were 2- to 6-fold upregulation on transcript (P < 0.01) and in part on protein level (P < 0.015) in GERD, while subgroup analysis of revealed this upregulation for ERD only. In both erosive and non-erosive reflux disease, expression levels of Occludin and ZO-1,-2 were not significantly affected. Notably, the induced expression of both claudins did not correlate with histopathological parameters (basal cell hyperplasia, dilated intercellular spaces) in patients with GERD.
Conclusions:
Taken together, the missing correlation between the expression of tight junction-related components and histomorphological GERD-specific alterations does not support a major role of the five proteins studied in the pathogenesis of GERD.
|
Background:
Gastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders in the world
[1,2]. Based on endoscopic findings GERD is differentiated in erosive (erosive reflux disease or ERD), non-erosive reflux disease (NERD) and Barrett’s esophagus (BE)
[3,4]. ERD is characterized by endoscopic visible breaks of esophageal mucosa integrity and classified according to various endoscopic classifications, most recently the Los Angeles classification
[5,6]. However, two thirds of patients with typical GERD symptoms do not exhibit visible mucosal changes in conventional esophagogastroduodenoscopy (EGD) and are thus diagnosed as having NERD
[6,7]. Although histology is not used in clinical practice for GERD diagnosis, frequent histological changes as basal cell hyperplasia, elongation of the papilla, inflammatory infiltrates and dilatation of the intercellular spaces are observed in the distal esophagus of patients with both ERD and NERD
[8-11]. Dilations of the intercellular spaces (ICS) are characteristic changes of the esophageal mucosa of patients with ERD and NERD. ICS were described by various others using electron microscopy and are even characterized by light microscopy. This feature is being more widely proposed as an additional morphological feature of acid-induced damage to the squamous epithelium
[10,12-14]. The widened ICS are supposed to permit the diffusion of molecules to the lamina propria where sensory nerve endings are located
[15]. Therefore, ICS dilation even in the absence of endoscopically visible mucosal damage may explain the occurrence of symptoms in patients with NERD
[16,17]. Furthermore, recent studies have provided evidence that the impaired barrier function of esophageal mucosa is a “hallmark” of GERD
[18-20]. The integrity of epithelial surfaces is based on various cell-cell contacts that provide the structural basis for barrier function by regulating the diffusion of molecules and sorting of transmembrane proteins to apical and basolateral surfaces. Tight junctions, adherens junction and desmosomes are the three major structural units mediating barrier and sorting function
[21,22]. Their structural composition, general functions, and pathophysiological relevance have been reviewed extensively by others
[21,23,24]. In line with the current concept in GERD, the role of molecules contributing to cell-cell contacts in esophageal mucosa in relation to GERD has been investigated in animal and human studies recently. Notably, the majority of studies were focused on the role of tight junction molecules (e.g. Claudin-2, -3, -4, -7 and −18) in Barrett’s metaplasia and carcinogenesis towards esophageal adenocarcinoma
[25-30]. In regard to the other 2 endoscopic entities (ERD, NERD), distinct alterations in the expression and or localization were described for Claudins 3 and 4 in GERD-related animal and in vitro models
[31-33]. Rat model revealed decreased expression of Claudin-3 and no change of Claudin-1 and 4
[31,32], while an in vitro model of esophageal-like squamous cells demonstrated a prominent role of Claudin-4
[33].
Here, we studied the expression patterns of five tight-junction related molecules (Occludin, Claudin-1, -2 and Zonula occludens-1-, 2) in the esophageal mucosa of a prospective cohort of patients with GERD as well as reflux-negative individuals. Gene expression was assessed both on transcriptional and protein level, and changes were studied in context to histopathological alterations associated with GERD.
Conclusions:
In summary, this study demonstrates a partial upregulation of tight junction-related components, in particular Claudin-1, in relation to GERD. Since identified molecular changes do not correlate with histomorphological alterations in general, a major role of Claudin-1 as of the other four tight junction-related proteins in the pathogenesis of GERD can not be concluded from our study.
|
Background:
Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function that is regulated by cell-cell contacts. The aim of the study was to investigate the expression pattern of selected components involved in the formation of tight junctions in relation to GERD.
Methods:
Eighty-four patients with GERD-related symptoms with endoscopic signs (erosive: n = 47) or without them (non-erosive: n = 37) as well as 26 patients lacking GERD-specific symptoms as controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and adapted Ismeil-Beigi criteria, respectively. Mucosal biopsies from distal esophagus were taken for analysis by histopathology, immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (RT-PCR) of five genes encoding tight junction components [Occludin, Claudin-1, -2, Zona occludens (ZO-1, -2)].
Results:
Histopathology confirmed GERD-specific alterations as dilated intercellular spaces in the esophageal mucosa of patients with GERD compared to controls (P < 0.05). Claudin-1 and -2 were 2- to 6-fold upregulation on transcript (P < 0.01) and in part on protein level (P < 0.015) in GERD, while subgroup analysis of revealed this upregulation for ERD only. In both erosive and non-erosive reflux disease, expression levels of Occludin and ZO-1,-2 were not significantly affected. Notably, the induced expression of both claudins did not correlate with histopathological parameters (basal cell hyperplasia, dilated intercellular spaces) in patients with GERD.
Conclusions:
Taken together, the missing correlation between the expression of tight junction-related components and histomorphological GERD-specific alterations does not support a major role of the five proteins studied in the pathogenesis of GERD.
| 11,448 | 346 | 19 |
[
"patients",
"claudin",
"expression",
"gerd",
"data",
"levels",
"table",
"gene",
"mucosa",
"controls"
] |
[
"test",
"test"
] |
[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]
|
[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]
|
[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]
|
[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]
|
[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]
|
[CONTENT] Gastroesophageal reflux disease | Tight junction | Claudins | Esophagitis | Inflammation [SUMMARY]
|
[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]
|
[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]
|
[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]
|
[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]
|
[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]
|
[CONTENT] Adult | Aged | Esophagitis | Esophagoscopy | Female | Gastroesophageal Reflux | Gastroscopy | Gene Expression Regulation | Humans | Immunohistochemistry | Male | Middle Aged | Tight Junction Proteins | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]
|
[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]
|
[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]
|
[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]
|
[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]
|
[CONTENT] patients | claudin | expression | gerd | data | levels | table | gene | mucosa | controls [SUMMARY]
|
[CONTENT] gerd | esophageal | molecules | ics | esophageal mucosa | visible | structural | nerd | claudin | cell [SUMMARY]
|
[CONTENT] patients | symptoms | reflux | reflux symptoms | performed | score | 80 | gene | study | defined [SUMMARY]
|
[CONTENT] claudin | expression | levels | table | transcript | correlations | panel | data | correlation | scores [SUMMARY]
|
[CONTENT] tight | junction related | tight junction related | tight junction | claudin | junction | related | alterations general major | general major | alterations general [SUMMARY]
|
[CONTENT] patients | claudin | expression | gerd | symptoms | levels | reflux | data | table | study [SUMMARY]
|
[CONTENT] patients | claudin | expression | gerd | symptoms | levels | reflux | data | table | study [SUMMARY]
|
[CONTENT] GERD ||| GERD [SUMMARY]
|
[CONTENT] Eighty-four | 47 | 37 | 26 | GERD ||| Los Angeles ||| Mucosal | RT-PCR | five ||| Zona [SUMMARY]
|
[CONTENT] GERD | 0.05 ||| 6-fold | 0.01 | 0.015 | GERD | ERD ||| Occludin ||| GERD [SUMMARY]
|
[CONTENT] five | GERD [SUMMARY]
|
[CONTENT] GERD ||| GERD ||| Eighty-four | 47 | 37 | 26 | GERD ||| Los Angeles ||| Mucosal | RT-PCR | five | Occludin | Zona ||| ||| GERD | 0.05 ||| 6-fold | 0.01 | 0.015 | GERD | ERD ||| Occludin ||| GERD ||| five | GERD [SUMMARY]
|
[CONTENT] GERD ||| GERD ||| Eighty-four | 47 | 37 | 26 | GERD ||| Los Angeles ||| Mucosal | RT-PCR | five | Occludin | Zona ||| ||| GERD | 0.05 ||| 6-fold | 0.01 | 0.015 | GERD | ERD ||| Occludin ||| GERD ||| five | GERD [SUMMARY]
|
||||||
Modification by hemochromatosis gene polymorphisms of the association between traffic-related air pollution and cognition in older men: a cohort study.
|
23413885
|
Previous studies found effect modification of associations between traffic-related air pollution and cardiovascular outcomes by polymorphisms in the hemochromatosis gene (HFE). As traffic-related air pollution may impact cognition through effects on cardiovascular health or through mechanisms which may also influence cardiovascular outcomes, we hypothesized that HFE polymorphisms would also modify a previously observed association between traffic-related air pollution exposure and cognition in older men.
|
BACKGROUND
|
We considered data from 628 participants of the VA Normative Aging Study. We estimated long term exposure to black carbon (BC), a marker of traffic related air pollution, using a spatio-temporal land use regression model. We assessed cognition using the Mini-Mental State Examination (MMSE), a test of global function, and performance on a battery of other tests, covering a wide range of domains. We investigated whether variants of HFE C282Y and H63D modified the association between BC and having a low MMSE score using logistic models with generalized estimating equations and multiplicative interaction terms. Similarly, we assessed whether HFE variants modified the association between BC and performance on the cognitive battery using linear mixed models with multiplicative interaction terms.
|
METHODS
|
Our results suggest modification of the BC-cognition association by HFE C282Y, although the test of interaction did not achieve statistical significance. In multivariable-adjusted models, participants who lacked a HFE C282Y variant (CC) exhibited an adverse association between BC and total cognition z-score (beta for a doubling in BC concentration: -0.061, 95% CI: -0.115, -0.007), while we did not observe an association in participants with at least one variant genotype (CY or YY) (beta for a doubling in BC concentration: 0.073, 95% CI: -0.081, 0.228; p-value for interaction: 0.11). The pattern of association was similar for analyses considering performance on the Mini-Mental State Examination. There was little evidence to support effect modification of the BC-cognition association by the HFE H63D genotype.
|
RESULTS
|
Our data suggest that older adults who lack an HFE C282Y variant may be more susceptible to an adverse effect of traffic-related air pollution exposure on cognition. This finding and the proposed biological mechanism require confirmation.
|
CONCLUSIONS
|
[
"Aged",
"Air Pollutants",
"Cognition",
"Cohort Studies",
"Genotype",
"Hemochromatosis Protein",
"Histocompatibility Antigens Class I",
"Humans",
"Male",
"Membrane Proteins",
"Middle Aged",
"Multiplex Polymerase Chain Reaction",
"Polymorphism, Single Nucleotide",
"Soot",
"United States",
"Vehicle Emissions"
] |
3599892
|
Background
|
While we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large
[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.
Traffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men
[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above
[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women
[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese
[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children
[6-11].
The hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease
[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium
[14-18].
Previous studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors
[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms.
|
Methods
|
Study sample The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis
[2] we refer readers to that paper for a more exhaustive description of our methods.
The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis
[2] we refer readers to that paper for a more exhaustive description of our methods.
Exposure assessment For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995
[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.
For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995
[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.
Cognitive testing The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.
The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.
HFE genotyping For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere
[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.
For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere
[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.
Statistical methods As the current dataset excludes participants from the previously reported analyses
[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.
We computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.
All analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere
[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).
As the current dataset excludes participants from the previously reported analyses
[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.
We computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.
All analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere
[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).
|
Results
|
Six hundred twenty eight (92%) of the 680 men with data on black carbon exposure, cognition, and all covariates were successfully genotyped for both HFE SNPs. Table
1 summarizes the sample characteristics for the current analysis in total and by HFE SNP genotypes. At baseline, our participants were, on average, 70 years old (SD: 7.1) and had 14.4 years of formal education (SD: 2.6). 540 (86%) of our participants lacked an HFE C282Y variant and 6 (1%) were homozygous for the C282Y variant. 469 (75%) lacked an HFE H63D variant and 18 (3%) were homozygous for the H63D variant. Both SNPs were in Hardy-Weinberg equilibrium (C282Y: χ2 = 2.05, p = 0.15; H63D: χ2 = 3.32, p = 0.07).
Baseline characteristics of the cohort (n = 628)
Abbreviations: HFE, hemochromatosis gene; SD, standard deviation; MET-hr/week, metabolic equivalent hours per week.
In the current study sample, the direction and magnitude of the association between black carbon and cognitive function in fully adjusted models was materially unchanged compared to previously reported analyses (Additional file
1: Table S1). As expected, there was little evidence to support a main effect of either HFE variant on cognition. The presence of an HFE C282Y variant did not appear to be associated with total cognition (beta: -0.063, 95% CI: -0.142, 0.017) or MMSE performance (odds ratio (OR): 0.92, 95% CI: 0.64, 1.35). Similarly, presence of an HFE H63D variant was not associated with either total cognition (beta: 0.022, 95% CI: -0.041, 0.085) or MMSE performance (OR: 0.98, 95% CI: 0.75, 1.28).
Consideration of regression models with interaction terms between HFE genotype and black carbon suggests that HFE C282Y, but not HFE H63D, modifies the association between black carbon concentrations and total cognitive function (Table
2). Although the p-values for interaction did not achieve the threshold for statistical significance in any model, the adverse association between black carbon exposure and cognition appears to be exclusive to those lacking the HFE C282Y variant. While participants who lacked an HFE C282Y variant exhibited strong adverse associations between BC and total cognition, a magnitude of effect that is equivalent to approximately 2.1 years of age in our data, we did not observe an adverse association for participants with at least one variant genotype. Similarly, we found elevated odds of low MMSE scores for a doubling in BC concentrations for participants who lacked an HFE C282Y variant but little evidence to support an association in participants with at least one copy of the HFE C282Y variant (Table
3).
The association between a doubling in BC concentration and total cognitive z-score by HFE polymorphisms
Abbreviations: HFE, hemochromatosis gene; BC, black carbon.
aAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.
The association between a doubling in BC concentration and low MMSE scores by HFE polymorphisms
Abbreviations: HFE, hemochromatosis gene; MMSE, Mini-Mental State Examination; BC, black carbon.
aAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.
We found little support for modification of the black carbon-cognition association by HFE H63D for either total cognition or MMSE performance. Although we observed statistically significant associations between black carbon and cognition within groups defined by HFE H63D genotype, the p-values for interaction did not achieve statistical significance in either model, and the pattern of association across those with and without the HFE H63D variant differed by outcome. In analyses of total cognition, we observed a statistically significant adverse association between black carbon concentrations among HFE H63D homozygous wild type participants, but not among those possessing at least one variant allele (Table
2). However, we observed the opposite pattern in analyses considering MMSE scores (Table
3), where we found a statistically significant adverse association in those with at least one H63D variant, but not in those who were homozygous wild type.
In sensitivity analyses, additional adjustment for hemoglobin, body mass index, smoking, or past lead exposure did not appreciably change effect estimates (data not shown).
|
Conclusions
|
In conclusion, our study suggests that persons who lack a HFE C282Y variant polymorphism may be more susceptible to adverse effects of exposure to traffic-related air pollution on cognition than those possessing a variant. The mechanism by which HFE modifies this association may involve metal transport and inflammation pathways. As with any candidate gene-environment interaction study, this analysis requires independent confirmation.
|
[
"Background",
"Study sample",
"Exposure assessment",
"Cognitive testing",
"HFE genotyping",
"Statistical methods",
"Abbreviations",
"Competing interests",
"Authors’ contributions"
] |
[
"While we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large\n[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.\nTraffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men\n[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above\n[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women\n[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese\n[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children\n[6-11].\nThe hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease\n[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium\n[14-18].\nPrevious studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors\n[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms.",
"The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.",
"For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.",
"The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.",
"For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.",
"As the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).",
"BC: Black carbon; BMI: Body mass index; HFE: Hemochromatosis gene; CI: Confidence interval; MET-hrs: Metabolic equivalent hours; MMSE: Mini-Mental State Examination; NAS: Normative Aging Study; OR: Odds ratio; QT interval: Heart-rate-corrected interval; VA: United States Department of Veterans Affairs.",
"The authors declare they have no competing interests.",
"MCP made substantial contributions to the study conception and design, analysis and interpretation of the data, and drafted the manuscript. MGW and JS made substantial contributions to the study conception and design, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. BAC, SEA, AS made substantial contributions to the acquisition of data and revised the manuscript critically for important intellectual content. ROW made substantial contributions to the analysis or interpretation of the data and revised the manuscript critically for important intellectual content. All authors have given final approval of the version to be published."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Study sample",
"Exposure assessment",
"Cognitive testing",
"HFE genotyping",
"Statistical methods",
"Results",
"Discussion",
"Conclusions",
"Abbreviations",
"Competing interests",
"Authors’ contributions",
"Supplementary Material"
] |
[
"While we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large\n[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.\nTraffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men\n[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above\n[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women\n[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese\n[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children\n[6-11].\nThe hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease\n[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium\n[14-18].\nPrevious studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors\n[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms.",
" Study sample The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.\nThe current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.\n Exposure assessment For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\nFor each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.\n Cognitive testing The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\nThe content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.\n HFE genotyping For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\nFor this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.\n Statistical methods As the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).\nAs the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).",
"The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis\n[2] we refer readers to that paper for a more exhaustive description of our methods.",
"For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995\n[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.",
"The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.",
"For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere\n[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.",
"As the current dataset excludes participants from the previously reported analyses\n[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.\nWe computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.\nAll analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere\n[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).",
"Six hundred twenty eight (92%) of the 680 men with data on black carbon exposure, cognition, and all covariates were successfully genotyped for both HFE SNPs. Table \n1 summarizes the sample characteristics for the current analysis in total and by HFE SNP genotypes. At baseline, our participants were, on average, 70 years old (SD: 7.1) and had 14.4 years of formal education (SD: 2.6). 540 (86%) of our participants lacked an HFE C282Y variant and 6 (1%) were homozygous for the C282Y variant. 469 (75%) lacked an HFE H63D variant and 18 (3%) were homozygous for the H63D variant. Both SNPs were in Hardy-Weinberg equilibrium (C282Y: χ2 = 2.05, p = 0.15; H63D: χ2 = 3.32, p = 0.07).\nBaseline characteristics of the cohort (n = 628)\nAbbreviations: HFE, hemochromatosis gene; SD, standard deviation; MET-hr/week, metabolic equivalent hours per week.\nIn the current study sample, the direction and magnitude of the association between black carbon and cognitive function in fully adjusted models was materially unchanged compared to previously reported analyses (Additional file\n1: Table S1). As expected, there was little evidence to support a main effect of either HFE variant on cognition. The presence of an HFE C282Y variant did not appear to be associated with total cognition (beta: -0.063, 95% CI: -0.142, 0.017) or MMSE performance (odds ratio (OR): 0.92, 95% CI: 0.64, 1.35). Similarly, presence of an HFE H63D variant was not associated with either total cognition (beta: 0.022, 95% CI: -0.041, 0.085) or MMSE performance (OR: 0.98, 95% CI: 0.75, 1.28).\nConsideration of regression models with interaction terms between HFE genotype and black carbon suggests that HFE C282Y, but not HFE H63D, modifies the association between black carbon concentrations and total cognitive function (Table \n2). Although the p-values for interaction did not achieve the threshold for statistical significance in any model, the adverse association between black carbon exposure and cognition appears to be exclusive to those lacking the HFE C282Y variant. While participants who lacked an HFE C282Y variant exhibited strong adverse associations between BC and total cognition, a magnitude of effect that is equivalent to approximately 2.1 years of age in our data, we did not observe an adverse association for participants with at least one variant genotype. Similarly, we found elevated odds of low MMSE scores for a doubling in BC concentrations for participants who lacked an HFE C282Y variant but little evidence to support an association in participants with at least one copy of the HFE C282Y variant (Table \n3).\nThe association between a doubling in BC concentration and total cognitive z-score by HFE polymorphisms\nAbbreviations: HFE, hemochromatosis gene; BC, black carbon.\naAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.\nThe association between a doubling in BC concentration and low MMSE scores by HFE polymorphisms\nAbbreviations: HFE, hemochromatosis gene; MMSE, Mini-Mental State Examination; BC, black carbon.\naAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.\nWe found little support for modification of the black carbon-cognition association by HFE H63D for either total cognition or MMSE performance. Although we observed statistically significant associations between black carbon and cognition within groups defined by HFE H63D genotype, the p-values for interaction did not achieve statistical significance in either model, and the pattern of association across those with and without the HFE H63D variant differed by outcome. In analyses of total cognition, we observed a statistically significant adverse association between black carbon concentrations among HFE H63D homozygous wild type participants, but not among those possessing at least one variant allele (Table \n2). However, we observed the opposite pattern in analyses considering MMSE scores (Table \n3), where we found a statistically significant adverse association in those with at least one H63D variant, but not in those who were homozygous wild type.\nIn sensitivity analyses, additional adjustment for hemoglobin, body mass index, smoking, or past lead exposure did not appreciably change effect estimates (data not shown).",
"Although the statistical evidence to support the claim that the HFE C282Y polymorphism modifies the association between traffic-related air pollution and cognitive function in our sample of older men is weak, the pattern of response across groups is intriguing and suggests the presence of effect modification by HFE C282Y. Participants possessing a variant HFE C282Y polymorphism appear to be protected from the adverse association between black carbon and cognition, while participants who lacked the C282Y variant exhibit strong adverse associations. We found little support for modification of the association between traffic-related air pollution and cognition by HFE H63D.\nThis is the first study to investigate effect modification by HFE of the air pollution-cognition relationship. These findings are in line with reported effect modification by HFE of the relationship between exposure to air pollution and cardiovascular outcomes or risk factors in the NAS cohort. Persons lacking HFE variant genotypes exhibit strong adverse associations between exposure to fine particulate matter (PM) and heart rate variability, while there is little evidence for an association among those possessing an HFE variant, and this pattern was driven primarily by the HFE C282Y SNP\n[20]. Similarly, higher exposures to black carbon or PM2.5 were associated with higher plasma homocysteine concentrations in participants who lacked the HFE C282Y variant, but no association was observed in participants possessing at least one HFE C282Y variant\n[19]. A similar pattern was observed for modification by HFE C282Y of the association between traffic-related air pollution and heart-rate-corrected (QT) interval, an electrocardiographic marker of ventricular repolarization that is associated with arrhythmia and cardiac death\n[21].\nThe mechanism by which exposure to traffic-related air pollution may contribute to cognitive impairment is unknown, but likely involves inhalation of particulates. In animal experiments, exposure to fine or ultrafine particulate matter appears to induce inflammation, lipid peroxidation, and neuronal degeneration in the central nervous system\n[25-28]. Metals, including iron, are often found adsorbed onto the surface of traffic-related particulates\n[29] and exposure to metals is associated with induction of oxidative stress and inflammation\n[30]. Oxidative stress and inflammation may contribute to the development of cognitive impairment directly or through inducement of cardiovascular problems.\nHFE modulates intracellular uptake of iron and other metals. Variants of HFE are associated with the disease hemochromatosis, which is characterized by iron overload due to excessive uptake of iron from the gastrointestinal tract\n[12]. Studies of HFE modulation of the health effects of air pollution, including the current study, generally find that variants in HFE protect against the adverse impact of air pollution exposure\n[19,20]. These findings may be attributable to HFE modulation of uptake of metals from the lung. Exposure to traffic-related air pollution occurs primarily through inhalation. In order for traffic-related air pollution to adversely impact human health, some component of traffic-related air pollution must enter the body, either through penetration of the respiratory epithelium, ingestion, or through translocation up the olfactory nerve. Variants of HFE C282Y and H63D are associated with increased iron uptake from the gut, and higher body stores of iron may down-regulate overall metal absorption, including absorption from the lung of metals adhered to inhaled air pollution. In animal models, rats fed a high-iron diet or exposed to iron oxide exhibited less absorption of manganese after intratracheal installation compared to those fed a regular diet\n[17,18]. This reduction in pulmonary absorption was paralleled by lower uptake by other organs, including the brain\n[18]. Therefore, HFE genotype, associated iron levels, and iron homeostatic mechanisms could influence the toxicity of exposure to traffic-related air pollution through modulation of metal uptake. Lower uptake of adsorbed metals would be expected to reduce the associated systemic and brain-based oxidative stress and inflammation and lessen any adverse effect of air pollution on cognition. This mechanistic hypothesis might explain why we did not see an adverse association between air pollution and cognition in carriers of the HFE C282Y variant.\nWe also note that the evidence to support effect modification of the association between air pollution and cognition was stronger when we considered the C282Y variant than when we considered the H63D variant, which is consistent with the relative degree of function of these SNPs. The HFE protein produced by the C282Y variant, but not the H63D variant protein, is often degraded before final processing and is not expressed on the cell surface\n[31] and does not associate with transferrin\n[32]. As such, the C282Y variant is associated with greater loss of function compared to the H63D variant, resulting in increased iron transfer in the gut. In this case, we may expect lower respiratory intake of adsorbed metals, and therefore lower levels of air pollution-related oxidative stress and inflammation, in those with the C282Y variant than in those with the H63D variant.\nWe acknowledge that our study has several limitations. Use of BC exposure estimates based on residential address may misclassify long-term exposure. However, given the age and residential stability of our cohort, misclassification of our exposure due to occupation, commuting, or relocation is expected to be minimal and largely unrelated to cognition or HFE status, suggesting bias towards the null. Non-differential misclassification of cognitive status is likely, but is mitigated by the use of all available cognitive data to consider the association between BC and total metrics of cognition. It is possible that residual confounding may bias our results. However, we were able to adjust for many known predictors of cognition, including several measures of socioeconomic status. Furthermore, we expect confounding by other classes of air pollution to be minimal, as the correlation between traffic-related air pollution and regional pollutants is relatively low due to differences in the spatial-temporal distribution of these types of pollutants. Selection bias is a concern, but because both poor cognition and exposure to traffic-related air pollution predict morbidity and mortality\n[33-36], we would expect selection bias to mask the expected adverse association or create a false protective association. While this may contribute to the absence of an adverse association among participants with HFE C282Y variants, it would not account for the strong adverse association observed within those who lacked HFE C282Y variants. While we are unable to concretely attribute our findings to a particular aspect of traffic-related exposure, modification of the BC-cognition association by HFE implicates metals adhered to particulate matter as a likely toxic agent. While population substructure may induce spurious associations in gene-environment interaction studies, this is unlikely to account for our findings given our homogeneous study sample of white men. Finally, our sample size and the number of participants with the variant HFE alleles are small, which makes it difficult to detect a subtle effect and increases the possibility that our findings are due to chance. This analysis requires replication in an independent sample.",
"In conclusion, our study suggests that persons who lack a HFE C282Y variant polymorphism may be more susceptible to adverse effects of exposure to traffic-related air pollution on cognition than those possessing a variant. The mechanism by which HFE modifies this association may involve metal transport and inflammation pathways. As with any candidate gene-environment interaction study, this analysis requires independent confirmation.",
"BC: Black carbon; BMI: Body mass index; HFE: Hemochromatosis gene; CI: Confidence interval; MET-hrs: Metabolic equivalent hours; MMSE: Mini-Mental State Examination; NAS: Normative Aging Study; OR: Odds ratio; QT interval: Heart-rate-corrected interval; VA: United States Department of Veterans Affairs.",
"The authors declare they have no competing interests.",
"MCP made substantial contributions to the study conception and design, analysis and interpretation of the data, and drafted the manuscript. MGW and JS made substantial contributions to the study conception and design, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. BAC, SEA, AS made substantial contributions to the acquisition of data and revised the manuscript critically for important intellectual content. ROW made substantial contributions to the analysis or interpretation of the data and revised the manuscript critically for important intellectual content. All authors have given final approval of the version to be published.",
"Comparison the association between a doubling in BC concentration and cognition in the HFE dataset to the previously reported association in the full dataset. Table showing the main effect of BC exposure in the full dataset used in the original analyses and the reduced dataset used in the current analyses.\nClick here for file"
] |
[
null,
"methods",
null,
null,
null,
null,
null,
"results",
"discussion",
"conclusions",
null,
null,
null,
"supplementary-material"
] |
[
"Aging",
"Black carbon",
"Cognitive dysfunction",
"Epidemiology",
"Particulate matter",
"HFE",
"Hemochromatosis",
"Gene-environment interaction",
"Susceptible group"
] |
Background:
While we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large
[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.
Traffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men
[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above
[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women
[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese
[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children
[6-11].
The hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease
[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium
[14-18].
Previous studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors
[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms.
Methods:
Study sample The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis
[2] we refer readers to that paper for a more exhaustive description of our methods.
The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis
[2] we refer readers to that paper for a more exhaustive description of our methods.
Exposure assessment For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995
[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.
For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995
[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.
Cognitive testing The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.
The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.
HFE genotyping For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere
[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.
For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere
[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.
Statistical methods As the current dataset excludes participants from the previously reported analyses
[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.
We computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.
All analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere
[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).
As the current dataset excludes participants from the previously reported analyses
[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.
We computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.
All analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere
[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).
Study sample:
The current study sample consists of participants from the United States Department of Veterans Affairs (VA) Normative Aging Study (NAS). This ongoing study of men was established in 1963, and since that time participants have been invited to complete an in-person examination every 3 years. At each visit the men provided written informed consent as approved by the VA Boston Healthcare System Institutional Review Board. The current study sample is limited to white participants with complete covariate data who completed at least one cognitive assessment between 1996 and 2007 and for whom we were able to obtain an estimate of long-term exposure to black carbon, a marker of traffic-related air pollution. For this study, we refer to the date of the first cognitive assessment completed during or after 1996 as the baseline visit. As this analysis is a straightforward extension of a previously reported analysis
[2] we refer readers to that paper for a more exhaustive description of our methods.
Exposure assessment:
For each participant, we used the average estimated black carbon concentration at their residential address in the year prior to their first cognitive assessment as a measure of long-term exposure to traffic-related air pollution. This estimate was derived using a validated spatio-temporal land use regression model that provides daily estimates of black carbon concentration throughout the greater Boston area starting in 1995
[22]. Estimates of black carbon concentration were log-transformed for use in all analyses.
Cognitive testing:
The content of the battery of cognitive tests used in the NAS has changed since cognitive assessment began in the mid-1990s. We focus on data from 7 cognitive tests administered at most cognitive assessments: the Mini-Mental State Examination (MMSE), the digit span backwards test, a verbal fluency task, constructional praxis, immediate recall of the ten word list, delayed recall of the ten word list, and a pattern comparison task. These seven tests were drawn from established batteries and assess a variety of domains; collectively, we use these tests to provide an assessment of total cognitive function. Most study participants have completed multiple waves of cognitive assessment, all of which were used in our analyses. As the MMSE exhibited a strong ceiling effect, with a large proportion of participants achieving scores at or near the maximum, MMSE scores were dichotomized at ≤25 and were considered in separate analyses (while a score of ≤24 is commonly used as a cutoff for dementia screening in research settings, very few of our participants met this criterion). The 7 scores produced by the remaining 6 cognitive tests (the pattern recognition task produces two unique scores that assess different aspects of cognition), were each standardized to a z-score using the mean and standard deviation of scores from the baseline cognitive assessment in our sample.
HFE genotyping:
For this study we focus on two single nucleotide polymorphisms (SNPs) known to produce missense mutations in HFE: HFE C282Y (rs1800562) and HFE H63D (rs1799945). We genotyped for these SNPs in a subset of NAS participants with archived blood samples using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA) by inputting sequences containing the SNP site and 100 bp of flanking sequence on either side. More detailed information on genotyping, including details of laboratory methods, primers, and quality control, can be found elsewhere
[20]. Participants for whom valid HFE SNP genotyping could not be obtained were excluded from all analyses. HFE SNP genotypes were coded as a dichotomous variable indicating presence or absence of a variant allele, given the small prevalence of homozygous variant persons in Caucasian populations.
Statistical methods:
As the current dataset excludes participants from the previously reported analyses
[2] who lacked valid HFE genotyping, we first reproduced the previously reported associations for the main effects of black carbon exposure on MMSE scores and total cognition in this reduced dataset. We used logistic regression with generalized estimating equations and empirical variance estimates to assess the association between black carbon and low MMSE scores to account for use of multiple waves of cognitive testing per participant. Using this model, we estimated the odds ratio for having a low MMSE score per doubling in black carbon concentrations. We also used a linear mixed model with random intercepts for individual and test to assess the association between black carbon and “total” cognitive function. As with the MMSE analysis, this analysis incorporated multiple waves of cognitive testing per participant for those with more than one assessment. It also accounted for the fact that we have multiple cognitive tests per assessment, as it effectively treats each cognitive test score as a repeat measure of underlying total cognition. Using this model, we estimated the difference in “total cognitive z-score” for a doubling in black carbon concentrations. Due to the small mean number of repeat assessments per person (2.15), we did not investigate the association between black carbon and cognitive trajectories in either the original or the current analysis.
We computed SNP frequencies and assessed Hardy-Weinberg equilibrium within our sample for both HFE SNPs. We began by evaluating the main effect of each of the two HFE SNPs, H63D and C282Y, on the odds of having a low MMSE score and on total cognition in four separate models. We then added multiplicative interaction terms between the pertinent HFE SNP and black carbon to each of these four models to assess the potential for effect modification of the black carbon-cognition association by each HFE SNP. We used Wald tests of the multiplicative interaction term and a p-value threshold of 0.05 to assess whether there was statistically significant support for effect modification of the black carbon-cognition relationship by HFE SNP in each model.
All analyses were adjusted for age at cognitive assessment and the following variables, assessed at baseline: education (≤12, 12-16, 16+ years), alcohol intake (<2/2+ drinks per day), physical activity (<12, 12-30, 30+ metabolic equivalent hours (MET-hrs) per week), diabetes (yes/no), dark fish consumption (less than once a week/once a week or more), computer experience (yes/no), first language (English/not English), percent of the participant’s census tract that is non-white, percent of the adult residents in the participant’s census tract with at least a college degree, an indicator for whether the cognitive data were from the participant’s first cognitive assessment (yes/no), and an indicator for whether the participant was a part-time resident of the greater Boston area (yes/no). In sensitivity analyses, we additionally adjusted all models for hemoglobin, body mass index, smoking status, and estimated long-term exposure to lead (as lead exposure has been associated with cognition in this cohort and the fact that traffic-related air pollution was a major source of lead exposure in the era of leaded gasoline). We used either measured (n = 345) or imputed (n = 270) bone lead concentrations as our estimate of long-term lead exposure, and excluded those without measured tibia lead levels who were missing essential covariates required for imputation (n = 13). Details regarding the measurement or imputation of bone lead concentrations are available elsewhere
[2,23,24]. All analyses were performed with R (version 2.10.1; R Development Core Team 2010) or SAS (version 9.2; SAS Institute Inc., Cary, NC).
Results:
Six hundred twenty eight (92%) of the 680 men with data on black carbon exposure, cognition, and all covariates were successfully genotyped for both HFE SNPs. Table
1 summarizes the sample characteristics for the current analysis in total and by HFE SNP genotypes. At baseline, our participants were, on average, 70 years old (SD: 7.1) and had 14.4 years of formal education (SD: 2.6). 540 (86%) of our participants lacked an HFE C282Y variant and 6 (1%) were homozygous for the C282Y variant. 469 (75%) lacked an HFE H63D variant and 18 (3%) were homozygous for the H63D variant. Both SNPs were in Hardy-Weinberg equilibrium (C282Y: χ2 = 2.05, p = 0.15; H63D: χ2 = 3.32, p = 0.07).
Baseline characteristics of the cohort (n = 628)
Abbreviations: HFE, hemochromatosis gene; SD, standard deviation; MET-hr/week, metabolic equivalent hours per week.
In the current study sample, the direction and magnitude of the association between black carbon and cognitive function in fully adjusted models was materially unchanged compared to previously reported analyses (Additional file
1: Table S1). As expected, there was little evidence to support a main effect of either HFE variant on cognition. The presence of an HFE C282Y variant did not appear to be associated with total cognition (beta: -0.063, 95% CI: -0.142, 0.017) or MMSE performance (odds ratio (OR): 0.92, 95% CI: 0.64, 1.35). Similarly, presence of an HFE H63D variant was not associated with either total cognition (beta: 0.022, 95% CI: -0.041, 0.085) or MMSE performance (OR: 0.98, 95% CI: 0.75, 1.28).
Consideration of regression models with interaction terms between HFE genotype and black carbon suggests that HFE C282Y, but not HFE H63D, modifies the association between black carbon concentrations and total cognitive function (Table
2). Although the p-values for interaction did not achieve the threshold for statistical significance in any model, the adverse association between black carbon exposure and cognition appears to be exclusive to those lacking the HFE C282Y variant. While participants who lacked an HFE C282Y variant exhibited strong adverse associations between BC and total cognition, a magnitude of effect that is equivalent to approximately 2.1 years of age in our data, we did not observe an adverse association for participants with at least one variant genotype. Similarly, we found elevated odds of low MMSE scores for a doubling in BC concentrations for participants who lacked an HFE C282Y variant but little evidence to support an association in participants with at least one copy of the HFE C282Y variant (Table
3).
The association between a doubling in BC concentration and total cognitive z-score by HFE polymorphisms
Abbreviations: HFE, hemochromatosis gene; BC, black carbon.
aAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.
The association between a doubling in BC concentration and low MMSE scores by HFE polymorphisms
Abbreviations: HFE, hemochromatosis gene; MMSE, Mini-Mental State Examination; BC, black carbon.
aAdjusted for age, education, first language, computer experience, physical activity, alcohol consumption, diabetes, dark fish consumption, percentage of residential census tract that is nonwhite, percentage of residential census tract adults with a college degree, indicator for first cognitive assessment, and indicator for part-time resident.
We found little support for modification of the black carbon-cognition association by HFE H63D for either total cognition or MMSE performance. Although we observed statistically significant associations between black carbon and cognition within groups defined by HFE H63D genotype, the p-values for interaction did not achieve statistical significance in either model, and the pattern of association across those with and without the HFE H63D variant differed by outcome. In analyses of total cognition, we observed a statistically significant adverse association between black carbon concentrations among HFE H63D homozygous wild type participants, but not among those possessing at least one variant allele (Table
2). However, we observed the opposite pattern in analyses considering MMSE scores (Table
3), where we found a statistically significant adverse association in those with at least one H63D variant, but not in those who were homozygous wild type.
In sensitivity analyses, additional adjustment for hemoglobin, body mass index, smoking, or past lead exposure did not appreciably change effect estimates (data not shown).
Discussion:
Although the statistical evidence to support the claim that the HFE C282Y polymorphism modifies the association between traffic-related air pollution and cognitive function in our sample of older men is weak, the pattern of response across groups is intriguing and suggests the presence of effect modification by HFE C282Y. Participants possessing a variant HFE C282Y polymorphism appear to be protected from the adverse association between black carbon and cognition, while participants who lacked the C282Y variant exhibit strong adverse associations. We found little support for modification of the association between traffic-related air pollution and cognition by HFE H63D.
This is the first study to investigate effect modification by HFE of the air pollution-cognition relationship. These findings are in line with reported effect modification by HFE of the relationship between exposure to air pollution and cardiovascular outcomes or risk factors in the NAS cohort. Persons lacking HFE variant genotypes exhibit strong adverse associations between exposure to fine particulate matter (PM) and heart rate variability, while there is little evidence for an association among those possessing an HFE variant, and this pattern was driven primarily by the HFE C282Y SNP
[20]. Similarly, higher exposures to black carbon or PM2.5 were associated with higher plasma homocysteine concentrations in participants who lacked the HFE C282Y variant, but no association was observed in participants possessing at least one HFE C282Y variant
[19]. A similar pattern was observed for modification by HFE C282Y of the association between traffic-related air pollution and heart-rate-corrected (QT) interval, an electrocardiographic marker of ventricular repolarization that is associated with arrhythmia and cardiac death
[21].
The mechanism by which exposure to traffic-related air pollution may contribute to cognitive impairment is unknown, but likely involves inhalation of particulates. In animal experiments, exposure to fine or ultrafine particulate matter appears to induce inflammation, lipid peroxidation, and neuronal degeneration in the central nervous system
[25-28]. Metals, including iron, are often found adsorbed onto the surface of traffic-related particulates
[29] and exposure to metals is associated with induction of oxidative stress and inflammation
[30]. Oxidative stress and inflammation may contribute to the development of cognitive impairment directly or through inducement of cardiovascular problems.
HFE modulates intracellular uptake of iron and other metals. Variants of HFE are associated with the disease hemochromatosis, which is characterized by iron overload due to excessive uptake of iron from the gastrointestinal tract
[12]. Studies of HFE modulation of the health effects of air pollution, including the current study, generally find that variants in HFE protect against the adverse impact of air pollution exposure
[19,20]. These findings may be attributable to HFE modulation of uptake of metals from the lung. Exposure to traffic-related air pollution occurs primarily through inhalation. In order for traffic-related air pollution to adversely impact human health, some component of traffic-related air pollution must enter the body, either through penetration of the respiratory epithelium, ingestion, or through translocation up the olfactory nerve. Variants of HFE C282Y and H63D are associated with increased iron uptake from the gut, and higher body stores of iron may down-regulate overall metal absorption, including absorption from the lung of metals adhered to inhaled air pollution. In animal models, rats fed a high-iron diet or exposed to iron oxide exhibited less absorption of manganese after intratracheal installation compared to those fed a regular diet
[17,18]. This reduction in pulmonary absorption was paralleled by lower uptake by other organs, including the brain
[18]. Therefore, HFE genotype, associated iron levels, and iron homeostatic mechanisms could influence the toxicity of exposure to traffic-related air pollution through modulation of metal uptake. Lower uptake of adsorbed metals would be expected to reduce the associated systemic and brain-based oxidative stress and inflammation and lessen any adverse effect of air pollution on cognition. This mechanistic hypothesis might explain why we did not see an adverse association between air pollution and cognition in carriers of the HFE C282Y variant.
We also note that the evidence to support effect modification of the association between air pollution and cognition was stronger when we considered the C282Y variant than when we considered the H63D variant, which is consistent with the relative degree of function of these SNPs. The HFE protein produced by the C282Y variant, but not the H63D variant protein, is often degraded before final processing and is not expressed on the cell surface
[31] and does not associate with transferrin
[32]. As such, the C282Y variant is associated with greater loss of function compared to the H63D variant, resulting in increased iron transfer in the gut. In this case, we may expect lower respiratory intake of adsorbed metals, and therefore lower levels of air pollution-related oxidative stress and inflammation, in those with the C282Y variant than in those with the H63D variant.
We acknowledge that our study has several limitations. Use of BC exposure estimates based on residential address may misclassify long-term exposure. However, given the age and residential stability of our cohort, misclassification of our exposure due to occupation, commuting, or relocation is expected to be minimal and largely unrelated to cognition or HFE status, suggesting bias towards the null. Non-differential misclassification of cognitive status is likely, but is mitigated by the use of all available cognitive data to consider the association between BC and total metrics of cognition. It is possible that residual confounding may bias our results. However, we were able to adjust for many known predictors of cognition, including several measures of socioeconomic status. Furthermore, we expect confounding by other classes of air pollution to be minimal, as the correlation between traffic-related air pollution and regional pollutants is relatively low due to differences in the spatial-temporal distribution of these types of pollutants. Selection bias is a concern, but because both poor cognition and exposure to traffic-related air pollution predict morbidity and mortality
[33-36], we would expect selection bias to mask the expected adverse association or create a false protective association. While this may contribute to the absence of an adverse association among participants with HFE C282Y variants, it would not account for the strong adverse association observed within those who lacked HFE C282Y variants. While we are unable to concretely attribute our findings to a particular aspect of traffic-related exposure, modification of the BC-cognition association by HFE implicates metals adhered to particulate matter as a likely toxic agent. While population substructure may induce spurious associations in gene-environment interaction studies, this is unlikely to account for our findings given our homogeneous study sample of white men. Finally, our sample size and the number of participants with the variant HFE alleles are small, which makes it difficult to detect a subtle effect and increases the possibility that our findings are due to chance. This analysis requires replication in an independent sample.
Conclusions:
In conclusion, our study suggests that persons who lack a HFE C282Y variant polymorphism may be more susceptible to adverse effects of exposure to traffic-related air pollution on cognition than those possessing a variant. The mechanism by which HFE modifies this association may involve metal transport and inflammation pathways. As with any candidate gene-environment interaction study, this analysis requires independent confirmation.
Abbreviations:
BC: Black carbon; BMI: Body mass index; HFE: Hemochromatosis gene; CI: Confidence interval; MET-hrs: Metabolic equivalent hours; MMSE: Mini-Mental State Examination; NAS: Normative Aging Study; OR: Odds ratio; QT interval: Heart-rate-corrected interval; VA: United States Department of Veterans Affairs.
Competing interests:
The authors declare they have no competing interests.
Authors’ contributions:
MCP made substantial contributions to the study conception and design, analysis and interpretation of the data, and drafted the manuscript. MGW and JS made substantial contributions to the study conception and design, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. BAC, SEA, AS made substantial contributions to the acquisition of data and revised the manuscript critically for important intellectual content. ROW made substantial contributions to the analysis or interpretation of the data and revised the manuscript critically for important intellectual content. All authors have given final approval of the version to be published.
Supplementary Material:
Comparison the association between a doubling in BC concentration and cognition in the HFE dataset to the previously reported association in the full dataset. Table showing the main effect of BC exposure in the full dataset used in the original analyses and the reduced dataset used in the current analyses.
Click here for file
|
Background:
Previous studies found effect modification of associations between traffic-related air pollution and cardiovascular outcomes by polymorphisms in the hemochromatosis gene (HFE). As traffic-related air pollution may impact cognition through effects on cardiovascular health or through mechanisms which may also influence cardiovascular outcomes, we hypothesized that HFE polymorphisms would also modify a previously observed association between traffic-related air pollution exposure and cognition in older men.
Methods:
We considered data from 628 participants of the VA Normative Aging Study. We estimated long term exposure to black carbon (BC), a marker of traffic related air pollution, using a spatio-temporal land use regression model. We assessed cognition using the Mini-Mental State Examination (MMSE), a test of global function, and performance on a battery of other tests, covering a wide range of domains. We investigated whether variants of HFE C282Y and H63D modified the association between BC and having a low MMSE score using logistic models with generalized estimating equations and multiplicative interaction terms. Similarly, we assessed whether HFE variants modified the association between BC and performance on the cognitive battery using linear mixed models with multiplicative interaction terms.
Results:
Our results suggest modification of the BC-cognition association by HFE C282Y, although the test of interaction did not achieve statistical significance. In multivariable-adjusted models, participants who lacked a HFE C282Y variant (CC) exhibited an adverse association between BC and total cognition z-score (beta for a doubling in BC concentration: -0.061, 95% CI: -0.115, -0.007), while we did not observe an association in participants with at least one variant genotype (CY or YY) (beta for a doubling in BC concentration: 0.073, 95% CI: -0.081, 0.228; p-value for interaction: 0.11). The pattern of association was similar for analyses considering performance on the Mini-Mental State Examination. There was little evidence to support effect modification of the BC-cognition association by the HFE H63D genotype.
Conclusions:
Our data suggest that older adults who lack an HFE C282Y variant may be more susceptible to an adverse effect of traffic-related air pollution exposure on cognition. This finding and the proposed biological mechanism require confirmation.
|
Background:
While we often focus on mean response to exposures in epidemiology, there are many reasons to be interested in identifying groups of individuals who are particularly susceptible to exposure-related health effects. First, identification of susceptible groups, particularly genetically susceptible groups, may provide insight into the mechanisms behind the association. Epidemiologic gene-environment interaction studies have some advantages over toxicology in this respect because we evaluate susceptibility of people, rather than laboratory animals or cell lines, at doses currently experienced in the population at large
[1]. Second, governmental agencies are sometimes charged with or prioritize protecting susceptible or otherwise sensitive groups. Identification and understanding of associations between environmental or occupational exposures and health effects within these groups is essential to understand the true risks associated with exposure in the population and to evaluate the appropriateness of existing or proposed regulatory standards.
Traffic-related air pollution may adversely impact cognition through induction of inflammation and oxidative stress. We previously found a significant association between long-term exposure to black carbon, a marker of traffic-related air pollution, and cognition in older men
[2]. Higher levels of black carbon exposure were associated with lower cognitive test performance. Similarly, in a cohort of older German women, living close to a major road, a marker of elevated exposure to traffic-related air pollution, was associated with lower cognitive test scores, especially among those 74 years old and above
[3]. Other types of air pollution may also impact cognition through similar mechanisms, as exposure to both coarse and fine particulate matter was associated with faster cognitive decline in a cohort of older women
[4] and worse air quality measures were associated with increased disability in activities of daily living and cognitive impairment in elderly Chinese
[5]. Finally, there may also be an adverse effect of air pollution exposure on cognition in younger adults or children
[6-11].
The hemochromatosis (HFE) gene regulates iron homeostatsis. Two common missense polymorphisms of this gene, HFE C282Y and H63D are associated with the disease state of hemochromatosis, an autosomal recessive genetic disease that causes an increase in absorption of ingested iron. Although the penetrance is low, this can, over time, lead to iron overload, manifesting in higher rates of diabetes, heart disease, and liver disease
[12,13]. Variation in these HFE polymorphisms may also appear to impact body burden of other metals, particularly divalent cations like manganese, lead and cadmium
[14-18].
Previous studies have reported that HFE polymorphisms modify the association between air pollution exposure and cardiovascular outcomes or risk factors
[19-21]. HFE polymorphisms may modify the uptake of metals adhered to particulate matter, altering the inflammatory response to external exposure, which has been proposed as a primary mechanism for the adverse association between air pollution and both cardiovascular endpoints and cognition. For the current study, we hypothesized that the previously observed adverse association between traffic-related air pollution and cognition would be weaker in participants with variant HFE polymorphisms.
Conclusions:
In conclusion, our study suggests that persons who lack a HFE C282Y variant polymorphism may be more susceptible to adverse effects of exposure to traffic-related air pollution on cognition than those possessing a variant. The mechanism by which HFE modifies this association may involve metal transport and inflammation pathways. As with any candidate gene-environment interaction study, this analysis requires independent confirmation.
|
Background:
Previous studies found effect modification of associations between traffic-related air pollution and cardiovascular outcomes by polymorphisms in the hemochromatosis gene (HFE). As traffic-related air pollution may impact cognition through effects on cardiovascular health or through mechanisms which may also influence cardiovascular outcomes, we hypothesized that HFE polymorphisms would also modify a previously observed association between traffic-related air pollution exposure and cognition in older men.
Methods:
We considered data from 628 participants of the VA Normative Aging Study. We estimated long term exposure to black carbon (BC), a marker of traffic related air pollution, using a spatio-temporal land use regression model. We assessed cognition using the Mini-Mental State Examination (MMSE), a test of global function, and performance on a battery of other tests, covering a wide range of domains. We investigated whether variants of HFE C282Y and H63D modified the association between BC and having a low MMSE score using logistic models with generalized estimating equations and multiplicative interaction terms. Similarly, we assessed whether HFE variants modified the association between BC and performance on the cognitive battery using linear mixed models with multiplicative interaction terms.
Results:
Our results suggest modification of the BC-cognition association by HFE C282Y, although the test of interaction did not achieve statistical significance. In multivariable-adjusted models, participants who lacked a HFE C282Y variant (CC) exhibited an adverse association between BC and total cognition z-score (beta for a doubling in BC concentration: -0.061, 95% CI: -0.115, -0.007), while we did not observe an association in participants with at least one variant genotype (CY or YY) (beta for a doubling in BC concentration: 0.073, 95% CI: -0.081, 0.228; p-value for interaction: 0.11). The pattern of association was similar for analyses considering performance on the Mini-Mental State Examination. There was little evidence to support effect modification of the BC-cognition association by the HFE H63D genotype.
Conclusions:
Our data suggest that older adults who lack an HFE C282Y variant may be more susceptible to an adverse effect of traffic-related air pollution exposure on cognition. This finding and the proposed biological mechanism require confirmation.
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[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]
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[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]
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[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]
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[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]
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[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]
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[CONTENT] Aging | Black carbon | Cognitive dysfunction | Epidemiology | Particulate matter | HFE | Hemochromatosis | Gene-environment interaction | Susceptible group [SUMMARY]
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[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]
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[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]
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[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]
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[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]
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[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]
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[CONTENT] Aged | Air Pollutants | Cognition | Cohort Studies | Genotype | Hemochromatosis Protein | Histocompatibility Antigens Class I | Humans | Male | Membrane Proteins | Middle Aged | Multiplex Polymerase Chain Reaction | Polymorphism, Single Nucleotide | Soot | United States | Vehicle Emissions [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]
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[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]
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[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]
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[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]
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[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]
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[CONTENT] hfe | cognitive | black | black carbon | carbon | cognition | exposure | association | participants | variant [SUMMARY]
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[CONTENT] air | pollution | air pollution | exposure | groups | associated | polymorphisms | hfe polymorphisms | disease | susceptible [SUMMARY]
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[CONTENT] cognitive | assessment | carbon | black carbon | black | participant | tests | hfe | mmse | lead [SUMMARY]
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[CONTENT] hfe | variant | association | h63d | c282y variant | table | total | black carbon | carbon | cognition [SUMMARY]
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[CONTENT] variant | hfe modifies | pollution cognition possessing variant | pollution cognition possessing | mechanism hfe modifies association | mechanism hfe modifies | mechanism hfe | hfe modifies association involve | hfe modifies association | effects exposure traffic [SUMMARY]
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[CONTENT] hfe | cognitive | black carbon | black | carbon | association | cognition | variant | exposure | air [SUMMARY]
|
[CONTENT] hfe | cognitive | black carbon | black | carbon | association | cognition | variant | exposure | air [SUMMARY]
|
[CONTENT] ||| [SUMMARY]
|
[CONTENT] 628 | the VA Normative Aging Study ||| BC ||| the Mini-Mental State Examination ||| H63D | BC ||| BC | linear [SUMMARY]
|
[CONTENT] BC ||| CC | BC | BC | 95% | CI | at least one | YY | BC | 0.073 | 95% | CI | 0.228 | 0.11 ||| the Mini-Mental State Examination ||| BC [SUMMARY]
|
[CONTENT] ||| [SUMMARY]
|
[CONTENT] ||| ||| 628 | the VA Normative Aging Study ||| BC ||| the Mini-Mental State Examination ||| H63D | BC ||| BC | linear ||| ||| BC ||| CC | BC | BC | 95% | CI | at least one | YY | BC | 0.073 | 95% | CI | 0.228 | 0.11 ||| the Mini-Mental State Examination ||| BC ||| ||| [SUMMARY]
|
[CONTENT] ||| ||| 628 | the VA Normative Aging Study ||| BC ||| the Mini-Mental State Examination ||| H63D | BC ||| BC | linear ||| ||| BC ||| CC | BC | BC | 95% | CI | at least one | YY | BC | 0.073 | 95% | CI | 0.228 | 0.11 ||| the Mini-Mental State Examination ||| BC ||| ||| [SUMMARY]
|
||||||
Atrioventricular block in coronary artery bypass surgery: perioperative predictors and impact on mortality.
|
26107447
|
Disturbances of the cardiac conduction system are frequent in the postoperative period of coronary artery bypass surgery. They are mostly reversible and associated with some injury of the conduction tissue, caused by the ischemic heart disease itself or by perioperative factors.
|
INTRODUCTION
|
Analysis of a retrospective cohort of patients submitted to coronary artery bypass surgery from the database of the Postoperative Heart Surgery Unit of the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande do Sul, using the logistic regression method.
|
METHODS
|
In the period from January 1996 to December 2012, 3532 coronary artery bypass surgery were carried out. Two hundred and eighty-eight (8.15% of the total sample) patients had atrioventricular block during the postoperative period of coronary artery bypass surgery, requiring temporary pacing. Eight of those who had atrioventricular block progressed to implantation of a permanent pacemaker (0.23% of the total sample). Multivariate analysis revealed a significant association of atrioventricular block with age above 60 years (OR=2.34; CI 95% 1.75-3.12; P<0.0001), female gender (OR=1.37; CI 95% 1.06-1.77; P=0.015), chronic kidney disease (OR=2.05; CI 95% 1.49-2.81; P<0.0001), atrial fibrillation (OR=2.06; CI 95% 1.16-3.66; P=0.014), functional class III and IV of the New York Heart Association (OR=1.43; CI 95% 1.03-1.98; P=0.031), perioperative acute myocardial infarction (OR=1.70; CI 95% 1.26-2.29; P<0.0001) and with the use of the intra-aortic balloon in the postoperative period of coronary artery bypass surgery (OR=1.92; CI 95% 1.21-3.05; P=0.006). The presence of atrioventricular block resulted in a significant increase in mortality (17.9% vs. 7.3% in those who did not develop atrioventricular block) (OR=2.09; CI 95% 1.46-2.99; P<0.0001) and a longer hospital stay (12.75 days x 10.53 days for those who didn't develop atrioventricular block) (OR=1.01; CI 95% 1.00-1.02; P=0.01).
|
RESULTS
|
In most cases, atrioventricular block in the postoperative period of coronary artery bypass surgery is transient and associated with several perioperative factors: age above 60 years, female sex, chronic kidney disease, atrial fibrillation, New York Heart Association functional class III or IV, perioperative acute myocardial infarction and use of an intra-aortic balloon. Its occurrence prolongs hospitalization and, above all, doubles the risk of mortality.
|
CONCLUSIONS
|
[
"Age Factors",
"Atrioventricular Block",
"Cardiopulmonary Bypass",
"Coronary Artery Bypass",
"Epidemiologic Methods",
"Female",
"Hospital Mortality",
"Humans",
"Length of Stay",
"Male",
"Middle Aged",
"Pacemaker, Artificial",
"Perioperative Period",
"Postoperative Complications",
"Risk Factors",
"Sex Factors",
"Time Factors",
"Treatment Outcome"
] |
4462961
|
INTRODUCTION
|
Disturbances of the cardiac conduction system are relatively frequent in the
postoperative period (PO) of coronary artery bypass grafting (CABG), with an
incidence ranging from 18 to 55% of cases[1-6].
Atrioventricular block (AVB) is one of these conduction disturbances and its
incidence ranges from 0.5 to 16%[3,5-9]. Most patients have transitory and
reversible conduction disorders that require temporary pacing (TP). 0.4 to 1.1% of
patients, however, when faced with the irreversibility of the condition, will have
to undergo a permanent pacemaker (PPM) implant during their hospital
stay[10].
This study, which is unprecedented in the national literature, tries to identify the
relationship between pre-, intra and postoperative (perioperative) factors
associated with the emergence of AVB, the need for TP and, if the case, the
implantation of a PPM in the PO of CABG.
|
METHODS
|
Population and sample Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass
(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic
University of Rio Grande do Sul (PUCRS).
Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass
(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic
University of Rio Grande do Sul (PUCRS).
Study Design Historical cohort observation study. Data were collected prospectively and
entered into the database of the Postoperative Heart Surgery Unit (POHS) of the
São Lucas Hospital of PUCRS.
Historical cohort observation study. Data were collected prospectively and
entered into the database of the Postoperative Heart Surgery Unit (POHS) of the
São Lucas Hospital of PUCRS.
Inclusion Criteria Patients with age equal to or greater than 18 years who were submitted to
isolated CABG.
Patients with age equal to or greater than 18 years who were submitted to
isolated CABG.
Exclusion Criteria Patients who had been undergo valvular surgery, for left ventricular
aneurysmectomy or correction of the interventricular communication associated
with CABG.
Patients who had been undergo valvular surgery, for left ventricular
aneurysmectomy or correction of the interventricular communication associated
with CABG.
Study Variables Age - the mean age was calculated and also divided into groups for analysis: less
than 60 years and greater than or equal to 60 years, according to the reference
in the literature[11,12]; gender (male and
female); left ventricular ejection fraction (EF) - evaluated by echocardiography
or radiocardiography, with the values being subdivided for analysis into
≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through
serum creatinine level > 1.5 mg/dl, according to the reference in the
literature[11,12]; Diabetes Mellitus
(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of
statins; previous use of other antiarrhythmics (propafenone and/or amiodarone);
acute myocardial infarction (AMI) prior to CABG; New York Heart Association
functional (NYHA) class; presence of calcification of the aorta; time of
cardiopulmonary bypass (CPB); aortic clamping time; need for the use of
intra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of
hospital stay and hospital death.
Age - the mean age was calculated and also divided into groups for analysis: less
than 60 years and greater than or equal to 60 years, according to the reference
in the literature[11,12]; gender (male and
female); left ventricular ejection fraction (EF) - evaluated by echocardiography
or radiocardiography, with the values being subdivided for analysis into
≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through
serum creatinine level > 1.5 mg/dl, according to the reference in the
literature[11,12]; Diabetes Mellitus
(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of
statins; previous use of other antiarrhythmics (propafenone and/or amiodarone);
acute myocardial infarction (AMI) prior to CABG; New York Heart Association
functional (NYHA) class; presence of calcification of the aorta; time of
cardiopulmonary bypass (CPB); aortic clamping time; need for the use of
intra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of
hospital stay and hospital death.
Outcome Development of AVB in the PO of CABG and the need for TP and implantation of a
PPM.
Development of AVB in the PO of CABG and the need for TP and implantation of a
PPM.
Procedures The CABGs were performed under general anesthesia. In all cases, a hyperkalemic
cardiac arrest was induced using a cold cardioplegic blood solution in the
anterograde flow, with the infusion being repeated every 20 minutes. A mild
systemic hypothermia (32ºC) was used. After surgery, all patients were
transferred to the ICU of the POHS with mechanical ventilation.
The CABGs were performed under general anesthesia. In all cases, a hyperkalemic
cardiac arrest was induced using a cold cardioplegic blood solution in the
anterograde flow, with the infusion being repeated every 20 minutes. A mild
systemic hypothermia (32ºC) was used. After surgery, all patients were
transferred to the ICU of the POHS with mechanical ventilation.
Statistical Analysis The data was plotted in a digital Microsoft Access® spreadsheet
and analyzed using version 17.0 of the statistical software SPSS. The
descriptive analysis was performed through frequency and mean ± standard
deviation analysis, according to the case. For the univariate analysis the
following tests were performed: chi-square and/or Fisher's Exact Test for
ordinal variables and the Student's T-test for quantitative data. The
multivariate analysis was performed using logistic regression (backward
conditional method). The difference was considered as statistically
significant for the value of P<0.05.
The data was plotted in a digital Microsoft Access® spreadsheet
and analyzed using version 17.0 of the statistical software SPSS. The
descriptive analysis was performed through frequency and mean ± standard
deviation analysis, according to the case. For the univariate analysis the
following tests were performed: chi-square and/or Fisher's Exact Test for
ordinal variables and the Student's T-test for quantitative data. The
multivariate analysis was performed using logistic regression (backward
conditional method). The difference was considered as statistically
significant for the value of P<0.05.
Ethical Considerations The design of this study was submitted to the Research Ethics Committee of the
Faculty of Medicine of the PUCRS, under registration number 060/3478.
The design of this study was submitted to the Research Ethics Committee of the
Faculty of Medicine of the PUCRS, under registration number 060/3478.
|
RESULTS
|
Of the 3532 patients undergoing CABG in the period under analysis, 288 (8.15%)
presented the clinical and electrocardiographic signs of AVB during the
postoperative period, with an indication for temporary pacing (TP).
Table 1 shows the demographic profile of the
patients studied. The univariate analysis of the preoperative data revealed a
greater need for TP in the PO of CABG in patients above 60 years of age (OR=2.48; CI
95% 1.90-3.24; P<0.0001), of the female gender (OR=1.03; CI 95%
1.00-1.05; P=0.012), with CKD (OR=2.03; CI 95% 1.55-2.65;
P<0.0001), the presence of AF (OR=2.38; CI 95% 1.49-3.72;
P<0.0001) and in patients with NYHA functional class III or
IV (OR=1.60; CI 95% 1.21-2.12; P=0.001).
Preoperative characteristics of the groups and univariate analysis.
AF=atrial fibrillation; AMI=acute myocardial infarction;
BB=beta-blockers; CI=confidence interval; CKD=chronic kidney disease;
DM=diabetes mellitus; EF=left ventricular ejection fraction;
FC=functional class; NYHA=New York Heart Association; NTP=no use of
temporary pacing; OR=odds ratio; P=statistical significance;
TP=temporary pacing
Table 2 presents the trans and postoperative
data, together with their univariate analysis. Here we can observe the association
of AVB with the need of TP in patients who presented calcification of the aorta,
perioperative AMI and the need for the use of an IAB. Of statistically significant
relevance, the univariate analysis also revealed the association of TP caused by AVB
with increased mortality (17.9% vs. 7.3%) and with a longer hospital stay (mean
hospitalization time of 12.75 days compared to 10.53 days for those who did not
require TP).
Trans and postoperative data of groups and univariate analysis.
Calcification Ao=calcification of the aorta; CPBT=cardiopulmonary bypass
time; IAB=intra-aortic balloon; Peri AMI=perioperative acute myocardial
infarction; Tclamping=aortic clamping time; Others: see Table 1
These data were submitted to multivariate analysis (Table 3), which revealed a higher risk of AVB in the PO of CABG in
patients with: age > 60 years, female sex, CKD, AF, NYHA functional class III or
IV, perioperative AMI and with the use of an IAB. Patients with EF≤40 %, DM,
the use of beta-blockers, statins and other antiarrhythmic drugs, prior AMI and CPB
and aortic clamping times didn't prove to be independent risk variables for the
development of AVB in the PO of CABG.
Multivariate analysis of the risk factors and outcomes of AVB in the PO of
CABG.
AMI=acute myocardial infarction; AVB=atrioventricular block; CABG=coronary
artery bypass grafting; CKD=chronic kidney disease; FC=functional class;
IAB=intra-aortic balloon; PO=postoperative
In the multivariate analysis, the presence of AVB resulted in a longer hospital stay
(12.75 days vs. 10.53 days for those who didn't develop AVB) (OR=1.01; CI 95%
1.00-1.02; P=0.01) and in a significant increase in the risk of
mortality (17.9% vs. 7.3% for patients without AVB) (OR=2.09; CI 95% 1.46-2.99;
P<0.0001).
In the subgroup of 288 patients who had AVB and who had undergone TP, 08 (2.78 %)
required a PPM implant, corresponding to 0.23% of the total cohort analyzed. The
average time elapsed since the surgery until the PPM implant was 12.25 days.
|
CONCLUSION
|
This work sheds light on the risk factors associated with the development of AVB in
the PO of CABG and the consequent need for TP and a definitive pacemaker. Based on
this we could establish that female patients, 60 years of age or more, with the
diagnosis of AF and CKD, in stages III and IV of the functional class, who had
perioperative AMI and required the use of an IAB, have a higher risk of developing
AVB in the PO of CABG. AVB determines a more prolonged hospitalization and, what is
more important, doubles the risk of mortality.
|
[
"Population and sample",
"Study Design",
"Inclusion Criteria",
"Exclusion Criteria",
"Study Variables",
"Outcome",
"Procedures",
"Statistical Analysis",
"Ethical Considerations",
"Limitations of the Study"
] |
[
"Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).",
"Historical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.",
"Patients with age equal to or greater than 18 years who were submitted to\nisolated CABG.",
"Patients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.",
"Age - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.",
"Development of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.",
"The CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.",
"The data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.",
"The design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.",
"The limitations of this study are those inherent to a retrospective database\nanalysis, but they reflect the significant years of experience of an academic\ninstitution. Within these limitations we can cite the relative difficulty of\naccessing the full data, which causes a potential risk of not measuring some\nrandom variables. The fact that the results come from the sample of a single\ncenter can also represent some degree of bias in the treatment. Another\nlimitation of this study is the absence of more precise information regarding\nthe height of the atrioventricular conduction disorder and the existence or not\nof any escape rhythm.\nRegarding the PPM implants performed in our study, they followed the\nrecommendations of Brazilian, American and European guidelines almost strictly.\nIn this small group of patients a more thorough analysis was compromised, but\nthis could be the target of a more detailed study to be developed in the\nfuture."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"METHODS",
"Population and sample",
"Study Design",
"Inclusion Criteria",
"Exclusion Criteria",
"Study Variables",
"Outcome",
"Procedures",
"Statistical Analysis",
"Ethical Considerations",
"RESULTS",
"DISCUSSION",
"Limitations of the Study",
"CONCLUSION"
] |
[
"Disturbances of the cardiac conduction system are relatively frequent in the\npostoperative period (PO) of coronary artery bypass grafting (CABG), with an\nincidence ranging from 18 to 55% of cases[1-6].\nAtrioventricular block (AVB) is one of these conduction disturbances and its\nincidence ranges from 0.5 to 16%[3,5-9]. Most patients have transitory and\nreversible conduction disorders that require temporary pacing (TP). 0.4 to 1.1% of\npatients, however, when faced with the irreversibility of the condition, will have\nto undergo a permanent pacemaker (PPM) implant during their hospital\nstay[10].\nThis study, which is unprecedented in the national literature, tries to identify the\nrelationship between pre-, intra and postoperative (perioperative) factors\nassociated with the emergence of AVB, the need for TP and, if the case, the\nimplantation of a PPM in the PO of CABG.",
" Population and sample Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).\nBetween January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).\n Study Design Historical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.\nHistorical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.\n Inclusion Criteria Patients with age equal to or greater than 18 years who were submitted to\nisolated CABG.\nPatients with age equal to or greater than 18 years who were submitted to\nisolated CABG.\n Exclusion Criteria Patients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.\nPatients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.\n Study Variables Age - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.\nAge - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.\n Outcome Development of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.\nDevelopment of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.\n Procedures The CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.\nThe CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.\n Statistical Analysis The data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.\nThe data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.\n Ethical Considerations The design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.\nThe design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.",
"Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass\n(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic\nUniversity of Rio Grande do Sul (PUCRS).",
"Historical cohort observation study. Data were collected prospectively and\nentered into the database of the Postoperative Heart Surgery Unit (POHS) of the\nSão Lucas Hospital of PUCRS.",
"Patients with age equal to or greater than 18 years who were submitted to\nisolated CABG.",
"Patients who had been undergo valvular surgery, for left ventricular\naneurysmectomy or correction of the interventricular communication associated\nwith CABG.",
"Age - the mean age was calculated and also divided into groups for analysis: less\nthan 60 years and greater than or equal to 60 years, according to the reference\nin the literature[11,12]; gender (male and\nfemale); left ventricular ejection fraction (EF) - evaluated by echocardiography\nor radiocardiography, with the values being subdivided for analysis into\n≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through\nserum creatinine level > 1.5 mg/dl, according to the reference in the\nliterature[11,12]; Diabetes Mellitus\n(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of\nstatins; previous use of other antiarrhythmics (propafenone and/or amiodarone);\nacute myocardial infarction (AMI) prior to CABG; New York Heart Association\nfunctional (NYHA) class; presence of calcification of the aorta; time of\ncardiopulmonary bypass (CPB); aortic clamping time; need for the use of\nintra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of\nhospital stay and hospital death.",
"Development of AVB in the PO of CABG and the need for TP and implantation of a\nPPM.",
"The CABGs were performed under general anesthesia. In all cases, a hyperkalemic\ncardiac arrest was induced using a cold cardioplegic blood solution in the\nanterograde flow, with the infusion being repeated every 20 minutes. A mild\nsystemic hypothermia (32ºC) was used. After surgery, all patients were\ntransferred to the ICU of the POHS with mechanical ventilation.",
"The data was plotted in a digital Microsoft Access® spreadsheet\nand analyzed using version 17.0 of the statistical software SPSS. The\ndescriptive analysis was performed through frequency and mean ± standard\ndeviation analysis, according to the case. For the univariate analysis the\nfollowing tests were performed: chi-square and/or Fisher's Exact Test for\nordinal variables and the Student's T-test for quantitative data. The\nmultivariate analysis was performed using logistic regression (backward\nconditional method). The difference was considered as statistically\nsignificant for the value of P<0.05.",
"The design of this study was submitted to the Research Ethics Committee of the\nFaculty of Medicine of the PUCRS, under registration number 060/3478.",
"Of the 3532 patients undergoing CABG in the period under analysis, 288 (8.15%)\npresented the clinical and electrocardiographic signs of AVB during the\npostoperative period, with an indication for temporary pacing (TP).\nTable 1 shows the demographic profile of the\npatients studied. The univariate analysis of the preoperative data revealed a\ngreater need for TP in the PO of CABG in patients above 60 years of age (OR=2.48; CI\n95% 1.90-3.24; P<0.0001), of the female gender (OR=1.03; CI 95%\n1.00-1.05; P=0.012), with CKD (OR=2.03; CI 95% 1.55-2.65;\nP<0.0001), the presence of AF (OR=2.38; CI 95% 1.49-3.72;\nP<0.0001) and in patients with NYHA functional class III or\nIV (OR=1.60; CI 95% 1.21-2.12; P=0.001).\nPreoperative characteristics of the groups and univariate analysis.\nAF=atrial fibrillation; AMI=acute myocardial infarction;\nBB=beta-blockers; CI=confidence interval; CKD=chronic kidney disease;\nDM=diabetes mellitus; EF=left ventricular ejection fraction;\nFC=functional class; NYHA=New York Heart Association; NTP=no use of\ntemporary pacing; OR=odds ratio; P=statistical significance;\nTP=temporary pacing\nTable 2 presents the trans and postoperative\ndata, together with their univariate analysis. Here we can observe the association\nof AVB with the need of TP in patients who presented calcification of the aorta,\nperioperative AMI and the need for the use of an IAB. Of statistically significant\nrelevance, the univariate analysis also revealed the association of TP caused by AVB\nwith increased mortality (17.9% vs. 7.3%) and with a longer hospital stay (mean\nhospitalization time of 12.75 days compared to 10.53 days for those who did not\nrequire TP).\nTrans and postoperative data of groups and univariate analysis.\nCalcification Ao=calcification of the aorta; CPBT=cardiopulmonary bypass\ntime; IAB=intra-aortic balloon; Peri AMI=perioperative acute myocardial\ninfarction; Tclamping=aortic clamping time; Others: see Table 1\nThese data were submitted to multivariate analysis (Table 3), which revealed a higher risk of AVB in the PO of CABG in\npatients with: age > 60 years, female sex, CKD, AF, NYHA functional class III or\nIV, perioperative AMI and with the use of an IAB. Patients with EF≤40 %, DM,\nthe use of beta-blockers, statins and other antiarrhythmic drugs, prior AMI and CPB\nand aortic clamping times didn't prove to be independent risk variables for the\ndevelopment of AVB in the PO of CABG.\nMultivariate analysis of the risk factors and outcomes of AVB in the PO of\nCABG.\nAMI=acute myocardial infarction; AVB=atrioventricular block; CABG=coronary\nartery bypass grafting; CKD=chronic kidney disease; FC=functional class;\nIAB=intra-aortic balloon; PO=postoperative\nIn the multivariate analysis, the presence of AVB resulted in a longer hospital stay\n(12.75 days vs. 10.53 days for those who didn't develop AVB) (OR=1.01; CI 95%\n1.00-1.02; P=0.01) and in a significant increase in the risk of\nmortality (17.9% vs. 7.3% for patients without AVB) (OR=2.09; CI 95% 1.46-2.99;\nP<0.0001).\nIn the subgroup of 288 patients who had AVB and who had undergone TP, 08 (2.78 %)\nrequired a PPM implant, corresponding to 0.23% of the total cohort analyzed. The\naverage time elapsed since the surgery until the PPM implant was 12.25 days.",
"CABG is a proven therapeutic strategy for the treatment of Coronary Artery Disease\n(CAD). Although it is well tolerated by most patients, perioperative complications\ncan occur, among which we find disturbances in the cardiac conduction system in\nvarying degrees, including AVB.\nPrevious studies have reported an incidence of conduction disturbances (CD) after\nCABG that varies from 18 to 55% of cases[1-6], with the\nright bundle branch block being the most common[4]. Atrioventricular block (AVB) is one of\nthese conduction disturbances and its incidence ranges from 0.5 to\n16%[3,5-9]. Our incidence of AVB is in line with this data, since\n8.15% of our patients developed AVB in the PO of CABG.\nThe etiology of AVB seems to be multifactorial. The patient's age (>60 years),\nhypertension, number of revascularized vessels, aortic clamping time, total time of\nCPB, use of digitalis and beta-blockers, type of cardioplegia and previously\nexisting left bundle branch block may be related to its appearance[1-4,8,9,13,14].\nMyocardial ischemia seems to be the factor that is most implicated in the emergence\nof AVB, since there is a correlation with coronary artery disease (CAD) and\npreoperative AMI[4].\nStudies[3,10] have demonstrated that\nperioperative AMI also increases the incidence of AVB in the PO of CABG. Caspi et\nal.[7]\nreported a higher occurrence of AVB in patients with AMI in the PO of CABG (12% vs.\n2 %, P<0.05).\nHowever, AMI before CABG was not a significant factor for the appearance of AVB in\nour study, which is consistent with the world literature[3,7,8,15,16].\nThis shows there is no difference in the incidence of AVB between patients who had\npreoperative AMI and those who didn't, regardless of its electrocardiographic\nlocation.\nCaspi et al.[7] have\nshown that the combination of left main disease and proximal obstruction of a\ndominant right coronary artery was more frequent in patients who exhibited AVB (32%)\nthan in those who without it (12%, P<0.05). The explanation for\nthis effect is the fact that the cardioplegic solution is not properly distributed\nto the coronary beds because of their high degree of obstruction, which compromises\nmyocardial protection and, in some cases, because of the impossibility of bypassing\nthe right coronary artery.\nThe impairment of myocardial irrigation gets worse with age, just as the frequency of\ndegenerative diseases of the conduction system, increasing the probability of\nAVB[9,11,15,17-19]. In this scenario, our patients above 60\nyears of age presented a significant risk (OR=2.34; CI 95% 1.75-3.12;\nP<0.0001) for the development of AVB in the PO of CABG,\ncorroborating the findings of other studies[7,8,13,14].\nThe electrical cardiac conduction tissue differs from cardiac myocytes by being less\ntolerant to the effects of ischemia, hyperkalemia and hypothermia (whether these are\nsystemic or, mostly, induced by a cardioplegic solution that is cold and rich in\npotassium). This may cause a transient block of the conduction\nsystem[11].\nThe advent of cold cardioplegia as a method of myocardial protection has increased\nthe incidence of CD from 20 to 58%[13]. The more significant incidence of conduction\ndisturbances occurred in patients who received cold cardioplegia, as opposed to warm\n(19.6% vs. 1.7 %, respectively)[13], a finding that has also been described by Sirlak\net al.[20].\nSpecifically with respect to AVB, the incidence was of 3.8% in the hypothermia group\nand zero in the normothermal group[13]. All patients in our study underwent surgery with\nthe myocardial protection performed by infusion of a cold cardioplegic blood\nsolution at the root of the aorta every 20 minutes, which contributed to the genesis\nof AVB cases.\nAs such, the perfusion injury determined by the myocardial ischemia and the\nhypothermic injury caused by the cardioplegic solution are the mechanisms that are\nmost involved in the genesis of AVB, acting on the proximal portions of the bundle\nof His, which are more sensitive to this type of aggression than the more distal\nconduction tissue, determining the emergence of bundle branch blocks and increasing\nthe risk of AVB[4].\nIn this scenario, the extent of the CAD, the duration of CPB and the aortic clamping\ntime could compromise myocardial protection during surgery, increasing the risk of\nan ischemic injury and of metabolic damage to the conduction\ntissue[11].\nHowever, our CPB time of ≥ 90 min and aortic clamping time of ≥ 40 min\nshowed no influence on the development of AVB, which is supported by the\nliterature[5-7,16,19]. Baerman\net al.[1], however,\ndemonstrated that patients with lower CPB (101±32min x 121±34min;\nP<0,01) and aortic clamping (44±19min x\n53±17min; P<0.05) times didn't show evidence of AVB in\nthe PO of CABG.\nOur study has shown that the female gender is a risk factor for the occurrence of AVB\n(OR=1.37, CI 95% 1.06-1.77; P=0.015), which contrasts with the\nresults of Gordon et al.[19] who observed a higher need for PPM implants in men\n(P=0.041). Other studies[3,8,15,20], however, didn't point to any of the genders as\nrisk factor. Cadore et al.[12] had already pointed to the female gender as a risk\npredictor for mortality in CABG, which can be an expression of the greater severity\nof the ischemic impairment in this gender and explain their greater tendency for\ndeveloping the block, as seen in our study.\nThe presence of CKD was also verified to be a risk factor for the development of AVB\n(OR = 2.05; CI 95% 1.49-2.81; P<0.0001). A previous\nstudy[19]\nindicated the presence of CKD as more significant among those patients who required\nPPM implantation in the PO of CABG. Like the female gender variable, CKD was also\nfound to be a predictor of mortality in patients underwent CABG according to the\nscore by Cadore et al.[12], expressing its potential for increasing the risk of\ncomplications in the PO of CABG.\nAnother risk predictor for the occurrence of AVB was the more advanced functional\nclass of the NYHA (III and IV) (OR = 1.43; CI 95% 1.03-1.98;\nP=0.031). Studies[18,19] have\ncorroborated this finding, indicating that patients who underwent heart surgery and\nneeding a PPM implant were in the more severe functional class of the NYHA (III and\nIV) when compared to patients who did not require such an implant (57% vs. 35%,\nrespectively, P<0.0001)[18]. Bateman et al.[6] showed that of those\npatients who passed away within the first 30 days of the PO of CABG and who had\ndeveloped some degree of blockage, 90% were into class IV of the NYHA in the\npreoperative period.\nThe patients in this study who had EF≤40% did not present a significant risk\nfor the appearance of AVB in the PO of CABG (OR=1.11; CI 95% 0.85-1.45;\nP=0.44), a finding supported by Gordon et al.[19] who didn't observe any\nsignificant impact of EF on the need for PPM implantation in the PO of isolated\nCABG. Caspi et al.[7], however, found a greater susceptibility to the\ndevelopment of atrioventricular block in patients submitted to CABG with a lower\nEF.\nAlthough Merin et al.[18] mention that the use of antiarrhythmic agents is more\nfrequent in the group of patients that develops blockage after heart surgery (CABG,\nvalve or combined), our data does not reflect this influence. Regarding the use of\nbeta-blockers, we also didn't find any association with the development of AVB,\nwhich has already been described by other authors[2,3,16].\nThe need for the use of an IAB in the PO of CABG occurred in 141 patients (4%) of the\ntotal sample of 3532 patients, of which 20.6% developed AVB, leading to the need for\nTP (OR=1.92; CI 95% 1.21-3.05; P=0.006). The need for the use of\nthe IAB has been associated with a greater probability of developing blocking and\nhas been indicated as a predictor of its occurrence and of the need for a PPM\nimplant[6,17,19]. Probably because its use is an expression of a\nmore significant ischemic cardiopathy, i.e., of patients with more severe\ncompromising. This finding is important because the patients did not have AVB in the\npreoperative period, presumably reflecting a greater perioperative myocardial\ninjury[6].\nPerioperative AMI was a risk factor for the emergence of AVB (OR=1.70; CI 95%\n1.26-2.29; P<0.0001), and this was corroborated by the study of\nCaspi et al.[7] who\nidentified the occurrence of low cardiac output (34% vs. 3%) and perioperative AMI\n(12% x 2%) as risk factors for AVB in the PO of CABG. Perioperative AMI also\nincreases the need for a PPM implant[10], reflecting acute ischemic damage of the conduction\ntissue.\nThe need for TP showed a significant association with mortality (OR=2.09; CI 95%\n1.46-2.99; P<0.0001), which was 17.7% for patients with AVB and\n7.2% for those who didn't develop it. Zeldis et al.[3] had already reported a\nmortality of 19.2% in the group of patients who developed block of the left\nconduction system (left bundle branch block or left anterior hemiblock or both),\ncompared with a 7% mortality rate in the group of patients without such block.\nSpecifically with respect to AVB, Caspi et al.[7] observed a significantly higher mortality\nin the group of patients who developed AVB (7% vs. 0.6%). On the other hand,\npatients who develop right bundle branch block or fascicular block have a more\nfavorable prognosis, because these are more transient disorders and because they do\nnot increase mortality[6,21].\nThe patients who developed AVB had a significantly longer hospital stay (mean\nhospitalization time of 12.75 days compared to 10.53 days for those who did not need\nTP for AVB (OR=1.01 CI 95% 1.00-1.02; P=0.01). Gordon et\nal.[19] have\nshown that the need for a PPM implant significantly increased hospital stay\n(23.3±18.7 days vs. 9.6±9.0 days for patients without need of implant,\nP=0.0001) and ICU stay (5.6±10.5 days vs. 2.2±3.3\ndays, P=0.0258). Other studies[11,18]\nalso corroborate this finding of a longer hospital stay in the presence of AVB and\nthe need of TP.\nIn our study, the need for a PPM implant occurred in 08 of the 3532 patients studied\n(0.23%), which is lower than the rate found in the literature, which points to the\nneed for PPM implants in 0.49% of AVB cases[8]. Gordon et al.[19] implanted PPMs in 50 of\ntheir 6859 patients submitted to CABG (0.73%). When other types of post-CABG\nconduction blocks are considered, the incidence of implants rises and ranges from\n0.4 to 1.1%[10]. The\ncalculated risk for need of a PPM implant in the PO of non-complicated CABG is\n0.9%[19].\nNascimento et al.[22]\ncouldn't identify any prognosis criterion for the reversibility of AVB in the PO of\nheart surgery. The ideal moment for the implantation of a PPM in the PO of CABG\nhasn't yet been properly established. According to the Brazilian Guidelines for\nImplantable Electronic Heart Devices[23], patients with asymptomatic AVB with wide QRS after\ncardiac surgery that persists after 15 days, are indicated for a PPM implantation\n(Class I, level of evidence C). In the cases of asymptomatic AVB persisting after 15\ndays, resulting from cardiac surgery, with narrow QRS or nodal escape rhythm and\ngood chronotropic response, and in those cases without the prospect of reversal\n(< 15 days) PPM implantation is also indicated (Class IIa, level C).\nAccording to the criteria of the American College of Cardiology and\nthe American Heart Association, a PPM implant is indicated in\n3rd and advanced 2nd degree AVB in the postoperative\nperiod of heart surgery, in addition to cases without expectation of resolution. The\ndecision regarding the time of the implant should be taken by the\nphysician[24].\nThe European Society of Cardiology recommends a waiting period of 5 to 7 days for the\nresolution of transient bradyarrhythmias after cardiac surgery, before the decision\nfor the implant is made[25].\nAccording to Pires et al.[13] and Merin et al.[18], the decision to perform the implant\nshould be taken between the 4th and 5th day of the PO, because\nif the AVB or dysfunction of the sinus node are still present up to this moment,\nthen they tend to be permanent. This would facilitate the early mobilization of\npatients and shorten their hospitalization time.\nOf the 288 patients in our study who had AVB, 08 received a PPM implant after an\naverage of 12.25 days into the PO, which is in line with the Brazilian (Class IIa,\nlevel of evidence C), American and European (Class I, level C) guidelines. Emlein et\nal.[8]\ndescribed a series of 8 patients who underwent a PPM implant after developing AVB\nwith an average of 10.5±6.5 days into the PO.\n Limitations of the Study The limitations of this study are those inherent to a retrospective database\nanalysis, but they reflect the significant years of experience of an academic\ninstitution. Within these limitations we can cite the relative difficulty of\naccessing the full data, which causes a potential risk of not measuring some\nrandom variables. The fact that the results come from the sample of a single\ncenter can also represent some degree of bias in the treatment. Another\nlimitation of this study is the absence of more precise information regarding\nthe height of the atrioventricular conduction disorder and the existence or not\nof any escape rhythm.\nRegarding the PPM implants performed in our study, they followed the\nrecommendations of Brazilian, American and European guidelines almost strictly.\nIn this small group of patients a more thorough analysis was compromised, but\nthis could be the target of a more detailed study to be developed in the\nfuture.\nThe limitations of this study are those inherent to a retrospective database\nanalysis, but they reflect the significant years of experience of an academic\ninstitution. Within these limitations we can cite the relative difficulty of\naccessing the full data, which causes a potential risk of not measuring some\nrandom variables. The fact that the results come from the sample of a single\ncenter can also represent some degree of bias in the treatment. Another\nlimitation of this study is the absence of more precise information regarding\nthe height of the atrioventricular conduction disorder and the existence or not\nof any escape rhythm.\nRegarding the PPM implants performed in our study, they followed the\nrecommendations of Brazilian, American and European guidelines almost strictly.\nIn this small group of patients a more thorough analysis was compromised, but\nthis could be the target of a more detailed study to be developed in the\nfuture.",
"The limitations of this study are those inherent to a retrospective database\nanalysis, but they reflect the significant years of experience of an academic\ninstitution. Within these limitations we can cite the relative difficulty of\naccessing the full data, which causes a potential risk of not measuring some\nrandom variables. The fact that the results come from the sample of a single\ncenter can also represent some degree of bias in the treatment. Another\nlimitation of this study is the absence of more precise information regarding\nthe height of the atrioventricular conduction disorder and the existence or not\nof any escape rhythm.\nRegarding the PPM implants performed in our study, they followed the\nrecommendations of Brazilian, American and European guidelines almost strictly.\nIn this small group of patients a more thorough analysis was compromised, but\nthis could be the target of a more detailed study to be developed in the\nfuture.",
"This work sheds light on the risk factors associated with the development of AVB in\nthe PO of CABG and the consequent need for TP and a definitive pacemaker. Based on\nthis we could establish that female patients, 60 years of age or more, with the\ndiagnosis of AF and CKD, in stages III and IV of the functional class, who had\nperioperative AMI and required the use of an IAB, have a higher risk of developing\nAVB in the PO of CABG. AVB determines a more prolonged hospitalization and, what is\nmore important, doubles the risk of mortality."
] |
[
"intro",
"methods",
null,
null,
null,
null,
null,
null,
null,
null,
null,
"results",
"discussion",
null,
"conclusions"
] |
[
"Atrioventricular block",
"Artificial Pacemaker",
"Coronary Artery Bypass",
"Postoperative Complications"
] |
INTRODUCTION:
Disturbances of the cardiac conduction system are relatively frequent in the
postoperative period (PO) of coronary artery bypass grafting (CABG), with an
incidence ranging from 18 to 55% of cases[1-6].
Atrioventricular block (AVB) is one of these conduction disturbances and its
incidence ranges from 0.5 to 16%[3,5-9]. Most patients have transitory and
reversible conduction disorders that require temporary pacing (TP). 0.4 to 1.1% of
patients, however, when faced with the irreversibility of the condition, will have
to undergo a permanent pacemaker (PPM) implant during their hospital
stay[10].
This study, which is unprecedented in the national literature, tries to identify the
relationship between pre-, intra and postoperative (perioperative) factors
associated with the emergence of AVB, the need for TP and, if the case, the
implantation of a PPM in the PO of CABG.
METHODS:
Population and sample Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass
(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic
University of Rio Grande do Sul (PUCRS).
Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass
(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic
University of Rio Grande do Sul (PUCRS).
Study Design Historical cohort observation study. Data were collected prospectively and
entered into the database of the Postoperative Heart Surgery Unit (POHS) of the
São Lucas Hospital of PUCRS.
Historical cohort observation study. Data were collected prospectively and
entered into the database of the Postoperative Heart Surgery Unit (POHS) of the
São Lucas Hospital of PUCRS.
Inclusion Criteria Patients with age equal to or greater than 18 years who were submitted to
isolated CABG.
Patients with age equal to or greater than 18 years who were submitted to
isolated CABG.
Exclusion Criteria Patients who had been undergo valvular surgery, for left ventricular
aneurysmectomy or correction of the interventricular communication associated
with CABG.
Patients who had been undergo valvular surgery, for left ventricular
aneurysmectomy or correction of the interventricular communication associated
with CABG.
Study Variables Age - the mean age was calculated and also divided into groups for analysis: less
than 60 years and greater than or equal to 60 years, according to the reference
in the literature[11,12]; gender (male and
female); left ventricular ejection fraction (EF) - evaluated by echocardiography
or radiocardiography, with the values being subdivided for analysis into
≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through
serum creatinine level > 1.5 mg/dl, according to the reference in the
literature[11,12]; Diabetes Mellitus
(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of
statins; previous use of other antiarrhythmics (propafenone and/or amiodarone);
acute myocardial infarction (AMI) prior to CABG; New York Heart Association
functional (NYHA) class; presence of calcification of the aorta; time of
cardiopulmonary bypass (CPB); aortic clamping time; need for the use of
intra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of
hospital stay and hospital death.
Age - the mean age was calculated and also divided into groups for analysis: less
than 60 years and greater than or equal to 60 years, according to the reference
in the literature[11,12]; gender (male and
female); left ventricular ejection fraction (EF) - evaluated by echocardiography
or radiocardiography, with the values being subdivided for analysis into
≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through
serum creatinine level > 1.5 mg/dl, according to the reference in the
literature[11,12]; Diabetes Mellitus
(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of
statins; previous use of other antiarrhythmics (propafenone and/or amiodarone);
acute myocardial infarction (AMI) prior to CABG; New York Heart Association
functional (NYHA) class; presence of calcification of the aorta; time of
cardiopulmonary bypass (CPB); aortic clamping time; need for the use of
intra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of
hospital stay and hospital death.
Outcome Development of AVB in the PO of CABG and the need for TP and implantation of a
PPM.
Development of AVB in the PO of CABG and the need for TP and implantation of a
PPM.
Procedures The CABGs were performed under general anesthesia. In all cases, a hyperkalemic
cardiac arrest was induced using a cold cardioplegic blood solution in the
anterograde flow, with the infusion being repeated every 20 minutes. A mild
systemic hypothermia (32ºC) was used. After surgery, all patients were
transferred to the ICU of the POHS with mechanical ventilation.
The CABGs were performed under general anesthesia. In all cases, a hyperkalemic
cardiac arrest was induced using a cold cardioplegic blood solution in the
anterograde flow, with the infusion being repeated every 20 minutes. A mild
systemic hypothermia (32ºC) was used. After surgery, all patients were
transferred to the ICU of the POHS with mechanical ventilation.
Statistical Analysis The data was plotted in a digital Microsoft Access® spreadsheet
and analyzed using version 17.0 of the statistical software SPSS. The
descriptive analysis was performed through frequency and mean ± standard
deviation analysis, according to the case. For the univariate analysis the
following tests were performed: chi-square and/or Fisher's Exact Test for
ordinal variables and the Student's T-test for quantitative data. The
multivariate analysis was performed using logistic regression (backward
conditional method). The difference was considered as statistically
significant for the value of P<0.05.
The data was plotted in a digital Microsoft Access® spreadsheet
and analyzed using version 17.0 of the statistical software SPSS. The
descriptive analysis was performed through frequency and mean ± standard
deviation analysis, according to the case. For the univariate analysis the
following tests were performed: chi-square and/or Fisher's Exact Test for
ordinal variables and the Student's T-test for quantitative data. The
multivariate analysis was performed using logistic regression (backward
conditional method). The difference was considered as statistically
significant for the value of P<0.05.
Ethical Considerations The design of this study was submitted to the Research Ethics Committee of the
Faculty of Medicine of the PUCRS, under registration number 060/3478.
The design of this study was submitted to the Research Ethics Committee of the
Faculty of Medicine of the PUCRS, under registration number 060/3478.
Population and sample:
Between January 1996 and December 2012, 3532 CABGs with cardiopulmonary bypass
(CPB) were performed at the Sao Lucas Hospital of the Pontifical Catholic
University of Rio Grande do Sul (PUCRS).
Study Design:
Historical cohort observation study. Data were collected prospectively and
entered into the database of the Postoperative Heart Surgery Unit (POHS) of the
São Lucas Hospital of PUCRS.
Inclusion Criteria:
Patients with age equal to or greater than 18 years who were submitted to
isolated CABG.
Exclusion Criteria:
Patients who had been undergo valvular surgery, for left ventricular
aneurysmectomy or correction of the interventricular communication associated
with CABG.
Study Variables:
Age - the mean age was calculated and also divided into groups for analysis: less
than 60 years and greater than or equal to 60 years, according to the reference
in the literature[11,12]; gender (male and
female); left ventricular ejection fraction (EF) - evaluated by echocardiography
or radiocardiography, with the values being subdivided for analysis into
≤ 40% and > 40 %; chronic kidney disease (CKD) - diagnosed through
serum creatinine level > 1.5 mg/dl, according to the reference in the
literature[11,12]; Diabetes Mellitus
(DM); atrial fibrillation (AF); previous use of beta-blockers; previous use of
statins; previous use of other antiarrhythmics (propafenone and/or amiodarone);
acute myocardial infarction (AMI) prior to CABG; New York Heart Association
functional (NYHA) class; presence of calcification of the aorta; time of
cardiopulmonary bypass (CPB); aortic clamping time; need for the use of
intra-aortic balloon (IAB) in the PO of CABG; perioperative AMI; length of
hospital stay and hospital death.
Outcome:
Development of AVB in the PO of CABG and the need for TP and implantation of a
PPM.
Procedures:
The CABGs were performed under general anesthesia. In all cases, a hyperkalemic
cardiac arrest was induced using a cold cardioplegic blood solution in the
anterograde flow, with the infusion being repeated every 20 minutes. A mild
systemic hypothermia (32ºC) was used. After surgery, all patients were
transferred to the ICU of the POHS with mechanical ventilation.
Statistical Analysis:
The data was plotted in a digital Microsoft Access® spreadsheet
and analyzed using version 17.0 of the statistical software SPSS. The
descriptive analysis was performed through frequency and mean ± standard
deviation analysis, according to the case. For the univariate analysis the
following tests were performed: chi-square and/or Fisher's Exact Test for
ordinal variables and the Student's T-test for quantitative data. The
multivariate analysis was performed using logistic regression (backward
conditional method). The difference was considered as statistically
significant for the value of P<0.05.
Ethical Considerations:
The design of this study was submitted to the Research Ethics Committee of the
Faculty of Medicine of the PUCRS, under registration number 060/3478.
RESULTS:
Of the 3532 patients undergoing CABG in the period under analysis, 288 (8.15%)
presented the clinical and electrocardiographic signs of AVB during the
postoperative period, with an indication for temporary pacing (TP).
Table 1 shows the demographic profile of the
patients studied. The univariate analysis of the preoperative data revealed a
greater need for TP in the PO of CABG in patients above 60 years of age (OR=2.48; CI
95% 1.90-3.24; P<0.0001), of the female gender (OR=1.03; CI 95%
1.00-1.05; P=0.012), with CKD (OR=2.03; CI 95% 1.55-2.65;
P<0.0001), the presence of AF (OR=2.38; CI 95% 1.49-3.72;
P<0.0001) and in patients with NYHA functional class III or
IV (OR=1.60; CI 95% 1.21-2.12; P=0.001).
Preoperative characteristics of the groups and univariate analysis.
AF=atrial fibrillation; AMI=acute myocardial infarction;
BB=beta-blockers; CI=confidence interval; CKD=chronic kidney disease;
DM=diabetes mellitus; EF=left ventricular ejection fraction;
FC=functional class; NYHA=New York Heart Association; NTP=no use of
temporary pacing; OR=odds ratio; P=statistical significance;
TP=temporary pacing
Table 2 presents the trans and postoperative
data, together with their univariate analysis. Here we can observe the association
of AVB with the need of TP in patients who presented calcification of the aorta,
perioperative AMI and the need for the use of an IAB. Of statistically significant
relevance, the univariate analysis also revealed the association of TP caused by AVB
with increased mortality (17.9% vs. 7.3%) and with a longer hospital stay (mean
hospitalization time of 12.75 days compared to 10.53 days for those who did not
require TP).
Trans and postoperative data of groups and univariate analysis.
Calcification Ao=calcification of the aorta; CPBT=cardiopulmonary bypass
time; IAB=intra-aortic balloon; Peri AMI=perioperative acute myocardial
infarction; Tclamping=aortic clamping time; Others: see Table 1
These data were submitted to multivariate analysis (Table 3), which revealed a higher risk of AVB in the PO of CABG in
patients with: age > 60 years, female sex, CKD, AF, NYHA functional class III or
IV, perioperative AMI and with the use of an IAB. Patients with EF≤40 %, DM,
the use of beta-blockers, statins and other antiarrhythmic drugs, prior AMI and CPB
and aortic clamping times didn't prove to be independent risk variables for the
development of AVB in the PO of CABG.
Multivariate analysis of the risk factors and outcomes of AVB in the PO of
CABG.
AMI=acute myocardial infarction; AVB=atrioventricular block; CABG=coronary
artery bypass grafting; CKD=chronic kidney disease; FC=functional class;
IAB=intra-aortic balloon; PO=postoperative
In the multivariate analysis, the presence of AVB resulted in a longer hospital stay
(12.75 days vs. 10.53 days for those who didn't develop AVB) (OR=1.01; CI 95%
1.00-1.02; P=0.01) and in a significant increase in the risk of
mortality (17.9% vs. 7.3% for patients without AVB) (OR=2.09; CI 95% 1.46-2.99;
P<0.0001).
In the subgroup of 288 patients who had AVB and who had undergone TP, 08 (2.78 %)
required a PPM implant, corresponding to 0.23% of the total cohort analyzed. The
average time elapsed since the surgery until the PPM implant was 12.25 days.
DISCUSSION:
CABG is a proven therapeutic strategy for the treatment of Coronary Artery Disease
(CAD). Although it is well tolerated by most patients, perioperative complications
can occur, among which we find disturbances in the cardiac conduction system in
varying degrees, including AVB.
Previous studies have reported an incidence of conduction disturbances (CD) after
CABG that varies from 18 to 55% of cases[1-6], with the
right bundle branch block being the most common[4]. Atrioventricular block (AVB) is one of
these conduction disturbances and its incidence ranges from 0.5 to
16%[3,5-9]. Our incidence of AVB is in line with this data, since
8.15% of our patients developed AVB in the PO of CABG.
The etiology of AVB seems to be multifactorial. The patient's age (>60 years),
hypertension, number of revascularized vessels, aortic clamping time, total time of
CPB, use of digitalis and beta-blockers, type of cardioplegia and previously
existing left bundle branch block may be related to its appearance[1-4,8,9,13,14].
Myocardial ischemia seems to be the factor that is most implicated in the emergence
of AVB, since there is a correlation with coronary artery disease (CAD) and
preoperative AMI[4].
Studies[3,10] have demonstrated that
perioperative AMI also increases the incidence of AVB in the PO of CABG. Caspi et
al.[7]
reported a higher occurrence of AVB in patients with AMI in the PO of CABG (12% vs.
2 %, P<0.05).
However, AMI before CABG was not a significant factor for the appearance of AVB in
our study, which is consistent with the world literature[3,7,8,15,16].
This shows there is no difference in the incidence of AVB between patients who had
preoperative AMI and those who didn't, regardless of its electrocardiographic
location.
Caspi et al.[7] have
shown that the combination of left main disease and proximal obstruction of a
dominant right coronary artery was more frequent in patients who exhibited AVB (32%)
than in those who without it (12%, P<0.05). The explanation for
this effect is the fact that the cardioplegic solution is not properly distributed
to the coronary beds because of their high degree of obstruction, which compromises
myocardial protection and, in some cases, because of the impossibility of bypassing
the right coronary artery.
The impairment of myocardial irrigation gets worse with age, just as the frequency of
degenerative diseases of the conduction system, increasing the probability of
AVB[9,11,15,17-19]. In this scenario, our patients above 60
years of age presented a significant risk (OR=2.34; CI 95% 1.75-3.12;
P<0.0001) for the development of AVB in the PO of CABG,
corroborating the findings of other studies[7,8,13,14].
The electrical cardiac conduction tissue differs from cardiac myocytes by being less
tolerant to the effects of ischemia, hyperkalemia and hypothermia (whether these are
systemic or, mostly, induced by a cardioplegic solution that is cold and rich in
potassium). This may cause a transient block of the conduction
system[11].
The advent of cold cardioplegia as a method of myocardial protection has increased
the incidence of CD from 20 to 58%[13]. The more significant incidence of conduction
disturbances occurred in patients who received cold cardioplegia, as opposed to warm
(19.6% vs. 1.7 %, respectively)[13], a finding that has also been described by Sirlak
et al.[20].
Specifically with respect to AVB, the incidence was of 3.8% in the hypothermia group
and zero in the normothermal group[13]. All patients in our study underwent surgery with
the myocardial protection performed by infusion of a cold cardioplegic blood
solution at the root of the aorta every 20 minutes, which contributed to the genesis
of AVB cases.
As such, the perfusion injury determined by the myocardial ischemia and the
hypothermic injury caused by the cardioplegic solution are the mechanisms that are
most involved in the genesis of AVB, acting on the proximal portions of the bundle
of His, which are more sensitive to this type of aggression than the more distal
conduction tissue, determining the emergence of bundle branch blocks and increasing
the risk of AVB[4].
In this scenario, the extent of the CAD, the duration of CPB and the aortic clamping
time could compromise myocardial protection during surgery, increasing the risk of
an ischemic injury and of metabolic damage to the conduction
tissue[11].
However, our CPB time of ≥ 90 min and aortic clamping time of ≥ 40 min
showed no influence on the development of AVB, which is supported by the
literature[5-7,16,19]. Baerman
et al.[1], however,
demonstrated that patients with lower CPB (101±32min x 121±34min;
P<0,01) and aortic clamping (44±19min x
53±17min; P<0.05) times didn't show evidence of AVB in
the PO of CABG.
Our study has shown that the female gender is a risk factor for the occurrence of AVB
(OR=1.37, CI 95% 1.06-1.77; P=0.015), which contrasts with the
results of Gordon et al.[19] who observed a higher need for PPM implants in men
(P=0.041). Other studies[3,8,15,20], however, didn't point to any of the genders as
risk factor. Cadore et al.[12] had already pointed to the female gender as a risk
predictor for mortality in CABG, which can be an expression of the greater severity
of the ischemic impairment in this gender and explain their greater tendency for
developing the block, as seen in our study.
The presence of CKD was also verified to be a risk factor for the development of AVB
(OR = 2.05; CI 95% 1.49-2.81; P<0.0001). A previous
study[19]
indicated the presence of CKD as more significant among those patients who required
PPM implantation in the PO of CABG. Like the female gender variable, CKD was also
found to be a predictor of mortality in patients underwent CABG according to the
score by Cadore et al.[12], expressing its potential for increasing the risk of
complications in the PO of CABG.
Another risk predictor for the occurrence of AVB was the more advanced functional
class of the NYHA (III and IV) (OR = 1.43; CI 95% 1.03-1.98;
P=0.031). Studies[18,19] have
corroborated this finding, indicating that patients who underwent heart surgery and
needing a PPM implant were in the more severe functional class of the NYHA (III and
IV) when compared to patients who did not require such an implant (57% vs. 35%,
respectively, P<0.0001)[18]. Bateman et al.[6] showed that of those
patients who passed away within the first 30 days of the PO of CABG and who had
developed some degree of blockage, 90% were into class IV of the NYHA in the
preoperative period.
The patients in this study who had EF≤40% did not present a significant risk
for the appearance of AVB in the PO of CABG (OR=1.11; CI 95% 0.85-1.45;
P=0.44), a finding supported by Gordon et al.[19] who didn't observe any
significant impact of EF on the need for PPM implantation in the PO of isolated
CABG. Caspi et al.[7], however, found a greater susceptibility to the
development of atrioventricular block in patients submitted to CABG with a lower
EF.
Although Merin et al.[18] mention that the use of antiarrhythmic agents is more
frequent in the group of patients that develops blockage after heart surgery (CABG,
valve or combined), our data does not reflect this influence. Regarding the use of
beta-blockers, we also didn't find any association with the development of AVB,
which has already been described by other authors[2,3,16].
The need for the use of an IAB in the PO of CABG occurred in 141 patients (4%) of the
total sample of 3532 patients, of which 20.6% developed AVB, leading to the need for
TP (OR=1.92; CI 95% 1.21-3.05; P=0.006). The need for the use of
the IAB has been associated with a greater probability of developing blocking and
has been indicated as a predictor of its occurrence and of the need for a PPM
implant[6,17,19]. Probably because its use is an expression of a
more significant ischemic cardiopathy, i.e., of patients with more severe
compromising. This finding is important because the patients did not have AVB in the
preoperative period, presumably reflecting a greater perioperative myocardial
injury[6].
Perioperative AMI was a risk factor for the emergence of AVB (OR=1.70; CI 95%
1.26-2.29; P<0.0001), and this was corroborated by the study of
Caspi et al.[7] who
identified the occurrence of low cardiac output (34% vs. 3%) and perioperative AMI
(12% x 2%) as risk factors for AVB in the PO of CABG. Perioperative AMI also
increases the need for a PPM implant[10], reflecting acute ischemic damage of the conduction
tissue.
The need for TP showed a significant association with mortality (OR=2.09; CI 95%
1.46-2.99; P<0.0001), which was 17.7% for patients with AVB and
7.2% for those who didn't develop it. Zeldis et al.[3] had already reported a
mortality of 19.2% in the group of patients who developed block of the left
conduction system (left bundle branch block or left anterior hemiblock or both),
compared with a 7% mortality rate in the group of patients without such block.
Specifically with respect to AVB, Caspi et al.[7] observed a significantly higher mortality
in the group of patients who developed AVB (7% vs. 0.6%). On the other hand,
patients who develop right bundle branch block or fascicular block have a more
favorable prognosis, because these are more transient disorders and because they do
not increase mortality[6,21].
The patients who developed AVB had a significantly longer hospital stay (mean
hospitalization time of 12.75 days compared to 10.53 days for those who did not need
TP for AVB (OR=1.01 CI 95% 1.00-1.02; P=0.01). Gordon et
al.[19] have
shown that the need for a PPM implant significantly increased hospital stay
(23.3±18.7 days vs. 9.6±9.0 days for patients without need of implant,
P=0.0001) and ICU stay (5.6±10.5 days vs. 2.2±3.3
days, P=0.0258). Other studies[11,18]
also corroborate this finding of a longer hospital stay in the presence of AVB and
the need of TP.
In our study, the need for a PPM implant occurred in 08 of the 3532 patients studied
(0.23%), which is lower than the rate found in the literature, which points to the
need for PPM implants in 0.49% of AVB cases[8]. Gordon et al.[19] implanted PPMs in 50 of
their 6859 patients submitted to CABG (0.73%). When other types of post-CABG
conduction blocks are considered, the incidence of implants rises and ranges from
0.4 to 1.1%[10]. The
calculated risk for need of a PPM implant in the PO of non-complicated CABG is
0.9%[19].
Nascimento et al.[22]
couldn't identify any prognosis criterion for the reversibility of AVB in the PO of
heart surgery. The ideal moment for the implantation of a PPM in the PO of CABG
hasn't yet been properly established. According to the Brazilian Guidelines for
Implantable Electronic Heart Devices[23], patients with asymptomatic AVB with wide QRS after
cardiac surgery that persists after 15 days, are indicated for a PPM implantation
(Class I, level of evidence C). In the cases of asymptomatic AVB persisting after 15
days, resulting from cardiac surgery, with narrow QRS or nodal escape rhythm and
good chronotropic response, and in those cases without the prospect of reversal
(< 15 days) PPM implantation is also indicated (Class IIa, level C).
According to the criteria of the American College of Cardiology and
the American Heart Association, a PPM implant is indicated in
3rd and advanced 2nd degree AVB in the postoperative
period of heart surgery, in addition to cases without expectation of resolution. The
decision regarding the time of the implant should be taken by the
physician[24].
The European Society of Cardiology recommends a waiting period of 5 to 7 days for the
resolution of transient bradyarrhythmias after cardiac surgery, before the decision
for the implant is made[25].
According to Pires et al.[13] and Merin et al.[18], the decision to perform the implant
should be taken between the 4th and 5th day of the PO, because
if the AVB or dysfunction of the sinus node are still present up to this moment,
then they tend to be permanent. This would facilitate the early mobilization of
patients and shorten their hospitalization time.
Of the 288 patients in our study who had AVB, 08 received a PPM implant after an
average of 12.25 days into the PO, which is in line with the Brazilian (Class IIa,
level of evidence C), American and European (Class I, level C) guidelines. Emlein et
al.[8]
described a series of 8 patients who underwent a PPM implant after developing AVB
with an average of 10.5±6.5 days into the PO.
Limitations of the Study The limitations of this study are those inherent to a retrospective database
analysis, but they reflect the significant years of experience of an academic
institution. Within these limitations we can cite the relative difficulty of
accessing the full data, which causes a potential risk of not measuring some
random variables. The fact that the results come from the sample of a single
center can also represent some degree of bias in the treatment. Another
limitation of this study is the absence of more precise information regarding
the height of the atrioventricular conduction disorder and the existence or not
of any escape rhythm.
Regarding the PPM implants performed in our study, they followed the
recommendations of Brazilian, American and European guidelines almost strictly.
In this small group of patients a more thorough analysis was compromised, but
this could be the target of a more detailed study to be developed in the
future.
The limitations of this study are those inherent to a retrospective database
analysis, but they reflect the significant years of experience of an academic
institution. Within these limitations we can cite the relative difficulty of
accessing the full data, which causes a potential risk of not measuring some
random variables. The fact that the results come from the sample of a single
center can also represent some degree of bias in the treatment. Another
limitation of this study is the absence of more precise information regarding
the height of the atrioventricular conduction disorder and the existence or not
of any escape rhythm.
Regarding the PPM implants performed in our study, they followed the
recommendations of Brazilian, American and European guidelines almost strictly.
In this small group of patients a more thorough analysis was compromised, but
this could be the target of a more detailed study to be developed in the
future.
Limitations of the Study:
The limitations of this study are those inherent to a retrospective database
analysis, but they reflect the significant years of experience of an academic
institution. Within these limitations we can cite the relative difficulty of
accessing the full data, which causes a potential risk of not measuring some
random variables. The fact that the results come from the sample of a single
center can also represent some degree of bias in the treatment. Another
limitation of this study is the absence of more precise information regarding
the height of the atrioventricular conduction disorder and the existence or not
of any escape rhythm.
Regarding the PPM implants performed in our study, they followed the
recommendations of Brazilian, American and European guidelines almost strictly.
In this small group of patients a more thorough analysis was compromised, but
this could be the target of a more detailed study to be developed in the
future.
CONCLUSION:
This work sheds light on the risk factors associated with the development of AVB in
the PO of CABG and the consequent need for TP and a definitive pacemaker. Based on
this we could establish that female patients, 60 years of age or more, with the
diagnosis of AF and CKD, in stages III and IV of the functional class, who had
perioperative AMI and required the use of an IAB, have a higher risk of developing
AVB in the PO of CABG. AVB determines a more prolonged hospitalization and, what is
more important, doubles the risk of mortality.
|
Background:
Disturbances of the cardiac conduction system are frequent in the postoperative period of coronary artery bypass surgery. They are mostly reversible and associated with some injury of the conduction tissue, caused by the ischemic heart disease itself or by perioperative factors.
Methods:
Analysis of a retrospective cohort of patients submitted to coronary artery bypass surgery from the database of the Postoperative Heart Surgery Unit of the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande do Sul, using the logistic regression method.
Results:
In the period from January 1996 to December 2012, 3532 coronary artery bypass surgery were carried out. Two hundred and eighty-eight (8.15% of the total sample) patients had atrioventricular block during the postoperative period of coronary artery bypass surgery, requiring temporary pacing. Eight of those who had atrioventricular block progressed to implantation of a permanent pacemaker (0.23% of the total sample). Multivariate analysis revealed a significant association of atrioventricular block with age above 60 years (OR=2.34; CI 95% 1.75-3.12; P<0.0001), female gender (OR=1.37; CI 95% 1.06-1.77; P=0.015), chronic kidney disease (OR=2.05; CI 95% 1.49-2.81; P<0.0001), atrial fibrillation (OR=2.06; CI 95% 1.16-3.66; P=0.014), functional class III and IV of the New York Heart Association (OR=1.43; CI 95% 1.03-1.98; P=0.031), perioperative acute myocardial infarction (OR=1.70; CI 95% 1.26-2.29; P<0.0001) and with the use of the intra-aortic balloon in the postoperative period of coronary artery bypass surgery (OR=1.92; CI 95% 1.21-3.05; P=0.006). The presence of atrioventricular block resulted in a significant increase in mortality (17.9% vs. 7.3% in those who did not develop atrioventricular block) (OR=2.09; CI 95% 1.46-2.99; P<0.0001) and a longer hospital stay (12.75 days x 10.53 days for those who didn't develop atrioventricular block) (OR=1.01; CI 95% 1.00-1.02; P=0.01).
Conclusions:
In most cases, atrioventricular block in the postoperative period of coronary artery bypass surgery is transient and associated with several perioperative factors: age above 60 years, female sex, chronic kidney disease, atrial fibrillation, New York Heart Association functional class III or IV, perioperative acute myocardial infarction and use of an intra-aortic balloon. Its occurrence prolongs hospitalization and, above all, doubles the risk of mortality.
|
INTRODUCTION:
Disturbances of the cardiac conduction system are relatively frequent in the
postoperative period (PO) of coronary artery bypass grafting (CABG), with an
incidence ranging from 18 to 55% of cases[1-6].
Atrioventricular block (AVB) is one of these conduction disturbances and its
incidence ranges from 0.5 to 16%[3,5-9]. Most patients have transitory and
reversible conduction disorders that require temporary pacing (TP). 0.4 to 1.1% of
patients, however, when faced with the irreversibility of the condition, will have
to undergo a permanent pacemaker (PPM) implant during their hospital
stay[10].
This study, which is unprecedented in the national literature, tries to identify the
relationship between pre-, intra and postoperative (perioperative) factors
associated with the emergence of AVB, the need for TP and, if the case, the
implantation of a PPM in the PO of CABG.
CONCLUSION:
This work sheds light on the risk factors associated with the development of AVB in
the PO of CABG and the consequent need for TP and a definitive pacemaker. Based on
this we could establish that female patients, 60 years of age or more, with the
diagnosis of AF and CKD, in stages III and IV of the functional class, who had
perioperative AMI and required the use of an IAB, have a higher risk of developing
AVB in the PO of CABG. AVB determines a more prolonged hospitalization and, what is
more important, doubles the risk of mortality.
|
Background:
Disturbances of the cardiac conduction system are frequent in the postoperative period of coronary artery bypass surgery. They are mostly reversible and associated with some injury of the conduction tissue, caused by the ischemic heart disease itself or by perioperative factors.
Methods:
Analysis of a retrospective cohort of patients submitted to coronary artery bypass surgery from the database of the Postoperative Heart Surgery Unit of the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande do Sul, using the logistic regression method.
Results:
In the period from January 1996 to December 2012, 3532 coronary artery bypass surgery were carried out. Two hundred and eighty-eight (8.15% of the total sample) patients had atrioventricular block during the postoperative period of coronary artery bypass surgery, requiring temporary pacing. Eight of those who had atrioventricular block progressed to implantation of a permanent pacemaker (0.23% of the total sample). Multivariate analysis revealed a significant association of atrioventricular block with age above 60 years (OR=2.34; CI 95% 1.75-3.12; P<0.0001), female gender (OR=1.37; CI 95% 1.06-1.77; P=0.015), chronic kidney disease (OR=2.05; CI 95% 1.49-2.81; P<0.0001), atrial fibrillation (OR=2.06; CI 95% 1.16-3.66; P=0.014), functional class III and IV of the New York Heart Association (OR=1.43; CI 95% 1.03-1.98; P=0.031), perioperative acute myocardial infarction (OR=1.70; CI 95% 1.26-2.29; P<0.0001) and with the use of the intra-aortic balloon in the postoperative period of coronary artery bypass surgery (OR=1.92; CI 95% 1.21-3.05; P=0.006). The presence of atrioventricular block resulted in a significant increase in mortality (17.9% vs. 7.3% in those who did not develop atrioventricular block) (OR=2.09; CI 95% 1.46-2.99; P<0.0001) and a longer hospital stay (12.75 days x 10.53 days for those who didn't develop atrioventricular block) (OR=1.01; CI 95% 1.00-1.02; P=0.01).
Conclusions:
In most cases, atrioventricular block in the postoperative period of coronary artery bypass surgery is transient and associated with several perioperative factors: age above 60 years, female sex, chronic kidney disease, atrial fibrillation, New York Heart Association functional class III or IV, perioperative acute myocardial infarction and use of an intra-aortic balloon. Its occurrence prolongs hospitalization and, above all, doubles the risk of mortality.
| 5,959 | 471 | 15 |
[
"avb",
"patients",
"cabg",
"analysis",
"study",
"po",
"need",
"ppm",
"po cabg",
"use"
] |
[
"test",
"test"
] |
[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]
|
[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]
|
[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]
|
[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]
|
[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]
|
[CONTENT] Atrioventricular block | Artificial Pacemaker | Coronary Artery Bypass | Postoperative Complications [SUMMARY]
|
[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] Age Factors | Atrioventricular Block | Cardiopulmonary Bypass | Coronary Artery Bypass | Epidemiologic Methods | Female | Hospital Mortality | Humans | Length of Stay | Male | Middle Aged | Pacemaker, Artificial | Perioperative Period | Postoperative Complications | Risk Factors | Sex Factors | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]
|
[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]
|
[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]
|
[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]
|
[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]
|
[CONTENT] avb | patients | cabg | analysis | study | po | need | ppm | po cabg | use [SUMMARY]
|
[CONTENT] conduction | incidence | disturbances | postoperative | ppm | tp | avb | po | period po coronary | period po [SUMMARY]
|
[CONTENT] analysis | performed | previous use | use | cabg | previous | hospital | according | pucrs | study [SUMMARY]
|
[CONTENT] ci | avb | 95 | ci 95 | analysis | days | patients | tp | table | univariate analysis [SUMMARY]
|
[CONTENT] risk | avb | avb po | avb po cabg | po cabg | po | use iab higher risk | avb determines prolonged hospitalization | ami required | ami required use [SUMMARY]
|
[CONTENT] avb | cabg | patients | analysis | study | po | po cabg | performed | ppm | tp [SUMMARY]
|
[CONTENT] avb | cabg | patients | analysis | study | po | po cabg | performed | ppm | tp [SUMMARY]
|
[CONTENT] ||| [SUMMARY]
|
[CONTENT] the Postoperative Heart Surgery Unit | the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande [SUMMARY]
|
[CONTENT] the period | January 1996 to December 2012 | 3532 ||| Two hundred and eighty-eight | 8.15% ||| Eight | 0.23% ||| age above 60 years | OR=2.34 | CI | 95% | 1.75 | CI | 95% | 1.06 | OR=2.05 | CI | 95% | 1.49 | OR=2.06 | CI | 95% | 1.16-3.66 | IV | the New York Heart Association | CI | 95% | 1.03 | OR=1.70 | CI | 95% | 1.26-2.29 | OR=1.92 | CI | 95% | 1.21 ||| 17.9% | 7.3% | CI | 95% | 1.46 | 12.75 days | 10.53 days | OR=1.01 | CI | 95% | 1.00-1.02 [SUMMARY]
|
[CONTENT] age above 60 years | New York Heart Association | IV ||| [SUMMARY]
|
[CONTENT] ||| ||| the Postoperative Heart Surgery Unit | the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande ||| ||| the period | January 1996 to December 2012 | 3532 ||| Two hundred and eighty-eight | 8.15% ||| Eight | 0.23% ||| age above 60 years | OR=2.34 | CI | 95% | 1.75 | CI | 95% | 1.06 | OR=2.05 | CI | 95% | 1.49 | OR=2.06 | CI | 95% | 1.16-3.66 | IV | the New York Heart Association | CI | 95% | 1.03 | OR=1.70 | CI | 95% | 1.26-2.29 | OR=1.92 | CI | 95% | 1.21 ||| 17.9% | 7.3% | CI | 95% | 1.46 | 12.75 days | 10.53 days | OR=1.01 | CI | 95% | 1.00-1.02 ||| age above 60 years | New York Heart Association | IV ||| [SUMMARY]
|
[CONTENT] ||| ||| the Postoperative Heart Surgery Unit | the Sao Lucas Hospital of the Pontifical Catholic University of Rio Grande ||| ||| the period | January 1996 to December 2012 | 3532 ||| Two hundred and eighty-eight | 8.15% ||| Eight | 0.23% ||| age above 60 years | OR=2.34 | CI | 95% | 1.75 | CI | 95% | 1.06 | OR=2.05 | CI | 95% | 1.49 | OR=2.06 | CI | 95% | 1.16-3.66 | IV | the New York Heart Association | CI | 95% | 1.03 | OR=1.70 | CI | 95% | 1.26-2.29 | OR=1.92 | CI | 95% | 1.21 ||| 17.9% | 7.3% | CI | 95% | 1.46 | 12.75 days | 10.53 days | OR=1.01 | CI | 95% | 1.00-1.02 ||| age above 60 years | New York Heart Association | IV ||| [SUMMARY]
|
||||||
Performance of two rapid diagnostic tests for malaria diagnosis at the China-Myanmar border area.
|
23433230
|
Rapid diagnostic tests (RDTs) have become an essential tool in the contemporary malaria control and management programmes in the world. This study aims to evaluate the performance of two commonly used RDTs for malaria diagnosis in the China-Myanmar border area.
|
BACKGROUND
|
A total 606 febrile patients in the China-Myanmar border were recruited to this study and were diagnosed for malaria infections by microscopy, two RDTs tests (Pf/Pan device, and Pv/Pf device) and nested PCR.
|
METHODS
|
Malaria parasites were found in 143 patients by microscopy, of which 51, 73, and 19 were Plasmodium falciparum, Plasmodium vivax and P. falciparum/P. vivax mixed infections, respectively. Compared to microscopy, the sensitivity of the Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4%. For a subset of 350 patients, the sensitivity of the Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively. The specificity of the Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively. Nested PCR detected malaria parasites in 174 of 606 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. Compared to nested PCR, all other methods had sensitivity below 80%, suggesting that a significant number of cases were missed.
|
RESULTS
|
Compared to PCR, both microscopy and RDTs had lower sensitivities. RDTs had similar performance to microscopy for P. falciparum diagnosis, but performed worse for P. vivax diagnosis. Other RDT products should be selected with higher sensitivity (and good specificity) for both P. falciparum and P. vivax diagnosis.
|
CONCLUSIONS
|
[
"Adolescent",
"Adult",
"Aged",
"Aged, 80 and over",
"Child",
"Child, Preschool",
"China",
"Clinical Laboratory Techniques",
"Diagnostic Tests, Routine",
"Female",
"Humans",
"Infant",
"Malaria",
"Male",
"Middle Aged",
"Myanmar",
"Parasitology",
"Plasmodium falciparum",
"Plasmodium ovale",
"Plasmodium vivax",
"Sensitivity and Specificity",
"Young Adult"
] |
3599043
|
Background
|
Malaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually
[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms
[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease
[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.
Microscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately
[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific
[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine
[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings
[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign
[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.
RDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results
[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported
[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria
[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria.
|
Methods
|
Study site and patients The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.
The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.
Microscopic examinations Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood
[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.
Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood
[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.
RDTs for malaria The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.
The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.
Diagnosis by PCR Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene
[20,21]. Plasmodium genus-specific primers were shown in Table
1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table
1.
Primers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections
Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene
[20,21]. Plasmodium genus-specific primers were shown in Table
1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table
1.
Primers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections
Statistical analysis The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table
2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.
Interpretation of the results for the Pf/Pan RDT
* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.
** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.
The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table
2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.
Interpretation of the results for the Pf/Pan RDT
* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.
** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.
|
Results
|
Blood samples were collected from a total of 606 patients. All samples were evaluated by the Pf/Pan test device, while a subset of 350 were also evaluated by the Pv/Pf test device. Of the 606 samples, malaria parasites were found in 143 by microscopy; 51, 73, and 19 were P. falciparum, P. vivax and P. falciparum/P. vivax mixed infections, respectively (Table
3). No other malaria parasite species were detected by microscopy. The parasite density ranges of P. falciparum were 40–105,920 parasites/μL. For P. vivax, the majority of cases had parasite density ranging from 80 to 17,800 parasites/μL. One P. vivax case had a deviant parasite density of >200,000 parasites/μL based on leucocyte number probably due to a low leucocyte count in this patient. Because P. falciparum single infections and mixed species infections containing P. falciparum cannot be differentiated by the Pf/Pan device, the microscopy results were grouped into “P. falciparum and mixed infections with P. falciparum” and “Non-falciparum”. Based on this interpretation, the Pf/Pan device detected 33 single P. falciparum infections (only the PfHRP2 line visible), 56 non-falciparum cases (only the pan-pLDH line visible) and 70 P. falciparum infections/potentially mixed infections (both lines visible) (Table
3). Compared with the results by microscopy, the Pf/Pan device detected 62 and 51 samples as true positives for P. falciparum and non-falciparum, respectively. From this result, the sensitivity of Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4% (Table
4).
Detection results of malaria infections by the Malaria Pf/Pan test in comparison with microscopy (N=606)
* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.
Performance of two RDTs for detection of
P. falciparum
and
P. vivax
with microscopy as the gold standard
Data are presented as percentage (95% confidence interval; CI).
For the subset of 350 samples, microscopy detected 37 samples containing P. falciparum, 50 samples containing P. vivax and 11 were diagnosed as mixed species infections (Table
5). Since the Pv/Pf device is able to differentiate P. falciparum and P. vivax infections, the microscopy results were grouped into “infections containing P. falciparum” and “infections containing P. vivax”. The Pv/Pf device detected 40 single P. falciparum infections, 42 P. vivax infections, and 8 mixed species infections (Table
5). Compared to microscopy, the Pv/Pf device detected 44 and 45 samples as true positives for P. falciparum and P. vivax, respectively, whereas the Pf/Pan device detected 42 and 36 samples as true positives for P. falciparum and P. vivax, respectively. The sensitivity of Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively (Table
4). The specificity of Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively (Table
4).
Detection results of malaria infections by Malaria Pv/Pf test and Malaria Pf/Pan test in comparison with the microscopic method (N=350)
* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.
It has been observed that some RDTs do not perform well when the parasite density is below 500 parasites/μL. For P. falciparum, both RDTs had significantly higher sensitivity for cases with a parasite density above 500 parasites/μL (>92%) than those with a density below 500 parasites/μL (<75.0%) (Table
6). However, for P. vivax malaria, regardless of the parasite density, both RDTs displayed sensitivity of below 79%. Both RDTs had similar detection limits; they were >240 and >320 parasites/μL for P. falciparum and P. vivax, respectively.
Comparative sensitivity of the two RDTs for detection of
P. falciparum
and
P. vivax
in comparison with the microscopic method, categorized according to parasite density
Sensitivity is presented as percentage (95% confidence interval; CI). TP true positive.
To further evaluate the performance of these two RDTs, all 606 samples were examined by nested PCR. Nested PCR detected malaria parasites in 174 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. No P. knowlesi infections were detected by PCR (Table
7). For the subset of 350 samples, 113 samples were identified by nested PCR as infection with malaria parasites. Of which, 40, 52, and 21 were P. falciparum, P. vivax, and P. falciparum/P. vivax mixed infections, respectively (Table
8). Among all detection methods, PCR was the most sensitive one. If PCR was used as the gold standard, the sensitivity of microscopy for P. falciparum and P. vivax was 71.0% and 73.3% in 606 detected samples, and 75.4% and 74.0% in the 350 subset samples, respectively (Table
9). The sensitivity of Pf/Pan test for P. falciparum and P. vivax was 81.7% and 64.6% in 606 detected samples, and 77.1% and 63.5% in the 350 subset samples, respectively (Table
9). For the subset of 350 samples analyzed by Pv/Pf devices, the sensitivity was 72.1% for P. falciparum and 58.9% for P. vivax, respectively (Table
9). Microscopy and the two RDTs had similar levels of specificity ranging from 94.7% to 96.3% (Table
9). It is noteworthy that although the Pf/Pan device is designed to identify all human malaria parasite species, the two P. ovale infections identified by PCR were missed by this device and by microscopy, possibly due to the low parasitaemia in the P. ovale infections (containing 120 parasites/μL and 320 parasites/μL, respectively). Re-examination of the P. ovale slides by microscopy did detect P. ovale-parasitized erythrocytes (Figure
1).
Blood smears showing parasitized erythrocytes by P. ovale in two malaria patients. The two patients with febrile illness attended a malaria clinic at the China-Myanmar border and were diagnosed for malaria. Both cases were only detected by PCR but missed by microscopy and RDTs.
Detection results of malaria infections by microscopy and Malaria Pf/Pan test in comparison with the Nested PCR (N=606)
* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.
Detection results of malaria infections by microscopy, Malaria Pf/Pan test and Malaria Pv/Pf test in comparison with the Nested PCR (N=350)
* For the Pf/Pan device, these cases had both test lines visible, suggesting they either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.
Performance of different diagnosis methods with the nested PCR method as the gold standard
Data are presented as percentage (95% confidence interval; CI).
According to the microscopy results corrected by PCR, four and eight cases were false negative in 606 samples examined by the Pf/Pan test device for P. falciparum and P. vivax, respectively (Table
3). When mixed infections were excluded, three out of four P. falciparum cases had lower parasite density (<480 parasites/μL). Only one P. falciparum case with higher parasite density (8,800 parasites/μL) was not detected by the Pf/Pan test device, but was positive by the Pv/Pf test device. Five out of eight P. vivax cases were lower parasite density (<560 parasites/μL). The remaining three P. vivax cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by the Pf/Pan test device. In the subset of 350 samples examined by the Pv/Pf test device (mixed infection not included, Table
5), two P. falciparum cases with lower parasite density (<480 parasites/μL) were false negative. Nine P. vivax cases were the false negative, of which three had lower parasite density (<320 parasites/μL), and three cases (parasite density of 1,320/μL, 2080/μL, 7,280/μL) were positive by the Pf/Pan test device. The other three cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by both RDTs.
False positive and wrong species identification were observed with both RDTs. In all 606 samples (identified by microscopy and corrected by PCR, mixed infection not included) examined by the Pf/Pan test device, one P. vivax case, one P. ovale case, and eight negative cases showed the PfHRP2 line. Ten P. vivax cases showed not only the pan-pLDH line, but the PfHRP2 line. Seven negative cases showed both PfHRP2 and pan-pLDH lines (Table
7). In the subset of 350 samples analysed by the Pv/Pf test, two negative cases showed the PfHRP2 line. One P. falciparum case showed the Pv-pLDH line only. Two P. vivax cases showed not only the Pv-pLDH line, but the PfHRP2-line. Six P. falciparum cases (parasite density ranging from 240 to 34,400 parasites/μL) showed not only the PfHRP2 line, but also the Pv-pLDH line (Table
8). It has been reported that P. falciparum infections with high parasite densities may generate a false positive Pv-pLDH line
[22-25]. For the Pv/Pf test devices, six out of 40 P. falciparum (corrected by PCR) with the Pv-pLDH line visible showed that cross reactions between different species occurred although parasite densities of some P. falciparum infections were not high. From these comparisons, the specificities of these devices need further improvement for areas with both P. falciparum and P. vivax malaria.
Since P. falciparum and P. vivax infections are treated with different drugs, it would be important to compare the different methods for detecting mixed species infections. PCR, microscopy and Pv/Pf test device detected 21, 11, and eight mixed-species infections in the 350 subset samples (Table
8), corresponding to 18.6%, 11.2% and 8.9% of positive cases detected by the respective methods.
|
Conclusions
|
The aim of this study was to evaluate the performance of two RDTs for malaria diagnosis at the China-Myanmar border area. PCR method was much more sensitive than microscopy and RDTs. Compared to microscopy, both RDTs demonstrated similar sensitivities for detecting P. falciparum infections but reduced sensitivity for detecting P. vivax infections. Neither of them had satisfactory results in detecting mixed species infections. Therefore, methods with improved sensitivity for diagnosing P. vivax malaria are needed. False negative results for the diagnosis of P. falciparum malaria call for further investigations on potential deletion of pfhrp2 gene in this malaria-endemic region.
|
[
"Background",
"Study site and patients",
"Microscopic examinations",
"RDTs for malaria",
"Diagnosis by PCR",
"Statistical analysis",
"Competing interests",
"Authors’ contributions"
] |
[
"Malaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually\n[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms\n[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease\n[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.\nMicroscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately\n[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific\n[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine\n[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings\n[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign\n[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.\nRDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results\n[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported\n[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria\n[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria.",
"The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.",
"Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.",
"The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.",
"Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections",
"The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.",
"The authors declare that they have no competing interests.",
"YJ, LN, WX, LP and ZZ carried out the experimental work and data analysis.WL, LS and ZG participated in data analysis. YJ performed manuscript writing. YC, QF and LC conceived the study and participated in the design of the study. All authors read and approved the final manuscript."
] |
[
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Study site and patients",
"Microscopic examinations",
"RDTs for malaria",
"Diagnosis by PCR",
"Statistical analysis",
"Results",
"Discussion",
"Conclusions",
"Competing interests",
"Authors’ contributions"
] |
[
"Malaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually\n[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms\n[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease\n[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.\nMicroscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately\n[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific\n[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine\n[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings\n[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign\n[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.\nRDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results\n[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported\n[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria\n[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria.",
" Study site and patients The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\nThe study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.\n Microscopic examinations Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\nThick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.\n RDTs for malaria The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\nThe RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.\n Diagnosis by PCR Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\nFresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections\n Statistical analysis The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.\nThe RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.",
"The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.",
"Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood\n[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.",
"The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.",
"Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene\n[20,21]. Plasmodium genus-specific primers were shown in Table \n1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table \n1.\nPrimers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections",
"The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table \n2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.\nInterpretation of the results for the Pf/Pan RDT\n* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.\n** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.",
"Blood samples were collected from a total of 606 patients. All samples were evaluated by the Pf/Pan test device, while a subset of 350 were also evaluated by the Pv/Pf test device. Of the 606 samples, malaria parasites were found in 143 by microscopy; 51, 73, and 19 were P. falciparum, P. vivax and P. falciparum/P. vivax mixed infections, respectively (Table \n3). No other malaria parasite species were detected by microscopy. The parasite density ranges of P. falciparum were 40–105,920 parasites/μL. For P. vivax, the majority of cases had parasite density ranging from 80 to 17,800 parasites/μL. One P. vivax case had a deviant parasite density of >200,000 parasites/μL based on leucocyte number probably due to a low leucocyte count in this patient. Because P. falciparum single infections and mixed species infections containing P. falciparum cannot be differentiated by the Pf/Pan device, the microscopy results were grouped into “P. falciparum and mixed infections with P. falciparum” and “Non-falciparum”. Based on this interpretation, the Pf/Pan device detected 33 single P. falciparum infections (only the PfHRP2 line visible), 56 non-falciparum cases (only the pan-pLDH line visible) and 70 P. falciparum infections/potentially mixed infections (both lines visible) (Table \n3). Compared with the results by microscopy, the Pf/Pan device detected 62 and 51 samples as true positives for P. falciparum and non-falciparum, respectively. From this result, the sensitivity of Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4% (Table \n4).\nDetection results of malaria infections by the Malaria Pf/Pan test in comparison with microscopy (N=606)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\n\nPerformance of two RDTs for detection of \n\nP. falciparum \n\nand \n\nP. vivax \n\nwith microscopy as the gold standard\n\nData are presented as percentage (95% confidence interval; CI).\nFor the subset of 350 samples, microscopy detected 37 samples containing P. falciparum, 50 samples containing P. vivax and 11 were diagnosed as mixed species infections (Table \n5). Since the Pv/Pf device is able to differentiate P. falciparum and P. vivax infections, the microscopy results were grouped into “infections containing P. falciparum” and “infections containing P. vivax”. The Pv/Pf device detected 40 single P. falciparum infections, 42 P. vivax infections, and 8 mixed species infections (Table \n5). Compared to microscopy, the Pv/Pf device detected 44 and 45 samples as true positives for P. falciparum and P. vivax, respectively, whereas the Pf/Pan device detected 42 and 36 samples as true positives for P. falciparum and P. vivax, respectively. The sensitivity of Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively (Table \n4). The specificity of Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively (Table \n4).\nDetection results of malaria infections by Malaria Pv/Pf test and Malaria Pf/Pan test in comparison with the microscopic method (N=350)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nIt has been observed that some RDTs do not perform well when the parasite density is below 500 parasites/μL. For P. falciparum, both RDTs had significantly higher sensitivity for cases with a parasite density above 500 parasites/μL (>92%) than those with a density below 500 parasites/μL (<75.0%) (Table \n6). However, for P. vivax malaria, regardless of the parasite density, both RDTs displayed sensitivity of below 79%. Both RDTs had similar detection limits; they were >240 and >320 parasites/μL for P. falciparum and P. vivax, respectively.\n\nComparative sensitivity of the two RDTs for detection of \n\nP. falciparum \n\nand \n\nP. vivax \n\nin comparison with the microscopic method, categorized according to parasite density\n\nSensitivity is presented as percentage (95% confidence interval; CI). TP true positive.\nTo further evaluate the performance of these two RDTs, all 606 samples were examined by nested PCR. Nested PCR detected malaria parasites in 174 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. No P. knowlesi infections were detected by PCR (Table \n7). For the subset of 350 samples, 113 samples were identified by nested PCR as infection with malaria parasites. Of which, 40, 52, and 21 were P. falciparum, P. vivax, and P. falciparum/P. vivax mixed infections, respectively (Table \n8). Among all detection methods, PCR was the most sensitive one. If PCR was used as the gold standard, the sensitivity of microscopy for P. falciparum and P. vivax was 71.0% and 73.3% in 606 detected samples, and 75.4% and 74.0% in the 350 subset samples, respectively (Table \n9). The sensitivity of Pf/Pan test for P. falciparum and P. vivax was 81.7% and 64.6% in 606 detected samples, and 77.1% and 63.5% in the 350 subset samples, respectively (Table \n9). For the subset of 350 samples analyzed by Pv/Pf devices, the sensitivity was 72.1% for P. falciparum and 58.9% for P. vivax, respectively (Table \n9). Microscopy and the two RDTs had similar levels of specificity ranging from 94.7% to 96.3% (Table \n9). It is noteworthy that although the Pf/Pan device is designed to identify all human malaria parasite species, the two P. ovale infections identified by PCR were missed by this device and by microscopy, possibly due to the low parasitaemia in the P. ovale infections (containing 120 parasites/μL and 320 parasites/μL, respectively). Re-examination of the P. ovale slides by microscopy did detect P. ovale-parasitized erythrocytes (Figure \n1).\nBlood smears showing parasitized erythrocytes by P. ovale in two malaria patients. The two patients with febrile illness attended a malaria clinic at the China-Myanmar border and were diagnosed for malaria. Both cases were only detected by PCR but missed by microscopy and RDTs.\nDetection results of malaria infections by microscopy and Malaria Pf/Pan test in comparison with the Nested PCR (N=606)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nDetection results of malaria infections by microscopy, Malaria Pf/Pan test and Malaria Pv/Pf test in comparison with the Nested PCR (N=350)\n* For the Pf/Pan device, these cases had both test lines visible, suggesting they either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.\nPerformance of different diagnosis methods with the nested PCR method as the gold standard\nData are presented as percentage (95% confidence interval; CI).\nAccording to the microscopy results corrected by PCR, four and eight cases were false negative in 606 samples examined by the Pf/Pan test device for P. falciparum and P. vivax, respectively (Table \n3). When mixed infections were excluded, three out of four P. falciparum cases had lower parasite density (<480 parasites/μL). Only one P. falciparum case with higher parasite density (8,800 parasites/μL) was not detected by the Pf/Pan test device, but was positive by the Pv/Pf test device. Five out of eight P. vivax cases were lower parasite density (<560 parasites/μL). The remaining three P. vivax cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by the Pf/Pan test device. In the subset of 350 samples examined by the Pv/Pf test device (mixed infection not included, Table \n5), two P. falciparum cases with lower parasite density (<480 parasites/μL) were false negative. Nine P. vivax cases were the false negative, of which three had lower parasite density (<320 parasites/μL), and three cases (parasite density of 1,320/μL, 2080/μL, 7,280/μL) were positive by the Pf/Pan test device. The other three cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by both RDTs.\nFalse positive and wrong species identification were observed with both RDTs. In all 606 samples (identified by microscopy and corrected by PCR, mixed infection not included) examined by the Pf/Pan test device, one P. vivax case, one P. ovale case, and eight negative cases showed the PfHRP2 line. Ten P. vivax cases showed not only the pan-pLDH line, but the PfHRP2 line. Seven negative cases showed both PfHRP2 and pan-pLDH lines (Table \n7). In the subset of 350 samples analysed by the Pv/Pf test, two negative cases showed the PfHRP2 line. One P. falciparum case showed the Pv-pLDH line only. Two P. vivax cases showed not only the Pv-pLDH line, but the PfHRP2-line. Six P. falciparum cases (parasite density ranging from 240 to 34,400 parasites/μL) showed not only the PfHRP2 line, but also the Pv-pLDH line (Table \n8). It has been reported that P. falciparum infections with high parasite densities may generate a false positive Pv-pLDH line\n[22-25]. For the Pv/Pf test devices, six out of 40 P. falciparum (corrected by PCR) with the Pv-pLDH line visible showed that cross reactions between different species occurred although parasite densities of some P. falciparum infections were not high. From these comparisons, the specificities of these devices need further improvement for areas with both P. falciparum and P. vivax malaria.\nSince P. falciparum and P. vivax infections are treated with different drugs, it would be important to compare the different methods for detecting mixed species infections. PCR, microscopy and Pv/Pf test device detected 21, 11, and eight mixed-species infections in the 350 subset samples (Table \n8), corresponding to 18.6%, 11.2% and 8.9% of positive cases detected by the respective methods.",
"RDTs are playing an essential role in the current malaria control/elimination campaign worldwide. In this study, the performance of two RDT devices commonly used at the China-Myanmar border area was evaluated. Compared to diagnosis by microscopy, both the Pf/Pan and Pv/Pf devices showed higher sensitivity (>87%) for detecting P. falciparum infections, whereas the sensitivity for detecting P. vivax infections was much lower (<74%). Both RDTs had similar levels of detection limits, and their performance was poor for infections with parasite densities below 500 parasites/μL for P. falciparum infections. Besides, since both devices rely on the PfHRP2 antigen for the detection of P. falciparum, pfhrp2 gene deletions will lead to false-negative results\n[26]. A recent report showing up to 40% of P. falciparum parasites with pfhrp2 deletions in South America\n[27] indicate that such RDTs would not perform well in these regions. It certainly warrants more detailed investigations in other malaria endemic regions.\nWhen PCR was used as the gold standard, the detection sensitivity of both RDTs was reduced especially for the detection of P. vivax infections (<64%), although the specificity of the all detection methods remained above 92%. In addition, the sensitivity of conventional microscopy was also low, ranging from 71.0% to 77.4% for detecting both parasite species. This result is worrisome; since nearly 30% of the malaria cases was not correctly diagnosed by microscopy or RDTs and subsequently treated, these patients may constitute an important reservoir for transmission. Whereas low parasite density might be the most important limiting factor for microscopic diagnosis\n[6,28], the varying skills and experience of microscopists may also be responsible for the lower sensitivity of diagnosis\n[29]. In the past, the Pf/Pan test (Wondfo) has been evaluated for diagnosis of P. falciparum only and showed highly satisfactory result\n[30]. Recently, a CareStart™ kit (Pf/Pan) was evaluated in the nearby Yunnan province of China, which showed comparable results to microscopy for diagnosing P. falciparum and P. vivax infections\n[31]. Therefore, further evaluations of RDTs are needed to select better diagnostic tests with improved accuracy in this region.\nOverall, both RDTs have good sensitivity for detecting P. falciparum infections, but the sensitivity for detecting P. vivax was much lower. This could be due to lower parasite density for P. vivax since this parasite selectively invades reticulocytes. However, comparison of the two RDTs showed that the sensitivities for P. vivax detection were not different between the high and low parasite densities (Table \n6). This observation is difficult to explain and could be due to the lower number of infections analyzed in the <500 parasites/μL group. Furthermore, the arbitrary selection of 500 parasites/μL as the border line between high and low parasite densities may not be appropriate for P. vivax. In addition, the sensitivity to P. vivax may be affected by other factors. Therefore, better RDTs with significantly improved sensitivity for P. vivax are needed.\nOne important criterion for the selection of RDTs in the GMS is the ability to detect other human malaria species in addition to P. falciparum. The advantage of the Pf/Pan test is that it is designed to detect four human parasite species, which co-exist in the GMS. However, this device failed to detect the two P. ovale infections, suggesting that further improvement in sensitivity for detecting other malaria species is needed. It is also worth to note that the PCR method failed to identify any P. knowlesi infections, which is in stark contrast to the eaerlier report of some 30% of malaria cases in this region as mixed infections with P. knowlesi[16]. Therefore, the significantly lower prevalence of other malaria parasite species in this region and the generally low sensitivity of the Pf/Pan test for diagnosis of P. ovale and P. malariae suggest that such an advantage is hardly of any importance in this region. In comparison, the Pv/Pf test is specific for P. falciparum and P. vivax, which make up the majority of malaria infections in the GMS, although it would certainly miss detecting P. malariae and P. ovale infections. Because P. falciparum and P. vivax require different antimalarial treatments, correct diagnosis of mixed infections should be an important criterion for the selection of an RDT. Even though the Pv/Pf device is able to detect P. falciparum and P. vivax mixed infection, the detection rate was low and the eight mix infections detected by this device were all misdiagnosed when compared to the PCR result. This result may be due to reduced density of one parasite species in the presence of another due to competition in the same host. Consequently, with the current anti-malarial drug policy of this region, misdiagnosis of mixed species infections as single species infections would lead to improper drug treatments.",
"The aim of this study was to evaluate the performance of two RDTs for malaria diagnosis at the China-Myanmar border area. PCR method was much more sensitive than microscopy and RDTs. Compared to microscopy, both RDTs demonstrated similar sensitivities for detecting P. falciparum infections but reduced sensitivity for detecting P. vivax infections. Neither of them had satisfactory results in detecting mixed species infections. Therefore, methods with improved sensitivity for diagnosing P. vivax malaria are needed. False negative results for the diagnosis of P. falciparum malaria call for further investigations on potential deletion of pfhrp2 gene in this malaria-endemic region.",
"The authors declare that they have no competing interests.",
"YJ, LN, WX, LP and ZZ carried out the experimental work and data analysis.WL, LS and ZG participated in data analysis. YJ performed manuscript writing. YC, QF and LC conceived the study and participated in the design of the study. All authors read and approved the final manuscript."
] |
[
null,
"methods",
null,
null,
null,
null,
null,
"results",
"discussion",
"conclusions",
null,
null
] |
[
"Rapid diagnostic tests (RDTs)",
"Malaria diagnosis",
"Microscopy",
"PCR",
"Sensitivity",
"Specificity"
] |
Background:
Malaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually
[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms
[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease
[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.
Microscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately
[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific
[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine
[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings
[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign
[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.
RDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results
[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported
[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria
[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria.
Methods:
Study site and patients The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.
The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.
Microscopic examinations Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood
[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.
Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood
[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.
RDTs for malaria The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.
The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.
Diagnosis by PCR Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene
[20,21]. Plasmodium genus-specific primers were shown in Table
1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table
1.
Primers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections
Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene
[20,21]. Plasmodium genus-specific primers were shown in Table
1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table
1.
Primers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections
Statistical analysis The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table
2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.
Interpretation of the results for the Pf/Pan RDT
* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.
** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.
The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table
2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.
Interpretation of the results for the Pf/Pan RDT
* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.
** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.
Study site and patients:
The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by P. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.
Microscopic examinations:
Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood
[19]. Blood films were examined by an experienced microscopist who was ‘blinded’ to the results of additional diagnostic tests. Smears were considered negative if no parasite was seen in 100 oil immersion fields on a thick blood film. All the slides were double checked blindly by a second, independent microscopist and the results were combined. Parasite density was calculated by determining the number of parasites per 200 white blood cells in a thick blood film.
RDTs for malaria:
The RDTs used in this study are One Step Malaria Pf/Pan test (Wondfo, China) and Malaria Pv/Pf test device (Cat. No. 200317, Tycolpharm Co., Limited, UK). The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for P. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.
Diagnosis by PCR:
Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene
[20,21]. Plasmodium genus-specific primers were shown in Table
1. For the primary PCR reactions, 2 μL of genomic DNA were used in a 25 μL reaction with outer primers rPLU5 and rPLU6, and 30 cycles (94°C for 1 min, 55°C for 2 min and 72°C for 2 min) were performed. For nested PCR, 2 μL of the primary PCR product were used as the template with species-specific primers for the four human malaria species and P. knowlesi in separate reaction tubes. After another 30 cycles of amplification (94°C for 40 s, 58°C for 1 min and 72°C for 2 min), the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table
1.
Primers based on the 18S rRNA gene in malaria parasites for nested PCR diagnosis of malaria infections
Statistical analysis:
The RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and (2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table
2. Sensitivity and specificity were calculated as TP/(TP+FN) and TN/(TN+FP), respectively. Statistical analysis was performed by the Chi-square test using the SPSS version 15.0 and the significance level was set as < 0.05.
Interpretation of the results for the Pf/Pan RDT
* P. falciparum or as a mixed infection with P. falciparum diagnosed as non-falciparum species.
** Non-falciparum species diagnosed as P. falciparum or as a mixed infection with P. falciparum.
Results:
Blood samples were collected from a total of 606 patients. All samples were evaluated by the Pf/Pan test device, while a subset of 350 were also evaluated by the Pv/Pf test device. Of the 606 samples, malaria parasites were found in 143 by microscopy; 51, 73, and 19 were P. falciparum, P. vivax and P. falciparum/P. vivax mixed infections, respectively (Table
3). No other malaria parasite species were detected by microscopy. The parasite density ranges of P. falciparum were 40–105,920 parasites/μL. For P. vivax, the majority of cases had parasite density ranging from 80 to 17,800 parasites/μL. One P. vivax case had a deviant parasite density of >200,000 parasites/μL based on leucocyte number probably due to a low leucocyte count in this patient. Because P. falciparum single infections and mixed species infections containing P. falciparum cannot be differentiated by the Pf/Pan device, the microscopy results were grouped into “P. falciparum and mixed infections with P. falciparum” and “Non-falciparum”. Based on this interpretation, the Pf/Pan device detected 33 single P. falciparum infections (only the PfHRP2 line visible), 56 non-falciparum cases (only the pan-pLDH line visible) and 70 P. falciparum infections/potentially mixed infections (both lines visible) (Table
3). Compared with the results by microscopy, the Pf/Pan device detected 62 and 51 samples as true positives for P. falciparum and non-falciparum, respectively. From this result, the sensitivity of Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4% (Table
4).
Detection results of malaria infections by the Malaria Pf/Pan test in comparison with microscopy (N=606)
* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.
Performance of two RDTs for detection of
P. falciparum
and
P. vivax
with microscopy as the gold standard
Data are presented as percentage (95% confidence interval; CI).
For the subset of 350 samples, microscopy detected 37 samples containing P. falciparum, 50 samples containing P. vivax and 11 were diagnosed as mixed species infections (Table
5). Since the Pv/Pf device is able to differentiate P. falciparum and P. vivax infections, the microscopy results were grouped into “infections containing P. falciparum” and “infections containing P. vivax”. The Pv/Pf device detected 40 single P. falciparum infections, 42 P. vivax infections, and 8 mixed species infections (Table
5). Compared to microscopy, the Pv/Pf device detected 44 and 45 samples as true positives for P. falciparum and P. vivax, respectively, whereas the Pf/Pan device detected 42 and 36 samples as true positives for P. falciparum and P. vivax, respectively. The sensitivity of Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively (Table
4). The specificity of Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively (Table
4).
Detection results of malaria infections by Malaria Pv/Pf test and Malaria Pf/Pan test in comparison with the microscopic method (N=350)
* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.
It has been observed that some RDTs do not perform well when the parasite density is below 500 parasites/μL. For P. falciparum, both RDTs had significantly higher sensitivity for cases with a parasite density above 500 parasites/μL (>92%) than those with a density below 500 parasites/μL (<75.0%) (Table
6). However, for P. vivax malaria, regardless of the parasite density, both RDTs displayed sensitivity of below 79%. Both RDTs had similar detection limits; they were >240 and >320 parasites/μL for P. falciparum and P. vivax, respectively.
Comparative sensitivity of the two RDTs for detection of
P. falciparum
and
P. vivax
in comparison with the microscopic method, categorized according to parasite density
Sensitivity is presented as percentage (95% confidence interval; CI). TP true positive.
To further evaluate the performance of these two RDTs, all 606 samples were examined by nested PCR. Nested PCR detected malaria parasites in 174 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. No P. knowlesi infections were detected by PCR (Table
7). For the subset of 350 samples, 113 samples were identified by nested PCR as infection with malaria parasites. Of which, 40, 52, and 21 were P. falciparum, P. vivax, and P. falciparum/P. vivax mixed infections, respectively (Table
8). Among all detection methods, PCR was the most sensitive one. If PCR was used as the gold standard, the sensitivity of microscopy for P. falciparum and P. vivax was 71.0% and 73.3% in 606 detected samples, and 75.4% and 74.0% in the 350 subset samples, respectively (Table
9). The sensitivity of Pf/Pan test for P. falciparum and P. vivax was 81.7% and 64.6% in 606 detected samples, and 77.1% and 63.5% in the 350 subset samples, respectively (Table
9). For the subset of 350 samples analyzed by Pv/Pf devices, the sensitivity was 72.1% for P. falciparum and 58.9% for P. vivax, respectively (Table
9). Microscopy and the two RDTs had similar levels of specificity ranging from 94.7% to 96.3% (Table
9). It is noteworthy that although the Pf/Pan device is designed to identify all human malaria parasite species, the two P. ovale infections identified by PCR were missed by this device and by microscopy, possibly due to the low parasitaemia in the P. ovale infections (containing 120 parasites/μL and 320 parasites/μL, respectively). Re-examination of the P. ovale slides by microscopy did detect P. ovale-parasitized erythrocytes (Figure
1).
Blood smears showing parasitized erythrocytes by P. ovale in two malaria patients. The two patients with febrile illness attended a malaria clinic at the China-Myanmar border and were diagnosed for malaria. Both cases were only detected by PCR but missed by microscopy and RDTs.
Detection results of malaria infections by microscopy and Malaria Pf/Pan test in comparison with the Nested PCR (N=606)
* For the Pf/Pan device, these cases had both test lines visible, suggesting they were either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.
Detection results of malaria infections by microscopy, Malaria Pf/Pan test and Malaria Pv/Pf test in comparison with the Nested PCR (N=350)
* For the Pf/Pan device, these cases had both test lines visible, suggesting they either P. falciparum single infections or P. falciparum/non-falciparum mixed infections.
Performance of different diagnosis methods with the nested PCR method as the gold standard
Data are presented as percentage (95% confidence interval; CI).
According to the microscopy results corrected by PCR, four and eight cases were false negative in 606 samples examined by the Pf/Pan test device for P. falciparum and P. vivax, respectively (Table
3). When mixed infections were excluded, three out of four P. falciparum cases had lower parasite density (<480 parasites/μL). Only one P. falciparum case with higher parasite density (8,800 parasites/μL) was not detected by the Pf/Pan test device, but was positive by the Pv/Pf test device. Five out of eight P. vivax cases were lower parasite density (<560 parasites/μL). The remaining three P. vivax cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by the Pf/Pan test device. In the subset of 350 samples examined by the Pv/Pf test device (mixed infection not included, Table
5), two P. falciparum cases with lower parasite density (<480 parasites/μL) were false negative. Nine P. vivax cases were the false negative, of which three had lower parasite density (<320 parasites/μL), and three cases (parasite density of 1,320/μL, 2080/μL, 7,280/μL) were positive by the Pf/Pan test device. The other three cases with higher parasite density (1,880/μL, 2,760/μL and 15,840/μL) were not detected by both RDTs.
False positive and wrong species identification were observed with both RDTs. In all 606 samples (identified by microscopy and corrected by PCR, mixed infection not included) examined by the Pf/Pan test device, one P. vivax case, one P. ovale case, and eight negative cases showed the PfHRP2 line. Ten P. vivax cases showed not only the pan-pLDH line, but the PfHRP2 line. Seven negative cases showed both PfHRP2 and pan-pLDH lines (Table
7). In the subset of 350 samples analysed by the Pv/Pf test, two negative cases showed the PfHRP2 line. One P. falciparum case showed the Pv-pLDH line only. Two P. vivax cases showed not only the Pv-pLDH line, but the PfHRP2-line. Six P. falciparum cases (parasite density ranging from 240 to 34,400 parasites/μL) showed not only the PfHRP2 line, but also the Pv-pLDH line (Table
8). It has been reported that P. falciparum infections with high parasite densities may generate a false positive Pv-pLDH line
[22-25]. For the Pv/Pf test devices, six out of 40 P. falciparum (corrected by PCR) with the Pv-pLDH line visible showed that cross reactions between different species occurred although parasite densities of some P. falciparum infections were not high. From these comparisons, the specificities of these devices need further improvement for areas with both P. falciparum and P. vivax malaria.
Since P. falciparum and P. vivax infections are treated with different drugs, it would be important to compare the different methods for detecting mixed species infections. PCR, microscopy and Pv/Pf test device detected 21, 11, and eight mixed-species infections in the 350 subset samples (Table
8), corresponding to 18.6%, 11.2% and 8.9% of positive cases detected by the respective methods.
Discussion:
RDTs are playing an essential role in the current malaria control/elimination campaign worldwide. In this study, the performance of two RDT devices commonly used at the China-Myanmar border area was evaluated. Compared to diagnosis by microscopy, both the Pf/Pan and Pv/Pf devices showed higher sensitivity (>87%) for detecting P. falciparum infections, whereas the sensitivity for detecting P. vivax infections was much lower (<74%). Both RDTs had similar levels of detection limits, and their performance was poor for infections with parasite densities below 500 parasites/μL for P. falciparum infections. Besides, since both devices rely on the PfHRP2 antigen for the detection of P. falciparum, pfhrp2 gene deletions will lead to false-negative results
[26]. A recent report showing up to 40% of P. falciparum parasites with pfhrp2 deletions in South America
[27] indicate that such RDTs would not perform well in these regions. It certainly warrants more detailed investigations in other malaria endemic regions.
When PCR was used as the gold standard, the detection sensitivity of both RDTs was reduced especially for the detection of P. vivax infections (<64%), although the specificity of the all detection methods remained above 92%. In addition, the sensitivity of conventional microscopy was also low, ranging from 71.0% to 77.4% for detecting both parasite species. This result is worrisome; since nearly 30% of the malaria cases was not correctly diagnosed by microscopy or RDTs and subsequently treated, these patients may constitute an important reservoir for transmission. Whereas low parasite density might be the most important limiting factor for microscopic diagnosis
[6,28], the varying skills and experience of microscopists may also be responsible for the lower sensitivity of diagnosis
[29]. In the past, the Pf/Pan test (Wondfo) has been evaluated for diagnosis of P. falciparum only and showed highly satisfactory result
[30]. Recently, a CareStart™ kit (Pf/Pan) was evaluated in the nearby Yunnan province of China, which showed comparable results to microscopy for diagnosing P. falciparum and P. vivax infections
[31]. Therefore, further evaluations of RDTs are needed to select better diagnostic tests with improved accuracy in this region.
Overall, both RDTs have good sensitivity for detecting P. falciparum infections, but the sensitivity for detecting P. vivax was much lower. This could be due to lower parasite density for P. vivax since this parasite selectively invades reticulocytes. However, comparison of the two RDTs showed that the sensitivities for P. vivax detection were not different between the high and low parasite densities (Table
6). This observation is difficult to explain and could be due to the lower number of infections analyzed in the <500 parasites/μL group. Furthermore, the arbitrary selection of 500 parasites/μL as the border line between high and low parasite densities may not be appropriate for P. vivax. In addition, the sensitivity to P. vivax may be affected by other factors. Therefore, better RDTs with significantly improved sensitivity for P. vivax are needed.
One important criterion for the selection of RDTs in the GMS is the ability to detect other human malaria species in addition to P. falciparum. The advantage of the Pf/Pan test is that it is designed to detect four human parasite species, which co-exist in the GMS. However, this device failed to detect the two P. ovale infections, suggesting that further improvement in sensitivity for detecting other malaria species is needed. It is also worth to note that the PCR method failed to identify any P. knowlesi infections, which is in stark contrast to the eaerlier report of some 30% of malaria cases in this region as mixed infections with P. knowlesi[16]. Therefore, the significantly lower prevalence of other malaria parasite species in this region and the generally low sensitivity of the Pf/Pan test for diagnosis of P. ovale and P. malariae suggest that such an advantage is hardly of any importance in this region. In comparison, the Pv/Pf test is specific for P. falciparum and P. vivax, which make up the majority of malaria infections in the GMS, although it would certainly miss detecting P. malariae and P. ovale infections. Because P. falciparum and P. vivax require different antimalarial treatments, correct diagnosis of mixed infections should be an important criterion for the selection of an RDT. Even though the Pv/Pf device is able to detect P. falciparum and P. vivax mixed infection, the detection rate was low and the eight mix infections detected by this device were all misdiagnosed when compared to the PCR result. This result may be due to reduced density of one parasite species in the presence of another due to competition in the same host. Consequently, with the current anti-malarial drug policy of this region, misdiagnosis of mixed species infections as single species infections would lead to improper drug treatments.
Conclusions:
The aim of this study was to evaluate the performance of two RDTs for malaria diagnosis at the China-Myanmar border area. PCR method was much more sensitive than microscopy and RDTs. Compared to microscopy, both RDTs demonstrated similar sensitivities for detecting P. falciparum infections but reduced sensitivity for detecting P. vivax infections. Neither of them had satisfactory results in detecting mixed species infections. Therefore, methods with improved sensitivity for diagnosing P. vivax malaria are needed. False negative results for the diagnosis of P. falciparum malaria call for further investigations on potential deletion of pfhrp2 gene in this malaria-endemic region.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
YJ, LN, WX, LP and ZZ carried out the experimental work and data analysis.WL, LS and ZG participated in data analysis. YJ performed manuscript writing. YC, QF and LC conceived the study and participated in the design of the study. All authors read and approved the final manuscript.
|
Background:
Rapid diagnostic tests (RDTs) have become an essential tool in the contemporary malaria control and management programmes in the world. This study aims to evaluate the performance of two commonly used RDTs for malaria diagnosis in the China-Myanmar border area.
Methods:
A total 606 febrile patients in the China-Myanmar border were recruited to this study and were diagnosed for malaria infections by microscopy, two RDTs tests (Pf/Pan device, and Pv/Pf device) and nested PCR.
Results:
Malaria parasites were found in 143 patients by microscopy, of which 51, 73, and 19 were Plasmodium falciparum, Plasmodium vivax and P. falciparum/P. vivax mixed infections, respectively. Compared to microscopy, the sensitivity of the Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4%. For a subset of 350 patients, the sensitivity of the Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively. The specificity of the Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively. Nested PCR detected malaria parasites in 174 of 606 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. Compared to nested PCR, all other methods had sensitivity below 80%, suggesting that a significant number of cases were missed.
Conclusions:
Compared to PCR, both microscopy and RDTs had lower sensitivities. RDTs had similar performance to microscopy for P. falciparum diagnosis, but performed worse for P. vivax diagnosis. Other RDT products should be selected with higher sensitivity (and good specificity) for both P. falciparum and P. vivax diagnosis.
|
Background:
Malaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually
[1]. Malaria in human can be caused by one of five malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms
[2], which changed the traditional view of this malaria as “benign tertian”. The great impact of the 130–435 million P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease
[3,4]. In many parts of the world, such as Southeast Asia, P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.
Microscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately
[5,6]. Furthermore, microscopic diagnosis of P. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific
[7,8], but drawbacks such as a requirement for equipment, higher cost, and a lengthy procedure limit their routine
[9-11]. Malaria rapid diagnostic tests (RDTs) have become very popular in various endemic settings
[12], especially in areas where microscopic expertise is lacking. They are now an essential tool in malaria management during the malaria elimination/eradication campaign
[13]. However, the wide variety of RDTs and their different performance under different endemic settings suggest that careful comparison of RDTs is needed before mass deployment for diagnosis.
RDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as P. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results
[14]. In malaria endemic areas of the Greater Mekong Subregion (GMS), the four human malaria species often co-exist, but with P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported
[15,16]. Several types of RDTs have been evaluated in these areas, and most of them had poor performance for low levels of parasitaemia (e.g., <500 parasites/μL), which is especially true for vivax malaria
[17,18]. In recent years, with extensive supports from the Global Fund to fight AIDS, Tuberculosis and Malaria, the malaria control programme at the China-Myanmar border area has been substantially strengthened. Among the control measures is the mass use of RDTs for malaria diagnosis. Specifically, two types of RDTs have been extensively used based on co-existence of P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria.
Conclusions:
The aim of this study was to evaluate the performance of two RDTs for malaria diagnosis at the China-Myanmar border area. PCR method was much more sensitive than microscopy and RDTs. Compared to microscopy, both RDTs demonstrated similar sensitivities for detecting P. falciparum infections but reduced sensitivity for detecting P. vivax infections. Neither of them had satisfactory results in detecting mixed species infections. Therefore, methods with improved sensitivity for diagnosing P. vivax malaria are needed. False negative results for the diagnosis of P. falciparum malaria call for further investigations on potential deletion of pfhrp2 gene in this malaria-endemic region.
|
Background:
Rapid diagnostic tests (RDTs) have become an essential tool in the contemporary malaria control and management programmes in the world. This study aims to evaluate the performance of two commonly used RDTs for malaria diagnosis in the China-Myanmar border area.
Methods:
A total 606 febrile patients in the China-Myanmar border were recruited to this study and were diagnosed for malaria infections by microscopy, two RDTs tests (Pf/Pan device, and Pv/Pf device) and nested PCR.
Results:
Malaria parasites were found in 143 patients by microscopy, of which 51, 73, and 19 were Plasmodium falciparum, Plasmodium vivax and P. falciparum/P. vivax mixed infections, respectively. Compared to microscopy, the sensitivity of the Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4%. For a subset of 350 patients, the sensitivity of the Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively. The specificity of the Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively. Nested PCR detected malaria parasites in 174 of 606 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. Compared to nested PCR, all other methods had sensitivity below 80%, suggesting that a significant number of cases were missed.
Conclusions:
Compared to PCR, both microscopy and RDTs had lower sensitivities. RDTs had similar performance to microscopy for P. falciparum diagnosis, but performed worse for P. vivax diagnosis. Other RDT products should be selected with higher sensitivity (and good specificity) for both P. falciparum and P. vivax diagnosis.
| 7,424 | 360 | 12 |
[
"falciparum",
"malaria",
"pf",
"vivax",
"infections",
"test",
"pcr",
"pan",
"device",
"rdts"
] |
[
"test",
"test"
] |
[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]
|
[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]
|
[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]
|
[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]
|
[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]
|
[CONTENT] Rapid diagnostic tests (RDTs) | Malaria diagnosis | Microscopy | PCR | Sensitivity | Specificity [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Child | Child, Preschool | China | Clinical Laboratory Techniques | Diagnostic Tests, Routine | Female | Humans | Infant | Malaria | Male | Middle Aged | Myanmar | Parasitology | Plasmodium falciparum | Plasmodium ovale | Plasmodium vivax | Sensitivity and Specificity | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Child | Child, Preschool | China | Clinical Laboratory Techniques | Diagnostic Tests, Routine | Female | Humans | Infant | Malaria | Male | Middle Aged | Myanmar | Parasitology | Plasmodium falciparum | Plasmodium ovale | Plasmodium vivax | Sensitivity and Specificity | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Child | Child, Preschool | China | Clinical Laboratory Techniques | Diagnostic Tests, Routine | Female | Humans | Infant | Malaria | Male | Middle Aged | Myanmar | Parasitology | Plasmodium falciparum | Plasmodium ovale | Plasmodium vivax | Sensitivity and Specificity | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Child | Child, Preschool | China | Clinical Laboratory Techniques | Diagnostic Tests, Routine | Female | Humans | Infant | Malaria | Male | Middle Aged | Myanmar | Parasitology | Plasmodium falciparum | Plasmodium ovale | Plasmodium vivax | Sensitivity and Specificity | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Child | Child, Preschool | China | Clinical Laboratory Techniques | Diagnostic Tests, Routine | Female | Humans | Infant | Malaria | Male | Middle Aged | Myanmar | Parasitology | Plasmodium falciparum | Plasmodium ovale | Plasmodium vivax | Sensitivity and Specificity | Young Adult [SUMMARY]
|
[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Child | Child, Preschool | China | Clinical Laboratory Techniques | Diagnostic Tests, Routine | Female | Humans | Infant | Malaria | Male | Middle Aged | Myanmar | Parasitology | Plasmodium falciparum | Plasmodium ovale | Plasmodium vivax | Sensitivity and Specificity | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] falciparum | malaria | pf | vivax | infections | test | pcr | pan | device | rdts [SUMMARY]
|
[CONTENT] falciparum | malaria | pf | vivax | infections | test | pcr | pan | device | rdts [SUMMARY]
|
[CONTENT] falciparum | malaria | pf | vivax | infections | test | pcr | pan | device | rdts [SUMMARY]
|
[CONTENT] falciparum | malaria | pf | vivax | infections | test | pcr | pan | device | rdts [SUMMARY]
|
[CONTENT] falciparum | malaria | pf | vivax | infections | test | pcr | pan | device | rdts [SUMMARY]
|
[CONTENT] falciparum | malaria | pf | vivax | infections | test | pcr | pan | device | rdts [SUMMARY]
|
[CONTENT] malaria | vivax | plasmodium | diagnosis | areas | rdts | falciparum | specific | endemic | different [SUMMARY]
|
[CONTENT] test | blood | falciparum | pcr | pf | malaria | positive | test device | min | pan [SUMMARY]
|
[CONTENT] falciparum | pf | infections | device | vivax | samples | pan | detected | cases | μl [SUMMARY]
|
[CONTENT] malaria | detecting | infections | microscopy rdts | rdts | sensitivity | diagnosis | vivax | microscopy | rdts demonstrated [SUMMARY]
|
[CONTENT] falciparum | malaria | pf | test | vivax | infections | rdts | blood | parasite | pcr [SUMMARY]
|
[CONTENT] falciparum | malaria | pf | test | vivax | infections | rdts | blood | parasite | pcr [SUMMARY]
|
[CONTENT] ||| two | China | Myanmar [SUMMARY]
|
[CONTENT] 606 | China | Myanmar | two | Pv/Pf | PCR [SUMMARY]
|
[CONTENT] Malaria | 143 | 51 | 73 | 19 | Plasmodium | Plasmodium | P. ||| the Pf/Pan | 88.6% | 69.9% | 90.4% ||| 350 | the Pf/Pan | Pv/Pf | 87.5% | 91.7% | 72.0% | 73.8% ||| Pv/Pf | 94.3% | 96.5% ||| 174 | 606 | 67 | 79 | two | 26 ||| PCR | 80% [SUMMARY]
|
[CONTENT] PCR ||| P. ||| RDT | P. [SUMMARY]
|
[CONTENT] ||| two | China | Myanmar ||| 606 | China | Myanmar | two | Pv/Pf | PCR ||| ||| Malaria | 143 | 51 | 73 | 19 | Plasmodium | Plasmodium | P. ||| the Pf/Pan | 88.6% | 69.9% | 90.4% ||| 350 | the Pf/Pan | Pv/Pf | 87.5% | 91.7% | 72.0% | 73.8% ||| Pv/Pf | 94.3% | 96.5% ||| 174 | 606 | 67 | 79 | two | 26 ||| PCR | 80% ||| PCR ||| P. ||| RDT | P. [SUMMARY]
|
[CONTENT] ||| two | China | Myanmar ||| 606 | China | Myanmar | two | Pv/Pf | PCR ||| ||| Malaria | 143 | 51 | 73 | 19 | Plasmodium | Plasmodium | P. ||| the Pf/Pan | 88.6% | 69.9% | 90.4% ||| 350 | the Pf/Pan | Pv/Pf | 87.5% | 91.7% | 72.0% | 73.8% ||| Pv/Pf | 94.3% | 96.5% ||| 174 | 606 | 67 | 79 | two | 26 ||| PCR | 80% ||| PCR ||| P. ||| RDT | P. [SUMMARY]
|
||||||
Time from Self-Detection of Symptoms to Seeking Definitive Care among Cervical Cancer Patients.
|
33247688
|
India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one-third of global cervical cancer deaths during the year 2018. Cervical cancer is the leading cause of cancer mortality in India. The present study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking care and different barriers for the possible time lag in seeking care.
|
BACKGROUND
|
A cross-sectional study was undertaken from April 2017 to September 2017 in a regional cancer centre in the south of India. The centre has both a population and a hospital-based cancer registry. Cervical cancer cases (N= 210) with histological confirmation were interviewed at the hospital using a pre-tested semi-structured questionnaire.
|
METHODS
|
The median time interval between the self-detection of cervical cancer symptoms and first contact with the general physician was 80 [IQR 45-150] days. The overall median time interval between the self-detection of symptoms to the initiation of primary treatment was 123[IQR 83-205] days. The major perceived reason for not seeking medical care was a lack of awareness in identifying cervical cancer symptoms in 183(92.9%) women.
|
RESULTS
|
The median time of 80 days was observed from the self-detection of cervical cancer symptoms to the first contact with a general physician. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care.<br />.
|
CONCLUSION
|
[
"Cross-Sectional Studies",
"Female",
"Follow-Up Studies",
"Health Knowledge, Attitudes, Practice",
"Humans",
"India",
"Middle Aged",
"Patient Acceptance of Health Care",
"Prognosis",
"Retrospective Studies",
"Self-Assessment",
"Surveys and Questionnaires",
"Time Factors",
"Time-to-Treatment",
"Uterine Cervical Neoplasms"
] |
8033105
|
Introduction
|
Globally, an estimated 570,000 new cases and 311,000 deaths has been reported due to cervical cancer in 2018 and it is the fourth most common cancer in women. The Incidence and mortality rank second behind breast cancer in lower human development index (HDI) settings. Eight out of 10 women diagnosed and nine of 10 women who die from cervical cancer live in a low- or middle-income countries (Bray et al., 2018) “ Cervical cancer continues to be a major public health problem affecting middle-aged women, particularly in less-resourced countries” (Arbyn et al., 2020). India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one third of global cervical cancer deaths during the year 2018 (“Globocan 2018,” 2018). Cervical cancer is the leading cause of cancer mortality in India which accounts for 17% of all cancer deaths among women aged 30 to 69 years.(Bobdey et al., 2016, “The Challenge Ahead,” 2014) One third of cervical cancer cases was reported among women aged 50 -59 years ( 27.4%) (Bobdey et al., 2016). The higher mortality and morbidity due to cancer is because most patients seek/get treatment at advanced stage and therefore have a higher risk of dying from the disease (Bray et al., 2018).
Cervical cancer is a preventable disease primarily because of its long lead time (women develop precancerous lesions years before it becomes cancerous). Cervical cancer can be detected in the pre-cancerous phase by screening and thereby the development of cancer prevented. Cervical cancer can also be treated successfully if diagnosed at an early stage. Routine screening by visual inspection of cervix through ‘See-and-treat’ approach among the female population has been suggested for early diagnosis and control of cervical cancer (Sankaranarayanan, 2012). Periodic Pap or HPV samples are difficult to organize in low- or middle-income countries like India and therefore large-scale routine screening would be difficult and not affordable (“HTA_CaCx Screening in India.pdf,” n.d.) Millions of cancer patients could be saved from premature death and disability, if they had timely access to early detection and treatment (“WHO,” n.d.). Studies have shown that it is possible to reduce the need of aggressive treatment of cancer is detected at an early stage. A long interval between start of disease symptoms to having a definite diagnosis is highly critical and associated with low survival among cervical cancer patients.(Chen et al., 2019)
In India, the present existing public health care delivery system is a three-level system. The primary level is primary health centres (PHCs). In India, PHCs focuses on eight elements of primary health care outlined in the Alma-Ata declaration and provides mainly outpatient services. In the secondary level the district hospitals provide outpatient, inpatient and emergency services. At the tertiary level, medical college hospitals, apex institutions and regional cancer centres provide specialized services. Under the programme of “National programme for prevention and control of cancer, diabetes, cardiovascular diseases and stroke (NPCDCS)”, cancer screening is done at the primary level by visual inspection of the cervix with acetic acid (VIA) and those who are suspected for cancer are referred to secondary or tertiary care level for further management. In addition, a health programme for non-communicable diseases is carried out with a cancer care component implemented at all the health care levels (Gulia et al., 2016)
This study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking for care and different barriers for the possible time lag in seeking care.
| null | null |
Results
|
The distribution of socio-demographic and clinical data of the 210 cervical cancer study subjects are presented along with data pertaining to the cases not enrolled for analysis (Table 1). Among 210 enrolled patients, the majority 106 (50.5%) of women were between the age 46 to 59 years where in not enrolled patients 89 (40.4%) were of same age group. It was noted 167 (63.0%) and 179 (81.4%) were not literate among enrolled and respectively. Among enrolled patients, 150 (71.4%) were from rural areas and 158 (71.4%) were financially dependent for livelihood on the family. Only 19 women (9.0%) had any health insurance policy. The majority of patients 197 (93.8%) were not aware of symptoms of cervical cancer and only 7 (3.3%) patients had a family history of cancer.
Of the symptoms which led for seeking medical care, the majority of women, 172 (81.9%), indicated lower abdominal pain, and 137 (65.2%) of the respondents experienced abnormal vaginal discharge/bleeding. Among all patients, 106 (50.5%) were diagnosed at an early stage of the disease (stages I, IIA and IIB).
The median time interval between self-detection of cervical cancer symptoms and first contact with general physician (T1) was 80 [IQR 45-150] days, while 74 patients (35.2%) had a time lag of 90 days or more. The median time taken for reaching diagnosis after the first contact (T2) was 25 [IQR 15 - 45] days. The overall median time between self-detection of symptoms to initiation of primary treatment (T5) was 123[IQR 83-205] days. The majority of patients, 145 (66.2%), had initiation of primary treatment only after 90 days from self-detection of cervical cancer symptoms (Table 2).
Factors associated with delay in seeking medical care of 90 days and more (patient delay) were analyzed in relation to socio-demographic characteristics and clinical symptoms (Table 3). In multivariate analysis after adjusting to all the variables in the study the odds of patient delay in the age group ≥ 60 years was 3.2 times higher compared to patients in the age group below 45 years (95% CI: 1.2 – 8.4). The other factors which were associated with patient delay were being not literate (OR 2.3, 95% CI: 1.0 – 5.4), from rural area (OR = 2.3, 95% CI: 1.1 – 4.9), being financially dependent for livelihood (OR = 4.5, 95% CI: 1.9 – 10.6) and not having any abnormal vaginal discharge / bleeding (OR = 2.4, 95% CI: 1.3 – 4.6). Family history of cancer, presence of any health insurance, knowledge regarding cancer cure by early seeking for care, knowledge of symptoms of cervical cancer and having lower abdominal pain was not statistically related to delay in seeking medical care.
Information was sought regarding perceived reasons from 197 (93.8%) patients who did not seek care within one week from self-detection of cancer symptoms. The major reasons (table 4) were lack of awareness in identifying cervical cancer symptoms in 183 (92.9%) women and 120 (60.9%) patients assuming that the symptom would resolve by itself. The other common reasons were absence of pain, changes in the body attributed to common illness and vague symptoms.
The most common reason for delay in seeking for definite diagnosis at tertiary care hospitals was due to non-affordability 82 (41.6%). The other common reasons were visiting multiple medical practitioners before presenting at tertiary care 60 (30.1%) and not suspected for cancer by physician at first contact.
The delay in seeking for treatment after diagnosis at tertiary care was due to long treatment procedure 57 (27.1%), fear of treatment 50 (23.8%), financial dependence on the family 48 (22.9%), fear of disfigurement of the body 26 (12.4%) and stigma attached with the disease (Table 4).
Distribution of Socio-Demographic, Clinical and Other Variables Among the Subject Enrolled and Not Enrolled to the Study
Distribution of Time Interval Between Self-Detection of Cancer Symptoms to Various Stages (T1 To T5) for Seeking Treatment
Factors Associated with Delay in Seeking Medical Care: Univariate and Multivariate Logistic Regression Results
Distribution of Perceived Reasons for Delay in Seeking Medical Care at Various Stages (T1 – T4)
*, Responses were elicited from those who had not sought specific care for more than one week from the onset of symptoms; #, Responses was elicited from those who had not sought treatment on specified time by doctor
Recruitment of Participants for the Study
| null | null |
[] |
[] |
[] |
[
"Introduction",
"Materials and Methods",
"Results",
"Discussion"
] |
[
"Globally, an estimated 570,000 new cases and 311,000 deaths has been reported due to cervical cancer in 2018 and it is the fourth most common cancer in women. The Incidence and mortality rank second behind breast cancer in lower human development index (HDI) settings. Eight out of 10 women diagnosed and nine of 10 women who die from cervical cancer live in a low- or middle-income countries (Bray et al., 2018) “ Cervical cancer continues to be a major public health problem affecting middle-aged women, particularly in less-resourced countries” (Arbyn et al., 2020). India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one third of global cervical cancer deaths during the year 2018 (“Globocan 2018,” 2018). Cervical cancer is the leading cause of cancer mortality in India which accounts for 17% of all cancer deaths among women aged 30 to 69 years.(Bobdey et al., 2016, “The Challenge Ahead,” 2014) One third of cervical cancer cases was reported among women aged 50 -59 years ( 27.4%) (Bobdey et al., 2016). The higher mortality and morbidity due to cancer is because most patients seek/get treatment at advanced stage and therefore have a higher risk of dying from the disease (Bray et al., 2018).\nCervical cancer is a preventable disease primarily because of its long lead time (women develop precancerous lesions years before it becomes cancerous). Cervical cancer can be detected in the pre-cancerous phase by screening and thereby the development of cancer prevented. Cervical cancer can also be treated successfully if diagnosed at an early stage. Routine screening by visual inspection of cervix through ‘See-and-treat’ approach among the female population has been suggested for early diagnosis and control of cervical cancer (Sankaranarayanan, 2012). Periodic Pap or HPV samples are difficult to organize in low- or middle-income countries like India and therefore large-scale routine screening would be difficult and not affordable (“HTA_CaCx Screening in India.pdf,” n.d.) Millions of cancer patients could be saved from premature death and disability, if they had timely access to early detection and treatment (“WHO,” n.d.). Studies have shown that it is possible to reduce the need of aggressive treatment of cancer is detected at an early stage. A long interval between start of disease symptoms to having a definite diagnosis is highly critical and associated with low survival among cervical cancer patients.(Chen et al., 2019) \nIn India, the present existing public health care delivery system is a three-level system. The primary level is primary health centres (PHCs). In India, PHCs focuses on eight elements of primary health care outlined in the Alma-Ata declaration and provides mainly outpatient services. In the secondary level the district hospitals provide outpatient, inpatient and emergency services. At the tertiary level, medical college hospitals, apex institutions and regional cancer centres provide specialized services. Under the programme of “National programme for prevention and control of cancer, diabetes, cardiovascular diseases and stroke (NPCDCS)”, cancer screening is done at the primary level by visual inspection of the cervix with acetic acid (VIA) and those who are suspected for cancer are referred to secondary or tertiary care level for further management. In addition, a health programme for non-communicable diseases is carried out with a cancer care component implemented at all the health care levels (Gulia et al., 2016) \nThis study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking for care and different barriers for the possible time lag in seeking care.",
"The data was collected from a regional cancer centre from south India, Bengaluru, which has a population-based cancer registry (PBCR) and a hospital-based cancer registry (HBCR). This tertiary care centre has state of art facilities for diagnosis, treatment and research. Cervical cancer cases with histological confirmation were recruited from the hospital-based cancer registry of this Institution during the time period of April 2017 to September 2017 using cross sectional study design. We identified 430 cervical cancer patients during the period from April 2017 to September 2017. Of these, 52 were ineligible due to referral to terminal/palliative care. Of 378 eligible women, 96 were not included for the analysis as patients moved to different hospital or not being available for interview, and 38 were not willing to participate in the study. In the 244 patients who gave their consent, 34 patient’s had incomplete information in the records. Finally, 210 patients were included for the present study (Figure 1) \nThe cervical cancers were mostly squamous cell carcinomas (SCC) and few adenocarcinomas (AC). They were divided in two stage based on FIGO classification (Bhatla et al., 2019). Stage I and II were categorized as early stage; III and IV were considered as late stage of cervical cancer. \nPatient delay in seeking for medical care refers to a long interval between detecting of first/initial symptoms and seeking for appointment with a general physician. The patients were further divided into two groups, those who sought medical care within 90 days and those who sought care 90 days and above (patient delay) after self-detection of the first symptoms of cervical cancer. \nSample size was calculated based on the study conducted by Rudd et al., (2017) where 39.3% patients had delayed diagnosis from the onset of self-detection of cervical cancer symptoms. In this study the sample size was estimated for patient related delay, from symptoms to first contact with a general practitioner, as it was our major concern. To get an absolute precision of 6.6% with 95% confidence level, the study required a minimum sample of 210 subjects. Based on hospital record collection, it was noted that six months’ time period was sufficient to achieve the required sample size. \nExpert opinion from oncologists, biostatisticians and epidemiologists were obtained using the Delphi technique (Skulmoski et al., 2007). After obtaining the written informed consent, patients were interviewed in person using the questionnaire and their medical records was used to collect the baseline information as well as key components of the time interval between self-detection of cervical cancer symptom to seeking medical care. More details of the collection of data and the different components of time intervals are described elsewhere.(Somanna et al., 2020)\nIndependent variables included socio-demographic characteristics, such as age at diagnosis, availability of health insurance, place of residence (rural/urban), history of cancer literacy, and financially dependent for livelihood and clinical variables, such as personal history of cancer, and family history of cancer.\nThe time interval between various stages was measured in days as reported by the patients and by review of hospital case sheets as indicated in earlier study.(Somanna et al., 2018) \na) Time interval between self-detection of symptoms and the first presentation at general practitioner (T1). \nb) Time interval from referral by the general practitioner to attending tertiary care centre (T2). \nc) Time interval from first consultation at tertiary care for diagnosis to definite diagnosis date (T3). \nd) Time interval from definite diagnosis to initiation of primary treatment (T4). \ne) Total time elapsed from self-detection of symptoms to initiation of primary treatment (T5).\n\nStatistical methods\n\nDescriptive statistics of time intervals were summarized with median and inter-quartile range as data were not normally distributed (Kolmogrov-Smirnov and Shapiro test). Univariate odds ratios (OR) with 95% confidence intervals (CI) were estimated between the factors and patient delay. The patient delay was categorised into < 90 days and ≥ 90 days. Considering ≥ 90 days as patient delay, the association of various factors were estimated considering the low risk group as the reference. Variables showing a univariate association with patient delay (at P < 0.20) were included in multivariate unconditional logistic stepwise forward regression model to adjust for confounders. The analysis was carried out using SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago. \nEthical approval was sought from the ethical review committee of the study Institution. ",
"The distribution of socio-demographic and clinical data of the 210 cervical cancer study subjects are presented along with data pertaining to the cases not enrolled for analysis (Table 1). Among 210 enrolled patients, the majority 106 (50.5%) of women were between the age 46 to 59 years where in not enrolled patients 89 (40.4%) were of same age group. It was noted 167 (63.0%) and 179 (81.4%) were not literate among enrolled and respectively. Among enrolled patients, 150 (71.4%) were from rural areas and 158 (71.4%) were financially dependent for livelihood on the family. Only 19 women (9.0%) had any health insurance policy. The majority of patients 197 (93.8%) were not aware of symptoms of cervical cancer and only 7 (3.3%) patients had a family history of cancer.\nOf the symptoms which led for seeking medical care, the majority of women, 172 (81.9%), indicated lower abdominal pain, and 137 (65.2%) of the respondents experienced abnormal vaginal discharge/bleeding. Among all patients, 106 (50.5%) were diagnosed at an early stage of the disease (stages I, IIA and IIB).\nThe median time interval between self-detection of cervical cancer symptoms and first contact with general physician (T1) was 80 [IQR 45-150] days, while 74 patients (35.2%) had a time lag of 90 days or more. The median time taken for reaching diagnosis after the first contact (T2) was 25 [IQR 15 - 45] days. The overall median time between self-detection of symptoms to initiation of primary treatment (T5) was 123[IQR 83-205] days. The majority of patients, 145 (66.2%), had initiation of primary treatment only after 90 days from self-detection of cervical cancer symptoms (Table 2). \nFactors associated with delay in seeking medical care of 90 days and more (patient delay) were analyzed in relation to socio-demographic characteristics and clinical symptoms (Table 3). In multivariate analysis after adjusting to all the variables in the study the odds of patient delay in the age group ≥ 60 years was 3.2 times higher compared to patients in the age group below 45 years (95% CI: 1.2 – 8.4). The other factors which were associated with patient delay were being not literate (OR 2.3, 95% CI: 1.0 – 5.4), from rural area (OR = 2.3, 95% CI: 1.1 – 4.9), being financially dependent for livelihood (OR = 4.5, 95% CI: 1.9 – 10.6) and not having any abnormal vaginal discharge / bleeding (OR = 2.4, 95% CI: 1.3 – 4.6). Family history of cancer, presence of any health insurance, knowledge regarding cancer cure by early seeking for care, knowledge of symptoms of cervical cancer and having lower abdominal pain was not statistically related to delay in seeking medical care. \nInformation was sought regarding perceived reasons from 197 (93.8%) patients who did not seek care within one week from self-detection of cancer symptoms. The major reasons (table 4) were lack of awareness in identifying cervical cancer symptoms in 183 (92.9%) women and 120 (60.9%) patients assuming that the symptom would resolve by itself. The other common reasons were absence of pain, changes in the body attributed to common illness and vague symptoms. \nThe most common reason for delay in seeking for definite diagnosis at tertiary care hospitals was due to non-affordability 82 (41.6%). The other common reasons were visiting multiple medical practitioners before presenting at tertiary care 60 (30.1%) and not suspected for cancer by physician at first contact. \nThe delay in seeking for treatment after diagnosis at tertiary care was due to long treatment procedure 57 (27.1%), fear of treatment 50 (23.8%), financial dependence on the family 48 (22.9%), fear of disfigurement of the body 26 (12.4%) and stigma attached with the disease (Table 4).\nDistribution of Socio-Demographic, Clinical and Other Variables Among the Subject Enrolled and Not Enrolled to the Study\nDistribution of Time Interval Between Self-Detection of Cancer Symptoms to Various Stages (T1 To T5) for Seeking Treatment\nFactors Associated with Delay in Seeking Medical Care: Univariate and Multivariate Logistic Regression Results\nDistribution of Perceived Reasons for Delay in Seeking Medical Care at Various Stages (T1 – T4)\n*, Responses were elicited from those who had not sought specific care for more than one week from the onset of symptoms; #, Responses was elicited from those who had not sought treatment on specified time by doctor \nRecruitment of Participants for the Study",
"The study centre was a regional cancer care centre having both population and hospital-based cancer registry and where a majority of cervix cancer patients from the entire state seek cancer care. The median age in enrolled and not enrolled patients was 50 and 56 years, respectively. Among all the patients, the proportion of not literate patients was higher (81%) in not enrolled patients compared to enrolled patients (63%). The study by Deshmukh and Rathod, (2017) observed that the average age of the patients was 57 years and 81% were not literate. Our result concur with the study by Panda et al., 2019 where the average age of the study participants was 52.1 ± 10.9 years. \nThe overall median time between the self-detection of cervical cancer symptoms and initiation of primary treatment was 123[IQR 83-205] days and the median time taken for first contact with general physician from self-detection of symptoms was 80 [IQR 45-150] days. Overall, 66% women delayed (≥ 90 days) for initiation of primary treatment from the onset of symptoms which is potentially concerning, because longer duration between diagnosis and treatment is related with higher risk of death. Among patients who started their treatment after an interval of >180 days, the risk of death was reported to be 36% higher compared to those who started treatment within 6 months from diagnosis (Chen et al., 2019) \nThe present study assessed the time interval between self-detection of symptoms to treatment of cervical cancer and patient delay. The median time interval between self-detection of symptoms and the first presentation at general practitioner (T1) was 80 days with 35.4% of the patients reporting 90 days or more. The present study observation was consistent with a study conducted in India on patients attending tertiary care centre, showing that 39% of patients had a diagnostic delay (Panda et al., 2019) The median time between symptoms and presenting at health care provider was 69 days from two tertiary care centres in Nepal (Gyenwali et al., 2014). It was noted from the study by Lim et al., (2014) that only 28% of patients had a delay of more than 3 months in all national health service (NHS) hospitals diagnosing cervical cancer in England. There was a mean delay of 23.1 weeks between onset of symptoms from the study in tertiary care centre of Sothern Malawi (Rudd et al., 2017). Results from our study were in line with studies from low- or middle-income countries but differed from studies in developed countries. This difference may be partially due to regular screening programs in many developed countries and differing rates of literacy (IARC, 2005). \nOverall median time from self-detection of symptoms to initiation of primary treatment (T5) was 123 days in the current study. Another study by Henriques, (2016) in India showed on an average six months duration from first symptom to cervical cancer treatment. Such a long time may be explained by the fact that lack of awareness of patients pertaining to cancer symptoms was high (93%) in present study. In another study, women who delayed, had mistaken the symptoms of cervix cancer as symptoms of sexually transmitted diseases (STD) and were scared to report (Deshmukh and Rathod, 2017). This shows that the lack of awareness of cervix cancer symptoms is a major factor and warrants improvement in public awareness. Our study results concur closely with other studies from similar settings and in low- or middle-income countries (Benemariya et al., 2018; Gyenwali et al., 2014).\nThe patients who were aged 60 years and above, residing in rural areas, not literate, financially dependent for livelihood and did not had abnormal vaginal discharge/ bleeding were independently associated with patient delay. Similar results were observed in a study from Nepal where not literate was found to be independently associated with late diagnosis and women having abnormal vaginal bleeding as symptom was protective factor for late diagnosis (Gyenwali et al., 2013). Previous studies from low- or middle-income countries also concur with our result (Dunyo et al., 2018)(Ouasmani et al., 2016)(Henriques, 2016).\nThe present study has been conducted in a population-based cancer registry (PBCR) where the data are included in International Agency for Research on Cancer (IARC) and data collection procedure involves standard quality control measures. The study included only patients who had been diagnosed with standard clinical and histopathological criteria. The information was obtained using pre-tested validated questionnaire by the investigator. Additional information pertaining to clinical evaluation and histological diagnosis were obtained from case sheets. All efforts were made to reduce the recall bias by local events calendar and standardization of terms employed in the questionnaire. \n\nLimitations\n\nPossible selection bias could have occurred as the subjects who were not enrolled for the study due to various reasons were older women and not literate compared to subjects in the study. Socio-demographic profile of population who availed the services at this centre may be different from private tertiary cancer care centre which would biases the result. The sample in study represent only those who availed the treatment at the centre and does not represent the entire population of cervical cancer patients. Thus, it may have some restrictions for external generalization. Possibility of recall bias exist as past history of cancer symptoms were assessed retrospectively. \nIn conclusion, the median time of 80 days was observed from self-detection of cervical cancer symptoms to first contact with general physician. Further, a median time of another 45 days was observed for obtaining primary treatment. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care. Interventions like raising awareness of cervical cancer symptoms and improving health seeking behavior of women should be focused."
] |
[
"intro",
"materials|methods",
"results",
"discussion"
] |
[
"Cervical cancer",
"time interval",
"cancer symptoms",
"seeking cancer care"
] |
Introduction:
Globally, an estimated 570,000 new cases and 311,000 deaths has been reported due to cervical cancer in 2018 and it is the fourth most common cancer in women. The Incidence and mortality rank second behind breast cancer in lower human development index (HDI) settings. Eight out of 10 women diagnosed and nine of 10 women who die from cervical cancer live in a low- or middle-income countries (Bray et al., 2018) “ Cervical cancer continues to be a major public health problem affecting middle-aged women, particularly in less-resourced countries” (Arbyn et al., 2020). India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one third of global cervical cancer deaths during the year 2018 (“Globocan 2018,” 2018). Cervical cancer is the leading cause of cancer mortality in India which accounts for 17% of all cancer deaths among women aged 30 to 69 years.(Bobdey et al., 2016, “The Challenge Ahead,” 2014) One third of cervical cancer cases was reported among women aged 50 -59 years ( 27.4%) (Bobdey et al., 2016). The higher mortality and morbidity due to cancer is because most patients seek/get treatment at advanced stage and therefore have a higher risk of dying from the disease (Bray et al., 2018).
Cervical cancer is a preventable disease primarily because of its long lead time (women develop precancerous lesions years before it becomes cancerous). Cervical cancer can be detected in the pre-cancerous phase by screening and thereby the development of cancer prevented. Cervical cancer can also be treated successfully if diagnosed at an early stage. Routine screening by visual inspection of cervix through ‘See-and-treat’ approach among the female population has been suggested for early diagnosis and control of cervical cancer (Sankaranarayanan, 2012). Periodic Pap or HPV samples are difficult to organize in low- or middle-income countries like India and therefore large-scale routine screening would be difficult and not affordable (“HTA_CaCx Screening in India.pdf,” n.d.) Millions of cancer patients could be saved from premature death and disability, if they had timely access to early detection and treatment (“WHO,” n.d.). Studies have shown that it is possible to reduce the need of aggressive treatment of cancer is detected at an early stage. A long interval between start of disease symptoms to having a definite diagnosis is highly critical and associated with low survival among cervical cancer patients.(Chen et al., 2019)
In India, the present existing public health care delivery system is a three-level system. The primary level is primary health centres (PHCs). In India, PHCs focuses on eight elements of primary health care outlined in the Alma-Ata declaration and provides mainly outpatient services. In the secondary level the district hospitals provide outpatient, inpatient and emergency services. At the tertiary level, medical college hospitals, apex institutions and regional cancer centres provide specialized services. Under the programme of “National programme for prevention and control of cancer, diabetes, cardiovascular diseases and stroke (NPCDCS)”, cancer screening is done at the primary level by visual inspection of the cervix with acetic acid (VIA) and those who are suspected for cancer are referred to secondary or tertiary care level for further management. In addition, a health programme for non-communicable diseases is carried out with a cancer care component implemented at all the health care levels (Gulia et al., 2016)
This study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking for care and different barriers for the possible time lag in seeking care.
Materials and Methods:
The data was collected from a regional cancer centre from south India, Bengaluru, which has a population-based cancer registry (PBCR) and a hospital-based cancer registry (HBCR). This tertiary care centre has state of art facilities for diagnosis, treatment and research. Cervical cancer cases with histological confirmation were recruited from the hospital-based cancer registry of this Institution during the time period of April 2017 to September 2017 using cross sectional study design. We identified 430 cervical cancer patients during the period from April 2017 to September 2017. Of these, 52 were ineligible due to referral to terminal/palliative care. Of 378 eligible women, 96 were not included for the analysis as patients moved to different hospital or not being available for interview, and 38 were not willing to participate in the study. In the 244 patients who gave their consent, 34 patient’s had incomplete information in the records. Finally, 210 patients were included for the present study (Figure 1)
The cervical cancers were mostly squamous cell carcinomas (SCC) and few adenocarcinomas (AC). They were divided in two stage based on FIGO classification (Bhatla et al., 2019). Stage I and II were categorized as early stage; III and IV were considered as late stage of cervical cancer.
Patient delay in seeking for medical care refers to a long interval between detecting of first/initial symptoms and seeking for appointment with a general physician. The patients were further divided into two groups, those who sought medical care within 90 days and those who sought care 90 days and above (patient delay) after self-detection of the first symptoms of cervical cancer.
Sample size was calculated based on the study conducted by Rudd et al., (2017) where 39.3% patients had delayed diagnosis from the onset of self-detection of cervical cancer symptoms. In this study the sample size was estimated for patient related delay, from symptoms to first contact with a general practitioner, as it was our major concern. To get an absolute precision of 6.6% with 95% confidence level, the study required a minimum sample of 210 subjects. Based on hospital record collection, it was noted that six months’ time period was sufficient to achieve the required sample size.
Expert opinion from oncologists, biostatisticians and epidemiologists were obtained using the Delphi technique (Skulmoski et al., 2007). After obtaining the written informed consent, patients were interviewed in person using the questionnaire and their medical records was used to collect the baseline information as well as key components of the time interval between self-detection of cervical cancer symptom to seeking medical care. More details of the collection of data and the different components of time intervals are described elsewhere.(Somanna et al., 2020)
Independent variables included socio-demographic characteristics, such as age at diagnosis, availability of health insurance, place of residence (rural/urban), history of cancer literacy, and financially dependent for livelihood and clinical variables, such as personal history of cancer, and family history of cancer.
The time interval between various stages was measured in days as reported by the patients and by review of hospital case sheets as indicated in earlier study.(Somanna et al., 2018)
a) Time interval between self-detection of symptoms and the first presentation at general practitioner (T1).
b) Time interval from referral by the general practitioner to attending tertiary care centre (T2).
c) Time interval from first consultation at tertiary care for diagnosis to definite diagnosis date (T3).
d) Time interval from definite diagnosis to initiation of primary treatment (T4).
e) Total time elapsed from self-detection of symptoms to initiation of primary treatment (T5).
Statistical methods
Descriptive statistics of time intervals were summarized with median and inter-quartile range as data were not normally distributed (Kolmogrov-Smirnov and Shapiro test). Univariate odds ratios (OR) with 95% confidence intervals (CI) were estimated between the factors and patient delay. The patient delay was categorised into < 90 days and ≥ 90 days. Considering ≥ 90 days as patient delay, the association of various factors were estimated considering the low risk group as the reference. Variables showing a univariate association with patient delay (at P < 0.20) were included in multivariate unconditional logistic stepwise forward regression model to adjust for confounders. The analysis was carried out using SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago.
Ethical approval was sought from the ethical review committee of the study Institution.
Results:
The distribution of socio-demographic and clinical data of the 210 cervical cancer study subjects are presented along with data pertaining to the cases not enrolled for analysis (Table 1). Among 210 enrolled patients, the majority 106 (50.5%) of women were between the age 46 to 59 years where in not enrolled patients 89 (40.4%) were of same age group. It was noted 167 (63.0%) and 179 (81.4%) were not literate among enrolled and respectively. Among enrolled patients, 150 (71.4%) were from rural areas and 158 (71.4%) were financially dependent for livelihood on the family. Only 19 women (9.0%) had any health insurance policy. The majority of patients 197 (93.8%) were not aware of symptoms of cervical cancer and only 7 (3.3%) patients had a family history of cancer.
Of the symptoms which led for seeking medical care, the majority of women, 172 (81.9%), indicated lower abdominal pain, and 137 (65.2%) of the respondents experienced abnormal vaginal discharge/bleeding. Among all patients, 106 (50.5%) were diagnosed at an early stage of the disease (stages I, IIA and IIB).
The median time interval between self-detection of cervical cancer symptoms and first contact with general physician (T1) was 80 [IQR 45-150] days, while 74 patients (35.2%) had a time lag of 90 days or more. The median time taken for reaching diagnosis after the first contact (T2) was 25 [IQR 15 - 45] days. The overall median time between self-detection of symptoms to initiation of primary treatment (T5) was 123[IQR 83-205] days. The majority of patients, 145 (66.2%), had initiation of primary treatment only after 90 days from self-detection of cervical cancer symptoms (Table 2).
Factors associated with delay in seeking medical care of 90 days and more (patient delay) were analyzed in relation to socio-demographic characteristics and clinical symptoms (Table 3). In multivariate analysis after adjusting to all the variables in the study the odds of patient delay in the age group ≥ 60 years was 3.2 times higher compared to patients in the age group below 45 years (95% CI: 1.2 – 8.4). The other factors which were associated with patient delay were being not literate (OR 2.3, 95% CI: 1.0 – 5.4), from rural area (OR = 2.3, 95% CI: 1.1 – 4.9), being financially dependent for livelihood (OR = 4.5, 95% CI: 1.9 – 10.6) and not having any abnormal vaginal discharge / bleeding (OR = 2.4, 95% CI: 1.3 – 4.6). Family history of cancer, presence of any health insurance, knowledge regarding cancer cure by early seeking for care, knowledge of symptoms of cervical cancer and having lower abdominal pain was not statistically related to delay in seeking medical care.
Information was sought regarding perceived reasons from 197 (93.8%) patients who did not seek care within one week from self-detection of cancer symptoms. The major reasons (table 4) were lack of awareness in identifying cervical cancer symptoms in 183 (92.9%) women and 120 (60.9%) patients assuming that the symptom would resolve by itself. The other common reasons were absence of pain, changes in the body attributed to common illness and vague symptoms.
The most common reason for delay in seeking for definite diagnosis at tertiary care hospitals was due to non-affordability 82 (41.6%). The other common reasons were visiting multiple medical practitioners before presenting at tertiary care 60 (30.1%) and not suspected for cancer by physician at first contact.
The delay in seeking for treatment after diagnosis at tertiary care was due to long treatment procedure 57 (27.1%), fear of treatment 50 (23.8%), financial dependence on the family 48 (22.9%), fear of disfigurement of the body 26 (12.4%) and stigma attached with the disease (Table 4).
Distribution of Socio-Demographic, Clinical and Other Variables Among the Subject Enrolled and Not Enrolled to the Study
Distribution of Time Interval Between Self-Detection of Cancer Symptoms to Various Stages (T1 To T5) for Seeking Treatment
Factors Associated with Delay in Seeking Medical Care: Univariate and Multivariate Logistic Regression Results
Distribution of Perceived Reasons for Delay in Seeking Medical Care at Various Stages (T1 – T4)
*, Responses were elicited from those who had not sought specific care for more than one week from the onset of symptoms; #, Responses was elicited from those who had not sought treatment on specified time by doctor
Recruitment of Participants for the Study
Discussion:
The study centre was a regional cancer care centre having both population and hospital-based cancer registry and where a majority of cervix cancer patients from the entire state seek cancer care. The median age in enrolled and not enrolled patients was 50 and 56 years, respectively. Among all the patients, the proportion of not literate patients was higher (81%) in not enrolled patients compared to enrolled patients (63%). The study by Deshmukh and Rathod, (2017) observed that the average age of the patients was 57 years and 81% were not literate. Our result concur with the study by Panda et al., 2019 where the average age of the study participants was 52.1 ± 10.9 years.
The overall median time between the self-detection of cervical cancer symptoms and initiation of primary treatment was 123[IQR 83-205] days and the median time taken for first contact with general physician from self-detection of symptoms was 80 [IQR 45-150] days. Overall, 66% women delayed (≥ 90 days) for initiation of primary treatment from the onset of symptoms which is potentially concerning, because longer duration between diagnosis and treatment is related with higher risk of death. Among patients who started their treatment after an interval of >180 days, the risk of death was reported to be 36% higher compared to those who started treatment within 6 months from diagnosis (Chen et al., 2019)
The present study assessed the time interval between self-detection of symptoms to treatment of cervical cancer and patient delay. The median time interval between self-detection of symptoms and the first presentation at general practitioner (T1) was 80 days with 35.4% of the patients reporting 90 days or more. The present study observation was consistent with a study conducted in India on patients attending tertiary care centre, showing that 39% of patients had a diagnostic delay (Panda et al., 2019) The median time between symptoms and presenting at health care provider was 69 days from two tertiary care centres in Nepal (Gyenwali et al., 2014). It was noted from the study by Lim et al., (2014) that only 28% of patients had a delay of more than 3 months in all national health service (NHS) hospitals diagnosing cervical cancer in England. There was a mean delay of 23.1 weeks between onset of symptoms from the study in tertiary care centre of Sothern Malawi (Rudd et al., 2017). Results from our study were in line with studies from low- or middle-income countries but differed from studies in developed countries. This difference may be partially due to regular screening programs in many developed countries and differing rates of literacy (IARC, 2005).
Overall median time from self-detection of symptoms to initiation of primary treatment (T5) was 123 days in the current study. Another study by Henriques, (2016) in India showed on an average six months duration from first symptom to cervical cancer treatment. Such a long time may be explained by the fact that lack of awareness of patients pertaining to cancer symptoms was high (93%) in present study. In another study, women who delayed, had mistaken the symptoms of cervix cancer as symptoms of sexually transmitted diseases (STD) and were scared to report (Deshmukh and Rathod, 2017). This shows that the lack of awareness of cervix cancer symptoms is a major factor and warrants improvement in public awareness. Our study results concur closely with other studies from similar settings and in low- or middle-income countries (Benemariya et al., 2018; Gyenwali et al., 2014).
The patients who were aged 60 years and above, residing in rural areas, not literate, financially dependent for livelihood and did not had abnormal vaginal discharge/ bleeding were independently associated with patient delay. Similar results were observed in a study from Nepal where not literate was found to be independently associated with late diagnosis and women having abnormal vaginal bleeding as symptom was protective factor for late diagnosis (Gyenwali et al., 2013). Previous studies from low- or middle-income countries also concur with our result (Dunyo et al., 2018)(Ouasmani et al., 2016)(Henriques, 2016).
The present study has been conducted in a population-based cancer registry (PBCR) where the data are included in International Agency for Research on Cancer (IARC) and data collection procedure involves standard quality control measures. The study included only patients who had been diagnosed with standard clinical and histopathological criteria. The information was obtained using pre-tested validated questionnaire by the investigator. Additional information pertaining to clinical evaluation and histological diagnosis were obtained from case sheets. All efforts were made to reduce the recall bias by local events calendar and standardization of terms employed in the questionnaire.
Limitations
Possible selection bias could have occurred as the subjects who were not enrolled for the study due to various reasons were older women and not literate compared to subjects in the study. Socio-demographic profile of population who availed the services at this centre may be different from private tertiary cancer care centre which would biases the result. The sample in study represent only those who availed the treatment at the centre and does not represent the entire population of cervical cancer patients. Thus, it may have some restrictions for external generalization. Possibility of recall bias exist as past history of cancer symptoms were assessed retrospectively.
In conclusion, the median time of 80 days was observed from self-detection of cervical cancer symptoms to first contact with general physician. Further, a median time of another 45 days was observed for obtaining primary treatment. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care. Interventions like raising awareness of cervical cancer symptoms and improving health seeking behavior of women should be focused.
|
Background:
India had the burden of 97,000 new cases of cervical cancer with 60,000 deaths accounting nearly one-third of global cervical cancer deaths during the year 2018. Cervical cancer is the leading cause of cancer mortality in India. The present study aims to estimate the time interval between self-detection of cervical cancer symptoms and seeking care and different barriers for the possible time lag in seeking care.
Methods:
A cross-sectional study was undertaken from April 2017 to September 2017 in a regional cancer centre in the south of India. The centre has both a population and a hospital-based cancer registry. Cervical cancer cases (N= 210) with histological confirmation were interviewed at the hospital using a pre-tested semi-structured questionnaire.
Results:
The median time interval between the self-detection of cervical cancer symptoms and first contact with the general physician was 80 [IQR 45-150] days. The overall median time interval between the self-detection of symptoms to the initiation of primary treatment was 123[IQR 83-205] days. The major perceived reason for not seeking medical care was a lack of awareness in identifying cervical cancer symptoms in 183(92.9%) women.
Conclusions:
The median time of 80 days was observed from the self-detection of cervical cancer symptoms to the first contact with a general physician. Lack of awareness of patients pertaining to cancer symptoms was the major concern in seeking cancer care.<br />.
| null | null | 3,637 | 278 | 4 |
[
"cancer",
"patients",
"symptoms",
"study",
"care",
"cervical",
"cervical cancer",
"time",
"treatment",
"days"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] Cervical cancer | time interval | cancer symptoms | seeking cancer care [SUMMARY]
| null |
[CONTENT] Cervical cancer | time interval | cancer symptoms | seeking cancer care [SUMMARY]
| null |
[CONTENT] Cervical cancer | time interval | cancer symptoms | seeking cancer care [SUMMARY]
| null |
[CONTENT] Cross-Sectional Studies | Female | Follow-Up Studies | Health Knowledge, Attitudes, Practice | Humans | India | Middle Aged | Patient Acceptance of Health Care | Prognosis | Retrospective Studies | Self-Assessment | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Uterine Cervical Neoplasms [SUMMARY]
| null |
[CONTENT] Cross-Sectional Studies | Female | Follow-Up Studies | Health Knowledge, Attitudes, Practice | Humans | India | Middle Aged | Patient Acceptance of Health Care | Prognosis | Retrospective Studies | Self-Assessment | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Uterine Cervical Neoplasms [SUMMARY]
| null |
[CONTENT] Cross-Sectional Studies | Female | Follow-Up Studies | Health Knowledge, Attitudes, Practice | Humans | India | Middle Aged | Patient Acceptance of Health Care | Prognosis | Retrospective Studies | Self-Assessment | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Uterine Cervical Neoplasms [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] cancer | patients | symptoms | study | care | cervical | cervical cancer | time | treatment | days [SUMMARY]
| null |
[CONTENT] cancer | patients | symptoms | study | care | cervical | cervical cancer | time | treatment | days [SUMMARY]
| null |
[CONTENT] cancer | patients | symptoms | study | care | cervical | cervical cancer | time | treatment | days [SUMMARY]
| null |
[CONTENT] cancer | cervical cancer | cervical | level | 000 | deaths | screening | 2018 | india | care [SUMMARY]
| null |
[CONTENT] cancer | symptoms | delay | patients | care | enrolled | 95 ci | table | delay seeking | seeking [SUMMARY]
| null |
[CONTENT] cancer | patients | symptoms | care | cervical | cervical cancer | study | time | days | delay [SUMMARY]
| null |
[CONTENT] India | 97,000 | 60,000 | nearly one-third | the year 2018 ||| India ||| [SUMMARY]
| null |
[CONTENT] first | 80 ||| ||| 123[IQR | 83 ||| 183(92.9% [SUMMARY]
| null |
[CONTENT] India | 97,000 | 60,000 | nearly one-third | the year 2018 ||| India ||| ||| April 2017 to September 2017 | India ||| ||| 210 ||| ||| first | 80 ||| ||| 123[IQR | 83 ||| 183(92.9% ||| 80 days | first ||| [SUMMARY]
| null |
|||
Alteration of Thyroid Hormone among Patients with Ischemic Stroke visiting a Tertiary Care Hospital: A Descriptive Cross-sectional Study.
|
34508483
|
Stroke is broadly classified as cerebral infarction, intracerebral hemorrhage and subarachnoid hemorrhage. Neuroendocrine profile is altered in acute ischemic stroke and there is a link between hypothyroidism and atherosclerosis which in turn may lead to stroke. The objective of this study was to find out the prevalence of alteration of thyroid hormones in patients with ischemic stroke in a tertiary care center.
|
INTRODUCTION
|
This descriptive cross-sectional study was conducted from June to December 2019 in a tertiary care center. Ethical approval was taken from Institutional review board of National Academy of Medical Sciences (reference number: IM 175). Patients with a diagnosis of stroke, without evidence of cardioembolic source, history of liver disease, renal failure and thyroid disease and who do not use thyroidal supplementation within 180 days prior the event were included. Convenience sampling was done. Data was entered in Microsoft Excel and analyzed using Statistical Package for the Social Sciences version 22. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.
|
METHODS
|
The prevalence of altered thyroid levels among 73 patients was 13 (17.8%) (90% Confidence Interval= 10.44-25.16). Among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Among severity of hypothyroid cases, subclinical hypothyroidism grade IA was seen in 51 (70%), subclinical hypothyroidism grade IB was seen in 22 (30%).
|
RESULTS
|
The prevalence of altered thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies.
|
CONCLUSIONS
|
[
"Brain Ischemia",
"Cross-Sectional Studies",
"Humans",
"Ischemic Stroke",
"Stroke",
"Tertiary Care Centers",
"Thyroid Hormones"
] |
9107842
|
INTRODUCTION
|
Stroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4
Thyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4
The aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.
|
METHODS
|
This descriptive cross-sectional study was conducted from June to December 2019 in National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu. Ethical approval from Institutional review board of NAMS (reference number. IM 175). Convenience sampling was done and the sample size of cases to be enrolled was calculated by using the formula,
n = Z2 × p × q / e2
= (1.645)2 × (0.5) × (1-0.5) / (0.1)2
= 67.65
Where,
n = minimum required sample size,
Z = 1.645 at 90% Confidence Interval (CI),
p = prevalence taken as 50% for maximum sample size,
q = 1-p
e = margin of error, 10%
Hence, the required sample size was 67.65. Adding a 10% nonresponse rate, we enrolled 73 patients in our study. Patients with clinically diagnosed stroke, presenting within 48 hours of onset of symptoms with CT or MRI findings were included in the study. Patients with prior use of thyroid supplements recently within last 180 days and with a history of liver disease, renal failure and known thyroid disease and who don't give consent were excluded from the study. Informed written consent was taken either in Nepali or in English language, in whichever they felt comfortable. Confidentiality was maintained to the utmost. For those who are not able to give consent because of their clinical state, informed consent was taken from their close relative.
Proforma was translated in Nepali verbally if needed depending upon the cases. For diagnosing the type of stroke, every patient underwent neuroimaging with non-contrast CT of the head. Cerebral infarction was diagnosed if neurological deficits are accompanied by a hypo dense lesion >15 mm in diameter in an appropriate area on a cranial CT scan. Lacunar infarction were diagnosed if the patient presented with a pure motor stroke, a pure sensory stroke, ataxic hemiparesis or a sensorimotor stroke in the absence of a visual field defect and evidence of higher cerebral dysfunction and a hypo dense lesion of < 15 mm in diameter or normal CT scan. In the laboratory, fully automatic analyzer VITROSECi/ECiQ Immunodiagnostic Systems, VITROSTSH was measured using a chemiluminescent immunometric assay with a detection range from 0.01 to 100 m IU/L. TSH values between 0.46 and 4.68 m IU/L were considered the normal reference range (euthyroid). Specimens with TSH values above or below the normal reference range were tested for free thyroxine (FT4). FT4 was measured using a competitive chemiluminescent assay with a detection range of 0.1-12.0 ng/dL. FT4 concentrations within 0.70-2.19 ng/dL was within the normal reference range. All samples included in the study met quality control criteria. Samples with TSH values > 4.69 m IU/L and normal FT4 levels were classified as indicating SCH.
The collected data was stored and analyzed with the Statistical Package for the Social Sciences (SPSS) version 22.0 for windows. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.
|
RESULTS
|
The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was 13 (17.8%) and among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Minimum age of patient in the study was 26 years and maximum age was 115 years. The mean age of presentation of cerebral infarction in this study was 63.30±19.217.
Among the total 73 cases, 49 were male and 24 were female. Among 49 males, 37 were euthyroid, 11 were hypothyroid and one male was hyperthyroid. Among 24 females, 23 were euthyroid and one female was hyperthyroid. On further categorization of altered thyroid status, 10 (13.7%) were subclinical hypothyroidism, 1 (1.4%) cases were of overt hypothyroidism and 2 (2.7%) cases had subclinical hyperthyroidism. With regards to the severity of subclinical hypothyroidism, out of the total 10 cases of subclinical hypothyroidism, 70% had grade IA, while the remaining 30% had grade IB severity of the status (Figure 1). In the study 49 (67.12%) of ischemic stroke were male and 24 (32.88%) were female.
Among euthyroid cases mean total cholesterol was 139.55 mg/dl which was higher than the mean cholesterol level in overt hypothyroid cases with 110 mg/dl. But mean total cholesterol level was lower among overt hypothyroid cases compared to euthyroid and overt hypothyroid cases. Mean TC was highest among subclinical hyperthyroid cases. Mean LDLc was highest among euthyroid cases with mean value of 73.94 mg/dl and lowest among overt hypothyroid cases with mean value of 41.00 mg/dl. TG was highest among overt hypothyroid cases with mean value of 199 mg/dl. HDLc was highest among euthyroid cases with mean value of 43.42 mg/dl. Subclinical hypothyroid cases had mean HDL value of 39.90 mg/dl (Table 1).
|
CONCLUSIONS
|
The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke.
|
[] |
[] |
[] |
[
"INTRODUCTION",
"METHODS",
"RESULTS",
"DISCUSSION",
"CONCLUSIONS"
] |
[
"Stroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4\nThyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4\nThe aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.",
"This descriptive cross-sectional study was conducted from June to December 2019 in National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu. Ethical approval from Institutional review board of NAMS (reference number. IM 175). Convenience sampling was done and the sample size of cases to be enrolled was calculated by using the formula,\nn = Z2 × p × q / e2\n = (1.645)2 × (0.5) × (1-0.5) / (0.1)2\n = 67.65\nWhere,\nn = minimum required sample size,\nZ = 1.645 at 90% Confidence Interval (CI),\np = prevalence taken as 50% for maximum sample size,\nq = 1-p\ne = margin of error, 10%\nHence, the required sample size was 67.65. Adding a 10% nonresponse rate, we enrolled 73 patients in our study. Patients with clinically diagnosed stroke, presenting within 48 hours of onset of symptoms with CT or MRI findings were included in the study. Patients with prior use of thyroid supplements recently within last 180 days and with a history of liver disease, renal failure and known thyroid disease and who don't give consent were excluded from the study. Informed written consent was taken either in Nepali or in English language, in whichever they felt comfortable. Confidentiality was maintained to the utmost. For those who are not able to give consent because of their clinical state, informed consent was taken from their close relative.\nProforma was translated in Nepali verbally if needed depending upon the cases. For diagnosing the type of stroke, every patient underwent neuroimaging with non-contrast CT of the head. Cerebral infarction was diagnosed if neurological deficits are accompanied by a hypo dense lesion >15 mm in diameter in an appropriate area on a cranial CT scan. Lacunar infarction were diagnosed if the patient presented with a pure motor stroke, a pure sensory stroke, ataxic hemiparesis or a sensorimotor stroke in the absence of a visual field defect and evidence of higher cerebral dysfunction and a hypo dense lesion of < 15 mm in diameter or normal CT scan. In the laboratory, fully automatic analyzer VITROSECi/ECiQ Immunodiagnostic Systems, VITROSTSH was measured using a chemiluminescent immunometric assay with a detection range from 0.01 to 100 m IU/L. TSH values between 0.46 and 4.68 m IU/L were considered the normal reference range (euthyroid). Specimens with TSH values above or below the normal reference range were tested for free thyroxine (FT4). FT4 was measured using a competitive chemiluminescent assay with a detection range of 0.1-12.0 ng/dL. FT4 concentrations within 0.70-2.19 ng/dL was within the normal reference range. All samples included in the study met quality control criteria. Samples with TSH values > 4.69 m IU/L and normal FT4 levels were classified as indicating SCH.\nThe collected data was stored and analyzed with the Statistical Package for the Social Sciences (SPSS) version 22.0 for windows. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.",
"The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was 13 (17.8%) and among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Minimum age of patient in the study was 26 years and maximum age was 115 years. The mean age of presentation of cerebral infarction in this study was 63.30±19.217.\nAmong the total 73 cases, 49 were male and 24 were female. Among 49 males, 37 were euthyroid, 11 were hypothyroid and one male was hyperthyroid. Among 24 females, 23 were euthyroid and one female was hyperthyroid. On further categorization of altered thyroid status, 10 (13.7%) were subclinical hypothyroidism, 1 (1.4%) cases were of overt hypothyroidism and 2 (2.7%) cases had subclinical hyperthyroidism. With regards to the severity of subclinical hypothyroidism, out of the total 10 cases of subclinical hypothyroidism, 70% had grade IA, while the remaining 30% had grade IB severity of the status (Figure 1). In the study 49 (67.12%) of ischemic stroke were male and 24 (32.88%) were female.\nAmong euthyroid cases mean total cholesterol was 139.55 mg/dl which was higher than the mean cholesterol level in overt hypothyroid cases with 110 mg/dl. But mean total cholesterol level was lower among overt hypothyroid cases compared to euthyroid and overt hypothyroid cases. Mean TC was highest among subclinical hyperthyroid cases. Mean LDLc was highest among euthyroid cases with mean value of 73.94 mg/dl and lowest among overt hypothyroid cases with mean value of 41.00 mg/dl. TG was highest among overt hypothyroid cases with mean value of 199 mg/dl. HDLc was highest among euthyroid cases with mean value of 43.42 mg/dl. Subclinical hypothyroid cases had mean HDL value of 39.90 mg/dl (Table 1).",
"Stroke is a form of acute stress with detrimental effect on various neurophysiological pathways.7 Hypothyroidism is a possible risk factor for stroke.8 A number of comorbidities have been associated with increased mortality in acute stroke patients. It is not known whether hypothyroidism (either clinical or subclinical) affects outcome in patients with acute cerebrovascular disease. A neuroprotective role of hypothyroidism has been shown in acute stroke patients.8 Low T3 appeared to be associated with stroke severity and short-term outcome.9 In addition, strokes of undetermined etiology accounted for one-third to one-quarter of ischemic strokes among young people and among cases of stroke of undetermined etiology hyperthyroidism could be the underlying cause.10,11 So, TFT screening is useful in patients with acute ischemic stroke (IS) because thyroid disorders are known risk factors for cerebrovascular diseases.6 In our study we observed 17.8% of the cases had thyroid dysfunction. Our finding is similar to the findings of the retrospective study done by Xu XY , et al. in 893 patients with acute ischemic stroke in which 19% of the cases had abnormal thyroid function tests with at least one of the thyroid related hormones below or above the normal ranges.12 Unknown overt or subclinical hyperthyroidism is associated with cardio-embolic stroke.13 Thyroid dysfunction in our study was lower than the study done by Dimopolou, et al. in 33 critically ill patients under mechanical ventilation due to acute stroke; study concluded 36% of the cases had thyroid dysfunction.14 In our study we found that 1.4% of the cases had overt hypothyroidism, this is comparable to the study done by O'keefe L.M , et al. in 129 patients with acute ischemic stroke. 2.32 % of the cases in the study had hypothyroidism.1513.7 % of our study population had subclinical hypothyroidism.\nOur result is similar to the study done by Pande, et al. to establish a relation between the variation in thyroid profile and ischemic CVAs in 75 patients within 48 hours of the event, which concluded that 12% of the cases had subclinical hypothyroidism.7 Prospective cohort study done by Chakler L, et al. on 47,573 adults (3451 subclinical hypothyroidism) from 17 cohorts concluded an increased risk of stroke on subjects younger than 65 years and those with higher TSH concentrations. Subclinical hypothyroidism (SCH) is postulated to increase stroke risk via atherogenic changes associated with abnormal thyroid function.16 The study concluded that hyperthyroidism is associated with an increased risk for ischemic stroke among young adults.11\nIn our study HDLc was low (<40 mg/dl) in all the case with thyroid dysfunction (18% cases) this is much lower compared to the study done by Pokharel , et al., among 281 stroke patients, in which 45% had their HDLc levels below 40 mg/dl.17 TG was elevated (>150 mg/dl) in cases with overt hypothyroidism (1.4%) this is consistent with the study done by Abrams JJ , et al. which concluded TG metabolism is not grossly deranged in hypothyroidism.18 In our study we found that stroke occurred more commonly among males (67.12%) compared to females (32.88%). Women have lower stroke incidence than men, it might be due to positive effects of estrogen on the cerebral circulation. A lifetime exposure to ovarian estrogens may protect against ischemic stroke.19 Seventy-two percent of the cases in our study were smokers. Wang, et al. reported that 48% of patients with stroke smoked.20 Tsai, et al. reported 38% of the cases with ischemic stroke were smokers.21\nOur results might not be generalized to all stroke patients as we recruited study participants from a single hospital only. As many of our patients presented to us after 48 hours of the occurrence of stroke events, we could not involve them in our study.",
"The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke."
] |
[
"intro",
"methods",
"results",
"discussion",
"conclusions"
] |
[
"\nhyperthyroidism\n",
"\nhypothyroidism\n",
"\nischemic stroke\n"
] |
INTRODUCTION:
Stroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4
Thyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4
The aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.
METHODS:
This descriptive cross-sectional study was conducted from June to December 2019 in National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu. Ethical approval from Institutional review board of NAMS (reference number. IM 175). Convenience sampling was done and the sample size of cases to be enrolled was calculated by using the formula,
n = Z2 × p × q / e2
= (1.645)2 × (0.5) × (1-0.5) / (0.1)2
= 67.65
Where,
n = minimum required sample size,
Z = 1.645 at 90% Confidence Interval (CI),
p = prevalence taken as 50% for maximum sample size,
q = 1-p
e = margin of error, 10%
Hence, the required sample size was 67.65. Adding a 10% nonresponse rate, we enrolled 73 patients in our study. Patients with clinically diagnosed stroke, presenting within 48 hours of onset of symptoms with CT or MRI findings were included in the study. Patients with prior use of thyroid supplements recently within last 180 days and with a history of liver disease, renal failure and known thyroid disease and who don't give consent were excluded from the study. Informed written consent was taken either in Nepali or in English language, in whichever they felt comfortable. Confidentiality was maintained to the utmost. For those who are not able to give consent because of their clinical state, informed consent was taken from their close relative.
Proforma was translated in Nepali verbally if needed depending upon the cases. For diagnosing the type of stroke, every patient underwent neuroimaging with non-contrast CT of the head. Cerebral infarction was diagnosed if neurological deficits are accompanied by a hypo dense lesion >15 mm in diameter in an appropriate area on a cranial CT scan. Lacunar infarction were diagnosed if the patient presented with a pure motor stroke, a pure sensory stroke, ataxic hemiparesis or a sensorimotor stroke in the absence of a visual field defect and evidence of higher cerebral dysfunction and a hypo dense lesion of < 15 mm in diameter or normal CT scan. In the laboratory, fully automatic analyzer VITROSECi/ECiQ Immunodiagnostic Systems, VITROSTSH was measured using a chemiluminescent immunometric assay with a detection range from 0.01 to 100 m IU/L. TSH values between 0.46 and 4.68 m IU/L were considered the normal reference range (euthyroid). Specimens with TSH values above or below the normal reference range were tested for free thyroxine (FT4). FT4 was measured using a competitive chemiluminescent assay with a detection range of 0.1-12.0 ng/dL. FT4 concentrations within 0.70-2.19 ng/dL was within the normal reference range. All samples included in the study met quality control criteria. Samples with TSH values > 4.69 m IU/L and normal FT4 levels were classified as indicating SCH.
The collected data was stored and analyzed with the Statistical Package for the Social Sciences (SPSS) version 22.0 for windows. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.
RESULTS:
The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was 13 (17.8%) and among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Minimum age of patient in the study was 26 years and maximum age was 115 years. The mean age of presentation of cerebral infarction in this study was 63.30±19.217.
Among the total 73 cases, 49 were male and 24 were female. Among 49 males, 37 were euthyroid, 11 were hypothyroid and one male was hyperthyroid. Among 24 females, 23 were euthyroid and one female was hyperthyroid. On further categorization of altered thyroid status, 10 (13.7%) were subclinical hypothyroidism, 1 (1.4%) cases were of overt hypothyroidism and 2 (2.7%) cases had subclinical hyperthyroidism. With regards to the severity of subclinical hypothyroidism, out of the total 10 cases of subclinical hypothyroidism, 70% had grade IA, while the remaining 30% had grade IB severity of the status (Figure 1). In the study 49 (67.12%) of ischemic stroke were male and 24 (32.88%) were female.
Among euthyroid cases mean total cholesterol was 139.55 mg/dl which was higher than the mean cholesterol level in overt hypothyroid cases with 110 mg/dl. But mean total cholesterol level was lower among overt hypothyroid cases compared to euthyroid and overt hypothyroid cases. Mean TC was highest among subclinical hyperthyroid cases. Mean LDLc was highest among euthyroid cases with mean value of 73.94 mg/dl and lowest among overt hypothyroid cases with mean value of 41.00 mg/dl. TG was highest among overt hypothyroid cases with mean value of 199 mg/dl. HDLc was highest among euthyroid cases with mean value of 43.42 mg/dl. Subclinical hypothyroid cases had mean HDL value of 39.90 mg/dl (Table 1).
DISCUSSION:
Stroke is a form of acute stress with detrimental effect on various neurophysiological pathways.7 Hypothyroidism is a possible risk factor for stroke.8 A number of comorbidities have been associated with increased mortality in acute stroke patients. It is not known whether hypothyroidism (either clinical or subclinical) affects outcome in patients with acute cerebrovascular disease. A neuroprotective role of hypothyroidism has been shown in acute stroke patients.8 Low T3 appeared to be associated with stroke severity and short-term outcome.9 In addition, strokes of undetermined etiology accounted for one-third to one-quarter of ischemic strokes among young people and among cases of stroke of undetermined etiology hyperthyroidism could be the underlying cause.10,11 So, TFT screening is useful in patients with acute ischemic stroke (IS) because thyroid disorders are known risk factors for cerebrovascular diseases.6 In our study we observed 17.8% of the cases had thyroid dysfunction. Our finding is similar to the findings of the retrospective study done by Xu XY , et al. in 893 patients with acute ischemic stroke in which 19% of the cases had abnormal thyroid function tests with at least one of the thyroid related hormones below or above the normal ranges.12 Unknown overt or subclinical hyperthyroidism is associated with cardio-embolic stroke.13 Thyroid dysfunction in our study was lower than the study done by Dimopolou, et al. in 33 critically ill patients under mechanical ventilation due to acute stroke; study concluded 36% of the cases had thyroid dysfunction.14 In our study we found that 1.4% of the cases had overt hypothyroidism, this is comparable to the study done by O'keefe L.M , et al. in 129 patients with acute ischemic stroke. 2.32 % of the cases in the study had hypothyroidism.1513.7 % of our study population had subclinical hypothyroidism.
Our result is similar to the study done by Pande, et al. to establish a relation between the variation in thyroid profile and ischemic CVAs in 75 patients within 48 hours of the event, which concluded that 12% of the cases had subclinical hypothyroidism.7 Prospective cohort study done by Chakler L, et al. on 47,573 adults (3451 subclinical hypothyroidism) from 17 cohorts concluded an increased risk of stroke on subjects younger than 65 years and those with higher TSH concentrations. Subclinical hypothyroidism (SCH) is postulated to increase stroke risk via atherogenic changes associated with abnormal thyroid function.16 The study concluded that hyperthyroidism is associated with an increased risk for ischemic stroke among young adults.11
In our study HDLc was low (<40 mg/dl) in all the case with thyroid dysfunction (18% cases) this is much lower compared to the study done by Pokharel , et al., among 281 stroke patients, in which 45% had their HDLc levels below 40 mg/dl.17 TG was elevated (>150 mg/dl) in cases with overt hypothyroidism (1.4%) this is consistent with the study done by Abrams JJ , et al. which concluded TG metabolism is not grossly deranged in hypothyroidism.18 In our study we found that stroke occurred more commonly among males (67.12%) compared to females (32.88%). Women have lower stroke incidence than men, it might be due to positive effects of estrogen on the cerebral circulation. A lifetime exposure to ovarian estrogens may protect against ischemic stroke.19 Seventy-two percent of the cases in our study were smokers. Wang, et al. reported that 48% of patients with stroke smoked.20 Tsai, et al. reported 38% of the cases with ischemic stroke were smokers.21
Our results might not be generalized to all stroke patients as we recruited study participants from a single hospital only. As many of our patients presented to us after 48 hours of the occurrence of stroke events, we could not involve them in our study.
CONCLUSIONS:
The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke.
|
Background:
Stroke is broadly classified as cerebral infarction, intracerebral hemorrhage and subarachnoid hemorrhage. Neuroendocrine profile is altered in acute ischemic stroke and there is a link between hypothyroidism and atherosclerosis which in turn may lead to stroke. The objective of this study was to find out the prevalence of alteration of thyroid hormones in patients with ischemic stroke in a tertiary care center.
Methods:
This descriptive cross-sectional study was conducted from June to December 2019 in a tertiary care center. Ethical approval was taken from Institutional review board of National Academy of Medical Sciences (reference number: IM 175). Patients with a diagnosis of stroke, without evidence of cardioembolic source, history of liver disease, renal failure and thyroid disease and who do not use thyroidal supplementation within 180 days prior the event were included. Convenience sampling was done. Data was entered in Microsoft Excel and analyzed using Statistical Package for the Social Sciences version 22. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.
Results:
The prevalence of altered thyroid levels among 73 patients was 13 (17.8%) (90% Confidence Interval= 10.44-25.16). Among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Among severity of hypothyroid cases, subclinical hypothyroidism grade IA was seen in 51 (70%), subclinical hypothyroidism grade IB was seen in 22 (30%).
Conclusions:
The prevalence of altered thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies.
|
INTRODUCTION:
Stroke is characterized as a neurological deficit including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) and is a major cause of disability and death worldwide.1 About 1,795,000 people experience a new or recurrent stroke each year.2 In developing countries, a majority of the stroke burden is observed, accounting for 75.2% of all stroke-related deaths and 81.0% of the associated DALYs lost.3 In Nepal, it was estimated that around 50,000 patients have stroke per year with annual death of around 15000. Of all the ischemic strokes, 77.4% are atherothrombotic occlusion of cerebral blood vessels.4
Thyroid dysfunction has been associated with cerebrovascular accidents (CVAs).5 Hyperthyroidism may cause ischemic stroke due to its relation to atrial fibrillation (AF).6 The main predisposing factors for stroke are hypertension, cigarette smoking, alcohol consumption and diabetes.4
The aim of this study was to find out the prevalence of altered thyroid levels among patients with ischemic CVAs in a tertiary care hospital.
CONCLUSIONS:
The prevalence of altered in thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies. The prevalence of stroke is increasing in developing countries like Nepal as there is rise in non-communicable diseases in this part of world. There are various risk factors for stroke which are modifiable and non-modifiable. So, early identification of these risk factors can have great impact in reduction of morbidity, mortality and burden associated with stroke.
|
Background:
Stroke is broadly classified as cerebral infarction, intracerebral hemorrhage and subarachnoid hemorrhage. Neuroendocrine profile is altered in acute ischemic stroke and there is a link between hypothyroidism and atherosclerosis which in turn may lead to stroke. The objective of this study was to find out the prevalence of alteration of thyroid hormones in patients with ischemic stroke in a tertiary care center.
Methods:
This descriptive cross-sectional study was conducted from June to December 2019 in a tertiary care center. Ethical approval was taken from Institutional review board of National Academy of Medical Sciences (reference number: IM 175). Patients with a diagnosis of stroke, without evidence of cardioembolic source, history of liver disease, renal failure and thyroid disease and who do not use thyroidal supplementation within 180 days prior the event were included. Convenience sampling was done. Data was entered in Microsoft Excel and analyzed using Statistical Package for the Social Sciences version 22. Point estimate at 90% Confidence Interval was calculated along with frequency and percentage for binary data.
Results:
The prevalence of altered thyroid levels among 73 patients was 13 (17.8%) (90% Confidence Interval= 10.44-25.16). Among them 11 (15.1%) were hypothyroid and 2 (2.7%) were hyperthyroid. Among severity of hypothyroid cases, subclinical hypothyroidism grade IA was seen in 51 (70%), subclinical hypothyroidism grade IB was seen in 22 (30%).
Conclusions:
The prevalence of altered thyroid levels among patients undergoing ischemic stroke was similar to the findings of other international studies.
| 1,919 | 298 | 5 |
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"patients",
"thyroid",
"hypothyroidism",
"ischemic",
"dl",
"subclinical",
"mean"
] |
[
"test",
"test"
] |
[CONTENT]
hyperthyroidism
|
hypothyroidism
|
ischemic stroke
[SUMMARY]
|
[CONTENT]
hyperthyroidism
|
hypothyroidism
|
ischemic stroke
[SUMMARY]
|
[CONTENT]
hyperthyroidism
|
hypothyroidism
|
ischemic stroke
[SUMMARY]
|
[CONTENT]
hyperthyroidism
|
hypothyroidism
|
ischemic stroke
[SUMMARY]
|
[CONTENT]
hyperthyroidism
|
hypothyroidism
|
ischemic stroke
[SUMMARY]
|
[CONTENT]
hyperthyroidism
|
hypothyroidism
|
ischemic stroke
[SUMMARY]
|
[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]
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[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]
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[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]
|
[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]
|
[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]
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[CONTENT] Brain Ischemia | Cross-Sectional Studies | Humans | Ischemic Stroke | Stroke | Tertiary Care Centers | Thyroid Hormones [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]
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[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]
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[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]
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[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]
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[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]
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[CONTENT] stroke | study | cases | patients | thyroid | hypothyroidism | ischemic | dl | subclinical | mean [SUMMARY]
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[CONTENT] stroke | death | year | 000 | stroke year | hemorrhage | ischemic | cause | cvas | associated [SUMMARY]
|
[CONTENT] range | normal | sample | ct | consent | size | ft4 | reference | sample size | iu [SUMMARY]
|
[CONTENT] mean | cases | cases mean | hypothyroid | hypothyroid cases | mg dl | mg | overt hypothyroid cases | overt hypothyroid | value [SUMMARY]
|
[CONTENT] modifiable | stroke | risk | non | risk factors | factors | prevalence | factors great | impact reduction morbidity | non communicable diseases world [SUMMARY]
|
[CONTENT] stroke | cases | study | mean | hypothyroidism | patients | ischemic | subclinical | thyroid | risk [SUMMARY]
|
[CONTENT] stroke | cases | study | mean | hypothyroidism | patients | ischemic | subclinical | thyroid | risk [SUMMARY]
|
[CONTENT] ||| ||| tertiary [SUMMARY]
|
[CONTENT] June to December 2019 | tertiary ||| Institutional review board | National Academy of Medical Sciences | IM | 175 ||| 180 days ||| ||| Microsoft Excel | Statistical Package | Social Sciences | 22 ||| Point | 90% [SUMMARY]
|
[CONTENT] 73 | 13 | 17.8% | 90% | 10.44-25.16 ||| 11 | 15.1% | 2 | 2.7% ||| IA | 51 | 70% | IB | 22 | 30% [SUMMARY]
|
[CONTENT] [SUMMARY]
|
[CONTENT] ||| ||| tertiary ||| June to December 2019 | tertiary ||| Institutional review board | National Academy of Medical Sciences | IM | 175 ||| 180 days ||| ||| Microsoft Excel | Statistical Package | Social Sciences | 22 ||| Point | 90% ||| ||| 73 | 13 | 17.8% | 90% | 10.44-25.16 ||| 11 | 15.1% | 2 | 2.7% ||| IA | 51 | 70% | IB | 22 | 30% ||| [SUMMARY]
|
[CONTENT] ||| ||| tertiary ||| June to December 2019 | tertiary ||| Institutional review board | National Academy of Medical Sciences | IM | 175 ||| 180 days ||| ||| Microsoft Excel | Statistical Package | Social Sciences | 22 ||| Point | 90% ||| ||| 73 | 13 | 17.8% | 90% | 10.44-25.16 ||| 11 | 15.1% | 2 | 2.7% ||| IA | 51 | 70% | IB | 22 | 30% ||| [SUMMARY]
|
||||||
Post-Transplant Lymphoproliferative Diseases in Pediatric Kidney Allograft Recipients with Epstein-Barr Virus Viremia.
|
31373185
|
Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, especially in children with Epstein-Barr virus (EBV) viremia (EV). We performed a retrospective study to evaluate risk factors for PTLD in children with EV.
|
BACKGROUND
|
Among 199 pediatric kidney transplantation (KT) recipients at our center from January 2001 to October 2015, records of those with EBV viral loads of > 1,000 copies/mL and/or PTLD were reviewed.
|
METHODS
|
Diagnosis of PTLD was made in seven patients (PTLD group), and 39 patients had EV only (EV only group). The median time from KT to EV and PTLD diagnosis was 6.7 (range 0.4-47.8) months and 8.2 (range, 2.8-98.9) months, respectively. There were no significant differences between the groups in terms of sex, age at transplantation, donor type, EBV viral load, or EV-free duration after KT. Higher tacrolimus level before EV (hazard ratio, 44.5; P = 0.003) was an independent risk factor for PTLD in multivariate Cox regression analysis. Six patients with a high EBV load (median 171,639 copies/mL) were treated with preemptive rituximab (RTX) therapy, resulting in transient reduction of EBV load. None of these patients developed PTLD (median follow-up 51.5 months); however, two had neutropenia and two developed infection requiring hospital admission.
|
RESULTS
|
In pediatric KT recipients, higher tacrolimus levels were associated with a higher incidence of PTLD. Conversely, those who received preemptive RTX for EV did not develop PTLD.
|
CONCLUSION
|
[
"Antineoplastic Agents, Immunological",
"Child, Preschool",
"Female",
"Herpesvirus 4, Human",
"Humans",
"Infant",
"Kidney Transplantation",
"Lymphoproliferative Disorders",
"Male",
"Neutropenia",
"Proportional Hazards Models",
"Retrospective Studies",
"Risk Factors",
"Rituximab",
"Transplantation, Homologous",
"Viral Load",
"Viremia"
] |
6676002
|
INTRODUCTION
|
Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, with an incidence ranging from 1% to 16% depending on the allograft organ.1 This incidence is 0.2%–2.5% in liver and kidney transplantation cases, which is relatively lower than that in other organ transplant cases.23 Incidence rates of PTLD are higher in pediatric kidney transplant recipients than in adult kidney transplant recipients, ranging from 1.2% to 10%.456 More than 90% of pediatric PTLDs are due to Epstein-Barr virus (EBV)-positive B-cell proliferation. Previously reported risk factors for EBV-associated PTLD include recipient EBV seronegativity, degree of immunosuppression, acute rejection episode, use of OKT3 or tacrolimus, recipient age and race, allograft type, host genetic variations, and especially Epstein-Barr virus viremia (EV).789101112 While adult allograft recipients usually have acquired-immunity to EBV at the time of transplantation, pediatric allograft recipients often experience primary EBV infection after transplantation. Although EBV-infected transformed B cells are highly immunogenic and rapidly eliminated by EBV-specific T cells in healthy hosts, if immunosuppressed pediatric patients have primary EBV infection, then an inadequate immune response may result in massive infection of B cells. Primary EBV infection increases the chance of developing PTLD 6–76 fold.13141516
EBV infection and/or reactivation, which can be detected as increasing copy numbers of EBV DNA in the peripheral blood, usually precede PTLD. Therefore, it is recommended that EBV titer be regularly monitored after solid-organ transplantation in patients at a higher risk for PTLD. The Kidney Disease Improving Global Outcomes clinical practice guideline for the care of kidney transplant recipients recommended the following monitoring regimen for EBV viral load monitoring in EBV-seronegative patients who received an allograft from a seropositive donor: every 1 week in the first 3 months after transplantation, at least monthly for 3–6 months, and then every 3 months for the rest of the first year.17 Once EBV infection and/or reactivation is noted, transplantation physicians reduce immunosuppression to prevent EBV-associated PTLD. However, EBV infection often persists and progresses despite reduction in immunosuppressive drug regimen, and there is no anti-viral agent with proven efficacy against EBV.8 In contrast, during stem cell transplantation, pre-emptive treatment with rituximab is often used to eradicate B lymphocytes, the reservoir of EBV, thereby to prevent PTLD.1819 A similar approach has been attempted in solid organ transplantation in patients at high-risk for EBV-associated PTLD.20
In this study, the risk factors for PTLD were assessed in pediatric kidney allograft recipients with EV, including the effect of pre-emptive rituximab treatment to prevent PTLD.
|
METHODS
|
Patients and data collection We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.
We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.
Immunosuppression In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.
In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.
EV monitoring Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.
Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.
Pre-emptive rituximab therapy Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.
Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.
Statistical analysis To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).
To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).
Ethics statement The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.
The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.
|
RESULTS
|
During the study period, 199 children underwent kidney transplantation in our center. Of these, 46 (23.1%) had viremia defined as an EBV load greater than 1,000 copies/mL in whole blood for 2 consecutive tests during a median follow-up period of 5.3 years (Fig. 1). Viremia of all patients (EBV > 1,000 copies/mL) was first detected at a median of 6.7 months (range, 0.4–47.8 months) after kidney transplantation. The diagnosis of PTLD was made in seven patients (PTLD group) at a median of 8.2 months (2.8–98.9 months) after transplantation. The other 39 patients had EV only (EV only group).
EBV = Epstein-Barr virus, PTLD = post-transplant lymphoproliferative disease, RTX = rituximab.
Clinical course of PTLD Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.
PTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.
aLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.
Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.
PTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.
aLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.
Risk factors for PTLD Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.
Values are expressed as numbers (%) and median (range).
PTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.
Tacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.
The Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.
HR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.
aFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.
Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.
Values are expressed as numbers (%) and median (range).
PTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.
Tacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.
The Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.
HR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.
aFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.
Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.
KT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.
aGenetic disorder caused by WT1 mutation.
EBV = Epstein-Barr virus, RTX = rituximab.
Regarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.
ANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.
Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.
KT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.
aGenetic disorder caused by WT1 mutation.
EBV = Epstein-Barr virus, RTX = rituximab.
Regarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.
ANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.
| null | null |
[
"Patients and data collection",
"Immunosuppression",
"EV monitoring",
"Pre-emptive rituximab therapy",
"Statistical analysis",
"Ethics statement",
"Clinical course of PTLD",
"Risk factors for PTLD",
"Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant"
] |
[
"We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.",
"In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.",
"Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.",
"Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.",
"To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).",
"The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.",
"Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.",
"Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.",
"Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"METHODS",
"Patients and data collection",
"Immunosuppression",
"EV monitoring",
"Pre-emptive rituximab therapy",
"Statistical analysis",
"Ethics statement",
"RESULTS",
"Clinical course of PTLD",
"Risk factors for PTLD",
"Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant",
"DISCUSSION"
] |
[
"Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, with an incidence ranging from 1% to 16% depending on the allograft organ.1 This incidence is 0.2%–2.5% in liver and kidney transplantation cases, which is relatively lower than that in other organ transplant cases.23 Incidence rates of PTLD are higher in pediatric kidney transplant recipients than in adult kidney transplant recipients, ranging from 1.2% to 10%.456 More than 90% of pediatric PTLDs are due to Epstein-Barr virus (EBV)-positive B-cell proliferation. Previously reported risk factors for EBV-associated PTLD include recipient EBV seronegativity, degree of immunosuppression, acute rejection episode, use of OKT3 or tacrolimus, recipient age and race, allograft type, host genetic variations, and especially Epstein-Barr virus viremia (EV).789101112 While adult allograft recipients usually have acquired-immunity to EBV at the time of transplantation, pediatric allograft recipients often experience primary EBV infection after transplantation. Although EBV-infected transformed B cells are highly immunogenic and rapidly eliminated by EBV-specific T cells in healthy hosts, if immunosuppressed pediatric patients have primary EBV infection, then an inadequate immune response may result in massive infection of B cells. Primary EBV infection increases the chance of developing PTLD 6–76 fold.13141516\nEBV infection and/or reactivation, which can be detected as increasing copy numbers of EBV DNA in the peripheral blood, usually precede PTLD. Therefore, it is recommended that EBV titer be regularly monitored after solid-organ transplantation in patients at a higher risk for PTLD. The Kidney Disease Improving Global Outcomes clinical practice guideline for the care of kidney transplant recipients recommended the following monitoring regimen for EBV viral load monitoring in EBV-seronegative patients who received an allograft from a seropositive donor: every 1 week in the first 3 months after transplantation, at least monthly for 3–6 months, and then every 3 months for the rest of the first year.17 Once EBV infection and/or reactivation is noted, transplantation physicians reduce immunosuppression to prevent EBV-associated PTLD. However, EBV infection often persists and progresses despite reduction in immunosuppressive drug regimen, and there is no anti-viral agent with proven efficacy against EBV.8 In contrast, during stem cell transplantation, pre-emptive treatment with rituximab is often used to eradicate B lymphocytes, the reservoir of EBV, thereby to prevent PTLD.1819 A similar approach has been attempted in solid organ transplantation in patients at high-risk for EBV-associated PTLD.20\nIn this study, the risk factors for PTLD were assessed in pediatric kidney allograft recipients with EV, including the effect of pre-emptive rituximab treatment to prevent PTLD.",
" Patients and data collection We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\nWe performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.\n Immunosuppression In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\nIn our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.\n EV monitoring Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\nPrincipally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.\n Pre-emptive rituximab therapy Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\nSome of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.\n Statistical analysis To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\nTo determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).\n Ethics statement The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.\nThe study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.",
"We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.",
"In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.",
"Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.",
"Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.",
"To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).",
"The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.",
"During the study period, 199 children underwent kidney transplantation in our center. Of these, 46 (23.1%) had viremia defined as an EBV load greater than 1,000 copies/mL in whole blood for 2 consecutive tests during a median follow-up period of 5.3 years (Fig. 1). Viremia of all patients (EBV > 1,000 copies/mL) was first detected at a median of 6.7 months (range, 0.4–47.8 months) after kidney transplantation. The diagnosis of PTLD was made in seven patients (PTLD group) at a median of 8.2 months (2.8–98.9 months) after transplantation. The other 39 patients had EV only (EV only group).\nEBV = Epstein-Barr virus, PTLD = post-transplant lymphoproliferative disease, RTX = rituximab.\n Clinical course of PTLD Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\nPatients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.\n Risk factors for PTLD Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\nTable 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.\n Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.\nSix patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.",
"Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.\nPTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.\naLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.",
"Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.\nValues are expressed as numbers (%) and median (range).\nPTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.\nTacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.\nThe Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.\nHR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.\naFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.",
"Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.\nKT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.\naGenetic disorder caused by WT1 mutation.\nEBV = Epstein-Barr virus, RTX = rituximab.\nRegarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.\nANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.",
"During the last 15 years, among the almost 200 pediatric kidney transplantation recipients at our center, seven developed EBV-associated PTLD (3.5%). In our study, the risk factor for PTLD in pediatric kidney transplant recipients with EV was a high tacrolimus level before EV. Twenty (43.4%) out of 46 recipients who had viremia were EBV seronegative, which is similar to the rates reported in previous studies in North America and Europe (19%–57%).212223 Pre-transplant recipient EBV seronegativity is a well-known risk factor for PTLD. In adult transplant studies, the rate of developing PTLD is 5–12 fold higher in EBV-seronegative patients than in EBV-seropositive patients.2324 McDonald et al.6 reported that EBV-seronegative pediatric subjects have a 4.7-fold higher relative HR than EBV-positive subjects. In our study, recipient EBV seronegativity did not increase the risk of PTLD in multivariate Cox regression analysis. While transplant from an EBV-seropositive donor to a seronegative recipient has been associated with the development of PTLD,25 there was no statistically significant difference in EBV serostatus (donor/recipient) in the present study. This finding was attributed mainly to the fact that there were no EBV seropositive recipients in PTLD group and we did not have serostatus information for the majority of donors (67.3%).\nThe majority of kidney allograft recipients in this study were given tacrolimus, and mycophenolate was used in more than 80% of patients instead of azathioprine.26 By the late 2000s, monoclonal interleukin-2 receptor antibodies were used as induction therapy in up to 80% of patients. Basiliximab, a monoclonal antibody which targets activated T lymphocytes, was not related to PTLD risk in our study, as previously reported.427 Several studies have suggested that higher tacrolimus levels are associated with higher risk for PTLD, and others have reported that the net state of immunosuppression, rather than any individual agent, increases the risk for PTLD.282930 In our study, all patients received tacrolimus for maintenance immunosuppression, and we found that a higher pre-EV tacrolimus level in the PTLD group compared with the EV only group was a risk factor for PTLD.\nRegular monitoring of EBV viral load and early recognition of recipients at high risk of PTLD have been identified as clinical priorities in recent years.31 Previous studies have shown that elevated levels of EBV DNA and persistent high EBV loads are risk factors for PTLD,122032 but no clear cut-off point of EBV viral load for the prediction of PTLD development has been determined. We did not find a significant relationship between EV and PTLD in this study; however, six patients with a high EBV titer were treated with preemptive RTX and they did not develop PTLD. Because they were included in analysis, this could have confounded the causality of high EBV titer and PTLD development.\nTreatment strategies for organ transplant recipients with EV include reduction of immunosuppression with/without antiviral agents, immunoglobulin, or RTX.33 These treatments are still undergoing clinical studies. Preemptive administration of RTX is widely used and has been demonstrated to reduce the incidence of PTLD in stem cell transplant recipients with a high EBV viral load.183435 Pre-emptive RTX therapy has been reported in 14.5% of global transplant programs, and in more than 60% of pediatric transplant patients worldwide.33 However, only one study reported the use of RTX in five pediatric renal allograft recipients,20 and there have been no prospective studies on the efficacy of pre-emptive RTX therapy in solid organ transplantation. Rituximab is a murine/human chimeric monoclonal anti-CD20 antibody and is able to deplete circulating B cells rapidly, including those infected with EBV. Although RTX was effective for reducing EBV viral load in our patients, this reduction was not permanent in line with previous reports.20 In addition, significant adverse effects, such as infection and neutropenia, accompanied RTX administration. In patients with stem cell transplant (SCT), preemptive RTX was not associated with an increase in infectious complications.1819 This is important because, while SCT recipients are able to discontinue their immunosuppressive agents within 6–9 months after SCT, recipients of solid organ transplantation have to be on immunosuppressive agents for as long as their allografts are functioning. Therefore, the long-term use of immunosuppressive agents may explain why infection and neutropenia are common clinical findings after RTX therapy in this patient population.\nAlthough the small sample size of the current study precludes us from drawing any definite conclusions, our observations suggest that preemptive RTX treatment may effectively reduce high EBV viral load in pediatric recipients of solid organ transplants. Because RTX therapy eradicates B-lymphocytes including transformed lymphocytes, the risk of PTLD might be reduced at least during the period of B cell depletion. However, one should take into account that this treatment significantly increases the risk of neutropenia and infection. More research into the influence of preemptive RTX therapy on PTLD development is needed.\nThe occurrence of PTLD in kidney transplant recipients follows a bimodal distribution, with one peak in the first year and the second in the later post-transplantation period. Early PTLD, occurring within the first year of transplantation, is associated with EBV infection and tends to occur more commonly in children than adults.123637 In this study, while six patients in the PTLD group were diagnosed with PTLD within the first year of renal transplantation, one patient with WT1 mutation developed PTLD later than 8.2 years after renal transplantation. We suspect that in this patient, the WT1 mutation of a tumor suppressor gene might have increased the risk of PTLD, and especially that of late-onset. The occurrence of PTLD in patients with WT1 mutation has been reported previously, within the first year in two cases and later than the first year in another.383940 Based on the limited availability of clinical data, the association between WT1 mutation and development of PTLD requires further investigation in future clinical studies and is beyond the scope of the current study.\nThere are several limitations of this study. First, this is a retrospective observational study of a single center and the number of the patients observed was therefore small. The limitations of this small sample size might have affected the outcome of multivariate analysis. In addition, data of donor EBV serology were not uniformly available in our study. Data on donor serology status was only available for 33% of participants. Therefore, our study did not show any association between PTLD and EBV-donor/recipient serostatus. Finally, the number of patients who were treated with RTX was not large enough to draw any definitive conclusions.\nIn summary, this study demonstrates that a higher tacrolimus level before EV is correlated with the development of PTLD. Preemptive RTX appears to be effective for reducing EBV viral load in pediatric kidney transplant recipients. However, the reduction of EBV viral load was not persistent, and adverse effects of RTX, namely infection and neutropenia, were clinically significant."
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null,
null,
null,
"discussion"
] |
[
"Post-Transplant Lymphoproliferative Disease",
"Kidney Transplantation",
"Epstein-Barr Virus",
"Rituximab"
] |
INTRODUCTION:
Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, with an incidence ranging from 1% to 16% depending on the allograft organ.1 This incidence is 0.2%–2.5% in liver and kidney transplantation cases, which is relatively lower than that in other organ transplant cases.23 Incidence rates of PTLD are higher in pediatric kidney transplant recipients than in adult kidney transplant recipients, ranging from 1.2% to 10%.456 More than 90% of pediatric PTLDs are due to Epstein-Barr virus (EBV)-positive B-cell proliferation. Previously reported risk factors for EBV-associated PTLD include recipient EBV seronegativity, degree of immunosuppression, acute rejection episode, use of OKT3 or tacrolimus, recipient age and race, allograft type, host genetic variations, and especially Epstein-Barr virus viremia (EV).789101112 While adult allograft recipients usually have acquired-immunity to EBV at the time of transplantation, pediatric allograft recipients often experience primary EBV infection after transplantation. Although EBV-infected transformed B cells are highly immunogenic and rapidly eliminated by EBV-specific T cells in healthy hosts, if immunosuppressed pediatric patients have primary EBV infection, then an inadequate immune response may result in massive infection of B cells. Primary EBV infection increases the chance of developing PTLD 6–76 fold.13141516
EBV infection and/or reactivation, which can be detected as increasing copy numbers of EBV DNA in the peripheral blood, usually precede PTLD. Therefore, it is recommended that EBV titer be regularly monitored after solid-organ transplantation in patients at a higher risk for PTLD. The Kidney Disease Improving Global Outcomes clinical practice guideline for the care of kidney transplant recipients recommended the following monitoring regimen for EBV viral load monitoring in EBV-seronegative patients who received an allograft from a seropositive donor: every 1 week in the first 3 months after transplantation, at least monthly for 3–6 months, and then every 3 months for the rest of the first year.17 Once EBV infection and/or reactivation is noted, transplantation physicians reduce immunosuppression to prevent EBV-associated PTLD. However, EBV infection often persists and progresses despite reduction in immunosuppressive drug regimen, and there is no anti-viral agent with proven efficacy against EBV.8 In contrast, during stem cell transplantation, pre-emptive treatment with rituximab is often used to eradicate B lymphocytes, the reservoir of EBV, thereby to prevent PTLD.1819 A similar approach has been attempted in solid organ transplantation in patients at high-risk for EBV-associated PTLD.20
In this study, the risk factors for PTLD were assessed in pediatric kidney allograft recipients with EV, including the effect of pre-emptive rituximab treatment to prevent PTLD.
METHODS:
Patients and data collection We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.
We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.
Immunosuppression In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.
In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.
EV monitoring Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.
Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.
Pre-emptive rituximab therapy Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.
Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.
Statistical analysis To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).
To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).
Ethics statement The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.
The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.
Patients and data collection:
We performed a retrospective study that included all patients aged 0–19 years who underwent kidney transplantation in Seoul National University Children's Hospital from January 2001 to October 2015. Patients were enrolled if their EBV load in whole blood was greater than 1,000 copies/mL for 2 consecutive tests. The EBV Q-PCR Alert kit (ELITech Group, Puteaux, France) was used to quantify the amount of Epstein-Barr virus nuclear antigen (EBNA)-1 in whole blood. The diagnosis of PTLD was made histologically after biopsy, and the association with EBV was assessed in tissue specimens by in-situ hybridization of Epstein-Barr virus-encoded RNA (EBER). Patients who received another solid organ transplantation before or after kidney transplantation were excluded. Data were obtained from electronic medical records. Data reviewed included underlying disease, sex, age at transplantation, EBV serologic status of the donor and the recipient at transplantation, donor source, type of induction therapy, maintenance immunosuppressive medication, rejection episodes, and tacrolimus level at EV onset and median levels before and after onset of EV. The median levels of tacrolimus before the onset of EV were calculated using all values measured from transplantation to the appearance of EV. EBV serostatus at the time of transplant was determined by viral capsid antigen (VCA)-IgM, VCA-IgG, and EBNA. Cytomegalovirus (CMV) viremia was defined as positive antigenemia or detection of CMV DNA determined by whole-blood PCR.
Immunosuppression:
In our center, induction immunosuppression therapy for kidney transplantation consisted of methylprednisolone with or without basiliximab or antithymocyte globulin. Steroids, tacrolimus, and mycophenolate mofetil were started perioperatively and continued as maintenance therapy. Methylprednisolone was administered as 10 mg/kg intravenous bolus dose at the time of surgery and was tapered gradually to a maintenance dose of prednisolone 0.3 mg/kg by 1 month after transplantation. The target tacrolimus trough level was 8–12 ng/mL for up to 3 months, 6–8 ng/mL between 3 and 6 months, and 4–6 ng/mL thereafter. Tacrolimus levels were monitored weekly for a month after discharge from operation, biweekly for the next 3 months, and monthly thereafter. From 2001 to 2008, basiliximab was used as induction therapy for high-risk patients with deceased-donor kidney transplant or higher number of human leukocyte antigen (HLA) mismatches. After 2008, most patients received basiliximab induction therapy.
EV monitoring:
Principally, EBV was monitored every month for the first three months following transplant, then every 3 months for the rest of the first year, and then yearly. For recipients who were positive for EBV VCA IgG, EBV was monitored every three months. When EV was detected, EBV was monitored again in 2 weeks, and if the titer was still high, immunosuppression was reduced, usually by reducing antimetabolites and then tacrolimus according to the judgment of the physicians.
Pre-emptive rituximab therapy:
Some of the patients who exhibited persistent high titers of EV (more than 1 × 104 copies/mL in whole blood for two consecutive weeks) despite immunosuppression reduction were treated with rituximab (RTX). These patients had prolonged high viremia over 1 year, a 3-fold or greater increase in EBV titer, or higher risk of malignancy (WT1 mutation). After confirming that the patients did not have active infection or neutropenia, a single dose of RTX therapy of 375 mg/m2 body surface area was administered.
Statistical analysis:
To determine statistical differences between groups, we used the chi-square test or Fischer's exact test for categorical variables and the t-test or Mann-Whitney test for continuous variables. Cox regression analysis was performed to identify risk factors for PTLD following EV. We performed the univariate Cox regression test to identify significant independent variables, and used independent variables with a univariate P value < 0.2 for multivariate Cox regression analysis. A P value < 0.05 was considered statistically significant. The statistical analysis was performed using IBM SPSS Statistics version 22.0 (IBM cooperation, Armonk, NY, USA).
Ethics statement:
The study was approved by the Institutional Review Board (IRB) of our center (IRB No. H-1312-068-541). The informed consent requirement was waived by the board.
RESULTS:
During the study period, 199 children underwent kidney transplantation in our center. Of these, 46 (23.1%) had viremia defined as an EBV load greater than 1,000 copies/mL in whole blood for 2 consecutive tests during a median follow-up period of 5.3 years (Fig. 1). Viremia of all patients (EBV > 1,000 copies/mL) was first detected at a median of 6.7 months (range, 0.4–47.8 months) after kidney transplantation. The diagnosis of PTLD was made in seven patients (PTLD group) at a median of 8.2 months (2.8–98.9 months) after transplantation. The other 39 patients had EV only (EV only group).
EBV = Epstein-Barr virus, PTLD = post-transplant lymphoproliferative disease, RTX = rituximab.
Clinical course of PTLD Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.
PTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.
aLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.
Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.
PTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.
aLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.
Risk factors for PTLD Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.
Values are expressed as numbers (%) and median (range).
PTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.
Tacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.
The Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.
HR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.
aFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.
Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.
Values are expressed as numbers (%) and median (range).
PTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.
Tacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.
The Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.
HR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.
aFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.
Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.
KT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.
aGenetic disorder caused by WT1 mutation.
EBV = Epstein-Barr virus, RTX = rituximab.
Regarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.
ANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.
Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.
KT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.
aGenetic disorder caused by WT1 mutation.
EBV = Epstein-Barr virus, RTX = rituximab.
Regarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.
ANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.
Clinical course of PTLD:
Patients with PTLD presented with fever, lymph node enlargement, or gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea (Table 1). Any gastrointestinal symptoms and/or lymph node enlargement raised suspicion for PTLD and prompted the clinician to perform further work-up to rule out PTLD. The majority of patients (n = 4) had gastrointestinal organ involvement, including small bowel and intraperitoneal lymph nodes. There was no extranodal PTLD. Pathologic diagnosis of PTLD revealed one case of early lesion, two cases of polymorphic PTLD, one case of Burkitt lymphoma, and three cases of diffuse large B-cell lymphoma. Upon diagnosis of PTLD, immunosuppressive medications were reduced, and RTX and/or chemotherapy were administered as appropriate. All patients achieved complete remission of PTLD after treatments. While one patient lost her allograft kidney due to complications of chemotherapy, six patients retained renal function after follow-up for 2.5–10.5 years.
PTLD = post-transplant lymphoproliferative disease, KT = kidney transplantation, EBV = Epstein-Barr virus, F = female, FSGS = focal segmental glomerulosclerosis, LNE = lymph node enlargement, LN = lymph node, D/R = donor/recipient, UK = unknown, RTX = rituximab, M = male, NS = nephrotic syndrome, MCDK = multicystic dysplastic kidney.
aLesion detected by imaging study (computed tomography or positron emission tomography); bEBV in-situ hybridization positive; cGenetic disorder caused by WT1 mutation.
Risk factors for PTLD:
Table 2 shows the comparison of clinical variables between the PTLD and EV only group by univariate analysis. There were no significant differences between the two groups in terms of sex, age at transplantation, donor type, interval between transplantation, and first appearance of EV. Although the peak median EBV titer was higher in the PTLD group (152,987 EBV copies/mL whole blood) than the EV only group (17,305 copies/mL whole blood), there was no statistical significance. There were also no significant differences between groups in terms of median EBV viral load and EV-free duration after kidney transplant. At the time of transplantation, six patients (85.7%) in the PTLD group and 14 patients (35.9%) in the EV only group were seronegative for EBV (P = 0.009). Data of donor EBV status before transplantation were available only in a few cases, with no statistically significant difference observed between the groups.
Values are expressed as numbers (%) and median (range).
PTLD = post-transplant lymphoproliferative disease, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus, CMV = cytomegalovirus, MMF = mycophenolate, AZA = azathioprine, RTX = rituximab.
Tacrolimus levels before EV tended to be higher in the PTLD group (9.5 ng/mL) than in the EV only group (7.7 ng/mL, P = 0.039). Maintenance immunosuppression regimen or history of rejection was not significantly different between the two groups. Six patients were treated with pre-emptive RTX, none of whom developed PTLD, while the number of RTX-treated patients was too small to be statistically significant.
The Cox proportional-hazard model was used to identify factors associated with an increased risk of developing PTLD after EV (Table 3). Values of 8.9 ng/mL for tacrolimus level and 35,900 copies/μL for peak EBV titer were determined as cutoff values based on the receiver operating characteristic curve analysis. The areas under curve of tacrolimus and peak EBV titer were 0.745 (95% confidence interval [CI] 0.522–0.969, sensitivity 85.7%, and specificity 79.5%) and 0.634 (95% CI 0.399–0.869, sensitivity 85.7%, and specificity 59.0%), respectively. A higher tacrolimus level (hazard ratio [HR], 13.7; 95% CI, 1.6–117.9; P = 0.017) was associated with PTLD. Basiliximab induction therapy, higher EBV titer, EBV seronegativity of recipients, and rejection history were not significant in multivariate Cox regression analysis.
HR = hazard ratio, CI = confidence interval, EV = Epstein-Barr virus viremia, EBV = Epstein-Barr virus.
aFactors with a value of P < 0.2 in the univariate Cox regression analysis were included in the multivariate analysis.
Efficacy and safety of pre-emptive rituximab treatment in pediatric kidney transplant:
Six patients in the EV only group (male: female, 2:4) who had a high EBV load received preemptive RTX therapy. Their median age at transplant was 4 years (1–14 years), and EBV infection was first detected at 4.3 months (3.9–9.9 months) after kidney transplantation. Administration of RTX was carried out at a median of 29.2 months (5.1–69.6 months) after transplantation. These six patients did not exhibit any symptoms such as fever, lymph node enlargement, or gastrointestinal problems; and no imaging studies were performed. Two patients were EBV seropositive and four patients were EBV seronegative (Table 4). The two EBV-seropositive patients were at low risk for the development of PTLD, but RTX was administered to these patients based on the clinician's decision; one patient had a mutation of WT1, and was therefore prone to tumor development, while the other had persistently high EBV load for 46 months despite the reduction of immunosuppression. Median EBV viral loads at the time of RTX treatment were 171,639 copies/mL (6,181–783,504 copies/mL). After a single dose of RTX therapy, a concordant decrease in EBV load and B lymphocytes was observed (Fig. 2). In five patients, EV disappeared within months; the other patient showed reduction of EBV titer but persistence of EV despite RTX treatment. Unfortunately, the patient lost the allograft due to rejection and concomitant infection within 8 months after RTX therapy, and EBV titer was not monitored after this adverse event. In the remaining five patients, EBV load rebounded along with recovery of B cells in a median 8 months. However, none of these five patients developed PTLD over a median follow-up of 51.5 months.
KT = kidney transplantation, EBV = Epstein-Barr virus, EV = Epstein-Barr virus viremia, RTX = rituximab, IS = immunosuppressant, F/U = follow up, F = female, D/R = donor/recipient, UK = unknown, TAC = tacrolimus, ESRD = end stage renal disease, CKD = chronic kidney disease, M = male.
aGenetic disorder caused by WT1 mutation.
EBV = Epstein-Barr virus, RTX = rituximab.
Regarding the safety of RTX therapy, only one patient complained of chest discomfort during RTX infusion. Two patients experienced neutropenia at 4 months and 1 month after RTX treatment (Fig. 3). Two patients were admitted for viral or bacterial infections at 1 month and 2 months after RTX treatment. In comparison, of 8 patients with high EV (EBV > 10,000 copies/mL) but without RTX treatment, three patients experienced 6 infection episodes and one patient developed neutropenia during 12 months after the occurrence of high EV. Infectious complications included Citrobacter freundii, CMV, influenza A, adenovirus and Pneumocystis jirovecii infections. There were no significant differences in the infectious complications and neutropenia between the RTX group and the non-RTX group in patients with high EV.
ANC = absolute neutrophil count, NF = neutropenic fever, RTX = rituximab.
DISCUSSION:
During the last 15 years, among the almost 200 pediatric kidney transplantation recipients at our center, seven developed EBV-associated PTLD (3.5%). In our study, the risk factor for PTLD in pediatric kidney transplant recipients with EV was a high tacrolimus level before EV. Twenty (43.4%) out of 46 recipients who had viremia were EBV seronegative, which is similar to the rates reported in previous studies in North America and Europe (19%–57%).212223 Pre-transplant recipient EBV seronegativity is a well-known risk factor for PTLD. In adult transplant studies, the rate of developing PTLD is 5–12 fold higher in EBV-seronegative patients than in EBV-seropositive patients.2324 McDonald et al.6 reported that EBV-seronegative pediatric subjects have a 4.7-fold higher relative HR than EBV-positive subjects. In our study, recipient EBV seronegativity did not increase the risk of PTLD in multivariate Cox regression analysis. While transplant from an EBV-seropositive donor to a seronegative recipient has been associated with the development of PTLD,25 there was no statistically significant difference in EBV serostatus (donor/recipient) in the present study. This finding was attributed mainly to the fact that there were no EBV seropositive recipients in PTLD group and we did not have serostatus information for the majority of donors (67.3%).
The majority of kidney allograft recipients in this study were given tacrolimus, and mycophenolate was used in more than 80% of patients instead of azathioprine.26 By the late 2000s, monoclonal interleukin-2 receptor antibodies were used as induction therapy in up to 80% of patients. Basiliximab, a monoclonal antibody which targets activated T lymphocytes, was not related to PTLD risk in our study, as previously reported.427 Several studies have suggested that higher tacrolimus levels are associated with higher risk for PTLD, and others have reported that the net state of immunosuppression, rather than any individual agent, increases the risk for PTLD.282930 In our study, all patients received tacrolimus for maintenance immunosuppression, and we found that a higher pre-EV tacrolimus level in the PTLD group compared with the EV only group was a risk factor for PTLD.
Regular monitoring of EBV viral load and early recognition of recipients at high risk of PTLD have been identified as clinical priorities in recent years.31 Previous studies have shown that elevated levels of EBV DNA and persistent high EBV loads are risk factors for PTLD,122032 but no clear cut-off point of EBV viral load for the prediction of PTLD development has been determined. We did not find a significant relationship between EV and PTLD in this study; however, six patients with a high EBV titer were treated with preemptive RTX and they did not develop PTLD. Because they were included in analysis, this could have confounded the causality of high EBV titer and PTLD development.
Treatment strategies for organ transplant recipients with EV include reduction of immunosuppression with/without antiviral agents, immunoglobulin, or RTX.33 These treatments are still undergoing clinical studies. Preemptive administration of RTX is widely used and has been demonstrated to reduce the incidence of PTLD in stem cell transplant recipients with a high EBV viral load.183435 Pre-emptive RTX therapy has been reported in 14.5% of global transplant programs, and in more than 60% of pediatric transplant patients worldwide.33 However, only one study reported the use of RTX in five pediatric renal allograft recipients,20 and there have been no prospective studies on the efficacy of pre-emptive RTX therapy in solid organ transplantation. Rituximab is a murine/human chimeric monoclonal anti-CD20 antibody and is able to deplete circulating B cells rapidly, including those infected with EBV. Although RTX was effective for reducing EBV viral load in our patients, this reduction was not permanent in line with previous reports.20 In addition, significant adverse effects, such as infection and neutropenia, accompanied RTX administration. In patients with stem cell transplant (SCT), preemptive RTX was not associated with an increase in infectious complications.1819 This is important because, while SCT recipients are able to discontinue their immunosuppressive agents within 6–9 months after SCT, recipients of solid organ transplantation have to be on immunosuppressive agents for as long as their allografts are functioning. Therefore, the long-term use of immunosuppressive agents may explain why infection and neutropenia are common clinical findings after RTX therapy in this patient population.
Although the small sample size of the current study precludes us from drawing any definite conclusions, our observations suggest that preemptive RTX treatment may effectively reduce high EBV viral load in pediatric recipients of solid organ transplants. Because RTX therapy eradicates B-lymphocytes including transformed lymphocytes, the risk of PTLD might be reduced at least during the period of B cell depletion. However, one should take into account that this treatment significantly increases the risk of neutropenia and infection. More research into the influence of preemptive RTX therapy on PTLD development is needed.
The occurrence of PTLD in kidney transplant recipients follows a bimodal distribution, with one peak in the first year and the second in the later post-transplantation period. Early PTLD, occurring within the first year of transplantation, is associated with EBV infection and tends to occur more commonly in children than adults.123637 In this study, while six patients in the PTLD group were diagnosed with PTLD within the first year of renal transplantation, one patient with WT1 mutation developed PTLD later than 8.2 years after renal transplantation. We suspect that in this patient, the WT1 mutation of a tumor suppressor gene might have increased the risk of PTLD, and especially that of late-onset. The occurrence of PTLD in patients with WT1 mutation has been reported previously, within the first year in two cases and later than the first year in another.383940 Based on the limited availability of clinical data, the association between WT1 mutation and development of PTLD requires further investigation in future clinical studies and is beyond the scope of the current study.
There are several limitations of this study. First, this is a retrospective observational study of a single center and the number of the patients observed was therefore small. The limitations of this small sample size might have affected the outcome of multivariate analysis. In addition, data of donor EBV serology were not uniformly available in our study. Data on donor serology status was only available for 33% of participants. Therefore, our study did not show any association between PTLD and EBV-donor/recipient serostatus. Finally, the number of patients who were treated with RTX was not large enough to draw any definitive conclusions.
In summary, this study demonstrates that a higher tacrolimus level before EV is correlated with the development of PTLD. Preemptive RTX appears to be effective for reducing EBV viral load in pediatric kidney transplant recipients. However, the reduction of EBV viral load was not persistent, and adverse effects of RTX, namely infection and neutropenia, were clinically significant.
|
Background:
Post-transplant lymphoproliferative disease (PTLD) is one of the major complications of organ transplantation, especially in children with Epstein-Barr virus (EBV) viremia (EV). We performed a retrospective study to evaluate risk factors for PTLD in children with EV.
Methods:
Among 199 pediatric kidney transplantation (KT) recipients at our center from January 2001 to October 2015, records of those with EBV viral loads of > 1,000 copies/mL and/or PTLD were reviewed.
Results:
Diagnosis of PTLD was made in seven patients (PTLD group), and 39 patients had EV only (EV only group). The median time from KT to EV and PTLD diagnosis was 6.7 (range 0.4-47.8) months and 8.2 (range, 2.8-98.9) months, respectively. There were no significant differences between the groups in terms of sex, age at transplantation, donor type, EBV viral load, or EV-free duration after KT. Higher tacrolimus level before EV (hazard ratio, 44.5; P = 0.003) was an independent risk factor for PTLD in multivariate Cox regression analysis. Six patients with a high EBV load (median 171,639 copies/mL) were treated with preemptive rituximab (RTX) therapy, resulting in transient reduction of EBV load. None of these patients developed PTLD (median follow-up 51.5 months); however, two had neutropenia and two developed infection requiring hospital admission.
Conclusions:
In pediatric KT recipients, higher tacrolimus levels were associated with a higher incidence of PTLD. Conversely, those who received preemptive RTX for EV did not develop PTLD.
| null | null | 8,529 | 310 | 13 |
[
"ebv",
"ptld",
"patients",
"rtx",
"ev",
"transplantation",
"months",
"kidney",
"therapy",
"ml"
] |
[
"test",
"test"
] | null | null |
[CONTENT] Post-Transplant Lymphoproliferative Disease | Kidney Transplantation | Epstein-Barr Virus | Rituximab [SUMMARY]
|
[CONTENT] Post-Transplant Lymphoproliferative Disease | Kidney Transplantation | Epstein-Barr Virus | Rituximab [SUMMARY]
|
[CONTENT] Post-Transplant Lymphoproliferative Disease | Kidney Transplantation | Epstein-Barr Virus | Rituximab [SUMMARY]
| null |
[CONTENT] Post-Transplant Lymphoproliferative Disease | Kidney Transplantation | Epstein-Barr Virus | Rituximab [SUMMARY]
| null |
[CONTENT] Antineoplastic Agents, Immunological | Child, Preschool | Female | Herpesvirus 4, Human | Humans | Infant | Kidney Transplantation | Lymphoproliferative Disorders | Male | Neutropenia | Proportional Hazards Models | Retrospective Studies | Risk Factors | Rituximab | Transplantation, Homologous | Viral Load | Viremia [SUMMARY]
|
[CONTENT] Antineoplastic Agents, Immunological | Child, Preschool | Female | Herpesvirus 4, Human | Humans | Infant | Kidney Transplantation | Lymphoproliferative Disorders | Male | Neutropenia | Proportional Hazards Models | Retrospective Studies | Risk Factors | Rituximab | Transplantation, Homologous | Viral Load | Viremia [SUMMARY]
|
[CONTENT] Antineoplastic Agents, Immunological | Child, Preschool | Female | Herpesvirus 4, Human | Humans | Infant | Kidney Transplantation | Lymphoproliferative Disorders | Male | Neutropenia | Proportional Hazards Models | Retrospective Studies | Risk Factors | Rituximab | Transplantation, Homologous | Viral Load | Viremia [SUMMARY]
| null |
[CONTENT] Antineoplastic Agents, Immunological | Child, Preschool | Female | Herpesvirus 4, Human | Humans | Infant | Kidney Transplantation | Lymphoproliferative Disorders | Male | Neutropenia | Proportional Hazards Models | Retrospective Studies | Risk Factors | Rituximab | Transplantation, Homologous | Viral Load | Viremia [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] ebv | ptld | patients | rtx | ev | transplantation | months | kidney | therapy | ml [SUMMARY]
|
[CONTENT] ebv | ptld | patients | rtx | ev | transplantation | months | kidney | therapy | ml [SUMMARY]
|
[CONTENT] ebv | ptld | patients | rtx | ev | transplantation | months | kidney | therapy | ml [SUMMARY]
| null |
[CONTENT] ebv | ptld | patients | rtx | ev | transplantation | months | kidney | therapy | ml [SUMMARY]
| null |
[CONTENT] ebv | ptld | ebv infection | transplantation | infection | pediatric | allograft | recipients | primary ebv infection | primary ebv [SUMMARY]
|
[CONTENT] test | ebv | transplantation | patients | therapy | months | ev | variables | tacrolimus | ml [SUMMARY]
|
[CONTENT] rtx | ebv | patients | ptld | ev | months | group | median | ml | lymph [SUMMARY]
| null |
[CONTENT] ebv | ptld | patients | rtx | months | ev | transplantation | ml | kidney | therapy [SUMMARY]
| null |
[CONTENT] Epstein-Barr | EBV | EV ||| EV [SUMMARY]
|
[CONTENT] 199 | KT | January 2001 to October 2015 | EBV | 1,000 [SUMMARY]
|
[CONTENT] seven | 39 | EV | EV ||| KT | EV | 6.7 | 0.4-47.8 | months | 8.2 | 2.8 | months ||| EV | KT ||| EV | 44.5 | P = | 0.003 ||| Six | EBV | 171,639 | EBV ||| 51.5 months | two | two [SUMMARY]
| null |
[CONTENT] Epstein-Barr | EBV | EV ||| EV ||| 199 | KT | January 2001 to October 2015 | EBV | 1,000 ||| seven | 39 | EV | EV ||| KT | EV | 6.7 | 0.4-47.8 | months | 8.2 | 2.8 | months ||| EV | KT ||| EV | 44.5 | P = | 0.003 ||| Six | EBV | 171,639 | EBV ||| 51.5 months | two | two ||| KT ||| RTX | EV [SUMMARY]
| null |
||||
Influence of ischemia before vein grafting on early hyperplasia of the graft and the dynamic changes of the intima after grafting.
|
23006637
|
To investigate both the influence of ischemia before grafting on early hyperplasia of the vein grafts, and the dynamic changes of the intima after grafting in a rabbit model of vein graft disease.
|
BACKGROUND
|
We performed paired vein graft experiments under different ischemic conditions (15 vs. 60 min; 15 vs. 90 min) in the neck of the rabbits and compared the differences between the grafts. Clopidogrel, an anti-platelet agent, was administered before and after surgery. Twenty-eight days after the grafting procedure, the veins were evaluated microscopically. The dynamic changes of the intima after grafting were evaluated by scanning electron microscopy over time.
|
METHODS
|
The vein grafts subjected to 60- or 90-min ischemia exhibited no differences compared to those subjected to 15-min ischemia in terms of the mean thickness of the intimal, medial, and adventitial layers of the graft. Similarly, there was no difference in the Ki-67 labeling index (proliferation marker) between the vein grafts. Vein grafts with 15-min ischemia lost endothelial cells (ECs) but healed by 3 days post graft, whereas vein grafts with 90-min ischemia suffered serious EC loss, which was restored with new ECs during days 2 to 14 post graft.
|
RESULTS
|
Ninety-minute ischemia before vein grafting can cause serious EC loss, but does not increase early intimal hyperplasia when clopidogrel is administered. Protecting the vein from ischemia and reperfusion injury preserves ECs.
|
CONCLUSIONS
|
[
"Animals",
"Blood Vessel Prosthesis",
"Blood Vessel Prosthesis Implantation",
"Cell Proliferation",
"Clopidogrel",
"Endothelial Cells",
"Histocytochemistry",
"Hyperplasia",
"Ischemia",
"Ki-67 Antigen",
"Microscopy, Electron, Scanning",
"Platelet Aggregation Inhibitors",
"Rabbits",
"Ticlopidine",
"Tunica Intima"
] |
3495780
|
Background
|
The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis
[1-3].
Endothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting
[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions
[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis
[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation
[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.
|
Methods
|
Animals and grouping New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.
New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.
Vein graft surgery Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.
To avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure
1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).
Surgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).
After 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).
To evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.
To observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.
Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.
To avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure
1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).
Surgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).
After 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).
To evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.
To observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.
Morphometric analysis The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.
The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.
Immunocytochemistry and cell proliferation analysis Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.
In each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.
Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.
In each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.
Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).
Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).
Statistical analysis All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.
All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.
|
Results
|
Graft patency All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.
All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.
Morphometric analysis Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table
1, Figure
2).
Comparison of vein grafts under different ischemic conditions
Values are mean ± standard deviation.
Histological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).
Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table
1, Figure
2).
Comparison of vein grafts under different ischemic conditions
Values are mean ± standard deviation.
Histological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).
Immunocytochemistry and cell proliferation analysis Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure
3), as in the mean thickness results.
The Ki-67 labeling index of the vein grafts.
Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure
3), as in the mean thickness results.
The Ki-67 labeling index of the vein grafts.
Scanning electron microscopy observation of intima SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure
4), likely due to the infusion pressure.
Scanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.
One hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure
5).
Scanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.
SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure
4), likely due to the infusion pressure.
Scanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.
One hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure
5).
Scanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.
|
Conclusions
|
In summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs.
|
[
"Background",
"Animals and grouping",
"Vein graft surgery",
"Morphometric analysis",
"Immunocytochemistry and cell proliferation analysis",
"Scanning electron microscopy",
"Statistical analysis",
"Graft patency",
"Morphometric analysis",
"Immunocytochemistry and cell proliferation analysis",
"Scanning electron microscopy observation of intima",
"Competing interest",
"Authors' contributions"
] |
[
"The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.",
"New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.",
"Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.",
"The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.",
"Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.",
"Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).",
"All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.",
"All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.",
"Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).",
"Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.",
"SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.",
"The authors declare that they have no competing interests.",
"RJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Animals and grouping",
"Vein graft surgery",
"Morphometric analysis",
"Immunocytochemistry and cell proliferation analysis",
"Scanning electron microscopy",
"Statistical analysis",
"Results",
"Graft patency",
"Morphometric analysis",
"Immunocytochemistry and cell proliferation analysis",
"Scanning electron microscopy observation of intima",
"Discussion",
"Conclusions",
"Competing interest",
"Authors' contributions"
] |
[
"The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis\n[1-3].\nEndothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting\n[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions\n[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis\n[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation\n[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.",
" Animals and grouping New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\nNew Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.\n Vein graft surgery Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nAnesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\n Morphometric analysis The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\nThe vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.\n Immunocytochemistry and cell proliferation analysis Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\nCell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.\n Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\nSamples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).\n Statistical analysis All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.\nAll data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.",
"New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.",
"Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.\nTo avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure\n1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).\nSurgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).\nAfter 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).\nTo evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.\nTo observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.",
"The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.",
"Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.\nIn each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.",
"Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).",
"All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.",
" Graft patency All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\nAll grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.\n Morphometric analysis Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\nUsing the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).\n Immunocytochemistry and cell proliferation analysis Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\nMany Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.\n Scanning electron microscopy observation of intima SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.\nSEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.",
"All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.",
"Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table\n1, Figure\n2).\nComparison of vein grafts under different ischemic conditions\nValues are mean ± standard deviation.\nHistological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).",
"Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure\n3), as in the mean thickness results.\nThe Ki-67 labeling index of the vein grafts.",
"SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure\n4), likely due to the infusion pressure.\nScanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.\nOne hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure\n5).\nScanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.",
"Ischemic injury before grafting and reperfusion injury after grafting may injure ECs, and subsequently activate platelets and trigger the coagulation cascade\n[10]. Various solutions and heparinized autologous blood have been used previously for vein preservation to reduce ischemia and reperfusion (IR) injury. For instance, Cavallari et al. used autologous whole blood (AWB), 0.9% normal saline solution (NS), and University of Wisconsin solution (UWs) individually to treat autogenous vein grafts for 45 min at 4°C before grafting, and found the veins stored in UWs had a similar intimal thickness with that of control samples, whereas the veins stored in AWB or NS had a thicker intima layer after grafting\n[11]. However, Solberg et al. found that veins stored in cell culture media at 4°C show a marked increase in EC dysjunction and an 18% loss of EC number, compared to only a 4% loss at 20°C, indicating that hypothermy may injure ECs\n[12]. In typical situations, the veins are placed in saline containing heparin sodium at 20°C after being removed. Therefore, we designed this study to investigate the influence of pre-grafting ischemia on early hyperplasia after grafting under these conditions, and found the ischemic tolerance of the vein. After the step, we planed to investigate how long the veins can be preserved in the solutions (such as UWs) or how long the AWB should be renewed. The cuff technique was used in this study for the vein graft model because it can limit the ischemic time caused by the procedure to 15 min, avoiding IR injury. In one side, the veins were grafted immediately after removal to offer controls, whereas, in the other side, the veins were stored before being used. We also included clopidogrel treatment to reduce the risk of thrombus or mural thrombus and to make the vein graft model more similar to the clinical situation.\nProspective controlled trials have demonstrated a graft patency benefit when aspirin was started 1, 7, or 24 h postoperatively, a benefit which was lost when started after 48 h\n[9]. Aspirin has been shown to significantly reduce postoperative mortality, myocardial infarction, stroke, renal failure, and bowel infarction when administered within 48 h of CABG\n[8]. However, in patients with atherosclerotic vascular disease, clopidogrel is more effective than aspirin in reducing the combined risk of stroke, myocardial infarction, and vascular death\n[13].\nBesides having anti-thrombotic properties, inhibitors of platelet aggregation attenuate platelet function, prevent platelet activation and release of peptide growth factors and nonpeptide substances, and thereby inhibit intimal hyperplasia\n[14]. Atherosclerosis is described as an inflammatory disease\n[15]. Clopidogrel inhibits the expression of platelet activation markers and the interaction of platelets and leukocytes\n[16], and is powerful in decreasing the levels of inflammatory factors\n[17].\nIn our present study, SEM revealed that 90-min ischemia caused serious injury of the intima, including the exposed subendothelial collagen fibers that may aggregate and activate platelets and the frequently adherent leukocytes, which may trigger vascular inflammation. Clopidogrel was used and avoided thrombus or mural thrombus successfully. Moreover, results show no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia in term of thickness and the hyperplasia intensity of the intima, media, or adventitia at 4 weeks after the grafting procedure. It seems ischemia up to 90 min does not increase early intimal hyperplasia of the vein graft when clopidogrel was used effectively. Approximately 90 min is a long enough time period for surgeons to complete 3 or 4 bypasses; therefore, when using clopidogrel, it is seemingly unnecessary to consider the influence of ischemia before grafting on early hyperplasia of the vein grafts.\nThe recovery of the intima was also observed post operation. EC coverage has been used in some studies\n[18,19], which is easily gained and facilitates statistical analysis. However, based on our study, it is apparently unreliable because fibrin and micro thrombi covered the surface of the intima when there was IR injury according to SEM analysis. Therefore, we only reported the characteristics of the intima in this study.\nAccording to our observations, the intimae of vein grafts with 15-min ischemia only lost a portion of their ECs and reintegrated at 3 days post operation. However, the intimae of vein grafts with 90-min ischemia lost all their ECs and redintegrated at 14 days post operation. Furthermore, the anterior had less risk of thrombosis. Reducing ischemia and reperfusion injury is valuable. It has been suggested previously that UWs preserves the vein before grafting because of its excellent effect on protecting the endothelium and its functions\n[20].\nHowever, vein grafts with 15-min ischemia, which had almost no IR injury, likely face a widely torn intima caused by systolic pressure 1 h after grafting. Furthermore, the vein grafts with 90-min ischemia likely face more serious loss of ECs until new ECs are present, which come predominantly from extrinsic cells such as bone marrow-derived cells\n[21], and form an intima layer with integrity. Thus, it seems antithrombotic therapy should be administered as early as possible post operation.\nGavaghan et al. found that administering aspirin within 1 h of CABG did not increase chest-tube blood loss, the red cell transfusion requirements, or the re-exploration rate\n[22]. However, Goldman et al. reported that aspirin use on the day before CABG has a similar effect on early graft patency within 6 h after surgery, and increased bleeding complications\n[23]. Considering that the number of platelets and the levels of blood coagulation factors vary and change with time after CABG, we recommend starting anti-platelet therapy when the postoperative chest tube drainage is ≤50 ml/h for 2 consecutive hours, as presented by both Halkos et al. and Chan et al.\n[24,25].",
"In summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs.",
"The authors declare that they have no competing interests.",
"RJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript."
] |
[
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"methods",
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
"conclusions",
null,
null
] |
[
"Vein graft",
"Ischemia",
"Intimal hyperplasia",
"Endothelial cell"
] |
Background:
The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis
[1-3].
Endothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting
[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions
[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis
[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation
[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.
Methods:
Animals and grouping New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.
New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.
Vein graft surgery Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.
To avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure
1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).
Surgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).
After 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).
To evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.
To observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.
Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.
To avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure
1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).
Surgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).
After 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).
To evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.
To observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.
Morphometric analysis The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.
The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.
Immunocytochemistry and cell proliferation analysis Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.
In each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.
Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.
In each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.
Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).
Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).
Statistical analysis All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.
All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.
Animals and grouping:
New Zealand white rabbits weighed between 2.5 and 3.0 kg were raised and provided by the Laboratory Animal Center of Shanghai No. 6 People's Hospital. The protocol for animal experiments was approved by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine, based on the Guidelines for Animal Experiments. The rabbits were randomly divided into two groups: (1) 15- vs. 60-min ischemia and (2) 15- vs. 90-min ischemia.
Vein graft surgery:
Anesthesia was induced in the rabbits with intravenous ethaminal sodium (15 to 30 mg/kg, depending on the response of the rabbit to the drug), allowing spontaneous ventilation throughout the procedure. In addition, heparin sodium (250 U/kg) and penicillin (400 kU) were administered intravenously before creating a skin incision. In each animal, a longitudinal incision was made in the neck over the region of the internal jugular vein. The internal jugular vein and the common carotid artery were dissected using the “no touch” technique, and the side branches were ligated with 5–0 silk sutures.
To avoid the operating difficulty caused by the spasm of vein, a intravenous remained trocar of 22 G was inserted into the distal internal jugular vein and secured in place with a ligature (Figure
1). A 2-cm portion of the internal jugular vein including the trocar was removed and rinsed with saline, then placed in saline containing heparin sodium (62.5 U/ml, 20°C) for 45 (group 1) or 75 min (group 2). A 1-cm segment of the common carotid artery between two vascular clamps was also removed. Polyvinyl chloride cuffs with 1-mm inner diameter were fixed to each end of the artery, around which the artery was everted and ligated. Subsequently, the vein with little protection in saline was sleeved over the cuffs and ligated. When the entire ischemic time was 60 (group 1) or 90 min (group 2), the vascular clamps were removed, pulsations and turbulent blood flow within the vein indicated successful grafting. On the contralateral side, a similar procedure was performed except the vein was sleeved immediately after being removed, and the entire ischemic time was 15 min. All animals received bilateral grafts to establish a paired comparison in the two groups consisting of 6 rabbits each (15- vs. 60-min ischemia; 15- vs. 90-min ischemia). Postoperatively, the rabbits were housed individually at 20°C and fed a normal diet with free access to water. Clopidogrel was administered to each rabbit the day before the surgery (6 mg/kg) and daily after surgery for 4 weeks (3 mg/kg).
Surgery. The vein was punctured with a intravenous remained trocar of 22G and was ligated on it (left image). Subsequently, the vein and trocar were removed. The infusing process was very simple and unproblematic, the vein did not spasm, and the procedure was carried out quickly (right image).
After 4 weeks, the grafts, each including a 0.5-cm segment of the proximal and distal common carotid artery, were harvested from the rabbits under anesthesia. The grafts were rinsed in saline and the vessel lumen was infused with 10% formalin at a pressure of 100 mmHg, simulating the systolic pressure for 12 h. Each of the grafts was divided into three equal parts. Each part was dehydrated, cleared, and paraffin-embedded. Approximately 5-μm transverse sections were taken from three points in each graft, mounted onto glass slides, and stained with hematoxylin/eosin (H/E) and Ponceau Red/Victoria Blue (P/VB).
To evaluate the influence of the infusion pressure on the intima by scanning electron microscopy (SEM), two sides of the ungrafted internal jugular veins were removed from 2 rabbits. After being rinsed in saline, the veins of one side were preserved in 2.5% glutaraldehyde (SIGMA, St. Louis, MO) for 12 h, and the veins of the other side were infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. All veins were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.
To observe dynamic changes in the intima by SEM, an additional 15- vs. 90-min ischemia group was also studied. Grafts were harvested at 1 h, and 1, 2, 3, 7, 14, and 28 days after grafting (2 rabbits at each time point). These grafts were rinsed in saline and infused intraluminally with 2.5% glutaraldehyde at a pressure of 100 mmHg for 12 h. Subsequently, the grafts were bisected and secured on a cork mounting board with 8–0 polypropylene sutures.
Morphometric analysis:
The vessel wall dimensions were captured under an LW200T light microscope (Shanghai Cewei Photoelectric Technology Co. Ltd., Shanghai, China) with a color video camera head (DH-HV3103UCUSB; Daheng Group, Inc., Beijing, China) and measured with YRMV image-analysis software (Shanghai Yinrui Information Technology Co. Ltd., Shanghai, China). The mean intimal, medial, and adventitial thickness measurements were derived by measuring the areas and perimeters of the borders between the intimal and medial sections of each graft.
Immunocytochemistry and cell proliferation analysis:
Cell proliferation was detected with immunohistochemistry using Ki-67 via a 3-step staining procedure. Briefly, 5-μm-thick sections were cut from all paraffin-embedded tissue samples, placed onto slides, dewaxed, and hydrated in graded alcohols. Microwave pretreatment with BD Retrievagen A (pH 6.5) (BD Biosciences, San Diego, CA) for 10 min was performed, and slides were left to cool for 30 min before being rinsed in 0.05 mol/L Tris-buffered saline (TBS). Sections were then incubated with rabbit serum for 30 min, followed by a 1-h incubation with mouse monoclonal antibody against Ki-67 (B56; BD Biosciences). Subsequently, sections were treated with a secondary biotinylated goat anti-mouse antibody (DAKO, Glostrup, Denmark) for 10 min. TBS was used as a washing buffer between the antibody incubation steps. The samples were then incubated with streptavidin-HRP (DAKO, Glostrup, Demmark) for 10 min and 0.05% 3,3-diaminobenzidine (DAB) for 10 min. A light counter stain with hematoxylin (30 sec) was applied to permit visualization of morphology.
In each tissue section, the total number of cells and the number of Ki-67-positive cells were counted in the intima, media, and adventitia in 6 microscopic fields of view with a 40× objective. The labeling index was calculated as the percentage of positive nuclei.
Scanning electron microscopy:
Samples were fixed in 2.5% glutaraldehyde, rinsed in 0.1 M phosphate buffer, fixed in 1% osmic acid (4°C for 2 h), and dehydrated through a graded series of ethanol (50% to 100%) and amyl acetate. The samples were critical point-dried (HCP-2; Hitachi, Tokyo, Japan), sputter coated with a thin layer of gold (IB-3; Eiko Engineering, Ibataki, Japan), and analyzed with SEM (S-520; Hitachi).
Statistical analysis:
All data were analyzed using SPSS 12.0 for Windows, and are expressed as the mean ± standard deviation (SD). A comparison between veins with different ischemic times in each group was performed using the paired Student’s t-test. P-values less than 0.05 were considered statistically significant.
Results:
Graft patency All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.
All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.
Morphometric analysis Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table
1, Figure
2).
Comparison of vein grafts under different ischemic conditions
Values are mean ± standard deviation.
Histological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).
Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table
1, Figure
2).
Comparison of vein grafts under different ischemic conditions
Values are mean ± standard deviation.
Histological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).
Immunocytochemistry and cell proliferation analysis Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure
3), as in the mean thickness results.
The Ki-67 labeling index of the vein grafts.
Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure
3), as in the mean thickness results.
The Ki-67 labeling index of the vein grafts.
Scanning electron microscopy observation of intima SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure
4), likely due to the infusion pressure.
Scanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.
One hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure
5).
Scanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.
SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure
4), likely due to the infusion pressure.
Scanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.
One hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure
5).
Scanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.
Graft patency:
All grafts remained patent after operation, and no cases of grossly visible thrombus or mural thrombus occurred. The grafts were adherent to surrounding tissues, and some regions were seemingly well incorporated.
Morphometric analysis:
Using the mean thickness of the 3 layers to compare graft hyperplasia, we found no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia at 4 weeks after the procedure (Table
1, Figure
2).
Comparison of vein grafts under different ischemic conditions
Values are mean ± standard deviation.
Histological appearance of the grafts (stained with P/VB; magnification ×3) 4 weeks after the grafting procedure.A: vein grafts with 15-min ischemia (group 1); B: vein grafts with 60-min ischemia (group 1); C: vein grafts with 15-min ischemia (group 2); D: vein grafts with 90-min ischemia (group 2).
Immunocytochemistry and cell proliferation analysis:
Many Ki-67-positive cells were found in the layers of the vein grafts, especially in the adventitia. There were no significant differences between the vein grafts that underwent different ischemic times in terms of the labeling index (Figure
3), as in the mean thickness results.
The Ki-67 labeling index of the vein grafts.
Scanning electron microscopy observation of intima:
SEM revealed that the intima of the ungrafted vein maintained its integrity before infusion but was notably torn after infusion (Figure
4), likely due to the infusion pressure.
Scanning electron micrographs of the intima surface of an ungrafted vein (original magnification ×400).A: uninfused vein; B: infused vein.
One hour after grafting, changes in the vein with 15-min ischemia included a widely torn intima with no loss of ECs. In the vein with 90-min ischemia, however, all ECs were lost, though the sub endothelial collagen fibers were not exposed. After 24 h, the intima of vein with 15-min ischemia showed little change, while in the vein with 90-min ischemia collagen fibers were thoroughly exposed. After 2 days, the vein with 15-min ischemia lost a portion of its ECs, which were disorderly arranged. In addition, the vein with 90-min ischemia began to show signs of restoration, with the collagen fibers covered by new ECs. The surface also showed infiltration of white blood cells (WBCs) and red blood cells (RBCs). Three days after grafting, the intima of vein with 15-min ischemia regained integrity, the ECs arranged orderly, whereas the intima of vein with 90-min ischemia had adherent surface fibrin and micro thrombi. After 7 days, the intima integrity of the vein with 15-min ischemia persisted and included folds, likely caused by hyperplasia. Similarly, the intima of the vein with 90-min ischemia also had folds, in addition to adherent fibrin and micro thrombi. After 14 days, both intimae showed integrity, including orderly arranged ECs and no fibrin adherence, WBCs, or RBCs. Twenty-eight days after grafting, both intimae with crowded ECs presented a “cobblestone” appearance (Figure
5).
Scanning electron micrographs of the intima surface of grafted veins (original magnification ×400) after 1 h and 1, 2, 3, 7, 14, or 28 days.A: vein grafts with 15-min ischemia; B: vein grafts with 90-min ischemia.
Discussion:
Ischemic injury before grafting and reperfusion injury after grafting may injure ECs, and subsequently activate platelets and trigger the coagulation cascade
[10]. Various solutions and heparinized autologous blood have been used previously for vein preservation to reduce ischemia and reperfusion (IR) injury. For instance, Cavallari et al. used autologous whole blood (AWB), 0.9% normal saline solution (NS), and University of Wisconsin solution (UWs) individually to treat autogenous vein grafts for 45 min at 4°C before grafting, and found the veins stored in UWs had a similar intimal thickness with that of control samples, whereas the veins stored in AWB or NS had a thicker intima layer after grafting
[11]. However, Solberg et al. found that veins stored in cell culture media at 4°C show a marked increase in EC dysjunction and an 18% loss of EC number, compared to only a 4% loss at 20°C, indicating that hypothermy may injure ECs
[12]. In typical situations, the veins are placed in saline containing heparin sodium at 20°C after being removed. Therefore, we designed this study to investigate the influence of pre-grafting ischemia on early hyperplasia after grafting under these conditions, and found the ischemic tolerance of the vein. After the step, we planed to investigate how long the veins can be preserved in the solutions (such as UWs) or how long the AWB should be renewed. The cuff technique was used in this study for the vein graft model because it can limit the ischemic time caused by the procedure to 15 min, avoiding IR injury. In one side, the veins were grafted immediately after removal to offer controls, whereas, in the other side, the veins were stored before being used. We also included clopidogrel treatment to reduce the risk of thrombus or mural thrombus and to make the vein graft model more similar to the clinical situation.
Prospective controlled trials have demonstrated a graft patency benefit when aspirin was started 1, 7, or 24 h postoperatively, a benefit which was lost when started after 48 h
[9]. Aspirin has been shown to significantly reduce postoperative mortality, myocardial infarction, stroke, renal failure, and bowel infarction when administered within 48 h of CABG
[8]. However, in patients with atherosclerotic vascular disease, clopidogrel is more effective than aspirin in reducing the combined risk of stroke, myocardial infarction, and vascular death
[13].
Besides having anti-thrombotic properties, inhibitors of platelet aggregation attenuate platelet function, prevent platelet activation and release of peptide growth factors and nonpeptide substances, and thereby inhibit intimal hyperplasia
[14]. Atherosclerosis is described as an inflammatory disease
[15]. Clopidogrel inhibits the expression of platelet activation markers and the interaction of platelets and leukocytes
[16], and is powerful in decreasing the levels of inflammatory factors
[17].
In our present study, SEM revealed that 90-min ischemia caused serious injury of the intima, including the exposed subendothelial collagen fibers that may aggregate and activate platelets and the frequently adherent leukocytes, which may trigger vascular inflammation. Clopidogrel was used and avoided thrombus or mural thrombus successfully. Moreover, results show no differences between vein grafts with 15- and 60-min ischemia or 15- and 90-min ischemia in term of thickness and the hyperplasia intensity of the intima, media, or adventitia at 4 weeks after the grafting procedure. It seems ischemia up to 90 min does not increase early intimal hyperplasia of the vein graft when clopidogrel was used effectively. Approximately 90 min is a long enough time period for surgeons to complete 3 or 4 bypasses; therefore, when using clopidogrel, it is seemingly unnecessary to consider the influence of ischemia before grafting on early hyperplasia of the vein grafts.
The recovery of the intima was also observed post operation. EC coverage has been used in some studies
[18,19], which is easily gained and facilitates statistical analysis. However, based on our study, it is apparently unreliable because fibrin and micro thrombi covered the surface of the intima when there was IR injury according to SEM analysis. Therefore, we only reported the characteristics of the intima in this study.
According to our observations, the intimae of vein grafts with 15-min ischemia only lost a portion of their ECs and reintegrated at 3 days post operation. However, the intimae of vein grafts with 90-min ischemia lost all their ECs and redintegrated at 14 days post operation. Furthermore, the anterior had less risk of thrombosis. Reducing ischemia and reperfusion injury is valuable. It has been suggested previously that UWs preserves the vein before grafting because of its excellent effect on protecting the endothelium and its functions
[20].
However, vein grafts with 15-min ischemia, which had almost no IR injury, likely face a widely torn intima caused by systolic pressure 1 h after grafting. Furthermore, the vein grafts with 90-min ischemia likely face more serious loss of ECs until new ECs are present, which come predominantly from extrinsic cells such as bone marrow-derived cells
[21], and form an intima layer with integrity. Thus, it seems antithrombotic therapy should be administered as early as possible post operation.
Gavaghan et al. found that administering aspirin within 1 h of CABG did not increase chest-tube blood loss, the red cell transfusion requirements, or the re-exploration rate
[22]. However, Goldman et al. reported that aspirin use on the day before CABG has a similar effect on early graft patency within 6 h after surgery, and increased bleeding complications
[23]. Considering that the number of platelets and the levels of blood coagulation factors vary and change with time after CABG, we recommend starting anti-platelet therapy when the postoperative chest tube drainage is ≤50 ml/h for 2 consecutive hours, as presented by both Halkos et al. and Chan et al.
[24,25].
Conclusions:
In summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs.
Competing interest:
The authors declare that they have no competing interests.
Authors' contributions:
RJZ carried out the whole study and drafted the manuscript. MJS: carried out the morphometric analysis, immunocytochemistry and cell proliferation analysis, scanning electron microscopy analysis. ZQL: designed and supported the study, revised the manuscript. QKG: carried out the surgery. All authors read and approved the final manuscript.
|
Background:
To investigate both the influence of ischemia before grafting on early hyperplasia of the vein grafts, and the dynamic changes of the intima after grafting in a rabbit model of vein graft disease.
Methods:
We performed paired vein graft experiments under different ischemic conditions (15 vs. 60 min; 15 vs. 90 min) in the neck of the rabbits and compared the differences between the grafts. Clopidogrel, an anti-platelet agent, was administered before and after surgery. Twenty-eight days after the grafting procedure, the veins were evaluated microscopically. The dynamic changes of the intima after grafting were evaluated by scanning electron microscopy over time.
Results:
The vein grafts subjected to 60- or 90-min ischemia exhibited no differences compared to those subjected to 15-min ischemia in terms of the mean thickness of the intimal, medial, and adventitial layers of the graft. Similarly, there was no difference in the Ki-67 labeling index (proliferation marker) between the vein grafts. Vein grafts with 15-min ischemia lost endothelial cells (ECs) but healed by 3 days post graft, whereas vein grafts with 90-min ischemia suffered serious EC loss, which was restored with new ECs during days 2 to 14 post graft.
Conclusions:
Ninety-minute ischemia before vein grafting can cause serious EC loss, but does not increase early intimal hyperplasia when clopidogrel is administered. Protecting the vein from ischemia and reperfusion injury preserves ECs.
|
Background:
The autologous saphenous vein is the most common conduit for coronary artery bypass grafting (CABG), despite of increased use of arterial grafts in cardiac surgery. After grafting, this vein is subjected to immediate increases in flow, resulting in longitudinal wall shear stress, circumferential deformation, and pulsatile stress. This can cause intimal hyperplasia and progressive thickening of the vein graft wall to occur. Approximately 60% of vein grafts remain patent long term, of which only 50% are free of significant stenosis
[1-3].
Endothelial cell (EC) injury plays a significant role in acute thrombosis after vein grafting
[4]. Heparinized autologous blood and other solutions are typically used before grafting to protect the endothelium and its functions
[5-7]. Another method for reducing acute thrombosis after grafting is the use of anti-platelet drugs to reduce the risk of thrombosis
[8]. Such drugs have been demonstrated to improve graft patency and have been used routinely post operation
[9]. When the risk of acute of thrombosis is reduced by such means, influence of ischemia before vein grafting on early hyperplasia of the graft can be studied to find out the ischemic time not increasing the hyperplasia under different preservation. In this study, we designed a series of paired trials to evaluate the effects of different ischemic times on hyperplasia associated with vein grafts when clopidogrel, an anti-platelet agent, was used.
Conclusions:
In summary, our study indicates that, when clopidogrel is administered effectively, ischemia up to 90 min does not increase early intimal hyperplasia of the vein grafts, although a long-term study is necessary for information on later time points after operation. Furthermore, IR injury can cause serious EC loss, avoiding the injury preserves ECs.
|
Background:
To investigate both the influence of ischemia before grafting on early hyperplasia of the vein grafts, and the dynamic changes of the intima after grafting in a rabbit model of vein graft disease.
Methods:
We performed paired vein graft experiments under different ischemic conditions (15 vs. 60 min; 15 vs. 90 min) in the neck of the rabbits and compared the differences between the grafts. Clopidogrel, an anti-platelet agent, was administered before and after surgery. Twenty-eight days after the grafting procedure, the veins were evaluated microscopically. The dynamic changes of the intima after grafting were evaluated by scanning electron microscopy over time.
Results:
The vein grafts subjected to 60- or 90-min ischemia exhibited no differences compared to those subjected to 15-min ischemia in terms of the mean thickness of the intimal, medial, and adventitial layers of the graft. Similarly, there was no difference in the Ki-67 labeling index (proliferation marker) between the vein grafts. Vein grafts with 15-min ischemia lost endothelial cells (ECs) but healed by 3 days post graft, whereas vein grafts with 90-min ischemia suffered serious EC loss, which was restored with new ECs during days 2 to 14 post graft.
Conclusions:
Ninety-minute ischemia before vein grafting can cause serious EC loss, but does not increase early intimal hyperplasia when clopidogrel is administered. Protecting the vein from ischemia and reperfusion injury preserves ECs.
| 7,886 | 278 | 17 |
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[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]
|
[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]
|
[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]
|
[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]
|
[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]
|
[CONTENT] Vein graft | Ischemia | Intimal hyperplasia | Endothelial cell [SUMMARY]
|
[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]
|
[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]
|
[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]
|
[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]
|
[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]
|
[CONTENT] Animals | Blood Vessel Prosthesis | Blood Vessel Prosthesis Implantation | Cell Proliferation | Clopidogrel | Endothelial Cells | Histocytochemistry | Hyperplasia | Ischemia | Ki-67 Antigen | Microscopy, Electron, Scanning | Platelet Aggregation Inhibitors | Rabbits | Ticlopidine | Tunica Intima [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]
|
[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]
|
[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]
|
[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]
|
[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]
|
[CONTENT] vein | min | ischemia | min ischemia | grafts | 15 | intima | vein grafts | 90 | 90 min [SUMMARY]
|
[CONTENT] thrombosis | vein | grafting | acute thrombosis | acute | hyperplasia | drugs | stress | use | risk [SUMMARY]
|
[CONTENT] min | rabbits | vein | saline | removed | shanghai | jugular | internal | internal jugular | rinsed [SUMMARY]
|
[CONTENT] vein | min ischemia | min | ischemia | grafts | vein grafts | intima | 15 min ischemia | 15 | ecs [SUMMARY]
|
[CONTENT] injury | study | study indicates clopidogrel administered | study indicates clopidogrel | cause ec loss avoiding | grafts long term study | grafts long term | grafts long | furthermore ir | loss avoiding [SUMMARY]
|
[CONTENT] vein | min | grafts | ischemia | min ischemia | vein grafts | 15 | 90 | 90 min | intima [SUMMARY]
|
[CONTENT] vein | min | grafts | ischemia | min ischemia | vein grafts | 15 | 90 | 90 min | intima [SUMMARY]
|
[CONTENT] [SUMMARY]
|
[CONTENT] 15 | 60 | 15 | 90 ||| Clopidogrel ||| Twenty-eight days ||| [SUMMARY]
|
[CONTENT] 60- | 90 | 15 ||| ||| 15 | 3 days | 90 | EC | days 2 | 14 [SUMMARY]
|
[CONTENT] Ninety-minute | EC ||| [SUMMARY]
|
[CONTENT] ||| 15 | 60 | 15 | 90 ||| Clopidogrel ||| Twenty-eight days ||| ||| ||| 60- | 90 | 15 ||| ||| 15 | 3 days | 90 | EC | days 2 | 14 ||| Ninety-minute | EC ||| [SUMMARY]
|
[CONTENT] ||| 15 | 60 | 15 | 90 ||| Clopidogrel ||| Twenty-eight days ||| ||| ||| 60- | 90 | 15 ||| ||| 15 | 3 days | 90 | EC | days 2 | 14 ||| Ninety-minute | EC ||| [SUMMARY]
|
||||||
Cognitive awareness of carbohydrate intake does not alter exercise-induced lymphocyte apoptosis.
|
21484033
|
Carbohydrate supplementation during aerobic exercise generally protects against the immunosuppressive effects of exercise. It is not currently known whether carbohydrate consumption or simply the knowledge of carbohydrate consumption also has that effect.
|
INTRODUCTION
|
Endurance trained male and female (N = 10) athletes were randomly assigned to one of two groups based on either a correct or incorrect cognitive awareness of carbohydrate intake. In the incorrect group, the subjects were informed that they were receiving the carbohydrate beverage but actually received the placebo beverage. Participants completed a 60-min ride on a cycle ergometer at 80% VO₂peak under carbohydrate and placebo supplemented conditions. Venous blood samples were collected at rest and immediately after exercise and were used to determine the plasma glucose concentration, lymphocyte count, and extent of lymphocyte apoptosis. Cognitive awareness, either correct or incorrect, did not have an effect on any of the measured variables.
|
METHODS
|
Carbohydrate supplementation during exercise did not have an effect on lymphocyte count or apoptotic index. Independent of drink type, exercise resulted in significant lymphocytosis and lymphocyte apoptosis (apoptotic index at rest = 6.3 ± 3% and apoptotic index following exercise = 11.6 ± 3%, P < 0.01).
|
RESULTS
|
Neither carbohydrate nor placebo supplementation altered the typical lymphocyte apoptotic response following exercise. While carbohydrate supplementation generally has an immune-boosting effect during exercise, it appears that this influence does not extend to the mechanisms that govern exercise-induced lymphocyte cell death.
|
CONCLUSION
|
[
"Adult",
"Analysis of Variance",
"Apoptosis",
"Awareness",
"Beverages",
"Blood Glucose",
"Cognition",
"Dietary Carbohydrates",
"Female",
"Humans",
"Lymphocytes",
"Male",
"Physical Endurance",
"Statistics, Nonparametric",
"Stress, Physiological"
] |
3059873
|
INTRODUCTION
|
Acute exercise has a marked effect on the immune system response. Increased neutrophil, monocyte, and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and short‐term swim exercise.3 Two hours of cycling at 55% VO2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30‐min at 60% VO2max increased lymphocyte proliferation and plasma interleukin‐6 concentration (IL‐6).2 Significant increases in tumor necrosis factor‐α (TNF‐α) have been reported after a 30‐min high‐intensity run at 85% VO2max4 and following 15‐min of moderate‐intensity swimming.5 Therefore, it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors.
While acute exercise significantly increases immune parameters, carbohydrate (CHO) supplementation has been shown to have an immunomodulating effect on many of these responses. CHO supplementation prior to 2.5 h of cycling at 65% VO2max resulted in reduced cell numbers of neutrophils, monocytes, and lymphocytes compared to placebo,6 as well as a significant decrease in plasma IL‐6 concentration.7 Similar reductions in immune cell counts and IL‐6 have been reported in trained individuals who received CHO supplementation during a 4‐h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8
Regarding the lymphocyte response to acute exercise, an increase in cell concentration is observed with the performance of high intensity exercise, followed by a reduction in concentration that is significantly lower than resting levels.9,10 A portion of this post‐exercise decrease is likely due to programmed cell death, or apoptosis. Previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 To our knowledge, only one previous study has assessed the effect that CHO supplementation has on lymphocyte apoptosis. Green et al. reported that CHO supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in CD4 and CD8 cells in culture taken from endurance‐trained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 This finding is consistent with previously cited research that CHO supplementation may reduce the immunoendocrine changes associated with exercise.6-8
A wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress (see review by Black).15 Neuroendocrine responses to stress are mediated by hypothalamic corticotropin‐releasing factor16 which acts on the hypothalamic‐pituitary‐adrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 In addition, there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 Therefore, it is possible that the immune system can be modified not only by nutritional supplementation as discussed above, but also independently by neural and cognitive factors. The influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume, suggesting potential deletion through a cell death mechanism (apoptosis).
While carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo, it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise. It was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise. This would hold true whether participants had a correct or incorrect knowledge of supplementation type. Therefore, the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exercise‐induced lymphocyte apoptotic response, independent of actual carbohydrate intake.
| null | null |
RESULTS
|
Glucose There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.
There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.
Lymphocyte Concentration No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).
No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).
3.3 Lymphocyte Apoptosis Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).
Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).
| null | null |
[
"Participants",
"2.2 Protocol",
"2.3 Statistical Analysis",
"Glucose",
"Lymphocyte Concentration",
"3.3 Lymphocyte Apoptosis",
"Acknowledgements"
] |
[
"Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.",
"Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.",
"Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.",
"There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.",
"No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).",
"Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).",
"The authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation."
] |
[
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"METHODS AND MATERIALS",
"Participants",
"2.2 Protocol",
"2.3 Statistical Analysis",
"RESULTS",
"Glucose",
"Lymphocyte Concentration",
"3.3 Lymphocyte Apoptosis",
"DISCUSSION",
"Acknowledgements"
] |
[
"Acute exercise has a marked effect on the immune system response. Increased neutrophil, monocyte, and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and short‐term swim exercise.3 Two hours of cycling at 55% VO2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30‐min at 60% VO2max increased lymphocyte proliferation and plasma interleukin‐6 concentration (IL‐6).2 Significant increases in tumor necrosis factor‐α (TNF‐α) have been reported after a 30‐min high‐intensity run at 85% VO2max4 and following 15‐min of moderate‐intensity swimming.5 Therefore, it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors.\nWhile acute exercise significantly increases immune parameters, carbohydrate (CHO) supplementation has been shown to have an immunomodulating effect on many of these responses. CHO supplementation prior to 2.5 h of cycling at 65% VO2max resulted in reduced cell numbers of neutrophils, monocytes, and lymphocytes compared to placebo,6 as well as a significant decrease in plasma IL‐6 concentration.7 Similar reductions in immune cell counts and IL‐6 have been reported in trained individuals who received CHO supplementation during a 4‐h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8 \nRegarding the lymphocyte response to acute exercise, an increase in cell concentration is observed with the performance of high intensity exercise, followed by a reduction in concentration that is significantly lower than resting levels.9,10 A portion of this post‐exercise decrease is likely due to programmed cell death, or apoptosis. Previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 To our knowledge, only one previous study has assessed the effect that CHO supplementation has on lymphocyte apoptosis. Green et al. reported that CHO supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in CD4 and CD8 cells in culture taken from endurance‐trained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 This finding is consistent with previously cited research that CHO supplementation may reduce the immunoendocrine changes associated with exercise.6-8 \nA wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress (see review by Black).15 Neuroendocrine responses to stress are mediated by hypothalamic corticotropin‐releasing factor16 which acts on the hypothalamic‐pituitary‐adrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 In addition, there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 Therefore, it is possible that the immune system can be modified not only by nutritional supplementation as discussed above, but also independently by neural and cognitive factors. The influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume, suggesting potential deletion through a cell death mechanism (apoptosis).\nWhile carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo, it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise. It was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise. This would hold true whether participants had a correct or incorrect knowledge of supplementation type. Therefore, the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exercise‐induced lymphocyte apoptotic response, independent of actual carbohydrate intake.",
" Participants Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\nEndurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.\n 2.2 Protocol Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\nPeak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.\n 2.3 Statistical Analysis Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.\nBased on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.",
"Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.",
"Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.\nSubjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.\nWith the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.\nImages of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.",
"Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.",
" Glucose There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\nThere was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.\n Lymphocyte Concentration No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\nNo interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).\n 3.3 Lymphocyte Apoptosis Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).\nCognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).",
"There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.",
"No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).",
"Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).",
"The main finding of this investigation was that cognitive awareness of drink type had no physiological effect on selected immune parameters following high‐intensity aerobic exercise. Individuals who had correct cognitive awareness of consumed supplementation (CHO or PLA) had statistically similar measures for plasma glucose, lymphocyte concentration, and lymphocyte apoptosis compared to individuals who had an incorrect cognitive awareness of the ingested supplement. In addition, we observed no effect of CHO supplementation on either lymphocyte concentration or apoptotic index following 60‐min of cycle exercise at 80% VO2peak.\nCarbohydrate supplementation significantly increased plasma glucose following exercise regardless of correct or incorrect cognitive awareness of drink type. The rise in glucose response has been confirmed by a number of other investigations.14,22-24 We are aware of only one other deception study in which subjects have received an incorrect cognitive awareness of carbohydrate or placebo supplementation prior to exercise.25 As we have shown, cognitive awareness of drink type (whether correct or incorrect) did not have an effect on the blood glucose response. The lack of a difference likely means that stimulating factors facilitating the release of glucose into the bloodstream (i.e. glucagon, epinephrine, etc.) are not affected directly by cognitive awareness during a bout high‐intensity aerobic exercise.\nAn exercise‐induced lymphocytosis was evident following both CHO and PLA supplemented bouts. An increase in lymphocyte concentration following exercise has been reported in numerous investigations.1-3,5-6 Thirty minutes of walking at 60% VO2max increased lymphocytosis by 28% in human subjects2 and 120‐min of cycling at 65% VO2max resulted in a 20% increase.6 Using an animal model, Prestes et al. observed increases in lymphocyte counts of 98% and 165% in male Wistar rats who performed unloaded swimming for 5 and 15‐min, and increases of 252% and 191% in animals who swam for 5 and 15‐min with a 5% load.5 The magnitude of lymphocytosis in the present study was 93% in endurance trained participants who completed 60‐min of cycle ergometry exercise at 80% VO2peak. An explanation for the lymphocytosis observed with exercise of increasing intensity is the greater expression of adhesion/activation molecules which can serve to facilitate movement of lymphocytes from the marginal pools into the circulation.26,27 \nWhile others have reported a significant decrease in lymphocyte count with CHO supplementation compared to PLA,6 our results did not show a difference. As part of an investigation to assess the effect of carbohydrate supplementation following caffeine ingestion, the participants of Walker et al. consumed either caffeine or placebo prior to exercise, and either carbohydrate or placebo during a 2 h cycle ride at 65% VO2max.6 Using only the trials in which placebo was consumed prior to exercise (and excluding the effect of caffeine), it was reported that supplementation with CHO during exercise reduced lymphocyte count by 21% compared with the trial in which placebo was administered during exercise.6 The common explanation for immunomodulation associated with CHO supplementation are effects through hypothalamic‐pituitary‐adrenal (HPA) activation and a muted stress hormone response.28 In the present study, CHO was only ingested prior to exercise and did not result in suppressed lymphocyte cell count compared to PLA. There are two possibilities to explain the differences between the study by Walker et al.6 and our findings. First, our participants completed only a 1h cycle ride compared to 2 h. It is possible that the extended exercise duration differentially affected the immune response through increased HPA activation compared to our subjects who completed only half of the duration. Second, the combination of supplemented glucose during the 2 h cycle exercise by the subjects of Walker et al. could have resulted in a greater prolonged effect toward reducing immune parameters compared to our participants who received a single bolus of carbohydrate prior to exercise, but did not receive supplementation at any other point during their exercise trial. Thus it is possible that blood glucose concentration plays a modulating role on cells of the immune system during exercise. In addition, it is likely that CHO has a different effect depending on the immune cell subset. While an overall decrease in leukocyte numbers have been reported in various CHO supplementation investigations,8,24-25 ingestion appears to affect neutrophils to a greater extent than lymphocytes. Nieman et al. and Sharhag et al. both found carbohydrate supplementation to reduce neutrophil count, but to have no effect on numbers of lymphocytes.8,24 \nNeither correct, nor incorrect, cognitive awareness of drink type altered immunity in terms of lymphocyte apoptosis following exercise. To our knowledge, only one other study has attempted to evaluate the effect of carbohydrate supplementation on exercise‐induced lymphocyte cell death.14 Green et al. used carboxyfluorescein succinimidylester fluorescence to estimate lymphocytes undergoing mitosis or cell death using iterative processes until all cells could be accounted for.14 They reported the estimated cell death rates of T‐cell subsets to be significantly reduced in CHO supplemented trials compared to PLA bouts in six well‐trained male cyclists. In the present study, carbohydrate supplementation did not have an effect on lymphocyte apoptosis. Recently, exercise‐induced lymphocyte apoptosis has been measured using either biochemical markers,4,29-30 or a morphological technique.11-13 In the current investigation, we employed the aforementioned morphological method which we have proposed is potentially more sensitive to cells displaying a wider range of apoptotic characteristics compared to a single apoptotic biomarker. We acknowledge that this method does have drawbacks (i.e. highly subjective, low number of cells that are evaluated per sample, etc.); however the methodology was the gold standard to which the current biomarker techniques were validated against.31 Using a direct evaluation of cells displaying morphological characteristics of apoptosis, we found that carbohydrate supplementation during exercise did not alter exercise‐induced cell death compared to the placebo supplemented trial. While carbohydrate supplementation may alter certain immune parameters, it appears that ingestion has no effect on the mechanisms governing cell death induction during exercise.\nWhile we were unable to ascertain differences in select immune parameters due to cognitive awareness of carbohydrate in the present investigation, our study design could be modified to make detection of such differences more likely. The utilization of a single group cross‐over intervention in which subjects randomly complete the conditions of exercise alone, exercise with carbohydrate supplementation, and exercise in conjunction with placebo, would represent an acceptable experimental design in this regard. It should also be noted that the findings of the present study can only be applied to young and middle‐aged individuals who are highly aerobically trained, and should not be extended to other populations. In this regard, other investigations are warranted.\nIn conclusion, our findings indicate that cognitive awareness (either correct or incorrect, of carbohydrate or placebo) has no effect on blood glucose concentration, lymphocyte concentration, or exercise‐induced lymphocyte apoptosis. While glucose intake itself may reduce the magnitude of various immune responses, knowledge of supplementation and indeed the carbohydrate supplement itself has no effect on altering immunity in terms of lymphocyte cell death following exercise.",
"The authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation."
] |
[
"intro",
"materials|methods",
null,
null,
null,
"results",
null,
null,
null,
"discussion",
null
] |
[
"Programmed cell death",
"High‐intensity aerobic exercise",
"Supplementation",
"Deception investigation",
"Immune system"
] |
INTRODUCTION:
Acute exercise has a marked effect on the immune system response. Increased neutrophil, monocyte, and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and short‐term swim exercise.3 Two hours of cycling at 55% VO2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30‐min at 60% VO2max increased lymphocyte proliferation and plasma interleukin‐6 concentration (IL‐6).2 Significant increases in tumor necrosis factor‐α (TNF‐α) have been reported after a 30‐min high‐intensity run at 85% VO2max4 and following 15‐min of moderate‐intensity swimming.5 Therefore, it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors.
While acute exercise significantly increases immune parameters, carbohydrate (CHO) supplementation has been shown to have an immunomodulating effect on many of these responses. CHO supplementation prior to 2.5 h of cycling at 65% VO2max resulted in reduced cell numbers of neutrophils, monocytes, and lymphocytes compared to placebo,6 as well as a significant decrease in plasma IL‐6 concentration.7 Similar reductions in immune cell counts and IL‐6 have been reported in trained individuals who received CHO supplementation during a 4‐h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8
Regarding the lymphocyte response to acute exercise, an increase in cell concentration is observed with the performance of high intensity exercise, followed by a reduction in concentration that is significantly lower than resting levels.9,10 A portion of this post‐exercise decrease is likely due to programmed cell death, or apoptosis. Previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 To our knowledge, only one previous study has assessed the effect that CHO supplementation has on lymphocyte apoptosis. Green et al. reported that CHO supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in CD4 and CD8 cells in culture taken from endurance‐trained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 This finding is consistent with previously cited research that CHO supplementation may reduce the immunoendocrine changes associated with exercise.6-8
A wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress (see review by Black).15 Neuroendocrine responses to stress are mediated by hypothalamic corticotropin‐releasing factor16 which acts on the hypothalamic‐pituitary‐adrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 In addition, there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 Therefore, it is possible that the immune system can be modified not only by nutritional supplementation as discussed above, but also independently by neural and cognitive factors. The influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume, suggesting potential deletion through a cell death mechanism (apoptosis).
While carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo, it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise. It was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise. This would hold true whether participants had a correct or incorrect knowledge of supplementation type. Therefore, the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exercise‐induced lymphocyte apoptotic response, independent of actual carbohydrate intake.
METHODS AND MATERIALS:
Participants Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.
Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.
2.2 Protocol Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.
Subjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.
With the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.
Images of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.
Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.
Subjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.
With the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.
Images of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.
2.3 Statistical Analysis Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.
Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.
Participants:
Endurance trained males and females (N = 10, female N = 6, male N = 4) were recruited from local running, cycling, and triathlon clubs voluntarily participated in this investigation. In order to meet inclusion criteria, participants were required to be classified in the “excellent” category for his or her age group for peak oxygen consumption (VO2peak), and were not currently taking any medications.20 Subjects' characteristics are presented in table 1. The Committee for the Protection of Human Subjects at the University of Houston reviewed and approved the testing procedures. Prior to testing, participants reported to the University of Houston's Laboratory of Integrated Physiology, where they were asked to read and sign an informed consent form and complete a medical history questionnaire.
2.2 Protocol:
Peak oxygen consumption (VO2peak) was determined using an incremental cycle test: stages 1‐3 were 3‐min in length with 50 watt increases in intensity; subsequent stages were 2‐min duration until volitional fatigue. VO2 was determined through automated analysis of expired respiratory gases (Cardio Coach Plus; Salt Lake City, UT). VO2peak was identified as the highest level of VO2 measured and sustained for at least 30‐sec. Exercise testing and experimental trials were completed on a Velotron Pro electronically‐braked cycle ergometer (RacerMate, Inc.; Seattle, WA) at a self‐selected pedal rate.
Subjects were randomly assigned into a group with correct cognitive awareness of supplementation type (female N = 3, male N = 2), or an incorrect knowledge group (female N = 3, male N = 2). For example in the incorrect knowledge group, subjects were informed they were receiving CHO, but actually received the placebo (PLA) beverage; whereas during the during the CHO trial they actually received PLA. The correct cognitive awareness group was informed of receiving CHO on the CHO supplemented trial, as well as placebo during the PLA trial. Each participant completed two experimental trials in a counterbalanced manner: CHO supplementation trial (250 mL every 15‐min, 6% CHO beverage) and control trial separated by at least seven days of recovery. Both the CHO and PLA drinks were designed and supplied by the Gatorade Sport Science Institute (Barrington, IL). Drinks were similar in composition, except that the PLA drink lacked carbohydrate. Regardless of supplementation type, participants completed a 60‐min ride at ∼80% VO2peak. The environmental conditions (room temperature and 60% humidity) were recorded daily and it was confirmed that there was no significant difference between testing days. All trials were completed between 0600‐0800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24‐h prior to testing. Participants were counseled not to alter their food intake in the days prior to the experimental trial. Food intake was documented by 24‐h food records that were completed by each participant prior to the experimental trial. No significant difference in total calorie and/or macronutrient intake was evident between subjects.
With the exception of the immediate post‐exercise sample, subjects were asked to rest for 20‐min prior to blood collection and sampling in a quiet, temperature controlled room. Venous blood samples were collected from a peripheral arm vein using a multi‐sample butterfly needle into an evacuated tube treated with sodium heparin (BD‐Biosciences; City, State). EDTA treated whole blood was used to determine complete blood counts (CBC), and to measure plasma glucose concentration using a YSI 2300 glucose analyzer (Yellow Springs, MO). Venous blood samples were collected before (PRE) and immediately after exercise (POST) and used to make whole blood films within 10‐min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 Briefly, smears were stained in May‐Grünwald stain (Sigma‐Aldrich, Inc; St. Louis, MO) for 3‐5 min, and then washed in phosphate buffered saline (PBS) for 3 min. Afterward, slides were placed in modified Giemsa stain (Sigma‐Aldrich, Inc) for 2 min, washed in deionized water, and allowed to air dry before evaluation.
Images of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera (MA88 Microscope Digital Camera, C&A Scientific Co., Inc.; Manassas, VA) mounted to a light microscope (CXL Plus, Labomed, India) under the oil objective lens. A single investigator evaluated all images in a blinded fashion. According to our previously described procedures, at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 Upon evaluation, lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane, while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 Cell death due to apoptosis was recorded as the apoptotic index (%), and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted. Slides were counted in duplicate and averaged to give the apoptotic index (AI). If duplicate counts varied by greater than 5%, the third slide was evaluated. The intraclass correlation for a single trained observer using the morphological method to determine exercise‐induced lymphocyte apoptosis in our laboratory is R = 0.96.
2.3 Statistical Analysis:
Based on previous studies from our laboratory,11-13 a large effect size was anticipated. In an effort to be conservative, our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise. The a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise. Post hoc power analysis indicated a statistical power of 92% for the present sample. For power analysis computations, a t‐test for the difference between two dependent means was calculated using G*Power software. Statistical analyses were carried out using SPSS 10.1 (SPSS Inc., Chicago, IL). Data were analyzed using a factorial 2 (cognitive awareness: correct or incorrect) × 2 (supplement: CHO or PLA) × 2 (test: PRE or POST) ANOVA with repeated measures on the second and third variable and significance accepted at P≤0.05. Data were tested for normality using the Shapiro‐Wilk test.
RESULTS:
Glucose There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.
There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.
Lymphocyte Concentration No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).
No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).
3.3 Lymphocyte Apoptosis Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).
Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).
Glucose:
There was a significant supplement x test interaction for blood glucose concentration (P = 0.04, Fig 1). Glucose values following exercise in the CHO supplemented conditions were 52% greater than following the PLA trials. Correct or incorrect cognitive awareness of supplement type did not affect glucose concentration.
Lymphocyte Concentration:
No interactions were evident with regard to lymphocyte counts. The only significant main effect was for test, where after exercise there were 93% more lymphocytes (P<0.001, Fig 2).
3.3 Lymphocyte Apoptosis:
Cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise (P = 0.43). There was a significant main effect of exercise on lymphocyte apoptosis, where after exercise there was an 84% increase in lymphocyte apoptotic index (P<0.01, Fig 3).
DISCUSSION:
The main finding of this investigation was that cognitive awareness of drink type had no physiological effect on selected immune parameters following high‐intensity aerobic exercise. Individuals who had correct cognitive awareness of consumed supplementation (CHO or PLA) had statistically similar measures for plasma glucose, lymphocyte concentration, and lymphocyte apoptosis compared to individuals who had an incorrect cognitive awareness of the ingested supplement. In addition, we observed no effect of CHO supplementation on either lymphocyte concentration or apoptotic index following 60‐min of cycle exercise at 80% VO2peak.
Carbohydrate supplementation significantly increased plasma glucose following exercise regardless of correct or incorrect cognitive awareness of drink type. The rise in glucose response has been confirmed by a number of other investigations.14,22-24 We are aware of only one other deception study in which subjects have received an incorrect cognitive awareness of carbohydrate or placebo supplementation prior to exercise.25 As we have shown, cognitive awareness of drink type (whether correct or incorrect) did not have an effect on the blood glucose response. The lack of a difference likely means that stimulating factors facilitating the release of glucose into the bloodstream (i.e. glucagon, epinephrine, etc.) are not affected directly by cognitive awareness during a bout high‐intensity aerobic exercise.
An exercise‐induced lymphocytosis was evident following both CHO and PLA supplemented bouts. An increase in lymphocyte concentration following exercise has been reported in numerous investigations.1-3,5-6 Thirty minutes of walking at 60% VO2max increased lymphocytosis by 28% in human subjects2 and 120‐min of cycling at 65% VO2max resulted in a 20% increase.6 Using an animal model, Prestes et al. observed increases in lymphocyte counts of 98% and 165% in male Wistar rats who performed unloaded swimming for 5 and 15‐min, and increases of 252% and 191% in animals who swam for 5 and 15‐min with a 5% load.5 The magnitude of lymphocytosis in the present study was 93% in endurance trained participants who completed 60‐min of cycle ergometry exercise at 80% VO2peak. An explanation for the lymphocytosis observed with exercise of increasing intensity is the greater expression of adhesion/activation molecules which can serve to facilitate movement of lymphocytes from the marginal pools into the circulation.26,27
While others have reported a significant decrease in lymphocyte count with CHO supplementation compared to PLA,6 our results did not show a difference. As part of an investigation to assess the effect of carbohydrate supplementation following caffeine ingestion, the participants of Walker et al. consumed either caffeine or placebo prior to exercise, and either carbohydrate or placebo during a 2 h cycle ride at 65% VO2max.6 Using only the trials in which placebo was consumed prior to exercise (and excluding the effect of caffeine), it was reported that supplementation with CHO during exercise reduced lymphocyte count by 21% compared with the trial in which placebo was administered during exercise.6 The common explanation for immunomodulation associated with CHO supplementation are effects through hypothalamic‐pituitary‐adrenal (HPA) activation and a muted stress hormone response.28 In the present study, CHO was only ingested prior to exercise and did not result in suppressed lymphocyte cell count compared to PLA. There are two possibilities to explain the differences between the study by Walker et al.6 and our findings. First, our participants completed only a 1h cycle ride compared to 2 h. It is possible that the extended exercise duration differentially affected the immune response through increased HPA activation compared to our subjects who completed only half of the duration. Second, the combination of supplemented glucose during the 2 h cycle exercise by the subjects of Walker et al. could have resulted in a greater prolonged effect toward reducing immune parameters compared to our participants who received a single bolus of carbohydrate prior to exercise, but did not receive supplementation at any other point during their exercise trial. Thus it is possible that blood glucose concentration plays a modulating role on cells of the immune system during exercise. In addition, it is likely that CHO has a different effect depending on the immune cell subset. While an overall decrease in leukocyte numbers have been reported in various CHO supplementation investigations,8,24-25 ingestion appears to affect neutrophils to a greater extent than lymphocytes. Nieman et al. and Sharhag et al. both found carbohydrate supplementation to reduce neutrophil count, but to have no effect on numbers of lymphocytes.8,24
Neither correct, nor incorrect, cognitive awareness of drink type altered immunity in terms of lymphocyte apoptosis following exercise. To our knowledge, only one other study has attempted to evaluate the effect of carbohydrate supplementation on exercise‐induced lymphocyte cell death.14 Green et al. used carboxyfluorescein succinimidylester fluorescence to estimate lymphocytes undergoing mitosis or cell death using iterative processes until all cells could be accounted for.14 They reported the estimated cell death rates of T‐cell subsets to be significantly reduced in CHO supplemented trials compared to PLA bouts in six well‐trained male cyclists. In the present study, carbohydrate supplementation did not have an effect on lymphocyte apoptosis. Recently, exercise‐induced lymphocyte apoptosis has been measured using either biochemical markers,4,29-30 or a morphological technique.11-13 In the current investigation, we employed the aforementioned morphological method which we have proposed is potentially more sensitive to cells displaying a wider range of apoptotic characteristics compared to a single apoptotic biomarker. We acknowledge that this method does have drawbacks (i.e. highly subjective, low number of cells that are evaluated per sample, etc.); however the methodology was the gold standard to which the current biomarker techniques were validated against.31 Using a direct evaluation of cells displaying morphological characteristics of apoptosis, we found that carbohydrate supplementation during exercise did not alter exercise‐induced cell death compared to the placebo supplemented trial. While carbohydrate supplementation may alter certain immune parameters, it appears that ingestion has no effect on the mechanisms governing cell death induction during exercise.
While we were unable to ascertain differences in select immune parameters due to cognitive awareness of carbohydrate in the present investigation, our study design could be modified to make detection of such differences more likely. The utilization of a single group cross‐over intervention in which subjects randomly complete the conditions of exercise alone, exercise with carbohydrate supplementation, and exercise in conjunction with placebo, would represent an acceptable experimental design in this regard. It should also be noted that the findings of the present study can only be applied to young and middle‐aged individuals who are highly aerobically trained, and should not be extended to other populations. In this regard, other investigations are warranted.
In conclusion, our findings indicate that cognitive awareness (either correct or incorrect, of carbohydrate or placebo) has no effect on blood glucose concentration, lymphocyte concentration, or exercise‐induced lymphocyte apoptosis. While glucose intake itself may reduce the magnitude of various immune responses, knowledge of supplementation and indeed the carbohydrate supplement itself has no effect on altering immunity in terms of lymphocyte cell death following exercise.
Acknowledgements:
The authors would like to recognize Western Kentucky University's Faculty Scholarship Council for support through a Summer Faculty Award. In addition, the authors would like to thank Michael Kueht and Nisa Dadjoo for their technical assistance with the investigation.
|
Background:
Carbohydrate supplementation during aerobic exercise generally protects against the immunosuppressive effects of exercise. It is not currently known whether carbohydrate consumption or simply the knowledge of carbohydrate consumption also has that effect.
Methods:
Endurance trained male and female (N = 10) athletes were randomly assigned to one of two groups based on either a correct or incorrect cognitive awareness of carbohydrate intake. In the incorrect group, the subjects were informed that they were receiving the carbohydrate beverage but actually received the placebo beverage. Participants completed a 60-min ride on a cycle ergometer at 80% VO₂peak under carbohydrate and placebo supplemented conditions. Venous blood samples were collected at rest and immediately after exercise and were used to determine the plasma glucose concentration, lymphocyte count, and extent of lymphocyte apoptosis. Cognitive awareness, either correct or incorrect, did not have an effect on any of the measured variables.
Results:
Carbohydrate supplementation during exercise did not have an effect on lymphocyte count or apoptotic index. Independent of drink type, exercise resulted in significant lymphocytosis and lymphocyte apoptosis (apoptotic index at rest = 6.3 ± 3% and apoptotic index following exercise = 11.6 ± 3%, P < 0.01).
Conclusions:
Neither carbohydrate nor placebo supplementation altered the typical lymphocyte apoptotic response following exercise. While carbohydrate supplementation generally has an immune-boosting effect during exercise, it appears that this influence does not extend to the mechanisms that govern exercise-induced lymphocyte cell death.
| null | null | 5,943 | 278 | 11 |
[
"exercise",
"lymphocyte",
"cho",
"min",
"supplementation",
"effect",
"apoptotic",
"apoptosis",
"cell",
"cognitive"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] Programmed cell death | High‐intensity aerobic exercise | Supplementation | Deception investigation | Immune system [SUMMARY]
| null |
[CONTENT] Programmed cell death | High‐intensity aerobic exercise | Supplementation | Deception investigation | Immune system [SUMMARY]
| null |
[CONTENT] Programmed cell death | High‐intensity aerobic exercise | Supplementation | Deception investigation | Immune system [SUMMARY]
| null |
[CONTENT] Adult | Analysis of Variance | Apoptosis | Awareness | Beverages | Blood Glucose | Cognition | Dietary Carbohydrates | Female | Humans | Lymphocytes | Male | Physical Endurance | Statistics, Nonparametric | Stress, Physiological [SUMMARY]
| null |
[CONTENT] Adult | Analysis of Variance | Apoptosis | Awareness | Beverages | Blood Glucose | Cognition | Dietary Carbohydrates | Female | Humans | Lymphocytes | Male | Physical Endurance | Statistics, Nonparametric | Stress, Physiological [SUMMARY]
| null |
[CONTENT] Adult | Analysis of Variance | Apoptosis | Awareness | Beverages | Blood Glucose | Cognition | Dietary Carbohydrates | Female | Humans | Lymphocytes | Male | Physical Endurance | Statistics, Nonparametric | Stress, Physiological [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] exercise | lymphocyte | cho | min | supplementation | effect | apoptotic | apoptosis | cell | cognitive [SUMMARY]
| null |
[CONTENT] exercise | lymphocyte | cho | min | supplementation | effect | apoptotic | apoptosis | cell | cognitive [SUMMARY]
| null |
[CONTENT] exercise | lymphocyte | cho | min | supplementation | effect | apoptotic | apoptosis | cell | cognitive [SUMMARY]
| null |
[CONTENT] exercise | immune | supplementation | system | carbohydrate | cho supplementation | acute | cell | lymphocyte | effect [SUMMARY]
| null |
[CONTENT] lymphocyte | glucose | exercise | fig | significant main | significant main effect | type affect | main effect | significant | concentration [SUMMARY]
| null |
[CONTENT] exercise | lymphocyte | glucose | effect | cho | fig | supplementation | test | significant | min [SUMMARY]
| null |
[CONTENT] ||| [SUMMARY]
| null |
[CONTENT] ||| 6.3 ± | 3% | 11.6 | 3% | 0.01 [SUMMARY]
| null |
[CONTENT] ||| ||| 10 | one | two ||| ||| 60 | 80% ||| ||| ||| ||| 6.3 ± | 3% | 11.6 | 3% | 0.01 ||| ||| [SUMMARY]
| null |
|||
Plasma Metabolite Profiles of Red Meat, Poultry, and Fish Consumption, and Their Associations with Colorectal Cancer Risk.
|
35267954
|
Red and processed meat consumption has been consistently associated with increased risk of colorectal cancer (CRC), but the association for fish intake is unclear. Evidence using objective dietary assessment approaches to evaluate these associations is sparse.
|
BACKGROUND
|
We measured plasma metabolites among 5269 participants from the Nurses' Health Study (NHS), NHSII, and Health Professionals Follow-Up study (HPFS). We calculated partial Spearman correlations between each metabolite and self-reported intake of seven red meat, poultry, and fish groups. Metabolite profile scores correlated to self-reported dietary intakes were developed using elastic net regression. Associations between self-reported intakes, metabolite profile scores, and subsequent CRC risk were further evaluated using conditional logistic regression among 559 matched (1:1) case-control pairs in NHS/HPFS and replicated among 266 pairs in Women's Health Study.
|
METHODS
|
Plasma metabolites, especially highly unsaturated lipids, were differentially associated with red meat and fish groups. Metabolite profile scores for each food group were significantly correlated with the corresponding self-reported dietary intake. A higher dietary intake of processed red meat was associated with a higher risk of CRC (pooled OR per 1 SD, 1.15; 95% CI: 1.03, 1.29). In contrast, higher metabolite profile scores for all fish groups, not dietary intakes, were consistently associated with a lower CRC risk: the pooled OR per 1 SD was 0.86 (95% CI: 0.78, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were found for other food groups.
|
RESULTS
|
Red meat and fish intake exhibited systematically different plasma metabolite profiles. Plasma metabolite profile of fish intake was inversely associated with CRC risk.
|
CONCLUSIONS
|
[
"Animals",
"Colorectal Neoplasms",
"Female",
"Follow-Up Studies",
"Logistic Models",
"Poultry",
"Red Meat"
] |
8912563
|
1. Introduction
|
Colorectal cancer (CRC) remains the second most commonly occurring cancer in women and the third in men worldwide [1]. CRC incidence is largely affected by screening and modifiable dietary and lifestyle factors [2]. Among these factors, meat and fish consumption has been the subject of many investigations. The consumption of red meat, especially processed red meat, is consistently associated with an increased risk of CRC [3]. However, there is still uncertainty regarding the association between fish intake and CRC risk. The World Cancer Research Fund and American Institute for Cancer Research (WCRF/AICR) concluded that there was only “limited evidence” for the beneficial effect of fish intake in CRC prevention [4]. Recent studies of fish intake and CRC risk also generated inconsistent results [5,6,7,8].
Previous epidemiological studies examining the association of meat and fish intake with CRC risk mainly used self-reported dietary data. Rapid developments in high-throughput metabolomics are leading to a new era in nutritional epidemiological research. By measuring the small-molecule metabolites in biological samples, metabolomics may provide an objective picture of food intake and its related biological consequences [9]. Feeding trials and observational studies have demonstrated that plasma and urinary metabolites differed between meat and fish consumption [10,11,12,13,14]. However, it is unknown whether the metabolite profiles related to meat and fish intake are associated with CRC risk and whether these profiles could be used as a complementary approach to evaluate the association between meat and fish intake and CRC risk.
Therefore, we examined the associations of red meat, poultry, and fish consumption with plasma metabolite profiles among participants from the Nurses’ Health Study (NHS), NHSII, and Health Professionals Follow-up Study (HPFS). We also developed metabolite profile scores that were correlated to the intakes of these meat and fish groups and evaluated the prospective associations of metabolite profile scores with CRC risk. The results for metabolite profile scores were further replicated in an external validation cohort (Women’s Health Study, WHS).
|
2. Methods
|
Study Population Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.
The external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.
Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.
The external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.
|
6. Results
|
In NHS/NHSII/HPFS, participants were predominately white and middle-aged (mean age 53 years), with an average BMI of 25.4 kg/m2 (Table 1). The percentage energy intake from protein was 18%, and animal protein was 13%. Participants with a higher total red meat intake were slightly more likely to have a lower total fish intake (Pearson r = −0.07). In the analysis of CRC, participants had similar demographic characteristics but were a little older (mean age 61 years) and had a lower proportion of females. Compared to control participants, CRC cases were less likely to use multivitamins and aspirin, receive endoscopy screening, and be physically active, but were more likely to smoke and have a family history of CRC. They also had a somewhat higher intake of total red meat (mainly processed red meat) and a lower fish intake. Similarly, CRC cases in WHS were less likely to be physically active and more likely to smoke and have a family history of CRC. However, they had a lower intake of total red meat (mainly unprocessed red meat) than control participants.
Of the 287 known metabolites, 85 were significantly correlated with total red meat intake, 36 with unprocessed red meat, 51 with processed red meat, 28 with poultry, 60 with total fish, 60 with dark meat fish, and 27 with canned tuna fish after Bonferroni correction (Figure 2). Similar metabolites correlated with total red meat intake, unprocessed red meat, and processed red meat. All three red meat groups were positively associated with creatine, hydroxyproline, coenzyme Q10, myristoleic acid, acylcarnitines, and plasmalogens with a number of double bonds ≤6 but were negatively associated with highly unsaturated lipid species including triglycerides (TAGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylcholines (LPCs). Poultry intake was positively correlated with creatine, ectoine, a few PCs, TAGs, and PE plasmalogens after Bonferroni correction. In contrast to red meat intake, total fish intake, dark meat fish, and canned tuna fish were all positively correlated with highly unsaturated lipid species. When we restricted the analysis to fasting participants and participants selected as controls in the original sub-studies, the correlation results were similar (Supplementary Figures S2 and S3).
Of the 2561 unknown metabolites, 702 were significantly correlated with total red meat intake after Bonferroni correction, 222 with unprocessed red meat, 462 with processed red meat, 65 with poultry, 284 with total fish intake, 414 with dark meat fish, and 119 with canned tuna fish (Supplementary Table S2). Among these, the absolute partial Spearman rho exceeded 0.3 for 11 unknown metabolites: one with total red meat intake, 10 with total fish intake, and 5 with dark meat fish intake. Pearson correlation analysis between these 11 unknown metabolites and those known metabolites (130 in total) that were significantly associated with total red meat, total fish, or dark meat fish consumption identified several unknown metabolites being strongly correlated with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC (all Pearson r > 0.8) (Supplementary Figure S4).
A total of 53 known metabolites were selected in the elastic net regression for total red meat intake, 55 for unprocessed red meat, 36 for processed red meat, 7 for poultry, 18 for total fish, 27 for dark meat fish, and 11 for canned tuna fish (Table 2). Each metabolite profile score based on the selected known metabolites was significantly correlated with the corresponding self-reported dietary intake in both NHS/NHSII/HPFS and WHS (r = 0.33–0.46 for total red meat; r = 0.36–0.42 for unprocessed red meat; r = 0.19–0.33 for processed red meat; r = 0.12–0.21 for poultry; r = 0.31–0.40 for total fish; r = 0.32–0.42 for dark meat fish; and r = 0.14–0.22 for canned tuna fish) (Table 2). Adding unknown metabolites into the metabolite profile scores did not materially improve their correlations with the dietary intake (Supplementary Table S3). As a sensitivity analysis, we adopted the random forest algorithm to impute the missingness and re-derived the metabolomic profile score. The newly derived scores were highly correlated to the original scores, with a Pearson r = 0.98 for the total red meat score, 0.98 for the unprocessed red meat, 0.96 for the processed red meat score, 0.92 for the poultry score, 0.99 for the total fish score, 0.99 for the dark meat fish score, and 0.97 for the canned tuna fish score.
After adjusting for potential confounders, dietary intake of processed red meat was significantly associated with CRC risk. The pooled OR for 1 SD higher intake was 1.15 (95% CI: 1.03, 1.29) (Table 3). We did not observe consistent associations for dietary intake of three fish groups across NHS/HPFS and WHS. An inverse association for total fish and canned tuna fish was only found in NHS/HPFS and for dark meat fish in WHS. In contrast, metabolite profile scores for these three fish groups were inversely associated with CRC risk in both NHS/HPFS and WHS (Table 3). The pooled OR per 1 SD higher was 0.86 (95% CI: 0.77, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were observed for other meat intakes or metabolite profile scores. The results remained similar after adjusting for menopausal status or ethnicity in NHS/HPFS (data not shown). In the analysis by tumor site, we observed that the above associations were stronger for rectal cancer than those for colon cancer (Supplementary Table S4).
Among metabolites selected in metabolite profile scores of all three fish groups, five metabolites (C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) were positively correlated to fish intake but inversely associated with CRC risk; two metabolites (C20:4 LPE and C22:5 LPC) were inversely correlated to fish intake but positively associated with CRC risk (Supplementary Figure S5).
| null | null |
[
"Study Population",
"3. Dietary Assessment",
"4. Metabolomics Measurement",
"5. Nondietary Covariates",
"Statistical Analyses"
] |
[
"Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.",
"Validated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.",
"Profiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.",
"In NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.",
"We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1."
] |
[
null,
null,
null,
null,
null
] |
[
"1. Introduction",
"2. Methods",
"Study Population",
"3. Dietary Assessment",
"4. Metabolomics Measurement",
"5. Nondietary Covariates",
"Statistical Analyses",
"6. Results",
"7. Discussion"
] |
[
"Colorectal cancer (CRC) remains the second most commonly occurring cancer in women and the third in men worldwide [1]. CRC incidence is largely affected by screening and modifiable dietary and lifestyle factors [2]. Among these factors, meat and fish consumption has been the subject of many investigations. The consumption of red meat, especially processed red meat, is consistently associated with an increased risk of CRC [3]. However, there is still uncertainty regarding the association between fish intake and CRC risk. The World Cancer Research Fund and American Institute for Cancer Research (WCRF/AICR) concluded that there was only “limited evidence” for the beneficial effect of fish intake in CRC prevention [4]. Recent studies of fish intake and CRC risk also generated inconsistent results [5,6,7,8].\nPrevious epidemiological studies examining the association of meat and fish intake with CRC risk mainly used self-reported dietary data. Rapid developments in high-throughput metabolomics are leading to a new era in nutritional epidemiological research. By measuring the small-molecule metabolites in biological samples, metabolomics may provide an objective picture of food intake and its related biological consequences [9]. Feeding trials and observational studies have demonstrated that plasma and urinary metabolites differed between meat and fish consumption [10,11,12,13,14]. However, it is unknown whether the metabolite profiles related to meat and fish intake are associated with CRC risk and whether these profiles could be used as a complementary approach to evaluate the association between meat and fish intake and CRC risk.\nTherefore, we examined the associations of red meat, poultry, and fish consumption with plasma metabolite profiles among participants from the Nurses’ Health Study (NHS), NHSII, and Health Professionals Follow-up Study (HPFS). We also developed metabolite profile scores that were correlated to the intakes of these meat and fish groups and evaluated the prospective associations of metabolite profile scores with CRC risk. The results for metabolite profile scores were further replicated in an external validation cohort (Women’s Health Study, WHS).",
" Study Population Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.\nOur primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.",
"Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.\nThe external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.",
"Validated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.",
"Profiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).\nWe excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.",
"In NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.\n Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.\nWe examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.",
"We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.\nTo develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].\nAssociations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.",
"In NHS/NHSII/HPFS, participants were predominately white and middle-aged (mean age 53 years), with an average BMI of 25.4 kg/m2 (Table 1). The percentage energy intake from protein was 18%, and animal protein was 13%. Participants with a higher total red meat intake were slightly more likely to have a lower total fish intake (Pearson r = −0.07). In the analysis of CRC, participants had similar demographic characteristics but were a little older (mean age 61 years) and had a lower proportion of females. Compared to control participants, CRC cases were less likely to use multivitamins and aspirin, receive endoscopy screening, and be physically active, but were more likely to smoke and have a family history of CRC. They also had a somewhat higher intake of total red meat (mainly processed red meat) and a lower fish intake. Similarly, CRC cases in WHS were less likely to be physically active and more likely to smoke and have a family history of CRC. However, they had a lower intake of total red meat (mainly unprocessed red meat) than control participants.\nOf the 287 known metabolites, 85 were significantly correlated with total red meat intake, 36 with unprocessed red meat, 51 with processed red meat, 28 with poultry, 60 with total fish, 60 with dark meat fish, and 27 with canned tuna fish after Bonferroni correction (Figure 2). Similar metabolites correlated with total red meat intake, unprocessed red meat, and processed red meat. All three red meat groups were positively associated with creatine, hydroxyproline, coenzyme Q10, myristoleic acid, acylcarnitines, and plasmalogens with a number of double bonds ≤6 but were negatively associated with highly unsaturated lipid species including triglycerides (TAGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylcholines (LPCs). Poultry intake was positively correlated with creatine, ectoine, a few PCs, TAGs, and PE plasmalogens after Bonferroni correction. In contrast to red meat intake, total fish intake, dark meat fish, and canned tuna fish were all positively correlated with highly unsaturated lipid species. When we restricted the analysis to fasting participants and participants selected as controls in the original sub-studies, the correlation results were similar (Supplementary Figures S2 and S3).\nOf the 2561 unknown metabolites, 702 were significantly correlated with total red meat intake after Bonferroni correction, 222 with unprocessed red meat, 462 with processed red meat, 65 with poultry, 284 with total fish intake, 414 with dark meat fish, and 119 with canned tuna fish (Supplementary Table S2). Among these, the absolute partial Spearman rho exceeded 0.3 for 11 unknown metabolites: one with total red meat intake, 10 with total fish intake, and 5 with dark meat fish intake. Pearson correlation analysis between these 11 unknown metabolites and those known metabolites (130 in total) that were significantly associated with total red meat, total fish, or dark meat fish consumption identified several unknown metabolites being strongly correlated with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC (all Pearson r > 0.8) (Supplementary Figure S4).\nA total of 53 known metabolites were selected in the elastic net regression for total red meat intake, 55 for unprocessed red meat, 36 for processed red meat, 7 for poultry, 18 for total fish, 27 for dark meat fish, and 11 for canned tuna fish (Table 2). Each metabolite profile score based on the selected known metabolites was significantly correlated with the corresponding self-reported dietary intake in both NHS/NHSII/HPFS and WHS (r = 0.33–0.46 for total red meat; r = 0.36–0.42 for unprocessed red meat; r = 0.19–0.33 for processed red meat; r = 0.12–0.21 for poultry; r = 0.31–0.40 for total fish; r = 0.32–0.42 for dark meat fish; and r = 0.14–0.22 for canned tuna fish) (Table 2). Adding unknown metabolites into the metabolite profile scores did not materially improve their correlations with the dietary intake (Supplementary Table S3). As a sensitivity analysis, we adopted the random forest algorithm to impute the missingness and re-derived the metabolomic profile score. The newly derived scores were highly correlated to the original scores, with a Pearson r = 0.98 for the total red meat score, 0.98 for the unprocessed red meat, 0.96 for the processed red meat score, 0.92 for the poultry score, 0.99 for the total fish score, 0.99 for the dark meat fish score, and 0.97 for the canned tuna fish score.\nAfter adjusting for potential confounders, dietary intake of processed red meat was significantly associated with CRC risk. The pooled OR for 1 SD higher intake was 1.15 (95% CI: 1.03, 1.29) (Table 3). We did not observe consistent associations for dietary intake of three fish groups across NHS/HPFS and WHS. An inverse association for total fish and canned tuna fish was only found in NHS/HPFS and for dark meat fish in WHS. In contrast, metabolite profile scores for these three fish groups were inversely associated with CRC risk in both NHS/HPFS and WHS (Table 3). The pooled OR per 1 SD higher was 0.86 (95% CI: 0.77, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were observed for other meat intakes or metabolite profile scores. The results remained similar after adjusting for menopausal status or ethnicity in NHS/HPFS (data not shown). In the analysis by tumor site, we observed that the above associations were stronger for rectal cancer than those for colon cancer (Supplementary Table S4).\nAmong metabolites selected in metabolite profile scores of all three fish groups, five metabolites (C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) were positively correlated to fish intake but inversely associated with CRC risk; two metabolites (C20:4 LPE and C22:5 LPC) were inversely correlated to fish intake but positively associated with CRC risk (Supplementary Figure S5).",
"Leveraging metabolomics data from four studies, we found systematic differences in plasma metabolite profiles according to various types of meat and fish consumption. We also developed metabolite profile scores that were correlated to the consumption of red meat, poultry, and fish. Higher metabolite profile scores for all three fish groups (total fish, dark meat fish, and canned tuna fish), rather than their self-reported dietary intake, were consistently associated with a lower CRC risk in both NHS/HPFS and WHS. These findings suggest the promising use of metabolomics in complementing traditional dietary assessments to evaluate the association between dietary exposure and health outcomes.\nConsistent with previous studies, we found that consumption of all three red meat groups was positively correlated with several PE and PC plasmalogens, all of which have a low degree of unsaturation and contain acid moieties derived from animal tissues [14,29]. In contrast, total fish intake was positively correlated with highly unsaturated lipids, including TAGs, PCs, PEs, LPCs, LPEs, CEs, and plasmalogens. Fish intake, especially marine fish, is the principal dietary contributor of highly n-3 polyunsaturated fatty acids [30], and thus, are dietary sources for these lipids and their downstream metabolites.\nApart from lipid metabolites, we also observed several other metabolites correlated with meat and fish consumption. Creatine, an amino acid metabolite abundant in skeletal muscle [31], was positively correlated with the intake of red meat, poultry, and fish. Hydroxyproline, an amino acid that forms part of collagen [32], was positively correlated with red meat and poultry intake. Additionally, we found a positive correlation between poultry intake and ectoine (an α-amino acid), in line with one previous study [14]. Ectoine can be synthesized and released by several species in the genus Halomonas [33], the dominant microbial genus in the chicken embryo [34], which might lead to the presence of ectoine in chicken.\nMost previous metabolomic studies mainly focused on known metabolites, which are only a small percent of the data obtained in a typical metabolomics measurement (~10%) [35]. In the present study, we analyzed the unknown metabolites and identified a set of unknown metabolites that were significantly correlated with meat or fish consumption. Several unknown metabolites even exhibited a higher correlation with meat and fish intake (partial Spearman rho > 0.3) compared to known metabolites. By further examining the correlation between these unknown metabolites and the known metabolites, strong correlations were found with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC, indicating their potential biological links. However, whether these unknown metabolites are metabolites of the known metabolites or unrelated molecules affected by the same food is not clear. A critical next step is to structurally identify the promising and influential unknown metabolites. It should also be noted that despite the many statistically significant correlations between meat and fish groups and the unknown metabolites, these unknown metabolites added minimally to the correlations between meat and fish intakes and metabolite profile score based only on the known metabolites. The possibility remains that other metabolomics platforms may add further independent prediction of intake.\nThe human metabolome reflects a holistic biological status, which can be influenced by various exogenous and endogenous factors, such as dietary intakes, gut microbiota, health status, and genotype [36,37]. The metabolites selected in the metabolite profile score for meat or fish consumption could represent, in part, the components of food, such as the eicosapentaenoic acid/docosahexaenoic acid-containing lipids for the fish intake and hydroxyproline for the intake of red meat and poultry. Other metabolites, for example, C20:4 LPE and C22:5 LPC, could be related to the metabolic processes after fish intake, as fish is one of the main sources of arachidonic acid (C20:4) [38] and docosapentaenoic acid (C22:5) [39], but it was inversely correlated with these two lipids. Thus, metabolite profile score can be a comprehensive way to reflect both genuine compounds from foods as well as markers of complex metabolomic responses to dietary exposures.\nNot surprisingly, we found a significant positive association between processed red meat intake and CRC risk. However, the metabolite profile score for processed red meat consumption was not significantly associated with CRC risk. One possible explanation could be that the increased CRC risk associated with high processed red meat intake was mainly due to the carcinogenic compounds [40], such as polycyclic aromatic hydrocarbons, heterocyclic amines, and N-nitroso compounds, which were not measured in our metabolomics platforms. On the contrary, we did not observe a consistent inverse association between dietary fish intake and CRC risk, but inverse associations were found for the metabolite profile scores of all three fish groups in both NHS/HPFS and WHS. Results from previous studies of fish intake and CRC risk were generally inconsistent, some indicating an inverse association [7,8], but others not [5,6]. Unlike self-reported dietary intake data widely used in previous studies, the metabolomics data objectively captures the metabolites related to dietary exposures in the human body, which might be more directly associated with health outcomes. The seven metabolites (C20:4 LPE, C22:5 LPC, C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) selected in metabolite profile scores of all three fish groups could represent the beneficial metabolomic response to fish intake, and this response led to a lower risk of CRC.\nThe main strengths of our study are the large sample size, comprehensive metabolite profiles including both known and unknown metabolites, prospectively collected dietary data before CRC diagnosis, and the use of the elastic net model, which performs well in high-dimensional data where there are high correlations between predictors [41]. Moreover, the robustness of our metabolite profile scores was validated in an independent study. Nevertheless, our results also have several limitations. First, we did not have data on the type of red meat. Red meat subtypes (beef, lamb, or pork) may differ in their associations with CRC risk [42]. Second, only one metabolomics measurement was performed for each participant. However, our previous pilot study indicated that most metabolites were highly stable over 1–2 years within individuals [26]. Third, the metabolites measured in our study focused on lipids, amino acids, and amino acid derivatives and did not include all established biomarkers of food intake. Finally, participants in our study were mainly white women; replication of our findings in men and other racial groups is needed.\nIn conclusion, we identified a panel of metabolites differentially correlated with the intake of red meat, poultry, and fish. We also developed metabolite profile scores associated with the consumption of meat and fish and observed consistent inverse associations between metabolite profile scores of fish intake and CRC risk. Our results suggest the potential utility of metabolomics in complementing traditional dietary assessments to investigate the diet-disease association and disentangle the metabolomic responses linking higher fish intake with reduced CRC risk."
] |
[
"intro",
"methods",
null,
null,
null,
null,
null,
"results",
"discussion"
] |
[
"red meat",
"fish",
"plasma metabolomics",
"colorectal cancer"
] |
1. Introduction:
Colorectal cancer (CRC) remains the second most commonly occurring cancer in women and the third in men worldwide [1]. CRC incidence is largely affected by screening and modifiable dietary and lifestyle factors [2]. Among these factors, meat and fish consumption has been the subject of many investigations. The consumption of red meat, especially processed red meat, is consistently associated with an increased risk of CRC [3]. However, there is still uncertainty regarding the association between fish intake and CRC risk. The World Cancer Research Fund and American Institute for Cancer Research (WCRF/AICR) concluded that there was only “limited evidence” for the beneficial effect of fish intake in CRC prevention [4]. Recent studies of fish intake and CRC risk also generated inconsistent results [5,6,7,8].
Previous epidemiological studies examining the association of meat and fish intake with CRC risk mainly used self-reported dietary data. Rapid developments in high-throughput metabolomics are leading to a new era in nutritional epidemiological research. By measuring the small-molecule metabolites in biological samples, metabolomics may provide an objective picture of food intake and its related biological consequences [9]. Feeding trials and observational studies have demonstrated that plasma and urinary metabolites differed between meat and fish consumption [10,11,12,13,14]. However, it is unknown whether the metabolite profiles related to meat and fish intake are associated with CRC risk and whether these profiles could be used as a complementary approach to evaluate the association between meat and fish intake and CRC risk.
Therefore, we examined the associations of red meat, poultry, and fish consumption with plasma metabolite profiles among participants from the Nurses’ Health Study (NHS), NHSII, and Health Professionals Follow-up Study (HPFS). We also developed metabolite profile scores that were correlated to the intakes of these meat and fish groups and evaluated the prospective associations of metabolite profile scores with CRC risk. The results for metabolite profile scores were further replicated in an external validation cohort (Women’s Health Study, WHS).
2. Methods:
Study Population Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.
The external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.
Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.
The external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.
Study Population:
Our primary analyses were based on three prospective cohorts: NHS, NHSII, and HPFS. The NHS started in 1976 among 121,700 female registered nurses aged 30–55 years, and the NHSII began in 1989 among 116,429 younger female registered nurses aged 25–42 years [15]. The HPFS was initiated in 1986 and enrolled 51,529 male health professionals aged 40–75 years [16]. Blood samples were collected from subsamples of the NHS between 1989 and 1990, NHSII between 1996 and 1999, and HPFS between 1993 and 1995 [17,18]. We included participants who provided blood samples and were previously selected for metabolomics sub-studies of breast cancer and CRC (all diagnosed after blood collection). After excluding participants with missing dietary data, 5269 participants (2627 from NHS, 2096 from NHSII, and 546 from HPFS) were included in the final analyses. Among them, we included 559 case-control pairs (404 pairs for colon cancer and 122 pairs for rectal cancer) in the analysis of CRC risk. Each CRC case was matched to one control by age, month, and fasting status at blood collection.
The external replication analysis was performed in WHS, a completed randomized controlled trial originally designed to examine the role of aspirin and Vitamin E in the prevention of cancer and cardiovascular disease [19]. From 1992 to 1995, 39,876 healthy women aged 45 years or older were recruited. Blood samples were provided by 71% of participants before randomization. The trial ended in 2004, but annual observational follow-up continued. We included samples from 266 CRC cases (diagnosed after blood collection) and matched controls with available dietary and metabolomics data. Each case was matched to a control by age, ethnicity, month, and fasting status at the time of blood collection and added follow-up time [20]. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health and those of participating registries as required.
3. Dietary Assessment:
Validated semi-quantitative food frequency questionnaires (FFQs) were administered to assess long-term intake of foods and nutrients in NHS/NHSII/HPFS every four years [21,22,23]. Participants were asked to report how often, on average, they consumed a standard portion size of each food in the past year. To better reflect dietary consumption at the time of blood draw, we calculated the average intakes from the two FFQs closest to the blood collection date for each cohort (1986 and 1990 in NHS, 1995 and 1999 in NHSII, and 1990 and 1994 in HPFS). We included seven meat and fish groups in the analyses: total red meat, unprocessed red meat, processed red meat, poultry, total fish, dark meat fish, and canned tuna fish. Consistent with previous studies from the same cohorts [24], unprocessed red meat included hamburgers; beef, pork, or lamb as a sandwich or mixed dish; and beef, pork, or lamb as a main dish. Processed red meat included bacon; hot dogs; and sausage, salami, bologna, or other processed meat. Total red meat was derived by summing consumption of unprocessed and processed red meat. Poultry included chicken or turkey with or without skin; chicken or turkey sandwiches; and chicken or turkey hot dogs. Total fish included dark meat fish (e.g., salmon); canned tuna fish; breaded fish cakes, pieces, or fish sticks; and other fish. In WHS, dietary information was collected using the same validated FFQ at trial baseline. The serving sizes for each food group are shown in Supplementary Table S1.
4. Metabolomics Measurement:
Profiles of plasma metabolites in all four studies (NHS, NHSII, HPFS, and WHS) were obtained using high-throughput liquid chromatography-mass spectrometry techniques at the Broad Institute of MIT and Harvard [25]. Hydrophilic interaction liquid chromatography with positive ion mode mass spectrometry detection was used to separate polar metabolites, and C8 chromatography with positive ion mode detection was used to profile lipids. Raw data were processed using TraceFinder software (Thermo Fisher Scientific Waltham, MA, USA) and Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK). Known metabolite identities were confirmed using authentic reference standards or reference samples. Unknown metabolites were aligned using an in-house alignment algorithm, m2Aligner. This tool identifies unambiguous shared peaks in datasets to be aligned and uses them as alignment vectors adjusting for deviations in retention time (RT), mass to charge ratio (m/z), and abundance for all the peaks in the datasets. The adjusted m/z and RTs are subsequently used to match peaks using a scoring system that takes into consideration their mass accuracy and their retention time deviation (the corresponding methodology manuscript is in preparation).
We excluded known or unknown metabolites whose intraclass correlation coefficient (ICC) across blinded quality control replicates (10% of study sample) were <0.4, had no between-person variation, or detection rate <75%. Metabolites that were not stable with the processing delay inherent in our cohort study blood collections were also excluded (n = 38) [26]. Metabolite levels were reported as measured LC-MS peak areas, which are proportional to metabolite concentration. Metabolite peak areas were then log-transformed and converted to z-scores with a mean of 0 and a standard deviation of 1 within each sub-study. A total of 287 known and 2561 unknown metabolites were included in the present analyses. Among these metabolites, 58 known metabolites had missing data, with a missing proportion ranging from 0.02% to 13.5%; 1206 unknown metabolites had missing data, with a missing proportion ranging from 0.02% to 22.1%. The missingness was imputed using ½ minimum value. The 287 known metabolites were primarily lipids (n = 206, including 85 glycerolipids, 31 glycerophospholipids, 22 plasmalogens, 21 carnitines, 21 lysophospholipids, 13 cholesterol & cholesterol esters, 5 sphingolipids, 4 steroids, and 4 ceramides), but also included amino acids related metabolites (n = 41) and other metabolites (n = 40) (Supplementary Figure S1A). The lipid metabolites were highly correlated with each other within categories (Supplementary Figure S1B). A majority of the known metabolites (254 out of 287) were qualified for replication analysis in WHS. Metabolite data were log-transformed and converted to z-scores within each sub-study.
5. Nondietary Covariates:
In NHS/NHSII/HPFS, we collected information on lifestyle factors, including smoking, physical activity, multivitamin use, aspirin use, history of previous endoscopy, and family history of CRC using the biennial follow-up questionnaires. BMI (in kg/m2) was calculated using height reported at baseline and body weight reported closest to the blood draw. Age and fasting status were collected via questionnaires completed at blood collection. In WHS, participants provided information on age, weight, height, and lifestyle factors at baseline.
Statistical Analyses We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.
To develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].
Associations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.
We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.
To develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].
Associations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.
Statistical Analyses:
We examined the associations between intake of the seven meat and fish groups and each known and unknown metabolite in NHS/NHSII/HPFS, using partial Spearman correlation analysis adjusting for age and fasting status at blood draw, endpoint, and case/control status in the original sub-study, smoking, BMI, physical activity, total energy intake, alcohol intake, and modified Alternate Healthy Eating Index (AHEI, a measure of diet quality; intakes of red meat, alcohol, trans fat, long-chain n-3 fats, and polyunsaturated fats were not included in the calculation). Intakes of red meat, poultry, and fish were mutually adjusted. The Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were used to account for multiple testing.
To develop metabolite profile scores that are correlated to the consumption of seven meat and fish groups, NHS/NHSII/HPFS participants were randomized to either the training set (n = 3688) or the testing set (n = 1581) in a 7 to 3 fashion (Figure 1). The dietary consumption data were inverse normal transformed to improve normality. We used an elastic net [27] with 10-fold cross-validation to regress the consumption of each meat and fish group on the 287 known metabolites in the training set. The trained model was then applied to calculate the metabolite profile score for the testing set and participants in WHS. The metabolomic score was calculated as the weighted sum of the selected metabolites with weights equal to coefficients from the elastic net regression. Metabolite profile scores in the training set were obtained using a leave-one-out approach to avoid overfitting. Pearson correlation coefficient between self-reported dietary consumption and the corresponding metabolite profile score was calculated to evaluate how well the score was correlated to the dietary consumption. Apart from the selected known metabolites in the metabolite profile scores, we added unknown metabolites into the elastic net model and developed a new score that included the unknown ones (Figure 1). The new metabolomic scores were applied to the testing set, and their Pearson correlation coefficients with the corresponding dietary consumption were calculated as well. We then compared the Pearson correlation coefficients before and after including unknown metabolites to assess the contribution of unknown metabolites to the correlation between metabolomic score and dietary consumption [28].
Associations of meat and fish consumption and their metabolite profile scores with CRC were assessed among 559 pairs of CRC cases and matched controls from NHS/HPFS and 266 pairs from WHS (Figure 1). Conditional logistic regression adjusting for BMI, family history of CRC, history of endoscopy, multivitamin use, aspirin use, smoking, physical activity, alcohol intake, total energy intake, and modified AHEI was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) for one SD increase in dietary intake or metabolite profile score. These covariates for adjustment were selected based on our subjective knowledge and previous analysis results in the cohorts. Results from NHS/HPFS and WHS were then pooled using a fixed-effect model. All statistical analyses were performed in R version 4.1.
6. Results:
In NHS/NHSII/HPFS, participants were predominately white and middle-aged (mean age 53 years), with an average BMI of 25.4 kg/m2 (Table 1). The percentage energy intake from protein was 18%, and animal protein was 13%. Participants with a higher total red meat intake were slightly more likely to have a lower total fish intake (Pearson r = −0.07). In the analysis of CRC, participants had similar demographic characteristics but were a little older (mean age 61 years) and had a lower proportion of females. Compared to control participants, CRC cases were less likely to use multivitamins and aspirin, receive endoscopy screening, and be physically active, but were more likely to smoke and have a family history of CRC. They also had a somewhat higher intake of total red meat (mainly processed red meat) and a lower fish intake. Similarly, CRC cases in WHS were less likely to be physically active and more likely to smoke and have a family history of CRC. However, they had a lower intake of total red meat (mainly unprocessed red meat) than control participants.
Of the 287 known metabolites, 85 were significantly correlated with total red meat intake, 36 with unprocessed red meat, 51 with processed red meat, 28 with poultry, 60 with total fish, 60 with dark meat fish, and 27 with canned tuna fish after Bonferroni correction (Figure 2). Similar metabolites correlated with total red meat intake, unprocessed red meat, and processed red meat. All three red meat groups were positively associated with creatine, hydroxyproline, coenzyme Q10, myristoleic acid, acylcarnitines, and plasmalogens with a number of double bonds ≤6 but were negatively associated with highly unsaturated lipid species including triglycerides (TAGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), lysophosphatidylethanolamines (LPEs), and lysophosphatidylcholines (LPCs). Poultry intake was positively correlated with creatine, ectoine, a few PCs, TAGs, and PE plasmalogens after Bonferroni correction. In contrast to red meat intake, total fish intake, dark meat fish, and canned tuna fish were all positively correlated with highly unsaturated lipid species. When we restricted the analysis to fasting participants and participants selected as controls in the original sub-studies, the correlation results were similar (Supplementary Figures S2 and S3).
Of the 2561 unknown metabolites, 702 were significantly correlated with total red meat intake after Bonferroni correction, 222 with unprocessed red meat, 462 with processed red meat, 65 with poultry, 284 with total fish intake, 414 with dark meat fish, and 119 with canned tuna fish (Supplementary Table S2). Among these, the absolute partial Spearman rho exceeded 0.3 for 11 unknown metabolites: one with total red meat intake, 10 with total fish intake, and 5 with dark meat fish intake. Pearson correlation analysis between these 11 unknown metabolites and those known metabolites (130 in total) that were significantly associated with total red meat, total fish, or dark meat fish consumption identified several unknown metabolites being strongly correlated with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC (all Pearson r > 0.8) (Supplementary Figure S4).
A total of 53 known metabolites were selected in the elastic net regression for total red meat intake, 55 for unprocessed red meat, 36 for processed red meat, 7 for poultry, 18 for total fish, 27 for dark meat fish, and 11 for canned tuna fish (Table 2). Each metabolite profile score based on the selected known metabolites was significantly correlated with the corresponding self-reported dietary intake in both NHS/NHSII/HPFS and WHS (r = 0.33–0.46 for total red meat; r = 0.36–0.42 for unprocessed red meat; r = 0.19–0.33 for processed red meat; r = 0.12–0.21 for poultry; r = 0.31–0.40 for total fish; r = 0.32–0.42 for dark meat fish; and r = 0.14–0.22 for canned tuna fish) (Table 2). Adding unknown metabolites into the metabolite profile scores did not materially improve their correlations with the dietary intake (Supplementary Table S3). As a sensitivity analysis, we adopted the random forest algorithm to impute the missingness and re-derived the metabolomic profile score. The newly derived scores were highly correlated to the original scores, with a Pearson r = 0.98 for the total red meat score, 0.98 for the unprocessed red meat, 0.96 for the processed red meat score, 0.92 for the poultry score, 0.99 for the total fish score, 0.99 for the dark meat fish score, and 0.97 for the canned tuna fish score.
After adjusting for potential confounders, dietary intake of processed red meat was significantly associated with CRC risk. The pooled OR for 1 SD higher intake was 1.15 (95% CI: 1.03, 1.29) (Table 3). We did not observe consistent associations for dietary intake of three fish groups across NHS/HPFS and WHS. An inverse association for total fish and canned tuna fish was only found in NHS/HPFS and for dark meat fish in WHS. In contrast, metabolite profile scores for these three fish groups were inversely associated with CRC risk in both NHS/HPFS and WHS (Table 3). The pooled OR per 1 SD higher was 0.86 (95% CI: 0.77, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were observed for other meat intakes or metabolite profile scores. The results remained similar after adjusting for menopausal status or ethnicity in NHS/HPFS (data not shown). In the analysis by tumor site, we observed that the above associations were stronger for rectal cancer than those for colon cancer (Supplementary Table S4).
Among metabolites selected in metabolite profile scores of all three fish groups, five metabolites (C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) were positively correlated to fish intake but inversely associated with CRC risk; two metabolites (C20:4 LPE and C22:5 LPC) were inversely correlated to fish intake but positively associated with CRC risk (Supplementary Figure S5).
7. Discussion:
Leveraging metabolomics data from four studies, we found systematic differences in plasma metabolite profiles according to various types of meat and fish consumption. We also developed metabolite profile scores that were correlated to the consumption of red meat, poultry, and fish. Higher metabolite profile scores for all three fish groups (total fish, dark meat fish, and canned tuna fish), rather than their self-reported dietary intake, were consistently associated with a lower CRC risk in both NHS/HPFS and WHS. These findings suggest the promising use of metabolomics in complementing traditional dietary assessments to evaluate the association between dietary exposure and health outcomes.
Consistent with previous studies, we found that consumption of all three red meat groups was positively correlated with several PE and PC plasmalogens, all of which have a low degree of unsaturation and contain acid moieties derived from animal tissues [14,29]. In contrast, total fish intake was positively correlated with highly unsaturated lipids, including TAGs, PCs, PEs, LPCs, LPEs, CEs, and plasmalogens. Fish intake, especially marine fish, is the principal dietary contributor of highly n-3 polyunsaturated fatty acids [30], and thus, are dietary sources for these lipids and their downstream metabolites.
Apart from lipid metabolites, we also observed several other metabolites correlated with meat and fish consumption. Creatine, an amino acid metabolite abundant in skeletal muscle [31], was positively correlated with the intake of red meat, poultry, and fish. Hydroxyproline, an amino acid that forms part of collagen [32], was positively correlated with red meat and poultry intake. Additionally, we found a positive correlation between poultry intake and ectoine (an α-amino acid), in line with one previous study [14]. Ectoine can be synthesized and released by several species in the genus Halomonas [33], the dominant microbial genus in the chicken embryo [34], which might lead to the presence of ectoine in chicken.
Most previous metabolomic studies mainly focused on known metabolites, which are only a small percent of the data obtained in a typical metabolomics measurement (~10%) [35]. In the present study, we analyzed the unknown metabolites and identified a set of unknown metabolites that were significantly correlated with meat or fish consumption. Several unknown metabolites even exhibited a higher correlation with meat and fish intake (partial Spearman rho > 0.3) compared to known metabolites. By further examining the correlation between these unknown metabolites and the known metabolites, strong correlations were found with C38:6 PC, C40:6 PC, C40:9 PC, and C22:6 LPC, indicating their potential biological links. However, whether these unknown metabolites are metabolites of the known metabolites or unrelated molecules affected by the same food is not clear. A critical next step is to structurally identify the promising and influential unknown metabolites. It should also be noted that despite the many statistically significant correlations between meat and fish groups and the unknown metabolites, these unknown metabolites added minimally to the correlations between meat and fish intakes and metabolite profile score based only on the known metabolites. The possibility remains that other metabolomics platforms may add further independent prediction of intake.
The human metabolome reflects a holistic biological status, which can be influenced by various exogenous and endogenous factors, such as dietary intakes, gut microbiota, health status, and genotype [36,37]. The metabolites selected in the metabolite profile score for meat or fish consumption could represent, in part, the components of food, such as the eicosapentaenoic acid/docosahexaenoic acid-containing lipids for the fish intake and hydroxyproline for the intake of red meat and poultry. Other metabolites, for example, C20:4 LPE and C22:5 LPC, could be related to the metabolic processes after fish intake, as fish is one of the main sources of arachidonic acid (C20:4) [38] and docosapentaenoic acid (C22:5) [39], but it was inversely correlated with these two lipids. Thus, metabolite profile score can be a comprehensive way to reflect both genuine compounds from foods as well as markers of complex metabolomic responses to dietary exposures.
Not surprisingly, we found a significant positive association between processed red meat intake and CRC risk. However, the metabolite profile score for processed red meat consumption was not significantly associated with CRC risk. One possible explanation could be that the increased CRC risk associated with high processed red meat intake was mainly due to the carcinogenic compounds [40], such as polycyclic aromatic hydrocarbons, heterocyclic amines, and N-nitroso compounds, which were not measured in our metabolomics platforms. On the contrary, we did not observe a consistent inverse association between dietary fish intake and CRC risk, but inverse associations were found for the metabolite profile scores of all three fish groups in both NHS/HPFS and WHS. Results from previous studies of fish intake and CRC risk were generally inconsistent, some indicating an inverse association [7,8], but others not [5,6]. Unlike self-reported dietary intake data widely used in previous studies, the metabolomics data objectively captures the metabolites related to dietary exposures in the human body, which might be more directly associated with health outcomes. The seven metabolites (C20:4 LPE, C22:5 LPC, C22:6 LPC, C22:6 LPE, C58:9 TAG, C60:12 TAG, and C38:7 plasmalogen) selected in metabolite profile scores of all three fish groups could represent the beneficial metabolomic response to fish intake, and this response led to a lower risk of CRC.
The main strengths of our study are the large sample size, comprehensive metabolite profiles including both known and unknown metabolites, prospectively collected dietary data before CRC diagnosis, and the use of the elastic net model, which performs well in high-dimensional data where there are high correlations between predictors [41]. Moreover, the robustness of our metabolite profile scores was validated in an independent study. Nevertheless, our results also have several limitations. First, we did not have data on the type of red meat. Red meat subtypes (beef, lamb, or pork) may differ in their associations with CRC risk [42]. Second, only one metabolomics measurement was performed for each participant. However, our previous pilot study indicated that most metabolites were highly stable over 1–2 years within individuals [26]. Third, the metabolites measured in our study focused on lipids, amino acids, and amino acid derivatives and did not include all established biomarkers of food intake. Finally, participants in our study were mainly white women; replication of our findings in men and other racial groups is needed.
In conclusion, we identified a panel of metabolites differentially correlated with the intake of red meat, poultry, and fish. We also developed metabolite profile scores associated with the consumption of meat and fish and observed consistent inverse associations between metabolite profile scores of fish intake and CRC risk. Our results suggest the potential utility of metabolomics in complementing traditional dietary assessments to investigate the diet-disease association and disentangle the metabolomic responses linking higher fish intake with reduced CRC risk.
|
Background:
Red and processed meat consumption has been consistently associated with increased risk of colorectal cancer (CRC), but the association for fish intake is unclear. Evidence using objective dietary assessment approaches to evaluate these associations is sparse.
Methods:
We measured plasma metabolites among 5269 participants from the Nurses' Health Study (NHS), NHSII, and Health Professionals Follow-Up study (HPFS). We calculated partial Spearman correlations between each metabolite and self-reported intake of seven red meat, poultry, and fish groups. Metabolite profile scores correlated to self-reported dietary intakes were developed using elastic net regression. Associations between self-reported intakes, metabolite profile scores, and subsequent CRC risk were further evaluated using conditional logistic regression among 559 matched (1:1) case-control pairs in NHS/HPFS and replicated among 266 pairs in Women's Health Study.
Results:
Plasma metabolites, especially highly unsaturated lipids, were differentially associated with red meat and fish groups. Metabolite profile scores for each food group were significantly correlated with the corresponding self-reported dietary intake. A higher dietary intake of processed red meat was associated with a higher risk of CRC (pooled OR per 1 SD, 1.15; 95% CI: 1.03, 1.29). In contrast, higher metabolite profile scores for all fish groups, not dietary intakes, were consistently associated with a lower CRC risk: the pooled OR per 1 SD was 0.86 (95% CI: 0.78, 0.96) for total fish, 0.86 (95% CI: 0.77, 0.96) for dark meat fish, and 0.87 (95% CI: 0.78, 0.97) for canned tuna fish. No significant associations were found for other food groups.
Conclusions:
Red meat and fish intake exhibited systematically different plasma metabolite profiles. Plasma metabolite profile of fish intake was inversely associated with CRC risk.
| null | null | 6,815 | 357 | 9 |
[
"fish",
"meat",
"metabolites",
"intake",
"red meat",
"red",
"metabolite",
"crc",
"dietary",
"profile"
] |
[
"test",
"test"
] | null | null |
[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]
|
[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]
|
[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]
| null |
[CONTENT] red meat | fish | plasma metabolomics | colorectal cancer [SUMMARY]
| null |
[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]
|
[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]
|
[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]
| null |
[CONTENT] Animals | Colorectal Neoplasms | Female | Follow-Up Studies | Logistic Models | Poultry | Red Meat [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]
|
[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]
|
[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]
| null |
[CONTENT] fish | meat | metabolites | intake | red meat | red | metabolite | crc | dietary | profile [SUMMARY]
| null |
[CONTENT] fish | crc | meat | fish intake | intake crc | fish intake crc | risk | intake | crc risk | intake crc risk [SUMMARY]
|
[CONTENT] blood | aged | cancer | blood collection | collection | samples | blood samples | years | included | matched [SUMMARY]
|
[CONTENT] meat | fish | red meat | red | intake | total | total red meat | total red | total fish | dark [SUMMARY]
| null |
[CONTENT] fish | meat | metabolites | intake | metabolite | red | red meat | crc | metabolite profile | meat fish [SUMMARY]
| null |
[CONTENT] CRC ||| [SUMMARY]
|
[CONTENT] 5269 | NHSII | Health Professionals Follow-Up ||| Spearman | seven ||| ||| CRC | 559 | 1:1 | NHS | 266 | Women's Health Study [SUMMARY]
|
[CONTENT] Plasma metabolites ||| ||| CRC | 1 SD | 1.15 | 95% | CI | 1.03 | 1.29 ||| CRC | 0.86 | 95% | CI | 0.78 | 0.96 | 0.86 | 95% | CI | 0.77 | 0.96 | 0.87 | 95% | CI | 0.78 | 0.97 ||| [SUMMARY]
| null |
[CONTENT] CRC ||| ||| 5269 | NHSII | Health Professionals Follow-Up ||| Spearman | seven ||| ||| CRC | 559 | 1:1 | NHS | 266 | Women's Health Study ||| ||| Plasma metabolites ||| ||| CRC | 1 SD | 1.15 | 95% | CI | 1.03 | 1.29 ||| CRC | 0.86 | 95% | CI | 0.78 | 0.96 | 0.86 | 95% | CI | 0.77 | 0.96 | 0.87 | 95% | CI | 0.78 | 0.97 ||| ||| ||| Plasma | CRC [SUMMARY]
| null |
||||
Sericin cream reduces pruritus in hemodialysis patients: a randomized, double-blind, placebo-controlled experimental study.
|
23006933
|
Uremic pruritus (UP) is a significant complication in ESRD patients and substantially impairs their quality of life. UP is considered to be a skin manifestation of chronic inflammation. Because sericin can suppress the release of pro-inflammatory cytokines, the purpose of this study was to investigate the short-term safety and efficacy of sericin cream for treating UP in hemodialysis patients.
|
BACKGROUND
|
This study used a double-blind design to investigate the effects of random topical administration of sericin cream and cream base (placebo) on either the right or left extremities of hemodialysis patients for 6 weeks. Skin hydration, irritation and pigmentation were evaluated every 2 weeks using Skin Diagnostic SD27. The visual analog scale for itching was also evaluated every 2 weeks, and the Kidney Disease Quality of Life Short Form was performed on the day of each patient's enrollment and after 6 weeks of treatment.
|
METHODS
|
Fifty dialysis patients were enrolled, 47 of which completed the study. The hydration of the skin of the patients' extremities increased significantly after administration of sericin cream; significant differences were found between sericin treatment and control after 6 weeks of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Moreover, a significant difference was also found in skin irritation between the two treatments (p = 0.013 for arms and p = 0.027 for legs, respectively). At the end of the study, the skin pigmentation level was significantly reduced on both the arms (p = 0.032) and legs (p = 0.021) of the sericin-treated side compared with the side treated with cream base. The mean itching score decreased significantly from moderate to severe at the time of enrollment to mild pruritus after 6 weeks of treatment (p = 0.002). A better quality of life was found in all domains tested although statistically significant differences before and after treatment was found only in the patients' pain scores, the effect of kidney disease on daily life, sleep quality and symptoms or problems related to kidney disease.
|
RESULTS
|
We conclude that sericin cream has a high potential for reducing UP in hemodialysis patients.The trial registration number of this study is ISRCTN16019033; its public title is "sericin cream reduces pruritus in hemodialysis patients".
|
CONCLUSIONS
|
[
"Administration, Topical",
"Double-Blind Method",
"Female",
"Humans",
"Male",
"Middle Aged",
"Placebo Effect",
"Pruritus",
"Quality of Life",
"Renal Dialysis",
"Sericins",
"Skin Cream",
"Treatment Outcome"
] |
3472272
|
Background
|
Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].
Pruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].
Sericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.
At present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.
|
Methods
|
Preparation of silk sericin cream Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.
Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.
Molecular weight determination of sericin To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.
To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.
Amino acid analysis of sericin The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.
The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.
Study design Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.
Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.
Study population Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.
All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.
Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.
Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.
Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.
All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.
Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.
Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.
Study treatment In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.
In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.
Measurement and outcome The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:
(1)
%
changes in each parameter
=
P
t
−
P
0
/
P
0
×
100
,
where P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.
Because itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].
The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:
(1)
%
changes in each parameter
=
P
t
−
P
0
/
P
0
×
100
,
where P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.
Because itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].
Patients’ quality of life The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].
The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].
Safety monitoring The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.
The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.
Statistical analysis The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.
The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.
|
Results
|
Molecular weight and amino acid composition of sericin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.
Amino acid composition of sericin
These mean values were obtained by triplicate analysis.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.
Amino acid composition of sericin
These mean values were obtained by triplicate analysis.
Study population Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base.
Baseline characteristics of subjects (N = 47)
BUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.
The level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).
Skin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment
* indicates a significant difference compared with the same treatment at baseline (p < 0.05).
SS = silk sericin.
Changes in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).
Changes in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).
Skin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.
The use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).
The mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008).
The mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).
Table 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).
Mean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)
* Indicates significant differences compared to baseline, p < 0.05.
Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base.
Baseline characteristics of subjects (N = 47)
BUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.
The level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).
Skin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment
* indicates a significant difference compared with the same treatment at baseline (p < 0.05).
SS = silk sericin.
Changes in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).
Changes in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).
Skin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.
The use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).
The mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008).
The mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).
Table 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).
Mean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)
* Indicates significant differences compared to baseline, p < 0.05.
|
Conclusions
|
Our study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients.
|
[
"Background",
"Molecular weight and amino acid composition of sericin",
"Study population",
"Preparation of silk sericin cream",
"Molecular weight determination of sericin",
"Amino acid analysis of sericin",
"Study design",
"Study population",
"Inclusion criteria",
"Exclusion criteria",
"Study treatment",
"Measurement and outcome",
"Patients’ quality of life",
"Safety monitoring",
"Statistical analysis",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.",
"Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.",
"Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.",
"Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.",
"To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.",
"The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.",
"Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.",
" Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.",
"All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.",
"Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.",
"In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.",
"The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].",
"The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].",
"The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.",
"The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.",
"The authors declare that they have no competing interests.",
"PA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\n"
] |
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[
"Background",
"Results",
"Molecular weight and amino acid composition of sericin",
"Study population",
"Discussion",
"Conclusions",
"Methods",
"Preparation of silk sericin cream",
"Molecular weight determination of sericin",
"Amino acid analysis of sericin",
"Study design",
"Study population",
"Inclusion criteria",
"Exclusion criteria",
"Study treatment",
"Measurement and outcome",
"Patients’ quality of life",
"Safety monitoring",
"Statistical analysis",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].\nPruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].\nSericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.\nAt present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.",
" Molecular weight and amino acid composition of sericin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.\n Study population Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.\nFifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.",
"Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.\nAmino acid composition of sericin\nThese mean values were obtained by triplicate analysis.",
"Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base. \nBaseline characteristics of subjects (N = 47)\nBUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.\nThe level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).\nSkin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment\n* indicates a significant difference compared with the same treatment at baseline (p < 0.05).\nSS = silk sericin.\nChanges in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).\nChanges in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).\nSkin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.\nThe use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).\nThe mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008). \nThe mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).\nTable 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).\nMean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)\n* Indicates significant differences compared to baseline, p < 0.05.",
"This study has shown that the use of sericin cream can reduce UP in hemodialysis patients according to their VAS scores. Sericin cream can also significantly increase skin hydration and reduce skin irritation and skin pigmentation in patients. The molecular weight analysis of the sericin used in this study gave results that agree with those reported by Sprague, who indicated that sericin is a mixture of at least 15 different polypeptide chains ranging in size from 20–220 kDa [19]. Similar to the earlier report by Kato et al., serine was the most abundant amino acid found in sericin [20].\nAlthough antihistamines, anti-allergic agents and topical corticosteroids are commonly used to treat UP in ESRD patients, their effectiveness is sometimes limited [2,8,21,22]. Alternative and effective treatments for intractable pruritus that generate low levels of adverse reactions, particularly in sensitive patients such as the ESRD group, must therefore be developed. This clinical study is the first to suggest that a sericin cream is beneficial for treating pruritus in hemodialysis patients. The benefits of this treatment apparently result from increases in skin hydration and suppression of pro-inflammatory cytokines, as shown in our earlier study [17,23], thereby resulting in less skin irritation without any allergic reactions.\nPruritus associated with chronic kidney disease has been shown to be associated with elevated levels of C-reactive protein and other inflammatory cytokines. This evidence suggests that there is an inflammatory component in this form of pruritus [24]. Some authors consider UP to be a skin manifestation of chronic inflammation [25]. Recently, the “persistent microinflammation” theory, in which it is suggested that inflammation may be related to the genesis of UP, was proposed [26]. The skin of ESRD patients with UP contains an increased number of mast cells, and these cells can release various substances such as histamine, interleukins (IL) and tumor necrosis factor (TNF) [27-30].\nPruritic skin can appear normal except for dryness; because the severity of pruritus is closely correlated with skin dryness [31], xerosis treatment normally begins with a topical agent such as a gentle moisturizing cream. Because sericin is well known for its moisturizing effect and its ability to reduce the generation of pro-inflammatory cytokines by fibroblasts, this study assessed the usefulness of sericin cream in treating UP. Evaluating the degree of pruritus was difficult because it is a subjective symptom; therefore, functional measurements of skin conditions known to be associated with pruritus, such as skin hydration and skin irritation, were considered to be objective indices. We found that both sericin cream and the cream base used to prepare the sericin cream increased the moisture content of the stratum corneum; however, the level of hydration was significantly higher in the skin treated with sericin cream. The side of the body treated with sericin cream had significantly lower levels of post-treatment dryness compared to pretreatment dryness and lower total dermatological severity scores in parameters such as skin pigmentation.\nThe pathogenesis of ESRD itself is associated with inflammation and oxidative stress, as reflected in studies of plasma biochemistry, including the levels of the cytokines IL and TNF-α [32-34], classic markers of inflammatory processes that are strongly affected by pathological conditions [35]. These cytokines are upregulated in the plasma of ESRD patients [32,36]. The skin inflammation associated with the release of IL and TNF-α is further complicated by the inflammatory lesions that occur secondary to scratching [37]. Our results indicate that improvement in skin hydration and suppression of the release of pro-inflammatory cytokines in skin might be the mechanism by which sericin improves UP. Skin irritation, which can be caused by either skin inflammation or by scratching, was substantially reduced in the sericin group compared with the cream base group after 6 weeks of treatment. This result confirmed that sericin reduces skin irritation in patients with UP and suggested that it might be due to suppression of the inflammatory cytokines in skin. Similar results have been obtained in animal studies [23].\nSecondary skin lesions that can result from the itch-scratch cycle include excoriations, hyperpigmentation, lichenification, prurigo nodules and scars [38]. Chronic renal failure is usually accompanied by a variety of cutaneous manifestations; dermal manifestations, including hyperpigmentation, have also been reported, and these can exact a considerable toll on the quality of life [39-41]. Although skin pigmentation is not related to specific clinical symptoms, it has an important effect on patient satisfaction. Sericin has been reported to have activity against tyrosinase [15], an enzyme related to melanin production; as this study shows, treatment with sericin results in significant reduction in the skin color of patients.\nThe pruritus/mortality relationship may be strongly attributed to sleep disturbances, as previously mentioned in the DOPPS [8,9,42]. Many previous studies have found a strong association between inflammation, as measured by C-reactive protein or inflammatory cytokines, and sleep disturbances in dialysis patients [43-45]. The significant effects of pruritus on sleep, mood and social functioning require further investigation with the goal of improving the available treatments for this serious ESRD complication. We found that reduction in itching intensity from moderate to severe pruritus at the time of enrollment to mild pruritus after 6 weeks of treatment was associated with a better quality of life in all of the measured domains, including the mental, physical and kidney disease components. The KDQOL-SF score in the ESRD patients indicated improvements in several domains regarding the patients’ quality of life, particularly pain and sleep, which are relevant to itching. A better quality of sleep may reflect some degree of relief from itching achieved through the treatments.\nThis study has certain limitations. First, the study design was an in-subject controlled study; it cannot be determined whether the improvements in the itching evaluation and the quality of life were from the sample (sericin cream) or the placebo (cream base). Moreover, due to the in-subject controlled design, the biochemical parameters could not be evaluated at the end of the study. Second, the study included a small number of patients, and these were followed for a relatively short time. A long-term study with a larger sample size is necessary. In addition to studies of hemodialysis patients, similar studies of other ESRD patients, such as peritoneal dialysis and kidney transplant patients, are also necessary to confirm the findings presented here.",
"Our study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients.",
" Preparation of silk sericin cream Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\nBecause commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.\n Molecular weight determination of sericin To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\nTo determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.\n Amino acid analysis of sericin The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\nThe amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.\n Study design Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\nSkin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.\n Study population Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\n Study treatment In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\nIn 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.\n Measurement and outcome The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\nThe level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].\n Patients’ quality of life The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\nThe patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].\n Safety monitoring The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\nThe occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.\n Statistical analysis The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.\nThe results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.",
"Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.",
"To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.",
"The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.",
"Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.",
" Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\nAll ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.\n Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.\nPruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.",
"All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.",
"Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.",
"In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.",
"The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:\n\n\n(1)\n\n\n%\nchanges in each parameter\n=\n\n\n\n\n\nP\nt\n\n−\n\nP\n0\n\n\n\n/\n\nP\n0\n\n\n\n×\n100\n,\n\n\n\n\nwhere P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.\nBecause itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].",
"The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].",
"The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.",
"The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.",
"The authors declare that they have no competing interests.",
"PA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2369/13/119/prepub\n"
] |
[
null,
"results",
null,
null,
"discussion",
"conclusions",
"methods",
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Pruritus",
"Hemodialysis",
"Sericin",
"Hydration",
"Inflammation",
"Pigmentation"
] |
Background:
Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].
Pruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].
Sericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.
At present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.
Results:
Molecular weight and amino acid composition of sericin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.
Amino acid composition of sericin
These mean values were obtained by triplicate analysis.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.
Amino acid composition of sericin
These mean values were obtained by triplicate analysis.
Study population Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base.
Baseline characteristics of subjects (N = 47)
BUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.
The level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).
Skin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment
* indicates a significant difference compared with the same treatment at baseline (p < 0.05).
SS = silk sericin.
Changes in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).
Changes in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).
Skin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.
The use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).
The mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008).
The mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).
Table 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).
Mean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)
* Indicates significant differences compared to baseline, p < 0.05.
Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base.
Baseline characteristics of subjects (N = 47)
BUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.
The level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).
Skin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment
* indicates a significant difference compared with the same treatment at baseline (p < 0.05).
SS = silk sericin.
Changes in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).
Changes in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).
Skin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.
The use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).
The mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008).
The mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).
Table 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).
Mean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)
* Indicates significant differences compared to baseline, p < 0.05.
Molecular weight and amino acid composition of sericin:
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sericin prepared by the high-temperature and high-pressure degumming technique showed continuous bands with molecular weights ranging from 50–150 kDa. The highest-intensity band had an apparent molecular weight of approximately 100 kDa. The amino acid composition of sericin extracted with this method is given in Table 1.
Amino acid composition of sericin
These mean values were obtained by triplicate analysis.
Study population:
Fifty hemodialysis patients with ESRD were enrolled in this study; however, only 47 subjects completed the treatment (3 patients were withdrawn due to relocation). Thirty of the 47 subjects were female (63.83%), and 17 were male (36.17%); the mean age was 49.6 ± 11.2 years. The average duration of dialysis was 24.6 ± 3.1 months. Table 2 shows the characteristics and biochemical parameters of the study population. Most of the biochemical parameters, such as calcium (9.87 ± 1.32 mg/dL), phosphorus (4.35 ± 1.02 mg/dL), albumin (3.96 ± 0.62 g/dL), total bilirubin (0.32 ± 0.11 mg/dL) and liver enzyme (alanine transaminase (ALT) 14.23 ± 5.98 IU/L, aspartate aminotransferase (AST) 15.32 ± 5.12 IU/L and alkaline phosphatase 96.58 ± 40.32 IU/L) levels, were within the normal range. The baseline characteristics of each parameter, including skin hydration, skin irritation and skin pigmentation on the right and left extremities, showed no significant differences (p = 0.819 and 0.982 for skin hydration on arms and legs, p = 0.892 and 0.857 for skin irritation on arms and legs, p = 0.834 and 0.901 for skin pigmentation on arms and legs, respectively). The average itching score of the subjects using the visual analogue scale (VAS) at baseline was 7.05, which was considered moderate to severe (VAS < 4.0 is considered to reflect mild pruritus, while VAS 4.0 - 6.9 indicates moderate pruritus, and VAS > 7.0 is considered to indicate severe pruritus [10]). At baseline, both the arms and legs of the subjects showed some degree of skin irritation. None of the subjects reported any allergy or dermatological symptoms caused by the sericin or the cream base.
Baseline characteristics of subjects (N = 47)
BUN = blood urea nitrogen, ALT = alanine transaminase, AST = aspartate aminotransferase.
The level of skin hydration in the patients’ extremities increased after treatment with either sericin or cream base. The same patients received sericin and placebo (cream base) treatment and that application of each compound were confined to one side of the body. On the sericin-treated side of the body, the skin hydration of the arms was 28.67 ± 7.11 at baseline, and it increased to 33.62 ± 6.93 after treatment for 6 weeks (p = 0.047), while the skin hydration of the legs, which was 25.10 ± 7.67 at baseline, increased to 29.05 ± 7.74 after 6 weeks of treatment (p = 0.025). On the cream-base-treated side of the body, the skin hydration of the arms was 27.55 ± 7.84 at baseline, and it increased to 29.40 ± 4.92 after treatment for 6 weeks (p = 0.593); the skin hydration of the legs, which was 23.29 ± 7.37 at baseline, changed to 26.02 ± 6.47 after 6 weeks of treatment (p = 0.276). The skin hydration changes were significantly higher on the side that received the sericin cream than on the side that received the cream base, and a significant difference between the sericin cream and cream base treatments was found in the level of skin hydration in both the arms and legs during the sixth week of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Table 3 shows the skin parameters for hydration (measured by the Corneometer) and for irritation and pigmentation (measured by the Mexameter) of the subjects’ extremities at weeks 2, 4 and 6 after treatment. Figure 1 and 2 illustrate the percent changes in the parameters of the skin that received the sericin cream or the cream base during weeks 2–6 compared to the baseline. Six weeks after treatment, the average level of skin hydration on the side of the body that received the sericin cream was significantly increased compared to the baseline (p = 0.047 for arms and p = 0.025 for legs), while the side that received the cream base showed no significant difference (p = 0.593 for arms and p = 0.276 for legs) in skin hydration. The arms of the patients who were treated with the sericin cream showed significant differences in the level of skin hydration four weeks after the treatment compared to baseline (p = 0.022).
Skin parameters of hydration, irritation and pigmentation on the subjects’ extremities after treatment
* indicates a significant difference compared with the same treatment at baseline (p < 0.05).
SS = silk sericin.
Changes in skin parameters of hydration, irritation and pigmentation on the arms. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ arms treated with sericin cream or with cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatments) (p < 0.05).
Changes in skin parameters of hydration and irritation and pigmentation on the legs. Detailed legend: The changes in the skin parameters of hydration (measured by Corneometer), irritation and pigmentation (measured by Mexameter) on patients’ legs treated with sericin cream or cream base at weeks 2, 4 and 6 of treatment compared to baseline (* indicates significant differences compared to baseline; # indicates significant differences between treatment) (p < 0.05).
Skin irritation was measured based on the redness of the skin; both the sericin cream and the cream base reduced the level of skin irritation throughout the study period. As shown in Table 3, the arms and legs that were treated with sericin cream showed statistically significantly reduced irritation or redness from the second week after treatment until the end of the study compared to the baseline (p = 0.031 for arms and p = 0.040 for legs). The degree of redness of the legs of the patients who were treated with the cream base during weeks 4 (p = 0.048) and week 6 (p = 0.036) was also significantly reduced compared to the baseline. In addition, a significant difference between treatment with sericin cream and cream base was found in the level of skin irritation of both arms (p = 0.013) and legs (p = 0.027) after 6 weeks of treatment.
The use of sericin cream significantly reduced the darkness of the skin on both the arms and legs of the patients, as shown in Table 3. In addition, in the sixth week of treatment, there was a significant difference in the level of skin pigmentation in the arms and legs treated with the sericin cream compared to those treated with the cream base (Figure 1 and 2, p = 0.032 for arms and p = 0.021 for legs, respectively). The cream base showed no statistically significant effect on the level of skin pigmentation, and no significant difference compared to baseline was found in the skin pigmentation of the patients’ arms and legs after use of the cream base for 6 weeks (p = 0.082 for arms and p = 0.067 for legs, respectively).
The mean VAS scores for itching at baseline and after 2, 4 and 6 weeks of treatment are shown in Figure 3; the scores shown are based on the overall symptoms without differentiating between the sericin cream and cream base treatments. The mean pruritus score gradually decreased from the beginning of the study and with increasing weeks of treatment. The mean pruritus score at baseline was 7.05 ± 2.17 (range 1–10, median 8), which is indicates moderate to severe pruritus [10,18]; the mean score decreased to 2.23 ± 1.73 (range 0–6, median 2), indicating mild pruritus [10,18], after 6 weeks of treatment (p = 0.008).
The mean VAS score for itching. Detailed legend: The mean VAS score for itching at baseline and after 2, 4 and 6 weeks of treatment (* indicates significant difference, p < 0.05).
Table 4 shows the mean score assigned to each domain of the Kidney Disease Quality of Life Short Form (KDQOL-SF) by the hemodialysis patients on the enrollment day and after 6 weeks of treatment. As above, the quality-of-life score was based on the patients’ overall symptoms without differentiating between the sericin cream and cream base treatments. On the enrollment day, the mean scores ranged from 43.05 for general health to 83.84 for the domain related to dialysis staff encouragement. After 6 weeks of treatment, the mean scores ranged from 44.21 for kidney disease burden to 87.50 for encouragement by the dialysis staff. We found a better quality of life in all the measured domains, including sleep and mood/emotional distress, after the treatment period. When the mean score on the enrollment day was compared with the mean score on the day after completion of treatment, significant differences were found in some domains, including pain (p = 0.001), the symptoms/problems list in kidney disease (p = 0.000), the effect of kidney disease on daily life (p = 0.008) and sleep, the most relevant parameter for itching (p = 0.014). The overall score increased from 60.00 at the time of enrollment to 61.95 after 6 weeks of treatment, although this difference was not statistically significant (p = 0.091).
Mean scores for each domain of the KDQOL-SF in hemodialysis patients on the enrollment day and after 6 weeks of treatment (N = 47)
* Indicates significant differences compared to baseline, p < 0.05.
Discussion:
This study has shown that the use of sericin cream can reduce UP in hemodialysis patients according to their VAS scores. Sericin cream can also significantly increase skin hydration and reduce skin irritation and skin pigmentation in patients. The molecular weight analysis of the sericin used in this study gave results that agree with those reported by Sprague, who indicated that sericin is a mixture of at least 15 different polypeptide chains ranging in size from 20–220 kDa [19]. Similar to the earlier report by Kato et al., serine was the most abundant amino acid found in sericin [20].
Although antihistamines, anti-allergic agents and topical corticosteroids are commonly used to treat UP in ESRD patients, their effectiveness is sometimes limited [2,8,21,22]. Alternative and effective treatments for intractable pruritus that generate low levels of adverse reactions, particularly in sensitive patients such as the ESRD group, must therefore be developed. This clinical study is the first to suggest that a sericin cream is beneficial for treating pruritus in hemodialysis patients. The benefits of this treatment apparently result from increases in skin hydration and suppression of pro-inflammatory cytokines, as shown in our earlier study [17,23], thereby resulting in less skin irritation without any allergic reactions.
Pruritus associated with chronic kidney disease has been shown to be associated with elevated levels of C-reactive protein and other inflammatory cytokines. This evidence suggests that there is an inflammatory component in this form of pruritus [24]. Some authors consider UP to be a skin manifestation of chronic inflammation [25]. Recently, the “persistent microinflammation” theory, in which it is suggested that inflammation may be related to the genesis of UP, was proposed [26]. The skin of ESRD patients with UP contains an increased number of mast cells, and these cells can release various substances such as histamine, interleukins (IL) and tumor necrosis factor (TNF) [27-30].
Pruritic skin can appear normal except for dryness; because the severity of pruritus is closely correlated with skin dryness [31], xerosis treatment normally begins with a topical agent such as a gentle moisturizing cream. Because sericin is well known for its moisturizing effect and its ability to reduce the generation of pro-inflammatory cytokines by fibroblasts, this study assessed the usefulness of sericin cream in treating UP. Evaluating the degree of pruritus was difficult because it is a subjective symptom; therefore, functional measurements of skin conditions known to be associated with pruritus, such as skin hydration and skin irritation, were considered to be objective indices. We found that both sericin cream and the cream base used to prepare the sericin cream increased the moisture content of the stratum corneum; however, the level of hydration was significantly higher in the skin treated with sericin cream. The side of the body treated with sericin cream had significantly lower levels of post-treatment dryness compared to pretreatment dryness and lower total dermatological severity scores in parameters such as skin pigmentation.
The pathogenesis of ESRD itself is associated with inflammation and oxidative stress, as reflected in studies of plasma biochemistry, including the levels of the cytokines IL and TNF-α [32-34], classic markers of inflammatory processes that are strongly affected by pathological conditions [35]. These cytokines are upregulated in the plasma of ESRD patients [32,36]. The skin inflammation associated with the release of IL and TNF-α is further complicated by the inflammatory lesions that occur secondary to scratching [37]. Our results indicate that improvement in skin hydration and suppression of the release of pro-inflammatory cytokines in skin might be the mechanism by which sericin improves UP. Skin irritation, which can be caused by either skin inflammation or by scratching, was substantially reduced in the sericin group compared with the cream base group after 6 weeks of treatment. This result confirmed that sericin reduces skin irritation in patients with UP and suggested that it might be due to suppression of the inflammatory cytokines in skin. Similar results have been obtained in animal studies [23].
Secondary skin lesions that can result from the itch-scratch cycle include excoriations, hyperpigmentation, lichenification, prurigo nodules and scars [38]. Chronic renal failure is usually accompanied by a variety of cutaneous manifestations; dermal manifestations, including hyperpigmentation, have also been reported, and these can exact a considerable toll on the quality of life [39-41]. Although skin pigmentation is not related to specific clinical symptoms, it has an important effect on patient satisfaction. Sericin has been reported to have activity against tyrosinase [15], an enzyme related to melanin production; as this study shows, treatment with sericin results in significant reduction in the skin color of patients.
The pruritus/mortality relationship may be strongly attributed to sleep disturbances, as previously mentioned in the DOPPS [8,9,42]. Many previous studies have found a strong association between inflammation, as measured by C-reactive protein or inflammatory cytokines, and sleep disturbances in dialysis patients [43-45]. The significant effects of pruritus on sleep, mood and social functioning require further investigation with the goal of improving the available treatments for this serious ESRD complication. We found that reduction in itching intensity from moderate to severe pruritus at the time of enrollment to mild pruritus after 6 weeks of treatment was associated with a better quality of life in all of the measured domains, including the mental, physical and kidney disease components. The KDQOL-SF score in the ESRD patients indicated improvements in several domains regarding the patients’ quality of life, particularly pain and sleep, which are relevant to itching. A better quality of sleep may reflect some degree of relief from itching achieved through the treatments.
This study has certain limitations. First, the study design was an in-subject controlled study; it cannot be determined whether the improvements in the itching evaluation and the quality of life were from the sample (sericin cream) or the placebo (cream base). Moreover, due to the in-subject controlled design, the biochemical parameters could not be evaluated at the end of the study. Second, the study included a small number of patients, and these were followed for a relatively short time. A long-term study with a larger sample size is necessary. In addition to studies of hemodialysis patients, similar studies of other ESRD patients, such as peritoneal dialysis and kidney transplant patients, are also necessary to confirm the findings presented here.
Conclusions:
Our study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients.
Methods:
Preparation of silk sericin cream Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.
Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.
Molecular weight determination of sericin To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.
To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.
Amino acid analysis of sericin The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.
The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.
Study design Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.
Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.
Study population Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.
All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.
Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.
Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.
Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.
All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.
Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.
Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.
Study treatment In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.
In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.
Measurement and outcome The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:
(1)
%
changes in each parameter
=
P
t
−
P
0
/
P
0
×
100
,
where P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.
Because itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].
The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:
(1)
%
changes in each parameter
=
P
t
−
P
0
/
P
0
×
100
,
where P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.
Because itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].
Patients’ quality of life The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].
The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].
Safety monitoring The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.
The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.
Statistical analysis The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.
The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.
Preparation of silk sericin cream:
Because commercial sericin cream is unavailable, the sericin cream used in this study was prepared from raw materials. Bombyx mori cocoons were purchased from Chul Thai Silk Co., Ltd. (Petchaboon province, Thailand). The cocoons were extracted with purified water (1 g of dry silk cocoon: 30 mL of water) using a high temperature and pressure degumming technique in an autoclave (SS-320, Tomy Seiko Co., Ltd., Tokyo, Japan) at 121°C and 15 psi for 60 min. This technique has been shown to be safe for the preparation of material used on keratinocyte and fibroblast cells and can activate high collagen production related to wound healing [46]. After filtration through a membrane (Whatman filter paper No. 1, Whatman PLC, Kent, United Kingdom) to remove fibroin, sericin powder was obtained by freezing and lyophilizing the sericin solution with a Heto LL3000 lyophilizer (Allrod, Denmark). Petroleum jelly, mineral oil, lanolin, glycerin, bisabolol, triethanolamine stearate, propylparaben and methylparaben were used to formulate a cream base. For an 8% sericin cream, a concentration that has been shown to be safe and effective in the treatment of second-degree burn wounds, the sericin powder was dissolved in warm water and then mixed with the other ingredients during the cream-forming process.
Molecular weight determination of sericin:
To determine the molecular weight of sericin, SDS-PAGE was performed as previously described, with some modifications [47]. Briefly, samples were prepared for SDS-PAGE by adding an equal volume of sample buffer (0.25 M Tris–HCl, pH 7.0 containing 4% SDS, 10% sucrose, 10% 2-mercaptoethanol and 0.025% bromophenol blue) to each protein solution. Each sample was then incubated at 98°C for 2–3 min and loaded onto a 5%-20% gradient gel (Atto Corporation, Tokyo, Japan). Electrophoresis was performed in 125 mM Tris base with 0.96 M glycine and 0.5% SDS, and the polypeptide bands were detected using silver staining.
Amino acid analysis of sericin:
The amino acid composition of sericin was determined using an amino acid analyzer (Hitachi L-8500A, Tokyo, Japan). Samples for analysis were hydrolyzed in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 100°C for 24 h under vacuum. The experiments were performed in triplicate.
Study design:
Skin hydration may be related to pruritus, and this parameter is sensitive to relative humidity, personal activities and diet. Therefore, an in-subject control using a split-body biometrological assessment (each patient received both treatments but on different sides of the body) was used to evaluate the safety and efficacy of the sericin cream for the treatment of UP in hemodialysis patients. Moreover, the distribution of pruritus between the patients was highly variable whereas the manifestation of mirror symmetry was an attribute they all shared [48]. An in-subject, randomized, double-blind, placebo-controlled experimental study was designed to investigate the effects of the sericin cream versus the cream base (placebo) in reducing the symptoms of UP (itching, dryness and redness) and skin pigmentation in stable maintenance hemodialysis patients. Each of the parameters, including skin hydration, skin irritation, skin pigmentation and itching score, was evaluated at baseline and at 2, 4 and 6 weeks after treatment intervention. The sericin cream and the cream base were identical in texture and scent. All of the products were packaged in containers that were label-free except for the treatment code number, and the packages were identical in shape, size and color; therefore, the treatment assignment remained unknown to the participants, the study investigators and the medical personnel. The subjects were recruited from December 2010 to February 2011, and the study was conducted between March 2011 and December 2011 at the Division of Nephrology, Phramongkutklao Hospital and at Priest Hospital, Thailand. Signed informed consent was obtained from all subjects after a thorough discussion of the protocol, its rationale and the potential risks. This study was approved by the Ethics Committee of the Institute Review Board at Phramongkutklao Hospital, Thailand and ended after the last participant completed the intervention.
Study population:
Inclusion criteria All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.
All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.
Exclusion criteria Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.
Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.
Inclusion criteria:
All ESRD patients at Phramongkutklao and Priest Hospitals over 18 years of age who had received hemodialysis for at least 3 months were screened for this study. Having mild to severe pruritus as measured by the VAS during the previous 6 weeks was also an inclusion criterion. The patients were required to refrain from using any antipruritic treatment (oral or topical) for a period of not less than 2 weeks prior to the start of the study. Patients of both genders, regardless of comorbidities or prescribed medications, were eligible. Any medication that had an antipruritic effect was discontinued 2 weeks before the study. No changes in the patients’ prescription medications were required during this study with the exception of a concomitant antipruritic treatment.
Exclusion criteria:
Pruritus caused by other skin diseases or medication was excluded by careful clinical assessment. Patients with a history of silk protein allergy, who were allergic to any compounds in the formula, or who had biliary atresia, liver problems, cancer, metabolic disorders or other diseases related to systemic pruritus were also excluded. Patients who had skin problems or rashes on their extremities (arms or legs) were also excluded from this study. Participants left the project when they could not comply with the treatment, when they were unwilling to continue with the study, or when the physician opined that other treatments were needed to relieve the symptoms. After reviewing patient profiles and explaining the protocol, 27 patients were excluded due to liver problems (N = 4), cancer (N = 3), metabolic disorder (N = 7), rashes on their extremities (N = 6) and refusal to participate in the study (N = 7). The remaining patients (N = 50) were enrolled in the study.
Study treatment:
In 2004, Okada and Matsumoto [49] evaluated the effect of an emollient containing a high water content on mild uremic pruritus; based on this study, the number of samples needed for a dependent sample was approximately 50 subjects. Split-body biometrological assessments were performed. The physician investigator enrolled the subjects into this study, and using a computer-generated block of four, another investigator generated the random allocation sequence that divided the patients into two groups. The identities of the patients in each group were concealed from both the investigators and the patients. The on-duty nurses assigned the participants to the intervention. The participants, investigators and those assessing the outcomes were blinded after assignment to the interventions. The patients in the first group received the sericin cream on their left extremities (left arm and left leg), while the other side of the body received the cream base. The patients in the second group received both the sericin cream and the cream base, but on opposite sides of the body from the first group. All of the patients were shown how to topically apply the assigned treatment evenly over the area indicated twice daily for a period of 6 weeks after showering.
Measurement and outcome:
The level of skin hydration on the arms and legs was assessed using a Corneometer® (KOKO Kosmetikvertrieb GmbH & Co., Leichlingen, Deutschland). The Corneometer® registers the moisture content in the surface layers of the skin as deep as 10–20 μm; the presence of capillary blood vessels and superficial skin fat do not influence this measurement. Skin irritation or erythema (measured by the redness of the skin) and skin pigmentation (measured by the melanin content) were assessed using a Mexameter linked to a Skin Diagnostic SD27 (Courage + Khazaka electronic GmbH, Köln, Germany). The measurement of melanin and the erythema readings are based on a light source with three specific wavelengths; the radiation is absorbed by the skin and reflected diffusely. A photodetector was used to analyze the diffuse reflection from the skin. The same measuring probe is used to quantify the skin redness (erythema) and to determine the skin pigmentation or the degree of skin darkness (melanin). The irritating effects of substances and the soothing effects of active agents can also be recorded by the investigator. Skin hydration, irritation and pigmentation values have no units because they are computer- generated based on the different dielectric constants of water (80.10 at 20°C) and other substances (typically < 7). The measuring capacitor shows changes of capacitance according to the moisture content of the samples (for hydration) and skin color (for irritation and pigmentation). Each parameter was measured at least three times in the same randomized area at patients’ extremities, and the mean value was used for the analysis. During the study, the patients were advised to consume similar types and amounts of food and beverages. Activities such as longer exposure to the sunlight and traveling were to be avoided to reduce any confounding factors. The percent changes in each parameter were calculated by subtracting the baseline score from the post-treatment scores at weeks 2, 4 and 6 according to the following equation:
(1)
%
changes in each parameter
=
P
t
−
P
0
/
P
0
×
100
,
where P0 is the value of each parameter at baseline (at the time of enrollment) and Pt is the value of each parameter during the follow-up period (2, 4 or 6 weeks). All of the measurements were performed in triplicate.
Because itching is a systemic symptom and likely to be generalized, most patients could not identify whether the itching occurred primarily on the right or left side of the body; therefore, itching was scored as an overall symptom. The severity of itching was systemically assessed on both the arms and legs of all patients using VAS on the enrollment day and every 2 weeks after treatment began. We used a VAS that consisted of a 10-cm horizontal line with no scale markings. The patients were asked to mark the intensity of their itching on the scale, with the strongest possible level of itching or unbearable pruritus marked on the right end of the line (10 cm) and no itching marked on the left end (0 cm) [50].
Patients’ quality of life:
The patients’ quality of life was assessed using the Thai version of the KDQOL-SF Version 1.3 [51]. Quality of life was evaluated on the enrollment day and after 6 weeks of treatment. The mean scores for the individual domain scores and for the three composite summary scores, which include the mental component score (MCS), the physical component score (PCS) and the kidney disease component score (KDCS), were compared as shown in Table 4. For the Hayes algorithm [52], the raw data obtained from the patients were first transformed into a pre-coded numeric value of 0–100; a higher transformed score reflected a better quality of life [51,53].
Safety monitoring:
The occurrence of allergic reactions during treatment with sericin cream was regularly evaluated by two dermatologists during each visit. The Naranjo algorithm was used to determine the likelihood of whether an adverse drug reaction was actually caused by the sericin cream or by other factors.
Statistical analysis:
The results are expressed as the mean ± SD unless otherwise indicated. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). A bidirectional α-level of significance was set at p = 0.05 for all of the measurements. From the baseline to weeks 2, 4 and 6, the VAS score changes and the levels of skin hydration, irritation and pigmentation were computed for each patient within the treatment group using a repeated measure analysis of variance (ANOVA). The paired t-test was used to analyze changes in the patients’ quality of life between baseline and 6 weeks after treatment. The differences in each parameter for the patients receiving the sericin cream and the cream base were compared at each time point using Student’s t-test.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
PA had full access to all of the data in the study, accepts responsibility for the integrity of the data and affirms that everyone who contributed significantly to the work has been listed. PA and OS developed the clinical study design and the experimental design. OK, TS and NB recruited the subjects and conducted the skin evaluation, patient consent, analysis and data interpretation. PA drafted the manuscript. All authors read the manuscript, provided critical input and approved the final version.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2369/13/119/prepub
|
Background:
Uremic pruritus (UP) is a significant complication in ESRD patients and substantially impairs their quality of life. UP is considered to be a skin manifestation of chronic inflammation. Because sericin can suppress the release of pro-inflammatory cytokines, the purpose of this study was to investigate the short-term safety and efficacy of sericin cream for treating UP in hemodialysis patients.
Methods:
This study used a double-blind design to investigate the effects of random topical administration of sericin cream and cream base (placebo) on either the right or left extremities of hemodialysis patients for 6 weeks. Skin hydration, irritation and pigmentation were evaluated every 2 weeks using Skin Diagnostic SD27. The visual analog scale for itching was also evaluated every 2 weeks, and the Kidney Disease Quality of Life Short Form was performed on the day of each patient's enrollment and after 6 weeks of treatment.
Results:
Fifty dialysis patients were enrolled, 47 of which completed the study. The hydration of the skin of the patients' extremities increased significantly after administration of sericin cream; significant differences were found between sericin treatment and control after 6 weeks of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Moreover, a significant difference was also found in skin irritation between the two treatments (p = 0.013 for arms and p = 0.027 for legs, respectively). At the end of the study, the skin pigmentation level was significantly reduced on both the arms (p = 0.032) and legs (p = 0.021) of the sericin-treated side compared with the side treated with cream base. The mean itching score decreased significantly from moderate to severe at the time of enrollment to mild pruritus after 6 weeks of treatment (p = 0.002). A better quality of life was found in all domains tested although statistically significant differences before and after treatment was found only in the patients' pain scores, the effect of kidney disease on daily life, sleep quality and symptoms or problems related to kidney disease.
Conclusions:
We conclude that sericin cream has a high potential for reducing UP in hemodialysis patients.The trial registration number of this study is ISRCTN16019033; its public title is "sericin cream reduces pruritus in hemodialysis patients".
|
Background:
Itching associated with end-stage renal disease (ESRD) or uremic pruritus (UP) affects between 20 and 50% of renal failure patients [1-3] who have no primary skin disease or systemic or psychological dysfunction that might cause pruritus. In addition, approximately 80% of patients undergoing hemodialysis were found to be affected by UP [4-6]. Unfortunately, dialysis has only a slight impact on pruritus [7].
Pruritus is an unpleasant symptom that negatively impacts a patient’s quality of life. The impact of moderate/severe uremic pruritus on the mortality of patients with ESRD seems to be associated with sleep disturbance rather than with uremic pruritus per se[8,9]. Moreover, in the recent Dialysis Outcomes and Practice Patterns Study II (DOPPS II) trial, pruritus was associated with depression, sleep disturbance and increased mortality risk [8,10]. The pathophysiology of UP is still unclear; many hypotheses have been proposed to explain its occurrence, including xerosis and hypohidrosis (a condition in which the skin is usually atrophic and dry), the presence of pruritogenic cytokines (histamine, kallikrein, interleukin [IL]-2, acetylcholine and other substances that are released by histamine-mediated mast cell stimulation and may lower the UP threshold), secondary hyperparathyroidism, immune-inflammatory reactions in which sericin cream is thought to play a central role in producing an anti-inflammatory effect, the uremic neuropathy hypothesis and the opioid hypothesis (k-opioid receptor understimulation and overexpression of μ-opioid receptors) [7,8].
Sericin, a biopolymer with a high molecular weight, is a water-soluble protein that is obtained from the silkworm (Bombyx mori) [11]. Sericin is characterized by the presence of 32% serine, which is the main amino acid of the natural moisture factor (NMF) in human skin; therefore, sericin has excellent moisturizing properties that may be helpful for treating hypohidrosis. Sericin also shows many biological activities and has been widely studied for potential use in medicines and biomaterials [12-16]. In 2009, Aramwit et al. showed that sericin significantly decreased the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in sericin-treated wounds in rats 7 days after an injury compared with the levels found in normal saline-soaked wounds and cream base-treated wounds [17]. As previously mentioned, the immune-inflammatory hypothesis considers UP a dermatologic manifestation of chronic inflammation and treats the condition as a possible result of derangements in the immune system that are based on a pro-inflammatory pattern. Based on this reasoning, sericin may help to relieve UP.
At present, there is no widely accepted treatment for UP. Topical products, such as moisturizers and emollients, are typically used to alleviate symptoms. Because sericin can suppress the release of pro-inflammatory cytokines, the present study was designed to investigate the short-term safety and efficacy of sericin cream for treating UP in ESRD patients. The quality of life of ESRD patients after using sericin cream was also evaluated.
Conclusions:
Our study shows that sericin reduces pruritus in patients with UP. The use of sericin cream significantly increased the level of skin hydration after 6 weeks of treatment compared to baseline and to the use of the cream base. The use of sericin cream also significantly reduced the level of skin irritation and pigmentation after 6 weeks of treatment compared to baseline, while use of the cream base reduced skin pigmentation slightly but not significantly. The results of this study suggest that sericin cream may be a good choice for treating pruritus in hemodialysis patients.
|
Background:
Uremic pruritus (UP) is a significant complication in ESRD patients and substantially impairs their quality of life. UP is considered to be a skin manifestation of chronic inflammation. Because sericin can suppress the release of pro-inflammatory cytokines, the purpose of this study was to investigate the short-term safety and efficacy of sericin cream for treating UP in hemodialysis patients.
Methods:
This study used a double-blind design to investigate the effects of random topical administration of sericin cream and cream base (placebo) on either the right or left extremities of hemodialysis patients for 6 weeks. Skin hydration, irritation and pigmentation were evaluated every 2 weeks using Skin Diagnostic SD27. The visual analog scale for itching was also evaluated every 2 weeks, and the Kidney Disease Quality of Life Short Form was performed on the day of each patient's enrollment and after 6 weeks of treatment.
Results:
Fifty dialysis patients were enrolled, 47 of which completed the study. The hydration of the skin of the patients' extremities increased significantly after administration of sericin cream; significant differences were found between sericin treatment and control after 6 weeks of treatment (p = 0.041 for arms and p = 0.022 for legs, respectively). Moreover, a significant difference was also found in skin irritation between the two treatments (p = 0.013 for arms and p = 0.027 for legs, respectively). At the end of the study, the skin pigmentation level was significantly reduced on both the arms (p = 0.032) and legs (p = 0.021) of the sericin-treated side compared with the side treated with cream base. The mean itching score decreased significantly from moderate to severe at the time of enrollment to mild pruritus after 6 weeks of treatment (p = 0.002). A better quality of life was found in all domains tested although statistically significant differences before and after treatment was found only in the patients' pain scores, the effect of kidney disease on daily life, sleep quality and symptoms or problems related to kidney disease.
Conclusions:
We conclude that sericin cream has a high potential for reducing UP in hemodialysis patients.The trial registration number of this study is ISRCTN16019033; its public title is "sericin cream reduces pruritus in hemodialysis patients".
| 16,613 | 447 | 22 |
[
"skin",
"cream",
"patients",
"treatment",
"sericin",
"study",
"weeks",
"sericin cream",
"baseline",
"base"
] |
[
"test",
"test"
] |
[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]
|
[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]
|
[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]
|
[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]
|
[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]
|
[CONTENT] Pruritus | Hemodialysis | Sericin | Hydration | Inflammation | Pigmentation [SUMMARY]
|
[CONTENT] Administration, Topical | Double-Blind Method | Female | Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome [SUMMARY]
|
[CONTENT] Administration, Topical | Double-Blind Method | Female | Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome [SUMMARY]
|
[CONTENT] Administration, Topical | Double-Blind Method | Female | Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome [SUMMARY]
|
[CONTENT] Administration, Topical | Double-Blind Method | Female | Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome [SUMMARY]
|
[CONTENT] Administration, Topical | Double-Blind Method | Female | Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome [SUMMARY]
|
[CONTENT] Administration, Topical | Double-Blind Method | Female | Humans | Male | Middle Aged | Placebo Effect | Pruritus | Quality of Life | Renal Dialysis | Sericins | Skin Cream | Treatment Outcome [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] skin | cream | patients | treatment | sericin | study | weeks | sericin cream | baseline | base [SUMMARY]
|
[CONTENT] skin | cream | patients | treatment | sericin | study | weeks | sericin cream | baseline | base [SUMMARY]
|
[CONTENT] skin | cream | patients | treatment | sericin | study | weeks | sericin cream | baseline | base [SUMMARY]
|
[CONTENT] skin | cream | patients | treatment | sericin | study | weeks | sericin cream | baseline | base [SUMMARY]
|
[CONTENT] skin | cream | patients | treatment | sericin | study | weeks | sericin cream | baseline | base [SUMMARY]
|
[CONTENT] skin | cream | patients | treatment | sericin | study | weeks | sericin cream | baseline | base [SUMMARY]
|
[CONTENT] inflammatory | sericin | uremic | pruritus | hypothesis | opioid | immune | associated | pro | pro inflammatory [SUMMARY]
|
[CONTENT] patients | skin | study | cream | sericin | treatment | excluded | weeks | sericin cream | itching [SUMMARY]
|
[CONTENT] cream | skin | baseline | significant | legs | arms | treatment | hydration | weeks | sericin [SUMMARY]
|
[CONTENT] use | cream | baseline use cream | baseline use cream base | compared baseline use | compared baseline use cream | treatment compared baseline use | baseline use | significantly | sericin [SUMMARY]
|
[CONTENT] cream | patients | skin | sericin | study | treatment | sericin cream | weeks | pruritus | baseline [SUMMARY]
|
[CONTENT] cream | patients | skin | sericin | study | treatment | sericin cream | weeks | pruritus | baseline [SUMMARY]
|
[CONTENT] ESRD ||| ||| [SUMMARY]
|
[CONTENT] 6 weeks ||| every 2 weeks | Skin Diagnostic ||| every 2 weeks | the Kidney Disease Quality of Life Short Form | the day | 6 weeks [SUMMARY]
|
[CONTENT] Fifty | 47 ||| 6 weeks | 0.041 | 0.022 ||| two | 0.013 | 0.027 ||| 0.032 | 0.021 ||| 6 weeks | 0.002 ||| [SUMMARY]
|
[CONTENT] ||| [SUMMARY]
|
[CONTENT] ESRD ||| ||| ||| 6 weeks ||| every 2 weeks | Skin Diagnostic ||| every 2 weeks | the Kidney Disease Quality of Life Short Form | the day | 6 weeks ||| ||| Fifty | 47 ||| 6 weeks | 0.041 | 0.022 ||| two | 0.013 | 0.027 ||| 0.032 | 0.021 ||| 6 weeks | 0.002 ||| ||| ||| [SUMMARY]
|
[CONTENT] ESRD ||| ||| ||| 6 weeks ||| every 2 weeks | Skin Diagnostic ||| every 2 weeks | the Kidney Disease Quality of Life Short Form | the day | 6 weeks ||| ||| Fifty | 47 ||| 6 weeks | 0.041 | 0.022 ||| two | 0.013 | 0.027 ||| 0.032 | 0.021 ||| 6 weeks | 0.002 ||| ||| ||| [SUMMARY]
|
||||||
Particulate matter air pollution and respiratory symptoms in individuals having either asthma or chronic obstructive pulmonary disease: a European multicentre panel study.
|
23039312
|
Particulate matter air pollution has been associated with adverse health effects. The fraction of ambient particles that are mainly responsible for the observed health effects is still a matter of controversy. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.The RUPIOH study, an EU-funded multicentre study, was designed to examine the distribution of various ambient particle metrics in four European cities (Amsterdam, Athens, Birmingham, Helsinki) and assess their health effects in participants with asthma or COPD, based on a detailed exposure assessment. In this paper the association of central site measurements with respiratory symptoms and restriction of activities is examined.
|
BACKGROUND
|
At each centre a panel of participants with either asthma or COPD recorded respiratory symptoms and restriction of activities in a diary for six months. Exposure assessment included simultaneous measurements of coarse, fine and ultrafine particles at a central site. Data on gaseous pollutants were also collected. The associations of the 24-hour average concentrations of air pollution indices with the health outcomes were assessed in a hierarchical modelling approach. A city specific analysis controlling for potential confounders was followed by a meta-analysis to provide overall effect estimates.
|
METHODS
|
A 10 μg/m3 increase in previous day coarse particles concentrations was positively associated with most symptoms (an increase of 0.6 to 0.7% in average) and limitation in walking (OR= 1.076, 95% CI: 1.026-1.128). Same day, previous day and previous two days ozone concentrations were positively associated with cough (OR= 1.061, 95% CI: 1.013-1.111; OR= 1.049, 95% CI: 1.016-1.083 and OR= 1.059, 95% CI: 1.027-1.091, respectively). No consistent associations were observed between fine particle concentrations, nitrogen dioxide and respiratory health effects. As for particle number concentrations negative association (mostly non-significant at the nominal level) was observed with most symptoms whilst the positive association with limitation of activities did not reach the nominal level of significance.
|
RESULTS
|
The observed associations with coarse particles are in agreement with the findings of toxicological studies. Together they suggest it is prudent to regulate also coarse particles in addition to fine particles.
|
CONCLUSIONS
|
[
"Adult",
"Aged",
"Aged, 80 and over",
"Air Pollutants",
"Air Pollution",
"Asthma",
"Cities",
"Europe",
"Female",
"Humans",
"Male",
"Middle Aged",
"Nitrogen Dioxide",
"Odds Ratio",
"Ozone",
"Particulate Matter",
"Pulmonary Disease, Chronic Obstructive",
"Respiration Disorders",
"Walking"
] |
3509003
|
Background
|
Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects
[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)
[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy
[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals
[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion
[9,12-14].
The only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked
[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.
The RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before
[16-20].
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Methods
|
Study design In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication
[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health
[21]. In all centres, participants were recruited between October 2002 and March 2004.
In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication
[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health
[21]. In all centres, participants were recruited between October 2002 and March 2004.
Study population Inclusion criteria and recruitment procedures have been described in detail before
[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months
[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)
[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.
Medical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.
Inclusion criteria and recruitment procedures have been described in detail before
[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months
[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)
[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.
Medical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.
Symptom diary The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study
[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops
[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.
During the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.
The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study
[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops
[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.
During the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.
Air pollution exposure Exposure assessment has been described in previous publications
[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels
[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.
Exposure assessment has been described in previous publications
[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels
[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.
Confounder data Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).
Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).
Quality assurance/quality control Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.
Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.
Statistical analysis Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.
A hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates
[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.
We applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.
In the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.
Because of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.
Effect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software
[27].
Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.
A hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates
[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.
We applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.
In the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.
Because of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.
Effect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software
[27].
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Results
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Panel characteristics A brief description of the study population is presented in Table
1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.
Characteristics of four European panels of asthmatic/COPD patients
a Total participants in panel.
b Asthma + COPD or chronic non-specific lung disease.
c Given as mean and [range].
d Chronic non-specific lung disease.
e Environmental tobacco smoke.
f Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.
A brief description of the study population is presented in Table
1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.
Characteristics of four European panels of asthmatic/COPD patients
a Total participants in panel.
b Asthma + COPD or chronic non-specific lung disease.
c Given as mean and [range].
d Chronic non-specific lung disease.
e Environmental tobacco smoke.
f Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.
Symptoms In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table
2).
Person days with symptoms in the diary (n = number of expected person days)
a due to breathing problems.
In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table
2).
Person days with symptoms in the diary (n = number of expected person days)
a due to breathing problems.
Air pollution concentrations Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table
3).
Daily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities
Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table
3).
Daily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities
Air pollution effects on symptoms-limitation in activities due to breathing problems Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables
4 and
5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table
6).
Associations of particulate matter indices, NO
2
and O
3
with prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of particulate matter indices, NO
2
and O
3
with limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of PM
10-2.5
and PM
2.5
with prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)
Bold are significant pooled effects.
The above-mentioned positive associations with PM10-2.5 (Tables
4 and
5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).
Ozone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables
4 and
5).
Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables
4 and
5).
Centre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure
1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.
Odds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m
3
in previous day (lag1) concentrations of each pollutant (10,000/cm
3
for PNC) in each participating city and overall estimate (random effects pooled estimates).
We observed very small differences in fixed and random effects combined estimates. In Tables
4 and
5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table
6).
Associations of particulate matter indices, NO
2
and O
3
with prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of particulate matter indices, NO
2
and O
3
with limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of PM
10-2.5
and PM
2.5
with prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)
Bold are significant pooled effects.
The above-mentioned positive associations with PM10-2.5 (Tables
4 and
5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).
Ozone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables
4 and
5).
Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables
4 and
5).
Centre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure
1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.
Odds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m
3
in previous day (lag1) concentrations of each pollutant (10,000/cm
3
for PNC) in each participating city and overall estimate (random effects pooled estimates).
Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables
4 and
5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table
6).
Associations of particulate matter indices, NO
2
and O
3
with prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of particulate matter indices, NO
2
and O
3
with limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of PM
10-2.5
and PM
2.5
with prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)
Bold are significant pooled effects.
The above-mentioned positive associations with PM10-2.5 (Tables
4 and
5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).
Ozone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables
4 and
5).
Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables
4 and
5).
Centre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure
1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.
Odds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m
3
in previous day (lag1) concentrations of each pollutant (10,000/cm
3
for PNC) in each participating city and overall estimate (random effects pooled estimates).
We observed very small differences in fixed and random effects combined estimates. In Tables
4 and
5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table
6).
Associations of particulate matter indices, NO
2
and O
3
with prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of particulate matter indices, NO
2
and O
3
with limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of PM
10-2.5
and PM
2.5
with prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)
Bold are significant pooled effects.
The above-mentioned positive associations with PM10-2.5 (Tables
4 and
5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).
Ozone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables
4 and
5).
Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables
4 and
5).
Centre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure
1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.
Odds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m
3
in previous day (lag1) concentrations of each pollutant (10,000/cm
3
for PNC) in each participating city and overall estimate (random effects pooled estimates).
Incidence analyses Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table
7).
Associations of particulate matter indices, NO
2
and O
3
with incidence of symptoms in the four panels (random effects pooled estimates)
Bold are significant pooled effects.
Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table
7).
Associations of particulate matter indices, NO
2
and O
3
with incidence of symptoms in the four panels (random effects pooled estimates)
Bold are significant pooled effects.
|
Conclusions
|
Our study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles.
|
[
"Background",
"Study design",
"Study population",
"Symptom diary",
"Air pollution exposure",
"Confounder data",
"Quality assurance/quality control",
"Statistical analysis",
"Panel characteristics",
"Symptoms",
"Air pollution concentrations",
"Air pollution effects on symptoms-limitation in activities due to breathing problems",
"Prevalence analyses",
"Incidence analyses",
"Abbreviations",
"Competing interests",
"Authors’ contributions"
] |
[
"Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20].",
"In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.",
"Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.",
"The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.",
"Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.",
"Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).",
"Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.",
"Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].",
"A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.",
"In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.",
"Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities",
" Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n",
"We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n",
"Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.",
"AIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.",
"The authors declare that they have no competing interests.",
"All authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions."
] |
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[
"Background",
"Methods",
"Study design",
"Study population",
"Symptom diary",
"Air pollution exposure",
"Confounder data",
"Quality assurance/quality control",
"Statistical analysis",
"Results",
"Panel characteristics",
"Symptoms",
"Air pollution concentrations",
"Air pollution effects on symptoms-limitation in activities due to breathing problems",
"Prevalence analyses",
"Incidence analyses",
"Discussion",
"Conclusions",
"Abbreviations",
"Competing interests",
"Authors’ contributions"
] |
[
"Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects\n[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)\n[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy\n[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals\n[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion\n[9,12-14].\nThe only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked\n[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.\nThe RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before\n[16-20].",
" Study design In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\nIn the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.\n Study population Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\nInclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.\n Symptom diary The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\nThe diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.\n Air pollution exposure Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\nExposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.\n Confounder data Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\nTime trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).\n Quality assurance/quality control Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\nAir pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.\n Statistical analysis Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].\nData analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].",
"In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication\n[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health\n[21]. In all centres, participants were recruited between October 2002 and March 2004.",
"Inclusion criteria and recruitment procedures have been described in detail before\n[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months\n[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)\n[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.\nMedical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.",
"The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study\n[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops\n[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.\nDuring the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.",
"Exposure assessment has been described in previous publications\n[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels\n[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.",
"Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).",
"Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.",
"Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.\nA hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates\n[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.\nWe applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.\nIn the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.\nBecause of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.\nEffect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software\n[27].",
" Panel characteristics A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\nA brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.\n Symptoms In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\nIn total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.\n Air pollution concentrations Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\nHelsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities\n Air pollution effects on symptoms-limitation in activities due to breathing problems Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\n Incidence analyses Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.\nPatterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.",
"A brief description of the study population is presented in Table\n1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.\nCharacteristics of four European panels of asthmatic/COPD patients\na Total participants in panel.\nb Asthma + COPD or chronic non-specific lung disease.\nc Given as mean and [range].\nd Chronic non-specific lung disease.\ne Environmental tobacco smoke.\nf Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.",
"In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table\n2).\nPerson days with symptoms in the diary (n = number of expected person days)\na due to breathing problems.",
"Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table\n3).\nDaily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities",
" Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n\nWe observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n",
"We observed very small differences in fixed and random effects combined estimates. In Tables\n4 and\n5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table\n6).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)\n\nBold are significant pooled effects.\n\nAssociations of PM\n\n10-2.5 \n\nand PM\n\n2.5 \n\nwith prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)\n\nBold are significant pooled effects.\nThe above-mentioned positive associations with PM10-2.5 (Tables\n4 and\n5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).\nOzone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables\n4 and\n5).\nNeither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables\n4 and\n5).\nCentre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure\n1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.\n\nOdds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m\n\n3 \n\nin previous day (lag1) concentrations of each pollutant (10,000/cm\n\n3 \n\nfor PNC) in each participating city and overall estimate (random effects pooled estimates).\n",
"Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table\n7).\n\nAssociations of particulate matter indices, NO\n\n2 \n\nand O\n\n3 \n\nwith incidence of symptoms in the four panels (random effects pooled estimates)\n\nBold are significant pooled effects.",
"In this multicentre study we found consistent positive associations between coarse particles central sites concentrations and prevalence of respiratory symptoms, as recorded in a 6-month diary, in four panels of participants with predominantly mild to moderate asthma or COPD in four European cities participating in the RUPIOH study. We also found a significant association of ozone with cough and woken with breathing problems, but not with other symptoms. Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst positive associations with woken with breathing problems and cough as well as with limitation of vigorous and moderate activities due to breathing problems, did not reach the nominal level of significance. Interestingly, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant at the nominal level. An analysis of the asthmatic subgroup showed generally lower odds ratios for PM10-2.5.\nOne particularity and strength of the RUPIOH study is the in depth assessment of particulate air pollution by measuring PM10, PM2.5 (then deriving coarse particles), filters absorbance as well as the number of ultrafine particles. Previous work from RUPIOH that included air pollution monitoring for one week inside and directly outside participants’ homes reported no association with lung function\n[19]. As the authors stated a potential explanation could be the high prevalence of medication use, the short period of measurements (one week) that limited the ability to assess lagged effects over several days or absence of an effect. The high prevalence of medication use may also have covered some associations in the present study.\nA limitation of the study is the inclusion of both COPD and asthma patients. COPD and asthma are two diseases with different underlying pathophysiological mechanisms and day to day variability in their symptoms\n[22,23]. Mixing of the two diseases does not create bias in the analysis in the full population as we adjusted for differences in health status between individuals. The generalizability of the size of the effect estimates is more affected by the population. Though asthma and COPD are different diseases, we are not aware of studies that have demonstrated differences in the magnitude of response to air pollution. In our recently accepted paper in the same panel we did not find any difference in the effect of PM10-2.5 on total nitrate and nitrite concentrations in exhaled breath condensate (EBC NOx), a marker of oxidative stress between asthma and COPD patients\n[28]. In that study we could evaluate disease status as the outcome was a continuous variable. Unfortunately, an analysis restricted to COPD patients was not possible due to the small number of COPD patients participating in Helsinki and Birmingham. Hence, we also could not test whether the smaller PM10-2.5 effect in the asthmatic subgroup differed significantly from the COPD subgroup.\nOur coarse particle findings are however consistent with the observation that in the RUPIOH study only the PM10-2.5 concentration at central sites was significantly associated with increased EBC NOx collected during the same week as the spirometry\n[28]. EBC NOx has been suggested as a reliable marker of oxidative stress\n[29-31]. The link between PM10-2.5 with oxidative stress and airway inflammation may explain the increase in respiratory symptoms we found.\nIn this study, we also report significant positive associations of ozone with cough throughout most of the examined lags both in the analysis of total participants and the subgroup of the asthmatics that are consistent with previous epidemiological and toxicological studies\n[32]. In addition, positive associations, but not significant in the nominal level, were observed with most of symptoms when total participants were included in the analysis. However, when we restricted the analysis to the subgroup of asthmatics, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2. Negative associations were also observed with woken with breathing problems, wheezing and with limitation in activities due to breathing problems, although non significant. Factors like high medication use, intrinsic differences in responsiveness to ozone among individuals, adaptation to ozone issues or other spurious effects may have been responsible for these findings\n[32].\nIn the last two decades a substantial body of literature has focused on the harmful health effects of PM10 and PM2.5[15,32]. As a result guideline values have been recommended by the U.S. Environmental Protection Agency and World Health Organization for both indicators of PM pollution to protect public health\n[2,3]. However, from recent studies there is increasing evidence that the health effects of coarse particles should not be underestimated. In a systematic review of epidemiological studies that have analyzed fine and coarse PM jointly, Brunekreef and Forsberg examined the epidemiological evidence for effects of coarse particles on health\n[15]. They concluded that the effects of PM10-2.5 were stronger than or as strong as PM2.5 on short-term respiratory morbidity. Furthermore, in a national multicity study, Zanobetti and Schwartz found a strong association of both fine and coarse particles with daily deaths in 112 U.S. cities\n[33]. A 10 μg/m3 increase in PM10-2.5 was significantly associated with total mortality, stroke, cardiovascular, and respiratory mortality, the latter of which showing the largest effect (a 1.2% increase). Mechanistically, these effects may be due either to biogenic factors or to metals carried by PM10-2.5 by activation of inflammatory and oxidative stress pathways\n[34-36]. The findings of our study support previous epidemiological and toxicological evidence that health effects due to the coarse fraction may be substantial\n[37].\nThe large number of calculations we have done could have given some statistically significant associations by chance. However, multiple testing is an unlikely explanation of the findings in the current study. In the full study population we found 14 significant associations of which 10 were positive; in the asthmatics subgroup we found 12 significant associations of which 8 were positive. The consistency of associations (e.g. for ozone and cough) further argues against chance as the main explanation for our findings. Additionally, in the full study population the significant associations for PM10-2.5 were supported by elevated though not nominally significant ORs for other lags and symptoms. Finally, ORs were mainly homogeneous across centers. Moreover, the modest correlations between PM10-2.5 and PM2.5 did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5.\nThe majority of studies that investigated health effects of particulate pollutants have expressed results on a mass basis. It has been suggested that when taking into consideration particle number or surface area, the pulmonary dose of toxic material related to PM2.5 may be much larger than the dose related to PM10-2.5 that for this reason alone, comparison on a mass basis may be less informative\n[15]. In our study we separately investigated the mass and the number effect. Neither central site PM2.5 nor PNC were consistently associated with symptoms. The association we observed with PM10-2.5, if not by chance, may also imply that a central measurement site is more appropriate for measurements of mass concentrations than for PNC. The analysis of RUPIOH data by Puustinen et al. showed generally high correlations between 24 hour average central site and residential outdoor concentrations for PM2.5 and soot with a lesser median correlation for PM10 and a lower correlation for PNC and PM10-2.5[17]. For PM10-2.5 correlations between central site and home outdoor measurements were 0.66, 0.74, 0.89 and 0.64 in Helsinki, Athens, Amsterdam and Birmingham respectively. A central site thus provides a reasonably good estimate of more local exposures even for coarse particles.\nThe relatively high divergence of PM10-2.5 concentrations between proximate sites in the UK has recently been confirmed by Liu and Harrison\n[38]. Consequently, for both PNC and PM10-2.5, there is a higher probability of exposure misclassification than for PM2.5 or soot. The finding of significant associations with respiratory health outcomes for PM10-2.5 but not for PNC is therefore quite striking but consistent with the recent findings of a time series study in London which found significant associations between PNC and cardiovascular health outcomes whilst PM mass metrics were associated with respiratory outcomes\n[39]. A plausible explanation could be the existence of different biological and pathophysiological mechanisms through which PM10-2.5 and PNC exert their adverse effects or different target organs. The results of recent toxicological studies support the theory that PM10-2.5 exert their effects at the site of deposition in the airways whereas PNC, after crossing the alveolar epithelial barrier, enter into the systemic circulation and affect cardiovascular function\n[40,41]. This theory could explain the positive associations we found between PM10-2.5 and respiratory symptoms.\nIn summary, our study contributes to the literature on the health effects of PM in respiratory patients. Moreover, the results of our study are in agreement with the findings of recent epidemiological and toxicological studies and provide enough evidence to conclude that it is prudent to keep PM10-2.5 regulated in addition to fine particles.",
"Our study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles.",
"AIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.",
"The authors declare that they have no competing interests.",
"All authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions."
] |
[
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"methods",
null,
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
null,
"discussion",
"conclusions",
null,
null,
null
] |
[
"Air pollution",
"Asthma",
"Chronic obstructive pulmonary disease",
"Coarse particles",
"Particle number concentration",
"Respiratory health"
] |
Background:
Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects
[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)
[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy
[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals
[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion
[9,12-14].
The only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked
[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.
The RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before
[16-20].
Methods:
Study design In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication
[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health
[21]. In all centres, participants were recruited between October 2002 and March 2004.
In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication
[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health
[21]. In all centres, participants were recruited between October 2002 and March 2004.
Study population Inclusion criteria and recruitment procedures have been described in detail before
[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months
[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)
[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.
Medical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.
Inclusion criteria and recruitment procedures have been described in detail before
[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months
[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)
[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.
Medical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.
Symptom diary The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study
[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops
[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.
During the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.
The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study
[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops
[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.
During the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.
Air pollution exposure Exposure assessment has been described in previous publications
[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels
[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.
Exposure assessment has been described in previous publications
[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels
[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.
Confounder data Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).
Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).
Quality assurance/quality control Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.
Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.
Statistical analysis Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.
A hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates
[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.
We applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.
In the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.
Because of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.
Effect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software
[27].
Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.
A hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates
[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.
We applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.
In the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.
Because of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.
Effect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software
[27].
Study design:
In the context of RUPIOH, a multicentre study was conducted from October 2002 to March 2004 in four European metropolitan areas, namely, Amsterdam (The Netherlands), Athens (Greece), Birmingham (United Kingdom) and Helsinki (Finland). During the whole study period a central site in each city was used to monitor particle mass and PNC on a daily basis. At various locations covering the entire metropolitan area, homes of participants with either asthma or COPD were selected. The criteria for the central site and homes selection have been described in detail in a previous publication
[17]. Respiratory health status of each participant was monitored for six months by a daily symptom diary. We used a staged entry of the participants (based on the real date the participants started to fill out the diaries) in order to increase the period of data collection and thus, decrease the likelihood for uncontrolled factors or unexpected events to influence the associations between air pollution and health
[21]. In all centres, participants were recruited between October 2002 and March 2004.
Study population:
Inclusion criteria and recruitment procedures have been described in detail before
[19]. Briefly, in each city the recruitment criteria for participants were age 35 or more, a doctor diagnosis of either asthma (as defined by Global Initiative for Asthma) or COPD (as defined by Global Initiative for Chronic Obstructive Lung Disease) and having had experienced respiratory symptoms in the past 12 months
[22,23]. Especially, in the Netherlands some patients who had not received a definite diagnosis of asthma or COPD were classified as chronic non-specific lung disease (CNSLD) as a relic of tradition (term previously used to indicate either asthma or COPD)
[24]. Severe patients defined as those using relief bronchodilating medications more than three times per day or using nebulised bronchodilators or long-term oxygen therapy as well as participants unable to perform a satisfactory spirometry test were excluded from the study. An attempt was made to select non-working, non-smoking patients living in a non-smoking household to eliminate potential confounding by occupational exposures to airborne particles and by environmental tobacco smoke. The same screening questionnaire was used across the four centres to ascertain eligibility. However, each centre was allowed to choose the optimal subject recruitment method. Specifically, in Amsterdam, the panelists were recruited through distribution of 10,000 information letters accompanied by screening questionnaires. Inclusion criteria were checked using the returned screening questionnaires followed by participants’ homes’ visits. In Athens, subjects recruited through local hospitals and pulmonary chest physicians were visited at home by a pulmonologist (A.K.) and one of the investigators of the exposure assessment team (I.K.) who checked whether inclusion criteria were met. In Finland, subjects were selected from the Helsinki Metropolitan Area (including cities of Helsinki, Espoo and Vantaa) by placing advertisement on two issues of the respiratory patient association magazine (circulation ~3500 households) and notice boards of pulmonary disease clinics of four major hospitals within the study area. Candidate subjects were interviewed and screened by telephone and invited to an information session when they met the criteria. In the United Kingdom, potential study subjects living in the greater area of Birmingham were selected from the Clinic for Respiratory illnesses (CRI) database of respiratory patients at the Heartlands Hospital. Privacy regulations restricted the selections to only those that had given their written consent to be approached for research studies.
Medical ethical clearance was acquired from the relevant local medical ethics committees in all centres before the start of the recruitment. Written informed consent was obtained from each subject.
Symptom diary:
The diary was based upon diaries used in previous studies of acute effects of air pollution such as the PEACE study
[21]. Although there is no real objective method of validating symptoms, a previous study by Hoek et al. provide evidence that symptoms, assessed with the same diary, are reflected in lung function drops
[25]. Participants were instructed to complete a daily record about respiratory symptoms and medication taken “as needed” for six months, grading shortness of breath, wheeze, cough, phlegm and woken with breathing problems as absent (0), slight (1), or moderate/severe (2). In addition, they were asked about any limitation in performing daily life activities categorized as vigorous (such as running, lifting heavy objects, participating in strenuous sports), moderate (such as moving a table, pushing a vacuum cleaner, bowling or playing golf), walking one block/climbing one flight of stairs and leaving one’s home, because of breathing problems. This limitation could be reported in three grades: no limitation (0), yes, did activity slowly (1) and yes, avoided activity completely (2). Questions on whether they have been outside the house or town and for how long have also been included.
During the study period there was personal contact with the participants once a month to collect the completed diary forms, discuss potential problems and keep the motivation at a good level.
Air pollution exposure:
Exposure assessment has been described in previous publications
[16-18,20]. In brief, during the entire study period (October 2002 to March 2004) in each city measurements of PM2.5, PM10 and PNC were performed continuously at a central site representing urban background levels
[17]. The same type of condensation particle counter (TSI 3022A, TSI Inc., St. Paul, MN, USA) was used in each city to monitor PNC. 24-hour average particle mass concentration was measured with Harvard impactors for PM2.5 and PM10. Coarse particles concentrations were calculated by subtracting PM2.5 from PM10. After weighing, the absorbance of the PM2.5 filters (a good surrogate for elemental carbon/soot) was determined using reflectometry. PNC was transformed to “noon-to-noon” 24-hour means to coincide with the PM2.5 measurements. Data on concentrations of other air pollutants (ozone, nitrogen dioxide) and meteorology (air temperature, relative humidity) were collected from existing national monitoring networks in each country. We did not replace missing values in exposures variables by imputation.
Confounder data:
Time trend in health endpoints (e.g. fatigue in reporting), weather (outdoor temperature, relative humidity), medication use and day of the week were taken into account as potential confounders. Because of the staged entry of participants, we evaluated two time variables: calendar date (proxy for unmeasured confounders) and day of study for a specific subject (possibly related to fatigue).
Quality assurance/quality control:
Air pollution and health measurements were performed according to standard operating procedures (SOPs). A training workshop was organized before the start of the fieldwork and site visits were implemented during the fieldwork to identify any deviations from SOPs.
Statistical analysis:
Data analysis was done according to a predefined analysis plan. The symptom variables, initially coded as 0 for no symptoms (absent), 1 for slight symptoms and 2 for moderate/severe symptoms, were dichotomised for the analysis by setting 0 for no symptoms and 1 for slight to moderate/severe symptoms. Each symptom was analysed separately either as prevalent (irrespective of its occurrence on the previous day) or incident (when that symptom was reported to be absent on the previous day). Medication use was coded as 0 (no medication) versus 1 (intake of one or more doses) independently of the initial medication group. Every person was included in the analysis regardless of how many diary entries were made. Moreover, diary entries were excluded when participants had left the study area during the measurement period. For every pollutant the following lags were evaluated: lag 0, 1, 2 and the average of lag 0–6 days. Lag 0 was defined as the 24-hour period starting from noon of the calendar day before the health response.
A hierarchical modelling approach was used. First, regression models were fitted in each city separately to allow specific control for seasonal effects, weather and other potential confounders. Results of the individual city analysis were used in a second stage analysis (meta-analysis) to provide overall estimates
[26]. We computed both fixed and random effects combined estimates. Furthermore, a chi-square test of heterogeneity of the four city-specific estimates was computed.
We applied logistic regression to obtain centre-specific effect estimates. A smooth function (natural splines with 6 degrees of freedom per year) of time was used to remove the seasonal patterns and long time trends from the data. Afterwards, same-day (lag 0) and previous-day (lag 1) mean daily temperatures were introduced simultaneously into the model. For both lags of temperature, a linear term was compared with a smoothed function (natural splines) with 2, 3 and 4 degrees of freedom and the model with the lowest Akaike’s Information Criterion (AIC) was selected. A linear term of relative humidity (lag 0) was added to the model as another indicator of weather. Finally, indicator variables for day of the week, medication use and individual differences in frequency of symptoms, were added to the model. After setting up the baseline model, the effects of the various lags of the pollutants were evaluated.
In the city specific analysis we fitted fixed effects models, described above, as well as random intercept logistic regression models using “glmmPQL” function from MASS library in R software, to take into account the correlation among each subject’s measurements. Results from the random effects analysis were very similar to those derived from fixed effects. In a few cases though, we faced convergence issues. This was even more the case when we tested a first order autoregressive correlation structure. The significance of the associations was similar between random intercept models and the models incorporating an autoregressive term.
Because of the heterogeneity of the study population, we repeated the analysis (for all air pollution measures) for the subgroup of asthmatic patients. There were not enough COPD patients to analyse these patients separately. We also fitted two pollutant models by including simultaneously PM2.5 and PM10-2.5 in order to better characterize which of the two components of PM10 (PM10-2.5 or PM2.5) was responsible for the observed health effects.
Effect estimates are expressed as odds ratios (OR) for an increase of 10 μg/m3 in PM10, 10 μg/m3 in PM2.5, 10 μg/m3 in PM10-2.5, 10,000 particles/cm3 for PNC and 1·10-5 m-1 for absorbance, in order to be comparable with other studies. For gaseous pollutants the effect estimates are expressed as OR for an increase of 10 μg/m3 in ozone and NO2 concentrations.All analyses were performed using R software
[27].
Results:
Panel characteristics A brief description of the study population is presented in Table
1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.
Characteristics of four European panels of asthmatic/COPD patients
a Total participants in panel.
b Asthma + COPD or chronic non-specific lung disease.
c Given as mean and [range].
d Chronic non-specific lung disease.
e Environmental tobacco smoke.
f Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.
A brief description of the study population is presented in Table
1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.
Characteristics of four European panels of asthmatic/COPD patients
a Total participants in panel.
b Asthma + COPD or chronic non-specific lung disease.
c Given as mean and [range].
d Chronic non-specific lung disease.
e Environmental tobacco smoke.
f Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.
Symptoms In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table
2).
Person days with symptoms in the diary (n = number of expected person days)
a due to breathing problems.
In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table
2).
Person days with symptoms in the diary (n = number of expected person days)
a due to breathing problems.
Air pollution concentrations Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table
3).
Daily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities
Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table
3).
Daily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities
Air pollution effects on symptoms-limitation in activities due to breathing problems Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables
4 and
5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table
6).
Associations of particulate matter indices, NO
2
and O
3
with prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of particulate matter indices, NO
2
and O
3
with limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of PM
10-2.5
and PM
2.5
with prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)
Bold are significant pooled effects.
The above-mentioned positive associations with PM10-2.5 (Tables
4 and
5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).
Ozone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables
4 and
5).
Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables
4 and
5).
Centre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure
1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.
Odds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m
3
in previous day (lag1) concentrations of each pollutant (10,000/cm
3
for PNC) in each participating city and overall estimate (random effects pooled estimates).
We observed very small differences in fixed and random effects combined estimates. In Tables
4 and
5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table
6).
Associations of particulate matter indices, NO
2
and O
3
with prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of particulate matter indices, NO
2
and O
3
with limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of PM
10-2.5
and PM
2.5
with prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)
Bold are significant pooled effects.
The above-mentioned positive associations with PM10-2.5 (Tables
4 and
5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).
Ozone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables
4 and
5).
Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables
4 and
5).
Centre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure
1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.
Odds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m
3
in previous day (lag1) concentrations of each pollutant (10,000/cm
3
for PNC) in each participating city and overall estimate (random effects pooled estimates).
Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables
4 and
5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table
6).
Associations of particulate matter indices, NO
2
and O
3
with prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of particulate matter indices, NO
2
and O
3
with limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of PM
10-2.5
and PM
2.5
with prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)
Bold are significant pooled effects.
The above-mentioned positive associations with PM10-2.5 (Tables
4 and
5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).
Ozone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables
4 and
5).
Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables
4 and
5).
Centre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure
1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.
Odds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m
3
in previous day (lag1) concentrations of each pollutant (10,000/cm
3
for PNC) in each participating city and overall estimate (random effects pooled estimates).
We observed very small differences in fixed and random effects combined estimates. In Tables
4 and
5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table
6).
Associations of particulate matter indices, NO
2
and O
3
with prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of particulate matter indices, NO
2
and O
3
with limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of PM
10-2.5
and PM
2.5
with prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)
Bold are significant pooled effects.
The above-mentioned positive associations with PM10-2.5 (Tables
4 and
5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).
Ozone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables
4 and
5).
Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables
4 and
5).
Centre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure
1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.
Odds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m
3
in previous day (lag1) concentrations of each pollutant (10,000/cm
3
for PNC) in each participating city and overall estimate (random effects pooled estimates).
Incidence analyses Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table
7).
Associations of particulate matter indices, NO
2
and O
3
with incidence of symptoms in the four panels (random effects pooled estimates)
Bold are significant pooled effects.
Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table
7).
Associations of particulate matter indices, NO
2
and O
3
with incidence of symptoms in the four panels (random effects pooled estimates)
Bold are significant pooled effects.
Panel characteristics:
A brief description of the study population is presented in Table
1. Mean age and age range were about the same in all cities. Three participants in Athens were slightly below of the recruitment criterion of ≥35 years. In Amsterdam a large group was reported to have CNSLD. Medication use was high in the panels. Seventy seven per cent of the participants (77%) used reliever medication. Use of “as needed medication” was recorded in 26.5% of total person days in Helsinki, 13.9% in Athens, 37.9% in Amsterdam and 59.7% in Birmingham. Twenty-nine participants (21%) worked outside their home especially from Amsterdam and Birmingham. Those who worked outside their home, worked on average 19 h/week.
Characteristics of four European panels of asthmatic/COPD patients
a Total participants in panel.
b Asthma + COPD or chronic non-specific lung disease.
c Given as mean and [range].
d Chronic non-specific lung disease.
e Environmental tobacco smoke.
f Includes short acting β2-agonist, long acting β2-agonist, anticholinergic drugs and combination of an anticholinergic drug and a β2-agonist.
Symptoms:
In total between 4,760 and 6,003 person days were available for analysis in the four cities. In Amsterdam, Athens and Birmingham participants filled out the diary from October 2002 to March 2004 whilst in Helsinki between October 2002 and February 2004. Missing values (person days) ranged between 9.4-15.1% in Amsterdam, 4.7-5.5% in Athens, 8.7-8.8% in Birmingham and 8.6-12.1% in Helsinki. Consistent with the composition of the panel, fairly high symptom prevalence occurred during the study period. Person days with severe symptoms were low, except for cough and phlegm. There were small differences between the cities (Table
2).
Person days with symptoms in the diary (n = number of expected person days)
a due to breathing problems.
Air pollution concentrations:
Helsinki had the lowest median concentrations for all PM components whilst Athens had the highest. However, maximum concentrations of PM2.5 were observed in Amsterdam (103.4 μg/m3) and of PM10-2.5 (152.6 μg/m3) in Helsinki (Table
3).
Daily (24 hours noon-to-noon, central site) median air pollution concentration and meteorology in the four cities
Air pollution effects on symptoms-limitation in activities due to breathing problems:
Prevalence analyses We observed very small differences in fixed and random effects combined estimates. In Tables
4 and
5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table
6).
Associations of particulate matter indices, NO
2
and O
3
with prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of particulate matter indices, NO
2
and O
3
with limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of PM
10-2.5
and PM
2.5
with prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)
Bold are significant pooled effects.
The above-mentioned positive associations with PM10-2.5 (Tables
4 and
5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).
Ozone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables
4 and
5).
Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables
4 and
5).
Centre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure
1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.
Odds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m
3
in previous day (lag1) concentrations of each pollutant (10,000/cm
3
for PNC) in each participating city and overall estimate (random effects pooled estimates).
We observed very small differences in fixed and random effects combined estimates. In Tables
4 and
5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table
6).
Associations of particulate matter indices, NO
2
and O
3
with prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of particulate matter indices, NO
2
and O
3
with limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of PM
10-2.5
and PM
2.5
with prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)
Bold are significant pooled effects.
The above-mentioned positive associations with PM10-2.5 (Tables
4 and
5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).
Ozone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables
4 and
5).
Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables
4 and
5).
Centre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure
1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.
Odds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m
3
in previous day (lag1) concentrations of each pollutant (10,000/cm
3
for PNC) in each participating city and overall estimate (random effects pooled estimates).
Prevalence analyses:
We observed very small differences in fixed and random effects combined estimates. In Tables
4 and
5 combined odds ratios for the association of particulate matter indices, NO2, ozone and prevalence of symptoms and limitation in activities are presented, using random effects models adjusting for the above mentioned confounders and “as needed” medication. When all participants were included in the analysis as a total, we found that a 10 μg/m3 increase in PM10 was significantly associated at the nominal level with shortness of breath in the lag 1 whilst the association in the lags 2 and 0 to 6 was of borderline significance. However, none of the associations was significant for the asthma group. Significant association was also observed for wheezing and limitation in walking due to breathing problems (lag 1). The association was driven by the PM10-2.5 component of PM10 and much less by PM2.5. Coarse particles concentrations were positively associated with most symptom and restriction of activities variables in lag1. In addition, the modest correlations between PM10-2.5 and PM2.5 (0.08, 0.40, 0.35 and 0.13 for Amsterdam, Athens, Birmingham and Helsinki respectively) did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5 (Table
6).
Associations of particulate matter indices, NO
2
and O
3
with prevalence of symptoms in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of particulate matter indices, NO
2
and O
3
with limitation in activities due to breathing problems in all participants and the subgroup of asthmatics (random effects pooled estimates)
Bold are significant pooled effects.
Associations of PM
10-2.5
and PM
2.5
with prevalence of symptoms and limitation in activities due to breathing problems after applying two pollutant models (random effects pooled estimates)
Bold are significant pooled effects.
The above-mentioned positive associations with PM10-2.5 (Tables
4 and
5) were reduced and no longer significant after restricting the analysis to the asthmatic only participants. A significant association remained with restricting walking activities and wheeze (borderline).
Ozone was significantly associated with cough at lag 0, lag 1, lag 2 and with woken with breathing problems at lag 0. Furthermore, the associations with wheezing, limitation in vigorous activities and walking due to breathing problems remained positive across all examined lags although, non significant. Negative but non significant associations were observed with shortness of breath across all examined lags. However, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2, in the asthma group. Moreover, in the asthmatics, negative associations were also observed for ozone with woken with breathing problems (lag 0), wheezing (lag 0 and lag 1), and with limitation in activities due to breathing problems (most of the lags), although non significant (Tables
4 and
5).
Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst the positive associations with woken with breathing problems and cough in lag 1 as well as in limitation of activities due to breathing problems (mainly vigorous and moderate) in lags 0, 1, 2 did not reach the nominal level of significance. Moreover, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant (Tables
4 and
5).
Centre specific and overall effect estimates with 95 percent confidence intervals (95% CI) for the association of each symptom and air pollutant in lag1 are presented in Figure
1. Odds ratios (OR) for the effect of PM10-2.5 were consistently above one in almost every city as well as in the pooled data using random effects meta-analysis.
Odds ratio (95% CI) for prevalence of symptoms and limitation in activities associated with an increase of 10 μg/m
3
in previous day (lag1) concentrations of each pollutant (10,000/cm
3
for PNC) in each participating city and overall estimate (random effects pooled estimates).
Incidence analyses:
Patterns similar to those in the combined prevalence analyses were observed for the associations of incident symptoms and particles especially the coarse fraction. Shortness of breath was consistently associated with PM10 and PM10-2.5 in lag 1 with no indication of heterogeneity between the centres (OR = 1.045, 95% CI: 1.008, 1.083 and OR = 1.065, 95% CI: 1.009, 1.124 respectively). There was also a tendency towards positive associations between PM10-2.5 and incidence of wheezing, cough and limitation in walking but none of the associations were statistically significant. Additionally, ozone was positively associated with cough in lags 1 and 2 as well as the average lag 0–6 days but only in lag 2 the association reached the nominal level of significance (Table
7).
Associations of particulate matter indices, NO
2
and O
3
with incidence of symptoms in the four panels (random effects pooled estimates)
Bold are significant pooled effects.
Discussion:
In this multicentre study we found consistent positive associations between coarse particles central sites concentrations and prevalence of respiratory symptoms, as recorded in a 6-month diary, in four panels of participants with predominantly mild to moderate asthma or COPD in four European cities participating in the RUPIOH study. We also found a significant association of ozone with cough and woken with breathing problems, but not with other symptoms. Neither PM2.5 nor NO2 were consistently associated with any symptom or limitation in activities variable. As for PNC a (mostly non-significant) negative association was observed with most symptoms whilst positive associations with woken with breathing problems and cough as well as with limitation of vigorous and moderate activities due to breathing problems, did not reach the nominal level of significance. Interestingly, for PNC a change of the negative associations with woken with breathing problems towards positive values, across all lags, was observed when the analysis was restricted to the asthmatic participants, although non significant at the nominal level. An analysis of the asthmatic subgroup showed generally lower odds ratios for PM10-2.5.
One particularity and strength of the RUPIOH study is the in depth assessment of particulate air pollution by measuring PM10, PM2.5 (then deriving coarse particles), filters absorbance as well as the number of ultrafine particles. Previous work from RUPIOH that included air pollution monitoring for one week inside and directly outside participants’ homes reported no association with lung function
[19]. As the authors stated a potential explanation could be the high prevalence of medication use, the short period of measurements (one week) that limited the ability to assess lagged effects over several days or absence of an effect. The high prevalence of medication use may also have covered some associations in the present study.
A limitation of the study is the inclusion of both COPD and asthma patients. COPD and asthma are two diseases with different underlying pathophysiological mechanisms and day to day variability in their symptoms
[22,23]. Mixing of the two diseases does not create bias in the analysis in the full population as we adjusted for differences in health status between individuals. The generalizability of the size of the effect estimates is more affected by the population. Though asthma and COPD are different diseases, we are not aware of studies that have demonstrated differences in the magnitude of response to air pollution. In our recently accepted paper in the same panel we did not find any difference in the effect of PM10-2.5 on total nitrate and nitrite concentrations in exhaled breath condensate (EBC NOx), a marker of oxidative stress between asthma and COPD patients
[28]. In that study we could evaluate disease status as the outcome was a continuous variable. Unfortunately, an analysis restricted to COPD patients was not possible due to the small number of COPD patients participating in Helsinki and Birmingham. Hence, we also could not test whether the smaller PM10-2.5 effect in the asthmatic subgroup differed significantly from the COPD subgroup.
Our coarse particle findings are however consistent with the observation that in the RUPIOH study only the PM10-2.5 concentration at central sites was significantly associated with increased EBC NOx collected during the same week as the spirometry
[28]. EBC NOx has been suggested as a reliable marker of oxidative stress
[29-31]. The link between PM10-2.5 with oxidative stress and airway inflammation may explain the increase in respiratory symptoms we found.
In this study, we also report significant positive associations of ozone with cough throughout most of the examined lags both in the analysis of total participants and the subgroup of the asthmatics that are consistent with previous epidemiological and toxicological studies
[32]. In addition, positive associations, but not significant in the nominal level, were observed with most of symptoms when total participants were included in the analysis. However, when we restricted the analysis to the subgroup of asthmatics, a significant preventive effect of ozone for shortness of breath was revealed for lags 1 and 2. Negative associations were also observed with woken with breathing problems, wheezing and with limitation in activities due to breathing problems, although non significant. Factors like high medication use, intrinsic differences in responsiveness to ozone among individuals, adaptation to ozone issues or other spurious effects may have been responsible for these findings
[32].
In the last two decades a substantial body of literature has focused on the harmful health effects of PM10 and PM2.5[15,32]. As a result guideline values have been recommended by the U.S. Environmental Protection Agency and World Health Organization for both indicators of PM pollution to protect public health
[2,3]. However, from recent studies there is increasing evidence that the health effects of coarse particles should not be underestimated. In a systematic review of epidemiological studies that have analyzed fine and coarse PM jointly, Brunekreef and Forsberg examined the epidemiological evidence for effects of coarse particles on health
[15]. They concluded that the effects of PM10-2.5 were stronger than or as strong as PM2.5 on short-term respiratory morbidity. Furthermore, in a national multicity study, Zanobetti and Schwartz found a strong association of both fine and coarse particles with daily deaths in 112 U.S. cities
[33]. A 10 μg/m3 increase in PM10-2.5 was significantly associated with total mortality, stroke, cardiovascular, and respiratory mortality, the latter of which showing the largest effect (a 1.2% increase). Mechanistically, these effects may be due either to biogenic factors or to metals carried by PM10-2.5 by activation of inflammatory and oxidative stress pathways
[34-36]. The findings of our study support previous epidemiological and toxicological evidence that health effects due to the coarse fraction may be substantial
[37].
The large number of calculations we have done could have given some statistically significant associations by chance. However, multiple testing is an unlikely explanation of the findings in the current study. In the full study population we found 14 significant associations of which 10 were positive; in the asthmatics subgroup we found 12 significant associations of which 8 were positive. The consistency of associations (e.g. for ozone and cough) further argues against chance as the main explanation for our findings. Additionally, in the full study population the significant associations for PM10-2.5 were supported by elevated though not nominally significant ORs for other lags and symptoms. Finally, ORs were mainly homogeneous across centers. Moreover, the modest correlations between PM10-2.5 and PM2.5 did allow us to apply a two-pollutant model in order to separate and further evaluate the effects of the two components of PM10. The magnitude of the associations for PM10-2.5 with prevalence of symptoms and restriction of activities remained approximately the same or increased when we applied a two-pollutant model with PM2.5.
The majority of studies that investigated health effects of particulate pollutants have expressed results on a mass basis. It has been suggested that when taking into consideration particle number or surface area, the pulmonary dose of toxic material related to PM2.5 may be much larger than the dose related to PM10-2.5 that for this reason alone, comparison on a mass basis may be less informative
[15]. In our study we separately investigated the mass and the number effect. Neither central site PM2.5 nor PNC were consistently associated with symptoms. The association we observed with PM10-2.5, if not by chance, may also imply that a central measurement site is more appropriate for measurements of mass concentrations than for PNC. The analysis of RUPIOH data by Puustinen et al. showed generally high correlations between 24 hour average central site and residential outdoor concentrations for PM2.5 and soot with a lesser median correlation for PM10 and a lower correlation for PNC and PM10-2.5[17]. For PM10-2.5 correlations between central site and home outdoor measurements were 0.66, 0.74, 0.89 and 0.64 in Helsinki, Athens, Amsterdam and Birmingham respectively. A central site thus provides a reasonably good estimate of more local exposures even for coarse particles.
The relatively high divergence of PM10-2.5 concentrations between proximate sites in the UK has recently been confirmed by Liu and Harrison
[38]. Consequently, for both PNC and PM10-2.5, there is a higher probability of exposure misclassification than for PM2.5 or soot. The finding of significant associations with respiratory health outcomes for PM10-2.5 but not for PNC is therefore quite striking but consistent with the recent findings of a time series study in London which found significant associations between PNC and cardiovascular health outcomes whilst PM mass metrics were associated with respiratory outcomes
[39]. A plausible explanation could be the existence of different biological and pathophysiological mechanisms through which PM10-2.5 and PNC exert their adverse effects or different target organs. The results of recent toxicological studies support the theory that PM10-2.5 exert their effects at the site of deposition in the airways whereas PNC, after crossing the alveolar epithelial barrier, enter into the systemic circulation and affect cardiovascular function
[40,41]. This theory could explain the positive associations we found between PM10-2.5 and respiratory symptoms.
In summary, our study contributes to the literature on the health effects of PM in respiratory patients. Moreover, the results of our study are in agreement with the findings of recent epidemiological and toxicological studies and provide enough evidence to conclude that it is prudent to keep PM10-2.5 regulated in addition to fine particles.
Conclusions:
Our study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles.
Abbreviations:
AIC: Akaike’s information criterion; CNSLD: Chronic non-specific lung disease; COPD: Chronic obstructive pulmonary disease; EBC NOx: Total nitrate and nitrite concentrations in exhaled breath condensate; OR: Odds ratio; PM: Particulate matter; PM10-2.5: Coarse particles; PM10: Mass concentration of particles less than 10 μm; PM2.5: Mass concentration of particles less than 2.5 μm; PNC: Particle number concentrations; RUPIOH: Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health; SOPs: Standard operating procedures; 95% CI: 95% confidence interval.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
All authors of this paper have critically read and approved the final version submitted. They have also made substantive intellectual contributions by directly participating either in the planning, execution, or analysis of the study. AK contributed to the development of the study design, acquisition and interpretation of data and drafted the paper. AA did the analysis, contributed to the interpretation of data and wrote the statistical analysis section of the paper. DP, IGK, JJdeH contributed substantially to acquisition and interpretation of data. JGA, RMH, AK, JP, KH, GPAK, KK contributed to the study design, interpretation of data and have been involved in drafting the manuscript. GH conceived and developed the study design, contributed to the interpretation of data and was involved in drafting the paper. All authors have revised drafts and contributed to the revisions.
|
Background:
Particulate matter air pollution has been associated with adverse health effects. The fraction of ambient particles that are mainly responsible for the observed health effects is still a matter of controversy. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.The RUPIOH study, an EU-funded multicentre study, was designed to examine the distribution of various ambient particle metrics in four European cities (Amsterdam, Athens, Birmingham, Helsinki) and assess their health effects in participants with asthma or COPD, based on a detailed exposure assessment. In this paper the association of central site measurements with respiratory symptoms and restriction of activities is examined.
Methods:
At each centre a panel of participants with either asthma or COPD recorded respiratory symptoms and restriction of activities in a diary for six months. Exposure assessment included simultaneous measurements of coarse, fine and ultrafine particles at a central site. Data on gaseous pollutants were also collected. The associations of the 24-hour average concentrations of air pollution indices with the health outcomes were assessed in a hierarchical modelling approach. A city specific analysis controlling for potential confounders was followed by a meta-analysis to provide overall effect estimates.
Results:
A 10 μg/m3 increase in previous day coarse particles concentrations was positively associated with most symptoms (an increase of 0.6 to 0.7% in average) and limitation in walking (OR= 1.076, 95% CI: 1.026-1.128). Same day, previous day and previous two days ozone concentrations were positively associated with cough (OR= 1.061, 95% CI: 1.013-1.111; OR= 1.049, 95% CI: 1.016-1.083 and OR= 1.059, 95% CI: 1.027-1.091, respectively). No consistent associations were observed between fine particle concentrations, nitrogen dioxide and respiratory health effects. As for particle number concentrations negative association (mostly non-significant at the nominal level) was observed with most symptoms whilst the positive association with limitation of activities did not reach the nominal level of significance.
Conclusions:
The observed associations with coarse particles are in agreement with the findings of toxicological studies. Together they suggest it is prudent to regulate also coarse particles in addition to fine particles.
|
Background:
Over the last decades numerous epidemiological studies have clearly shown that urban air pollution can produce a variety of adverse health effects
[1,2]. Ambient particulate matter (PM) either characterized as the mass concentration of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5) are considered to be the major culprit. Therefore, current air quality standards or guidelines refer to PM10 and/or PM2.5[3,4]. However, in reality ambient PM is a mixture of coarse (2.5-10 μm), PM2.5 (named also fine particles) and ultrafine (<0.1 μm) particles generated from different processes, having variable chemical composition and atmospheric behavior. It should also be noted that although the ultrafine fraction accounts for less than 1% of the mass of particulate matter, it represents the greatest proportion in terms of number of particles (typically >80%)
[5-7]. Furthermore, the mechanism and the fraction of PM that are mainly responsible for the observed health effects is a matter of controversy
[1]. In 1995 Seaton hypothesized that the number of ultrafine particles may be a more health relevant property than the usually measured mass of inhaled PM10 and PM2.5[8]. This is because of the greater surface area available to react with epithelial and inflammatory cells in the lung and because of the capacity of ultrafine particles to penetrate deeper in the lung parenchyma, potentially reaching the circulation and exerting adverse biological effects by releasing toxic free radicals
[8-11]. In meantime other studies were published, however, the role of ultrafine particles is still under discussion
[9,12-14].
The only systematic review of studies that have analysed fine and coarse PM jointly demonstrates that the health effects of coarse particles are significant and should not be overlooked
[15]. Thus, special consideration should be given to each fraction of the particles and their effects on health. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.
The RUPIOH (Relationship between Ultrafine and fine Particulate matter in Indoor and Outdoor air and respiratory Health) is an EU-funded multicentre study designed to examine the distribution of various particle metrics both indoors and outdoors in four European cities and assess their health effects in individuals with asthma or chronic obstructive pulmonary disease (COPD), based on a detailed exposure assessment. The study consisted of two parts: i) the diary study in which participants were asked to complete a daily diary for six months while exposure was assessed based on a central site measurements and ii) the intensive week measurements during which, for each subject, more intensive health and exposure measurements were conducted. In this paper, we report the association of ambient PM10, PM2.5, coarse particle mass (PM10-2.5) and particle number concentrations (PNC), measured at the central site, with respiratory symptoms and limitation in activities due to breathing problems in participants having either asthma or COPD who have been followed for six months. Associations of the health outcomes with gaseous air pollutants were also examined based on data collected from existing national monitoring networks in each country. The relationships between central site outdoor, residential outdoor and indoor concentrations, as well as the association between outdoor and indoor exposure to fine and ultrafine particles and lung function in the same participants but based on the intensive week measurements have been published before
[16-20].
Conclusions:
Our study adds to the limited existing evidence of recent epidemiological and toxicological studies that health effects due to the coarse fraction of ambient PM may be substantial. Further studies are needed to clarify possible different effects of PM on COPD and asthmatic patients. The observed associations suggest it is prudent to regulate also coarse particles in addition to fine particles.
|
Background:
Particulate matter air pollution has been associated with adverse health effects. The fraction of ambient particles that are mainly responsible for the observed health effects is still a matter of controversy. Better characterization of the health relevant particle fraction will have major implications for air quality policy since it will determine which sources should be controlled.The RUPIOH study, an EU-funded multicentre study, was designed to examine the distribution of various ambient particle metrics in four European cities (Amsterdam, Athens, Birmingham, Helsinki) and assess their health effects in participants with asthma or COPD, based on a detailed exposure assessment. In this paper the association of central site measurements with respiratory symptoms and restriction of activities is examined.
Methods:
At each centre a panel of participants with either asthma or COPD recorded respiratory symptoms and restriction of activities in a diary for six months. Exposure assessment included simultaneous measurements of coarse, fine and ultrafine particles at a central site. Data on gaseous pollutants were also collected. The associations of the 24-hour average concentrations of air pollution indices with the health outcomes were assessed in a hierarchical modelling approach. A city specific analysis controlling for potential confounders was followed by a meta-analysis to provide overall effect estimates.
Results:
A 10 μg/m3 increase in previous day coarse particles concentrations was positively associated with most symptoms (an increase of 0.6 to 0.7% in average) and limitation in walking (OR= 1.076, 95% CI: 1.026-1.128). Same day, previous day and previous two days ozone concentrations were positively associated with cough (OR= 1.061, 95% CI: 1.013-1.111; OR= 1.049, 95% CI: 1.016-1.083 and OR= 1.059, 95% CI: 1.027-1.091, respectively). No consistent associations were observed between fine particle concentrations, nitrogen dioxide and respiratory health effects. As for particle number concentrations negative association (mostly non-significant at the nominal level) was observed with most symptoms whilst the positive association with limitation of activities did not reach the nominal level of significance.
Conclusions:
The observed associations with coarse particles are in agreement with the findings of toxicological studies. Together they suggest it is prudent to regulate also coarse particles in addition to fine particles.
| 17,122 | 432 | 21 |
[
"effects",
"pm10",
"associations",
"significant",
"symptoms",
"lag",
"participants",
"problems",
"breathing",
"breathing problems"
] |
[
"test",
"test"
] |
[CONTENT] Air pollution | Asthma | Chronic obstructive pulmonary disease | Coarse particles | Particle number concentration | Respiratory health [SUMMARY]
|
[CONTENT] Air pollution | Asthma | Chronic obstructive pulmonary disease | Coarse particles | Particle number concentration | Respiratory health [SUMMARY]
|
[CONTENT] Air pollution | Asthma | Chronic obstructive pulmonary disease | Coarse particles | Particle number concentration | Respiratory health [SUMMARY]
|
[CONTENT] Air pollution | Asthma | Chronic obstructive pulmonary disease | Coarse particles | Particle number concentration | Respiratory health [SUMMARY]
|
[CONTENT] Air pollution | Asthma | Chronic obstructive pulmonary disease | Coarse particles | Particle number concentration | Respiratory health [SUMMARY]
|
[CONTENT] Air pollution | Asthma | Chronic obstructive pulmonary disease | Coarse particles | Particle number concentration | Respiratory health [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]
|
[CONTENT] Adult | Aged | Aged, 80 and over | Air Pollutants | Air Pollution | Asthma | Cities | Europe | Female | Humans | Male | Middle Aged | Nitrogen Dioxide | Odds Ratio | Ozone | Particulate Matter | Pulmonary Disease, Chronic Obstructive | Respiration Disorders | Walking [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]
|
[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]
|
[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]
|
[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]
|
[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]
|
[CONTENT] effects | pm10 | associations | significant | symptoms | lag | participants | problems | breathing | breathing problems [SUMMARY]
|
[CONTENT] ultrafine | health | particles | μm | ultrafine particles | intensive | effects | based | fine | outdoor [SUMMARY]
|
[CONTENT] analysis | study | day | criteria | symptoms | participants | city | patients | models | effects [SUMMARY]
|
[CONTENT] significant | lag | activities | pooled | associations | effects | problems | breathing problems | breathing | limitation [SUMMARY]
|
[CONTENT] studies | pm | clarify possible different | toxicological studies health effects | limited existing evidence recent | limited existing evidence | limited existing | effects coarse fraction ambient | coarse particles addition | coarse particles addition fine [SUMMARY]
|
[CONTENT] pm10 | effects | significant | associations | lag | symptoms | participants | study | problems | pm2 [SUMMARY]
|
[CONTENT] pm10 | effects | significant | associations | lag | symptoms | participants | study | problems | pm2 [SUMMARY]
|
[CONTENT] ||| ||| ||| EU | four | European | Amsterdam | Athens | Birmingham | Helsinki | COPD ||| [SUMMARY]
|
[CONTENT] six months ||| ||| ||| 24-hour ||| [SUMMARY]
|
[CONTENT] 10 | previous day | 0.6 to 0.7% | 1.076 | 95% | CI | 1.026 ||| previous day | two days | 1.061 | 95% | CI | 1.013 | 1.049 | 95% | CI | 1.016-1.083 | 1.059 | 95% | CI | 1.027 ||| ||| [SUMMARY]
|
[CONTENT] ||| [SUMMARY]
|
[CONTENT] ||| ||| ||| EU | four | European | Amsterdam | Athens | Birmingham | Helsinki | COPD ||| ||| six months ||| ||| ||| 24-hour ||| ||| ||| 10 | previous day | 0.6 to 0.7% | 1.076 | 95% | CI | 1.026 ||| previous day | two days | 1.061 | 95% | CI | 1.013 | 1.049 | 95% | CI | 1.016-1.083 | 1.059 | 95% | CI | 1.027 ||| ||| ||| ||| [SUMMARY]
|
[CONTENT] ||| ||| ||| EU | four | European | Amsterdam | Athens | Birmingham | Helsinki | COPD ||| ||| six months ||| ||| ||| 24-hour ||| ||| ||| 10 | previous day | 0.6 to 0.7% | 1.076 | 95% | CI | 1.026 ||| previous day | two days | 1.061 | 95% | CI | 1.013 | 1.049 | 95% | CI | 1.016-1.083 | 1.059 | 95% | CI | 1.027 ||| ||| ||| ||| [SUMMARY]
|
||||||
DRAIN AMYLASE ON THE FIRST POSTOPERATIVE DAY OF WHIPPLE SURGERY: WHAT VALUE IS THE BEST PREDICTOR FOR EARLY DRAIN REMOVAL?
|
29513806
|
The value of drain amylase on the first postoperative day after pancreatic resections has been described as an efficient predictor of pancreatic fistula. In spite of this, the cut-off point below which the drains can be removed early remains controversial.
|
BACKGROUND
|
Were included patients undergoing Whipple surgery in the period of 2007 to 2016. Group 1 enrolled the ones who did not develop fistula and those who developed biochemical fistula for less than seven days postoperatively and group 2 included patients who developed persistent biochemical fistula between seven and 21 days and those with grade B and C fistula.
|
METHOD
|
Sixty-one patients were included, 41 comprised group 1 and 20 group 2. The incidence of abdominal collections, need for reoperation and time of hospitalization were for group 1 and 2, respectively: 17.1%, 17.1% and 9.5 days, and 65%, 40% and 21.1 days. The median of the amylase from the drain at 1st postoperative day was in group 1 and 2, respectively: 175 U/l and 3172.5 U/l (p=0.001). Using a cut-off of 180 to predict the group to which the patient would belong there was obtained sensitivity, specificity, positive predictive value and negative predictive value of 100%, 48.8%, 50% and 100% respectively.
|
RESULTS
|
It was validated the cut-off value of 180 U/l as appropriate to early drain removal.
|
CONCLUSION
|
[
"Amylases",
"Drainage",
"Female",
"Humans",
"Male",
"Middle Aged",
"Pancreatic Fistula",
"Pancreaticoduodenectomy",
"Postoperative Care",
"Postoperative Complications",
"Predictive Value of Tests",
"Prospective Studies"
] |
5863991
|
INTRODUCTION
|
Historically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them
16
. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis
13
,
11
. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results
8
,
18
,
19
, which has been a limiting factor in the practice of not using drains in this type of procedure.
Description of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L
6
and 90 U/l
14
have been suggested.
The objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.
|
METHOD
|
The project was submitted and approved by the Human Research Ethics Committee, under nº 59716616.9.0000.5292 in the Plataforma Brasil.
Sixty-three patients were analyzed from a prospective database, who had undergone Kausch-Whipple surgery performed by the 1st author of this study (ECA), for the treatment of pancreatic or peripancreatic diseases. The time period was between June 2007 and September 2016, in the following hospitals located in the city of Natal: Hospital Universitário Onofre Lopes, Liga Norte Riograndense Contra o Câncer, Casa de Saúde São Lucas and Natal Hospital Center.
The technical steps for the Whipple technique as performed by the author are detailed in previous studies
1
,
2
. Three techniques for pancreatojejunal anastomosis were utilized. For pancreas with main pancreatic duct ≥5 mm, two-layer pancreatojejunal terminolateral duct to mucosa anastomosis was performed. For pancreas with normal or slightly dilated (diameter <5 mm) main pancreatic duct, terminoterminal pancreatojejunal anastomosis (“telescopic”) or terminolateral pancreatojejunal anastomosis (“invagination”) were carried out, the latter performed systematically after the 33rd patient from the casebook. At the end of the surgery, two drains, preferably laminar silicone drains, were placed in the cavity and exteriorized at each flank.
In the majority of patients, subcutaneous octreotide was utilized as prophylaxis for the prevention of PF, at 0.3 mg/day, fractioned in 8 h intervals, during seven days. Debits were recorded along with amylase dosage of the drained liquid on the 1st, 3rd, 5th, 7th and sometimes, on the 9th postoperative day. The value of AD1PO vas defined from the highest amylase value of both drains, measured from a sample of liquid obtained on the first day after surgery.
PF diagnosis utilized the 2016 revised criterion of GIEDFP
3
. PF was defined when on the 3rd postoperative (PO) day the value of amylase in the drained liquid was three times higher than the normal upper limit for serum amylase. Patients that did not develop fistula or those with transient biochemical fistula (<7 days PO) were characterized as group 1, and patients with persistent biochemical fistula (between 7 and 21 days PO) and fistula grade B and C were characterized as group 2. In most cases, on the 9th day after surgery, control ultrasound or tomography tests were carried out. Removal of drains occurred in those cases with low amylase values for the drained liquid (under three times the upper limit for normal serum amylase) and when image tests did not show abdominal collections. Intra-hospital mortality was defined as death occurring within 90 days of surgery.
The Receiver Operating Characteristic (ROC) curve was utilized to identify an adequate cut-off point for AD1PO and verify its predictive characteristic in patients of both groups. The ROC value was generated after sensitivity, specificity and accuracy calculations. Additionally, the cut-off point 578 U/l was also selected because of its closeness to the value utilized by Fong e cols
9
, for comparison purposes.
Statistical analysis After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests.
After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests.
|
RESULTS
|
From the initial sample of 63 patients, two were excluded. One had AD1PO collected incorrectly on the 2nd day after surgery, and the other passed within the 1st week PO. Therefore analysis covered 61 patients. Group 1 was constituted of 41 patients, of which 36 did not develop fistula and five transient biochemical fistula (grade A). Group 2 was constituted of 20 patients, of which six presented persistent biochemical fistula (grade A), eight degree B fistula, and six grade C fistula. The index of clinical fistula (grades B + C) in the overall sample was 22.9%.
The demographic and clinical characteristics of the patients, as well as respective tests, are shown in Table 1. There was no statistical difference between groups regarding age, gender and type of illness. However, regarding the size of main pancreatic duct and type of anastomosis, a statistically significant difference was detected. All patients with dilated main pancreatic ducts as well as those submitted to duct to mucosa anastomosis belonged to group 1.
TABLE 1Associations between demographic and clinical characteristics and patient groups Variables Group 1 Group 2 pAge 58.0±13.06I
57.1±12.1I
0.706II
n % n %
Gender
Male 19 63.3 11 36.7 0.525III
Female 22 71.0 9 29.0Disease
Pancreatic adenocarcinoma 17 65.4 9 34.6 0.264III
Papillary adenocarcinoma 12 60.0 8 40.0Duodenal adenocarcinoma 1 33.3 2 66.7Colangiocarcinoma 4 100.0 0 0.0Frantz 5 100.0 0 0.0Others 2 66.7 1 33.3Size of duct
< 5 31 60.8 20 39.2 0.023IV
> 5 10 100.0 0 0.0Anastomosis*
T-L (Invagination) 14 50.0 14 50.0 0.011III
T-T 17 73.9 6 26.1T-L (DM) 10 100.0 0 0.0I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)
I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)
Regarding postoperative evolution, group 2 was associated with a higher level of general complications, postoperative collections, longer permanence of abdominal drain and hospitalization time. There was also an association between high resurgery index and patients of group 2. Nevertheless, despite all these worse result factors related to group 2, there was no statistically significant difference when comparing mortality across groups (Table 2).
TABLE 2Associations between postoperative clinical findings and patient groups, along with results for drain and hospitalization times, per group Variable Group 1 Group 2 p n % n %
Collection
Yes733.31466.7 <0.001I
No3485.0615.0Postoperative complications
Yes2050.020.050.0 <0.001II
No21100.000.0Resurgery
Yes746.7853.3 0.051I
No3473.91226.1Time with drain (days) 9.5±2.60III29.1±13.58III <0.001IV
Hospitalization time (days) 16.7±17.9III34.7±16.7III <0.001IV
Mortality49.815.0 1.000II
I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test
I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test
Mortality of the series was 8.19%: four patients belonged to group 1 and one patient to group 2. The mortality causes in group 1 were: kidney insufficiency due to coagulopathy; dehiscence of gastrojejunal anastomosis and sepsis, pneumonia due to broncoaspiration and necrosis of the pancreaticojejunostomy loop associated with sepsis. The only patient of group 2 passed due to necrotic pancreatitis associated with pancreatic fistula and sepsis.
Regarding the AD1PO value, the median was 175 U/l (48.5-954) in group 1 and 2172.5 U/l (833.5-6421.0) in group 2. This difference was statistically significant (p=0.001).
The area under the ROC curve presented an exactness index of 83.0% (p=0.001). When considering a 180 U/l cut-off point for AD1PO, the following results were obtained: sensitivity 100%, specificity 48.8%, positive predictor value 50%, and negative predictor value 100%. There was an association between the arbitrated AD1PO value from the ROC curve (>180 U/l) with groups 1 and 2 (p<0.001). With a cut-off point of 578 U/l, the results were: sensitivity 80%, specificity 39%, positive predictor value 50% and negative predictor value 86.2%. There was also an association between the arbitrated AD1PO value from the ROC curve (>578 U/l) with groups 1 and 2 (p<0.003). Patients presenting AD1PO values higher than 180 U/l and 578 U/l were 2.0 and 6.25 times more likely to belong to group 2, and therefore require postoperative drainage. Table 3 shows the comparison between the two AD1PO values.
TABLE 3Precision and exactness measurements, per AD1PO cut-off point Parameters Cut-off point (U/l) 180 578Sensitivity 100% 80%Specificity 48.8% 39%VPP 50% 50%VPN 100% 86.2%
|
CONCLUSION
|
Was validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients.
|
[
"Statistical analysis"
] |
[
"After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. "
] |
[
null
] |
[
"INTRODUCTION",
"METHOD",
"Statistical analysis",
"RESULTS",
"DISCUSSION",
"CONCLUSION"
] |
[
"Historically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them\n16\n. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis\n13\n\n,\n\n11\n. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results\n8\n\n,\n\n18\n\n,\n\n19\n, which has been a limiting factor in the practice of not using drains in this type of procedure. \nDescription of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L\n6\n and 90 U/l\n14\n have been suggested. \nThe objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.",
"The project was submitted and approved by the Human Research Ethics Committee, under nº 59716616.9.0000.5292 in the Plataforma Brasil.\nSixty-three patients were analyzed from a prospective database, who had undergone Kausch-Whipple surgery performed by the 1st author of this study (ECA), for the treatment of pancreatic or peripancreatic diseases. The time period was between June 2007 and September 2016, in the following hospitals located in the city of Natal: Hospital Universitário Onofre Lopes, Liga Norte Riograndense Contra o Câncer, Casa de Saúde São Lucas and Natal Hospital Center.\nThe technical steps for the Whipple technique as performed by the author are detailed in previous studies\n1\n\n,\n\n2\n. Three techniques for pancreatojejunal anastomosis were utilized. For pancreas with main pancreatic duct ≥5 mm, two-layer pancreatojejunal terminolateral duct to mucosa anastomosis was performed. For pancreas with normal or slightly dilated (diameter <5 mm) main pancreatic duct, terminoterminal pancreatojejunal anastomosis (“telescopic”) or terminolateral pancreatojejunal anastomosis (“invagination”) were carried out, the latter performed systematically after the 33rd patient from the casebook. At the end of the surgery, two drains, preferably laminar silicone drains, were placed in the cavity and exteriorized at each flank. \nIn the majority of patients, subcutaneous octreotide was utilized as prophylaxis for the prevention of PF, at 0.3 mg/day, fractioned in 8 h intervals, during seven days. Debits were recorded along with amylase dosage of the drained liquid on the 1st, 3rd, 5th, 7th and sometimes, on the 9th postoperative day. The value of AD1PO vas defined from the highest amylase value of both drains, measured from a sample of liquid obtained on the first day after surgery. \nPF diagnosis utilized the 2016 revised criterion of GIEDFP\n3\n. PF was defined when on the 3rd postoperative (PO) day the value of amylase in the drained liquid was three times higher than the normal upper limit for serum amylase. Patients that did not develop fistula or those with transient biochemical fistula (<7 days PO) were characterized as group 1, and patients with persistent biochemical fistula (between 7 and 21 days PO) and fistula grade B and C were characterized as group 2. In most cases, on the 9th day after surgery, control ultrasound or tomography tests were carried out. Removal of drains occurred in those cases with low amylase values for the drained liquid (under three times the upper limit for normal serum amylase) and when image tests did not show abdominal collections. Intra-hospital mortality was defined as death occurring within 90 days of surgery.\nThe Receiver Operating Characteristic (ROC) curve was utilized to identify an adequate cut-off point for AD1PO and verify its predictive characteristic in patients of both groups. The ROC value was generated after sensitivity, specificity and accuracy calculations. Additionally, the cut-off point 578 U/l was also selected because of its closeness to the value utilized by Fong e cols \n9\n, for comparison purposes. \n Statistical analysis After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. \nAfter determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. ",
"After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests. ",
"From the initial sample of 63 patients, two were excluded. One had AD1PO collected incorrectly on the 2nd day after surgery, and the other passed within the 1st week PO. Therefore analysis covered 61 patients. Group 1 was constituted of 41 patients, of which 36 did not develop fistula and five transient biochemical fistula (grade A). Group 2 was constituted of 20 patients, of which six presented persistent biochemical fistula (grade A), eight degree B fistula, and six grade C fistula. The index of clinical fistula (grades B + C) in the overall sample was 22.9%. \nThe demographic and clinical characteristics of the patients, as well as respective tests, are shown in Table 1. There was no statistical difference between groups regarding age, gender and type of illness. However, regarding the size of main pancreatic duct and type of anastomosis, a statistically significant difference was detected. All patients with dilated main pancreatic ducts as well as those submitted to duct to mucosa anastomosis belonged to group 1.\n\nTABLE 1Associations between demographic and clinical characteristics and patient groups Variables Group 1 Group 2 pAge 58.0±13.06I\n 57.1±12.1I\n 0.706II\n\n n % n %\nGender\n\n\n\n\nMale 19 63.3 11 36.7 0.525III\nFemale 22 71.0 9 29.0Disease\n\n\n\n\nPancreatic adenocarcinoma 17 65.4 9 34.6 0.264III\nPapillary adenocarcinoma 12 60.0 8 40.0Duodenal adenocarcinoma 1 33.3 2 66.7Colangiocarcinoma 4 100.0 0 0.0Frantz 5 100.0 0 0.0Others 2 66.7 1 33.3Size of duct\n\n\n\n\n< 5 31 60.8 20 39.2 0.023IV\n> 5 10 100.0 0 0.0Anastomosis*\n\n\n\n\nT-L (Invagination) 14 50.0 14 50.0 0.011III\nT-T 17 73.9 6 26.1T-L (DM) 10 100.0 0 0.0I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\n\nI=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)\nRegarding postoperative evolution, group 2 was associated with a higher level of general complications, postoperative collections, longer permanence of abdominal drain and hospitalization time. There was also an association between high resurgery index and patients of group 2. Nevertheless, despite all these worse result factors related to group 2, there was no statistically significant difference when comparing mortality across groups (Table 2). \n\nTABLE 2Associations between postoperative clinical findings and patient groups, along with results for drain and hospitalization times, per group Variable Group 1 Group 2 p n % n %\nCollection\n\n\n\n\nYes733.31466.7 <0.001I\nNo3485.0615.0Postoperative complications\n\n\n\n\nYes2050.020.050.0 <0.001II\nNo21100.000.0Resurgery\n\n\n\n\nYes746.7853.3 0.051I\nNo3473.91226.1Time with drain (days) 9.5±2.60III29.1±13.58III <0.001IV\nHospitalization time (days) 16.7±17.9III34.7±16.7III <0.001IV\nMortality49.815.0 1.000II\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\n\n I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test\nMortality of the series was 8.19%: four patients belonged to group 1 and one patient to group 2. The mortality causes in group 1 were: kidney insufficiency due to coagulopathy; dehiscence of gastrojejunal anastomosis and sepsis, pneumonia due to broncoaspiration and necrosis of the pancreaticojejunostomy loop associated with sepsis. The only patient of group 2 passed due to necrotic pancreatitis associated with pancreatic fistula and sepsis. \nRegarding the AD1PO value, the median was 175 U/l (48.5-954) in group 1 and 2172.5 U/l (833.5-6421.0) in group 2. This difference was statistically significant (p=0.001).\nThe area under the ROC curve presented an exactness index of 83.0% (p=0.001). When considering a 180 U/l cut-off point for AD1PO, the following results were obtained: sensitivity 100%, specificity 48.8%, positive predictor value 50%, and negative predictor value 100%. There was an association between the arbitrated AD1PO value from the ROC curve (>180 U/l) with groups 1 and 2 (p<0.001). With a cut-off point of 578 U/l, the results were: sensitivity 80%, specificity 39%, positive predictor value 50% and negative predictor value 86.2%. There was also an association between the arbitrated AD1PO value from the ROC curve (>578 U/l) with groups 1 and 2 (p<0.003). Patients presenting AD1PO values higher than 180 U/l and 578 U/l were 2.0 and 6.25 times more likely to belong to group 2, and therefore require postoperative drainage. Table 3 shows the comparison between the two AD1PO values. \n\nTABLE 3Precision and exactness measurements, per AD1PO cut-off point Parameters Cut-off point (U/l) 180 578Sensitivity 100% 80%Specificity 48.8% 39%VPP 50% 50%VPN 100% 86.2%\n",
"AD1PO as a predictor of post-pancreatectomy PF was described by Yamaguchi e cols in 2003\n20\n. The authors analyzed the amylase values measured in abdominal drains of 26 patients submitted to pancreatectomy, and observed that values were already high on the first day PO in those who developed clinical PF after. At least three meta-analysis\n10\n\n,\n\n15\n\n,\n\n21\n have validated the correlation between high AD1PO values and the development of PF. However, there is still controversy on the adequate cut-off point for AD1PO to predict PF, and on its applicability to the removal of prophylactic drains inserted after pancreatic resections. Verona’s group was the first to establish a strategy for the early removal of abdominal drains based on AD1PO values\n6\n. The authors carried out a prospective study on 114 patients submitted to pancreatic resection and randomized those with AD1PO <5000 U/l to remove the drain on the 3rd or 5th day PO. Early removal of drains was associated with a lower rate of pancreatic fistula (1.8% vs. 26%), lower rate of abdominal complications (12.2% vs. 52.6%), lower rate of pulmonary complications (26.3% vs. 52.6%), shorter hospitalization times (8.7 (±4) vs. 10.8 (±6.9) and lower hospital readmission rates (0% vs. 8.8%). Although the study suggested that early removal of abdominal drains was safe with AD1PO under 5000 U/l, other authors reported hypothetical PF indexes between 25-48% if drains were removed according to this suggestion\n7\n\n,\n\n12\n.\nFor those patients submitted to duodenopancreatectomy and that received prophylactic drains, the most important question is to know which patients will not develop PF - and not the other way around. On one hand, knowing which patients will not develop PF is useful because it allows for early drain removal, which leads to shorter hospitalization times. On the other hand, predicting which patients will develop PF after surgery, when the abdominal cavity is being drained, is not as relevant because PF can be easily diagnosed by measuring amylase in the drained liquid. In most cases, treatment is simple maintenance of drains, associated with fasting and nutritional support. For this reason the study herein presented analyzed two low AD1PO cut-off points (180 and 578) that were associated with higher specificity and consequently, with a high negative predictive factor, in detriment to a high cut-off point associated with higher sensitivity to PF diagnosis. Another reason for the selection is related to the impossibility of obtaining daily interventionist radiology services in the public hospital where the majority of the patients studied underwent surgery. These services include percutaneous drainage of abdominal collections/abscesses, which is indicated in urgent cases where there are abdominal complications as a consequence of undrained anastomotic leaks. \nThe methodology of this study utilized the PF definition based on the revised classification by ISGPS\n5\n, which no longer considers grade A of the previous classification to be a true PF\n4\n. However, as this study aimed at identifying a group of patients in which early removal of drains could be beneficial, it was important to include not only the concept of PF, but also grades B and C and those patients with biochemical fistula that persisted with amylase-rich debit between 7-21 days PO. In these patients, early removal of drains would increase susceptibility to abdominal collections or abscesses. With the 180 U/l cut-off point, an excellent correlation was obtained between AD1PO and absence of fistula. Therefore it was possible to identify 1/3 of patients that would have been benefitted by early drain removal. Besides, with the same cut-off, none of the patients that developed clinically relevant fistula or even persistent biochemical fistula would have had their drains removed early. \nData obtained herein were capable of validating previous publications that utilized low cut-off points\n7\n\n,\n\n12\n\n,\n\n14\n\n,\n\n17\n. Differently from these studies, however, the first series was entirely prospective herein, including only patients submitted consecutively to duodenopancreatectomy by a single team of surgeons, where PF definition followed ISGPS criteria. The cut-off point 180 U/l obtained herein was lower than the one found by Fong et al\n9\n at the Massachusetts General Hospital. They divided the study in two cohorts: the first involved 126 patients, and the 612 U/l cut-off point was defined as the one presenting best accuracy, sensitivity and specificity; the second had the objective of validating a 600 U/l cut-off point, involving 369 patients submitted to duodenopancreatectomy within January 2009 and December 2012. Almost two-thirds of patients (62.1%) presented AD1PO lower than 600 U/l and only two PF cases (0.9%) were diagnosed. This was different than the other patients with AD1PO of at least 600 U/l, with PF incidence of 31.4%. When considering herein a cut-off point close to Fong et al., 578 U/l, 13.8% of patients with PF diagnosis would have been affected by early removal of drains (negative predictor value: 86.2%). In the reality considered herein, there is no non-invasive treatment for intracavitary collections/abscesses available all the time at the public hospital, and therefore increasing the cut-off point would be a high price to pay. \nThis study presents some limitations. The first concerns the low number of included patients, which did not hinder statistical analysis. The second is related to the non-verification of the real benefits of drains in patients who developed PF, as all patients received drains. The high index of abdominal collections (65%) and necessity of re-surgery (40%) in group 2 suggested that, for some patients, drains were not totally efficient; nevertheless, the objective of the study was to find a group of patients in which drains could be removed early and not confirm the non-efficiency of drains in the group that developed PF.",
"Was validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients. "
] |
[
"intro",
"methods",
null,
"results",
"discussion",
"conclusions"
] |
[
"Amylase",
"Postoperative complications",
"Pancreatic fistula",
"Pancreatoduodenectomy",
"Amilase",
"Complicações pós-operatórias",
"Fístula pancreática",
"Pancreatoduodenectomia"
] |
INTRODUCTION:
Historically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them
16
. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis
13
,
11
. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results
8
,
18
,
19
, which has been a limiting factor in the practice of not using drains in this type of procedure.
Description of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L
6
and 90 U/l
14
have been suggested.
The objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.
METHOD:
The project was submitted and approved by the Human Research Ethics Committee, under nº 59716616.9.0000.5292 in the Plataforma Brasil.
Sixty-three patients were analyzed from a prospective database, who had undergone Kausch-Whipple surgery performed by the 1st author of this study (ECA), for the treatment of pancreatic or peripancreatic diseases. The time period was between June 2007 and September 2016, in the following hospitals located in the city of Natal: Hospital Universitário Onofre Lopes, Liga Norte Riograndense Contra o Câncer, Casa de Saúde São Lucas and Natal Hospital Center.
The technical steps for the Whipple technique as performed by the author are detailed in previous studies
1
,
2
. Three techniques for pancreatojejunal anastomosis were utilized. For pancreas with main pancreatic duct ≥5 mm, two-layer pancreatojejunal terminolateral duct to mucosa anastomosis was performed. For pancreas with normal or slightly dilated (diameter <5 mm) main pancreatic duct, terminoterminal pancreatojejunal anastomosis (“telescopic”) or terminolateral pancreatojejunal anastomosis (“invagination”) were carried out, the latter performed systematically after the 33rd patient from the casebook. At the end of the surgery, two drains, preferably laminar silicone drains, were placed in the cavity and exteriorized at each flank.
In the majority of patients, subcutaneous octreotide was utilized as prophylaxis for the prevention of PF, at 0.3 mg/day, fractioned in 8 h intervals, during seven days. Debits were recorded along with amylase dosage of the drained liquid on the 1st, 3rd, 5th, 7th and sometimes, on the 9th postoperative day. The value of AD1PO vas defined from the highest amylase value of both drains, measured from a sample of liquid obtained on the first day after surgery.
PF diagnosis utilized the 2016 revised criterion of GIEDFP
3
. PF was defined when on the 3rd postoperative (PO) day the value of amylase in the drained liquid was three times higher than the normal upper limit for serum amylase. Patients that did not develop fistula or those with transient biochemical fistula (<7 days PO) were characterized as group 1, and patients with persistent biochemical fistula (between 7 and 21 days PO) and fistula grade B and C were characterized as group 2. In most cases, on the 9th day after surgery, control ultrasound or tomography tests were carried out. Removal of drains occurred in those cases with low amylase values for the drained liquid (under three times the upper limit for normal serum amylase) and when image tests did not show abdominal collections. Intra-hospital mortality was defined as death occurring within 90 days of surgery.
The Receiver Operating Characteristic (ROC) curve was utilized to identify an adequate cut-off point for AD1PO and verify its predictive characteristic in patients of both groups. The ROC value was generated after sensitivity, specificity and accuracy calculations. Additionally, the cut-off point 578 U/l was also selected because of its closeness to the value utilized by Fong e cols
9
, for comparison purposes.
Statistical analysis After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests.
After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests.
Statistical analysis:
After determination of the cut-off point, the association was verified by Fisher’s Exact test. The odds ratio (OR) was also calculated. The hypothesis tested was that AD1PO levels were different in groups 1 and 2, utilizing Mann-Whitney’s test. Chi-squared and Fisher’s tests were applied to verify the association between demographic and clinical variables with different groups. Statistical package SPSS®21 was utilized. A 5% significance level was applied to all tests.
RESULTS:
From the initial sample of 63 patients, two were excluded. One had AD1PO collected incorrectly on the 2nd day after surgery, and the other passed within the 1st week PO. Therefore analysis covered 61 patients. Group 1 was constituted of 41 patients, of which 36 did not develop fistula and five transient biochemical fistula (grade A). Group 2 was constituted of 20 patients, of which six presented persistent biochemical fistula (grade A), eight degree B fistula, and six grade C fistula. The index of clinical fistula (grades B + C) in the overall sample was 22.9%.
The demographic and clinical characteristics of the patients, as well as respective tests, are shown in Table 1. There was no statistical difference between groups regarding age, gender and type of illness. However, regarding the size of main pancreatic duct and type of anastomosis, a statistically significant difference was detected. All patients with dilated main pancreatic ducts as well as those submitted to duct to mucosa anastomosis belonged to group 1.
TABLE 1Associations between demographic and clinical characteristics and patient groups Variables Group 1 Group 2 pAge 58.0±13.06I
57.1±12.1I
0.706II
n % n %
Gender
Male 19 63.3 11 36.7 0.525III
Female 22 71.0 9 29.0Disease
Pancreatic adenocarcinoma 17 65.4 9 34.6 0.264III
Papillary adenocarcinoma 12 60.0 8 40.0Duodenal adenocarcinoma 1 33.3 2 66.7Colangiocarcinoma 4 100.0 0 0.0Frantz 5 100.0 0 0.0Others 2 66.7 1 33.3Size of duct
< 5 31 60.8 20 39.2 0.023IV
> 5 10 100.0 0 0.0Anastomosis*
T-L (Invagination) 14 50.0 14 50.0 0.011III
T-T 17 73.9 6 26.1T-L (DM) 10 100.0 0 0.0I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)
I=mean±standard deviation; II=Mann-Whitney’s test; III= Chi-squared test; IV= Fisher’s exact test; * T-L=terminolateral; T-T=terminoterminal; T-L (DM)=terminolateral (duct to mucosa)
Regarding postoperative evolution, group 2 was associated with a higher level of general complications, postoperative collections, longer permanence of abdominal drain and hospitalization time. There was also an association between high resurgery index and patients of group 2. Nevertheless, despite all these worse result factors related to group 2, there was no statistically significant difference when comparing mortality across groups (Table 2).
TABLE 2Associations between postoperative clinical findings and patient groups, along with results for drain and hospitalization times, per group Variable Group 1 Group 2 p n % n %
Collection
Yes733.31466.7 <0.001I
No3485.0615.0Postoperative complications
Yes2050.020.050.0 <0.001II
No21100.000.0Resurgery
Yes746.7853.3 0.051I
No3473.91226.1Time with drain (days) 9.5±2.60III29.1±13.58III <0.001IV
Hospitalization time (days) 16.7±17.9III34.7±16.7III <0.001IV
Mortality49.815.0 1.000II
I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test
I=Chi-squared test; II=Fisher’s exact test; III= mean±standard deviation; IV=Mann-Whitney’s test
Mortality of the series was 8.19%: four patients belonged to group 1 and one patient to group 2. The mortality causes in group 1 were: kidney insufficiency due to coagulopathy; dehiscence of gastrojejunal anastomosis and sepsis, pneumonia due to broncoaspiration and necrosis of the pancreaticojejunostomy loop associated with sepsis. The only patient of group 2 passed due to necrotic pancreatitis associated with pancreatic fistula and sepsis.
Regarding the AD1PO value, the median was 175 U/l (48.5-954) in group 1 and 2172.5 U/l (833.5-6421.0) in group 2. This difference was statistically significant (p=0.001).
The area under the ROC curve presented an exactness index of 83.0% (p=0.001). When considering a 180 U/l cut-off point for AD1PO, the following results were obtained: sensitivity 100%, specificity 48.8%, positive predictor value 50%, and negative predictor value 100%. There was an association between the arbitrated AD1PO value from the ROC curve (>180 U/l) with groups 1 and 2 (p<0.001). With a cut-off point of 578 U/l, the results were: sensitivity 80%, specificity 39%, positive predictor value 50% and negative predictor value 86.2%. There was also an association between the arbitrated AD1PO value from the ROC curve (>578 U/l) with groups 1 and 2 (p<0.003). Patients presenting AD1PO values higher than 180 U/l and 578 U/l were 2.0 and 6.25 times more likely to belong to group 2, and therefore require postoperative drainage. Table 3 shows the comparison between the two AD1PO values.
TABLE 3Precision and exactness measurements, per AD1PO cut-off point Parameters Cut-off point (U/l) 180 578Sensitivity 100% 80%Specificity 48.8% 39%VPP 50% 50%VPN 100% 86.2%
DISCUSSION:
AD1PO as a predictor of post-pancreatectomy PF was described by Yamaguchi e cols in 2003
20
. The authors analyzed the amylase values measured in abdominal drains of 26 patients submitted to pancreatectomy, and observed that values were already high on the first day PO in those who developed clinical PF after. At least three meta-analysis
10
,
15
,
21
have validated the correlation between high AD1PO values and the development of PF. However, there is still controversy on the adequate cut-off point for AD1PO to predict PF, and on its applicability to the removal of prophylactic drains inserted after pancreatic resections. Verona’s group was the first to establish a strategy for the early removal of abdominal drains based on AD1PO values
6
. The authors carried out a prospective study on 114 patients submitted to pancreatic resection and randomized those with AD1PO <5000 U/l to remove the drain on the 3rd or 5th day PO. Early removal of drains was associated with a lower rate of pancreatic fistula (1.8% vs. 26%), lower rate of abdominal complications (12.2% vs. 52.6%), lower rate of pulmonary complications (26.3% vs. 52.6%), shorter hospitalization times (8.7 (±4) vs. 10.8 (±6.9) and lower hospital readmission rates (0% vs. 8.8%). Although the study suggested that early removal of abdominal drains was safe with AD1PO under 5000 U/l, other authors reported hypothetical PF indexes between 25-48% if drains were removed according to this suggestion
7
,
12
.
For those patients submitted to duodenopancreatectomy and that received prophylactic drains, the most important question is to know which patients will not develop PF - and not the other way around. On one hand, knowing which patients will not develop PF is useful because it allows for early drain removal, which leads to shorter hospitalization times. On the other hand, predicting which patients will develop PF after surgery, when the abdominal cavity is being drained, is not as relevant because PF can be easily diagnosed by measuring amylase in the drained liquid. In most cases, treatment is simple maintenance of drains, associated with fasting and nutritional support. For this reason the study herein presented analyzed two low AD1PO cut-off points (180 and 578) that were associated with higher specificity and consequently, with a high negative predictive factor, in detriment to a high cut-off point associated with higher sensitivity to PF diagnosis. Another reason for the selection is related to the impossibility of obtaining daily interventionist radiology services in the public hospital where the majority of the patients studied underwent surgery. These services include percutaneous drainage of abdominal collections/abscesses, which is indicated in urgent cases where there are abdominal complications as a consequence of undrained anastomotic leaks.
The methodology of this study utilized the PF definition based on the revised classification by ISGPS
5
, which no longer considers grade A of the previous classification to be a true PF
4
. However, as this study aimed at identifying a group of patients in which early removal of drains could be beneficial, it was important to include not only the concept of PF, but also grades B and C and those patients with biochemical fistula that persisted with amylase-rich debit between 7-21 days PO. In these patients, early removal of drains would increase susceptibility to abdominal collections or abscesses. With the 180 U/l cut-off point, an excellent correlation was obtained between AD1PO and absence of fistula. Therefore it was possible to identify 1/3 of patients that would have been benefitted by early drain removal. Besides, with the same cut-off, none of the patients that developed clinically relevant fistula or even persistent biochemical fistula would have had their drains removed early.
Data obtained herein were capable of validating previous publications that utilized low cut-off points
7
,
12
,
14
,
17
. Differently from these studies, however, the first series was entirely prospective herein, including only patients submitted consecutively to duodenopancreatectomy by a single team of surgeons, where PF definition followed ISGPS criteria. The cut-off point 180 U/l obtained herein was lower than the one found by Fong et al
9
at the Massachusetts General Hospital. They divided the study in two cohorts: the first involved 126 patients, and the 612 U/l cut-off point was defined as the one presenting best accuracy, sensitivity and specificity; the second had the objective of validating a 600 U/l cut-off point, involving 369 patients submitted to duodenopancreatectomy within January 2009 and December 2012. Almost two-thirds of patients (62.1%) presented AD1PO lower than 600 U/l and only two PF cases (0.9%) were diagnosed. This was different than the other patients with AD1PO of at least 600 U/l, with PF incidence of 31.4%. When considering herein a cut-off point close to Fong et al., 578 U/l, 13.8% of patients with PF diagnosis would have been affected by early removal of drains (negative predictor value: 86.2%). In the reality considered herein, there is no non-invasive treatment for intracavitary collections/abscesses available all the time at the public hospital, and therefore increasing the cut-off point would be a high price to pay.
This study presents some limitations. The first concerns the low number of included patients, which did not hinder statistical analysis. The second is related to the non-verification of the real benefits of drains in patients who developed PF, as all patients received drains. The high index of abdominal collections (65%) and necessity of re-surgery (40%) in group 2 suggested that, for some patients, drains were not totally efficient; nevertheless, the objective of the study was to find a group of patients in which drains could be removed early and not confirm the non-efficiency of drains in the group that developed PF.
CONCLUSION:
Was validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients.
|
Background:
The value of drain amylase on the first postoperative day after pancreatic resections has been described as an efficient predictor of pancreatic fistula. In spite of this, the cut-off point below which the drains can be removed early remains controversial.
Methods:
Were included patients undergoing Whipple surgery in the period of 2007 to 2016. Group 1 enrolled the ones who did not develop fistula and those who developed biochemical fistula for less than seven days postoperatively and group 2 included patients who developed persistent biochemical fistula between seven and 21 days and those with grade B and C fistula.
Results:
Sixty-one patients were included, 41 comprised group 1 and 20 group 2. The incidence of abdominal collections, need for reoperation and time of hospitalization were for group 1 and 2, respectively: 17.1%, 17.1% and 9.5 days, and 65%, 40% and 21.1 days. The median of the amylase from the drain at 1st postoperative day was in group 1 and 2, respectively: 175 U/l and 3172.5 U/l (p=0.001). Using a cut-off of 180 to predict the group to which the patient would belong there was obtained sensitivity, specificity, positive predictive value and negative predictive value of 100%, 48.8%, 50% and 100% respectively.
Conclusions:
It was validated the cut-off value of 180 U/l as appropriate to early drain removal.
|
INTRODUCTION:
Historically, abdominal drains have been utilized at the end of gastrointestinal surgeries with the objective of removing blood, pancreatic juices, lymph, and other secretions that could be present. Besides, it is an efficient manner to identify pancreatic fistulas (PF) and even treat them
16
. In the last 25 years, some studies have suggested that abdominal drains placed after duodenopancreatectomy can cause negative effects due to the risk of contamination of the abdominal cavity or even due to direct lesions of the intestinal loops or anastomosis
13
,
11
. There are few studies that have randomized the use (or not) of systematic prophylactic drains in pancreatic resections, with conflicting results
8
,
18
,
19
, which has been a limiting factor in the practice of not using drains in this type of procedure.
Description of correlation between drain amylase in the 1st postoperative day (AD1PO) after pancreatic resections and development of PF has stimulated a new approach to manage these patients. The idea is to substitute the non-utilization of drains in a systematic manner for early removal in those cases where AD1PO dosage is considered low. Several studies have attempted to define an ideal cut-off point for this scenario and variations are as wide as 5.000 U/L
6
and 90 U/l
14
have been suggested.
The objective of the study presented herein is, from a cohort of patients submitted prospectively to duodenopancreatectomy, validate the use of AD1PO in the correlation with PF and define the most adequate cut-off point for early removal of drains in a group of patients.
CONCLUSION:
Was validated the 180 U/l cut-off point as adequate to define those patients in which abdominal drains can be removed early after duodenopancreatectomy. At the same time it is recognized that there is no ideal cut-off point to be utilized uniformly in all services. The ideal cut-off point must vary in accordance with the specifications of the service and compensate for the risk of undrained PF with the benefits of a faster recovery in a higher number of patients.
|
Background:
The value of drain amylase on the first postoperative day after pancreatic resections has been described as an efficient predictor of pancreatic fistula. In spite of this, the cut-off point below which the drains can be removed early remains controversial.
Methods:
Were included patients undergoing Whipple surgery in the period of 2007 to 2016. Group 1 enrolled the ones who did not develop fistula and those who developed biochemical fistula for less than seven days postoperatively and group 2 included patients who developed persistent biochemical fistula between seven and 21 days and those with grade B and C fistula.
Results:
Sixty-one patients were included, 41 comprised group 1 and 20 group 2. The incidence of abdominal collections, need for reoperation and time of hospitalization were for group 1 and 2, respectively: 17.1%, 17.1% and 9.5 days, and 65%, 40% and 21.1 days. The median of the amylase from the drain at 1st postoperative day was in group 1 and 2, respectively: 175 U/l and 3172.5 U/l (p=0.001). Using a cut-off of 180 to predict the group to which the patient would belong there was obtained sensitivity, specificity, positive predictive value and negative predictive value of 100%, 48.8%, 50% and 100% respectively.
Conclusions:
It was validated the cut-off value of 180 U/l as appropriate to early drain removal.
| 3,414 | 276 | 6 |
[
"patients",
"drains",
"ad1po",
"pf",
"group",
"cut",
"cut point",
"point",
"test",
"fistula"
] |
[
"test",
"test"
] |
[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]
|
[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]
|
[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]
|
[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]
|
[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]
|
[CONTENT] Amylase | Postoperative complications | Pancreatic fistula | Pancreatoduodenectomy | Amilase | Complicações pós-operatórias | Fístula pancreática | Pancreatoduodenectomia [SUMMARY]
|
[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]
|
[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]
|
[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]
|
[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]
|
[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]
|
[CONTENT] Amylases | Drainage | Female | Humans | Male | Middle Aged | Pancreatic Fistula | Pancreaticoduodenectomy | Postoperative Care | Postoperative Complications | Predictive Value of Tests | Prospective Studies [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]
|
[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]
|
[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]
|
[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]
|
[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]
|
[CONTENT] patients | drains | ad1po | pf | group | cut | cut point | point | test | fistula [SUMMARY]
|
[CONTENT] drains | pancreatic | studies | systematic | manner | use | pf | correlation | objective | resections [SUMMARY]
|
[CONTENT] amylase | tests | performed | pancreatojejunal | utilized | value | surgery | groups | liquid | applied [SUMMARY]
|
[CONTENT] group | test | 100 | table | 50 | fistula | value | patients | groups | duct [SUMMARY]
|
[CONTENT] ideal cut | ideal | ideal cut point | cut | point | cut point | patients | pf benefits faster | service | compensate risk [SUMMARY]
|
[CONTENT] patients | drains | pf | cut | test | cut point | point | ad1po | group | groups [SUMMARY]
|
[CONTENT] patients | drains | pf | cut | test | cut point | point | ad1po | group | groups [SUMMARY]
|
[CONTENT] the first postoperative day ||| [SUMMARY]
|
[CONTENT] Whipple | the period of 2007 to 2016 ||| Group 1 | less than seven days | 2 | between seven and 21 days [SUMMARY]
|
[CONTENT] Sixty-one | 41 | 1 | 20 | 2 ||| 1 | 2 | 17.1% | 17.1% | 9.5 days | 65% | 40% | 21.1 days ||| 1st postoperative day | 1 | 2 | 175 | 3172.5 | U/l (p=0.001 ||| 180 | 100% | 48.8% | 50% and 100% [SUMMARY]
|
[CONTENT] 180 [SUMMARY]
|
[CONTENT] the first postoperative day ||| ||| Whipple | the period of 2007 to 2016 ||| Group 1 | less than seven days | 2 | between seven and 21 days ||| ||| Sixty-one | 41 | 1 | 20 | 2 ||| 1 | 2 | 17.1% | 17.1% | 9.5 days | 65% | 40% | 21.1 days ||| 1st postoperative day | 1 | 2 | 175 | 3172.5 | U/l (p=0.001 ||| 180 | 100% | 48.8% | 50% and 100% ||| 180 [SUMMARY]
|
[CONTENT] the first postoperative day ||| ||| Whipple | the period of 2007 to 2016 ||| Group 1 | less than seven days | 2 | between seven and 21 days ||| ||| Sixty-one | 41 | 1 | 20 | 2 ||| 1 | 2 | 17.1% | 17.1% | 9.5 days | 65% | 40% | 21.1 days ||| 1st postoperative day | 1 | 2 | 175 | 3172.5 | U/l (p=0.001 ||| 180 | 100% | 48.8% | 50% and 100% ||| 180 [SUMMARY]
|
||||||
Does a continuous local anaesthetic pain treatment after immediate tissue expander reconstruction in breast carcinoma patients more efficiently reduce acute postoperative pain--a prospective randomised study.
|
24433317
|
Immediate breast reconstruction with an expander is a reasonable option for properly selected patients. After reconstruction, patients have severe postoperative pain, which responds poorly to opioids. Our aim was to evaluate if continuous wound infusion of a local anaesthetic into the surgical wound reduces postoperative pain, consumption of opioids and incidence of chronic pain compared to standard intravenous piritramide after primary breast reconstruction in breast carcinoma patients.
|
BACKGROUND
|
Altogether, 60 patients were enrolled in our study; one half in the group with wound infusion of a local anaesthetic, and the other half in the standard (piritramide) group. Parameters measured included: pain intensity (visual analogue scale), drug requirements, alertness, hospitalisation, side-effects and late complications. A p-value of < 0.05 was considered statistically significant.
|
METHODS
|
In the recovery room, the test group reported less acute pain at rest (P = 0.03) and at activity (P = 0.01), and on the day of the surgical procedure they reported less pain at activity (P = 0.003). Consumption of piritramide and metoclopramide was lower in this group (P < 0.0001), but their alertness after the surgical procedure was higher compared to the standard group (P < 0.001). After three months, the test group reported less chronic pain (P = 0.01).
|
RESULTS
|
After primary tissue expander breast reconstruction, wound infusion of a local anaesthetic significantly reduces acute pain and enables reduced opioid consumption, resulting in less postoperative sedation and reduced need for antiemetic drugs. Wound infusion of a local anaesthetic reduces chronic pain.
|
CONCLUSIONS
|
[
"Acute Disease",
"Aged",
"Anesthetics, Local",
"Breast Neoplasms",
"Carcinoma, Ductal, Breast",
"Carcinoma, Intraductal, Noninfiltrating",
"Carcinoma, Lobular",
"Case-Control Studies",
"Catheters, Indwelling",
"Female",
"Follow-Up Studies",
"Humans",
"Lymphatic Metastasis",
"Mammaplasty",
"Mastectomy",
"Middle Aged",
"Neoplasm Grading",
"Neoplasm Staging",
"Pain, Postoperative",
"Postoperative Complications",
"Prognosis",
"Prospective Studies",
"Tissue Expansion Devices"
] |
3899444
|
Background
|
Breast cancer is the most common cancer in women both in the developed and the developing countries
[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer
[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option
[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010
[4].
As a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery
[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids
[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies
[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids
[6].
Use of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction
[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized
[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.
We decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma
[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile
[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve
[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty
[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy
[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.
|
Methods
|
Altogether, 60 breast carcinoma patients were enrolled in the study protocol between January 2011 and May 2012: 72 patients who were eligible for inclusion were offered the opportunity to participate in our study during the recruitment period; 12 of them declined participation, and the remaining 60 were included in the study. Patients listed for mastectomy and primary reconstruction with a tissue expander because of the presence of breast carcinoma or for prophylactic mastectomy, were randomized during the routine preoperative anaesthetic assessment. Anaesthesia for the procedure was the same for all study subjects. We used the same protocol as already reported for patients with axillary dissection
[14]. The first study arm was treated with continuous local analgesia, and the other (control) arm received standard intravenous analgesia. Allergies to local anaesthetics and chronic pre-operative opioid consumption were the criteria to exclude patients from our study.
After informed consent was obtained the randomization was made by research nurses from the Clinical Research Unit of the Institute of Oncology Ljubljana. The research nurse performed randomization using random numbers generated by a computer and then informed the principal investigator about the treatment allocation of the patient.
Surgical procedures were performed by ten experienced oncology surgeons and ten plastic surgeons. Sentinel lymph node biopsy was performed in 55 patients. In one patient with positive imprint cytology of the sentinel lymph node, axillary lymphadenectomy was performed immediately.
The study was reviewed by the The National Medical Ethics Committee of the Republic of Slovenia and performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. Our study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and conducted with the understanding and consent of all subjects involved. Study was entirely financed by the Institute of Oncology as a part of public service.
Test group of patients Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.
Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.
Control group of patients The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.
The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.
Both groups of patients Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.
From the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.
Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.
From the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.
Pain measurement Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.
Three months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale
[19] in a composite way, as we did in our previous study
[14].
After the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.
Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.
Three months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale
[19] in a composite way, as we did in our previous study
[14].
After the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.
Statistical analysis In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.
The Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.
In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.
The Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.
|
Results
|
The mean age of patients was 47.8 years (range 25 to 64 y), height was 166 cm (range 153 to 178 cm), weight was 61.7 kg (range 45 to 85 kg), and the body mass index (BMI) was 22.4 (range 17 to 31.2). There were no significant differences between the study groups in BMI, American Society of Anesthesiology (ASA) score, comorbidities (Table
1), type and length of surgical procedure, extent of lymph node dissection, adjuvant therapy, complications, or hospital stay (Table
2).
Patient characteristics
ASA, American Society of Anesthesiology; HER-2, human epidermal growth factor receptor-2; n, number of patients.
Treatment of patients, adjuvant therapy, hospital stay and complications
Values are numbers of patients unless stated otherwise.
Acute pain Data on postoperative pain are presented in Table
3.
Pain in the local anaesthetic group and the standard group of patients
Data are presented as median visual analogue scale (VAS) score unless stated otherwise.
Data on postoperative pain are presented in Table
3.
Pain in the local anaesthetic group and the standard group of patients
Data are presented as median visual analogue scale (VAS) score unless stated otherwise.
Opioid consumption Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table
4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).
Mean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients
OAA/S - observer's assessment of alertness/sedation.
Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table
4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).
Mean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients
OAA/S - observer's assessment of alertness/sedation.
Nausea Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).
Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).
Complications No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.
No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.
Hospital stay The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.
The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.
Unilateral and bilateral tissue expander In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).
In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).
Late complications (three months after primary reconstruction) Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.
Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.
|
Conclusions
|
Our current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia.
|
[
"Background",
"Test group of patients",
"Control group of patients",
"Both groups of patients",
"Pain measurement",
"Statistical analysis",
"Acute pain",
"Opioid consumption",
"Nausea",
"Complications",
"Hospital stay",
"Unilateral and bilateral tissue expander",
"Late complications (three months after primary reconstruction)",
"Abbreviations",
"Competing interests",
"Authors’ contributions"
] |
[
"Breast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.",
"Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.",
"The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.",
"Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.",
"Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.",
"In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.",
"Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.",
"Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.",
"Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).",
"No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.",
"The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.",
"In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).",
"Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.",
"BMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.",
"The authors declare that they have no competing interests.",
"BS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Test group of patients",
"Control group of patients",
"Both groups of patients",
"Pain measurement",
"Statistical analysis",
"Results",
"Acute pain",
"Opioid consumption",
"Nausea",
"Complications",
"Hospital stay",
"Unilateral and bilateral tissue expander",
"Late complications (three months after primary reconstruction)",
"Discussion",
"Conclusions",
"Abbreviations",
"Competing interests",
"Authors’ contributions"
] |
[
"Breast cancer is the most common cancer in women both in the developed and the developing countries\n[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer\n[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option\n[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010\n[4].\nAs a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery\n[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids\n[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies\n[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids\n[6].\nUse of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction\n[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized\n[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.\nWe decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma\n[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile\n[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve\n[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty\n[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy\n[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.",
"Altogether, 60 breast carcinoma patients were enrolled in the study protocol between January 2011 and May 2012: 72 patients who were eligible for inclusion were offered the opportunity to participate in our study during the recruitment period; 12 of them declined participation, and the remaining 60 were included in the study. Patients listed for mastectomy and primary reconstruction with a tissue expander because of the presence of breast carcinoma or for prophylactic mastectomy, were randomized during the routine preoperative anaesthetic assessment. Anaesthesia for the procedure was the same for all study subjects. We used the same protocol as already reported for patients with axillary dissection\n[14]. The first study arm was treated with continuous local analgesia, and the other (control) arm received standard intravenous analgesia. Allergies to local anaesthetics and chronic pre-operative opioid consumption were the criteria to exclude patients from our study.\nAfter informed consent was obtained the randomization was made by research nurses from the Clinical Research Unit of the Institute of Oncology Ljubljana. The research nurse performed randomization using random numbers generated by a computer and then informed the principal investigator about the treatment allocation of the patient.\nSurgical procedures were performed by ten experienced oncology surgeons and ten plastic surgeons. Sentinel lymph node biopsy was performed in 55 patients. In one patient with positive imprint cytology of the sentinel lymph node, axillary lymphadenectomy was performed immediately.\nThe study was reviewed by the The National Medical Ethics Committee of the Republic of Slovenia and performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. Our study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and conducted with the understanding and consent of all subjects involved. Study was entirely financed by the Institute of Oncology as a part of public service.\n Test group of patients Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\nBefore wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.\n Control group of patients The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\nThe control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.\n Both groups of patients Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\nPatient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.\n Pain measurement Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\nData about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.\n Statistical analysis In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.\nIn order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.",
"Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.",
"The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.",
"Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.\nFrom the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.",
"Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.\nThree months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale\n[19] in a composite way, as we did in our previous study\n[14].\nAfter the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.",
"In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.\nThe Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.",
"The mean age of patients was 47.8 years (range 25 to 64 y), height was 166 cm (range 153 to 178 cm), weight was 61.7 kg (range 45 to 85 kg), and the body mass index (BMI) was 22.4 (range 17 to 31.2). There were no significant differences between the study groups in BMI, American Society of Anesthesiology (ASA) score, comorbidities (Table \n1), type and length of surgical procedure, extent of lymph node dissection, adjuvant therapy, complications, or hospital stay (Table \n2).\nPatient characteristics\nASA, American Society of Anesthesiology; HER-2, human epidermal growth factor receptor-2; n, number of patients.\nTreatment of patients, adjuvant therapy, hospital stay and complications\nValues are numbers of patients unless stated otherwise.\n Acute pain Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\nData on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.\n Opioid consumption Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\nConsumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.\n Nausea Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\nPatients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).\n Complications No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\nNo local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.\n Hospital stay The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\nThe average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.\n Unilateral and bilateral tissue expander In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\nIn order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).\n Late complications (three months after primary reconstruction) Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.\nThree months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.",
"Data on postoperative pain are presented in Table \n3.\nPain in the local anaesthetic group and the standard group of patients\nData are presented as median visual analogue scale (VAS) score unless stated otherwise.",
"Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table \n4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).\nMean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients\nOAA/S - observer's assessment of alertness/sedation.",
"Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).",
"No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.",
"The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.",
"In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).",
"Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.",
"Ideally the study should have been placebo double-blinded to eliminate all biases. However, the cost of the elastomeric pump and wound catheter was 175 euro, so we were not able to perform a double-blind study. However, the cost of an elastomeric pump and wound catheter was not the only reason that precluded us from performing such a study. We had also ethical reasons for not carrying out a placebo-controlled study. Namely, a reconstruction with a tissue expander is a very painful procedure for the majority of patients, so we felt that it is not ethical to have a control group treated by placebo only. Instead, we offered our control group of patients the best available treatment, that is, intravenous analgesia. Based on the good results of our preliminary data on the use of continuous local anaesthetics for treatment of pain, our patients were willing to participate in our study because they were offered state-of-the-art pain treatment versus potentially even more effective pain treatment.\nSub-pectoral breast augmentation and breast reconstruction with an expander or an implant are associated with considerable pain in the early postoperative period\n[6,9,10,20-24]. Legeby et al.\n[6] found that pain in the first 3 h after immediate breast reconstruction with a tissue expander was more severe than pain after mastectomy or axillary dissection. The mean VAS value for pain in the first 3 h after breast reconstruction was 4.9 and 5.0, if axillary dissection was also performed, whereas the values were 2.1 for mastectomy and 3.0 for mastectomy with axillary dissection\n[6]. Similarly, after tissue-expander implantation, our standard analgesia group of patients also suffered pain in the recovery room, which was by about 3.0 VAS units larger than in patients who underwent breast surgery with axillary dissection without reconstruction, as reported by Strazisar et al.\n[14]. The mean VAS score after immediate reconstruction was 4.9 in the standard analgesia group, whereas it was only 3.4 in the local anaesthetic group. In our previous study, the mean VAS score in recovery after breast cancer surgery with axillary dissection was 2.0 in the standard control group and 0.5 in the local anaesthetic group\n[14]. In both our studies, the local anaesthetic reduced pain by 1.5 VAS units more than standard treatment alone.\nThe most positive result of our study is the observation that 77% of patients treated with local anaesthetics did not experience severe pain. To our knowledge, there are no reports in the literature about similarly effective early postoperative analgesia after the implantation of a breast-tissue expander. Legeby et al. reported that even 48% of patients treated with local anaesthetics experienced unacceptably severe pain (VAS 4.7 to 10.0) in the early postoperative period\n[6]. Legeby et al. used an epidural catheter with only a few holes at its tip and instilled local anaesthetic every 3 h. They probably did not establish an equal distribution of the local anaesthetic along the entire length of the muscle and the wound. On the other hand, we used quality wound-catheters that were perforated 15 cm at the distal end, which enabled a better distribution of the local anaesthetic. Furthermore, we used an elastomeric pump, which enabled a continuous infusion of the local anaesthetic. That is probably the reason why only seven patients (23.1%) in the test group reported pain above a VAS score of 5.0, whereas in the control group, such pain levels were reported by thirteen patients (42.9%).\nLegeby et al. found that the use of opioids was about three times higher after the implantation of a tissue expander compared to other breast surgical procedures\n[6]. After the tissue-expander implantation with or without axillary dissection, the consumption of opioids was 12.8 mg and 11.8 mg, whereas after mastectomy or mastectomy with axillary dissection, the mean consumption of opioids was 3.2 mg and 5.5 mg, respectively\n[6]. Our patients with standard analgesia after the tissue-expander implantation needed 5.6 times more opioids than patients who underwent axillary dissection alone\n[14]. Local anaesthetics can reduce consumption of opioids after mastectomy and axillary dissection\n[11,13,14] and also after breast augmentation with implants\n[7]. Our study shows that the use of continuous local anaesthesia can effectively reduce opioid consumption. Our patients treated with a local anaesthetic needed about three times less opioids than the standard analgesia group. Subsequently, patients from the local anaesthesia group experienced nausea less often, thus they had a lower consumption of metoclopramide than the standard analgesia group. Furthermore, the use of local anaesthetics resulted in less sedated and more alert patients in the recovery room and 6 h after the operation. The complication rate did not differ between the two groups of patients. This is in agreement with the reports of other authors who used local anaesthetics\n[11-13,18,21,25-30]. Some authors report that local anaesthetics appear to reduce the incidence of inflammation\n[31-33].\nThere was no difference in pain on the first postoperative day between the study and control group of patients. It means that local anaesthesia was no longer as effective as it was on the day of surgery. A possible explanation is that muscle overstretching caused by a tissue expander is no longer as painful as it was. Other explanations are that in some patients the position of the wound catheter might have changed during movement or that there was a different perfusion region in the erect, prone or supine position, or that continuous infusion was flawed because of the wound drainage. Unfortunately, in our study we did not test a rescue bolus-injection of levobupivacaine in the case of severe pain. However, we have good experience with application of a rescue bolus of levobupivacaine in patients with neuropathic pain after axillary lymphadenectomy. Many times we have observed that injection of a bolus of 15 mL of 0.25% levobupivacaine into a drain and clamping of wound drainage inhibited neuropathic pain for a couple of hours.\nMemory of severe postoperative pain is the most important risk factor for the development of chronic pain\n[34]. Patients who were treated with patient-controlled analgesia had three times less chronic pain than the traditionally nurse-administered patients\n[6]. The results of our study support this observation. Only 17% of patients treated with a local anaesthetic experienced chronic pain. This is significantly less than in the standard analgesia group of patients, of whom 50% suffered chronic pain. The proportion of patients treated with axillary lymphadenectomy, chemotherapy, hormone therapy and/or irradiation was similar in both study arms. Hence, we think that inadequately treated acute pain had an impact on the frequency of chronic pain in the standard analgesia group.",
"Our current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia.",
"BMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.",
"The authors declare that they have no competing interests.",
"BS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript."
] |
[
null,
"methods",
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
null,
null,
"discussion",
"conclusions",
null,
null,
null
] |
[
"Breast carcinoma",
"Primary reconstruction with tissue expander",
"Pain treatment",
"Wound infusion of local anaesthetic",
"Elastomeric pump"
] |
Background:
Breast cancer is the most common cancer in women both in the developed and the developing countries
[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer
[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option
[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010
[4].
As a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery
[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids
[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies
[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids
[6].
Use of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction
[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized
[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.
We decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma
[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile
[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve
[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty
[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy
[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.
Methods:
Altogether, 60 breast carcinoma patients were enrolled in the study protocol between January 2011 and May 2012: 72 patients who were eligible for inclusion were offered the opportunity to participate in our study during the recruitment period; 12 of them declined participation, and the remaining 60 were included in the study. Patients listed for mastectomy and primary reconstruction with a tissue expander because of the presence of breast carcinoma or for prophylactic mastectomy, were randomized during the routine preoperative anaesthetic assessment. Anaesthesia for the procedure was the same for all study subjects. We used the same protocol as already reported for patients with axillary dissection
[14]. The first study arm was treated with continuous local analgesia, and the other (control) arm received standard intravenous analgesia. Allergies to local anaesthetics and chronic pre-operative opioid consumption were the criteria to exclude patients from our study.
After informed consent was obtained the randomization was made by research nurses from the Clinical Research Unit of the Institute of Oncology Ljubljana. The research nurse performed randomization using random numbers generated by a computer and then informed the principal investigator about the treatment allocation of the patient.
Surgical procedures were performed by ten experienced oncology surgeons and ten plastic surgeons. Sentinel lymph node biopsy was performed in 55 patients. In one patient with positive imprint cytology of the sentinel lymph node, axillary lymphadenectomy was performed immediately.
The study was reviewed by the The National Medical Ethics Committee of the Republic of Slovenia and performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. Our study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and conducted with the understanding and consent of all subjects involved. Study was entirely financed by the Institute of Oncology as a part of public service.
Test group of patients Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.
Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.
Control group of patients The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.
The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.
Both groups of patients Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.
From the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.
Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.
From the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.
Pain measurement Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.
Three months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale
[19] in a composite way, as we did in our previous study
[14].
After the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.
Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.
Three months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale
[19] in a composite way, as we did in our previous study
[14].
After the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.
Statistical analysis In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.
The Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.
In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.
The Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.
Test group of patients:
Before wound closure, a fenestrated wound catheter was placed by a plastic surgeon into the wound cavity under the musculus pectoralis major and upon the entire length over the upper side of the wound. The wound catheter was fenestrated along 15 cm in the distal part. Nine patients undergoing bilateral reconstruction received a catheter into the wound on both sides. A bolus of 15 mL of 0.25% levobupivacaine was injected into the wound through the catheter immediately after wound closure. Surgical drains and the fenestrated catheter were clamped for 5 minutes after the administration of levobupivacaine to enable its absorption. After the administration of the bolus, the elastomeric pump was connected to the wound catheter. Clamps on the surgical drains were released after 5 minutes, and the clamp on the fenestrated catheter was released after 20 minutes, enabling a continuous infusion of 0.25% levobupivacaine. The elastomeric pump contained 100 mL of 0.25% levobupivacaine, and the flow rate was 2 mL per h. The catheter was removed after 50 h.
Control group of patients:
The control patients, that is, those who received the standard intravenous piritramide treatment, were on a continuous intravenous infusion with piritramide (30 mg), metoclopramide (20 mg) and metamizole (2.5 g) in 100 mL of 0.9% sodium chloride (3 mL/h - 6 mL/h) until the next morning. The nursing staff were instructed to maintain the lowest drip rate of infusion, which relieved the pain. Our experience is that after the first postoperative day the severity of pain decreases and becomes bearable. In order to avoid the side effects of opioids the patients from our control group were treated with continuous intravenous infusion only during the first 24 h as is the standard of care in our department.
Both groups of patients:
Patient-controlled analgesia was not used in our patients. A rescue dose of analgesics was administered by nursing stuff on the demand of a patient. Whenever needed, patients from both groups could get an intravenous bolus of a rescue analgesic (3 mg of piritramide) and/or an antiemetic drug. In the case of nausea or vomiting, the patient received a bolus of an antiemetic drug, the first one being metoclopramide. If no relief was achieved, 1 mg of granisetron or, with persistent nausea 1.25 mg of droperidol, was administered intravenously.
From the first postoperative day, all patients received analgesics in the form of tablets. They were administered one tablet of 100 mg of diclofenac and a combination of paracetamol 2,600 mg per day and tramadol 300 mg per day. The consumption of drugs (analgesia infusion, intravenous drugs and tablets) during hospitalisation was registered.
Pain measurement:
Data about pain (visual analogue scale (VAS) score) were collected by nursing staff, that is, by an independent observer. Pain was measured using the standard VAS score, ranging from 0 to 10. The first measurement was made in the recovery room. Pain was also measured 3, 6 and 9 h after the surgical procedure. Thereafter, pain was measured every 8 h over the next 4 days. All VAS measurements were performed twice, at rest and on activity of an upper extremity. Median VAS score was calculated on the day of surgery and on the first postoperative day from all eight and six measurements, respectively.
Three months after the surgical procedure, that is, before the tissue expander was replaced by an implant, the patients were asked about pain in the postoperative area or upper extremities. Chronic pain after 3 months was defined as pain that bothered patients during normal daytime activities or during rest or sleep. In the majority of patients with persistent pain it occurred during movement of an upper extremity or in a specific position of the extremity. Pain was permanently present when at rest in only a minority of patients. Six hours after the surgical procedure, we measured patients' alertness using the observer’s assessment of alertness/sedation (OAA/S) scale
[19] in a composite way, as we did in our previous study
[14].
After the surgical procedure, the patients received adjuvant therapy according to the tumour stage and guidelines. Adjuvant chemotherapy, external irradiation and/or hormone therapy was used in 38%, 20% and/or 60% of the patients, respectively. The occurrence of complications (inflammation, haematoma and other) was recorded.
Statistical analysis:
In order to estimate the probability (power) to reject the null hypothesis, PS: Power and Sample Size Calculations Software (Version 3.0, January 2009) was used. Prior data indicated that the failure rate among controls was 0.5. If the true failure rate for experimental subjects is 0.15, 27 experimental subjects and 27 control subjects are needed to be able to reject the null hypothesis that the failure rates for experimental and control subjects are equal, with probability (power) of 0.8. The type-I error probability associated with this test of the null hypothesis is 0.05.
The Student t-test or the Mann–Whitney U-test was used according to data distribution. The association between categorical variables was tested by the chi-square test or Fisher's exact test, as appropriate. All comparisons were two-sided and a P-value <0.05 was considered statistically significant. The statistical packages PASW 18 (SPSS Inc., Chicago, IL, USA) and R 2.11.1 (R Foundation for Statistical Computing, Vienna, Austria) were used for the analysis.
Results:
The mean age of patients was 47.8 years (range 25 to 64 y), height was 166 cm (range 153 to 178 cm), weight was 61.7 kg (range 45 to 85 kg), and the body mass index (BMI) was 22.4 (range 17 to 31.2). There were no significant differences between the study groups in BMI, American Society of Anesthesiology (ASA) score, comorbidities (Table
1), type and length of surgical procedure, extent of lymph node dissection, adjuvant therapy, complications, or hospital stay (Table
2).
Patient characteristics
ASA, American Society of Anesthesiology; HER-2, human epidermal growth factor receptor-2; n, number of patients.
Treatment of patients, adjuvant therapy, hospital stay and complications
Values are numbers of patients unless stated otherwise.
Acute pain Data on postoperative pain are presented in Table
3.
Pain in the local anaesthetic group and the standard group of patients
Data are presented as median visual analogue scale (VAS) score unless stated otherwise.
Data on postoperative pain are presented in Table
3.
Pain in the local anaesthetic group and the standard group of patients
Data are presented as median visual analogue scale (VAS) score unless stated otherwise.
Opioid consumption Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table
4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).
Mean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients
OAA/S - observer's assessment of alertness/sedation.
Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table
4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).
Mean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients
OAA/S - observer's assessment of alertness/sedation.
Nausea Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).
Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).
Complications No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.
No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.
Hospital stay The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.
The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.
Unilateral and bilateral tissue expander In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).
In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).
Late complications (three months after primary reconstruction) Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.
Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.
Acute pain:
Data on postoperative pain are presented in Table
3.
Pain in the local anaesthetic group and the standard group of patients
Data are presented as median visual analogue scale (VAS) score unless stated otherwise.
Opioid consumption:
Consumption of piritramide during the first 24 h after the surgical procedure was lower in the test group compared to the control group (P <0.0001) (Table
4). Alertness, as measured 6 h after the surgical procedure, was higher in the test group compared to the control group (P <0.001).
Mean consumption of drugs, and alertness, in the local anaesthetic group and the control group of patients
OAA/S - observer's assessment of alertness/sedation.
Nausea:
Patients in the test group reported less nausea than patients in the standard group. Consumption of metoclopramide during the first 24 h after the surgical procedure was also lower in the test group compared to the standard group (P <0.0001).
Complications:
No local signs of an infection were observed in the area where the wound catheter was inserted. All microbiological samples taken were negative. There were no significant differences in the complications following the surgical procedure between the two groups. Altogether, two patients (3.3%) underwent another surgical procedure because of haematoma, one from the test group and one from the standard group. Inflammation after the surgical procedure occurred in four patients, one in the test group and three in the standard group. After antibiotic therapy, all patients recovered, and the tissue expander stayed in place.
Hospital stay:
The average postoperative hospital stay was 5.3 days. There was no significant difference in the duration of the hospital stay between the two groups.
Unilateral and bilateral tissue expander:
In order to collect a sufficient number of patients all cases with a unilateral or bilateral tissue expander were included in our study. As a result of randomization the number of the patients with a bilateral and unilateral tissue expander reconstruction in the local anaesthetic and control group was nine and ten, respectively. Patients with bilateral tissue expander reconstruction received 2 mL/hour of 0.25% levobupivacaine on each side. Thus, the total daily amount of levobupivacaine was 240 mg, which is within the recommended range and does not exceed the maximum tolerated dose. Patients from the control group with a bilateral reconstruction had more severe pain than patients with a unilateral reconstruction, whereas pain in the local anaesthetic group was not different after a bilateral or unilateral reconstruction. In the control group the mean VAS score in the recovery room after a bilateral and unilateral reconstruction was 6.45 and 4.09, whereas on the day of surgery it was 5.36 and 3.13, respectively (P <0.05).
Late complications (three months after primary reconstruction):
Three months after primary reconstruction, the patients completed a questionnaire on pain. In the test and the control groups of patients, pain was reported in 16.7% and 50% (P = 0.01), respectively. Unilateral lymphoedema of the arm was present in two patients, one from the control group and one from the test group. The patient with oedema from the test group underwent a dissection of the axilla; the patient from the control group underwent a bilateral sentinel lymph node biopsy.
Discussion:
Ideally the study should have been placebo double-blinded to eliminate all biases. However, the cost of the elastomeric pump and wound catheter was 175 euro, so we were not able to perform a double-blind study. However, the cost of an elastomeric pump and wound catheter was not the only reason that precluded us from performing such a study. We had also ethical reasons for not carrying out a placebo-controlled study. Namely, a reconstruction with a tissue expander is a very painful procedure for the majority of patients, so we felt that it is not ethical to have a control group treated by placebo only. Instead, we offered our control group of patients the best available treatment, that is, intravenous analgesia. Based on the good results of our preliminary data on the use of continuous local anaesthetics for treatment of pain, our patients were willing to participate in our study because they were offered state-of-the-art pain treatment versus potentially even more effective pain treatment.
Sub-pectoral breast augmentation and breast reconstruction with an expander or an implant are associated with considerable pain in the early postoperative period
[6,9,10,20-24]. Legeby et al.
[6] found that pain in the first 3 h after immediate breast reconstruction with a tissue expander was more severe than pain after mastectomy or axillary dissection. The mean VAS value for pain in the first 3 h after breast reconstruction was 4.9 and 5.0, if axillary dissection was also performed, whereas the values were 2.1 for mastectomy and 3.0 for mastectomy with axillary dissection
[6]. Similarly, after tissue-expander implantation, our standard analgesia group of patients also suffered pain in the recovery room, which was by about 3.0 VAS units larger than in patients who underwent breast surgery with axillary dissection without reconstruction, as reported by Strazisar et al.
[14]. The mean VAS score after immediate reconstruction was 4.9 in the standard analgesia group, whereas it was only 3.4 in the local anaesthetic group. In our previous study, the mean VAS score in recovery after breast cancer surgery with axillary dissection was 2.0 in the standard control group and 0.5 in the local anaesthetic group
[14]. In both our studies, the local anaesthetic reduced pain by 1.5 VAS units more than standard treatment alone.
The most positive result of our study is the observation that 77% of patients treated with local anaesthetics did not experience severe pain. To our knowledge, there are no reports in the literature about similarly effective early postoperative analgesia after the implantation of a breast-tissue expander. Legeby et al. reported that even 48% of patients treated with local anaesthetics experienced unacceptably severe pain (VAS 4.7 to 10.0) in the early postoperative period
[6]. Legeby et al. used an epidural catheter with only a few holes at its tip and instilled local anaesthetic every 3 h. They probably did not establish an equal distribution of the local anaesthetic along the entire length of the muscle and the wound. On the other hand, we used quality wound-catheters that were perforated 15 cm at the distal end, which enabled a better distribution of the local anaesthetic. Furthermore, we used an elastomeric pump, which enabled a continuous infusion of the local anaesthetic. That is probably the reason why only seven patients (23.1%) in the test group reported pain above a VAS score of 5.0, whereas in the control group, such pain levels were reported by thirteen patients (42.9%).
Legeby et al. found that the use of opioids was about three times higher after the implantation of a tissue expander compared to other breast surgical procedures
[6]. After the tissue-expander implantation with or without axillary dissection, the consumption of opioids was 12.8 mg and 11.8 mg, whereas after mastectomy or mastectomy with axillary dissection, the mean consumption of opioids was 3.2 mg and 5.5 mg, respectively
[6]. Our patients with standard analgesia after the tissue-expander implantation needed 5.6 times more opioids than patients who underwent axillary dissection alone
[14]. Local anaesthetics can reduce consumption of opioids after mastectomy and axillary dissection
[11,13,14] and also after breast augmentation with implants
[7]. Our study shows that the use of continuous local anaesthesia can effectively reduce opioid consumption. Our patients treated with a local anaesthetic needed about three times less opioids than the standard analgesia group. Subsequently, patients from the local anaesthesia group experienced nausea less often, thus they had a lower consumption of metoclopramide than the standard analgesia group. Furthermore, the use of local anaesthetics resulted in less sedated and more alert patients in the recovery room and 6 h after the operation. The complication rate did not differ between the two groups of patients. This is in agreement with the reports of other authors who used local anaesthetics
[11-13,18,21,25-30]. Some authors report that local anaesthetics appear to reduce the incidence of inflammation
[31-33].
There was no difference in pain on the first postoperative day between the study and control group of patients. It means that local anaesthesia was no longer as effective as it was on the day of surgery. A possible explanation is that muscle overstretching caused by a tissue expander is no longer as painful as it was. Other explanations are that in some patients the position of the wound catheter might have changed during movement or that there was a different perfusion region in the erect, prone or supine position, or that continuous infusion was flawed because of the wound drainage. Unfortunately, in our study we did not test a rescue bolus-injection of levobupivacaine in the case of severe pain. However, we have good experience with application of a rescue bolus of levobupivacaine in patients with neuropathic pain after axillary lymphadenectomy. Many times we have observed that injection of a bolus of 15 mL of 0.25% levobupivacaine into a drain and clamping of wound drainage inhibited neuropathic pain for a couple of hours.
Memory of severe postoperative pain is the most important risk factor for the development of chronic pain
[34]. Patients who were treated with patient-controlled analgesia had three times less chronic pain than the traditionally nurse-administered patients
[6]. The results of our study support this observation. Only 17% of patients treated with a local anaesthetic experienced chronic pain. This is significantly less than in the standard analgesia group of patients, of whom 50% suffered chronic pain. The proportion of patients treated with axillary lymphadenectomy, chemotherapy, hormone therapy and/or irradiation was similar in both study arms. Hence, we think that inadequately treated acute pain had an impact on the frequency of chronic pain in the standard analgesia group.
Conclusions:
Our current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia.
Abbreviations:
BMI: body mass index; ASA: American Society of Anesthesiology; VAS: visual analogue scale; OAA/S: observer’s assessment of alertness/sedation.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
BS - made the study design, carried out the study and drafted the manuscript, NB - co author of the study design and co drafted the manuscript, UA - consultant and co drafted the manuscript in the part of Synopsis. All authors read and approved the final manuscript.
|
Background:
Immediate breast reconstruction with an expander is a reasonable option for properly selected patients. After reconstruction, patients have severe postoperative pain, which responds poorly to opioids. Our aim was to evaluate if continuous wound infusion of a local anaesthetic into the surgical wound reduces postoperative pain, consumption of opioids and incidence of chronic pain compared to standard intravenous piritramide after primary breast reconstruction in breast carcinoma patients.
Methods:
Altogether, 60 patients were enrolled in our study; one half in the group with wound infusion of a local anaesthetic, and the other half in the standard (piritramide) group. Parameters measured included: pain intensity (visual analogue scale), drug requirements, alertness, hospitalisation, side-effects and late complications. A p-value of < 0.05 was considered statistically significant.
Results:
In the recovery room, the test group reported less acute pain at rest (P = 0.03) and at activity (P = 0.01), and on the day of the surgical procedure they reported less pain at activity (P = 0.003). Consumption of piritramide and metoclopramide was lower in this group (P < 0.0001), but their alertness after the surgical procedure was higher compared to the standard group (P < 0.001). After three months, the test group reported less chronic pain (P = 0.01).
Conclusions:
After primary tissue expander breast reconstruction, wound infusion of a local anaesthetic significantly reduces acute pain and enables reduced opioid consumption, resulting in less postoperative sedation and reduced need for antiemetic drugs. Wound infusion of a local anaesthetic reduces chronic pain.
|
Background:
Breast cancer is the most common cancer in women both in the developed and the developing countries
[1]. Although systemic therapy and conservative surgery are recommended treatments, mastectomy is still the standard surgical procedure for the local treatment of all stages of breast cancer
[2]. In the United States, one third of newly discovered breast cancer patients decide to undergo a mastectomy, mostly because of fear of recurrence. For those women who choose mastectomy as part of their approach to breast-cancer therapy or prevention, reconstruction may be offered as an option
[3]. In Breast Center Humanitas in Milan, breast reconstruction after mastectomy was performed in 82% of patients in 2010
[4].
As a reconstruction method, an expander or an implant provides a reasonable option in properly selected patients. Patients with small, minimally ptotic breasts are good candidates. The advantages of this method are simple operation, no morbidity at a distant donor site, use of tissue of similar colour, texture and sensation, reduced operating time, and faster postoperative recovery
[5]. Although breast reconstruction with a tissue expander is simple, it causes severe postoperative pain that may respond poorly to opioids
[6]. Severe postoperative pain after breast implant insertion is well-known from breast augmentation studies
[7]. Pain relief was poor despite the progressive increase in the opioid dose and presence of adverse effects of opioids
[6].
Use of local anaesthetics is one of the possible methods for the treatment of acute postoperative pain. To our knowledge, only three studies have been published on the use of local anaesthetics for the treatment of acute postoperative pain after breast reconstruction
[8-10]. Use of local anaesthetics administered into the surgical wound has been shown to be an effective treatment for postoperative pain control and decreased opioid consumption. However, only a small number of patients were included in these studies. Only one of the studies was prospective and randomized
[8] but it was performed on patients with delayed breast reconstruction after mastectomy. We failed to find any prospective randomized trial on the use of local anaesthetics for the treatment of postoperative pain after primary breast reconstruction with a tissue expander.
We decided to conduct study because of the good results obtained in previous studies on axillary lymph node dissection and mastectomy due to breast carcinoma
[11-14]. Levobupivacaine and ropivacaine are long-lasting and very effective local anaesthetics with a favourable toxic profile
[15]. Levobupivacaine was chosen for use in our patients because it was available on our market at the time of our study, while ropivacaine was not. The duration of action of local anaesthetic is proportional to the length of time that it is in contact with the nerve
[16]. Pacik found that a continuous flow as well as intermittent bolus anaesthesia was effective in controlling postoperative pain in augmentation mammoplasty
[17]. But according to Talbot et al., a bolus application of local anaesthetic was not a successful pain treatment after mastectomy
[18], so we decided to use a continuous infusion of local anaesthetic. The aim of our prospective randomized study was to evaluate the effectiveness of a local anaesthetic administered into the surgical wound as a continuous infusion after immediate breast reconstruction with a tissue expander. Our hypothesis was that local anaesthetics are more effective than standard intravenous analgesics for pain relief. We also wanted to determine that continuous infusion of the local anaesthetic enables lower consumption of opioids, reducing the need for antiemetic drugs and sedation compared to standard opioid-based analgesia.
Conclusions:
Our current prospective randomized study shows that a continuous local anaesthetic infusion with the use of analgesic catheters inserted into the surgical wound after primary breast reconstruction with a tissue expander reduces acute pain immediately after the surgical procedure and also on the operative day. Because of that, it represents an effective postoperative pain treatment without side effects. It enables lower opioid consumption in the first 24 h after the surgical procedure, higher alertness and less nausea. We observed that patients treated with a local anaesthetic experienced a lower frequency of chronic pain compared to patients treated with standard analgesia.
|
Background:
Immediate breast reconstruction with an expander is a reasonable option for properly selected patients. After reconstruction, patients have severe postoperative pain, which responds poorly to opioids. Our aim was to evaluate if continuous wound infusion of a local anaesthetic into the surgical wound reduces postoperative pain, consumption of opioids and incidence of chronic pain compared to standard intravenous piritramide after primary breast reconstruction in breast carcinoma patients.
Methods:
Altogether, 60 patients were enrolled in our study; one half in the group with wound infusion of a local anaesthetic, and the other half in the standard (piritramide) group. Parameters measured included: pain intensity (visual analogue scale), drug requirements, alertness, hospitalisation, side-effects and late complications. A p-value of < 0.05 was considered statistically significant.
Results:
In the recovery room, the test group reported less acute pain at rest (P = 0.03) and at activity (P = 0.01), and on the day of the surgical procedure they reported less pain at activity (P = 0.003). Consumption of piritramide and metoclopramide was lower in this group (P < 0.0001), but their alertness after the surgical procedure was higher compared to the standard group (P < 0.001). After three months, the test group reported less chronic pain (P = 0.01).
Conclusions:
After primary tissue expander breast reconstruction, wound infusion of a local anaesthetic significantly reduces acute pain and enables reduced opioid consumption, resulting in less postoperative sedation and reduced need for antiemetic drugs. Wound infusion of a local anaesthetic reduces chronic pain.
| 7,759 | 307 | 20 |
[
"patients",
"group",
"pain",
"local",
"control",
"test",
"surgical",
"reconstruction",
"wound",
"standard"
] |
[
"test",
"test"
] |
[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]
|
[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]
|
[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]
|
[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]
|
[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]
|
[CONTENT] Breast carcinoma | Primary reconstruction with tissue expander | Pain treatment | Wound infusion of local anaesthetic | Elastomeric pump [SUMMARY]
|
[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]
|
[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]
|
[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]
|
[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]
|
[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]
|
[CONTENT] Acute Disease | Aged | Anesthetics, Local | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Intraductal, Noninfiltrating | Carcinoma, Lobular | Case-Control Studies | Catheters, Indwelling | Female | Follow-Up Studies | Humans | Lymphatic Metastasis | Mammaplasty | Mastectomy | Middle Aged | Neoplasm Grading | Neoplasm Staging | Pain, Postoperative | Postoperative Complications | Prognosis | Prospective Studies | Tissue Expansion Devices [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]
|
[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]
|
[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]
|
[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]
|
[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]
|
[CONTENT] patients | group | pain | local | control | test | surgical | reconstruction | wound | standard [SUMMARY]
|
[CONTENT] breast | local | use | mastectomy | pain | anaesthetics | local anaesthetics | breast reconstruction | postoperative pain | reconstruction [SUMMARY]
|
[CONTENT] patients | pain | catheter | wound | mg | subjects | ml | intravenous | fenestrated | day [SUMMARY]
|
[CONTENT] group | patients | unilateral | bilateral | test | control group | test group | control | reconstruction | standard group [SUMMARY]
|
[CONTENT] patients treated | surgical | pain | treated | lower | local anaesthetic | anaesthetic | surgical procedure | procedure | local [SUMMARY]
|
[CONTENT] group | patients | pain | test | control | surgical | local | surgical procedure | standard | procedure [SUMMARY]
|
[CONTENT] group | patients | pain | test | control | surgical | local | surgical procedure | standard | procedure [SUMMARY]
|
[CONTENT] ||| ||| [SUMMARY]
|
[CONTENT] 60 | one half | half ||| ||| [SUMMARY]
|
[CONTENT] 0.03 | 0.01 | the day | 0.003 ||| ||| three months | 0.01 [SUMMARY]
|
[CONTENT] ||| [SUMMARY]
|
[CONTENT] ||| ||| ||| 60 | one half | half ||| ||| ||| ||| 0.03 | 0.01 | the day | 0.003 ||| ||| three months | 0.01 ||| ||| [SUMMARY]
|
[CONTENT] ||| ||| ||| 60 | one half | half ||| ||| ||| ||| 0.03 | 0.01 | the day | 0.003 ||| ||| three months | 0.01 ||| ||| [SUMMARY]
|
||||||
Lung transplantation as therapeutic option in acute respiratory distress syndrome for coronavirus disease 2019-related pulmonary fibrosis.
|
32251003
|
Critical patients with the coronavirus disease 2019 (COVID-19), even those whose nucleic acid test results had turned negative and those receiving maximal medical support, have been noted to progress to irreversible fatal respiratory failure. Lung transplantation (LT) as the sole therapy for end-stage pulmonary fibrosis related to acute respiratory distress syndrome has been considered as the ultimate rescue therapy for these patients.
|
BACKGROUND
|
From February 10 to March 10, 2020, three male patients were urgently assessed and listed for transplantation. After conducting a full ethical review and after obtaining assent from the family of the patients, we performed three LT procedures for COVID-19 patients with illness durations of more than one month and extremely high sequential organ failure assessment scores.
|
METHODS
|
Two of the three recipients survived post-LT and started participating in a rehabilitation program. Pearls of the LT team collaboration and perioperative logistics were summarized and continually improved. The pathological results of the explanted lungs were concordant with the critical clinical manifestation, and provided insight towards better understanding of the disease. Government health affair systems, virology detection tools, and modern communication technology all play key roles towards the survival of the patients and their rehabilitation.
|
RESULTS
|
LT can be performed in end-stage patients with respiratory failure due to COVID-19-related pulmonary fibrosis. If confirmed positive-turned-negative virology status without organ dysfunction that could contraindicate LT, LT provided the final option for these patients to avoid certain death, with proper protection of transplant surgeons and medical staffs. By ensuring instant seamless care for both patients and medical teams, the goal of reducing the mortality rate and salvaging the lives of patients with COVID-19 can be attained.
|
CONCLUSIONS
|
[
"Aged",
"Betacoronavirus",
"COVID-19",
"Coronavirus Infections",
"Extracorporeal Membrane Oxygenation",
"Humans",
"Lung Transplantation",
"Male",
"Middle Aged",
"Pandemics",
"Pneumonia, Viral",
"Pulmonary Fibrosis",
"Respiratory Distress Syndrome",
"SARS-CoV-2"
] |
7339336
|
Introduction
|
The outbreak of the coronavirus disease 2019 (COVID-19) caused more than 200,000 reported cases and more than 8000 people have lost their lives,[1] and thus, was declared a global pandemic by the World Health Organization.[2] As reported by Zhong et al,[3] 5.0% of 1099 patients were admitted to the intensive care unit (ICU) and 2.3% received invasive mechanical ventilation (MV). Publications have reported the general characteristics of disease progression with analysis of outcomes, including those of the recovered and mortality cases.[4] Some patients have prolonged ICU stays due to severe complications, such as acute respiratory distress syndrome (ARDS) and related pulmonary fibrosis, even after results of repeated virological tests are confirmed to be negative. Even with maximal support with MV and extra-corporeal membrane oxygenation (ECMO), such patients showed irreversible deterioration of pulmonary function. We performed lung transplantation (LT) for end-stage patients and hereby present the early results of LT in these recipients. Issues related to the urgent candidate listing of post-COVID-19 critical patients and peri-LT management are further discussed.
|
Methods
|
Ethical approval The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]
The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]
Patient information and data collection This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.
The chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.
This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.
The chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.
Construction of surgical facilities with high protection level In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].
Transplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.
Patient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.
In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].
Transplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.
Patient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.
|
Results
|
General characteristics before LT All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.
General characteristics of the three urgently listed LT recipients.
All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.
General characteristics of the three urgently listed LT recipients.
Pearls of peri-operative management All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.
All the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).
For Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.
In Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.
All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.
All the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).
For Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.
In Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.
Post-LT survival status and explant pathology Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.
Post-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.
When examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.
Representative comparison of explant pathologies.
Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.
Post-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.
When examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.
Representative comparison of explant pathologies.
| null | null |
[
"Ethical approval",
"Patient information and data collection",
"Construction of surgical facilities with high protection level",
"General characteristics before LT",
"Pearls of peri-operative management",
"Post-LT survival status and explant pathology",
"Key points on determination of LT candidacy",
"Best practices for the protection of the medical team involved",
"Factors important for post-LT survival",
"Perspective",
"Funding"
] |
[
"The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]",
"This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.",
"In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.",
"All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.",
"All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.",
"Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.",
"The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.",
"For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.",
"During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.",
"With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.",
"This study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City."
] |
[
null,
"subjects",
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Introduction",
"Methods",
"Ethical approval",
"Patient information and data collection",
"Construction of surgical facilities with high protection level",
"Results",
"General characteristics before LT",
"Pearls of peri-operative management",
"Post-LT survival status and explant pathology",
"Discussion",
"Best time to perform transplantation in critical patients",
"Key points on determination of LT candidacy",
"Best practices for the protection of the medical team involved",
"Factors important for post-LT survival",
"Perspective",
"Funding",
"Conflicts of interest"
] |
[
"The outbreak of the coronavirus disease 2019 (COVID-19) caused more than 200,000 reported cases and more than 8000 people have lost their lives,[1] and thus, was declared a global pandemic by the World Health Organization.[2] As reported by Zhong et al,[3] 5.0% of 1099 patients were admitted to the intensive care unit (ICU) and 2.3% received invasive mechanical ventilation (MV). Publications have reported the general characteristics of disease progression with analysis of outcomes, including those of the recovered and mortality cases.[4] Some patients have prolonged ICU stays due to severe complications, such as acute respiratory distress syndrome (ARDS) and related pulmonary fibrosis, even after results of repeated virological tests are confirmed to be negative. Even with maximal support with MV and extra-corporeal membrane oxygenation (ECMO), such patients showed irreversible deterioration of pulmonary function. We performed lung transplantation (LT) for end-stage patients and hereby present the early results of LT in these recipients. Issues related to the urgent candidate listing of post-COVID-19 critical patients and peri-LT management are further discussed.",
" Ethical approval The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\nThe lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]\n Patient information and data collection This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\nThis is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.\n Construction of surgical facilities with high protection level In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.\nIn view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.",
"The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]",
"This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.\nThe chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.",
"In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].\nTransplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.\nPatient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.",
" General characteristics before LT All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\nAll the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.\n Pearls of peri-operative management All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\nAll the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.\n Post-LT survival status and explant pathology Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.\nBoth patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.",
"All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.\nGeneral characteristics of the three urgently listed LT recipients.",
"All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.\nAll the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).\nFor Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.\nIn Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.",
"Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.\nPost-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.\nWhen examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.\nRepresentative comparison of explant pathologies.",
" Best time to perform transplantation in critical patients This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\nThis is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.\n Key points on determination of LT candidacy The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\nThe following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.\n Best practices for the protection of the medical team involved For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\nFor the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.\n Factors important for post-LT survival During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\nDuring the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.\n Perspective With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.\nWith careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.",
"This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.",
"The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.",
"For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.\nConsiderations and comparisons of surgical preparations.\nDuring the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.\nBy gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.",
"During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]\nRehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.\nWith negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.\nIn the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.",
"With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.",
"This study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City.",
"None."
] |
[
"intro",
"methods",
null,
"subjects",
null,
"results",
null,
null,
null,
"discussion",
"subjects",
null,
null,
null,
null,
null,
"COI-statement"
] |
[
"Coronavirus disease 2019",
"Lung transplantation",
"Acute respiratory distress syndrome",
"Pulmonary fibrosis",
"Sequential Organ Failure Assessment score"
] |
Introduction:
The outbreak of the coronavirus disease 2019 (COVID-19) caused more than 200,000 reported cases and more than 8000 people have lost their lives,[1] and thus, was declared a global pandemic by the World Health Organization.[2] As reported by Zhong et al,[3] 5.0% of 1099 patients were admitted to the intensive care unit (ICU) and 2.3% received invasive mechanical ventilation (MV). Publications have reported the general characteristics of disease progression with analysis of outcomes, including those of the recovered and mortality cases.[4] Some patients have prolonged ICU stays due to severe complications, such as acute respiratory distress syndrome (ARDS) and related pulmonary fibrosis, even after results of repeated virological tests are confirmed to be negative. Even with maximal support with MV and extra-corporeal membrane oxygenation (ECMO), such patients showed irreversible deterioration of pulmonary function. We performed lung transplantation (LT) for end-stage patients and hereby present the early results of LT in these recipients. Issues related to the urgent candidate listing of post-COVID-19 critical patients and peri-LT management are further discussed.
Methods:
Ethical approval The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]
The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]
Patient information and data collection This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.
The chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.
This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.
The chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.
Construction of surgical facilities with high protection level In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].
Transplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.
Patient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.
In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].
Transplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.
Patient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.
Ethical approval:
The lung transplant review and institutional ethics committees of the Shenzhen Third People's Hospital (No. 2020-014) and the Wuxi People's Hospital (No. F2020013) were consulted for urgent LT. Numerous discussions with the patients’ family members ensued regarding goals of survival and care while the patients were sedated. All the procedures were approved by the institutional ethics committees. The multidisciplinary team included transplant surgeons, anesthesiologists, physicians, nurses, epidemiologists, physical therapists, respiratory therapists, pathologists, and perfusionists. Volunteer donors were registered in the Chinese organ donation system. Donated lungs were allocated to the urgently listed candidates in accordance with the national organ allocation principles[5] while considering the priority of urgency related to disease severity. Organ procurement were performed according to the standard protocol.[6]
Patient information and data collection:
This is a case series-based report of the initial experience on LT for post-COVID-19 patients. Post-COVID-19 patients with pulmonary fibrosis-related ARDS that led to an irreversible pulmonary injury were assessed for LT. From February 10 to March 10, 2020, three male patients were urgently listed, and underwent transplantation. Pre-LT chest imaging confirmed pulmonary consolidation with fibrotic change [Figure 1]. Anti-viral drugs and supportive care were provided as routine protocols to the patients. Tracheostomies have been performed to the three patients. The perioperative demographic and clinical data of the patients were collected and analyzed.
The chest image obtained before LT shows extensive pulmonary consolidation with effusions. Patient 1: (A) radiographic image on pre-LT day 1 and (B) chest CT image on pre-LT day 5. Patient 2: (C) radiographic image on pre-LT day 1 and (D) chest CT image on pre-LT day 5. Patient 3: (E) radiographic image on pre-LT day 2, (F) chest CT image on pre-LT day 1. CT: Computed tomography; LT: Lung transplantation.
Construction of surgical facilities with high protection level:
In view of the high risk of disease transmission and contagion to the surrounding environment by the 2019-novel coronavirus (2019-nCoV), special arrangements were made in 48 h before LT, to guarantee that the appropriate operation room set-up and the necessary protection facilities were available. The hospital where patient 1 was admitted is a regional center for infectious diseases. A special work flow was established for post-COVID-19 LT, and an isolated operation area was created [Figure 2A].
Transplantation logistics and post-LT rehabilitation. (A) The standardized negative-pressure operating room with the medical team fully equipped and remote monitoring for patient 1. (B) Intercity transportation of patient 2 by a protected medical team. (C) Improved operation setup and team collaboration for patient 3. (D) Intra-operative image obtained when the right lung was transplanted and the left lung was about to be explanted in patient 3. (E) Illustration of explanted lungs from patient 3. (F) Early initiation of the rehabilitation program for patient 2, with psychological support from family and friends (blessing cards on the wall). LT: Lung transplantation.
Patient 2 was transported from Lianyungang City to Wuxi City, over a distance of 450 km, escorted by a professional team experienced in handling critical high infectious-risk patients [Figure 2B]. Patient 3 was from Hubei Province and traveled to Wuxi City. He was diagnosed as COVID-19 and admitted directly to the local hospital for communicable diseases. With the full support of the Provincial Headquarters for Disease Prevention, Control and Treatment, the LT procedure-related operation room setup was constructed in the hospital for communicable diseases in Wuxi City. Wuxi People's Hospital, the largest LT center in China, technically supported the construction of all the facilities, with upgrading of the protection and care systems.
Results:
General characteristics before LT All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.
General characteristics of the three urgently listed LT recipients.
All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.
General characteristics of the three urgently listed LT recipients.
Pearls of peri-operative management All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.
All the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).
For Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.
In Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.
All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.
All the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).
For Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.
In Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.
Post-LT survival status and explant pathology Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.
Post-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.
When examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.
Representative comparison of explant pathologies.
Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.
Post-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.
When examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.
Representative comparison of explant pathologies.
General characteristics before LT:
All the recipients were male and aged from 58 to 73 years [Table 1]. Convalescent plasma infusion was performed for all the patients before LT. Coagulopathy was a common manifestation of patients with severe COVID-19 infection. Patient 1 and patient 2 suffered from uncontrolled intra-pulmonary bleeding which could not be controlled by conventional therapy. Thus, urgent assessment for LT was warranted while the patients were using MV and ECMO (veno-venous [VV]-ECMO, cannulated via the right jugular and femoral veins; veno-arterio-venous ECMO for patient 1, cannulated via the right jugular vein, right femoral artery and vein). We cautiously repeated the nucleic acid tests with samples from different sites and confirmed the positive-turned-negative status. However, we intermittently observed some mildly positive results from fecal samples of patient 2. All the patients ultimately showed extremely high pre-LT sequential organ failure assessment scores[7] and D-dimer values, as shown in Table 1. Comorbidities, as shown in Table 1, were present before COVID-19. Donated grafts were from deceased brain death donors and negative for 2019-nCoV, fully matched with the recipients. Transportation distance of donated organs were 128, 790, and 1470 km, respectively. The shortest ischemic time could still be guaranteed by steering of green channel for human organ transportation in China.
General characteristics of the three urgently listed LT recipients.
Pearls of peri-operative management:
All the patients were tested negative for a panel of reactive antibodies. Bilateral LT was taken into consideration as the primary choice of treatment. For Patient 1, an additional heart transplantation was also discussed when the medical team anticipated an unstable hemodynamic status with escalation of the inotrope dose. En bloc heart and lung procurement was performed pre-LT with allocation approval, giving the transplant team the option to decide, following intra-operative assessment of the recipient, whether heart and LT to be performed.
All the recipients were escorted to the standard negative pressure operating rooms, and the medical team members were fully equipped [Figure 2C]. The patients remained on MV and ECMO, while the exchange of the endotracheal tube to left- sided double-lumen tube was performed by the anesthesiologists. Owing to pulmonary hypertension and related cardiac dysfunction, intra-operative central cannulated veno-arterial (VA)-ECMO was established for all three patients, to support the circulation through the ascending aorta and right atrium at a flow of 3 to 4 L. The right lung was explanted first for all three cases. For patient 2 and 3, the same procedure was performed for the left side [Figure 2D]. Consolidation, extreme pulmonary tissue edema, and congestion with intra-pulmonary hematoma could be observed in all three cases (Figure 2E showed the explanted lungs from patient 3).
For Patient 1, the right lung was transplanted uneventfully. During the left lung transplant procedure, ventricular fibrillation developed abruptly and the heart arrested. Cardiac massage was commenced and cardiopulmonary bypass was established with cannulation via the superior, inferior venae cava and ascending aorta, instead of VA-ECMO. Emergent heart transplant was performed. The heart was resuscitated to normal rhythm with strength; however, bleeding from the chest cavity and anastomosis could not be managed with sutures and coagulation in the following 5 h. The transplanted heart arrested again, and the patient was pronounced dead.
In Patient 2, a relatively narrow space in the left chest cavity was observed, and a lobar lung transplant using the left upper lobe was performed. As for patient 3, when the left lung was explanted, the patient developed recurrent atrial fibrillation, causing hemodynamic instability. With intensive intra-operative medical management, the left LT was performed as planned. Both patients 2 and 3 were weaned off their intra-operative VA-ECMO support, and the pre-LT VV-ECMO was maintained with low-dose inotropes. The patients were escorted and monitored in isolated negative pressure ICUs.
Post-LT survival status and explant pathology:
Both patients 2 and 3 survived post-LT and regained consciousness on post-operative day (POD) 1. Continuous renal replacement therapy was commenced to achieve negative fluid balance and restoration of renal function. The patients were weaned from VV-ECMO 37 h (patient 2) and 40 h (patient 3) after LT, with grafts fully expanded [Figure 3]. Cyclosporine A was prescribed at a dose (100 mg/d) lower than the conventional initial dose, with gradual tapering according to immune status. Ganciclovir (200 mg/d), antibiotics, and anti-fungal agents were administered per institutional protocol without special treatment for 2019-nCoV. The rehabilitation programs for both patients were initiated early after LT and involved swallowing, limb movements, sitting, and muscle strength training. As the patients had pre-LT tracheostomy, respiratory rehabilitation was initiated with intermittent weaning off MV and respiratory muscle training on POD 3 for patient 2, who gradually progressed to standing balance training on POD 13. Rehabilitation was initiated for patient 3 on POD 2. Patient 2 could be weaned off MV up to 8 h a day on POD 22 and 3 h for patient 3 on POD 12. Bronchoscopy was performed consecutively for 3 days post-LT and every other day from POD 4 on. There were no abnormal findings but a routine clearing of airways. Chest tubes were removed on POD 5 for both patients. Follow-up days were showed in Table 1. Patients 2 and 3 are alive and still admitted in isolated negative-pressure wards. Regularly nucleic acid tests results from samples of bronchoalveolar lavage fluid, nasopharynx, serum, and fecal were all negative. We keep on monitoring the virus tests. If still negative up to 30 days post-LT, we are planning to move the patients to general ward with routine care.
Post-LT images demonstrated fully expanded lung grafts. (A) Radiographic imaging on post-LT day 20 of patient 2. (B) Radiographic image on post-LT day 11 of patient 3. LT: Lung transplantation; POD: Post-operative day.
When examining the explanted lungs from patients 1 and 2 and comparing the pathological characteristics recorded in the national guidelines for COVID-19,[8] we observed a more severe pulmonary injury [Table 2]. Generally, extensive hemorrhage and fibrosis occurs in patients with end-stage ARDS post-COVID-19.[9] Effusions and sputum bolts in small airways, extensive thrombosis, and secondary hemorrhage had further impaired the gas exchange, accelerating the irreversible deterioration of the lung function.
Representative comparison of explant pathologies.
Discussion:
Best time to perform transplantation in critical patients This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.
This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.
Key points on determination of LT candidacy The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.
The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.
Best practices for the protection of the medical team involved For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.
Considerations and comparisons of surgical preparations.
During the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.
By gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.
For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.
Considerations and comparisons of surgical preparations.
During the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.
By gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.
Factors important for post-LT survival During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]
Rehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.
With negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.
In the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.
During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]
Rehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.
With negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.
In the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.
Perspective With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.
With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.
Best time to perform transplantation in critical patients:
This is the first case series report on LT for COVID-19 patients who could not be weaned off ECMO support. Issues related to viral infection course, clinical course of recipients in the long term and staff protection are worthy of further exploration. In the acute care setting, the patients described herein were confirmed to have consecutive negative nucleic acid test results but presented with severe ARDS without lung function recovery. The physicians who treated the patients had difficulty waiting and merely observing the inevitable fatal outcome. LT in severe acute respiratory syndrome (SARS) patients with ARDS has not been reported. However, LT has been performed for patients receiving ECMO for >45 days after H1N1 infection.[10] ECMO is believed to contribute to lung function recovery in severely infected patients and it serves as a bridge treatment before LT. LT has been shown to be beneficial for patients with pneumonia-induced ARDS and post-ARDS pulmonary fibrosis receiving long-term MV.[11] The fact that young individuals receiving ECMO for a short time could be considered as candidates for LT after deliberate pre-transplant consultation is generally accepted.[12] However, our transplanted patients had higher SOFA scores than those reported in critical COVID-19 populations, indicating a high risk of mortality. LT provided the final option for these patients to avoid certain death.
Key points on determination of LT candidacy:
The following three critical points should be thoroughly evaluated and confirmed before decision-making regarding LT candidacy: (1) confirmed irreversibility of refractory respiratory failure despite maximal medical support; (2) confirmed positive-turned-negative virology status by performing consecutive nucleic acid tests with samples derived from multiple sites; and (3) confirmed absence of other organ system dysfunction that could contraindicate LT. LT was regarded as an urgently needed salvage therapy after full evaluation of the pathological condition of the patients. We further discussed the plan for bilateral LT or heart and LT. The consideration of heart transplantation and LT for patient 1 involved the concern that the prolonged procedure time would add to the risks of bleeding and death. Further, 2019-nCoV may attack the cardiac tissue, and indeed, we observed that all the patients had an elevated pulmonary artery pressure related to cardiac dysfunction. Thus, assessment of cardiac function using ultrasonography and monitoring B-type natriuretic peptide levels are crucial steps that can provide information regarding procedural decision making.
Best practices for the protection of the medical team involved:
For the protection of the medical team to avoid infection, we provide a detailed and specialized plan based on grade 3 (highest level of protection) protection requirement [Table 3]. Suggestions for protection are as follows: (1) head covers with positive pressure are necessary for surgeons, nurses, anesthesiologists, and cardiopulmonary physicians; (2) head covers will help surgeons keep their field of view clear without fogging of eye protectors; however, these will negatively impact sound conduction, including communication between physicians and alerts from monitors; (3) considering the physical demands and challenges for surgeons in full protective clothing, an intra-procedure rotation plan is necessary to guarantee optimal performance during surgery.
Considerations and comparisons of surgical preparations.
During the transplantation procedure for patient 1, we used a remote video communication tool to connect the surgical team in the isolated operating room and experts outside the contaminated regions. However, there were still blind-spots and delay for instantaneous communication. As we transplanted patients 2 and 3, coordinators who were responsible for communicating with the staff inside the operating room and supplying medical materials, played crucial roles. Instant voice messages and video images could be transmitted outside the isolated operating room, thereby ensuring the full-scale monitoring and service for surgery. Furthermore, all the operation-related medical staffs rehearsed the procedure before surgery. Considering the difficulty of direct communication while wearing head covers during surgery, gestures, and actions that indicated the need for assistance, the passing of sutures or instruments between surgeons and nurses allowed for non-verbal communication.
By gaining more experience regarding the comprehensive logistics of LT for patient 2 in Wuxi, the Wuxi group managed the key steps and thoroughfares that led to the successful LT. LT for these patients was seemingly unimaginable due to high risk of contagion and challenges on team collaboration and psychological strength. However, all the obstacles were overcome within a shorter operation time of less than 5 hours than that in conventional LT. So far, no infection event has been noted among the members of the medical team.
Factors important for post-LT survival:
During the SARS infection outbreak, medical resources in China, such as experienced ECMO teams and fully protective negative pressure operating rooms, were scarce. Further, transportation of donated lungs over long distances for urgently listed patients post-ARDS was impossible. The volunteer organ donation volume in China has reached up to 6000 cases per year recently. However, the usage percentage of donated lungs is around 5%, significantly lower than that of roughly 25% in western countries. All the three recipients were transplanted with the officially allocated organs through national allocation system. Currently, the Chinese organ donation system and green channel for human organ transportation play pivotal roles in achieving patient survival after LT.[13]
Rehabilitation procedures have been well established for patients who have undergone a conventional LT. Early mobilization for our post-COVID-19 patients provided enormous strength to pursue a good quality of life [Figure 2F]. The patient started advanced rehabilitation with fully functional grafts. As can be inferred from our current experience, patients with irreversible pulmonary fibrosis related to ARDS post-COVID-19 can be scrutinized for the possibility of LT. However, the duration of isolated ward admission was extremely long in our patients. Moreover, the medical team involved was instructed not to leave the isolated area and was separated from their families. Taken together, psychological assistance, including modern social media platform and communication tools, would best enhance the confidence of both the patients and medical teams while striving for survival.
With negative nucleic acid test results, positive IgG were detected in patients 2 and 3. No virological relapse was noted. Although a mild positive nucleic result could be observed from fecal sample of patient 2, this patient had confirmed negative results in fecal samples post-LT. We speculated that the positive result could be derived from nucleic acid segments or components from residue virus without further infectivity, thus providing an explanation of the mild positive results from explants. This still warrants further close follow-ups and research.
In the future, patients’ immune status regulated by therapeutic regimens and interaction with potential infection events should be evaluated. A previous report indicated that the cytokine profiles of ICU-admitted COVID-19 patients were greatly altered,[14] which might contribute to immune imbalances and multi-organ dysfunction. LT and immunosuppressive regimens could ameliorate these severe pathological conditions. More data and evidence are still urgently needed to evaluate the long-term benefits of this salvage therapy.
Perspective:
With careful protection and complete preparation of the patients and surgeons, the success of LT can be ensured, thereby reducing the high mortality rate of end-stage COVID-19 patients. The success of LT in our post-COVID-19 patients was credited not only to the medical team but also to the dedication of the public health and pathology teams with P3 laboratory-based research capability. A more precise pathophysiological knowledge prompts clinicians to establish a targeted treatment plan. Overall teamwork supported by government systems played fundamental roles in the LT of our patients. The swift response of regulatory departments for approval of nucleic acid test tools would further ensure credible disease monitoring and the protection of health-care providers.
Funding:
This study was supported by grants from the Chen Jingyu team of “Sanming Project of Medicine” in Shenzhen (No. SZSM201812058), and the Foundation for Special Projects of COVID-19 Prevention and Control in Wuxi City.
Conflicts of interest:
None.
|
Background:
Critical patients with the coronavirus disease 2019 (COVID-19), even those whose nucleic acid test results had turned negative and those receiving maximal medical support, have been noted to progress to irreversible fatal respiratory failure. Lung transplantation (LT) as the sole therapy for end-stage pulmonary fibrosis related to acute respiratory distress syndrome has been considered as the ultimate rescue therapy for these patients.
Methods:
From February 10 to March 10, 2020, three male patients were urgently assessed and listed for transplantation. After conducting a full ethical review and after obtaining assent from the family of the patients, we performed three LT procedures for COVID-19 patients with illness durations of more than one month and extremely high sequential organ failure assessment scores.
Results:
Two of the three recipients survived post-LT and started participating in a rehabilitation program. Pearls of the LT team collaboration and perioperative logistics were summarized and continually improved. The pathological results of the explanted lungs were concordant with the critical clinical manifestation, and provided insight towards better understanding of the disease. Government health affair systems, virology detection tools, and modern communication technology all play key roles towards the survival of the patients and their rehabilitation.
Conclusions:
LT can be performed in end-stage patients with respiratory failure due to COVID-19-related pulmonary fibrosis. If confirmed positive-turned-negative virology status without organ dysfunction that could contraindicate LT, LT provided the final option for these patients to avoid certain death, with proper protection of transplant surgeons and medical staffs. By ensuring instant seamless care for both patients and medical teams, the goal of reducing the mortality rate and salvaging the lives of patients with COVID-19 can be attained.
| null | null | 10,633 | 324 | 17 |
[
"lt",
"patients",
"patient",
"post",
"lung",
"covid",
"covid 19",
"19",
"negative",
"team"
] |
[
"test",
"test"
] | null | null |
[CONTENT] Coronavirus disease 2019 | Lung transplantation | Acute respiratory distress syndrome | Pulmonary fibrosis | Sequential Organ Failure Assessment score [SUMMARY]
|
[CONTENT] Coronavirus disease 2019 | Lung transplantation | Acute respiratory distress syndrome | Pulmonary fibrosis | Sequential Organ Failure Assessment score [SUMMARY]
|
[CONTENT] Coronavirus disease 2019 | Lung transplantation | Acute respiratory distress syndrome | Pulmonary fibrosis | Sequential Organ Failure Assessment score [SUMMARY]
| null |
[CONTENT] Coronavirus disease 2019 | Lung transplantation | Acute respiratory distress syndrome | Pulmonary fibrosis | Sequential Organ Failure Assessment score [SUMMARY]
| null |
[CONTENT] Aged | Betacoronavirus | COVID-19 | Coronavirus Infections | Extracorporeal Membrane Oxygenation | Humans | Lung Transplantation | Male | Middle Aged | Pandemics | Pneumonia, Viral | Pulmonary Fibrosis | Respiratory Distress Syndrome | SARS-CoV-2 [SUMMARY]
|
[CONTENT] Aged | Betacoronavirus | COVID-19 | Coronavirus Infections | Extracorporeal Membrane Oxygenation | Humans | Lung Transplantation | Male | Middle Aged | Pandemics | Pneumonia, Viral | Pulmonary Fibrosis | Respiratory Distress Syndrome | SARS-CoV-2 [SUMMARY]
|
[CONTENT] Aged | Betacoronavirus | COVID-19 | Coronavirus Infections | Extracorporeal Membrane Oxygenation | Humans | Lung Transplantation | Male | Middle Aged | Pandemics | Pneumonia, Viral | Pulmonary Fibrosis | Respiratory Distress Syndrome | SARS-CoV-2 [SUMMARY]
| null |
[CONTENT] Aged | Betacoronavirus | COVID-19 | Coronavirus Infections | Extracorporeal Membrane Oxygenation | Humans | Lung Transplantation | Male | Middle Aged | Pandemics | Pneumonia, Viral | Pulmonary Fibrosis | Respiratory Distress Syndrome | SARS-CoV-2 [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] lt | patients | patient | post | lung | covid | covid 19 | 19 | negative | team [SUMMARY]
|
[CONTENT] lt | patients | patient | post | lung | covid | covid 19 | 19 | negative | team [SUMMARY]
|
[CONTENT] lt | patients | patient | post | lung | covid | covid 19 | 19 | negative | team [SUMMARY]
| null |
[CONTENT] lt | patients | patient | post | lung | covid | covid 19 | 19 | negative | team [SUMMARY]
| null |
[CONTENT] reported | patients | icu | cases | disease | results | mv | lt | peri lt | patients peri [SUMMARY]
|
[CONTENT] lt | patient | image | image pre lt day | pre lt day | image pre | image pre lt | hospital | day | lt day [SUMMARY]
|
[CONTENT] patient | pod | lt | patients | left | heart | ecmo | post | day | operative [SUMMARY]
| null |
[CONTENT] lt | patients | patient | post | day | 19 | covid | covid 19 | lung | ecmo [SUMMARY]
| null |
[CONTENT] 2019 | COVID-19 ||| Lung [SUMMARY]
|
[CONTENT] February 10 to March 10, 2020 | three ||| three | COVID-19 | more than one month [SUMMARY]
|
[CONTENT] Two | three ||| ||| ||| [SUMMARY]
| null |
[CONTENT] 2019 | COVID-19 ||| Lung ||| February 10 to March 10, 2020 | three ||| three | COVID-19 | more than one month ||| Two | three ||| ||| ||| ||| COVID-19 ||| ||| COVID-19 [SUMMARY]
| null |
||||
A proposed adaptation of the European Foundation for Quality Management Excellence Model to physical activity programmes for the elderly - development of a quality self-assessment tool using a modified Delphi process.
|
21958203
|
There has been a growing concern in designing physical activity (PA) programmes for elderly people, since evidence suggests that such health promotion interventions may reduce the deleterious effects of the ageing process. Complete programme evaluations are a necessary prerequisite to continuous quality improvements. Being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly in order to support its continuous improvement is, therefore, crucial. Thus, the aim of this study was to develop a self-assessment tool for PA programmes for the elderly.
|
BACKGROUND
|
A 3-round Delphi process was conducted via the Internet with 43 national experts in PA for the elderly, management and delivery of PA programmes for the elderly, sports management, quality management and gerontology, asking experts to identify the propositions that they considered relevant for inclusion in the self-assessment tool. Experts reviewed a list of proposed statements, based on the criteria and sub-criteria from the European Foundation for Quality Management Excellence Model (EFQM) and PA guidelines for older adults and rated each proposition from 1 to 8 (disagree to agree) and modified and/or added propositions. Propositions receiving either bottom or top scores of greater than 70% were considered to have achieved consensus to drop or retain, respectively.
|
METHODS
|
In round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93. In round 2, a total of 104 propositions were presented, of which experts modified 39 and achieved consensus on 53. In the last round, of 51 proposed statements, the experts achieved consensus on 19. After 3 rounds of rating, experts had not achieved consensus on 32 propositions. The resulting tool consisted of 165 statements that assess nine management areas involved in the development of PA programmes for the elderly.
|
RESULTS
|
Based on experts' opinions, a self-assessment tool was found in order to access quality of PA programmes for the elderly. Information obtained with evaluations would be useful to organizations seeking to improve their services, customer satisfaction and, consequently, adherence to PA programmes, targeting the ageing population.
|
CONCLUSION
|
[
"Aged",
"Aging",
"Consensus",
"Delivery of Health Care",
"Delphi Technique",
"Europe",
"Exercise",
"Geriatrics",
"Health Promotion",
"Humans",
"Internet",
"Practice Guidelines as Topic",
"Program Evaluation",
"Quality Control",
"Sports"
] |
3278362
|
Background
|
Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.
The 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].
Evidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.
While numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.
|
Methods
|
A modified Delphi process was conducted using the Internet, from October 2009 to September 2010. The Delphi technique was developed in the 1950s by scientists at the Rand Corporation as a method of making informed decisions based on expert opinion [23]. Since then, it has been used to clarify a variety of problems in different sectors [24-29]. Despite having undergone some modifications, it remains a viable approach for gathering expert opinions through a structured iterative process that builds consensus [30]. This process involves multiple interactions with participants who usually complete two or more rounds in a reasonable amount of time [31] - even when participants are in geographically-distinct locations, since rounds can be conducted by mail or email [32,33]. The results of previous iterations can be modified by participants in later iterations, as they are able to review comments and feedbacks provided by other experts in earlier rounds [31]. Furthermore, the Delphi technique offers a number of specific advantages and is particularly helpful because it avoids the barriers commonly observed in other group discussions, such as interpersonal influence, time pressure and group demands [31,34,35]. This is due to the fact that respondents are not aware of the identities of other respondents and are, therefore, freed of personal and social constraints [30]. They are also able to complete the Delphi rounds in ways that suit them best because they participate in the rounds asynchronously [36]. The Delphi technique is also advantageous because a variety of statistical analysis techniques can be used to interpret the data its generates [37].
The Delphi process was conducted in three rounds [38,39] (Figure 1). Following each step listed in the previous figure, our main question was: Which quality practices must be included in a quality self-assessment tool for PA programmes for the elderly?
Steps of the modified Delphi process used in the present study.
Using criteria and sub-criteria from the EFQM Excellence Model [13] and PA guidelines for older adults [3,40] as a starting point, we reviewed the literature to identify best practice principles and generate a list of statements. Our review was undertaken using PubMed (1980-2010), B-On (1980-2010), and Google™. We searched a variety of combinations of key words related to PA programmes for the elderly, quality management and the EFQM Excellence Model, such as: 'evaluation', 'guidelines', 'recommendations', 'exercise', 'physical activity', 'programmes', 'elderly', 'old', 'review', 'framework', 'EFQM', 'assess' and 'quality'.
After identifying a list of statements, an online questionnaire was developed and tested with 5 PA programme coordinators for comments on readability and functionality. Some adjustments were made to make the affirmations included in the questionnaire clearer and more relevant to this case. We established that statements that received greater than 70% of experts' votes had achieved consensus [41-43] in both the bottom scores (i.e., reached consensus to drop) and top scores (i.e., reached consensus to include/retain). Statements that were dropped were not included in subsequent rounds of ratings. The remaining items were included in the next rounds, until a consensus was achieved to either drop or retain. At the end of three rounds, the statements on which experts had not reached consensus were also not included in the output list.
The fourth phase of the process involved nominating experts to participate in the Delphi rounds. National experts in research on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology were identified. Our decisions were based on expertise or/and breadth of scientific work [44]. The DeGóis Curricula Platform1 assisted us in this process. A list of 63 potential participants was generated, along with key contacts for each. This group included 34 PhD scientists and academics (11 in PA for the elderly, 4 in sports management, 18 in quality management and 1 in gerontology), 3 non-PhD academics (1 in PA for the elderly and 2 in sports management) and 26 senior technicians (22 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). Previous information containing details about the EFQM Excellence Model, the Delphi process and the purpose of our study was provided. Of those invited to participate, 5 did not respond and 3 declined, due to lack of time (all PhD scientists and academics in quality management). Thus, 55 experts (30 females and 25 males) responded to our initial invitation and agreed to participate. Those who accepted our invitation were informed that they were required to respond to three online rounds of ratings.
The rounds were performed using Survey Monkey, a web-based survey and data collection system. In every round, participants were asked to rate their level of agreement with each proposition, from 1 to 8 ('strongly disagree' to 'strongly agree'), suggest modifications to proposed definitions and/or add propositions that would be useful in a quality self-assessment tool for PA programmes for the elderly. The 8-point Likert scale was selected to bring out more variability in responses [45]. After each round, the frequency and mean of the panel's ratings and the percentage of scores ≥ 7 were calculated. Based on this data, a new questionnaire was designed and placed online for the next round. We asked participants to review all the information sent and re-rate each statement.
After round 3, we gathered all our data and developed a list of statements that did and did not reach consensus.
| null | null |
Conclusion
|
1 It is an instrument for gathering, supplying and analyze the intellectual and scientific production of the Portuguese researchers.
|
[
"Background",
"Results",
"Discussion",
"Strengths and Limitations",
"Conclusion"
] |
[
"Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.",
"Eight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\nResults of the three rounds by criterion\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\" to \"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).",
"The main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\" and \"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\nThroughout the Delphi process, it was suggested that the proposition \"The programme involves a multidisciplinary team of professionals\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \"external partnerships\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\" and \"Regular and formal communication procedures are established with partners\".\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \"Market research is used to determine the needs and expectations of future customers\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \"The programme has measures of perception and/or performance indicators regarding employees' performance\" and \"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.",
"To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.",
"Our Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results)."
] |
[
null,
null,
null,
null,
null
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[
"Background",
"Methods",
"Results",
"Discussion",
"Strengths and Limitations",
"Conclusion",
"Supplementary Material"
] |
[
"Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.\nThe 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].\nEvidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.\nWhile numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.",
"A modified Delphi process was conducted using the Internet, from October 2009 to September 2010. The Delphi technique was developed in the 1950s by scientists at the Rand Corporation as a method of making informed decisions based on expert opinion [23]. Since then, it has been used to clarify a variety of problems in different sectors [24-29]. Despite having undergone some modifications, it remains a viable approach for gathering expert opinions through a structured iterative process that builds consensus [30]. This process involves multiple interactions with participants who usually complete two or more rounds in a reasonable amount of time [31] - even when participants are in geographically-distinct locations, since rounds can be conducted by mail or email [32,33]. The results of previous iterations can be modified by participants in later iterations, as they are able to review comments and feedbacks provided by other experts in earlier rounds [31]. Furthermore, the Delphi technique offers a number of specific advantages and is particularly helpful because it avoids the barriers commonly observed in other group discussions, such as interpersonal influence, time pressure and group demands [31,34,35]. This is due to the fact that respondents are not aware of the identities of other respondents and are, therefore, freed of personal and social constraints [30]. They are also able to complete the Delphi rounds in ways that suit them best because they participate in the rounds asynchronously [36]. The Delphi technique is also advantageous because a variety of statistical analysis techniques can be used to interpret the data its generates [37].\nThe Delphi process was conducted in three rounds [38,39] (Figure 1). Following each step listed in the previous figure, our main question was: Which quality practices must be included in a quality self-assessment tool for PA programmes for the elderly?\nSteps of the modified Delphi process used in the present study.\nUsing criteria and sub-criteria from the EFQM Excellence Model [13] and PA guidelines for older adults [3,40] as a starting point, we reviewed the literature to identify best practice principles and generate a list of statements. Our review was undertaken using PubMed (1980-2010), B-On (1980-2010), and Google™. We searched a variety of combinations of key words related to PA programmes for the elderly, quality management and the EFQM Excellence Model, such as: 'evaluation', 'guidelines', 'recommendations', 'exercise', 'physical activity', 'programmes', 'elderly', 'old', 'review', 'framework', 'EFQM', 'assess' and 'quality'.\nAfter identifying a list of statements, an online questionnaire was developed and tested with 5 PA programme coordinators for comments on readability and functionality. Some adjustments were made to make the affirmations included in the questionnaire clearer and more relevant to this case. We established that statements that received greater than 70% of experts' votes had achieved consensus [41-43] in both the bottom scores (i.e., reached consensus to drop) and top scores (i.e., reached consensus to include/retain). Statements that were dropped were not included in subsequent rounds of ratings. The remaining items were included in the next rounds, until a consensus was achieved to either drop or retain. At the end of three rounds, the statements on which experts had not reached consensus were also not included in the output list.\nThe fourth phase of the process involved nominating experts to participate in the Delphi rounds. National experts in research on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology were identified. Our decisions were based on expertise or/and breadth of scientific work [44]. The DeGóis Curricula Platform1 assisted us in this process. A list of 63 potential participants was generated, along with key contacts for each. This group included 34 PhD scientists and academics (11 in PA for the elderly, 4 in sports management, 18 in quality management and 1 in gerontology), 3 non-PhD academics (1 in PA for the elderly and 2 in sports management) and 26 senior technicians (22 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). Previous information containing details about the EFQM Excellence Model, the Delphi process and the purpose of our study was provided. Of those invited to participate, 5 did not respond and 3 declined, due to lack of time (all PhD scientists and academics in quality management). Thus, 55 experts (30 females and 25 males) responded to our initial invitation and agreed to participate. Those who accepted our invitation were informed that they were required to respond to three online rounds of ratings.\nThe rounds were performed using Survey Monkey, a web-based survey and data collection system. In every round, participants were asked to rate their level of agreement with each proposition, from 1 to 8 ('strongly disagree' to 'strongly agree'), suggest modifications to proposed definitions and/or add propositions that would be useful in a quality self-assessment tool for PA programmes for the elderly. The 8-point Likert scale was selected to bring out more variability in responses [45]. After each round, the frequency and mean of the panel's ratings and the percentage of scores ≥ 7 were calculated. Based on this data, a new questionnaire was designed and placed online for the next round. We asked participants to review all the information sent and re-rate each statement.\nAfter round 3, we gathered all our data and developed a list of statements that did and did not reach consensus.",
"Eight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.\nThe results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.\nResults of the three rounds by criterion\nIn round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change \"Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes\" to \"Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes\". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.\nBased on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.\nIn the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.\nAdditional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).",
"The main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.\nAlthough there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.\nThe 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include \"The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility\" and \"The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)\". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].\nLeaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.\nExperts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.\nThe statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].\nThroughout the Delphi process, it was suggested that the proposition \"The programme involves a multidisciplinary team of professionals\" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was \"Emphasis is placed on recruiting employees whose profile matches the needs of the programme\". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.\nDuring the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning \"external partnerships\", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: \"Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes\" and \"Regular and formal communication procedures are established with partners\".\nConsensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, \"Market research is used to determine the needs and expectations of future customers\" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to \"Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers\", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].\nConcerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: \"The programme has measures of perception and/or performance indicators regarding employees' performance\" and \"The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork\". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].\nThe tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.\n Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.\nTo the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.",
"To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.\nHowever, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.",
"Our Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).",
"Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors). the file presents the resulting tool - named Q-STEPS - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly.\nClick here for file"
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[
null,
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null,
null,
null,
null,
"supplementary-material"
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[
"physical activity",
"programmes",
"elderly",
"tool",
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] |
Background:
Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.
The 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].
Evidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.
While numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.
Methods:
A modified Delphi process was conducted using the Internet, from October 2009 to September 2010. The Delphi technique was developed in the 1950s by scientists at the Rand Corporation as a method of making informed decisions based on expert opinion [23]. Since then, it has been used to clarify a variety of problems in different sectors [24-29]. Despite having undergone some modifications, it remains a viable approach for gathering expert opinions through a structured iterative process that builds consensus [30]. This process involves multiple interactions with participants who usually complete two or more rounds in a reasonable amount of time [31] - even when participants are in geographically-distinct locations, since rounds can be conducted by mail or email [32,33]. The results of previous iterations can be modified by participants in later iterations, as they are able to review comments and feedbacks provided by other experts in earlier rounds [31]. Furthermore, the Delphi technique offers a number of specific advantages and is particularly helpful because it avoids the barriers commonly observed in other group discussions, such as interpersonal influence, time pressure and group demands [31,34,35]. This is due to the fact that respondents are not aware of the identities of other respondents and are, therefore, freed of personal and social constraints [30]. They are also able to complete the Delphi rounds in ways that suit them best because they participate in the rounds asynchronously [36]. The Delphi technique is also advantageous because a variety of statistical analysis techniques can be used to interpret the data its generates [37].
The Delphi process was conducted in three rounds [38,39] (Figure 1). Following each step listed in the previous figure, our main question was: Which quality practices must be included in a quality self-assessment tool for PA programmes for the elderly?
Steps of the modified Delphi process used in the present study.
Using criteria and sub-criteria from the EFQM Excellence Model [13] and PA guidelines for older adults [3,40] as a starting point, we reviewed the literature to identify best practice principles and generate a list of statements. Our review was undertaken using PubMed (1980-2010), B-On (1980-2010), and Google™. We searched a variety of combinations of key words related to PA programmes for the elderly, quality management and the EFQM Excellence Model, such as: 'evaluation', 'guidelines', 'recommendations', 'exercise', 'physical activity', 'programmes', 'elderly', 'old', 'review', 'framework', 'EFQM', 'assess' and 'quality'.
After identifying a list of statements, an online questionnaire was developed and tested with 5 PA programme coordinators for comments on readability and functionality. Some adjustments were made to make the affirmations included in the questionnaire clearer and more relevant to this case. We established that statements that received greater than 70% of experts' votes had achieved consensus [41-43] in both the bottom scores (i.e., reached consensus to drop) and top scores (i.e., reached consensus to include/retain). Statements that were dropped were not included in subsequent rounds of ratings. The remaining items were included in the next rounds, until a consensus was achieved to either drop or retain. At the end of three rounds, the statements on which experts had not reached consensus were also not included in the output list.
The fourth phase of the process involved nominating experts to participate in the Delphi rounds. National experts in research on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology were identified. Our decisions were based on expertise or/and breadth of scientific work [44]. The DeGóis Curricula Platform1 assisted us in this process. A list of 63 potential participants was generated, along with key contacts for each. This group included 34 PhD scientists and academics (11 in PA for the elderly, 4 in sports management, 18 in quality management and 1 in gerontology), 3 non-PhD academics (1 in PA for the elderly and 2 in sports management) and 26 senior technicians (22 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). Previous information containing details about the EFQM Excellence Model, the Delphi process and the purpose of our study was provided. Of those invited to participate, 5 did not respond and 3 declined, due to lack of time (all PhD scientists and academics in quality management). Thus, 55 experts (30 females and 25 males) responded to our initial invitation and agreed to participate. Those who accepted our invitation were informed that they were required to respond to three online rounds of ratings.
The rounds were performed using Survey Monkey, a web-based survey and data collection system. In every round, participants were asked to rate their level of agreement with each proposition, from 1 to 8 ('strongly disagree' to 'strongly agree'), suggest modifications to proposed definitions and/or add propositions that would be useful in a quality self-assessment tool for PA programmes for the elderly. The 8-point Likert scale was selected to bring out more variability in responses [45]. After each round, the frequency and mean of the panel's ratings and the percentage of scores ≥ 7 were calculated. Based on this data, a new questionnaire was designed and placed online for the next round. We asked participants to review all the information sent and re-rate each statement.
After round 3, we gathered all our data and developed a list of statements that did and did not reach consensus.
Results:
Eight of the 63 invited experts, did not respond or declined. Of the 55 who agreed to participate in this process, 43 responded to round 1 and were invited to participate in the subsequent rounds. This group included 25 females and 18 males and was comprised of 20 PhD scientists and academics (9 in PA for the elderly, 2 in sports management, 8 in quality management and 1 in gerontology), 2 non-PhD academics (1 in PA for the elderly and 1 in sports management) and 21 senior technicians (17 in PA programmes for elderly management and delivery, 3 in quality management and 1 in gerontology). The 12 experts who did not respond to round 1 were not involved in subsequent rounds.
The results of the three rounds (total number of statements, statements approved by consensus, statements without consensus, statements modified by experts and new statements proposed by experts) for the nine criteria are presented in Table 1.
Results of the three rounds by criterion
In round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93, which were retained for inclusion in the self-assessment tool. Of the 41 suggested modifications, 14 were related to Leadership (38,39%), 9 to Policy & strategy (32,14%), 7 to People (18,92%), 7 to Processes (14,89%), 1 to Customer results and People results (16,67% and 11,11 respectively) and 2 to Key performance results (50%). Some modifications consisted of minor changes to words or sentence structures, while others were about content (e.g., change "Higher education qualification, with specialization in physical activity and aging, is required for instructors'/teachers' programmes" to "Higher education qualification, with specialization in physical activity and aging, or relevant experience in this field, is required for instructors'/teachers' programmes". The addition was related to the People criterion. Generally, experts made the greatest number of suggestions to Leadership and the fewest (0 in this case) to Partnership & resources and Society results. The best practice principles that were retained were mostly in Partnership & resources (15 out of 26, i.e. 57,69%), Processes (27 out of 47, i.e. 57,45%) and Customer results (3 out of 6, i.e. 50%). The criterion on which least consensus was reached was Key performance results (1 out of 4, i.e. 25%). No proposition was dropped in round 1, i.e. none received greater than 70% of the experts' votes in both the bottom scores.
Based on the results of round 1, 104 propositions were presented in round 2. At this stage, experts modified 39 and achieved consensus on 53 propositions. Most of the suggestions were made on Policy & strategy, Partnership & resources and Processes, with none suggestions to Results' criteria. The best practice principles that were retained were mostly in People (14 out of 20, i.e. 70%), Leadership (14 out of 23, i.e. 60,87%) and Processes (12 out of 20, i.e. 60%). The criterion on which there was least consensus was Society results, on which there was no agreement. Once more, no proposition was dropped. Forty one of the 43 experts responded to round 2.
In the last round, of the 51 statements proposed, the experts achieved consensus on 19, mostly in Policy & strategy (5 out of 11, i.e. 45,45%), Processes (4 out of 8, i.e. 50%) and Partnership & resources (4 out of 9, i.e. 44,44%). After 3 rounds of rating, they had not achieved consensus on 32 propositions. Most of these statements were concerned with Leadership (7, i.e. 21,88%), Policy & strategy (6, i.e. 18,75%) and Partnership & resources (5, i.e. 15,63%). One expert who had not responded to round 2 was willing to participate in round 3; thus, 42 of the 43 experts responded to round 3.
Additional file 1 presents the resulting tool - named Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly. Five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).
Discussion:
The main goal of this study was to describe the development of a quality self-assessment tool for PA programmes for the elderly. To the best of our knowledge, no previous studies have sought expert opinions on PA for the elderly, PA programmes for elderly management and delivery, sports management, quality management and gerontology, with the aim of identifying practices that must be observed when assessing the quality of such programmes.
Although there are recommendations and guidelines for promoting the physical activity of older people [3,40] and recommendations about the need to evaluate these interventions [14,46], the literature is scarce [47], if not absent, on how to integrate these recommendations into PA programmes. No framework or tool has yet been developed to identify or influence the enablers and outcomes of PA programmes for the elderly.
The 43 national experts who participated in the Delphi process were quite engaged throughout, as evidenced by the number of their suggestions (one addition and 53 modifications) and the greater than 97% response rate to all three rounds of ratings. Most of their suggestions pertained to Leadership, while they made no suggestions on Society results. We presume that these results are related to the fact that many experts are programme leaders and thus, are more aware of practices that pertain to Leadership. Also, experts may have been aware of the fact that Leadership is understood by some authors [48-50] as the key to driving quality improvement. Our data indicate a high degree of consensus on the retention of all propositions concerning the development of vision and mission and the enhancement of a culture of communication by programme coordinators. These are considered fundamental to quality management [51-53], since the physical presence of leaders - their visibility and concern for quality improvement - are associated with transformational leadership [54], i.e. leadership that creates valuable and positive change in its followers. Of the seven statements on Leadership on which experts did not achieve consensus, five belong to the sub-criteria that concern the interaction of programme coordinators with politicians, customers, partners and representatives of society. While our study revealed that most of the statements concerning interaction with customers, partners and representatives of society achieved consensus, propositions concerning relationships with politicians or political affairs did not achieve consensus. This may be related to popular negative perceptions of the political class [55]. Examples of statements that touched on the relationship between leadership and politics include "The coordinator manages relations with politicians and other stakeholders to ensure shared responsibility" and "The coordinator interacts regularly and proactively with policy makers from relevant executive areas (e.g. Alderman of Sport)". The British Heart Foundation (BHF) has stated that participants or other stakeholders must be actively involved in all aspects of programme development, including planning, promotion and evaluation [40]. The ACSM also recognizes that PA leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56].
Leaders unanimously agreed to retain statements about the importance of leaders identifying and championing organizational change. Fostering change is increasingly seen as part of a leader's role [57], and the EFQM Fundamental Concepts upon which the Model is based [58] include standard recommendations such as planning change, communicating reasons for it, enabling people to manage change and reviewing the effectiveness of change.
Experts also suggested modifications to about 30% of the original propositions on Policy & strategy. A high degree of consensus was achieved on the retention of all propositions concerning the development, review and updating of policy and strategy.
The statement that received the greatest degree of consensus was related to the development of annual reports. Data from such reports helps improve the annual planning cycles of PA programmes. These procedures are in agreement with those found in other studies [59,60] or with different documents, such as those that outline the planning and evaluation of PA programmes [61,62] and health promotion programmes [63].
Throughout the Delphi process, it was suggested that the proposition "The programme involves a multidisciplinary team of professionals" be added to the People criterion. In fact, the teams that run PA programmes for seniors should include not only exercise and sports professionals, but general practitioners, practice nurses and care and residential managers [40]. Of the propositions on the planning, management and improvement of human resources that the experts agreed to retain, the one on which there was greatest consensus was "Emphasis is placed on recruiting employees whose profile matches the needs of the programme". The Physical Activity and Health Branch (PAHB) of the CDC has established that PA programmes should be run by highly-skilled PA practitioners [14]. The Cross-National Expert Survey Report on Physical Activity Programmes and Physical Activity Promotion Strategies for Older People [64] also notes the importance of recruiting teachers who are highly qualified and reinforces the importance of continuous professional development.
During the first round, a high level of consensus was immediately reached on propositions related to the management of finances and maintenance of facilities, equipment and materials (Partnership & resources criterion). The management of financial resources is key to consolidating programmes' financial structure and ensuring that programmes can fulfil their missions in the present and the future, as well as periodically provide maintenance plans for equipment and buildings [65,66]. Experts did not achieve consensus on half the propositions concerning "external partnerships", although the development and sustainment of community partnerships is the first public health benchmark for PA programmes established by the PAHB [14]. Particularly with regards to PA programmes for the elderly, some organizations have reinforced the importance and strength of these partnerships, which provide additional resources in the form of funding, facilities and equipment, as well as access to wide-ranging abilities and knowledge [40,67]. Indeed, one of the propositions that did not reach consensus was the one that pointed the participation in networks in order to exchange knowledge and to improve relationships. However, of the propositions on which experts did not achieve consensus, most were similar to other statements that were retained. Examples include: "Appropriate partnership agreements are established, defining roles, responsibilities and expected outcomes" and "Regular and formal communication procedures are established with partners".
Consensus was not reached on only four of the 47 statements about Processes. Once more, most were similar to other statements that were retained. For example, "Market research is used to determine the needs and expectations of future customers" -- a proposition that only received 64,29% of votes equal to or greater than 7 -- is comparable to "Surveys and other ways of obtaining feedback are used to determine the needs and expectations of current and future customers", a retained proposition. Physical activity leaders should work closely with individuals to design PA regimens that reflect personal preferences and capabilities [56]. The BHF recommends that participants should be involved in this process [40]. Moreover, tailoring exercise programmes to the needs and interests of participants has been associated with higher programme attendance [68,69].
Concerning the four Results' criteria, the highest level of consensus was achieved on Customer results, in which all propositions were accepted. Indeed, organizations must measure and achieve customer results [13]. Similarly, both the processes by which PA interventions are conducted and the outcomes of such interventions should be evaluated [47]. The experts achieved a high degree of consensus on all propositions related to client assessment, i.e. customer satisfaction, customer loyalty, communication, complaints handling and management and outcomes (physical fitness evaluations and psychological/mental evaluations). By contrast, they displayed relatively little consensus on the criterion People results (4 out of 9). In fact, the experts were unable to reach consensus on whether or not to retain propositions related to employee involvement, motivation, initiative and loyalty. However, it should be emphasised that similar statements were retained. Examples include: "The programme has measures of perception and/or performance indicators regarding employees' performance" and "The programme has measures of perception and/or performance indicators regarding employees' involvement in teamwork". In actuality, to achieve excellence, organisations must also focus on People results [13], since employee involvement is one of the most important drivers of continuous improvement [58]. Furthermore, without satisfied and motivated employees, it is impossible to create satisfied and loyal customers [70].
The tool that resulted from this process provides a framework tailored to evaluating PA programmes for the elderly, applicable to a variety of settings, namely community-based programmes and/or those developed by the Public Local Administration. The information obtained through such evaluations would be useful for organizations seeking to improve their services. It would help them guide interventions toward excellence, in order to improve customer satisfaction and adherence to PA programmes targeting the ageing population.
Strengths and Limitations To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.
However, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.
To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.
However, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.
Strengths and Limitations:
To the best of our knowledge, this is the first study to gather expert opinions with the aim of identifying practices that must be observed when assessing the quality of PA programmes for the elderly. Because of the heterogeneity of their interests, panel members were able to cover a broad range of topics. In addition, they were able to submit comments on each sub-criterion in every round, enabling us to use their expertise to develop or modify new statements. This also guaranteed that the process did not neglect to include any pertinent issues in subsequent rounds of rating.
However, this study has certain limitations. Our results should not be interpreted as representing the views of all experts in the field of quality management, physical activity for older adults or gerontology, due to the process used to collect the sample. It is also important to note that the tool suggested by our consensus process may not be applicable to certain PA programmes, including those for special population subgroups, such as: the most elderly, the frail, older adults with chronic illnesses or varying degrees of medical co-morbidity. Likewise, our consensus-informed quality practices do not reflect possible differences in PA programmes that were developed in institutional elderly care settings. Additional research is necessary to provide the feasibility analysis of this assessment and to adapt and replicate our tool to other circumstances.
Conclusion:
Our Delphi process identified 165 quality practices that 43 experts consider essential to assessments of the quality of PA programmes for the elderly. The Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors) tool assesses nine areas involved in the development of PA programmes for the elderly: five criteria assess Enablers (Leadership, Policy & strategy, People, Partnership & resources, and Processes) and four criteria assess the Results (Customer results, People results, Society results, and Key performance results).
Supplementary Material:
Q-STEPS (Quality Self-assessment Tool for Exercise Programmes for Seniors). the file presents the resulting tool - named Q-STEPS - which consists of 165 statements that assess nine areas involved in the development of PA programmes for the elderly.
Click here for file
|
Background:
There has been a growing concern in designing physical activity (PA) programmes for elderly people, since evidence suggests that such health promotion interventions may reduce the deleterious effects of the ageing process. Complete programme evaluations are a necessary prerequisite to continuous quality improvements. Being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly in order to support its continuous improvement is, therefore, crucial. Thus, the aim of this study was to develop a self-assessment tool for PA programmes for the elderly.
Methods:
A 3-round Delphi process was conducted via the Internet with 43 national experts in PA for the elderly, management and delivery of PA programmes for the elderly, sports management, quality management and gerontology, asking experts to identify the propositions that they considered relevant for inclusion in the self-assessment tool. Experts reviewed a list of proposed statements, based on the criteria and sub-criteria from the European Foundation for Quality Management Excellence Model (EFQM) and PA guidelines for older adults and rated each proposition from 1 to 8 (disagree to agree) and modified and/or added propositions. Propositions receiving either bottom or top scores of greater than 70% were considered to have achieved consensus to drop or retain, respectively.
Results:
In round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93. In round 2, a total of 104 propositions were presented, of which experts modified 39 and achieved consensus on 53. In the last round, of 51 proposed statements, the experts achieved consensus on 19. After 3 rounds of rating, experts had not achieved consensus on 32 propositions. The resulting tool consisted of 165 statements that assess nine management areas involved in the development of PA programmes for the elderly.
Conclusions:
Based on experts' opinions, a self-assessment tool was found in order to access quality of PA programmes for the elderly. Information obtained with evaluations would be useful to organizations seeking to improve their services, customer satisfaction and, consequently, adherence to PA programmes, targeting the ageing population.
|
Background:
Physical activity (PA) programmes play a significant role in senior citizens' health, autonomy and ability to face daily tasks, being particularly important to prevent and minimize the deleterious effects of the ageing process [1,2] and to improve quality of life [1-4]. It is widely accepted that the benefits of such programmes depend on adherence to exercise, which is influenced by degree of enjoyment and satisfaction [5-10]. One of the most important factors in customer satisfaction is quality of service [11-13]. Therefore, continual improvements in PA programmes for the elderly are important to elderly satisfaction and adherence to PA.
The 3rd Benchmark from the Physical Activity and Health Branch of the Centers for Disease Control and Prevention (CDC) [14] holds that complete programme evaluations are an important and desired prerequisite to continuous quality improvements. Similarly, World Health Organization (WHO) guidelines for the evaluation of health promotion emphasize the need to evaluate and propose the allocation of adequate evaluative resources [15].
Evidence shows that quality matters, is measurable, moveable and malleable [16], but also has costs [17]. However, literature also shows that the costs of not doing so are far greater [18,19]. Several studies have focused on the advantages of quality schemes [20-22]. With the aim of helping organizations improve the quality of their services, the European Foundation for Quality Management (EFQM) introduced the EFQM Excellence Model in 1991. The EFQM Excellence Model is a non-prescriptive framework that is based on nine criteria divided into 32 sub-criteria [13]. It promotes the use of management methodologies based on objective criteria that are applicable to all areas of business or services and constitutes an exercise in self-assessment. Self-assessment sheds light on areas requiring improvement, as well as on the processes and actions necessary to generate improvement.
While numerous PA programmes have been designed for the elderly in recent years - especially by the Public Local Administration - their evaluation has been scarce. In fact, few details are available on how these programmes have been developed, how they have been structured, how service delivery is conducted and how results are being achieved. The lack of a standard approach to assessing PA programmes for the elderly makes it difficult to compare the quality of both the planning and the delivery of such programmes. In this way, being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly, and that improve these programmes, is therefore important, not only to help programmes evaluate their ability to perform public health functions, but to address local health needs and guide community health-planning efforts. Thus, the aim of this study is to describe the development of a quality self-assessment tool for PA programmes for the elderly.
Conclusion:
1 It is an instrument for gathering, supplying and analyze the intellectual and scientific production of the Portuguese researchers.
|
Background:
There has been a growing concern in designing physical activity (PA) programmes for elderly people, since evidence suggests that such health promotion interventions may reduce the deleterious effects of the ageing process. Complete programme evaluations are a necessary prerequisite to continuous quality improvements. Being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly in order to support its continuous improvement is, therefore, crucial. Thus, the aim of this study was to develop a self-assessment tool for PA programmes for the elderly.
Methods:
A 3-round Delphi process was conducted via the Internet with 43 national experts in PA for the elderly, management and delivery of PA programmes for the elderly, sports management, quality management and gerontology, asking experts to identify the propositions that they considered relevant for inclusion in the self-assessment tool. Experts reviewed a list of proposed statements, based on the criteria and sub-criteria from the European Foundation for Quality Management Excellence Model (EFQM) and PA guidelines for older adults and rated each proposition from 1 to 8 (disagree to agree) and modified and/or added propositions. Propositions receiving either bottom or top scores of greater than 70% were considered to have achieved consensus to drop or retain, respectively.
Results:
In round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93. In round 2, a total of 104 propositions were presented, of which experts modified 39 and achieved consensus on 53. In the last round, of 51 proposed statements, the experts achieved consensus on 19. After 3 rounds of rating, experts had not achieved consensus on 32 propositions. The resulting tool consisted of 165 statements that assess nine management areas involved in the development of PA programmes for the elderly.
Conclusions:
Based on experts' opinions, a self-assessment tool was found in order to access quality of PA programmes for the elderly. Information obtained with evaluations would be useful to organizations seeking to improve their services, customer satisfaction and, consequently, adherence to PA programmes, targeting the ageing population.
| 5,170 | 421 | 7 |
[
"programmes",
"pa",
"quality",
"consensus",
"elderly",
"pa programmes",
"results",
"experts",
"management",
"statements"
] |
[
"test",
"test"
] | null |
[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]
|
[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]
| null |
[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]
|
[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]
|
[CONTENT] physical activity | programmes | elderly | tool | evaluation | quality | adherence [SUMMARY]
|
[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]
|
[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]
| null |
[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]
|
[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]
|
[CONTENT] Aged | Aging | Consensus | Delivery of Health Care | Delphi Technique | Europe | Exercise | Geriatrics | Health Promotion | Humans | Internet | Practice Guidelines as Topic | Program Evaluation | Quality Control | Sports [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]
|
[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]
| null |
[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]
|
[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]
|
[CONTENT] programmes | pa | quality | consensus | elderly | pa programmes | results | experts | management | statements [SUMMARY]
|
[CONTENT] health | programmes | quality | important | pa | elderly | pa programmes | improve | satisfaction | efqm [SUMMARY]
|
[CONTENT] rounds | delphi | management | list | included | participants | consensus | pa | process | quality [SUMMARY]
| null |
[CONTENT] results | criteria assess | people | quality | programmes | assess | criteria | essential assessments quality pa | tool assesses areas | essential assessments quality [SUMMARY]
|
[CONTENT] programmes | pa | quality | results | elderly | consensus | pa programmes | experts | statements | tool [SUMMARY]
|
[CONTENT] programmes | pa | quality | results | elderly | consensus | pa programmes | experts | statements | tool [SUMMARY]
|
[CONTENT] ||| ||| ||| [SUMMARY]
|
[CONTENT] 3 | Delphi | 43 | PA ||| the European Foundation for Quality Management Excellence Model | PA | 1 to 8 ||| greater than 70% [SUMMARY]
| null |
[CONTENT] ||| [SUMMARY]
|
[CONTENT] ||| ||| ||| ||| 3 | Delphi | 43 | PA ||| the European Foundation for Quality Management Excellence Model | PA | 1 to 8 ||| greater than 70% ||| ||| 1 | 196 | 41 | 1 | 93 ||| 2 | 104 | 39 | 53 ||| 51 | 19 ||| 3 | 32 ||| 165 | nine ||| ||| [SUMMARY]
|
[CONTENT] ||| ||| ||| ||| 3 | Delphi | 43 | PA ||| the European Foundation for Quality Management Excellence Model | PA | 1 to 8 ||| greater than 70% ||| ||| 1 | 196 | 41 | 1 | 93 ||| 2 | 104 | 39 | 53 ||| 51 | 19 ||| 3 | 32 ||| 165 | nine ||| ||| [SUMMARY]
|
|||||
miR-223 increases gallbladder cancer cell sensitivity to docetaxel by downregulating STMN1.
|
27577078
|
MicroRNAs (miRs) are involved in cancer carcinogenesis, and certain regulatory miRs could provide promising therapeutic methods for refractory malignancies, such as gallbladder cancer (GBC). miR-223 was found to play a pivotal role in enhancing chemotherapeutic effects, therefore evoking interest in the role of miR-223 in GBC.
|
BACKGROUND
|
We examined miR-223 expression in GBC tissue and GBC cell lines using qRT-PCR. The effects of modulated miR-223 expression in GBC cells were assayed using Cell Counting Kit-8 (CCK8), flow cytometry, and wound-healing and invasion assays. Susceptibility to docetaxel was evaluated in miR-223/STMN1-modulated GBC cells and xenograft tumor models. The protein expression of relevant genes was examined by Western blotting.
|
METHODS
|
miR-223 was decreased in GBC tissues and cell lines, and ectopic miR- 223 expression exhibited multiple anti-tumorigenic effects in GBC cells, including decreased proliferation, migration and invasion in vitro. However, treatment with a miR-223 inhibitor increased cell viability. We determined that STMN1 was negatively correlated with and regulated by miR-223 in GBC. miR-223 increased GBC sensitivity to docetaxel in vitro and in vivo, and the induced sensitivity to docetaxel was suppressed by the restoration of STMN1 expression.
|
RESULTS
|
These findings indicated that miR-223 might serve as an onco-suppressor that enhances susceptibility to docetaxel by downregulating STMN1 in GBC, highlighting its promising therapeutic value.
|
CONCLUSIONS
|
[
"Animals",
"Antineoplastic Agents",
"Cell Line, Tumor",
"Cell Proliferation",
"Cell Survival",
"Docetaxel",
"Down-Regulation",
"Flow Cytometry",
"Gallbladder",
"Gallbladder Neoplasms",
"Gene Expression Regulation, Neoplastic",
"Genes, Tumor Suppressor",
"Humans",
"Immunohistochemistry",
"Mice",
"Mice, Nude",
"MicroRNAs",
"Real-Time Polymerase Chain Reaction",
"Stathmin",
"Taxoids",
"Xenograft Model Antitumor Assays"
] |
5308733
|
INTRODUCTION
|
Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.
MicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].
miR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].
The present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.
|
MATERIALS AND METHODS
|
Reagents The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).
The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).
GBC tissue samples and cell lines Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.
The human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.
Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.
The human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.
Tissue immunohistochemistry for STMN1 and in situ hybridization for miRNA Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.
The paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.
Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.
The paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.
Western blotting After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.
After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.
RNA isolation and qRT-PCR Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:
miR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;
miR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;
STMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;
STMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;
GAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;
GAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;
U6_F 5′-CTCGCTTCGGCAGCACA-3′′ and
U6_R 5′-AACGCTTCACGAATTTGCGT-3′.
PCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT
STMN1 - CT
GAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.
Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:
miR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;
miR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;
STMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;
STMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;
GAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;
GAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;
U6_F 5′-CTCGCTTCGGCAGCACA-3′′ and
U6_R 5′-AACGCTTCACGAATTTGCGT-3′.
PCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT
STMN1 - CT
GAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.
Cell biology experimental procedures In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].
In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].
DAPI staining cell apoptosis test Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.
Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.
Animal experiments To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.
To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.
Statistical analyses Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).
Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).
| null | null |
DISCUSSION
|
Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).
|
[
"INTRODUCTION",
"RESULTS",
"Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation",
"miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression",
"Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation",
"miR-223 overexpression inhibits GBC cell migration and invasion",
"GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics",
"The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid",
"DISCUSSION"
] |
[
"Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.",
" Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.",
"To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.",
"To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.",
"To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.",
"Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).",
"GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).",
"The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.",
"GBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach."
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[
"INTRODUCTION",
"RESULTS",
"Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation",
"miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression",
"Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation",
"miR-223 overexpression inhibits GBC cell migration and invasion",
"GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics",
"The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid",
"DISCUSSION",
"MATERIALS AND METHODS",
"SUPPLEMENTARY MATERIALS FIGURES"
] |
[
"Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.\nMicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].\nmiR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].\nThe present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.",
" Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\nTo determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.\n miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\nTo observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.\n Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\nTo investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.\n miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\nWound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).\n GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\nGBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).\n The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.\nThe above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.",
"To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).\n(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.",
"To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).\n(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.",
"To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).\n(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.",
"Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.\n(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).",
"GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).\n(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).",
"The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).\n(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.",
"GBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].\nAccumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.\nTo date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.\nIn the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.\nIn summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach.",
" Reagents The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\nThe CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).\n GBC tissue samples and cell lines Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\nFresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.\nThe human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.\n Tissue immunohistochemistry for STMN1 and in situ hybridization for miRNA Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\nImmunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.\nThe paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.\n Western blotting After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\nAfter treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.\n RNA isolation and qRT-PCR Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\nTotal RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:\nmiR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;\nmiR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;\nSTMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;\nSTMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;\nGAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;\nGAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;\nU6_F 5′-CTCGCTTCGGCAGCACA-3′′ and\nU6_R 5′-AACGCTTCACGAATTTGCGT-3′.\nPCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT\nSTMN1 - CT\nGAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.\n Cell biology experimental procedures In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\nIn these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].\n DAPI staining cell apoptosis test Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\nTreated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.\n Animal experiments To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\nTo investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.\n Statistical analyses Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).\nStatistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).",
""
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null,
null,
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null,
null,
"methods",
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[
"miR-223",
"gallbladder cancer",
"malignancy",
"STMN1"
] |
INTRODUCTION:
Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.
MicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].
miR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].
The present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.
RESULTS:
Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).
(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.
To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).
(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.
miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).
(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.
To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).
(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.
Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).
(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.
To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).
(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.
miR-223 overexpression inhibits GBC cell migration and invasion Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.
(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).
Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.
(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).
GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).
(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).
GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).
(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).
The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).
(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.
The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).
(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.
Aberrant miR-223 expression in GBC tissue samples and correlation with STMN1 upregulation:
To determine the mRNA and protein expression levels of miR-223 in GBC, quantitative real-time RT-PCR (qRT-PCR) and Western blot assays were performed, respectively. The expression of miR-223 and STMN1 mRNA in the surgically resected tissue samples from 5 cholecystolithiasis patients and 16 GBC patients, and 2 GBC cell lines was investigated. miR-223 was highly expressed in non-cancerous gallbladder tissues and in pericarcinous gallbladder tissues from GBC patients but was downregulated in GBC tissues and cell lines (Figure 1A). The expression of STMN1 mRNA and miR- 223 in 16 GBC cancer tissues was also examined. STMN1 mRNA expression was significantly negatively correlated with miR-223 expression in GBC patients (Figure 1B). Therefore, we further examined STMN1 protein levels by Western blotting and qRT-PCR in 5 pairs of GBC tissues with their pericarcinous gallbladder tissues and observed that the expression levels of STMN1 protein were elevated in GBC tissues (Figure 1C and Supplementary Figure S2). These results suggested that miR-223 was downregulated in GBC and correlated with elevated STMN1 expression. We then performed a bioinformatic analysis of potential miR-223 target genes using the online miRBase sequence database provided by the University of Manchester [36]. STMN1 was predicted as one of the target genes with a PicTar score of 3.12; the putative target sites for miR-223 in the 3′ UTR of the STMN1 mRNA are shown in Figure 1D. miR-223 was downregulated in GBC tissue compared with normal gallbladder tissue as determined using in situ hybridization (Figure 1E).
(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.
miR-223 mimics and inhibitors efficiently elevate and decrease miR-223 levels, respectively, in GBC cells to modulate STMN1 expression:
To observe the effect of modulating the miR-233 levels and STMN1 expression in GBC cells, we used miR-223 mimics, a miR-223 inhibitor and an STMN1 expression plasmid to transfect GBC-SD and NOZ cells. In the GBC-SD and NOZ cell lines, qRT-PCR analysis showed that miR-223 expression was efficiently elevated or decreased 24 h after transfection of miR-223 mimics or miR-223 inhibitor, respectively, compared with the control group (Figure 2A). The STMN1 mRNA and protein levels were simultaneously modulated with miR-223 mimics, miR-223 inhibitor and the STMN1 expression plasmid in GBC cells (Figure 2B–2D and Supplementary Figure S1).
(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.
Ectopic miR-223 suppresses GBC cell proliferation, whereas a miR-223 inhibitor promotes GBC proliferation:
To investigate the biological function of miR-223 in GBC development and progression, we examined cell proliferation using the Cell Counting Kit-8 (CCK8) assay. At 2 days after the introduction of exogenous miR-223, GBC-SD and NOZ cell proliferation was significantly lower in cells treated with miR-223 mimics compared with that of the scramble controls by 32.9% and 27.5%, respectively, (P < 0.05, Figure 3A). By contrast, GBC-SD and NOZ cell proliferation was significantly higher upon treatment with the miR-223 inhibitor compared with that of the scramble controls by 15.2% and 10.4%, respectively (P < 0.05, Figure 3B). The growth curve of the GBC cells after transfection with either exogenous miR-223 mimics or inhibitor was evaluated in GBC-SD and NOZ cells. GBC cell growth was significantly faster when transfected with miR-223 inhibitor but was significantly slowed in the presence of the miR-223 inhibitor compared with the cells transfected with control vector (P < 0.001 for both, Figure 3C and 3D).
(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.
miR-223 overexpression inhibits GBC cell migration and invasion:
Wound-healing and invasion assays were performed in GBC cells to evaluate the effect of miR-223 on cell migration and invasion. The results showed that transfection of miR-223 mimics reduced the wound-healing distance at 24 h compared with the scramble control (78.3% of GBC-SD cells and 45.2% of NOZ cells, P < 0.001, Figure 4A). In the cell invasion transwell assay, the number of cells that migrated through the Matrigel-coated membrane into the lower chamber (invasion assay) was significantly lower among cells transfected with miR-223 mimics than in scramble-transfected cells. The transfection of miR-223 mimics inhibited 95.1% of GBC cell (P < 0.001) and 91.3% NOZ cell (P < 0.001) migration compared with the scramble control (Figure 4B). These data suggest that exogenous miR-223 might inhibit GBC cell metastasis.
(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).
GBC-SD and NOZ cell resistance to docetaxel was sensitized by transfection of miR-223 mimics:
GBC is a refractory malignancy that is resistant to most chemotherapy agents. The literature has failed to provide strong evidence that patients with advanced GBC could benefit from neoadjuvant chemotherapy [7]. Upon discovering that miR-223 might downregulate STMN1 and inhibit GBC proliferation, STMN1 activity was reported to be correlated with certain chemotherapy agents [37], suggesting that the downregulation of STMN1 could sensitize malignant cells to docetaxel [38]. Therefore, we hypothesized that miR-223 could downregulate STMN1 expression and subsequently increase the sensitivity of GBC cells to chemotherapy agents that target microtubules, such as docetaxel. We first investigated the sensitivity of GBC-SD and NOZ cells to docetaxel using the CCK8 proliferation assay. The results showed that both cell lines were resistant to docetaxel below a concentration of 50 μM, with an IC50 value of 82.43 μM for GBC- SD cells (95% CI, 75.66 to 89.81) and an IC50 value of 68.5 for NOZ cells (95% CI, 63.90 to 73.43) as shown in Figure 5A. Next, we investigated the sensitivity of GBC cells transfected with either miR-223 mimics or an inhibitor using the CCK8 proliferation assay. We observed that the GBC cells were sensitized to 10 μM docetaxel upon transfection of miR-223 mimics. We also compared the cytostatic effect of 10 μM docetaxel in conjunction with miR-223 mimic transfection to docetaxel treatment alone, and the results showed that GBC cells transfected with miR-223 mimics had a significantly higher cytostatic effect upon treatment with 10 μM docetaxel compared with that of the GBC cells transfected with scramble. The inhibition rate was increased by 72.80% in the GBC-SD cells (P < 0.001) and 77.31% in the NOZ cells (P < 0.001). Moreover, the inhibition rate was significantly higher in cells transfected with miR-223 mimics treated with 10 μM docetaxel than in cells that were only transfected (73.92% vs. 36.37%, respectively, for the GBC-SD cells (P < 0.001) and 77.78% vs. 34.34%, respectively, for the NOZ cells (P < 0.001)). No significant differences were detected in the cells transfected with the miR-223 inhibitor. The possibility of synergy was evaluated using the formula IR(A+B)>IR(A)+IR(B)-IR(A) × IR(B), where IR is the inhibition rate. Figure 5B shows synergistic effects were observed in the GBC-SD and NOZ cells. We further investigated the growth curve of GBC-SD and NOZ cells after transfection with miR- 223 mimics and in the presence or absence of docetaxel. GBC cell growth was significantly suppressed when treated with 10 μM docetaxel and transfected with miR-223 mimics compared with that of the other groups for both cell lines (P < 0.001, Figure 5C and 5D). These data suggest that exogenous miR-223 could increase GBC cell sensitivity to cancer chemotherapy. Next, we observed the chemosensitizing effect of the miR- 223 mimics vector in vivo using a NOZ xenograft model. The tumor volume growth was significantly slower in animals grafted with NOZ cells transduced with lentivirus expressing miR- 223 expression compared with that of the other animals (P < 0.001, Figure 5E). The measured tumors were significantly smaller in the miR- 223-expressing NOZ- cell group than those in the scramble vector group (Figure 5F). STMN1 expression in the tumor was examined by immunohistochemistry and showed weaker staining in miR-223-expressing tumors than in scramble control tumors (Figure 5G).
(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).
The chemosensitizing effects of miR-223 mimics were neutralized by restoring STMN1 expression using a transfected STMN1 overexpression plasmid:
The above results demonstrated that ectopic miR-223 expression exerts a chemosensitizing effect by downregulating STMN1. Therefore, we used an STMN1 vector to restore its expression in GBC cells. The response of the treated GBC-SD and NOZ cells to docetaxel was evaluated using the CCK8 assay, with cell apoptosis quantified by DAPI and Annexin V/PI staining and flow cytometry and by evaluating the expression level of cleaved caspase 3 by Western blotting. Cellular resistance to docetaxel was restored after transfection of the STMN1 expression plasmid (Figure 6A). The number of apoptotic cells was increased in groups treated with docetaxel and expressing ectopic miR-223 compared with those expressing the scramble control, but cells co-transfected with STMN1 vector showed no significant change in the levels of apoptosis (Figure 6B and 6C). We also detected the expression of cleaved caspase 3 to evaluate the apoptotic activity of GBC-SD cells ectopically expressing miR-223 treated with docetaxel. Cleaved caspase 3 levels were increased in docetaxel-treated cells transfected with miR-223 but not in scramble-transfected cells; furthermore, STMN1 transfection suppressed this effect (Figure 6D).
(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.
DISCUSSION:
GBC is one of the intractable malignancies of the digestive system. Radical surgical resection is the sole promising treatment for GBC available to patients in the early stage of this disease. However, due to embryology origination and anatomy, GBC is difficult to diagnose in the early stages. Moreover, GBC barely responds to available anticancer regimens or radiotherapy due to its biological nature. miRs are small non-coding RNA molecules that function as negative regulators of mRNA and have been reported to exert important regulatory effects in carcinogenesis [39–44].
Accumulating evidence has supported important roles for miRs as either tumor suppressors or oncogenes [15]. The recent development of miR-based therapeutics has provided a new strategy in cancer treatment [45]. We observed that miR-223 was involved in diverse malignancies and induced a prominent reversal in chemotherapy resistance, which has triggered an interest in investigating the role of miR-223 in GBC.
To date, the consensus is that miR-223 expression is aberrant in multiple cancer types; however, the expression and function of miR-223 in tumors remains controversial. There are reports identifying miR-223 overexpression in gastric cancer [46], colorectal cancer [47] and esophageal squamous cell carcinoma [48], but with unknown mechanisms. These reports indicated the complicated role of miR-223 in carcinogenesis. Microtubules are dynamic α/GAPDH heterodimers that play key roles in cell division, morphology, motility, and intracellular transport [49]. STMN1 is a microtubule-regulatory protein that modulates microtubule dynamics by preventing tubulin polymerization and promoting the destabilization and disassembly of microtubules during interphase and late mitosis during the cell cycle progression; this process is regulated by changes in the phosphorylation status of STMN1 [50]. STMN1 also plays a role in a variety of other biological processes such as cell proliferation, mobility, metastasis, differentiation, and resistance to antimicrotubule therapy [51]. One previous study confirmed that STMN1 is a target of miR-223 and that downregulation of miR-223 contributes to chemoresistance in various cultured tumor cells [52]. Thus, we hypothesized that the ectopic expression of miR-223 could induce the chemosensitivity of GBC cells.
In the present study, we revealed that miR-223 is downregulated in human GBC and demonstrated that exogenous miR-223 expression inhibits GBC cell proliferation, induces GBC cell apoptosis, and suppresses GBC cell migration and invasion. Moreover, exogenous miR-223 sensitized GBC cells to chemotherapy reagents. miR-223 has multiple target genes, including STMN1, that function as microtubule modulators. Therefore, we further investigated and observed that STMN1 expression was modulated by transfection of either miR-223 mimics or an inhibitor in a GBC cell line. These results suggest that ectopic miR-223 expression may induce an anticancer effect by downregulating STMN1 expression in GBC cells. These data demonstrate for the first time that miR-223 functions as a tumor suppressor in GBC and that the miR-223/STMN1 pathway in GBC carcinogenesis is worthy of further investigation.
In summary, our study indicated that the introduction of ectopic miR-223 in GBC cells inhibited proliferation and reduced invasiveness and metastasis, thus enhancing the sensitivity of GBC to chemotherapy in vitro and in vivo and suggesting the possible application of miR- 223 as a therapeutic target for GBC. Further studies are required to fully understand the detailed mechanisms of miR-223 in GBC carcinogenesis and as a potential therapeutic approach.
MATERIALS AND METHODS:
Reagents The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).
The CCK8 assay was purchased from Dojindo Laboratories (Kumamoto, Japan). Puromycin, docetaxel (DTX), anti-STMN1 antibody and anti-GAPDH antibody were purchased from Sigma. Anti-Digoxin IgG monoclonal antibody was obtained from Invitrogen. Fetal bovine serum, DMEM and William's medium E cell culture medium were purchased from Gibco. The RIPA cell lysate buffer, bicinchoninic acid (BCA) assay and enhanced chemiluminescent (ECL) detection reagent were purchase from Cell Signaling, Thermo Scientific and Pierce, respectively. The oligonucleotides encoding the hsa-miR-223 mimics (miR-223), mimics control (miR-control), hsa-miR-223 inhibitor (anti-miR-223) and inhibitor control (anti-miR-control), as well as the STMN1 expression plasmid, were obtained from ZoonBio (Nanjin, China). The hsa-miR-223 overexpression lentivirus and digoxin-labeled miR-223 probe were purchased from Exiqon (Vedbaek, Denmark). An anti-caspase 3 antibody was purchased from Abcam. An apoptosis assay kit was purchased from Biotium Inc. Lipofectamine 2000 was obtained from Invitrogen. TRIzol, OPTI-MEM, M-MLV Reverse Transcriptase, pre-miR-223 and antisense nucleotides of miR-223 were purchased from Life Technologies. The qPCR primers were purchased from BBI Life Science Corporation, the TaqMan human MicroRNA Assay kits from Qiagen and SYBR® Green PCR Master Mix from Applied Biosystems. The DAPI stain was purchased from Beyotime Biotechnology (Nantong, China), and Triton™ X-100 was obtained from Takara Bio (Dalian, China).
GBC tissue samples and cell lines Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.
The human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.
Fresh samples of GBC tissues stored in liquid nitrogen and formalin-fixed as well as paraffin-embedded cancerous gallbladder tissue samples with a validated pathology diagnosis were obtained from the tissue sample library of the Department of General Surgery at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). Written consent was obtained after approval by the local Ethics Committee. None of the enrolled patients underwent either preoperative chemotherapy or radiotherapy.
The human GBC cell lines GBC-SD and NOZ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The GBC-SD cells were cultured in DMEM and the NOZ cells in William's medium E. The media for both cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.
Tissue immunohistochemistry for STMN1 and in situ hybridization for miRNA Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.
The paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.
Immunohistochemistry was performed to investigate STMN1 expression in the GBC tissues harvested from a xenograft mice model as previously described [53]. Briefly, the tissues were formalin-fixed, paraffin-embedded and cut into tissue sections. The tissue sections were then dehydrated with ethanol, washed three times with phosphate-buffered saline (PBS) and boiled for 8 min in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide for 10 min at 26°C. The sections were further blocked with 3% normal goat serum for 10 min. After the serum was discarded, the sections were incubated overnight with primary rabbit anti-human STMN1 antibody in a humidified chamber at 4°C. The following day, the sections were incubated with secondary antibody-coated polymer peroxidase complexes (Abcam, Cambridge, UK) for 30 min at room temperature. After three 3-min washes with PBS, the sections were developed using diaminobenzidine (Abcam), and the slides were counterstained with hematoxylin for long-term storage. Negative controls were treated identically but without the primary antibody treatment.
The paraffin-embedded tissue section tissue sections were dehydrated with ethanol, washed 3 times with PBS and boiled for 15 min in a sodium citrate solution. Then, digested with 5 μg/ml proteinase K at 37°C for 5 min and washed 3 times with PBS for 3 min followed by incubation in 0.1 mol/L glycine PBS for 10 min and 0.25% acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 min. The sections were then pre-hybridized with 0.2× SSC and 50% formamide for 60 min at 37°C. Hybridization was performed with the probe in the hybridization solution for 18 h. Finally, the sections were washed using SSC and detected by immunohistochemistry with an anti-Digoxin IgG monoclonal antibody and horseradish peroxidase-conjugated secondary antibodies to detect miR-223 expression.
Western blotting After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.
After treatment, the cells were collected and lysed in RIPA buffer. After centrifugation at 14,000 × g for 30 min, the protein concentration of the harvested supernatant was determined using the BCA assay. The protein lysates (20 μg/lane) were separated by 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then probed with the primary antibodies against either STMN1 or GAPDH. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunocomplex was visualized using an enhanced chemiluminescent (ECL) detection reagent.
RNA isolation and qRT-PCR Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:
miR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;
miR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;
STMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;
STMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;
GAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;
GAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;
U6_F 5′-CTCGCTTCGGCAGCACA-3′′ and
U6_R 5′-AACGCTTCACGAATTTGCGT-3′.
PCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT
STMN1 - CT
GAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.
Total RNA was isolated from tissue samples and cell lines using TRIzol reagent. cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase. The expression levels of the miRNAs and STMN1 mRNA were evaluated by qRT-PCR, with the U6 small nuclear RNA and GAPDH, respectively used for normalization. The following primers were used for the detection of miR-223, STMN1 and GAPDH expression according to the human STMN1 and GAPDH cDNA sequences in GenBank:
miR-223_F: 5′- GCGTGTATTTGACAAGCTGAG TT -3′;
miR-223_R: 5′- GTGTCAGTTTGTCAAATACCC CA -3′;
STMN1_F: 5′- GCCTGTCGCTTGTCTTCT -3′;
STMN1_R: 5′- TCATGGGACTTGCGTCTT -3′;
GAPDH_F: 5′- CAACAGCCTCAAGATCATCAGC -3′;
GAPDH_R: 5′-TTCTAGACGGCAGGTCAGGTC -3′;
U6_F 5′-CTCGCTTCGGCAGCACA-3′′ and
U6_R 5′-AACGCTTCACGAATTTGCGT-3′.
PCRs of each sample were conducted in triplicate. The relative expression level of the target gene was calculated using 2−ΔCT (ΔCT = CT
STMN1 - CT
GAPDH) and normalized to the relative expression detected in the corresponding control tissue or cells. miR-223 expression was defined as upregulated when the relative expression ratio was > 1 and downregulated when the relative expression ratio was < 1.
Cell biology experimental procedures In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].
In these studies, the following cell biology experiments were performed: cell viability assay; transient transfection; flow cytometric cell cycle analysis; Annexin V/PI staining assay; wound-healing migration assay; transwell migration assay; and invasion assay. Generally, these experiments were performed as previously described [54].
DAPI staining cell apoptosis test Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.
Treated NOZ cells (1 × 105 cells/well) were seeded in 24-well plates with round coverslips and exposed to docetaxel for 24 h. Following washing with PBS, the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 at room temperature for 10 min. The cells were treated with DNase-Free RNase (50 mg/ml) for 2 h at 37°C and then stained with 5 μM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope. The cells with a condensed nucleus were defined as apoptotic cells.
Animal experiments To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.
To investigate the effects of the indicated genes in tumor growth, NOZ cells were transfected with either the miR-223 overexpression lentivirus or scramble control lentivirus and selected with puromycin. Approximately 36 h after transfection, 5 × 106 cells were suspended in serum-free medium and injected s.c. into nude mice (n = 8/group). At 10 days after the injection (when the subcutaneous tumor reached approximately 0.5 cm in diameter), either 100 μg of docetaxel (approximately 5 mg/kg) or the same volume of PBS was injected i.v. every week for 3 weeks. The tumor volume was measured every 3 days. The mice were sacrificed 21 days later, and the tumors were weighed. The animal experiments were conducted in strict accordance with the experimental animal guidelines and protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University.
Statistical analyses Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).
Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).
SUPPLEMENTARY MATERIALS FIGURES:
|
Background:
MicroRNAs (miRs) are involved in cancer carcinogenesis, and certain regulatory miRs could provide promising therapeutic methods for refractory malignancies, such as gallbladder cancer (GBC). miR-223 was found to play a pivotal role in enhancing chemotherapeutic effects, therefore evoking interest in the role of miR-223 in GBC.
Methods:
We examined miR-223 expression in GBC tissue and GBC cell lines using qRT-PCR. The effects of modulated miR-223 expression in GBC cells were assayed using Cell Counting Kit-8 (CCK8), flow cytometry, and wound-healing and invasion assays. Susceptibility to docetaxel was evaluated in miR-223/STMN1-modulated GBC cells and xenograft tumor models. The protein expression of relevant genes was examined by Western blotting.
Results:
miR-223 was decreased in GBC tissues and cell lines, and ectopic miR- 223 expression exhibited multiple anti-tumorigenic effects in GBC cells, including decreased proliferation, migration and invasion in vitro. However, treatment with a miR-223 inhibitor increased cell viability. We determined that STMN1 was negatively correlated with and regulated by miR-223 in GBC. miR-223 increased GBC sensitivity to docetaxel in vitro and in vivo, and the induced sensitivity to docetaxel was suppressed by the restoration of STMN1 expression.
Conclusions:
These findings indicated that miR-223 might serve as an onco-suppressor that enhances susceptibility to docetaxel by downregulating STMN1 in GBC, highlighting its promising therapeutic value.
|
INTRODUCTION:
Gallbladder cancer (GBC) is rare; however, it is the most common malignancy of the biliary tract [1]. Cancers within this tract are difficult to treat because of their highly lethal nature [2, 3]. Patients with GBC have a reported overall 5-year survival of less than 5% and a mean survival of only 6 months [4], and their initial clinic presentation is often at an advanced stage of the disease [5, 6]. Moreover, studies reporting the benefit of neoadjuvant therapy for patients with advanced GBC failed to provide sufficient power to show significant improvements [7]. Controlling the progression of advanced GBC remains unfavorable, which contributes to the current poor prognosis of patients with GBC.
MicroRNAs (miRs) are noncoding 17 to 25 nucleotide RNAs that post-transcriptionally regulate gene expression [8]. These RNAs are believed to be expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [9] as well as oncogenesis and tumor metastasis [10, 11]. Aberrant expression of certain miRs have been shown to promote cancer initiation and progression by modulating their target genes [12], thus designating these miRs as cancer-related miRs [13–15]. It was hypothesized that manipulating miR expression would change their biological behaviors and contribute to the treatment of corresponding malignancies [16, 17]. Some preclinical studies have demonstrated that modulating miR expression levels could increase chemotherapy efficacy [18, 19] and have highlighted potential applications to improve the treatment of certain chemo-resistant malignancies [20].
miR-223 was first reported to be involved in the regulation of human granulocyte proliferation and function [21, 22]. Some studies have demonstrated that miR-223 plays a complicated role in leukemia [23, 24]. miR-223 was shown to be significantly downregulated in progressive chronic lymphocytic leukemia [25–27]. Recent studies further investigated the downregulation of miR- 223 in chronic lymphocyte leukemia [28], nasopharyngeal carcinoma [29], hepatocellular carcinoma [30], gastric cancer [31], malignant pleural mesothelioma [32] and prostate cancer [33] via different mechanisms. Moreover, miR-223 was demonstrated to rescue the cellular response to anticancer drugs by modulating ABCB1 (ATP-Binding Cassette Sub-Family B Member 1) in hepatocellular carcinoma [34] and targeting PARP1 (poly(ADP-ribose) polymerase 1) in esophageal adenocarcinoma [35].
The present study was performed to investigate the role of targeted miR-223 treatments on the invasion and metastasis of GBC cells and to explore the potential chemo-sensitizing effect of miR-223. Our results indicate that miR-223 may be involved in GBC development, whereby GBC cells overexpressing miR-223 exhibit increased sensitivity to chemotherapy agents. These results provide valuable information for potential clinical applications.
DISCUSSION:
Statistical analysis was performed using a GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Differences between subgroups were tested using Student's t-test. Parameter correlation was tested using nonparametric Spearman correlation analysis and line fitting by linear regression. A P-value of less than 0.05 (denoted by *) was considered significant (**P < 0.01, ***P < 0.001).
|
Background:
MicroRNAs (miRs) are involved in cancer carcinogenesis, and certain regulatory miRs could provide promising therapeutic methods for refractory malignancies, such as gallbladder cancer (GBC). miR-223 was found to play a pivotal role in enhancing chemotherapeutic effects, therefore evoking interest in the role of miR-223 in GBC.
Methods:
We examined miR-223 expression in GBC tissue and GBC cell lines using qRT-PCR. The effects of modulated miR-223 expression in GBC cells were assayed using Cell Counting Kit-8 (CCK8), flow cytometry, and wound-healing and invasion assays. Susceptibility to docetaxel was evaluated in miR-223/STMN1-modulated GBC cells and xenograft tumor models. The protein expression of relevant genes was examined by Western blotting.
Results:
miR-223 was decreased in GBC tissues and cell lines, and ectopic miR- 223 expression exhibited multiple anti-tumorigenic effects in GBC cells, including decreased proliferation, migration and invasion in vitro. However, treatment with a miR-223 inhibitor increased cell viability. We determined that STMN1 was negatively correlated with and regulated by miR-223 in GBC. miR-223 increased GBC sensitivity to docetaxel in vitro and in vivo, and the induced sensitivity to docetaxel was suppressed by the restoration of STMN1 expression.
Conclusions:
These findings indicated that miR-223 might serve as an onco-suppressor that enhances susceptibility to docetaxel by downregulating STMN1 in GBC, highlighting its promising therapeutic value.
| 12,789 | 263 | 11 |
[
"mir",
"mir 223",
"223",
"gbc",
"cells",
"cell",
"stmn1",
"expression",
"sd",
"noz"
] |
[
"test",
"test"
] | null |
[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]
|
[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]
| null |
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[CONTENT] miR-223 | gallbladder cancer | malignancy | STMN1 [SUMMARY]
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[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]
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[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]
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[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]
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[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]
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[CONTENT] Animals | Antineoplastic Agents | Cell Line, Tumor | Cell Proliferation | Cell Survival | Docetaxel | Down-Regulation | Flow Cytometry | Gallbladder | Gallbladder Neoplasms | Gene Expression Regulation, Neoplastic | Genes, Tumor Suppressor | Humans | Immunohistochemistry | Mice | Mice, Nude | MicroRNAs | Real-Time Polymerase Chain Reaction | Stathmin | Taxoids | Xenograft Model Antitumor Assays [SUMMARY]
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[CONTENT] mir | mir 223 | 223 | gbc | cells | cell | stmn1 | expression | sd | noz [SUMMARY]
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[CONTENT] mir | mir 223 | 223 | gbc | cells | cell | stmn1 | expression | sd | noz [SUMMARY]
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[CONTENT] mir | mir 223 | 223 | gbc | cells | cell | stmn1 | expression | sd | noz [SUMMARY]
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[CONTENT] mir | 223 | mir 223 | mirs | gbc | studies | leukemia | cancer | carcinoma | advanced [SUMMARY]
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[CONTENT] min | purchased | sections | antibody | anti | mir | tissue | obtained | assay | 10 [SUMMARY]
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[CONTENT] mir | mir 223 | 223 | gbc | stmn1 | carcinogenesis | expression | cell | microtubule | role [SUMMARY]
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[CONTENT] mir | 223 | mir 223 | gbc | cells | stmn1 | expression | sd | cell | gbc sd [SUMMARY]
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Harm avoidance is associated with progression of parkinsonism in community-dwelling older adults: a prospective cohort study.
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24754876
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We tested the hypothesis that harm avoidance, a trait associated with behavioral inhibition, is associated with the rate of change in parkinsonism in older adults.
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BACKGROUND
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At baseline harm avoidance was assessed with a standard self-report instrument in 969 older people without dementia participating in the Rush Memory and Aging Project, a longitudinal community-based cohort study. Parkinsonism was assessed annually with a modified version of the motor section of the Unified Parkinson's Disease Rating Scale (mUPDRS).
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METHODS
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Average follow-up was 5 years. A linear mixed-effects model controlling for age, sex and education showed that for an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10), the overall severity of parkinsonism increased by about 0.05 unit/ year (Estimate, 0.054, S.E., 0.007, p <0.001) and that the level of harm avoidance was associated with the progression of parkinsonism (Estimate, 0.004, S.E., 0.001, p <0.001). Thus, for an average participant, every 6 point (~1 SD) increase in harm avoidance score at baseline, the rate of progression of parkinsonism increased about 50% compared to an individual with an average harm avoidance score. This amount of change in parkinsonism over the course of the study was associated with about a 5% increased risk of death. The association between harm avoidance and progression of parkinsonism persisted when controlling for cognitive function, depressive symptoms, loneliness, neuroticism, late-life cognitive, social and physical activities and chronic health conditions.
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RESULTS
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A higher level of the harm avoidance trait is associated with a more rapid progression of parkinsonism in older adults.
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CONCLUSION
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[
"Aged",
"Aged, 80 and over",
"Cohort Studies",
"Disease Progression",
"Female",
"Follow-Up Studies",
"Harm Reduction",
"Humans",
"Longitudinal Studies",
"Male",
"Parkinsonian Disorders",
"Prospective Studies",
"Residence Characteristics"
] |
4022545
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Background
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Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.
There is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].
To examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.
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Methods
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Participants Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].
The Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.
Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].
The Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.
Clinical diagnoses Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].
Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].
Assessment of harm avoidance Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].
Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].
Assessment of parkinsonism Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].
Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].
Assessment of other covariates Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].
Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].
Statistical analyses The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.
In order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.
In subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].
The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.
In order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.
In subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].
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Results
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Descriptive properties of harm avoidance measure The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].
Clinical characteristics of the participants included in these analyses at this study’s baseline
*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.
Harm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).
The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].
Clinical characteristics of the participants included in these analyses at this study’s baseline
*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.
Harm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).
Harm avoidance and change in parkinsonism Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.
A model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*
*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.
Person-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.
Harm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.
Since baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].
In sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).
The harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).
Association of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*
*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].
Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.
A model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*
*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.
Person-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.
Harm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.
Since baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].
In sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).
The harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).
Association of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*
*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].
Potential confounders of harm avoidance and change in parkinsonism Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).
Psychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).
In further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).
Since higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).
Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).
Psychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).
In further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).
Since higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).
Clinical significance of the loss of motor function associated with personality To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).
To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).
|
Conclusions
|
The growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.
|
[
"Background",
"Participants",
"Clinical diagnoses",
"Assessment of harm avoidance",
"Assessment of parkinsonism",
"Assessment of other covariates",
"Statistical analyses",
"Descriptive properties of harm avoidance measure",
"Harm avoidance and change in parkinsonism",
"Potential confounders of harm avoidance and change in parkinsonism",
"Clinical significance of the loss of motor function associated with personality",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.",
"Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.",
"Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].",
"Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].",
"Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].",
"Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].",
"The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].",
"The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).",
"Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].",
"Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).",
"To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).",
"The authors declare that they have no competing interests.",
"ASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\n"
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Participants",
"Clinical diagnoses",
"Assessment of harm avoidance",
"Assessment of parkinsonism",
"Assessment of other covariates",
"Statistical analyses",
"Results",
"Descriptive properties of harm avoidance measure",
"Harm avoidance and change in parkinsonism",
"Potential confounders of harm avoidance and change in parkinsonism",
"Clinical significance of the loss of motor function associated with personality",
"Discussion",
"Conclusions",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.\nThere is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].\nTo examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.",
" Participants Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\nParticipants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.\n Clinical diagnoses Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\nClinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].\n Assessment of harm avoidance Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\nStudy participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].\n Assessment of parkinsonism Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\nTrained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].\n Assessment of other covariates Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\nAge in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].\n Statistical analyses The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].\nThe global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].",
"Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].\nThe Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.",
"Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].",
"Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].",
"Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].",
"Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].",
"The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.\nIn order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.\nIn subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].",
" Descriptive properties of harm avoidance measure The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\nThe clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).\n Harm avoidance and change in parkinsonism Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\nBaseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].\n Potential confounders of harm avoidance and change in parkinsonism Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\nPrior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).\n Clinical significance of the loss of motor function associated with personality To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).\nTo determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).",
"The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].\nClinical characteristics of the participants included in these analyses at this study’s baseline\n*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.\nHarm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).",
"Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.\nA model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*\n*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.\nPerson-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.\nHarm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.\nSince baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].\nIn sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).\nThe harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).\nAssociation of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*\n*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].",
"Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).\nPsychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).\nIn further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).\nSince higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).",
"To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).",
"Harm avoidance is a broad anxiety-related trait. People with a high level of the trait tend to be pessimistic, apprehensive, shy, and easily fatigued, behaviorally inhibited and to avoid new and potentially aversive situations [3]. In a cohort of more than 900 older adults, those with a high level of the trait showed a more rapid rate of progression of parkinsonism as compared to older adults with a low level of the trait. This association persisted after controlling for other psychosocial factors including depressive symptoms, loneliness and neuroticism, late-life activities including physical, social and cognitive activities, global cognitive function and chronic health conditions. Together these data suggest that the level of the trait of harm avoidance, may identify older individuals at higher risk for more rapidly progressive parkinsonism and provides evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.\nPrior studies have reported that higher levels of harm avoidance are associated with adverse health consequences in older adults including: incident disability, [6] and late-life cognitive impairments including incident mild cognitive impairment (MCI) and Alzheimer’s disease (AD) as well as cognitive decline [7,24,25]. This study extends these findings by showing that harm avoidance is also associated with both the level and rate of progression of parkinsonism in older adults without overt neurologic diseases including dementia, stroke and Parkinson’s disease (PD). Analyses of the sub-scale components of harm avoidance showed that all of its constituent factors are associated with the rate of progressive parkinsonism. The association of harm avoidance and parkinsonism, was robust and remained significant after accounting for related personality traits and psychosocial factors, global cognition, a wide range of late-life activities and chronic health conditions. The results of the current study have important translational implications as they suggest that personality traits need to be considered for our understanding of individual differences in the progression of parkinsonism in older adults. Furthermore, understanding the biologic basis of the association has the potential to lead to new therapeutic targets to reduce the burden of late-life motor impairment.\nThe basis of the association between harm avoidance and parkinsonism is likely to be complex. While, harm avoidance is related to other personality traits and psychosocial factors, [26] the association of harm avoidance and parkinsonism was unchanged when we adjusted for several of these factors in the current study (Table 3) [27]. Personality traits may affect for lifestyle choices i.e. physical and other late-life activities and could thus link harm avoidance with parkinsonism. In contrast to other personality traits, [17,28] statistical adjustment for late-life physical, cognitive and social activities in the current study did not affect the association of harm avoidance with progressive parkinsonism. Both harm avoidance and motor function in older people are preferentially associated with structural changes in specific brain regions including cortical and cerebellar structures which might account for their association in this study [29-33]. Thus, higher levels of harm avoidance may be lead to stress-related changes in neurotransmitters (e.g., cortisol or dopamine) causing brain atrophy, damaging motor-related brain regions or decreasing the brain’s capacity (motor reserve) to tolerate ongoing neurodegeneration and the accumulation of other neuropathologies [34-37].\nOver the years there have been many reports suggesting that PD may be associated with a distinctive personality which may manifest many years before clinical evidence of parkinsonism, but empirical studies have failed to support this suggestion [38,39]. Nonethelss, building on imaging data linking harm avoidance with brain dopamine receptors, recent studies have reported higher levels of harm avoidance occur in individuals with early clinical manifestations of PD such as REM sleep disorder which can manifest years before a clinical diagnosis of PD [40-43]. Studies in the current cohort have suggested that PD pathology is found in up to 40% of older adults without a clinical diagnoses of PD, and these lesions are associated with the severity of parkinsonism proximate to death [14]. Further work is needed to determine if the accumulation of subclinical PD pathology might contribute to the association between harm avoidance and the rate of change in the severity of parkinsonism. Alternatively, lower harm avoidance is associated with resilience, optimism, composure, and energy which may facilitate adaptation to accumulating neurodegeneration and the accumulation of neuropathology in older adults [10,44]. Further work to understand the neurobiologic basis for the current findings has the potential to identify new targets and pathways, for interventions to decrease the growing burden of progressive parkinsonism in older adults.\nOur study has some limitations. While some studies have suggested that personality traits such as harm avoidance are stable even in old age, others suggest that personality changes may occur with age but there are few studies which have focused on individuals older than 80 years [45-48]. Further, it is possible that a third unmeasured variable is related to both harm avoidance and progressive parkinsonism. A precondition for participation in the current study was consent to annual exam and organ donation at death, so given the selected nature of the cohort, our findings will need replication in other cohorts. This study was large since on an individual level the effect sizes for the association of harm avoidance and progressive parkinsonism are small. Similar effect sizes have been reported in prior reports of other psychosocial factors with motor decline [17]. Nonetheless, from a public policy perspective even the modest effect sizes observed in the current study are likely to be important.\nHowever, several factors increase confidence in our findings. Perhaps most importantly, the study enjoys high follow-up participation reducing bias due to attrition. In addition, personality traits were assessed among people without dementia and parkinsonism was evaluated as part of a uniform clinical evaluation which incorporated other widely accepted personality, affect and cognitive measures. In addition, a relatively large number of older people were studied, so that there was adequate statistical power to identify the association of interest while controlling for several potentially confounding variables. Results were similar with total score and subscores of the trait of harm avoidance.",
"The growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.",
"The authors declare that they have no competing interests.",
"ASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2318/14/54/prepub\n"
] |
[
null,
"methods",
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
"conclusions",
null,
null,
null
] |
[
"Late-life motor impairment",
"Aging",
"Parkinsonism",
"Harm avoidance"
] |
Background:
Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.
There is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].
To examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.
Methods:
Participants Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].
The Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.
Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].
The Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.
Clinical diagnoses Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].
Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].
Assessment of harm avoidance Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].
Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].
Assessment of parkinsonism Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].
Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].
Assessment of other covariates Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].
Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].
Statistical analyses The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.
In order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.
In subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].
The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.
In order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.
In subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].
Participants:
Participants were recruited from about 40 retirement facilities and subsidized housing facilities, as well as from church groups and social service agencies in northeastern Illinois. All participants signed an informed consent agreeing to annual clinical evaluation. The study was in accordance with the latest version of the Declaration of Helsinki and was approved by the Rush University Medical Center institutional review board [9].
The Memory and Aging Project began in 1997 and the overall follow-up rate is about 95% of survivors. Because of the rolling admission and mortality, the length of follow-up and number of examinations varies across participants. Further, because the collection of harm avoidance data was not added until 2004, it was only available on a subset of Memory and Aging Project participants. Baseline for these analyses was considered the first cycle at which harm avoidance was assessed. Eligibility for these analyses required: 1) a valid assessment of harm avoidance without evidence of clinical dementia and 2) a valid measure of parkinsonism at the time harm avoidance was assessed and at least one or more follow-up evaluations of parkinsonism in order to assess change in parkinsonism. There were 1,241 participants with harm avoidance assessment and 63 with evidence of clinical dementia were excluded. Of the remaining 1178 participants, there were 209 participants with incomplete parkinsonism data who were excluded from these analyses. These included 19 (1.6%) without any parkinsonism data and 190 (16.1%) who had a single valid assessment of parkinsonism but did not have a second evaluation either because they died before their first follow-up examination or because they had not been in the study long enough for follow-up evaluation leaving 969 participants for these analyses.
Clinical diagnoses:
Clinical diagnoses were made using a multi-step process as previously described [9]. Cognitive function testing included 19 performance tests were summarized into a summary measure of global cognition [9]. Participants were then evaluated in person by an experienced clinician who diagnosed dementia, stroke, or Parkinson’s disease, or other neurological and psychiatric disorders based on published criteria [11-13].
Assessment of harm avoidance:
Study participants completed the 35-items Harm Avoidance scale from the Temperament and Character Inventory assessing harm avoidance, but not the remaining 205 items [3,10]. Items from four subscales were rated as true or false: anticipatory worry (11 items; e.g., “Things often go wrong for me unless I’m careful”; range), fear of uncertainty (7 items; e.g., “I usually feel tense and worried when I have to do something new and unfamiliar”), shyness (8 items; e.g., “I am more shy than most people”), and fatigability (9 items; e.g., “I have less energy and tire more quickly than most people”). The score for the full scale (range, 0–35) and each subscale is the number of item responses indicative of the trait in question. These continuous trait measures were used in analyses as described in a prior study in this cohort [6].
Assessment of parkinsonism:
Trained nurse clinicians administered a 26-item modified motor UPDRS [14]. Four previously established parkinsonian sign scores were derived and scaled from 0 to 100 and a global parkinsonian sign score which was constructed by averaging these four scores was the primary outcome measure in these analyses [14].
Assessment of other covariates:
Age in years was computed from self-reported date of birth, and date of the baseline examination. Sex was recorded at the baseline interview. Education (reported highest grade or years of education) was obtained at baseline testing. Depressive symptoms were assessed with the Center for Epidemiological Studies Depression Scale, using a 10-item (e.g., “I felt sad”) version [15]. Loneliness was assessed with a 5-item (e.g., “I miss having people around”) form of the deJong-Gierveld Loneliness Scale [16]. The neuroticism trait was measured with the standard 48-item (e.g., “I have a low opinion of myself) scale from the NEO Personality Inventory [17]. BMI was calculated based on measured weight and height. Chronic health conditions include 3 vascular risk factors (i.e. hypertension, diabetes mellitus, and smoking), and 4 vascular diseases (i.e., myocardial infarction, congestive heart failure, claudication and stroke) [18]. Frequency of participation in cognitively stimulating activities was quantified with a scale, wherein people rated how often they had participated in each of 7 cognitive activities (e.g., reading a newspaper) over the past year [19]. Frequency of participation in social activity was based on 6 items about activities involving social interaction over the past year [20]. Physical activity was assessed using questions adapted from the 1985 National Health Interview Survey. Minutes spent engaged in each activity were summed and expressed as hours of activity/week [21].
Statistical analyses:
The global parkinsonian sign score had a positively skewed distribution and was subjected to a square root transformation, and the transformed scores were used as outcome variables in all analyses. We first examined pairwise correlations of harm avoidance with several other covariates. Then we used a series of linear mixed-effects models to examine the association of baseline harm avoidance score with the rate of change in severity of parkinsonism during the study period [22]. In these models, repeated measures of square root transformed global parkinsonian sign scores were used as the longitudinal outcome. The primary model predictors included a term for Time in years since the baseline as well as terms for harm avoidance at baseline and a term for its interaction with Time. To control for the effect of demographic variables, we also included terms for age, sex and education and their interaction with Time. The model predicted values, as described in the result section, were derived from the observed values of the independent variables and the corresponding model coefficients and then squared to back-transform these values to the original scale.
In order to contextualize the association of harm avoidance with parkinsonism, we centered baseline age, education, and harm avoidance score, such that all the model coefficients were interpreted with respect to a typical participant, that is, a female 80 years old at baseline, 14 years of education and a harm avoidance score of 10. Specifically, the coefficient for Time was interpreted as the annual rate of change in parkinsonism for a typical participant with the characteristics mentioned above. Similarly, the coefficients for harm avoidance and its interaction with Time estimated the average differences in baseline parkinsonism and rate of change in parkinsonism with a 1-point change in baseline harm avoidance score for a typical participant.
In subsequent models, we examined whether other covariates might account for the association between harm avoidance and parkinsonism. Next, we repeated the global measure of harm avoidance with trait subscores. To determine the clinical significance of the amount of change in the severity of parkinsonism, we first estimated the amount of increase in the rate of progression of parkinsonism with a 1SD increase in harm avoidance score, about 6 points, in a mixed effects model. Subsequently we constructed Cox proportional hazards models examining adverse health consequences of change in parkinsonism and estimated the hazard ratios associated with the amount of increase in rate of progression of parkinsonism, as given above.. These models controlled for age, sex and education. Models were examined graphically and analytically and assumptions were judged to be adequately met. A priori level of statistical significance was 0.05. Programming was done in SAS version 9.3 (SAS Institute Inc, Cary, NC) [23].
Results:
Descriptive properties of harm avoidance measure The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].
Clinical characteristics of the participants included in these analyses at this study’s baseline
*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.
Harm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).
The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].
Clinical characteristics of the participants included in these analyses at this study’s baseline
*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.
Harm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).
Harm avoidance and change in parkinsonism Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.
A model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*
*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.
Person-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.
Harm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.
Since baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].
In sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).
The harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).
Association of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*
*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].
Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.
A model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*
*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.
Person-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.
Harm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.
Since baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].
In sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).
The harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).
Association of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*
*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].
Potential confounders of harm avoidance and change in parkinsonism Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).
Psychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).
In further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).
Since higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).
Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).
Psychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).
In further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).
Since higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).
Clinical significance of the loss of motor function associated with personality To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).
To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).
Descriptive properties of harm avoidance measure:
The clinical characteristics of the participants included in these analyses at baseline are included in Table 1. Baseline harm avoidance scores ranged from 0 to 34 with higher values indicating higher level of this trait. Harm avoidance scores were approximately normally distributed (mean =10.3; SD = 6.47). Harm avoidance was higher in women (mean = 10.6; SD = 6.56) versus men (mean = 9.1; SD = 6.06) [t[923] = 3.14, p = 0.002].
Clinical characteristics of the participants included in these analyses at this study’s baseline
*BMI: Body mass index: weight in kilograms divided by height in meters squared. Global Cognition: composite measure of 19 tests. Depressive Symptoms: Modified 10 item Center for Epidemiological Studies Depression Scale, a higher score indicates greater depressive symptomatology. Loneliness: Loneliness was assessed with a 5-item form of the deJong-Gierveld Loneliness Scale. Neuroticism: was measured with the standard 48-item scale from the NEO Personality Inventory. Vascular Risk Factors: sum of smoking, diabetes, and hypertension self-reported. Vascular Diseases: sum of myocardial infarction, congestive heart failure, claudication and stroke self-reported. Physical Activity: Self-reported frequency of participation in 5 physical activities (hours/week), a higher score indicates more frequent participation. Cognitive Activity: Self reported frequency of participation in 7 cognitive activities, a higher score indicates more frequent participation. Social Activity: Self-reported frequency of participation in 6 items about activities involving social interaction, a higher score indicates more frequent participation.
Harm avoidance was associated with neuroticism (r = 0.50, p < 0.001) depressive symptoms (r = 0.38, p < 0.001), loneliness (r = 0.30, p < 0.001), physical activities (r = -0.13, p < 0.001), cognitive activities (r = -0.16, p < 0.001) and social activities (r = 0.-0.23, p < 0.001), global cognition (r = -0.16, p < 0.001), BMI (r = -0.09, p = 0.005) and vascular diseases (r = 0.06, p < 0.05) but was not associated with vascular risk factors (r = 0.044, p = 0.174).
Harm avoidance and change in parkinsonism:
Baseline global parkinsonism ranged from 0 to 43 (mean = 7.3; SD = 6.58). We used a linear mixed - effects model controlled for age, sex, and education to test the hypothesis that the baseline level of harm avoidance was associated with the progression of parkinsonism. During an average follow-up of 5 years (mean = 4.8; SD = 2.59 years), for an average participant (female, 80 years old at baseline with 14 years of education and a harm avoidance score of 10) the overall severity of parkinsonism increased by about 0.05 unit/year (Rate of Change, Table 2). Figure 1 illustrates the heterogeneity of the rate of change in parkinsonism for a 25% random sample of the participants included in these analyses. Each line in the figure shows the person-specific change in the rate of parkinsonism during the study. Most participants (59.8%) showed increasing severity of parkinsonism, slope >0, with the remainder exhibiting either less severe parkinsonism, (slope <0, (38.0%) or no change in parkinsonism (2.3%) during the study.
A model examining the association of baseline harm avoidance with the level and annual rate of change in parkinsonism, adjusting for demographic variables*
*Based on a linear mixed effect model. The model coefficients are interpreted with respect to a female participant 80 years old at baseline, with14 years of education and a harm avoidance score of 10. This model shows the cross sectional association of an average baseline harm avoidance score with parkinsonism at baseline as well as the association of the average harm avoidance score with the annual rate of change in parkinsonism . The model has a total of 9 terms listed in the left column. It contained a term which show the annual rate of change of parkinsonism (Time), the cross-sectional association of in the level of harm avoidance with level of parkinsonism (Harm Avoidance) and its association with the rate of change in parkinsonism (Harm avoidance X Rate of change in parkinsonism). In addition, the model also included 6 additional terms to control for the association of demographic variables (age, sex, education) with level of parkinsonism and their interaction with the annual rate of change in parkinsonism. For each term its Estimate (Standard Error, p Value) is shown in the right column.
Person-specific paths of progressive parkinsonism. The figure is organized according to the age of the participant at each evaluation; the length of each line relative to the x-axis indicates the total years of observation for that individual. The figure is estimated for a 25% random sample of the cohort and shows smoothed person-specific paths estimated from a random-effects model which included a term for time and controlled for age, sex, education and their interaction with time. The left Y axis shows the square root of global parkinsonian scores and the right Y axis shows the untransformed global parkinsonian scores.
Harm avoidance was associated with both the level of baseline parkinsonism (Harm Avoidance, Table 2) and the annual rate of change in parkinsonism (Rate of Change in Parkinsonism, Table 2). Thus, an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10) had an untransformed global parkinsonian score of 6.15 at baseline and an increase of 0.27 units in their parkinsonian score from study entry to their 1 year follow-up assessment. In contrast a similar participant whose baseline harm avoidance score was increased by a 6-point (~1 SD), had an untransformed parkinsonian score of 6.87 at study entry and an increase 0.42 units during their first year in the study, a difference of about 1.6 times larger than the typical participant with the average harm avoidance score.
Since baseline age was also associated with the annual rate of change in parkinsonism in this model (Table 2), we can contextualize the annual rate of change in the severity of parkinsonism associated with harm avoidance by comparing it with the increased severity of parkinsonism associated with a more common metric increased age. Comparing their respective coefficients in this model (Table 2) indicates that a female 80 years old at baseline with14 years of education and a harm avoidance score of 16 (about 1 SD above the mean) had a rate of increasing severity of parkinsonism equivalent to a female 85 years old at baseline, with 14 years of education and a harm avoidance score of 10. [(6 point increase in Harm avoidance score × Estimate for harm avoidance interaction of 0.004)/ Estimate for Age interaction of 0.005) = 4.8 years].
In sensitivity analyses, the association of harm avoidance and the rate of change in parkinsonism remained significant when we excluded individuals with a history of stroke (N = 102, 11.6%) or Parkinson’s disease (N = 13, 1.4%) which might cause more severe parkinsonism (Stroke: Estimate = 0.006, S.E. = 0.001, p < 0.001); PD: Estimate = 0.005, S.E. = 0.001, p < 0.001).
The harm avoidance subscores ranges showed substantial variation: anticipatory worry (range: 0, 11), fear of uncertainty (range: 0,7), shyness (range: 0,8) and fatigability (range: 0,9). To determine whether the subscores showed different associations with the rate of change in parkinsonism, we analyzed each in a separate model. Higher levels of all four subscores were associated with a more rapid progression of parkinsonism (Table 3).
Association of harm avoidance subscores in a typical participant with the level and the annual rate of change in parkinsonism, adjusted for demographic variables*
*We repeated the model shown in Table 2, four times replacing harm avoidance with each of its 4 subscores. Each row shows the results for a separate linear mixed effect models for a different subscore. The first term is the annual rate of change in parkinsonism; the 2nd term is the relationship between the subscore and level of parkinsonism and the 3rd columns shows the relationship between the subscore and the annual rate of change in parkinsonism. Each model also included terms (not shown) which controlled for age, sex, education and their interaction with the rate of change in parkinsonism [Estimate (Standard Error, p Value)].
Potential confounders of harm avoidance and change in parkinsonism:
Prior work has linked harm avoidance with cognition, we found that controlling for baseline global cognition, did not affect the association of harm avoidance and progression of parkinsonism (Estimate, 0.004, S.E. 0.001, p < 0.001).
Psychosocial factors can affect late-life motor impairment [16,17]. Therefore we examined whether depressive symptoms, loneliness or the personality trait of neuroticism might affect the association of harm avoidance and increasing parkinsonism. The association of harm avoidance with the rate of change in parkinsonism was not affected when we added terms for depressive symptoms, loneliness or neuroticism (results not shown). With all 3 of these correlated covariates in a single model, the association of harm avoidance and the progression of parkinsonism remained significant (Estimate = 0.003, S.E. = 0.001, p = 0.005).
In further analyses, we considered whether chronic health conditions might have affected our results. Adding terms for BMI and BMI squared (because both very low and very high body mass affect health) as well as for vascular risk factors and vascular diseases, first in separate analyses (results not shown) and then together in a single model did not affect the association of harm avoidance and the progression of parkinsonism (Estimate = 0.005, S.E. = 0.001, p = 0.005).
Since higher levels of late-life activities are associated with a slower rate of motor decline, [20] we examined whether the level of a range of different activities might account for the association of harm avoidance with progression in parkinsonism. In separate analyses (results not shown) as well as together, late-life physical, social and cognitive activities did not affect the association of harm avoidance and progression of parkinsonism (Estimate = 0.003, S.E. = 0.001, p = 0.003).
Clinical significance of the loss of motor function associated with personality:
To determine the clinical significance of the more rapid progression of parkinsonism associated with a 6-point (about 1 SD) increased harm avoidance score at baseline (Harm Avoidance X Time, Table 2), we constructed Cox proportional hazards model examining the association of change in parkinsonism with death and subsequently estimated the hazard ratios associated with the amount of the increased rate of change in parkinsonism attributable to a 6-point increase in baseline harm avoidance score (~1 SD) for an average participant (female 80 years old with 14 years of education). From these models (data not shown), we calculated that a 6-point increased harm avoidance score at baseline for a typical participant was associated with more than a 5% increased risk of death over the course of the study (Hazard Ratio: 1.053, 95% CI: 1.030, 1.077).
Discussion:
Harm avoidance is a broad anxiety-related trait. People with a high level of the trait tend to be pessimistic, apprehensive, shy, and easily fatigued, behaviorally inhibited and to avoid new and potentially aversive situations [3]. In a cohort of more than 900 older adults, those with a high level of the trait showed a more rapid rate of progression of parkinsonism as compared to older adults with a low level of the trait. This association persisted after controlling for other psychosocial factors including depressive symptoms, loneliness and neuroticism, late-life activities including physical, social and cognitive activities, global cognitive function and chronic health conditions. Together these data suggest that the level of the trait of harm avoidance, may identify older individuals at higher risk for more rapidly progressive parkinsonism and provides evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.
Prior studies have reported that higher levels of harm avoidance are associated with adverse health consequences in older adults including: incident disability, [6] and late-life cognitive impairments including incident mild cognitive impairment (MCI) and Alzheimer’s disease (AD) as well as cognitive decline [7,24,25]. This study extends these findings by showing that harm avoidance is also associated with both the level and rate of progression of parkinsonism in older adults without overt neurologic diseases including dementia, stroke and Parkinson’s disease (PD). Analyses of the sub-scale components of harm avoidance showed that all of its constituent factors are associated with the rate of progressive parkinsonism. The association of harm avoidance and parkinsonism, was robust and remained significant after accounting for related personality traits and psychosocial factors, global cognition, a wide range of late-life activities and chronic health conditions. The results of the current study have important translational implications as they suggest that personality traits need to be considered for our understanding of individual differences in the progression of parkinsonism in older adults. Furthermore, understanding the biologic basis of the association has the potential to lead to new therapeutic targets to reduce the burden of late-life motor impairment.
The basis of the association between harm avoidance and parkinsonism is likely to be complex. While, harm avoidance is related to other personality traits and psychosocial factors, [26] the association of harm avoidance and parkinsonism was unchanged when we adjusted for several of these factors in the current study (Table 3) [27]. Personality traits may affect for lifestyle choices i.e. physical and other late-life activities and could thus link harm avoidance with parkinsonism. In contrast to other personality traits, [17,28] statistical adjustment for late-life physical, cognitive and social activities in the current study did not affect the association of harm avoidance with progressive parkinsonism. Both harm avoidance and motor function in older people are preferentially associated with structural changes in specific brain regions including cortical and cerebellar structures which might account for their association in this study [29-33]. Thus, higher levels of harm avoidance may be lead to stress-related changes in neurotransmitters (e.g., cortisol or dopamine) causing brain atrophy, damaging motor-related brain regions or decreasing the brain’s capacity (motor reserve) to tolerate ongoing neurodegeneration and the accumulation of other neuropathologies [34-37].
Over the years there have been many reports suggesting that PD may be associated with a distinctive personality which may manifest many years before clinical evidence of parkinsonism, but empirical studies have failed to support this suggestion [38,39]. Nonethelss, building on imaging data linking harm avoidance with brain dopamine receptors, recent studies have reported higher levels of harm avoidance occur in individuals with early clinical manifestations of PD such as REM sleep disorder which can manifest years before a clinical diagnosis of PD [40-43]. Studies in the current cohort have suggested that PD pathology is found in up to 40% of older adults without a clinical diagnoses of PD, and these lesions are associated with the severity of parkinsonism proximate to death [14]. Further work is needed to determine if the accumulation of subclinical PD pathology might contribute to the association between harm avoidance and the rate of change in the severity of parkinsonism. Alternatively, lower harm avoidance is associated with resilience, optimism, composure, and energy which may facilitate adaptation to accumulating neurodegeneration and the accumulation of neuropathology in older adults [10,44]. Further work to understand the neurobiologic basis for the current findings has the potential to identify new targets and pathways, for interventions to decrease the growing burden of progressive parkinsonism in older adults.
Our study has some limitations. While some studies have suggested that personality traits such as harm avoidance are stable even in old age, others suggest that personality changes may occur with age but there are few studies which have focused on individuals older than 80 years [45-48]. Further, it is possible that a third unmeasured variable is related to both harm avoidance and progressive parkinsonism. A precondition for participation in the current study was consent to annual exam and organ donation at death, so given the selected nature of the cohort, our findings will need replication in other cohorts. This study was large since on an individual level the effect sizes for the association of harm avoidance and progressive parkinsonism are small. Similar effect sizes have been reported in prior reports of other psychosocial factors with motor decline [17]. Nonetheless, from a public policy perspective even the modest effect sizes observed in the current study are likely to be important.
However, several factors increase confidence in our findings. Perhaps most importantly, the study enjoys high follow-up participation reducing bias due to attrition. In addition, personality traits were assessed among people without dementia and parkinsonism was evaluated as part of a uniform clinical evaluation which incorporated other widely accepted personality, affect and cognitive measures. In addition, a relatively large number of older people were studied, so that there was adequate statistical power to identify the association of interest while controlling for several potentially confounding variables. Results were similar with total score and subscores of the trait of harm avoidance.
Conclusions:
The growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
ASB, RSW, LY, JMS, PAB, and DAB were involved in the conception, organization and execution of the project. ASB, LY and RSW were involved in the design, execution and review of the statistical analyses. ASB wrote the first draft and RSW, LY, JMS, PAB, and DAB reviewed and critiqued this and subsequent drafts of the manuscript. All authors read and approved the final manuscript. ASB and co-authors had full access to the data, have the right to publish all the data, and have had the right to obtain independent statistical analyses of the data. ASB takes responsibility for the integrity of the data and the accuracy of the data analysis.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2318/14/54/prepub
|
Background:
We tested the hypothesis that harm avoidance, a trait associated with behavioral inhibition, is associated with the rate of change in parkinsonism in older adults.
Methods:
At baseline harm avoidance was assessed with a standard self-report instrument in 969 older people without dementia participating in the Rush Memory and Aging Project, a longitudinal community-based cohort study. Parkinsonism was assessed annually with a modified version of the motor section of the Unified Parkinson's Disease Rating Scale (mUPDRS).
Results:
Average follow-up was 5 years. A linear mixed-effects model controlling for age, sex and education showed that for an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10), the overall severity of parkinsonism increased by about 0.05 unit/ year (Estimate, 0.054, S.E., 0.007, p <0.001) and that the level of harm avoidance was associated with the progression of parkinsonism (Estimate, 0.004, S.E., 0.001, p <0.001). Thus, for an average participant, every 6 point (~1 SD) increase in harm avoidance score at baseline, the rate of progression of parkinsonism increased about 50% compared to an individual with an average harm avoidance score. This amount of change in parkinsonism over the course of the study was associated with about a 5% increased risk of death. The association between harm avoidance and progression of parkinsonism persisted when controlling for cognitive function, depressive symptoms, loneliness, neuroticism, late-life cognitive, social and physical activities and chronic health conditions.
Conclusions:
A higher level of the harm avoidance trait is associated with a more rapid progression of parkinsonism in older adults.
|
Background:
Late-life parkinsonism including motor slowing (bradykinesia), posture and gait disturbances, rigidity and tremor may be present in up to 50% of community-dwelling older adults without known neurologic disease by age of 85 years. Parkinsonism is associated with a wide range of adverse health outcomes including morbidity, mortality, cognitive decline and dementia [1]. Thus, parkinsonism as part of the spectrum of late-life motor impairment is an important barrier to the maintenance of independence and well-being in old age [2]. Identifying risk factors for the progression of parkinsonism in older adults is an essential step in efforts to develop interventions which decrease its growing burden.
There is increasing recognition that personality traits are important determinants of healthy aging. Harm avoidance is a personality trait indicative of behavioral inhibition [3]. People with a high level of the trait tend to be pessimistic, apprehensive, shy, easily fatigued and risk averse. In prospective studies of children and young adults, low harm avoidance has been associated with worse health-related behavior and health outcomes, possibly because young people with a low level of harm avoidance trait tend to engage in risky behaviors [4,5]. By contrast, in prior works we have shown that in older people, high harm avoidance is associated with incident disability and dementia [6,7]. While there is some evidence to suggest that the level of harm avoidance is related to the level of physical activity, it is not known if or to what extent this trait is associated with other age-related conditions such as parkinsonism [8].
To examine the association of harm avoidance and progression of parkinsonism, we used clinical data from older adults without dementia participating in the Rush Memory and Aging Project [9]. Participants completed a standard self report measure of the trait based on the Harm Avoidance scale from the Temperament and Character Inventory [10]. At baseline and at annual intervals thereafter, they had structured evaluations that included a modified version of the motor section of the Unified Parkinson Disease Rating Scale (mUPDRS) [9]. We tested the hypothesis that a higher level of the harm avoidance trait is associated with the rate of progression of parkinsonism. In further analyses, we examined whether this association was confounded by cognition, other personality traits, psychosocial factors, chronic health conditions and lifestyles.
Conclusions:
The growing personal and social burden of late-life motor impairment in our aging population is a public health challenge. In a cohort of nearly 1000 older adults, individuals with a higher level of the trait of harm avoidance showed a more rapid rate of progressive parkinsonism as compared to older adults with a low level of the trait. The results of the current study suggest that the level of the trait of harm avoidance may identify older adults at higher risk for more rapid motor decline and underscore that personality traits need to be considered in studies of individual differences of late-life motor impairments. Finally, this study provides additional evidence for the importance of personality traits as one of the growing number of factors which may contribute to healthy aging.
|
Background:
We tested the hypothesis that harm avoidance, a trait associated with behavioral inhibition, is associated with the rate of change in parkinsonism in older adults.
Methods:
At baseline harm avoidance was assessed with a standard self-report instrument in 969 older people without dementia participating in the Rush Memory and Aging Project, a longitudinal community-based cohort study. Parkinsonism was assessed annually with a modified version of the motor section of the Unified Parkinson's Disease Rating Scale (mUPDRS).
Results:
Average follow-up was 5 years. A linear mixed-effects model controlling for age, sex and education showed that for an average participant (female, 80 years old at baseline, with 14 years of education and a harm avoidance score of 10), the overall severity of parkinsonism increased by about 0.05 unit/ year (Estimate, 0.054, S.E., 0.007, p <0.001) and that the level of harm avoidance was associated with the progression of parkinsonism (Estimate, 0.004, S.E., 0.001, p <0.001). Thus, for an average participant, every 6 point (~1 SD) increase in harm avoidance score at baseline, the rate of progression of parkinsonism increased about 50% compared to an individual with an average harm avoidance score. This amount of change in parkinsonism over the course of the study was associated with about a 5% increased risk of death. The association between harm avoidance and progression of parkinsonism persisted when controlling for cognitive function, depressive symptoms, loneliness, neuroticism, late-life cognitive, social and physical activities and chronic health conditions.
Conclusions:
A higher level of the harm avoidance trait is associated with a more rapid progression of parkinsonism in older adults.
| 13,063 | 330 | 18 |
[
"parkinsonism",
"harm avoidance",
"harm",
"avoidance",
"baseline",
"change",
"rate",
"score",
"change parkinsonism",
"rate change"
] |
[
"test",
"test"
] |
[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]
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[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]
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[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]
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[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]
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[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]
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[CONTENT] Late-life motor impairment | Aging | Parkinsonism | Harm avoidance [SUMMARY]
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[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]
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[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]
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[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]
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[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]
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[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]
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[CONTENT] Aged | Aged, 80 and over | Cohort Studies | Disease Progression | Female | Follow-Up Studies | Harm Reduction | Humans | Longitudinal Studies | Male | Parkinsonian Disorders | Prospective Studies | Residence Characteristics [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]
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[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]
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[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]
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[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]
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[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]
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[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | change | rate | score | change parkinsonism | rate change [SUMMARY]
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[CONTENT] harm | avoidance | harm avoidance | parkinsonism | adults | older | trait | related | associated | level [SUMMARY]
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[CONTENT] avoidance | harm avoidance | harm | parkinsonism | participants | items | baseline | score | models | change [SUMMARY]
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[CONTENT] parkinsonism | avoidance | harm avoidance | harm | change | change parkinsonism | 001 | rate change | rate change parkinsonism | rate [SUMMARY]
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[CONTENT] adults | older | older adults | level trait | motor | traits | growing | personality traits | level | level trait harm [SUMMARY]
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[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | score | change | rate | association | change parkinsonism [SUMMARY]
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[CONTENT] parkinsonism | harm avoidance | harm | avoidance | baseline | score | change | rate | association | change parkinsonism [SUMMARY]
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[CONTENT] [SUMMARY]
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[CONTENT] 969 | the Rush Memory and Aging Project ||| annually | the Unified Parkinson's [SUMMARY]
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[CONTENT] 5 years ||| 80 years old | 14 years | 10 | about 0.05 unit/ year | Estimate | 0.054 | S.E. | 0.007 | p <0.001 | Estimate | 0.004 | S.E. | 0.001 | p <0.001 ||| 6 | about 50% ||| 5% ||| [SUMMARY]
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[CONTENT] [SUMMARY]
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[CONTENT] ||| 969 | the Rush Memory and Aging Project ||| annually | the Unified Parkinson's ||| 5 years ||| 80 years old | 14 years | 10 | about 0.05 unit/ year | Estimate | 0.054 | S.E. | 0.007 | p <0.001 | Estimate | 0.004 | S.E. | 0.001 | p <0.001 ||| 6 | about 50% ||| 5% ||| ||| [SUMMARY]
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[CONTENT] ||| 969 | the Rush Memory and Aging Project ||| annually | the Unified Parkinson's ||| 5 years ||| 80 years old | 14 years | 10 | about 0.05 unit/ year | Estimate | 0.054 | S.E. | 0.007 | p <0.001 | Estimate | 0.004 | S.E. | 0.001 | p <0.001 ||| 6 | about 50% ||| 5% ||| ||| [SUMMARY]
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Secular Trends in Dietary Intake over a 20-Year Period in People with Type 2 Diabetes in Japan: A Comparative Study of Two Nationwide Registries; Japan Diabetes Complications Study (JDCS) and Japan Diabetes Clinical Data Management Study (JDDM).
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34684444
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In order to provide effective dietary guidance, it is necessary to consider dietary intake, which can change over time. This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period.
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BACKGROUND
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We compared the results of two dietary surveys that used the food frequency questionnaire format. The first was conducted in 1996 by the Japan Diabetes Complications Study (JDCS) (n = 1509; males 53.3%), and the second in 2014-2018 by the Japan Diabetes Clinical Data Management Study (JDDM) (n = 1145; males 65.6%). Both are nationwide representative registries of outpatients with type 2 diabetes in Japan.
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METHODS
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Over a 20-year period, both men and women with type 2 diabetes had a significant increase in body mass index (BMI). Nonetheless, there was only a small change in energy intake. Conversely, there was a significant increase in fat intake and thus in the fat-to-energy ratio. With regard to food groups, there was a significant increase in meat intake and a decrease in the intake of fish, soybeans/soy products, vegetables, and fruits, with a particularly significant decrease in vegetables.
|
RESULTS
|
Even in Japan, an industrialized country with a stable socioeconomic environment, there were many significant changes in the dietary intake of patients with type 2 diabetes over the 20-year period.
|
CONCLUSIONS
|
[
"Aged",
"Biomarkers",
"Body Mass Index",
"Diabetes Mellitus, Type 2",
"Diet",
"Disease Susceptibility",
"Energy Intake",
"Feeding Behavior",
"Female",
"Humans",
"Japan",
"Male",
"Middle Aged",
"Public Health Surveillance",
"Registries"
] |
8538089
|
1. Introduction
|
Diet plays a central role in diabetes self-management education and support [1]. To provide effective dietary guidance and/or to establish contemporary dietary guidelines, we must understand and take into account the changes in dietary intake and pattern over time. Dietary habits, including dietary content, are strongly influenced by changes in socioeconomic conditions and the environment [2]. The National Health and Nutrition Survey in Japan revealed decreases in intakes of 151 kcal (−7.4%) of energy, 24.9 g (−8.9%) of carbohydrates, 12.7 g (−15.5%) of protein, and 3.8 g (−27.7%) of salt (as sodium chloride) over a period of 20 years (1996–2016).
People with diabetes often receive nutritional guidance in the early stages of the disease, so their knowledge, as well as dietary content and habits, may differ from those who do not have diabetes. Thus, dietary changes in people with type 2 diabetes may differ from the general population. However, very few studies have investigated this change. In a study reporting on the dietary changes made by people with type 2 diabetes in the United States over the past 25 years, Casagrande et al. [3] reported 206 kcal (+12.2%) and 339 mg (+11.2%) increases in energy and sodium, respectively, as well as a 0.9 g (−8.7%) decrease in dietary fiber. In addition, according to the Dietary Survey of Type 2 Diabetes in Spain [4], although the sample size was small at 200, protein intake decreased by 6.9% in men and 1.4% in women, and fat intake increased by 3.2% in men and 5.5% in women in 2000 compared to 1993. However, as these studies focused on nutrient changes, it was unclear what changes in foods contributed to these nutrient changes. Dietary intake by food groups in those with type 2 diabetes has been previously reported [5], but to the best of our knowledge, there have been no studies on changes in dietary intake over time.
The diabetic population in the Asian region is rapidly increasing, with those in East Asia accounting for approximately a quarter of the world diabetes population [6]. Therefore, from the perspective of global epidemiology, it is important to elucidate changes in the nutrient intake of Asian people with diabetes over time. Previously, we reported [7] on the nutritional status of Japanese patients with type 2 diabetes from a survey conducted in 1996. However, no study thus far has examined changes in nutrient intake in the Asian diabetic population over time on a nationwide scale.
The purpose of this study was to reveal the detailed changes in dietary intake in Japanese patients with type 2 diabetes over a 20-year period beginning in 1996. In the present study, we conducted a nutritional survey, almost identical to our previous survey, which focused on a newly established nationwide registry of Japanese patients with type 2 diabetes. We then compared the results with those of our previous study. The results of this study reveal the importance of understanding which areas need to be focused on for efficient and effective dietary guidance, and thus for the creation of contemporary dietary guidelines. In addition, the results may provide fundamental insights into the differences in dietary advice received by patients of Asian and Western origins.
| null | null |
3. Results
|
Table 1 shows the clinical features of participants according to registry and sex. Although statistically significant differences were found between JDCS and JDDM patients in most variables, the differences in age and diabetes duration were too small to be considered clinically different. Variables that were considered to be clinically different between registries included BMI, HbA1c, blood pressure, and smoking rate among men. The BMI was significantly higher (+3.1 kg/m2 in men and +2.7 kg/m2 in women) in JDDM participants compared to JDCS participants. Comparing the mean BMI between JDCS and JDDM participants, the proportion of obese patients defined by BMI ≥ 25 kg/m2 according to Japanese criterion was significantly greater in JDDM participants, being approximately 20% to 50% higher in men and approximately 30% to 50% higher in women. HbA1c was significantly lower by approximately 1% and systolic blood pressure by approximately 5 mmHg for both men and women among JDDM participants, and the difference in the smoking rate for men was approximately 15% lower in JDDM participants than in JDCS participants.
When participants were divided into two groups based on the median age of 60 years, the characteristics were similar to those identified in the comparison of all groups, regardless of age. However, there was a dramatic increase in the BMI of JDDM participants younger than 60 years compared to JDCS participants in the same age group (Supplementary Table S1).
Table 2 shows a comparison of daily nutrient intake between patients in both studies. In Model 2, which was adjusted for age, HbA1c, BMI, insulin use, and oral medication use, there was no significant difference in energy intake in men between the two studies, but in women, there was a slight, but significant, increase of approximately 5% in the JDDM compared to the JDCS. Regarding proportions of macronutrients, the fat-to-energy ratio increased significantly, by about 3%, and the carbohydrate-to-energy ratio and protein-to-energy ratio decreased significantly for both men and women from the JDCS to the JDDM. Regarding changes in fat intake, the proportion of energy derived from saturated fatty acids increased significantly from 7.6% to 9.0% in men and from 8.3% to 9.4% in women in the interval between the surveys, whereas the proportion of energy derived from polyunsaturated fatty acids decreased significantly. Other variables that showed nutritionally meaningful differences between the two studies included significant decreases in dietary fiber, by 1.7 g for men and 1.6 g for women, and in salt, by 1.9 g for men and 2.7 g for women. The results were similar to the comparison of all groups, with fat-to-energy ratio and saturated fatty acid-to-energy ratio significantly increased in JDDM patients regardless of age, and polyunsaturated fatty acid-to-energy ratio, dietary fiber, and salt significantly decreased in JDDM patients (Supplementary Table S2).
Table 3 shows a comparison of daily food intakes categorized by food groups. In terms of grains intake, from the JDCS to the JDDM, there was a slight, but significant, decrease of 7.4% in men, but no significant difference was found in women. Meat intake by JDDM patients was significantly increased, by about 60%, for both men and women compared to JDCS patients, and intake of fish and soybean/soy products was significantly decreased in JDDM patients. In particular, changes in meat and fish intake were remarkable, with an increase of about 30 g of meat in the JDDM participants and a decrease of about 30 g of fish in the JDDM participants, for both men and women. In summary, the JDCS patients consumed more fish and soybean/soy products than meat, while the JDDM patients consumed more meat than fish and soybean/soy products; thus, the composition of the major protein food sources was reversed over the 20-year period. In addition, dairy intake decreased by about 20%, and intake of sweets and snacks was about three times higher in that interval. On the other hand, the intake of vegetables and fruits among JDDM patients was dramatically reduced by about 30% in both men and women compared with JDCS patients. Both intakes of green-yellow vegetables and other vegetables significantly decreased from the time of the JDCS survey to the JDDM survey. In particular, intake of green-yellow vegetables was markedly reduced between the two surveys by 42.0% for men and 35.0% for women. Regardless of age, meat intake increased in JDDM patients, while fish and soybean/soy products showed a decrease. However, there was no trend toward a reversal of the composition of the major protein food sources in patients aged 60 years and older. In both the under 60 and over 60 age groups, there was an increase in sweets and snacks and a decrease in fruits and total vegetable intake (Supplementary Table S3).
| null | null |
[
"2. Materials and Methods"
] |
[
"This study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study."
] |
[
null
] |
[
"1. Introduction",
"2. Materials and Methods",
"3. Results",
"4. Discussion"
] |
[
"Diet plays a central role in diabetes self-management education and support [1]. To provide effective dietary guidance and/or to establish contemporary dietary guidelines, we must understand and take into account the changes in dietary intake and pattern over time. Dietary habits, including dietary content, are strongly influenced by changes in socioeconomic conditions and the environment [2]. The National Health and Nutrition Survey in Japan revealed decreases in intakes of 151 kcal (−7.4%) of energy, 24.9 g (−8.9%) of carbohydrates, 12.7 g (−15.5%) of protein, and 3.8 g (−27.7%) of salt (as sodium chloride) over a period of 20 years (1996–2016).\nPeople with diabetes often receive nutritional guidance in the early stages of the disease, so their knowledge, as well as dietary content and habits, may differ from those who do not have diabetes. Thus, dietary changes in people with type 2 diabetes may differ from the general population. However, very few studies have investigated this change. In a study reporting on the dietary changes made by people with type 2 diabetes in the United States over the past 25 years, Casagrande et al. [3] reported 206 kcal (+12.2%) and 339 mg (+11.2%) increases in energy and sodium, respectively, as well as a 0.9 g (−8.7%) decrease in dietary fiber. In addition, according to the Dietary Survey of Type 2 Diabetes in Spain [4], although the sample size was small at 200, protein intake decreased by 6.9% in men and 1.4% in women, and fat intake increased by 3.2% in men and 5.5% in women in 2000 compared to 1993. However, as these studies focused on nutrient changes, it was unclear what changes in foods contributed to these nutrient changes. Dietary intake by food groups in those with type 2 diabetes has been previously reported [5], but to the best of our knowledge, there have been no studies on changes in dietary intake over time.\nThe diabetic population in the Asian region is rapidly increasing, with those in East Asia accounting for approximately a quarter of the world diabetes population [6]. Therefore, from the perspective of global epidemiology, it is important to elucidate changes in the nutrient intake of Asian people with diabetes over time. Previously, we reported [7] on the nutritional status of Japanese patients with type 2 diabetes from a survey conducted in 1996. However, no study thus far has examined changes in nutrient intake in the Asian diabetic population over time on a nationwide scale.\nThe purpose of this study was to reveal the detailed changes in dietary intake in Japanese patients with type 2 diabetes over a 20-year period beginning in 1996. In the present study, we conducted a nutritional survey, almost identical to our previous survey, which focused on a newly established nationwide registry of Japanese patients with type 2 diabetes. We then compared the results with those of our previous study. The results of this study reveal the importance of understanding which areas need to be focused on for efficient and effective dietary guidance, and thus for the creation of contemporary dietary guidelines. In addition, the results may provide fundamental insights into the differences in dietary advice received by patients of Asian and Western origins.",
"This study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].\nThe nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.\nIn addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.\nThis study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study.",
"Table 1 shows the clinical features of participants according to registry and sex. Although statistically significant differences were found between JDCS and JDDM patients in most variables, the differences in age and diabetes duration were too small to be considered clinically different. Variables that were considered to be clinically different between registries included BMI, HbA1c, blood pressure, and smoking rate among men. The BMI was significantly higher (+3.1 kg/m2 in men and +2.7 kg/m2 in women) in JDDM participants compared to JDCS participants. Comparing the mean BMI between JDCS and JDDM participants, the proportion of obese patients defined by BMI ≥ 25 kg/m2 according to Japanese criterion was significantly greater in JDDM participants, being approximately 20% to 50% higher in men and approximately 30% to 50% higher in women. HbA1c was significantly lower by approximately 1% and systolic blood pressure by approximately 5 mmHg for both men and women among JDDM participants, and the difference in the smoking rate for men was approximately 15% lower in JDDM participants than in JDCS participants.\nWhen participants were divided into two groups based on the median age of 60 years, the characteristics were similar to those identified in the comparison of all groups, regardless of age. However, there was a dramatic increase in the BMI of JDDM participants younger than 60 years compared to JDCS participants in the same age group (Supplementary Table S1).\nTable 2 shows a comparison of daily nutrient intake between patients in both studies. In Model 2, which was adjusted for age, HbA1c, BMI, insulin use, and oral medication use, there was no significant difference in energy intake in men between the two studies, but in women, there was a slight, but significant, increase of approximately 5% in the JDDM compared to the JDCS. Regarding proportions of macronutrients, the fat-to-energy ratio increased significantly, by about 3%, and the carbohydrate-to-energy ratio and protein-to-energy ratio decreased significantly for both men and women from the JDCS to the JDDM. Regarding changes in fat intake, the proportion of energy derived from saturated fatty acids increased significantly from 7.6% to 9.0% in men and from 8.3% to 9.4% in women in the interval between the surveys, whereas the proportion of energy derived from polyunsaturated fatty acids decreased significantly. Other variables that showed nutritionally meaningful differences between the two studies included significant decreases in dietary fiber, by 1.7 g for men and 1.6 g for women, and in salt, by 1.9 g for men and 2.7 g for women. The results were similar to the comparison of all groups, with fat-to-energy ratio and saturated fatty acid-to-energy ratio significantly increased in JDDM patients regardless of age, and polyunsaturated fatty acid-to-energy ratio, dietary fiber, and salt significantly decreased in JDDM patients (Supplementary Table S2).\nTable 3 shows a comparison of daily food intakes categorized by food groups. In terms of grains intake, from the JDCS to the JDDM, there was a slight, but significant, decrease of 7.4% in men, but no significant difference was found in women. Meat intake by JDDM patients was significantly increased, by about 60%, for both men and women compared to JDCS patients, and intake of fish and soybean/soy products was significantly decreased in JDDM patients. In particular, changes in meat and fish intake were remarkable, with an increase of about 30 g of meat in the JDDM participants and a decrease of about 30 g of fish in the JDDM participants, for both men and women. In summary, the JDCS patients consumed more fish and soybean/soy products than meat, while the JDDM patients consumed more meat than fish and soybean/soy products; thus, the composition of the major protein food sources was reversed over the 20-year period. In addition, dairy intake decreased by about 20%, and intake of sweets and snacks was about three times higher in that interval. On the other hand, the intake of vegetables and fruits among JDDM patients was dramatically reduced by about 30% in both men and women compared with JDCS patients. Both intakes of green-yellow vegetables and other vegetables significantly decreased from the time of the JDCS survey to the JDDM survey. In particular, intake of green-yellow vegetables was markedly reduced between the two surveys by 42.0% for men and 35.0% for women. Regardless of age, meat intake increased in JDDM patients, while fish and soybean/soy products showed a decrease. However, there was no trend toward a reversal of the composition of the major protein food sources in patients aged 60 years and older. In both the under 60 and over 60 age groups, there was an increase in sweets and snacks and a decrease in fruits and total vegetable intake (Supplementary Table S3).",
"This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period using two surveys with essentially the same format. Although the backgrounds of the patients, including age and duration of diabetes, were quite similar between the two studies, BMI and obesity rates were markedly increased in the later study. Regarding dietary content, fat intake increased, including saturated fatty acids, along with significant increases in the consumption of meat and sweets and snacks, and a significant decrease in vegetable and fish intake. To date, there have been no studies on the changes in dietary intake by persons with type 2 diabetes in Asia, except for a report on adherence to dietary recommendations in Korea [12]. To our knowledge, the changes in nutrient and food intake have not been reported. Therefore, this is the first study to clarify the secular changes in the dietary status of patients with type 2 diabetes in Asia.\nSelf-management education and support for diet and physical activity are essential in the care of overweight or obese people with type 2 diabetes. A previous study observing the secular trends in the United States from 1988 to 2012 [3] showed a trend of increases in BMI and energy intake in patients with type 2 diabetes, which was also the case even in adults without diabetes. On the other hand, in our study of East Asians, the BMI markedly increased from 22.7 kg/m2 to 25.8 kg/m2 in men and 23.2 kg/m2 to 25.9 kg/m2 in women, although no such remarkable changes were found in the Japanese general population according to the Japan National Health and Nutrition Survey performed during a similar period (1996 and 2016) (from 23.3 kg/ m2 to 24.0 kg/m2 in men and 23.2 kg m2 to 22.6 kg/m2 in women) [13]. This suggests that secular changes in BMI and obesity were different between people with and without type 2 diabetes and between the East and the West. According to the National Health and Nutrition Surveys, from 1996 to 2016, the energy intake by the general population aged 40–69 years mildly declined from 2284 kcal to 2145 kcal (−139 kcal) for men and from 1880 kcal to 1722 kcal (−158 kcal) for women, although the dietary survey method was different from our method [14].\nWe previously reported, using JDCS baseline data, that Western populations with diabetes are mostly obese and are also more obese than the general population, whereas Japanese people with diabetes not only have a mean BMI within the normal range, but also it is almost the same as in the general population [15]. The results of the current study, including the JDDM results 20 years after the initial JDCS survey, strongly support the fact that obesity among those with type 2 diabetes has recently increased in Japan, as is the case in Western countries [16]. On the other hand, it is not clear from this study alone why the increase in BMI in Japanese patients with type 2 diabetes was significantly greater than in the general population, even though there was no significant increase in energy intake between the two studies. Our previous study [17] suggested that there are racial differences in the relationship between energy intake and obesity, but several other hypotheses are also possible.\nThe first possible explanation for the significant increase in BMI without a significant increase in energy intake in Japanese patients with type 2 diabetes may be the effect of decreased energy expenditure due to decreased physical activity. A comparison of the activity levels between people with diabetes and the general population found no significant difference in physical activity levels and leisure-time physical activity patterns, and reported that neither groups achieved the recommended physical activity goals [18,19]. The decrease in the number of steps by the general Japanese population from 1996 to 2016 was remarkable for both men and women [13], and it was pointed out that the decrease in physical activity was due to the development of transportation [20]. In this study, direct comparisons were difficult because of the differences in the studies’ survey methods regarding the amount of physical activity; however, 44.4% of patients in the JDDM reported low levels of overall physical activity in their daily lives, which was calculated from activity time and activity intensity. Future research on secular trends in the amount of physical activity in patients with type 2 diabetes is expected.\nThe second theory as to why BMI increased without an increase in energy intake is that it could be due to the effect of smoking rates and medications. The BMI of smokers was reportedly lower than that of non-smokers [21,22], and the smoking rate of men in the JDDM with a high BMI was significantly lower than that in the JDCS. In addition, the increase in the rate of insulin use over time that was observed between these studies may be a factor in weight gain. The fact that there was a significant decrease in the rate of use of oral hypoglycemic agents may have contributed to weight gain or weight loss, depending on the type of oral hypoglycemic agent [23]. Further studies, including a detailed follow-up of the status of the medications, are necessary.\nAs a third conjecture, the ratio of macronutrients may have affected these findings. Regarding the energy-producing nutrients, both men and women in the JDDM had higher fat-to-energy ratios than those in the JDCS. In the JDDM compared to the JDCS, men had a lower carbohydrate-to-energy ratio and protein-to-energy ratio and women had a lower protein-to-energy ratio. Women in the JDDM had an increase in energy intake, which could be attributed to an increased fat intake as their carbohydrate and protein intakes did not increase. In the Japanese National Health and Nutrition Survey, which was of a similar duration (1996 and 2016), the fat-to-energy ratio increased (men: +2.6%, women: +2.9%), but carbohydrate and protein intakes tended to decrease (carbohydrate: −22.6 g/day for men, −33.4 g/day for women, protein: −15.9 g/day for men, −13.0 g/day for women), and fat intake was almost unchanged, suggesting that the fat-to-energy ratio was relatively increased. Increased fat-to-energy ratios due to an increased fat intake in patients with type 2 diabetes may have contributed to the increased BMI. However, previous studies have not provided a unified consensus on the association between the fat-to-energy ratio and obesity, which needs to be clarified in the future [24].\nIt is well known that, regardless of body weight, a high-fat diet, especially an excessive intake of saturated fatty acids and a high saturated fatty acid-to-energy ratio, is significantly associated with a higher risk of cardiovascular events in patients with type 2 diabetes [25]. According to the Japanese practice guidelines for diabetes [26], a reduction in saturated fatty acids is recommended when the lipid-to-energy ratio exceeds 25%. In addition, Dietary Reference Intakes for Japanese [27] set a target saturated fatty acid-to-energy ratio of ≤7%. Among our patients with a fat-to-energy ratio of ≥25%, only 9.5% of JDCS patients and 4.4% of JDDM patients had a saturated fatty acid-to-energy ratio of ≤7%, suggesting the necessity for enhanced guidance to reduce the saturated fatty acid-to-energy ratio. The saturated fatty acid-to-energy ratio for Americans with type 2 diabetes was approximately 11%, which was still higher than the level (<10%) recommended by the U.S. Dietary Guidelines, but it did not increase between 1988 and 2012 [28].\nIn contrast, in our Japanese patients, the saturated fatty acid-to-energy ratio increased significantly over the 20 years to approximately 9% in both men and women, and the percentage of patients with a saturated fatty acid-to-energy ratio of more than 10% increased significantly from 8.2% in men and 13.3% in women in the JDCS to 28.4% in men and 36.6% in women in the JDDM. This suggests that the fat intake pattern of Japanese people with type 2 diabetes is approaching that of Western patients. Therefore, correcting excessive lipid intake while taking into account the content and quality of lipids is considered to be a crucial issue.\nIn terms of secular changes in food group intake patterns, an increase in meat intake and a decrease in the consumption of fish and dairy products were observed as sources of protein intake. JDCS patients had higher intakes of fish and soybeans/soy products than meat, whereas JDDM patients had higher intakes of meat than fish and soybeans/soy products. The National Health and Nutrition Survey [29] also showed the same secular changes in food group intake patterns as this study.\nIt is indicated that patients with type 2 diabetes in Japan had a major change in the composition of their protein sources, as did the general Japanese population, which was a change to a meat-centered diet. In general, among people in Western countries, the circumstances in which meat intake is higher than fish intake continues. It is inferred that the diet of Japanese patients with type 2 diabetes has been transformed into a diet centered on meat due to the influx of various lifestyle patterns and dietary cultures from Western countries, as is the case with the general Japanese population. It has been reported that meat was associated with cardiovascular complications and mortality in people with type 2 diabetes in a study in the US [30] and in our previous study [31]. Thus, it is considered necessary to strengthen the guidance on protein sources to prevent complications in the future.\nMeat is the food with the highest contribution to fat intake by Japanese people. Therefore, it is thought that an increased meat intake may also affect fat-to-energy ratios and saturated fatty acid increases in JDDM patients. Aside from meat, saturated fatty acids are found in the Japanese diet in milk, butter, and coconut oil, which are commonly used as ingredients in Western sweets rather than in traditional Japanese ones (which are mostly made with sugar, rice, and beans [32]). Although the type of sweets could not be identified in this survey, as their intake more than doubled from the JDCS to the JDDM, it is inferred that in conjunction with meat, the increase in the intake of sweets contributed to the increase in saturated fatty acid consumption.\nContrary to meat, the intake of fruits and vegetables decreased significantly between the two studies. As vegetables and fruits are good sources of dietary fiber, fiber intake decreased significantly as vegetable and fruit intakes decreased. A higher intake of dietary fiber is known to improve glycemic control, and we previously reported that it was significantly associated with a reduced risk of stroke [33] and retinopathy [34] in JDCS patients. However, despite its importance, the intake of dietary fiber was lower than the recommended goal of 20 g/day or higher in 1996, and even declined in the following 20 years. A decreased intake of fruits and vegetables is apparent in the Japanese general population [29], and is considered to be an important issue in dietary guidance.\nAs to the background of the decrease in the consumption of vegetables and fruits, survey reports have suggested that differences in lifestyle, such as economic conditions [35] and living alone, exist [36] and that diabetic patients who work may choose a less diversified diet [37]. It was also reported that the consumption of vegetables and fruits tends to be higher with a lower frequency of eating meals away from home [38]. As to the proportions of food expenditure in Japan from 1995 to 2015, the consumption of fresh foods has decreased, and eating out and consuming processed foods have increased from 65.4% to 72.6% [39]. This suggests that the increase in eating out and buying processed foods may influence the decrease in fruit and vegetable intake, not only in the general population, but also in those with type 2 diabetes. This Westernization of dietary habits has been widely adopted, and advice on the dietary treatment of diabetes should consider the current dietary situation.\nThis study has several limitations. First, both the JDCS and the JDDM were comprised of patients from different cohorts, although they were both multicenter studies. However, both studies were conducted at outpatient clinics throughout Japan specializing in diabetes, and the clinical settings were similar except for the time period studied. An additional limitation is that the number of participants that were included in both surveys was different from the potential candidates. Of the 2033 JDCS patients, 1588 responded to a baseline dietary survey, and 524 patients who had missing values in the dietary survey were excluded. In the JDDM patients, 1145 of 2337 patients met the eligibility criteria for the JDCS and responded to the baseline dietary survey. However, there were no notable differences in baseline characteristics between the responders and non-responders [8], and consequently, no selection bias was found. Finally, the participants’ dietary intake was obtained from a self-administered questionnaire, which could be an underestimation. As for the long-term comparison of 20 years, changes had been made in the food composition tables and in the FFQg version used in both studies. However, those changes corresponded to changes in dietary habits and food ingredients due to secular trends, and there was little effect on energy intake and intake of macronutrients due to the changes in the food composition tables.\nThe results of this study clarify the changes in the dietary intake of Japanese patients with type 2 diabetes over 20 years, beginning in 1996. Despite a lack of significant change in the total energy intake during the period, both men and women with type 2 diabetes had a large increase in BMI. A rise in the fat-to-energy ratio due to an increase in fat intake was also observed, particularly in men. In terms of intake by food group, both men and women had clearly unfavorable changes in terms of complication prevention. These included an increase in the intake of meat and a decrease in the intake of fish, soybean/soy products, vegetables, and fruits. The results of our study strongly suggest that, in order to prevent complications in Japanese patients with type 2 diabetes in the future, the provision of nutritional guidance, in line with these chronological changes in dietary intake, is necessary."
] |
[
"intro",
null,
"results",
"discussion"
] |
[
"food intake",
"type 2 diabetes",
"obesity",
"diabetes mellitus",
"Asia"
] |
1. Introduction:
Diet plays a central role in diabetes self-management education and support [1]. To provide effective dietary guidance and/or to establish contemporary dietary guidelines, we must understand and take into account the changes in dietary intake and pattern over time. Dietary habits, including dietary content, are strongly influenced by changes in socioeconomic conditions and the environment [2]. The National Health and Nutrition Survey in Japan revealed decreases in intakes of 151 kcal (−7.4%) of energy, 24.9 g (−8.9%) of carbohydrates, 12.7 g (−15.5%) of protein, and 3.8 g (−27.7%) of salt (as sodium chloride) over a period of 20 years (1996–2016).
People with diabetes often receive nutritional guidance in the early stages of the disease, so their knowledge, as well as dietary content and habits, may differ from those who do not have diabetes. Thus, dietary changes in people with type 2 diabetes may differ from the general population. However, very few studies have investigated this change. In a study reporting on the dietary changes made by people with type 2 diabetes in the United States over the past 25 years, Casagrande et al. [3] reported 206 kcal (+12.2%) and 339 mg (+11.2%) increases in energy and sodium, respectively, as well as a 0.9 g (−8.7%) decrease in dietary fiber. In addition, according to the Dietary Survey of Type 2 Diabetes in Spain [4], although the sample size was small at 200, protein intake decreased by 6.9% in men and 1.4% in women, and fat intake increased by 3.2% in men and 5.5% in women in 2000 compared to 1993. However, as these studies focused on nutrient changes, it was unclear what changes in foods contributed to these nutrient changes. Dietary intake by food groups in those with type 2 diabetes has been previously reported [5], but to the best of our knowledge, there have been no studies on changes in dietary intake over time.
The diabetic population in the Asian region is rapidly increasing, with those in East Asia accounting for approximately a quarter of the world diabetes population [6]. Therefore, from the perspective of global epidemiology, it is important to elucidate changes in the nutrient intake of Asian people with diabetes over time. Previously, we reported [7] on the nutritional status of Japanese patients with type 2 diabetes from a survey conducted in 1996. However, no study thus far has examined changes in nutrient intake in the Asian diabetic population over time on a nationwide scale.
The purpose of this study was to reveal the detailed changes in dietary intake in Japanese patients with type 2 diabetes over a 20-year period beginning in 1996. In the present study, we conducted a nutritional survey, almost identical to our previous survey, which focused on a newly established nationwide registry of Japanese patients with type 2 diabetes. We then compared the results with those of our previous study. The results of this study reveal the importance of understanding which areas need to be focused on for efficient and effective dietary guidance, and thus for the creation of contemporary dietary guidelines. In addition, the results may provide fundamental insights into the differences in dietary advice received by patients of Asian and Western origins.
2. Materials and Methods:
This study compared the results of two dietary surveys and the associated clinical variables regarding participants in nationwide registries of outpatients with type 2 diabetes in Japan, namely the Japan Diabetes Complications Study (JDCS) [8] and the Japan Diabetes Clinical Data Management Study (JDDM). For the dietary surveys, both used an almost identical format to the food frequency questionnaire based on food groups (FFQg) [9]. The JDCS is a nationwide cohort study of patients with type 2 diabetes aged 40–70 years, with HbA1c levels of 6.5% or higher, and who attended outpatient clinics at 59 hospitals [8]. Of the 2033 enrollees, 1509 who responded to the self-administered dietary survey in 1996 were included in the analysis. The JDDM is another nationwide registry of outpatients with type 2 diabetes, but which started later than the JDCS. The JDDM conducted its dietary survey in 2014–2018 with essentially the same format as that used by the JDCS. We used the survey results from 1145 patients aged 40–70 years [10].
The nutrient and food intakes in both studies were assessed using the FFQg (JDCS: FFQg 1st edition, JDDM: FFQg v3.5). FFQg is a questionnaire that solicits information on the intake of 29 food groups and 10 cooking methods. An evaluation can be made on the average weekly intake for each food or food group in commonly used units or portion sizes. The validity of the FFQ was shown by comparison with consecutive 7-day weighed dietary records of 66 individuals aged 19–60 years [11]. We calculated the nutrient and food group intakes using standardized nutrition software (EIYO-KUN (JDCS: v4.5, JDDM: v6.0)) that is used in surveys and nutrition counseling in Japan. The FFQ v3.5 used in the JDDM calculates grains and seaweed in a rehydrated condition, while the JDCS calculates these products as a dry food. Therefore, in the present analysis, the values from the JDDM survey were converted to dry matter values for comparison of food conditions with the JDCS survey. The differences between EIYO-KUN v4.5 and v6.0 consist of additional listings of food composition, so the method for the calculation of nutrients was not changed.
In addition to dietary surveys, information from physical examinations and blood tests was also evaluated. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Blood tests were carried out at each clinic. All data are presented as mean ± standard deviation or percentage. The comparisons of participants’ characteristics, nutrient intakes, and intakes by food group were examined by the unpaired Student’s t-test, chi-square (χ2) tests, and an analysis of covariance. In the analysis of covariance, each of the nutrients was used as a dependent variable, and age, HBA1c, BMI, treated by insulin (yes or no), and treated by OHA (yes or no) were used as adjustment variables. All statistical analyses were carried out using SPSS software (v26.0) and the significance level was 0.05.
This study was in accordance with the Declaration of Helsinki and the ethical guidelines for clinical-epidemiological studies by the Japanese Ministry of Health, Labour and Welfare; the JDCS was approved by the review boards of all participating institutions; and the JDDM was approved by the ethical review boards of the JDDM and Niigata University (2015–1648). Informed consent was obtained from all individual participants included in the study.
3. Results:
Table 1 shows the clinical features of participants according to registry and sex. Although statistically significant differences were found between JDCS and JDDM patients in most variables, the differences in age and diabetes duration were too small to be considered clinically different. Variables that were considered to be clinically different between registries included BMI, HbA1c, blood pressure, and smoking rate among men. The BMI was significantly higher (+3.1 kg/m2 in men and +2.7 kg/m2 in women) in JDDM participants compared to JDCS participants. Comparing the mean BMI between JDCS and JDDM participants, the proportion of obese patients defined by BMI ≥ 25 kg/m2 according to Japanese criterion was significantly greater in JDDM participants, being approximately 20% to 50% higher in men and approximately 30% to 50% higher in women. HbA1c was significantly lower by approximately 1% and systolic blood pressure by approximately 5 mmHg for both men and women among JDDM participants, and the difference in the smoking rate for men was approximately 15% lower in JDDM participants than in JDCS participants.
When participants were divided into two groups based on the median age of 60 years, the characteristics were similar to those identified in the comparison of all groups, regardless of age. However, there was a dramatic increase in the BMI of JDDM participants younger than 60 years compared to JDCS participants in the same age group (Supplementary Table S1).
Table 2 shows a comparison of daily nutrient intake between patients in both studies. In Model 2, which was adjusted for age, HbA1c, BMI, insulin use, and oral medication use, there was no significant difference in energy intake in men between the two studies, but in women, there was a slight, but significant, increase of approximately 5% in the JDDM compared to the JDCS. Regarding proportions of macronutrients, the fat-to-energy ratio increased significantly, by about 3%, and the carbohydrate-to-energy ratio and protein-to-energy ratio decreased significantly for both men and women from the JDCS to the JDDM. Regarding changes in fat intake, the proportion of energy derived from saturated fatty acids increased significantly from 7.6% to 9.0% in men and from 8.3% to 9.4% in women in the interval between the surveys, whereas the proportion of energy derived from polyunsaturated fatty acids decreased significantly. Other variables that showed nutritionally meaningful differences between the two studies included significant decreases in dietary fiber, by 1.7 g for men and 1.6 g for women, and in salt, by 1.9 g for men and 2.7 g for women. The results were similar to the comparison of all groups, with fat-to-energy ratio and saturated fatty acid-to-energy ratio significantly increased in JDDM patients regardless of age, and polyunsaturated fatty acid-to-energy ratio, dietary fiber, and salt significantly decreased in JDDM patients (Supplementary Table S2).
Table 3 shows a comparison of daily food intakes categorized by food groups. In terms of grains intake, from the JDCS to the JDDM, there was a slight, but significant, decrease of 7.4% in men, but no significant difference was found in women. Meat intake by JDDM patients was significantly increased, by about 60%, for both men and women compared to JDCS patients, and intake of fish and soybean/soy products was significantly decreased in JDDM patients. In particular, changes in meat and fish intake were remarkable, with an increase of about 30 g of meat in the JDDM participants and a decrease of about 30 g of fish in the JDDM participants, for both men and women. In summary, the JDCS patients consumed more fish and soybean/soy products than meat, while the JDDM patients consumed more meat than fish and soybean/soy products; thus, the composition of the major protein food sources was reversed over the 20-year period. In addition, dairy intake decreased by about 20%, and intake of sweets and snacks was about three times higher in that interval. On the other hand, the intake of vegetables and fruits among JDDM patients was dramatically reduced by about 30% in both men and women compared with JDCS patients. Both intakes of green-yellow vegetables and other vegetables significantly decreased from the time of the JDCS survey to the JDDM survey. In particular, intake of green-yellow vegetables was markedly reduced between the two surveys by 42.0% for men and 35.0% for women. Regardless of age, meat intake increased in JDDM patients, while fish and soybean/soy products showed a decrease. However, there was no trend toward a reversal of the composition of the major protein food sources in patients aged 60 years and older. In both the under 60 and over 60 age groups, there was an increase in sweets and snacks and a decrease in fruits and total vegetable intake (Supplementary Table S3).
4. Discussion:
This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period using two surveys with essentially the same format. Although the backgrounds of the patients, including age and duration of diabetes, were quite similar between the two studies, BMI and obesity rates were markedly increased in the later study. Regarding dietary content, fat intake increased, including saturated fatty acids, along with significant increases in the consumption of meat and sweets and snacks, and a significant decrease in vegetable and fish intake. To date, there have been no studies on the changes in dietary intake by persons with type 2 diabetes in Asia, except for a report on adherence to dietary recommendations in Korea [12]. To our knowledge, the changes in nutrient and food intake have not been reported. Therefore, this is the first study to clarify the secular changes in the dietary status of patients with type 2 diabetes in Asia.
Self-management education and support for diet and physical activity are essential in the care of overweight or obese people with type 2 diabetes. A previous study observing the secular trends in the United States from 1988 to 2012 [3] showed a trend of increases in BMI and energy intake in patients with type 2 diabetes, which was also the case even in adults without diabetes. On the other hand, in our study of East Asians, the BMI markedly increased from 22.7 kg/m2 to 25.8 kg/m2 in men and 23.2 kg/m2 to 25.9 kg/m2 in women, although no such remarkable changes were found in the Japanese general population according to the Japan National Health and Nutrition Survey performed during a similar period (1996 and 2016) (from 23.3 kg/ m2 to 24.0 kg/m2 in men and 23.2 kg m2 to 22.6 kg/m2 in women) [13]. This suggests that secular changes in BMI and obesity were different between people with and without type 2 diabetes and between the East and the West. According to the National Health and Nutrition Surveys, from 1996 to 2016, the energy intake by the general population aged 40–69 years mildly declined from 2284 kcal to 2145 kcal (−139 kcal) for men and from 1880 kcal to 1722 kcal (−158 kcal) for women, although the dietary survey method was different from our method [14].
We previously reported, using JDCS baseline data, that Western populations with diabetes are mostly obese and are also more obese than the general population, whereas Japanese people with diabetes not only have a mean BMI within the normal range, but also it is almost the same as in the general population [15]. The results of the current study, including the JDDM results 20 years after the initial JDCS survey, strongly support the fact that obesity among those with type 2 diabetes has recently increased in Japan, as is the case in Western countries [16]. On the other hand, it is not clear from this study alone why the increase in BMI in Japanese patients with type 2 diabetes was significantly greater than in the general population, even though there was no significant increase in energy intake between the two studies. Our previous study [17] suggested that there are racial differences in the relationship between energy intake and obesity, but several other hypotheses are also possible.
The first possible explanation for the significant increase in BMI without a significant increase in energy intake in Japanese patients with type 2 diabetes may be the effect of decreased energy expenditure due to decreased physical activity. A comparison of the activity levels between people with diabetes and the general population found no significant difference in physical activity levels and leisure-time physical activity patterns, and reported that neither groups achieved the recommended physical activity goals [18,19]. The decrease in the number of steps by the general Japanese population from 1996 to 2016 was remarkable for both men and women [13], and it was pointed out that the decrease in physical activity was due to the development of transportation [20]. In this study, direct comparisons were difficult because of the differences in the studies’ survey methods regarding the amount of physical activity; however, 44.4% of patients in the JDDM reported low levels of overall physical activity in their daily lives, which was calculated from activity time and activity intensity. Future research on secular trends in the amount of physical activity in patients with type 2 diabetes is expected.
The second theory as to why BMI increased without an increase in energy intake is that it could be due to the effect of smoking rates and medications. The BMI of smokers was reportedly lower than that of non-smokers [21,22], and the smoking rate of men in the JDDM with a high BMI was significantly lower than that in the JDCS. In addition, the increase in the rate of insulin use over time that was observed between these studies may be a factor in weight gain. The fact that there was a significant decrease in the rate of use of oral hypoglycemic agents may have contributed to weight gain or weight loss, depending on the type of oral hypoglycemic agent [23]. Further studies, including a detailed follow-up of the status of the medications, are necessary.
As a third conjecture, the ratio of macronutrients may have affected these findings. Regarding the energy-producing nutrients, both men and women in the JDDM had higher fat-to-energy ratios than those in the JDCS. In the JDDM compared to the JDCS, men had a lower carbohydrate-to-energy ratio and protein-to-energy ratio and women had a lower protein-to-energy ratio. Women in the JDDM had an increase in energy intake, which could be attributed to an increased fat intake as their carbohydrate and protein intakes did not increase. In the Japanese National Health and Nutrition Survey, which was of a similar duration (1996 and 2016), the fat-to-energy ratio increased (men: +2.6%, women: +2.9%), but carbohydrate and protein intakes tended to decrease (carbohydrate: −22.6 g/day for men, −33.4 g/day for women, protein: −15.9 g/day for men, −13.0 g/day for women), and fat intake was almost unchanged, suggesting that the fat-to-energy ratio was relatively increased. Increased fat-to-energy ratios due to an increased fat intake in patients with type 2 diabetes may have contributed to the increased BMI. However, previous studies have not provided a unified consensus on the association between the fat-to-energy ratio and obesity, which needs to be clarified in the future [24].
It is well known that, regardless of body weight, a high-fat diet, especially an excessive intake of saturated fatty acids and a high saturated fatty acid-to-energy ratio, is significantly associated with a higher risk of cardiovascular events in patients with type 2 diabetes [25]. According to the Japanese practice guidelines for diabetes [26], a reduction in saturated fatty acids is recommended when the lipid-to-energy ratio exceeds 25%. In addition, Dietary Reference Intakes for Japanese [27] set a target saturated fatty acid-to-energy ratio of ≤7%. Among our patients with a fat-to-energy ratio of ≥25%, only 9.5% of JDCS patients and 4.4% of JDDM patients had a saturated fatty acid-to-energy ratio of ≤7%, suggesting the necessity for enhanced guidance to reduce the saturated fatty acid-to-energy ratio. The saturated fatty acid-to-energy ratio for Americans with type 2 diabetes was approximately 11%, which was still higher than the level (<10%) recommended by the U.S. Dietary Guidelines, but it did not increase between 1988 and 2012 [28].
In contrast, in our Japanese patients, the saturated fatty acid-to-energy ratio increased significantly over the 20 years to approximately 9% in both men and women, and the percentage of patients with a saturated fatty acid-to-energy ratio of more than 10% increased significantly from 8.2% in men and 13.3% in women in the JDCS to 28.4% in men and 36.6% in women in the JDDM. This suggests that the fat intake pattern of Japanese people with type 2 diabetes is approaching that of Western patients. Therefore, correcting excessive lipid intake while taking into account the content and quality of lipids is considered to be a crucial issue.
In terms of secular changes in food group intake patterns, an increase in meat intake and a decrease in the consumption of fish and dairy products were observed as sources of protein intake. JDCS patients had higher intakes of fish and soybeans/soy products than meat, whereas JDDM patients had higher intakes of meat than fish and soybeans/soy products. The National Health and Nutrition Survey [29] also showed the same secular changes in food group intake patterns as this study.
It is indicated that patients with type 2 diabetes in Japan had a major change in the composition of their protein sources, as did the general Japanese population, which was a change to a meat-centered diet. In general, among people in Western countries, the circumstances in which meat intake is higher than fish intake continues. It is inferred that the diet of Japanese patients with type 2 diabetes has been transformed into a diet centered on meat due to the influx of various lifestyle patterns and dietary cultures from Western countries, as is the case with the general Japanese population. It has been reported that meat was associated with cardiovascular complications and mortality in people with type 2 diabetes in a study in the US [30] and in our previous study [31]. Thus, it is considered necessary to strengthen the guidance on protein sources to prevent complications in the future.
Meat is the food with the highest contribution to fat intake by Japanese people. Therefore, it is thought that an increased meat intake may also affect fat-to-energy ratios and saturated fatty acid increases in JDDM patients. Aside from meat, saturated fatty acids are found in the Japanese diet in milk, butter, and coconut oil, which are commonly used as ingredients in Western sweets rather than in traditional Japanese ones (which are mostly made with sugar, rice, and beans [32]). Although the type of sweets could not be identified in this survey, as their intake more than doubled from the JDCS to the JDDM, it is inferred that in conjunction with meat, the increase in the intake of sweets contributed to the increase in saturated fatty acid consumption.
Contrary to meat, the intake of fruits and vegetables decreased significantly between the two studies. As vegetables and fruits are good sources of dietary fiber, fiber intake decreased significantly as vegetable and fruit intakes decreased. A higher intake of dietary fiber is known to improve glycemic control, and we previously reported that it was significantly associated with a reduced risk of stroke [33] and retinopathy [34] in JDCS patients. However, despite its importance, the intake of dietary fiber was lower than the recommended goal of 20 g/day or higher in 1996, and even declined in the following 20 years. A decreased intake of fruits and vegetables is apparent in the Japanese general population [29], and is considered to be an important issue in dietary guidance.
As to the background of the decrease in the consumption of vegetables and fruits, survey reports have suggested that differences in lifestyle, such as economic conditions [35] and living alone, exist [36] and that diabetic patients who work may choose a less diversified diet [37]. It was also reported that the consumption of vegetables and fruits tends to be higher with a lower frequency of eating meals away from home [38]. As to the proportions of food expenditure in Japan from 1995 to 2015, the consumption of fresh foods has decreased, and eating out and consuming processed foods have increased from 65.4% to 72.6% [39]. This suggests that the increase in eating out and buying processed foods may influence the decrease in fruit and vegetable intake, not only in the general population, but also in those with type 2 diabetes. This Westernization of dietary habits has been widely adopted, and advice on the dietary treatment of diabetes should consider the current dietary situation.
This study has several limitations. First, both the JDCS and the JDDM were comprised of patients from different cohorts, although they were both multicenter studies. However, both studies were conducted at outpatient clinics throughout Japan specializing in diabetes, and the clinical settings were similar except for the time period studied. An additional limitation is that the number of participants that were included in both surveys was different from the potential candidates. Of the 2033 JDCS patients, 1588 responded to a baseline dietary survey, and 524 patients who had missing values in the dietary survey were excluded. In the JDDM patients, 1145 of 2337 patients met the eligibility criteria for the JDCS and responded to the baseline dietary survey. However, there were no notable differences in baseline characteristics between the responders and non-responders [8], and consequently, no selection bias was found. Finally, the participants’ dietary intake was obtained from a self-administered questionnaire, which could be an underestimation. As for the long-term comparison of 20 years, changes had been made in the food composition tables and in the FFQg version used in both studies. However, those changes corresponded to changes in dietary habits and food ingredients due to secular trends, and there was little effect on energy intake and intake of macronutrients due to the changes in the food composition tables.
The results of this study clarify the changes in the dietary intake of Japanese patients with type 2 diabetes over 20 years, beginning in 1996. Despite a lack of significant change in the total energy intake during the period, both men and women with type 2 diabetes had a large increase in BMI. A rise in the fat-to-energy ratio due to an increase in fat intake was also observed, particularly in men. In terms of intake by food group, both men and women had clearly unfavorable changes in terms of complication prevention. These included an increase in the intake of meat and a decrease in the intake of fish, soybean/soy products, vegetables, and fruits. The results of our study strongly suggest that, in order to prevent complications in Japanese patients with type 2 diabetes in the future, the provision of nutritional guidance, in line with these chronological changes in dietary intake, is necessary.
|
Background:
In order to provide effective dietary guidance, it is necessary to consider dietary intake, which can change over time. This study analyzed changes in the diet of Japanese patients with type 2 diabetes over a 20-year period.
Methods:
We compared the results of two dietary surveys that used the food frequency questionnaire format. The first was conducted in 1996 by the Japan Diabetes Complications Study (JDCS) (n = 1509; males 53.3%), and the second in 2014-2018 by the Japan Diabetes Clinical Data Management Study (JDDM) (n = 1145; males 65.6%). Both are nationwide representative registries of outpatients with type 2 diabetes in Japan.
Results:
Over a 20-year period, both men and women with type 2 diabetes had a significant increase in body mass index (BMI). Nonetheless, there was only a small change in energy intake. Conversely, there was a significant increase in fat intake and thus in the fat-to-energy ratio. With regard to food groups, there was a significant increase in meat intake and a decrease in the intake of fish, soybeans/soy products, vegetables, and fruits, with a particularly significant decrease in vegetables.
Conclusions:
Even in Japan, an industrialized country with a stable socioeconomic environment, there were many significant changes in the dietary intake of patients with type 2 diabetes over the 20-year period.
| null | null | 5,052 | 277 | 4 |
[
"intake",
"patients",
"diabetes",
"dietary",
"jddm",
"energy",
"jdcs",
"men",
"type",
"type diabetes"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] food intake | type 2 diabetes | obesity | diabetes mellitus | Asia [SUMMARY]
| null |
[CONTENT] food intake | type 2 diabetes | obesity | diabetes mellitus | Asia [SUMMARY]
| null |
[CONTENT] food intake | type 2 diabetes | obesity | diabetes mellitus | Asia [SUMMARY]
| null |
[CONTENT] Aged | Biomarkers | Body Mass Index | Diabetes Mellitus, Type 2 | Diet | Disease Susceptibility | Energy Intake | Feeding Behavior | Female | Humans | Japan | Male | Middle Aged | Public Health Surveillance | Registries [SUMMARY]
| null |
[CONTENT] Aged | Biomarkers | Body Mass Index | Diabetes Mellitus, Type 2 | Diet | Disease Susceptibility | Energy Intake | Feeding Behavior | Female | Humans | Japan | Male | Middle Aged | Public Health Surveillance | Registries [SUMMARY]
| null |
[CONTENT] Aged | Biomarkers | Body Mass Index | Diabetes Mellitus, Type 2 | Diet | Disease Susceptibility | Energy Intake | Feeding Behavior | Female | Humans | Japan | Male | Middle Aged | Public Health Surveillance | Registries [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] intake | patients | diabetes | dietary | jddm | energy | jdcs | men | type | type diabetes [SUMMARY]
| null |
[CONTENT] intake | patients | diabetes | dietary | jddm | energy | jdcs | men | type | type diabetes [SUMMARY]
| null |
[CONTENT] intake | patients | diabetes | dietary | jddm | energy | jdcs | men | type | type diabetes [SUMMARY]
| null |
[CONTENT] dietary | changes | diabetes | type diabetes | type | intake | asian | study | changes dietary | people [SUMMARY]
| null |
[CONTENT] jddm | men | significantly | women | participants | jddm participants | jdcs | patients | intake | jddm patients [SUMMARY]
| null |
[CONTENT] intake | jddm | dietary | diabetes | jdcs | patients | energy | type | men | changes [SUMMARY]
| null |
[CONTENT] ||| Japanese | 2 | 20-year [SUMMARY]
| null |
[CONTENT] 20-year | 2 | BMI ||| ||| ||| [SUMMARY]
| null |
[CONTENT] ||| Japanese | 2 | 20-year ||| two ||| first | 1996 | the Japan Diabetes Complications Study | JDCS | 1509 | 53.3% | second | 2014-2018 | the Japan Diabetes Clinical Data Management Study | 1145 | 65.6% ||| 2 | Japan ||| 20-year | 2 | BMI ||| ||| ||| ||| Japan | 2 | 20-year [SUMMARY]
| null |
|||
Are pre-existing markers of chronic kidney disease associated with short-term mortality following acute community-acquired pneumonia and sepsis? A cohort study among older people with diabetes using electronic health records.
|
25605811
|
We aimed to examine whether pre-existing impaired estimated glomerular filtration rate (eGFR) and proteinuria were associated with mortality following community-acquired pneumonia or sepsis among people aged ≥ 65 years with diabetes mellitus, without end-stage renal disease.
|
BACKGROUND
|
Patients were followed up from onset of first community-acquired pneumonia or sepsis episode in a cohort study using large, linked electronic health databases. Follow-up was for up to 90 days, unlimited by hospital discharge. We used generalized linear models with log link, normal distribution and robust standard errors to calculate risk ratios (RRs) for all-cause 28- and 90-day mortality according to two markers of chronic kidney disease: eGFR and proteinuria.
|
METHODS
|
All-cause mortality among the 4743 patients with pneumonia was 29.6% after 28 days and 37.4% after 90 days. Among the 1058 patients with sepsis, all-cause 28- and 90-day mortality were 35.6 and 44.2%, respectively. eGFR <30 mL/min/1.73 m(2) was a risk marker of higher 28-day mortality for pneumonia (RR 1.27: 95% CI 1.12-1.43) and sepsis (RR 1.32: 95% CI 1.07-1.64), adjusted for age, sex, socio-economic status, smoking status and co-morbidities. Neither moderately impaired eGFR nor proteinuria were associated with short-term mortality following either infection.
|
RESULTS
|
People with pre-existing low eGFR but not on dialysis are at higher risk of death following pneumonia and sepsis. This association was not explained by existing co-morbidities. These patients need to be carefully monitored to prevent modifiable causes of death.
|
CONCLUSIONS
|
[
"Age Factors",
"Aged",
"Aged, 80 and over",
"Biomarkers",
"Cohort Studies",
"Community-Acquired Infections",
"Comorbidity",
"Diabetes Mellitus",
"Electronic Health Records",
"Female",
"Humans",
"Kidney Failure, Chronic",
"Male",
"Pneumonia",
"Proteinuria",
"Renal Dialysis",
"Risk Factors",
"Sepsis",
"Survival Rate"
] |
4438741
|
INTRODUCTION
|
Chronic kidney disease (CKD) affects an estimated 1.8 million people in England, 98% of whom do not require renal replacement therapy [1]. CKD is defined by reduced estimated glomerular filtration rate (eGFR) or evidence of kidney damage such as proteinuria and is commonest among older people [2, 3]. Most patients with CKD are managed in primary care [4].
Infection is an important cause of mortality among older people [5, 6]. Both reduced eGFR and proteinuria are associated with an increased rate of infection-related mortality, which could be partly explained by increased incidence of infection [7–9]. It is less clear whether CKD is also associated with poorer prognosis following infection. When clinicians assess patients with community-acquired infection, developing complications such as acute kidney injury (AKI) may not yet be apparent, but they will know which patients have pre-existing CKD. If pre-existing CKD is a risk marker for short-term mortality, this would be useful for risk stratification and clinical management of patients with infections, especially in primary care where clinicians may not have access to immediate laboratory tests. While the implications of acute changes in eGFR during infection are a focus of current research, few studies have investigated the role of pre-existing CKD [10]. Low baseline eGFR has been found to be associated with mortality following sepsis and community-acquired pneumonia, but rarely examined according to clinically meaningful categories of eGFR [11–15]. To the best of our knowledge, proteinuria has not been examined as a potential risk marker for mortality following infection [12, 15, 16].
Among older people, CKD frequently co-exists with other co-morbidities [3]. An association of CKD with poor prognosis of infections could thus be due to confounding from these co-morbidities. For example, CKD is strongly associated with cardiovascular disease, which may be complicated by infection, resulting in post-infection mortality driven by the underlying cardiovascular disease [17]. Such deaths would largely follow hospitalization for cardiovascular events. CKD is associated with healthcare-associated pneumonia, which carries a worse prognosis than community-acquired pneumonia [18, 19]. Focusing on community-acquired infections should exclude infections arising as short-term sequelae of cardiovascular events and improve understanding of the relationship between pre-existing CKD markers and infection prognosis.
Older people with diabetes mellitus form a large and growing population in primary care who suffer a high incidence of community-acquired pneumonia and sepsis [20]. Forty per cent of adults with diabetes have CKD, of whom three-quarters have proteinuria, and CKD among these patients is associated with a greater all-cause excess mortality than among patients without diabetes [21]. If proteinuria is a risk marker for mortality among older patients with diabetes who develop community-acquired pneumonia or sepsis, this could inform clinical management of a large primary care patient population with appreciable mortality following infection [20].
This study aimed to examine whether baseline eGFR and proteinuria were independent risk markers for short-term mortality following community-acquired pneumonia or sepsis among older people with diabetes mellitus, using large, linked electronic health record databases.
| null | null |
RESULTS
|
We identified 4957 individuals with community-acquired pneumonia and 1114 individuals with community-acquired sepsis. Data were missing (for smoking status and/or HbA1C results) for 212/4957 individuals with pneumonia (4.3%) and 56/1114 individuals with sepsis (5.0%). These patients were excluded. Among patients with pneumonia, patients with missing data were older (median age 83 years, IQR: 78–88) compared with those included (median age 80 years, IQR: 74–85), with a higher 28-day mortality (excluded 88/214, 41.1%: included 1406/4743, 29.6%) but a similar distribution of baseline eGFR. A similar pattern was seen for sepsis.
Estimated GFR was based on the higher of two results for 4029 patients with pneumonia (84.9%) and 919 patients with sepsis (86.9%); for the remaining patients, only a single valid serum creatinine result was available. CKD prevalence was high: almost half of the patients had eGFR <60 mL/min/1.73 m2 and a third had proteinuria. Patients with eGFR <60 mL/min/1.73 m2 were older, with a higher prevalence of ischaemic heart disease and congestive heart failure than patients with eGFR ≥60 mL/min/1.73 m2 (Table 1).Table 1.Baseline characteristics of the study populationPneumoniaSepsiseGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2eGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2Age (years) Median (IQR)82 (77–87)78 (72–83)81 (75–86)76 (71–82)n (col %)n (col %)n (col %)n (col %)Gender Female1231 (53.0)1106 (42.0)294 (55.7)268 (45.7)Onset prior to 1 April 2004584 (25.1)527 (20.0)124 (23.5)120 (20.5)Socio-economic status (IMD quintile)a 1 (least deprived)383 (16.5)438 (16.6)109 (20.6)106 (18.1) 2554 (23.8)595 (22.6)116 (22.0)127 (21.7) 3494 (21.3)582 (22.1)120 (22.7)137 (23.4) 4502 (21.6)574 (21.8)110 (20.8)108 (18.4) 5 (most deprived)391 (16.8)444 (16.9)73 (13.8)108 (18.4)Smoking status Current321 (13.8)549 (20.9)70 (13.3)103 (17.6) Ex-smoker1185 (51.0)1351 (51.3)235 (44.5)284 (48.5) Non-smoker776 (33.4)698 (26.5)207 (39.2)193 (32.9) Missing42 (1.8)35 (1.3)16 (3.0)6 (1.0)Comorbidities Chronic lung disease503 (21.6)686 (26.1)59 (11.2)95 (16.2) Hypertension1623 (69.8)1662 (63.1)372 (70.5)381 (65.0) Congestive heart failure734 (31.6)430 (16.3)147 (27.8)95 (16.2) Ischaemic heart disease966 (41.6)887 (33.7)223 (42.2)207 (35.3) Cerebrovascular disease692 (29.8)638 (24.2)152 (28.8)145 (24.7) Other dementia174 (7.5)190 (7.2)42 (8.0)23 (3.9) Cancer382 (16.4)474 (18.0)99 (18.8)120 (20.5) Connective tissue disorders228 (9.8)215 (8.2)36 (6.8)52 (8.9)HbA1C Good <7%1176 (50.6)1401 (53.2)251 (47.5)308 (52.6) Borderline 7–10%966 (41.6)1033 (39.2)213 (40.3)230 (39.3) Poor >10%108 (4.7)131 (5.0)41 (7.8)34 (5.8) None recorded74 (3.2)68 (2.6)23 (4.4)14 (2.4)Prior antidiabetes medication Insulin122 (5.3)126 (4.8)34 (6.4)23 (3.9) Oral medications1139 (49.0)1426 (54.2)247 (46.8)312 (53.2) Both462 (19.9)384 (14.6)130 (24.6)114 (19.5) None601 (25.9)697 (26.5)117 (22.2)137 (23.4) Total23242633528586eGFR, estimated glomerular filtration rate.aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.
Baseline characteristics of the study population
eGFR, estimated glomerular filtration rate.
aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.
Patients with pneumonia experienced 29.6% 28-day all-cause mortality (1406 deaths). Patients with sepsis experienced 35.6% 28-day all-cause mortality (377 deaths) (Table 2). Survival curves showed high mortality at infection onset, declining over ∼30 days to a more stable rate for the next 60 days following both pneumonia and sepsis (Figure 1). RRs for 28-day mortality were higher among people with eGFR <30 mL/min/1.73 m2 compared with people with eGFR ≥60 mL/min/1.73 m2 for pneumonia (RR = 1.27; 95% CI 1.10–1.47) and sepsis (RR = 1.42; 1.10–1.84), adjusted for age, sex, socio-economic status and onset prior to April 2004. Adjustment for smoking status, co-morbidities and characteristics of diabetes had minimal effect on these RRs for pneumonia (fully adjusted RR = 1.27; 1.12–1.43) or sepsis (fully adjusted RR = 1.32; 1.07–1.64). There was no evidence of associations between intermediate levels of eGFR and 28-day mortality for either infection, nor for an association between proteinuria and 28-day mortality. The pattern of associations of eGFR and proteinuria with 90-day mortality was similar to those for 28-day mortality (Table 2). Results were unchanged by additional adjustment for peripheral vascular disease. There was no good evidence of effect modification between eGFR and proteinuria, and in particular no evidence of any association of proteinuria with 28-day mortality within any category of eGFR status (data not shown).Table 2.Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)aNumber (column %)28-Day mortality (row %)90-Day mortality (row %)RRs for 28-day mortality (95% CI)RRs for 90-day mortality (95% CI)Adjusted for demographicsbadjusted for non-CVD co-morbiditiescFully adjusteddAdjusted for demographicsbAdjusted for non-CVD co-morbiditiescFully adjusteddPneumonia Proteinuria (adjusted for eGFR) Yes1611 (34.0)499 (31.0)625 (38.8)1.07 (0.97–1.17)1.07 (0.98–1.18)1.07 (0.98–1.18)1.05 (0.97–1.13)1.06 (0.98–1.14)1.05 (0.98–1.14) No3132 (66.0)907 (29.0)1150 (36.7)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<1523 (0.5)12 (52.2)12 (52.2)1.27 (1.10–1.47)1.31 (1.13–1.52)1.30 (1.12–1.52)1.26 (1.12–1.42)1.29 (1.14–1.45)1.27 (1.12–1.43)15–29263 (5.6)110 (41.8)139 (52.9) 30–44764 (16.1)265 (34.7)336 (44.0)0.98 (0.86–1.10)0.99 (0.88–1.13)0.99 (0.88–1.12)1.02 (0.93–1.13)1.04 (0.94–1.15)1.03 (0.94–1.14) 45–601162 (24.5)332 (28.6)418 (36.0)0.91 (0.82–1.02)0.94 (0.84–1.04)0.93 (0.84–1.04)0.91 (0.83–1.00)0.93 (0.85–1.02)0.92 (0.84–1.01) ≥602531 (53.4)687 (27.1)870 (34.4)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total47431406 (29.6)1775 (37.4)Sepsis Proteinuria (adjusted for eGFR) Yes358 (33.8)128 (35.8)159 (44.4)0.98 (0.82–1.17)1.01 (0.85–1.21)1.01 (0.85–1.21)0.98 (0.86–1.13)0.96 (0.84–1.10)0.96 (0.84–1.10) No700 (66.2)249 (35.6)309 (44.1)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<158 (0.8)2 (25.0)5 (62.5)1.42 (1.10–1.84)1.41 (1.08–1.84)1.37 (1.05–1.79)1.39 (1.14–1.70)1.32 (1.07–1.63)1.32 (1.07–1.64)15–2962 (5.9)32 (51.6)39 (62.9) 30–44190 (18.0)88 (46.3)103 (54.2)1.25 (1.01–1.55)1.24 (0.99–1.55)1.24 (0.99–1.54)1.14 (0.96–1.36)1.12 (0.94–1.33)1.11 (0.94–1.32) 45–60232 (21.9)76 (32.8)95 (41.0)0.95 (0.75–1.19)0.91 (0.72–1.15)0.91 (0.72–1.14)0.92 (0.77–1.11)0.89 (0.74–1.07)0.89 (0.75–1.07) ≥60566 (53.5)179 (31.6)226 (39.9)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total1058377 (35.6)468 (44.2)aExcluding patients with missing smoking or HbA1C data.bAge, gender, socio-economic status, onset prior to 1 April 2004.cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.FIGURE 1:Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.
Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)a
aExcluding patients with missing smoking or HbA1C data.
bAge, gender, socio-economic status, onset prior to 1 April 2004.
cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.
dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.
Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.
The underlying causes of death following pneumonia and sepsis were similar for patients with eGFR above and below 60 mL/min/1.73 m2. Causes of 28-day mortality following sepsis were predominantly sources of infection (Table 3). Following pneumonia onset, pneumonia was recorded as an underlying or contributory cause of death for 83.9% (1191/1419) of deaths within 28 days and 76.3% (1366/1790) of deaths within 90 days. Among patients with eGFR <60 mL/min/1.73 m2, renal disease was recorded as an underlying or contributory cause for 10.6% (77/724) of those who died within 28 days of pneumonia onset and 16.6% (152/913) of those who died within 90 days. Recording of CKD as a cause of death increased with lower eGFR (Table 4).Table 3.Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)aeGFReGFR <60 mL/min/1.73 m2eGFR ≥60 mL/min/1.73 m228-Day mortality following pneumonia, n = 1419J18 Pneumonia, organism unspecified, n = 256, 35.4%J44 Other chronic obstructive pulmonary disease, n = 63, 8.7%I25 Chronic ischaemic heart disease, n = 47, 6.5%I50 Heart failure, n = 43, 5.9%I64 Stroke, not specified as haemorrhage or infarction, n = 37, 5.1%J18 Pneumonia, organism unspecified, n = 216, 31.1%J44 Other chronic obstructive pulmonary disease, n = 63, 9.1%I64 Stroke, not specified as haemorrhage or infarction, n = 35, 5.0%C34 Malignant neoplasm of bronchus and lung, n = 34, 4.9%I25 Chronic ischaemic heart disease, n = 29, 4.2%Total = 724Total = 69529–90 Day mortality following pneumonia, n = 371J18 Pneumonia, organism unspecified, n = 38, 20.1%I25 Chronic ischaemic heart disease, n = 24, 12.7%J44 Other chronic obstructive pulmonary disease, n = 13, 6.9%C34 Malignant neoplasm of bronchus and lung, n = 12, 6.4%I21 Acute myocardial infarction, n = 12, 6.4%J18 Pneumonia, organism unspecified, n = 20, 11.0%C34 Malignant neoplasm of bronchus and lung, n = 20, 11.0%J44 Other chronic obstructive pulmonary disease, n = 19, 10.4%I25 Chronic ischaemic heart disease, n = 9, 5.0%I64 Stroke, not specified as haemorrhage or infarction, n = 9, 5.0%Total = 189Total = 18228-Day mortality following sepsis, n = 387N39 Other disorders of urinary systemb, n = 33, 15.9%J18 Pneumonia, organism unspecified, n = 30, 14.4%E14 Unspecified diabetes mellitus, n = 12, 5.8%A41 Other sepsis, n = 11, 5.3%L03 Cellulitis, n = 11, 5.3%N39 Other disorders of urinary systemb, n = 20, 11.2%A41 Other sepsis, n = 18, 10.1%J18 Pneumonia, organism unspecified, n = 18, 10.1%E14 Unspecified diabetes mellitus, n = 10, 5.6%J44 Other chronic obstructive pulmonary disease, n = 6, 3.4%=K55 Vascular disorders of intestine, n = 6, 3.4%=L03 Cellulitis, n = 6, 3.4%Total = 208Total = 179aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.Table 4.Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFReGFR (mL/min/1.73 m2)Deaths within 28 days of pneumonia onsetDeaths within 90 days of pneumonia onsetTotalN18 CKD, n (%)Renal disease,b
n (%)TotalN18 CKD, n (%)Renal disease,b
n (%)<1586 (75.0)7 (87.5)86 (75.0)7 (87.5)15–2910919 (17.4)41 (37.6)13926 (18.7)53 (38.1)30–4427514 (5.1)2 (15.3)34720 (5.8)56 (16.1)35–593328 (2.4)27 (8.1)41910 (2.4)36 (8.6)Total72447 (6.5)77 (10.6)91362 (6.8)152 (16.6)aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.
Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)a
aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.
bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.
Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFR
aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.
bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.
| null | null |
[
"Data sources",
"Study population",
"Definition of infections",
"Study outcomes",
"Definition of CKD",
"Other variables",
"Data analysis",
"Ethics"
] |
[
"The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].",
"The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.",
"Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.",
"The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.",
"CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.",
"Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.",
"Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.",
"The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116)."
] |
[
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"MATERIALS AND METHODS",
"Data sources",
"Study population",
"Definition of infections",
"Study outcomes",
"Definition of CKD",
"Other variables",
"Data analysis",
"Ethics",
"RESULTS",
"DISCUSSION"
] |
[
"Chronic kidney disease (CKD) affects an estimated 1.8 million people in England, 98% of whom do not require renal replacement therapy [1]. CKD is defined by reduced estimated glomerular filtration rate (eGFR) or evidence of kidney damage such as proteinuria and is commonest among older people [2, 3]. Most patients with CKD are managed in primary care [4].\nInfection is an important cause of mortality among older people [5, 6]. Both reduced eGFR and proteinuria are associated with an increased rate of infection-related mortality, which could be partly explained by increased incidence of infection [7–9]. It is less clear whether CKD is also associated with poorer prognosis following infection. When clinicians assess patients with community-acquired infection, developing complications such as acute kidney injury (AKI) may not yet be apparent, but they will know which patients have pre-existing CKD. If pre-existing CKD is a risk marker for short-term mortality, this would be useful for risk stratification and clinical management of patients with infections, especially in primary care where clinicians may not have access to immediate laboratory tests. While the implications of acute changes in eGFR during infection are a focus of current research, few studies have investigated the role of pre-existing CKD [10]. Low baseline eGFR has been found to be associated with mortality following sepsis and community-acquired pneumonia, but rarely examined according to clinically meaningful categories of eGFR [11–15]. To the best of our knowledge, proteinuria has not been examined as a potential risk marker for mortality following infection [12, 15, 16].\nAmong older people, CKD frequently co-exists with other co-morbidities [3]. An association of CKD with poor prognosis of infections could thus be due to confounding from these co-morbidities. For example, CKD is strongly associated with cardiovascular disease, which may be complicated by infection, resulting in post-infection mortality driven by the underlying cardiovascular disease [17]. Such deaths would largely follow hospitalization for cardiovascular events. CKD is associated with healthcare-associated pneumonia, which carries a worse prognosis than community-acquired pneumonia [18, 19]. Focusing on community-acquired infections should exclude infections arising as short-term sequelae of cardiovascular events and improve understanding of the relationship between pre-existing CKD markers and infection prognosis.\nOlder people with diabetes mellitus form a large and growing population in primary care who suffer a high incidence of community-acquired pneumonia and sepsis [20]. Forty per cent of adults with diabetes have CKD, of whom three-quarters have proteinuria, and CKD among these patients is associated with a greater all-cause excess mortality than among patients without diabetes [21]. If proteinuria is a risk marker for mortality among older patients with diabetes who develop community-acquired pneumonia or sepsis, this could inform clinical management of a large primary care patient population with appreciable mortality following infection [20].\nThis study aimed to examine whether baseline eGFR and proteinuria were independent risk markers for short-term mortality following community-acquired pneumonia or sepsis among older people with diabetes mellitus, using large, linked electronic health record databases.",
" Data sources The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\nThe Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].\n Study population The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\nThe study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.\n Definition of infections Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\nInfection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.\n Study outcomes The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\nThe outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.\n Definition of CKD CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\nCKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.\n Other variables Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\nAge was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.\n Data analysis Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\nPneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.\n Ethics The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).\nThe study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).",
"The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].\nData linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].",
"The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.\nA valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.",
"Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.\nHospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.",
"The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.",
"CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.\nClinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].\nWe aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].\nA history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.",
"Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.",
"Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.\nWe described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.\nWe focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].\nCauses of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.\nStata version 13.1 was used for data analysis. All code lists are available on request.",
"The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).",
"We identified 4957 individuals with community-acquired pneumonia and 1114 individuals with community-acquired sepsis. Data were missing (for smoking status and/or HbA1C results) for 212/4957 individuals with pneumonia (4.3%) and 56/1114 individuals with sepsis (5.0%). These patients were excluded. Among patients with pneumonia, patients with missing data were older (median age 83 years, IQR: 78–88) compared with those included (median age 80 years, IQR: 74–85), with a higher 28-day mortality (excluded 88/214, 41.1%: included 1406/4743, 29.6%) but a similar distribution of baseline eGFR. A similar pattern was seen for sepsis.\nEstimated GFR was based on the higher of two results for 4029 patients with pneumonia (84.9%) and 919 patients with sepsis (86.9%); for the remaining patients, only a single valid serum creatinine result was available. CKD prevalence was high: almost half of the patients had eGFR <60 mL/min/1.73 m2 and a third had proteinuria. Patients with eGFR <60 mL/min/1.73 m2 were older, with a higher prevalence of ischaemic heart disease and congestive heart failure than patients with eGFR ≥60 mL/min/1.73 m2 (Table 1).Table 1.Baseline characteristics of the study populationPneumoniaSepsiseGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2eGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2Age (years) Median (IQR)82 (77–87)78 (72–83)81 (75–86)76 (71–82)n (col %)n (col %)n (col %)n (col %)Gender Female1231 (53.0)1106 (42.0)294 (55.7)268 (45.7)Onset prior to 1 April 2004584 (25.1)527 (20.0)124 (23.5)120 (20.5)Socio-economic status (IMD quintile)a 1 (least deprived)383 (16.5)438 (16.6)109 (20.6)106 (18.1) 2554 (23.8)595 (22.6)116 (22.0)127 (21.7) 3494 (21.3)582 (22.1)120 (22.7)137 (23.4) 4502 (21.6)574 (21.8)110 (20.8)108 (18.4) 5 (most deprived)391 (16.8)444 (16.9)73 (13.8)108 (18.4)Smoking status Current321 (13.8)549 (20.9)70 (13.3)103 (17.6) Ex-smoker1185 (51.0)1351 (51.3)235 (44.5)284 (48.5) Non-smoker776 (33.4)698 (26.5)207 (39.2)193 (32.9) Missing42 (1.8)35 (1.3)16 (3.0)6 (1.0)Comorbidities Chronic lung disease503 (21.6)686 (26.1)59 (11.2)95 (16.2) Hypertension1623 (69.8)1662 (63.1)372 (70.5)381 (65.0) Congestive heart failure734 (31.6)430 (16.3)147 (27.8)95 (16.2) Ischaemic heart disease966 (41.6)887 (33.7)223 (42.2)207 (35.3) Cerebrovascular disease692 (29.8)638 (24.2)152 (28.8)145 (24.7) Other dementia174 (7.5)190 (7.2)42 (8.0)23 (3.9) Cancer382 (16.4)474 (18.0)99 (18.8)120 (20.5) Connective tissue disorders228 (9.8)215 (8.2)36 (6.8)52 (8.9)HbA1C Good <7%1176 (50.6)1401 (53.2)251 (47.5)308 (52.6) Borderline 7–10%966 (41.6)1033 (39.2)213 (40.3)230 (39.3) Poor >10%108 (4.7)131 (5.0)41 (7.8)34 (5.8) None recorded74 (3.2)68 (2.6)23 (4.4)14 (2.4)Prior antidiabetes medication Insulin122 (5.3)126 (4.8)34 (6.4)23 (3.9) Oral medications1139 (49.0)1426 (54.2)247 (46.8)312 (53.2) Both462 (19.9)384 (14.6)130 (24.6)114 (19.5) None601 (25.9)697 (26.5)117 (22.2)137 (23.4) Total23242633528586eGFR, estimated glomerular filtration rate.aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nBaseline characteristics of the study population\neGFR, estimated glomerular filtration rate.\naIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.\nPatients with pneumonia experienced 29.6% 28-day all-cause mortality (1406 deaths). Patients with sepsis experienced 35.6% 28-day all-cause mortality (377 deaths) (Table 2). Survival curves showed high mortality at infection onset, declining over ∼30 days to a more stable rate for the next 60 days following both pneumonia and sepsis (Figure 1). RRs for 28-day mortality were higher among people with eGFR <30 mL/min/1.73 m2 compared with people with eGFR ≥60 mL/min/1.73 m2 for pneumonia (RR = 1.27; 95% CI 1.10–1.47) and sepsis (RR = 1.42; 1.10–1.84), adjusted for age, sex, socio-economic status and onset prior to April 2004. Adjustment for smoking status, co-morbidities and characteristics of diabetes had minimal effect on these RRs for pneumonia (fully adjusted RR = 1.27; 1.12–1.43) or sepsis (fully adjusted RR = 1.32; 1.07–1.64). There was no evidence of associations between intermediate levels of eGFR and 28-day mortality for either infection, nor for an association between proteinuria and 28-day mortality. The pattern of associations of eGFR and proteinuria with 90-day mortality was similar to those for 28-day mortality (Table 2). Results were unchanged by additional adjustment for peripheral vascular disease. There was no good evidence of effect modification between eGFR and proteinuria, and in particular no evidence of any association of proteinuria with 28-day mortality within any category of eGFR status (data not shown).Table 2.Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)aNumber (column %)28-Day mortality (row %)90-Day mortality (row %)RRs for 28-day mortality (95% CI)RRs for 90-day mortality (95% CI)Adjusted for demographicsbadjusted for non-CVD co-morbiditiescFully adjusteddAdjusted for demographicsbAdjusted for non-CVD co-morbiditiescFully adjusteddPneumonia Proteinuria (adjusted for eGFR) Yes1611 (34.0)499 (31.0)625 (38.8)1.07 (0.97–1.17)1.07 (0.98–1.18)1.07 (0.98–1.18)1.05 (0.97–1.13)1.06 (0.98–1.14)1.05 (0.98–1.14) No3132 (66.0)907 (29.0)1150 (36.7)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<1523 (0.5)12 (52.2)12 (52.2)1.27 (1.10–1.47)1.31 (1.13–1.52)1.30 (1.12–1.52)1.26 (1.12–1.42)1.29 (1.14–1.45)1.27 (1.12–1.43)15–29263 (5.6)110 (41.8)139 (52.9) 30–44764 (16.1)265 (34.7)336 (44.0)0.98 (0.86–1.10)0.99 (0.88–1.13)0.99 (0.88–1.12)1.02 (0.93–1.13)1.04 (0.94–1.15)1.03 (0.94–1.14) 45–601162 (24.5)332 (28.6)418 (36.0)0.91 (0.82–1.02)0.94 (0.84–1.04)0.93 (0.84–1.04)0.91 (0.83–1.00)0.93 (0.85–1.02)0.92 (0.84–1.01) ≥602531 (53.4)687 (27.1)870 (34.4)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total47431406 (29.6)1775 (37.4)Sepsis Proteinuria (adjusted for eGFR) Yes358 (33.8)128 (35.8)159 (44.4)0.98 (0.82–1.17)1.01 (0.85–1.21)1.01 (0.85–1.21)0.98 (0.86–1.13)0.96 (0.84–1.10)0.96 (0.84–1.10) No700 (66.2)249 (35.6)309 (44.1)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<158 (0.8)2 (25.0)5 (62.5)1.42 (1.10–1.84)1.41 (1.08–1.84)1.37 (1.05–1.79)1.39 (1.14–1.70)1.32 (1.07–1.63)1.32 (1.07–1.64)15–2962 (5.9)32 (51.6)39 (62.9) 30–44190 (18.0)88 (46.3)103 (54.2)1.25 (1.01–1.55)1.24 (0.99–1.55)1.24 (0.99–1.54)1.14 (0.96–1.36)1.12 (0.94–1.33)1.11 (0.94–1.32) 45–60232 (21.9)76 (32.8)95 (41.0)0.95 (0.75–1.19)0.91 (0.72–1.15)0.91 (0.72–1.14)0.92 (0.77–1.11)0.89 (0.74–1.07)0.89 (0.75–1.07) ≥60566 (53.5)179 (31.6)226 (39.9)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total1058377 (35.6)468 (44.2)aExcluding patients with missing smoking or HbA1C data.bAge, gender, socio-economic status, onset prior to 1 April 2004.cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.FIGURE 1:Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nShort-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)a\naExcluding patients with missing smoking or HbA1C data.\nbAge, gender, socio-economic status, onset prior to 1 April 2004.\ncAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.\ndAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.\nSurvival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.\nThe underlying causes of death following pneumonia and sepsis were similar for patients with eGFR above and below 60 mL/min/1.73 m2. Causes of 28-day mortality following sepsis were predominantly sources of infection (Table 3). Following pneumonia onset, pneumonia was recorded as an underlying or contributory cause of death for 83.9% (1191/1419) of deaths within 28 days and 76.3% (1366/1790) of deaths within 90 days. Among patients with eGFR <60 mL/min/1.73 m2, renal disease was recorded as an underlying or contributory cause for 10.6% (77/724) of those who died within 28 days of pneumonia onset and 16.6% (152/913) of those who died within 90 days. Recording of CKD as a cause of death increased with lower eGFR (Table 4).Table 3.Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)aeGFReGFR <60 mL/min/1.73 m2eGFR ≥60 mL/min/1.73 m228-Day mortality following pneumonia, n = 1419J18 Pneumonia, organism unspecified, n = 256, 35.4%J44 Other chronic obstructive pulmonary disease, n = 63, 8.7%I25 Chronic ischaemic heart disease, n = 47, 6.5%I50 Heart failure, n = 43, 5.9%I64 Stroke, not specified as haemorrhage or infarction, n = 37, 5.1%J18 Pneumonia, organism unspecified, n = 216, 31.1%J44 Other chronic obstructive pulmonary disease, n = 63, 9.1%I64 Stroke, not specified as haemorrhage or infarction, n = 35, 5.0%C34 Malignant neoplasm of bronchus and lung, n = 34, 4.9%I25 Chronic ischaemic heart disease, n = 29, 4.2%Total = 724Total = 69529–90 Day mortality following pneumonia, n = 371J18 Pneumonia, organism unspecified, n = 38, 20.1%I25 Chronic ischaemic heart disease, n = 24, 12.7%J44 Other chronic obstructive pulmonary disease, n = 13, 6.9%C34 Malignant neoplasm of bronchus and lung, n = 12, 6.4%I21 Acute myocardial infarction, n = 12, 6.4%J18 Pneumonia, organism unspecified, n = 20, 11.0%C34 Malignant neoplasm of bronchus and lung, n = 20, 11.0%J44 Other chronic obstructive pulmonary disease, n = 19, 10.4%I25 Chronic ischaemic heart disease, n = 9, 5.0%I64 Stroke, not specified as haemorrhage or infarction, n = 9, 5.0%Total = 189Total = 18228-Day mortality following sepsis, n = 387N39 Other disorders of urinary systemb, n = 33, 15.9%J18 Pneumonia, organism unspecified, n = 30, 14.4%E14 Unspecified diabetes mellitus, n = 12, 5.8%A41 Other sepsis, n = 11, 5.3%L03 Cellulitis, n = 11, 5.3%N39 Other disorders of urinary systemb, n = 20, 11.2%A41 Other sepsis, n = 18, 10.1%J18 Pneumonia, organism unspecified, n = 18, 10.1%E14 Unspecified diabetes mellitus, n = 10, 5.6%J44 Other chronic obstructive pulmonary disease, n = 6, 3.4%=K55 Vascular disorders of intestine, n = 6, 3.4%=L03 Cellulitis, n = 6, 3.4%Total = 208Total = 179aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.Table 4.Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFReGFR (mL/min/1.73 m2)Deaths within 28 days of pneumonia onsetDeaths within 90 days of pneumonia onsetTotalN18 CKD, n (%)Renal disease,b\nn (%)TotalN18 CKD, n (%)Renal disease,b\nn (%)<1586 (75.0)7 (87.5)86 (75.0)7 (87.5)15–2910919 (17.4)41 (37.6)13926 (18.7)53 (38.1)30–4427514 (5.1)2 (15.3)34720 (5.8)56 (16.1)35–593328 (2.4)27 (8.1)41910 (2.4)36 (8.6)Total72447 (6.5)77 (10.6)91362 (6.8)152 (16.6)aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.\nTop five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)a\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.\nRecording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFR\naDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.\nbAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.",
"Among this population of older people with diabetes mellitus, eGFR <30 mL/min/1.73 m2 was a risk marker of higher 28- and 90-day mortality following community-acquired pneumonia and sepsis, compared with patients with eGFR ≥60 mL/min/1.73 m2. The relationship between eGFR and mortality did not change with adjustment for co-morbidities. Neither moderately impaired eGFR nor proteinuria was associated with higher short-term mortality following either infection.\nThe strengths of this study follow from the analysis of a focused question using large, linked datasets for a highly monitored primary care population with a cohort study design. Our study identifies that the association between eGFR and post-infection mortality persists when patients with end-stage renal disease (ESRD) are excluded (and is not explained by renal replacement therapy), when considering fixed-term rather than in-hospital mortality (thus is not due to differences in hospital stay) and when exclusively community-acquired infections are considered (so does not result from increased risk of healthcare-associated infections). The linked datasets allowed us to identify infections both among patients presenting directly to hospital and those managed in the community, maximized ascertainment of mortality and enabled description of the causes of death. The highly monitored population allowed good ascertainment of CKD status. The cohort study design has less potential for selection bias than an equivalent case–control study.\nA limitation is our assumption that the absence of a record implies a negative status for proteinuria and co-morbidities. Under-ascertainment of co-morbidities could result in residual confounding, with unpredictable effects, but the high prevalence of co-morbidities observed suggests that ascertainment was not markedly incomplete. We observed a high prevalence of proteinuria, and this is a highly monitored population (with financial incentives for standardized recording of proteinuria since 2004), but under-ascertainment of proteinuria could result in underestimation of any association between proteinuria and mortality [24]. Residual confounding from undiagnosed cardiovascular disease should have been minimized by adjustment for cardiovascular disease risk factors including smoking, hypertension and characteristics of diabetes.\nOur findings for eGFR provide further detail to build on previous findings that baseline eGFR <60 mL/min/1.73 m2 or renal disease are risk factors for short-term mortality following (hospital- or community-acquired) sepsis and for in-hospital mortality following community-acquired pneumonia (including patients receiving dialysis) [11–15]. A more comparable Canadian study examined the associations between eGFR and 30-day mortality following community-acquired pneumonia among the general population aged ≥65 years, excluding patients with ESRD [16]. Fully adjusted hazard ratios for 30-day mortality were 1.22 (95% CI 1.01–1.49) for eGFR 45–59, 2.03 (1.64–2.50) for eGFR 30–44 and 4.94 (3.94–6.19) for eGFR < 30, compared with eGFR 60–104 mL/min/1.73 m2. These are somewhat greater than the associations we observed. The difference may be explained by the different study populations. Both studies required a baseline serum creatinine result for inclusion. Our study population of older people with diabetes were routinely monitored for CKD (with financial incentivization in primary care) [32]. Creatinine testing of the Canadian study population may have been encouraged by co-morbidities or health-behaviours associated with CKD (such as smoking), which increase post-infection mortality, resulting in over-estimation of the association of eGFR and post-infection mortality. Our study population is less vulnerable to differential ascertainment of CKD. Alternatively, the association between eGFR and post-infection mortality may be smaller among patients with diabetes.\nTo the best of our knowledge, our examination of any association between proteinuria and mortality following community-acquired pneumonia and sepsis is novel. A history of proteinuria, although a marker for mortality in general, does not appear to be a risk marker for short-term mortality following community-acquired infection. This is unlikely to be due to chance, as the study was large, with findings consistent across both infections. We designed our study to produce conservative estimates and may have under-estimated the association between proteinuria and post-infection mortality due to under-ascertainment of proteinuria. Alternatively, any potential relationship between proteinuria and mortality may have been mitigated by clinical care of patients with infection who had pre-existing proteinuria, for example, through swift recognition of AKI.\nThe survival curves demonstrate a steep initial mortality following infection onset, and a high proportion of deaths had the underlying cause assigned to infection. Since 2001 in England, co-morbidities are assigned as the underlying cause of death when pneumonia has occurred in the context of, for example, malignancy or respiratory disease [25]. This suggests that the associations we observed are driven by an association between eGFR and infection prognosis, not merely high underlying baseline mortality among patients with impaired eGFR. This is supported by previous research which found 7.7-fold elevated mortality in the 30 days following community-acquired pneumonia [19]. Estimates for associations between eGFR and mortality were not substantially altered by adjustment for co-morbidities, suggesting that any causal relationship between eGFR and mortality is not mediated through co-morbidities.\nOur findings apply to the large population of older patients with community-acquired pneumonia or sepsis with diabetes mellitus who do not have ESRD. Inclusion criteria are unlikely to have limited generalizability appreciably. Practices which consent to data linkage could be more research oriented, providing good primary care management of risk factors for infection-related mortality (such as smoking cessation), but this is unlikely to affect the relationship between CKD and short-term mortality post-infection. Lack of pre-existing creatinine test results is likely to reflect limited time potentially eligible for the study rather than CKD status among this highly monitored population. Missing data on smoking status and HbA1C may be a marker of low patient engagement: caution should be used in generalizing our results to patients who are not actively managed in primary care.\nWe found that CKD is a useful clinical risk marker for post-infectious mortality. Whether this relationship is causal is less clear; but the association does not appear to be explained by age, co-morbidities or hospital attendance. Potential mechanisms include immune system dysfunction, but also more preventable complications such as AKI. Combinations of risk factors may be important: for example, patients with post-operative AKI have higher mortality if they also have pre-existing CKD [30].\nOur results have implications for patient management and future research. Patients with baseline eGFR <30 mL/min/1.73 m2 and community-acquired infection need careful monitoring, particularly in the 28 days following infection. Future research should investigate preventable mechanisms by which low baseline eGFR could be related to post-infection mortality, for example, fluid management, AKI and drug dosage in patients with low renal clearance."
] |
[
"intro",
"materials|methods",
null,
null,
null,
null,
null,
null,
null,
null,
"results",
"discussion"
] |
[
"chronic kidney disease",
"community-acquired infections",
"electronic health records",
"infection/mortality",
"proteinuria"
] |
INTRODUCTION:
Chronic kidney disease (CKD) affects an estimated 1.8 million people in England, 98% of whom do not require renal replacement therapy [1]. CKD is defined by reduced estimated glomerular filtration rate (eGFR) or evidence of kidney damage such as proteinuria and is commonest among older people [2, 3]. Most patients with CKD are managed in primary care [4].
Infection is an important cause of mortality among older people [5, 6]. Both reduced eGFR and proteinuria are associated with an increased rate of infection-related mortality, which could be partly explained by increased incidence of infection [7–9]. It is less clear whether CKD is also associated with poorer prognosis following infection. When clinicians assess patients with community-acquired infection, developing complications such as acute kidney injury (AKI) may not yet be apparent, but they will know which patients have pre-existing CKD. If pre-existing CKD is a risk marker for short-term mortality, this would be useful for risk stratification and clinical management of patients with infections, especially in primary care where clinicians may not have access to immediate laboratory tests. While the implications of acute changes in eGFR during infection are a focus of current research, few studies have investigated the role of pre-existing CKD [10]. Low baseline eGFR has been found to be associated with mortality following sepsis and community-acquired pneumonia, but rarely examined according to clinically meaningful categories of eGFR [11–15]. To the best of our knowledge, proteinuria has not been examined as a potential risk marker for mortality following infection [12, 15, 16].
Among older people, CKD frequently co-exists with other co-morbidities [3]. An association of CKD with poor prognosis of infections could thus be due to confounding from these co-morbidities. For example, CKD is strongly associated with cardiovascular disease, which may be complicated by infection, resulting in post-infection mortality driven by the underlying cardiovascular disease [17]. Such deaths would largely follow hospitalization for cardiovascular events. CKD is associated with healthcare-associated pneumonia, which carries a worse prognosis than community-acquired pneumonia [18, 19]. Focusing on community-acquired infections should exclude infections arising as short-term sequelae of cardiovascular events and improve understanding of the relationship between pre-existing CKD markers and infection prognosis.
Older people with diabetes mellitus form a large and growing population in primary care who suffer a high incidence of community-acquired pneumonia and sepsis [20]. Forty per cent of adults with diabetes have CKD, of whom three-quarters have proteinuria, and CKD among these patients is associated with a greater all-cause excess mortality than among patients without diabetes [21]. If proteinuria is a risk marker for mortality among older patients with diabetes who develop community-acquired pneumonia or sepsis, this could inform clinical management of a large primary care patient population with appreciable mortality following infection [20].
This study aimed to examine whether baseline eGFR and proteinuria were independent risk markers for short-term mortality following community-acquired pneumonia or sepsis among older people with diabetes mellitus, using large, linked electronic health record databases.
MATERIALS AND METHODS:
Data sources The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].
Data linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].
The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].
Data linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].
Study population The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.
A valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.
The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.
A valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.
Definition of infections Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.
Hospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.
Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.
Hospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.
Study outcomes The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.
The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.
Definition of CKD CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.
Clinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].
We aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].
A history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.
CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.
Clinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].
We aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].
A history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.
Other variables Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.
Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.
Data analysis Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.
We described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.
We focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].
Causes of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.
Stata version 13.1 was used for data analysis. All code lists are available on request.
Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.
We described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.
We focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].
Causes of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.
Stata version 13.1 was used for data analysis. All code lists are available on request.
Ethics The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).
The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).
Data sources:
The Clinical Practice Research Datalink (CPRD) is an anonymized UK dataset, comprising primary care records (including diagnoses, prescriptions and test results) for 12.8 million patients in May 2011 when data were extracted. The CPRD population is representative of the general UK population and validity of recorded diagnoses is generally high [22, 23]. Monitoring of eGFR and proteinuria in primary care is standard practice for people with diabetes and has been financially incentivized by the Quality Outcomes Framework since April 2004 [24].
Data linkage is available within England subject to practice-level consent. Records of all patients in CPRD with available linkage to Office for National Statistics (ONS) mortality data formed the study dataset [25]. We additionally used linked Hospital Episodes Statistics admissions data, which were available for all patients [26].
Study population:
The study population was a subset of a population described in more detail previously [20]. It comprised people aged ≥65 years with diabetes mellitus who experienced a first community-acquired pneumonia or sepsis, with a valid serum creatinine result and no history of renal replacement therapy.
A valid serum creatinine result was one recorded in primary care after the latest time-point of diabetes diagnosis, 65th birthday, 1 year after patients' practice registration, date the practice reached CPRD quality control standards or 1 January 1998. Study exit occurred at the first time-point of death, patient leaving the practice, last data collection from the practice, last ONS data linkage date, renal replacement therapy (kidney transplant or dialysis) or 31 March 2011. Patients with a history of renal replacement therapy were excluded.
Definition of infections:
Infection was identified by a diagnostic Read code in primary care records, or a diagnostic International Classification of Disease 10 (ICD-10) code as the primary cause of hospital admission in secondary care records. The first consultation for infection was treated as the date of infection onset. Any community-acquired infection with onset at least 28 days after the first valid serum creatinine result, and before study exit, was included in the study.
Hospital-acquired infections were identified and excluded as described previously [20]. Briefly, infections were designated as hospital-acquired if onset was during or within 14 days of discharge from a hospitalization. Hospital-acquired infections continued until 28 days had passed without a diagnostic code for the infection or 28 days after hospital discharge, whichever was the later. After this, patients re-entered follow-up for community-acquired infection.
Study outcomes:
The outcomes were death from any cause recorded in ONS mortality data within 28 days (primary outcome) or within 90 days (secondary outcome) of infection onset.
Definition of CKD:
CKD was described in terms of eGFR and proteinuria, using primary care records. We estimated eGFR from serum creatinine test results using the CKD Epidemiology Collaboration (CKD-EPI) equation including adjustment for ethnicity [27]. We excluded serum creatinine results <28 days prior to infection onset to avoid misclassification of CKD status, as a developing infection could disrupt serum creatinine levels.
Clinically, CKD diagnosis is based on two GFR estimates at least 3 months apart [2]. Using a single GFR estimate can result in over-ascertainment of CKD due to creatinine fluctuation [28]. If more than one serum creatinine result was recorded between the start of patient follow-up and 28 days prior to infection onset, we used the higher eGFR from the latest two results that were at least 3 months apart, to obtain conservative estimates of eGFR [28].
We aimed to categorize eGFR according to thresholds corresponding to those used in diagnosing CKD stage. Due to the small number of outcomes in the category eGFR < 15 mL/min/1.73 m2, we collapsed Stages 4 and 5 to categorize eGFR as < 30, 30–44, 45–59 and ≥60 mL/min/1.73 m2 [2].
A history of proteinuria was defined by either a positive urine protein test result (excluding results on the same day as a urinary tract infection diagnosis) or a diagnosis of proteinuric renal disease. We did not count trace results as positive.
Other variables:
Age was defined in 5-year age-bands up to a final category of ≥85 years. Socio-economic status was assigned by quintile at an individual level, using 2007 ONS estimates of the Index of Multiple Deprivation, a composite area-level marker of deprivation [25]. If this was not available, it was supplemented by the socio-economic status for the patient's primary care practice. Smoking status was defined by the most recent record before infection onset when available, otherwise by the first subsequent record. Non-cardiovascular co-morbidities (chronic lung disease, dementia, cancer, connective tissue disorders, hypertension and cerebrovascular disease) and cardiovascular co-morbidities (congestive heart failure and ischaemic heart disease) were defined by diagnostic CPRD Read codes, and diabetic medication history by CPRD prescription records prior to infection onset. HbA1C test results within 28 days before infection were excluded as these could reflect disturbed glycaemic control during the early stages of infection.
Data analysis:
Pneumonia and sepsis analyses were conducted separately. A patient could be included in both the pneumonia and sepsis analyses if they experienced both infections, but not for multiple episodes of either pneumonia or sepsis. We excluded patients with no smoking status or HbA1C result available.
We described mortality using Kaplan–Meier survival curves stratified by eGFR status. We calculated risk ratios (RRs) using a generalized linear model with log link, normal distribution and robust standard errors, according to a pre-specified analysis plan [29]. Our first model adjusted for age, sex, socio-economic status and infection onset prior to 1 April 2004 (when Quality Outcomes Framework guidelines financially incentivizing recording of CKD status among people with diabetes in primary care were introduced) [24]. Our second model adjusted for confounding by smoking status, characteristics of diabetes (HbA1C and diabetic medication history) and non-cardiovascular co-morbidities. Our final model additionally adjusted for congestive heart failure and ischaemic heart disease, which could confound or mediate an association between CKD and post-infection mortality. We repeated the final model with additional adjustment for peripheral vascular disease as a sensitivity analysis. We looked for effect modification between eGFR and proteinuria in the final model.
We focused on whether pre-existing CKD was a risk marker for short-term mortality following infection. Data on acute electrolyte changes during infection were not routinely available, and so potential causal mechanisms such as AKI could not be explored [30].
Causes of death in ONS mortality data are recorded using ICD-10 codes from 1 January 2001, and ICD-9 codes prior to this, which are not easily comparable [31]. We therefore described cause of death among patients who died after 1 January 2001.
Stata version 13.1 was used for data analysis. All code lists are available on request.
Ethics:
The study was approved by the Independent Scientific Advisory Group of the CPRD (ISAC reference 11_033A) and the London School of Hygiene & Tropical Medicine Ethics Committee (LSHTM reference 6116).
RESULTS:
We identified 4957 individuals with community-acquired pneumonia and 1114 individuals with community-acquired sepsis. Data were missing (for smoking status and/or HbA1C results) for 212/4957 individuals with pneumonia (4.3%) and 56/1114 individuals with sepsis (5.0%). These patients were excluded. Among patients with pneumonia, patients with missing data were older (median age 83 years, IQR: 78–88) compared with those included (median age 80 years, IQR: 74–85), with a higher 28-day mortality (excluded 88/214, 41.1%: included 1406/4743, 29.6%) but a similar distribution of baseline eGFR. A similar pattern was seen for sepsis.
Estimated GFR was based on the higher of two results for 4029 patients with pneumonia (84.9%) and 919 patients with sepsis (86.9%); for the remaining patients, only a single valid serum creatinine result was available. CKD prevalence was high: almost half of the patients had eGFR <60 mL/min/1.73 m2 and a third had proteinuria. Patients with eGFR <60 mL/min/1.73 m2 were older, with a higher prevalence of ischaemic heart disease and congestive heart failure than patients with eGFR ≥60 mL/min/1.73 m2 (Table 1).Table 1.Baseline characteristics of the study populationPneumoniaSepsiseGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2eGFR < 60 mL/min/1.73 m2eGFR ≥ 60 mL/min/1.73 m2Age (years) Median (IQR)82 (77–87)78 (72–83)81 (75–86)76 (71–82)n (col %)n (col %)n (col %)n (col %)Gender Female1231 (53.0)1106 (42.0)294 (55.7)268 (45.7)Onset prior to 1 April 2004584 (25.1)527 (20.0)124 (23.5)120 (20.5)Socio-economic status (IMD quintile)a 1 (least deprived)383 (16.5)438 (16.6)109 (20.6)106 (18.1) 2554 (23.8)595 (22.6)116 (22.0)127 (21.7) 3494 (21.3)582 (22.1)120 (22.7)137 (23.4) 4502 (21.6)574 (21.8)110 (20.8)108 (18.4) 5 (most deprived)391 (16.8)444 (16.9)73 (13.8)108 (18.4)Smoking status Current321 (13.8)549 (20.9)70 (13.3)103 (17.6) Ex-smoker1185 (51.0)1351 (51.3)235 (44.5)284 (48.5) Non-smoker776 (33.4)698 (26.5)207 (39.2)193 (32.9) Missing42 (1.8)35 (1.3)16 (3.0)6 (1.0)Comorbidities Chronic lung disease503 (21.6)686 (26.1)59 (11.2)95 (16.2) Hypertension1623 (69.8)1662 (63.1)372 (70.5)381 (65.0) Congestive heart failure734 (31.6)430 (16.3)147 (27.8)95 (16.2) Ischaemic heart disease966 (41.6)887 (33.7)223 (42.2)207 (35.3) Cerebrovascular disease692 (29.8)638 (24.2)152 (28.8)145 (24.7) Other dementia174 (7.5)190 (7.2)42 (8.0)23 (3.9) Cancer382 (16.4)474 (18.0)99 (18.8)120 (20.5) Connective tissue disorders228 (9.8)215 (8.2)36 (6.8)52 (8.9)HbA1C Good <7%1176 (50.6)1401 (53.2)251 (47.5)308 (52.6) Borderline 7–10%966 (41.6)1033 (39.2)213 (40.3)230 (39.3) Poor >10%108 (4.7)131 (5.0)41 (7.8)34 (5.8) None recorded74 (3.2)68 (2.6)23 (4.4)14 (2.4)Prior antidiabetes medication Insulin122 (5.3)126 (4.8)34 (6.4)23 (3.9) Oral medications1139 (49.0)1426 (54.2)247 (46.8)312 (53.2) Both462 (19.9)384 (14.6)130 (24.6)114 (19.5) None601 (25.9)697 (26.5)117 (22.2)137 (23.4) Total23242633528586eGFR, estimated glomerular filtration rate.aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.
Baseline characteristics of the study population
eGFR, estimated glomerular filtration rate.
aIndex of multiple deprivation (IMD) score for patient's postcode where available, otherwise, practice-level IMD score.
Patients with pneumonia experienced 29.6% 28-day all-cause mortality (1406 deaths). Patients with sepsis experienced 35.6% 28-day all-cause mortality (377 deaths) (Table 2). Survival curves showed high mortality at infection onset, declining over ∼30 days to a more stable rate for the next 60 days following both pneumonia and sepsis (Figure 1). RRs for 28-day mortality were higher among people with eGFR <30 mL/min/1.73 m2 compared with people with eGFR ≥60 mL/min/1.73 m2 for pneumonia (RR = 1.27; 95% CI 1.10–1.47) and sepsis (RR = 1.42; 1.10–1.84), adjusted for age, sex, socio-economic status and onset prior to April 2004. Adjustment for smoking status, co-morbidities and characteristics of diabetes had minimal effect on these RRs for pneumonia (fully adjusted RR = 1.27; 1.12–1.43) or sepsis (fully adjusted RR = 1.32; 1.07–1.64). There was no evidence of associations between intermediate levels of eGFR and 28-day mortality for either infection, nor for an association between proteinuria and 28-day mortality. The pattern of associations of eGFR and proteinuria with 90-day mortality was similar to those for 28-day mortality (Table 2). Results were unchanged by additional adjustment for peripheral vascular disease. There was no good evidence of effect modification between eGFR and proteinuria, and in particular no evidence of any association of proteinuria with 28-day mortality within any category of eGFR status (data not shown).Table 2.Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)aNumber (column %)28-Day mortality (row %)90-Day mortality (row %)RRs for 28-day mortality (95% CI)RRs for 90-day mortality (95% CI)Adjusted for demographicsbadjusted for non-CVD co-morbiditiescFully adjusteddAdjusted for demographicsbAdjusted for non-CVD co-morbiditiescFully adjusteddPneumonia Proteinuria (adjusted for eGFR) Yes1611 (34.0)499 (31.0)625 (38.8)1.07 (0.97–1.17)1.07 (0.98–1.18)1.07 (0.98–1.18)1.05 (0.97–1.13)1.06 (0.98–1.14)1.05 (0.98–1.14) No3132 (66.0)907 (29.0)1150 (36.7)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<1523 (0.5)12 (52.2)12 (52.2)1.27 (1.10–1.47)1.31 (1.13–1.52)1.30 (1.12–1.52)1.26 (1.12–1.42)1.29 (1.14–1.45)1.27 (1.12–1.43)15–29263 (5.6)110 (41.8)139 (52.9) 30–44764 (16.1)265 (34.7)336 (44.0)0.98 (0.86–1.10)0.99 (0.88–1.13)0.99 (0.88–1.12)1.02 (0.93–1.13)1.04 (0.94–1.15)1.03 (0.94–1.14) 45–601162 (24.5)332 (28.6)418 (36.0)0.91 (0.82–1.02)0.94 (0.84–1.04)0.93 (0.84–1.04)0.91 (0.83–1.00)0.93 (0.85–1.02)0.92 (0.84–1.01) ≥602531 (53.4)687 (27.1)870 (34.4)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total47431406 (29.6)1775 (37.4)Sepsis Proteinuria (adjusted for eGFR) Yes358 (33.8)128 (35.8)159 (44.4)0.98 (0.82–1.17)1.01 (0.85–1.21)1.01 (0.85–1.21)0.98 (0.86–1.13)0.96 (0.84–1.10)0.96 (0.84–1.10) No700 (66.2)249 (35.6)309 (44.1)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) eGFR (mL/min/1.73 m2) (adjusted for proteinuria) <30<158 (0.8)2 (25.0)5 (62.5)1.42 (1.10–1.84)1.41 (1.08–1.84)1.37 (1.05–1.79)1.39 (1.14–1.70)1.32 (1.07–1.63)1.32 (1.07–1.64)15–2962 (5.9)32 (51.6)39 (62.9) 30–44190 (18.0)88 (46.3)103 (54.2)1.25 (1.01–1.55)1.24 (0.99–1.55)1.24 (0.99–1.54)1.14 (0.96–1.36)1.12 (0.94–1.33)1.11 (0.94–1.32) 45–60232 (21.9)76 (32.8)95 (41.0)0.95 (0.75–1.19)0.91 (0.72–1.15)0.91 (0.72–1.14)0.92 (0.77–1.11)0.89 (0.74–1.07)0.89 (0.75–1.07) ≥60566 (53.5)179 (31.6)226 (39.9)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference)1 (reference) Total1058377 (35.6)468 (44.2)aExcluding patients with missing smoking or HbA1C data.bAge, gender, socio-economic status, onset prior to 1 April 2004.cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.FIGURE 1:Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.
Short-term mortality by CKD status (n = 4743 for pneumonia, n = 1058 for sepsis)a
aExcluding patients with missing smoking or HbA1C data.
bAge, gender, socio-economic status, onset prior to 1 April 2004.
cAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C.
dAge, gender, socio-economic status, onset prior to 1 April 2004, smoking status, chronic lung disease, dementia, cancer, connective tissue disorders, hypertension, cerebrovascular disease, diabetes medications, latest HbA1C, congestive heart failure, ischaemic heart disease.
Survival curves of short-term mortality following infection onset by eGFR status for (A) pneumonia and (B) sepsis.
The underlying causes of death following pneumonia and sepsis were similar for patients with eGFR above and below 60 mL/min/1.73 m2. Causes of 28-day mortality following sepsis were predominantly sources of infection (Table 3). Following pneumonia onset, pneumonia was recorded as an underlying or contributory cause of death for 83.9% (1191/1419) of deaths within 28 days and 76.3% (1366/1790) of deaths within 90 days. Among patients with eGFR <60 mL/min/1.73 m2, renal disease was recorded as an underlying or contributory cause for 10.6% (77/724) of those who died within 28 days of pneumonia onset and 16.6% (152/913) of those who died within 90 days. Recording of CKD as a cause of death increased with lower eGFR (Table 4).Table 3.Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)aeGFReGFR <60 mL/min/1.73 m2eGFR ≥60 mL/min/1.73 m228-Day mortality following pneumonia, n = 1419J18 Pneumonia, organism unspecified, n = 256, 35.4%J44 Other chronic obstructive pulmonary disease, n = 63, 8.7%I25 Chronic ischaemic heart disease, n = 47, 6.5%I50 Heart failure, n = 43, 5.9%I64 Stroke, not specified as haemorrhage or infarction, n = 37, 5.1%J18 Pneumonia, organism unspecified, n = 216, 31.1%J44 Other chronic obstructive pulmonary disease, n = 63, 9.1%I64 Stroke, not specified as haemorrhage or infarction, n = 35, 5.0%C34 Malignant neoplasm of bronchus and lung, n = 34, 4.9%I25 Chronic ischaemic heart disease, n = 29, 4.2%Total = 724Total = 69529–90 Day mortality following pneumonia, n = 371J18 Pneumonia, organism unspecified, n = 38, 20.1%I25 Chronic ischaemic heart disease, n = 24, 12.7%J44 Other chronic obstructive pulmonary disease, n = 13, 6.9%C34 Malignant neoplasm of bronchus and lung, n = 12, 6.4%I21 Acute myocardial infarction, n = 12, 6.4%J18 Pneumonia, organism unspecified, n = 20, 11.0%C34 Malignant neoplasm of bronchus and lung, n = 20, 11.0%J44 Other chronic obstructive pulmonary disease, n = 19, 10.4%I25 Chronic ischaemic heart disease, n = 9, 5.0%I64 Stroke, not specified as haemorrhage or infarction, n = 9, 5.0%Total = 189Total = 18228-Day mortality following sepsis, n = 387N39 Other disorders of urinary systemb, n = 33, 15.9%J18 Pneumonia, organism unspecified, n = 30, 14.4%E14 Unspecified diabetes mellitus, n = 12, 5.8%A41 Other sepsis, n = 11, 5.3%L03 Cellulitis, n = 11, 5.3%N39 Other disorders of urinary systemb, n = 20, 11.2%A41 Other sepsis, n = 18, 10.1%J18 Pneumonia, organism unspecified, n = 18, 10.1%E14 Unspecified diabetes mellitus, n = 10, 5.6%J44 Other chronic obstructive pulmonary disease, n = 6, 3.4%=K55 Vascular disorders of intestine, n = 6, 3.4%=L03 Cellulitis, n = 6, 3.4%Total = 208Total = 179aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.Table 4.Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFReGFR (mL/min/1.73 m2)Deaths within 28 days of pneumonia onsetDeaths within 90 days of pneumonia onsetTotalN18 CKD, n (%)Renal disease,b
n (%)TotalN18 CKD, n (%)Renal disease,b
n (%)<1586 (75.0)7 (87.5)86 (75.0)7 (87.5)15–2910919 (17.4)41 (37.6)13926 (18.7)53 (38.1)30–4427514 (5.1)2 (15.3)34720 (5.8)56 (16.1)35–593328 (2.4)27 (8.1)41910 (2.4)36 (8.6)Total72447 (6.5)77 (10.6)91362 (6.8)152 (16.6)aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.
Top five underlying causes of death by ICD-10 code for short-term mortality following pneumonia and sepsis (deaths after 2001)a
aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.
bAll incidences of code N39 were N39.0 Urinary tract infection, site not specified.
Recording of renal disease as a cause of deatha following pneumonia among patients with reduced eGFR
aDeaths prior to 2001 were recorded using ICD-9 codes and have not been included.
bAny ICD-10 code from Chapter XIV ‘Diseases of the genitourinary system’ except N10 ‘Acute tubule-interstitial nephritis’, which is used for pyelonephritis, N30 ‘Cystitis’, N34 ‘Urethritis’ or N39.0 ‘Urinary tract infection, site not specified’.
DISCUSSION:
Among this population of older people with diabetes mellitus, eGFR <30 mL/min/1.73 m2 was a risk marker of higher 28- and 90-day mortality following community-acquired pneumonia and sepsis, compared with patients with eGFR ≥60 mL/min/1.73 m2. The relationship between eGFR and mortality did not change with adjustment for co-morbidities. Neither moderately impaired eGFR nor proteinuria was associated with higher short-term mortality following either infection.
The strengths of this study follow from the analysis of a focused question using large, linked datasets for a highly monitored primary care population with a cohort study design. Our study identifies that the association between eGFR and post-infection mortality persists when patients with end-stage renal disease (ESRD) are excluded (and is not explained by renal replacement therapy), when considering fixed-term rather than in-hospital mortality (thus is not due to differences in hospital stay) and when exclusively community-acquired infections are considered (so does not result from increased risk of healthcare-associated infections). The linked datasets allowed us to identify infections both among patients presenting directly to hospital and those managed in the community, maximized ascertainment of mortality and enabled description of the causes of death. The highly monitored population allowed good ascertainment of CKD status. The cohort study design has less potential for selection bias than an equivalent case–control study.
A limitation is our assumption that the absence of a record implies a negative status for proteinuria and co-morbidities. Under-ascertainment of co-morbidities could result in residual confounding, with unpredictable effects, but the high prevalence of co-morbidities observed suggests that ascertainment was not markedly incomplete. We observed a high prevalence of proteinuria, and this is a highly monitored population (with financial incentives for standardized recording of proteinuria since 2004), but under-ascertainment of proteinuria could result in underestimation of any association between proteinuria and mortality [24]. Residual confounding from undiagnosed cardiovascular disease should have been minimized by adjustment for cardiovascular disease risk factors including smoking, hypertension and characteristics of diabetes.
Our findings for eGFR provide further detail to build on previous findings that baseline eGFR <60 mL/min/1.73 m2 or renal disease are risk factors for short-term mortality following (hospital- or community-acquired) sepsis and for in-hospital mortality following community-acquired pneumonia (including patients receiving dialysis) [11–15]. A more comparable Canadian study examined the associations between eGFR and 30-day mortality following community-acquired pneumonia among the general population aged ≥65 years, excluding patients with ESRD [16]. Fully adjusted hazard ratios for 30-day mortality were 1.22 (95% CI 1.01–1.49) for eGFR 45–59, 2.03 (1.64–2.50) for eGFR 30–44 and 4.94 (3.94–6.19) for eGFR < 30, compared with eGFR 60–104 mL/min/1.73 m2. These are somewhat greater than the associations we observed. The difference may be explained by the different study populations. Both studies required a baseline serum creatinine result for inclusion. Our study population of older people with diabetes were routinely monitored for CKD (with financial incentivization in primary care) [32]. Creatinine testing of the Canadian study population may have been encouraged by co-morbidities or health-behaviours associated with CKD (such as smoking), which increase post-infection mortality, resulting in over-estimation of the association of eGFR and post-infection mortality. Our study population is less vulnerable to differential ascertainment of CKD. Alternatively, the association between eGFR and post-infection mortality may be smaller among patients with diabetes.
To the best of our knowledge, our examination of any association between proteinuria and mortality following community-acquired pneumonia and sepsis is novel. A history of proteinuria, although a marker for mortality in general, does not appear to be a risk marker for short-term mortality following community-acquired infection. This is unlikely to be due to chance, as the study was large, with findings consistent across both infections. We designed our study to produce conservative estimates and may have under-estimated the association between proteinuria and post-infection mortality due to under-ascertainment of proteinuria. Alternatively, any potential relationship between proteinuria and mortality may have been mitigated by clinical care of patients with infection who had pre-existing proteinuria, for example, through swift recognition of AKI.
The survival curves demonstrate a steep initial mortality following infection onset, and a high proportion of deaths had the underlying cause assigned to infection. Since 2001 in England, co-morbidities are assigned as the underlying cause of death when pneumonia has occurred in the context of, for example, malignancy or respiratory disease [25]. This suggests that the associations we observed are driven by an association between eGFR and infection prognosis, not merely high underlying baseline mortality among patients with impaired eGFR. This is supported by previous research which found 7.7-fold elevated mortality in the 30 days following community-acquired pneumonia [19]. Estimates for associations between eGFR and mortality were not substantially altered by adjustment for co-morbidities, suggesting that any causal relationship between eGFR and mortality is not mediated through co-morbidities.
Our findings apply to the large population of older patients with community-acquired pneumonia or sepsis with diabetes mellitus who do not have ESRD. Inclusion criteria are unlikely to have limited generalizability appreciably. Practices which consent to data linkage could be more research oriented, providing good primary care management of risk factors for infection-related mortality (such as smoking cessation), but this is unlikely to affect the relationship between CKD and short-term mortality post-infection. Lack of pre-existing creatinine test results is likely to reflect limited time potentially eligible for the study rather than CKD status among this highly monitored population. Missing data on smoking status and HbA1C may be a marker of low patient engagement: caution should be used in generalizing our results to patients who are not actively managed in primary care.
We found that CKD is a useful clinical risk marker for post-infectious mortality. Whether this relationship is causal is less clear; but the association does not appear to be explained by age, co-morbidities or hospital attendance. Potential mechanisms include immune system dysfunction, but also more preventable complications such as AKI. Combinations of risk factors may be important: for example, patients with post-operative AKI have higher mortality if they also have pre-existing CKD [30].
Our results have implications for patient management and future research. Patients with baseline eGFR <30 mL/min/1.73 m2 and community-acquired infection need careful monitoring, particularly in the 28 days following infection. Future research should investigate preventable mechanisms by which low baseline eGFR could be related to post-infection mortality, for example, fluid management, AKI and drug dosage in patients with low renal clearance.
|
Background:
We aimed to examine whether pre-existing impaired estimated glomerular filtration rate (eGFR) and proteinuria were associated with mortality following community-acquired pneumonia or sepsis among people aged ≥ 65 years with diabetes mellitus, without end-stage renal disease.
Methods:
Patients were followed up from onset of first community-acquired pneumonia or sepsis episode in a cohort study using large, linked electronic health databases. Follow-up was for up to 90 days, unlimited by hospital discharge. We used generalized linear models with log link, normal distribution and robust standard errors to calculate risk ratios (RRs) for all-cause 28- and 90-day mortality according to two markers of chronic kidney disease: eGFR and proteinuria.
Results:
All-cause mortality among the 4743 patients with pneumonia was 29.6% after 28 days and 37.4% after 90 days. Among the 1058 patients with sepsis, all-cause 28- and 90-day mortality were 35.6 and 44.2%, respectively. eGFR <30 mL/min/1.73 m(2) was a risk marker of higher 28-day mortality for pneumonia (RR 1.27: 95% CI 1.12-1.43) and sepsis (RR 1.32: 95% CI 1.07-1.64), adjusted for age, sex, socio-economic status, smoking status and co-morbidities. Neither moderately impaired eGFR nor proteinuria were associated with short-term mortality following either infection.
Conclusions:
People with pre-existing low eGFR but not on dialysis are at higher risk of death following pneumonia and sepsis. This association was not explained by existing co-morbidities. These patients need to be carefully monitored to prevent modifiable causes of death.
| null | null | 8,630 | 322 | 12 |
[
"infection",
"mortality",
"egfr",
"patients",
"ckd",
"pneumonia",
"disease",
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"28",
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[
"test",
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] | null | null | null |
[CONTENT] chronic kidney disease | community-acquired infections | electronic health records | infection/mortality | proteinuria [SUMMARY]
| null |
[CONTENT] chronic kidney disease | community-acquired infections | electronic health records | infection/mortality | proteinuria [SUMMARY]
| null |
[CONTENT] chronic kidney disease | community-acquired infections | electronic health records | infection/mortality | proteinuria [SUMMARY]
| null |
[CONTENT] Age Factors | Aged | Aged, 80 and over | Biomarkers | Cohort Studies | Community-Acquired Infections | Comorbidity | Diabetes Mellitus | Electronic Health Records | Female | Humans | Kidney Failure, Chronic | Male | Pneumonia | Proteinuria | Renal Dialysis | Risk Factors | Sepsis | Survival Rate [SUMMARY]
| null |
[CONTENT] Age Factors | Aged | Aged, 80 and over | Biomarkers | Cohort Studies | Community-Acquired Infections | Comorbidity | Diabetes Mellitus | Electronic Health Records | Female | Humans | Kidney Failure, Chronic | Male | Pneumonia | Proteinuria | Renal Dialysis | Risk Factors | Sepsis | Survival Rate [SUMMARY]
| null |
[CONTENT] Age Factors | Aged | Aged, 80 and over | Biomarkers | Cohort Studies | Community-Acquired Infections | Comorbidity | Diabetes Mellitus | Electronic Health Records | Female | Humans | Kidney Failure, Chronic | Male | Pneumonia | Proteinuria | Renal Dialysis | Risk Factors | Sepsis | Survival Rate [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] infection | mortality | egfr | patients | ckd | pneumonia | disease | status | 28 | onset [SUMMARY]
| null |
[CONTENT] infection | mortality | egfr | patients | ckd | pneumonia | disease | status | 28 | onset [SUMMARY]
| null |
[CONTENT] infection | mortality | egfr | patients | ckd | pneumonia | disease | status | 28 | onset [SUMMARY]
| null |
[CONTENT] ckd | associated | mortality | infection | older | older people | community | community acquired | acquired | prognosis [SUMMARY]
| null |
[CONTENT] reference reference | reference | pneumonia | reference reference reference | day mortality | mortality | disease | reference reference reference reference | 10 | 73 [SUMMARY]
| null |
[CONTENT] infection | mortality | egfr | ckd | patients | days | data | 28 | acquired | status [SUMMARY]
| null |
[CONTENT] ≥ | 65 years [SUMMARY]
| null |
[CONTENT] 4743 | 29.6% | 28 days | 37.4% | 90 days ||| 1058 | 28- | 90-day | 35.6 | 44.2% ||| m(2 | 28-day | 1.27 | 95% | CI | 1.12-1.43 | 1.32 | 95% | CI | 1.07-1.64 ||| [SUMMARY]
| null |
[CONTENT] ≥ | 65 years ||| first ||| up to 90 days ||| linear | 28- | 90-day | two | proteinuria ||| 4743 | 29.6% | 28 days | 37.4% | 90 days ||| 1058 | 28- | 90-day | 35.6 | 44.2% ||| m(2 | 28-day | 1.27 | 95% | CI | 1.12-1.43 | 1.32 | 95% | CI | 1.07-1.64 ||| ||| ||| ||| [SUMMARY]
| null |
|||
Endometrial intraepithelial carcinoma in association with polyp: review of eight cases.
|
23414240
|
The uterine endometrial polyp (EMP) has a potential risk of developing malignant tumors especially in postmenopausal women. These malignancies include endometrial intraepithelial carcinoma (EIC).
|
BACKGROUND
|
Eight patients with EIC in the EMP, who were postmenopausal with ages ranging from 49 to 76 years (av. 62), were cytologically reviewed in comparison with histological findings.
|
PATIENTS AND METHODS
|
The endometrial cytological findings were summarized as follows: mucous and watery diathesis as a background lacking or with little necrotic inflammatory changes; micropapillary cluster formation; abrupt transition between carcinoma cells and normal cells; nuclear enlargement; high N/C ratio; and single or a few prominent nucleoli. Histologically, one case had EIC alone in the EMP; three cases had EIC with stromal invasion confined to the EMP; and four cases had EIC in the atrophic endometrium in addition to EIC in the EMP. Seven patients have taken a disease-free course after surgical resection, but one patient died 44 months following the initial diagnosis because of the massive tumor extending over her peritoneal cavity.
|
RESULTS
|
Endometrial cytology may be helpful for the detection of early endometrial adenocarcinomas with serous features including EIC. Some early stage endometrial adenocarcinomas represented by EIC exceptionally take an aggressive clinical course irrespective of a lack of extrauterine lesions.
|
CONCLUSIONS
|
[
"Adenocarcinoma",
"Aged",
"Carcinoma in Situ",
"Disease Progression",
"Early Detection of Cancer",
"Endometrial Neoplasms",
"Female",
"Humans",
"Middle Aged",
"Polyps",
"Predictive Value of Tests",
"Time Factors",
"Treatment Outcome"
] |
3599099
|
Introduction
|
The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].
Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.
The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.
| null | null |
Results
|
The EMPs including EIC with / without stromal invasion measured less than 1 cm in the great diameter in all of the patients. The cytological features are summarized in Table 1 and representatively depicted in Figures 1, 2, and 3, including histological findings of three patients. The tumor diathesis was characterized as being predominantly mucous for 3 patients, watery for 3 patients, and bloody for 2 patients, but with no prominent necrotic inflammatory changes. The tumor cell clusters predominantly took a micropapillary pattern and occasionally a sheet-like pattern. Overlapping of the tumor cell nuclei was noted in a variable degree: often for 2 patients, and occasionally for 5 patients. There were varying amounts of normal epithelial cells, which served as a hallmark in distinction from the malignancy because of a significant difference in the nuclear size. Using Isis ver. 5 (MetaSystems GmbH, Altlussheim, Germany), the nuclear size (greatest diameter) of tumor cells were measured as well as normal atrophic cells in the cytological preparations. The numbers of counted cells for the former and the latter ranged from 50 to 70 in each case. On the total average of 8 cases, the tumor cell size was 12.5 μm and the relative ratio to the corresponding normal atrophic cells was 2.3 (Table 1). The chromatin was considerably dense, coarse or granular. The number of prominent nucleoli was as follows: 1 for 4 patients; 1 or 2 for 4 patients. Calcified deposits of psammoma body were not apparently observed in any patient.
Cytological features of endometrial intraepithelial carcinomas
a: average of largest diameter of tumor cells (relative value compared to normal nucleus).
Case 2 possessing EIC in both endometrial polyp and endometrium (A. Pap × 20, B. Pap × 60, C. loupe view, D. HE × 20, E. HE × 60, F. HE × 60). A. Papillary-structured carcinoma cell cluster is accompanied by a watery background (Pap. ×20). B. The N/C ratio is fairly high and the chromatin is coarsely granular (Pap. ×60). C. Loupe findings show an endometrial polyp arising in the uterine fundus, measuring 1.5 cm in longitudinal length. The endometrium is markedly thin and atrophic. The area marked by an arrow and that marked by an arrowhead are depicted respectively in E and F. D. In the polyp, the glands composed of carcinoma cells are found to be aggregated in the fibrotic stroma. E. Carcinoma cells are replacing the glands in the polyp and stratified into micropapillary protrusions, lacking apparent stromal invasion (HE × 20). F. Carcinoma nests similar to those in the endometrial polyp (D) are also present in the endometrial gland (HE × 60).
Case 5 possessing EIC in both endometrial polyp and endometrium, but with stromal invasion in the polyp (A. Pap × 20, B. Pap × 60, C. HE × 20, D. HE × 60). A. A carcinoma cell cluster is accompanied by a bloody background. B. Carcinoma cells having enlarged nuclei appear to be transitional with normal atrophic cells having small-sized nuclei (arrows). C. The polyp has many atypical glands with intraluminal papillary protrusion of the carcinoma cells. D. The border between the carcinoma cells and normal cells is clearly discernible (arrows). There are small carcinoma nests showing stromal invasion (arrowheads).
Case 6 possessing EIC confined to endometrial polyp (A. Pap × 20, B. Pap × 60, C. HE × 60, D. HE × 60). A. Large-sized carcinoma cell clusters are accompanied by a non-necroinflammatory background. B. Carcinoma cells mainly appear in a flat and sheet-like cluster. The nucleus has an enlarged eosinophilic nucleolus. C. The atypical glands are normal-sized, accompanied by cystic dilatation of atrophic normal glands. D. Carcinoma cells are replacing pre-existing glands and are arranged in a single layer (compare with normal gland marked by an arrow).
The clinicopathological profiles are summarized in Table 2. All the patients had an EMP in which atypical tumor cells were arranged predominantly in a tubular structure with micropapillary protrusions, replacing the pre-existing endometrial glands. Micropapillary protrusions and small tufts were accompanied with or without thin fibrovascular cores. These tumor cells showed conspicuous nuclear pleomorphism and hyperchromatism, frequent mitotic figures, high N/C ratio, and also one or two prominent nucleoli. The tumor front was abruptly made into normal glandular epithelium and surface-lining epithelium in the EMP. Minimal stromal invasion of the tumor cells limited to the EMP was recognized in 3 patients. In addition, similar tumor cells also were also found to replace some atrophic endometrial glands in 4 patients, lacking stromal invasion. In the background, cystic dilatation of atrophic normal glands was evident especially in the EMP.
Clinicopathological profiles of endometrial intraepithelial carcinomas
a:+, strongly positive cells more than 80%.
b: index of positive cells(%), c: +, positive cells more than 80%.
d: +, positive cells more than 80%.
e: since initial diagnosis.
f: history of breast cancer with Tamoxifen administration.
g: autopsy performed.
h: with stromal invasion, i: disease free, j: death on disease.
Immunohistochemical expressions of p53 (DO7, 1:50, Dako, Glostrup, Denmark), Ki-67 (MIB-1, 1:100, Dako, Glostrup, Denmark), ER (SP-1, 1:1, Ventana, AZ, USA), and PgR (1E2, 1:1, Ventana, AZ, USA) were examined (Figure 4). Six patients showed marked p53 expression with a positive ratio of more than 80%. The labeling index of Ki-67 ranged from 25 to 70% (av. approximately 40). ER was mildly expressed in one patient and PgR expression also was mildly observed in 2 patients.
Immunohistochemical staining profiles represented by Case 5 (A. p53 × 40, B. MIB-1 × 40, C. ER × 20, D. PgR × 20). A. Almost all of the carcinoma cells are strongly positive. B. More than half the carcinoma cells are labeled. C. The expression is observed in the cystically dilated normal glands and stromal cells but not in the carcinoma cells. D. The expression behavior is basically similar to the ER expression.
FIGO stage was determined at IA for all the patients. Neither adjuvant chemotherapy nor irradiation was performed. Seven patients have been taking an uneventful clinical course (follow-up period ranging from 12 to 66 months, av. 41), but one patient died of the recurrent disease massively occupying the abdominal cavity 44 months after the initial diagnosis.
| null | null |
[
"Introduction",
"Patients",
"Patients’ consents",
"Ethical approval",
"Competing interests",
"Authors’ contributions"
] |
[
"The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.",
"The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.",
"Written informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.",
"This review was performed based on compliance with the Helsinki Declaration.",
"All of the authors declare that they have no competing interests.",
"MY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript."
] |
[
null,
null,
null,
null,
null,
null
] |
[
"Introduction",
"Patients",
"Results",
"Discussion",
"Patients’ consents",
"Ethical approval",
"Competing interests",
"Authors’ contributions"
] |
[
"The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].\n Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.\nThe eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.",
"The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.",
"The EMPs including EIC with / without stromal invasion measured less than 1 cm in the great diameter in all of the patients. The cytological features are summarized in Table 1 and representatively depicted in Figures 1, 2, and 3, including histological findings of three patients. The tumor diathesis was characterized as being predominantly mucous for 3 patients, watery for 3 patients, and bloody for 2 patients, but with no prominent necrotic inflammatory changes. The tumor cell clusters predominantly took a micropapillary pattern and occasionally a sheet-like pattern. Overlapping of the tumor cell nuclei was noted in a variable degree: often for 2 patients, and occasionally for 5 patients. There were varying amounts of normal epithelial cells, which served as a hallmark in distinction from the malignancy because of a significant difference in the nuclear size. Using Isis ver. 5 (MetaSystems GmbH, Altlussheim, Germany), the nuclear size (greatest diameter) of tumor cells were measured as well as normal atrophic cells in the cytological preparations. The numbers of counted cells for the former and the latter ranged from 50 to 70 in each case. On the total average of 8 cases, the tumor cell size was 12.5 μm and the relative ratio to the corresponding normal atrophic cells was 2.3 (Table 1). The chromatin was considerably dense, coarse or granular. The number of prominent nucleoli was as follows: 1 for 4 patients; 1 or 2 for 4 patients. Calcified deposits of psammoma body were not apparently observed in any patient.\nCytological features of endometrial intraepithelial carcinomas\na: average of largest diameter of tumor cells (relative value compared to normal nucleus).\nCase 2 possessing EIC in both endometrial polyp and endometrium (A. Pap × 20, B. Pap × 60, C. loupe view, D. HE × 20, E. HE × 60, F. HE × 60). A. Papillary-structured carcinoma cell cluster is accompanied by a watery background (Pap. ×20). B. The N/C ratio is fairly high and the chromatin is coarsely granular (Pap. ×60). C. Loupe findings show an endometrial polyp arising in the uterine fundus, measuring 1.5 cm in longitudinal length. The endometrium is markedly thin and atrophic. The area marked by an arrow and that marked by an arrowhead are depicted respectively in E and F. D. In the polyp, the glands composed of carcinoma cells are found to be aggregated in the fibrotic stroma. E. Carcinoma cells are replacing the glands in the polyp and stratified into micropapillary protrusions, lacking apparent stromal invasion (HE × 20). F. Carcinoma nests similar to those in the endometrial polyp (D) are also present in the endometrial gland (HE × 60).\nCase 5 possessing EIC in both endometrial polyp and endometrium, but with stromal invasion in the polyp (A. Pap × 20, B. Pap × 60, C. HE × 20, D. HE × 60). A. A carcinoma cell cluster is accompanied by a bloody background. B. Carcinoma cells having enlarged nuclei appear to be transitional with normal atrophic cells having small-sized nuclei (arrows). C. The polyp has many atypical glands with intraluminal papillary protrusion of the carcinoma cells. D. The border between the carcinoma cells and normal cells is clearly discernible (arrows). There are small carcinoma nests showing stromal invasion (arrowheads).\nCase 6 possessing EIC confined to endometrial polyp (A. Pap × 20, B. Pap × 60, C. HE × 60, D. HE × 60). A. Large-sized carcinoma cell clusters are accompanied by a non-necroinflammatory background. B. Carcinoma cells mainly appear in a flat and sheet-like cluster. The nucleus has an enlarged eosinophilic nucleolus. C. The atypical glands are normal-sized, accompanied by cystic dilatation of atrophic normal glands. D. Carcinoma cells are replacing pre-existing glands and are arranged in a single layer (compare with normal gland marked by an arrow).\nThe clinicopathological profiles are summarized in Table 2. All the patients had an EMP in which atypical tumor cells were arranged predominantly in a tubular structure with micropapillary protrusions, replacing the pre-existing endometrial glands. Micropapillary protrusions and small tufts were accompanied with or without thin fibrovascular cores. These tumor cells showed conspicuous nuclear pleomorphism and hyperchromatism, frequent mitotic figures, high N/C ratio, and also one or two prominent nucleoli. The tumor front was abruptly made into normal glandular epithelium and surface-lining epithelium in the EMP. Minimal stromal invasion of the tumor cells limited to the EMP was recognized in 3 patients. In addition, similar tumor cells also were also found to replace some atrophic endometrial glands in 4 patients, lacking stromal invasion. In the background, cystic dilatation of atrophic normal glands was evident especially in the EMP.\nClinicopathological profiles of endometrial intraepithelial carcinomas\na:+, strongly positive cells more than 80%.\nb: index of positive cells(%), c: +, positive cells more than 80%.\nd: +, positive cells more than 80%.\ne: since initial diagnosis.\nf: history of breast cancer with Tamoxifen administration.\ng: autopsy performed.\nh: with stromal invasion, i: disease free, j: death on disease.\nImmunohistochemical expressions of p53 (DO7, 1:50, Dako, Glostrup, Denmark), Ki-67 (MIB-1, 1:100, Dako, Glostrup, Denmark), ER (SP-1, 1:1, Ventana, AZ, USA), and PgR (1E2, 1:1, Ventana, AZ, USA) were examined (Figure 4). Six patients showed marked p53 expression with a positive ratio of more than 80%. The labeling index of Ki-67 ranged from 25 to 70% (av. approximately 40). ER was mildly expressed in one patient and PgR expression also was mildly observed in 2 patients.\nImmunohistochemical staining profiles represented by Case 5 (A. p53 × 40, B. MIB-1 × 40, C. ER × 20, D. PgR × 20). A. Almost all of the carcinoma cells are strongly positive. B. More than half the carcinoma cells are labeled. C. The expression is observed in the cystically dilated normal glands and stromal cells but not in the carcinoma cells. D. The expression behavior is basically similar to the ER expression.\nFIGO stage was determined at IA for all the patients. Neither adjuvant chemotherapy nor irradiation was performed. Seven patients have been taking an uneventful clinical course (follow-up period ranging from 12 to 66 months, av. 41), but one patient died of the recurrent disease massively occupying the abdominal cavity 44 months after the initial diagnosis.",
"Endometrial polyp is a common benign disease of the uterus. The overall incidence where cancer arises in the endometrial polyp has been reported as only a few percent, while postmenopausal women with EMP are at an increased risk of malignancy, compared to premenopausal women [10,13-15]. As risk factors for development of the malignancy in EMP, hypertension, obesity, and unopposed estrogen therapy have been indicated in addition to postmenopausal status [15]. There are unique or specific pathological and clinical features for EIC as a putative precursor of uterine body serous adenocarcinoma, as follows [1,4,5,7,16-20]: close association with an EMP; multicentric occurrence in the inactive or resting thin endometrium, lacking alteration in the architecture in the endometrium lacking an intervening phase of endometrial hyperplasia; extrauterine invasive extent, irrespective of an absence of stromal invasion of the uterus; and controversial outcome, whether taking a favorable or unfavorable clinical course. Development of EIC associated with an EMP may also be explained in part by Tamoxifen administration following breast cancer treatment [6,17]. However, little has been documented about the endometrial cytological features of minimal serous adenocarcinoma of the uterine body including EIC, mainly due to the fact that the disease is not frequently encountered in the routine practice. To the best of our knowledge, there is a case report cytologically referring to serous adenocarcinoma of the uterine body associated with an EMP [21], but here the patient already showed peritoneal dissemination at the initial presentation. In another report, serous adenocarcinomas of the uterine body were clinico-cytologically reviewed but they were all at advanced stages [22]. The latter report mentions that cytologically diagnostic findings of uterine body serous adenocarcinoma would be based on the following features: papillary cluster as well as tubular cluster, arborescent structure, frequent exfoliation, psammoma body, eccentric enlarged nuclei, coarsely granular chromatin, numerous nucleoli, and diameter of greater than G1 endometrioid adenocarcinoma [22]. In our review, 5 patients were purely non-invasive as they were composed of only EIC existing in an EMP, with few or no necroinflammatory changes. The background was found to be characteristically watery or mucous (Figure 1) in a way of simulating the immigration of extrauterine carcinoma cells via the fallopian tubes [12]. Many clusters were arranged in micropapillary architecture without fine vascular stroma, and others were occasionally tubular. An abrupt transition between the EIC cells and atrophic endometrial cells was noted by front formation (Figure 2). Conspicuous nucleoli were found in 3 patients (Figure 3). Even though it seems difficult to reach a definite diagnosis, these findings may serve as a diagnostic indicator for the uterine body serous adenocarcinoma, whether being at an early stage or an advanced stage.\nDifferential diagnoses for atypical epithelial sheets in the normal-appearing endometrial background include variable interpretations as follows: EIC, endometrial glandular dysplasia, endometrial intraepithelial neoplasia, hyperplastic polyp, and metastatic carcinoma [9,23]. In addition to these, isolated atypical glands with morphological and immunohistochemical features of a typical endometrial hyperplasia or type I endometrial adenocarcinoma may be encountered in grossly normal postmenopausal endometrium of asymptomatic patients [24]. Especially in the postmenopausal state, thin and smooth endometrium with atrophy is composed of hypocellular and flattened glands lined by cells with reduced or absent mitosis and sparse fibrous stroma [25]. It is supposed that the abrupt transition of the atrophic endometrial cells to atypical cells may indicate EIC in postmenopausal women.\nOverexpression of p53 is closely linked to the rapid growth of uterine body serous adenocarcinoma [6]. In EIC, too, frequent p53 mutation is reported to be associated with LOH of 17p [26]. Thus, immunohistochemistry for p53 is also adjunctively available in the diagnosis of uterine body serous adenocarcinoma, irrespective of whether it is at an early stage or advanced stage [27], in the discrimination between uterine body serous adenocarcinoma and benign morphologic mimics [28]. Negativity of ER and PgR staining is more reasonable with the diagnosis of serous adenocarcinoma including EIC.\nIn summary, EMP arising in postmenopausal women is at a high risk of giving birth to the tumorigenetic background for endometrial carcinomas such as endometrioid adenocarcinoma and serous adenocarcinoma including EIC. Endometrial cytology may become a helpful diagnostic approach for serous adenocarcinoma, whether it is at early or advanced stage, from the viewpoint of the distinctive cytological features. Above all, it should be noted that some EIC behaves in a more aggressive fashion regardless of whether or not it lacks apparent stroma invasion and extrauterine extent.",
"Written informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.",
"This review was performed based on compliance with the Helsinki Declaration.",
"All of the authors declare that they have no competing interests.",
"MY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript."
] |
[
null,
null,
"results",
"discussion",
null,
null,
null,
null
] |
[
"Endometrial intraepithelial carcinoma (EIC)",
"Endometrial polyp",
"Cytology"
] |
Introduction:
The uterine body type II cancers are histogenetically distinguished from type I cancers with a background of glandular hyperplasia which is in association with the genetic alterations represented by PTEN inactivation [1]. Tumor development of uterine body serous adenocarcinoma, categorized as the type II group, has been clarified to be linked with a putative precursor lesion designated as endometrial intraepithelial carcinoma (EIC) [2-4]. EIC has been alternatively regarded as in situ serous adenocarcinoma [5,6] and is considered to usually occur in the setting of inactive or resting endometrium and frequently involves endometrial polyp (EMP) [3]. Many minimal serous adenocarcinomas, defined as limited to the endometrium and less than 1 cm [7], are also found to frequently have EIC and involve EMP [8]. However, the nomenclature of EIC remains controversial because there are morphological variations in the endometrial intraepithelial precancerous lesion, and EIC is known to be potentially complicated with the extrauterine lesion. Therefore, instead of EIC, a newly defined terminology of endometrial glandular dysplasia has been proposed [9]. Reportedly, 10–34% of endometrial cancers in postmenopausal women have been associated with EMPs [10]. Usually, the major adenocarcinomas are the endometrioid type and the second major adenocarcinomas are the serous type [10]. The eight cases with early endometrial carcinoma of the uterine body, characterized by EIC being associated with EMP, were subjected to close cytological review as well as histological review. For them, histological diagnostic confirmation was limited due to the inner location of tumors at the fundus and their small size. However, the endometrial cytological approach is supposed to be of help for the detection of these early lesions. The candidates of differential cytological diagnosis for these cases are advanced serous adenocarcinoma, clear cell adenocarcinoma, high grade endometrioid adenocarcinoma and also low grade endometrioid adenocarcinoma [11]. Immigration of ovarian carcinoma via the fallopian tube, instead of metastasizing to the endometrium, is also included in the differential diagnoses [12].
Patients The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.
The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.
Patients:
The eight patients were postmenopausal, with ages ranging from 49 to 76 years (av. 62) when the cytological abnormalities were initially recognized. All of them visited their neighboring hospitals with a chief complaint of irregular genital bleeding. One patient had received surgical treatment for breast cancer 6 years before and had been administered an anti-estrogenic drug (Tamoxifen). There was no past history of other malignancies in the remaining patients. The endometrial cytology using Endocyte® (MSD Co., Tokyo, Japan) suspected adenocarcinoma which was consistent with being at the early stage, including EIC, for all the patients. However, the endometrial biopsy did not lead to the confirmation of malignancy except in two patients for whom the diagnosis was adenocarcinoma, although the adenocarcinoma could not be specified because the specimen was too small. They all underwent total abdominal hysterectomy and bilateral adnexectomy. Regional lymph node dissection was performed in three of the patients, resulting in no evidence of metastatic lesions. In two patients for whom polypectomy with hysteroscopic assistance had been performed previously, no residual carcinoma was found in the hysterectomy specimen.
Results:
The EMPs including EIC with / without stromal invasion measured less than 1 cm in the great diameter in all of the patients. The cytological features are summarized in Table 1 and representatively depicted in Figures 1, 2, and 3, including histological findings of three patients. The tumor diathesis was characterized as being predominantly mucous for 3 patients, watery for 3 patients, and bloody for 2 patients, but with no prominent necrotic inflammatory changes. The tumor cell clusters predominantly took a micropapillary pattern and occasionally a sheet-like pattern. Overlapping of the tumor cell nuclei was noted in a variable degree: often for 2 patients, and occasionally for 5 patients. There were varying amounts of normal epithelial cells, which served as a hallmark in distinction from the malignancy because of a significant difference in the nuclear size. Using Isis ver. 5 (MetaSystems GmbH, Altlussheim, Germany), the nuclear size (greatest diameter) of tumor cells were measured as well as normal atrophic cells in the cytological preparations. The numbers of counted cells for the former and the latter ranged from 50 to 70 in each case. On the total average of 8 cases, the tumor cell size was 12.5 μm and the relative ratio to the corresponding normal atrophic cells was 2.3 (Table 1). The chromatin was considerably dense, coarse or granular. The number of prominent nucleoli was as follows: 1 for 4 patients; 1 or 2 for 4 patients. Calcified deposits of psammoma body were not apparently observed in any patient.
Cytological features of endometrial intraepithelial carcinomas
a: average of largest diameter of tumor cells (relative value compared to normal nucleus).
Case 2 possessing EIC in both endometrial polyp and endometrium (A. Pap × 20, B. Pap × 60, C. loupe view, D. HE × 20, E. HE × 60, F. HE × 60). A. Papillary-structured carcinoma cell cluster is accompanied by a watery background (Pap. ×20). B. The N/C ratio is fairly high and the chromatin is coarsely granular (Pap. ×60). C. Loupe findings show an endometrial polyp arising in the uterine fundus, measuring 1.5 cm in longitudinal length. The endometrium is markedly thin and atrophic. The area marked by an arrow and that marked by an arrowhead are depicted respectively in E and F. D. In the polyp, the glands composed of carcinoma cells are found to be aggregated in the fibrotic stroma. E. Carcinoma cells are replacing the glands in the polyp and stratified into micropapillary protrusions, lacking apparent stromal invasion (HE × 20). F. Carcinoma nests similar to those in the endometrial polyp (D) are also present in the endometrial gland (HE × 60).
Case 5 possessing EIC in both endometrial polyp and endometrium, but with stromal invasion in the polyp (A. Pap × 20, B. Pap × 60, C. HE × 20, D. HE × 60). A. A carcinoma cell cluster is accompanied by a bloody background. B. Carcinoma cells having enlarged nuclei appear to be transitional with normal atrophic cells having small-sized nuclei (arrows). C. The polyp has many atypical glands with intraluminal papillary protrusion of the carcinoma cells. D. The border between the carcinoma cells and normal cells is clearly discernible (arrows). There are small carcinoma nests showing stromal invasion (arrowheads).
Case 6 possessing EIC confined to endometrial polyp (A. Pap × 20, B. Pap × 60, C. HE × 60, D. HE × 60). A. Large-sized carcinoma cell clusters are accompanied by a non-necroinflammatory background. B. Carcinoma cells mainly appear in a flat and sheet-like cluster. The nucleus has an enlarged eosinophilic nucleolus. C. The atypical glands are normal-sized, accompanied by cystic dilatation of atrophic normal glands. D. Carcinoma cells are replacing pre-existing glands and are arranged in a single layer (compare with normal gland marked by an arrow).
The clinicopathological profiles are summarized in Table 2. All the patients had an EMP in which atypical tumor cells were arranged predominantly in a tubular structure with micropapillary protrusions, replacing the pre-existing endometrial glands. Micropapillary protrusions and small tufts were accompanied with or without thin fibrovascular cores. These tumor cells showed conspicuous nuclear pleomorphism and hyperchromatism, frequent mitotic figures, high N/C ratio, and also one or two prominent nucleoli. The tumor front was abruptly made into normal glandular epithelium and surface-lining epithelium in the EMP. Minimal stromal invasion of the tumor cells limited to the EMP was recognized in 3 patients. In addition, similar tumor cells also were also found to replace some atrophic endometrial glands in 4 patients, lacking stromal invasion. In the background, cystic dilatation of atrophic normal glands was evident especially in the EMP.
Clinicopathological profiles of endometrial intraepithelial carcinomas
a:+, strongly positive cells more than 80%.
b: index of positive cells(%), c: +, positive cells more than 80%.
d: +, positive cells more than 80%.
e: since initial diagnosis.
f: history of breast cancer with Tamoxifen administration.
g: autopsy performed.
h: with stromal invasion, i: disease free, j: death on disease.
Immunohistochemical expressions of p53 (DO7, 1:50, Dako, Glostrup, Denmark), Ki-67 (MIB-1, 1:100, Dako, Glostrup, Denmark), ER (SP-1, 1:1, Ventana, AZ, USA), and PgR (1E2, 1:1, Ventana, AZ, USA) were examined (Figure 4). Six patients showed marked p53 expression with a positive ratio of more than 80%. The labeling index of Ki-67 ranged from 25 to 70% (av. approximately 40). ER was mildly expressed in one patient and PgR expression also was mildly observed in 2 patients.
Immunohistochemical staining profiles represented by Case 5 (A. p53 × 40, B. MIB-1 × 40, C. ER × 20, D. PgR × 20). A. Almost all of the carcinoma cells are strongly positive. B. More than half the carcinoma cells are labeled. C. The expression is observed in the cystically dilated normal glands and stromal cells but not in the carcinoma cells. D. The expression behavior is basically similar to the ER expression.
FIGO stage was determined at IA for all the patients. Neither adjuvant chemotherapy nor irradiation was performed. Seven patients have been taking an uneventful clinical course (follow-up period ranging from 12 to 66 months, av. 41), but one patient died of the recurrent disease massively occupying the abdominal cavity 44 months after the initial diagnosis.
Discussion:
Endometrial polyp is a common benign disease of the uterus. The overall incidence where cancer arises in the endometrial polyp has been reported as only a few percent, while postmenopausal women with EMP are at an increased risk of malignancy, compared to premenopausal women [10,13-15]. As risk factors for development of the malignancy in EMP, hypertension, obesity, and unopposed estrogen therapy have been indicated in addition to postmenopausal status [15]. There are unique or specific pathological and clinical features for EIC as a putative precursor of uterine body serous adenocarcinoma, as follows [1,4,5,7,16-20]: close association with an EMP; multicentric occurrence in the inactive or resting thin endometrium, lacking alteration in the architecture in the endometrium lacking an intervening phase of endometrial hyperplasia; extrauterine invasive extent, irrespective of an absence of stromal invasion of the uterus; and controversial outcome, whether taking a favorable or unfavorable clinical course. Development of EIC associated with an EMP may also be explained in part by Tamoxifen administration following breast cancer treatment [6,17]. However, little has been documented about the endometrial cytological features of minimal serous adenocarcinoma of the uterine body including EIC, mainly due to the fact that the disease is not frequently encountered in the routine practice. To the best of our knowledge, there is a case report cytologically referring to serous adenocarcinoma of the uterine body associated with an EMP [21], but here the patient already showed peritoneal dissemination at the initial presentation. In another report, serous adenocarcinomas of the uterine body were clinico-cytologically reviewed but they were all at advanced stages [22]. The latter report mentions that cytologically diagnostic findings of uterine body serous adenocarcinoma would be based on the following features: papillary cluster as well as tubular cluster, arborescent structure, frequent exfoliation, psammoma body, eccentric enlarged nuclei, coarsely granular chromatin, numerous nucleoli, and diameter of greater than G1 endometrioid adenocarcinoma [22]. In our review, 5 patients were purely non-invasive as they were composed of only EIC existing in an EMP, with few or no necroinflammatory changes. The background was found to be characteristically watery or mucous (Figure 1) in a way of simulating the immigration of extrauterine carcinoma cells via the fallopian tubes [12]. Many clusters were arranged in micropapillary architecture without fine vascular stroma, and others were occasionally tubular. An abrupt transition between the EIC cells and atrophic endometrial cells was noted by front formation (Figure 2). Conspicuous nucleoli were found in 3 patients (Figure 3). Even though it seems difficult to reach a definite diagnosis, these findings may serve as a diagnostic indicator for the uterine body serous adenocarcinoma, whether being at an early stage or an advanced stage.
Differential diagnoses for atypical epithelial sheets in the normal-appearing endometrial background include variable interpretations as follows: EIC, endometrial glandular dysplasia, endometrial intraepithelial neoplasia, hyperplastic polyp, and metastatic carcinoma [9,23]. In addition to these, isolated atypical glands with morphological and immunohistochemical features of a typical endometrial hyperplasia or type I endometrial adenocarcinoma may be encountered in grossly normal postmenopausal endometrium of asymptomatic patients [24]. Especially in the postmenopausal state, thin and smooth endometrium with atrophy is composed of hypocellular and flattened glands lined by cells with reduced or absent mitosis and sparse fibrous stroma [25]. It is supposed that the abrupt transition of the atrophic endometrial cells to atypical cells may indicate EIC in postmenopausal women.
Overexpression of p53 is closely linked to the rapid growth of uterine body serous adenocarcinoma [6]. In EIC, too, frequent p53 mutation is reported to be associated with LOH of 17p [26]. Thus, immunohistochemistry for p53 is also adjunctively available in the diagnosis of uterine body serous adenocarcinoma, irrespective of whether it is at an early stage or advanced stage [27], in the discrimination between uterine body serous adenocarcinoma and benign morphologic mimics [28]. Negativity of ER and PgR staining is more reasonable with the diagnosis of serous adenocarcinoma including EIC.
In summary, EMP arising in postmenopausal women is at a high risk of giving birth to the tumorigenetic background for endometrial carcinomas such as endometrioid adenocarcinoma and serous adenocarcinoma including EIC. Endometrial cytology may become a helpful diagnostic approach for serous adenocarcinoma, whether it is at early or advanced stage, from the viewpoint of the distinctive cytological features. Above all, it should be noted that some EIC behaves in a more aggressive fashion regardless of whether or not it lacks apparent stroma invasion and extrauterine extent.
Patients’ consents:
Written informed consents were obtained from the reviewed patients for publication of this report and any accompanying images.
Ethical approval:
This review was performed based on compliance with the Helsinki Declaration.
Competing interests:
All of the authors declare that they have no competing interests.
Authors’ contributions:
MY conducted the method of reviewing the cases. TK took part in analysis of cytological findings. SH analyzed the clinical information. KS took part in measurement of the nuclear size. KS took part in measurement of the nuclear size. MM analyzed the clinical information. MN performed immunohistochemistry. SM assisted in immunohistochemistry. ON designed tables and figures. YK searched the literature. All authors read and approved the final manuscript.
|
Background:
The uterine endometrial polyp (EMP) has a potential risk of developing malignant tumors especially in postmenopausal women. These malignancies include endometrial intraepithelial carcinoma (EIC).
Methods:
Eight patients with EIC in the EMP, who were postmenopausal with ages ranging from 49 to 76 years (av. 62), were cytologically reviewed in comparison with histological findings.
Results:
The endometrial cytological findings were summarized as follows: mucous and watery diathesis as a background lacking or with little necrotic inflammatory changes; micropapillary cluster formation; abrupt transition between carcinoma cells and normal cells; nuclear enlargement; high N/C ratio; and single or a few prominent nucleoli. Histologically, one case had EIC alone in the EMP; three cases had EIC with stromal invasion confined to the EMP; and four cases had EIC in the atrophic endometrium in addition to EIC in the EMP. Seven patients have taken a disease-free course after surgical resection, but one patient died 44 months following the initial diagnosis because of the massive tumor extending over her peritoneal cavity.
Conclusions:
Endometrial cytology may be helpful for the detection of early endometrial adenocarcinomas with serous features including EIC. Some early stage endometrial adenocarcinomas represented by EIC exceptionally take an aggressive clinical course irrespective of a lack of extrauterine lesions.
| null | null | 3,319 | 247 | 8 |
[
"patients",
"endometrial",
"cells",
"adenocarcinoma",
"eic",
"carcinoma",
"serous",
"serous adenocarcinoma",
"body",
"normal"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] Endometrial intraepithelial carcinoma (EIC) | Endometrial polyp | Cytology [SUMMARY]
| null |
[CONTENT] Endometrial intraepithelial carcinoma (EIC) | Endometrial polyp | Cytology [SUMMARY]
| null |
[CONTENT] Endometrial intraepithelial carcinoma (EIC) | Endometrial polyp | Cytology [SUMMARY]
| null |
[CONTENT] Adenocarcinoma | Aged | Carcinoma in Situ | Disease Progression | Early Detection of Cancer | Endometrial Neoplasms | Female | Humans | Middle Aged | Polyps | Predictive Value of Tests | Time Factors | Treatment Outcome [SUMMARY]
| null |
[CONTENT] Adenocarcinoma | Aged | Carcinoma in Situ | Disease Progression | Early Detection of Cancer | Endometrial Neoplasms | Female | Humans | Middle Aged | Polyps | Predictive Value of Tests | Time Factors | Treatment Outcome [SUMMARY]
| null |
[CONTENT] Adenocarcinoma | Aged | Carcinoma in Situ | Disease Progression | Early Detection of Cancer | Endometrial Neoplasms | Female | Humans | Middle Aged | Polyps | Predictive Value of Tests | Time Factors | Treatment Outcome [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] patients | endometrial | cells | adenocarcinoma | eic | carcinoma | serous | serous adenocarcinoma | body | normal [SUMMARY]
| null |
[CONTENT] patients | endometrial | cells | adenocarcinoma | eic | carcinoma | serous | serous adenocarcinoma | body | normal [SUMMARY]
| null |
[CONTENT] patients | endometrial | cells | adenocarcinoma | eic | carcinoma | serous | serous adenocarcinoma | body | normal [SUMMARY]
| null |
[CONTENT] adenocarcinoma | patients | endometrial | eic | serous | type | specimen | patients endometrial | hysterectomy | years [SUMMARY]
| null |
[CONTENT] cells | normal | 60 | carcinoma | tumor | patients | carcinoma cells | glands | pap | 20 [SUMMARY]
| null |
[CONTENT] patients | adenocarcinoma | endometrial | cells | eic | authors | authors declare competing | competing interests | competing | declare [SUMMARY]
| null |
[CONTENT] EMP ||| EIC [SUMMARY]
| null |
[CONTENT] ||| one | EIC | EMP | three | EIC | EMP | four | EIC | EIC | EMP ||| Seven | 44 months [SUMMARY]
| null |
[CONTENT] EMP ||| EIC ||| Eight | EIC | EMP | 49 to 76 years | 62 ||| ||| ||| one | EIC | EMP | three | EIC | EMP | four | EIC | EIC | EMP ||| Seven | 44 months ||| EIC ||| EIC [SUMMARY]
| null |
|||
Changes in specialized blood vessels in lymph nodes and their role in cancer metastasis.
|
23035663
|
High endothelial venules (HEV) have been recognized to play a role in metastasis by its changes seen in the cancer microenvironment of lymph nodes (LN) and solid cancers. Squamous cell carcinoma (SCC) of the tongue is a prevalent tumor of the head and neck region with high propensity for LN metastasis. The extent of LN metastasis is the most reliable adverse prognostic factor. Primary tumors can induce vasculature reorganization within sentinel LN before the arrival of tumor cells and HEV represents these remodelled vessels. This study aims to evaluate the cancer induced vascular changes in regional lymph nodes (LN) of patients by studying the morphological and functional alterations of HEV and its correlation with clinical outcome and pathological features.
|
BACKGROUND
|
This study was based on 65 patients with SCC tongue who underwent primary surgical treatment including neck dissection. The patients were categorized into 2 groups based on the presence of malignancy in their cervical lymph nodes. A review of the patients' pathological and clinical data was performed from a prospective database. Immunohistochemical staining of the tissue blocks for HEV and high-power-field image analysis were performed and analyzed with correlation to the patients' clinical and pathological features.
|
METHODS
|
The total number of HEV was found to be significantly associated to disease-free interval. There was a similar association comparing the HEV parameters to overall survival. The density of abnormal HEV was significantly higher in patients with established metastases in their lymph nodes and HEV was shown to be a better prognosis factor than conventional tumor staging. The HEV morphological metamorphosis demonstrates a spectrum that correlates well with disease progression and clinical outcome.
|
RESULTS
|
The results suggest that the HEV displays a spectrum of morphological changes in the presence of cancer and LN metastasis, and that HEV is possibly involved in the process of cancer metastasis. We revealed the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environments in regional lymph nodes of tongue cancer patients in relation to clinical outcomes. The significant observation of modified dilated HEV containing red blood cells in lymph nodal basin of a cancer suggests the shifting of its function from one primarily of immune response to that of a blood carrying vessel. It also demonstrated potential prognostic value. More studies are needed to elucidate its potential role in cancer immunotherapy and as a potential novel therapeutic approach to preventing metastasis by manipulating the remodelling processes of HEV.
|
CONCLUSIONS
|
[
"Case-Control Studies",
"Disease-Free Survival",
"Endothelium, Vascular",
"Humans",
"Kaplan-Meier Estimate",
"Lymph Nodes",
"Lymphatic Metastasis",
"Neoplasms",
"Venules"
] |
3551724
|
Background
|
Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.
Oropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States
[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy
[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.
The first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin
[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)
[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised
[6,7].
High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy
[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance
[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors
[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically
[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ
[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses
[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”
[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis
[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases
[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route
[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients
[6].
In addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit
[5,10].
There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy
[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance
[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors
[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically
[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ
[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses
[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”
[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis
[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases
[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route
[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients
[6].
In addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit
[5,10].
Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis
[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.
We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis
[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.
|
Methods
|
Approval was obtained from the Institutional Review Boards of both institutions involved in the study (2007/464/B). The study population consisted of 175 consecutive patients with SCC of the head and neck who had received primary surgical treatment at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Center, Singapore, from January 2001 through to December 2005. The patients’ pathological and clinical data including follow-up information was reviewed from a prospective database. The inclusion criteria included all surgically treated patients with histologically proven SCC tongue who underwent a neck dissection, with a minimum of 2-year follow up period. Patients with a second primary cancer and patients who did not have a neck dissection as part of their primary treament were excluded. There were a total 65 patients that met the study inclusion criteria. There were 35 patients in the group with primary SCC tongue without pathologically proven lymph node metastases in their neck dissection specimen who were designated as “cases”, and 30 patients in the group with primary SCC tongue with pathologically proven lymph node metastases in the neck dissection specimens, who were designated as “controls”. All patients included had radical excision of the primary tongue lesion and a neck dissection according to departmental protocol. In both groups, all patients had radical enbloc excision of the primary tongue lesion with or without resection of adjacent structures with either a unilateral or a bilateral, supraomohyoid neck dissection or a modified radical neck dissection. Tumours were classified according to the AJCC TNM Staging Classification, based on a pre-operative clinical evaluation and this determined the type of neck dissection performed by the primary surgeon
[19]. All cases were discussed at a multi-discplinary head and neck tumor board. Intra-operative frozen sections were performed for margins of the tongue lesion and further resection of the tongue was performed in event that the surgical margins were involved. Upon discharge from the hospital, they were followed up at regular intervals of monthly, 3-monthly, 6-monthly and yearly at a progessive rate. Inpatient and outpatient clincal data, pathological, radiological and operative records were retrieved from a central medical records office and from the Department of Pathology, Singapore General Hospital. Mortality data was obtained and confirmed from the hospital central medical records office as well as the Singapore Registry of Births & Deaths.
Immunohistochemistry A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed
[5,20].
A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed
[5,20].
Computer assisted image analysis The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)
[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules
[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.
The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)
[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules
[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.
Definitions of the HEV’s parameters and ratios This is represented by the following alphabets A, B and C (Figure
1).
• number of all HEV : A
• number of dilated HEV (defined as lumen size more than 80 square micron) : B
• number of dilated HEV with red blood cells (RBC) within its lumen : C
The different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).
In addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as
• Ratio of dilated HEV to the total number of HEV : B/A
• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B
• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A
This is represented by the following alphabets A, B and C (Figure
1).
• number of all HEV : A
• number of dilated HEV (defined as lumen size more than 80 square micron) : B
• number of dilated HEV with red blood cells (RBC) within its lumen : C
The different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).
In addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as
• Ratio of dilated HEV to the total number of HEV : B/A
• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B
• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A
Statistical analysis Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.
Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.
|
Results
|
The association of HEV and clinical outcome was studied by initially analyzing the OS and DFI of the 2 groups prior to individually analyzing the various HEV parameters (A, B, C, B/A, C/B, C/A) against OS and DFI. The details of the preliminary results comparing HEV parameters and patient outcomes have been previously published in an earlier paper
[20]. In the secondary analysis, we further analyzed the tumor pathological characteristics (i.e. primary tumor volume, the stage of the disease and grade of the tumor) against clinical outcome (OS and DFI), to determine if these conventional tumor factors were prognostic in nature. We further investigated the role of HEV and its prognostic value by comparing and analyzing the HEV parameters with respect to the tumor volume, grade and stage. These analyses were repeated without controlling for the group factor (i.e. taking the sample population as a single cohort). Finally, we investigated the HEV parameters in each group to determine if there was a difference in the HEV values in the presence or absence of metastasis in the LN.
We analyzed the DFI and OS of the 65 patients with respect to their groups (35 cases versus 30 controls) to estimate the relative risk of DFI and OS associated with the group.
Overall survival analysis The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure
2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table
1).
Kaplan Meier overall survival curves for the two groups (Cases vs. Controls).
Summary of results
The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure
2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table
1).
Kaplan Meier overall survival curves for the two groups (Cases vs. Controls).
Summary of results
Disease free interval analysis Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure
3).
Disease free interval curves for the two groups (Cases vs. Controls).
There was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table
1).
Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure
3).
Disease free interval curves for the two groups (Cases vs. Controls).
There was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table
1).
Secondary analysis Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table
2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table
3).
Summary of secondary analysis results
Comparing high endothelial venules parameters in the 2 patient groups
Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table
2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table
3).
Summary of secondary analysis results
Comparing high endothelial venules parameters in the 2 patient groups
|
Conclusion
|
This study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets.
|
[
"Background",
"High endothelial venules and its role in cancer metastasis",
"Objectives of study",
"Immunohistochemistry",
"Computer assisted image analysis",
"Definitions of the HEV’s parameters and ratios",
"Statistical analysis",
"Overall survival analysis",
"Disease free interval analysis",
"Secondary analysis",
"Limitations",
"Abbreviations",
"Competing interests",
"Authors’ contribution"
] |
[
"Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.",
"There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].",
"We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.",
"A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].",
"The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.",
"This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A",
"Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.",
"The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results",
"Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).",
"Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups",
"Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.",
"HEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.",
"The authors declare that they have no competing interests, financial or non-financial.",
"LSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"High endothelial venules and its role in cancer metastasis",
"Objectives of study",
"Methods",
"Immunohistochemistry",
"Computer assisted image analysis",
"Definitions of the HEV’s parameters and ratios",
"Statistical analysis",
"Results",
"Overall survival analysis",
"Disease free interval analysis",
"Secondary analysis",
"Discussion",
"Limitations",
"Conclusion",
"Abbreviations",
"Competing interests",
"Authors’ contribution"
] |
[
"Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.\nOropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States\n[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy\n[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.\nThe first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin\n[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)\n[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised\n[6,7].\n High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\nThere is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].\n Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.\nWe aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.",
"There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy\n[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance\n[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors\n[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically\n[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ\n[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses\n[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”\n[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis\n[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases\n[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route\n[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients\n[6].\nIn addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit\n[5,10].",
"We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis\n[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.",
"Approval was obtained from the Institutional Review Boards of both institutions involved in the study (2007/464/B). The study population consisted of 175 consecutive patients with SCC of the head and neck who had received primary surgical treatment at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Center, Singapore, from January 2001 through to December 2005. The patients’ pathological and clinical data including follow-up information was reviewed from a prospective database. The inclusion criteria included all surgically treated patients with histologically proven SCC tongue who underwent a neck dissection, with a minimum of 2-year follow up period. Patients with a second primary cancer and patients who did not have a neck dissection as part of their primary treament were excluded. There were a total 65 patients that met the study inclusion criteria. There were 35 patients in the group with primary SCC tongue without pathologically proven lymph node metastases in their neck dissection specimen who were designated as “cases”, and 30 patients in the group with primary SCC tongue with pathologically proven lymph node metastases in the neck dissection specimens, who were designated as “controls”. All patients included had radical excision of the primary tongue lesion and a neck dissection according to departmental protocol. In both groups, all patients had radical enbloc excision of the primary tongue lesion with or without resection of adjacent structures with either a unilateral or a bilateral, supraomohyoid neck dissection or a modified radical neck dissection. Tumours were classified according to the AJCC TNM Staging Classification, based on a pre-operative clinical evaluation and this determined the type of neck dissection performed by the primary surgeon\n[19]. All cases were discussed at a multi-discplinary head and neck tumor board. Intra-operative frozen sections were performed for margins of the tongue lesion and further resection of the tongue was performed in event that the surgical margins were involved. Upon discharge from the hospital, they were followed up at regular intervals of monthly, 3-monthly, 6-monthly and yearly at a progessive rate. Inpatient and outpatient clincal data, pathological, radiological and operative records were retrieved from a central medical records office and from the Department of Pathology, Singapore General Hospital. Mortality data was obtained and confirmed from the hospital central medical records office as well as the Singapore Registry of Births & Deaths.\n Immunohistochemistry A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\nA histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].\n Computer assisted image analysis The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\nThe digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.\n Definitions of the HEV’s parameters and ratios This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\nThis is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A\n Statistical analysis Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.\nCorrelating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.",
"A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed\n[5,20].",
"The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)\n[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules\n[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.",
"This is represented by the following alphabets A, B and C (Figure\n1).\n• number of all HEV : A\n• number of dilated HEV (defined as lumen size more than 80 square micron) : B\n• number of dilated HEV with red blood cells (RBC) within its lumen : C\nThe different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).\nIn addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as\n• Ratio of dilated HEV to the total number of HEV : B/A\n• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B\n• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A",
"Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.",
"The association of HEV and clinical outcome was studied by initially analyzing the OS and DFI of the 2 groups prior to individually analyzing the various HEV parameters (A, B, C, B/A, C/B, C/A) against OS and DFI. The details of the preliminary results comparing HEV parameters and patient outcomes have been previously published in an earlier paper\n[20]. In the secondary analysis, we further analyzed the tumor pathological characteristics (i.e. primary tumor volume, the stage of the disease and grade of the tumor) against clinical outcome (OS and DFI), to determine if these conventional tumor factors were prognostic in nature. We further investigated the role of HEV and its prognostic value by comparing and analyzing the HEV parameters with respect to the tumor volume, grade and stage. These analyses were repeated without controlling for the group factor (i.e. taking the sample population as a single cohort). Finally, we investigated the HEV parameters in each group to determine if there was a difference in the HEV values in the presence or absence of metastasis in the LN.\nWe analyzed the DFI and OS of the 65 patients with respect to their groups (35 cases versus 30 controls) to estimate the relative risk of DFI and OS associated with the group.\n Overall survival analysis The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\nThe relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results\n Disease free interval analysis Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\nSimilarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).\n Secondary analysis Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups\nTumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups",
"The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure\n2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table\n1).\nKaplan Meier overall survival curves for the two groups (Cases vs. Controls).\nSummary of results",
"Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure\n3).\nDisease free interval curves for the two groups (Cases vs. Controls).\nThere was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table\n1).",
"Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table\n2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table\n3).\nSummary of secondary analysis results\nComparing high endothelial venules parameters in the 2 patient groups",
"Squamous cell carcinoma of the tongue has one of the fastest rising rate today affecting the young with a high propensity for LN metastases.\n[22] The presence of metastatic cells in regional LN, along with extracapsular spread are the most important prognostic factors in patients with squamous cell carcinoma (SCC) of the tongue\n[22-25]. The prognosis is worse compared to other equivalent carcinomas of the head and neck region.\nTumor cell metastasis to regional LN marks one of the initial steps in the cascade of tumor metastasis after tumor cell progression in the primary tumor. The lymphatic metastatic cascade is a series of complex interrelated steps and processes\n[26]. As the tumor grows and enlarges, cytokines are secreted to promote lymphangiogenesis\n[27]. As malignant cells invade the extracellular matrix, they enter the lumen of the lymphatics and they move in clusters or as single cells to the first echelon of regional LN, otherwise known as the SLN\n[28,29]. The structural properties, such as the lack of basement membrane in terminal lymphatics, the presence of the intercellular clefts, the traumatic environment in the blood including shear forces of blood turbulence and tumor antigenicity detection by host immune cells, contribute and account for the observation that in many solid cancers, lymphatic metastasis precedes metastasis via the vascular system\n[26,30]. The relationship between angiogenesis and lymphangiogenesis has been extensively studied in cancer research\n[14,26,31,32]. This is pertinent in the biology of LN metastasis where the two systems literally lie side by side. There is extensive evidence showing members of the Vascular endothelial growth factor (VEGF) family, namely VEGF-C, VEGF-D and VEGF-A\n[33], are not only important regulators of lymph vessel growth in vivo, but may also be involved in the process of promoting lymphatic metastasis, such as VEGF-C\n[27,34-36]. This is significant because there is evidence that tumor can activate both LN lymphangiogenesis and angiogenesis before metastasis, and this would translate into logical efforts in the field of anti-cancer research specifically targeting pathways of tumor lymphangiogenesis and angiogenesis. There have been encouraging results from therapeutic targeting of the various VEGF member pathways to inhibit lymph node metastasis, reduce lymphatic and vascular density in LN as well as to reduce overall tumor burden in the lymph nodes and distant metastases\n[34,37,38].\nTargeted therapy such as anti-angiogenesis drugs has shown positive results in clinical studies. Bevacizumab (AvastinTM), a monoclonal antibody to VEGF-A, was approved by United States Food and Drug Administration (FDA) in 2004 and has proven clinical benefits in treatment of metastatic colorectal cancer when the drug was added to standard chemotherapy and also has positive results in metastatic kidney cancer and advanced lung cancer. There are many ongoing trials including Phase III clinical trials in advanced or metastatic renal cell carcinoma, pancreatic cancer, and ovarian cancer for AvastinTM[39]. Other molecular pathways that harbor potential targets which have varying degrees of anti-angiogenesis and anti-lymphangiogenesis properties, are ligands such as hepatocyte growth factor (HGF), receptors like Neuropilin 2, PDGFRα/β and others such as Angiostatin, interferon α/β and platelet-factor 4\n[40]. Two other antiangiogenic drugs, Sorafenib (Nexavar™, Bayer) and Sunitinib (Sutent™, Pfizer), have also been approved by the FDA for various aspects of cancer treatment, for example, Sorafenib and Sunitinib have been beneficial in the treatment of metastatic renal-cell cancer and advanced hepatocellular carcinoma\n[41,42]. They target multiple receptor tyrosine kinases, including VEGF receptors and platelet-derived growth factor (PDGF) receptors\n[43].\nIn our series, we detected a statistical significance when we analyzed the OS but not in DFI in the patients with or without LN metastasis (cases vs. controls) (Figures\n2 and\n3). This may be attributed to our limited sample size. It has been proposed and is currently being validated, that SLN biopsy may play the next stage in the evolution in the neck management and treatment of tongue SCC\n[44]. Exploration and understanding of this concept coupled with advances and optimistic results in anti-angiogenesis therapy involving VEGF anti-metabolites translates the next logical exploration to be invested in the study of the pathogenesis of lymph node metastasis. It was shown that the lumen of the lymphatic sinuses and blood vessels were dilated in SLN before metastasis\n[5]. We have demonstrated that lymph nodes are transformed by the primary tongue tumor to become a functional blood vessel–enriched organ before and independent of metastasis, with the changes in the morphology of the HEV to become main blood flow carrier in the lymph node (Figure\n4). This process of vascularization in the LN (HEV morphological alterations) appears similar and consistent in human tissues and previous animal models\n[5,20]. This study presents functional and structural data that the primary tumor can possibly manipulate the microenvironment and biology of lymph nodes to potentially facilitate the proliferation of subsequent tumor deposits leading to established metastases. The morphological alteration of HEV in the presence of a cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shift and phenotypic change of HEV from its known function as a lymphocyte recruitment modulator to one as a blood carrier.\nDilated HEVs with red blood cells in its lumen (high power field).\nIn previous studies, the degree of lymphatic dilation in the SLN significantly correlated with the primary tumor weight\n[5,10]. This observation suggests that the lymphatic fluid from the primary tumor induced the persistent alteration of the lymph channel in SLN. These results are consistent with findings that demonstrate, in contrast to angiogenesis, in which blood flow proceeds only after the vessel develops, lymphangiogenesis can be induced by interstitial fluid channeling\n[45]. In this aspect, this is analogous to immunology studies showing the alteration of lymph channels in SLN can also occur during an inflammatory reaction, which facilitates the migration of inflammatory cells\n[46,47] The difference in cancer metastasis biology and inflammatory process starts here. In endotoxin induced experiments and immunology studies, the dilated lymphatic sinuses and vessels in the associated reactive LN were full of lymphocytes. In contrast in tumor-reactive lymphadenopathy, the lymphatics contained minimum or no cellular material, suggesting different roles of the SLN lymphatic channels in different pathologic processes. It has been well characterized that the HEV of lymph nodes play an important role in recruiting lymphocytes for the generation of immune responses\n[9,11]. By expressing homing receptors on their surface, which blood lymphocytes can recognize as they pass in circulation, HEV provide a unique location where naive lymphocytes can enter the lymph node\n[47,48].\nIn our previous study\n[5], we found that the role of HEV was transformed from a lymphocyte recruiter to become the main blood flow carrier in the SLN prior to metastasis. We have shown in this study that in the regional cervical LN, not only could the morphology of individual HEV change dramatically to increase blood flow, but the proliferation rate of HEV endothelial cells was also increased before metastasis. This transformation of HEV as a lymphocytic carrier in LN to a blood-flow carrier can be seen in the large quantity of red blood cells visible in the HEV even in pre-metastatic regional LN. More significantly, this phenomenon is also more pronounced in the regional LN of the patients with established LN metastasis. This is consistent with the findings of Chung and colleagues demonstrating the increased HEV density in SLN of oral SCC before the arrival of tumor cells, the HEV density in metastatic SLN was also higher than that of non-metastatic SLN\n[6]. We have shown this in our supplementary data that patients with lymph node metastases have more dilated HEV with RBC in their LN as compared to patients without LN metastasis. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV(C/B) in the control (pN+) group is statistically significantly higher than that in case group (p-value = 0.0318).\nIn addition, with the benefit of patients’ pathological and follow-up information, we have demonstrated that even with just an increase of 1 HEV in a high-power-field (HPF), regardless of the group, the risk is 1.024 times worse in terms of overall survival. This is consistent with clinical knowledge and natural history of the disease. It is well established that patients without LN metastases in their regional LN (cases in our cohort) have a significantly better prognosis, and; this fact is also reflected in our analysis\n[22,49-51] (Figure\n2). Cases had a 0.428 times the risk of Controls with regards to overall survival if they had the same number of HEV. It is crucial to understand that this risk is exponential in nature. To illustrate the significance of this result, an example will be used: Patient A with tongue SCC has proven LN metastases in the cervical LN. On further immunohistochemical staining, it is found that he has 0 HEV in a random high-power-field. Comparing this to patient B who has the same grade, stage of tumor, degree of LN metastases as patient A but has 100 HEV in a HPF, patient B will have a [ (1.024)100 = 10.715 ] 10 times worse prognosis in OS as compared to patient A. We will then consider patient C who has tongue SCC with no LN metastasis. If the HEV amount in a HPF is the same as either patient A or B, patient C’s risk as compared to either patient A or B will be 2.34 times (1/0.428= 2.34) better in terms of overall survival (Table\n4). Although this did not reach statistical significance (p=0.471), we believe this to be a factor of a small sample size.\nDifferences in OS and DFI relative risk in the 2 groups\nAssuming that our hypothesis is true, HEV transformation from a normal immunological mediator to a tumor metastasis mediator is reflected by morphological changes from a normal appearing HEV, to a dilated HEV, and then to a dilated HEV containing RBC. We believe that this metamorphosis occurs on a spectrum, and this process begins with the HEV increasing in absolute numbers, then each becoming more dilated before progressing into a functional vessel carrying blood (Figure\n5). Hence we analyzed our data looking at the 3 different stages of HEV transformation and the ratio comparing each parameter in a systematic way to demonstrate this spectrum (Figure\n1).\nMetamorphosis of High endothelial venules in a tumor microenvironment. This process begins with the high endothelial venules increasing in absolute numbers, subsequently each of them becoming more dilated and lastly every one of them will become a function vessel carrying blood.\nWe recall that that in the last example we illustrated that a patient’s OS risk is 1.024 times worse than an equivalent patient if there is 1 more HEV in one HPF of his regional LN, the risk is increased to 1.071 if the HEV is a dilated HEV. The risk is even higher at 1.116 times if the HEV is a dilated HEV containing RBC (Table\n1)\n[20]. This trend is also seen when all patients are considered in a single cohort. A patient’s OS risk is worsened by 1.004 times if there is an increase of 1 dilated HEV in a HPF. If that dilated HEV contains RBC, the OS risk is increased to 1.008 times.\nIn the treatment of tongue carcinomas, DFI is important as it signifies the failure of loco-regional control for which there is little effective therapy. Further surgery e.g. neck dissection to remove loco-regional recurrences is plagued with high morbidity and mortality. This, coupled with poor chemotherapy and radiotherapy response rates in recurrences, often equates to rapid deterioration of the patient’s condition. Disease free interval was also analyzed in the same manner to the three different HEV morphological phenotypes.\nWe found statistical significance in the relationship between the total number of HEV (A) and DFI when we controlled for the group (i.e. taking in the presence of LN metastasis as a factor). (p=0.022). This significance is preserved when we analyzed all the patients as a cohort (p=0.023)\n[20]. This means that the quantity of HEV is inversely related to DFI. If you consider the LN status and take it into statistical consideration, if there is one more HEV in a HPF, the DFI is 1.051 times worse than zero HEV. It is again important to note that the relationship of the quantity of HEV with respect to DFI is exponential in nature. For example, a patient (patient B) with 100 more HEV (A) in one HPF has a {(1.051)100 =144.634} 144 times shorter DFI as compared to an equivalent patient with only one HEV per HPF (patient A) (Table\n4). Excluding the group effect and considering all the patients as a cohort, a patient with 1 more HEV per HPF will have a DFI that is 1.051 times worse than a patient with zero HEV in a HPF (p=0.023).\nWe looked at the different ratios of abnormal HEV and compared them to several clinic-pathologic parameters namely overall survival, disease–free interval, tumor volume, stage and grade of the tumor. The details are specified in the secondary results (Table\n2).\nThere is a general trend observed. The more advanced the disease, the higher the ratio/percentage of abnormality of HEV. This can be seen in when we analyzed OS and the 2 ratios. The OS relative risk worsens by 1.078 if the ratio B/A (ratio of dilated HEV to the total number of HEV) increased by 1, while the OS relative risk worsens by 3.624 times if the ratio of dilated HEVs with RBC to the total number of HEV increases by 1. Most importantly, if we consider the most abnormal form of HEV (dilated HEV with RBC within its lumen, C) and look at it as a ratio to the total no. of HEV, a patient’s OS relative risk worsens by 17.884 times if this ratio (C/A) increases by a factor of 1. This observation approaches marginal significance. (p = 0.171) (Figure\n6).\nOverall survival relative risk with respect to the different high endothelial venules ratios.\nIn this study, we have shown the relationship of HEV and their transformation in a cancerous environment. We also note that in previous studies the HEV morphology did not alter at all in endotoxin-induced lymphadenopathy, implying a selective reaction of HEV in the cancerous condition\n[5]. Distinct, differentiated gene expression has also been reported when the endothelial cells respond to the changing of their microenvironment\n[52]. In a previous study, it was shown that the cellular morphology of the tall endothelial cells forming HEV changed dramatically to become flat endothelial cells in the presence of cancer. As a consequence, the HEV was remodelled from a thick-walled, endothelial vessel with a small lumen to a thin walled, large-lumen vessel shifting its function from recruitment of lymphocytes to becoming a blood vessel (Figure\n4)\n[10]. These facts indicate that the blood vessel endothelium has tremendous potential to adapt its environment. The confined lymph and blood channel alterations within the SLN, but not in the next station lymph node, imply that an inducer from the primary tumor is functioning locally with the existence of an active primary cancer. VEGF-A has been found to be an inducer of lymphangiogenesis in SLN\n[33]. Other studies have shown that serum level of VEGF-A was elevated in patients with late stage NPC\n[21] VEGF-A is a secreted protein factor that can travel in blood to other lymph nodes, these findings suggest that there may be other inducers involved. It is also of interest to explore the role of HEV after the establishment of a metastatic tumor nest. The enlarged, remodelled HEV could integrate into the metastatic tumor vasculature with further differentiation, characterized by the gradual loss of their specific marker MECA-79 from the tumor margin to the central part of the metastatic tumor nest.\n[5,10] It has been explained that, compared with primary tumors, the more rapid growth of metastatic lesions in the cervical lymph nodes of NPC patients was due to clonal selection of the cancer cells during metastases, with highly proliferative clones disseminated to the cervical nodes. However, based on our findings, the metastatic tumor vasculature in lymph nodes consists of many large blood vessels derived from normal HEVs, suggesting that the efficiency of nutrition and oxygen supplies could be better for the metastatic tumor cells in the involved lymph node. The enrichment of the blood supply in the lymph node before and after metastasis may favor the growth of newly arriving metastatic cancer cells. Consequently, as evident clinically in the long term follow-up of cancer patients, the involved regional lymph nodes may become manifest first, whereas the primary tumor remain clinically occult for years\n[53]. Moreover, the high density of functioning blood vessels in lymph nodes may subsequently facilitate the metastasis of cancer cell to distant organs. Conversely, consistent with its role in lymphocyte trafficking, if the HEV density is increased in the tumor themselves as opposed to LN, it has favorable prognostic value, Martinet et al. reported that in 146 breast cancers, the density of tumor HEV was an independent prognostic risk factor for DFI and OS\n[8]. This further suggests the tumor HEV function as major gateway for lymphocyte infiltration into tumors and represents potential targets for cancer immunotherapy. It will also be important to elucidate if the highly vascularized premetastatic SLN is associated with an increased metastatic potential. Control of lymphatic fluid movement may also be a target to consider in preventing metastases.\nAssuming our hypothesis is true with regards to the role of HEV in cancer metastasis, a potential therapeutic approach is to interrupt or block this remodelling process and evaluate if there is an effect on metastasis. This will confirm the HEV’s central role in the pathogenesis of metastasis.\nFurther studies need to designed and performed to elucidate the pathways of HEV transformation. A control gene for the growth and differentiation of HEV remains to be identified\n[9]. With regards to this, a recently identified nuclear factor (NF), NF-HEV has potential because it is preferentially expressed by HEV\n[54]. Dendritic cells, well known for their antigen presentation roles has recently been shown to modulate the phenotype of HEV and control entry of naive lymphocytes to LN, their role in a cancer microenvironment is also an area of potential research\n[13]. There are recent experiments in mouse models investigating conditional gene targeting of HEV as well\n[55]. Identification and characterization the molecular pathways and the control genes would have considerable clinical applications. For example, it would enable the design of experiments and studies to induce or retard the formation of HEV in various tissues, including tumors and their LN, improving vaccination strategies against pathogens and cancers. Therefore, there is a wealth of translational applications with regards to targeting HEV as a ligand for cancer investigational therapeutics. It paves the journey in the discovery of many unknown genes and molecules with potential clinical importance. Lastly, more work can be performed to validate our findings in other solid cancers.\n Limitations Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.\nOur study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.",
"Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.\nIt is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.\nSecondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV\n[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.\nA major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.\nFinally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.",
"This study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets.",
"HEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.",
"The authors declare that they have no competing interests, financial or non-financial.",
"LSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript."
] |
[
null,
null,
null,
"methods",
null,
null,
null,
null,
"results",
null,
null,
null,
"discussion",
null,
"conclusions",
null,
null,
null
] |
[
"High endothelial venules",
"Cancer metastasis",
"Angiogenesis",
"Lymph nodes"
] |
Background:
Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.
Oropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States
[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy
[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.
The first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin
[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)
[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised
[6,7].
High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy
[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance
[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors
[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically
[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ
[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses
[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”
[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis
[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases
[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route
[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients
[6].
In addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit
[5,10].
There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy
[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance
[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors
[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically
[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ
[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses
[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”
[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis
[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases
[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route
[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients
[6].
In addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit
[5,10].
Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis
[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.
We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis
[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.
High endothelial venules and its role in cancer metastasis:
There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy
[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance
[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors
[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically
[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ
[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses
[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”
[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis
[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases
[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route
[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients
[6].
In addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit
[5,10].
Objectives of study:
We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis
[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.
Methods:
Approval was obtained from the Institutional Review Boards of both institutions involved in the study (2007/464/B). The study population consisted of 175 consecutive patients with SCC of the head and neck who had received primary surgical treatment at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Center, Singapore, from January 2001 through to December 2005. The patients’ pathological and clinical data including follow-up information was reviewed from a prospective database. The inclusion criteria included all surgically treated patients with histologically proven SCC tongue who underwent a neck dissection, with a minimum of 2-year follow up period. Patients with a second primary cancer and patients who did not have a neck dissection as part of their primary treament were excluded. There were a total 65 patients that met the study inclusion criteria. There were 35 patients in the group with primary SCC tongue without pathologically proven lymph node metastases in their neck dissection specimen who were designated as “cases”, and 30 patients in the group with primary SCC tongue with pathologically proven lymph node metastases in the neck dissection specimens, who were designated as “controls”. All patients included had radical excision of the primary tongue lesion and a neck dissection according to departmental protocol. In both groups, all patients had radical enbloc excision of the primary tongue lesion with or without resection of adjacent structures with either a unilateral or a bilateral, supraomohyoid neck dissection or a modified radical neck dissection. Tumours were classified according to the AJCC TNM Staging Classification, based on a pre-operative clinical evaluation and this determined the type of neck dissection performed by the primary surgeon
[19]. All cases were discussed at a multi-discplinary head and neck tumor board. Intra-operative frozen sections were performed for margins of the tongue lesion and further resection of the tongue was performed in event that the surgical margins were involved. Upon discharge from the hospital, they were followed up at regular intervals of monthly, 3-monthly, 6-monthly and yearly at a progessive rate. Inpatient and outpatient clincal data, pathological, radiological and operative records were retrieved from a central medical records office and from the Department of Pathology, Singapore General Hospital. Mortality data was obtained and confirmed from the hospital central medical records office as well as the Singapore Registry of Births & Deaths.
Immunohistochemistry A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed
[5,20].
A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed
[5,20].
Computer assisted image analysis The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)
[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules
[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.
The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)
[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules
[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.
Definitions of the HEV’s parameters and ratios This is represented by the following alphabets A, B and C (Figure
1).
• number of all HEV : A
• number of dilated HEV (defined as lumen size more than 80 square micron) : B
• number of dilated HEV with red blood cells (RBC) within its lumen : C
The different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).
In addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as
• Ratio of dilated HEV to the total number of HEV : B/A
• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B
• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A
This is represented by the following alphabets A, B and C (Figure
1).
• number of all HEV : A
• number of dilated HEV (defined as lumen size more than 80 square micron) : B
• number of dilated HEV with red blood cells (RBC) within its lumen : C
The different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).
In addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as
• Ratio of dilated HEV to the total number of HEV : B/A
• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B
• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A
Statistical analysis Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.
Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.
Immunohistochemistry:
A histological review and analysis of the primary SCC tongue lesion and their respective lymph node tissues in the neck dissection specimen from the 65 cases of tongue cancer patients was conducted. The lymph node tissues in each case were separated into levels according to their anatomical locations. The tumor and the neck dissection paraffin tissue blocks from the selected patients were retrieved from the Department of Pathology, Singapore General Hospital. An independent head and neck pathologist reviewed the histological specimens and selected the 3 largest lymph node in both groups for staining of the HEV. The largest 3 LN were chosen as the size of a lymph node correlates with the degree of tumor lymphadenopathy and in the cases of the non-metastatic neck, the degree of reactive lymphadenopathy. Optimisation of the immunohistochemical staining protocol and subsequent staining of the HEV using the purified rat anti-mouse MECA-79 (PNAd) antibody (BD PharMingen, CA, U.S.A), which is specific for HEV, was performed
[5,20].
Computer assisted image analysis:
The digital capture of the HEV images and the quantitative image analyses were performed using the slide scanner (ScanScope, Aperio T3; ScanScope Console v. 9.0. Aperio Technologies, Vista, CA U.S.A) utilising an illuminator(Fiber-Lite DC-950, Dolan Jenner, Boxborough, MA, U.S.A). We analyzed snapshots of the largest 3 lymph nodes under 10x magnification. The total number of HEV, abnormal HEV (defined as having a lumen area of 80 square microns) and the HEV with presence of red blood cells were counted with the aid of imaging software (Image J programme with cell counter plug-in, NIH, Bethesda, MD U.S.A)
[21]. The lumen size of 80 square microns was selected as it is the minimum luminal cross-sectional area for functional vessels, corresponding to the minimum caliber of physiologic venules
[5]. An average value for each of these parameters was calculated for each LN. These 3 values were correlated to the size of its respective LN and the patient’s clinical outcomes, namely the overall survival, disease-free interval and recurrence rate.
Definitions of the HEV’s parameters and ratios:
This is represented by the following alphabets A, B and C (Figure
1).
• number of all HEV : A
• number of dilated HEV (defined as lumen size more than 80 square micron) : B
• number of dilated HEV with red blood cells (RBC) within its lumen : C
The different High endothelial venules parameters. Venn diagram illustrating the relationship between the different HEV parameters (A, B, C).
In addition to these three parameters, we also analyzed the ratios of these abnormal HEV (B and C) with respect to the total number of HEV (A). This is summarized below as
• Ratio of dilated HEV to the total number of HEV : B/A
• Ratio of dilated HEV with RBC within its lumen to total number of dilated HEV : C/B
• Ratio of dilated HEV with RBC within its lumen with respect to total number HEV : C/A
Statistical analysis:
Correlating the clinical information from our prospective database, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with respect to several clinical parameters, namely Disease-Free Interval (DFI) and Overall Survival (OS) in the 2 groups (Cases vs. Controls) and as a single cohort. We used Cox’s Proportional Hazard Model for analysis. In the secondary analysis, we analyzed the 3 parameters (A, B, C) and the 3 ratios (B/A, C/B, C/A) with tumor characteristics. A non-parametric test, Wilcoxon-rank-sum test, was performed to test the difference on HEV and its parameters as well as 3 ratios between the 2 groups of patients. A non-parametric test, Kruskal-Wallis test, was performed to test the difference on the HEV as well as 3 ratios among different stages. All p-values of < 0.05 were considered statistically significant.
Results:
The association of HEV and clinical outcome was studied by initially analyzing the OS and DFI of the 2 groups prior to individually analyzing the various HEV parameters (A, B, C, B/A, C/B, C/A) against OS and DFI. The details of the preliminary results comparing HEV parameters and patient outcomes have been previously published in an earlier paper
[20]. In the secondary analysis, we further analyzed the tumor pathological characteristics (i.e. primary tumor volume, the stage of the disease and grade of the tumor) against clinical outcome (OS and DFI), to determine if these conventional tumor factors were prognostic in nature. We further investigated the role of HEV and its prognostic value by comparing and analyzing the HEV parameters with respect to the tumor volume, grade and stage. These analyses were repeated without controlling for the group factor (i.e. taking the sample population as a single cohort). Finally, we investigated the HEV parameters in each group to determine if there was a difference in the HEV values in the presence or absence of metastasis in the LN.
We analyzed the DFI and OS of the 65 patients with respect to their groups (35 cases versus 30 controls) to estimate the relative risk of DFI and OS associated with the group.
Overall survival analysis The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure
2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table
1).
Kaplan Meier overall survival curves for the two groups (Cases vs. Controls).
Summary of results
The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure
2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table
1).
Kaplan Meier overall survival curves for the two groups (Cases vs. Controls).
Summary of results
Disease free interval analysis Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure
3).
Disease free interval curves for the two groups (Cases vs. Controls).
There was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table
1).
Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure
3).
Disease free interval curves for the two groups (Cases vs. Controls).
There was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table
1).
Secondary analysis Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table
2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table
3).
Summary of secondary analysis results
Comparing high endothelial venules parameters in the 2 patient groups
Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table
2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table
3).
Summary of secondary analysis results
Comparing high endothelial venules parameters in the 2 patient groups
Overall survival analysis:
The relative risk of OS of cases was estimated to be 0.229 times of the risk of control group based on the study sample (95% C.I. 0.048 ~ 1.102). Patients with presence of metastases in their regional cervical LNs had a 4.367 times risk of mortality as compared to patients without metastasis. The Kaplan -Meier curves were significant in terms of overall survival time between controls and cases (p = 0.046) (Figure
2). There was no significance detected found in the total no. of HEV (A); no. of dilated HEV (B); no. of dilated HEV with RBC inside its lumen(C) or any ratios(B/A, C/B, C/A) with respect to the overall survival (Table
1).
Kaplan Meier overall survival curves for the two groups (Cases vs. Controls).
Summary of results
Disease free interval analysis:
Similarly, we compared the DFI between the 2 cases and controls. The risk of cases was estimated to be 0.75 times of the risk of control group based on the sample (95% C.I. 0.342 ~ 1.644). This implied that in patients without LN metastases, if recurrences occur, the DFI tend to be longer. However, this did not achieve statistical significance (p = 0.472) (Figure
3).
Disease free interval curves for the two groups (Cases vs. Controls).
There was a significant association of number of HEVs (A) to the disease free interval (DFI). This was statistically significant when controlling for the group factor (p-value = 0.022), and when analyzing as a single cohort (p-value = 0.023). However, there was no significance detected found in the other HEV parameters in relation to DFI, namely no. of dilated HEVs (B); no. of dilated HEVs with RBC inside its lumen (C) or any of the HEV ratios(B/A, C/B, C/A) with respect to DFI (Table
1).
Secondary analysis:
Tumor pathological data, namely primary tumor volume, the stage of the disease and grade of the tumor were analyzed against OS and DFI, to determine if these conventional tumor factors were prognostic in nature. We did not detect any prognostic value of tumor volume; stage or grade that reached statistical significance (Table
2). As part of the secondary analysis, we compared the HEV parameters in the 2 groups as well as the HEV parameters to the tumor characteristics (tumor stage and its grade only; tumor volume being a continuous variable was excluded). There was no significance detected in either stage or grade of tumor with regards to the HEV parameters. In the final part, we investigated the various HEV parameters in each groups to if there was a difference in the HEV values in the presence or absence of a metastasis in the LN. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV (C/B) in control groups was statistically significantly higher than that in case group (p = 0.0318) (Table
3).
Summary of secondary analysis results
Comparing high endothelial venules parameters in the 2 patient groups
Discussion:
Squamous cell carcinoma of the tongue has one of the fastest rising rate today affecting the young with a high propensity for LN metastases.
[22] The presence of metastatic cells in regional LN, along with extracapsular spread are the most important prognostic factors in patients with squamous cell carcinoma (SCC) of the tongue
[22-25]. The prognosis is worse compared to other equivalent carcinomas of the head and neck region.
Tumor cell metastasis to regional LN marks one of the initial steps in the cascade of tumor metastasis after tumor cell progression in the primary tumor. The lymphatic metastatic cascade is a series of complex interrelated steps and processes
[26]. As the tumor grows and enlarges, cytokines are secreted to promote lymphangiogenesis
[27]. As malignant cells invade the extracellular matrix, they enter the lumen of the lymphatics and they move in clusters or as single cells to the first echelon of regional LN, otherwise known as the SLN
[28,29]. The structural properties, such as the lack of basement membrane in terminal lymphatics, the presence of the intercellular clefts, the traumatic environment in the blood including shear forces of blood turbulence and tumor antigenicity detection by host immune cells, contribute and account for the observation that in many solid cancers, lymphatic metastasis precedes metastasis via the vascular system
[26,30]. The relationship between angiogenesis and lymphangiogenesis has been extensively studied in cancer research
[14,26,31,32]. This is pertinent in the biology of LN metastasis where the two systems literally lie side by side. There is extensive evidence showing members of the Vascular endothelial growth factor (VEGF) family, namely VEGF-C, VEGF-D and VEGF-A
[33], are not only important regulators of lymph vessel growth in vivo, but may also be involved in the process of promoting lymphatic metastasis, such as VEGF-C
[27,34-36]. This is significant because there is evidence that tumor can activate both LN lymphangiogenesis and angiogenesis before metastasis, and this would translate into logical efforts in the field of anti-cancer research specifically targeting pathways of tumor lymphangiogenesis and angiogenesis. There have been encouraging results from therapeutic targeting of the various VEGF member pathways to inhibit lymph node metastasis, reduce lymphatic and vascular density in LN as well as to reduce overall tumor burden in the lymph nodes and distant metastases
[34,37,38].
Targeted therapy such as anti-angiogenesis drugs has shown positive results in clinical studies. Bevacizumab (AvastinTM), a monoclonal antibody to VEGF-A, was approved by United States Food and Drug Administration (FDA) in 2004 and has proven clinical benefits in treatment of metastatic colorectal cancer when the drug was added to standard chemotherapy and also has positive results in metastatic kidney cancer and advanced lung cancer. There are many ongoing trials including Phase III clinical trials in advanced or metastatic renal cell carcinoma, pancreatic cancer, and ovarian cancer for AvastinTM[39]. Other molecular pathways that harbor potential targets which have varying degrees of anti-angiogenesis and anti-lymphangiogenesis properties, are ligands such as hepatocyte growth factor (HGF), receptors like Neuropilin 2, PDGFRα/β and others such as Angiostatin, interferon α/β and platelet-factor 4
[40]. Two other antiangiogenic drugs, Sorafenib (Nexavar™, Bayer) and Sunitinib (Sutent™, Pfizer), have also been approved by the FDA for various aspects of cancer treatment, for example, Sorafenib and Sunitinib have been beneficial in the treatment of metastatic renal-cell cancer and advanced hepatocellular carcinoma
[41,42]. They target multiple receptor tyrosine kinases, including VEGF receptors and platelet-derived growth factor (PDGF) receptors
[43].
In our series, we detected a statistical significance when we analyzed the OS but not in DFI in the patients with or without LN metastasis (cases vs. controls) (Figures
2 and
3). This may be attributed to our limited sample size. It has been proposed and is currently being validated, that SLN biopsy may play the next stage in the evolution in the neck management and treatment of tongue SCC
[44]. Exploration and understanding of this concept coupled with advances and optimistic results in anti-angiogenesis therapy involving VEGF anti-metabolites translates the next logical exploration to be invested in the study of the pathogenesis of lymph node metastasis. It was shown that the lumen of the lymphatic sinuses and blood vessels were dilated in SLN before metastasis
[5]. We have demonstrated that lymph nodes are transformed by the primary tongue tumor to become a functional blood vessel–enriched organ before and independent of metastasis, with the changes in the morphology of the HEV to become main blood flow carrier in the lymph node (Figure
4). This process of vascularization in the LN (HEV morphological alterations) appears similar and consistent in human tissues and previous animal models
[5,20]. This study presents functional and structural data that the primary tumor can possibly manipulate the microenvironment and biology of lymph nodes to potentially facilitate the proliferation of subsequent tumor deposits leading to established metastases. The morphological alteration of HEV in the presence of a cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shift and phenotypic change of HEV from its known function as a lymphocyte recruitment modulator to one as a blood carrier.
Dilated HEVs with red blood cells in its lumen (high power field).
In previous studies, the degree of lymphatic dilation in the SLN significantly correlated with the primary tumor weight
[5,10]. This observation suggests that the lymphatic fluid from the primary tumor induced the persistent alteration of the lymph channel in SLN. These results are consistent with findings that demonstrate, in contrast to angiogenesis, in which blood flow proceeds only after the vessel develops, lymphangiogenesis can be induced by interstitial fluid channeling
[45]. In this aspect, this is analogous to immunology studies showing the alteration of lymph channels in SLN can also occur during an inflammatory reaction, which facilitates the migration of inflammatory cells
[46,47] The difference in cancer metastasis biology and inflammatory process starts here. In endotoxin induced experiments and immunology studies, the dilated lymphatic sinuses and vessels in the associated reactive LN were full of lymphocytes. In contrast in tumor-reactive lymphadenopathy, the lymphatics contained minimum or no cellular material, suggesting different roles of the SLN lymphatic channels in different pathologic processes. It has been well characterized that the HEV of lymph nodes play an important role in recruiting lymphocytes for the generation of immune responses
[9,11]. By expressing homing receptors on their surface, which blood lymphocytes can recognize as they pass in circulation, HEV provide a unique location where naive lymphocytes can enter the lymph node
[47,48].
In our previous study
[5], we found that the role of HEV was transformed from a lymphocyte recruiter to become the main blood flow carrier in the SLN prior to metastasis. We have shown in this study that in the regional cervical LN, not only could the morphology of individual HEV change dramatically to increase blood flow, but the proliferation rate of HEV endothelial cells was also increased before metastasis. This transformation of HEV as a lymphocytic carrier in LN to a blood-flow carrier can be seen in the large quantity of red blood cells visible in the HEV even in pre-metastatic regional LN. More significantly, this phenomenon is also more pronounced in the regional LN of the patients with established LN metastasis. This is consistent with the findings of Chung and colleagues demonstrating the increased HEV density in SLN of oral SCC before the arrival of tumor cells, the HEV density in metastatic SLN was also higher than that of non-metastatic SLN
[6]. We have shown this in our supplementary data that patients with lymph node metastases have more dilated HEV with RBC in their LN as compared to patients without LN metastasis. The average ratio of dilated HEV with RBC within its lumen to total no. of dilated HEV(C/B) in the control (pN+) group is statistically significantly higher than that in case group (p-value = 0.0318).
In addition, with the benefit of patients’ pathological and follow-up information, we have demonstrated that even with just an increase of 1 HEV in a high-power-field (HPF), regardless of the group, the risk is 1.024 times worse in terms of overall survival. This is consistent with clinical knowledge and natural history of the disease. It is well established that patients without LN metastases in their regional LN (cases in our cohort) have a significantly better prognosis, and; this fact is also reflected in our analysis
[22,49-51] (Figure
2). Cases had a 0.428 times the risk of Controls with regards to overall survival if they had the same number of HEV. It is crucial to understand that this risk is exponential in nature. To illustrate the significance of this result, an example will be used: Patient A with tongue SCC has proven LN metastases in the cervical LN. On further immunohistochemical staining, it is found that he has 0 HEV in a random high-power-field. Comparing this to patient B who has the same grade, stage of tumor, degree of LN metastases as patient A but has 100 HEV in a HPF, patient B will have a [ (1.024)100 = 10.715 ] 10 times worse prognosis in OS as compared to patient A. We will then consider patient C who has tongue SCC with no LN metastasis. If the HEV amount in a HPF is the same as either patient A or B, patient C’s risk as compared to either patient A or B will be 2.34 times (1/0.428= 2.34) better in terms of overall survival (Table
4). Although this did not reach statistical significance (p=0.471), we believe this to be a factor of a small sample size.
Differences in OS and DFI relative risk in the 2 groups
Assuming that our hypothesis is true, HEV transformation from a normal immunological mediator to a tumor metastasis mediator is reflected by morphological changes from a normal appearing HEV, to a dilated HEV, and then to a dilated HEV containing RBC. We believe that this metamorphosis occurs on a spectrum, and this process begins with the HEV increasing in absolute numbers, then each becoming more dilated before progressing into a functional vessel carrying blood (Figure
5). Hence we analyzed our data looking at the 3 different stages of HEV transformation and the ratio comparing each parameter in a systematic way to demonstrate this spectrum (Figure
1).
Metamorphosis of High endothelial venules in a tumor microenvironment. This process begins with the high endothelial venules increasing in absolute numbers, subsequently each of them becoming more dilated and lastly every one of them will become a function vessel carrying blood.
We recall that that in the last example we illustrated that a patient’s OS risk is 1.024 times worse than an equivalent patient if there is 1 more HEV in one HPF of his regional LN, the risk is increased to 1.071 if the HEV is a dilated HEV. The risk is even higher at 1.116 times if the HEV is a dilated HEV containing RBC (Table
1)
[20]. This trend is also seen when all patients are considered in a single cohort. A patient’s OS risk is worsened by 1.004 times if there is an increase of 1 dilated HEV in a HPF. If that dilated HEV contains RBC, the OS risk is increased to 1.008 times.
In the treatment of tongue carcinomas, DFI is important as it signifies the failure of loco-regional control for which there is little effective therapy. Further surgery e.g. neck dissection to remove loco-regional recurrences is plagued with high morbidity and mortality. This, coupled with poor chemotherapy and radiotherapy response rates in recurrences, often equates to rapid deterioration of the patient’s condition. Disease free interval was also analyzed in the same manner to the three different HEV morphological phenotypes.
We found statistical significance in the relationship between the total number of HEV (A) and DFI when we controlled for the group (i.e. taking in the presence of LN metastasis as a factor). (p=0.022). This significance is preserved when we analyzed all the patients as a cohort (p=0.023)
[20]. This means that the quantity of HEV is inversely related to DFI. If you consider the LN status and take it into statistical consideration, if there is one more HEV in a HPF, the DFI is 1.051 times worse than zero HEV. It is again important to note that the relationship of the quantity of HEV with respect to DFI is exponential in nature. For example, a patient (patient B) with 100 more HEV (A) in one HPF has a {(1.051)100 =144.634} 144 times shorter DFI as compared to an equivalent patient with only one HEV per HPF (patient A) (Table
4). Excluding the group effect and considering all the patients as a cohort, a patient with 1 more HEV per HPF will have a DFI that is 1.051 times worse than a patient with zero HEV in a HPF (p=0.023).
We looked at the different ratios of abnormal HEV and compared them to several clinic-pathologic parameters namely overall survival, disease–free interval, tumor volume, stage and grade of the tumor. The details are specified in the secondary results (Table
2).
There is a general trend observed. The more advanced the disease, the higher the ratio/percentage of abnormality of HEV. This can be seen in when we analyzed OS and the 2 ratios. The OS relative risk worsens by 1.078 if the ratio B/A (ratio of dilated HEV to the total number of HEV) increased by 1, while the OS relative risk worsens by 3.624 times if the ratio of dilated HEVs with RBC to the total number of HEV increases by 1. Most importantly, if we consider the most abnormal form of HEV (dilated HEV with RBC within its lumen, C) and look at it as a ratio to the total no. of HEV, a patient’s OS relative risk worsens by 17.884 times if this ratio (C/A) increases by a factor of 1. This observation approaches marginal significance. (p = 0.171) (Figure
6).
Overall survival relative risk with respect to the different high endothelial venules ratios.
In this study, we have shown the relationship of HEV and their transformation in a cancerous environment. We also note that in previous studies the HEV morphology did not alter at all in endotoxin-induced lymphadenopathy, implying a selective reaction of HEV in the cancerous condition
[5]. Distinct, differentiated gene expression has also been reported when the endothelial cells respond to the changing of their microenvironment
[52]. In a previous study, it was shown that the cellular morphology of the tall endothelial cells forming HEV changed dramatically to become flat endothelial cells in the presence of cancer. As a consequence, the HEV was remodelled from a thick-walled, endothelial vessel with a small lumen to a thin walled, large-lumen vessel shifting its function from recruitment of lymphocytes to becoming a blood vessel (Figure
4)
[10]. These facts indicate that the blood vessel endothelium has tremendous potential to adapt its environment. The confined lymph and blood channel alterations within the SLN, but not in the next station lymph node, imply that an inducer from the primary tumor is functioning locally with the existence of an active primary cancer. VEGF-A has been found to be an inducer of lymphangiogenesis in SLN
[33]. Other studies have shown that serum level of VEGF-A was elevated in patients with late stage NPC
[21] VEGF-A is a secreted protein factor that can travel in blood to other lymph nodes, these findings suggest that there may be other inducers involved. It is also of interest to explore the role of HEV after the establishment of a metastatic tumor nest. The enlarged, remodelled HEV could integrate into the metastatic tumor vasculature with further differentiation, characterized by the gradual loss of their specific marker MECA-79 from the tumor margin to the central part of the metastatic tumor nest.
[5,10] It has been explained that, compared with primary tumors, the more rapid growth of metastatic lesions in the cervical lymph nodes of NPC patients was due to clonal selection of the cancer cells during metastases, with highly proliferative clones disseminated to the cervical nodes. However, based on our findings, the metastatic tumor vasculature in lymph nodes consists of many large blood vessels derived from normal HEVs, suggesting that the efficiency of nutrition and oxygen supplies could be better for the metastatic tumor cells in the involved lymph node. The enrichment of the blood supply in the lymph node before and after metastasis may favor the growth of newly arriving metastatic cancer cells. Consequently, as evident clinically in the long term follow-up of cancer patients, the involved regional lymph nodes may become manifest first, whereas the primary tumor remain clinically occult for years
[53]. Moreover, the high density of functioning blood vessels in lymph nodes may subsequently facilitate the metastasis of cancer cell to distant organs. Conversely, consistent with its role in lymphocyte trafficking, if the HEV density is increased in the tumor themselves as opposed to LN, it has favorable prognostic value, Martinet et al. reported that in 146 breast cancers, the density of tumor HEV was an independent prognostic risk factor for DFI and OS
[8]. This further suggests the tumor HEV function as major gateway for lymphocyte infiltration into tumors and represents potential targets for cancer immunotherapy. It will also be important to elucidate if the highly vascularized premetastatic SLN is associated with an increased metastatic potential. Control of lymphatic fluid movement may also be a target to consider in preventing metastases.
Assuming our hypothesis is true with regards to the role of HEV in cancer metastasis, a potential therapeutic approach is to interrupt or block this remodelling process and evaluate if there is an effect on metastasis. This will confirm the HEV’s central role in the pathogenesis of metastasis.
Further studies need to designed and performed to elucidate the pathways of HEV transformation. A control gene for the growth and differentiation of HEV remains to be identified
[9]. With regards to this, a recently identified nuclear factor (NF), NF-HEV has potential because it is preferentially expressed by HEV
[54]. Dendritic cells, well known for their antigen presentation roles has recently been shown to modulate the phenotype of HEV and control entry of naive lymphocytes to LN, their role in a cancer microenvironment is also an area of potential research
[13]. There are recent experiments in mouse models investigating conditional gene targeting of HEV as well
[55]. Identification and characterization the molecular pathways and the control genes would have considerable clinical applications. For example, it would enable the design of experiments and studies to induce or retard the formation of HEV in various tissues, including tumors and their LN, improving vaccination strategies against pathogens and cancers. Therefore, there is a wealth of translational applications with regards to targeting HEV as a ligand for cancer investigational therapeutics. It paves the journey in the discovery of many unknown genes and molecules with potential clinical importance. Lastly, more work can be performed to validate our findings in other solid cancers.
Limitations Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.
It is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.
Secondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV
[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.
A major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.
Finally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.
Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.
It is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.
Secondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV
[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.
A major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.
Finally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.
Limitations:
Our study did find some preliminary associations in HEV with regards disease pathology and clinical correlations and demonstrated the HEV phenotype changes in cancer; however, it is premature to draw any strong conclusions. Our study has limitations and there are mainly associated with 2 major factors. It is a retrospective study with a limited sample size and its lacks a molecular experimental component.
It is an investigative and proof-of-concept study as the exact role of HEV role in cancer metastasis is still under investigation. The limited sample size of 65 patients is derived over a period of 5 years and it is a subset of a large group selected from the Head and Neck service database in 2 large tertiary institutions. This sample population size was deemed to be sufficient to show a significant difference in their overall survival between the 2 study groups. Nonetheless, in an effort to reduce confounders and errors in the interpretation of our results, a strict selection criteria was applied to obtain a more homogenous group. Due to the small sample size, we believe that some of the associations in our hypothesis did not reach statistical significance. The retrospective nature of the study is no doubt a limitation but as the nature of this study does not involve therapeutics or a comparison of factors, the retrospective nature actually works to our advantage. We are able to collate a large sample study size with confirmatory clinic-pathological data within a short period of time and concentrate our time and effort in the investigative and analytical aspects.
Secondly, one main investigative feature of our study concentrated largely on the histopathological assessment of the patients’ LN status preserved tissue paraffin blocks in storage. There are limitations to using tissue paraffin sections and the available techniques have their limitations. There are deficiencies in the consistency of the quality of the tissue paraffin blocks, some tissue blocks are less well preserved than others, especially the older blocks may not be stored in optimal conditions over time. We use an optimized technique for our immunohistochemistry using anti-MECA-79 as our sole antibody for HEV
[5]. Ideally we would like to use at least 2 different HEV markers to confirm our findings in event of specificity and sensitivity inaccuracies in the antibody, however, there is no other commercially available antibody for HEV to date. One assumption in the study is that MECA-79’s specificity and sensitivity is high enough and representative of all HEV in our tissues. Ultimately, analysis at the level of molecular genetics will be the critical factor in confirming the value of immunohistochemical stains in the assessment of biological behavior and prognosis. A considerable problem to be overcome is the marked variation in tissue staining that can be encountered, both in different, patients, neoplasms and in different laboratories. These differences reflect the varied biology of neoplasms, as well as differences in fixation and technique. These variations make comprehensive interpretation of the data and results a worthwhile challenge.
A major limitation in the study is the lack of experimental evidence and results to strengthen our observatory data. The next step in proving our hypothesis is to establish molecular pathway experiments and studies to elucidate the pathways and the molecular mechanisms underlying the phases of HEV’s metamorphosis peri-metastasis. In order to systematically study the role of the modified HEV as a blood vessel and a shortcut mechanism for metastasis, appropriate tracer molecules with real time imaging technology and xenograft experiments studying the mechanism of the lymph node microenvironment changes correlating to the spreading of cancer cells should be pursued. As the objective of this study is to establish the presence of a correlation between HEV and clinico-pathological features in cancer patients, the molecular experiments are not included but are planned for as stated in our future directions.
Finally, ideally the best controls should include neck LN dissection specimens in patients without cancer to optimally illustrate the spectrum of HEV changes from a normal to a pre-metastatic stage and finally to a metastatic one however in reality, it is rare to obtain such specimens and thus not included in the study.
Conclusion:
This study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets.
Abbreviations:
HEV: High endothelial venules; LN: Lymph nodes; RBC: Red blood cells; SLN: Sentinel lymph node; SCC: Squamous cell carcinoma; OS: Overall survival; DFS: Disease free survival; PNAd: Peripheral node addressin; VEGF: Vascular endothelial growth factor; EC: Endothelial cells; FDA: Food and drug administration; HGF: Hepatocyte growth factor; PDGF: Platelet-derived growth factor; NPC: Nasopharyngeal carcinomas; HPF: High-power-field; NF-HEV: Nuclear factor - High endothelial venules.
Competing interests:
The authors declare that they have no competing interests, financial or non-financial.
Authors’ contribution:
LSY, QCN, SKC conceived the concept, study design and designed the experiments. LSY, OAS, WJC, WHM, MSS collected the data and performed the experiments. SKH, LSY performed the histopathology review: LSY, CP, QCN analyzed the data. LSY, QCN wrote the manuscript. QCN, SKC supervised and critically reviewed the manuscript: All authors have contributed significantly, read and approved the final manuscript.
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Background:
High endothelial venules (HEV) have been recognized to play a role in metastasis by its changes seen in the cancer microenvironment of lymph nodes (LN) and solid cancers. Squamous cell carcinoma (SCC) of the tongue is a prevalent tumor of the head and neck region with high propensity for LN metastasis. The extent of LN metastasis is the most reliable adverse prognostic factor. Primary tumors can induce vasculature reorganization within sentinel LN before the arrival of tumor cells and HEV represents these remodelled vessels. This study aims to evaluate the cancer induced vascular changes in regional lymph nodes (LN) of patients by studying the morphological and functional alterations of HEV and its correlation with clinical outcome and pathological features.
Methods:
This study was based on 65 patients with SCC tongue who underwent primary surgical treatment including neck dissection. The patients were categorized into 2 groups based on the presence of malignancy in their cervical lymph nodes. A review of the patients' pathological and clinical data was performed from a prospective database. Immunohistochemical staining of the tissue blocks for HEV and high-power-field image analysis were performed and analyzed with correlation to the patients' clinical and pathological features.
Results:
The total number of HEV was found to be significantly associated to disease-free interval. There was a similar association comparing the HEV parameters to overall survival. The density of abnormal HEV was significantly higher in patients with established metastases in their lymph nodes and HEV was shown to be a better prognosis factor than conventional tumor staging. The HEV morphological metamorphosis demonstrates a spectrum that correlates well with disease progression and clinical outcome.
Conclusions:
The results suggest that the HEV displays a spectrum of morphological changes in the presence of cancer and LN metastasis, and that HEV is possibly involved in the process of cancer metastasis. We revealed the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environments in regional lymph nodes of tongue cancer patients in relation to clinical outcomes. The significant observation of modified dilated HEV containing red blood cells in lymph nodal basin of a cancer suggests the shifting of its function from one primarily of immune response to that of a blood carrying vessel. It also demonstrated potential prognostic value. More studies are needed to elucidate its potential role in cancer immunotherapy and as a potential novel therapeutic approach to preventing metastasis by manipulating the remodelling processes of HEV.
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Background:
Cancer research has focused significantly on the pathogenesis of metastasis as the presence of metastases often translate to a poor prognosis with relatively few effective therapeutic measures.
Oropharyngeal carcinoma is ranked among the top ten most common cancer diagnosed in men in the United States
[1]. Of all the carcinomas of the head and neck, tongue is the most prevalent site. The 5-year survival rate for oral cancer has not improved significantly over the past several decades and remains at 50–55%, despite current advances in surgery and radiation therapy
[2,3]. This is primarily because mortality results from metastatic disease and local recurrence.
The first draining lymph node was coined the “sentinel node” 3 decades ago by Cabanas, who defined the concept of the sentinel node being the doorway to the regional node basin
[4]. Sentinel lymph node (SLN) metastasis is the preliminary process in the spread of cancer in many malignancies. The sentinel lymph nodes undergo morphological and functional changes induced by the primary tumor. Some of these changes are brought into effect by vasculature and lymph channel reorganizations before the arrival of cancer cells and the key blood vessels in such lymph nodes (LN) that are remodeled are identified as high endothelial venules (HEV)
[5]. Tumor-reactive lymphadenopathy in SLN has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain poorly characterised
[6,7].
High endothelial venules and its role in cancer metastasis There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy
[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance
[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors
[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically
[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ
[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses
[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”
[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis
[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases
[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route
[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients
[6].
In addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit
[5,10].
There is emerging data elucidating the potential role of specialized blood vessels called HEV in cancer, either as a prognostic factor or as a potential modulator or target in tumor immunotherapy
[5,6,8-10]. High endothelial venules are specialized post-capillary venules found in lymphoid tissues, located mainly in the T-call zones such as the para-cortical areas of LN. They are morphologically and functionally distinct from ordinary venules and after the vascular endothelial cells (EC) of the blood–brain barrier, the EC of HEV are the next most well characterized. Each HEV has a prominent peri-vascular sheath; a thick basal lamina and the layer of ECs are tall and plump in appearance
[9,11]. High endothelial venules were first characterized and studied extensively in the field of immunology. The evidence suggests that HEV has a central role in lymphocyte trafficking to LN, allowing the entrance of native L-selectin high cells in to the LN parenchyma and this is largely mediated by chemokines produced in and around HEV such as the peripheral node addressins (PNAds). There is a synchrony between HEV and lymphatic vessels as seen in immunization studies, which reveal that the lymphoid tissue microenvironment is crucial for the maintenance of HEV characteristics during homeostasis and the close association in time, function and space between lymphatics and HEV in the remodeling process after immunization indicate that the two systems are closely related and engage in cross-talk through various factors
[9,12-14]. The LN undergo remodeling with changes in complex kinetics of lymph flow, cell content flow, blood flow, HEV gene expression as well as morphologically
[12]. Recently, with animal models, it was shown that before the arrival of metastasis in the SLN, there are reorganizations of vasculature and lymphatic channels resulting in the SLN becoming a functional blood vessel enriched organ
[5]. These prominent blood vessels are identified as remodeled HEV. The role of HEV in immune function and in a cancer metastasis microenvironment has some differences. In inflammatory conditions, the chief role of HEV in the traffic control of lymphocytes is evident from the presence of lymphocytes in the dilated lymphatic sinuses
[9,13]; whereas in tumor-reactive lymphadenopathy, there are few cells, suggesting a different process. There are also studies in murine models to suggest that the movement of tumor cells to LN resembles the normal migration of dendritic cells during immune stimulation, resulting in the term “Tumor cell trafficking”
[9,15]. This observation, coupled with the knowledge of the intimate relationship between HEV and lymphatic vessels in LN leads to our proposed hypothesis that HEV might provide the shortcut or a bypass route connecting the vascular and lymphatic system at the level of the SLN. This shortcut route is a speculative hypothesis and requires further research. This study aims to provide some preliminary observations in the changes of HEV morphology in a cancer environment and to provide some consistent clinical and morphological data to support our hypothesis
[10]. This is in contrast to the traditional Halstedian philosophy that an enbloc resection of the primary cancer and its regional lymphatic basin will achieve cure as its assumption is that tumor cells follow a stepwise pathway, from the primary tumor to the regional lymph nodes and to the next echelon and then to the systematic circulation through distal lymphaticovenous connections such as the thoracic duct. While this orderly fashion is true in the majority of the time, this stepwise progression is not strictly followed as shown in clinical follow-up studies, where up to 20% of women with node-negative breast cancer go on to develop distant metastases
[16,17]. This is consistent with the analysis of sentinel lymphadenectomies suggesting that 20% of systemic metastases are derived from cancer cells that bypass this orderly lymphatic route
[18]. The HEV providing the vehicle of this shortcut route for cancer cells might account for this observation in this subset of these patients
[6].
In addition to this hypothetical role of providing the physical shortcut route in the LN, evident from the presence of significant abundance of red blood cells within the HEV and significant increase size of its lumen in the presence of a tumor, hints that HEV presumably functions like a blood vessel in anticipation to supply the needs for an accelerating growth of a soon-to-arrive tumor deposit
[5,10].
Objectives of study We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis
[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.
We aim to evaluate the tumor-induced vascularization in regional LN of the patients with tongue cancer, specifically looking at HEV. We also seek to confirm the morphological and functional alterations of HEV in the regional lymph nodes of the patients with carcinomas of the tongue and correlate these findings with clinical outcome. The key blood vessels involved in LN in a cancer setting are HEV, with its function shifting from immune response to one as a blood flow carrier. We hypothesize that the transformation of HEV in the presence of cancer is a spectrum and these morphological features of altered HEV correlate with clinical outcome of patients, establishing its pivotal role in the pathogenesis of metastasis
[6]. This association will serve HEV as a novel prognostic marker and a potential candidate for therapeutic targeting.
Conclusion:
This study demonstrates morphologic and functional alterations of the HEV to become the main blood flow carrier in the lymph node. The analysis reveals the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environment in regional lymph nodes of tongue cancer patients in close relation to clinical outcomes. Our findings coupled with studies elucidating the basis of lymphangiogesis and angiogenesis within SLN support our hypothesis that HEV play a pivotal role and may be the elusive junction providing the lymph flow shortcut route into the systemic circulation at the level of the SLN. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets.
|
Background:
High endothelial venules (HEV) have been recognized to play a role in metastasis by its changes seen in the cancer microenvironment of lymph nodes (LN) and solid cancers. Squamous cell carcinoma (SCC) of the tongue is a prevalent tumor of the head and neck region with high propensity for LN metastasis. The extent of LN metastasis is the most reliable adverse prognostic factor. Primary tumors can induce vasculature reorganization within sentinel LN before the arrival of tumor cells and HEV represents these remodelled vessels. This study aims to evaluate the cancer induced vascular changes in regional lymph nodes (LN) of patients by studying the morphological and functional alterations of HEV and its correlation with clinical outcome and pathological features.
Methods:
This study was based on 65 patients with SCC tongue who underwent primary surgical treatment including neck dissection. The patients were categorized into 2 groups based on the presence of malignancy in their cervical lymph nodes. A review of the patients' pathological and clinical data was performed from a prospective database. Immunohistochemical staining of the tissue blocks for HEV and high-power-field image analysis were performed and analyzed with correlation to the patients' clinical and pathological features.
Results:
The total number of HEV was found to be significantly associated to disease-free interval. There was a similar association comparing the HEV parameters to overall survival. The density of abnormal HEV was significantly higher in patients with established metastases in their lymph nodes and HEV was shown to be a better prognosis factor than conventional tumor staging. The HEV morphological metamorphosis demonstrates a spectrum that correlates well with disease progression and clinical outcome.
Conclusions:
The results suggest that the HEV displays a spectrum of morphological changes in the presence of cancer and LN metastasis, and that HEV is possibly involved in the process of cancer metastasis. We revealed the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environments in regional lymph nodes of tongue cancer patients in relation to clinical outcomes. The significant observation of modified dilated HEV containing red blood cells in lymph nodal basin of a cancer suggests the shifting of its function from one primarily of immune response to that of a blood carrying vessel. It also demonstrated potential prognostic value. More studies are needed to elucidate its potential role in cancer immunotherapy and as a potential novel therapeutic approach to preventing metastasis by manipulating the remodelling processes of HEV.
| 14,568 | 451 | 18 |
[
"hev",
"tumor",
"ln",
"cancer",
"patients",
"blood",
"study",
"lymph",
"cells",
"dilated"
] |
[
"test",
"test"
] |
[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]
|
[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]
|
[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]
|
[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]
|
[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]
|
[CONTENT] High endothelial venules | Cancer metastasis | Angiogenesis | Lymph nodes [SUMMARY]
|
[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]
|
[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]
|
[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]
|
[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]
|
[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]
|
[CONTENT] Case-Control Studies | Disease-Free Survival | Endothelium, Vascular | Humans | Kaplan-Meier Estimate | Lymph Nodes | Lymphatic Metastasis | Neoplasms | Venules [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]
|
[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]
|
[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]
|
[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]
|
[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]
|
[CONTENT] hev | tumor | ln | cancer | patients | blood | study | lymph | cells | dilated [SUMMARY]
|
[CONTENT] hev | cancer | lymphatic | cells | tumor | blood | ln | route | role | vessels [SUMMARY]
|
[CONTENT] hev | neck | test | number | neck dissection | parameters | dissection | dilated hev | total number | number hev [SUMMARY]
|
[CONTENT] tumor | hev | dfi | hev parameters | parameters | risk | grade | cases | stage | groups [SUMMARY]
|
[CONTENT] shortcut | lymph | circulation | sln | flow | metastatic | hev | role elusive junction | role elusive junction providing | pre metastatic metastatic environment [SUMMARY]
|
[CONTENT] hev | tumor | parameters | cancer | dilated | lymph | dilated hev | patients | ln | cases [SUMMARY]
|
[CONTENT] hev | tumor | parameters | cancer | dilated | lymph | dilated hev | patients | ln | cases [SUMMARY]
|
[CONTENT] LN ||| LN ||| LN ||| ||| LN | HEV [SUMMARY]
|
[CONTENT] 65 ||| 2 ||| ||| HEV [SUMMARY]
|
[CONTENT] HEV ||| HEV ||| HEV | HEV ||| HEV [SUMMARY]
|
[CONTENT] HEV | LN | HEV ||| HEV ||| ||| ||| HEV [SUMMARY]
|
[CONTENT] LN ||| LN ||| LN ||| ||| LN | HEV ||| 65 ||| 2 ||| ||| HEV ||| ||| HEV ||| HEV ||| HEV | HEV ||| HEV ||| HEV | LN | HEV ||| HEV ||| ||| ||| HEV [SUMMARY]
|
[CONTENT] LN ||| LN ||| LN ||| ||| LN | HEV ||| 65 ||| 2 ||| ||| HEV ||| ||| HEV ||| HEV ||| HEV | HEV ||| HEV ||| HEV | LN | HEV ||| HEV ||| ||| ||| HEV [SUMMARY]
|
||||||
Preoperative therapy restores ventilatory parameters and reduces length of stay in patients undergoing myocardial revascularization.
|
25140472
|
The frequency of surgical procedures has increased steadily in recent decades, including the myocardial revascularization.
|
INTRODUCTION
|
We conducted a prospective study with patients undergoing myocardial revascularization, the Hospital das Clínicas da Universidade Estadual Paulista (UNESP)/Botucatu - SP. We evaluated 70 patients of both genders, aged between 40 and 75 years, subdivided into two groups: group I - 35 patients of both genders, who received a written protocol guidance, breathing exercises and respiratory muscle training in the preoperative period and group II - 35 patients of both genders, who received only orientation of the ward on the day of surgery. This study was approved by the Ethics Committee of UNESP / Botucatu - SP.
|
METHODS
|
Maximal inspiratory pressure in third postoperative day and fifth postoperative day and significant difference between groups, being better for the intervention group. Expiratory pressure was significant in fifth postoperative day in the intervention group compared to controls. The difference of length of hospital stay in the postoperative was found between the groups with shorter hospital stay in the group receiving preoperative therapy.
|
RESULTS
|
Physical therapy plays an important role in the preoperative period, so that individuals in the intervention group more readily restored the parameters evaluated before surgery, in addition, there was a decrease in the time of the postoperative hospital stay. Thus, it is thought the cost-effectiveness of a program of preoperative physiotherapy.
|
CONCLUSION
|
[
"Adult",
"Aged",
"Breathing Exercises",
"Female",
"Humans",
"Inspiratory Capacity",
"Length of Stay",
"Male",
"Middle Aged",
"Muscle Strength",
"Muscle Stretching Exercises",
"Myocardial Revascularization",
"Preoperative Care",
"Preoperative Period",
"Prospective Studies",
"Reference Values",
"Reproducibility of Results",
"Respiratory Muscles",
"Respiratory Rate",
"Statistics, Nonparametric",
"Tidal Volume",
"Time Factors",
"Treatment Outcome"
] |
4389447
|
INTRODUCTION
|
The cardiac surgery was always clothed with great interest, curiosity, and in some
moments, mysticism, and fact understandable when we analyzed the noble importance of
this component for the proper functioning of the human body[1].
In recent decades the frequency of surgical procedures has progressively increased,
among them the myocardial revascularization (MR). Cardiovascular diseases are a serious
public health problem in Brazil[2].
In individuals over 70 years old it is estimated an incidence of 70% of coronary artery
disease. As the age is a determinant of coronary atherosclerosis, a growing number of
elderly people will be subjected to MR and/or other therapeutic methods[3,4].
The main risk factors that contribute to cardiac diseases are: smoking, high levels of
low-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density
lipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,
family history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors
related to atherosclerosis and its clinical manifestations[4,5].
The coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment
of ischemic heart disease that is manifested clinically by angina and acute myocardial
infarction. The CABG surgery creates a new route for the blood flow and, is used for
making a diversion of blood to the portions of the coronary artery distal to the
obstructive atherosclerotic involvement[1,5].
With the advancements in care of physical therapy in the preoperative period, surgical
techniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,
anesthesia and intensive care in the post-operative period, there was a decrease in
morbidity and mortality of MR which caused the surgical indication in groups of patients
increasingly complex[6,7].
Observing the fact that they are common pulmonary complications of CABG with CPB was
justified to carry out this research, there is great importance in properly carrying out
evaluations, guidelines and breathing exercises in the period before surgery, and
addition, observe the influence and duration of physical therapy assistance in the
preoperative period and also its behavior in the postoperative period.
Objective To demonstrate the importance of the role of physical therapy in the preoperative
period of cardiac surgery with extracorporeal circulation, in relation to the
respiratory muscle strength, pulmonary volumes and duration of hospital stay after
surgery.
To demonstrate the importance of the role of physical therapy in the preoperative
period of cardiac surgery with extracorporeal circulation, in relation to the
respiratory muscle strength, pulmonary volumes and duration of hospital stay after
surgery.
|
METHODS
|
We performed a prospective clinical study, simple blind, in the period from July 2009 to
September 2011 in the ward of cardiac and thoracic surgery at Hospital das Clínicas da
Universidade Estadual Paulista (UNESP), Botucatu-SP. We evaluated 70 patients of both
gender, age ranging from 40 to 75 years, undergoing CABG with use of CPB, randomized by
means of a computerized software (STATTREK) and delivered in a sealed envelope to the
physical therapist responsible for primary health care, subdividing into two groups:
- Group I (GI) - Thirty-five patients, undergoing physiotherapeutic intervention,
performed under supervision, once a day, during the period that preceded the surgery.
The protocol consisted of breathing exercises (breathing in time, deep breathing
followed by prolonged expiration, sustained maximal inspiration with apnea of 6 seconds,
and diaphragmatic breathing associated with the mobilization of the upper limbs) and
breathing exercises with threshold - IMT® (Threshold - IMT®
Inspiratory Muscle Trainer, HealthscanProducts Inc.) at an intensity of 40% of the
initial MIP with three sets of ten repetitions, respecting two-minute intervals between
each series.
- Group II (GII) - thirty-five patients who did not undergo the physical therapy
protocol during the preoperative period, receiving only guidelines ward routine.
We included patients of both sexes, aged 40-75 years with coronary artery disease
admitted for cardiac surgery with cardiopulmonary bypass (CPB) MR, who agreed to
participate in the study. We excluded patients who require the use of intra-aortic
balloon, carrying pulmonary pathology, musculoskeletal impairment and severe
neurological surgery performed without CPB, emergency surgery, homodynamic instability,
and any event that threatens the integrity of patient or the fidelity of the measures
analyzed.
Delimitation All patients were evaluated by the same evaluator (preoperative period - PRE), third
postoperative (3PO) and fifth postoperative days (5PO), as well as received
physiotherapeutical treatment, according to their needs in the postoperative period
by physical therapy team in the hospital and only Group I received physiotherapy in
the preoperative period.
The variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI
> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days
before surgery and for former smokers those with a prior history, 30 days ago) as
well as the time of surgery, CPB, anoxia, IOT (defined by the sum between the
anesthesia time and the time that the patient remained with the endotracheal tube in
the intensive care unit - ICU), VM (IOT time defined by the time of ventilator
disconnection (when the patient was placed in spontaneous ventilation in the T-tube),
ICU stay (when the patient arrived in the intensive care unit until discharged to the
ward bed) and length of stay in the OP (obtained from the time that the patient
arrived in the ICU of the Cardio chest until the time of discharge.) All these data
were obtained by means of the daily developments of medical and nursing staff.
The assessment of respiratory muscle strength was obtained by measurement of
inspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative
period (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand
spread over -120 to 120 cmH2O equipped with adapter buccal and nasal
forceps according to the technique described by Nephew et al. (2008). The MIP
measurement was performed from residual volume, and MEP was obtained from total lung
capacity. Three measurements were taken technically satisfactory, and if there were
more than 20% difference between the measurements, a fourth and even fifth
measurement would be performed, being always considered the highest value obtained.
The MIP was performed for evaluation of inspiratory muscle strength in three
different stages of the study (Pre, 3PO and 5PO), as well as to adjust the training
load (40% MIP) using the Threshold - IMT® (Threshold - IMT®
inspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one
millimeter (mm) was performed to prevent occlusion of the glottis and influence of
the buccinator muscles during MIP maneuver[9].
Mark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to
prevent lung volumes. The minute volume was obtained with the patient breathing for a
minute for a mouth connected to the spirometer. Tidal volume was obtained indirectly
by the relationship between the respiratory rate and minute volume (MV/FR), the two
volumes measured in milliliters (ml) and respiratory rate was measured in cycles per
minute (cpm) and obtained by direct observation of respiratory cycles during the
evaluation of the minute volume[7].
All patients were evaluated by the same evaluator (preoperative period - PRE), third
postoperative (3PO) and fifth postoperative days (5PO), as well as received
physiotherapeutical treatment, according to their needs in the postoperative period
by physical therapy team in the hospital and only Group I received physiotherapy in
the preoperative period.
The variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI
> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days
before surgery and for former smokers those with a prior history, 30 days ago) as
well as the time of surgery, CPB, anoxia, IOT (defined by the sum between the
anesthesia time and the time that the patient remained with the endotracheal tube in
the intensive care unit - ICU), VM (IOT time defined by the time of ventilator
disconnection (when the patient was placed in spontaneous ventilation in the T-tube),
ICU stay (when the patient arrived in the intensive care unit until discharged to the
ward bed) and length of stay in the OP (obtained from the time that the patient
arrived in the ICU of the Cardio chest until the time of discharge.) All these data
were obtained by means of the daily developments of medical and nursing staff.
The assessment of respiratory muscle strength was obtained by measurement of
inspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative
period (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand
spread over -120 to 120 cmH2O equipped with adapter buccal and nasal
forceps according to the technique described by Nephew et al. (2008). The MIP
measurement was performed from residual volume, and MEP was obtained from total lung
capacity. Three measurements were taken technically satisfactory, and if there were
more than 20% difference between the measurements, a fourth and even fifth
measurement would be performed, being always considered the highest value obtained.
The MIP was performed for evaluation of inspiratory muscle strength in three
different stages of the study (Pre, 3PO and 5PO), as well as to adjust the training
load (40% MIP) using the Threshold - IMT® (Threshold - IMT®
inspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one
millimeter (mm) was performed to prevent occlusion of the glottis and influence of
the buccinator muscles during MIP maneuver[9].
Mark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to
prevent lung volumes. The minute volume was obtained with the patient breathing for a
minute for a mouth connected to the spirometer. Tidal volume was obtained indirectly
by the relationship between the respiratory rate and minute volume (MV/FR), the two
volumes measured in milliliters (ml) and respiratory rate was measured in cycles per
minute (cpm) and obtained by direct observation of respiratory cycles during the
evaluation of the minute volume[7].
Procedures/treatment Patients allocated to the GI protocol underwent preoperative physiotherapy consisting
of breathing exercises (breathing in time, deep breath and then exhaling long,
sustained maximal inspiration with a 6-second apnea, diaphragmatic breathing
associated with mobilization of the upper limb, with three series out of ten for each
breathing exercise) and breathing exercises with the device Threshold -
IMT®. We conducted three sets of ten repetitions with an interval of
two minutes each repetition, once a day for every day of hospitalization in the PRE,
with a load of 40% of the initial MIP, obtained by manovacuometry[8-10].
The GII patients received only guidelines ward routine before surgery, but
postoperatively, both groups performed physical therapy as needed by staff
physiotherapy service.
Patients allocated to the GI protocol underwent preoperative physiotherapy consisting
of breathing exercises (breathing in time, deep breath and then exhaling long,
sustained maximal inspiration with a 6-second apnea, diaphragmatic breathing
associated with mobilization of the upper limb, with three series out of ten for each
breathing exercise) and breathing exercises with the device Threshold -
IMT®. We conducted three sets of ten repetitions with an interval of
two minutes each repetition, once a day for every day of hospitalization in the PRE,
with a load of 40% of the initial MIP, obtained by manovacuometry[8-10].
The GII patients received only guidelines ward routine before surgery, but
postoperatively, both groups performed physical therapy as needed by staff
physiotherapy service.
Statistical Analysis The sample should be made with at least 26 patients who were randomly divided into
two study groups (with physical therapy preoperatively and control patients who did
not receive preoperative physiotherapy). We considered the level of significance of
5% and a test power in the order of 80% for a minimum difference in the order of
two.
The comparison between the groups for age and body mass index (BMI) was performed by
Wilcoxon test for independent samples. The smoking variable was analyzed using
Goldman to compare proportions within and among multinomial populations. The
perfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative
hospital stay were obtained in minutes and analyzed by Student's t test or the
nonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman
tests for comparison of the moments in each group and Mann Whitney test to compare
groups at each time, adopting a significance level of 5% probability of rejecting
null hypothesis.
This study was approved by the Research Ethics Committee of Universidade Estadual
Paulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,
protocol 3223) and all patients signed an informed consent form.
The sample should be made with at least 26 patients who were randomly divided into
two study groups (with physical therapy preoperatively and control patients who did
not receive preoperative physiotherapy). We considered the level of significance of
5% and a test power in the order of 80% for a minimum difference in the order of
two.
The comparison between the groups for age and body mass index (BMI) was performed by
Wilcoxon test for independent samples. The smoking variable was analyzed using
Goldman to compare proportions within and among multinomial populations. The
perfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative
hospital stay were obtained in minutes and analyzed by Student's t test or the
nonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman
tests for comparison of the moments in each group and Mann Whitney test to compare
groups at each time, adopting a significance level of 5% probability of rejecting
null hypothesis.
This study was approved by the Research Ethics Committee of Universidade Estadual
Paulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,
protocol 3223) and all patients signed an informed consent form.
|
RESULTS
|
Seventy patients were divided into two groups, Group I composed of 35 patients (12
female and 23 male) and Group II, with 35 patients (six female and 29 male), as
illustrated in Figure 1.
Characterization of groups by gender
No significant difference was noticed between groups for the variables age (GI 58.9±9.53
years and GII 61.4±8.43 years; P=0.26) and body mass index (GI
26.8±3.96 kg/m2 and G II 26±3.86 kg/m2) (Table 1).
Patient characteristics regarding age and BMI.
BMI: body mass index; Wilcoxon test. * P<0.05
The groups were homogenous concerning presence of active smoking, ex-smokers and
nonsmokers, as illustrated in Table 2.
Number and proportion of patients in each group according to the presence of
active smoking, ex-smokers and nonsmokers.
Two proportions followed by the same lowercase letter do not differ in groups
(rows). Two proportions followed by the same capital letter do not differ in
the types of smoking (P>0.05). Goldman test to compare proportions between
and within populations multinomina.
Between the GI and GII, no significant difference regarding the proportion of
ex-smokers, smokers and nonsmokers, in other words, the groups were homogeneous as
demonstrated below (Table 2).
Maximum Inspiratory Pressure (MIP) The mean values the for maximum inspiratory pressures evaluated in the preoperative
period and 3PO showed no significant difference between groups
(P=0.276 and 0.065; respectively), however, we observed greater
inspiratory muscle strength for GI in relation to GII in 5PO
(P=0.001) (Figure 2).
Median maximum inspiratory pressure (MIP) variable in different moments of the
operation of each group analyzed (with and without physical therapy
preoperatively). Friedman test for comparison of moments in each group and Mann
Whitney test to compare the groups at each time, *P<0.05.
The mean values the for maximum inspiratory pressures evaluated in the preoperative
period and 3PO showed no significant difference between groups
(P=0.276 and 0.065; respectively), however, we observed greater
inspiratory muscle strength for GI in relation to GII in 5PO
(P=0.001) (Figure 2).
Median maximum inspiratory pressure (MIP) variable in different moments of the
operation of each group analyzed (with and without physical therapy
preoperatively). Friedman test for comparison of moments in each group and Mann
Whitney test to compare the groups at each time, *P<0.05.
Maximum Expiratory Pressure (MEP) The mean value for the maximum expiratory pressure obtained in the preoperative
period for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80
cmH2O) for GII, not presenting significant difference between groups
(P=0.704). However, the values referring MEP measured in the 5PO
showed significant difference between groups (P=0.001), as
illustrated in Figure 3.
Median for maximum expiratory pressure (MEP) variable at different times of the
operation in the different groups. Friedman test for comparison of moments in
each group and Mann Whitney test to compare the groups at each time,
*P<0.05.
The mean value for the maximum expiratory pressure obtained in the preoperative
period for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80
cmH2O) for GII, not presenting significant difference between groups
(P=0.704). However, the values referring MEP measured in the 5PO
showed significant difference between groups (P=0.001), as
illustrated in Figure 3.
Median for maximum expiratory pressure (MEP) variable at different times of the
operation in the different groups. Friedman test for comparison of moments in
each group and Mann Whitney test to compare the groups at each time,
*P<0.05.
Minute Volume (MV) The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period
presented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;
P=0.001). However, these values have not showed significative
difference in the 3PO and 5PO (P=0.585 and P=0.329;
respectively).
Median minute volume (VM ) variable at different times of the operation of each
group analyzed. Friedman test for comparison of the moments in each group and
the Mann Whitney test for comparison between groups at any time,
*P<0.05.
The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period
presented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;
P=0.001). However, these values have not showed significative
difference in the 3PO and 5PO (P=0.585 and P=0.329;
respectively).
Median minute volume (VM ) variable at different times of the operation of each
group analyzed. Friedman test for comparison of the moments in each group and
the Mann Whitney test for comparison between groups at any time,
*P<0.05.
Tidal Volume Figure 5 illustrates the analysis of variable
volume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between
groups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these
values were not significantly different for the 3PO and 5 PO
(P=0.059 and P=0.549; respectively).
Median tidal volume variable at different times for the group treated with
preoperative physiotherapy and without physical therapy preoperatively.
Friedman test for comparison of moments in each group and Mann Whitney test to
compare the groups at each time, *P<0.05.
Figure 5 illustrates the analysis of variable
volume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between
groups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these
values were not significantly different for the 3PO and 5 PO
(P=0.059 and P=0.549; respectively).
Median tidal volume variable at different times for the group treated with
preoperative physiotherapy and without physical therapy preoperatively.
Friedman test for comparison of moments in each group and Mann Whitney test to
compare the groups at each time, *P<0.05.
Respiratory rate (RR) The median refers to the respiratory rate obtained preoperatively for GI and GII
showed no significant difference (16 cpm and 18 cpm, respectively,
P=0.602). Likewise, the measurements were made in the 5PO 3PO and
showed no significant difference between groups (P=0.090 and
P=0.886, respectively), as shown in Figure 6.
Median variable respiratory rate at different times of the operation of each
group analyzed (with and without physical therapy preoperatively). Friedman
test for comparison of moments in each group and Mann Whitney test to compare
the groups at each time, *P<0.05.
Infusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and
hospital stay after surgery.
Variables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU
stay did not significantly differ between groups, however, significant difference was
observed for the length of stay variable in the postoperative period
(P=0.001), as shown in Table
3.
Values for the infusion time, anoxia, mechanical ventilation, anesthesia,
surgery, ICU stay and hospital stay after surgery for preoperative, 3PO and
5PO.
Values are shown as mean ± standard deviation or median (minimum and maximum
values).
Mann and Whitney
Student's t test.
Significant difference between groups (P <0.05). ICU: intensive care
unit; PO: postoperative
The median refers to the respiratory rate obtained preoperatively for GI and GII
showed no significant difference (16 cpm and 18 cpm, respectively,
P=0.602). Likewise, the measurements were made in the 5PO 3PO and
showed no significant difference between groups (P=0.090 and
P=0.886, respectively), as shown in Figure 6.
Median variable respiratory rate at different times of the operation of each
group analyzed (with and without physical therapy preoperatively). Friedman
test for comparison of moments in each group and Mann Whitney test to compare
the groups at each time, *P<0.05.
Infusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and
hospital stay after surgery.
Variables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU
stay did not significantly differ between groups, however, significant difference was
observed for the length of stay variable in the postoperative period
(P=0.001), as shown in Table
3.
Values for the infusion time, anoxia, mechanical ventilation, anesthesia,
surgery, ICU stay and hospital stay after surgery for preoperative, 3PO and
5PO.
Values are shown as mean ± standard deviation or median (minimum and maximum
values).
Mann and Whitney
Student's t test.
Significant difference between groups (P <0.05). ICU: intensive care
unit; PO: postoperative
|
CONCLUSIONS
|
The physical therapy has an important role in the preoperative period, restoring greater
readiness ventilatory parameters of patients undergoing coronary artery bypass grafting
with cardiopulmonary bypass, resulting in a decrease in length of hospital stay after
surgery.
|
[
"Objective",
"Delimitation",
"Procedures/treatment",
"Statistical Analysis",
"Maximum Inspiratory Pressure (MIP)",
"Maximum Expiratory Pressure (MEP)",
"Minute Volume (MV)",
"Tidal Volume",
"Respiratory rate (RR)",
"Limitation of the study"
] |
[
"To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.",
"All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].",
"Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.",
"The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.",
"The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.",
"The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.",
"The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.",
"Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.",
"The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative",
"The major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"Objective",
"METHODS",
"Delimitation",
"Procedures/treatment",
"Statistical Analysis",
"RESULTS",
"Maximum Inspiratory Pressure (MIP)",
"Maximum Expiratory Pressure (MEP)",
"Minute Volume (MV)",
"Tidal Volume",
"Respiratory rate (RR)",
"DISCUSSION",
"Limitation of the study",
"CONCLUSIONS"
] |
[
"The cardiac surgery was always clothed with great interest, curiosity, and in some\nmoments, mysticism, and fact understandable when we analyzed the noble importance of\nthis component for the proper functioning of the human body[1].\nIn recent decades the frequency of surgical procedures has progressively increased,\namong them the myocardial revascularization (MR). Cardiovascular diseases are a serious\npublic health problem in Brazil[2].\nIn individuals over 70 years old it is estimated an incidence of 70% of coronary artery\ndisease. As the age is a determinant of coronary atherosclerosis, a growing number of\nelderly people will be subjected to MR and/or other therapeutic methods[3,4].\nThe main risk factors that contribute to cardiac diseases are: smoking, high levels of\nlow-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density\nlipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,\nfamily history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors\nrelated to atherosclerosis and its clinical manifestations[4,5].\nThe coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment\nof ischemic heart disease that is manifested clinically by angina and acute myocardial\ninfarction. The CABG surgery creates a new route for the blood flow and, is used for\nmaking a diversion of blood to the portions of the coronary artery distal to the\nobstructive atherosclerotic involvement[1,5].\nWith the advancements in care of physical therapy in the preoperative period, surgical\ntechniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,\nanesthesia and intensive care in the post-operative period, there was a decrease in\nmorbidity and mortality of MR which caused the surgical indication in groups of patients\nincreasingly complex[6,7].\nObserving the fact that they are common pulmonary complications of CABG with CPB was\njustified to carry out this research, there is great importance in properly carrying out\nevaluations, guidelines and breathing exercises in the period before surgery, and\naddition, observe the influence and duration of physical therapy assistance in the\npreoperative period and also its behavior in the postoperative period.\n Objective To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.\nTo demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.",
"To demonstrate the importance of the role of physical therapy in the preoperative\nperiod of cardiac surgery with extracorporeal circulation, in relation to the\nrespiratory muscle strength, pulmonary volumes and duration of hospital stay after\nsurgery.",
"We performed a prospective clinical study, simple blind, in the period from July 2009 to\nSeptember 2011 in the ward of cardiac and thoracic surgery at Hospital das Clínicas da\nUniversidade Estadual Paulista (UNESP), Botucatu-SP. We evaluated 70 patients of both\ngender, age ranging from 40 to 75 years, undergoing CABG with use of CPB, randomized by\nmeans of a computerized software (STATTREK) and delivered in a sealed envelope to the\nphysical therapist responsible for primary health care, subdividing into two groups:\n- Group I (GI) - Thirty-five patients, undergoing physiotherapeutic intervention,\nperformed under supervision, once a day, during the period that preceded the surgery.\nThe protocol consisted of breathing exercises (breathing in time, deep breathing\nfollowed by prolonged expiration, sustained maximal inspiration with apnea of 6 seconds,\nand diaphragmatic breathing associated with the mobilization of the upper limbs) and\nbreathing exercises with threshold - IMT® (Threshold - IMT®\nInspiratory Muscle Trainer, HealthscanProducts Inc.) at an intensity of 40% of the\ninitial MIP with three sets of ten repetitions, respecting two-minute intervals between\neach series.\n- Group II (GII) - thirty-five patients who did not undergo the physical therapy\nprotocol during the preoperative period, receiving only guidelines ward routine.\nWe included patients of both sexes, aged 40-75 years with coronary artery disease\nadmitted for cardiac surgery with cardiopulmonary bypass (CPB) MR, who agreed to\nparticipate in the study. We excluded patients who require the use of intra-aortic\nballoon, carrying pulmonary pathology, musculoskeletal impairment and severe\nneurological surgery performed without CPB, emergency surgery, homodynamic instability,\nand any event that threatens the integrity of patient or the fidelity of the measures\nanalyzed.\n Delimitation All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\nAll patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].\n Procedures/treatment Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\nPatients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.\n Statistical Analysis The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.\nThe sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.",
"All patients were evaluated by the same evaluator (preoperative period - PRE), third\npostoperative (3PO) and fifth postoperative days (5PO), as well as received\nphysiotherapeutical treatment, according to their needs in the postoperative period\nby physical therapy team in the hospital and only Group I received physiotherapy in\nthe preoperative period.\nThe variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI\n> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days\nbefore surgery and for former smokers those with a prior history, 30 days ago) as\nwell as the time of surgery, CPB, anoxia, IOT (defined by the sum between the\nanesthesia time and the time that the patient remained with the endotracheal tube in\nthe intensive care unit - ICU), VM (IOT time defined by the time of ventilator\ndisconnection (when the patient was placed in spontaneous ventilation in the T-tube),\nICU stay (when the patient arrived in the intensive care unit until discharged to the\nward bed) and length of stay in the OP (obtained from the time that the patient\narrived in the ICU of the Cardio chest until the time of discharge.) All these data\nwere obtained by means of the daily developments of medical and nursing staff.\nThe assessment of respiratory muscle strength was obtained by measurement of\ninspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative\nperiod (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand\nspread over -120 to 120 cmH2O equipped with adapter buccal and nasal\nforceps according to the technique described by Nephew et al. (2008). The MIP\nmeasurement was performed from residual volume, and MEP was obtained from total lung\ncapacity. Three measurements were taken technically satisfactory, and if there were\nmore than 20% difference between the measurements, a fourth and even fifth\nmeasurement would be performed, being always considered the highest value obtained.\nThe MIP was performed for evaluation of inspiratory muscle strength in three\ndifferent stages of the study (Pre, 3PO and 5PO), as well as to adjust the training\nload (40% MIP) using the Threshold - IMT® (Threshold - IMT®\ninspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one\nmillimeter (mm) was performed to prevent occlusion of the glottis and influence of\nthe buccinator muscles during MIP maneuver[9].\nMark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to\nprevent lung volumes. The minute volume was obtained with the patient breathing for a\nminute for a mouth connected to the spirometer. Tidal volume was obtained indirectly\nby the relationship between the respiratory rate and minute volume (MV/FR), the two\nvolumes measured in milliliters (ml) and respiratory rate was measured in cycles per\nminute (cpm) and obtained by direct observation of respiratory cycles during the\nevaluation of the minute volume[7].",
"Patients allocated to the GI protocol underwent preoperative physiotherapy consisting\nof breathing exercises (breathing in time, deep breath and then exhaling long,\nsustained maximal inspiration with a 6-second apnea, diaphragmatic breathing\nassociated with mobilization of the upper limb, with three series out of ten for each\nbreathing exercise) and breathing exercises with the device Threshold -\nIMT®. We conducted three sets of ten repetitions with an interval of\ntwo minutes each repetition, once a day for every day of hospitalization in the PRE,\nwith a load of 40% of the initial MIP, obtained by manovacuometry[8-10].\nThe GII patients received only guidelines ward routine before surgery, but\npostoperatively, both groups performed physical therapy as needed by staff\nphysiotherapy service.",
"The sample should be made with at least 26 patients who were randomly divided into\ntwo study groups (with physical therapy preoperatively and control patients who did\nnot receive preoperative physiotherapy). We considered the level of significance of\n5% and a test power in the order of 80% for a minimum difference in the order of\ntwo.\nThe comparison between the groups for age and body mass index (BMI) was performed by\nWilcoxon test for independent samples. The smoking variable was analyzed using\nGoldman to compare proportions within and among multinomial populations. The\nperfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative\nhospital stay were obtained in minutes and analyzed by Student's t test or the\nnonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman\ntests for comparison of the moments in each group and Mann Whitney test to compare\ngroups at each time, adopting a significance level of 5% probability of rejecting\nnull hypothesis.\nThis study was approved by the Research Ethics Committee of Universidade Estadual\nPaulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,\nprotocol 3223) and all patients signed an informed consent form.",
"Seventy patients were divided into two groups, Group I composed of 35 patients (12\nfemale and 23 male) and Group II, with 35 patients (six female and 29 male), as\nillustrated in Figure 1.\nCharacterization of groups by gender\nNo significant difference was noticed between groups for the variables age (GI 58.9±9.53\nyears and GII 61.4±8.43 years; P=0.26) and body mass index (GI\n26.8±3.96 kg/m2 and G II 26±3.86 kg/m2) (Table 1).\nPatient characteristics regarding age and BMI.\nBMI: body mass index; Wilcoxon test. * P<0.05\nThe groups were homogenous concerning presence of active smoking, ex-smokers and\nnonsmokers, as illustrated in Table 2.\nNumber and proportion of patients in each group according to the presence of\nactive smoking, ex-smokers and nonsmokers.\nTwo proportions followed by the same lowercase letter do not differ in groups\n(rows). Two proportions followed by the same capital letter do not differ in\nthe types of smoking (P>0.05). Goldman test to compare proportions between\nand within populations multinomina.\nBetween the GI and GII, no significant difference regarding the proportion of\nex-smokers, smokers and nonsmokers, in other words, the groups were homogeneous as\ndemonstrated below (Table 2).\n Maximum Inspiratory Pressure (MIP) The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\nThe mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.\n Maximum Expiratory Pressure (MEP) The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\nThe mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.\n Minute Volume (MV) The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\nThe analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.\n Tidal Volume Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\nFigure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.\n Respiratory rate (RR) The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative\nThe median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative",
"The mean values the for maximum inspiratory pressures evaluated in the preoperative\nperiod and 3PO showed no significant difference between groups\n(P=0.276 and 0.065; respectively), however, we observed greater\ninspiratory muscle strength for GI in relation to GII in 5PO\n(P=0.001) (Figure 2).\nMedian maximum inspiratory pressure (MIP) variable in different moments of the\noperation of each group analyzed (with and without physical therapy\npreoperatively). Friedman test for comparison of moments in each group and Mann\nWhitney test to compare the groups at each time, *P<0.05.",
"The mean value for the maximum expiratory pressure obtained in the preoperative\nperiod for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80\ncmH2O) for GII, not presenting significant difference between groups\n(P=0.704). However, the values referring MEP measured in the 5PO\nshowed significant difference between groups (P=0.001), as\nillustrated in Figure 3.\nMedian for maximum expiratory pressure (MEP) variable at different times of the\noperation in the different groups. Friedman test for comparison of moments in\neach group and Mann Whitney test to compare the groups at each time,\n*P<0.05.",
"The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period\npresented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;\nP=0.001). However, these values have not showed significative\ndifference in the 3PO and 5PO (P=0.585 and P=0.329;\nrespectively).\nMedian minute volume (VM ) variable at different times of the operation of each\ngroup analyzed. Friedman test for comparison of the moments in each group and\nthe Mann Whitney test for comparison between groups at any time,\n*P<0.05.",
"Figure 5 illustrates the analysis of variable\nvolume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between\ngroups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these\nvalues were not significantly different for the 3PO and 5 PO\n(P=0.059 and P=0.549; respectively).\nMedian tidal volume variable at different times for the group treated with\npreoperative physiotherapy and without physical therapy preoperatively.\nFriedman test for comparison of moments in each group and Mann Whitney test to\ncompare the groups at each time, *P<0.05.",
"The median refers to the respiratory rate obtained preoperatively for GI and GII\nshowed no significant difference (16 cpm and 18 cpm, respectively,\nP=0.602). Likewise, the measurements were made in the 5PO 3PO and\nshowed no significant difference between groups (P=0.090 and\nP=0.886, respectively), as shown in Figure 6.\nMedian variable respiratory rate at different times of the operation of each\ngroup analyzed (with and without physical therapy preoperatively). Friedman\ntest for comparison of moments in each group and Mann Whitney test to compare\nthe groups at each time, *P<0.05.\nInfusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and\nhospital stay after surgery.\nVariables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU\nstay did not significantly differ between groups, however, significant difference was\nobserved for the length of stay variable in the postoperative period\n(P=0.001), as shown in Table\n3.\nValues for the infusion time, anoxia, mechanical ventilation, anesthesia,\nsurgery, ICU stay and hospital stay after surgery for preoperative, 3PO and\n5PO.\nValues are shown as mean ± standard deviation or median (minimum and maximum\nvalues).\nMann and Whitney\nStudent's t test.\nSignificant difference between groups (P <0.05). ICU: intensive care\nunit; PO: postoperative",
"Studies by Feltrim et al.[10] show\nthat performing preoperative physiotherapy is more effective in reducing respiratory\ncomplications in patients with moderate or higher risk than those whose risk was\nlow.\nIn the present study, we observed no significant difference between the subjects of\nGroup I and Group II as the characteristics (age, BMI, smoking and number of co\nmorbidities), which suggests that the study subjects in both groups were\nhomogeneous.\nIn the study by Leguisamo et al.[9],\nwith 86 patients undergoing CABG, divided into two groups: the intervention group (44\npatients) who were assessed and received physiotherapeutic guidance at least 15 days\nbefore surgery. The control group received routine care on the day of\nhospitalization.\nThe average MIP between groups showed neither significant difference in the preoperative\nperiod nor in the 1PO and 6PO.\nIn the present study it was observed that there was no significant difference between\ngroups to measure MIP in the preoperative period, but now in times of 3PO and 5PO\nsignificant difference between groups was found. Similarly, no significant difference in\nthe amount of MEP preoperatively in the 3PO and between groups. However in 5PO, there\nwas significant difference between the groups with the best values of the group that\nperformed physical therapy before surgery.\nBarros et al.[11] evaluated the MIP\nand MEP in the preoperative period, the first day after surgery and in 38 patients\nundergoing CABG with CPB who were divided into two groups: 23 patients in group I\n(respiratory muscle training) and 15 in group II (control). Group I received\nconventional physiotherapy over respiratory muscle training and Group II the\nconventional physiotherapy. The authors found that the values obtained for MIP and MEP\nin hospital discharge were higher in group I.\nThere was no decrease in maximal respiratory pressures, transdiaphragmatic pressure and\ndiaphragm pressure, indicating weakness of respiratory muscle strength[12,13].\nAgreeing with the authors cited above, we observed in the present study, after cardiac\nsurgery, MIP values in GI and GII which decreased in 3PO, however the value of MIP in\n5PO increase was observed for GI values compared to those in PRE and 3PO since the GII\nvalues remained stable in 3PO. The authors noted the drop in MEP in 3PO in both groups,\nhowever, in 5PO MEP has increased in GI. Regarding PRE and 3PO in GII no return value\nwas observed in PRE.\nArcêncio et al.[12] conducted a study\nwith 30 patients, aged 50 years-old and candidates for CABG and / or heart valve,\ndividing into two groups. Intervention group comprised 15 patients who underwent at\nleast a two weeks inspiratory muscle training using incentive spirometry (\"Threshold -\nIMT®) with 40% charge of the MIP and control group with 15 patients who\nreceived only general guidelines. The authors compared the spirometric values before and\nafter training and showed no significant difference in the values of MIP and\nMEP[14].\nElias et al.[15] studied 42 patients\naged 18 to 75 years-old with coronary artery disease, in the pre-and postoperative\nperiod, divided into two groups. Intervention group received TMR with Threshold -\nIMT® and control group received only guidelines. The TMR both pre and\npost-operative period consisted of three sets of 10 inspiratory exercises once a day for\nthree consecutive days. It was observed that after cardiac surgery there was a\nsignificant reduction in MIP and MEP in both groups.\nMorsch et al.[13] conducted a study to\nevaluate 108 patients undergoing elective CABG surgery from April 2006 to February 2007\n(preoperatively and postoperatively).\nThe average values of MIP in the preoperative period were significantly decreased\ncompared to 6 days after the surgery (P<0.001). The authors also\nobserved a significant decrease in the values of MEP obtained on the 6th\npostoperative day compared to the preoperative period (P<0.001).\nPatients with respiratory muscle weakness have higher risk of postoperative pulmonary\ncomplications. The respiratory muscle training in the pre-operative may prevent\npulmonary complications in patients undergoing thoracic surgery[16]. There may be a reduction of all lung\nvolumes resulting from factors such as diaphragmatic dysfunction, pain, absence of deep\nbreaths, pulmonary changes and ribcage. The functional residual capacity (FRC) decreases\nbecause of the reduction of both the residual volume (RV) and expiratory reserve volume.\nThe ventilation is affected by VC reduction by approximately 20%, and the increase in\nFR(22-24). The sum of these factors cause changes in the mechanics of\nrespiration, which leads to a shallow breathing pattern of decreased lung\nvolume[13].\nPatients undergoing CABG develop mostly PO pulmonary dysfunction with a significant\nreduction in lung volumes, impairments in respiratory function, decreased lung\ncompliance and increased work of breathing. The reduction in lung volumes and capacities\ncontributes to changes in gas exchange, resulting in hypoxemia[17].\nIn this study, the VM showed significant difference regarding the groups I and II in the\npreoperative period but not significant between groups in 3PO. Likewise, the mean value\nof the VM for the GI 5PO, there was no significant difference in the GII. The mean value\nof VC in PRE in GI was significant, as the mean value of the VC in GII patients, however\nin GI and GII we observed in 5PO and 3PO that there was no significant difference. It\nwas observed in GI at different times that the increase of VM will be very likely due to\nincreased FR, since the current VC remained without many changes between different\ntimes.\nBarros et al.[11] demonstrated a\nsignificant decrease in lung volumes in patients undergoing cardiac surgery in the\npostoperative period.\nCarvalho et al.[18] observed a\ndecrease in the values of minute volume and tidal volume measured on the 2nd\npostoperative day, however, these values showed gradual improvement returning to the\nvalues obtained at baseline (preoperative period) at the time of hospital discharge.\nIn a study, Guizilini et al.[19]\nevaluated 30 patients with a mean age of 56 years and divided into two groups, group A\n(n=15) without CPB and group B (n=15) with CPB. All patients underwent pulmonary\nfunction. In both groups, there was a decrease in FVC until the fifth postoperative day\n(P<0.05).\nRegarding the respiratory rate in the present study, we can verify that there was an\nincrease in both groups, with a higher peak on the 3rd postoperative day and\ndecreased on the 6th postoperative day, but the respiratory rate did not\nreturn to the preoperative values, Carvalho et al.[18] already mentioned above that they concluded in their studies\nthat the mean respiratory frequency had a significant increase between pre and\n2nd PO, 3rd PO, decreasing on the 4th postoperative\nday and increased again on the 5th postoperative day and decreased in\nhospital discharge, but patients did not return to the preoperative values.\nAccording to the results obtained in this study, we can affirm that there was no\nsignificant difference between the groups regarding the time variables: anesthesia,\nsurgery, perfusion, anoxia, mechanical ventilation, intubation. This suggests that the\ngroups were quite homogeneous. As for the time of postoperative hospital stay, there was\na decrease of approximately 25 hours, comparing patients who underwent physical therapy\nbefore surgery with patients who did not.\nIn contrast to the findings of this study, Patman et al.[20] performed a randomized study of 236 patients to\nevaluate the effects of physical therapy interventions performed early versus only\nstarted after extubation in patients undergoing cardiac surgery and no significant\ndifferences were found in the time of intubation, ICU stay and length of hospital stay\nbetween the groups.\nHowever, Celli et al.[21] conducted a\nstudy with 172 patients and showed a decrease in length of hospital stay in the group\nthat had received guidelines breathing exercises (9.6±3.2 days) compared to the control\ngroup (13±5 days).\nCorroborating these findings, as well as those obtained in the present study, Leguisamo\net al.[9] performed a prospective\nstudy to evaluate the effectiveness of a physiotherapy program start in the preoperative\nperiod in reducing the length of hospital stay and showed a significant reduction in\nlength of hospital stay for the intervention group compared to the control group\n(P<0.05).\nThese data suggest that physical therapy initiated before surgery can improve conditions\nof patients, reestablishing early respiratory pressures, reducing the length of stay and\ndecreased hospital costs for the period of hospitalization.\n Limitation of the study The major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.\nThe major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.",
"The major limitation was the short period of time that patients received preoperative\nphysiotherapy; however, we understand that physiotherapeutic treatment had a\nrelevance even in a short period, so the study may add knowledge of response to\ntherapy performed during intraoperative CABG surgery.\nNot performing spirometry can be mentioned as a limitation on the analysis of\nventilation parameters, however, due to the conditions offered by our service, we\ncould not perform this evaluation.\nOn the other hand, the results showed that even with limited time monitoring in the\npreoperative period and respiratory variables obtained only by respirometry and\nmanometry in physical therapy in the preoperative period, proved to be beneficial for\npatients undergoing surgery CABG.\nWe understand that the results may open up new avenues of future research related to\nthe role of physiotherapy in preoperative myocardial revascularization, in addition,\nfurther studies should be conducted using tools such as spirometry, to assess and\nquantify the volume and lung capacity more accurately.",
"The physical therapy has an important role in the preoperative period, restoring greater\nreadiness ventilatory parameters of patients undergoing coronary artery bypass grafting\nwith cardiopulmonary bypass, resulting in a decrease in length of hospital stay after\nsurgery."
] |
[
"intro",
null,
"methods",
null,
null,
null,
"results",
null,
null,
null,
null,
null,
"discussion",
null,
"conclusions"
] |
[
"Thoracic Surgery",
"Breathing Exercises",
"Physical Therapy (Specialty)"
] |
INTRODUCTION:
The cardiac surgery was always clothed with great interest, curiosity, and in some
moments, mysticism, and fact understandable when we analyzed the noble importance of
this component for the proper functioning of the human body[1].
In recent decades the frequency of surgical procedures has progressively increased,
among them the myocardial revascularization (MR). Cardiovascular diseases are a serious
public health problem in Brazil[2].
In individuals over 70 years old it is estimated an incidence of 70% of coronary artery
disease. As the age is a determinant of coronary atherosclerosis, a growing number of
elderly people will be subjected to MR and/or other therapeutic methods[3,4].
The main risk factors that contribute to cardiac diseases are: smoking, high levels of
low-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density
lipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,
family history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors
related to atherosclerosis and its clinical manifestations[4,5].
The coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment
of ischemic heart disease that is manifested clinically by angina and acute myocardial
infarction. The CABG surgery creates a new route for the blood flow and, is used for
making a diversion of blood to the portions of the coronary artery distal to the
obstructive atherosclerotic involvement[1,5].
With the advancements in care of physical therapy in the preoperative period, surgical
techniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,
anesthesia and intensive care in the post-operative period, there was a decrease in
morbidity and mortality of MR which caused the surgical indication in groups of patients
increasingly complex[6,7].
Observing the fact that they are common pulmonary complications of CABG with CPB was
justified to carry out this research, there is great importance in properly carrying out
evaluations, guidelines and breathing exercises in the period before surgery, and
addition, observe the influence and duration of physical therapy assistance in the
preoperative period and also its behavior in the postoperative period.
Objective To demonstrate the importance of the role of physical therapy in the preoperative
period of cardiac surgery with extracorporeal circulation, in relation to the
respiratory muscle strength, pulmonary volumes and duration of hospital stay after
surgery.
To demonstrate the importance of the role of physical therapy in the preoperative
period of cardiac surgery with extracorporeal circulation, in relation to the
respiratory muscle strength, pulmonary volumes and duration of hospital stay after
surgery.
Objective:
To demonstrate the importance of the role of physical therapy in the preoperative
period of cardiac surgery with extracorporeal circulation, in relation to the
respiratory muscle strength, pulmonary volumes and duration of hospital stay after
surgery.
METHODS:
We performed a prospective clinical study, simple blind, in the period from July 2009 to
September 2011 in the ward of cardiac and thoracic surgery at Hospital das Clínicas da
Universidade Estadual Paulista (UNESP), Botucatu-SP. We evaluated 70 patients of both
gender, age ranging from 40 to 75 years, undergoing CABG with use of CPB, randomized by
means of a computerized software (STATTREK) and delivered in a sealed envelope to the
physical therapist responsible for primary health care, subdividing into two groups:
- Group I (GI) - Thirty-five patients, undergoing physiotherapeutic intervention,
performed under supervision, once a day, during the period that preceded the surgery.
The protocol consisted of breathing exercises (breathing in time, deep breathing
followed by prolonged expiration, sustained maximal inspiration with apnea of 6 seconds,
and diaphragmatic breathing associated with the mobilization of the upper limbs) and
breathing exercises with threshold - IMT® (Threshold - IMT®
Inspiratory Muscle Trainer, HealthscanProducts Inc.) at an intensity of 40% of the
initial MIP with three sets of ten repetitions, respecting two-minute intervals between
each series.
- Group II (GII) - thirty-five patients who did not undergo the physical therapy
protocol during the preoperative period, receiving only guidelines ward routine.
We included patients of both sexes, aged 40-75 years with coronary artery disease
admitted for cardiac surgery with cardiopulmonary bypass (CPB) MR, who agreed to
participate in the study. We excluded patients who require the use of intra-aortic
balloon, carrying pulmonary pathology, musculoskeletal impairment and severe
neurological surgery performed without CPB, emergency surgery, homodynamic instability,
and any event that threatens the integrity of patient or the fidelity of the measures
analyzed.
Delimitation All patients were evaluated by the same evaluator (preoperative period - PRE), third
postoperative (3PO) and fifth postoperative days (5PO), as well as received
physiotherapeutical treatment, according to their needs in the postoperative period
by physical therapy team in the hospital and only Group I received physiotherapy in
the preoperative period.
The variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI
> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days
before surgery and for former smokers those with a prior history, 30 days ago) as
well as the time of surgery, CPB, anoxia, IOT (defined by the sum between the
anesthesia time and the time that the patient remained with the endotracheal tube in
the intensive care unit - ICU), VM (IOT time defined by the time of ventilator
disconnection (when the patient was placed in spontaneous ventilation in the T-tube),
ICU stay (when the patient arrived in the intensive care unit until discharged to the
ward bed) and length of stay in the OP (obtained from the time that the patient
arrived in the ICU of the Cardio chest until the time of discharge.) All these data
were obtained by means of the daily developments of medical and nursing staff.
The assessment of respiratory muscle strength was obtained by measurement of
inspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative
period (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand
spread over -120 to 120 cmH2O equipped with adapter buccal and nasal
forceps according to the technique described by Nephew et al. (2008). The MIP
measurement was performed from residual volume, and MEP was obtained from total lung
capacity. Three measurements were taken technically satisfactory, and if there were
more than 20% difference between the measurements, a fourth and even fifth
measurement would be performed, being always considered the highest value obtained.
The MIP was performed for evaluation of inspiratory muscle strength in three
different stages of the study (Pre, 3PO and 5PO), as well as to adjust the training
load (40% MIP) using the Threshold - IMT® (Threshold - IMT®
inspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one
millimeter (mm) was performed to prevent occlusion of the glottis and influence of
the buccinator muscles during MIP maneuver[9].
Mark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to
prevent lung volumes. The minute volume was obtained with the patient breathing for a
minute for a mouth connected to the spirometer. Tidal volume was obtained indirectly
by the relationship between the respiratory rate and minute volume (MV/FR), the two
volumes measured in milliliters (ml) and respiratory rate was measured in cycles per
minute (cpm) and obtained by direct observation of respiratory cycles during the
evaluation of the minute volume[7].
All patients were evaluated by the same evaluator (preoperative period - PRE), third
postoperative (3PO) and fifth postoperative days (5PO), as well as received
physiotherapeutical treatment, according to their needs in the postoperative period
by physical therapy team in the hospital and only Group I received physiotherapy in
the preoperative period.
The variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI
> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days
before surgery and for former smokers those with a prior history, 30 days ago) as
well as the time of surgery, CPB, anoxia, IOT (defined by the sum between the
anesthesia time and the time that the patient remained with the endotracheal tube in
the intensive care unit - ICU), VM (IOT time defined by the time of ventilator
disconnection (when the patient was placed in spontaneous ventilation in the T-tube),
ICU stay (when the patient arrived in the intensive care unit until discharged to the
ward bed) and length of stay in the OP (obtained from the time that the patient
arrived in the ICU of the Cardio chest until the time of discharge.) All these data
were obtained by means of the daily developments of medical and nursing staff.
The assessment of respiratory muscle strength was obtained by measurement of
inspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative
period (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand
spread over -120 to 120 cmH2O equipped with adapter buccal and nasal
forceps according to the technique described by Nephew et al. (2008). The MIP
measurement was performed from residual volume, and MEP was obtained from total lung
capacity. Three measurements were taken technically satisfactory, and if there were
more than 20% difference between the measurements, a fourth and even fifth
measurement would be performed, being always considered the highest value obtained.
The MIP was performed for evaluation of inspiratory muscle strength in three
different stages of the study (Pre, 3PO and 5PO), as well as to adjust the training
load (40% MIP) using the Threshold - IMT® (Threshold - IMT®
inspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one
millimeter (mm) was performed to prevent occlusion of the glottis and influence of
the buccinator muscles during MIP maneuver[9].
Mark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to
prevent lung volumes. The minute volume was obtained with the patient breathing for a
minute for a mouth connected to the spirometer. Tidal volume was obtained indirectly
by the relationship between the respiratory rate and minute volume (MV/FR), the two
volumes measured in milliliters (ml) and respiratory rate was measured in cycles per
minute (cpm) and obtained by direct observation of respiratory cycles during the
evaluation of the minute volume[7].
Procedures/treatment Patients allocated to the GI protocol underwent preoperative physiotherapy consisting
of breathing exercises (breathing in time, deep breath and then exhaling long,
sustained maximal inspiration with a 6-second apnea, diaphragmatic breathing
associated with mobilization of the upper limb, with three series out of ten for each
breathing exercise) and breathing exercises with the device Threshold -
IMT®. We conducted three sets of ten repetitions with an interval of
two minutes each repetition, once a day for every day of hospitalization in the PRE,
with a load of 40% of the initial MIP, obtained by manovacuometry[8-10].
The GII patients received only guidelines ward routine before surgery, but
postoperatively, both groups performed physical therapy as needed by staff
physiotherapy service.
Patients allocated to the GI protocol underwent preoperative physiotherapy consisting
of breathing exercises (breathing in time, deep breath and then exhaling long,
sustained maximal inspiration with a 6-second apnea, diaphragmatic breathing
associated with mobilization of the upper limb, with three series out of ten for each
breathing exercise) and breathing exercises with the device Threshold -
IMT®. We conducted three sets of ten repetitions with an interval of
two minutes each repetition, once a day for every day of hospitalization in the PRE,
with a load of 40% of the initial MIP, obtained by manovacuometry[8-10].
The GII patients received only guidelines ward routine before surgery, but
postoperatively, both groups performed physical therapy as needed by staff
physiotherapy service.
Statistical Analysis The sample should be made with at least 26 patients who were randomly divided into
two study groups (with physical therapy preoperatively and control patients who did
not receive preoperative physiotherapy). We considered the level of significance of
5% and a test power in the order of 80% for a minimum difference in the order of
two.
The comparison between the groups for age and body mass index (BMI) was performed by
Wilcoxon test for independent samples. The smoking variable was analyzed using
Goldman to compare proportions within and among multinomial populations. The
perfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative
hospital stay were obtained in minutes and analyzed by Student's t test or the
nonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman
tests for comparison of the moments in each group and Mann Whitney test to compare
groups at each time, adopting a significance level of 5% probability of rejecting
null hypothesis.
This study was approved by the Research Ethics Committee of Universidade Estadual
Paulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,
protocol 3223) and all patients signed an informed consent form.
The sample should be made with at least 26 patients who were randomly divided into
two study groups (with physical therapy preoperatively and control patients who did
not receive preoperative physiotherapy). We considered the level of significance of
5% and a test power in the order of 80% for a minimum difference in the order of
two.
The comparison between the groups for age and body mass index (BMI) was performed by
Wilcoxon test for independent samples. The smoking variable was analyzed using
Goldman to compare proportions within and among multinomial populations. The
perfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative
hospital stay were obtained in minutes and analyzed by Student's t test or the
nonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman
tests for comparison of the moments in each group and Mann Whitney test to compare
groups at each time, adopting a significance level of 5% probability of rejecting
null hypothesis.
This study was approved by the Research Ethics Committee of Universidade Estadual
Paulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,
protocol 3223) and all patients signed an informed consent form.
Delimitation:
All patients were evaluated by the same evaluator (preoperative period - PRE), third
postoperative (3PO) and fifth postoperative days (5PO), as well as received
physiotherapeutical treatment, according to their needs in the postoperative period
by physical therapy team in the hospital and only Group I received physiotherapy in
the preoperative period.
The variables evaluated were: body mass index (BMI) - criterion for obesity was a BMI
> 30 kg/m2, smoking (patients who smoke or have smoked up to 30 days
before surgery and for former smokers those with a prior history, 30 days ago) as
well as the time of surgery, CPB, anoxia, IOT (defined by the sum between the
anesthesia time and the time that the patient remained with the endotracheal tube in
the intensive care unit - ICU), VM (IOT time defined by the time of ventilator
disconnection (when the patient was placed in spontaneous ventilation in the T-tube),
ICU stay (when the patient arrived in the intensive care unit until discharged to the
ward bed) and length of stay in the OP (obtained from the time that the patient
arrived in the ICU of the Cardio chest until the time of discharge.) All these data
were obtained by means of the daily developments of medical and nursing staff.
The assessment of respiratory muscle strength was obtained by measurement of
inspiratory pressure (MIP) and maximal voluntary expiratory (MEP) in the preoperative
period (PRE), 3PO and 5PO using analog manometer Commercial Medical® brand
spread over -120 to 120 cmH2O equipped with adapter buccal and nasal
forceps according to the technique described by Nephew et al. (2008). The MIP
measurement was performed from residual volume, and MEP was obtained from total lung
capacity. Three measurements were taken technically satisfactory, and if there were
more than 20% difference between the measurements, a fourth and even fifth
measurement would be performed, being always considered the highest value obtained.
The MIP was performed for evaluation of inspiratory muscle strength in three
different stages of the study (Pre, 3PO and 5PO), as well as to adjust the training
load (40% MIP) using the Threshold - IMT® (Threshold - IMT®
inspiratory Muscle Trainer, HealthscanProducts Inc.). A small hole about one
millimeter (mm) was performed to prevent occlusion of the glottis and influence of
the buccinator muscles during MIP maneuver[9].
Mark Wright 8 display with 35 mm (FERRARIS) in PRE moments, 3PO and 5PO were used to
prevent lung volumes. The minute volume was obtained with the patient breathing for a
minute for a mouth connected to the spirometer. Tidal volume was obtained indirectly
by the relationship between the respiratory rate and minute volume (MV/FR), the two
volumes measured in milliliters (ml) and respiratory rate was measured in cycles per
minute (cpm) and obtained by direct observation of respiratory cycles during the
evaluation of the minute volume[7].
Procedures/treatment:
Patients allocated to the GI protocol underwent preoperative physiotherapy consisting
of breathing exercises (breathing in time, deep breath and then exhaling long,
sustained maximal inspiration with a 6-second apnea, diaphragmatic breathing
associated with mobilization of the upper limb, with three series out of ten for each
breathing exercise) and breathing exercises with the device Threshold -
IMT®. We conducted three sets of ten repetitions with an interval of
two minutes each repetition, once a day for every day of hospitalization in the PRE,
with a load of 40% of the initial MIP, obtained by manovacuometry[8-10].
The GII patients received only guidelines ward routine before surgery, but
postoperatively, both groups performed physical therapy as needed by staff
physiotherapy service.
Statistical Analysis:
The sample should be made with at least 26 patients who were randomly divided into
two study groups (with physical therapy preoperatively and control patients who did
not receive preoperative physiotherapy). We considered the level of significance of
5% and a test power in the order of 80% for a minimum difference in the order of
two.
The comparison between the groups for age and body mass index (BMI) was performed by
Wilcoxon test for independent samples. The smoking variable was analyzed using
Goldman to compare proportions within and among multinomial populations. The
perfusion, anoxia, mechanical ventilation, anesthesia, surgery, ICU and postoperative
hospital stay were obtained in minutes and analyzed by Student's t test or the
nonparametric Mann-Whitney test. MIP, MEP, TV, MV and FR were assessed by Friedman
tests for comparison of the moments in each group and Mann Whitney test to compare
groups at each time, adopting a significance level of 5% probability of rejecting
null hypothesis.
This study was approved by the Research Ethics Committee of Universidade Estadual
Paulista (UNESP)/Faculty of Medicine of Botucatu (registration number 236/2009,
protocol 3223) and all patients signed an informed consent form.
RESULTS:
Seventy patients were divided into two groups, Group I composed of 35 patients (12
female and 23 male) and Group II, with 35 patients (six female and 29 male), as
illustrated in Figure 1.
Characterization of groups by gender
No significant difference was noticed between groups for the variables age (GI 58.9±9.53
years and GII 61.4±8.43 years; P=0.26) and body mass index (GI
26.8±3.96 kg/m2 and G II 26±3.86 kg/m2) (Table 1).
Patient characteristics regarding age and BMI.
BMI: body mass index; Wilcoxon test. * P<0.05
The groups were homogenous concerning presence of active smoking, ex-smokers and
nonsmokers, as illustrated in Table 2.
Number and proportion of patients in each group according to the presence of
active smoking, ex-smokers and nonsmokers.
Two proportions followed by the same lowercase letter do not differ in groups
(rows). Two proportions followed by the same capital letter do not differ in
the types of smoking (P>0.05). Goldman test to compare proportions between
and within populations multinomina.
Between the GI and GII, no significant difference regarding the proportion of
ex-smokers, smokers and nonsmokers, in other words, the groups were homogeneous as
demonstrated below (Table 2).
Maximum Inspiratory Pressure (MIP) The mean values the for maximum inspiratory pressures evaluated in the preoperative
period and 3PO showed no significant difference between groups
(P=0.276 and 0.065; respectively), however, we observed greater
inspiratory muscle strength for GI in relation to GII in 5PO
(P=0.001) (Figure 2).
Median maximum inspiratory pressure (MIP) variable in different moments of the
operation of each group analyzed (with and without physical therapy
preoperatively). Friedman test for comparison of moments in each group and Mann
Whitney test to compare the groups at each time, *P<0.05.
The mean values the for maximum inspiratory pressures evaluated in the preoperative
period and 3PO showed no significant difference between groups
(P=0.276 and 0.065; respectively), however, we observed greater
inspiratory muscle strength for GI in relation to GII in 5PO
(P=0.001) (Figure 2).
Median maximum inspiratory pressure (MIP) variable in different moments of the
operation of each group analyzed (with and without physical therapy
preoperatively). Friedman test for comparison of moments in each group and Mann
Whitney test to compare the groups at each time, *P<0.05.
Maximum Expiratory Pressure (MEP) The mean value for the maximum expiratory pressure obtained in the preoperative
period for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80
cmH2O) for GII, not presenting significant difference between groups
(P=0.704). However, the values referring MEP measured in the 5PO
showed significant difference between groups (P=0.001), as
illustrated in Figure 3.
Median for maximum expiratory pressure (MEP) variable at different times of the
operation in the different groups. Friedman test for comparison of moments in
each group and Mann Whitney test to compare the groups at each time,
*P<0.05.
The mean value for the maximum expiratory pressure obtained in the preoperative
period for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80
cmH2O) for GII, not presenting significant difference between groups
(P=0.704). However, the values referring MEP measured in the 5PO
showed significant difference between groups (P=0.001), as
illustrated in Figure 3.
Median for maximum expiratory pressure (MEP) variable at different times of the
operation in the different groups. Friedman test for comparison of moments in
each group and Mann Whitney test to compare the groups at each time,
*P<0.05.
Minute Volume (MV) The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period
presented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;
P=0.001). However, these values have not showed significative
difference in the 3PO and 5PO (P=0.585 and P=0.329;
respectively).
Median minute volume (VM ) variable at different times of the operation of each
group analyzed. Friedman test for comparison of the moments in each group and
the Mann Whitney test for comparison between groups at any time,
*P<0.05.
The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period
presented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;
P=0.001). However, these values have not showed significative
difference in the 3PO and 5PO (P=0.585 and P=0.329;
respectively).
Median minute volume (VM ) variable at different times of the operation of each
group analyzed. Friedman test for comparison of the moments in each group and
the Mann Whitney test for comparison between groups at any time,
*P<0.05.
Tidal Volume Figure 5 illustrates the analysis of variable
volume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between
groups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these
values were not significantly different for the 3PO and 5 PO
(P=0.059 and P=0.549; respectively).
Median tidal volume variable at different times for the group treated with
preoperative physiotherapy and without physical therapy preoperatively.
Friedman test for comparison of moments in each group and Mann Whitney test to
compare the groups at each time, *P<0.05.
Figure 5 illustrates the analysis of variable
volume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between
groups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these
values were not significantly different for the 3PO and 5 PO
(P=0.059 and P=0.549; respectively).
Median tidal volume variable at different times for the group treated with
preoperative physiotherapy and without physical therapy preoperatively.
Friedman test for comparison of moments in each group and Mann Whitney test to
compare the groups at each time, *P<0.05.
Respiratory rate (RR) The median refers to the respiratory rate obtained preoperatively for GI and GII
showed no significant difference (16 cpm and 18 cpm, respectively,
P=0.602). Likewise, the measurements were made in the 5PO 3PO and
showed no significant difference between groups (P=0.090 and
P=0.886, respectively), as shown in Figure 6.
Median variable respiratory rate at different times of the operation of each
group analyzed (with and without physical therapy preoperatively). Friedman
test for comparison of moments in each group and Mann Whitney test to compare
the groups at each time, *P<0.05.
Infusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and
hospital stay after surgery.
Variables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU
stay did not significantly differ between groups, however, significant difference was
observed for the length of stay variable in the postoperative period
(P=0.001), as shown in Table
3.
Values for the infusion time, anoxia, mechanical ventilation, anesthesia,
surgery, ICU stay and hospital stay after surgery for preoperative, 3PO and
5PO.
Values are shown as mean ± standard deviation or median (minimum and maximum
values).
Mann and Whitney
Student's t test.
Significant difference between groups (P <0.05). ICU: intensive care
unit; PO: postoperative
The median refers to the respiratory rate obtained preoperatively for GI and GII
showed no significant difference (16 cpm and 18 cpm, respectively,
P=0.602). Likewise, the measurements were made in the 5PO 3PO and
showed no significant difference between groups (P=0.090 and
P=0.886, respectively), as shown in Figure 6.
Median variable respiratory rate at different times of the operation of each
group analyzed (with and without physical therapy preoperatively). Friedman
test for comparison of moments in each group and Mann Whitney test to compare
the groups at each time, *P<0.05.
Infusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and
hospital stay after surgery.
Variables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU
stay did not significantly differ between groups, however, significant difference was
observed for the length of stay variable in the postoperative period
(P=0.001), as shown in Table
3.
Values for the infusion time, anoxia, mechanical ventilation, anesthesia,
surgery, ICU stay and hospital stay after surgery for preoperative, 3PO and
5PO.
Values are shown as mean ± standard deviation or median (minimum and maximum
values).
Mann and Whitney
Student's t test.
Significant difference between groups (P <0.05). ICU: intensive care
unit; PO: postoperative
Maximum Inspiratory Pressure (MIP):
The mean values the for maximum inspiratory pressures evaluated in the preoperative
period and 3PO showed no significant difference between groups
(P=0.276 and 0.065; respectively), however, we observed greater
inspiratory muscle strength for GI in relation to GII in 5PO
(P=0.001) (Figure 2).
Median maximum inspiratory pressure (MIP) variable in different moments of the
operation of each group analyzed (with and without physical therapy
preoperatively). Friedman test for comparison of moments in each group and Mann
Whitney test to compare the groups at each time, *P<0.05.
Maximum Expiratory Pressure (MEP):
The mean value for the maximum expiratory pressure obtained in the preoperative
period for GI was 110 (120/50 cmH2O) and 110 cmH2O (120/80
cmH2O) for GII, not presenting significant difference between groups
(P=0.704). However, the values referring MEP measured in the 5PO
showed significant difference between groups (P=0.001), as
illustrated in Figure 3.
Median for maximum expiratory pressure (MEP) variable at different times of the
operation in the different groups. Friedman test for comparison of moments in
each group and Mann Whitney test to compare the groups at each time,
*P<0.05.
Minute Volume (MV):
The analysis of the minute volume variable is presented in Figure 4. The values the MV obtained in the preoperative period
presented significant difference between groups (GI: 10,110 ml and GII: 12,660 ml;
P=0.001). However, these values have not showed significative
difference in the 3PO and 5PO (P=0.585 and P=0.329;
respectively).
Median minute volume (VM ) variable at different times of the operation of each
group analyzed. Friedman test for comparison of the moments in each group and
the Mann Whitney test for comparison between groups at any time,
*P<0.05.
Tidal Volume:
Figure 5 illustrates the analysis of variable
volume obtained in the PRE, 3PO and 5PO. Upon PRE, a significant difference between
groups was detected (GI: 607 ml and GII: 769 ml, P=0.003), but these
values were not significantly different for the 3PO and 5 PO
(P=0.059 and P=0.549; respectively).
Median tidal volume variable at different times for the group treated with
preoperative physiotherapy and without physical therapy preoperatively.
Friedman test for comparison of moments in each group and Mann Whitney test to
compare the groups at each time, *P<0.05.
Respiratory rate (RR):
The median refers to the respiratory rate obtained preoperatively for GI and GII
showed no significant difference (16 cpm and 18 cpm, respectively,
P=0.602). Likewise, the measurements were made in the 5PO 3PO and
showed no significant difference between groups (P=0.090 and
P=0.886, respectively), as shown in Figure 6.
Median variable respiratory rate at different times of the operation of each
group analyzed (with and without physical therapy preoperatively). Friedman
test for comparison of moments in each group and Mann Whitney test to compare
the groups at each time, *P<0.05.
Infusion time, anoxia, mechanical ventilation, anesthesia, surgery, ICU stay and
hospital stay after surgery.
Variables infusion time, anoxia, mechanical ventilation, anesthesia, surgery and ICU
stay did not significantly differ between groups, however, significant difference was
observed for the length of stay variable in the postoperative period
(P=0.001), as shown in Table
3.
Values for the infusion time, anoxia, mechanical ventilation, anesthesia,
surgery, ICU stay and hospital stay after surgery for preoperative, 3PO and
5PO.
Values are shown as mean ± standard deviation or median (minimum and maximum
values).
Mann and Whitney
Student's t test.
Significant difference between groups (P <0.05). ICU: intensive care
unit; PO: postoperative
DISCUSSION:
Studies by Feltrim et al.[10] show
that performing preoperative physiotherapy is more effective in reducing respiratory
complications in patients with moderate or higher risk than those whose risk was
low.
In the present study, we observed no significant difference between the subjects of
Group I and Group II as the characteristics (age, BMI, smoking and number of co
morbidities), which suggests that the study subjects in both groups were
homogeneous.
In the study by Leguisamo et al.[9],
with 86 patients undergoing CABG, divided into two groups: the intervention group (44
patients) who were assessed and received physiotherapeutic guidance at least 15 days
before surgery. The control group received routine care on the day of
hospitalization.
The average MIP between groups showed neither significant difference in the preoperative
period nor in the 1PO and 6PO.
In the present study it was observed that there was no significant difference between
groups to measure MIP in the preoperative period, but now in times of 3PO and 5PO
significant difference between groups was found. Similarly, no significant difference in
the amount of MEP preoperatively in the 3PO and between groups. However in 5PO, there
was significant difference between the groups with the best values of the group that
performed physical therapy before surgery.
Barros et al.[11] evaluated the MIP
and MEP in the preoperative period, the first day after surgery and in 38 patients
undergoing CABG with CPB who were divided into two groups: 23 patients in group I
(respiratory muscle training) and 15 in group II (control). Group I received
conventional physiotherapy over respiratory muscle training and Group II the
conventional physiotherapy. The authors found that the values obtained for MIP and MEP
in hospital discharge were higher in group I.
There was no decrease in maximal respiratory pressures, transdiaphragmatic pressure and
diaphragm pressure, indicating weakness of respiratory muscle strength[12,13].
Agreeing with the authors cited above, we observed in the present study, after cardiac
surgery, MIP values in GI and GII which decreased in 3PO, however the value of MIP in
5PO increase was observed for GI values compared to those in PRE and 3PO since the GII
values remained stable in 3PO. The authors noted the drop in MEP in 3PO in both groups,
however, in 5PO MEP has increased in GI. Regarding PRE and 3PO in GII no return value
was observed in PRE.
Arcêncio et al.[12] conducted a study
with 30 patients, aged 50 years-old and candidates for CABG and / or heart valve,
dividing into two groups. Intervention group comprised 15 patients who underwent at
least a two weeks inspiratory muscle training using incentive spirometry ("Threshold -
IMT®) with 40% charge of the MIP and control group with 15 patients who
received only general guidelines. The authors compared the spirometric values before and
after training and showed no significant difference in the values of MIP and
MEP[14].
Elias et al.[15] studied 42 patients
aged 18 to 75 years-old with coronary artery disease, in the pre-and postoperative
period, divided into two groups. Intervention group received TMR with Threshold -
IMT® and control group received only guidelines. The TMR both pre and
post-operative period consisted of three sets of 10 inspiratory exercises once a day for
three consecutive days. It was observed that after cardiac surgery there was a
significant reduction in MIP and MEP in both groups.
Morsch et al.[13] conducted a study to
evaluate 108 patients undergoing elective CABG surgery from April 2006 to February 2007
(preoperatively and postoperatively).
The average values of MIP in the preoperative period were significantly decreased
compared to 6 days after the surgery (P<0.001). The authors also
observed a significant decrease in the values of MEP obtained on the 6th
postoperative day compared to the preoperative period (P<0.001).
Patients with respiratory muscle weakness have higher risk of postoperative pulmonary
complications. The respiratory muscle training in the pre-operative may prevent
pulmonary complications in patients undergoing thoracic surgery[16]. There may be a reduction of all lung
volumes resulting from factors such as diaphragmatic dysfunction, pain, absence of deep
breaths, pulmonary changes and ribcage. The functional residual capacity (FRC) decreases
because of the reduction of both the residual volume (RV) and expiratory reserve volume.
The ventilation is affected by VC reduction by approximately 20%, and the increase in
FR(22-24). The sum of these factors cause changes in the mechanics of
respiration, which leads to a shallow breathing pattern of decreased lung
volume[13].
Patients undergoing CABG develop mostly PO pulmonary dysfunction with a significant
reduction in lung volumes, impairments in respiratory function, decreased lung
compliance and increased work of breathing. The reduction in lung volumes and capacities
contributes to changes in gas exchange, resulting in hypoxemia[17].
In this study, the VM showed significant difference regarding the groups I and II in the
preoperative period but not significant between groups in 3PO. Likewise, the mean value
of the VM for the GI 5PO, there was no significant difference in the GII. The mean value
of VC in PRE in GI was significant, as the mean value of the VC in GII patients, however
in GI and GII we observed in 5PO and 3PO that there was no significant difference. It
was observed in GI at different times that the increase of VM will be very likely due to
increased FR, since the current VC remained without many changes between different
times.
Barros et al.[11] demonstrated a
significant decrease in lung volumes in patients undergoing cardiac surgery in the
postoperative period.
Carvalho et al.[18] observed a
decrease in the values of minute volume and tidal volume measured on the 2nd
postoperative day, however, these values showed gradual improvement returning to the
values obtained at baseline (preoperative period) at the time of hospital discharge.
In a study, Guizilini et al.[19]
evaluated 30 patients with a mean age of 56 years and divided into two groups, group A
(n=15) without CPB and group B (n=15) with CPB. All patients underwent pulmonary
function. In both groups, there was a decrease in FVC until the fifth postoperative day
(P<0.05).
Regarding the respiratory rate in the present study, we can verify that there was an
increase in both groups, with a higher peak on the 3rd postoperative day and
decreased on the 6th postoperative day, but the respiratory rate did not
return to the preoperative values, Carvalho et al.[18] already mentioned above that they concluded in their studies
that the mean respiratory frequency had a significant increase between pre and
2nd PO, 3rd PO, decreasing on the 4th postoperative
day and increased again on the 5th postoperative day and decreased in
hospital discharge, but patients did not return to the preoperative values.
According to the results obtained in this study, we can affirm that there was no
significant difference between the groups regarding the time variables: anesthesia,
surgery, perfusion, anoxia, mechanical ventilation, intubation. This suggests that the
groups were quite homogeneous. As for the time of postoperative hospital stay, there was
a decrease of approximately 25 hours, comparing patients who underwent physical therapy
before surgery with patients who did not.
In contrast to the findings of this study, Patman et al.[20] performed a randomized study of 236 patients to
evaluate the effects of physical therapy interventions performed early versus only
started after extubation in patients undergoing cardiac surgery and no significant
differences were found in the time of intubation, ICU stay and length of hospital stay
between the groups.
However, Celli et al.[21] conducted a
study with 172 patients and showed a decrease in length of hospital stay in the group
that had received guidelines breathing exercises (9.6±3.2 days) compared to the control
group (13±5 days).
Corroborating these findings, as well as those obtained in the present study, Leguisamo
et al.[9] performed a prospective
study to evaluate the effectiveness of a physiotherapy program start in the preoperative
period in reducing the length of hospital stay and showed a significant reduction in
length of hospital stay for the intervention group compared to the control group
(P<0.05).
These data suggest that physical therapy initiated before surgery can improve conditions
of patients, reestablishing early respiratory pressures, reducing the length of stay and
decreased hospital costs for the period of hospitalization.
Limitation of the study The major limitation was the short period of time that patients received preoperative
physiotherapy; however, we understand that physiotherapeutic treatment had a
relevance even in a short period, so the study may add knowledge of response to
therapy performed during intraoperative CABG surgery.
Not performing spirometry can be mentioned as a limitation on the analysis of
ventilation parameters, however, due to the conditions offered by our service, we
could not perform this evaluation.
On the other hand, the results showed that even with limited time monitoring in the
preoperative period and respiratory variables obtained only by respirometry and
manometry in physical therapy in the preoperative period, proved to be beneficial for
patients undergoing surgery CABG.
We understand that the results may open up new avenues of future research related to
the role of physiotherapy in preoperative myocardial revascularization, in addition,
further studies should be conducted using tools such as spirometry, to assess and
quantify the volume and lung capacity more accurately.
The major limitation was the short period of time that patients received preoperative
physiotherapy; however, we understand that physiotherapeutic treatment had a
relevance even in a short period, so the study may add knowledge of response to
therapy performed during intraoperative CABG surgery.
Not performing spirometry can be mentioned as a limitation on the analysis of
ventilation parameters, however, due to the conditions offered by our service, we
could not perform this evaluation.
On the other hand, the results showed that even with limited time monitoring in the
preoperative period and respiratory variables obtained only by respirometry and
manometry in physical therapy in the preoperative period, proved to be beneficial for
patients undergoing surgery CABG.
We understand that the results may open up new avenues of future research related to
the role of physiotherapy in preoperative myocardial revascularization, in addition,
further studies should be conducted using tools such as spirometry, to assess and
quantify the volume and lung capacity more accurately.
Limitation of the study:
The major limitation was the short period of time that patients received preoperative
physiotherapy; however, we understand that physiotherapeutic treatment had a
relevance even in a short period, so the study may add knowledge of response to
therapy performed during intraoperative CABG surgery.
Not performing spirometry can be mentioned as a limitation on the analysis of
ventilation parameters, however, due to the conditions offered by our service, we
could not perform this evaluation.
On the other hand, the results showed that even with limited time monitoring in the
preoperative period and respiratory variables obtained only by respirometry and
manometry in physical therapy in the preoperative period, proved to be beneficial for
patients undergoing surgery CABG.
We understand that the results may open up new avenues of future research related to
the role of physiotherapy in preoperative myocardial revascularization, in addition,
further studies should be conducted using tools such as spirometry, to assess and
quantify the volume and lung capacity more accurately.
CONCLUSIONS:
The physical therapy has an important role in the preoperative period, restoring greater
readiness ventilatory parameters of patients undergoing coronary artery bypass grafting
with cardiopulmonary bypass, resulting in a decrease in length of hospital stay after
surgery.
|
Background:
The frequency of surgical procedures has increased steadily in recent decades, including the myocardial revascularization.
Methods:
We conducted a prospective study with patients undergoing myocardial revascularization, the Hospital das Clínicas da Universidade Estadual Paulista (UNESP)/Botucatu - SP. We evaluated 70 patients of both genders, aged between 40 and 75 years, subdivided into two groups: group I - 35 patients of both genders, who received a written protocol guidance, breathing exercises and respiratory muscle training in the preoperative period and group II - 35 patients of both genders, who received only orientation of the ward on the day of surgery. This study was approved by the Ethics Committee of UNESP / Botucatu - SP.
Results:
Maximal inspiratory pressure in third postoperative day and fifth postoperative day and significant difference between groups, being better for the intervention group. Expiratory pressure was significant in fifth postoperative day in the intervention group compared to controls. The difference of length of hospital stay in the postoperative was found between the groups with shorter hospital stay in the group receiving preoperative therapy.
Conclusions:
Physical therapy plays an important role in the preoperative period, so that individuals in the intervention group more readily restored the parameters evaluated before surgery, in addition, there was a decrease in the time of the postoperative hospital stay. Thus, it is thought the cost-effectiveness of a program of preoperative physiotherapy.
|
INTRODUCTION:
The cardiac surgery was always clothed with great interest, curiosity, and in some
moments, mysticism, and fact understandable when we analyzed the noble importance of
this component for the proper functioning of the human body[1].
In recent decades the frequency of surgical procedures has progressively increased,
among them the myocardial revascularization (MR). Cardiovascular diseases are a serious
public health problem in Brazil[2].
In individuals over 70 years old it is estimated an incidence of 70% of coronary artery
disease. As the age is a determinant of coronary atherosclerosis, a growing number of
elderly people will be subjected to MR and/or other therapeutic methods[3,4].
The main risk factors that contribute to cardiac diseases are: smoking, high levels of
low-density lipoprotein cholesterol (LDL-cholesterol), low level of high-density
lipoprotein cholesterol (HDL-cholesterol), diabetes mellitus, systemic hypertension,
family history, lifestyle, obesity, sedentary lifestyle and alcohol intake as factors
related to atherosclerosis and its clinical manifestations[4,5].
The coronary artery bypass graft surgery (CABG) procedure is indicated for the treatment
of ischemic heart disease that is manifested clinically by angina and acute myocardial
infarction. The CABG surgery creates a new route for the blood flow and, is used for
making a diversion of blood to the portions of the coronary artery distal to the
obstructive atherosclerotic involvement[1,5].
With the advancements in care of physical therapy in the preoperative period, surgical
techniques, cardiopulmonary bypass (CPB), techniques for myocardial protection,
anesthesia and intensive care in the post-operative period, there was a decrease in
morbidity and mortality of MR which caused the surgical indication in groups of patients
increasingly complex[6,7].
Observing the fact that they are common pulmonary complications of CABG with CPB was
justified to carry out this research, there is great importance in properly carrying out
evaluations, guidelines and breathing exercises in the period before surgery, and
addition, observe the influence and duration of physical therapy assistance in the
preoperative period and also its behavior in the postoperative period.
Objective To demonstrate the importance of the role of physical therapy in the preoperative
period of cardiac surgery with extracorporeal circulation, in relation to the
respiratory muscle strength, pulmonary volumes and duration of hospital stay after
surgery.
To demonstrate the importance of the role of physical therapy in the preoperative
period of cardiac surgery with extracorporeal circulation, in relation to the
respiratory muscle strength, pulmonary volumes and duration of hospital stay after
surgery.
CONCLUSIONS:
The physical therapy has an important role in the preoperative period, restoring greater
readiness ventilatory parameters of patients undergoing coronary artery bypass grafting
with cardiopulmonary bypass, resulting in a decrease in length of hospital stay after
surgery.
|
Background:
The frequency of surgical procedures has increased steadily in recent decades, including the myocardial revascularization.
Methods:
We conducted a prospective study with patients undergoing myocardial revascularization, the Hospital das Clínicas da Universidade Estadual Paulista (UNESP)/Botucatu - SP. We evaluated 70 patients of both genders, aged between 40 and 75 years, subdivided into two groups: group I - 35 patients of both genders, who received a written protocol guidance, breathing exercises and respiratory muscle training in the preoperative period and group II - 35 patients of both genders, who received only orientation of the ward on the day of surgery. This study was approved by the Ethics Committee of UNESP / Botucatu - SP.
Results:
Maximal inspiratory pressure in third postoperative day and fifth postoperative day and significant difference between groups, being better for the intervention group. Expiratory pressure was significant in fifth postoperative day in the intervention group compared to controls. The difference of length of hospital stay in the postoperative was found between the groups with shorter hospital stay in the group receiving preoperative therapy.
Conclusions:
Physical therapy plays an important role in the preoperative period, so that individuals in the intervention group more readily restored the parameters evaluated before surgery, in addition, there was a decrease in the time of the postoperative hospital stay. Thus, it is thought the cost-effectiveness of a program of preoperative physiotherapy.
| 8,719 | 265 | 15 |
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"group",
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[
"test",
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[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]
|
[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]
|
[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]
|
[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]
|
[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]
|
[CONTENT] Thoracic Surgery | Breathing Exercises | Physical Therapy (Specialty) [SUMMARY]
|
[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] Adult | Aged | Breathing Exercises | Female | Humans | Inspiratory Capacity | Length of Stay | Male | Middle Aged | Muscle Strength | Muscle Stretching Exercises | Myocardial Revascularization | Preoperative Care | Preoperative Period | Prospective Studies | Reference Values | Reproducibility of Results | Respiratory Muscles | Respiratory Rate | Statistics, Nonparametric | Tidal Volume | Time Factors | Treatment Outcome [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]
|
[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]
|
[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]
|
[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]
|
[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]
|
[CONTENT] groups | surgery | time | patients | period | preoperative | group | obtained | test | significant [SUMMARY]
|
[CONTENT] cholesterol | importance | surgery | surgical | cardiac | coronary | period | duration | mr | myocardial [SUMMARY]
|
[CONTENT] breathing | patients | performed | patient | obtained | mip | time | minute | pre | threshold [SUMMARY]
|
[CONTENT] groups | test | significant | significant difference | difference | group | values | maximum | median | variable [SUMMARY]
|
[CONTENT] bypass | coronary artery bypass grafting | grafting cardiopulmonary bypass resulting | undergoing coronary | role preoperative period restoring | grafting | grafting cardiopulmonary | grafting cardiopulmonary bypass | ventilatory parameters patients undergoing | undergoing coronary artery bypass [SUMMARY]
|
[CONTENT] groups | test | surgery | period | group | patients | significant | time | preoperative | difference [SUMMARY]
|
[CONTENT] groups | test | surgery | period | group | patients | significant | time | preoperative | difference [SUMMARY]
|
[CONTENT] recent decades [SUMMARY]
|
[CONTENT] Estadual Paulista | UNESP)/Botucatu - SP ||| 70 | between 40 and 75 years | two | II - 35 | the day ||| the Ethics Committee of UNESP / Botucatu - SP [SUMMARY]
|
[CONTENT] third postoperative day | fifth ||| fifth ||| [SUMMARY]
|
[CONTENT] ||| [SUMMARY]
|
[CONTENT] recent decades ||| Estadual Paulista | UNESP)/Botucatu - SP ||| 70 | between 40 and 75 years | two | II - 35 | the day ||| the Ethics Committee of UNESP / Botucatu - SP ||| ||| third postoperative day | fifth ||| fifth ||| ||| ||| [SUMMARY]
|
[CONTENT] recent decades ||| Estadual Paulista | UNESP)/Botucatu - SP ||| 70 | between 40 and 75 years | two | II - 35 | the day ||| the Ethics Committee of UNESP / Botucatu - SP ||| ||| third postoperative day | fifth ||| fifth ||| ||| ||| [SUMMARY]
|
||||||
DNA Methylation status of Wnt antagonist SFRP5 can predict the response to the EGFR-tyrosine kinase inhibitor therapy in non-small cell lung cancer.
|
23009178
|
It is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy.
|
BACKGROUND
|
Tumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC).
|
METHODS
|
We found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype.
|
RESULTS
|
Patients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy.
|
CONCLUSIONS
|
[
"Adaptor Proteins, Signal Transducing",
"Aged",
"Biomarkers, Pharmacological",
"Carcinoma, Non-Small-Cell Lung",
"DNA Methylation",
"Disease-Free Survival",
"ErbB Receptors",
"Eye Proteins",
"Female",
"Humans",
"Male",
"Membrane Proteins",
"Middle Aged",
"Mutation",
"Neoplasm Staging",
"Protein Kinase Inhibitors",
"Treatment Outcome",
"Wnt Signaling Pathway"
] |
3524045
|
Background
|
Lung cancer is the leading cause of cancer death worldwide
[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases
[2,3]. The efficacy of traditional chemotherapy has reached a plateau
[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations
[7-11].
EGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients
[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes
[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy
[10,16,17]. In addition, previous studies reported that both T790M mutation
[18] and c-MET amplification
[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.
The Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development
[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors
[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al
[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.
In current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.
|
Methods
|
Patients 155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.
The smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)
[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.
155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.
The smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)
[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.
DNA extraction and methylation-specific PCR Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously
[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:
sFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.
Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously
[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:
sFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.
Mutation detection The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously
[28].
The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously
[28].
Statistical analysis All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.
All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.
|
Results
|
Characteristics of study patients Table
1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).
Methylation and mutation profile of NSCLC
*The frequency of this group is significantly higher than their counterparts.
Table
1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).
Methylation and mutation profile of NSCLC
*The frequency of this group is significantly higher than their counterparts.
Epigenotype of Wnt antagonists in NSCLC Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file
1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table
1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.
Using DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file
1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table
1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table
2).
P value among methylated genes and EGFR mutation
We next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure
1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.
Hierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.
Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file
1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table
1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.
Using DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file
1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table
1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table
2).
P value among methylated genes and EGFR mutation
We next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure
1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.
Hierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.
Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table
3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.
Multivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)
Previous studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table
3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.
Next, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).
The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table
3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.
Multivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)
Previous studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table
3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.
Next, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).
Epigenotype of Wnt antagonists and progression-free survival (PFS) We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure
2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure
2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file
1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table
4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.
Kaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of
SFRP5
(A),
WIF1
(B), different genotype of
EGFR
(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).
Cox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)
Similar to the previous discovery
[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure
2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure
2D).
We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure
2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure
2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file
1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table
4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.
Kaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of
SFRP5
(A),
WIF1
(B), different genotype of
EGFR
(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).
Cox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)
Similar to the previous discovery
[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure
2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure
2D).
Epigenotype of Wnt antagonists and overall survival rate (OS) To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure
3B), while the epigenotypes of SFRP5 (Figure
3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file
1: Figure S3 A-E), as well as the genotype of EGFR (Figure
3C) were not associated with OS in our patients.
Kaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of
SFRP5
(A),
WIF1
(B), or different genotype of
EGFR
(C).
To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure
3B), while the epigenotypes of SFRP5 (Figure
3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file
1: Figure S3 A-E), as well as the genotype of EGFR (Figure
3C) were not associated with OS in our patients.
Kaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of
SFRP5
(A),
WIF1
(B), or different genotype of
EGFR
(C).
Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.
In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.
|
Conclusions
|
In conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development
[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC.
|
[
"Background",
"Patients",
"DNA extraction and methylation-specific PCR",
"Mutation detection",
"Statistical analysis",
"Characteristics of study patients",
"Epigenotype of Wnt antagonists in NSCLC",
"Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy",
"Epigenotype of Wnt antagonists and progression-free survival (PFS)",
"Epigenotype of Wnt antagonists and overall survival rate (OS)",
"Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy",
"Abbreviations",
"Competing interests",
"Authors’ contributions",
"Authors’ informations"
] |
[
"Lung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.",
"155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.",
"Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.",
"The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].",
"All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.",
"Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.",
"Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.",
"The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).",
"We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).",
"To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n",
"In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.",
"EGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.",
"The authors declare that they have no competing interests.",
"JZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.",
"Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22)."
] |
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[
"Background",
"Methods",
"Patients",
"DNA extraction and methylation-specific PCR",
"Mutation detection",
"Statistical analysis",
"Results",
"Characteristics of study patients",
"Epigenotype of Wnt antagonists in NSCLC",
"Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy",
"Epigenotype of Wnt antagonists and progression-free survival (PFS)",
"Epigenotype of Wnt antagonists and overall survival rate (OS)",
"Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy",
"Discussion",
"Conclusions",
"Abbreviations",
"Competing interests",
"Authors’ contributions",
"Authors’ informations",
"Supplementary Material"
] |
[
"Lung cancer is the leading cause of cancer death worldwide\n[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases\n[2,3]. The efficacy of traditional chemotherapy has reached a plateau\n[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations\n[7-11].\nEGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients\n[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes\n[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy\n[10,16,17]. In addition, previous studies reported that both T790M mutation\n[18] and c-MET amplification\n[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.\nThe Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development\n[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors\n[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al\n[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.\nIn current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.",
" Patients 155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.\n DNA extraction and methylation-specific PCR Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\nGenomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.\n Mutation detection The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\nThe denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].\n Statistical analysis All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.\nAll data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.",
"155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.\nThe smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)\n[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.",
"Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously\n[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:\nsFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.",
"The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously\n[28].",
"All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.",
" Characteristics of study patients Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\nTable\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.\n Epigenotype of Wnt antagonists in NSCLC Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\nGenomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.\n Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\nThe RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).\n Epigenotype of Wnt antagonists and progression-free survival (PFS) We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\nWe next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).\n Epigenotype of Wnt antagonists and overall survival rate (OS) To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\nTo test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n\n Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.\nIn order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.",
"Table\n1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).\nMethylation and mutation profile of NSCLC\n*The frequency of this group is significantly higher than their counterparts.",
"Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file\n1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table\n1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.\nUsing DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file\n1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table\n1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table\n2).\nP value among methylated genes and EGFR mutation\nWe next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure \n1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.\nHierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.",
"The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table\n3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.\nMultivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)\nPrevious studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table\n3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.\nNext, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).",
"We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure \n2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure \n2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file\n1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table\n4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.\n\nKaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), different genotype of \n\nEGFR \n\n(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).\n\nCox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)\nSimilar to the previous discovery\n[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure \n2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure \n2D).",
"To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure \n3B), while the epigenotypes of SFRP5 (Figure \n3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file\n1: Figure S3 A-E), as well as the genotype of EGFR (Figure \n3C) were not associated with OS in our patients.\n\nKaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of \n\nSFRP5 \n\n(A), \n\nWIF1 \n\n(B), or different genotype of \n\nEGFR \n\n(C).\n",
"In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.",
"Recent studies have demonstrated that cancer is as much an epigenetic disease as it is a genetic disease (Iacobuzio-Donahue). Therefore, in addition to genetic alterations, changes in epigenetic features such as CpG DNA methylation status of specific gene loci also mark the progress of cancers. Our current study showed that methylation of Wnt antagonist SFRP5 gene before treatment, independent of the genotype of EGFR gene, correlated with decreased progression free survival rate in NSCLC patients in response to the EGFR-TKI therapy. To our knowledge, this is the first report indicating that DNA methylation at specific gene loci in patient may predict drug response to the EGFT-TKI therapy.\nBoth genetic and epigenetic risk factors for NSCLC have been studied extensively. Suzuki et al\n[23] has reported that methylation of the Wnt antagonist DKK3 correlated with low survival rate in NSCLC patients, despite of the different therapies patients received. However, in our study, we did not find significant difference in the EGFR-TKI responses between patient groups with or without methylated DKK3 (Additional file\n1: Figure S2 and S3). In contrast, our results suggested epigenotype of SFRP5 provide better prognostic estimation for the EGFR-TKI response, comparing to other Wnt antagonists.\nSFRP5 is a member of the SFRP protein family containing a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins. It acts as soluble antagonist of Wnt signaling and is highly expressed in the retinal pigment epithelium, and moderately expressed in the pancreas (\"Entrez Gene: SFRP5 secreted frizzled-related protein 5\"). Previous studies has identified association of SFRP5 promoter hypermethylation with Acute myeloid leukemia\n[29], ovarian cancer\n[30], gastric cancer\n[31], oral squamous cell carcinoma\n[32], pancreatic cancer\n[33] and breast cancer\n[34].\nWe found that hypermethylation of SFRP5 predicted worse outcomes of the EGFR-TKI therapy. Therefore, SFRP5 DNA methylation status may serve as a prognostic molecular marker for appropriately predicting whether NSCLC patients would benefit from the EGFR-TKI therapy. Especially, it is interesting that in the subgroup with adenocarcinoma and EGFR mutation, patients with sFRP5 methylation have a significantly shorter PFS than those without sFRP5 methylation, While in nonsmokers without EGFR mutation, patients without sFRP1 methylation have a longer PFS compared with patients with its methylation(9.7 ms vs 2.0 ms, p = 0.05). Based on these results, we can make a hypothesis that activation of Wnt signaling by antagonist methylation could confer tumors the characters of stem cell, which consequently causes tumors resistant to EGFR TKIs therapy by generating acquired resistance, such as MET amplification or changes of PTEN tumor suppressor activity and so on. Further study is needed to validate this hypothesis.",
"In conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development\n[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC.",
"EGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.",
"The authors declare that they have no competing interests.",
"JZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.",
"Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22).",
"Figure S1. Methylated and unmethyalted bands of Wnt antagonist genes and wild/mutant EGFR. S1: The example graphs of methylated and unmethyalted bands of Wnt antagonist genes (A) and EGFR wild (B) and mutation types (C, D) by methylation specific PCR and DHPLC respectively. Figure S2 PFS with different epigenotypes of Wnt antagonist genes. Figure2S A-F.Kaplan-Meier curves of comparing the progression free survival of patients with different epigenotypes of SFRP1(A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F). Figure S3 OS with different epigenotypes of Wnt antagonist genes. Figure3S A-F. Kaplan-Meier curves of comparing the overall survival of patients with different epigenotypes of SFRP1 (A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F).\nClick here for file"
] |
[
null,
"methods",
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
null,
"discussion",
"conclusions",
null,
null,
null,
null,
"supplementary-material"
] |
[
"DNA methylation",
"EGFR-TKI",
"Wnt antagonists",
"Non-small cell lung cancer"
] |
Background:
Lung cancer is the leading cause of cancer death worldwide
[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases
[2,3]. The efficacy of traditional chemotherapy has reached a plateau
[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations
[7-11].
EGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients
[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes
[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy
[10,16,17]. In addition, previous studies reported that both T790M mutation
[18] and c-MET amplification
[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.
The Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development
[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors
[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al
[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.
In current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.
Methods:
Patients 155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.
The smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)
[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.
155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.
The smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)
[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.
DNA extraction and methylation-specific PCR Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously
[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:
sFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.
Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously
[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:
sFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.
Mutation detection The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously
[28].
The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously
[28].
Statistical analysis All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.
All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.
Patients:
155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.
The smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)
[24] including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.
DNA extraction and methylation-specific PCR:
Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously
[25-27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:
sFRP1:MethylatedF:5’-GTTTTCGGAGTTAGTGTCGCGC-3’,R:5’-ACGATCGAAAACGACGCGAACG-3’,UnmethylatedF:5’-GTAGTTTTTGGAGTTAGTGTTGTGT-3’,R:5’-ACCTACAATCAAAAACAACACAAACA-3’;sFRP2:MethylatedF:5’-TCGGAGTTTTTCGGAGTTGCGC-3’,R:5’-GCTCTCTTCGCTAAATACGACTCG-3’,UnmethylatedF:5’-GGTTGGAGTTTTTTGGAGTTGTGT-3’,R:5-CCCACTCTCTTCACTAAATACAACTCA-3’;sFRP5:MethylatedF:5’-TGGCGTTGGGCGGGACGTTC-3’,R:5’-AACCCGAACCTCGCCGTACG-3’,UnmethylatedF:5’-TGGTGTTGGGTGGGATGTTTG-3’,R:5’-CAACCCAAACCTCACCATACAC-3’;DKK3MethylatedF:5’-GGGGCGGGCGGCGGGGC-3’,R:5’-ACATCTCCGCTCTACGCCCG-3’,UnmethylatedF:5’-TTAGGGGTGGGTGGTGGGGT-3’,R:5’-CTACATCTCCACTCTACACCCA-3’;WIF-1MethylatedF:5’-CGTTTTATTGGGCGTATCGT-3’,R:5’-ACTAACGCGAACGAAATACGA-3’,UnmethylatedF:5’-GGGTGTTTTATTGGGTGTATTGT-3’,R:5’-AAAAACTAACACAAACAAAATACAAAC-3’;APCMethylatedF:5’-TATTGCGGAGTGCGGGTC-3’,R:5’-TCGACGAACTCCCGACGA-3’,UnmethylatedF:5’-GTGTTTTATTGTGGAGTGTGGGTT-3’,R:5’-CCAATCAACAAACTCCCAACAA-3’;CDH-1MethylatedF:5’-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3’,R:5’-CGAATACGATCGAATCGAACCG-3’,UnmethylatedF:5’-TGGTTGTAGTTATGTATTTGTTTTTAGTGG-3’,R:5’-ACACCAAATACAATCAAATCAAACCAAA-3’.
Mutation detection:
The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously
[28].
Statistical analysis:
All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.
Results:
Characteristics of study patients Table
1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).
Methylation and mutation profile of NSCLC
*The frequency of this group is significantly higher than their counterparts.
Table
1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).
Methylation and mutation profile of NSCLC
*The frequency of this group is significantly higher than their counterparts.
Epigenotype of Wnt antagonists in NSCLC Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file
1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table
1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.
Using DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file
1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table
1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table
2).
P value among methylated genes and EGFR mutation
We next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure
1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.
Hierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.
Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file
1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table
1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.
Using DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file
1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table
1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table
2).
P value among methylated genes and EGFR mutation
We next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure
1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.
Hierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.
Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table
3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.
Multivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)
Previous studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table
3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.
Next, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).
The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table
3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.
Multivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)
Previous studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table
3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.
Next, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).
Epigenotype of Wnt antagonists and progression-free survival (PFS) We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure
2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure
2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file
1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table
4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.
Kaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of
SFRP5
(A),
WIF1
(B), different genotype of
EGFR
(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).
Cox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)
Similar to the previous discovery
[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure
2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure
2D).
We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure
2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure
2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file
1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table
4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.
Kaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of
SFRP5
(A),
WIF1
(B), different genotype of
EGFR
(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).
Cox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)
Similar to the previous discovery
[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure
2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure
2D).
Epigenotype of Wnt antagonists and overall survival rate (OS) To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure
3B), while the epigenotypes of SFRP5 (Figure
3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file
1: Figure S3 A-E), as well as the genotype of EGFR (Figure
3C) were not associated with OS in our patients.
Kaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of
SFRP5
(A),
WIF1
(B), or different genotype of
EGFR
(C).
To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure
3B), while the epigenotypes of SFRP5 (Figure
3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file
1: Figure S3 A-E), as well as the genotype of EGFR (Figure
3C) were not associated with OS in our patients.
Kaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of
SFRP5
(A),
WIF1
(B), or different genotype of
EGFR
(C).
Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.
In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.
Characteristics of study patients:
Table
1 summarized the demographic characteristics of 155 study patients, among which 118 cases were adenocarcinoma and 37 cases were non- adenocarcinoma (29 squamous carcinoma, 5 large cell carcinoma, and 3 adeno- squamous carcinoma cases). 60 of all patients received EGFR-TKI as the first-line therapy, while the rest had EGFR-TKI as the second- or more-line treatment. Among those 95 patients who had EGFR-TKI as the second- or more-line treatment, 63 patients took platinum-based chemotherapy as the first-line treatment. The median follow-up time for all patients was 22.4 months (from 2.4 to 77.2 months).
Methylation and mutation profile of NSCLC
*The frequency of this group is significantly higher than their counterparts.
Epigenotype of Wnt antagonists in NSCLC:
Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file
1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table
1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.
Using DHPLC, we also detected EGFR activating mutations in exon 19 or 21 (the examples of wild type, mutated exon 19, and mutated exon 21 were shown in Additional file
1: Figure S1B, 1C, and 1D). Among the 155 patients, 85 (55.4%) carried mutations in either exon 19 or 21 of the EGFR genes (Table
1).Similar to the previous studies, we found that EGFR mutation rates were significantly increased among the patients younger than 65 years old (P = 0.02, Fisher’s exact test) and the patients who are nonsmokers (P = 0.04, Fisher’s exact test). EGFR mutation reversely correlates with sFPR1 methylation (P = 0.005) and sFRP5 (P = 0.011). We fail to find methylation of other wnt antagonist genes correlated with EGFR mutation (Table
2).
P value among methylated genes and EGFR mutation
We next investigated whether the epigenotype of any Wnt antagonist genes correlated with the genotype of EGFR. Hierarchical clustering of the epigenotype of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, as well as the genotype of EGFR (defined as “1” if mutation was detected in the exon 19 or 21, and as “0” if no mutation was detected) was generated using Partek Genomics Suite 6.5 (Partek Inc., MO). As shown in Figure
1, the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR. Therefore, our results suggested that the DNA methylation of Wnt antagonist might be independently regulated from the genotype of EGFR.
Hierarchical clustering of Wnt antagonist DNA methylation status and EGFR genotype in 155 patients received EGFR-TKI therapy. Red represents methylated gene or mutated EGFR, while blue represents unmethylated gene or wild-type EGFR, The figure of hierarchical clustering showed that the epigenotype of Wnt antagonist genes had similar patterns, which were different from the genotype of EGFR.
Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy:
The RECIST was used to evaluate the clinical response of all patients to the TKI therapy. By the end of our study, 59 (38.1%), 53 (33.2%), 43 (27.7%) patients were defined with PD, SD, or PR, respectively. We then calculated the ORR and DCR and analyzed the difference between patient groups with different demographic characteristics, as well as with different genotypes of EGFR and epigenotypes of Wnt antagonist genes. As shown in Table
3, when only single factor was considered, the histology of the cancer (adenocarcinoma/nonadenocarcinoma), line treatment of TKI therapy (first line/not- first line), as well as smoking status (smoker/nonsmoker) significantly affected the ORR to the TKI therapy. Similarly, the gender (male/female), the histology of the cancer (adenocarcinoma/nonadenocarcinoma) as well as smo-king status (smoker/nonsmoker) were found to significantly affect the DCR of the TKI therapy. However, when all demographic characteristics were considered, only the histology of the cancer (P = 0.006, 95% CI, 1.712-26.057, multivariate logistic regression) was associated with ORR.
Multivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)
Previous studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table
3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.
Next, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).
Epigenotype of Wnt antagonists and progression-free survival (PFS):
We next analyzed whether the epigenotypes of Wnt antagonists could predict the PFS in response to the TKI therapy. The median PFS time in all patients was 5.1 months (ranging from 0.4 month to 38 months). Interestingly, as shown in Figure
2A, patients with methylated SFRP5 gene had significantly shorter median PFS time (1.2 months, 95% CI, 0.5-1.9) as compared to those with unmethylated SFRP5 gene (6.1 months, 95% CI, 4.4-7.8) (P = 0.002, Logrank Test). Similarly, patients with methylated WIF1 gene had significantly shorter median PFS time (1.1 months, 95% CI, 95% CI, 1.0-1.2) as compared to those with unmethylated WIF1 gene (5.4 months, 95% CI, 3.5-7.4) (P = 0.006, Logrank Test) (Figure
2B). We did not find association between epigenotype of other Wnt antagonists and PFS in response to the TKI therapy (Additional file
1: Figure S2 A-F). Moreover, after adjusted by age, gender, histology of the cancer, smoking status, and line of treatment, the methylation of SFRP5 gene was still significantly associated with a shorter PFS (P = 0.008; harzard ratio, 2.165, 95% CI, 1.2-3.8; Cox proportional hazards models of survival analysis), while the methylation of WIF1 gene was no longer associated with a shorter PFS (P = 0.224; hazard ratio, 1.804, 95% CI, 0.7-4.7; Cox proportional hazards models of survival analysis) (Table
4). Taken together, our results suggested that the methylation status of SFRP5 might be able to predict the PFS in response to the TKI therapy.
Kaplan-Meier curves are shown comparing the progression free survival of patients with different epigenotypes of
SFRP5
(A),
WIF1
(B), different genotype of
EGFR
(C), or SFRP5 in adenocarcinoma with EGFR mutation group (D).
Cox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)
Similar to the previous discovery
[27], we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure
2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure
2D).
Epigenotype of Wnt antagonists and overall survival rate (OS):
To test whether the epigenotype of Wnt antagonists can predict the clinical outcome of the TKI therapy, we first investigated the association of DNA methylation of the Wnt antagonists and overall survival rate in our patient cohort. Nine patients (6.5%) were lost during the follow-up period of our study. The median OS time was 27.4 months (ranging from 3.0 to 93.1 months). Interestingly, patients with methylated WIF1 genes had significantly reduced overall survival time (P = 0.006, Logrank Test) (Figure
3B), while the epigenotypes of SFRP5 (Figure
3A), SFRP1, SFRP2, DKK3, APC, and CDH1 (Additional file
1: Figure S3 A-E), as well as the genotype of EGFR (Figure
3C) were not associated with OS in our patients.
Kaplan-Meier curves are shown comparing the overall survival of patients with different epigenotypes of
SFRP5
(A),
WIF1
(B), or different genotype of
EGFR
(C).
Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy:
In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.
Discussion:
Recent studies have demonstrated that cancer is as much an epigenetic disease as it is a genetic disease (Iacobuzio-Donahue). Therefore, in addition to genetic alterations, changes in epigenetic features such as CpG DNA methylation status of specific gene loci also mark the progress of cancers. Our current study showed that methylation of Wnt antagonist SFRP5 gene before treatment, independent of the genotype of EGFR gene, correlated with decreased progression free survival rate in NSCLC patients in response to the EGFR-TKI therapy. To our knowledge, this is the first report indicating that DNA methylation at specific gene loci in patient may predict drug response to the EGFT-TKI therapy.
Both genetic and epigenetic risk factors for NSCLC have been studied extensively. Suzuki et al
[23] has reported that methylation of the Wnt antagonist DKK3 correlated with low survival rate in NSCLC patients, despite of the different therapies patients received. However, in our study, we did not find significant difference in the EGFR-TKI responses between patient groups with or without methylated DKK3 (Additional file
1: Figure S2 and S3). In contrast, our results suggested epigenotype of SFRP5 provide better prognostic estimation for the EGFR-TKI response, comparing to other Wnt antagonists.
SFRP5 is a member of the SFRP protein family containing a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins. It acts as soluble antagonist of Wnt signaling and is highly expressed in the retinal pigment epithelium, and moderately expressed in the pancreas ("Entrez Gene: SFRP5 secreted frizzled-related protein 5"). Previous studies has identified association of SFRP5 promoter hypermethylation with Acute myeloid leukemia
[29], ovarian cancer
[30], gastric cancer
[31], oral squamous cell carcinoma
[32], pancreatic cancer
[33] and breast cancer
[34].
We found that hypermethylation of SFRP5 predicted worse outcomes of the EGFR-TKI therapy. Therefore, SFRP5 DNA methylation status may serve as a prognostic molecular marker for appropriately predicting whether NSCLC patients would benefit from the EGFR-TKI therapy. Especially, it is interesting that in the subgroup with adenocarcinoma and EGFR mutation, patients with sFRP5 methylation have a significantly shorter PFS than those without sFRP5 methylation, While in nonsmokers without EGFR mutation, patients without sFRP1 methylation have a longer PFS compared with patients with its methylation(9.7 ms vs 2.0 ms, p = 0.05). Based on these results, we can make a hypothesis that activation of Wnt signaling by antagonist methylation could confer tumors the characters of stem cell, which consequently causes tumors resistant to EGFR TKIs therapy by generating acquired resistance, such as MET amplification or changes of PTEN tumor suppressor activity and so on. Further study is needed to validate this hypothesis.
Conclusions:
In conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development
[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC.
Abbreviations:
EGFR: Epidermal growth factor receptor; EGFR-TKI: Epidermal growth factor receptor -tyrosine kinase inhibitors; MSP: Methylation specific PCR; Wnt: Wingless-type; ECOG: Eastern cooperative oncology group; ORR: Objective response rate; DCR: Disease control rate; PFS: Progression-free survival; OS: Overall survival; PD: Disease progression; CR: Complete response; PR: Partial response; SD: Stable disease; RECIST: Response evaluation criteria in solid tumors; HR: Hazard ratio.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
JZ, YW carried out the molecular genetic studies; JD, MZ, ZW, JZ, SW, LY, TA, MW participated in Provision of study materials or patients and collection and assembly of data; LW, JZ, YW, HB and JW analyzed final data and JZ, YW, JW drafted the manuscript. All authors read and approved the final manuscript.
Authors’ informations:
Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22).
Supplementary Material:
Figure S1. Methylated and unmethyalted bands of Wnt antagonist genes and wild/mutant EGFR. S1: The example graphs of methylated and unmethyalted bands of Wnt antagonist genes (A) and EGFR wild (B) and mutation types (C, D) by methylation specific PCR and DHPLC respectively. Figure S2 PFS with different epigenotypes of Wnt antagonist genes. Figure2S A-F.Kaplan-Meier curves of comparing the progression free survival of patients with different epigenotypes of SFRP1(A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F). Figure S3 OS with different epigenotypes of Wnt antagonist genes. Figure3S A-F. Kaplan-Meier curves of comparing the overall survival of patients with different epigenotypes of SFRP1 (A), SFRP2 (B), DKK3 (C), APC (D), CDH1 (E) and combination analysis (F).
Click here for file
|
Background:
It is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy.
Methods:
Tumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC).
Results:
We found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype.
Conclusions:
Patients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy.
|
Background:
Lung cancer is the leading cause of cancer death worldwide
[1]. NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases
[2,3]. The efficacy of traditional chemotherapy has reached a plateau
[4-6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations
[7-11].
EGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients
[12-14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes
[15], lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy
[10,16,17]. In addition, previous studies reported that both T790M mutation
[18] and c-MET amplification
[19] involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.
The Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development
[20]. Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors
[21], which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR[22]; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al
[23] analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.
In current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.
Conclusions:
In conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development
[35,36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC.
|
Background:
It is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy.
Methods:
Tumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC).
Results:
We found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype.
Conclusions:
Patients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy.
| 10,366 | 266 | 20 |
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[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]
|
[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]
|
[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]
|
[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]
|
[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]
|
[CONTENT] DNA methylation | EGFR-TKI | Wnt antagonists | Non-small cell lung cancer [SUMMARY]
|
[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]
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[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]
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[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]
|
[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]
|
[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]
|
[CONTENT] Adaptor Proteins, Signal Transducing | Aged | Biomarkers, Pharmacological | Carcinoma, Non-Small-Cell Lung | DNA Methylation | Disease-Free Survival | ErbB Receptors | Eye Proteins | Female | Humans | Male | Membrane Proteins | Middle Aged | Mutation | Neoplasm Staging | Protein Kinase Inhibitors | Treatment Outcome | Wnt Signaling Pathway [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]
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[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]
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[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]
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[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]
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[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]
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[CONTENT] egfr | patients | methylation | wnt | sfrp5 | tki | therapy | pfs | 95 | 95 ci [SUMMARY]
|
[CONTENT] egfr | lung | lung cancer | cancer | therapy | wnt | tki | signaling | egfr tki | tki therapy [SUMMARY]
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[CONTENT] unmethylatedf | dna | defined | disease | pcr | pairs | methylatedf | primer pairs | primer | specific [SUMMARY]
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[CONTENT] ci | 95 ci | 95 | patients | egfr | sfrp5 | pfs | months | wnt | significantly [SUMMARY]
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[CONTENT] drugs | detection | dna methylation | dna | egfr tki | protein rna | sfrp5 independent | pfs long | conclusion study revealed sfrp5 | conclusion study revealed [SUMMARY]
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[CONTENT] egfr | patients | wnt | methylation | tki | sfrp5 | 95 | 95 ci | ci | therapy [SUMMARY]
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[CONTENT] egfr | patients | wnt | methylation | tki | sfrp5 | 95 | 95 ci | ci | therapy [SUMMARY]
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[CONTENT] EGFR | EGFR ||| EGFR [SUMMARY]
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[CONTENT] 155 | EGFR | Wnt | SFRP1 | SFRP5 | DKK3 | APC | PCR | MSP ||| EGFR | Denaturing High Performance Liquid Chromatography (DHPLC [SUMMARY]
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[CONTENT] Wnt ||| EGFR | 0.005 | 0.011 ||| Wnt | EGFR ||| SFRP5 | SFRP5 | EGFR | 0.002 | EGFR [SUMMARY]
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[CONTENT] SFRP5 | EGFR [SUMMARY]
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[CONTENT] EGFR | EGFR ||| EGFR ||| 155 | EGFR | Wnt | SFRP1 | SFRP5 | DKK3 | APC | PCR | MSP ||| EGFR | Denaturing High Performance Liquid Chromatography (DHPLC ||| ||| Wnt ||| EGFR | 0.005 | 0.011 ||| Wnt | EGFR ||| SFRP5 | SFRP5 | EGFR | 0.002 | EGFR ||| SFRP5 | EGFR [SUMMARY]
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[CONTENT] EGFR | EGFR ||| EGFR ||| 155 | EGFR | Wnt | SFRP1 | SFRP5 | DKK3 | APC | PCR | MSP ||| EGFR | Denaturing High Performance Liquid Chromatography (DHPLC ||| ||| Wnt ||| EGFR | 0.005 | 0.011 ||| Wnt | EGFR ||| SFRP5 | SFRP5 | EGFR | 0.002 | EGFR ||| SFRP5 | EGFR [SUMMARY]
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Factors Associated with Increased Alpha-Tocopherol Content in Milk in Response to Maternal Supplementation with 800 IU of Vitamin E.
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31013594
|
Vitamin E supplementation might represent an efficient strategy to increase the vitamin E content in milk. The present study aimed to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol content of milk and the factors associated with the increase in vitamin E.
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BACKGROUND
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Randomized clinical trial with 79 lactating women from Brazil, who were assigned to the control group, or to the supplemented group (800 IU of RRR-alpha-tocopherol). Milk and serum were collected between 30 and 90 days after delivery (collection 1), and on the next day (collection 2). Alpha-tocopherol was analyzed using high-performance liquid chromatography.
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METHODS
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In the supplemented group, the alpha-tocopherol content in serum and milk increased after supplementation (p < 0.001). In the multivariate analysis, only alpha-tocopherol in milk (collection 1) was associated with the level of this vitamin in milk after supplementation (β = 0.927, p < 0.001), and binary logistic regression showed that the dietary intake was the only determinant for the greater effect of supplementation in milk.
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RESULTS
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The pre-existing vitamin level in milk and diet are determinants for the efficacy of supplementation in milk, suggesting that in populations with vitamin E deficiency, high-dose supplementation can be used to restore its level in milk.
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CONCLUSION
|
[
"Adult",
"Breast Feeding",
"Dietary Supplements",
"Female",
"Humans",
"Logistic Models",
"Maternal Nutritional Physiological Phenomena",
"Milk, Human",
"Vitamin E",
"Young Adult",
"alpha-Tocopherol"
] |
6520676
|
1. Introduction
|
Breast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].
Studies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].
One strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].
Single-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.
Interestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.
Thus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period.
| null | null |
3. Results
|
3.1. General Characteristics of the Population The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).
The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).
3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).
After supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).
For the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.
At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).
After supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).
For the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.
3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.
When dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).
In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.
When dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).
|
5. Conclusions
|
Vitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E.
|
[
"2. Materials and Methods",
"2.2. Data Collection",
"2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples",
"2.4. Dietary Intake of Vitamin E",
"2.5. Statistical Analysis",
"3.1. General Characteristics of the Population",
"3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk",
"3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation"
] |
[
" 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.",
"A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.",
"The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].",
"Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.",
"Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.",
"The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).",
"At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.",
"In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively)."
] |
[
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"1. Introduction",
"2. Materials and Methods",
"2.1. Participants and Intervention",
"2.2. Data Collection",
"2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples",
"2.4. Dietary Intake of Vitamin E",
"2.5. Statistical Analysis",
"3. Results",
"3.1. General Characteristics of the Population",
"3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk",
"3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation",
"4. Discussion",
"5. Conclusions"
] |
[
"Breast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].\nStudies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].\nOne strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].\nSingle-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.\nInterestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.\nThus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period.",
" 2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\nThe study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).\n 2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\nA semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.\n 2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\nThe extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].\n 2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\nVitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.\n 2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.\nStatistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.",
"The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.\nThe present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.\nThe sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.\nThe eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.\nThe eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).",
"A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).\nMilk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.\nA 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.\nThe dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.",
"The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).\nHPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].\nThe transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].",
"Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.",
"Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.",
" 3.1. General Characteristics of the Population The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\nThe socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).\n 3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\nAt collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.\n 3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).\nIn the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).",
"The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).",
"At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).\nAfter supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).\nFor the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.",
"In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.\nWhen dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).",
"Mature milk is the most stable stage of lactation, in which the content of alpha-tocopherol is not influenced by pregnancy-related factors, as occurs in the colostrum [2,7]. It should be emphasized that the mature milk presents a higher concentration of lipids and in contrast, there is a lower secretion of alpha-tocopherol, suggesting that there are distinct mechanisms involved in the transfer of this vitamin into breast milk [2,35,36].\nIn the present study, the lactating women had adequate vitamin E status, in accordance with other studies considering the same stage of lactation [8,21]. However, a low dietary intake of vitamin E was noted (Table 1), which could trigger the mobilization of alpha-tocopherol from maternal reserves, such as the adipose tissue, into breast milk [15,19]. This low consumption of vitamin E was also reported in other populations in Brazil, Greece and Poland [21,25,36], which suggests a frequent inadequacy in vitamin E consumption during lactation.\nEven in situations of inadequate consumption, the vitamin E concentration in milk was not influenced by diet and circulating maternal levels [8,20,21,37,38,39,40]. However, this present study was the first to identify a positive association between alpha-tocopherol levels in milk and ingested vitamin E (diet + supplementation) and with serum alpha-tocopherol (Figure 2d,c). This suggested that in high-consumption situations, the ingested and circulating maternal levels are the main factors responsible for the vitamin level in milk. It is likely that in situations of low vitamin E consumption (as found in the studies cited), milk vitamin E might originate from other sources, such as the body’s reserve [4], explaining the absence of a relationship between those variables.\nTo prevent of VED in infants, it is necessary for breast milk to contain adequate levels of vitamin E, so that children can obtain the benefits of the micronutrient, through the creation of vitamin reserves in the body and its antioxidant action [4,19]. Some studies that evaluated this vitamin in mature milk observed values below the nutritional requirements of infants [20,23,36,37], which suggested that maternal vitamin E supplementation could be an important strategy to increase milk vitamin contents [17,20,21].\nIn this study, supplementation with 800 IU of alpha-tocopherol caused a 183% increase in serum alpha-tocopherol, and a 124% increase in breast milk alpha-tocopherol (Table 2). Other clinical trials using a lower dose (400 IU alpha-tocopherol) found an increase of 60% to 80% in the vitamin content in milk after supplementation [18,20,21,22], showing a reduced effect compared with that shown in the present study. These findings suggested that the response to supplementation might be influenced by both the dosage used and the determinant factors. However, trials have not evaluated the factors associated with the different responses to large vitamin E doses [17,20], being important to understand how this vitamin is transferred into the mammary gland.\nWhen analyzing the factors associated with a better response in milk after supplementation, it is important to highlight that only the content of this vitamin in the basal milk (before supplementation) and the dietary intake were demonstrated to increase the levels of this micronutrient in milk (Table 3), which suggested that the higher the consumption of vitamin E and its levels in milk, the greater the transfer of alpha-tocopherol from the supplement to the mammary gland.\nAssessment of the profile of lactating women in the supplemented group, showed that the participants with the highest supplementation effect (quartiles 2–4) had a higher intake of calories, alpha-tocopherol and total fat (Table 4). These findings suggested that the amount of fat available in the diet may improve the bioavailability of the vitamin in the body, such as its absorption and distribution to tissues, and in this case, to the mammary gland, as reported in [41,42,43].\nSuch evidence also demonstrated that in mature milk, the Michaelis–Menten kinetic theory could not be applied, as it proposes that the transfer of vitamin to milk occurs through active transport, characterized by a saturation of the lipoprotein receptors in the breast tissue in situations of large contents of vitamin E, which would prevent the continuous transfer of the vitamin into the breast milk after supplementation [38,44]. Notably, in a study of dairy cows, Weiss and Wyatt (2003) [45] suggested that the ability of lipoproteins to carry alpha-tocopherol could determine the uptake of this vitamin by the breast tissue, and that the limiting factor for this mechanism would be the maximum content of vitamin E in the lipoproteins.\nTo further investigate this relationship between lipoproteins and vitamin E transport, we determined the circulating lipoproteins and the serum cholesterol and triglycerides profiles; however, no relation between them and the response to supplementation was found (Table 2). In fact, the mechanism of transport of vitamin E to the mammary gland is poorly understood [2,46,47]. Circulating lipoproteins are responsible for this transfer, with LDL being the main carrier [44,48]; however, transport may occur in the presence or absence of its receptors in the mammary gland [2,44]. Other receptors are found in breast tissue, such as Scavenger Receptor B-1 (SR-B1), which has binding sites for both LDL and HDL, and CD36, which has high affinity binding sites for HDL, LDL, and very low-density lipoprotein (VLDL) [2,48]. It has also been suggested the participation of lipoprotein lipase (LPL), which may show increased activity during lactation, contributes to the greater circulation of alpha-tocopherol and its uptake [49].\nThese findings provide important information to understand the mechanisms by which vitamin E is transferred into the mammary gland, demonstrating that, in situations of supplementation with 800 IU of vitamin E and its effect in mature milk, the better the vitamin E status (considering the milk and dietary intake), the more effective uptake into the mammary gland will be, regardless of receptor saturation. Further investigation into how this transport occurs is required, by means of in vitro and in vivo studies and using labeled isotopes of alpha-tocopherol, for example, to investigate its biotransformation. Notably, in populations with dietary inadequacy and low contents of vitamin E in milk, supplementation with higher doses of the vitamin, such as 800 IU alpha-tocopherol, might be required to obtain a more effective intervention. The analysis of a single dose during the day allowed us to investigate possible factors that could interfere with the response to supplementation; however, it is necessary to analyze how long the effect of this supplementation could be sustained, its contribution to maternal and infant nutritional status, and the use of smaller daily doses.\nTherefore, supplementation associated with an adequate intake of vitamin E is an effective strategy to increase vitamin E levels in breast milk and prevent cases of vitamin E deficiency in infants, especially premature infants [2,4,11].",
"Vitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E."
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[
"clinical trial",
"lactation",
"infants",
"breastfeeding",
"lactating women"
] |
1. Introduction:
Breast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].
Studies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].
One strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].
Single-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.
Interestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.
Thus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period.
2. Materials and Methods:
2.1. Participants and Intervention The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.
The present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.
The sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.
The eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.
The eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).
The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.
The present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.
The sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.
The eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.
The eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).
2.2. Data Collection A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).
Milk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.
A 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.
The dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.
A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).
Milk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.
A 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.
The dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.
2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).
HPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].
The transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].
The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).
HPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].
The transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].
2.4. Dietary Intake of Vitamin E Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.
Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.
2.5. Statistical Analysis Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.
Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.
2.1. Participants and Intervention:
The study was a randomized, parallel-group trial. Participants were recruited at the Pediatric Ambulatory Care of the Onofre Lopes University Hospital (HUOL), Natal-RN, Brazil, and data collection took place between October 2017 and July 2018.
The present study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), under the protocol number 2.327.614, CAAE 76779217.1.0000.5537, and was also registered in the Brazilian Registry of Clinical Trials—ReBec, under the code RBR-38nfg2, available at http://www.ensaiosclinicos.gov.br/rg/RBR-38nfg2/.
The sample calculation was performed using GPower software, Version 3.1.9 [26] considering two independent groups tested using one way analysis of variance (ANOVA) for repeated measures among factors, with alpha parameters equal to 5%, expected power at 80%, and the effect measure value equal to 0.25 [27]. The analysis showed that each group should have at least 33 individuals, totaling 66 participants.
The eligibility criteria included women between 30 and 90 days after delivery; who were breastfeeding their children, either exclusively or partially; who were residents of Natal, RN and its metropolitan regions; who were not diagnosed with a diseases (hypertension, diabetes, neoplasms, heart disease, diseases of the gastrointestinal and hepatic tract, syphilis or were HIV-positive); who were non-smokers; no multiple births and whose infants were not malformed. Exclusion criteria were women who did not have sufficient milk or blood for analysis of vitamin levels, users of illicit drugs, and those who made daily use of vitamin supplements containing vitamin E during lactation.
The eligible participants were informed of the study’s objectives and those who agreed to participate signed the consent form. At recruitment, they were allocated in one of the study groups, depending on the day of the week: Monday and Thursday for the supplemented group and Tuesday and Wednesday for the control group, where only the supplemented group ingested two capsules containing 400 IU of RRR-alpha-tocopherol consecutively, totaling 800 IU (588 mg of alpha-tocopherol). The capsule contained 98% RRR-alpha-tocopherol acetate, as assessed according to the method of Lira (2017) [21]. The study complied with the Consolidated Standards of Reporting Trials—CONSORT (Figure 1).
2.2. Data Collection:
A semi-structured questionnaire was used to collect data on socioeconomic aspects, such as family income, schooling and maternal age, as well as information on gestational age and type of delivery. Maternal height and current weight were also assessed and used to calculate the body mass index (BMI).
Milk and serum were collected from the participants at two time points. Collection 1 was performed at the hospital and collection 2 was performed at the participant’s home the day after collection 1. In the supplemented group, supplementation with 800 IU of RRR-alpha-tocopherol was performed immediately after collection 1 of milk and serum.
A 2 mL sample of breast milk was collected by manual expression from a single breast that had not breastfed recently, and 5 mL of blood were collected by venipuncture. All the biological samples were collected after a 4 to 6 h fast, stored in polypropylene tubes packed in aluminum foil, and transported in refrigerated units. The breast milk was stored at −20 °C until the time of analysis. Before storage, the blood samples were centrifuged for 10 min (at 4000 rpm), to separate the serum for analysis of vitamin E and lipoproteins.
The dietary intake of vitamin E was evaluated by means of the 24 h dietary recall (24HR) applied at the two collection time points. Participants were asked about all foods, supplements and beverages consumed the day before the interview.
2.3. Determination of Alpha-Tocopherol and Lipid Profile in Biological Samples:
The extraction of alpha-tocopherol from milk and serum was performed according to the method adapted by Lira et al. (2013) [28]. Ethanol (95%) was used to precipitate proteins (Vetec, Rio de Janeiro, Brazil), and hexane PA (Vetec, Rio de Janeiro, Brazil) was used as an extraction reagent. After evaporation in nitrogen, serum and milk residues were dissolved, respectively, in 250 μL of absolute ethanol (Vetec, Rio de Janeiro, Brazil) and 250 mL of dichloromethane (Vetec, Rio de Janeiro, Brazil): methanol (Sigma-Aldrich, St. Louis, Missouri, EUA) (2:1; v/v). The aliquots were then analyzed using high-performance liquid chromatography (HPLC).
HPLC consisted of an LC-20AT (Shimadzu, Kyoto, Japan) pump coupled to a CBM 20A communicator and an SPD-10A UV-VIS detector (Shimadzu, Kyoto, Japan). AC18 reversed phase column (LiChroCART 250-4, Merck, Darmstadt, Germany) was used for chromatographic separation. The mobile phase was 100% methanol in an isocratic system, with a flow rate of 1 mL/min and a wavelength of 292 nm was used to detect alpha-tocopherol. The identification and quantification of the vitamin in the samples were established by comparing the area of the peak obtained in the chromatogram with the area of the alpha-tocopherol standard (Sigma-Aldrich, São Paulo, Brazil). The concentration of the standard was confirmed by the specific extinction coefficient for alpha-tocopherol (e1%, alpha-tocopherol, 1 cm = 75.8 to 292 nm) in absolute ethanol (Vetec, Rio de Janeiro, Brazil) [29]. Women with serum alpha-tocopherol values less than 12 μmol/L were considered as deficient in vitamin E [30].
The transport of vitamin E from serum to breast milk involves lipoproteins; therefore, serum cholesterol and high-density lipoprotein (HDL) levels were analyzed with using a commercial kit (Labtest) and enzymatic colorimetric methods, by using an automatic biochemistry analyzer (Labmax plenno). Low-density lipoprotein (LDL) was quantified using the equation proposed by Martin et al. (2013) [31].
2.4. Dietary Intake of Vitamin E:
Vitamin E intake was obtained using two 24 h dietary recall (24HR), applied using face-to-face interviews at the two data collection times in both groups. During this interview, the participants were asked about the food (and its preparation), supplements and beverages consumed in the last 24 h before the interview, in which the home measures described were converted to grams or milliliters [32,33] and the amount of vitamin E consumed was analyzed using the software Virtual Nutri Plus [34], from the database constructed by Rodrigues (2016) [8]. The dietary intake of vitamin E was corrected for total energy intake. The resulting values were obtained using SPSS, version 21.0 for Windows (SPSS Inc., Chicago, IL, USA) employing the residual method.
2.5. Statistical Analysis:
Statistical analysis was performed using the statistical software IBM SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov normality test was applied. Numerical data were expressed as the mean (standard deviation, SD), and categorical results were reported as absolute and relative frequencies. Student’s t-test for dependent samples was used to verify intragroup differences, and the t-test for independent samples was used to analyze the differences between the groups. To evaluate the relationship between serum, breast milk and dietary vitamin E intake, the Pearson correlation coefficient was calculated. Linear multiple regression analysis was used to verify the ratio between alpha-tocopherol in milk after vitamin supplementation and in serum, the lipid profile, vitamin E intake and other maternal factors. The factors associated with the effect of supplementation on milk were also investigated. For this, the lactating women in the supplemented group were divided into quartiles according to the percentage increase in the milk alpha-tocopherol content between collection 1 and collection 2, being classified into a smaller effect (quartile 1) and greater effect (quartiles 2–4). The quartile categorization was used to identify the participants who presented lower effect and greater effects, because all participants should present higher alpha-tocopherol in milk values after supplementation. In addition to providing an analysis of the possible determinants for the milk supplementation response. The association of maternal variables with the effect of supplementation was evaluated according to binary multiple regression. All differences were considered significant when p ≤ 0.05.
3. Results:
3.1. General Characteristics of the Population The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).
The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).
3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).
After supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).
For the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.
At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).
After supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).
For the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.
3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.
When dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).
In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.
When dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).
3.1. General Characteristics of the Population:
The socioeconomic characteristics of the lactating women are presented in Table 1. The mean age of the participants was 27 years, and the majority had completed high school. About 40% of the women were overweight according to their BMI values, and exclusive breastfeeding was predominant (>84%) in both groups. The dietary intake of vitamin E was equivalent to 8.7 mg/day in the control, which was below the recommended intake (16 mg/day) [30] and there was no difference between the groups in terms of dietary intake of vitamin E (p = 0.901).
3.2. Effect of Vitamin E Supplementation on Serum and Breast Milk:
At collection 1, the maternal serum alpha-tocopherol concentrations were similar between the control and supplemented groups, at 26.37 (4.6) μmol/L and 26.38 (5.4) μmol/L, respectively (p = 0.996). In the control group, there was no difference in the alpha-tocopherol concentrations between collection 1 and collection 2 (p > 0.05). Neither group contained cases of VED (<12 μmol/L). In addition, the lipid profiles were similar between the collections and between the groups (p > 0.05) (Table 2).
After supplementation with 800 IU RRR-alpha-tocopherol, a 183% increase in serum alpha-tocopherol was observed in the supplemented group (collection 2), reaching 48.27 μmol/L (p < 0.001) (Table 2).
For the alpha-tocopherol content in mature milk, the control group presented 6.91 (1.81) μmol/L and the supplemented group presented 6.98 (2.18) μmol/L (p = 0.883). One day after supplementation (collection 2), milk from the supplemented group presented higher levels of alpha-tocopherol (15 μmol/L) compared with that in the control group (6.94 μmol/L) (p < 0.001), an increase equivalent to 124% in the post-supplementation milk.
3.3. Factors Associated with Alpha-Tocopherol in Breast Milk after Supplementation:
In the supplemented group, after Pearson correlation analysis, milk from collection 1, dietary intake of vitamin E, and alpha-tocopherol in serum from collection 2 were identified as positively related to alpha-tocopherol levels in milk from collection 2 (Figure 2). These variables were included in the multiple linear regression analysis to evaluate the factors associated with the alpha-tocopherol concentration in breast milk after supplementation. Only alpha-tocopherol in the milk before supplementation was a determinant for the increase in the vitamin content in the milk after administration of 800 IU alpha-tocopherol (β = 0.927, p < 0.001, 95% CI 1.925–2.396). Thus, the higher the vitamin concentration in milk, the greater the transfer of the vitamin to the mammary gland.
When dividing the participants of the supplemented group according to the effect of supplementation (quartile 1 and quartiles 2–4), where quartile 1 is equivalent to 83% of the vitamin E increase percentage in milk after supplementation, we observed that the dietary intake of vitamin E was a determinant that caused a greater response to supplementation (p = 0.020, 95% CI 0.209–0.877), which suggested that the higher the intake, the greater the effect of supplementation (Table 3). The characteristics of the participants divided by the effect of supplementation are described in Table 4, which showed that the consumption of calories, alpha-tocopherol and total fat was higher in the group showing a higher effect of supplementation (p = 0.001, p = 0.013, p = 0.033, respectively).
4. Discussion:
Mature milk is the most stable stage of lactation, in which the content of alpha-tocopherol is not influenced by pregnancy-related factors, as occurs in the colostrum [2,7]. It should be emphasized that the mature milk presents a higher concentration of lipids and in contrast, there is a lower secretion of alpha-tocopherol, suggesting that there are distinct mechanisms involved in the transfer of this vitamin into breast milk [2,35,36].
In the present study, the lactating women had adequate vitamin E status, in accordance with other studies considering the same stage of lactation [8,21]. However, a low dietary intake of vitamin E was noted (Table 1), which could trigger the mobilization of alpha-tocopherol from maternal reserves, such as the adipose tissue, into breast milk [15,19]. This low consumption of vitamin E was also reported in other populations in Brazil, Greece and Poland [21,25,36], which suggests a frequent inadequacy in vitamin E consumption during lactation.
Even in situations of inadequate consumption, the vitamin E concentration in milk was not influenced by diet and circulating maternal levels [8,20,21,37,38,39,40]. However, this present study was the first to identify a positive association between alpha-tocopherol levels in milk and ingested vitamin E (diet + supplementation) and with serum alpha-tocopherol (Figure 2d,c). This suggested that in high-consumption situations, the ingested and circulating maternal levels are the main factors responsible for the vitamin level in milk. It is likely that in situations of low vitamin E consumption (as found in the studies cited), milk vitamin E might originate from other sources, such as the body’s reserve [4], explaining the absence of a relationship between those variables.
To prevent of VED in infants, it is necessary for breast milk to contain adequate levels of vitamin E, so that children can obtain the benefits of the micronutrient, through the creation of vitamin reserves in the body and its antioxidant action [4,19]. Some studies that evaluated this vitamin in mature milk observed values below the nutritional requirements of infants [20,23,36,37], which suggested that maternal vitamin E supplementation could be an important strategy to increase milk vitamin contents [17,20,21].
In this study, supplementation with 800 IU of alpha-tocopherol caused a 183% increase in serum alpha-tocopherol, and a 124% increase in breast milk alpha-tocopherol (Table 2). Other clinical trials using a lower dose (400 IU alpha-tocopherol) found an increase of 60% to 80% in the vitamin content in milk after supplementation [18,20,21,22], showing a reduced effect compared with that shown in the present study. These findings suggested that the response to supplementation might be influenced by both the dosage used and the determinant factors. However, trials have not evaluated the factors associated with the different responses to large vitamin E doses [17,20], being important to understand how this vitamin is transferred into the mammary gland.
When analyzing the factors associated with a better response in milk after supplementation, it is important to highlight that only the content of this vitamin in the basal milk (before supplementation) and the dietary intake were demonstrated to increase the levels of this micronutrient in milk (Table 3), which suggested that the higher the consumption of vitamin E and its levels in milk, the greater the transfer of alpha-tocopherol from the supplement to the mammary gland.
Assessment of the profile of lactating women in the supplemented group, showed that the participants with the highest supplementation effect (quartiles 2–4) had a higher intake of calories, alpha-tocopherol and total fat (Table 4). These findings suggested that the amount of fat available in the diet may improve the bioavailability of the vitamin in the body, such as its absorption and distribution to tissues, and in this case, to the mammary gland, as reported in [41,42,43].
Such evidence also demonstrated that in mature milk, the Michaelis–Menten kinetic theory could not be applied, as it proposes that the transfer of vitamin to milk occurs through active transport, characterized by a saturation of the lipoprotein receptors in the breast tissue in situations of large contents of vitamin E, which would prevent the continuous transfer of the vitamin into the breast milk after supplementation [38,44]. Notably, in a study of dairy cows, Weiss and Wyatt (2003) [45] suggested that the ability of lipoproteins to carry alpha-tocopherol could determine the uptake of this vitamin by the breast tissue, and that the limiting factor for this mechanism would be the maximum content of vitamin E in the lipoproteins.
To further investigate this relationship between lipoproteins and vitamin E transport, we determined the circulating lipoproteins and the serum cholesterol and triglycerides profiles; however, no relation between them and the response to supplementation was found (Table 2). In fact, the mechanism of transport of vitamin E to the mammary gland is poorly understood [2,46,47]. Circulating lipoproteins are responsible for this transfer, with LDL being the main carrier [44,48]; however, transport may occur in the presence or absence of its receptors in the mammary gland [2,44]. Other receptors are found in breast tissue, such as Scavenger Receptor B-1 (SR-B1), which has binding sites for both LDL and HDL, and CD36, which has high affinity binding sites for HDL, LDL, and very low-density lipoprotein (VLDL) [2,48]. It has also been suggested the participation of lipoprotein lipase (LPL), which may show increased activity during lactation, contributes to the greater circulation of alpha-tocopherol and its uptake [49].
These findings provide important information to understand the mechanisms by which vitamin E is transferred into the mammary gland, demonstrating that, in situations of supplementation with 800 IU of vitamin E and its effect in mature milk, the better the vitamin E status (considering the milk and dietary intake), the more effective uptake into the mammary gland will be, regardless of receptor saturation. Further investigation into how this transport occurs is required, by means of in vitro and in vivo studies and using labeled isotopes of alpha-tocopherol, for example, to investigate its biotransformation. Notably, in populations with dietary inadequacy and low contents of vitamin E in milk, supplementation with higher doses of the vitamin, such as 800 IU alpha-tocopherol, might be required to obtain a more effective intervention. The analysis of a single dose during the day allowed us to investigate possible factors that could interfere with the response to supplementation; however, it is necessary to analyze how long the effect of this supplementation could be sustained, its contribution to maternal and infant nutritional status, and the use of smaller daily doses.
Therefore, supplementation associated with an adequate intake of vitamin E is an effective strategy to increase vitamin E levels in breast milk and prevent cases of vitamin E deficiency in infants, especially premature infants [2,4,11].
5. Conclusions:
Vitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E.
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Background:
Vitamin E supplementation might represent an efficient strategy to increase the vitamin E content in milk. The present study aimed to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol content of milk and the factors associated with the increase in vitamin E.
Methods:
Randomized clinical trial with 79 lactating women from Brazil, who were assigned to the control group, or to the supplemented group (800 IU of RRR-alpha-tocopherol). Milk and serum were collected between 30 and 90 days after delivery (collection 1), and on the next day (collection 2). Alpha-tocopherol was analyzed using high-performance liquid chromatography.
Results:
In the supplemented group, the alpha-tocopherol content in serum and milk increased after supplementation (p < 0.001). In the multivariate analysis, only alpha-tocopherol in milk (collection 1) was associated with the level of this vitamin in milk after supplementation (β = 0.927, p < 0.001), and binary logistic regression showed that the dietary intake was the only determinant for the greater effect of supplementation in milk.
Conclusions:
The pre-existing vitamin level in milk and diet are determinants for the efficacy of supplementation in milk, suggesting that in populations with vitamin E deficiency, high-dose supplementation can be used to restore its level in milk.
|
1. Introduction:
Breast milk contains all the essential nutrients and factors for the growth and development of the infant’s gastrointestinal, cerebral and immune system [1,2]. Thus, exclusive breastfeeding is recommended during the first six months of life [3]. Among the vitamins present in milk, vitamin E, is an antioxidant responsible for protecting the lipoproteins and polyunsaturated fatty acids present in the cellular membranes against peroxidation [4]. Vitamin E deficiency in children and newborns, including preterm infants (birth <37 gestational weeks), can lead to intracranial hemorrhage, chronic pulmonary diseases, hemolytic anemia, retinopathy and childhood cognitive deficits [4]. The prevalence of vitamin E deficiency (SVD) in newborns can be up to 77% [5,6,7] and in Brazil, a study found low vitamin levels (<500 μg/dL) in 90% of newborns [8]. The transfer of vitamin E to breast milk depends on circulating lipoproteins, and this mechanism can be influenced by maternal factors, both intrinsic and extrinsic [2,9,10]. In colostrum milk, the actions of pregnancy hormones, such as estrogen, contributes to the increase in circulating lipoproteins, ensuring a greater transfer of vitamin E into milk [2,11]. However, in mature milk the vitamin E content decreases because of changes in fat globules, and other characteristics, such as maternal age, gestational age of delivery, and the fatty acid profile might that influence the vitamin E content in milk [8,9,12,13].
Studies analyzing this micronutrient in mature milk observed that even in lactating women with vitamin E deficiency, its concentration in milk was maintained, which suggested a possible mobilization of alpha-tocopherol from the adipose tissue, which is considered the largest extrahepatic vitamin E reserve [2,8,14,15].
One strategy to increase the concentrations of vitamin E in milk is maternal supplementation [9,10]. Garcia et al. [16] found that at 24 h after supplementation, alpha-tocopherol levels in the colostrum increased. Other studies [17,18] found that vitamin E supplementation in its naturally occurring form (RRR-alpha-tocopherol) is more efficient to increase its content in milk compared with supplementation with the synthetic form or with a blend of natural and synthetic forms. In the natural form, the lateral chain has the RRR conformation, whereas the synthetic form can present isomers with 2R- (RRR-, RSR-, RSS- and RRS-) and 2S- (SRR-, SRS-, SSR-, SSS-) conformations. This structural difference results in increased bioavailability of the RRR form because of its higher affinity for the liver alpha-tocopherol transfer protein (alpha-TPP) [18,19].
Single-dose supplementation with 400 IU RRR-alpha-tocopherol in the immediate postpartum period caused an increase in the vitamin in the transitional milk (between 7 and 15 days after delivery), but not in the mature milk [20,21]. The authors suggested that a higher dose of vitamin E could influence the duration of the response. This identified the need to investigate the effect of higher doses, because the studies only used 400 IU of alpha-tocopherol, and suggested that this supplementation should be provided in the mature milk phase, which comprises a period of greater stability in milk nutritional composition.
Interestingly, different responses to supplementation have been noted, where the same treatment caused a greater increase of the vitamin in the milk in some studies [20,21,22] and a smaller effect in others [17], however, these studies lacked an analysis of the factors that influenced this response. These observations should be considered, because maternal milk with a low alpha-tocopherol content has been found, which could expose infants to vitamin E deficiency (VED) [20,21,23,24,25]. By contrast, studies of vitamin E supplementation in a single dose and in greater quantity could reveal the previously unknown mechanism of vitamin transfer to the mammary gland.
Thus, given that maternal supplementation with vitamin E is an effective measure to increase this vitamin content in milk [21,22], the mother-child binomial should be protected from the adverse effects of VED, and that there are differences in the response to this supplementation, but no understanding of which characteristics contribute to this response. The objective of the present study was to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol level in mature milk and the factors associated with the increase, with the aim of improving our understanding of the mechanism the transfer of vitamin E in the lactation period.
5. Conclusions:
Vitamin E supplementation increased vitamin levels in milk and in maternal serum, and a positive relationship was found between alpha-tocopherol levels in milk, serum and dietary intake of vitamin E. Factors associated with the increase in alpha-tocopherol contents in milk after maternal supplementation with 800 IU of vitamin E were the basal levels of alpha-tocopherol in milk and the dietary intake of vitamin E.
|
Background:
Vitamin E supplementation might represent an efficient strategy to increase the vitamin E content in milk. The present study aimed to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol content of milk and the factors associated with the increase in vitamin E.
Methods:
Randomized clinical trial with 79 lactating women from Brazil, who were assigned to the control group, or to the supplemented group (800 IU of RRR-alpha-tocopherol). Milk and serum were collected between 30 and 90 days after delivery (collection 1), and on the next day (collection 2). Alpha-tocopherol was analyzed using high-performance liquid chromatography.
Results:
In the supplemented group, the alpha-tocopherol content in serum and milk increased after supplementation (p < 0.001). In the multivariate analysis, only alpha-tocopherol in milk (collection 1) was associated with the level of this vitamin in milk after supplementation (β = 0.927, p < 0.001), and binary logistic regression showed that the dietary intake was the only determinant for the greater effect of supplementation in milk.
Conclusions:
The pre-existing vitamin level in milk and diet are determinants for the efficacy of supplementation in milk, suggesting that in populations with vitamin E deficiency, high-dose supplementation can be used to restore its level in milk.
| 9,199 | 266 | 13 |
[
"vitamin",
"milk",
"alpha",
"tocopherol",
"alpha tocopherol",
"supplementation",
"collection",
"group",
"serum",
"intake"
] |
[
"test",
"test"
] | null |
[CONTENT] clinical trial | lactation | infants | breastfeeding | lactating women [SUMMARY]
| null |
[CONTENT] clinical trial | lactation | infants | breastfeeding | lactating women [SUMMARY]
|
[CONTENT] clinical trial | lactation | infants | breastfeeding | lactating women [SUMMARY]
|
[CONTENT] clinical trial | lactation | infants | breastfeeding | lactating women [SUMMARY]
|
[CONTENT] clinical trial | lactation | infants | breastfeeding | lactating women [SUMMARY]
|
[CONTENT] Adult | Breast Feeding | Dietary Supplements | Female | Humans | Logistic Models | Maternal Nutritional Physiological Phenomena | Milk, Human | Vitamin E | Young Adult | alpha-Tocopherol [SUMMARY]
| null |
[CONTENT] Adult | Breast Feeding | Dietary Supplements | Female | Humans | Logistic Models | Maternal Nutritional Physiological Phenomena | Milk, Human | Vitamin E | Young Adult | alpha-Tocopherol [SUMMARY]
|
[CONTENT] Adult | Breast Feeding | Dietary Supplements | Female | Humans | Logistic Models | Maternal Nutritional Physiological Phenomena | Milk, Human | Vitamin E | Young Adult | alpha-Tocopherol [SUMMARY]
|
[CONTENT] Adult | Breast Feeding | Dietary Supplements | Female | Humans | Logistic Models | Maternal Nutritional Physiological Phenomena | Milk, Human | Vitamin E | Young Adult | alpha-Tocopherol [SUMMARY]
|
[CONTENT] Adult | Breast Feeding | Dietary Supplements | Female | Humans | Logistic Models | Maternal Nutritional Physiological Phenomena | Milk, Human | Vitamin E | Young Adult | alpha-Tocopherol [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] vitamin | milk | alpha | tocopherol | alpha tocopherol | supplementation | collection | group | serum | intake [SUMMARY]
| null |
[CONTENT] vitamin | milk | alpha | tocopherol | alpha tocopherol | supplementation | collection | group | serum | intake [SUMMARY]
|
[CONTENT] vitamin | milk | alpha | tocopherol | alpha tocopherol | supplementation | collection | group | serum | intake [SUMMARY]
|
[CONTENT] vitamin | milk | alpha | tocopherol | alpha tocopherol | supplementation | collection | group | serum | intake [SUMMARY]
|
[CONTENT] vitamin | milk | alpha | tocopherol | alpha tocopherol | supplementation | collection | group | serum | intake [SUMMARY]
|
[CONTENT] vitamin | milk | studies | supplementation | alpha | form | alpha tocopherol | tocopherol | vitamin deficiency | deficiency [SUMMARY]
| null |
[CONTENT] μmol | supplementation | tocopherol | alpha tocopherol | alpha | group | milk | collection | control | table [SUMMARY]
|
[CONTENT] vitamin | milk maternal | milk | levels milk | levels | tocopherol | alpha | alpha tocopherol | contents milk maternal | increased vitamin levels [SUMMARY]
|
[CONTENT] vitamin | milk | alpha | alpha tocopherol | tocopherol | supplementation | collection | group | intake | serum [SUMMARY]
|
[CONTENT] vitamin | milk | alpha | alpha tocopherol | tocopherol | supplementation | collection | group | intake | serum [SUMMARY]
|
[CONTENT] Vitamin ||| [SUMMARY]
| null |
[CONTENT] ||| 1 | 0.927 [SUMMARY]
|
[CONTENT] [SUMMARY]
|
[CONTENT] Vitamin ||| 79 | Brazil ||| between 30 and 90 days | 1 | the next day | 2 ||| Alpha ||| ||| ||| 1 | 0.927 ||| [SUMMARY]
|
[CONTENT] Vitamin ||| 79 | Brazil ||| between 30 and 90 days | 1 | the next day | 2 ||| Alpha ||| ||| ||| 1 | 0.927 ||| [SUMMARY]
|
|||||
Mobile Phone Apps to Promote Weight Loss and Increase Physical Activity: A Systematic Review and Meta-Analysis.
|
26554314
|
To our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity.
|
BACKGROUND
|
We conducted a systematic review and meta-analysis of relevant studies identified by a search of PubMed, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), and Scopus from their inception through to August 2015. Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted data. We included all controlled studies that assessed a mobile phone app intervention with weight-related health measures (ie, body weight, body mass index, or waist circumference) or physical activity outcomes. Net change estimates comparing the intervention group with the control group were pooled across studies using random-effects models.
|
METHODS
|
We included 12 articles in this systematic review and meta-analysis. Compared with the control group, use of a mobile phone app was associated with significant changes in body weight (kg) and body mass index (kg/m(2)) of -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) and -0.43 kg/m(2) (95% CI -0.74 to -0.13; I2 = 50%), respectively. Moreover, a nonsignificant difference in physical activity was observed between the two groups (standardized mean difference 0.40, 95% CI -0.07 to 0.87; I2 = 93%). These findings were remarkably robust in the sensitivity analysis. No publication bias was shown.
|
RESULTS
|
Evidence from this study shows that mobile phone app-based interventions may be useful tools for weight loss.
|
CONCLUSIONS
|
[
"Cell Phone",
"Humans",
"Mobile Applications",
"Motor Activity",
"Obesity",
"Weight Loss"
] |
4704965
|
Introduction
|
Overweight and obesity are a global public health issue and an important feature in discussions about strategies for primary and secondary health care. Developing since the 1960s and now gathering pace rapidly, the issue is contributing, together with population aging, to major increases in the prevalence of high blood pressure and cholesterol levels, type 2 diabetes, and cancers [1]. Mortality rates are increasing with increasing degrees of overweight, as measured by body mass index (BMI) [2].
In 2008, 35% of adults older than 20 years were overweight (BMI ≥ 25 kg/m2) and the worldwide prevalence of obesity (BMI ≥30 kg/m2) had nearly doubled since 1980, from 5% of men and 8% of women to 10% and 14%, respectively [2]. An estimated 205 million men and 297 million women were obese—a total of more than half a billion adults worldwide [3]. For these reasons, identifying effective interventions is an important component in public health efforts to curb obesity, but the most effective strategies for weight loss remain unclear.
With the extensive market penetration of mobile phones, the International Telecommunications Union (ITU) reports that as of 2015, there are more than 7 billion mobile cellular subscriptions worldwide, corresponding to a 97% penetration rate—defined by ITU as mobile cellular telephone subscribers per 100 inhabitants [4]. Advanced-feature mobile phones (those with computer operating systems) have broadened the functions of mobile phones considerably. Mobile phone apps meet a variety of user needs, and are designed and adapted for each type of mobile device; therefore, they are applicable in nearly all social and economic sectors and environments. At present, these apps, apart from their recreational function, are becoming instruments of patient education and support and are also helpful to health care professionals [5]. Nonetheless, the market for health care apps is very fragmented because many of them are very specific or directed at minority diseases or specialties. The world market for medical apps for mobile phones and tablets multiplied seven times over in 2011 alone, reaching a total of US $718 million according to a market analysis by the American firm research2guidance [6]. A recent analysis of app store catalogs identified more than 97,000 mHealth apps, most of them dealing with general health and physical fitness; in general, they facilitate the monitoring of various parameters by individual users and provide general information and support related to those topics [5]. Previous research has suggested that mobile apps may be beneficial in asthma control [7] and diabetes management [8,9].
To our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity. The objective of this study was to perform a systematic review and meta-analysis of published studies to evaluate the efficacy of interventions that included mobile phone apps compared with other interventions to reduce weight and increase physical activity in populations of children and adults.
|
Methods
|
Search Strategy We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.
We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.
Study Selection Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.
Flowchart for the selection of the articles in this meta-analysis.
Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.
Flowchart for the selection of the articles in this meta-analysis.
Data Extraction and Quality Assessment Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.
Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.
Statistical Methods For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:
SD2
diff= SD2
pre+ SD2
post- 2×ρ×SDpre×SDpost
Where SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.
For body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).
For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:
SD2
diff= SD2
pre+ SD2
post- 2×ρ×SDpre×SDpost
Where SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.
For body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).
|
Results
|
Study Selection The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).
Characteristics of included clinical trials.
aSS: sample size.
bCCS: case-control study.
cN/A: not applicable.
dBMI: body mass index.
eRCT: randomized controlled trial.
fMCCT: matched case-control trial.
gMPA: moderate physical activity.
hVPA: vigorous physical activity.
Characteristics of intervention types and description of apps.
aBMI: body mass index.
bMPA: moderate physical activity.
cVPA: vigorous physical activity.
dSMS: short message service.
The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).
Characteristics of included clinical trials.
aSS: sample size.
bCCS: case-control study.
cN/A: not applicable.
dBMI: body mass index.
eRCT: randomized controlled trial.
fMCCT: matched case-control trial.
gMPA: moderate physical activity.
hVPA: vigorous physical activity.
Characteristics of intervention types and description of apps.
aBMI: body mass index.
bMPA: moderate physical activity.
cVPA: vigorous physical activity.
dSMS: short message service.
Meta-Analysis of Mobile App Intervention and Body Weight Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.
Meta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.
Meta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Meta-Analysis of Mobile App Intervention and Body Mass Index Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.
Meta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.
Meta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Meta-Analysis of Mobile App Intervention and Physical Activity Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.
Meta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.
Meta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Risk of Bias in Included Studies Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.
Summary of review authors’ assessments of risk of bias for each Cochrane item and each included study.
Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.
Summary of review authors’ assessments of risk of bias for each Cochrane item and each included study.
| null | null |
[
"Search Strategy",
"Study Selection",
"Data Extraction and Quality Assessment",
"Statistical Methods",
"Study Selection",
"Meta-Analysis of Mobile App Intervention and Body Weight",
"Meta-Analysis of Mobile App Intervention and Body Mass Index",
"Meta-Analysis of Mobile App Intervention and Physical Activity",
"Risk of Bias in Included Studies"
] |
[
"We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.",
"Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.",
"Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.",
"For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).",
"The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.",
"Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.",
"Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.",
"Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.",
"Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Introduction",
"Methods",
"Search Strategy",
"Study Selection",
"Data Extraction and Quality Assessment",
"Statistical Methods",
"Results",
"Study Selection",
"Meta-Analysis of Mobile App Intervention and Body Weight",
"Meta-Analysis of Mobile App Intervention and Body Mass Index",
"Meta-Analysis of Mobile App Intervention and Physical Activity",
"Risk of Bias in Included Studies",
"Discussion"
] |
[
"Overweight and obesity are a global public health issue and an important feature in discussions about strategies for primary and secondary health care. Developing since the 1960s and now gathering pace rapidly, the issue is contributing, together with population aging, to major increases in the prevalence of high blood pressure and cholesterol levels, type 2 diabetes, and cancers [1]. Mortality rates are increasing with increasing degrees of overweight, as measured by body mass index (BMI) [2].\nIn 2008, 35% of adults older than 20 years were overweight (BMI ≥ 25 kg/m2) and the worldwide prevalence of obesity (BMI ≥30 kg/m2) had nearly doubled since 1980, from 5% of men and 8% of women to 10% and 14%, respectively [2]. An estimated 205 million men and 297 million women were obese—a total of more than half a billion adults worldwide [3]. For these reasons, identifying effective interventions is an important component in public health efforts to curb obesity, but the most effective strategies for weight loss remain unclear.\nWith the extensive market penetration of mobile phones, the International Telecommunications Union (ITU) reports that as of 2015, there are more than 7 billion mobile cellular subscriptions worldwide, corresponding to a 97% penetration rate—defined by ITU as mobile cellular telephone subscribers per 100 inhabitants [4]. Advanced-feature mobile phones (those with computer operating systems) have broadened the functions of mobile phones considerably. Mobile phone apps meet a variety of user needs, and are designed and adapted for each type of mobile device; therefore, they are applicable in nearly all social and economic sectors and environments. At present, these apps, apart from their recreational function, are becoming instruments of patient education and support and are also helpful to health care professionals [5]. Nonetheless, the market for health care apps is very fragmented because many of them are very specific or directed at minority diseases or specialties. The world market for medical apps for mobile phones and tablets multiplied seven times over in 2011 alone, reaching a total of US $718 million according to a market analysis by the American firm research2guidance [6]. A recent analysis of app store catalogs identified more than 97,000 mHealth apps, most of them dealing with general health and physical fitness; in general, they facilitate the monitoring of various parameters by individual users and provide general information and support related to those topics [5]. Previous research has suggested that mobile apps may be beneficial in asthma control [7] and diabetes management [8,9].\nTo our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity. The objective of this study was to perform a systematic review and meta-analysis of published studies to evaluate the efficacy of interventions that included mobile phone apps compared with other interventions to reduce weight and increase physical activity in populations of children and adults.",
" Search Strategy We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\nWe conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.\n Study Selection Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\nTwo members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.\n Data Extraction and Quality Assessment Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\nTwo investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.\n Statistical Methods For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).\nFor each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).",
"We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.",
"Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.\nFlowchart for the selection of the articles in this meta-analysis.",
"Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.",
"For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:\nSD2\ndiff= SD2\npre+ SD2\npost- 2×ρ×SDpre×SDpost\n\nWhere SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.\nFor body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).",
" Study Selection The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\nThe search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.\n Meta-Analysis of Mobile App Intervention and Body Weight Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Body Mass Index Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Meta-Analysis of Mobile App Intervention and Physical Activity Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\nData from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.\n Risk of Bias in Included Studies Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.\nRandomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.",
"The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).\nCharacteristics of included clinical trials.\n\naSS: sample size.\n\nbCCS: case-control study.\n\ncN/A: not applicable.\n\ndBMI: body mass index.\n\neRCT: randomized controlled trial.\n\nfMCCT: matched case-control trial.\n\ngMPA: moderate physical activity.\n\nhVPA: vigorous physical activity.\nCharacteristics of intervention types and description of apps.\n\naBMI: body mass index.\n\nbMPA: moderate physical activity.\n\ncVPA: vigorous physical activity.\n\ndSMS: short message service.",
"Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.\nMeta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.",
"Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.\nMeta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.",
"Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).\nThe funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.\nMeta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.",
"Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.\nSummary of review authors’ assessments of risk of bias for each Cochrane item and each included study.",
"The current meta-analysis suggested that mobile phone app interventions compared with various control interventions significantly reduced body weight by 1.04 kg, reduced BMI by 0.43 kg/m2, and nonsignificantly increased physical activity by an SMD of 0.40. Our findings were robust across sensitivity analyses. Although the mean reductions in body weight and BMI were modest, it would not be expected for a single change in weight-loss interventions, such as mobile phone apps, to cause clinically meaningful weight loss compared with other control interventions [25]. Many of the control group treatments were other interventions. This could dilute the analysis, as it is possible that in some of the studies the treatment group showed a significant change, while the control group also showed a similar significant result. In our sensitivity analyses, the results were not modified when we excluded one study that did not describe if the control group had received any intervention [13].\nSome of the most recognizable research in mobile interventions has focused on text-messaging interventions, or SMS. A previous meta-analysis [26] found that mobile phone interventions were associated with significant changes in body weight and BMI compared with the control group (-1.44 kg and -0.24 units, respectively). This meta-analysis included only mobile interventions based on contacts by SMS and multimedia message services (MMS). A previous systematic review found strong evidence from the included RCT that weight loss occurs in the short term because of mobile technology interventions [27]. Another systematic review that included seven articles demonstrated a beneficial impact of text messaging or a mobile app for reducing physical inactivity and/or overweight/obesity [28]. Finally, a recent systematic review found that most apps that are focused on weight loss have inconsistent outcomes [29].\nTo our knowledge, this study is the first meta-analysis to summarize the evidence to date regarding effects of mobile phone app interventions compared with various control interventions. We excluded from our meta-analysis interventions based only on text messaging and focused solely on mobile phone apps because text-message interventions do not utilize the full potential of mobile phone technologies. Well-designed apps expand the potential for technology-based health interventions to impact populations in ways that previously were not possible and cannot be achieved without the capabilities of mobile phone software. Therefore, the need to regulate this growing market is becoming a concern, with increased advertising claims about effectiveness and researchers emphasizing the need for studies that will contribute scientific evidence about the true impact of these types of apps. The portability of mobile phones enables users to have access 24 hours a day, making possible the long-term management and reinforcement of health behaviors through a variety of communications and apps. Fitness and weight-loss mobile phone apps allow for the tracking of diet, weight, and physical activity; making grocery and restaurant decisions; cooking healthy meals [28]; or gamification of the intervention. Moreover, participants do not need to carry an extra piece of purchased technology, such as a pedometer, to track physical activity.\nSeveral limitations have been noted. Only a small number of available studies assess the effectiveness of mobile phone apps in weight-loss programs, and they included small sample sizes and short follow-up periods. The use of apps for improving physical activity and reducing anthropometric measures is relatively new. More randomized controlled trials with larger sample sizes and longer follow-up periods are needed to determine the effectiveness of mobile apps in improving health outcomes.\nA recent study aimed to evaluate diet/nutrition and anthropometric apps based on incorporation of features consistent with theories of behavior change; all apps were found to be very low in theoretical content or use of theory to guide behavior change [28]. The studies included in this meta-analysis also varied in the content and theoretical basis of the intervention. Further investigation into the effective features of the mobile phone apps and the interventions’ consistencies with theories of behavior change was not possible; this should be considered an area for future research.\nThe risk of bias was high in most of the studies and future research should improve on several issues, such as the use of blinding or improving attrition rate. All studies, except for one [19], failed to conceal the blinding of participants and personnel, and only one study [24] blinded the research staff collecting the data on outcomes as to the allocation of participants. Given the nature of the intervention, blinding of participants and personnel is difficult. However, it is important to recognize the possible influence of patient and personnel expectations. Therefore, adoption of blinding techniques, such as the use of sham procedures, blinding participants to the study hypothesis, or using a blinded centralized assessment of primary outcomes, will improve the quality of the evidence [30].\nA large attrition rate was noted in some of the studies [17,20] included in this meta-analysis. High attrition rates are common in weight-loss interventions, and the reasons for this are likely complex and varied [31]. Attrition has an obvious impact on the validity of results obtained and can introduce bias; for example, those more motivated to reduce their weight or increase physical activity may remain in the trial. Moreover, several studies found that most participants rarely used the app after the first month of the study [20,23]. As with other weight-loss interventions, the most effective app may be one that can engage people for the longest period. It is known that adherence to self-monitoring of food intake is associated with twice as much weight loss as infrequent monitoring [32]. Without the participant’s active engagement, the app is not likely to be used and as a result will not be effective [28]. The most highly ranked engagement strategies identified are (in order of preference) ease of use, design aesthetic, feedback, function, ability to change design to suit own preference, tailored information, and unique mobile phone features [33]. Weight-loss apps may need to be substantially more engaging or less time-consuming to produce weight reduction in the average individual. It would be useful to design strategies to increase “app appeal” before implementing this type of intervention. Gamification of the app, financial incentives, or delivering the app in a setting of group competition could be important adjuncts to increase motivation to use the app and lose weight [20,34].\nTo our knowledge, this is the first meta-analysis to summarize the effectiveness of mobile apps designed to improve physical activity and reduce anthropometric measures. Our meta-analysis highlighted the need to perform larger, high-quality, randomized controlled clinical trials with longer follow-up. The number of available mobile phone apps is growing steadily, and mobile phones are constantly undergoing updates so the features have changed over time. Incorporating features consistent with theories of behavior change into health-related apps would be useful to improve weight-loss outcomes [35]. We searched several databases in order to avoid publication bias, which is a concern in meta-analyses that only include published studies. Using funnel plots in our meta-analysis made it possible to exclude publication bias with some confidence.\nAs the world’s understanding of health and how to empower individuals to take better care of their health changes, health professionals must treat this change as progress. However, we must ensure that the patient is using a mobile app with appropriate quality guarantees. This meta-analysis aimed to provide a rigorous, systematic, and quantitative review of the studies that have analyzed the effectiveness of mobile apps and attempted to measure their influence on lifestyle changes. Bombarded by information overload in all arenas, health professionals and managers have a need for the insights provided by a tool like the meta-analysis; this could help them make decisions and decide what direction we should be moving in our efforts to promote weight loss, increase physical activity, and confront the public health crisis presented by overweight and obesity.\nIn summary, the results from this meta-analysis demonstrated that interventions based on mobile phone apps are associated with more weight loss than other types of interventions. Furthermore, a nonsignificant increase in physical activity was detected. Evidence form this meta-analysis shows that mobile phone app-based intervention may be useful tools for weight loss."
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Introduction:
Overweight and obesity are a global public health issue and an important feature in discussions about strategies for primary and secondary health care. Developing since the 1960s and now gathering pace rapidly, the issue is contributing, together with population aging, to major increases in the prevalence of high blood pressure and cholesterol levels, type 2 diabetes, and cancers [1]. Mortality rates are increasing with increasing degrees of overweight, as measured by body mass index (BMI) [2].
In 2008, 35% of adults older than 20 years were overweight (BMI ≥ 25 kg/m2) and the worldwide prevalence of obesity (BMI ≥30 kg/m2) had nearly doubled since 1980, from 5% of men and 8% of women to 10% and 14%, respectively [2]. An estimated 205 million men and 297 million women were obese—a total of more than half a billion adults worldwide [3]. For these reasons, identifying effective interventions is an important component in public health efforts to curb obesity, but the most effective strategies for weight loss remain unclear.
With the extensive market penetration of mobile phones, the International Telecommunications Union (ITU) reports that as of 2015, there are more than 7 billion mobile cellular subscriptions worldwide, corresponding to a 97% penetration rate—defined by ITU as mobile cellular telephone subscribers per 100 inhabitants [4]. Advanced-feature mobile phones (those with computer operating systems) have broadened the functions of mobile phones considerably. Mobile phone apps meet a variety of user needs, and are designed and adapted for each type of mobile device; therefore, they are applicable in nearly all social and economic sectors and environments. At present, these apps, apart from their recreational function, are becoming instruments of patient education and support and are also helpful to health care professionals [5]. Nonetheless, the market for health care apps is very fragmented because many of them are very specific or directed at minority diseases or specialties. The world market for medical apps for mobile phones and tablets multiplied seven times over in 2011 alone, reaching a total of US $718 million according to a market analysis by the American firm research2guidance [6]. A recent analysis of app store catalogs identified more than 97,000 mHealth apps, most of them dealing with general health and physical fitness; in general, they facilitate the monitoring of various parameters by individual users and provide general information and support related to those topics [5]. Previous research has suggested that mobile apps may be beneficial in asthma control [7] and diabetes management [8,9].
To our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity. The objective of this study was to perform a systematic review and meta-analysis of published studies to evaluate the efficacy of interventions that included mobile phone apps compared with other interventions to reduce weight and increase physical activity in populations of children and adults.
Methods:
Search Strategy We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.
We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.
Study Selection Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.
Flowchart for the selection of the articles in this meta-analysis.
Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.
Flowchart for the selection of the articles in this meta-analysis.
Data Extraction and Quality Assessment Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.
Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.
Statistical Methods For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:
SD2
diff= SD2
pre+ SD2
post- 2×ρ×SDpre×SDpost
Where SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.
For body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).
For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:
SD2
diff= SD2
pre+ SD2
post- 2×ρ×SDpre×SDpost
Where SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.
For body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).
Search Strategy:
We conducted a systematic literature search of three databases from their inception through August 30, 2015, to identify studies examining the effectiveness of a mobile app intervention compared with a control intervention in achieving anthropometric or physical activity changes: Medline (via PubMed; National Library of Medicine, Bethesda, MD; started in 1966), Scopus (Elsevier; started in 1995), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL; started in 1960). Details on the search strategy are presented in Multimedia Appendix 1. Briefly, our literature search strategy combined synonyms for mobile app (the intervention of interest) with synonyms for the three outcomes: weight, body mass index, and exercise. The search period was all-inclusive up to August 2015. There were no language restrictions. In addition, we manually reviewed reference lists from relevant original research and review articles.
Study Selection:
Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted the data. We included all studies that assessed a mobile app intervention, compared to a control group, with weight-related health measures (ie, body weight, BMI, or waist circumference) or physical activity outcomes. We included studies performed in populations of children and of adults. Exclusion criteria were as follows: (1) no original research (ie, reviews, editorials, or nonresearch letters), (2) case reports and case series, (3) data on body weight, BMI, waist circumference, or physical activity not reported, (4) no control group, (5) participants with any disease except a diagnosis of obesity, (6) mobile telephone intervention based on text messaging, such as short message service (SMS), and (7) intervention used or included personal digital assistants (PDAs). Ethics approval was not required because only published data were analyzed in this review. The study selection process is summarized in Figure 1.
Flowchart for the selection of the articles in this meta-analysis.
Data Extraction and Quality Assessment:
Two investigators (EG-F, GF-M) independently abstracted articles that met the selection criteria and resolved discrepancies by consensus. A form developed in Microsoft Word was used to extract data from eligible research papers, including author, country of study, age of participants, length of follow-up, sample size, and study outcomes. Study outcomes recorded were mean and standard deviation (SD) body weight, BMI, waist circumference, and/or physical activity. These values were captured as mean changes from baseline to the end of the intervention, with variations reported as SD, standard error (SE), or 95% CI. When there were several publications from the same cohort, the study with the longest follow-up was selected; when the follow-up was equivalent, we selected the study with the largest number of cases, the publication that used internal comparisons, or the most recent study. An intention-to-treat analysis was used wherever possible. The risk of bias was assessed following Cochrane recommendations, considering random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting [10]. Each criterion was categorized as clearly yes, not sure, or clearly no. Criteria for which there were differences between the two evaluators were discussed until a consensus decision was reached.
Statistical Methods:
For each study, the net effect size was calculated as the change in body weight-related and physical activity measures resulting from treatment from baseline to the end of the intervention in the intervention group, minus the change in body weight-related and physical activity measures in the control group during the same time period. The SEs and CIs were converted to SDs for analysis. For studies without SD data, we calculated the variance from CIs or test statistics. If the SD for change between baseline and the end of the intervention was not reported, it was calculated using the following equation [11]:
SD2
diff= SD2
pre+ SD2
post- 2×ρ×SDpre×SDpost
Where SDprecorresponds to the SD at baseline, SDpostcorresponds to the SD at the end of intervention, and ρ is the correlation coefficient for correlations between measurements taken at baseline and at the end of the intervention.
For body weight and BMI, weighted mean differences (WMDs) were estimated using random-effects models. For physical activity outcomes, standardized mean differences (SMDs) were estimated using random-effects models. Heterogeneity was quantified with the I2 statistic, which describes the proportion of total variation in study estimates as a result of heterogeneity [12]. To further assess the robustness of our findings, we performed several sensitivity analyses by excluding nonrandomized studies, or studies that did not report the intervention in the control group. We also assessed the relative influence of each study on pooled estimates by omitting one study at a time. Finally, we assessed the publication bias by using Egger's test and funnel plots. Statistical analyses were performed with Review Manager software, version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration).
Results:
Study Selection The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).
Characteristics of included clinical trials.
aSS: sample size.
bCCS: case-control study.
cN/A: not applicable.
dBMI: body mass index.
eRCT: randomized controlled trial.
fMCCT: matched case-control trial.
gMPA: moderate physical activity.
hVPA: vigorous physical activity.
Characteristics of intervention types and description of apps.
aBMI: body mass index.
bMPA: moderate physical activity.
cVPA: vigorous physical activity.
dSMS: short message service.
The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).
Characteristics of included clinical trials.
aSS: sample size.
bCCS: case-control study.
cN/A: not applicable.
dBMI: body mass index.
eRCT: randomized controlled trial.
fMCCT: matched case-control trial.
gMPA: moderate physical activity.
hVPA: vigorous physical activity.
Characteristics of intervention types and description of apps.
aBMI: body mass index.
bMPA: moderate physical activity.
cVPA: vigorous physical activity.
dSMS: short message service.
Meta-Analysis of Mobile App Intervention and Body Weight Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.
Meta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.
Meta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Meta-Analysis of Mobile App Intervention and Body Mass Index Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.
Meta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.
Meta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Meta-Analysis of Mobile App Intervention and Physical Activity Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.
Meta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.
Meta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Risk of Bias in Included Studies Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.
Summary of review authors’ assessments of risk of bias for each Cochrane item and each included study.
Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.
Summary of review authors’ assessments of risk of bias for each Cochrane item and each included study.
Study Selection:
The search strategy retrieved 1124 articles from different sources and 12 articles were included in this meta-analysis [13-24] (see Figure 1 and Table 1). One study contributed two articles [14,15]. We used BMI data from the 2011 Turner-McGrievy and Tate study [14], but because that report did not include physical activity measurements, we took the physical activity data from a 2013 publication by Turner-McGrievy et al [15]. Studies were published between 2010 and 2015, and sample sizes ranged from 35 [18] to 361 [22]. There were two nonrandomized controlled trials [13,16], but the rest of the studies were randomized controlled trials. The interventions in many control groups were ones such as traditional interventions or intensive counseling. Only one study did not specify the type of intervention in the control group [13] (see Table 2).
Characteristics of included clinical trials.
aSS: sample size.
bCCS: case-control study.
cN/A: not applicable.
dBMI: body mass index.
eRCT: randomized controlled trial.
fMCCT: matched case-control trial.
gMPA: moderate physical activity.
hVPA: vigorous physical activity.
Characteristics of intervention types and description of apps.
aBMI: body mass index.
bMPA: moderate physical activity.
cVPA: vigorous physical activity.
dSMS: short message service.
Meta-Analysis of Mobile App Intervention and Body Weight:
Data from 913 participants were analyzed in nine clinical trials [13,14,17-21,23,24]. Compared with the control group, mobile phone app interventions resulted in significant decreases in body weight, with the pooled estimates of the net change in body weight being -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) (see Figure 2). In the sensitivity analysis, we excluded the Lee study [13] because it was not a randomized study and did not include any intervention in the control group. The exclusion of this study did not modify the results (WMD -1.04 kg, 95% CI -1.80 to -0.27 kg; I2 = 48%).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.63 to -1.20 kg.
Meta-analysis of the net change in body weight (kg) associated with mobile phone app intervention, expressed as the change during the mobile phone app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Meta-Analysis of Mobile App Intervention and Body Mass Index:
Data from 1047 participants were analyzed in eight clinical trials [14,17,18,21-24]. Pooled results indicated a significant net difference in BMI between mobile phone app and control intervention groups (WMD -0.43 kg/m2, 95% CI -0.74 to -0.13; I2 = 50%) (see Figure 3). The exclusion of the Lee study [13] did not modify the results (WMD -0.42 kg/m2, 95% CI -0.76 to -0.07; I2 = 54%).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps for weight loss (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not substantially modify estimates; the pooled WMDs ranged from -0.36 to -0.59 kg/m2.
Meta-analysis of the net change in BMI (kg/m2) associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control diet. The area of each square is proportional to the inverse of the variance of the weighted mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Meta-Analysis of Mobile App Intervention and Physical Activity:
Data from 1243 participants were analyzed in seven clinical trials [14,16,18,20-22,24]. Pooled results indicated a nonsignificant difference in physical activity between mobile app and control intervention groups (SMD 0.40, 95% CI -0.07 to 0.87; I2 = 93%) (see Figure 4). The sensitivity analysis indicated that no single study was the main origin of heterogeneity between studies. Next, we excluded any two studies in turn and pooled the data of the remaining studies. The heterogeneity was decreased (I2 = 33%) after two studies—Kirwan et al [16] and Smith et al [22]—were excluded (SMD 0.27, 95% CI 0.08-0.47).
The funnel plot showed reasonable symmetry, which suggested no evidence of publication bias in the clinical trials of mobile apps designed to increase physical activity (see Multimedia Appendix 2). In the sensitivity analysis, the exclusion of individual studies did not modify the estimates; the pooled SMDs ranged from 0.17 to 0.51.
Meta-analysis of the net change in physical activity associated with mobile phone app intervention, expressed as the change during the mobile app intervention minus the change during the control intervention. The area of each square is proportional to the inverse of the variance of the standardized mean difference. Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from inverse variance (IV) weighted random-effects models.
Risk of Bias in Included Studies:
Randomization was considered adequate in most of the studies (see Figure 5). Only one study's participants were blinded as to their allocations [19], and in another study [24] the research staff collecting data on outcomes were blinded to the allocation of participants. For most of the studies we located the original study protocols [15,17,19-24]. Since we found no discrepancies between the outcomes that authors originally intended to measure and those reported in this study, we judged the risk of reporting bias to be low for this domain.
Summary of review authors’ assessments of risk of bias for each Cochrane item and each included study.
Discussion:
The current meta-analysis suggested that mobile phone app interventions compared with various control interventions significantly reduced body weight by 1.04 kg, reduced BMI by 0.43 kg/m2, and nonsignificantly increased physical activity by an SMD of 0.40. Our findings were robust across sensitivity analyses. Although the mean reductions in body weight and BMI were modest, it would not be expected for a single change in weight-loss interventions, such as mobile phone apps, to cause clinically meaningful weight loss compared with other control interventions [25]. Many of the control group treatments were other interventions. This could dilute the analysis, as it is possible that in some of the studies the treatment group showed a significant change, while the control group also showed a similar significant result. In our sensitivity analyses, the results were not modified when we excluded one study that did not describe if the control group had received any intervention [13].
Some of the most recognizable research in mobile interventions has focused on text-messaging interventions, or SMS. A previous meta-analysis [26] found that mobile phone interventions were associated with significant changes in body weight and BMI compared with the control group (-1.44 kg and -0.24 units, respectively). This meta-analysis included only mobile interventions based on contacts by SMS and multimedia message services (MMS). A previous systematic review found strong evidence from the included RCT that weight loss occurs in the short term because of mobile technology interventions [27]. Another systematic review that included seven articles demonstrated a beneficial impact of text messaging or a mobile app for reducing physical inactivity and/or overweight/obesity [28]. Finally, a recent systematic review found that most apps that are focused on weight loss have inconsistent outcomes [29].
To our knowledge, this study is the first meta-analysis to summarize the evidence to date regarding effects of mobile phone app interventions compared with various control interventions. We excluded from our meta-analysis interventions based only on text messaging and focused solely on mobile phone apps because text-message interventions do not utilize the full potential of mobile phone technologies. Well-designed apps expand the potential for technology-based health interventions to impact populations in ways that previously were not possible and cannot be achieved without the capabilities of mobile phone software. Therefore, the need to regulate this growing market is becoming a concern, with increased advertising claims about effectiveness and researchers emphasizing the need for studies that will contribute scientific evidence about the true impact of these types of apps. The portability of mobile phones enables users to have access 24 hours a day, making possible the long-term management and reinforcement of health behaviors through a variety of communications and apps. Fitness and weight-loss mobile phone apps allow for the tracking of diet, weight, and physical activity; making grocery and restaurant decisions; cooking healthy meals [28]; or gamification of the intervention. Moreover, participants do not need to carry an extra piece of purchased technology, such as a pedometer, to track physical activity.
Several limitations have been noted. Only a small number of available studies assess the effectiveness of mobile phone apps in weight-loss programs, and they included small sample sizes and short follow-up periods. The use of apps for improving physical activity and reducing anthropometric measures is relatively new. More randomized controlled trials with larger sample sizes and longer follow-up periods are needed to determine the effectiveness of mobile apps in improving health outcomes.
A recent study aimed to evaluate diet/nutrition and anthropometric apps based on incorporation of features consistent with theories of behavior change; all apps were found to be very low in theoretical content or use of theory to guide behavior change [28]. The studies included in this meta-analysis also varied in the content and theoretical basis of the intervention. Further investigation into the effective features of the mobile phone apps and the interventions’ consistencies with theories of behavior change was not possible; this should be considered an area for future research.
The risk of bias was high in most of the studies and future research should improve on several issues, such as the use of blinding or improving attrition rate. All studies, except for one [19], failed to conceal the blinding of participants and personnel, and only one study [24] blinded the research staff collecting the data on outcomes as to the allocation of participants. Given the nature of the intervention, blinding of participants and personnel is difficult. However, it is important to recognize the possible influence of patient and personnel expectations. Therefore, adoption of blinding techniques, such as the use of sham procedures, blinding participants to the study hypothesis, or using a blinded centralized assessment of primary outcomes, will improve the quality of the evidence [30].
A large attrition rate was noted in some of the studies [17,20] included in this meta-analysis. High attrition rates are common in weight-loss interventions, and the reasons for this are likely complex and varied [31]. Attrition has an obvious impact on the validity of results obtained and can introduce bias; for example, those more motivated to reduce their weight or increase physical activity may remain in the trial. Moreover, several studies found that most participants rarely used the app after the first month of the study [20,23]. As with other weight-loss interventions, the most effective app may be one that can engage people for the longest period. It is known that adherence to self-monitoring of food intake is associated with twice as much weight loss as infrequent monitoring [32]. Without the participant’s active engagement, the app is not likely to be used and as a result will not be effective [28]. The most highly ranked engagement strategies identified are (in order of preference) ease of use, design aesthetic, feedback, function, ability to change design to suit own preference, tailored information, and unique mobile phone features [33]. Weight-loss apps may need to be substantially more engaging or less time-consuming to produce weight reduction in the average individual. It would be useful to design strategies to increase “app appeal” before implementing this type of intervention. Gamification of the app, financial incentives, or delivering the app in a setting of group competition could be important adjuncts to increase motivation to use the app and lose weight [20,34].
To our knowledge, this is the first meta-analysis to summarize the effectiveness of mobile apps designed to improve physical activity and reduce anthropometric measures. Our meta-analysis highlighted the need to perform larger, high-quality, randomized controlled clinical trials with longer follow-up. The number of available mobile phone apps is growing steadily, and mobile phones are constantly undergoing updates so the features have changed over time. Incorporating features consistent with theories of behavior change into health-related apps would be useful to improve weight-loss outcomes [35]. We searched several databases in order to avoid publication bias, which is a concern in meta-analyses that only include published studies. Using funnel plots in our meta-analysis made it possible to exclude publication bias with some confidence.
As the world’s understanding of health and how to empower individuals to take better care of their health changes, health professionals must treat this change as progress. However, we must ensure that the patient is using a mobile app with appropriate quality guarantees. This meta-analysis aimed to provide a rigorous, systematic, and quantitative review of the studies that have analyzed the effectiveness of mobile apps and attempted to measure their influence on lifestyle changes. Bombarded by information overload in all arenas, health professionals and managers have a need for the insights provided by a tool like the meta-analysis; this could help them make decisions and decide what direction we should be moving in our efforts to promote weight loss, increase physical activity, and confront the public health crisis presented by overweight and obesity.
In summary, the results from this meta-analysis demonstrated that interventions based on mobile phone apps are associated with more weight loss than other types of interventions. Furthermore, a nonsignificant increase in physical activity was detected. Evidence form this meta-analysis shows that mobile phone app-based intervention may be useful tools for weight loss.
|
Background:
To our knowledge, no meta-analysis to date has assessed the efficacy of mobile phone apps to promote weight loss and increase physical activity.
Methods:
We conducted a systematic review and meta-analysis of relevant studies identified by a search of PubMed, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), and Scopus from their inception through to August 2015. Two members of the study team (EG-F, GF-M) independently screened studies for inclusion criteria and extracted data. We included all controlled studies that assessed a mobile phone app intervention with weight-related health measures (ie, body weight, body mass index, or waist circumference) or physical activity outcomes. Net change estimates comparing the intervention group with the control group were pooled across studies using random-effects models.
Results:
We included 12 articles in this systematic review and meta-analysis. Compared with the control group, use of a mobile phone app was associated with significant changes in body weight (kg) and body mass index (kg/m(2)) of -1.04 kg (95% CI -1.75 to -0.34; I2 = 41%) and -0.43 kg/m(2) (95% CI -0.74 to -0.13; I2 = 50%), respectively. Moreover, a nonsignificant difference in physical activity was observed between the two groups (standardized mean difference 0.40, 95% CI -0.07 to 0.87; I2 = 93%). These findings were remarkably robust in the sensitivity analysis. No publication bias was shown.
Conclusions:
Evidence from this study shows that mobile phone app-based interventions may be useful tools for weight loss.
| null | null | 8,752 | 320 | 13 |
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[CONTENT] mHealth | mobile phone | apps | obesity | physical activity | intervention [SUMMARY]
|
[CONTENT] mHealth | mobile phone | apps | obesity | physical activity | intervention [SUMMARY]
|
[CONTENT] mHealth | mobile phone | apps | obesity | physical activity | intervention [SUMMARY]
| null |
[CONTENT] mHealth | mobile phone | apps | obesity | physical activity | intervention [SUMMARY]
| null |
[CONTENT] Cell Phone | Humans | Mobile Applications | Motor Activity | Obesity | Weight Loss [SUMMARY]
|
[CONTENT] Cell Phone | Humans | Mobile Applications | Motor Activity | Obesity | Weight Loss [SUMMARY]
|
[CONTENT] Cell Phone | Humans | Mobile Applications | Motor Activity | Obesity | Weight Loss [SUMMARY]
| null |
[CONTENT] Cell Phone | Humans | Mobile Applications | Motor Activity | Obesity | Weight Loss [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] study | mobile | intervention | studies | weight | physical | analysis | activity | physical activity | control [SUMMARY]
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[CONTENT] study | mobile | intervention | studies | weight | physical | analysis | activity | physical activity | control [SUMMARY]
|
[CONTENT] study | mobile | intervention | studies | weight | physical | analysis | activity | physical activity | control [SUMMARY]
| null |
[CONTENT] study | mobile | intervention | studies | weight | physical | analysis | activity | physical activity | control [SUMMARY]
| null |
[CONTENT] mobile | apps | health | market | mobile phones | phones | health care | general | worldwide | million [SUMMARY]
|
[CONTENT] intervention | sd | study | baseline | end | end intervention | weight | body weight | search | baseline end intervention [SUMMARY]
|
[CONTENT] mobile | pooled | kg | change | intervention | study | trials | 95 | app | 13 [SUMMARY]
| null |
[CONTENT] mobile | study | intervention | physical | weight | studies | physical activity | activity | change | app [SUMMARY]
| null |
[CONTENT] [SUMMARY]
|
[CONTENT] PubMed | Allied Health Literature (CINAHL | August 2015 ||| Two ||| ||| [SUMMARY]
|
[CONTENT] 12 ||| kg/m(2 | -1.04 kg | 95% | CI | I2 | 41% | 95% | CI | I2 | 50% ||| two | 0.40 | 95% | CI | 0.87 | I2 | 93% ||| ||| [SUMMARY]
| null |
[CONTENT] ||| PubMed | Allied Health Literature (CINAHL | August 2015 ||| Two ||| ||| ||| ||| 12 ||| kg/m(2 | -1.04 kg | 95% | CI | I2 | 41% | 95% | CI | I2 | 50% ||| two | 0.40 | 95% | CI | 0.87 | I2 | 93% ||| ||| ||| [SUMMARY]
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Association of simultaneously measured four-limb blood pressures with cardiovascular function: a cross-sectional study.
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28155695
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Simultaneous measurement of four-limb blood pressures can improve the accuracy of cardiovascular disease diagnosis. This study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the non-invasive diagnostic method of cardiovascular disease in primary care.
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BACKGROUND
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229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled. Four-limb blood pressure measurements were simultaneously performed using a blood pressure and pulse monitor device in the supine position. Cardiac functional parameters were also measured by using a cardiac hemodynamic detector in the same position. Data were statistically analyzed with SPSS15.0.
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METHODS
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The mean age of the 229 subjects was 60.56 ± 8.68 years. Cardiovascular functional parameters decreased with age and body mass index (BMI), only the total peripheral resistance (TPR) was in contrast. Age, BMI, left ankle diastolic pressure (LADP), high arm mean arterial pressure (HARMAP), left arm diastolic pressure (LARDP) and right ankle diastolic pressure (RADP) were significantly correlated with cardiovascular functional parameters. Cardiovascular functional parameters have significant differences with inter-arm difference in systolic blood pressure (SBP) between ≥10 and <10 mmHg, inter-ankle difference in SBP between ≥15 and ≥20 mmHg, inter-ankle difference in SBP between ≥15 and <10 mmHg and right ankle brachial index (RABI) between ≤0.9 and ≥1.0. After excluding 99 hypertensive patients, a part of cardiovascular functional parameters has still significant differences with inter-arm difference in SBP between ≥10 and ≥15 mmHg and RABI between ≤0.9 and ≥1.0.
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RESULTS
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Age, BMI, LADP, HARMAP, LARDP and RADP were the determinants of cardiovascular functional parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in SBP ≥10 mmHg, inter-ankle difference in SBP ≥15 mmHg and RABI ≤0.9, while these differences still existed after excluding 99 hypertensive patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the non-invasive diagnostic method of cardiovascular disease in primary care.
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CONCLUSION
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[
"Adult",
"Aged",
"Ankle Brachial Index",
"Arterial Pressure",
"Blood Pressure",
"Blood Pressure Determination",
"Cardiovascular Diseases",
"Cardiovascular System",
"Computer Simulation",
"Cross-Sectional Studies",
"Female",
"Hemodynamics",
"Humans",
"Hypertension",
"Linear Models",
"Male",
"Middle Aged",
"Models, Statistical",
"Primary Health Care",
"Risk Assessment"
] |
5259996
|
Background
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Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].
A blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.
Accordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.
| null | null |
Results
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Baseline characteristics of subjects The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2
24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525
Baseline characteristics of the study participants
The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2
24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525
Baseline characteristics of the study participants
Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.
Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.
Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)
Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters
Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)
Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters
Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects
Fig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences
Fig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences
The distribution of four-limb blood pressure difference in the subjects
The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences
The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences
In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects
Fig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences
Fig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences
The distribution of four-limb blood pressure difference in the subjects
The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences
The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences
Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.
Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.
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Conclusion
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LADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care.
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[
"Background",
"Subjects",
"Four-limb blood pressure measurements",
"Cardiovascular function measurements",
"Statistical analysis",
"Baseline characteristics of subjects",
"Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters",
"Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters",
"Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters",
"Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup"
] |
[
"Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.",
"This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.",
"Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.",
"Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.",
"Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.",
"The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants",
"Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.",
"Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters",
"In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences",
"Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0."
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[
"Background",
"Methods",
"Subjects",
"Four-limb blood pressure measurements",
"Cardiovascular function measurements",
"Statistical analysis",
"Results",
"Baseline characteristics of subjects",
"Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters",
"Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters",
"Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters",
"Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup",
"Discussion",
"Conclusion"
] |
[
"Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].\nA blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.\nAccordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.",
" Subjects This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\nThis study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.\n Four-limb blood pressure measurements Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\nFour-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.\n Cardiovascular function measurements Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\nCardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.\n Statistical analysis Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.\nData were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.",
"This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.",
"Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.\nBased on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.",
"Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\n\nSpecific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device\nCardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).\nIn addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.",
"Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.",
" Baseline characteristics of subjects The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\nThe mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants\n Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\nPearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.\n Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\nMultiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters\n Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\nIn additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.\nSince cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.",
"The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2\n24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525\n\nBaseline characteristics of the study participants",
"Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.",
"Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)\n\nMultiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters",
"In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects\nFig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nFig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences\n\nThe distribution of four-limb blood pressure difference in the subjects\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences\nThe mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences",
"Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.",
"In this cross-sectional study, by using a simultaneous measurement technique, the association between four-limb blood pressure and cardiovascular function and related risk factors was evaluated. Cardiovascular functional parameters illustrated the cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity. Thus, this study has more systematicness and pertinence than previous studies. This study suggested that cardiovascular functional parameters (CO, SV, CI, SVI, EF, IC, HI, EDV, LVEDP, SWI, AC and TPR) have all a significant difference with HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP, while there was a negative correlation between them except TPR. Meanwhile, the determinants of cardiac functional parameter were age, BMI, LADP, HARMAP LARDP, HARPPI ADBPD, RARSP, RADP and LARSP, while the TPR was positively correlated with age, BMI, LADP and HARMAP.\nPrevious studies demonstrated that population aging has become the uncontrolled risk factor of cardiovascular disease [16–18]. It is a physiology problem that the effect between blood pressure and cardiovascular function is mutual. The cardiovascular function decreased gradually with age, especially arteries can produce physiological degeneration. The thickening of vessels wall, the decrease or even rupture and calcification of elastic fibers, the increase of peripheral resistance, the decrease of blood vessel compliance, all these factors will result in the systolic blood pressure and diastolic blood pressure increasing with age. In addition, it is a fluid mechanics problem that different arteries away from heart have different resistance and pressure. According to the Poiseuille’s law, brachial artery resistance is relatively small and the blood pressure is low because heart is close to the upper limb, while the phenomenon of the ankle artery is in contrast. Hence blood pressures in four limbs are different. In addition, the distance from the heart affects pressure wave propagation and reflection because of the geometry tapper and elasticity tapper, and augments the pressure of peripheral vascular compared with that of upper limb.\nPrevious studies have reported that inter-arm or inter-ankle blood pressure difference predicted cardiovascular mortality [1, 2, 8–13, 15]. These results suggested that the simultaneous measurement of four-limb blood pressure is needed to improve diagnostic accuracy between cardiovascular disease and blood pressure difference [2–5]. The present study demonstrated the significant correlation between cardiovascular functional parameters and four-limb blood pressure, while cardiovascular functional parameters had no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI. However, the mean plots of cardiovascular functional parameters present obvious change trend in four-limb systolic blood pressure difference and diastolic blood pressure difference ≥10 or ≥15 mmHg. These results were different form previous studies. Verberk et al. [19] reported that the prevalence of inter-arm difference in systolic blood pressure of 10 mmHg or more was roughly doubled when diagnosis measurements method used a sequential approach, or used manual approach rather than automated measurements approach. Thus, simultaneous measurement of four-limb blood pressure and calculation of four-limb blood pressure difference may be helpful and necessary in evaluating the heart function and predicting patients with cardiovascular disease. Further study, the subgroup analysis showed that the predictive value of four limbs blood pressure for cardiovascular function. Subgroup analysis found that a part of cardiovascular functional parameter has still significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and with RABI between ≤0.9 and ≥1.0. However, the exact reason is unclear now, hypertension is the influence factors of cardiovascular function parameters, and four-limb blood pressure differences are also applicable to diagnose hypertensive patients. As mentioned above, four limbs blood pressures were not only influence factors of cardiovascular function parameters, but a predictor for cardiovascular disease, evaluating cardiovascular function parameters was important in clinical practice and epidemiological studies. In addition, the better way for cardiovascular function parameters was simultaneous blood pressures for four limbs.\nA clinical device, the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China), has been developed to automatically and simultaneously measure blood pressure in four limbs, and the measurement can be easily obtained. The similar device was reported by previous studies, for example, VP-1000 ABI—form device (Colin Co. Ltd., Komaki, Japan) and VP-1000 device (Omron, Kyoto, Japan) were used to automatically and simultaneously measure four limbs’ blood pressures. The validation in measurement technology of device (Boundless Horizon Company, Shandong, China) might be prone for measurement errors, especially in multinomial detection of cardiac function parameters rather than single detection. Although it could not give very precise measurements for the clinical value of cardiac function parameters, it can provide objective evaluation of health in the early stage of cardiovascular diseases. Thus, these devices can be applied for the epidemiological evaluation between four limbs’ blood pressures and cardiac function parameters.\nIn this study, by using a simultaneous and noninvasive measurement technique, we measured four limbs blood pressures. But this technique was not a popular one to measure blood pressure in daily clinical practice. Hence, although four limbs blood pressure with simultaneous measurement could improve the predictive value for cardiovascular disease, our results might be changed if daily clinical measurement was used. In addition, the subjects were mainly from retired people, whose health care consciousness is better than the serving officer. The factors of smoking, drinking, salting and movement have no significant difference in this study subjects. Hence, the clinical utility of this study may be limited in community people.",
"LADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care."
] |
[
null,
"materials|methods",
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
"discussion",
"conclusion"
] |
[
"Cardiovascular function",
"Four limbs",
"Blood pressure difference",
"Simultaneous measurement"
] |
Background:
Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].
A blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.
Accordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.
Methods:
Subjects This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.
This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.
Four-limb blood pressure measurements Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.
Based on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.
Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.
Based on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.
Cardiovascular function measurements Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device
Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device
Cardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).
In addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.
Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device
Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device
Cardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).
In addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.
Statistical analysis Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.
Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.
Subjects:
This study was approved by the Ethics Committee of Hospital in Beijing University of Technology, and College of Life Science and Bioengineering in Beijing University of Technology. All subjects gave written informed consent. From September 2015 to January 2016, staffs of Beijing University of Technology took part in comprehensive examinations of cardiovascular disease and its risk evaluation. Subjects with limb disability, hemiplegia, congenital heart disease, heart failure, and the history of artery intervention were excluded. Finally, 229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled in this study.
Four-limb blood pressure measurements:
Four-limb blood pressure was measured in an air-conditioned room at a temperature of 22–23 °C by using the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China). Trained technicians placed the blood pressure cuffs on both arms and both ankles and performed the measurements, after each subject had bared four limbs and taken 10-min rest in supine position. The device simultaneously and automatically measured the supine blood pressure of four limbs, and automatically calculated the ankle-brachial index (ABI) [ABI include right ankle-brachial index (RABI) and left ankle-brachial index (LABI)], and then stored the measurement data in a database.
Based on the systolic and diastolic blood pressure, we calculated the inter-arm and inter-ankle blood pressure differences as the absolute value of the difference between the right and left arm blood pressure and between the right and left ankle blood pressure, respectively. Pulse pressure (PP) was the absolute value of difference between systolic and diastolic blood pressure. Pulse pressure index (PPI) was calculated as the ratio of PP divided by systolic blood pressure. Mean arterial pressure (MAP) was two-thirds diastolic pressure plus one-third systolic pressure.
Cardiovascular function measurements:
Cardiac functional parameters were also measured by using a cardiac hemodynamic detector device (Boundless Horizon Company, Shandong, China) in the supine position and in the same air-conditioned room after obtaining four limbs blood pressure. Before cardiovascular function measurement, each subject had bared abdomen and neck, stuck to electrode slices and taken 10-min rest in supine position. Trained technicians placed the red/black electrode holders on abdomen and neck, placed one yellow electrode holder on the fifth rib left anterior axillary line, respectively. The specific measurement positions were shown in Fig. 1. The device automatically measured the cardiac functional parameters, and then stored the measurement data in a database. Cardiovascular functional parameters related mainly to five parameters in this study: cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity.Fig. 1Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device
Specific measurement positions of cardiovascular function parameters. a Measurement positions of electrode holder. b Electrode holder measuring device
Cardiac pump function parameters include cardiac output per minute (CO), stroke volume (SV), cardiac index (CI), stroke volume index (SVI) and ejection fraction (EF). Cardiac systolic function parameters have function index of left ventricular (LFVI), index of contractility (IC) and heather index (HI). Cardiac diastolic function parameters have end diastolic volume (EDV) and left ventricular end diastolic pressure (LVEDP). Cardiac efficiency function parameters have stroke work (SW), cardiac work (CW), stroke work index (SWI) and cardiac work index (CWI). Vascular elasticity function parameters have aortic compliance (AC) and total peripheral resistance (TPR).
In addition, the observer also administered a standardized questionnaire to collect information of subjects on age, sex, height, weight, medical history, lifestyle, use of medications, drinking and smoking history. The body mass index (BMI) was calculated as the ratio of weight in kilograms divided by the square of height in meters.
Statistical analysis:
Data were stored in Excel 2013 and statistically analyzed with SPSS15.0. Data were expressed as percentages and mean ± SD. The differences of inter-arm and inter-ankle were divide into five groups (<5, 5–9, 10–14, 15–19 and ≥20), and ABI were divide into three groups (≤0.9, 0.91–0.99, ≥1.0). The differences between groups were checked by the analysis of variance for continuous variables or by Chi square test for categorical variables. Pearson correlation analysis was used to determine the correlation degree between cardiac functional parameters and four-limb blood pressure. Multiple linear regression analysis was used to determine the relationship between cardiac functional parameters and four-limb blood pressure. A difference was considered significant if the P value was less than 0.05.
Results:
Baseline characteristics of subjects The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2
24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525
Baseline characteristics of the study participants
The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2
24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525
Baseline characteristics of the study participants
Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.
Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.
Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)
Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters
Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)
Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters
Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects
Fig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences
Fig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences
The distribution of four-limb blood pressure difference in the subjects
The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences
The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences
In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects
Fig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences
Fig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences
The distribution of four-limb blood pressure difference in the subjects
The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences
The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences
Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.
Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.
Baseline characteristics of subjects:
The mean age of the 229 subjects was 60.56 ± 8.68 years, 9 subjects were younger than 45 years, 31 subjects between 45 and 54 years, 136 subjects between 55 and 64 years, 39 subjects between 65 and 74 years, and 14 subjects were aged 75 years or older. Table 1 presents the clinical characteristics of the subjects by gender. The cardiovascular functional parameter difference in CI, EF, LFVI, IC, HI, EDV was significance between male and female (P < 0.05). Also, independent-samples T test was performed between hypertension patients and the general population, and it was found that the differences of cardiovascular functional parameters as CI, SV, CO, SVI, EF, LFVI, IC, HI, LVEDP, EDV, AC and TPR were significant (P < 0.05) between them. The mean values of these parameters in hypertension patients were lower than those of the general population except TPR.Table 1Baseline characteristics of the study participantsCharacteristicsMale (n = 62)Female (n = 167)PAge, years63.50 ± 11.1359.47 ± 7.330.002Body mass index, kg/m2
24.54 ± 3.1525.75 ± 3.700.023Simultaneous four-limb BP measurement, mmHg Left armSystolic pressure (LARSP)136.69 ± 19.01135.78 ± 18.220.740Diastolic pressure (LARDP)82.50 ± 10.9681.02 ± 10.280.342 Right armSystolic pressure (RARSP)136.89 ± 18.47135.78 ± 18.580.688Diastolic pressure (RARDP)83.21 ± 11.8781.32 ± 10.060.230 Left ankleSystolic pressure (LASP)151.48 ± 26.19150.38 ± 22.770.754Diastolic pressure (LADP)79.82 ± 10.8777.25 ± 8.300.057 Right ankleSystolic pressure (RASP)150.45 ± 25.93150.47 ± 22.780.997Diastolic pressure (RADP)77.05 ± 11.5075.16 ± 7.580.150BP on the higher arm/ankle side of systolic pressure, mmHg ArmSystolic pressure (HARSP)140.27 ± 18.79139.03 ± 18.440.652Diastolic pressure (HARDP)83.94 ± 11.5681.72 ± 10.380.166Pulse pressure (HARPP)56.34 ± 14.1957.31 ± 10.380.647Pulse pressure index (HARPPI)0.40 ± 0.060.41 ± 0.060.299Mean arterial pressure (HARMAP)102.72 ± 12.73100.03 ± 11.850.295 AnkleSystolic pressure (HASP)154.50 ± 25.99153.79 ± 22.510.839Diastolic pressure (HADP)80.02 ± 11.8177.11 ± 8.500.041Pulse pressure (HAPP)74.48 ± 18.2776.68 ± 17.930.414Pulse pressure index (HAPPI)0.48 ± 0.070.49 ± 0.060.046Inter-arm BP difference, mmHg Systolic pressuremean ± SD6.97 ± 7.666.63 ± 5.980.725 Diastolic pressuremean ± SD4.35 ± 3.303.95 ± 3.840.466 Systolic pressure≥10 mmHg, n (%)9 (14.5)23 (13.7)0.705 Systolic pressure≥15 mmHg, n (%)9 (14.5)17 (10.2)0.528 Diastolic pressure≥10 mmHg, n (%)3 (4.8)7 (4.1)0.362 Diastolic pressure≥15 mmHg, n (%)1 (1.6)4 (2.4)0.702Inter-ankle BP difference, mmHg Systolic pressuremean ± SD7.06 ± 7.876.74 ± 5.200.715 Diastolic pressuremean ± SD4.87 ± 4.074.11 ± 3.480.165 Systolic pressure≥10 mmHg, n (%)5 (8.1)26 (15.6)0.712 Systolic pressure≥15 mmHg, n (%)9 (14.5)16 (9.6)0.001 Diastolic pressure≥10 mmHg, n (%)4 (6.5)18 (10.8)0.054 Diastolic pressure≥15 mmHg, n (%)3 (4.8)0 (0)–Arm-ankle BP difference, mmHg L-ABImean ± SD1.08 ± 0.151.08 ± 0.100.979 R-ABImean ± SD1.08 ± 0.161.08 ± 0.090.734 L-ABI≤0.9, n (%)4 (6.1)6 (3.6)0.0150.91–0.99, n (%)6 (9.7)30 (18.0)0.852≥1.00, n (%)52 (83.9)131 (78.4)0.497 R-ABI≤0.9, n (%)5 (8.1)5 (3.0)0.1760.91–0.99, n (%)7 (11.3)26 (15.6)0.992≥1.00, n (%)50 (80.6)136 (81.4)0.251Cardiac pump function CO5.27 ± 1.165.26 ± 1.190.969 SV78.90 ± 17.9876.81 ± 18.190.439 CI2.98 ± 0.763.23 ± 0.820.034 SVI44.69 ± 11.8447.34 ± 12.630.152 EF0.64 ± 0.070.69 ± 0.070.000Cardiac systolic function LFVI0.17 ± 0.020.16 ± 0.010.002 IC0.05 ± 0.010.06 ± 0.020.000 HI17.02 ± 4.8422.03 ± 6.830.000Cardiac diastolic function EDV121.41 ± 17.57110.23 ± 18.140.000 LVEDP10.30 ± 3.419.46 ± 2.980.067Cardiac efficiency SW0.11 ± 0.020.10 ± 0.020.140 CW7.09 ± 1.556.93 ± 1.670.511 SWI0.06 ± 0.010.06 ± 0.020.303 CWI4.00 ± 0.964.26 ± 1.120.109Vascular elasticity AC1.70 ± 0.601.74 ± 0.830.705 TPR1598.87 ± 453.211558.99 ± 408.930.525
Baseline characteristics of the study participants
Pearson correlation analysis between four-limb blood pressures and cardiovascular functional parameters:
Pearson correlation analysis presents the correlation degree between four-limb blood pressures and cardiovascular functional parameters as shown in Table 1. Cardiac pump function parameters (CO, SV, CI, SVI, and EF) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac systolic function parameters (IC and HI) have significant differences (P < 0.05) and a negative correlation with age, BMI, HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP. Cardiac diastolic function parameters (EDV and LVEDP) have significant differences (P < 0.05) and a negative correlation with BMI, HARSP, HARDP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Cardiac efficiency function parameters (SW, CW, SWI, and CWI) have significant differences (P < 0.05) and a negative correlation with age and BMI, SWI also has significant differences (P < 0.05) and a negative correlation with HARSP, HARDP, HARMAP, HASP, HADP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP. Vascular elasticity function parameter (AC and TPR) have significant differences (P < 0.05) with age, BMI, HARSP, HARDP, HARPP, HARMAP, HASP, HADP, HAPP, RARSP, RARDP, LARSP, LARDP, RASP, RADP, LASP, and LADP, AC has a negative correlation with these parameters, and TPR has a position correlation with these parameters. Cardiovascular functional parameters have no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI.
Multiple linear regression analysis between four-limb blood pressures and cardiovascular functional parameters:
Multiple linear regression stepwise analysis presents the determinants of cardiovascular functional parameter as shown in Table 2. The independent factors negatively correlated with CO were found to be age, BMI and LADP (β = −0.250, −0.332, −0.190; all P < 0.05). The independent factors negatively correlated with SV were found to be age, BMI, LADP and HARMAP (β = −0.250, −0.318, −0.186, −0.157; all P < 0.05). The independent factors negatively correlated with CI were found to be age, BMI and LADP (β = −0.240, −0.482, −0.204; all P < 0.05). The independent factors negatively correlated with SVI were found to be age, BMI and LARDP (β = −0.255, −0.457, −0.185; all P < 0.05). The independent factors negatively correlated with EF were found to be age, BMI, LARDP and LADP (β = −0.297, −0.252, −0.173, −0.216; all P < 0.05). The independent factors negatively and positively correlated with LFVI were found to be HARPP (β = −0.280; P < 0.05) and age (β = 0.186; P < 0.05) respectively. The independent factors negatively and positively correlated with IC were found to be age, BMI (β = −0.313, −0.290; all P < 0.05) and HARPPI (β = 0.148; P < 0.05) respectively. The independent factors negatively and positively correlated with HI were found to be age, BMI, LADP (β = −0.363, −0.230, −0.255; all P < 0.05) and HARPPI (β = 0.161; P < 0.05) respectively. The independent factors negatively correlated with EDV were found to be age, BMI and HARMAP (β = −0.148, −0.308, −0.240; all P < 0.05). The independent factors negatively and positively correlated with LVEDP were found to be BMI, LASP (β = −0.198, −0.224; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SW were found to be age and BMI (β = −0.203, −0.344; all P < 0.05). The independent factors negatively and positively correlated with CW were found to be age, BMI, LADP (β = −0.178, −0.313, −0.194; all P < 0.05) and ADBPD (β = 0.131; P < 0.05) respectively. The independent factors negatively correlated with SWI were found to be age and BMI (β = −0.201, −0.504; all P < 0.05). The independent factors negatively and positively correlated with CWI were found to be age, BMI, LADP (β = −0.211, −0.484, −0.242; all P < 0.05) and RARSP, RADP (β = 0.184, 0.209; P < 0.05) respectively. The independent factors negatively correlated with AC were found to be age, BMI and LARSP (β = −0.167, −0.175, −0.434; all P < 0.05). The independent factors positively correlated with TPR were found to be age, BMI, LADP and HARMAP (β = 0.238, 0.296, 0.191, 0.260; all P < 0.05).Table 2Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parametersDependent variableRVariableβP95% CICO0.500Age−0.2500.000(−0.050 to −0.018)BMI−0.3320.000(−0.148 to −0.070)LADP−0.1900.002(−0.040 to −0.009)SV0.586Age−0.2500.000(−0.749 to −0.292)BMI−0.3180.000(−2.161 to −1.044)HARMAP−0.1570.046(−0.465 to −0.004)LADP−0.1860.016(−0.670 to −0.070)CI0.625Age−0.2400.000(−0.032 to −0.013)BMI−0.4820.000(−0.132 to −0.085)LADP−0.2040.000(−0.028 to −0.009)SVI0.688Age−0.2550.000(−0.504 to −0.288)BMI−0.4570.000(−1.925 to −1.240)LARDP−0.1850.006(−0.377 to −0.063)LADP−0.1690.013(−0.411 to −0.050)EF0.591Age−0.2970.000(−0.003 to −0.002)BMI−0.2520.000(−0.007 to −0.003)LARDP−0.1730.021(−0.002 to 0.000)LADP−0.2160.004(−0.003 to −0.001)LFVI0.294Age0.1860.005(0.000 to 0.001)HARPP−0.2800.000(0.000 to 0.000)IC0.430Age−0.3130.000(−0.001 to 0.000)BMI−0.2900.000(−0.002 to −0.001)HARPPI0.1480.016(0.007 to 0.071)HI0.554Age−0.3630.000(−0.367 to −0.194)BMI−0.2300.000(−0.643 to −0.218)HARPPI0.1610.005(5.143 to 28.907)LADP−0.2550.000(−0.273 to 0.0104)EDV0.475Age−0.1480.014(−0.572 to −0.064)BMI−0.3080.000(−2.215 to −0.979)HARMAP−0.2400.000(−0.557 to −0.181)LVEDP0.327BMI−0.1980.003(−0.283 to −0.061)LASP−0.2240.001(−0.046 to −0.013)ADBPD0.1310.044(0.003 to 0.220)SW0.400Age−0.2030.001(−0.001 to 0.000)BMI−0.3440.000(−0.003 to −0.001)CW0.415Age−0.1780.004(−0.056 to −0.011)BMI−0.3130.000(−0.199 to −0.086)RARDP0.3470.000(0.028 to 0.080)LADP−0.1940.026(−0.065 to −0.004)SWI0.544Age−0.2010.000(−0.001 to 0.000)BMI−0.5040.000(−0.003 to −0.002)CWI0.547Age−0.2110.000(−0.041 to −0.012)BMI−0.4840.000(−0.180 to −0.111)RARSP0.1840.027(0.001 to 0.020)LADP−0.2420.003(−0.048 to −0.010)RADP0.2090.020(0.003 to 0.039)AC0.562Age−0.1670.004(−0.025 to −0.005)BMI−0.1750.002(−0.062 to −0.014)LARSP−0.4340.000(−0.023 to −0.005)TPR0.644Age0.2380.000(6.506 to 16.539)BMI0.2960.000(22.357 to 46.836)HARMAP0.1910.001(1.587 to 11.696)LADP0.2600.000(5.432 to 18.592)
Multiple linear regression stepwise analysis between four-limb blood pressure and cardiovascular functional parameters
Variance analysis between four-limb blood pressure differences and cardiovascular functional parameters:
In additional, variance analysis was performed to check the difference among the cardiovascular functional parameters, four-limb blood pressure differences (<5, 5–9, 10–14, 15–19 and ≥20) and ABI (≤0.9, 0.91–0.99, ≥1.0). The distribution of four-limb blood pressure differences as shown in Fig. 2. There were 11.35, 4.37, 10.92, and 9.61% of subjects with an inter-arm difference in systolic blood pressure of >15 mmHg, inter-arm difference in diastolic blood pressure of >10 mmHg, inter-ankle difference in systolic blood pressure of >15 mmHg and inter-ankle difference in diastolic blood pressure of >10 mmHg, respectively. The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference (<5, 5–9, 10–14, 15–19 and ≥20) as shown in Figs. 3 and 4. Cardiovascular functional parameters (CI, SV, CO, SVI, EDV, TPR, SW, SWI and CWI) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and <10 mmHg. Cardiovascular functional parameters (EF, HI and TPR) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and ≥20 mmHg. Cardiovascular functional parameters (LFVI and AC) have significant differences (P < 0.05) with inter-ankle difference in systolic blood pressure between ≥15 and <10 mmHg. CI, CO, LVEDP, CW, CWI have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.Fig. 2The distribution of four-limb blood pressure difference in the subjects
Fig. 3The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences
Fig. 4The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences
The distribution of four-limb blood pressure difference in the subjects
The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of CI among four-limb blood pressure differences. b Mean distribution of SV among four-limb blood pressure differences. c Mean distribution of CO among four-limb blood pressure differences. d Mean distribution of SVI among four-limb blood pressure differences. e Mean distribution of EF among four-limb blood pressure differences. f Mean distribution of LFVI among four-limb blood pressure differences. g Mean distribution of IC among four-limb blood pressure differences. h Mean distribution of HI among four-limb blood pressure differences. i Mean distribution of LVEDP among four-limb blood pressure differences. j Mean distribution of EDV among four-limb blood pressure differences. k Mean distribution of AC among four-limb blood pressure differences. l Mean distribution of TPR among four-limb blood pressure differences
The mean distribution of cardiovascular functional parameters among four-limb blood pressure difference in this study subjects. a Mean distribution of SW among four-limb blood pressure differences. b Mean distribution of CW among four-limb blood pressure differences. c Mean distribution of SWI among four-limb blood pressure differences. d Mean distribution of CWI among four-limb blood pressure differences
Analysis between four-limb blood pressures and cardiovascular functional parameters in subgroup:
Since cardiovascular function has been associated with hypertension, we also performed a subgroup analysis after excluding 99 hypertension patients. After Pearson correlation analysis in subgroup, we found that a part of blood pressure parameters correlation with cardiovascular functional parameters disappeared, and the correlation coefficient reduced. In addition, after analysis of variance in subgroup, we also found that cardiovascular functional parameters (SV, EDV and SW) have significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and cardiovascular functional parameters (CI, CO, TPR, CWI and HI) have significant differences (P < 0.05) with RABI between ≤0.9 and ≥1.0.
Discussion:
In this cross-sectional study, by using a simultaneous measurement technique, the association between four-limb blood pressure and cardiovascular function and related risk factors was evaluated. Cardiovascular functional parameters illustrated the cardiac pump function, cardiac systolic, cardiac diastolic, cardiac efficiency and vascular elasticity. Thus, this study has more systematicness and pertinence than previous studies. This study suggested that cardiovascular functional parameters (CO, SV, CI, SVI, EF, IC, HI, EDV, LVEDP, SWI, AC and TPR) have all a significant difference with HARDP, HARMAP, HASP, HADP, LARDP, RADP, LASP, and LADP, while there was a negative correlation between them except TPR. Meanwhile, the determinants of cardiac functional parameter were age, BMI, LADP, HARMAP LARDP, HARPPI ADBPD, RARSP, RADP and LARSP, while the TPR was positively correlated with age, BMI, LADP and HARMAP.
Previous studies demonstrated that population aging has become the uncontrolled risk factor of cardiovascular disease [16–18]. It is a physiology problem that the effect between blood pressure and cardiovascular function is mutual. The cardiovascular function decreased gradually with age, especially arteries can produce physiological degeneration. The thickening of vessels wall, the decrease or even rupture and calcification of elastic fibers, the increase of peripheral resistance, the decrease of blood vessel compliance, all these factors will result in the systolic blood pressure and diastolic blood pressure increasing with age. In addition, it is a fluid mechanics problem that different arteries away from heart have different resistance and pressure. According to the Poiseuille’s law, brachial artery resistance is relatively small and the blood pressure is low because heart is close to the upper limb, while the phenomenon of the ankle artery is in contrast. Hence blood pressures in four limbs are different. In addition, the distance from the heart affects pressure wave propagation and reflection because of the geometry tapper and elasticity tapper, and augments the pressure of peripheral vascular compared with that of upper limb.
Previous studies have reported that inter-arm or inter-ankle blood pressure difference predicted cardiovascular mortality [1, 2, 8–13, 15]. These results suggested that the simultaneous measurement of four-limb blood pressure is needed to improve diagnostic accuracy between cardiovascular disease and blood pressure difference [2–5]. The present study demonstrated the significant correlation between cardiovascular functional parameters and four-limb blood pressure, while cardiovascular functional parameters had no significant differences with inter-arm blood pressure difference, inter-ankle blood pressure difference and ABI. However, the mean plots of cardiovascular functional parameters present obvious change trend in four-limb systolic blood pressure difference and diastolic blood pressure difference ≥10 or ≥15 mmHg. These results were different form previous studies. Verberk et al. [19] reported that the prevalence of inter-arm difference in systolic blood pressure of 10 mmHg or more was roughly doubled when diagnosis measurements method used a sequential approach, or used manual approach rather than automated measurements approach. Thus, simultaneous measurement of four-limb blood pressure and calculation of four-limb blood pressure difference may be helpful and necessary in evaluating the heart function and predicting patients with cardiovascular disease. Further study, the subgroup analysis showed that the predictive value of four limbs blood pressure for cardiovascular function. Subgroup analysis found that a part of cardiovascular functional parameter has still significant differences (P < 0.05) with inter-arm difference in systolic blood pressure between ≥10 and ≥15 mmHg, and with RABI between ≤0.9 and ≥1.0. However, the exact reason is unclear now, hypertension is the influence factors of cardiovascular function parameters, and four-limb blood pressure differences are also applicable to diagnose hypertensive patients. As mentioned above, four limbs blood pressures were not only influence factors of cardiovascular function parameters, but a predictor for cardiovascular disease, evaluating cardiovascular function parameters was important in clinical practice and epidemiological studies. In addition, the better way for cardiovascular function parameters was simultaneous blood pressures for four limbs.
A clinical device, the VS-1500 blood pressure and pulse monitor device (Fukuda Company, Beijing, China), has been developed to automatically and simultaneously measure blood pressure in four limbs, and the measurement can be easily obtained. The similar device was reported by previous studies, for example, VP-1000 ABI—form device (Colin Co. Ltd., Komaki, Japan) and VP-1000 device (Omron, Kyoto, Japan) were used to automatically and simultaneously measure four limbs’ blood pressures. The validation in measurement technology of device (Boundless Horizon Company, Shandong, China) might be prone for measurement errors, especially in multinomial detection of cardiac function parameters rather than single detection. Although it could not give very precise measurements for the clinical value of cardiac function parameters, it can provide objective evaluation of health in the early stage of cardiovascular diseases. Thus, these devices can be applied for the epidemiological evaluation between four limbs’ blood pressures and cardiac function parameters.
In this study, by using a simultaneous and noninvasive measurement technique, we measured four limbs blood pressures. But this technique was not a popular one to measure blood pressure in daily clinical practice. Hence, although four limbs blood pressure with simultaneous measurement could improve the predictive value for cardiovascular disease, our results might be changed if daily clinical measurement was used. In addition, the subjects were mainly from retired people, whose health care consciousness is better than the serving officer. The factors of smoking, drinking, salting and movement have no significant difference in this study subjects. Hence, the clinical utility of this study may be limited in community people.
Conclusion:
LADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care.
|
Background:
Simultaneous measurement of four-limb blood pressures can improve the accuracy of cardiovascular disease diagnosis. This study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the non-invasive diagnostic method of cardiovascular disease in primary care.
Methods:
229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled. Four-limb blood pressure measurements were simultaneously performed using a blood pressure and pulse monitor device in the supine position. Cardiac functional parameters were also measured by using a cardiac hemodynamic detector in the same position. Data were statistically analyzed with SPSS15.0.
Results:
The mean age of the 229 subjects was 60.56 ± 8.68 years. Cardiovascular functional parameters decreased with age and body mass index (BMI), only the total peripheral resistance (TPR) was in contrast. Age, BMI, left ankle diastolic pressure (LADP), high arm mean arterial pressure (HARMAP), left arm diastolic pressure (LARDP) and right ankle diastolic pressure (RADP) were significantly correlated with cardiovascular functional parameters. Cardiovascular functional parameters have significant differences with inter-arm difference in systolic blood pressure (SBP) between ≥10 and <10 mmHg, inter-ankle difference in SBP between ≥15 and ≥20 mmHg, inter-ankle difference in SBP between ≥15 and <10 mmHg and right ankle brachial index (RABI) between ≤0.9 and ≥1.0. After excluding 99 hypertensive patients, a part of cardiovascular functional parameters has still significant differences with inter-arm difference in SBP between ≥10 and ≥15 mmHg and RABI between ≤0.9 and ≥1.0.
Conclusions:
Age, BMI, LADP, HARMAP, LARDP and RADP were the determinants of cardiovascular functional parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in SBP ≥10 mmHg, inter-ankle difference in SBP ≥15 mmHg and RABI ≤0.9, while these differences still existed after excluding 99 hypertensive patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the non-invasive diagnostic method of cardiovascular disease in primary care.
|
Background:
Accurate measurement of blood pressure and scientific evaluation are the precondition for the early detection of cardiovascular disease. The studies found that four-limb blood pressure simultaneous measurement can improve the accuracy of blood pressure for cardiovascular disease diagnosis [1–3]. Therefore, it is an important that four-limb blood pressure should be simultaneously measured to identify and manage the cardiovascular disease. However, most evidences on cardiovascular disease from these studies are obtained by either measuring single limb blood pressure or performing sequence measurement instead of simultaneous four limbs measurement [4–6]. Current technology has allowed to measure four-limb blood pressure simultaneously [7], which could generate accurate blood pressure differences between four limbs, provide a comprehensive evaluation of blood pressure and improve the accuracy of blood pressure for cardiovascular disease diagnosis [2, 4].
A blood pressure difference between arms has been associated with subclavian stenosis, peripheral artery disease, cardiovascular mortality and all-cause mortality [1, 8–11], meanwhile recent studies on inter-leg systolic blood pressure difference have added a new evidence to this concept [12–14]. The meta-analysis reported by Cao showed that inter-arm systolic blood pressure difference ≥15 mmHg might help to predict increased cardiovascular mortality (HR 1.94, 95% CI 1.12–3.35, P < 0.05) in the community populations [15]. However, the other meta-analysis reported by Singh showed that there was not statistically direct association of cardiovascular mortality with inter-arm systolic blood pressure difference of 10 mmHg or more (OR 1.82; CI 0.68–4.88; P = 0.23), 15 mmHg or more (OR 1.66; CI 0.68–4.07; P = 0.27), and inter-leg systolic blood pressure difference of 15 mmHg or more (OR 1.97; CI 0.72–5.34; P = 0.19) [2]. Although the importance of blood pressure difference between arms or between legs is sometimes already recognized [1, 8–14], association of four limbs blood pressure differences with cardiovascular mortality and morbidity remains controversial.
Accordingly, this study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the current non-invasive diagnostic method of cardiovascular disease in primary care.
Conclusion:
LADP, HARMAP, LARDP, and RADP were all significantly correlated with cardiovascular functional parameter. Age and body mass index are all the major risk factors for cardiovascular function parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in systolic blood ≥10 mmHg, inter-ankle difference in systolic blood pressure ≥15 mmHg and RABI ≤0.9, while these differences still exist after excluding 99 hypertension patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the current non-invasive method of cardiovascular disease in primary care.
|
Background:
Simultaneous measurement of four-limb blood pressures can improve the accuracy of cardiovascular disease diagnosis. This study aims to investigate the association of simultaneously measured four-limb blood pressures with cardiovascular function as the non-invasive diagnostic method of cardiovascular disease in primary care.
Methods:
229 subjects (62 males, mean age, 63.50 ± 11.13 years; 167 females, mean age, 59.47 ± 7.33 years) were enrolled. Four-limb blood pressure measurements were simultaneously performed using a blood pressure and pulse monitor device in the supine position. Cardiac functional parameters were also measured by using a cardiac hemodynamic detector in the same position. Data were statistically analyzed with SPSS15.0.
Results:
The mean age of the 229 subjects was 60.56 ± 8.68 years. Cardiovascular functional parameters decreased with age and body mass index (BMI), only the total peripheral resistance (TPR) was in contrast. Age, BMI, left ankle diastolic pressure (LADP), high arm mean arterial pressure (HARMAP), left arm diastolic pressure (LARDP) and right ankle diastolic pressure (RADP) were significantly correlated with cardiovascular functional parameters. Cardiovascular functional parameters have significant differences with inter-arm difference in systolic blood pressure (SBP) between ≥10 and <10 mmHg, inter-ankle difference in SBP between ≥15 and ≥20 mmHg, inter-ankle difference in SBP between ≥15 and <10 mmHg and right ankle brachial index (RABI) between ≤0.9 and ≥1.0. After excluding 99 hypertensive patients, a part of cardiovascular functional parameters has still significant differences with inter-arm difference in SBP between ≥10 and ≥15 mmHg and RABI between ≤0.9 and ≥1.0.
Conclusions:
Age, BMI, LADP, HARMAP, LARDP and RADP were the determinants of cardiovascular functional parameters. In addition, a part of cardiovascular functional parameter is associated with inter-arm difference in SBP ≥10 mmHg, inter-ankle difference in SBP ≥15 mmHg and RABI ≤0.9, while these differences still existed after excluding 99 hypertensive patients. Hence, simultaneous measurement of four-limb blood pressures has become feasible and useful approach to the non-invasive diagnostic method of cardiovascular disease in primary care.
| 13,813 | 427 | 14 |
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"limb blood pressure",
"parameters",
"mean",
"cardiovascular"
] |
[
"test",
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] | null |
[CONTENT] Cardiovascular function | Four limbs | Blood pressure difference | Simultaneous measurement [SUMMARY]
| null |
[CONTENT] Cardiovascular function | Four limbs | Blood pressure difference | Simultaneous measurement [SUMMARY]
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[CONTENT] Cardiovascular function | Four limbs | Blood pressure difference | Simultaneous measurement [SUMMARY]
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[CONTENT] Cardiovascular function | Four limbs | Blood pressure difference | Simultaneous measurement [SUMMARY]
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[CONTENT] Cardiovascular function | Four limbs | Blood pressure difference | Simultaneous measurement [SUMMARY]
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[CONTENT] Adult | Aged | Ankle Brachial Index | Arterial Pressure | Blood Pressure | Blood Pressure Determination | Cardiovascular Diseases | Cardiovascular System | Computer Simulation | Cross-Sectional Studies | Female | Hemodynamics | Humans | Hypertension | Linear Models | Male | Middle Aged | Models, Statistical | Primary Health Care | Risk Assessment [SUMMARY]
| null |
[CONTENT] Adult | Aged | Ankle Brachial Index | Arterial Pressure | Blood Pressure | Blood Pressure Determination | Cardiovascular Diseases | Cardiovascular System | Computer Simulation | Cross-Sectional Studies | Female | Hemodynamics | Humans | Hypertension | Linear Models | Male | Middle Aged | Models, Statistical | Primary Health Care | Risk Assessment [SUMMARY]
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[CONTENT] Adult | Aged | Ankle Brachial Index | Arterial Pressure | Blood Pressure | Blood Pressure Determination | Cardiovascular Diseases | Cardiovascular System | Computer Simulation | Cross-Sectional Studies | Female | Hemodynamics | Humans | Hypertension | Linear Models | Male | Middle Aged | Models, Statistical | Primary Health Care | Risk Assessment [SUMMARY]
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[CONTENT] Adult | Aged | Ankle Brachial Index | Arterial Pressure | Blood Pressure | Blood Pressure Determination | Cardiovascular Diseases | Cardiovascular System | Computer Simulation | Cross-Sectional Studies | Female | Hemodynamics | Humans | Hypertension | Linear Models | Male | Middle Aged | Models, Statistical | Primary Health Care | Risk Assessment [SUMMARY]
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[CONTENT] Adult | Aged | Ankle Brachial Index | Arterial Pressure | Blood Pressure | Blood Pressure Determination | Cardiovascular Diseases | Cardiovascular System | Computer Simulation | Cross-Sectional Studies | Female | Hemodynamics | Humans | Hypertension | Linear Models | Male | Middle Aged | Models, Statistical | Primary Health Care | Risk Assessment [SUMMARY]
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[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] pressure | blood | blood pressure | limb | limb blood | differences | limb blood pressure | parameters | mean | cardiovascular [SUMMARY]
| null |
[CONTENT] pressure | blood | blood pressure | limb | limb blood | differences | limb blood pressure | parameters | mean | cardiovascular [SUMMARY]
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[CONTENT] pressure | blood | blood pressure | limb | limb blood | differences | limb blood pressure | parameters | mean | cardiovascular [SUMMARY]
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[CONTENT] pressure | blood | blood pressure | limb | limb blood | differences | limb blood pressure | parameters | mean | cardiovascular [SUMMARY]
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[CONTENT] pressure | blood | blood pressure | limb | limb blood | differences | limb blood pressure | parameters | mean | cardiovascular [SUMMARY]
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[CONTENT] blood | blood pressure | pressure | disease | mortality | cardiovascular | cardiovascular disease | cardiovascular mortality | systolic blood pressure difference | pressure difference [SUMMARY]
| null |
[CONTENT] distribution | mean distribution | 000 | pressure | limb blood pressure differences | pressure differences mean distribution | pressure differences mean | differences mean distribution | blood pressure differences mean | differences mean [SUMMARY]
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[CONTENT] cardiovascular | difference systolic blood | functional parameter | cardiovascular functional parameter | parameter | blood | mmhg | difference systolic | systolic blood | age body mass index [SUMMARY]
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[CONTENT] pressure | blood | blood pressure | parameters | cardiovascular | cardiac | differences | 000 | limb blood pressure | limb blood [SUMMARY]
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[CONTENT] pressure | blood | blood pressure | parameters | cardiovascular | cardiac | differences | 000 | limb blood pressure | limb blood [SUMMARY]
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[CONTENT] four ||| four [SUMMARY]
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[CONTENT] 229 | 60.56 | 8.68 years ||| BMI | TPR ||| BMI | RADP ||| SBP | 10 | SBP | SBP | 10 | RABI | ≥1.0 ||| 99 | SBP | RABI | ≥1.0 [SUMMARY]
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[CONTENT] BMI | HARMAP | LARDP | RADP ||| SBP | SBP | RABI | 99 ||| four [SUMMARY]
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[CONTENT] four ||| four ||| 229 | 62 | 63.50 | 11.13 years | 167 | 59.47 | 7.33 years ||| Four ||| Cardiac ||| ||| ||| 229 | 60.56 | 8.68 years ||| BMI | TPR ||| BMI | RADP ||| SBP | 10 | SBP | SBP | 10 | RABI | ≥1.0 ||| 99 | SBP | RABI | ≥1.0 ||| BMI | HARMAP | LARDP | RADP ||| SBP | SBP | RABI | 99 ||| four [SUMMARY]
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[CONTENT] four ||| four ||| 229 | 62 | 63.50 | 11.13 years | 167 | 59.47 | 7.33 years ||| Four ||| Cardiac ||| ||| ||| 229 | 60.56 | 8.68 years ||| BMI | TPR ||| BMI | RADP ||| SBP | 10 | SBP | SBP | 10 | RABI | ≥1.0 ||| 99 | SBP | RABI | ≥1.0 ||| BMI | HARMAP | LARDP | RADP ||| SBP | SBP | RABI | 99 ||| four [SUMMARY]
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Incidence of symptoms and accidents during baths and showers among the Japanese general public.
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21478641
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Bathing is a deeply ingrained custom among Japanese; however, data on the incidence rate of symptoms and accidents during bathing have not yet been reported for the Japanese general public.
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BACKGROUND
|
We conducted a population-based cross-sectional study of 617 Japanese adults who attended a specialized health checkup. Participants completed a self-administered questionnaire to assess weekly frequencies of bathtub bathing and showering and the frequency of symptoms/accidents (falling, loss of consciousness, and other) during these activities in the past year. We calculated the incidence rates of accidents per 10 000 baths/showers and 95% confidence intervals (CIs) and compared the clinical characteristics of participants who had symptoms/accidents with those who did not.
|
METHODS
|
The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22-0.84) and 0.24 (95% CI: 0.04-1.37). Although these rates are low, there were 740 000 bathtub bathing-related accidents in Japan, due to the fact that bathing is an almost-daily habit. There was no significant difference in clinical characteristics between groups.
|
RESULTS
|
We collected basic information on the incidence of bathing-related accidents in Japan. Falls and loss of consciousness during bathing or showering can potentially lead to a serious accident, so the general public should be educated about the possibility of such accidents during bathing.
|
CONCLUSIONS
|
[
"Accidental Falls",
"Accidents, Home",
"Adult",
"Aged",
"Baths",
"Confidence Intervals",
"Cross-Sectional Studies",
"Female",
"Health Surveys",
"Humans",
"Incidence",
"Japan",
"Male",
"Middle Aged",
"Public Health",
"Self-Assessment",
"Surveys and Questionnaires"
] |
3899424
|
INTRODUCTION
|
Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public.
|
METHODS
|
This cross-sectional study enrolled adults aged 40 to 74 years who underwent a specialized health checkup provided in October 2008 in the district of Kawane in Shimada city, Shizuoka. The investigation utilized self-administered questionnaires, measurement of height and weight, blood pressure examination, and blood testing for triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, L-aspartate aminotransferase, L-alanine aminotransferase, and hemoglobin A1c, based on a program standardized by the Japanese Ministry of Health, Labour and Welfare.9 From a total of 1319 eligible adults, 617 who actually completed the health checkup and gave consent participated in the study. Participants completed a self-administered questionnaire to assess the weekly frequencies of bathtub bathing and showering during summer (July through September) and winter (December through February). In addition, we investigated the frequency of symptoms/accidents (falling, loss of consciousness, pallor, nausea/vomiting, and other) during bathtub bathing and showering in the past year: the list of symptoms/accidents consisted of those frequently identified in previous research.7
First, we calculated the weekly frequencies of bathtub bathing and showering by averaging the frequencies between the 2 seasons, after which we assessed the incidence of each type of accident during bathtub bathing and showering in the past year for all participants and in 2 groups divided by the median age of the participants (ie, 66 years). Next, based on the weekly frequencies of bathtub bathing and showering, we estimated the overall frequencies of bathtub bathing and showering during the past year for all participants. We then divided the total number of accidents by the annual frequencies of bathtub bathing and showering for all participants and calculated the incidence rates and 95% confidence intervals (CIs) of accidents per 10 000 baths/showers. We calculated 95% CIs using a binomial distribution according to Wilson’s method.10 Then, to estimate the annual frequencies of bathtub bathing and showering among Japanese aged 40 to 74 years, we used the 2008 Vital Statistics of Japan to determine how many Japanese were included in this age group.11 By multiplying these estimates by the incidence rates for accidents (which were obtained earlier), we estimated the total annual numbers of accidents that were associated with bathtub bathing and showering in Japanese aged 40 to 74 years.
After dividing the participants into 2 groups according to their accident status, ie, cases with and without bath-related accidents, we used the t-test to compare mean values for age, body mass index, blood pressure, and blood chemistry findings and Fisher’s exact test to compare differences in sex, medication use, and past medical history.
The study protocol was approved by the institutional review board (IRB) of Hamamatsu University School of Medicine (No. 20-55). The nature of the study was explained to the participants using written statements approved by the IRB. Voluntary informed consent was obtained from all participants before beginning the study. Completion of the questionnaire was deemed as indicating consent to participate in the study. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (SPSS Japan Inc., version 17.0, Tokyo, Japan).
|
RESULTS
|
All 617 individuals (266 men, 351 women) who had completed the health checkup and were recruited for the study agreed to participate. The frequencies of bathtub bathing and showering were calculated based on summer and winter frequencies (mean ± SD) of 5.82 ± 2.09 and 1.29 ± 1.81 times per week, respectively (Table 1). The overall annual numbers of bathtub baths and showers were estimated based on the weekly frequencies and were approximately 190 000 and 41 000, respectively. Of the 617 individuals, 588 completed a self-administered questionnaire about accidents. Of these, 5 reported a total of 8 bathtub bathing-related accidents over the past year, including 5 cases of facial pallor, 2 cases of loss of consciousness, and 1 case of falling. In contrast, there was only 1 shower-related accident (loss of consciousness). The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22–0.84) and 0.24 (95% CI: 0.04–1.37), respectively. According to estimates based on the Japanese population of adults aged 40 to 74 years, people in this age group bathe a total of 1.7 × 1010 times per year and have a total of 740 000 (95% CI: 380 000 to 1 500 000) accidents. They shower 3.8 × 109 times per year and have a total of 93 000 (95% CI: 16 000 to 530 000) accidents.
SD: standard deviation; CI: confidence interval.
A comparison of the characteristics of participants who did and did not have bath-related accidents are shown in Table 2. There was no significant difference in the characteristics of the 5 participants who had accidents and those who did not.
BMI: Body mass index; SBP: Systolic blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; AST: L-aspartate aminotransferase; ALT: L-alanine aminotransferase.
| null | null |
[
"INTRODUCTION"
] |
[
"Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public."
] |
[
null
] |
[
"INTRODUCTION",
"METHODS",
"RESULTS",
"DISCUSSION"
] |
[
"Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public.",
"This cross-sectional study enrolled adults aged 40 to 74 years who underwent a specialized health checkup provided in October 2008 in the district of Kawane in Shimada city, Shizuoka. The investigation utilized self-administered questionnaires, measurement of height and weight, blood pressure examination, and blood testing for triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, L-aspartate aminotransferase, L-alanine aminotransferase, and hemoglobin A1c, based on a program standardized by the Japanese Ministry of Health, Labour and Welfare.9 From a total of 1319 eligible adults, 617 who actually completed the health checkup and gave consent participated in the study. Participants completed a self-administered questionnaire to assess the weekly frequencies of bathtub bathing and showering during summer (July through September) and winter (December through February). In addition, we investigated the frequency of symptoms/accidents (falling, loss of consciousness, pallor, nausea/vomiting, and other) during bathtub bathing and showering in the past year: the list of symptoms/accidents consisted of those frequently identified in previous research.7\nFirst, we calculated the weekly frequencies of bathtub bathing and showering by averaging the frequencies between the 2 seasons, after which we assessed the incidence of each type of accident during bathtub bathing and showering in the past year for all participants and in 2 groups divided by the median age of the participants (ie, 66 years). Next, based on the weekly frequencies of bathtub bathing and showering, we estimated the overall frequencies of bathtub bathing and showering during the past year for all participants. We then divided the total number of accidents by the annual frequencies of bathtub bathing and showering for all participants and calculated the incidence rates and 95% confidence intervals (CIs) of accidents per 10 000 baths/showers. We calculated 95% CIs using a binomial distribution according to Wilson’s method.10 Then, to estimate the annual frequencies of bathtub bathing and showering among Japanese aged 40 to 74 years, we used the 2008 Vital Statistics of Japan to determine how many Japanese were included in this age group.11 By multiplying these estimates by the incidence rates for accidents (which were obtained earlier), we estimated the total annual numbers of accidents that were associated with bathtub bathing and showering in Japanese aged 40 to 74 years.\nAfter dividing the participants into 2 groups according to their accident status, ie, cases with and without bath-related accidents, we used the t-test to compare mean values for age, body mass index, blood pressure, and blood chemistry findings and Fisher’s exact test to compare differences in sex, medication use, and past medical history.\nThe study protocol was approved by the institutional review board (IRB) of Hamamatsu University School of Medicine (No. 20-55). The nature of the study was explained to the participants using written statements approved by the IRB. Voluntary informed consent was obtained from all participants before beginning the study. Completion of the questionnaire was deemed as indicating consent to participate in the study. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (SPSS Japan Inc., version 17.0, Tokyo, Japan).",
"All 617 individuals (266 men, 351 women) who had completed the health checkup and were recruited for the study agreed to participate. The frequencies of bathtub bathing and showering were calculated based on summer and winter frequencies (mean ± SD) of 5.82 ± 2.09 and 1.29 ± 1.81 times per week, respectively (Table 1). The overall annual numbers of bathtub baths and showers were estimated based on the weekly frequencies and were approximately 190 000 and 41 000, respectively. Of the 617 individuals, 588 completed a self-administered questionnaire about accidents. Of these, 5 reported a total of 8 bathtub bathing-related accidents over the past year, including 5 cases of facial pallor, 2 cases of loss of consciousness, and 1 case of falling. In contrast, there was only 1 shower-related accident (loss of consciousness). The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22–0.84) and 0.24 (95% CI: 0.04–1.37), respectively. According to estimates based on the Japanese population of adults aged 40 to 74 years, people in this age group bathe a total of 1.7 × 1010 times per year and have a total of 740 000 (95% CI: 380 000 to 1 500 000) accidents. They shower 3.8 × 109 times per year and have a total of 93 000 (95% CI: 16 000 to 530 000) accidents.\nSD: standard deviation; CI: confidence interval.\nA comparison of the characteristics of participants who did and did not have bath-related accidents are shown in Table 2. There was no significant difference in the characteristics of the 5 participants who had accidents and those who did not.\nBMI: Body mass index; SBP: Systolic blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; AST: L-aspartate aminotransferase; ALT: L-alanine aminotransferase.",
"The present study is the first to ascertain the incidence rate of bathing-related accidents among the Japanese general public. The incidence rate in the present study was somewhat higher than that noted in an investigation of public welfare facilities for the aged (0.067 and 0.204 per 10 000 in-facility baths and home-visit baths, respectively).8 The somewhat lower incidence rate of accidents in bathing services provided by welfare facilities may be attributable to the following facts. First, in welfare services, a physical checkup is routinely performed before bathing, which may prevent accidents. Second, bathing in the presence of a caregiver may allow for early detection of potential accidents and serious symptoms. Third, the incidence rate was likely to have been underreported in the previous study,8 as it was based only on the objective findings of caregivers.\nIn the present study, the incidence rate of accidents during showering was lower than that during bathtub bathing. It has been suggested that, as compared with bathtub bathing, the lower water pressure used during showering imposes a lower burden on the body.12 However, as habitual bathing is reported to be associated with good self-rated health and sleep quality,13 it would not be wise to suggest that bathtub bathing is, in general, more hazardous to general health than showering.\nWhile the incidence rate of accidents in the present study is small, the total number of bathtub bathing-related accidents in Japan was as high as 740 000, because bathing is an almost-daily habit. Although no accidents resulted in death in this study, falls and loss of consciousness could potentially lead to a serious accident.14 One participant who reported falling in the bath lost consciousness twice. Therefore, the general public must be educated about accidents such as falls during bathing.\nThis study has several limitations. First, the use of self-report questionnaires may have caused underreporting due to obscured memory. Second, the sample size was relatively small, and the study was conducted within 1 geographic region. It is very difficult to use a small sample in a specific area to estimate the incidence for the general population. To determine the characteristics of the accident group, we analyzed the relationship between disease history and the results of the health checkup using additional data from the health checkup. However, there were no characteristic findings. Third, participants had all sought a health checkup, suggesting possible selection bias. A previous study reported high incidence rates of drowning among the very old.3 It is possible that, in the present study, recall bias explains why incidence rates were lower among the very old than among middle-aged adults. Although there were some limitations in the present report, it is the first to collect information on the incidence of bathing-related accidents among the Japanese general public."
] |
[
null,
"methods",
"results",
"discussion"
] |
[
"baths",
"shower",
"incidence",
"accidents",
"population-based study"
] |
INTRODUCTION:
Bathing is a custom that is deeply ingrained in the life of Japanese people. Unlike their European and American counterparts,1 many Japanese are thought to bathe in a bathtub almost daily; however, there are few reports on the present bathing habits of the Japanese general public. Bathing-related symptoms and accidents are frequently reported, which has led allied medical associations to highlight the dangers associated with bathing and submit an official statement to the Japanese Ministry of Health, Labour and Welfare regarding the need for a systematic investigation of the causes of such accidents.2 Previous reports on bathing-related accidents include a study that used existing data on accidental bathtub drowning deaths and found that approximately 3500 people drown in household bathtubs every year,3 a medicolegal study of deaths during bathing,4 an investigation of accidents occurring at hot spring resorts,5 an investigation of cases involving emergency medical transport,6 and a report on accidents occurring in welfare service settings.7 The only available incidence rate for bathing-related accidents is one based on such welfare services, as reported by us previously.8 Basic data on the incidence rate among the general public have not been reported in Japan. The present study aimed to investigate bathing habits and the incidence rate of bathing-related symptoms/accidents among the Japanese general public.
METHODS:
This cross-sectional study enrolled adults aged 40 to 74 years who underwent a specialized health checkup provided in October 2008 in the district of Kawane in Shimada city, Shizuoka. The investigation utilized self-administered questionnaires, measurement of height and weight, blood pressure examination, and blood testing for triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, L-aspartate aminotransferase, L-alanine aminotransferase, and hemoglobin A1c, based on a program standardized by the Japanese Ministry of Health, Labour and Welfare.9 From a total of 1319 eligible adults, 617 who actually completed the health checkup and gave consent participated in the study. Participants completed a self-administered questionnaire to assess the weekly frequencies of bathtub bathing and showering during summer (July through September) and winter (December through February). In addition, we investigated the frequency of symptoms/accidents (falling, loss of consciousness, pallor, nausea/vomiting, and other) during bathtub bathing and showering in the past year: the list of symptoms/accidents consisted of those frequently identified in previous research.7
First, we calculated the weekly frequencies of bathtub bathing and showering by averaging the frequencies between the 2 seasons, after which we assessed the incidence of each type of accident during bathtub bathing and showering in the past year for all participants and in 2 groups divided by the median age of the participants (ie, 66 years). Next, based on the weekly frequencies of bathtub bathing and showering, we estimated the overall frequencies of bathtub bathing and showering during the past year for all participants. We then divided the total number of accidents by the annual frequencies of bathtub bathing and showering for all participants and calculated the incidence rates and 95% confidence intervals (CIs) of accidents per 10 000 baths/showers. We calculated 95% CIs using a binomial distribution according to Wilson’s method.10 Then, to estimate the annual frequencies of bathtub bathing and showering among Japanese aged 40 to 74 years, we used the 2008 Vital Statistics of Japan to determine how many Japanese were included in this age group.11 By multiplying these estimates by the incidence rates for accidents (which were obtained earlier), we estimated the total annual numbers of accidents that were associated with bathtub bathing and showering in Japanese aged 40 to 74 years.
After dividing the participants into 2 groups according to their accident status, ie, cases with and without bath-related accidents, we used the t-test to compare mean values for age, body mass index, blood pressure, and blood chemistry findings and Fisher’s exact test to compare differences in sex, medication use, and past medical history.
The study protocol was approved by the institutional review board (IRB) of Hamamatsu University School of Medicine (No. 20-55). The nature of the study was explained to the participants using written statements approved by the IRB. Voluntary informed consent was obtained from all participants before beginning the study. Completion of the questionnaire was deemed as indicating consent to participate in the study. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) for Windows (SPSS Japan Inc., version 17.0, Tokyo, Japan).
RESULTS:
All 617 individuals (266 men, 351 women) who had completed the health checkup and were recruited for the study agreed to participate. The frequencies of bathtub bathing and showering were calculated based on summer and winter frequencies (mean ± SD) of 5.82 ± 2.09 and 1.29 ± 1.81 times per week, respectively (Table 1). The overall annual numbers of bathtub baths and showers were estimated based on the weekly frequencies and were approximately 190 000 and 41 000, respectively. Of the 617 individuals, 588 completed a self-administered questionnaire about accidents. Of these, 5 reported a total of 8 bathtub bathing-related accidents over the past year, including 5 cases of facial pallor, 2 cases of loss of consciousness, and 1 case of falling. In contrast, there was only 1 shower-related accident (loss of consciousness). The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22–0.84) and 0.24 (95% CI: 0.04–1.37), respectively. According to estimates based on the Japanese population of adults aged 40 to 74 years, people in this age group bathe a total of 1.7 × 1010 times per year and have a total of 740 000 (95% CI: 380 000 to 1 500 000) accidents. They shower 3.8 × 109 times per year and have a total of 93 000 (95% CI: 16 000 to 530 000) accidents.
SD: standard deviation; CI: confidence interval.
A comparison of the characteristics of participants who did and did not have bath-related accidents are shown in Table 2. There was no significant difference in the characteristics of the 5 participants who had accidents and those who did not.
BMI: Body mass index; SBP: Systolic blood pressure; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; AST: L-aspartate aminotransferase; ALT: L-alanine aminotransferase.
DISCUSSION:
The present study is the first to ascertain the incidence rate of bathing-related accidents among the Japanese general public. The incidence rate in the present study was somewhat higher than that noted in an investigation of public welfare facilities for the aged (0.067 and 0.204 per 10 000 in-facility baths and home-visit baths, respectively).8 The somewhat lower incidence rate of accidents in bathing services provided by welfare facilities may be attributable to the following facts. First, in welfare services, a physical checkup is routinely performed before bathing, which may prevent accidents. Second, bathing in the presence of a caregiver may allow for early detection of potential accidents and serious symptoms. Third, the incidence rate was likely to have been underreported in the previous study,8 as it was based only on the objective findings of caregivers.
In the present study, the incidence rate of accidents during showering was lower than that during bathtub bathing. It has been suggested that, as compared with bathtub bathing, the lower water pressure used during showering imposes a lower burden on the body.12 However, as habitual bathing is reported to be associated with good self-rated health and sleep quality,13 it would not be wise to suggest that bathtub bathing is, in general, more hazardous to general health than showering.
While the incidence rate of accidents in the present study is small, the total number of bathtub bathing-related accidents in Japan was as high as 740 000, because bathing is an almost-daily habit. Although no accidents resulted in death in this study, falls and loss of consciousness could potentially lead to a serious accident.14 One participant who reported falling in the bath lost consciousness twice. Therefore, the general public must be educated about accidents such as falls during bathing.
This study has several limitations. First, the use of self-report questionnaires may have caused underreporting due to obscured memory. Second, the sample size was relatively small, and the study was conducted within 1 geographic region. It is very difficult to use a small sample in a specific area to estimate the incidence for the general population. To determine the characteristics of the accident group, we analyzed the relationship between disease history and the results of the health checkup using additional data from the health checkup. However, there were no characteristic findings. Third, participants had all sought a health checkup, suggesting possible selection bias. A previous study reported high incidence rates of drowning among the very old.3 It is possible that, in the present study, recall bias explains why incidence rates were lower among the very old than among middle-aged adults. Although there were some limitations in the present report, it is the first to collect information on the incidence of bathing-related accidents among the Japanese general public.
|
Background:
Bathing is a deeply ingrained custom among Japanese; however, data on the incidence rate of symptoms and accidents during bathing have not yet been reported for the Japanese general public.
Methods:
We conducted a population-based cross-sectional study of 617 Japanese adults who attended a specialized health checkup. Participants completed a self-administered questionnaire to assess weekly frequencies of bathtub bathing and showering and the frequency of symptoms/accidents (falling, loss of consciousness, and other) during these activities in the past year. We calculated the incidence rates of accidents per 10 000 baths/showers and 95% confidence intervals (CIs) and compared the clinical characteristics of participants who had symptoms/accidents with those who did not.
Results:
The incidence rates of accidents per 10 000 bathtub baths and showers were 0.43 (95% CI: 0.22-0.84) and 0.24 (95% CI: 0.04-1.37). Although these rates are low, there were 740 000 bathtub bathing-related accidents in Japan, due to the fact that bathing is an almost-daily habit. There was no significant difference in clinical characteristics between groups.
Conclusions:
We collected basic information on the incidence of bathing-related accidents in Japan. Falls and loss of consciousness during bathing or showering can potentially lead to a serious accident, so the general public should be educated about the possibility of such accidents during bathing.
| null | null | 1,754 | 274 | 4 |
[
"bathing",
"accidents",
"study",
"bathtub",
"incidence",
"bathtub bathing",
"showering",
"000",
"japanese",
"participants"
] |
[
"test",
"test"
] | null | null |
[CONTENT] baths | shower | incidence | accidents | population-based study [SUMMARY]
|
[CONTENT] baths | shower | incidence | accidents | population-based study [SUMMARY]
|
[CONTENT] baths | shower | incidence | accidents | population-based study [SUMMARY]
| null |
[CONTENT] baths | shower | incidence | accidents | population-based study [SUMMARY]
| null |
[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]
|
[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]
|
[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]
| null |
[CONTENT] Accidental Falls | Accidents, Home | Adult | Aged | Baths | Confidence Intervals | Cross-Sectional Studies | Female | Health Surveys | Humans | Incidence | Japan | Male | Middle Aged | Public Health | Self-Assessment | Surveys and Questionnaires [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]
|
[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]
|
[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]
| null |
[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | showering | 000 | japanese | participants [SUMMARY]
| null |
[CONTENT] bathing | accidents | japanese | bathing related | incidence rate | rate | public | general public | general | related [SUMMARY]
|
[CONTENT] bathing showering | bathtub bathing showering | showering | bathtub bathing | frequencies | participants | frequencies bathtub bathing | frequencies bathtub bathing showering | frequencies bathtub | bathing [SUMMARY]
|
[CONTENT] 000 | ci | 95 ci | accidents | 95 | times | total | respectively | frequencies | bathtub [SUMMARY]
| null |
[CONTENT] bathing | accidents | study | bathtub | incidence | bathtub bathing | 000 | general | rate | incidence rate [SUMMARY]
| null |
[CONTENT] Japanese | Japanese [SUMMARY]
|
[CONTENT] 617 | Japanese ||| weekly | the past year ||| 10 000 | 95% [SUMMARY]
|
[CONTENT] 10 | 95% | 0.22-0.84 | 0.24 | 95% | CI | 0.04 ||| 740 | Japan ||| [SUMMARY]
| null |
[CONTENT] Japanese | Japanese ||| 617 | Japanese ||| weekly | the past year ||| 10 000 | 95% ||| ||| 10 | 95% | 0.22-0.84 | 0.24 | 95% | CI | 0.04 ||| 740 | Japan ||| ||| Japan ||| [SUMMARY]
| null |
||||
Effect of probiotics on length of hospitalization in mild acute pancreatitis: A randomized, double-blind, placebo-controlled trial.
|
33510561
|
Acute pancreatitis is the leading cause of hospitalization for acute gastrointestinal disease worldwide. The effects of probiotics in mild acute pancreatitis have not been studied. We hypothesized that the administration of probiotics may accelerate the recovery of intestinal function and shorten the length of hospital stay (LOS) in patients with mild pancreatitis.
|
BACKGROUND
|
We conducted a double-blind randomized clinical trial to evaluate the effects of probiotics administered to patients with mild acute pancreatitis at a tertiary medical center. The patients were given probiotics capsules (a mixed preparation of Bacillus subtilis and Enterococcus faecium) or placebo. The primary study endpoint was the LOS. The secondary endpoints included time to abdominal pain relief, recurrent abdominal pain, and time to successful oral feeding.
|
METHODS
|
A total of 128 patients were included, with 64 patients in each arm. The severity of illness and the etiological distribution of disease were similar in the two groups. There was a significant reduction in the LOS in the probiotics treatment group vs the placebo group (5.36 ± 0.15 vs 6.02 ± 0.17 d, P < 0.05). The probiotics group was associated with a shorter time to abdominal pain relief and time to successful oral feeding (P < 0.01 for both) than the placebo group. No statistical difference was found in recurrent abdominal pain between the two groups.
|
RESULTS
|
The study results showed that the administration of probiotics capsules is associated with a shorter duration of hospitalization in patients with mild acute pancreatitis.
|
CONCLUSION
|
[
"Acute Disease",
"Double-Blind Method",
"Hospitalization",
"Humans",
"Pancreatitis",
"Probiotics",
"Treatment Outcome"
] |
7807297
|
INTRODUCTION
|
Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.
More and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.
|
MATERIALS AND METHODS
|
Study design This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.
Patients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.
This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.
Patients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.
Inclusion and exclusion criteria The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.
The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.
Data collection We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15].
We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15].
Outcomes We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol.
We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol.
Sample size estimation According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.
According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.
Statistical analysis Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).
Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).
| null | null |
CONCLUSION
|
Probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases.
|
[
"INTRODUCTION",
"Study design",
"Inclusion and exclusion criteria",
"Data collection",
"Outcomes",
"Sample size estimation",
"Statistical analysis",
"RESULTS",
"Participant characteristics ",
"Primary endpoint",
"Secondary end points",
"Safety and harm",
"DISCUSSION",
"CONCLUSION"
] |
[
"Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.",
"This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.",
"The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.",
"We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. ",
"We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. ",
"According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.",
"Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).",
" Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups",
"We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.",
"The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.",
"The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups",
"The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups",
"The present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.",
"In conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"MATERIALS AND METHODS",
"Study design",
"Inclusion and exclusion criteria",
"Data collection",
"Outcomes",
"Sample size estimation",
"Statistical analysis",
"RESULTS",
"Participant characteristics ",
"Primary endpoint",
"Secondary end points",
"Safety and harm",
"DISCUSSION",
"CONCLUSION"
] |
[
"Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.\nMore and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.",
" Study design This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\nThis 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.\n Inclusion and exclusion criteria The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\nThe inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.\n Data collection We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \nWe extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. \n Outcomes We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \nWe used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. \n Sample size estimation According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\nAccording to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.\n Statistical analysis Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).\nContinuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).",
"This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.\nPatients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.",
"The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.",
"We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15]. ",
"We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol. ",
"According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.",
"Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).",
" Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\nWe assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.\n Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\nThe mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.\n Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\nThe study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups\n Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups\nThe adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups",
"We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response. \nCONSORT diagram.\nThus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%). \nBaseline characteristics of participants\nBMI: Body mass index; CRP: C-reactive protein.",
"The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.\nComparison of length of hospital stay between probiotics and placebo groups.",
"The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.\nComparison of secondary endpoints between probiotics and placebo groups",
"The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.\nComparison of side effects between probiotics and placebo groups",
"The present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.\nAt present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.\nIn our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.\nAdverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating. \nThe present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.",
"In conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases."
] |
[
null,
"methods",
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Acute pancreatitis",
"Probiotics",
"Length of hospitalization",
"Randomized study",
"Placebo",
"Mild"
] |
INTRODUCTION:
Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.
More and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.
MATERIALS AND METHODS:
Study design This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.
Patients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.
This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.
Patients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.
Inclusion and exclusion criteria The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.
The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.
Data collection We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15].
We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15].
Outcomes We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol.
We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol.
Sample size estimation According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.
According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.
Statistical analysis Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).
Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).
Study design:
This 1:1 randomized, controlled, double-blind clinical trial was conducted at the emergency ward of a tertiary teaching hospital with 5186 beds in China from July 2017 to July 2019. The study protocol was defined in accordance with the declaration of Helsinki and was approved by the Ethics Committee of our institution. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2000030425) and is reported in accordance with the CONSORT guidelines. All patients provided written informed consent before study participation. All study authors had access to the data and approved the final manuscript.
Patients were randomly allocated to a probiotics capsules or placebo group, and probiotics capsules or placebo were given orally from study inclusion to discharge. Considering the safety and efficacy shown in other clinical research[11], we chose a probiotics commercial preparation, a mixed preparation of Bacillus subtilis and Enterococcus faecium, which has been widely used in China (Meichangan, national medicine permission number S20030087). A computer-generated random-number table was prepared by statisticians to assign patients to receive either the probiotics capsules or placebo. The random-number table was unknown to the investigators, who enrolled the patients. To ensure blinding, placebo capsules identical to the probiotics capsules were manufactured (same appearance, color, and odor). The patients, clinicians, evaluators, and statisticians were blinded to the study treatment.
Inclusion and exclusion criteria:
The inclusion criteria[12] were patients who (1) were 18-75 years old; and (2) met two of the following three diagnostic criteria for acute pancreatitis, including typical upper abdominal pain, serum amylase and/or lipase activity more than three times, and imaging manifestations of pancreatitis. Patients who presented with any indication of severe acute pancreatitis were excluded from our study. The indications of severe acute pancreatitis included: (1) Evidence of organ failure (oxygen saturation less than 90% on room air, mean arterial pressure less than 70 mmHg, the requirement for vasopressors or inotropic support, serum creatinine greater than 2.0 mg/dL, and Glasgow coma score less than 15)[12]; (2) Suspected or confirmed infected pancreatic necrosis; (3) C-reactive protein (CRP) over 150 mg/L[8]; and (4) Acute Physiology and Chronic Health Evaluation score ≥ 8[8]. Patients were included only for the first episode of mild pancreatitis. Patients receiving probiotics therapy during the 3 mo preceding the index admission were excluded. Additionally, pregnant or breastfeeding women were not allowed to participate.
Data collection:
We extracted clinical data from the hospital electronic medical record systems. We collected the clinical data including basic information at admission such as sex, age, CRP, body mass index (BMI), Charlson score, disease etiology (gallstones-related, hypertriglyceridemia, alcohol-related, others, or unknown), relevant diagnostic and therapeutic interventions, and outcomes including LOS, time to successful oral feeding, rate of recurrent abdominal pain, time to abdominal pain relief, 30-d readmissions, and mortality. Successful oral feeding means that patients could take food through the mouth without any abdominal pain or other discomforts. Rate of recurrent abdominal pain means the failure rate of the patient's initial attempt to take food orally characterized as abdominal pain, nausea, vomiting, or other abdominal discomforts after the attempt. Abdominal pain relief means the time when abdominal pain symptoms completely disappeared for the first time. Previously reported adverse effects of probiotics in our study were closely monitored, including exacerbating abdominal discomfort (abdominal pain, nausea, vomiting, flatulence, or diarrhea)[13], anaphylactic reaction[14], probiotic-related infections[13], and lactic acidosis[15].
Outcomes:
We used LOS as our primary endpoint. It is generally believed that patients with mild pancreatitis, characterized by complete abdominal pain relief, no abdominal discomfort (such as nausea and vomiting), and successfully eating solid or semi-liquid foods, could be discharged from the hospital[16]. Thus, we chose the following indexes as our secondary endpoint: Time to first abdominal pain relief, rate of recurrent abdominal pain, and time to successful oral feeding. Patients were followed for 30 d for mortality and 30-d readmissions. All these outcomes were prespecified in the protocol.
Sample size estimation:
According to a previous study[16,17], assuming the median LOS of mild pancreatitis patients was 5 d, we needed 128 patients (64 in each group) to obtain 80% power for detecting a 1-d relative decrease in the LOS with the one-sided alpha risk of 0.05.
Statistical analysis:
Continuous variables are expressed as the mean ± SD if they were normally distributed. Categorical variables are expressed as percentages. The Kolmogorov–Smirnov test was used to assess whether continuous data were normally distributed (P > 0.05). For continuous variables, differences between groups were tested by Student’s t-test for normally distributed data or Mann–Whitney U test for non-normally distributed data and the c2 test for categorical variables. All analyses were done according to the intention-to-treat principle. Results were considered significant at P < 0.05. Statistical analyses were carried out using SPSS version 23.0 (IBM Inc., Chicago, Illinois, United States).
RESULTS:
Participant characteristics We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response.
CONSORT diagram.
Thus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%).
Baseline characteristics of participants
BMI: Body mass index; CRP: C-reactive protein.
We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response.
CONSORT diagram.
Thus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%).
Baseline characteristics of participants
BMI: Body mass index; CRP: C-reactive protein.
Primary endpoint The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.
Comparison of length of hospital stay between probiotics and placebo groups.
The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.
Comparison of length of hospital stay between probiotics and placebo groups.
Secondary end points The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.
Comparison of secondary endpoints between probiotics and placebo groups
The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.
Comparison of secondary endpoints between probiotics and placebo groups
Safety and harm The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.
Comparison of side effects between probiotics and placebo groups
The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.
Comparison of side effects between probiotics and placebo groups
Participant characteristics :
We assessed 234 patients for eligibility, and 106 were excluded. The patients were excluded for having severe acute pancreatitis, suspected or confirmed infected pancreatic necrosis, and Acute Physiology and Chronic Health Evaluation score ≥ 8. Fifty-one patients refused to participate in the study, mainly because of concerns about the safety of the drugs. The remaining 128 patients were randomized, and 64 were allocated to each group. The CONSORT standardized flow diagram shows the enrollment process (Figure 1). We followed the patients for 30 d for mortality or readmissions through telephone calls, of whom eight were lost to follow-up due to change of contact number, or non-response.
CONSORT diagram.
Thus, 128 patients were included in the intention-to-treat analysis. The baseline demographics of the study population are shown in Table 1. The average age of the collective group was 52.73 years, with 64.1% male. The mean CRP level was 54.9 mg/L. There were no significant differences in age, sex, BMI, CRP, Charlson score, or disease etiology distribution between the two groups. The most common etiology overall for acute pancreatitis in this study was gallstones-related (50%).
Baseline characteristics of participants
BMI: Body mass index; CRP: C-reactive protein.
Primary endpoint:
The mean LOS in the probiotics group was 5.36 ± 0.15 d, and in the placebo group was 6.02 ± 0.17 d. Patients in the probiotics group had a significantly shorter LOS than the placebo group by about 0.65 d (P < 0.05). The result are shown in Figure 2.
Comparison of length of hospital stay between probiotics and placebo groups.
Secondary end points:
The study endpoints between the probiotics and placebo groups are listed in Table 2. Recurrent abdominal pain was noted in six patients in the probiotics group and nine patients in the placebo group, with no significant difference between the two groups (P = 0.41). Notably, the time to abdominal pain relief in the probiotics group was significantly shorter than that in the placebo group by about 1 d (2.98 ± 0.74 d vs 4.14 ± 1.32 d, P < 0.01). Additionally, the probiotics group had a significantly shorter time to successful oral feeding than the placebo group (4.56 ± 1.13 d vs 5.37 ± 1.13 d, P < 0.01). No patient mortality was noted in our study. One patient was re-hospitalized within 30 d of discharge because of hyperlipidemic pancreatitis, which was caused by repeated overeating in the probiotics group; and one patient in the placebo group was re-admitted for pancreatitis secondary to repeated heavy alcohol drinking.
Comparison of secondary endpoints between probiotics and placebo groups
Safety and harm:
The adverse effects of probiotics administration in our study were closely monitored. The results are shown in Table 3. The most common adverse effect was exacerbating abdominal discomfort. In the probiotics group, about 18.7% (12/64) of patients experienced transient abdominal discomfort, such as abdominal pain (4.7%), flatulence (7.8%), and nausea (4.7%). The proportion was lower in that of the placebo group [14.1% (9/64)]. There was no significant difference between the two groups in the rate of exacerbating abdominal discomfort (P = 0.47). The symptoms were mild and transient despite the high abdominal discomfort rate. No anaphylactic reaction, probiotic-related infections, or lactic acidosis secondary to probiotics administration was found in our study.
Comparison of side effects between probiotics and placebo groups
DISCUSSION:
The present study investigated the effect of the administration of probiotics on patients with mild pancreatitis. We found that probiotics were associated with decreased LOS. The possible reasons were that probiotics may accelerate successful oral feeding and abdominal pain relief. The safety of probiotics in the treatment of mild pancreatitis in our study was also considered. Transient abdominal discomfort was the most common adverse effect found in our study, but no significant difference was found between the probiotics and placebo groups.
At present, studies on pancreatitis tend to focus on severe cases, including management of early acute inflammatory response, fluid resuscitation, and management of late infectious complications[18]. There are relatively fewer studies on mild acute pancreatitis because of its low mortality rate. However, mild acute pancreatitis accounts for up to 80% of all pancreatitis cases[3]. With the increasing pressure of medical costs due to mild acute pancreatitis, shortening the LOS and reducing the medical cost have become the primary target of acute pancreatitis treatment. At present, probiotics are mainly used to treat diarrhea, constipation, enteritis, abdominal distension, dyspepsia, and loss of appetite caused by intestinal flora imbalance. Some studies[10,19] suggested that probiotics were associated with decreased LOS, but these reports were of low quality and included a small sample of patients with severe acute pancreatitis. Besselink et al[8] conducted a study with the largest sample size of 298 randomized patients with severe acute pancreatitis; they found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality. No difference was found in intensive care stay and hospital stay between their probiotics and non-probiotics groups.
In our randomized placebo-controlled study, probiotics were associated with a decreased LOS in patients with mild acute pancreatitis. In mild acute pancreatitis, rapid relief of abdominal pain and successful oral feeding were the main indications for hospital discharge. Based on previous gastrointestinal disease studies[20,21], we hypothesized that probiotics may improve intestinal microecology and food tolerance, reduce the inflammatory response, and consequently improve the possibility of successful oral feeding. Consistent with previous research results, our study showed accelerated successful oral feeding and abdominal pain relief after the administration of probiotics capsules consisting of Bacillus subtilis and Enterococcus faecium.
Adverse reactions to probiotics were closely observed in this study. The most frequent adverse effect was exacerbating abdominal discomfort (7%–9%), mostly experienced as flatulence. The reasons may be as follows: First, probiotic supplements are packaged as capsules that need to be taken orally with water, which contradicts the usual practice of diet prohibition before abdominal pain relief. As recommended in the 2013 International Association of Pancreatology and the American Pancreatic Association Evidence-Based Guidelines[22], and the 2018 American Gastroenterological Association Institute Guideline[23], oral feeding in mild pancreatitis can be restarted only when abdominal pain is decreasing and inflammatory markers are improving. The early administration of probiotic capsules may aggravate the gastrointestinal condition. Second, the role and target of probiotics on the intestinal flora in patients with disease states are not yet fully understood[24]. Rao et al[25] concluded that probiotics-fermented carbohydrates in the proximal small bowel induce intestinal bacterial overgrowth, resulting in increased gas output, and abdominal bloating.
The present study had several limitations. First, our results may not be generalizable to all patients with acute pancreatitis because we only included patients with mild acute pancreatitis. Although we showed that probiotics may be beneficial to patients with mild pancreatitis, the adverse reactions of probiotics should prompt clinician concern. Second, the results of this study are not generalizable to all probiotics since we only used Bacillus subtilis and Enterococcus faecium probiotic strains. At present, there are many types of probiotics with different clinical therapeutic and adverse effects that may be different or opposite to the findings of our study. Therefore, caution is advised in considering the results of this study.
CONCLUSION:
In conclusion, probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases.
|
Background:
Acute pancreatitis is the leading cause of hospitalization for acute gastrointestinal disease worldwide. The effects of probiotics in mild acute pancreatitis have not been studied. We hypothesized that the administration of probiotics may accelerate the recovery of intestinal function and shorten the length of hospital stay (LOS) in patients with mild pancreatitis.
Methods:
We conducted a double-blind randomized clinical trial to evaluate the effects of probiotics administered to patients with mild acute pancreatitis at a tertiary medical center. The patients were given probiotics capsules (a mixed preparation of Bacillus subtilis and Enterococcus faecium) or placebo. The primary study endpoint was the LOS. The secondary endpoints included time to abdominal pain relief, recurrent abdominal pain, and time to successful oral feeding.
Results:
A total of 128 patients were included, with 64 patients in each arm. The severity of illness and the etiological distribution of disease were similar in the two groups. There was a significant reduction in the LOS in the probiotics treatment group vs the placebo group (5.36 ± 0.15 vs 6.02 ± 0.17 d, P < 0.05). The probiotics group was associated with a shorter time to abdominal pain relief and time to successful oral feeding (P < 0.01 for both) than the placebo group. No statistical difference was found in recurrent abdominal pain between the two groups.
Conclusions:
The study results showed that the administration of probiotics capsules is associated with a shorter duration of hospitalization in patients with mild acute pancreatitis.
|
INTRODUCTION:
Acute pancreatitis is one of the most common causes of acute abdominal pain, resulting in a huge clinical and financial burden worldwide. A nationwide retrospective analysis involving two million acute pancreatitis patients from the United States found that there was a 13.2% increase in admissions in the years 2009-2012 compared to that in 2002–2005[1]. In 2012, the annual cost of acute pancreatitis hospitalizations was about $2.6 billion[2]. Most patients (80%) with acute pancreatitis present with mild disease, which is characterized by a short hospital stay, no or few complications, and rapid resumption of oral feeding[3]. Allowing for the very low mortality rate (about 0.79%) of mild acute pancreatitis[1], shortening the length of hospital stay (LOS), and reducing medical financial expenditure have become the research priority.
More and more studies have shown that intestinal microbial communities are closely related to the occurrence and progression of gastrointestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer[4,5]. Altering the microbiota to decrease the severity of disease or even cure the disease has become a sought-after research direction. Probiotics play an important role in a variety of microecologic interventions. Probiotics were defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”[6]. The mechanisms of probiotics in the treatment of intestinal disease include improving barrier function, modulating cell-mediated and humoral immune functions, interacting with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and supporting microbiota stability, etc.[7]. In a multicenter randomized, placebo-controlled trial, 298 patients predicted with severe acute pancreatitis were randomly assigned to receive a multispecies probiotic or placebo; it was found that probiotic prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality[8]. Subsequently, a meta-analysis involving six trials with 536 patients showed that probiotics supplementation did not significantly affect the pancreatic infection rate, LOS, or mortality[9]. In contrast, another meta-analysis showed reduced LOS in Chinese patients with severe acute pancreatitis[10]. However, the effect of probiotics in mild acute pancreatitis has not been studied. Considering the potential benefits of probiotics in the treatment of gastrointestinal disease, we hypothesized that probiotics may accelerate the recovery of intestinal function and shorten the LOS in patients with mild pancreatitis. Thus, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the effect of the administration of probiotics in patients with mild pancreatitis.
CONCLUSION:
Probiotics supplementation appears to be safe and effective in reducing the length of hospitalization for patients with mild acute pancreatitis in this double-blinded randomized clinical trial. However, the sample size is small and the follow-up time is short in this study. In future studies, a randomized study with a larger sample using different types and doses of probiotics can be conducted to further clarify the role of probiotics in acute pancreatitis and other uninvestigated gastrointestinal diseases.
|
Background:
Acute pancreatitis is the leading cause of hospitalization for acute gastrointestinal disease worldwide. The effects of probiotics in mild acute pancreatitis have not been studied. We hypothesized that the administration of probiotics may accelerate the recovery of intestinal function and shorten the length of hospital stay (LOS) in patients with mild pancreatitis.
Methods:
We conducted a double-blind randomized clinical trial to evaluate the effects of probiotics administered to patients with mild acute pancreatitis at a tertiary medical center. The patients were given probiotics capsules (a mixed preparation of Bacillus subtilis and Enterococcus faecium) or placebo. The primary study endpoint was the LOS. The secondary endpoints included time to abdominal pain relief, recurrent abdominal pain, and time to successful oral feeding.
Results:
A total of 128 patients were included, with 64 patients in each arm. The severity of illness and the etiological distribution of disease were similar in the two groups. There was a significant reduction in the LOS in the probiotics treatment group vs the placebo group (5.36 ± 0.15 vs 6.02 ± 0.17 d, P < 0.05). The probiotics group was associated with a shorter time to abdominal pain relief and time to successful oral feeding (P < 0.01 for both) than the placebo group. No statistical difference was found in recurrent abdominal pain between the two groups.
Conclusions:
The study results showed that the administration of probiotics capsules is associated with a shorter duration of hospitalization in patients with mild acute pancreatitis.
| 6,334 | 283 | 15 |
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[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]
|
[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]
| null |
[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]
|
[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]
|
[CONTENT] Acute pancreatitis | Probiotics | Length of hospitalization | Randomized study | Placebo | Mild [SUMMARY]
|
[CONTENT] Acute Disease | Double-Blind Method | Hospitalization | Humans | Pancreatitis | Probiotics | Treatment Outcome [SUMMARY]
|
[CONTENT] Acute Disease | Double-Blind Method | Hospitalization | Humans | Pancreatitis | Probiotics | Treatment Outcome [SUMMARY]
| null |
[CONTENT] Acute Disease | Double-Blind Method | Hospitalization | Humans | Pancreatitis | Probiotics | Treatment Outcome [SUMMARY]
|
[CONTENT] Acute Disease | Double-Blind Method | Hospitalization | Humans | Pancreatitis | Probiotics | Treatment Outcome [SUMMARY]
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[CONTENT] Acute Disease | Double-Blind Method | Hospitalization | Humans | Pancreatitis | Probiotics | Treatment Outcome [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]
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[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]
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[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]
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[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]
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[CONTENT] probiotics | patients | abdominal | pancreatitis | study | group | pain | abdominal pain | placebo | acute [SUMMARY]
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[CONTENT] acute | pancreatitis | acute pancreatitis | disease | probiotics | microbiota | intestinal | patients | mild | analysis [SUMMARY]
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[CONTENT] abdominal | abdominal pain | pain | patients | data | capsules | time | study | normally distributed | test [SUMMARY]
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[CONTENT] sample | probiotics | randomized | acute | acute pancreatitis | pancreatitis double blinded randomized | follow time short | time short study future | time short study | time short [SUMMARY]
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[CONTENT] probiotics | abdominal | group | patients | pancreatitis | placebo | study | acute | pain | abdominal pain [SUMMARY]
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[CONTENT] probiotics | abdominal | group | patients | pancreatitis | placebo | study | acute | pain | abdominal pain [SUMMARY]
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[CONTENT] tertiary ||| Enterococcus ||| ||| [SUMMARY]
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[CONTENT] ||| ||| ||| tertiary ||| Enterococcus ||| ||| ||| ||| 128 | 64 ||| two ||| 5.36 ± | 0.15 | 6.02 | 0.17 ||| ||| two ||| [SUMMARY]
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[CONTENT] ||| ||| ||| tertiary ||| Enterococcus ||| ||| ||| ||| 128 | 64 ||| two ||| 5.36 ± | 0.15 | 6.02 | 0.17 ||| ||| two ||| [SUMMARY]
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Extract of Corallodiscus flabellata attenuates renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS signaling pathway.
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35227255
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Fibrosis is one of the most common pathological features of the aging process of the kidney, and fibrosis in aging kidneys also aggravates the process of chronic kidney disease (CKD). Corallodiscus flabellata B. L. Burtt (C. flabellata, CF) is a commonly used botanical drug in Chinese folklore. However, few studies have reported its pharmacological effects. This study aimed to explore the effect of CF ethanol extract on renal fibrosis in SAMP8 mice and identify potentially active compounds.
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BACKGROUND
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Senescence-accelerated mouse-prone 8 (SAMP8) were used as animal models, and different doses of CF were given by gavage for one month. To observe the degree of renal aging in mice using β-galactosidase staining. Masson staining and the expression levels of Col-I, α-SMA, and FN were used to evaluate the renal fibrosis in mice. The protein expression levels of Nrf2 pathway and Wnt/β-catenin/RAS pathway in the kidney were measured. And β-galactosidase (β-gal) induced NRK-52E cells as an in vitro model to screen the active components of CF.
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METHODS
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The CF ethanol extract significantly inhibited the activity of renal β-galactosidase and the expression levels of Col-I, α-SMA, and FN in SAMP8 mice, and improved Masson staining in SAMP8 mice. CF remarkably reduced urinary protein, creatinine, urea nitrogen and serum levels of TNF-α and IL-1β in SAMP8 mice, and significantly increased the levels of SOD and GSH-Px. Moreover, CF activated the Nrf2 pathway and blocked the Wnt/β-catenin/RAS pathway in the kidneys of mice. Besides, 3,4-dihydroxyphenylethanol (SDC-0-14, 16) and (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (SDC-1-8) were isolated from CF, which reduced the senescence of NRK-52E cells, and maybe the active ingredients of CF playing the anti-aging role.
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RESULTS
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Our experiments illuminated that CF ethanol extract may ameliorate renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS pathway. And SDC-0-14,16 and SDC-1-8 may be the material basis for CF to exert anti-renal senescence-related effects.
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CONCLUSIONS
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[
"Animals",
"Fibrosis",
"Mice",
"Plant Extracts",
"Renal Insufficiency, Chronic",
"Wnt Signaling Pathway",
"beta Catenin"
] |
8887028
|
Introduction
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The global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.
CF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books.
| null | null |
Results
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CF improved the aging and fibrosis of the kidneys in SAMP8 mice To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).
Fig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)
CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)
To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).
Fig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)
CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)
CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.
Fig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)
CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)
Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.
Fig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)
CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)
CF activated the Nrf2 pathway in the kidney of SAMP8 mice The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).
Fig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)
CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)
The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).
Fig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)
CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)
CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).
Fig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)
CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)
In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).
Fig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)
CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)
CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).
Fig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group
CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group
D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).
Fig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group
CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group
CF improved PCA analysis of kidney tissue in SAMP8 mice Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.
Fig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;
PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;
Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.
Fig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;
PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;
|
Conclusions
|
In conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF.
|
[
"Collection and extraction of the plant material",
"Animals and administration",
"Sample collection",
"Cell culture and in vitro study",
"Senescence-associated β-galactosidase staining",
"Histological analysis",
"Biochemical measurements",
"Immunohistochemical analysis",
"Western blot analysis",
"UPLC-Q-TOF-MS analysis for kidney samples",
"Statistical analysis",
"CF improved the aging and fibrosis of the kidneys in SAMP8 mice",
"CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice",
"CF activated the Nrf2 pathway in the kidney of SAMP8 mice",
"CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice",
"CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal",
"CF improved PCA analysis of kidney tissue in SAMP8 mice",
""
] |
[
"“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).",
"Six-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.",
"At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.",
"NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.",
"Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.",
"The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.",
"The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.",
"The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.",
"Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.",
"The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.",
"The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.",
"To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)",
"Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)",
"The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)",
"In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)",
"D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group",
"Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;",
"\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3."
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[
"Introduction",
"Materials and methods",
"Collection and extraction of the plant material",
"Animals and administration",
"Sample collection",
"Cell culture and in vitro study",
"Senescence-associated β-galactosidase staining",
"Histological analysis",
"Biochemical measurements",
"Immunohistochemical analysis",
"Western blot analysis",
"UPLC-Q-TOF-MS analysis for kidney samples",
"Statistical analysis",
"Results",
"CF improved the aging and fibrosis of the kidneys in SAMP8 mice",
"CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice",
"CF activated the Nrf2 pathway in the kidney of SAMP8 mice",
"CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice",
"CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal",
"CF improved PCA analysis of kidney tissue in SAMP8 mice",
"Discussion",
"Conclusions",
"Supplementary Information",
""
] |
[
"The global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.\nCF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books.",
" Collection and extraction of the plant material “Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).\n“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).",
"“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.\nAnother separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).",
"Six-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.\n Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\nAt the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.\n Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\nNRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.\n Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\nFrozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.\n Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\nThe kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.\n Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\nThe serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.\n Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\nThe immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.\n Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\nWestern blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.\n UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\nThe renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.\n Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.\nThe acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.",
"At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.",
"NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.",
"Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.",
"The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.",
"The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.",
"The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.",
"Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.",
"The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.",
"The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.",
" CF improved the aging and fibrosis of the kidneys in SAMP8 mice To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nTo investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\n CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nBesides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\n CF activated the Nrf2 pathway in the kidney of SAMP8 mice The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nThe expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\n CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nIn order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\n CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nD-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\n CF improved PCA analysis of kidney tissue in SAMP8 mice Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPrincipal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;",
"To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).\n\nFig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)\nCF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)",
"Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.\n\nFig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)\nCF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)",
"The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).\n\nFig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)\nCF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)",
"In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).\n\nFig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)\nCF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)",
"D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).\n\nFig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group\nCF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group",
"Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.\n\nFig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;\nPCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;",
"Aging plays an important role in the progression of CKD [19]. As CKD fibrosis progresses, senescent cells express and secrete pro-fibrotic factors (TGF-β, CTGF, and so forth) and pro-inflammatory factors (IL-1β, IL-6, TNF-α, and so forth), which are senescence-associated secretory phenotype factors, thereby accelerating renal fibrosis [20, 21]. At present, traditional Chinese medicine has achieved good results in treating renal fibrosis with fewer side effects [22]. This study explored the interventional effects of CF extract on renal fibrosis in SAMP8 mice.\nThe mouse strain selected for this study was SAMP8, which was developed based on the lifespan, senescence, and pathological phenotypic grading scores of AKR/J mice and was an accelerated aging model solely of genetic origin [23, 24]. Studies have shown that the changes in the renal pathology of SAMP8 mice (9 months) include tubulointerstitial fibrosis and focal segmental glomerulosclerosis [25]. And it was found that renal fibrosis in SAMP8 mice was age-related [6]. Therefore, we tested the activity of β-galactosidase and the level of renal fibrosis in SAMP8 mice, and found that CF-L and CF-M improved kidney fibrosis and β-galactosidase activity in SAMP8 mice (Fig. 1). And we also tested the urine output of the mice. Consistent with previously published studies indicating that CF obtained by two different processes had diuretic effects [13], the extract significantly increased the urine volume of SAMP8 mice. Also, the present study showed that supplementation with CF improved the renal function, the activity of antioxidant enzymes, and levels of inflammatory factors in SAMP8 mice (Fig. 2).\nThe Keap1–Nrf2 system gained the attention of many scholars in recent years due to its antioxidant and anti-inflammatory properties. Its pharmacological potential in treating kidney diseases had been extensively studied in nonclinical and clinical studies [26]. Nrf2 is a master transcriptional regulator for genes related to redox status and antioxidant effects [27]. Studies have shown that phosphorylation is required for Nrf2 activation and target gene induction [28]. Activating Nrf2 improved CKD progression by preventing oxidative stress and maintaining cellular redox homeostasis [29]. As a key transcription factor, Nrf2 plays a crucial role in defense against oxidative stress by regulating its downstream antioxidants and detoxification enzymes [30]. Kim et al. reported that resveratrol, as a potent Nrf2 activator, ameliorated aging-related progressive renal injury [31]. In the present study, CF improved the attenuation of Nrf2 in the kidney of SAMP8 mice without affecting the expression level of Keap1, suggesting that CF crude extract might improve oxidative damage in the kidneys by activating the Nrf2 pathway (Fig. 3).\nRenal fibrosis is characterized by excessive extracellular matrix (ECM) deposition leading to the formation of scars in the renal parenchyma [32]. Transforming growth factor-β (TGF-β) is thought to be a key cytokine in fibroblast overactivation [33, 34]. Of course, targeting only the TGF-β signaling pathway is insufficient to reduce renal fibrosis. Some studies indicated that CTGF, Wnt/β-catenin, renin-angiotensin system, oxidative stress, and so forth, were implicated in renal fibrosis [35–37]. Wnt/β-catenin is an evolutionarily conserved signaling pathway involved in the regulation of tissue homeostasis, organ development, and injury repair [38]. Cisternas et al. discussed the pro-fibrotic effect of Wnt signaling in both skeletal muscle and kidney [39]. Mounting evidence established that the Wnt/β-catenin signaling pathway plays a crucial role in regulating the development and progression of renal fibrotic lesions following injury [40–42]. The Wnt/β-catenin signal is relatively silent in the kidneys of healthy adults and is activated once the kidneys are subjected to various kinds of damages [43]. In mammals, the Wnt family has at least 19 family members critical for kidney development. And at least 15 of these family members are differentially upregulated in the aging kidney, including Wnt4 [6]. The results of the present study showed that the expression level of Wnt4 protein in the kidneys of SAMP8 mice was significantly higher than that in SAMR1 mice, which was verified by both Western blot and immunohistochemical analyses (Fig. 4a, d and f). Coincidentally, the expression level of β-catenin protein was also upregulated. Also, both Wnt4 and β-catenin were expressed in renal tubular epithelial cells, as revealed by the immunohistochemical analysis (Fig. 4a and f). Wnt/β-catenin elicited renal fibrosis by inducing multiple fibrogenic genes such as RAS components, matrix metalloproteinase-7 (MMP-7), plasminogen activator inhibitor 1 (PAI-1), and Snail1 [37]. Zhou et al. described Wnt/β-catenin as the major upstream regulator, which controls the expression of all tested RAS components in the kidneys [44]. Zhou et al. used a 5/6 nephrectomy (5/6 NX) rat model to show that the expression levels of major components of RAS in the brain and kidneys, such as angiotensinogen, angiotensin-converting enzyme, and angiotensin II AT1-receptor, was significantly upregulated. The upregulated expression level was inhibited by a central blocker of Wnt, which was an adeno-associated virus vector overexpressing the DKK1 gene [45]. Similarly, the present study found that AGTR1 was still expressed in the renal tubular epithelium, and the expression levels of renin and AGTR1 in the kidney tissues of SAMP8 mice were significantly greater than those in SAMR1 mice (Fig. 4a and d g). These results indicated that the Wnt/β-catenin/RAS signaling pathway was activated in the kidneys of SAMP8 mice, and the CF effectively inhibited the activation of this pathway.\nCTGF exerts multiple biological functions, including promoting mitosis of chemotactic cells, inducing adhesion, and promoting cell proliferation and ECM synthesis [35, 46]. CCN2 (CTGF) modulated Wnt signaling by binding to low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to further mediate fibrosis [39, 47]. Other studies used gene silencing to discover and confirm that Nrf2 regulated the Wnt pathway by regulating the expression level of CTGF, affecting renal interstitial disease. Also, Nrf2 regulated the CTGF transcription level mainly via CTGF transcriptional regulator c-Fos [48]. The immunohistochemical analysis revealed that CTGF and p-c-Fos were expressed mainly in renal tubular epithelial cells, and p-c-Fos was also distributed in glomeruli. The quantitative analysis showed that the expression levels of both proteins was inhibited by CF (Figs. 3b and e and 4a and c). Finally, the PCA analysis was used to further verify that CF improved kidney damage in SAMP8 mice (Fig. 6). The present study provided evidence that the CF not only activated Nrf2 signaling but also relied on Nrf2 to balance oxidative stress and inflammation, inhibited Wnt/β-catenin/RAS signaling, and improved kidney aging and renal fibrogenesis. However, we also found inconsistent improvement in the effect of CF on Masson and SA-β-gal staining in this experiment. It is speculated that this may be due to the inconsistent progression of renal aging and renal fibrosis in SAMP8 mice. The degree of renal aging in the mice in this experiment may be more serious than fibrosis. Therefore, the ameliorating effect of CF-H on renal aging in SAMP8 mice was not as obvious as that of renal fibrosis.\nAs a reducing monosaccharide, D-galactose is widely used in various age-related diseases in vivo and in vitro. D-gal was found to cause senescence and injury in NRK-52E cells [49], induce senescence of human kidney proximal tubular epithelial cells (HKC-8 cells), and increase the expression levels of two renal fibrosis marker proteins FN and α-SMA [6]. In this study, D-gal was used to induce NRK-52E cells in vitro. Using MTT assay and β-galactosidase staining to detect the compounds isolated from CF, it was found that SDC-0-14,16 and SDC-1-8 enhanced the cell viability induced by D-gal and inhibited D-gal induced cell senescence (Fig. 5). These suggested that SDC-0-14,16 and SDC-1-8 may be the material basis for CF to delay kidney aging. SDC-1-8 is one of the phenylethanoid glycosides. phenylethanoid glycosides were found to extend the life span of Caenorhabditis Elegans [50], with anti-aging, [51] and neuroprotective effects [52–54]. These provide a direction for our follow-up study on the pharmacological effects of CF.",
"In conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF.",
" \nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.\n\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3.",
"\nAdditional file 1.\nAdditional file 1.\n\nAdditional file 2.\nAdditional file 2.\n\nAdditional file 3.\nAdditional file 3."
] |
[
"introduction",
"materials|methods",
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
null,
"discussion",
"conclusion",
"supplementary-material",
null
] |
[
"Aging",
"\nCorallodiscus flabellata\n",
"Kidney",
"Renal fibrosis",
"SAMP8",
"Senescence",
"Wnt/β-catenin/RAS"
] |
Introduction:
The global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.
CF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books.
Materials and methods:
Collection and extraction of the plant material “Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.
Another separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).
“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.
Another separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).
Collection and extraction of the plant material:
“Yunnan Chinese Herbal Medicine” records that CF can be harvested throughout the year. The CF plants used in this experiment was harvested in Xixia County, Henan Province, China in September. It was identified by Professor Suiqing Chen from Henan University of Chinese Medicine, and the specimens were stored in the laboratory specimen library. The plants (1 kg) were refluxed with 50% ethanol (3 × 12 L, each 1 h), and the mixture was filtered with 16 layers of gauze. The combined filtrates were dried by rotary evaporation using a freeze drier. Finally, the percentage yield of 50% ethanol crude extract of CF was 15.5%. The dried extract was kept in a fridge until further use. The supplementary materials list the relevant data on the ingredient designation of CF extract.
Another separation process previously reported was used in this study to obtain the water elution fraction, 20% ethanol elution fraction, 30% ethanol elution fraction, and 40% ethanol elution fraction from CF [13]. The 40% ethanol fraction was separated using Sephadex LH-20 and silica gel column and purified by semi-preparative high-performance liquid chromatography (HPLC) to finally obtain compounds such as SDC-0-14,16, SDC-1-8, SDC-0-60 (p-hydroxybenzyl alcohol).
Animals and administration:
Six-month-old male SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China) were used in this study. The animals were housed under controlled light (12-h light/dark cycle), temperature (23-25 °C), and humidity (45-55%) conditions and received a standard diet and water ad libitum. A total of 48 SAMP8 mice were divided into four experimental groups (n = 12/group): SAMP8 model mice (M), low-dose CF ethanol extract-treated SAMP8 mice (CF-L, 387.5 mg/kg, intragastrically), medium-dose CF ethanol extract-treated SAMP8 mice (CF-M, 775 mg/kg, intragastrically), and high-dose CF ethanol extract-treated SAMP8 mice (CF-H, 1550 mg/kg, intragastrically). The mice in the SAMR1 control (Con, n = 10) and SAMP8 model groups (M) were treated with physiological saline (0.9%). All mice were treated orally for 1 month. All animal experiments were approved by the ethics committee of Henan University of Chinese Medicine and performed under the institutional guidelines (Henan, China Approval Number: HACTCM-2018009060-19). During the experiment, there was one mouse death in each of the Con and M group, and two mice died in the CF-H group.
Sample collection At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.
At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.
Cell culture and in vitro study NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.
NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.
Senescence-associated β-galactosidase staining Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.
Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.
Histological analysis The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.
The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.
Biochemical measurements The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.
The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.
Immunohistochemical analysis The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.
The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.
Western blot analysis Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.
Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.
UPLC-Q-TOF-MS analysis for kidney samples The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.
The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.
Statistical analysis The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.
The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.
Sample collection:
At the end of the experiment, the mice were housed individually in metabolic cages for 12-h urinary collection. The mice were then anesthetized with isoflurane, and blood samples were collected through retro-orbital bleeding. The kidneys from each mouse were then surgically removed and kept at -80 °C until the analyses.
Cell culture and in vitro study:
NRK-52E cells purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured to investigate the effect of CF extract on cell aging caused by D-galactose (D-gal, S11050, Yuanye, Shanghai, China). The NRK-52E cells were grown in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM, 12,100,046, Thermo Fisher, Massachusetts, USA) supplied with 10% fetal bovine serum in an incubator at 37 °C and in the presence of 5% CO2. Cells were grown in 96-well plates or 6-well plates to 80–85% confluence and then treated with growth media containing different drug combinations: Media + D-gal (20 mg/mL), Media + D-gal + CF (10, 25, 50, 100 µg/mL) or Media + D-gal + monomeric compound (10 µM) respectively for 48 h. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and β-galactosidase staining was used to observe cell senescence.
Senescence-associated β-galactosidase staining:
Frozen kidneys from mice sliced into 10-µm-thick sections and NRK-52E cells were stained with senescence-associated β-galactosidase (SA-β-gal, C0602, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols.
Histological analysis:
The kidney sections were fixed with 4% buffered paraformaldehyde, and 10-µm-thick paraffin-embedded sections were stained with Masson’s trichrome (G1006, Servicebio, Wuhan, China) and observed microscopically. The blue-colored areas in Masson’s trichrome–stained sections were measured quantitatively from six randomly selected fields and analyzed by Image-Pro Plus 6.0 software.
Biochemical measurements:
The serum and kidney homogenate samples were thawed to room temperature, and the levels of superoxide dismutase (SOD, CSB-E08556m, Wuhan Huamei, Wuhan, China), glutathione peroxidase (GSH-Px, A005-1-2, Nanjing Jiancheng, Nanjing, China), interleukin-1β (IL-1β, RK00006, ABclonal, Wuhan, China), tumor necrosis factor-α (TNF-α, RK00027, ABclonal, Wuhan, China), urea nitrogen (C013-2-1, Nanjing Jiancheng, Nanjing, China), creatinine (Cr, C011-2-1, Nanjing Jiancheng, Nanjing, China) in mouse serum, total urinary protein (C035-2-1, Nanjing Jiancheng, Nanjing, China) in mouse urine and the expression levels of collagen type I (Col-I, MU30364, Bio-Swamp, Wuhan, China), α-smooth muscle actin (α-SMA, MU30359, Bio-Swamp, Wuhan, China), fibronectin (FN, MU30179, Bio-Swamp, Wuhan, China) in mouse kidney tissues were measured sequentially according to the protocol of the assay kit manufacturer.
Immunohistochemical analysis:
The immunohistochemical analysis was performed using the routine method [17]. The antibodies used included the following: Wnt4 (14371-1-AP, Proteintech, Chicago, USA), β-catenin (17565-1-AP, Proteintech, Chicago, USA), type 1 angiotensin II receptors (AGTR1, 25343-1-AP, Proteintech, Chicago, USA), p-nuclear factor erythroid 2–related factor 2 (p-Nrf2, ab76026, Abcam, Cambridge, UK), p-c-Fos (ab27793, Abcam, Cambridge, UK), connective tissue growth factor (CTGF, GB11078, Servicebio, Wuhan, China). The sections were observed under a microscope (Olympus, Tokyo, Japan). Measure the area and integrated optical density (IOD) of the area with tan expression using Image-Pro Plus 6.0 software, and calculate the mean optical density (MOD, MOD = IOD/area) for semi-quantitative analysis.
Western blot analysis:
Western blot assay was conducted as described in a previous study [18]. Briefly, the kidney tissues were homogenized in lysis buffer and quantified using a Bradford Protein Assay Kit (AR0197, Boster Biological Technology, Wuhan, China). The homogenates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in blocking buffer (4% nonfat dry milk) for 90 min. They were then incubated with primary antibodies (Wnt4; renin; AGTR1; p-Nrf2; p-c-Fos; Kelch like Ech associated protein 1 (Keap1, GB11847, Servicebio, Wuhan, China); β-actin (AC026, Abclonal, Wuhan, China); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AC033, Abclonal, Wuhan, China)) overnight at 4℃, followed by incubation with an appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. The proteins of interest were scanned with an Odyssey IR scanner (LI-COR Biosciences, Nebraska, USA), and the signal intensities were quantified using Image Studio software. The protein levels were normalized against the β-actin or GAPDH.
UPLC-Q-TOF-MS analysis for kidney samples:
The renal tissues were weighed and homogenized in ice-cold physiological saline (w/v = 1:1). Then, 1 mL of acetonitrile was added to 200 µL of tissue homogenate samples, followed by ultrasonic extraction for 30 min. The extract was centrifuged at 12,000 g and 4℃ for 10 min. The supernatant was taken into the vial for analysis. Chromatographic separation was carried out on ultraperformance liquid chromatography (UPLC) (Dionex UltiMate 3000 System, Thermo Scientific, Massachusetts, USA), with the LC system comprising an Acclaim RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm; Thermo Scientific). The mobile phase consisted of solvent A (acetonitrile) and water with 0.1% formic acid (B). The separation was performed by gradient elution as follows: 10-70% A from 0 to 3 min, 70-78% A from 4 to 13 min, 78-90% A from 14 to 15 min, 90%-10% A from 15 to 16 min, and 10% A from 16 to 20 min. The injection volume of the test sample was 2 µL. The mass spectrometry (MS) analysis was performed by using an ESI source under the following conditions: the capillary voltage in the positive mode was 3.5 kV, and the capillary voltage in the negative mode was 3.2 kV. The pressure of the nebulizer was 2.0 bar, the temperature of the dry gas was 230 ℃, and the flow rate was 8 L/min.
Statistical analysis:
The acquired raw data from ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) analysis were first preprocessed using profile analysis (version 2.1, Bruker, Germany). The “bucket table” was obtained and imported into the SIMCA-P software (version13.0 Umetrics AB, Sweden) for principal component analysis (PCA). Other results were presented as mean ± standard deviation (SD). The data were processed using IBM SPSS Stastic 26.0. The normal distribution of the data used the Levene test, which required the average value, P > 0.05; the one-way analysis of variance was used for comparison between groups. A P value less than 0.05 was considered statistically significant.
Results:
CF improved the aging and fibrosis of the kidneys in SAMP8 mice To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).
Fig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)
CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)
To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).
Fig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)
CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)
CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.
Fig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)
CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)
Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.
Fig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)
CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)
CF activated the Nrf2 pathway in the kidney of SAMP8 mice The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).
Fig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)
CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)
The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).
Fig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)
CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)
CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).
Fig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)
CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)
In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).
Fig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)
CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)
CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).
Fig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group
CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group
D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).
Fig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group
CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group
CF improved PCA analysis of kidney tissue in SAMP8 mice Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.
Fig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;
PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;
Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.
Fig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;
PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;
CF improved the aging and fibrosis of the kidneys in SAMP8 mice:
To investigate the effect of CF on the kidneys of SAMP8 mice, SA-β-gal activity was measured in the kidneys. In Fig. 1a, The SA-β-gal activity in kidney tissues significantly increased in the M group (blue enhancement). Administration of CF-L and CF-M significantly inhibited β-galactosidase activity in the kidneys of SAMP8 mice while the improvement by CF-H was not significant. Also, the kidney index of the model group was significantly lower than that of the control group (Fig. 1b, P < 0.05). After treatment with CF-M, the kidney index of SAMP8 mice were improved (P < 0.01). Next, the results of Masson’s trichrome staining (Fig. 1c, P < 0.01) and the expression of fibrosis indicators Col-I, α-SMA, and FN revealed that M-group mice had more severe renal fibrosis than Con-group mice, and CF-L and CF-M significantly attenuated renal fibrosis in SAMP8 mice (Fig. 1e-g, P < 0.01).
Fig. 1CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)
CF improved the aging and fibrosis of the kidneys in SAMP8 mice. a The activity of SA-β-gal in frozen sections of kidneys. Scale bars: 50 μm, magnification 200×. b Changes in the mouse kidney index (kidney index = kidney weight/mouse weight × 100%). c Masson’s trichrome staining of kidneys. Renal fibrosis was expressed by collagen deposition (blue color area) in Masson’s trichrome staining. Scale bars: 50 μm, magnification 400×. d Semi-quantification of renal interstitial fibrosis. The ratio of the blue color area to the field area from Masson’s trichrome-stained sections. e-g The expression levels of Col-I, α-SMA, and FN in kidney tissue were detected by ELISA. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 6)
CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice:
Besides, the renal function indicators of each group of mice were tested, which included urine volume in mice for 12 h, Cr and urea nitrogen levels in the serum, and urine protein levels. As shown in Fig. 2a-d, the urine protein, serum Cr and serum urea nitrogen levels of the model group were significantly higher than those of the control group (Fig. 2a, P < 0.01), while the urine volume of the model group was significantly lower than that of the control group (Fig. 2b-d, P < 0.01). CF supplementation down-regulated the levels of urine protein, serum Cr and urea nitrogen in the model group (Fig. 2b-d, P < 0.05 or P < 0.01) while increasing the urine output of model mice. Collectively, the results indicated that SAMP8 mice had kidney damage associated with aging, and treatment with CF effectively ameliorated this injury. Next, whether CF beneficially modulated oxidative stress and inflammation in the kidneys of rapidly aging mice was explored. As shown in Fig. 2e-h, the levels of SOD and GSH-Px in the kidneys of the M group were significantly lower than those of the Con group, and the levels of IL-1β were significantly higher than those of the Con group. After CF treatment, the above indicators have been improved, especially the improvement effect of CF-L and CF-M is better.
Fig. 2CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)
CF improved renal function, oxidative stress and inflammation levels in SAMP8 mice. a-d Effect of CF on the urine volume, urine protein levels, and serum Cr and urea nitrogen levels, respectively. e-h The level of SOD, GSH-Px, TNF-α and IL-1β in serum, respectively. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Values expressed as mean ± SD; *P < 0.05 and **P < 0.01 vs. M group (n = 5–12)
CF activated the Nrf2 pathway in the kidney of SAMP8 mice:
The expression level of p-Nrf2 was measured in kidney tissues to investigate whether the Nrf2 pathway was involved in the protective effect of CF (Fig. 3a-c) in the present study. The expression level of p-Nrf2 significantly decreased in the SAMP8 group alone (P < 0.01, Fig. 3c), and the downregulation was reversed to some extent by the treatment of CF crude extract. There was no significant change in the expression level of Keap1 among the groups (Fig. 3b and d). Further, the expression level of p-c-Fos was detected and analyzed by immunohistochemical and Western blot analyses. It was found that the expression level of p-c-Fos in the kidneys of mice in the M group was significantly higher than that in the Con group; while CF treatment significantly reduced the expression levels of p-c-Fos in the kidneys of the mice (Fig. 3a-b and e).
Fig. 3CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)
CF activated the Nrf2 pathway in the kidney of SAMP8 mice. Scale bars: 50 μm. a The localization of p-Nrf2, p-c-Fos in the kidney was detected by immunohistochemistry. b-e Protein strip and quantification of p-Nrf2, Keap1, and p-c-Fos in the kidney were detected by western blotting. Arrows indicate positive expression. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. M group (n = 3 or n = 6)
CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice:
In order to further explored the potential mechanism of CF exerting anti-fibrosis effect. The localization of protein was performed using immunohistochemistry. As shown in Fig. 4a, the expression level of Wnt4 and β-catenin was induced predominantly in renal tubular cells. Similar results were observed when AGTR1, the downstream pathway targets of Wnt signaling, were assessed (Fig. 4a). After that, the protein expression level was quantified, and the results showed that Wnt4, β-catenin, renin, and AGTR1 proteins were accumulated in the kidney tissue of mice in the model group, whereas CF significantly inhibited those alterations (Fig. 4b-f ). Furthermore, the expression level of CTGF was also located and quantitatively analyzed. Consistent with the aforementioned results, CTGF was mainly expressed in the renal tubules, and CF markedly inhibited the activity of CTGF (Fig. 4d g).
Fig. 4CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)
CF attenuated Wnt/β-catenin/RAS signaling activation in the kidney of SAMP8 mice. Scale bars: 50 μm. The localization of AGTR1, Wnt4, β-catenin, and CTGF in the kidney was detected by immunohistochemistry (a), and β-catenin (b) and CTGF (c) were semi-quantitatively analyzed. Protein strip (d) and quantification of renin (e), Wnt4 (f), and AGTR1 (g) in the kidney were detected by western blotting. Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract; Arrows indicate positive expression. Data represent the mean values ± SD. *P < 0.05 and **P < 0.01 vs. the M group (n = 3)
CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal:
D-gal-induced NRK-52E cells were used to screen certain active ingredients in CF, to determine the material basis for the treatment of senile kidney by the extract of CF. The results showed that the CF enhanced cell viability in a dose-dependent manner, and the compounds SDC-0-14,16 and SDC-1-8 isolated from CF had better effects on cell proliferation. Besides, 25 µg/mL CF and the compounds SDC-0-14,16 and SDC-1-8 all displayed the effect of inhibiting β-galactosidase activity (Fig. 5).
Fig. 5CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group
CF and its compounds inhibited the senescence of NRK-52E cells induced by D-gal. a Detection of cell viability of CF and its chemical components. b β-galactosidase staining of NRK-52E cells. Scale bars: 50 μm. CF: C. flabellate extract; D-gal: D-galactose; SDC-0-14,16: 3,4-dihydroxyphenylethanol; SDC-1-8: (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside; SDC-0-60: p-hydroxybenzyl alcohol. Data represent the mean values ± SD from three independent experiments. *P < 0.05 and **P < 0.01 vs. the D-gal group
CF improved PCA analysis of kidney tissue in SAMP8 mice:
Principal components analysis (PCA) was performed to explore the effects of CF on SAMP8 mice. As shown in Fig. 6a, there was an obvious grouping between the control and model groups (R2X = 0.542; Q2 = 0.38), suggesting that the endogenous metabolites of SAMP8 mice were different from SAMR1 mice, which causes them to deviate from the SAMR1 group. As shown in Fig. 6b, the different CF groups also clustered into different classes from the control and model groups, and the CF groups moved closer to the control group, indicating that the endogenous metabolites of the CF-intervened SAMP8 mice were similar to those of the SAMR1 mice.
Fig. 6PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;
PCA score plot of kidney homogenate extract in positive modes. a PCA score plot for the Control and M groups (R2X = 0.542, Q2 = 0.38); b PCA score plot for all groups (R2X = 0.63, Q2 = 0.489). Con: SAMR1 mice; M: SAMP8 mice; CF-L: Treated with low-dose CF ethanol extract; CF-M: Treated with medium-dose CF ethanol extract; CF-H: Treated with high-dose CF ethanol extract;
Discussion:
Aging plays an important role in the progression of CKD [19]. As CKD fibrosis progresses, senescent cells express and secrete pro-fibrotic factors (TGF-β, CTGF, and so forth) and pro-inflammatory factors (IL-1β, IL-6, TNF-α, and so forth), which are senescence-associated secretory phenotype factors, thereby accelerating renal fibrosis [20, 21]. At present, traditional Chinese medicine has achieved good results in treating renal fibrosis with fewer side effects [22]. This study explored the interventional effects of CF extract on renal fibrosis in SAMP8 mice.
The mouse strain selected for this study was SAMP8, which was developed based on the lifespan, senescence, and pathological phenotypic grading scores of AKR/J mice and was an accelerated aging model solely of genetic origin [23, 24]. Studies have shown that the changes in the renal pathology of SAMP8 mice (9 months) include tubulointerstitial fibrosis and focal segmental glomerulosclerosis [25]. And it was found that renal fibrosis in SAMP8 mice was age-related [6]. Therefore, we tested the activity of β-galactosidase and the level of renal fibrosis in SAMP8 mice, and found that CF-L and CF-M improved kidney fibrosis and β-galactosidase activity in SAMP8 mice (Fig. 1). And we also tested the urine output of the mice. Consistent with previously published studies indicating that CF obtained by two different processes had diuretic effects [13], the extract significantly increased the urine volume of SAMP8 mice. Also, the present study showed that supplementation with CF improved the renal function, the activity of antioxidant enzymes, and levels of inflammatory factors in SAMP8 mice (Fig. 2).
The Keap1–Nrf2 system gained the attention of many scholars in recent years due to its antioxidant and anti-inflammatory properties. Its pharmacological potential in treating kidney diseases had been extensively studied in nonclinical and clinical studies [26]. Nrf2 is a master transcriptional regulator for genes related to redox status and antioxidant effects [27]. Studies have shown that phosphorylation is required for Nrf2 activation and target gene induction [28]. Activating Nrf2 improved CKD progression by preventing oxidative stress and maintaining cellular redox homeostasis [29]. As a key transcription factor, Nrf2 plays a crucial role in defense against oxidative stress by regulating its downstream antioxidants and detoxification enzymes [30]. Kim et al. reported that resveratrol, as a potent Nrf2 activator, ameliorated aging-related progressive renal injury [31]. In the present study, CF improved the attenuation of Nrf2 in the kidney of SAMP8 mice without affecting the expression level of Keap1, suggesting that CF crude extract might improve oxidative damage in the kidneys by activating the Nrf2 pathway (Fig. 3).
Renal fibrosis is characterized by excessive extracellular matrix (ECM) deposition leading to the formation of scars in the renal parenchyma [32]. Transforming growth factor-β (TGF-β) is thought to be a key cytokine in fibroblast overactivation [33, 34]. Of course, targeting only the TGF-β signaling pathway is insufficient to reduce renal fibrosis. Some studies indicated that CTGF, Wnt/β-catenin, renin-angiotensin system, oxidative stress, and so forth, were implicated in renal fibrosis [35–37]. Wnt/β-catenin is an evolutionarily conserved signaling pathway involved in the regulation of tissue homeostasis, organ development, and injury repair [38]. Cisternas et al. discussed the pro-fibrotic effect of Wnt signaling in both skeletal muscle and kidney [39]. Mounting evidence established that the Wnt/β-catenin signaling pathway plays a crucial role in regulating the development and progression of renal fibrotic lesions following injury [40–42]. The Wnt/β-catenin signal is relatively silent in the kidneys of healthy adults and is activated once the kidneys are subjected to various kinds of damages [43]. In mammals, the Wnt family has at least 19 family members critical for kidney development. And at least 15 of these family members are differentially upregulated in the aging kidney, including Wnt4 [6]. The results of the present study showed that the expression level of Wnt4 protein in the kidneys of SAMP8 mice was significantly higher than that in SAMR1 mice, which was verified by both Western blot and immunohistochemical analyses (Fig. 4a, d and f). Coincidentally, the expression level of β-catenin protein was also upregulated. Also, both Wnt4 and β-catenin were expressed in renal tubular epithelial cells, as revealed by the immunohistochemical analysis (Fig. 4a and f). Wnt/β-catenin elicited renal fibrosis by inducing multiple fibrogenic genes such as RAS components, matrix metalloproteinase-7 (MMP-7), plasminogen activator inhibitor 1 (PAI-1), and Snail1 [37]. Zhou et al. described Wnt/β-catenin as the major upstream regulator, which controls the expression of all tested RAS components in the kidneys [44]. Zhou et al. used a 5/6 nephrectomy (5/6 NX) rat model to show that the expression levels of major components of RAS in the brain and kidneys, such as angiotensinogen, angiotensin-converting enzyme, and angiotensin II AT1-receptor, was significantly upregulated. The upregulated expression level was inhibited by a central blocker of Wnt, which was an adeno-associated virus vector overexpressing the DKK1 gene [45]. Similarly, the present study found that AGTR1 was still expressed in the renal tubular epithelium, and the expression levels of renin and AGTR1 in the kidney tissues of SAMP8 mice were significantly greater than those in SAMR1 mice (Fig. 4a and d g). These results indicated that the Wnt/β-catenin/RAS signaling pathway was activated in the kidneys of SAMP8 mice, and the CF effectively inhibited the activation of this pathway.
CTGF exerts multiple biological functions, including promoting mitosis of chemotactic cells, inducing adhesion, and promoting cell proliferation and ECM synthesis [35, 46]. CCN2 (CTGF) modulated Wnt signaling by binding to low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to further mediate fibrosis [39, 47]. Other studies used gene silencing to discover and confirm that Nrf2 regulated the Wnt pathway by regulating the expression level of CTGF, affecting renal interstitial disease. Also, Nrf2 regulated the CTGF transcription level mainly via CTGF transcriptional regulator c-Fos [48]. The immunohistochemical analysis revealed that CTGF and p-c-Fos were expressed mainly in renal tubular epithelial cells, and p-c-Fos was also distributed in glomeruli. The quantitative analysis showed that the expression levels of both proteins was inhibited by CF (Figs. 3b and e and 4a and c). Finally, the PCA analysis was used to further verify that CF improved kidney damage in SAMP8 mice (Fig. 6). The present study provided evidence that the CF not only activated Nrf2 signaling but also relied on Nrf2 to balance oxidative stress and inflammation, inhibited Wnt/β-catenin/RAS signaling, and improved kidney aging and renal fibrogenesis. However, we also found inconsistent improvement in the effect of CF on Masson and SA-β-gal staining in this experiment. It is speculated that this may be due to the inconsistent progression of renal aging and renal fibrosis in SAMP8 mice. The degree of renal aging in the mice in this experiment may be more serious than fibrosis. Therefore, the ameliorating effect of CF-H on renal aging in SAMP8 mice was not as obvious as that of renal fibrosis.
As a reducing monosaccharide, D-galactose is widely used in various age-related diseases in vivo and in vitro. D-gal was found to cause senescence and injury in NRK-52E cells [49], induce senescence of human kidney proximal tubular epithelial cells (HKC-8 cells), and increase the expression levels of two renal fibrosis marker proteins FN and α-SMA [6]. In this study, D-gal was used to induce NRK-52E cells in vitro. Using MTT assay and β-galactosidase staining to detect the compounds isolated from CF, it was found that SDC-0-14,16 and SDC-1-8 enhanced the cell viability induced by D-gal and inhibited D-gal induced cell senescence (Fig. 5). These suggested that SDC-0-14,16 and SDC-1-8 may be the material basis for CF to delay kidney aging. SDC-1-8 is one of the phenylethanoid glycosides. phenylethanoid glycosides were found to extend the life span of Caenorhabditis Elegans [50], with anti-aging, [51] and neuroprotective effects [52–54]. These provide a direction for our follow-up study on the pharmacological effects of CF.
Conclusions:
In conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF.
Supplementary Information:
Additional file 1.
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Additional file 1.
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Background:
Fibrosis is one of the most common pathological features of the aging process of the kidney, and fibrosis in aging kidneys also aggravates the process of chronic kidney disease (CKD). Corallodiscus flabellata B. L. Burtt (C. flabellata, CF) is a commonly used botanical drug in Chinese folklore. However, few studies have reported its pharmacological effects. This study aimed to explore the effect of CF ethanol extract on renal fibrosis in SAMP8 mice and identify potentially active compounds.
Methods:
Senescence-accelerated mouse-prone 8 (SAMP8) were used as animal models, and different doses of CF were given by gavage for one month. To observe the degree of renal aging in mice using β-galactosidase staining. Masson staining and the expression levels of Col-I, α-SMA, and FN were used to evaluate the renal fibrosis in mice. The protein expression levels of Nrf2 pathway and Wnt/β-catenin/RAS pathway in the kidney were measured. And β-galactosidase (β-gal) induced NRK-52E cells as an in vitro model to screen the active components of CF.
Results:
The CF ethanol extract significantly inhibited the activity of renal β-galactosidase and the expression levels of Col-I, α-SMA, and FN in SAMP8 mice, and improved Masson staining in SAMP8 mice. CF remarkably reduced urinary protein, creatinine, urea nitrogen and serum levels of TNF-α and IL-1β in SAMP8 mice, and significantly increased the levels of SOD and GSH-Px. Moreover, CF activated the Nrf2 pathway and blocked the Wnt/β-catenin/RAS pathway in the kidneys of mice. Besides, 3,4-dihydroxyphenylethanol (SDC-0-14, 16) and (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (SDC-1-8) were isolated from CF, which reduced the senescence of NRK-52E cells, and maybe the active ingredients of CF playing the anti-aging role.
Conclusions:
Our experiments illuminated that CF ethanol extract may ameliorate renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS pathway. And SDC-0-14,16 and SDC-1-8 may be the material basis for CF to exert anti-renal senescence-related effects.
|
Introduction:
The global prevalence of chronic kidney disease (CKD) is increasing with the aging of the population and poses a significant burden to society [1–3]. The most common pathological manifestation of CKD is some form of renal fibrosis [4]. Likewise, renal fibrosis is also one of the signs of kidney aging [2]. Studies have shown that oxidative stress, inflammation, Wnt/β-catenin, renin angiotensin aldosterone system (RAS) and rapamycin (m-TOR) signaling are all related to CKD induced by senescence [5]. Renal fibrosis in aging kidneys is closely related to the activation of Wnt/β-catenin and RAS signaling pathway [6]. Upon activation of Wnts, β-catenin in the cytoplasm is translocated to the nucleus, where it binds to the lymphoid enhancer-binding factor (LEF)/the T-cell factor (TCF) transcription factor family and initiates the transcription of downstream target genes. Interestingly, the promoter region of the RAS gene also contains LEF/TCF binding sites, which allows β-catenin to promote the binding of LEF-1 to these sites [7]. Therefore, targeting the Wnt/β-catenin/RAS signaling pathway could be a potential therapeutic strategy for renal fibrosis [8, 9]. In recent years, the replacement therapy of renal fibrosis by natural products has attracted the attention of many scholars [10]. Xiaoyan Shen et al. found that ginsenoside Rg1 ameliorated glomerular fibrosis during kidney aging by inhibiting the activation of NLRP3 inflammasome in SAMP8 mice [11]. This study explored the effect of C. flabellate (CF) extract on renal fibrosis in SAMP8 mice from the Wnt/β-catenin/RAS pathway.
CF as a medicinal plant in China, was first recorded in the “Dian Nan Ben Cao.” The whole plant is commonly used for treating dysentery, premature ejaculation, seminal vesicle disease, and kidney disease in ethnic minority areas of China [12]. At present, there are few reports about CF in modern research. The pharmacological studies in our laboratory found that CF extract had diuretic effects and ameliorated lipopolysaccharide/D-galactosamine-induced liver failure and brain damage in rats [13, 14]. And phytochemical studies on it revealed that phenylethanoid glycosides and flavonoids were the main chemical components of CF [15, 16]. The aim of this study was to investigate the effect of CF extract on renal fibrosis in SAMP8 mice and to elucidate its possible mechanism, so as to provide experimental evidence for the treatment of kidney disease with CF described in ancient books.
Conclusions:
In conclusion, in vivo studies showed that CF reduced renal fibrosis in elderly mice. Some potential active ingredients were found in in vitro experiments. These findings provided pharmacological support for treating kidney disease using CF and a direction for further research on the active ingredients of CF.
|
Background:
Fibrosis is one of the most common pathological features of the aging process of the kidney, and fibrosis in aging kidneys also aggravates the process of chronic kidney disease (CKD). Corallodiscus flabellata B. L. Burtt (C. flabellata, CF) is a commonly used botanical drug in Chinese folklore. However, few studies have reported its pharmacological effects. This study aimed to explore the effect of CF ethanol extract on renal fibrosis in SAMP8 mice and identify potentially active compounds.
Methods:
Senescence-accelerated mouse-prone 8 (SAMP8) were used as animal models, and different doses of CF were given by gavage for one month. To observe the degree of renal aging in mice using β-galactosidase staining. Masson staining and the expression levels of Col-I, α-SMA, and FN were used to evaluate the renal fibrosis in mice. The protein expression levels of Nrf2 pathway and Wnt/β-catenin/RAS pathway in the kidney were measured. And β-galactosidase (β-gal) induced NRK-52E cells as an in vitro model to screen the active components of CF.
Results:
The CF ethanol extract significantly inhibited the activity of renal β-galactosidase and the expression levels of Col-I, α-SMA, and FN in SAMP8 mice, and improved Masson staining in SAMP8 mice. CF remarkably reduced urinary protein, creatinine, urea nitrogen and serum levels of TNF-α and IL-1β in SAMP8 mice, and significantly increased the levels of SOD and GSH-Px. Moreover, CF activated the Nrf2 pathway and blocked the Wnt/β-catenin/RAS pathway in the kidneys of mice. Besides, 3,4-dihydroxyphenylethanol (SDC-0-14, 16) and (3,4-dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-β-D-apiofuranosyl-(1→3)-β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (SDC-1-8) were isolated from CF, which reduced the senescence of NRK-52E cells, and maybe the active ingredients of CF playing the anti-aging role.
Conclusions:
Our experiments illuminated that CF ethanol extract may ameliorate renal fibrosis in SAMP8 mice via the Wnt/β-catenin/RAS pathway. And SDC-0-14,16 and SDC-1-8 may be the material basis for CF to exert anti-renal senescence-related effects.
| 17,396 | 442 | 24 |
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[CONTENT] Aging |
Corallodiscus flabellata
| Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]
| null |
[CONTENT] Aging |
Corallodiscus flabellata
| Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]
|
[CONTENT] Aging |
Corallodiscus flabellata
| Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]
|
[CONTENT] Aging |
Corallodiscus flabellata
| Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]
|
[CONTENT] Aging |
Corallodiscus flabellata
| Kidney | Renal fibrosis | SAMP8 | Senescence | Wnt/β-catenin/RAS [SUMMARY]
|
[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]
| null |
[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]
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[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]
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[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]
|
[CONTENT] Animals | Fibrosis | Mice | Plant Extracts | Renal Insufficiency, Chronic | Wnt Signaling Pathway | beta Catenin [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
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[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]
| null |
[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]
|
[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]
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[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]
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[CONTENT] cf | mice | extract | kidney | samp8 | ethanol | samp8 mice | treated | dose | ethanol extract [SUMMARY]
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[CONTENT] fibrosis | renal fibrosis | catenin | renal | ras | disease | lef | cf | wnt catenin | wnt [SUMMARY]
| null |
[CONTENT] cf | mice | cf treated | dose cf ethanol | dose cf ethanol extract | cf ethanol extract | dose cf | ethanol extract | cf ethanol | dose [SUMMARY]
|
[CONTENT] ingredients | active | active ingredients | cf | pharmacological support treating | research active | experiments findings provided pharmacological | experiments findings provided | experiments findings | ingredients found [SUMMARY]
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[CONTENT] cf | mice | ethanol | extract | china | additional | additional file | file | kidney | samp8 [SUMMARY]
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[CONTENT] cf | mice | ethanol | extract | china | additional | additional file | file | kidney | samp8 [SUMMARY]
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[CONTENT] ||| B. L. Burtt | CF | Chinese ||| ||| CF | SAMP8 [SUMMARY]
| null |
[CONTENT] CF | SAMP8 | Masson | SAMP8 ||| TNF | IL-1β | SAMP8 | SOD | GSH-Px ||| Nrf2 | Wnt ||| 3,4 | SDC-0-14 | 16 | 3,4 | SDC-1-8) | CF | NRK-52E | CF [SUMMARY]
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[CONTENT] CF | SAMP8 | Wnt ||| SDC-0-14,16 | SDC-1-8 | CF [SUMMARY]
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[CONTENT] ||| B. L. Burtt | CF | Chinese ||| ||| CF | SAMP8 ||| CF | one month ||| ||| Masson ||| Nrf2 | Wnt ||| NRK-52E | CF ||| ||| CF | SAMP8 | Masson | SAMP8 ||| TNF | IL-1β | SAMP8 | SOD | GSH-Px ||| Nrf2 | Wnt ||| 3,4 | SDC-0-14 | 16 | 3,4 | SDC-1-8) | CF | NRK-52E | CF ||| CF | SAMP8 | Wnt ||| SDC-0-14,16 | SDC-1-8 | CF [SUMMARY]
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[CONTENT] ||| B. L. Burtt | CF | Chinese ||| ||| CF | SAMP8 ||| CF | one month ||| ||| Masson ||| Nrf2 | Wnt ||| NRK-52E | CF ||| ||| CF | SAMP8 | Masson | SAMP8 ||| TNF | IL-1β | SAMP8 | SOD | GSH-Px ||| Nrf2 | Wnt ||| 3,4 | SDC-0-14 | 16 | 3,4 | SDC-1-8) | CF | NRK-52E | CF ||| CF | SAMP8 | Wnt ||| SDC-0-14,16 | SDC-1-8 | CF [SUMMARY]
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The plant coumarins auraptene and lacinartin as potential multifunctional therapeutic agents for treating periodontal disease.
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22742512
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Periodontal diseases are bacterial infections leading to chronic inflammation disorders that are frequently observed in adults. In the present study, we evaluated the effect of auraptene and lacinartin, two natural oxyprenylated coumarins, on the growth, adherence properties, and collagenase activity of Porphyromonas gingivalis. We also investigated the capacity of these compounds to reduce cytokine and matrix metalloproteinase (MMP) secretion by lipopolysaccharide (LPS)-stimulated macrophages and to inhibit MMP-9 activity.
|
BACKGROUND
|
Microplate dilution assays were performed to determine the effect of auraptene and lacinartin on P. gingivalis growth as well as biofilm formation stained with crystal violet. Adhesion of FITC-labeled P. gingivalis to oral epithelial cells was monitored by fluorometry. The effects of auraptene and lacinartin on LPS-induced cytokine and MMP secretion by macrophages were determined by immunological assays. Fluorogenic assays were used to evaluate the capacity of the two coumarins to inhibit the activity of P. gingivalis collagenase and MMP-9.
|
METHODS
|
Only lacinartin completely inhibited P. gingivalis growth in a complex culture medium. However, under iron-limiting conditions, auraptene and lacinartin both inhibited the growth of P. gingivalis. Lacinartin also inhibited biofilm formation by P. gingivalis and promoted biofilm desorption. Both compounds prevented the adherence of P. gingivalis to oral epithelial cells, dose-dependently reduced the secretion of cytokines (IL-8 and TNF-α) and MMP-8 and MMP-9 by LPS-stimulated macrophages, and inhibited MMP-9 activity. Lacinartin also inhibited P. gingivalis collagenase activity.
|
RESULTS
|
By acting on multiple targets, including pathogenic bacteria, tissue-destructive enzymes, and the host inflammatory response, auraptene and lacinartin may be promising natural compounds for preventing and treating periodontal diseases.
|
CONCLUSIONS
|
[
"Bacterial Adhesion",
"Biofilms",
"Cell Line",
"Coumarins",
"Epithelial Cells",
"Humans",
"Macrophages",
"Periodontal Diseases",
"Plant Extracts",
"Porphyromonas gingivalis"
] |
3489859
|
Introduction
|
Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues
[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults
[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens
[3]. Of the over 700 bacterial species that have been identified in the oral cavity
[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction
[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process
[6,7].
Over the past two decades, there has been increasing interest in the potential human health benefits of natural compounds
[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress
[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans
[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.
Auraptene and lacinartin are polyphenols that belong to the coumarin family
[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure
1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.
[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits
[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties
[18,19] while little is known about lacinartin.
Chemical structures of auraptene (A) and lacinartin (B).
To the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.
| null | null |
Results
|
Lacinartin (50 and 100 μg/ml) almost completely inhibited the growth of P. gingivalis in THB-HK (Figure.
2B), while the highest concentration of auraptene tested (100 μg/ml) only reduced growth by 42% (Figure.
2A). Bacterial growth was slightly lower in MBB-H (OD660 = 0.59), which represents an iron poor condition for P. gingivalis, than in THB-HK (OD660 = 0.78) (Figure.
2). The lowest concentration of auraptene tested (12.5 μg/ml) inhibited growth by 58% in MBB-H, while such a concentration had no significant inhibitory effect in THB-HK (Figure.
2A). Almost complete inhibition was observed at higher concentrations (50 and 100 μg/ml). As for auraptene, lacinartin seemed to be more effective for inhibiting growth of P. gingivalis in iron-limiting conditions.
Effect of auraptene (A) and lacinartin (B) on the growth of P. gingivalis in a complex medium (THB-HK) and under iron-limiting conditions in MBB-H. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed using the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).
Auraptene had no obvious inhibitory effect on P. gingivalis biofilm formation or desorption (Figure.
3). On the contrary, high concentrations (50 and 100 μg/ml) of auraptene appear to increase biofilm formation (Figure.
3A). Lacinartin at 50 and 100 μg/ml inhibited biofilm formation by P. gingivalis by approximately 75% (Figure.
3A). In addition, lacinartin (12.5-100 μg/ml) caused approximately one-third of the biofilm to desorb (Figure.
3B).
Effect of auraptene and lacinartin on P. gingivalis biofilm formation (A) and on desorption of a pre-formed P. gingivalis biofilm (B). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).
Auraptene and lacinartin both dose-dependently inhibited bacterial adhesion to oral epithelial cells (Figure.
4). At the lowest concentration tested (12.5 μg/ml), auraptene and lacinartin reduced the adherence of P. gingivalis to epithelial cells by 33% and 43%, respectively, while 100 μg/ml of auraptene and lacinartin reduced adherence by 37% and 71%, respectively (Figure.
4).
Effect of auraptene and lacinartin on the adherence of P. gingivalis to human oral epithelial cells. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. A value of 100% was assigned to the control (no auraptene; no lacinartin). Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).
The highest non-cytotoxic concentrations of auraptene and lacinartin that can be used to evaluate their effect on the inflammatory response of a human macrophage model stimulated with LPS were 25 and 50 μg/ml, respectively (data not shown). Following a 2-h pretreatment of the model with auraptene (6.25, 12.5, and 25 μg/ml) or lacinartin (12.5, 25, and 50 μg/ml), macrophages were stimulated with LPS to induce an inflammatory response (cytokine and MMP secretion). Auraptene and lacinartin both had a significant inhibitory effect on IL-8 and TNF-α secretion. At the lowest concentration tested (6.25 μg/ml), auraptene reduced IL-8 and TNF-α secretion by 22% and 37%, respectively, compared to untreated cells, while at the highest concentration tested (25 μg/ml), it inhibited IL-8 and TNF-α secretion by 92% and 85%, respectively (Figure.
5). As shown in Fig.
5, the highest concentration of lacinartin tested (50 μg/ml) reduced IL-8 and TNF-α secretion by 95% and 99%, respectively, while 12.5 and 25 μg/ml of lacinartin increased the secretion of both cytokines, likely due to a synergistic effect of LPS and lacinartin, since lacinartin alone had no effect (data not shown). Auraptene and lacinartin both reduced MMP-8 and MMP-9 secretion, sometimes below basal levels (Figure.
6). Auraptene reduced MMP-8 secretion by 63% (6.25 μg/ml), 69% (12.5 μg/ml), and 73% (25 μg/ml), while lacinartin reduced MMP-8 secretion by 79% (12.5 μg/ml), 83% (25 μg/ml), and 89% (50 μg/ml) (Figure.
6A). At their highest concentrations tested, auraptene (25 μg/ml) reduced MMP-9 secretion by 76% (25 μg/ml) (Figure.
6B), while lacinartin (50 μg/ml) reduced MMP-9 secretion by 82% (Figure.
6B).
Effect of auraptene and lacinartin on the secretion of IL-8 (A) and TNF-α (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control, †: p ≤ 0.05 vs. control without auraptene or lacinartin).
Effect of auraptene and lacinartin on the secretion of MMP-8 (A) and MMP-9 (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control).
After demonstrating that auraptene and lacinartin can decrease MMP secretion in a macrophage model, we evaluated their effect on proteinase activity. Both auraptene and lacinartin (12.5 μg/ml) reduced MMP-9 activity by 74% (Table
1). Auraptene had no inhibitory effect on P. gingivalis collagenase activity while 12.5 μg/ml and 100 μg/ml of lacinartin reduced collagenase activity by 20% and 64%, respectively (Table
1).
Effect of auraptene and lacinartin on the activity of MMP-9 and
P. gingivalis
collagenase
1Inhibitor of MMP-9: GM6001 (0.025 μM); inhibitor of P. gingivalis collagenase: leupeptin (1 μM).
|
Conclusions
|
In conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity.
|
[
"Introduction",
"Compounds",
"Effect on Porphyromonas gingivalis growth",
"Effect on P. gingivalis biofilm formation/desorption",
"Effect on P. gingivalis adherence to oral epithelial cells",
"Anti-inflammatory properties in a macrophage model",
"Inhibition of MMP-9 and P. gingivalis collagenase activity",
"Statistical analysis",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.",
"Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.",
"P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.",
"P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.",
"P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.",
"U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.",
"Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.",
"Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.",
"The authors declare that they have no competing interests.",
"All authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\n"
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Introduction",
"Materials and methods",
"Compounds",
"Effect on Porphyromonas gingivalis growth",
"Effect on P. gingivalis biofilm formation/desorption",
"Effect on P. gingivalis adherence to oral epithelial cells",
"Anti-inflammatory properties in a macrophage model",
"Inhibition of MMP-9 and P. gingivalis collagenase activity",
"Statistical analysis",
"Results",
"Discussion",
"Conclusions",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] |
[
"Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues\n[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults\n[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens\n[3]. Of the over 700 bacterial species that have been identified in the oral cavity\n[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction\n[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process\n[6,7].\nOver the past two decades, there has been increasing interest in the potential human health benefits of natural compounds\n[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress\n[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans\n[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.\nAuraptene and lacinartin are polyphenols that belong to the coumarin family\n[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure\n1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.\n[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits\n[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties\n[18,19] while little is known about lacinartin. \n Chemical structures of auraptene (A) and lacinartin (B).\nTo the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.",
" Compounds Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\nAuraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.\n Effect on Porphyromonas gingivalis growth P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\nP. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.\n Effect on P. gingivalis biofilm formation/desorption P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\nP. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.\n Effect on P. gingivalis adherence to oral epithelial cells P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\nP. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.\n Anti-inflammatory properties in a macrophage model U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\nU937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.\n Inhibition of MMP-9 and P. gingivalis collagenase activity Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\nHuman recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.\n Statistical analysis Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.\nResults are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.",
"Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure\n[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.",
"P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.",
"P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.",
"P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.",
"U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock\n[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.",
"Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.",
"Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.",
"Lacinartin (50 and 100 μg/ml) almost completely inhibited the growth of P. gingivalis in THB-HK (Figure.\n2B), while the highest concentration of auraptene tested (100 μg/ml) only reduced growth by 42% (Figure.\n2A). Bacterial growth was slightly lower in MBB-H (OD660 = 0.59), which represents an iron poor condition for P. gingivalis, than in THB-HK (OD660 = 0.78) (Figure.\n2). The lowest concentration of auraptene tested (12.5 μg/ml) inhibited growth by 58% in MBB-H, while such a concentration had no significant inhibitory effect in THB-HK (Figure.\n2A). Almost complete inhibition was observed at higher concentrations (50 and 100 μg/ml). As for auraptene, lacinartin seemed to be more effective for inhibiting growth of P. gingivalis in iron-limiting conditions.\n Effect of auraptene (A) and lacinartin (B) on the growth of P. gingivalis in a complex medium (THB-HK) and under iron-limiting conditions in MBB-H. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed using the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene had no obvious inhibitory effect on P. gingivalis biofilm formation or desorption (Figure.\n3). On the contrary, high concentrations (50 and 100 μg/ml) of auraptene appear to increase biofilm formation (Figure.\n3A). Lacinartin at 50 and 100 μg/ml inhibited biofilm formation by P. gingivalis by approximately 75% (Figure.\n3A). In addition, lacinartin (12.5-100 μg/ml) caused approximately one-third of the biofilm to desorb (Figure.\n3B).\n Effect of auraptene and lacinartin on P. gingivalis biofilm formation (A) and on desorption of a pre-formed P. gingivalis biofilm (B). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nAuraptene and lacinartin both dose-dependently inhibited bacterial adhesion to oral epithelial cells (Figure.\n4). At the lowest concentration tested (12.5 μg/ml), auraptene and lacinartin reduced the adherence of P. gingivalis to epithelial cells by 33% and 43%, respectively, while 100 μg/ml of auraptene and lacinartin reduced adherence by 37% and 71%, respectively (Figure.\n4).\n Effect of auraptene and lacinartin on the adherence of P. gingivalis to human oral epithelial cells. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. A value of 100% was assigned to the control (no auraptene; no lacinartin). Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).\nThe highest non-cytotoxic concentrations of auraptene and lacinartin that can be used to evaluate their effect on the inflammatory response of a human macrophage model stimulated with LPS were 25 and 50 μg/ml, respectively (data not shown). Following a 2-h pretreatment of the model with auraptene (6.25, 12.5, and 25 μg/ml) or lacinartin (12.5, 25, and 50 μg/ml), macrophages were stimulated with LPS to induce an inflammatory response (cytokine and MMP secretion). Auraptene and lacinartin both had a significant inhibitory effect on IL-8 and TNF-α secretion. At the lowest concentration tested (6.25 μg/ml), auraptene reduced IL-8 and TNF-α secretion by 22% and 37%, respectively, compared to untreated cells, while at the highest concentration tested (25 μg/ml), it inhibited IL-8 and TNF-α secretion by 92% and 85%, respectively (Figure.\n5). As shown in Fig.\n5, the highest concentration of lacinartin tested (50 μg/ml) reduced IL-8 and TNF-α secretion by 95% and 99%, respectively, while 12.5 and 25 μg/ml of lacinartin increased the secretion of both cytokines, likely due to a synergistic effect of LPS and lacinartin, since lacinartin alone had no effect (data not shown). Auraptene and lacinartin both reduced MMP-8 and MMP-9 secretion, sometimes below basal levels (Figure.\n6). Auraptene reduced MMP-8 secretion by 63% (6.25 μg/ml), 69% (12.5 μg/ml), and 73% (25 μg/ml), while lacinartin reduced MMP-8 secretion by 79% (12.5 μg/ml), 83% (25 μg/ml), and 89% (50 μg/ml) (Figure.\n6A). At their highest concentrations tested, auraptene (25 μg/ml) reduced MMP-9 secretion by 76% (25 μg/ml) (Figure.\n6B), while lacinartin (50 μg/ml) reduced MMP-9 secretion by 82% (Figure.\n6B).\n Effect of auraptene and lacinartin on the secretion of IL-8 (A) and TNF-α (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control, †: p ≤ 0.05 vs. control without auraptene or lacinartin).\n Effect of auraptene and lacinartin on the secretion of MMP-8 (A) and MMP-9 (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control).\nAfter demonstrating that auraptene and lacinartin can decrease MMP secretion in a macrophage model, we evaluated their effect on proteinase activity. Both auraptene and lacinartin (12.5 μg/ml) reduced MMP-9 activity by 74% (Table\n1). Auraptene had no inhibitory effect on P. gingivalis collagenase activity while 12.5 μg/ml and 100 μg/ml of lacinartin reduced collagenase activity by 20% and 64%, respectively (Table\n1).\n\nEffect of auraptene and lacinartin on the activity of MMP-9 and \n\nP. gingivalis\n\n collagenase\n\n1Inhibitor of MMP-9: GM6001 (0.025 μM); inhibitor of P. gingivalis collagenase: leupeptin (1 μM).",
"Periodontal diseases are polymicrobial infections and are the most common chronic inflammatory disorders in adults\n[22]. Periodontitis is induced by a specific group of Gram-negative anaerobic bacteria and is the major cause of tooth loss in adults\n[23]. Over the past two decades, natural compounds with antibacterial and anti-inflammatory properties have received considerable attention as new therapeutic agents for the treatment of periodontal infections. In this study, we investigated the potential of auraptene and lacinartin for preventing and treating periodontal diseases.\nWe first showed that lacinartin and to a lesser extent auraptene reduced P. gingivalis growth. This is the first report indicating that lacinartin possesses anti-bacterial properties. Previous studies have shown that auraptene has antibacterial properties against Helicobacter pylori[24,25]. The exact mechanism by which lacinartin and auraptene inhibit bacterial growth is unknown. However, other natural coumarins (novobiocin and clorobiocin) inhibit deoxyribonuclease gyrase activity, which results in bacteria death\n[14,26]. In addition, we showed that auraptene and lacinartin inhibit growth more effectively under iron-limiting conditions, requiring much lower concentrations to significantly reduce the growth of P. gingivalis. Our results are in agreement with those of Mladnka et al.\n[27], who showed that coumarins possess iron-chelating properties. Additional studies are required to investigate interactions between iron and auraptene and lacinartin.\nWe also showed that lacinartin, but not auraptene, inhibits biofilm formation by P. gingivalis. Lacinartin also caused the desorption of a pre-formed P. gingivalis biofilm. To the best of our knowledge, this is the first report regarding the inhibitory effects of lacinartin on bacterial biofilms. Auraptene and lacinartin prevented the adherence of P. gingivalis to oral epithelial cells to a significant degree. Epithelial cells act as a physical barrier, and bacterial adherence to these host cells may be a critical step for the initiation of periodontal diseases\n[28]. Given its ability to reduce growth of P. gingivalis and its adherence to epithelial cells, lacinartin may be a promising therapeutic candidate through its action on different targets.\nPolyphenols reduce inflammatory mediator secretion and, as such, inflammation-mediated damage\n[29]. We showed that auraptene markedly reduces IL-8 and TNF-α secretion by LPS-stimulated macrophages. Our results are in agreement with those of Genovese et al., who reported that auraptene inhibits the release of TNF-α by RAW 264.7 macrophages\n[30]. To our knowledge, no one has investigated the anti-inflammatory properties of lacinartin. We showed that 12.5 μg/ml of lacinartin induced IL-8 and TNF-α secretion, likely due to a synergistic interaction between LPS and lacinartin. On the other hand, 50 μg/ml of lacinartin significantly inhibited IL-8 and TNF-α secretion. We also showed that auraptene and lacinartin reduced MMP-8 and MMP-9 secretion. These results are in agreement with those of a study by Epifano et al., who reported that auraptene inhibits MMP-7 secretion by HT-29 epithelial cells\n[18]. Since MMP release and cytokine secretion are associated with tooth-supporting tissue destruction, our results suggested that both compounds may contribute to reducing host cell damage, including bone resorption\n[5,31]. The mechanisms by which auraptene and lacinartin reduce inflammatory mediator secretion are unknown, but previous studies have shown that coumarins can block the activation of nuclear factor-κB and inhibit kinase pathways (Akt/PKB)\n[26]. Considering that gingival fibroblasts may also play a significant role in periodontal tissue destruction through cytokine-inducible MMP secretion, future studies should investigate the effects of auraptene and lacinartin on this cell type.\nWe further showed that auraptene and lacinartin reduce MMP-9 activity while only lacinartin inhibits P. gingivalis collagenase activity. Auraptene has previously been shown to inhibit MMP-7 activity\n[32]. These observations suggest that these coumarins may contribute to reducing tissue destruction.",
"In conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity.",
"The authors declare that they have no competing interests.",
"All authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/12/80/prepub\n"
] |
[
null,
"materials|methods",
null,
null,
null,
null,
null,
null,
null,
"results",
"discussion",
"conclusions",
null,
null,
null
] |
[
"Coumarins",
"Auraptene",
"Lacinartin",
"Antibacterial",
"Anti-adherence",
"Anti-inflammatory"
] |
Introduction:
Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues
[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults
[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens
[3]. Of the over 700 bacterial species that have been identified in the oral cavity
[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction
[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process
[6,7].
Over the past two decades, there has been increasing interest in the potential human health benefits of natural compounds
[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress
[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans
[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.
Auraptene and lacinartin are polyphenols that belong to the coumarin family
[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure
1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.
[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits
[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties
[18,19] while little is known about lacinartin.
Chemical structures of auraptene (A) and lacinartin (B).
To the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.
Materials and methods:
Compounds Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure
[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.
Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure
[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.
Effect on Porphyromonas gingivalis growth P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.
P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.
Effect on P. gingivalis biofilm formation/desorption P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.
P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.
Effect on P. gingivalis adherence to oral epithelial cells P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.
P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.
Anti-inflammatory properties in a macrophage model U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock
[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.
U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock
[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.
Inhibition of MMP-9 and P. gingivalis collagenase activity Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.
Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.
Statistical analysis Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.
Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.
Compounds:
Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure
[20]. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.
Effect on Porphyromonas gingivalis growth:
P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.
Effect on P. gingivalis biofilm formation/desorption:
P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.
Effect on P. gingivalis adherence to oral epithelial cells:
P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.
Anti-inflammatory properties in a macrophage model:
U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock
[21]. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.
Inhibition of MMP-9 and P. gingivalis collagenase activity:
Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.
Statistical analysis:
Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.
Results:
Lacinartin (50 and 100 μg/ml) almost completely inhibited the growth of P. gingivalis in THB-HK (Figure.
2B), while the highest concentration of auraptene tested (100 μg/ml) only reduced growth by 42% (Figure.
2A). Bacterial growth was slightly lower in MBB-H (OD660 = 0.59), which represents an iron poor condition for P. gingivalis, than in THB-HK (OD660 = 0.78) (Figure.
2). The lowest concentration of auraptene tested (12.5 μg/ml) inhibited growth by 58% in MBB-H, while such a concentration had no significant inhibitory effect in THB-HK (Figure.
2A). Almost complete inhibition was observed at higher concentrations (50 and 100 μg/ml). As for auraptene, lacinartin seemed to be more effective for inhibiting growth of P. gingivalis in iron-limiting conditions.
Effect of auraptene (A) and lacinartin (B) on the growth of P. gingivalis in a complex medium (THB-HK) and under iron-limiting conditions in MBB-H. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed using the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).
Auraptene had no obvious inhibitory effect on P. gingivalis biofilm formation or desorption (Figure.
3). On the contrary, high concentrations (50 and 100 μg/ml) of auraptene appear to increase biofilm formation (Figure.
3A). Lacinartin at 50 and 100 μg/ml inhibited biofilm formation by P. gingivalis by approximately 75% (Figure.
3A). In addition, lacinartin (12.5-100 μg/ml) caused approximately one-third of the biofilm to desorb (Figure.
3B).
Effect of auraptene and lacinartin on P. gingivalis biofilm formation (A) and on desorption of a pre-formed P. gingivalis biofilm (B). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).
Auraptene and lacinartin both dose-dependently inhibited bacterial adhesion to oral epithelial cells (Figure.
4). At the lowest concentration tested (12.5 μg/ml), auraptene and lacinartin reduced the adherence of P. gingivalis to epithelial cells by 33% and 43%, respectively, while 100 μg/ml of auraptene and lacinartin reduced adherence by 37% and 71%, respectively (Figure.
4).
Effect of auraptene and lacinartin on the adherence of P. gingivalis to human oral epithelial cells. Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. A value of 100% was assigned to the control (no auraptene; no lacinartin). Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. control without auraptene or lacinartin).
The highest non-cytotoxic concentrations of auraptene and lacinartin that can be used to evaluate their effect on the inflammatory response of a human macrophage model stimulated with LPS were 25 and 50 μg/ml, respectively (data not shown). Following a 2-h pretreatment of the model with auraptene (6.25, 12.5, and 25 μg/ml) or lacinartin (12.5, 25, and 50 μg/ml), macrophages were stimulated with LPS to induce an inflammatory response (cytokine and MMP secretion). Auraptene and lacinartin both had a significant inhibitory effect on IL-8 and TNF-α secretion. At the lowest concentration tested (6.25 μg/ml), auraptene reduced IL-8 and TNF-α secretion by 22% and 37%, respectively, compared to untreated cells, while at the highest concentration tested (25 μg/ml), it inhibited IL-8 and TNF-α secretion by 92% and 85%, respectively (Figure.
5). As shown in Fig.
5, the highest concentration of lacinartin tested (50 μg/ml) reduced IL-8 and TNF-α secretion by 95% and 99%, respectively, while 12.5 and 25 μg/ml of lacinartin increased the secretion of both cytokines, likely due to a synergistic effect of LPS and lacinartin, since lacinartin alone had no effect (data not shown). Auraptene and lacinartin both reduced MMP-8 and MMP-9 secretion, sometimes below basal levels (Figure.
6). Auraptene reduced MMP-8 secretion by 63% (6.25 μg/ml), 69% (12.5 μg/ml), and 73% (25 μg/ml), while lacinartin reduced MMP-8 secretion by 79% (12.5 μg/ml), 83% (25 μg/ml), and 89% (50 μg/ml) (Figure.
6A). At their highest concentrations tested, auraptene (25 μg/ml) reduced MMP-9 secretion by 76% (25 μg/ml) (Figure.
6B), while lacinartin (50 μg/ml) reduced MMP-9 secretion by 82% (Figure.
6B).
Effect of auraptene and lacinartin on the secretion of IL-8 (A) and TNF-α (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control, †: p ≤ 0.05 vs. control without auraptene or lacinartin).
Effect of auraptene and lacinartin on the secretion of MMP-8 (A) and MMP-9 (B) by human macrophages stimulated with A. actinomycetemcomitans LPS (1 μg/ml). Values are expressed as means ± standard deviations of triplicate assays for a minimum of three independent experiments. Data were analyzed with the Student’s t-test (*: p ≤ 0.05 vs. untreated control).
After demonstrating that auraptene and lacinartin can decrease MMP secretion in a macrophage model, we evaluated their effect on proteinase activity. Both auraptene and lacinartin (12.5 μg/ml) reduced MMP-9 activity by 74% (Table
1). Auraptene had no inhibitory effect on P. gingivalis collagenase activity while 12.5 μg/ml and 100 μg/ml of lacinartin reduced collagenase activity by 20% and 64%, respectively (Table
1).
Effect of auraptene and lacinartin on the activity of MMP-9 and
P. gingivalis
collagenase
1Inhibitor of MMP-9: GM6001 (0.025 μM); inhibitor of P. gingivalis collagenase: leupeptin (1 μM).
Discussion:
Periodontal diseases are polymicrobial infections and are the most common chronic inflammatory disorders in adults
[22]. Periodontitis is induced by a specific group of Gram-negative anaerobic bacteria and is the major cause of tooth loss in adults
[23]. Over the past two decades, natural compounds with antibacterial and anti-inflammatory properties have received considerable attention as new therapeutic agents for the treatment of periodontal infections. In this study, we investigated the potential of auraptene and lacinartin for preventing and treating periodontal diseases.
We first showed that lacinartin and to a lesser extent auraptene reduced P. gingivalis growth. This is the first report indicating that lacinartin possesses anti-bacterial properties. Previous studies have shown that auraptene has antibacterial properties against Helicobacter pylori[24,25]. The exact mechanism by which lacinartin and auraptene inhibit bacterial growth is unknown. However, other natural coumarins (novobiocin and clorobiocin) inhibit deoxyribonuclease gyrase activity, which results in bacteria death
[14,26]. In addition, we showed that auraptene and lacinartin inhibit growth more effectively under iron-limiting conditions, requiring much lower concentrations to significantly reduce the growth of P. gingivalis. Our results are in agreement with those of Mladnka et al.
[27], who showed that coumarins possess iron-chelating properties. Additional studies are required to investigate interactions between iron and auraptene and lacinartin.
We also showed that lacinartin, but not auraptene, inhibits biofilm formation by P. gingivalis. Lacinartin also caused the desorption of a pre-formed P. gingivalis biofilm. To the best of our knowledge, this is the first report regarding the inhibitory effects of lacinartin on bacterial biofilms. Auraptene and lacinartin prevented the adherence of P. gingivalis to oral epithelial cells to a significant degree. Epithelial cells act as a physical barrier, and bacterial adherence to these host cells may be a critical step for the initiation of periodontal diseases
[28]. Given its ability to reduce growth of P. gingivalis and its adherence to epithelial cells, lacinartin may be a promising therapeutic candidate through its action on different targets.
Polyphenols reduce inflammatory mediator secretion and, as such, inflammation-mediated damage
[29]. We showed that auraptene markedly reduces IL-8 and TNF-α secretion by LPS-stimulated macrophages. Our results are in agreement with those of Genovese et al., who reported that auraptene inhibits the release of TNF-α by RAW 264.7 macrophages
[30]. To our knowledge, no one has investigated the anti-inflammatory properties of lacinartin. We showed that 12.5 μg/ml of lacinartin induced IL-8 and TNF-α secretion, likely due to a synergistic interaction between LPS and lacinartin. On the other hand, 50 μg/ml of lacinartin significantly inhibited IL-8 and TNF-α secretion. We also showed that auraptene and lacinartin reduced MMP-8 and MMP-9 secretion. These results are in agreement with those of a study by Epifano et al., who reported that auraptene inhibits MMP-7 secretion by HT-29 epithelial cells
[18]. Since MMP release and cytokine secretion are associated with tooth-supporting tissue destruction, our results suggested that both compounds may contribute to reducing host cell damage, including bone resorption
[5,31]. The mechanisms by which auraptene and lacinartin reduce inflammatory mediator secretion are unknown, but previous studies have shown that coumarins can block the activation of nuclear factor-κB and inhibit kinase pathways (Akt/PKB)
[26]. Considering that gingival fibroblasts may also play a significant role in periodontal tissue destruction through cytokine-inducible MMP secretion, future studies should investigate the effects of auraptene and lacinartin on this cell type.
We further showed that auraptene and lacinartin reduce MMP-9 activity while only lacinartin inhibits P. gingivalis collagenase activity. Auraptene has previously been shown to inhibit MMP-7 activity
[32]. These observations suggest that these coumarins may contribute to reducing tissue destruction.
Conclusions:
In conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
All authors contributed equally in data acquisition and in writing of the manuscript. All the authors read and approved the final version of the manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1472-6882/12/80/prepub
|
Background:
Periodontal diseases are bacterial infections leading to chronic inflammation disorders that are frequently observed in adults. In the present study, we evaluated the effect of auraptene and lacinartin, two natural oxyprenylated coumarins, on the growth, adherence properties, and collagenase activity of Porphyromonas gingivalis. We also investigated the capacity of these compounds to reduce cytokine and matrix metalloproteinase (MMP) secretion by lipopolysaccharide (LPS)-stimulated macrophages and to inhibit MMP-9 activity.
Methods:
Microplate dilution assays were performed to determine the effect of auraptene and lacinartin on P. gingivalis growth as well as biofilm formation stained with crystal violet. Adhesion of FITC-labeled P. gingivalis to oral epithelial cells was monitored by fluorometry. The effects of auraptene and lacinartin on LPS-induced cytokine and MMP secretion by macrophages were determined by immunological assays. Fluorogenic assays were used to evaluate the capacity of the two coumarins to inhibit the activity of P. gingivalis collagenase and MMP-9.
Results:
Only lacinartin completely inhibited P. gingivalis growth in a complex culture medium. However, under iron-limiting conditions, auraptene and lacinartin both inhibited the growth of P. gingivalis. Lacinartin also inhibited biofilm formation by P. gingivalis and promoted biofilm desorption. Both compounds prevented the adherence of P. gingivalis to oral epithelial cells, dose-dependently reduced the secretion of cytokines (IL-8 and TNF-α) and MMP-8 and MMP-9 by LPS-stimulated macrophages, and inhibited MMP-9 activity. Lacinartin also inhibited P. gingivalis collagenase activity.
Conclusions:
By acting on multiple targets, including pathogenic bacteria, tissue-destructive enzymes, and the host inflammatory response, auraptene and lacinartin may be promising natural compounds for preventing and treating periodontal diseases.
|
Introduction:
Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues
[1]. It is estimated that 5% to 20% of any population suffers from severe, generalized periodontitis, while mild to moderate periodontitis affects a majority of adults
[2]. These diseases are mixed infections induced by a specific group of Gram-negative anaerobic bacteria called periodontopathogens
[3]. Of the over 700 bacterial species that have been identified in the oral cavity
[4] only a few are associated with periodontitis, including Porphyromonas gingivalis[5]. This bacterial species produces a number of virulence factors that contribute to host colonization, immune defense system neutralization, and periodontal tissue destruction
[5]. High numbers of P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process
[6,7].
Over the past two decades, there has been increasing interest in the potential human health benefits of natural compounds
[8]. Polyphenols, which are well known for their antioxidant properties, contribute to the protection of deoxyribonucleic acid (DNA) and macromolecules (lipids and proteins) and can prevent some types of cancers, cardiovascular diseases, and other disorders associated with oxidative stress
[9,10]. These natural compounds are members of a large class of organic molecules that are widely distributed in the plant kingdom and, as such, are an integral part of the daily diet of humans
[11,12]. Since polyphenols have been reported to possess antimicrobial and anti-inflammatory properties, they may be of interest as therapeutic agents for controlling periodontal diseases, which involve both pathogenic bacteria and host immune responses.
Auraptene and lacinartin are polyphenols that belong to the coumarin family
[13,14]. While the chemical structures of these two compounds are similar, lacinartin has a methoxy group on the benzene ring and an isopentenyloxy side chain (Figure
1). Auraptene, which is also known as 7-geranyloxycoumarin, was first isolated in the 1930s by Komatsu et al.
[15]. It is the most abundant naturally occurring prenyloxycoumarin and is mostly found in Citrus fruits
[13,16,17]. Auraptene has been reported to possess antioxidant, anti-inflammatory, antibacterial, and anti-cancer properties
[18,19] while little is known about lacinartin.
Chemical structures of auraptene (A) and lacinartin (B).
To the best of our knowledge, no one has investigated the potential beneficial effects of auraptene and lacinartin on oral health. We hypothesized that auraptene and lacinartin may be promising natural compounds that could be used to prevent and treat periodontal diseases. We thus evaluated the effects of these compounds on the growth, biofilm formation/desorption, and adherence to human oral epithelial cells of P. gingivalis. We also investigated their anti-inflammatory properties using a macrophage model as well as their ability to inhibit MMP-9 and P. gingivalis collagenase.
Conclusions:
In conclusion, our study provided new information on auraptene and lacinartin indicating that they possess an array of interesting antimicrobial, anti-adhesion, anti-inflammatory and anti-protease properties that may be useful for the prevention and treatment of periodontal diseases. Since auraptene and lacinartin act on both etiologic factors of periodontal diseases (periodontopathogens and the host inflammatory response), they may be an alternative to traditional antimicrobials. Further studies are required to investigate the mechanisms of these coumarins, especially the mechanisms involved in their anti-inflammatory activity.
|
Background:
Periodontal diseases are bacterial infections leading to chronic inflammation disorders that are frequently observed in adults. In the present study, we evaluated the effect of auraptene and lacinartin, two natural oxyprenylated coumarins, on the growth, adherence properties, and collagenase activity of Porphyromonas gingivalis. We also investigated the capacity of these compounds to reduce cytokine and matrix metalloproteinase (MMP) secretion by lipopolysaccharide (LPS)-stimulated macrophages and to inhibit MMP-9 activity.
Methods:
Microplate dilution assays were performed to determine the effect of auraptene and lacinartin on P. gingivalis growth as well as biofilm formation stained with crystal violet. Adhesion of FITC-labeled P. gingivalis to oral epithelial cells was monitored by fluorometry. The effects of auraptene and lacinartin on LPS-induced cytokine and MMP secretion by macrophages were determined by immunological assays. Fluorogenic assays were used to evaluate the capacity of the two coumarins to inhibit the activity of P. gingivalis collagenase and MMP-9.
Results:
Only lacinartin completely inhibited P. gingivalis growth in a complex culture medium. However, under iron-limiting conditions, auraptene and lacinartin both inhibited the growth of P. gingivalis. Lacinartin also inhibited biofilm formation by P. gingivalis and promoted biofilm desorption. Both compounds prevented the adherence of P. gingivalis to oral epithelial cells, dose-dependently reduced the secretion of cytokines (IL-8 and TNF-α) and MMP-8 and MMP-9 by LPS-stimulated macrophages, and inhibited MMP-9 activity. Lacinartin also inhibited P. gingivalis collagenase activity.
Conclusions:
By acting on multiple targets, including pathogenic bacteria, tissue-destructive enzymes, and the host inflammatory response, auraptene and lacinartin may be promising natural compounds for preventing and treating periodontal diseases.
| 8,512 | 314 | 15 |
[
"lacinartin",
"auraptene",
"ml",
"auraptene lacinartin",
"gingivalis",
"μg",
"μg ml",
"100",
"cells",
"mmp"
] |
[
"test",
"test"
] | null |
[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]
| null |
[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]
|
[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]
|
[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]
|
[CONTENT] Coumarins | Auraptene | Lacinartin | Antibacterial | Anti-adherence | Anti-inflammatory [SUMMARY]
|
[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]
| null |
[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]
|
[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]
|
[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]
|
[CONTENT] Bacterial Adhesion | Biofilms | Cell Line | Coumarins | Epithelial Cells | Humans | Macrophages | Periodontal Diseases | Plant Extracts | Porphyromonas gingivalis [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]
| null |
[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]
|
[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]
|
[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]
|
[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | μg | μg ml | 100 | cells | mmp [SUMMARY]
|
[CONTENT] diseases | compounds | inflammatory | periodontal | known | immune | properties | anti | natural | polyphenols [SUMMARY]
| null |
[CONTENT] μg | μg ml | ml | figure | lacinartin | secretion | auraptene | reduced | effect | 25 μg ml [SUMMARY]
|
[CONTENT] anti | inflammatory | mechanisms | periodontal | periodontal diseases | diseases | anti inflammatory | protease | properties useful prevention treatment | properties useful prevention [SUMMARY]
|
[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | 100 | μg | μg ml | authors | wells [SUMMARY]
|
[CONTENT] lacinartin | auraptene | ml | auraptene lacinartin | gingivalis | 100 | μg | μg ml | authors | wells [SUMMARY]
|
[CONTENT] ||| lacinartin | two | Porphyromonas ||| [SUMMARY]
| null |
[CONTENT] lacinartin ||| lacinartin ||| Lacinartin ||| TNF | MMP-8 | MMP-9 | LPS ||| Lacinartin [SUMMARY]
|
[CONTENT] lacinartin [SUMMARY]
|
[CONTENT] ||| lacinartin | two | Porphyromonas ||| ||| ||| ||| lacinartin | LPS ||| two | MMP-9 ||| ||| lacinartin ||| Lacinartin ||| TNF | MMP-8 | MMP-9 | LPS ||| Lacinartin ||| lacinartin [SUMMARY]
|
[CONTENT] ||| lacinartin | two | Porphyromonas ||| ||| ||| ||| lacinartin | LPS ||| two | MMP-9 ||| ||| lacinartin ||| Lacinartin ||| TNF | MMP-8 | MMP-9 | LPS ||| Lacinartin ||| lacinartin [SUMMARY]
|
|||||
A pilot prospective study to evaluate whether the bladder morphology in cystography and/or urodynamic may help predict the response to botulinum toxin a injection in neurogenic bladder refractory to anticholinergics.
|
25123234
|
We have observed different clinical responses to botulinum toxin A (BTX-A) in patients who had similar urodynamic parameters before the procedure. Furthermore, some bladders evaluated by cystography and cystoscopy during the procedure had different characteristics that could influence the outcome of the treatment. The aim of this study was to assess whether cystography and urodynamic parameters could help predict which patients with neurogenic detrusor overactivity (NDO) refractory to anticholinergics respond better to treatment with injection of BTX-A.
|
BACKGROUND
|
In total, 34 patients with spinal cord injury were prospectively evaluated. All patients emptied their bladder by clean intermittent catheterization (CIC) and had incontinence and NDO, despite using 40 mg or more of intravesical oxybutynin and undergoing detrusor injection of BTX-A (300 IU). Pretreatment evaluation included urodynamic, and cystography. Follow-up consisted of urodynamic and ambulatory visits four months after treatment. The cystography parameters used were bladder shape, capacity and presence of diverticula. Urodynamic parameters used for assessment were maximum cystometric capacity (MCC), maximum detrusor pressure (MDP), compliance and reflex volume (RV).
|
METHODS
|
After injection of BTX-A, 70% of the patients had success, with 4 months or more of continence. Before the treatment, there were significant differences in most urodynamic parameters between those who responded successfully compared to those who did not. Patients who responded successfully had greater MCC (p = 0.019), higher RV (p = 0.041), and greater compliance (p = 0.043). There was no significant difference in the MDP (0.691). The cystography parameters were not significantly different between these groups bladder shape (p = 0.271), capacity (p > 0.720) and presence of diverticula (p > 0.999). Statistical analyses were performed using SPSS (version 20.0) and included Student's t-test for two paired samples and Fisher's exact test, with a significance threshold of 0.05.
|
RESULTS
|
This study suggests that the cystography parameters evaluated cannot be used to help predict the response to injection of BTX-A in the treatment of refractory NDO. However, the urodynamic parameters were significantly different in patients who responded to the treatment, with the exception of the MDP.
|
CONCLUSIONS
|
[
"Adult",
"Botulinum Toxins, Type A",
"Female",
"Humans",
"Male",
"Mandelic Acids",
"Middle Aged",
"Muscarinic Antagonists",
"Neuromuscular Agents",
"Pilot Projects",
"Prospective Studies",
"Radiography",
"Urinary Bladder",
"Urinary Bladder, Neurogenic",
"Urinary Bladder, Overactive",
"Urodynamics",
"Young Adult"
] |
4139716
|
Background
|
Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.
The treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].
In previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.
|
Methods
|
Thirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7 [8] for two dependent groups (matched pairs). Parameters used in the calculations were as follows: effect size, 0.50; significance probability, 0.05; and power test, 0.80. Of the 34 patients, 23 were male and 11 were female. All methods and definitions were based on the standardization of terminology of lower urinary tract function, as described by Abrams et al. [9]. The study was approved by the ethics committee of Sarah Hospital in Brasilia (CAAE 24188413.3.0000.0022). Informed consent was obtained from all patients participating in the study.The inclusion criteria for the injection of BTX-A were those patients who emptied their bladder by CIC and had urinary incontinence due to hyperactivity refractory to intravesical oxybutynin doses equal or greater than 40 mg. An evaluation was conducted before the procedure and included a clinical history, physical examination, ultrasonography of the kidneys and urinary tract, cystography and urodynamic tests (Multichannel Urodynamics - Medtronic Duet systems, version 8.20, Minneapolis). The following urodynamic parameters were measured: reflex volume (RV), maximum detrusor pressure (MDP), bladder compliance, and maximum cystometric capacity (MCC). For better assessment of compliance, only the study bladders with a capacity ≥ 150 ml were used and compliance was measured five minutes after the end of bladder filling to allow for stabilization of the detrusor pressure. During cystography, the bladder filling was stopped when the maximum capacity had been reached, urinary leakage started, or there was supra pubic discomfort. Outcome was assessed in relation to the bladder shape and capacity and the presence of diverticula. The shape of the bladder was characterized as either “rounded” or “pear-shaped” and/or “pine” (see Figure 1). The cystometric capacity was measured in milliliters. Diverticula were characterized as absent or present, with one group consisting of patients with a diverticula number <10 and another group with a diverticula number ≥ 10 (Figure 1).
The shape of the bladder (“rounded” shape, “pear-shaped” or “pine”).
All procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A (Westport Allergan Pharmaceuticals Ireland - Ireland) was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. [4]. Patients were instructed to continue using the anticholinergic medication and to gradually reduce the dose after the procedure, if it was successful and suspend its use if possible. The treatment was considered successful if the patient remained continent for four months or more, with complete absence of urinary leak, regardless of the use of the anticholinergic medication. Clinical and urodynamic control assessments were performed 4 months after treatment. Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test with a significance threshold of 0.05. Before each t-test, the hypothesis of equality of variances was checked using Levene’s test. Differences between the groups were compared with the Mann–Whitney U test for two independent samples.
|
Results
|
Of the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366) (Table 1). There were significant differences in most of the pretreatment urodynamic parameters in patients with a successful response compared with those with an unsuccessful response. The MCC (p = 0.019), RV (0.041) and compliance (p = 0.043) were higher in patients with a successful response. The MDP was not significantly different between groups (p = 0.691) (Table 2).
Urodynamic assessment before and after botulinum toxin injection
*Paired, two-sided Student’s t-test.
Urodynamic parameters before botulinum toxin injection
*Mann–Whitney U Test for two independent samples.
Regarding cystography, before the procedure, 24 patients (70%) had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) (Table 3).
Results and cystography parameters
*Mann–Whitney U Test for two independent samples.
After 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 of these patients were able to decrease the dose of the anticholinergic drugs after the procedure (Table 5).
Anticholinergic use after botulinum toxin injection (Total)
Anticholinergic use after botulinum toxin injection (success)
|
Conclusion
|
This study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work.
|
[
"Background",
"Abbreviations",
"Competing interests",
"Author’s contributions",
"Pre-publication history"
] |
[
"Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.",
"NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.",
"The authors declare that they have no competing interests.",
"RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\n"
] |
[
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Results",
"Discussion",
"Conclusion",
"Abbreviations",
"Competing interests",
"Author’s contributions",
"Pre-publication history"
] |
[
"Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.\nThe treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].\nIn previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.",
"Thirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7 [8] for two dependent groups (matched pairs). Parameters used in the calculations were as follows: effect size, 0.50; significance probability, 0.05; and power test, 0.80. Of the 34 patients, 23 were male and 11 were female. All methods and definitions were based on the standardization of terminology of lower urinary tract function, as described by Abrams et al. [9]. The study was approved by the ethics committee of Sarah Hospital in Brasilia (CAAE 24188413.3.0000.0022). Informed consent was obtained from all patients participating in the study.The inclusion criteria for the injection of BTX-A were those patients who emptied their bladder by CIC and had urinary incontinence due to hyperactivity refractory to intravesical oxybutynin doses equal or greater than 40 mg. An evaluation was conducted before the procedure and included a clinical history, physical examination, ultrasonography of the kidneys and urinary tract, cystography and urodynamic tests (Multichannel Urodynamics - Medtronic Duet systems, version 8.20, Minneapolis). The following urodynamic parameters were measured: reflex volume (RV), maximum detrusor pressure (MDP), bladder compliance, and maximum cystometric capacity (MCC). For better assessment of compliance, only the study bladders with a capacity ≥ 150 ml were used and compliance was measured five minutes after the end of bladder filling to allow for stabilization of the detrusor pressure. During cystography, the bladder filling was stopped when the maximum capacity had been reached, urinary leakage started, or there was supra pubic discomfort. Outcome was assessed in relation to the bladder shape and capacity and the presence of diverticula. The shape of the bladder was characterized as either “rounded” or “pear-shaped” and/or “pine” (see Figure 1). The cystometric capacity was measured in milliliters. Diverticula were characterized as absent or present, with one group consisting of patients with a diverticula number <10 and another group with a diverticula number ≥ 10 (Figure 1).\nThe shape of the bladder (“rounded” shape, “pear-shaped” or “pine”).\nAll procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A (Westport Allergan Pharmaceuticals Ireland - Ireland) was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. [4]. Patients were instructed to continue using the anticholinergic medication and to gradually reduce the dose after the procedure, if it was successful and suspend its use if possible. The treatment was considered successful if the patient remained continent for four months or more, with complete absence of urinary leak, regardless of the use of the anticholinergic medication. Clinical and urodynamic control assessments were performed 4 months after treatment. Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test with a significance threshold of 0.05. Before each t-test, the hypothesis of equality of variances was checked using Levene’s test. Differences between the groups were compared with the Mann–Whitney U test for two independent samples.",
"Of the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366) (Table 1). There were significant differences in most of the pretreatment urodynamic parameters in patients with a successful response compared with those with an unsuccessful response. The MCC (p = 0.019), RV (0.041) and compliance (p = 0.043) were higher in patients with a successful response. The MDP was not significantly different between groups (p = 0.691) (Table 2).\nUrodynamic assessment before and after botulinum toxin injection\n*Paired, two-sided Student’s t-test.\nUrodynamic parameters before botulinum toxin injection\n*Mann–Whitney U Test for two independent samples.\nRegarding cystography, before the procedure, 24 patients (70%) had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) (Table 3).\nResults and cystography parameters\n*Mann–Whitney U Test for two independent samples.\nAfter 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 of these patients were able to decrease the dose of the anticholinergic drugs after the procedure (Table 5).\nAnticholinergic use after botulinum toxin injection (Total)\nAnticholinergic use after botulinum toxin injection (success)",
"BTX-A injections into the detrusor muscle provide a clinically significant improvement in patients with NDO refractory to anticholinergics and are very well tolerated (5;6). In this study, continence was observed over a period of more than 4 months in 70% of the patients undergoing treatment with BTX-A. In studies with similar populations of patients, the percentage of patients with continence after injection of the toxin ranged from 42 to 87% [10]. Karsenty et al. reported that anticholinergic agents may be discontinued in 28% to 58% of patients after treatment with BTX-A and the dose can be substantially reduced in the remaining patients [10]. In this study, after 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) discontinued the use and nine (26.5%) did not change the dose of medication (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 patients were able to decrease the dose of anticholinergic drugs after the procedure (Table 5). We note that some of these patients did not reduce the dose because they were afraid that the urinary losses would return after being continent. According to previous studies, some factors that may be related to the efficacy of BTX-A injections into the detrusor muscle include, for example, whether the doses of anticholinergics used before the procedure were considered high (refractory bladder) [7], what the optimal dose of BTX-A is [11,12], the formulations used [13,14] and the injection technique [11]. These factors may explain some of the differences observed in the results across different studies. The results obtained in this study were similar to earlier studies. Among patients presenting with NDO that is refractory to anticholinergic agents, there was a percentage with unsuccessful results.\nAlthough it has been reported that alterations can occur in the detrusor muscle of the NDO [15,16], it was postulated that the morphology of the bladder (shape and presence of diverticula) could be a result of these alterations, and could, in turn, influence the response to BTX-A injections. These changes in bladder shape are most likely the result of smooth muscle hypertrophy and changes in the connective tissue matrix that do not respond to conservative treatment. However, the present study did not demonstrate that the cystography parameters could predict which cases would be more likely to have a better response to BTX-A injections.\nRegarding urodynamic parameters, we observed in this study that the bladders that had better compliance, greater capacity and increased reflex volume before treatment showed a better response to treatment. Furthermore, there was no significant change in compliance after surgery (p = 0.366), suggesting that compliance is related to alterations in the bladder wall, is unresponsive to drug therapy, and is, therefore, directly related to the treatment response. This is in contrast to a study conducted by Klaphajone J [17], in which a small number of patients did not show this relationship.",
"This study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work.",
"NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.",
"The authors declare that they have no competing interests.",
"RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2490/14/66/prepub\n"
] |
[
null,
"methods",
"results",
"discussion",
"conclusions",
null,
null,
null,
null
] |
[
"Neurogenic detrusor overactivity",
"Botulinum toxin A",
"Cystography",
"Urodynamic",
"Neurogenic bladder"
] |
Background:
Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.
The treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].
In previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.
Methods:
Thirty-four patients with spinal cord injury who received injections of BTX-A between January 2012 and July 2013 participated of this prospective observational study. The sample size was determined by G*Power software, version 3.1.7 [8] for two dependent groups (matched pairs). Parameters used in the calculations were as follows: effect size, 0.50; significance probability, 0.05; and power test, 0.80. Of the 34 patients, 23 were male and 11 were female. All methods and definitions were based on the standardization of terminology of lower urinary tract function, as described by Abrams et al. [9]. The study was approved by the ethics committee of Sarah Hospital in Brasilia (CAAE 24188413.3.0000.0022). Informed consent was obtained from all patients participating in the study.The inclusion criteria for the injection of BTX-A were those patients who emptied their bladder by CIC and had urinary incontinence due to hyperactivity refractory to intravesical oxybutynin doses equal or greater than 40 mg. An evaluation was conducted before the procedure and included a clinical history, physical examination, ultrasonography of the kidneys and urinary tract, cystography and urodynamic tests (Multichannel Urodynamics - Medtronic Duet systems, version 8.20, Minneapolis). The following urodynamic parameters were measured: reflex volume (RV), maximum detrusor pressure (MDP), bladder compliance, and maximum cystometric capacity (MCC). For better assessment of compliance, only the study bladders with a capacity ≥ 150 ml were used and compliance was measured five minutes after the end of bladder filling to allow for stabilization of the detrusor pressure. During cystography, the bladder filling was stopped when the maximum capacity had been reached, urinary leakage started, or there was supra pubic discomfort. Outcome was assessed in relation to the bladder shape and capacity and the presence of diverticula. The shape of the bladder was characterized as either “rounded” or “pear-shaped” and/or “pine” (see Figure 1). The cystometric capacity was measured in milliliters. Diverticula were characterized as absent or present, with one group consisting of patients with a diverticula number <10 and another group with a diverticula number ≥ 10 (Figure 1).
The shape of the bladder (“rounded” shape, “pear-shaped” or “pine”).
All procedures were performed in a hospital under general anesthesia. Antibiotics were administered orally, according to a urine culture, for seven days. The procedure performed on the fifth day of the antibiotic, all patients tested positive for bacteria. BTX- A (Westport Allergan Pharmaceuticals Ireland - Ireland) was diluted in sterile saline to a final concentration of 10 units/ml. Using a 19-Fr Storz cystoscope and 5 FR needle, a total of 300 IU (30 ml) was injected into 30 sites of the detrusor muscle, sparing the trigone region, as described in Schurch et al. [4]. Patients were instructed to continue using the anticholinergic medication and to gradually reduce the dose after the procedure, if it was successful and suspend its use if possible. The treatment was considered successful if the patient remained continent for four months or more, with complete absence of urinary leak, regardless of the use of the anticholinergic medication. Clinical and urodynamic control assessments were performed 4 months after treatment. Statistical analyses were performed using SPSS (version 20.0) and included Student’s t-test for two paired samples and Fisher’s exact test with a significance threshold of 0.05. Before each t-test, the hypothesis of equality of variances was checked using Levene’s test. Differences between the groups were compared with the Mann–Whitney U test for two independent samples.
Results:
Of the 34 patients, 23 were male and 11 were female. The mean age was 31.2 ± 10.3 years (mean ± standard deviation), with a range 19–55 years. There were 28 paraplegic and 6 tetraplegic patients and 25 traumatic and 9 non-traumatic cases. The mean time since SCI was 5.9 ± 4.4 years, with a maximum of 19 years and a minimum of 1 year. The patients tolerated all of the procedures and showed no acute complications related to the injections. Thirty patients (88.2%) were completely continent after the procedure. Six of these patients, however, showed little clinical response at four months and were considered unsuccessful. Four patients (11.8%) remained incontinent four months after treatment, although there were improvements in most urodynamic parameters and urinary losses. Twenty-four (70%) patients remained continent for more than 4 months and were considered successful. Twelve patients (35.2%) had an acontractile detrusor bladder after surgery. After four months, the following changes in the urodynamic parameters were observed: increased MCC (P <0.001), increased RV (p <0.001) and decreased MDP (p <0.001). The compliance did not change significantly (p = 0.366) (Table 1). There were significant differences in most of the pretreatment urodynamic parameters in patients with a successful response compared with those with an unsuccessful response. The MCC (p = 0.019), RV (0.041) and compliance (p = 0.043) were higher in patients with a successful response. The MDP was not significantly different between groups (p = 0.691) (Table 2).
Urodynamic assessment before and after botulinum toxin injection
*Paired, two-sided Student’s t-test.
Urodynamic parameters before botulinum toxin injection
*Mann–Whitney U Test for two independent samples.
Regarding cystography, before the procedure, 24 patients (70%) had no diverticula, eight patients had between 1 and 10 diverticula and 2 patients had more than 10 diverticula. By Fisher’s exact test, there was no association between the response and the presence of diverticula (p > 0.999), bladder shape (p <0.271) or bladder capacity (p = 0.720) (Table 3).
Results and cystography parameters
*Mann–Whitney U Test for two independent samples.
After 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) had discontinued their use and nine (26.5%) had not changed the dose (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 of these patients were able to decrease the dose of the anticholinergic drugs after the procedure (Table 5).
Anticholinergic use after botulinum toxin injection (Total)
Anticholinergic use after botulinum toxin injection (success)
Discussion:
BTX-A injections into the detrusor muscle provide a clinically significant improvement in patients with NDO refractory to anticholinergics and are very well tolerated (5;6). In this study, continence was observed over a period of more than 4 months in 70% of the patients undergoing treatment with BTX-A. In studies with similar populations of patients, the percentage of patients with continence after injection of the toxin ranged from 42 to 87% [10]. Karsenty et al. reported that anticholinergic agents may be discontinued in 28% to 58% of patients after treatment with BTX-A and the dose can be substantially reduced in the remaining patients [10]. In this study, after 4 months of follow-up, twenty patients (58.8%) had reduced the dose of anticholinergics, five (14.7%) discontinued the use and nine (26.5%) did not change the dose of medication (Table 4). Among the 24 patients with successful results, only 5 had discontinued the use of anticholinergics. However, 16 patients were able to decrease the dose of anticholinergic drugs after the procedure (Table 5). We note that some of these patients did not reduce the dose because they were afraid that the urinary losses would return after being continent. According to previous studies, some factors that may be related to the efficacy of BTX-A injections into the detrusor muscle include, for example, whether the doses of anticholinergics used before the procedure were considered high (refractory bladder) [7], what the optimal dose of BTX-A is [11,12], the formulations used [13,14] and the injection technique [11]. These factors may explain some of the differences observed in the results across different studies. The results obtained in this study were similar to earlier studies. Among patients presenting with NDO that is refractory to anticholinergic agents, there was a percentage with unsuccessful results.
Although it has been reported that alterations can occur in the detrusor muscle of the NDO [15,16], it was postulated that the morphology of the bladder (shape and presence of diverticula) could be a result of these alterations, and could, in turn, influence the response to BTX-A injections. These changes in bladder shape are most likely the result of smooth muscle hypertrophy and changes in the connective tissue matrix that do not respond to conservative treatment. However, the present study did not demonstrate that the cystography parameters could predict which cases would be more likely to have a better response to BTX-A injections.
Regarding urodynamic parameters, we observed in this study that the bladders that had better compliance, greater capacity and increased reflex volume before treatment showed a better response to treatment. Furthermore, there was no significant change in compliance after surgery (p = 0.366), suggesting that compliance is related to alterations in the bladder wall, is unresponsive to drug therapy, and is, therefore, directly related to the treatment response. This is in contrast to a study conducted by Klaphajone J [17], in which a small number of patients did not show this relationship.
Conclusion:
This study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work.
Abbreviations:
NDO: Neurogenic detrusor overactivity; BTX-A: Botulinum toxin A; BTX: Botulinum toxin; CIC: Clean intermittent catheterization; RV: Reflex volume; MDP: Maximum detrusor pressure; MCC: Maximum cystometric capacity.
Competing interests:
The authors declare that they have no competing interests.
Author’s contributions:
RAA conceived of the study and carried out the acquisition, analysis and interpretation of data and was involved in drafting the manuscript. IDA provided substantial contributions to the conception and design and revised the manuscript critically for important intellectual content. MDS participated in the study design and coordination. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2490/14/66/prepub
|
Background:
We have observed different clinical responses to botulinum toxin A (BTX-A) in patients who had similar urodynamic parameters before the procedure. Furthermore, some bladders evaluated by cystography and cystoscopy during the procedure had different characteristics that could influence the outcome of the treatment. The aim of this study was to assess whether cystography and urodynamic parameters could help predict which patients with neurogenic detrusor overactivity (NDO) refractory to anticholinergics respond better to treatment with injection of BTX-A.
Methods:
In total, 34 patients with spinal cord injury were prospectively evaluated. All patients emptied their bladder by clean intermittent catheterization (CIC) and had incontinence and NDO, despite using 40 mg or more of intravesical oxybutynin and undergoing detrusor injection of BTX-A (300 IU). Pretreatment evaluation included urodynamic, and cystography. Follow-up consisted of urodynamic and ambulatory visits four months after treatment. The cystography parameters used were bladder shape, capacity and presence of diverticula. Urodynamic parameters used for assessment were maximum cystometric capacity (MCC), maximum detrusor pressure (MDP), compliance and reflex volume (RV).
Results:
After injection of BTX-A, 70% of the patients had success, with 4 months or more of continence. Before the treatment, there were significant differences in most urodynamic parameters between those who responded successfully compared to those who did not. Patients who responded successfully had greater MCC (p = 0.019), higher RV (p = 0.041), and greater compliance (p = 0.043). There was no significant difference in the MDP (0.691). The cystography parameters were not significantly different between these groups bladder shape (p = 0.271), capacity (p > 0.720) and presence of diverticula (p > 0.999). Statistical analyses were performed using SPSS (version 20.0) and included Student's t-test for two paired samples and Fisher's exact test, with a significance threshold of 0.05.
Conclusions:
This study suggests that the cystography parameters evaluated cannot be used to help predict the response to injection of BTX-A in the treatment of refractory NDO. However, the urodynamic parameters were significantly different in patients who responded to the treatment, with the exception of the MDP.
|
Background:
Botulinum toxin (BTX), which was described by Van Ermengem [1] in 1897, exists as serotypes A, B, C, D, E, F and G [2]. Currently, serotypes A and B are available for clinical use. When injected into the muscle, BTX causes flaccid paralysis by inhibiting acetylcholine release at the presynaptic cholinergic junction. This effect is transient and dose-related. In smooth muscle, it was shown by Smith et al. [3] that BTX-A affects the release of acetylcholine and norepinephrine in the bladder and urethra, respectively.
The treatment of neurogenic detrusor overactivity (NDO) with an injection of BTX-A into the detrusor muscle was introduced in 2000 [4]. This therapy is a minimally invasive treatment option, and, although more invasive than oral treatment with anticholinergic, is less invasive than surgery [4]. Its safety and efficacy has been confirmed in a randomized, placebo-controlled clinical trial [5]. Some studies have evaluated the use of BTX-A injections in the detrusor muscle of patients with spinal cord injury to reduce NDO, increase bladder capacity, reduce incontinence and improve the quality of life of these patients [5,6].
In previous studies, we have observed different clinical responses to BTX-A in patients who had similar urodynamic parameters before the procedure [7]. This study was motivated by the observation that some bladders evaluated by cystography and cystoscopy during the procedure revealed different characteristics that could influence the outcome of the treatment. With respect to the morphology observed in cystography, we investigated whether differences in diverticula, shape, and capacity could influence the results of treatment. Similarly, we assessed whether the urodynamic parameters evaluated before the procedure could help predict the results of detrusor BTX-A injection for the treatment of NDO refractory to anticholinergics.
Conclusion:
This study suggests that the cystography parameters evaluated cannot be used to predict the response to BTX-A injection for the treatment of refractory NDO. It was observed in the urodynamic parameters, that patients whose bladders had higher cystometric capacity, greater reflex volume and greater compliance showed better results after treatment. The bladder compliance showed no significant improvement after the procedure, which is an important factor to be noted. A larger study with a multivariate analysis would be appropriate to clarify the results of this work.
|
Background:
We have observed different clinical responses to botulinum toxin A (BTX-A) in patients who had similar urodynamic parameters before the procedure. Furthermore, some bladders evaluated by cystography and cystoscopy during the procedure had different characteristics that could influence the outcome of the treatment. The aim of this study was to assess whether cystography and urodynamic parameters could help predict which patients with neurogenic detrusor overactivity (NDO) refractory to anticholinergics respond better to treatment with injection of BTX-A.
Methods:
In total, 34 patients with spinal cord injury were prospectively evaluated. All patients emptied their bladder by clean intermittent catheterization (CIC) and had incontinence and NDO, despite using 40 mg or more of intravesical oxybutynin and undergoing detrusor injection of BTX-A (300 IU). Pretreatment evaluation included urodynamic, and cystography. Follow-up consisted of urodynamic and ambulatory visits four months after treatment. The cystography parameters used were bladder shape, capacity and presence of diverticula. Urodynamic parameters used for assessment were maximum cystometric capacity (MCC), maximum detrusor pressure (MDP), compliance and reflex volume (RV).
Results:
After injection of BTX-A, 70% of the patients had success, with 4 months or more of continence. Before the treatment, there were significant differences in most urodynamic parameters between those who responded successfully compared to those who did not. Patients who responded successfully had greater MCC (p = 0.019), higher RV (p = 0.041), and greater compliance (p = 0.043). There was no significant difference in the MDP (0.691). The cystography parameters were not significantly different between these groups bladder shape (p = 0.271), capacity (p > 0.720) and presence of diverticula (p > 0.999). Statistical analyses were performed using SPSS (version 20.0) and included Student's t-test for two paired samples and Fisher's exact test, with a significance threshold of 0.05.
Conclusions:
This study suggests that the cystography parameters evaluated cannot be used to help predict the response to injection of BTX-A in the treatment of refractory NDO. However, the urodynamic parameters were significantly different in patients who responded to the treatment, with the exception of the MDP.
| 2,504 | 430 | 9 |
[
"patients",
"btx",
"treatment",
"bladder",
"study",
"parameters",
"detrusor",
"urodynamic",
"procedure",
"capacity"
] |
[
"test",
"test"
] |
[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]
|
[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]
|
[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]
|
[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]
|
[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]
|
[CONTENT] Neurogenic detrusor overactivity | Botulinum toxin A | Cystography | Urodynamic | Neurogenic bladder [SUMMARY]
|
[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]
|
[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]
|
[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]
|
[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]
|
[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]
|
[CONTENT] Adult | Botulinum Toxins, Type A | Female | Humans | Male | Mandelic Acids | Middle Aged | Muscarinic Antagonists | Neuromuscular Agents | Pilot Projects | Prospective Studies | Radiography | Urinary Bladder | Urinary Bladder, Neurogenic | Urinary Bladder, Overactive | Urodynamics | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]
|
[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]
|
[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]
|
[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]
|
[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]
|
[CONTENT] patients | btx | treatment | bladder | study | parameters | detrusor | urodynamic | procedure | capacity [SUMMARY]
|
[CONTENT] btx | treatment | invasive | muscle | evaluated | detrusor | clinical | release | acetylcholine | serotypes [SUMMARY]
|
[CONTENT] test | patients | bladder | performed | urinary | measured | ml | version | capacity | diverticula [SUMMARY]
|
[CONTENT] patients | table | toxin injection | years | botulinum toxin injection | response | months | test | 001 | mean [SUMMARY]
|
[CONTENT] compliance showed | showed | greater | compliance | results | treatment | study | parameters | analysis appropriate clarify results | bladders higher cystometric capacity [SUMMARY]
|
[CONTENT] patients | btx | treatment | study | authors | detrusor | bladder | authors declare competing interests | competing interests | declare competing interests [SUMMARY]
|
[CONTENT] patients | btx | treatment | study | authors | detrusor | bladder | authors declare competing interests | competing interests | declare competing interests [SUMMARY]
|
[CONTENT] ||| ||| NDO | BTX-A. [SUMMARY]
|
[CONTENT] 34 ||| CIC | NDO | 40 | BTX-A ||| ||| four months ||| ||| MDP | RV [SUMMARY]
|
[CONTENT] BTX-A | 70% | 4 months ||| ||| MCC | 0.019 | RV | 0.041 | 0.043 ||| MDP | 0.691 ||| 0.271 | 0.720 | 0.999 ||| SPSS | 20.0 | Student | two | Fisher | 0.05 [SUMMARY]
|
[CONTENT] BTX-A | NDO ||| MDP [SUMMARY]
|
[CONTENT] ||| ||| NDO | BTX-A. Methods | 34 ||| CIC | NDO | 40 | BTX-A ||| ||| four months ||| ||| MDP | RV ||| ||| BTX-A | 70% | 4 months ||| ||| MCC | 0.019 | RV | 0.041 | 0.043 ||| MDP | 0.691 ||| 0.271 | 0.720 | 0.999 ||| SPSS | 20.0 | Student | two | Fisher | 0.05 ||| BTX-A | NDO ||| MDP [SUMMARY]
|
[CONTENT] ||| ||| NDO | BTX-A. Methods | 34 ||| CIC | NDO | 40 | BTX-A ||| ||| four months ||| ||| MDP | RV ||| ||| BTX-A | 70% | 4 months ||| ||| MCC | 0.019 | RV | 0.041 | 0.043 ||| MDP | 0.691 ||| 0.271 | 0.720 | 0.999 ||| SPSS | 20.0 | Student | two | Fisher | 0.05 ||| BTX-A | NDO ||| MDP [SUMMARY]
|
||||||
Efficacy and safety of risedronate 150-mg once a month in the treatment of postmenopausal osteoporosis: 2-year data.
|
22752050
|
Risedronate is effective in the treatment of postmenopausal osteoporosis in oral daily, weekly, or on two consecutive days per month doses. This 2-year randomized, double-blind, multicenter study assesses the efficacy and safety of a single risedronate 150-mg once-a-month oral dose compared with the 5-mg daily regimen.
|
INTRODUCTION
|
Women with postmenopausal osteoporosis were randomly assigned to receive risedronate 5-mg daily (n = 642) or 150-mg once a month (n = 650) for 2 years. Bone mineral density (BMD), bone turnover markers, new vertebral fractures, and adverse events were evaluated. The primary efficacy endpoint was the mean percent change from baseline in lumbar spine BMD after 1 year.
|
METHODS
|
Four hundred ninety-eight subjects in the daily group (77.6 %) and 513 subjects in the once-a-month group (78.9 %) completed the study. After 24 months, the mean percent change in lumbar spine BMD was 3.9 % (95 % confidence interval [CI], 3.43 to 4.42 %) and 4.2 % (95 % CI, 3.68 to 4.65 %) in the daily and once-a-month groups, respectively. The once-a-month regimen was determined to be non-inferior to the daily regimen. The mean percent changes in BMD at the hip were similar in both dose groups, as were changes in biochemical markers of bone turnover. The incidence of adverse events, adverse events leading to withdrawal, and upper gastrointestinal tract adverse events were similar in the two treatment groups.
|
RESULTS
|
After 2 years, treatment with risedronate 150-mg once a month provided similar efficacy and tolerability to daily dosing and provides an alternative for patients who prefer once-a-month oral dosing.
|
CONCLUSIONS
|
[
"Administration, Oral",
"Aged",
"Biomarkers",
"Bone Density",
"Bone Density Conservation Agents",
"Double-Blind Method",
"Drug Administration Schedule",
"Etidronic Acid",
"Female",
"Femur",
"Humans",
"Lumbar Vertebrae",
"Middle Aged",
"Osteoporosis, Postmenopausal",
"Risedronic Acid",
"Treatment Outcome"
] |
3536944
|
Introduction
|
Risedronate is a pyridinyl bisphosphonate that has been shown in prospective studies to reduce the risk of vertebral, nonvertebral, and hip fractures [1–3]. Like other bisphosphonates, risedronate remains active on the surface of bone for long periods after dosing, providing the opportunity to develop a range of dosing schedules.
The original risedronate dosing regimen for postmenopausal osteoporosis was an oral dose of 5-mg daily [1–3]. It was later demonstrated that risedronate 35-mg once a week and 75-mg each day for two consecutive days a month provided similar efficacy and safety to the daily regimen [4, 5]. The efficacy and tolerability of risedronate once-a-month dosing (150-mg) was compared with risedronate daily dosing (5-mg) in women with osteoporosis with changes in lumbar spine bone mineral density (BMD) as the primary endpoint. After 1 year of treatment, published previously, the efficacy of risedronate 150-mg once-a-month regimen was non-inferior to the 5-mg daily regimen [6]. The once-a-month regimen also had a similar tolerability profile as the daily regimen after 1 year of treatment. This study continued for an additional year of treatment, and the results of the complete study over 2 years are presented here.
| null | null |
Results
|
Subjects From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density
Disposition of subjects. BMD bone mineral density
From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density
Disposition of subjects. BMD bone mineral density
Efficacy assessments The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month
Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month
There was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.
Significant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)
Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)
The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month
Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month
There was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.
Significant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)
Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)
Safety assessments Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)
n (%)
n (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa
172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb
23 (3.6)25 (3.8)
aIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain
bIncludes benign and malignant neoplasms, polyps, and cysts
AE adverse event
Summary of adverse events
aIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain
bIncludes benign and malignant neoplasms, polyps, and cysts
AE adverse event
Adverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.
Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)
n (%)
n (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa
172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb
23 (3.6)25 (3.8)
aIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain
bIncludes benign and malignant neoplasms, polyps, and cysts
AE adverse event
Summary of adverse events
aIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain
bIncludes benign and malignant neoplasms, polyps, and cysts
AE adverse event
Adverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.
| null | null |
[
"Study design",
"Patients",
"Treatments",
"Efficacy assessments",
"Safety assessments",
"Statistical analysis",
"Subjects",
"Efficacy assessments",
"Safety assessments"
] |
[
"This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.",
"Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].",
"Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.",
"Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.",
"Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).",
"The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.",
"From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density",
"The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)",
"Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Introduction",
"Materials and methods",
"Study design",
"Patients",
"Treatments",
"Efficacy assessments",
"Safety assessments",
"Statistical analysis",
"Results",
"Subjects",
"Efficacy assessments",
"Safety assessments",
"Discussion"
] |
[
"Risedronate is a pyridinyl bisphosphonate that has been shown in prospective studies to reduce the risk of vertebral, nonvertebral, and hip fractures [1–3]. Like other bisphosphonates, risedronate remains active on the surface of bone for long periods after dosing, providing the opportunity to develop a range of dosing schedules.\nThe original risedronate dosing regimen for postmenopausal osteoporosis was an oral dose of 5-mg daily [1–3]. It was later demonstrated that risedronate 35-mg once a week and 75-mg each day for two consecutive days a month provided similar efficacy and safety to the daily regimen [4, 5]. The efficacy and tolerability of risedronate once-a-month dosing (150-mg) was compared with risedronate daily dosing (5-mg) in women with osteoporosis with changes in lumbar spine bone mineral density (BMD) as the primary endpoint. After 1 year of treatment, published previously, the efficacy of risedronate 150-mg once-a-month regimen was non-inferior to the 5-mg daily regimen [6]. The once-a-month regimen also had a similar tolerability profile as the daily regimen after 1 year of treatment. This study continued for an additional year of treatment, and the results of the complete study over 2 years are presented here.",
" Study design This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\nThis randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.\n Patients Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\nEligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].\n Treatments Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\nSubjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.\n Efficacy assessments Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\nDual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.\n Safety assessments Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\nPhysical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).\n Statistical analysis The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.\nThe primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.",
"This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.",
"Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].",
"Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.",
"Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.",
"Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).",
"The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].\nSecondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.\nAfter 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.\nThe treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.",
" Subjects From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\nFrom the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density\n Efficacy assessments The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\nThe within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n Safety assessments Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.\nOverall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.",
"From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density\n\nDisposition of subjects. BMD bone mineral density",
"The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\n\nMean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month\nThere was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.\nSignificant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)\n\nMean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)",
"Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)\nn (%)\nn (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa\n172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb\n23 (3.6)25 (3.8)\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\nbIncludes benign and malignant neoplasms, polyps, and cysts\nAE adverse event\n\nSummary of adverse events\n\naIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain\n\nbIncludes benign and malignant neoplasms, polyps, and cysts\n\nAE adverse event\nAdverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.",
"Risedronate is a widely used osteoporosis treatment with proven vertebral and nonvertebral antifracture efficacy and a minimum wait of 30 min after dosing before eating or drinking anything other than water. A 5-mg daily regimen was developed originally, but less frequent dose regimens have now been developed. This study was a preplanned 2-year study comparing a dose of risedronate 150-mg once a month to the 5-mg daily dose. These 2-year data show that the 150-mg once-a-month dose continues to produce clinical effects that are similar to those seen with the 5-mg daily dose. Specifically, the mean percent change in lumbar spine BMD at 24 months in the monthly group was non-inferior to the mean percent change in lumbar spine BMD in the daily group. Changes in secondary efficacy parameters, including BMD at the hip, bone turnover markers at endpoint, and morphometric vertebral fractures, were also similar in both groups. Statistically significant differences between treatment groups were observed for all three bone turnover markers at month 3, but persisted at months 6 and 12 for CTX only. No statistically significant differences between groups were observed for any marker at month 24 or endpoint. That the CTX response did not differ between treatment groups at month 24 might be explained by the small number of subjects at month 24 that would limit statistical power to observe difference. It is not likely that these small differences between groups in bone turnover markers are clinically meaningful.\nThe risedronate 150-mg once-a-month dose was well tolerated over 2 years, with a safety profile similar to that seen with the 5-mg daily regimen. The low incidences of subjects with vertebral and nonvertebral clinical fractures were similar between groups and consistent with rates previously observed with the 5-mg daily dose [1–3].\nChange in BMD is an appropriate endpoint when evaluating a new dosing schedule of a bisphosphonate for which a fracture benefit has already been established. Similar non-inferiority trials have been conducted previously to evaluate new dosing regimens of oral bisphosphonates [4, 8, 9], and this approach has been accepted by both the US Food and Drug Administration and the European Medicines Agency [10] for approval of new regimens of established agents. The magnitude of BMD change associated with the vertebral and nonvertebral antifracture efficacy of risedronate has been established in multiple large studies that had fracture as the primary endpoint [1–3]. This study has demonstrated that the 150-mg once-a-month dose reduces bone turnover and increases BMD to a degree comparable to that observed with the 5-mg daily dose in these fracture studies.\nThe results of this study after 2 years are consistent with the findings at month 12 [6], demonstrating the persistent similarity between risedronate 150-mg once-a-month and the 5-mg daily dosing regimens. Additionally, these results are consistent with the favorable tolerability and efficacy profiles observed in large placebo-controlled clinical trials of the risedronate 5-mg daily regimen [1–3]. The findings are also consistent with previous studies of less frequent dosing with risedronate. Such studies showed that the treatment effects of risedronate 35-mg weekly and 75-mg on two consecutive days each month were similar to the effects of daily dosing [4, 5].\nRisedronate 150-mg once a month, taken for 2 years, is similar in efficacy and tolerability to the 5-mg daily dosing regimen that had been proven to reduce the incidence of vertebral and nonvertebral fractures. The addition of this dosing regimen to the therapeutic armamentarium will provide women with postmenopausal osteoporosis a full range of risedronate oral dosing options, from daily to weekly to monthly."
] |
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"introduction",
"materials|methods",
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"results",
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"discussion"
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[
"Bone mineral density",
"Fracture risk",
"Monthly",
"Osteoporosis",
"Risedronate"
] |
Introduction:
Risedronate is a pyridinyl bisphosphonate that has been shown in prospective studies to reduce the risk of vertebral, nonvertebral, and hip fractures [1–3]. Like other bisphosphonates, risedronate remains active on the surface of bone for long periods after dosing, providing the opportunity to develop a range of dosing schedules.
The original risedronate dosing regimen for postmenopausal osteoporosis was an oral dose of 5-mg daily [1–3]. It was later demonstrated that risedronate 35-mg once a week and 75-mg each day for two consecutive days a month provided similar efficacy and safety to the daily regimen [4, 5]. The efficacy and tolerability of risedronate once-a-month dosing (150-mg) was compared with risedronate daily dosing (5-mg) in women with osteoporosis with changes in lumbar spine bone mineral density (BMD) as the primary endpoint. After 1 year of treatment, published previously, the efficacy of risedronate 150-mg once-a-month regimen was non-inferior to the 5-mg daily regimen [6]. The once-a-month regimen also had a similar tolerability profile as the daily regimen after 1 year of treatment. This study continued for an additional year of treatment, and the results of the complete study over 2 years are presented here.
Materials and methods:
Study design This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.
This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.
Patients Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].
Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].
Treatments Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.
Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.
Efficacy assessments Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.
Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.
Safety assessments Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).
Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).
Statistical analysis The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].
Secondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.
After 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.
The treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.
The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].
Secondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.
After 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.
The treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.
Study design:
This randomized, double-blind, active-controlled, parallel-group non-inferiority study was conducted at 47 study centers in the Americas, Europe, Australia, and Asia (Appendix). The first subject was screened in October 2005, and the last subject observation took place in March 2008. The study was performed in accordance with good clinical practice and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the appropriate institutional review boards or ethics committees, and the subjects gave written, informed consent to participate.
Patients:
Eligible subjects who gave consent were randomly assigned in a 1:1 ratio to the two treatment groups. Women were eligible to enroll in the study if they were at least 50 years of age, ambulatory, in generally good health, postmenopausal (at least 5 years since last menses), had at least three vertebral bodies in the lumbar spine (L1 to L4) that were evaluable by densitometry (i.e., without fracture or degenerative disease), and had a lumbar spine BMD T-score of less than −2.5 or a T-score of less than −2.0 with at least one prevalent vertebral fracture (T4 to L4). Specific details of the inclusion criteria and methods have been previously published [6].
Treatments:
Subjects received oral risedronate 5-mg daily or 150-mg once a month (i.e., a single 150-mg tablet on the same calendar day each month, followed by a placebo tablet daily for the rest of the month). All tablets were identical in appearance and supplied in identical blister cards. Tablets were taken on an empty stomach in the morning at least 30 min before the first food or drink of the day, with at least 4 oz of plain water. Subjects were instructed to remain in an upright position for at least 30 min after dosing. Subjects were considered compliant if they took at least 80 % of the study tablets. Calcium (1,000-mg/day) and vitamin D (400–500 IU/day) were supplied to all subjects, although they were allowed to take up to 1,000 IU/day of vitamin D. These supplements were taken with a meal other than breakfast and not with the study medication.
Efficacy assessments:
Dual x-ray absorptiometry (DXA) measurements of the lumbar spine and proximal femur were obtained at baseline and after 6, 12, and 24 months using instruments manufactured by Lunar Corporation (General Electric, Madison, WI, USA) or Hologic (Waltham, MA, USA). DXA scans collected at the clinical sites were sent to a central facility for quality control and analysis (Synarc, Copenhagen/Hamburg). Lateral thoracic and lumbar spine radiographs collected at screening and at 12 and 24 months were analyzed for vertebral fractures by semi-quantitative analysis [7] at a central radiology site (Synarc, Copenhagen/Hamburg). Biochemical markers of bone turnover were assessed at 3, 6, 12, and 24 months. Serum bone-specific alkaline phosphatase (BALP) was measured using an immunochemiluminescence assay on an automatic analyzer (Ostase, Access, Beckman Coulter, LaBrea, CA, USA). The intra- and interassay coefficients of variation for this measurement were less than 4 and 10 %, respectively. The detection limit of the test was 0.07 ng/mL, and the limit of quantitation was 0.28 ng/mL. Urinary N-terminal crosslinking telopeptide of type I collagen (NTX) was measured with an electrochemiluminescent immunoassay on an automated machine (Vitros ECi, Johnson and Johnson, Rochester, NY, USA). The intra- and interassay coefficients of variation were below 7 and 6 %, respectively. The detection limit of the test was 4 nM, and the limit of quantitation was 22 nM. This measurement was corrected for creatinine (NTX/Cr). Serum C-terminal crosslinking telopeptide of type I collagen (CTX) was measured using an enzyme immunoassay kit (Serum CrossLaps®, Nordic Bioscience Diagnostics, Herlev, Denmark). The intra- and interassay coefficients of variation were below 8 and 6 %, respectively. The lower limit of detection was 0.044 ng/mL. Bone turnover marker assays were performed at a central laboratory (Synarc SAS, Lyon, France). The samples for the 24-month study visit were measured at a different time than the samples for all previous visits.
Safety assessments:
Physical examinations were performed at baseline and after 12 and 24 months. Vital signs, concomitant medications, and adverse event reports were recorded at regular clinic visits throughout the study. Adverse event reports were captured using the Medical Dictionary for Regulatory Activities (MedDRA) system. Blood and urine samples for clinical chemistry and other standard laboratory measurements were collected at baseline and after 3, 6, 9, 12, 18, and 24 months of treatment. Specimens were analyzed by Quintiles Laboratories (Smyrna, GA, USA).
Statistical analysis:
The primary endpoint analysis was a test of non-inferiority comparing the least squares mean percent change from baseline in lumbar spine BMD in the 150-mg once-a-month and 5-mg daily groups after 12 months. This test employed a predefined non-inferiority margin of 1.5 % and a one-sided type I error of 2.5 %. The results of this analysis have been published previously [6].
Secondary endpoints included the percent change from baseline in lumbar spine BMD at months 6 and 24, and at endpoint; the percent change from baseline in BMD of the total proximal femur, femoral neck, and femoral trochanter at months 6, 12, and 24, and at endpoint; the percentage of patients with new vertebral fractures at year 1 and 2; and the percent change from baseline in biochemical markers of bone turnover (NTX/Cr, CTX, and BALP) at months 3, 6, 12, and 24, and at endpoint. All data reported here are based upon cumulative data collected over the entire 2-year treatment period.
After 2 years of treatment, a non-inferiority analysis was performed based on the one-sided 97.5 % confidence interval (CI) for the difference in mean percent change from baseline to month 24 in lumbar spine BMD. The CIs were constructed using an ANOVA model with fixed effects for treatment and pooled investigative center. If the upper bound of the 97.5 % one-sided CI did not exceed 2.0 %, then the once-a-month treatment was considered non-inferior to the daily treatment. This analysis was based on all subjects who were randomized and took at least one dose of study medication and who had evaluable lumbar spine BMD measurements at both baseline and at least one postbaseline time point (last observation carried forward; this is the intent-to-treat [ITT] population). The month 24 non-inferiority “delta” was selected using the same rationale used to select the month 12 non-inferiority margin. In previous studies contrasting risedronate 5-mg daily and placebo for the treatment of postmenopausal osteoporosis, the mean percent change difference between the treatment groups in lumbar spine BMD from baseline to month 24 ranged from 4.1 to 5.4 %. Thus, using a “delta” of 2.0 % would maintain approximately 50 % of the effect size of the risedronate 5-mg daily dose relative to placebo at month 24.
The treatment group differences at month 24 in percent changes in proximal femur BMD and bone turnover markers were analyzed using an ANOVA model; two-sided 95 % CIs for the treatment differences were constructed using the ITT population. The incidence of new vertebral fractures over 24 months was analyzed using Fisher's exact test. Adverse events were summarized as frequency distribution tables and reported by treatment group.
Results:
Subjects From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density
Disposition of subjects. BMD bone mineral density
From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density
Disposition of subjects. BMD bone mineral density
Efficacy assessments The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month
Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month
There was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.
Significant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)
Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)
The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month
Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month
There was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.
Significant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)
Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)
Safety assessments Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)
n (%)
n (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa
172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb
23 (3.6)25 (3.8)
aIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain
bIncludes benign and malignant neoplasms, polyps, and cysts
AE adverse event
Summary of adverse events
aIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain
bIncludes benign and malignant neoplasms, polyps, and cysts
AE adverse event
Adverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.
Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)
n (%)
n (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa
172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb
23 (3.6)25 (3.8)
aIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain
bIncludes benign and malignant neoplasms, polyps, and cysts
AE adverse event
Summary of adverse events
aIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain
bIncludes benign and malignant neoplasms, polyps, and cysts
AE adverse event
Adverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.
Subjects:
From the total of 2,221 women who were screened, 1,294 subjects were randomized, and 1,292 subjects received at least one dose of study drug (Fig. 1). Overall, baseline characteristics were similar in both treatment groups. Demographics of the subjects in each treatment group have been reported previously [6]. A similar percentage of subjects in each treatment group completed 24 months of the study (5-mg daily group, 77.6 %; 150-mg once-a-month group, 78.9 %). The most common reasons given for withdrawal during year 2 were adverse event and voluntary withdrawal, which occurred at similar incidences in both treatment groups. A high percentage of subjects in both groups (95.5 % of subjects in the 5-mg daily group and 95.7 % of subjects in the 150-mg once-a-month group) took at least 80 % of the study tablets.Fig. 1Disposition of subjects. BMD bone mineral density
Disposition of subjects. BMD bone mineral density
Efficacy assessments:
The within-group mean percent changes from baseline in lumbar spine BMD were statistically significant in both treatment groups at each time point (Fig. 2). The mean percent changes at 24 months (95 % CI) were 3.9 % (3.43 to 4.42 %) for the 5-mg daily group and 4.2 % (3.68 to 4.65 %) for the 150-mg once-a-month group. The difference from the 5-mg daily group (daily minus once a month) in mean percent change from baseline in lumbar spine BMD at month 24 was –0.24 % (95 % upper confidence bound, 0.25 %). This upper boundary was well below the 2.0 % needed to establish non-inferiority; thus, the 150-mg once-a-month regimen was determined to be non-inferior to the 5-mg daily regimen at 24 months. Significant increases from baseline in BMD were observed at 6, 12, and 24 months in both treatment groups (Fig. 2). There was no statistically significant difference between treatment groups in mean percent change in BMD at the lumbar spine or regions of the proximal femur (total proximal femur, femoral neck, and femoral trochanter) at any time point.Fig. 2Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month
Mean percent change (±SEM) from baseline in bone mineral density in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. There were no statistically significant differences between treatment groups at any time point at any of these sites. OAM once a month
There was no difference between treatment groups in the occurrence of new incident vertebral fracture as determined by morphometric measurement during the study; 14 subjects (2.5 %) in the 5-mg daily group and 15 subjects (2.6 %) in the 150-mg once-a-month group experienced such a fracture.
Significant decreases from baseline in NTX/Cr, CTX, and BALP were observed at 3, 6, 12, and 24 months in both treatment groups (Fig. 3). In general, changes from baseline in these biochemical markers were similar in both treatment groups. The small difference in CTX between groups was statistically significant at months 3, 6, and 12 but not at month 24. There was no statistically significant difference between treatment groups at endpoint (the last-observation-carried forward values at month 24) for any of the biochemical markers of bone turnover.Fig. 3Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)
Mean percent change (±SEM) from baseline in biochemical markers of bone turnover in women receiving risedronate 5-mg daily (dashed line with triangles) or 150-mg once a month (solid line with circles). Endpoint refers to the value calculated using the last observation carried forward at month 24. CTX C-terminal crosslinking telopeptide of type I collagen, NTX N-terminal crosslinking telopeptide of type I collagen, OAM once a month. *p < 0.05 indicates a statistically significant difference between treatment groups (unadjusted for multiple comparisons)
Safety assessments:
Overall, the frequency of adverse events was similar in both treatment groups (Table 1). Among the most common adverse events, only diarrhea and influenza were more frequent in the monthly group compared with the daily group after 24 months. The difference between groups in these adverse events was primarily driven by events reported during the first few months of the study. Most events of diarrhea were mild or moderate in severity. One subject (0.2 %) in the 5-mg daily group and six subjects (0.9 %) in the 150-mg once-a-month group withdrew from the study as a result of diarrhea. All events of influenza were mild or moderate in severity, most occurred more than 90 days after the start of treatment, and none of the subjects withdrew because of influenza. More patients in the 150-mg once-a-month group reported serious adverse events than in the 5-mg daily group (Table 1). This difference was predominantly due to small differences between the two groups in several MedDRA System Organ Class categories, and none of the differences in any of the individual System Organ Class categories reached statistical significance.Table 1Summary of adverse eventsRisedronate5-mg daily150-mg once a month(N = 642)(N = 650)
n (%)
n (%)AEs554 (86.3)578 (88.9)Serious AEs51 (7.9)77 (11.8)Deaths4 (0.6)0Withdrawn due to an AE84 (13.1)80 (12.3)Most common AE associated with withdrawal Gastrointestinal disorder49 (7.6)47 (7.2)Most common AEs Influenza57 (8.9)94 (14.5) Nasopharyngitis62 (9.7)70 (10.8) Diarrhea43 (6.7)69 (10.6) Arthralgia68 (10.6)65 (10.0) Back pain80 (12.5)65 (10.0) Bronchitis68 (10.6)57 (8.8)AEs of special interest Clinical vertebral fracture6 (0.9)4 (0.6) Nonvertebral fracture25 (3.9)28 (4.3) Upper gastrointestinal tract AEs148 (23.1)169 (26.0) Selected musculoskeletal AEsa
172 (26.8)163 (25.1) Atrial fibrillation1 (0.2)3 (0.5) Neoplasmsb
23 (3.6)25 (3.8)
aIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain
bIncludes benign and malignant neoplasms, polyps, and cysts
AE adverse event
Summary of adverse events
aIncludes arthralgia, back pain, bone pain, musculoskeletal pain, musculoskeletal discomfort, myalgia, and neck pain
bIncludes benign and malignant neoplasms, polyps, and cysts
AE adverse event
Adverse events of special interest for bisphosphonates (clinical vertebral and nonvertebral fractures, upper gastrointestinal tract adverse events, and musculoskeletal adverse events) were reported by similar proportions of subjects in both treatment groups (Table 1). The incidence of atrial fibrillation reported as either an adverse event or a serious adverse event was low and similar between groups (Table 1). There were no reported cases of osteonecrosis of the jaw. The number of subjects who developed a neoplasm did not differ by treatment group (Table 1). Results of clinical chemistry and other laboratory measurements, including measures of hepatic and renal function, were similar in both treatment groups.
Discussion:
Risedronate is a widely used osteoporosis treatment with proven vertebral and nonvertebral antifracture efficacy and a minimum wait of 30 min after dosing before eating or drinking anything other than water. A 5-mg daily regimen was developed originally, but less frequent dose regimens have now been developed. This study was a preplanned 2-year study comparing a dose of risedronate 150-mg once a month to the 5-mg daily dose. These 2-year data show that the 150-mg once-a-month dose continues to produce clinical effects that are similar to those seen with the 5-mg daily dose. Specifically, the mean percent change in lumbar spine BMD at 24 months in the monthly group was non-inferior to the mean percent change in lumbar spine BMD in the daily group. Changes in secondary efficacy parameters, including BMD at the hip, bone turnover markers at endpoint, and morphometric vertebral fractures, were also similar in both groups. Statistically significant differences between treatment groups were observed for all three bone turnover markers at month 3, but persisted at months 6 and 12 for CTX only. No statistically significant differences between groups were observed for any marker at month 24 or endpoint. That the CTX response did not differ between treatment groups at month 24 might be explained by the small number of subjects at month 24 that would limit statistical power to observe difference. It is not likely that these small differences between groups in bone turnover markers are clinically meaningful.
The risedronate 150-mg once-a-month dose was well tolerated over 2 years, with a safety profile similar to that seen with the 5-mg daily regimen. The low incidences of subjects with vertebral and nonvertebral clinical fractures were similar between groups and consistent with rates previously observed with the 5-mg daily dose [1–3].
Change in BMD is an appropriate endpoint when evaluating a new dosing schedule of a bisphosphonate for which a fracture benefit has already been established. Similar non-inferiority trials have been conducted previously to evaluate new dosing regimens of oral bisphosphonates [4, 8, 9], and this approach has been accepted by both the US Food and Drug Administration and the European Medicines Agency [10] for approval of new regimens of established agents. The magnitude of BMD change associated with the vertebral and nonvertebral antifracture efficacy of risedronate has been established in multiple large studies that had fracture as the primary endpoint [1–3]. This study has demonstrated that the 150-mg once-a-month dose reduces bone turnover and increases BMD to a degree comparable to that observed with the 5-mg daily dose in these fracture studies.
The results of this study after 2 years are consistent with the findings at month 12 [6], demonstrating the persistent similarity between risedronate 150-mg once-a-month and the 5-mg daily dosing regimens. Additionally, these results are consistent with the favorable tolerability and efficacy profiles observed in large placebo-controlled clinical trials of the risedronate 5-mg daily regimen [1–3]. The findings are also consistent with previous studies of less frequent dosing with risedronate. Such studies showed that the treatment effects of risedronate 35-mg weekly and 75-mg on two consecutive days each month were similar to the effects of daily dosing [4, 5].
Risedronate 150-mg once a month, taken for 2 years, is similar in efficacy and tolerability to the 5-mg daily dosing regimen that had been proven to reduce the incidence of vertebral and nonvertebral fractures. The addition of this dosing regimen to the therapeutic armamentarium will provide women with postmenopausal osteoporosis a full range of risedronate oral dosing options, from daily to weekly to monthly.
|
Background:
Risedronate is effective in the treatment of postmenopausal osteoporosis in oral daily, weekly, or on two consecutive days per month doses. This 2-year randomized, double-blind, multicenter study assesses the efficacy and safety of a single risedronate 150-mg once-a-month oral dose compared with the 5-mg daily regimen.
Methods:
Women with postmenopausal osteoporosis were randomly assigned to receive risedronate 5-mg daily (n = 642) or 150-mg once a month (n = 650) for 2 years. Bone mineral density (BMD), bone turnover markers, new vertebral fractures, and adverse events were evaluated. The primary efficacy endpoint was the mean percent change from baseline in lumbar spine BMD after 1 year.
Results:
Four hundred ninety-eight subjects in the daily group (77.6 %) and 513 subjects in the once-a-month group (78.9 %) completed the study. After 24 months, the mean percent change in lumbar spine BMD was 3.9 % (95 % confidence interval [CI], 3.43 to 4.42 %) and 4.2 % (95 % CI, 3.68 to 4.65 %) in the daily and once-a-month groups, respectively. The once-a-month regimen was determined to be non-inferior to the daily regimen. The mean percent changes in BMD at the hip were similar in both dose groups, as were changes in biochemical markers of bone turnover. The incidence of adverse events, adverse events leading to withdrawal, and upper gastrointestinal tract adverse events were similar in the two treatment groups.
Conclusions:
After 2 years, treatment with risedronate 150-mg once a month provided similar efficacy and tolerability to daily dosing and provides an alternative for patients who prefer once-a-month oral dosing.
| null | null | 10,390 | 365 | 13 |
[
"month",
"mg",
"treatment",
"24",
"groups",
"daily",
"subjects",
"group",
"baseline",
"mg daily"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] Bone mineral density | Fracture risk | Monthly | Osteoporosis | Risedronate [SUMMARY]
| null |
[CONTENT] Bone mineral density | Fracture risk | Monthly | Osteoporosis | Risedronate [SUMMARY]
| null |
[CONTENT] Bone mineral density | Fracture risk | Monthly | Osteoporosis | Risedronate [SUMMARY]
| null |
[CONTENT] Administration, Oral | Aged | Biomarkers | Bone Density | Bone Density Conservation Agents | Double-Blind Method | Drug Administration Schedule | Etidronic Acid | Female | Femur | Humans | Lumbar Vertebrae | Middle Aged | Osteoporosis, Postmenopausal | Risedronic Acid | Treatment Outcome [SUMMARY]
| null |
[CONTENT] Administration, Oral | Aged | Biomarkers | Bone Density | Bone Density Conservation Agents | Double-Blind Method | Drug Administration Schedule | Etidronic Acid | Female | Femur | Humans | Lumbar Vertebrae | Middle Aged | Osteoporosis, Postmenopausal | Risedronic Acid | Treatment Outcome [SUMMARY]
| null |
[CONTENT] Administration, Oral | Aged | Biomarkers | Bone Density | Bone Density Conservation Agents | Double-Blind Method | Drug Administration Schedule | Etidronic Acid | Female | Femur | Humans | Lumbar Vertebrae | Middle Aged | Osteoporosis, Postmenopausal | Risedronic Acid | Treatment Outcome [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] month | mg | treatment | 24 | groups | daily | subjects | group | baseline | mg daily [SUMMARY]
| null |
[CONTENT] month | mg | treatment | 24 | groups | daily | subjects | group | baseline | mg daily [SUMMARY]
| null |
[CONTENT] month | mg | treatment | 24 | groups | daily | subjects | group | baseline | mg daily [SUMMARY]
| null |
[CONTENT] regimen | risedronate | dosing | mg | year treatment | daily | efficacy | daily regimen | tolerability | year [SUMMARY]
| null |
[CONTENT] mg | groups | month | group | treatment | adverse | treatment groups | significant | events | line [SUMMARY]
| null |
[CONTENT] mg | month | treatment | 24 | daily | subjects | groups | group | baseline | adverse [SUMMARY]
| null |
[CONTENT] daily | two consecutive days per month ||| 2-year | 150 | 5 | daily [SUMMARY]
| null |
[CONTENT] Four hundred ninety-eight | daily | 77.6 % | 513 | 78.9 % ||| 24 months | the mean percent | BMD | 3.9 % | 95 % | CI | 3.43 | 4.42 % | 4.2 % | 95 % | CI | 3.68 | 4.65 % | daily ||| daily ||| The mean percent | BMD ||| two [SUMMARY]
| null |
[CONTENT] daily | two consecutive days per month ||| 2-year | 150 | 5 | daily ||| 5 | daily | 642 | 150 | 650 | 2 years ||| BMD ||| the mean percent | BMD | 1 year ||| Four hundred ninety-eight | daily | 77.6 % | 513 | 78.9 % ||| 24 months | the mean percent | BMD | 3.9 % | 95 % | CI | 3.43 | 4.42 % | 4.2 % | 95 % | CI | 3.68 | 4.65 % | daily ||| daily ||| The mean percent | BMD ||| two ||| 2 years | 150 | daily [SUMMARY]
| null |
|||
Survival Trend of Tuberculosis Patients and Risk Factors Associated with Mortality and Developing Drug-Resistant Tuberculosis in Hospital Pulau Pinang, Malaysia: A Retrospective Study.
|
36412638
|
Multidrug resistance TB (MDR-TB) has emerged as a public health issue worldwide, and the mortality rate is worrying. Therefore, this study was conducted to investigate the factors related to MDR-TB occurrence and the survival experience of TB patients.
|
BACKGROUND
|
A retrospective cohort study was conducted at Hospital Pulau Pinang in Malaysia. Medical records of active TB patients from 2014-2018 were reviewed. Cox regression was used to identify the factors associated with MDR-TB development and mortality among TB patients.
|
METHODS
|
The patients had a mean age of 48.84 ± 16.713 years, and a majority of the Chinese race (46.4%). Out of 351 TB patients, 325 (92.6%) were drug-susceptible TB, and 26 (7.4%) were diagnosed with MDR-TB. Among drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, and 73 (22.5%) passed away. In multivariable Cox regression, drug addiction, levels of white blood cells, urea, platelets, and albumin were significantly associated with death. Relapsed TB, alcohol consumption, and being single were significant risk factors for MDR-TB development.
|
RESULTS
|
Patients achieved a success rate of 75.4%, which is encouraging but still far below the WHO target (at least an 85% success rate) and has room for further improvement.
|
CONCLUSION
|
[
"Humans",
"Adult",
"Middle Aged",
"Aged",
"Retrospective Studies",
"Antitubercular Agents",
"Malaysia",
"Tuberculosis, Multidrug-Resistant",
"Risk Factors",
"Hospitals"
] |
9774739
|
1. Introduction
|
Tuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients.
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3. Results
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3.1. Sociodemographic and Clinical Characteristics At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).
At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).
3.2. Drug Resistant Patterns of TB Patients The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).
The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).
3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).
The observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.
In univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).
In multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).
Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).
The observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.
In univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).
In multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).
3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).
In univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).
In multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).
Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).
In univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).
In multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).
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5. Conclusions
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Eventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single.
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[
"2. Materials and Methods",
"2.1. Study Design and Population",
"2.2. Diagnosis and Treatment",
"2.3. Data Collection",
"2.4. Statistical Analysis",
"3.1. Sociodemographic and Clinical Characteristics",
"3.2. Drug Resistant Patterns of TB Patients",
"3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB"
] |
[
" 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. ",
"A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. ",
"Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. ",
"Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. ",
"To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. ",
"At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).",
"The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).",
"Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10)."
] |
[
null,
null,
null,
null,
null,
null,
"subjects",
null
] |
[
"1. Introduction",
"2. Materials and Methods",
"2.1. Study Design and Population",
"2.2. Diagnosis and Treatment",
"2.3. Data Collection",
"2.4. Statistical Analysis",
"3. Results",
"3.1. Sociodemographic and Clinical Characteristics",
"3.2. Drug Resistant Patterns of TB Patients",
"3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients",
"3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB",
"4. Discussion",
"5. Conclusions"
] |
[
"Tuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients.",
" 2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \nA retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. \n 2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \nPatients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. \n 2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \nData of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. \n 2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. \nTo analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. ",
"A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).\nSample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis. ",
"Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7]. \nThe diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB. \nEPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism. \nPatients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen. \nIn Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR). \nTreatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1. ",
"Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns. \nPatients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2. ",
"To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables. ",
" 3.1. Sociodemographic and Clinical Characteristics At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\nAt the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).\n 3.2. Drug Resistant Patterns of TB Patients The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\nThe study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).\n 3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\nOf 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).\n 3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).\nAmong the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).",
"At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).",
"The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).",
"Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).\nThe observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.\nIn univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).\nIn multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).",
"Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).\nIn univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).\nIn multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).",
"Our research looked into the factors that contribute to mortality among TB patients, as well as the factors associated with MDR-TB development. Since then, tuberculosis treatment has been problematic and difficult to apply properly because of long duration and compliance issues due to anti-tubercular drugs’ side effects. Some of the side effects are self-limiting, but others necessitate treatment discontinuation. Although rare, thrombocytopenia is one of the most common and serious life-threatening side effects. Thrombocytopenia is caused by the formation of antibodies that bind to platelets and initiate the complement cascade, causing these cells to lyse. These antibodies also suppress the production of platelets [8]. This study demonstrated that thrombocytopenia significantly increased the risk of mortality among TB patients. [9] reported a low platelet count as a significant risk factor for mortality.\nMortality among patients suffering from any infection has been linked to WBC count [10]. As a result, elevated WBC counts in TB patients may be related to the severity of lung involvement and provide an estimated risk of mortality [11]. During TB infection, the body produces leukocytes and macrophages as part of the immune mechanism to defend the body against invading Mycobacterium tuberculosis, which raises the total number of WBC [12]. The current study findings documented that every 103 cells/µL increment in WBC above the normal range increased the risk of death by 1.102. In line with our results, a study conducted in Malaysia pointed out that the risk of death increased by 1.12 times for every 103 cells per microliter unit increase in total WBC [13]. Other studies have indicated that WBC levels are a significant risk factor correlated with TB mortality [11,14]. Pneumonia ranked the second cause of death among the study population. The WBC count elevation has been reported as a predictor of the severity of pneumonia, one of the leading causes of death in TB patients [11].\nThe present findings indicated that the level of urea was a strong significant predictor of death. Increased levels of urea were found to be significantly associated with mortality [11]. We can relate elevated urea levels to kidney dysfunction, where renal clearance is reduced due to renal impairment/failure or chronic kidney disease (CKD). Urea levels may also increase in other conditions, such as upper GI bleeding [15]. As the prevalence of CKD in our cohort was 5.69%, where 20 patients developed CKD while receiving anti-tuberculosis treatment, further research is needed to determine the relationship between CKD and TB. Strategies for the prevention, detection, and treatment of TB-CKD cannot be refined without advanced understanding.\nHypoalbuminemia showed a statistically significant relationship with death; patients who had hypoalbuminemia were more likely to pass away than patients who did not. Matching our results, lower albumin levels showed an association with non-survivors (p< 0.001) compared to survivors. Low levels of albumin were found to be significantly correlated with the hazard of death [9,16]. Furthermore, hypoalbuminemia was considered a predictor of mortality in community-acquired pneumonia (CAP), which is not surprising considering that albumin is involved in inflammation and indicates malnutrition [17]. The initial value of serum albumin might also be a result of malnutrition or an underlying disease that can worsen the nutritional status of the patient [18]. As a result of malnutrition, deceased patients’ immune systems were suppressed and were therefore sicker upon initial presentation.\nAs for drug addiction characteristics, the findings of this study revealed that patients who had a history of addiction or were current addicts had a 1.895 times higher risk of death than those who had never abused drugs. In line with our findings, deceased patients were more likely to abuse heroin IV than living patients, and addiction was correlated with all causes of death in tuberculosis patients [19,20]. Supporting our results, a history of addiction was a statistically significant risk factor for death within the first year after diagnosis in Hong Kong, Russia, and Iran [21,22,23]. Poor adherence of drug users, default treatment, and poor access to health services can create challenges for the treatment of the drug addict population [24]. As drug users have a higher likelihood of default treatment than non-drug users, the increased hazard of death can be attributed to the increased prevalence of alcohol misuse, chronic viral hepatitis, and anti-tubercular drug-induced hepatotoxicity that injectable drug users exhibit [25,26].\nDrug resistance TB has emerged as a serious health issue undermining global efforts to control TB and reduce the spread of the disease, in addition to making TB treatment more complicated. Thus, we aimed to study the risk factors associated with the development of MDR-TB in TB patients. In the current study, relapsed TB cases were more likely to develop MDR-TB than newly registered cases that had never had exposure to anti-TB treatment. In agreement with our findings, a history of treatment with anti-tubercular drugs has been mentioned in the previous literature as a risk factor for MDR-TB development in common with other risk factors [27,28,29,30]. Relapsed TB cases were found to be more likely to achieve unsuccessful treatment outcomes [31,32]. As the history of being treated with anti-TB drugs raises the hazard of resistance to many drugs, more attention must be paid to identifying the underlying causes of treatment failure and default treatment, which may decrease the incidence of retreatment among tuberculosis patients [28]. The link between prior anti-TB exposure and the emergence of drug resistance could be due to the fact that prior anti-TB exposure suppresses only the growth of susceptible bacilli. On the other hand, it may allow for the multiplication of pre-existing drug-resistant mutants [33].\nOne of the sociodemographic characteristics related to tuberculosis is marital status. Our findings reported that single patients were at 6.817 times higher risk of developing MDR-TB than married patients, which was in agreement with other studies that revealed that single individuals were at higher risk of developing MDR-TB than married individuals [34,35]. Even though there is no biological relationship between the marital status of the patients and TB, when compared to married people, single people are more likely to be infected with tuberculosis or MDR-TB strains. Being at high risk can still be due to a lack of social support or participation in high-risk behaviors, such as alcohol and drug addiction [36].\nAside from the previously mentioned risk factors, alcohol consumption has been significantly associated with developing MDR-TB. Patients who had a history of drinking or were current drinkers had a 7.591 times higher risk of MDR-TB than those who had never drank. Drinkers had a higher risk of MDR-TB, according to the results of a study conducted in China and Uganda [37,38]. Alcohol use was a contributing factor in TB treatment interruption, which is the main factor in MDR-TB development [39]. Many explanations could stand behind the impact of alcohol consumption on MDR-TB. Poorer treatment outcomes, including default treatment, mortality, and treatment failure, have been observed in TB patients who have consumed alcohol and MDR-TB, while treatment failure has been identified as a significant risk factor for drug resistance [38]. In addition, the consequences of alcohol ingestion can result in liver damage, weakened immunity, and nutritional deficiency, contributing to sensitive and resistant TB infection [40,41,42].\nThe findings of the current study highlighted patients’ groups that are at high risk of achieving unsuccessful TB treatment outcomes. As MDR-TB has proven to be a major influencer on patients’ health and safety, more attention must be paid to the factors leading to relapse and overcoming them in order to minimize the risk of MDR-TB occurrence in TB patients. A must improve public awareness about the worsening effect of alcohol on TB treatment and the importance of psychological support from the patient’s family to adhere to TB treatment. Moreover, the government should implement more restricted rules to minimize the availability of drugs in the population hands and to increase the awareness of people regarding the hazard of drugs. Additionally, malnutrition status was a risk factor for death among TB patients, so monthly counseling and material support in the form of food is suggested.\nThe study has many limitations that must be recognized. This study targeted only patients registered with HPP, which may not cover the true proportion of the Malaysian population. Being a unicentral study and a convenient sample technique limits the generalization of the results to the entire Malaysian population. An unequal proportionality among several variables, including age, ethnicity, and gender, might influence the final results in comparison with randomized control trials (selection bias). Due to the retrospective nature of the present study, it was impossible to record certain sociodemographic characteristics of the patients who passed away and default treatment (information bias), which might enhance the findings of the study. It was difficult to set a control group that needed further clinical trials to support the study results. The aforementioned limitations might affect the extent to which the current findings are accurate and consistent and can be reproduced under the same methodology.",
"Eventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single."
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1. Introduction:
Tuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients.
2. Materials and Methods:
2.1. Study Design and Population A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).
Sample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis.
A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).
Sample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis.
2.2. Diagnosis and Treatment Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7].
The diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB.
EPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism.
Patients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen.
In Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR).
Treatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1.
Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7].
The diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB.
EPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism.
Patients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen.
In Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR).
Treatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1.
2.3. Data Collection Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns.
Patients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2.
Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns.
Patients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2.
2.4. Statistical Analysis To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables.
To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables.
2.1. Study Design and Population:
A retrospective cohort study was conducted at the respiratory clinic of Hospital Pulau Pinang (HPP), a public sector tertiary care referral hospital that covers a large proportion of the Pinang population (1.7 million), the 3rd populated state in Malaysia [6]. It is currently the second-largest tertiary health care public hospital with a capacity of 1107 beds in 1961 in the northern region of Malaysia. Medical records of patients with confirmed TB diagnoses from 2014–2018 were screened, and a total of 351 patients were included in the final analysis. Patients aged 18 years or above, diagnosed with active pulmonary tuberculosis (PTB) and/or extrapulmonary tuberculosis (EPTB), patients diagnosed with MDR-TB, and patients with co-morbidities with TB, including diabetes mellitus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, were included in the study, whereas patients aged less than 18 years, patients with latent TB, cancer patients receiving chemotherapy treatment, pregnant and lactating women, patients transferred out who did not complete their treatment in HPP, patients who were diagnosed with TB than the diagnosis has been changed and patients with incomplete records were excluded (Figure 1).
Sample size calculation: A convenient sampling technique was adopted for the current study. All TB patients’ records registered at chest clinics (2914 records) from 2014–2018 were screened, and patients whose records complied with the inclusion and inclusion criteria (351 records) were included in the final analysis.
2.2. Diagnosis and Treatment:
Patients suspected of having TB were initially hospitalized to confirm or exclude the diagnosis. Once the patients’ diagnosis is confirmed, they have been discharged for treatment continuation unless they have medical complications that need monitoring. TB diagnosis was based on the guidelines of the Ministry of Health (MOH) Malaysia, which used the same procedure WHO raised [4,7].
The diagnosis of PTB and/or EPTB was based on the patient’s history reviewed by clinical expertise and clinical examination of the patient’s signs and symptoms, further supported by imaging (Chest X-ray) and laboratory tests. The diagnosis was confirmed by isolating Mycobacterium tuberculosis from clinical samples. Mycobacterial cultures were sent at the initiation of TB treatment to confirm the presence of Mycobacterium tuberculosis and to exclude drug-resistant TB. In the case of smear negative PTB, a chest X-ray is a sensitive tool used for identifying and excluding the diagnosis, as the clinical symptoms of smear negative PTB are similar to smear positive PTB.
EPTB diagnosis depended on obtaining samples for Mycobacterium tuberculosis culture from the affected sites, such as cerebrospinal fluid (CSF), pleural fluid, fine needle aspiration (FNA), and/or biopsy from lymph nodes, pleura, and any other infected sites, to confirm the diagnosis and provide the drug susceptibility profile of the organism.
Patients suspected of having MDR-TB should be initially evaluated for the presence of acid-fast bacilli (AFB) and rifampicin resistance using smear microscopy and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay. Upon positive sputum smear microscopy and rifampicin resistance, the patient should be enrolled in MDR-TB treatment with an empirical treatment regimen, and the specimen sample should be sent to a reference laboratory for a culture and drug sensitivity test (DST) against both first- and second-line anti-TB drugs (LJ Media and Automated/liquid media). Upon reception of DST results, patients should be switched from an empirical regimen to a standardized or DST-based individualized regimen.
In Malaysia, TB treatment is fully under the umbrella of the national TB control program. Patients were treated with recommended standardized regimens following WHO TB guidelines. Patients diagnosed with PTB were treated with two months of intensive phase followed by 4 months for PTB and 4–12 months of maintenance phase for EPTB patients. The intensive phase regimen included isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE), whereas the maintenance phase regimen included isoniazid and rifampicin (HR).
Treatment of MDR-TB depended either on a standard MDR-TB regimen (standardized approach) or an individually tailored regimen based on DST for second-line drugs. Intensive phase regimens consisted of 4 s-line anti-TB drugs that were proven to be more effective, including fluoroquinolones, aminoglycosides, ethionamide, and cycloserine with pyrazinamide. WHO recommended 8 months of the intensive phase for most patients. The anti-TB drugs used in treatment and their doses are shown in Table 1.
2.3. Data Collection:
Data of patients with confirmed active PTB and/or EPTB that were being followed up at the Chest Clinic were extracted manually from patients’ records in the chest clinic. A designed data sheet was used to collect patients’ sociodemographic and baseline clinical characteristics (age, gender, race, marital status, occupation, smoking, drinking, drug addiction, comorbidity, TB site, and TB history), pharmacotherapeutic options (receiving second-line anti-tuberculosis drugs, intensive and maintenance phase regimens, treatment duration, number of drugs taken, and patients’ compliance), microbiological (TB smear, TB culture, and sensitivity at baseline and Gene Xpert test), laboratory values, and drug-resistant patterns.
Patients were given ID numbers (1–351) for coding, and then their data were entered into SPSS software as categorical and continuous variables for analysis. Based on WHO guidelines, patients’ treatment outcomes have been categorized into successful outcomes, including cured and treatment completed, and unsuccessful outcomes, including death, treatment failure, treatment failure, and default treatment [1]. Definitions of TB treatment outcomes are shown in Table 2.
2.4. Statistical Analysis:
To analyze the data, SPSS (version 23, IBM Corp., Armonk, NY, USA) was used. Descriptive statistics were conducted to analyze the data regarding the descriptive statistics for continuous data and categorical data. Continuous variables were presented as either mean ± standard deviation if they were normally distributed or median with their interquartile range (IQR) if not normally distributed using histogram and the Kolmogrov–Smirnov test. A variable is not normally distributed if “Sig.” < 0.05. Categorical variables are presented as percentages. The Cox proportional hazard regression model was used to determine the factors that are associated with time to mortality, having satisfied the assumptions of mutually exclusive events (dead and censored) and constant hazard ratios over time. Time (months) from TB diagnosis confirmed at hospital Pulau Pinang until death occurred, defined as time to event of interest. Additionally, Cox regression was used to determine the factors associated with MDR-TB development over time. Time to event of interest here is defined as time from TB diagnosis confirmed at hospital until MDR-TB diagnosis confirmed on the basis of drug sensitivity test results. Univariable Cox regression was performed by entering independent variables one by one to examine the association between these variables and MDR-TB development, as well as death, along with treatment duration. The entry of the independent variables into univariate analysis was based on the previously published studies, their possible relationship with the treatment outcomes, and recommendations from the clinical team and supervisors of the current study. Independent variables with a significant p-value (≤0.05) were included in a multivariable Cox regression analysis to see the independent risk factors of mortality and developing MDR-TB among TB patients. The Kaplan–Meier survival curve and log-rank test were performed to estimate the cumulative probability of survival of different variables.
3. Results:
3.1. Sociodemographic and Clinical Characteristics At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).
At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).
3.2. Drug Resistant Patterns of TB Patients The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).
The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).
3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).
The observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.
In univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).
In multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).
Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).
The observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.
In univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).
In multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).
3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).
In univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).
In multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).
Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).
In univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).
In multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).
3.1. Sociodemographic and Clinical Characteristics:
At the study site, a total number of 351 TB patients received their treatment and had been followed up for treatment duration. The mean age of the patients was 48.84 ± 16.71 years. The majority of patients were males (74.4%), aged between 54–64 years (20.8%), Chinese (46.4%), married (64.4%), non-drinkers (86.0%), and non-drug addicts (83.2%). The majority of patients were registered as new cases (72.1%) and diagnosed with PTB (81.2%). Of the 285 PTB patients, 229 (65.2%) were smear positive upon initiation of treatment (Table 3).
3.2. Drug Resistant Patterns of TB Patients:
The study participants were resistant to a median of one drug (range 1–4 drugs). At the baseline visit, the minority of participants were resistant to isoniazid (2.8%), rifampicin (4.0%), ethambutol (0.9%), pyrazinamide (0.9%), and streptomycin (1.7%) (Table 4).
3.3. Treatment Outcomes and Associated Factors of Mortality among TB Patients:
Of 325 drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, 73 (22.5%) passed away, 6 (1.8%) defaulted, and one patient failed treatment. The median duration of drug-susceptible TB treatment was 6 months. The median time to death was 1 month (Table 5).
The observed difference in survival experiences in different patient groups was assessed using the log-rank test. The results of the log-rank test show that the differences in the cumulative probability of survival of various factors, such as smoking, drug addiction, intensive phase regimen, and maintenance phase regimen, are statistically significant (p < 0.05), as shown in Figure 2, Figure 3, Figure 4 and Figure 5 and Table 6.
In univariable analysis, smoking (HR = 1.723, 95% CI = 1.081–2.746), drug addiction (HR = 1.652, 95% CI = 0.960–2.843), level of white blood cells at baseline (HR = 1.071, 95% CI = 1.026–1.118), neutrophils % (HR = 1.017, 95% CI = 1.006–1.029), urea (HR = 1.034, 95% CI = 1.020–1.048), and creatinine (HR = 1.001, 95% CI = 1.000–1.003) were significantly associated with increase risk of mortality. In addition, level of hemoglobin at baseline (HR = 0.809, 95% CI = 0.736–0.890), level of platelets (HR = 0.997, 95% CI = 0.996–0.999), lymphocytes % (HR = 0.943, 95% CI = 0.919–0.968), and albumin (HR = 0.957, 95% CI = 0.939–0.976) were significantly associated with decreased risk of mortality (Table 7).
In multivariable analysis, drug addiction (HR = 1.836, 95% CI = 1.019–3.309), levels of white blood cells at baseline (HR = 1.102, 95% CI = 1.057–1.148), and urea (HR = 1.029, 95% CI = 1.011–1.047) were independently associated with an increased risk of mortality. The level of platelets (HR = 0.996, 95% CI = 0.995–0.998) and albumin (HR= 0.964, 95% CI = 0.940–0.990) were associated with a decreased risk of mortality (Table 8).
3.4. Treatment Outcomes and Associated Factors for Developing Multidrug Resistant TB:
Among the 351 patients included in the final analysis, 26 (7.4%) were diagnosed with MDR-TB. Of the 21 (80.8%) patients who achieved successful outcomes, 5 (19.2%) passed away. The median duration of treatment was 12 months (range 2–20 months) (Table 5).
In univariable analysis, the variables that had a statistically significant association with increased risk of MDR-TB were being single (HR = 0.457, 95% CI = 1.960–10.548), alcohol consumption (HR = 7.452, 95% CI = 3.206–17.324), and relapsed TB cases (HR = 3.035, 95% CI = 1.306–10.196). In addition, smoking (HR = 0.088, 95% CI = 0.025–0.301), not being a prisoner (HR = 0.270, 95% CI = 0.075–0.978), not having a history of TB (HR = 0.400, 95% CI = 0.168–0.952), no growth of MTB (HR = 0.300, 95% CI = 0.093–0.971) and receiving an anti-TB regimen as separated therapy (HR = 0.101, 95% CI = 0.029–0.355) were associated with decrease risk of MDR-TB development (Table 9).
In multivariable analysis, the variables that emerged as independent risk factors associated with the development of MDR-TB were drinking (HR = 7.591, 95% CI = 3.097–18.610), relapsed cases (HR = 3.035, 95% CI = 1.028–8.957) and being single (HR = 6.817, 95% CI = 2.599–17.879) (Table 10).
4. Discussion:
Our research looked into the factors that contribute to mortality among TB patients, as well as the factors associated with MDR-TB development. Since then, tuberculosis treatment has been problematic and difficult to apply properly because of long duration and compliance issues due to anti-tubercular drugs’ side effects. Some of the side effects are self-limiting, but others necessitate treatment discontinuation. Although rare, thrombocytopenia is one of the most common and serious life-threatening side effects. Thrombocytopenia is caused by the formation of antibodies that bind to platelets and initiate the complement cascade, causing these cells to lyse. These antibodies also suppress the production of platelets [8]. This study demonstrated that thrombocytopenia significantly increased the risk of mortality among TB patients. [9] reported a low platelet count as a significant risk factor for mortality.
Mortality among patients suffering from any infection has been linked to WBC count [10]. As a result, elevated WBC counts in TB patients may be related to the severity of lung involvement and provide an estimated risk of mortality [11]. During TB infection, the body produces leukocytes and macrophages as part of the immune mechanism to defend the body against invading Mycobacterium tuberculosis, which raises the total number of WBC [12]. The current study findings documented that every 103 cells/µL increment in WBC above the normal range increased the risk of death by 1.102. In line with our results, a study conducted in Malaysia pointed out that the risk of death increased by 1.12 times for every 103 cells per microliter unit increase in total WBC [13]. Other studies have indicated that WBC levels are a significant risk factor correlated with TB mortality [11,14]. Pneumonia ranked the second cause of death among the study population. The WBC count elevation has been reported as a predictor of the severity of pneumonia, one of the leading causes of death in TB patients [11].
The present findings indicated that the level of urea was a strong significant predictor of death. Increased levels of urea were found to be significantly associated with mortality [11]. We can relate elevated urea levels to kidney dysfunction, where renal clearance is reduced due to renal impairment/failure or chronic kidney disease (CKD). Urea levels may also increase in other conditions, such as upper GI bleeding [15]. As the prevalence of CKD in our cohort was 5.69%, where 20 patients developed CKD while receiving anti-tuberculosis treatment, further research is needed to determine the relationship between CKD and TB. Strategies for the prevention, detection, and treatment of TB-CKD cannot be refined without advanced understanding.
Hypoalbuminemia showed a statistically significant relationship with death; patients who had hypoalbuminemia were more likely to pass away than patients who did not. Matching our results, lower albumin levels showed an association with non-survivors (p< 0.001) compared to survivors. Low levels of albumin were found to be significantly correlated with the hazard of death [9,16]. Furthermore, hypoalbuminemia was considered a predictor of mortality in community-acquired pneumonia (CAP), which is not surprising considering that albumin is involved in inflammation and indicates malnutrition [17]. The initial value of serum albumin might also be a result of malnutrition or an underlying disease that can worsen the nutritional status of the patient [18]. As a result of malnutrition, deceased patients’ immune systems were suppressed and were therefore sicker upon initial presentation.
As for drug addiction characteristics, the findings of this study revealed that patients who had a history of addiction or were current addicts had a 1.895 times higher risk of death than those who had never abused drugs. In line with our findings, deceased patients were more likely to abuse heroin IV than living patients, and addiction was correlated with all causes of death in tuberculosis patients [19,20]. Supporting our results, a history of addiction was a statistically significant risk factor for death within the first year after diagnosis in Hong Kong, Russia, and Iran [21,22,23]. Poor adherence of drug users, default treatment, and poor access to health services can create challenges for the treatment of the drug addict population [24]. As drug users have a higher likelihood of default treatment than non-drug users, the increased hazard of death can be attributed to the increased prevalence of alcohol misuse, chronic viral hepatitis, and anti-tubercular drug-induced hepatotoxicity that injectable drug users exhibit [25,26].
Drug resistance TB has emerged as a serious health issue undermining global efforts to control TB and reduce the spread of the disease, in addition to making TB treatment more complicated. Thus, we aimed to study the risk factors associated with the development of MDR-TB in TB patients. In the current study, relapsed TB cases were more likely to develop MDR-TB than newly registered cases that had never had exposure to anti-TB treatment. In agreement with our findings, a history of treatment with anti-tubercular drugs has been mentioned in the previous literature as a risk factor for MDR-TB development in common with other risk factors [27,28,29,30]. Relapsed TB cases were found to be more likely to achieve unsuccessful treatment outcomes [31,32]. As the history of being treated with anti-TB drugs raises the hazard of resistance to many drugs, more attention must be paid to identifying the underlying causes of treatment failure and default treatment, which may decrease the incidence of retreatment among tuberculosis patients [28]. The link between prior anti-TB exposure and the emergence of drug resistance could be due to the fact that prior anti-TB exposure suppresses only the growth of susceptible bacilli. On the other hand, it may allow for the multiplication of pre-existing drug-resistant mutants [33].
One of the sociodemographic characteristics related to tuberculosis is marital status. Our findings reported that single patients were at 6.817 times higher risk of developing MDR-TB than married patients, which was in agreement with other studies that revealed that single individuals were at higher risk of developing MDR-TB than married individuals [34,35]. Even though there is no biological relationship between the marital status of the patients and TB, when compared to married people, single people are more likely to be infected with tuberculosis or MDR-TB strains. Being at high risk can still be due to a lack of social support or participation in high-risk behaviors, such as alcohol and drug addiction [36].
Aside from the previously mentioned risk factors, alcohol consumption has been significantly associated with developing MDR-TB. Patients who had a history of drinking or were current drinkers had a 7.591 times higher risk of MDR-TB than those who had never drank. Drinkers had a higher risk of MDR-TB, according to the results of a study conducted in China and Uganda [37,38]. Alcohol use was a contributing factor in TB treatment interruption, which is the main factor in MDR-TB development [39]. Many explanations could stand behind the impact of alcohol consumption on MDR-TB. Poorer treatment outcomes, including default treatment, mortality, and treatment failure, have been observed in TB patients who have consumed alcohol and MDR-TB, while treatment failure has been identified as a significant risk factor for drug resistance [38]. In addition, the consequences of alcohol ingestion can result in liver damage, weakened immunity, and nutritional deficiency, contributing to sensitive and resistant TB infection [40,41,42].
The findings of the current study highlighted patients’ groups that are at high risk of achieving unsuccessful TB treatment outcomes. As MDR-TB has proven to be a major influencer on patients’ health and safety, more attention must be paid to the factors leading to relapse and overcoming them in order to minimize the risk of MDR-TB occurrence in TB patients. A must improve public awareness about the worsening effect of alcohol on TB treatment and the importance of psychological support from the patient’s family to adhere to TB treatment. Moreover, the government should implement more restricted rules to minimize the availability of drugs in the population hands and to increase the awareness of people regarding the hazard of drugs. Additionally, malnutrition status was a risk factor for death among TB patients, so monthly counseling and material support in the form of food is suggested.
The study has many limitations that must be recognized. This study targeted only patients registered with HPP, which may not cover the true proportion of the Malaysian population. Being a unicentral study and a convenient sample technique limits the generalization of the results to the entire Malaysian population. An unequal proportionality among several variables, including age, ethnicity, and gender, might influence the final results in comparison with randomized control trials (selection bias). Due to the retrospective nature of the present study, it was impossible to record certain sociodemographic characteristics of the patients who passed away and default treatment (information bias), which might enhance the findings of the study. It was difficult to set a control group that needed further clinical trials to support the study results. The aforementioned limitations might affect the extent to which the current findings are accurate and consistent and can be reproduced under the same methodology.
5. Conclusions:
Eventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single.
|
Background:
Multidrug resistance TB (MDR-TB) has emerged as a public health issue worldwide, and the mortality rate is worrying. Therefore, this study was conducted to investigate the factors related to MDR-TB occurrence and the survival experience of TB patients.
Methods:
A retrospective cohort study was conducted at Hospital Pulau Pinang in Malaysia. Medical records of active TB patients from 2014-2018 were reviewed. Cox regression was used to identify the factors associated with MDR-TB development and mortality among TB patients.
Results:
The patients had a mean age of 48.84 ± 16.713 years, and a majority of the Chinese race (46.4%). Out of 351 TB patients, 325 (92.6%) were drug-susceptible TB, and 26 (7.4%) were diagnosed with MDR-TB. Among drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, and 73 (22.5%) passed away. In multivariable Cox regression, drug addiction, levels of white blood cells, urea, platelets, and albumin were significantly associated with death. Relapsed TB, alcohol consumption, and being single were significant risk factors for MDR-TB development.
Conclusions:
Patients achieved a success rate of 75.4%, which is encouraging but still far below the WHO target (at least an 85% success rate) and has room for further improvement.
|
1. Introduction:
Tuberculosis (TB) is still a disease that has a strong impact on global public health. Efforts are made to control the disease transmission, especially in high TB burden, and to face the emerged multidrug-resistant TB (MDR-TB), which plays a key role in achieving unsuccessful TB treatment outcomes. Globally, TB is the leading infectious disease killer and one of the top ten causes of death, with 10.0 million people falling ill with TB in 2019 [1,2]. The impact of COVID-19 has resulted in a large drop in TB incidence due to the increase in mortality among TB patients, from 1.2 million in 2019 to 1.4 million in 2020 [2]. In Malaysia, an intermediate–TB burden country, the TB mortality rate is the highest death rate in comparison with other infectious diseases, showing that transmission of TB is still active [3]. It increased from 5.5/per 100,000 people in 2014 to 7.1 per 100,000 people in 2020 [4].MDR-TB, which is defined as the resistance of Mycobacterium tuberculosis to at least isoniazid and rifampicin, the most potent first-line anti-tubercular drugs, has appeared to compromise patients’ health and to challenge national TB control programs. A total estimate of 465,000 new MDR-TB cases were reported worldwide in 2019 [1]. Even though MDR-TB is relatively uncommon in Malaysia, the number of MDR-TB cases climbed from 101 to 370 between 2015 and 2017, which highlighted the need for further research into management and treatment outcomes [5]. The previous literature shows a scarcity of data on MDR-TB outcomes and related risk factors in Malaysia, especially after 2015. Moreover, this is the first study in the country to evaluate the effect of laboratory values (WBC, platelets, neutrophils, lymphocytes, ALT, albumin, urea, and creatinine) on TB-related mortality. The current study aimed to determine the factors that contribute to the development of MDR-TB, as well as the associated factors of mortality among TB patients.
5. Conclusions:
Eventually, patients with a history of addiction or who were currently addicted had a higher mortality rate than non-addict patients. Among TB patients, drug addiction, white blood cell count, urea levels, platelet counts, and albumin levels were all risk factors for death. Multidrug-resistant tuberculosis developed in 26 (6.0%) out of 351 patients. MDR-TB development was significantly related to relapsed cases, alcohol consumption, and being single.
|
Background:
Multidrug resistance TB (MDR-TB) has emerged as a public health issue worldwide, and the mortality rate is worrying. Therefore, this study was conducted to investigate the factors related to MDR-TB occurrence and the survival experience of TB patients.
Methods:
A retrospective cohort study was conducted at Hospital Pulau Pinang in Malaysia. Medical records of active TB patients from 2014-2018 were reviewed. Cox regression was used to identify the factors associated with MDR-TB development and mortality among TB patients.
Results:
The patients had a mean age of 48.84 ± 16.713 years, and a majority of the Chinese race (46.4%). Out of 351 TB patients, 325 (92.6%) were drug-susceptible TB, and 26 (7.4%) were diagnosed with MDR-TB. Among drug-susceptible TB patients, 245 (75.4%) achieved successful outcomes, and 73 (22.5%) passed away. In multivariable Cox regression, drug addiction, levels of white blood cells, urea, platelets, and albumin were significantly associated with death. Relapsed TB, alcohol consumption, and being single were significant risk factors for MDR-TB development.
Conclusions:
Patients achieved a success rate of 75.4%, which is encouraging but still far below the WHO target (at least an 85% success rate) and has room for further improvement.
| 9,383 | 268 | 13 |
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[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]
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[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]
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[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]
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[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]
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[CONTENT] multidrug-resistant | mortality | death | treatment outcomes | survival | unsuccessful treatment [SUMMARY]
|
[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]
| null |
[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]
|
[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]
|
[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]
|
[CONTENT] Humans | Adult | Middle Aged | Aged | Retrospective Studies | Antitubercular Agents | Malaysia | Tuberculosis, Multidrug-Resistant | Risk Factors | Hospitals [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
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[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
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[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]
| null |
[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]
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[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]
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[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]
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[CONTENT] tb | patients | treatment | hr | ci | 95 ci | 95 | mdr tb | mdr | drug [SUMMARY]
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[CONTENT] tb | mdr tb | mdr | 2019 | disease | people | 000 | million | mortality | 2020 [SUMMARY]
| null |
[CONTENT] 95 | 95 ci | ci | hr | table | tb | associated | risk | patients | drug [SUMMARY]
|
[CONTENT] patients | levels | addiction | eventually patients history addiction | patients drug addiction | related relapsed cases alcohol | tuberculosis developed | tuberculosis developed 26 | tuberculosis developed 26 351 | patients drug addiction white [SUMMARY]
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[CONTENT] tb | patients | 95 | ci | 95 ci | hr | treatment | mdr tb | mdr | drug [SUMMARY]
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[CONTENT] tb | patients | 95 | ci | 95 ci | hr | treatment | mdr tb | mdr | drug [SUMMARY]
|
[CONTENT] TB ||| MDR | TB [SUMMARY]
| null |
[CONTENT] 16.713 years | Chinese | 46.4% ||| 351 | 325 | 92.6% | TB | 26 | 7.4% | MDR ||| 245 | 75.4% | 73 | 22.5% ||| ||| TB | MDR [SUMMARY]
|
[CONTENT] 75.4% | WHO | at least an | 85% [SUMMARY]
|
[CONTENT] TB ||| MDR | TB ||| Hospital Pulau Pinang | Malaysia ||| TB | 2014-2018 ||| MDR | TB ||| ||| 16.713 years | Chinese | 46.4% ||| 351 | 325 | 92.6% | TB | 26 | 7.4% | MDR ||| 245 | 75.4% | 73 | 22.5% ||| ||| TB | MDR ||| 75.4% | WHO | at least an | 85% [SUMMARY]
|
[CONTENT] TB ||| MDR | TB ||| Hospital Pulau Pinang | Malaysia ||| TB | 2014-2018 ||| MDR | TB ||| ||| 16.713 years | Chinese | 46.4% ||| 351 | 325 | 92.6% | TB | 26 | 7.4% | MDR ||| 245 | 75.4% | 73 | 22.5% ||| ||| TB | MDR ||| 75.4% | WHO | at least an | 85% [SUMMARY]
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|||||
Is the Association between Age and Fertility Problems Modified by Diet Quality? Findings from a National Study of Reproductive Age Women in Australia.
|
36297039
|
Increasing age is a strong risk factor for infertility, and there is accumulating evidence of the importance of a healthier diet for fertility. Whether a healthier diet modifies the association between increasing age and infertility has not been investigated. This study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems.
|
BACKGROUND
|
Data were from Surveys 3 and 5 of the 1973-1978 birth cohort of the Australian Longitudinal Study on Women's Health. Cross-sectional analysis with multivariable generalized linear models were used to examine the association between age and fertility status, adjusted for various confounders. Multiplicative and additive effect modification by diet quality was assessed, with additive effect modification evaluated with the relative risk for interaction (RERI).
|
METHODS
|
In total, 3387 women were included from Survey 3 (age range 24-31 years) and 5614 women from Survey 5 (age range 30-38 years); 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems in the respective surveys. In Survey 3, compared to younger women with a good-quality diet, older women with a poor-quality diet had a 43% increased risk for fertility problems, with risk increasing after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62). In Survey 5 in younger women (<33.9 years), there was no association between diet quality and risk for infertility problems. The RERI (across different adjusted models) was between -0.08 (-0.70, 0.55) to -0.39 (-1.40, 0.62) in survey 3 and 0.07 (-0.17, 0.31) to 0.08 (-0.17, 0.32) in Survey 5.
|
RESULTS
|
There is little evidence to suggest effect modification on the effect of age and fertility problems with diet quality.
|
CONCLUSIONS
|
[
"Female",
"Humans",
"Aged",
"Young Adult",
"Adult",
"Infertility, Female",
"Longitudinal Studies",
"Cross-Sectional Studies",
"Australia",
"Fertility",
"Diet",
"Risk Factors"
] |
9606952
|
1. Introduction
|
Infertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].
Increasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].
Although increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].
There is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality.
| null | null |
3. Results
|
3.1. Participants A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.
A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.
3.2. Effect Modification of Diet on Age and Fertility Problems Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).
Table 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.
At Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.
Table 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.
Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).
Table 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.
At Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.
Table 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.
3.3. Dietary Change and Fertility Status Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).
Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).
|
5. Conclusions
|
In conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age.
|
[
"2. Materials and Methods",
"2.1. Study Design",
"2.2. Study Population",
"2.3. Exposure Variables",
"2.4. Outcome Variable",
"2.5. Confounding Variables",
"2.6. Statistical Analysis",
"3.2. Effect Modification of Diet on Age and Fertility Problems",
"3.3. Dietary Change and Fertility Status"
] |
[
" 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).",
"Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.",
"This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).",
"The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.",
"Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.",
"A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].",
"Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).",
"Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.",
"Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5)."
] |
[
null,
null,
null,
null,
null,
null,
null,
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null
] |
[
"1. Introduction",
"2. Materials and Methods",
"2.1. Study Design",
"2.2. Study Population",
"2.3. Exposure Variables",
"2.4. Outcome Variable",
"2.5. Confounding Variables",
"2.6. Statistical Analysis",
"3. Results",
"3.1. Participants",
"3.2. Effect Modification of Diet on Age and Fertility Problems",
"3.3. Dietary Change and Fertility Status",
"4. Discussion",
"5. Conclusions"
] |
[
"Infertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].\nIncreasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].\nAlthough increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].\nThere is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality.",
" 2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\nOur primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.\n 2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\nThis study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).\n 2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\nThe exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.\n 2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\nWomen were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.\n 2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\nA range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].\n 2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).\nDescriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).",
"Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.",
"This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.\nThis study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).",
"The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.",
"Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.",
"A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].",
"Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).\nEffect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].\nThe secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).",
" 3.1. Participants A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\nA total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.\n 3.2. Effect Modification of Diet on Age and Fertility Problems Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\nTable 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.\n 3.3. Dietary Change and Fertility Status Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).\nTable 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).",
"A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.",
"Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).\nTable 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.\nAt Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.\nTable 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.",
"Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).",
"Among a large cohort of Australian women, we examined whether diet modified the relationship between age and infertility, and whether a change in diet impacts fertility. Our results do not support our hypothesis; that is, in both Survey 3 and in Survey 5, there was little evidence that diet quality modified the association between age and fertility problems. Although increasing age (from Survey 3 to Survey 5) was associated with an approximate 5% higher rate of fertility problems, a change in diet quality over this time was not associated with fertility problems at Survey 5.\nAmong Survey 3 participants who were aged between 24–31 years, independent associations were found, such that older woman (28.2 to 30.8 years) who had a poor-quality diet had higher risk for infertility problems compared to the younger women (24.4 to <28.2 years) with a good-quality diet. At Survey 5 when women were aged 30–38 years, there was no difference in risk for fertility problems by diet quality or age. This was an unexpected finding. At Survey 5, there was an approximate 10% higher percentage of women with a good-quality diet; thus, with increasing age, the importance of a healthier diet may be realised, irrespective of fertility status. Yet, the older women at Survey 5 did appear to drink slightly more alcohol and perform less metabolic minutes of exercise, which are both associated with higher likelihood for infertility [6,36]. Interestingly, the mean BMI among women with and without fertility problems was consistent over the 5-year period from Survey 3 to Survey 5, with the women with infertility problems maintaining the approximate two-unit higher BMI than the women without infertility problems. The combination of some infertility risk factors increasing over time, but others remaining consistent, may partly explain the lack of association in this slightly older age group of women. Nevertheless, our results might suggest a small window of opportunity in women aged 28–31 years to optimise their diet to support fertility, whereas after this age, the current data do not support this. Although many studies have reported on the independent effects of increasing age with infertility, and increasing evidence shows the importance of a healthy diet for reproductive health, qualitative data reveals that women do not have a clear understanding of the age at which fertility begins to decline [37]. There are also no specific recommendations for an appropriate diet to optimise fertility. The knowledge gaps and misconceptions surrounding reproductive health and nutrition, highlights a critical opportunity that is needed to support patient awareness, counselling to support dietary behaviours, and that involves pregnancy planning.\nOur findings provide little evidence of effect modification by diet at either survey. Whilst at Survey 3 there was a suggestion of a negative effect measure modification, such that the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet, the wide 95% CIs are consistent with positive effect modification by diet. At Survey 5, the RERI was positive, suggesting that the effect of age interacting with diet quality was higher than the sum of the independent effects of age and diet, but the wide confidence intervals are also consistent with positive effect modification. Thus, we cannot conclude that better quality diet could attenuate the decline in fertility with age over the observed periods. These findings suggest that the effect of a poor-quality diet and older age on fertility may not be as pronounced as expected, but also that a poor diet quality is not appreciably leading to reduced fertility in older women.\nWhilst our results are unexpected, they build the foundation for further studies to assess the interactions between age and diet quality. Although the additive effect modification RERI is the more relevant public health measure, and we would theorise that the targeted group for intervention would be older women with a poor-quality diet, in this instance, we cannot confidently draw this conclusion. Importantly, our results do not mean that a good-quality diet is adverse in any way but implies that a good-quality diet does not attenuate the relationship between age and infertility, and that the joint effect of the two exposures is less than their main effects combined. The indication of a positive RERI at Survey 5 supports the promotion of improved diet quality in older women.\nOverall diet quality of women was low in both surveys, which is consistent with previous studies reporting that a poor-quality diet is common amongst women of reproductive age [23,38,39,40,41]. Despite the slight increase in proportion of women having a good-quality diet from Survey 3 to Survey 5, there was no evidence that a change in diet, whether that was an improvement or worsening of diet quality over time, was associated with fertility problems. However, we acknowledge that this change in diet quality is very small and may be equivalent to only replacing white bread with brown (whole meal or multigrain) as the usual bread choice. In line with our findings, a review of studies assessing pregnancy intentions and diet or physical activity behaviours in the preconception and antenatal periods found that women intending to become pregnant do not report different preconception dietary or physical activity behaviours compared with women without pregnancy intentions [42]. This reflects the common barriers observed in women of reproductive age to achieve successful health behaviour change [43]. In our study, the lack of association between a change in diet and fertility may also relate to reverse causation. That is, following a diagnosis of fertility problems, women may alter their behaviours towards healthier choices, or their improvement in dietary intake may not be sufficient to alter the effect on fertility problems.\nTo the best of our knowledge, this is the first study to assess whether better diet quality can reduce the age-related decline in fertility. A strength of this study is the use of data from a nationally representative cohort of Australian women at two different time points, increasing the generalisability of our findings to all Australian women. We used robust statistical measures, including calculation of the RERI, whereby effect measure modification of diet on the additive scale provides a clear comparison of the effect of diet on the association between one of the major risk factors of fertility (i.e., age) and fertility problems among women. Some limitations are also worth mentioning. A validated, semi-quantitative FFQ was used to capture women’s dietary consumption, which was designed for use in the Australian community. Although the ARFS is an acceptable tool for assessing diet quality, for most foods the scoring was based on ‘at least once per week’. Additional response options might be helpful to quantify diet quality more accurately. While the analysis included a large cohort of women, it may not be sufficiently powered to identify interactions; thus, further work in this area, with larger populations and covering wider age ranges, is warranted. Lastly, the current study included self-reported fertility problems and dietary intakes that could be subject to recall bias.",
"In conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age."
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1. Introduction:
Infertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].
Increasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].
Although increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].
There is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality.
2. Materials and Methods:
2.1. Study Design Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.
Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.
2.2. Study Population This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.
This study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).
This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.
This study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).
2.3. Exposure Variables The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.
The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.
2.4. Outcome Variable Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.
Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.
2.5. Confounding Variables A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].
A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].
2.6. Statistical Analysis Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).
Effect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].
The secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).
Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).
Effect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].
The secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).
2.1. Study Design:
Our primary aim utilises a cross-sectional analysis at Survey 3 and at Survey 5 to determine whether diet modifies the association between age and fertility problems at each survey, with consideration of a range of confounding factors including BMI. Our secondary aim utilises a longitudinal design to explore whether a change in diet quality from Survey 3 to Survey 5 is associated with fertility problems at Survey 5.
2.2. Study Population:
This study is based on data from the Australian Longitudinal Study on Women’s Health (ALSWH), a prospective cohort study of women’s physical and mental health, psychosocial aspects of health (such as sociodemographic and lifestyle factors), and use of health services [25]. In 1996, three cohorts of young (born 1973–1978, aged 18–23 years; n = 14,247), middle-aged (born 1946–1951, aged 45–50 years; n = 13,715), and older (born 1921–1926, aged 70–75 years; n = 12,432) women were recruited. Women were randomly selected from the national health insurance scheme (Medicare) database which includes women who are permanent residents of Australia, with national recruitment and intentional over-sampling from rural and remote areas [26]. The women completed a mailed survey every three years, with additional details reported elsewhere [25,26]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The Human Research Ethics Committees of the University of Newcastle and the University of Queensland.
This study uses data from the cohort of young women who completed survey 3 in 2003 (25–30 years) and Survey 5 in 2009 (31–36 years). Inclusion criteria were women who completed the FFQ and had known fertility status at either survey (n = 2169). The fertility status of 5523 women in Survey 3 (61.3%) and 2464 women in Survey 5 (30.2%) was unknown so they were excluded. We then excluded those with an incomplete FFQ (>10% of items missing responses; n = 5) or a reported daily energy intake >14,700 kJ/day (3513.4 Kcal/day) or <2100 kJ/day (501.9 Kcal/day) [27] (n = 95 in Survey 3 and n = 77 in Survey 5).
2.3. Exposure Variables:
The exposure variable is age in years, split at the median value, and categorised as younger vs. older women. That is, for Survey 3, the median age was 28.2 years (younger age <28.2 years, older age ≥28.2 years); and for Survey 5, the median age was 33.9 years (younger age <33.9 years, older age ≥33.9 years). The potential effect modifier is diet quality, estimated by the Australian Recommended Food Score (ARFS) [28], obtained from version 2 of the Dietary Questionnaire for Epidemiological Studies (DQES), a validated, semi-quantitative food frequency questionnaire [29]. The DQES uses a 10-point frequency option for participants to report their usual frequency of consumption of 74 foods and beverages over the past 12 months. Serving sizes are adjusted using portion size photographs. Respondents are asked further questions about the total number of daily serves of fruit, vegetables, bread, dairy products, eggs, fat spreads and sugars. The ARFS was derived as previously described [28]. For the alcohol variable, however, we modified the score for pregnancy based on Australian alcohol guidelines “To prevent harm from alcohol to their unborn child, women who are pregnant or planning a pregnancy should not drink alcohol” [30]. Therefore, any alcohol consumption scored 0 points and no alcohol scored 1 point. The highest ARFS is a score of 73, with higher scores indicating better diet quality.
2.4. Outcome Variable:
Women were asked to report their fertility status in each survey using the following question: “Have you and your partner (current or previous) ever had problems with infertility (that is, tried unsuccessfully to get pregnant for 12 months or more)?” Responses included “no, never tried to get pregnant”, “no, had no problems with fertility”, “yes, but have not sought help/treatment” or “yes, and have sought help/treatment”. Women who responded to “no, never tried to get pregnant” were excluded and women who had problems with fertility, with or without seeking treatment were combined as “yes had fertility problems”. Data were binary coded as “fertility problems’ (coded 1) and “no fertility problems” (coded 0). The resulting sample size in Survey 3 was n = 3387, Survey 5 n = 5614; and 2169 women were included across both surveys.
2.5. Confounding Variables:
A range of sociodemographic and health variables were collected at each of the surveys and were identified and included, a priori, as confounders, based on directed acyclic graphs. Surveys 3 and 5 collected information on BMI, country of birth (collected from survey 1), education (highest qualification), SEIFA (socio-economic advantage/disadvantage), physical activity (MET/mins/week), sedentary activity (minutes sitting/week), cigarette smoking, household income, irregular monthly periods, total energy intake (kJ/day), alcohol intake (g/day). Survey 5 additionally collected information on vitamin and mineral supplement usage, and Polycystic Ovary Syndrome (PCOS) status. PCOS is an enduring diagnosis, so we extrapolated the diagnosis from Survey 5 to Survey 3 [31].
2.6. Statistical Analysis:
Descriptive statistics for Survey 3 and 5 are presented as means and standard deviations for normally distributed continuous variables, medians and interquartile ranges for non-normal continuous variables, and frequencies and percentages for categorical variables. Multivariable generalized linear models (GLM) of Poisson regression with robust variance were fitted to examine the association between age and fertility status for Survey 3 and 5, with adjustment for diet quality, as measured by ARFS diet score (dichotomized at ARFS = 39). Unadjusted models were first assessed, and then model 1 adjusted for ARFS diet score and age, then Model 2 was: Model 1 plus country of birth, education level, SEIFA, physical activity, sedentary activity, cigarette smoking, household income, irregular monthly periods, total energy intake, alcohol intake, vitamin/mineral supplements (Survey 5 only), and the interaction between age and diet score; Model 3 was Model 2 plus BMI (kg/m2); and Model 4 was Model 3 plus PCOS status. The estimated effects were quantified as risk ratios (RR) and 95% confidence intervals (CI).
Effect modification by diet quality was evaluated on both the multiplicative and additive scales. Effect measure modification evaluates whether the relationship between one exposure varies within strata of another exposure of interest, whereas the joint exposure effect evaluates the combined effect of two exposures of interest [32]. This is distinct to analyses for interaction, where there are two hypothetical interventions under consideration whereby for effect modification there is only one [33]. We present an effect measure modification analysis because our main interest is to examine the protective role of diet quality on the effect of age on fertility problems, and not the combined effect of diet and age. Tests for multiplicative effect modification were assessed by including the cross product of diet quality and age in the models. Additive effect modification was evaluated with the relative risk for interaction (RERI) using the methods described by Knol et al. [34]. A RERI higher than 0 indicates effect modification greater than the expected additive effect; a RERI of 0 suggests no effect modification; and a negative value indicates effect modification in a negative direction. The RERI is interpreted according to the direction of effect measure modification, as opposed to its size, also recommended by Knol and VanderWeele [32]. The 95% CI of RERI was estimated using the bootstrap method [34].
The secondary aim examined the effect of change in diet between Survey 3 and 5 and fertility status in Survey 5, with the ARFS categorised as ‘good-quality diet (≥39)’ or ‘poor-quality diet (<39)’ [35]. Change in diet was categorized as ‘no change—healthy’ (good-quality diet in both Surveys), ‘no change—unhealthy (poor-quality diet in both surveys), ‘diet improved’ (poor-quality diet in Survey 3, good-quality diet in Survey 5) and ‘worse diet’ (good-quality diet in Survey 3, poor-quality diet in Survey 5)’. Models were fitted with the same adjustment covariates as those in the primary analysis. All statistical analyses performed using Stata version 17 (StataCorp, College Station, TX, USA).
3. Results:
3.1. Participants A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.
A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.
3.2. Effect Modification of Diet on Age and Fertility Problems Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).
Table 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.
At Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.
Table 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.
Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).
Table 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.
At Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.
Table 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.
3.3. Dietary Change and Fertility Status Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).
Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).
3.1. Participants:
A total of 3387 women were included in the analysis at Survey 3 (age range 24–31 years) and 5614 women at Survey 5 (age range 30–38 years), of whom 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems at the respective survey (Table 1). Across both surveys, the mean Australian Recommended Food Score did not substantially change from Survey 3 to Survey 5, increasing by around 3 units. At both surveys, women with fertility problems had a 1–2 unit higher BMI, and there was a higher percentage of women with irregular periods and with PCOS. At Survey 5, energy intake and metabolic minutes of exercise were slightly lower than at Survey 3, but alcohol intake was higher by around 1 g/day. Between 23–29% of women ever smoked in Survey 3, which fell to 12–13% at Survey 5.
3.2. Effect Modification of Diet on Age and Fertility Problems:
Table 2 reports the unadjusted and adjusted relative risks for diet and fertility problems in younger and older women at Survey 3. The reference group was young women (<28.2 years) with a good-quality diet (≥39 on the ARFS). Compared to the reference group, younger women with a poor-quality diet (<39) had an increased risk for fertility problems from 20–40%; however, the confidence intervals were wide. Older women with a good-quality diet had an adjusted risk of fertility problems of 1.09 to 1.34 across models 1 to 3, but again, the confidence intervals were wide and did not reach statistical significance. Model 4 that additionally included PCOS demonstrated a 1.69 (0.98, 2.91) increased risk for fertility problems. Compared to the reference group, older women with a poor-quality diet had a 43% increased risk for fertility problems, with increasing risk after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62).
Table 2 also includes the effect measure modification of diet on the effect of age on fertility problems. A RERI of −0.08 to −0.39 suggests a negative effect measure modification on the additive scale; thus, the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet. However, the 95% CIs overlap 0; thus, additive interaction is absent and little evidence of effect modification of diet in women aged 24–31 years.
At Survey 5, compared to the reference group of younger women [<33.9 years] with a good-quality diet, younger women with a poor-quality diet had an unadjusted increased risk for fertility problems by 18% (95% CI: 1.05, 1.32). However, after adjusting for a series of confounders, the effect estimates were small and not significant (Table 3). Compared to the reference group of younger women [<33.9 years] with a good-quality diet, there was no increased risk for fertility problems in the older women, regardless of diet quality.
Table 3 also shows the effect measure modification of diet on the effect of age on fertility problems. A RERI higher than 0 suggests that the effects of the two exposures operating together is higher than that of each added together (the effect measure modification is positive). However, the 95% CIs overlap 0; thus, additive interaction is not supported in women aged 30–38 years.
3.3. Dietary Change and Fertility Status:
Table 4 shows the frequency and percentage of women in the cohort by change in diet quality and whether they had fertility problems in Survey 5. There were no important differences in the proportion of women who improved or had a decrease in diet quality, or had no change in diet quality, across fertility status groups. There was no evidence that fertility was associated with a change in diet from Survey 3 to Survey 5 (Table 5).
4. Discussion:
Among a large cohort of Australian women, we examined whether diet modified the relationship between age and infertility, and whether a change in diet impacts fertility. Our results do not support our hypothesis; that is, in both Survey 3 and in Survey 5, there was little evidence that diet quality modified the association between age and fertility problems. Although increasing age (from Survey 3 to Survey 5) was associated with an approximate 5% higher rate of fertility problems, a change in diet quality over this time was not associated with fertility problems at Survey 5.
Among Survey 3 participants who were aged between 24–31 years, independent associations were found, such that older woman (28.2 to 30.8 years) who had a poor-quality diet had higher risk for infertility problems compared to the younger women (24.4 to <28.2 years) with a good-quality diet. At Survey 5 when women were aged 30–38 years, there was no difference in risk for fertility problems by diet quality or age. This was an unexpected finding. At Survey 5, there was an approximate 10% higher percentage of women with a good-quality diet; thus, with increasing age, the importance of a healthier diet may be realised, irrespective of fertility status. Yet, the older women at Survey 5 did appear to drink slightly more alcohol and perform less metabolic minutes of exercise, which are both associated with higher likelihood for infertility [6,36]. Interestingly, the mean BMI among women with and without fertility problems was consistent over the 5-year period from Survey 3 to Survey 5, with the women with infertility problems maintaining the approximate two-unit higher BMI than the women without infertility problems. The combination of some infertility risk factors increasing over time, but others remaining consistent, may partly explain the lack of association in this slightly older age group of women. Nevertheless, our results might suggest a small window of opportunity in women aged 28–31 years to optimise their diet to support fertility, whereas after this age, the current data do not support this. Although many studies have reported on the independent effects of increasing age with infertility, and increasing evidence shows the importance of a healthy diet for reproductive health, qualitative data reveals that women do not have a clear understanding of the age at which fertility begins to decline [37]. There are also no specific recommendations for an appropriate diet to optimise fertility. The knowledge gaps and misconceptions surrounding reproductive health and nutrition, highlights a critical opportunity that is needed to support patient awareness, counselling to support dietary behaviours, and that involves pregnancy planning.
Our findings provide little evidence of effect modification by diet at either survey. Whilst at Survey 3 there was a suggestion of a negative effect measure modification, such that the effect of age and a good-quality diet was lower than the expected sum of the individual effects of age and diet, the wide 95% CIs are consistent with positive effect modification by diet. At Survey 5, the RERI was positive, suggesting that the effect of age interacting with diet quality was higher than the sum of the independent effects of age and diet, but the wide confidence intervals are also consistent with positive effect modification. Thus, we cannot conclude that better quality diet could attenuate the decline in fertility with age over the observed periods. These findings suggest that the effect of a poor-quality diet and older age on fertility may not be as pronounced as expected, but also that a poor diet quality is not appreciably leading to reduced fertility in older women.
Whilst our results are unexpected, they build the foundation for further studies to assess the interactions between age and diet quality. Although the additive effect modification RERI is the more relevant public health measure, and we would theorise that the targeted group for intervention would be older women with a poor-quality diet, in this instance, we cannot confidently draw this conclusion. Importantly, our results do not mean that a good-quality diet is adverse in any way but implies that a good-quality diet does not attenuate the relationship between age and infertility, and that the joint effect of the two exposures is less than their main effects combined. The indication of a positive RERI at Survey 5 supports the promotion of improved diet quality in older women.
Overall diet quality of women was low in both surveys, which is consistent with previous studies reporting that a poor-quality diet is common amongst women of reproductive age [23,38,39,40,41]. Despite the slight increase in proportion of women having a good-quality diet from Survey 3 to Survey 5, there was no evidence that a change in diet, whether that was an improvement or worsening of diet quality over time, was associated with fertility problems. However, we acknowledge that this change in diet quality is very small and may be equivalent to only replacing white bread with brown (whole meal or multigrain) as the usual bread choice. In line with our findings, a review of studies assessing pregnancy intentions and diet or physical activity behaviours in the preconception and antenatal periods found that women intending to become pregnant do not report different preconception dietary or physical activity behaviours compared with women without pregnancy intentions [42]. This reflects the common barriers observed in women of reproductive age to achieve successful health behaviour change [43]. In our study, the lack of association between a change in diet and fertility may also relate to reverse causation. That is, following a diagnosis of fertility problems, women may alter their behaviours towards healthier choices, or their improvement in dietary intake may not be sufficient to alter the effect on fertility problems.
To the best of our knowledge, this is the first study to assess whether better diet quality can reduce the age-related decline in fertility. A strength of this study is the use of data from a nationally representative cohort of Australian women at two different time points, increasing the generalisability of our findings to all Australian women. We used robust statistical measures, including calculation of the RERI, whereby effect measure modification of diet on the additive scale provides a clear comparison of the effect of diet on the association between one of the major risk factors of fertility (i.e., age) and fertility problems among women. Some limitations are also worth mentioning. A validated, semi-quantitative FFQ was used to capture women’s dietary consumption, which was designed for use in the Australian community. Although the ARFS is an acceptable tool for assessing diet quality, for most foods the scoring was based on ‘at least once per week’. Additional response options might be helpful to quantify diet quality more accurately. While the analysis included a large cohort of women, it may not be sufficiently powered to identify interactions; thus, further work in this area, with larger populations and covering wider age ranges, is warranted. Lastly, the current study included self-reported fertility problems and dietary intakes that could be subject to recall bias.
5. Conclusions:
In conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age.
|
Background:
Increasing age is a strong risk factor for infertility, and there is accumulating evidence of the importance of a healthier diet for fertility. Whether a healthier diet modifies the association between increasing age and infertility has not been investigated. This study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems.
Methods:
Data were from Surveys 3 and 5 of the 1973-1978 birth cohort of the Australian Longitudinal Study on Women's Health. Cross-sectional analysis with multivariable generalized linear models were used to examine the association between age and fertility status, adjusted for various confounders. Multiplicative and additive effect modification by diet quality was assessed, with additive effect modification evaluated with the relative risk for interaction (RERI).
Results:
In total, 3387 women were included from Survey 3 (age range 24-31 years) and 5614 women from Survey 5 (age range 30-38 years); 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems in the respective surveys. In Survey 3, compared to younger women with a good-quality diet, older women with a poor-quality diet had a 43% increased risk for fertility problems, with risk increasing after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62). In Survey 5 in younger women (<33.9 years), there was no association between diet quality and risk for infertility problems. The RERI (across different adjusted models) was between -0.08 (-0.70, 0.55) to -0.39 (-1.40, 0.62) in survey 3 and 0.07 (-0.17, 0.31) to 0.08 (-0.17, 0.32) in Survey 5.
Conclusions:
There is little evidence to suggest effect modification on the effect of age and fertility problems with diet quality.
|
1. Introduction:
Infertility is characterised by the inability of couples to establish pregnancy after a year of regular and unprotected intercourse [1]. Worldwide, infertility impacts up to 15% of reproductive-aged couples [2,3], with prevalence rates varying across countries [4]. Data from the Australian Longitudinal Study on Women’s Health (ALSWH) indicate that one-in-six women aged 28–33 years in 2006 had experienced infertility [5], with longitudinal data across 15 years (2000–2015) from the same study showing the cumulative incidence of fertility problems in women at 14.4% [6]. Infertility contributes considerable public health burden including emotional and psychological distress, social stigmatization, and economic burden [3].
Increasing maternal age is a recognised and well-established risk factor for infertility [7], with age 35 years considered advanced maternal age [8]. In a review of natural fertility populations comprising 58,051 women in developed countries, age-related loss of fertility increased from 4.5% at 25 years of age to 20% at 38 years of age [9]. Global data demonstrate fertility rates declining over time, with greatest declines in younger women and in regions of higher income and education [4]. In Australia, the percentage of women having their first child over the age of 30 has risen from 23% in 1991 to 48% in 2016 [10]. Current data suggest several reasons for this with many women deciding to postpone childbearing to pursue a career, not having a partner, or being unaware of the impact of age on fertility [9,10,11].
Although increasing maternal age is a well-known risk factor for infertility, the fact that age cannot be modified supports the need to examine increasing age in the context of modifiable factors. Obesity is a modifiable factor, consistently associated with infertility [12]. Increasing age is associated with increasing body mass index (BMI), with the rise in obesity prevalence most prominent in women of reproductive age [13]. Whether weight loss in the preconception period is critical to pregnancy success is a topic of current debate [14]. Dietary intake is also a modifiable factor. Research investigating nutritional intake and fertility has flourished over the last two decades, indicating a healthier diet during the preconception period is associated with overall improved fertility outcomes [15,16,17]. Healthier dietary patterns characterised by high consumption of beans, wholegrains, vegetables and fruits [18], greater adherence to a Mediterranean diet [19,20], higher consumption of fruits and lower intakes of fast foods [21], and lower intake of sugar sweetened beverages [22] were associated with shorter time to pregnancy, higher fecundability, higher rates of clinical pregnancy and live birth, and a reduced risk of ovulatory disorder infertility. Interestingly, the study by Chavarro et al. found that the association between the “fertility diet” score and ovulatory infertility was not modified by age, parity, or BMI, with diet composition having a greater apparent impact on fertility than BMI [18]. The study by Karayiannis et al. found that in non-obese women aged <35 years, a 5-point increase in the MedDietScore was associated with ~2.7 times higher likelihood of achieving clinical pregnancy and live birth, but not among women ≥35 years [19].
There is limited information examining dietary intakes across women of different ages, with inconsistent evidence for differences in diet quality between younger and older women [23,24]. Furthermore, whether diet quality modifies the association between age and infertility has not been assessed. Therefore, this study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems. Our hypothesis is that the effects of age on the risk for fertility problems will be less among women with better diet quality.
5. Conclusions:
In conclusion, our findings indicate that diet quality and older age are independently associated with infertility problems at the younger age range of 24–31 years but not at the older age range of 30–38 years. The present observations provide little evidence that better diet quality could attenuate the decline in fertility associated with age. Investigation of other cohorts with longer follow-up times will be helpful to confirm or refute our findings. Intervention studies to assess the effect of improved diet quality in older and younger women and their effect on fertility may also be helpful to better understand the role that diet has in the context of minimising infertility with increasing age.
|
Background:
Increasing age is a strong risk factor for infertility, and there is accumulating evidence of the importance of a healthier diet for fertility. Whether a healthier diet modifies the association between increasing age and infertility has not been investigated. This study aimed to (i) examine if better diet quality could help reduce age-related infertility; and (ii) assess whether changes in diet quality over time are associated with fertility problems.
Methods:
Data were from Surveys 3 and 5 of the 1973-1978 birth cohort of the Australian Longitudinal Study on Women's Health. Cross-sectional analysis with multivariable generalized linear models were used to examine the association between age and fertility status, adjusted for various confounders. Multiplicative and additive effect modification by diet quality was assessed, with additive effect modification evaluated with the relative risk for interaction (RERI).
Results:
In total, 3387 women were included from Survey 3 (age range 24-31 years) and 5614 women from Survey 5 (age range 30-38 years); 588 (17.4%) and 1321 (23.4%) self-reported to have fertility problems in the respective surveys. In Survey 3, compared to younger women with a good-quality diet, older women with a poor-quality diet had a 43% increased risk for fertility problems, with risk increasing after further adjustment for BMI (RR: 1.59; 95% CI: 1.07, 2.37) and PCOS (RR: 1.74; 95% CI: 1.15, 2.62). In Survey 5 in younger women (<33.9 years), there was no association between diet quality and risk for infertility problems. The RERI (across different adjusted models) was between -0.08 (-0.70, 0.55) to -0.39 (-1.40, 0.62) in survey 3 and 0.07 (-0.17, 0.31) to 0.08 (-0.17, 0.32) in Survey 5.
Conclusions:
There is little evidence to suggest effect modification on the effect of age and fertility problems with diet quality.
| 9,634 | 386 | 14 |
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"survey",
"women",
"fertility",
"quality",
"age",
"effect",
"problems",
"years",
"fertility problems"
] |
[
"test",
"test"
] | null |
[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]
| null |
[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]
|
[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]
|
[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]
|
[CONTENT] age | diet quality | reproductive age | Australia | survey | infertility | women [SUMMARY]
|
[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]
| null |
[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]
|
[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]
|
[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]
|
[CONTENT] Female | Humans | Aged | Young Adult | Adult | Infertility, Female | Longitudinal Studies | Cross-Sectional Studies | Australia | Fertility | Diet | Risk Factors [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]
| null |
[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]
|
[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]
|
[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]
|
[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]
|
[CONTENT] age | infertility | women | fertility | factor | diet | pregnancy | study | years | associated [SUMMARY]
| null |
[CONTENT] diet | women | fertility | fertility problems | survey | quality | effect | quality diet | problems | table [SUMMARY]
|
[CONTENT] age | diet | findings | helpful | diet quality older | quality older | age range | older | older age | quality [SUMMARY]
|
[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]
|
[CONTENT] diet | survey | women | fertility | quality | age | effect | problems | years | fertility problems [SUMMARY]
|
[CONTENT] ||| ||| [SUMMARY]
| null |
[CONTENT] 3387 | 3 | 24-31 years | 5614 | Survey 5 | 30-38 years | 588 | 17.4% | 1321 | 23.4% ||| Survey 3 | 43% | BMI | 1.59 | 95% | CI | 1.07 | 2.37 | PCOS (RR: 1.74 | 95% | CI | 1.15 | 2.62 ||| Survey 5 ||| -0.70 | 0.55 | 0.62 | 3 | 0.31 | 0.08 | 0.32 | Survey 5 [SUMMARY]
|
[CONTENT] [SUMMARY]
|
[CONTENT] ||| ||| ||| Surveys 3 and 5 | 1973-1978 | the Australian Longitudinal Study on Women's Health ||| linear ||| ||| ||| 3387 | 3 | 24-31 years | 5614 | Survey 5 | 30-38 years | 588 | 17.4% | 1321 | 23.4% ||| Survey 3 | 43% | BMI | 1.59 | 95% | CI | 1.07 | 2.37 | PCOS (RR: 1.74 | 95% | CI | 1.15 | 2.62 ||| Survey 5 ||| -0.70 | 0.55 | 0.62 | 3 | 0.31 | 0.08 | 0.32 | Survey 5 [SUMMARY]
|
[CONTENT] ||| ||| ||| Surveys 3 and 5 | 1973-1978 | the Australian Longitudinal Study on Women's Health ||| linear ||| ||| ||| 3387 | 3 | 24-31 years | 5614 | Survey 5 | 30-38 years | 588 | 17.4% | 1321 | 23.4% ||| Survey 3 | 43% | BMI | 1.59 | 95% | CI | 1.07 | 2.37 | PCOS (RR: 1.74 | 95% | CI | 1.15 | 2.62 ||| Survey 5 ||| -0.70 | 0.55 | 0.62 | 3 | 0.31 | 0.08 | 0.32 | Survey 5 [SUMMARY]
|
|||||
The I405V and Taq1B polymorphisms of the CETP gene differentially affect sub-clinical carotid atherosclerosis.
|
23039379
|
Cholesteryl ester transfer protein (CETP) plays a major role in lipid metabolism, but studies on the association of CETP polymorphisms with risks of cardiovascular disease are inconsistent. This study investigated whether the CETP gene I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian population sample.
|
BACKGROUND
|
The polymorphisms were analyzed using polymerase chain reaction in 207 adult volunteers. Serum lipid profiles, oxLDL Ab titers, C-reactive protein and tumor necrosis factor-α concentrations and CETP and phospholipid transfer protein (PLTP) activities were determined, and common carotid artery intima-media thickness (cIMT) was measured using ultrasonography.
|
METHODS
|
No differences in cIMT were observed between the presence or absence of the minor B2 and V alleles in either polymorphism. However, inverse correlations between mean cIMT and CETP activity in the presence of these polymorphisms were observed, and positive correlations of these polymorphisms with PLTP activity and oxLDL Ab titers were identified. Moreover, logistic multivariate analysis revealed that the presence of the B2 allele was associated with a 5.1-fold (CI 95%, OR: 1.26 - 21.06) increased risk for cIMT, which was equal and above the 66th percentile and positively interacted with age. However, no associations with the V allele or CETP and PLTP activities were observed.
|
RESULTS
|
None of the studied parameters, including CETP activity, explained the different relationships between these polymorphisms and cIMT, suggesting that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.
|
CONCLUSIONS
|
[
"Adult",
"Aged",
"Autoantibodies",
"Brazil",
"C-Reactive Protein",
"Carotid Artery Diseases",
"Carotid Intima-Media Thickness",
"Cholesterol Ester Transfer Proteins",
"Female",
"Gene Frequency",
"Humans",
"Lipids",
"Lipoproteins, LDL",
"Male",
"Middle Aged",
"Phospholipid Transfer Proteins",
"Polymorphism, Genetic",
"Tumor Necrosis Factor-alpha",
"Young Adult"
] |
3503625
|
Background
|
Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall
[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism
[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis
[3].
The I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels
[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent
[5], and their association with subclinical CVD are less well characterized.
Therefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.
|
Methods
|
Study subjects A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.
The following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)
[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).
The local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.
Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).
HDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation
[7].
CETP
[8] and phospholipid transfer protein (PLTP) activities
[9] in plasma were determined using radioassays with exogenous substrates.
Antibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA
[10].
The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).
HDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation
[7].
CETP
[8] and phospholipid transfer protein (PLTP) activities
[9] in plasma were determined using radioassays with exogenous substrates.
Antibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA
[10].
A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.
The following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)
[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).
The local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.
Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).
HDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation
[7].
CETP
[8] and phospholipid transfer protein (PLTP) activities
[9] in plasma were determined using radioassays with exogenous substrates.
Antibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA
[10].
The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).
HDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation
[7].
CETP
[8] and phospholipid transfer protein (PLTP) activities
[9] in plasma were determined using radioassays with exogenous substrates.
Antibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA
[10].
Polymorphism detection Genomic DNA was extracted
[11] and amplified using polymerase chain reaction to identify the Taq1B
[12] and I405V
[13] polymorphisms.
Genomic DNA was extracted
[11] and amplified using polymerase chain reaction to identify the Taq1B
[12] and I405V
[13] polymorphisms.
Measurement of cIMT A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method
[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.
A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method
[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.
Statistical analysis Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.
All the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis
[15].
Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.
All the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis
[15].
|
Results
|
The frequencies of I405V and Taq1B genotypes in our population sample, represented by the presence (VV, IV) and absence (II) of the V allele and the presence (B2B2, B1B2) and absence (B1B1) of the B2 allele, are presented in Table
1. The frequencies of the II, IV and VV genotypes were 28%, 53% and 19%, respectively, and the frequencies of the B1B1, B1B2 and B2B2 were 33%, 41% and 26%, respectively.
Biochemical parameters in the presence and absence of minor alleles
Data are presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values: Mann–Whitney U test for comparisons between the presence and absence of the minor alleles (V and B2). Significant differences: p≤0.05 in bold. CETP (%) = activity of cholesterol ester transfer protein; HDL-C = high-density lipoprotein cholesterol; PLTP (%) = activity of phospholipid transfer protein; oxLDL Ab = antibodies against oxidized LDL titers; (OD)= optical density.
Allele frequencies were consistent with Hardy–Weinberg equilibrium for the I405V polymorphism (p=0.36) but not the Taq1B (p=0.02), and the MAFs (minor allele frequency) were <45% and <46%, respectively. The linkage disequilibrium between the two polymorphisms was very weak, as evidenced by the D`=0.17.
Clinical and anthropometric characteristics and biochemical data of the investigated population are presented in Tables
2 and
1.
Clinical and anthropometric characteristics in the presence and absence of minor alleles
Data presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values = Mann–Whitney U test for comparisons between the presence and absence of the alleles (V and B2); gender comparisons, Chi-Square test. Significant differences: p≤0.05 in bold. BMI= Body Mass Index; WC= waist circumference; SBP= systolic blood pressure; DBP= diastolic blood pressure; cIMT= carotid intima-media thickness.
Table
3 presents the significant correlations between mean cIMT, oxLDL Ab titers and CETP and PLTP activities.
Significant correlations between cIMT, oxLDL Ab, CETP (%) and PLTP (%) in the presence of minor alleles
r= Spearman´s coefficient; Significant differences: p≤0.05 in bold. cIMT= carotid intima-media thickness; oxLDL Ab= antibodies against oxidized LDL titers; (OD) = optical density; CETP(%) = activity of cholesteryl ester transfer protein; PLTP (%) = activity of phospholipid transfer protein.
The univariate analyses (Table
4) indicated that the female gender, age, HDL-C, BMI and SBP were significantly associated (p value ≤0.05) with higher cIMT.
Univariate Logistic Regression analyses for higher cIMT (above 66 percentile) (
n=
118)
cIMT below and above the 66th percentile, <0.795 (n=76) and >0.795 (n=42), respectively. (ref).=reference level. (cont).= continue variable. OR= Odds ratio for higher cIMT, above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for the Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism, V = minor allele of I405V polymorphism, HDL-C = high-density lipoprotein cholesterol, BMI: body mass index, SBP: systolic blood pressure.
The multivariate analysis (Table
5) demonstrated that age and the presence of the B2 allele were positively related to higher cIMT. Individuals with increased risk exhibited the B2 allele (5.1-fold increase) and higher age (21.2% increase/each year of age).
Multivariate Logistic Regression* analyses for higher cIMT (above 66
th
percentile) (
n=
114)
*Stepwise criteria for selection of variables. cIMT below and above 66th percentile, <0.795 (n=73) and >0.795 (n=41), respectively. (ref).= reference level; (cont).=continue variable. OR= Odds ratio for higher cIMT above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism.
A Chi-Square analysis indicated an interaction between the presence of the B2 allele and age. The combination between age, categorized in ≥ and <50 years (median value), and the presence or absence B2 allele formed 4 groups of individuals who were crossed with cIMTs above and below 0.795 mm (data not shown).
|
Conclusions
|
The I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved.
|
[
"Background",
"Study subjects",
"Biochemical analysis",
"Polymorphism detection",
"Measurement of cIMT",
"Statistical analysis",
"Abbreviations",
"Misc",
"Competing interests",
"Authors’ contributions"
] |
[
"Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.",
"A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].",
"The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].",
"Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.",
"A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.",
"Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].",
"CETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.",
"Eliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.",
"The authors state that they have no conflicts of interest.",
"ESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"Background",
"Methods",
"Study subjects",
"Biochemical analysis",
"Polymorphism detection",
"Measurement of cIMT",
"Statistical analysis",
"Results",
"Discussion",
"Conclusions",
"Abbreviations",
"Misc",
"Competing interests",
"Authors’ contributions"
] |
[
"Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall\n[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism\n[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis\n[3].\nThe I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels\n[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent\n[5], and their association with subclinical CVD are less well characterized.\nTherefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.",
" Study subjects A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nA total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\n Polymorphism detection Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\nGenomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.\n Measurement of cIMT A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\nA single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.\n Statistical analysis Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].\nMann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].",
"A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.\nThe following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)\n[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).\nThe local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.\n Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].\nThe following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].",
"The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).\nHDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation\n[7].\nCETP\n[8] and phospholipid transfer protein (PLTP) activities\n[9] in plasma were determined using radioassays with exogenous substrates.\nAntibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA\n[10].",
"Genomic DNA was extracted\n[11] and amplified using polymerase chain reaction to identify the Taq1B\n[12] and I405V\n[13] polymorphisms.",
"A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method\n[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.",
"Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.\nAll the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis\n[15].",
"The frequencies of I405V and Taq1B genotypes in our population sample, represented by the presence (VV, IV) and absence (II) of the V allele and the presence (B2B2, B1B2) and absence (B1B1) of the B2 allele, are presented in Table \n1. The frequencies of the II, IV and VV genotypes were 28%, 53% and 19%, respectively, and the frequencies of the B1B1, B1B2 and B2B2 were 33%, 41% and 26%, respectively.\nBiochemical parameters in the presence and absence of minor alleles\nData are presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values: Mann–Whitney U test for comparisons between the presence and absence of the minor alleles (V and B2). Significant differences: p≤0.05 in bold. CETP (%) = activity of cholesterol ester transfer protein; HDL-C = high-density lipoprotein cholesterol; PLTP (%) = activity of phospholipid transfer protein; oxLDL Ab = antibodies against oxidized LDL titers; (OD)= optical density.\nAllele frequencies were consistent with Hardy–Weinberg equilibrium for the I405V polymorphism (p=0.36) but not the Taq1B (p=0.02), and the MAFs (minor allele frequency) were <45% and <46%, respectively. The linkage disequilibrium between the two polymorphisms was very weak, as evidenced by the D`=0.17.\nClinical and anthropometric characteristics and biochemical data of the investigated population are presented in Tables \n2 and\n1.\nClinical and anthropometric characteristics in the presence and absence of minor alleles\nData presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values = Mann–Whitney U test for comparisons between the presence and absence of the alleles (V and B2); gender comparisons, Chi-Square test. Significant differences: p≤0.05 in bold. BMI= Body Mass Index; WC= waist circumference; SBP= systolic blood pressure; DBP= diastolic blood pressure; cIMT= carotid intima-media thickness.\nTable \n3 presents the significant correlations between mean cIMT, oxLDL Ab titers and CETP and PLTP activities.\nSignificant correlations between cIMT, oxLDL Ab, CETP (%) and PLTP (%) in the presence of minor alleles\nr= Spearman´s coefficient; Significant differences: p≤0.05 in bold. cIMT= carotid intima-media thickness; oxLDL Ab= antibodies against oxidized LDL titers; (OD) = optical density; CETP(%) = activity of cholesteryl ester transfer protein; PLTP (%) = activity of phospholipid transfer protein.\nThe univariate analyses (Table \n4) indicated that the female gender, age, HDL-C, BMI and SBP were significantly associated (p value ≤0.05) with higher cIMT.\n\nUnivariate Logistic Regression analyses for higher cIMT (above 66 percentile) (\n\nn=\n\n118)\n\ncIMT below and above the 66th percentile, <0.795 (n=76) and >0.795 (n=42), respectively. (ref).=reference level. (cont).= continue variable. OR= Odds ratio for higher cIMT, above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for the Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism, V = minor allele of I405V polymorphism, HDL-C = high-density lipoprotein cholesterol, BMI: body mass index, SBP: systolic blood pressure.\nThe multivariate analysis (Table \n5) demonstrated that age and the presence of the B2 allele were positively related to higher cIMT. Individuals with increased risk exhibited the B2 allele (5.1-fold increase) and higher age (21.2% increase/each year of age).\n\nMultivariate Logistic Regression* analyses for higher cIMT (above 66\n\nth\n\npercentile) (\n\nn=\n\n114)\n\n*Stepwise criteria for selection of variables. cIMT below and above 66th percentile, <0.795 (n=73) and >0.795 (n=41), respectively. (ref).= reference level; (cont).=continue variable. OR= Odds ratio for higher cIMT above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism.\nA Chi-Square analysis indicated an interaction between the presence of the B2 allele and age. The combination between age, categorized in ≥ and <50 years (median value), and the presence or absence B2 allele formed 4 groups of individuals who were crossed with cIMTs above and below 0.795 mm (data not shown).",
"This study investigated whether the I405V and Taq1B polymorphisms modify subclinical atherosclerosis in an asymptomatic population sample.\nPrevious studies present controversial data on the role of Taq1B and I405V in atherosclerosis and CVD. The B2 allele is associated with lower internal carotid artery IMT in men\n[16], but other studies do not identify associations between the Taq1B polymorphism and cIMT\n[17,18]. The present study demonstrated that cIMT did not differ between either allele of the polymorphisms. However, inverse correlations between cIMT and CETP activity were demonstrated in both polymorphisms (Table \n3), which suggest a potential atheroprotective role for CETP in these individuals. A potential anti-inflammatory role for CETP was demonstrated previously\n[19] wherein the induction of acute inflammation produced lower mortality due to sepsis and lower cytokine circulation in mice that express human CETP. Reductions in CETP concentrations in human patients with sepsis are more pronounced in non-survivors\n[20].\nWe identified positive correlations between cIMT and PLTP and oxLDL Ab in the presence of the V and B2 alleles. Previous studies have suggested that PLTP increases the risk of cardiovascular disease in humans\n[21], and the balance of evidence suggests a pro-atherogenic role for oxLDL Ab titers\n[22] in the development of atherosclerosis. These positive correlations and the lack of correlations of these variables in both common B1B1 and II allele homozygotes (CETP p≤0.318 and 0.091, oxLDL Ab p≤ 0.709 and 0.386 and PLTP p≤0.823 and 0.470, respectively) (data not shown) suggest a possible pro-atherogenic role of these markers associated with the polymorphisms. Our sample size was relatively small and different between groups. Therefore, further studies are underway in our lab to further confirm these correlations.\nThe presence of the V allele was associated with lower CETP (24%) and PLTP (29%) activities and higher HDL2-C (13%) concentrations. Patients with coronary disease typically exhibit fewer large HDL particles (HDL2), and the concentration of these large particles is a better predictor of future cardiovascular events than HDL-C concentration\n[23].\nLipoprotein (a) plasma levels were also significantly increased in the presence of the B2 allele. High levels of lipoprotein (a) are significantly associated with the presence, severity and extent of carotid atherosclerosis\n[24], but this increase was not observed as a possible pro-atherogenic factor.\nNo significant differences in CETP activity and HDL-C levels were observed in Taq1B polymorphism, despite a 15.4% reduction in CETP activity in presence of the B2 allele. These results are consistent with a previous study in another Brazilian population, in which no association between Taq1B genotypes and HDL-C levels was observed\n[25].\nA univariate analysis (Table \n4) followed by a multivariate logistic stepwise analysis (Table \n5) demonstrated that the presence of the B2 allele was independently associated with a mean 5.1-fold increased risk for higher cIMT and age interacted with the allele effect. The presence of the B2 allele in the ≥ 50 years age group exhibited the highest risk of higher cIMT in the Chi-Square analysis (data not shown).\nThe effects of the Taq1B variant on circulating plasma lipids may be related to other functional mutations within the CETP gene locus that are in LD\n[26], which may explain the association in our study. However, other factors than the CETP activity may exert different atherosclerotic repercussions in Taq1B and I405V polymorphisms. A multivariate analysis was performed to verify the independence of the associations between the qualitative binary variable cIMT above or below 66th percentile (0.795mm), CETP and PLTP plasma activities and the potential confounding factors, including gender, age, BMI, SBP and HDL-C. Only age exhibited a significant statistical association (OR: 1.247, CI 95%OR: 1.129 – 1.377), which was a strong determinant of cIMT (data not shown). The absence of interactions of other tested parameters with higher cIMT in this study suggests that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.\nOur results focused on several biomarkers that were assessed for the first time in the Brazilian population, and the results were partially based on the correlations between a relatively small numbers of individuals. These results merit further exploration due to the high frequency of the investigated polymorphisms in the present Brazilian sample. Identifying the impact of these polymorphisms is an ongoing aim of our research group.",
"The I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved.",
"CETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.",
"Eliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.",
"The authors state that they have no conflicts of interest.",
"ESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript."
] |
[
null,
"methods",
null,
null,
null,
null,
null,
"results",
"discussion",
"conclusions",
null,
null,
null,
null
] |
[
"Carotid atherosclerosis",
"Carotid intima-media thickness",
"Cholesteryl ester transfer protein",
"Genetic polymorphism"
] |
Background:
Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall
[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism
[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis
[3].
The I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels
[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent
[5], and their association with subclinical CVD are less well characterized.
Therefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.
Methods:
Study subjects A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.
The following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)
[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).
The local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.
Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).
HDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation
[7].
CETP
[8] and phospholipid transfer protein (PLTP) activities
[9] in plasma were determined using radioassays with exogenous substrates.
Antibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA
[10].
The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).
HDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation
[7].
CETP
[8] and phospholipid transfer protein (PLTP) activities
[9] in plasma were determined using radioassays with exogenous substrates.
Antibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA
[10].
A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.
The following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)
[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).
The local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.
Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).
HDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation
[7].
CETP
[8] and phospholipid transfer protein (PLTP) activities
[9] in plasma were determined using radioassays with exogenous substrates.
Antibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA
[10].
The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).
HDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation
[7].
CETP
[8] and phospholipid transfer protein (PLTP) activities
[9] in plasma were determined using radioassays with exogenous substrates.
Antibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA
[10].
Polymorphism detection Genomic DNA was extracted
[11] and amplified using polymerase chain reaction to identify the Taq1B
[12] and I405V
[13] polymorphisms.
Genomic DNA was extracted
[11] and amplified using polymerase chain reaction to identify the Taq1B
[12] and I405V
[13] polymorphisms.
Measurement of cIMT A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method
[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.
A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method
[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.
Statistical analysis Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.
All the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis
[15].
Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.
All the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis
[15].
Study subjects:
A total of 207 asymptomatic volunteers of both sexes (20–75 years) were recruited at the Lipid Outpatient Clinic at the State University (Unicamp) Hospital in Campinas, SP, Brazil.
The following exclusion criteria were used: body mass index (BMI) ≥ 30 kg/m2; a diagnosis of endocrinological, liver or kidney disease; use of drugs that alter the lipid profile; excessive use of alcohol (>75 g/day)
[6]; meeting the “present CVD” criteria (acute myocardial infarction and angina pectoris, as established by electrocardiogram, cardiac-specific enzyme levels, coronary angiography and/or a history of coronary angioplasty or myocardial revascularization).
The local Medical School Ethics Committee approved this study, and all participants signed an informed consent form.
Biochemical analysis The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).
HDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation
[7].
CETP
[8] and phospholipid transfer protein (PLTP) activities
[9] in plasma were determined using radioassays with exogenous substrates.
Antibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA
[10].
The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).
HDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation
[7].
CETP
[8] and phospholipid transfer protein (PLTP) activities
[9] in plasma were determined using radioassays with exogenous substrates.
Antibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA
[10].
Biochemical analysis:
The following blood measurements were performed using Roche Diagnostics (Mannheim, Germany) reagents in an automated system: total cholesterol (CHOD-PAP), triglycerides (TG, GPO-PAP), HDL-C (HDL-C plus 3rd generation), low-density lipoprotein cholesterol (LDL-C plus 2nd generation for TG >400 mg/dL and Friedewald’s equation for TG ≤ 400 mg/dL). Apolipoproteins AI (NAS-ApoA1), B100 (NAS-ApoB) and lipoprotein (a) were obtained from Siemens (USA). C-reactive protein (CRP) was measured using the Tina-quant® CRP (latex) high sensitivity assay (Roche Diagnostics, Switzerland). Plasma levels of tumor necrosis factor α (TNF-α) were measured using ELISA (Cayman Chemical, USA).
HDL2 and HDL3 sub-fractions were obtained using sequential micro-ultracentrifugation
[7].
CETP
[8] and phospholipid transfer protein (PLTP) activities
[9] in plasma were determined using radioassays with exogenous substrates.
Antibodies against oxidized LDL (oxLDL Ab) titers were determined using an in-house ELISA
[10].
Polymorphism detection:
Genomic DNA was extracted
[11] and amplified using polymerase chain reaction to identify the Taq1B
[12] and I405V
[13] polymorphisms.
Measurement of cIMT:
A single trained sonographer, who was blind to the subject’s identity, performed high-resolution B-mode common carotid ultrasonography using a 6–9 MHz linear array ultrasound imaging system (ATL HDI 1500 Ultrasound System, Advanced Technology Laboratories Ultrasound, USA). Longitudinal measurements of segments of the common carotid arteries at the distal wall and 1 cm from the bifurcation were performed according to a standardized method
[14]. The mean common carotid artery intima-media thickness (cIMT) was calculated as the average of five measurements on each side (right and left) and expressed in millimeters.
Statistical analysis:
Mann–Whitney U and Chi-Squared tests compared variables, and the Spearman test was performed for correlations. Univariate and multivariate logistic regression analyses, with stepwise criteria of the variables of interest, the presence of the V and B2 allele, gender, age, HDL-C, BMI and systolic blood pressure (SBP), were performed to verify the relationships of these variables with higher cIMT values, which was defined as above or below the established cutoff value of 0.795 mm and corresponding to cIMT equal and above the 66th percentile values of the studied population.
All the analyses were performed using the SAS®, version 9.1.3 and SPSS 11.5 for Windows. Haploview, v 4.2© was used for the linkage disequilibrium (LD) analysis
[15].
Results:
The frequencies of I405V and Taq1B genotypes in our population sample, represented by the presence (VV, IV) and absence (II) of the V allele and the presence (B2B2, B1B2) and absence (B1B1) of the B2 allele, are presented in Table
1. The frequencies of the II, IV and VV genotypes were 28%, 53% and 19%, respectively, and the frequencies of the B1B1, B1B2 and B2B2 were 33%, 41% and 26%, respectively.
Biochemical parameters in the presence and absence of minor alleles
Data are presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values: Mann–Whitney U test for comparisons between the presence and absence of the minor alleles (V and B2). Significant differences: p≤0.05 in bold. CETP (%) = activity of cholesterol ester transfer protein; HDL-C = high-density lipoprotein cholesterol; PLTP (%) = activity of phospholipid transfer protein; oxLDL Ab = antibodies against oxidized LDL titers; (OD)= optical density.
Allele frequencies were consistent with Hardy–Weinberg equilibrium for the I405V polymorphism (p=0.36) but not the Taq1B (p=0.02), and the MAFs (minor allele frequency) were <45% and <46%, respectively. The linkage disequilibrium between the two polymorphisms was very weak, as evidenced by the D`=0.17.
Clinical and anthropometric characteristics and biochemical data of the investigated population are presented in Tables
2 and
1.
Clinical and anthropometric characteristics in the presence and absence of minor alleles
Data presented as means ± standard deviation; (sample number): n = minimum and maximum of subjects with determined parameters; p values = Mann–Whitney U test for comparisons between the presence and absence of the alleles (V and B2); gender comparisons, Chi-Square test. Significant differences: p≤0.05 in bold. BMI= Body Mass Index; WC= waist circumference; SBP= systolic blood pressure; DBP= diastolic blood pressure; cIMT= carotid intima-media thickness.
Table
3 presents the significant correlations between mean cIMT, oxLDL Ab titers and CETP and PLTP activities.
Significant correlations between cIMT, oxLDL Ab, CETP (%) and PLTP (%) in the presence of minor alleles
r= Spearman´s coefficient; Significant differences: p≤0.05 in bold. cIMT= carotid intima-media thickness; oxLDL Ab= antibodies against oxidized LDL titers; (OD) = optical density; CETP(%) = activity of cholesteryl ester transfer protein; PLTP (%) = activity of phospholipid transfer protein.
The univariate analyses (Table
4) indicated that the female gender, age, HDL-C, BMI and SBP were significantly associated (p value ≤0.05) with higher cIMT.
Univariate Logistic Regression analyses for higher cIMT (above 66 percentile) (
n=
118)
cIMT below and above the 66th percentile, <0.795 (n=76) and >0.795 (n=42), respectively. (ref).=reference level. (cont).= continue variable. OR= Odds ratio for higher cIMT, above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for the Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism, V = minor allele of I405V polymorphism, HDL-C = high-density lipoprotein cholesterol, BMI: body mass index, SBP: systolic blood pressure.
The multivariate analysis (Table
5) demonstrated that age and the presence of the B2 allele were positively related to higher cIMT. Individuals with increased risk exhibited the B2 allele (5.1-fold increase) and higher age (21.2% increase/each year of age).
Multivariate Logistic Regression* analyses for higher cIMT (above 66
th
percentile) (
n=
114)
*Stepwise criteria for selection of variables. cIMT below and above 66th percentile, <0.795 (n=73) and >0.795 (n=41), respectively. (ref).= reference level; (cont).=continue variable. OR= Odds ratio for higher cIMT above the 66th percentile value (0.795 mm), CI 95% OR= Confidence interval of 95% for Odds ratio. Significant differences: p≤0.05 in bold; B2 = minor allele of Taq1B polymorphism.
A Chi-Square analysis indicated an interaction between the presence of the B2 allele and age. The combination between age, categorized in ≥ and <50 years (median value), and the presence or absence B2 allele formed 4 groups of individuals who were crossed with cIMTs above and below 0.795 mm (data not shown).
Discussion:
This study investigated whether the I405V and Taq1B polymorphisms modify subclinical atherosclerosis in an asymptomatic population sample.
Previous studies present controversial data on the role of Taq1B and I405V in atherosclerosis and CVD. The B2 allele is associated with lower internal carotid artery IMT in men
[16], but other studies do not identify associations between the Taq1B polymorphism and cIMT
[17,18]. The present study demonstrated that cIMT did not differ between either allele of the polymorphisms. However, inverse correlations between cIMT and CETP activity were demonstrated in both polymorphisms (Table
3), which suggest a potential atheroprotective role for CETP in these individuals. A potential anti-inflammatory role for CETP was demonstrated previously
[19] wherein the induction of acute inflammation produced lower mortality due to sepsis and lower cytokine circulation in mice that express human CETP. Reductions in CETP concentrations in human patients with sepsis are more pronounced in non-survivors
[20].
We identified positive correlations between cIMT and PLTP and oxLDL Ab in the presence of the V and B2 alleles. Previous studies have suggested that PLTP increases the risk of cardiovascular disease in humans
[21], and the balance of evidence suggests a pro-atherogenic role for oxLDL Ab titers
[22] in the development of atherosclerosis. These positive correlations and the lack of correlations of these variables in both common B1B1 and II allele homozygotes (CETP p≤0.318 and 0.091, oxLDL Ab p≤ 0.709 and 0.386 and PLTP p≤0.823 and 0.470, respectively) (data not shown) suggest a possible pro-atherogenic role of these markers associated with the polymorphisms. Our sample size was relatively small and different between groups. Therefore, further studies are underway in our lab to further confirm these correlations.
The presence of the V allele was associated with lower CETP (24%) and PLTP (29%) activities and higher HDL2-C (13%) concentrations. Patients with coronary disease typically exhibit fewer large HDL particles (HDL2), and the concentration of these large particles is a better predictor of future cardiovascular events than HDL-C concentration
[23].
Lipoprotein (a) plasma levels were also significantly increased in the presence of the B2 allele. High levels of lipoprotein (a) are significantly associated with the presence, severity and extent of carotid atherosclerosis
[24], but this increase was not observed as a possible pro-atherogenic factor.
No significant differences in CETP activity and HDL-C levels were observed in Taq1B polymorphism, despite a 15.4% reduction in CETP activity in presence of the B2 allele. These results are consistent with a previous study in another Brazilian population, in which no association between Taq1B genotypes and HDL-C levels was observed
[25].
A univariate analysis (Table
4) followed by a multivariate logistic stepwise analysis (Table
5) demonstrated that the presence of the B2 allele was independently associated with a mean 5.1-fold increased risk for higher cIMT and age interacted with the allele effect. The presence of the B2 allele in the ≥ 50 years age group exhibited the highest risk of higher cIMT in the Chi-Square analysis (data not shown).
The effects of the Taq1B variant on circulating plasma lipids may be related to other functional mutations within the CETP gene locus that are in LD
[26], which may explain the association in our study. However, other factors than the CETP activity may exert different atherosclerotic repercussions in Taq1B and I405V polymorphisms. A multivariate analysis was performed to verify the independence of the associations between the qualitative binary variable cIMT above or below 66th percentile (0.795mm), CETP and PLTP plasma activities and the potential confounding factors, including gender, age, BMI, SBP and HDL-C. Only age exhibited a significant statistical association (OR: 1.247, CI 95%OR: 1.129 – 1.377), which was a strong determinant of cIMT (data not shown). The absence of interactions of other tested parameters with higher cIMT in this study suggests that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.
Our results focused on several biomarkers that were assessed for the first time in the Brazilian population, and the results were partially based on the correlations between a relatively small numbers of individuals. These results merit further exploration due to the high frequency of the investigated polymorphisms in the present Brazilian sample. Identifying the impact of these polymorphisms is an ongoing aim of our research group.
Conclusions:
The I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved.
Abbreviations:
CETP: Cholesteryl Ester Transfer Protein; HDL-C: High-Density Lipoproteins Cholesterol; CVD: Cardiovascular Disease; BMI: Body Mass Index; TG: TriGlycerides; LDL-C: Low-Density Lipoprotein Cholesterol; Apo: Apolipoprotein; CRP: C-Reactive Protein; TNF-α: Tumor Necrosis Factor-α; PLTP: Phospholipid Transfer Protein; oxLDL Ab: Antibodies titers against oxidized LDL; cIMT: common Carotid Artery Intima-Media Thickness; SBP: Systolic Blood Pressure; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency.
Misc:
Eliane Soler Parra and Natália Baratella Panzoldo contributed equally to this manuscript.
Competing interests:
The authors state that they have no conflicts of interest.
Authors’ contributions:
ESP worked hard on the data analysis, manuscript preparation, and editing. NBP wrote and reviewed the final manuscript. DK participated in the selection of participants, collection of all clinical and laboratory data and clinical examinations. HCFO and JES assisted in the execution of the experimental protocol for the polymorphism detection. LSFC and ACS performed additional statistical analyses and critical discussion. MG conducted the immunological experiments. RTN performed the carotid ultrasonography. VHSZ, ERN and ECRQ critically revised all text and complemented the discussion. ECF designed the study, implemented the clinical arm and coordinated the work. All authors read an approved the manuscript.
|
Background:
Cholesteryl ester transfer protein (CETP) plays a major role in lipid metabolism, but studies on the association of CETP polymorphisms with risks of cardiovascular disease are inconsistent. This study investigated whether the CETP gene I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian population sample.
Methods:
The polymorphisms were analyzed using polymerase chain reaction in 207 adult volunteers. Serum lipid profiles, oxLDL Ab titers, C-reactive protein and tumor necrosis factor-α concentrations and CETP and phospholipid transfer protein (PLTP) activities were determined, and common carotid artery intima-media thickness (cIMT) was measured using ultrasonography.
Results:
No differences in cIMT were observed between the presence or absence of the minor B2 and V alleles in either polymorphism. However, inverse correlations between mean cIMT and CETP activity in the presence of these polymorphisms were observed, and positive correlations of these polymorphisms with PLTP activity and oxLDL Ab titers were identified. Moreover, logistic multivariate analysis revealed that the presence of the B2 allele was associated with a 5.1-fold (CI 95%, OR: 1.26 - 21.06) increased risk for cIMT, which was equal and above the 66th percentile and positively interacted with age. However, no associations with the V allele or CETP and PLTP activities were observed.
Conclusions:
None of the studied parameters, including CETP activity, explained the different relationships between these polymorphisms and cIMT, suggesting that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.
|
Background:
Atherosclerotic disease is a complex multifactorial process that leads to the deposition and accumulation of cholesterol along the arterial wall
[1]. Cholesteryl ester transfer protein (CETP) plays a major role in cholesterol metabolism
[2], and polymorphisms within this gene may underlie the susceptibility to atherosclerosis
[3].
The I405V (rs5882) and Taq1B (rs708272) polymorphisms and their respective minor alleles, V and B2, are associated with decreased CETP activity and increased high-density lipoprotein cholesterol (HDL-C) levels
[4]. However, studies on the association of these polymorphisms with risks of cardiovascular disease (CVD) are inconsistent
[5], and their association with subclinical CVD are less well characterized.
Therefore, this study investigated whether I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian adult sample population.
Conclusions:
The I405V and Taq1B polymorphisms were differently associated with subclinical atherosclerosis because only the presence of the minor B2 allele, but not the V allele, was independently associated with higher cIMT. We demonstrated that none of the studied parameters explained the different relationships between these polymorphisms and cIMT, which leads us to speculate that the association between the B2 allele and cIMT is due to other non-studied factors. Ongoing studies are underway in our laboratory to determine the modulation factors that are involved.
|
Background:
Cholesteryl ester transfer protein (CETP) plays a major role in lipid metabolism, but studies on the association of CETP polymorphisms with risks of cardiovascular disease are inconsistent. This study investigated whether the CETP gene I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian population sample.
Methods:
The polymorphisms were analyzed using polymerase chain reaction in 207 adult volunteers. Serum lipid profiles, oxLDL Ab titers, C-reactive protein and tumor necrosis factor-α concentrations and CETP and phospholipid transfer protein (PLTP) activities were determined, and common carotid artery intima-media thickness (cIMT) was measured using ultrasonography.
Results:
No differences in cIMT were observed between the presence or absence of the minor B2 and V alleles in either polymorphism. However, inverse correlations between mean cIMT and CETP activity in the presence of these polymorphisms were observed, and positive correlations of these polymorphisms with PLTP activity and oxLDL Ab titers were identified. Moreover, logistic multivariate analysis revealed that the presence of the B2 allele was associated with a 5.1-fold (CI 95%, OR: 1.26 - 21.06) increased risk for cIMT, which was equal and above the 66th percentile and positively interacted with age. However, no associations with the V allele or CETP and PLTP activities were observed.
Conclusions:
None of the studied parameters, including CETP activity, explained the different relationships between these polymorphisms and cIMT, suggesting that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.
| 5,263 | 290 | 14 |
[
"cimt",
"allele",
"hdl",
"cetp",
"performed",
"tg",
"protein",
"cholesterol",
"b2",
"presence"
] |
[
"test",
"test"
] |
[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]
|
[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]
|
[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]
|
[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]
|
[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]
|
[CONTENT] Carotid atherosclerosis | Carotid intima-media thickness | Cholesteryl ester transfer protein | Genetic polymorphism [SUMMARY]
|
[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]
|
[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]
|
[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]
|
[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]
|
[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]
|
[CONTENT] Adult | Aged | Autoantibodies | Brazil | C-Reactive Protein | Carotid Artery Diseases | Carotid Intima-Media Thickness | Cholesterol Ester Transfer Proteins | Female | Gene Frequency | Humans | Lipids | Lipoproteins, LDL | Male | Middle Aged | Phospholipid Transfer Proteins | Polymorphism, Genetic | Tumor Necrosis Factor-alpha | Young Adult [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]
|
[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]
|
[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]
|
[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]
|
[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]
|
[CONTENT] cimt | allele | hdl | cetp | performed | tg | protein | cholesterol | b2 | presence [SUMMARY]
|
[CONTENT] polymorphisms | cholesterol | association | atherosclerosis | subclinical | disease | cvd | i405v | taq1b | cetp [SUMMARY]
|
[CONTENT] tg | performed | usa | mg dl | dl | plus | diagnostics | pap | obtained | nas [SUMMARY]
|
[CONTENT] cimt | allele | presence | significant | absence | 05 | minor | 795 | b2 | significant differences 05 bold [SUMMARY]
|
[CONTENT] allele | factors | cimt | studied | associated | b2 allele | polymorphisms | b2 | explained different | explained different relationships [SUMMARY]
|
[CONTENT] cimt | allele | polymorphisms | performed | cholesterol | protein | tg | cetp | b2 | taq1b [SUMMARY]
|
[CONTENT] cimt | allele | polymorphisms | performed | cholesterol | protein | tg | cetp | b2 | taq1b [SUMMARY]
|
[CONTENT] ||| CETP | Brazilian [SUMMARY]
|
[CONTENT] 207 ||| Ab | CETP | PLTP [SUMMARY]
|
[CONTENT] ||| CETP | PLTP ||| ||| 5.1-fold | CI | 95% | 1.26 - 21.06 | 66th ||| CETP | PLTP [SUMMARY]
|
[CONTENT] CETP [SUMMARY]
|
[CONTENT] Cholesteryl ||| CETP | Brazilian ||| 207 ||| Ab | CETP | PLTP ||| ||| CETP | PLTP ||| ||| 5.1-fold | CI | 95% | 1.26 - 21.06 | 66th ||| CETP | PLTP ||| CETP [SUMMARY]
|
[CONTENT] Cholesteryl ||| CETP | Brazilian ||| 207 ||| Ab | CETP | PLTP ||| ||| CETP | PLTP ||| ||| 5.1-fold | CI | 95% | 1.26 - 21.06 | 66th ||| CETP | PLTP ||| CETP [SUMMARY]
|
||||||
Subjective Evaluation of the Results of Injectable Hyaluronic Acid Fillers for the Face.
|
32021131
|
Skin ageing is a physiological process, progressive and irreversible. Hyaluronic acid injection treatments are used to correct the signs of skin ageing.
|
BACKGROUND
|
Hyaluronic acid was implanted in the area of the cheek and the forehead aesthetic units in 57 women, aged 35-55 years. Apart from the clinical observation, self-assessment of the therapeutic results was conducted. The "My skin" questionnaire was used for subjective evaluation of the treatment results.
|
MATERIAL AND METHODS
|
Mean wrinkle score in the pre-menopausal group changed after the treatment, from 3.2±0.6 to 1.1±0.3 and from 3.2±0.6 to 0.8±0.6 for the forehead and the cheek esthetic units, respectively. In the post-menopausal group, the score decreased from 3.8±0.4 to 1.7± 0.7 and from 3.2±0.617 to 0.8± 0.6 for the forehead and the cheek esthetic units, respectively. The changes were age-dependent. Improved appearance of the facial skin - higher satisfaction with skin tone and scent - was reported after hyaluronic acid injections. Higher subjective perception of improvement corresponded to older age, irrespectively of the menopausal status. Correlations between age and the effect of the treatment on maintaining proper skin hydration as well as between improved appearance of the forehead area and feelings of autonomy and well-being were found.
|
RESULTS
|
Hyaluronic acid injections significantly improved the subjective perception and overall assessment of the scent and appearance of the facial skin.
|
CONCLUSION
|
[
"Adult",
"Cosmetic Techniques",
"Face",
"Female",
"Humans",
"Hyaluronic Acid",
"Injections",
"Middle Aged",
"Patient Satisfaction",
"Rejuvenation",
"Skin Aging",
"Treatment Outcome",
"Viscoelastic Substances"
] |
6968800
|
Introduction
|
The skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.
Skin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.
| null | null | null | null |
Conclusions
|
HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being.
|
[
"Objectives",
"Materials and Methods",
"Results",
"Discussion",
"Conclusions"
] |
[
"The aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.",
"A total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).",
"Mean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\n\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.",
"The condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47",
"HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being."
] |
[
null,
null,
null,
null,
null
] |
[
"Introduction",
"Objectives",
"Materials and Methods",
"Results",
"Discussion",
"Conclusions"
] |
[
"The skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.\nSkin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.",
"The aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.",
"A total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.\nHyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.\nAn original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.\nWrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.\nThe study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.\nAs both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.\nWritten informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).",
"Mean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).\nMean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.\nThe main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nMean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.\nSubjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.\nThe respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314\n\nChanges in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF\nNo statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nSubjective evaluation of skin functions depending on patient age before and 30 days after HAF.\nAt the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.\nIn both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.",
"The condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20\nAppleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23\nA gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26\nHA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28\nIn our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.\nIn our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43\nA properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.\nSince the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47",
"HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being."
] |
[
"intro",
null,
null,
null,
null,
null
] |
[
"hyaluronic acid fillers",
"skin",
"women",
"subjective evaluation"
] |
Introduction:
The skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.
Skin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.
Objectives:
The aim of the study was to evaluate the effects of injectable HAF on patient satisfaction with skin appearance, tone and scent, as well as perception of skin function and its connection with physical and psychosocial well-being, before and after treatment, among females undergoing esthetic dermatology treatment.
Materials and Methods:
A total of 57 women (aged 35–55 years), pre-menopausal and post-menopausal, who reported for esthetic dermatology treatment were randomly recruited for the study. The difference in mean patient age was considerable (d=1.8) and statistically significant (t(10.1) = 4.3, p < 0.001). All subjects were healthy and did not take any medicine throughout the entire observation period (30 days±1 day). The patients received detailed information about the aim and the scope of the treatment, possible adverse effects, and the study protocol. Written informed consent was obtained. The inclusion criteria were as follows: informed consent, no history of esthetic dermatology or plastic surgery facial treatments, and no contraindications to treatment. Non-animal stabilized hyaluronic acid (NASHA) was used for facial skin rejuvenation. Restylane® Lyft (formerly Perlane) with Lidocaine is a sterile gel of hyaluronic acid generated by Streptococcus species of bacteria, chemically cross-linked with BDDE, stabilized and suspended in phosphate-buffered saline at pH=7 and concentration of 20 mg/mL with 0.3% lidocaine.
Hyaluronic acid (HA) was injected into facial esthetic units: the cheeks (0.2 mL in 5 lines; 1 mL for each cheek) and the forehead (0.1 mL in 10 lines, 1 mL in total, symmetrically on both sides). HA injections were free of charge.
An original Polish questionnaire known as “My skin”,7 a popular tool in esthetic dermatology, was used for subjective evaluation of the treatment results. Section AB focused on the satisfaction with the following: 1. skin condition in various areas of the body; 2. skin tone, scent and structure; 3. appearance and condition of the hair and the nails. Section C included questions about the perception of physiological and psychosocial skin functions, as well as the relationship between the condition of the skin and physical and emotional well-being. A 5-point Likert scale was used (section AB: “I do not like it at all”, “I do not like it”, “No opinion”, “I like it”, “I absolutely like it”; section C: “I disagree”, “I absolutely disagree”, “No opinion”, “I agree”, “I absolutely agree”). The questionnaire was completed twice – before and 30 days after the treatment. A questionnaire “My skin” is included in the Supplementary materials.
Wrinkle formation on the cheeks and the forehead was measured using the scale from 1 to 5, as proposed by Lemperle et al,8 and assessed before and 30 days after the treatment.
The study was designed to investigate changes in the perception of skin appearance after HA administration. The control variables included age and menstruation. As these variables are connected, a mixed model analysis of covariance (ANCOVA), with age as the covariate, was applied. Jamovi v. 0.8.1.5 [jamovi project (2017)]9 was used for the analysis. Type II sum of squares was used in the analysis due to unequal sample size.
As both groups were unequal in size and age and the variables in section C of the questionnaire are expressed on the weak end of the ordinal scale (the most common scores were 4 or 5), Pearson’s chi-square test with Yule’s correction was used to analyze the differences in the evaluation of skin function after HA injections among pre-menopausal and post-menopausal women. For the same reason, Spearman’s rank was used to evaluate the strength of the relationship between the age of the respondents and their perception of skin function.
Written informed consent was obtained from all subjects. The study was conducted in accordance with the Declaration of Helsinki. Bioethics Committee at the Poznan University of Medical Sciences approved of the study protocol (No. 740/11).
Results:
Mean age of the pre-menopausal participants (n=48; 84%) was 41.8 ± 3.9 years (range: 35–51 years) and was significantly lower (g = 1.77, CI.95[0.99–2.56]) as compared to their post-menopausal peers (n=9; 16%) – 49 ± 4.7 years (range: 39–55 years).
Mean wrinkle score changed after HAF from 3.8 ± 0.4 to 1.7 ± 0.7 for the forehead esthetic unit in the post-menopausal group and from 3.2 ±0.6 to 1.1 ±0.3 in the pre-menopausal group. Mean wrinkle score decreased after HAF from 3.8 ± 0.4 to 1.9 ±0.3 in the post-menopausal group and from 3.2±0.6 to 0.8 ± 0.6 in the pre-menopausal group. A strong relationship was observed between age and the scores of the wrinkle scale for the forehead unit (before treatment: r = 0.610, p < 0.001; after treatment: r = 0.429, p < 0.001) and the cheek (before treatment: r = 0.527, p < 0.001; after treatment: r = 0.588, p < 0.001). Repeated measures ANOVA revealed a statistically significant change in the forehead scores (F(1, 54) = 803.5, p < 0.001, η2p = 0.937). The change was age-related. Higher age corresponded to higher improvement, irrespectively of the menopausal status (F(1. 54) = 1.35, p = 0.251). A similar set of results was observed for the cheek scores. The change was high and statistically significant (F(1, 54) = 1009, p < 0.001, η2p = 0.949), although no relationship with age (rho = −0.163, p = 0.225) or the menopausal status (F(1, 54) = 3.14, p = 0.082) was found.
The main outcome of the treatment, i.e. improved skin appearance in the subjective evaluation of the study participants – proved to be statistically significant (F(1.54) = 350.2, p < 0.001, eta2part = 0.866, CI.95[0.785–0.897]). Higher level of satisfaction with skin appearance in different areas of the body was observed in both groups, demonstrating that entering the period of menopause does not affect the perception of large improvement in skin appearance after injectable HAF therapy (F(1.54) = 0.86, p = 0.358) (Figure 1). Age turned out to be a significant factor affecting the mean scores of skin appearance (F(1.54) = 6.77, p = 0.012). Based on the benchmarks suggested by Cohen (1988), we found that age variable has a significant effect (eta2part = 0.11, CI.95[0.005–0.265]) on the average score of skin appearance, and that the score decreased with age (rbefore = −0.40, p = 0.001; rafter = −0.32, p = 0.007) (Figure 2).Figure 1Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.Figure 2Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.
Mean subjective evaluation of the satisfaction with the skin appearance before and 30 days after HAF among pre-menopausal and post-menopausal women.
Subjective evaluation of the skin appearance depending on the patient age before and 30 days after the HAF.
The respondents evaluated the skin tone and scent. A significantly larger number of the participants reported higher satisfaction with their skin tone and scent after HAF, and fewer women were satisfied with the appearance of their hair. No relationship was found between the treatment and the appearance of the fingernails, but more women reported improved appearance of the toenails after treatment (Table 1).Table 1Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAFParameterDeteriorationNo ChangeImprovementSignificanceEffect SizeSkin tone2 (4%)22 (39%)33 (58%)W = 827.50, z = 5.22, p < 0.001r = 0.691Skin scent3 (5%)22 (39%)32 (56%)W = 844.50, z = 4.93, p < 0.001r = 0.653Hair23 (40%)28 (49%)6 (11%)W = 911.00, z = 5.31, p < 0.001r = −0.703Fingernails5 (9%)40 (70%)12 (21%)W = 1490.00, z = 1.62, p = 0.127–Toenails4 (7%)39 (68%)14 (25%)W = 1480.50, z = 2.37, p = 0.026r = 0.314
Changes in the Evaluation of Individual Skin Parameters and Its Appendages Before and 30 Days After HAF
No statistically significant relationship between the treatment and the total subjective score of skin functions (F(1.54) = 2.52, p = 0.118) was found. Age also did not significantly lower the scores (F(1.54) = 2.31, p = 0.135), although minimal correlations between the variables were observed (rhobefore = −0.24, p = 0.036; rhoafter = −0.28, p = 0.017) (Figure 3).Figure 3Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.
Subjective evaluation of skin functions depending on patient age before and 30 days after HAF.
At the same time, a correlation was found between improved appearance of the forehead area and psychological functions of the skin. The change in skin appearance was positively correlated with the feelings of autonomy (My skin makes me unique; Spearman’s Rho −0.292; p = 0.028) and well-being (My skin affects my well-being; Spearman’s Rho −0.347; p = 0.008).Attempts have been made to investigate whether pre-menopausal and post-menopausal women differed in their perception of skin function after HA treatment among. No significant changes in the perception of the physiological, psychological and social skin functions, responsible for physical and mental well-being, reaction to external stimuli, a sense of autonomy, detachment, and social acceptance, were observed in the majority of cases. Most women, pre-menopausal as well as post-menopausal, did not differ significantly in their perception of the relationship between skin condition and physical and mental well-being before and after the treatment.
In both groups of women, significant differences were found in the perception of the following skin functions: maintaining proper hydration (p < 0.001), and the ability to regenerate (p < 0.001). The distribution in the post-menopausal group was even – the treatment did not affect the pre- and post-test scores and the same number of women reported deterioration, or no change, or improved skin appearance, while in the pre-menopausal group, a significant majority of the subjects reported no change after the treatment. Importantly, it needs to be emphasized that the results are necessarily speculative due to lack of sample size balance (the post-menopausal group was relatively small).A similar analysis was conducted in relation to age, with 45 years as the point of division. A statistically significant correlation was found between age and the effect of the treatment on maintaining proper skin hydration (p < 0.001), with younger women, more frequently reporting deteriorated skin hydration. In the group of the older women, 3 categories (improvement, no change, deterioration) were identified, with no statistically significant differences in the frequency of the scores. As far as the remaining skin functions are concerned, no significant changes between pre- and post-treatment scores were observed.
Discussion:
The condition of the skin reflects the functioning of the entire body as changes in skin temperature, tone, muscle tension and hydration level is not only a source of information about somatic health, but also about the emotional well-being.1,2 Signals transmitted by the appearance of the skin remain beyond the conscious control of the host. Psychological and social functions connected with creating attachment in the childhood and autonomy and identity in the adolescence, shaping self-image, establishing intimate bonds, developing social communication, as well as accepting age-related changes, have long been linked with the condition of the skin.3,4,10 Middle and late adulthood are typically associated with characteristic changes in skin appearance, which are related to the perception of biological age of an individual. The ageing of skin is a multifactorial and progressive process which includes skeletal reabsorption, ligament loosening, muscle atrophy as well as fat pad displacement, consequently leading to overall facial laxity and contour change. Changes in skin appearance may constitute a source of considerable psychological stress, which in turn adversely affects the quality of life in late adulthood.11,12 These changes are usually perceived as unfavorable and have a negative impact on self-esteem and psychosocial well-being. They also have a symbolic significance as they place an individual in the group of “the elderly”, stereotypically associated with undesirable physical, mental and behavioral features. The face is the key determinant of the overall physical attractiveness for both, men and women. Faces with average13,14 values of anthropometric characteristics of a given population as well as symmetric faces15,16 are perceived as highly attractive. Moreover, a positive relationship has been reported between attractiveness and lack of fluctuating asymmetry (FA) in case of both sexes,17 and – in case of women – strongly feminized facial features,18 as well as clear and bright skin.19 Susceptibility to FA is connected with age and varies for different parameters. FA is associated with pigmentation and skin wrinkles as well as with adipose tissue, and develops throughout the entire life, whereas FA of mimic muscle tone is characteristic of ageing and old age.20
Appleton et al,19 found a positive correlation between attractiveness of the female facial skin and healthy (unblemished) skin and facial symmetry. Skin tone and texture also play key roles in the perceived attractiveness of the face. Matts et al,21 revealed a relationship between skin tone and color homogeneity and health, age and attractiveness of the female facial skin. According to Fink et al,22 homogeneous skin, i.e. smooth and with even color and regular texture, correlates positively with attractiveness of a woman as perceived by men. Faces with even skin color were also perceived as healthier and younger.21,22 Skin lacking color and texture homogeneity, with visible hypo or hyper-pigmentations, negatively affects the perceived physical attractiveness of a female.20,23
A gradual decrease in HA concentration and structural changes of collagen fibers, resulting in dehydration and loss of elasticity, accompany the ageing process of the skin.6 HA concentrations among women aged 19–47, or 60, or 70 years have been estimated at approximately 0.03%, 0.015% and 0.007%, respectively.24 The most noticeable changes include loss of skin elasticity and volume, and the appearance of facial fossa and wrinkles. Moreover, up to 80% of the visible signs of skin ageing (dryness with scaling and wrinkling,25 impaired pigmentation and photoaging) are the results of exposure to ultraviolet radiation (UV) and correlate with cancer risk.26
HA injections are one of the most commonly used treatments to correct the age-related skin changes. In photo-aged skin, they stimulate fibroblasts, collagen and elastin production, HA synthesis, extracellular matrix production and epithelial regeneration in the areas undergoing treatment.27 Intradermal injections of stabilized HA also decrease skin porosity and have a beneficial effect on skin elasticity.28
In our study, we found that HA treatment positively influenced the perceived attractiveness of the facial skin, which is consistent with the findings of other authors.29,30 In a study by Baumann et al, the majority of the patients undergoing injectable HA gel therapy for facial fossa reported “a younger look” after treatment.30 According to Wilson et al,31 over 80% of the participants with age-related volume loss of the facial skin reported significant improvement, even up to 12 months after treatment. A high level of satisfaction with the appearance of the facial skin after tear trough and temporal fossa augmentation was noted by Tung et al, and Berguiga et al32,33 Jegasothy et al,34 also reported beneficial effects (wrinkle depth reduction, improved skin hydration, firmness and elasticity) of low molecular nano-hyaluronic acid injections. Positive changes in the appearance of the facial skin after HA treatment (improved firmness and pigmentation) were observed by Landi et al35 Trong et al36 also documented improved skin elasticity and wrinkle depth reduction, while Baspeyras et al,37 observed significant improvement in skin hydration, firmness and viscoelasticity after HA microinjection treatment. A statistically significantly improved radiance, pigmentation and hydration were observed by Sparavigna et al,38 in the process of treating skin with symptoms of ageing and photoaging.
In our study, we found significantly improved skin appearance after HA injections, not only in the areas where the preparation was administered, but also on the torso and the limbs. At the same time, improved appearance of the facial skin after the treatment negatively affected the perceived appearance of the neck and cleavage skin, possibly due to the disproportion between smoothed facial skin and untreated skin of the neck and the cleavage, with visible ageing signs. It remains a challenge to explain the reasons behind improved self-perceived appearance of the untreated torso and limb skin. To the best of our knowledge, no literature sources have reported similar findings. The results of our study proved that subjective satisfaction after HA injections with skin tone and scent, as well as with the appearance of the toenails, was higher. Baspeyras et al,37 and Qian et al,39 also reported improved satisfaction with facial skin tone after HA injections. However, we were not able to find any reports on changes in the level of satisfaction with skin scent and the appearance of the toenails after such treatment. The scent of the body changes in the course of a person’s life.40 Mitro et al,40 proved that their study participants were able to differentiate between scent samples of younger (20–30 and 45–55 years) and older (75–95 years) people. The 2-Nonenal – an unsaturated aldehyde with greasy and grassy odor which is absent in scent samples from younger people but identified in scent samples from people >40 years of age are considered to be a potential biomarker of the ageing process.41,42 Furthermore, the scent of a woman’s body changes depending on the phase of the menstrual cycle and its attractiveness, as perceived by men, is connected with high levels of estradiol and low levels of progesterone.43
A properly hydrated skin is perceived as healthier, and its ageing is definitely slower.44 The positive effect after stabilized HA injections, i.e. healthier looking facial skin, can be sustained for over half a year.45 Marrakchi and Maibach46 found that skin hydration in people over 66 years of age is lower than in younger individuals. In our study, we found the self-reported level of skin hydration after HA injections to be correlated with the age variable – significantly higher differentiation (improvement, no change, deterioration) was observed in the answers of the older women (post-menopausal) as compared to their younger (pre-menopausal) peers, who more frequently reported no change or even deteriorated parameters.
Since the dawn of time, human beings have been altering their appearance. In doing so, they took into consideration – to a lesser or greater degree – the cultural requirements of their times.46 Modern surgical dermatology and plastic surgery have offered various possibilities to alter and modify one’s own appearance. In the past, changes in the body were mainly connected with the passage of time and, consequently, loss of physical and sexual attractiveness. Currently, there is a visible tendency to treat the body as a stage of sorts, a space where individuals display themselves.47
Conclusions:
HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being.
|
Background:
Skin ageing is a physiological process, progressive and irreversible. Hyaluronic acid injection treatments are used to correct the signs of skin ageing.
Methods:
Hyaluronic acid was implanted in the area of the cheek and the forehead aesthetic units in 57 women, aged 35-55 years. Apart from the clinical observation, self-assessment of the therapeutic results was conducted. The "My skin" questionnaire was used for subjective evaluation of the treatment results.
Results:
Mean wrinkle score in the pre-menopausal group changed after the treatment, from 3.2±0.6 to 1.1±0.3 and from 3.2±0.6 to 0.8±0.6 for the forehead and the cheek esthetic units, respectively. In the post-menopausal group, the score decreased from 3.8±0.4 to 1.7± 0.7 and from 3.2±0.617 to 0.8± 0.6 for the forehead and the cheek esthetic units, respectively. The changes were age-dependent. Improved appearance of the facial skin - higher satisfaction with skin tone and scent - was reported after hyaluronic acid injections. Higher subjective perception of improvement corresponded to older age, irrespectively of the menopausal status. Correlations between age and the effect of the treatment on maintaining proper skin hydration as well as between improved appearance of the forehead area and feelings of autonomy and well-being were found.
Conclusions:
Hyaluronic acid injections significantly improved the subjective perception and overall assessment of the scent and appearance of the facial skin.
|
Introduction:
The skin performs a number of vital physiological functions and its condition is a source of reliable, albeit indirect, information about the well-being of the entire body.1,2 Dermal changes (temperature, tone, muscle tension, and hydration) are not only indicative of the somatic well-being, but also the emotional condition of an individual, as signs manifested by the skin are outside their conscious control. The skin is also believed to play a role in a number of psychological and social functions,3,4 which are associated with the realization of developmental tasks in the course of a lifespan.
Skin ageing is a physiological, progressive and irreversible process which is associated with biochemical, morphological and biophysical changes in the body.5 Esthetic dermatology offers a variety of skin treatments to correct age-related changes, including injectable hyaluronic acid-based fillers (HAFs).6 Satisfaction with skin appearance in different areas of the face and the body may increase after esthetic dermatology treatments, thus being indicative of their effectiveness. To the best of our knowledge, only a few studies so far have focused on the subjective evaluation of HAF effectiveness by patients, as most researchers investigate objective and measurable changes in the biophysical parameters.
Conclusions:
HA injections are connected with significant positive changes in the subjective perception and overall evaluation of the appearance and scent of the facial skin. Numerous authors have confirmed a positive influence of the treatment on patient self-esteem. Self-evaluation of the skin appearance after HA treatment is age-related. HA injections are connected with a change in the subjective evaluation of such skin functions as maintaining proper hydration, ability to regenerate, as well as one’s autonomy and well-being.
|
Background:
Skin ageing is a physiological process, progressive and irreversible. Hyaluronic acid injection treatments are used to correct the signs of skin ageing.
Methods:
Hyaluronic acid was implanted in the area of the cheek and the forehead aesthetic units in 57 women, aged 35-55 years. Apart from the clinical observation, self-assessment of the therapeutic results was conducted. The "My skin" questionnaire was used for subjective evaluation of the treatment results.
Results:
Mean wrinkle score in the pre-menopausal group changed after the treatment, from 3.2±0.6 to 1.1±0.3 and from 3.2±0.6 to 0.8±0.6 for the forehead and the cheek esthetic units, respectively. In the post-menopausal group, the score decreased from 3.8±0.4 to 1.7± 0.7 and from 3.2±0.617 to 0.8± 0.6 for the forehead and the cheek esthetic units, respectively. The changes were age-dependent. Improved appearance of the facial skin - higher satisfaction with skin tone and scent - was reported after hyaluronic acid injections. Higher subjective perception of improvement corresponded to older age, irrespectively of the menopausal status. Correlations between age and the effect of the treatment on maintaining proper skin hydration as well as between improved appearance of the forehead area and feelings of autonomy and well-being were found.
Conclusions:
Hyaluronic acid injections significantly improved the subjective perception and overall assessment of the scent and appearance of the facial skin.
| 3,976 | 264 | 6 |
[
"skin",
"appearance",
"treatment",
"age",
"menopausal",
"changes",
"ha",
"facial",
"skin appearance",
"significant"
] |
[
"test",
"test"
] | null | null |
[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]
| null | null |
[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]
|
[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]
|
[CONTENT] hyaluronic acid fillers | skin | women | subjective evaluation [SUMMARY]
|
[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]
| null | null |
[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]
|
[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]
|
[CONTENT] Adult | Cosmetic Techniques | Face | Female | Humans | Hyaluronic Acid | Injections | Middle Aged | Patient Satisfaction | Rejuvenation | Skin Aging | Treatment Outcome | Viscoelastic Substances [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
| null | null |
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] test | test [SUMMARY]
|
[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]
| null | null |
[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]
|
[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]
|
[CONTENT] skin | appearance | treatment | age | menopausal | changes | ha | facial | skin appearance | significant [SUMMARY]
|
[CONTENT] skin | changes | effectiveness | indicative | biophysical | body | number | associated | physiological | treatments [SUMMARY]
| null | null |
[CONTENT] ha injections connected | injections connected | evaluation | ha | positive | self | injections | evaluation skin | ha injections | connected [SUMMARY]
|
[CONTENT] skin | treatment | appearance | ha | age | menopausal | evaluation | changes | facial | haf [SUMMARY]
|
[CONTENT] skin | treatment | appearance | ha | age | menopausal | evaluation | changes | facial | haf [SUMMARY]
|
[CONTENT] ||| [SUMMARY]
| null | null |
[CONTENT] [SUMMARY]
|
[CONTENT] ||| ||| 57 | 35-55 years ||| ||| ||| 3.2±0.6 | 1.1±0.3 | 3.2±0.6 ||| 3.8±0.4 | 0.7 | 0.8± 0.6 ||| ||| ||| ||| ||| [SUMMARY]
|
[CONTENT] ||| ||| 57 | 35-55 years ||| ||| ||| 3.2±0.6 | 1.1±0.3 | 3.2±0.6 ||| 3.8±0.4 | 0.7 | 0.8± 0.6 ||| ||| ||| ||| ||| [SUMMARY]
|
||||
Function and regulation annotation of up-regulated long non-coding RNA LINC01234 in gastric cancer.
|
32011780
|
Accumulated evidences indicate that long non-coding RNAs (lncRNAs) participate in many biological mechanisms. Moreover, it acts as an essential regulator in various human diseases such as gastric cancer (GC). Nevertheless, the comprehensive regulatory roles and clinical significance of most lncRNAs in GC are not fully understood.
|
BACKGROUND
|
In this research, our aim was to investigate the underlying mechanism of lncRNA LINC01234 in GC. Firstly, the usage of qRT-PCR helped to establish expression pattern of LINC01234 in GC tissues. Following this, appropriate statistical tests were applied to analyze the relation between expression level and clinicopathological factors. Ultimately, potential functions and regulatory network of LINC01234 were concluded via GSEA and a series of bioinformatics tools or databases, respectively.
|
METHODS
|
Consequently, at the end of research we found LINC01234 is up-regulated in GC tissues in comparison with adjacent normal tissues. Furthermore, its expression level is correlated with differentiation of patients with GC. It is also important to highlight bioinformatics analysis revealed that LINC01234 is involved in cancer-associated pathways such as cell cycle and mismatch repair. Also, regulatory network of LINC01234 presented a probability in the involvement of tumorigenesis through regulating cancer-associated genes.
|
RESULTS
|
Overall, our results suggested that LINC01234 may play a crucial role in GC.
|
CONCLUSION
|
[
"Adenocarcinoma",
"Aged",
"Biomarkers, Tumor",
"Female",
"Gene Expression Regulation, Neoplastic",
"Gene Regulatory Networks",
"Humans",
"Male",
"MicroRNAs",
"Middle Aged",
"RNA, Long Noncoding",
"Stomach Neoplasms",
"Up-Regulation"
] |
7246363
|
INTRODUCTION
|
Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.
Long non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006
12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.
Over the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.
In this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.
| null | null |
RESULTS
|
Experimental verification of LINC01234 up‐regulation in GC tissues Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).
A, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues
Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).
A, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues
Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.
Relationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients
Abbreviation: SE, standard error.
The sample size is so small that the result may be unbelievable even though P < .05.
P < .05.
Diagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR
In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.
Relationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients
Abbreviation: SE, standard error.
The sample size is so small that the result may be unbelievable even though P < .05.
P < .05.
Diagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR
Potential functions of LINC01234
To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.
Potential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)
To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.
Potential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)
Regulatory network of LINC01234
LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).
Regulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues
TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).
Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).
miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32
A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32
RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).
A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).
LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).
Regulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues
TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).
Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).
miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32
A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32
RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).
A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).
| null | null |
[
"INTRODUCTION",
"Differently expression analysis of lncRNAs in STAD from TCGA",
"Collection of GC samples and patients' clinical information",
"Total RNA extraction and qRT‐PCR",
"Gene set enrichment analysis of LINC01234\n",
"Construction of LINC01234 regulatory network",
"TF‐LINC01234 regulation",
"miRNA‐LINC01234 interactions",
"RBP‐LINC01234 interactions",
"Statistical analysis",
"Experimental verification of LINC01234 up‐regulation in GC tissues",
"Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients",
"Potential functions of LINC01234\n",
"Regulatory network of LINC01234\n",
"TF‐LINC01234 regulation",
"miRNA‐LINC01234 regulation",
"RBP‐LINC01234 regulation"
] |
[
"Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.",
"Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.",
"Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.",
"The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.",
"By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.",
" TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.",
"We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.",
"The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n",
"Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.",
"IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.",
"Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues",
"In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR",
"To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)",
"LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).",
"Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).",
"A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n",
"A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4)."
] |
[
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] |
[
"INTRODUCTION",
"MATERIALS AND METHODS",
"Differently expression analysis of lncRNAs in STAD from TCGA",
"Collection of GC samples and patients' clinical information",
"Total RNA extraction and qRT‐PCR",
"Gene set enrichment analysis of LINC01234\n",
"Construction of LINC01234 regulatory network",
"TF‐LINC01234 regulation",
"miRNA‐LINC01234 interactions",
"RBP‐LINC01234 interactions",
"Statistical analysis",
"RESULTS",
"Experimental verification of LINC01234 up‐regulation in GC tissues",
"Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients",
"Potential functions of LINC01234\n",
"Regulatory network of LINC01234\n",
"TF‐LINC01234 regulation",
"miRNA‐LINC01234 regulation",
"RBP‐LINC01234 regulation",
"DISCUSSION",
"Supporting information"
] |
[
"Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.\nLong non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006\n12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.\nOver the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.\nIn this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.",
" Differently expression analysis of lncRNAs in STAD from TCGA Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\nFragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.\n Collection of GC samples and patients' clinical information Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\nPaired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.\n Total RNA extraction and qRT‐PCR The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\nThe methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.\n Gene set enrichment analysis of LINC01234\n By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\nBy using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.\n Construction of LINC01234 regulatory network TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\n Statistical analysis IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.\nIBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.",
"Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.",
"Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.",
"The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.",
"By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.",
" TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\nWe downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.\n miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\nThe conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n\n RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.\nLikewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.",
"We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234\n, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.",
"The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23\n",
"Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.",
"IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.",
" Experimental verification of LINC01234 up‐regulation in GC tissues Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\nFirstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues\n Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\nIn the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR\n Potential functions of LINC01234\n To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\nTo explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)\n Regulatory network of LINC01234\n LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nLncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).",
"Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).\nA, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues",
"In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.\nRelationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients\nAbbreviation: SE, standard error.\nThe sample size is so small that the result may be unbelievable even though P < .05.\n\nP < .05.\nDiagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR",
"To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.\nPotential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)",
"LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).\nRegulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues\n TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\nSome of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).\n miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\nA total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n\n RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).\nA total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).",
"Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).",
"A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32\n",
"A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).",
"LncRNA is a type of non‐coding RNAs without the ability of encoding proteins; despite this, it has a regulating gene expression at chromatin modification, transcriptional, or post‐transcriptional levels.35 In addition, the polymorphisms in lncRNAs could be a risk of disease or cancer.36 In fact, an increasing number of lncRNAs have been identified to be related to numerous kinds of diseases,37 including GC. For example, through the expression analysis of metabolic pathway‐related lncRNAs and protein‐coding genes, a dozen of lncRNAs were functionally annotated and discovered to be important in GC.38\nDGCR9, another lncRNA up‐regulated in GC, was shown to promote the tumorigenesis of GC.\n39 Some of lncRNAs were even indeed considered as biomarkers for diagnosis or prognosis of GC. For instance, a metabolism‐related lncRNA, RP11‐555H23.1, was found to be a potential diagnostic biomarker in GC.40 Also, the expression level of H19 in plasma could be served as a biomarker for patients with GC.41 Due to the regulatory role of lncRNAs, exploring new lncRNA biomarkers can help to explain the initiation and progression mechanism of GC. Nevertheless, still there are many unknown lncRNAs in GC nowadays.\nThe previous study found LINC01234 is highly expressed in esophageal squamous cell carcinoma (ESCC). Also, it is one of the three lncRNAs that can be a signature to predict the survival time of ESCC patients accurately.42, 43, 44 Besides, it was likewise discovered to be up‐regulated in the GC in prior study.26 However, the functions and regulatory role of LINC01234 need to be studied.\nIn this research, we identified that LINC01234 was also highly expressed in GC tissues compared with adjacent non‐cancerous tissues. Later, we explored the associations between the expression level of LINC01234 and clinical features through which we found LINC01234 was correlated with differentiation of GC. LncRNAs usually interact with other kinds of molecules to involve in multiple biological processes. For example, the binding of lncRNA OLC8 and IL‐11 will impair the degradation of IL‐11 mRNAs to accelerate GC development.45 Besides, combination of lncRNA and its target may increase the diagnostic value of lncRNA. Just as it was found by previous report that the combined use of RP11‐19P22.6‐001 and its target NOS2 may be useful to diagnose patients with GC.46 Thus, we further explored the potential functions and regulatory network of LINC01234. We identified 218 relationships of LINC01234 in total. Among them, 17 associations including two pairs of TF regulation and 17 pairs of RBP interaction were found to be co‐expressed. One of 2 TFs, ELK1, is an important regulator and known to activate many lncRNAs including TRPM2‐AS, MIR100HG, and HOXA10‐AS in cancer until now,47, 48, 49 indicating ELK1 may induce expression of LINC01234 to promote tumor progression in GC too.\nIn conclusion, the results of this present study indicated that LINC01234 expression is linked with the diagnostics of patients with GC and similarly may be involved in differentiation in GC through cell cycle or other cancer‐related pathways.",
" \nClick here for additional data file.\n \nClick here for additional data file.\n \nClick here for additional data file.\n \nClick here for additional data file."
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"materials-and-methods",
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"results",
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[
"gastric cancer",
"\nLINC01234\n",
"long non‐coding RNA",
"regulatory network"
] |
INTRODUCTION:
Gastric cancer (GC) is a complex disease caused by accumulation of both genetic and epigenetic factors and imposes a considerable global health burden.1 In fact, in 2016, there were 1.2 million cases of GC with 834 000 deaths worldwide and 18.3 million DALYs (disability‐adjusted life years).2 Even though scientists are making great efforts and there is a steady decline in GC incidence and mortality rates, it is still hard to diagnose GC patients at early stage. To illustrate, this means most GC patients are missing their opportunity for radical gastrectomy, which is currently the best way to cure GC when diagnosed.3, 4 Additionally, many patients have a significant risk of metastasis and low survival time even after curative resection. Thus, it is vital to identify new effective biomarkers and therapeutic target agents for the treatment and early diagnosis of GC.
Long non‐coding RNAs (lncRNAs) are a kind of non‐coding RNAs (lacking the ability of encoding protein) with a length larger than 200 nt. Even though the same as mRNAs, lncRNAs are also transcribed out of DNA by RNA polymerase II; they were initially thought to be a noise in transcriptome when first found.5 Despite this, accumulated studies established that lncRNAs function as regulators of gene expression, stability, and location at the epigenetic, transcriptional, and post‐transcriptional levels.6, 7 Thus, aberrance of lncRNA expression is involved in numerous biological processes such as cell cycle, cell differentiation, proliferation, apoptosis, metastasis, invasion, and migration in several kinds of cancer including GC.8, 9 Also, some lncRNAs can indeed be used as a biomarker for the diagnosis and prognosis in many kinds of cancers such as breast cancer10 and GC.11 For example, LINC1006
12 was found to be a novel biomarker for GC previously. Above all, lncRNAs are important in both the initiation and development of GC.
Over the last decades, numerous experimental researches have identified several lncRNAs that play crucial role in GC such as imprinted maternally expressed transcript (H19),13 small nucleolar RNA host gene 5 (SNHG5),14 homeobox transcript antisense RNA (HOTAIR),15 AGAP2 antisense RNA 1 (AGAP2‐AS1),16 and Pvt1 oncogene (PVT1).17 Yet they were only a tip of iceberg, there remain a large number of lncRNAs with unknown functions and regulation mechanism in GC. Due to the advances of sequencing technology, more and more high‐throughput data of transcriptome in GC were carried out. The Cancer Genome Atlas (TCGA) collects sequencing data of genome, transcriptome, and epigenome from many patients with various kinds of cancer including stomach adenocarcinoma (STAD). It provides an opportunity to dig out unknown genes especially for those lncRNAs in GC.
In this research, we first analyzed gene expression profiles of STAD patients in TCGA and found a number of lncRNAs differently expressed in cancerous tissues compared with adjacent non‐cancerous tissues. Then, we verified one of the up‐regulated lncRNA, LINC01234, in GC tissues compared with adjacent non‐cancerous tissues by real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Also, the association between expression level of LINC01234 and clinicopathological factors was analyzed. Subsequently, we annotated the functions of LINC01234 using Gene Set Enrichment Analysis (GSEA) method and constructed the LINC01234 regulatory network to well interpret the regulation mechanism of LINC01234 in GC.
MATERIALS AND METHODS:
Differently expression analysis of lncRNAs in STAD from TCGA Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.
Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.
Collection of GC samples and patients' clinical information Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.
Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.
Total RNA extraction and qRT‐PCR The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.
The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.
Gene set enrichment analysis of LINC01234
By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.
By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.
Construction of LINC01234 regulatory network TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234
, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.
We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234
, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.
miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23
The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23
RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.
Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.
TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234
, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.
We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234
, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.
miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23
The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23
RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.
Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.
Statistical analysis IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.
IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.
Differently expression analysis of lncRNAs in STAD from TCGA:
Fragments per Kilobase of transcript per Million fragments mapped (FPKM) expression profiles and clinical information of STAD patients were downloaded from TCGA website (https://portal.gdc.cancer.gov/). There are 375 cancerous samples and 32 adjacent normal samples (Table S1). Long intergenic non‐coding RNA (lincRNA) and antisense RNAs were selected as lncRNAs and were analyzed by t test. False discovery rate (FDR) method was used to correct P values. Those with FDR < 0.05 and fold change larger than 1.5 were considered to be as differently expressed lncRNAs.
Collection of GC samples and patients' clinical information:
Paired cancerous and adjacent normal tissues of 83 GC patients were collected during surgery in the span of 2010 to 2015 at Zhejiang Cancer Hospital. The adjacent normal tissues were defined as those tissues located 5 cm away from the edge of the tumor. All the samples with a size of around 0.1 cm3 were immediately preserved in RNA fixer (BioTeke) and stored at −80°C until use. For each GC patient, the clinical information consisted of age, gender, invasion depth, differentiation, lymphatic metastasis, distal metastasis, and TNM stage. It is important to state no patient had undergone preoperative radiotherapy or chemotherapy. Also, each patient had handed over a written consent with a signed name indicating they are willing to participate in this research and the ethics committee of Ningbo University approved for this investigation.
Total RNA extraction and qRT‐PCR:
The methods for total RNA preparation and qRT‐PCR were analogous to our previous study.18 For instance, we extracted the total RNA using TRIzol reagent (Thermo Fisher Scientific) from each cancer tissue and adjacent normal tissue. From here, we were able to detect total RNA by using a protein‐nucleic acid spectrophotometer according to A260/280 ratio. Hereafter, 2 μg RNA was reverse‐transcribed into cDNA with GoTaq qPCR Master Mix (Promega) and the process of qRT‐PCR was performed on LightCycler 480 (Roche). The sequences of PCR primers for β‐actin were 5′‐CATGTACGTTGCTATCCAGGC‐3′ (forward) and 5′‐CTCCTTAATGTCACGCACGAT‐3′ (reverse). On the other hand, the sequences of PCR primers for LINC01234 were 5‐TCTACTAGAGCCTCCAGAAGG‐3′ (forward) and 5‐CTACTCTTCACGCAGAGGA‐3′ (reverse). Importantly, the conditions of thermal cycling were as follows: predegeneration at 95°C in 10 minutes, after which 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The expression level of LINC01234 was calculated using the ΔCt method with β‐actin expression value as control, which was calculated by subtracting the Ct values of β‐actin from the Ct values of LINC01234. Then, ΔΔCt of LINC01234 was calculated by subtracting ΔCt of adjacent non‐cancerous tissue from that of the paired cancer tissue. At last, the fold change of LINC01234 was calculated by the equation 2−ΔΔCt. All results were described as the expression of mean ± standard deviation of three independent experiments.
Gene set enrichment analysis of LINC01234
:
By using the median expression level of LINC01234 as cutoff, STAD patients were divided into two groups: with low expression and high expression of LINC01234, respectively. Subsequently, FPKM expression profiles for STAD patients and group labels of samples were put into GSEA software.19 Gene Ontology (GO) Biological Process (BP) term and KEGG pathway datasets were selected to calculate the enriched functions and pathways associated with LINC01234. Adjusted P‐value < .05 was considered to be statistically significant.
Construction of LINC01234 regulatory network:
TF‐LINC01234 regulation We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234
, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.
We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234
, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.
miRNA‐LINC01234 interactions The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23
The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23
RBP‐LINC01234 interactions Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.
Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.
TF‐LINC01234 regulation:
We downloaded the genomic location of peaks of transcription factor (TF) from Cistrome databases,20 which re‐calculated ChIP‐seq datasets for TF and histone modification from GEO database.21 Next, we compared the chromosome position of these binding regions with that of LINC01234
, only those with the binding sites locating promoters of LINC01234 were considered as TF‐LINC01234 regulation relationships. Then, TFs were filtered by differently expressed protein‐coding genes in GC identified from STAD expression profiles from TCGA by using t test. False discovery rate (FDR) method was used to correct P‐values for multiple comparisons, and .05 was set as a cutoff.
miRNA‐LINC01234 interactions:
The conclusion of miRNA‐LINC01234 interactions was established upon reliable miRNA target prediction tool known as miRanda set on default parameters.22 Due to the up‐regulation of LINC01234 in GC, only those miRNAs down‐regulated in GC were obtained according to miRCancer database.23
RBP‐LINC01234 interactions:
Likewise, prediction of RBP‐LINC01234 interactions was set by utilizing a model called lncPro24 using sequence information downloaded from UniProt.25 Afterward, RBP was also filtered by removing non‐differently expressed protein‐coding genes in STAD from TCGA.
Statistical analysis:
IBM SPSS 21.0 software (SPSS) and R 3.3.3 were the two software used to perform statistical analysis. Comparison of “expression values” among three or more groups was analyzed by one‐way analyses of variance (ANOVAs), while that between two groups was performed by Student's t test. Statistical differences were set at *P < .05, **P < .01, and ***P < .001. P < .05 was set to analyze the statistical significances.
RESULTS:
Experimental verification of LINC01234 up‐regulation in GC tissues Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).
A, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues
Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).
A, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues
Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.
Relationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients
Abbreviation: SE, standard error.
The sample size is so small that the result may be unbelievable even though P < .05.
P < .05.
Diagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR
In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.
Relationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients
Abbreviation: SE, standard error.
The sample size is so small that the result may be unbelievable even though P < .05.
P < .05.
Diagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR
Potential functions of LINC01234
To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.
Potential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)
To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.
Potential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)
Regulatory network of LINC01234
LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).
Regulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues
TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).
Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).
miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32
A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32
RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).
A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).
LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).
Regulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues
TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).
Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).
miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32
A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32
RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).
A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).
Experimental verification of LINC01234 up‐regulation in GC tissues:
Firstly, we downloaded the expression profiles of STAD from TCGA and investigated the differently expressed lncRNAs. Consequently, 1016 up‐regulated and 140 down‐regulated lncRNAs in GC compared with non‐cancer tissues were found (Figure 1A, Table S2). Among them, one of up‐regulated lncRNAs, LINC01234, was selected to study deeply because of poor knowledge of it in GC. We then verified the disorder expression pattern of LINC01234 using qRT‐PCR in 83 GC tissues and adjacent normal tissues (Figure 1B). Hence, by comparing the adjacent non‐cancerous tissues, it is concluded that LINC01234 is strictly up‐regulated in 61 of 83 GC tissues (73.5%, Figure 1C, P < .001).
A, Differently expressed lncRNAs in STAD. B, Expression level (FPKM) of LINC01234 in STAD tissues compared with adjacent normal tissues. C, Expression level (△Ct value) of LINC01234 in GC tissues compared with adjacent normal tissues
Association analysis between expression level of LINC01234 and clinicopathological factors in GC patients:
In the previous study, LINC01234 was considered to be a potential diagnostic marker in GC based on the data of TCGA.26 Consequently, we evaluated the likely diagnostic value of LINC01234 based on our own dataset. Initially, we performed a statistical analysis to examine the relationship between the clinicopathological factors and the expression level of LINC01234. As a result, we found differentiation of GC was associated with LINC01234 expression, that means the lower the LINC01234 expression is, the more the possibility for poor differentiation of GC tissues is (P < .05, Table 1). Besides, P value of the test for association between distal metastasis and LINC01234 expression is <0.05. However, the sample size of GC patients with M1 stage is not enough (n = 5) that the result may be unbelievable. Other clinicopathological factors including age, gender, greatest tumor dimension, invasion depth, lymphatic metastasis, and TNM stage are not related to LINC01234 level. In addition, we further explored the ability of differentiation of GC tissues from the normal adjacent tissues by a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was 0.888 for TCGA dataset (95% CI, 0.848‐0.929; P < .05, Figure 2A) while 0.664 for our qRT‐PCR results (95% CI, 0.581‐0.748; P < .05, Figure 2B), indicating that LINC01234 plays a prominent role in GC tumorigenesis.
Relationship between LINC01234 expression level (ΔCt value) and clinicopathological factors of GC patients
Abbreviation: SE, standard error.
The sample size is so small that the result may be unbelievable even though P < .05.
P < .05.
Diagnostic value of LINC01234 in GC. A, ROC curve of LINC01234 predicting STAD samples using FPKM value from TCGA. B, ROC curve of LINC01234 predicting GC samples using △Ct value detected by qRT‐PCR
Potential functions of LINC01234
:
To explore the potential functions of LINC01234 in GC, we firstly divided the STAD patients from TCGA into two groups, low expression and high expression of LINC01234 in cancer tissues, respectively. Secondly, GSEA was performed to investigate biological processes or pathways that were associated with LINC01234. Thus, the results showed LINC01234 may be involved in cancer and immune‐related pathways such as cell cycle (Figure 3A), mismatch repair (Figure 3B), intestinal immune network for IgA production (Figure 3C), and B‐cell receptor signaling pathway (Figure 3D). In the case of GO BP, cancer‐associated functions were found such as negative regulation of tumor factor–mediated signaling pathway (Figure 3E) and positive regulation of cell migration are involved in sprouting angiogenesis (Figure 3F). These findings present a strong evidence that LINC01234 has a major role in GC formation.
Potential functions of LINC01234 in GC. GSEA showed that aberrant expression of LINC01234 would affect the genes involved in cancer‐related pathways such as cell cycle (A), mismatch repair (B), intestinal immune network for IgA production (C), B‐cell receptor signaling pathway (D), negative regulation of tumor factor–mediated signaling pathway (E), and positive regulation of cell migration involved in sprouting angiogenesis (F)
Regulatory network of LINC01234
:
LncRNAs have been discovered to interact with various types of molecules including DNA, miRNA, mRNA, and protein. To analyze the regulation mechanism of LINC01234 in GC, we constructed a regulatory network of LINC01234 by utilizing a series of bioinformatics tools and databases. This network included TF‐lncRNA regulation, miRNA‐lncRNA relationship, as well as lncRNA‐RBP interactions. In total, 31 TFs, 49 miRNAs, and 138 RBPs associated with LINC01234 were achieved (Table S3, Figure 4).
Regulatory network of LINC01234 in GC. The central node is LINC01234. Rectangle nodes represent TF, circle nodes represent RBP, and green triangle nodes represent miRNA. Red color represents high expression in GC, while blue color represents low expression in GC. Color strength represents log2 value of fold change in GC tissues to adjacent normal tissues
TF‐LINC01234 regulation Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).
Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).
miRNA‐LINC01234 regulation A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32
A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32
RBP‐LINC01234 regulation A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).
A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).
TF‐LINC01234 regulation:
Some of the 31 TFs have thoroughly participated in GC development. For example, FOXK2 inhibited the proliferation, invasion, and migration of GC cells, and its down‐regulation is related to poor prognosis in GC patients.27 Besides, HDAC2 was significantly up‐regulated in various histopathologic grades of human GC, and the inactivation of HDAC2 has been confirmed to reduce cell motility, cell invasion, clonal expansion, and tumor growth.28 Specifically, 2 TFs were found to co‐express with LINC01234 according to the co‐expression network we previously constructed.29 They are ELK1 and ZNF664 (Table S4).
miRNA‐LINC01234 regulation:
A total of 49 miRNAs were predicted to regulate LINC01234 in GC. Several of them have already been accepted to be correlated with GC progression. Also, the overexpression of miR‐1284 was reported to be a suppressor for GC by controlling over cell proliferation and apoptosis.30 In fact, a prior study showed miR‐1284 might modulate multidrug resistance of GC cells by targeting specific genes.31 The miR‐1297 expression found to be remarkably lower in GC tissue and suppress GC cell growth by inhibiting the expression of CREB1.32
RBP‐LINC01234 regulation:
A total of 138 RBPs were predicted to likely interact with LINC01234. Among them, a part of RBPs was already shown to be related to GC. For example, reports revealed DDX21 could affect the proliferation of GC cells by up‐regulating levels of cyclin D1 and CDK2.33 Likewise, the suppression of EIF3B inhibits the proliferation and metastasis of GC by effectively modulating the expression of cancer‐related genes.34 Likewise, we also found 15 co‐expression relationships in RBP interactions such as CPSF3, DDX18, DKC1, and FUS (Table S4).
DISCUSSION:
LncRNA is a type of non‐coding RNAs without the ability of encoding proteins; despite this, it has a regulating gene expression at chromatin modification, transcriptional, or post‐transcriptional levels.35 In addition, the polymorphisms in lncRNAs could be a risk of disease or cancer.36 In fact, an increasing number of lncRNAs have been identified to be related to numerous kinds of diseases,37 including GC. For example, through the expression analysis of metabolic pathway‐related lncRNAs and protein‐coding genes, a dozen of lncRNAs were functionally annotated and discovered to be important in GC.38
DGCR9, another lncRNA up‐regulated in GC, was shown to promote the tumorigenesis of GC.
39 Some of lncRNAs were even indeed considered as biomarkers for diagnosis or prognosis of GC. For instance, a metabolism‐related lncRNA, RP11‐555H23.1, was found to be a potential diagnostic biomarker in GC.40 Also, the expression level of H19 in plasma could be served as a biomarker for patients with GC.41 Due to the regulatory role of lncRNAs, exploring new lncRNA biomarkers can help to explain the initiation and progression mechanism of GC. Nevertheless, still there are many unknown lncRNAs in GC nowadays.
The previous study found LINC01234 is highly expressed in esophageal squamous cell carcinoma (ESCC). Also, it is one of the three lncRNAs that can be a signature to predict the survival time of ESCC patients accurately.42, 43, 44 Besides, it was likewise discovered to be up‐regulated in the GC in prior study.26 However, the functions and regulatory role of LINC01234 need to be studied.
In this research, we identified that LINC01234 was also highly expressed in GC tissues compared with adjacent non‐cancerous tissues. Later, we explored the associations between the expression level of LINC01234 and clinical features through which we found LINC01234 was correlated with differentiation of GC. LncRNAs usually interact with other kinds of molecules to involve in multiple biological processes. For example, the binding of lncRNA OLC8 and IL‐11 will impair the degradation of IL‐11 mRNAs to accelerate GC development.45 Besides, combination of lncRNA and its target may increase the diagnostic value of lncRNA. Just as it was found by previous report that the combined use of RP11‐19P22.6‐001 and its target NOS2 may be useful to diagnose patients with GC.46 Thus, we further explored the potential functions and regulatory network of LINC01234. We identified 218 relationships of LINC01234 in total. Among them, 17 associations including two pairs of TF regulation and 17 pairs of RBP interaction were found to be co‐expressed. One of 2 TFs, ELK1, is an important regulator and known to activate many lncRNAs including TRPM2‐AS, MIR100HG, and HOXA10‐AS in cancer until now,47, 48, 49 indicating ELK1 may induce expression of LINC01234 to promote tumor progression in GC too.
In conclusion, the results of this present study indicated that LINC01234 expression is linked with the diagnostics of patients with GC and similarly may be involved in differentiation in GC through cell cycle or other cancer‐related pathways.
Supporting information:
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Background:
Accumulated evidences indicate that long non-coding RNAs (lncRNAs) participate in many biological mechanisms. Moreover, it acts as an essential regulator in various human diseases such as gastric cancer (GC). Nevertheless, the comprehensive regulatory roles and clinical significance of most lncRNAs in GC are not fully understood.
Methods:
In this research, our aim was to investigate the underlying mechanism of lncRNA LINC01234 in GC. Firstly, the usage of qRT-PCR helped to establish expression pattern of LINC01234 in GC tissues. Following this, appropriate statistical tests were applied to analyze the relation between expression level and clinicopathological factors. Ultimately, potential functions and regulatory network of LINC01234 were concluded via GSEA and a series of bioinformatics tools or databases, respectively.
Results:
Consequently, at the end of research we found LINC01234 is up-regulated in GC tissues in comparison with adjacent normal tissues. Furthermore, its expression level is correlated with differentiation of patients with GC. It is also important to highlight bioinformatics analysis revealed that LINC01234 is involved in cancer-associated pathways such as cell cycle and mismatch repair. Also, regulatory network of LINC01234 presented a probability in the involvement of tumorigenesis through regulating cancer-associated genes.
Conclusions:
Overall, our results suggested that LINC01234 may play a crucial role in GC.
| null | null | 9,795 | 252 | 21 |
[
"gc",
"linc01234",
"expression",
"tissues",
"regulation",
"cell",
"stad",
"lncrnas",
"patients",
"cancer"
] |
[
"test",
"test"
] | null | null | null |
[CONTENT] gastric cancer |
LINC01234
| long non‐coding RNA | regulatory network [SUMMARY]
| null |
[CONTENT] gastric cancer |
LINC01234
| long non‐coding RNA | regulatory network [SUMMARY]
| null |
[CONTENT] gastric cancer |
LINC01234
| long non‐coding RNA | regulatory network [SUMMARY]
| null |
[CONTENT] Adenocarcinoma | Aged | Biomarkers, Tumor | Female | Gene Expression Regulation, Neoplastic | Gene Regulatory Networks | Humans | Male | MicroRNAs | Middle Aged | RNA, Long Noncoding | Stomach Neoplasms | Up-Regulation [SUMMARY]
| null |
[CONTENT] Adenocarcinoma | Aged | Biomarkers, Tumor | Female | Gene Expression Regulation, Neoplastic | Gene Regulatory Networks | Humans | Male | MicroRNAs | Middle Aged | RNA, Long Noncoding | Stomach Neoplasms | Up-Regulation [SUMMARY]
| null |
[CONTENT] Adenocarcinoma | Aged | Biomarkers, Tumor | Female | Gene Expression Regulation, Neoplastic | Gene Regulatory Networks | Humans | Male | MicroRNAs | Middle Aged | RNA, Long Noncoding | Stomach Neoplasms | Up-Regulation [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] test | test [SUMMARY]
| null |
[CONTENT] gc | linc01234 | expression | tissues | regulation | cell | stad | lncrnas | patients | cancer [SUMMARY]
| null |
[CONTENT] gc | linc01234 | expression | tissues | regulation | cell | stad | lncrnas | patients | cancer [SUMMARY]
| null |
[CONTENT] gc | linc01234 | expression | tissues | regulation | cell | stad | lncrnas | patients | cancer [SUMMARY]
| null |
[CONTENT] gc | lncrnas | transcriptome | gene | rna | kinds | patients | cancerous tissues | tissues | non [SUMMARY]
| null |
[CONTENT] gc | linc01234 | expression | cell | tissues | figure | regulation | related | proliferation | mir [SUMMARY]
| null |
[CONTENT] gc | linc01234 | expression | tissues | cell | lncrnas | regulation | set | 05 | stad [SUMMARY]
| null |
[CONTENT] ||| GC ||| GC [SUMMARY]
| null |
[CONTENT] GC ||| GC ||| ||| [SUMMARY]
| null |
[CONTENT] ||| GC ||| GC ||| GC ||| Firstly | GC ||| ||| ||| ||| GC ||| GC ||| ||| ||| GC [SUMMARY]
| null |
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